editorial 1 screening for prostate cancer: what are the benefits? murad lesmana department of microbiology medical faculty, trisakti university univ med vol. 31 no.1 in men of advanced age, the epithelial cells of the prostate gland undergo substantial stressassociated changes that may result in dna damage. the aging prostate gland acquires several cancer cell foci originating from special clonal transformation. most of these foci never develop into clinically detectable cancer.(1) in the united states, prostate cancer ranks second as the cause of death of all cancers affecting men. with the increasing use of screening for prostate-spesific antigen (psa), the mortality rate from prostate cancer has decreased. for example, the mortality rate in 1997 was far below that recorded for 1986 when psa tests were seldom performed. there is a consensus of opinion among clinicians that the psa test has the highest predictive value for prostate cancer, and that psa screening may help detect prostate cancer in its early stages. however, there is no consensus on the level of psa that may be used as an indicator for performing biopsy. this difference in opinion is based on the following: defining a higher psa threshold carries the risk of loss of the probability of detecting prostate cancer until too late, while a lower threshold unnecessarily increases such measures as biopsies. the use of a psa threshold value of 4.0 ng/ml for men older than 50 years has been accepted by most clinicians as a middle way for the above issue. however, there is a lack of information on the prevalence of prostate cancer detectable by biopsy among persons with a psa value of > 4.0 ng/ml without indications for biopsy.(2) recently the u.s. preventive services task force (uspstf) has issued a recommendation statement against the use of psa tests as the basis for prostate cancer screening, after this test had been extensively applied for that purpose in the last couple of decades.(3) all guidelines recommend that clinicians discuss beforehand with the patients the advantages and disadvantages of this test and to leave the decision to the patients individually in accordance with their choice. to help the patients make their choice, clinicians are recommended to provide information on the uncertainties and risks, in addition to the potential benefits of this test. among the benefits may be that screening may lead to early detection of prostate cancer, whereas one of the risks is that screening may result in serious complications for the patients due to the effect of interventions. however, for both clinicians and patients, it is not easy to reach a decision on this issue, because it includes a number of unanswered possibilities, for example, what is the best cut-off point for psa? at which concentration or value is it considered necessary to perform repeat psa tests, or is there a need for performing biopsy? should a repeat test be indicated, when should it be performed? how high is the probability that psa tests in combination with biopsy can detect cancer, assuming the existence of prostate cancer? the most important question is: can screening 2 univ med reduce the risk of death from prostate cancer? because of the high number of possibilities evaluated on the pros and contras, reaching informed decisions may be said to be difficult; as most of these decisions reflect the patients’ general concern about cancer or their inclination to accept (or reject) medical intervention.(4) since 2009, clinicians’ considerations about the need for performing psa tests are becoming increasingly more difficult to deal with, because of the indefinite results of 2 large studies and the fact that experts in this field are still not agreed on their interpretation.(5) discussion with patients on the advantages and disadvantages of psa tests has becomes increasingly more difficult, because now clinicians must explain to patients why there is uncertainty about the results of psa screening and why two large studies are still incapable of providng certainty. another variable causing problems is the management of psa values in primary health care. many psa values are near the threshold commonly used to determine measures, this being in the range of 2.5 – 4.0 ng per ml. the patients are then to be retested a number of times at arbitrary intervals. frequently cven the increase in psa values causes the patients to be prescribed quinolones that are intended to reduce the psa values in these asymptomatic patients who are assumed to have prostatitis. these attitudes reflect either a fundamental misunderstanding or an unwillingness to accept the limitations of the psa test as a marker for early detection of prostate cancer. observational studies clearly indicate that psa values fluctuate spontaneously above or below any threshold level, while random biopsies may detect prostate cancer in 12% of men with psa values of < 2 ng/ml and in 25% of men whose psa values are between 2.1 and 4.0 ng/ml. the latter percentage almost equals the prevalence of prostate cancer frequently found in men with psa values of 4.0 – 10.0 ng/ml. one problem that is of no less importance and is frequently ignored is the economic factor, i.e. the costs to be paid by individuals in connection with psa screening. this includes repeat screenings in cases of equivocal psa results or interpretation of fluctuating results of psa screening. in addition the patients have to return many times to the clinicians for discussion of the equivocal test results. it is the required time and costs that frequently receive less attention in the management of psa screening. according to the european randomized study of screening for prostate cancer (erspc) in estimating the risk of prostate cancer, the probability of finding cancer by biopsy is 21% in a 65-year old man who has decided to undergo biopsy on the basis of a psa value of 4.0 ng/ml. if this man has a routine urological examination, comprising digital rectal examination (dre) and estimation of prostatic volume by transrectal ultrasonography (tu) or other methods, the expected value of biopsy results changes dramatically. if a normal dre is found without any suspicious lesions being found on tu, and the prostatic volume is 55 ml, the risk of a positive biopsy result decreases to 8%. if dre and tu results are abnormal, even with a prostatic volume of 25 ml, the risk of positive biopsy is 65%. therefore, inclusion of other results dramatically changes the indications for biopsy on the basis of psa tests. in view of the abovementioned factors, presumably the paradigm of psa screening is less in step with the priorities of health management. nevertheless, consideration for performing psa screening psa should not be ignored by assuming that it is of no benefit at all. however, the decision to perform psa screening should be made individually by patients who are clearly informed and after discussing it with their physicians, with consideration of various relevant factors. 3vol. 31 no.1 references 1. gelmann ep. complexities of prostate cancer risk. n engl j med 2008;358:961-3. 2. carter h. prostate cancers in men with low psa levels – must we find them? n engl j med 2004;350: 2292-4. 3. u.s. preventive services task force. screening for prostate cancer: u.s. preventive services task force recommendation statement. ann intern med 2008;149:185-91. 4. barry mj. screening for prostate cancer among men 75 years of age or older. n engl j med 2008;350:25156. 5. schwarttz lm, woloshin s, fowler fj jr, welch hg. enthusiasm for cancer screening in the united states. jama 2004;291:71-8. c:\users\universa medicina\docu 1 primary health care for all: lessons from its failure to achieve its goal and covid-19 erlangga yusuf department of medical microbiology and infectious diseases, erasmus mc university medical center, rotterdam, the netherlands according to the world health organization (who), primary health care (phc) can be defined as a whole-of-society approach to health and well-being, centered on the needs and preferences of individuals, f amilies and communities.(1) in the last five years, several major events occurred that emphasize the importance of and the need for phc. first, the publication of the united nations sustainable development goals (sdgs) in 2015.(2) one of the sdgs is to achieve universal health care (uhc). uhc means that all people can get access to health service without it resulting in financial hardship, and the who believes that phc is fundamental to achieving uhc.(3) second, a new international declaration on phc has been released in astana, kazakhstan, in 2018.(4) this is a new declaration and global commitment on phc, four decennia after the first declaration on phc in alma-ata, in the former soviet union. despite the great beginning that culminated in the alma-ata declaration, phc failed to reach the aim of 'health for all by 2000'. in the year 2000, there was still inequity in the access to health care.(5) part of this failure can be explained by the rivalry between the phc and the 'selective' phc proponents. the 'selective' phc concept was launched almost immediately after alma-ata in 1978. as the word 'selective' implies, selective phc covered only a narrow package of low-cost technical interventions to tackle the main disease problems of poor countries, for example by the introduction of growth monitoring, or al rehydration, breastfeeding, and immunization program (gobi). this was the reaction of the 'selective' phc supporters who considered that the broad phc was slow, and even impossible to achieve because it was too comprehensive and too optimistic. the division was even more difficult to reconcile since the choice of public health priorities in the 'selective' phc was done by policy makers (top down), which was exactly the contrary of what the phc was aiming, i.e. public participation. despite the phc failure to achieve health for all and the success of gobi, in the four decades since alma-ata, the recognition of the importance of the declaration's original vision for phc never really faded.(6) yet, it is clear that the commitment to phc from the government is needed more than ever, to make phc a success. regarding 'broad' phc versus 'selective' phc, it should be realized that broad phc can go hand in hand with 'selective' phc because they share many important beliefs. they both consider poverty, deprivation, malnutrition, lack of education, and a gross maldistribution of resources as important problems causing ill health.(7) similarly, they believe that cheap interventions could prevent many infant, child, and maternal deaths and some other illnesses and deaths. supporters of 'selective' phc put too much emphasis on economic aspects and costefficiency while they may forget that holistic approach and putting permanent infrastructure (as strived for in the original version of phc) has universa medicina january-april 2021 vol.40no.1 *email: angga.yusuf@gmail.com orcid id: 0000-0003-4779-2191 2 many other effects that cannot be measured using money and cur renc ie s, such as selfempowerment and happiness. it is important to end the discussion and to decide to choose one direction. broad phc is indeed ambitious. therefore, it can be expected that achieving properly functioning phc is a long, and painstaking process. let it grow at its own speed, and accept that the process is more an evolution than a revolution. at this moment, we are at the dawn of economic recession due to the corona virus disease of 2019 (covid-19) pandemic. the recession will hit many countries, including developing countries. it is not unthinkable that these countries are going to knock on the door of the international monetary fund (imf) in the future for a loan. in the past, the imf that is an institutional embodiment of neoliberalism (an idea that is shared by several supporters of 'selective' phc), requested structural adjustment policies to the countries that needed loans from it.(8) this approach may be counterproductive to the phc. the neoliberal idea puts emphasis on the minimum influence of governments and is associated with free-market capitalism and often with austerity. struc tural adj ustmen t policies r equired governments to reduce spending, including subsidies and investment in health care. the imf considered health as a commodity, for which fees should be paid for services. consequently, access to health care became limited. many countries have overlooked primary care services and the primary health-care strategy as the vehicle for tackling disease outbreaks.(9) in conclusion, phc failed to achieve 'health for all by 2000' but it has led to an unexpected twist with the success of 'selective' phc. the recent renewal of commitment from many governments of the world on phc in astana, kazakhstan in 2018 is the first important step in reaching uhc. the possible economic impact due to the covid-19 pandemic may pose a thr eat to phc and uhc, a nd should be anticipated. references 1. world health organization. primary health care. geneva: world health organization; 2019. 2. barredo l, agyepong i, liu g, reddy s. ensure healthy lives and promote well-being for all at all ages. un chronicle 2015;51:4. https://doi.org/ 10.18356/3bfe3cfa-en. 3. van weel c, kidd mr. why strengthening primary health care is essential to achieving universal health coverage. cmaj 2018; 190:e4636. doi: 10.1503/cmaj.170784. 4. world health organization. declaration on primary health care. astana: world health organization; 2018. 5. haider w. why health for all by 2000 policy (hfa 2000) failed to achieve its goal? ann king edw med univ 2019;25:1-3. 6. world health organization regional office for europe. from alma-ata to astana: primary health care reflecting on the past, transforming for the future. copenhagen, denmark: who regional office for europe; 2018. 7. world health organization. why is primary health care important? geneva: world health organization; 2019. https://www.who.int/newsroom/fact-sheets/detail/primary-health-care. 8. clift b. the imf and the politics of austerity in the wake of the global financial crisis. oxford: oxford university press; 2018. 9. rasanathana k, evans tg. primary health care, the declaration of astana and covid-19. bull world health organ 2020;98:801-8. doi: http:// dx.doi.org/10.2471/blt.20.252932. universa medicina january-april 2021 vol.40no.1 univ med 2021;40:1-2. doi: http://dx.doi.org/10.18051/univmed.2021.v40.1-2 c:\users\user\documents\41 no 2 101 reflections on the concept of sedentary behavior during the covid-19 pandemic leandro dragueta delfino* postgraduate program in movement sciences, são paulo state university/unesp, brazil the topic of sedentar y behavior has permeated the scientific community for some time now; however, the covid-19 pandemic resulted in increased sedentary time irrespective of loc kdown conditi ons or popula tion. (1 ) therefore, it is opportune to br ing some reflections on the concept of sedentary behavior, taking into account its literal, historical and teleological aspects. the acts of conceptualizing, which consists of defining something, and that of classifying, which is nothing more than separating and organizing by classes using some methodology or system, are human actions carried out to select significant data on a given theme. in addition to having a relevant didactic character, concepts and classifications deserve special attention in their elaboration, as they will compose a whole systematized set. according to young et al.,(2) the daily estimate of time spent by adults in different contexts of energy expenditure is: 8.3 hours in sleeping; 7.7 hours in sedentary behavior; 7.8 hours in light activities and 0.2 hours in moderate or vigorous physical activities. the importance of understanding the meanings of the concepts of sedentary behavior and light physical activity is evident, since 98% of an adult’s daily waking time is spent on these activities. sedentary behaviors are typically defined by physical activity with low energy expenditure, with a metabolic rate generally less than 1.5 metabolic equivalents of tasks (mets), and in a sitting or reclining posture.(3) the sedentary be h a vi o r re s e a r c h ne t wo r k ( sbrn) ( 4 ) suggested that journal editors formally define sedentary behavior as any waking behavior characterized by an energy expenditure of less than 1.5 met’s in a sitting, reclining or lying position. complementing the classification, there is physical activity of light intensity defined as any activity with energy expenditure between 1.5 and 3.0 mets; moderate intensity physical activity, any activity with a met value between 3.0 and 5.9; and that of vigorous intensity with values 6 met’s.(5) these consensus definitions, as presented here, were derived to assist with the standardization, or at least harmonization, of measurement procedures, data processing, and data analytics. this editorial aims to reflect the need to understand sedentary time and behavior, and their relationship(s) with health outcomes, and may be more important than ever with the emergence of the novel coronavirus 2019 disease (covid-19). coronavirus disease (covid-19) has severely impacted lifestyles worldwide. responses to covid-19 have intentionally been restricted to the factors that encourage regular and frequent physical activity (pa), namely opportunity, c apability and motivation. there is a universal need to address the low levels of physical activities postcovid-19. the consequences of decreased physical activity across all intensities has powerful, potentially recoverable impacts. it is evident that sedentary behavior is present day after day in the contemporary lifestyle, characterized by the high availability universa medicina may-august 2022 vol.41no.2 *email: ledragueta@hotmail.com orcid id: 0000-0002-5359-8910 102 of technologies that leads the individual to develop a monotonous life, with little movement and minimum physical effort;(4) in view of this, the fact that the concept of sedentary behavior is restricted to an activity with reduced energy expenditure only, and only in the lying, sitting or reclining position, not only seems inappropriate, but completely disregards the etymological and historical interpretation of the concept. in april 2020, over 50% of the global population were subj ect to some form of government restrictions, many of which may h a ve h a d un i nt e n d e d d e l e t e r io us h e a l t h consequences.(6) more specifically, homestay strategies may have increased sitting and screen time, due to children participating in online learning and adults working from home, whilst decreasing opportunities to break-up prolonged periods of sedentary time or behavior.(7) the “compendium of physical activities: classification of energy costs of human physical activities” provides a comprehensive list of physical activity met values for use in scoring physical activity questionnaires.(8) once a met value is obtained for a physical activity performed, an activity score can be computed. a physical activity score can be a simple ordinal number, with higher numbers reflecting greater levels of activity, or a volume score computed by multiplying the frequency in sessions per week (or month), minutes per session, and intensity of the activity recalled. the intensity often is expressed as mets. several activities are being classified as sedentary, simply because they are performed in the sitting position, not observing the energy expenditure, and at the same time there are activities that according to the criterion of energy expenditure are considered sedentary, but because they are performed in the orthostatic position, do not fit the concept due to the postural requirement. concepts and classifications must not give rise to any kind of uncertainty, inaccuracy and imprecision; in addition, the criteria and methods used to classify must not be applied to just one subject under any justification, as they distort the system; and the interpretation, to be well performed, must take into account the literal context of the word, as well as the historical (its representation in the past) and teleological context. for all the above the most plausible thing is to understand the concept of sedentary behavior as “any waking behavior characterized by an energy expenditure of 1.5 met’s or less”. acknowledgements funding source: this study was supported in part by the coordenação de aperfeiçoamento de pessoal de nível superior – capes (finance code 001). references 1. runacres a, mackintosh ka, knight rl, et al. impact of the covid-19 pandemic on sedentary time and behaviour in children and adults: a systematic review and meta-analysis. int j environ res public health 2021;18:11286. doi: 10.3390/ ijerph182111286. 2. young dr, hivert mf, alhassan s, et al. sedentary behavior and cardiovascular morbidity and mortality: a science advisory from the american heart association. circulation 2016; 134:e262-279. doi: 10.1161/cir.0000000000000440. 3. saunders tj, chaput jp, tremblay ms. sedentary behaviour as an emerging risk factor for cardiometabolic diseases in children and youth. can j diabetes 2014;38:53–61. doi: 10.1016/ j.jcjd.2013.08.266. 4. tremblay ms, aubert s, barnes jd, et al. sedentary behavior research network (sbrn) – terminology consensus project process and outcome. int j behav nutr phys act 2017;14: 75. doi: 10.1186/s12966-017-0525-8. 5. van der ploeg hp, hillsdon m. is sedentary behaviour just physical inactivity by another name? int j behav nutr phys act 2017;14:142. doi : 10.1186/s12966-017-0601-0. 6. world health organisation. coronavirus disease 2019 (covid-19) situation report 51; geneva (switzerland): world health organization;2020. 7. unicef for every children. rethinking screentime in the time of covid-19. new york: unicef; 2020. universa medicina may-august 2022 vol.41no.2 103 univ med 2022;41:101-3. doi: http://dx.doi.org/10.18051/univmed.2022.v41.101-103 8. ainsworth be, haskell wl, herrmann sd, et al. 2011 compendium of physical activities: a second update of codes and met values. med sci sports exerc 2011;43:1575-81. doi: 10.1249/mss. 0b013e31821ece12. universa medicina may-august 2022 vol.41no.2 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1298 c:\users\universa medicina\docu 187 being aware of the neurological manifestations of covid-19 infection basak atalay* department of radiology, istanbul medeniyet university, school of medicine the novel cor onavirus disease-2019 (covid-19) was first reported from china, but is unfortunately no longer restricted and has spread over all geographic areas.(1) the main symptoms of covid-19 infection are respiratory tract related. infected patients usually have symptoms such as fever, sore throat, and fatigue, and they may have atypical symptoms like headache, seizures, anosmia, stroke, and even impaired consciousness.(2,3) although most patients with covid-19 have a mild or moderate illness, up to 5-10% have a life-threatening disease course, with a mortality rate of about 2%.(4) strong evidence of an association between covid-19 infection and neur ological manifestations has been reported.(5) besides, the neurologic manifestations may be the initial presentation of covid-19 infection.(6) although it is now accepted that neurological involvement and complications may accompany, studies on this topic are being added to the literature every day. one of the most extensive systematic r eviews about neur ological manifestations of covid-19 infection involved 6335 patients from 49 studies. acute ischemic and hemorrhagic strokes or cerebral sinus venous thrombosis were the most common neurological complications, whereas headache, dizziness, nausea, vomiting, and confusion were the most common neurological symptoms. encephalopathy, encephalitis, oculomotor nerve palsy, anosmia, and guillain-barré syndrome were the other covid19 relevant neurological disorders.(5) while there have been attempts to explain the increased stroke rates in covid-19 patients by hypercoagulopathy that may have been induced by inflammation, this issue remains open to research.(7) other neurological findings can be explained by the passage of infected leukocytes through the blood-brain barrier or the direct olfactory pathway of the neurotropic virus.(8) once the body is in contact with the virus, a cytokine storm can be triggered by a dysregulated immune host response. this cytokine storm may lead to neurological diseases such as encephalitis or acute necrotizing encephalopathy.(9) with disseminated intravascular coagulation arterial and venous thromboembolic phenomena in the severely ill covid-19 patients have been reported.(10) also isolated sudden onset of anosmia has been reported as an early symptom of covid-19 infection. the interesting point is that covid-19 patients can have anosmia without signs of nasal obstruction and rhinorrhea. whether this is caused by damage to epithelial tissue or by retrograde transmission to the central nervous system is not clear yet.(11) de sp i t e e x t e n si ve d a t a on t ypi c a l respiratory signs of covid-19 infection, radiological data on neurological signs that may accompany or may be an initial finding are still lacking. in addition, many patients have not been evaluated by imaging and some have normal imaging findings. it is also difficult to obtain imaging, especially for patients in intensive care units. universa medicina september-december 2021 vol.40no.3 *e-mail: basak_hosgoren@yahoo.com orcid id: 0000-0003-3318-3555 invited editorial 188 the fact that patients with neurological findings or with worsening conditions during follow-up have been reported increasingly, awareness of complications such as stroke, encephalitis, and seizures, which are of vital importance, raises the importance of brain imaging and the role of the radiologist in terms of not only the detection of pulmonary infection findings but also brain involvement. at the beginning of the pandemic, people were asked not to visit hospitals, unless it was required, in our country as in the whole world, according to the restrictions and recommendations of the health ministry and social media to avoid covid-19 transmission and preserve hospital capacity. as a consequence, we observed a dec rease in both the numb er of hospi tal admissions and imaging. this situation, which we initially observed in non-covid hospital admissions, has gained a different dimension in this dynamic period where varied data are constantly flowing. with the increasing cases of covid-19 infection, a significant rise in the number of inpatients and intensive care patients was observed. in our practice, one of turkey's largest university hospitals, we also observed stroke, spontaneous intracerebral hemorrhage, meningitis, encephalitis, and signal intensity c h a n ge s amon g i ma gi n g mod a li t i e s. we experienced that the most common neurological c o mp l i c a ti on wa s s t r o ke a n d t ha t bo t h hemorrhagic and ischemic strokes could be seen. co ns i de r ing t h a t min i ma l o r t r a ns i e n t neurological findings such as transient ischemic attack (tia) can be neglected by patients and overlooked by clinicians, it is expected that we have seen more frequent stroke complications caused by large vessel occlusions. also, patients referred to the radiology department with a prediagnosis of headache, anosmia, tremor, move me nt d i so r d e r s , a nd s e izur e s w e r e evaluated for any abnormality on computed tomography (ct) or magnetic resonance (mr) imaging. nevertheless, most of the patients with these symptoms did not show abnormal findings on imaging. although the justification of neurovirulent potential seems to be speculative, the referring clinicians and radiologists should consider the possibility of central nervous system transmission and its possible sequelae in covid-19 patients with neurological symptoms. although the reasons for the tendency to thromboembolism and stroke, and also to the neurological manifestations are still not obvious, both the clinicians and radiologists should be aware of this possibility. the necessity of imaging of the brain is essential for an exact diagnosis. regardless of any direct or indirect neurological manifestations, the covid-19 pandemic has a huge impact on the management of neurological patients. using a global network with standardized protocols and common data elements is critical to facilitate further studies to under stand covid-19 neurological manifestations. references 1. zhu n, zhang d, wang w, et al. a novel coronavirus from patients with pneumonia in china, 2019. n engl j med 2020;382:727-33. doi: 10.1056/nejmoa2001017. 2. guan wj, ni zy, hu y, et al. on behalf of the china medical treatment expert group for covid19. clinical characteristics of coronavirus disease 2019 in china. n engl j med 2020;382:1708-20. doi: 10.1056/nejmoa2002032. 3. jiang f, deng l, zhang l, cai y, cheung cw, xia z. review of the clinical characteristics of coronavirus disease 2019 (covid-19). j gen intern med 2020;35:1545-9. doi: 10.1007/s11606020-05762-w. 4. gavriatopoulou m, ntanasis-stathopoulos i, korompoki e, et al. emerging treatment strategies for covid-19 infection. clin exp med 2021;21:167-79. doi: 10.1007/s10238-020-00671-y. 5. collantes mev, espiritu ai, sy mcc, anlacan vmm, jamora rdg. neurological manifestations in covid-19 infection: a systematic review and meta-analysis. can j neurol sci 2021;48:66-76. doi: 10.1017/cjn.2020.146. 6. singhania n, bansal s, singhania g. an atypical presentation of novel coronavirus disease 2019 (covid-19). am j med 2020;133:e365-e6. doi: 10.1016/j.amjmed.2020.03.026. 7. katal s, balakrishnan s, gholamrezanezhad a. neuroimaging and neurologic findings in covidatalay neurological manifestations of covid-19 infection 189 universa medicina vol.40no.3 19 and other coronavirus infections: a systematic review in 116 patients. j neuroradiol 2021;48:4350. doi: 10.1016/j.neurad.2020.06.007. 8. mankad k, perry md, mirsky dm, rossi a. covid-19: a primer for neuroradiologists. neuroradiology 2020;62:647-8. doi: 10.1007/ s00234-020-02437-5. 9. mehta p, mcauley df, brown m, et al. on behalf of the hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression. lancet 2020;395:10334. doi: 10.1016/s0140-6736(20)30628-0. 10. ma j, xia p, zhou y, et al. potential effect of blood purification therapy in reducing cytokine storm as a late complication of critically ill covid-19. clin immunol 2020 ;214:108408. doi: 10.1016/ j.clim.2020.108408. 11. aghagoli g, gallo marin b, katchur nj, chavessell f, asaad wf, murphy sa. neurological involvement in covid-19 and potential mechanisms: a review. neurocrit care 2020:1-10. doi: 10.1007/s12028-020-01049-4. univ med 2021;40:187-9. doi: http://dx.doi.org/10.18051/univmed.2021.v40.187-189 c:\users\universa medicina\docu 151 emerging zoonoses in indonesia: implication for the future toni wandra* directorate of postgraduate, sari mutiara indonesia university, medan, indonesia the world health organization (who) defines zoonotic diseases (zoonoses) as those diseases and infections which are naturally transmitted between vertebrate animals and humans.(1) more than 250 zoonoses have been described, over 60% of pathogens that cause diseases in humans are zoonoses, comprising 75% of emerging infectious diseases.(2) not all zoonotic diseases become pandemics, but most pandemics are caused by zoonoses and they have become characteristic of the anthropocene era.(3) joint consultation on emerging zoonotic diseases was provided by who, food and agr i c u l t ur e orga ni za t i on , a n d wor l d organization for animal health in geneva in 2004, and defined an emerging zoonosis as a pathogen that is newly recognized or newly evolved, or that has occurred previously but shows an increase in incidence or expansion in geographical, host or vector range. some agents causing such diseases may evolve further and become transmissible from human to human.(3) fa c t o r s in f l u e n c i ng zoo no s es c ompr i se ecological changes in the environment, intensive livestock farming and agriculture, international trade of exotic animals, the rapid increase in movements of humans and transportation, and urbanization that has disrupted the humananimal-environment interface.(4) pervasive evidence has shown that severe acute respiratory syndrome (sars), middle east respiratory syndrome (mers) and severe acute respiratory syndrome coronavirus-2 (sars-cov-2) have a bat origin and are transmitted to humans via intermediate hosts.(5) co nt r a r i ly, t h e r e c e n t p and e mi c o f cov id-1 9 h a s s h own t he p o te nt i a l of microorganisms from animal reservoirs to adapt to human hosts, and evolve further and become e f f e c t i ve ly a n d e s se nt i a l l y exc l u s ive l y transmissible from human to human.(3) as of 26 august 2020, the government of indonesia announced 160,165 confirmed cases of covid-19, 6,944 deaths and 115, 409 recovered cases from 485 districts across all 34 provinces. as of 26 august, most confirmed cases were in java: dki jakarta followed by east java, central java, and west java. south sulawesi is the only province outside java that is among the top five provinces in terms of the reported number of confirmed cases. java c o nt r ib u t ed 5 8. 4% o f t he t o t al c a se s i n indonesia.(6) since covid-19 was declared as a global pandemic on 11 march, 2020, social lives have been upended, and economic activities disrupted around the world, including in indonesia. key sectors such as the travel and tourism sector, and other services sectors have been affected.(7) covid-19 will presumably not be the last zoonotic pandemic,(3) such that reducing the risk of zoonotic outbreaks should be prioritized at all levels. therefore in 2005, the who western pacific region and the south-east asian region created asia pacific strategy for emerging diseases a nd public health emergencies (apsed). there must be close multisectoral universa medicina september-december 2020 vol.39no.3 *email: tony_wdr2009@yahoo.com orcid id: 0000-0002-4606-5411 152 cooperation, particularly between the health and agricultural sector at each level to prevent and control zoonoses.(8) on the other hand, one health is a concept that human and animal health are linked as important now as ever. the term one health was first introduced in 2003–2004 and was associated with the emergence of severe acute respiratory disease (sars) in early 2003 and by the spread of highly pathogenic avian influenza.(9) in indonesia, the presidential decree no 7/2020 on the acceleration of the country’s covid-19 handling, through a task force st e e r in g co u nc i l , a nd w a s a me n d e d b y presidential decree, no. 9/2020. the new decree mandates governors as members of the task force steering council to form a task force at the provincial levels to monitor and evaluate the implementation of the control program.(10) however, synergies in implementing the program and people’s awareness to adhere to the health protocols are needed as well as law e n f o r c e men t . t h e cov id -1 9 pa n d e mi c reminds us of the devastating effect that previous emerging diseases had in our history until the development of effective vaccines that greatly reduced the burden caused by those i nf e c ti on s . ( 1 1 ) in do n e si a mu st i mpr ove biosecurity measures and good hygiene at the human–animals interface and thus may impact the human health in the future. references 1. world health organization. zoonoses. available at: https://www.who.int/topics/zoonoses/en/. accessed july 30, 2020. 2. dahal r, kahn l. zoonotic diseases and one health approach. epidemiol 2014; 4: e115. doi: 10.4172/2161-1165.1000e115. 3. zoonoses: beyond the human–animal– environment interface. lancet 2020;396:1. [editorial]. 4. hassell jm, begon m, ward mj, fèvre em. urbanization and disease emergence: dynamics at the wildlife–livestock–human interface. trends ecol evol 2017;32:55–67. doi: 10.1016/j.tree.2016. 09.012: 10.1016/j.tree.2016.09.012. 5. ye zw, yuan s, yuen ks, fung sy, chan cp, jin dy. zoonotic origins of human coronaviruses. int j biol sci 2020;16:1686–97. doi: 10.7150/ ijbs.45472. 6. world health organization. coronavirus disease 2019 (covid-19): indonesia situation report – 22. available at: https://www.who.int/indonesia. accessed august 31, 2020. 7. asean integration monitoring directorate at the asean secretariat. asean policy brief: economic impact of covid-19 outbreak on asean. analysis and monitoring on finance and socio-economic;2020. 8. world health organization. asia pacific strategy for emerging diseases and public health emergencies (apsed iii): advancing implementation of the international health regulations (2005). manila, philippines: world health organization regional office for the western pacific; 2017. 9. mackenzie js, jeggo m. the one health approach – why is it so important? trop med infect dis 2019;4:88. doi: 10.3390/tropicalmed4020088. 10. office of assistant to deputy cabinet secretary for state documents & translation. gov’t declares covid-19 pandemic as national disaster. jakarta pusat: cabinet secretariat of the republic of indonesia; 2020. 11. florindo hf, kleiner r, vaskovich-koubi d, et al. immune-mediated approaches against covid-19. nature nanotechnol 2020;15:630–45. https:// doi.org/10.1038/s41565-020-0732-3. universa medicina september-december 2020 vol.39no.3 univ med 2020;39:151-2. doi: http://dx.doi.org/10.18051/univmed.2020.v39.151-152 c:\users\user\documents\editori 71 real-time polymerase chain reaction for detecting sars-cov-2 in indonesia: are the results reliable? monica dwi hartanti* department of medical biology, faculty of medicine, trisakti university since it was first reported in wuhan, china, in december 2019, coronavirus disease 2019 ( co v id-19 ) ha s b ee n c o n si d e r e d a s a pandemic. (1 ) it is caused by sever e acute respiratory syndrome coronavirus 2 (sarscov-2), a member of the coronavirus family.(1) in indonesia, this disease was first detected in the beginning of march 2020 and has been spreading in 34 provinces and more than 417 districts all over indonesia, with more than 27,000 confirmed cases and a case fatality rate of around 6%.(2-3) the indonesian government has implemented several strategies in order to slow d own t he s pr e a d o f cov id -19, s uc h a s promoting social distancing, implementing largescale social restriction, providing personal protection equipment for health workers, adding hospitals for treating covid-19 patients, and improving diagnostic management. currently the most widely used method of confirming covid19 cases is real-time pcr. the term "polymerase chain reaction" was first introduced in 1985 in a paper written by saiki et al.(4) the general concept of pcr involves primers, dna polymerase, nucleotides, ions, and dna template and consists of cycles t ha t c o mpr i s e 3 s te ps , wh i ch a r e dna denaturation, primer annealing, and extension. pcr h a s ind e e d p r o vi d e d a si gn i f i c a n t contribution to the development of biomedical research. however, the most fundamental change in pcr application was the introduction of the concept of real-time pcr. real-time pcr or quantitative pcr (qpcr) is a method of quantifying the amplified nucleic acid present in a sample in real time using a fluorescent dye or fluorescent-labeled oligos. the fluorescence will be measured after each cycle and the fluorescent signal intensity denotes the amount of transient dna amplicons in a sample at that particular time. the intensity of the fluorescent signal will be below the detectable level at the beginning of cycles due to background signals. once it increases above the level, the intensity corresponds proportionally to the preliminary number of dna molecules in the sample. this is the point where the absolute quantity of target dna in the sample is measured based on a calibration curve created by serial diluted standard s amples with known concentrations. this point is known as the quantification cycle (cq) or the cycle threshold (ct). the quantitative real-time pcr is known to play a critical role in detecting and quantifying viral pathogens due to its appropriate sensitivity and specificity, as well as fast and highthroughput results compared to other methods, s uc h a s e lisa a n d c u lt u r e me t ho ds . ( 5 ) additionally, cross-contamination can be avoided because qpcr does not require further sample ma ni p u la ti o n a f t e r th e a mp li f i c a t i on . furthermore, its ability to amplify several targets in a single reaction makes that users can include internal amplification controls in one run. the multiplexing option is essential for detection and universa medicina may-august 2020 vol.39no.2 *department of medical biology, faculty of medicine, trisakti university jl. kyai tapa no.260, grogol, jakarta barat email: mdhartanti@trisakti.ac.id orcid id: 0000-0001-7542-6434 72 quantification in diagnostic qpcr assays that rely on the inclusion of internal amplification controls. ac c or din g t o t h e wor ld he a lt h organization (who), one-step quantitative realtime pcr (qrt-pcr) assays have become a standard assessment for identifying sars-cov2 in suspected patients.(6) covid-19 cases are confirmed by detecting unique sequences of viral rna by nucleic acid amplification tests such as qrt-pcr. the targeted viral genes so far include the three genes that encode structural proteins; n (nucleocapsid), e (envelope), and s (spike), as well as the rna-dependent rna polymerase (rdrp) gene that plays critical roles in viral rna synthesis. rna extraction should be done in a biosafety cabinet. it seems that qrt-pcr is the most widely used and preferred method for confirming covid-19 cases. the only critical problem regarding the real-time pcr test would be any possibility of false negative or false positive results. it has been reported that a patient in wuhan, china, had three negative sars-cov2 rt-pcr testing results within 3 weeks before showing positive results for both rt-qpcr and next generation sequencing (ngs) testing from bronchoalveolar lavage fluid (balf) samples.(7) another study in wuhan reported that 21 of 70 hospitalized covid-19 patients with two consecutive negative results had positive results for the third testing.(8) what would be causing these false negative results? there are several factors that are thought to be related to the inconsistency of qpcr results. firstly, the primers. those used in qrt-pcr can be influenced by the variant of viral rna sequences, leading to the discrepancy of the results. there are 3 strains of sars-cov-2 that have been identified, type a, b, and c.(9) a recent study shows that the strain of sars-cov-2 in indonesia is different from those three strains, with mutation sites in the s protein and open reading frame (orf) protein.(10) primers that are able to detect the indonesian sars-cov-2 strains are needed to produce accurate and consistent results. secondly, the laboratory practice standards and personnel skills. qrt-pcr is a method that requires skills in the relevant technical procedures, such as pipetting skills. additionally, safety procedures must be implemented in running this method in at least a biosafety level 2 laboratory. there are 148 laboratories across indonesia that have been certified to perform this test and more than 200,000 samples have been tested so far (as of 2 june 2020).(2) lastly, sampling procedures, such as sample types, collecting timing and tools, and sampling transfer. the sample should be collected from the upper (nasopharyngeal and oropharyngeal swab) and/or lower respiratory tracts (sputum and/or endotracheal aspirate or bronchoalveolar lavage).(11) the collection time must be carefully determined due to different viral load kinetics of sars-cov-2 in different patients, leading to different results.(7) dacron or polyester flocked swabs should be used for collecting samples and the latter must be transferred to the laboratory at 2-8°c as soon as possible after collection to prevent interference by inhibitors in the sample.(11) indonesia has been able to identify more than 27,000 sars-cov-2 positive samples from 237,947 total samples (as of 2 june 2020) using qrt-pcr methods (equal to 0.03 test per thousand inhabitants) and the recovery numbers have been increased to almost 8,000 cases.(2,3) thus, the results of qrt-pcr have been used to confirm many suspected covid-19 cases in indonesia. however, the interpretation of the results must be made cautiously. negative results with clinical characteristics of covid19 must be followed by multiple sample types at various points of time. it is worth noting that the combination of qrt-pcr results and clinical feat ures s hould a lways be c on sidere d in managing the disease. good laboratory practice standards must be implemented in testing samples of covid-19 cases to improve the resulting quality and reduce the inconsistency. universa medicina september-december 2019 vol.38no.3 73 references 1. world health organization. covid 19 public health emergency of international concern (pheic). geneva: world health organization; 2020. 2. corona disease mitigation acceleration task force. covid-19 in indonesia (2 june 2020). jakarta: corona disease mitigation acceleration task force;2020. 3. hasell j, ortiz-ospina e, mathieu e, et al. coronavirus (covid-19) testing. our world in data;2020. 4. saiki rk, scharf s, faloona f, et al. enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. science 1985;230:1350-4. doi: 10.1126/ science.2999980. 5. shen m, zhou y, ye j, et al. recent advances and perspectives of nucleic acid detection for coronavirus. j pharm anal 2020;10:97-101. https:/ /doi.org/10.1016/j.jpha.2020.02.010. 6. world health organization. laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases: interim guidance. geneva: world health organization;2020. 7. wang y, kang h, liu x, tong z. combination of rt-qpcr testing and clinical features for diagnosis of covid-19 facilitates management of sars-cov-2 outbreak. j med virol 2020;1-2. doi: 10.1002/jmv.25721. 8. xiao at, tong yx, gao c, et al. dynamic profile of rt-pcr findings from 301 covid-19 patients in wuhan, china: a descriptive study. j clin virol 2020;104346.https://doi.org/10.1016/j.jcv.2020. 104346. 9. forster p, forster l, renfrew c, forster m. phylogenetic network analysis of sars-cov-2 genomes. proc natl acad sci usa 2020; 117:92413. doi: 10.1073/pnas.2004999117. 10. global initiative on sharing all influenza data. genomic epidemiology of hcov-19. bonn: global initiative on sharing all influenza data (gisaid), 2020. 11. pengurus pusat perhimpunan dokter spesialis patologi klinik dan kedokteran laboratorium indonesia. manajemen spesimen dan diagnosis laboratorium kasus suspek 2019-ncov;2020. univ med 2020;39:71-3. doi: http://dx.doi.org/10.18051/univmed.2020.v39.71-73 universa medicina january-april vol.42no.1, 2023 invited editorial potential role of probiotic supplementation in cognitive improvement and other metabolic biomarkers phakkharawat sittiprapaporn head, neuropsychological research laboratory head, neuroscience research center school of anti-aging and regenerative medicine, mae fah luang university, bangkok, thailand gut dysbiosis and changes in gut homeostasis, which are linked to a number of physical and mental problems, affect the connection between the gut and the brain. probiotics are considered dietary supplements that can be used to treat mental illnesses and cognitive issues. probiotics are live bacteria that, when consumed in sufficient quantities, benefit the host's health. probiotics can improve cognitive performance by improving the gut-brain axis and the microbial environment in the gut.(1) cognitive development refers to a child's ability to think, reason, and use language, all of which are necessary for their overall development. attention deficit/ hyperactivity disorder (adhd) is the most common problem with a child's brain development, which occurs in parallel to the child's cognition. attention deficit/hyperactivity disorder is caused by a number of factors, of which gut homeostasis and gut microbiota are two of the most important ones.(1) fukngoen et al.(1) recently investigated the effect of lactobacillus paracasei hii01 on the attention state of healthy children as well as changes in representative neuroinflammatory markers. they predicted that taking lactobacillus paracasei hii01 could reduce the risk of developing neuropsychiatric disorders. ten healthy young thai children were given 109 cfu of lactobacillus paracasei hii01 daily for 12 weeks in this study. the go/no-go activities were used to assess changes in attention state. changes in brain waves were also tracked using electroencephalographic (eeg) recordings. quinolinic acid (qa, a tryptophan metabolite), and 5-hydroxyindoleacetic acid (5-hiaa, a serotonin metabolite), were measured in urine at the start of the study and 12 weeks later. both the qa and 5-hiaa concentrations significantly decreased and increased, respectively. the qa/5hiaa ratio also decreased significantly. according to the go/no-go test, the percentages of go accuracy and go error increased and decreased dramatically, respectively. in terms of eeg data, theta, alpha, and beta waves were significantly changed between the study's baseline and 12th week. the data revealed that lactobacillus paracasei hii01 may improve gut flora and oscillatory brain activity, hence preserving the attention states of persons.(1) urbanization influences our way of life, especially in fast-paced environments where we are more prone to stress. it is believed that stress management increases longevity. the use of probiotics for psychological treatment is supported by a small body of diversely established evidence.(2) there are currently few studies on the use of synbiotics to reduce stress in stressed subjects. lalitsuradej et al.(2) recently investigated how synbiotics affected stress levels in a thai population. a thai stress test (tst) was used to screen 32 people and determine how stressed they were. participants were divided into two groups: stressed and *email: wichian.sit@mfu.ac.th orcid id: 0000-0002-4103-9396 1 mailto:wichian.sit@mfu.ac.th https://orcid.org/0000-0002-4103-9396 2 non-stressed. synbiotic preparation contained 10g of prebiotics (5g galacto-oligosaccharides (gos) and 5g oligofructose (fos)), and a combination of 2 probiotic strains at a total concentration of 1 x 1010 cfu/day (5.0 x 109 cfu of lactobacillus paracasei hii01 and 5.0 x 10 9 cfu of bifidobacterium animalis subsp. lactis). all parameters were measured at the start of the trial and again after the 12th week. synbiotics significantly reduced the negative and positive tst scale scores in the stressed group, but increased the corresponding scores in the nonstressed group (p<0.05).(2) synbiotics significantly decreased tryptophan levels in the non-stressed group (p<0.05), but significantly increased levels of dehydroepiandrosterone sulfate (dhea-s), tumor necrosis factor-α ( tnf-α), 5hydroxyindoleacetic acid (5-hiaa), short-chain fatty acids (scfas), acetate, and propionate. both gr oups had lower levels of cortisol and lipopolysaccharide (lps), but higher levels of interleukin-10 (il-10) and immunoglobulin a (iga). the synbiotics administration slightly improved stress status in the stressed group but not in the non-stressed group.(2) obesity, dyslipidemia, hypertension, and glucose intolerance are metabolic conditions that incr ease the risk of type 2 dia betes and cardiovascular disease. according to emerging evidence, microbiota may play an important role in human health and disease, including digestion, energy and gluc ose metabolism, immunomodulation, and brain function. obesity is becoming more common, and the primary causes are overly processed diets and sedentary lifestyles. furthermore, hypercholesterolemia is a major contributor to cardiovascular disease. probiotics may ben efit patients with hypercholesterolemia by lowering their high blood lipid levels.(3) chaiyasut et al.(3) recently proposed a method for comprehending and elucidating the synergistic role of prebiotics and probiotics in the treatment of obesity. obese thai people were tested for biomarkers of obesity such as cholesterol, gut permeability, oxidative stress, bacterial toxins, cytokines, and short-chain fatty acids after being given a synbiotic supplement containing lac tobacillus paracasei, bifidobacterium longum, bifidobacterium breve, inulin, and fructooligosaccharide. the results showed that including synbiotics in one's diet had a significant and positive effect on biomarkers linked to obesity.(3) in another study, chaiyasut et al.(4) investigated 52 subjects randomly divided into two gr oups, the lactobacillus paracasei hii01 treatment group and the placebo group, to understand how lac tobacillus paracasei hii01 affec ts cholesterol, oxidative stress, and other indicators. the study was conducted over a 12-week supplemental intervention period. lactobacillus paracasei hii01 significantly improved the subjects' high density lipoprotein (hdl), total antioxidant capacity (tac), and propionic acid levels while significantly lowering their total cholesterol (tch), triglycerides (tgs), tumor necrosis factor-α (tnf), and lipopolysaccharide (lps). the findings suggest that lactobacillus paracasei hii01 improves blood lipid profiles and reduce s oxidative stress in thai hypercholesterolemic subjects.(4) bacteria, poisons, and allergens are prevented from diffusing into surrounding tissue and the circulatory system by the integrity of the intestines. inflammation, gastrointestinal issues, neurological diseases, and neurodegenerative disorders are just a few of the minor to major health issues that can arise as a result of intestinal integrity damage.(5) it is essential to maintain a healthy gut barrier function in order to preserve health. protecting and restoring intestinal permeability in vitro and in vivo are known features of probiotics. multiple strains of probiotics are more effective in protecting the body than a single strain. the e ff ects of pr obiotic supplementation on intestinal permeability, lipid profile, obesity index, and metabolic indicators in elderly thai participants were recently analyzed by chaiyasut et al. (5 ) t he pr obiotic supplementation included lac toba cillus paracasei hii01, bifidobacterium breve, and bifidobacterium longum. the r esults universa medicina 3 univ med 2023;42:1-3. doi: http://dx.doi.org/10.18051/univmed.2023.v42.1-3 demonstrated that supplementation with the investigated probiotics significantly boosted highdensity lipoprotein (hdl) cholesterol (up to 48%) and enhanced the function of the intestinal barrier. furthermore, the intervention increased shortchain fatty acid levels as well as anthropometric indicators associated with obesity in humans. this study began with the goal of developing probioticbased health supplements as an adjunctive therapy for a variety of metabolic diseases.(5) in contrast, a randomised, double-blind, placebocontrolled trial to assess whether a 12-week b. breve a1 supplementation could affect the cognitive function of elderly subjects with memory complaints, showed that no significant intergroup difference was observed in terms of changes in scores from the baseline scores.(6) the recent studies' restricted sample size, the questionnaire about eating habits, exercise, work activity, and overcoming the disease, the lack of extended follow-up, and microbiome analysis, must be supported by further studies. to prove probiotics' benefits and develop health supplements to treat metabolic illnesses, more research is needed. references 1. fukngoen, p, sivamaruthi, bs, sirilun, s, et al. the influence of lactobacillus paracasei hii01 supplementation on performance in attention (go/ no-go) tasks and quinolinic acid and 5hydroxyindoleacetic acid levels in thai childrena preliminary study. appl sci 2022;12:5658. https:/ /doi.org/10.3390/app12115658. 2. lalitsuradej e, sirilun s, sittiprapaporn p, et al. the effects of synbiotics administration on stress related parameters in thai subjects: a preliminary study. foods 2022;11:759. https://doi.org/10.3390/ foods11050759. 3. chaiyasut c, sivamaruthi bs, kesika p, et al. synbiotic supplementation improves obesity index and metabolic biomarkers in thai obese adults: a randomized clinical trial. foods 2021; 10:1580. https://doi.org/10.3390/foods10071580. 4. chaiyasut c, tirawat y, sivamaruthi bs, et al. effect of lactobacillus paracasei hii01 supplementation on total cholesterol, and on the parameters of lipid and carbohydrate metabolism, oxidative stress, inflammation and digestion in thai hypercholesterolemic subjects. appl sci 2021;11:4333. https://doi.org/10.3390/ app11104333. 5. chaiyasut c, sivamaruthi bs, lailerd n, et al. probiotics supplementation improves intestinal permeability, obesity index and metabolic biomarkers in elderly thai subjects: a randomized controlled trial. foods 2022;11:268. https:// doi.org/10.3390/foods11030268. 6. kobayashi y, kuhara t, oki m, xiao jz. effects of bifidobacterium breve a1 on the cognitive function of older adults with memory complaints: a randomised, double-blind, placebo-controlled trial. benef microbes 2019;10:511-20. https:// doi.org/10.3920/bm2018.0170. january-april 2023 vol.42 no.1 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1410 january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 effect of hypnotherapy on prolactin level in women after cesarean section supriyadi hari respati1 *, risang bhaskoro1 , eric edwin yuliantara1 , uki retno budihastuti1 , and debree septiawan2 abstract background postpartum depression occurs in women after cesarean section, due to increased cortisol and decreased prolactin levels, leading to delayed breast milk production. hypnotherapy is one of the most effective and efficient complementary therapy options for reducing postpartum depression. hypnotherapy increases oxytocin secretion from the paraventricular nucleus of the anterior pituitary gland, decreases cortisol levels, and increases prolactin, thereby increasing breast milk production. this study evaluates hypnotherapy’s effect on prolactin levels after a cesarean section. methods this study was an experimental non-blinded randomized controlled trial with a post-test-only control group design conducted at dr. moewardi general hospital and uns hospital. a total of 20 post-cesarean -section women were included in the study. they were randomized into two groups, a group i: given hypnotherapy, and group 2: the control group (without hypnotherapy). both groups were tested for prolactin levels. group i received hypnotherapy 6 to 12 hours post-cesarean section, and study participants were confirmed to be unaffected by the anesthetic. data analysis used the independent t-test and p<0.05. results at baseline, there was no significant difference in age, parity, and indication for cesarean section between the hypnotherapy and the control groups (p>0.05). after intervention, the mean prolactin level was higher in the hypnotherapy group (247.6 ± 81.1 ng/ml) compared to the control group (120.1 ± 55.4 ng/ml) (p<0.001). conclusion hypnotherapy increases post-cesarean prolactin levels compared to the controls. our findings open up a wide range of potential hypnotic applications among women with postpartum depression. keywords: cesarean section, hypnotherapy, prolactin, breastfeeding 1department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ dr. moewardi hospital, jawa tengah, indonesia 2department of psychiatry, faculty of medicine, universitas sebelas maret/ dr. moewardi hospital, jawa tengah, indonesia *correspondence: supriyadi hari respati department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ dr. moewardi hospital, jawa tengah, indonesia email: sp.obgin@staff.uns.ac.id orcid id: 0000-0001-7210-996x date of first submission, september 13, 2022 date of final revised submission, january 7, 2023 date of acceptance, january 16, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 6 doi: http://dx.doi.org/10.18051/univmed.2023.v42:6-11 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1373 cite this article as: respati sh, b has koro r, yuliantara ee, budihastuti ur, septiawan d. effect of hypnotherapy on prolactin level in women after cesarean section. univ med 2023 ;42:6-11. doi: 10.18051/ univmed.2023.v42:6-11. mailto:sp.obgin@staff.uns.ac.id https://orcid.org/0000-0001-7210-996x https://orcid.org/0000-0002-7722-4016 https://orcid.org/0000-0001-7622-3380 https://orcid.org/0000-0003-0611-7464 https://orcid.org/0000-0002-4631-6268 7 univ med vol. 42 no. 1 introduction a cesarean section is the birth of an infant by surgery on the abdomen (laparotomy) and the uterus (hysterotomy). cesarean sections are increasing significantly worldwide.(1,2) between 1990 and 2014, an analysis of 121 countries found that cesarean section rates increased by 12.4% (from 6.7% to 19.1%), with an average annual increase rate of 4.4%.(3) in 2018, indonesia’s cesarean section rate was 17.6%.(4) cesarean delivery carries a higher risk of postpartum mood disorders for women than does vaginal delivery.(5,6) in one study, postpartum depression occurred in 21.1% (43 of 205) of patients who had undergone elective cesarean section. increased risk of postpartum depression is associated with increased preoperative anxiety, pain scores, central sensitization, and decreased need for analgesics.(5) maternal psychological stress is associated with an increase in postpartum cortisol levels, interfering with the regulation of oxytocin and prolactin, which can affect the frequency and duration of breastfeeding.(7) breastfeeding mothers who had undergone elective cesarean section had lower l actation rates than breastfeeding mothers who gave birth vaginally.(8) the combination of depressive symptoms and difficulty in breastfeeding can ultimately affect the duration and su ccess of exclusive breastfeeding.(7) hypnotherapy significantly affects the optimization of postpartum maternal colostrum on the first day due to the release of endorphins (hormones that support prolactin secretion).(9) a previous study stated that there were significant differences in depression and anxiety symptoms in the two months postpartum, with control group results being higher than those of the experimental gr oup who underwen t hypnosis during pregnancy.(10) one study reported that the combination of hypnotherapy and acupressure reduced anxiety and pain levels significantly in post-c esar ean mothers, ther eby implying increased breast milk production.(11) prolactin levels increased significantly in postpartum mothers who received oxytocin massage and breast care compared to the control group.(12) psychological symptoms during pregnancy, such as depression, can potentially cause postpartum depression.(13) a systematic review showed that the hypnosis-based interventions improved women’s emotional experiences and outlook towards birth, with less anxiety, increased satisfaction, fewer birth interventions, more postnatal well-being, and better childbirth experience. (1 4) however, recent literature suggests the effectiveness of hypnosis in alleviating postpartum psychological symptoms, such as anxiety and depression.(10) although a correlation between the amount of milk produced and the maternal prolactin level is not yet well established, it remains high in nursing mothers, with peaks in response to breastfeeding.(15) considering all this, this study aims to evaluate the effects of hypnotherapy in raising prolactin levels in post-cesarean patients. methods research design this study used an experimental non-blinded randomized controlled trial with a post-test-only control group design to determine the effects of hypnotherapy on prolactin levels in post-cesarean patients. the study was conducted at dr. moewardi general hospital and uns hospital. the research duration was from march 2022 to may 2022. study subjects the study population was post-cesarean section patients treated at dr. mewardi general hospital and uns hospital. research subjects were taken from those who met the inclusion and exclusion criteria. inclusion criteria: post-cesarean -section patients, patients undergoing cesarean section under spinal anesthesia, patients without hearing loss, and patients who can attend hypnotherapy sessions. exclusion criteria: patients with complications of other diseases and 8 a previous history of mental disorders. twenty subjects involved in the study were divided randomly into two groups (10 subjects in the hypnotherapy group and 10 subjects in the control group). the sample size was calculated using comparing two means formula with effect size=0.9, α=0.05, =0.2, and the optimum number of subjects was 10 for each group. intervention the treatment gr oup underwent a hypnotherapy intervention 6-12 hours after the cesarean section to confirm that they were unaffected by anesthesia. hypnotherapy is carried out once, after which audio recordings are used before bedtime and in the morning after waking up. hypnotherapy interventions are carried out by a psychiatrist with expertise in hypnotherapy. hypnotherapy is carried out through the following procedures: providing information to the patient to obtain her informed consent for hypnotherapy, explaining hypnothe r apy, indications, contraindications, and side ef fects of hypnotherapy so that the patient understands the action to be performed, conducting a suggestibility test to test/find out the patient’s response to specific suggestions, and choosing the strategy to be used and training to enter a trance/hypnosis condition, induction (method for transferring the patient from the conscious state of mind to the subconscious state). during this phase, the patient must organize physical and psychological relaxation so that the patient is in a trance), trance deepening (to bring the patient to a deeper subconscious state), suggestion (to induce a feeling of calmness and reduce anxiety), termination (r eturn the patie nt to full consciousness). the control group did not receive hypnotherapy treatment. laboratory analysis prolactin hormone levels were evaluated in the hypnotherapy group and the control group. first, a 5-ml blood sample was taken. then, it is allowed to stand for 15 minutes and centrifuged at 3000 rpm for 15 minutes. next, it is sent in a cooler bag within 24 hours to the laboratory to measure the level of prolactin in the blood on the condition that the sample should not be frozen. statistical analysis the research data were analyzed using the ibm spss version 22 statistical application. in addition, normality tests were carried out. finally, because the data distribution was normal, an independent t-test was used to analyze the data. ethical clearance this study was approved by the ethical health research commission of the dr. moewardi hospital, surakarta, jawa tengah, indonesia, under no. 352/ iii/ hrec/ 2022, dated march 18, 2022. results a total of 20 subjects participated in the study and were randomized into the hypnotherapy (n=10) a nd control gr oups ( n=10). t he hypnotherapy subjects were given hypnotherapy intervention 6-12 hours after the cesarean section to confirm that they were unaffe cted by anesthesia (figure 1). at baseline, there was no significant difference in age, education, occupation, parity, history, indication for cesarean section, height, weight, and bmi between the hypnotherapy and the control groups (p>0.05) (table 1). after treatment mean prolactin level in the hypnotherapy group (247.6 ± 81.1 mg/dl) was significantly higher than that in the control group (120.1 ± 55.4 mg/dl) (p=0.001) (data not shown). discussion this study showed that hypnotherapy may increase prolactin levels in patients who have undergone cesarean section, compared to those not receiving hypnotherapy. this is consistent with a study by anuhgera et al.,(9) which states that hypnotherapy is more effective than acupressure in stimulating the production of the hormone prolactin and the production of breast milk in women who give birth by cesarean section. respati , bhaskoro, yuliantara, et al post-cesarean hypnotherapy 9 univ med vol. 42 no. 1 variable hypnotherapy (n=10) control (n=10) p value age (years) 28.90 ±7.23 29.60 ± 3.77 0.789 parity primipara 2 (20) 6 (60) 0.068 multipara 8 (80) 4 (40) history cesarean section history 6 (60) 3 (30) 0.178 primary 4 (40) 7 (70) indication for cesarean section elective 4 (40) 2 (20) 0.329 emergency 6 (60) 8 (80) weight (kg) 58.20 ±18.22 60.70 ±13.16 0.729 height (cm) 152.70 ± 5.77 152.20 ± 6.00 0.851 bmi (kg/m2) 20.95 ± 7.22 26.55 ± 4.89 0.631 education junior high school 2 (20) 1 (10) 0.480 senior high school 4 (40) 3 (30) diploma 3 3 (30) 2 (20) bachelor 1 (10) 4 (40) occupation civil servant 0 (0) 2 (20) 0.315 private employee 3 (30) 4 (40) housewife 6 (60) 4 (40) farmer 1 (10) 0 (0) table 1. distribution of several characteristics of the research subjects by treatment groups at baseline *significant at p<0.05; bmi: body mass index; data presented as mean ± sd and n (%) figure 1. flow diagram of the participants 10 based on research conducted by findik et al.,(13) post-cesarean pain and anxiety adversely affect milk production. postpartum pain and anxiety were lower with vaginal delivery than with cesarean delivery. epinephrine release increases during cesarean section and reduces the release of oxytocin, which in turn inhibits milk production and delays the onset of lactation. stress activates the hypothalamic–pituitary– adrenal (hpa) axis and triggers the release of corticotrophin releasing hormone (crh) in the paraventricular nucleus (pvn), which will increase the secretion of adrenocorticotrophin (act h) from the a nterior pituitary. adrenocorticotrophin will induce the release of cortisol and reduce prolactin secretion.(16–18) in addition, stress can indirectly inhibit prolactin through sympathetic activation. prola ctin deficiency can cause lactation problems.(19,20) hypnotherapy is therapy that uses hypnosis to work on the subconscious mind. hypnotherapy brings joy, contentment, relief, and peace to patients after cesarean delivery. in addition, hypnosis can also reduce stress. cortisol levels can drop very low during hypnotherapy,(21–23) showing that there is a relationship between hypnotherapy and the hpa axis. hypnotherapy has a statistically significant effect in lowering serum cortisol levels in patients.(19) hypnotherapy may affect maternal beliefs, although temporarily, in the form of effective attention focus control, especially for breastfeeding. this effect will reduce depression and affect the hormonal axis. our study did not measure prolactin levels at baseline because research funding was limited, and this was not a routine post-cesarean test. therefore, there was no data on prolactin levels before administering hypnotherapy. high prolactin levels increase breast milk production, reduce low birth weight infants and infant mortality, and support exclusive breastfeeding. we suggest to reduce the use of drugs to raise prolactin levels, make patients more relaxed, and reduce trauma after cesarean delivery. conclusions this study demonstrated that hypnotherapy after cesarean delivery increased prolactin levels. in addition, hypnosis-based interventions improve women’s experience after cesarean section. conflict of interest no relevant disclosures. acknowledgment we thank the director of dr. moewardi general hospital f or his permission and cooperation. contributors shr drafted the manuscript. rb contributed to data collection. all authors contributed to the analysis and interpretation of the data. shr, eey, urb, and ds contributed to critically revising the manuscript for critical intellectual content. all authors have read and approved the final manuscript. references 1. mengesha mb, adhanu hh, weldegeorges da, et al. maternal and fetal outcomes of cesarean delivery and factors associated with its unfavorable management outcomes in ayder specialized comprehensive hospital, mekelle, tigray, ethiopia, 2017. bmc res notes 2019;12:4– 9. doi: 10.1186/s13104-019-4690-5. 2. boerma t, ronsmans c, melesse dy, et al. global epidemiology of use of and disparities in caesarean sections. lancet 2018;392:1341–8. doi: 10.1016/s0140-6736(18)31928-7. 3. betrán ap, ye j, moller ab, zhang j, gülmezoglu am, torloni mr. the increasing trend in caesarean section rates: global, regional and national estimates: 1990-2014. plos one 2016;11:1–12. doi: 10.1371/journal.pone.0148343. 4. kementrian kesehatan republik indonesia/ kemenkes ri. laporan nasional riskesdas respati , bhaskoro, yuliantara, et al post-cesarean hypnotherapy 11 univ med vol. 42 no. 1 2018. badan penelitian dan pengembangan kesehatan. jakarta: badan penelitian dan pengembangan kesehatan; 2018. 5. chan clj, tan cw, chan jji, et al. factors associated with the development of postnatal depression after cesarean delivery: a prospective study. neuropsychiatr dis treat 2020;16:715–27. doi: 10.2147/ndt.s241984. 6. benton m, salter a, tape n, wilkinson c, turnbull d. women’s psychosocial outcomes following an emergency caesarean section: a systematic literature review. bmc pregnancy childbirth 2019;19. doi: 10.1186/s12884-019-2687-7. 7. mohd shukri nh, wells jck, fewtrell m. the effectiveness of interventions using relaxation therapy to improve breastfeeding outcomes: a systematic review. matern child nutr 2018;14: e12563. doi: 10.1111/mcn.12563. 8. ýsik y, dag zo, tulmac ob, pek e. early postpartum lactation effects of cesarean and vaginal birth. ginekol pol 2016;87:426–30. doi: 10.5603/gp.2016.0020. 9. anuhgera de, kuncoro t, sumarni s, mardiyono m, suwondo a. hypnotherapy is more effective than acupressure in the production of prolactin hormone and breast milk among women having given birth with caesarean section. med sci int med j 2017;1. doi: 10.5455/medscience.2017. 06.8659. 10. beevi z, low wy, hassan j. the effectiveness of hypnosis intervention in alleviating postpartum psychological symptoms. am j clin hypn 2019; 61:409–25. doi: 10.1080/00029157.2018.1538870. 11. anuhgera de, kuncoro t, sumarni s, mardiyono, suwondo a. effect of combination of hypnobreastfeeding and acupressure on anxiety and wound pain in post-caesarean mothers. belitung nurs j 2017;3:525–32. doi: 10.33546/ bnj.191. 12. aini yn, hadi, rahayu s, pramono n, mulyantoro dk. effect of combination of oxytocin massage and hypnobreastfeeding on uterine involution and prolactin levels in postpartum mothers. belitung nurs j 2017;3:784-90. doi: 10.33546/ bnj.293. 13. redshaw m, henderson j. from antenatal to postnatal depression: associated factors and mitigating influences. j womens health (larchmt) 2013;22:518-25. doi: 10.1089/jwh.2012.4152. 14. catsaros s, wendland j. hypnosis-based interventions during pregnancy and childbirth and their impact on women’s childbirth experience: a systematic review. midwifery 2020;84: 102666. doi: 10.1016/j.midw.2020.102666. 15. mcguire tm. drugs affecting milk supply during lactation. aust prescr 2018;41:7–9. doi: 10.18773/ austprescr.2018.002. 16. torner l. actions of prolactin in the brain: from physiological adaptations to stress and neurogenesis to psychopathology. front endocrinol (lausanne) 2016;7:25. doi: 10.3389/ fendo.2016.00025. 17. yuan y, wu w, chen m, et al. reward inhibits paraventricular crh neurons to relieve stress. curr biol 2019;29:1243-51.e4. doi: 10.1016/ j.cub.2019.02.048. 18. stephens mac, mccaul me, wand gs. the potential role of glucocorticoids and the hpa axis in alcohol dependence. in : noronha abc, cui c, harris ra, crabbe jc, editors. neurobiology of alcohol dependence. academic press;2014. p.429-50. https://doi.org/10.1016/c2012-0-02501x. 19. begum t, rahman a, nababan h, et al. indications and determinants of caesarean section delivery: evidence from a populationbased study in matlab, bangladesh. plos one 2017;12:1–16. doi: 10.1371/journal.pone. 0188074. 20. bernard v, young j, binart n. prolactin a pleiotropic factor in health and disease. nat rev endocrinol 2019;15:356–65. doi:10.1038/s41574019-0194-6. 21. mahandaru ah, respati sh, sulistyowati s, laqif a, prasetya h. the effect of hypnotherapy on postpartum pain and depression in women with post caesarean delivery. indones j med 2021; 6:194–205. doi: 10.26911/theijmed.2021.06.02.08. 22. alizamar a, ifdil i, fadli rp, et al. the effectiveness of hypnotherapy in reducing stress levels. addict disord trea 2018;17:191–5. doi: 10.1097/adt. 0000000000000140. 23. fisch s, trivaković-thiel s, roll s, et al. group hypnosis for stress reduction and improved stress coping: a multicenter randomized controlled trial. bmc complement med ther 2020; 20:344. doi: 10.1186/s12906-017-1806-0. black sugarcane decoction reduces rat brain ischemia 40 *anatomy department of medical faculty, islamic university of indonesia **physiology department of medical faculty, islamic university of indonesia ***histology department of medical faculty, islamic university of indonesia ****medical profession program, medical faculty, islamic university of indonesia correspondence : dr. ety sari handayani, m.kes department of anatomy, faculty of medicine, islamic university of indonesia jalan kaliurang km 14,5 yogyakarta indonesia phone: +62858 7819 2619 email: eshyasser@yahoo.co.id univ med 2016;35:40-5 doi: 10.18051/univmed.2016.v35.40-45 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 black sugarcane decoction reduces rat brain ischemia ety s. handayani*, zainuri s. nugraha*, titis nurmasitoh**, kuswati*, dwi n. ahsani***, and ajeng g. nanda**** background there are people in yogyakarta, who use black sugarcane decoction (bsd) to prevent stroke. bsd contains policosanol and antioxidants. it has been proven that policosanol can reduce global ischemia in mongolian gerbils. this study aims to evaluate the effect of bsd on brain ischemia in a rat stroke model. methods a laboratory experiment using eighteen 3-month old male wistar rats without any defects, of 175-250 g body weight. brain ischemia was produced by a 20-minute bilateral carotid communis artery oclusion (bccao). using a rat stroke model, brain ischemia was produced by a 20-minute bccao. the rats were randomized into three groups: bsd treated stroke model rats (group 1), non treated stroke model rats (group 2), and sham operated rats (group 3). bsd was administered by gavage for 1 week before bccao. decapitation of rats was performed two hours post bccao. brain tissues were stained with 2,3,5-triphenyltetrazolium chloride (ttc). ischemic areas were analyzed using image j softwere. statistical analysis was conducted by one way anova test. results the mean percentages of rat brain ischemic area differed between group 3 (0.0 ± 0.0%), group 2 (3.13 ± 0.59%) and group 1 (1.15 ± 0.47%) p =0.001). post hoc test showed that there was no difference between group 3 with group 1. instead, there was a significant difference between group 2 and the other groups. conclusion the administration of bsd reduced rat brain ischemia after bilateral carotid artery ligation. keywords: sugarcane, brain ischemia, bilateral carotid communis artery oclusion, rats doi: http://dx.doi.org/10.18051/univmed.2016.v35.40-45 41 univ med vol. 35 no.1 introduction scientific research shows that sugarcane contains policosanol, a mixture of long-chain alcohols, including mainly octacosanol.(1) preclinical trials on policosanol showed that the compound had the ability to reduce the incidence of stroke through the mechanism of inhibition of platelet aggregation and a decrease in blood cholesterol levels.(1,2) it was the question of the benefits of policosanol as a stroke therapy that was still debatable. a clinical study showed that administration of policosanol immediately after the incidence of ischemic stroke would reduce subsequent ischemic strokes.(3) an experimental study showed that oral policosanol pre-treatment (50– 400 mg/kg) protected against blood-brain barrier damage induced by cerebral ischemia/ reperfusion in rats.(4) in addition to policosanol, sugarcane also contains antioxidants, especially quercetin. in vivo, the ability of quercetin in reducing cerebral ischemia is still debated. there are controversies about the ability of quercetin to pass through the blood brain barrier.(5) in contrast, antioxidant compounds in sugarcane could reduce oxidative stress in human hepg2 cells in vitro.(6,7) based on the fact that sugarcane contains policosanol and quercetin, the usage of black sugar cane decoction (bsd) as a stroke therapy in the community, and the development of a rat stroke model, in which brain ischemia was produced by a 20-minute bilateral carotid communis artery oclusion (bccao).the objective of this study was evaluate the effect of bsd in decreasing bccao-induced rat brain ischemia. methods research design this research applied a quasi-experimental design, using a post-test for the control group. the study was conducted from september until november 2015 at the pharmacy laboratory, islamic university of indonesia. animals and experimental procedure the animals used in this study were 18 male rats (rattus norvegicus of the wistar strain) that had met the inclusion and exclusion criteria. the rats were reared in the pharmacy laboratory, inslamic university of indonesia. inclusion criteria for this study were healthy 3-month old male rats without any defects, of 175-250 g body weight. determination of healthy rats was based on the physical state of the rats, i.e. those with clean, not wet or sticky bristles, active movements, and appropriate cycle of eating, drinking and sleeping. exclusion criteria of this research were sick and dying rats during the study. from day 1 to 7, the experimental animals were kept in cages of 40 x 20 x 20 cm for adaptation, at a density of one rat per cage. the inside temperature of the cage was set at room temperature. lighting was arranged with a 12hour light-dark cycle. the light cycle began at 06.00 am and dark cycle at 06:00 pm. pellets were given every day in the morning at 06.00 am. drinking water was provided ad libitum. subjects were randomized into three groups, of which each consisted of 6 rats. the groups were as follows: group 1 consisted of bsd treated stroke model rats (rats with the stroke model receiving bsd by gavage for 1 week before bccao), group 2 contained non-treated stroke model rats, and group 3 comprised sham operated rats (rats undergoing the same operation without bccao). brain ischemia was produced by a 20minute bccao. bccao procedure bccao was performed on the 15th day. stages of the ligation are as follows: a). anesthesia. during surgery, the rats were anesthetized using 80-100 mg/kg i.m. ketamine. the rats were placed on a sterile platform and the rat rectal temperature was kept at 37 ± 1° c. b). disinfection stage. this stage aims to prevent infection. surgical swipes are done with betadine from the center of surgical site outwards (anterior surface of the rat neck). c). incision stage. the anterior neck was opened with a midline vertical 42 incision. the underlying submandibular gland was then dissected, as was also the medial side of the right sternocleidomastoid muscle to expose the common carotid artery (cca). the arteries were then carefully separated from the vagus nerve and connective tissue. d. ligation stage. using a sterile nylon suture, both carotid arteries were ligated for 20 minutes. e. subsequently analgesic therapy was administered as 0.1 ml 0.25% bupivacaine, at the frequency of one time/ day (bupivacaine is the analgesic recommended for the rat stroke model). preparation of bsd the bsd was made based according to a modification of asikin’s protocol. (7) the policosanol dose was 200 mg/kg/day. (4) since 100 g fresh sugarcane peel contains 500 mg policosanol, the sugarcane peel dose was 40 g/ kg/day. the number of boiled sugarcane stalks were determined by the daily volume of the solution that was required. before boiling, the b l a c k s u g a r c a n e s h o u l d b e s t o r e d a t a temperature of 10° c.(8) preparation of the brain the rat brain tissues were taken at day 15, two hours post bccao. decapitation of rats was performed with a transcardial perfusion technique. brain tissues were stained with 2,3,5triphenyltetrazolium chloride (ttc) as follows: a 2% ttc solution in 1x phosphate-buffered solution (pbs) was prepared (i.e. 2 g ttc in 100 ml 1x pbs). the brain was cut in the coronal plane at 2 mm thickness, and the sliced brain was incubated in the 2% ttc solution in black boxes for 15-20 min. the ttc solution was carefully aspirated and fresh 10% pfa solution was added. the ttc solution should be p r o t e c t e d f r o m l i g h t a n d k e p t a t r o o m temperature. the parts of the rat brains that were studied comprised the cerebral cortex and striatum at the first slice of brain. statistical analysis the relative sizes of the ischemic areas were analyzed using image j. statistical analysis was performed by using one-way anova test . ethical clearance this study was reviewed by the ethical clearance committee for preclinical research, faculty of medicine, islamic university of indonesia. results the ischemic brain area appeared white in each group using image j software (figure 1). the size of the ischemic area was expressed as a percentage, i.e. the ratio of the ischemic area to the total area of the brain. figure 1. ttc staining results. a. sham-operated rats, b. non-treated stroke model rats, c. bsd-treated stroke model rats handayani, nugraha, nurmasitoh, et al black sugarcane and rat brain inchemia 43 univ med vol. 35 no.1 table 1. mean percentages of rat brain ischemic areas by treatment groups bsd=black sugarcane decoction; *statistically significant table 2. post hoc test bsd=black sugarcane decoction; *statistically significant these results indicated that there were differences in the mean percentage of rat brain ischemic area between sham-operated rats (0.00 ± 0.00%), non-treated stroke model rats (3.13 ± 0.59%) and bsd-treated stroke model rats (1.15 ± 0.47%) (p=0.001) (table 1). post hoc test showed that there was no difference between the percentage of ischemic brain area in shamoperated rats and in the bsd-treated stroke model rats. meanwhile, there was a significant difference between the group of non-treated stroke model rats and the other groups (table 2). discussion our results show that a 20-minute bccao can induce the occurrence of ischemic areas of the cortex and striatum. ttc staining shows ischemic areas of the brains of non-treated stroke model rats, occurring 2 hours post bccao. the results support the other research studies, stating that bilateral ligation of the carotid arteries for 30 minutes and reperfusion for 1 hour caused global ischemia in sprague-dawley rat brains and infarcted areas on ttc staining.(9) 2,3,5-triphenyltetrazolium chloride is a water-soluble dye that binds to dehydrogenases and nicotinamide cofactors (nad) in the mitochondria. a healthy tissue is dark red whereas damaged mitochondria in ischemic areas of the brain are white because they will not be stained by ttc.(9) bilateral carotid artery ligation causes brain ischemia, resulting in necrosis and s w e l l i n g o f t h e c e l l s , b r e a k d o w n o f t h e intracellular organelles and plasma membrane, releasing enzymes such as lactate dehydrogenase (ldh) into the plasma,(10) where its concentration can be measured. compared to other enzymes, lactate dehydrogenase is a more sensitive marker of brain ischemia and can be used to assess the incidence of stroke.(11) the period in which the enzyme levels increase in the blood varies from 8 hours to several days after the onset of stroke. lactate dehydrogenase levels in brain tissue may reach a peak at 48-120 hours post stroke. in the first hours of stroke there are differences in lactate dehydrogenase levels between cerebral cortical and subcortical ischemic lesions.(11) in one study, an increase was found in rat brain ldh activity under occlusion of the carotid artery for 1 hour.(12) similarly, in another study, there was an increase in the ldh activity in pyramidal neurons of the hippocampus and layers ii, iii and v of the cerebral cortex of the mongolian gerbils after 7 minutes of ischemia. the ldh activity was variable, ldh levels beginning to appear at the 7th minute of ligation, declining in the first 2 hours post-ligation, and returning to normal 7 days after the onset of ischemia (reperfusion period).(11) 44 the ischemic areas of the bsd-treated stroke model rats are smaller than those of the non-treated stroke model rats. these results show that black sugarcane can be used as a neuroprotectant, since sugarcane is a source of antioxidants.(13) the phenolic concentration in molasses is 381 µg/g molasses. the phenolic compounds contain cathechin (16.42 mg/g molasses) and quercetin 3-o-glucosyl-xyloxide (25.27 mg/g molasses). catechin functions as an antioxidant,(14) while it is known that quercetin has anti-tumoral, antithrombotic, anti-inflammatory and anti-apoptotic effects. quercetin plays an active role in the cellular mechanisms, by acting as an inhibitor of phosphatidylinositol-3 kinase, protein kinase c, xanthine oxidase and nadph diaphorase, causing quercetin to have a neuroprotective effect.(5) the high levels of these phenolics in molasses allow the use of these compounds in the prevention and treatment of diseases caused by oxidative stress, because molasses is able to protect the cells, being even more protective than á-tocopherol.(6,7) phenolics in black sugarcane can inhibit lipid peroxidation in the brain so that black sugarcane can be used as a neuroprotectant.(15) in addition, there are higher primary aliphatic acids derived from sugarcane wax that are called d-003. these compounds have antioxidant properties and mainly contain octacosanoic acid (c28). d-003 is able to inhibit plasma lipid peroxidase.(16,17) toxicity tests of sugarcane molasses have proven that d-003 is safe since it does not increase ldh levels.(18) one study found that 50–400 mg/kg policosanol protects effectively against cerebral ischemia in gerbils.(19) the administration of 200 mg/kg d-003 reduces cerebral edema and clinical signs in mongolian gerbils with bilateral carotid artery ligation.(17) policosanol, a mixture of high molecular weight alcohols purified from sugarcane wax, has been shown to produce antiplatelet and antioxidant effects.(20) one limitation of this study is the use of catgut threads to tie the carotid arteries. it is necessary to ensure that the ligatures on both sides are of equal strength. the results of this study can be used as a scientific basis for the community that the black sugarcane can be used as a neuroprotectant to prevent stroke. further research is necessary to determine the therapeutic dose, toxicity of black sugarcane decoction, and development of the ligation technique using artery clamps. conclusion this study demonstrated the neuroprotective effects of bsd in experimental brain ischaemia rats. conflict of interest competing interests: no relevant disclosures. acknowledgement this study used a grant from uppm, faculty medicine, islamic university of indonesia. references 1. gnanaraj ra. applications of sugarcane wax and it’s products: a review. int j chemtech res 2012;4:705-12 2. taylor j, rapport l, lockwood g.b. octacosanol in human health. nutrition 2003;19:192–5. 3. sánchez j, illnait j, mas r, et al. effects of policosanol plus aspirin therapy on the neurological recovery and plasma oxidative markers of patients with ischemic stroke. j pharm 2013;3:31-40. 4. guerra yp, cuevas vm, ferreiro rm, et al. effects of policosanol pre-treatment on bloodbrain barrier damage induced by ischemiareperfusion in rats. int j pharm sci rev res 2015;32:1-6. 5. dajas f, rivera-megret f, blasina f, et al. neuroprotection by flavonoids. braz j med biol res 2003;36:1613-20. 6. valli v, caravaca a, nunzio m, et al. sugar cane and sugar beet molasses, antioxidant-rich alternatives to refined sugar. j agric food chem 2012;60:12508-15. 7. asikin y. flavor characteristics and biological functions of okinawan sugary and citrus materials (thesis). united graduate school of handayani, nugraha, nurmasitoh, et al black sugarcane and rat brain inchemia 45 univ med vol. 35 no.1 agricultural sciences: kagoshima university, japan;2014. 8. krishnakumar t, thamilselvi c, devadas ct. effect of delayed extraction and storage on quality of sugarcane juice. afr j agric res 2013; 8:930-5. 9. rekabi md, hussein fh, mosawi a, et al. histopathological effects of im methionine in rat cerebral ischemia reperfusion i/r injury. br j med health res 2015;2:2394-67. 10. chan f, moriwaki k, rosa mj. detection of necrosis by release of lactate dehydrogenase (ldh) activity. methods mol biol 2013;979:65– 70. doi:10.1007/978-1-62703-290-2_7. 11. shcherbak ns, galagudza mm, ovchinnikov da, et al. lactate dehydrogenase activity in the cerebral cortex and hippocampus of mongolian gerbils in ischemic and reperfusion injuries. neurosci behav physiol 2013;43:941-5. 12. khalilov ra, dzhafarova am, dzhabrailova rn, et al. analysis of the kinetic characteristics of lactate dehydrogenase from the rat brain during ischemia and reperfusion.neurochem j 2014;8:265-70. 13. khan sk, tahir m, lone kp, et al. protective effect of saccharum officinarum l. (sugar cane) juice on isoniazid induced hepatotoxicity in male albino mice. j ayub med coll abbottabad 2015; 27:2. 14. chacko sm, thambi pt, kuttan r, et al. beneficial effects of green tea: a literature review. chinese med 2010;5:13. 15. almeida jm, novoa av, linares af, et al. antioxidant activity of phenolics compounds from sugar cane (saccharum officinarum l.) juice. plant foods hum nutr 2006;61:187-92. 16. perez y, molina v, mas r, et al. comparison of in vivo effects of d-003, a mixture of high molecular weight sugarcane wax acids and grape seed extract on lipid peroxidation markers in rats. lat am j pharm 2008;27:498-504. 17. molina v, noa m, arruzazabala l, et al. effect of d-003, a mixture of very-long-chain aliphatic acids purified from sugarcane wax on cerebral ischemia in mongolian gerbils. j med food 2005;8:482-7. doi:10.1089/jmf.2005.8.482. 18. rahiman f, pool ej. preliminary study on the effect of sugar cane (saccharum officinarum) molasses on steroidogenesis in testicular cell cultures. african j food sci 2010;4:37-40. 19. molina v, ravelo y, noa m, et al. therapeutic effects of policosanol and atorvastatin against global brain ischaemia-reperfusion injury in gerbils. indian j pharm sci 2013;75:635-41. 20. molina v, carbajal d, arruzazabala ml, et al. comparative study of policosanol and grape seed extract on platelet aggregation in rats. rev cienc biol 2011;42:3-6. oktavianus 165 *center for the study of oncology, faculty of medicine, universitas padjadjaran **department of pathology, faculty of medicine, universitas padjadjaran/ dr. hasan sadikin general hospital, bandung ***department of biochemistry and molecular biology, faculty of medicine, universitas padjadjaran ****department of obstetrics and gynecology, faculty of medicine, universitas padjadjaran/ dr. hasan sadikin general hospital, bandung corresponding: afandi charles center for the study of oncology faculty of medicine, universitas padjadjaran jl. raya bandung sumedang km.21, jatinangor, west java, indonesia. phone and fax number: +6282297446690 email: afandi.charles@gmail.com univ med 2016;35:165-70 doi: 10.18051/univmed.2016.v35.165-170 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 paclitaxel-carboplatin chemotherapy induced hematologic toxicities among epithelial ovarian cancer patients afandi charles*, birgitta m. dewayani**, edhyana sahiratmadja***, gatot n.a. winarno****, and herman susanto**** background epithelial ovarian cancer (eoc) is one of the most common cancers diagnosed in indonesian women. a combination of paclitaxel and carboplatin is used to treat eoc as standard chemotherapy which is known to have hematologic toxicities. this study aimed to investigate the effect of combined paclitaxel-carboplatin chemotherapy on hematologic status in eoc patients managed at dr. hasan sadikin general hospital, bandung, west java. methods all patients with confirmed pathological diagnosis of eoc at dr. hasan sadikin general hospital in the period of 2013 to 2014 were registered. only patients with complete hematologic data before and after chemotherapy were collected and compared using the paired nonparametric wilcoxon and mcnemar tests. results in total there were 147 patients with eoc (median age 46 ± 12 years), with the most dominant pathological diagnosis of mucinous (32.7%) and serous (29.3%) types. only 33 patients had hematologic data before the initiation of chemotherapy. there was a significant decrease after chemotherapy including hemoglobin level (12.0 vs 10.9 g/dl, p=0.013), erythrocyte count (4.53 vs 3.74 million/µl, p<0.001), leukocyte count (7,700 vs 4,000/mm3 p<0.001) and platelet count (343,000 vs 215,000/ mm3, p<0.001). interestingly, anemia cases after chemotherapy were predominant (87.9%) compared with erythopenia, leukopenia, thrombocytopenia i.e. 39.4%, 57.6%, and 27.3% respectively. conclusions this study confirmed the hematologic toxicities after paclitaxelcarboplatin chemotherapy in eoc patients treated in hasan sadikin general hospital, west java. the hemoglobin concentration may serve as prognostic factor. further studies directed to other factors such as genetic factor for polymorphisms may be encouraged to explore the decrease of the hematologic indices. keywords: anemia, erythropenia, leukopenia, thrombocytopenia, epithelial ovarian cancer, paclitaxel-carboplatin doi: http://dx.doi.org/10.18051/univmed.2016.v35.165-170 166 charles, dewayani, sahiratmadja, et al hematologic toxicities by paclitaxel-carboplatin chemotherapy introduction epithelial ovarian cancers (eoc) are still burdening female health nowadays. the world health organization estimated that this type of cancer accounted for 151,905 female deaths worldwide in 2012.(1) in indonesia, eoc, together with other ovarian cancers are placed in the third rank of the most prevalent gynecologic cancers, next to breast and cervical cancers.(2) there are several risk factors associated with eoc, for example age older than 50 years, no pregnancy and lactation history and family history of breast and ovarian cancers.(3) management of eoc consists of explorative surgery and chemotherapy.(3) the standard regimen for eoc is intravenous paclitaxel 175 mg/m2 and carboplatin area under curve is 7.5 for six cycles.(3) these regimen components, especially carboplatin have several side effects in bone marrow, resulting in suppression of hematopoiesis. common hematologic toxicities developing in patients include anemia, thrombocytopenia, and leukopenia.(4) these side effects are mediated by the formation of dnaprotein cross-linking that further disrupts cellcycle regulation and suppresses cell growth.(5) chemotherapy is a standardized process performed at dr. hasan sadikin hospital, bandung. there is a need for research on the hematologic impact of the paclitaxelcarboplatin combined therapy in eoc patients. therefore, the aim of this study was to explore eoc patients treated in dr. hasan sadikin general hospital, bandung and to investigate the effect of the paclitaxel-carboplatin regimen. methods research design this study employed an analytical retrospective cohort study design conducted using medical records of patients treated in dr. hasan sadikin general hospital, bandung. the medical records were collected in the period of january 2013 to december 2014. research subjects all subjects with a pathological diagnosis of eoc were recruited for this study. other types of ovarian cancer including malignant granulosa cell tumor and teratoma were excluded. clinical, laboratory and chemotherapy data including the number of chemotherapy cycles were recorded. laboratory analysis as for laboratory data, hematologic findings were collected before the initiation of chemotherapy and after chemotherapy of 6 cycles. anemia was defined at a hb concentration of <12.0 g/dl, erythropenia at an erythrocyte count of <3.6 million/µl, leukopenia at a leukocyte count of <4400/mm3 and thrombocytopenia at a platelet count of <150,000/mm3. only patients with complete hematologic data and number of chemotherapy cycles were included. statistical analysis descriptive statistics for clinical data and histopathological category of eoc were shown as frequencies. mcnemar and wilcoxon tests were used to determine the association of the h e m a t o l o g i c s t a t u s b e f o r e a n d a f t e r chemotherapy. for the statistical analyses, the spss 22.0 software package (spss inc. chicago. il) was used. ethical clearance the ethical clearance required for this study was granted by the health research ethics committee, faculty of medicine, universitas padjadjaran dr. hasan sadikin general hospital, bandung (no. 3010/un6.c.c1/pp/ 2015). results in total, there were 147 women with a median age of 46 years and an age range of 14 to 81 years, among whom more than half (59.2%) were younger than 50 years, i.e. the cut-off point of menopausal age in indonesia. the most common histopathological types found in these 167 univ med vol. 35 no.3 table 1. characteristics of epithelial ovarian cancer patients with complete hematologic data (n=33) *figo=international federation of gynecology and obstetrics table 2. difference of hematologic findings between preand post-chemotherapy *p values were statistically significant. analysis conducted using wilcoxon test. significance level p=0.05 **values were x1.000 patients were the mucinous and serous types, comprising 32.7% and 29.3%, respectively. many of the subjects were lost to follow up; some of them refused to undergo chemotherapy m a n a g e m e n t o r d i d n o t h a v e c o m p l e t e hematologic laboratory data, resulting in only 33 patients with complete laboratory and chemotherapy data who were further analyzed in this study. of these eoc patients, most (54.5%) were diagnosed before the age of 50 years and were already in stage iii (51.5%). only 12 of 33 patients completed the six cycles of chemotherapy, while others underwent only three t o f i v e c y c l e s ( ta b l e 1 ) . t h e r e w a s n o documentation in the medical records regarding t h e c a u s e o f e a r l y t e r m i n a t i o n o f t h e chemotherapy. the hematologic findings were not normally distributed, except for erythrocyte counts. therefore the hemoglobin, leukocyte and platelet counts were presented as mean and standard deviation, whereas the erythrocyte count was presented as median and range. the hemoglobin level and the erythrocyte, leukocyte and platelet c o u n t s d e c r e a s e d s i g n i f i c a n t l y a f t e r chemotherapy as depicted in table 2. using categorical laboratory references, the distribution of patients with anemia, erythropenia, leukopenia, and thrombocytopenia also decreased significantly as shown in table 3. interestingly, after chemotherapy there was a predominant increase in anemia in 20 of 33 patients (60.6%), while erythropenia, leukopenia, and thrombocytopenia increased only in 13/33 (39.4%), 19/33 (57.6%), and 9/33 (27.3%) of patients, respectively (table 3). the age at clinical presentation of eoc was analyzed to determine whether it was associated with the decrease in all post-chemotherapy hematologic findings, but the results showed no association (data not shown). 168 charles, dewayani, sahiratmadja, et al hematologic toxicities by paclitaxel-carboplatin chemotherapy table 3. chemotherapy induced anemia, erythropenia, leukopenia and thrombocytopenia in adults *p values were statistically different. analysis conducted using mcnemar test. significance level p=0.05 **low hematologic counts after chemotherapy discussion our study explored the hematologic findings before and after paclitaxel-carboplatin chemotherapy among patients with epithelial ovarian cancer (eoc) and confirmed that there was a significant decrease in hematologic findings such as anemia, erythropenia, leukopenia, and thrombocytopenia. these findings are coherent with studies in advanced eoc cases, either using conventional or dose-dense paclitaxel-carboplatin regimen.(4,7) the paclitaxel-carboplatin regimen is known to have a lower incidence of high-grade neutropenia in eoc chemotherapy compared to its alternative regimen, docetaxel and carboplatin.(8) in our study we did not perform neutrophil counts; however, the hemoglobin level was significantly reduced after chemotherapy. hemoglobin levels before chemotherapy have proposed to have prognostic value in terms of progression-free survival time for eoc patients.(9) the paclitaxel-carboplatin regimen is also used in other neoplasm cases, notably non-small cell lung cancer (nsclc) and urothelial cancer, and has been associated with febrile neutropenia.(10) furthermore, anemia, neutropenia, and thrombocytopenia are the most frequently observed toxicities after paclitaxel-carboplatin chemotherapy in advanced urothelial cancer patients in japan.(11) these hematologic toxicities are also observed in nsclc, and in addition, the paclitaxel-carboplatin regimen has been revealed to produce more severe anemia and neutropenia in asian than in non-asian patients.(12) as hematopoietic stem cells undergo senescence, many lesions and mutations are accumulated in their dna, producing exhaustion in stem cell division.(7) this underlying mechanism may be responsible for the more severe hematologic toxicities of cytotoxic chemotherapy regimens, for example cyclophosphamidedoxorubicin in breast cancer and bevacizumabpaclitaxel-carboplatin in nsclc.(13,14) however, an association between age and severity was not documented in these studies, and another study in nsclc cases with paclitaxel-carboplatin chemotherapy also yielded similar results.(15) the hematologic toxicities occurring in eoc patients after having received paclitaxelcarboplatin chemotherapy are influenced by several factors, i.e. from the regimen itself and patient background. paclitaxel exerts its c y t o t o x i c e ff e c t s b y d e p o l y m e r i z i n g microtubules, thus arresting the cell cycle and inducing apoptosis.(16) however, paclitaxel only induces minimal hematologic toxicities when administered alone.(16) carboplatin used in this regimen has a myelosuppressive effect via the mechanism of dna-protein damage in bone marrow by means of crosslinking between them, t h u s r e s u l t i n g i n d e c r e a s e d n u m b e r s o f erythrocytes, leukocytes, and platelets, as well 169 univ med vol. 35 no.3 as other toxic effects (neurotoxicity, ototoxicity, and nephrotoxicity).(17,18) it also seems that carboplatin may increase serum tnf-α, suppressing the development of hematologic progenitor cells.(19) currently, genetic polymorphisms are being investigated to determine their association with paclitaxel-carboplatin hematologic toxicity, depending on race and ethnicity. the single nucleotide polymorphisms 2677g>t/a and 3435c>t of abcb1 genes have been associated with neutropenia in paclitaxel chemotherapy in e o c c a s e s i n s o m e s t u d i e s , ( 2 0 , 2 1 ) b u t n o association has been found in others.(22,23) some studies in nsclc have demonstrated that polymorphisms in drug-transporter genes (abcb1 and abcg2), dna-repair pathway genes ( ercc4 and xcc ), and apoptosis pathway genes (casp 8 and casp10) are associated with thrombocytopenia, neutropenia, and severe hematologic toxicity, respectively, due to paclitaxel-carboplatin effects.(24-26) one limitation of this study is the small sample size which was not appropriate to the estimated sample size due to incomplete data. another limitation is inadequate hematologic data in each of the chemotherapy cycles, which l i m i t s s t a t i s t i c a l a n a l y s i s ; h o w e v e r, t h e decreasing trend in the hematologic data might be used to generalize the findings. conclusion in conclusion, the decrease in hematologic data such as anemia, leukopenia, and thrombocytopenia after paclitaxel-carboplatin chemotherapy were confirmed in eoc patients treated in dr. hasan sadikin general hospital. the hemoglobin concentration may serve as prognostic factor. in addition, further studies are needed on genetic background factors that may play a role in the decrease in hematologic data. conflict of interest competing interest: no relevant disclosures. acknowledgement this research is part of a progress grants 2014 study conducted under the oncology working group, faculty of medicine, universitas padjadjaran. references 1. union for international cancer control. epithelial ovarian cancer. geneva: world health organization;2014. 2. wahidin m, noviani r, hermawan s, et al. population-based cancer registration in indonesia. asian pac j cancer prev 2012;13: 1709-10. 3. jelovac d, armstrong dk. recent progress in the diagnosis and treatment of ovarian cancer. ca 2011;61:183-203. 4. katsumata n, yasuda m, takahashi f, et al. dose-dense paclitaxel once a week in combination with carboplatin every 3 weeks for advanced ovarian cancer: a phase 3, openlabel, randomised controlled trial. lancet 2009;374:1331-8. 5. barker s, weinfeld m, murray d. dna– protein crosslinks: their induction, repair, and biological consequences. mutat res 2005;589:111-35. 6. siregar mfg. perimenopausal and postmenopausal complaints in paramedics assesed by menopause rating scale in indonesia. j dental med sci 2009;13:38:42. 7. van der burg m, vergote i, onstenk w, et al. long-term results of weekly paclitaxel carboplatin induction therapy: an effective and well-tolerated treatment in patients with platinum-resistant ovarian cancer. eur j cancer 2013;49:1254-63. 8. pignata s, cannella l, leopardo d, et al. chemotherapy in epithelial ovarian cancer. cancer lett 2011;303:73-83. 9. eichbaum mh, weiss lm, bruckner t, et al. prognostic impact of hemoglobin levels before and during carboplatin/taxane-based chemotherapy in patients with primary invasive epithelial ovarian cancer. med sci monit basic res 2009;15:cr156-cr63. 10. aapro ms, cameron da, pettengell r, et al. eortc guidelines for the use of granulocyte-colony stimulating factor to reduce the incidence of chemotherapyinduced febrile neutropenia in adult patients 170 charles, dewayani, sahiratmadja, et al hematologic toxicities by paclitaxel-carboplatin chemotherapy with lymphomas and solid tumours. eur j cancer 2006;42:2433-53. 11. soga n, onishi t, arima k, et al. paclitaxel carboplatin chemotherapy as a second line chemotherapy for advanced platinum resistant urothelial cancer in japanese cases. int j urol 2007;14:828-32. 12. hasegawa y, kawaguchi t, kubo a, et al. ethnic difference in hematological toxicity in patients with non-small cell lung cancer treated with chemotherapy: a pooled analysis on asian versus non-asian in phase ii and iii clinical trials. j thorac oncol 2011;6: 1881-8. 13. ramalingam ss, dahlberg se, langer cj, et al. outcomes for elderly, advanced-stage non–small-cell lung cancer patients treated with bevacizumab in combination with carboplatin and paclitaxel: analysis of eastern cooperative oncology group trial 4599. j clin oncol 2008;26:60-5. 14. muss hb, berry da, cirrincione c, et al. toxicity of older and younger patients treated with adjuvant chemotherapy for nodepositive breast cancer: the cancer and leukemia group b experience. j clin oncol 2007;25:3699-704. 15. ganti ak, loberiza fr jr, kessinger a. factors affecting bone marrow toxicity following administration of carboplatin and paclitaxel in patients with non-small cell lung cancer. anticancer res 2010;30:1365-9. 16. kampan nc, madondo mt, mcnally om, et al. paclitaxel and its evolving role in the management of ovarian cancer. biomed res int 2015;2015:413076. 17. hildebrandt ma, gu j, wu x. pharmacogenomics of platinum-based chemotherapy in nsclc. expert opin drug metab toxicol 2009;5:745-55. 18. joerger m, huitema ad, richel dj, et al. population pharmacokinetics and pharmacodynamics of paclitaxel and carboplatin in ovarian cancer patients: a study by the european organization for research and treatment of cancer-pharmacology and molecular mechanisms group and new drug development group. clin cancer res 2007; 13:6410-8. 19. arafa hm, ismail rs, nabil n, et al. carnitine deficiency: a causative clue or a sequel in carboplatin myelosuppression. j cancer res updates 2014;3:226-35. 20. sissung tm, mross k, steinberg sm, et al. association of abcb1 genotypes with paclitaxel-mediated peripheral neuropathy and neutropenia. eur j cancer 2006;42:28936. 21. bergmann tk, brasch andersen c, gréen h, et al. impact of abcb1 variants on neutrophil depression: a pharmacogenomic study of paclitaxel in 92 women with ovarian cancer. basic clin pharmacol toxicol 2012; 110:199-204. 22. bergmann tk, gréen h, brasch-andersen c, et al. retrospective study of the impact of pharmacogenetic variants on paclitaxel toxicity and survival in patients with ovarian cancer. eur j clin pharmacol 2011;67:693700. 23. marsh s, paul j, king cr, et al. pharmacogenetic assessment of toxicity and outcome after platinum plus taxane chemotherapy in ovarian cancer: the scottish randomised trial in ovarian cancer. j clin oncol 2007;25:4528-35. 24. qian j, qu h-q, yang l, et al. association between casp8 and casp10 polymorphisms and toxicity outcomes with platinum-based chemotherapy in chinese patients with non-small cell lung cancer. oncologist 2012;17:1551-61. 25. lamba jk, fridley bl, ghosh tm, et al. genetic variation in platinating agent and taxane pathway genes as predictors of outcome and toxicity in advanced non-smallcell lung cancer. pharmacogenomics 2014;15:1565-74. 26. gu s, wu q, zhao x, et al. association of casp3 polymorphism with hematologic toxicity in patients with advanced non small cell lung carcinoma treated with platinum based chemotherapy. cancer sci 2012;103: 1451-9. c:\users\universa medicina\docu 263 abstract universa medicina marasmus and stunting in 2-month-old boy with pneumatocele: a case report maya susanti1 and aidah juliaty1* case report pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.263-269 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1142 september-december 2021 vol.40no.3 cite this article as: susanti m, juliaty a. marasmus and stunting in 2-monthold boy with pneumatocele: a case report. univ med 2021;40:263-9. doi: 1 0 . 1 8 0 5 1 / un i v m ed . 2 0 2 1 .v4 0 . 2 6 3 2 6 9 1department of pediatrics, faculty of medicine, hasanuddin university/ wahidin sudirohusodo hospital, makassar, indonesia correspondence: *aidah juliaty, md, ph.d department of pediatrics, faculty of medicine, hasanuddin university jl. perintis kemerdekaan km. 10, tamalanrea 90245, makassarindonesia email: juliatyaidah07@gmail.com orcid id: 0000-0001-9927-1940 date of first submission, february 22, 2020 date of final revised submission, october 6, 2021 date of acceptance, october 11, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license background nutrition is essential for humans and if inadequate may lead to undernutrition. undernourished children are very susceptible to infections, such as pneumonia, one of its complications being pneumatocele, which is a more severe condition. pulmonary pneumatoceles are thin-walled, air-filled cysts that develop within the lung parenchyma and have been found in 2.4 percent of 493 infants and children with pneumonia. however, in staphylococcal pneumonias, the frequency of pneumatocele can reach as high as 85 percent. infants and young children are more likely to have pneumatoceles. it is critical to distinguish marasmus with pneumatocele from many other similar diagnoses. failure to recognize can lead to incorrect diagnosis and treatment, causing more harm than good to patient care. this case highlights the importance of maintaining a high suspicion of pneumonia in neonates even with normal radiological findings and of searching for pneumatoceles. case description we report a case of marasmus and stunting accompanied by pneumatocele in a 2-month-old boy. the diagnosis was made based on history, physical examination, anthropometric examination using the who child growth standards, laboratory tests, and radiological imaging. management of the patient was according to a multidisciplinary system including antibiotics administration and wasting management. conclusion pneumatoceles are serious complications after pneumonia. pneumonia is common in children with marasmus. children with marasmus are caused by various underlying factors. the progression begins in the womb and continues through the first 1000 days of life. keywords: stunting, pneumonia, pneumatocele, marasmus 264 susanti, juliaty marasmus and stunting with pneumatocele introduction pulmonary pneumatoceles are thin-walled, air-filled cysts that develop within the lung parenchyma as a complication of pneumonia. they can also result from non-infectious etiologies such as trauma and positive pressure ventilation.(1) in most patients, pneumatoceles are asymptomatic and do not require surgical treatment. hussain et al.(2) in the post surfactant era, including 1053 infants with a gestational age o f d ” 30 w ee ks a nd r e spi ra tor y d i st r ess syndrome (rds) requiring positive pressure ventilation, identified pneumatoceles in 19 infants, giving an incidence of 1.8% and a male to fema le ratio of 12:7. pneumatocele is commonly seen in infants and young children as a c omplication of staphylococcus aureus pneumonia,(3) its frequency can be as high as 85% and its predilection site depends on the susceptibility of the individual to infection. the high incidence was attributed to the high level of undernutrition in children.(4) nutrition is essential and plays a role in the quality of life, because inadequate dietary intake will cause undernutrition, which then induces long-term effects on child physical and mental development and quality of life.(5,6) under nutr ition occurs as a result of malabsorption or metabolic failure. clinical signs of undernutrition are identified by indicators measuring body weight (bw), body length (bl), and upper arm circumference (uac) that are significantly lower than the mean. undernutrition can have long-term consequences for child physical and mental development and quality of life.(6) the 2018 global nutrition report stated that the case burden of severe wasting reached 7.5% or 50.5 million children under five in 2017.(7) indonesia ranks second highest (12.1%) in prevalence of wasting among 17 countries in the asian region. in south sulawesi, the prevalence of wasting reaches 9.6%.(8) stunting affected 159 million children younger than 5 years of age in 2014.(9) toddler s with unde rnutrition have a decreased immune system, thus they are prone to infections, such as pneumonia, which is one of the most common causes of death in children under five.(10) pneumonia accounts for 15% of all deaths of children under 5 ye ars old. (1 1) pneumatocele is a complication of communityacquired pneumonia (cap) and increases the risk of death in children under five years.(3) the purpose of this case is to highlight the importance of adequate treatment when neonatal pneumonia is highly suspected with normal radiological findings, to search for pneumatocele as a complication of marasmus and stunting and to rule out congenital malformations in case of pulmonary cyst. case report a two-month-old boy was transferred from the jaury jusuf putera academic hospital, makassar, south sulawesi, to the emergency unit of our hospital with the main complaint of shortness of breath that had been noticed for 17 days and was getting worse a day before admission. there was no cyanosis and no cough but there was a history of cough two weeks before being admitted to the hospital. oral intake was stopped in the last three days before the patient was transferred. he was treated for 11 days and received antibiotic therapy (cefotaxime and gentamicin). there were no abnormalities during delivery. the patient’s birth weight was 3500 grams and his body length was 50 cm. his condition after birth was good. he only received hepatitis b immunization and had formula milk since birth. the mother never visited the health care center after being discharged after birth. physical examination revealed tachypnea, which had been present for 17 days prior to hospital admission and had worsened one day before admission. there was no fever and the oxygen saturation was 98% with nasal cannula. on examination of the head, the patient had an old man’s face. chest examination revealed intercosta l and subc ostal r etr action and 265 univ med vol. 40 no 3 bronchovesicular breathing sounds with rales were heard in both lung fields. wasting was found on the extremities. skin examination showed no bcg scars. the examination of other regions was within normal limits (figure 1). anthropometric examination showed that body weight (bw) was 3000 grams, body length (bl) 52 cm, head circumference (hc) 39 cm (normal 38-42 cm), and upper arm circumference (uac) 8.5 cm. weight-for-length was below -2 figure 1. clinical examination on the 1st day of care: the patient appeared very wasted, with wasting on chest and extremities; on the 5th day, the patient began to regain weight figure 2. child growth: weight-for-length was below -2 sd (a), weight-for-age was below -3 sd (b), length-for-age was below -3 sd (c), body mass index (bmi)-for-age was below-3 sd (d), using who child growth standards 266 susanti, juliaty marasmus and stunting with pneumatocele sd (wasting), weight-for-age was below -3 sd (severely underweight), and length-for-age was below -3 sd (severely stunted), all based on the who child growth standards (figure 2). based on the patient’s anthropometric and clinical status, it was concluded that the child had nutritional marasmus. laboratory examin ation fr om j aury academic hospital showed hb 10.9 g/dl, ht 34.8%, leukocytes 23,560/ul, platelets 353,000/ ul. laboratory examination from our hospital showed hb 8.2g/dl, mcv 91 fl (femtoliter), mch 30 pg, ht 25%, leukocytes 6,300/ul, platelets 251,000/ul, lymphocytes 29.9%, neutrophils 55.7%, monocytes 8.5%, glucose 174 mg/dl, sodium 133 mmol/l, potassium 4.3 mmol/ ll, ureum 8 mg/dl, creatinine 0.31 mg/dl, sgot 132 u/l, sgpt 71 u/l, albumin 2.8 g/dl, crp 2.0 mg/l, procalcitonin 2.03 ng/ml, ferritin 1018.22 ng/ml. blood and urine cultures show no growth of aerobic bacteria. radiological examination (multi slice computed tomography/ msct of thorax without contrast) showed bilateral pneumonia accompanied by multiple left pulmonary cavities suspected as pneumatoceles and bilateral pleural effusions (figure 3a). figure 3a. thoracic msct (without contrast) july, 23 2020 showed bilateral pneumonia accompanied by multiple left pulmonary cavities suspected of being pneumatocele and bilateral pleural effusion figure 3b. thoracic msct (without contrast) after 2 months of observation, september 20, 2020 showed bilateral pneumonia and no multiple pulmonary cavities 267 the patient was diagnosed as having nutritional marasmus, with an additional diagnosis of inadequate intake, stunting, communityacquired pneumonia, pneumatocele, bilateral pleural effusions, anemia of chronic disease, hypoalbuminemia, elevated transaminases, and delayed immunization. the patient was treated with nasal oxygen and antibiotics (ceftazidime and amikacin) . we also per for med gastric decompression and gave the nutrition via parenteral route. the patient was planned for tuberculosis work-up. the stabilization phase of wasting management was temporarily postponed. the patient’s fluid requirement on the first day after the restriction was 240 ml/day with an energy requirement (schofield) of 257.85 kcal/ day. after treatment, laboratory examination showed hb 9.9 g/dl, mcv 84 fl, mch 29 pg, ht 22%, leukocytes 9,600/ul, platelets 312,000/ ul,%, glucose 155 mg/dl, sodium 130 mmol/l, potassium 4.3 mmol/l, ureum 12 mg/dl, creatinine 0.26 mg/dl, sgot 23 u/l , sgpt 14 u/l, albumin 3.3 g/dl, crp 0.0 mg/l, procalcitonin 0.25 ng/ml, and it ratio 10%. on the third day, the child was given enteral priming at 10 ml/kg/day through a nasogastric tube while still being given parenteral nutrition. on the 4th and 5th days, enteral priming was increased gradually to 30 ml/kg/day. on the 6th day, the child’s condition improved, dyspnea had decrease d, entera l administra tion via the nasogastric tube was given to 50% of energy requirement with high-calorie formula milk, and the remaining 50% with parenteral nutrition. on the 8th day of treatment, the patient was started on the stabilization phase of wasting treatment on day one using high-calorie formula milk at 12 x 25 ml (240 kcal) through the nasogastric tube, with supplementation. on the 9th, 10th, and 11th days, the treatment was followed by the transition and rehabilitation phase of wasting management. after 2 months of observation, radiological examination (msct thorax without contrast) showed bilateral pneumonia with no evidence of multiple pulmonary cavities (figure 3b). written informed consent was obtained from the patient’s parents for publication of this case report and accompanying images. discussion the nutritional status of undernutrition children under the age of five influences the risk of pneumonia in these children. undernourished children under the age of five have a weakened immune system, making them vulnerable to infectious diseases. wasting is a nutritional disorder that can have a negative impact on the health of children under the age of five and will stifle their growth if not handled properly.(12) our case report is consistent with the findings of a case of pulmonary pneumatocele in postpneumonic empyema in a 4-year-old female.(13) following pneumonia, inf lammation and narrowing of the bronchi cause the formation of an endobronchial ball valve mechanism, resulting in distal dilatation of the bronchi and alveoli. the obstr uction is belie ved to be caused by inflammatory exudates within the airway lumen or wall, allowing air to enter but not to leave the cystic space. there is little information available about infective pulmonary cysts in infants. the high incidence is attributed to undernutrition in children. it is hypothesized that undernutrition would delay the development of kohn’s pores, resulting in undernourished children having lungs similar to newborns. when there is a valvular obstruction, air continues to enter with no way out due to the absence of kohn’s pores, resulting in pneumatocele.(4) the majority of children with severe pneumonia ar e malnour ished and unhealthy. the decrease in immunity is caused by a decrease in the activity of leukocytes to phagocytize and kill germs. according to a study, there is a link between infant pneumonia and nutritional status. the toddlers do not have appetite, which leads to undernutrition.(6) in the present case, the patient had a weightfor-height value below -2 sd. he had clinical symptoms of marasmus. we also found that the patient’s length-for-age was below -3 sd with the growth rate in these patients for ages 0-12 univ med vol. 40 no 3 268 susanti, juliaty marasmus and stunting with pneumatocele months being 23-27 cm/year or 1.9-2.2 cm/month. the patient met the diagnostic criteria for stunting. according to epidemiological studies, an estimated 155 million children under the age of five were stunted in 2016, with 52 million suffering from wasting and 17 million suffering from severe wasting. marasmus is equally distributed between the sexes; however, due to cultural differences in some parts of the world, women may be at greater risk of developing marasmus. meanwhile, the causes of stunting are classified as direct and indirect. food intake and infectious diseases have a direct impact on stunting, while the indirect causes are food accessibility, parenting, drinking water availability, sanita tion, and health services.(14) inadequate total caloric intake is the main cause of marasmus. however, it is critical to understand what causes the decrease in calorie intake in a marasmic patient. severe caloric restriction increases susceptibility to infection, which occurs as a result of secondary immune deficiency. prolonged caloric restriction is associated with impaired mucosal barrier integrity in the respiratory and gastrointestinal systems. inflammatory cytokines such as interleukin 1 (il1), il-6, and il-12 disrupt growth hormone function, contributing to short stature. as a result of t cell dysfunction and decreased neutrophil microbicidal activity, there is an increased susceptibility to infection.(14,15) infection with gram-negative organisms, in particular, has been linked to marasmus. in addition, marasmus is linked to urinary, gastrointestinal, and respiratory tract infections.(10) pneumatocele is one of the complications of pneumonia that are found in children under five. it occurs due to inflammation and narrowing of the bronc hi causi ng the for mation of endobronchial ball valves which initiate dilatation of the distal part of the bronchi and alveoli. the obstr uction is belie ved to be caused by inflammatory exudate within the lumen or airway walls, allowing air to enter the cystic space but not to escape. subsequent enlargement of the pneumatocele occurs either due to pressure from the adjacent pneumatocele or intraluminal inflammatory exudate.(5) in our case, the patient complained of dyspnea, and a radiological examination revealed that the patient had pneumonia with pneumatocele. as described in the theory above, this patient has several risk factors, including: low socioeconomic status, residence in a densely populated area, non-exclusive breastfeeding, and malnutrition. due to the high incidence of infection in malnourished children and to the patient’s complaints, we also performed radiological examinations to determine the patient’s source of infection. the chest ct scan results revealed bilateral pneumonia with multiple left pulmonary cavities suspected of being a pneumatocele. pneumatoceles can be treated using a variety of methods, including: i) observation; 90% of pneumatoceles resolve spontaneously within a few weeks up to 6 to 12 months without intervention. however, antimicrobials were still administered during the observation period; ii) utilization of unilateral ventilation with highfrequency oscillation (hfo); iii) percutaneous drainage in the decubitus position with the affected side down, followed by selective intubation of the unaffected side; iv) surgical resection; including video-assisted thoracoscopic surgery (vats), lobectomy, pneumatocele consideration/deroofing, and pneumonectomy.(16) in this case, we planned to perform observation by giving antibiotics and to do thoracic msct 3 months later to observe the progress. one of the contraindications for enteral nutrition is severe respiratory distress. therefore, we used parenteral nutrition (pn). when the condition is stable, children with pn may possibly be given enteral or oral nutrition. in this transition period, pn was given cyclically to prevent hyperinsulinism and hypoglycemia. therefore, even though we had enteral priming on the second day, pn was continued and decreased slowly.(17) parenteral nutrition should be stopped gradually and malnutrition management should be initiated. the stabilization phase should be started when the children receive full enteral nutrition.(17) 269 patients with good oro-motor function should have oral nutrition directly. the patient was discharged on the eleventh day to continue outpatient care with the consideration of improved clinical condition, being conscious, without medical problems or edema, the baby getting adequate intake, and the weight gain being sufficient (>5 g/kg/day) for three consecutive days.(17) however, adequate nutrition for children is essential for growth, development, and defense of the body, ensuring that they are not susceptible to infection. conclusion this case highlights the importance of maintaining a high suspicion of pneumonia in marasmic and stunted infants with multiple left pulmona ry cavities suspected of being pneumatoceles and bilateral pleural effusions in radiological findings with a focus on the diagnostic value and sputum cultures and viral testing to complete the investigation on infectious etiology. clinicians should search for pneumatoceles and other complications when pneumonia is diagnosed, especially in cases accompanied by malnutrition. conflict of interest the authors declare no conflict of interest. contributors all authors critic ally reviewed the manuscript and approved the final version submitted for publication. references 1. kesieme eb, kesieme cn, akpede go, et al. tension pneumatocele due to enterobacter gergoviae pneumonia: a case report. case rep med 2012;2012:808630. 2. hussain n, noce t, sharma p, et al. pneumatoceles in preterm infants: incidenceand outcome in the post surfactant era. j perinatol 2010;30:330-6. https://doi.org/10.1038/jp.2009.162. 3. choueiry e, diab s, el haber c, torbey ph, ghorayeb z, gerbaka b. incomplete treatment of neonatal pneumonia causing giant pneumatocele in a 45-day infant. int j clin pediatr 2017;6:24-7. 4. jamil a, kasi a. pneumatocele. treasure island (fl): statpearls publishing llc;2021 5. lelijveld n, seal a, wells jc, et al. chronic disease outcomes after severe acute malnutrition in malawian children (chrosam): a cohort study. lancet glob heal 2016;4:e654–62. doi: 10.1016/ s2214-109x(16)30133-4. 6. direktorat gizi masyarakat. kebijakan pencegahan dan tata laksana gizi buruk pada balita. jakarta: direktorat gizi masyarakat; 2019. 7. global nutrition report. 2018 global nutrition report. new york : global nutrition report;2018 8. kementerian kesehatan republik indonesia. status gizi indonesia alami perbaikan. jakarta; kementerian kesehatan republik indonesia;2019. 9. international food policy research institute. global nutrition report 2016. international food policy research institute, washington, dc;2016 10. ruwandasari n. correlation between severe malnutrition and pneumonia among under-five children in east java. j berk epidemiol 2019;7:120. 11. world health organization. pneumonia. geneva: world health organization;2019. 12. artawan a, purniti ps, sidiartha igl. the relationship between nutritional status with pneumonia severity in children at sanglah hospital, bali, indonesia. sari pediatr 2016;17:418. 13. hsairi m, louati h, abid h, et al. pneumatocele in postpneumonic empyema in children: a case report. int j pediatr 2017; 5:6539-40. doi:10.22038/ ijp.2017.27673.2393. 14. ibrahim mk, zambruni m, melby cl, melby pc. impact of childhood malnutrition on host defense and infection. clin microbiol rev 2017;30:919– 71. 15. mortaz e, alipoor sd, adcock im,mumby s,koenderman l. update on neutrophil function in severe inflammation. front immunol 2018; 9: 2171. doi: 10.3389/fimmu.2018.02171. 16. takele y, adem e, getahun m, et al. malnutrition in healthy individuals results in increased mixed cytokine profiles, altered neutrophil subsets and function. plos one 2016;11:e0157919. https:// doi.org/10.1371/journal.pone.0157919. 17. munoz a, moores dc, khan fa, baerg j, radulescu a. successful treatment of postinfectious pneumatocele via percutaneous drainage in a premature infant. j pediatr surg case reports 2019;47:101235. https://doi.org/10.1016/ j.epsc.2019.101235. univ med vol. 40 no 3 c:\users\user\documents\afifah. 114 abstract universa medicina the protective effect of celery ethanol extract on oxidative stress in chronic kidney disease rat model afifah1*, fajar wahyu pribadi1, aulia salsabiela2, dimo hari anggara2, zahra muthmainnah komara2, and robby al fauzy2 background chronic kidney disease (ckd) is a serious health problem in which oxidative stress plays an important role. oxidative stress is an imbalance of reactive oxygen species (ros) production and antioxidant defense, where antioxidants have the potential to inhibit ckd progression. celery contains several substances that have an antioxidant effect. this study aimed to evaluate the administration of celery ethanol extract in the prevention of the progressive damage in ckd caused by oxidative stress in male rats. methods twenty male sprague-dawley rats were randomly divided into 5 groups: sham operation (so, n=4), subtotal nephrectomy (sn, n=4), sn+celery ethanol extract 200 mg/kg bw (sn+s1, n=4), sn+celery ethanol extract 250 mg/kg bw (sn+s2, n=4), sn+celery ethanol extract 300 mg/kg bw (sn+s3, n=4). the celery ethanol extract was given for 14 days before induction of ckd and 21 days after induction of the ckd rat model. serum creatinine, malondialdehyde (mda), superoxide dismutase (sod) and glutathione (gsh) were examined in this study. data were analyzed by one way anova followed by lsd test for creatinine, mda, sod, and kruskal wallis test for gsh. results there were significant between-group differences in serum creatinine, sod, and mda (p<0.05), but not in gsh (p>0.05). the administration of celery ethanol extract at 250 mg/kg bw was the most effective in preventing an increase in mda and a decrease in sod and gsh. conclusion celery ethanol extract has the potential to prevent oxidative stress in the ckd rat model. keywords: celery, chronic kidney disease, oxidative stress, nephrectomy, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.114-120 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1265 may-august 2022 vol.41no.2 1department of pharmacology, faculty of medicine, universitas jenderal soedirman, purwokerto, indonesia 2medical profession study program, faculty of medicine, universitas jenderal soedirman, purwokerto, indonesia correspondence: afifah jalan sidodadi 3 rt1 rw7, sokaraja tengah, sokaraja, banyumas, central java, indonesia phone: +6282242637171 email: afifah@unsoed.ac.id orcid id: 0000-0002-5703-7061 date of first submission, december 14, 2021 date of final revised submission, may 9, 2022 date of acceptance, may 18, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: afifah, pribadi fw, salsabiela a, anggara dh, komara zm, al fauzy r. the protective effect of celery ethanol extract on oxidative stress in chronic kidney disease rat model. univ med 2022;41:104-13. doi: 10.18051/univmed.2022.v41. 114-120. 115 univ med vol. 41 no 2 introduction chronic kidney disease (ckd) is a serious public health probl em characterized by progressive loss in renal function that is growing in incidence. the worldwide prevalence of ckd is 11%-13%.(1) there are more than 2 million people with ckd glo bally and most are undergoing hemodialysis or need other forms of renal replacement therapy. oxidative stress has an essential role in ckd.(2) oxidative stress, which is an overproduction of reactive oxygen species (ros) and/or a reduction in antioxidant defense capacity,(2) is known to occur in ckd patients and contributes to inflammation, endothelial dysfunction, risk of atherosclerosis, and progression of ckd. oxidative stress causes oxidative damage and results in the production of various compounds such as malondialdehyde (mda) which is a marker of oxidative injury. antioxidants such as superoxide dismutase (sod) and glutathione (gsh) decrease with the increase in renal dysfunction. ther efore, antioxidants have the potential for inhibiting ckd progression.(3) inhibition of ckd progression is important to prevent complications and mortality in ckd. using natural ingredients as a preventive agent is one effort that can be done because it is widely available in the community. celery (apium graveolens l.) is a vegetable that has many health benefits, functioning as antioxidant and anti-inflammatory agent and preventing cardiovascular disease.(4– 6) celery contains phenols, flavonoids, alkaloids, steroids, furocoumarins, volatile oils, sesquiterpene alcohols, and fatty acids.(7) celery ethanol extract is known to prevent increased renal dysfunction.(8) li et al.(9) investigated the in vivo and in vitro effect of apiin, an antioxidant flavonoid isolated from celery, and showed that apiin has significant inhibitory activity on mda, and dramatically enhances the activity of sod, glutathione (gsh) peroxidase, and catalase.(9) our previous study about the protective effect of celery ethanol extract at doses of 250, 500, and 1000 mg/kgbw, administered for 14 days before and 14 days after induction of 5/6 subtotal nephrectomy on anemia in the 5/6 subtotal nephrectomy rat model showed that celery ethanol extract at a dose 250 mg/kgbw is the most effective in preventing decreases in hemoglobin, red blood cells, and hematocrit and also in preventing decreases in serum creatinine. however, the prevention of anemia in that study was not significantly different between groups, therefore in the present study we try to extend the time after surgery. the administration of celery ethanol extract at doses of 500 mg/kgbw and 1000 mg/kgbw resulted in a higher serum creatinine level and in lower levels of hemoglobin, red blood cell count, and hematocrit. (8) other our previous study about protective effect of ethanol extract of celery (apium graveolens l.) on kidney damage in an ischemia/reperfusion injury rat model showed that celery ethanol extract at a dose of 1000 mg/kgbw was the most effective in preventing kidney damage in the ischemia/ reperfusion injury rat model. the difference between our previous study and our present study is that the present study uses a chronic model of kidney disease and longer administration times of celery ethanol extract, i.e. 14 days before and 21 days after operation . the pr esent study investigated the effect of celery ethanol extract on oxidative stress in the ckd rat model. methods research design a study of experimental design with a posttest-only control group was conducted in the animal house of the pharmacology laboratory, faculty of medicine, universitas jenderal soedirman, while laboratory measurements of the variables were performed in the research laboratory, faculty of medicine, universitas jenderal soedirman from july until october 2020. the preparation of celery ethanol extract was conducted in the lansida herbal technology laboratory, yogyakarta. 116 collection of plant material and extraction celery stems and leaves were collected from pratin, purbalingga, central java, indonesia. the stems and leaves were washed and dried before being crushed in a disk mill with a sieve of 60 mesh. this material was then added to 70% ethanol in an extractor and extracted using an ultra-turrax® disperser at 1000 rpm and macerated for 24 hours. filtration of the mixture was performed with a buchner funnel attached to a vacuum pump. a vacuum rotary evaporator was used for evaporation of the filtrate at 45oc and 90 rpm. the extract was transferred into a porcelain dish and placed in a digital oven at 45oc, then dried 3 times to a constant weight. the extract was prepared with distilled water. animals and treatment twenty sprague dawley male rats, 190-210 g, 2-3 months old were used in this study. the minimum total number of subjects in this study was 15 based on the ‘resource equation’ approach.(10) we used four animals per group in this study. the study subjects were selected through a simple random sampling technique. the rats were randomized into 5 groups: sham operation (so, n=4), subtotal nephrectomy (sn, n=4), sn+celery ethanol extract 200 mg/kg bw (sn+s1, n=4), sn+celery ethanol extract 250 mg/ kg bw (sn+s2, n=4), sn+celery ethanol extract 300 mg/kg bw (sn+s3, n=4). celery ethanol extract was administered orally 14 days before and 21 days after induction of experimental ckd by 5/6 subtotal nephrectomy. the rats were housed in 12-h light-dark cycle, 25oc ambient temperature, 40-60% humidity with free access to water and food. induction of experimental chronic kidney disease the 5/6 subtotal nephr ectomy was performed to induce ckd. the procedure performed on the animals was either 5/6 subtotal nephrectomy or a sham operation, both under ke tamine anesthesi a (100 mg/kg bw). nephrectomy was started on the left kidney followed by the removal of the upper and lower poles of the right kidney. the peritoneum and skin were then closed and sutured using 3/0 silk.(8) the rats were sacrificed by cervical dislocation. laboratory analysis blood serum was obtained from the retroorbital vein for serum creatinine, sod, gsh, and mda level measurement. serum creatinine levels, measured in the medico laboratory, purwokerto, were used to evaluate renal function. the antioxidant parameters were the sod and gsh levels. for sod measurement serum samples and the wst-1 cell viability and proliferation assay (manufacturer, cat. no. 0000) were used. the absorbance was assessed using a spectrophotometer at 595 nm wavelength. measurement of glutathione was carried out using an elisa kit, prepared by the research laboratory, faculty of medicine, universitas jenderal soedirman. the oxidant parameter was the measurement of the mda level, using a colorimetric method with a spectrophotometer at 532 nm wavelength. the measurement was carr ied out in the research labora tory, universitas jenderal soedirman. statistical analysis we used the shapiro-wilk data normality test and levene’s test for homogeneity. the statistical analysis was performed using one-way anova followed by lsd test f or serum creatinine, sod, and mda, and the kruskalwallis test for glutathione at a level of significance of 0.05. ethical clearance this study has obtained ethical clearance from the ethics committee of the medical faculty, universitas jenderal soedirman, purwokerto, indonesia, under no. 138/kepk/vii/2020. results serum creatinine levels after sn and administration of celery ethanol extract are afifah, pribadi, salsabiela, et al celery ethanol extract on oxidative stress 117 univ med vol. 41 no 2 a b presented in table 1. the level of serum creatinine in the sn group (0.95 ± 0.07 mg/dl) was higher than in the so group (0.75 ± 0.08 mg/dl) (p<0.05). this study used celery ethanol extract at 200, 250, and 300 mg/kg bw once daily for 14 days befor e and 21 days after 5/6 sn. administration of celery ethanol extract at a dose of 200 mg/kg bw prevented an increase in serum creatinine level, but the effect was not significantly different from the sn group (p=0.268). administration of celery ethanol extract at 250 and 300 mg/kg bw for 14 days before and 21 days after 5/6 sn prevented an increase in serum creatinine level compared to the sn group (p=0.049 and p=0.441, respectively), but there were no significant differences between the 5/6 sn+celery 250 mg/kg bw group and the 5/6 sn+celery 300 mg/kg bw group (p=0.959). the mean mda level in the sn group (33.41 ± 12.99 nmol/ml) was significantly higher than in the so group (16.36 ± 0.94 nmol/ml) (p<0.021) (table 1), whereas the mean mda level in the treatment groups was lower than that in the sn group. the mda level after administration of celery ethanol extract at doses of 200, 250, and 300 mg/kgbw was 17.2 ± 1.48 nmol/ml, 18.21 ± 3.42 nmol/ml, and 28.64 ± 3.68 nmol/ml, respectively. the mean glutathione and sod levels in the sn group were lower than in the so group (table 1). the mean sod levels in the treatment groups were 22.01 ± 1.22 u/ml, 22.91 ± 0.76 u/ml, and 23.92 ± 1.5 u/ml, after administration of celery ethanol extract at doses of 200, 250, and 300 mg/kgbw, respectively. discussion induction of the ckd rat model by means of 5/6 subtotal nephrectomy in this study was successful, which can be seen from the mean serum creatinine level, this being significantly higher in the sn group than in the so group. according to sari et al.,(11) 14 days after 5/6 sn operation the serum creatinine level has increased significantly in the rats. the reduction in renal mass in 5/6 sn led to decreased renal function and resulted in progressive hypertension, hype rtrophy and f ocal segmental glomerulosclerosis. according to afifah et al.,(8) celery at a dose of 250 mg/kg bw can inhibit the increase in serum creatinine level for 14 days before and after 5/6 sn operation. however, although 14 days after sn operation there was an increase in serum creatinine, the operation was not able to cause anemia as a result of ckd. therefore, the present study increased the time period after 5/6 sn operation to 21 days. creatinine is produced from creatine, the important molecule in the energy production process in the muscle. this molecule is carried into the kidneys and filtered out in the urine, and is a reliable marker of renal function.(12) oxidative stress, the imbalance of ros production and antioxidant defense, is an important mechanism in ckd. even in the early stage of ckd, there is an increase in ros production mainly caused by hyperactivation of nicotinamide adeninedinucleotide phosphate ( nadph) oxidase, and increased synthesis of oxidative stress markers and of uremic toxins.(13) in line with layal et al. ( 14 ) the mda level was significantly higher in the 5/6 sn rat model than in the control group. malondialdehyde, which is the product of lipid peroxidation, is used as an indicator of the intensity of oxidative stress. the increase of mda in ckd patients indicates increased oxidative stress and its progressive increase with worsening renal function implies that oxidative stress increases with disease progression.(3) oxidative stress arises when there is an imbalance between free radical production and antioxidant defense. oxidative stress is involved in the progression of renal injury.(2) the oxidative stress further causes oxidative damage to important biomolecules such as lipids, proteins, and nucleic acids resulting in the formation of various compounds such as mda that have relatively longer half-lives and are used as general markers of oxidative injury.(3) in the early stages of ckd, nutritional and pharmacological 118 t a b le 1 . d is tr ib u ti o n o f cr ea ti n in e, g lu ta th io n e, m d a , a n d s o d l ev el b y t re at m en t gr o u p s n o te : m d a : m al o nd ia ld eh yd e; s o d : su p er o xi de d is m ut as e; d at a p re se nt ed a s m ea n ± s d , e xc ep t gl u ta th io n e [m ed ia n ( q 1q 4 ) a = p < 0. 0 5 i n a n o v a a n al ys is . b : p > 0 .0 5 i n k ru sk al w al li s an al ys is . * = p < 0. 0 5 v s s n , # = p < 0. 0 5 v s s o . s o ( s h am o p er at io n ), s n ( s ub to ta l n ep h re ct o m y) , s n + s 1 (s u b to ta l n ep h re ct o m y+ ce le ry 2 0 0 m g /k g b w ), s n + s 2 ( s ub to ta l n ep h re ct o m y+ ce le ry 2 5 0 m g/ kg b w ), s n + s 3 (s u b to ta l n ep h re ct om y+ ce le ry 3 0 0 m g /k g b w ) afifah, pribadi, salsabiela, et al celery ethanol extract on oxidative stress 119 univ med vol. 41 no 2 conclusions this study demonstrated that celery ethanol extract has the potential to prevent oxidative stress in the ckd rat model. celery ethanol extract at 250 mg/kg bw has the greatest effect in preventing oxidative stress in the ckd rat model. conflict of interest the authors declared no conflict of interests. acknowledgement the authors would like to tha nk the research institutions and community service, universitas jenderal soedirman. contributors af, fwp interpreted the data and prepared the manuscript for publication. af, as, dha, zk, raf collected the data and conducted the study. all of the authors have read and approved this manuscript. references 1. hill nr, fatoba st, oke jl, et al. global prevalence of chronic kidney disease – a systematic review and meta-analysis. plos one 2016;11:1–18. https://doi.org/10.1371/journal.pone.0158765. 2. ling xc, kuo k. oxidative stress in chronic kidney disease. ren replace ther 2018;4:1–9. https://doi.org/10.1186/s41100-018-0195-2 3. sridhar avssn, rao pvlns, sivakumar v, et al. study of oxidant and anti oxidant status in patients with chronic kidney disease. j clin sci res 2018;7:124–30. doi: 10.4103/jcsr.jcsr_ 45_18. 4. sukketsiri w, chonpathompikunlert p, tanasawet s, choosri n, wongtawatchai t. effects of apium graveolens extract on the oxidative stress in the liver of adjuvant-induced arthritic rats. prev nutr food sci 2016;21:79–84. http://dx.doi.org/10.3746/ pnf.2016.21.2.79 5. zhu lh, bao th, deng y, li h, chen lx. constituents from apium graveolens and their anti-inflammatory effects. j asian nat prod res 2017;19:1079-86. doi: 10.1080/10286020.2017. 1381687. intervention is needed to prevent the progressive damage in ckd.(13) in our present study, the administration of celery ethanol extract at doses of 200, 250 mg/kg bw significantly prevents the increase in mda level. celery ethanol extract at 250 mg/kg bw is the most effective in preventing the increase in mda level in the 5/6 subtotal nephrectomy rat model. chronic kidney disease increases ros formation in the cells. the high production of ros accompanies an incr ease in endogenous antioxidants. the increase in ros production for an extended period will disturb the antioxidant activity against oxidative stress. glutathione and sod are the antioxidant enzymes that act as a defense against oxidative stress.(2) celery is a widely used plant in traditional medicine because of its content of phytochemical compounds.(15) celery ethanol extract has an antioxidant effect. celery contains phenols, flavonoids, alkaloids, steroids, glycosides, fatty acids, and a wide range of trace elements, limonene, salinene, furocoumarine glycosides, vitamin a and vitamin c.(16,17) phenols and flavonoids have an antioxidant effect.(18) the antioxidant capacity of flavonoids is derived from their ability to scavenge the ros directly. flavonoids can chelate free radicals quickly by donating a hydrogen atom or by single-electron transfer.(12,19) flavonoids can also stimulate antioxidant enzymes.(20) one type of flavonoids are the flavones, of which apigenin is the most commonly found in celery.(21) in addition to flavonoids, celery also contains high amounts of vitamin c, which scavenges ros and reactive nitrogen species by forming semidehydroascorbic acid and thus protec ts aga inst oxidative damage.(13) the limitation of this study is that it did not measure the antioxidant content of the celery ethanol extract. the clinical implication of this study is that celery has the potential to prevent the progressive damage in chronic kidney disease via its antioxidant effect. further research is recommended to determine the contents of celery ethanol extract a nd examine other pathomechanisms in the ckd rat model. 120 6. kooti w, ali-akbari s, asadi-samani m, ghadery h, ashtary-larky da. review on medicinal plant of apium graveolens. adv herb med 2014;1:48– 59. 7. khairullah ar, solikhah ti, ansori anm, et al. review on the pharmacological and health aspects of apium graveolens l or celery: an update. sys rev pharm 2021;12:606–12. 8. afifah a, muflikhah k, lestari t, sutrisna e, kirana as, prastiwi sd. the protective effect of celery (apium graveolens l.) ethanol extract on anemia in 5/6 subtotal nephrectomy rat model. univ med 2020;39:12–8. doi: http://dx.doi.org/ 10.18051/univmed.2020.v39.12-18. 9. li p, jia j, zhang d, xie j, xu x, wei d. in vitro and in vivo antioxidant activities of a flavonoid isolated from celery (apium graveolens l. var. dulce). food funct 2014;5:50–6. doi: 10.1039/ c3fo60273g. 10. arifin wn, zahiruddin wm. sample size calculation in animal studies using resource equation approach. malays j med sci 2017;24: 101–5. https://doi.org/10.21315/mjms2017.24.5.11. 11. sari dcr, putri mw, leksono tp, et al. calcitriol ameliorates kidney injury through reducing podocytopathy, tubular injury, inflammation and fibrosis in 5/6 subtotal nephrectomy model in rats. kobe j med sci 2020;65:153–63. 12. nainggolan m, sinaga kr, sinaga sm, nugraha se. activity of ethyl acetate fraction of celery herb (apium graveolens l.) on creatinine and urea level in ethylene glycol induced rats. int res j pharm 2018;9:70–2. doi: 10.7897/2230-8407. 0910228. 13. granata s, dalla gassa a, tomei p, lupo a, zaza g. mitochondria: a new therapeutic target in chronic kidney disease. nutr metab 2015;12:1– 21. http://dx.doi.org/10.1186/s12986-015-0044-z. 14. layal k, perdhana is, louisa m, estuningtyas a, soetikno v. the effects of quercetin on oxidative stress and fibrosis markers in chronic kidney disease rat model. med j indones 2017;26:169– 77. http://dx.doi.org/10.13181/mji.v26i3.1462. 15. kooti w, daraei n. a review of the antioxidant activity of celery (apium graveolens l). j evid based complementary altern med 2017;22:1029– 34. doi: 10.1177/2156587217717415. 16. ravichandran h, magesh a. phytochemical analysis of two medicinal plants dicorea bulbifera and apium graveooslons. int j pharm sci rev res 2015;33:344–7. 17. jung ws, chung im, kim sh, kim my, ahmad a, praveen n. in vitro antioxidant activity, total phenolics and flavonoids from celery (apium graveolens) leaves. j med plants res 2011;5: 7022–30. doi: 10.5897/jmpr11.1129. 18. huyut z, beydemir ª, gülçin i. antioxidant and antiradical properties of selected flavonoids and phenolic compounds. biochem res int 2017;2017. https://doi.org/10.1155/2017/7616791. 19. banjarnahor sds, artanti n. antioxidant properties of flavonoids. med j indones 2014;23: 239–44. http://dx.doi.org/10.13181/mji.v23i4.1015. 20. panche an, diwan ad, chandra sr. flavonoids: an overview. j nutr sc 2016;5:e47. doi: 10.1017/ jns.2016.41. 21. shahidi f, ambigaipalan p. phenolics and polyphenolics in foods, beverages and spices: antioxidant activity and health effects a review. j funct foods 2015;18:820–97. http://dx.doi.org/ 10.1016/j.jff.2015.06.018. afifah, pribadi, salsabiela, et al celery ethanol extract on oxidative stress henie12 88 *department of pharmacy, faculty of medicine and health sciences, jenderal soedirman university **center of excellence for translational research in oncology (centro) correspondence heny ekowati, m.sc., apt department of pharmacy, faculty of medicine and health sciences, jenderal soedirman university jl. dr. soeparno, karangwangkal purwokerto 53122 phone:+62281-642840 purwokerto 53123 email: heny240377@gmail.com univ med 2012;31:88-95 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background liver cancer is the third most common cause of death from cancer worldwide. recently, natural products have been widely used as an alternative therapy for liver cancer. previous studies have reported that nigella sativa has chemopreventive activity in vitro and in vivo. the objective of this study was to evaluate the effect of a chloroform extract of nigella sativa seeds (nss) on female rat hepatocytes after administration of 7,12-dimethylbenz [á] anthracene (dmba). methods the experimental design comprised five groups of rats. group i (dbma control group) received oral dmba at a dosage of 20 mg/kgbw twice weekly for five weeks, while group v (solvent control group) was given corn oil only. the other three groups received dmba + nss at dosages of 250 mg/kgbw, 500 mg/kgbw, and 750 mg/kgbw, respectively. each group consisted 12 rats. the nss extract dissolved in corn oil was administered daily by the oral route for 2 weeks before and subsequenyly during dmba tumor induction. at the end of the study, rat livers were collected and stained with hematoxylin and eosin (h&e) and silver staining by the the agnor method. results there was a difference in liver tissue histopathological profile between the nss, dmba control, and the solvent control group. agnor counts in the dmba control group, the dmba+nss 250 mg/kgbw group, dmba+nss 500 mg/kgbw group, and dmba+nss 750 mg/kgbw group were 1.79, 1.51, 1.41, and 1.35, respectively. conclusion nigella sativa seed extract was able to reduce the liver damage and proliferation in rats induced by dmba administration. keywords: nigella sativa, liver, dmba, cancer, female rats protective effects of nigella sativa against 7,12-dimethylbenz [á] anthracene (dmba) induced carcinogenesis in rats nur fatmi alisah*,**, hanif nasiatul baroroh* and heny ekowati*,** abstract 89 univ med vol. 31 no.2 efek protektif nigella sativa terhadap karsinogenesis yang diinduksi 7,12-dimetilbenz [á] antrasena (dmba) pada tikus abstrak latar belakang kanker hati merupakan penyebab ketiga kematian di dunia yang disebabkan kanker. produk bahan alam digunakan secara luas akhir-akhir ini sebagai terapi alternatif untuk kanker hati. penelitian sebelumnya melaporkan bahwa nigella sativa mempunyai aktivitas kemopreventif in vitro dan in vivo. penelitian ini bertujuan untuk menilai efek dari ekstrak kloroform biji n. sativa (nss) terhadap sel hepar tikus betina setelah diinisiasi dengan 7,12dimetilbenz(á)antrasene (dmba). metode sebuah rancangan eksperimental mengikutsertakan lima kelompok tikus. grup i adalah kelompok kontrol dmba yang diberi dmba oral dengan dosis 20 mg/kgbb dua kali seminggu selama lima minggu,, sedangkan grup v adalah kelompok kontrol pelarut yang diberi minyak jagung semata. ketiga grup lainnya diberi dmba + nss, dengan dosis masing-masing 250 mg/kgbb, 500 mg/kgbb dan 750 mg/kgbb. setiap grup terdiri dari 12 ekor tikus. ekstrak nss yang dilarutkan dalam minyak jagung diberikan tiap hari dengan rute peroral dua minggu sebelum dan selanjutnya selama inisiasi dmba. pada akhir percobaan, tikus dinekropsi dan dilakukan pengecatan hematoksilin eosin (h&e) dan perak dengan metode agnor. hasil terdapat perbedaan pada profil histopatologi jaringan hati antara grup perlakuan ekstrak, grup dmba dan grup minyak jagung. hasil pewarnaan agnor menunjukkan bahwa pemberian nss dapat menginhibisi proliferasi sel hepar pada kanker hati pada tikus betina yang diinduksi dmba. nilai rerata agnor pada grup kontrol dmba; dmba+nss 250 mg/kgbb, 500 mg/kgbb dan 750 mg/kgbb berturut-turut adalah 1,79; 1,51; 1,41 dan 1,35. kesimpulan penelitian ini mengindikasikan bahwa n. sativa dapat mengurangi kerusakan dan proliferasi hati pada kanker hati yang diinduksi dmba. kata kunci: nigella sativa, hati, dmba, kanker, tikus introduction cancer is the second most common cause of death in the united states,(1) and liver cancer is the third most common cause of death from cancer worldwide. liver cancer is extremely difficult to treat and the overall 5-year survival rate (for all stages) is only about 7% in the united states and even lower in developing countries.(2) several methods for liver cancer therapy such as surgery, chemotherapy, hormonal therapy, and radiotherapy have been established. however, there are no recognized methods for prevention of liver cancer, and only recently has there been e x p l o r a t i o n o f n a t u r a l p r o d u c t s t o f i n d chemopreventive agents, in an attempt to reduce the number of cancer patients. chemoprevention is defined as the use of substances of natural origin, phytochemical agents, synthetic, or chemical compounds to prevent cancer or suppress cancer progression, effect a reversal to normal physiological functions, and perform e a r l y d e t e c t i o n o f p a t h o l o g i c a l c a n c e r conditions.(3) one of the medicinal plants that 90 has been extensively investigated is black cumin (nigella sativa seeds), which contain fixed and essential (volatile) oils, that presumably have anticancer activity. the main components of the fixed oil are linoleic acid, oleic acid, and palmitic acid.(4) the effects of n. sativa have been evaluated in animal studies as well as in vitro. chloroform extracts of black cumin have cytotoxic activity against t47d cells with ic 50 of 124.21 ìg/ml.(5) there are many reports in the literature on the biological activities of n. sativa, i n c l u d i n g i m m u n o p o t e n t i a t i n g , a n t i inflammatory, analgesic, antihypertensive, antiulcerogenic, respiratory stimulation, antibacterial, antifungal, anticestode, antinematode, a n t i g l y c e m i c , a n t i t u m o r, a n d a n t i c a n c e r effects.(6-10) an experimental study showed that orally administered n. sativa had hepatoprotective effects against dimethylaminoazobenzene (dab)-induced cholangiocarcinoma in male swiss albino mice.(11) n. sativa essential oils injected into tumors grafted in dba2 mice presumably showed either antimetastatic activity, or an inhibitory or delaying effect on metastasis through rapid reduction of primary tumor volume at the site of induction.(12) another study indicated that an ethanol extract of n. sativa effected a significant decrease in cell proliferation, dna synthesis, mitosis and the extension of the percentage of live rats.(13) the administration of ns reduced the carcinogenic effects of dmba in skin carcinoma, suggesting a protective effect.(14) cancers have a high proliferation rate that i s p o s i t i v e l y r e l a t e d t o t h e a m o u n t o f argyrophilic proteins (agnor proteins) in the nucleolar organizer regions (nors), which are dna loops located on acrocentric chromosomes responsible for their synthesis. agnor proteins are visualized as black-brown dots by their binding to silver nitrate according the agnor method. a higher agnor count indicates a correspondingly higher proliferation rate (as in c a n c e r c e l l s ) , a n d c o n v e r s e l y, a l o w e r proliferation rate (as in normal cells) is shown by a low agnor count.(15) the objective of our s t u d y w a s t o e v a l u a t e t h e a c t i v i t y o f a chloroform extract of ns seeds (nss) on 7,12dimethylbenz [á ] anthracene (dmba)-induced liver carcinogenesis in rats by examination of h i s t o p a t h o l o g i c s e c t i o n s s t a i n e d w i t h hematoxylin-eosin and with silver-staining by the agnor method. methods animals thirty-day old female sprague dawley rats weighing from 100 to 150 g were obtained from the laboratory of pharmacology and toxicology, faculty of pharmacy, gadjah mada university, yogyakarta, indonesia. prior to the experiments, the rats were fed a standard pellet diet and water ad libitum, and acclimatized to a 1 2 h l i g h t d a r k c y c l e . t h e s t u d y w a s conducted from march – october 2011. study design a total of 60 female rats was randomized into 5 groups (control and experimental groups) of twelve rats each. the minimum group size as derived from the formula (t-1) (r-1) >15, where t = number of treatments and r = number of rats, was 4 rats per group. however, in this study we used 12 rats in each group. group i ( d m b a c o n t r o l g r o u p ) r e c e i v e d d m b a dissolved in corn oil (sigma) at a dosage of 20 mg/kg bw for 5 weeks. in group ii, iii and iv, two weeks before dmba initiation, the animals were given a single oral dose of nss daily at a dosage of 250 mg/kg bw, 500 mg/kg bw, and 750 mg/kg bw, respectively, followed by dbma administration at a dosage of 20 mg/ kg bw for 5 weeks. group v (solvent control group) received corn oil for 5 weeks. after 16 weeks, all animals were sacrificed by ether as scheduled. body weights of the animals were recorded weekly throughout the study. alisah, baroroh, , ekowati n. sativa protective effects in vivo 91 univ med vol. 31 no.2 histopathologic examination at autopsy, the rat livers were removed and fixed in 10% buffered formalin. after 12–24 h of fixation, paraffin-embedded 3–5 ìm sections were stained with hematoxylin-eosin (h&e) and agnor for histopathologic examination. mean agnor scores were calculated by counting at l east 100 cells per par aff in section at a magnification of 1000 x. ethical clearance animals were handled according to the rules and regulations of the animal care committee of the faculty of medicine, gadjah mada university. statistical analysis a statistically significant difference in the mean number of black dots (mean agnor score) was evaluated by one way anova, continued with tukey hsd. a p value of <0.05 between groups was considered statistically significant using spss. results this study showed that the ns chloroform extract was capable of reducing cellular proliferation in dmba-induced liver cancers in rats. as may be seen from table 1, the mean a g n o r v a l u e s i n t h e f i v e g r o u p s w e r e statistically significantly different (p<0.05) by one way anova continued with tukey hsd. the cellular proliferation rate was found to be highest i n t h e d m b a c o n t r o l g r o u p a n d t o b e significantly decreased in the rats of group iv receiving 750 mg/kgbw nss extract and corn o i l . t h e n s s e x t r a c t h a d a n o p t i m a l antiproliferation activity at a concentration of 750 mg/kg with a mean agnor value of 1.35 ± 0.038. anova was followed by tukey hsd to differentiate between groups, and the results showed significant differences in mean agnor between nss extract groups (at dosages of 250 mg/kg, 500 mg/kg, and 750 mg/kg) and dmba 20 mg/kg. by contrast, mean agnor values of the nss extract groups and the corn oil control group showed no significant difference. observation was terminated at 8 w e e k s a f t e r t h e l a s t d m b a i n i t i a t i o n . macroscopic observation showed morphological differences of the livers in the five groups. the livers from the dmba control group (group i) were nodular in appearance, while the livers in groups ii-v did not show any nodules. the histopathological appearance of liver tumors in dmba and dmba+ns treated rats are depicted in figure 1. h&e staining showed necrosis (figure 2). treatment with corn oil did not induce cancer in the livers of the rats. the agnors appeared as intranuclear black dots (figure 3). discussion this study explored the effect of ns seed chloroform extract on dmba-induced liver cancers in female rats. nss extract was given two weeks before and also during tumor table 1. magnor value of n. sativa on proliferation of dmba-induced liver female rats magnor=mean agnor; dmba=7,12-dimethylbenz[á ]anthracene; values are means ± sd; *p<0.05 compared with dmba 92 induction by dmba. nss treatment was designed to prevent metabolic activation of dmba and suppress liver cancer progression. the liver is exceedingly vulnerable to damage by chemical carcinogens, which is thought to result from its central role in the metabolism of foreign substances (xenobiotics), including potential carcinogens.(16) dmba is a polycyclic aromatic hydrocarbon (pah) carcinogen that is oxidized to 7,12-dmba-3,4-oxide by cytochrome p450 enzymes (cyps), then hydrolyzed to its corresponding diol and finally figure 2. histological evaluation in liver tissues of control and experimental group. rats were divided into 5 groups, (a) corn oil; (b) dmba control group (20 mg/kgbw in corn oil); (c) dmba+ 250 mg/kgbw nss; (d) dmba+500 mg/kgbw nss, (e) dmba+750 mg/kgbw nss; (�) necrosis magnification x600 alisah, baroroh, , ekowati n. sativa protective effects in vivo figure 1. morphological difference of liver treated with nss in dmba-induced female rats liver cancer. (a) dmba control group; (b) dmba+250 mg/kgbw nss; (c) dmba+500 mg/kgbw nss; (d) dmba+750 mg/kgbw nss; (e) corn oil; (�) nodule 93 univ med vol. 31 no.2 oxidized by cyps to 7,12-dmba-3,4-oxided i o l 1 , 2 e p o x i d e , w h i c h i s t h e u l t i m a t e carcinogen.(17) individual pahs may affect their own metabolism, which is catalyzed by cyp 1a1, 1a2, and 1b1.(18) dmba metabolites are toxic and cause oxidative stress, leading to structural damage to cells and possibly cell necrosis.(19) dmba administration to experimental animals causes an increase in lactate dehydrogenase (ldh), followed by necrosis.(20) figure 1 shows that the livers in the dmba control group were nodular in appearnce. h&e staining showed necrosis in the dmba control and dmba + nss groups, with the group treated with nss at a dosage of 750 mg/kgbw showing negligible necrosis on h&e sections, if at all, which was similar to the solvent control (corn oil-only) group. necrosis is defined as a type of cell death without apoptosis and autophagy, and is usually considered out of control.(21) damage due to oxidative stress in sixty swiss albino rats weighing about 40 ± 5 g, which were orally a d m i n i s t e r e d o f 0 . 5 % d m b a s o l u t i o n demonstrated the presence of necrosis. (16) necrotic hepatocytes release factors (damage signals or alarmins) that activate kupffer cells (kcs), which in turn produce cytokines, such as interleukin 6 (il-6), that promote compensatory hepatocyte proliferation.(22) fractionation of the nss chloroform extract yields linoleic and palmitic acids, and an indole compound which is an alkaloid that is tryptamine.(5) linoleic acid (omega-3) and á-linoleic acid (omega-6) can influence gene expression in experimental animals, have antiinflammatory activity, and can suppress interleukin-1â (il-1â), tumor necrosis factor-á (tnfá) and interleukin-6 (il-6).(23) the linoleic acid content of ns seeds can inhibit cell proliferation. linoleic acid reduces cancer cell proliferation in lung, breast, prostate and colon cancers.(24) the linoleic and palmitic acid content of nss has antiproliferative activity by induction of apoptosis in liver cells.(25) cancer is a complex disease that involves many regulatory proteins that differ with the figure 3. agno3 stained liver tissues of control and experimental group. rats were divided into 5 groups, (a) corn oil; (b) dmba control group (20 mg/kgbw in corn oil); (c) dmba+ 250 mg/kgbw nss; (d) dmba+500 mg/kgbw nss; (e) dmba+750 mg/kgbw nss. argyrophilic nucleolar organiser regions (agnors) are visible as dark dots within the plasma cell nuclei (pointed by black arrow); magnification x1000 94 t y p e o f c a n c e r. t h i s s t u d y p r o v i d e s t h e information that nss inhibits liver cancer growth. in the present study, nss inhibited tumor incidence based on histopathological profile and mean agnor value. however, the proposed molecular mechanism involving cyp and glutathione s-transferases (gst) by the nss extract needs to be further explored. gsts may be potential targets on which to base the development of new antitumor compounds. these results suggest that nss may be a promising new anticancer therapeutic agent and a potential candidate to be further evaluated. conclusions ns seed extract was able to reduce the hepatic cellular damage and proliferation in dnba-induced liver cancers. this study indicated that ns can be developed into a chemopreventive agent for liver cancer. acknowledgements the authors whish to express their sincere g r a t i t u d e t o t h e d i r e c t o r a t e o f h i g h e r education, ministry of education and culture, and to jenderal soedirman university for f i n a n c i a l s u p p o r t o f t h i s s t u d y t h r o u g h competitive research grant 2010-2011. references 1. hope s. rugo md. cancer. in: current medical diagnosis and treatment. new york: mcgrawhill;2008.p.1387-458. 2. ruddon rw. the epidemiology of human cancer. in: cancer biology, 4nd ed. new york: oxford university press inc;2007.p.70-116. 3. greenwald p. cancer chemoprevention. bmj 2002;324:714–8. 4. nickavar b, mojab f, javidnia k, amoli mar. chemical composition of the fixed and volatile oils of nigella sativa l. from iran. z naturforsch 2003;58c:629-31. 5. ekowati h, prasasti e, rastuti u. the active fraction from nigella sativa and its activity against t47d cell line. indo j chem 2011;20: 217-22. 6. nazrul sk, begum f, ahsan t, saiful hma. immunosuppressive and cytotoxic properties of nigella sativa. phytother res 2004;18:395-8. 7. al-naggar tb, serranillos gmp, carreto me, villar am. neuropharmacological activity of nigella sativa l. extract. j ethnopharmacol 2003;88:63-8. 8. mohtashami r, amini m, huseini f, ghamarchehre m, sadeqhi z, hajiagaee r, et al. blood glucose lowering effects of nigella sativa l. seeds oil in healthy volunteers: a randomized, double-blind, placebo-controlled clinical trial. j med plants 2011;10:1-5. 9. rooney s, ryan mf. effects of alpha-hederin and thymoquinone – a constituent of nigella sativa, on human cancer cell lines. anticancer res 2005;25:2197-204. 10. salim ei, fukushima s. chemopreventive potential of volatile oil from black cumin (nigella sativa) seeds against rat colon carcinogenesis. nutr cancer 2003;45:195-202. 11. mohamed ha, el-sayed ih, moawad m. protective effect of nigella sativa seeds against dimethylaminoazobenzene (dab) induced liver carcinogenesis. nat sci 2010;8:80-7. 12. mbarek la, mouse ha, elabbadi n, bensalah m, gamouh a, aboufatima, r, et al. anti-tumor properties of blackseed (nigella sativa l.) extracts. braz j med biol res 2007;40:839-47. 13. daoud m, nihat d, hatice g, gulruh u, muharrem b. antitumor activity of an ethanol extract of n. sativa seeds. biol brat 2004;59:73540. 14. shoieb am, elgayyari m, dudrick ps, bell jl, tithof pk. in vitro inhibition of growth and induction of apoptosis in cancer cell lines by thymoquinone. int j oncol 2003;22:107-13. 15. silva bak, silva is, pereira dm, aydos rd, carvalho ptc. usefulness of argyrophilic nucleolar organizer regions in detection of lung cells alterations after benzo[a]pyrene instillation. acta cir bras 2006;21 suppl 4:s36-9. 16. muqbil i, banu n. enchancement of pro-oxodant effect of 7,12-dimethylbenz (α ) anthracene (dmba) in rats by pre-exposure to restraint stress. cancer lett 2006;240:213-20. 17. shimada t, kuriyama fy. metabolic activation of polycyclic aromatic hydrocarbons to carcinogens by cytochromes p450 1a1 and 1b1. cancer sci 2004;95:1-6. 18. shimada t, guengerich fp. inhibition of human cytochrome p450 1a1-, 1a2-, and 1b1-mediated activation of procarcinogens to genotoxic metabolites by polycyclic aromatic hydrocarbons. chem res toxicol 2006;19:228-94. alisah, baroroh, , ekowati n. sativa protective effects in vivo 95 univ med vol. 31 no.2 19. patri m, padmini p. polycyclic aromatic hydrocarbons in air and their neurotoxic potency in assosiation with oxidative stress: a brief perspective. ann neurosci 2009;16:340-9. 20. al-attar ma. the influence of dietary grapeseed oil on dmba-induced liver enzymes disturbance in the frog, rana ridibunda. pakistan j nutr 2004;3:304-9. 21. golstein p, kroemer g. cell death by necrosis: towards a molecular definition. sci dir 2006;32: 37-43. 22. naugler we, sakurai t, kim s, maeda s, kim k, elsharkawy am, et al. gender disparity in liver cancer due to sex differences in myd88dependent il-6 production. sci 2007;317:121– 4. 23. simopoulos ap. the omega-6/omega-3 fatty acid ratio, genetic variation, and cardiovascular disease. asia pac j clin nutr 2008;17:131-4. 24. maggiora m, bologna m, ceru mp, possati l, angelucci a, cimini a, et al. an overview of the effect of linoleic and conjugated linoleic acids on the growth of several human tumor cell lines. j int cancer 2004;112:909-19. 25. zhang y, xue r, zhang z, yang x, shi h. palmitic and linoleic acids induce er stress and apoptosis in hepatoma cells. lipids health dis 2012;11:1-8. oktavianus 12 *department of medical pharmaceutics, faculty of medicine, diponegoro university **department of pharmacology and clinical pharmacy, faculty of pharmacy, gajah mada university correspondence dra. endang sri sunarsih, m.kes., apt. department of medical pharmaceutics, faculty of medicine, diponegoro university e-mail : endss2007@yahoo.co.id univ med 2012;31:12-19 abstract universa medicina january-april, 2012january-april, 2012january-april, 2012january-april, 2012january-april, 2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background indole and its derivatives, such as indole acetic acid are active constituents of cauliflower (brassica oleracea var. botrytis l.) with are known to function as plant growth hormones. drug metabolism mainly occurs in the liver, so that the possibility of damage to this organ is considerable. the purpose of this study was to evaluate the effect of cauliflower extract on alanine transaminase (alt) levels and on the hepatocyte morphology induced by theophylline in wistar rats. methods twenty wistar rats were divided randomly into four groups. group i was given only theophylline 20 mg/kg bw. groups ii, iii, and iv were given cauliflower extract 200 g/kg bw, for 5, 10, 15 days, and on the last day were given theophylline 20 mg/kg bw. on days 5, 10, and 15 the animals were terminated, their livers taken and observed for microscopic appearance, while blood samples were taken for measuring alt levels. results cauliflower affected a reduction in alt levels, mean serum alt concentration in rats given cauliflower for 5 days (109.86 ± 2.20 iu/l was significant higher compared to rats given cauliflower for 15 days (71.58 ± 3.17 iu/l) (p=0.000). mean values of histopathologic liver cells in rats given cauliflower for 15 days (1.17 ± 0.01) significant lower compared to rats given cauliflower for 5 days (1.45 ± 0.06) (p=0.000). conclusions cauliflower extract reduced the level of alt and improved the microscopic appearance of hepatic cells in the rats. cauliflower extract could lead to the development of potent anti-tumor agents for hepatic cancer treatment. keywords: cauliflower extract, theophylline, liver microscopic appearance, wistar rats protective effect of brassica oleracea var. botrytis l. against theophylline-induced hepatocellular abnormalities in rats endang sri sunarsih*, lukman hakim**, sugiyanto** and sumantri** 13 univ med vol. 31 no. 1 efek protektif dari brassica oleracea var. botrytis l. terhadap kelainan hepatoseliler pada tikus diinduksi teofilin latar belakang kubis bunga (brassica oleracea l. var. botrytis l.) mengandung senyawa aktif indole acetic acid (iaa) memiliki fungsi sebagai hormon pertumbuhan pada tumbuh-tumbuhan. interaksi cauliflower dengan teofilin metabolismenya terjadi di hepar, kemungkinan terjadinya perubahan komposisi sel hepar menjadi sangat besar. penelitian ini bertujuan untuk menilai pengaruh pemberian ekstrak cauliflower terhadap perubahan kadar alanine transaminase (alt) dan gambaran mikroskopis sel hepar pada tikus wistar yang diberi teofilin. metode duapuluh ekor tikus wistar dibagi secara acak menjadi 4 kelompok. kelompok i berupa kelompok kontrol hanya diberi teofilin 20 mg/kgbb/1x pemberian. kelompok ii, iii, dan iv adalah kelompok perlakuan diberi ekstrak cauliflower dengan dosis 200g/kgbb selama 5, 10, dan 15 hari, pada hari terakhir perlakuan diberi teofilin 20 mg/ kgbb/1x pemberian. pada hari ke-5, 10, 15 dilakukan terminasi, organ hepar diamati gambaran mikroskopis dan sampel darah untuk pengukuran kadar alt. hasil hasil penelitian menunjukkan bahwa rata-rata kadar enzim alt pada pemberian kubis bunga selama 5 hari (109,86 ± 2,20 iu/l) lebih tinggi secara bermakna dibandingkan pemberian ekstrak cauliflower selama 15 hari (71,58 ± 3,17 iu/l) (p=0,000). rata-rata nilai histopatologi sel hepar pada tikus yang diberi ekstrak kubis bunga selama 15 hari (1,17 ± 0,01) lebih rendah secara bermakna dibandingkan pemberian ekstrak kubis bunga selama 15 hari (1,45 ± 0,06) (p=0,000). kesimpulan pemberian ekstrak kubis bunga mampu menurunkan kadar enzym alt dan memperbaiki sel hepar menuju normal pada tikur yng diinduksi dengan teofiline. ekstrak kubis bunga cauliflower perlu dikembangkan lebih lanjut sebagai agen anti kanker yang bermanfaat untuk pengobatan kanker hati. kata kunci: ekstrak bunga kubis, teofilin, mikroskopik hepar, tikus wistar introduction cauliflower is a common vegetable in the diet of many communities. this plant belongs to the family brassicaceae and its scientific name is brassica oleracea l., comprising a large number of varieties, including broccoli. the regular consumption of cauliflower results in a high probability that this vegetable is taken concurrently with drugs. cauliflower is known to contain compounds capable of accelerating the biotransformation of concurrently ingested drugs,(1) as it contains the active compounds indole and sulforaphane. indole has been recognized as an inducer of enzymes for the biotransformation of drugs. both compounds are capable of inducing cytochrome p-450 and s e v e r a l e n z y m e s c a t a l y z i n g c o n j u g a t i o n reactions.(2,3) in addition, both components have definite anticancer effects.(4) cauliflower also has antioxidant properties, since it contains vitamin a, vitamin c, and several minerals. cauliflower is capable of repairing the cytoplasmic membrane of hepatocytes and one of the mechanisms involved is through its antioxidant effect. one study reported that progoitrin was one of the abstrak 14 predominant glucosinolates in brassica oleracea (cabbage). glucosinolates and their derived products have been reported to have health beneficial effects by reducing the risk of certain cancers in humans.(5) theophylline is one of the antiasthma drugs that is still frequently utilized. in addition to its relatively low cost, this drug is also much prescribed by doctors. furthermore, the side effects of theophylline are known, and may therefore be anticipated by doctors, while it is clinically beneficial as a bronchodilator.(6) theophylline has even been recommended by the ministry of health of the republic of indonesia, and in (2007/2008) year it was classified as a drug for respiratory tract complaints obtainable without prescription in the “one thousand drugs program” (“program obat seribu”). however, because theophylline is metabolized in the liver and has a narrow therapeutic range, it may become relatively dangerous if not consumed according to need. drugs that are metabolized in the liver may have potential hepatotoxic effects. xenobiotics or drugs that are given in high doses and/or for long periods result in an i n t i m a t e a n d l o n g e r c o n t a c t w i t h t h e hepatocytes.(6) drug interactions are considered to be clinically important if they increase the toxicity or decrease the effectiveness of the i n t e r a c t i n g d r u g s . . i n t h e h e p a t o c e l l u l a r environment the effect of these interactions may r e s u l t i n i n f l a m m a t i o n , d e g e n e r a t i o n , intracellular deposits, necrosis, and fibrosis. these effects depend on the xenobiotics in question, but are also affected by their dose and duration of contact.(6) one simple and routine test for determining liver functions is the measurement of alanine a m i n o t r a n s f e r a s e ( a lt ) a n d a s p a r t a t e aminotransferase (ast). alt is bound to the cytoplasm of hepatocytes, while ast is bound to their organelles. hepatocytes undergoing n e c r o s i s w i l l c a u s e a r i s e i n t h e s e r u m concentrations of both enzymes. although alt and ast are frequently considered to be liver enzymes, because of the high concentration of both enzymes in hepatocytes, only alt is specific to the liver. in contrast, ast is also found in cardiac muscle, skeletal muscle, the kidneys, and the pancreas.(7) the purpose of this study was to evaluate the effect of administration of cauliflower extract on alt concentrations and hepatocyte morphology in wistar rats given theophylline. methods study design this was an experimental laboratory study using a post test only with control group design to evaluate the effect of cauliflower extract on alt concentrations and hepatocyte morphology in wistar rats given theophylline. the study was conducted from january until june 2011. preparation of cauliflower extract the cauliflower sample (brassica oleracea v a r. botr ytis l . ) c a m e f r o m t h e a r e a o f bandungan in central java. to prepare the extract, the vegetable was macerated in water at low temperature, to obtain a concentration and volume corresponding to the desired dose. experimental animals the experimental animals used were male wistar albino rats, 2-3 months old and weighing 190-250 grams, obtained from the integrated research and testing institute (lembaga penelitian dan pengujian terpadu, lppt), gajah mada university, yogyakarta. the study used a total of twenty animals, based on a sample size of 5 animals per group. the inclusion criteria were: wistar rats, male, age 2-3 months, weight 190-250 grams, in healthy condition. rats with anatomical deformity were excluded from the study. the twenty male wistar rats were assigned to 4 groups by simple randomization, resulting in 5 animals in each group. group i was given a single oral theophylline dose of 20 mg/kgbw,(5) while groups ii-iv received cauliflower extract orally for 5 days at a concentration of 200g/ sunarsih, hakim, sugiyanto, et al brassica oleracea var botrytis. l 15 univ med vol. 31 no. 1 kgbw, for 5, 10, and 15 days, respectively, followed by a single oral theophyilline dose of 20mg/kgbw on the last day of intervention. on days 5, 10, and 15 the animals were terminated and the livers taken and observed for abnormal hepatocyte morphology, which was scored by the method of maretnowati, (8) blood samples was a l s o t a k e n f o r d e t e r m i n a t i o n o f a lt concentrations. the use of a single oral theophylline dose of 20 mg/kgbb was based on the therapeutic dose generally used in medical practice, and the cauliflower extract was given for 5, 10 and 15 days to protect the hepatocytes against theophylline-induced abnormalities. measurement of alt blood samples were collected from the lateral ocular vein for determination of post test alt concentrations and analyzed by means of a 4010 photometer and gpt ready-made reagents. the enzyme concentration was measured by putting 100 µl serum into a cuvette, adding 1000 µl of the enzyme reagent r1, letting the mixture stand for 5 minutes, then adding 200 µl of the substrate reagent r2, mixing, and reading the result in a spectrophotometer at 340 nm wavelength. preparation of histopathology slides liver tissue was fixed in formalin 10%, made into blocks and sectioned with a microtome at a thickness of ± 3 mm, then stained with hematoxylin-eosin, and observed under the microscope at a magnification of 400x. the degree of hepatocyte abnormality was scored according to the method used by maretnowati (table 1).(8) the scoring results were obtained from observation of 20 hepatocytes in five different fields of view, thus obtaining a total of 100 cells, at a magnification of 400x, then the scores were used to calculate the histopathologic value of the hepatocytes, expressed as the mean (± standard deviation) of 100 observations for each group. ethical clearance ethical clearance was obtained from the committee for animal research ethics at the faculty of public health, diponegoro university. data analysis the obtained results in the form of data on serum alt activity and histopathologic value of hepatocytes were tested by one way anova at confidence level of 95%, using spss version 13. results administration of cauliflower extract for 5, 10 and 15 days resulted in measurable changes in alt concentrations, determined on the last day of intervention. the resulting serum alt concentrations are shown in table 2. these demonstrate the occurrence of interactions between theophylline and cauliflower, with degree of abnormality score normal cellular morphology 1 paren chymatous degeneration 2 hydropic degeneration 3 necrosis 4 table 1. scoring of hepatocyte abnormality (8) table 2. mean serum alt* concentrations by intervention group *alt=alanine aminotransferase intervention p theophylline (n=5) caulifl ower extract for 5 days + theophylline (n=5) cauliflower extract for 1 0 days + theophylline (n=5) cauliflower extract for 10 days + theophylline (n=5) alt level (iu/l) 176.26 ± 5.48 109.86 ± 2.20 85.10 ± 3.89 71.58 ± 3.17 0.000 16 i n c r e a s i n g l e n g t h o f a d m i n i s t r a t i o n o f cauliflower, leading to a reduction in serum alt concentrations in the rats. mean serum alt concentration in rats given cauliflower for 5 days (109.86 ± 2.20 iu/l was significant higher compared to rats given cauliflower for 15 days (71.58 ± 3.17 iu/l) (p=0.000). the results of tukey multiple comparison test indicated significant differences in alt concentrations between theophylline and cauliflower administration for 5, 10 and 15 days followed by theophylline (p=0.000). h i s t o p a t h o l o g i c a n a l y s i s o f t h e hepatocytes was performed qualitatively and quantitatively to determine the hepatocyte m o r p h o l o g i c a b n o r m a l i t i e s i n d u c e d b y i n t e r a c t i o n s b e t w e e n t h e o p h y l l i n e a n d c a u l i f l o w e r, w i t h i n c r e a s i n g l e n g t h o f administration of cauliflower. the data on microscopic observation of the hepatocytes representative of each intervention group, may be seen in table 3 and figures 1-4. table 3 shows that the histopathologic value of the hepatocyte after administration of cauliflower extract for 5, 10, and 15 days, d e c r e a s e d w i t h i n c r e a s i n g l e n g t h o f administration. mean values of histopathologic liver cells in rats given cauliflower for 15 days (1.17 ± 0.01) significant lower compared to rats given cauliflower for 5 days (1.45 ± 0.06) (p=0.000) this may be explained by the presence in cauliflower of sulforaphane and indole, which are capable of restoring the cytoplasmic membrane of hepatocytes, to accelerate their regeneration. the histopathologic morphology representing the various histopathologic values obtained in this study may be seen in figures 1 – 4. . sunarsih, hakim, sugiyanto, et al brassica oleracea var. botrytis l. figure 1. hepatocyte morphology, magnification 400x , he stain. (!) : congested cells (2.778) table 3. mean values of histopathologic of hepatocytes, by intervention group 17 univ med vol. 31 no. 1 figure 2 hepatocyte morphology, magnification 400x , he stain.(!) : cells with parenchymatous degeneration (1.456) figure 3. hepatocyte morphology, magnification 400x, he stain.(!) : cells with parenchymatous degeneration. (1.266) figure 4. hepatocyte morphology, magnification 400x , he stain.(!) : normal cells (1.176) 18 discussion the present study demonstrates that administration of cauliflower extract for 5, 10, and 15 days was capable of reducing serum alt concentrations in rats given a single dose of theophylline. the results of this study are c o n s i s t e n t w i t h t h o s e o f a s t u d y o n t h e administration of brassica oleracea var. botrytis l. for 30 days to female rats followed by intraperitoneal diethylnitrosamine (den) for 7 days.(9,10) essentially similar results were obtained in another study, in which oral administration of broccoli caused a significant decrease in serum alt levels in rats with hepatotoxic lesions induced by n-nitrosodiethylamine (ndea) and carbon tetrachloride (ccl4).(11) the biomarkers used in this study provide the measures of carcinogen exposure in rats as an area of high risk for development of hepatocellular carcinoma. this study revealed that oral administration of cauliflower extracts caused a significant decrease in serum levels of a lt. b r o c c o l i i s a p l a n t o f t h e f a m i l y brassicaceae. the observed enhancement level by brassica vegetables was due to their content o f g l u c o s i n o l a t e s , f l a v o n o i d s a n d o t h e r phenolics.(12) the anticarcinogenic activity is related to the presence of biologically active components that modulate the activity of phase i and ii detoxification enzymes and other mechanisms triggered by glucosinolates, which are formed as a result of hydrolysis and catalyzed by the enzyme myrosinase. hepatotoxic abnormalities may be evaluated from the aspects of both cellular structure and function. evaluation of hepatocellular structure was performed by light microscopy. cauliflower with its indole and sulforaphane content is capable of inducing conjugases through phase ii drug metabolism, as demonstrated by its ability to reduce alt concentrations.(9) in addition, its high antioxidant content also plays a role in improving liver functions. hepatotoxic effects may take the form of inflammation, degeneration, necrosis, and fibrosis. the observed prophylactic effect of cauliflower extract on hepatocyte morphology of the experimental animals, indicate the occurrence of significant between-group differences in histopathologic picture. the hepatocyte abnormalities may take the from of p a r e n c h y m a t o u s d e g e n e r a t i o n , h y d r o p i c degeneration, as well as necrosis. on average, histopathologic picture of the hepatocytes tended to decrease in proportion to the length of administration of the cauliflower extract. these hepatocellular changes may be due to decreased atp levels caused by covalent binding of theophylline to intracellular proteins, resulting in abnormalities of action. failure of actin fibril assembly on the hepatocyte surface causes cytoplasmic membrane rupture.(13) the reduction in alt concentrations and improvement in hepatocyte morphology by cauliflower extract in this study are due to the central role of the liver in the detoxification of drugs such as theophylline. the high content of antioxidants in cauliflower, such as vitamin a, vitamin c, and several minerals, which may act synergistically, is capable of restoring cellular functions and repairing hepatocyte menbrane damage. in addition, cauliflower contains indole and sulforaphane, which are capable of restoring hepatocellular functions to their original status.(4,12,13) this is also supported by previous studies demonstrating that cauliflower (brassica oleracea var. botrytis l.) is also capable of reducing ldl and increasing hdl cholesterol in hyperlipidemic rats, as well as decreasing total cholesterol and mda (malondialdehyde).(14) a phase i clinical study have characterized the metabolism and excretion of glucosinolates and isothiocyanates of broccoli sprouts in humans to assist in the design of dosing regimens for studies of efficacy.(15) studies of cultured cancer cells and animal models of cancer provide the most compelling evidence that the nonnutrient phytochemical composition of brassica reduces tumor incidence.(16,17) sunarsih, hakim, sugiyanto, et al brassica oleracea var. botrytis l 19 univ med vol. 31 no. 1 conclusions administration of cauliflower (brassica oleracea var. botrytis l.) has a protective effect on hepatocyte abnormalities and reduced the alt concentrations induced by theophylline in r a t s . t h e r e f o r e t h i s s t u d y r e c o m m e n d s increasing the dietary intake of cauliflower (brassica oleracea var. botrytis l), as a complementary medicines for patients with liver cancer. references 1. gibson gg, martínez-sánchez a, gil-izquierdo a, gil mi, ferreres f. a comparative study of flavonoid compounds, vitamin c, and antioxidant properties of baby leaf brassicaceae species. j agricul food chem 2008;56:2330-40. 2. perocco p, bronzetti g, canistro d. glucoraphanin, the bioprecursor of the widely extolled chemopreventive agent sulforaphane found in broccoli, induces phase-i xenobiotic metabolizing enzymes and increases free radical generation in rat liver. mutat res 2006;595:12536. 3. lee sa, fowke jh, lu w, ye c, zheng y, cai q, et al. cruciferous vegetables, the gstp1 ile105val genetic polymorphism,and breast cancer risk. am j clin nutr 2008;87:753-60. 4. myzak mc, tong p, dashwood wm, dashwood rh. sulforaphane retards the growth of human pc-3 xenografts and inhibits hdac activity in human subjects. exp biol med 2007;232:22734. 5. kumar s, andy a. health promoting bioactive phytochemicals from brassica. int food res j 2012;19:141-52. 6. darmansjah i, wiria mss. dasar toksikologi. in: gunawan sg, setiabudi r, nafrialdi, editors. farmakologi dan terapi. 5th ed. jakarta: departemen farmakologi dan terapeutik fakultas kedokteran universitas indonesia; 2007.p.820-5. 7. giannini e, risso d, botta f, chiarbonello b, fasoli a, malfatti f, et al. validity and clinical utility of the aspartatea–alanine aminotransferase ratio in assessing disease severity and prognosis in patients with hepatitis c virus–related chronic liver disease. arch intern med 2003;163:21824. 8. maretnowati n, widyawaruyanti a, santosa mh. uji toksisitas akut dan subakut ekstrak etanol dan ekstrak air kulit batang artocarpus champeden spreng dengan parameter histopatologi hati mencit. majalah farmasi airlangga 2005;5:91-5. 9. hamed ma, aly hf, ali sa, metwalley ns, hassan sa, ahmed sa. in vitro and in vivo assessment of some functional foods against initiation of hepatocellular carcinoma. j basic appl sci res 2012;2:471-83. 10. hecht ss, carmella sg, kenney pm, low sh, arakawa k, yu mc. effects of cruciferous vegetable consumption on urinary metabolites of the tobacco-specific lung carcinogen 4(methylnitrosamino)-1-(3-pyridyl)-1-butanone in singapore chinese. cancer epidemiol biomark prev 2004;13:997–1004. 11. morsy af, ibrahima hs, shalabyb ma. protective effect of broccoli and red cabbage against hepatocellular carcinoma induced by nnitrosodiethylamine in rats. j am sci 2010;6: 1136-44. 12. galati g, o’brien pj. potential toxicity of flavonoids and other dietary phenolics: significance for their chemopreventive and anticancer properties. free rad biol med 2004; 37:287-330. 13. richard a, matthew r. clinical diagnosis and management by laboratory methods. new york: saunders elsevier;2007. 14. sunarsih es, cecilia mns, fajaryanti n. pengaruh pemberian jus bunga kubis (brassica oleracea var. botrytis) terhadap kadar mda dan kadar kholesterol pada tikus putih jantan dengan hiperlipidemia. majalah farmasi indonesia 2009;1:35-41. 15. shapiro t, fahey jw, wade kl, stephenson kk, talalay p. chemoprotective glucosinolates and isothiocyanates of broccoli sprouts: metabolism and excretion in humans. cancer epidemiol biomark prev 2001;10:501–8. 16. isbir t, yaylim i, aydin m, ozturk o, koyuncu h, zeybek u. the effect of brassica oleracea var capitata on epidermal glutathione and lipid peroxides in dmba-initated-tpa-promoted mice. anticancer res 2000;20:219–24. 17. fowke jh, morrow jd, motley s, bostick rm, ness rm. brassica vegetable consumption reduces urinary f2-isoprostane levels independent of micronutrient intake. carcinogenesis 2006;27:2096–102. c:\users\universa medicina\docu 11 abstract universa medicina pre-operative intraocular pressure as a predictor of post-operative intraocular pressure after phacoemulsification in non-glaucomatous patients titiek ernawati1,2,*, kevin anggakusuma hendrawan1, wilson christianto khudrati1, and kevin samsudin1 background cataract has been known to cause high intraocular pressure which may lead to secondary glaucoma. some anatomical changes in cataract patients are assumed to be factors contributing to increased intraocular pressure (iop). the changes in iop after cataract surgery tend to help surgeons to predict clinical outcomes. therefore, iop control is very important in these patients. this study aimed to determine the ocular biometric parameters and pressure-to-depth (pd) ratio associated with iop in non-glaucomatous patients who undergo cataract surgery. methods a prospective study using secondary clinical data collected from 81 nonglaucomatous patients. data were collected by examining each subject preand post-operatively. the changes in ocular biometry parameters and iop were measured one week before surgery and 8 weeks after the surgery. univariate and multivariate linear regression were performed to analyze the data. results the mean anterior chamber depth (acd) change was 0.73 ± 0.16 mm, mean pd ratio was 5.04 ± 1.16, and the mean pre-operative iop was 16.07 ± 2.92 mmhg, decreasing by 2.35 mm hg (14.6 %) to 13.72 ± 3.42 mm hg at 8 weeks postoperatively. univariate linear regression results showed a significant correlation between pd ratio and post-operative iop (p=0.000), but no significant association was observed between pd ratio and post-operative iop in multiple linear regression (p=0.126). however, pre-operative iop was significantly associated with post-operative iop (beta=1.244; p=0.004) conclusions our data demonstrated that pre-operative iop was the most influential risk factor of iop reduction after phacoemulsification in non-glaucomatous patients. keywords : biometry, intraocular pressure, phacoemulsification, pressure to depth ratio, nonglaucomatous patients original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.11-17 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1209 january-april, 2022 vol.41no.1 1ophthalmology department, faculty of medicine, widya mandala catholic university surabaya 2ophthalmology department primasataya husada citra hospital surabaya correspondence: *titiek ernawati, ph.d ophthalmology department, faculty of medicine, widya mandala catholic university surabaya phone and fax +6281 2300 6365 email: titiek@ukwms.ac.id orcid id: 0000-0002-8638-672x date of first submission, august 8, 2021 date of final revised submission, february 5, 2022 date of acceptance, february 8, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: ernawati t, hendrawan ka, khudrati wc, samsudin k. preop erat ive intr aocu lar pressure as a predictor of post-operative intraocular pressure after phacoemulsification in nonglaucomatous patients. univ med 2022; 41:11-7. doi: 10.18051/univmed. 2022. v41 . 11 -1 7 12 ernawati, hendrawan, khudrati, et al pre-operative intraocular pressure introduction rapid assessment of avoidable blindness data shows that the frequency of blindness in people aged >50 years in indonesia in the year 2018 was 3%, with cataract as the leading cause of blindness in indonesia. cataract causes visual impairment which can be prevented and treated through cataract extraction surgery.(1) cataract surgery has become one of the most frequently performed surgical procedure in the world. the high cataract prevalence increases the need for cataract surgery to reduce the cataract backlog.(2) biometric measurements are important in cataract surgery to achieve the targeted post-operative refraction.(3,4) unaddressed cataract can lead to further complications, one of them being the increase in intraocular pressure (iop) which can cause glaucoma. phacoemulsification is one of the cataract surgery techniques that has been proposed as a potential procedure to lower iop.(5) the specific ocular parameters which can be used to predict the iop lowering effect following phacoemulsification, remain unclear.(6) the pressure-to-depth (pd) ratio is the ratio of the pre-operative intraocular pressure to preoperative anterior chamber depth. it has been shown that pd ratio is a useful predictor of changes in the iop post-surgery.(7) the pd ratio is a simple indicator to check because acd and iop are standard pre-operative examinations that must be performed in all cataract patients. if the pd ratio can be used as a predictor of iop reduction, it would be great if it can be applied in clinical practice. the study of hsu et al. (8) reported a diurnal fluctuation in iop, which makes other predictors, such as pre-operative iop and pd ratio, become less reliable. furthermore, the study of mar kic e t al. (9 ) showed t hat the predictability of the pd ratio parameter was low in post-operative iop changes. on the other hand, ramakrishman et al.(10) demonstrated that the pd ratio is a valuable prognostic indicator to predict postoperative iop outcome. dhamankar et al.(11) also found that pre-operative pd ratio was consistently associated with changes in the iop among glaucomatous eyes undergoing cataract surgery. the contradictory results reported by previous studies provided us the opportunity for further research. the difference between present study and the previous ones are that our study employed only non-glaucomatous patients. the objective of this study was to determine the ocular biometric parameters and pressure-to-depth (pd) ratio associated with iop in non-glaucomatous patients undergoing cataract surgery. methods research design a longitudinal analysis using secondary clinical data was performed at primasatya husada citra hospital surabaya, from december 2019 until march 2020. study subjects a total of 81 non-glaucomatous subjects were enrolled in this study. patients were monitored until 2 months after the cataract surgery to evaluate the post-operative biometry parameter changes and post-operative iop. the inclusion criteria in this study were: 1) non-glaucomatous patients; 2) age more than 40 years old; 3) male and female; 4) iop of less than 21 mmhg; 5) no history of ocular intervention or trauma; 6) undergoing cataract extraction surgery using phacoemulsification and willing to participate in the study. the exclusion criteria were: patients who were absent on the scheduled biometry and intraocular pressure examinations, patients with co-morbidities, except cataract, patients who were absent on the scheduled follow-up and patients with complications during and after the surgery. sample size determination 𝑛 = [ (𝑍𝛼 + 𝑍𝛽) 0.5 ln 1 + 𝑟 1 − 𝑟 ]2 + 3 = [ (1.960 + 1.282) 0.5 ln 1 + 0.56 1 − 0.56 ]2 + 3 = 47 subjects 13 notes: n: minimum number of subjects z : 1.96 with α = 0.05 z :1.282 with β= 0.2 r: correlation coefficient, the reference r=0.56 from ramezani et al. (7) that shows the correlation between pre-operative iop and post-operative iop. the anticipated drop-out was 15% and the optimal sample size was 54. surgical technique phacoemulsification was conducted under topical anesthesia by a skilled surgeon. eyes were prepared for surgery by instilling tropicamide 0.5 % and phenylephrine 10 % for pupil dilation. after making a 2.75 mm incision in the temporal cornea, viscoelastic injection was performed, then continuous curvilinear capsulorrhexis, that creates access to the cata ract, f ollowed by phacoemulsification and intraocular lens implantation. study assessment pressure-to-depth (pd) ratio, anterior chamber depth, lens thickness, and ocular axial length were measured with alcon ocuscan rxp ophthalmic ultrasound; iop was measured using a canon tx-20p full auto non-contact tonometer. pre-operative biometry and iop measurements were performed one week prior to the surgery while the post-operative measurements were determined 8 weeks after the surgery. statistical analysis we analyzed the data using univariate regression analyses, in which the associations between independent variables and the postoperative iop change were evaluated; these variables included gender, age, pre-operative iop, underlying disease, pd ratio, anterior chamber depth, lens thickness, and axial length. all variables showing a significant association in the univariate analysis at p<0.25 were entered into a multivariate linear regression model of risk factors associated with the post-operative iop. a value of p<0.05 was considered statistically significant. ethical clearance the ethical clearance protocol of this study was approved by the health research ethics committee of the medical faculty, widya mandala surabaya catholic university, under no. 070/wm12/kepk/dosen/t/2019 in a c c or d an c e w i th th e who-cio m s international ethical guidelines for health related research. results eighty-one patients were enrolled into the study. in table 1, the baseline characteristics show that most of the patients suffered from diabetes mellitus as co-morbidity. sex and history of hypertension did not exhibit any significant differences in the number of patients. all subjects were non-glaucomatous. baseline characteristic n (%) sex male female 30 (37.0) 51 (63.0) age (years) 65.21  7.41 history of dm yes no 19 (23.5) 62 (76.5) history of ht yes no 40 (49.4) 41 (50.6) acd (mm) pre-operative post-operative 3.24 ± 0.44 3.97 ± 0.28 al (mm) pre-operative post-operative 23.82 ± 0.91 23.62 ± 0.95 lt (mm) pre-operative 4.47 ± 0.55 iop (mmhg) pre-operative post-operative 16.07 ± 2.92 13.72 ± 3.42 pressure to depth ratio 5.04 ± 1.16 table 1. baseline demographics and clinical features of non-glaucomatous subjects (n=81) note: data presented as mean ± sd, except for sex, history of dm and history of ht; dm = diabetes mellitus; ht = hypertension; acd = anterior chamber depth; al = axial length; lt = lens thickness; iop = intraocular pressure 14 ernawati, hendrawan, khudrati, et al pre-operative intraocular pressure c table 1 shows us some data about baseline demographics and clinical features of age, sex, history of diabetes mellitus, history of hypertension, acd, axial length (al), lens thickness (lt ), and iop. t he se da ta are descr iptive pr e-oper ative values in nonglaucomatous patients. the mean intraocular pressure reduction was 2.35 mmhg. table 2 shows analysis of univariate and multivariate linear regression of baseline demographics, pre-operative clinical features and post-operative iop. the data show that the univariate linear regression analysis gave significant results e xcept for history of hypertension (p=0.729), pre-operative acd (p=0.085) and pre-operative al (p=0.561). the multivariate linear regression analysis showed that the variables of sex (p=0.014), pre-operative al (p=0.036) and pre-operative iop (p=0.004) were significantly associated with post-operative iop. pre-operative iop was the most influential predictive factor of post-operative iop (beta = 1.244; p=0.004) discussion this study showed that the mean value of post-operative iop was 13.72 ± 3.42 mmhg. this is in agreement with the average post-operative iop value (14.47±2.39 mmhg), as reported by a study in malaysia.(12) another study conducted by ramakrishnan et al.,(10) reported the mean 3month post-operative iop to be 19.23±2.2 mmhg. our study showed that the post-operative acd increased from 3.24 ±0.44 mm to 3.97 ±0.28 mm. this is similar to that of the study by huang et al.(13) where mean acd increased from 2.52 ± 0.43 mm before surgery to 3.84 ± 0.29 mm after surgery. our study showed that there were no significant roles of acd and lt in postoperative iop after phacoemulsification. many studies have been conducted in order to analyze the role of many parameters which can be used to predict post-op erative iop after phacoemulsification.(14-17) most studies which have been carried out prior to our study used preoperative parameters in predicting post-operative iop. the results of our study are in agreement with a study performed in malaysia, reporting that there was no significant correlation between postoperative iop reduction and the parameters of acd and lt.(12) on the contrary, huang et al.(14) observed a significant role of acd widening and lens vault in predicting the iop reduction after phacoemulsification surgery.(14) deepening of the acd is postulated as the mechanism of iop reduction. although there was a significant change in acd in our study, this was not significantly associated with iop reduction. we assumed that this might be associated with other factors such table 2. univariate and multivariate linear regression analyses of baseline demographics, preoperative clinical features, and post -operative iop variables univariate linear regression multivariate linear regression* β 95% ci p value β beta 95% ci p value age sex history of dm history of ht pre-operative acd pre-operative al pre-operative lt pre-operative iop pre-operative pd ratio -0.418 0.217 0.263 -0.039 0.193 -0.066 -0.288 0.643 0.431 -0.584, -0.221 0.008, 0.425 0.055, 0.449 -0.269, 0.171 -0.049, 0.415 -0.288, 0.151 -0.491, -0.074 0.500, 0.741 0.203, 0.608 0.000 0.052 0.018 0.729 0.085 0.561 0.009 0.000 0.000 -0.048 1.632 0.432 -1.286 1.206 1.460 -2.307 -0.104 0.232 0.054 -0.166 0.194 1.244 -0.784 -0.142, 0.045 0.344, 2.919 -1.070, 1.934 -7.273, 4.700 -0.497, 2.909 0.474, 2.446 -5.283, 0.668 0.308 0.014 0.568 0.670 0.162 0.004 0.126 *including age, sex and parameters with p<0.25 in univariate linear regression analysis note : β : regression coefficient; ci : confidence interval; dm : diabetes mellitus; ht: hypertension; acd : anterior chamber depth; al = axial length; lt = lens thickness; iop = intraocular pressure; pd : pressure to depth 15 as angle configuration which was not being investigated and observed in our study. the differences might be caused by the different measurement methods involved in this study. a usg scan was utilized in measuring the biometry parameters in this study, whereas huang et al.(14) u se d a n t er i o r s e gme n t oc u l ar c o mpu t e d tomography (as-oct) as their modality. in this study, we found that the preoperative iop value was the determinant factor in predicting the post-operative iop and also the reduction in iop. similarly, beato et al. (5) and picoto et al. (18) found that a higher preoperative iop is significantly associated with the reduction in iop, measured 6 months after surgery. our study found that the pd ratio played an important role in determining both the posto pe r a t ive iop. ou r r e su l t s w e r e a l s o in agreement with the study by ramakrishman et al.(10) showing a significant correlation between reduction in iop and pd ratio. furthermore yoo et al.(19) stated that the pd ratio was found to be a significant predictor of absolute iop change following cataract surgery in patients with or at risk of primary open angle glaucoma (poag). additionally dhamankar et al.(11) found that the p r e -o pe r a t i ve pd r at i o w a s c o ns i s t ent l y associated with changes in iop. however, our multivariate analysis of the pd ratio shows no significant relation with post-operative iop. this finding can indirectly explain the importance of acd in iop reduction. calculation of the pd ratio can help the clinician predict iop reduction after cataract extraction surgery. this study also shows that age and iop reduction have a significant inverse correlation in univariate linear regression, thus supporting the findings of zhao et al.(20) that age and iop reduction in cataract surgery have inverse longitudinal and cross-sectional associations. structural changes in the trabecular meshwork can explain the inverse association because an aging tra becular meshwork increases the resistance to aqueous humor outflow, resulting in increased iop.(20) sex-related post-operative iop reduction has been inconsistent across studies. in our study, men had lower iop reduction than women. in contrast, the kangbuk samsung health (20) and gutenberg health studies(21) f oun d t h at w ome n h a d l ow e r iop. t he y hypothesized that higher iop in men could be due to a higher prevalence of cardiovascular risk factors. also, estrogen may affect the inflow of aqueous humor; an indian study(22) showed that the iop was higher in postmenopausal than in premenopausal women and attributed this difference to higher levels of testosterone and decrease of estrogen and progesterone levels with onset of menopause. however, our study f o u nd t h a t s e x -r e la te d iop r ed u c t i on i s significant in univariate and multivariate a n a l ys i s . fu r t h e r r e se a r c h i s n e e d e d to under sta nd the se x-r ela ted di f fe ren ce in intraocular pressure. as the limitation of our study, the iop measurement was done using non-contact tonometry instead of the goldmann applanation tonometer which is the gold standard in measuring iop value. the follow-up period in our study was also limited to two months only. a longer follow-up period needs to be used to further evaluate the iop changes. the clinical implication of this study is that the clinicians will know that phacoemulsification can decrease intraocular pressure. in nongla uc o ma tou s p a t i e n t s t h e pd r a t i o i s significantly associated with postoperative iop in univariate analysis and non-significantly associated with iop in multivariate analysis so that it has been shown that the pd ratio can be used independently as a predictive factor of iop after phacoemulsification. conclusions this study demonstrated that pre-operative iop is an independent and good predictor of iop reduction after phacoemulsification surgery in non-glaucomatous patients. univ med vol. 41 no 1 16 ernawati, hendrawan, khudrati, et al pre-operative intraocular pressure conflict of interest there is no conflict of interest in this study. acknowledgement we would like to acknowledge the support of medical faculty, widya mandala surabaya catholic university, for this study as an approved research proposal, and primasatya husada citra hospital for their support and assistance. contributors et and hka contributed to study concept and design, provision of study material or patients. et, hka and kwc contributed to data analysis and interpretation. et and ks contributed to administrative support and data collection and assembly. et, hka, kwc and ks contributed to manuscript writing and reviewing. all authors approved the final manuscript and will take public responsibility for the content of the manuscript submitted to universa medicina.. references 1. direktorat pencegahan dan pengendalian penyakit tidak menular direktorat jenderal pencegahan dan pengendalian penyakit. peta jalan penanggulangan gangguan penglihatan di indonesia tahun 2017-2030. jakarta: direktorat pencegahan dan pengendalian penyakit tidak menular direktorat jenderal pencegahan dan pengendalian penyakit; 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97: e12881. doi: 10.1097/ md.0000000000012881. univ med vol. 41 no 1 20. zhao d, kim mh, pastor-barriuso r, et al. a longitudinal study of age-related changes in intraocular pressure: the kangbuk samsung health study. invest ophthalmol vis sci 2014; 55: 6244-50. doi: 10.1167/iovs.14-14151. 21. hoehn r, mirshahi a, hoffmann em. distribution of intraocular pressure and its association with ocular features and cardiovascular risk factors: the gutenberg health study. ophthalmology 2013;120:961-8. doi: 10.1016/j.ophtha.2012.10.031. 22. panchami, pai sr, shenoy jp, shivakumar j, kole sb. postmenopausal intraocular pressure changes in south indian females. j clin diagn res 2013;7:1322-4. doi: 10.7860/jcdr/2013/ 5325.3145. c:\users\user\documents\39 no 2 121 abstract universa medicina body mass index increases cd4+ count in hiv/aids patients on first-line therapy mirna widiyanti*, moch irfan hadi**, mei lina fitri kumalasari***, evi iriani natalia*, dedi purba*, and setyo adiningsih* background the body mass index (bmi) may contribute somewhat to drug metabolism, thus affecting the efficacy of antiretroviral therapy (art). cd4+ counts in people infected with hiv are essential in determining the stage of the disease, initiation of antiretroviral therapy, opportunistic infections and evaluating treatment outcomes. the aim of this study was to determine the association of bmi and clinical stage with cd4+ counts in hiv patients seeking treatment using first-line antiretroviral therapy (art). methods an analytic study with a cross-sectional approach was conducted involving 251 hiv/aids patients who had received first-line antiretrovirals over six months. bmi, clinical staging according to who and cd4 + were collected. multiple linear regression was used to evaluate the relationship between bmi, clinical stage and cd4+. results among the enrolled patients, the median age was 36 years, 135 (55%) of the patients was female, 102 (40.6%) was overweight/obese, 161 (64.1%) was in stage 3 of the disease, and the median cd4+ count was 389 cells/ mm3. multiple linear regression test showed two variables with a significant effect on cd4+ count, namely bmi (b=69.247; 95 % ci : 42.886-95.608) and clinical stage (b=61.590; 28.910-94.270). bmi was the most influencing factor for cd4+ count (=0.307) compared to clinical stage (=0.216). conclusions body mass index was the most influencing factor for cd4 + counts of hiv/ aids patients. regular art can increase cd4+ counts and maintain the health of hiv/aids patients. keywords: cd4 + count, body mass index, clinical staging, hiv patients original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.121-127 may-august 2020 vol.39no.2 cite this article as: widiyanti m, hadi mi, kumalasari mlf, natalia ei, purba d, adiningsih s. body mass index increases cd4+ count in hiv/aids patients on first-line therapy. univ med 202 0;39 :1 2 1 -7 . doi : 10 .18 05 1/ univmed.2020. v39.121-127 *centre for papua health research and development, ministry of health, republic of indonesia **faculty of science and technology, universitas islam negeri sunan ampel, surabaya ***faculty of psychology and health sciences, universitas islam negeri sunan ampel, surabaya correspondence: mirna widiyanti centre for papua health research and development, ministry of health, republic of indonesia phone: +6281344388000 email: ninawidhy@gmail.com orcid id: 0000-0001-8246-7672 date of first submission, june 19, 2020 date of final revised submission, august 12, 2020 date of acceptance, august 13, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 122 widiyanti, hadi, kumalasari, et al bmi increases cd4+ count in hiv/aids patients introduction hiv/aids is a global health problem, with the number of cases to date reaching 32 million. incr eased access to effective prevention, diagnosis, treatment and care makes this disease manageable and allows people living with hiv (plhiv) to live healthier lives. at the end of 2018 there were around 37.9 million people living with hiv and 1.7 million new cases of infection.(1) the latest data in 2019 show that the numbers of hiv and aids cases in papua province were 34,473 and 22,554 respectively, while in the province of west papua there were 5,243 and 1,741 cases, respectively. (2) this remains a challenge in the prevention and control of hiv/aids in papua, especially in we st pa pua . west pa pua provinc e a s of september 2016 had 5,839 cases, where the highest number of cases was in manokwari district with 1,422 cases, followed by sorong city with 1,902 cases, sorong district with 1,026 cases, and fak-fak district with 423 cases.(3) this led us to consider making manokwari regency, sorong city and fak-fak regency our research sites in west papua province. the measurement of body weight is an important evaluation in diagnosing hiv. low body mass index (bmi) is the first criteria in defining aids. in addition, bmi also plays a role in determining who clinical stage, which includes weight loss, where weight loss of <10% is categorized as stage 2, while a weight loss of >10% or a body mass index of 18.5 kg/m2 is included in stage 3, and the hiv wasting syndrome falls into the clinical category of stage 4.(4) the body mass index of hiv patients is an important predictor of antiretroviral outcomes(57) including prediction of cd4 cell changes (8) and death.(9-11) negative changes in a patient’s bmi are also independent predictors of dropout from hiv care cases.(12) the bmi of hiv patients is influenced by gender, who stage (13), duration of treatment,(14-15) and cd4 cell count.(16) world health organization (who) classifies body mass index by dividing the bmi value into underweight (<18.5 kg/m2), normal weight (18.5-24.9 kg/m2), pre-obesity (25-29.9 kg/ m2), obesity (30-39.9 kg/m2), and morbid obesity (>40kg/m2).(17) one study found that a higher bmi is associated with more robust cd4+ t-cell recovery in haart-treated patients.(18) crumcianflone et al.(19) discovered that following haart, obese hiv-infected patients acquired fewer cd4+ t lymphocytes compared to normal weight hiv-infected patients, indicating that the potentially adverse immune response is associated with excess weight.(19) however, the study of tedaldi et al.(20) found that higher bmi is not correlated with immunological and viral responses to antiretroviral therapy.(20) thus the effects of bmi on immune reconstitution after initiating haart remain inconsistent. the present study focused on patients with first-line art and used bmi data that are easily calculated but important for predicting the outcome of art treatment, treatment drop-out and cd4 recovery and death. the aim of this study was to determine the association of demographic factors, bmi and clinical staging with cd4+ counts that illustrate the progression of the disease in hiv patients seeking treatment using first-line antiretrovirals. methods research design an analytic research of cross-sectional design was conducted in west papua (manokwari regency, sorong regency and fak-fak regency) from may to july 2019. study subjects the sample size was estimated using a 3% level precision (d), estimated population proportion 0.98 with 95% confidence interval. based on formula calculations the total sample to be taken in the three regencies was 251 persons. the respondents were hiv or aids patients who 123 went to the voluntary counseling testing (vct) clinics of manokwari hospital, sele be solu hospital (sorong city), and fak-fak regional hospital in 2019 and who had received first-line art for 6 months. the inclusion criteria of the study were availability of cd4 data and complete medical records, and having received art for a minimum of 6 months and a maximum of 60 months. female patients who were pregnant at baseline or during the follow-up visits were excluded from the study. measurements body mass index (bmi) was calculated using the formula weight (kilograms)/height (meters squared). the body mass index (bmi) in this study was also analyzed based on groupings from the western pacific region of the who criteria pertaining to obesity (wpro criteria, 2000), namely underweight (bmi <18.5), normal bmi (18.5–22.9), overweight (bmi 2324,) obese i (25-29.9) and obese ii (bmi=30). world health organization clinical staging: stage 1, patients who are asymptomatic or have persistent generali zed lympha denopathy (lymphadenopathy of at least two sites [not including inguinal] for longer than 6 months) are categorized as being in stage 1, where they may remain for several years. stage 2, even in early hiv infection, patients may demonstrate several clinical manifestations. clinical findings included in stage 2 (mildly symptomatic stage) are unexplained weight loss of less than 10 percent of total body weight and recurrent respiratory infections (such as sinusitis, bronchitis, otitis media, and pharyngitis). stage 3, as disease progresses, additional clinical manifestations may appear. those encompassed by the who clinical stage 3 category (the moderately symptomatic stage) are weight loss of greater than 10 percent of total body weight, prolonged (more than 1 month) unexpla ined diarr hea, pulmonary tuberculosis, and severe systemic bacterial infections including pneumonia, pyelonephritis, empyema, pyomyositis, meningitis, bone and joint infections, and bacteremia. stage 4, the who clinical stage 4 (the severely symptomatic stage) designation includes all of the aids-defining illnesses.(4) laboratory analysis cd4 + cell examination uses the pima analyzer (alere), pima bead count and the pima cd4 cartridge. the cd4 value that comes out of the pima tool is an absolute value without a percentage value. as a priority, art should be initiated in all individuals with severe or advanced hiv clinical disease (who clinical stages 3 or 4) and individuals with cd4 count 350 cells/ mm3 (strong recommendation, moderate-quality evidence). the art should be initiated in all individuals with hiv with a cd4 count of >350 cells/mm3 and 500 cells/mm3, regardless of who clinical stage (strong recommendation, moderate-quality evidence).(21) statistical analysis the independent variables were age (years), gender (m/f), education (years), occupation (yes/ no), body mass index and clinical stage, while the dependent (outcome) variable was the cd cell count. data were analyzed using multiple linear regression with 95% confidence interval to quantify the strength of association between cd4 as the dependent variable and the independent variables. a p-value of less than 0.05 was considered significant. ethical clearance this study has been approved by the health research ethics committee of the board of health research and development, ministry of health, republic of indonesia, under number lb.02.01/2/ke.008/2019. results demographic char a cteristics of hiv research subjects include age, sex, occupation and education, while clinical characteristics include bmi, clinical stage and cd4+ count. univ med vol. 39 no.2 124 widiyanti, hadi, kumalasari, et al bmi increases cd4+ count in hiv/aids patients a b c table 1 shows that the subjects were of productive age, that the highest level of education was high school, and that almost half were private employees. many hiv subjects were of normal weight, and had hiv/aids stage 3. table 2 shows the results of the multiple linear regression analysis showing that the two variables that had a significant relationship with cd4+ count were bmi (b=69.247;95% ci= 42.886-95.608) and clinical stage (b=61.590; 95% ci=28.910-94.270). bmi was the most influencing factor for cd4+ (β=0.307) compared to clinical stage (β=0.216). discussion demogr aphic char a cteristics of hiv respondents in west papua indicate that the characteristics of gender, age, education and occupation are still dominated by women of productive age, secondary education level and private employment. in a previous study in papua province it was also found that most of the subjects were women, were of productive a ge ( 1 7 -35 ye a r s ) a n d ha d hi gh s c ho o l education.(22) as to the clinical characteristics of west papuan patients, the majority of patients have a cd4 count of >350 cells/mm3, are in clinical stage 3 and have a normal body mass index of 18.5-22.9 kg/m2. this is in contrast to a study in timika (mimika regency) where more patients in stages 1-2 were found.(23) from the results of the analysis it was found that there is a significant association between cd4+ count and body mass index of hiv patients in west papua. the greater the bmi value, the higher the cd4+ count. the profile of nutritional changes in hiv patients has been reported to have changed since starting therapy, characteristic n (%) age (median-range in years) 36 (15-72) sex (n%) male 113 (45) female 138 (55) education (n%) no schooling primary school junior high school senior high school bachelor employment (n%) government employee military/police private employee sailor farmer laborer unemployed body mass index (n%) underweight normal weight overweight obese clinical stage (n%) stage i stage ii stage iii stage iv cd4 count (median range) 2 (8) 24 (9.6) 40 (15.9) 106 (42.2) 79 (31.5) 41 (16.3) 3 (1.2) 110 (43.8) 3 (1.2) 8 (3.2) 7 (2.8) 79 (31.5) 49 (19.5) 100 (39.8) 37 (14.7) 65 (25.9) 50 (19.9) 30 (12) 161 (64.1) 10 (4) 389 (10-1500) table 1. demographic and clinical characteristics of the subjects (n=251) characteristics b β p value 95% ci lower upper sex 6.304 0.156 0.120 -13.632 16.975 age 0.239 0.009 0.877 -2.795 3.274 education 12.568 0.050 0.432 -44.048 18.912 employment 2.493 0.024 0.718 -11.112 16.098 body mass index 69.247 0.307 0.000* 42.886 95.608 clinical stage 61.590 0.216 0.000* 28.910 94.270 table 2. factors associated with cd4+ lymphocyte resulting from multiple linear regression b: unstandardized regression coefficient; β: standardized regression coefficient 125 univ med vol. 39 no.2 with an increase in the prevalence of obesity and weight loss. several studies have shown that the level of overweight found in people with hiv/ aids does not diffe r f rom the gener al population.(24) changes in body fat distribution, dyslipidemia and insulin resistance are common conditions in adult hiv patients undergoing antiretroviral ther apy and be ing o verweight can cause complications.(25-26) a decrease in bmi is associated with a worse prognosis in people with hiv. body mass index is related to nutritional status of hiv sufferers, while nutritional status influences the function and immunity status of hiv patients.(27-28). the poor immune function due to hiv/aids causes nutritional deficiencies resulting in immune dysfunction and accelerated disease progression to aids.(29) besides body mass index, another factor associated with cd4 + is the clinical stage of the disease. the analysis of hiv patients in west papua found that the higher the clinical stage, the lower the cd4 + value. as many as 61.4% of hiv patients in west papua are in clinical stage 3. like cd4 counts, recognition of these clinical findings included in the who system is an important method for identifying hiv-infected individuals at high risk for morbidity and mortality. remaining aware of the natural course of hiv infection allows one to base management decisions on the patient’s clinical presentation. according to the who, advanced hiv/aids disease is defined for surveillance purposes as any clinical stage-3 or stage-4 disease or any clinical stage with a cd4 count greater than 350 per cubic mm, and this information can be used to calculate the burden of disease and the demand for antiretroviral therapy.(5) there is strong evidence supporting the clinical benefit of antiretroviral medications for adults with advanced hiv/aids as determined clinically or immunologically, with the who recommending definitive initiation of antiretroviral therapy in adults and adolescents in clinical stage 4, consideration of therapy initiation for those in clinical stage 3, and antiretroviral use for those in clinical stage 1 or 2 only if the cd4 count is greater than 200 per cubic mm. for patients taking antiretroviral therapy for more than 24 weeks, new or recurrent clinical staging events can be a guide to decision-making. prior to 24 weeks of antiretroviral treatment, clinical events are largely influenced by immune reconstitution or treatment toxicity and may not accurately reflect immune deterioration.(21) who guidelines report that the appearance of new or recurrent who clinical stage 3 and 4 conditions beyond 24 weeks after initiation of therapy suggests treatment failure. the hiv/aids epidemic clearly has broad and significant implications for individuals living around the globe. populations in developing nations are especially hard-hit by hiv infection and, at the same time, frequently lack access to technological advances and other resources for diagnosing and managing care. screening strategies, such as the who clinical staging system, allow for efficient identification of early infection and aggressive management when clinicians are equipped with the knowledge to apply them, and can therefore be useful tools for improving access to and implementation of care. a limitation of our study was its cross-sectional design. staging at a single visit may not adequately capture events that might have occurred prior to the study evaluation. also, the who clinical staging depends largely on gathering relevant clinical information from the patient and is therefore subjective. the clinical implication of this study is that an absolute cd4 cell count below 200 cells/mm3 indicates severe damage to the body’s immune system. absolute cd4 cell counts can still be used to determine the initiation of antiretroviral therapy. future studies should perform a cd4 percentage check in addition to an absolute cd4 count, because cd4 percentage is more stable than the absolute value. conclusions clinical stage and body mass index affect the absolute cd4+ count; a greater bmi value 126 widiyanti, hadi, kumalasari, et al bmi increases cd4+ count in hiv/aids patients will increase the cd4+ count and a higher clinical hiv stage will decrease the cd4+ count. conflict of interest the authors declare that there is no conflict of interest. acknowledgement this work was partially supported by a grant f r om th e bo a r d of he a lth re se a r c h a n d development, ministry of health, republic of indonesia. the authors are grateful to the vcts of manokwari, sorong and fak-fak health agencies for their generous support. contributors mw contributed to design of the study, sampling and performing the experiment; mih and mlf interpreted the data. mw and ei contributed to writing the draft of the manuscript , dp and sa contributed to the revision and finalization the manuscript. all authors have read and approved the final manuscript. references 1. world health organization. hiv/aids : key facts. geneva : world health organization;2020. 2. kementrian kesehatan ri. general situation of hiv/aids and hiv test. jakarta: pusat data dan informasi kementrian kesehatan ri; 2018. 3. dinas kesehatan provinsi papua barat. jumlah kasus hiv aids papua barat; 2016. 4. weinberg jl, kovarik cl. the who clinical staging system for hiv/aids. virtual mentor 2010;12:202-6. doi: 10.1001/virtualmentor2010. 12.3.cprl1-1003. 5. tsegaye at, wubshet m, awoke t, alene ka. predictors of treatment failure on second-line antiretroviral therapy among adults in northwest ethiopia: a multicentre retrospective follow-up study. bmj open 2016;6:e012537. doi: 10.1136/ bmjopen-2016-012537. 6. seyoum a, ndlovu p, zewotir t. quasi-poisson versus negative binomial regression models in identifying factors affecting initial cd4 cell count change due to antiretroviral therapy administered to hiv-positive adults in north-west ethiopia (amhara region). aids res ther 2016;13:36. doi: 10.1186/s12981-016-0119-6. 7. dalhatu i, onotu d, odafe s, et al. outcomes of nigeria’s hiv/aids treatment program for patients initiated on antiretroviral treatment between 2004-2012. plos one 2016;11:e0165528. doi: 10.1371/journal.pone.0165528. 8. koethe jr, jenkins ca, lau b, et al. body mass index and early cd4+ t cell revovery among adults initiating antiretroviral therapy in north america,1998-2010. hiv med 2015;16:572–7. doi: 10.1111/hiv.12259. 8. chakravarty j, sundar s, chourasia a, et al. outcome of patients on second line antiretroviral therapy under programmatic condition in india. bmc infect dis 2015;15: 517. doi: 10.1186/s12879015-1270-8. 10. thao vp, quang vm, wolbers m, et al. secondline hiv therapy outcomes and determinants of mortality at the largest hiv referral center in southern vietnam. medicne (baltimore) 2015;94:e1715. doi: 10.1097/md.000000000000 1715. 11. swain pk, grover g. determination of predictors associated with hiv/aids patients on art using accelerated failure time model for interval censored survival aata. am j biostat 2016;6:12–9. doi: 10.3844/ajbssp.2016. 12. asiimwe sb, kanyesigye m, bwana b, okello s, muyindike w. predictors of dropout from care among hiv-infected patients initiating antiretroviral therapy at a public sector hiv treatment clinic in sub-saharan africa. bmc infect dis 2016;16: 43. doi: 10.1186/s12879-0161392-7. 13. in ‘t veld dh, balestre e, buyze j, et al. determinants of weight evolution among hivpositive patients initiating antiretroviral treatment in low resource settings. j acquir immune defic syndr 2015;70:146–54. doi: 10.1097/qai. 0000000000000691. 14. baraki ag,gezie ld, zeleke eg, awoke t, tsegaye at . body mass index variation over time and associated factors among hiv-positive adults on second-line art in northwest ethiopia: a retrospective follow-up study. bmj open 2019; 9:e033393. doi:10.1136/bmjopen-2019-033393. 15. tang am, sheehan hb, jordan mr, et al. predictors of weight change in male hiv-positive injection drug users initiating antiretroviral therapy in hanoi, vietnam. aids res treat 2011;2011:890308. doi: 10.1155/2011/890308. 127 univ med vol. 39 no.2 16. li n, spiegelman d, drain p, et al. predictors of weight loss after haart initiation among hivinfected adults in tanzania. aids 2012 ;26:577– 85. doi: 10.1097/qad.0b013e32834f9851. 17. world health organization regional office for europe. body mass index bmi. copenhagen, denmark : world health organization regional office for europe;2020. 18. koethe jr, jenkins ca, lau b, et al. higher timeupdated body mass index: association with improved cd4+ cell recovery on hiv treatment. j acquir immune defic syndr 2016;73:197–204. doi: 10.1097/qai.0000000000001035. 19. crum-cianflone nf, roediger m, eberly le, et al. impact of weight on immune cell counts among hiv-infected persons. clin vaccine immunol 2011;18:940–6. doi:10.1128/cvi.00020-11. 20. tedaldi em, brooks jt, weidle pj, et al. increased body mass index does not alter response to initial highly active antiretroviral therapy in hiv-1infected patients. j acquir immune defic syndr 2006;43:35–41. doi: 10.1097/01.qai.0000234084. 11291.d4. 21. world health organization. consolidated guidelines on the use of antiretroviral drugs for treatning and preventing hiv infection: recommendation for a public health approach. 2nd ed. geneva: world health organization;2016. 22. widiyanti m, hadi mi, adiningsih s, et al. demographic characteristics of the people living with hiv in papua. j heal sci prev 2019;3:10–5. doi: http://doi.org/10.29080/jhsp.v3i1.175. 23. widiyanti m, ubra r, fitriana e. low body mass index increases risk of anemia in patients with hiv-aids receiving antiretroviral therapy. univ med 2017;36:221–7. doi: 10.18051/univmed. 2017.v36.221-227. 24. de araújo mariz c, de albuquerque mfpm, de alencar ximenes ra, et al. body mass index in individuals with hiv infection and factors associated with thinness and overweight/obesity. cad saude publica 2011;27:1997–2008. doi: 10.1590/s0102-311x2011001000013. 25. lake je, currier js. metabolic disease in hiv infection. lancet infect dis 2013;13:964–75. doi: 10.1016/s1473-3099(13)70271-8. 26. duncan ad, goff lm, peters bs. type 2 diabetes prevalence and its risk factors in hiv: a crosssectional study. plos one 2018;13:e0194199. doi: 10.1371/journal.pone.0194199 27. crum-cianflone n, roediger mp, eberly l, et al. increasing rates of obesity among hiv-infected persons during the hiv epidemic. plos one 2010;5: e10106. doi:10.1371/journal.pone.0010106. 28. thimmapuram r, lanka s, esswein a, dall l. correlation of nutrition with immune status in human immunodeficiency virus outpatients. mo med 2019;116:336–9. 29. sicotte m, langlois év, aho j, ziegler d, zunzunegui mv. association between nutritional status and the immune response in hiv + patients under haart: protocol for a systematic review. systematic reviews 2014 3:9. doi: 10.1186/20464053-3-9 oktavianus 63 *department of physical and rehabilitation medicine faculty of medicine, trisakti university, jakarta correspondence dr. dr. maria regina rachmawati, sprm department of physical and rehabilitation medicine, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: +6221-5672731 ext.2101 email: ati_dr@yahoo.com univ med 2012;31:63-70 universa medicina january-april, 2012january-april, 2012january-april, 2012january-april, 2012january-april, 2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background menopause is an aging process of the female reproductive system characterized by reduced estrogen levels. this results in increased osteoclastic activity, causing increased bone resorption and thus reduced bone mineral density (bmd). in addition to being influenced by osteoclastic activity, bmd of the lumbar vertebrae is also affected by the erector spinae muscle. the purpose of this study was to determine an association between postural scoliosis and erector spinae muscle endurance and its relation to bmd in postmenopausal women. methods this was a cross-sectional study conducted on postmenopausal women, who were selected by simple random sampling among residents of mampang prapatan subdistrict. the postural abnormality of scoliosis was determined by physical examnation, while erector spinae muscle endurance time was assessed using a modified biering-sorensen technique, and bmd was measured by bone mineral densitometry, to categorize into normal, osteopenia, and osteoporosis. results a total of 213 postmenopausal women with mean age of 53.52 ± 3.64 years participated in the study. the prevalence of scoliosis was 54.0%, and osteoporosis was 38.1%. the prevalence of osteoporosis was higher in women with scoliosis (48.7%) in comparison with those without scoliosis (31.6%) (p=0.411). in postmenopausal women with strong erector spinae muscle endurance the risk of of scoliosis was lower (0.76;95% confidence interval = 0.58 0.99) conclusions in post menopausal women with postural scoliosis found a higher incidence of osteoporosis. high endurance of erector spinae muscle lowers the risk of scoliosis. exercise to improve posture and increase endurance of erector spinae muscle need to be done to prevent decline of bmd. key words: scoliosis, erector spinae muscle, endurance, postmenopausal women prevalence of osteoporosis increased in postmenopausal women with postural scoliosis maria regina rachmawati*, nuryani sidarta* and yefta d. bastian* abstract 64 rachmawati, sidarta, bastian osteoporosis in postural scoliosis prevalensi osteoporosis meningkat pada perempuan pascamenopause dengan skoliosis postural latar belakang menopause adalah proses penuaan sistem reproduksi perempuan, yang ditandai dengan terjadinya penurunan hormon esterogen. penurunan hormon esterogen akan menstimulasi peningkatan aktivitas osteoklas, yang akan menyebabkan terjadinya peningkatan aktivitas resorbsi tulang, sehingga terjadi penurunan densitas masa tulang. densitas masa tulang vertebra lumbal selain dipengaruhi oleh aktivitas osteoklas, juga dipengaruhi oleh tekanan tonus otot yang bekerja di sekitar vertebra. kelemahan otot erektor spina juga diperkirakan menyebabkan skoliosis postural. penelitian ini bertujuan untuk menentukan adanya hubungan antara skolisis dan ketahanan otot erektor spina serta kaitannya dengan densitas masa tulang pada perempuan pascamenopause. metode rancangan penelitian jenis potong lintang digunakan pada subjek perempuan pasca menopause, yang dipilih secara random sederhana pada kecamatan mampang prapatan jakarta selatan. abnormalitas postur berupa skoliosis ditentukan berdasarkan pemeriksaan fisik, ketahanan otot erektor spina ditentukan dengan modifikasi metode biering-sorensen. densitas masa tulang ditentukan dengan bone mineral densitometry (bmd), dengan menilai skor t kemudian dibuat kategori densitas tulang normal, osteopenia, dan osteoporosis. hasil sebanyak 213 perempuan pasca menopause dengan rata-rata usia 53,52 ± 3,64 tahun ikut serta pada penelitian. prevalensi osteoporosis (48.7%) lebih tinggi pada pada perempuan pascamenopause yang mengalami skoliosis postural dibandingkan yang tidak mengalami skoliosis (31,6%) (p=0,411). pada perempuan pascamenopause yang memiliki ketahanan otot erektor spina yang kuat risiko terjadinya skolisis lebih kecil sebesar 0,76 (95% interval keperayaan 0,580,99). kesimpulan pada perempuan pascamenopause yang mengalami skoliosis postural dijumpai kejadian osteoporosis yang lebih tinggi. ketahanan erektor spina yang kuat menurunkan risiko terjadinya skoliosis pada perempuan pascamenopause. perlu dilakukan pelatihan perbaikan postur dan peningkatan ketahanan otot erektor spina untuk mencegah terjadinya penurunan densitas masa tulang. kata kunci: skoliosis, ketahanan, otot erektor spina, perempuan pasca menopause abstrak introduction menopause is an irreversible aging process of the female reproductive system, leading to cessation of menstruation. per definition menopause starts 12 months after the last menstruational period. at this point in time no graaffian follicles are to be found in the ovaries, thus reducing the sinthesis of estrogens. the reduction in estrogen concentrations gives rise to numerous symptoms, including signs of vasomotor instability such as tachycardia and 65 facial hot flushes, urogenital abnormalities such as vaginal dryness and dyspareunia, and disturbances of bone metabolism.(1-3) as a result of repeated microtraumas, bony tissues are constantly undergoing dynamic remodeling, in which bone formation is followed by resorption. the cells playing a role in the process of bone remodeling are the osteoblasts, which give rise to the formation of osteoid cells, and the osteoclasts, which function in bone resorption. bone consists of two types of tissue, i.e. trabecular or cancellous bone and compact or cortical bone. trabecular bone undergoes an annual rate of bone formation and resorpstion of approximately 25%, wheras cortical has an annual turnover rate of only 3%.(4-6) in menopause there is a decrease in gonadal functioning, leading to increased activity of osteoclastic progenitor cells. the reduction in estrogen synthesis initiates e x c e s s i v e b o n e r e s o r p t i o n , f o l l o w e d b y inadequate bone formation. all osteoblasts, osteocytes, and osteoclasts possess estrogen receptors. estrogens influence bone cells indirectly through cytokines growth factors. high estrogen levels increase apoptosis of osteoclasts through production of transforming growth factor (tgf-â). in conditions of estrogen deficiency, t cells increase the recruitment, differensiation, and survival of osteoclasts through interleukin-1 (il-1), il-6, and tumor necrosis factor alpha (tnf-á). one s t u d y o n o v a r i e c t o m i z e d m i c e f o u n d significantly higher levels of il-6 and higher numbers of macrophages and granulocytes. these findings demonstrate that estrogens play a role in inhibiting the secreetion of il-6, and t h a t i l 6 p l a y s a r o l e i n r e c r u i t m e n t o f osteoclasts via monocytes.(2,5,6) the excessive activity of osteoclasts leads to imbalance of the bone formation and resorption processes, with a higher resorption rate relative to the rate of bone formation, thus resulting in a reduction of bone mass. in a cross-sectional study on 203 postmenopausal women aged 47-60 years a prevalence of osteoporosis of 30% was found, while according to the national osteopororosis foundation 55% of postmenopausal women aged 50 years or older have osteoporosis.(7,8) i n t h e b o n e r e m o d e l i n g p r o c e s s , contraction of muscles is also believed to play a role, where stresses and strains on the bones stimulate bone remodeling. decreased muscular a c t i v i t y o r c o n t r a c t i o n m a y p r e s u m a b l y decrease bone formation and is thought to play a role in reducing bone mass. the muscles responsible for maintaining vertebral alignment are the erector spinae. adequate functioning of the erector spinae is thought to be capable of preventing postural abnormalities and reduction in vertebral bone mass. one of the most frequent postural abnormality occurring as a result of faulty alignment is postural scoliosis. another postural abnormality frequently found in patients with osteoporosis is kyphosis. the postural abnormalities in postmenopausal women with osteoporosis are caused by thinning of the vertebral bodies and/or weakness of erector spinae muscles.(9,10) erector spinae muscles are type i muscles, which are adapted for long-term low-intensity activities and serve to support the spinal column in a given posture, to control spinal motion, and to particularly protect the spinal column in truncal flexion. these supporting, control, and protective functions may be disturbed when the erector spinae muscles are fatigued.(11,12) t h e d e g e n e r a t i v e p r o c e s s e s i n postmenopausal women commonly lead to a decreased work capacity or endurance of the muscles, including the erector spinae. the decreased erector spinae endurance could be explained as a result of prolonged low back pain.(12) in women with osteoporosis, tasks that challenge balance may cause associated coc o n t r a c t i o n o f t r u n k m u s c l e s , ( 1 3 ) t h u s considerably increasing spinal loading. in this connection, vertebral fractures in individuals with fragile bone may lead to further fractures, as a result of aforementioned spinal loading. in other studies, scoliosis was assessed by measuring cobb’s angle on x-ray radiographs univ med vol. 31 no.1 66 rachmawati, sidarta, bastian osteoporosis in postural scoliosis a n d e n d u r a n c e o f t r u n c a l m u s c l e s b y electromyopgraphy. (14,15) in view of these facts, this study was conducted with the purpose to determine an association between scoliosis and bone mineral density in postmenopausal women and its relation to erector spinae muscle endurance. methods study design this study used a cross-sectional design and was conducted from december 2009 to march 2010 at mampang prapatan subdistrict in south jakarta. study subjects a total of 213 postmenopausal women who had menopause for 5 years were recruited for participation in this study. the inclusion c r i t e r i o n w a s a c u t e l y o r c h r o n i c a l l y i l l postmenopausal women. the subjects were selected by simple random sampling from among residents of the catchment area of the mampang prapatan subdistrict health center in south jakarta. data collection d a t a c o l l e c t i o n w a s d o n e b y p h y s i o t h e r a p i s t s w h o h a d b e e n t r a i n e d previously by the investigators. the subjects w e r e i n t e r v i e w e d u s i n g a q u e s t i o n n a i r e containing items on age, education, occupation, number of past pregnancies, and duration of menopause. physical examination physical examination was performed to determine body weight, height, presence of p o s t u r a l s c o l i o s i s , a n d e r e c t o r s p i n a e endurance. the presence of scoliosis was determined by a simple physical examination, in which the minimally clothed subjects standing in a relaxed posture were inspected for symmetry of shoulders and hips. vertebral alignment was determined by comparison with a plumb line made from a cord with a suspended weight. the subjects were initially palpated to find the spinal process of the seventh cervical vertebra. subsequently the plumb line was suspended without touching the subjects, and the alignment of the vertebrae was compared against the plumb line, to find any lateral deviation of the spine.(16) the procedure for determination of postural scoliosis is shown in figure 1. modified biering-sorensen test for erector spinae endurance the strength of the erector spinae muscles was assessed by placing the subjects in a position of horizontal truncal extension on a special examination table and is illustrated in figure 2. the biering-sorensen test has a demonstrated validity and reliability for determining erector spinae endurance.(17) figure 1. postural scoliosis examination 67 figure 2. horizontal truncal extension test based on the results of a preliminary study using our modified biering-sorensen test, n o r m a l m u s c l e e n d u r a n c e t i m e f o r postmenopausal women was set at 30 seconds for the present study.(12) measurement of bone mineral density measurements of bone mineral density (bmd) were expressed as t scores of the lumbar spine. the bmd was determined at budi jaya hospital, jakarta, using the lunar dpx bravo nomusa densitometer (ge medical systems), which is a dual-energy x-ray (dxa) absorptiometer. bmd was measured for the carpal bones, lumbar vertebrae, and femoral head. the t score criteria for determining bone mineral density used the follwing who standards: normal bmd if the t score is > -1, osteopenia if the t score is < -1 and > -2.5, and osteoporosis if the t score is < -2.5.(18) ethical clearance this study was obtained approval and ethical clearance from the research ethical committee, faculty of medicine, trisakti university. all participating subjects agreed to a written and signed informed consent. statistical analysis chi-square test was used to compare the bmd between normal subjects and those with postural scoliosis. the relationship between postural scoliosis and erector spinae endurance w a s d e t e r m i n e d f r o m c a l c u l a t i o n o f t h e prevalence ratio. the alpha level was set at 0.05 for all statistics. results mean age of the subjects was 53.51 ± 3.62 years, the majority (50.7%) of them had finished primary school, and most of them (66.6%) were unemployed. mean body mass index (bmi) was 26.21 ± 5.53, indicating overweight nutritional status. (table 1) *mean ± sd; #classification: normal: t score >-1; osteopenia t score >-2.5 <-1; osteoporosis t score <-2.5 table 1. distribution of subject characterisitics, posture, erector spinae endurance, and vertebral bone mineral density (n=213) characteristic n (% ) age (years)* 53.51 ± 3.62 educational level 21 (9.8) no formal educat ion 20 (9.3) pr imary sch ool (not finished) 109 (50.7) pr imary sch ool 40 (18.6) junior high school 19 (8.8) senior high sch ool 4 (1.8) university/college 21 (9.8) employment entrepren eur 19 (8.9) government official 2 (0.01) trader 49 (23.0) unemployed 143 (66.6) postural abnormalities normal 98 (46.0) postural scoliosis 115 (54.0) erector spinae endurance more than or equal to 30 seconds 90 (42.3) less than 30 seconds 123 (57.7) duration of menopau se (years)* 4.00 ± 2.20 body weight (kg)* 59.14 ± 11.33 height (cm)* 148.86 ± 5.21 body mass index(kg/m2)* 26.21 ± 5.53 bone mineral density t score* -1.61 ± 1.05 classification # normal 26 (12.2) osteopenia 100 (46.9) osteopor osis 87 (40.9) univ med vol. 31 no.1 68 rachmawati, sidarta, bastian osteoporosis in postural scoliosis post ural scoli osis bone mineral density category p normal (n,%) osteopenia (n,%) osteoporosis (n,%) yes 12 (10.4) 47 (40.9) 56 (48.7) 0.0411 no 14 (14.3) 53 (54.1) 31 (31.6) table 2. bone mineral density category by postural scoliosis postural (n=213) most of the subjects (54.0%) had postural scoliosis, while 57.7% was unable to perform horzontal contraction of the erector sipnae muscles for 30 seconds. the majority of bmd scores of the subjects (46.9% of all subjects) was in the osteopenia category. in subjects with postural scoliosis, the p r o p o r t i o n o f o s t e o p o r o s i s ( 4 8 . 7 % ) w a s significantly higher than that of subjects without postural scoliosis (31.6%) (p=0.041) (table 2). the data on the relationship between erector spinae endurance and postural scoliosis is shown in table 3. t h e r e w a s a s i g n i f i c a n t a s s o c i a t i o n between erector spinae muscle endurance and postural scolisosis. in subjects with high erector spinae muscle endurance (> 30 seconds), the risk of postural scoliosis was 0.76 (95% confidence interval 0.58 – 0.99) (p=0.0351) which was lower than that in subjects with poor erector spinae muscle endurance (table 3). discussion our study shows that the majority of postmenopausal women have postural scoliosis, which is similar to the results of a previous study conducted by birkes et al., who found an increased scoliosis prevalence in individuals older than 60 years. our results are also similar to those of schwab et al., showing a mean cobb’s angle of 17º at age 70.5 years.(14,19) differing results were found in a study involving 380 postmenopausal women aged 50 years and older, where the prevalence of lumbar scoliosis was 12.9%.(20) this difference between this study and ours was due to the differing methods used for the assessment of scoliosis. in the study by urrutia,(20) the presence or absence of scoliosis was based on lumbar curvature magnitude in the coronal plane, as measured in dxa images with cobb’s method. a lumbar curvature of 10° or above was taken as indicating the presence of scoliosis. in our study the prevalence of osteoporosis was higher in postmenopausal women with scoliosis, as compared to subjects without postural scoliosis. the results of a retrospective study of radiographs of 454 consecutive adult patients were consistent with our results, as they showed that scoliosis was common among the osteoporotic subjects and that lumbar scoliosis is a useful clinical marker for osteoporosis.(21) the study conducted by cheng et al. showed a l o w e r b m d i n s u b j e c t s w i t h i d i o p a t h i c scoliosis.(15) the amount of bone in the vertebral bodies is directly related to their load-bearing strength. in osteoporosis, the reduction in bone mass of the vertebral bodies may result in their failure *pr : prevalence ratio table 3. relationship between between erector spinae endurance and postural scoliosis (n=231) erector spinae endurance postural scoliosis pr* (95% c.i.) p yes no > 30 seconds 41 49 0.76 (0.580.99) 0,0351* < 30 seconds 74 49 69 under the load of the body, as they are essential for supporting the weight of the body .(22) epidemiological studies indicate that adults with osteoporosis or osteomalacia have a six-fold higher risk of scoliosis, which is associated with significant morbidity, including low back pain and radicular symptoms.(23) our study shows that there is a lower risk of postural scoliosis in subjects with high erector spinae muscle endurance in comparison with subjects low erector spinae muscle endurance. these results support those of a previous study conducted by kawahara et al. in patients with osteoid osteoma and scoliosis, indicating that the occurrence of scoliosis was associated with w e a k n e s s o f t h e e r e c t o r s p i n a e m u s c l e endurance.(24) another study on patients with progressive idiopathic scoliosis, who underwent electromyography (emg) examination, also found a higher degree of paravertebral muscle imbalance in these patients.(25) this study demonstrated that subjects with postural scoliosis have lower bone mineral densities and that the occurrence of postural scoliosis is influenced by weakness of the erector spinae muscles. erector spinae muscles of adequate strength exert strains and stresses on the spinal column, thus stimulating balanced b o n e r e m o d e l i n g a n d p l a y i n g a r o l e i n maintaining aligment of the spinal column and preventing postural scoliosis. a limitation of this study was the absence of an analysis of the relation between type of activity and sports habits of the subjcts and erector spinae endurance. conclusions postmenopausal women with postural s c o l i o s i s h a v e a s i g n i f i c a n t l y i n c r e a s e d prevalence of osteoporosis. high erector spinae muscle endurance reduces the risk of postural scoliosis postural. to reduce the risk of osteoporosis in postmenopausal women with scoliosis, it is recommended that postmenopausal women perform exercises for improving their posture and erector spinae muscle endurance. acknowledgements we hereby wish to express our profoundest gratitude to the dean of the faculty of medicine, trisakti university, who provided the funds for conducting this study, to the head of the mampang prapatan subdistrict health center, s o u t h j a k a r t a , f o r c o o r d i n a t i o n a n d accommodation during the study, and to the participants of this study for their cooperation. references 1. freedman rr. hot flash trends and mechanisms. menopause 2002;9:151-2. 2. etgen am, ansonoff ma, quesada a. mechanisms of ovarian steroid regulation of norepinephrine receptor mediated signal transduction in the hypothalamus: implications for female reproductive physiology. horm behav 2001;40:169-77. 3. recker r, lappe j, davies km, heaney r. bone remodeling increases substantially in the years after menopause and remains increased in older osteoporosis patients. j bone miner res 2004;19: 1628-33. 4. johnell o, kanis j. epidemiology of osteoporotic fractures. osteoporos int 2005;16 (suppl 2): s37. 5. seeman e, delmas pd. bone quality-the material and structural basis of bone strength and fragility. n engl j med 2006;354:2250-61. 6. raisz lg. pathogenesis of osteoporosis: concepts, conflicts, and prospects. j clin invest. 2005;115: 3318-25. 7. meiyanti. epidemiology of osteoporosis in postmenopausal women aged 47 to 60 years. univ med 2010;29:169-76. 8. national osteoporosis foundation. prevalence report. available at: http://www.nof.org/advocacy /resources/prevalencereport. accessed december 2, 2011. 9. bono cm, einhorn ta. overview of osteoporosis: pathophysiology and determinants of bone strength. eur spine j 2003;12 suppl 2:s90-6. 10. knutson ga, owens e. erector spinae and quadratus lumborum muscle endurance tests and supine leg-length alignment asymmetry: an observational study. j manipulative physiol ther 2005;28:575-81. 11. suden e, ereline j, gapeyeva h, pasuke m. low back muscle fatigue during sorensen endurance univ med vol. 31 no.1 70 rachmawati, sidarta, bastian osteoporosis in postural scoliosis test in patients with chronic low back pain: relationship between electromyographic spectral compression and anthropometric characteristics. electromyogr clin neurophysiol 2008;48:18592. 12. rachmawati mr. high erector spinae endurance reduced low back pain in postmenopausal women. univ med 2011;30:111-9. 13. greiga am, bennella kl, briggsa am, hodges pw. postural taping decreases thoracic kyphosis but does not influence trunk muscle electromyographic activity or balance in women with osteoporosis. man ther 2007; doi:10.1016/ j.math.2007.01.011. 14. schwab f, dubey a, gamez l, el fegoun ab, hwang k, pagala m, et al. adult scoliosis: prevalence, sf-36, and nutritional parameters in an elderly volunteer population. spine 2005;30:1082-5. 15. cheng jcy, qin l, cheung csk, sher ahl, lee km, ng swe, et al. generalized low areal and volumetric bone mineral density in adolescent idiopathic scoliosis. j bone miner res 2000;15:1587–95. 16. gross jm, fetto j, rosen e. musculoskeletal examination. 3rd ed. singapore: wiley-blackwell; 2009. 17. demoulin c, vanderthommen m, duysens c, crielaard jm. spinal muscle evaluation using the sorensen test: a critical appraisal of the literature. joint bone spine 2006;73:43-50. 18. kanis ja, burlet n, cooper c. european guidance for the diagnosis and management of osteoporosis in postmenopausal women. osteoporos int 2008;19:399-428. 19. birkes jk, white ap, albert tj. adult degenerative scoliosis: a review. neurosurgery 2008;63:a94-a103. 20. urrutia j, diaz-ledezma c, espinosa j, berven sh. lumbar scoliosis in postmenopausal women: prevalence and relationship with bone density, age, and body mass index. spine 2011;36:73740. 21. pappou ip, girardi fp, sandhu hs, parvataneni hk, cammisa fp jr, schneider r, et al. discordantly high spinal bone mineral density values in patients with adult lumbar scoliosis. spine 2006;31:1614-20. 22. leone a, guglielmi g, cassar-pullicino vn, bonomo l. lumbar intervertebral instability: a review. radiology 2007;245:62-77. 23. daffner sd, vaccaro ar. adult degenerative lumbar scoliosis. am j orthop 2003;32:77–82. 24. kawahara c, tanaka y, kato h, watanabe s, kokobun s. myolysis of the erector spinae muscles as the cause of scoliosis in osteoid osteoma. spine 2002;27:e313-15. 25. cheung j, halbertsma jpk, veldhuizen ag, sluiter wj, maurits nm, cool jc, et al. a preliminary study on electromyographic analysis of the paraspinal musculature in idiopathic scoliosis. eur spine j 2005;14:130-7. oktavianus 3 *health analyst, faculty of nursing and health, muhammadiyah university, semarang **department of nutrition, dr. kariadi hospital/ diponegoro university, semarang ***department of pathology, dr. kariadi hospital/ diponegoro university, semarang ****department of pharmacy, dr. kariadi hospital/ diponegoro university, semarang correspondence dr. budi santosa, s.km, m.si.med health analyst, faculty of nursing and health, muhammadiyah university, semarang jl. kedungmundu raya 18 semarang mobile: +6281805867211 email: budisantosa.unimus@gmail.com univ med 2015;34:3-9 doi: 10.18051/univmed.2016.v35.3-9 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background lead acetate (pb) inhibits heme biosynthesis through inhibition of δaminolevulinic acid dehydrogenase (δ-alad), copro porphyrinogen oxidase, and ferro chelatase. zinc supplementation increases lead-binding metallothionein proteins. the purpose of this study was to find evidence that zinc supplementation prior to lead exposure improves heme biosynthesis in rats. methods this was a randomized post-test only control-group design study involving 28 rats assigned to 4 groups (1 control and 3 treatment groups). the treatment groups were supplemented with zinc at doses of 0.2, 0.4, and 0.8 mg daily by gavage for 3 weeks. from week 4 to 13, all groups were exposed to lead 0.5 g/kg bw/day by gavage. at the end of week 13, δalad, erythrocyte protoporphyrin (epp), and heme concentrations were determined by means of elisa. one-way anova, followed by bonferroni’s test was used to analyse the data. results mean δ-alad concentrations decreased from the control group down to treatment group 3 (0.24 ± 0.20; 0.15 ± 0.15; 0.12 ± 0.11; 0.05 ± 0.06 ng/ mean per unit). mean epp concentrations decreased from the control group down to treatment group 3 (1.96 ± 0.50; 1.24 ± 0.24; 1.03 ± 0.05; 0.62 ± 0.16 ng/ml). mean heme concentrations increased from the control group up to treatment group 3 (8.07 ± 2.64; 10.11 ± 2.27; 10.04 ± 1.65; 11.41 ± 2.58 µm). anova followed by bonferroni showed that epp concentrations differed significantly between the control group and treatment group 3 (p=0.00). conclusion zinc supplementation prior to lead exposure improves heme biosynthesis in rats exposed to lead. keywords: zinc, δ-alad, epp, heme zinc supplementation improves heme biosynthesis in rats exposed to lead budi santosa*, hertanto wahyu subagio**, lisyani suromo***, and henna rya sunoko**** doi: http://dx.doi.org/10.18051/univmed.2015.v34.3-9 4 santosa, subagio, suromo, et al zinc improves heme biosynthesis suplementasi seng memperbaiki biosintesis heme pada tikus yang terpajan pb latar belakang plumbum (pb) asetat menghambat proses biosintesis heme melalui inhibisi enzim δ-alad, coproporfirinogen oksidase, ferrokhelatase. suplementasi seng meningkatkan protein metallothionein yang mengikat pb. tujuan penelitian ini untuk membuktikan suplementasi seng sebelum pajanan pb memperbaiki proses biosintesis heme pada tikus. metoda penelitian eksperimental dengan rancangan pra-pasca dengan menggunakan kontrol dilakukan pada 28 ekor tikus yang secara acak sederhana dibagi menjadi 4 kelompok yaitu 1 kelompok kontrol dan 3 kelompok perlakuan. pada kelompok perlakuan disuplementasi seng 0,2; 0,4; 0,8 mg setiap hari melalui sonde sampai minggu ke-3, awal minggui ke-4 kelompok kontrol dan perlakuan dipajan pb 0,5 gr/kg bb/hari melalui sonde sampai minggu ke-13. akhir minggu ke-13 diperiksa δ-alad, epp, heme menggunakan elisa. analisa data menggunakan kruskal wallis, kadar epp dan heme dengan uji one way anova, dilanjutkan bonferroni. hasil rerata δ-alad menurun mulai kelompok kontrol sampai perlakuan ke-3 (0,24 ± 0,20; 0,15 ± 0,15; 0,12 ± 0,11; 0,05 ± 0,06 ng rerata per unit). rerata epp menurun dari kelompok kontrol sampai perlakuan ke-3 (1,96 ± 0,50; 1,24 ± 0,24; 1,03 ± 0,05; 0,62 ± 0,16 ng/ml). rerata heme meningkat dari kelompok kontrol sampai perlakuan ke3 (8,07± 2,64; 10,11 ± 2,27; 10,04 ± 1,65; 11,41 ± 2,58 µm). uji anova diteruskan bonferroni menunjukkan epp berbeda bermakna antara kelompok kontrol dengan ke-3 kelompok perlakuan (p=0,00). kesimpulan suplementasi seng sebelum pajanan pb mampu memperbaiki biosisntesis pada tikus terpajan pb. kata kunci: seng, δ−alad, epp, dan heme abstrak introduction health problems may have various causes; one of these are heavy metals such as lead, which is a hazardous air pollutant. the environment can be polluted by lead through increased human acitivities, such as mining, smelting, combustion of gasoline, and the use of lead for commercial purposes.(1) some of the main sources of lead poisoning are vegetables, batteries, paints, cosmetics, jewelry, toys, gasoline, and others. in west and central java the increasing use of phosphate fertilizers, pesticides, and herbicides have the potential to increase lead exposure. the study conducted by shah f et al.(2) showed that in general anemia occurs in children between 15 years old, who have higher lead levels as compared with children without anemia. among children with blood lead levels of > 10 µg/dl there are 2.87-fold more anemia cases in comparison with children with blood lead levels of <10 µg/dl.(3) lead intoxication may occur through inhalation, ingestion of contaminated foods and drinks, and transdermally.(4) lead accumulates in the mitochondria, resulting in the predominant 5 effects of lead, i.e. abnormal heme biosynthesis and hematopoiesis.(5) lead binds and inhibits the sulfhydryl-rich enzymes involved in heme b i o s y n t h e s i s , i . e . δa m i n o l e v u l i n i c a c i d dehydrogenase (δ-alad) in the cytosol, and coproporphyrinogen oxidase and ferrochelatase in the mitochondria.(6) the inhibition of these enzymes in heme biosynthesis as a result of lead exposure occurs at the starting point, at midpoint, and at the endpoint of heme biosynthesis. deltaalad is the initial enzyme inhibited by lead. inhibition of δ-alad prevents the conversion o f δa m i n o l e v u l i n i c a c i d (δa l a ) i n t o porphobilinogen (pbg), causing increased δala and δ-alad concentrations in the blood. the enzyme inhibited at midpoint of heme biosynthesis is coproporphyrinogen oxidase, resulting in increased coproporphyrinogen concentrations. the last enzyme inhibited by lead in heme biosynthesis is ferrochelatase, causing increased protoporphyrin concentrations in the erythrocytes (free erythrocyte protoporphyrin/ epp). (7) t h e s e a b n o r m a l i t i e s o f h e m e biosynthesis result in a reduction in heme concentrations, leading to decreased hemoglobin concentrations and anemia. various attempts have been made to treat lead intoxication, such as the use of edta as chelating agent.(8) a chelating agent binds lead to form a polar (hydrophilic) complex that is excreted from the body through the kidneys. according to studies that have been conducted on the use of edta, this is a therapeutic or curative agent. however, edta treatment is far f r o m m a x i m a l , a n d i t i s a d m i n i s t e r e d intravenously. therefore preventive agents should be studied to reduce lead toxicity, one of these being metallothionein proteins, since these play important roles in poisoning by heavy metals, including lead.(9) metallothioneins are rich in sulfhydryl groups, which form covalent bonds with heavy metals such as lead. studies on metallothioneins have found correlations with zinc levels in the body.(10) the study by santosa et al.(11) showed that zinc supplementation in stepwise increasing doses, i.e. at 0.2 mg, 0.4 mg, and 0.8 mg daily to rats (rattus norvegicus) is capable of increasing metallothioneins. these d o s e s h a v e b e e n c a l c u l a t e d b y t a k i n g a conversion factor from humans to rats into consideration. preventive zinc supplementation has been proven to enhance hematopoesis by reducing basophilic stippling and hematocrit values in lead-exposed rats.(12) the mechanism of the prevention of lead intoxication by zinc through heme biosynthesis is by blocking enzyme action, competition between lead and zinc, and covalent bonding. the blocking mechanism is due to the binding of lead to enzyme/protein active groups, thus inactivating the enzyme. competition between l e a d a n d z i n c a t o m s m a y o c c u r f o r t h e metallothionein sulfhydryl groups. for each bond, the higher the cationic charge of the metal, the stronger the bond. the nearer the metal is to the noble metals the stronger its affinity. in the periodic system of elements, lead belongs to period vii and zinc to period iv. therefore, the cationic charge of lead is greater than that of zinc, favoring lead in the competition for the metallothionein sulfhydryl groups.(13) the covalent bonding of pb2+ with the metallothionein sulfhydryl groups is stable and irreversible. the formation of irreversible covalent bonds between l e a d a n d m e t a l l o t h i o n e i n c a u s e s h e m e biosynthesis to be disturbed by lead exposure. therefore zinc supplementation should be studied as a preventive alternative against inhibition of heme biosynthesis by lead exposure. the aim of this study was to evaluate the effect of zinc supplementation on heme biosynthesis in lead-exposed rats. methods study design the present study was carried out according to an experimental randomized posttest only control-group design. the time period from selection of experimental animals and their housing up to the interventions was 100 days, the study being conducted between december univ med vol. 34 no.1 6 santosa, subagio, suromo, et al zinc improves heme biosynthesis 2012 and march 2013. the study took place in the integrated research and testing laboratory (laboratorium penelitian dan pengujian terpadu, lppt), gadjah mada university, yogyakarta. experimental animals the experimental animals used were 28 15week old male black rats (rattus norvegicus. the sampled size was determined by means of the formula: sample size = (t – 1) (r – 1) > 15 where t = number of groups, r = number of experimental animals per treatment group. the study used 3 treatment groups and and 1 control group, so that t=4, (4-1) (r-1) > 15, r > 6. the number of rats used was 6 per group (3 treatment groups and 1 control group) so that total sample size to be used in this study was 24. to the number of rats per group, one animal was added as reserve, in anticipation of deaths, so that the total sample size comprised 28 rats. zinc supplementation zinc supplementtion was administered to the treatment groups in stepwise increasing doses of 0.2, 0.4, and 0.8 mg/day by gavage, whereas t h e c o n t r o l g r o u p d i d n o t r e c e i v e z i n c supplementation. the supplementation period was from the first week to the third week. the 7 animals per group were assigned by simple random sampling.(13) lead exposure lead exposure was by administration of 0.5g/kgbw/day (acute dose) for 10 weeks, from the third week to the thirteenth week. the amount of lead exposure was based on a previous study on the effect of lead acetate administration to rattus norvegicus.(14) lead exposure was given in the form of a solution of 0.5 g/ml. measurement of blood parameters for determination of heme biosynthesis, the parameters investigated were serum δ-alad, epp, and heme concentrations. this was performed by means of the elisa assay in the gaki laboratory, diponegoro university, semarang, for all experimental animals of the control and treatment groups on the last day of the thirteenth week. data analysis differences in δ-alad concentrations between control and treatment groups were analyzed by the kruskal-wallis test, whereas differences in epp and heme concentrations were tested by one way anova, followed by bonferroni’s test. ethical clearance we conducted this study after receiving ethical clearance from the ethics commission, faculty of medicine, diponegoro university/dr. kariadi hospital, semarang, under no. 339/ec/ fk/rsdk/2012. the head of the integrated research and testing laboratory, gadjah mada university, yogyakarta, permitted the study to b e c a r r i e d o u t a f t e r h a v i n g r e c e i v e d communications regarding the ethical clearance. results improvement of heme biosynthesis was determined from δ-alad, epp, and heme concentrations. the concentrations of δ-alad a n d e p p w e r e d e c r e a s e d w i t h z i n c supplementation, in comparison with the control group, whereas the heme concentrations were increased. the details of the mean differences and statistical test results for δ-alad, epp, and heme concentrations after the thirteenth week by treatment group are presented in tables 1 and 2 below. there was a difference in mean δ-alad concentrations between control and treatment groups. the highest mean was in the control groups (0.24 ± 0.20 ng/ml) and the lowest in the zinc iii treatment group (0.05 ± 0.06 ng/ ml). the kruskal-wallis test did not find any significant differences between control and treatment groups (p=0.16). 7 table 1. mean δ-alad, epp, and heme concentrations after the thirteenth week by treatment group there were differences in mean epp concentrations between the control and treatment groups. the highest mean (1.96 ± 0.50 ng/ml) w a s i n t h e c o n t r o l g r o u p w i t h o u t z i n c supplementation, and the lowest mean (0.62 ± 0.16 ng/ml) was in the treatment group receiving zinc supplementation at a dose of 0.8 mg. a n o va t e s t r e s u l t s s h o w e d s i g n i f i c a n t differences between control and treatment groups (p=0.00). there was no significant difference in mean heme concentrations (p=0.13). the results of bonferroni’s muliple comparison test showed significant differences in mean epp concentrations between the control group versus the zinc i, zinc ii and zinc ii treatment groups (table 2). discussion from the results of this study, there was a reduction in δ-alad concentrations in the treatment groups receiving zinc supplementation. however, laboratory investigations showed a downward trend in δ-alad concentrations, starting from the control group to the treatment groups of the lead-exposed rats. higher zinc doses resulted in lower δ-alad concentrations in the lead-exposed rats. lead can inhibit precursors of heme biosynthesis, such as δalad. the activity of δ-alad is disturbed by lead, causing δ-ala not to be converted into porphobilinogen so that there is an increase in δ-alad concentrations. these changes due to lead exposure are acute or toxic. further studies are needed on the possibility of compensatory mechanisms of the body in the form of increases in δ-alad concentrations to convert δ-ala into porphobilinogen. the elisa assay used in the present study was capable of measuring all δalad concentrations, both those from lead inhibition as well as those excreted by the body as a compensatory mechanism, so that δ-alad concentrations increased after lead exposure. lead exposure of rats that have previously received zinc supplementation resulted in decreased δ-alad concentrations. this may be because the metallothioneins that are formed will bind the lead, while the metallothionein-bound zinc is released and activates δ-alad. because of activation, δ-alad again functions normally, being capable of converting δ-ala into porphobilinogen, so that the body does no longer i n c r e a s e δa l a d c o n c e n t r a t i o n s i n compensation, so decreasing its concentrations. this δ-alad activation improves heme biosynthesis. several studies support our results stating that lead is capable of increasing δ-alad and some of these studies have been conducted on zinc and δ-alad.(14-16) the epp concentrations were reduced in the treatment groups receiving zinc supplementation in comparison with the control group that were only exposed to lead. this may be explained by the fact that the last enzyme to be inhibited by lead in heme biosynthesis is ferrochelatase. this table 2. results of bonferroni’s test for epp after thirteenth week by treatment group univ med vol. 34 no.1 8 santosa, subagio, suromo, et al zinc improves heme biosynthesis enzyme is present in the mitochondria and plays a r o l e i n t h e c o m p l e x i n g o f f e t o t h e protoporphyrin ring. ( 1 7 ) i n h i b i t i o n o f f e r r o c h e l a t a s e i n c r e a s e s e r y t h r o c y t e protoporphyrin/epp. several studies have found evidence for a correlation between lead exposure and erythrocyte protoporphyrin. lowry et al.(18) s t u d i e d t h e e ff e c t s o f l e a d e x p o s u r e o n cytochrome 450 (cyp) and found that lowintensity lead exposure had no effects on cytochrome 450 metabolism, but affected several pathways in hemoglobin synthesis. the epp concentrations increased on exposure to lead of >10 µg/dl. epp concentrations increased significantly in the exposed group (battery factory workers) as compared with controls.(15) shah f et al.(2) studied blood lead levels and the prevalence of iron deficiency anemia in children aged between 1 to 5 years, and found that children with severe iron deficiency anemia had higher lead levels in the blood as compared with children with mild iron deficiency anemia. there was a significant negative correlation between blood lead levels and iron deficiency anemia. these results are similar to those of zinc supplementation, but are based on different mechanisms. iron complexes with porphyrin to form heme, whereas zinc supplementation competes with lead for metallothionein sulfhydryl groups so that there is no accumulation of epp. in the present study, heme concentrations were low after lead exposure without zinc supplementation, whereas heme concentrations increased, although not significantly, after lead exposure with zinc supplementation. this proves t h a t z i n c s u p p l e m e n t a t i o n i s c a p a b l e o f increasing heme synthesis in lead-exposed rats. to increase heme concentrations significantly in lead-exposed rats, the zinc supplementation dose should be increased. zinc is an important micromineral in biological processes in the body. the role of zinc in heme biosynthesis is with regard to alad, which is a zinc-containing enzyme that plays a role in catalyzing the condensation of two molecules of ala into two molecules of water and one molecule of porphobilinogen (pbg). zinc deficiency inhibits this reaction and influences heme formation.(3) in heme synthesis, the second enzyme that plays a role is δ-alad. this enzyme is exceedingly rich in sulfhydryl groups with a stronger affinity for lead in comparison with zinc. full enzyme activity requires intact or complete sulfhydryl groups and one zinc atom for each subunit. enzyme activity is inhibited if the zinc is replaced by other metals, such as lead.(3) addition of zinc may reverse the inhibition of δ-alad, but this requires higher zinc concentrations.(14) according to santosa et al.(12) zinc supplementation may increase metallothioneins that are rich in sulfhydryl groups capable of binding lead, causing no interference with delta alad (3) and no inhibition of heme biosynthesis. various studies of the effect of lead exposure on heme biosynthesis have supported the results of the present study. several studies on lead exposure in battery factory workers showed increases in the heme biosynthesis parameters δ-alad and epp, but showed decreases in heme and hemoglobin concentrations.(18) lead exposure in 30 automobile workers caused increases in δalad, epp, porphobilinogen and decreases in heme and hemoglobin concentrations.(19) heme is an integral part of proteins involved in several electron transport chains for energy renewal that are found in nearly all forms of life. in addition, heme is a cofactor of enzymes, including catalase, peroxidase, cytochrome p450, and is a part of sensor molecules.(20) one limitation of this study was that the elisa assay for assessment of δ-alad concentrations will measure all forms of the enzyme, and cannot distinguish between leadinhibited and noninhibited forms. the clinical i m p l i c a t i o n o f t h i s s t u d y i s t h a t z i n c supplementation has been proved to reduce δalad and epp concentrations and to increase heme concentrations, indicating that zinc is capable of repairing heme biosynthesis in rats exposed to lead, so that further studies are necessary to apply these findings to humans. 9 conclusion zinc supplementation at doses of 0.4 mg and 0.8 mg significantly improves heme biosynthesis, marked by reduced δ-alad and epp concentratioins and increased heme concentrations. acknowledgments we thank the directorate-general of the department of higher education (dirjen dikti), ministry of education and culture (kemendikbud), for the funding of this study. references 1. markowitz m. lead poisoning. in: behrman re, kliegman rm, jenson hb, editors. nelson textbook of pediatrics. 18th ed. philadelphia: saunders; 2010.p.2913-7. 2. shah f, kazi tg, afridi hi, et al. environmental exposure of lead and iron deficit anemia in children age range 1-5 years: a cross sectional study. sci total environ 2010;408:5325-30. doi: 10.1016/j.scitotenv.2010.07.091. 3. ahamed m, singh s, behari jr, et al. interaction of lead with some essential trace metals in the blood of anemic children from lucknow, india. clin chim acta 2007;2:92-7. 4. hamilton wj, ledvina ps, lopez ra, et al. childhood lead poisoning in galveston, texas: background health effects hot spots – intervention. version 1. texas: baylor college of medicine; 2007. 5. manahan se. toxicological chemistry and biochemistry. 3rd ed. new york washington: lewis publishers; 2003. 6. world health organization. childhood lead poisoning. geneva: world health organization; 2010. 7. kabe y, ohmori m, shinouchi k, et al. porphyrin accumulation in mitochondria is mediated by 2oxoglutarate carrier. j biol chem 2006;281: 31729-35. 8. american academy of pediatrics, committee on environmental health. lead exposure in children: prevention, detection, and management. pediatrics 2005;16:1036-46. 9. zuo p, qu w, cooper rn, et al. potential role of a-synuclein metallothionein in lead-induced inclusion body formation. tox sci 2009;111:1008. 10. lee sm, mclaughlin jn, frederick dr, et al. metallothionein-induced zinc partitioning exacerbates hyperoxic acute lung injury. am j physiol lung cell mol physiol 2013;304:35060. 11. santosa b, subagio ws, suromo l, et al. zinc supplementation dosage variations to metallothionein protein level of rattus norvegicus. int j sci eng 2013;5:15-7. 12. santosa b, subagio hw, suromo l, et al. zinc supplementation decreases basophilic stippling in rats exposed to lead. univ med 2014;33:118. 13. krêzel a, hao q, maret w. the zinc/thiolate redox biochemistry of metallothionein and the control of zinc ion fluctuations in cell signaling. arch biochem biophys 2007;463:188-200. 14. hariono b. efek pemberian plumbum (timah hitam) pada tikus putih anorganik (rattus norvegicus). j sain vet 2006;24:125-33. 15. shaik ap, jamil k. a study on the alad gene polymorphisms associated with lead exposure. toxicol ind health 2008;24:501-06. 16. yang y, wu j, sun p. effects of deltaaminolevulinic acid dehydratase polymorphisms on susceptibility to lead in han subjects from southwestern china. int j environ res public health 2012;9:2326-38. 17. atamna h, killilea dw, killilea an, et al. heme deficiency may be a factor in the mitochondrial and neuronal decay of aging. pnas 2002;99: 14807-12. 18. lowry ja, pearce re, gaedigk a, et al. lead and its effects on cytochromes p450. j drug metab toxicol 2012;5:1-4. 19. dongre nn, suryakar an, patil aj, et al. biochemical effects of lead exposure on systolic & diastolic blood pressure, heme biosynthesis and hematological parameters in automobile workers of north karnataka (india). ind j clin biochem 2011;26:400-6. 20. heinemann iu, jahn m, jahn d. the biochemistry of heme biosynthesis. arch biochem biophys 2008;474:238-51. univ med vol. 34 no.1 central obesity increases the risk of type 2 diabetes mellitus among urban adults 187 *public health intervention technology center, hrda-moh ri **clinical epidemiology and applied health technology center, hrdamoh ri correspondence: dr. dr. julianty pradono, ms. jl.dewi sartika no.292 jakarta timur mobile: +62812 100 4523 email: jpradono@yahoo.com univ med 2015;34:187-96 doi: 10.18051/univmed.2016.v35.187-196 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background in indonesia the number of persons with type 2 diabetes mellitus (t2dm) was 8.5 million in 2013, and is estimated to be 11.8 million in 2030. the aim of this study was to determine the major risk factors associated with t2dm prevalence in urban aged >15 years. methods an observational study with cross-sectional design was used with the data from riskesdas 2007 and 2013 on population aged >15 years. total respondents were 294,352 (2007) and 368,281 (2013). data were collected by trained personnel through interviews, blood pressure and anthropometry measurements, and blood glucose and lipid tests. odds ratio (or) was used to test the relationship between t2dm and a number of other variables. multiple logistic regression analysis was obtained to determine the main risk factor associated with t2dm prevalence. results type 2 diabetes mellitus prevalence increased from 2.3% (2007) to 2.5% (2013). t2dm tended to be increased in the younger age groups. low education, middle-to-high economic status, less physical activity, smoking >200 cigarettes, and high risk blood lipid levels were strongly associated with t2dm prevalence. the main risk factors were inadequate physical activity (2007: or 1.9;95% ci:1.16–2.98 and 2013: or 2.44;95% ci: 1.57-3.78) and central obesity (2007: or 1.8;95% ci:0.99–3.10, and 2013:or 3.84; 95% ci: 2.49-5.93) after controlling for age, gender, employment and economic status. conclusions type 2 diabetes mellitus prevalence in the population aged >15 years increased within 5 years. lack of physical activity and central obesity were the major risk factors of t2dm prevalence in urban adults. keywords: physical activity, central obesity, type 2 diabetes mellitus, adults, urban areas central obesity increases the risk of type 2 diabetes mellitus among urban adults julianty pradono*, nunik kusumawardani*, and delima** doi: http://dx.doi.org/10.18051/univmed.2015.v34.187-196 188 pradono, kusumawardani, delima central obesity risk diabetes mellitus obesitas sentral meningkatkan kejadian diabetes melitus tipe 2 pada orang dewasa di perkotaan latar belakang di indonesia jumlah penduduk dengan diabetes melitus tipe 2 (dm2) adalah 8,5 juta orang pada tahun 2013, dan diperkirakan menjadi 11,8 juta pada tahun 2030. tujuan penelitian ini untuk mendapatkan faktor risiko utama yang berhubungan dengan kejadian dm pada penduduk umur >15 tahun di perkotaan. metode penelitian observasional dengan desain potong lintang digunakan dengan data riskesdas 2007 dan 2013 pada penduduk umur >15 tahun. jumlah sampel sebanyak 294.352 (2007) dan 368.281 responden (2013). pengumpulan data oleh enumerator terlatih melalui wawancara, pengukuran tekanan darah, antropometri, dan pemeriksaan darah (kadar glukosa darah dan lipid darah). odds rasio (or) digunakan untuk menetukan hubungan antara t2dm dan beberapa variabel. regresi logistik ganda digunakan untuk menentukan faktor risiko utama yang mempunyai hubungan dengan kejadian dm2. hasil prevalensi dm2 meningkat dari 2,3% (2007) menjadi 2,5% (2013). ada kecendrungan peningkatan dm2 pada umur lebih muda. penduduk dengan pendidikan rendah, status ekonomi mampu, kurang aktivitas fisik, merokok >200 batang, dan kadar lipid darah berisiko mempunyai hubungan kuat dengan kejadian dm2. sedangkan faktor risiko utama adalah kurang aktivitas fisik (2007: or 1,9;95% ci:1,16–2,98 dan 2013: or 2,44;95% ci: 1,57-3,78) dan obesitas sentral (2007: or 1;8; 95% ci:0,99–3,10, dan 2013: or 3,84;95% ci: 2,49-5,93) setelah dikontrol oleh umur, jenis kelamin, pekerjaan dan status ekonomi. kesimpulan terjadi peningkatan prevalensi dm2 pada penduduk umur >15 tahun dalam kurun waktu 5 tahun di perkotaan. kurang aktivitas fisik dan obesitas sentral merupakan faktor risiko utama dari kejadian dm2 pada orang dewasa di perkotaan. kata kunci: aktivitas fisik, obesitas sentral, diabetes melitus tipe 2, orang dewasa, perkotaan abstrak introduction the number of persons with type 2 diabetes mellitus (t2dm) is expected to become a burden in adults, particularly in especially developing countries. in 2013, there were 382 million people with diabetes, and this number is projected to become 592 million in 2035. the majority (77%) of people with diabetes live in low and middle income countries.(1) the international diabetes federation (idf) estimates that in 2030 a total of 398 million (7.1%) will have impaired glucose tolerance (igt) and that dm will cause 4.9 million deaths in 2014, or that every seven seconds one person will die from dm.(2) i n t h e a s i a p a c i f i c r e g i o n , t 2 d m constitutes a health problem. undiagnosed cases or case with delayed diagnosis and low levels of monitoring are frequently associated with i n c r e a s e d r i s k o f m i c r o v a s c u l a r a n d c a r d i o v a s c u l a r d i s e a s e , d i s a b i l i t i e s , a n d premature deaths.(3,4) 189 univ med vol. 34 no.3 although indonesia does not belong to the 10 countries with the highest diabetes prevalence in the world, as a country with a large population, in 2013 it was number 7 in rank, with 8.5 million persons with t2dm, which is projected to become 11.8 million in 2030.(5) the results of the basic health research (riset kesehatan dasar, riskesdas) of 2007 and 2013 showed that the prevalence of t2dm in indonesia, as obtained by interviews, also tended to increase from 1.1% (6) to 2.1%.(7) diabetes mellitus is a life-long chronic metabolic disease that is a risk factor for cardiovascular disease, a major cause of death. uncontrolled diabetes mellitus results in various complications, such as diabetic foot, neuropathy, retinopathy, chronic renal disease, coronary heart disease, and stroke.(8) the results of several studies have indicated a number of unmodifiable and modifiable factors that increase the risk of dm. unmodifiable risk factors include ethnicity, family history of diabetes, age, history of delivery of infants of >4kg body weight, and history of gestational dm. modifiable risk factors comprise overweight, low physical activity level, hypertension, dyslipidemia, and unhealthy diet (high in sugar and low in fiber).(6,9) awareness of the increased incidence of t2dm and measures to prevent t2dm by controlling the risk factors need to be promoted. for this purpose, the prevalence and the determining risk factors of t2dm should be continuously investigated and analyzed from year to year. to date no information was available on studies showing a tendency of increased t2dm incidence in indonesia adults residing in urban areas. the objective of this study was to determine a tendency of increased prevalence of t2dm and its determining risk factors in urban adults. methods research design this research was part of the riskesdas 2007 and 2013 studies. this was a noninterventional cross-sectional study conducted from september to november 2007 and from august to october 2013. study subjects the respondents were household members aged >15 years, residing in urban areas, of households selected as samples in riskesdas 2007 or riskesdas 2013. the inclusion criteria were residents >15 years old, males and females, agreeing to be interviewed and to be subjected to blood sampling. the number of respondents fullfilling the inclusion criteria in 2007 and 2013 were 294,352 and 368,281 respectively. interviews interviews were performed using a questionnaire comprising items on sociodemographic characteristics, past history of disease, and high risk behavioral factors. the questions focused on the respondents having been diagnosed by health personnel as suffering from t2dm, on the t2dm symptom triad (polyphagia, polydipsia, and polyuria) and on current use of antidiabetics or insulin injections. measurements of the anthropometrical measurements, weight was measured in “kg” using personal scales, and height was measured in “cm” using a stadiometer. abdominal circumference was measured in “cm” using a measuring tape. body mass index (bmi) was calculated from the formula: bmi (kg/m2) = weight (kg)/[height (m)2]. blood pressure was measured using a digital sphygmomanometer. laboratory analysis blood samples from the cubital vein were drawn after the respondents had fasted for 10-12 hours. fasting and 2-hour postload blood glucose concentrations were determined by electroenzymatic methods. blood lipid concentration was determined at the health research and development agency (hrda, balitbangkes) integrated laboratory, using the 190 pradono, kusumawardani, delima central obesity risk diabetes mellitus cobas 6000 roche analyzer and the c501module kit on enzymatic colorimetric principles.(10,11) blood examinations consisted of fasting glucose measurement and determination of blood glucose 2 hours after loading with a solution of 75 g glucose in 250 ml water, total cholesterol, high density lipoprotein (hdl), low density lipoprotein (ldl), and triglyceride concentrations. diabetes mellitus type 2 was diagnosed according to the 2011 guidelines of the american diabetes association (ada).(12) the t2dm diagnostic criteria were considered to be met if: 1) random plasma glucose concentration was >200 mg/dl and the classical symptoms of t2dm were present, such as polyuria, polydipsia, polyphagia, and unexplained weight loss; 2) fasting plasma glucose of >126 mg% with classical t2dm symptoms; 3) 2-hour postload glucose >200 mg/dl. blood lipid concentration was referenced to the normal values according to the national cholesterol education program (ncep)-adult treatment panel (atp) iii.(13) normal total cholesterol was taken as <200 mg/dl, and abnormal total cholesterol concentrations included the criteria borderline (200-239 mg/dl) and high (>240 mg/dl). in the analysis, the limit of 240 mg/dl was taken. the normal hdl value in males and females was >40 mg/dl and the optimal ldl value was <100 mg/dl. based on the reference, the category of near optimal/above optimal was 100-129 mg/dl, borderline high was 130-159 mg/ dl, high had a value of 160-189 mg/dl, and very high had a value of >190 mg/dl. in the analysis, ldl values were categorized as <160 mgdl and >160 mg/dl. the normal triglyceride value was <150 mg/dl. there were several triglyceride categories, i.e. borderline high with the values of 150-199 mg/dl, high with 200-499 mg/dl and very high with >500 mg/dl. in the analysis, the triglyceride limits were taken as <200 mg/dl and >200 mg/dl. data analysis data analysis was performed descriptively to describe the prevalence distribution of t2dm based on several variables. the odds ratio was used to test for a relationship between t2dm and a number of other variables. furthermore, multiple logistic regression analysis was used to obtain models of t2dm risk factors. ethical clearance the conduct of riskesdas 2007 and riskesdas 2013 had obtained ethical approval from the ethical commission of the health research and development agency, ministry of health, republic of indonesia (nhrd-moh ri). results t h e p r e v a l e n c e o f t 2 d m b a s e d o n symptoms (dm triad) and blood glucose testing (fasting and 2 hour postload) in persons aged >15 years increased from 2.3% in 2007 to 2.5% in 2013. the prevalence of t2dm increased with advancing age up to 55-64 years, then tended to decrease up to 75 years or above. this was found in both the findings of the year 2007 and 2013. when comparing these two studies, there was a tendency for the t2dm prevalence to increase, particularly in the younger age groups. the prevalence of t2dm was higher in persons with low education and middle-to-high economic status (quintiles 3-5) in comparison with high education and low economic status (quintiles 1-2). working males had more f r e q u e n t l y t 2 d m i n c o m p a r i s o n w i t h nonworking females in 2007, but the opposite occurred in 2013 (table 1). the prevalence of t2dm was higher in persons with inadequate physical activity, in smokers with a brinkman index of 200 cigarettes or more, in ex-smokers, consumers of fruits and vegetables of 3 servings or more per day, consumers of sweets and consumers of coffee of less than once daily in 2007 and 2013. on the other hand, the prevalence of t2dm was higher in 2007, but lower in 2013, in persons who consumed fatty foods once daily or more (table 1). 191 table 1. relationship between t2dm and sociodemographic characteristics and behavior of respondents or = odds ratio; t2dm = type 2 diabetes mellitus in general, higher fasting and 2-hour postload blood glucose concentrations were strongly associated with the incidence of t2dm. in 2007 no fasting blood glucose concentrations were collected, so that this finding could not be compared. highrisk blood lipid concentrations (total cholesterol, hdl, ldl, triglycerides) had a strong association with the incidence of t2dm univ med vol. 34 no.3 192 pradono, kusumawardani, delima central obesity risk diabetes mellitus table 2. relationship between t2dm and biological risk factor or : odds ratio; t2dm : type 2 diabetes mellitus in the studies of 2007 and 2013. the prevalence of total cholesterol and ldl versus t2dm showed an increase in comparison with 5 years previously (2007). similar, in the obese, body mass index and abdominal obesity carried a higher risk than in the non-obese. the prevalence of hypertension and mental-emotional disturbances in persons with t2dm increased within the 5year period (table 2). riskesdas 2007 showed that the factors associated with t2dm, in addition to age, also comprised inadequate physical activity, consumption of sweets, and central obesity, after controlling for the variables gender, employment, and economic status. higher age was strongly associated with incidence of t2dm. persons with inadequate physical activity had a 1.9 fold higher risk (95% ci:1.16-2.98) for t2dm, in comparison with those with adequate physical activity. persons with central obesity had a 1.8 fold higher risk (95% ci:0.99-3.10) in comparison with those without central obesity. the models had a 97.9 percent concordance with the true classification (table 3). the factors associated with t2dm in riskesdas 2013 showed a 97.5 percent concordance with the true classification, except for inadequate physical activity with or 2.4 (95% 193 table 3. determining factors of t2dm prevalence in 2007 *adjusted odds ratio controlled for variables in this table ci: 1,57-3,78) and central obesity with or 3.8 (95% ci: 2.49-5.93) (table 4). active smokers and ex-smokers had a 4.3 fold higher risk (95% ci: 2.63-6.96) of suffering from t2dm than non-smokers. persons with emotional disturbances had a 3.4 fold higher risk (95% ci: 2.03–5.57) of suffering from t2dm than those without mental emotional disturbances. persons with inadequate physical activity (less active) had a 2.4 fold higher risk (95% ci 1.573.78) than those with adequate physical activity. persons with ldl cholesterol concentrations of >200 mg/dl had a 1.9 fold higher risk (95% ci 1.08– 3.38) than those with ldl cholesterol concentrations of <200 mg/dl. persons who smoked >200 cigarettes had a 1.7 fold higher risk (95% ci 1.06-2.56) than those who smoked <200 cigarettes. persons with hypertension had a 1.5 fold higher risk (95% ci 0.99-2.37) than those without hypertension. persons with fasting blood glucose concentrations of 100-125 mg/dl had a 2.5 fold higher risk (95% ci 1.45–4.23), but those with fasting blood glucose concentrations of >126 mg/dl had a 9.6 higher risk (95% ci 5.17-17.85), whereas persons with 2-hour postload blood glucose concentrations of >200 mg/dl had a 2.4 fold higher risk (95% ci 1.32-4.34), after controlling for the variables age, gender, and economic status. discussion diabetes mellitus is a health problem that has most rapidly developed as a consequence of changes in life style. the prevalence of t2dm based on symptoms and blood glucose testing in persons aged >15 years within a period of 5 years increased by 0.2% (2.3% in 2007 vs 2.5% in 2013). the results of analysis showed that the determining factors of t2dm incidence are inadequate physical activity and central obesity in both 2007 and 2013. in the bivariate analysis of the 2007 data, the incidence of t2dm had a significant association with bmi scores of >27 table 4. determining factors of t2dm prevalence in 2013 *adjusted odds ratio controlled for variables in this table univ med vol. 34 no.3 194 pradono, kusumawardani, delima central obesity risk diabetes mellitus kg/m2, smokers with a brinkman index of >200 cigarettes, ldl cholesterol concentrations of >200 mg%, emotional disturbances, and hypertension, but had no significant association in the multivariate analysis. however, a cohort study showed that central obesity was not a risk factor for the o c c u r r e n c e o f n e w o n s e t t 2 d m . ( 1 4 ) t h e occurrence of new onset t2dm consists of increases in positive components of the metabolic syndrome. even accumulation of triglycerides and free fatty acids may be associated with insulin resistance and beta cell dysfunction, so that monitoring of the blood glucose and triglyceride concentrations is very important in patients with t2dm, particularly those with central obesity.(15) as a developing country with increasingly improved conditions, changes in dietary structure and life style result in altered food consumption, with more high caloric and high fat foods, while the requirement for physical exertion in occupational and daily activities has been reduced. these factors have led to increased obesity, thus causing a serious increase in the risks of comorbidity such as t2dm, heart disease, certain cancers, and shorter life expectancy. longitudinal studies are in support of the fact that a diet high in fat and calories and inadequate physical activity are risk factors for obesity and that the diabetes incidence of 8095% may be associated with obesity with fat distribution in the abdominal region. however, there are still many people who tolerate obesity. the underlying mechanism of genetic diversity i n t h e o x i d a t i o n o f s u b s t r a t e s i s p o o r l y understood, and may explain the occurrence of inter-individual variability in weight gain from l i f e s t y l e . ( 1 6 ) t h e n e g a t i v e i n f l u e n c e o f urbanization (living in metropolitan areas, having at least one car) on the level of activity, thus increasing the risk of t2dm, has been demonstrated in a lebanese study in adults.(17) in addition, the significantly decreased physical activity in the older age groups and in females does not reach the recommended targets, in comparison with males.(18) a study in china showed that the prevalence of diabetes increased with increasing age and weight on the basis of body mass index. the prevalence of diabetes was also found to be higher in the u r b a n t h a n i n t h e r u r a l p o p u l a t i o n . ( 1 9 ) furthermore, the cognitive factor of emotion, which affects dietary intake, may explain why some individuals are able to survive while having obesity.(20) whatever the reasons associated with it, obesity has contributed to non-communicable disease, especially diabetes mellitus.(21) a study of the national institutes of health in the us found that moderate activity (30 minutes daily per week) or vigorous exercise (20 minutes three times weekly) is associated with overall reductions in the risk of death by 27% and 32%, respectively. the study concluded that regular physical activity is the most important part of diabetes management planning and this finding underlines the importance of the regular application of physical activity as part of life style measures.(22) blomster et al.(23) in their study concluded that moderate and vigorous activity are significantly associated with the reduction of cardiovascular and microvascular incidence, and of all-cause mortality in patients with t2dm. another study showed a significant association of physical activity with age, gender, and ethnicity. with increasing age the level of physical activity was lower than recommended. females (49%) had less physical activity than recommended, in comparison with males (62.4%).(19) a cohort study involving 6,997 respondents showed that metabolic dysfunction increased the risk of t2dm. in the group with normal weight the risk was 4.7 fold higher, in the group with overweight the risk was 8.5 fold higher, and in the group with obesity 16.3 fold higher, in comparison with the group with normal weight without metabolic dysfunction.(24) one of the limitations of this study was its cross-sectional design, which takes a momentary snapshot of the conditions of the subjects, so that it cannot as yet provide an answer to the causes and effects 195 of central obesity in increasing the incidence of t2dm in urban adults. conclusions within a five-year period there was an increase in the prevalence of t2dm. the major risk factors associated with the prevalence of t2dm was central obesity. this may be of note in the institution of preventive and promotive measures to inhibit or prevent t2dm events, particularly in urban areas. acknowledgements we wish to express our gratitude to the head and secretariat of the health research and development agency, ministry of health, republic of indonesia [badan penelitian dan p e n g e m b a n g a n k e s e h a t a n , k e m e n t e r i a n kesehatan ri] who provided us with the opportunity to perform further analyses of the data from riskesdas 2007 and 2013. thanks are also due to the data laboratory for providing the data for analysis. conflict of interest the authors had no conflict of interest to declare in the conduct of this study. references 1. guariguata l, whiting dr, hambleton i, et al. global estimates of diabetes prevalence for 2013 and projections for 2035. diabetes res clin pract 2014;103:137-49. 2. international 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10.1186/1471-245813-1002. 18. baldew ss, krishnadath is, smits cc, et al. self-reported physical activity behavior of a multi-ethnic adult population within the urban and rural setting in suriname.bmc public health 2015;15:485. doi: 10.1186/s12889-0151807-1. 19. yang w, lu j, weng j, et al. china national diabetes and metabolic disorders study group: p revalence of diabetes among men and women in china. n engl j med 2010;362:1090-101. doi: 10.1056/nejmoa0908292. 20. galgani j, ravussin e. energy metabolism, fuel selection and body weight regulation. int j obes 2008;32:s109–19. doi: 10.1038/ijo.2008.246. 21. australian institute of health and welfare. overweight and obesity;2015.available at : http:/ /www.aihw.gov.au/overweight-and-obesity. accessed april 7, 2005. 22. zethelius b, gudbjornsdottir s, eliasson b, et al. level of physical activity associated with risk of cardiovascular diseases and mortality in patients with type-2 diabetes: report from the swedish national diabetes register. eur j prev cardiol 2013;2047487313510893. 23. blomster ji, chow c k, zoungas s, et al. the influence of physical activity on vascular complications and mortality in patients with type 2 diabetes mellitus. diabetes obes metab 2013; 15:1008–12. 24. franssens bt, van der graaf y, kappelle lj, et al. body weight, metabolic dysfunction, and risk of type 2 diabetes in patients at high risk for cardiovasculare events or with manifest cardiovascular disease: acohort study. diabetes care 2015;38:1945-51. c:\users\user\documents\41 no 2 219 abstract universa medicina knowledge levels of fetal rights in turkish pediatric nurses selen ozakar akca1*, ahu pinar turan1, and dilek kalkan yalcin2 background fetal rights are violated due to acts such as negligence, ignorance, false beliefs and substance addiction. pediatric nurses, as defenders of the fetus, should inform pregnant women and the family about the rights of the fetus and assist the family in the decision-making process. this study aims to determine the knowledge levels of pediatric nurses on fetal rights. methods this cross-sectional study involved 121 nurses working in the pediatric clinic of a training and research hospital. data about characteristics of pediatric nurses and knowledge levels about fetal rights were collected. statistical analyses were made with spss package program. mann whitney u test and kruskal wallis test was used to analyse the data. results in this study, it was found that 76.9% of the pediatric nurses participating in the study did not have prior knowledge about fetal rights. there was a statistically significant difference between the participants’ mean scores of knowledge levels about fetal rights and their age, educational status, status of prior knowledge about fetal rights and where this knowledge was gained (p<0.05). conclusion as a result, it was found that most of the pediatric nurses did not have prior knowledge about fetal rights, and that their knowledge about fetal rights was lacking. considering that pediatric nurses, as the defenders of the fetus, have a duty to inform the family about fetal rights, it is recommended to plan trainings on fetal rights for pediatric nurses. keywords: fetus, rights, pediatric nurse, education, birth original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.219-227 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1345 1hitit university faculty of health sciences, department of child health and diseases nursing. corum, turkey. 2 çorum private elitpark hospital, department of gynecology and obstetrics. corum, turkey *correspondence: selen ozakar akca. hitit university faculty of health sciences, corum, turkey email:selenozakar@hotmail.com, selenozakar@gmail.com, selenozakar@hitit.edu.tr phone: +903642230730; fax: +9036422230731 orcid id: 0000-0002-6943-6713 date of first submission, may 18, 2022 date of final revised submission, august 28, 2022 date of acceptance, september 2, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: ozakar akca s, turan ap, kalkan yalcin d. knowledge levels of fetal rights in turkish pediatric nurses. univ med 202 2;41 :2 1 9 -2 2 7 . doi : 10 .18 05 1/ univmed.2022.v41.219-227. september-december 2022 vol.41no.3 220 ozakarakca, turan, kalkan yalcin fetal rights introduction the rights of the fetus, based on the right to live, include the rights to life, health, nutrition, shelter and protection.(1) fetal rights are violated due to acts such as negligence, ignorance, false beliefs and substance addiction.(2,3) in addition, the fetus might be harmed by the pathological conditions that occur in the fetus before birth and the failure to perform the diagnosis and treatment required for the health of the fetus or by the family who do not want the baby(2,4), which leads to the rise of questions such as “are there fetal rights? does the pregnant woman have the right to terminate the pregnancy? can a pregnant woman terminate the pregnancy at any time? until when can a pregnancy be legally terminated? is there a penalty for illegal actions to terminate a pregnancy?”(5,6) although many rights, especially human rights, are guaranteed in the world with laws and codes, there is no legal regulation directly related to the rights of the fetus. however, there are legal decisions on the position of the fetus.(7) the decisions made by the legal system to protect the fetus are important for the rights of the fetus. these decisions differ from country to country. in the canadian legal system, the pregnant woman is not held responsible for the damage she causes to the fetus.(1) in contrast, while the australian and uk legal systems do not accept the fetus as an individual and do not give the fetus a right in the prenatal period, those who cause fetal harm with live birth are held legally responsible for this.(8) according to the “congenital disabilities act” enacted in england in 1976, the baby can sue those who harm him or her after birth and can be taken from his or her family for protection purposes if deemed necessary.(1,8) in the united states of america, the status of being held responsible for the harm caused to the fetus differs according to states.(8) in turkey, it is understood from the entailments of articles in the turkish civil code and turkish penal code that the fetus is taken under protection from the very first days of the pregnancy.(9-11) the first person a pregnant woman encounters and gets information from about prenatal care is the nurse.(12) for this reason, fetal rights come to the fore intensely in the field of nursing, which causes nurses from time to time to come into conflict with the rights of the fetus. although the fetus has the right to live and be treated, both the family and the nurses should be able to make ethical decisions in case of possible dilemmas.(13) pediatric nurses as defenders of the fetus, should inform pregnant women and the family about the rights of the fetus and assist the family in the decision-making process.(12) a study to assess the knowledge regarding fetal well-being among the staff nurses working in obstetrics and gynecological wards showed that 72.5% of staff nurses have average knowledge and 15% have poor knowledge regarding assessment of fetal well-being.(14) a quasi-experimental study showed that after participants completed a prenatal genetic nursing education program (pgnep) workshop, the mean score of total information needs about prenatal genetic testing and nursing (i-pgtn) decreased from 95.00 ± 8.86 to 91.53 ± 15.16. but this change was not statistically significant (p>0.05).(15) however, current nursing education does not sufficiently prepare nurses for the full range of competencies needed for effective prenatal fetal well-being.(16) in this direction, the purpose of this study was to determine the level of knowledge of pediatric nurses on fetal rights and to form the basis for training programs on fetal rights. methods research design the study was of cross-sectional design and conducted in the pediatric clinic of a training and research hospital in corum, turkey from october 2020 to march 2021. research subjects the study subjects were composed of pediatric nurses working in the pediatric clinic of a training and research hospital in corum, 221 turkey. no sample selection was made and the sample consisted of nurses (n=121), who were working between the dates (15.10.20/15.03.21) of the study and participated voluntarily. in the study, it was aimed to reach the whole population, so no sample size calculation was performed. the study was completed with 121 nurses. the inclusion criteria were being a pediatric nurse, accepting to participate in the study, not having a problem that would prevent communication, and willingness and volunteering to participate in the study. nurses who did not work in the pediatric clinic were excluded. data collection tools the data were obtained through a data collection form developed by the principal investigator in line with the literature and expert opinions.(1,7,10,11) the data collection form consists of two parts. in the first part, there are 7 q ue s t i on s to d e t e r mi ne t h e de s c r i pt i ve characteristics of pediatric nurses, and in the second part there are 8 statements to determine the pediatric nurses’ knowledge levels about fetal rights. the questions about fetal rights were as follows: 1. turkey has legislations directly dealing with the fetal rights; 2. it is legal to terminate the pregnancy within 10 weeks, depending on the mother ’s conse nt; 3. when anomalies i nc omp a t ibl e wi t h l i f e a r e e nc o un t e r e d , termination of pregnancy between 10-22 weeks of gestation is medically, legally and ethically appropriate; 4.no one has the right to take away the right of the fetus to be delivered alive and healthy, at the right time and with the right method after the 22nd gestational week, for any reason; 5. any person who performs an abortion of a child upon a woman without her consent shall be sentenced to a penalty of imprisonment for a term of five to ten years; 6. a person who, in the absence of medical necessity, performs an abortion of a child, upon a woman, who is more than ten weeks pregnant and with her consent, shall be sentenced to imprisonment for a term of two to four years; 7. where a woman is pregnant due to an offence that she was a victim of, no penalty shall be imposed upon any person who terminates such pregnancy, where the term of pregnancy is not more than 20 weeks and there is consent from the woman; however this requires the termination of a pregnancy by expert doctors in a hospital; 8. a woman who willingly aborts a child where the term of pregnancy is more than ten weeks shall be sentenced to a penalty of imprisonment for a term up to one year and a judicial fine. those who gave a correct answer to each statement to determine the knowledge level of the participants about fetal rights were given 1 point, and 0 point was given to those who answered incorrectly or answered “do not know”. the knowledge levels of the participants were calculated by adding these scores. a maximum of 8 points and a minimum of 0 point can be obtained from these statements. a high score indicates that the nurse has a high level of knowledge about fetal rights. content validity and non-additive analyses were conducted regarding the validity of the form for determining the knowledge level of pediatric nurses about fetal rights. in order to evaluate the content validity of this form, it was submitted to the opinion of 5 experts in the field of child health and diseases nursing. the experts were asked to rate the questions as “item appropriate”, “item not appropriate” and “item should be revised”. necessary changes were made in the form in line with the experts’ opinions, the data collection tool was made ready for application, and reliability analyses of the questionnaire and noncollectibility analyses were carried out for 8 items determined by the experts. the additiveness of the questionnaire was tested using tukey nonadditive analysis of variance. according to the results of the analysis of variance, 8 items are additive and there is a significant difference in t he c ha nge b e t we en t he me a su r e me n t s (p<0.001).(17,18) according to these results, the lowest and the highest score that can be obtained from the 8-item form is 0 and 8, respectively. univ med vol. 41 no. 3 222 ozakarakca, turan, kalkan yalcin fetal rights a b data collection the data of the study were collected from pediatric nurses working in a training and research hospital between october 2020 and march 2021 and agreeing to participate in the study. the study was explained to the participants by the researchers and written informed consent statements were obtained from the nurses. data collection forms were distributed to the pediatric nurses in october 2020 and the nurses were asked to fill in the data collection forms in 10-15 minutes in the hospital environment. the research data were collected by face-to-face interviews under the supervision of the researchers in a total of five days. ethical dimension of the study in order to conduct the study, approval was obtained from the ethics committee (30.09.2020/ 2020-96) and written permission was obtained from the training and research hospital, the institution where the study was planned to be conducted. informed consent statements were obtained from the nurses who agreed to participate in the study upon informing them about the purpose of the study and the confidentiality of the information they gave. statistical analysis statistical analyses were made with the spss package program. numerical variables were presented as mean and standard deviation, and categorical variables as numbers and percentages. in statistical analysis, normal distribution of data for test selection was evaluated by kolmogorov-smirnov and shapiro-wilk tests. as a result, it wa s obser ved that all the questionnaire score data were not normally distributed. since the data did not show a normal distribution, the mann whitney u test was used for two independent groups and kruskal wallis test was used for mor e than two groups. bonferroni-corrected mann-whitney u test was used to determine the between-group differences. questionnaire scores were presented as mean and standard deviation. statistically, a value of p<0.05 was considered significant. results a total of 39.7% of the pediatric nurses participating in the study were in the age group of 26-31 years, 53.7% were married and 71.1% were undergraduate. it was determined that 55.4% of the nurses had incomes equal to their expenses, 45.5% worked as a nurse for 0-5 years, 23.1% had prior knowledge about fetal rights and 71.4% received this knowledge during undergraduate and graduate education. the distribution of the answers given to the questions about fetal rights by the pediatric nurses participating in the study is given in table 1. when the relationship between pediatric nurses’ knowledge levels of fetal rights and their descriptive characteristics (ta ble 2) was examined, it was found that there was a statistically significant difference between the participants’ mean scores of knowledge levels about fetal rights with their age, educational status, status of prior knowledge about fetal rights and where this knowledge was gained (p<0.05). it was determined that there was no statistically significant difference between the mean scores of knowledge level about fetal rights with the pediatric nurses’ marital status, monthly income, and working years as a nurse (p>0.05). discussion the findings which we have obtained from this study show that most of the pediatric nurses did not receive any education about fetal rights, that the maj ority ob tained it during the undergraduate or graduate education, and that the knowledge level of those who had knowledge on the subject was found to be insufficient. fetal rights are one of the issues that have not been defined precisely and are not sufficiently raised in many countries.(1) unlike patients’ rights, the topic of fetal rights is not one of the subjects 223 t ab le 1 .d is tr ib u ti o n o f th e an sw er s gi ve n b y th e p ed ia tr ic n u rs es t o t h e q u es ti o n s ab o u t fe ta l ri gh ts ( n = 1 2 1 ) univ med vol. 41 no. 3 224 ozakarakca, turan, kalkan yalcin fetal rights undergraduate and graduate education. these results show that these pediatric nurses lack information about fetal rights and that they are not sufficiently informed about it in the hospital where they work. although rights such as human rights, women’s rights and children’s r ights are guaranteed by laws in the world, there is no legal regulation directly related to the rights of the fetus.(1) this situation is no different in turkey. however, the penalties are available in the penal code of turkey that can be applied in case the fetus is harmed by the mother or others.(10) in our study, the majority of the pediatric nurses was found to have given the response “do not know”to the statement that “turkey has legal regulations directly related to fetal rights” (55.4; table 1). the fact that turkey has no regulation directly related to fetal rights implies that pediatric nurses lack information about this question. on the agenda in many countries such as uk, netherlands, sweden, canada etc. as well as in turkey.(1,7,12,19) the fetus can be harmed due to ignorance, insensibility and false beliefs of the mother and family. (7,20) when the mother and family have false information or insufficient knowledge, the first persons that they can get information from and can trust in are the nurses. considering that nurses have the advocacy role for the patients and that pediatric nurses are the defenders of the fetus, (7,12,13) pediatric nurses are expected to have knowledge about this issue. while training on patient rights for nurses is included in the quality program of almost all hospitals, services such as education, training and consultancy on fetal rights are not common. in this study, it was determined that most of the pediatric nurses did not have prior knowledge about fetal rights (76.9%), and 71.4% of those who received information received it during table 2.knowledge levels on fetal rights according to the several variables in pediatric nurses note: mwu: mann whitney u test; kw: kruskal wallis test; *p<0.05 variables n % median (q1-q3) test p value age (years) 20-25 26-31 32-37 38-43 35 48 16 22 28.9 39.7 13.2 18.2 0.40 (0.13-0.93) 0.53 (0.00-1.00) 0.53 (0.27-0.80) 0.36 (0.00-0.93) kw 0.018* marital status married single 65 56 53.7 46.3 0.46 (0.00-0.93) 0.47 (0.00-1.00) mw u 0.591 educational status (graduation) high school undergraduate graduate 16 86 19 13.2 71,1 15.7 0.40 (0.00-0.80) 0.46 (0.00-0.93) 0.60 (0.07-1.00) kw 0.022* monthly income income<expense income=expense income>expense 32 67 22 26.4 55.4 18.2 0.40 (0.00-0.93) 0.53 (0.00-0.93) 0.53 (0.07-1.00) kw 0.302 years of work as a nurse 0-5 6-10 11-15 16 years and more 55 35 16 15 45.5 28.9 13.2 12.4 0.53 (0.00-1.00) 0.46 (0.00-0.80) 0.53 (0.13-0.80) 0.33 (0.00-0.93) kw 0.545 prior knowledge about fetal rights yes no 28 93 23.1 76.9 0.53 (0.20-0.93) 0.40 (0.00-1.00) mw u 0.012* where prior knowledge about fetal right was gained undergraduate/graduate education workplace (the hospital) 20 8 71.4 28.6 0.60 (0.20-1.00) 0.36 (0.20-0.93) mw u 0.016* 225 studies show that termination of pregnancy in the first 10 weeks is safe for health(22), and this is recognized by law in many countries such as turkey, germany, france, spain, and the netherlands.(20) according to turkish population planning law no. 2827, the legal termination limit of pregnancy is within 10 weeks, depending on the mother’s consent.(23) in our study, it was determined that most of the pediatric nurses knew that termination of pregnancy within 10 weeks is legal (71.9%; table 1), which might be due to the fact that pregnancy termination is one the most common situations that pediatric nurses should face in the hospitals.(24) article 99/1 of the turkish penal code states that “any person who performs an abortion of a child upon a woman without her consent shall be sentenced to a penalty of imprisonment for a term of five to ten years.”, article 99/2 states that “a person who, in the absence of medical necessity, performs an abortion of a child, upon a woman, who is more than ten weeks pregnant and with her consent, shall be sentenced to imprisonment for a term of two to four years. “ and article 99/ 6 states that “where a woman is pregnant due to an offence that she was a victim of, no penalty shall be imposed upon any person who terminates such pregnancy, where the term of pregnancy is not more than 20 weeks and there is consent from the woman. however this requires the termination of the pregnancy by expert doctors in a hospital environment”. article 100/1 of the same law states that “a woman who willingly aborts a child where the term of pregnancy is more than ten weeks shall be sentenced to a penalty of imprisonment for a term of up to one year and a judicial fine.”(10) in the present study, it was determined that more than half of the pediatric nurses gave to the questions about fetal rights (5,6,7 and 8) the incorrect answers of “false” and “do not know” (table 1). in line with all these results, it is seen that pediatric nurses have minimal knowledge of legal codes regarding abortion and miscarriage. in this study, it was determined that most of the pediatric nurses did not have prior knowledge about fetal rights (76.9%), and that 71.4% of those who received information received it during undergraduate and graduate education. in this study, mean scores of pediatric nurses’ knowledge level were found to be higher in the age group of 26-31 years compared to those in the other age groups, in graduates compared to high school graduates and undergraduates, in nurses receiving information about fetal rights compared to those who did not, and in those who received information during undergraduate / graduate education compared to those receiving education in the hospital where they work. the differences between the mean scores of the knowledge level were found to be statistically significant (p<0.05; table 2). unlike patients’ rights, fetal rights is not one of the subjects on the agenda in many countries such as uk, netherlands, sweden, canada etc. as well as in turkey.(1,7,12,19) while training on patient rights for nurses is included in the quality program of almost all hospitals, services such as education, training and consultancy on fetal rights are not common. these results show that pediatric nurses lack information about fetal rights and that education and level of education increase the level of knowledge about the subject.(25) the research data collected to determine the knowledge level of the participants about fetal rights is limited to the answers given by nurses. caution is thus needed in generalizing the study’s finding. conclusion as a result of this study, it was found that most of the pediatric nurses did have prior knowledge about fetal rights, that the majority of those had obtained it during the undergraduate / graduate education, and that knowledge level of those who had knowledge on the subject was found insufficient. in this direction, there is a need for studies on fetal rights, which play an important role in the development of healthcare services. the effectiveness of in-service trainings for personnel by training units should be increased, and knowledge, attitude and behavior change univ med vol. 41 no. 3 226 ozakarakca, turan, kalkan yalcin fetal rights should be created in pediatric nurses. activities and programs should be planned to establish the culture and to educate nurses and patient’s relatives about fetal rights. conflict of interest there is no conflict of interest authorship contributions soa, apt and dky contributed to writing the manuscript, soa and apt contributed to design and data collection. soa and apt contributed to analyzing the data. soa contributed to revising the manuscript. all authors have read and approved the final manuscript. acknowledgments we thank the pedia tr ic nurses who participated in this study. funding the author (s) received no financial support for the research, authorship and/or publication of this article. references 1. atar s, yalým y. 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death of the fetus. bioethics 2017;31:697-702. https://doi.org/10.1111/bioe.12404. 7. anolak h, thornton c, davis d. what’s wrong with using the f word? a systematic integrative review of how the fetus is talked about in situations of fetal demise or high risk of fetal loss. midwifery 2019;79:102537. https://doi.org/ 10.1016/j.midw.2019.102537. 8. wilkinson d, skene l, de crespigny l, savulescu j. protecting future children from ýn utero harm. bioethics 2016;30:425-32. https://doi.org/10.1111/ bioe.12238. 9. mathison e, davis j. is there a right to the death of the foetus? bioethics 2017;31:313-20. https:// doi.org/10.1111/bioe.12331. 10. european commission for democracy through law (venice commission). penal code of turkey. strasbourg: council of europe;2016. 11. republic of turkey. turkish civil code; 2001. 12. hutti mh. a comparison of the caring processes used by obstetric, surgical, and emergency nurses when caring for the woman with a fetal loss. j obstet gynecol neonatal nurs 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abortion laws; 2021. 23. laws turkey. turkish population planning law; 1983. 24. mccallum c, menezes g, reis apd. the dilemma of a practice: experiences of abortion in a public maternity hospital in the city of salvador, bahia. hist ciên saúde manguinhos 2016;23:37-56. https://doi.org/10.1590/s0104-597020160001000 04. 25. diaz-quijano fa, martínez-vega ra, rodriguezmorales aj, rojas-calero ra, luna-gonzález ml, díaz-quijano rg. association between the level of education and knowledge, attitudes and practices regarding dengue in the caribbean region of colombia. bmc public health 2018;18:110. doi: 10.1186/s12889-018-5055-z. univ med vol. 41 no. 3 september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 pleural plaques and pleural changes among lung cancer patients exposed to asbestos aziza ghanie icksan1 , canti widharisastra2, anna suraya3* , and martina ferstl4 abstract background indonesia is one of the world's largest asbestos importing countries. while asbestos is the main contributor to work-related lung cancer, studies or reports regarding the ct findings of asbestos-related lung cancer have been limited in the country. the objective of this study was to compare ct findings of the size of the tumor and pleural and lung parenchymal changes between lung cancer patients exposed to asbestosis and those not exposed. methods this cross-sectional study involved 96 lung cancer patients consisting of 48 subjects who had been exposed to asbestos and 48 who had not been exposed. they underwent thoracic ct scans at the radiology department of persahabatan hospital. asbestos exposure was determined using interviews that followed a protocol similar to that of a previous study about asbestos-related lung cancer. senior radiologists investigated the existence of pleural and parenchymal changes. an independent t-test and chi-square test to compare ct scan features between the two groups. results the mean age was 57.75 ± 8.56 years in the asbestos-exposed group and 58.56 ± 7.99 years in the unexposed group. the proportion of tumor sizes of more than five cm, pleural plaques, and subpleural dotlike or branching opacities were significantly higher among asbestosexposed subjects compared to the non-exposed group (p=0.044; p=0.37; p=0.041, respectively). conclusions asbestos exposure is significantly related to the size of the tumor and the existence of pleural plaques and asbestosis. these findings may help further management of lung cancer patients and the policy of asbestos use in indonesia. keywords: pleural plaque, asbestosis, lung cancer, asbestos, adults 1department of radiology, persahabatan hospital, jakarta, medical faculty of universitas pembangunan nasional veteran jakarta, indonesia 2bhayangkara lemdiklat polri hospital, jakarta, indonesia 3occupational health and safety department, binawan university, jakarta, indonesia 4münchen klinik schwabing, institut für diagnostische und interventionelle radiologie und kinderradiologie, munich, germany *correspondence: anna suraya jl. sudirman no. 45 rt 03/01 harapan mulya, bekasi, 17143, indonesia phone: 08569813835 fax: +62-21-88966141 annasuraya13@gmail.com, anna.suraya@binawan.ac.id orcid id: 0000-0002-5862-3521 date of first submission, february 28, 2022 date of final revised submission, august 18, 2022 date of acceptance, august 22, 2022 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license 210 doi: http://dx.doi.org/10.18051/univmed.2022.v41.210-218 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1307 original article cite this article as: icksan ag, widharisastra c, suraya a, ferstl m. pleural plaques and pleural changes among lung cancer patients exposed to asbestos. univ med 2022;41:210 8. doi: 10.18051/univmed.2022.v41. 210-218. mailto:annasuraya13@gmail.com mailto:anna.suraya@binawan.ac.id https://orcid.org/0000-0001-8064-1526 https://orcid.org/0000-0002-5862-3521 http://dx.doi.org/10.18051/univmed.2022.v41.210-218 211 univ med vol. 41 no. 3 introduction lung cancer is the most commonly diagnosed cancer in men and the leading cause of cancer death worldwide. in indonesia, lung cancer ranks first in men and third in both sexes in terms of incidence, with 34,783 new cases and accounting for 30,843 deaths in 2020.(1) even though the cause of lung cancer is mainly attributed to tobacco smoking, other agents also play a role in lung cancer.(2) studies found that the incidence of lung cancer also rises among non-smokers, indicating that environmental and occupational exposure to other agents contribute to lung cancer as the world is becoming more industrialized.(3) asbestos, radon, and arsenic are widely known non-t obac co carcinogens associated strongly with lung cancer.(2,4,5) occupational exposure contributes 17% to 29% of all lung cancers in men. out of all work-related lung cancers, asbestos has been proclaimed the primary contributor, responsible for around 85% of all occupational deaths.(6) according to the world health organization (who), at least 125 million workers are exposed to asbestos in the workplace, and 107,000 people die each year from asbestos-related diseases.(7) asbestos is a group of natural fibrous minerals that can cause a spectrum of benign to malignant pulmonary diseases, such as asbestosis, pleural fibrosis and plaques, benign asbestos pleural effusion, and malignant mesothelioma, besides lung cancer.(8) asbestos gained popularity in the late nineteenth century due to its fireproof and insulation properties. it was widely used for commercial applications, predominantly in the construction industry throughout the twentieth century.(9) at present, around 65 countries have completely banned all forms of asbestos.(10) unfortunately, the use of asbestos is increasing in eastern europe, asia, and south america.(11) indonesia has imported asbestos since 1950 and is currently one of the world’s largest importers of asbestos, after india and china.(12) the most common asbe stos-related malignancy with high mortality is lung cancer.(13) chest ct is the best imaging modality to diagnose lung cancer. it can also detect abnormalities in the lung parenchyma and pleura, such as pleural plaques or diffuse pleural thickening, likely to be found in individuals with asbestos exposure.(14,15) several studies showed that pleural plaques are early manifestations of asbestos exposure, and that asbestos exposure is associated with an increased risk of malignancies. therefore studies on ct imaging of asbestos-related lung cancers have been conducted in various countries.(16–19) lung cancer caused by asbestos exposure is not always preceded by asbestosis. the presence of asbestosis and pleural plaques on ct imaging of l ung ca nce r pa tie nt s ma y sugge st t he possibility of asbestos exposure as the cause of cancer. therefore, it should not be ignored. however, a case report on the risk of lung cancer and radiology imaging in a construction worker have proved the existence of asbestosrelated lung cancer in indonesia.(20) some studies evaluated the tumor location or histopathological type of lung cancers in asbestos-exposed and unexposed patients.(21) several studies have also demonstrated an increase in lung cancer risk, although no consistent dose-response relationship was observed while considering occupational exposure to specific lung carcinogens such as asbestos or silica.(22,23) although there is still no uniformity of results, the majority of studies reported no significant difference between asbestos-exposed and unexposed lung cancers based on tumor location and histopathology. therefore further research must be done. the objective of this study was to compare ct findings of tumor size and pleural and lung parenchymal changes between lung cancer patients exposed to asbestosis and those not exposed. methods research design a cross-sectional study analyzed the presence of asbestos-related parenchymal and pleural abnormalities and cancer morphology in 212 subjects who had histologically confirmed lung cancer. the study was conducted in jakarta, indonesia, from september 2020 to january 2021 at the radiology department of persahabatan hospital. research subjects subjects were recruited from secondary data of a previous study entitled ”asbestos-related lung cancer: a hospital-based case-control study in indonesia”.(5) the inclusion criteria were patients with primary lung cancer who had chest cts and a clear history of asbestos exposure (exposed or unexposed). asbestos exposure was considered to be positive if there was a history of occupational exposure to asbestos at least ten years prior to the interview. subjects who had chemothera py and inc omplete data were excluded. a total of 48 subjects met the asbestosexposure criteria, and 48 subjects with no history of asbestos exposure were enrolled as controls. both groups were similar in age, sex, and smoking history. image interpretation this study used 128-slice siemens ct scanner detector configurations with reference protocol, radiation dose of 80–160 mas and 120 kvp tube current time, as well as gantry rotation time of 0.33–0.50 seconds. the images obtained were interpreted by a general radiologist from indonesia and 2 thoracic consultant radiologists with more than 10-year e xperience from germany. the features of lung cancer assessed included size and margin of the mass, enlarged lymph nodes, and metastases, based on the eighth edition of the tnm staging system.(24) the pleural abnormalities assessed were pleural effusion, pleural plaques, diffuse pleural thickening, and rounded atelectasis. other lung parenchyma abnormalities evaluated were linear opacities and subpleural nodularities, curvilinear lines, parenchymal bands, interstitial intralobular thickening, interlobular septal thickening, groundglass opacities, and honeycomb appearance.(25) statistical analysis the data were processed using the statistical package for the social sciences (spss) version 25. univariate analysis was carried out to describe the characteristics of the research subjects, including age, gender, ct findings of lung cancer characteristics. categorical data were presented as proportions while numerical data were presented as mean and standard deviation. using the independent -t-test and chi-squared test, bivariate analysis was done to compare ct scan features between as bestos-exposed and unexposed lung cancer. ethical clearance the ethics committee of persahabatan hospital has approved this study under no. 18/ kepk-rsupp/03/2018. results almost all subjects were male (97.2%), with mean age of 57.75 ± 8.56 years in the asbestosexposed group and 58.56 ±7.99 years in the unexposed group. the commonest smoking history for both asbestos-exposed and unexposed groups was 501 to 1,000 pack-years. the distribution of smoking rate is shown in table 1. in asbestos-exposed subjects, it was found that the tumor mass was more likely to be found in the right lung (58.3%), upper lobe (52.1%), and central (66.7%) in location. the mass was mostly more than 7 cm (60.4%) and had regular edges (62.5%). ipsilateral mediastinal and subcarinal lymph node enlargement (n2) was the most typical (16.3%). metastasis was primarily found in the lung (20.8%). other conditions caused by the tumors such as rib fracture were seen in 6.25% of patients and pleural effusion in 52% of patients. the comparison of the tumor mass imaging characteristics between the exposed and unexposed groups is shown in table 1. there was a significant difference in tumor size in which the proportion of tumor sizes of 5–7 cm and >7 cm was higher in the asbestos-exposed group (p=0.044). icksan, widharisastra, suraya pleural plaques and pleural changes 213 univ med vol. 41 no. 3 a b the histological features of the lung cancers in all subjects can be seen in table 2. the most common type of cancer found in this study was adenocarcinoma. using the chi-squared test, there was no significant difference in histological type between the two groups of subjects. figure 1 and figure 2 present the pleural and the lung parenchyma abnormalities on ct of asbestos-exposed subjects and table 3 describes the proportion of these abnormalities in both asbestos-exposed and unexposed groups. the proportion of pleural effusion in both groups of subjects was identical. diffuse pleural thickening and pleural plaque were more frequent in the exposed group (p<0.05). among pulmonary parenchymal abnormalities related to asbestosis, table 1. selected characteristics on chest ct of the study subjects by asbestos exposure data presented as n (%) except for age (mean ± sd); * chi-squared test was used for categorical variables; ¶ independent-t-test asbestos exposure p value* exposed (n=48) unexposed (n=48) n (%) n (%) age (years) [mean ± sd]¶ 57.75 ± 8.56 58.56 ± 7.99 0.623 sex 1.000 male 47 (97.92) 47 (97.92) female 1 (2.08) 1 (2.08) smoking history (pack-years) 0 – 200 9 (18.8) 10 (20.8) 201 – 500 13 (27.1) 12 (25) 0.992 501 – 1.000 20 (41.7) 21 (43.8) 1001 – 1.500 4 (8.3) 3 (6.3) >1501 – 2.000 2 (4.2) 2 (4.2) tumor location location a right left location b superior inferior medially/lingula multiple lobes location c peripheral central size (cm) < 3 3 -5 5 -7 > 7 edge regular irregular lymph node enlargement n1 n2 n3 metastasis lung pericardial effusion lung and pericardial effusion others 28 (58.3) 20 (41.7) 25 (52.1) 14 (29.2) 1 (2.2) 8 (16.7) 16 (33.3) 32 (66.7) 4 (8.3) 4 (8.3) 11 (22.9) 29 (60.4) 30 (62.5) 18 (37.5) 2 (4.2) 16 (33.3) 5 (10.4) 10 (20.8) 6 (12.5) 4 (8.3) 2 (4.2) 31 (64.6) 17 (35.4) 27 (56.25) 12 (25) 4 (8.33) 5 (10.41) 12 (25) 36 (75) 4 (8.33) 14 (29.16) 5 (10.41) 25 (52.08) 26 (54.2) 22 (45.8) 1 (2.1) 15 (31.3) 7 (14.6) 11(22.9) 7 (14.6) 3 (6.2) 1 (2.1) 0.529 0.436 0.369 0.044 0.408 0.873 0.749 214 the proportions of parenchymal bands, subpleural dot-like or branching opacities, and subpleural curvilinear lines were significantly higher in the asbestos-exposed group. discussion several studies on ct imaging of asbestosrelated lung cancers have been conducted in various countries.(16-19) some studies evaluated the tumor locations or histopathological type of lung cancers in asbestos-exposed and unexposed patients and although there is still no uniformity of results, the majority of these studies reported no significant difference between asbestosexposed and unexposed lung cancers based on tumor location and histopathology. lung cancer caused by asbestos exposure is not always preceded by asbestosis. however, asbestosis may be associated with an increased risk of lung cancer due to fibrotic reaction.(26) this study pr ovid es an overview of differences in ct scan findings between asbestos-exposed and unexposed lung cancer patients. parenchymal and pleural changes such as pleural plaques, parenchymal bands, subpleural dot-like or branching nodularities, and subpleural curvilinear lines are significantly found in asbestos exposure p-value * exposed unexposed n (%) n (%) non-small cell carcinoma squamous cell carcinoma 14 (29.2) 13 (27.1) adenocarcinoma 16 (33.3) 17 (35.4) 0.843 large cell carcinoma 1 (2.1) 0 others 1 (2.1) 2 (4.2) small cell carcinoma 0 2 (4.2) neuroendocrine carcinoma 0 1 (2.1) unidentified 16 (33.3) 13 (27.1) table 2. comparison of histological features of lung cancer in asbestos-exposed and unexposed subjects * chi-squared test was used for categorical variables asbestos exposure p-value *exposed unexposed n (%) n (%) pleural abnormality pleural effusion 25 (52.1) 25 (52.1) 1.000 diffuse pleural thickening 14 (29.2) 9 (18.8) 0.232 pleural plaques 10 (20.8) 3 (6.3) 0.037 pulmonary parenchymal abnormality round atelectasis 0 0 parenchymal band 11 (22.9) 4 (8.3) 0.049 subpleural dot-like or branching opacities 4 (8.3) 0 0.041 interlobular septal thickening 8 (16.7) 3 (6.3) 0.109 intralobular interstitial septal thickening 5 (10.4) 2 (4.2) 0.239 subpleural curvilinear lines 4 (8.3) 0 0.041 traction bronchiectasis/bronchiectasis/ bronchiectasis 6 (12.5) 9 (18.8) 0.399 honeycomb 1 (2.1) 0 0.315 ground glass opacities 2 (4.2) 2 (4.2) 1.000 table 3. comparison of pleural and lung parenchymal abnormalities on chest ct in asbestosexposed and unexposed lung cancer patients * chi-squared test was used for categorical variables icksan, widharisastra, suraya pleural plaques and pleural changes 215 univ med vol. 41 no. 3 dot-like structures, subpleural lines, intralobular interstitial thickening, interlobular septal thickening, ground-glass opac ities, and parenchymal bands.(27) this is in accordance with our study which found that parenchymal bands, subpleural nodularities, and subpleural curvilinear opacities were more frequent and statistically asbestos-exposed lung cancer patients. according to cha et al.,(14) the most common parenchymal features in korean patients with asbestosis are subpleural dot-like or branching opacities (97.5%), parenchymal bands (50.5%) and pleural plaques (98.5%). . in the early stage of asbestosis, the high resolution ct findings include subpleural figure 1. pleural abnormalities found in the study subjects: pleural plaques (a,b), calcified pleural plaques (c), and pleural effusion with pleural thickening (d) figure 2. lung parenchymal abnormalities found in the study subjects: bronchiectasis (a), traction bronchiectasis (b), thickening of interlobular and intralobular interstitial septa (c), nodular opacity/branching in the subpleura (d) and curvelinear lines (e, f) 216 more significant in the asbestos-exposed group. asbestosis is not necessarily found in asbestosrelated lung cancer, but it is associated with an increased risk of lung cancer.(16,20) detection of these abnormalities might indicate previous asbestos exposure and help radiologists assess the possibility of asbestos-related lung cancer. therefore, these abnormalities should not be ignored. pleural plaque is considered a marker of asbestos exposure.(26) the presence of pleural plaque increases the risk of lung cancer. therefore, the attribution of lung cancer to asbestos exposure has to be supported by occupational history.(28) attention should be given particularly to individuals with a history of employment in the construction industry, as this sector has the highest prevalence of pleural plaques.(17) other parenchymal and pleural changes, namely interlobular septal thickening, interstitial intralobular septal thickening, traction bronchiectasis, honeycomb appearance, groundglass opacity, pleural effusion, and diffuse pleural thickening were not significantly different in asbestos-exposed and unexposed groups. those abnormalities can also be caused by other conditions, such as post-infection or interstitial pulmonary fibrosis (ipf).(14,29) pleural plaques occur more commonly in subjects with increasing time since first exposure to asbestos and with greater cumulative exposure, especially to amphibole varieties of asbestos.(30) although cumulative asbestos exposure is associated with an increased risk of lung cancer, contradictory data from other studies raise questions as to whether subjects with pleural plaques have an additional higher risk of lung cancer.(31,32) according to ct features in the lung cancer patients, the difference in tumor size between asbestos-exposed and nonexposed groups is statistically significant. to the best of our knowledge, there is no study reporting that tumor size in lung cancer is significantly associated with asbestos exposure. the findings of histological features in this study are in accordance with previous studies in which the major histological types of lung cancer are found in asbestosexposed and nonexposed individuals with no significant d if ferences. (1 9,,33) theref ore, histological type has no value in determining asbestos exposure in lung cancer. seve ral fea tures of the pleur al and lung parenchymal abnormalities may be caused by other diseases such as tuberculosis which also causes fibrotic changes and is very common in indonesia. unfortunately, this study did not perform histopathological examination to exclude other possible causes. in order to overcome the issue, this study referred to existing studies that may explain those lesions.(14,29) in addition, all ct scans were interpreted by two senior radiologists who had more than ten years experience as a thoracal consultant. the limitation of this study is that it was conducted in one center, although that center is a national referral hospital for lung disease in indonesia. in indonesia there is a lack of studies such as the present study and as far as the author knows, this is the first study so it cannot be compared to other studies. further studies are needed and it would be better if these studies were conducted in multiple centers because it would better represent the population. conclusions this study revealed that the proportions of pleural plaques and asbestosis are significantly higher in asbestos-exposed lung cancer subjects than in the nonexposed ones. these findings may help further management of lung cancer patients and the policy of asbestos use in indonesia. conflict of interest the authors declare no conflict of interest related to the subject matter discussed in this manuscript. icksan, widharisastra, suraya pleural plaques and pleural changes 217 univ med vol. 41 no. 3 acknowledgements the authors are grateful to have some support from prof. dennis nowak, dr. stephan boese-o’reilly, and all staff members of persahabatan hospital. author contributions conceptualization and methodology agi, cw, as; data collection as, agi, cw, mf; data analysis agi, cw, as, mf; writing—original draft preparation, agi, cw, as; writing—review and 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pleural plaques, and the risk of death from lung cancer. am j respir crit care med 2014;190:1413–20. doi: 10.1164/rccm.201406-1074. 32. silva m, sverzellati n, colombi d, et al. pleural plaques in lung cancer screening by low-dose computed tomography: prevalence, association with lung cancer and mortality. bmc pulm med 2017;17:155. doi: 10.1186/s12890-017-0506-3. 33. nielsen ls, bælum j, rasmussen j, et al. occupational asbestos exposure and lung cancer—a systematic review of the literature. arch environ occup health 2014;69:191–206. arch environ occup health 2014;69:191-206. doi: 10.1080/19338244.2013.863752. icksan, widharisastra, suraya pleural plaques and pleural changes c:\users\universa medicina\docu 96 abstract universa medicina effective perioperative scoring in orthopedic surgery to prevent covid-19 infection komang agung irianto1* and taufan adityawardhana2 background the pandemic has placed health-care workers (hcw) under an unprecedented strain where surgeries are being delayed and health care workers’ deaths because of covid-19 are prominent and causing staff shortages. an effective, fast, and concise approach to preoperative screening is very much needed. hospitals are suggested to develop their own algorithm to minimize delays and complications. the objective of this study was to optimize hcw safety by developing a working system based on the hospital’s service capability in treating orthopedic surgeries. methods a retrospective study was conducted involving 305 hcw. data were collected on the application of a novel elective and emergency orthopedic surgery clinical pathway and scoring method based on questionnaires, and laboratory and radiographic chest examinations for several months to ensure the safety of hcw. hcw covid-19 status was routinely checked as the indicator of hcw’s safety. results mean age of all patients was 47.0 ± 22.41 years. the screening process was done in a series of questionnaires and laboratory examinations, with the majority of patients (82 or 35.9%) having a score of 0. patients with a score of 3 or more and testing positive for covid-19 were immediately referred to a hospital having adequate facilities to prevent delays. there were 51 patients (16.7%) with positive rapid test results, and 21 patients (6.8%) with positive pcr tests. there were 4 (1.3%) covid-19 positive hcw. conclusions despite covid-19 being a new threat, we have shown that developing an effective perioperative clinical pathway could eventually optimize healthcare worker safety. keywords: covid-19, pandemic, screening, scoring, orthopedic surgery, healthcare workers original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.99-109 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1122 may-august 2021 vol.40no.2 ci te this art icle as: ir iant o ka, ad itya ward hana t. effecti ve perioperative scoring in orthopedic surgery to prevent covid-19 infection. uni v med 20 21;4 0:9 6 -1 0 6 . doi: 10.18051/univmed.2021.v40.99-109. 1surabaya orthopedic and traumatology hospital, department of orthopedic and traumatology, faculty of medicine, universitas airlangga, surabaya, indonesia 2medical education research and staff development unit, faculty of medicine, universitas airlangga, surabaya, indonesia correspondence: *komang agung irianto surabaya orthopedic and traumatology hospital, department of orthopedic and traumatology, faculty of medicine, universitas airlangga, jl. mayjend.prof.dr. moestopo 6-8 surabaya,60286 fax: +6231-5030406 surabaya, indonesia email: komang168@yahoo.com orcid id: 0000-0001-6625-970x date of first submission, january 31, 2021 date of final revised submission, june 27, 2021 date of acceptance, july 7, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 97 univ med vol. 40 no 2 introduction covid-19 has been a catastrophe since 2019 and is classified as a global pandemic.(1) since march 2020 orthopedic surgeons have been facing a dilemma on whether or not to postpone elective surgeries; however, it is not easy to delay the emergency surgeries (2) the pandemic also placed health-care workers (hcw) under an unprecedented strain, with more than 100,000 hcw deaths worldwide as of april 2020.(3) as reported in indonesia, in june 2020 2.6% of covid-19 confirmed patients were indonesian hcw (1,077 persons).(4) at least 282 hcw have died of covid-19 ever since the start of the outbreak in indonesia until november 10th based on the statement of the indonesian medical association (idi).(5) it has even been supposed that the actual number may have been much higher.(4) staff shortages in healthcare eventually became a significant conflict worldwide. the pandemic has forced hcw to have more specific strategic deployment towards their clinical services as a response to their service d e ma nd s in t e r ms o f b ot h e le c t i ve a nd emergency clinical services.(6) thus, the question arises as to the best medical practice for patients who suffer from both fracture and covid-19 infection at the time of presentation.(1) however, the covid-19 screening is time-consuming, which delays surgery and may result in malunion, disability of an extremity, or a more serious complication. therefore, maintaining a balance between adequate screening and timely surgery is essential for perioperative management of traumatic fractures.(2) a standardized approach to perioperative covid-19 testing is based on the patient’s symptoms, and evaluating the risk of the procedure is crucial to ensure maximal safety of patients, their families, and hcw. a healthy hcw is an asset to the prolonged response in this pandemic and helps to reduce burnout.(7) a previously published guideline was more focused on laboratory examination to predict covid-19 infection and was proven to be effective,(7) while another guideline was issued t o mi n i mize d e l a ys a nd pe r iop e r a ti ve complications in orthopedic surgery but required several rooms and much space for proper covid-19 screening, which we were unable to contemplate. (2) hospitals were then suggested to develop their own protocol based on their own resources and facilities to prevent covid-19 and optimize what they had.(9) the aim of this study is to ensure hcw safety with the application of novel clinical p a t hw a ys b a se d o n qu e s t i on na i r e s a nd laboratory and radiographic chest examinations in all orthopedic surgeries, including elective and e me r ge n c y s ur ge r ie s, a nd obs e r ve t h e effectiveness of the clinical pathway as indicated by the number of infected hcw which could be emulated by hospitals with limited space or resources. methods research design this was a retrospective cross-sectional study of the orthopedic division’s medical records of patients that underwent surgeries from march 2020 to october 2020 at the orthopedic & traumatology hospital, surabaya. study subjects a total of 305 medical records from march 2020 to october 2020 had adequate data and were included in this stud y. patients’ clinical presentation, laboratory examination and radiogr aphic exa mination were recorded, analyzed statistically and grouped. instr uments and o rthopedic surgery classification since april 2020, an emergency specialized task force at the orthopedic & traumatology hospital was tasked to develop instruments for a perioperative clinical pathway for personal protective equipmen t (ppe) to be used specifically for orthopedics surgery in the hospital. 98 the task force agreed to focus more on the screening process to decide what type of ppe should be used in the surgery, with the goal of ensuring the safety of hcw, non-medical staff and patients. the clinical pathway used in elective orthopedic surgery differs from that in emergency orthopedic surgery. types of surgery were classified further, elective orthopedic surgery consisted of one-day surgery and non-emergency inpatient care, whereas emergency orthopedic surgery consisted of immediate, acute and urgent orthopedic cases. the surgery classification is described further in table 1. emergency acute urgent nonemergency surgery one day surgery time to surgery < 24 hrs 24-48 hrs 48 hrs-1 week > 1 week definition life threatening/ limb threatening. delay in treatment potentially causes disability or mortality delay in treatment may cause severe infection, disability or mortality. estimate significant ongoing blood loss delay in treatment potentially causes complications due to bleeding, infection, joint disruption and prolonged immobilization delay may not cause significant morbidities/ complication without hospital admission case compartment syndrome femoral shaft fracture every type of closed fracture including spine and femoral shaft every implant removal surgery every surgery requiring local anaesthetic open fracture pelvic fracture every acute ligament and tendon injury whereas end-to-end repair is planned total joint arthroplasty ctev cases avn injury multiple long bone fracture every pain management cases that requires inpatient care curettage and bone graft dislocation open degloving injury neglected open fracture minimally invasive spine surgery, including interspinous device insertion and cervical disc arthroplasty table 1. orthopedic surgery classification irianto, adityawardhana scoring in orthopedic surgery and covid-19 99 univ med vol. 40 no 2 a b c clinical pathway and scoring system for covid-19 prevention this study combined, compared and was modele d a fter published guidelines from international and national suggestions. this eventually resulted in a scoring based on questionnaires and laboratory and radiographic chest examinations (7,8) and periope rative assessment,(9-11) the scoring was made by the hospital’s service capability and is described in table 2. these screening results will be taken into consideration, thus influencing the clinical pathway, surgical process, and level of personal protective equipment (ppe) for the surgery. the clinical pathway was compared to previous published clinical pathways for emerge ncy (2 ,8,12 ) and ele ctive orthopedic surgery.(13) the proper ppe suggestions and training for hcw was also considered based on other publications. (9,14) the clinical pathways for elective orthopedic surgery and emergency orthopedic surgery are described in figures 1 and 2, respectively. h e a l t hc a r e w or ke r s ( h c w) s af e t y indicators the covid-19 status of the hcw was routinely checked by means of the pcr test. it acute septic arthritis with sirs/sepsis osteomyelitis with subperiosteal abscess musculoskeletal malignancies amputation every soft tissue related case, including tendon lengthening and tenotomy crush injury cellulitis with sirs/sepsis osteomyelitis/tb infection debridement every arthroscopic procedure and ligament reconstruction damage control surgeries fracture dislocation of spine with cord compression/ spinal cord injury musculoskeletal abscess without sepsis acromioplasty bursectomy suppurative tenosynovitis acute joint infection without sepsis tumor excision displaced talar neck fracture bony and soft tissue reconstruction unstable scfe every case requiring general anesthesia that is not included in emergency, acute or urgent group closed fracture dislocation abbreviation : avn: avascular necrosis, also called osteonecrosis; sirs: systemic inflammatory response syndrome; scfe: slipped capital femoral epiphysis; ctev: congenital talipes equinovarus table 1. continued 100 criteria score history of last 14 days fever (>37.8) 0 = none 1 = one of the criteria 2 = ≥ 2 criteria 3 = close contact (+) cough / flu / sore throat breathing difficulty abdominal pain / diarrhea / nausea/ vomiting anosmia / cephalgia myalgia / arthralgia returned from abroad close contact with suspected or confirmed covid-19 patients clinical symptoms fever (>37.8) 0 = none 1 = one of the criteria 2 = ≥ 2 criteria 3 = desaturation (+) cough/sneeze/sore throat breathing difficulty (respiration rate ≥ 24 per minute) abdominal pain/diarrhea/nausea vomiting anosmia/cephalgia myalgia/arthralgia rhonchi / wheezing desaturation (spo2 ≤ 95% without oxygen supply) laboratory results leukopenia (< 5000/mm3) or leukocytosis (>10000/mm3) 0 = none 1 = one of the criteria 2 = ≥ 2 criteria lymphopenia (alc < 1500/mm3) nlr ≥ 5.8 crp > 10 with normal procalcitonine thrombocytopenia of unknown origin increased bun/serum creatinine or sgot/sgpt of unknown origin radiology consolidation or multifocal infiltrates observed on x-ray, unilateral or bilateral 0 = none 3 = positive total score = table 2. scoring based on questionnaire, laboratory and radiographic chest examination results abbreviations: alc: absolute lymphocyte counts; nlr: neutrophil-to-lymphocyte ratio; crp: c-reactive protein became the indicator for the caregivers’ safety and was routinely reported to evaluate the application of the clinical pathway. the hcw with positive results for covid-19 were to be traced until the virus transmission was clearly pointed out. data analysis descriptive analysis was presented as frequency for categorical data and as mean (± standard deviation) for numerical data. ethical clearance this study was approved by the health research ethics commission of the surabaya orthopedic & traumatology hospital under no. 01/pers-ec/rsot/v/2021. results a total of 305 medical records from march 2020 to october 2020 containing adequate data were included in this study. around 55.8% of the records were of female patients and 44.2% of male patients. mean age of all patients was 47.0 ± 22.41 years. due to the continuous development of the screening process not all the records contained adequate data for all three parts of screening. unfortunately only 228 medical records had adequate data for scoring and 289 medical records for orthopedic surgery classification. thankfully, preoperative rapid tests wer e done to all orthopedics patients —with swab tests done accordingly. irianto, adityawardhana scoring in orthopedic surgery and covid-19 101 univ med vol. 40 no 2 figure 1. elective orthopedic surgery clinical pathway figure 2. emergency orthopedic surgery clinical pathway emergency orthopedic surgery (immediate, acute, urgent) screening with scoring system during admission score ≥ 3 rapid test (-) score < 3 rapid test (-) score ≥ 3 rapid test (+) or score < 3 rapid test (+) surgery  • ppe level 2 • head box for patients if using general anaesthetic • pre-operation and after operation at different room • normal room for patient post-operation internal discussion between surgeon, anaesthesiologist and internist refer to referral hospital surgery  • ppe level 3 • head box for patients if using general anaesthetic • pre operation and after operation at same room • isolation room for patient post-operation allowed not allowed non-emergency surgery one-day surgery • pre-operation lab • chest radiograph • covid-19 rapid test rapid test (+) rapid test (-) • delay surgery • referred for covid -19 pcr test surgery  • covid-19 pcr test • pre-operation lab • chest radiograph • patients self-isolation at home while waiting for pcr test result pcr (+) pcr (-) rapid test before surgery rapid test (+) rapid test (-) refer to referral hospital surgery  elective orthopedic surgery 102 the screening process was done in a series of questionnaires and laboratory examinations and 82 patients (35.9%) had a score of 0. due to the hospital’s inability to provide a negative pressure operating theater, some patients having a score of 3 or more were immediately referred to a hospital with a negative pressure operating theater and were therefore not included in this research. the same procedure was followed for emergency patients that tested positive with the rapid test, which resulted in their data not being recorded properly to be included in this study. overall, there were 51 patients (16.7%) with positive rapid test results, and 21 patients (6.8%) with positive pcr tests. every patient who received a pcr test had already shown positive rapid test results. based on the surgery classification, the majority of 163 patients (56.4%) underwent elective surgery and 86 patients (29.7%) were classified into urgent surgery. the summary of covid-19 screening score, patients’ rapid test and swab test results; and surgery classification are presented in table 3. routine checkup was done for the hcw. there were four (1.3%) hospital staff with covid-19 positive results from march 2020 to october 2020, namely one anesthesiologist, one nurse, one lab officer and one marketing officer. after being traced, none of them had picked up the virus from the hospital environment. the anesthesiologist had caught it from a colleague at another hospital, the lab officer had it from family members, and the nurse had got the virus from her parents and had transmitted it to the marketing officer. there was no hcw that had caught the virus from the patients. discussion we were able to formulate the screening method, apply it to the clinical pathway for elective and emergency orthopedic patients, determine the time of surgery and refer the patient to avoid patient delays and to pr otect the hcw. unfortunately there were 4 (1.3%) hospital staff with covid-19. it is consistent with previous publications that using an easy-to-follow decision tree algorithm for the interventional platforms, early screening and triage,(15) if implemented with proper training and self-discipline, hcw safety will be optimized.(11,12) this is accomplished by minimizing perioperative complications and reducing delay of surgery (2,9) as an alternative method based on hospital capabilities.(7) strict infection-control measures should be implemented to orthopedic patients particularly patients receiving surgical intervention.(1) the importance of covid-19 is undeniable and further steps have to be taken more carefully in order to not worsen the contamination that eventually may endanger the hcw.(2) it is the role of hospitals to develop protocol and workflow guidelines for diagnosis, management and precaution.(10) the toughest problem is the emergency orthopedic surgery, where delays could bring significant harm to the patient or the outcome.(6) maintaining the balance between adequate screening and time-saving is essential, especially covid-19 screening score (n=228) n (%) score of 0 82 (35.9) score of 1 52 (22.8) score of 2 89 (39.1 score of 3 4 (1.7) score of 4 1(0.4) patients’ rapid and swab test (n=305) positive rapid test 51 (16.7) positive swab test 21 (6.8) surgery classification (289) one day surgery 13(4.5) elective 163 (56.4) urgent 86 (29.7) acute 3 (1.1) emergency 24 (8.3) table 3. distribution of covid-19 screening score, rapid and swab test and surgery classification irianto, adityawardhana scoring in orthopedic surgery and covid-19 103 univ med vol. 40 no 2 in emergency cases.(2) by screening with a scoring system during admission the response time could be faster, the preparation could be started as soon as possible and the referral process could be started sooner if necessary. with proper training and discipline, this definitive scoring system and clinical pathway results in shortening of hcw’s contact time with the patient, thus lowering the risk of covid-19 transmission. due to the demand for elective surgeries, to stop them altogether for a long period of time is very unlikely. by march 2020 it was suggested to stop elective surgery altogether for a period of time and by then it was commonly agreed that the covid-19 incidence in the hospital should decrease for at least 2 weeks before the elective surgery.(2) the goal is to make the hospital stay as short as possible, as long as the roadmap of elective surgery is able to clearly sort out the problems, resumption seems possible.(6) we applied the same approach as described in figure 1 for elective surgery and in fact, most of our included medical records are about elective surgery. granted the number of emergency surgeries included in this study could be higher but the fact that elective surgeries were done in high numbers suggests that it is possible to do. the key in our hospital was to be able to determine when to do the surgery, when to delay or to refer the patient because of the hospital’s limitations. e mergency specialized task forces were suggested to be assembled with the sole purpose of developing a perioperative scoring system based on the hospital’s service capability because there is lack of proper and standardized scoring for covid-19 screening, especially in surgical patients.(11) a combination of the patient’s history for the last 14 days, clinical findings, laboratory data, and radiological pattern based on the institutional or national policy are sufficient and are interpreted as a scoring based on published scoring guidelines.(7,8) the scoring then suggested the appropriate management (9) with appropriate ppe(11) and perioperative preparation (11,14) which can also be used for elective surgery.(13) this was modeled into a clinical pathway for an easy-to-follow guideline as suggested.(12) this was simplified into a scoring system that was proven to be effective, because hcw could make faster and more objective decisions that can reduce covid-19 transmission by shortening contact time. with a proper clinical pathway, training and discipline preparation, the patient’s management could be started sooner. it is very unfortunate that there were limitations with the study design being retrospective, the data were secondary data and this scoring only applied to orthopedic surgery. however, it was shown to be effective with only 4 (1.3%) hcw becoming covid-19 positive and it can potentially be used in all other types of surgery, whether an emergency or elective surgery, with promising results. it is recommended that other hospitals develop a scoring system and risk assessment based on their capabilities and limitations with the sole purpose of protecting t he p a t i ent s a n d t he hcw a t l e a s t f or emergency management during the covid-19 pandemic.(15) ideally a negative pressure operating room should be provided for covid-19 patients. but in some hospitals, including our own hospital where a negative pressure operating theater is not available, the surgeries had to be done in a positive pressure operating room. but not all orthopedic patients could be referred, especially with the hospital that has a negative pressure operating room being overloaded. emergency cases still have to be treated if the hospital is capable and due to the demand for elective surgeries, to stop them altogether for a long period of time is very unlikely.(2,6) fortunately there were several guidelines, both international (10,12,13) and national,(11) that are also specific for orthopedic surgery (14) on how to manage the surgical process without negative pressure operating room. for the latter to succeed, the positive pressure system and the air conditioners have 104 to be turned off.(11) we used a portable highefficiency particulate air or hepa machine to help maintain the proper air ventilation that is important to reduce the number of viruses in the circulating air.(11,13,14) in the case of elective surgery, surgery can be performed in rooms at the normal positive pressure but there have to be modalities to maintain air ventilation.(14) it is also recommended to minimize the number of people in the operating room, and if possible the patient’s recovery in the post-operative phase should also be done in the operating room.(11,14) hospitals have to develop their own screening system based on their capabilities.(13) to be able to decide which patient to operate on and which to refer is essential to reduce covid-19 transmission both inside a nd outside the operating room. on top of all, hcw protection should become a priority. while there is a shortage of staff, protecting hcw has to be prioritized while the hcw themselves have to adapt to the new situation.(17) with social distancing not essentially effective in controlling covid-19 incidence,(18) the stressor among hcw in dealing with the possibility of a prolonged pandemic could cause burnouts.(19) supplies of personal protective e q ui p me nt ( ppe) ma y be li mi t e d , a n d unfortunately this requires a more restricted access to ppe to make sure it is being used properly.(10) routine testing and tracing are suggested in the hospital environment because the infection rate is still high, but unfortunately the number of pcr swab tests for covid-19 is still limited. proper management to maintain the balance between screening needs and swab test supplies is required. the hcw or other hospital staff who have been proved positive should be isolated and their contacts traced.(16) proper training and self-discipline once again has to be applied with the already existing clinical pathways and regulations.(10) fortunately we were able to routinely check the hcw. four of them were swab positive from march 2020 to october 2020 and only two of the four (an anesthesiologist and a nurse) were in close contact with patients, whereas the other two (a lab officer and a marketing officer) were not. after tracing was done as suggested,(16,17) it was found that the anesthesiologist caught it from a colleague in another hospital, the lab officer had it from family members, and the nurse got the virus from her parents and transmitted it to the marketing officer. there were no hcw that caught the virus from the patients. hcw with a positive swab test were to be self-quarantined for at least 2 weeks and if showing signs and symptoms were to be admitted to hospital. these results may be an indicator that the use of a proper clinical pathway, proper scoring for immediate screening and proper surgery regulation combined with proper training and discipline may minimize the chance of hcw contracting covid-19 from their patients. in addition, with the possibility of economic impact on primary and universal healthcare the simple alterations to our daily practice may benefit the hcw and their patients.(20) the limitation of this study is that this is a retrospective review study and all the data for this study are secondary data. the score has also to be validated in larger populations. hopefully the screening methods, clinical pathways and results of this research could be a model for hospital service that can be used for a ll orthopedic surger y interve ntional platforms and trigger hospitals in underdeveloped and developing countries to devise their own easy-to-use algorithms. conclusion the covid-19 pandemic has forced rapid development of novel hospital guidelines across a spectrum of clinical environments, including orthopedics. despite covid-19 being a new threat, we have shown that developing an effective perioperative clinical pathway could eventually optimize healthcare worker safety. irianto, adityawardhana scoring in orthopedic surgery and covid-19 105 univ med vol. 40 no 2 conflict of interest the authors declared no potential conflicts o f in te re st w it h re sp e ct to th e re se ar ch , authorship and/or publication of this article. funding the authors received no financial support for the research, authorship and/or publication of this article. contributors bo th k ai a n d ta we re in vol ve d i n conception and design of study, data acquisition, drafting of the manuscript and revising the ma nu sc ri pt . bot h auth or s have re ad a nd approved the final manuscript. references 1. mi b, chen l, xiong y, xue h, zhou w. liu g. characteristics and early prognosis of covid-19 infection in fracture patients. j bone joint surg am 2020;102:750-8. doi: 10.2106/jbjs.20.00390. 2. meng y, leng k, shan l, et al. a clinical pathway for pre-operative screening of covid-19 and its influence on clinical outcome in patients with traumatic fractures. int orthop 2020; 44:1549-55. doi:10.1007/s00264-020-04645-3. 3. black jr, bailey c, przewrocka j, et al. covid-19: the case for health-care worker screening to prevent hospital transmission. lancet 2020; 395:1418-20. doi: 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19. windarwati hd, ati na, paraswati md, et al. stressor, coping mechanism, and motivation among health care workers in dealing with stress due to the covid-19 pandemic in indonesia. asian j psychiat 2021;56:102470.j.ajp.2020. 102470. 20. yusuf e. primary health care for all: lessons from its failure to achieve its goal and covid-19. univ med 2021;40:1-2. doi: 10.18051/univmed.2021. v40. 1-2. irianto, adityawardhana scoring in orthopedic surgery and covid-19 increased knowledge of thalassemia promotes early carrier status examination among medical students 220 *department of biochemistry, faculty of medicine, universitas padjadjaran, bandung indonesia **department of public health, faculty of medicine, universitas padjadjaran, bandung indonesia ***working group of genetics, faculty of medicine, universitas padjadjaran, bandung indonesia correspondence: dr. julius broto dewanto department of biochemistry, faculty of medicine, universitas padjadjaran jl. raya sumedang km 121 jatinangor jawa barat mobile: 0812-1459 6149 email: juliusdewanto@yahoo.com univ med 2015;34:220-8 doi: 10.18051/univmed.2016.v35.220-228 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background thalassemia is an autosomal recessive genetic disorder, in which the patient requires life-long blood transfusion. as indonesia harbors 6 to 10% thalassemia carriers, thalassemia prevention measures such as early screening and education in the community are urgently needed. the aim of this study was to explore the knowledge, attitude and practice about thalassemia among young medical students. methods a cross-sectional analytic observational study was conducted on 179 subjects in 2015, using a questionnaire with items on knowledge, attitude and practice about thalassemia for data collection. after signing informed consent, the questionnaire was filled in by the students and a blood test was performed when the students agreed to be examined. detection of probable thalassemia carrier status was done by determination of hemoglobin, mean corpuscular volume and mean corpuscular hemoglobin. results the knowledge about thalassemia of the first year medical students (n=179) was good (21.1%), moderate (70.9%) and poor (21.1%). only 67 (38.3%) of the students agreed to a blood examination for determination of their carrier status after filling-in the questionnaire. the knowledge of thalassemia among first year medical students was statistically related to the timing when they would agree to have their thalassemia carrier status examined (p=0.021, one way anova test). conclusion a higher thalassemia knowledge score causes medical students to be willing to undergo thalassemia carrier status examination at an earlier point in timing. a well-organized educational program focusing on thalassemia and early screening in young adults may enhance the thalassemia prevention program. keywords: early detection, knowledge, thalassemia, carrier status, medical students increased knowledge of thalassemia promotes early carrier status examination among medical students julius broto dewanto*, haryono tansah*, sari puspa dewi**, helena napitu***, ramdan panigoro*, and edhyana sahiratmadja* doi: http://dx.doi.org/10.18051/univmed.2015.v34.220-228 221 univ med vol. 34 no.3 pengetahuan thalassemia yang lebih baik berdampak pada pemeriksaan dini status karir thalessemia mahasiswa kedokteran latar belakang thalassemia merupakan kelainan autosomal resesif yang penderitanya membutuhkan transfusi darah sepanjang hidupnya. di indonesia terdapat 6 sampai 10% penduduk dengan karir thalassemia, sehingga tindakan pencegahan penyakit thalassemia seperti deteksi dini terhadap karir dan edukasi pada masyarakat mengenai beban penyakit thalassemia sangat diperlukan. tujuan studi ini adalah untuk mendalami pengetahuan, sikap dan perilaku mengenai thalassemia pada mahasiswa kedokteran. metoda penelitian potong lintang studi analitik observasional yang dilakukan pada 179 subjek pada tahun 2015. kuesioner yang meliputi pertanyaan mengenai pengetahuan, sikap dan perilaku tentang thalassemia digunakan untuk pengumpulan data. setelah mendatangani surat pernyataan persetujuan penelitian, mahasiswa mengisi kuesioner dan diperiksa darahnya bila berkenan. deteksi untuk kemungkinan awal status karir thalassemia dilakukan dengan memeriksa nilai hemoglobin (hb), mean corpuscular volume (mcv) dan mean cospuscular hemoglobin (mch). hasil pengetahuan mahasiswa kedokteran tingkat pertama (n=179) mengenai thalassemia dapat dikategorikan baik (21,1%), sedang (70,9%) dan buruk (21,1%). hanya 67 (38.3%) mahasiswa yang setuju untuk dilakukan pemeriksaan darah dalam menentukan status karir thalassemianya setelah pengisian kuesioner. pengetahuan thalassemia diantara mahasiswa fakultas kedokteran ini secara statistik berelasi dengan waktu mereka mau diperiksa status karir thalassemianya (p=0,021, oneway anova test). kesimpulan nilai pengetahuan thalassemia yang lebih baik membuat mahasiswa kedokteran melakukan pemeriksaaan status karir thalasemianya lebih awal. program edukasi yang organisasinya baik dan berfokus kepada thalassemia serta deteksi dini status karir thalassemia pada dewasa muda dapat meningkatkan program pencegahan penyakit thalassemia. kata kunci: deteksi dini, pengetahuan, status karir thalassemia, mahasiswa kedokteran abstrak introduction thalassemia, an autosomal recessive genetic disorder involving hemoglobin (hb) chain synthesis, is prevalent among populations in mediterranean, middle eastern, south asian and south east asian countries.(1-4) the disease is not curable and the patients require life-long blood transfusion; however, new technologies o ff e r t h e p o s s i b i l i t y f o r b o n e m a r r o w transplantation. (5) due to life-long blood transfusion, thalassemia patients may have complications of iron overload leading to abnormalities associated with iron deposition in tissues, ultimately resulting in death, thus necessitating iron chelation therapy.(6) infants with thalassemia major are born from parents who both harbor heterozygous mutations or are carriers, therefore their probability of having a child with thalassemia is one out of four pregnancies. 222 there are several tests in the laboratory to diagnose thalassemia carrier status. early screening for possible thalassemia carrier status does not require special examinations, only routine blood examination for hb, mean c o r p u s c u l a r v o l u m e ( m c v ) , a n d m e a n c o r p u s c u l a r h e m o g l o b i n ( m c h ) . ( 7 ) t h e heterozygous form of thalassemia carrier may manifest in healthy young adults as a low hb. since iron deficiency anemia due to dietary foods that are poor in iron accounts for approximately 75% of all anemia types in the developing countries,(8) healthy young adults with low hb need to be investigated accordingly. when apparently healthy young adults have low hb, mcv and mch, further examinations are necessary, including iron parameters to exclude iron deficiency, and hb electrophoresis to measure the hba2 level. a hba2 value over 3.5% confirms β-thalassemia carrier status and a value less than 3.5% may indicate the presence of iron deficiency, α-thalassemia, or rare forms of β-thalassemia. however, the definitive diagnosis can only be determined following dna analysis. a combination of hb analysis and dna testing seems to be the best way to confirm carrier status in a region with high frequency of thalassemia.(9) the knowledge about thalassemia in some areas with high thalassemia carrier state varies. unknown thalassemia carrier status may lead to h i g h p r e v a l e n c e o f t h a l a s s e m i a d i s e a s e . thalassemia can be prevented by premarital screening for early thalassemia carrier status or by prenatal diagnosis.(10) a pregnancy with an affected fetus can be terminated after the carrier parents opt for the prenatal diagnosis; however, in some religions abortion of a fetus is definitely not an option.(11) young adults from an area with high prevalence of thalassemia are prospective parents, and ideally they should be aware of their carrier status before pregnancy or even before marriage.(12) since indonesia harbors 6 to 10% of thalassemia carriers, early screening and education of young adults and the community is of interest.(13) therefore, early detection of thalassemia carriers in the general population, especially in school students and young adults is encouraged.(14) the aim of the present study was to explore the knowledge and perception of thalassemia among first year medical students and their attitudes towards informed consent for thalassemia carrier testing. these students may be representative of young adults on their attitude towards managing their thalassemia carrier status. methods study design this study was of cross-sectional analytic observational design, and was conducted in march 2015 at the faculty of medicine, universitas padjadjaran, bandung. research subjects this was a study exploring the knowledge, attitude and practice of young adults using one questionnaire. total sampling was used for sample size determination, therefore, all first year medical students were asked to participate. there were no exclusion criteria in this study. questionnaire about knowledge, attitude and practice of thalassemia the questionnaire was comprised of 25 questions and divided into 3 parts. the first part was to explore the knowledge about thalassemia, consisting of 18 questions. in brief, participants were given closed questions with three answers of ‘yes, no and don’t know’. the answer was checked with the open questions to ensure that the questions were rightor wrong answered. for each question, the correct answer was given a score of one, and the incorrect or “don’t know” answer was scored as zero, with a total possible score of 0 to 18. a score of 0-6 indicates poor knowledge, a score of 7-12 indicates moderate a n d a s c o r e o f 1 3 1 8 g o o d k n o w l e d g e . cronbach’s alpha was used to assess the reliability of the knowledge scale, with values dewanto, tansah, dewi, et al knowledge of thalassemia early carrier 223 univ med vol. 34 no.3 of at least 0.7 indicating acceptability of the internal consistency. the second part of the questionnaire was to assess the attitude toward thalassemia, and consisted of 5 questions. participants were asked for their agreement on premarital screening, marriage between individuals who are both carriers, pregnancy of carrier couples, and termination of pregnancies with a fetus affected by thalassemia major. in the third part of the questionnaire with 2 questions, the practice or willingness to screen for possible thalassemia status were asked. the questionnaire was adapted and modified from a published article (11) and translated into bahasa indonesia. early screening for possible thalassemia carrier status students who had agreed to take part in the screening as stated in the third part of the questionnaire were invited to have their blood e x a m i n e d . e a r l y s c r e e n i n g f o r p o s s i b l e thalassemia carrier status was performed by routine blood examination, using as indicators l o w h b o r h y p o c h r o m i c a n e m i a ( m e a n corpuscular hemoglobin/mch<27pg) and m i c r o c y t i c a n e m i a ( m e a n c o r p u s c u l a r mcv<80fl).(9) the venous blood samples were collected in 3 ml edta vacuum tubes by welltrained laboratory technicians (laboratorium p r o d i a , b a n d u n g ) . a n e m i a w a s d e f i n e d according to who criteria,(8) corrected by gender, as a hb concentration of <12g/dl and <13g/dl, for females and males, respectively. the severity of anemia was classified into three stages: mild (10-11g/dl), moderate (7-10/dl), or severe (hb<7 g/dl). the laboratory results were distributed to the students in closed e n v e l o p e s . st u d e n t s w h o h a d p r o b a b l e thalassemia carrier status were counselled individually. statistical analyses responses to the questionnaire were expressed as percentages. knowledge was scored and categorized as good, moderate, or poor; and comparison between genders was analyzed using pearson chi-square or anova-test where needed. hematological data were presented and analyzed for probable thalassemia carriers. the description and statistical analyses were conducted using spss 16.0 for windows. ethical clearance information about the study had been given prior to the filling-in of the questionnaire, both verbally and written in the informed consent form that was signed, ensuring the confidentiality of the study results. the study protocol was approved by the ethical committee of the medical faculty, universitas padjadjaran, bandung, indonesia, under no. 715/un6.c2.1.2/ kepk/pn/2014. results questionnaires were distributed to 219 firstyear medical students, but only 175 (70.9%) s t u d e n t s r e s p o n d e d a n d r e t u r n e d t h e questionnaire. these students consisted of 45 (25.7%) males and 130 (74.3%) females. the mean age of the students was 18.1 years (sd = 0.71), and the age range was from 16 to 21 years. the students identified themselves as sundanese (n 55; 31.4%) or javanese (n 47; 26.8%), the two dominant ethnicities on java. the reliability of the knowledge scale (cronbach’s alpha) for 18 questions was tested, resulting in high score of 0.86. therefore, the questionnaire can be used to assess thalassemia knowledge. the data distribution of the knowledge scores among the students was not normal (shaphiro-wilk score 0.012). the median knowledge score among the students was 9 and thus categorized as moderate knowledge. there was no difference between genders in knowledge about thalassemia (p=0.781, pearson chi-square) as depicted in table 1. w h e n e x p l o r i n g e a c h i t e m o f t h e questionnaire, only 37.7% knew the definition of thalassemia as a disease with low numbers of red blood cells. this was in line with the low 224 number of students who successfully mentioned the types of thalassemia (30.9%) and thalassemia as an autosomal recessive inherited disease (57.1%). despite their low percentage of general knowledge of thalassemia, most of the students knew that thalassemia was not infectious (93.1%) and required life-long blood transfusion (90.3%). they knew that thalassemia major is not curable (77.1%), but they did not know that iron chelators are needed for the iron overload (92.6%). the students knew that infants born as thalassemia carriers will remain carriers and do not develop into thalassemia major later in life (72.6%), but the majority of the students (81.1%) could not tell about the prevention of thalassemia carriers. furthermore, they did not know the estimated prevalence rate of thalassemia in indonesia (100%) and in west java (97.1%). when stratified by gender, there was no statistically significant difference in all of these answers between female and male students (data not shown). the majority of the students were of the opinion that premarital screening for thalassemia is necessary (99.4%). should they know that their partner is also a carrier, they would agree not to marry their partner (77.1%), and that premarital counseling would therefore be necessary (98.9%). termination of a pregnancy affected with thalassaemia major or abortion of the fetus was agreed to by 62.3%. among moslem students, 94 of 151 (62.2%) were for termination. we could not further compare the religious background for the termination of an affected fetus as the percentage of non-moslem students was low, with 13 of 17 protestants and 1 of 5 roman catholics being for termination. when the students were asked whether they would screen for thalassemia carrier status, 2 (1.6%) reported that they had indeed been screened for thalassaemia status, while 3 (1.7%) rejected screening and thought that it was not necessary as no one in the direct family line was affected. the majority of students (n=170; 95.6%), who had not been screened for thalassemia carrier status, indicated their willingness to undergo screening, but the timing of screening varied from soon after filling-in the questionnaire (n=67; 39.4%), some timing before getting married (n=69; 40.6%) to some timing in the future (n=34; 20.0%). when the willingness to undergo carrier status examination was compared between male and female students, no statistical difference was found between genders (p=0.584, pearson chi-square test, table 1). the results of further analyses of the knowledge and the timing of thalassemia carrier status examination indicated that the knowledge of these students had a statistically significant association with the timing of thalassemia status screening. therefore, higher thalassemia knowledge scores signify earlier thalassemia status examination (p=0.021, one way anova test) as depicted in table 2. on post-hoc analysis, there was a significant difference between mean table 1. knowledge among subjects about thalassemia and timing of thalassemia carrier status examination by gender note. knowledge criteria: good (>12 good answers), moderate (7-12 good answers) and poor (<6 good answers) out of 18 questions. statistical significance was set at p<0.05 (pearson chi-square) dewanto, tansah, dewi, et al knowledge of thalassemia early carrier 225 univ med vol. 34 no.3 knowledge scores of individuals who were willing to get their status examined directly after fillingin the questionnaire and mean knowledge scores of individuals who would postpone the examination until a later period in life (bonferroni p=0.025). students above 18 years old were considered to be young adults, but most students stated that they needed their parents’ consent before undergoing definitive thalassemia screening (84.6%), and should they finally know that they were carriers, most of them (95.4%) would persuade their siblings and parents to check for their thalassemia status to prevent the disease and decrease the number of thalassemia cases. one month after filling-in the questionnaire, the students were invited to have their blood examined for probable thalassemia carrier status. we expected that 67 students (table 1) would come for direct blood examination as stated in the questionnaire. surprisingly, the participation rate was higher, as there were 87 students who came up for the thalassemia carrier screening. laboratory hematology findings showed that 7 female students, corrected by gender according to who criteria, were identified as having mild anemia (hb 10-11 g/dl). on the basis of hb concentration only, the prevalence of anemia in these apparently healthy first year medical students in bandung was 8% (7 out of 87 students). interestingly, some of those who had normal hb, had low mcv (<80 fl) and mch (<27 pg) (table 3), so that 24 of 87 students (27.5%) had probable thalassemia carrier status, and therefore needed further examination for determination of definitive thalassemia carrier status. discussion thalassemia is prevalent in indonesia,(13) but studies on knowledge, attitude and practice related to thalassemia are relatively scarce. first year medical students in our medical faculty have lectures about ‘fundamentals of basic sciences’ including the central dogma of dna/rna/ table 2. relation between knowledge and timing of thalassemia carrier status examination among first year medical students in bandung note: statistical significance was set at p<0.05 (one-way anova) table 3. probable thalassemia carrier status among first year medical students in bandung stratified by gender, according to triple parameters of hb, mcv and mch note: * low hb corrected by gender according to who(8), low mcv <80fl, mch <27pg; hb hemoglobin; mch mean cospuscular hemoglobin; mcv mean corpuscular volume. in total there were 24 out of 87 (27.5%) students with probable thalassemia carrier status 226 protein, and thalassemia is introduced as one of the cases in the problem-based learning program. it is shown that most of the first year medical students (70.9%) have a moderate knowledge about thalassemia after the lecture, but the knowledge of a substantial percentage still remains poor. although the thalassemia knowledge level among the first year students is moderate, the median knowledge score of 9 out of 18 may reflect a general lack of knowledge among the students. the responses also indicated that specific knowledge on thalassemia was poor, with regard to the existence of different types of thalassemia, the genetic nature of the disease, and its pattern of inheritance. the limitation here is that the questions in the questionnaire may not be very understandable for first year students, therefore, the questionnaire may need further revision. owing to the genetic complexity of the disorder, it remains a challenge to educate the young adult. regular educational efforts are needed to raise awareness of thalassemia in young adults at school and at the university.(14) it is suggested to screen the hb in young adults so they can become aware of their overall health condition, specifically with the extended mcv and mch when hb is low. genetic counselling for thalassemia needs to be provided alongside with confirmation of the thalassemia carrier status, so the young adult carriers are reassured of their condition. the awareness of carrier status in the indonesian community has not yet been explored and this is reflected among the students as they do not know that the prevalence of the carrier status is quite high. in general, studies have shown that the awareness of thalassemia is higher in the higher educational and professional or managerial categories.(11) as for our students, this study confirmed that the knowledge is associated with the timing of examination for carrier status, thus, the better the thalassemia knowledge score, the sooner will they have their thalassemia status examined. in some countries, students and young adults have good responses for thalassemia screening.(4,14) in our study, a very positive attitude was expressed by the majority of the students toward screening for thalassemia, but what is of concern is that only one-third of the students would come for thalassemia status screening after the lecture, while two-thirds of the students would only want to be screened later in the future. therefore, the thalassemia carrier screening program for young and unmarried women should be included in the national programs, and this may dramatically reduce the incidence of infants born with thalassemia major. for example, a screening program in iran resulted in a 70% reduction in the annual birth rate of infants with thalassemia,(15) and interestingly it has been reported from italy that screening can reduce the number of births of affected infants in the latium native population to zero.(14) identification of carriers is also offered early in pregnancy in most developed countries due to high migration rates of populations from high thalassemia prevalence areas to west european countries.(16) undoubtedly these studies have shown that thalassemia prevention programs including premarital and prenatal screening have been cost-effective in reducing the thalassemia prevalence.(17) one should bear in mind that it is of utmost importance to dispel the misconception about thalassemia carriers, so that this genetic disorder is not stigmatized by society. the general public should be educated on the fairly high prevalence of thalassemia in indonesia. health care providers are encouraged to discuss thalassemia as a public health problem and therefore may enhance public awareness to prevent prejudice and discrimination against carriers. at the end of the study, a sharing experience about thalassemia and carriers was given by one of the researchers who is also a carrier. with her personal sharing, communication among the students was experienced as effective. the study by wong (2011) showed that audio-visual aids and personal experience sharing have been s u g g e s t e d a s a n e ff e c t i v e m e a n s f o r communication and education among young adults.(18) therefore, efforts to promote screening are of great interest to young adults. dewanto, tansah, dewi, et al knowledge of thalassemia early carrier 227 univ med vol. 34 no.3 thalassemia can be prevented by prenatal diagnosis, but selective abortion is not widely accepted by the public. the majority of the first year students in our university were of the opinion that premarital screening for thalassaemia is necessary and when they know that their partner is also a carrier, then they agree not to marry their partner, so that premarital counseling is needed. these findings confirm those of studies that premarital screening is necessary as one of the early screening strategies or genetic counselling,(2) and this can have an impact on families with thalassemia major. early screening in the extended family is also very important, especially where resources and budgets for screening are limited.(19) undoubtedly public education in the community regarding thalassemia can impact on the prevalence of thalassemia and can direct the policy in the population.(14) religion has a significant impact on the decision for screening and may also be associated with refusal for prenatal diagnosis and termination of an affected fetus.(11) in indonesian law, an abortion is only permitted if the pregnancy is likely to result in danger to the mother’s physical and mental health.(20) we did not analyze this issue further since the religious issue is sensitive in the society. given diversity in attitudes toward termination of a fetus in different religions, thalassemia prevention programs should consider the beliefs and preferences of individuals in the multiethnic society. when thalassemia prevention is offered to the population through carrier screening and premarital or prenatal diagnosis, then socio-economic, cultural and religious factors must be carefully taken into account.(11) this study has several limitations. this current study limits in-depth exploration of the reasons for delaying thalassemia carrier screening. further investigations in future qualitative studies may be of great benefit to explore the reason for this delay. although the questionnaire was adapted from a published article,(11) the translated questionnaire has not yet been validated. the small numbers of participants are not representative of first year medical students in indonesia per se, thus larger numbers from various regions in indonesia are needed to represent the general thalassemia knowledge of young adults from differing ethnic groups and religions in indonesia. despite the limitations of this study, there was a good response rate for the willingness to undergo laboratory screening. our study has increased awareness of thalassemia among these young adults and their education needs to be enhanced. carrier status screening at an early age in school and premarital screening programs aimed at identifying individuals before marriage has been proven to be effective and culturally acceptable when compared to prenatal diagnosis. the implication of this study is to increase the knowledge of young adults, especially first year medical students. it is therefore recommended that young first year medical students as future health care professionals need to have good knowledge for this disease, and in turn they can give better education in thalassemia prevention to the next generation. conclusions this study has identified that thalassemia is not well known among our young adults. a higher thalassemia knowledge score causes medical students to be willing to undergo thalassemia carrier status examination at an earlier point in timing. the results of this study warrant further evidence-based surveys on a larger scale to validate these findings and to eventually set a wellorganized educational program targeting young adult awareness of thalassemia and the complications of thalassemia therapy. conflict of interest the authors had no conflict of interest to declare. acknowledgements we are grateful to alvinsyah pramono for his help in this study. this work was supported 228 by research grant hibah internal fakultas kedokteran universitas padjadjaran 2014 no. 4142/un6.c/hk/2014. references 1. cao a, kan yw. the prevention of thalassemia. cold spring harb perspect med 2013;3:a011775. doi: 10.1101/cshperspect. a011775. 2. saffi m, howard n. exploring the effectiveness of mandatory premarital screening and genetic counselling programmes for â-thalassaemia in the middle east: a scoping review. public health genomics 2015;18:193-203. 3. verma ic, saxena r, kohli s. past, present & future scenario of thalassaemic care & control in india. indian j med res 2011;134:507-21. 4. rahimah an, nisha s, safiah b, et al. distribution of alpha thalassaemia in 16 year old malaysian students in penang, melaka and sabah. med j malaysia 2012;67:565-70. 5. gaziev j, marziali m, isgro a, et al. bone marrow transplantation for thalassemia from alternative related donors: improved outcomes with a new approach. blood 2013;122:2751-6. 6. saliba an, harb ar, taher at. iron chelation therapy in transfusion-dependent thalassemia patients: current strategies and future directions. j blood med 2015;6:197-209. 7. schoorl m, schoorl m, van pelt j, et al. application of innovative hemocytometric parameters and algorithms for improvement of microcytic anemia discrimination. hematol rep 2015;7:5843. doi: 10.4081/hr.2015.5843 8. benoist b, mclean e, egll i, et al. worldwide prevalence of anaemia 1993–2005: who global database on anaemia. geneva: world health organization; 2008. 9. viprakasit v, limwongse c, sukpanichnant s, et al. problems in determining thalassemia carrier status in a program for prevention and control of severe thalassemia syndromes: a lesson from thailand. clin chem lab med 2013;51:1605-14. 10. ip hw, so cc. diagnosis and prevention of thalassemia. crit rev clin lab sci 2013;50: 125-41. 11. wong lp, george e, tan ja. public perceptions and attitudes toward thalassaemia: influencing factors in a multi-racial population. bmc public health 2011;11:193. doi: 10.1186/1471-245811-193. 12. hashemizadeh h, noori r. premarital screening of beta thalassemia minor in north-east of iran. iran j ped hematol oncol 2013;3:210-5. 13. widayanti cg, ediati a, tamam m, et al. feasibility of preconception screening for thalassaemia in indonesia: exploring the opinion of javanese mothers. ethn health 2011; 16:483-99. 14. amato a, cappabianca mp, lerone m, et al. carrier screening for inherited haemoglobin disorders among secondary school students and young adults in latium, italy. j community genet 2014;5:265-8. 15. miri-moghaddam e, motaharitabar e, erfannia l, et al. high school knowledge and attitudes towards thalassemia in southeastern iran. int j hematol oncol stem cell res 2014;8:24-30. 16. giordano pc, harteveld cl, bakker e. genetic epidemiology and preventive healthcare in multiethnic societies: the hemoglobinopathies. int j environ res public health 2014;11:613646. 17. ahmadnezhad e, sepehrvand n, jahani ff, et al. evaluation and cost analysis of national health policy of thalassaemia screening in westazerbaijan province of iran. int j prev med 2012;3:687-92. 18. wong lp, george e, tan ja. a holistic approach to education programs in thalassemia for a multiethnic population: consideration of perspectives, attitudes, and perceived needs. j community genet 2011;2:71-9. 19. ansari sh, baig n, shamsi ts, et al. screening immediate family members for carrier identification and counseling: a cost-effective and practical approach. j pak med assoc 2012;62: 1314-7. 20. kementerian kesehatan republik indonesia. peraturan pemerintah republik indonesia nomor 61 tahun 2014 tentang kesehatan reproduksi. available at: http://www.kemenpppa.go.id/jdih/ peraturan/pp-nomor-61-tahun-2014-tentangkesehatan-reproduksi.pdf. accessed may 11, 2015. dewanto, tansah, dewi, et al knowledge of thalassemia early carrier c:\users\universa medicina\docu 29 abstract universa medicina increased oxidative stress in adult women with iron deficiency anemia alpaslan karabulut1, gulcin alp avci2, and emre avci3* background iron deficiency anemia (ida), a type of anemia with an increasing global frequency, is more common in women than men in the population. in ida, the sensitivity of erythrocytes to oxidants is increased and their lifespan is shortened. oxidative stress is an imbalance between free radicals and antioxidant molecules which is one of the potential biochemical mechanisms involved in the pathogenesis of ida. in our study, we aimed to determine the levels of oxidant and antioxidant markers by assessing the levels of total antioxidant status (tas), total oxidant status (tos), oxidative stress index (osi), paraoxonase-1 (pon-1), and myeloperoxidase (mpo) in women with ida. methods this was a cross-sectional study involving 47 women with ida aged 40 years and 47 women volunteers. the levels of tas, tos, osi, pon-1, and mpo were determined spectrophotometrically using appropriate kits. nonparametric mann whitney-u tests were used to analyze the data. results the levels of antioxidants tas (1.42 mmol trolox equiv./l) and mpo (54.00 u/l) in the ida group were significantly lower than in the control group [tas (1.67 mmol trolox equiv./l) and mpo (89.00 u/l)] (p=0.000 and p= 0.019, respectively). however, tos (6.25 μmol h 2 o 2 equiv./l) level in the ida group was significantly higher than in the control group (4.13 μmol h 2 o 2 equiv./l) (p=0.000), but pon-1 was not significantly different between the two groups (p=0.375). conclusion in women with ida, the oxidant-antioxidant balance is impaired, resulting in oxidative stress. therefore, ida in adult women must receive adequate attention in clinical practice. keywords: iron deficiency anemia, antioxidants, paraoxonase-1, myeloperoxidase, oxidative stress, women. original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.29-36 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1250 january-april, 2022 vol.41no.1 1hitit university, faculty of medicine, department of internal medicine, corum, turkey 2health sciences university, gülhane faculty of dentistry, department of basic medical sciences, medical microbiology, ankara, turkey 3health sciences university, gülhane faculty of pharmacy, department of biochemistry, ankara, turkey *correspondence: emre avci, university of health sciences, gülhane faculty of pharmacy, department of biochemistry, ankara, turkey, avci.emre@yahoo.com, emre.avci@sbu.edu.tr orcid id: 0000-0003-1046-8208 date of first submission, november 11, 2021 date of final revised submission, february 21 2022 date of acceptance, maret 3, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: karabulut a, alpavci g, avci e. increased oxidative stress in ad ult women with i ron defi cien cy anemia. univ med 2022;41:29-36. doi: 10.18051/univmed.2022.v41.29-36 30 karabulut a, alpavci g, avci e anemia in women and oxidative stress introduction anemia is called iron deficiency anemia (ida) when the iron that the body loses daily cannot be compensated for by the iron taken with food. iron deficiency is the most common cause of anemia in the world and is more common in women than men.(1,2) despite the decline in the prevalence of anemia worldwide, the burden of anemia remains consistently high in many regions. greater focus on interventions is needed to accelerate progress towards the global nutrition target 2, which is to reduce anemia in women of reproductive age by 50% by 2025 and to alleviate significant health losses in children under five.(3) the total amount of iron in the body is 4-5 grams, of which about 65% is in hemoglobin, 4% in myoglobin, 1% in heme compounds that facilitate intracellular oxidation, and 0.1% is associated with transferrin protein in blood plasma. in addition, 15-30% of iron is stored as ferritin in the reticuloendothelial system and liver parenchymal cells.(4) a fine balance exists between dietary intake and loss of iron and is normally maintained in a healthy person. the iron loss is approximately 1 mg daily through the shedd in g of ce ll s f ro m mu cosal a nd cutaneous surfaces, but in premenopausal female adults it may increase to about 2 mg t hr ou gh mens t r u a t i on . in a d dit i o n, i r on requirements are temporarily greater during neonatal and childhood growth spurts because of increased body mass.(5,6) with the formation of free radicals under normal physiological conditions, the antioxidant defense system is in a continuous state of alertness. antioxidants retain reactive oxygen species (ros) within normal limits and reduce their harmful effects.(7) if this equilibrium slips to oxidants, oxidative stress occurs, and it creates ros and damages the body.(8) reactive oxygen species plays a role in the pathogenesis of various diseases such as malignancies, systemic lupus erythematosus, coronary artery disease, rheumatic diseases, diabetes mellitus, and bechet’s disease.(9) recently, studies were performed on oxidant/antioxidant molecules. it is possible to measure the plasma levels of the oxidizing molecules separately, but this may not be appropriate, as these molecules can affect each other. therefore, total oxidant status (tos) level reflecting the oxidant status is used.(10-11) th e mea su remen t of t os a lon e is mo re enlightening than the individual measurements of the oxidants. for the same reason, instead of individual antioxidant measurements, total antioxidant status (tas) measurements are more widely used.(10) besides, the enzyme myeloperoxidase (mpo) is a lysosomal enzyme secreted from leukocytes in response to oxidative s tr es s. m ye lop er oxi da s e is a te t ra mer ic , glyc osylated, heme-containing protein.(1 2) myeloperoxidase produces free radicals and oxidants with its antimicrobial effects, thus causing oxidative damage in the main tissues in regions of inflammation.(13,14) an antioxidant, paraoxonase-1 (pon-1), which is synthesized by the liver, is transported in plasma depending on high-density lipoprotein (hdl). it ensures the anti-atherogenic features of hdl, by a voi d i ng lo w-d e n si t y l i p op r ot e i n ( l dl ) oxidation.(15) iron deficiency is an important health problem of women over the age of 40, which is frequently encountered in our country as well as all over the world and creates a certain burden on the health economy of a country. a cross-sectional study showed that mean paraoxonase-1 (pon-1) in the iron-deficiency anemia group was significantly lower than in the control group (102.4 ±19.2 u/l and 163.3 ±13.68 u/ l ; p =0. 00 0 ) . ( 1 6 ) a c r o ss -s e c t i o na l observational study among men and women with anemia and without anemia (i.e., healthy controls), showed that tas levels did not vary significantly among the categories (p=009).(17) however, another study among 26 subjects with ida and 20 healthy controls showed different results, the serum tas being significantly lower in patients with iron deficiency anemia than in controls (p<0.05).(18) there were inconsistent results of the previous studies, therefore, a study 31 was performed to compare tas between ida and healthy controls. in our study, it was aimed to evaluate more than one oxidative stress parameter together in female patients diagnosed with iron deficiency anemia. in this study, we planned to compare a number of oxidative stress markers (tas, tos, serum mpo, and pon-1 levels) between patients with ida and healthy controls. methods research design the present cross-sectional observational study was conducted at the internal medicine o ut p a t i e nt c l i n i c be t we e n j a nu a r y a n d december 2020. research subjects healthy female individuals who were not diagnosed with ida were included as the control group. the sample size determination comparing the means of pon-1 between two groups with effect size =0.6; α=0.05 and β=0,1, yielded a minimum sample size for each group of 45.(16) the most important clinical indicators in the diagnosis of ida for the inclusion of patients in t he s t u dy we r e : a ge 40 ye a r s a nd ove r, hemoglobin concentration hb <12 g/dl in women, transferrin saturation 15%, mean red blood cell volume <80 fl, serum iron concentration <45 g/ dl, and serum ferritin concentration <15 ng/ml as an internationally valid criterion.(19) the healthy females, who did not have the exclusion criteria and anemia, were included in the study as the control group. exclusion criteria were: i) usage of any medication, ii) chronic disease, iii) oral or parenteral iron treatment in the last 3 months, iv) presence of dimorphic anemia, v) history of blood transfusion within 3 months before the study, vi) systemic or local infection, and liver and renal diseases. blood sample collection the blood samples of the individuals included in the study were taken after 8 hours of fasting. the biochemistry panel was studied the same day, the blood sample taken for other markers was centrifuged at 5000 rpm for 5 minutes and the serum was separated and transferred to eppendorf tubes. the separated sera were stored at -20o c until the studies were carried out to determine the parameters that are indicators of oxidative stress. laboratory analysis complete blood counts of the individuals in the study group were studied with the beckman coulter lh 780 (miami, florida, usa) device. iron, iron-binding capacity, and ferritin from the collected samples were determined by the e n zyma t i c c ol o r i me tr i c me t h od i n a n autoanalyzer (olympus au 2700, mishima ol ymp u s co. lt d. j a p a n ) . tr a ns f e r r i n saturation was calculated using the formula: serum iron / serum iron-binding capacity x 100. folic acid and vitamin b12 levels were measured in all patients to exclude vitamin b12 and folic acid deficiency. de t e r m i na t i o n o f oxi dat iv e st r e s s parameters oxidant and antioxidant markers (tas. tos, osi, pon-1, and mpo levels) were measured in the individuals constituting the study groups. the total antioxidant status (tas) the tas of all individuals in the study group was determined spectrophotometrically using the automatic measurement method. with this test, the antioxidants in the samples reduce the dark blue-green, 2'-azinobis-(3-ethylbenzothiazoline6-sulfonic acid radical cation (abts(*+)), abts radical to the colorless reduced abts form. the change in absorbance at 660 nm observed in this reaction is associated with the total antioxidant level of the sample. the results are expressed as millimolar hydrogen peroxide equivalents per liter (mmol trolox equiv./l). 32 karabulut a, alpavci g, avci e anemia in women and oxidative stress c the total oxidant status (tos) the tos in serum samples of individuals belonging to the study groups was determined spectrophotometrically using an automatic measurement method.(10) with this test, the oxidants in the sample oxidize the ferrous iono-dianisidine complex to the ferric ion, and the oxidation reaction here is prolonged by the glycerol molecules in the reaction medium. the ferric ion forms a colored complex with the c h r o mo ge n x yl e n ol o r a n ge i n a n a c i d ic environment. here, a measurable color intensity occurs and the total amount of oxidant molecules in the sample is determined with the help of a spectrophotometer. the results are expressed as micromolar hydrogen peroxide equivalents per liter (μmol h 2 o 2 equiv./l). the oxidative stress index (osi) the osi, which is the indicator of oxidative load, was calculated according to the formula below and the results are shown in arbitrary units (au).(19) the paraoxonase-1 the pon-1 activity was measured using a kit, which consists of two different sequential reagents, namely tris-buffer as a cofactor of pon-1 enzyme and a stable substrate solution. the sample is mixed with reagent 1 and the substrate solution is added. the linear increase in absorbance of p-nitrophenol produced from paraoxon is examined. the non-enzymatic hydrolysis of paraoxon is subtracted from the total hydrolysis rate. based on the molar absorptivity rate of p-nitrophenol of 18,290 m-1 cm-1, one unit of pon-1 activity is equal to 1 mole of paraoxon hydrolyzed per liter per minute at 37°c.(20) the pon-1 results were expressed as u/l in serum. the myeloperoxidase (mpo) the spectrophotometric method was used to determine mpo activity. myeloperoxidase activity is evaluated by measuring the h 2 o 2 dependent oxidation of o-dianisidine. with this method, one unit of enzymatic activity is defined as the amount of mpo that causes a 1.0/min change in absorbance at 410 nm at 37oc.(19) statistical analysis statistical analysis was performed using the ibm spss statistics 20.0 software (ibm spss inc., usa). the normality of the resulting data distribution was investigated by the shapiro-wilk test. since the significance level of each parameter with the shapiro-wilk test was p<0.05, it was decided that the distribution was not normal (p=0.000-0.016). for this reason, nonparametric mann whitney-u tests were used. the critical significance level was determined at 0.05. all data are reported as median and interquartile range (iqr). ethical clearance for this study, we obtained written consent from the patients and approval from the local ethics committee of our hospital under no. 28.08.2019-32. results all demographic data belonging to patient and control groups are shown in table 1. according to the findings, age parameters were not found to be significantly different between ida patients and healthy individuals (u=262.500, p=0.211, p>0.05). however, for other all parameters, statistically significant differences between the patient group and control group were found (p<0.05). in our study, serum iron, transferrin saturation, and ferritin data were found to be significantly lower in patients with ida compared to healthy individuals in the control group. in addition, there was a significant increase in iron-binding capacity among the r e l a te d gr ou p s. w h e n w e c o mp a r e d t he complete blood count (cbc) parameters, mcv, mch, and mchc were found to be significantly lower in the patients with ida compared to the healthy individuals (table 1). oxidative stress parameter data of patients and controls are given 33 in table 2 together with the statistical analysis data. a significant between-group difference was observed for tas, tos, and mpo using the mann-whitney u test (p<0.005). the tas (1.67 mmol trolox equiv./l) and mpo (89.0 u/ l) levels in the control group were significantly higher than those in the ida patient group [1.42 mmol trolox equiv./l (p=0.000) and 54.00 u/l (p=0.019), respectively] (table 2). however, the tos level (6.44 μmol h 2 o 2 equiv./l) and the osi level (0.42 arbitrary unit) of the patient group was significantly higher than that of the control group (4.13 μmol h 2 o 2 equiv./l) and (0.24 arbitrary unit , respectively (p=0.000). moreover, for pon-1 data, a statistically nonsignificant difference was observed among these groups (p=0.375, which is greater than 0.05) (table 2). discussion iron deficiency anemia, one of the most common causes of anemia, is an important public health problem, especially in developing countries where nutritional problems are much more common.(20,21) this disease is mostly seen in women. menstruation, pregnancy, abortion, and curettage are the most frequent causes in women. in menopausal women, the reason for the emergence of iron deficiency anemia is assumed to be located in the digestive system until proven otherwise.(22) in this study, we planned to compare some oxidative stress markers (tas, tos, serum mpo, and pon-1 levels) between patients with iron deficiency anemia and healthy controls. in our study, tas levels were found to be statistically lower in the patient group. univ med vol. 41 no 1 parameters ida (n:47) (median, iqr) control (n:47) (median, iqr) p-value age (year) 37.0 (28.0-44.0) 31.0 (24.0-41.0) 0.211* hemoglobin (g/dl) 9.6 (7.2-11.0) 12.8 (12.5-13.8) 0.000 mcv (fl) 67.4 (60.4-77.5) 81.4 (78.783.3) 0.000 mch (pg) 20.9 (16.8-23.9) 26.9 (24.5-28.0) 0000 mchc (g/l) 30.1 (27.8-32.3) 33.2 (31.3-34.1) 0.000 serum iron (ug/l) 18.0 (16.0-25.0) 57.0 (41.0-93.0) 0.000 tibc (ug/dl) 446.0 (382.0-477.0) 370.0 (324.0-431.0) 0.001 transferrin saturation (%) 4.0 (3.0-6.0) 23.0 (13.0-34.0) 0.000 ferritin (ng/ml) 4.6 (2.2-8.7) 18.7 (9.9-42.2) 0.000 table 1. comparison of demographic and clinical parameters between patients with ida and control group ida: iron deficiency anemia; iqr: inter quartile range. critical statistical significance level at p>0.05. mcv : mean corpuscular volume ;mch : mean corpuscular hemoglobin ;mchc : mean corpuscular hemoglobin concentration; tibc : total ironbinding capacity variables ida (n=47) (median, iqr) control (n=47) (median, iqr) p-value tas (mmol trolox equiv./l) 1.42 (1.39-1.54) 1.67 (1.55-1.81) 0.000 tos (µmol h2o2 equiv./l) 6.25 (5.30-8.16) 4.13 (3.60-5.75) 0.000 osi (arbitrary unit) 0.42 (0.38-0.55) 0.24 (0.20-0.37) 0.000 mpo (u/l) 54.00 (38.0-74.0) 89.0 (53.0-120.0) 0.019 pon-1 (u/l) 272.00 (97.0-382.0) 147.0 (113.0-298.0) 0.375* table 2. median values of oxidative and antioxidants stress markers in the iron-deficiency anemia (ida) and control group iqr: inter quartile range; tas, total antioxidant capacity; tos, total oxidant status; osi, oxidative stress index; mpo, myeloperoxidase; pon-1, paraoxonase-1 activity; p=0.05, critical statistical significance level at p>0.05 34 karabulut a, alpavci g, avci e anemia in women and oxidative stress so me s tud i es h ave inc lud e d s i mil a r parameters. it is noteworthy that in most of the s t ud i e s , oxi d a t i ve s t r e s s i nc re a s e d , a n d antioxidant parameters decreased in these patients. in the case of oxidative stress, while sod, glutathione peroxidase (gsh-px), and glutathione reductase increase, antioxidant vitamins decrease, and tas can determine the r e a l e f f ec t . (2 3 ,2 4) in t he c a se o f ida, th e antioxidant capacities of erythrocytes are d e c r e a se d, a nd l i p id p e r o xid a t i on i s accelerated.(25) in ida, increased oxidative stress, an inadequate antioxidant system (such as sod, gsh-px) and hypoxic conditions may be attributed to increased superoxide release because of mitochondrial function disorders in skeletal muscle, heart, liver, and blood cells.(26) in the study of zaka-ur-rab et al.(27) levels of antioxidant enzymes were significantly lower in children with ida as compared to controls. in the study of aslan et al.(18) oxidative stress was investigated in 26 patients with ida and 20 healthy individuals. in this study, serum tas was found to be significantly lower in ida compared to healthy subjects. myeloperoxidase is considered an important oxidant marker in many diseases. in the studies conducted, it is accepted that lipid peroxidation plays a role in the pathogenesis of various disorders and diseases.(28) inhibition of lipid peroxidation, which causes changes in fluidity and permeability, inhibition of metabolic processes, and changes in ion transport and membrane organization, are important for antioxidants.(29) in our study, a significant between-group difference was observe d f or tas, tos, osi, and mpo. another antioxidant has been included in the studies recently, namely pon-1, which is a protective enzyme and an important molecule in the pathogenesis of many diseases.(30) in some studies, it has been observed that the level of pon-1, which is an important antioxidant marker of oxidant balance, is low in some diseases such as cardiovascular diseases, insulin resistance, and diabetes.(31,32) in a study by okuturlar et al.(17) pon-1 activity was investigated in 50 patients with iron deficiency anemia (ida) and 40 healthy individuals. they found that the mean pon-1 activity in the iron-deficiency anemia group was significantly lower than that of the control group, which was also found by merono et al.(33) similarly, in our study we observed a decreased level of pon-1 activity in patients with ida. a statistically significant difference in pon-1 data was not observed among these groups. in another study, it was stated that the oxidant-antioxidant balance was disturbed, and oxidative stress occurred during the anemia process in individuals with iron deficiency. the investigators also stated that the inadequacy of the high antioxidant activity of erythrocytes in patients with iron deficiency in the process of anemia may be attributed to the release of superoxide, which cr eate s mitoc hondrial dysfunctions in skeletal muscle, liver, heart, and blood cells.(34) an increase in oxidative stress may play a role in the pathophysiology of iron deficiency anemia. it has been stated that in individuals with iron de fic iency anemia, antioxidant supplementation together with iron therapy may provide better responses and early improvement of the symptoms related to iron deficiency anemia.(18) the present study has broad implications. low hemoglobin and hematocrit associated with iron deficiency anemia are closely associated with poor quality of life and performance. this is important in terms of demonstrating the effectiveness of oxidative stress, which plays a role in the processes affecting activities of daily living, and of the role of antioxidant support in overcoming this stress. in addition, anemia also influences the cardiovascular system. therefore, although these patients have risk factors for cardiovascular diseases, some biomarkers are important to obtain information about the process. on the other hand, low ferritin levels or transferrin saturation indicate an absolute or functional iron deficiency state. the difficulty of distinguishing chronic diseases accompanying iron deficiency anemia from the anemia itself limits this study. in future studies adding different 35 markers specific to iron deficiency anemia and oxidative stress is important in terms of the p at ho physi olo gy of t his di se a se a n d the prevention of the development of other diseases. conclusion this study demonstrated that adult women with ida are subject to chronic oxidative stress. therefore, it is important to determine the oxidative stress status from important markers to minimize the risk of ida in adult women. conflict of interest the authors declare that they have no conflicts of interest for this work. author contributions ea contributed to writing the manuscript, ea and gaa contributed to design and data collection. ak and gaa contributed to analyzing the da ta. ea contributed to revising the manuscript. all authors have read and approved the final manuscript. references 1. paternostro r, kapzan l, mandorfer m, et al. anemia and iron deficiency in compensated and decompensated cirrhosis: prevalence and impact on clinical outcomes. j gastroenterol hepatol 2020; 35: 1619-27. doi:10.1111/jgh.14988. 2. owaidah t, al-numair, n, al-suliman a, et al. 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gender-based analysis a 36 karabulut a, alpavci g, avci e anemia in women and oxidative stress pilot study. j evolution med dent sci 2020;9:373942. doi:10.14260/jemds/2020/821. 18. aslan m, horoz m, çelik h. evaluation of oxidative status in iron deficiency anemia through total antioxidant capacity measured using an automated method. turk j hematol 2011;28:42-6. doi: 10.5152/tjh.2011.04. 19. calim a, kanat e, mazi ee, oygen s, karabay u, borlu f. evaluation of in-patients with iron deficiency anemia in terms of etiology. med bull sisli etfal hosp 2020;54:428-32. doi: 10.14744/ semb.2018.47354. 20. jimenez k, lang m. diagnostic approach to iron deficiency anemia. wien med wochenschr 2016;166:402-10. doi: 10.1007/ s10354-016-0495-5. 21. chandyo rk, henjum s, ulak m, et al. prevalence of anemia and iron deficiency is more common in breastfed infants than their mothers in bhaktapur, nepal. eur j clin nutr 2016;70:456-62. doi: 10.1038/ ejcn.2015.199. 22. bayen s, le grand c, bayen m, 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relation to oxidative stress and response to iron therapy. pediatr nephrol 2015;30:1835-42. doi: 10.1007/s00467015-3122-6. 32. meneses mj, silvestre r, sousa-lima i, macedo mp. paraoxonase-1 as a regulator of glucose and lipid homeostasis: impact on the onset and progression of metabolic disorders. int j mol sci 2019;20:4049. doi: 10.3390/ijms20164049 33. meroño t, dauteuille c, tetzlaff w, et al. oxidative stress, hdl functionality and effects of intravenous iron administration in women with iron deficiency anemia. clin nutr 2017;36:552-8. doi: 10.1016/j.clnu.2016.02.003. 34. asci d, ekerbicer n, guvenc demiragci y, yilmaz r. oxidative stress investigation in anemia. j health med nur 2020;75:16-25. doi: 10.7176/ jhmn/75-03. c:\users\universa medicina\docu 190 abstract universa medicina epidemiological aspects of colorectal cancer in east azerbaijan, northwest iran: five year survival analysis ramin barnous1, mohammad hossein somi1, zohreh sanaat2, pooneh jabbaripoor1, neda dolatkhah3, and roya dolatkhah2* background colorectal cancer (crc) is the third most common cancer and the second leading cause of death from cancer in the world. currently, crc is the fourth most common cancer in men and the second common cancer in women of all ages in iran. the aim of this study was to determine the epidemiologic profile of crc along with crc specific survival analysis. methods this was an analytical cross-sectional study using the east azerbaijan population based cancer registry database (ea-pbcr) as a source for data related to patients with a diagnosis of crc. colorectal cancer specific 1to 5-year survival analysis and mortality rates were calculated. log-rank test and cox regression analysis was performed to test the equality of survival function and mortality hazard. results a total of 2,366 newly diagnosed crcs were registered during 3 years, with a male: female ratio of 1.31. overall survival rate was 49.8%. oneto 5-year survival rates were 96.21%, 56.94%, 48.62%, 47.88% and 46.76% respectively. at multivariate level, after adjusting for all variables, regression analysis showed that the hazard of mortality in stage iv cancers was 46.44 times higher than that in stage i cancers (hr=46.44, 95% ci: 14.86-145.14, p=0.000). however, differences in patients’ age group and sex and the subsite of cancer did not create any statistically significant variation between groups in regards to mortality hazards (p>0.05). conclusion this study demonstrated that the stage and grade of crc were important prognostic factors and that early screening and diagnosis of crc were essential. keywords: colorectal cancer, epidemiology, survival, prognosis, east azerbaijan original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.190-199 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1171 september-december 2021 vol.40no.3 cite this article as: barnous r, somi m h, sa na a t z, j a b b a rip oor p, dolatkhah n, dolatkhah r. epidemiological aspects of colorectal cancer in east azerbaijan, northwest iran: five yea r su rviva l an alysis. univ m ed 2021 ;40 :1 9 0-9 . doi: 10. 1805 1/ univmed.2021.v40.190-199 1liver and gastrointestinal diseases research center, tabriz university of medical sciences, tabriz, iran 2hematology and oncology research center, tabriz university of medical sciences, tabriz, iran 3physical medicine and rehabilitation research center, aging research institute, tabriz university of medical sciences, tabriz, iran *correspondence : roya dolatkhah, md phd postal address: hematology and oncology research center, tabriz university of medical sciences, tabriz, iran postal code/ p.o. box: 5166614731 telephone number: +98 4133361358 email: royadolatkhah@yahoo.com orcid id: 0000-0002-6897-7120 date of first submission, april 7, 2021 date of final revised submission, august 25, 2021 date of acceptance, august 30, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 191 univ med vol. 40 no 3 introduction according to the latest report of the international agency for research on cancer in 2020, colorectal cancer (crc) is the third most common cancer and the second leading cause of death from cancer in the world. (1) the incidence rate is three times higher in developed countries, but due to higher mortality in developing countries, there is a smaller gap in mortality rates.(2) currently, colorectal cancer is the fourth most common cancer in men and the second common cancer in women of all ages in iran. (3) globally three patterns are identifiable in relation to development when it comes to crc mortality and incidence trends: 1) an increase in incidence and mortality trends; 2) increasing incidence but decreasing mortality trends; 3) a decrease in mortality and incidence trends. (2,4) in iran there was an increase in the agestandardized incidence rates (asrs) for crc in 2012-2015, and their incidence is moving in an upward manner.(5-7) in east azerbaij an province crc is the second most common cancer in both sexes, with an overall incidence rate of 20.2 and 16.2 per 100000 respectively in men and women.(8) east azerbaijan province has the 4th place among iran’s provinces when it comes to crc asrs, and is the only province in north/northwest iran with an asr higher than 20 (asr= 20.77). however, the highest asr was reported from semnan (asr= 24.29), a province located in central iran, and the lowest is for kohkiloye-boyerahmad (asr= 6.39), in central iran.(6) comprehensive overviews revealed a significant increasing trend in crc incidence in iran, (5) but systematic analysis of survival rates in different regions of the country revealed that the prognosis of crc patients in iran was better than that in similar asian countries. the pooled 5-year survival rates were reported to be 33.6% in india and 34.3% in malaysia, which 5-year survival rate was estimated at 0.54 (95% ci: 0.490.58) in iran.(9) the highest 5-year survival rate was reported to be 84.6% from kermanshah (west iran), and the lowest was 27.02 from shira z ( south iran). on the othe r hand, multivariate model analysis revealed a significant improvement in the prognosis of crc patients during the study period.(9-11) since crc has a high incidence and cancer-related mortality in iran and east azerbaijan province, it is necessary to have an epidemiological study of the incidence, sex, age distribution and morphological types of colorectal cancer. the aim of the present study was to determine the most recent epidemiological profile of colorectal cancer along with 1to 5year colorectal cancer specific survival in east azerbaijan, northwest iran. methods research design this was an analytical cross-sectional study using the east azerbaijan population based cancer registry database (ea-pbcr) as a source for data related to patients with a diagnosis of colorectal cancer in east azerbaijan province. the ea-pbcr database includes the data from all newlydiagnosed cancer patients, from march 20th 2015 to march 19th 2017 (3 iranian solar years); encompassing the data collected from cancer related databases, including reports from pathology laboratorie s, hospital records, radiotherapy centers, and the death registry database. study subjects east azerbaijan is a province in northwest iran and is one of the most populated provinces in iran with a population of around 4 million people -at the time of performing this studymost of whom are of azeri ethnicity. as we used the eapbcr database, all newly diagnosed colorectal cancer patients during three years of study were included in the study, so we did not have any sample size determination. we included all cases with confirmed primary and newly diagnosed colorectal cancer for each year of the study from diff erent da ta sourc es. we excluded any duplicated cases by checking the patients’ national 192 identification number (nid), first name, family name and fathers’ name. also we evaluated any cancer cases with invasion, metastasis, or recurrence according to the morphology and behavior of the cancers, to avoid any missed and/ or duplicated cancer data. data collection this database includes patients’ data from three main aspects namely patient, tumor and data source. patient demographic information included name, nid, birth date, sex, place of residence, date of first diagnosis and fathers’ name. tumor information included the morphology (histology, behavior, stage and grade) and topography (primary site of tumor), the latter being coded and collected in accordance with the international classification of disease for oncology (icd-o3). also contact information and data sources were recorded. we performed statistical analysis and consistency check to ensure that the data were non-duplicated. follow-up data and the data related to patient outcomes were obtained through contacting patients/relatives, and from hospital information system (his), although these data were not available for all patients. outcome of the study the primary outcome of interest was colorectal cancer specific overall survival (os), defined as the time from crc diagnosis to date of death or date of last follow-up. also overall mortality rate and oneto 5-year survival rates were obtained. statistical analysis colorectal cancer specific 1to 5-year survival and mortality rates were calculated using stata mp 14.2 (stata corp lp, college station, texas 77845 usa). survival was calculated from the date of diagnosis to the date of cancer-related death, and/or the last follow-up time. the final follow-up date of the patients was september 30th, 2020. survival analysis was performed using the kaplan-meier method and life tables for 1to 5-year survival data. logrank test and cox regression were used to test the equality of survival function and mortality ha za rd (hr, with 95% c.i.) by age, se x, morphology, subsite, stage and grade. statistical significance was set at a value of p < 0.05. ethical clearance the ethics committee of tabriz university of medical sciences approved this study as a confirmed researc h proj ect (code: ir.tbzmed.rec.1398.1002). in line with the ethics rules of ea-pbcr, all patient information and records were kept confidential. results characteristics of the studied population the total number of crc patients diagnosed in this 3-year period was 2,366; of these, 701 (29.63%), 870 (36.77%) and 795 (33.60%) were diagnosed during 2 015, 2016 and 2017 respectively. the male: female ratio was 1.31 and 1344 patients (56.80%) were male and 1022 (43.20%) female. the mean age of the patients was 63.37 ± 14.26 years, with the youngest and oldest patients being 14 and 96 years old respectively, while 601 patients (25.40%) were in the 7th decade of their lives. data about the stage and the grade of the cancers wer e available in 1 722 and 1832 ca ses, respectively; 0.41% (7 patients) were in stage 0, 7.90 % (136 patients) in stage i, 23.98 % (413 patients) in stage ii, 35.71 % (615 patients) in stage iii and 32% (551 patients) were in stage iv. as for grading, 1023 cases (55.84%) were in grade i, 671 cases (36.63%) in grade ii and 138 cases (7.53%) were in grade iii of colorectal cancer. the most common morphological type was adenocarcinoma (ac) (n=1681, 71.05%). as for topography, the majority of cases (101843.03%) were not otherwise specified (nos), followed by cancers of the anus and rectum (72630.68%) (table 1). barnous, somi, sanaat, et al colorectal cancer in east azerbaijan 193 univ med vol. 40 no 3 a b c characteristic total cohort (n=2366) cohort with follow-up (n=1425) age (years) mean (±sd) range ≤50 >50 63.37 ± 14.26 14-96 318(13.44%) 2048 (86.56%) 62.48 ± 13.959 14-96 179 (12.56%) 1246 (87.44%) sex male female 1344 (56.80%) 1022 (43.20%) 821(57.61%) 604 (42.39%) subsite proximal colon distal colon rectum, anal canal nos 239 (10.10%) 383 (16.19%) 726(30.68%) 1018 (43.03%) 161(11.30%) 257(18.04%) 462 (32.42%) 545 (38.25%) morphology ac mucinous ac nos others 1681 (71.05%) 68 (2.87%) 530(22.40%) 87 (3.68) 996 (69.89%) 59 (4.14%) 311 (21.82%) 59 (4.14%) grade i ii iii 1023 (55.84%) 671 (36.63%) 138 (7.53%) total (n=1832) 739 (54.10%) 512 (37.48%) 115 (8.42%) total (n=1366) stage i ii iii iv 143 (8.30%) 413 (23.98%) 615 (35.71%) 551 (32.00%) total (n=1722) 70 (5.22%) 286 (21.34%) 495 (36.94%) 489 (36.49%) total (n=1340) table1. distribution of characteristics and pathologic parameters of total cohort and cohort with follow-up of studied population ac: adenocarcinoma; nos: not otherwise specified survival analysis due to lack of contact information for 941 patients, we were able to perform survival analysis for 1,425 cases only. of these, in the 5year follow-up period, 715 (50.2%) patients died, giving an overall survival rate of 49.8%. oneto 5-year survival rates were 96.21%, 56.94%, 48.62%, 47.88% and 46.76% respectively. the overall mortality rate was 19.99 per 100,000 people, and mean follow-up time was 35.29 months (95% ci: 34.21-36.38) (table 2). years of study case (%) no. of events* (%) mortality rate/100,000 overall survival (%) mean follow up time 2015 190 (13.33) 108 (15.10) 19.84 43.2 28.64 2016 566 (39.71) 254 (35.52) 16.96 55.1 34.55 2017 669 (46.94) 353 (49.37) 22.99 47.2 25.94 total 1425 715 19.99 49.8 35.29 table 2. summary of statistics of 1425 cases included in survival analysis during three years of study * event=death 194 as for survival functions, there was a significant difference in regard to the grade and stage of the cancers (p log-rank=0.000for both the grade and the stage of the cancer). also, the effects of morphological type (p logrank=0.000) and the subsite of tumor (p logrank=0.012) on survival functions, showed a statistical significance. however, the effects of age group (50 years and >50 years) and sex on survival functions was not statistically significant. (p log-rank= 0.538 and 0.562 respectively) (figure-1) detailed results of univariate and multivariate cox regression analysis are presented in table 3. figure 1: kaplan-meier survival curve, results for the test of equality of colorectal cancer-speciûc survival functions for the diûerent variables in northwest iran in (a) sex (b) age groups, (c) subsite groups, (d) grades, (e) stages and (f) morphologic types (a) (b) (c) (d) (e) (f) barnous, somi, sanaat, et al colorectal cancer in east azerbaijan 195 univ med vol. 40 no 3 cox regression analysis at univariate level showed that as expected, in comparison with lowe r cancer stages, higher stages had a significantly higher mortality hazard (hr=53.05; 95% ci:17.01-165.342; p=0.000) for stage iv vs. stage i. these effects are the same for cancer grading, as the mortality hazard for grade iii was 2.25 times higher than for grade i. (hr=2.25; 95% ci: 1.78-2.83; p=0.000) at multivariate level, after adjusting for all variables, regression analysis showed that the hazard of mortality in stage iv cancers were 46.44 times higher (hr=46.44; 95% ci:14.86145.14; p=0.000), in stage iii cancers 10.06 time higher (hr=10.09;95% ci:3.22-3160, p=0.000), and in stage ii cancers 3.35 times higher (hr=3.35;95% ci:1.04-10.83, p=0.043) than in stage i cancers. however, differences in age group, sex and subsite of cancer among patients did not create any statistically significant variation between groups in regards to mortality hazards. discussion the number of newly diagnosed colorectal cancers in east azerbaijan province during the 3-year period of stu dy was 2,366 ca ses. adenocarcinoma had the highest proportion (1681, 71.05%); cancers of the rectum and anal canal (726, 30.68%) were the most frequent. in east azerbaijan colorectal cancer ranked second among most common cancers both in men and women with an asr of 18.2 and 13.7 respectively.(8) this, alongside the increasing trend of the incidence of colorectal cancer in iran shows the importan ce of an extended epidemiologic analysis on colorectal cancer.(12) recently colorectal cancer was the third highest variable univariate cox regression multivariate cox regression* n (%) hr† 95% confidence interval p value hr* 95% confidence interval p value age (years) ≤50 179 (12.56) ref. ref. >50 1246 (87.44) 0.93 0.75 1.16 0.578 1.01 0.80 1.26 0.908 sex male 821 (57.61) ref. ref. female 604 (42.39) 0.96 0.82 1.11 0.601 0.86 0.74 1.00 0.058 subsite proximal colon 161 (11.30) ref. ref. distal colon 257 (18.04) 0.90 0.67 1.22 0.526 0.92 0.68 1.25 0.626 rectum, anal canal 462 (32.42) 1.05 0.80 1.37 0.693 0.92 0.68 1.17 0.443 nos 545 (38.25) 1.45 1.12 1.87 0.004 0.99 0.76 1.30 0.993 stage i 70 (5.22) ref. ref. ii 286 (21.34) 3.54 1.09 11.44 0.034 3.35 1.04 10.83 0.043 iii 495 (36.94) 10.77 17.01 33.71 0.000 10.09 3.22 31.60 0.000 iv 489 (36.94) 53.05 17.01 165.42 0.000 46.44 14.86 3.33 0.000 grade i 739 (54.10) ref. ref. ii 512 (37.48) 1.14 0.97 1.34 0.089 1.07 0.91 1.25 0.394 iii 115 (8.42) 2.25 1.78 2.83 0.000 1.24 0.98 1.57 0.069 morphology nos 311 (21.82) ref. ref. others 59 (4.14) 0.69 0.49 0.97 0.033 0.75 0.53 1.06 0.111 ac 996 (69.89) 0.38 0.32 0.45 0.000 0.54 0.49 0.71 0.000 mucinous ac 59 (4.14) 0.42 0.28 0.63 0.000 0.59 0.39 0.90 0.014 table 3. univariate and multivariate analysis with significant baseline characteristics (cox proportional hazards regression, breslow method for ties) notes: †unadjusted hazard ratio; *adjusted hazard ratio; **total p-value; abbreviations: hr: hazard ratio; ci: confidence interval; cox regression — breslow method for ties, p value 0.05; ac: adenocarcinoma; nos: not otherwise specified 196 incidence cancer in iran in terms of incidence, crude rate and age-standardized incidence rate (asir), which had a 41% increase since 2000, with a sex transition in the incidence pattern which was higher in women than men.(13) we aimed to determine the epidemiologic profile of colorectal cancer along with 1to 5-year colorectal cancer specific survival analysis in east azerbaijan, iran. colorectal cancer is the second among cancer-related deaths and occupies the third place in terms of worldwide incidence. as reported in globocan 2020, colorectal cancer deaths account for 9.4% of all cancer deaths in the world (935,000 deaths).(1) colorectal cancer has a higher incidence in developed countries(2) and there is also an increase in the incidence rate of several types of cancer, including colorectal, prostate and breast cancer in lo w and middle-income countries.(1,14) in iran, as a developing country, cancers are the third leading cause of death; there has been an increase in the incidence of colorectal cancers in iran ove r the past decade. (1 3) colorectal cancers have a higher incidence in central iran and the highest asr in both men and women has been reported from semnan, a province in central iran (24.29 and 16.64 per 100000 respectively), closely followed by east azerbaijan province which also has a high asr for colorectal cancer (20.77 and 15.69 for men and women respectively),(15) being second to stomach cancer in men and breast cancer in women.(8) as shown in figure 1-e colorectal cancer 5-year survival is greatly affected by the stage of the cancer, with survival rates for stages i and iv being at the extremes of the survival spectrum. similar results were found in different studies, from 90% 5-year survival rates for cancers being diagnosed in localized stages to nearly 10% survival for metastatic cancers.(16) survival rates for colorectal cancers have increased throughout the years.(16) even though the incidence is higher in developed countries, due to still lower survival rates in developing countries the overall mortality in developed and developing countries are somewhat close.(2) in asian countries, the overall 5-year survival rates of colorectal cancer is around 60%,(17) with china having recently reported 1-, 2-, 3-, 4-, and 5-year survival rates of 80.73, 74.12, 64.74, 61.37 and 52.37% for all crc patients, r espectively.(18 ) as per the surveillance, epidemiology, and end results (seer) database, 5-year relative survival rates for colon and rectal cancer for all stages combined were 63% and 67% respectively, in the united states.(19) our study yielded a 5-year survival rate of 49.8%, which was lower than in most developed countries but similar to other asian countries.(18) around 39% of colorectal cancers were diagnosed in the early stages.(16) the current study yielded somewhat similar results as the percentage of patients being diagnosed with colorectal cancers in stage 0, i, ii was 32.28%. these results show the importance of early screening and diagnosis of colorectal cancers. yet the lower percentage of patients in the earlier stages may be the reason for the somewhat higher mortality rates in the present study compared to china and the united states. stage, grade and morphology of the cancer are factors that seem to have an effect on the survival rate, as the mortality hazard for stage iv was 53.05 times higher than for stage i cancers. although the effect of grade was not as strong as that of the stage of cancer, still, grade iii cancers had a 2.25 fold higher mortality hazard than grade i. similar results were reported both in iran and in different parts of the world.(10, 20-24) as saadati et al. (10) and hassan et al. ( 23 ) repor ted, sex was a prognostic factor in colorectal cancers both in iran and malaysia, but in the majority of studies, sex did not have a prognostic value.(20, 25-27) we did not find a significant relationship between sex and mortality either; yet the incidence was higher in men than in women, with a male: female ratio of 1.31. this was similar to previous reports from east azerbaijan,(8) iran (15) and the world.(2) age was a prognostic factor mentioned in some studies, as higher age relates to poor prognosis and lower 5-year survival rate.(10,28,29) applying additive hazard models revealed that barnous, somi, sanaat, et al colorectal cancer in east azerbaijan 197 univ med vol. 40 no 3 increasing age of crc patients significantly increased the mortality hazard rate and remained in effect over the entire time of the period, signifying that age at first diagnosis of crc was not dependent on the time of diagnosis.(29) also during a systematic review of 28 studies from different parts of iran, 12 studies revealed an association between age and crc prognosis, in that about 12% of these patients were younger than 40 years.(11) however in another group of studies age was not reported as an important prognostic factor. (30,31) it has been elucidated that age at diagnosis has no impact on colorectal cancer specific survival rates, but has an impact on deaths due to other causes and on hazard ratio of age at first diagnosis of colorectal cancer.(32) however our results in relation to effects of age were similar to those of the latter group, as there was no significant difference between patients younger than 50 years and older than 50 years of age in terms of mortality hazard. the most important limitation of this study was the lack of contact information on some of the patients that limited the survival analysis to 60% percent of the diagnosed patients. another limitation was due to the stage of the disease not being a mandatory factor in the recent iranian national cancer registry program, and so staging was not available for all of the colorectal cancers. however, with the aim to achieve a more complete data set, another national program has recently been initiated and is ongoing in iran for cancer staging. the strength of our study lies in the fact that our work is based on the east azerbaijan populationbased cancer registry (ea-pbcr) which is one of the best and most reliable population-based cancer databases created in recent years in iran. overall, population-based cancer registries are a great resource for epidemiological studies about cancers and ea-pbcr is an ongoing project, helping to improve the quality of data available on cancer patients. our aim is to improve the quality indicators and coverage of ea-pbcr in the near future. in view of the recent improvement of prognosis and overall survival rates in most developed countries, due to effective and population-based early diagnosis and prevention programs, it is necessary to thoroughly determine the total impact of clinico-pathological factors on survival rates and prognosis of colorectal cancer patients. a priority of iranian health policy must be to develop an executive plan to identify the most appropriate risk assessment tool, screening method, and target age group for colorectal cancer. conclusion our data showed that the stage and grade of colorectal cancers are important prognostic factors. because the patients’ age and sex and the subsite of the cancers were not significant prognostic factors in this study, additional studies are needed, as they will be most helpful in determining the effect of cancer morphology and other clinic-pathological factors. conflict of interest the authors report no conflicts of interest in this work. acknowledgments we would like to acknowledge the support of the hematology and oncology research center for this study as an approved research proposal and dr ramin barnous’ doctoral thesis (thesis no: 60473; ir.tbzmed.rec.1398. 1002). contributors rb and rd contributed to conception and design, provision of study material or patients, data ana lysis and interpretation. mhs and zs contributed to administrative support. rb and pj contributed to collection and assembly of data. rb, rd, and nd contributed to manuscript 198 writing and reviewing. all authors approved the final manuscript. all authors take public responsibility for the content of the manuscript submitted to universa medicina. funding this study was supported and funded based on population based cancer registry program of east azerbaijan from the ministry of health and medical education, deputy of research technology (grant number: 700/1480,1395.10.4). data availability data are openly available in a public repository that issues datasets with the responsibility of the corresponding author. consent for publication as per the ethics rules of ea-pbcr, all patient information and records are confidential. availability of data and material the datasets analyzed and presented in this study are available from the corresponding author on reasonable request. references 1. sung h, ferlay j, siegel rl, et al. global cancer statistics 2020: globocan estimates of incidence and mortality 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cancer 2020;51:401-11. doi: 10.1007/ s12029-019-00275-0. 11. tahmasbi b, abedi g, moosazadeh m, et al. determining the survival rate of colorectal cancer in iran: a systematic review and meta-analysis. asian pac j cancer prev 2018;19:3009-18. doi: 10.31557/apjcp.2018.19.11.3009. 12. dolatkhah r, somi mh, kermani ia, et al. increased colorectal cancer incidence in iran: a systematic review and meta-analysis. bmc public health 2015;15:997. doi: 10.1186/s12889-015-23429. 13. khanali j, kolahi aa. national and subnational cancer incidence for 22 cancer groups, 2000 to 2016: a study based on cancer registration data of iran. j cancer epidemiol 2021;2021:6676666. doi:10.1155/2021/6676666. 14. young c, wood hm, seshadri ra, et al. the colorectal cancer-associated faecal microbiome of developing countries resembles that of developed countries. genome med 2021;13:27. doi: 10.1186/s13073-021-00844-8. 15. roshandel g, ghanbari-motlagh a, partovipour e, et al. cancer incidence in iran in 2014: 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nomogram to predict the overall survival of colorectal cancer patients: a multicenter national study. int j environ res public health 2021;18: 7734. doi:10.3390/ijerph18157734. 29. madadizadeh f, ghanbarnejad a, ghavami v, bandamiri mz, mohammadianpanah m. applying additive hazards models for analyzing survival in patients with colorectal cancer in fars province, southern iran. asian pac j cancer prev 2017;18:1077-83. doi: 10.22034/apjcp.2017. 18.4.1077. 30. hosseini sv, rezaianzadeh a, rahimikazerooni s, et al. prognostic factors of rectal cancer in southern iran. j gastrointest cancer 2020. doi: 10.1007/s12029-020-00553-2. 31. jalaeikhoo h, khajeh-mehrizi a, zokaasadi m, et al. sixteen years of experience with the treatment of advanced colorectal cancer in iran; a report from three institutions. middle east j dig dis 2018;10:160-8. doi: 10.15171/mejdd.2018.105. 32. moamer s, baghestani a, pourhoseingholi ma, hajizadeh n, ahmadi f, norouzinia m. evaluation of prognostic factors effect on survival time in patients with colorectal cancer, based on weibull competing-risks model. gastroenterol hepatol bed bench 2017;10:54-9. january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 nurses' knowledge, attitudes and practices related to physical restraint: the case from turkey selen ozakar akca1* and ahu pinar turan1 abstract background nurses are primarily for the care of patients and should ensure that physical restraint, which has many negative consequences, be applied according to standard practice. therefore, they should have knowledge about physical restraint and apply physical restraint by observing existing laws and ethical principles. this study aimed to determine the knowledge level, attitude and practices of pediatric nurses on physical restraint and identify the factors influencing physical restraint use. methods a cross-sectional study was conducted involving 150 pediatric nurses working in the pediatric clinic of a training and research hospital. the data were collected with an information form containing the introductory characteristics of the nurses and the level of knowledge, attitudes and practices of staff regarding physical restraints questionnaire. data were analyzed using the mann-whitney u test and the kruskal-wallis test. results overall, nurses had good restraint-related knowledge with positive attitudes, although the physical restraint practices of nurses were not the best and there were some deficiencies. a statistically non-significant difference was found between age, gender, education status, nurses' work unit, and training status regarding physical restraint on the one hand and knowledge, attitude and practice scores relating to the use of physical restraints on the other (p>0.05). conclusion pediatric nurses' level of knowledge about physical restraint was quite good and their attitudes were positive. however, there were some deficiencies in their practices. future training should therefore be emphasized to enhance nurses' ethical and sensitive thinking, increase their competence in applying physical restraint and working with patients. keywords: physical restraint, patient rights, child nursing, knowledge, attitude, practices 1hitit university faculty of health sciences, department of child health and diseases nursing. corum, turkey *correspondence: assoc. prof. selen ozakar akca hitit university faculty of health sciences, corum, turkey email: selenozakar@hotmail.com, selenozakar@gmail.com, selenozakar@hitit.edu.tr phone: +903642230730; fax: +9036422230731 orcid id: 0000-0002-6943-6713 date of first submission, july 20, 2022 date of final revised submission, january 24, 2023 date of acceptance, january 31, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 12 doi: http://dx.doi.org/10.18051/univmed.2023.v42:6-12-20 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1352 cite this article as: ozakar akca s, turan ap. nurses' knowledge, at titudes and practices related to physical restraint: the case from turkey. univ med 2023;42:12 -20. doi: 10.18051/ univmed.2023.v42:12-20. mailto:selenozakar@hotmail.com mailto:selenozakar@gmail.com mailto:selenozakar@hitit.edu.tr https://orcid.org/0000-0002-6943-6713 https://orcid.org/0000-0001-6560-4153 13 univ med vol. 42 no. 1 introduction physical restraint is defined as applying physical force to the patient’s body for a short time or using mechanical devices, materials and tools that the individual cannot easily remove.(1-3) physical restraint is applied when the patient’s behaviors are physically harmful and alternative methods are insufficient to protect the patient or the people around her or him.(3,4) physical restraint is used to support the proper posture of patients at risk of falling out of bed who are demented, delirious, agitated, or confused.(5) in addition, this practice is performed to keep patients under control, to prevent them from pulling out the connections such as tubes, drains and medical devices and to prevent them from harming themselves, to facilitate the application of medical treatments, and to help relieve them.(6) therefore, it is recommended to use physical restraint widely, especially in intensive care, neurology, psychiatry and pediatric clinics.(7) in the studies conducted in egypt (5) and czechia,(8) it is seen that physical restraint is widely used in health institutions to ensure patient safety. it is reported that physical restraint is widely used in the management of agitated patients, especially in intensive care units, in austra lia, (9 ) the usa (1 0) and eu ropean countries.(11) quality standards are mentioned in the health standards issued by the ministry of health of turkey. according to these standards, it is emphasized that the decision of physical restraint should be made by the physician and should be reviewed every 24 hours.(12) in physical restraint practices, nurses should seek physicians’ requests, show due care in keeping records, and know the physical and psychological complications of the practice.(7,13) it is emphasized that physical restraint can be started and terminated by the decision of the physician or in cooperation with the physician, and that physical restraint should be considered as a last resort in ensuring patient safety and continuity of treatment. therefore, before deciding on the application of physical restraint, nurses should consider alternatives that better comply with human rights than existing policies and procedures and should be aware of the psychological and physical effects that may occur in the patient due to physical restraint.(14-16) however, studies conducted in our country (17,18) reported that nurses do not receive a physician’s request in applying physical restraints and that the necessary care is not shown in this regard, the number of nurses who know the complications associated with physical restraint is low, and most of them resort to physical restraint practice in clinics.(14) these deficiencies indicate that more studies should be conducted on the use of physical restraints. physical restraint practice of nurses working in these clinics is seen as a mandatory intervention, especially since children in pediatric clinics have a risk of harming themselves and their environment. for this reason, the importance of studies on the knowledge, attitude and practices of nurses working in the pediatric clinic about physical restraint comes to the fore.(14,19,20) in the present study, it was aimed to determine the knowledge, attitudes and practices of pediatric nurses regarding physical restraint. methods research design the cross-sectional study was carried out in a training and research hospital between january and november 2019. research subjects the population of the study was composed of pediatric nurses working in a training and research hospital (n=161). in the study, it was aimed to reach the whole population, so the sample size calculation was not made. a total of 11 nurses were not included in the study, of whom 1 was not willing to participate in the study, 5 were on shift leave and 5 participated in our data collection pilot study. the study was completed with 150 nurses and 93.1% of the population was reached. 14 study inclusion c ri te ria are tha t the participant should work between january and november 2019, be a pediatric nurse, accept to participate in the study, should not have any communication prob lems, be eager and volunteering to participate in the study. nurses who did not work in the pediatric clinic and did not agree to participate in the study were excluded. instruments an introductory characteristics form and the level of knowledge, attitudes and practices of staff regarding ph ysical restr aints questionnaire were used to collect data in the study. the introductory characteristics form was prepared by the researchers in line with the literature and expert opinions.(1-4) this form deals with the nurses’ age, gender, education status, the unit they work in, the number of working years in nursing, and physical restraint training status. the level of knowledge, attitudes and practices of staff regarding physical restraints questionnaire was developed by by janelli (1994) and improved by suen (1999) and adapted for turkish society by kaya et al.(21) in the first section of this three-part scale, there are 11 items that measure nurses’ knowledge of physical restraint. in this section, 10 items are true and one item is false. correct responses are given a score of 1 point and incorrect responses a score of 0, with the scores ranging from 0 to 11. a high score indicates a “high level of knowledge”. the second section includes 12 items that deal with nurses’ attitudes towards physical restraint. in this section, participants are asked to respond on a four point likert scale as to whether they “strongly agree” 4 points, “agree” 3 points, “disagree “ 2 points, or “strongly disagree” 1 point with the 12 statements. in this section, the score varies between 12 and 48 points, the high score reflecting the positive attitude, the low score the negative attitude. section 3 contains 14 items that deal with the practices of nurses regarding physical restraint. in this section, responses are evaluated on a three-point likert scale with a score of 3 for “always”, 2 for “sometimes” and 1 for “never”. the section’s score limit is between 14 and 42. the highest score obtained from the section indicates the best practice. the test-retest total correlation coefficient of the original scale was found to be between 0.85-0.99, and in this study, cronbach’s alpha value for the total scale was 0.95. data collection the data of the study were collected from pediatric nurses who worked at the training and research hospital and agreed to participate in the study. explanations about the study were made by the researchers and written informed consent was obtained from the nurses. data collection forms were distributed to the nurses, who were asked to fill the data collection forms in 10-15 minutes in the hospital environment. in order to determine the intelligibility and application time of the questionnaire and the scale prepared for the research, a pilot study was made on 5 of the pediatric nurses, in which it was found that no changes were needed on the data collection forms. the data collected in the pilot application were excluded from the sample. ethical considerations in order to conduct the study, approval from ethics committee (28.02.2018/2018-27) and written permission from the training and research hospital, which was the institution where the research was to be conducted, were obtained. informed consent was obtained from the nurses who agreed to participate in the study, by providing information about the purpose of the study and the confidentiality of the information. statistical analysis statistical analysis was performed with spss (ve rsion 22.0) package program. categorical variables are presented as numbers and percentages, while numerical variables are presented as median and minimum-maximum scores. since the data did not show a normal distribution, in analyzing the median (min-max) scores of the scale according to the descriptive ozakar akca, turan physical restraint 15 univ med vol. 42 no. 1 characteristics of the nurses, the mann-whitney u test was used in the comparisons of two groups, and the kruskal-wallis h test was used in the comparison of more than two groups. statistically, the value of p<0.05 was considered significant. results the age of the nurses in the study was 22 to 29 years (48.0%) , 80.0% were women, 78.0% had a bachelor’s degree, 42.0% worked in pediatric clinics and 52.0% did not receive training in physical restraint. nurses’ sub-scale median scores from the level of knowledge, attitudes and practices of staff regarding physical restr aints questionnaire based on their introductory characteristics are provided in table 1. a statistically non-significant difference was found between age, gender, education status, the unit the nurse work in, and training status regarding physical restraint on the one hand and knowledge, attitude and practice scores relating to the use of physical restraints on the other (p>0.05) (table 1). according to nurses’ sub-scale median scores of the level of knowledge, attitudes and practices of staff regarding physical restraints questionnaire, their knowledge level median score was found to be 8.0, and the minimum and maximum scores were found to be 1 to 11. considering that the highest score that pediatric nurses can get is 11, it has been concluded that their knowledge on the use of physical restraint is quite good (table 2). it was determined that the attitude sub-scale median score of the level of knowledge, attitudes and practices of staff regarding physical restraints questionnaire was 31.0, and the minimum and maximum scores were 24 and 48, respectively (scale score range 12-48). the attitudes of pediatric nurses were found to be positive (table 3) considering that high scores in the attitude sub-scale show a positive attitude. characteristics n (%) knowledge attitudes practices age (years) 22-29 30-37 38-45 p value * gender female male p value** educational status high school diploma bachelor’s degree master’s degree or above p value * the unit the nurse work in pediatrics clinics child/ neonatal intensive care child emergency p value * training status regarding physical restraint received training did not receive training p value ** 72 (48.0) 57 (38.0) 21 (14.0) 120 (80.0) 30 (20.0) 18 (12.0) 117 (78.0) 15 (10.0) 63 (42.0) 48 (32.0) 39 (26.0) 72 (48.0) 78 (52.0) md (min-max) 8.0 (1-11) 9.0 (5-11) 8.0 (7-11) 0.671 9.0 (1-11) 8.0 (5-10) 0.291 7.5 (5-10) 9.0 (5-11) 8.0 (1-9) 0.662 8.0 (7-11) 9.0 (6-11) 7.0 (1-11) 0.104 8.5 (5-11) 8.0 (1-11) 0.541 md (min-max) 31.0 (26-48) 31.0 (25-48) 30.0 (24-40) 0.713 31.0 (24-38) 33.0 (27-39) 0.4041 29.0 (24-35) 31.0 (25-48) 29.0 (27-32) 0.481 30.0 (24-33) 31.0 (27-39) 32.0 (25-48) 0.411 30.5 (26-39) 31.0 (24-48) 0.552 md (min-max) 39.0 (29-42) 39.0 (32-42) 38.0 (30-39) 0.122 39.0 (29-42) 38.0 (32-42) 0.43 38.5 (37-40) 39.0 (30-42) 34.0 (29-39) 0.261 39.0 (32-41) 39.0 (30-42) 38.0 (29-42) 0.572 39.0 (30-42) 39.0 (29-42) 0.434 table 1. distribution for average scores of nurses’ levels of knowledge, attitudes, and practices regarding physical restraints based on some characteristics (n=150) note : data presented as median (min-max), *kruskal wallis test **mann whitney u test 16 scale division median minimum maximum scale limits knowledge 8.0 1 11 0-11 knowledge items i agree n (%) i disagree n (%) 1informed consent must be obtained from a family member when a restraint is applied to the patient. 108 (72.0)* 42 (28.0) 2restraint is practiced by professionals only. 108 (72.0)* 42 (28.0) 3the appropriate restraint should be determined based on the patient's condition. 102 (68.0)* 48 (32.0) 4the restraint should be attached to the edges of the bed. 135 (90.0)* 15 (10.0) 5the restraint should be loosened in 2 hours. 117 (78.0)* 33 (22.0) 6when the restraint is applied to the patient, the risk of skin integrity deterioration increases. 135 (90.0)* 15 (10.0) 7the patient should never be restrained face down because there may be a suffocation risk 114 (76.0)* 36 (24.0) 8there is no restraint method or device that we can say works very well in every condition. 129 (86.0)* 21 (14.0) 9when practiced, type of restraint, time of application, and reason for using restraint should be recorded in the nursing notes. 141 (94.0)* 9 (6.0) 10the patient has the right to appeal use of restraints. 87 (58.0)* 63 (42.0) 11restraint is practiced when the patient cannot be closely monitored by nurses. 27 (18.0) 123 (82.0)* table 2. pediatric nurses’ knowledge levels of physical restraint use *correct response table 3. pediatric nurses’ attitudes of physical restraint use scale division median minimum maximum scale limits attitude 31.0 24 48 12 48 attitude items i strongly agree n (%) i agree n(%) i do not agree n (%) i strongly disagree n (%) 1if i were the patient, i would have had the right to accept or refuse application of a restraint 0 (0.0) 33 (22.0) 84 (56.0) 33 (22.0) 2i feel guilty if i apply the restraint to the patient myself. 0 (0.0) 15 (10.0) 78 (52.0) 57 (38.0) 3i feel bad when one of the family members enters the room of a patient to whom a restraint was applied 15 (10.0) 108 (72.0) 24 (16.0) 3 (2.0) 4i feel bad when the patient is worse / angry after application of a restraint 39 (26.0) 69 (46.0) 18 (12.0) 24 (16.0) 5the patient experiences a decrease in selfconfidence after application of a restraint 15 (10.0) 90 (60.0) 36 (24.0) 9 (6.0) 6the application of a restraint reduces the duration of nursing care 6 (4.0) 78 (52.0) 51 (34.0) 15 (10.0) 7i think restraints decrease the rate of falls in patients 6 (4.0) 72 (48.0) 60 (40.0) 12 (8.0) 8i think that family members have the right to oppose application of a restraint 3 (2.0) 54 (36.0) 63 (42.0) 30 (20.0) 9the reason for using restraints in intensive care unit is the inadequate number of nurses 0 (0.0) 27 (18.0) 78 (52.0) 45 (30.0) 10i feel bad when the patient’s orientation deteriorates after application of a restraint. 15 (10.0) 69 (46.0) 78 (52.0) 18 (10.0) 11it is important for me and for my institution to apply the restraint while observing the legal measures. 9 (6.0) 90 (60.0) 45 (30.0) 6 (4.0) 12i think restraints increases the risk of suffocation in patients 0 (0.0) 15 (10.0) 93 (62.0) 42 (28.0) ozakar akca, turan physical restraint 17 univ med vol. 42 no. 1 in table 4, it was determined that the practice median score of pediatric nurses was 39.0 (29-42). considering that the minimum score that pediatric nurses can get is 14 and the highest score is 42, it has been determined that the physical restraint practices of nurses are not the best and that there are some deficiencies. discussion physical restraint is among the practice standards of various clinics in order to ensure patient safety and to create a therapeutic envi ronment.( 2 2 ,2 3 ) si n c e n ur se s ha ve a n important role in eliminating the negative c o ns e q u e nce s of p hysi c a l r e st r a i n t a n d preventing the physical and psychological complications that may develop, they must have sufficient knowledge, attitude and practical skills in this regard. in this study, conducted to determine the knowledge, attitudes and practices of pediatric nurses on physical restraint, it was determined that the majority (52.0%) of pediatric nurses do not receive training on physical restraint (table 1). it was determined that correct responses were given to most of the questions that evaluate the information level; however, they were not at a perfect level. for example, it is a sad finding that 42.0% of the pediatric nurses in the study said they do not agree with the statement “residents are allowed to refuse to be placed in a restraint”. it is a sad finding that 32.0% said they do not agree with the item that “the physical restraint suitable for the patient’s condition should be determined.” (table 2). although pediatric nurses’ knowledge level median scores regarding the use of physical restraints are at a desired level, it is still not the best, which indicates that nurses need in-service training. the knowledge level of nurses was scale division median minimum maximum scale limits practice 39.0 29 42 14 42 practice items always n (%) sometimes n (%) never n (%) 1i try different nursing interventions to prevent the patient from falling before applying a restraint to the patient. 87 (58.0) 60 (40.0) 3 (2.0) 2i apply a restraint to the patient only by physician’s directive 102 (68.0) 39 (26.0) 9 (6.0) 3i share my idea with the physician when i think the patient does not need to be restrained. 102 (68.0) 48 (32.0) 0 (0.0) 4if the patient has been restrained, i respond to the patient’s calls as soon as possible. 135 (90.0) 15 (10.0) 0 (0.0) 5i check the restraint every two hours to determine whether it is in the correct position. 120 (80.0) 30 (20.0) 0 (0.0) 6i check the skin of the patient with restraint in terms of friction or irritation. 132 (88.0) 18 (12.0) 0 (0.0) 7i inform family members why the patient was restrained. 138 (92.0) 12 (8.0) 0 (0.0) 8i inform the patient why the restrain is necessary. 120 (80.0) 27 (18.0) 3 (2.0) 9i inform the patient when the restraint will be removed. 111 (74.0) 36 (24.0) 3 (2.0) 10i check the restraint frequently to determine whether it has opened automatically 45 (30.0) 63 (42.0) 42 (28.0) 11when the restraint is applied, i record the type of the restraint, the reason for using restraint, application hour, and relevant nursing interventions in nursing notes. 60 (40.0) 87 (58.0) 3 (2.0) 12when the restraint is applied, i frequently check, evaluate, and record its effects. 117 (78.0) 27 (18.0) 6 (4.0) 13as the number of colleagues decreases, the number of patients with restraints increases. 0 (0.0) 27 (18.0) 123 (82.0) 14we try to find different ways of controlling the patient's movements in our institution rather than applying restraint. 132 (88.0) 18 (12.0) 0 (0.0) table 4. pediatric nurses’ practices of physical restraint use 18 found to be good in the studies by abd elhameed and elemam(24) and karagozoglu et al.(17) who investigated the knowledge, attitudes and practices of nurses regarding the use of physical restraints. however, in two similar studies conducted by kisacik et al.(18) and cui et al.(25) it was determined that the knowledge level of nurses about physical restraint was low. it is thought that this difference may be due to the importance given to physical fixation in countries and hospitals and the differences in education. the findings of our study have similarities to those of abd elhameed and elemam(24 ) and kar agozoglu et al. (17) however, in line with the findings of all these studies and the present research, it is necessary to increase the knowledge level of all nurses in order to correctly fulfill the knowledge level, attitude and practices of nurses regarding physical restraint. in our study, it was determined that the pediatric nurses’ attitudes towards the use of physical restraint were positive. the attitude of the person about the subject-matter is one of the most basic elements of the use of physical restraints. in this study, it is a promising finding that pediatric nurses’ positive attitudes towards the use of physical restraints will also reflect on the quality of nursing care practices. in the literature, it is seen that nurses’ attitudes towards the use of physical restraints differ. in this study in which nurses’ attitudes towards the use of physical restraints were investigated and in the studies by karagozoglu et al.(17) and kassew et al.(26) it was determined that nurses have a positive attitude towards the use of physical restraint. in contrast, mehrok et al.(22) and wang et al.(27) observed that nurses’ attitudes towards the use of physical restraint were negative. in addition, in our study, the fact that nurses did not feel guilty when applying restraints to the patient, did not see the lack of staff as the main reason for physical restraint, and took on the roles of caregivers and decision makers imply that they displayed a professional attitude rather than an emotional approach to the psychological consequences of physical restraint use. however, the fact that pediatric nurses were found to feel bad when a family member entered the room of a patient with restraint, and that nurses’ attitudes were found to be negative about the thought that self-confidence would decrease in patients with restraints suggest that nurses have dilemmas about the practice of physical restraint. it is a saddening finding that 90% of pediatric nurses stated that they do not agree and strongly disagree in response to the statement that “i believe that restraints increase the risk of strangulation.” which is an indication of how serious the nurses’ lack of knowledge on the subject was in this study. in the study, it was determined that the practices of pediatric nurses regarding the use of physical restraint were positive. in this study, showed that nurses did not attach the necessary importance to the process of recording, which is a legal assur ance f or nurses. in ma ny studies(3,24,27) parallel to this study, it was observed that the nurses’ physical restraint practice scores were positive, but were not perfect, and it was found that nurses’ physical restraint practices were inadequate considering the distribution of practice regarding the use of physical restraint by pediatric nurses. for example, it was found that nurses paid less attention to checking frequently in order to determine whether the restraint could be released, and when the restrainer was applied, to record the type of the restraint in the nursing note, the reason for using it, the time of practice, and the necessary nursing interventions. in the literature(7,24) it is seen that there are results of studies that are in line with those of this study. there are limitations in this study that need to be taken into consideration. first, the participants were working in pediatric wards. theref ore, the study results may not be generalizable to other settings. second, the design of our study was cross-sectional, and therefore no causal relationship can be inferred based on our r esults. further, collecting data via questionnaires may be subject to social desirability response bias. ozakar akca, turan physical restraint 19 univ med vol. 42 no. 1 in this direction, it is suggested that more studies should be conducted to determine the needs and shortcomings of nurses regarding physical restraint, and to support nurses with training programs. it is recommended to develop a standard implementation procedure and guideline for physical restraint in health institutions. conclusions this study demonstrated that pediatric nurses’ level of knowledge about physical restraint was quite good and their attitudes were positive, and they mostly reflected these in their practices. however, it was observed that the nurses within the scope of the study had shortcomings in checking the restraint and recording it in the nursing note when the restraint was applied. conflict of interest there is no conflict of interest. authorship contributions soa and apt contributed to writing the manuscript, soa and apt contributed to design and data collection. soa and apt contributed to analyzing the data. soa contributed to revising the manuscript. all authors have read and approved the final manuscript. acknowledgments we thank the pedia tr ic nurses who participated in this study. funding the author (s) received no financial support for the research, authorship and/or publication of this 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is the most influential risk factor of cardiopulmonary endurance in older women rita khairani1 , donna adriani2, and patwa amani3 background pulmonary function decreases with age. similarly, physical exercise capacity measured by maximal oxygen uptake, which is one indicator to measure the level of cardiac and pulmonary vascular resistance, decreases by >1% per year. this results in many respiratory diseases that occur in older persons, especially in obese older persons. pulmonary function is a basic component of cardiopulmonary endurance, besides other factors such as age, hemodynamics, metabolism, and lifestyle. the objective of this study was to determine whether body mass index (bmi) and pulmonary function have a significant association with cardiopulmonary endurance in older women. methods the study used a cross-sectional design involving 66 subjects aged 60 years and older in south jakarta. data was collected by measuring body mass index (bmi), pulmonary function (fev1, fvc, fev 1 /fvc) and maximal oxygen uptake (vo 2 max) for cardiopulmonary endurance. data analysis used simple and multiple linear regression tests in spss v.25, with p<0.05. results of the 66 older persons, 57.6% was obese. pulmonary function of subjects showed restriction (33.3%), obstruction (6.1%) and a combination of restriction and obstruction (1.5%). furthermore, mean vo 2 max was 12.2 ml/kg/min. multiple linear regression analysis showed a significant association of bmi and fev 1 /fvc with vo 2 max (β =-0.56; 95% ci=-0.81 (-) 0.31; p=0.000; β =0.09; 95% ci=0.01-0.18; p=0.028). bmi was the most influential risk factor of vo 2 max (beta = -0.46). conclusions there was a significant association of bmi and fev 1 /fvc with vo 2 max, bmi being the most influential risk factor of cardiopulmonary endurance in older women. keywords : pulmonary function, body mass index, maximal oxygen uptake, obesity, elderly women original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.254-262 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1215 september-december 2021 vol.40no.3 cite this article as: khairani r, adriani d, amani p. obesity is the most influential risk factor of cardiopulmonary endurance in older women. univ med 202 1;40 :2 5 4-6 2 . doi: 10 .18 05 1/ univmed.2021.v40.254-262. 1department of internal medicine, faculty of medicine universitas trisakti, jakarta 2department of physiology, faculty of medicine universitas trisakti, jakarta 3department of physiology, faculty of medicine universitas trisakti, jakarta correspondence: rita khairani faculty of medicine universitas trisakti jl kyai tapa 260 grogol jakarta, indonesia phone: +62 82124004364 email : rita.khairani@trisakti.ac.id orcid id : 0000-0001-7831-0816 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license date of first submission, august 14, 2021 date of final revised submission, december 2, 2021 date of acceptance, december 6, 2021 255 univ med vol. 40 no 3 introduction the population of older persons is increasing rapidly in the world, including in indonesia. within a span of five decades, the percentage of the older population (aged >60 years) in indonesia has doubled to 9.6%, or around 25 million people from 1971 to 2019.(1) in old age, the body experience changes in biologic al and psychological processes due to aging, which results in susceptibility to degenerative diseases, including those related to the respiratory system. in healthy aging, changes in respiration usually do not lead to serious pulmonary problems.(1) the changes in old age related to the respiratory system include changes in lung parenchymal tissue and structural changes of the thoracic cage, pulmonary function abnormalities, air ventilation and diffusion problems, and decreased respiratory muscle strength leading to low exercise capacity, but these will not cause serious problems, such as airway obstruction or pulmonary parenchymal disease, due to the sufficient functioning of pulmonary reserves capacity.(2,3) in addition, older persons may also experience a decrease in lung stretch capacity caused by a decrease in the elasticity or recoil of the lung parenchyma and chest cavity. this condition is associated with a decrease in energy expenditur e of the r espiratory system. (2 ) pulmonary function tests measure lung volume, capacity and rate of flow, and some measurements such as forced expiratory volume in one second (fev 1 ) and forced vital capacity (fvc) which decreases along with increasing age. meanwhile, pulmonary capacity remains the same. in addition, forced vital capacity may decrease due to an increase in residual volume and decreased diffusion capacity.(2,4) cardiopulmonary endurance is defined as the ability of the circulatory, respiratory, and muscular systems to efficiently deliver oxygen and nutrients into the cells through the blood vessels during physical activity. maximal oxygen uptake (vo 2 max) is one indicator to measure the level of cardiac and pulmonary vascular resistance, which can determine a person’s physical exercise ability.(5) aging is associated with a progressive decrease in physical activity capacity. in addition, maximal oxygen uptake ( v o2 ma x) d e c r e a se s by > 1% pe r ye a r depending on the individual capacity of physical activity.(3,6) furthermore, the main problem is a decrease in the maximal oxygen uptake rate or vo2 max, caused by an increase in the ratio of dead space to ventilation volume and the differences in oxygen levels between the alveoli and the arteries that occur during physical activity.(7) pulmonary function is a basic component of cardiopulmonary endurance although several other factors also are of influence, such as age, hemodynamics, metabolism, and lifestyle. several studies have been conducted to assess the correlation between age and pulmonary function, such as the study conducted by roman et al.,(8) which found that age can cause a reduction in pulmonary function that cannot be overcome by physical exercise. decreased pulmonary function can cause limitations in ventilation and reduce the capacity for physical activity. a study by park (9) showed opposite findings, where high-intensity aerobic exercise had a positive effect on pulmonary function in older women. low birth weight, ambient air pollution, obesity, a nd asthma were confirmed as determinants of lung function impairment among never-smokers.(10) several studies have reported a reduction in lung volume and capacity in obese individuals as compared to the non-obese.(11-13) one cross-sectional study by liu et al.(14) showed that there was no evidence that body mass index (bmi) is associated with vital capacity in college students. a study of white morbidly obese subjects with normal lung function demonstrated that increasing levels of bmi were significantly associated with decreasing po2 and increasing pco2 even after adjustments for age, gender, and smoking habit (pack-years). this study also found no association between alterations of vital capacity and bmi.(15) 256 another study involving 60 young healthy male subjects in the age group of 18 to 22 years showed a highly significant negative correlation between obe sity an d v o2 max (r=-0.88 p<0.05).(16) the epi health cohort study involving 12.791 women, aged 45–75 years, showed that in the absence of central obesity, a high bmi is not related to restrictive lung function impairment but rather to higher lung function.(17) because the results of these previous studies were inconsistent, therefore a new study was deemed necessary to determine which variables were correlated with cardiopulmonary endurance. the present study aimed to assess the characteristics of older persons and to find out whether bmi and pulmona ry function are assoc iated with cardiopulmonary endurance in the elderly. methods research design this observational analytic study using a cross-sectional desi gn was conducted in mampang prapatan district, south jakarta, from january–may 2018. research subjects the sample size was determined based on the results of a preliminary study showing that the pearson correlation coefficient (r) between bmi and vo2 max was 0.49 (data not shown). using r=0.49, α=0.05 and β=0.1 plus anticipated dropout rate of 15%, the optimum sample size was 54. the inclusion criteria were: i). men and women aged 60-76 years; ii) independent; able to walk without assistive devices; iii) and having signed the consent form. the exclusion criteria were: i) having a history of cardiovascular diseases ii) unable to complete the 6-minute walking test. a consecutive non-random sampling method was used to select the research subjects. written informed consent was obtained from all subjects prior to participation. measurements demographic data, including age and gender, wer e obtained through inter views. other parameters measured were bmi, blood pressure, pulmonary function, and the 6-minute walking test. body height was measured using a height measuring device (microtoise), with an accuracy of 0.1 cm, while body weight was measured using a stamping scale, with an accuracy of 0.1 kg. bmi was calculated as body mass divided by height squared (kg/m2). based on bmi, the subjects were categorized as underweight (bmi <18 kg/m2), normal weight (bmi 18.5 – 22.9 kg/ m2), overweight (bmi 23 – 24.9 kg/m2), obesity i (bmi 25 – 29.9 kg/m2), and obesity ii (bmi 30 kg/m2).(18) furthermore, blood pressure was measured using a c alibrated mercury sphygmomanometer. the measurement of blood pressure was conducted with the subject in a sitting position following standard procedures after taking a rest for at least 5 minutes before the test. blood pressure was measured in millimeters of mercury (mghg), consisting of systolic blood pressure (sbp) and diastolic blood pressure (dbp).(19) pulmonary function tests pulmonary function tests including forced expiratory volume in one second (fev 1 ), forced vital capacity (fvc) and the ratio of fev 1 to fvc (fev 1 /fvc) were performed using a mir spirolab spirometer, based on american thoracic society criteria. briefly, the pulmonary function test was conducted with the subject in a standing position following standard procedures and at least three maneuvers were performed, with the largest fvc and the largest fev 1 observed from all of the acceptable values being reported. fev 1 is forced expiratory volume in one second, i.e. the volume of air exhaled in the first second under force after a maximal inhalation, while fvc is forced vital capacity, i.e. the total volume of air that can be exhaled during a maximal forced expiration effort. the fev 1 /fvc ratio is the percentage of the fvc expired in one second.(20) cardiopulmonary endurance the data on maximal oxygen uptake were collected using a predictive formula by calculating the results of a 6-minute walking test, and utilizing khairani, adriani, amani obesity and cardiopulmonary endurance 257 univ med vol. 40 no 3 a b the nury formula for healthy indonesian adults (mongoloid). this test is performed on a straight 15 meter long track with markers every 3 meters, 30 centimeters wide to the right and left from the center line. the subject are to go straight forward as close to the center line as possible. when the subject has arrived at both ends, the subject rotates with the three-step method. the nury formula which consists of five variables is more applicable because it does not require any measurement tools or specific competency.(21) statistical analysis the collected data were analyzed with the shapiro-wilk normality test and the results showed tha t all va ri ables were normally distributed. simple and multiple linear regression were used to analyze the relationship between severa l independe nt var ia bles and cardiopulmonary endurance as the dependent variable using spss v.25 program. statistical significance was set at p<0.05. ethical clearance the research was approved by the faculty of medicine researc h ethics committee universitas trisakti under no.119/ker/fk/xii/ 2017. results a total of 66 subjects was recruited during the study. table 1 shows that the majority of subjects were older women (98.5%) with mean age of 64.8 ± 4.1 years. based on bmi, it was found that 57.6% were obese with bmi of 25, that around 24.2% had normal weight and 16.7% were overweight, and that only 1.5% were underweight. blood pressure assessment showed that the majority of subjects had normal blood pressure (90.9%). the average pulmonary function was still above 70% in fev 1 , fvc, and the ratio of fev 1 / fvc parameters, thus the average subject still had normal pulmonary function. in addition, around 33.3% had restriction (fvc<70%) and 6.1% had obstruction (fev1/fvc<70), and only 1.5% had a combination of restriction and obstruction. on cardiopulmonary endurance, the obtained vo2 max was a minimum of 1.3 ml/kg/min and a maximum of 21.1 ml/kg/min with a mean of 12.2 ml/kg/min. in simple regression analysis, bmi, dbp, fev1 and fev1/fvc were significantly associated with vo2 max (β=-0.67; 95%ci=0.92–(-)0.49; p=0.000; β=0.08; 95%ci=0.010.11; p=0.016; β=0.06; 95%ci=0.01–0.10; p=0.013; β=0.15; 95%ci=0.06–0.25; p=0.02; respectively), but in multiple linear regression after adjustment for age, only bmi and fev1/fvc were significantly associated with vo2 max (β=0.56; 95%ci=-0.81–(-)0.31; p=0.000; β=0.09; 95%ci=0.01-0.18; p=0.028; respectively). bmi was the most influential risk factor of vo2 max (beta=-0.46)) compared to fev 1 /fvc (beta =0.23) (table 2). discussion in this study, the majority of subjects were older women with a mean age of 64.8 years. according to data from the central bureau of statistics, the number of older women is one percent higher than that of older men (10.10%:9.10%). in addition, the majority (63.82%) in the cohort were the younger old (aged 60-69 years).(1) table 1. characteristics of the subjects (n=66) notes: § data are presented as n (%); afev 1 = forced expiratory volume in one second; bfvc = forced vital capacity characteristics mean ± sd age (years) 64.8 ± 4.1 body weight (kg) 59.2 ± 9.6 body height (cm) 152.2 ± 6.8 body mass index (kg/m2) 25.5± 3.8 systolic blood pressure (mmhg) 124.8 ± 16.1 diastolic blood pressure (mmhg) 79.0 ± 15.3 fev1a § 75.9 ± 8.8 fvcb § 74.7 ± 10.2 fev1/fvc § 84.5 ± 9.1 vo2 max (ml/kg/min) § 12.2 ± 3.6 normal § 39 (59.1) restriction (fvc<70%) § 22 (33.3) obstruction (fev1/fvc < 70) § 4 (6.1) combination of restriction and obstruction § 1 (1.5) 258 based on bmi, more than half (57.6%) of the subjects were obese (bmi 25). physiological functions in older persons will decrease due to the aging process promoting the occurrence of degenerative diseases. factors affecting the health of older persons include unhealthy eating habits since early in life and lack of exercise. these main factors will affect susceptibility to diseases in old age.(22) our study showed that obesity is the most influential risk factor of vo2 max in the elderly and that obesity significantly decreases cardiopulmonary endurance. this finding confirms a previous report showing that bmi was negatively associated with cardiorespiratory fitness among 151 adolescent girls in south africa.(23 ) in the obese, excess fat in the abdominal cavity and chest limits the two primary inspiratory movements: contraction of the diaphragm propelling the abdominal contents downwards and forwards, and increased chest diameter by means of rib movement.(24) the results of this study confirm the presence of a restrictive pattern in obese individuals when they demonstrate a decrease in lung volume and capacity. furthermore, age, obesity and comorbidity were identified as a significant predictor of functional decline in future. a previous study has reported that bmi could be one of the independent factors for functional limitations and disabilities due to reduced muscle mass and strength (in those with sarcopenic obesity), such as increased joint dysfunction, limited activities, being prone to falls, table 2. factors associated with vo 2 max in elderly women using simple and multiple regression analysis (n=66) note : multiple linear regression adjusted for age; β: regression coefficient; beta: standardized regression coefficient; ci: confidence interval having chronic pain, resulting in low quality of life.(25) both obesity and aging are characterized by low-grade systemic inflammatory conditions and endocrine changes. aging is associated with increased adipose tissue. this is the primary cause of low-grade systemic inflammation signified by cytokin e elevation, r educed respiratory capacity and its resultant decrease in body immune status. this is the major cause of chronically elevated levels of pro-inflammatory cytokines and is associated with reduced muscle strength and decreased immune function.(26) this study showed that most of the subjects still had good pulmonary function. about 40.9% experienced either restriction, obstruction, or a combination of both, as recorded by spirometry. due to the aging process, pulmonary function gradually decreases, as recorded by either fev 1 , fvc, or peak expiratory flow. pulmonary function will peak at the age of 20–36 years and will begin to decline along with age, namely about 20 ml/ year, starting at the age of 25 years. in addition, it will decrease even more to 38 ml/year after the age of 65 years.(27) the results of a cohort study, conducted for more than 10 years in subjects having no previous history of pulmonary disease, indicated that the decrease in fev 1 ranged from 17.7 to 46.4 ml/ year with a median of 22.4 ml/year. in addition, in men the decrease is more rapid (median of 43.5 ml/year) than in women (median of 30.5 ml/year).(18) furthermore, results of another study on subjects aged 25–75 years that examined the various factors affecting pulmonary function khairani, adriani, amani obesity and cardiopulmonary endurance variables simple linear regression multiple linear regression β 95% ci p value β beta 95 % ci p value age bmi sbp dbp fev1 fvc fev1/fvc -0.58 -0.67 0.04 0.08 0.06 0.01 0.15 -2.64 – 1.48 -0.92 -0.49 -0.05 – 0.06 0.010.11 0.01 – 0.10 -0.030.03 0.06 – 0,25 0.577 0.000* 0.884 0.016* 0.013* 0.978 0.002* -0.56 0.18 0.03 0.09 -0.46 0.06 0.17 0.23 -0.81 -0.31 -0.420.07 0.010.07 0.010.18 0.000* 0.547 0.100 0.028* 259 univ med vol. 40 no 3 indicated that pulmonary function significantly decreased along with increase in age. the decline can occur more rapidly in subjects aged over 60 years and remain constant throughout ages 60– 90 years.(28,29) cardiac and pulmonary endurance is defined as the ability of the heart and lungs to supply oxygen throughout the body in the long term. it is measured by vo2 max, the maximal value of oxygen uptake, distribution, and utilization in the body during physical activity. the vo2 max is an indicator to assess health status including the risk of heart disease in older persons.(30) the present study found a mean vo2 max of 12.25 ml/kg/min as measured by the 6-minute walking test. when compared with the mean vo2 max based on age, the vo2 max value obtained was very poor for subjects aged 56–65 years or >65 years.(2) the subjects were mostly older women who have 40% lower vo2 max than older men due to their lower percentage of muscle tissue and higher percentage of body fat from their total body weight. furthermore, hemoglobin levels are different between older men and women. lower hemoglobin levels in older women lead to lower blood transport capacity and less oxygen uptake in women so that their physical activity capacity is lower than that of older men.(9,30) the blood oxygen-carrying capacity declines slightly with age, around 10% from 30 to 80 years and in females the decline is much lower than in males.(31) it contributes to the decline in vo2 max in old age. furthermore, if a person does not do regular exercise, the value decreases more drastically. the combination of aging and sedentary lifestyle dramatically affects oxygen delivery and utilization, and influences vo2 max capacity. regular exercise improves oxygen extraction by muscle activity and increases vo2 max. furthermore, the 30% increase in vo2 max found in the elderly is due to an increase in cardiac output (70%) and oxygen extraction (30%) by skeletal muscles.(6,32) the results of our study indicated that fev 1 / fvc was significantly associated with vo2 max. these results are similar to those of the study conducted by fatemi et al.,(33) in which 51 subjects divided into three groups of vo2 max level (high, medium, and low) suggested a significant difference (p<0.001) in pulmonary function as assessed by measuring the values of fev 1 , fvc and fev 1 /fvc, where the lower the pulmonary function, the lower the level of vo2 max. the measurement of pulmonary function can be influenced by several factors, such as height and weight, measurement position, respiratory muscle strength, the condition of the lung, and thoracic cavity expansion. several factors that can affect vital capacity are age, sex, height, physical exercise, and possibly ethnicity and comorbidities. the spirometry results also depend on the effort and consistency of the subject.(34) doewes et al.(35) assessed the correlation of pulmonary function, hemoglobin, and heart rate with vo2 max in athletes of several sports. they showed that the contribution of the respiratory system (tidal volume) to vo2 max was 12.32%, which was far below that of the cardiovascular system (36.97%) and the oxygen transport system (49.29%). the contribution of the respiratory system depends on the type of sport performed. the largest contribution was found in swimming, followed by badminton. meanwhile, the smallest was found in volleyball. the insignificant association between other pulmonary function parameters and vo2 max may also have been caused by obesity because the mean bmi of the subjects was in the obesity category. the results of the multiple regression analysis between bmi and vo2 max indicated a significant negative association, meaning that the higher the bmi, the lower the vo2 max. body mass index also has its limitations in assessing body composition because it cannot distinguish between body fat and lean mass. furthermore, it cannot provide the adiposity index, weak distribution, and metabolic risk. in addition, the bmi score can be influenced by age, gender, and ethnicity.(36) 260 there are some other factors that affect lung compliance in obesity. the position of the diaphragm in the thoracic cavity is obviously elevated when the individual gains weight. the change will result in a decline in pulmonary function and in the extra work of breathing. fat accumulation on the chest wall will impede the movement of the thoracic cage by a direct resistance or the abnormal function of the intercostal muscles. obesity increases the release of inflammatory markers in the lung, with slight effect on airway diameter, is related to the lung volumes not airway obstruction.(12) an increase in age and bmi may cause a decrease in vo2 max. decreased pulmonary function is associated with decreased respiratory muscle strength and decreased elasticity of pulmonary tissue. physical exercise will increase vo2 max due to oxygen consumption and concurrent increase in muscle performance. the ability of muscles to generate aerobic energy optimally will increase the maximal oxygen uptake capacity.(6,35) the beneficial effects of 12 weeks sprint interval training among obese subjects on vo2 max while concurrently decreasing body fat distribution was found by bagley et al.(36) after completion of the 12-week exercise, there was an increase in vo2 max of 18.7% in women and 6.0% in men. men and women both improved their vo2 max, but the increases among women were greater than in men. a higher amount of lean body mass was found in men as compared to women, which partially explains the differences between men and women in maximal oxygen capacity. maintaining physical activities in older persons is an integral part of the healthy aging process and minimizes multi-pathologies. decreased pulmonary function may cause limitations in physical activity that contribute to low activity tolerance and compliance in healthy aging, especially in older persons who experience gradual lowering of their internal capacity.(8) this study has several imitations. first, bmi takes no account of body fat distribution as an indicator of obesity. differences in fat distribution may cause changes in lung function. second, the examination of pulmonary function is also influenced by the effort and consistency of the subjects following the aforementioned procedures with all their limited internal capacities. in addition to the respiratory system that affects vo2 max, other factors, such as the cardiovascular and oxygen transport systems, were not investigated in the study. this study showed that obesity in older persons especially women and decreased pulmonary function may cause limitations in physical activity that c ontr ib ut e to l ow a ct ivit y to le r a nc e an d compliance in healthy aging, especially in older persons who experience gradual lowering of their internal capacity. in future, it is recommended that chest, abdominal and hip circumferences or their ratios are used instead of bmi. other factors that affect cardiopulmonary endurance in older persons should also be investigated. conclusions the study demonstrated that increased bmi correlates with decreased cardiopulmonary enduran ce i n th e eld erly women. it a lso reinforced the tendency that women in the older p op ul a t i o n wi t h sa r c o pe ni c obe s i t y a n d decreased pulmonary capacity will be more susceptible to both non-communicable and communicable diseases. conflict of interests the author(s) declared no potential conflicts o f in te re st w it h re sp e ct to th e re se ar ch , authorship, and/or publication of this article. acknowledgement the authors wish to express their gratitude to the faculty of medicine, universitas trisakti, for the funding of this study. khairani, adriani, amani obesity and cardiopulmonary endurance 261 univ med vol. 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and the rates of fatty acid oxidation and vo2 max during exercise. bmj open sport exerc med 2016;2: e000056. doi:10.1136/bmjsem-2015-000056. c:\users\universa medicina\docu 104 abstract universa medicina relationship between depression and physical disability by gender among elderly in indonesia ana satria1, yeni1, hairil akbar2, henny kaseger3, linda suwarni4, abubakar yakubu abbani5,6, and maretalinia5,7 background the increasing population of older people can be a positive impact if they could be productive, or it could be negative as a burden if they depend on others. elderly with physical disabilities can be influenced by various factors, including depression, which can be caused by functional changes in the body due to aging, loss of work, or even the loss of a loved one. this study aimed to determine the relationship between depression and physical disabilities among the elderly in indonesia who were stratified by gender. methods a cross-sectional study using a national survey dataset namely basic health survey and socio-economic survey for 2018 involving 85,427 elderly. depression and disability were assessed using mini international neuropsychiatric interview and barthel index. chi-square test and multiple binary logistic regression were used to analyze the data. results the results revealed that 24.75% of elderly people experienced physical disabilities. the probability of males and females with depression having physical disabilities was respectively 2.95 (95% ci = 2.74-3.17) and 2.49 (95% ci = 2.36-2.64) times higher compared to those without depression. the interaction between depression and gender was statistically significant, in that females with depression had an 11% probability of having physical disability. conclusion this study reaffirmed that disability is a risk factor for depression in the elderly and female gender is the effect modifier rather than the risk factor. the male depression group showed more physical disability symptoms than the female depression group. there is collaboration from various sectors to prevent depression and physical disabilities. keywords: depression, physical disability, elderly, 2018 basic health survey original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.104-113 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1277 may-august 2022 vol.41 no.2 1department of public health, faculty of public health, universitas sriwijaya, indonesia 2department of public health, faculty of health science, institut kesehatan dan teknologi graha medika, indonesia 3nursing program, faculty of health science, institut kesehatan dan teknologi graha medika, indonesia 4department of public health, faculty of health science, university of muhammadiyah pontianak, indonesia 5ph.d. program in demography, institute for population and social research, mahidol university, thailand 6department of demography and social statistics, federal university birnin kebbi, nigeria 7department of public health, university of mohammad husni thamrin, jakarta, indonesia correspondence: maretalinia faculty of public health, university of mohammad husni thamrin jl. raya pondok gede no.23-25 jakarta timur 13550, indonesia phone: (+62)82376690768 email: mareta.linia.21@gmail.com orcid id: 0000-0003-0250-7721 date of first submission, january 9, 2022 date of final revised submission, april 22, 2022 date of acceptance, may 5, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: satria a, yeni, akbar h, kaseger h, suwarni l, abbani ay, maretalinia. relationship between depression and physical disability by gender among elderly in indonesi a. uni v med 20 22 ;41 :1 0 41 3 . doi: 10 .18 05 1/ univmed.2022.v41.104-113. 105 univ med vol. 41 no 2 introduction aging is the decreased ability of biological organs to maintain and rebuild their structure and function normally.(1) one of the health issues among the elderly is psychological, including depression. depression is a disability of human feeling which disturbs the sleeping pattern and appetite, as shown by decreased concentration, anhedonia, and attempt at suicide.(2) physical disability among the elderly is the condition when the elderly cannot independently, routinely, and universally do their activities of daily living.(3) life expectancy is one indicator to assess the public health index. from 2000 to 2016, global data showed that the combined life expectancy for males and females has increased by 5.5 years to 72 years.(4) the elderly population is increasing due to the high quality of life.(5) however, the dependency ratio increased to 18.4 in 2019.(6) as a result of demographic transition in indonesia, several impacts have occurred, such as increasing numbers of those aged 60 years and over, declining fertility and mortality, modernization in all aspects of life, the practice of good disease prevention programs, and increasing life expectancy.(6) the indonesian population pyramid in 1950, 1990, and 2020 showed an increased number of elderly, while the projections for 2030, 2050, and 2100 are showing a rapid increase in elderly.(7) population aging is defined as a significant decline of fertility level in a certain group of people, that leads to infertility and mortality, and the increased proportion of elderly is calculated by comparing the number of the older population and the total population.(8) indonesia has an aging population, since the proportion of elderly was 9.6% in 2019 or very close to 10%. the structure of the older population shows that the proportion of older people has increased over time. the quantity and well-being of the elderly can indicate the quality of health, economic, and social status in a country. the adults of reproductive age are also important to support the economic development as well as the elderly’s quality of life. the indonesian ministry of health has categorized indonesia as an aging c ountry sinc e 2017 because the proportion of elderly has reached 9.03%.(9) in the past five decades, the population of elderly in indonesia had increased twice from 1971 to 2019, from 9.6% or around 25 million which consisted of 1% more e lderly wome n than men. (1 0) according to ministry of social affair data, as of 31 march 2020, there were 6,452 staff supporting 176,283 older individuals, of which 20,000 individuals were receiving home care; 2,655 receiving therapy services, and 7,000 were under family support.(11) overall, indonesia had 9.6% elderly in 2019 which had increased from 9.03% in 2017.(12) in 2015, every 100 persons of working age had to support 13 persons aged less than 15 or more than 60 years.(10) in the same year, the indonesian ministry of health reported that 28.62% of the elderly were likely to be at higher risk of morbidity due to non-communicable diseases.(9) one of the human development indexes in indonesia is the life expectancy, which increased to 71.2 years in 2018.(6) the increasing life expectancy also affects the number of elderly. according to data from statistics indonesia, the population of elderly in indonesia in 2019 was 25,901.9 million. (1 0) t he worl d health organization reported that about 280 million elderly in the world are depressed at an average age of 60 years.(13) in indonesia, the prevalence of adults more than 15 years old is 6.1% and 91% do not have access to health services.(14) the dependency ratio is also related to the vulnerable physical condition to perform the activities of daily living.(15) the common result found from previous studies was age as the mediator of correlation between depression and physical disabilities. it was revealed that the risk of disabilities is higher for older people because of their declining physical function. the correlation between depression and age may arise from medical reasons, because higher age leads to a higher risk of cognitive function and dementia. however, depression and age are not exactly positively linked since one 106 study found that younger participants endorsed a higher level of depression.(16) in contrast with previous studies, one indonesian study found that severe functional status has the greatest influence on depression in old age.(17) the attractiveness of this new topic is shown by the fact that there is a study that examined the correlation between gender, depression, and disabilities,(18) the results of which are still controversial because some researchers assert that the correlation between gender and depression is variable throughout the life span.(18) the lack of studies examining the association between depression and physical disabilities among the elderly stratified by gender led the authors to conduct this study. this study aimed to examine the relationship between depression and physical disability by gender among the elderly in indonesia. methods research design this was a quantitative study of crosssectional analytic design. the basic health research is the official representative survey which is conducted every 5 years in 34 provinces, 416 districts, and 98 cities. the original survey on which the present study was based, was conducted from april to may 2018. research subjects the subjects of this study were persons aged 60 years or older. the study sample was selected from the sampling designed by susenas (survei sosial ekonomi nasional). the original survey was the cross-sectional primary health research (riskesdas 2018) that had been conducted by the ministry of health, indonesia. the data are closed national data from: https:// labmandat.litbang.kemkes.go.id/menu-layan. the primary health research 2018 study was a largescale multi-topic household and community survey that covered 34 provinces on a national scale, in which were visited 300,000 households from 30,000 census blocks selected by probability proportional to size sampling. this sampling method has been selected by two-stage linear systematic sampling. the total number of respondents in this study was 85,427 elderly aged more than 60 years. the inclusion criteria for the sample used in this study were: elderly 60 years or older who completely answered the questions about depression and physical disabilities. the exclusion criterion was elderly who did not answer the questions completely. the present study used supporting variables such as age, gender, marital status, place of residence, employment status, and history of chronic diseases. instruments the physical disabilities variable is defined by using the barthel index questionnaire for activities of daily living.(19) this index uses the ordinal scale with 10 items (d24 to d33) to measure the functional indepe ndence for activities of daily living and mobility. the total score of this variable is 20. the interpretation of assessment is as follows: 20 (independent), 1219 (slight dependen ce), 9-11 ( moder ate dependence), 5-8 (severe dependence), and 0-4 (total dependence). the depression variable was defined by using the mini international neuropsychiatric interview questionnaire (mini icd-10).(20) this questionnaire is a tool to diagnose the main psyc hiatric pr oblems in the international classification of diseases and consists of 10 questions (items c01-c10) with a total score of 10. interpretation of the assessment: a score of 4 or higher is defined as depression and a score of less than 4 is defined as not having depression. statistical analysis this study conducted the univariate, bivariate, and multivar iate analyses. t he univar iate analysis aims to describe the sociodemographic information of the subject by using frequency, percentage, median, and mean. the bivariate analysis aims to determine the association between stress and disability and the association between confounders and disability. satria, yeni, akbar, et al relationship between depression and physical disability 107 univ med vol. 41 no 2 a b the bivariate analysis was done by using the chisquare test. finally, the multivariate analysis aims to examine the association between depression and disability by controlling the confounders, namely age, gender, educational level, occupation, and place of residence. the analysis was done using spss software. ethical clearance ethical approval for the use of secondary data was obtained from the health research ethics committee, faculty of public health, universitas sriwijaya under no. 019/un9.1.10/ kke/2020. results the results of this study were categorized into bivariate, and multivariate analysis. table 1 below describes the cross-tabulation by gender and the bivariate results. the data were presented as frequency. among 85,427 elderly who were eligible to be included in this study, about onefourth of them had physical disabilities. in total, 24.75% of the study sample experienced physical disabilities. the crude odds ratio (cor) explains the correlation between each independent variable and physical disabilities. the results were categorized by gender to explore the differences between males and females who have depression regarding the physical disabilities. the probability of those males and females with depression of having physical disabilities was respectively 2.95 (95% c.i. 2.74-3.17) and 2.49 (95% c.i. 2.362.64) times higher than those without depression. in terms of age, male and female elderly who were older than 70 years had a higher probability of respectively 1.97 (85% c.i. 1.87-2.06) and 2.10 (95% c.i. 2.02-2.19) times for having physical disabilities compared to those aged less than 70 years. males with low educational level have a higher probability of physical disabilities compared to fe male s (1.24 and 1.33, respectively). males without a job had a higher probability of physical disability (2.45 and 1.92, respectively). females who lived in urban areas had a higher probability of physical disabilities (1.07 and 0.95, respectively). females who were single or divorced had a higher probability of physical disability than did males (1.50 and 1.39, respectively). females with a history of chronic diseases had a higher probability of physical disability than did males (1.54 and 1.47, respe ctively). ge ne ra lly, among seven independent variables, five of them showed that the probability of physical disability was higher for females than males. the multiple binary logistic regression has been done to examine the relationship between depression and physical disability by controlling the confounders. table 2 below presents the results of the multivariate analysis. it shows in detail that depression is significantly associated with physical activity, after controlling for other independent variables. compared with the elderly without depression, the elderly with depression significantly 2.64 (95 % c.i. 2.45 2.84) times more likely to have physical disabilities. compared with elderly aged 61 to 69 years, elderly aged 70 or above tend to be 1.74 ( 95% c.i. 1.68 1.80) times more likely to have a physical disability. in terms of educational level, compared to the elderly who had high educational levels, the elderly who had low educational levels were 1.15 times more likely to have physical disabilities. after controlling for other independent variables, compared to elderly who worked, elderly who did not work were 1.78 times more likely to have physical disabilities. additionally, compared with married ones, single or divorced elderly were 1.13 times more likely to have physical disabilities. then, compared to the elderly who did not have a history of chronic disease, the elderly who did have a history of chronic disease were 1.42 times more likely to have physical disabilities. the interaction between gender and depression was included in this study, and it was found that compared with males without depression, females with depression had a decreased probability of 11% (aor= 0.89;95% c.i. 0.81-0.98) of having a physical disability. however, the variables of gender and place of residence did not show a significant association with physical disability. 108 t ab le 1 . t h e ch ar ac te ri st ic s o f re sp o n d en ts a n d e ac h i n d ep en d en t v ar ia b le r el at ed t o p h y si ca l d is ab il it y s tr at if ie d b y g en d er satria, yeni, akbar, et al relationship between depression and physical disability c o r : cr u d e o d d s ra ti o ;c i : c o n fi d en ce in te rv al 109 univ med vol. 41 no 2 table 2. the multiple binary logistic regression of physical disability and the interaction between females and depression lr chi2 (9) = 5618.24, prob > chi2 = 0.000, pseudo r2 = 0.0588, log likelihood = -44988.776 aor: adjusted odds ratio; ci: confidence interval discussion depression is a mental illness that can be coped with by spiritual practices as shown by the study that found a relationship between spirituality and depression.(21) in an aging society, social participation is imp ortant for cognitive functioning.(22) a study in korea found that comorbidity such as anxiety and depression is increasing the risk of physical disorders and disability after a follow-up of over two years.(23) the covid-19 situation also influences the emotional and social experience of older people in england and as a result, it also affects physical disabilities.(24) physical disability itself is also inf luenc ed by seve ra l f actors including malignancies, female gender, and depressive mood.(25) in line with the previous study, the study by lamonica et al. (16) found that depressive symptoms were the most significant variable related to disability.click or tap here to enter text. the role of gender was found by the study of mahwati (21) in indonesia who revealed that females comprise the largest population that is prone to depression. similar to the results of our study, mahwati (21)also found that the variables age 70 years, low educational level, unemployed, unmarried, and multimorbidity were risk factors of depression.) however, mahwati’s (21)study did not include physical disabilities as one of the dependent variables. in contrast to mahwati’s study, a study in brazil found that severe depression is increasing among males, rather than females.(24) however, according to girgus and independent variables aor 95% ci for aor p-value lower upper depression absent 1 present 2.64 2.45 2.84 0.000 age (years) 61-69 1 >70 1.74 1.68 1.80 0.000 gender male 1 female 1.02 0.98 1.06 0.460 educational level high 1 low 1.15 1.09 1.21 0.000 employment status employed 1 unemployed 1.78 1.72 1.85 0.000 place of residence rural 1 urban 0.99 0.96 1.03 0.630 marital status married 1 single/and divorce 1.13 1.09 1.17 0.000 history of chronic disease absent 1 present 1.42 1.37 1.47 0.000 depression x gender no x male 1 yes x female 0.89 0.81 0.98 0.017 110 yang,(18) females are twice more likely to be diagnosed with depression.click or tap here to enter text. vice versa to our study, a study in four south asian countries found that lower frequencies of physical activity lead the higher rates of depression.(25) another study also put depression as a dependent variable instead of a predictor. it was shown by a study in korea that disability is a risk factor for depression and that female gender is the effect modifier.(26) the recommendation for decreasing depression and physical disability among the elderly is by human-animal interac tion. (2 7) a study in china that put depression as the dependent variable found that the mobility problem among the elderly is one factor that increases the risk of depressive symptoms. (2 8) previous studie s a lso put depression as the outcome, and found that the number of physical activities was negatively associated with depression.(29,30) the finding by a study in germany was that physical activity as shown by sports activity was associated with a lower occurrence of depression.(31) the manner in which physical activity can influence depression may be explained by changes in neuroplasticity, inflammation, oxidative stress, the endocrine system, self-esteem, social support, and selfefficacy.(32) social support has negatively mediated the relationship between physical disability and depressive symptoms.(33) our study found depression as the risk factor for physical disabilities among the elderly aged >60 years. the findings from our study are in line with those of previous studies, in that psychological aspects could influence physical disabilities. two studies found a relationship between depression or anxiety and physical condition.(34,35) mental health is also found to be the predictor of the physical condition among the elderly in the united states of america.(36) the physical disorders were found to be associated with depression and other medical conditions. depression in late life had a relationship with worse physical health, while dementia was also found to be associated with physical health and accommodation. studies in china and india found a strong association between depression and physical disabilities that also affect social support.(33,37) in indonesia, a strong association between depression and physical disabilities was also found.(38) two studies showed partial dependence among almost all elderly with physical disabilities.(39,40) depression itself is stimulated by several factors including social networks.(41) overall, most of the findings from previous studies found an association of stress or depression or other kinds of mental health problems with physical disabilities. the rationale of the findings might be that health status is the combination of 3 health conditions namely mental health, social health, and physical health, each of which needs the other. many other factor s might have an association with depression and physical disabilities. a study in spain found that noncommunicable diseases could affect depression status.(42) the psychological health problem is also linked with crohn’s disease.(43) in terms of the age of the elderly, one study found age and low education as the risk factors for disability of community mobility.(44) a study in japan found that living with children tends to reduce the risk of having physical disabilities.(45) based on one study, the factors of older age, unmarried status, and having ncds are associated with physical disabilities.(46) physical disabilities have been investigated by previous studies, most of which included physical health problems. for example, one study has found that diabetes mellitus is the major risk factor for physical disabilities. this study brought the mental health instead of the physical health problem to examine the risk factors of physical disabilities. therefore, it can be concluded that age, education, employment status, marital status, and history of chronic diseases could inf luenc e the assoc iation between depression and physical disability. our study may need more assessment to determine the role of non-communicable diseases since it was based on self-report data. the present study only includes depression as the main predictor instead satria, yeni, akbar, et al relationship between depression and physical disability 111 univ med vol. 41 no 2 of other psychological health problems. this is due to the limitation of variables in the secondary data. our study did not include some important variables such as smoking behavior and accident history. the clinical implications of this study are that physical disability might also influence other chronic and acute diseases. future research might include the mediator of the association between depression and physical disability to obtain a clearer result. the recommendation for future study includes the role of other independent variables, mediators, and confounding factors to build the fixed model. the variable of depression was assessed by self-report and needs to be checked by the researcher. this study has included mental and physical health; the next study could add the social aspects in assessing the health status of the elderly. conclusion this study confirms the role of depression as the risk factor of physical disability and female gender is the effect modifier rather than the risk factor. female elderly with depression had a lower risk of physical disabilities. conflict of interest the authors declare no potential conflicts of interest concerning the research, authorship, and/or publication of this article. acknowledgment the authors wish to express their gratitude to the ministry of health indonesia which permitted us to use the raw data 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http://dx.doi.org/ 10.1111/jgs.15801. 42. rohaedi s, putri st, kharimah ad. tingkat kemandirian lansia dalam activities daily livingdi panti sosial tresna werdha senja rawi. j pendidikan keperawatan indonesia 2016;2:16–21. http:// dx.doi.org/10.17509/jpki.v2i1.2848. 43. tough h, siegrist j, fekete c. social relationships, mental health and wellbeing in physical disability: a systematic review. bmc public health 2017;17:1–18. http://dx.doi.org/10.1186/s12889017-4308-6. 44. amboage-paz mt, díaz-peromingo ja, díazperomingo ja. comorbidity between physical disorders and depression: prevalence and risk factors. int j med sci health res 2020;4:56-67. 45. ananthakrishnan an, khalili h, pan a, et al. association between depressive symptoms and incidence of crohn’s disease and ulcerative colitis: results from the nurses’ health study. clin gastroenterol hepatol 2013;11:57–62. http:// dx.doi.org/10.1016/j.cgh.2012.08.032. 46. nunes jd, de oliveira sm, nunes bp, et al. functional disability indicators and associated factors in the elderly: a population-based study in bagé, rio grande do sul, brazil. epidemiol serv saude brasília 2017;26:295–304. doi: 10.5123/ s1679-49742017000200007. 47. saito e, ueki s, yasuda n, yamazaki s, yasumura s. risk factors of functional disability among community-dwelling elderly people by household in japan: a prospective cohort study. bmc geriatr 2014;14:1-9. http://dx.doi.org/10.1186/1471-231814-93. 48. schuch fb, stubbs b, meyer j, et al. physical activity protects from incident anxiety: a meta analysis of prospective cohort studies. depress anxiety 2019;36:846–58. http://dx.doi.org/ 10.1002/da.22915. oktavianus 120 *department of pharmacology, faculty of medicine trisakti university **department of pharmacology, faculty of medicine university of indonesia ***department of internal medicine, faculty of medicine university of indonesia §department of obstetrics and gynecology, faculty of medicine university of indonesia §§department of community medicine, faculty of medicine, trisakti university correspondence dr. dr. elly herwana, m.biomed department of pharmacology, faculty of medicine trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: +6221-5672731 ext.2801 email:elly_herwana@cbn.net.id univ med 2012;31:120-30 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background isoflavone containing soy protein has been associated with beneficial health effects. equol, a gut bacterial metabolite of the isoflavone daidzein, has been hypothesized to be the cause of the effectiveness of isoflavones. the objective of this study was to evaluate the effect of 100 mg/day soy isoflavone supplementation for 3 months on the ability to produce equol in postmenopausal women with osteopenia. method a pre-post experimental study was conducted to assess the effects of 100 mg/ day soy isoflavone supplementation on equol production capability and the proportion of equol producers to non-equol producers in postmenopausal women with osteopenia. a total of 39 subjects received 1 supplement tablet containing 100 mg soy isoflavones (genistein, daidzein) for 3 months. determination of serum genistein, daidzein, and equol concentrations was performed twice at baseline and at 3 months post-supplementation using high-performance liquid chromatography mass spectrophotometry (hplc-ms). equol producer status was determined by the detection of a serum equol concentration of 5 ì g/l. results mean genistein and daidzein concentrations at baseline were 86.2 ± 68.4 ì g/ l and 16.7 ± 18.6 ìg/l, respectively. the proportion of equol producers was 69.2%. after 3 months of soy isoflavone supplementation the serum concentrations of genistein and daidzein significantly increased to 161.0 ± 5.8 ì g / l (p=0.000) and 49.9 ± 40.4 ìg/l (p=0.000), respectively, and the proportion of equol producers also significantly increased (100.0%). conclusion soy isoflavone supplementation was capable of increasing the serum concentrations of isoflavones (genistein and daidzein) and the equol-producing capacity of postmenopausal women with osteopenia. key words: soy isoflavone, equol, postmenopausal, osteopenia soy isoflavone supplementation increases equol-producing capability in postmenopausal women with osteopenia elly herwana*, rianto setiabudy**, sidartawan soegondo***, ali baziad§ and adi hidayat§§ abstract 121 univ med vol. 31 no.2 suplementasi isoflavon kedelai meningkatkan produksi equol pada perempuan pascamenopause dengan osteopenia latar belakang protein kedelai yang mengandung isoflavon bermanfaat bagi kesehatan. equol, yaitu metabolit bakteri usus dari isoflavon daidzein, diperkirakan merupakan penyebab efektivitas isoflavon. penelitian ini bertujuan untuk menilai efek suplementasi isoflavon kedelai 100 mg/hari selama 3 bulan terhadap kemampuan memproduksi equol pada perempuan pascamenopause dengan osteopenia. metode penelitian ini menggunakan desain pre-post experimental study untuk menilai efek suplementasi isoflavon kedelai 100 mg/hari pada kadar isoflavon dalam serum, kadar equol, dan proporsi equol producer terhadap non-equol producer pada perempuan pascamenopause dengan osteopenia. sebanyak 39 subjek setiap hari menerima 1 tablet suplemen yang berisi 100 mg isoflavon kedelai (genistein, daidzein) selama 3 bulan. pengukuran kadar genistein, daidzein, dan equol dalam serum dilakukan 2 kali pada kadar awal dan pada 3 bulan pascasuplementasi menggunakan high performance liquid chromatography mass spectro-photometry (hplc-ms). status equol producer ditetapkan bilamana dapat terdeteksi kadar equol serum sebesar 5µg/l. hasil rerata asupan genistein dan daidzein masing-masing besarnya 149,0 ± 138,2 mg/hari dan 239,5 ± 175,8 mg/hari. proporsi kelompok yang memproduksi equol besarnya 69,2%. setelah suplementasi isoflavon kedelai selama 3 bulan, kadar genistein dan daidzein meningkat menjadi 161,0 ± 5,8 ì g/l (p=0,000) dan 49,9 ± 40,4 ì g/l (p=0,000) serta proporsi kelompok yang memproduksi equol naik menjadi 100,0%. kesimpulan suplementasi isoflavon kedelai 100 mg/hari selama 3 bulan mampu meningkatkan kadar isoflavon dalam serum (genistein dan daidzein) dan kemampuan memproduksi equol pada perempuan pascamenopause dengan osteopenia. kata kunci: isoflavon kedelai, equol, pasacamenopause, osteopenia abstrak introduction menopause is the most important risk factor for osteopenia and osteoporosis. (1) p o s t m e n o p a u s a l w o m e n e x p e r i e n c e a permanently decreased secretion of the ovarian hormones estrogen and progesterone as a result of depletion of ovarian follicles.(1) the higher prevalence of osteopenia, osteoporosis, and cardiovascular disease in post-menopausal women is presumably due to decreased estrogen c o n c e n t r a t i o n s . ( 2 , 3 ) a l t h o u g h i t h a s b e e n demonstrated that the decrease in estrogen concentrations may actually reduce bone density, numerous studies are being conducted to clarify the latter process. studies using tissue cultures have demonstrated direct effects of estrogen on bone cells via estrogen receptors.(4) several of the most recent studies indicate that estrogen plays a role in regulation of bone homeostasis through regulatory effects on the immune system, oxidative stress, and direct effects on bone cells.(5) the direct effects of estrogen on bone cells comprise effects on 122 osteoblasts, osteocytes, and osteoclasts, mediated through estrogen receptors (ers). ers are of two types, erá and erâ , both of which a r e p r e s e n t o n b o n e c e l l s , a l t h o u g h n o t uniformly distributed. erá being predominantly in cortical bone, while erâ is mainly found in trabecular bone.(4,6) accelerated postmenopausal osteoporosis requires effective management to prevent the o c c u r r e n c e o f f r a c t u r e s , w h i c h a r e characteristic of osteoporosis due to reduced b o n e d e n s i t y. h o r m o n a l r e p l a c e m e n t b y administration of synthetic estrogens or selective estrogen receptor modulator (serm) has been demonstrated to be an effective t r e a t m e n t o p t i o n f o r p o s t m e n o p a u s a l osteoporosis.(7-10) the clinical trial conducted by the women’s health initiative (whi) revealed that hormonal replacement reduced the r i s k o f a l l f r a c t u r e s a s s o c i a t e d w i t h osteoporosis, including femoral neck and vertebral fractures, although only in patients with a low risk of fractures. (8) however, hormonal replacement also shows an increased risk of cardiovascular disorders such as venous thromboembolism and breast and endometrial malignancies, such that the benefits to risks ratio should be considered. in japanese and asian women with a high consumption of dietary products derived from isoflavone-containing soy beans, the incidence of osteoporosis was found to be lower than in western women with a low consumption of isoflavones.(11,12) this reinforces the idea that isoflavones in soy beans serve to inhibit postmenopausal osteoporosis, because soy isoflavones have been identified to possess phytoestrogenic effects.(13,14) the major class of phytoestrogens attracting attention from the nutritional and medical viewpoint are the ligans and isoflavones. genistein, daidzein, and g l y c i t e i n , w h i c h a r e f r e e f o r m s o f s o y isoflavones, have a molecular structure similar to estradiol and display estrogenic effects (figure 1).(15) there are many pharmacokinetic studies on isoflavones in connection with equol, which is an active metabolite of daidzein. equol enters the enterohepatic circulation and is actively secreted in the renal tubules, such that it is detectable in the blood and urine. equol has a greater estrogenic effect than genistein or daidzein.(16-18) various studies also demonstrate an association of equol concentrations with the variable biologic effects as well as the clinical benefits of isoflavone administration.(18,19) figure 1. molecular structure of isoflavone derivatives and estradiol.(15) herwana, setiabudy, soegondo, et al isoflavone increases equal capability 123 univ med vol. 31 no.2 individuals differ in their capacity to metabolize daidzein into equol, and therefore can be classified into equol producers and non-equol producers.(21,22) numerous in vitro studies have found evidence that the characteristics of intestinal bacteria play a role in the metabolism of daidzein into equol, with daidzein being hydrolyzed into equol by bacterial endo-â glycosidases.(20-22) there is a wide variation in the capacity of individuals to produce equol, with a proportion of equol producers of 30-40% in western countries, (23,24) while several studies have reported a higher proportion of equol producers i n a s i a n a s c o m p a r e d w i t h we s t e r n populations.(20,25) lampe et al.(26) reported that genetic variation may affect phytochemical metabolic pathways such as to potentially induce changes in their biological response. vergne et al.(27) reported that isoflavone pharmacokinetics and bioavailability are influenced by ethnicity and dietary history of isoflavone intakes. in our study, the criterion of osteopenia w a s u s e d o n t h e c o n s i d e r a t i o n t h a t p o s t m e n o p a u s a l w o m e n w i t h o s t e o p e n i a comprise the largest group compared to normal bone mineral density (bmd) and osteoporosis groups.(28) there are few reports of studies on e q u o l p r o d u c e r s i n i n d o n e s i a , a l t h o u g h indonesian communities consume a high proportion of soy-based foods, usually in the form of tofu, tempeh, soy sauce, and soy milk. the aim of the present study was to evaluate the effects of soy isoflavone supplementation on serum isoflavone concentrations and the potential to stimulate equol-producing capacity in postmenopausal women with osteopenia. methods research design this study was a study of pre-post test experimental design conducted between january and july 2010 to assess the effects of soy isoflavone supplementation on equol-producing capacity. study subjects the study subjects were 48-60 year old postmenopausal women with osteopenia, who were recruited by consecutive random sampling from the mampang parapatan district, south jakarta. screening was performed by means of questionnaires, physical examination, and measurement of bmd. the inclusion criteria were as follows: postmenopausal women 48-60 years of age, duration of menopause of more than one year but not exceeding 10 years, osteopenia (t score between –1 and –2.5), without a history of medications in the past 6 months, such as hormonal replacement, isoflavone-containing supplements, antidiabetics, antihyper-lipidemic agents and anticoagulants, agreeing to participate in the study and signing informed consent. laboratory investigations: bilirubin < 2.0 mg/ dl, glutamic pyruvic transaminase (sgpt) and serum glutamic oxaloacetic transaminase (sgot) within normal limits, creatinine < 1.5 mg/dl, capable of walking unaided and capable of communication. exclusion criteria were: women with a past history of hysterectomy or oophorectomy, irradiation, chemotherapy, malignant disease (such as mammary, cervical or endometrial carcinomas), and chronic disease (such as hepatic cirrhosis, renal failure, diabetes mellitus, cardiac disorders, hyperthyroidism, or stroke). intervention soybean extract was imported from huisong pharmaceuticals, china. each 100 mg of soy extract contained 40% soy isoflavones. the supplement tablets used in this study were prepared and packaged by pt ikapharmindo putramas. the supplements contained 250 mg s o y b e a n e x t r a c t e q u i v a l e n t t o 1 0 0 m g isoflavones (composed of genistein 56%, d a i d z e i n 4 1 % , a n d g l y c i t e i n 3 % ) . a l l postmenopausal women took 1 tablet daily for 3 m o n t h s . t h e s u p p l e m e n t a t i o n w a s administered at daily home visits under direct observation by the cadre on duty and recorded in a check list. 124 measurements s u b j e c t s m e e t i n g t h e i n c l u s i o n a n d exclusion criteria on the basis of interviews, were screened by physical examination, laboratory t e s t s , a n d b m d m e a s u r e m e n t . p h y s i c a l examination comprised determination of pulse rate, body weight, and height. dual-energy xray absorptiometry (dxa) was used for bmd screening, with the subjects with t-scores b e t w e e n – 1 a n d – 2 . 5 b e i n g s e l e c t e d f o r participation in the study.(7) the bmd was determined at budi jaya hospital, jakarta, using the lunar dpx bravo nomusa densitometer (ge medical systems). laboratory screening tests were done on fasting subjects and comprised blood glucose, bilirubin, sgpt, sgot, and creatinine. soy intake measurement intake of soybean-containing foods was assessed at baseline by means of food records and food recall. the subjects recorded their complete dietary intake per day for 3 days (2 work days and 1 holiday), to be confirmed by food recall. the dietary intake was calculated semi-quantitively using the soy food frequency questionnaire (ffq) and the results of an analysis of the soy isoflavone content of foods frequently consumed by the subjects. isoflavone analysis the serum concentrations of genistein, daidzein, and equol were determined at baseline a n d a t 3 m o n t h s p o s t s u p p l e m e n t a t i o n . measurement of isoflavone concentrations was performed by means of the high-performance liquid chromatography mass spectrophotometer (hplc-ms) using the waters 2690 separation module-alliance-ms (micromass quattro lc triple quadropole). a c18 column with a photo diode array detector was used at 200-350 nm.(29) the detection limit was 0.5 ng/ml, with an individual being classified as equol producer if the serum equol concentration was > 5 ìg/l.(23) the coefficients of variation of daidzein, genistein and equol were 1.51%, 1.53%, and 2.67%, respectively. compliance measures subject compliance was assessed by means of the supplementation checklist and by counting the remaining tablets in the bottle, and was monitored by the investigators in periodic meetings with the subjects. the subjects were categorized as dropouts if they failed to take the supplements for a total of more than 10% of the tablets (9 tablets) within 3 months of supplementation. statistical analysis subject characteristics such as age, body weight, height, body mass index (bmi), blood pressure, and laboratory screening test results were presented as descriptive statistics, with the values given as mean ± standard deviation (sd). the paired t-test and mcnemar test were used to compare the differences in isoflavone concentration and equol producer status between baseline and post-supplementation, pvalue of <0.05 being considered significant. ethical clearance the study protocol was approved by the committee of medical research ethics of the faculty of medicine, university of indonesia. figure 2. flowchart of study subjects herwana, setiabudy, soegondo, et al isoflavone increases equal capability 125 univ med vol. 31 no.2 table 1. subject characteristics at baseline (n=39) a) values are mean ± standard deviation; b) number of subjects (%) isoflavone intake, and serum isoflavone concentration are presented as mean ± sd. the subjects were postmenopausal women aged 4860 years with osteopenia. among the 39 subjects who completed the study, the youngest was 48 years and the eldest 60 years of age, with mean age of 53.0 ± 3.5 years. the shortest duration of menopause was 1 year and the longest 9 years, with a mean duration of 4.4 ± 2.1 years. marital status was categorized as married with living husband 27 (69.2%) and unmarried (single) or widow 26 (30.8%). most of the subjects (25 or 64.2%) had a low educational l e v e l ( n o e d u c a t i o n – p r i m a r y s c h o o l ) . employment status was divided into employed (48.7%) and unemployed (51.3%). mean bmi was 27 ± 4.4 kg/m 2, and estradiol concentration was 7.3 ± 5.6 pg/ml. isoflavone intake was assessed by means of the ffq method for the isoflavone derivatives genistein and daidzein. daily genistein intake was 149.0 ± 138.2 mg, while daidzein intake was higher at 239.5 ± 175.8 mg. i n t h i s s t u d y, s e r u m i s o f l a v o n e concentrations, comprising genistein, daidzein, and equol concentrations, were determined twice, namely at baseline and at 3 months postsupplementation. baseline isoflavone concentrations, presented in table 1, showed mean genistein concentration of 86.2 ± 68.4 ìg/ l, mean daidzein concentration of 16.7 ±18.6 ìg/l, and mean equol concentration of 7.5 ± 4.6 ìg/l, with 27 (69.2%) equol producers and 12 (30.8%) non-equol producers. a f t e r 3 m o n t h s o f i s o f l a v o n e supplementation there was a significant increase in the concentrations of genistein (86.2 ± 68.4 vs. 161 ± 5.8 ìg/l; p = 0.000), daidzein (16.7 ± 18.6 vs. 49.3 ± 40.4 ìg/l; p=0.000), and equol (7.5 ± 4.5 vs 13.0 ± 5.9; p =0.000) (table 2). as presented in table 3, the results of mcnemar’s test showed a significant difference in equol-producer status at baseline and at 3 months post-supplementation (p=0.000). results study subjects in this study, 44 subjects fulfilled the inclusion and exclusion criteria. five subjects dropped out for the following reasons: 3 subjects declined to continue participation, 1 subject had a change of residence, and 1 subject discontinued participation due to diarrhea in the third month. a total of 39 subjects participated until completion of the study, and were analyzed as per protocol (figure 1). baseline characteristics of the study subjects are listed in table 1. age, years since m e n o p a u s e , b o d y w e i g h t , h e i g h t , b m i , 126 discussion the study subjects were postmenopausal women with osteopenia, which is a condition that without intervention is at risk of deteriorating to osteoporosis. the results of the present study indicate high genistein and daidzein daily intakes of 149.0 ± 138.2 mg and 239.5 ± 175.8 mg, respectively, which are higher than those of postmenopausal women in texas (58.7 ± 21 mg/ d for genistein and 53.4 ± 10.7 mg/d for daidzein).(30) a study in japan(31) showed that older japanese adults consume approximately 611 g of soy protein and 25-50 mg of isoflavones per day (expressed as aglycone equivalents), which are lower than the isoflavone intakes among postmenopausal women in our study. in indonesia, soy beans are on the daily menu in processed form as tempeh, tofu, soy sauce, and soy milk, which are much consumed by the subjects of this study in particular and by indonesian communities in general, in view of their low cost and ready availability in traditional markets. dietary isoflavone intakes show considerable individual variation in many countries. i n o u r s t u d y, s e r u m i s o f l a v o n e concentrations were already high at baseline, before the administration of the isoflavone supplements, while post-supplementation serum isoflavone concentrations increased significantly (p=0.000). these data may be considered as o b j e c t i v e e v i d e n c e c o n f i r m i n g s u b j e c t compliance in taking the supplements and at the same time demonstrating that the administered supplementation was able to reach the systemic circulation. pharmacokinetics deals with the fate of drugs in the body and comprises 4 processes, i.e. absorption, distribution, metabolism, and e x c r e t i o n . i n t e ri n d i v i d u a l v a r i a t i o n i n metabolizing capacity for a drug may influence the effectiveness of the drug. modification of soy isoflavones by intestinal bacteria may produce metabolites that differ in biological activity from the parent compounds. bacterial metabolism of soy isoflavone varies among individuals.(32) usually, further degradation and transformation of aglycones produce less active compounds, but hydrolysis of daidzein glycosides results in the more active compound equol [7-hydroxy-3-(4’hydroxyphenyl)-[chroman]]. although the herwana, setiabudy, soegondo, et al isoflavone increases equal capability table 3. comparison of equol producer status before and after administration of soy isoflavones for 3 months (n=39) a) number of subjects; b) p value calculated by mc nemars test tabel 2. mean isoflavone at baseline and at 3 months post-supplementation a) values are mean standard deviation; b) p value calculated by paired t-test 127 univ med vol. 31 no.2 factors that influence equol-producing capacity are not clearly established, intestinal physiology, host genetics, and diet appear to contribute to interindividual differences in the conversion of daidzein to equol.(32) other studies state that intake of fructo-oligosaccharidies (fos)(21) or lactobacillus casei with isoflavone may increase equol production.(17) a high carbohydrate diet may also increase the production of equol.(13) in contrast, administration of antibiotics has the opposite effect, modifying the numbers and characteristics of intestinal bacteria and thus inhibiting the metabolic conversion of daidzein into equol. assessment of equol may be done on serum or urinary samples. on the basis of their equolp r o d u c i n g c a p a c i t y, i n d i v i d u a l s c a n b e classified into equol producers and non-equol producers. there is as yet no standard criterion for this classification, as many studies use different criteria. a number of studies consider their subjects to be equol producers if their serum equol concentration is >5 ìg/l or their urinary equol is >20 ìg/l.(23) in the present study, a value of serum equol of >5 µg/l was used, while the limit of detection of the measuring instrument used for determining equol concentration was 0.5 ìg/l.(23) the proportions of equol producers between countries show considerable variation, with the proportion of equol producers in western countries being approximately 33%, while japan and korea, with their high dietary soy isoflavone c o n s u m p t i o n , s h o w a p r o p o r t i o n o f approximately 55-60%.(33) in indonesia there are not many study reports on the proportions of equol producers and non-equol producers. in the present study, serum isoflavone concentrations were determined twice, namely at baseline and at 3 months post-supplementation. at baseline, mean serum equol concentration was 7.5 ± 4.6 ìg/l and the proportion of equol producers was 27/39 (69.2%). although no supplementation had yet been administered, the proportion of equol producers was already relatively high, because soy beans are much consumed by the subjects of this study in particular and by indonesian communities in general. at 3 months post-supplementation the proportion of equol producers increased to 39/39 (100%). this indicates that mean equol concentration and the proportion of equol producers was already high since the start of the study, while the proportion increased at 3 months post-supplementation. differing results were found in the study by vedrine et al.,(20) who reported that soy isoflavone supplementation for one month was unable to convert non-equol producers into equol producers. the modification of individual equolproducing status is as yet controversial and affected by a variety of factors. in contrast, liu et al.(34) reported the existence of an association between age, isoflavone intake, and proportion o f e q u o l p r o d u c e r s i n e t h n i c c h i n e s e . supplementation of 41 mg genistein for 3 days was capable of raising the proportion of equol producers from 26.8% to 60.4%. vergne et al.(27) reported that ethnic differences and dietary history play a role in the differing bioavailability of isoflavones in ethnic asians and caucasians. a one-time dose of isoflavone led to an increase in maximal isoflavone concentration (c max ) and a larger area under the plasma concentration time c u r v e ( a u c ) i n a s i a n s a s c o m p a r e d t o caucasians. on isoflavone administration for 10 days, c max and auc were raised in caucasians but not in asians.(27) the factor of dietary background in asian ethnic groups with a prior high consumption of isoflavones plays a role in inter-ethnic differences in bioavailability. setchell (23) reported a proportion of equol producers of 33.3% in non-vegetarians and a higher proportion (51.7%) in vegetarians. lampe et al.(32) have explained interi n d i v i d u a l d i ff e r e n c e s i n m e t a b o l i s m o f naturally occurring compounds. intake of naturally occurring compounds does not always accurately reflect their tissue concentrations. inter-individual differences may be due to genetic variability involving pharmacokinetic factors, such as absorption, distribution, and metabolism, thus resulting in different tissue 128 concentrations and modification of disease risk. plant polyphenols including the isoflavones as phytoestrogens are extensively metabolized by intestinal bacteria. inter-individual differences influencing the host bacteria are associated with isoflavone metabolism. soy isoflavones contain flavonoids in the form of glycosides that are difficult to absorb. under the influence of âglycosidases from bacteria as well as the i n t e s t i n a l m u c o s a , t h e s e g l y c o s i d e s a r e hydrolyzed into readily absorbable aglycones, such that their absorption rate is influenced by the hydrolyzing capacity. similarly to other naturally occurring compounds or xenobiotics, isoflavones also undergo biotransformation. polymorphisms in biotransforming enzymes, such as glutathione s-transferase (gst), uridine 5’-diphosphate (udp)-glucuronosyltransferase (ugt), sulfotransferase (sult), cytochrome p450, also play a role in the varying effects on natural compounds. pharmacodynamic factors such as changes in target receptor sensitivity or mutations may affect the binding site of drug receptors, resulting in modification of drug effects. the study conducted by pusparini(35) on a population of indonesian postmenopausal women irrespective of osteopenic status, showed a lower proportion of equol producers of 60.4%. in the present study, there exists the possibility that the specific subject characteristic of osteopenia in postmenopausal women also contributed to a more homogenous group of equol producers. it is apparent that there is a need for further extensive studies on the effects of dietary patterns on daidzein metabolizing capacity in various indonesian communities, considering that equol producer status is highly dependent on the characteristics of intestinal bacteria, which are in turn affected by individual dietary patterns. a history of high isoflavone intake in all study subjects since the start of the study has an important role in the equol-producing capacity of the subjects. high carbohydrate intake and a history of high longterm dietary isoflavone intakes in indonesian communities in general, also constitute factors that affect isoflavone metabolism in the subjects of this study. there is a need for further studies to acquire a more c o m p r e h e n s i v e i n s i g h t i n t o t h e f a c t o r s influencing equol-metablizing capacity. this study has a number of limitations, the first being that the isoflavone intake levels of t h e s t u d y p a r t i c i p a n t s w e r e p r o b a b l y underestimated, because the estimation was based on a questionnaire. secondly, the isoflavone content of some modern processed protein-containing foods (e.g. white bread) could not be estimated because the ingredients and composition were not available. finally, the effect of isoflavone supplementation may have been influenced by the fact that the dietary soy isoflavone intake of the study subjects could not be controlled, since they lived at their own homes. conclusion the increase in equol producer status following isoflavone supplementation in the present study demonstrates that administration of soy isoflavones 100 mg/day for 3 months resulted in increased equol-producing capacity. acknowledgements the authors thank the postmenopausal women who volunteered for partication in this study. thanks are also due to the dean and vice deans of the faculty of medicine, trisakti university, for funding this study. references 1. nelson hd. menopause. the lancet 2008;371: 760-70. 2. raisz lg. pathogenesis of osteoporosis: concepts, conflict, and prospects. j clin invest 2005;115: 3318-25. 3. finkelstein js, brockwell se, mehta v, greendalega, sowers mr, ettinger b, et al. bone mineral density changes during the menopause transition in a multiethnic cohort of herwana, setiabudy, soegondo, et al isoflavone increases equal capability 129 univ med vol. 31 no.2 women. j clin endocrinol metab 2008;93:8618. 4. deroo bj, korachks. estrogen receptor and human disease. j clin invest 2006;116:561-70. 5. weitzmann mn, pacifici r. estrogen deficiency and bone loss: an inflammatory tale. j clin invest 2006;116:1186-94. 6. gustafsson ja. erâ scientific visions translate to clinical uses. climacteric 2006;9:156-60. 7. kanis ja, burlet n, cooper c, delmas pd, reginster jy, borgstrom f,et al. european guidance for diagnosis and management of osteoporosis in postmenopausal women. osteoporosis int 2008;19:399-428. 8. de villiers t. the role of menopausal hormone therapy in osteoporosis. climacteric 2007;10:713. 9. barlow dh. osteoporosis guidelines. climacteric 2007;10(suppl 2):79-82. 10. jordan n, barry m, murphy e. comparative effects of antiresorptive agents on bone mineral density and bone turnover in postmenopausal women. clin invest aging 2006;1:377-87. 11. kronenberg f, fugh-berman a. complementary and alternative medicine for menopausal symptoms: a review of randomized, controlled trials. annals intern med 2002;137:805-13. 12. chanteranne b, branca f, kaardinal a, wahala k, braesco v, ladroite p, et al. food matrix and isoflavones bioavailability in early post menopausal women: a european clinical study. clin interv aging 2008;3:711-8. 13. setchell kdr, lydeking-olsen e. dietary phytoestrogen and their effect on bone: evidence from in-vitro and in-vivo, human observational, and dietary intervention studies. am j clin nutr 2003;78:593s-609s. 14. vincent a, fitzpatrick la. soy isoflavones: are they useful in menopause? mayo clin proc 2000; 75:1174-83. 15. wood ce, appt se, clarkson tb, franke aa, lees cj, doerge dr, et al. effect of high-dose soy isoflavones and equol on reproductive tissues in female cynomolgus monkeys. biol reprod 2006;75:477-86. 16. setchell kdr. soy isoflafone-benefit and risk from narure’s selective estrogen receptor modulators (serms). j am coll nutr 2001;20: 354s-62s. 17. mathey j, mardon j, fokialakis n, puel c, katicoulibaly s, mitakou s, et al. modulation of soy isoflavones bioavailability and subsequent effects on bone health in ovariectomized rats: the case for equol. osteoporos int 2007;18:671-9. 18. fujioka m, uehara m, wu j, adlercreutz h, suzuki k, kanasawa k, et al. equol, a metabolite of daidzein, inhibits bone loss in ovariectomized mice. j nutr 2004;134:2623-7. 19. setchell kdr, brown nm, olsen el. the clinical importance of the metabolite equol –a clue to the effectiveness of soy and its isoflavones. j nutr 2002;132:3577-85. 20. vedrine n, mathey j, morand c, brandolini m, davicco mj, guy l, et al. one-month exposure to soy isoflavones did not induce the ability to produce equol in postmenopausal women. eur j clin nutr 2006;60:1039-45. 21. bolca s, possemiers s, herregat a, huybrechts i, heyerick a, de vriese s, et al. microbial and dietary factors are associated with the equol producer phenotype in healthy postmenopausal women. j nutr 2007;137:2242-6. 22. decroos k, eeckhaut e, possemiers s, verstraete w. administration of equol-producing bacteria alters the equol production status in the simulator of the gastrointestinal microbial ecosystem (shime). j nutr 2006;136:946-52. 23. setchell kdr, cole sj. method of defining equol-producer status and its frequency among vegetarians. j nutr 2006;136:2188-93. 24. frankenfeld cl, atkinson c, thomas wk, gonzales a, jokela t, wahala k, et al. high concordance of daidzein metabolizing phenotypes in individuals measured 1 to 3 years apart. br j nutr 2005;94:873-6. 25. mascarinec g, yamakawa r, hebshi s, franke aa. urinary isoflavonoid excretion and soy consumption in three generation of japanese women in hawaii. eur j clin nutr 2007;61:25561. 26. lampe jw, chang jl. interindividual differences in phytochemical metabolism and disposition. semin cancer biol 2007;17:347-53. 27. vergne s, sauvant p, lamothe v, chantre p, asselineau j, perez p, et al. influence of ethnic origin (asian vs. caucasian) and background diet on the bioavailability of dietary isoflavones. br j nutr 2009;102:1642-53. 28. meiyanti. epidemiology of osteoporosis in postmenopausal women aged 47 to 70 years. univ med 2010;29:169-76. 29. lee kj, row kh, jun ic. preparative separation of isoflavones from korean soy bean be hplc. asian j scien res 2008;3:288-92. 30. huang y, cao s, nagamani m, anderson ke, grady jj, lu ljw. decreased circulating levels of tumor necrosis factor-α in postmenopausal women during consumption of soy-containing 130 isoflavones. j clin endocrinol metab 2005;90: 3956–62. 31. messina m, nagata c, wu ah. estimated asian adult soy protein and isoflavone intakes. nutr cancer 2006;55:1–12. 32. lampe jw. is equol the key to the efficacy of soy foods? am j clin nutr 2009;89 suppl:s1664-7. 33. akaza h, miyanaga n, takashima n, naito s, hirao y, tsukamoto t, et al. comparisons of percent equol producer between prostate cancer patients and controls: case controlled studies of isoflavones in japanese, korean and american residents. jpn clin oncol 2004:34:86-9. 34. liu b, qin l, liu a, uchiyama s, ueno t, li x, et al. prevalence of equol-producer phenotype and its relationship with dietary isoflavone and serum lipids in healthy chinese adult. j epidemiol 2010;20:377-84. 35. pusparini. pengaruh suplementasi isoflavon kedelai terhadap penanda fungsi endotel pada perempuan pascamenopause (disertasi). jakarta: universitas indonesia; 2011. herwana, setiabudy, soegondo, et al isoflavone increases equal capability c:\users\universa medicina\docu 163 abstract universa medicina chronic limb ischemia manifestation in covid-19 infection: awareness and treatment in primary care sidhi laksono1,2*, reynaldo halomoan siregar3, and hillary kusharsamita4 review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.166-175 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1038 cite this article as: laksono s, reynaldo halomoan r, kusharsamita k. chronic limb ischemia manifestation in covid19 infection: awareness and treatment in prima ry ca re. un iv m ed 2 021 ; 40 :1 63 -72 . doi: 10.18051/ univmed. 2021 .v40.16 6-1 75 1head of cardiac catheterization laboratory, department of cardiology and vascular medicine, rsud pasar rebo, east jakarta 2faculty of medicine, universitas muhammadiyah prof. dr. hamka, tangerang 3 faculty of medicine of universitas katolik indonesia atma jaya 4 faculty of medicine, universitas diponegoro, semarang *correspondence: sidhi laksono faculty of medicine, universitas muhammadiyah prof. dr. hamka jl. raden patah no.01, rt.002/ rw.006, parung serab, kec. ciledug, kota tangerang, banten 13460 phone number: +622127564161 email: sidhilaksono@uhamka.ac.id orcid id: 0000-0002-2959-8937 date of first submission, october 20, 2020 date of final revised submission, may 4, 2021 date of acceptance, may 24, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license chronic limb ischemia (cli) is a type of peripheral arterial disease (pad) that is still underdiagnosed and undertreated despite the increasing incidence, thus becoming a global health burden. and cli reflects the local manifestations of a lethal systemic disease — atherosclerosis. if left untreated, chronic limb ischemia can result in major limb loss. in this pandemic era, limb ischemia has become one of several clinical manifestations that occur in patients with covid-19 infection. systemic inflammation in covid-19 infection, direct viral infection, hypercoagulable state, and hyperinflammatory response are responsible for damage to the arterial system, causing endothelial dysfunction. diagnosing pad has become a challenge especially in the early stage and in the asymptomatic phase. the untreated condition could lead to the development of cli. the primary physicians in the primary health facilities hold an important role in the early diagnosis and management of patients with cli symptoms or with risk factors of cli, especially in patients who have experienced covid-19 infection. due to the limitation of diagnostic testing modalities at primary health facilities, the physician can assess the ankle-brachial index (abi) to determine the presence of cli. management of the disease is different for every patient and is customized based on the other comorbidities. risk factors should be controlled in order to achieve a better outcome. a good management strategy will improve the quality of life of the patient. this review will discuss the occurrence of cli in covid19 infection. keywords: covid-19, ankle-brachial index, chronic limb ischemia, management 164 laksono, siregar, kusharsamita cli in covid-19 infection introduction peripheral arteria l disease (pad) is becoming a burden for the health system globally. pad is closely related to atherosclerotic disease. the disease has affected more than 200 million people around the world and causes 12-15% mortality in europe.(1,2) in the united states, the disease affects 1.3% of men and 1.7% of women (ages 40-49 years). t he prevalence was significantly increased in the older population with 29.5% of men and 24.7% of women older than 80 years.(3) chronic limb ischemia (cli) is a type of pad that is underdiagnosed and undertreated because about 20-50% of the patients may be asymptomatic at the beginning.(4,5) the burden is even higher due to unawareness of the physicians (70%) that the diagnosis had already been established during pad screening.(6) however, during the progression of the disease, cli would manifest in several symptoms that are related to the impairment of the quality of life. cli is mainly associated with the presence of diabetes mellitus, dyslipidemia, hypertension, smoking, and also older age. these risk factors would damage the arterial system, reducing the blood flow causing poor perfusion to the tissues.(7) although the patient may experience mild symptoms, the prevention of further progression is crucial. the well-known severe form of cli is critical limb ischemia. this manifestation could put someone at a higher risk of limb loss and cardiovascular disease.(8) critical limb ischemia affects 12% of the adult population and is related to a quite high mortality rate, 25% at 1 year, and 60% at 5 years. the prognosis may become poorer for patients with critical limb ischemia, thus there is a need for establishing a rapid diagnosis and early management.(9) critical limb ischemia is also more common in the older population.(10) the ongoing spr ea d of severe ac ute respiratory coronavirus syndrome-2 (sars-cov2) has resulted in a coronavirus disease (covid19) pandemic, raising major challenges in all medical specialties. limb and digit ischemia have recently been linked to covid-19 infection. systemic inflammation in covid-19 infection may play a role in developing limb ischemia due to endothelial dysfunction related to the ace2 receptor.(11) moreover, critical limb ischemia remains a life-thre a tening disease that necessitate s prompt treatment to prevent mortality and limb loss.(12) diagnostic modalities and management strategies in the hospital are able to treat the patient thoroughly.(4) however, patients may come to the primary health facilities at the beginning to treat the symptoms. due to the limited modalities in the primary health facilities, necessarily the physician should be able to perform the simple yet reliable examination that is needed to establish the diagnosis of cli. a literature search was conducted from pubmed on topic-related articles from 2011 to 2020 using the keywords “covid-19”, “sars-cov-2”, “coronavirus”, and “chronic limb ischemia”. all types of article such as observational studies, systematic reviews and meta-analyses, guidelines, and other narrative reviews were included. the present review will highlight the curr ent knowledge on covid-19 pathophysiology, with a focus on cli. def inition of chronic limb ischemia chronic limb ischemia (cli) is often also defined as critical limb ischemia, thus both share a common definition. the american heart association/american college of cardiology (aha/acc) guideline defines this disease as a chronic condition (14 days) with the characteristics of ischemic pain at rest, nonhealing ulcers, and the presence of gangrene. these characteristics may occur in 1 or both legs with the presence of arterial occlusive disease objectively proven.(13) these symptoms are the result of poor peripheral perfusion caused by the chronic pathological process in the arterial system. critical limb ischemia is also defined as a chronic limb-threatening ischemia (clti) by another guideline.(14) it affects 11.08% of the pad population annually. (15) 165 pathophysiology of limb ischemia in covid-19 infection the mechanisms underlying thrombosis in covid-19 infection are not fully understood yet, although var ious me c hanisms have bee n proposed, including direct infection of the virus to the vascular system, hyperinflammatory responses, and coagulation abnormalities.(16) in patients with covid-19, there are higher levels of some hypercoagulability biomarkers such as d-dimer, f ibrinogen , factor v iii, and antiphospolipid antibodies and lower protein c, protein s, and antithrombin levels. these abnormalities are related to peripheral arterial ischemia.(17) along with an increased proinflammatory cytokine production (interleukin, tumor necrosis factor, interferon) , these mechanisms will lead to the formation of microthrombi. covid-19 infection result in higher levels of helper t lymphocytes, triggering interleukin-6 (il-6) production, which is responsible for the production of acute-phase reactants such as c-reactive protein (crp) and fibrinogen. both crp and fibrinogen cause hypercoagulopathy, leading to thrombosis.(18,19) direct infection of sars-cov-2 through the ace2 receptor (which is expressed in endothelial cells) and inflammatory cells induce endothelitis, which is characterized by disruption of the circulation.(20) another well-known endothelial dysfunction-based mechanism is oxidative stressmediated endothelial dysfunction (figure 1), (21) endothelial cells (ecs) are found mostly in the inner layer of blood vessels and are covered by pericytes, which help to maintain the vessel structure.(22) depending on the tissues and muscles, ecs have various functions and structures. controlling vascular permeability and regulating vascular tone are two widely known roles of ecs in body physiology homeostasis.(22) ecs can produce and release endothelial-derived relaxing factors such as nitric oxide (no) and prostaglandin (pg), as well as contractile factors such as endothelin (et), thromboxane a2 (txa2), reactive oxygen species (ros), and angiotensin ii (ang ii), all of which play important roles in the control of vascular tone.(23) when activated, ecs se cre te che moattr acta nts, cytokines, and adhesion molecules, resulting in increased blood vessel permeability.(22) in resting ecs, no can suppress the synthesis of these molecules. no has a direct effect on leukocytes, inhibiting them from activating into motile forms capable of penetrating tissues.(24) a dysfunctional endothelial response to damage or infection, on the other hand, cannot produce sufficient no.(25) covid-19 patients have been shown to have a nitric oxide deficiency.(26) as a result, a decrease in no bioavailability always indicates endothelial dysfunction. reduced endothelial nitric oxide synthase (enos) production, a loss of enos substrates, enos inactivation, and accelerated no degradation are thought to induce a decrease in no bioavailability.(27) as previously mentioned, the serum level of no in covid-19 patients is reduced, suggesting oxidative stress (oxs).(26) oxidative stress plays an important role in the development of a variety of arterial diseases. according to several studies of oxs in pad, there is a series of reac tive oxygen species (ros) formation, mitochondrial injury, endothelial dysfunction, and selective damage to muscle myofibers. thus, oxs functions as a critical mechanism in both the creation and progression of pad.(24) risk factors associated with the develop ment of chronic limb ischemia the presence of cli is highly associated with the atherosclerotic process which is related to several risk factors such as smoking, diabetes mellitus, dyslipidemia, and hypertension. a study by joosten et al. (28) showed that smoking is one of the leading risk factors for cli, affecting 44% of the population. an active smoker has a 4.3 times higher risk for developing symptomatic pad (including cli) while a former smoker has a 2.3 times higher risk.(29) while it is clear that an active smoker has an increased risk of developing may-august 2021 vol.40no.2 166 laksono, siregar, kusharsamita cli in covid-19 infection pad, studies on the passive smoker are lacking. however, two studie s showed a similar association between passive smoking and pad. the first study showed that 2.4% (134 of 5686) of the population who never smokes, had pad.(30) the second study showed that 3.2% of the population had intermittent claudication.(31) diabetes mellitus increases the risk of pad. increased hba1c level in diabetic patients also accounts for a 26% risk for pad. (2) diabetic patients with pad are also at a 5-times higher risk of amputation.(32) infection rates are higher in the diabetic patients and this can increase the possibility of amputation in the patients.(33) hypertension is an important risk factor after smoking and diabetes. itoga et al. (34) found that a systolic blood pressure of 160 mmhg was related to a 21% higher risk of pad. however, this study figure 1. mechanisms of endothelial activation and dysfunction during covid-19. (21) 167 also found a relation between lower systolic blood pressure (< 120 mmhg) and pad. the result of the itoga study is different from other cohort studies that found an association of a higher but not a lower systolic blood pressure with the risk of pad.(35,36) the difference might come from the fact that itoga et al.(34) conducted their study on a population that had hypertension and other cardiovascular risk factors, while the other two studies were conducted in healthy populations. dyslipidemia is a ssoc iated with the progression of pad and its complication. the increased ratio between total cholesterol and highdensity lipoprotein cholesterol (hdl-c), triglyceride-rich lipoprotein, and also a decreased hdl level are associated with the incidence of pad. (37) diagnostic confirmation of chronic lim b ischemia in primary care chronic limb ischemia is diagnosed based on the clinical condition, and supporting investigations. while some cases might need advanced imaging mod alities, the initial examination is important and can be done by the primary physician at the primary health care facility. history taking the primary physician must address the related signs and symptoms and also risk factors. the patient may have symptoms of claudication, pain at rest, walking impairment, and even ulcers. claudication is defined as pain that occurs with activity or exercise and resolves after 10 minutes of rest. it usually includes pain in the hip, buttock, calf, thigh, and foot. (39) the ischemic rest pain is usually felt in the forefoot and metatarsal area.(4) physical examination in the cli suspected patient, assessment of the arterial system of the limb is very important. the physician must palpate and address any abnormal pulses of the arteries (femoral, popliteal, dorsalis pedis, and posterior tibialis).(4) the absence of dorsalis pedis pulsation might happen in 12% of patients, but the absence of a posterior tibial pulse is always abnormal. other physical findings are audible bruits, nonhealing wounds or ulcers, gangrenous tissue, pallor at elevation, delayed capillary refill, and cool limbs. (4,9) besides the examination of the affected limb, we must also examine the blood pressure on both arms to evaluate if there is subclavian artery disease. a 20 mmhg difference between both arms is sugges tive for subclavian disease.(40,41) diagnostic testing suspicion of cli must be supported with the presence of occlusive disease. however, there are limitations in the primary health care facilities to perform a more advanced examination such as treadmill test, duplex ultrasound, computed tomography angiography/magnetic resonance angiography (cta/mra). the resting anklebrachial index (abi) is an initial diagnostic test that can be done to confirm cli in primary health facilities. abi is the highest systolic blood pressure of the foot (dorsalis pedis or posterior tibial) divided by the highest systolic pressure of the arm.(42) the resting abi is defined as abnormal (0.90), borderline (0.91-0.99), normal (1.00-1.40), and noncompressible (>1.40).(43,44) age  65 years old age 50-64 years old along with risk factors of atherosclerosis: diabetes mellitus, hypertension, cigarette smoking, or dyslipidemia. age < 50 years old with diabetes mellitus and an additional risk factor for atherosclerosis atherosclerotic disease in other vascular beds table 1. risk factors of cli(38) may-august 2021 vol.40no.2 168 laksono, siregar, kusharsamita cli in covid-19 infection an abi score of 0.90 shows reduced peripheral blood flow and is suggestive for the diagnosis with 95% sensitivity and 99% specificity. abi <0.4 is indicative of severe ischemia. a lower abi indicates a more severe ischemia.(45) however, an abi of more than 1.40 may be associated with calcified, rigid, and non-compressible arterial walls; a risk of having cardiovascular events, and impairment of quality of life. (4 6,4 7) it is recommended to measure abi in patients with a history and physical examination suggestive of cli and in patients with an increased risk for cli without features in the history and physical examination. management strategy of chronic limb ischemia once the diagnosis of cli is established, the management strategy should be initiated to prevent complications, reduce the risk of cardiovascular events, and improve the functional capacity. the early management strategy in primary health care includes pharmacological therapy and lifestyle modification. pharmacological therapy the treatment using pharmacological agents in patients with cli is customized, based on the comorbidities (hypertension, dyslipidemia, diabetes mellitus). antiplatelet therapy using aspirin (75325 mg/day) or clopidogrel (75 mg/day) can be given in symptomatic patients. (class: i, level of evidence: a). antiplatelet therapy is indicated as secondary prevention in patients at high risk of cardiovascular events.(13) in pad patients who are asymptomatic, the use of aspirin may reduce the risk of cardiovascular events even though a systematic review conducted by schmit et al.(48) showed no benefit of aspirin in asymptomatic pad patients. the use of clopidogrel is highlighted in a study by katsanos et al.(49) where clopidogrel was able to reduce the risk of major adverse cardiovascular events (mace). clopidogrel can be used as a single therapy for pad patients to prevent the risk of bleeding if combined with aspirin. in covid-19 infection, early medical ther apy to revascula rize can be done by administering anticoagulants with therapeutic doses of heparin or enoxaparin. however, patients should be referred to hospitals with availability of tissue plasminogen activator (thrombolytic therapy) and interventional facilities if needed.(11) other comorbidities must be tr eated according to the guidelines. statin therapy is indicated in all patients with pad (asymptomatic and symptomatic) and it is associated with reduced cardiovascular events and mortality. this is supported by the data from the reduction of atherothrombosis fo r continued health (reach) registry which showed a 17% reduction of the adverse cardiovascular events. in cli patients, the low-density lipoprotein cholesterol (ldl-c) should be maintained at <70mg/dl or 50% from the initial ldl-c levels (if baseline: 70-135 mg/dl).(14,50,51) for the hypertensive condition, administration of angiotensin-converting enzyme inhibitor (acei) and angiotensin ii receptor blocker (arb) are also associated with a decreased cardiovascular events with the goal to maintain the blood pressure at <140/90 mmhg.(14) in diabetic patients, the hba1c levels should be maintained at <7% to prevent further complication.(52) symptomatic treatment for pain is also important to improve the quality of life. physician could prescribe paracetamol or non-steroidal antiinflammatory drugs (nsaid) to eliminate the pain, although those medications might not be sufficient.(47,53) however, we have to be aware giving the nsaid because it can disrupt the wound healing process.(54,55) lifestyle modification smoking has been a major risk factor in cli development. smoking cessation must be advised to the patient. smoking cessation is associated with smoking abstinence and therefore decreases the risk of cli.(56) stopping of smoking will lower the mortality of the patient and also will increase amputation-free survival.(57) some additional therapy could be given to help the smoking 169 cessation program, such as varenicline, bupropion, and nicotine replacement therapy.(13) physical activity is important for cli patients because it reduces the role of risk factors in the progression of the disease. it is recommended for patients to do structured exercise therapy, which is also able to reduce the claudication symptoms. several studies were able to show the beneficial effect of exercise on claudication.(58,59) structured exercise therapy (set) can be done in a health facility or at home. the set program at the health facility (class: i, level of evidence: a) includes an intermittent walking exercise which is supervised by the healthcare provider. the exercise is performed with a minimum duration of 30-45 minutes/session, with 3 sessions/ week for a minimum of 12 weeks. the patient can do the self-directed set program at home or the set can be community-based (class: iia, level of evidence: a) with the guidance and exercise regimen prescription from the healthcare provider. (1 3) patien ts who do no t have improvements after therapy and even experience limb-threatening conditions should be referred to the adva nced health facilities to undergo revasc ularization ther apy and wound management by the wound specialist team.(60-62) patients with chronic limb-threatening ischemia (clti) need a more adequate approach with a multi-specialist team. the goals are: to control the symptoms, increase the quality of life, and prevent any complications (medical goals); revasc ularization t o preserve the limb (interventional goal); and proper monitoring after treatment (surveillance goal). primary care physicians have an important role and should be able to give care to achieve the medical goals.(63) preventive strategy of cli reducing the risk of cli progression is the goal especially in the vulnerable population. physical activity can prevent the development of the disease. it is related to its effect on controlling the atherosclerotic factor in the patient. the benefit of physical activity is also related to its function on increasing nitric oxide, thus preventing endothelial dysfunction.(64) other preventive strategies include the maintenance or control of lipid level, blood pressure, and also blood glucose level.(65) conclusion endothelial dysfunction due to covid-19 infection puts patients at higher risk for developing cli. resting abi is a reliable diagnostic test for patients with suspected cli. an abi of 0.90 is suggestive of the presence of ischemia. the management includes pharmacological therapy and lifestyle modification to reduce cardiovascular risks and prevent further complications. conflict of interest there is no potential conflict of interest acknowledgement the authors thank all who supported this manuscript. contributors sb contributed significantly in writing the review. rh and hk all wrote the revision. all authors participated in identifying the relevant studies for inclusion. manuscript. all authors read and approved 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f, et al. physical activity in the prevention of peripheral artery disease in the elderly. front physiol 2014;5:12. doi:10.3389/fphys.2014.00012. 65. aggarwal s, loomba rs, arora r. preventive aspects in peripheral artery disease. ther adv cardiovasc dis 2012;6:53-70. doi: 10.1177/ 1753944712437359. january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 resveratrol protects against copper and iron toxicity in drosophila melanogaster osaretin godwin igharo1* , lucky osemu ebaluegbeifoh2 , godwin aigbedo aikpitanyiiduitua3 , henry uzor oshilonyah4 , and idris babatunde momodu5 abstract background copper (cu) and iron (fe) are essential trace elements that when in excess are capable of causing cytotoxic effects leading to lipid peroxidation and promoting oxidative stress. resveratrol (res) is a natural polyphenol with antioxidant and anti-inflammatory properties. this study was carried out to evaluate the protective role of res in fe and cu sulphate-induced oxidative stress in drosophila melanogaster. methods adult wild type flies were fed cu2+ and fe2+ (1 mm each) and/or res (30 and 60 mg/kg diet) for 7 days. survival, negative geotaxis and emergence rate were evaluated by daily recording of fruit fly mortality and final analysis. fruit flies were anaesthetized using co 2 gas, homogenized and centrifuged at 4,000 rpm for 10 minutes at 4°c. aliquots of the supernatants were used for the estimation of biochemical markers using spectrophotometry. results fruit flies co-treated with feso4 + cuso4 (1 mm each) + res (30 and 60 mg/kg) significantly elevated h2o2, no, lipid peroxidation, acetylcholinesterase as well as gsh, gst, catalase and total thiols (p<0.05) compared with the cu2+ + fe2+ (1mm each) treated group. flies co-treated with feso4 + cuso4 (1mm each) + res (30 and 60mg/kg) also had significantly improved (p< 0.05) eclosion and climbing rates compared with the cu2+ + fe2+ (1mm each) treated group. 1biomedical toxicology and chemical safety research group, department of medical laboratory sciences, school of basic medical sciences, college of medical sciences, university of benin, benin city, nigeria 2centre of excellence in reproductive health innovative, university of benin, benin city, nigeria 3education department, medical laboratory science council of nigeria, abuja, nigeria 4department of microbiology, faculty of science, university of delta, agbor, delta state, nigeria 5department of science laboratory technology, federal polytechnic, kaltungo, gombe state *correspondence: dr. osaretin godwin igharo university of benin, benin city, nigeria tel.: +2348038664896 email: osaretin.igharo@uniben.edu orcid id: 0000-0002-7086-534 conclusion this study demonstrated that res reduced cu2+ and fe2+-induced radical generation in d. melanogaster and improved the antioxidant buffering capability of the flies. therefore, res could be used in management of disorders involving oxidative stress. keywords: resveratrol, reactive oxygen species, antioxidant, drosophila melanogaster date of first submission, october 31, 2022 date of final revised submission, march 1, 2023 date of acceptance, march 8, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article cite this article as: igharo og, ebaluegbeifoh lo, aikpitanyi-iduitua ga, oshilonyah hu, momodu ib. resveratrol protects against copper and iron toxicity in drosophila melanogaster. univ med 2023;42:29-40. doi: 10.18051/ univmed.2023.v42:29-40 29 doi: http://dx.doi.org/10.18051/univmed.2023.v42:29-40 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1385 https://orcid.org/0000-0002-7086-5347 https://orcid.org/0000-0001-5382-428x https://orcid.org/0000-0002-8914-1998 https://orcid.org/0000-0003-2202-2713 https://orcid.org/0000-0002-8877-1998 30 igharo, ebaluegbeifoh, aikpitanyi-iduitua. et al drosophila melanogaster introduction iron (fe2+)) and copper (cu2+) are essential trace elements (ete) for biological processes and vital functions such as cell respiration, maturation of erythrocytes, antioxidant defence, and enzyme cofactors.(1) iron and copper are cofactors required by enzymes for structural and catalytic activities in oxidative phosphorylation, protein synthesis, and neurotransmission modulation.(2) the routes for copper and iron administration are through inhalation, consumption of contaminated food and water, and dermal contact with polluted air, water, and soil. once inside the body, copper and iron are first deposited in the liver after which liver detoxification activities occur. these metals are present in trace amounts because a variety of homeostatic mec hanisms in normal circumstances maintains a physiologically essential amount of these metals.(1) however, when in excess, dyshomeostatic mechanisms of iron and copper are capable of causing cytotoxic effects and production of highly damaging free radicals by fenton or haber-weiss reactions, thus causing brain lipid peroxidation and protein oxidation which promotes oxidative stress.(3,4) reactive oxygen species (ros) are formed by the ability of copper to change states, from cupric (cu2+) to cuprous (cu+) by cuproenzymes which are involved in redox reactions. cuprous ions can catalyse hydrogen peroxide decomposition, through the fenton reaction, which forms hydroxyl radicals (oh) capable of reacting with biological molecules such as proteins, lipids and dna, thereby damaging them.(5) the ability of iron to accept and donate electrons can lead to the formation of reactive nitrogen and oxygen species which trigger the oxidative attack of the brain, thereby contributing to brain disease and perhaps aging itself. the buildup of copper or iron in the liver, kidneys, brain, and eyes results in possible cell death, permanent nerve damage, oxidative stress, and reduced cell proliferation.(6,7) re s ve r a tr o l ( re s) i s a po l yp h e no l commonly found in grapes, wine, peanuts, and soy, and is known to possess anti-oxidative, anticarcinogenic, and anti-inflammatory properties.(8, 9) a study showed that res can alleviate liver injury in iron-overloaded mice. the mechanism may be related to improving antioxidant capacity and reducing excess iron in the liver.(10) interestingly, drosophila melanogaster models have been used in vivo to investigate disease mechanisms. (8-10) drosophila has a genetic makeup of about 60% homology to that of humans. also, the fruit fly possesses 75% of the genes responsible for human diseases as homologues, thereby making it a very good model organism.(11) as a result of ros production which links oxidative stress, resveratrol mitigates ros generation through (i) the prevention of molecular oxygen and/or peroxide reactions and the chelation of iron and copper ion; (ii) the chelation of iron and copper to the redox state being kept, making the iron and copper incapable to reduce molecular oxygen; (iii) the trapping of formed radicals.(12) previous studies have shown that res has the potency to increase glutathione peroxidase, superoxide dismutase as well as glutathione levels. futher more, res also dec reases lipi d pero xidati on and prot ein oxidation. thus, a decrease in the amount of lipid peroxides also confirms the decrease in oxidative stress.(8, 9) one study showed that res can alleviate liver injury in iron-overloaded mice. the mechanism may be related to improvement of antioxidant capacity and reduction of excess iron i n t he l ive r. ( 13 ) st u di e s on t h e us e of d. melanogaster in iron and copper-induced oxidative stress and the preventive effects of r e s ve r a t r ol a r e s c a r c e i n t h e l i t e r a t u r e . therefore, as an alternative to conventional rodent models, a study is needed to investigate the cu2+ and fe2+-induced toxicity and the r e l e va n c e o f res p r o t e c t i ve r ol e in d. me la n og as t e r. in t hi s r e s e a r c h , d. melanogaster was employed as a model to examine cu2+ and fe2+-induced toxicity and the ameliorative impact of res. 31 univ med vol. 42 no. 1 methods research design the experimental study was carried out at the drosophila laboratory, department of biochemistry, university of ibadan, nigeria, between january and august, 2021. drosophila melanogaster stock and culture dros o ph i l a me l a no g as t e r ( ha r wi c h strain) of both genders (1-3 days old) were cultured and maintained in the drosophila me la n og as t e r re se a r c h la bo r a t o r y, de pa rtment of bioc he mistry, col le ge of medicine, university of ibadan, oyo state, nigeria. the flies were allowed to mate in vials monitored at regulated temperature and humidity (22–24°c; 60–70% relative humidity) until the eggs hatched into young adult fruit flies under 12 hours’ light and 12 hours’ dark daily cycles for the period of administration of the chemical compound under investigation. chemical compounds re sve ra tr ol wa s p ro cur e d fro m a k scientific, 30023 ahern ave, union city, ca 94587, united states of america at a percentage purity of 95%. copper sulphate and ferrous sulphate were procured from sigma aldrich. all chemicals used were of analytical grade. 1chloro-2,4-d initr obenzene ( cdnb), 5, 5'd it h i ob i s-2-n i t r o -be nzo i c a ci d ( dt n b) , acetylthiocholine iodide, and hydrogen peroxide (30% solution [w/w]) were purchased from sigma usa. other reagents were commercial products of high analytical grade. treatment of d. melanogaster with cuso 4 and feso 4 for oxidative stress inducement the flies were fed on diet mixed with cuso 4 and feso 4 at a final dose of feso 4 0.5 mm, feso 4 1 mm, feso 4 0.5 mm + cuso 4 0.5 mm a n d fe so 4 1 mm + cuso 4 1 mm , respectively. the flies were monitored for 7 days, harvested and homogenized, after which free radical generation was determined. treatment of d. melanogaster with cuso 4 , feso 4 and resveratrol the flies were fed on a diet mixed with cuso 4 , feso 4 , and/or res, while the final group (g5) received edta instead of res, at a dose of 50mg/kg diet. the flies were divided into six groups, each containing five replicates/groups with 40 flies/vial: positive control: 50 mg edta /kg diet, g1: 30 mg res/kg diet), g2: 60 mg res/kg diet), g3: 1mm cuso 4 + 1mm feso 4 + 30 mg res/kg diet), g4: 1mmcuso 4 + 1mm feso 4 + 60 mg res/kg diet and g5: (1mm cuso 4 + 1mm feso 4 + 50mg edta/kg diet). the flies were monitored for 7 days, harvested and homogenized, followed by determination of free radical generation. preparation of samples for biochemical assays for the determination of biochemical assays, 50 flies (of both genders) were exposed to final concentrations of feso 4 0.5 mm, feso4 1 mm, feso 4 0.5 mm + cuso 4 0.5 mm and feso 4 1 mm + cuso 4 1mm and res (30 and 60 mg/kg diet) for 7 days. at the end of the treatment period, flies were anaesthetized using co 2 , weighed, homogenized in 0.1m phosphate buffer, ph 7.0 (ratio of 1 mg:10 ml), and centrifuged at 4000 g for 10 min at 4 æ%c in a t he r mo sc i e n t i f i c so r va l mi c r o 1 7 r refrigerated centrifuge. subsequently, the supernatants were separated from the pellets into labelled eppendorf tubes, stored at -20 o c a n d u s e d f or t he d e t e r min a t io n of acetylcholinesterase (ache), gst and catalase activities as well as cell viability and tsh and h 2 o 2 levels. all the assays were carried out in duplicate for each of the five replicates of feso 4 + cuso 4 and resveratrol concentrations. in vivo assays survival and negative geotaxis (climbing) rate of flies the flies were exposed to a diet mixed with cuso 4 and feso 4 , namely feso 4 0.5 mm, 32 feso 4 1 mm, feso 4 0.5 mm + cuso 4 0.5 mm and feso 4 1 mm + cuso 4 1mm for 21 days. the survival rates were evaluated by daily recording of fly mortality after which the data were analysed.(14) we determined negative ge ot a x i s a n d e me r ge nc e r a t e o f d. melanogaster offspring after being fed for a period of seven days on a diet mixed with cuso 4 , feso 4 , and/or res and edta, respectively, in the positive control group and groups g1–g5, as stated in a previous section. ex vivo assays sample preparation for biochemical assays drosophila melanogaster treated with cuso 4 , feso 4 , and res for 7 days were anaesthetized using co 2 gas. the fruit flies were weighed and homogenized in 0.1 m potassium phosphate buffer (ph 7.4, 1 mg: 10 μl buffer). the homogenates were centrifuged at 4,000g for 10 minutes at 4 °c in a thermo scientific sorval legend micro 7r centrifuge. the aliquots of the supernatants were separated from the pellets into eppendorf tubes, kept at “20°c in a freezer and were used for the estimation of biochemical markers, ie. lipid peroxidation, hydrogen peroxide (h 2 o 2 ), total thiols (tsh), glutathione (gsh), protein, and nitric oxide (no, nitrate and nitrite) levels as well as the activity of enzymes such as acetylcholinesterase, catalase and glutathione-s-transferase (gst). in addition, behavioural parameters such as negative geotaxis were also determined. biochemical assays protein determination the principle of the assay is based on the reaction of cu+, produced by the oxidation of peptide bonds, with folin-ciocalteu reagent (a mi xt u r e of ph o sp hot u ngs ti c a c i d a n d phosphomolybdic acid. the reaction mechanism involves reduction of the folin-ciocalteu reagent and oxidation of aromatic residues (mainly tryptophan, also tyrosine). cysteine residues in p r o te in p ro ba b l y a l s o c o nt r i but e t o t he absorbance seen in the lowry assay. the concentration of the reduced folin reagent was measured by absorbance at 750nm. de t e r m ina t i o n o f c a t al as e an d acetylcholinesterase activity catalase activity was determined by the method descri bed by a ebi .(15) the reaction mi xt u r e co ns i s t e d o f 5 0 mm po t a s si u m phosphate buffer (ph 7.0), 300 mm h 2 o 2 and sample (1:50 dilution). the loss in absorbance of h 2 o 2 was monitored for 2 min at 240 nm and thence used for the calculation of catalase activity expressed as μmol of h 2 o 2 consumed per minutes per milligram of protein. the reaction was carried out in 0.1 m potassium phosphate buffer of ph 7.4, 1 mm dtnb and 0.8 mm acetylthiocholine as the initiator. the reaction was monitored for 2 min (at 30 s intervals) at 412 nm. the enzyme activity was determined as μmol of acetylthiocholine hydrolysed/minute/mg protein. the activity of acetylcholinesterase was determined according to the method described by ellman et al.(16) determination of total thiols, hydrogen peroxide, and nitric oxide (nitrate/nitrite) le ve l the reaction mixture contained 510 μl of 0.1 m phosphate buffer (ph 7.4), 20 μl of sample, 35 μl of 1 mm dtnb and 35 μl of distilled water. then, incubation was carried out at room temperature for 30 min. the absorbance was measured at 412 nm. hydrogen peroxide level was determined using the method of wolff.(17) the reaction mixture consisted of fox 1 (10 ml of 100 mm xylenol orange, 50 ml of 250 mm ammonium ferrous sulphate, 10 ml of 100 mm sorbitol, 5 ml of 25 mm h 2 so 4 and 30 ml of distilled water) and was reacted with the sample. after 30 min incubation at room temperature, the absorbance was measured at 560 nm and the values were extrapolated from the standard curve and expressed in micromole per milligram (μm/mg) protein. igharo, ebaluegbeifoh, aikpitanyi-iduitua. et al drosophila melanogaster 33 univ med vol. 42 no. 1 nitric oxide (nitrate and nitrite) level was estimated according to the method of the griess reaction. 250 μl of sample was incubated with 250 μl of griess reagent [0.1 % n-(1-naphthyl) ethylenediamine di hydrochloride; 1% sulfanilamide in 5% phosphoric acid; 1:1 ratio] at room temperature for 20 min. the absorbance was measured spectrophotometrically at 550 nm (od 550) and nitrite concentration was quantified by comparison with the od 550 of a standard sodium nitrite solution of known concentration. determination of glutathione s-transferase activity the activity of gst was quantified based on the method of habig and jakoby (18) where 1chloro-2, 4-dinitrobenzene (cdnb) was used as substrate. the reaction mixture contained 270 μl of a solution made up of 20 μl of 0.25 m potassium phosphate buffer, ph 7.0, with 2.5 mm edta, 10.5 μl of distilled water and 500 μl of 0.1 m gsh at 25 °c),10 μl of 25 mm cdnb and 20 μl of sample (1:5 dilution). the mixture was monitored for 5 min (at 10 s intervals) and absorbance was read at 340 nm using a spectrophotometer. determination of lipid peroxidation lipid peroxidation was estimated by measuring the formation of thiobarbituric acid reactive substances (tbars) following the method of varshney and kale. (19) under acidic condition, malondialdehyde (mda) produced from peroxidation of membrane fatty acids and food products react with the chromogenic reagent, 2-thiobarbituric acid (tba). a pink coloured complex is generated and absorbance is read at 532 nm. determination of reduced glutathione level the reduced glutathione (gsh) level was measured according to the method of beutler et al. (20) the reduced form of glutathione comprises in most instances the bulk of cellular non-protein sulphhydryl groups. this technique depends on the development of a relatively stable (yellow) colour when 5´, 5´-dithiobis-(2-nitrobenzoic acid) (ellman’s reagent) is added to sulphhydryl compounds. the chromophoric product resulting from the reaction of ellman’s reagent with the reduced glutathione is measured at 412nm. estimation of glutathione (gsh) content glutathione (gsh) content was determined colorimetrically using ellman’s reagent (dtnb) according to the procedure described by jollow et al.(21) the supernatant was precipitated with 4% sulphosalicylic acid (4%) in the ratio of 1:1. the samples were preserved at 4 æ%c for 1 h and then subjected to centrifugation at 5000 rpm for 10 min at 4 æ%c. the assay mixtures consisted of 550 µl of 0.1 m phosphate buffer, 100 µl of supernatant and 100 µl of dtnb. the od was read at 412 nm and the results were expressed as moles of gsh/gram tissue. statistical analysis data were expressed as mean  standard error of mean. treated and control groups were compared using one-way anova followed by tukey’s post hoc test. in all the groups, differences with a p-value < 0.05 were considered statistically significant. results survival rate and selected biochemical markers af ter exposure to fe rrous and copper sulphate induced oxidative stress in drosophila melanogaster figure 1 shows the effects of ferrous and copper sulphate on the survival biochemical markers in of d. melanogaster. all doses of feso 4 and cuso 4 induced a toxic effect thus, reducing the survival of flies (figure 1). at varied concentration, feso 4 and cuso 4 significantly (p<0.05) depleted t-sh, gst, gsh levels and catalase activities except 1mm feso 4 where there was no significant difference for both gst and catalase after 7 days of treatment when compared to the control group (figure. 1b, 1c, 1e and 1g) (p>0.05). furthermore, significant 34 (a) (b) (c) (d) (e) (f) (f) (g) figure.1. effects of feso 4 and cuso 4 on survival rate and biochemical parameters in d. melanogaster. data are presented as mean ± se of 40 flies/ vial with 5 replicates per treatment group. significant differences when compared with the control group are indicated by (p<0.05) igharo, ebaluegbeifoh, aikpitanyi-iduitua. et al drosophila melanogaster 35 univ med vol. 42 no. 1 t ab le 1 . c o m p ar is o n o f am el io ra ti ve i m p ac t o f re sv er at ro l o n f er ro u s an d c o p p er s u lp h at ein d u ce d o x id at iv e st re ss i n d ro so p h il a m el a n o g a st er *s ig n if ic an t d if fe re n ce c o m p ar ed w it h c o n tr o l g ro u p ( p < 0 .0 5 ) t ab le 2 : c o m p ar is o n o f am el io ra ti ve im p ac t o f re sv er at ro l o n c li m b in g ra te , e cl o si o n r at e, a n d s el ec te d b io ch em ic al m ar ke rs a ft er e x p o su re o f d . m el a n o g a st er t o v ar ie d c o n ce n tr at io n s o f fe rr o u s an d c o p p er s u lp h at e k ey : v al u es e xp re ss ed i n m ea n + s ta nd ar d e rr o r o f m ea n ( n = 5 ), p < 0 .0 5 = si gn if ic an t, f es o 4 = f er ro u s su lp h at e, c u s o 4 = co pp er s u lp h at e, r e s = r es ve ra tr o l, e d t a = et h yl en ed ia m in et et ra ac et ic a ci d , a c h e = a ce ty lc h o li n es te ra se 36 increase was observed in no (nitrate and nitrite, figure. 1d) and h 2 o 2 (figure 1f) levels at varied concentration of feso 4 and cuso 4 when compared to the control goup (p<0.05). resveratrol reverses ferrous and copper sulphate induced oxidative stre ss in drosophila melanogaster we assessed hydrogen peroxide and nitric oxide levels in d. melanogaster after 7 days of ferrous and copper sulphate exposure and observed that res restored ferrous and copper sulphate -induced elevations of h 2 o 2 (p < 0.05), and no levels (p<0.05). we also evaluated selected markers of antioxidants such as tbars, tsh, gsh, gst, and catalase (table 1). the levels of total thiols (tsh) in cu + fe and resexposed flies were not significantly different when compared with the controls (p>0.05) as shown in table 1. in addition, res reversed cu + feinduced inhibition of catalase activity and improved gst activity that was inhibited by cu + fe in d. melanogaster (p<0.05). for tbars, a significant increase (p<0.05) was observed in the flies when comparisons between the control group and the various treatment groups were made. however, flies that were treated with 60mg res /kg diet were closer in values to that of the control group. it could be inferred that resveratrol restored ferrous and copper sulphate-induced elevated h 2 o 2 , no and tbars levels in drosophila melanogaster. compar ison of amel ior ative impact of resveratrol on negative geotaxis (climbing rate), eclosion and selected biochemical markers after exposure of d. melanogaster table 2 shows th e c omparison of ameliorative impact of res on climbing rate, eclosion and selected biochemical marker after exposure of d. melanogaster to var ied concentration of ferrous and copper sulphate after seven days of treatment. there was a significant reduction in the climbing rate (p<0.05) in cu + fe challenged flies when compared with other groups; however, resveratrol significantly increased the climbing rate (p<0.05). a significant reduction in the eclosion rate (table 2, p<0.05) in cu + fe challenged flies when compared with the control and other groups was also observed. however, resveratrol significantly increased the eclosion rate of flies as shown in table 2. for acetylcholine sterase enzyme, a significant increase (p<0.05) was observed when comparison between control and cu + fe challenged flies were made. interestingly, a significant decrease (p<0.05) was observed in the acetylcholinesterase activities of flies treated with resveratrol. therefore, resveratrol significantly reduced the activities of acetylcholinesterase after co-treatment with toxicants to a level comparable to the control group as well as improved the climbing rate and the eclosion rate in drosophila melanogaster. effects of ferrous sulphate (feso 4 ) and copper sulphate (cuso 4 ) on survival rate of drosophila melanogaster the effect of ferrous sulphate (feso 4 ) and copper sulphate (cuso 4 ) on sur viva l of drosophila melanogaster is shown in figure 2 below. selected concentrations of feso 4 and combination of feso 4 and cuso 4 doses were used as shown in figure 2., 7 days exposure duration was carried out and daily mortality was recorded in the survival study. it was observed that feso 4 and cuso 4 induced toxic effect at all doses used when compared with the control. hence, feso 4 and cuso 4 reduced the survival rate of the flies. discussion this study showed that ferrous and copper sulphate reduced the sur vival rate of d. melanogaster, suggesting that these metal ions are harmful at 1mm. excessive ros formation, which is an unavoidable by-product of cellular breakdown, leads to oxidative damage to macromolecules, increasing the susceptibility to degenerative disorders.(22) the significant increase in the levels of h 2 o 2 , nitric oxide, lipid peroxidation (tbars) with reduced total thiols igharo, ebaluegbeifoh, aikpitanyi-iduitua. et al drosophila melanogaster 37 univ med vol. 42 no. 1 and glutathione as well as catalase inhibition across flies treated with ferrous and copper sulphates (1mm each) in this study suggests that the formation of oxidants in the metal-treated flies outpaced the antioxidant buffering capability of the flies, resulting in ineffective oxidant detoxification. elevated nitric oxide (no) serves as a pro-inflammatory mediator.(14,23) therefore, both the ineffective oxidant detoxification and inflammation directly reduced the survival rate of the flies in the face of oxidative stress. remarkably, treati ng flie s with j ust resveratrol (30 or 60 mg/kg) increased their antioxidant and anti-inflammatory capacity, as seen by the lowering of hydrogen peroxide, nitric oxide, and lipid peroxidation, while increasing catalase activity and elevating glutathione, glutathione-s-transferase and total thiols. our study agrees with a previous study done by konyalioglu et al.,(24) which suggested that resveratrol partially protected embryonic neural stem cells from hydrogen peroxide-induced toxicity. these findings also agreed with a previous study carried out by rege et al.,(25) which postulated that resveratrol inhibited membrane lipid peroxidation and decreased the toxic effects produced by ros. in living organisms, glutathione-s-transferases (gsts) as detoxifying enzymes catalyse the conjugation of gsh to exogenous and endogenous electrophilic sites.(26) glutathione-s-transferase is a component of the first line of antioxidant defence. it aids in the prevention of free radical chain reactions and the detoxification of harmful ros when used in conjunction with gsh-dependent enzymes.(27 indeed, resveratrol corrected the depletion of gsh and total thiols caused by copper and ferrous sulphate in the flies. total thiols (including nonprotein and protein thiols) protect the body from oxidative stress.(28) in addition, resveratrol shielded gst activity from copper and iron-induced reduction, and the flies’ response to oxidative stress and heavy metal detoxification was activated. this finding led us to believe that resveratrol’s protective mechanism against copper and iron-induced toxicity is improved further by the enhancement of thiol-containing proteins other than the gsh molecule alone. these findings agree with a previous report that res restored cigarette smoke extract (cse)de pleted gsh levels by up regulation of antioxidant genes such as glutamate-cysteine ligase (gcl) through activation of nuclear erythroid-related factor 2 (nrf2) and also quenched cse-induced release of reactive oxygen species.(29) the antioxidative effects of rsv determined in the current study were consistent with the results of a study by li et al.,(30) which indicate the role of rsv in protecting against oxidative stress by decreasing ros levels and increasing the expression of cat, gsh-px, gsh and t-sod in obese-asthmatic rat models. increased catalase by resveratrol catalyses the dismutation of h 2 o 2 to molecular oxygen and water to reduce oxidative stress-induced toxicity.(31) the fact that resveratrol significantly decreases ferrous and copper sulphate-induced nitric oxide increase to levels comparable to the control group, shows that the flies’ basal level of no, which is essential for physiological functions, was restored. this finding is in line with man et al.,(32) who suggested that resveratrol inhibits the acetylation of endogenous enos on lysine residues in vitro and postulated that sirt 1 gene activation may play a fundamental role in regulating endothelial no and endotheliumdependent vascular tone by deacetylating nitric oxide synthase (enos). also, oh and yun (33) state that activation of sirt 1 through resveratrol in rinm5f cells (a pancreatic islet cell line) or isolated rat islets also prevented the proinflammatory stress induced by il-1β and ifn-γ by inhibiting inos and nitric oxide production likely through the inhibition of the nfκb signalling pathway. this observation also agrees with a previous report that resveratrol is effective in reducing the inflammatory status in vitro and in vivo settings of neuroinflammation.(34,35) the levels of tbars, the end result of lipid peroxidation, were also measured to support the idea that ferrous and copper sulphate cause oxidative stress. the copperand ferrous-sulphate 38 treated groups had significantly higher levels of tbars, indicating ox idative stress, but resveratrol treatment was able to ameliorate this impact. this observation is in agreement with a previous study, in that resveratrol prevents irondriven mitochondrial dysfunction by inhibiting glycogen synthase kinase-3 beta activity (a mechanism useful also to prevent tau hyper phosphorylation),(36) and by reducing peroxidation of lipoproteins and lipids through its activity as scavenger. therefore, the protective effects of resveratrol on ferrous and copper sulphateinduced oxidative stress can be related to its ability to scavenge free radicals and restore the cellular redox balance as well as control physiological activities of the flies. due to certain constraints, elucidation of the possible mechanism by which these metals induce their oxidative effects on drosophila melanogaster could not be ascertained. however, the ability of resveratrol in ameliorating these effects may present it as a suitable antioxidant agent in the prevention of heavy metal-associated diseases. conclusion we demonstrated that feso 4 and cuso 4 were able to induce oxidative stress. interestingly, resveratrol was able to ameliorate free radical generation. as a result, resveratrol protects against copper and iron oxidant toxicity in vivo when studied in combination. because of the greatly complex chemical composition and multiple pharmacological effects of res, further research on its protective mechanism is required. conflict of interest the authors declare that there is no conflict of interests. acknowledgement the authors sincerely appreciate all staff members of the drosophila unit, department of biochemistry, university of ibadan. contributors all authors take public responsibility for the contents of the manuscript submitted to universa medicina. the concept of this research was developed by ogi and loe. ogi, loe, and gaaii performed the animal experiments. huo and ibm worked on the concept and draft, and prepared the manuscript. all authors have read and approved the final manuscript. references 1. pavelková m, vysloužil j, kubová k, vetchý d. biologická role mědi jako základního stopového prvku v lidském organismu. 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aikpitanyi-iduitua. et al drosophila melanogaster september-december 2022 vol. 41 no. 2 no.3 universa medicina cite this article as: sulfiana, iswanti fc. chemokines in allergic asthma inflammation. univ med 2022;41: ........doi: 10.18051/univmed.2022. v41..... pissn: 1907-3062 / eissn: 2407-2230 chemokines in allergic asthma inflammation sulfiana1 and febriana catur iswanti2* abstract asthma is the most frequent noncommunicable disease and one of the leading causes of years lived with disability. asthma has a severe impact on a patient's life, being able to disturb the activities of both children and adults. the morbidity and mortality of asthma may depend on the severity and progressiveness of the symptoms experienced by the patient. different and complex pathomechanisms underline the pathology of asthma, in which the regulation of innate and adaptive immune responses plays a role. there is a complex interaction between immune cells including chemokines involved in the pathogenesis of asthma. immune cell trafficking is orchestrated by a family of small proteins called chemokines. leukocytes express cell-surface receptors that bind to chemokines and trigger transendothelial migration. this review article outlines the main role of chemokines in inflammatory reactions that occur in allergic asthma, based on the latest literature studies that have been published previously. the allergic reaction in asthma expresses various chemokines and their receptors. chemokines including eotaxins (ccl11, ccl24, and ccl26), ccl2, ccl5, ccl17, and ccl22 regulate immune cells that under pathological conditions travel to the inflammatory site, mainly in the lung, to protect the body from pathogen invasion. chemokines are released by a number of immune cells such as monocytes, dendritic cells, mast cells, and epithelial cells in the airway. the biological effects of chemokine production are enhanced by secreted cytokines when an allergic reaction occurs in asthma, such as il-4, il-5, and il-13. chemokines cause an accumulation of different inflammatory cells at the site of inflammation, which ultimately results in tissue damage to the airway. the inhibition of the reactions evoked by the interaction between chemokines and their receptors is considered a candidate for the development of potent therapeutic drugs for asthma in the future. keywords: asthma, allergic inflammation, chemokines, immune cells. abbreviations: asm : airway smooth muscle cells; ba: bronchial asthma; bams: bronchoalveolar macrophages; ccl: chemokine (c-c motif) ligand; ccr: cc chemokine receptor; cd: cluster of differentiation 2; cdc42 : cell division cycle 42; crs: chemokine recognition site; crth2: chemoattractant receptor-homologous molecule expressed on th2 cells; cxcl: chemokine (c-x-c motif) ligand; cxcr : cxc chemokine receptor; cys-lts: cysteinyl leukotrienes; dag: 1,2-diacylglycerol; dalys: disability-adjusted life years; dc: dendritic cell; ec: epithelial cell; ecl: extracellular loop; egfr: epidermal 1master's programme in biomedical sciences, faculty of medicine, universitas indonesia, jakarta, indonesia 2department of biochemistry and molecular biology, faculty of medicine, universitas indonesia, jakarta, indonesia *correspondence: dr. dr. febriana catur iswanti, m. biomed department of biochemistry and molecular biology, faculty of medicine, universitas indonesia, jakarta, indonesia phone: +628151665231 email: febriana.iswanti@ui.ac.id orcid id: 0000-0001-7593-2716 date of first submission, april 18, 2022 date of final revised submission, september 6, 2022 date of acceptance, september 19, 2022 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2022.v41.................... copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1332 review article mailto:febriana.iswanti@ui.ac.id https://orcid.org/0000-0001-9164-049x https://orcid.org/0000-0001-7593-2716 http://dx.doi.org/10.18051/univmed.2022.v41................. sulfiana, iswanti allergic asthma inflammation introduction allergic disorders are a significant public health concern. ec ological changes, industrialization, and immunologic associations increase the prevalence of allergic disease such as bronchial asthma.(1,2) asthma is a chronic respiratory disease characterized by reversible airway obstruction, increasing mucus production, bronchospasm, and underlying airwa y inflammation.(3) the global initiative for asthma (gina) defined asthma as “a heterogeneous disease, usually characterized by chronic airway inflammation. it is defined by the history of respiratory symptoms such as wheeze, shortness of breath, chest tightness and cough, that vary over time and intensity, together with variable expiratory airflow limitation”.(4) the prevalence of asthma has been increasing consistently for several decades.(5) about 339 million individuals have asthma globally.(6) asthma is positioned 16th among the leading causes of years lived with disability and 28th among the main causes of disease burden, as estimated by disability-adjusted life years (dalys) universally. the percentage of total dalys due to asthma in 0-9-year-olds ranked 19th in 2019.(7) around 418,000 people die every year because of asthma, and dalys attributable to asthma account for 24.8 million.(1) the percentage of asthma in the u.s. population has increased over time. current asthma prevalence increased from 7.3% in 2001 to 7.9% in 2017. in 2019, the total prevalence of asthma in the u.s was about 7.8%, while the percentage of asthma in the child population (<18 years) and in adults (18 years) was 7.0% and 8.0 %, respectively.(6) according to the 2018 indonesian basic health research, the prevalence of asthma at all ages in indonesia is about 2.4%, while the highest percentage of asthma is for yogyakarta (4.5%), followed by east kalimantan (4.0%), bali (3.9%), centra l kalimantan (3.4%), and nor th kalimantan (3.3%).(8) based on research utilizing the international study of asthma and allergies in childhood (isaac) questionnaire, the asthma prevalence in indonesia varies by region, ranging from 4-11% in 6-7-year-olds and 6-13% in 1314-year-olds.(9) under pathologic conditions, the immune responses of the ind ividual r equire the coordination of various cells and secreted factors to defend the body against invasion by pathogens. complex interactions between cells, cytokines, and chemokines are involved in asthma pathogenesis. leukocytes express cell surface receptors (chemokine ligands) capable of binding to chemokines on endothelial cells, resulting in growth factor receptor; er: endoplasmic reticulum; fcεri: high-affinity ige receptor; fcεrii: low-affinity ige receptor; feno : fraction of exhaled nitric oxide; gag: glycosaminoglycan; gata3: gata binding protein 3; gdp: guanosine diphosphate; gefs: guanine nucleotide exchange factors; gina: global initiative for asthma; gm-csf: granulocyte-macrophage colony-stimulating factor; gpcrs: g protein-coupled receptors; gtp: guanosine triphosphate; gtpases: guanosine triphosphatases; icl: intracellular loop; icos: inducible co-stimulator; ifnγ: interferon gamma; ige: immunoglobulin e; il: interleukin; ilc2: innate lymphoid cells 2; ip-10: interferon gammainducible protein 10; ip3: inositol 1,4,5-trisphosphate; isaac: international study of asthma and allergies in childhood; jak: janus kinase; mapk: mitogen-activated protein kinase; mbp: major basic protein; mcp-1: monocyte chemoattractant protein-1; mdc: macrophage-derived chemokine; mhc: major histocompatibility complex; nap-1: neutrophil activating protein-1; nf-κb : nuclear factor κb; nk: natural killer; pak1: p21-activated kinase 1; pbmcs: peripheral blood mononuclear cells; pga: paucigranulocytic asthma; pgd2: prostaglandin d2; ph: pleckstrin homology; pi3kγ: phosphatidylinositol-3-kinase γ; pip2: phosphatidylinositol (4,5)-bisphosphate; plc-: phospholipase c-; ptks: protein tyrosine kinases; px : phox homology; rantes: regulated upon activation normal t-cell expressed and presumably secreted; ros: reactive oxygen species; stat6: signal transducer and activator of transcription 6; tarc: thymus and activation-regulated chemokine; tgf-β1: transforming growth factor β1; th: t-helper; tlrs: toll-like receptors; tnfα: tumor necrosis factor-α; tslp: thymic stromal lymphopoietin; yld: years lived with disability; 7tm: 7-transmembrane. transendothelial migration of the leukocytes.(10– 12) tissue-resident cells release inflammatory chemokines during an inflammatory response. interaction between chemokines and types of lymphocytes in some allergic diseases has been presented in a previous literature study.(13) here, this r eview aims to descri be the role of chemokines especially in the allergic inflammation of asthma. an understanding of the cellular process of chemokines in asthma is essential for developing advanced asthma therapy. we used search engines including pubmed and google scholar to explore research articles, reviews, reports, e-books, and other data sources, published in 2013-2022. the search terms used include allergic inflammation, chemokine-receptor interactions, asthma endotypes, eosinophilic and non-eosinophilic asthma, and therapeutic strategies. classif icat ion of asthm a and clinical manifestation asthma is a common heterogeneous chronic respiratory disorder and has a variety of phenotypes. classically, asthma has been divided into extrinsic and intrinsic asthma. extrinsic asthma or atopic asthma is commonly associated with allergy, begins in childhood, the patient has relatives with allergy, and starts after an individual has been exposed to the allergen. in intrinsic asthma, there is no allergy or family history of asthma and the disorder begins in adulthood. the global initiative for asthma has classified asthma according to the frequency and severity of symptoms into intermittent, mild persistent, moderate persistent, and severe persistent asthma.(14,15) however, based on the underlying pathological mechanism, asthma is classified as type 2 and non-type 2 asthma. this classification is widely accepted because it is considered to reflect better the endotypes.(16) type 2 is the most common type of asthma, in which t helper 2 (th2) lymphocytes are related to airway inflammation. the involvement of th2 lymphocytes causes eosinophilic inflammation of the airway by producing interleukin (il)-4, il-5, il-13, and immunoglobulin e (ige). innate lymphoid cells 2 (ilc2) also add to the airway inflammatory responses in type 2 asthma.(17) frequently, patients with this type have a level of blood eosinophilia of 150/µl and fraction of exhaled nitric oxide (feno) of 20 ppb. type 2 asthma has been classified into early-onset allergic eosinophilic airway inflammation and late-onset nonallergic eosinophilic airway inflammation. the allergic form of type 2 asthma can be triggered by exposure to an allergen, but in late-onset nonallergic eosinophilic airway inflammation, there is no allergy involved. microbes, viruses, air pollutants, glycolipids, and irritants can trigger this airway inflammation.(18,19) non-type 2 asthma is characterized as a non-eosinophilic asthma without the presence of type 2 inflammatory markers and consists of neutrophilic and paucigranulocytic inflammation, depending on the cellular findings in sputum specimens. th1 and th17 cells are involved in its pathobiology. other cytokines implicated in this non-type 2 asthma include il 1ß, il 6, il 8, il 17a/f, tumor necrosis factor-α (tnf α), and interferon-gamma (ifn γ).(20) neutrophilic inflammation is characterized by the proportion of neutrophils of 40%–60% i n sputum specimens. infection, tobacco smoke, and irritants can trigger this type of inflammation. neutrophilic inflammation is often associated with th1 and th17-related cytokines, il-8, and granulocytemacrophage colony-stimulating factor (gm-csf) that induce neutrophils to migrate to the airways. paucigranulocytic asthma (pga) is the least common and milder disorder, in which the patients have normal eosinophils and neutrophils in the sputum.(18–20) the frequency of asthma symptoms varies greatly, as various triggers can exacerbate the symptoms. some patients have infrequent, brief attacks of asthma a nd some can suffer continuous symptoms. asthma is characterized by excessive sputum production, shortness of breath, cough, and wheezing, sometimes accompanied by the inability to lie flat, insomnia, and fatigue. symptoms often deteriorate at night. univ med vol. 41 no 3 sulfiana, iswanti allergic asthma inflammation a b circadian rhythms of bronchomotor tone and bronchial reactivity reach the lowest point between 3-4 in the morning, increasing bronchoconstriction symptoms. the presence of tachypnea, wheezing, chest hyperinflation, tachycardia, diaphoresis, inability to speak, and difficulty lying are the signs that arise in severe asthma.(21,22) pathomechanism of asthma when the body is exposed to allergens, generally aeroallergens such as pollen and house dust mites, these attach to the toll-like receptors (tlrs) and promote their activation. activated tlrs induce epithelial cells to produce the cytokines thymic stromal lymphopoietin (tslp), il-25, and il-33, which are able to promote type2 asthma adaptive immune responses. tlr activation also triggers the secretion of chemokines such as ccl2 and ccl20, that aim to promote the maturation of dendritic cells (dc). the dendritic cells then head to the lumen of the airway to take up aeroallergens and produce allergen peptide fragments to be served to type ii major histocompatibility complex (mhc) proteins. activation of th2-mediated immune responses and inflammatory cytokine generation are induced by il-33.(14,23,24) naive t cell stimulation requir es costimulatory molecules such as cd28, inducible co-stimulator (icos), and ox40 with ligands that exist in dc (cd80/b7.1, cd86/b7.2, ox40 ligands, and icos ligands). differentiation of t lymphocytes is also heavily influenced by the cytokine environment. th2 polarization implies high amounts of il-4 and low levels of il-12.(14) activation of th2 cells through the main transcription factor, namely gata binding protein 3 (gata3), induces the secretion of il-5, il-4, and il-13 by these cells. il-5 plays a role in the maturation and survi val of eosinophils. furthermore, these cells will migrate to the epithelium of the bronchus and induce chemokines such as eotaxins (ccl11, ccl24, ccl26) and ccl5 which are paired with ccr3 receptors. immunoglobulin isotypes changes in b cells are mediated by il-5, il-4, and il-13, the latter two promoting ige synthesis through il-4rα.(12,19) ige recognizes two types of receptors, namely highaffinity receptors (fcεri) and low-affinity receptors (fcεrii/cd23). fcεri receptors are expressed by mast cells, basophils, dendritic cells, and eosinophils. in addition, fcεri is found in other cells such as airway smooth muscle cells (asm), and epithelial and endothelial cells. in antigen presentation, dendritic cells are induced by bonds between ige and fcεri receptors to activate th2 cells. through cd23, ige receptors are activated on epithelial cells of the airway and are involved in the transportation of the igeallergen complex across the airway mucosal barrier. eosinophils actively release mediators, such as histamine, lipid mediators, cysteinyl leukotrienes (cys-lts), il-8, major basic protein (mbp), gm-csf, and reactive oxygen species (ros). besides eosinophils, histamine is also released by basophils and mast cells. the role of histamine in asthma is inducing bronchial smooth muscle contractions, increasing capillary permeability, disruption of the epithelial barrier, and hypersecretion of mucus. cys-lts and prostaglandin d2 (pgd2) are also produced by mast and eosinophil cells that can trigger eosinophil chemotaxis via the chemoattractant receptor-homologous molecule expressed on th2 cells (crth2), which are expressed in ilc2, th2, eosinophils, and basophils. crth2 activation escalates airway inflammation. epithelial damage in asthma is caused by mbp mediators. as for remodeling of the airway, this is mediated by leukotrienes. il-8 plays a role in neutrophil recruitment.(12,24) classification of chemokines and chemokine receptors the purpose of the immune system is to protect the body against pathogens. chemotactic trafficking of the immune response is governed by 20 g protein-coupled receptors (gpcrs) and more than 40 chemokines that escort the immune cells to the right site at the right time.(25) chemokines are small soluble proteins (<10 kda), consisting of 70–80 amino acid residues with conserved sequence, are structurally related to cytokines, and play the main role in immunity and inf lammation. chemo kines contain four conserved cysteine residues forming two disulfide bonds.(26,27) the term chemokine is derived from the wor d “chemotaxis”, meaning the cell movement triggered by a chemical stimulus. “taxis” in classical greek means “arrangement”. the stimulus will attract the cells towards chemoattractants including chemokines, which are arranged in concentration gradients in the microenvironment. cells migrate from one location in the body to another, where they may invade tissues or recirculate through the blood or lymph. chemokines are a broad category of molecules with similar fundamental structures and activities. chemokines comprise almost 50 similar proteins with a molecular weight of 8 to 10 kda.(28) chemokines contain a three-stranded β-sheet and a c-terminal α-helix domain lying across one face of the β-sheet, and flanked by an n-terminal domain (usually ~10 residues). chemokines are classified into four subgroups based on the position in the molecule of the first two cys residues, which may be adjacent (cc, n=28), separated by one amino acid (cxc, n=17), separated by three amino acids (cx3c, n=1), or only the first cysteine group is present (c, n=2). most chemokines are chemotactic agonists.(28,29) chemokines are cytokines that stimulate cell migration toward sites with higher ligand concentrations. the ligands adhere to the extracellular matrix and generate a gradient of different expansion in tissues. leukocytes that possess chemokine receptors, a class of g protein-coupled receptors expressed on the cell surf ace, can detect this concentration gradient.(28,30) the chemokine receptor consists of seven transmemb rane helices ( 7t m) connected by 3 intracellular loops (icl1-3) and 3 extracellular loops (ecl1-3), an extracellular n-terminal domain, and an intracellular c-terminal domain, which includes an α-helix (helix 8). a disulfide bond connects the n-terminus to ecl1 and ecl3 to ecl2. chemokine receptors are classified into 2 groups, namely the g proteincoupled chemotactic chemokine receptors (typical receptors) and the atypical chemokine receptors. chemokine receptors are involved in biological processes, including cell adhesion and migration to inflammatory sites by binding to their ligands.(29) there are 19 chemokine receptors in humans, namely ccr1 – ccr10, cxcr1 – cxcr6, cxcr8, xcr1, and cx3cr1, that are activated by different chemokines.(31) chemokine signaling pathway all members of the chemokine family have the same tertiary structure, which consists of a flexible n-terminal and an irregular n-loop, followed by a three-stranded antiparallel β-sheet onto which a folded c-terminal α-helix is attached. the n-terminal is widely recognized as being essential for receptor activation.(27,32) chemokine receptors and their chemokine ligands interact via a two-step binding process. first, the chemokine’s c-terminal domain binds to the receptor’s nterminal domain and extracellular loops (ecl)/ crs [chemokine recognition site 1 (crs1)]. the unstructured n-terminus of the chemokine can then target the 7tm helical bundle (crs2) and fix the receptor in an active conformation, thereby inducing intracellular signal transmission (figure 1).(31) the chemokine signaling pathway is transduced by g-protein coupled receptors expressed on the surface of immune cells. interaction of the chemokine with the chemokine receptors which are expressed on the cell surface as 7-transmembrane proteins coupled with gprotein will promote cell signal transmission following ligand binding. after activation, gpcrs as guanine nucleotide exchange factors (gefs) for the gα subunit produce the exchange of guanosine diphosphate (gdp) to guanosine triphosphate (gtp), leading to dissociation of the gt p-bound gα subunit from the gβγ heterodimers.(33) protein dissociation into subunits α and βγ will initiate diverse downstream g proteindependent effectors including phospholipases a2, univ med vol. 41 no 3 sulfiana, iswanti allergic asthma inflammation cell adhesion, chemotaxis, and myosin contraction, rac controls the formation of lamellipodia, while cdc42 controls the formation of filopodia. p21activated kinase 1 (pak1) as downstream targets of a small guanosine triphosphatase also has a role in myosin contraction. cellular regulatory mechanisms control the expression, activation, and signaling of gpcrs.(29,33) role of c he mokines in aller gic asthma inflammation the role of chemokines is critical in the immune system. the ability of dendritic cells to migrate to the lymph nodes is mediated by chemokines at the beginning of an adaptive immune response.(28) chemokine receptors are activated when chemokines are secreted into blood vessels, where they promote leukocyte adhesion to vascular endothelium, reorganization of the ac tin cytoske leton, and leukocyte extravasation or migration into the tissues. in the tissue extracellular matrix, chemokines form defined concentration gradients by binding glycosaminoglycans (gag), which will eventually c (subtypes β2 and β3) and d, protein tyrosine kinase s (pt ks) a nd phosphata ses, phosphatidylinositol-3-kinase γ (pi3kγ), low molecular weight guanosine triphosphatases (gtpases), and mitogen-activated protein kinases (mapks). phospholipase c-β (plc-β) hydrolyzes phosphatidylinositol (4,5)-bisphosphate (pip2) to form inositol 1,4,5-trisphosphate (ip3) and 1,2-diacylglycerol (dag). ip3 releases ca2+ from intracellular stores in the endoplasmic reticulum (er), which acts with dag to activate protein kinase c (pkc), which involves regulating receptors via phosphorylation and desensitization. pi3kγ phosphorylates pip2 to form pip3 at the cell membr ane, which causes loca lized recruitment of proteins containing pleckstrin homology (ph) domains or phox (px) domains, leading to actin polymerization and shape changes at the leading edge of the cell. these processes result in the movement toward the site of highest concentration of the chemokine. ph domaincontaining targets (protein kinase b (akt), rhogef, racgef, and cdc42gef) modulate cell movement. rho is involved in the regulation of figure 1. chemokine–receptor interactions. the chemokine c-terminal domain (-helix) interacts with the nh 2 terminal domain and extracellular loops of the chemokine receptor (light blue). following the targeting of the 7tm helical bundle by the nh 2 -terminus of the chemokine, the receptor is fixed in an active conformation, resulting in the induction of intracellular signal transmission. 7-tm (7-transmembrane) direct leukocyte migration to the location of tissue damage.(27) resident cells in the airway include epithelial cells and alveolar macrophages, and are cellular sources of chemokines.(34,35) the presence of a leaky epithelium due to damage to barrier integrity has been shown in asthma. damage to the epithelial cells causes secretion of alarmins, chemokines, and other cytokines. the epithelial cell (ec)-derived cytokines such as tslp, il-5, il-33, and gm-csf can induce the differentiation and activation of th2 cells, ilc2s, basophils, eosinophils, and alt ernatively activated macrophages.(35) some chemokines have a crucial role in the pathophysiology of asthma. the chemokines and their receptors involved in asthma are shown in figure 2. eotaxin eotaxins are proteins that belong to the chemokine family, consisting of eotaxin-1 (ccl11), eotaxin-2 (ccl24), and eotaxin-3 (ccl26), each of which attracts and activates ccr3-bearing cells. eotaxin-1 was the first eosinophil-specific chemoattractant to be discovered, followed by eotaxin-2 and eotaxin-3. according to a study, eotaxin-1 levels are correlated with the number of eosinophils found in samples of pig blood and lung, being crucial for eosinophil mobilization from the bone marrow. children with stable asthma reported greater amounts of eotaxin-1 compared to controls, and they also had sputum eosinophilia. to respond to inflammation in the early stages, eotaxin-1 is required, while eotaxin-2 and eotaxin-3 are required later to keep eosinophils alive. eotaxin1 has also been found as a chemokine that has a centr al r ole in producing airway hyperreactivity.(36,37) a recent study also showed that serum levels of eotaxin-2 and transforming growth factor β1 (tgf-β1) were substantially greater in patients with severe eosinophilic univ med vol. 41 no 3 figure 2. human chemokine and chemokine receptors are involved in the pathophysiology of asthma. ccl2 causes intracellular signal transduction by interacting with the ccr2 receptor. ccl5 interacts with ccr1, ccr3, and ccr5. ccl5 is a chemoattractant for a wide spectrum of immune cells (dendritic cells, t-cells, eosinophils, monocytes, basophils, nk cells), guiding them to the site of inflammation in the airway. some chemokines have the same chemokine receptor, including eotaxin 1,2, and 3 (bind ccr3); ccl17 and ccl22 (bind ccr4). cxcl8 and cxcl10 bind cxcr2 and cxcr3, respectively sulfiana, iswanti allergic asthma inflammation asthma, there being a significant association between these two cytokines.(35) after stimulation with il-4 and il-13, eotaxins are produced by lung epithelial cells, eosinophils, lymphocytes, mast cells, vascular endothelial cells, dermal fibroblasts, alveolar macrophages, and in smaller amounts by smooth muscle cells of the airways. cutaneous and nasal epithelial cells, fibroblasts, and macrophages are the principal sources of eotaxin2 (ccl24) in the human body, while eotaxin-3 is produced primarily by endothelial cells and dermal fibroblasts. when eotaxin activates the ccr3 receptor, the ligand is internalized, and chemotaxis is induced via calcium mobilization and actin polymerization. with il-5, eotaxin primarily attracts eosinophils to the lungs.(36–38) ccl17 and ccl22 epithelial cell -derived chemokines bind their receptors ccr3 and ccr4 in th2 cells. the chemokine receptor ccl13 interacts with ccr3, while ligands for ccr4 (that is highly expressed by th2 cells) are ccl17, also referred to as tarc (thymus and ac tivation-regulated chemokine) and ccl22, a macrophage-derived chemokine. both ccl17 and ccl22 are chemoattractants for th2 cells, induce the differentiation of naive t cells into th2 cells, and are considered a marker of human and murine m2a macrophages.(37,39) ccl17 is one of the chemokines that has been linked to type 2 immunological responses. human ccl17/tarc is an 8-kda, 71-aminoacid protein that is encoded on chromosome 16q13. mature myeloid dendritic cells and langerhans cells are the main sources of this chemokine. ccl17 has a biological effect in the trafficking of th2 cells in eosinophil-associated disorders, such as allergic asthma, by binding to ccr4. as a result, increased levels of ccl17 in serum and/or tissue, as well as cellular ccr4 expression, may serve as biomarkers for disease severity. ccl17 production is stimulated by il-4 in immune cells, which synergizes with various cytokines. ccl17 production is initiated also by il-3, tnf-α, and ifn-γ, which o per ate synergistically in an nfκb-dependent manner in bronchial and alveolar epithelial cells.(13,40) by connecting to the ccl17 gene promoter directly via two binding sites, activation of signal transducer and activator of transcription 6 (stat6) is a crucial step in il-4-induced ccl17 production. in research on asthma, the ccl17ccr4 axis plays a role in th2 cell migration to the lungs. in individuals with asthma, ex vivo allergen exposure of human bronchial explants has been found to stimulate the activation of ccl17.(40) a clinical study showed a significant association between sputum eosinophilia and overexpr ession of ccl17 mrna in the epithelium and submucosa of bronchial biopsies of patients with asthma compared to controls.(13) ccl22, also known as macrophage-derived chemokine (mdc), has a 37% amino acid sequence homology with ccl17/tarc, but has a greater affinity (2 to 3-fold) for its receptor (ccr4) than ccl17. in alle rgic a sthma, pulmonary ccl22 level is elevated. ccl22 is produced by immune cells such as dendritic cells, monocytes, natural killer (nk) cells, and macrophages. it is more effective in inducing lymphocyte vcam-1 integrin-dependent arrest. ccl22 stimulates activated ccr4-positive th2 cells, which keep the allergic process going.(40,41) the interaction of dendritic cells with naive t cells involves a binding between ox40l on dendritic cells with ox40 on naive t cells. after activation and differentiation of t cells, th2 releases cytokines tnf-α, il 4, il-5, and il-13 which cause excessive mucus secretion, disruption of the epithelial barrier, inflammation, and airway hyperresponsiveness. secretion of ccl17 by ecs is stimulated by il-4 and il-13. expression of ccl17 in bronchial epithelial cells is mediated by metalloproteinase-dependent phosphorylation of epidermal growth factor receptor (egfr), mapk, and nuclear factor κb (nf-κb). il-13 also induces ccl22 secretion. expression of ccl17 and ccl22 is upregulated together with ccr4 to recruit th2 cells.(37,39) ccl2 ccl2, also called mcp-1 (monocyte chemoattractant protein-1), is an important 13 kda protein with 76 amino acids that is encoded on chromosome 17q11.2 and is involved in monocyte and macrophage migration, particularly in the lungs, where these cells turn into bronchoalveolar macrophages (bams).(42,43) monocytes and macrophages are the most common sources of ccl2, but it is also produced by endothelial cells, mast cells, fibroblasts, epithelial cells, and smooth muscle cells. ccl2 causes intracellular signal transduction by interacting with the ccr2 receptor (a g-protein-coupled receptor), which is expressed by immune cells such as t cells, b cells, monocytes, and nk cells.(42–44) ccl2 may play a role in allergic airway responses, resulting in airway hyper-responsiveness by promoting mast cell activation and the production of inflammatory mediators such as leukotriene c4. ccl2 that is released from bronchial epithelial cells via upregulated gene expression is enhanced by cytokines il-4 and il-13.(43,44) in the study by velikova et al., mcp-1/ccl2 levels were shown to be considerably greater in children with bronchial asthma (ba) and cystic fibrosis than in controls.(42) ccl5 ccl5 from the cc chemokine family, with a molecular weight of 7.5 kda, is a powerful leukocyte chemoattractant that plays a key function in inflammation. it is also known as regulated upon activation, normal t-cell expressed and presumably secreted (rantes), that activates and trafficks a wide spectrum of immune cells, including t-cells, eosinophils, dendritic cells monocytes, basophils, nk cells, to the site of inflammation when it interacts with its receptors, ccr1, ccr3, and ccr5. ccl5 levels have been found to be higher in asthma patients. furthermore, targeting ccl5 with antibodies in the allergic mouse model inhibited inflammation of the airway.(45) a study suggested that ccl5 is linked to allergic inflammation. ccl5 was significantly higher in patients with atopic asthma than in controls, and it was positively correlated with absolute eosinophil counts and total serum ige in juvenile asthma patients. in asian and caucasian populations, two ccl5 snps (located at -403g/ a and -28c/g, respectively) were linked to the risk of asthma.(13) eosinophils are key effector cells in the airway inflammation of type 2 asthma, (37) and also has an important effect in airway remodeling. interaction of eosinophils with mast cells in an in-vitro study showed that secretion of fibrogenic factors was followed by the release of tgf-β1 from eosinophils, while interaction of eosinophils with epithelial cells promoted the release of epithelial-derived cytokines such as ccl5, cxcl8, and ccl11. these chemokines will induce eosinophil infiltration into the airway tissues. eosinophil granules contain a variety of cytokines, enzymes, extracellular matrix, growth factors, and chemokines, including ccl3, ccl5, ccl7, ccl8, ccl1, ccl13, ccl17, ccl22 and cx cl1, cx cl8 , cx cl9, cx cl10, cxcl12,(46) among which ccl5 and ccl11 are potent eosinophil chemoattractants. ccl11 induces eosinophil chemotaxis through the mapk and extracellular regulated protein kinase (erk) pathways, while cxcl8 and cxcl10 induces eosinophil chemotaxis by binding to cxcr2 and cxcr3, respectively. the eosinophil invasion is associated with asthma severity.(37,47) cxcl8 and cxcl10 neutrophils are involved in non-type 2 asthma, in which the patients show a neutrophilpredominant phenotype without evident th2 cytokines. potent c hemoattra cta nts for neutrophils are cxcl8 and cxcl10. a study by takaku et al. identified that both cxcl8 and cxcl10 are increased in asthma phenotypes with increased neutrophils and eosinophils. cxcl8 binds to cxcr2, a specific surface receptor on neutrophils.(11,37) in addition, cxcl1 and cxcl5 also bind to cxcr2. cxcl8/interleukin-8/neutrophil activating protein-1 (nap-1), is a potent neutrophil chemoattractant and activator that is released by univ med vol. 41 no 3 sulfiana, iswanti allergic asthma inflammation neutrophils, macrophages, epithelial cells, and bronchial smooth muscle cells. neutrophils can force their own recruitment by producing il-8 when an inflammatory response occurs.(48,49) like many other chemoattractants, cxcl8 causes cytoskeleton reorganization, intracellular ca2+ alterations, integrin activation, granule protein exocytosis, and respiratory burst.(48) cxcl8 also attracts circulating monocytes, which develop into macrophages in the lungs and are assumed to be responsible for neutrophilic inflammation.(50) the binding of cxcl1 and cxcl5 to cxcr2 activates ne utrophils, stimulates adhesion molecule expression, and cell homing. the cytokine il-17 induces cxcl1 in bronchial ec mainly via mpk and erk pathways. il-17 also promotes cxcl5 expression in ec. while cxcl10 secretion in ec cells is stimulated via the janus kinase (jak) pathway in synergy with tnf-α and interferon-γ, ccr6 is expressed by various cell subsets, including dendritic cells, t cells, and b cells. in the t-cell subset, the th17 cell has the highest ccr6 expression. ccr6 binds to its receptor, ccl20, which results in th17 migration to the lungs. a study in patients wi t h a s thma s h ow e d e n ha nc e d ccr6 expression on th cells in peripheral blood mononuclear cells ( pbmcs) compared to control subjects.(11,37) eosinophils are thought to have a role in asthma exacerbation through a variety of processes, including activation of cxcl10 and c o nt a c t wi t h n e ut r op hi ls . ( 5 1 ) cx cl1 0 o r interferon gamma-inducible protein (ip)-10 is produced by activated t cells, while its ligand (cxcr3) is highly expressed by th1 cells. a study on virus-induced pediatric asthma showed elevated serum cxcl10 levels, implying that viral infections can trigger th1-chemokine responses in asthmatic patients.(52) ip levels in pe-diatric asthma are positively correlated with the severity of airway obstruction. thus, cxcl10 (ip-10) may be of benefit as a marker of inflammatory asthma severity.(53) cxcl10 with proinflammatory cytokines/chemokines such as tnf-α, il-6, cxcl8, act together in the development and worsening of allergic asthma.(52) through cxcr3, which is expressed on eosinophils, cxcl10 causes numerous effects including eosinophil adhesion, o 2 generation, and in vitro cytokine production.(51) a summary of the role of chemokines in allergic inflammation of asthma is listed in table 1. conclusion as thma is a co mmon he ter oge neo us chronic respiratory disorder with a variety of phenotypes and endotypes. chemokines have a prominent contribution to the process of allergic inflammation in asthma and their trafficking is driven by g protein-coupled receptors (gpcrs). the main sources of chemokines that are associated with allergic inflammation in the pathogenesis of asthma are epithelial cells, dendritic cells, eosinophils, and macrophages. m a n y c h e moki ne s ar e i nvol ve d in t he recruitment of inflammatory cells in the allergic reactions of asthma, particularly eotaxin, ccl17 (tarc), ccl22 (mdc), ccl2, and ccl5 (rantes). interaction of chemokines with chemokine receptors and their activation eventually direct leukocyte migration to the site of tissue inflammation. the inhibition of the interaction between chemokines and their receptors could be useful in the development of potent therapeutic drugs for asthma. conflict of interest all authors declare no conflict of interest. acknowledgments we would like to thank the master ’s programme in biomedical sciences, faculty of medicine, universitas indonesia, for the similarity test (turnitin) checker. contributors fc and sa contributed to conceptualization. sa contributed to drafting the article and the manuscr ipt’s r evision. fc contr ibuted to supervision and review. all authors have read and approved the final manuscript. references 1. almatroudi a, mousa am, vinnakota d, et al. prevalence and associated factors of respiratory allergies in the kingdom of saudi arabia: a crossuniv med vol. 41 no 3 references type of article chemokines mechanism of action zajkowska, et al.(36) literature review eotaxin 1, eotaxin 2, eotaxin 3 mobilization of eosinophils, mast cells, and th2 lymphocytes to the inflammation site. liu, et al.(37) literature review eotaxin 1 crucial chemoattractant for eosinophils. bakakos, et al.(38) literature review eotaxin 1 eotaxin 2, eotaxin 3 mobilization of eosinophils from the bone marrow. keeps eosinophils alive. abdelaziz, et al.(39) literature review ccl17, ccl22 induce the differentiation of naive t cells into th2 cells and chemotaxis for th2 cells to the lungs. catherine, et al.(40) literature review ccl17 chemotaxis for th2 cells to the lungs. rapp, et al.(41) original article ccl22 maintains the allergic reaction by activating ccr4-positive th2 cells. velikova , et al.(42) original article; a cross-sectional study ccl2 attracts monocytes and macrophages to the lungs. wang, et al.(43) literature review ccl2 attracts monocytes and eosinophils, and induces mast cell activation and leukotriene c4 secretion. bawazeer, et al,(44) original article ccl2 promotes mast cell migration to the inflammation site. alturaiki, et al. 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7:17–28. doi: 10.32598/jpr.7.1.17. c:\users\universa medicina\docu 29 abstract universa medicina suppression of transforming growth factor- by mesenchymal stem-cells accelerates liver regeneration in liver fibrosis animal model nur anna c sa'dyah1,4, agung putra2,4,5*, bayu tirta dirja3, nurul hidayah2, salma yasmine azzahara6, and risky chandra satria irawan2 introduction liver fibrosis (lf) results from the unregulated chronic wound healing process in liver tissue. transforming growth factor-beta (tgf-β) is the major contributing cytokine of lf promotion through activation of quiescent hepatic stellate cells (hscs) into myofibroblasts (mfs) and increased extracellular matrix (ecm) deposition such as collagen leading to scar tissue development. mesenchymal stem cells (mscs) have an immunomodulatory capability that could be used as a new treatment for repairing and regenerating lf through suppression of tgf-β. this study aimed to examine the role of mscs in liver fibrosis animal models through suppression of tgf-β levels without scar formation particularly in the proliferation phase. methods in this study, a completely randomized design was used with sample size of 24. male sprague dawley rats were injected intraperitoneally (ip) with carbon tetrachloride (ccl 4 ), twice weekly, for eight weeks to induce lf. rats were randomly assigned to four groups: negative control, ccl 4 group , and ccl 4 + msc-treated groups t1 and t2, at doses of 1 x 106 and 2x106 cells, respectively. tgf-β levels were analyzed by enzyme-linked immunosorbent assay (elisa). one-way anova and a least significant difference (lsd) was used to analyse the data. results the tgf levels of lf rat models decreased on day 7 after msc administration. the levels of tgf-β in both msc groups t1 and t2 decreased significantly compared with the control group (p<0.05). the tgf-β suppression capability of t2 was optimal and more significant than that of t1. conclusion mscs can suppress tgf levels in liver fibrosis induced rats. keywords: liver fibrosis, mesenchymal stem cells, transforming growth factor-, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.29-35 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1079 january-april, 2021 vol.40no.1 1department of internal medicine, medical faculty, sultan agung islamic university semarang 2stem cell and cancer research (sccr), medical faculty, sultan agung islamic university semarang 3department of microbiology, medical faculty, mataram university, west nusa tenggara 4department of postgraduate biomedical science, medical faculty, sultan agung islamic university, semarang 5department of pathological anatomy, medical faculty, sultan agung islamic university, semarang 6undergraduate student of medical faculty, diponegoro university semarang *correspondence: assoc. prof. dr. dr. agung putra m.si. med. kaligawe raya km. 4 semarang, central java, 50112, indonesia po box 1054/sm telp. (+6224) 6583584 email: dr.agungptr@gmail.com orcid id : 0000-0003-4261-9437 date of first submission, december 17, 2020 date of final revised submission, february 12, 2021 date of acceptance, february 26, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: sa'diyah nac, putra a, dirja bt, hidayah n, azzahara sy, candra r. suppression of transforming growth factor- by mesenchymal stem-cells accelerates liver regeneration in liver fibrosis animal model. univ med 2021;40:29-35. doi: 10.18051/univmed.2021.v40.29-35 30 sa’dyah, putra, dirja, et al mesenchymal stem-cells accelerates liver regeneration introduction liver fibrosis (lf), a most common chronic liver injury, results from unregulated wound healing.(1,2) transforming growth factor-beta (tgf-β), a cytokine released by inflammatory cells (for example macrophages), parenchymal cells, and platelets during the healing process, is the main contributor of lf promotion through activation of quiescent hepatic stellate cells (hscs) into myofibroblasts (mfs).(3) the active mfs are characterized by expression of α-smooth muscle actin (α-sma) and increased synthesis of the extracellular matrix (ecm) such as collagen, potentially inducing scar tissue development.(4–6) therefore, novel treatments to reverse fibrogenesis via tgf-β suppression remain to be explored. on the other hand, recent studies have reported that mesenchymal stem cells (mscs) offer a new alternative pattern in the repair and regeneration of lf that results in improved hepatocyte survival and inhibition of hsc activation.(7–9) however, the role of mscs in inactivating hscs through tgf-β has not been evaluated. mesenchymal stem ce lls (mscs) are identified as plastic-adherent cells, which have multipotent differentiation capacity into several specific mature tissue cells including osteocytes, chondrocytes, adipocytes, and neurocytes. (10) mesenchymal stem cells can also express several surface markers such as cd73, cd90, cd105, cd44, while lacking the expression of cd45, cd34, cd14 or cd11b, cd79a, or cd19, and human leucocyte antigen (hla) class ii. (11,12) a previous study revealed that mscs could preserve immunoregulatory pr operties including suppressing the prolonged release of tgf in liver injury.(5) furthermore, the improvement of lf is associated with a decreased level of tgf-β.(13) these statements indicate that mscs may decrease inflammatory cell-released tgf-β expression to induce optimum liver regeneration in lf. transforming growth factor β , belonging to a superfamily of cytokines and molecular agents, has a prime role in immune regulation and wound healing, including lf regeneration. (13,14) previous studies have demonstrated that tgfβ1 can activate hscs into mfs leading to the synthesis of excessive amounts of matrix proteins resulting in accelerated wound healing.(15,16) furthermore, tgf-β expressed by kupffer and other proinflammatory immune cells during liver injury plays essential roles in regulating tissue development and homeostasis.(17) however, a prolonged wound healing process could cause the aggregation of proinflammatory immune cells in liver injury, resulting in a continuous release of tgf β that is correlated with the accumulation of collagen deposition leading to lf formation.(7) therefore, there is a need for the suppression of tgf-β levels released by inflammatory cells, at the appropriate time, specifically on day 7, that indicates the proliferation phase, by using mscs to modulate liver regeneration without scar formation. here lies the difference between our study and the previous studies. the aim of this study was to examine the role of mscs in suppressing tgf-β levels to accelerate liver regeneration in lf. methods research design this experimental study was of completely randomized design to compare the treatment effects on the experimental and control groups at the end of the treatment. the research was conducted in june – august 2020 at the stem cell and cancer research (sccr) laboratory, faculty of medicine, sultan agung islamic university, semarang. isolation of mscs mesenchymal stem cells were obtained from a single pregnant sprague-dawley (sd) rat under anesthesia and transplanted into an lf rat model. in brief, after removal of the blood vessels the umbilical cord was cut into pieces and then transferred to a t25 culture flask containing complete dulbecco’s modified eagle’s medium 31 (dmem) (sigma-aldrich, st louis, mo) mixed with 10% fetal bovine serum (fbs) (gibco(tm) invitrogen, ny, usa), and 100 iu/ml penicillin/ streptomycin (sigma-aldrich). isolated cells were incubated in a 5% co 2 , 37ºc incubator and the medium was changed every 3 days. after the cells had attained 80% confluence, the msc-like cells were passaged with trypsin. cells from the 4th passage were used for the experiments. animal model for liver fibrosis in order to develop the liver fibrosis model, twenty-four male sd r ats wer e inj ected intraperitoneally with carbon tetrachloride (ccl 4 ) (sigma–aldrich, usa) at 1 ml/kg body weight, twice weekly for 8 weeks. administration of mscs we isolated mscs from a single rat umbilical cord and transplanted the uc-mscs into other rats (allogeneic transplantation). the sample size in this research was determined using the federer formula (t-1) (r-1) 15 (t=number of treatments; r=number of replications). therefore the size of the sample used in this study was 4x4=16 animals in which there were 4 animals in every treatment group. however, replication is required at every treatment as an alternative. therefore the total number of animals needed was 24, such that there were 6 animals for each treatment. the experimental rats were randomly assigned into the negative control (n=6), ccl 4 (n=6), and ccl 4 +t1 (n=6) and ccl 4 +t2 (n=6) groups. in addition, the negative controls received nacl 0.1 ml twice for a week, and the treatment groups t1 and t2 received ccl 4. plus mscs at doses of 1x106 and 2x106 cells in 500 µl nacl, respectively. the treatment doses were administered in 0.1 ml saline via tail vein injection twice for a week. flow cytometric immunophenotyping of mscs the msc immunophenotypes were analyzed in the fourth passage. mscs were stained with conjugated antibodies, namely fluorescein isothiocyanate (fitc)-conjugated cd90, allophycocyanin (apc)-conjugated cd73, peridinin chlorophyll protein complex (percp)conjugated cd105 and phycoerythrin (pe)conjugated lin monoclonal antibodies, for 30 min at 40c in the dark. the fluorescence intensity of the cells was evaluated through flow cytometry (bd bioscience, franklin lakes, nj, usa). in vitro differentiation mesenchymal stem cell differentiation potential was determined in order to characterize the isolated cells. these cells were cultured in dmem medium supplemented with 10% fbs, 10 mmol/l β-glycerophospate, 107mol/l/ 0.1 µm dexamethasone, 50 µmol/l ascorbate-2phosphate (all from sigma-aldrich, louis st, mo), at 370c and 5% co 2 . the fixed cells were stained with 0.2% alizarin red solution (sigma-aldrich) to demonstrate calcium deposition (cells used were from the fourth passage). enzym e-linked imm unosorbent assay (elisa) rat blood was harvested via periorbital venous plexus bleeding under general anesthesia on day 7 after msc administration and the serum was collected by centrifugation at 40c. the tgfβ levels were measured by means of enzymelinked immunosorbent assay (elisa) kits, based on the manufacturer’s instructions (abbkine) and according to a standard curve constructed for each assay. the colorimetric absorbance was recorded at a wavelength of 450 nm. statistical analysis all data were presented as mean ± standard deviation with differences between groups analyzed by a one-way anova and a least significant difference (lsd) comparison post hoc test. this was obtained using a p < 0.05 statistical significant value. ethical clearance this study was approved by the institutional review board of the ethics committee, faculty univ med vol. 40 no 1 32 sa’dyah, putra, dirja, et al mesenchymal stem-cells accelerates liver regeneration a b c of medicine, sultan agung islamic university, semarang, indonesia, under number 607/vi/2020/ komisi bioetik. results characteristics and differentiation of mscs the immunophenotype characteristics of mscs were evaluated using flow cytometric analysis to determine and verify the specific markers of mscs. the results showed that the isolated cells expressed an msc-specific marker profile, such as the presence of cd105 (96.7%), cd73 (99.2%), and cd90 (96.7%), and lack of lin (0.03%) (figure 1). in line with the expression of msc specific markers, we also analyzed msc morphology and differentiation. mscs were isolated by their plastic adherence capability under standard culture conditions (37°c, 5% co 2 ) and their peculiar fibroblast-like (spindle shape) morphology (magnification x200, scale bar 100 μm). the differentiation assay indicated that the multipotency of cultured mscs was wellmaintained which was identified as calcium deposits (red color appearance) in alizarin red staining (figure 2). this method corresponds to the minimal criteria for mscs of the international society of cellular therapy (isct). mesenchymal stem cell suppression of tgfβ levels in lf in lf, tgf-β plays a crucial role through induction of hsc activation, whereas reduction of tgf-β expression is correlated with lf improvement. to determine the role of mscs in lf regeneration, we measured the tgf-β levels using elisa. the levels of tgf-β in both the low and high dose msc groups were significantly figure 1. msc characteristics by flow cytometry. mscs expressed positive markers (cd105, cd73, and cd90) and lack of lin (lin ). figure 2. morphology and differentiation of mscs. (a) msc morphology. mscs showed a red color with alizarin red staining (magnification x40, scale bar 50 μm). (b) msc differentiation. after the fourth passage, msc differentiation showed as homogeneous, spindle-shaped, fibroblast-like cells (200× magnification, scale bar 100 μm) 33 univ med vol. 40 no.1 decreased (t1=64.07 ± 27.53; t2=34.91 ± 0.74) compared with the control group (184.3 ± 8.26) (p<0.05). the suppression capability of high dose mscs was optimal and more significant than that of low dose mscs. in addition, the level of tgfβ1 expression in the ccl 4 group was high due to continuous inflammation. discussion this study showed that the tgf-β levels of the lf rat models decreased on day 7 after msc administration. several studies have widely demonstrated that the potential of mscs to regenerate diseased livers, including lf, is by inducing hepatocyte proliferation, in addition to reducing the activation of hepatocyte stellate cells (hscs).(18–20) the activation and differentiation of hscs into mfs are induced by main molecular agents, particularly tgf-β.(14,21) tgf-β is the most potent profibrotic cytokine released and activated after tissue injury, thus suppressing its expression is important in fibrotic disease.(21) previous studies have shown that mscs can reduce the expression of tgf-β1 level in fibrotic diseases.(13,22) however, the decrease in tgf-β expression by mscs in lf has not been clearly explained. therefore, in this study we investigated the mscs’ pivotal role in suppressing tgf-β levels, leading to the decrease in collagen density in lf regeneration. to analyze the roles of mscs in suppressing tgf-β levels associated with collagen density in lf, we used carbon tetrachloride (ccl 4 )-injected sprague-dawley rats to induce an established lf animal model, according to previous protocols. in our study, the decrease in tgf-β1 levels occurring in lf might be controlled by msc administration through the release of antiinflammatory cytokines, primarily interleukin 10 (il-10). as a potent anti-inflammatory cytokine, il-10 could prevent fibrosis progression by competitively binding to tgf-β receptors resulting in the reduction of tgf-β expression. currently, mscs can suppress tgf-β levels released by m2 macrophages through release of il-10.(9,23) we suggest that the decrease in tgf by il-10 occurs through the binding of il-10 to kupffer receptors that activate janus tyrosine kinase 1 (jak1) and tyrosine kinase-2, leading to signal transduction and activation of transcription 3 (stat3) which then migrates to the nucleus and binds to the target gene promoter, resulting in the suppression of tgf-β expression.(9,24,25) in group tgf-β1 (pg/ml)(mean ± sd) nc 44.38 ± 8.60 ccl4 184.31 ± 28.55* t1 64.07 ± 27.53* t2 34.1 ± 0.74* table 1. levels of tgf-β1 by treatment group on day 7 data were presented as the mean ± standard deviation. *p<0.05. nc = negative control; ccl 4 = carbon tetrachloride; t1 = ccl 4 + msc dose of 1 x 106 cells; t2 = ccl 4 + msc dose of 2 x 106 cells; tgf-β1 = transforming growth factor-β1. group comparison group significance 95% confidence interval lower limit upper limit nc ccl4* 0.01 -178.06 -101.77 t1 0.27 -57.83 18.46 t2 0.58 -28.67 47.62 ccl4 t1* 0.01 82.09 158.38 t2* 0.01 111.25 187.54 t1 t2* 0.04 -8.98 67.31 table 2. post-hoc lsd test for levels of tgf-β1 expression between study groups significance p < 0.05; nc = negative control; ccl4 = carbon tetrachloride; t1 = ccl4+ msc dose of 1 x 106 cells; t2 = ccl4+ msc dose of 2 x 106 cells 34 sa’dyah, putra, dirja, et al mesenchymal stem-cells accelerates liver regeneration addition, the increased tgf-β levels in the control group may be due to the lack of il-10 that is released by mscs to suppress the tgf-β levels, consequently, the tgf level increases because the inflammation has not been controlled. on the other hand, the decrease in tgf may also be effected by blocking the smad pathway.(24) in regard to the janus tyrosine kinase 1 (jak1) pathway to suppress tgf, il-10 also blocks the smad pathway. the interaction of il-10 with the tgfβ-receptor induces serine/ threonine kinase activity, leading to the induction of the downstream signaling proteins smad2 or smad3. this results in the inhibition of the expression of fibrotic genes, such as α-sma, collagen and fibronectin.(26–28) this is supported by a previous study that reported that increased il-10 induces smad7, re sulting in downregulation of the smad2/smad3 pathway.(29) thus, we believe that blocking the binding of tgfβ-receptors by il-10 released mscs may be associated with decreased tgfβ expression leading to lf improvement. overall, the implication of this study may be useful for research on the potential of stem cells to accelerate and direct target screening through molecular mechanisms to treat fibrosis in chronic liver injury. a limitation of this study is that we did not examine il-10 as a competitive receptor to tgf-β associated with jak1 and smads. in addition, we also did not analyze the collagen density as a fibrosis indicator in the lf animal model. t here for e, f or f uture dir ections, understanding the role of il-10 secreted by mscs to suppress tgf-β expr ession i n association with the decrease in collagen remains to be explored. conclusions we conclude that mscs can suppress the tgf-β levels in lf induced rats. this study provides new insights into the benefits of ucmscs administration in most injured tissues, particularly in the proliferation phase in liver fibrosis. conflict of interest the authors report no conflicts of interest. the authors are responsible for the content and writing of this article. acknowledgment we would like to thank the stem cell and cancer research (sccr) laboratory, the faculty of medicine, sultan agung islamic university (unissula), semarang, indonesia and all who contributed to this research. contributors nacs and ap did the concept and design of the work. btd and nh built the database and did the data collection. sya and rcsi did all the analysis and interpretation. ap supervised the 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mutation and variants of the sars-cov-2 gene edy parwanto* department of biology, faculty of medicine, universitas trisakti, indonesia since the onset of the covid-19 pandemic, our society has come to understand that the cause is the corona virus (2019-ncov) (1) or more popularly known as the severe acute respiratory syndrome coronavirus 2 (sars-cov-2).(2) further developments showed that sars-cov2 has mutated. it was reported in the uk on 18 dece mber 2020 tha t sars-cov-2 gene mutations resulted in a new variant called b.1.1.7 or “variant under investigation, year 2020, month 12, variant 01” (vui 202012/01).(3) the name b.1.1.7 is derived from its phylogenetic heritage. since the new viral variants are under study, they are also called “variant under investigation” or “variant of concern”.(4) for this reason, the sars-cov-2 lineage variant b.1.1.7 is also referred to as “variant of concern 202012/01” (voc-202012/01).(5) the b.1.1.7 variant of sars-cov-2 is also called 20i/501y.v1.(6) moreover, it has been demonstrated that line b.1.1.7 is associated with significant changes in the viral phenotype.(7) the sars-cov-2 lineage variant b.1.1.7 is characterized by 8 gene mutations in the spike protein. for example, sars-cov-2 variant b.1.1.7 contains the mutations d614g and n501y, that appear to increase the interaction between the spike protein and the angiotensin converting enzyme 2 (ace2) receptor.(8) other researchers have also stated that the new variant of sars-cov-2 has an n501y mutation in the spike protein. the new variant “may be associated” with the recent rise in cases in southeast england. even so it is stated that the new variant of sars-cov-2 which is more contagious does not necessarily makes it more dangerous. it is further stated that a number of new variants have been detected in the uk, for example the d614g variant. in more detail, it is stated that the new variant vui-202012/01 is defined by multiple spike protein mutations, including deletion 69-70, deletion 144, n501y, a570d, d614g, p681h, t716i, s982a, d1118h. subsequently it was reported that it was 14 days from 16th december 2020 there has been a spike in covid-19 cases in south east england, uk.(9) apart from type b.1.1.7 / 20i / 501y.v1 of sars-cov-2 which appeared in the uk there is a new sars-cov-2 lineage 501y.v2 which was found in southern africa and which emerged after the first epidemic wave in a severely affected metropolitan area, nelson mandela bay, located on the coast of the eastern cape province. this lineage spread rapidly, becoming within weeks the dominant lineage in the eastern cape and western cape provinces.(10) it was further stated that two of the substitutions in the 501y.v2 lineage (e484k and n501y) are within the receptor binding motif (rbm) associated with the function of binding with the human ace2 (hace2) receptor. apart from the uk and south africa, it has been reported that the b.1.1.7 variant of sars-cov-2 shows rapid transmission in the united states.(5) furthermore, a new variant of sars-cov-2, namely b.1.429 (also known as cal.20c or 452r.v1), has been identified in california, usa. this strain is characterized by the presence of 5 mutations, namely orf1a: i4205v, orf1b: d1183y, s: s13i; w152c; l452r.(11) in response to the new variants of sarscov-2, various types of vaccines have been tested. at the community level, it is imperative that public health authorities communicate what the variants mean for people’s day-to-day risk and explain why masking and physical distancing remain the best strategy for preventing disease universa medicina may-august 2021 vol.40no.2 *email: edyparwanto@trisakti.ac.id; edy.parwanto@gmail.com orcid id : 0000-0002-0797-6925 78 and viral evolution. to overcome the new variant of sars-cov-2 which is based on the substitution of n501y on the spike protein, isogenic n501 and y501 of sars-cov-2 have been made. the mrna-ba sed cov id-19 vaccine namely bnt162b2 has been tested and has a neutralizing titer that is equivalent to the n501 and y501 viruses. it shows the neutralizing effect of the bnt162b2 vaccine for mutant n501y of sarscov-2. neutralization of the sars-cov-2 n501y mutant by bnt162b2 vaccine-elicited sera.(12) apart from these vaccines, it has been reported that the adenoviral vector vaccine namely chadox1 ncov-19 (azd1222) is clinically effective in neutralizing the activity of the b.1.1.7 and non b.1.1.7 variants of sars-cov-2. moreover, it wa s stated that the vaccine decreased the viral load of both b.1.1.7 and nonb.1.1.7 variants of sars-cov-2.(13) as the world works to vaccinate, isolate, and treat c ommuniti es to choke of f the transmission, the virus is working to evade our scientific arsenal. coordinated international efforts in genomic surveillance and phenotypic characterization of new strains will be critical if we hope to stay one step ahead of the virus. references 1. zhou p, yang x, wang x, et al. a pneumonia outbreak associated with a new coronavirus of probable bat origin. nature 2020;579:270–3. doi: 10.1038/s41586-020-2012-7. 2. world health organization. naming the coronavirus disease (covid-19) and the virus that causes it. geneva: world health organization; 2020 3. mahase e. covid-19: what have we learnt about the new variant in the uk? bmj 2020;371:m4944. http://dx.doi.org/10.1136/bmj.m4944. 4. mascola jr, graham bs, anthony s, et al. sarscov-2 viral variants-tackling a moving target. jama;2021:325:1261-2. doi: 10.1001/jama.2021. 2088. 5. washington nl, gangavarapu k, zeller m, et al. genomic epidemiology identifies emergence and rapid transmission of sars-cov-2 b.1.1.7 in the united states. medrxiv preprint 2021. doi: 10.1101/ 2021.02.06.21251159 . 6. galloway se, paul p, maccannell dr, et al. emergence of sars-cov-2 b.1.1.7 lineage — united states, december 29, 2020–january 12, 2021. morb mortal wkly rep 2021;70:95–9. doi: http://dx.doi.org/10.15585/mmwr.mm7003e2. 7. volz e, mishra s, meera cm, et al. transmission of sars-cov-2 lineage b.1.1.7 in england: insights from linking epidemiological and genetic data. medrxiv preprint 2021. doi : https://doi.org/ 10.1101/2020.12.30.20249034. 8. villoutreix bo, calvez v, marcelin ag, khatib am. in silico investigation of the new uk (b.1.1.7) and south african (501y.v2) sars-cov-2 variants with a focus at the ace2–spike rbd interface. int j mol sci 2021;22:1695. https:// doi.org/10.3390/ijms22041695. 9. european centre for disease prevention and control. rapid increase of a sars-cov-2 variant with multiple spike protein mutations observed in the united kingdom – 20 december 2020. ecdc: stockholm; 2020. 10. tegally h, wilkinson e, giovanetti m, et al. detection of a sars-cov-2 variant of concern in south africa. nature 2021;592:438–43. https:// doi.org/10.1038/s41586-021-03402-9. 11. zhang w, davis bd, chen ss, et al. emergence of a novel sars-cov-2 strain in southern california, usa. medrxiv preprint 2021. doi: https://doi.org/10.1101/2021.01.18.21249786. 12. xie x, liuy, liu j, et al. neutralization of sarscov-2 spike 69/70 deletion, e484k and n501y variants by bnt162b2 vaccine-elicited sera. nat med 2021;27: 620–1. https://doi.org/10.1038/ s41591-021-01270-4. 13. moore jp. approaches for optimal use of different covid-19 vaccines: issues of viral variants and vaccine efficacy. jama 2021;325:1251-2. doi: 10.1001/jama.2021.3465. universa medicina may-august 2021 vol.40no.2 univ med 2021;40:77-8. doi: http://dx.doi.org/10.18051/univmed.2021.v40.77-78 oktavianus 131 *tebet subdistrict health center **professional medical study program, faculty of medicine, trisakti university corespondence acitta raras wimala, s.ked. professional medical study program, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 email: acittararaswimala@yahoo.com univ med 2012;31:131-8 abstract universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background nutritional deficits have been linked to poor cognitive function and are highly prevalent in the elderly. several factors associated with cognitive function have been studied, but the results were inconclusive. the objective of this study was to determine the relationship between blood glucose level and cognitive impairment in the elderly. methods a cross-sectional study was conducted and a total of 109 elderly were included in the study. research subjects were selected using consecutive non-random sampling from the tebet sub-district in south jakarta. random blood glucose level was assessed using glucose strips (nesco). cognitive function was measured with the montreal cognitive assessment (moca) and informant questionnaire on cognitive decline in elderly (iqcode) questionnaire. the relationship between blood glucose levels and cognitive function was analyzed by means of multiple linear regression analysis. results the mean age of the elderly was 67.95 ± 6.42 years, length of formal education was 10.12 ± 5.88 years, and mean random blood glucose level was 137.41 ± 70.25 mg/dl. multiple regression analysis showed that length of formal education (â= 0.769; p=0.000) and random blood glucose levels (â=0.016; p=0.014) were significantly associated with cognitive function. conclusion cognitive function is negatively affected by high blood glucose, thus random blood glucose level can be used to predict cognitive impairment. keywords: random blood glucose level, cognitive impairment, elderly random blood glucose level as predictor of cognitive impairment in elderly amnur r. kayo*, acitta raras wimala**, natalya angela**, and izzura binti abdul rashid** 132 kayo, wimala, angela, et al blood glucose level predict cognitive kadar gula darah sewaktu sebagai prediktor gangguan fungsi kognitif pada lanjut usia latar belakang pada lanjut usia seringkali terjadi defisiensi nutrisi yang dapat mengakibatkan gangguan fungsi kognitif. prevalensi gangguan fungsi kognitif pada lanjut usia sangat tinggi dan faktor yang dapat mempengaruhi fungsi kognitif adalah multifaktorial. sejumlah penelitian tentang hubungan antara diabetes dan terjadnya gangguan kognitif pada lanjut usia masih belum konsisten. penelitian ini bertujuan untuk menentukan adanya hubungan antara kadar gula darah sewaktu dalam darah dan gangguan fungsi kognitif pada lanjut usia. metode sebuah rancangan penelitian potong silang digunakan dengan mengikut sertakan 109 subjek lanjut usia. sampel dipilih dengan menggunakan cara consecutive non-random sampling di wilayah kecamatan tebet jakarta selatan. kadar gula darah sewaktu diukur menggunakan alat pengukur gula darah dan strip glukosa (nesco). fungsi kognitif diperoleh dengan cara wawancara menggunakan kuesioner montreal cognitive assessment (moca) dan informant quessioner on cognitive decline in elderly (iqcode). data dianalisis mengunakan uji regresi linear ganda. hasil rata-rata umur lansia besarnya 67,95 ± 6,42 tahun, lama pendidikan formal yang dialami besarnya 10,12 ± 5,88 tahun, dan kadar gula darah sewaktu 137,41 ± 70,25 mg/dl. uji regresi linear ganda menunjukkan variabel yang berhubungan dengan fungsi kognitif adalah lama pendidikan (â=0,769; p=0,000) dan kadar gula darah sewaktu (â=0,016; p=0,014). kesimpulan kadar gula darah yang semakin meningkat menurunkan fungsi kognitif pada lanjut usia. lama pendidikan merupakan faktor yang tidak dapat diubah. pengaturan kadar gula darah sangat diperlukan untuk mencegah terjadinya penurunan fungsi kognitif pada lanjut usia. kata kunci: kadar gula darah sewaktu, fungsi kognitif, lanjut usia introduction there is no agreement on a definition of old age, as there are too many opinions on what exactly constitutes old age (elderly). the definition of old age (elderly) according to the world health organization (who) includes middle age (between 45 and 59 years), elderly age (60-74 years), old age (75–90 years), and very old age (above 90 years). the currently accepted definition of old age in indonesia is framed in the law no. 13 of the year 1998 on older persons’ welfare, stating that an elderly person is an individual aged 60 years and over.(1) the increasing number of elderly people in indonesia is unavoidable, concomitantly with the increase in life span. the population of elderly indonesian persons is projected to increase between the years 1990-2025 by 414%, which is the highest in the world. a number of biological-physical, psychological, and social problems will appear in the elderly as a result of the aging process or degenerative diseases, concomitantly with the advancing age of these abstrak 133 individuals.(2) according to the most recent census data from the year 2010, there are in indonesia 18,037,009 elderly or approximately 7.59% of the total indonesian population. in the special province of jakarta (dki jakarta) there are 495,024 persons older than 60 years, o r a p p r o x i m a t e l y 5 . 1 5 % o f t h e t o t a l population.(3) i n t h e e l d e r l y t h e r e a r e f r e q u e n t l y n u t r i t i o n a l d e f i c i e n c i e s , p a r t i c u l a r l y o f micronutrients, and metabolic disorders (such as hyperlipidemia and type 2 diabetes mellitus), that are frequently associated with cognitive functional disorders that are very prevalent in the elderly.(4) a study showed that the prevalence of diabetes mellitus was 15.8%, and high blood glucose levels decreased quality of life of the elderly.(5) several factors associated with cognitive function have been studied, among others diabetes mellitus, vascular disease, hypertension, and also individual characteristics and habits.(6,7) in elderly persons with type 2 diabetes there is an accelerated decline of cognitive function, as has been demonstrated in several large population-based longitudinal studies,(8) but the factors involved have not been fully determined. it has been suggested by some investigators that hypertension may play an essential role,(9) but others have found associations with glycemic control.(10) recently it was found that up to 80% of alzheimer patients have type 2 diabetes or impaired fasting glucose.(11) the aim of the present study was to determine the risk factors (diabetes mellitus, hypertension, hyperlipidemia, nutritional status, and individual characteristics) associated with the occurrence of cognitive dysfunction in the elderly. methods research design an observational-analytical study using a cross-sectional design approach was conducted at the tebet subdistrict, south jakarta, from september to october 2011. study subjects elderly persons aged >60 years, who were cooperative and capable of communication, constituted the subjects of this study. exclusion criteria were elderly with severe chronic disease (e.g. stroke), and severe disabilities that could prevent them from participating. the subjects were selected by consecutive non-random sampling of patients attending the primary health center (puskesmas) of tebet subdistrict, south jakarta. data collection data on the characteristics of the subjects, comprising age, gender, marital status, number of children, educational level, occupation, income, smoking, alcohol consumption, past history of illness, were collected by means of questionnaire-based interviews. laboratory investigations capillary blood samples were collected by the finger-prick method from the left third finger f o r d e t e r m i n a t i o n o f b l o o d g l u c o s e a n d c h o l e s t e r o l c o n c e n t r a t i o n s . c h o l e s t e r o l concentrations, expressed in mg/dl, were determined by means of a total blood cholesterol meter and cholesterol strips (nesco). random blood glucose concentrations, also expressed in mg/dl, were determined using a blood glucose meter and glucose strips (nesco). measurements of blood pressure, body weight and height systolic and diastolic blood pressures were measured by means of a sphygmomanometer with the subjects seated in front of the examiner and the manchette around the right upper arm, using a littman stethoscope applied to the cubital region, with the first and fifth korotkoff s o u n d s d e n o t i n g s y s t o l i c a n d d i a s t o l i c pressures, respectively. both systolic and d i a s t o l i c p r e s s u r e s w e r e e x p r e s s e d i n millimeters of mercury (mmhg). subjects were weighed without footwear using tanita weighing scales and weight was univ med vol. 31 no.2 134 kayo, wimala, angela, et al blood glucose level predict cognitive expressed in kilograms (kg) to the nearest decimal. knee height of the subjects was measured with the subjects in the sitting position without footwear, using a wooden knee height meter and was expressed in centimeters (cm) to the nearest decimal. the height of the subjects was obtained from knee height by means of the following mathematical equation. for men: height = 64.65 + 1.87 x knee height (cm); for women: height = 53.80 + 2.10 knee height (cm) – 0.09 x age (years).(12) body mass index (bmi) was calculated as weight (kg) divided by height squared (m2). for asian populations bmi is categorized as underweight (<18.5 kg/m2), normal weight (18.5 – 23.0 kg/m2), overweight (23.0 – 27.5 kg/m2), and obese (>27.6 kg/ m2).(13) cognitive function cognitive function is a person’s way of thinking and how a person’s intrapsychical functions prepares him or her to react to external reality and is measured by means of the montreal cognitive assesment (moca) questionnaire and the informant questionnaire on cognitive decline in elderly (iqcode). the moca questionnaire evaluates short term memory (5 points), visuospatial ability (4 points), executive function (4 points), attention, memory, and concentration (6 points), language ability (5 points) and orientation (6 points).(14) the moca is a useful screening tool for the detection of mild dementia and mild cognitive impairment. the iqcode questionnaire consists of 16 everyday life situations where a person has to use memory and intelligence. each situation is evaluated by the respondents’ nearest family members regarding changes in the last 10 years, using the scale of “far better”, “somewhat better”, “not much changed”, “somewhat worse” and “far more worse”.(15) based on the iqcode instrument, a score b e t w e e n 3 . 3 a n d 3 . 6 i s t h e c u t t o ff f o r determining early cognitive impairment. the iqcode is a reliable informant questionnaire that is unaffected by education or language.(16) moca scores range from 1-30, while iqcode scores are in the range 1.0 – 5.0. ethical clearance ethical clearance was issued by the commission on research ethics of the faculty of medicine, trisakti university and all subjects were asked to give written informed consent. data analysis the computer software program used for data analysis was the spss version 15.0. data are presented as proportions (%) and mean ± sd. simple linear regression analysis was used to determine the presence of an association betwen independent variables and cognitive function variables. to determine independent variables with the highest impact on cognitive functions of the elderly, multiple linear regression analysis was used. the level of significance used in this study was 0.05. results a total of 109 subjects participated in this study, consisting of 98 (89.9%) females and 11 (10.1%) males, with a mean age of 67.95 ± 6.42 years. the mean level of education (indicated by length of formal education) was 10.12 ± 5.88 years. most of the subjects, totalling 60 (56.9%) were widowers/widows. only 11 (10.1%) of the subjects were smokers, and 2 (1.8%) regularly consumed alcohol. a total of 36 (33.0%) subjects had a history of diabetes mellitus and 54 (49.5%) had hypertension. mean random blood glucose level was 137.41 ± 70.25 mg/dl, and mean blood cholesterol level was 68.13 ± 48.69 mg/dl. mean cognitive function as measured by the moca and iqcode instruments was 19.01 ± 6.21 and 3.69 ± 0.39, respectively. mean bmi of the participants was 22.84 ± 4.21 kg/m² (table 1). the results of simple linear regression analysis showed that educational level, random blood glucose level, and bmi were significantly 135 associated with cognitive function, with regression coefficient values of 0.703 (95% ci 0.552 – 0.854), -0.044 (95% ci -0.053 – -0.035), -0.019 (95% ci -0.036 – -0.003), 0.001 (95% ci 0.000 – 0.002), 0.421 (95% confidence interval 0.150 – 0.693) and -0.026 – -0.009), respectively (table 2). these three variables with a significant association with cognitive function, were input into a multiple linear regression model. the level of education of the elderly subjects and random blood glucose level were significantly associated with cognitive function. it is apparent from table 3 that educational level (beta = 0.652 for both moca and iqcode) and random blood glucose (beta = 0.118 for moca and beta = 0.181 for iqcode) were of influence for cognitive function. higher educational level had the greatest positive influence on cognitive function. in contrast, increased random blood glucose level had a lowering effect on cognitive function of the elderly. low iqcode and high moca scores both signify better cognitive function (table 3). table 1. distribution of several important variables in the elderly (n= 109) *moca=montreal cognitive assesment; @iqcode= informant questionnaire on cognitive decline in elderly univ med vol. 31 no.2 table 2. simple linear regression between age, educational level, blood glucose level, total cholesterol, blood pressure, body mass index and cognitive function (n=109) *b=regression coefficient; @ moca=montreal cognitive assesment; # iqcode=informant questionnaire on cognitive decline in elderly table 3. multiple linear regression between educational level, blood glucose level, body mass index, and cognitive function *b=regression coefficient; **beta=standardized regression coefficient; @moca=montreal cognitive assesment; #iqcode=informant questionnaire on cognitive decline in elderly 136 kayo, wimala, angela, et al blood glucose level predict cognitive discussion this study involving elderly subjects showed that educational level and random blood glucose level were significantly associated with cognitive function. of these two variables, it is only the random blood glucose level that may be modified to improve cognitive function of the elderly. essentially similar results were found in two large population-based data sets in sweden, using a random sample from the population aged 35–85 years, where an increase i n p l a s m a g l u c o s e w a s a s s o c i a t e d w i t h impairment in episodic memory in women. this could be explained by a negative effect on the hippocampus caused by the raised plasma glucose levels.(17) glucose increases the risk of atherosclerosis, thereby increasing the risk of vascular dementia. hyperglycemia is often coupled to other metabolic abnormalities that could be linked to cognitive impairment.(18) regarding the well-known sex differences in memory, there are several studies suggesting a major role for glucose levels. according to awad et al.(19) improving metabolic control in diabetic patients has been reported to improve cognitive function. differing results were found in the study conducted by euser et al, indicating that increased blood glucose level was not associated with impaired cognitive function in elderly persons without diabetes.(20) however, the nurse health study reported that increased insulin concentration was associated with decreased cognitive function in nondiabetic female elderly.(21) these differing study results indicate the need for further studies on the relationship between glucose metabolism and cognitive function in the elderly. there are several biological mechanisms that are possibly involved in the metabolism of amyloid b, such as accumulation of advanced glycation end products and acceleration of cerebrovascular disease. in addition to vascular pathways, several p o s s i b l e p a t h o p h y s i o l o g i c m e c h a n i s m s , including hyperglycemia, insulin resistance, oxidative stress, advanced glycation end products, and inflammatory cytokines, may explain the effect of glucose deregulation on dementia risk. therefore, an increased risk of developing dementia resulting from diabetes can be expected in people with mild cognitive impairment.(22) the results of our study showed that bmi was not associated with impaired cognitive function in the elderly. a cohort study on subjects aged >70 years with diabetes also showed that bmi was not associated with impaired cognitive function in the elderly.(16) a population-based cross-sectional study in singapore among older adults aged 55 and above showed that low bmi by itself was not significantly associated with poor cognitive performance, but low bmi with chronic comorbidity (or 1.73; 95% ci 1.02–2.95) was independently associated with poor cognitive performance.(23) results of the relationship between high bmi or obesity and cognitive status are also inconsistent,(24,25) but most studies suggested that low bmi was related to dementia risk.(26,27) in our study two instruments were used to assess cognitive function in the elderly, namely iqcode and moca, because one study has demonstrated that the use of two instruments is capable of providing better information than the use of one instrument only.(28) the information obtained may increase the accuracy of screening and diagnosis. there are numerous tests that have been studied for their efficacy in assessing individuals for the presence of cognitive impairment. at this time there is no one test that is clearly better than all of the others. there are two test-related criteria that must be considered when evaluating the accuracy of a test, sensitivity and specificity. variation in the specificity and sensitivity of a test can have a substantial impact on the cost of a screening test.(29) in spite of the above, our study has several limitations that should be noted. first, we used 137 random blood glucose to measure the blood glucose levels, which might result in an attenuation of the associations. second, as there are no specific recommended tools to diagnose cognitive dysfunction, the operationalization of the criteria may differ slightly from those in other studies. third, our study was of crosssectional design and thus cannot demonstrate the presence of a causal relationship between random blood glucose level and impaired cognitive function in the elderly. conclusions elevated blood glucose level is associated with cognitive impairment in older people. longer duration of education increases the cognitve performance of older people. our findings underline the importance of regularly monitoring the serum blood glucose levels to reduce the risk of cognitive impairment in the elderly. acknowledgment the investigators wish to express their thanks to all elderly who were willing to participate in this study. references 1. world health organization. active ageing: a policy framework. geneva: world health organization;2002. 2. soejono ch, setiati s, nasrun mws, silaswati s. pedoman pengelolaan kesehatan pasien geriatri untuk dokter dan perawat. edisi pertama. jakarta: pusat informasi dan penerbit bagian ilmu penyakit dalam fkui; 2004. 3. badan pusat statistik. penduduk menurut kelompok umur dan jenis kelamin 2010. available at: http://www.bps.go.id/aboutus.php? sp=1. accessed october 1, 2011. 4. greenwood ce. dietary carbohydrate, glucose regulation, and cognitive performance in elderly persons. nutr rev 2003;61:s68-74. 5. khairani r. prevalence of diabetes mellitus and the relationship with quality of life of older people in the community. univ med 2007;26: 18-26. 6. durga j, van boxtel mp, schouten eg. effect of 3-year folic acid 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b, bäckman l, qiu c, et al. accelerated progression from mild cognitive impairment to dementia in people with diabetes. diabetes 2010;59:2928– 35. 23. ng tp, feng l, niti m, yap kb. albumin, haemoglobin, bmi and cognitive performance in older adults. age ageing 2008;37:423–9. 24. gustafson d. adiposity indices and dementia. lancet neurol 2006;5:713–20. 25. kuo hk, jones rn, milberg wp. cognitive function in normal-weight, overweight, and obese older adults: an analysis of the advanced cognitive training for independent and vital elderly cohort. j am geriatr soc 2006;54:97– 103. 26. stewart r, masaki k, xue ql. a 32-year prospective study of change in body weight and incident dementia: the honolulu-asia aging study. arch neurol 2005;62:55–60. 27. buchman as, wilson rs, bienias jl. change in body mass index and risk of incident alzheimer disease. neurology 2005;65:892–7. 28. mackinnon a, mulligan r. combining cognitive testing and informant report to increase accuracy in screening for dementia. am j psychiatry 1998; 155:1529-35. 29. ashford jw. screening for memory disorders, dementia and alzheimer’s disease. aging health 2008;4:399-432. ethanolic periwinkle leaf extract reduces telomerase expression in t47d cancer cells 79 *department of anatomy, faculty of medicine, yarsi university, jakarta **department of biochemistry, faculty of medicine, yarsi university, jakarta correspondence: prof. dr. endang purwaningsih, ms, pa department of anatomy, faculty of medicine, university of yarsi jln. letjend suprapto, cempaka putih, jakarta10510 phone: +6221-4206674/76 email: endpurwaning@gmail.com/ endang.purwaningsih@yarsi.ac.id univ med 2015;34:79-86 doi: 10.18051/univmed.2016.v35.79-86 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background cancer cells have a relatively high telomerase activity and a lower p53 protein expression than normal cells, so that cancer cells have the ability to continue to proliferate and do not undergo apoptosis. one of the cancer treatments is chemotherapy using bioactive ingredients from synthesis or isolation of natural materials. one of the plants that have potential as anticancer agent is periwinkle (catharanthus roseus l). the research objective was to evaluate the effect of ethanolic periwinkle leaf extract against p53 protein and telomerase expression in t47d cancer cells. methods an experimental study with controls was conducted involving t47d breast cancer cells. they were divided into 3 groups (control, ½ dose of ic50/ 26.849 µg/ml, and one dose of ic50/53.699 µg/ml) at a cell density of 1 x 104 cells/well. expression of p53 and telomerase was measured by the immunohistochemistry method. data were analyzed using one-way anova followed by a multiple comparison test. results periwinkle leaf extract significantly increased p53 protein expression (p<0.05) at both treatment doses, ½ ic50 and ic50, compared to the control group and it highly significantly reduced telomerase expression (p<0.01), in comparison with the control group at both treatment doses. conclusion periwinkle leaf extract has potential as an anti-breast cancer agent by increasing p53 protein expression and inhibiting telomerase expression. keywords: periwinkle, p53, telomerase, t47d cells ethanolic periwinkle leaf extract reduces telomerase expression in t47d cancer cells endang purwaningsih*, etty widayanti*, and yulia suciati** doi: http://dx.doi.org/10.18051/univmed.2015.v34.79-86 80 purwaningsih, widayanti, suciati periwinkle leaf extract reduces telomerase ekstrak etanol daun tapak dara menurunkan ekspresi telomerase pada sel kanker t47d latar belakang kanker merupakan penyakit yang mendapat perhatian serius di dunia kedokteran. hal ini disebabkan oleh jumlah penderitanya yang semakin meningkat dan belum ada cara efektif untuk mengobatinya. sel kanker memiliki aktivitas telomerase yang relatif tinggi dan menunjukkan ekspresi protein p53 yang lebih rendah dibandingkan sel normal sehingga sel kanker memiliki kemampuan untuk terus berproliferasi dan tidak mengalami apoptosis. salah satu pengobatan kanker adalah dengan kemoterapi menggunakan bahan-bahan bioaktif dari hasil sintesis atau isolasi bahan alam. salah satu tanaman yang memiliki potensi sebagai antikanker adalah tapak dara (catharanthus roseus l). tujuan penelitian adalah untuk menilai efek ekstrak etanol daun tapak dara terhadap ekspresi protein p53 dan telomerase pada kultur sel kanker t47d. metode sebuah penelitian eksperimental dengan kontrol dilakukan deengan mengikut sertakan kultur sel kanker payudara t47d (cell line). mereka secara acak dibagi menjadi 3 kelompok (kontrol, ½ dosis ic50/26.849 µg/ml, dan satu dosis ic50/53.699 µg/ml) dengan kepadatan sel 1 x 104 sel/sumuran. ekspresi p53 dan telomerase diukur dengan metode immunohistochemistry. data dianalisis menggunakan anova satu jalan dilanjutkan dengan uji perbandingan berganda. hasil ekstrak daun tapak dara meningkatkan ekspresi protein p53 secara bermakna pada kedua dosis perlakuan (p<0,05) baik ½ ic 50 maupun 1 ic50 dibandingkan kelompok kontrol (p<0,05), dan menurunkan ekspresi telomerase secara sangat bermakna pada kedua dosis perlakuan (p<0,01). kesimpulan ekstrak daun tapak dara memiliki potensi sebagai anti kanker payudara melalui peningkatan ekspresi protein p53 dan hambatan telomerase. kata kunci : tapak dara, p53, telomerase, sel t47d abstrak introduction cancer is a health problem in various countries throughout the world and is a disease that attracts serious attention in the medical field. this is because the number of victims is increasing from year to year and no effective treatment method has been found. medical cancer treatments that have been currently used are surgery, irradiation, and chemotherapy. the latter, which is currently of interest, comprises the use of synthetic or natural bioactive substances such as those from the rat tail rhizome (typhonium flagelliforme) and periwinkle (catharanthus roseus).(1-4) according to data from the world health o rg a n i z a t i o n ( w h o ) , 8 0 % o f t h e w o r l d population still depends on plant-derived traditional medications for its health needs. a total of 25% of the commercially available modern drugs in the world are derived from active substances that have been isolated and developed from plants. at present the use of traditional medications is on the increase, despite the 81 univ med vol. 34 no.2 substantial advances in modern medical therapeutics. the indonesian community has recognized various medicinal plants, because indonesia is known as one of the countries with the greatest biodiversity and as such has potential for the development of plant-based medications, particularly anticancer drugs.(5) one of the plants that contain potential anticancer compounds is the periwinkle plant (catharanthus roseus (l) g don), formerly known as vinca rosea l. the leaves of this plant contain a number of alkaloids that are potential anticancer agents, i.e. vincristine, vinblastine, vindesine, vindoline, tabersonine, leurosine, catharanthine, and lochnerine.(4,6) periwinkle leaf extract may be used as anticancer or antitumor agent, according to a number of previous in vivo studies on rats as well as in vitro studies using cultures of the mcf7 cancer cell line.(7,8) in addition, periwinkle leaf extract at a dose of 1.0 ml/kg has been shown to be able to significantly reduce serum hdl (high density lipoprotein) and ldl (low density lipoprotein) cholesterols and triglycerides in normal rats.(7) furthermore it is known that there are more than 70 types of alkaloid in the roots, stems, leaves, and seeds of the periwinkle plant, including 28 bi-indole alkaloids. in addition to anticancer alkaloids, the periwinkle plant also contains blood glucose reducing alkaloids, i.e. leurosine, lochnerine, tetrahydroalstonine, vindoline and vindolinine.(6,9) an extract of its flowers also has hyperglycemic effects in diabetic rats and functions as antioxidant, antibacterial, anthelminthic, and antidiarrheal agent.(9-11) a number of cancer cells have a high telomerase activity and each type of cancer cell may have different degrees of telomerase expression according to the type or location of the cancer. this can be used as a basis for the treatment of cancer cells if there are substances that can inhibit the activity of the telomerase enzyme. in addition, cancer cells possess a high mitotic activity since they undergo uncontrolled cell division as a result of low p53 tumor suppressor protein expression. the p53 protein is responsible for the mechanism of apoptosis of cancer cells and has become the latest cancer treatment strategy.(12,13) a number of scientific data have demonstrated the benefits of periwinkle, such as inhibition of proliferation and triggering of apoptosis in cancer cells (11,14,15) as well as cytotoxicity against cancer cells.(8) the antiapoptotic effect may be determined by counting the percentage of cells undergoing apoptosis by flow cytometer. the effect of the periwinkle plant on p53 or telomerase protein expression in cancer cells is still unknown. the objective of this study was to evaluate the effect of periwinkle (catharanthus roseus l) leaf extract on p53 and telomerase expression in t47d breast cancer cells. methods design of study this laboratory experimental study was carried out from june 2013 until november 2013 at the department of biochemistry, faculty of medicine, yarsi university. study subjects the study subjects were cultures of the t47d cancer cell line, obtained from the parasitology laboratory, faculty of medicine, gadjah mada university. the cells were cultured on 24-well microplates using rpmi (roswell park memorial institute) medium, with two replications for each group. the cultures were incubated for 72 hours or until confluence was reached, with a density of 1 x 104 cells/well. upon confluence, the cultures were treated with the respective doses. preparation of periwinkle leaf extract periwinkle leaf extract was prepared as follows: fresh leaves were cleansed, cut into small strips, and air-dried. the dried strips were then blenderized into a powder. the powder was macerated in 80% ethanol in an erlenmeyer flask, under continuous stirring. the ethanolic extract was then transferred to a rotary flask to 82 purwaningsih, widayanti, suciati periwinkle leaf extract reduces telomerase be concentrated in a rotary evaporator. the concentrated periwinkle leaf extract was dissolved in dmso, diluted to give a number of concentrations, and filtered using a 0.2 µm filter before being placed on the 24-well plates. intervention the cultures of the t47d cancer cell line were assigned to 3 groups, i.e. one control group that did not receive extract, one group receiving extract at a dose of one half the maximal inhibitory concentration (ic50) (53.699 µg/ml), and one group receiving a dose of ½ ic50 (26.849 µg/ ml). before treatment with the extract, a test was performed to determine the ic50 of the periwinkle leaf extract against t47d cell cultures. the result of ic50 testing was used to determine the treatment dose to be used. the treated cell cultures on coverslips were incubated under 5% co 2 at 37oc for 24 hours. preparation of cell culture and cell growth media preparation of roswell park memorial institute (rpmi) culture medium was by dissolving rpmi 1640 powder for one liter into approximately 800 ml of twice distilled water. after adding 2.0 grams of sodium bicarbonate and 2.0 grams of hepes, twice distilled water was added to make up a volume of one liter. the solution was mixed with a magnetic stirrer for about 10 minutes until homogenous, then neutralized with 1 n hcl to give a ph of 7.2 – 7.4. growth medium was prepared by mixing fbs 19 ml, penicilline streptomycin 2 ml, fungizone 0.5 ml, then diluted with rpmi 1640 culture medium to a volume of 100 ml. the solution was then aseptically filtered using a sterile 0.2 µm polyethylene sulfone filter and afterwards stored in the refrigerator in stoppered bottles. immunohistochemical determination of p53 protein and telomerase expression t47d cell cultures at a density of 1x 104/ well that had attached to the coverslips were stained immunohistochemically according to standard procedures of the parasitology laboratory, faculty of medicine, gadjah mada university. immunohistochemical determination of p53 protein and telomerase expression was as follows: the cell cultures on the coverslips, after being given the extract and incubated under 5% co 2 at 37oc for 24 hours, were placed on poly-llysine slides and fixed in acetone or methanol for 10 minutes at -200c. the slides were then washed with pbs for 3 x 5 minutes, then 0.3% h 2 o 2 was dripped 20 minutes. normal mouse serum (1:50) was dripped for 15 minutes and the liquid was discarded (without washing). then p53 or telomerase antibody was dripped for 60 minutes. the slides were then washed with pbs for 3 x 5 minutes, incubated with biotinylated secondary antibody for 5-10 minutes, and again washed with pbs for 3 x 5 minutes. this was followed by incubating the slides with streptavidin-peroxidase for 5-10 minutes, washing with pbs for 3 x 5 minutes, incubating with the chromogen deaminobenzidine tetrahydrochloride (dab) for 5-10 minutes (at a chromogen to substrate ratio of 1:20), and washing with distilled water. the slides were immersed in hematoxyllin for 3-5 minutes for counterstaining, washed with distilled water, dehydrated in 95% ethanol and immersed in xylene, for 10 minutes respectively. the slides were given a drop of canada balsam as mounting medium and covered with a coverslip. the expression of p53 protein and telomerase was observed under the light microscope. cells expressing p53 protein or telomerase showed brownish colored nuclei and cytoplasms, whereas cells without protein expression showed blueviolet nuclei and cytoplasms. expression-positive cells were counted per 100 observed cells and counting was done in triplicate. the results were expressed as percentages. data analysis the p53 protein expression and telomerase percentages were analyzed by one-way anova, followed by least significant difference (lsd) multiple comparisons, using spss version 17. 83 univ med vol. 34 no.2 results b e f o r e e v a l u a t i n g t h e e ff e c t o f t h e periwinkle leaf extract on the expression of p53 and telomerase, toxicity testing was performed on periwinkle leaf extract against cultures of t47d breast cancer cells. the toxicity tests yielded an ic50 value of 53.699 µg/ml. assessment of periwinkle leaf extract effects on p53 protein and telomerase expression by immunohistochemistry was done by counting the percentages of expression-positive cells. the percentages of p53 and telomerase positive cells in the treatment groups receiving extract (at doses of ½ ic50 or 26.849 µg/ml and 1 ic50 or 53.699 µg/ml, respectively) are shown in table 1. from the percentages of the expressionpositive cells it is apparent that periwinkle leaf extract tended to increase p53 protein expression and to reduce telomerase expression. according to the results of anova analysis on the expression of p53 protein and telomerase, there were substantially significant differences (p=0.000) between groups. between-group lsd m u l t i p l e c o m p a r i s o n t e s t r e s u l t s o n t h e expression of p53 and telomerase are presented in table 2. the lsd multiple comparison test results on p53 protein expression showed significant differences (p<0.05) between the control group and the group receiving a dose of one-half ic50, and also between the group receiving a dose of one-half ic50 and the group receiving a dose of one ic50. there were also highly significant differences (p<0.01) between the control group and the group receiving a dose of one ic50. the lsd multiple comparison test results on telomerase expression showed highly significant differences between the control group and the one-half ic50 group, between the control group and the one ic50 group, and between the onehalf ic50 group and the one ic50 group. discussion from the results of this study, it is apparent that ethanolic periwinkle leaf extract is able to reduce telomerase expression and increase p53 protein expression in t47d breast cancer cells. t h e r e f o r e t h e c o m p o u n d s c o n t a i n e d i n periwinkle leaf extract are able to inhibit telomerase expression in breast cancer cells, or in other words, the extract may act as a telomerase inhibitor. previous investigators have reported that chloroform and methanolic extracts of the periwinkle (catharanthus roseus l) possess cytotoxic effects against hct-116 colon cancer cells.(16) in addition crude aqueous periwinkle extract also has cytotoxic effects against leukemic t cells and induces the proliferation of normal blood cells.(17) there have been no reports on the effect of periwinkle extract on telomerase expression in colon cancer cells and leukemic t cells. it has also been reported that table 1. expression of p53 protein and telomerase in t47d cell cultures by treatment group ic50 : one half the maximal inhibitory concentration table 2. results of lsd test on expression of p53 and telomerase, by treatment group ic50: one half the maximal inhibitory concentration 84 purwaningsih, widayanti, suciati periwinkle leaf extract reduces telomerase curcumin, a component of turmeric (curcuma longa l) rhizomes, has in vitro anti-proliferative activity against bel7402, hl60 and sgc7901 cancer cell lines and demonstrates in vivo antitumor effects in laboratory rats. in addition, curcumin at a dose of 1 ì m is able to inhibit telomerase activity in cancer cells in vitro and to induce apoptosis.(18) it is known that various cancer cells have relatively higher telomerase activities than normal cells and that there is a correlation between telomerase activity and the development of cancer cells. telomerase is the principal key to cellular immortality and tumorogenesis. telomerase is activated in 80–90% of human cancer cells, but not in somatic cells (normal cells). furthermore it has been reported that telomerase may be a biomarker for early detection of cancers and cancer monitoring, and has sufficient potential for a basis of development of cancer treatment.(19) the p53 protein is a tumor suppressor protein that is responsible for the mechanism of apoptosis in cancer cells. the mechanism comprises the repair of cells that have been induced to become cancerous. expression of specific p53 protein occurs in abnormally replicating cells and requires programmed cell death called apoptosis. higher p53 protein expression is associated with a higher tendency to apoptosis. the p53 protein is expressed upon the occurrence of dna damage.(12,13) the p53 protein is a tumor suppressor protein, with a molecular weight of 53 kilodalton, that is activated upon the occurrence of dna damage or certain types of stress in the cells. this protein can promote apoptosis by increased expression of the bax gene, which codes for the bax protein that plays a role in apoptosis. detection of damaged dna is regulated by the p53 tumor suppressor. when dna damage occurs, p53 prevent the cells from entering the next phase of cell division and gives time to the dna to repair itself, or if the damage is sufficiently severe, p53 will initiate programmed cell death (apoptosis). loss of a number of molecular checkpoints are to be found in the development of several tumors or cancers.(20) administration of the leaf extract of periwinkle (catharantus roseus) can increase p53 protein expression, such that this extract may be used to increase apoptosis of breast cancer cells. the periwinkle plant produces secondary metabolites that are active as alkaloid anticancer agents. (9,14) on the other hand, periwinkle extract has been shown to reduce telomerase expression, as has been shown in t47d cell cultures in the treatment group at ½ i c 5 0 a n d 1 i c 5 0 . te l o m e r a s e i s a ribonucleoprotein enzyme that maintains the protective structures at the ends of eukaryotic chromosomes, called telomers. telomerase is a structure that is responsible for the maintenance of telomers, being a ttagggn nucleotide replication. in most human somatic cells, such a s f i b r o b l a s t s , t e l o m e r a s e e x p r e s s i o n i s repressed, the telomers become progressively shorter and at each somatic cell division the cells undergo programmed cell death or apoptosis. in contrast, the majority of human tumor or cancer cells express telomerase, such that the telomer length is stable and the cells do not die by apoptosis. this observation shows that telomer maintenance is essential for tumor or cancer cell proliferation.(21) telomerase activity determines cell proliferation, both of cancer and normal cells, in vitro as well as in vivo conditions.(22,23) telomerase activity in cancer cells increase after certain stages, i.e. when there is a loss of a number of substances or telomer subunits in the cells. finally the telomerase is activated to stabilize the telomers, the cancer cells become immortal and proliferate continuously. (24) telomerase plays a role in cell replication, since it is able to maintain telomer length in the cell progeny. therefore cancer cells without sufficient telomerase will cease to grow when this enzyme cannot any longer continuously maintain telomer length as a result of abnormal control of proliferation. lack of telomerase may inhibit 85 univ med vol. 34 no.2 cancer growth by continuous cell division such that the telomers become shorter and the cells die before the occurrence of further damage. however, if the cancer cells are able to synthesize telomerase then these cells will proliferate continuously and become immortal.(23) periwinkle leaf extract contains more than 100 monoterpenoids that exert cytotoxic effects and are used in cancer chemotherapy. alkaloids such as vincristin and vinblastin in periwinkle leaf extract may function as potent antineoplastic agents (25) by inhibiting dna synthesis and blocking mitosis in metaphase or anaphase.(26) a study using periwinkle leaf extract on t47 d cancer cells found that the extract had anticancer effects through induction of apoptosis, but that it did not show antioxidant activity in t47d cells.(11) one limitation of this study is that it did not investigate other proteins that play a role in apoptosis, such as bax and bcl-2, after administration of periwinkle leaf extract to t47d breast cancer cells. as a result of this study, a natural raw material with chemopreventive actions against breast cancer cells will be available from the periwinkle plant (catharanthus roseus). conflict of interest there are no conflict of interest. conclusion compounds in periwinkle leaf extract are potential agents against breast cancer by increasing p53 protein expression leading to apoptosis and by telomerase inhibition. acknowledgement the authors wish to express their profound gratitude to the directorate general of the ministry of higher education (direktorat jendral dikti) for support of this study through the hpeq research fund, allowing us to conduct this study. references 1. hamid is, nazar ds, ratnani. hambatan ekspresi vascular endothelial growth factor oleh ekstrak daun sambung nyawa pada endotel membran korioalantois. j veteriner 2013;14:8590. 2. chadidjah, nasihun t, widayati e, et al. typhonium flagelliforme decreases protein expression in murine breast cancer. univ med 2014;33:161-70. 3. purwaningsih e, widayanti e, suciati y. cytotoxicity assay of typhonium flagelliforme lodd against breast and cervical cancer cells. univ med 2014;33:75-82. 4. sain m, sharma v. catharanthus roseus (an anticancerous drug yielding plant): a review of potential theurapeutic properties. int j pure app biosci 2013;1:139-42. 5. farida y, rahayu l, faizatun. aktivitas antioksidan serbuk n-heksana dan metanol hasil pengeringan semprot ekstrak keladi tikus (typhonium flagelliforme l) decue. kongres ilmiah xviii ikatan apoteker indonesia 2010. makasar, 10–12 desember 2010. 6. man s, gao w, wei c, et al. anticancer drugs from traditional toxic chinese medicines: review. phytother res 2012;26:1449-65. 7. antia bs, otokan je. effect of leaf juice of catharanthus roseus linn on cholesterol, triglyceride, and lipoprotein levels in normal rats. indian j pharmacol 2005;37:401-2. 8. ruskin s, aruna sr. in vitro and in vivo antitumor activity of catharanthus roseus. irjpas 2014;4:1-4. 9. tiong sh, yeng looi c, hazni h, et al. antidiabetic and antioxidant properties of alkaloids from catharanthus roseus (l) g. don. molecules 2013;18:9770-84. 10. natarajan a, syed ahmed ksz, sundaram s, et al. effect of aqueous flower extract of catharanthus roseus on alloxan induced diabetes in male albino rats. ijpsdr 2012;4:150-3. 11. widowati w, mozef tj, risdian c, et al. apoptosis and antioxidant activities of catharanthus roseus (l) g. don extract on breast cancer cell line. ind j cancr chem 2010;1:99107. 12. bai l, zhu wg. p53: structure, function and therapeutic applications. j cancer mol 2006;2: 141-53. 13. amaral jd, xavier jm, steer cj, et al. the role of p53 in apoptosis. discovery med 2010;9:14552. 86 purwaningsih, widayanti, suciati periwinkle leaf extract reduces telomerase 14. widowati w, mozef t, risdian c, et al. anticancer and free radical scavenging potency of catharanthus roseus, dendropthoe petandra, piper betle and curcuma mangga extracts in breast cancer cell lines. oxid antioxid med sci 2013;2:137-42. 15. looi cy, arya a, cheah fk, et al. induction of apoptosis in human breast cancer cells via caspase pathway by vernodalin isolated from centratherum anthelminticum (l.) seeds. plos one 2013;8:e56643. doi:10.1371/journal.pone. 005664. 16. siddiqui mj, ismail z, aisha afa, et al. cytotoxic activity of catharanthus roseus (apocynaceae) crude extracts and pure compounds against human colorectal carcinoma cell line. int j pharmacol 2010;6:43-7. 17. ahmad nh, rahim ra, mat i. chatarhanthus roseus aqueous extract is cytotoxic to jurkat leukaemic t cells but induce the proliferation of normal peripheral blood mononuclear cells. trop life sci 2010;21:105-15. 18. chui sx, qu kj, xie yy, et al. curcumin inhibits telomerase activity in human cancer cell lines. int j mol med 2006;18:227-31. 19. chen ch, cheng rj. prevalence of telomerase activity in human cancer. j formos med assoc 2011;110:275-89. 20. rivlin n, brosh r, oren m, et al. mutations in the p53 tumor suppressor gene: important milestones at the various steps of tumorogenesis. genes cancer 2011;2:466-74. 21. quellette mw, wright we, shay jw. targeting telomerase-expressing cancer cells. j cell mol med 2011;15:1433-42. 22. shay jw, wright we. senescence and immortalization: role of telomere and telomerase. carcinogenesis 2005;26:867-74. 23. artandi se, depinho ra. telomeres and telomerase in cancer. carcinogenesis 2010;3:918. 24. blackburn e. telomerase and cancer. mol cancer res 2005;3:9. doi: 110.1158/1541-7786 -mcr-05-0147. 25. gajalaksmi s, vijayalaksmi s, rajeswari d. pharmacological activities of catharanthus roseus: a perspective. int j pharm bio sci 2013;4: 431-9. 26. aqubi ae, al-naimi rs, al taee eh. comparative pathological and cytogenetical study of ethanolic extract vinca rosea l and vinblastine in treating mammary gland adenocarcinoma implanted mice. kufa j vet med sci 2011;2:146-63. c:\users\user\documents\41 no 2 universa medicina september-december 2022 vol.41no.3 invited editorial genetics of circadian rhythm in disease and aging process juhi aggarwal*, niharika singh, and jyoti batra department of biochemistry, santosh medical college and hospital, sdtu, ghaziabad, uttar pradesh there are multiple peripheral clocks located in various tissues and organs of the body, which cause variations in biological activities and are involved in the generation of circadian rhythms. in mammals, the circadian rhythm is generated by an endogenous timing system known as the circadian clock, which is 24 hours long. during each day, the circadian clock coordinates with environmental cues to regulate many cellular, behavioral, and physiological processes. during the day, a rise in heart rate, blood pressure, temperature and body activity is observed, while in the evening there is a decrease in these parameters. circadian rhythm disruptions and altered sleep-wake cycles can significantly affect work performance and behavior. there has been a growing understanding that the circadian system has a major role in regulating human physiological processes, since recent studies have shown that around 80% of protein coding genes in many tissues and organs exhibit circadian oscillations.(1) the suprachiasmatic nucleus (scn) in the mammalian hypothalamus has been identified as the site controlling circadian behavioral rhythmicity. lesions of the scn abolish locomotor rhythms, and scn transplants reinstate rhythmic behavior with the circadian properties. the oscillatory mechanism of the clock is i ntracell ular and can be monitored. the role of circadian rhythm genes in aging mechanisms, as confirmed by studies, state that 20-43% of genes in every human cell depend on circadian regulators.(2) circadian rhythm genes are regular genes which regulate the cell cycle and inhibit cellular aging (an irreversible exit from the cell cycle). aging is primarily a gradual loss of cellular and whole body capacity of resp o nd ing to stress fo llo wed by reestablishment of the body to a normal state. there are some defined interventions, which give promising results in animal models or even in human studies, resulting in lifespan elongation or health span improvement. one of the most promising targets for anti-aging approaches is proteins belonging to the sirtuin family. sirtuin (sirt)-mediated deacetylation of histone h3 in k9/k14 was observed adjacent to promoters of transcription factors such as forkhead box ''o'' (foxo), e2f1, p300/cbp and ppar.(3) soon it was discovered that the main activity of sirtuins is deacetylation of lysine residues. this is a two-step reaction-firstly, sirtuins cleave nicotinamide adenine dinucleotide (nad) to nicotinamide (nam). in the salvage pathway, nam is converted to nicotinamide mononucleotid e (nmn) by nicotinam ide phosphoribosyl transferase (nampt), a limiting enzyme for the whole pathway. nampt is regulated by a complex consisting of circadian locomotor output cycles kaput (clock) and brain and muscle arylhydrocarbon receptor nuclear translocator-like 1 (bmal1).(4) the circadian clock controls the cellular process that involves the regulation of inflammation and energy hemostasis. mice deficient in the bmal1 circadian transcription factor have impaired circadian behavior and *email: jaggarwal38@gmail.com orcid id: 0000-0003-2883-6785 207 mailto:jaggarwal38@gmail.com https://orcid.org/0000-0003-2883-6785 https://orcid.org/0000-0002-8192-295x https://orcid.org/0000-0002-5391-9593 universa medicina september-december 2022 vol.41no.3 demonstrate loss of rhythmicity in the expression of target genes. these mice significantly earlier manifested age related pathologies such as inflamm atio n of the co rnea, cataracts, sarcopenia, osteoporosis, ptosis of internal organs, alopecia, reduction of visceral and subcutaneous adipose tissue, and alteration in blood composition.(5) it was shown that sirt1 can be found in the telomere and pericentromere regions. telomerase is known for its ability to provide eukaryotic cells with immortality via restoring chromosome ends, which is considered a canonical function of the enzyme. telomeres are perceived as a molecular clock that determines the longevity of cells allowing them to divide up to approximately 50 times (hayflick limit) and that number is limited due to a replication-end problem.(6) deletion of the clock gene was associated with development of cataracts and extensive dermatitis rather early compared to wild type. in clock deficient mice, a decrease in lifespan by 10% was observed. mice with induced deficiency in clock expression exhibit low activity of telomerase which could represent a key reason for the observed low lifespan. telomere length undoubtedly is important for cellular aging.(7) recently, hitherto different studies have deciphered the roles of several clock genes like period (per1, per2, per3), timeless (tim), and cryptochrome (cry1, cry2) in regulating dna damage response (ddr).(8) timeless gene can be associated with cell cycle regulators which control aging. timeless interacts with a cell cycle checkpoint kinase chk1 and the atr-atrip, both of which are linked to cry2. the loss of the tim gene results in decreased production of per2. it was suggested that tim is responsible for regulation of replication in the absence of dna damage influences.(9) the circadian clock particip ates in tumorigenesis by regulating downstream tumor related genes. the period circadian regulator 1 (per1) and period circadian regulator 2 (per2) have been reported to be associated with the upregulation of proliferation gene ki-67, the proto oncogene mdm2, the antiapoptotic gene bcl-2, the invasion and metastasis gene mmp9, and bax and the down regulation of bcl-2, c-myc, and p53 in lung, mammary, pancreatic, hepatocellular, and oral carcinoma cell lines.(10) disruption of circadian rhythms as a consequence of iterative alterations in lifestyle may affect well-being and normal physiological processes in humans and represents a well-known risk factor for promoting various cardio-metabolic disorders like metabolic syndrome, diabetes, and cerebrovascular disease (cvd). humans have a significant reduction in sleeping efficiency due to circadian misalignment (shift work and eating at night), that is associated with elevated blood pressure, lower leptin levels, and higher glucose and insulin levels. many studies have reported a strong association between evening chronotype (the tendency of a person to wake up late and sleep late) and the metabolic disease.( 11) amyloid-beta (a?) peptide levels in mouse hippocampus interstitial fluid exhibit robust daily oscillations due to alzheimer's disease (ad) production and deposition. this peptide is responsible for the onset of cognitive decline in alzheimer's disease patients. the circadian clock may play an important role in determining a? levels.(12) with an increase in neurological activity, we appear to lose the ability to regulate our circadian rhythms as we age, and the master clock in the scn appears to decline with age. the circadian system is composed of a hierarchically organized set of structures which are responsible for generating circadian rhythms and synchronizing them to the environmental conditions. thus, circadian rhythms can be used as an aging predictor. with the advent of biobanks to ensure vast genotyping for the masses, the mapping of circadian genomics is useful to measure an account of its effect on metabolic diseases, immune system, and as an introduction to translational medicine. the widespread use of anti-oxidants must be thoroughly correlated in order to demonstrate their utility in anti-aging processes. 208 universa medicina september-december 2022 vol.41no.3 references 1. ruben md, wu g, smith df, et al. a database of tissue-specific rhythmically expressed human genes has potential applications in circadian medicine. sci transl med 2018;10:eaat8806. doi: 10.1126/scitranslmed.aat8806. 2. zhang r, lahens nf, ballance hi, hughes me, hogenesch jb. a circadian gene expression atlas in mammals: implications for biology and medicine. proc natlacad sci usa2014;111:16219 24. doi: 10.1073/pnas.1408886111. 3. edatt l, poyyakkara a, raji gr, ramachandran v, shankar ss, kumar vbs. role of sirtuins in tumor angiogenesis. front oncol 2020;9:1516. doi: 10.3389/fonc.2019.01516. 4. grabowska w, sikora e, bielak-zmijewska a. sirtuins, a promising target in slowing down the ageing process. biogerontology 2017;18:447-76. doi 10.1007/s10522-017-9685-9. 5. hoylaerts mf. animal models of aging research. blood 2015;126:sci-4. http://doi.org/10.1182/ blood.v126.23.sci-4.sci-4. 6. romaniuk a, paszel-jaworska a, toton e, et al. the non-canonical functions of telomerase: to turn off or not to turn off. mol biol reports 2019;46:1401-11. https://doi.org/10.1007/s11033 018-4496-x. 7. vaiserman a,krasnienkov d. telomere length as a marker of biological age: state-of-the-art, open issues, and future perspectives. front genet 2021; 11:630186. doi: 10.3389/fgene.2020.630186. 8. kolinjivadi am, chong st, ngeow j. molecular connections between circadian rhythm and genome maintenance pathways. endocrine related cancer 2020;2:r55-r66. https://doi.org/ 10.1530/erc-20-0372. 9. guilliam ta. mechanisms for maintaining eukaryotic replisome progression in the presence of dna damage. front mol biosci 2021;8:712971. doi: 10.3389/fmolb.2021.712971. 10. li hx. the role of circadian clock genes in tumors. oncotargets ther 2019;12:3645-60. doi: 10.2147/ ott.s203144. 11. baldanzi g, hammar u, fall t, et al. evening chronot ype is asso ciat ed with elevated biomarkers of cardiometabolic risk in the epi health cohort: a cross-sectional study. sleep j 2022;45:1-10. doi: 10.1093/sleep/zsab226. 12. kress gj, liao f, dimitry j, et al. regulation of amyloid- dynamics and pathology by the circadian clock. j exp med. 2018;215:1059-68. univ med 2022;41:207-9. doi: http://dx.doi.org/10.18051/univmed.2022.v41.207-209 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1356 209 http://doi.org/10.1182/ http://dx.doi.org/10.18051/univmed.2022.v41.207-209 c:\users\universa medicina\docu 184 abstract universa medicina nephrolepis exaltata hibiscus rosa-sinensis combination herbal mask decreases levels of reactive oxygen species in nasal wash awal prasetyo*, resti ariani**, arlita leniseptaria antari***, udadi sadhana†, ika pawitra miranti†, and tri nur kristina*** background poor air and mask quality in the work environment increase the risks of inflammation, reactive oxygen species (ros) production, ongoing oxidative stress, and superoxide dismutase 3 (sod3) levels in the airways. the study aimed to investigate the effect of the nephrolepis exaltata hibiscus rosa-sinensis (ne-hrs) combination herbal mask on the airways, as measured by ros and sod3 levels in the nasal wash of textile industry workers. methods this was a preand post-test randomized controlled trial, consisting of 30 subjects with 5-year working time, conducted for eight weeks and monitored every ten days. subjects were randomized into 3 groups, regular surgical mask group (n=10), ne-hrs combination herbal mask group (n=10) and factory mask group (n=10). sod3 and ros levels were determined using elisa. data were analyzed with one-way anova and multiple comparison tests. results ros levels decreased significantly in all groups (p=0.004). regular surgical masks decreased ros levels by 67.90 ± 50.66 pg/ml, ne-hrs combination herbal masks by 49.91 ± 45.06 pg/ml, and factory masks by 193.96 ± 128.36 pg/ml. sod3 levels also decreased but not significantly (p=0.053). differences in ros levels between surgical mask and ne-hrs combination herbal mask was not significant (p=0.796). conclusion herbal masks laminated with ne-hrs combination significantly reduced ros levels among textile industry workers. ne-hrs combination herbal masks have a similar protective effect to the regular surgical mask. keywords: herbal mask, ros, sod3, textile industry workers original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.184-191 september-december 2020 vol.39no.3 cite this article as: prasetyo a, ariani r, antari al, sadhana u, miranti ip, kristina tn. nephrolepis exaltata-hibiscus rosasinensis combination herbal mask decreases levels of reactive oxygen species in nasal wash univ med 2020;39:184-91. doi: 10.18051/univmed.2020.v39.184-191 *department of biomedical science, faculty of medicine, diponegoro university, semarang, indonesia **blood bank technology, bina trada polytechnic (polbitrada), semarang, indonesia ***department of microbiology, faculty of medicine, diponegoro university/kariadi hospital, semarang, indonesia †department of anatomic pathology, faculty of medicine, diponegoro university/kariadi hospital, semarang, indonesia correspondence: awal prasetyo department of biomedical science, faculty of medicine, diponegoro university, semarang, indonesia email: awalpras@fk.undip.ac.id, awalpras@yahoo.com mobile phone: +62-8122810954 orcid id : 0000-0002-1187-1245 date of first submission, may 14, 2020 date of final revised submission, november 12, 2020 date of acceptance, november 16, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 185 univ med vol. 39 no.3 introduction poor air condition due to exposure to p ol lut an t s ca n c aus e se r iou s re spi r at or y problems, especially if the pollutants are reactive chemicals that easily initiate inflammation. the body will respond continuously with a series of dramatic biological processes if the pollutants are inhaled continuously for a long period of time. the initial risk can result from excessive mucus production, damage to the protective epithelium of the respiratory tract, and malignancy.(1,2) ea r ly p r e vent i o n of d i sea se s of t h e respiratory tract consists in the use of good quality masks. thus, the chance of acute and chronic inflammation in the respiratory tract can be reduced and the inflammatory response including the secretion of pro-inflammatory cytokines, chemokine up-regulation, and other complex transduction signals that lead to proliferation and excessive cell activation can be controlled.(3) another important focus that attracts attention is the changing balance of reactive oxygen species (ros) and superoxide d is mu ta se 3 ( sod3) i n t h e b od y whe n inflammation occurs. inhaled air pollutants deposited in the respiratory tract cause chemical reactions in the body that produce ros (oh-, o 2 -, ho 2 , and h 2 o 2 ) in the respiratory tract. excessive production of ros results in oxidative stress which can injure cells and tissues.(4) masks with nephrolepis exaltata (ne) herbal laminate are reported to be able to protect the respiratory tract better. the use of ne herbal masks significantly inhibits the production of the pro-inflammatory cytokine tumor necrosis factor α (tnf-α) in tests of a limited population of textile industry workers exposed to reactive chemicals. similar studies also show that ne herbal masks work actively in suppressing high free ros and result in decreased superoxide dismutase 3 sod3, leading to an oxidant-antioxidant imbalance.(5) an experimental study showed that the ne herbal mask is better in increasing iga and improving the pulmonary function in textile workers compared to the regular mask.(6) the ethanol extract of hibiscus rosasinensis (hrs) contains flavonoids, saponins and steroids and might be responsible for significant inhibition of inflammation, which may involve inhibition of prostaglandins,(7) as well as blocking of the oxidative stress. our previous study on different subjects recommended the further study of the potency of the ne herbal mask on the sinonasal cellular immune system (6) and the present study added the measurement variables to prove the effect of the ne-hrs combination herbal mask on sinonasal oxidative stress. this study was expected to prove the effectiveness of the nehrs combination herbal mask in decreasing the levels of ros and sod3 in textile dyeing factory workers. methods research design this study used a non-blinded experimental design with pre-and post-test randomized controlled trial and was conducted in pt. apac inti corpora, bawen, semarang, between september-october 2019. research subjects the study recruited 36 subjects that fulfilled the inclusion criteria, but only 30 subjects were included (2 subjects not meeting the inclusion criteria and 4 subjects declining to participate). the study sample consisted of employees of a garment factory in bawen, semarang, who had been exposed to occupational chemicals in the dyeing process section. in total, 30 employees were selected based on the inclusion criteria i.e., 25–50 years old, healthy, and willing to be a research subject; and the exclusion criteria i.e., having a history of alcohol consumption, smoking, liver disease, autoimmune disease, cancer, pulmonary and heart disease and/or being pregnant;. the sample was not similar to that of our previous study.(6) the sample comprised 30 male textile factory workers, 25-50 years old, and working 186 prasetyo, ariani, antari herbal mask and reactive oxygen species in the dyeing and sizing areas. the sample size determination was based on the total number of male workers in the dangerously polluted area of the factory (dyeing area). the inclusion criteria also included subjects having complaints of mucus or watery discharge out of the nose and a working time (in a textile factory) of 5 years. the allocation of the groups was done using the randomized block method with block size=6 (using randomly generated numbers). the subjects were randomized into the groups wearing i) regular medical/surgical masks (n=10), ii) ne-hrs combination herbal masks (regular medical/surgical masks that were laminated with ne and hrs extracts) (n=10) and iii) factory masks (single-ply cloth masks) (n=10). to the subjects it was explained how to use the masks correctly. the researchers gave the masks to the supervisor of the subjects every 10 days, then the supervisor distributed the ma s ks t o t h e s u bj e c t s a nd en s ur e d t he compliance of mask wearing. the masks were used in the work area and were replaced daily for 8 weeks. the duration of wearing masks per day was around 6-7 hours on work days. compliance in using the masks was ensured by daily supervision using monitoring cards and follow-up by the researchers was done every 10 days to evaluate the usage of masks and the health condition of the research subjects. after 8 weeks, a second measurement of ros and sod3 was done. the present study determined the protective effect of the masks from the ros and sod3 levels, but did not report on the evaluation of the usage of masks and health conditions of the subjects. preparation of plant extracts and herbal masks t he pr oce s s o f ma ki ng e xt r a c t s o f nephrolepis exaltata and hibiscus rosasinensis was carried out at the laboratory of sc i e n c e a nd m a t h ema t ic s, d i po ne go r o university, semarang, while the herbal masks as well as the regular surgical masks were made by cv. beauty kasatama, surabaya. the herbal masks were prepared by a lamination technique (three-ply surgical masks were laminated with ne-hrs extracts) to maximize the quality and comfort of the mask design. the single-ply cloth masks were produced by the factory itself. details of the procedures are as specified in indonesian simple patents nos. s00201909 40 9 an d s00 20 19 09412, b ot h published on january 21, 2020. laboratory analysis for examination of sod3 and ros levels nasal wash samples were used. subjects were instructed to sit with the head extended at 45o, then to take a deep breath and hold their breath. a syringe filled with 5 ml of isotonic solution at 37oc was inserted into one nostril (with the other nostril closed). the subjects were to hold their position for a few seconds, then look down and slowly drain the liquid into a container. the procedure was to be repeated for other nostril and the whole procedure done in duplicate. all nasal wash fluid samples were stored at 4oc. each sample was processed for testing by the elisa method. the elisa test for sod3 levels used human sod3 (extracellular superoxide dismutase [cu-zn]) elisa kit) while for ros levels the human reactive oxygen species (ros) elisa kit was used. statistical analysis the data were analyzed with spss for windows version 25. primary data on sod3 and ros were analyzed by the shapiro-wilk test to determine the data distribution, which turned out to be normal. the one-way anova test was used to analyze the data. if significant the a n a l ys i s wa s c o nt i nu e d us i ng mul t i p le comparison tests. ethical clearance this study was approved by the health research ethics commission of the faculty of medicine, undip and dr. kariadi hospital, se ma r a ng, un d e r no . 1 94 / ec/ k e pk / fk undip/v/2019. 187 univ med vol. 39 no.3 a b results the study included 36 subjects that fulfilled the inclusion criteria, but only 30 subjects were included (2 subjects not meeting the inclusion criteria and 4 subjects declining to participate). during the study period, 30 male subjects were randomized into three groups. ten subjects were in the regular surgical mask group, 10 subjects in the ne-hrs combination herbal mask group and 10 subjects in the factory mask group. they were followed-up for 8 weeks (figure 1). at base line, there were no significant differences in age, work duration, and sod3 level, respectively, between the three groups (p=0.318; p=0.626; p=0.29), but there was a significant difference in ros level between the three groups (table 1). this means that the randomization process was unable to equally distribute all variables among the three groups. after exposure for 8 weeks, it appears that ros showed significant results (table 2). the ros levels between groups were significantly different, in that the preand posttest multiple comparison showed significant results between regular sur gica l mask, ne-hrs combination herbal mask, and factory mask (p<0.05), but no significant difference between regular surgical mask and ne-hrs combination herbal mask (table 3). this indicates that the assessed for eligibility (n=36) excluded (n=6): . not meeting inclusion criteria (n=2) . declined to participate (n=4) randomized (n=30) allocated to regular surgical mask (n=10) allocated to ne-hrs combination herbal mask (n=10) allocated to factory mask (n=10) lost to follow up (n=0) lost to follow up (n=0) lost to follow up (n=0) analyzed (n=10) analyzed (n=10) analyzed (n=10) follow-up 8 weeks variables regular surgical mask (n=10) ne-hrs herbal mask (n=10) factory mask (n=10) p value age (yrs) length of work (yrs) ros (pg /ml) sod3 (ng /ml) 38.25 ± 6.76 15.00 ± 9.74 147.00 ± 79.89 4.02 ± 2.18 38.13 ± 7.81 16.25 ± 9.74 134.66 ± 73.33 4.26 ± 1.62 41.63 ± 8.57 19.25 ± 7.65 329.44 ± 122.28 3.27 ± 0.96 0.318 0.626 0.001 0.239 table 1. demographic characteristics, ros and sod3 by treatment group at base-line note: ros: reactive oxygen species; sod3: superoxide dismutase 3 figure 1. flow diagram of the participants 188 protective effect of the ne-hrs combination herbal mask was similar to that of the regular surgical mask. discussion the study showed the effectiveness of the ne-hrs combination herbal mask in reducing the levels of ros significantly, but failed to prove a significant decreasing effect on sod3 level. all treatment groups showed a decrease in sod3 levels with differ ent protective powers. statistically, sod3 levels after wearing the masks did not show any significant differences, although the greatest decline was found in the herbal mask group. it has been known that volatile organic compound (voc), azo dyes, anthraquinone derivatives, ch3cooh, hcooh, naoh, nahs, and formaldehyde are some of the ingredients that are often involved in the coloring process in the textile industry. the heating process at high temperatures (60°-79°c) to strengthen covalent bonds with fabric fibers causes the reactive dyes to evaporate and emit a sharp odor.(8) the fumes released into the air during the coloring process contains h 2 s and no which are easily inhaled by workers and become inflammatory agents in the respiratory tract.(9) microscopic observations show that airway inflammation due to exposure to formaldehyde (h 2 co) and voc complexes with nox in the air is characterized by erosion of airway epithelial cells, goblet cell metaplasia and hyperplasia of flat epithelial cells.(10,11) inhaled pollutants deposited in the respiratory tract cause inflammation and chemical reactions in the body and produce reactive oxygen species (ros, oh-, o 2 -, ho 2 , and h 2 o 2 ) that cover the respiratory tract. some studies have shown that excessive ros production in oxidative stress can injure cells and tissues in the respiratory tract. this condition then disturbs the oxidantantioxidant balance in the body. n. exaltata extract was chosen as a coating on herbal masks because of its strong antioxidant content, inhibition of human bronchial epithelial carcinoma (beas2b) cells and counteracting of oxidative stress due to inhaled vocs.(12) in this study, ne herbs were enriched with the addition of hrs to maximize its protective function in suppressing inflammation and oxidative stress due to an imbalance between ros and antioxidants.(13-15) the analysis of ros levels of the workers showed that all three types of masks used were protective. all study groups (regular, herbal mask or factory mask groups) were capable of reducing workers’ ros levels significantly. at base-line, the distribution of all variables between groups was homogenous, except for the ros level. after 8 weeks exposure the factory mask group surprisingly showed an extreme decrease in ros level. theoretically, the reactive oxygen species levels in the factory mask group should have been the highest compared to the other two groups. this is because in factory masks, the filter note: ros: reactive oxygen species; sod3: superoxide dismutase 3 table 2. ros and sod3 levels after 8 weeks of exposure, by treatment group table 3. multiple comparison of preand post-test of ros levels the mean difference was significant at the 0.05 level treatment groups mean differences p value regular surgical mask ne-hrs combination herbal mask factory mask ne-hrs combination herbal mask factory mask 12.34 ± 47.18 -182.44 ± 47.18 -194.78 ± 47.18 0.796 0.001 0.000 prasetyo, ariani, antari herbal mask and reactive oxygen species characteristics regular surgical mask (n=10) ne-hrs herbal mask (n=10) factory mask (n=10) p value ros (pg/ml) sod3 (ng /ml) 79.10 ± 32.89 0.81 ± 0.12 84.76 ± 40.99 0.67 ± 0.10 135.48 ± 20.98 0.77 ± 0.12 0.004* 0.053 189 univ med vol. 39 no.3 density used tends to be more lax and more likely to pass inhaled pollutants into the respiratory tract. exogenous agents (reactive chemicals, ozone, nitrogen dioxide, cigarette smoke, pollutants, radiation, dr ugs, nut ritional inta ke) and endogenous agents (inflammation, organelle damage, peroxisomes, mitochondria and cell metabolism) are the components that are involved in causing the levels of ros in the body.(14) the mechanisms of interaction of pollutants and ros components with the body structure are complex, but one hypothesis is the involvement of reduction-oxidation (redox) reaction cycle in the epithelial lining of the respiratory tract.(4) this process is initiated by the transfer of electrons from antioxidants to quinone transition metal ions to form reduced metal ions or semiquinones. this process is further rearranged by the reaction with o 2 to form the oxygen free radical which is then converted to h 2 o 2 which is the center of the cycle of radical reactions and oxidative stress in the respiratory tract.(15) basically, oxidant production is neutralized by several antioxidants through cellular mechanisms in the respiratory tract. intracellular antioxidants in the respiratory tract include catalase, sod, and glutathione, formed by the enzymes γ-glutamyl cysteine synthetase and glutathione synthetase. the sod3 test results of the workers after using a mask showed better data (because the improving of sod3 levels) and these indicated protective functions; however, the differences among groups were not significant. all treatment groups showed a decrease in sod3 levels with a different protective power. statistically, the difference in the sod3 levels after wearing the masks did not show any significant difference, but the greatest decline was found in the herbal mask group. one possible reason for this finding is that the herbal mask gives double protection to the factory workers, as antioxidant as well as antiinflammatory agent, to prevent pulmonary edema.(16) this study found that using masks prevented the decrease in the level of sod3 and the increase of ros, although the results were not significant. this is in line with a previous study, that found that using a mask protected the respiratory system from harmfull pollutants.(17) these results also represent the concept of an oxidantantioxidant balance, where good health status allows one to be able to adapt to external stimuli and maintain the immune system, so the ups and downs of sod3 levels are closely related to the deposition of high and low ros in the body.(18) this result was also in line with a previous study stating that sod3 levels in the extracellular matrix decreased with the alveolar injury condition of animals which gradually improved.(19) previously, observations of the level of the cytokine tnf-α as inflammatory marker showed a significant decrease in levels after routine wearing of masks by the workers.(20) this condition turned out to be synergistic with sod3’s position as an antioxidant agent that plays a role in reducing the migration of inflammatory cells with regulation of adhesion molecules such as tumor necrosis factor (tnf)-α, interleukin (il)1α, il6, macrophage inflammatory protein (mip)2, monocyte chemoattractant protein (mcp)-1, va scular adhesion molecule ( vcam), intercellular adhesion molecule (icam), pselectin, and e-selectin in inflammatory tissues. the balance between antioxidants and ros is also supported by research showing that antioxidant levels increase with increasing ros levels in the healthy control group. conversely, antioxidants drop when ros levels are reduced. this will be different in pathological conditions in which ros accumulate in many cells and tissues. the addition of exogenous antioxidants to a certain extent actually stimulates the production of higher levels of endogenous antioxidants because it balances excessive ros levels in the body.(21) some factors influenced the results of this study, both internal and external factors. the dyeing area was chosen as the location of the study because it involved the greatest number of chemicals in the coloring process. the risk of inhalation of these chemicals is due to the low degree of awareness of the workers who lack discipline in using personal protective 190 equipment. in fact, the coloring process takes place at high temperatures and emits strongsmelling hot steam. in addition to the location and discipline of workers, smoking habits, good nutritional intake, age and sex are thought to have participated in influencing ros and sod levels throughout the study. further study is needed about the use of the ne-hrs combination herbal mask in other industries, the prolonged use of the ne-hrs combination herbal mask, and other combinations of herbal mask composition. the limitation of this study was that the time for data collection could not be equated to all samples, and may have been aûected by the physical and mental conditions of the subjects. the limited population of subjects was also suggested to be another limitation of the study, and may be attributed to differences in the conditions of the work environment. the clinical implication of the study is the importance of wearing a mask, especially in polluted areas, that should become a daily habit. the further studies should be directed to prove the potency of the ne-hrs combination herbal mask on the cellular immune system as well as lung functions in acute exposure, compared to the n95 mask. conclusion this study demonstrated that the ne-hrs combination herbal mask was ca pable of reducing the ros levels among industrial textile workers. the protective effect of the ne-hrs combination herbal mask was similar to that of the regular surgical mask. conflict of interest the authors declare that they have no conûicts of interest concerning this article. acknowledgement we thank all laboratory and nursing personnel and the funding agency for this study. references 1. watanabe m, noma h, kurai j, et al. effects of short-term exposure to particulate air pollutants on the inflammatory response and respiratory symptoms: a panel study in schoolchildren from rural areas of japan. int j environ res public health 2016;13:983. doi: 10.3390/ijerph13100983. 2. wang h, song l, ju w, et al. the acute airway inflammation induced by pm 2.5 exposure and the treatment of essential oils in balb/c mice. sci rep 2017;7:44256. doi: 10.1038/srep44256. 3. shakya km, noyes a, kallin r, peltier re. evaluating the efficacy of cloth facemasks in 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http://dx.doi.org/10.1016/j.fsi.2014. 10.027. january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 effect of spinal anesthesia with sufentanil on length of stages i and ii of labor in singleton pregnant women: a randomized controlled trial mina yadollahi1 , kambiz sadegi2 , and batool shahraki mojahed1* abstract background labor pain is one of the most painful experiences in a women's life. one of the methods of pain relief is spinal anesthesia. the purpose of this study was to evaluate the effect of spinal anesthesia with sufentanil on the length of the first and second stages of labor in singleton pregnant women. methods a randomized clinical trial was conducted involving 56 women who had to be at least 37 weeks pregnant with a singleton pregnancy and 20 to 35 years of age, as well as seeking a spontaneous natural delivery. they were randomized into an intervention group (spinal anesthesia with sufentanil) of 28 subjects and a control group (no spinal anesthesia) of 28 subjects. statistical analysis was performed using spss software program 20.0. results mean duration of stage i of labor was 152.32 ± 92.01 and 187.68 ± 121.01 minutes in the intervention and control groups, respectively (p=0.34), whereas mean duration of stage ii of labor in the 2 groups was 15.96 ± 14.26 and 26.43 ± 20.90 minutes, respectively (p=0.06). twenty five percent of women in the intervention group and 35.71% of women in the control group experienced a long stage i, whereas 21.43% of women in the intervention group and 35.71% of women in the control group experienced a long stage ii (p>0.05). conclusion this study suggests that spinal anesthesia with sufentanil does not increase the duration of labor stages. it is recommended that more studies be performed in the future using larger sample sizes to allow for the drawing of solid conclusions. keywords: spinal anesthesia, neuraxial analgesia, long labor, duration of labor, singleton pregnancy 1department of gynecology and medical sciences, zabol, iran 2department of anesthesiology, zabol university of medical sciences, zabol, iran *correspondence: batool shahraki mojahed assistant professor of gynecology and obstetrics, department of gynecology and obstetrics, zabol university of medical sciences, zabol, iran email: 30biran@gmail.com date of first submission, february 1, 2023 date of final revised submission, april 3, 2023 date of acceptance, april 17, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 94 doi: http://dx.doi.org/10.18051/univmed.2023.v42:94-100 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1430 cite this article as: yadollahi m, kambiz sadegi k, afshari m, et al. effect of spinal anesthesia with sufentanil on length of stages i and ii of labor in singleton pregnant women: a randomized con trolled trial. univ med 2023;42:94-100. doi: 10.18051/univmed.2023.v42:94 100 https://orcid.org/0000-0002-7839-7752 https://orcid.org/0000-0002-3271-1937 https://orcid.org/0000-0003-3989-1093 95 univ med vol. 42 no. 1 introduction one of a woma n’s most agonizing experiences is going through labor. the best treatment for reducing pain during labor is thought to be neuraxial analgesia. the most popular type of pain treatment during childbirth is epidural analgesia.(1) a self-administered questionnairebased study on the effects of epidural anesthesia received responses from 324 nige rian obstetricians, who mentioned high expenses and having not enough skills as the reasons for not employing the epidural approach.(2) in addition, some medical professionals prefer spinal anesthesia over epidural anesthesia. low amounts of local anesthetic are used during the latter treatment, mostly because the drug can migrate into the spinal fluid. while still being carried out similarly as epidural anesthesia, spinal analgesia calls for a thinner needle to generate a spinal block, which entails making a tiny hole in the dura. furthermore, the spinal block method may take less time than an epidural block.(3) according to several studies, spinal analgesia can be provided more quickly, more affordably, and more effectively than epidural analgesia.(4-6) to prevent undesirable effects such as hypotension in this procedure, it is usual practice to combine local anesthetics and opioid medications in nerve blocks that do not impact sympathetic activity.(7-9) an elongated stage ii of labor is associated with some negative outcomes including chorioamnionitis, perineal rupture, and postpartum hemorrhage. professionals will thus be interested in any treatment that influences the course and outcome of stage ii of labor.(10) a retrospective cohort study on 120 pregnant women who were divided into 2 groups, with one group receiving spinal anesthesia (n=60) and the other without spinal anesthesia (n=60), showed that the duration of stage ii of labor was significantly longer in the women who rec eived spinal anesthesia (p=0.008).(11) the anesthetic drug sufentanil has high solubility, short latency and about 7 hours of action, which makes it a potential effective option for providing fast analgesia.(12) in addition, sufentanil has been reported to cause less respiratory depression than morphine and fentanyl.(13) the analgesic properties of sufentanil have been reported previously and studies suggested its use in both spinal and epidural analgesia.(14) a study to compare the application and efficacy of ropivacaine combined with sufentanil for continuous epidural anesthesia (cea) and combined spinal-epidural anesthesia (csea) in labor analgesia, showed that the use of the combination for csea achieved a shorter onset time and labor period (p<0.01).(15) another study by manouchehrian et al.(16) compared the effects of sufentanil and fentanyl for labor analgesia and reported a similar analgesic effect as well as longer analgesia time for sufentanil. single-dose spinal analgesia can be useful in some cases, such as rapid delivery in primiparous and multiparous individuals and in places where the use of an epidural catheter is restricted. since finding the best option for ana lgesia with the least complications is crucial, the aim of the present study was to determine whether or not spinal anesthesia using a low dose of sufentanil lengthens labor stages i and ii. methods research design this non-blinded, prospective, randomized, clinical trial was conducted in the department of gynaecology and obstetrics, amir-al momenin hospital, zabol, iran from april 1, 2018 to april 1, 2019. research subjects the study subjects were pregnant women who were sent to amir-al momenin hospital in 2019 for a natural birth and agreed to participate in the study. to be eligible for inclusion, a woman had to be at least 37 weeks pregnant with a singleton pregnancy, between 20 and 35 years old, and seeking a spontaneous natural delivery. the exclusion criteria included a history of opiate use, sensitivity to anesthesia, gestational diabetes, 96 gestational hypertension, and indications for cesarean delivery. sample size the required sample size was estimated based on najafi’s study,(17) using a type 1 error of 0.01, a statistical power of 0.90, and a 20% possible dropout rate. the sample size for comparing the means was calculated using the formula; n= f(α, β) 2σ 2 /2δ2, where δ is the true difference and σ is the standard deviation of the outcome. it was estimated that there should be 28 subjects in each of the intervention and control groups. in the study a total of 56 pregnant women, who satisfied the requirements for entry were included. all these subjects as well as their spouses gave their written informed consent after being informed of the study’s goals and conditions. based on a table of random numbers, these participants were randomized using simple randomization into the intervention (spinal anesthesia) and the control group (no anesthesia). intervention in this research, a single dosage of 2.5–5 micrograms of sufentanil was injected in the midline of the l3–l4 or l4–l5 intervertebral regions in the subarachnoid space to induce spinal anesthesia while the patient was seated in a standard operating room. following the injection, the patient remained seated for three minutes befor e lying down. all procedures were performed in the sitting position under aseptic conditions by an expert anesthesiologist, who was to administer the intervention. outcomes measurement vital signs, uterine contractions, and fetal heart sounds were monitored throughout the trial in both groups every 10-15 minutes in the first stage and every 5 minutes in the second stage. the patient was dropped out of the study if any of the following conditions were met: the need for a cesarean section, anesthetic allergy, or cessation of the stages of labor. the gynecology and obstetrics resident who carried out the study kept track of the time. the lengths of the first and second stages of labor were then evaluated and recorded in the two groups, following which the data was imported into spss for statistical analysis. the normal length for the first stage was considered to be 10 hours in nulliparous and 5 hours in multiparous women. the corresponding lengths for the second stage were 3 and 2 hours. the patients were followed up for one month after discharge for hospital in order to be checked for any possible complications. data analysis using spss software version 20.0 and student’s t-test, the descriptive statistics (such as mean and standard deviation) were evaluated. data were compared between the two groups using the t-test for quantitative variables and the chi-square test for categorical variables. a pvalue less than 0.05 was regarded as significant. ethical clearance the zabol university of medical sciences ethics committee authorized this prospective, randomized trial (e thics approval code: ir.zbmu.rec.1397.212). this clinical trial has been registered in the iranian registry of clinical trials (irct). in addition, all participants, together with their spouses, gave written consent before participating. results we enrolled 56 participants in the active phase of labor. twenty eight women were assigned to the intervention group (with spinal anesthesia) and 28 to the control group (without spinal anesthesia). all 56 participants completed their enrolment to the study. no technical difficulty was found in any patient (figure 1). the mean age of the subjects was 26.59 ± 5.58 years in the age range of 18 to 39 years. the mean gestational age was 38.74 ± 1.17 weeks in the range of 35 to 41 weeks. thirty-nine women (69.64%) were multiparous, while 17 were yadollahi, sadegi, afshari, et al spinal anesthesia with sufentanil 97 univ med vol. 42 no. 1 figure 1. flowchart of the participants nulliparous (30.36%). there was no significant difference in any of the demographic factors between the intervention and control groups (table 1). the mean durations of first and second stages of labor were 170.0 ± 108.0 (ranging from 35 to 480 minutes) and 21.20 ± 18.50 (ranging from 2 to 60 minutes), respectively. the mean duration of the first stage of labor was 152.32 minutes in the intervention group compared to 187.68 minutes in the control group (p=0.341), while the mean duration of the second stage was 15.96 minutes in the intervention group compared to 26.43 minutes in the control group (p=0.062), with the means showing no statistically significant difference between the two groups (table 1). overall, 28.57% of women had a long second phase, whereas 30.36% of women had a long first phase. twenty five percent of the women in the intervention group and 35.71% in the control group experienced a long first stage, while 21.43% of women in the intervention group and 35.71 % of women in the control group experienced an elongated second stage. for both stages of labor, there was no significant difference between the spinal group and the control group (table 2). discussion the main finding of our study was that we observed no significant difference in the length of either of stages i and ii between the group who received spinal analgesia with sufentanil and the group who did not. sufentanil is a liposoluble opioid which has a fast onset and a low risk of hypoventilation.(18) 98 a study in india showed that combined spinal epidural using sufentanil and fentanyl achieved high patient satisfaction and excellent labour analgesia without serious maternal or neonatal side-effects. sufentanil provided a significantly longer duration of labour analgesia compared with fentanyl.(19) it is suggested that spinal analgesia lengthens labor by causing the uterine muscle to generate inefficient electrical activity. this idea states that a breakdown in the transmission of contractions to the lower section of the uterus inhibits the primary genesis of uterine contractions. other researchers reported no significant increase in the length of stages of labor by singledose spinal analgesia with 2.5mg hyperbaric bupivacaine plus 50 μg f entanyl. (2 0) t he heterogeneity between studies can be due to the fact that each of these studies used a specific dosage and type of medication for anesthesia. rahmati et al.(20) in their trial used a 0.5 ml (2.5 mg) dosage of hyperbaric bupivacaine 0.5% combined with 50 μg fentanyl and suggested this combination as a safe method for spinal analgesia with no significant complication. our results showed that the control group’s mean lengths of both first and second stages were even longer than those of the spinal anesthesia group (121 ± 187.68 min vs. 92 ± 152.33 min, and 20.90 ± 26.42 min vs. 14.26 ± 15.96 min, respectively). when compared to the contr ol group, even the prevalence of the prolonged second stage was higher in the control group, fairly close to significant levels. zhi et al.(21) in a systematic review and meta-analysis compared the efficacy parameters spinal group (n=28) control group (n=28) p value age (years) weight (kg) height (cm) gestational age (weeks) parity nulliparous multiparous stage i duration (min) stage ii duration (min) cervical dilation before analgesia (cm) 26 ± 5.68 78.6 ± 8.3 1.63 ± 0.05 38.73 ± 1.13 7 21 152.33 ± 92.01 15.96 ± 14.26 2.3 ± 0.3 27.18 ± 5.52 76.1 ± 7.4 1.59 ± 0.09 38.74 ± 1.23 10 18 187.68 ± 121.01 26.42 ± 20.90 2.5 ± 0.4 0.431 0.843 0.612 0.871 0.743 0.341 0.062 0.511 table1. demographic and clinical characteristics of subjects at base-line *values are expressed as mean ±_sd, except for parity as n (%); p-value was obtained from independent sample t-test; p<0.05 considered statistically significant parameters spinal group (n=28) control group (n=28) p-value stage i duration (min) stage ii duration (min) side effect (pruritus) cesarean section postpartum hemorrhage apgar scores at 1 min apgar scores at 5 min 152.33 ± 92.01 15.96 ± 14.26 1 0 2 9.68 ± 0.61 9.53 ± 0.21 187.68±121.01 26.42 ± 20.90 0 0 3 9.73 ± 0.41 9.62 ± 0.56 0.341 0.062 0.451 0.523 0.712 0.832 *values are expressed as mean ±_sd, except for side effects, cesarean section and postpartum hemorrhage as n (%) table 2. comparison of the length of labor and side effects in spinal group vs. control group yadollahi, sadegi, afshari, et al spinal anesthesia with sufentanil 99 univ med vol. 42 no. 1 of sufentanil and fentanyl in combined spinalepidural analgesia and reported that sufentanil was more effective in pain management as well as in extending the analgesia time. we only observed post-dural puncture headache in one patient which was consistent with the results of sharpe et al.(3) which reported postdural puncture headache as the only complication in 2.1% of spinal analgesia patients. another study found that, especially in scenarios with limited resources, spinal anesthesia is one of the most effective methods for treating pregnant women who are extremely restless owing to pain in the later stages of labor, with a success rate of 98%. (2 2) they r eported that a low-dose combination of 250 µg of morphine, 2.5 mg of bupivacaine, and 25 µg of fentanyl can be effective for up to 4 hours. however, due to the unpredictable nature of labor and the particular nature of the labor process, a second spinal block (fentanyl 25 µg + bupivacaine 2.5 mg) may be necessary once the first dose’s effects wear off. another recent study also reported that using opioids can extend the anesthesia time up to 2 hours or even longer.(23) our study had some limitations. the main one was that because of the type of injection technique needed, we could not perform a sham injection as placebo for the control group. however, we believe that this limitation did not cause differential effects on the outcomes. our study did not find a significant increase in labor duration among the patients who received analgesia. since this pain control is a crucial component of labor, professionals should continue to evaluate the available data in order to develop the recommendations for labor pain management. until then, based on our study and previous studies, the use of spinal sufentanil for anesthesia in labor seems to be a safe method that does not increase the length of labor. conclusion this study demonst rated that spinal anesthesia with sufentanil does not increase the duration of the labor stages. it seems that the effect of this method on the duration of labor stages requires further studies. conflict of interest all authors declare that they have no conflict on interests. funding no funding was received for conducting this study. author contribution my and bsm: conception and design, library searches, and assembling relevant literature, critical review of the paper, supervising the wr iting of the paper, and database management. ks: data collection, library searches, assembling relevant literature, writing the paper, critical review of the paper ma: data analysis. all authors have read and approved the final manuscript. 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doi: 10.1016/j.ogc.2022.10.016. yadollahi, sadegi, afshari, et al spinal anesthesia with sufentanil c:\users\universa medicina\docu 207 abstract universa medicina propranolol significantly reduced dna polymerase  expression in patients with essential tremor nefise kandemir1,2, sercan kenanoglu1 , murat gultekin3, nuriye gokce1, hilal akalin1,4, nazife tascioglu1, meral mirza3, emel koseoglu3, and munis dundar1* background essential tremor (et) is the most common movement disorder. propranolol is a first-line medication for et. we aimed to evaluate the effect of propranolol on the expression of poly (adp-ribose) polymerase 1 (parp1) and dna polymerase beta (polb) genes, which are known to be related to neurodegenerative diseases, in patients with et. methods thirty-five healthy volunteers and thirty-five patients followed up with essential tremors were included in a non-randomized control experimental study. expressions of parp1 and polb genes were compared between the control group and the patient group. in addition, preand post-treatment gene expression levels and fahn-tolosa-marin tremor scale values of the patient group were compared after 8 weeks of propranolol treatment. the wilcoxon rank and mann whitney u tests were used to analyze the data. results at baseline, parp1 expression was significantly lower in the et group than in the control group. (p<0.001). polb gene expression was significantly higher in the pre-treatment et group than in the controls (p<0.05). there was no significant difference in parp1 expression levels before and after 8 weeks of propranolol treatment. polb gene expression was significantly higher in the pre-treatment group than in the post-treatment group (p<0.001). conclusion propranolol significantly decreased polb gene expression but there was no significant difference in parp1 gene expression levels in the patient group, after 8 weeks of propranolol treatment. keywords: tremor, genes, propranolol, essential tremor original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.207-215 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1197 september-december 2021 vol.40no.3 ci te t his art icle as: ka ndemir n, kenanoglu s, gultekin m, et al. propranolol significantly reduced dna polymerase  expression in patients with essential tremor. univ med 2021;40: 207-15. doi: 10.18051/univmed.2021. v40.207-215 1department of medical genetics, erciyes university faculty of medicine, kayseri, turkey 2department of medical genetics, diskapi yildirim beyazit training and research hospital, ankara, turkey 3department of neurology, erciyes university facu lty o f medicine, kayseri, turkey 4department of medical genetics, ahi evran university faculty of medicine, kirsehir, turkey *correspondence: munis dundar, professor erciyes university medical school, department of medical genetic, 38039 kayseri/turkey mobile: +905326486116 e-mail: dundar@erciyes.edu.tr orcid id :0000-0003-0969-4611 date of first submission, june 21, 2021 date of final revised submission, september 29, 2021 date of acceptance, october 1, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 208 kandemir n, kenanoglu s, gultekin m, et al gene expressions in essential tremor introduction essential tremor (et) that provokes uncontrolled and rhythmical contractions and relaxations (shaking, twitching movements) is one of the progressive neur ologic movement disorders. the disease is also called idiopathic tremor or benign tremor. even though the disease can occur at any period of life, it is more frequently seen in the older age group. the prevalence of et has a wide range, from 1% in the general population to 5% in the elderly.(1, 2) the main motor dysfunction of et is a kinetic tremor that occurs during voluntary movements of the hands and arms and over time spreads to the head, vocal cords, jaw, and other anatomical parts. another common sign of et is bilateral postural tremor. essential tremor is a clinically heterogeneous disease and also comprises many disorders such as cognitive disorders, dementia and sensory and psychiatric disorders. age and family history can be considered as major risk factors for et.(3) the diagnosis is made based on the typical pattern of tremor and the exclusion of other diseases with tremor. despite its high prevalence, especially in the elderly, the pathophysiology of the disease is still unclear, although many cases seem to be familial.(3-5) there are still several parts in the etiology of et which need to be clarified but the most known substantial etiological factors are genetic and environmental. many large family group samples show an autosomal-dominant pattern. first-degree relatives of et patients have a greater risk of et and the risk increases if the tremor starts at an early age.(5) considering that more than half of the affected individuals have a family history, genetics plays a substantial role in the disease. in addition, there is higher concordance in monozygotic twins than in dizygotic twins. (6) some studies showed that concordance among monozygotic twins is approximately 60% but reported studies about sporadic cases and variable age of onset in some familial cases are arguments for nongenetic factors.(7) a positive family history is quite frequent in patients with et, among whom a significant number of families presumably have autosomal dominant inheritance, while patients with a positive family history have a younger age of onset for the disease.(8) essential tr e mor and some neurodegenerative diseases such as parkinson’s disease (pd), dystonia, and progressive supranuclear palsy (psp) overlap in clinical and pathological features. some post-mor tem examinations have shown that there are tau aggregates in brain samples of patients with et, similar to those in psp. besides, it has been reported that tremors are present also in pd as well as in et.(9) there are several reported responsible genes for the inheritance of the disease, for example, hs1-binding protein-3 (hs1bp3), essential tremor 1-4 (etm1, etm2, etm3, etm4), dopamine receptor d3 (drd3), glial high-affinity glutamate transporter member 2 (slc1a2), hightemperature requirement a serine peptidase 2 (htra2), fused in sarcoma (fus), leucine-rich repeat and ig domain containing nogo receptorinteracting protein 1 (lingo1), and teneurin transmembrane protein 4 (tenm4).(10,11) patients with et have medical and surgical treatment options. propranolol, primidone, topiramate, and gabapentin are generally first choice medicines and another treatment option is surgery that includes thalamotomy, tumoral surgery, and deep brain stimulation.(12) poly (adp-ribose) polymerase 1 (parp1) is an adp-ribosylating enzyme essential for initiating various forms of dna repair. in the pathological condition, extensive dna damage in cells results in parp1 cleavage (inactive), preventing dna repair and thereby leading to cell apoptosis or necrosis. (13,14) poly (adp-ribose) polymerase 1 is a gene encoding the poly (adp-ribosyl) transferase enzyme and is localized in human chromosome 1 (1q42.12). parp1 encoded by this gene can be activated by dna damage; it is a nuclear enzyme involved in dna repair, genomic stability, and many physiological processes such as cell 209 apoptosis. parp1 can act as a sensor for dna damage. after binding to damaged dna, parp1 binds covalently to nuclear acceptor proteins by forming homodimers and catalyzes the cleavage of nad + to nicotinamide and adp-ribose to synthesize long-branching poly (adp ribose) polymers. (15) the dna polymerase beta (polb) is a gene localized on the 8th chromosome 8p11.21 region, and is an important type of dna polymerase that participates in gap-filling dna synthesis. the protein is normally found in the cytoplasm and functions as a monomer but migrates to the nuc leus in case of dna damage.(16) in the literature, many studies observed changes in the expression levels of parp1 and polb genes in diseases with pathological features like those of et. however, no studies have been conducted to evaluate the effect of propranolol on parp1 and polb genes in et patients. in this study we therefore investigated the effect of propranolol on parp1 and polb genes in patients with essential tremors (et), which are known to be related to neurodegenerative diseases. methods research design this was a non-ra ndomized control experimental study that was conducted on 35 patients diagnosed with et in erciyes university medical genetics and neurology departments between july 2016 and january 2019. research subjects and intervention thirty-five healthy volunteers and thirty-five patients who were followed up with essential tremors were included in the study after they were assessed by the erciyes university adult neurology and medical genetics departments. volunteers and patients were assessed by neurological examination and the fahn-tolosamarin clinical rating scale. as a result, those without neurological disorders or chronic medical diseases were selected as the control group. power analysis was performed for 35 patients and 35 healthy volunteers to determine sample size. the effect size was 0.082, 1-beta was 0.924 and alpha 0.05. the inclusion criteria of et patients were age between 18 and 60 years, diagnosed with et by a specialist neurologist, having no additional disease and receiving 40 mg propranolol twice a day for eight weeks. exclusion criteria include patients with hyperthyroidism, diabetes mellitus, psychiatric diseases, tremors (neuropathic, dystonic, and orthostatic), persons receiving treatment that increases tremor and patients under the age of 18. the control group consisted of volunteers aged between 18 and 60 who were medically healthy, did not have any neurological diagnosis and did not have a chronic disease, as a result of the evaluation made by a specialist neurologist. measurements the fahn-tolosa-marin clinical rating scale for tremor (ftm) was used to assess the essential tremor. (17) fahn-tolosa-marin is a scale used to evaluate resting, postural and action tremors. there are up to five ratings representing its severity. tremor severity is evaluated by giving an ftm score between 0 and 4. briefly, the ftm procedure is as follows: the patients are given a device which they hold for 10 seconds to define the tremor severity and frequency. the degree of the scale is directly proportional to the severity of the disease. ftm was measured only in the patient group before and after propranolol treatment, and the two groups were compared statistically. laboratory analysis after peripheral blood samples had been taken from each of the patients and healthy volunteers into 10 ml edta tubes, leukocyte isolation was performed in the laboratory of the erciyes university medical genetics department with the red cell lysis method. leukocytes taken univ med vol. 40 no 3 210 kandemir n, kenanoglu s, gultekin m, et al gene expressions in essential tremor a b in trizol™ reagent were stored at -20°c until rna isolation (invitrogen, thermo fisher scientific, california, united states). rna isolation was performed using the phenolchloroform method. cdna synthesis was done using the evoscript universal cdna master mix kit. expressions of parp1 and polb genes were performed by using the quantitative real-time pcr method on the light cycler 480 ii device (roche diagnostics ltd., rotkreuz, switzerland) using real-time ready catalog primary-probe kits (parp1 with assay id 111143) and polb with assay id 147200)) (roche diagnostic gmbh, mannheim, germany). the reference gene was the β-actin housekeeping gene. the expression analyses were performed using the 2-δδct formula with light cycler 480 software program (version 1.5.0 sp4) (roche diagnostics ltd., rotkreuz, switzerland). gene expression levels were measured in the control group, pre-treatment et group, and post-treatment et group (who had received 8 weeks of propranolol treatment). statistical analysis statistical analysis was performed with the ibm spss statistics 22 software (ibm spss statistics for windows, version22.0. armonk, ny: ibm corp. released 2013). histograms, quantile-quantile plots, and shapiro-wilks tests were used for evaluating the normal distribution of the data. the wilcoxon rank test was used fo r pre -post t rea tme nt eval uat io n. ma nn whitney u test was used to evaluate the difference between the control group and the patient group. the chi-square test was used to compare the distribution according to the gender of the participants. a p<0.05 significance level was accepted. ethical clearance informed consent was obtained from all subjects who agreed to participate in the study. ethics committee approval was obtained from erciyes university ethical committee of clinical studies (2019/82). figure 1. flow diagram of the participants 211 results seventy people were stratified into two groups, namely the et patient group (n=35) and the healthy control group (n=35). the et subjects were given propranolol for 8 weeks (figure 1). at baseline, there was no significant difference in age and sex (gender) between the et and control groups (table 1). a significantly lower parp1 gene expression level was found in the pretreatment et group than in the control group (p<0.001) . polb ge ne expre ssion was significantly higher in the pre-treatment et group compared to the control group (p<0.05) (table 1). polb gene expression was also significantly higher in the pre-treatment group than in the posttreatment group (p<0.001) (table 2). propranolol significantly reduced polb expression, but the decrease in parp1 gene expression was not significant. discussion in our study, the et patient group showed statistically significant changes in parp1 and polb expression levels, in the values compared with the healthy control group. propranolol significantly reduced polb expression, but the reduction in expression of the parp1 gene was not significant. essential tremor, affecting 4% of individuals aged 40 and over, is one of the most common tremor diseases within the globe. the disease can be either a component of different neurodegenerative diseases or an isolated disease.(18) the molecular mechanism of et is still unclear because there is no specific diagnostic marker for et. the disease has clinical similarities with some diseases such as dystonia and parkinson’s disease (pd) and can also be seen together with other neurological diseases such as restless leg syndrome.(19, 20) coexistence with other diseases causes difficulties in the diagnosis of et. twin studies have shown that not only genetic factors, but also environmental factors take part in the etiology of the disease. it was shown in one study that monozygotic twins (6093%) have greater concordance compared to dizygotic twins (27-29%). according to twin studies, the inheritance of et has been reported to be between 45% and 90%.(20, 21) the genetic structure of et is quite complex. the genetic etiology of et comprises both monogenic and multifactorial inheritance. only a few regions of sensitivity have been identified in the etiology of et. variables essential tremor group (n=35) control group (n=35) p value age (years) gender male female parp1 polb 36.71 ± 11.55 24 11 0.32 ± 0.18 0.71 ± 0.58 35.11 ± 10.59 16 19 0.46 ± 0.16 0.46 ± 0.13 >0.05@ >0.05# <0.001@ <0.05@ table 1. distribution of demographic characteristics, parp1 and polb at base line note: parp1: poly (adp-ribose) polymerase 1; polb: dna polymerase beta; @ mann-whitney u test; # chi-square test variables before treatment (n=35) after treatment (n=35) p value parp1 polb ftm-trs grade 0.32 ± 0.18 0.71 ± 0.58 2.4 ± 0.9 0.32 ± 0.21 0.35 ± 0.20 1.9±0.7 > 0.05 $ < 0.001$ <0.01$ table 2. parp1 and polb expression before and after treatment in the et group note: parp1: poly (adp-ribose) polymerase 1; polb: dna polymerase beta; ftm-trs: fahn tolosa marin tremor rating scale; $ wilcoxon rank test univ med vol. 40 no 3 212 kandemir n, kenanoglu s, gultekin m, et al gene expressions in essential tremor genome-wide association studies (gwas) revealed the complex inheritance of et. the etm1, etm2 and etm3 genes were found to be more effective in familial et. on the other h a nd , t h e sl c1a 2, l ing o1 , ct n na3 , ppargc1a and stk32b genes were observed to be effective in sporadic et. in whole genome sequencing (wes) studies, rare variants in the fus, ht ra2 , t e nm 4 , no s3, k cns2 , hapln4, usp6 genes have been associated with et with monogenic inheritance.(22) liao et al.(23) demonstrated with phewas, a phenomewide association study, that propranolol treatment can alter the expression level of genes. propranolol is one of the recommended first-line treatment options for essential tremors. this drug is a non-selective beta-adrenergic receptor antagonist and is given in divided doses three times a day. (24) β-adrenoceptors belong to the family of g-protein-coupled receptors, with the β1 and β2-adrenoceptors being expressed in the brain. central β2-adrenoreceptor blockage is postulated to have favorable effects on tremors.(25) poly (adp-ribose) polymerase-1 (parp1) has dna binding and adp-ribosylation effects. through these features, parp1 constitutes the main part of the cellular repair and defense program mechanism. poly (adp-ribose) may also affect some transcription factors, which c o nt r ol t he e xp r e s s i on o f i nf l a mma t o r y mediators, such as nuclear factor kappa b (nfκb). on the other hand, increased parp1 activation may cause fatal effects on cells by the release of apoptosis-inducing factor (aif) and nad+ and atp reduction. even though parp1 i s par t of t he ge nome pro te c ti on mechanisms, hyperactivity of parp1 can be harmful to the cell. excessive activation of parp1 has been linked to several biological processes including aging, neurodegeneration, and parthanatosis. parthanatos is a caspaseindependent programmed cell death pathway b e c a u se o f n uc le a r t r a n s lo c a ti o n o f t he mitochondria-associated aif.(26) excessive activation of parp1 has been implicated in several neurodegenerative premature aging disorders such as xpa, csb, and atm, and the etiology of stroke.(27,28) poly (adp-ribose) polymerase-1 has a role in the pathogenesis of parkinson’s disease (pd). this information can be supported by the presence of much parp in the nuclei of dopaminergic neurons of the substantia nigra in patients with pd.(29) excessive parp1 activation, which causes dna damage, occurs with age in wildtype c. elegans.(28) multiple studies have shown that increased activation is associated with aging, neurodegeneration, and metabolic disorders.(27) inhibition of parp1 provides prominent neuroprotection in animal models; furthermore, it has been dete rmined that parp1 has a physiological role in the regulation of alphasynuclein expression. it has also been shown that variants involved in the parp1 gene promoter decrease the risk of pd and postpone the initial age of the disease.(30) the ability of parp1 to function simultaneously as both catalytic and acceptor proteins has led to conflicting data.(31) α-synuclein (α-syn) is a presynaptic protein that accumulates and causes loss of neuronal function in pd. injection of this protein inhibits parp-1 activity in the cerebral cortex and hippocampus of rat models.(32-34) the nitric oxide pool that is released after the application of α -syn and no-mediated caspase-3 activation may play a role in the reduction of parp-1 activity.(35,36) both pd and essen tial tremor s are neurodegenerative diseases with tremors. in our results, the expression level of the parp1 gene in et was lower than in the control group. the reason for this result can be related to the fact that parp1 has a significant role in the dna repair mechanism and the loss of functionality of this gene will be effective in the development of et. after propranolol treatment, no significant difference was observed in the expression of parp1. it is particularly important to conduct studies considering parp targeted therapy to understand the details of the parp1 catalytic mechanism and regulation. 213 dna polymerase β is essential for the base excision dna repair (ber) pathway, which clears the genome of apurinic/apyrimidinic (ap) regions.(37) apurinic/apyrimidinic sites could be detrimental for cells because they have mutagenic and/or cytotoxic effects. during the repair of ap regions, dna pol-β participates in synthesis and lyase activities. due to the crucial role of polb in sequence conservation, polb is considered critical for cellular survival. the role of polb has been confirmed with polb null mouse cells, that were more sensitive to agents which damage dna.(38) publications ar e proving that endoreduplication occurs during cell division in patients with pd and alzheimer’s disease (ad). the molecula r cont rol mecha nisms of endoreduplication in the neurodegeneration process are still confused. excessive expression of polb promotes rot enone-induced endoreduplication and this is associated with maintenance of the g2 state. polb i s overexpressed during dopaminergic neuronal death, which is associated with cell cycle re-entry in rotenone-based animal models. therefore, excessive polb expression caused by rotenone increases genome instability in dopaminergic cells, through induction of endoreduplication. increased polb and re-entry of the cell cycle may be associated with the loss of dopaminergic neurons. to better understand the relationship between polb and endoreduplication, we need more detailed studies, especially using in vivo models.(39) in our study, the polb expression level was observed to be higher in et patients compared to the control group. these results are like those seen in the literature in patients with ad and pd. after propranolol treatment, the polb expression level was lower than in the pre-treatment group. this clearly shows that propranolol treatment reduces the effects of polb in et, considering the recovery status of the patients. in our study, we examined parp1 and polb gene expressions in the blood of et pa tients. considering that et affects the cerebellar, brain stem, thalamic, and cortical pathways, animal studies can be conducted, and our results can be compared by looking at the expression differences between blood and brain tissue. the number of patients is one of the important limitations of our study. in future studies, looking at the expression levels of these genes with more subjects and in patients treated with different concentrations of propranolol will increase the reliability. conclusion this study demonstrated that polb gene expression was significantly higher in the pretreatment group than in the post-treatment group. gene expression levels and recovery status of patients can be followed by creating groups treated with different propranolol levels, and these are conceived as commencing strategies for emerging novel treatments. acknowledgment erciyes university supported the study as project tsa-2019-8891. conflict of interest the authors declare no competing interests. contributors nk and md developed the theory and concept of the study. sk and ha designed the study. md, mm, mg, and ek investigated and supervised the findings of this work. nk wrote the manuscript with support from ng, nt, sk, and md. ng and nk performed the statistical analyses. md, ha and sk verified the analytical methods. nt, ng, and sk made the literature search. mg, mm and ek performed clinical studies. nk analyzed and interpreted the patient data. all authors discussed the results and contributed to the final manuscript. all authors read and approved the final manuscript. univ med vol. 40 no 3 214 kandemir n, kenanoglu s, gultekin m, et al gene expressions in essential tremor references 1. louis ed. 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10.1016/bs.enz.2019.08.002. 39. wang h, chen y, chen j, cell cycle regulation of dna polymerase beta in rotenone-based parkinson’s disease models. plos one 2014;9: e109697. doi: 10.1371/journal.pone.0109697. univ med vol. 40 no 3 zairin noor 96 *department of orthopedics, ulin general hospital, faculty of medicine, lambung mangkurat university, banjarmasin **department of orthopedics, saiful anwar regional general hospital, faculty of medicine, brawijaya university, malang ***department of orthopedics, hasan sadikin hospital, faculty of medicine, padjadjaran university, bandung §department of biology, faculty of mathematics and natural sciences, brawijaya university, malang correspondence dr. dr. zairin noor, spot(k), mm., fics department of orthopedics, ulin general hospital, faculty of medicine, lambung mangkurat university, banjarmasin jl. a. yani km 2 no.43 banjarmasin 70233 email: noorzairin@yahoo.com mobile phone:+62811511130 univ med 2012;31:96-104 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background both cortical and cancellous bone display a complex, porous microstructure whose properties depend on the macrostructure of bone as well as age and health of the individual. the aim of this study was to compare the microstructure and characteristics of mineral atoms in osteoporotic and normal bone. methods a prospective laboratory experimental study was conducted from august to december 2010 at several hospitals in banjarmasin. twenty patients with osteoporosis and twenty six normal patients were involved in this study. bone obtained from surgery was analyzed for microstructure by scanning electron microscopy (sem), while mapping of mineral atoms was performed by means of sem-energy dispersive x-ray spectroscopy (sem-edax) at the brawijaya university, malang. results the osteoporotic subjects with mean age of 64.65 ± 16.41 years were older than the normal bone subjects with mean age of 39.38 ± 17.16 years. the body mass index was similar in both groups of subjects. from sem-edax results, three patterns of mineral atoms were apparent in osteoporotic and normal bone. in osteoporotic bone, these patterns were indicative of a disordered substitution or incorporation process. sem results showed degeneration of microarchitecture (resorption cavities, perforations, and prominent granules) in osteoporotic but not in normal bone. there was a significant difference in microstructure between osteoporotic and normal bone, which was caused by differences in atomic properties. conclusions microstructural abnormalities of bone and disharmonization of mineral atoms in the periodic system were found in osteoporosis. keywords: mineral atoms, composite, microstructure, osteoporosis bone microstructure and atomic periodic disharmonization in osteoporosis zairin noor*, mohammad hidayat**, agus hadian rahim*** and sutiman b. sumitro§ abstract 97 univ med vol. 31 no.2 mikrostruktur dan disharmonisasi periodik atom pada osteoporosis latar belakang pada osteoporosis terjadi degenerasi mikrostruktur yang meningkatkan fragilitas dan risiko terjadinya fraktur. insidens osteoporosis meningkat sesuai bertambahnya umur dan terjadinya fraktur biasanya relatif disebabkan oleh trauma minor. penelitian ini bertujuan untuk membedakan mikrostruktur dan karakteristik atom mineral antara tulang osteoporosis dan tulang normal. metode sebuah penelitian prospektif dengan uji eksperimental laboratorium telah dilakukan antara bulan agustus-desember 2010 di beberapa rumah sakit di banjarmasin. dua puluh pasien osteoporosis dan dua puluh enam pasien normal terlibat dalam penelitian ini. tulang yang diambil ketika dilakukan operasi fraktur, kemudian dianalisis mikrostruktur dengan menggunakan scanning electron microscope (sem) dan mapping atom dengan sem-energy dispersive x-ray spectroscopy (sem-edax) di laboratorium fisika universitas negeri malang. hasil subjek osteoporosis berusia rata-rata 64,65 ± 16,41 tahun jauh lebih tinggi dibandingkan subjek dengan tulang normal yang berusia rata-rata 39,38 ± 17,16 tahun. indeks massa tubuh antara kedua kelompok tidak berbeda. gambaran sem menunjukkan degenerasi mikroarsitektur (kavitas resorpsi, lubang, dan granula yang tinggi) pada osteopotosis dibandingkan tulang normal. hasil sem-edax ditemukan tiga pola mapping atom pada osteoporosis dan tulang normal. pada osteoporosis pola mapping atom mengindikasikan ketidakteraturan dalam sistem periodik akibat substitusi atau inkorporasi. terdapat perbedaan mikrostruktur yang bermakna antara osteoporosis dan tulang normal, yang mengakibatkan adanya perbedaan pada pola mineral atom. kesimpulan mikrostruktur tulang yang disertai adanya disharmonisasi atom mineral dalam sistem periodik ditemukan pada osteoporosis. kata kunci: atom mineral, komposit, mikrostruktur, osteoporosis abstrak introduction bone is multifunctional in character, c o m p r i s i n g s u p p o r t f o r b o d y s t r u c t u r e s , protection and storage of regenerative cells, and homeostasis of mineral ions.(1) this material is non-uniform and heterogenous. increasing age results in increased mineralization of bone tissue and the development of localized hypermineralization, such that it is prone to develop cracks that affect its overall mechanical characteristics. the effects of its mineralization pattern are more complex than that observable f r o m a n o v e r a l l i n c r e a s e i n t i s s u e mineralization. with overall increase in its mineral content, bone becomes harder and stronger. when the optimum point or limit of mineralization for a given mineral is exceeded, the material loses its strength.(2) osteoporosis is characterterized by low b o n e m i n e r a l d e n s i t y ( b m d ) a n d microarchitectural degradation, both of which i n c r e a s e b o n e f r a g i l i t y a n d t h e r i s k o f fractures.(3-5) the incidence of osteoporosis increases with age and fractures commonly occur from relatively minor injury. several studies have stressed the role of environmental and social factors as predisposing factors of osteoporosis.(6) 98 bone mineralization changes are strongly influenced by the properties of atoms that are capable of substitution to form composites. previous studies have determined the atomic composition of bone,(7-9) which determines the dimensions of the hydoxyapatite crystals, which are larger(10) or smaller11 in osteoporotic bone than in normal bone. to date the various atomic compositions of bone have only been assessed quantitatively, and there have been no studies for detection of atomic mapping in bone structures. the purpose of the present study was to compare atomic mapping and microstructural aspects of normal and osteoporotic bone. assessment of atomic mapping of bone may supply further information on the determiner of bone quality, which should not be based solely on the quantitative aspect of mineral atomic content. methods design of the study a prospective laboratory experimental study was conducted from august to december 2010 at several hospitals in banjarmasin. study subjects the subjects of this study were women undergoing surgery in the department of orthopedics of ulin general hospital in b a n j a r m a s i n a n d o t h e r c e n t e r s , w i t h a s i n c l u s i o n c r i t e r i a m e n o p a u s a l a n d n o n m e n o p a u s a l w o m e n ; w i t h t r a b e c u l a r fracture of the proximal femur; and osteoporotic and normal bmd scores. exclusion criteria: concomitant disease (diabetes mellitus, stroke, and chronic renal failure) and use of steroids. measurements of bone microstructure at the time of surgery, a sample of the fermoral head was taken for determination of bone microstructure by means of scanning electron microscopy (sem) and mineral atomic mapping of the bone using sem-energydispersive x-ray spectroscopy (sem-edax). sem has been routinely used for the nondestructive study of bone surface structure and morphology. in conjunction with edax, sem is capable of analyzing the atomic composition of materials by detecting atomic structures from levels of about 0.1 wt. the subjects were divided according to bmd scores into a group with normal bone and a group with osteoporotic bone. for the diagnosis of osteoporosis we used bmd data (t scores) for young adults. according to these c r i t e r i a , a t s c o r e o f ≤ 2 . 5 i n d i c a t e s osteoporotic bone, while a t score of > -1 indicates normal bone.(5) sem and edax-sem examinations were performed at the physics laboratory of brawijaya university in malang. analysis of edax-sem data was based on a t o m i c d i s t r i b u t i o n p a t t e r n s o n s e m micrographs, classified according to the position of the respective elements in the periodic table of mendeleev. statistical analysis t h e c h a r a c t e r i s t i c s o f t h e s u b j e c t s , comprising age, weight, height, bmi and p r o x i m a l f e m o r a l b m d , w e r e c o m p a r e d between the osteoporosis and normal groups, using independent t-test. the sem and semedax results were analyzed by visually comparing their respective images. semedax results were compared by evaluating the distribution of the colored points on the bone s u r f a c e , i n d i c a t i n g t h e m i n e r a l a t o m i c distribution. ethical clearance this study obtained ethical clearance from the research ethics commission of the faculty of medicine, brawijaya university, malang. results this study involved 20 patients with osteoporotic bmd and 26 patients with normal bmd, whose characteristics are presented in table 1. the osteoporotic subjects with mean noor, hidayat, rahim, et al disharmonization in osteoporosis 99 univ med vol. 31 no.2 age of 64.65 ± 16.41 years were older than the normal subjects with mean age of 39.38 ± 17.16 years. the bmi of both groups of subjects were comparable. as presented in table 2, this study found deviations in atomic patterns of osteoporotic bone in relation to the periodic system of mendeleev, while the atoms in normal bone still followed the regularity of the mendeleev periodic system. calcium (ca), potassium (k), and titanium (ti) atoms, which showed a homogenous pattern in normal bone (period iv), became non-homogenous as a result of the presence of phosphorus (p) (period iii). the atoms of al, p, mg, and s, with a homogenous pattern in normal bone (period iii), showed a non-homogenous pattern due to the presence of k, ti and fe from period iv. on the basis of sem analysis, this study found microstructural differences between o s t e o p o r o t i c a n d n o r m a l b o n e . a t 5 0 x magnification, sem images of osteoporotic bone showed trabecular structural degradation, with cavities due to excessive resorption of the trabecular arches. the degraded bone was marked by trabeculae with thin and flat walls and numerous cracks, and the beginings of a g r a n u l a r s t r u c t u r e . i n n o r m a l b o n e t h e t r a b e c u l a e w e r e s t i l l m a s s i v e , w i t h interconnected thick-walled arches and rare instances of trabecular cracks, without apparent granular structure (figures 1a and 1b). figure 1. appearance of trabecular structures in osteoporotic (1a) and normal (1b) bone at 50 x magnification 1a 1b table 2. atomic mapping patterns in osteoporotic and normal bone according to period table 1. characteristics of osteoporotic and normal subjects bmi=body mass index; bmd=bone mineral density 100 at 100 x magnification, the sem images of osteoporotic bone showed the presence of perforations at several locations, surrounded by resorption cavities and trabecular stumps. the surface of the remaining trabecular structures appeared flattened and thin. in contrast, normal bone showed resorption cavities that had not yet become large perforations; the trabecular walls were still thick with a knobby surface, without any cracks (figures 2a and 2b). at 3000 x magnification osteoporotic bone s h o w e d g r a n u l a r s t r u c t u r e s o f v a r i a b l e dimensions surrounding resorption cavities and trabecular stumps. the formation of granules resulted in increased porosity of the osteoporotic bone. in normal bone no granular structures were yet apparent and its dominant feature was the presence of a network of interconnected fibrillar strands (figures 3a and 3b). on edax-sem analysis there were differences in patterns of mineral atoms between osteoporotic and normal bone. ca and p had similar atomic mapping patterns in osteoporotic bone. however, in normal bone the ca mapping pattern differed from the p mapping pattern, and resembled the k and ti mapping patterns (figure 4). t h e s e c o n d m a p p i n g p a t t e r n i n osteoporotic bone was that shown by zn and cu. in normal bone the mapping patterns of cu and zn resembled that of fe (figure 5). the third mapping pattern in osteoporotic bone was the pattern shown by al, k, mg, s, ti, and fe, while in normal bone the mapping patterns of al, p, mg, and s were similar (figure 6). figure 2. appearance of trabecular structures in osteoporotic (2a) and normal (2b) bone at 100 x magnification figure 3. appearance of trabecular structures in osteoporotic (3a) and normal (3b) bone at 3000 x magnification 3a 3b 2a 2b noor, hidayat, rahim, et al disharmonization in osteoporosis 101 univ med vol. 31 no.2 discussion bone undergoes continuous changes throughout life under the influence of hormonal and physical factors. trabecular bone is sensitive to hormonal or biological factors involved in its metabolism. the mechanical properties of bone are determined by the microarchitecture of the trabeculae, comprising their numbers and thickness, and the presence of cavities.(12) figure 4. mapping patterns of ca and p in osteoporotic bone (top) and mapping patterns of ca, k, and ti in normal bone (bottom). zn fecuzn cu figure 5. mapping patterns of zn and cu in osteoporotic bone (top) and mapping patterns of zn, cu and fe in normal bone (bottom) ca ti p kca 102 the microstructure of osteoporotic bone shows a diminished amount of bone and a d e g r a d e d m i c r o a r c h i t e c t u r e . a t 5 0 x magnification osteoporotic bone shows a thinning and reduction of bone structures, and the presence of cracks and perforations, resulting in loss of integrity of the trabecular arches. at 100 x magnification osteoporotic bone displays flattened trabeculae with perforations, while at 3000 x magnification granules are found. qualitatively, these results are similar to those obtained by shen et al(12) and jasiuk.(13) differences were also found between osteoporotic and normal bone with regard to a t o m i c m a p p i n g p a t t e r n s , a s a r e s u l t o f differences in subsitutions or incorporations in the atomic structure of the bone. substitution is the replacement of one type of atom with another type, as a result of similarities in the length of the radii and electric charges of these atoms. incorporation is the joining of one type of atom into the structure/system of other types of atoms, thus affecting the overall integrity of the molecule. in addition, the presence of molecular cavities surrounded by functional groups with affinities to certain mineral atoms kai spai fetis mg mg figure 6. mapping patterns of al, k, mg, s, ti, and fe in osteoporotic bone (top) and mapping patterns of al, p, mg, and s in normal bone (bottom) also determines the type of atom involved in substitution or incorporation. the affinity of a functional group may attract or repel one or more types of mineral atoms. overall, the role of substitution and incorporation determines the growth of the bone matrix.(14) this study found that in osteoporotic bone there were deviations in atomic patterns in relation to the periodic system of mendeleev, whereas in normal bone the regulatity of the periodic system was still present. these abnormalities were presumably caused by substitution or incorporation of mineral atoms. in addition, these patterns also determine the potential for substitution or function of each atom within the patterns. in normal bone, the first pattern showed a similar mapping between ca, k, and ti. these three elements are in the same period iv in the periodic system of mendeleev. the potassium (k) atom substitutes for calcium (ca) in the hydroxyapatite crystal, given the presence of a molecular cavity, because the k atom has a longer ionic radius than ca.(15) the titanium atom imparts hardness and strength to the bone. in the periodic system, the position of ti is adjacent to that of ca, in other words, ti noor, hidayat, rahim, et al disharmonization in osteoporosis 103 univ med vol. 31 no.2 is homogenous with ca. the smaller ionic radius (2.23 å) of the ti atom enables it to substitute for ca, or to reach homeostasis to support the strenght of bone. the periodic disharmonization in osteoporosis is marked by similar mapping patterns of ca atoms (period iv) and p atoms (period iii). this disharmonization is due to atomic dishomeostasis, porosity, or decreased size of the hydroxyapatite crystal in osteoporosis. apparently the first pattern is related to the strength of bone. the second pattern of normal bone indicates the potentials for substitution between zn, cu, and fe atoms as a result of their adjacent positions in the periodic system. the ionic radius of fe (1.73 å) is longer than that of cu (1.57 å) and zn (1.53 å), such that fe may be substituted by both cu and zn. zn has been proved to be important in organic structures as well as in crystals. cu plays a role in the cross-linking of bone with collagen. both zn and cu are mutually antagonistic in bone, where a decrease in zn levels increases the cu content of bone.(16-18) regarding the role of these three mineral atoms as connectors to the organic structures of bone, all three (fe, cu, zn) are involved in this function in normal bone, whereas in osteoporotic bone only cu and zn are involved. apparently the second pattern is related to binding anorganic to organic structures. the third pattern in normal bone involves al, p, mg, and s in period iii of the periodic system of mendeleev, while in osteoporotic bone there are periodic irregularities comprising al, k, mg, s, ti, and fe. presumably fe forms a complex with s, which hampers the function of fe as a connector with the organic structure. in addition, the remote position of ti relative to ca potentially decreases substitution of ca by ti. p r e v i o u s s t u d i e s h a v e r e v e a l e d a n association between bone microstructure and t h e s t r u c t u r e a n d d i m e n s i o n s o f i t s hydroxyapatite crystals. the present study explores another aspect of microstructure development, i.e. the mapping of mineral atoms on the microstructural surfaces involved in the development of osteoporosis. this study revealed disharmonization of mineral atoms in t h e p e r i o d i c s y s t e m o f m e n d e l e e v i n osteoporotic bone based on substitution and incorporation. on the other hand, in normal bone there is still a mendeleev periodic harmonization (the atoms within one period form a homogenous pattern in the structure of bone). these results extend the findings of busse (19) on ca and p hypermineralization in osteoporotic bone. one limitation of this study was the qualitative nature of the data analysis, the study of the detailed mechanism of substitution and incorporation in the development of osteoporosis as a result of exposure to metals is the subject of further studies. conclusions t h i s s t u d y d e m o n s t r a t e s t h a t t h e microstructure of trabecular bone in osteoporosis differs from that of normal bone, which is associated with disharmonization of mineral a t o m s i n t h e p e r i o d i c s y s t e m . a t o m i c disharmonization indicates that osteoporosis is a process leading to amorphous state. acknowledgements we thank drs. abdulloh fuad, m.si for analysis of sem and sem-edax results. daftar pustaka 1. mano jf. viscoelastic properties of bone: mechanical spectroscopy studies on a chicken model. mater sci eng 2005;25:145-52. 2. tho jy, zioupos p, currey jd, pharr gm. microstructural elasticity and regional heterogeneity in human femoral bone of various ages examined by nano-indentation. j biomech 2002;35:189-98. 3. huang q, kung awc. genetic of osteoporosis. mol genet metab 2006;88:295-306. 4. duncan el, brown ma. genetic studies in osteoporosis – the end of the beginning. arthritis res ther 2008;10:214. 104 5. brandao cmr, lima mg, da silva al, silva gd, guerra aa jr, acurcio fa. treatment of postmenopausal osteoporosis in women: a systematic review. cad saude publica 2008; 24supl4:592-606. 6. handa r, kalla aa, maalouf g. osteoporosis in developing countries. best pract res clin rheumatol 2008;22:693-708. 7. wiechula d, jurkiewicz a, loska k. an assessment of natural concentrations of selected metals in the bone tissue of the femur head. sci total environ 2008;406:261-7. 8. nielsen fh, stoecker bj. boron and fish oil have different beneficial effects on strength and trabecular microarchitecture of bone. j trace elem med biol 2009;23:195-203. 9. odabasi e, turan m, aydin a, akay c, kutlu m. magnesium, zinc, copper, manganese, and selenium levels in postmenopausal women with osteoporosis: can magnesium play key role in osteoporosis. ann acad med singapore 2008; 37:564-7. 10. noor z, sumitro sb, hidayat m, rahim ah, taufiq a. assesment of microarchitecture and crystal structure of hydroxyapatite in osteoporosis. univ med 2011;30:29-35. 11. sastry tp, chandrasekaran a, sundaraseelan j, ramasastry m, sreedhar r. comparative study of some physico-chemical characteristics of osteoporosis and normal human femur heads. clin biochem 2007;40:907-912. 12. shen y, zhang z, jiang s, jiang l, dai l. postmenopausal woman with osteoarthritis and osteoporosis show different ultrastructural characteristics of trabecular bone of the femoral head. bmc musculoskeletal disord 2009;10:35. doi:10.1186/1471-2474-10-35. 13. jasiuk im. analysis of trabecular bone as a hierarchial material. xxi ictam, warsaw, poland, 15-21 august, 2004. 14. noor z, sumitro sb, hidayat m, rahim ah, sabarudin a, umemura t. atomic mineral characteristics of indonesian osteoporosis by high-resolution inductively coupled plasma mass spectrometry. the sci world j 2012. doi: 10.1100/2012/372972. 15. vallet-regi m, arcos d. biomimetic nanoceramics in clinical use: from materials to applications. cambridge: the royal society of chemistry, thomas graham house, science park;2008. 16. ovesen j, moller-madsen b, nielsen pt, christensen ph, simonsen o, hoeck hc, et al. differences in zinc status between patients with osteoarthritis and osteoporosis. j trace elem med biol 2009;23:1-8. 17. holloway wr, collier fm, herbst re. hodge jm, nicholson gc. osteoblast-mediated effects of zinc on isolated rat osteoclasts: inhibition of bone resorption and enhancement of osteoclast number. bone 1996;19:137–42. 18. aina v, perardi a, bergandi l. cytotoxicity of zinc-containing bioactive glasses in contact with human osteoblasts. chem biol interact 2007; 167:207-18. 19. busse d, hahn m, soltau m, zustin j, puschel k, duda gn, et al. increased calcium content and inhomogeneity of mineralization render bone toughness in osteoporosis: mineralization, morphology and biomechanics of human single trabeculae. bone 2009;45:10341-3. noor, hidayat, rahim, et al disharmonization in osteoporosis oktavianus 27 *neurologist and interventional neurologist, fatmawati general hospital, jakarta **neurologist and interventional neurologist, dr. soetomo general hospital, surabaya ***director, department of interventional neurology and stroke therapy, max super specialty hospital, saket-new delhi, india correspondence dr. fritz sumantri usman, sr, sps, fins neurologist and interventional neurologist, fatmawati general hospital, jakarta email : fritzsumantri@yahoo.com univ med 2012;31:27-33 abstract universa medicina january-april ,2012january-april ,2012january-april ,2012january-april ,2012january-april ,2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background cerebral digital subtraction angiography (dsa) continues to be used for the examination of patients with cerebrovascular diseases. in the past decade, safer contrast agents have been used and there have been important technical advances including smaller catheters, hydrophylic guide wires, and digital imaging systems. the objective of this study was to determine the neurological complication rates of cerebral angiography performed for inpatients. methods a prospective study was conducted from january 2009 until december 2011. the patient’s demographic characteristics, the procedural details as well as complications appearing during and after the procedure were documented. neurological complications are classified based on the international classification: (a) transient, disappearing within 24 hours; (b) reversible, lasting more than 24 hours but less than 7 days; (c) permanent, if the complication last for more than 7 days. the complications were examined by a neurologist. results the patients comprised 82 (41%) women and 118 (59%) men, ranging from 11 to 86 years of age. from 200 patients who underwent the procedure, permanent neurological complications were found in 1 (0.50 %) patient. neither reversible nor transient neurological complications were found. conclusion the cerebral digital subtraction angiography procedure, when conducted by a neuro interventionist, is relatively save, both from the aspect of neurological and non-neurological complications, and from the number of deaths. the overall neurological complication rate fell within the limits recommended by quality improvement and safe practice guidelines. keywords: cerebral angiography, safety, neurology complications safety of cerebral digital subtraction angiography : complication rate analysis fritz sumantri usman*, achmad firdaus sani** and shakir husain*** 28 usman, sani, husain cerebral digital subtraction angiography cerebral digital substraction angiography : analisis komplikasi latar belakang digital subtraction angiography (dsa) serebral masih sering digunakan untuk memeriksa penderita dengan gangguan serebrovaskular. selama decade terakhir banyak kemajuan yang telah diperoleh untuk meningkatkan keamanan penggunaan dsa serebral, seperti kontras yang lebih aman, kateter yang lebih kecil, kawat (hydrophilic guides), dan sistem pengamatan digital yang semakin baik. penelitian ini bertujuan untuk menentukan besarnya kompliklasi neurologi yang terjadi pada penggunaan dsa serebral. metode penelitian prospektif telah dilakukan mulai dari januari 2009 hingga desember 2011. data yang dikumpulkan meliputi karakteristik demografi pasien, rincian proses prosedur, termasuk ada tidaknya komplikasi yang timbul, baik selama atau setelah dilakukan prosedur. komplikasi neurologi dikelompokkan menjadi: (a) transient, yang akan menghilang setelah 24 jam, (b) reversibel, bila terjadi lebih dari 24 jam tetapi kurang dari 7 hari, (c) permanen, bila komplikasi terjadi lebih dari 7 hari. penilain terjadinya komplikasi dilakukan oleh seorang ahli neurologi. hasil subjek terdiri dari 84 (41%) perempuan dan 118 (59%) laki-laki, dengan usia berkisar antara 11 sampai 86 tahun. sebanyak 200 pasien telah menjalani prosedur dsa serebral, dan komplikasi neurologi yang bersifat permanen terjadi pada 1 kasus (0,50%). tidak didapatkan komplikasi yang bersifat reversibel ataupun transient dalam penelitian ini. kesimpulan dsa serebral yang dilakukan oleh seorang ahli neuro-intervensi adalah relatif aman, baik dalam hal komplikasi neurologi, komplikasi non-neurologi maupun terjadinya kematian. komplikasi neurologi yang terjadi masih dalam batas yang direkomendasikan sesuai dengan petunjuk keamanan untuk melakukan dsa serebral. kata kunci: angiografi serebral, keamanan, komplikasi, neurologi introduction r e c e n t a d v a n c e s i n n o n i n v a s i v e neurovascular imaging techniques, including magnetic resonance angiography (mra) and computed tomography angiography (cta), have reduced the number of catheter-based cerebral angiograms performed for purely diagnostic reasons. cerebral digital subtraction angiography (dsa) remains, however the gold standard to find vascular abnormalities of the brain, such as arterial stenosis, arteriovenous malformation (avm) and brain aneurysm.(1,2) in the past decade, safer contrast agents have been used and there have been important technical advances including smaller catheters, hydrophylic guide wires, and digital imaging systems.(3) however, the usage of cerebral dsa for those cases, particularly in indonesia, seems slowly developed. some neurologists are still considered that cerebral dsa as an expensive and invasive procedure that able to cause morbidity as well as mortality. clinician has expressed concerns about the complications, p a r t i c u l a r l y n e u r o l o g i c a l c o m p l i c a t i o n a s s o c i a t e d w i t h c e r e b r a l a n g i o g r a p h y. abstrak 29 knowledge of the complication rates and/or risk factors is important to improve the examination procedure and patient selection. t h e r e w e r e n o t a n y r e p o r t s a b o u t characteristic and safety of cerebral dsa done by indonesian interventional neurologist as well as other interventionist. the objective of this study was to determine the complication rates related to digital subtraction angiography (dsa) in patients with stroke and non stroke. methods research design a prospective study was conducted from january 2009 to december 2011 in four institutions in jakarta. subjects two hundred consecutive diagnostic cerebral angiograms obtained at the four institutions were studied prospectively with institutional review board approval and patient informed consent. all studies were performed on the basis of accepted clinical indications for treatment. angiographic protocol the medical history of the subjects was collected by interviewing the patients as well as their relatives. the subjects were also examined for vital and neurological signs before a n d a f t e r t h e p r o c e d u r e s . p a t i e n t s w e r e restricted from having solid foods 6 hours before the procedure, but were allowed clear fluids. in each patients, an intravenous catheter w a s p l a c e d p r i o r t o a n g i o g r a p h y. electrocardiography, pulse oximetry, and vital signs were monitored throughout the procedure. femoral arterial puncture, using a 5-f sheath, was performed in all procedures. the sheath was constantly flushed with heparinized saline (1,000 iu of heparin in 1,000 ml of normal saline). a similar heparinized saline solution was used for intermittent flushing of the catheter. a bolus of 40-60 iu/kg of heparin was administered at the beginning of the procedure.(4) for diagnostic purposes, guide wires and normal diagnostic catheters of the head hunter type 0.035 inch of width, were used. the catheter was flushed intermittently with isotonic heparinized saline solution to prevent blood clots. non-ionic contrast mixed with saline at a ratio of 2 : 1 was used. contrast was injected manually in the following volumes: 8–10 ml at a rate of 4-5 ml/second into the main and internal carotid arteries, 5 ml at 3-4 ml/second into the vertebral artery, and 6 -8 ml at 3-4 ml/ second into the subclavian artery. on completion of the procedure, the femoral sheath was pulled and the patient’s groin at the location of the puncture site, was compressed for 15–20 minutes. neurological examination was performed by a neurologist after completion of the procedure. after the patient’s condition was stable, the patient was transferred to the recovery room for 4-6 hours, during which the patient was observed. the p a t i e n t w o u l d b e h o s p i t a l i z e d u p o n t h e occurrence of any complications. data collection for each procedure, information was collected about the patient’s demographic characteristics, contrast volume, fluoroscopic time, date of the procedure, operator ’s name, patient’s status, indication(s) for the procedure, size and type of catheter, type and number of injected blood vessels, type of anesthesia (local, general), heparin bolus dosage, emerging complications after the procedure. neurological complications were classified based on the international classification as follows; (a) transient, disappearing within 24 hours; (b) reversible, lasting more than 24 hours but less than 7 days; (c) permanent, if the complication lasted for more than 7 days. any occurring hematomas were classified as small hematomas if the hematomas were less than 5 cm in univ med vol. 31 no.1 30 usman, sani, husain cerebral digital subtraction angiography diameter, and large hematomas, if the diameter was more than 5 cm. statistical analysis statistical analysis was conducted by descriptive statistics (spss 16). after data c o l l e c t i o n , t h e d a t a w e r e c l a s s i f i e d i n accordance to particular characteristics, which are sex, case type, age, number of catheters used, co-morbidity and fluoroscopy time. ethical clearance this study was approved by the ethical committee of research ethical commission and development resources of fatmawati general hospital jakarta indonesia. results a total of 200 cerebral dsa procedures w e r e c o n d u c t e d f o r t h i s s t u d y a n d a r e summarized in table 1. the proportion of stroke and non-stroke cases was 73% and 27%, respectively. the subjects comprised 82 (41%) women and 118 (59%) men, in the age range of 11-86 years of age (mean age 51.14 ± 16.08). most of the procedures in this study (97%) used one catheter, while fluoroscopy time was mostly l e s s t h a n 3 0 m i n u t e s ( 9 5 % ) . a l l o f t h e angiograms were technically successful. there was no intraprocedural complications, and in particular, no occurrence of iatrogenic vessel injury (arterial dissection). there was no instance of contrast agent allergy and no evidence of contrast nephrotoxicity. there was o n l y o n e n e u r o l o g i c a l p o s t p r o c e d u r a l complication (0.5 %). comorbidities of all the patients are shown in table 2. table 3 shows that the cause of most nons t r o k e c a s e s w a s b r a i n a r t e r i o v e n o u s malformation (33.3 %). on the other hand, most stroke cases were caused by recurrent ischemic stroke (45.2%), ischemic stroke (24.7%) and recurrent transient ischemic stroke (23.3%), respectively. characteristics n (% ) sex male 118 (59.0) female 82 (4 1.0) cases stroke 146 (73.0) non stroke 54 (2 7.9) age < 30 years old 27 (1 3.5) 31-50 years old 48 (2 4.0) 51-70 years old 110 (55.0) > 70 years old 15 (7.5) number of catheters used 1 catheter 194 (97.0) > 1 cath eters 6 (3.0) complications no complication 199 (99.5) complication(s) present 1 (0.5) flu oroscopy time < 30 minutes 190 (95.0) 30-60 minutes 8 (4.0) > 60 minutes 2 (1.0) table 1. characteristics of patients and procedures (n=200) comorbidity n (%) hypertension 82 (41.0) hyperlipidemia 96 (48.0) heart diseases 24 (12.0) diabetes mellitus 32 (16.0) *avm=arteriovenous malformation cases n (% ) non-stroke 54 vascular headache 12 (22.2) brain avm* 18 (33.3) intracranial tum or 8 (14.8) other (ptosis, serious head injur y) 16 (29.7) stroke 146 recurrent transi ent ischemic 34 (23.3) isch emic 36 (24.7) recurrent ischemic 66 (45.2) hemorrhagic 6 (4.1) recurrent hemorrhagic 1 (0.7) subarachnoi d hemorrhage 3 (2.0) table 3. underlying diseases of patients table 2. patients’ comorbidity (n=200)* *a number of patients had multiple comorbidities 31 in this study there was one patient with c o m o r b i d i t i e s s u c h a s h y p e r t e n s i o n , hyperlipidemia, diabetes mellitus and stroke, who experienced neurological complications in the form of motor weakness (deterioration of motor strength from 3 to 1 degree). the patient was 72 years old and the indication for dsa was recurrent ischemic stroke. during the dsa procedure for this patient, 2 catheters were used and fluoroscopy time was 25 minutes and 38 seconds. the disease findings in all 200 cerebral dsa procedures in this study are summarized in table 4. among 200 patients, extracranial stenosis/occlusion was found in 36% patients and intracranial stenosis/occlusion in 32.5% (table 4). discussion cerebral dsa is an invasive procedure using catheters, guide wires, contrast with the i m a g e b y a n g i o g r a p h y m a c h i n e . ( 5 ) t h e procedure is performed to find any cerebral vascular abnormality (such as aneurysm, artery-venous malformation, stenosis) as well as to determine blood-flow and vascular conditions (such as vasospasm, vasculitis, vascularization of brain tumor). by doing this procedure, optimal therapy can be achieved for any brain vascular abnormality.(5,6) cerebral dsa is still used as the gold standard for detection of brain vascular abnormalities, particularly aneurysm and artery-venous malformation.(7,8) although imaging technology has developed rapidly, cerebral dsa is still important because the results of this examination are highly comprehensive in providing information about brain vascularization.(9,10) a number of studies about the risks of cerebral dsa in the past ten years have shown that the proportion of neurological complications that may occur during the procedure is 0.05% 2.9%, while the proportion of non-neurological complications is 0.05% 14.7%. the mortality risk of the procedure is around 0.05 %0.08%.(1,2) some modifications and approaches have been performed to minimize the number of complications occurring. it turns out that the duration of the procedure, high co-morbidity, patient’s age, and the vessel anatomy.(11) t h e c o m p l i c a t i o n s , i n p a r t i c u l a r neurological complications, are predicted to be the most common complications occurring as a result of thromboembolism from the use of catheters and guide wires.(3) thromboembolism occurs in the catheter during the guide wire manipulation (when the catheter is pulled out and the guide wire is inserted). the skill of the operator has a major influence on the number of this complication occurring.(11) it is caused by the insertion of the guide wire into the catheter, leading to blood clots in the empty space inside the catheter.(12,13) in daily practice, the operator tries to minimize the empty space inside the catheter by minimizing the frequency and duration of use of guide wires in the procedure. furthermore, the continuous flux of heparin in the 3-way stopcock will decrease the risk of thromboembolism.(14,15) atherosclerotic plaque disruption by catheters and wires has often resulted in stroke.(15) the operator will perform a minimum of selective intra-arterial examination into the blood vessel if on initial examination the operator has found plaque disruption that may c a u s e f a t a l c o m p l i c a t i o n s o n p r o l o n g e d manipulation of the catheter in the area.(16,17) other causes of complications are arterial dissection, platelet activation, coagulation univ med vol. 31 no.1 dsa findings n (%) normal 37 (18.5) intr acranial avms** 18 (9.0) intr acranial aneurysm 8 (4.0) extracranial stenosis/occlusion 72 (36.0) intr acranial stenosis/occlusion 65 (32.5) table 4. distribution of dsa* findings *dsa=digital subtraction angiography **avm=arteriovenous malformation 32 usman, sani, husain cerebral digital subtraction angiography modifying factors, and neurotoxicity of the contrast agent.(18) in this study, we found that the percentage of angiographic procedure complications in the form of neurological complications was 0.5%. there were no deaths in this study. several previous studies yielded similar results, with the proportion of neurological complications between 0.4 – 1.2% for combined permanent, reversible and transient.(10,19) a study to evaluate the proportion of complications in cerebral digital angiography procedures for detection of aneurysms and aneurysm malformations, involving 155 subjects, showed consistent results, with 1.5% of permanent neurological complications (95% confidence interval 0.34.3%).(6) computed tomographic angiography (cta) h a s r e c e n t l y e m e rg e d a s a n o n i n v a s i v e alternative to digital subtraction angiography (dsa) for the detection of residual cerebral aneurysms (ra). this meta-analysis supports cta as an acceptable modality for postoperative detection of ra, although dsa remains the gold standard. by implementing multidetector cta technology in experienced centers, the sensitivity and specificity of cta may approach that of traditional dsa for detecting ra. as a costeffective, non-invasive modality, cta is a promising alternative to dsa for initial and longt e r m e v a l u a t i o n o f r a . ( 2 0 ) a n a d e q u a t e knowledge of the risk factors associated with cerebral angiography may assist clinical decision making and closer observation of patients at high risk of complications. conclusion we conclude that cerebral dsa procedures are relatively safe and have a low number of complications. there were no temporary or reversible neurological and non-neurological c o m p l i c a t i o n s . t h e o v e r a l l n e u r o l o g i c a l c o m p l i c a t i o n r a t e w a s w i t h i n t h e l i m i t s recommended for quality improvement and safe practice guidelines. acknowledgement t h e r e s e a r c h e r s a c k n o w l e d g e t h e i r indebtedness to all who helped during the research. the researchers highly appreciate fitri octaviana md,mmed (for manuscript correction and statistical analysis), and all neuro-intervention practitioners in indonesia ( yu w o n o m d , tr i w a h y u d i m d , to m m y setyawan md, riri sarisanti md, hermanto swatan md, suwito pantoro md, ashari bahar md, and antonius adinatha md). furthermore, the study would never have been adequately conducted without the help of prof. hasan sjahrir md, prof. hasan machfoed md, hastari soekardi md, wijoto md, chaerul r. nasution md.finasim, and andi w. attas md. references 1. okahara m, kiyosue h, yamashita m, nagatomi h, hata h, saginoya t, et al. diagnostic accuracy of magnetic resonance angiography for cerebral aneurysms in correlation with 3d-digital subtraction angiographic images: a study of 133 aneurysms. stroke 2002;33:1803-8. 2. van rooij wj, sprengers me, de gast an. 3drotational angiography: the new gold standard in the detection of additional intracranial aneurysms. am j neuradiol 2008;29:976 –9. 3. willinsky ra, taylor sm, terbrugge k, farb ri, omlinson g, montanera w. neurologic complications of cerebral angiography: prospective analysis of 2,899 procedures and review of the literature. radiology 2003;227: 522–8. 4. morris p. performing a cerebral or spinal arteriogram. in: practical neuroangiography. new york: lippicott william & wilkins;2007. 5. johnston dc, champman km, goldstein lb. low rate of complications of cerebral angiographin routine clinical practice. neurology 2001;57:2012-4. 6. guo y, piza m, rubin gl, dorsch n, young n,wong kp. neurological complication of cerebral angiography performed for hospital inpatients. j hk coll radiol 2007;10:9-15. 7. nguyen-huynh mn, wintermark m, english j, lam j, vittinghoff e, smith ws, et al. how accurate is ct angiography in evaluating 33 intracranial atherosclerotic disease. stroke 2008; 39:1184-8. 8. latchaw re, alberts mj, lev mh, connors jj, harbaugh re, higashida rt, et al. recommendations for imaging of acute ischemic stroke: a scientific statement from the american heart association. stroke 2009;40:3646-78. 9. dankbaar jw, de rooij nk, rijsdijk m, velthuis bk, frijns cjm, rinkel gje, et al. diagnostic threshold values of cerebral perfusion measured with computed tomography for delayed cerebral ischemia after aneurysmal subarachnoid hemorrhage. stroke 2010;41:1927-32. 10. gounis mj, de leo mj, wakhloo ak. advances in interventional neuroradiology. stroke 2010; 41:e80-e7. 11. kaufmann tj, houston j, mandrekaar jn. complication of diagnostic cerebral angiography: evaluation of 19,826 consecutive patients. radiology 2007;3:812-9. 12. jurga j, nyman j, tornvall p, mannila mn, svenarud p, van der linden j, et al. cerebral microembolism during coronary angiography: a randomized comparison between femoral and radial arterial access. stroke 2011;42:1475-7. 13. kwon ok, oh cw, park h. is fasting necessary for elective cerebral angiography?. am j neuroradiol 2011;32:908-10. 14. madrid mm, barret ea, winstead fry p. a study of the feasibility of introducing therapeutic touch tnto the operative environment with patients undergoing cerebral angiography. j holist nurs 2010;28:168-74. 15. amarenco p, lavallée pc, labreuche j, ducrocq g, juliard jm, feldman l, et al. prevalence of coronary atherosclerosis in patients with cerebral infarction. stroke 2011;42:22–9. 16. kelly me, furlan aj, fiorella d. recanalization of an acute middle cerebral artery occlusion using a self-expanding, reconstrainable, intracranial microstent as a temporary endovascular bypass. stroke 2008;39:1770-3. 17. short jl majid a, hussain si. endovascular treatment of symptomatic intracranial atherosclerotic desease. front neurol 2011;1:18. doi:10.3389/fneur.2010.00160. 18. nguyen tn, babikian vl, romero l, pikula a, kase cs, jovin tg. intra arterial treatment methods in acute ischaemic stroke. front neurol 2011;2:1-10. doi: 10.3389/fneur.2011.00009. 19. connors jj, sacks d, furlan aj. for the neurovascular coalition writing group. training, competency, and credentialing standards for diagnostic cervicocerebral angiography, carotid stenting, and cerebrovascular intervention. ajnr 2004;25: 1732-41. 20. thaker ng, turner jd, cobb ws, hussain i, janjua n, he w, et al. computed tomographic angiography versus digital subtraction angiography for the postoperative detection of residual aneurysms: a single-institution series and meta-analysis. j neurointervent surg 2012; 4:219-225. doi:10.1136/neurintsurg-2011010025. univ med vol. 31 no.1 c:\users\universa medicina\docu 79 abstract universa medicina low covid-19-related practice increases the risk of poor health literacy in international students nergiz sevinc1* and burcu korkut2 background health literacy (hl) is the ability to access, understand, appraise, and apply health information, making it crucial for navigating coronavirus and covid-19 information environments. the objective of this study was to determine the health literacy of international students who could not return to their countries after announcement of the coronavirus disease (covid19) pandemic and their perspective and behavior about covid-19. methods a cross-sectional study was conducted involving 399 international students. a 73-item questionnaire consisting of health literacy (23 items), knowledge, attitude and practice about covid-19 (37 items), and sociodemographic characteristics (13 items) was answered by the students. the relationship between the hl score and various variables was determined using univariate and multiple binary logistic regression. results of the 399 international students, 322 (80.7%) participated. the hl of the international students was found to be poor (78.0%) and good (22.0%). participants’ covid-19-related behaviors: 8.4% of the participants’ knowledge level was good, and 38.5% had high covid-19 related practice. multivariate models showed that hl was independently and significantly associated with covid-19 related practice (or= 6.27; 95% ci: 2.45 15.79; p=0.000). conclusion this study revealed that international students’ hl was poor and that low covid-19 related practice increased the risk of low hl in international students. according to the findings, further efforts should be made to enhance levels of hl and covid-19-related behavior of international students. keywords : international student, health literacy, covid-19, behavior original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.79-86 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1144 may-august 2021 vol.40no.2 cite this article as: servic n, korkut b. low covid-19-related practice increases the risk of poor health literacy in international students. univ med 2021 ;40 :7 9 -8 6 . doi: 10. 1805 1/ univmed.2021.v40.79-86 1department of public health, school of medicine, karabük university, turkey 2karabük public health directorate correspondence: *nergiz sevinc karabuk university faculty of medicine, baliklar kayasi mevkii, demir çelik campus, karabuk, turkey. email: dr.nergizsevinc@gmail.com; phone: +905468211834 orcid id: 0000-0003-4763-1902 date of first submission, february 25, 2021 date of final revised submission, april 22, 2021 date of acceptance, may 4, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 80 sevinc, korkut covid-19 and poor health literacy introduction the novel human coronavirus disease (covid-19) first emerged in wuhan city of china and then spread all over the world, becoming a global public health problem.(1-4) the world health organization (who) reported that about 15 million people were infected with covid-19 by july 2020, and 620 thousand people died.(5) the number of cases has now exceeded 15 million showing how quickly the virus spreads considering the lower number of cases reported in previous studies.(6,7) soon after the covid-19 outbreak turned into a pandemic, the who warned all the countries regarding the required measures to decelerate the spread of the virus and to minimize the possible risks.(8) the measures taken such as travel restriction, quarantine and social distancing have caused the individuals to have psychological problems and reduced quality of life.(9-12) following the first death case in china, passengers were screened with thermal cameras at airports in turkey, flights to china were canceled, the iran border gates were closed, and flights to italy and south korea were canceled. after the first coronavirus case was confirmed on 11 march 2020 in turkey, some restrictions were imposed gradually to prevent and minimize the spread of the virus across the country. data received by the turkish ministry of health on october 25, 2020, showed that nationally 362.800 were positive, 163.093 recovered, and 9.799 died.(13) health literacy (hl) is the use of a wide range of skills including reading, writing, speaking, listening, numeracy, critical analysis and communication in order to improve an individual’s ability to act on knowledge to lead healthier lives.(14) the effects of socio-economic factors such as age, gender, and economic status on the level of health literacy have been known for a long time.(15) today, low hl is accepted as an important determinant of morbidity and mortality among the elderly in many developed countries.(16) besides age, low income and language problems can have a negative impact on healt h lite racy. in a stu dy evaluat ing i mmi gr a n t s’ h l, r e s e a r c h e r s f o u nd t h a t immigrants with language problems have lower levels of hl.(17) in a recent study, it was found that people with poor literacy skills have low health behavior.(18) given that hl plays a key role in the ability of individuals to correctly understand information and warnings on health as well as properly use the information that they have obtained, hl has become a defining concept for the countries in tackling the outbreak.(19,20) improving the hl level of society by following the public health guidelines will obviously play an important role in tackling the covid-19 pandemic. while the countries sending students abroad for higher education have preferred western countries for many years, in recent years a growing number of international students have opted for turkey. the number of international students studying in turkey in the last decade has risen from 15 893 students to 125 138 students in 2018. (21) during the covid-19 pandemic, the responsibilities and roles of the international students are determined by the c a mp us c ommi tt e e s i n l i ne wi t h t h e recommendations of the turkish ministry of health. hand-washing, wearing masks, keeping a safe distance and awareness of quarantine and s e l f -i s ol at i o n a r e de f i ne d a s t he ir ma in responsibilities.(22) although many studies have been conducted to investigate the hl levels in different age and occupation segments of the community within a very short period since the first day of the covid-19 outbreak, there are only a small number of studies which have explored the hl levels of university students on covid-19. a study investigating the levels of health literacy among pakistani university students in the cov id-19 pandemic and infodemic revealed that their hl was not at an optimal level as these participants expressed difficulty for half of the items of the health li te ra cy s cale . (2 3) anoth er study showed different results, in that medical students had b e t t e r knowl e dge ( 41 .8 % vs 3 3 .2 % , p 81 value=0.000) and health protocol compliance (6.3% vs 2.4%, p-value=0.049) than non-medical students.(24) in a study conducted in indonesia on the covid-19 hl level of biology teacher candidate students, it was determined that most of the students did not have sufficient knowledge a b ou t cov id-1 9 s ympt o ms, r o u te s of transmission and the role of vaccines, and it was also suggested to provide these students with comprehensive education about covid-19.(25) similarly, in a study conducted on medical students, the relationship between the level of hl and fear of covid-19 was investigated. the results of the study revealed that hl showed a protective effect on fear and that smoking and drinking appeared to have a negative effect on fear of covid-19.(26) however, no study has been conducted so far on international students. the present study aimed to determine the behavior on covid-19 and the hl level of international students who were studying in karabük university and who could not return to their countries due to the outbreak. methods research design this cross-sectional study was conducted between may and august 2020 on international students studying in karabük university who could not return to their countries due to the covid-19 outbreak. study subjects a total of 399 international students were recruited into the study. they were informed about the study before the administration of the questionnaire and the participants were chosen among those who had been living in turkey for at least 2 years. however, 26 students who gave incomplete answers to questions and 51 students who refused to participate in the study were excluded from the study. verbal informed consent was obtained from the participants before data collection. prior to the interview, participants and interviewers washed their hands. participants and interviewers wore face masks and did not make any physical contact with each other. paper and pens were not shared among the participants. in observance of the social distancing rules, the interview was conducted outdoors, with the participants seated at least 2 meters from each other. a face-toface survey was administered to 322 students who agreed to participate in the study after receiving their verbal consent. measurements the questionnaire was prepared based on published literature and information available on t he web s i t e s of t he w ho . ( 5 ,2 7-2 9 ) t h e questionnaire was initially structured in english. it was reviewed by public health experts (two a s s oc i a t e p r o f e s s or s a n d t h r e e a s si s t a n t professors) and revised according to their recommendations. thereafter the questionnaire was translated into turkish. the data collection form consisted of a total of 73 questions. the first part contained 13 questions about sociodemographic characteristics such as age, sex, n a t i on a l i t y, e c ono mi c si t u a t i on a n d accommodation. the second part contained 37 yes-no questions. to evaluate knowledge on covid-19, 15 questions were prepared. the participants were asked about symptoms, risk factors, transmission routes, vaccination and treatment of covid-19. to determine the behavior of the participants, 12 questions were prepared about how to protect oneself against coronavirus. a score of 1 was assigned for a correct answer and 0 for a wrong answer for knowledge and practice questions, with a total p os s i bl e kno wl e d ge sc or e of 0– 1 5. in accor dance with a similar study, level of knowledge was categorized into “low” for participants who scored <10, and “high” for those who scored >10.(27) as to the scoring of practice, the total possible score was 0-12 and participants with scores of <9 and >9 were considered to have “low” and “high” practice, respectively. to assess attitudes toward covid-19, the participants were asked 5 questions including univ med vol. 40 no 2 82 sevinc, korkut covid-19 and poor health literacy a b c statements on the sources of information about the coronavirus, whether it may be a fatal d is e a se a nd wh e t he r t h e d is e a se w a s exaggerated or not. each positive attitude received 1 point, with a total possible attitude score of 0–5; participants with a score <3 and 3> were considered to have “low” and “high” attitude, respectively. the third part, adult health literacy scale ( a hls ) , c on s i st e d o f a h e a l th l it e r a c y questionnaire with 23 questions. the ahls developed by sezer et al.,(29) the reliability and validity of which had been tested, consists of 1 diagram and questions about health information and drug use to determine the competency of adult individuals in hl. thirteen of the questions were yes/no questions, 4 were gap-filling questions, 4 were multiple-choice questions and 2 were matching questions. each question was individually scored. those who marked the positive expressions in the yes/no questions were given 1 point, those who marked the negative expressions were given 0 point, those who marked the correct answer in the gap-filling questions were given 1 point, and those who marked the wrong answer were given 0 point. those who marked two or more correct answers in the multiple-choice questions were given 1 point, and those who marked no correct answer or both correct and wrong answers were given 0 point. those who matched more than two correct answers in the matching questions were given 1 point, and the others were given 0 point. as a result of reliability and validity analysis, the reported cronbach alpha coefficient of ahls was found to be 0.77 and the test-retest reliability coefficient was found to be 0.87. the content validity index of ahls that consists of 23 items was found to be 90.71%. although the cutoff score of the scale was not calculated, it was considered that the higher scores in the questionnaire were deemed to indicate a higher level of health literacy. with a total ahls score of 0–23, participants with scores of 0-11 and 12-23 were considered to have poor and good hl, respectively. based on these findings, sezer et al.(29) suggested that the ahls is reliable and can be used in the adult group. statistical analysis the data were evaluated in a computer environment using spss package software ver sion 22.0. t he c a te go ri c al d a ta wer e e x pr e s se d a s f r e q ue nc y a n d pe r c e n t a ge distribution. knowledge, practice and attitude scores were categorized into low and high. t he relationship of hl with socio-demographic and further characteristics was tested by univariate and multiple logistic regression analysis. the pvalues <0.05 were considered statistically significant. ethical clearance the ethics committee approval (no. 2020/ 230) was obtained from karabük university and permission was also obtained from the ministry of health. results three hundred twenty-two international students were included in the study; 85.1% of them were male, while 14.9% were female, and the median age was 22.0. 68.3% of them were african, while 31.7% were asian. 92.9% of the participants were single, 59.3% had been living in turkey for at least 2 years, and 39.1% had been studying in engineering. because of the pandemic 65.5% of them had to stay in turkey, and 77.3% of them had been living at residence during the pandemic. also, 77.6% of the participants had not received any previous health training (table 1). as for the behaviors of the international students related to covid-19, 91.6 % of the participants had a low knowledge level, 61.5% had a low level of practice and 66.1 % had low level of attitude. wi t h r e ga rd t o t he hl sc or e o f t he international students, 78.0% was found to have poor hl and 22.0 % good hl. the results of 83 univ med vol. 40 no.2 bivariate logistic regression analysis showed that duration of living in turkey and receiving training on health and knowledge on covid-19 were not significantly associated with hl, but that attitude a nd pr actice on covid-19 were significantly associated with hl (table 2). the multivariate logistic regression showed that practice was the risk factor of hl among international students. low levels of covid19-related practice will increase the risk of low hl (or= 6.27;95 % ci: 2.45 15.79;p=0.000) (table 2). discussion the present study was focused on hl and the related behavior including knowledge, attitude and practice on covid-19 of the international students who have studied in karabuk university, turkey. findings from this study revealed that the hl scor es of the international students were low, and their covid-19-related knowledge and practices were also low. in our study the attitude and practice about covid-19 were associated significantly with hl. this result was in line with the study among 256 students in the us showing that hl was independently associated with pr eventive practices about covid-19.(3 0) students with lower hl use social media more frequently and thus are at greater risk of exposure to false and misleading information, possibly becoming additional vectors in the propagation of poor information, encouragement of harmful health practices, or resistance to public health guidance. characteristics n (%) age (years) 18-24 ≥ 25 sex female male economic status good moderate bad duration of living in turkey (years) 2 3 ≥ 4 have you received a training on health? yes no knowledge low high attitude low high practice low high health literacy poor good 245 (76.1) 77 (23.9) 48 (14.9) 274 (85.1) 122(37.9) 190(59.0) 10(3.1) 191 (59.3) 106 (32.9) 25 (7.8) 72 (22.4) 250 (77.6) 295 (91.6) 27 (8.4) 213 (66.1)) 109 (33.9) 208 (66.1) 124 (38.5) 251 (78.0) 71 (22.0) table 1. characteristics of the participants (n=322) 84 sevinc, korkut covid-19 and poor health literacy characteristics bivariate logistic regression multiple logistic regression or 95 % ci p value or 95% ci p value age (years) 18-24 ≥ 25 sex female male economic status low high duration of living in turkey (years) 2 3≥ have you received a training on health? no yes knowledge low high attitude low high practice low high 1 6.86 1 0.20 1 0.32 1 0.68 1 0.73 1 0.79 1 0.43 1 1.96 2.41-19.53 0.06-0.67 0.18-0.55 0.39-1.19 0.39-1.34 0.32-1.95 0.25-0.73 1.10-3.52 0.000 0.009 0.000 0.183 0.315 0.612 0.002 0.023 1 11.75 1 0.29 1 0.20 1 0.45 1 0.50 1 0.78 1 0.14 1 6.27 3.08-44.75 0.07-1.10 0.08-0.51 0.19-1.04 0.21-1.17 0.27-2.22 0.06-0.33 2.49-15.79 0.000 0.070 0.001 0.063 0.112 0.645 0.000 0.000 table 2. bivariate and multiple binary logistic regression of socio-demographic and further characteristics with health literacy (n=322) threaten public health, such as pandemics.(34,35) according to paakkari,(6) health literacy should be seen in relation to social responsibility and solidarity. in a study evaluating the relationship between the level of hl and the level of fear of covid-19, health literacy was found to protect medical students from fear, (26) while sun et al. found that health literacy is a direct influencing factor of health behavior.(36) in our study, the hl levels were poor. fauzi et al.(26) conducted a study on 290 biology teacher candidate university students and reported that most of the students had a poor level of hl. the fact that the covid-19 related practices of the students were inadequate may b e a s s o c ia t e d wi t h t h e p o or hl l e ve l . universities and public institutions can provide information via electronic boards, announcement posters and e-mails so that these students can access reliable information sources in pandemic situations and overcome the negative effects of the pandemic. there are some limitations of the study. the first limitation is the lack of an outbreak-specific hl scale as the study was conducted at the beginning of the pandemic. the second limitation is that most of the participants were male (85.1%) due to the unwillingness of female students to participate in the study. therefore, the results of this study cannot be generalized since it is a single-center study and male participation is higher than female participation. moreover, the researchers did not analyze any potential moderating effect or association between the field of study and hl. further research must be done in several centers with a better design such as a cohort study. 85 univ med vol. 40 no.2 conclusion the hl of international students was poor. in addition, their low cov id-19-related practices will increase the risk of poor hl among international students. continuing public and p rof es si ona l e du c a ti o n c ampa ign s a b ou t covid-19 should be expanded to include international students. conflict of interest none declared. contributors ns contributed to writing the original draft. ns and bk contributed to review and editing. ns and bk contributed to data collection and analysis. both authors have read and approved the final manuscript. references 1. ren ll, wang ym, wu zq, et al. identification of a novel coronavirus causing severe pneumonia in human: a descriptive study. chinese med j 2020;133:1015-24. doi: 10.1097/ cm9.0000000000000722. 2. wang c, horby p, hayden fg, et al. a novel coronavirus outbreak of global health concern. the lancet 2020;395:470-3. https://doi.org/ 10.1016/ s0140-6736(20)30185-9. 3. wu jt, leung k, leung gm. nowcasting and forecasting the potential domestic and international spread of the 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promot int 2012;27: 117-26. doi: 10.1093/heapro/dar043. 15. øimnáèová z, kajanová a, bøízová b. selected socio-economic factors of health literacy of the poor. human affairs 2018; 28: 461-70. doi:10.1515/ humaff-2018-003.7 16. chesser ak, woods kn, smothers k, et al. health literacy and older adults: a systematic review. gerontol geriatr med 2016;2:1-13. doi: 10.1177/ 2333721416630492. 17. mantwill s, schulz pj. does acculturation narrow the health literacy gap between immigrants and non-immigrants—an explorative study patient educ couns 2017;4:760-7 doi: 10.1016/ j.pec.2016.10.021. 18. easton p, entwistle va, wýllýams b. health in the ‘hidden population’ of people with low literacy. a systematic review of the literature. bmc public health 2010;10:1-10 doi:10.1186/14712458-10-459. 86 sevinc, korkut covid-19 and poor health literacy 19. van den broucke s. why health promotion matters to the covid-19 pandemic, and vice versa. health promot int 2020; 35: 181-6. doi:10.1093/heapro/daaa042. 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implications for interventions. int j environ res public health 2021; 18:3301. https:// doi.org/10.3390/ijerph18063301. 31. ikhlaq a, hunniya be, riaz ib, et al. awareness and attitude of undergraduate medical students towards 2019-novel corona virus. pakistan j med sci 2020; 36(covid19-s4), 32. doi: 10.12669/ pjms.36.covid19-s4.2636. 32. alzoubi h, alnawaiseh n, al-mnayyis a, et al. covid-19-knowledge, attitude and practice among medical and non-medical university students in jordan. j pure appl microbiol 2020; 14, 17-24. doi:10.22207/jpam.14.1.04. 33. cheng vcc, wong sc, chuang vwm, et al. the role of community-wide wearing of face mask for control of coronavirus disease 2019 (covid-19) epidemic due to sars-cov-2. j infect 2020;81:107-14. doi: 10.1016/j.jinf.2020.04.024. 34. sentell t, vamos s, okan o. interdisciplinary perspectives on health literacy research around the world: more important than ever in a time of covid-19. int j environ res public health 2020 ; 17: 3010. doi: 10.3390/ijerph17093010. 35. abel t, mcqueen d. critical health literacy and the covid-19 crisis. health promot int 2020; 35:1612-3. doi: 10.1093/heapro/daaa040. 36. sun x, shi y, zeng q, et al. determinants of health literacy and health behavior regarding infectious respiratory diseases: a pathway model. bmc public health 2013; 13: 261. doi: 10.1186/14712458-13-261. january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 influence of interspecies interactions on biomass and extracellular polymeric substances of bacterial biofilms mercedes baltazara schettini alava1 and naga raju maddela2* abstract background studies with emphasis on pure and mixed-species biofilms are of significant importance in understanding biofilm formation mechanisms during microbial infections. this research aims to evaluate pureand dual-species biofilms of escherichia coli (code a), staphylococcus aureus (code b), klebsiella pneumoniae (code c) and pseudomonas aeruginosa (code d) pathogenic bacterial species and their production of biofilm exopolysaccharides at laboratory scale. methods biofilm biomass (a595) of pureand dual-species cultures was determined by means of a microtiter plate assay in triplicate using a microplate photometer (fisher scientific, type-357). extracellular polymeric substances (eps) and soluble microbial products (smp) were extracted from the biofilm cells (pureand dual-species cultures) using the alkaline-heat extraction method. dry weights (g/l) of eps and smp were determined by drying the samples at 105 °c for 8 hours. results klebsiella pneumoniae biofilm biomass accounted for a 28-72% greater biofilm biomass than the other bacteria. experimental values of dualspecies biofilm biomasses were in the range of 6% to 30% over theoretical values. the experimental value of one dual-species (bacteria b + d) biofilm biomass was 30% higher than its expected value. decrease or increase in the dual-species biofilm biomass of either bacteria a+c or bacteria b+c combinations was totally dependent on the cell density of bacteria c. conclusions biofilm biomass of pure-species cultures was totally species-dependent, and the biofilm biomass of four species was in the following order: bacteria c > d > a > b. relation between biofilm biomasses and smp or eps was inconsistent. keywords: biofilms, bacteria, polysaccharides, pure biofilms, dual-species biofilms, eps 1masters program in biomedicine with mention in special tests and biomedical diagnosis, postgraduate institute, universidad técnica de manabí, portoviejo-ecuador 2department of biological sciences, faculty of health science, universidad técnica de manabí, portoviejo ecuador correspondence: dr. naga raju maddela, ph.d, professor department of biological sciences, faculty of health science, universidad técnica de manabí, portoviejo 130105, ecuador. mobile: +593 0962981508 email: raju.maddela@utm.edu.ec orcid id: 0000-0002-7893-0844 date of first submission, february 13, 2023 date of final revised submission, april 5, 2023 date of acceptance, april 10, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 84 doi: http://dx.doi.org/10.18051/univmed.2023.v42:84-93 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1418 cite this article as: alava mbs, maddela nr. influence of interspecies interac tions on biomass and extracellular poly meric substances of bacterial biofilms. univ med 2023 ;42 :84 -93. doi: 10.18051/univmed.2023.v42:84-93 mailto:raju.maddela@utm.edu.ec https://orcid.org/0000-0003-3928-1463 https://orcid.org/0000-0002-7893-0844 http://dx.doi.org/10.18051/univmed.2023.v42:84-93 https://univmed.org/ejurnal/index.php/medicina/article/view/1418 85 univ med vol. 42 no. 1 introduction biofilms are self-organized communities of one or more types of microorganisms embedded in an extracellular matrix. they collectively represent the largest biomass and activity center on the planet, playing a major role in the biology and chemistry of the environment (both natural and engineered) and in maintaining public health.(1,2) over 500 bacterial species have been identified in typical dental plaque biofilms. biofilms are held together by molecular strands of nucleic acids, collectively termed extracellular polymeric substances or exopolymeric substances (eps), which are highly hydrated polymers that are mainly composed of polysaccharides, proteins, and dna. the bacterial cells produce eps and are held together by these eps strands, allowing them to develop complex three-dimensional, resilient, attached communities. biofilms can be as thin as a few cell layers or many inches thick, depending on environmental conditions.(3,4) “pathogenic biofilms” is one of the hot topics in the world of biomedical research. several methodologies such as in vitro assays, in vitro catheter-infection assays, and in vivo mouse catheter infection models have been used to assess biofilm formation. the capacity for biofilm formation was found in 59% to 100% of different bacterial and fungal species isolated from intravenous catheters, medical devic es, a nd c linic al sites. (5 ) pseudomonas aeruginosa is an opportunistic human pathogen and has been used as a model organism for the study of bacterial biofilm formation. the bacterium produces at least three exopolysaccharides (alginate, psl and pel) for biofilm formation. the polysaccharide psl is rich in mannose and galactose, while pel is a glucoserich, cellulose-like polymer. mutants deficient in the production of these polysaccharides have been constructed to investigate the interactive contribution of alginate, psl and pel to the formation of biofilms. (6 ) t he c ommon opportunistic pathogens candida albicans and staphylococcus aureus frequently co-exist as dual-species biofilms that cause nosocomial infections associated with medical devices and are more resistant to antibiotics and host immune responses than mono-species biofilms.(7) in a study on dual-species biofilm formation by b. cereus and p. fluorescens, the concentrations of which were determined by real-time pcr, it was found that in comparison to their respective monospecies biofilms, the dual-species biofilms produced a significantly higher protein content in the eps and were mor e resistant to the antibacterial effects of grapefruit seed extract (gse). (8 ) in dual-speci es biofilms of pseudomonas fragi and e. coli growing on meat, competition for limited adhesion surface was the principal interplay between the two species, but the competition did not affect the spatial structure and microbial composition of the mature dual-species biofilms. the study also found that e. coli was the more aggressive species in regard with surface colonization.(9) bacterial exopolys accharides are an essential group of compounds secreted by bacteria, and these versatile eps are used individually or in combination with different materials for a wide range of functions in the biomedical field, being commonly used for their biosynthesis pathways and characteristics.(10) microbial exopolysaccharides have found outstanding medical applications since the midtwentieth century, with the first clinical trials on dextran solutions as plasma expanders, or other exopolysaccharides entering medicine first as conventional pharmaceutical excipients (e.g., xanthan, as a suspension stabilizer, or pullulan, in capsules and oral care products). while pathogenic biofilms contribute to states of c hronic inflammation, probiotic lactobacillus biofilms cause a negligible immune response and, in states of inflammation, exhibit robust anti-inflammatory properties. these probiotic biofilms colonize and protect the gut and vagina and have been implicated in improved healing of damaged skin. overall, biofilms stimulate a unique immune response that we are only beginning to understand.(11) most eps are exopolysaccharides i.e. extracellular carbohydrate 86 polymers produce d and se creted by microorganisms which accumulate outside cells and are capable of being released into the surrounding environment.(12) exopolysaccharides are carbohydrate macromolecules, usually together with proteins, which are synthesized by bacterial enzymes and excreted and accumulated extracellularly giving the culture a mucilaginous appearance. the main monosaccharides present in exopolysaccharides are: rhamnose: 28-45%, glucosamine: 12-20% , glucose: 20-30%, galactose: 15-25%.(13) bacterial eps also have extensive commer cial applications in the pharmaceutical and food industries. owing to the structural and functional diversity, genetic and metabolic engineering strategies are currently employed to increase eps production.(14) in regard to the biofilm formation potentials of different pathogenic bacteria, strains of staphylococcus au reus, pseudomonas aeruginosa, and acinetobacter baumannii were found to produce strong biofilms, whereas moderately strong biofilms were reportedly produced by strains of staphyl ococcus saproph yticus and staphyloco ccus epidermidis. (1 5) biofilm formation allows otherwise unicellular organisms to assume a temporary multicellular communal lifestyle, which affords protection from harsh environmental conditions and provision of centralized and concentrated resources, such as nutrients and genetic exchange opportunities.(16) during biofilm formation, sev eral species of bacteria communicate with one another, employing quorum sensing (qs) by monitoring the number of small molecular autoinducers.(17) in pathogens, this qs circuit allows the expression of specific virulence factors, with consequent physiological changes associated with the infection. it is noteworthy that in e. coli the qs circuit makes it possible to coordinate the expression of genes that participate in some important metabolic pathways, and this is probably one of the reasons why the carbon storage regulator a (csra) protein is essential in this bacterium.(18) s. aureus secretes various toxins that harm the host, one of them being the poreforming protein, alpha toxin. based on the lytic activity of alpha toxin in red blood cells, a complex mechanism of action has been suggested for the intoxication of nucleated cells. previously, it has been proposed that alpha toxin plays an essential role in the pathogenesis of pneumonia caused by s. aureus.(19) the influence of the cell density of one strain on the dual-species biofilm biomass is demonstrated by the fact that cooperative interactions in dual-species biofilms lead to higher biomass and higher tolerance to disinfectants. however, in real food processing environments, the presence of many other microbial species clearly adds additional complexity to the behavior of mixed-species biofilms; all incorporated microorganisms may compete, cooperate, and communicate with each other. one problem that has been poorly addressed with respect to pathogenic bacterial species, is whether biofilm biomasses are influenced by soluble microbial products (smps) and eps.(20) a wide range of micr oorganisms pro duce eps that are fundamental for microbial life and provide an ideal environment for chemical reactions, nutrient entrapment, and protection against environmental stresses such as salinity and drought.(21) investigating dual-species biofilms containing different densities of partner strains is vital in understanding pathogenic biofilms and their disease mechanism. most investigations have been focused on the pure-species biofilms; but such results are far from complete for understanding of biofilms of natural environments. to the best of our knowledge, investigations that are based on the analysis and characterization of biofilm biomasses in interactions of two species with each other are very few in number. the present investigation focused on an interactive approach of four bacterial species. therefore, in the present investigation, we aimed to address fundamental questions in order to understand more about the pureand duals-species biofilms of four different clinically important bacterial species, i.e. escherichia coli, staphylococcus aureus, kle bsiella pneumoniae and pseudomonas aeruginosa. alava, maddela bacterial biofilms 87 univ med vol. 42 no. 1 methods bacterial cultures cultures of four pure species of bacteria (table 1) and their combinations were investigated for their respective biofilm interactions. the biofilms of the same cultures were used to extract their eps. quantif ication of pur e-spe cies biof ilm biomass prior to developing biofilms, 4 bacterial species were sub-cultured separately (5 ml each in 10 ml test tubes) using nutrient broth sterilized at 121°c for 20 minutes and incubated at 37°c. aliquots of these suspensions were stored at 4°c in the refrigerator for regular use and as glycerol stocks at –20°c for future use. to each of four wells in a 96-well microtiter plate, 90 μl of nutrient broth and 10 μl of a pure bacterial suspension were added to develop purespecies biofilms. this step was repeated with the 3 other bacterial suspensions. in addition, 4 wells were kept separate for water and medium control. plates were incubated for 48 h at 37°c in an incubator without agitation. after 48 h, the wells were washed twice with 200 μl of sterile distilled water. the biofilms were then fixed with 200 μl of 99% methanol per well. after 15 min, excess methanol was removed from each well and the biofilms were completely air-dried. finally, 200 μl of 0.1% crystal violet was added to each well and the microtiter plates were incubated at room temperature (~28°c) for 20 min. the biofilm layer was then dissolved by adding 150 μl of 33% acetic acid to each well. the acetic acid samples were then diluted 50-fold in 33% acetic acid 96-well microtiter plates, and absorbance was measured in triplicate at 590 nm using a microplate reader (fisher scientific thermo scientific multiskan fc microplate photometer type 357).(22) dual-species biofilms dual-species biofilms were prepared in triplicate by using different combinations of the four bacterial species at 3 different cell densities (inoculum sizes) i.e. 1:1, 1:9 and 9:1. for the development of 1:1 dual-species biofilms, 5 μl of culture-1 + 5 μl of culture-2 + 90 μl of nutrient broth were placed in a microtiter plate well. to grow 1:9 dual-species biofilm, 1 μl of culture-1 + 9 μl of culture-2 + 90 μl of nutrient broth were placed in the well, whereas 9:1 dualspecies biofilm was cultured by placing 9 μl of culture-1 + 1 μl of culture-2 + 90 μl of nutrient broth in the well. culturing conditions, harvesting, staining, and measuring of dual-species biofilms were similar to that of pure-species biofilms described in the preceding subsection. charact erizati on of biof ilm exopolysaccharides the bound eps was obtained by removing the smp by centrifugation of the culture broth (25 ml of the medium stationary phase, i.e., 48 h) at 3500 rpm and 4°c for 15 min. the cell sediment with intact bound eps was then dissolved in 25 ml of 0.5% nacl solution and placed in a water bath at 60°c for 30 min. the suspension was then centrifuged again at 12,000 rpm and 4°c for 15 min, and the supernatant was considered to be bound eps. the dry weight of the gross eps (in grams per liter) was measured by drying at 105°c for 8 h in an oven.(23) statistical analysis percent coefficient of variation (%cv) was calculated for the data of pure species biofilm biomasses as well as for theor etical and experimental biofilm biomasses. the onesample t-test (95% ci, alpha = 0.05) was used to calculate the column means (experimental and theoretical biofilm biomasses) which were strain name code escherichia coli a staphylococcus aureus b klebsiella pneumoniae c pseudomonas aeruginosa d table 1. bacterial strains used in this study and assigned codes 88 significantly different from the hypothetical value. the above statistical analyses were done by using the graphpad prism 7.00 program. ethical considerations the project was authorized by the bioethics committee of the technical university of manabí, portoviejo, ecuador (volume: 021-10 folio: 2110-017). results out of four pure species bacterial cultures (i.e. bacteria a, b, c and d), bacteria c showed the highest amount of biofilm biomass (a 590 = 0.1015) (figure 1), followed by bacteria d (a 590 =0.0793), bacteria a (a 590 =0.0735), and bacteria b (a 590 =0.059). the increment in the biofilm biomass of bacteria c was 28%, 38%, and 72% higher than bacteria d, bacteria a and bacteria b, respectively. highest (72%) and lowest (8%) biofilm biomass differences were found between bacteria b versus bacteria c and bacteria a versus bacteria d, respectively. with respect to pure-species biofilm biomasses, nevertheless, 8-72% of variation was found among the 4 bacterial species studied (figure 1). the coefficient variation percent (cv%) values of bacteria b, a, d, and c biofilm biomasses were 1.857, 3.726, 1.029, and 22.125, respectively. dual-species (1:1) biofilm biomasses (a 590 ) of 6 different combinations (bacteria a+b, a+c, a+d, b+c, b+d, and c+d) of four studied species were in the range of 0.0665-0.0905 (figure 2), where the combinations of bacteria a + bacteria b and bacteria c + bacteria d showed the lowest and highest amount of biofilm biomasses, respectively. based on the data prese nted in the figure. 1, the expected theoretical values of the biofilm biomasses were calculated for those 6 combinations of dualspecies cultures and compared with the experimental biofilm biomass values (figure 2). in four out of six du al-species cultures, experimental biofilm biomass values were higher (1-30%) than the theoretical values, but were lower (3-6%) than the theoretical values in the remaining two dual-species cultures (i.e. bacteria a+c and c+d) (figure 2). nonetheless, the highest difference between experimental and theoretical values was shown by the dual-species culture b+d, where the experimental value was 30% higher than the expected theoretical value. the lowest and highest %cv values were found to be 0.5297 and 18.6783 for the combinations a+b and b+d, respectively. in conclusion, a significant difference (p<0.0001) was found between the experimental and theoretical values of biofilm biomasses of different combinations. in a dual-species biofilm biomass, bacterial cell density or inoculum size (i.e. 10% (1:9), 50% (1:1), and 90% (9:1)) played a significant role in influencing the final biofilm biomass. this was investigated with dual-species biofilms, either of two gram-negative bacteria (a+c) (figure 3) or of a mixed culture of gram-negative and grampositive bacteria (b+c) (figure 4). experimental values of biofilm biomasses of the three different cell densities (1:9, 1:1, 9:1) were compared with the theoretical values calculated for the respective cultures based on the data in fig.1. when bacteria a density was 10% in a dual-species culture figure 1. pure species biofilm biomasses (a 595 ) of four different bacterial strains [escherichia coli (code a), staphylococcus aureus (code b), klebsiella pneumoniae (code c) and pseudomonas aeruginosa (code d)] as determined on 96-well microtiter plates. alava, maddela bacterial biofilms 89 univ med vol. 42 no. 1 containing bacteria a+c, the experimental biofilm biomass was 12.5% higher than the theoretical value. however, when the density of bacteria a was increased to higher values (i.e. 50% and 90%), the experimental biofilm biomasses declined significantly, and this decline was dosedependent on bacteria a culture. for instance, the decrements in the experimental biofilm biomass were ~6% and ~22% when the densities of bacteria a were 50% and 90%, respectively (figure 3). similarly, by increasing the percentage of bacteria b in a dual-species culture containing bacteria b + c, the experimental dual-species biofilm biomass was significantly reduced (figure 4). for instance, when bacteria b densities were 10% (1:9), 50% (1:1), and 90% (9:1), the experimental biofilm biomasses of the dualspecies culture of bacteria b + c were reduced by 19% and 4%, for bacteria b densities of 50% and 90%, respectively, and increased by 11% for the bacteria b density of 10%, over the theoretical biofilm biomass values for the same combinations (figure 1). finally, smps and epss were extracted from the pureand dual-species biofilms and their dry weights were determined (table 2), and the figure 2. theoretical and experimental values of biofilm biomasses (a595) of mixed culture bacterial species. stimulatory and inhibitory experimental values in relation to expected values were in the range of 1-30% and 36%, respectively. only bacteria b (staphylococcus aureus) showed stimulatory interaction with either bacteria a (escherichia coli), c (klebsiella pneumoniae) or d (pseudomonas aeruginosa) during biofilm formation. figure 3. deviation (per cent of decrease or increase) of experimental biofilm biomass of a dual-species (a+c at 1:1, 1:9 and 9:1 ratio) culture from theoretical value (a+c at 1:1). when bacteria a cell density was increased from 50% (1:1) to 90% (9:1), dual-species biofilm biomass was decreased by 28%, but biofilm biomass was increased when bacteria a cell density was decreased to 10% (1:9). (bacteria a = escherichia coli, bacteria c = klebsiella pneumoniae) different concentrations were determined in mixed species: 1:1 (culture 1 = 50% and culture 2 = 50% in the mixture); 1:9 (culture 1 = 10% and culture 2 = 90% in the mixture), and 9:1 (culture 1 = 90% and culture 2 = 10%). 90 discussion in the present investigation on the biomass of biofilms of four pure species (figure 1), it was determined that there was a greater formation of biofilms in the gra m negative bac ter ia escherichia coli and pseudomonas sp. there was a particularly greater biofilm biomass formation of 40% in klebsiella pneumoniae, while in the biofilm biomass of gram-positive bacteria the expected results were minimal. in the research directed by surgers et al.,(24) 57.1% of the bacteria were strong biofilm producers (biofilm formation index [bfi] < 2), while 13.4% lacked biofilm production (bfi >18). standard biofilm production (bfi <7) was common in e. coli isolates (61.9%). although almost all (90.2%) of k. pneumoniae have standard biofilm production, there was a 2.4-fold greater chance of observing biofilm. cangui-panchi et al.(25) determined that almost all staphylococcal species (such as s. epidermidis, s. saprophyticus, s. capitis, s. cohnii, s. hominis, s. aureus, and s. haemolyticus) showed a high prevalence of figure 4. deviation (per cent of decrease or increase) of experimental biofilm biomass of a dual-species culture (b+c at 1:1, 1:9 and 9:1 ratios) from theoretical value (b+c at 1:1). when bacteria b cell density was either 10% (1:9) or 50% (1:1), dual-species biofilm biomass was increased by 5%-20%, but biofilm biomass was decreased by ~10% when bacteria b cell density was increased to 90% (9:1). [bacteria b = staphylococcus aureus; bacteria c = klebsiella pneumoniae] pureor dual-species bacterial biofilm smp (g/l) eps (g/l) a 5.2 13 b 9.2 13 c 13 6.8 d 9.3 7.7 a+b (1:1) 10.6 10.0 a+c (1:1) 5.7 55.3 a+d (1:1) 6.4 7.1 b+c (1:1) 11.4 13.1 b+d (1:1) 12.1 9.2 c+d (1:1) 6.9 7.7 a+c (9:1) 6.2 77.6 b+c (9:1) 6.7 12.3 c+d (9:1) 12.9 8.2 a+c (1:9) 18.1 14.9 b+c (1:9) 7.3 6.0 b+d (1:9) 11.4 11.6 table 2. dry weight (g/l) of soluble microbial products (smp) and exopolymeric substances (eps) of pureand dual-species biofilms notes: a = escherichia coli; b = staphylococcus aureus; c = klebsiella pneumoniae; d = pseudomonas aeruginosa; smp: soluble microbial products; eps: exopolymeric substances biofilm-associated infections. furthermore, all gram-negative bacteria (e. coli, p. aeruginosa, k. pneumoniae, and p. mirabilis) reached 100% of biofilm prevalence except for a. baumannii evidencing 77.8% biofilm prevalence obtained in only one study. finally, candida. albicans and corynebacterium striatum also demonstrated a 100% biofilm prevalence, being the only yeast and gram-positive bacillus, respectively. (5) in the theoretical and experimental values of biofilm biomass obtained from mixed species (figure 2), between a and b there is a difference of 1%, while between a and d there is a difference of 2%, these representing minimum percentages. however, it was found that the difference between b and d is 30%, which is gr eater than in the other mixed species combinations. in the tables analyzed, the biomass species of mixe d biof ilms de ter mine the relationship between bacterial densities of 50% and 90%, indicating that the interaction and stimulation are greater, that is, the greater the bacterial density, the less will be the formation of biofilms. we char act erized the in vitro alava, maddela bacterial biofilms 91 univ med vol. 42 no. 1 polymicrobial biofilm of c. albicans and s. aureus, as they have been found together in biofilm-re lated infe ctions. we found the interesting the fact that fetal bovine serum (fbs) affects the adhesion of both microorganisms, as well as the formation of s. aureus biofilms and of polymicrobial biofilms. we suggest that plasma proteins, such as albumin and fibrinogen, may act as an interface, binding to biofilm-derived microorganisms on colonized catheters. c. albicans contributed the most to the overall biofilm density, in which β-1,6-linked polysaccharides app ear to be important components. finally, c. albicans adhered preferentially to all tested surfaces, while s. aureus mainly adhered to the surface of c. albicans. the nutrient requirements that may be involved in all steps of biofilm formation, such as adhesion, cell growth, extracellular matrix formation, and dispersal, need to be analyzed in the future.(26) during the interactions of p. fluorescens and s. aureus a s multispecies biofilms, as well as their behavior in the presence of carvacrol, it was found that p. fluorescens in monoand dual-species cultures exhibited similar numbers of planktonic cells (free flowing bacteria in suspension) and biofilm cells.(27) extracellular polymeric substances are of importance for the functions and characteristics of microbial aggregates in biological wastewater treatment, among which eps are involved in both beneficial and detrimental characteristics of microbial aggregates. specifically, eps are involved in biofilm formation and stability, sludge behaviors as well as sbr granulation, whereas they are also responsible for membrane fouling in membrane bioreactors (mbrs).(28) chitosan has the potential to be used in an immobilization technique for retaining biomass of caldicellulosiruptor species at a lower concentration of 1 mg l. this property was associated with natural biofilm produced by c. owensensis in the chemostat at higher maximum dilution rate (d).(29) unlike the present research, 200 μl of 0.1% crystal violet is added to each well and the microtiter plates are incubated at room temperature for 20 min. the biofilm layer is then dissolved by adding 150 μl of 33% acetic acid to each well. finally, acetic acid samples are diluted 50-fold (plus 33% acetic acid) on 96-well microtiter plates with transparent bottoms and black walls, and absorbance is measured at 590 nm in triplicate in a plate reader. the role of the biofilm three-dimensional structure in this tolerant phenotype has been studied extensively; however, the impact of small molecules released from biofilm bacteria in modulating host immune function is less well understood. a model of mixed-species biofilms composed of salmonella enteritidis (se) in the presence of the food-processing environmental bacterium bacillus paramycoides b5 responses to bioactive molecules released from either biofilm or planktonic bacteria. (30) the behavior of salmonella enteritidis (se) in the presence of the food-proces sing envir onmental bacterium bacillus paramycoides b5 (bp5) during dual-species biofilm formation was investigated by means of illumina rna-seq transcriptome analysis combined with phenotype validation. the results showed that se initial adhesion was significantly enhanced with large microcolony formation when cocultured with bp5.(30) this research describes how bacteria of mixed species interact with each other, and what leads to the formation of the biofilms that they can produce. in addition, raising awareness that bacteria are currently the main sources of diseases, therefore “one health” implies the interrelationship between public, animal and environmental health actors, taking into account that bacteria have the capacity and ability to adapt to the environment in which they f ind themselves.(30) to reach this goal it is important that day by day training and innovating technology is be ing updated to determine how the developmental process of bacteria changes according to the e nvironme nt a nd the circumstances in which they live. the major limitation of this study was our studying only four bacterial species. in order understand more about 92 the biofilm formation potentials of pathogenic bacteria, following future directions of research are greatly warranted – mixed species biofilms containing a wide range of non-model pathogenic bacteria; quantification of each bacterial species in mature mixed-species biofilms by using the sta ndard plate coun t (spc) method; and characterization of eps and smp by fourier transform infrared (ftir) spectroscopy for functional groups. conclusion in our investigation, klebsiella pneumoniae showed higher biofilm biomass (> 28-72%) than three other bacterial species. experimental values of dual-species biofilm biomasses decreased (by 6%) or increased (b y 30%) compared to theoretical values. we also found that cell density of a particular species significantly affects the biomass of dual-species biofilm. we did not find any correlation between either smp or eps with the biofilm biomasses of the four studied bacterial species. future research should be more in-depth and analytical on the interactions of mixed bacterial strains. acknowledgements mbsa thanks the instituto de posgrado, universidad técnica de manabí for providing laboratory facilities. declaration the authors declare that they do not have any type of conflict of interest. author contribution mbsa performed the work, prepared the draft of first submission and revised draft as per the editor/reviewers’ comments. nrm designed, supervised the work and edited the first and revised draft. all authors have read and approved the final manuscript. references 1. sánchez rea. complicaciones frecuentes en pacientes adultos relacionado con la traqueotomía percutánea en el área de cuidados intensivos del hospital alfredo noboa montenegro. 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dual-species biofilms. lwt 2022;156:113053. doi: 10.1016/j.lwt.2021.113053. oktavianus 171 *department of biochemistry, school of medicine, atma jaya catholic university of indonesia **department of parasitology, school of medicine, atma jaya catholic university of indonesia correspondence dr. jenny hidayat, m.biomed. department of biochemistry, school of medicine, atma jaya catholic university of indonesia jl. pluit raya no.2, north of jakarta, 14440 phone : +62-21 6693168 ext 203, 204, fax : +62-21 6606123 email: jenny.hidayat@atmajaya.ac.id univ med 2014;33:171-8 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background aspergillus flavus is one of the causes of aspergillosis, with a high virulence and resistance to standard antifungals, resulting in a high mortality rate. medicinal plants are increasingly used as they are relatively safer with minimal side effects. previously we found that the ethanol extract of neem (azadirachta indica a juss) leaves inhibits a. flavus growth in vitro. however, most chemical compounds with antifungal effect are nonpolar. the purpose of this research was to compare the antifungal effect of neem leaves extracted in a nonpolar solvent to that of leaves extracted in a polar solvent. methods an in vitro experimental research was conducted between october 2013 and january 2014. neem leaves were extracted in ethanol or hexane at various concentrations. a macrodilution test with 48-hour incubation time was done in triplicate on 8 groups of samples. these comprised the neem leaf ethanol extract (nlee) at 0.5, 1.0, and 2.0 g/dl, neem leaf hexane extract (nlhe) at 0.5, 1.0, and 2.0 g/dl, positive control, and negative control groups. fungal growth was detected on sabouroud dextrose agar. statistical analysis used chi square and fisher’s exact test. results nlhe had a higher, but statistically non-significant, inhibitory effect on a. flavus than nlee (p=0.996). at higher concentrations, the antifungal effect of nlhe is better than that of nlee. conclusion there is no significant difference in in-vitro inhibitory effectivity on a. flavus of neem leaves between extracts in polar and nonpolar solvents. keywords : azadirachta indica, neem, a. flavus, ethanol, hexane, antifungal hexane neem leaf extract more potent than ethanol extract against aspergillus flavus jenny hidayat*, ay ly margaret*, hanna yolanda**, and lies k. wibisono* 172 hidayat, margaret, yolanda, et al hexane neem leaf extract more potent ekstrak heksana daun mimba lebih potensial dibandingkan ekstrak etanol terhadap aspergillus flavus latar belakang aspergillus flavus merupakan salah satu penyebab aspergilosis. virulensi a. flavus yang tinggi dan resistensi terhadap antifungal standar menyebabkan angka mortalitas yang tinggi. penggunaan tanaman obat semakin meningkat karena relatif lebih aman dan minimal efek samping. hasil penelitian kami sebelumnya mendapatkan ekstrak etanol daun mimba menghambat pertumbuhan a. flavus in vitro. namun, senyawa kimia yang berefek antifungal lebih banyak bersifat nonpolar. oleh sebab itu, penelitian ini bertujuan untuk menilai efek ekstrak daun mimba dalam pelarut nonpolar lebih baik dibandingkan pelarut polar terhadap a. flavus. metode penelitian ini merupakan penelitian eksperimental in vitro yang dilakukan antara bulan oktober 2013 – januari 2014. daun mimba diekstrak dalam pelarut etanol dan heksana pada berbagai konsentrasi. uji makrodilusi dilakukan pada 8 kelompok percobaan, yang diulang sebanyak 3 kali, dan diinkubasi selama 48 jam. kelompok terdiri dari ekstrak etanol daun mimba (eedm 0,5; 1,0; 2,0 g/dl), ekstrak heksana daun mimba (ehdm 0,5; 1,0; 2,0 g/dl), kontrol positif, dan kontrol negatif. deteksi pertumbuhan jamur dinilai dengan menanam sedikit dari media – media uji tersebut pada agar dekstrosa sabouroud. uji statistik yang digunakan adalah uji chi square dan fisher’s exact test. hasil ekstrak heksana daun mimba memiliki efek penghambatan terhadap a. flavus yang lebih baik dibandingkan eedm namun tidak bermakna (p=0,996). pada konsentrasi yang lebih tinggi, efek antifungal ehdm lebih baik dibanding eedm. kesimpulan tidak ada perbedaan efektivitas yang bermakna antara ekstrak daun mimba dalam pelarut polar dan nonpolar terhadap pertumbuhan a. flavus in vitro. kata kunci : a. indica, neem, a. flavus, etanol, heksana, antifungal abstrak introduction aspergillus flavus is the second most common cause of aspergillosis, after aspergillus fumigatus. fungal infection caused by a. flavus is 100 times more invasive than that by a. fumigatus.(1) infection in aspergillosis occurs through inhalation of fungal spores. the clinical manifestations of aspergillosis are miscellaneous, from non invasive to invasive.(2) the incidence of invasive aspergillosis has been increasing.(3) the mortality rate of invasive aspergillosis in treated patients is 50%. standard antifungals is reported to have various side effects.(4) this encourages researchers to find natural sources of antifungal alternative treatment.(5) in recent years natural herbal compounds with antifungal properties have received the attention of researchers because they are natural and low risk s u b s t a n c e s f o r h u m a n h e a l t h a n d t h e environment.(6) medicinal plants are considered to be safer and have fewer side effects than chemical drugs.(3) neem (azadirachta indica a 173 juss) is known as one of the medicinal plants that can inhibit aspergillus growth. neem leaves have in vitro antifungal activity towards a. fumigatus and aspergillus niger.(3) for more than 2000 years, neem has been known as a medicinal plant with broad aspects of biological activity.(7) neem leaf extract can be used as an immunomodulatory, antiinflamatory, antihyperglycemic, antiulcer, antimalarial, a n t i f u n g a l , a n t i b a c t e r i a l , a n t i o x i d a n t , antimutagenic, and anticarcinogenic agent.(8,9) neem leaf extract has been reported to be effective for superficial fungal infection.(9) however, the information on the effectivity of neem leaf extract against a. flavus in systemic fungal infection is still limited. the result of our previous study was that the minimal inhibitory concentration of neem leaf ethanol extract (nlee) against a. flavus is 0.5 g/dl.(10) ethanol is a polar solvent. the biological compounds that can be extracted by polar and nonpolar solvent are different.(11) one of the nonpolar solvents is hexane. some studies have revealed that neem leaf extracts both in polar a n d n o n p o l a r s o l v e n t s h a v e a n t i f u n g a l activities.(11-13) however, the antifungal activities o f t h e e x t r a c t e d c o m p o u n d s m a y d i ff e r, depending on the nature of the compounds.(13) based on these considerations, the purpose of this study was to evaluate the comparative effectivity of neem leaves extracted by hexane and ethanol on a. flavus. methods research design this study was an experimental research study conducted at the biochemistry and parasitology laboratories of the medical school, atma jaya catholic university of indonesia, from october 2013 to january 2014. preparation of neem leaf extract neem leaves were obtained from the karyasari botanical garden, indonesia. fresh neem leaves were cut into small pieces and then allowed to dry for at least 2-3 weeks (no direct sunlight), then blended into powder. each of fifty grams of dried neem leaf powder was extracted with 200 ml of 95% ethanol and 200 ml of hexane by maceration for 72 hours. the extracted solution was evaporated by rotary evaporator with set temperature of 60-650c for hexane and 70-750c for 95% ethanol. the ethanol extract that still contained small amounts of alcohol was dried in a dessicator with silica gel. the extracts were stored in the refrigerator at 40c. fungal preparation a. flavus was cultured on sabouroud dextrose agar (sda) (difco, france) for 7 days at 37oc.(14,15) fungal colonies were suspended in 0.9% sterile nacl. the turbidity of this suspension was adjusted by spectrophotometry to obtain an optical density of 0.09-0.11 at 530 nm. this adjusted suspension was diluted 1:50 in sabouroud dextrose broth (sdb).(14,15) macrodilution assay the concentrations of neem leaf extract used in this study were 0.5 g/dl, 1.0 g/dl, and 2.0 g/dl, both for the ethanol and hexane extracts. the total volume of each group was 4 m l , c o n s i s t i n g o f 1 m l s d b , 1 m l dimethylsulfoxide (dmso) 1%, 2 ml fungal suspension of 0.09-0.11 od at 530 nm, 10 µl chloramphenicol 1 mg/ml (to prevent bacterial contamination from neem leaf extract), and neem leaf extract. the positive control was a solution consisting of 1 ml sdb, 1 ml dmso 1%, 2 m l f u n g a l s u s p e n s i o n , a n d 1 0 µ l chloramphenicol 1 mg/ml. the positive control was used as a standard of fungal growth without neem leaves. the negative control was a solution consisting of 1 ml sdb, 1 ml dmso 1%, and 10 µl chloramphenicol 1 mg/ml. all of these solutions were incubated for 48 hours at 37oc. antifungal activity of different extracts of neem leaves fungal growth was detected by culturing on sda after serial dilution.(16) serial dilution univ med vol. 33 no.3 174 hidayat, margaret, yolanda, et al hexane neem leaf extract more potent was conducted by diluting 0.1 ml solution in 0.9 ml 0.9% sterile nacl, and then taking 0.1 ml from the first dilution into 0.9 ml 0.9% sterile nacl. a volume of 20 µl from the second dilution was cultured on sda, with an incubation time of 72 hours at 37æ%c. the area that was covered by a. flavus was compared to the area of the petri dish. the results were defined as: no growth, growth of less than half of the petri dish, and growth of more than half of the petri dish area. the tests were done in triplicate. data analysis data was analyzed by spss 17, with chi square and fisher’s exact test. results fifty grams of dried neem leaf powder produced about 7.85 grams of nlee in 95% ethanol and 0.55 grams of neem leaf extract in hexane (nlhe). nlee was dark green in color, and nlhe was yellowish green. figures 1 and 2 show the effect of nlee and nlhe on growth of a. flavus at various concentrations. figure 3 shows the positive and negative controls. statistically, there were no significant differences between nlhe and nlee at identical concentrations (p=0.996) (table 1). there were also no significant differences among concentrations in nlee and nlhe (table 2). figure 1. a. flavus colonies in various concentrations of nlee a = nlee 0.5 g/dl (1) ; b = nlee 0.5 g/dl (2); c = nlee 0.5 g/dl (3); d = nlee 1.0 g/dl (1); e = nlee 1.0 g/dl (2); f = nlee 1.0 g/dl (3); g = nlee 2.0 g/dl (1); h = nlee 2.0 g/dl (2); i = nlee 2.0 g/dl (3) a cb fed g h i 175 figure 3. negative control (a); positive control (b) figure 2. a.flavus colonies in various concentrations of nlhe a = nlhe 0.5 g/dl (1); b = nlhe 0.5 g/dl (2); c = nlhe 0.5 g/dl (3); d = nlhe 1.0 g/dl (1); e = nlhe 1.0 g/dl (2); f = nlhe 1.0 g/dl (3); g = nlhe 2.0 g/dl (1); h = nlhe 2.0 g/dl (2); i = nlhe 2.0 g/dl (3) cb fed g h i a discussion according to some studies, neem leaf extract has antifungal effects.(11-13) neem leaves a b contain nimbin, nimbidin, azadirachtin, and tannins that have antifungal activity. in this study, nlee concentration was inversely proportional to the growth of a. flavus, although not univ med vol. 33 no.3 176 hidayat, margaret, yolanda, et al hexane neem leaf extract more potent table 1. differences on a.flavus growth between nlee and nlhe the experiments were done in tripilicate. n = number of replications with results denoted as “no of fungal growth”, “fungal growth on <50% of petri dish area”, or “fungal growth on >=50% of petri dish area”. nlee : neem leaf ethanol extract; nlhe : neem leaf hexane extract table 2. differences on a. flavus growth between several concentrations of nlee and nlhe nlee : neem leaf ethanol extract; nlhe : neem leaf hexane extract statistically significant (p=0.996). fungal colonies in nlee 0.5 g/dl were more abundant than in nlee 1.0 g/dl. at higher concentrations, the antifungal effect of nlee also increased. the density of the fungal growth at nlee 0.5 g/dl was higher than at nlee 2 g/dl. this may be due to uneven spread of fungal growth, because the growth was only at the edge of the dish and the number of colonies were less in nlee 2 g/ dl than nlee 0.5 g/dl. t h i s s t u d y u s e d t h e a n t i b i o t i c chloramphenicol at 1 mg/ml to prevent bacterial growth, which is consistent with the research of akpuaka et al.(12) this dose should be sufficient to inhibit the growth of bacteria. radhika et al.(8) used chloramphenicol 0.05 mg/ml, while jabeen (9) u s e d c h l o r a m p h e n i c o l 0 . 5 m g / m l . nevertheless, in all media there was still visible growth of bacterial colonies, although the media were given antibiotics. this may be due to the p r e s e n c e o f b a c t e r i a l r e s i s t a n c e t o chloramphenicol. at higher concentrations of nlee, there was less growth of bacterial colonies, with smaller diameters, than at lower concentration of nlee. this finding supports the research of biswas et al.(17) which stated that neem leaf extract has antibacterial properties. the solubility of nlee and nlhe in the m e d i a i s i m p r o v e d b y u s i n g d m s o . dimethylsulfoxide will bind to the fungal cell membrane and increase its permeability. in this study, we used 1% dmso, since according to hazen’s research the final concentrations of dmso used in in vitro antifungal experiments should be 0.5% and 1%.(18,19) randhawa (19) stated that dmso concentrations of 1% and below were safe to use in antifungal experiments. rauf et al.(20) showed that 0.45% dmso still can be used in antifungal tests. however, radhika et al.(8) used a higher concentration of dmso (5%) than the recommended ones. it has been reported that dmso concentrations of 2% and above slow the fungal kinetic growth.(18) therefore, there is the possibility that the fungal growth is 177 influenced by the concentration of dmso used, apart from the extract itself. in this study, the number of a. flavus colonies in nlhe were inversely proportional to the concentration. fungal growth was visible in nlhe 0.5 g/dl but not in nlhe 1.0 g/dl and 2.0 g/dl. bacterial growth was also inversely proportional to the concentration of nlhe. this may be due to the antibacterial effect of the neem leaves.(12,21) in this study, nlhe gave better results in inhibiting a. flavus compared with nlee, although the increase was not statistically significant. at nlhe 0.5 g/dl, fungal growth was less than nlee. at nlhe 2.0 g/dl, the diameters of bacterial colonies were slightly smaller than those at nlhe 0.5 g/dl and 1.0 g/ dl. according to verma et al.(21) neem leaf contains compounds that are antibacterial and antifungal.this allows an increase in the active antibacterial and antifungal compounds with increasing concentrations of neem leaf extract. in the present study, the inhibitory activity on the growth of a. flavus appeared to be higher for nlhe compared to nlee at various concentrations. however, no statistically significant differences were found between nlee and nlhe inhibitory activity. nlhe contains many bioactive compounds that have antifungal activity.(12) according jeyasakthy et al.(22) and kumar et al.,(23) neem leave have tannins, flavonoids, terpenoids, and alkaloids that are antifungal. these compounds can be found in neem leaf extracts using different solvents. mondali study showed that the antifungal compounds of neem leaf extract in non-polar solvents were better than in polar solvents.(11) akpuaka et al. identified 33 bioactive compounds in the hexane extract of neem leaves which have antifungal activity. our study found that nlhe activity in inhibiting the growth of a. flavus was better than that of nlee. in contrast, radhika et al.(8) stated that nlee was more effective in inhibiting the growth of fungi compared to nlhe. nlhe contains more terpenoids and diterpenoids than nlee.(12) terpenoid and diterpenoid compounds allegedly have high antifungal activity. rauf et al.(20) stated that the hexane fraction of some medicinal plants from pakistan has the highest antifungal activity, compared to ethyl acetate and methanol fractions. the results of our study differ from the results of radhika et al.(8) this may be caused by the difference in fungal species used. radhika et al.(8) used a superficial dermatophyte as test fungus, whereas our study used a. flavus. the growth of a. flavus in nlhe was less than the growth of a. flavus in nlee although not statistically significant. this may because of the inadequate number of trials, or because of contamination in the preparation of dmso and neem leaf extract. the clinical implication of this study is to provide information to clinicians and the community about the potential of neem leaves as systemic antifungal agent, especially about the discovery of a new natural and low-risk antifungal drug. future directions of this study are to investigate the potential of neem leaf extract as alternative treatment for lung cancer with pulmonary aspergillosis. conclusion there is no significant difference in effectivity of neem leaves to inhibit a. flavus in vitro, between leaves extracted in polar and nonpolar solvents. acknowledgement the research was funded by the atma jaya catholic university of indonesia (unika atma jaya 2013). references 1. rasmin m, syarifuddin pk, setiawati a, soemitro d, hamid a. sistem pernapasan. in: budimulyati u, bramono k, menaldi sl, editors. konsensus fkui dan pmki tentang univ med vol. 33 no.3 178 hidayat, margaret, yolanda, et al hexane neem leaf extract more potent tatalaksana mikosis sistemik. jakarta: balai penerbit fkui;2001.p.5-16. 2. thompson gr, patterson tf. pulmonary aspergillosis. semin respir crit care med 2008; 29:103-10. 3. bansod s, rai m. antifungal activity of essential oils from indian medicinal plants against human pathogenic aspergillus fumigatus and a. niger. world j med sci 2008;3:81-8. 4. hedayati mt, pasqualotto ac, warn pa, bowyer p, denning dw. aspergillus flavus: human pathogen, allergen and mycotoxin producer. microbiol 2007;153:1677-92. 5. radojevic id, stankovic ms, stefanovic od, topuzovic md, comic lr, ostojic am. antiaspergillus properties of different extracts from selected plants. afr j microbiol res 2011;5:398690. 6. ziveai f, afshari h, moghadam mm, sobhanipur a. study on antifungal effects of herbal essences on aspergillus flavus. bull env pharmacol life sci 2013;2:100-5. 7. paul d, kevin k. pulmonary aspergillosis: clinical presentation, diagnosis, and therapy. brit j biomed sci 2001;58:197-205. 8. radhika sm, michael a. in vitro antifungal activity of leaf extracts of azadirachta indica. int j pharm pharm sci 2013;5:723-5. 9. jabeen k. antifungal activity of azadirachta indica against alternaria solani. j life sci technol 2013;1:89-92. 10. li ma, yolanda h, wibisono lk. antifungal activity of neem leaves ethanol extract on aspergillus flavus. univ med 2013;32:104-9. 11. mondali nk, mojumdar a, chatterje sk, banerjee a, datta jk, gupta s. antifungal activities and chemical characterization of neem leaf extracts on the growth of some selected fungal species in vitro culture medium. j appl sci environ manage 2009;13:49-53. 12. akpuaka a, ekwenchi mm, dashak da, dildar a. biological activities of characterized isolates of n-hexane extract of azadirachta indica a juss (neem) leaves. n y sci j 2013;6:119-24. 13. moslem ma, el-kholie em. effect of neem (azardirachta indica a.juss) seeds and leaves extract on some plant pathogenic fungi. pak j biol sci 2009;12:1045-8. 14. clinical and laboratory standard institute. reference method for broth dilution antifungal susceptibility testing of yeasts; document m27a2. 2nd ed. pennsylvania: clsi;2002. 15. khan s, singhal s, mathur t, upadhyay dj, rattan a. antifungal susceptibility testing method for resource constrained laboratories. indian j med microbiol 2006;24:171-6. 16. canton e, espinel-ingroff a, peman j. trends in antifungal susceptibility testing using clsi reference and commercial methods. expert rev anti infect ther 2009;7:107-19. 17. biswas k, chattopadhyay i, benerjee rk, bandyopadhyay u. biological activity and medical properties of neem (azadirachta indica). curr sci 2002;82:701-11. 18. hazen kc. influence of dmso antifungal activity during susceptibility testing in vitro. diagn microbiol infect dis 2013;75:60-3. 19. randhawa ma. the effect of dimethyl sulphoxide (dmso) on the growth of dermatophytes. japanese j med mycol 2006;47: 313-8. 20. rauf a, khan a, rasool s, shah za, saleem m. in-vitro antifungal activity of three selected pakistani medicinal plants. middle-east j med pl res 2012;1:41-3. 21. verma a, joshi p, arya a. screening of eight plant extracts for their antimicrobial properties. int j curr microbiol app sci 2013;2:315-20. 22. jeyasakthy s, jeyadevan jp, thavaranjit ac, manoranjan t, srikaran r, krishnapillai n. antifungal activity and qualitative phytochemical analysis of extracts obtained by sequential extraction of some medicinal plants in jaffna peninsula. arch appl sci res 2013;5: 214-21. 23. kumar ps, mishra d, ghosh g, panda cs. biological action and medicinal properties of various constituent of azadirachta indica (meliaceae): an overview. ann biol res 2010;1: 24-34. c:\users\universa medicina\docu 52 abstract universa medicina pediatric skull bone defect due to epidermoid cyst robert sinurat* and fajar lamhot gultom** background tumors of the skull usually affect adult patients and less than twenty percent of pediatric patients. as the tumors grow, the surrounding bone may undergo destruction and erosion. when the tumors are located in the fontanel, the timely closure of the fontanel may be inhibited. epidermoid cysts are benign tumors that are intracranially located and very rarely in the midline of the cranium. meanwhile about 32% of stratified-squamous epithelial epidermoid cysts affect the head and neck region and only 6.7% occur in the scalp. we report an unusual epidermoid cyst located and growing in the anterior fontanel of a pediatric patient and inhibiting fontanel closure. case description a 21-month-old boy and his parents visited our polyclinic because there was a lump on his anterior fontanel since four months before they came to the hospital. the lump was initially the size of a peanut and had grown to become as large as a quail’s egg. the physical examination was normal and the brain ct-scan showed bone destruction without any intracranial lesion. the tumor was excised on the preoperative diagnosis of dermoid cyst. histopathology examination showed the characteristics of an epidermoid cyst. the cyst had a wall and was full of keratin flakes without hair follicles or sebaceous glands. follow-up one year after complete excision did not reveal any recurrence. conclusion epidermoid cysts in the skull of pediatric patients must be completely excised as soon as possible because their growth can damage the bone and inhibit the closure of the sutures. keywords: bone defect, fontanel, epidermoid cyst, malignancy, boy case report pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.52-56 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1053 cite this article as: sinurat r, gultom fl. pediatric skull bone defect due to epidermoid cyst adults. univ med 2021; 40:5 2 -6 . doi: 10 .18 051/uni vmed. 2021.v40.52-56 * surgery department, medical faculty, universitas kristen indonesia ** anatomic pathology department, medical faculty, universitas kristen indonesia correspondence: dr. dr. robert sinurat, spbs(k) medical faculty, universitas kristen indonesia jln mayjen sutoyo, cawang, jakarta timur, indonesia, 13630 tel: +62 21 8092425 email: robertsinurat@yahoo.com orchid id: 0000-0001-5511-3430 date of first submission, october 10, 2020 date of final revised submission, january 29, 2021 date of acceptance, february 6, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license univ med vol. 40 no 1 53 introduction the majority of patients with skull bone tumors are adults and only about 19% are pediatric.(1) most of the lesions are malignant, with the epidermoid cyst being one of the benign pathologic lesions. seventy five percent of epidermoid cysts are located intracranially, and the remaining 25 percent are within the diploic space of the skull.(2,3) nigam et al.(4) found in their study that 32% of all cutaneous and subcutaneous epidermoid cysts were located in the region of the head and neck, while 6.7% were in the scalp. around 23.3% of the patients was in the age range of 21-30 years, while 20.4% was in the age range of 41-50 years. patients aged under 10 years comprised less than 7%, while there were no patients under two years of age. epidermoid cysts in pediatric patients below two years of age and located in the midline of the skull are very rare. when the epidermoid cyst grows, the bone will be destroyed, thus inhibiting fontanel closure.(5) these cysts are often insidious and go unnoticed for years, if ever discovered. the commonest presenting complaint is the lump without neurological deficits. erythema, swelling and pain may occur because of inflammation.(2) the pathophysiology of these lesions is classically attributed to incomplete neural tissue cleavage as congenital defects of neural tube closure, leading to ectodermal cell inclusion.(3,6) early diagnosis and proper treatment will avoid the destruction of the bone. in addition to destruction of the bone or soft tissue surrounding the tumor, many authors also have reported that the epidermoid cyst can be transformed into a malignancy with an incidence rate of 1%.(2) before surgery, if the lump occurs in the scalp and does not mobilize when palpated or if there is a sign of pulsation, a brain computed tomography (ct) scan or magnetic resonance imaging (mri) examination must be performed to rule out intracranial lesions. on ct scan, the epidermoid cyst shows fluid density (hypodense), while there is low signal intensity on mri t1-weighted images but high intensity on t2-weighted images.(7) to d a t e , t he r e a re no r e p o r t s o f a n epidermoid cyst in a very young patient’s fontanel and inhibiting closure of the crown because of erosion of the inner and outer tables of the skull. here, we present a pediatric case of an unusual epidermoid cyst which is located in the fontanel, with invasion of the surrounding bone by the growing tumor. case report a 21-month-old boy “k” visited our outpatient clinic with his parents because there was a growing mass on his fontanel. his parents had noticed that the lump was the size of a peanut since four months earlier before they came to the hospital. the lump was becoming larger than before, and was now as large as a quail’s egg. the vital signs were normal and no neurologic deficits were found. in the anterior fontanel region, there was a mass under the intact skin that was 2x3cm in size, soft, elastic, and not mobile when palpated. blood examination results were within normal limits with no signs of infection. the brain ct-scan without contrast showed a bone defect (low density) in the midline (fontanel) region. there was no intracranial lesion (figure 1). figure 1. brain ct-scan shows bone defect in the midline univ med vol. 40 no 1 54 based on the physical examination, tumor location, laboratory and brain ct-scan results, excision of the tumor was performed on preoperative diagnosis of dermoid cyst. during surgery, after incision and dissection of the skin and fascia, the tumor was exposed, looking yellowish within the capsule inside the bone defect (figure 2). the dura mater was intact and the tumor was removed completely with unbroken capsule. histopathology examination showed that the cyst wall was formed by stratified-squamous epithelial cells. the cyst was full of keratin flakes without hair follicles and sebaceous glands (figure 3), which matches the diagnosis of epidermoid cyst. two days after surgery, the patient was discharged and observed for one year, when there was no recurrence of the tumor. discussion the authors report on a young patient with an epidermoid cyst on his fontanel that inhibited timely closure of the crown because of erosion of the inner and outer tables of his calvarium. the diagnosis of this case before surgery was a dermoid cyst because it was located in the midline of the head.(8) however, the results of histopathology examination after surgery showed the characteristics of an epidermoid cyst. epidermoid cysts are generally located at the cerebellopontine angle and parasellar region, with less than 5% be ing intra diploic and intraparenchymal.(9) both epidermoid and dermoid cysts are lined with stratified squamous epithelial cells. epidermoid cysts contain keratin, cholesterol and cellular debris, whereas dermoid cysts in addition contain skin appendages, such as hairs or glands.(10,11) epidermoid cysts generally occur in the third and fourth decades of life. this patient was under two years old and the cyst was not present at birth. bharti et al.(12) have reported three cystic cases in pediatric fontanel patients. two o f th em we re der mo i d cyst s a nd o ne a n epidermoid cyst, but all of the children presented the cysts from the time of birth. their patient with epidermoid cyst was 6 months old. a study on intradiploic epidermoid cysts by arko et al.(13) reviewed published case reports and series that can be obtained from an internet search engine. there were 169 cases with a mean age of 38.1 years. the pathophysiology of epidermoid cysts especially in children is still being debated. one widely accepted theory states that it is caused by inclusion of ectodermal remnants during the process of neural tube closure. another theory proposed for acquired-origin cases is that these cysts probably originate from implantation of epidermoid fragments into connective tissue by previous trauma. severe spinal or head trauma i s b e l i e ved t o b e th e c a u s e of a c q u ir e d intracr anial epider moid cysts. (3 ,6 ) lumbar epidermoid cysts can also occur because of skin figure 2. tumor lies inside the bone defect, looks yellowish and encapsulated figure 3. histopathology result shows keratin flakes (arrow) without hair follicle or sebaceous gland sinurat, gultom pediatric skull bone defect 55 c i mpl a n t a t i on d ur in g l umb a r pu nc tu r e procedures.(6) in the present case the epidermoid cyst did not appear at birth, therefore the acquired cyst theory is the most appropriate even though the patient’s parents do not remember any history of head trauma. epidermoid tumors on the brain ct scan show fluid density, while dermoid cysts show fat density.(11,14) on magnetic resonance imaging, epidermoid cysts are circumscribed lesions and well demarcated. the cyst wall has low signal intensities on t1or t2-weighted images. the mri signal of an epidermoid cyst depends on the contents, being intermediate to slightly increased on t2-weighted images, while t1weighted images show high signal intensity. when compared to muscle, the epidermoid cyst has higher signal intensities on t1-weighted images, which is characteristic of the cyst.(15) in the present case, the brain ct scan showed low density with bone defect without any intracranial lesions. the bone erosion is believed to be due to long-term pressure as the cyst slowly enlarges and damages the surrounding tissues, including the bone.(7) therefore, complete excision surgery is the treatment of choice.(5,11) complete excision of epidermoid cysts should also be performed effective and usually prevents c yst s f r o m r e c ur r i ng. ( 1 6) ho we ve r, i f t h e epidermoid cysts has an active infection the procedure must be delayed until the resolution of infection.(2) in most cases, epidermoid cysts managed by complete surgical resection have been cured, but 1% of stratified squamous epithelial epidermoid cysts become squamous cell or basal cell carc inomas (2) among all intracr anial epidermoid cysts cases, around 5.8% of patients have experienced recurrences due to incomplete rese ction, with an e stimate d 44% of the recurrences having been transformed into squamous-cell carcinoma.(13) several authors have also found intracranial epidermoid cysts that initially had been partially resected and s ub s e qu e nt l y t r a n s f o r me d i n t o t h i s malignancy.(17,18) the signs and symptoms of rapid progression of an epidermoid cyst must be considered as marking the transformation into malignancy. the mechanism of epidermoid cyst transformation into a malignant tumor is still unclear, but some authors suspect that chronic inflammation due to cyst rupture and also partial resection of the cyst are the risk factors.(19) the incidence of epidermoid cysts in a very young patient’s scalp and skull is indeed low, but every parent should be vigilant when finding a lump on their children’s head especially if it has a tendency to enlarge. conclusion epidermoid cysts in the skull of pediatric patients must be completely excised as soon as possible because their growth can damage the b on e a nd i nhi bit c losu r e of the sut ur es . regardless of differing courses of treatment, one thing is quite clear: the exact pathogenesis and behavior of epidermoid cysts in the central nervous system remains largely a mystery. acknowledgment i thank the patient and his parents for their approval and agreement to sign the consent forms. funding this research did not receive grants from any agency in the public or commercial sectors. conflict of interest none declared contributors ds contributed to preparing and writing the original draft. rs and flg did the review and editing. both authors have read and approved the final manuscript. univ med vol. 40 no 1 56 references 1. liu h, zhang x, zhang m, et al. skull bone tumor: a review of clinicopathological and neuroimaging characteristics of 426 cases at a single center. cancer commun 2019;39:8. doi:10.1186/s40880019-0353-0. 2. hoang vt, trinh ct, nguyen ch, chansomphow v, chansomphow v, tran ttt. overview of epidermoid cyst. eur j radiol open 2019;6:291301. doi: 10.1016/j.ejro.2019.08.003. 3. enchev y, kamenov b, william a, karakostov v. posttraumatic giant extradural intradiploic epidermoid cysts of posterior cranial fossa: case report and review of the literature. j korean neurosurg soc 2011;49:53-7. doi: 10.3340/ jkns.2011.49.1.53. 4. nigam js, bharti jn, nair v, et al. epidermal cysts: a clinicopathological analysis with emphasis on unusual findings. int j trichol 2017;9:108-12. doi: 10.4103/ijt.ijt_16_17. 5. oommen a, govindan j, peroor ds, azeez cr, rashmi r, jalal mja. giant occipital intradiploic epidermoid cyst. asian j neurosurg 2018;13:51417. doi:10.4103/1793-5482.181146. 6. kalfas f, ramanathan d, mai j, schwartz s, sekhar ln. petrous bone epidermoid cyst caused by penetrating injury to the external ear: case report and review of literature. asian j neurosurg 2012;7: 93-7. doi: 10.4103/1793-5482.98656. 7. lim j, cho k. epidermoid cyst with unusual magnetic resonance characteristics and spinal extension. world j surg oncol 2015; 13:240. doi 10.1186/s12957-015-0651-1. 8. lakhsmi v, thirumurthy ks, rabia s, chaitra v. intradiploic epidermoid cyst-a pearl within the skull. j diagn pathol 2015;9:38-42. doi.org: 10.4038/jdp.v9i2.7660. 9. law ekc, lee rkl, ng awh, siu dyw, ng hk. atypical intracranial epidermoid epidermoid cysts: rare anomalies with unique radiological features. case rep radiol 2015;2015:528632. doi.org:10.1155/528632. 10. reissis d, pfaff mj, patel a, steinbacher dm. craniofacial dermoid cysts: histological analysis and inter-site comparison. yale j biol med 2014;87:349-57. pmid:25191150. 11. gaivas s, rotariu d, dumitrescu g, iliescu b. intradiploic cyst of the skul : case report. romanian neurosurg 2011;xviii:1-5. 12. bharti p, gupta u, bharti sk. rare case of anterior fontanelle epidermoid/dermoid. j pediatr neurosci 2015;10:247-9. doi:10.4103/1817-1745.165679. 13. arko l, berry ct, desai as, weaver m. intradiploic epidermoid tumors of the cranium: case report with review of the literature. j neurol surg a cent eur neurosurg 2017;78:167-79. doi: 10.1055/s-0036-1585544. 14. choudhary g, udayasankar u, saade c, winegar b, maroun g, chokr j. a systematic approach in the diagnosis of paediatric skulls lesions: what radiologists need to know. pol j radiol 2019;84;e92-111. doi: 10.5114/pjr.2019.83101. 15. sakamoto a, okamoto t, matsuda s. characteristic mri findings of epidermal cysts categorized by size. open orthop j 2018;12:4628. doi: 10.2174/1874325001812010462. 16. di giustino f, pecci r, giannoni b, vannucchi p. cerebellopontine angle epidermoid cyst: case report. int j otorhinolaryngol head neck surg 2013;2:5-7. doi: 10.4236/ijohns/2013.21002. 17. agarwal s, rishi a, suri v, et al. primary intracranial squamous cell carcinoma arising in an epidermoid cyst-a case report and review of the literature. clin neurol neurosurg 2007;109:888-91. doi.org: 10.1016/j.clineuro.2007.07.026. 18. mascarenhas a, parsons a, smith c, molloy c, jukes a. malignant squamous cell carcinoma arising in a previously resected cerebellopontine angle epidermoid. surg neurol int 2017;8:186. doi: 10.4103/1817-1745.165679. 19. ding s, jin y, jiang j. malignant transformation of an epidermoid cyst in the temporal and parapontine region: report of a case and differential diagnosis. oncol lett 2016;11:3097100. doi: 10.3892/ol.2016.4368. sinurat, gultom pediatric skull bone defect cite this article as: wija ya nti ias, mahadewi npap, sudira pg, tini k, susilawathi nm, adnyana imo. head ache in healthcare workers related to personal protective equipment use in covid-19 referral hospital. univ med 2023 ;42 :52-60 . doi: 10.18051/ univmed.2023.v42:52-60 january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 headache in healthcare workers related to personal protective equipment use in covid-19 referral hospital ida ayu sri wijayanti1* , ni putu ayu putri mahadewi1 , putu gede sudira1 , kumara tini1 , ni made susilawathi1 , and i made oka adnyana1,2 abstract background studies show that wearing personal protective equipment (ppe) for long periods of time can lead to discomfort such as headaches, which could affect the performance of healthcare workers. the aim of this study was to determine the prevalence and risk factors of headaches related to ppe in healthcare workers at a covid-19 referral hospital. methods a cross-sectional study was conducted involving 174 healthcare workers in a covid-19 referral hospital in bali. we conducted interviews using a questionnaire that consisted of three main parts: characteristics of the subjects, ppe usage, and ppe-associated headaches. a multiple logistic regression was used to analyze the data. results the analysis results showed that the ppe-associated headaches had a prevalence of 63.8% and were gradual in onset, pressure-like in quality (46%), and mild in intensity (80.1%). ppe level iii-associated headache was the most common type. the majority of the participants had headaches up to 6 hours after using the protective gear, but improving within 15-30 minutes of removal and/or after pharmacotherapy. a chi squared analysis showed a statistically significant association between duration of ppe use, working units, and ppe levels (p<0.05). a logistic regression analysis found a significant relationship between ppe level and headache occurrence (or=4.826;95%ci: 2.433-9.572; p<0.001). conclusion the frequency of ppe-associated headache was high and the ppe level was a risk factor of headache among healthcare workers. better strategies are needed to reduce the duration of ppe exposure so that the work performance and quality of life of healthcare workers are not significantly affected. keywords: covid-19, personal protective equipment, headaches, healthcare workers 1department of neurology, faculty of medicine, udayana university/ udayana hospital, bali, indonesia 2prof.dr.dr.igng ngoerah/sanglah general hospital, bali, indonesia *correspondence: ida ayu sri wijayanti department of neurology, faculty of medicine, udayana university/ udayana hospital, bali, indonesia email: wijayanti@unud.ac.id orcid: 0000-0003-0995-3126 date of first submission, october 1, 2022 date of final revised submission, april 2, 2023 date of acceptance, april 6, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license list of abbreviations covid-19: corona virus disease 2019; ppe: personal protective equipment; nrs: numeric rating scale; n-95 mask: non-oil 95 mask; vas: visual analog scale 52 doi: http://dx.doi.org/10.18051/univmed.2023.v42:52-60 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1379 original article mailto:wijayanti@unud.ac.id https://orcid.org/0000-0003-0995-3126 https://orcid.org/0000-0003-4464-1419 https://orcid.org/0000-0002-3086-0496 https://orcid.org/0000-0002-5102-8033 https://orcid.org/0000-0002-3166-2440 https://orcid.org/0000-0003-3772-8970 53 univ med vol. 42 no. 1 introduction the use of personal protective equipment (ppe) is obligatory for the entire community, specifically healthcare workers who are treating covid-19 patients. hospitals require their healthcare workers and visitors to always wear a mask when in the healthcare facility. typically, healthcare personnel and visitors wear surgical masks, but while directly caring for covid-19 patients, healthcare personnel wear n95 masks. furthermore, the level of protection to be used is determined by the type of activity they carry out as well as the setting of their working units. they are advised to wear n95 aprons and masks instead of surgical masks while carrying out aerosolgenerating procedures. high-risk areas include intensive care units, special isolation rooms for infectious patients, emergency units, and operating rooms.(1) healthcare workers are people devoted to the health sector with knowledge and/or skills to provide healthcare services to patients. they are classified by law into several clusters and subgroups, namely medical staff, clinical psychologists, nursing staff, midwifery staff, pharmaceutical staff, public health workers, environmental worke rs, nutritionists, physiotherapists, medical technicians, biomedical engineering staff, traditional health workers and others.(2) the impact of prolonged wear of ppe is physiologic and psychologic distress and reduced work efficiency, such as difficulty breathing, headache, rashes, acne, and the disequilibrium syndrome. ppe-associated headaches can be attributed to mechanical factors, hypercapnia, and hypoxemia. tight straps and pr essure on superficial facial and cervical ner ves are mechanical f actors c apable of inflicting complications. cervical neck pressure, sleep problems, abnormal mealtimes, and emotional distress are among the different sources of headache in healthcare workers during ppe usage.(3) a previous study showed that ppe use by healthcare workers caused various complaints, such as skin damage, cognitive impairment, and headaches.(3) this finding is consistent with 2 previous studies in casablanca (morocco) and tehran (iran), where 62% and 72.4% of the respe ctive study s amples e xperienced headaches.(4,5) furthermore, the use of masks, specifically n95, increased the incidence of the side effects, which was then aggravated in 81% of worker s without a previous history of headaches. discomf ort around the ear accompanied by lack of hydration as well as conditions related to hypoxia/hypercapnia can cause headaches. the side effect is often experienced <60 minutes after use of ppe and improvement occurs within 30 minutes of removal.(6) previous studies showed that users can also feel pain due to external pressure in the neck (cervical) area, which then develops into a cervicogenic or tension-type headache.(6-9) a study among 268 healthcare workers from a covid-19 referral center in mexico, showed that 78.4% of participants were found to have ppe-associated headaches, and that the majority reported using mixed ppe, with stress as predictive risk factor for its conversion into chronic headache. (1 0) in contr ast, the aforementioned study in tehran (iran), which was a cross-sectional study involving 243 medical staff at four referral hospitals for covid-19, showed no relationship of ppe headache with bmi, marital status, age, blood pressure, or history of systemic or psychiatric disorders.(5) the 'de novo' headache occurrence, related to filter masks common among certain healthcare workers, has greater professional, familial, personal and social impact.(1,6) because of the need for ppe use by healthcare worker, especially during the covid19 pandemic, it is necessary to identify and eliminate the factors that lead to intolerance for these devices to help improve the performance of the healthcare workers. our objective in this study was to determine the prevalence and risk factors of ppe-associated headache in healthcare workers. 54 methods research design a cross-sectional study was conducted in udayana university hospital, which was used as a covid-19 referral hospital in bali, indonesia, from january to february 2022. research subjects a total of 174 participants in this study were recruited among healthcare workers providing healthcare services directly and indirectly to the patients in the hospital. they included doctors, nurses, pharmacists, midwives, radiology officers, and laboratory, and nutrition workers. healthcare workers who reported having regular usage of ppe during the covid-19 pandemic in their shift work, age between 20-40 years old, and able and willing to provide written informed consent, were included in this study. data collection demographic and headache characteristics of the participants were collected using a structured questionnaire. the questionnaire was written in the indonesian language and consisted of three main parts, namely: 1) characteristics of the participants, such as age, gender, working unit, working time, history and types of previous headaches; 2) characteristics of ppe usage including level, duration, location, type of ppe, and intensity; 3) characteristics of the headache experienced, such as onset, duration, type, persistence after ppe removal, reducing factors, and management. in this section, we used openended questions for participants to describe any headaches they might have. headache was classified ac cording to the international classification of headache disorders (ichd3).(11) headache severity was expressed in the numeric rating scale (nrs), which was used to assess transient pain severity using a 0-10 scale, with zero meaning "no pain" and 10 meaning "the worst pain imaginable". the scale is more practical than a vas and easier to understand for most people.(12) validation of the screening questionnaire was carried out in the neurology department, udayana university hospital. we used 8 hours as the cut-off point for the working time in accordance with the time regulation of work shifts in the hospitals that divides a working day into two or three work shifts. statistical analysis descriptive analysis was used to study baseline subject characteristics. categorical data were reported in frequencies and percentages. continuous data were reported as mean ± standard deviation if normally distributed and as median (interquartile range) if not normally distributed. the data were analyzed using the chisquare test, for differences in proportions and crude odds ratio. a multivariate logistic regression analysis was used to assess the main determinant associated with headache in healthcare workers after controlling for other determinants and calculating the adjusted odds ratio. a p-value of less than 0.05 was considered statistically significant. the data analysis was performed using the statistical package for social sciences (ibm spss statistics) for windows, version 22.0 (ibm corp., armonk, ny, usa). ethical approval this study was approved by the ethics commission of the faculty of medicine, udayana university, under r efer ence number 71/ un14.2.2.vii.14/lt/2022. the study was conducted in accordance with the principles of the declaration of helsinski. results this study included 174 healthcare workers as the participants, whose mean age was 28.18 years. eighty-four (48.3%) of the participants were male and 90 (51.7%) of them were female. the working time was up to 8 hours in 107 (61.49%) of the study participants. a total of 143 (82.18%) of them were working in a medical unit (isolation room, emergency room, outpatient clinic, mate rnity ward), whe reas 31 (17.82%) wijayanti, mahadewi , sudira headache in healthcare workers 55 univ med vol. 42 no. 1 variable n % mean age (years) 28.18 ± 3.86 gender male 84 48.3 female 90 51.7 working unit* medical 143 82.18 non-medical 31 17.82 working time (hours) up to 8 hours 107 61.49 more than 8 hours 67 38.51 previous headaches yes 25 14.4 no 149 85.6 type of the previous headaches migraine 15 60.0 tension-type headache 6 24.0 others 4 16.0 ppe level ppe levels i and ii 89 51.1 ppe level iii 85 48.9 ppe usage time (hours) 6.02 ± 3.83 headache during ppe usage yes 111 63.8 no 63 36.2 location of headache forehead 49 23.0 temple area 27 12.7 behind the eyes 14 6.6 back of the head 34 16.0 right side 36 16.9 left side 34 16.0 neck 19 8.9 type of headaches¥ pressure 51 46.0 stabbing 7 6.3 pulsating 29 26.1 rebound 19 17.1 achy 5 4.5 intensity of headaches mild 89 80.1 moderate 21 18.9 severe 1 0.9 type of headaches during ppe usage migraine 3 2.7 tension-type headaches 108 97.3 onset of headaches gradually 59 53.2 suddenly 11 9.9 mixed onset type 41 36.9 duration onset of headaches while ppe usage up to 6 hours 87 78.38 more than 6 hours 24 21.62 improving headaches after removed table 1. characteristics demographics of healthcare workers, ppe usage and headaches (n=174) data presented as n (%), except for age and ppe usage time; *medical unit = isolation room, emergency room, outpatient clinic, maternity ward; nonmedical unit = laboratory, radiology, laundry, pharmacy, nutrition; ¥ more than one answer; ppe: personal protective equipment 56 participants were working in a non-medical unit (laboratory, radiology, laundry, pharmacy, nutrition). pre-existing headache diagnosis was recorded in 25 (14.4%) participants, with the predominant type of previous headache being migraine in 15 (60%) of participants as shown in table 1. eighty-five (48.9%) of participants used ppe at level iii, 89 (51.1%) used ppe at levels ii and i, while mean ppe use by participants was 6 hours per working shift. the present study focuses on participants' experience of headaches when using ppe while working in a hospital in the covid-19 period. a total of 111 (63.8%) participants experi enc ed he adaches and predominantly had tension-type headache (97.3%). the majority described the headache as being in the forehead (23%), with pressurelike quality (46%), mild in intensity (80.1%), gradual in onset (53.2%), and up to 6 hours in duration (78.38%), while lying down or sleeping reduce d the headac he (51.4%). table 1 summarizes the demographic characteristics of the healthcare workers and the headache characteristics during ppe usage. from bivariate analysis (table 2) it was found that participants were divided into two groups, namely the group with headache and ppe usage that had 111 (63.8%) participants and the group without headache with 63 (36.2%) participants. the chi-square analysis showed that the working units, ppe levels, and duration of ppe use were significantly different between the two groups of participants, as shown in table 2. there were more subjects in the group who experienced headaches while wearing ppe who were working in medical units (p=0.01), had ppe level iii usage (p=0.001), and were using ppe for up to 6 hours per day (p=0.001). a logistic regression analysis was used to examine the association of the ppe levels, working units, and duration of ppe use with the headaches (table 3). the analysis found a significant relationship between the ppe level and headache occurrence (or=4.83;95%ci:2.439.57; p<0.001). these results indicate that when healthcare workers use ppe at level iii, they were four times more likely to experience headaches than those using ppe at lower levels. variables headaches (+) headaches (-) or (95% ci) p value age 1.07 (0.572.01) 0.833 up to 28 years old 67 64.4 37 35.6 >28 years old 44 62.9 26 37.1 gender 1.04 (8.36-56.66) 0.891 male 54 64.3 30 35.7 female 57 63.3 33 36.7 working unit** 4.0 (1.3012.30) 0.001* medical 106 66.7 53 33.3 non-medical 5 33.3 10 66.7 shift duration 1.08 (0.572.04) 0.810 up to 8 hours 69 64.5 38 35.5 >8 hours 42 62.7 25 37.3 ppe level** 0.21 (0.10 0.41) 0.001* ppe levels i&ii 42 47.2 47 52.8 ppe level iii 69 81.2 16 18.8 ppe duration** 3.09 (1.586.05) 0.001* up to 6 hours 87 71.9 34 28.1 >6 hours 24 45.3 29 54.7 previous headaches 1.55 (0.613.94) 0.356 yes 18 72.0 7 28.0 no 93 62.4 56 37.6 table 2. bivariate analysis of several risk factors of headache *p-values <0.05 considered statistically significant; data presented as n (%); or: odds ratio; ppe: personal protective equipment wijayanti, mahadewi , sudira headache in healthcare workers 57 univ med vol. 42 no. 1 final pdf artikel jurnal dan upload di web table 3. multivariable logistic regression analysis of independent factors and ppe usage patterns associated with the development of de novo headache note: ci: confidence interval; or: odds ratio; ppe: personal protective equipment; *p<0.05 considered significant discussion nearly 65% of the participants reported the occurrence of headache with the use of ppe at all levels (i, ii or iii). the ppe level i protection consists of the use of a mask (3-ply cotton cloth mask) with or without disposable gloves. the types of ppe used in level ii protection consist of a 3-ply surgical mask, gown, disposable rubber gloves, eye protection/face shield, and headcap. a radiographer's coat is used for healthcare workers who work in the radiology department. the ppe level iii protection consists of a 3-ply surgical mask, gown (when at risk of splashing body fluids), disposable rubber gloves, eye protection/face shield (when at risk of splashing body fluids) and head cap. (1, 13,14) the present study was based on the previous studies conducted by hajjij et al.(4) and ong et al.(6) both studies found that increased use of ppe during the covid-19 pandemic is responsible for generating headaches in frontline healthcare workers, whereas in our study, only individuals without a previous history of pre-existing headaches were found to be more susceptible to 'de novo' ppe-induced headaches. furthermore, the majority of healthcare workers were female, and this is consistent with indonesian 2019 and 2017 data on the distribution of the nurses' population, which is dominated by women.(2) similar studies in spain, korea, mexico and italy also reported that the majority of healthcare worke r s are female s. (1 ,1 0,13 ,1 5) progesterone and estrogen aff ect neurotransmitter systems and have been suggested to be dir e ctly involved in the pathophysiology of some primary headaches. this could be an important mechanism explaining why women are more susceptible than men.(10) the participants were grouped according to their work area, and this is in line with a previous study that the risk attached to their working unit determines the type of personal protective equipment. high-risk areas include intensive care units, special isolation rooms for infectious patients, emergency units, and operating rooms.(1) current guidelines require that those caring for patients, regardless of the patients' status, should wear ppe, resulting in an additional amount of time during which healthcare workers must wear protective equipment and obey the who protocols in work settings.(14) this study describes 'de novo' ppeassociated headache s amongst fr ontline healthcare workers throughout the current covid-19 outbreak. following ppe use, nearly 63.8% of the participants experienced headaches with different characteristics, pressure-like in quality (46%) and mild in intensity (80.1%), which were related to tension-type headache (97.3%). a similar result was obtained by a previous study of hajjij et al.(4) in marocco showing that 62% of healthcare workers suffered from headache associated with the use of ppe. in the study of jafari et al.(5) in iran, a prevalence rate of 72.4% was recorded among healthcare workers after the use of masks. the type of mask most commonly associated with headache was the n95 type. other studies revealed that facial pain or discomfort around the ear, lack of hydration as well as conditions related to hypoxia/hypercapnia can cause headaches. our study associated the incidence of headache with ppe usage, based on the prevailing ppe level in the hospital. we found that the use of ppe at level iii (81.2%) was associated with the occurrence of pperelated headaches and this was the type of protection used by 48.9% of the participants in our hospital. the distribution of protection levels 58 in this study was based on the type of health service and the actions taken by the workers. the n95 mask was used by healthcare and supporting personnel on duty in hospital. it was also used while performing aerosol-generating activities and respiratory sampling for suspected and confirmed covid-19 patients.(16) the abovementioned study by jafari et al.(5) in iran reported that 41% of all headaches experienced were related to the use of n95 masks. discomfort or pain is caused by the strong pressure exerted by the mask straps, specifically while using kn95 masks for a long time.(16,17) similar results were obtained by previous studies, namely that headaches can be caused by the external pressure from the ppe used.(9,10,18,19) another study also reported that external pressure was responsible for 25.1% of side effects related to the use of protective gear by healthcare workers.(5) pressure in the cervical area while using ppe causes pa in, whic h lea ds to cervicogenic or tension-type headaches.(7,8,13,20) furthermore, mask straps can cause local tissue damage as well as superficial nerve irritation in the face, head, and cervical region and an abnormal scalp sensitivity or discomfort during headache attacks elicited by non-noxious stimuli (allodynia), including the elastic head straps.(9,13) a mean ppe usage duration of 6 hours per working shift was obtained along with a mean working time of 12.46 ± 8.82 hours. a previous study reported that healthcare workers on duty for >12 hours per day are more susceptible to severe headaches.(4) the several research studies about ppe usage for >4 hours per day and those who had pre-existing headache analysis had a gr eater probability of developing such headaches.(6) the ratio of working time and usage duration in each study has a relationship with the emergence of headaches that are experienced after the use of ppe. about 24.3% of the respondents stated that the side effects appeared >120 minutes after they used the protective gear, while 28.8% revealed that the headache improved 15-30 minutes after ppe removal. a similar study reported that most of the headaches were felt <60 minutes after using ppe and pain relief occurred within 30 minutes of removal.(6) the differences in the results obtained were caused by the patients' condition as well as the type of ppe used. this study found a significant difference between the occurrence of headache related to ppe usage on the one hand, and the ppe levels, working units, and duration of ppe usage on the other. the problem of ppe-usagerelated headaches may also worsen if the covid-19 outbreak continues for a longer time. shorter duty shifts and the resultant shorter duration of ppe use is probably a better approach to avoid the adverse effects of ppe usage. various activities were carried out by the participants to reduce the headaches while using ppe, such as lying down or sleeping, massaging the head, continue with their duties, and applying cold or warm compr esses to the head. meanwhile, some healthcare workers did not take analgesics to relieve the adverse effects, although a previous study reported that 61.4% of the subjects who experienced headaches responded positively to the pain relief.(1) this study is not without limitations. the retrospective assessment of previous headaches and headaches while using ppe can lead to a recall bias and although a similar study in singapore (21) reported that an increase in the frequency and duration of severe headaches after using ppe was also associated with sleep deprivation, physical stress, emotional stress, late eating, and inadequate intake of water, these factors were not assessed in the present study. however, our study has its strengths, since it was conducted by filling out the questionnaire directly between doctor and participants, which ensures a good understanding of the questions and the correct interpretation of data results. our study was performed in a special hospital for covid19 isolation, where almost all healthcare workers on duty are involved in treating covid-19 patients and adapting to the ppe level. the results from this study are expected to serve as basic data for future studies with a broader sample population, considering that healthcare workers wijayanti, mahadewi , sudira headache in healthcare workers 59 univ med vol. 42 no. 1 in covid-19 referral hospitals have different workloads and stress levels. conclusion the frequency of ppe-related headache in covid-19 healthcare workers is high, with the healthcare workers being at risk to evolve to chronic and medication overuse headaches. the magnitude of this condition is clinically significant and might worsen if the current outbreak spreads widely and stays for a longer time, affecting the work performance of healthcare workers. better strategies are needed to reduce the duration of ppe exposure so that the work performance and quality of life of healthcare workers are not significantly affected. competing interests no relevant disclosures. acknowledgement the authors would like to thank all participants in this study and all the staffs of udayana university hospital in jimbaran, bali for their cooperation throughout the study. contributors iasw conceived the original idea of this research and proof outline. iasw wrote the manuscript with support, help and input from npapm, ipgs, km, nms and imoa. iasw, npapm, and km collected the samples and input the data. iasw with help from ipgs, nms, and npapm analyzed the data. iasw, imoa, npapm and ipgs also did the copyediting, proofreading and revised the final manuscript. all authors were involved in drafting the article or revising it critically for important intellectual content. all authors have read and approved the final manuscript to be published. references 1. ramirez-moreno jm, ceberino d, 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(covid-19). who interim guidance. geneva: world health organization;2020. 21. ong jjy, chan acy, bharatendu c, teoh hl, chan yc, sharma vk. headache related to ppe use during the covid-19 pandemic. curr pain headache rep 2021;25:53. doi: 10.1007/s11916021-00968-x. wijayanti, mahadewi , sudira headache in healthcare workers c:\users\user\documents\39 no 3 192 abstract universa medicina nephroprotective effect of soy (glycine max (l.) merr.) flour against diazinon-induced renal toxicity in rats desie dwi wisudanti*@, rena normasari**, and firman herdiana*** background diazinon is a widely used pesticide to eradicate agricultural pests globally with the potential to cause harmful effects on humans by generating free radicals and depleting endogenous antioxidants. soy flour possesses high antioxidant effect. this study evaluated the nephroprotective effect of soy flour on diazinon-induced nephrotoxicity in rats. methods an experimental laboratory study was carried out involving 30 male wistar rats were randomly divided into five groups of 6 rats. the normal control (kn) and the negative control (k-) groups received normal saline, the treatment groups (k1, k2, k3) received 10%, 15%, and 20% soy flour in distilled water, respectively, on day 1 to day 28. subsequently, for five days, all groups received diazinon 40 mg/kg, except for the normal control group. blood urea nitrogen (bun) and creatinine levels were measured at the end of the study. the kruskal wallis test with the post hoc mann whitney u test were used to analyze the data. results blood urea nitrogen and creatinine levels were lower in all treatment groups (k1, k2, k3) compared to the negative control group (k-), while the k3 level was the lowest (24.28 ± 1.56 mg/dl and 0.93 ± 0.14 mg/dl). there was a significant decrease in bun and creatinine levels in the experimental rats of the 15% and 20% soy flour groups (k2, k3) compared to the negative control group (k-). conclusion administration of soy flour (glycine max (l.) merr.) had a nephroprotective effect in preventing increases in bun and creatinine levels of diazinoninduced nephrotoxic male wistar rats. keywords: antioxidant, kidney, pesticide, functional food, bun, creatinine, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.192-198 september-december 2020 vol.39no.3 cit e th is article as: wi sud ant i dd, normas ari r, h erdi ana f. nephroprotective effect of soy (glycine max (l.) merr.) flour against diazinoninduced renal toxicity in rats. univ med 2020;39:192-8. doi: 10.18051/univmed. 2020.v39.192-198 *department of pharmacology, faculty of medicine, university of jember **department of pathology anatomy, faculty of medicine, university of jember ***undergraduate program, faculty of medicine, university of jember correspondence: @desie dwi wisudanti jl. kalimantan no.37, kampus bumi tegal boto, jember, jawa timur, 68121 phone. +62331 337877 fax. +62331 337877 email: desie.fk@unej.ac.id orcid id: 0000-0002-1636-7734 date of first submission, june 9, 2020 date of final revised submission, december 8, 2020 date of acceptance, december 10, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 193 univ med vol. 39 no.3 introduction organophosphate pesticides are a type of pesticide that is widely used in indonesia and is favored by farmers because it decomposes faster than the other groups of pesticides, and has a fast and significant power in repelling p e st s . ( 1 ) on e o f t h e mos t wi de ly u se d organophosphates is diazinon. it has been known that diazinon can be decomposed into other d er i va t ive f orms , l ea ves re sid ues in th e environment,(2) and has a high level of toxicity.(3) diazinon was reported to be able to inhibit acetylcholinesterase activity and other organic functions in animals.(4,5) the kidney is one of the organs affected because of its role in the excretion of diazinon metabolites, as evidenced by several animal studies that found an alteration in blood urea nitrogen (bun) and creatinine levels, as well as histopathological changes in rat kidneys.(4,5) in addition to reactive oxygen species (ros) accumulation, diazinon can cause deoxyribonucleic acid (dna) and cell damage through the apoptotic pathway.(6) continuous exposure to diazinon can affect kidney function,(7) therefore a substance that can inhibit ros activity and cell apoptosis in the kidney is needed. isoflavone is a class of flavonoid that has been known as an antioxidant that can inhibit cell oxidation reactions. (8) soy (glycine max (l.) merr.) has a high isoflavone content, which has been proven to reduce the concentration of ros and malondialdehyde (mda), as well as to activate superoxide dismutase (sod) and glutathione peroxidase (gpx) in several organs. (9) in addition, a previous study has proven that the soy isoflavones, namely daidzein and genistein, also have antiapoptotic properties.(10) there was also a study showing that bread made from 10% soy-flour-enriched wheat flour had a better effect on the kidney of rats than bread without the addition of soy flour. (11) in this study, rats were not induced by substances that could damage the kidney, so a little addition of soy flour could have a good effect on the kidney. another study on the effect of soy flour on the liver has been conducted in rats induced by carbon tetrachloride (ccl 4 ), (12) but only its effect on the liver was studied. the results showed a significant decrease in liver enzyme markers, but the value was still higher than in the normal group. the investigators used a wide dose range between treatment groups and a relatively short time of soy flour administration (7 days). therefore, a longer administration period with a narrower dose range needs to be evaluated and assessed based on kidney function examination. the present study was aimed at evaluating the protective effect of soy flour against diazinon nephrotoxicity in rats. methods research design the study of experimental design was conducted in the physiology and pharmacology laboratories, faculty of medicine, university of jember, from october to december 2018. animals and treatment physically active adult male wistar rats (rattus norvegicus), around three months of age and weighing 150-300 g, were purchased from “wistar farm” in malang, east java, indonesia. the rats were kept under constant conditions (temperature of 25 ± 2 °c and humidity of 60 ± 5% ) and were given rodent feed and water ad libitum. a total of 30 rats were involved in this study. the resource equation method (13) was used to calculate the sample size, where the number of samples per group was 5 for this study. the final sample size after the addition of an estimated 10% of animal mortality due to diazinon toxicity was 6 animals per group.(13) after acclimatization to the laboratory conditions, the animals were randomly divided into five groups (six rats in each): normal control group (kn), negative control group (k-), and treatment groups (k1, k2, k3). on day 1 to day 28, the normal control group (kn) and the negative control group (k-) received normal saline, and 194 wisudanti, normasari, herdiana soy against diazinon nephrotoxicity the treatment groups (k1, k2, k3) received 10%, 15% and 20% soy flour in distilled water, respectively. on day 29 to day 33, all groups were given diazinon 40 mg/kg, except for the normal control group. all treatments were performed using a gastric tube and diazinon was administered to those groups dissolved in corn o il . o n th e 3 4 t h d a y, t h e a n i ma ls we r e anesthetized, and blood samples were taken out by cardiac puncture to measure bun and creatinine levels. soy flour sample the soy flour was made from the baluran variety of glycine max (l.) merr. and was determined at the agronomy laboratory, faculty of agriculture, university of jember. the grinding method of soy flour used in this research wa s t h e wet me th o d, c a r r i e d o ut a t t he agricultural product process engineering laboratory, faculty of agriculture, university of jember. the grinding process was carried out on soy beans that had been soaked for 3 hours, then boiled for 5 minutes, drained, and sun-dried for 4 hours. the beans were then oven-dried at 50oc for 24 hours and ground to flour using a hammer mill, then sifted through an 80 mesh sieve. the sieving process was repeated twice to obtain an optimal quality of soy flour. then the soy flour was mixed with distilled water to obtain concentrations of 10%, 15%, and 20%. measurement of bun and creatinine levels the blood samples were centrifuged for 15 minutes at 3500 rpm to separate serum from blood cells, then bun and creatinine levels were d et e rmi ne d u s in g di a sys ® r ea gen t kits , according to the manufacturer ’s instruction. bun level was measured based on the method of urease-gldh (glutamate dehydrogenase) enzymatic uv test, and creatinine level was determined by a non-deproteinized kinetic test using jaffe’s method. the solution was read using an analyticon biolyzer 100 that had been set to an incubation time of 2 minutes and a wavelength of 340 nm for measuring bun level and a wavelength of 492 nm to determine creatinine level. the bun and creatinine serum levels were expressed in mg/dl units.(5) statistical analysis statistical analysis was performed using the spss 16 application for windows software. descriptive data were expressed as mean ± standard deviation. in the case the data obtained were not normally distributed, the nonparametric kruskal wallis data analysis was used for the comparative test, followed by post hoc mann whitney u. all data were displayed as the means ± standard deviation. the p-value <0.05 indicated the significance of the data. ethical clearance all experimental protocols have been approved by the ethics committee of the faculty of medicine, university of jember, no.1.268/ h25.1.11/ke/2018. results the mean bun and creatinine levels from the lowest to the highest were the kn group of 24.09 ± 1.34 mg/dl and 0.87 ± 0.4 mg/dl, k3 24.28 ± 1.56 mg/dl and 0.93 ± 0.14 mg/dl, k2 24.88 ± 1.91 mg/dl and 1.1 ± 0.09 mg/dl, k1 26.45 ± 1.93 mg/dl and 1.27 ± 0.19 mg/dl, and k28.77 ± 3.27 mg/dl and 1.31 ± 0.13 mg/dl. the lowest mean bun and creatinine levels were found in the normal control group (kn), indicating the smallest damage to kidney function, while the highest mean bun level was found in the negative control group (k-), indicating the most severe damage of kidney function. the mean bun and creatinine levels can be seen in table 1. statistical analysis was performed using the kruskal wallis test, and showed that the levels of bun (p=0.014) and creatinine (p=0.01) in the five groups were significantly different. the test was followed by the mann whitney u post hoc test as shown in table 2. the normal control group (kn) showed significantly lower 195 univ med vol. 39 no.3 a b bun and creatinine levels (p<0.05) compared to the negative control group (k-). the levels of bun and creatinine in the 15% and 20% soy flour supplementation groups (k2 and k3) decreased significantly when compared to the negative control group (p<0.05). between k2 and k3, there was a significant difference in creatinine level, but not in bun level. this study showed that giving 20% soy flour was more effective in reducing creatinine level. discussion in our study, the levels of bun and creatinine in the normal control group were significantly different from the negative control group. this indicates that the 40 mg/kg diazinon induction for five days was able to increase the mean bun and creatinine levels. diazinon can increase bun and creatinine serum levels because it has a pro-oxidant effect that induces oxidative stress and lipid peroxidation in the kidney. the result of this research is in line with several previous studies,(4,5,14) which proved that giving diazinon orally for several days in vivo can significantly increase the levels of bun and creatinine. shah and iqbal proved that giving diazinon 15 mg/kg orally for 8 weeks in mice can significantly increase bun and creatinine levels.(14) other similar studies proved that giving diazinon 20 mg/kg orally for 2 weeks in experimental rabbits can significantly increase bun and creatinine levels.(4) diazinon 40 mg/ kg orally given for 5 days increased bun and creatinine levels significantly.(5) the results indicated that diazinon exposure could decrease renal gluta thione , ca use the depletion of a n t i ox i dan t e n zyme s a nd t h e e xc e ss i ve production of oxidants. diazinon is a bs or bed from the gastrointestinal tract and metabolized rapidly. diazinon induces biochemical and histopathological changes in several organs.(4,5) one of the targeted organs is the kidney, which plays a major role in excretion. diazinon causes formation of ros and induces oxidative stress.(14) as mitochondria play an important role in apoptosis, mitochondrial dysfunction by oxidative stress will release cytochrome c and activate caspase that leads to apoptotic cells.(10) diazinon exposure may cause toxic effects through inhibition of acetylcholinesterase (ache) in the the nervous system.(15) this inhibition causes ach to remain at its receptors, which increase the treatment groups p value kn (n=6) k(-) (n=6) k1 (n=6) k2 (n=6) k3 (n=6) bun (mg/dl) 24.07 ± 1.34 28.76 ± 3.27 26.44 ± 1.93 24.88 ± 1.90 24.28 ± 1.56 0.014 creatinine (mg/dl) 0.86 ± 0.39 1.30 ± 0.13 1.26 ± 0.18 1.10 ± 0.09 0.93 ± 0.14 0.011 table 1. distribution of mean bun and creatinine levels by treatment groups after intervention data presented as mean ± sd; bun : blood urea nitrogen; kn: control; k (-): negative control; k1: 10% soy flour; k2: 15% soy flour; k3: 20% soy flour treatment groups kn kk1 k2 k3 bun creatinine bun creatinine bun creatinine bun creatinine bun creatinine kn 0.009 0.036 0.059 0.090 0.401 0.389 0.751 0.674 k0.009 0.036 0.175 0.523 0.012 0.008 0.012 0.009 k1 0.059 0.090 0.175 0.523 0.402 0.159 0.076 0.020 k2 0.401 0.389 0.012 0.008 0.402 0.159 0.462 0.033 k3 0.751 0.674 0.012 0.009 0.076 0.020 0.462 0.033 table 2. the results of mann whitney u test of bun and creatinine level kn: control; k (-): negative control; k1: 10% soy flour; k2: 15% soy flour; k3: 20% soy flour 196 p r o du c t i on o f no f r e e r a d i c a l s . t he accumulated no will be bound by gsh to gsn o, th er e by r e d uc i n g g sh l e ve l s . decreasing levels of gsh result in free radicals such as hydroxyl radicals (oh.), superoxide anion radicals (o 2 .-), and peroxyl radicals (roo) that cannot be neutralized. these free radicals r e a c t wi th u ns a t u r a t e d f a t ty a c id s or polyunsaturated fatty acids (pufas) which are found in many cell membranes, resulting in lipid peroxidation.(16) diazinon can also interf ere with the mitochondrial respiration pathway resulting in disruption of adenosine triphosphate (atp) production in the electron transfer pathway in the mitochondria by inducing the bh3-only protein that activates allosteric bax and bak, wh i c h i nt e r f e r e s wi t h mi t o c h on dr i a l p er me a bi l it y. ( 17 ) impai r ed mito c hon dr ia l membrane permeability releases proapoptotic f a c t or s su c h a s c yto c h r o me c , se c on d mitochondria-derived activator of caspase (smac), and apoptosis inducing factor (aif), which can lead to kidney cell degeneration and decreased kidney function.(18) ou r s t ud y s ho wed t ha t s oy f l ou r supplementation at concentrations of 10%, 15%, and 20% was able to prevent the increase in mean bun and creatinine levels in male wistar rats induced by diazinon. however, bun and creatinine levels of the 15% and 20% soy flour groups decreased significantly compared to the negative controls. this result was in accordance with a previous study on the nephroprotective effect of soy flour on the histopathological change in rat kidney induced by diazinon.(19) the aforementioned study proved that soy flour supplementation at a dose of 1 g/ml, 1.5 g/ml and 2 g/ml for 28 days had a significantly lower histopathological score compared to the group without diazinon. the group on 2 g/ml soy flour had the lowest renal histopathological score, approaching the normal control group score. in another study,(11) a diet of bread made from a mixture of wheat and soy flour at concentrations of 10%, 20%, 30%, and 40% for 28 days was able to prevent an increase in bun and creatinine levels compared to bread made from pure wheat flour. in that research, the rats were not exposed to nephrotoxic substances, wh e r e a s in t h e p r e s e n t s t udy, t he 10 % concentration and the negative control groups were not significantly different, indicating that the 10% concentration was not yet effective in preventing the increase in bun and creatinine levels. decreasing bun and creatinine levels oc cur due to th e pr esenc e of is ofl avone compounds, supported by vitamin e and other antioxidant substances such as phenol and linoleic acid found in soy flour.(11) these substances can prevent ros accumulation, lipid peroxidation, and cell apoptosis. soy flour is one of the types of processed soy containing high isoflavone levels.(20) isoflavone has biochemical activities as antioxidants by suppressing free radical activity and donating electrons and hydrogen atoms to inhibit cell oxidation reactions.(8) the two main isoflavones in soy are genistein and daidzein, which have antioxidant and anti-apoptotic activities by inhibiting the activity of bak so that they can inhibit cell apoptotic pathways.(10) vitamin e has a role in pr otecting unsaturated fatty acids in cell membranes, and inhibits the lipid autoxidation process by forming relatively stable tocopheroxyl radicals.(21) in this study, soy flour was given before diazinon induction to determine the effectiveness of soy flour protection against kidney damage before exposure to diazinon. based on the results of this study, the prevention of increased bun and creatinine levels occurred in the groups given soy flour. the results proved that the antioxidant and anti-apoptotic contents of soy flour could prevent diazinon-induced increases in bun and creatinine levels. these results are in line with research on the nephroprotective effects of pomegranate (punica granatum) seed oil (22) and juniper (juniperus procera) leaf extract.(23) these studies showed that the antioxidant-rich plants acted as protective agents wisudanti, normasari, herdiana soy against diazinon nephrotoxicity 197 univ med vol. 39 no.3 for renal function by preventing increased bun and serum creatinine levels in experimental animals induced by diazinon in rats and by thioacetamide in mice, respectively. t he a dmi nis t r a t i o n of s oy f lo u r a t concentrations of 15% and 20% showed a nephroprotective potential, as evidenced by the bun a n d cr e a t i ni n e l e ve l s , w hi c h we r e statistically significant compared to the negative control approaching the normal control group. however, 20% soy flour supplementation significantly reduced creatinine level compared to the 15% soy flour group, but not the bun levels, which were not significantly different between the 15% and 20% soy flour groups. these findings prove that administration of 20% soy flour is the most effective way to prevent the increase in creatinine levels induced by diazinon in rats. the results show that soy flour intake provides significant benefits for rats with nephrotoxicity induced by diazinon. the research results will have a major impact on improving the nutritional status and health of the community through regular consumption of functional foods such as soy flour. therefore, further research is needed to identify edible food forms that have a similar effect as potential nephroprotective agents. the limitations of this study are the uncontrolled external factors that can affect bun and creatinine levels. these factors include activity and diet. high activity with increased muscle mass will affect creatinine levels in the blood, as a result of the breakdown of phosphocreatine that occurs in skeletal muscles. meanwhile, urea is excreted as a result of protein metabolism, so that its levels in the blood are influenced by protein consumption and protein catabolism. overweight or obesity in rats can also affect kidney function, thereby affecting l e ve l s o f bun a n d s e r um c r ea ti n i ne . ( 2 4 ) therefore, the use of rats in this study should be carried out in a weight range that is not too wide and varied. the difficulty in this study is that there is a rapid increase in rat body weight during the adaptation period, so that the range of rat body weight is quite wide, namely 150300 grams. conclusions this study demonstrated that soy (glycine max (l.) merr.) flour has a nephroprotective effect on diazinon-induced nephrotoxicity in wistar rats by preventing the increase in bun and creatinine serum levels. apart from that, it was also concluded that 20% of soy flour provided the best overall quality. conflict of interest the authors declare that there are no conflicts of interest regarding the publication of this manuscript. acknowledgment the researchers would like to thank the faculty of medicine, university of jember, for the opportunity given in this research. contributors ddw contributed to the basic concept and design of the study. ddw and fh contributed t o pe r f 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risk factor dewi kristanti*, ekowati rahajeng*, eva sulistiowati*, nunik kusumawardani*, and frans dany* background type 2 diabetes mellitus (dm) is a non-communicable disease that constitutes a huge health burden, with the presence of comorbidities of dm adding to it. this study aimed to obtain the main determinants of the combined incidence of dm and its main comorbidities in adults. methods this was a further analysis of the non-communicable disease risk factor cohort study 2011 – 2018 involving 3730 subjects. data of diabetes-free respondents at baseline were followed up every 2 years for 6 years. data collection was carried out through interviews and health examinations. all subjects were assayed for blood glucose and lipid parameters. chi-square test and cox regression were implemented for data analysis. results during 6 years of follow-up, dm incidence occurred in 567 (15.2%) subjects. the most common comorbidities were increased low density lipoprotein (ldl), central obesity, increased total cholesterol, obesity and hypertension. most of the comorbidities occurred before the diagnosis of dm incidence. the determinants of the combined dm incidence–increased ldl are obesity, hypertension, and a family history of dm. the determinants of the combined dm incidence–central obesity are increased triglycerides, hypertension, male gender, and family history of dm. while the determinants of the combined dm incidence–hypertension are obesity and increased triglycerides. conclusion this study demonstrated a high burden of diabetes incidence with comorbidities among adults. knowledge of the magnitude of the diabetes comorbidity determinants emphasizes the role of non pharmacological intervention such as weight reduction and dietary modification. keywords: diabetes mellitus incidence, comorbidity, ncdrf cohort study, indonesia original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.3-13 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1035 january-april, 2021 vol.40no.1 ci te t his art icle as: kr ista nti d, ra hajeng e , su lis tiowati e, kusumawardani n, dany f. determinants of comorbidities of diabetes in indonesia: a cohort study of non-communicable disease risk factor. univ med 202 1;40:3-13 . doi : 10 .18 05 1/ univmed.2021.v40.3-13 *national institute of health research and development, republic of indonesia correspondence: dewi kristanti national institute of health research and development, republic of indonesia jalan percetakan negara no 29 jakarta pusat phone (021)4241921 email: dewikris@litbang.kemkes.go.id, find.dewi@gmail.com. orcid id: 0000-0002-9694-2064. date of first submission, september 1, 2020 date of final revised submission, december 24, 2020 date of acceptance, january 5 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 4 kristanti, rahajeng, sulistiowati, et al comorbidities of type 2 dm: ccohort study introduction diabetes mellitus (dm) constitutes a global and national health problem. throughout the world, dm occurs in 463 million individuals aged between 20 and 79 years. around 79.4 percent of them live in low and middle-income countries.(1) indonesia occupies seventh rank among the countries, with the highest number of dm patients of 10.7 million.(1) according to the results of the indonesian basic health research (riset keseh atan dasar, riskes das) the prevalence of dm based on blood sugar examination of persons aged 15 years was 6.9 % in 2013, increasing to 8.5 % in 2018.(2,3) the highest prevalence of dm was in persons aged 55 years and over, were of female gender, had low education, and resided in rural areas.(3) comorbidities of dm are one or more chronic conditions occurring in patients with dm or are other diagno sed diseases found concomitantly with dm.(4) all cases of chronic disease are considered to be comorbidities, even though occurring before or after the diagnosis of dm incidence.(5) comorbidities may be found at the time of diagnosis or at the time of followup.(6) according to the results of several studies worldwide, hypertension is the main comorbidity in dm, with a proportion of around 34.9 – 71.0%.(7-9) according to the indonesian society of endocrinology (perkumpulan endokrinologi indonesia), in indonesia the main comorbidities of dm can be in the form of hypertension, dyslipidemia, obesity and coagulation disorders.(10) based on a cross-sectional study involving patients with dm who were participants of the chr onic diseases manage ment program (program pe ngelolaan penyaki t kronik, prolanis) in a community health center (puskesmas) in jakarta, it was found that the propor tion of patie nts with dm and the comorbidity of obesity attained 63.9%.(11) based on the results of a retrospective observational study, concordant as well as discordant comorbidities in patients with dm were proven to increase healthcare utilization and costs.(12) comorbidities, particularly depression, also cause a decrease in the quality of life.(13) in addition, comorbidities increase the risk of complications, such as renal failure, and interfere with diabetic control.(14) the presence of comorbidities in diabetes mellitus and its risk factors have been reported in various studies.(15-17) however, in contrast with a previous study on the influence of the principal comorbidities such as hypertension, obesity and dyslipidemia, our study investigated their association with the time of occurrence of dm incidence in adults. the present study aimed to find the determinants of dm incidence and its main comorbidities in adult subjects. methods research design this study was a further analysis of the cohort study on ncd risk factors (studi kohor faktor risiko ptm), hereafter designated ncdrf cohort study, that was conducted by the national institute of health research and development for the years 2011–2018 in bogor city, west java. baseline data were collected in three stages, namely in the years 2011, 2012 and 2015, whereas periodic follow-up was performed every four months for a limited number of parameter s (data on blood pre ssur e and anthropometrics) and every two years for the complete set of parameters.(18) study subjects the study population comprised the subjects of stages 1 and 2 of the ncdrf cohort study (2011 and 2012 baseline data), whereas the study sample consisted of subjects who were free of dm on initial observation. the inclusion criteria were subjects who participated fully in the baseline data collection (in terviews and health examinations) and in the biennial follow-ups. each of the groups of subjects had been followed-up for 6 years, where the subjects of stage 1 were followed up in the years 2013, 2015 and 2017, 5 while the subjects of stage 2 were followed up in the years 2014, 2016 and 2018. the exclusion criteria were subjects with incomplete data for the study variables of interest. measurements dm incidence were defined as subjects who had ever been diagnosed with dm by health personnel, or who had a fasting blood glucose concentration of 126 mg/dl or a blood glucose concentration of 200 mg/dl two hours after loading with 75 g glucose in 250 ml drinking water. t he de finiti on of blood glucose concentration was according to the 2019 criteria of the indonesian society of endocrinology (perkumpul an endokrinologi i ndonesia, perkeni).(10) the comorbidities evaluated in this study were obesity, central obesity, hypertension, dyslipidemia, coronary heart disease, and stroke. the comorbidities could occur before, at the time of, or after the diagnosis of dm incidence and were determined from interviews, measurements, and ancillary examinations. obesity was defined as a body mass index (bmi) of 25 kg/m2 based on the criteria of obesity in asian populations according to the perkeni 2019 guidelines for dyslipidemia management,(19) whereas central obesity was defined as an abdominal circumference of >90 cm for males or >80 cm for females based on the perkeni 2019 criteria.(10) hypertension was defined according to the jnc viii criteria as a systolic blood pressure of 140 mmhg or diastolic blood pressure of  90 mmhg at rest. (2 0) dyslipidemia was defined as abnormal lipid metabolism, marked by an increase or a reduction in the plasma lipid fraction. the main lipid fraction abnormalities were increased total cholesterol (200 mg/dl), ldl (100 mg/dl), increased triglycerides (150 mg/dl), or decreased hdl (<40 mg/dl for males and <50 mg/dl for females).(10,19) coronary heart disease (chd) was defined as ischemic heart disease or infarction on the resting or active/stressed electrocardiogram (ekg).(18) stroke was defined as a history of stroke based on interviews and the finding of sequelae in the form of neurological deficits.(18) the independent variables consisted of the sociodemographic characteristics (age, gender, marital status, highest level of education, and family history of dm). age was in the form of continuous data, gender consisted of male and female, highest level of education consisted of low education (unschooled to finished elementary school), middle (finished junior and/or senior high school), and high (university graduate), marital status consisted of married, single, and divorced, and family history of dm was based on presence of family members who had been diagnosed with dm or had died as a result of dm. (18) the behavioral risk factors investigated were smoking status, physical activity, and mentalemotional abnormality. smoking status was defined as the smoking behavior in the last month, and was categorized into non-smoker, ex-smoker, and active smoker. physical activity was defined as the intensity of daily activities of living and was calculated from the weight of the type of physical activity and its duration. physical activity was categorized as adequate (600 met per week) and poor (<600 met per week).(21) mental-emotional abnormality was defined as the psychological condition of an individual that has an influence on mood, thoughts, and behavior and was measured by answering 20 items in a selfreported questionnaire (srq) that described the condition in the last two weeks. (22) data collection procedures data collection in the ncdrf cohort study was conducted according to the who steps appr oach for sur vei llance of ncd r isk factors.(23) the steps instrument covers three dif ferent levels of “steps” of risk factor assessment, consisting of questionnaires on demographic information and behavior al measurements, physical measurements, and biochemical measurements. collection of data on socio-demographics, behavior, and medical history was performed by trained enumerators in step univ med vol. 40 no 1 6 kristanti, rahajeng, sulistiowati, et al comorbidities of type 2 dm: ccohort study a b c 1, using a questionnaire that was specially developed for the indonesian ncdrf cohort study. in step 2 the blood pressure was measured 2–3 times with an interval of 5 minutes between measurements, using an and digital blood pressure monitor on the left upper arm with the subject in the sitting position. anthropometric measures were determined by measuring height with a fiberglass stadiometer, weight with and digital scales, and abdominal circumference using measuring tape. the determination of blood pressure and anthropometric measures was performed by trained health personnel from the bogor municipal health service. the neurological examinations to determine stroke sequelae was done by a team of neurologists. the ekg was recorded using electrocardiograph was provided with the minnesota code, and the ekg results were read by a cardiologist and validated by two cardiology consulta nts. t he labora tory examinations comprised fasting and 2-hour postprandial blood glucose, total cholesterol, triglycerides, low density lipoprotein (ldl), and high density lipoprotein (hdl) using enzymatic clinical chemistry methods and were performed in step 3 by a certified medical laboratory. statistical analysis descriptive analysis was used to for the subject characteristics at baseline and follow-up in the second, fourth and sixth years. bivariate analysis using the chi-square test was done to determine the distribution of dm incidence based on the presence of comorbidities and the time of comorbidity onset. multivariate analysis using the cox regression test was done to evaluate the determinants that play a role in the combined incidence of dm with the main comorbidities. variables with p values of <0.25 in the bivariate analysis were selected for further multivariate analysis. in the multivariate analysis, the variables that were statistically non-significant and were not confounders, were removed one by one from the model, so that the final model contained the adjusted hazard ratios of the independent variables that were determinants. a 95% confidence interval and a 5% level of significance was used to interpret statistical significance. research ethics the ncdrf cohort study was submitted annually for review by and ethical approval from the commission of health research ethics of the national institute of health research and development. all requirements for the subjects’ informed consent had been met and had been approved prior to data collection. the numbers of the ethical approvals were as follows: no.ke.01.08/ec/485/2011; no.ke.01.05/ec/ 394/2012; no.lb.02.01/5.2/ke.215/2013; no.lb-02.01/5.2/ke.143/2014; no.lb.02.01/ 5.2/ke.135/2015; no.lb.02.01/5.2/ke.042/ 2016; no.lb.02.01/2/ke.108/2017; and no. lb.02.01/2/ke.076/2018. results flow of subject selection the baseline samples in stages 1 and 2 comprised 5296 subjects. of this number, 467 subjects had dm at baseline and were removed from the samples, so there were 4829 subjects without dm. there were 1099 subjects that did not meet the criteria, so that the final sample for analysis was 3730 subjects. the flow of subject selection is depicted in figure 1. subject characteristics the subject characteristics are presented in table 1. initially there were 3730 subjects, then the attendance rates (response rates) decreased to 91.59%, 86.06% and 82.5%, respectively, during the follow-ups in the second, fourth and sixth years. the cumulative incidence of dm was 567 subjects (15.2%) in the six years of follow-up. according to socio-demographic characteristics, the mean age of the subjects at baseline was 43.7 ± 10 years, the majority were females, married, and had a medium educational level. the most frequent types of comorbidities found at baseline as well as at biennial followups were obesity, central obesity and increased 7 univ med vol. 40 no.1 ldl. based on the number of comorbidities, from the start of the data collection only less than 5 percent of subjects were free from comorbidities, more than half of the subjects had 1-3 comorbidities and almost 40 % of subjects had 4-6 comorbidities. during follow-up the proportion of subjects with 4-6 comorbidities increased to 51.4 % in the sixth year of followup. for the subjects with dm incidence, bivariate analysis was performed to determine the proportion of comorbidities occurring at each follow-up, as presented in table 2. it was found that ldl increased in nearly all subjects. obesity, central obesity and increased total cholesterol occurr ed in ar ound 80% of subjec ts. hyper tension, low ldl and increased triglycerides occurred in 57-78% of subjects, while comorbidities in the form of chd and stroke occurred in a minority of subjects. based on the time of appearance of the comorbidities, in table 2 are presented the proportions of occurrences of each of the comorbidities that are categorized as before, during, or after diagnosis of dm incidence. particularly for the follow-up results in the sixth ye ar, the proportio ns of occ urr ence of comorbidities after diagnosis of dm incidence could not be filled in because the available data were only up to the sixth year of follow-up. it was found that for obesity, central obesity, and hypertension, the largest proportion occurred before the diagnosis of dm incidence. the largest proportion of the increased total cholesterol, increased ldl and low hdl concentration occurred before diagnosis of dm incidence. meanwhile, the largest proportion of increased triglyceride concentrations occurred at the time of diagnosis of dm incidence. the comorbidity chd occurred most frequently at the time of figure 1. flow of sample selection in cohort study 8 kristanti, rahajeng, sulistiowati, et al comorbidities of type 2 dm: ccohort study diagnosis of dm incidence,, whereas stroke had different patterns at each follow-up. the principal comorbidities that were evaluated in multivariate analysis were the three types of comorbidities having the highest proportion in bivariate analysis, namely increased ldl, central obesity, and hypertension. the independent variables that were entered into the initial model were age, gender, educational level, marital status, family history of dm, smoking status, physical activity, mental-emotional abnormalities, obesity, increased triglycerides, and hypertension. all independent variables were taken from the baseline data. variables that were identical to the types of comorbidities under current analysis were not included. central obesity was not entered simultaneously into the initial model because it had a lower crude hazard ratio (hr) than obesity, while from among the parameters of dyslipidemia only the triglyceride concentration was taken because it had the largest crude hr as compared with the other parameters. the results of the multivariate analysis can be seen in table 3. the determinants in the combined dm incidence and increased ldl were obesity, hypertension, and family history of dm. the determinants in the combined dm incidence and central obesity were increased triglycerides, hypertension, male gender and family history of dm, whereas the determinants in the combined dm incidence and hypertension were obesity and increased triglycerides. table 1. distribution of subject characteristics by follow-up period data presented as n (%), except for age (mean ± sd) subject characteristic follow-up baseline n=3730 year 2 n=3416 year 4 n=3210 year 6 n=3077 incidence of dm (n) 157 129 281 age (yr) 43.7 ± 10.0 45.7 ± 9.9 47.6 ± 9.8 49.6 ± 9.9 gender female 68.5 70 70.7 71.1 male 31.5 30 29.3 28.9 marital status married 85.1 85.1 83.9 80.8 single 5.8 5.3 4.2 3.5 divorced 9.1 9.6 11.9 15.7 educational level high 6.4 6.3 6.5 5.8 middle 57.6 57.8 57.9 58.8 low 36 35.9 35.6 35.4 comorbidities obesity 45.5 50.7 51.8 55.3 central obesity 41.1 49.3 58.1 62.2 hypertension 29.4 29.7 28.5 37.5 increased total cholesterol 27.4 45.1 50.2 61.2 increased triglycerides 16.5 18.8 19.9 25.5 increased ldl 82.3 81.9 81.7 82.9 low hdl 39.4 38.7 38.8 35.1 chd 16 18.6 18.9 20.1 stroke 1.8 2.8 3.3 3.8 number of comorbidities none 3.9 4.3 4.6 3.3 1-3 53.1 51.3 48.8 41.6 4-6 39.9 41.9 44.7 51.4 7-9 2.7 2.5 1.8 3.7 9 univ med vol. 40 no.1 comorbidities time of appearance of comorbidities incidence of dm in year 2 (n=157) incidence of dm in year 4 (n=129) incidence of dm in year 6 (n=281) n % n % n % obesity none 26 1.7 23 1.6 59 4.6 before diagnosis 128 6.1 106 4.7 221 9.1 at time of diagnosis 2 6.1 0 0 1 4.8 after diagnosis 1 2.7 0 0 central obesity none 13 1 15 1.4 39 4 before diagnosis 141 6 112 4.3 240 8.8 at time of diagnosis 2 3.7 2 5.7 2 7.7 after diagnosis 60 1 0 0 hypertension none 37 1.9 34 2 60 4.3 before diagnosis 113 7 93 4.8 211 9.2 at time of diagnosis 4 5.5 1 2.7 10 20.8 after diagnosis 3 3.5 1 2.1 increased total cholesterol none 16 1.5 19 1.8 48 4.6 before diagnosis 107 5.8 90 4.2 209 8.7 at time of diagnosis 16 5.3 13 5.6 24 8.1 after diagnosis 18 3.4 7 2.4 increased triglycerides none 60 2.5 54 2.2 120 5 before diagnosis 58 9.4 46 5.2 131 12.1 at time of diagnosis 29 10.6 14 7.1 30 12.4 after diagnosis 10 2.3 15 6.2 increased ldl none 6 2.2 5 1.9 12 4.5 before diagnosis 145 4.7 117 3.6 262 7.8 at time of diagnosis 5 2.5 5 5.1 7 7.5 after diagnosis 1 0.5 2 2.2 low hdl none 44 2.9 38 2.5 94 6.2 before diagnosis 81 5.5 73 4 172 8.7 at time of diagnosis 23 6.1 6 4.5 15 6.2 after diagnosis 9 2.4 12 5 chd none 147 4.1 119 3.3 262 7.4 before diagnosis 0 0 6 7.3 10 8.1 at time of diagnosis 5 6.1 3 7.1 9 19.1 after diagnosis 5 5.6 1 2.1 stroke none 138 3.8 114 3.2 274 7.6 before diagnosis 8 15.4 10 12.5 4 4 at time of diagnosis 5 17.9 1 5.3 3 8.8 after diagnosis 6 11.3 4 11.8 table 2. type and time of appearance of comorbidities related to incident dm dm–comorbidity determinant adjusted* p value hr 95% ci dm–increased ldl obesity 2.37 1.95 – 2.87 <0.001 hypertension 1.51 1.25 – 1.83 <0.001 family history of dm 1.47 1.17 – 1.84 <0,001 dm–central obesity increased triglycerides 2.32 1.89 – 2.84 <0.001 male gender 2.23 1.74 – 2.85 <0.001 hypertension 1.77 1.45 – 2.16 <0.001 family history of dm 1.65 1.31 – 2.08 <0.001 dm– hypertension obesity 3.19 2.46 – 4.15 <0.001 increased triglycerides 1.78 1.39 – 2.27 <0.001 table 3. determinants of combined incidence of dm and main comorbidity *controlled for age at baseline 10 kristanti, rahajeng, sulistiowati, et al comorbidities of type 2 dm: ccohort study obesity increased the risk of occurrence of the combined dm incidence–hypertension and dm incidence–increased ldl, with hr values of 3.19 (95% ci: 2.45–4.15) and 2.37 (95% ci: 1.95–2.87), respectively, in comparison with the non-obesiity groups. males had a risk of 2.23 (hr=2.23; 95% ci: 1.74–2.85) for the combined dm incidence–central obesity when compared with females. for the lipid profiles, although increased triglycerides were not the main comorbidity in the incidence of dm, they posed an important risk for the occurrence of the combined dm incidence–central obesity as well as that of dm incidence–hypertension. discussion type 2 dm is one of the principal ncds in indonesia. the presence of the comorbidity factor in patients with dm increases the risk of future complications, interferes with diabetic control, and reduces the patient’s quality of life.(1,13,14) in the present study, only less than 5% of subjects did not have comorbidities from the start of the study up to the 6-year follow-up, signifying that nearly all subjects had comorbidities. this proportion is higher than that of other studies. for example in croatia, according to lang and markovic, in subjects with dm in the healthcare service, a total of 77.7% had comorbidities, with mean comorbi di ty of 1.6 ± 1.28.(24) in the netherlands, a cohort study found that according to the continuous morbidity registration data, ther e were almost 8 5% of patients with comorbidities at initial diagnosis of dm. around one-fourth of subjects without comorbidities at initial diagnosis of dm will suffer from at least one comorbidity in one year of follow-up.(5) based on the type of comorbidities, it was found that increased ldl occurred in nearly all of the subjects. obesity, central obesity and increased total cholesterol occurred in around 80% of subjects, whereas hypertension occurred in more than 70% of subjects. this finding associated with hypertension was similar to other study results, but for dyslipidemia and obesity the results were different. according to ekoru et al.,(9) the proportion of hypertension was 71%, but that of dyslipidemia was only 34% and that of obesity 27%. in other studies, hypertension was generally the main comorbidity in patients with dm. in korea, from the results of further analysis of patients’ medical records at keimyung university, dongsan, it was also found that the most common comorbidity was hypertension at 34.9%, followed by gastritis at 15.6% and senile cataract at 15.4%.(7) hypertension as the most frequent comorbidity was also found in the most recent studies, such as the study in england by nowakowska et al.,(6) the study on women of reproductive age in the us by britton,(8) and the study in sub-saharan africa by ekoru.(9) the finding associated with the high proportion of dyslipidemia merits special attention, in view of the fact that dyslipidemia is the primary risk for chd and may play a role before the appearance of the other main risk factors. in patients with dm, dyslipidemia results in a higher risk of coronary abnormalities.(20) the finding of chd as comorbidity of dm in the present study differs from the results of previous studies. only 6.4–7.8% of subjects with dm incidence had chd as comorbidity. in contrast, based on the results of a study in italy, from further analysis of several healthcare databases it was found that chd was one of the most frequent comorbidities of dm with a proportion of 21.7%.(4) the results of the study in croatia also state that the most frequent comorbidities as complications of dm were polyneuropathy, chd, retinopathy, nephropathy, non-healing wounds, and amputations.(24) based on the results of the multivariate analysis, the determinants of the combined dm incidence–incr eased ldl wer e obesity, hypertension, and family history of dm. the determinants of the combined dm incidence– central obesity were increased triglycerides, hypertension, male gender, and family history of dm, whereas the determinants of the combined dm incidence–hypertension were obesity and increased triglycerides. regarding the combined 11 dm incidence–increased ldl as well as the combined dm incidence–hypertension, the findings agreed with those of previous other studies that found increased bmi as one of the determinants. however, for the determinants of the combined dm incidence–central obesitythe present study found that the group of males had the higher risks, which is contrary to the other studies.(9) in the present study it was found that obesity increased the risk of combined dm incidence– hype rtension. obesity as deter min ant of hypertension in patients with dm is in line with the guidelines on the care of the comorbidities of dm by the american diabetes association.(25) in persons with diabetes there is a tendency of increased bmi as compared with healthy persons. the results of the nhanes longitudinal study in the us state that in the 20 years of study, among subjects with undiagnosed dm there was an increase in bmi from 31.5 kg/m2 to 34.3 kg/m2, whereas in subjects with diagnosed dm there was an increase in bmi from 28.1 kg/m2 to 32.6 kg/m2.(26) the presence of comorbidities of dm impacts on the occurrence of complications, such as cardiovascular disease. the accumulation of metabolic and cardiovascular risk factors in patients with dm may aggravate the condition and impede the attainment of therapeutic targets, which increases the risk of cardiovascular morbidity and mortality.(6) this is in line with the results of a systematic review conducted by einarson et al., (27) indicating that the proportion of patients with dm and chd was higher in the obese than in the non-obese and that there was a positive correlation between increased bmi and incr ease d prevalence of heart disease in individuals with dm. patients with dm have dysfunction of the blood vessel walls that may be triggered by environmental factors, such as sedentary life style, metabolic factors (chronic hyperglycemia, obesity) that is associated with increased regulation of reactive oxygen specie (ros), chronic i nf lammation, and univ med vol. 40 no.1 hypercoagulation, leading to atherosclerosis and cardiovascular disease.(6) based on the findings of the present study, a more comprehensive treatment of dm is necessary in patients with dm, i.e. regular taking of medications and having an increasingly healthy life style so that the treatment reaches its targets and reduces the risk factors of comorbidity. to this end, the follow-up of patients with dm at the community level should be increased through the ncd integrated health post (posbindu ptm) and household visits in the healthy indonesia through family approach program (program indonesia sehat melalui pendekatan keluarga, pis-pk). one of the limitations of the present study is the use of community-level data, so that there may be differences in results with those of other studies that use data from healthcare facilities. another limitation is the short follow-up period (only 6 years), and the dearth of data on other comorbidities, i.e. the complications of dm, the study of which is still in the planning stage. by knowing the extent of the problem of the comorbidities of dm and their determinants, it is hoped that public health interventions may be instituted to prevent the occurrence of these comorbidities of dm. it is necessary to conduct further cohort studies to determine the influence of comorbidities and the risk factors of the complications of dm, with the involvement of a larger number of associated determinants and longer observation periods. conclusions in the six years of follow-up, the incidence of dm was 567 (15.2%) subjects. the most frequent comorbidities were increased ldl, central obesity, increased total cholesterol, obesity and hypertension. these findings again highlight the importance of implementing an integrated approach for managing all these conditions since they are responsible for a substantial proportion of the burden of dm. 12 kristanti, rahajeng, sulistiowati, et al comorbidities of type 2 dm: ccohort study conflict of interest none declared acknowledgments we hereby extend our gratitude to the national institute of health research and development (badan pene litian and pengembangan kesehatan), of the bogor municipal health service (dinas kesehatan kota bogor), the teams of experts/consultants, investigators, data collectors, and all subjects of the ncdrf cohort study. contributors dk was the main contributor who was responsible for the initial design and data analysis. er and nk were supporting contributors in directing the data analysis and final manuscript. es and fd were supporting contributors in putting of the substance of the manuscript and its revision. all authors have read and approved the final version. references 1. international diabetes federation. idf diabetes atlas 9th 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dugas l, luke a, cooper r, durazo-arvizu r. increasing bmi and waist circumference and prevalence of obesity among adults with type 2 diabetes: the national health and nutrition examination surveys. j diabetes complications 2010;24:368–74. doi: 10.1016/ j.jdiacomp.2009.10.001. 27. einarson tr, acs a, ludwig c, panton uh. prevalence of cardiovascular disease in type 2 diabetes: a systematic literature review of scientific evidence from across the world in 2007– 2017. cardiovasc diabetol 2018;17:83. doi: 10.1186/s12933-018-0728-6 september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 anal human papillomavirus genotype distribution and its associations with abnormal anal cytology among men who have sex with men i ketut agus somia1 , tuti parwati merati1 , dewi dian sukmawati1 , ni wayan winarti2 , igaa elis indira3 , ida bagus dwija putra4 , and made yogi oktavian prasetia5 abstrak background anal hpv is associated with the severity of anal cytologic abnormalities that are precancerous lesions. knowledge of human papillomavirus (hpv) type distribution in populations at risk for anal cancer is needed. this study investigated anal hpv infections and cytological abnormalities among men who have sex with men (msm). methods a cross-sectional study was conducted involving 90 men aged > 30 years with a history of anal sexual intercourse with men. demographic characteristics and sexual behaviors were collected by using a selfcompleted questionnaire. anal cytological results were examined, and hpv genotyping was performed by the linear array hpv genotyping test. descriptive analyses of subject characteristics, prevalence, and 95% confidence intervals (ci) were performed. logistic regression was used to estimate risk factors and their associations with high-risk hpv infection and cytological abnormalities. results the overall prevalence of abnormal cytology was 32% (24/75), atypical squamous cells of undetermined significance (ascus) 17.56 % (13/75), 14.66% (11/75) were classified as low-grade sil (lsil) and no participant with high-grade sil (hsil). prevalence hpv infection in normal cytology was 86.27% (44/51), ascus 92.30% (12/13), and lsil 100% (11/11). the most common types of anal hpv in participants with cytological abnormalities are hpv 16, hpv 18 for high-risk hpv, and hpv 11, hpv 6 for low-risk hpv. conclusion high prevalence hpv infection in msm with abnormal anal cytology. a routine anal pap smear program and vaccination are needed to prevent hpv infection and anal dysplasia in msm. keywords: anal human papillomavirus, genotype, anal cytology, men who have sex with men. 1division of tropical and infectious diseases, department of internal medicine, 2department of pathology anatomy, 3department of dermatology and veneorology, 4departement of clinical microbiology, udayana university prof.dr,i.g.n.g ngoerah general hospital, denpasar, bali, indonesia 5nusa indah vct clinic, prof,dr,i.g.n.g ngoerah general hospital, denpasar, bali, indonesia *correspondence: i ketut agus somia diponegoro street, denpasar, bali phone/fax : 089617587075 email : agus.somia@unud.ac.id orcid id: 0000-0003-4168-9572 date of first submission, september 1, 2022 date of final revised submission, december 2, 2022 date of acceptance, december 13, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 246 doi: http://dx.doi.org/10.18051/univmed.2022.v41.246-253 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1368 original article cite this article as: somia ika, merati tp, sukmawati dd, winarti nw, igaa indira e, putra ibd, and prasetia myo. anal human papillomavirus genotype distribution and its associations with abnormal anal cytology among men who have sex with men. univ med 2022;41:246-53 http://dx.doi.org/10.18051/univmed.2022.v41.246-253 https://univmed.org/ejurnal/index.php/medicina/article/view/1368 https://orcid.org/0000-0003-4168-9572 https://orcid.org/0000-0001-9296-0428 https://orcid.org/0000-0001-7380-0437 https://orcid.org/0000-0002-4778-4848 https://orcid.org/0000-0002-4968-2319 https://orcid.org/0000-0001-6032-0610 https://orcid.org/0000-0002-8342-3433 247 univ med vol. 41 no 2 introduction anal intraepithelial neoplasia (ain) is a premalignant lesion of the anal mucosa that is a precursor of anal cancer. the risk factors for ain and anal cancer consist of clinical and behavioral factors associated with the acquisition and persistence of human papilloma virus (hpv) infection. risky anal intercourse behaviors include receptive anal intercourse,(1, 2) number of sexual partners,(3,4) and age at first anal intercourse.(1,5) a recent meta-analysis reported the prevalence of hpv in relation to the severity of anal cytological abnormalities.(6,7) in addition, it was also reported that the incidence of ain was higher in patients with hiv than in those without hiv.(8,9) a study among patients with hiv infection showed that 74.8% (294/393) were negative for intraepithelial malignancies (nilm) and 22.9% (90/393) exhibited squamous intraepithelial lesions (sil). in the latter category, 13.7% of samples (54/393) contained atypical squamous cells of undetermined significance (ascus), 6.9% (27/ 393) were classified as low-grade sil (lsil) and 2.3% (9/393) as high-grade sil (hsil).(10) a study in 714 hiv-infected men showed that on anal cytology, 24.5% (175/741) subjects had atypical squamous cells of undetermined significance (ascus) or higher grades of dysplasia, including 49.7% (87/714) with ascus, 41.7% (73/714) with low-grade squamous intraepithelial lesions (lsils) and 8.6% (15/714) with high-grade squamous intraepithelial lesions (hsils).(11) a cross-sectional study in china showed that the prevalence of any type hpv was 30.6% for heterosexual men, 74.1% for homosexual and 63.6% for bisexual men among hiv-positive participants, while the prevalence was 8.3%, 29.2% and 23.8% respectively among hivnegatives.(12) anal hpv infection is mainly transmitted through anal intercourse, which is usually considered as a major high-risk sexual behavior in men who have sex with men (msm).(13) among 163 msm, the overall prevalence of hpv was 66.2%. cytological findings showed that 15.3% had atypical squamous cells of undetermined significance, 16.6% had low-grade squamous intraepithelial lesion, 4.9% had atypical squamous cells that cannot exclude high-grade squamous intraepithelial lesions and 17% had highgrade squamous intraepithelial lesions.(14) however, most of the previous studies were mainly confined to hiv-seropositive msm. data comparing hpv prevalence among both hiv seropositive and seronegative msm is limited. therefore, this study aims first to determine the prevalence of anal hpv among msm with and without hiv, and second, to determine the prevalence of anal hpv among msm with and without anal cytological abnormalities in bali, indonesia. methods research design a cross-sectional study was conducted at nusa indah vct clinic of prof,dr,i.g.n.g ngoerah general hospital, which is a hiv and sexual transmitted diseases referral center in bali, indonesia, from june 22nd 2011 to february 13rd 2012. research subjects we screened all male patients who came to nusa indah vct clinic of prof. dr. i.g.n.g ngoerah general hospital from june 22nd 2011 to february 13rd 2012. males at least 18 years of age who reported having regular sex with men in the past 6 months were enrolled. eligibility criteria were as follows: (1) willing to provide blood samples for hiv and anal swab specimens for hpv genotype and cytology examination, and (2) able and willing to provide written informed consent. a total of 90 male patients who came to nusa indah vct clinic of prof. dr. i.g.n.g ngoerah general hospital were included in the study. data collection study subjects were interviewed at study entry using a structured questionnaire. the information collected included demographic 248 characteristics (age, educational level, and employment), and sexual behavior within the previous year. we used 30 years as cut-off point for age since the median age was 31 years. we used senior high school (grade 9) as cut-off point for education because of the 9-year compulsory education program. hiv serological determination blood samples were collected for hiv serological testing (anti-hiv). anti-hiv tests were conducted with 3 different rapid test methods i.e.vikia® hiv 1/2, hiv 1 & 2 antibody rapid test oncoprobe, and hiv intec ½ rapid test. hiv-positive status was defined by three reactive rapid tests results. positive samples were run in duplicate and verified by western blot hiv-1 and hiv-2 assays (new lav blot-i and ii; bio-rad fujirebio, tokyo, japan). anal specimen collection for cytology and hpv genotyping a trained physician collected anal swab samples for hpv genotyping and cytology examination by rotating a moistened and nonlubricated flocked swab in the anal canal without direct visualization. the collected samples were stored in the liquid cytology media (thin prep pap test preservcyt® solution) . anal swab specimens were then stored at -80o c in the molecular biology laboratory, faculty of medicine, udayana university, bali, indonesia. human papilloma virus genotyping was done using the linear array hpv genotyping test (roche molecular systems, inc., alameda, ca) in the clinical pathology laboratory of dharmais cancer hospital, jakarta, indonesia. the test amplified the target dna within the polymerase l1 region of the hpv genome (450 base pairs) by the polymerase chain reaction (pcr). the test then utilized nucleic acid hybridization to independently identify 37 anogenital hpv dna genotypes (6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69,70,71,72,73 (mm9), 81, 82 (mm4), 83 (mm7), 84 (mm8), is39 and cp6108). the anal cytology slides were interpreted by two pathologists. the cytological results were interpreted according to the bethesda system terminology as negative, atypical squamous cells of undetermined significance (ascus), lowgrade squamous intraepithelial lesion (lsil), atypical-squamous cells that cannot exclude high grade squamous intraepithelial lesion (asch) and high grade squamous intraepithelial lesion (hsil). hpv prevalence was defined as having a specific hpv genotype at enrollment visit. high risk (hr) hpv types (oncogenic hpv) are hpv with genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68.(11) low risk (lr) hpv types (nononcogenic hpv) are hpv with genotypes 6, 11, 26, 40, 42, 53, 54, 55, 61, 64, 66, 67, 69, 70, 71, 72, 73, 81, 82, 83, 84, is39, and cp6108.(11) statistical analysis we compared baseline characteristics between hiv seropositive and seronegative msm and tested differences in categorical variables using chi-squared or fisher’s exact tests, while continuous variables were analyzed using student-t or mann-whitney u tests. frequency distributions and descriptive statistics were used to characterize the study sample. data were analyzed using ibm spss statistics for windows, version 25.0 (spss inc., chicago, ill., usa). ethical clearance the study was approved by the institutional review board of kerti praja foundation with ethical clearance no 027/irb-ykp/2011. results there were 90 participants enrolled in this study; 11 out of 90 subjects did not attend when the specimen collection was performed. out of 79 specimens, 2 (2.5%) were invalid and 2 (2.5%) specimens were un-isolated dna specimens, i.e. their dna could not be isolated, and 75 (95.0%) were adequate specimens for data analysis. the mean and median ages of the participants were somia, merati, sukmawati hpv among msm with abnormal anal cytology 249 univ med vol. 41 no 2 a b table 1. demographic characteristics of hiv-infected and hiv-uninfected msm (n=75) note: hpv = human papilloma virus, msm : men who have sex with men table 2. anal intraepithelial neoplasia among 75 participants data presented as n (%) except for age and number of detected high-risk men (mean ± sd) note: hiv = human immunodeficiency virus; hpv = human papillomavirus characteristics hiv infected (n=47) hiv uninfected (n=28) p value age (years) < 30 17 (48.6 %) 18 (51.4%) 0.017 ≥ 30 30 (75 %) 10 (25%) education level low ( < 9 years) 20 (76.9%) 6 (23.1%) 0.052 high (≥9 years) 27 (55.1%) 22 (44.9%) age at first time anal sexual debut (years) < 18 30 (57.7%) 22 (42.3%) 0.149 ≥18 17 (73.9%) 6 (26.1%) number of sex partners in the last 6 months (median (min-max) 4 (160) 3 (1 75) 0.03 receptive anal intercourse in the last 6 months yes 9 (52.9%) 8 (47.1%) 0.25 no 38 (65.5%) 20 (34.5%) condom use in the last 6 months yes 22 (88%) 2 (12%) 0.001 no 25 (50%) 25 (50%) hpv positive 21 (31.3%) 46 (68.7%) 0.03 negative 26 (87.5 %) 1 (12.5%) cytology normal 30 (63.8%) 17 (36.2%) 0.229 abnormal 21 (75.0%) 7 (25.0%) characteristics normal cytology (n=51) abnormal cytology (n=24) p value age (years) 31.75 ( ± 7.82) 30.54 (±7.49) 0.53 positive hiv status (%) 30 (63.8%) 17 (36.2%) 0.229 education level high ( ≥ 9 years) 31 (63.3%) 18 (36.7%) 0.17 partnership status single/fixed partner 10 (55.6%) 8 (44.4%) 0.157 condom use 35 (70%) 15 (30%) 0.392 hpv dna detection 44 (65.7%) 23 (34.3%) 0.202 number of detected high-risk (mean) 1.61 (±1.297) 1.50 (± 1.31) 0.739 hpv high risk genotypes hpv16 15 (75.0%) 5 (25.0%) 0.312 hpv18 8 (50%) 8 (50%) 0.077 hpv 31 1 (50%) 1 (50%) 0.54 hpv 33 2 (40%) 3 (60%) 0.18 hpv 39 3 (42.9%) 4 (57.1%) 0.14 hpv 45 4 (80%) 1 (20%) 0.48 hpv 51 11 (57.9%) 8 (42.1%) 0.28 hpv 52 12 (70.6%) 5 (29.4%) 0.522 hpv 56 5 (100%) 0 (0%) 0.136 hpv 58 7 (70%) 3 (30.0%) 0.59 hpv 59 8 (88.9%) 1 (11.1%) 0.146 hpv 68 6 (85.7%) 1 (14.3%) 0.27 250 31.36 ±7.69 and 31 years, respectively. the demographic characteristics of the participant are shown in table 1, comprising data on age (<30 years), number of sex partners in the last 6 months, condom usage in the last 6 months, and hpv infection status as predictors for hiv infections. overall, 75 participants received anal cytology examinations. figure 1 shows the anal cytology findings. normal cytology findings were found in 51 participants (68%), ascus in 13 participants (17.33%), and lsil in 11 participants (14.66%). no hsil and asch was found. the prevalence of hpv inf ection in participants with normal and abnormal cytology was 86.27% (44/51) and 23 (34.3%), respectively. among the latter, there were 92.30% (12/13) ascus and 100% (11/11) lsil (table 2). the 6 commonest low risk anal hpv types seen in each cytology result group were as follows: normal cytology: hpv-70 (7/51; 13.72%), hpv62 (6/51; 9.8%), hpv-6 (5/51; 9.8%), hpv-53 (5/51; 9.8%), hpv-55 (5/51; 9.8%) , and hpv61 (5/51; 9.8%). low risk hpv infection in ascus: hpv-11 (3/13; 23.07%), hpv-54 (3/ 13; 23.07%), hpv-61 (3/13; 23.07%), hpv-6 (2/ 13; 20.37%), hpv-62 (2/13; 20.37%), hpv-72 (2/13; 20.37%) and hpv-73 (2/13; 20.37%). low risk hpv infection in lsil: hpv-62 (6/11; 54.54%), hpv-6 (5/11; 45.45%), hpv-11 (4/11; 36.36%), hpv-54 (3/11; 27.27%) , hpv-72 (3/ 11; 27.27%) and hpv-84. (3/11; 27.27%). in addition, the 6 commonest high risk anal hpv types seen in each cytology result group were as follows: normal cytology: hpv-16 (15/ 51), hpv-52 (12/51: 25,33%) , hpv-51 (11/51: 68%), hpv-18 (8/51: 21.33%), hpv-59 (8/51: 13.33%), and hpv-58 (7/51:12%) t he prevalence of low risk hpv infection in ascus was as follows: hpv-16 (3/13;23.07%), hpv18 (3/13;23.07%), hpv-51 (3/13;23.07%), hpv52 (3/13; 23.07%), hpv-39 (1/13; 7.69%), and hpv-45 (1/13; 7.69%). the prevalence of low risk hpv infection in lsil was as follows: hpv18 (5/11; 45.45%), hpv-51 (5/11; 45.45%), hpv-39 (3/11; 27.27%), hpv-35 (3/11; 27.27%), figure 1. prevalence of anal cytology results among men who have sex with men with and without human immunodeficiency virus infection note: ascus = atypical squamous cells of undetermined significance; lsil = low-grade squamous intraepithelial lesion somia, merati, sukmawati hpv among msm with abnormal anal cytology 251 univ med vol. 41 no 2 hpv-16 (2/11; 18.18%), hpv-52 (2/11; 18.18%) and hpv-58 (2/11; 18.18%). as can be seen in table 2, no characteristic can be used to determine abnormal cytology. discussion this study was conducted in a voluntary counseling and testing (vct) clinic setting in a referral hospital in bali, indonesia, that receives a mixed population, some of whose individuals are self-referred, whereas others are referred by their health-care providers for specialist care. the overall prevalence of hpv dna, low risk hpv and high risk hpv was 89.3%, 69.33%, and 81.33% , respe ctively. t he prevalence of abnormal cytology was 32%, of ascus 17.56% and of lsil 14.66%, but hsil was not identified. the absence of hsil detection in this study may be due to the limited sensitivity/ specificity for cytological detection of hsil,(4) to the high false negative rate in patients with hiv infection (5) or to sampling factors. in this study, the proportion of participants with hiv was 62.66% with 70.8% having cytological abnormalities. however, in high-risk groups such as msm, hiv or the immunocompromised, screening with anal cytology is still recommended.(5) the prevalence of abnormal cytology found in this study was lower than that of studies in india,(15) taiwan,(11,14) pakistan,(16) thailand,(17) australia,(18) and the united states of america.(19) in our study, we found that the prevalence of high-risk hpv was 82.35% for normal cytology, 76.92% for ascus and 81.81% for lsil. these figures are higher than in other studies.(6,7,11,14,20) in our study, the types of hr hpv most often found in all of the abnormal cytological categories were hpv-16, 18, 51 and 52. similar results have been reported in other studies, with hpv 16 and hpv 18 being the most common hr hpv found in abnormal cytological categories.(11,14,15) the prevalence of hpv16 and hpv51 increases with the severity of abnormal anal cytological findings.(14) of interest in this study was the large number of hpv 52 in abnormal cytology findings. the prevalence of low-risk anal hpv was 60.78% for normal cytology, 84.61% for ascus and 90.90% for lsil. this is similar to the results of a study in taiwan.(11) in our study, the commonest low-risk types seen in all abnormal cytology categories were hpv-11, 6, 62, 54. this is consistent with other reports stating that hpv 6 and hpv 11 are most often found in cytological abnormalities.(4,15) there are limitations to this study that should be considered. due to the heterogeneity in hpv types, larger sample sizes are needed to further elucidate the influence of specific hpv types or number of anal hpv infections on abnormal anal cytological lesions. lastly, due to its crosssectional design, from the study results it may be difficult to infer causality, since we could not establish causal temporality. this study has shown the high prevalence of abnormal cytology in those with high-risk hpv. therefore, this study has shown the importance of hpv vaccination to prevent abnormal anal cytology, such as was done in scotland.(21) conclusions this study demonstrated a high prevalence of hpv infection and abnormal anal cytology in msm. the most common types of hpv in participants with cytological abnormalities are high risk hpv (hpv 16, hpv 18) and low risk hpv (hpv 11, hpv 6). therefore, it is advisable to recommend routine anal pap smears and hpv vaccination in this group using the quadrivalent vaccine type (hpv 6, 11, 16, 18). conflict of interest none to declare. acknowledgement we would like to thank all participants in this study, the nurses at nusa indah clinic of prof.dr.i.g.n.g ngoerah general hospital, and 252 the staffs of the molecular biology laboratory and the pathology anatomy laboratory, both of udayana university – prof.dr.i.g.n.g ngoerah general hospital in denpasar, bali, as well as ms siriporn ninoy for facilitating team training, site visits, monitoring of anal swab specimen storage, and ms nittaya phanuphak and dr. evy yunihastuti for their cooperation and 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clinical epidemiology jl. percetakan negara 29 jakarta pusat 10560 phone: +6221 4259860, fax: +6221 4244375 email: dr.eva_s@yahoo.com univ med 2016;35:46-55 doi: 10.18051/univmed.2016.v35.46-55 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 ncep-atp iii and idf criteria for metabolic syndrome predict type 2 diabetes mellitus eva sulistiowati* and marice sihombing* background subjects with metabolic syndrome (mets) have a greater risk for acquiring type 2 diabetes mellitus (type 2 dm). the mets criteria usually used are those of the national cholesterol education program expert panel (ncep) and adult treatment panel iii (atp iii) and of the international diabetes federation (idf). this study aimed to evaluate the modified ncep-atp iii and idf criteria as predictor of type 2 dm among subjects with mets. methods a cohort study was conducted among 4240 subjects with mets. mets was determined according to the modified ncep-atp iii and idf criteria. the study followed up 3324 non-diabetic subjects of the cohort study of non-communicable disease (ncd) risk factors (ncd study) during a 2year period. type 2 dm was determined from the diagnosis by health personnel or from fasting blood glucose of >126 mg/dl or blood glucose of >200 mg/dl, 2 hours after 75g glucose loading. results the mets prevalence based on modified ncep atp iii and idf criteria in non-dm subjects was 17.1% and 15.6%, respectively. the risk for dm in subjects with mets using modified ncep atp iii and idf criteria was 4.7 (ci 95%: 3.4-6.5) and 4.1 (ci 95%: 3.0-5.7), respectively. conclusions both mets criteria can be used as predictors of the occurrence of dm type 2, but the modified ncep-atp iii is more properly applied than the idf criteria in subjects with mets. screening programs and routine monitoring of mets components are required for early detection of type 2 dm. keywords: metabolic syndrome, national cholesterol education program expert panel and adult treatment panel iii, type 2 diabetes mellitus doi: http://dx.doi.org/10.18051/univmed.2016.v35.46-55 47 univ med vol. 35 no.1 introduction the metabolic syndrome (mets) is one of a cluster of metabolic risk factors that include central obesity, hyperglycemia /glucose intolerance, hypertension, low hdl cholesterol levels, and high triglyceride levels.(1-3) the factors that affect the occurrence of mets are among others gender, age, race/ethnicity, genetic factors, obesity, food intake, physical activity, alcohol consumption and smoking habit.(4) the criteria for the definition of mets are variable, since there are different definitions of mets, depending on the respective health organizations, such as the world health organization, the national cholesterol education program expert panel (ncep) and adult treatment panel iii (atp iii), the european group for study of insulin resistance (egir) and the international diabetes federation (idf).(1-3) the who criteria emphasize the occurrence of insulin resistance with impaired fasting glucose (ifg), where fasting glucose is 100-125 mg/dl, or impaired glucose tolerance (igt) where blood glucose level 2 hours after loading with 75 g glucose is 140-199 mg/dl, or on measuring the homeostatic model assessment of insulin resistance (homa-ir) value, in which fasting insulin and fasting glucose are compared.(1) the ncep-atp iii criteria for the year 2004 are a combination of hyperglycemia, central obesity, atherogenic dyslipidemia and hypertension. these criteria are more frequently used because they are more simple and reliable compared with other criteria,(5) whereas the idf focus on the presence of central obesity, even in the absence of insulin resistance, together with 2 or more components. the cut-off point for central obesity depends on the population, ethnic group, and gender.(1) the mets prevalence is currently steadily increasing in many countries concomitant with the high rates of general and central obesity in the community.(6) in individuals with mets, the morbidity and mortality risks increase as a result of cardiovascular disease and diabetes mellitus (dm). several studies have shown that subjects with mets have a three-fold greater risk for experiencing myocardial infarction or stroke and a five-fold greater risk for suffering from type 2 dm.(7-13) the british regional heart study (brhs) and the prospective study of pravastatin in elderly at risk (prosper) in the uk have also shown that mets carries a 7.5and 4.4 times greater risk for the occurrence of dm.(10) a cohort study conducted by dekker et al.(11) for 10 years on non-dm subjects showed that mets has a 2fold increased morbidity and mortality risk as a result of cardiovascular disease. individuals having >3 mets components and central obesity have a 10-fold greater risk for dm.(12) these research results found different risks for dm, depending on the mets criteria used.(13) the mets prevalence based on the ncepatp iii criteria in jakarta is 28.4%,(5) whereas that based on the idf criteria among subjects of the cohort study of non-communicable disease (ncd) risk factors (3945 subjects) is 14.1%.(14) the risk of dm in non-dm subjects with mets in the cohort study of ncd risk factors have not been further analyzed. therefore, this study aimed to find the prevalence of mets based on the ncepatp iii and idf criteria and the risk of type 2 dm in respondents with mets among non-dm subjects. methods study design an observational study using a cohort approach was conducted from 2011 to 2014 in five kelurahan [villages], i.e. kebon kalapa, ciwaringin, panaragan, babakan and babakan pasar), central bogor district, bogor city. study subjects the study subjects were 25-65 year-old respondents of the cohort study of ncd risk factors (baseline data for the years 2011 and 2012), who were permanent residents of five kelurahan in central bogor district, bogor city, and whose data were complete. these baseline data had been collected with the who steps 48 method, comprising interviews, physical examination, and laboratory investigations.(14) the respondents with these baseline data were 5280 in number, but 468 of these individuals had type 2 dm (161 persons had been diagnosed as having dm by health personnel, 307 persons had undiagnosed diabetes mellitus based on their blood glucose levels), and 472 persons had not undergone laboratory tests or had incomplete tests. the respondents with type 2 dm who had not undergone laboratory tests were removed from the baseline data and the final number of respondents was 4340 persons (1518 males and 2822 females). the respondents with mets on the baseline data who had a follow-up in the second year, were to be subjected to an analysis of their risk factors for type 2 dm. the evaluation in the second year could only be performed on 81% respondents with mets (601 persons with mets according to the modified ncep-atp iii criteria and 549 persons with mets according to idf criteria). laboratory investigations venous blood samples to a volume of 8 ml were collected by experienced laboratory technicians from the respondents after a 10-12 hour fast. the collection of the blood samples was conducted in the center for applied health technology and clinical epidemiology in bogor. the blood samples were centrifuged at 5000 rpm for 3 minutes and subsequently the plasma was separated from the serum. from the serum samples, the blood glucose, total cholesterol, low density lipoprotein (ldl), high density lipoprotein (hdl) and triglyceride concentrations were determined at prodia laboratories, bogor. on the other hand, the plasma samples were sent to the biomedical and health technology center for determination of other laboratory indicators. b l o o d g l u c o s e c o n c e n t r a t i o n w a s determined with the glucose hexokinase ii (gluh) method, total cholesterol with a standard enzymatic method, ldl and hdl cholesterol was determined by a standard homogeneous method, while for triglyceride measurement the glycerol-3-phosphate oxidase (gpo) method was used. these investigations were performed with the hitachi model 747 automatic analyzer. the subjects were said to be at risk if they had a total cholesterol concentration of >200 mg/dl, triglyceride concentration of >150 mg/dl, ldl cholesterol >100 mg/dl, and hdl cholesterol <40 mg/dl for males and <50 mg/dl for females. a second blood sample of 1 ml was collected 2 hours post loading with 75 g glucose. these blood samples were handled similarly as in the blood glucose determination.(18) ncep-atp iii and idf criteria for the metabolic syndrome the metabolic syndrome was diagnosed according to the criteria of the national cholesterol education program expert panel (ncep) and adult treatment panel iii (atp iii), that were modified in 2004 and adapted for asians, and of the international diabetes federation (idf) for 2005. according to the modified ncep-atp iii criteria for asians, the definition of mets comprises the clinical condition meeting at least 3 or more than 5 risk factors, i.e. central obesity (waist circumference >90 cm for males and >80 cm for females), low hdl cholesterol (males <40 mg/dl and females <50 mg/dl, or under treatment), high serum triglycerides (>150 mg/dl, or under treatment), increased blood pressure (>130/85 mmhg or under treatment), and fasting blood glucose (>100 mg/dl or under treatment).(4) in contrast, according to the idf, the metabolic syndrome must have central obesity (modification for asians: waist circumference >90 cm in males and >80 cm in females), with 2 additional criteria from among the following: triglyceride level >150 mg/dl, hdl cholesterol (<40 mg/dl in males and <50 mg/dl in females), blood pressure >130/85 mmhg and fasting blood glucose >100 mg/dl.(16) criteria for type 2 dm type 2 diabetes mellitus was determined based on the results of interviews, in which the sulistiowati, sihombing metabolic syndrome predict type 2 diabetes mellitus 49 univ med vol. 35 no.1 subjects knew that they suffered from dm or had been diagnosed by health personnel and/or by laboratory determination of fasting blood glucose of >126 mg/dl and/or blood glucose level of >200 mg/dl, at 2 hours after loading with 75g glucose (ada criteria, 2003).(17) measurements the waist circumference was measured by means of measuring tape. the respondents were asked to stand upright with the feet together, and avoid wearing heavy clothing. the waist circumference was measured by extending the measuring tape around the abdomen from the midpoint between the lower ribs and the iliac crest. for a prominent abdomen, the most protruding part was taken for measurement. obesity was determined based on body mass index (bmi) calculated with the formula: weight (kg)/height (m)2. the subject was said to be obese if the bmi was >25.0 (according to recommendations of the indonesian ministry of health).(18) hypertension was determined based on interview results, in which the subjects knew that they had hypertension or had been diagnosed by health personnel, while their blood pressure measurements indicated hypertension according to jnc vii, and the subjects had a history of consuming anti-hypertensive drugs. measurement of blood pressure was performed in the sitting position on the right arm using a digital sphygmomanometer. the measurement was performed twice with an interval of ± 3 minutes, and if there was a difference of >10 mmhg between the two blood pressure measurements, either systolic or diastolic, then a third measurement was performed after a 10-minute resting period.(18) total cholesterol, triglyceride, low density lipoprotein (ldl) and high density lipoprotein (hdl) cholesterol concentrations were determined after a fast of 10-12 hours. the subjects were said to be at risk if the total cholesterol was >200 mg/dl, the triglyceride concentration was >150 mg/dl, ldl cholesterol >100 mg/dl, and hdl cholesterol <40 mg/dl for males and <50 mg/dl for females.(18) ethical clearance the baseline data collection and follow-up program of the cohort study of ncd risk factors obtained ethical clearance from the commission of health research ethics, health research and development agency, ministry of health, republic of indonesia. data analysis multivariate analysis was performed and agreement between ncep atp iii and idf criteria was determined using the kappa (κ) statistic. the interpretation of the kappa agreement was as follows: slight if κ= 0-0.20; fair if κ= 0.21-0.40; moderate if κ= 0.41-0.60; substantial if κ= 0.610.80; almost perfect if κ>0.80.(12,19) results non-dm respondents with mets in the baseline data based on the ncep-atp iii criteria were 743 in number (17.1%), while those based on idf criteria were 676 in number (15.6%). females had a higher proportion of mets than males, based on ncep-atp iii (21.1% and 9.7%) as well as on idf criteria (11.2% dan 5.9%). the metabolic syndrome occurred at the mean age of 47 years. mean waist circumference, bmi, ldl cholesterol, total cholesterol, and systolic and diastolic pressures in respondents with mets were greater as compared with residents without mets, either based on ncep-atp iii or idf criteria, whereas hdl cholesterol was lower (table 1). all respondents with mets based on idf criteria were also found to have mets based on ncep-atp iii criteria, while 9% of respondents who were said to have mets according to ncepatp iii criteria were considered to have no mets according to idf criteria. kappa agreement between ncep-atp iii and idf criteria was 0.94 (p<0.001; ci 95%: 0.93-0.96) or very good (almost perfect). figure 1 shows that the percentage of mets based on ncep-atp iii as well as on idf criteria increased concomitantly with increasing age, as was also the case with the number of mets 50 table 1. distribution of characteristics of non-dm subjects at base-line (n=4340) components. based on ncep-atp iii criteria, the respondents in the age group of 25-35 years had only 3-4 mets components, but starting with the age of >36 years the respondents had 5 mets components. similarly with the idf criteria, the age group of 25-35 years had central obesity with 2-3 mets components only. respondents with mets who participated in the follow-up accounted for 81%. the observational results showed that there were 78 cases with type 2 dm (13.0%) among the respondents with mets according to ncep-atp iii criteria, while among the respondents with mets according to idf criteria, there were 69 cases with type 2 dm (12.6%). respondents who experienced type 2 dm had significantly higher mean values for age, triglyceride, hdl, ldl, total cholesterol, fasting blood glucose and 2-hour pp blood glucose, compared with subjects without dm (p<0.05). on the other hand, waist circumference and bmi did not show significant differences. these results can be seen in table 2. figure 1. percentages of metabolic syndrome components by age group atp iii criteria idf criteria sulistiowati, sihombing metabolic syndrome predict type 2 diabetes mellitus 51 univ med vol. 35 no.1 a s m a y b e s e e n i n ta b l e 3 , a f t e r multivariate analysis, respondents with mets according to ncep-atp iii criteria had a 4.7 times greater risk for experiencing type 2 dm when under observation in the second year (ci 95%: 3.4-6.5), while respondents with mets based on idf criteria had a 4.1 times greater risk (ci 95%: 3.0-5.7). the risk of type 2 dm increased with increasing age. the age group of 35-43 years had a 2.6 times greater risk (ci 95% 1.2-5.6), while the age group of >44 years had a 4.3 times risk (ci 95%: 2.1-8.9). the sm component having the greatest influence on the occurrence of type 2 dm was the blood glucose level of >100 mg/dl. respondents with fasting glucose level of >100 mg/dl had a 4.6 times greater risk of dm (ci 95%:3.0-7.1). other mets components that carried a risk of type 2 dm were central obesity, blood pressure of >130/ 85 mmhg, hypertriglyceridemia, and low hdl cholesterol level. discussion the results of the analysis showed that the prevalence of mets in non-dm subjects based on idf criteria was 1.5% lower compared with ncep-atp iii criteria. these results agree with previously conducted studies in several asian countries. the hong kong cardiovascular risk factor prevalence study (crisps) in china showed that the mets prevalence in 1679 subjects according to ncep-atp iii criteria (14.5%) was also higher than that based on idf (11.4%).(20) a table 2. distribution of characteristics of subjects with the metabolic syndrome who had type 2 dm after 2 years table 3. risk factors for type 2 dm after 2 years 52 population study in bangladesh showed that mets based on ncep-atp iii criteria was higher (30.7%) compared with idf (24.5%).(21) similarly a study in kuala lumpur, malaysia, showed that mets based on ncep-atp iii was higher (41.4%) than that based on idf (38.2%).(22) however, our results are different from those of adam et al.(3) who reported that idf-based mets prevalence in the european population of adelaide, australia, was higher (22.8%) compared with the mets prevalence based on ncep-atp iii (15%). similar results were also shown by nilson (23) who reported that idf-based mets prevalence of 37.2% was higher than the mets prevalence of 24.7% based on ncep-atp iii criteria. these differences in the results were due to the fact that idf criteria require the presence of central obesity for determination of mets occurrence. even though the values of the other components exceed the normal values, in the absence of central obesity, the subjects will not be categorized as having mets. in addition, there is the difference in the ethnicity of the populations between asia and australia/europe, for which the cut-off points of waist circumference are different.(4) the mets prevalence in females was higher than in males, both according to ncep-atp iii and idf criteria. these results are in line with the results of a study in bali,(24) where the mets prevalence was 18.2% (16.6% for males and 20.0% for females). similarly, in an american study, a higher mets prevalence was obtained for females (24%), whereas for males it was 23.4%. (25) females are more at risk for experiencing mets, because physically they have a greater chance for increasing their body mass index. the premenstrual syndrome and the postmenopausal period give rise to an easier accumulation of body fat as a result of hormonal processes, which interfere with the action of insulin.(25) furthermore, the mets prevalence obtained also depends on the population under study. in the present study, there was a higher proportion of females among the participating subjects. respondents with mets have greater waist circumference, bmi, ldl, total cholesterol, and mean systolic and diastolic pressures, in comparison with those without mets, according to ncep-atp iii criteria as well as idf criteria. the mechanism causing the occurrence of the metabolic syndrome is based on insulin resistance and central obesity (visceral fat). visceral fat is metabolically more active than peripheral fat. the accumulation of adipocytes will increase the free fatty acids resulting from lipolysis, which will reduce the sensitivity to insulin. this insulin resistance and hyperinsulinemia in turn will cause metabolic changes, leading to hypertension, dyslipidemia, increased inflammatory responses and coagulation, by means of complex mechanisms such as endothelial dysfunction and oxidative stress.(26,27) the proportion of mets, both that based on ncep-atp iii criteria and on idf criteria increases with advancing age. the number of components associated with mets also increases, and at a more advanced age the components are greater in number. the occurrence of mets at a younger age requires close attention. uncontrolled mets occurring in younger persons will at a future date carry the risk of atherosclerosis that is associated with mets, resulting in the occurrence of cardiovascular disease and dm.(26) the mets criteria, whether based on ncepatp iii or idf, were excellent for determining the occurrence of mets in our present study. similar study results were found by moy and bulgiba,(22) who stated that although there was excellent agreement between ncep-atp iii and idf, 7.6% of the respondents were not diagnosed as having mets when using the idf criteria. the study of chackrewarthy et al.(28) in ceylon also showed that the idf criteria failed to identify 21% of males and 7% of females who had been declared as having mets based on the ncepatp iii criteria. this was because respondents who had no central obesity, but had high cardiometabolic risk factors such as blood pressure of >130/85 mmhg, fasting blood glucose of >100 mg/dl, and low hdl cholesterol levels, were sulistiowati, sihombing metabolic syndrome predict type 2 diabetes mellitus 53 univ med vol. 35 no.1 not diagnosed with mets according to the idf criteria requiring the presence of central obesity plus two other mets components.(22,28) on follow up observations, type 2 dm was found in 78 (13.0%) of the respondents with ncep-atp iii based mets and in 69 (12.6%) of the respondents with idf-based mets. these respondents with dm had a greater mean number o f m e t s c o m p o n e n t s t h a n t h e n o n d m r e s p o n d e n t s . t h e r i s k o f t y p e 2 d m i n respondents with mets according to ncep-atp iii criteria was 4.7 times greater, whereas respondents with mets according to idf was 4.1 greater. these results show that mets is closely associated with type 2 dm. similar studies in china showed that mets according to both ncep-atp iii and idf can predict the dm incidence, with risks of 4.1 (95% ci 2.8–6.0) and 3.5 (2.3–5.2), respectively.(20) this is in line with the results of a review of 13 cohort studies that was conducted by ford, li and sattar,(13) also showing that subjects with mets by ncep atp iii criteria have a 3.53 times higher risk, and by idf criteria a 4.42 times higher risk for the occurrence of dm. cohort studies conducted by wilson et al.(29) also showed that the risk of dm was 6.9 times higher in subjects with mets within an 8-year period. the more mets components, the higher the risk. the factors affecting the occurrence of type 2 dm, in addition to age, include mets components such as central obesity, fasting glucose level of >100 mg/dl, blood pressure of >130/85 mmhg, hypertriglyceridemia and low hdl cholesterol level. fasting glucose level of >100 mg/dl is the component with the greatest risk (4.6 times greater) for occurrence of type 2 dm after 2 years. the aging process causes a reduction in the capacity of pancreatic β cells to produce insulin. in individuals with mets, insulin resistance is increasingly more severe and finally the insulin secretion decreases, so that there is hyperglycemia and manifestations of type 2 dm.(26,27) the results of the above analysis show that mets prevalence is relatively high in the five kelurahan in bogor. the metabolic syndrome can be used as predictor of type 2 dm. screening programs and routine monitoring of mets components are necessary for detecting the onset of type 2 dm, in addition to being supported with changes in life style, dietary pattern, and increased physical activity. the analysis has s e v e r a l l i m i t a t i o n s , b e c a u s e i t d i d n o t differentiate between genders, although it is known that there is a difference in waist circumference between females and males. this was because there were more females than males among the subjects. conclusions both ncep-atp iii and idf criteria can be used as predictors of type 2 dm in subjects with mets. however, the ncep-atp iii criteria are more properly used for determining mets as compared with the idf criteria. screening programs and routine monitoring of mets components are necessary for detecting the onset of type 2 dm. conflict of interests the authors declare that they have no conflict of interests. acknowledgments we wish to express our gratitude to the head of the bogor city health service and staff, all respondents of the cohort study of ncd risk factors and all parties who helped in the conduct of this study. this study was financed by funds from the dipa (daftar isian penggunaan anggaran) badan litbangkes for 2011-2012. references 1. huang p. a comprehensive definition for metabolic syndrome. dis models mech 2009; 2:231-7. 2. deepa m, farooq s, datta m, et al. prevalence of metabolic syndrome using who, atp iii and 54 idf definition in asian indians: the chennai urban rural epidemiology study (cures-34). diabetes metab res rev 2007;23:127-34. 3. adams rj, appleton s, wilson dh, et al. population comparison of two clinical approaches to the metabolic syndrome. diabetes care 2005;28:2777-9. 4. grundy ms, cleemen ji, daniels sr, et al. diagnosis and management of the metabolic syndrome: an american heart association/ national heart, lung and blood institute scientific statement. circulation 2005;112: 2375-2. 5. soewondo p, purnamasari d, oemardi m, et al. prevalence of metabolic syndrome using ncep/ atp iii criteria in jakarta, indonesia: the 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2004;24:816-23. 28. chackrewarthy s, gunasekera d, pathmeswaren a, et al. a comparison between revised ncep atp iii and idf definitions in diagnosing metabolic syndrome in an urban sri lankan population: the ragama health study. isrn endocrinol 2013; article id 320176,7 pages. 29. wilson p, d’agostino r, parise h, et al. metabolic syndrome as a precursor of cardiovascular disease and type 2 diabetes mellitus. circulation 2005;112:3066-72. c:\users\universa medicina\docu 64 abstract universa medicina microscopic examination using negative staining for rapid diagnosis of syphilis yeva rosana1*, ida effendi2,3, wresti indriatmi4, and andi yasmon1 background syphilis is a global health problem, especially in developing countries including indonesia. treponema pallidum, the etiologic agent of syphilis, cannot be cultured in vitro. syphilis has several clinical manifestations, making laboratory testing a very important aspect of diagnosis. microscopic examination may support the diagnosis but is rarely used in indonesia. the aim of this study was to evaluate negative staining using the light microscope to detect t. pallidum in syphilitic lesions. methods a cross-sectional study was conducted involving 27 subjects who came to several dermato-venereology clinics in jakarta. exudates were collected from genital ulcers, condylomata lata, and dry mucocutaneous rash on palms and soles of syphilis patients. negative staining using one drop of indian ink was used to examine for treponemas under the light microscope at 10x100 magnification. results microscopic examination using negative staining showed a few clusters of small and spiral shaped bacteria. of the 39 specimens from 27 subjects, microscopic examinations were successfully done on 10 specimens. observations could only be conducted on 5 specimens, 3 (60.0%) of which showed the morphology of spirochetes. this examination is the easiest method for detecting the bacteria. moreover, the bacteria that were isolated from painless genital ulcers could be observed more clearly than those from erythematous maculopapular lesions. conclusion treponema pallidum was successfully detected by microscopic examination in all moist lesions, but was difficult to detect in dry lesions. negative staining under the light microscope appears to be simple, affordable, and available in most microbiology laboratories in indonesia. keywords: microscopic examination, light microscope, negative staining, treponema pallidum original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.64-70 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1254 january-april 2022 vol.41no.1 cite this article as: rosana y, effendi i, indriatmi w, yasmon a. microscopic examination using negative staining for rapid diagnosis of syphilis. univ med 2022 ;41 :6 4 -7 0 . doi: 10. 1805 1/ univmed.2022.v41.64-70. 1department of microbiology, medical faculty, universitas indonesia/ cipto mangunkusumo hospital, jakarta 2clinical microbiologist program, faculty of medicine, universitas indonesia, jakarta 3department of microbiology, faculty of medicine, universitas trisakti, jakarta 4department of dermato-venereology, medical faculty, universitas indonesia /cip to mangunkusu mo hospital, jakarta *correspondence: yeva rosana department of microbiology, medical faculty, universitas indonesia cipto mangu nkusumo hosp ital, jakarta, indonesia phone: +62 3160492 fax: +62 3100810 email: yeva.rosana@ui.ac.id orcid id : 0000-0002-5502-5593 date of first submission, november 18, 2021 date of final revised submission, march 23, 2022 date of acceptance, march 30, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 65 univ med vol. 41 no 1 introduction syphilis, known in indonesian as the “lion king”, is a disease that is transmitted primarily through sexual contact. the disease shows multiple clinical manifestations, a long course of disease and serious complications. the course of syphilis in patients who do not receive treatment or receive inadequate treatment will develop from a local infection (primary stage), after which the infection enters the circulation and spreads throughout the body, causing a severe inflammatory reaction (secondary stage), leading to complications in the central nervous system and the vital organs, including the heart and eyes (tertiary stage). this disease may be without any symptoms at the latent stage, so that the patient can go undiagnosed. syphilis can also be transmitted through the placenta from an infected mother to her fetus, who is at risk of developing fetal defects and intrauterine fetal death.(1,2) syphilis is a global health problem, especially in developing countries including indonesia.(3,4) the diagnosis of syphilis is difficult to establish. limitations in laboratory facilities become an obstacle in detecting t. pallidum. moreover, this bacteria cannot survive outside the human body, has a long generation time (3033 hours), minimal metabolic ability, and is highly dependent on the host. t. pallidum cannot be cultured on bacteriological media outside the human body.(2,5) serology can only be used for the detection of antibodies with the risk of getting false negative and false positive results.(6,7) the simplest and most rapid diagnostic test for syphilis is microscopic examination that directly detects t. pallidum bacteria from the lesions. this examination requires a special type of microscope, such as the darkfield microscope, f l u or e s c enc e mic r os c o p e , o r e l e c t r on microscope, or needs special stains such as the silver stain.(8-10) currently, the availability of microscopic examination facilities for syphilis in the laboratory is very limited. there has been no recent study about negative staining for syphilis because most patients come with no specific symptoms, therefore it is not easy to collect specimens. also, due to the limited number of facilities and competent technicians, microscopic examination for the detection of t. pallidum in indonesia is rarely performed. since most microbiology laboratories in indonesia use the light microscope for dire ct specimen examination, negative staining with light microscopy was applied in this study. this method is simple, affordable, and possible to use in most microbiology laboratories in indonesia. the aim of this study was to evaluate negative staining using the light microscope to detect t. pallidum in syphilitic lesions. methods research design a cross-sectional laboratory experimental study was conducted in 2015 at the clinical microbiology laboratory, faculty of medicine, universitas indonesiacipto mangunkusumo hospital, jakarta. this study was part of a larger study to evaluate treponema pallidum drug resistance which was finished in 2019. we published this study because as far as we know there has been no recent study about this topic using a cheap and simple method that is important to be applied in indonesia. study subjects the study subjects were collected using consecutive sampling from patients who came to dermato-venereology clinics in jakarta, including the dermato-venereology clinic of ci pt o ma ngu n ku s umo h os pi t a l ( ce n t r a l jakarta), the sexually transmitted disease (std) clinic in pasar rebo district, east jakarta, the std clinic in tambora district, west jakarta, and the indonesian family planning association procare clinic in jatinegara district, east jakarta. syphilis patients with signs and symptoms of primary or secondary syphilis, and willing to become study subjects by filling out an informed-consent form were included in this 66 study. specimens were taken from adult patients, more than 18 years of age. patients with latent and tertiary stage syphilis, neurosyphilis, or congenital syphilis were excluded from the study. patients who received antibiotics in the three days before specimen collection were also excluded. sample size determination th e sa mpl e s ize w as d ete rmine d by c o ns i d e r i ng t he se ns i t ivi t y o f d a r kf i e l d microscopic examinations to be 80% (11) and using the following formula (12): where n= sample size zα= 1.96 s = sensitivity of dark-field microscopy= 80% d = desired level of absolute accuracy = 15% so that the minimal sample size was 27. intervention sterile normal saline was used to clean the clinical lesion of syphilis. the exudate was collected using a cotton swab by gently pressing and rotating it on the moist lesion. the exudates from dry lesions were collected by gentle abrasion using a scalpel and pressing until the exudate was seen. serous fluid was put directly on a glass slide, or taken using a swab or a bacteriologic loop. if necessary, a drop of physiologic saline was placed on the slide to prevent drying of the specimen. one drop of indian ink was mixed with the exudate and spread out on the slide using another glass slide. the glass slide was covered using a cover glass and examined under the microscope at 10x100 magnification using immersion oil. measurements microscopic examinations were performed qualitatively to observe t. pallidum. due to the difficulty of visualizing its typical motility, the negative staining was focused more on its morphology. the observation was started by rotating the objective lens to the lowest power, and placing the slide on the stage, with the coverslip centered. the objective lens was focused using the coarse focus knob a nd continued using fine focus. when nothing was seen, the slide was slightly moved while viewing and focusing. a negative result was reported if no spiral shape was found after observing all fields of the slide. laboratory analysis proper specimen collection and handling was critical for optimizing the sensitivity of negative staining. after obtaining the serous exudate, minimizing contamination with blood or pus would incr ease the sensiti vity of this method. microscopy visualization at 10x100 magnification and immersion oil showed that t. pallidum was a delicate, tightly spiraled, corkscrew-shaped organism, which was occasionally bent in the middle when obstructed by cellular elements or debris in the field. on negative staining, against the black background on the glass slide, the treponemes appeared colorless or pale. statistical analysis the results of microscopic examination using negative staining were recorded and photographed. descriptive methods were used to analyze the data. ethical clearance this study was approved by the ethics committee, faculty of medicine, university of indonesia cipto mangunkusumo hospital, under no. 158/un2.f1/etik/2015, and all patients gave their written consent before enrollment into the study. results the study subjects were dominated by men with a median age of 26 years and an overall rosana, effendi, indriatmi, et al negative staining for rapid diagnosis of syphilis n = (zα)2s(1 − s) d2 n = (1.96)20.80(1 − 0.80) 0.152 67 univ med vol. 41 no 1 a b age range of 18 to 40 years, among whom 88.5% were men who have sex with men (msm), and most of them (54.10%) were hiv patients. in this study, many clinical manifestations were found. the most frequent was redness of the skin in the form of maculopapular erythema (100%) that was found in almost all parts of the body, particularly on the palms and soles. other clinical manifestations found were genital ulcers in the area of the shaft and the dorsum of the penis (18.03%). most of the genital ulcers found were solitary, but some were multiple with an ulcer base of granulomatous tissue while the ulcer margins were not raised. the ulcers ranged from painless to mildly painful. complaints of hair loss were found in 6.56% of patients with features of moth-eaten alopecia (alopecia syphilitica) and thinning eyebrows. among the patients 4.92% complained of mild visual disturbances in the form of blurred vision. condylomata lata were found in 3.28% of female subjects. microscopic examination was conducted in this study using negative staining and observed u nd er th e light mic r osc o pe . mi c r osc op y visualization at 10x100 magnification using immersion oil showed a few clusters of small and spiral-shaped bacteria (figure 1). of the 39 specimens, microscopic examinations were done on 10 specimens, but reliable observations could only be made on 5 specimens, with 3 (60.0%) of them showing spirochete morphology. discussion syphilis is a disease that is transmitted mainly through sexual contact with infected mucosal lesions. other body fluids are also infectious when patients have bacteremia. syphilis can cause early complications such as irreversible loss of vision, so rapid diagnosis of this infection is important for primary care clinicians.(13) primary syphilis classically presents as a si ngle, pain le ss, i ndu ra ted geni ta l u lc er (chancre), but this presentation is only 31% sensitive; lesions can be painful, multiple, and extra-genital. since the symptoms appear 1090 days (mean = 21 days) after exposure, (14) the patients often do not consult a doctor. mostly patients come to a clinic at the secondary stage of syphilis. the clinical manifestations of secondary stage of syphilis are very diverse and confusing. therefore, syphilis has been known as “the great imitator” as it may cause symptoms similar to many other diseases.(15-17) bacterial dissemination in secondary syphilis classically presents as a diffuse, symmetric, coppercolored, maculopapular, possibly pruritic rash of any morphology except vesicular. mucous figure 1. treponema pallidum microscopic picture using negative staining of exudate specimens 68 lesions, patchy alopecia, fever, headaches, and generalized painless adenopathy may also occur.(18,19) similar to previous reports, many clinical manifestation were found in this study, including maculopapular erythema in nearly all parts of the body, particularly on the palms and soles, painless to mildly painful genital ulcers, alopecia, thinning eyebrows, blurred vision and c o nd yl o ma t a l a t a . t h e re f o r e, l abo r a to r y examination is needed to support the diagnosis of syphilis. t he r e h a s b e e n ve r y l i mi te d u s e o f laboratory examinations supporting a definite diagnosis of syphilis. the incapability of culturing treponema pallidum outside the human body becomes an obstacle in detecting this spirochete.(5,20) the routine method is serology, but this gives many false negative and false positive results.(6,7) moreover, the diagnosis of syphilis is more difficult in hiv-positive patients, because hiv can alter the clinical picture of syphilis and can cause more serious complications. unusual serological responses such as high titers as well as false negative reactions have been reported for vdrl tests in hiv-reactive patients.(6,21) therefore specific tests will be needed particularly in all hivreactive patients. microscopic examination may support the diagnosis of syphilis by directly detecting t. pallidum bacteria in the lesions. this method is simple and rapid but rarely used in indonesia. it requires a special type of microscope such as t he d a r kf ie l d mi c r o sc op e , f lu o r e s c e n c e microscope, or electron microscope, or needs special stains such as the silver stain. darkfield microscopy requires a special microscope and a trained microscopist in close proximity to where patients are examined. the fluorescence microscope or the electron microscope are also of limited availability in most indonesian laboratories. special stains are expensive and need more skill in interpreting the results. this study investigated the use of a simple microscopic examination to support the diagnosis of syphilis using the light microscope. negative staining with the light microscopy is simple, affordable, and available in most laboratories in indonesia. in our study, negative staining successfully detected treponema pallidum in all moist lesions, but had difficulty in detecting the bacterium in dry lesions such as mucocutaneus rash on the palms and soles (figure 2). this method is simple and reliable for detecting t. pallidum directly in exudates and fluids from lesions, and needs only one drop of indian ink figure 2. clinical picture of secondary syphilis. a,b,c genital ulcers; d,e maculopapular rash rosana, effendi, indriatmi, et al negative staining for rapid diagnosis of syphilis 69 univ med vol. 41 no 1 and the light microscope. it can overcome the limitations of darkfield microscopy as the gold standard for syphilis microscopic examination which is not available in most laboratories in indonesia. there are no recent studies about negative staining for syphilis using the light microscope. compared to darkfield microscopy which is the gold standard for microscopic examination of syphilis, negative staining is simpler and presumably more reliable for detecting t. pallidum directly in exudates and fluids from lesions, when darkfield microscopy and expertise is not available. while the wet mount method using the darkfield microscope can examine the morphology and motility of the spirochete,(8,11) microscopic examinations using negative staining has difficulty in visualizing its typical motility and is more focused on its morphology. however, negative staining is simpler, cheaper, and more applicable, because the examination uses the light microscope which is available in most laboratories in indonesia. the present study was able to show the possibility of using negative staining to assist clinicians in the rapid diagnosis of syphilis. one of the limitations of this study is the fact that a limited number of microscopic slides were examined for the diagnosis of syphilis due to the difficulties in collecting exudates from the lesions of syphilis in all patients. further studies are required to examine more specimens and explain the benefit of negative staining for routine laboratory examination to support the diagnosis of syphilis. conclusion microscopic examination using negative staining can be used to support the early diagnosis of syphilis in moist lesions, but the results should be carefully interpreted in the case of dry lesions. negative staining using the light microscopy appears to be a simple, affordable, and highly available method in most microbiology laboratories in indonesia. conflict of interest the authors declare that they have no conflict of interest. acknowledgment this study was funded by hibah pupt of the directorate of higher education indonesia 2015-2016. we thank the dermato-venereology clinic of cipto mangunkusumo hospital, the std clinics of pasar rebo and tambora, and the indonesian family planning association procare clinic in jatinegara, for all assistance and cooperation in this study. our deepest appreciation is extended to our dedicated team from the faculty of medicine, universitas indonesia-cipto mangunkusumo hospital and universitas trisakti. contributors yr and ie designed the study. wi and ay supervised fieldwork and collected data and samples. yr and ie analyzed the results. yr and ie wrote the first draft of the report, with revisions and input from wi and ay. all authors contributed to revisions and approved the final version. references 1. world health organization. global incidence and prevalence of selected curable sexually transmitted infections-2008. switzerland: who library cataloguing-in-publication data; 2012. 2. tramont ec. treponema pallidum (syphilis). in: bennett je, dolin r, blaser mj, eds. mandell, douglas, and bennett’s principles and practice of infectious diseases. philadelphia: elsevier saunders; 2015. pp.3035-55. 3. kementerian kesehatan republik indonesia. p e d o ma n ta ta l a ksa na s ifil is u nt uk pengendalian sifilis di layanan kesehatan dasar; 2017. 4. kementerian kesehatan republik indonesia. p e d o ma n n a si on a l p e n a n ga na n i n fe ks i menular seksual; 2016. 70 5. edmondson dg, hu b, norris sj. long-term in v it r o c ul tur e o f th e syp h il is s p i r o c h et e treponema pallidum subsp. pallidum. mbio 2018;9:e01153-18. https://doi.org/10.1128/ mbio.01153-18. 6. sharma s, chaudhary j, hans c. vdrl v/s tpha for diagnosis of syphilis among hiv sero-reactive patients in a tertiary care hospital. int j curr microbiol app sci 2014;3:726-30. 7. shah d, marfatia ys. serological tests for syphilis. indian j sex transm dis aids 2019; 40: 186–91. doi: 10.4103/ijstd.ijstd_86_19. 8. theel es, katz ss, pillay a. molecular and direct detection tests for treponema pallidum subspecies pallidum: a review of the literature, 1964–2017. cid 2020;71(suppl 1):s1-s12. doi: 10.1093/cid/ciaa176. 9. luo y, xie y, xiao y. laboratory diagnostic tools fo r syp h il is : c ur r en t st a t us a nd f ut ur e prospects. front cell infect microbiol 2021;10: 574806. doi: 10.3389/fcimb.2020.574806. 10. workowski ka, bolan ga. sexually transmitted diseases treatment guidelines, 2015. mmwr recomm rep 2015;64;1-140. 11. pierce ef, katz ka. darkfield microscopy for point-of-care syphilis diagnosis. med lab obs 2011;43:30-1. 12. hajian-tilaki k. sample size estimation in d i a g no st ic t es t st ud ie s o f b io me di ca l informatics. j biomed informatics 2014;48:193– 204. http://dx.doi.org/10.1016/j.jbi.2014.02.013. 13. department of health and human services centers for disease control and prevention. sexually transmitted disease: summary of cdc treatment guidelines 2015 us; 2015. 14. byrne po, macpherson p. syphilis. bmj 2019; 366:l4746. doi: 10.1136/bmj.l4746. 15. çakmak sk, tamer e, karadað as, waugh m. syphilis: a great imitator. clin dermatol 2019; 37:182-91. doi: 10.1016/j.clindermatol.2019. 01.007. 16. nyatsanza f, tipple c. syphilis: presentations i n ge ne r a l me d i c i ne . cl in m e d ( lo nd ) 2016;16:184-8. 10.7861/clinmedicine.16-2-184. 17. klausner jd. the great imitator revealed: syphilis. top antivir med 2019;27:71-4. 18. cruz ar, pillay a, zuluaga av, et al. secondary syphilis in cali, colombia: new concepts in disease pathogenesis. plos negl trop dis 2010;4:e690. 19. tuddenham s, ghanem kg. emerging trends and persistent challenges in the management of adult syphilis. bmc infect dis 2015;15:351. doi: 10.1186/s12879-015-1028-3. 20. sato ns. laboratorial diagnosis of syphilis. in: sato ns (editor). syphilis recognition, description and diagnosis. croatia: intech; 2011.pp.87-108. 21. arando m, naval cf, foix mm, et al. early syphilis: risk factors and clinical manifestations focusing on hiv-positive patients. bmc infect dis 2019;19:727. https://doi.org/10.1186/ s12879-019-4269-8. rosana, effendi, indriatmi, et al negative staining for rapid diagnosis of syphilis c:\users\universa medicina\docu 4 abstract universa medicina bone metastases tend to increase in non-small cell lung cancer with epidermal growth factor receptor mutation reginald maleachi1*, dini rachma erawati2, suryanti dwi pratiwi3, and sri andarini4 background increased understanding in molecular pathology of advanced non-small cell lung cancer (nsclc) over the past decades has led to personalized treatment approaches being advocated. epidermal growth factor receptor (egfr) mutation that often occurs in nsclc can be identified using immunohistochemical examinations. moreover, clarifying the relationship between computed tomography (ct) and egfr mutation of nsclc might inform therapeutic decision-making. the purpose of this study was to determine the relationship between metastatic sites on primary chest ctscan and egfr mutation in nsclc lung cancer patients. methods an cross-sectional design using secondary data was conducted, involving 76 nsclc patients. egfr mutations were determined by immunohistochemical examination and metastatic sites by chest ct-scan with contrast. the collected metastatic sites comprised hilar and mediastinal lymphadenopathy, pulmonary nodules, and bone, liver, spleen and suprarenal metastases. a chi square test was used to analyze the data. results this study revealed that the highest nsclc stage was ivb, found in 39 samples (51.3%), while 34 (44.7%) subjects had egfr mutation. there was no statistically significant difference between metastatic site and positive egfr mutation, although positive bone metastases (54.8%) tend to have more numerous positive egfr mutations compared to negative bone metastases (37.7%) (p=0.142). conclusions patients with positive bone metastases tend to have higher positive egfr mutation compared to negative bone metastases in nsclc lung cancer patients. prospective studies evaluating patients with egfr mutation for bone metastases should be considered. this can provide information on therapeutic decision-making to obtain good clinical outcomes. keywords : chest ct-scan, egfr mutation, bone metastasis, lung cancer original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.4-10 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1180 january-april, 2022 vol.41no.1 1radiology specialist education study p rogram, faculty o f medicine, universitas brawijaya/dr. saiful anwar malang hospital 2department of radiology, faculty of medicine, universitas brawijaya/dr. saiful anwar malang hospital 3department of p ulmonology and respiratory medicin e, faculty of medicine, universitas brawijaya/dr. saiful anwar malang hospital 4department of ikm-kp, faculty of medicine, universitas brawijaya correspondence: *reginald maleachi faculty of medicine, universitas brawijaya / dr. saiful anwar malang hospital phone and fax: +6281282289972 email: reginaldmaleachi@gmail.com orcid id: 0000-0002-8830-9568 date of first submission, may 1, 2021 date of final revised submission, january 24, 2022 date of acceptance, january 27, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: maleachi r, erawati dr, pratiwi sd, andarini s. bone metastases tend to increase in non-small cell lung cancer with epidermal growth factor receptor mutation. univ med 2022;41:4-10. doi: 10.18051/univmed. 2022 .v41. 4-10 univ med vol. 41 no 1 5 introduction epidemiological studies in the world show that lung cancer is the leading cause of death, with more than half of patients diagnosed with lung cancer dying within one year of diagnosis, while the 5-year survival rate is around 17.8%.(1) currently for patients with non-small cell lung cancer (nsclc), evaluation of egfr mutation status by immunohistochemical examination is recommended, because the presence of egfr mutation greatly influences the choice of therapy.(2-4) unfortunately, this type of examination is still quite expensive and the facilities are not always available. adequate samples must be obtained for this examination,(5) but sometimes these are difficult to obtain. therefore, the prediction of egfr mutation from clinical characteristics and imaging tests such as chest ct-scan is important. as we know, the primary diagnostic imaging and staging of a lung mass is performed using a chest ct-scan with contrast.(6,7) the chest ct-scan can be used to assess the extent of the occurring metastatic process. on the c hest ct-sc an we can examine metastatic sites in hilar and mediastinal lymph node s, pulmonar y nodules, liver, spleen, suprarenal gland, and bone especially vertebrae. several studies have investigated the process of metastasis in nsclc with egfr mutation. as is well known, bone is one of the common sites of nsclc metastases. previous studies have investigated the morphological relationship between lung masses and egfr mutations. in the studies of hasegawa et al.(8) and qiu et al.,(9) tumor size was not correlated with egfr mutations, but liu et al.(10) stated that small tumors could predict the presence of egfr mutations. in the research of qiu et al.(9) and han et al.(11) there was a correlation between the density variable and egfr mutations. a retrospective study involving 57 patients with primary lung adenocarcinoma identified ct imaging-based histogram features of bone metastases with and without egfr mutation, which may contribute to diagnosis and prediction of egfr mutation status.(12) a systematic review and meta-analysis showed that the overall discordance rates in egfr mutation status between primary nsclc and distant metastatic tumors is low but varied largely between studies.(13) due to the inconsistency between several studies on metastatic tumor, the aim of the present study was to determine the relationship between nsclc metastatic site in chest ct-scan and egfr mutation in nsclc patients. methods research design a study of cross-sectional design using secondary data was conducted in the ct-scan room of the radiology installation and clinical data were collected at the pulmonology department of dr. saiful anwar malang hospital from september 2020 to december 2020. study subjects a total of 76 patients recruited into the study were taken from data forms over the time period of 2018 to 2019. the study population comprised patients diagnosed with lung cancer by a pulmonary specialist who had conducted an egfr immunohistochemical examination and performed a chest ct-scan with contrast at the dr. saiful anwar malang hospital from january 2018 to december 2019. the inclusion criteria for this study were all male and female patients of any age with lung cancer who had already undergone an immunohistochemical examination for egfr and chest ct-scan with contrast at the dr. saiful anwar malang hospital. the exclusion criteria were patients with lung cancer whose primary mass was not found on the chest ct-scan and patients with lung cancer who did not have a chest ct-scan at rsud dr. saiful anwar malang. measurements the chest ct-scan was performed using a 128-slice toshiba aquilion tsx-101a ct 6 scanner at the central radiology installation of dr. saiful anwar malang hospital. the dicom file was evaluated by three radiologists using the radiant dicom viewer software version 2020.1.1. egfr mutation was determined by immunohistochemical examination of tumor tissue specimens. analysis of the 76 samples was carried out by the three radiologists who had more than 5 years experience in measuring metastatic sites. the assessed me tastases included contralateral pulmonary nodules as seen on chest ct scan. metastases to the liver were determined as the presence of multiple lesions that intensify after addition of contrast. with regard to other sites, nodular lesions were found in the spleen and osteoblastic or osteolytic lesions, or even mixed lesions, were found in the bones. in addition, nodules or masses may be found in the adrenals (suprarenals). statistical analysis the data obtained were categorical data, so the analysis used a non-parametric chi-square test. all analyses used the spss 20 software. a p value <0.05 was considered significant. ethical clearance this study has been declared ethical based on the approval letter by the ethics and research commission of dr. saiful anwar hospital no.400/ 190/k.3/302/2020. results the study sample consisted of 76 patients, namely 47 men and 29 women. the majority of them were in the age range of 51–60 years, totaling 25 persons (32.9%), while 37 persons (48.7%) had adenocarcinoma on histopathological examination. in this study sample, it was revealed that the highest stage was ivb, found in 39 samples (51.3%). thirty four subjects (40.7%) had egfr mutation. the characteristics of the samples are shown in table 1. there was no statistically significant association between metastatic site and egfr mutation, but patients with positive bone metastases (54.8%) tended to have higher efgfpositive compared to negative bone metastases (37.7%) (p=0.142). the results shown in table 2. discussion lung cancer with mediastinal lymph node metastasis is more likely to recur and metastasize after complete resection: therefore, targeted therapy is a promising treatment strategy.(8) the r e s u l t s of t he h i l a r a n d medi a s t i n a l lymphadenopathy analysis indicate that there is no significant relationship with the egfr mutation. the samples are dominated by hilar and mediastinal lymphadenopathy. this could be due to the predominance of advanced stadium samples, so that patients with stage n tend to be numerous. research conducted by zhang et al.(14) in a cohort of 280 patients with 133 men table 1. demography and tumor characteristics of the study subjects (n= 76) maleachi, erawati, pratiwi, et al bone metastasis in nsclc with egfr mutation variables n (%) gender male 47 (61.8) female 29 (38.2) age (year) 31-40 5 (6.6) 41-50 13 (17.1) 51-60 25 (32.9) 61-70 21 (27.6) >70 12 (15.8) histopathology adenocarcinoma 37 (48.7) adeno-squamous carcinoma 9 (11.8) squamous cell carcinoma 2 (2.6) no data 28 (36.8) staging ia 1 (1.3) ib 1 (1.3) iia 0 (0) iib 2 (2.6) iiia 6 (7.9) iiib 2 (2.6) iiic 5 (6.6) iva 20 (26.3) ivb 39 (51.3) univ med vol. 41 no 1 7 c and 147 women showed that 120 (42.9%) patients with lung cancer had positive egfr mutation in lymph node metastases. in contrast, the study conducted by liu z et al.(15) found that the incidence of egfr mutation in patients with lymphadenopathy was only 15.3%. this is because there are other genotypic factors besides egfr that affect the incidence of metastatic lymphadenopathy. diffuse lung metastases have been reported in nsclc with positive egfr mutation. in a cohort study conducted by digumarthy et al.(16) involving 217 patients, the results showed that the finding of a primary pulmonary tumor with diffuse lung metastases in nsclc should point to the possibility of egfr mutation. however, it was said that this feature does not replace molecular genotyping. research conducted by wu et al.(17) showed that patien ts who had miliary carcinomatosis had high egfr mutation rates. the study of hsu et al.(18) showed that lung metastases often occur in nsclc with egfr mutation. after analyzing our study, we found that egfr mutation was positive in 22 (47.8%) pulmonary nodule metastases and negative in 24 (52.2%), but the difference was statistically not significant (table 2). further studies are needed to find out a clear mechanism for the occurrence of pulmonary nodules metastasis with egfr mutation. bone is a common site of metastasis of advanced nsclc. among patients with advanced lung cancer, 30–40% have bone metastases (bom) at the time of diagnosis.(19) patients with bone metastatic nsclc and egfr mutation, when treated with egfr tyrosine kinase inhibitors (tkis), have a relatively long survival expectancy.(20) in our study, there was no statistically significant association between bone metastases and positive egfr mutation, although patients with positive bone metastases (17 or 54.8%) tend to have higher positive egfr mutation rates than do those with negative bone metastases (14 or 37.7%). a retrospective study by shen et al.(12) involving 57 patients with primary lung adenocarcinoma identified in these patients ct imaging-based histogram features of bone metastases with and without egfr mutation, that may contribute to diagnosis and prediction of egfr mutation status. the study table 2. relationship between metastatic site and egfr mutation in nsclc patients metastatic site efgr mutation p value positive egfr mutation (n=34) (n,%) negative egfr mutation (n=42) (n,%) hilar and mediastinal lymphadenopathy positive 25 (40.9) 36 (59.1) 0.184 negative 9 (60.0) 6 (40.0) metastatic pulmonary nodule positive 22 (47.8) 24 (52.2) 0.502 negative 12 (4.0) 18 (60.0) bone metastasis positive 17 (54.8) 14 (45.2) 0.142 negative 17 (37.7) 28 (62.3) liver metastasis positive 5 (33.3) 10 (66.7) 0.321 negative 29 (47.5) 32 (52.5) spleen metastasis positive 0 (0.0) 1 (100.0) 0.365 negative 34 (45.3) 41 (54.7) suprarenal metastasis positive 1 (50.0) 1 (50.0) 1.000 negative 33 (44.6) 41 (55.4) 8 conducted by krawczyk et al.(21) found that nsclc with egfr mutation had more metastases to the bones than to the brain. these investigators also found that the activating mutation in the egfr gene was significantly more frequent in adenocarcinoma bone metastases than in the primary lung adenocarcinoma. kuijpers et al.(22) have confirmed that patients with positive egfr mutation have a high incidence of bone metastases, since 54% of patients with positive egfr mutation in stage iv nsclc had bone metastases at diagnosis. beypinar et al,(23) using a retrospective case-control study from 2013 to 2019, totaling 1085 samples, examined the site of metastases with egfr mutation and without egfr mutation, and found no significant association between the two. prospective studies evaluating egfr mutation patients for bone metastases should be considered. this can provide faster therapy to obtain good clinical outcomes. in our study. the assessment of liver metastases showed no significant association between metastatic site and egfr mutation. similarly, beypinar et al.(23) concluded that there was no difference in the incidence of liver metastases with and without egfr mutation. our study results may be due to the sample size being dominated by the presence of pulmonary metastases with positive and negative egfr mutations of 47.5% and 52.5%, respectively. the spleen is a rare site of metastasis in nsclc.(24) however, we also found metastases in the spleen although only in 1 sample with negative egfr mutation which showed no significance. in the study by mitsimponas et al.(25) on their case of spleen metastasis, also no egfr mutation was found. adrenal metastases in nsclc are also frequent.(26) however, in our study we only found 2 nsclc samples with adrenal metastases but no significant association with egfr mutation. the limita tion of this study is the heterogeneity of the samples due to the absence of data in the interval between the time of chest ct-scan and the time of first diagnosis. the majority of samples were at an advanced stage. for this study, it is suggested that it is necessary to detect lung cancer at an early stage to reduce the predominance of samples with an advanced stage. conclusions there was no significant relationship between metastatic site and egfr mutation in nsclc lung cancer, based on chest ct-scan with contrast, but patients with positive bone metastases tend to have higher positive egfr mutation compared to patients with negative bone metastases. prospective studies should be considered evaluating egfr mutation patients for bone metastases. this can provide faster decisions on therapy to obtain good clinical outcomes. conflict of interest compe ting inter e sts: no relevant disclosures. acknowledgement the authors thank bayhaqi nasir aslam and habiba aurora who helped to assess the results of ct-scan examination. contributors dre, sdp, sa contributed to conception and design; rm, dre, sdp, sa contributed to analyse and interpretation of the data; rm contributed to drafting of the article; dre, sdp, sa contributed to revise the manuscript. all authors have read and approved the final manuscript. references 1. zappa c, mousa sa. non-small cell lung cancer: current treatment and future advances. transl lung cancer res 2016;5:288–300. doi: 10.21037/ tlcr.2016.06.07. maleachi, erawati, pratiwi, et al bone metastasis in nsclc with egfr mutation univ med vol. 41 no 1 9 2. grigoriu b, berghmans t, meert ap. management of egfr mutated nonsmall cell lung carcinoma patients. eur respir j 2015;45:1132–41. doi: 10.1183/09031936.00156614. 3. yoneda k, imanishi n, ichiki y, tanaka f. treatment of non-small cell lung cancer with egfr-mutations. j uoeh 2019;41:153-63. doi: 10.7888/juoeh.41.153. 4. choi yl, sun jm, cho j, et al. egfr mutation testing in patients with advanced non-small cell lung cancer: a comprehensive evaluation of realworld practice in an east asian tertiary hospital. plos one 2013;8:e56011. doi: 10.1371/journal. pone.0056011. 5. haralsingh a, west m. tissue yields for epidermal growth factor receptor analysis in non-small cell lung cancer patients in trinidad and tobago. cureus 2021;13:e12531. doi: 10.7759/cureus.12531. 6. purandare nc, rangarajan v. imaging of lung cancer: implications on staging and management. indian j radiol imaging 2015;25:109–20. doi: 10.4103/0971-3026.155831. 7. jonas de, reuland ds, reddy sm, et al. screening for lung cancer with low-dose computed tomography: updated evidence report and systematic review for the us preventive services task force. j am med assoc 2021;325:971–87. doi: 10.1001/jama.2021.0377. 8. hasegawa m, sakai f, ishikawa r, kimura f, ishida h, kobayashi k. ct features of epidermal growth factor receptor-mutated adenocarcinoma of the lung: comparison with nonmutated adenocarcinoma. j 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respir j 2013;41:417–24. doi: 10.1183/09031936. 00006912. 18. hsu f, de caluwe a, anderson d, nichol a, toriumi t, ho c. patterns of spread and prognostic implications of lung cancer metastasis in an era of driver mutations. curr oncol 2017;24:228–33. doi: 10.3747/co.24.3496. 19. santini d, barni s, intagliata s, et al. natural history of non-small-cell lung cancer with bone metastases. sci rep 2015; 5 :18670. https://doi.org/ 10.1038/srep18670 20. laganà m, gurizzan c, roca e, et al. high prevalence and early occurrence of skeletal complications in egfr mutated nsclc patients with bone metastases. front oncol 2020;10: 588862. doi: 10.3389/fonc.2020.588862. 21. krawczyk p, kowalski dm, ramlau r, et al. comparison of the effectiveness of erlotinib, gefitinib, and afatinib for treatment of non-small cell lung cancer in patients with common and rare egfr gene mutations. oncol lett 2017;13:4433– 44. doi: 10.3892/ol.2017.5980. 22. kuijpers cchj, hendriks lel, derks jl, et al. association of molecular status and metastatic organs at diagnosis in patients with stage iv nonsquamous non-small cell lung cancer. lung cancer 2018;121:76–81. https://doi.org/10.1016/ j.lungcan.2018.05.006. 23. beypinar i, demir h, araz m, uysal m. the relationship between egfr mutation and metastasis pattern in lung adenocarcinoma. j oncol sci 2019;5:65–9. https://doi.org/10.1016/ j.jons.2019.08.002. 10 24. belli a, de luca g, bianco f, et al. an unusual metastatic site for primary lung cancer: the spleen. j thorac oncol 2016;11:128–9. https://dx.doi.org/ 10.1016/j.jtho.2015.08.005. 25. mitsimponas n, mitsogianni m, crespo f, et al. isolated splenic metastasis from non-small-cell lung cancer: a case report and review of the maleachi, erawati, pratiwi, et al bone metastasis in nsclc with egfr mutation literature. case rep oncol 2017;10:638–43. https:/ /doi.org/10.1159/000478002. 26. bazhenova l, newton p, mason j, bethel k, nieva j, kuhn p. adrenal metastases in lung cancer: clinical implications of a mathematical model. j thorac oncol 2014;9:442-6. doi: 10.1097/ jto.0000000000000133. oktavianus 113 *department of physiology, faculty of medicine, trisakti university **department of obstetrics and gynecology, faculty of medicine, trisakti university correspondence dr. martiem mawi, ms department of physiology, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: +6221-5672731 ext.2803 email: martiemmawi@yahoo.com univ med 2012;31:113-9 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background increasing age and decreased testosterone concentrations in males influence muscle strength and muscle mass, particularly in skeletal muscle. there have been few studies on decreased lung function resulting from reduced mass and strength of respiratory muscles. the aim of the present study was to investigate the existence of an association between free testosterone index (fti) and lung function in males aged between 40 and 80 years. methods this cross-sectional study involved 167 males aged between 40 and 80 years in cilandak subdistrict, south jakarta. total serum testosterone and sex hormone-binding globulin (shbg) concentrations were determined by electrochemiluminescence immunoassay (eclia) using roche elecsys reagent kit cat 11776061 and elecsys 2010 reagent (cobas e601), respectively fti was calculated using the formula free testosterone/shbg x 100%. forced expiratory volume in 1 second (vep1) was assessed by means of an as 500 spirometer. results mean age of the subjects was 53.32 ± 8.26 years, mean total serum testosterone concentration was 532.59 ± 206.92 ng/dl, mean shbg concentration 41.26 ± 21.14 nmol/l, mean fti 48.22 ± 14.34 %, and mean vep1 was 1.63 ± 0.54 l. there was a significant association between both shbg and fti on the one hand and vep1 on the other, with pearson correlation coefficients of -0.199 (p=0.010) and 0.271 (p=0.000), respectively. linear multiple regression analysis indicated that fti was the most influential variable on lung function (vep1), higher fti values indicating higher vep1 (â=0.008: p=0.004). conclusion in males aged 40-80 years, higher fti values indicate better lung function as determined by means of vep1. keywords: testosterone, lung function, andropause, adult males high free testosterone index increases lung function in adult males martiem mawi* and rully ayu nirmalasari** abstract 114 mawi, nirmalasari testosterone increases lung function indeks testosteron bebas yang tinggi meningkatkan fungsi paru pada laki-laki dewasa latar belakang peningkatan usia dan penurunan kadar hormon testosteron pada laki-laki akan mempengaruhi kekuatan dan massa otot terutama otot rangka. penurunan fungsi paru akibat pengurangan massa dan kekuatan otot pernapasan belum banyak diketahui. penelitian ini bertujuan untuk menentukan hubungan antara indeks testosteron dan fungsi paru pada laki-laki berusia antara 40 sampai 80 tahun. metode penelitian ini menggunakan desain potong lintang mengikut sertakan 167 laki-laki yang berusia antara 40 sampai 80 tahun di kecamatan cilandak jakarta selatan. pemeriksaan kadar testosteron total dilakukan dengan electrochemiluminescence immunoassay (eclia) dengan roche elecsys reagen kit cat 11776061. sex hormonebinding globulin (shbg) diukur dengan metode eclia dengan reagen elecsys 2010 (calos e601). indeks testosteron bebas (itb) dihitung berdasarkan kadar testosteron bebas/shbg x 100%. volume ekspirasi paksa satu detik pertama (vep1) diperiksa dengan memakai spirometer as 500. hasil usia rata-rata subjek adalah 53,32 ± 8,26 tahun, kadar testosteron total serum rata-rata adalah 532,59 ± 206,92 ng/dl, kadar shbg rata-rata adalah 41,26 ± 21,14 nmol/l, nilai itb rata-rata adalah 48,22 ± 14,34 %, dan vep1 rata-rata adalah 1,63 ± 0,54 l. terdapat hubungan bermakna antara kadar shbg dan itb dengan vep1 dengan koefisien korelasi pearson masing-masing sebesar -0,199 (p=0,010) dan 0,271 (p=0,000). analisis regresi ganda linier menunjukkan itb yang paling berpengaruh terhadap fungsi paru (vep1), semakin tinggi nilai itb semakin tinggi vep1 (â=0,008: p=0,004). kesimpulan pada laki-laki yang berusia antara 40-80 tahun, peningkatan itb akan meningkatkan fungsi paru yang diukur dengan menggunakan vep1. keywords: testosteron, fungsi paru, andropause, laki-laki dewasa abstrak introduction increased health status and longevity in indonesia have resulted in a rapid increase in the number of older persons, which in 2010 was estimated to equal the number of under-fives at around 24 million or 9.77% of the total population. in 2020 there will be an estimated 28.8 million or 11.34% of the total population.(1) in males, increasing age is associated with a reduction in serum androgenic hormones, particularly testosterone, with a concomitant signs of andropause, such as decreased libido, increased incidence of erectile dysfunction, and decreased muscle mass and muscle strength. the reduction in testosterone levels is caused by decreased testicular production, decreased luteinezing hormone (lh) levels(2) and decreased capacity of the brain to stimulate testosterone release. (3) the andropause has also been a s s o c i a t e d w i t h e m o t i o n a l a n d p h y s i c a l changes.(4) androgenic hormones comprise testosterone, dehydrotestosterone (dht), dehydro-epiandrosterone (dhea), dehydro115 e p i a n d r o s t e r o n e s u l f a t e ( d h e a s ) , a n d androstenedione. although dhea levels follow the circadian rhythm, attaining peak levels in the morning hours, dheas levels are relatively stable throughout the day. dhea and dheas are intermediate metabolites in the synthesis of other s t e r o i d s , s u c h a s t h e m a l e s e x h o r m o n e testosterons.(5) of the circulatory testosterone, 40% is bound to sex hormone binding globulin (shbg) and inactive, 58% is bound to albumin and is the active form in various tissues (bioavailable testosterone), while 2% is in a free active form. testosterone not bound to shbg diffuses passively through the cell membrane into the target cells, where it binds androgen receptors (ar). it is the free serum testosterone and the testosterone bound to albumin that have biological functions.(2) symptoms of andropause appear at the age of 40-55 years. from the age of 30 years onwards there is a 10% reduction in testosterone levels for each decade, such that at the age of 50 years there will be a 30% reduction in testosterone levels, leading to the appearance of symptoms.(6) there are 10 signs of andropause, i.e. irritability, sleep disturbances, decreased libido, erectile dysfunction, decreased muscle mass, increased body weight, loss of memory, thinning of scalp hair, reduced bone density, and depression.(6) the respiratory changes are associated with increasing age are important because they may i n c r e a s e t h e d e v e l o p m e n t o f r e s p i r a t o r y disorders. this is associated with increased pulmonary compliance, such as pulmonary elastic recoil, and decreased compliance of the chest wall. both of these result in a progressive decrease in forced vital capacity (fvc), forced expiratory volume in 1 second (vep1), and forced expiratory flow, and an increase in r e s i d u a l f u n c t i o n a l c a p a c i t y. (7) r e d u c e d testosterone levels with increasing age is associated with decreased muscle strength and muscle mass.(8) the massachusetts male aging study found an association between low serum testosterone concentration and mortality from respiratory disease.(9) patients with chronic bronchitis, which is associated high mortality r i s k , h a v e l o w t o t a l s e r u m t e s t o s t e r o n e concentrations.(10) the reduced testosterone levels in older males are receiving increasing attention, particularly with regard to their effects on lung function. therefore the aim of the present study was to determine any association existing between testosterone level and lung function in males aged between 40 and 80 years. methods research design t h i s w a s a c r o s s s e c t i o n a l s t u d y t o determine whether there is an association between testosterone level and lung function in males aged between 40 and 80 years. the study was conducted from july to october 2011. study subjects the subjects of the study were males aged 40 80 years who were residents of 5 kelurahan (villages) in cilandak subdistrict, south jakarta, comprising cilandak barat, pondok labu, lebak bulus, cipete selatan and gandaria selatan. inclusion criteria in this study were: males aged 40-80 years, not suffering from pulmonary, cardiac or renal diseases and diabetes mellitus, willing to participate in the study on the basis of i n f o r m e d c o n s e n t , c a p a b l e o f a c t i v e communication, mobile (not requiring walking aids). the exclusion criterion was consumption of any hormonal medication in the last three years. all respondents gave signed informed consent. anthropometric measurements height was measured to the nearest 0.1 cm using a portable microtoise, with the respondents standing without shoes. weight was measured to the nearest 0.1 kg using sage portable scales, in lightly clothed respondents without shoes. body mass index (bmi) was determined by dividing weight in kg by height in square meters. univ med vol. 31 no.2 116 mawi, nirmalasari testosterone increases lung function bmi was divided into 4 categories as follows: underweight (<18.5 kg/m2), normal (18.5 22.9 kg/m2), overweight (23.0-27.5 kg/m2), and obesity (>27.6 kg/m2). laboratory assays a fasting blood sample was drawn from the median cubital vein of each subject, with the subject in the supine position, between 8.00 a.m. and 10.00 a.m. the sample was collected in a plain tube, left to clot for 30-45 minutes, and subsequently centrifuged at 3000 rpm for 15 minutes to separate the serum. measurement of t o t a l t e s t o s t e r o n e a n d s h b g w a s b y e l e c t r o c h e m i l u m i n e s cenc e i m m u n o a s s a y (eclia), respectively using an elecsys 2010/ cobas e601 and roche elecsys reagent kit cat. 11776061 (r1: anti-testosterone-ad-biotin, r2: testosterone-ruthenium) and roche elycsys reagen kit cat. 03052001 (r1: anti-shbg-adbiotin, r2: anti shbg-ruthenium). the intraassay coefficients of variation of testosterone and shbg were 5.9% and 5.8%, respectively. the free testosterone index (fti) was calculated according to the formula total serum testosterone/ shbg) x 100. fasting blood glucose was measured by spectrophotometry using an advia 1800 instrument and bayer advia 180074024 glucose reagent (r1: atp, nad, sodium azide; r2: hexokinase, g6pd). detection limit: 0.6mg/ dl, linearity: 0-700 mg/dl. conversion factor: mg/dl x 0.05555 mmol/l. measurement of lung function in this study the vep1 was used as an indicator of lung function. data on height, weight, age and gender of the subjects were entered in the minicomputer of the as 500 spirometer. the standing subjects faced the instrument and took one inspiration and then performed a single forced expiration into the mouth piece attached to the spirometer. the examination was performed three times, after which the mean of the three values was calculated. research ethics the present study obtained ethical clearance from the research ethics commission of the faculty of medicine, trisakti university. data analysis all variables were checked for normal distribution using the kolmogorov-smirnov onesample test for goodness-of-fit. the pearson correlation test was used to determine a relationship between vep1 and age, bmi, fasting blood glucose, total serum testosterone, shbg, and fti as covariates. covariates with a significant association with vep1 were entered into a linear multiple regression model to determine the covariates with the greatest influence on lung function. two-sided p-values of less then 0.05 were considered significant. data processing was by means of spss for windows version 17. results of the 167 subjects participating in this study the mean age was 53.32 ± 8.26 years. there were 72 (43.1%) subjects with senior high s c h o o l e d u c a t i o n , 1 2 4 ( 7 4 . 3 % ) w e r e unemployed, mean bmi was 24.23 ± 4.01 kg/ m2 and vep1 was 1.63 ± 0.54 l. mean fasting blood glucose was 108.58 ± 42.96 mg/dl, total testosterone 532.38 ± 206.92 nmol/l, shbg 41.04 ± 21.12 nmol/l, and fti 48.13 ± 14.28 % (table 1). the results of the pearson correlation a n a l y s i s b e t w e e n v e p 1 a n d h o r m o n e concentration and other covariates showed that shgb and fti were significantly correlated with vep1, the pearson correlation coefficients being -0.199 (p=0.010) and 0.271 (p=0.000), r e s p e c t i v e l y ( ta b l e 2 ) . b o t h s i g n i f i c a n t variables were entered into a linear multiple regression model with vep1 as the dependent variable. the results of the linear multiple regression showed that fti was of greatest influence on 117 table 1. characteristics of participating subjects (n=167) *frequency (percentage); bmi=body mass index; shbg=sex hormone binding globulin; vep1=forced expiratory volume in 1 second; fti=free testosterone index table 2. relationship between hormone concentration and other covariates and lung function *p<0.05; bmi=body mass index; shbg=sex hormone binding globulin; fti=free testosterone index; vep1=forced expiratory volume in 1 second table 3. linear multiple regression with vep1 as dependent variable β=regression coefficient; beta=standardized beta coefficient; shbg=sex hormone binding globulin; fti=free testosterone index; vep1=forced expiratory volume in 1 second univ med vol. 31 no.2 lung function (vep1), with higher fti values indicating high vep1 (β=0.008; p=0.004) (table 3). discussion the results of this study showed that fti was of greatest influence on lung function in males aged 40–80 years. our findings were consistent with those of a study conducted on 2197 male subjects aged 29 years and older, where free testosterone level and fti were significantly associated with lung function, measured from forced vital capacity (fvc) and f o r c e d e x p i r a t o r y v o l u m e i n 1 s e c o n d (fev1).(11) our study determined only fti and not serum free testosterone concentrations. fti is capable of describing androgen status of an i n d i v i d u a l , b e c a u s e f t i r e a s o n a b l y approximates bioavailable testosterone with the use of equilibrium dialysis and has been shown to be suitable for clinical use.(12) t h e p r e s e n t s t u d y d e t e r m i n e d t h e physiologic serum levels of total testosterone and shbg, and calculated the fti. the lack of association between total testosterone and lung function may be explained by the fact that there is no sufficiently significant variation in physiologic testosterone levels across different age groups for clearly differentiating between individual lung functions. in addition, serum testosterone levels might only play a minor role in lung functions of healthy males. it is debatable whether it is only older males with low testosterone levels who can experience 118 mawi, nirmalasari testosterone increases lung function improved lung function. in the massachusetts male aging study an association was found between low total serum testosterone levels and respiratory disease mortality,(11) observable only with calculated free testosterone, and not with total testosterone. patients with chronic bronchitis and associated high mortality risk, h a v e b e e n s h o w n t o h a v e l o w e r s e r u m testosterone levels.(10) furthermore, lower serum testosterone levels have been associated with an increased morbidity not only in people suffering from respiratory diseases but have been shown to predict an increased mortality in both middleaged and old men owing to cardiovascular disease and metabolic syndrome.(13,14) increasing age in males is associated with decreasing capacity for testosterone synthesis, caused by decreased hypophyseal production of luteinizing hormone (lh), which is necesary f o r s t i m u l a t i n g l e y d i g c e l l s t o p r o d u c e testosterone. in addition, older males have higher concentrations of shbg, which binds to testosterone and decreases the availability of circulatory free testosterone to various tissues, such as the muscles. advanced age also cause a flattening of the daily testosterone concentration curve, in contrast to younger males, where testosterone production has a morning peak. however, the decrease in testosterone levels in men occurs at a slower rate in comparison with the decrease in estrogen levels in postmenopausal women.(2) testosterone serves to regulate various physiological processes in the body, comprising muscle protein metabolism, various sexual aspects, cognitive functions, secondary sexual characteristics, erythropoiesis, plasma lipids, and bone metabolism.(15,16) the reduction in testosterone levels affect the functioning of various organs and tissues, such as the mass of the respiratory muscles. this study demonstrated that fti was of great influence on lung function (vep1). several investigators report that in patients with chronic obstructive pulmonary disease (copd) there is a reduction in testosterone due to hypoxia, leading to a decrease in muscle mass and muscle strength.(17,18) administration of testosterone to copd patients may result in an increase in the mass and strength of the skeletal muscles, particulary the respiratory muscles, resulting in improved lung function.(19,20) one limitation of the present study is that it did not measure free serum testosterone concentrations, but only calculated fti, even t h o u g h f t i h a s b e e n u s e d b y s e v e r a l investigators and has yielded reliable results in studies of cross-sectional design.(11,21) another limitation of this study is its cross-sectional community-based design, which limits the interpretation of the results and the conclusions that can be drawn as to direction of causality. conclusion in males aged 40-80 years, raised fti increases lung function as determined by means of vep1. acknowledgements we thank the study subjects and the staff at puskesmas kecamatan and puskesmas kelurahan of cilandak subdistrict, south jakarta. thanks are also due to the faculty of medicine, trisakti university, for providing the funding and facilities for this study. references 1. kementerian kesehatan r.i. menuju tua: sehat mandiri dan produktif. panduan peringatan hari kesehatan sedunia, 7 april 2012. available at: http://promkes.depkes.go.id/download/panduan _hks_2012.pdf. access april 22,2012. 2. cunningham gr, matsumoto am, swerdloff r. low testosterone and men. healthy j clin endocrinol metab 2004;89:2-23. 3. morales a, buvat j, googren lj, guay at, kaufman jm. endocrine aspects of sexual dysfunction in men. j sexual med 2004;4:6981. 4. howe cl. hypogonadism: evaluation and management in the older male. arizona geriatr j 2008;13:18-9. 5. friedrich n, volzke h, rosskopf d, stevelling a, krebs a, nauck m, et al. reference ranges 119 for serum dehydroepiandrosterone sulfate and testosterone in adult men. j androl 2008;29:6107. 6. kaufman jm, vermeulen a. the decline of androgen levels in elderly men and its clinical and therapeutic implications. endocr rev 2005;26:833–76. doi: 10.1210/er.2004-0013. 7. laghi f. tobin mj. disorders of the respiratory muscles. am j respir crit care med 2003;168: 10-48. 8. katznelson l, robinson mw, coyle cl, lee h, farrel ce. effect of modest testosterone supplementation and exercise for 12 weeks on body composition and quality of life in elderly men. eur j endocrinol 2006;155:867-75. 9. aranyo ab, kupelian v, page st, handelsman dj, bremner wj, mckinlay jb. steroids and all cause and cause-specific mortality in men. arch intern med 2007;167:1252-60. 10. van vliet m, spruit ma, varleden g, kasran a, van herck e, pitta f, et al. hypogonadism, quadriceps weakness, and exercise intolerance in chronic obstructive pulmonary disease. am j respir crit care med 2005;172:1105-11. 11. svartberg j, schirmer h, medbo a, melbye h, aasebo u. reduced pulmonary function is associated with lower levels of endogenous total and free testosterone: the tromso study. eur j epidemiol 2007;22:107–12. 12. roy ta, blackman mr, harman sm, tobin jd, schrager m, metter ej. interrelationships of serum testosterone and free testosterone index with ffm and strength in aging men. am j physiol endocrinol metab 2002;283:e284-e94. 13. khaw kt, dowsett m, folkerd e, bingham s, wareham n, luben r, et al. endogenous testosterone and mortality due to all causes, cardiovascular disease, and cancer in men: european prospective investigation into cancer in norfolk (epic-norfolk) prospective population study. circulation 2007;116:2694– 701. 14. laughlin ga, barrett-connor e, bergstrom j. low serum testosterone and mortality in older men. j clin endocrinol metab 2008;93:68–75. 15. isodori am, giannetta e, greco ea, gianfrilli d, bonifacio v. effects of testosterone on body composition, bone metabolism and serum lipid profile in middle-aged men: a meta-analysis. clin endocrinol 2005;63:280-93. 16. witter ga, chapman im, haren mt, mackuitosh s, coates p. oral testosterone supplementation increase muscle and decrease fat mass in healthy elderly with low-normal gonad status. j gerontol a biol sci med sci 2003;58:618-25. 17. cruetzberg ec, casaburi r. endocrinological disturbances in chronic obstructive pulmonary disease. eur respir j suppl 2003;22:s76-80. 18. gupta b, kant s, pant n. chronic obstructive pulmonary disease: beyond respiratory system. internet j pulm med 2008;10:4-14. 19. rhoden el, morgentaler a. risk of testosteronereplacement therapy and recommendations for monitoring. n eng j med 2004;350:482-92. 20. cosabury r, shalender b, cosentino l, porszasz j, somfray a. effects of testosterone and resistance training in men with chronic obstructive pulmonary disease. am j respir crit care med 2004;170:870-8. 21. morley je, patrick p, perry hm. evaluation of assays available to measure free testosterone. metabolism 2002;51:554–9. univ med vol. 31 no.2 oktavianus 43 *fetomaternal division, department of obstetrics and gynecology, faculty of medicine, diponegoro university / dr. kariadi hospital semarang correspondence dr. besari adi pramono, msi, med, spog(k) fetomaternal division, department of obstetrics and gynecology, faculty of medicine, diponegoro university/ dr. kariadi hospital semarang email: babas1504@yahoo.com univ med 2012;31:43-51 abstract universa medicina january-april, 2012january-april, 2012january-april, 2012january-april, 2012january-april, 2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background the main causes of maternal mortality in indonesia are pre-eclampsia, hemorrhage, and infection. oxidative stress has a primary role in pre-eclampsia and one of its parameters is 8-isoprostane serum level. the objective of this study is to measure 8-isoprostane and to analyze the relationship between 8isoprostane level and birth weight in pre-eclampsia. methods a cross-sectional study involving 23 pre-eclampsia and 29 normotensive pregnant women with normal or cesarean delivery at dr. kariadi hospital semarang from january to may 2011. collected maternal blood samples were assessed for 8-isoprostane levels by means of a specific elisa kit. neonatal birth weight was measured immediately after delivery by means of calibrated baby scales. differences in 8-isoprostane levels between pre-eclampsia and normotensive women were assessed using independent t-test for normal distributed data, and the mann-whitney test for non-normally distributed data. results mean 8-isoprostane level was significantly higher in women with pre-eclampsia than in normotensive women (62.52 ± 12.19 pg/ml vs 28.64 ± 8.81 pg/ml) (p<0.05). low birth weight was twice as frequent in pre-eclampsia than in normotensives. there was no correlation between 8-isoprostane serum level and neonatal birth weight in pre-eclampsia. conclusion the level of 8-isoprostane was higher in pre-eclampsia than in normotensives. it is recommended to conduct further studies to determine whether 8-isoprostane may be used as a predictive marker of pre-eclampsia. keywords: 8-isoprostane level, birth weight, pre-eclampsia serum 8-isoprostane increased in pre-eclampsia besari adi pramono* and herman kristanto* 44 pramono, kristanto isoprostane increased in pre-eclampsia kadar isoprostan-8 meningkat pada pre-eklampsia latar belakang stres oksidatif berperan penting pada pre-eklampsia, dan salah satu parameter stres oksidatif yang dapat diukur adalah isoprostan-8.tujuan penelitian ini adalah membandingkan kadar isoprostan-8 pada pre-eklampsia dengan kehamilan normotensi dan menganalisis hubungan antara kadar isoprostan-8 dengan berat lahir bayi pada preeklampsia. metode penelitian belah lintang mengikutsertakan 23 ibu pre-eklampsia dan 29 ibu hamil normotensi yang melahirkan baik pervaginam/perabdominam di rsup dr. kariadi semarang selama januari–mei 2011. sampel darah vena diambil pada saat persalinan kemudian diukur kadar isoprostan-8 dan berat badan bayi diukur setelah bayi lahir. isoprostan-8 serum kemudian dianalisis korelasinya dengan berat badan lahir bayi. analisis statistik menggunakan uji korelasi, uji beda dengan t test, dan uji mann whitney. hasil rerata isoprostan-8 pada ibu hamil dengan pre-eklampsia lebih tinggi dari pada kehamilan normal (62,52 ± 12,19 pg/ml dibanding 28,64 ± 8,81 pg/ml) dan bermakna secara statistik (p<0,05). berat badan lahir bayi yang dilahirkan oleh ibu hamil dengan pre-eklampsia yang <2500 gram 2 kali lebih banyak daripada yang dilahirkan ibu normotensi. tidak terdapat hubungan antara kadar isoprostan-8 dengan berat badan lahir bayi pada ibu dengan pre-eklampsia. kesimpulan kadar isoprostan-8 ibu hamil dengan pre-eklampsia lebih tinggi dibanding dengan ibu hamil normotensi. tidak terdapat hubungan antara isoprostan-8 dengan berat badan lahir bayi pada ibu dengan pre-eklampsia. kata kunci: isoprostan-8, berat badan lahir, pre-eklampsia abstrak introduction to date pre-eclampsia and eclampsia are still one of the complications of pregnancy, labor/delivery, or the postpartum period, and is also the main cause of maternal and fetal mortality and morbidity.(1) one of the targets of “making pregnancy saver” in indonesia for the years 2001-2010 is to reduce maternal and neonatal mortality rates to 125 per 100,000 live b i r t h s a n d 1 6 p e r 1 0 0 ,0 0 0 l i v e b i r t h s , respectively. current maternal and neonatal mortality rates in indonesia are still high, being 334 per 100,000 live births and 21 per 100,000 live births, respectively. the main causes of maternal mortality in indonesia are pree c l a m p s i a , h e m o r r h a g e , a n d i n f e c t i o n . (2 ) worldwide, approximately 585,000 women die every year from pregnancies, 95% of them occurring in developing countries and 13% of maternal deaths is caused by hypertension d u r i n g p r e g n a n c y, p a r t i c u l a r l y i n p r e eclampsia.(2,3) pre-eclampsia is a pregnancy-specific syndrome marked by increased blood pressure and proteinuria in pregnancies of more than 20 weeks.(4) pre-eclampsia is still a disease of theories, as its pathogenesis is still not known with certainty, although all experts agree on the occurrence of vascular endothelial dysfunction and acute atherosis involving multiple organ s y s t e m s . (1 ) n e w e r c o n c e p t s f o c u s o n 45 involvement of free radicals presumably associated with endothelial dysfunction and increased lipid level due to peroxidation of the free radicals. the increase in lipid levels also increases the susceptibility of fatty acids to damage by free radicals from oxidative stress, and is marked by increased levels of 8isoprostane.(5) 8-isoprostane is a prostaglandinlike compound produced within the tissues mainly through esterification of arachidonic acid by non-enzymatic reactions catalyzed by free radicals, and its level is also raised in preeclampsia. 8-isoprostane is believed to be an indicator of oxidative stress. oxidative stress plays an important role in pre-eclampsia, in which an imbalance between pro-oxidants and antioxidants leads to endothelial dysfunction. one of the results of endothelial damage is i n c r e a s e d v a s c u l a r p e r m e a b i l i t y w i t h concomitant vascular leakage of micronutrients that are essential for the fetus.(6) among these micronutrients are amino acids that act as fetal growth factors and affect birth weight. various studies on the difference in isoprostane levels between women with normal pregnancies and t h o s e w i t h p r e -e c l a m p s i a h a v e s h o w n inconsistent results. a cross-sectional study involving normotensive pregnant subjects and those with pre-eclampsia showed total plasma 8-iso-prostane levels were not significantly elevated in pre-eclamptic women compared with control subjects during pregnancy.(7) other studies showed different results. women with pre-eclampsia had higher 8-isoprostane plasma levels than controls.(8) as there are no data on the relationship between serum 8-isoprostane levels and infant birth weight, it is deemed necessary to conduct a study on a possible association between serum 8-isoprostane levels and post-eclampsia birth weight. the purpose of the present study was to analyze and compare serum 8-isoprostane levels in pregnant women with pre-eclampsia and women with normal pregnancy and their association with infant birth weight. methods research design a cross-sectional study was undertaken between january and july 2011 at the dr. kariadi hospital in semarang. research subjects the study subjects were pregnant women with pre-eclampsia and normotensive pregnant women who had deliveries in the delivery ward of dr. kariadi hospital, met the inclusion criteria, and agreed to participate in this study. the inclusion criteria were: normotensive women or women with pre-eclampsia, single live intra-uterine pregnancy, without chronic systemic disorders, and non-smokers. based on a significance level of 0.05 and a correlation coefficient and power of 80%, the size of the s a m p l e w a s 2 2 . p r e g n a n c y s t a t u s w a s differentiated into 2 categories: a normotensive pregnancy was defined as a pregnancy with normal blood pressure, while pre-eclampsia is a hypertensive condition in a pregnancy of 20 weeks accompanied by proteinuria. isoprostane measurement serum 8-isoprostane is a lipid oxidation product used to evaluate the results of oxidative stress, determined in maternal serum by means of a specific elisa kit (cayman). the serum levels were expressed in pg/ml (bio-rad model 680). birth weight measument infant birth weight is the weight in grams of the unclothed baby immediately after birth, determined by means of calibrated baby scales (tanita). statistical analysis d i f f e r e n c e s i n 8 i s o p r o s t a n e l e v e l s between pre-eclampsia and normotensive women were assessed using independent t-test for normal distributed data, and the mannuniv med vol. 31 no.1 46 pramono, kristanto isoprostane increased in pre-eclampsia whitney test for non-normally distributed data. all tests were two-tailed with statistical significance assessed at the p-value of <0.05. all computations were done with spss software version 15.0. ethical clearance this study was approved by the ethics and research commission, faculty of medicine, diponegoro university, dr. kariadi central hospital, semarang. results characteristics of normal pregnant women and preeclamptic patients are shown in table 1. table 1 shows that the mean age of normotensive pregnant women was slightly higher than women with severe pre-eclampsia, but according to statistical analysis this age difference was not significant (p=0.71). in table 1 it is also apparent that mean systolic pressure in women with preeclampsia was over 160 mmhg, being 165.61 ± 23.51 mmhg, whereas in normotensive pregnant women it was within normal limits, being 120.73 ± 11.00 mmhg. mean diastolic pressure in women with pre-eclampsia was 107.45 ± 13.89 mmhg, while in the group of normotensive pregnant women it was 78.28 ± 5.39 mmhg. characteristics of infants born from women with pre-eclampsia and from normotensive women are shown in table 2. table 2. characteristics of infants of preeclamptic and normotensive pregnant women characteri stics pregnancy status p pre-ecclampsia (n=23) normotensive (n=29) gender* male 12 (52.2%) 17 (58.6%) 0.64 female 11 (47.8%) 12 (41.4%) bir th weight (g) ‡ 2832.60 ± 685.36 2984.11 ± 453.57 0.31 * χ2 test; ‡ mean ± sd table 1. characteristics of preeclamptic and normotensive pregnant women ¥mann-whitney test; *χ2 test; § unpaired t-test; ‡ mean ± standard deviation characteristics pregnancy status p pre-eclampsia (n=23) normotensive (n=29) age (years) ‡ 29.37 ± 7.96 29.77 ± 6.48 0.71¥ age of pregnancy (weeks) ‡ 39.11 ± 1.08 39.01 ± 1.25 0.62¥ pregnancy categor y 1 12 (52.2%) 11 (37.9%) 2 – 4 8 (34.8%) 15 (51.7%) ≥ 5 3 (13.0%) 3 (10.4%) 0.51 body weight (kg) ‡ 68.73 ± 1 2.61 61.42 ± 9.10 0.03¥ height (cm) ‡ 156.82 ± 4.48 154.13 ± 6.70 0.11§ body mass index ‡ 28.19 ± 5.72 25.95 ± 3.33 0.13¥ blood pressure (mmhg)‡ systolic 1 65.61 ± 23.51 120.73 ± 11.00 diastolic 1 07.45 ± 13.89 78.28 ± 5.39 hemoglobin con centration 12.26 ± 1.43 11.38 ± 1.39 0.06 47 in table 2 it is apparent that both in women with severe pre-eclampsia and in normotensive pregnant women, the majority of babies were o f m a l e g e n d e r. h o w e v e r, a c c o r d i n g t o statistical analysis, the difference in gender was statistically not significant (p=0.64). similarly, birth weight of infants from pre-ecclamptic mothers was lower in comparison with infants from normotensive mothers, although statistical test results showed non-significant differences in weight distribution (p=0.31). s e r u m 8 i s o p r o s t a n e l e v e l s o f preeclamptic and normal pregnant women are shown in table 3. table 3 shows that mean serum 8-isoprostane level of women with pree c l a m p s i a w a s 6 2 . 5 2 ± 1 2 . 1 9 , w h i c h i s significantly higher than mean serum 8isoprostane level of normotensive pregnant women of 28.64 ± 8.81 (p=0.04). table 4 presents a comparison of maternal serum 8-isoprostane levels by infant birth weight category. from table 4 it is apparent that serum 8-isoprostane levels of women with pre-eclampsia whose infants had a birth weight of ≥ 2500 gram were higher compared to those with infants of < 2500 gram birth weight. on the other hand, serum 8-isoprostane levels in the group of normotensive pregnant women whose infants had a birth weight of ≥ 2500 gram were similar to those whose infants weighed <2500 gram at birth. however, in both cases the differences were not statistically significant, at p=0.5 and p=0.9, respectively. discussion the present study found that serum 8isoprostane levels of women with pre-eclampsia were higher than those of normotensive pregnant women. these results are concurrent with a previous study by mandang et al., who also found an increased expression of in the placenta of women with pre-eclampsia.(8) this indicates that oxidative stress in pre-eclampsia is not only present in placental tissues, but also systemically. study results concomitant with the present study have also been reported by ouyang et al.,(9) who also found increased serum 8-isoprostane levels in women with preeclampsia. 8-isoprostane is considered to be a better marker for oxidative stress and tissue damage due to free radicals, as compared with other oxidative stress parameters, such as malondialdehyde (mda). in addition, 8i s o p r o s t a n e a l s o h a s a h i g h e r d e g r e e o f correlation with proinflammatory cytokines, in c o m p a r i s o n w i t h o t h e r o x i d a t i v e s t r e s s parameters.(9) the increased 8-isoprostane in p r e -e c l a m p s i a i s c a u s e d b y i n c r e a s e d phospholipase a2 activity, especially in table 3. serum 8-isoprostane levels (pg/ml) of preeclamptic and normal pregnant women *mann-whitney test table 4. maternal serum 8-isoprostane levels by (pg/ml) infant birth weight category *mann-whitney test univ med vol. 31 no.1 48 pramono, kristanto isoprostane increased in pre-eclampsia placental tissues.(10) moreover, leukocytes are r e p o r t e d l y c a p a b l e o f s y n t h e s i z i n g 8 isoprostane. incubation of leukocytes with plasma of pre-eclampsia patients yielded higher amounts of isoprostans-8 than did incubation of leukocytes with plasma of normotensive pregnant women.(11) the study of tsukimori et al. demonstrated that leukocytes of patients with pre-eclampsia produced significantly higher amounts of free oxigen radicals than leukocytes of normotensive pregnant women.(12) the results of the latter study and of previous studies support the hypothesis of increases in oxidative stress, both systemically and locally in the placenta, to explain the pathogenesis of pre-eclampsia. however, in contrast to the results of the present study, a previous study by ishihara reported that there was no significant difference between serum 8isoprostane levels of women with pre-eclampsia and normotensive pregnant women.(13) these d i ff e r i n g r e s u l t s a r e p r e s u m a b l y d u e t o differences in gestational age range of the study subjects. in ishihara’s study the gestational age range was 27-40 weeks, while in the present study the age range was 37-42 weeks. the difference in gestational age presumably affects the production of 8-isoprostane, because of differences in placental developmental stage at different gestational ages. in addition, there is the possibility that 8-isoprostane synthesis is influenced by the degree of pre-eclampsia suffered by the study subjects. in the study by ishihara, serum 8-isoprostane levels were not differentiated by degree of pre-eclampsia, in contrast with the present study, where all p a t i e n t s h a d s e v e r e p r e e c l a m p s i a . p r e eclampsia does not only affect the mothers, but also fetal intrauterine growth of their infants. previous studies have reported finding an increased risk of low birth weight and smallfor-gestational-age birth weight in infants born from women with pre-eclampsia in comparison w i t h t h o s e b o r n f r o m n o r m a l p r e g n a n t women.(13) in this study there was no significant difference between birth weight of infants born from women with pre-eclampsia and those born from normotensive pregnant women. the proportion of low infant birth weight in the group of women with pre-eclampsia in the present study was 11%, which is lower than that found in previous studies of low birth weight and small-for-gestational-age birth weight in infants born from women with pree c l a m p s i a , r a n g i n g f r o m 2 0 8 0 % . t h e difference is caused by the fact that in those previous studies, the majority of infants were preterm, whereas in the present study all infants w e r e f u l l t e r m . g u p t a e t a l . ( 1 4 ) f o u n d n o significant difference in markers of oxidative stress between patients with pre-eclampsia and normotensive pregnant women, and consider this to be due to differences in assessment methods, inadequate assessment methods, and to differences in sensitivity and specificity of t h e a s s e s s m e n t m e t h o d s . a s m e n t i o n e d p r e v i o u s l y, p r e e c l a m p s i a a l s o a ff e c t s intrauterine fetal growth. one of the parameters of intrauterine infant growth is infant birth weight, which is determined by length of pregnancy and fetal growth rate. szymonowicz et al.(15) have reported on the occurrence of an increased risk of infants with small-forgestational age birth weight in women with pree c l a m p s i a a s c o m p a r e d w i t h t h e r i s k i n normotensive pregnant women. in addition, these studies found a proportion of 20-80% for small-for-gestational-age infants in preeclampsia, whereas the present study found the smaller proportion of small-for-gestational-age infants in women with pre-eclampsia to be lower, namely 11.54%. this study also found no significant difference between infant birth weight from women with pre-eclampsia and that f r o m n o r m o t e n s i v e p r e g n a n t w o m e n . furthermore, the results of the present study are consistent with those of the study conducted by xiong et al.,(16) who also found no significant difference in infant birth weight from women 49 with pre-eclampsia and infants of gestational age of ≥ 37 weeks. a significantly lower infant birth weight in comparison with that of infants from normotensive pregnant women was only found in women with pre-eclampsia with infants of gestational age of <37 weeks. these findings m a y e x p l a i n t h e r e s u l t s o f t h e s t u d y b y szymonowicz et al., who found a lower birth weight in infants born from women with preeclampsia than that of infants born from normotensive pregnant women. the gestational a g e o f w o m e n w i t h p r e e c l a m p s i a a n d normotensive pregnant women in the study of szymonowicz et al.(14) was 28 weeks. in contrast to the study of szymonowicz et al., in the present study the gestational age for all subjects was 3742 weeks. the reason for these differing results is as yet unclear. based on the theory of placental ischemia as the underlying cause of preeclampsia, a decrease in uteroplacental and fetoplacental perfusion leads to a chronic reduction in fetal blood supply, resulting in impairment of intrauterine growth.(17,18) h o w e v e r, o t h e r s t u d i e s r e p o r t t h a t uteroplacental perfusion in women with third trimester pre-eclampsia does not differ from that in normotensive pregnant women, and may occasionally even be higher. the higher perfusion may be due to an increased cardiac output.(19) isoprostane is one of the factors responsible for placental vasoconstriction. the present study also found a slightly negative correlation between serum 8-isoprostane levels and infant birth weight. in addition, this study also found no significant difference in maternal serum 8-isoprostane levels between the group of women with infant birth weight of ≥2500 gram and those with infant birth weight of <2500 gram. this may be caused by the fact that infant birth weight is not solely the result of placental perfusion. sebire et al.(20) reported an association between placental histology and degree of pre-eclampsia, despite a reduction in placental perfusion, leading to the supposition of differences in compensatory mechanisms in uteroplacental perfusion between preterm and f u l l t e r m g e s t a t i o n a l a g e . o n e o f t h e compensatory mechanisms may be an increased n i t r i c o x i d e ( n o ) s e c r e t i o n r e s u l t i n g i n improved uteroplacental circulation through vasodilation in fullterm pregnancy,(21) but this has still to be confirmed by further studies. another possibility is that an earlier diagnosis of pre-eclampsia causes the fetus to be exposed to a longer period of hypoxia due to reduced p l a c e n t a l p e r f u s i o n f r o m i n c r e a s e d 8 i s o p r o s t a n e l e v e l s . i n w o m e n w i t h p r e eclampsia in the third trimester of pregnancy, increased isoprostane levels do not significantly affect fetal growth any more. the results of the present study shows the occurrence of increased oxidative stress in pre-eclampsia. this opens the possibility of administration of antioxidants for the management of pre-eclampsia. vadillo-ortega et al.(22) reported that administration of l-arginine and antioxidant vitamins may decrease the prevalence of preeclampsia in high-risk populations. based on this report, it may presumably be necessary to conduct further studies on the use of serum 8isoprostane levels in the first trimester of pregnancy as predictor of pre-eclampsia. the availablity of accurate markers in combination with administration of antioxidants may be expected to reduce both maternal and infant mortality and morbidity from pre-eclampsia. a limitation of this study is the determination of serum 8-isoprostane levels only, without determination of urinary 8-isoprostane levels. measurement of serum 8-isoprostane levels only does not give reflect actual 8-isoprostane levels in the body. determination of urinary 8isoprostane levels can provide a measure of 8isoprostane clearance, which may be used to explain the influence of high 8-isoprostane levels on the uteroplacental circulation. another limitation of this study is that it did not evaluate intakes of antioxidant vitamins nor blood antioxidant levels, such that it was not possible to evaluate the simultaneous effects of oxidants univ med vol. 31 no.1 50 and antioxidants on intrauterine fetal growth in pre-eclampsia. conclusion mean serum 8-isoprostane levels in preeclampsia are higher than those in normotensive pregnant women. it is recommended to conduct f u r t h e r s t u d i e s t o d e t e r m i n e w h e t h e r 8 isoprostane may be used as a predictive marker of preeclamsia. references 1. gupta s, aziz n, sekhon l, agarwal r, mansour g, li j, et al. lipid peroxidation and antioxidant status in pre-eclampsia: a systematic review. obstet gynecol surv 2009;64:750-9. 2. andrian g, wikjosastro gh, waspodo d. buku acuan nasional pelayanan kesehatan maternal dan neonatal. jakarta. yayasan bina pustaka sarwono prawiroharjo;2001. 3. staff ac, dechend r, pijnenborg r. learning from the placenta: acute atherosis and vascular remodeling in pre-eclampsia–novel aspects for atherosclerosis and future cardiovascular health. hypertension 2010;56:1026-34. 4. american college of obstetricians and gynecologists. acog practice bulletin: diagnosis and management of pre-eclampsia and eclampsia. int j gynaecol obstet 2002;77:6775. 5. pasouglu h, bulduk g, ogus e. nitric oxide, lipid peroxides (mda), an uric acid levels in pre-eclampsia and eclampsia. tohoku j exp med 2004;202:87-92. 6. desai p, rathod sp, garge v, mansuri z. evaluation of pro-oxidants and antioxidants in pre-eklampsia. j obstet gynecol ind 2003;53: 445-8. 7. barden a, beilin lj, ritchie j, croft kd, walters bn, michael ca. plasma and urinary 8-isoprostane as an indicator of lipid peroxidation in pre-eclampsia and normal pregnancy. clin sci 1996;91:711-8. 8. mandang s, manuelpillai u, wallace em. oxidative stress increases placental and endothelial cell activin a secretion. j endocrinol 2007;192:485–93. 9. ouyang yq, li sj, zhang q, cai hb, chen hp. interactions between inflammatory and oxidative stress in pre-eclampsia. hypertens pregnancy 2009;28:56-62. 10. sharma jb, sharma a, bahadur a, vimala n, satyam a, mittal s. oxidative stress markers and antioxidant levels in normal pregnancy and pre-eclampsia. int j gynaecol obstet 2006;94: 23-7. 11. zhao s, gu y, lewis df, wang y. predominant basal directional release of thromboxane, but not prostacyclin, by placental trophoblasts from normal and preeclamptic pregnancies. placenta 2008;29:81-8. 12. tsukimori k, nakano h, wake n. difference in neutrophil superoxide generation during pregnancy between pre-eclampsia and essential hypertension. hypertension 2007;49:1436–41. 13. ishihara o, hayashi m, osawa h, kobayashi k, takeda s, vessby b, et al. isoprostanes, prostaglandins and tocopherols in pre-eclampsia, normal pregnancy and non-pregnancy. free rad res 2004;913-8. 14. gupta s, agarwal a, sharma rk. the role of placental oxidative stress and lipid peroxidation in pre-eclampsia. obstet gynecol surv 2005;60: 807-16. 15. szymonowicz w, yu vyh. severe pre-eclampsia and infants of very low birth weight. arch dis child 1987;62:712-6. 16. xiong x, demianczuk nn, saunders ld, wang fl, fraser wd. impact of pre-eclampsia and gestational hypertension on birth weight by gestational age. am j epidemiol 2002;155:2039. 17. granger jp, alexander bt, linas mt, bennett wa, khalil ra. pathophysiology of preeclampsia: linking placental ischemia/hypoxia with microvascular dysfunction. microcirculation 2002;9:147-60. 18. zahumensky j. doppler flowmetry in preeclampsia. bratisl lek listy 2009;110:432-5. 19. gant nf, hutchinson ht, siiteri pk, macdonald pc. study of the metabolic clearance rate of dehydroisoandrosterone sulfate in pregnancy. am j obstet gynecol 1971;111:555-63. 20. sebire nj, goldin rd, regan l. term preeclampsia is associated with minimal histopathological placental features regardless of clinical severity. j obstet gynaecol 2005;25: 117-8. 21. rodrigo r, parra m, bosco c, fernández v, barja p, guajardo j, et al. pathophysiological basis for the prophylaxis of pre-eclampsia through early supplementation with antioxidant vitamins. pharmacol ther 2005;107:177-97. pramono, kristanto isoprostane increased in pre-eclampsia 51 22. vadillo-ortega f, perichart-perera o, espino s, avila-vergara ma, ibarra i, ahued r, et al. effect of supplementation during pregnancy with l-arginine and antioxidant vitamins in medical food on pre-eclampsia in high risk population: randomised controlled trial. bmj 2011;342: d2901.doi:10.1136/bmj.d2901. univ med vol. 31 no.1 c:\users\universa medicina\docu 37 abstract universa medicina interleukin-22-induced β defensin-2 expression by intranasal immunization with streptococcus pneumoniae rrgb epitopes diana chusna mufida1*, antonius dwi saputra2, bagus hermansyah3, dini agustina1, muhammad ali shodikin1, and yunita armiyanti3 background streptococcus pneumoniae causes pneumococcal disease, which is responsible for millions of deaths worldwide. various pneumococcal vaccine candidates have been developed to prevent s. pneumoniae infection, one of which is an epitope-based vaccine. this study aimed to prove that intranasal immunization with each of the five s. pneumoniae rrgb epitopes can induce a mucosal immune response by increasing the -defensin-2 concentration through upregulation of interleukin (il)-22 expression. methods an experimental laboratory study was conducted using 28 male wistar rats aged 3-4 months, that were randomly divided into 7 groups containing four rats each. group 1 was given 40 ml of phosphate-buffered saline (pbs) only (control group). group 2 was the adjuvant group that received 40 ml pbs containing 2 μg cholera toxin b (ctb), and groups 3-7 were immunized with 40 ml pbs containing a combination of adjuvant and one of the five different s. pneumoniae rrgb epitopes. the concentrations of il-22 and -defensin-2 from nasal rinse examination were measured by means of elisa. the kruskal-wallis test, followed by the mann-whitney post-hoc test were used for statistical analysis. results rats immunized with the adjuvant-epitope combination had significantly higher -defensin-2 and il-22 levels than the control group (p=0.030; p=0.018, respectively), according to the kruskal-wallis test. and the mannwhitney statistical test, showed there was a significant increase in bdefensin-2 and il-22 levels. conclusions intranasal immunization with epitope 1 of the s. pneumoniae rrgb can increase -defensin-2 expression significantly and has a greater potential to be developed into a pneumococcal vaccine. keywords: intranasal immunization, epitope-based vaccine, streptococcus pneumoniae rrgb protein, -defensin-2 original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.37-46 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1237 january-april, 2022 vol.41no.1 1department of microbiology, faculty of medicine, university of jember, jember 2undergraduate program, faculty of medicine, university of jember, jember 3department of parasitology, faculty of medicine, university of jember, jember *correspondence: diana chusna mufida department of microbiology, faculty of medicine, university of jember jl. kalimantan no.37, kampus bumi tegal boto, jember, jawa timur, indonesia 68121 phone and fax number: +6281334605168 /+62331-337877 email: chusna.fk@unej.ac.id orcid id: 0000-0001-7197-9816 date of first submission, october 3, 2021 date of final revised submission, march 3, 2022 date of acceptance, maret 8, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: mufida dc, saputra ad, hermansyah b, agustina d, shodikin ma, armiyanti y. interleu ki n-2 2-in duced  defensin-2 expression by intranasal immunization with streptococcus pneumoniae rrgb epitopes. univ med 2022;41:37-46. doi: 10.18051/univmed. 2022 .v41. 37-46 38 mufida, saputra, hermansyah, et al defensin-2 by intranasal immunization introduction streptococcus pneumoniae (pneumococcus) is a gram-positive opportunistic pathogen that colonizes the human upper respiratory tract (urt ), spe cif ica lly the nasopharynx. s. pneumoniae can cause pneumococcal disease, including community-acquired pneumonia (cap), sinusitis, acute otitis media, sepsis or bacteremia, and meningitis.(1) in 2017, the world health organization (who) included s. pneumoniae in the 12 priority pathogens responsible for millions of deaths worldwide. pneumococcal pneumonia is a lung infection of high morbidity and mortality, especially in children under five years old, the elder ly above 65 years old, and immunocompromized individuals.(2) the primary transmission mode of s. pneumoniae is direct contact with secretions from the respiratory system. it is more frequent during the colder months with dry air, when respiratory secretions are excessive, and there often is coinfection of influenza and respiratory syncytial virus (rsv) with s. pneumoniae.(3) various pneumococcal vaccine candidates have been developed. currently, two types of licensed pneumococcal vaccines are available to prevent pneumococcal infection, namely a 23valent pneumococcal polysaccharide-based vaccine (ppv23) and a 13-valent pneumococcal conj ugate vaccine ( pcv 13). ppv23 is a capsular polysaccharide vaccine containing 23 serotypes that cause 90% of invasive pneumococcal disease. however, ppv23 only induces a t cell-independent immune response that is less effective in children under two years. pcv13 is a capsular polysaccharide vaccine conjugated with the non-toxic diphtheria toxin cross-reactive material 197 (crm 197 ). pcv13 can induce good antibody response, t celldependent response, and immunological memory even in infancy. nevertheless, this vaccine is more expensive and has limited serotype coverage.(4) both vaccines are invasive because they are administrated intramuscularly. until now, there are 98 serotypes of s. pneumoniae. these vaccines have limited coverage of pneumococcal serotypes and c ann ot prote ct against unencapsulated or non-vaccine serotypes of s. pneumoniae. this condition can lead to a rapid increase in antibiotic-resistant non-vaccine serotypes.(5) advances in prevention or treatment of pneumonia have been made with the development of vaccines and antibiotics. however, it is certain that such strategy may not be able to cover all of the 98 known serotypes of s. pneumoniae.(6) another vaccine candidate is an intranasal epitope-based vaccine that has more advantages than conventional vaccines, including high specificity, good safety and stability, and ease of production and storage.(7) the nasal mucosa is suitable for administering vaccines because it is an entry site for pathogens. besides, it has nasalassociated lymphoid tissue (nalt), which can help induce the mucosal and systemic immune systems. the intranasal epitope-based vaccine is expected to be more effective in protecting against s. pneumoniae infection with a broader spectrum of serotypes, to induce mucosal and systemic immunity, and to be affor dable and noninvasive.(7,8) previous in-silico studies have identified five epitopes of the s. pneumoniae rrgb (pilus backbone) protein with peptide lengths between 13-23 amino acids as a potential candidate for the pneumococcal vaccine.(9) the intranasal immunization epitope of the s. pneumoniae rrgb protein will be received by antigen-presenting cells (apc) and presented to naïve t cells through the t-cell receptor (tcr). furthermore, naïve t cells will differentiate into th1, th2, th17, and t reg. th17 will express ccr6 and produce various cytokines such as interleukin-22 (il-22), interleukin 17a (il-17a), interleukin-17f (il-17f), interleukin-26 (il-26), tumor necrosis factor-α (tnf-α), chemokine (cc motif) ligand 20 (ccl20), and granulocyte macrophage-colony stimulating factor (gmcsf). interleukin-22 is an important cytokine in mucosal immunity that can increase complement 3 (c3) deposition on s. pneumoniae, thereby increasing the phagocytosis of this pathogen by neutrophils. besides, il-22 can also increase 39 proliferation and repair of pulmonary epithelial cells after injury.(10) moreover, il-22 and il-17a are the most important cytokines that will synergistically induce the respiratory epithelial cells to produce antimicrobials like β-defensin-2, lipocalin-2 (lcn2), mucin (muc5b), and various chemokines such as cxcl5, cxcl9, ccl3, ccl-20, and g-csf which can prevent s. pneumoniae infection.(11,12) in a previous study by widiatmaja et al.,(13) there was no significant difference in il-4 levels of wistar rats induced with s. pneumoniae rrgb 225-270 protein. another indicator of immune response that we want to measure is β-defensin2 which is also an agent of innate, mucosal, and adaptive immunity that plays a role in preventing s. pneumoniae infection. in this connection, another study on wistar rats showed that intranasal immunization with the 54 kda hemagglutinin pilus proteins of s. pneumoniae enhanced the expression of β defensin 2.(14) we can use the measurement of β-defensin-2 expression in the respiratory mucosa to assess the mucosal immune response, which is important in protecting cells from s. pneumoniae infection.(15,16) there were inconsistent results in the previous studies, therefore, a study was performed to evaluate the effect of intranasal immunization on β defensin-2 through the upregulation of il-22 expression. the purpose of this study was to evaluate the effect of intranasal immuniza tion of the five s. pneumoniae rrgb epitopes as vaccine candidates for induction of the mucosal immune response by incre asi ng the β defensin-2 concentration through the upregulation of il-22 expression. methods research design this study was of an experimental in-vivo post-test only control group design and was carried out in the microbiology, pharmacology, and biochemistry laboratories, faculty of medicine, university of jember, from january to december 2020. epitopes of s. pneumoniae rrgb protein this study used the five epitopes of s. pneumoniae rrgb protein identified from previous in-silico studies as potential candidates for a pneumococcal vaccine. epitope 1 contains 23 amino acids (vnhqvgdvveyeivtkipa lany), epitope 2 comprises 16 amino acids (tvkvtvddvaleagdy), epitope 3 has 19 amino acids (tfdlvnaqagkvvqtvtlt), epitope 4 consists of 14 amino ac ids (pk vvt ygk kfvk vn) , and epitope 5 comprises 13 amino acids (dravaaynal taq).(8) these epitopes were designed by and purchased from pt. genetika science indonesia. intranasal immunization the sample of 28 male wistar strain rats aged 3-4 months was randomly divided into 7 groups, each consisting of 4 animals. group 1 was the control group and received 40 ml of phosphate buffered saline (pbs). group 2 was the adjuvant group and received 40 ml pbs containing 2 μg cholera toxin b (ctb). groups 3-7 were immunized with 40 ml pbs containing a combination of adjuvant and an antigen of the five different s. pneumoniae rrgb epitopes. a 20ml volume of the adjuvant-epitope combination was administered in each nostril. all rats were immunized on days 0, 7, and 14.(16) all animal studies were conducted following guidelines and approval from the institutional biosafety committee and institutional animal care and use committee, faculty of medicine, university of jember. nasal rinse isolation seven days after the third immunization, the rats were anesthetized with 10% ether by inhalation. after that, the rats were sacrificed by decapitation. nasal rinse isolation was performed on a retrograde basis by inserting 1–2 ml of sterile saline through the trachea. we accumulated the univ med vol. 41 no 1 40 mufida, saputra, hermansyah, et al defensin-2 by intranasal immunization c droplets of saline solution from the nostrils into a sterile eppendorf tube. il 22 and β defensin 2 concentrations il 22 and β defensin 2 concentrations in the nasal rinse were measured with an enzyme linked immunosorbe nt assa y (elisa) kit from elabsc ience ® acc ording to the specified procedure. 100 µl samples were loaded into the wells, incubated for 90 minutes at 37oc, then the nasal rinse liquid and 100 µl of il 22 or β defensin 2 antibodies were added. after having been left to stand for 1 hour at 37oc, the plate was rinsed with the washing solution. then, 100 µl of horseradish peroxidase (hrp) conjugate was added and the plate incubated for 30 minutes at 37°c. next, the plate was rinsed with the washing solution and 50 ml of substrate reagent was added and the plate incubated for 15 minutes at 37°c. the reaction was then quenched with 50 µl of the stop solution. color expression was measured by optical density at a wavelength of 450 nm with an elisa reader. statistical analysis descriptive data were presented as mean ± standa rd deviation. the between-group differences in il 22 and β defensin 2 mean concentrations were assessed by the kruskalwallis test, followed by the mann-whitney posthoc test. the significance of the data is indicated by a p-value of <0.05.(17) ethical clearance the ethics committee of the faculty of medicine, university of jember, approved this study under no. 1313/h25.1.11/ke/2019. results the concentrations of il 22 and β defensin 2 in the nasal rinse were measured by the elisa method and the results are presented in table 1. rats immunized with the adjuvant-antigen combination had a higher concentration of βdefensin-2 than the control group, but there was no significant increase in il-22 concentrations. the kruskal-wallis test showed that the mean concentrations of il-22 (p=0.030) and βdefensin-2 (p=0.018) were significantly different between all groups. the test was followed by the mann-whitney post-hoc test to determine the groups with a significant difference. the mann-whitney test results for the il 22 concentrations are presented in table 2, showing that the epitope 4 group had the lowest concentration that was significantly different from the other groups. the results of the mannwhitney test for the β defensin 2 concentrations are shown in table 3. there was a statistically significant difference between the mean βdefensin-2 concentration in the control and the other treatment groups. these results prove that intranasal immunization, both with adjuvant alone and with a combination of one of the five epitopes and adjuvant, can significantly increase the concentration of β-defensin-2 in the respiratory tract mucosa of wistar rats. we can also see that the mean concentration of β-defensin-2 in the epitope 1 group was significantly different from that in the control group and the epitope 3, epitope 4, and epitope 5 groups. these results indicate that epitope 1 of the s. pneumoniae rrgb 214236 pilus consisting of the 23 amino acids vnhqvgdvveyeivtkipalany as the antigen in combination with the adjuvant can increase β-defensin-2 expression significantly compared to the other epitopes. discussion β-defe nsin-2 is a very important antimicrobial in innate and adaptive immunity to prevent s. pneumoniae inf e ction. t he mechanism of β defensin 2 as an antimicrobial is through several ways: (a) bacterial membrane depolarization; (b) the formation of holes in the membranes of microorganisms resulting in cell leakage; (c) activating the enzymes and pathways for the degradation of bacterial walls; (d) damaging the cell wall; and (e) preventing attachment of bacter ia to the host cell 41 univ med vol. 41 no 1 t ab le 1 . d is tr ib u ti o n o f il -2 2 a n d β -d ef en si n -2 l ev el s in t h e n as al r in se o f ra ts a ft er in tr an as al i m m u n iz at io n , b y t re at m en t gr o u p s n o te : c : co n tr o l. a : ad ju va n t; e 1 : ep it o p e 1 ; e 2 : ep it o p e 2 ; e 3 : ep it o p e 3 ; e 4 : ep it o p e 4 ; an d e 5 : ei to p e 5 ; il : in te rl eu ki n e p it o p e 1 v n h q v g d v v e y e iv t k ip a l a e p it o p e 2 t v k v t v d d v a l e a g d e p it o p e 3 t f d l v n a q a g k v v q t v e p it o p e 4 p k v v t y g k k f v k v n e p it o p e 5 d r a v a a y n a l t a q 42 mufida, saputra, hermansyah, et al defensin-2 by intranasal immunization treatment group control adjuvant epitope 1 epitope 2 epitope 3 epitope 4 epitope 5 control adjuvant 0.02* epitope 1 0.02* 0.08 epitope 2 0.04* 0.56 0.08 epitope 3 0.02* 0.39 0.04* 0.77 epitope 4 0.02* 0.39 0.02* 0.77 0.39 epitope 5 0.02* 0.77 0.04* 1.00 0.77 0.77 (*) means that there is a significant difference (p <0.05) epitope 1: vnhqvgdvveyeivtkipala; epitope 2: tvkvtvddvaleagd; epitope 3: tfdlvnaqagkvvqtv; epitope 4: pkvvtygkkfvkvn; epitope 5: dravaaynaltaq table 3. results of the mann-whitney test of β defensin 2 concentrations treatment group control adjuvant epitope 1 epitope 2 epitope 3 epitope 4 epitope 5 control adjuvant 0.56 epitope 1 0.77 0.24 epitope 2 0.25 0.08 0.39 epitope 3 0.15 0.15 0.08 0.04* epitope 4 0.02* 0.02* 0.02* 0.02* 0.04* epitope 5 0.47 0.24 0.56 0.39 0.25 0.15 table 2. results of the mann-whitney test of il 22 concentrations (*) means that there is a significant difference (p <0.05) epitope 1: vnhqvgdvveyeivtkipala; epitope 2: tvkvtvddvaleagd; epitope 3: tfdlvnaqagkvvqtv; epitope 4: pkvvtygkkfvkvn; epitope 5: dravaaynaltaq epithelium.(15) β-defensin-2 also has a role in adaptive immunity: (a) chemotactic activity to recruit immature dendritic cells and memory t cells from the circulation through the ccr1 and ccr5 and ccr6 receptors; (b) formation of defensin-a ntigen complexes that aid the presentation to dendritic cells; (c) induction of the maturation of dendritic cells by β-defensin-2, resulting in the production of il-2, tnf-α, and il-1 by macrophages and monocytes.(18) the regulation of β-defensin-2 expression in the respiratory epithelial cells involves multiple signaling pathways, including phosphatidylinositol3-kinase (pi3k), mitogen-activated protein kinases (mapks), and protein kinase c (pkc). the bacterial antigens will induce the expression of β-defensin-2 in the respiratory epithelial cells through nuclear factor interleukin 6 (nf-il6), the transcription factor nf-κb, myeloid elf-1-like factor (mef), or activated protein 1–3 (ap1–3). other factors that induce β-defensin-2 expression are the toll-like receptors (tlrs) 1–6 in intracellular vesicles and on the epithelial surface, and tlr 3,7,8,9 in endosomes or the endoplasmic reticulum. a previous study found that pulmonary epithelial cells inf ected with legionella pneumophila produced def ensin-2. its expression was mediated by tlr2 and tlr5 receptors, then activated via the mapk (p38, jnk), transcription factor nf-κb, and ap-1 pathways.(18) β-defensin-2 is expressed mainly in all epithelial layers of the human body, including the conjunctiva, respiratory tract, digestive tract, urogenital tract, mucous membranes, skin, and peripheral blood. in the respiratory tract, βdefensin-2 is induced by bacterial, viral, or fungal infection and proinflammatory cytokines such as interferon-gamma (ifn-γ), tumor necrosis factor-alpha (tnf-α), interleukin 1α (il-1α), 43 interleukin 1β (il-1β), and interleukin 6 (il-6). β-defensin-2 is expressed in the mucous membrane from the oral cavity to the epithelium of the lungs.(18) a previous study showed that cytokines il-17 and il-22 produced by th17 cells are more powerful in controlling the expression of β-defensin-2 on mucosal surfaces than are other cytokines.(14) interleukin-22 is an il-10 family cytokine produced by the lymphoid lineage cells cd4+ and cd8+ t cells, nk cells, γδ t cells, and innate lymphoid cells (ilcs). in mucosal immunity, th17 cells are a significant source of il-22. il-22 has two heterodimeric transmembrane receptors, namely il-10r2 an d il-22r1, which subsequently activate the erk, jak/stat3, and jnk pathways that induce the expression of βdefensin-2. various factors regulate il-22 secretion. the main inducers of il-22 are il-23 and il-1β, which are produced by dendritic cells and macrophages. on the contrary, the expression of il-22 is inhibited by tgf-β, inducible costimulator (icos), and il-27.(19) interleukin-22 is essential for human immunity against extracellular pathogens, such as s. pneumoniae. the function of il-22 can be categorized into three major areas. first, il-22 initiates epithelial proliferation that maintains and restores the integrity of the epithelial barrier function during the invasion of pathogens. second, il-22 induces the production of inflammatory mediators such as il-1β, il-6, serum amyloid a, granulocyte-colony stimulating factor (g-csf), and lipopolysaccharide-binding protein. third, il22 synergistically with other cytokines, such as il-17 or tnf-α, induces the expression of antimicrobial peptides (amps), including βdefensin-2, β-defensin-3, s100a7, s100a8, and s100a9, that are critical for mucosal immunity in the airways, the skin and the intestine.(20) in our study, there was a statistically significant difference between the mean βdefensin-2 concentrations in the control group compared to the other treatment groups. this signifies that intranasal immunization of adjuvant alone or a combination of each of the five different epitopes of s. pneumoniae rrgb pilus with an adjuvant can significantly increase the concentration of β-defensin-2 in the respiratory tract mucosa of wistar rats. the mean βdefensin-2 concentration in the epitope 1 group was the highest and significantly different from that in the control group and the epitope 3, epitope 4, and epitope 5 groups. these results indicate that a combination of adjuvant and epitope 1 of the s. pneumoniae rrgb 214-236 p il u s c o nt a i n i ng t h e 2 3 a min o a c i d s vnhqvgdvveyeivtkipalany as the antigen can increase β-defensin-2 expression significantly and has a greater potential to be developed into a pneumococcal vaccine than have the other epitopes. previous studies have shown that synthetic epitope-based vaccines usually must consist of 20-30 amino acids to induce a good immune response. in addition, the longer cytotoxic t lymphocyte epitope can expand the target range to increase the peptide’s i mmu no ge ni c i t y. in t hi s s t ud y, t h e se characteristics are found in epitope 1, which has 23 amino acids and is the longest epitope compared to the other four epitopes.(21) there has been no research about the effect of intranasal immunization with rrgb epitopes on β-defensin-2 concentrations. a previous study that can be used for comparison is the research by mufida et al.,(14) in which there was an increase in β-defensin-2 concentration through the upregulation of il-22 and il-17a expression in the nasal rinses of wistar rats immunized intranasally with the 54 kda s. pneumoniae hemagglutinin pilus which is identical to the rrgb protein. in that study, the concentration of β-defensin-2 in the adjuvant group was not significantly different from the control group, therefore it was concluded that the cholera toxin subunit b (ctb) adjuvant does not increase β-defensin-2 expression and only acts as an immunomodulator. these results differ from those of the present study, where the β-defensin-2 concentration in the adjuvant group was relatively high and significantly different from that of the control group. univ med vol. 41 no 1 44 mufida, saputra, hermansyah, et al defensin-2 by intranasal immunization cholera toxin subunit b is a non-toxic homopentamer protein antigen, that is often used as an adjuvant for mucosal vaccines, both intranasal and oral. cholera toxin subunit bbased vaccines can elicit a stronger mucosal immunity because ctb has high affinity for monosia lotetra hexosylganglioside (gm1) receptors on leukocytes and mucosal epithelium, es pec ial ly m ce lls . (2 2) be si de s, ct b c a n increase antigen uptake in the mucosa and stimulate the expression of cd40, cd80, and cd86 and the secretion of il-1 and il-6 by apcs such as b cells, dendritic cells, and macrophages. this feature is intended to reduce the minimum antigen dose for causing an siga and igg adaptive immune response. cholera toxin subunit b has a low direct effect on th17 cells, but other studies have shown that ctb stimulates the secretion of il-17a by cd4 + t cells and the differentiation of th17 cells. mature th17 cells do not express il-22 but express il-17f and il-17a. ctb via the camp-pka pathway can induce the secretion of il-6, tgf-β, il-17a, and gm-csf.(11) il17a is an essential cytokine that induces the secretion of β-defensin-2 in the epithelial cells of the respiratory tract mucosa. the ability of il-17a to stimulate β-defensin-2 expression has b e e n i nve s t iga t e d i n -vi t r o i n h uma n tracheobronchial tissue. it is proven that il-17a induces a stronger β-defensin-2 expression than other cytokines, especially tnfα, il-1α, il-1β, and il-6.(16) in our study, ctb administration as an adjuvant may have increased il-17a, il6, and tgf-β secretion that in turn may have raised β-defensin-2 concentration. the increase in tgf-β can inhibit il-22 expression. a previous study found that increased tgf-β leads to the loss of th22 cells that produce il-22 in the intestinal mucosa.(23) similarly, in our study there was an increase in β-defensin-2 concentration in the adjuvant group, but no increased il-22 concentration in all groups. the limitations of this study are that the me c h a ni s m a nd p a t h wa y un de rl yi n g t he improvement of β-defensin-2 expression after immunization with s. pneumoniae rrgb pilus epitopes are still unclear. bacterial antigens such as the epitopes of the s. pneumoniae rrgb pilus can induce the expression of β-defensin-2 in the respiratory epithelial cells to involve multiple signaling pathways through the nf-il6, nf-κb, mef, ap1–3, pi3k, mapks, or pkc and stimulate proinflammatory cytokines such as il22, il-17a, il-1α, il-1β, il-6, or tnfα, and ifn-γ. (1 8) to kn ow the mechanism of the increase in β-defensin-2 expression by the rrgb epitopes, further research is needed to measure the concentration of other proinflammatory cytokines that induce β-defensin-2 expression. another limitation is that there is no group immunized with antigen alone; in further studies it is necessary to include such a group to ensure that the increase in β-defensin-2 is caused only by antigen. external factors such as inflammation and infection can affect the expression of βdefensin-2, such that routine health examinations on the experimental animals need to be carried out. the selection of adjuvant also needs to be considered, because ctb as an adjuvant may stimulate il-17a, il6, and tgf-β secretion that increases β-defensin-2 concentration.(11) all these factors can affect the results of the study. the study by gentile et al.(24) also proved that intraperitoneal immunization with the s. p ne u mo ni a e rr gb pr ot e i n s i gni f i c a nt l y increased serum igg and protected experimental animals from bacteremia. these results indicate that intranasal immuniza tion with the s. pneumoniae rrgb pilus epitope in our study also may have the potential to increase mucosal and systemic immunity. the high concentration of β-defensin-2 in nasal rinses of wistar rats, especially in the epitope-1 group indica tes that intranasal immunization with the s. pneumoniae rrgb protein epitope can increase mucosal immunity and potentially protect against s. pneumoniae infection. in future studies to prove the rrgb pr otein epitope as a ca ndidate for a new pneumococcal vaccine, the siga concentration in the respiratory tract mucosa and the serum 45 igm or igg concentrations should be measured to determine the adaptive mucosal immune response and the systemic immune response, respectively. our present study is expected to be the beginning of a new breakthrough to solve the problem of pneumococcal vaccines and s. pneumoniae infection. conclusions this study demonstrated that intranasal immunization with the s. pneumoniae rrgb epitope could induce a mucosal immune response by increasing β-defensin-2 expression in the nasal rinse of wistar rats. combining adjuvant and the s. pneumoniae rrgb 214-236 pilus epitope-1 as antigen comprising the 23 amino ac ids vnhqvgdvv eyeiv t kipalany can increase β-defensin-2 expression significantly and has a greater potential to be developed into a pneumococcal vaccine compared to the other epitopes. conflict of interest there is no conflict of interest in the writing and publishing of this manuscript. acknowledgement t he r e s ea r c h e r s t h a nk t he is la mi c development bank (idb) that has funded this study and the faculty of medicine, university of jember, for the opportunity given in this research. contributors dcm, bh, da, mas, and ya contributed to the design of this study. dcm, bh, and ads contributed to performing experiments and data collection and analysis. dcm, bh, ads, da, mas, and ya contributed to the article’s writing and critical revision, and approved the final manuscript. references 1. eze ua, mustapha a, nworie a. streptococcus pneumoniae: virulence factors and their role in pathogenesis. cont j med res 2013;7:27–45. doi: 10.5707/cjmedres.2013.7.1.27.45. 2. weiser jn, ferreira dm, paton jc. streptococcus pneumoniae: transmission, colonization and invasion. nat rev microbiol 2018;16:355-67. doi: 10.1038/s41579-018-0001-8. 3. brooks lrk, mias gi. streptococcus pneumoniae’s virulence and host immunity: aging, diagnostics, and prevention. front immunol 2018;9 : 1-29. https://doi.org/10.3389/ fimmu.2018.01366. 4. chang ms, woo jh. t he prevention of pneumococcal infections. clin exp vaccine res 2016;5:3. doi: 10.7774/cevr.2016.5.1.3. 5. masomian m, ahmad z, gew lt, poh cl. development of next generation streptococcus pneumoniae vaccines conferring broad protection. vaccines 2020;8:1–23. doi: 10.3390/ vaccines8010132. 6. geno ka, gilbert gl, song jy, et al. 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mucosal vaccine-induced protection against h. pylori in mice. mucosal immunol 2017;10:271-81. doi: 10.1038/mi.2016.38. 21. yang h, kim ds. peptide immunotherapy in vaccine development: from epitope to adjuvant. adv protein chem struct biol 2015;99:1-14. doi: 10.1016/bs.apcsb.2015.03.001. 22. stratmann t. cholera toxin subunit b as adjuvant—an accelerator in protective immunity and a break in autoimmunity. vaccines 2015;3:579– 96. doi: 10.3390/vaccines3030579. 23. leung jm, davenport m, wolff mj, et al. il-22producing cd4+ cells are depleted in actively inflamed colitis tissue. mucosal immunol 2014;7: 124–33. https://doi.org/10.1038/mi.2013.31. 24. gentile ma, melchiorre s, emolo c, et al. structural and functional characterization of the streptococcus pneumoniae rrgb pilus backbone d1 domain. j biol chem 2011; 286: 14588–97. doi: 10.1074/jbc.m110.202739. frailty decreases physical health domain of quality of life in nursing home elderly 213 *department of public health, faculty of medicine and center for health research, atma jaya catholic university of indonesia, jakarta **department of neurology, faculty of medicine and center for health research, atma jaya catholic university of indonesia, jakarta correspondence prof dr. drg. yvonne suzy handajani, mkm department of public health, atma jaya catholic university of indonesia, jakarta jl. pluit raya no.2, jakarta utara 14440 mobile: +62816 135 3738 email: yvonne.sh@gmail.com univ med 2015;34:213-9 doi: 10.18051/univmed.2016.v35.213-219 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background approximately 10-27% of the population aged >65 years suffers from frailty. the percentage increases with age so that the prevalence of frailty in the population aged >85 years reaches 45%. the objective of this study was to determine the relationship between frailty and quality of life (qol) in nursing home elderly. methods this was a cross-sectional study of 138 subjects aged >60 years who were recruited from 4 nursing homes in west jakarta. participants with frailty status were evaluated by the survey of health, ageing and retirement in europe (share) instrument and qol was evaluated by the whoqol-bref questionnaire. one-way anova and chi-square tests were used to find relations between the frailty syndrome and qol. results the percentages of respondents with pre-frail, frail, and non-frail status were 30.4%, 52.2%, and 17.4%, respectively. a decline in qol scores of pre-frail and frail respondents was found for almost all qol domains (physical, psychological and environment domains), except social relationships. the subdomains most influenced were “energy and fatigue” in the physical health domain, “thinking, learning, memory and concentration” in psychological health, and “opportunities for acquiring new information and skills” in the environment domain. conclusions more than half of the nursing home elderly were frail and one-third were pre-frail. the main factor of frailty was weakness. the frailty syndrome in the elderly has a negative impact on qol, especially in the physical health, psychological and environment domains in nursing home elderly. keywords: frailty syndrome, quality of life, elderly, nursing homes frailty decreases physical health domain of quality of life in nursing home elderly yvonne suzy handajani*, nelly tina widjaja*, and yuda turana** doi: http://dx.doi.org/10.18051/univmed.2015.v34.213-219 214 handajani, widjaja, turana frailty decreases physical health frailty menurunkan kualitas hidup pada domain kesehatan fisik pada lanjut-usia di panti werdha latar belakang sekitar 10-27% populasi lanjut usia (lansia) berusia >65 tahun menderita frailty. presentasenya meningkat dengan bertambahnya usia sehingga prevalensi frailty pada populasi berusia >85 tahun mencapai 45%. tujuan dari penelitian ini adalah untuk menentukan hubungan antara frailty kualitas hidup pada lansia di panti werdha. metode penelitian ini merupakan studi cross-sectional pada 138 subyek dengan usia >60 tahun yang didapatkan dari 4 panti werdha di jakarta barat. status frailty subyek diukur dengan survey of health, ageing and retirement in europe (share) dan kualitas hidup dilakukan penilaian dengan kuesioner whoqol-bref. uji one way anova dan uji chi-square digunakan untuk mendapatkan hubungan antara sindrom frailty dan kualitas hidup. hasil persentase responden dengan status pre-frail (30,4%), frail (52,2%) dan normal (17,4%). penurunan skor kualitas hidup lansia dengan status frailty dan pre-frail ditemukan hampir di semua domain kualitas hidup (domain fisik, psikologis dan lingkungan), kecuali domain hubungan sosial. subdomain yang paling dipengaruhi adalah “energi dan kelelahan” pada domain kesehatan fisik, “berpikir, belajar, memori dan konsentrasi” pada domain kesehatan psikologis, serta “peluang untuk memperoleh informasi dan keterampilan baru” pada domain lingkungan. kesimpulan lebih dari setengah lansia mengalami frailty dan sepertiga lansia dengan status pre-frail di panti werdha. faktor utama frailty adalah kelemahan. sindrom frailty pada lansia berdampak negatif pada kualitas hidup, khususnya pada domain kesehatan fisik, psikologis dan lingkungan pada lansia di panti werdha. kata kunci: sindrom frailty, kualitas hidup, lansia, panti werdha abstrak introduction an aging population is a challenge that affects both the developed and developing countries. the growth of the elderly population needs resources and health services to take care of.(1) lately, geriatricians and gerontologists have been focusing their attention on frailty in the elderly, which is increasing significantly. approximately 10-27% of the population aged >65 years is suffering from frailty (2) and the percentage increases with age, so that the prevalence of frailty in the population aged >85 years reaches 45%.(3) several studies in europe found that 61.8% of the elderly population were s u ff e r i n g f r o m f r a i l t y. (2) f r a i l t y i s a heterogeneous clinical syndrome that may include several different medical conditions, such as cardiovascular disease, musculoskeletal disorders (arthritis, osteoporosis and fractures), gastrointestinal disease and cognitive disorders.(4) muscular strength, physical performance, nutritional status and psychological status are the parameters that are useful to evaluate the frailty status of elderly.(5) several studies have found a significant relationship between frailty and quality of life (qol), with lower qol scores in respondents with frailty.(6) similar results were also found in the taiwanese elderly population.(7) 215 there has been little research on the effect of frailty on the qol of elderly residing in nursing homes. the objective of the present study was to determine the relationship between frailty and qol in nursing home elderly. methods research design the design of this study was cross sectional and the study was conducted between april 2014 and december 2015 at four nursing homes in west jakarta. research subjects the size of the sample was calculated based on the formula of the sample size to test a planned proportion at 95% confidence level to achieve a 5% margin of error for the study. from previous studies, the prevalence of frailty was known to be 24.74% in elderly <85 years old and 45% in elderly >85 years old. from the results of these calculations, the minimum sample size was 132. this study comprised 138 subjects aged 60-95 years and living in four nursing homes in west jakarta (panti sasana tresna werdha budi mulia jelambar, panti usila santa anna, panti sosial tresna budi mulia 2, panti sosial tresna werdha usada mulia 5). the subjects were recruited through the head of each nursing home, according to the inclusion and exclusion criteria. all of them gave signed informed consent. measurements frailty was measured by means of the survey of health, ageing and retirement in europe (share) instrument. (8) t h e computations were done using two share-fi calculators, one for males and one for females, which assessed the following five factors: 1) fatigue; 2) loss of appetite; 3) grip strength; 4) functional difficulties (walking 100 m and climbing stairs) and 5) physical activity. based on these factors, frailty was categorized into three groups, i.e. normal, pre-frail and frail. quality of life measurement was performed w i t h w h o q o l b r e f c o n s i s t i n g o f 2 6 questions and 4 domains: 1) physical health; 2) psychological; 3) social relationships; 4) environment and also to assess overall quality of life and satisfaction about health. this is a valid and reliable instrument to measure qol in the elderly.(9,10) statistical analysis the one-way anova test was used to analyze the relationship between qol and frailty status. the chi square test was used to analyze the effect of overall qol, satisfaction about health and the characteristics of respondents on frailty status. a p-value lower or equal to 0.05 (<0.05) was considered as statistically significant. ethical clearance the research had been approved by ethical clearance committee, faculty of medicine, atma jaya catholic university of indonesia on 3 april 2014. results based on the characteristics, 51.4% of the respondents were female, 81.2% were >65 years old, 64.5% had elementary education or lower, and 55.8% were divorced or widowed. this study found that the percentages of normal or non-frail, pre-frail, and frail respondents were 17.4%, 30.4%, and 52.2%, respectively. regarding overall qol and satisfaction about health, 16.6% of respondents had poor qol, 47.1% had sufficient qol, and 36.3% good qol, while 28.3% was not satisfied, 28.3% moderately satisfied and 43.5% satisfied about their health. the assessment of the five components of frailty resulted in 34.8% with exhaustion, 22.5% with loss of appetite, 53.6% were weaker on the right handgrip and 49.3% on the left handgrip, 56.5% had difficulty in walking and climbing stairs, and 36.2% had never done physical activity (table 1). univ med vol. 34 no.3 216 handajani, widjaja, turana frailty decreases physical health the results of the analysis between frailty status and each of the qol domains showed that respondents who were pre-frail had a qol score for physical health of 2.10 points, lower than the qol score in normal (non-frail) respondents (p=0.018). similarly, respondents who were frail had a qol lower than that in normal respondents (p=0.018). from these results we can conclude that pre-frail and frail respondents had worse qol scores for the physical health domain than normal respondents. table 2. distribution of the means of four qol domains and total quality of life by frailty status in elderly the pre-frail respondents had lower qol scores for the psychological domain of 1.58 points, as compared to those who were normal (p=0.024). this means that the pre-frail respondents did not have better qol for the psychological domain as compared to those who were normal. respondents with pre-fail or frail health status did not show a significant relationship with qol for the domain of social relations (p=0.228). the respondents with frail health status had qol scores for the environment domain of 2.79 points, lower than those in the normal (non-frail) respondents (p=0.007). it can be concluded that frail respondents have worse qol scores for the environment domain compared to the normal (non-frail) respondents (table 2). post-hoc analysis showed that in elderly who were frail the physical health, environment and psychological domain was significantly declined compared to normal elderly (table 3). the chi square test for frailty on overall qol and satisfaction about health, resulted in significant relationships (p=0.035; p=0.009). the largest percentage of those with low scores for qol and satisfaction about health of 53.4% and 55.1%, respectively, was found in frail respondents, followed by those who were pre-frail, with respective scores of 35.2% and 34.6% (table 4). discussion the respondents in this study comprised 138 elderly living in nursing homes in west jakarta. they were mostly women aged >65 years, had elementary education or lower, and most of them were divorced or widowed. frailty is a health condition that deals with aging and dependence. table 1. demographic characteristics, frailty status, overall quality of life, and satisfaction about health in elderly 217 table 3. post-hoc analysis of physical health, psychological and environment domain with frailty status table 4. frailty, overall quality of life and satisfaction about health in elderly a reduction or delay in frailty status can improve the quality of life of elderly. in this research, the percentages of respondents with pre-frail, frail, and non-frail status were 30.4%, 52.2%, and 17.4%, respectively. research conducted in taiwanese communities found that 9.9% elderly were frail, 44.5% pre-frail and 45.6% non-frail. a crosssectional study showed that about 7% of elderly aged 65 years was suffering from frailty and the number would have increased to over 45 % after the age of 85 years.(11) based on fried’s criteria about frailty, there were 5.9% frail, 62.8% prefrail and 31.3% non-frail subjects among elderly who received health services in taiwan.(12) based on the findings above, the number of elderly with frailty in this research was found to be greater than that in other studies. this is because the respondents were living in nursing homes so that their daily activities were less than those of elderly who are living in the community. this statement is supported by barthalos et al (2012) who informed that lifestyle and nursinghome dwelling with slight variations in daily activities negatively affect the status of physical fitness, body composition, and quality of life. selfmotivation, active lifestyle, regular and varied programs seem to have a major role in the quality of life of the elderly population.(13) an advanced age was predicted relating to sensory, motoric and cognitive changes that potentially prevent the elderly to function effectively.(14) in advanced age, the physiological system will have abnormalities in structure and function. age-related physiological changes influence many tissues, organ systems and functions, and cumulatively can impact on activities of daily living (adl).(15) the findings of fried et al.(16) showed that frailty was associated with a significant reduction in qol. the results of other studies are consistent with their findings. other investigators discovered that frail subjects had worse overall qol than pre-frail and non-frail subjects.(17,18) lin et al. reported that elderly who did not experience weakness (frailty) significantly had better health compared to the elderly with pre-frail and frail conditions at all scales. similarly, those with prefrail status had reportedly better qol than those with a frail condition. similarly, frail elderly had significantly worse health related quality of life (hrqol) than non-frail elderly in the same population.(7) other findings reported that outpatient subjects at health centers in taiwan with frail status had significantly lower qol scores (on physical and mental health scale) as compared to the non-frail subjects.(12) bilotta et al.(19) discovered a negative relationship between frailty status and qol of older subjects, measured using the older people’s quality of life (opqol) questionnaire. nearly all qol dimensions correlated inversely with frailty, except for “social univ med vol. 34 no.3 218 handajani, widjaja, turana frailty decreases physical health relations and participation” as well as “financial circumstances”. the findings of the present study indicate that respondents suffering from pre-frail and frail condition had lower qol scores for the physical health domain, from the normal (non-frail) respondents. likewise, the psychological domain score was lower in pre-frail respondents. in the environment domain score was lower in frail respondents, while in the domain of social relationships, the relationship was not statistically significant. fontecha et al.(20) stated that functional judgment was the most important factor in determinating frailty. masel et al.(6) found that in mexican communities pre-frail and frail respondents were significantly associated with lower qol scores on physical and mental health domains than elderly who were not frail. the frailty syndrome is closely related to hrqol of the elderly community in taiwan who are using the taipei health services. for frailty phenotypes, slowness is a major factor in the sf36 physical component scale, and fatigue is a major factor in the mental component scale.(14) the results of the present study are consistent with other findings stating that the qol score in the physical and mental health domains was lower in frail or pre-frail respondents. bilotta et al.(19) also found consistent findings of no association between “social relationships and participation” with frailty. a study consisting of 590 patients aged 65 years or older showed that frailty score was not associated with qol in nursing home residents.(20) one of the limitations of this study is related to the cross-sectional design that does not allow establishing of cause-and-effect relations. another limitation is the self-report nature of several key variables. on the other hand, the results could guide gerontological nursing care professionals in their practice with frail, pre-frail and non-frail elderly. conclusions the frailty status in the elderly has a poor impact on quality of life, particularly in the physical health, psychological, and environment domains. the subdomain most influenced was “energy and fatigue” in the physical health domain, “thinking, learning, memory and concentration” in the psychological health domain, and “opportunities for acquiring new information and skills” in the environment domain. conflict of interest none declared. acknowledgment the authors would like to thank the sasana tresna werdha budi mulia jelambar, santa anna, tresna budi mulia 2, and tresna werdha usada mulia nursing homes for the opportunity to carry out this study. references 1. topinkova e. getting older facts about a biological process: aging, disability and frailty. ann nutr metab 2008;52:6–11. 2. eggimann bs, cuenoud p, spagnoli j, et al. prevalence of frailty in middle-aged and older community-dwelling europeans living in 10 countries. j gerontol 2009;64:675-81. 3. walston j, mcburnie ma, newman a, et al. frailty and activation of the inflammation and coagulation systems with and without clinical comorbidities: results from the cardiovascular health study. arch intern med 2002;162:2333– 41. 4. rockwood k, howlett se, macknight c, et al. prevalence, attributes, and outcomes of fitness and frailty in community-dwelling older adults: report from the canadian study 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referred to an outpatient geriatric service in italy. health qual life outcomes 2010;8:56. 20. fougère b, kelaiditi e, hoogendijk eo, et al. frailty index and quality of life in nursing home residents: results from incur study. j gerontol a biol sci med sci 2015. doi: 10.1093/gerona/ glv098. univ med vol. 34 no.3 c:\users\universa medicina\docu 216 abstract universa medicina breastfeeding self-efficacy and related factors during early postpartum period senay topuz1, nuriye buyukkayaci duman2, gulzade uysal3, and dilek ocalan4 background breastfeeding self-efficacy (bse) could be an indicator of the performance of mothers in breastfeeding during the immediate postpartum time period. the factor most affecting the duration and success of breastfeeding is the self-efficacy perception of the mothers. this study was carried out to determine the bse and the factors related to it in the early postpartum period. methods a cross-sectional study was performed involving 264 mothers. data were collected by using a questionnaire and the bse scale (bses). the questionnaire consisted of questions regarding descriptive characteristics (age, educational status, family type, income level, etc.) and obstetric characteristics regarding pregnancy, delivery and breastfeeding status (number of pregnancies, number of births, type of delivery, planned and desired pregnancy, birth weight, first breast-feeding time after birth, etc.). simple and multiple linear regression were performed to examine factors related to bse. results the mean bse score of the mothers participating in the study was 65.20 ± 9.3. simple and multiple linear regression models indicated that significant determinants of bse score include having nuclear family type, having social security, a greater number of pregnancies, a greater number of births, the status of antenatal visits in pregnancy, the status of getting information about breastfeeding, and short first breastfeeding time. getting information about breastfeeding was the most influential factor of bse (beta=3.432; 95% ci : 32.771-51.626; p=0.000). conclusions in this study, getting information about breastfeeding was the most influential factor of bse in the early postpartum period. a woman's level of breastfeeding self-efficacy should be determined during the early postpartum period. keywords: breastfeeding, self-efficacy, early postpartum period original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.216-225 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1150 september-december 2021 vol.40no.3 cite this article as: topuz s, duman nb, uysal g, ocalan d. breastfeeding selfefficacy and related factors during early postpartum period. univ med 2021;40: 216-25. doi: 10.18051/univmed.2021. v40.21 6-22 5. 1ankara university, faculty of health sciences, department of midwifery, ankara, turkey 2hitit university, faculty of health sciences, department of nursing, çorum, turkey 3okan university, faculty of health sciences, department of nursing, istanbul, turkey 4afyonkarahisar university of health sciences, faculty of health sciences, department of nursing, afyonkarahisar, turkey correspondence: nuriye buyukkayaci duman hitit university, faculty of health sciences, department of nursing, corum, turkey, tr 19100 phone: +905055015752 email: nurfatihh@hotmail.com orcid id: 0000-0002-4246-3607 date of first submission, march 9, 2021 date of final revised submission, october 31, 2021 date of acceptance, november 10, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 217 univ med vol. 40 no 3 introduction breast milk is the most important and main source of food for the baby, and breastfeeding is the best type of feeding. the world health organization and the united nations international children’s emergency fund recommend to feed babies only with breast milk within the first 6 months after birth (exclusive breastfeeding), and besides, the innocenti declaration emphasizes that the breastfeeding period should be two years or even more.(1,2) however, it was found that the rates of breastfeeding are high in turkey but the rates of exclusive breastfeeding (giving only breast milk) within the first six months are low.(3) while the rate of breastfeeding in the first hour after birth in turkey is 71%, the rate of breastfeeding within the first day after birth is 86%, while the rate of giving infant formula before breastfeeding is 42%.(3) it is well known that starting and continuing breastfeeding and exclusive breastfeeding within the first six months are affected by many factors.(4-6) in many studies evaluating the factors affecting breastfeeding, it was emphasized that the factor most affecting the duration and success of breastfeeding was the self-efficacy perception of the mothers.(4-6) according to the breastfeeding self-efficacy (bse) theory that was developed by dennis,(7) mothers who have a high selfefficacy prefer breastfeeding, behave more positively when faced with difficulties and try to solve the problems by thinking positively. dennis (7) defined the perception of bse as “mother’s feeling of self-efficacy for breastfeeding” and emphasized that mothers with a high self-efficacy for breastfeeding could cope with the difficulties during the breastfeeding period more strongly and that mothers with a low self-efficacy left breastfeeding earlier. it was stated that the breastfeeding success of the mothers, whose bse was low, was low and that the mother could not continue breastfeeding.(7,8) studies on the subject show that mothers with high breastfeeding selfefficacy have higher breastfeeding success and breastfeed their babies for a longer period of time.(9-12) mother’s age, educational and income levels, family structure, status of desiring pregnancy and experiencing health problems during pregnancy, previous experiences regarding breastfeeding, employment status, type of delivery, starting time for breastfeeding, mother’s feeling that her breast milk is insufficient and support from the spouse can be counted as other factors that affect bse.(9-12) when the literature is examined, different research results are found on the factors affecting mothers’ bse. in the studies of dodt et al.(13) it was determined that as the age of the mother increased, the mean bse score also increased. in the same study, it was determined that the mother’s educational level, profession, marital status, income level and number of pregnancies did not affect breastfeeding self-efficacy. in the studies of glassman et al.,(14) it was reported that there is a positive relationship between high educational level, breastfeeding for more than 6 months, exclusive breastfeeding and bse. in the studies of yang et al.,(15) no relationship was found between socio-demographic and perinatal characteristics such as age, educational level, occupation, monthly income level, time of decision to breastfeed, mode of delivery and bse score averages. it has been determined that multiparity and participation in breastfeeding education classes have a positive effect on bse. in the studies of zhu et al.,(16) it was reported that mothers who had breastfeeding experience had higher bse. in the studies of cantürk and kostak (17) and of akkoyun and arslan,(18) it was reported that as age, number of pregnancies and number of living children increased, the bse of mothers increased, while in the studies of yol and tezel,(19) the bse of multiparous mothers was higher than that of primiparous mothers, but age did not affect bse. in the studies of akkoyun and arslan,(18) fata and ustun akan,(20) and canturk and kostak,(17) it was determined that there is a positive relationship between educational level and bse. in the studies of uludad(21) and comert and ege,(22) it was reported that family type did not affect the bse of mothers, while in the studies of 218 yol and tezel,(19) and cantürk and kostak(17), it was reported that the bse of those with a nuclear family structure was high. in the studies of bolat et al,(23) küçükoðlu et al.,(24) cömert and ege,(22) as well as of cantürk and kostak,(17) it was determined that mothers with social security and medium and high income levels had higher bse. in the studies of akkoyun and arslan,(18) it was reported that income status did not affect bse. there are different study results regarding the relationship of experiencing problems during pregnancy and bse of mothers. in some studies it was reported that mothers who did not have problems during pregnancy had higher bse and that having problems during pregnancy affected bse negatively (17,25) while in other studies no relationship was found.(22) according to the literature, one of the factors affecting bse is early skin-to-skin contact and breastfeeding within the first 30 minutes after birth. studies on the subject show that mothers who feed their babies with breast milk (colostrum) within the first 30 minutes have higher bse.(25-28) when the literature is examined, it is seen that there are many studies on breastfeeding status of infants and breastfeeding, but the results are still inconsistent. this situation was the determining factor in the planning of our study. this study was carried out to determine the bse and its predictors in the early postpartum period. methods research design a cross-sectional study was performed between may 2017-may 2018 in kirikkale university medical faculty hospital. research subjects the universe of the study was composed of all mothers who delivered a baby in kirikkale university medical faculty hospital. the sample size of the study was based on the total score of breastfeeding self-efficacy scale of 68.08 ±14.48 in the study of konukodlu and pasinliodlu (29) using the 2019 statistics, graphics, power analysis & sample size( ncss-pass) program (type i error 0.05, type ii error 0.20), such that the sample was composed of 264 mothers who were eligible according to the inclusion criteria and were approved to participate in the study. included in this study were women 18 years of age or older, who had a single birth at term (38–42 weeks), had a healthy newborn (birth weight of 2500-4000 g, no congenital disease, no history of hospitalization in the neonatal intensive care unit), had no chronic disease that prevented breastfeeding, and breastfeeding women who did not have communication problems. data collection mothers who delivered a baby were given a face-to-face questionnaire and the bse scale. the questionna ir e w as pr epar ed by the researchers based on the literature and the bse scale, and consisted of questions regarding descriptive characteristics of the mothers (age, educational status, family type, income level, having social security) and their obstetric characteristics regarding pregnancy, delivery and breastfeeding status (number of pregnancies, number of births, type of delivery, planned and desired pregnancy, birth weight, first breastfeeding time after birth, getting information about breastfeeding, etc.). the content of the questionnaire form, operability and clarity of the questions were evaluated by taking an expert opinion; and some revisions were made in the questionnaire form in accordance with the opinions and suggestions of the expert. breastfeeding self-efficacy scale (bses) this is a scale comprising 33 items that was developed by dennis and faux(8) in order to measure beliefs and thoughts re garding breastfeeding and breastfeeding efficacy of the mothers. a validity and reliability study of the turkish version of the scale was conducted by eksioglu and ceber.(30) breastfeeding efficacies of the mothers were evaluated based on a 5-point likert scale. the items were scored as (1) i never topuz, duman, uysal , et al breastfeeding self-efficacy and related factors 219 univ med vol. 40 no 3 a b trust myself, (2) i do not trust myself very much, (3) i sometimes trust myself, (4) i trust myself most of the time, (5) i always trust myself. the lowest score is 14, and the highest score is 70.(8) as the score obtained from the scale increases, breastfeeding self-efficacy also increases. the average application time of the scale takes 10-15 minutes.(8) statistical analysis the data were evaluated using the statistical package for the social sciences (spss) program version 20.0. descriptive statistics were shown with number (n), percentage (%) and mean ± standard deviation in the analysis of the data. simple linear regression analysis was used to find associations between the independent variables and the bse. all variables showing a significant association in the bivariate analysis at p<0.25 were entered into a multivariate linear regression model to determine the risk factors associated with the bse. the results were evaluated at 95.0% confidence interval, p<0.05 significance level. ethical consent ethical approval was obtained from local clinical research ethics committee kirikkale university (15/12/2015-28-4). all participants provided written informed consent. results the mean age of the women was 28.39 ± 5.82 years and approximately half of them were between the ages of 26-35 years; 41.1% were high school graduates, 82.5% had a nuclear family structure, 78% had middle income and 90.2% had social security (table 1). it was determined that 42.7% of the women participating in the study had their first pregnancy and 52.8% had their first birth. the majority of women (87.4%) had planned and desired the pregnancy and 72.8% stated that they did not have any health problems during pregnancy. furthermore, 94.3% stated that they regularly went to antenatal visits and 54.9% of the women gave birth by cesarean. when the birth weight of the babies was examined, 83.7% of the babies weighed 2500-4000 g. it was determined that 58.9% of women breastfed their babies within 30-60 minutes following birth. while 56.1% of women stated that they received information about breastfeeding during pregnancy, 84.1% stated that it is necessary to give information to mothers about breastfeeding during pregnancy (table 2). the mean bse score of the mothers participating in the study was 65.20 ± 9.3. in view of this score average, it can be said that the mean bses score of the mothers participating in the study was high (table 2). the simple and multiple linear regression models indicated that significant determinants of bse score include increased educational status, having nuclear family type, having social security, a greater number of pregnancies, a greater number of births, the status of antenatal visits in pregnancy, the status of getting information about breastfeeding, short first breastfeeding time and no health problems in pregnancy. the most characteristics n (%) age*(years) 28.39 ± 5.82 18-25 26-35 36 years and over 90 (36.6) 129 (52.4) 27 (11.0) educational status primary school high school university master degree 15 (6.1) 101 (41.1) 95 (38.6) 35 (14.2) family type nuclear family extended family 203 (82.5) 43 (17.5) income level high middle low 28 (11.4) 192 (78.0) 26 (10.6) having social security yes no 222 (90.2) 24 (9.8) table 1. distribution of the mothers according to their sociodemographic characteristics (n=246) *mean ± sd 220 influential determinant factor of bse was the status of getting information about breastfeeding (beta=3.432; 95% c.i.: 32.77151.626;p=0.000) (table 3). discussion the breastfeeding self-efficacy scale mean score of the mothers in the study was found to be 65.20 ± 9.3. based on this mean score, it can be said that the bse of the mothers participating in the study was high. when the studies on the subject were examined, different research results were found. unlike the results of our study, mothers’ bses mean scores were found to be low in some studies. (24,31) in the studies of faridvand et al.,(9) yenal et al.(32) and akkoyun and arslan (18), mothers’ bses mean scores were found to be lower, namely 59.18 ± 9.56, 59.49 ± 8.46, and 59.18 ± 9.46, respectively. the finding of our study differs from the results of the aforementioned studies. this difference may have been due to the fact that our sample group has differ ent sociodemographic and obstetric characteristics from living in a different region. according to the literature, mothers’ bse, age, socio-cultural level, economic status, having health insurance, access to health services, number of pregnancies and births, mode of delivery, past breas tf eeding e xperience, breastf eeding educat ion, pr epar ation for pregnancy and participation in prenatal education classes, are affected by many factors such as the condition of pregnancy or the status of having problems during pregnancy.(31,33,34) similarly, in our study, significant determinants of bses include increased educational status, having nuclear family type, having social security, a greater number of pregnancies, a greater number of births, the status of antenatal visits in pregnancy, the status of ge ttin g information about breastfeeding, short first breastfeeding time and no health problems in pregnancy. in our study, the status of getting information about breastfeeding (beta=3.432) was the most influential determinant factor of bse (table 3). it was also observed that mean bses scores of the mothers who got information about breastfeeding during pregnancy and thought that it was necessary, were higher than that of the ones who did not. similar to the results of our characteristics n (%) number of pregnancies 1 2 3 4 and over 105 (42.7) 72 (29.3) 52 (21.1) 17 (6.9) number of births 1 2 3 4 and over 130 (52.8) 55 (22.4) 50 (20.3) 11 (4.5) type of delivery vaginal cesarean 111 (45.1) 135 (54.9) planned and desired pregnancy yes no 215 (87.4) 31 (12.6) having a health problem in pregnancy yes no 67 (27.2) 179 (72.8) the status of antenatal visits in pregnancy yes no 232 (94.3) 14 (5.7) birth weight (grams) under 2500 2500-4000 4000 and over 19 (7.7) 206 (83.7) 21 (8.5) first breast-feeding time after birth within the first 30-60 minutes 2-3 hours 1 day 2 days and over 145 (58.9) 42 (17.1) 24 (9.8) 35 (14,2) getting information about breastfeeding yes no 138 (56.1) 108 (43.9) thinking that mothers need to be informed during pregnancy yes no 207 (84.1) 39 (15.9) bse score* 65.20 ±9.3 table 2 . distribution of the mothers according to pregnancy-related features, history of births, and the bse score *mean ± sd; bse : breastfeeding self-efficacy topuz, duman, uysal , et al breastfeeding self-efficacy and related factors 221 univ med vol. 40 no 3 study, some studies have reported that the status of getting information about breastfeeding or breastfeeding education positively affects bse.(1417) in support of the outcome of our study, rosscowdery et al.(35) emphasized in their study that breastfeeding training given during pregnancy was very important in early breastfeeding and in providing the continuity of breastfeeding. mccoy et al.(36) also detected that training that was based on bse theory was very effective in improving self-efficacy percep tion a nd suc cess of breastfeeding. on the contrary, yilmaz et al.(37) found the rate of exclusive breastfeeding within the first 6 months to be 33.3% in the group which had training during pregnancy and 37.5% in the control group. in our study, it was found that regular checkups during pregnancy and not having any health problems during pregnancy were associated with high bse. similarly, it was seen that mean bse scores of the mothers who did not experience any problems during pregnancy was higher than that of the mothers who had problems. going to antenatal visits regularly during pregnancy and getting consultations on breastfeeding are important since this increases the success of breastfeeding and provides early detection and resolution of possible problems.(35,36) completion of this period without having problems can affect the success of breastfeeding since it enables the women to enter the postpartum period with less stress. the results of our study support these outcomes. in the studies of faridvand et al.(9) and yol and tezel,(19) no relationship was found between the age of the mothers and the mean bses scores, whereas the mean bses scores of multiparous mothers were higher than those of primiparous mothers. in the studies of cantürk and akgun kostak,(17) it was reported that as the age of the mothers, the number of pregnancies and the number of children increased, the bse levels also increased. in the studies of yang et al.,(15) while no relationship was found between other socio-demogra phic a nd perinatal characteristics such as age, educational level, characteristics simple linear regression multiple linear regression β 95 % c.i p value beta 95 % c.i p value age (years) 0.296 (0.9771.945) 0.494 educational status 1.324 (2.3574.393) 0.000* 1.673 (3.4125.221) 0.000* nuclear family type 1.287 (7.23412.432) 0.000* 1.778 (6.81016.220) 0.000* income level 0.312 (0.4181.324) 0.381 having social security 0.820 (2.435: 5.348) 0.000* 1.118 (3.1706.857) 0.000* number of pregnancy 1.673 (0.0005-0.003) 0.000* 1.431 (0.00031.009) 0.000* number of births 1.433 (6.25510.278) 0.000* 1.345 (5.1498. 530) 0.000* planned and desired pregnancy 0.334 (0.6892.346) 0.392 type of delivery 0.128 (0.5781.345) 0.421 the status of having no health problem in pregnancy 1.478 (7.67512.238) 0.000* 1.562 (7.44313.338) 0.000* the status of antenatal visits in pregnancy 1.423 (0.4440.620) 0.000* 1.423 (0.6560.778) 0.000* birth weight (grams) 0.446 (0.5421.978) 0.296 first breast-feeding time after birth 2.687 (18.34532.123) 0.000* 2.546 (16.23228.223) 0.000* getting information about breastfeeding 2.458 (28.67846.316) 0.000* 3.432 (32.77151.626) 0.000* table 3. results of simple and multiple linear regression for predicting breastfeeding self-efficacy in postpartum women (n=246) β: regression coeffficient; beta: standardized regression coefficient 222 occupation, monthly income level, time of decision to breastfeed, mode of delivery and the mean bse scale scores, it was found that multiparity and participation in breastfeeding education classes had a positive effect on bse.(15) in the studies of zhu et al.,(16) it was reported that mothers who had breastfeeding experience had higher bse. in the study by muelbert and giugliani (38) evaluating the factors that cause breastfeeding to continue for 6, 12 and 24 months in adolescent mothers, it was determined that adolescent mothers had more negative behaviors and attitudes compared to the adult mothers regarding breastfeeding; and they required more information and help. the reason of the decrease in mean bse score as age decreased in our study may be the high incidence of early age marriages and the presence of a strong family support in the marriages.(39) according to the results of the study, the increase in bse with the number of pregnancies and births, regardless of the age of the mothers, can be explained by the mothers having knowledge an d exper ience about breastfeeding. educational level is an important factor that increases awareness of breastfeeding. the rates of breastfeeding are expected to be higher among the mothers as educational level increases.(38-41) the findings of our study agree with the results of these prior studies. family type is also one of the factors affecting the success of breastfeeding (38,42) also in our study, it was detected that mean bse scores of the mothers who were living in a core family were higher than those of the ones who were living in a large family, the difference being statistically significant. similar to the results of our study, in the study by faridvand et al.(9) it was reported that mean bse scores of the mothers who were living in a core family were higher than the ones who were living in a large family and a statistically significant difference was found between both groups. the fact that pregnancy is planned and desired is one of th e factors af fecting breastfeeding behavior.(42-44) in our study, it was determined that planned and voluntary pregnancy did not affect mothers’ bse. similarly, in the studies of faridvand et al.(9), the relationship between planned desired pregnancy and the bse mean scores was found to be statistically insignificant. according to the literature, it is reported that the situation of having problems during pregnancy may affect the bse of mothers.(17,22) similarly, in our study, it was determined that as the mothers. in the literature, the type of delivery has been reported to constitute one of the factors problems in pregnancy increased, the bses mean scores decreased. when the studies on the subject were examined, it was determined that comert and ege’s (22) study did not affect the status of having health problems during pregnancy and the bses mean scores. supporting the results of our study, it was reported that mothers who had an uneventful pregnancy had high bse levels in the studies of cantürk and kostak.(17) affecting the success of breastfeeding and beginning and duration of breastfeeding.(42,45) when the studies on the subject are examined, it is reported that cesarean delivery negatively affects the mean starting breastfeeding, continuing breastfeeding and total breastfeeding times.(22,23) in our study, it was determined that the mode of delivery did not affect the bse of the mothers. similarly, in the studies of cantürk and akgün kostak,(17) aluþ tokat et al.,(28) cömert and ege,(22) gokbulut,(25) akkoyun and arslan(18) yol and tezel (19) and uludad,(21) no relationship was found between the mode of delivery and the mothers’ bses mean scores. in our study, it is thought that the mode of delivery does not affect breastfeeding proficiency, since most of the mothers breastfeed their babies in the first hour regardless of the mode of delivery. the characteristics of the newborn such as birth weight are also among the factors affecting success of breastfeeding.(46,47) it is an expected situation that mothers of the newborns who have a low birth weight experience problems with cuddling the baby and with the breastfeeding position. the same problems may be experienced by the mothers of newborns who have a birth topuz, duman, uysal , et al breastfeeding self-efficacy and related factors 223 univ med vol. 40 no 3 weight above normal.(46,47) differently, in our study, it was determined that birth weight did not relate to maternal bse. this can be explained by the fact that the majority of the babies (83.7%) included in the sample were of normal birth weight. because this study was limited to the bse of mothers in a university hospital in kýrýkkale province, the results can only be generalized to this group. for this reason, the numerical smallness of the sample size in the study and the homogeneous distr ibution of the sociodemographic characteristics of the mothers can be considered as the limitations of the study. based on the results of the study, it is thought that the standard evaluation of mothers’ bse and the affecting factors in the postpartum period and the determination of breastfeeding counseling needs can positively affect mothers’ bse and breastfeeding success. conclusion in this study, the status of getting information about breastfeeding was the most influential determinant factor of bse after analysis of the standardized regression coefficient (beta). it is suggested that further studies to be conducted on the subject should be planned as cohort studies. conflicts of interest there are no conflicts of interest. acknowledgment we thank the mothers in the postpartum period who supported the study by accepting to participate in the study. contributors st, nbd, and gu designed this study. st and nbd carried out the data 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10.1186/s12884-020-2732-6. 46. parker mg, melvin p, graham da, et al. timing of first milk expression to maximize breastfeeding continuation among mothers of very low-birthweight infants. obstet gynecol 2019;133:1208-15. doi: 10.1097/aog.0000000000003258. 47. mohammadi f, kiani a, gholamzadeh s, asadi noghabi f, sadeghi t. the factors affecting successful breast-feeding. iranian j neonatol 2018;9:73-82. doi:10.22038/ijn.2018.24904.1322. c:\users\user\documents\al rasy 270 abstract universa medicina morning hypertension for stroke and cardiovascular: clinical pearls for primary care al rasyid1* and elvan wiyarta2 hypertension is the world’s leading cause of mortality and morbidity. one of the phenomena that commonly occur in hypertensive as well as normotensive patients, is morning hypertension. blood pressure (bp) follows a diurnal rhythm, reaching its highest level during the morning hours and dropping to the lowest level at midnight. transient increases in bp in morning hypertension plus persistent stressors within 24 hours are thought to increase target organ damage and trigger cardiovascular events. therefore, ambulatory bp monitoring or morning home bp monitoring is recommended as a strong predictor of cardiovascular events. there are two types of morning hypertension according to its underlying mechanisms; the first one is called nocturnal hypertensive morning hypertension, and the other one is morning-surge hypertension. numerous studies have proved that this phenomenon often leads to several acute cardiovascular events, such as stroke, coronary artery disease, and peripheral artery disease. to prevent these complications, cost-effective management is needed, especially for identifying accurate diagnostic tools, as well as creating specific regimens. therefore, to achieve appropriate management of hypertension, including morning hypertension, long-acting antihypertensive drugs should be used, at full doses and in the form of combination therapy. the clinical usefulness of antihypertensive drugs with specific mechanisms for morning bp or split or timed dosing of longacting drugs in controlling morning bp remains under investigation. more studies are needed, especially looking for other clinical evidence of the benefits of lowering bp in the morning. home bp monitoring is recommended as a good choice for bp measurements, especially in the primary care setting. keywords: morning hypertension, cardiovascular events, primary care, awareness, prevention review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.270-278 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1217 september-december 2021 vol.40no.3 cite this article as: rasyid a, wiyarta e. morning hypertension for stroke and cardiovascular : clinical pearls for primary care. univ med 2021;40:270-8. doi: 10.18051/univmed.2021.v40.2702 7 8 1department of neurology, cipto mangunkusumo hospital, faculty of medicine, universitas indonesia, jakarta, indonesia 2medical education study program faculty of medicine, universitas indonesia *correspondence: al rasyid, md, phd, stroke consultant department of neurology, cipto mangunkusumo hospital, faculty of medicine, universitas indonesia, 10430 jakarta, indonesia tel: +62 81218676230 email: alrasyid50@yahoo.com orcid id: 0000-0002-7568-8124 date of first submission, august 17, 2021 date of final revised submission, november 25, 2021 date of acceptance, december 6, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 271 univ med vol. 40 no 3 introduction hypertension remains the leading cause of cardiovascular morbidity and mortality, including stroke, coronary heart disease, heart failure, chronic kidney failure, and central and peripheral vascular disease.(1-3) blood pressure (bp) values for diagnosis and therapy are taken from the average of two measurements, although there are diurnal variation factors that influence it.(4) blood pressure tends to decrease during sleep and rise again in the morning.(5) this is evidenced by the results of 24-hour ambulatory bp monitoring (abpm).(6,7) based on various epidemiological studies, it was found that the prevalence of acute cardiovascular manifestations that have the potential to cause death, such as myocardial infarction, stroke, or sudden cardiac death, peaks in the morning, usually within 4-6 hours after awakening.(8,9) transient increases in bp, known as morning bp surge (mbps), in morning hypertension plus persistent stressors within 24 hours are thought to increase target organ damage and trigger these cardiovascular events.(10-12) therefore, bp monitoring using outof-clinic abpm or morning home bp monitoring (hbpm), is recommended as a strong predictor of cardiovascular events.(13-15) through these methods, the phenomenon of the white-coat effect and masked hypertension can be eliminated, and the measurements are more reproducible than in clinical examinations. this review will discuss more about morning hypertension and the things you need to know in primary care. morning hypertension is a problem that is often taken for granted, but this condition can be potentially fatal, especially if not diagnosed early. the primary care physician as the front line in health services has an important role in diagnosing morning hypertension. morning hypertension and morning blood pressure surge available studies use several different definitions of mbps as seen in figure 1.(10) assessment of mbps is generally carried out with 24-hour abpm. according to kario (10) there are two definitions in use, namely sleep-troughsurge which is the value of bp in the morning (2 hours after waking) minus the lowest bp value during sleep, and pre awakening surge which is the value of bp in the morning minus the value of bp 2 hours before waking up. in other studies, mbps is referred to as a rising surge, which is the difference in the value of bp that increases in the morning minus bp in the supine position as measured <30 minutes earlier.(17) in another study, the difference in bp between the morning and evening values is called the morning and evening (me) difference.(2) in the jichi medical schoolambulatory blood pressure monitoring study and the international database on ambulatory blood pressure monitoring in relation to cardiovascular outcomes, pathological morning bp increases were in the 10th percentile class of these bp values.(10) however, there is no official consensus on a definition of this phenomenon or a threshold for pathological elevation. in contrast to mbps, morning hypertension is a bp value of 135/85 mmhg in the morning, either through abpm or hbpm. in the japanese society of hypertension 2014 guidelines for the management of hypertension, the term masked morning hypertension was introduced, which refers to a home morning bp of 135/85 mmhg, but with normal clinic bp (<140/90 mm hg).(17) regar dless of the mechanism, either pathophysiological or as a result of inadequate antihypertensive therapy, morning hypertension is associated with more frequently occurring significant cardiovascular events in the post-wake period.(14) the prevalence of this phenomenon is quite high in the population of patients who have been diagnosed with hypertension, namely 15.9– 43.6%.(18) cases of masked morning hypertension are still found at a high rate in the controlled hypertension population, which is 23.1 – 60.7%.(19) morning hypertension is classified into 2 types, namely nocturnal hypertensive morning hypertension and morning -surge hypertension.(20,21) the first type includes nondippers (i.e. bp that does not decrease at night) 272 and risers (i.e. bp at night that is higher than during the day).(22) meanwhile, the second type is related to the phenomenon of morning surge, namely an increase in bp that starts 2 hours before waking up and increases after waking up.(22) from the explanation above, it can be concluded that morning hypertension and mbps are two different entities. a morning bp surge can still occur even without morning hypertension, for example in cases of excessively decreased nocturnal bp. on the other hand, morning hypertension can occur even though there is no significant mbps, for example in the case of bp that remains high from night to morning. mechanism of morning hypertension morning hypertension is influenced by the sympathetic nervous system, the r eninangiotensin system, and cortisol secretion.(23,24) in normal individuals, this occurs physiologically, causing incr ease d b lood flow and shear stress.(25,26) if there is vascular stenosis due to ather osclerosis, plat elet aggr egation and activation of plasminogen activator inhibitor 1 (pai-1) occur.(27) this mechanism is illustrated in figure 2.(28) figure 2 explains the mechanism of damage to cardiovascular organs in this phenomenon. there are two genes that play a role, namely the central and peripheral clock genes.(28) the central clock gene regulates variations in blood pressure in the morning, including sleep-wake cycles, body position, and physical activity.(28) these things affect the body’s physiology which is regulated by peripheral clock genes, namely the sympathetic nervous system and the renin-angiotensin system.(29) both of these factors contribute to an increase in mbps in morning hypertension.(27,28) furthermore, mbps also causes endothelial dysfunction and spasm, and platelet activation.(28) endothelial dysfunction further leads to plaque rupture in the blood vessels. when combined with increased activity of pai-1 so that fibrinolytic activity decreases, it can put a person at high risk for these cardiovascular events.(27,28) several other studies have hypothesized that cardiovascular events in morning hypertension are associated with structural damage to large arteries, which in turn can lead to tearing of elastic fibers, hypertrophy and structural abnormalities of smooth muscle, and plaque rupture.(30-33) this mechanism mainly occurs in atherosclerotic figure 1. definition of morning surge in bp (10) rasyid, wiyarta morning hypertension clinical pearls 273 univ med vol. 40 no 3 a b arteries, under conditions of high oxidative stress, and in the geriatric population with baroreflex dysfunction.(34) cardiovascular risk of morning hypertension in previous studies, morning hypertension was considered a predictor of organ damage or cardiovascular outcome.(2) in a study in japan on a population of hypertensive patients, it was found that the group with morning and night bp differences in the highest quartile had a higher risk of developing concentric left ventricular hypertrophy than the group in the lowest quartile.(35) in another study, the risk of stroke was found to be significantly higher in the morning hypertensive population than in the population with chronic hypertension.(36) the japan morning surge-home blood pressure (j-hop) study showed that morning home systolic bp (sbp) itself should be evaluated to ensure best stroke prediction in clinical practice, at least in japan.(37) a recent study from the international database on ambulatory blood pressure in relation to cardiovascular outcome even shows that there is an increased risk of cardiovascular events by 30-40% in the population with mbps.(38) the mbps group in the highest decile class was associated with the prognosis of stroke or brain infarction twice as high in later life.(10) sleep-trough mbps was significantly associated with a 25% increased risk of stroke.(10) the study of the international database on ambulatory blood pressure in relation to cardiovascular outcome concluded that the highest decile group in sleep trough (37 mmhg) and pre awakening (28 mmhg) mbps had a significantly higher risk of all-cause mortality and total cardiovascular events.(39) recommendations f or management of morning hypertension in primary care despite the important clinical implications, specific guidelines for the management of morning hypertension have not been published to date, although several guidelines on hypertension have ge neral rec ommendat ions regarding the management of morning hypertension, such as the british nice (40) and canadian guidelines.(13) both guidelines recommend out-of-office bp measurements, such as the abpm or hbpm, for diagnostic and therapeutic purposes. however, specific recommendations regarding morning hypertension have not been listed in either. the importance of measuring bp regularly, especially in the morning, is also supported by guidelines from several other countries, such as china,(11) figure 2. organ damage mechanism in morning hypertension (28) 274 table 1. commonly used oral antihypertensive drugs from cooper et al.(48) note: aextemporaneously prepared infusion; bapproved by the us food and drug administration (fda) for patients >6 years old; cfda approved for patients >12 years old; dfda approved for patients >1 year old class medication initial dose dosing interval maximal dose peripheral antagonists prazosin 5 μg/kg 2 or 3 times per day 0.4 mg/kg (15 mg) terazosin 1 mg daily 20 mg doxazosin 1 mg daily 4 mg central antagonists clonidinea 5-10 μg/kg 2 or 3 times per day 0.9 mg methyldopaa,b 10 mg/kg 2-4 times per day 3 g (65 mg/kg/d) guanfacinec 1 mg nightly 2 mg nonselective β antagonists propranolola 1-2 mg/kg 2 or 3 times per day 4 mg/kg selective β1 antagonists metoprolol a,b immediate release 1-2 mg/kg 2 times per day 6 mg/kg (200 mg/d) extended release 1 mg/kg/d daily 2 mg/kg (200 mg/d) atenolola 0.5-1 mg/kg daily, 2 times per day 2 mg/kg (100 mg/d) mixed α/β antagonists labetalola oral 4 mg/kg 2 times daily 40 mg/kg intermittent bolus 0.2-1 mg/kg 20 mg carvedilola immediate release 6.25 mg 2 times daily 50 mg extended release 20 mg daily 80 mg angiotensin converting enzyme inhibitors captoprila 0.15-0.5 mg/kg 2-4 times per day 6 mg/kg enalaprila,b 0.1-0.5 mg/kg daily, 2 times per day 0.5 mg/kg/ (40 mg)) fosinoprilb >50 kg, 5-10 mg daily 40 mg lisinoprila,b 0.07 mg/kg daily 40 mg benazeprila,b 0.1-0.6 mg/kg daily 40 mg angiotensin ii receptor blockers candesartana,b 0.2 mg/kg daily, 2 times per day 0.4 mg/kg (32 mg) valsartana,b 1.3 mg/kg daily 40 mg losartana,b 0.7 mg/kg daily 100 mg olmesartana,d 20-35 kg 10 mg daily 20 mg >35 kg 20 mg daily 40 mg calcium channel blockers amlodipine a,b 0.05-0.1 mg/kg daily, 2 times per day 10 mg felodipine 2.5 mg daily 10 mg nifedipine immediate release 0.2-0.5 mg/kg every 4-6 h 10 mg extended release 0.2-0.5 mg/kg daily, 2 times per day 3 mg/kg (120 mg) isradipinea immediate release 0.05-0.15 mg/kg every 4-6 h 0.8 mg/kg/d (20 mg) extended release 0.05-0.15 mg/kg daily, 2 times per day 20 mg diuretics chlorothiazidea,b oral 20 mg/kg 2 times per day 1 g iv 40 mg/kg daily, 2 times per day 20 mg/kg hydrochlorothiazideb 1 mg/kg daily 3 mg/kg (50 mg) chlorthalidonea 0.3 mg/kg daily 2 mg/kg (50mg) metolazonea 0.2-0.4 mg/kg daily, 2 times per day 6 mg/kg furosemidea 1-2 mg/kg daily, 4 times per day 100 mg spironolactoneb 1-3 mg/kg 2-4 times per day vasodilators hydralazine,b oral 0.7-1 mg/kg 2-4 times per day 25 mg (200 mg/d) rasyid, wiyarta morning hypertension clinical pearls 275 univ med vol. 40 no 3 korea (41) and taiwan,(42) although they do not emphasize strict monitoring through abpm and hbpm . t he 20 13 e ur op e a n so c i e t y of hypertension guidelines,(43) the 2014 guideline of the us eight joint national committee (jnc8) (44) and the 2020 guidelines of the international society of hypertension (45) have not discussed this phenomenon. the selection of the right drug in the management of morning hypertension raises a question mark among clinicians. in one study, long-acting antihypertensive drugs have the advantage of controlling the blood pressure for 24 hours.(46) in another study, bp reductions were greater in the group receiving long-acting drugs, such as amlodipine and telmisartan.(47) in addition, morning bp control was also better when antihypertensive drugs were given at full/ maximum doses compared to low doses, either in the form of monotherapy or combination t he r a p y. ( 1 9) t he c ommon ly u s e d or a l antihypertensive drugs can be seen in table 1 which is taken from cooper et al.(48) several studies have also compared the e f f e c t o f a n t ih ype r t e ns i ve s gi ve n a l o ne (monotherapy) or in combination on 24-hour bp control. combination therapy of an angiotensinr e c e pt o r bl o c ke r ( arb) o r a ngi o te ns i nconverting enzyme inhibitor (acei) with a calcium-channel blocker (ccb) or diuretic can improve bp control within 24 hours.(19) in a japanese study, the combination of losartan and hydrochlorothiazide was able to significantly improve blood pressure control compared to high-dose losartan.(50) another study using a combination of arb-ccb and arb-acei showed similar results.(50) in several other studies, the increase in morning blood pressure is associated with activation of the sympathetic nervous system and the renin-angiotensin system, therefore several hypotheses suggest the use of drugs that act specifically on this pathway.(51,52) however, there have been no studies comparing the potential of each of the antihypertensive drugs separately to lower blood pressure in the morning. although clinical evidence of the effect of morning hypertension is abundant, studies on the relationship of morning blood pressure control for preventing cardiovascular events have not been widely carried out. the anglo-scandinavian cardiac outcomes trial compared hypertensive patients receiving amlodipine and atenolol. as predicted, the decrease in blood pressure in the amlodipine group was greater at the time of measurement in the clinic and in the subject’s home at night.(53) after analysis, the hazard ratio for cardiovascular events in both groups was 0.74 (increased to 0.81 after adjusting for clinical systolic blood pressure measurements, and 0.85 after adjusting for night-time systolic blood pressure measurements).(53) these results suggest that the long-acting effects of amlodipine may be beneficial to cardiovascular outcomes. conclusion morning hypertension is one of the important factors for various cardiovascular events that often occur in post-wake times. this is important for clinicians to know for the management of hypertension and the prevention of cardiovascular complications. uncontrolled bp in the morning is a sign that the antihypertensive regimen given is not appropriate, either due to inadequate duration of action, inadequate dosage, or not using combination therapy. therefore, to achieve appropriate management of hypertension, including morning hypertension, long-acting antihypertensive drugs should be used, at full doses and in the form of combination therapy. more studies are needed, especially looking for other clinical evidence of the benefits of lowering bp in the morning, compared to other times, on cardiovascular outcomes. more research is also needed to find the most accurate blood pressure monitoring method in detecting the morning blood pressure surge, as well as its specific therapy. conflict of interest the authors declare no conflict of interest. 276 funding no funding to declare. contributors ar contributed to writing the manuscript. ew contributed to revising the manuscript. all authors have read and approved the final manuscript. references 1. bilo g, grillo a, guida v, parati g. morning blood pressure surge: pathophysiology, clinical relevance and therapeutic aspects. integr blood press control 2018;11:47-56. doi: 10.2147/ ibpc.s130277. 2. oh j, lee cj, kim ic, et al. association of morning hypertension subtype with vascular target organ damage and ventral hemodynamics. j am heart assoc 2017;6:e005424. doi: 10.1161/jaha. 116.005424. 3. wang jg, kario k, chen ch, et al. management of morning hypertension: a consensus statement of an asian expert panel. j clin hypertens (greenwich) 2018;20:39-44. doi: 10.1111/jch.13140. 4. oparil s, acelajado mc, bakris gl, et al. hypertension. nat rev dis primers 2018;4:18014. doi: 10.1038/nrdp.2018.14. 5. ncd risk factor collaboration (ncd-risc). worldwide trends in blood pressure from 1975 to 2015: a pooled analysis of 1479 population-based measurement studies with 19·1 million participants. lancet 2017;389:37-55. doi: https:// doi.org/10.1016/s0140-6736(16)31919-5. 6. pena-hernandez c, nugent k, tuncel m. twentyfour-hour ambulatory blood pressure monitoring. j prim care community health 2020;11: 2150132720940519. doi: 10.1177/21501327209 40519. 7. turner jr, viera aj, shimbo d. ambulatory blood pressure monitoring in clinical practice: a review. am j med 2015;128:14-20. doi: 10.1016/j.amjmed. 2014.07.021. 8. katritsis dg, gersh bj, camm aj. a clinical perspective on sudden cardiac death. arrhythm electrophysiol rev 2016;5:177-82. doi: 10.15420/ aer.2016:11:2. 9. marijon e, uy-evanado a, dumas f, et al. warning symptoms are associated with survival from sudden cardiac arrest. ann intern med 2016; 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2nd ed. mount prospect, illinois, usa: society of critical care medicine;2016.p. 951-61. 49. hanayama y, uchida ha, nakamura y, makino h. losartan/hydrochlorothiazide combination therapy surpasses high-dose angiotensin receptor blocker in the reduction of morning home blood pressure in patients with morning hypertension. acta med okayama 2012;66:44959. doi: 10.18926/amo/49041. 50. omboni s, volpe m. angiotensin receptor blockers versus angiotensin converting enzyme inhibitors for the treatment of arterial hypertension and the role of olmesartan. adv ther 2019;36:27897. doi: 10.1007/s12325-018-0859-x. 51. miller aj, arnold ac. the renin-angiotensin system in cardiovascular autonomic control: recent developments and clinical implications. clin auton res 2019;29:231-43. doi: 10.1007/ s10286-018-0572-5. 52. vian j, pereira c, chavarria v, et al. the renin– angiotensin system: a possible new target for depression. bmc med 2017;15:144. doi: 10.1186/ s12916-017-0916-3. 53. gupta a, mackay j, whitehouse a, et al. longterm mortality after blood pressure-lowering and lipid-lowering treatment in patients with hypertension in the anglo-scandinavian cardiac outcomes trial (ascot) legacy study: 16-year follow-up results of a randomised factorial trial. lancet 2018;392:1127-37. doi: 10.1016/s01406736(18)31776-8. rasyid, wiyarta morning hypertension clinical pearls january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 determinants of coronary heart disease among adults: a case-control study sudikno1 , srilaning driyah2 , and julianty pradono2* abstract background according to recent world health organization data, coronary heart disease (chd) remains the leading cause of death worldwide. although the risk factors of this disease are well known, the strength of these factors varies in different populations. the aim of the study was to assess the determinants of chd in indonesian adults aged 25 years and over. methods a case–control study was carried out involving 592 subjects aged 25 years and over (444 controls and 148 cases). participants were interviewed using validated questionnaires. physical examinations and supporting examinations were conducted. the chi square test, fisher’s exact test, and independent t test were used to analyze the data. to determine independent predictors of chd, the odds ratio (or) was determined via the multiple logistic regressions test. results the chd subjects (case group) showed higher blood sugar and systolic pressure than non-chd subjects (controls), with mean fasting blood sugar of 92.53±27.05 mg/dl vs 88.29 ±23.43 mg/dl (p=0.038), 2-hour postprandial blood sugar of 133.15±65.09 mg/dl vs 120.87±44.60 mg/dl (p=0.000), and systolic blood pressure of 89±27.62 mmhg vs 129.98±10.58 mmhg (p=0.002). the logistic regression analysis in the case group showed that higher education (college) had a 2.32-fold greater effect (95% ci. 1.01-5.35) on chd incidence compared to the control group. conclusions this study has demonstrated that the most frequent risk factor for cvd in adults aged 25 years and over is higher education. control and prevention of chd need to be done with regular control of blood sugar levels and blood pressure to stabilize them within normal limits. keywords: coronary heart disease, blood sugar, blood pressure, adults 1national research and innovation agency, jakarta, indonesia 2community research program, faculty of medicine, university of malahayati lampung, indonesia *correspondence: julianty pradono community research program, faculty of medicine, university of malahayati lampung jl. percetakan negara 29 jakarta pusat email: jpradono@yahoo.com orcid id: 0000-0002-6730-3175 date of first submission, may 24, 2022 date of final revised submission, march 7, 2023 date of acceptance, march 16, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 41 doi: http://dx.doi.org/10.18051/univmed.2023.v42:41-51 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1347 cite this article as: sudikno, driyah s, pradono j. determinants of coronary heart disease among adults: a case-control study. univ med 2023;42:41-51. doi: 10.18051/univmed.2023.v42:41-51 mailto:jpradono@yahoo.com https://orcid.org/0000-0002-5007-5575 https://orcid.org/0000-0001-6765-797x https://orcid.org/0000-0002-6730-3175 42 sudikno, driyah, pradono coronary heart disease among adults introduction about three-quarters of the proportion of deaths from non-communicable diseases (ncds) occurs in lowand middle-income countries, with no difference in mortality between men and women. (1 ) t he gl obal prevale nce of cardiovascular hear t disease (chd) has increased over the last 20 years and the disease is now recognized as a leading cause of death and disability worldwide.(2) data from the american heart association (aha) statistics in 2016 reported that 15.5 million individuals aged 20 year s had chd. its prevalence increases with age in women as well as in men. it is estimated that every 42 seconds there will be one more chd sufferer in the us.(3) in addition to the reported increase in cases, the pattern of the ten main causes of death in 2017 is identical to that in 2016, namely heart disease, cancer, injury, chronic lower respiratory disease, stroke, alzheimer’s disease, diabetes mellitus (dm), influenza and pneumonia, kidney disease, and suicide.(4) in indonesia, from the 2013 basic health research (riskesdas) data it is apparent that heart disease sufferers increase with age, the highest number being in the 65-74 year age group, which is about 2 percent of the population aged 15 years and over. (5 ) basic health resear ch data (riskesdas) for 2018 show that west java ranks 5th highest in indonesia (1.6%) with 186,809 individuals with heart disease from all age groups.(5) there are various risk factors for heart disease. population studies over the last 20 years have proven that overweight (overweight and obesity), hypertension, and hypercholesterolemia are the four risk factors that have the highest contribution to cardiovascular disease.(6) the results of further analysis of the 2011 noncommunicable disease cohort study showed that hypertension, ldl cholesterol, hdl cholesterol, triglycerides, and obesity are also risks for chd with a probability of 64.84 percent.(7) diabetes mellitus (including hyperglycemia), abnormal lipid profile, changes in inflammatory mediators, and coagulation or thrombolytic parameters, as well as other risk factors that are closely related to insulin resistance, are reported to have a risk of chd. diabetes mellitus increases the risk of chd 2 to 4 times.(8) an unmatched case-control study that was conducted on 143 newly diagnosed patients with cvd and 286 controls demonstrated that the most frequent risk factors for cvd were older age, smoking history, low level of physical activity, and greater waist circumference.(9) however, this study is inconsistent with the finding from india with the same study design, demonstrating that no significant association was observed between physical inactivity and coronary artery disease (cad) among females.(10) a matched case control study conducted on 1000 male military patients [250 with cad and 750 without cad] indicated that the risk factors, including diabetes, hyperlipidemia, smoking, hypertension, and positive family history of cad, increase the probability of cad.(11) although these risk factors have been identified, cad is still highly prevalent. diabetes, smoking, hypertension, hyperlipidemia, and positive family history are well-known factors for cardiovascular disease, but it is unclear what their precise effects are in different populations.(11) previous studies have shown that several factors are associated with coronary heart disease, such as coffee drinking habits, high carbohydrate diet, fasting blood sugar levels, systolic blood pressure, and education.(12–14) considering these cases, the present study aimed to estimate the proportion of the determinants of chd in adults aged 25 years and over. methods research design this study was a further analysis with a matched case-control design using sub-sample data from the “cohort study of ncd risk factors” which was conducted in five villages of central bogor district, bogor city, in 2011-2018. the study was performed between january 2011 43 and december 2018 in the aforementioned five villages of central bogor district, bogor city. research subjects the sample determination for the casecontrol study used a 1:3 ratio of cases and controls, after conducting a pilot study on the percentage of exposed controls in the study setting considering hypertension as a risk factor. the proportion of chd in subjects with and without hypertension was 33.1% and 16.5%, respectively; (12) using the following case-control formula with a power of 80% (zβ = 0.84); 5% significance level (zα/2 = 1.96), and a ratio of controls to cases of 3, a minimum sample size of 133 per group was obtained. the sample population was all individuals in the age group of 25 years and over. the study sample was individuals with chd cumulatively from 2011-2018,(15) and the controls were individuals who did not suffer from chd, during the 8 years of the study and did not suffer from other diseases such as diabetes mellitus. individuals in the two selected groups were matched for age and gender. there were 148 chd cases identified from 2011-2018, while the control group consisted of 444 individuals, which is 3 times the number of individuals with chd in the cases group.(16) the chart of the chd sample size can be seen below (figure 1). selection of cases cases were adult patients who had a confirmed diagnosis of chd. the criteria for chd are individuals who are declared to have coronary heart disease based on the results of electrocardiography (ecg) examinations which are carried out at a follow-up examination every two years. the results of the ecg were evaluated by a consultant cardiologist (mentioned by his/ her initials). in addition, deaths of individuals from chd were confirmed through verbal autopsy by a trained puskesmas medical doctor. therefore the diagnosis of chd is based on an electrocardiogram (ecg) for living persons or verbal autopsy result for deceased individuals.(5) selection of controls controls were defined as individuals who had not experienced chd based on a doctor’s diagnosis and were selected by simple random sampling, taking into account gender and age matching. in addition, we excluded outpatients with hypertension and diabetes mellitus because the risk factors for these diseases may be similar to those of cases. figure 1. individuals with and without chd non-chd baseline (n=4506) chd (n=148) cumulative incidence of chd 2011-2018 (n=200) non-chd (n=444) cases with matching age and gender controls with matching age and gender univ med vol. 42 no. 1 44 sudikno, driyah, pradono coronary heart disease among adults data sources and case handling the data come from structured interviews, physical examinatio ns, and supporting examinations that are carried out every two years. interviews included socio-demographic data, past illnesses, diet, medication history, and behavior (physical activity, smoking, etc.). physical examinations carried out were anthropometric measurements (weight, height, abdominal circumference, and blood pressure [systolic and diastolic pressure]). investigations include clinical chemistry examination (lipid profile, blood sugar, kidney function, and hba1c), and supporting electrocardiograms (ecg). individuals with abnormalities in the results of physical and supporting examinations are referred to health facilities, namely puskesmas and referral hospitals for treatment and health control.(5) the laboratory results use the third report of the national cholesterol education program (ncep) or adult treatment panel iii as reference,(17) which has been adapted to asian individual conditions. the risk limits used were waist circumference for men of >90 cm and for women of >80 cm, triglycerides >150 mg/dl, hdl cholesterol of <40 mg/dl and <50 mg/dl for men and women, respectively, fasting blood glucose of 110 mg/dl., and blood pressure of 130/85 mmhg.(18) data quality assurance before data collection, the questionnaire was pretested for feasibility in 10% of study subjects. data recording quality was checked by the end of each day of data collection, for consistency, completeness, clarity, and accuracy; also during data processing, the information was checked for completeness and internal consistency. data collection was done by four clinical nurses. a oneday training and practical demonstration session on interview techniques and measurement procedures was given to the data collectors by supervisors. data analysis this further analysis uses spss software version 16, (serial number 5061284). the kolmogorov–smirnov test was used to determine the data distribution, which proved to be normal. because of the normal data distribution (p>0.05), the chi square test, fisher’s exact test, and independent t test were used. bivariate and multiple logistic regression was used to determine the predictors of chd within 8 years. factors with p values of <0.25 in the bivariate analysis were selected and used in multiple logistic regression analysis to adjust the confounders. statistical significance was set at a p value of < 0.05. research ethics the study protocol was reviewed and approved by the hea lth research ethics committee, health research and development agency, when collecting data from 2011 to 2018 under the following numbers: ke.01.08/ec/485/ 2011; ke.01.05/ec/394/2012; lb.02.01/5.2/ ke.215/2013; lb.02.01/5.2/ke. 143/2014; lb.02.01/5.2/ke.135/2015; lb.02.01/5.2/ ke.042/2016; lb.02.01/2/ke.108/2017; and lb.02.01/2/ke.076/2018. informed consent was obtained from all participants. results in the initial data collected in 2011, there were 4,506 respondents who were not diagnosed with chd. the cumulative incidence of chd cases from the results of follow-up examinations from 2013 to 2018 was identified, but only 148 cases with and 444 cases without chd could be further analyzed by case-control (figure 1). characteristics of study subjects at baseline generally described case subjects as having higher mean lipid levels, blood sugar levels, body mass index, abdominal circumference, and blood pressure than control individuals (table 1). however, only the mean fasting blood sugar, postprandial blood glucose, and systolic blood pressure of case subjects were significantly higher than those of control subjects (p<0.05). the education of individuals with and without chd was mostly low and secondary education, 45 but higher education was two and a half times greater in the case group than in the control group. most of the individuals in both groups had obesity, hypercholesterolemia, and high ldl levels. although the majority of fasting and post-prandial blood glucose levels in the case and control groups were within normal limits, fasting and post-prandial blood glucose levels in the case group were 1.5 – 2 times higher than in the control group. similarly, systolic blood pressures of 130 mmhg and diastolic blood pressures of 85 mmhg were more frequent in the case group than in the control group (table 2). from the results of logistic regression analysis after controlling for other risk factors, it appears that only higher education was a predictor of chd. subjects with higher education were 2.32 times more likely to suffer from chd than those with low education. other risk factors such as physical activity, smoking behavior, mental-emotional disorders, routine medications, consumption of sodium, fat, and sugar, central obesity, bmi, blood pressure, and lipid profile had no effect on the incidence of chd in the analysis (tables 2 and 3). discussion the results showed that a high level of education was a predictor of chd. the results of this study did not show a relationship of blood pressure and lipid profile with chd, although several previous studies have shown such a relationship. a study in china found a positive association between blood pressure and cardiovascular disease.(19) the results of our analysis show the same results as previous studies, in that systolic blood pressure is a predictor of chd.(20) the same result was also reported by the study of qi et al. (21) namely the higher the blood pressure, the greater the risk of chd. a systolic pressure of 130–139 mmhg and/or a diastolic pressure of 80–89 mmhg has a 1.7 times risk, and a systolic pressure of 140–159 mmhg and/or a diastolic pressure of 90–99 mmhg has a 2.4 times risk of developing chd. the study characteristics case (n=148) control (n= 444) p value gender male 35 (23.6) 105 (23.6) 1.000 @ female 113 (76.4) 339 (76.4) age groups (years) 40-49 67 (45.3) 201 (45.3) 1.000 @ 50-59 64 (43.2) 192 (43.2) 60 + 17 (11.5) 51 (11.5) age (years) 50.37±6.49 50.37±6.47 1.000 # total cholesterol (mg/dl) 213.46±35.07 207.70±35.88 0.968 # triglycerides (mg/dl) 119.47±60.54 113.49±67.23 0.659 # ldl-c (mg/dl) 135.81±31.02 131.97±31.01 0.733 # hdl-c (mg/dl) 53.06±11.32 51.52 ±11.28 0.811# fasting blood glucose (mg/dl) 92.53±27.05 88.29±23.43 0.038 # post-prandial blood glucose (mg/dl) 133.15±65.09 120.87±44.60 0.000 # bmi (kg/m2) 25.58±4.16 25.08±4.46 0.461 # waist circumference (cm) 81.42±10.98 80.32±8.88 0.501# blood pressure diastolic (mmhg) 85.64±13.11 80.74±12.23 0.418 # systolic (mmhg) 140.89±27.62 129.98±10.58 0.002 # table 1. characteristics of study subjects at baseline note: data presented as n (%) and mean ± sd, ldl-c = low density lipoprotein cholesterol, hdl-c = high density lipoprotein cholesterol, bmi = body mass index; @ chi-square test; # independent t-test univ med vol. 42 no. 1 46 sudikno, driyah, pradono coronary heart disease among adults risk factors case (n=148) control (n= 444) or (ci 95%) p value education low (elementary school and no schooling) 72 (48.6) 207 (46.6) 1 middle (middle school and high school) 61 (41.2) 219 (49.3) 0.33 (0.15-0.70) 0.004 high 15 (10.1) 18 (4.1) 0.41 (0.20-0.87) 0.020 physical activity sufficiently 94 (63.5) 298 (67.3) 1 less 54 (36.5) 145 (32.7) 1.18 (0.80-1.74) 0.403 smoking never smoker 87 (65.9) 232 (62.4) 1 light smoker 16 (12.1) 75 (20.2) 0.56 (0.31-1.03) 0.062 moderate smoker 19 (14.4) 45 (12.1) 1.12 (0.62-2.03) 0.694 heavy smoker 10 (7.6) 20 (5.4) 1.33 (0.60-2.96) 0.480 emotional mental disorder no 137 (92.6) 401 (90.5) 1 yes 11 (7.4) 42 (9.5) 0.76 (0.38-1.53) 0.451 treatment* regularly 65 (62.9) 118 (95.2) 1 no regularly 5 (7.1) 6 (4.8) 1.51 (0.44-5.14) 0.508 sodium < 2000 mg 69 (73.4) 208 (71.5) 1 >= 2000 mg 25 (26.6) 83 (28.5) 0.90 (0.53-1.53) 0.718 fat <67 g 84 (56.8) 250 (56.4) 1 >= 67 g 64 (43.2) 193 (43.6) 0.98 (0.67-1.43) 0.945 sugar <50 g 111 (92.5) 324 (90.0) 1 >= 50 g 9 (7.5) 36 (10.0) 0.73 (0.34-1.56) 0.417 central obesity normal 46 (31.1) 142 (32.0) 1 obese 102 (68.9) 302 (68.0) 1.04 (0.69-1.55) 0.838 bmi normal 35 (23.6) 112 (25.3) 1 underweight 4 (2.7) 17 (3.8) 0.75 (0.23-2.38) 0.630 overweight 18 (12.2) 66 (14.9) 0.87 (0.45-1.66) 0.679 obese 91 (61.5) 248 (56.0) 1.16 (0.74-1.83) 0.494 total cholesterol normal<200 mg/dl 38 (25.7) 142 (32.1) 1 high >=200 mg/dl 110 (74.3) 301 (67.9) 1.36 (0.89-2.07) 0.145 triglycerides normal<150 mg/dl 97 (65.5) 320 (72.1) 1 high >=150 mg/dl 51 (34.5) 124 (27.9) 1.35 (0.91-2.01) 0.132 ldl-c normal 19 (12.8) 54 (12.2) 1 high 129 (87.2) 390 (87.8) 0.94 (0.53-1.64) 0.829 hdl-c normal 89 (60.1) 256 (57.7) 1 low 59 (39.9) 188 (42.3) 0.90 (0.61-1.31) 0.597 fasting blood glucose <126 mg/dl 119 (80.4) 401 (90.3) 1 >=126 mg/dl 29 (19.6) 43 (9.7) 2.27 (1.36-3.79) 0.002 post-prandial blood glucose <200 mg/dl 119 (80.4) 389 (87.6) 1 >=200 mg/dl 29 (19.6) 55 (12.4) 1.72 (1.05-2.82) 0.031 systolic blood pressure <130 mmhg 54 (36.5) 241 (54.3) 1 >=130 mmhg 94 (63.5) 203 (45.7) 2.06 (1.40-3.03) 0.000 diastolic blood pressure <85 mmhg 62 (41.9) 260 (58.6) 1 >=85 mmhg 86 (58.1) 184 (41.4) 1.96 (1.34-2.85) 0.000 note : data presented as n (%);or : odds ratio; c.i, : confidence interval; ldl-c = low density lipoprotein cholesterol; hdl-c = high density lipoprotein cholesterol; bmi = body mass index table 2. relationship between risk factors and coronary heart disease 47 risk factors or 95%ci p value education low (elementary school and no schooling) 1 middle (middle school and high school) 0.80 0.52-1.24 0.333 high 2.32 1.01-5.35 0.047 smoking never smoker 1 light smoker 0.66 0.36-1.22 0.193 moderate smoker 1.26 0.68-2.32 0.459 heavy smoker 1.05 0.44-2.50 0.898 total cholesterol normal<200 mg/dl 1 high >=200 mg/dl 1.05 0.65-1.69 0.823 triglycerides normal<150 mg/dl 1 high >=150 mg/dl 1.12 0.70-1.80 0.617 fasting blood glucose <126 mg/dl 1 >=126 mg/dl 1.96 0.97-3.95 0.060 post-prandial blood glucose <200 mg/dl 1 >=200 mg/dl 1.15 0.57-2.32 0.678 systolic blood pressure <130 mmhg 1 >=130 mmhg 1.35 0.76-2.41 0.298 diastolic blood pressure <85 mmhg 1 >=85 mmhg 1.69 0.95-3.00 0.073 table 3. the results of multiple logistic regression analysis of chd risk factors note: factors with p < 0.25 in the bivariate analysis were included in the multivariate models by tur in et al. (2 2) showed tha t chd was influenced by hypertension in both men and women, who had a 2-times higher risk of chd than did normotensives. similarly for blood sugar levels. the result of this study did not show a relationship between lipid profile and chd, and was therefore different from previous studies. the results of a meta-analysis of 32 articles in the middle east, with a sample size of 191,979 individuals, reported that the highest risk factors for heart disease were dyslipidemia, hypertension and diabetes. the proportion of dyslipidemia (43.3%) in this region is higher than that of hypertension (26.2%) and diabetes mellitus (16%).(23) research on chd risk factors was also conducted in shenzhen, china, on 3,395 hypertensive respondents aged between 30 and 79 years. it was found that 1,153 individuals had chd within 3 years. the reported risk factors for coronary heart disease are age, body mass index, diabetes, dyslipidemia, and chronic kidney disease as predictors in individuals with hypertension. the chinese study also found that the odds ratio of mental and emotional disorders is 1.54 times and sleep disturbance is 1.69 times (95%1ci. 1.21-2.34) with an area under curve (auc) of 0.839. this shows that individuals with hypertension need to maintain psychological health and good quality of sleep to help prevent coronary heart disease.(24) blood pressure has a consistent relationship with chd, the higher the blood pressure, the higher the likelihood of chd. hypertension triggers atherosclerosis which can damage the endothelium through oxidative stress, resulting in increased endothelial cell synthesis of collagen and fibronectin, and its reduction is dependent on nitric oxide and increased permeability to lipoproteins.(25) univ med vol. 42 no. 1 48 sudikno, driyah, pradono coronary heart disease among adults meta-analysis studies show that a decrease in glycemic index and glycemic load is associated with the incidence of chd in women. the pooled analysis showed increased chd risk for the highest compared with the lowest glycemic index quintile.(26) other studies have reported that type 1 and type 2 diabetes lead to an increased incidence of ischemi c heart disease and congestive heart failure. coronary heart disease accounts for up to 80 percent of mortality in type 2 diabetes patients, especially in women with diabetes.(27) the underlying factors for diabetic heart disease are coronary artery disease, hypertension, metabolic disorders such as hyperglycemia and dyslipidemia, microvascular disease, and autonomic neuropathy. the increase in diabetes-related sudden death may be due to autonomic neuropathy.(28) other studies have shown that chd is more common in women. stroke, hypertension, and hyperglycemia (fasting blood sugar level 100 mg/ dl) are determinant factors f or chd. (2 9) meanwhile, metabolic syndrome contributes to an increased risk of chd and type 2 dm, which is twice the risk of chd and five times the risk of dm in individuals without metabolic syndrome. diabetes mellitus will also increase the risk of chd up to three ti me s tha t of non-dm disorders.(30) a study by jiang et al.(31) showed that postprandial hyperglycemia correlated with chd, and suggested fasting blood sugar to be used to screen for chd. fasting sugar levels that are higher than normal are also associated with levels of arterial stiffness, coronary artery hypercalcemia, and risk of cardiovascular disease. conversely, hypoglycemia or rapid changes in plasma glucose levels can cause an increase in counterregulatory hormones, such as epinephrine and norepinephrine. an increase in this hormone induce s vasoconstri ction and platelet aggregation. ( 32 ) likewise, p eople with hypercholesterolemia and hyperlipidemia ge nerally ha ve a bn ormal c arbohydr ate metabolism with elevated insulin levels.(33) insulin resistance predicts future cardiovascular risk.(34) thus, any dietary factor that worsens glucose tolerance or increases insulin resistance is likely to increase the risk of death from chd. the result of this study showed that higher education had a 2.32-fold risk of getting chd compared to low education. this finding is interesting and differs from the results of studies of genetic data with the hypothesis that low education is a causa l r isk factor in the development of chd. similarly, research by maharani et al.(35) reported that individuals with lower education suffered from chd. the higher the level of education, the fewer people suffer from chd and from above-normal blood sugar. this finding is also similar to the research of tillmann et al.(36), in that low education is a risk factor in the development of chd. so it is possible that the cause of chd is more complex in individuals with higher education than in those with low education because there are other factors that need to be investigated more deeply, including diet, life demands, work status, and environmental influences. moreover, level of education is considered to affect the individual’s ability to turn information into practical measures and behavior, affects access to recourses, employment-related problems and social exclusion if unemployed. a limitation of this case-control study was that food consumption or eating patterns could not be accurately measured, due to difficulties in measuring food recall. as is well known, chd is affected by the consumption of macronutrients such as carbohydrates, fats, protein, and fiber. it is also influenced by micronutrients such as sodium and vitamins. khazanah’s et al.(37) proves that excess sodium has an effect on blood pressure which will increase heart risk, and that consumption patterns of high carbohydrates, fat, cholesterol, and low fiber consumption. based on the results of this study, it is still necessary to control chd risk factors, especially those related to increased blood glucose and blood pressure. subjects with higher education need to get proper nutritional knowledge so that they are expected to have alternatives in choosing healthy foods. in addition, it is necessary to routinely 49 monitor the health of respondents by health workers at posyandu and primary health facilities. conclusion higher education is a predictor of chd among adults. suggested chd prevention and control programs can be carried out by regular control of blood sugar levels and blood pressure. it is necessary to investigate further to find the reason why higher education is a predictor of chd in this study. conflict of interest none. acknowledgment we would like to thank the health research and development agency for providing us with the opportunity to conduct a cohort study of risk factors for non-communicable diseases. thanks are also due to the cohort study team who have collaborated on cohort activities so that this paper can be written. our special thanks to prof. dr. dr. emiliana tjitra who has provided guidance and direction in writing this article. author’s contribution s and sd contributed to literature search, data analysis and writing of the draft. jp contributed to literature search and analysis of participant characteristics. all authors have read and approved the final manuscript references 1. roth ga, johnson c, abajobir a, et al. global, regional, and national burden of cardiovascular diseases for 10 causes , 1990 to 2015. j am coll cardiol 2017;70:1-25. doi: 10.1016/j.jacc.2017.04. 052. 2. finucane mm, paciorek cj, danaei g, ezzati m.bayesian estimation of population-level trends in measures of health status. stat sci 2014;29:18– 25. doi: 10.1214/13-sts427. 3. sanchis-gomar f, perez-quilis c, leischik r, lucia a. epidemiology of coronary heart disease and acute coronary syndrome. ann transl med 2016; 4:256. doi: 10.21037/atm.2016.06.33 4. benjamin ej, muntner p, alonso a, et al. on behalf of the american heart association council on epidemiology and prevention statistics committee and stroke statistics subcommittee. heart disease and stroke statistics — 2019 update: a report from the american heart association. circulation 2019;139:e56-e528. https://doi.org/ 10.1161/cir.0000000000000659. 5. indonesian health research and development agency. hasil utama laporan riskesdas 2018. 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[consumption of sodium, saturated fats, and fiber is associated with incidence of coronary heart disease in dr. zainoel abidin hospital, banda aceh]. j kesehat 2019;7:40–4. univ med vol. 42 no. 1 c:\users\user\documents\41 no 2 157 abstract universa medicina diabetes mellitus patients in indonesia: management in a tertiary hospital compared to primary health care hikmat permana1, raspati cundarani koesoemadinata2,3*, nanny natalia mulyani soetedjo1,2, nury fitria dewi2, novi jayanti2, sofia imaculata2, rovina ruslami2,4, bachti alisjahbana1,2, and susan margaret mcallister5 background the increasing prevalence of diabetes mellitus (dm) requires that patients have greater access to care, which is yet lacking in many lowand middleincome countries and the quality of which varies between health care facilities. we compare the characteristics, complications, and risk profile of diabetes in patients receiving care in primary and tertiary level health facilities in bandung, indonesia. methods adult dm patients were recruited from 25 community health centres (chcs) and the outpatient clinic at one referral hospital. key data collected and compared to national guidelines were dm history, treatment, complications, blood pressure, height, weight, and laboratory examinations on glycated haemoglobin (hba1c), lipid profile, and creatinine. data analyses were conducted using chi-square or fisher's exact test for categorical variables and student's t-test or mann-whitney test for numerical variables. results of the 809 dm patients (median age 59 years, 63% female, 98% type 2 dm), 318 (39%) were from chcs and 491 (61%) from the hospital. overall median hba1c was 8.3%, with no difference between chc and hospital patients. only 32% of patients with hba1c 10% were on insulin (chcs 5.9%, hospital 42.9%), and only 18% of those on insulin had a good glycaemic control (<7%). hypertension was common (chcs 62%, hospital 51%, p<0.001), and only 44% of chc and 34% of hospital patients received antihypertensive therapy. among those with macrovascular complications, only 32% (chcs) and 26% (hospital) were receiving aspirin. the numbers reaching the treatment targets were low for those on antihypertensives and lipid-lowering medications (80/251 and 11/105, respectively). conclusion glycaemic control and management of complications of dm patients at both health care levels need considerable improvement. original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.157-168 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1313 cite this article as: permana h, koesoemadinata rc, soetedjo nnm, dewi nf, jayanti n, imaculata s, ruslami r, alisjahbana b, mcallister sm. diabetes mellitus patients in indonesia: management in a tertiary hospital compared to primary health care. univ med 2022;41: 157-68. doi: 10.18051/univmed.2021.v40.157-168. 1department of internal medicine, faculty of medicine, universitas padjadjaran/dr hasan sadikin general hospital, bandung, indonesia 2research center for care and control of infectious disease, universitas padjadjaran, bandung, indonesia 3department of internal medicine, radboud institute for health sciences, radboud university medical center, nijmegen, the netherlands 4department of biomedical sciences, faculty of medicine, universitas padjadjaran, bandung, indonesia 5centre for international health, department of preventive and social medicine, university of otago medical school, dunedin, new zealand correspondence: *raspati cundarani koesoemadinata research center for care and control of infectious disease universitas padjadjaran, bandung, indonesia 40161 phone/fax: 022-2044128 email: r.c.koesoemadinata@unpad.ac.id orcid id: 0000-0002-3817-4589 date of first submission, april 25, 2022 date of final revised submission, july 26, 2022 date of acceptance, august 1, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license may-august 2022 vol.41 no.2 158 permana, koesoemadinata, mulyani , et al diabetes mellitus management, indonesia introduction diabetes mellitus (dm) is one of the most extensive global health problems of the 21st century. in 2019, there were 463 million adults estimated to be living with dm. this number is predicted to increase to 700 million by 2045.(1) an estimated 79% of these people are thought to be living in lowor middle-income countries, the majority of whom have type 2 diabetes.(2) patients with dm o ften present with cardiovascular and other complications that require more careful and often specialist care.(3) diabetes mellitus and its complications often constitute a significant financial burden to patients and their families.(4) the care of dm patients in low-resource countries is challenging as health care systems are less equipped to manage people with chronic conditions. with an estimated 10.7 million people with dm, indonesia is ranked 7th in the world.(1) the provision of quality care for dm patients, from early diagnosis to treatment, and prevention of complications, is hampered by a fragile and poorly resourced health system. currently, only 113 endocrinologists provide specialist care for dm patients, mainly concentrated at secondary and tertiary care levels in the main urban centres.(5) at the primary level, there is one community health centre (chc) for every 27,000 people.(6) however, the number of general physicians is 3.8 per 10,000 population, far below the world average of 17.4,(7) and there is a significant geographical variation with poorer access and a limited number of general practitioners and nurses in remote areas. dm patients are primarily managed in chcs, where they can be diagnosed through random or fasting blood glucose, treated with oral dm medications, and receive regular monitoring. in 2014, the indonesian government started an insurance system to enable universal health coverage to strengthen primary care capacity, particularly in chronic diseases – including dm. (3 ) since then, it has been recommended that patients with dm who have no uncontrollable hype rglyc aemia or complications should be referred to a secondary or tertiary hospital. while the indonesian society of endocrinology has established guidelines for the management of dm patients,(8) the definitions and processes for referral are not always clear or easy to understand. further studies urgently need to be carried out in indonesia because current dm therapy is focused almost solely on the clinical aspects. furthermore, the present study can be used as a scientific base for the government of indonesia in organizing dm programs, especially in supporting the government program to strengthen the primary service in indonesia. our study aims to describe the severity and complications of dm, the risk factors for cardiovascular disease, and the medication management of dm patients, and to investigate any difference in these characteristics between patients cared for at the tertiary level hospital compared to primary health care level. methods research design this cross-sectional study was part of the tandem research project on tuberculosis and diabetes (www.tandem-fp7.eu) (9) and was conducted at dr hasan sadikin general hospital and community health centres (chcs) situated throughout the city between december 2013 and february 2015. study participants a total of 809 dm patients were eligible and consented to participate in this study, consisting of 491 subjects from the hospital and 318 subjects from the chcs). patients eligible to participate were aged 18 years and over with known dm (either under care for dm or on dm medication). patients who had gestational or steroid-induced diabetes were excluded. recruitment was conducted among patients from the outpatient endocrine clinic of dr hasan sadikin general hospital – a tertiary level public referral hospital for the west java region of indonesia, located in 159 bandung city, or from 25 chcs situated throughout the city. data collection the research physicians interviewed each patient asking about their socio-demographic characteristics (age, gender, and education), behavioural characteristics such as smoking status and alcohol consumption, and diabetic characteristics such as dm history, complications, medication, and management. the research nurses followed a standard operating procedure measuring height, weight (using digital scales), and waist circumference to calculate body mass index (bmi) and central obesity. patients’ weight and height were classified based on the asia pacific bmi criteria,(10) namely underweight (<18.5 kg/m2); normal (18.5-22.9 kg/m2); overweight (23.0-24.9 kg/m2); obese i (25.0-29.9 kg/m2); obese ii (30 kg/m2). central obesity was categorized as a waist circumference (wc) of 80 cm for females and 90 cm for males. laboratory analysis the research nurses followed a standard operating procedure for taking patients’ blood pressure using a digital device. blood was taken for laboratory glycated haemoglobin (hba1c) examinations and urine for albumin creatinine ratio (acr) examinations. lipid profile and creatinine concentrations, tested in dr hasan sadikin general hospital laboratory, as the most recent test undertaken within the previous month, were obtained from electronic medical records for a subset of patients under regular care in the endocrine clinic. laboratory hba1c was categorized into three groups: <7.0%; 7.0-9.9%; and 10% for analysis and also dichotomized into two groups: <10% and 10%. albumin creatinine ratio categories were: n ormal (<30 mg/g) ; micr oalbuminuria ( 30-300 mg/g) , and macroalbuminuria (>300 mg/g).(8,11) low-density lipoprotein (ldl) was categorized as uncontrolled if the result was 100 mg/dl.(8,11) estimated glomerular filtration rate (egfr) was calculated according to the ckd-epi creatinine equation 2009 (12) which was then categorized according to the us national kidney foundation guidelines for chronic kidney disease (ckd): stage 1 (egfr 90), stage 2 (egfr 60-89), stage 3 (egfr 3059), stage 4 (egfr 15-29), stage 5 (egfr <15).(13) key measures key mea sures sought are r elate d to guidelines set by the indonesian society of endocrinology as follows: 1) patients with hba1c 10% should have insulin added to their medications; 2) patients with systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg should be managed with antihypertensive medication; 3) patients with macrovascular complications should be managed with aspirin; and 4) patients with ldl 100 mg/ dl should be managed with statin therapy.(8) blood pressure was categorized according to the indonesian guidelines (box 1) and also the jnc vii(14) i.e. normal (systolic and diastolic <120/80 mmhg); pre-hypertension (systolic 120139 or diastolic 80 -89 mmhg) ; stage i hypertension (systolic 140-159 or diastolic 90-99 mmhg); stage ii hypertension (systolic 160 or diastolic 100 mmhg). complications complications were self-reported by patients and cross-checked for patients who had a medical record in the hospital. complications were then classified according to the charlson morbidity index (cmi).(15) macrovascular complications included a history of heart disease: heart attack, angina pectoris, coronary artery bypass grafting (cabg), percutaneous transluminal coronary intervention (ptci), history of cardiovascular disease, or history of other vascular diseases. microvascular complications included a history of renal disease or eye problems (blindness, decreased visual acuity, cataract operation or laser treatment, glaucoma). univ med vol. 41 no. 2 160 permana, koesoemadinata, mulyani , et al diabetes mellitus management, indonesia a b data management demographic and clinical data were entered onto a case report form and then into a secure, centr ally mana ged e lectronic database (redcap).(16) data such as laboratory results, diabetes history, smoking status, and complications were entered directly into redcap. data quality was checked monthl y for acc uracy and completeness. statistical analysis descriptive data are presented as mean and standard deviation for normally distributed data, median and inter-quartile range (iqr) for abnormally distributed data, and proportions for categorical data. chi-square or fisher’s exact tests of association were used to compare groups where appropriate, and student’s t-test was used to compare mean values. statistical analyses were performed using stata v13 (statacorp, college station, tx, usa). ethical considerations all patients provided written informed consent prior to inclusion. ethical approval was received from the observational/interventions research ethics committee, london school of hygiene and tropical medicine on 18 december 2013 (lsht m ethics ref : 6449, lsht m amendment no: a473) and from the health research ethics committee, faculty of medicine, universitas padjadjaran, bandung on 5 february 2014 (no: 05/un6.c2.1.2/kepk/pn/2014). table 1. patient characteristics according by recruitment site abbreviations: chc=community health center; dm=diabetes mellitus; nd=no data; sd=standard deviation ï dm type was only recorded from medical records for 491 patients recruited in the hospital *chi-square test; # independent-t-test characteristics total n=809 n (%) recruitment site p-value* chc n=318 n (%) hospital n=491 n (%) sex, female 511 (63.2) 231(72.6) 280 (57.0) <0.001 mean age (sd) – years# 59 (10) 60 (9) 58 (11) 0.006 age group (years) <40 33 (4.1) 8 (2.5) 25 (5.1) 0.008 40-49 100 (12.4) 32 (10.0) 68 (13.9) 50-59 285 (35.2) 101 (31.8) 184 (37.5) 60-69 276 (34.1) 129 (40.6) 147 (29.9) ≥70 115 (14.2) 48 (15.1) 67 (13.6) education no formal education/ primary 249 (30.8) 105 (33.0) 144 (29.3) 0.64 junior high school 153 (18.9) 58 (18.2) 95 (19.3) senior high school 229 (28.3) 84 (26.4) 145 (29.5) higher education 178 (22.0) 71 (22.3) 107 (21.8) smoking status current 117 (14.5) 43 (13.5) 74 (15.1) 0.003 past 235 (29.0) 73 (23.0) 162 (33.0) never 457 (56.5) 202 (63.5) 255 (51.9) alcohol consumption (current/past) 17 (2.1) 3 (0.9) 14 (2.8) 0.114 duration of diabetes (years) <1 144 (17.8) 58 (18.2) 86 (17.5) 0.191 1-5 311 (38.4) 134 (42.1) 177 (36.0) 6-15 286 (35.4) 105 (33.0) 181 (36.9) >15 68 (8.4) 21 (6.6) 47 (9.6) dm type ϯ type 1 nd 12 (2.4) type 2 nd 479 (97.6) 161 results description of study participants the mean age was 59 ± 10 years, and 63.2% were female. the majority of patients had type 2 dm (97.6%) and had dm for more than one year (82.2%). a total of 491 (61%) patients were recruited in the hospital and 318 (39%) from chc. patients recruited in the hospital were slightly younger (58 vs. 60 years (p=0.006), a smaller proportion were females (57.0% vs. 72.6%, p<0.001), and had never smoked (51.9% vs. 63.5%, p=0.003) (table 1). dm severity and complications the overall median hba1c was 8.3% (iqr 6 .8 -1 0. 3) , a n d t h e r e w a s n o si gni f i c a nt difference according to the recruitment site. a history of any macrovascular complications was r ep o r te d b y 20 .3% of p at i ent s , a nd t hi s proportion was higher in patients recruited in the hospital than in chcs (23.2% vs 15.7%, p=0.010). the most common macrovascular compl ica tion re ported wa s hear t disease (34.1%). fifteen (1.9%) patients had undergone foot amputation (table 2). table 2. diabetes severity and complications according by recruitment site abbreviations: chc=community health center; acr=albumin to creatinine ratio; cabg=coronary artery bypass grafting; egfr=estimated glomerular filtration rate; iqr=interquartile range; nd=no data; ptci=percutaneous transluminal coronary intervention a egfr ml/min/1.73 m2 *chi-square-test; ¶ mann-whitney test univ med vol. 41 no. 2 characteristics total n=809 n (%) recruitment site p-value* chc n=318 n (%) hospital n=491 n (%) median hba1c (iqr) %¶ 8.3 (6.8-10.3) 8.4 (6.9-10.3) 8.2 (6.7-10.2) 0.663 history of macrovascular complications any macrovascular complications 164 (20.3) 50 (15.7) 114 (23.2) 0.010 heart disease: myocardial infarction 110 (13.6) 27 (8.5) 83 (16.9) 0.001 cabg or ptci 35 (4.3) 4 (1.3) 31 (6.3) 0.001 angina pectoris or heart failure 131 (16.2) 33 (10.4) 98 (20.0) 0.001 stroke 80 (9.9) 27 (8.5) 53 (10.8) 0.284 foot amputation 15 (1.9) 6 (1.9) 9 (1.8) 0.956 history of microvascular complications any microvascular complications 542 (67.0) 218 (68.6) 324 (66.0) 0.448 renal failure 28 (3.5) 4 (1.3) 24 (4.9) 0.022 eye problems: blindness 34 (4.2) 5 (1.6) 29 (6.0) 0.003 decreased visual acuity 232 (28.7) 113 (35.6) 119 (24.2) 0.001 cataract operation/laser treatment 130 (16.1) 45 (14.2) 85 (17.3) 0.349 glaucoma 7 (0.9) 2 (0.6) 5 (1.0) 0.439 chronic kidney disease classification (n=301) stage 1 (egfra ≥90) nd 217 (44.2) stage 2 (egfra 60-89) nd 147 (29.9) stage 3 (egfra 30-59) nd 91 (18.6) stage 4 (egfra 15-29) nd 30 (6.0) stage 5 (egfra <15) nd 6 (1.3) albuminuria normal (acr <30 ug/mg) 402 (49.7) 160 (50.3) 242 (49.3) 0.746 micro-albuminuria (acr 30-300 ug/mg) 241 (29.8) 97 (30.5) 144 (29.3) macro-albuminuria (acr >300 ug/mg) 166 (20.5) 61 (19.2) 105 (21.4) 162 permana, koesoemadinata, mulyani , et al diabetes mellitus management, indonesia microvascular complications were reported by 67% of patients, with similar proportions in patients in the hospital and chcs (66.0% vs 68.6%, p=0.448). t he most common microvascular complication reported was decreased visual acuity (28.7%). even though only 3.5% of patients reported renal failure, 29.8% patients had microalbuminuria, and 20.5% had macroalbuminuria. there was no significant dif ference between r ecruitment sites for albuminuria (p=0.746). the mean egfr was 78.24 ± 30.05 for the 491 patients recruited in the hospital. almost half of the patients had a normal egfr indicating preserved kidney function (stage 1 ckd classification). patients indicated for dialysis or kidney replacement table 3. cardiovascular risk profile and medication management of patients according by recruitment site abbreviations: chc=community health centre; bmi=body mass index; dm=diabetes mellitus; wc=waist circumference; hdl=high density lipoprotein; ldl=low density lipoprotein; nd=no data data were complete (n=809) except for ldl /hdl, only measured in 272 patients recruited in the hospital. a blood pressure classification according to jnc vii:  normal = systolic and diastolic <120/80 mmhg;  pre-hypertension = systolic or diastolic 120/80-139/89 mmhg;  stage i hypertension = systolic or diastolic 140/90-159/99 mmhg;  stage ii hypertension = systolic or diastolic e”160/100 mmhg b twenty-three patients with no medication (35.4%) had been diagnosed within the last 12 months. c using sulfonylurea, thiazolidinediones, acarbose, or dpp-4 inhibitors d using metformin, sulfonylurea, thiazolidinediones, acarbose, and/or dpp-4 inhibitor besides insulin e using sulfonylurea, thiazolidinediones, acarbose, and/or dpp-4 inhibitor beside using metformin *chi-square test characteristics total n=809 n (%) recruitment site p-value* chc n=318 n (%) hospital n=491 n (%) body mass index (bmi, kg/m2) under weight (<18.5) 29 (3.6) 7 (2.2) 22 (4.5) 0.145 normal (18.5-22.9) 205 (25.3) 75 (23.6) 130 (26.5) overweight (23.0-24.9) 150 (18.5) 68 (21.4) 82 (16.7) obese i (25.0-29.9) 312 (38.6) 128 (40.2) 184 (37.5) obese ii (≥30) 113 (14.0) 40 (12.6) 73 (14.9) central obesity females wc ≥80 cm yes 352 (68.9) 156 (67.5) 196 (70.0) 0.549 males wc ≥90 cm yes 110 (36.9) 30 (34.5) 80 (37.9) 0.577 blood pressure classificationa normal 123 (15.2) 29 (9.1) 94 (19.1) <0.001 pre-hypertension 237 (29.3) 92 (28.9) 145 (29.5) stage i hypertension 242 (29.9) 94 (29.6) 148 (30.1) stage ii hypertension 207 (25.6) 103 (32.4) 104 (21.2) hyperlipidemia (mg/dl) ldl ≥100 nd 222 (81.9) hdl ≤40 (male); ≤50 (female) nd 124 (45.6) diabetes medication no medicationb 65 (8.0) 28 (8.8) 37 (7.5) <0.001 insulin alone 172 (21.3) 10 (3.1) 162 (33.0) insulin & any oral dm drugsd 58 (7.2) 9 (2.8) 49 (10.0) metformin alone 166 (20.5) 96 (30.2) 70 (14.3) metformin & other oral dm drugse 233 (28.8) 111 (34.9) 122 (24.9) other oral dm drugs alonec 115 (14.2) 64 (20.1) 51 (10.4) anti-hypertensive drugs 251 (31.0) 114 (35.9) 137 (27.9) 0.017 lipid-lowering drugs (statins) 194 (24.0) 55 (17.3) 139 (28.3) <0.001 anti-platelet drugs (aspirin) 67 (8.3) 25 (7.9) 42 (8.6) 0.727 163 therapy (stages 4 and 5 ckd classification) accounted for less than 10% (table 2). cardiovascular risk profile and medication management overall, more than half of the dm patients were in the bmi obese i and ii categories (38.6% and 14.0%, respectively) and were centrally obese (57.1%), and this was similar in people recruited in both sites. in add ition, stage i and ii hypertension were similarly reported in more than half of the patients (55.5%), with more chc patients having severe stage ii hypertension (p<0.001) (table 3). of all dm patients in this study, half (56%) were on monotherapy for dm control (insulin 21.3%; metformin 20.5%, other oral dm medications 14.2%). almost a third (28.8%) were on combined therapy of metformin with other oral dm medications, and 7.2% were on insulin combination therapy. the proportion of hospital patients on insulin was 43.0%, while most chc patients were on oral dm medication (85.2%) (table 3). the indonesian treatment guidelines recommend that patients with hba1c 10% have insulin added to their medications, but in our study, only 32.4% (n=77/238) of such patients were on insulin. of the patients who were considered hypertensive according to guidelines, only 38.1% (n=171/449) were receiving antihypertensive therapy, and this proportion was higher in chc patients (43.6%) compared to hospital patients (33.7%) (p=0.032). one hundred and sixty-four (20.3%) patients reported macrovascular complications, of whom only 28.1% (n=46/164) were receiving aspirin. this proportion was higher in chc patients (32.0%) compared to hospital patients (26.3%) but not statistically significant (p=0.456). of the patients with ldl 100 mg/ dl, 42.3% (n=94/222) were on statin (figure 1). similarly, there were very small proportions of patients on treatment who were achieving the figure 1. treatment indication and actual treatment with insulin, antihypertensive, antiplatelet, and lipidlowering medication the proportion of diabetes patients with an indication for insulin, antihypertensive, antiplatelet, and lipidlowering medication according to the indonesian diabetes guideline. the grey bar represents those patients with a treatment indication actually receiving insulin (32.4%), antihypertensive drugs (38.1%), aspirin (28.0%) and statins (42.3%). data were available for 809 patients, except for ldl (n=272) univ med vol. 41 no. 2 164 permana, koesoemadinata, mulyani , et al diabetes mellitus management, indonesia expected treatment targets. of the 230 patients on insulin, only 41 (17.8%) had an hba1c of <7%. of the 251 patients on antihypertensive medication, 80 (31.9%) reached the treatment target of systolic and diastolic blood pressure of <140 mmhg and <90 mmhg, respectively. of the 105 patients who were on statins and for whom we had ldl information on, only 11 (10.5%) had an ldl <100 mg/dl (figure 2). discussion in this study of patients under care for dm in an urban setting in indonesia, a large proportion had uncontrolled blood glucose, macrovascular and microvascular complications, risk factors for cardiovascular disease, and unmet medication needs. while blood glucose levels, microvascular complications, and albuminuria in patients recruited from the hospital and chc were similar, this was not so for macrovascular complications, hypertension, and obesity which were more common in patients recruited from the hospital. medication management was also different in the two recruitment sites, with fewer hospital patients on antihypertensive medication and more on lipidlowering statins than patients from chc. the median hba1c in our study was 8.3%, with almost three-quarters of patients not reaching the target set in the indonesian type 2 dm management guidelines (8) of having an hba1c less than 7%. a similarly high mean hba1c was reported by soewondo et al.(17) (mean hba1c 8.1%) and cholil et al.(18) (mean hba1c 8.3%) in their extensive studies of patients with diabetes in indonesia. there was no difference in median hba1c in hospital and chc patients, despite routine and regular hba1c monitoring in the hospital. however, in-depth reasons for this could not be explored. still, our results which showed such a small number being on insulin as recommended and then those on insulin not achieving the treatment target, would indicate an overall lack of blood glucose monitoring, referral, and prescribing of appropriate medications from the health system perspective. from the patients’ figure 2. patients receiving insulin, antihypertensives, and lipid-lowering medication and those who have reached the treatment target the proportion of diabetes patients receiving insulin, antihypertensives, and lipid-lowering medication. the grey bar represents those patients who have reached the treatment target of hba1c <7% (17.8%), systolic blood pressure <140 mmhg and diastolic blood pressure <90 mmhg (31.9%), and ldl <100 mg/dl (10.5%). data were available for 809 patients, except for ldl (n=272) 165 perspective, further research is required to understand and estimate their knowledge about dm, medication adherence, and other barriers to their dm care. similar poor glycaemic and metabolic control among type 2 dm patients has also been reported in south-east asian countries.(19–21) misra et al.(22) categorized clinical management challenges of type 2 dm as patient-related challenges, physician-related challenges, and health care, infrastructure, and drug-related factors. patient-related challenges included poor awareness, unhealthy choice of food, and low socioeconomic status. physic ian-related challenges were inadequate skills in chronic disease management, poor communication with patients, too few specialized diabetes nurses, and the non-suitability of guidelines from high-income countries. for many years, public health services in many south asian countries have primarily been designed to focus on infectious diseases. it is only recently that non-communicable conditions have been consider ed and appr opriate programmes created. overcrowded facilities at the primary health care level in indonesia may contribute to poor diabetes care, as is also seen across the western pacific region.(23) almost a quarter of our patients had macrovascular complications, the most prevalent of which was heart disease, with a higher prevalence than that reported in an earlier study (2012) on dm patients in indonesia.(18) many lowand middle-income countries, including indonesia, are undergoing a rapid transition in lifestyle, including work patterns, diet, and eating habits, with a subsequent increase in obesity, heart disease, and other chronic conditions.(24–26) a greater proportion of patients in the hospital had a history of macrovascular complications than did those in chcs. this condition was expected because diabetes patients with complications are more likely to be referred to secondary and tertiary hospitals. despite the larger number of patients with macrovascular complications, only a small proportion was using antiplatelet drugs (aspirin). the high proportion of patients in our study with microvascular complications such as blindness, macro albuminuria, and microalbuminuria is comparable to the 2012 indonesian study. in addition,(18) microvascular complications r equ ire more intensive measurement by physicians, which is not routinely carried out in either site. hence there was no difference between hospital and chc patients. also, patients do not usually present with signs and symptoms of microvascular complications, especially for microalbuminuria. more than half of the patients in our study were obese, combined with hypertension and a high ldl, which together increases the risk for cardiovascular complications, with only a small proportion receiving the relevant antihypertensive, lipid-lowering (statin), or antiplatelet (aspirin) drugs. this limited use of preventive therapy was found at both hospital and chc levels. at the chc level, there is often a limited supply of medications which may contribute to this finding. in the hospital, the national insurance system requires that patients using insurance be referred to the relevant clinic to obtain their medication. for example, those with hypertension are referred to the renal and hypertension clinic to get antihypertensive drugs, and those needing statins as dyslipidaemia drugs must be referred to the cardiology clinic. in each clinic, patients have to queue, often for a long time which is likely to impact their medication uptake and ongoing adherence. of those receiving antihypertensive and dyslipidaemia drugs, only a small proportion reached the blood pressure control target according to indonesian type 2 dm management guidelines.(8) reasons for this are unclear and need to be explored further, but possible explanations could be that medications are not regularly taken because of their side effects, or patients have too many drugs to take daily (polypharmacy). other reasons may be that in a busy clinic, physicians may not be aware that the dose they are prescribing is inadequate. in addition, the number of drugs allowed to be prescribed at univ med vol. 41 no. 2 166 permana, koesoemadinata, mulyani , et al diabetes mellitus management, indonesia one time is limited by the insurance restrictions, or there may be a limited drug supply. in keeping with national guidelines, most of the patients on insulin were under care in the hospital. however, it appeared that more referrals were necessary, with many of the patients with an hba1c 10% not being referred and not receiving insulin. introducing insulin is not an easy task, with many patients being reluctant due to the insulin needing to be injected. even amongst those receiving insulin, many did not achieve the hba1c target of below 7%. this is likely due to a combination of patient, prescriber, and health system management issues as mentioned earlier, but requires further studies to explore this in more detail. on the other hand, many patients with hba1c <7% and without complications were still receiving their medication in the hospital while they were supposed to be referred back to primary care. this may cause an extra burden for clinicians, preventing them from being focused on individual treatment indicators of patients. to our knowledge, this is the first study comparing diabetes care in primary and tertiary health care in indonesia at the individual patient level and with relatively large sample size. however, the study’s limitation was that much of the data was self-reported, particularly that of dm complications, rather than having been collected and confirmed from medical records. also, some data was only available for also, some data was only available for patients recruited in the hospital and could not be compared to those of chc patients. based on the resul ts of this study, complications in people living with dm may be avoided by improving care, i.e., routine monitoring of key laboratory indicators and medication adjustments. chronic disease management trainings may be required for the physicians at the primary health care levels. our study has identified gaps in care, but further research is required to determine the gaps, the key difficulties and barriers for patients and those in the health care system, and how best to address these gaps. moreover, data obtained for this study was from the early stages of the national insurance scheme implementation. since then, several new regulations have been added,(27,28) to ensure better treatment pathways. further evaluation should be done to see whether the improvements have led to better patient care. regular auditing and benchmarking against evidence-based guidelines will be essential to track progress. (26) conclusions overall, the control of diabetes and its complications appears to be poor for patients cared for at both health care levels. many patients need to use insulin but have no access in primary care, and even though hypertensive drugs are more used in primary care, their blood pressure control remained poor. these conditions suggest a need for input and improvement within the health system, and better integration of services both within the hospital and between the primary and tertiary levels of care, particularly for those using national insurance. conflict of interest all authors declare that there was no conflict of interests relevant to the subject of this study. acknowledgement the authors would like to acknowledge the assistance of the staff in the endocrine clinic at dr hasan sadikin general hospital, of dr ahyani raksanagara at the bandung city health office, and of the staff at the community health centres for their role in patient recruitment. prodia laboratory, bandu ng, did the hba1c measurements. funding sources this study was part of the tandem research proj ect (www.tande m-fp7.eu), supported by the european union’s seventh 167 framework programme (fp7/2007-2013) under grant agreement number 305279. contributors conception or design: hp, nnms, smm acquisition, analysis, interpretation of data: nfd, nj, si, nnms, rck, smm writing – original draft: hp, rck writing – review and editing: nnms, smm supervision: ba, rr project administration: ba, rck all authors have read and approved the final manuscript. references 1. international diabetes federation. idf diabetes atlas 9th edition. brussels, belgium: international diabetes federation; 2019. 2. zimmet pz, magliano dj, herman wh, shaw je. diabetes: a 21st century challenge. lancet diabetes endocrinol 2014;2:56-64. doi:10.1016/ s2213-8587(13)70112-8. 3. soewondo p, ferrario a, tahapary dl. challenges in diabetes management in indonesia: a literature review. global health 2013;9:1-17. 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qaim m. obesity trends and determinants in indonesia. appetite 2012;58:100513. doi:10.1016/j.appet.2012.02.053. 26. shivashankar r, kirk k, kim wc, et al. quality of diabetes care in lowand middle-income asian and middle eastern countries (1993-2012) a 20year systematic review. diabetes res clin pract 2015;107:203-23. doi:10.1016/j.diabres.2014.11. 004. 27. kementerian kesehatan republik indonesia. keputusan menteri kesehatan republik indonesia nomor hk.01.07/menkes/813/2019 tentang formularium nasional. jakarta: kementerian kesehatan republik indonesia;2019. 28. kementerian kesehatan republik indonesia. keputusan menteri kesehatan republik indonesia nomor hk.01.07/menkes/603/2020 tentang pedoman nasional pelayanan kedokteran tata laksana diabetes melitus tipe 2 dewasa. jakarta: kementerian kesehatan republik indonesia;2020. c:\users\user\documents\41 no 2 149 abstract universa medicina anger control is the most influential risk factor of mobile phone addiction among nursing and midwifery students of zahedan university of medical sciences sadegh dehghanmehr1, fatemeh kordsalarzehi2*, najmeh ghiamikeshtgar3, nahid mir4, and mahsima banaei heravan5, and farhad shafeie6 background religious attitude and anger management are two psychopathological constructs receiving little empirical scrutiny in relation to smart phone addiction, but theoretically should demonstrate significant relationships. today one of these new media that is used by many people around the world, is the mobile phone. students are one of the most important groups that are affected by mobile social networks. the aim of this study was to determine religious attitudes and anger management as risk factors of mobile phone addiction in nursing and midwifery students. methods this study was a cross-sectional study involving 200 nursing and midwifery students. relevant data were collected through demographic information questionnaire, anger management skills questionnaire, religious attitude questionnaire, and mobile phone addiction questionnaire. a multiple regression model was used to examine the relationship between variables. results the mean age of the research subjects was 22.04 ± 3.30 years. anger control and religious attitude were a significant risk factors of smartphone addiction (β=-0.500; p=0.000; β= -0.069; p=0.004, respectively). the variables of anger ýcontrol and spiritual attitude can predict ýand explain 33.6% and 2.7% (36.3% in total) of the ýchanges in the mobile addiction score. anger control is the most influential risk factor of mobile phone addiction among nursing and midwifery students (beta=-0.385) conclusion findings indicate the importance of controlling anger and strengthening religious attitude in reducing the rate of mobile phone addiction in students. this provides guidance to the future development of smartphone addiction prevention programs for students. keywords: anger control, religious, attitude, nursing and midwifery students, mobile phone addiction original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.149-156 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1334 may-august 2022 vol.41no.2 1medical surgical nursing department of nursing, school of nursing and midwifery iranshahr university of medical sciences, iranshahr, iran 2medical surgical nursing department of nursing, school of nursing, iranshahr university of medical sciences,chabahar,iran 3operating room department of operating room, school of nursing and midwifery zahedan university of medical sciences, zahedan, iran 4intensive care nursing department, iranshahr university of medical sciences, iranshahr, iran 5department of midwifery, pregnancy health research center, zahedan university of medical sciences, zahedan, iran 6operating room department, faculty of medical sciences, islamic azad university, garmsar branch, garmsar, iran *correspondence: fatemeh kordsalarzehi iranshahr university of medical sciences, chabahar, iran phone: 09901478645 email: fa.salarzehi20@gmail.com orcid: 0000-0001-9103-5759 date of first submission, april 19, 2022 date of final revised submission, july 11, 2022 date of acceptance, july 23, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: dehghanmehr s, kordsalarzehi f, ghiamikeshtgar n, mir n, heravan mb, shafeie f. anger control is the most influential risk factor of mobile phone addiction among nursing and midwifery students of zahedan university of medical sciences. univ med 2022;41: 149-156. doi: 10.18051/univmed.2022.v41.149-156. 150 dehghanmehr, kordsalarzehi , ghiamikeshtgar, et al anger control is the most influential introduction mobile phones have entered the world as a communication tool, but gradually, its developing capacities and capabilities have elevated the use of this communication tool as a media or multimedia.(1) mobile phones have found a powerful place in human social and personal life.(2) today, the mobile phone is not just a voice device among users; rather, through i nt er ne t a cc es s , te xt i ng, vid eo s, ph ot os, cyberspace, and watching movies online has become an important part of people’s daily lives.(3) because people can communicate with each other, mobile phones increase the sense of security. in addition, studies have shown that the mobile phone can also be a useful teaching a i d . ( 4) a l t ho ugh th e s e c o nn e c t i o ns h a ve accelerated tasks and reduced distances,(5) this new communication technology can cause many disadvantages and problems.(6) researchers believe that the unreasonable and excessive use of mobile phones has led to the emergence of a phenomenon called mobile phone addiction.(3) mobile phone addiction is a non-drug and behavioral addiction in which sometimes excessive use of this device is done without awareness. in the present century, its use has been mentioned as an obsessive-compulsive disorder and addiction.(7) among the important users of this technology are students. features and facilities in mobile phones, such as quick access to resources and information, and the existence of social networks, have transformed students’ learning.(8) students are among the users who in addition to facing problems of educational, economic, and cultural and social changes during their study period, are exposed to the harms caused by excessive use of mobile phones.(5) international studies showed that 6% of mobile phone users are addicted to this device.(9) in iran, the prevalence of mobile phone addiction is relatively high among students (prevalence of 10% among students of the university of tehran and 18.5% among students of the university of birjand).(10,11) people addicted to mobile phones feel depressed, lost, and isolated compared to ordinary people without mobile phones. sometimes their work and life are disrupted due to the abundance of contacts, text messages, web browsing, and online chats.(12) mobile phone addiction has been introduced in the fifth international classification of mental disorders (dsm-v), as a technologyrelated disorder. studies have shown that this addiction leads to mental health problems.(13,14) in the meantime, nursing students should be given special attention, as society’s health depends on their knowledge, attitude, and behavior, and they will ultimately be in occupations that convey health messages. the results of a study in turkey showed that smartphone addiction has a negative effect on nursing students’ empathy with patients. (15) studies have shown that there is a positive relationship between problematic use of mobile phones and aggression.(16) studies show that people with aggressive characteristics are addicted to smartphones.(17) trust in technology to increase social c o mmu ni c a ti o n ma y h a ve ps ycho l ogi c a l consequences, such as creating technological dependencies, extroverted personality traits (deepening interpersonal interactions), and anger. the results of the kim study showed that s ma r t ph o ne a d d ic t io n i s i n fl ue n c e d by psychosocial factors such as aggression.(18) this is because one of the topics that has attracted a lot of empirical and theoretical attention in the last two decades, is the discussion of emotions which can be due to the role of emotional regulation in external behaviors (individual). anger is a fundamental emotion and an inner state that regulates the type of reaction against the environment. in fact, anger is characteristic of the initial stage of the development of hostility and aggression.(19) research has shown that t he re i s a po s it i ve r e l a t i onsh i p b e twe e n problematic cell phone use and aggression.(20) the religious attitude has an effective role in cognition, emotions, and behavior of people 151 univ med vol. 41 no 2 towards internal and environmental factors, and it can affect the mental and social health of people. however, adlipour and kianpour (21) showed that interaction in cyberspace of internet social networks affects the religiosity of young people. various aspects of people’s lives have been comprehensively influenced by new media, including the internet and mobile phone social networks, so that the effects and consequences of these new information and communication technologies cannot be denied. therefore, this study aimed to determine the relationship of religious attitudes and anger control with mobile phone addiction among nursing and midwifery students of zahedan university of medical sciences. methods research design this was cross-sectional study that was conducted at zahedan school of nursing and midwifery from january to march 2021. research subjects this study was performed on 200 nursing and midwifery students. the formula (n= 50 + 8) for the minimum sample size in regression analysis was used,(22) where n is the number of predictor variables. considering that the number of predictor variables in the present study was 3, and using the zanjani study, the minimum size of the required sample was 74 people, which was increased to 200 people to ensure the adequacy of the sample.(23) the inclusion criteria were: 1student is studying in the field of nursing or midwifery at the bachelor or master level. 2student must have a smartphone. 3he/she must not suffer from mental illness and chronic diseases. 4he/she does not use drugs that affect the nervous and mental system. exclusion criteria: 1dissatisfaction to participate in the study or cancelling the continuation of the study for any reason. 2occurrence of acute problems and diseases during the study. evaluative tools the instruments of this research included: a demographic questionnaire, religious attitude questionnaire, standard questionnaire of anger management skills (positive adolescence choices training), and mobile phone addiction questionnaire. demographic questionnaire the researcher-made demographic questionnaire includes age, gender, education, course of study, field of study, marital status, occupation, housing, and economic status. religious attitude questionnaire the religious attitude questionnaire was developed by khodayarifard et al.(24) and includes 40 questions, and 6 dimensions of worship, ethics, values, influence on behavior and life, social factors, worldview, beliefs, science, and religion. this questionnaire is based on the likert scale, and is arranged and scored in 5 options in the range of strongly agree to strongly disagree. khodayarifard et al.(24) in a study reported the split-half reliability coefficient of the questionnaire by the spearman and guttman methods to be 0.93 and 0.92, respectively, while the alpha coefficient was reported to be 0.95. anger management skills questionnaire st a n da r d q u e s tio nn a i r e of a nge r management skills (positive adolescence choices training) was developed by hamond, warfield and young,(25) and is used as one of the authoritative programs in the field of anger control. this questionnaire has 8 questions, and its scoring is based on the likert scale. subjects should rate their answers within 5 degrees (very disagree, disagree, no comment, agree, very agree. smartphone addiction self-report scale sevari (26) in the study “construction and validation of a mobile addiction questionnaire” to measure the variable of mobile phone addiction, 152 dehghanmehr, kordsalarzehi , ghiamikeshtgar, et al anger control is the most influential a b used some foreign questionnaires and some findings of foreign research. first, with their help, she made 15 questions, and after presenting them to some expert professors in psychological issues, two questions were removed from them, and a preliminary questionnaire consisting of 13 questions was used. then, to extract the causes of cell phone addiction, exploratory factor analysis was used. the analysis showed that after twentyfive experimental rotations on the data, 13 questions and three factors called de-creativity (7 questions), desire (3 questions), and loneliness (3 questions) were obtained questionnaire questions are graded on a five-point scale from never (1) to most of the time (5). the minimum possible score will be 0 and the maximum will be 52. the score between 0 and 13: low sense of cohesion. the score between 13 and 26: medium sense of cohesion. a score above 26: high sense of cohesion. the total reliability of the questionnaire was 0.87, the de-creativity factor was 0.78, the desire factor was 0.76, and the loneliness factor was 0.84. the validity of the questionnaire was determined by confirmatory studies. ethical considerations after approval of the study by the ethics committee of the university (ethics code: ir.zaums.rec.1399.509), and obtaining permission from the officials of the school of nursing and midwifery, the investigators conducted the study. statistical analysis we used the statistical package for the social sciences (spss) for windows version 22.0 (ibm, armonk, ny, usa) and a p-value of <0.05 was considered statistically significant. data were normally distributed according to the kolmogorovsmirnov test. therefore, it was possible to use parametric tests such as regression. descriptive statistics were used to analyze the demographic characteristics, and simple and multiple regression analysis models were used to examine the relationship between variables. results table 1 shows t he demographic characteristics of the subjects. the mean and standard deviation of the age of the research subjects were 22.04 and 3.30, respectively. in addition, 45.5% (91 subjects) were boys and 54.5% (109 subjects) were girls, 88% (176 subjects) were single and 12% (24 subjects) were married, 92% (184 subjects) and 8% (16 subjects) were taking the bachelor and master course, respectively. the mean and standard deviation of mobile phone addiction scores in this study were 36.9 and 13.14, respectively, indicating the high dependence of the subjects on mobile phones. the mean and standard deviation of anger control scores were 20.77 and 7.70, respectively; which indicates the average ability of the subjects to table 1. demographic characteristics of the participants (n=200) data presented as n (%), except for age, smartphone addiction, anger control and spiritual attitude as mean ± sd variables n ( %) age (years) gender female male marital status single married course of study bachelor master field of study nursing midwifery employment status unemployed employed housing dorm with family private house interest in chosen field of study interesting no comment uninteresting smartphone addiction anger control spiritual attitude 22.04 ± 3.30 91 (45.50) 109 (54.50) 176 (88.0) 24 (12.0) 184 (92.0) 16 (8.0) 134 (67.0) 66 (33.0) 179 (89.5) 21 (10.5) 101 (50.5) 90 (45.0) 9 (4.5) 136 (68.0) 47 (23.5) 17 (8.5) 36.90 ±13.14 20.77 ± 7.70 104.56 ± 47.94 153 univ med vol. 41 no 2 control their anger. also, the mean and standard deviation of the religious attitude scores were 104.56 and 47.94, respectively, indicating low religious attitude. to predict the score of mobile phone addiction from scores of anger control and religious attitude, and some demographic characteristics, the regression model was used. the results showed that there was a significant correlation between mobile phone addiction score and predictor variables (p=0.001, f=56.13). based on the final model, the variables of religious attitude and anger control remained in the model. so that, the variables of anger control and religious attitude can predict and explain 33.6% and 2.7% (36.3% in total) of the changes in the mobile addiction score, respectively. anger control was the most influential risk factor of smart phone addiction (table 2). discussion the results of this study showed that there is a significant and negative statistical correlation of the variables of religious attitude and anger control with mobile phone addiction, and these two variables together can predict 36.3% of the changes in the mobile phone addiction score, but there was no statistically significant correlation between any of the demographic variables and mobile phone addiction. khazaei e t al. (1 0) in a study fo und a significant correlation between mobile phone dependence and the incidence of aggression in students, and also, no significant correlation was found between demographic characteristics and mobile phone addiction. ahangari (27) in a study among students in kashmar found that there is an inverse and significant correlation between the use of mobile phone social networks and adherence to religious beliefs. hariri et al.(28) in their study on students of kharazmi university, found a negative and significant correlation between internet addiction and religious attitudes. kim et al.(29) conducted a study titled “smartphone addiction: (focusing on the three variables of depression, aggression, and motivation) among students”. the results of the study showed that there is a positive and significant correlation of smartphone addiction with depression and aggression. knabb (30) conducted a study among christian students and concluded that connection with god has a significant inverse effect on internet dependence, and neglect of tasks and relationships due to internet use. the results of the above studies are consistent with the results of the present study. charlton et al.(31) concluded in their study that there is a negative correlation between religiosity and internet addiction in the four religions of islam, hinduism, buddhism, and christianity, which is consistent with the results of the present study. however, they found that there is a significant correlation between gender and mobile phone addiction, and women who are more committed to religious norms than men, have a stronger negative correlation between religiosity and mobile phone addiction, which is not consistent with the present study. the reason can be attributed to the definitions of religiosity in different religions, differences in societies, etc. the results of the present study also showed that the students participating in this study have high mobile phone addiction, moderate anger control, and low spiritual attitude. najafisharjabad (32) in a study on 300 students concluded that half of the students are unable to control their anger. pavithra et al.(33) in a study in bangalore concluded that nomophobia or mobile phone addiction is very common among students. the table 2. multiple linear regression analysis of several predictor variables and smartphone addiction β = regression coefficient, beta = standardized regression coefficient predictor variables β beta 95% c.i p value r2 age anger control religious attitude 0.199 -0.56 -0.069 -0.050 -0.385 -0.252 0.865 – 0.878 -4.386 – 14.422 -2.878 – -2.904 0.880 0.000 0.004 0.336 0.027 154 dehghanmehr, kordsalarzehi , ghiamikeshtgar, et al anger control is the most influential results of these two studies are consistent with the present study. mamashli et al.(34) found in a study that nursing students are in a relatively good condition in terms of internet addiction and mobile phone injuries, which is not consistent with the results of the pre sent study. the re ason for this discrepancy can be attributed to the method of data collection, the research community (which in our study comprised only nursing students), and climatic characteristics. one of the limitations of the research was the lack of cooperation of some students in completing the questionnaires. due to the high dependence of students on mobile phones and the negative impact of mobile phone addiction on spiritual attitudes and anger management, longterm and effective planning should be done in the field of students’ optimal use of mass media. future research should also use longitudinal research designs to follow participants over time in studying associations between psychopathology and smartphone addiction severity. conclusion according to the results of the present study and the high dependence of students on mobile phone use, anger control was the most influential risk factor of smartphone addiction. because of the destructive psychological effects of mobile phone overuse, and the resulting disorder in the education of students, long-term and effective planning should be done in the field of optimal use of mass media by students, and education in this regard should be started at an early age, in the family as well as at school. conflict of interest authors have no conflict of interest. acknowledgement we thank all the students of the school of nursing and midwifery, and the research deputy of zahedan university of medical sciences who helped us in this research. contributors conceptualization: sd. data curation: nm. formal analysis: fks. funding acquisition: zahedan university of medical sciences. methodology: sd, fs. project administration: ngk. writing of original draft: mb, fs. writing of review and editing: sd. all authors have read and approved the final manuscript. references 1. arbaabafzali h, naghibalsadat r, kia a. mobile media threats and features and strategies for improving 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these factors make its precise diagnosis more difficult. approximately 25% of hccs harbor actionable mutations, which are yet to be translated into clinical practice. in the era of precision medicine, molecular or genomic information are indispensable for hcc diagnosis and prognosis. exploring genomic alterations has become a requirement for identifying the molecular subtypes of hcc. recent studies have introduced molecular markers to help identify early hcc and to clarify its multistep process of carcinogenesis. the subclassification of tumors into proliferation class and nonproliferation class hccs gives pointers to the hcc phenotype and facilitates the selection of appropriate treatments. in this review, we broadly summarize some of the latest insights into hcc subclassification from the perspective of molecular pathology. immunohistochemistrybased subclassification allows improved characterization of hcc in daily clinical practice. moreover, analysis of the immune microenvironment, intra-tumoral morphological heterogeneity, and imaging features gives additional information regarding the classification of hcc. combinations of these approaches are expected to inform and advance the precision diagnosis and management of hcc. keywords: hepatocellular carcinoma, molecular subclassification, molecular pathology, immune subtypes review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.194-206 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1273 may-august 2022 vol.41no.2 cite this article as: effendi k, kwa wt, uen o a, saka mot o m . the role of molecular pathology in the precision dia gnos is a nd s ubclassi fica tion of hepatocellular carcinoma. univ med 2022 ;41 :1 9 4 -2 0 6 . doi : 10 .18 05 1/ univmed.2022.v41.194-206 1department of pathology, keio university school of medicine, tokyo, japan 2department of medicine, yong loo lin school of medicine, national university of singapore, singapore *correspondence: kathryn effendi, md, phd department of pathology, keio university school of medicine, 35 shinanomachi, shinjuku-ku, tokyo 160-8582, japan tel.: +81-3-5363-3764 fax: +81-3-3353-3290 e-mail: kathryn@a8.keio.jp orcid id: 0000-0001-7844-1170 date of first submission, december 27, 2021 date of final revised submission, may 24, 2022 date of acceptance, june 13, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abbreviations bmi-1, b lymphoma mo-mlv insertion region 1 homolog; cap2, cyclase-associated protein 2; ck19, cytokeratin19; ctnnb1, catenin b1; emt, epithelial-mesenchymal transition ; eob-mri, gadoxetic acid-enhanced mri; epcam, epithelial cell adhesion molecule; gpc3, glypican-3; gs, glutamine synthetase; hcc, hepatocellular carcinoma; hsp70, heat-shock protein 70; hgehcc, high-grade early hcc; ldgn, low-grade dysplastic nodules; lgehcc , low-grade early hcc; hgdn, high-grade dyspalstic nodule; icis, immune checkpoint inhibitors; lgr5, leucine-rich repeat containing g protein-coupled receptor 5; oatp1b3, anion transporting polypeptide 1b3; pd1, programmed cell death 1; sall4, sal-like protein 4; tert, telomerase reverse transcriptase; tpms, tert promoter mutations 195 univ med vol. 41 no 2 introduction in 2020, liver cancer was the sixth most common cancer and the third leading cause of cancer-related deaths worldwide. incidence rates and mortality are two to three times higher in men than in women, and liver cancer ranks fifth in terms of global incidence and second in terms of mortality for men. hepatocellular carcinoma (hcc) is the most common form of liver cancers, accounting for ~90% of all liver cancers. it is still the most common cancer in east and southeast asia and is the leading cause of cancer death in mongolia, thailand, cambodia, egypt, and guatemala among both men and women.(1) a study in indonesia showed that there had been no improvement in the median survival rate of hcc for patients diagnosed in 2013–2014 compared with those diagnosed in 1998–1999.(2) hepatocellular carcinoma develops in association with major risk factors such as chronic infection with hepatitis b virus (hbv) or hepatitis c virus (hcv), consumption of aflatoxin b1-contaminated food, heavy alcohol intake, and metabolic syndrome (type 2 diabetes, obesity, and non-alcoholic fatty liver disease).(3) despite recent advances in the surveillance and therapeutic management of hcc, prognosis is still poor and the disease remains a global health challenge.(4) although the background conditions vary from country to country, hcc itself has diverse predisposing factor s and genetic susceptibilities. moreover, hepatocarcinogenesis is known to be a multistep process in which many genetic signaling pathways are involved. these characteristics result in hcc being an extremely heterogeneous solid cancer; indeed, among the tumor types so far identified, hcc is one of the most highly heterogeneous.(5) in the era of targeted molecular treatments as promising therapies for advanced cancer, it is important to understand the deta iled nature of the heter ogeneous f eatures of hcc. ( 3,6 ) consequently, a comprehensive analysis of the morphologic, immunohistochemical, immune, and/ or mutational status of hcc is crucial to enhance our understanding of hepatocarcinogenesis and to improve the clinical management of hcc. clinical pathological assessment of hcc is now possible based on its molecular landscape and having a precise diagnosis has led to improved therapeutic strategies.(7) here, we discuss a broad range of molecular targets that have been (or could be) used to help define a more precise hcc diagnosis and to more accurately reflect its features based on pathological perspectives. the understanding of molecular features that underline hcc onset and progression is crucial for more efficacious management approaches in the future. molecular markers of early hccs hepatocellular carcinoma is characterized by a clear multista ge pr ocess of tumor development. many cases of hcc develop in damaged livers related to chronic hbv or hcv infection. histopathologically, hcc starts as premalignant dysplastic nodules (dn) and early hcc (ehcc) before it develops to progressed hcc.(8) the concept of ehcc, i.e., small nodules with indistinct margins, has been accepted internationally since 2009 and was officially adopted in the fourth edition of the world health organization (who) classification of tumors of the digestive system in 2010.(9,10) however, the diagnosis of ehcc is often challenging because it generally lacks obvious histological atypia, its molecular mechanism is unclear, and it possesses quite heterogeneous clinical behavior. nonetheless, rec ent advances in immunohistochemical analysis using various molecular markers have allowed us to carefully evaluate ehccs. the heat-shock protein 70 (hsp70) gene is a housekeeping gene that assists with a variety of vital intracellular chaperoning functions. expression of hsp70 radually increases with the stepwise progression of hepatocarcinogenesis, but this is not observed in benign nodular lesions; this fact makes hsp70 a useful marker for ehcc and could clearly distinguish hcc from chronic hepatitis or cirrhosis.(11) the expression profile analyses of hsp70s from multiple databases such 196 many solid and non-solid cancers; moreover, bmi1 was recently identified in association with the epithelial–mesenchymal transition in cancer cells.(20-23) another study suggested that the forced expression of bmi-1 promotes the malignant transformation of hepatic progenitor cells, thereby providing a link to its “stemness” properties and neoplastic proliferation.(24) cyclase-associated protein 2 (cap2) is also involved in the progression of hcc. cyclaseassociated protein 2 is a bifunctional protein in which the n-terminal domain binds to adenylyl cyclase and the c-terminal domain binds to monomeric actin. expression of cap2 is not seen in normal liver tissue but its expression increases during hcc progression from dn, through ehcc, to progressed hcc. positive cap2 expression was frequently seen in stromal invasion, a characteristic feature of ehcc, and high expression of cap2 was correlated with poor prognosis of patients with hcc.(25) indeed, we found that cap2 was not only associated with the process of skeletal muscle development, but it was also significantly associated with tumor size, poor differentiation, portal vein invasion, and intrahepatic metastasis.(26) in the zebrafish, a ve rte brate model, the silencing of cap2 expression resulted in the development of a shorter body, which may have resulted from the downregulation of actin by cap2. expression of cap2 was also found to be colocalized with actin in the leading edge of the lamellipodium overlap of hcc cells; moreover, migration assays showed that cap2 knockdown reduced cell motility. these findings support the functional association of high cap2 expression and explain how it could help promote invasive behavior in hcc cells. the role of cap2 in tumor progression is not observed only in hcc: we found that cap2 expression also increased stepwise during the progression of malignant melanoma and is upregulated in the aggressive histologic type of epithelial ovarian cancer.(27,28) extending the evaluation of detailed histological f eature s, such as scirrhous components, with specific molecular markers, as the cancer genome atlas (tcga) and oncomine show a significant increase in hsp70s expression in hcc tissues.(12) glypican3 (gpc3), a member of the heparan sulfate proteoglycan family, is known to be upregulated in hcc but is not detected in normal liver tissues or benign liver lesions. the combination of gpc3 and alpha fetoprotein as markers for ehcc increases the sensitivity to 76% for early-stage tumors <3 cm in size.(13,14) glutamine synthetase (gs) catalyzes the synthesis of glutamine, which is the major energy source for tumor cells. expression of gs was found to increase in a stepwise manner from early to advanced hcc and promotes invasion of hcc cells through mediating the epithelial-mesenchymal transition (emt ). (1 5) a rece nt report showed that combination biomarkers significantly improved the sensitivity of the panel for ehcc detection and currently, the use of hsp70, gpc3, and gs is established as a useful marker combination for ehcc diagnosis.(16,17) this three-marker panel can increase the diagnostic accuracy of liver biopsies and was described in the fifth edition of the who classification of tumors of the digestive system.(18) additional markers have also proven useful in the diagnosis of ehcc. we previously reported the upregulation of the polycomb group gene member, b-lymphoma mo-mlv insertion region 1 (bmi-1), in early-stage hcc.(19) the signaling pathway of bmi-1 may allow cells to maintain their self-renewal ability and may thereby link to neoplastic proliferation by acting as a negative regulator of the ink4a/arf locus that encodes two important tumor suppressor proteins in human cancer, p16 and p19. we observed positive expression of bmi-1 in hcc as high-intensity dot patterns within the nucleus that may reflect bmi-1 activity as a transcriptional repressor by regulating chromatin silencing.(8) expression of bmi-1 was particularly observed in early and well-diff erentiated hccs, but not in the surrounding liver tissue, a fact that should facilitate the identification ehcc. the role of bmi-1 in carcinogenesis has been reported for effendi, kwa, ueno, et al molecular pathology in the precision diagnosis 197 univ med vol. 41 no 2 a b such as hsp70, bmi-1, and cap2, showed that small vaguely nodular lesions could be further subclassified as low-grade ehcc (lgehcc), high-grade e hcc (h gehcc), low-grade dysplastic nodules (lgdn), or high-grade dysplastic nodules (hgdn).(29) the investigation of 66 small vaguely nodular lesions showed that 20 nodules were dn and 46 nodules (69.8%) were ehcc. among these 46 ehccs, 18 nodules (39.1%) showed marked stromal invasion and/or the presence of a scirrhous component and were subclassified as hgehcc, wher eas the remaining 28 nodules (60.9%) did not show these kinds of features and were subclassified as lgehcc. positive immunohistochemical expression of bmi-1 was found at lower rates in lgdn but at higher rates in lgehcc. the difference between them was significant and should prove useful to support differentiation between premalignant dysplastic nodules and ehcc. a stepwise incr ease in the immunohistochemical expression of cap2 was also seen from lgdn to hgehcc. a significant expression of cap2 in hgehcc may already indicate the malignant potential of hgehcc nodules, and that the transitional stage to advanced hcc may already be present in hgehcc; as a result, the re-evaluation of treatment strategies for ehcc may be needed. molecular subclassification of hccs recent advanc es in mole cular subclassification by gene expression analysis have facilitated a new understanding of the molecular landscape of hcc. during the past two decades, many studies ha ve proposed hcc subclassifications based on the molecular features of the tumor. molecular classification of hcc proposed by boyault, and hoshida are widely accepted. (3 0) genome -wide transc riptome microarray analysis and quantitative reversetranscription polymerase chain reaction data in a series of 120 hccs and 3 hepatocellular adenomas by boyault et al.(31) resulted in the proposal of six robust subgroups of hcc, termed g1–g6. tumors classified in the g1–g3 subgroups were associated with high chromosomal instability compared to tumors in the g4–g6 subgroups. a previous study conducted by hoshida et al. (3 2) ident if ied a ce rtain commonality between subclasses defined by a meta-analysis that encompassed 603 hcc patients from both western and eastern countries. they revealed three robust hcc subclasses (termed s1, s2, and s3) that could be associated with clinical parameters and distinct biological processes in hepatocarcinogenesis.(33) subclasses s1 and s2 are associated with large tumors and poor histological differentiation. characteristics found to be similar in the s1 and s2 subgroups, such as activation of the akt pathway and frequent p53 mutations, were also found in boyault’s g1–g3 groups. the less-aggressive s3 subclass (which retains a hepatocyte-like phenotype) also resembles the g5 and g6 subgroups, showing associations with the wnt/ β-catenin (ctnnb1) signaling pathway. these concepts of hcc subclassification are increasingly accepted worldwide, and many gene alterations in hcc now can be associated with molecular subclassifications. lgr5, also known as g-protein-coupled r eceptor 49 (grp49), has been well established as a target of the w nt/β-caten in signaling pathway, particularly in colorectal cancer.(34) we also found that lgr-5 is notably overexpressed in hcc carrying β-catenin mutations. stable clones of lgr5-overexpressing hcc cells formed nodular tumors with a tightly aggregated morphology, whereas downregulation of lgr5 changed the cells into a loosely associated morphology resulting in enhanced infiltration and increased cell motility.(35) our findings regarding lgr5 function in hcc cells seem to be an exemplar of hoshida’s s3 subclass and boyault’s g5–g6 subgroups.(36) the typical morphological and biological features of lgr5 expression in hcc may indicate a subset of the less-aggressive hcc phenotype. taken together, these studies suggest that, despite highly variable clinical backgrounds and molecular heterogeneity, hccs may share a similar gene-expression pattern. at present, data 198 based on genomic profiling studies show that hccs can be roughly divided into two major molecular clusters, namely the proliferation class and the nonproliferation class. patients with hcc features consistent with the proliferation class may have aggressive tumors, higher alpha fetoprotein levels, moderate/poor cell differentiation, and frequent vascular invasion. in contrast, the nonproliferation class is characterized by a less aggressive phenotype , be tter histologic differentiation, and lower alpha fetoprotein levels.(37,38) characterizing hcc based on its distinctive molecular and clinical features is a prerequisite for precision medicine because it provides more accurate classification and diagnosis which allow more effective treatment. clinical trials for hcc have suggested that targeted agents have different efficacy in diverse populations because different molecular pathways are involved in different hcc patients.(39,40) however, performing genomic profiling for ever y hcc case is not straightforward in daily clinical practice. consequently, we have proposed an hcc subclassification based on immunohistochemical staining. immunohistochemical analysis was carried out for panels of several molecular markers commonly used in hcc, and our findings indicated that hccs could be broadly divided into three subclasses. the biliary/stem cell marker positive subclass (b/s subclass) is indicated by positive staining for cytokeratin 19 (ck19, krt19), sal-like protein 4 (sall4), or epithelial cell adhesion molecule (epcam); the wnt/βcatenin signaling-related marker positive subclass (w/b subclass) is indicated by positive staining for β-catenin or glutamine synthetase (gs); and the negative subclass (-/subclass) is indicated when all markers are negative.(41) representative hcc cases associated with b/s or w/b subclass are shown in figure 1. the b/s subclass can be further divided into two subgroups: ck19 and/or sall4 positive (b/s1 subgroup) and epcam only positive ( b/s2 subgroup) . t he b/s subclasses, particularly the b/s1 subgroup, exhibit poor tumor differentiation, increased frequency figure 1. representative hcc cases with positive immunohistochemical expression for ck19, sall4, epcam, -catenin, and gs. hcc case with ck19+, sall4+, and epcam+ was included as b/s subclass (corresponds to negative gs staining); while hcc case with -catenin+ and gs+ was included as w/b subclass. the two groups have been associated with different clinicopathological features. (41) he: hematoxylin and eosin staining. scale bar = 100 µm. effendi, kwa, ueno, et al molecular pathology in the precision diagnosis 199 univ med vol. 41 no 2 of portal vein invasion and/or intrahepatic metastases, and highly proliferative activity. they are also associated with the shortest recurrence time. in contrast, the w/b subclass exhibits better tumor differentiation, a decreased frequency of portal vein invasion, and less proliferative activity.(41) these subclasses partly overlap with previous molecular subgroups from transcriptomic methods: the b/s subclass shares some features with the s2 and g1 subgroups, and the w/b subclass roughly corresponds to the s3 and g5-6 subgroups (7) (table 1). the hcc phenotype was shown to be related to its molecular alterations and underlying oncogenic pathways.(42) furthermore, recent studies showed that cancer heterogeneity is also shaped by active immune responses. immune cells are important elements of tumor tissues, and the amount of immune cell infiltration considerably differs among tumor types and histological subtypes. most of the hallmarks of cancer are enabled and sustained to varying degrees by a tumorsupporting microenvironment.(43,44) hepatocellular carcinoma is known to result from a complex interplay between genetic and environmental factors. inte rvent ion in the immune microenvironment of hcc may benefit the management of hcc.(37,45) recently, immune checkpoint inhibitors (icis) have shown potential as treatments for various cancers including hcc. a study showed that nivolumab, a programmed cell death protein-1 (pd-1) inhibitor, exhibited favorable efficacy and safety as a treatment for advanced hcc.(46) nivolumab has since gained approval from the u.s. food and dr ug administration for hcc patients previously treated with sorafenib.(47) although the use of icis has been considered a promising approach to treat advanced hccs, some patients have shown a limited response to such treatment. this again suggests that identifying hcc subclasses based on a combination of immune response and table 1. the concordance between different molecular subtyping methods for hcc note: icis = immune checkpoint inhibitors; number in parentheses = reference number in body text 200 its association with clinicopathological findings is important for predicting prognosis and therapeutic efficacy.(45,48) ou r gr oup s howe d tha t the i mmu ne microenvironment of hcc can be classified into three immune subtypes (namely, immune-high, immune-mid, and immune-low) that have additional prognostic impacts on histological and mol e c ul a r c l a s s i f i c a t i o ns of h cc. ( 4 9 ) comprehensive investigation of immune cells through multiplex immunohistochemical analysis of 919 regions of 158 hccs indicated that incr eased b-cell, plasma-cell, and t-cell infiltrations were hallmarks of the immune-high hcc subtype. immune-high-predominant hcc wa s f o un d i n nea r l y 20 % of t he immunohistochemistry-based b/s1 subclass and i n l e ss tha n 1 0% of t he w / b s u bc l a s s . interestingly, the immune-high subtype indicates hccs with better prognosis among poorly ck19-positive and/or sall4-positive highgrade hccs. however, the immunosubtypes were not prognostically significant in w/b hcc.(49) the immune-high subtype was also significantly enriched in hoshida’s s1 and boyault’s g2 subclasses, whereas the immunelow subtype was observed in hoshida’s s3 and boyault’s g6 subclasses.(7) recently, immune factors have been shown to be involved in the characterization of “cold tumor” phenotypes which are more resistant to immunotherapies.(50) hccs with cold phenotype were associated with w n t / β-c a t e nin mu t a t i on s a n d dec r e a s e d infiltration of cd8+ t-cells into the tumor tissues.(51) this is a great challenge since it can promote immune escape and is less likely to r e s p on d to icis th e r a py. a s d e s c r i b e d previously, around 30% of hccs belong to the immune exclusion class and are associated with hoshida’s s3, boyault’s g5-6, and kurebayashi’s immune-low subtypes (table1).(50-52) these r e s u l t s h i gh l i gh t t he imp or ta nc e of comprehensive pathological evaluation of the immune microenvironment in addition to the standard histopathological and molecular classification of hccs. intratumoral morphological heterogeneity in hccs the heterogeneous nature of hcc clearly represents a challenge to the establishment of a robust hcc classification system and hcc treatments. intratumor heterogeneity is defined as distinct genetic alterations and phenotypes between cancer cells within the same tumor nodule. (5 3) intratumor het erogene ity was reportedly detectable in most hcc cases (20 of 23, 87%), and heterogeneity solely at the level of morphology was found in 6 of 23 (26%) hcc cases.(6) the extent of intratumor heterogeneity varies considerably among hcc patients. individuals with primary lesions larger than 5 cm showed a significantly higher extent of intratumor heterogeneity.(54,55) intratumor heterogeneity can have major clinical consequences because the different genetic alterations and phenotypes between cancer cells, or between different tumor nodules, may require diff erent tr eatment decisions.(53) unlike many other cancers, hcc has not seen the benefit of individualized treatment due in par t to intratumor heterogeneity.(54) therefore, from the perspective of pathology, elucidating the relationship between morphological pattern and genetic alterations in hccs is one way to gain further insight into hepatocarcinogenesis and to attain the goal of precision medicine. telomerase reverse transcriptase (tert) is a catalytic subunit of the enzyme telomerase that is crucial for maintaining telomere elongation; furthermore, tert activation is considered to be a fundamental step in tumorigenesis. mutation in the tert promoter regions that could result in increased tert expression have been identified in many cancers, including hccs.(56,57) in hccs, tert promoter mutations (tpms) reportedly occur at two hot spots: –124 base pairs (c228t), and –146 base pairs (c250t) upstream of the atg translation start site. tpms are also the most frequently found somatic genetic alteration in hcc, with an overall frequency of around 60%.(58-60) interestingly, tpms frequently occur early in the development of hcc and are highly effendi, kwa, ueno, et al molecular pathology in the precision diagnosis 201 univ med vol. 41 no 2 related to the st epwise process of hepatocarcinogenesis. from a series of 168 liver samples, tpms were already identified in 6% of lgdn, 19% of hgdn, and 61% of ehcc; in contrast, other common gene mutations in hcc, such as those of ctnnb1 or tp53, occurred at a later stage.(61) these results show that tpms are early events involved in the transformation of premalignant lesions to hcc; these findings also provide evidence that ehcc should get more attention as “hcc”.(7,62) our group analyzed a total of 189 freshfrozen liver tissue samples and found that 55% had tpms.(63) in a retrospective analysis, all cross-sectional slides containing the whole tumor area we r e re -assesse d for morpho logical histological patterns. the distribution percentage of each histological pattern in the tumor area for each hcc case was semi-quantitatively calculated using a homogeneity index. we found that hccs with tpms (tpm-positive hccs) clearly exhibited intratumoral morphological heterogeneity as indicated by the smaller mean homogeneity index (0.800 ± 0.117 vs 0.927 ± 0 .0 96 , p< 0 . 00 01 ) . t p m-p o si t i ve hccs displayed more diverse differentiation patterns, i.e., usually characterized by two or more histological differentiation patterns in each hcc nodule; in contrast, tpm-negative hccs more often displayed single dominant patterns. furthermore, early, or well-differentiated histological patterns were more commonly seen in tpm-positive hccs than in tpm-negative hccs (63) (figure 2). we also noted that most of our hccs with heterogeneous patterns had an hcv-related background and were likely to be tpm-positive. previously, multiplex molecular profiling of hcc patients also revealed that patients with a tert promoter mutation were more likely to be hcv positive rather than hbv positive and likely corresponded to hoshida’s s3 subtype (table 1).(39,64) hcv-induced hcc development is thought to occur in a multistep process that involves the initiation of neoplastic clones accompanied by irreversible somatic genetic/epigenetic alterations and progression in figure 2. intratumoral morphological heterogeneity based on the presence of tert promoter mutations. shown are representative gross specimens of a tert promoter mutation (tpm)-positive hcc and a tpmnegative hcc. histological differentiation patterns in the whole tumor area were evaluated as area percentages for each case of tpm-positive and negative hcc. tpm-positive hcc cases frequently displayed more variety in differentiation patterns than tpm-negative cases.(63) scale bar = 100 µm 202 a carcinogenic tissue microenvironment.(65,66) although additional genetic alterations appear t o b e r e qu i r e d to de ve l op cl i ni c a l l y heterogeneous hccs, tpms may act as a precursor lesion in the early stage, as supported by our observation indicating a possible role of tpms in hccs with intratumoral heterogeneity. the heterogeneous features of hcc might also be investigated through patho-radiological correlation studies. several imaging modalities are commonly used for diagnosing hcc, such as ultrasound, computed tomography, and magnetic resonance imaging (mri). gadoxetic acid (synonymous with gadolinium ethoxybenzyl diethylenetriaminepentaacetic acid, gd-eobdtpa), a gadolinium-based mri contrast agent, is a liver-specific (hepatobiliary) contrast agent t ha t h a s e x p a nd e d t h e r o le o f mri i n simultaneously providing morphologic and functional information about the hepatobiliary system. gadoxetic acid enters hepatocytes via active transport by organic anion transporting polypeptides (oatp1b1/3), and the resulting i ns i ght s in t o t h e c e l l ul a r mec h a n i sm of transportation have led to a better understanding of the correlation between radiologic and histologic features.(67,68) pr e vi o us s t u di e s h a ve s how n t ha t oatp1b3 expression could be important for evaluating the hcc tumor enhancement seen i n ga do x et ic a c id -e n h a nc e d m ri ( eo bmri).(69,70) we have observed that oatp1b3 expression decreases gradually during multistep hepatocarcinogenesis from the dysplastic nodule stage to progressed hcc. moreover, different nodules in hcc may exhibit different features on eob-mri. hcc nodules with low intensity on the hep at obi lia ry-p ha se of eob-m ri indicate a negative uptake of gadoxetic acid, whereas high-intensity nodules indicate a positive uptake. these nodules differentiated on eob-mri likely have different gene signaling alterations and hcc features. it has been reported that oat p1b3 is a downstream molecule of the wnt/β-catenin signaling pathway, a n d o at p1b3 -u pr e gu l a t e d hcc l ike l y represents a specific subgroup of wnt/β-cateninactivated hcc. (70) hccs with mutations in ctnnb1 are likely to be well-differentiated tumors, as previously described in boyault’s g5 and g6 subclasses or hoshida’s s3 subclass. indeed, hccs with aggressive phenotypes are likely to have decreased oatp1b3 expression. ctnnb1-mutated hcc is likely associated with innate resistance to icis because it lacks inflammatory cell infiltration. because recent studies have indicated that characterization of the immune microenvironment of hcc is important for predicting the effectiveness of treatments, performing eob-mri may be helpful in cases where immunotherapy is considered.(71,72) over the past few decades, considerable progress has been made in understanding the epidemiology, risk factors, and particularly the molecular features of hcc. however, the incidence rates and cancer-specific mortality of hcc continue to increase in many countries, and by 2025 more than 1 million individuals are predicted to be affected by hcc annually.(73-75) there is no doubt that continuous investigations on hcc he ter ogene ity features with comprehensive and integrative multidisciplinary approaches are still necessary to overcome hcc. conclusions hepatocellular carcinoma has variable molecular features that affect the diagnosis, treatment, and clinical outcome. comprehensive analysis at the level of pathological features, including morphology, immune microenvironment, and genomic status, combined with imaging analysis is impor ta nt to elucida te the heterogeneous features of hcc. integrated analysis from pathological and molecular studies will help elucida te the development of hepatocarcinogenesis and facilitate precise diagnosis as the fundamental basis on which to develop the precision treatment of hcc. effendi, kwa, ueno, et al molecular pathology in the precision diagnosis 203 univ med vol. 41 no 2 conflict of interest there is no potential conflict of interest. acknowledgments the authors would like to express sincere thanks to dr. yutaka kurebayashi and dr. hanako tsujikawa for their continuous support and helpf ul suggestions in preparing the manuscript. contributors ke contributed to the writing and design of the manuscript, and drafting of the figures and tables. wtk drafted the figures. au and ms reviewed the manuscript. all authors have read and approved the final manuscript. references 1. sung h, ferlay j, siegel rl, et al. global cancer statistics 2020: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries. ca cancer j clin 2021;71:209– 49. https://doi.org/10.3322/caac.21660. 2. loho im, hasan i, lesmana cr, dewiasty e, gani ra. hepatocellular carcinoma in a tertiary referral hospital in indonesia: lack of improvement of oneyear survival rates between 1998-1999 and 20132014. asian pac j cancer prev 2016;17:2165–70. doi: 10.7314/apjcp.2016.17.4.2165. 3. mazzanti r, arena u, tassi r. hepatocellular carcinoma: where are we? world j exp med 2016;6:21–36. doi: 10.5493/wjem.v6.i1.21. 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for research on cancer; 2018. effendi, kwa, ueno, et al molecular pathology in the precision diagnosis c:\users\universa medicina\docu 130 abstract universa medicina sensitivity and specificity of cell block method of transthoracic fine needle aspiration in diagnosis of lung cancer yusup subagio sutanto1*, nur santi1, brian wasita1, ana rima1, and hendra kurniawan1 background lung cancer is still the main cause of cancer deaths. the high lung cancer mortality rate is caused by a diagnosis factor or therapy selection. the cell block cytology technique using fine needle aspiration (fna) samples can provide immunocytochemical material that plays an important role in the differential diagnosis of lung cancer subtypes and in determining immunotherapy administration. this study aimed to determine the sensitivity and specificity of transthoracic fna (ttfna) cell block cytology in comparison with bronchial washing smears and ttfna smears in diagnosing lung cancer. methods this was a cross-sectional diagnostic study involving 26 subjects. all subjects had undergone bronchial washing and ct scan-guided fine needle aspiration followed by cell block preparation. both direct fna smears and cell blocks are useful in the diagnostic work-up of patients. comparative statistical analysis of ttfna cell block versus bronchial washing smear and ttfna smear cytology was carried out using the mcnemar test. results lung cancer was found in 15 patients (57.7%) using the ttfna cell block technique. the sensitivity and specificity of the ttfna cell block technique were 85.7% and 75%, respectively. there was no difference in the positivity value between ttfna cell block technique of bronchial wash smear technique, and ttfna smear on lung cancer diagnosis (p>0.05). conclusions transthoracic fine-needle aspiration in combination with the cell block technique has good sensitivity and specificity. the ttfna can be used for immunocytochemical examinations in lung cancer diagnosis and therapy. this approach is valuable for providing individualized treatment and prognostic evaluations. keywords: bronchial washing, cell block, lung cancer, smear, ttfna original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.133-140 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1091 may-august 2021 vol.40no.2 cite this article as: sutanto ys, santi n, wasita b, rima a, kurni awan h. sensitivity and specificity of cell block method of transthoracic fine needle aspiration in diagnosis of lung cancer. univ med 202 1;40 :13 0 7 . doi: 10. 1805 1/ univmed.2021. v40.133-140. 1department of pulmonology and respiratory medicine faculty of medicine, sebelas maret university / dr. moewardi general hospital surakarta *correspondence: yusup subagio sutanto department of pulmonology and respiratory medicine, faculty of medicine, sebelas maret university surakarta, indonesia/dr. moewardi general hospital surakarta rsud dr. moewardi gedung a lt. ii jl. kolonel sutarto no.132, jebres, surakarta, jawa tengah 57126 email: dr_yusupsubagio@yahoo.com mobile: +62811284165 orcid id: 0000-0001-9392-7603 date of first submission, january 11, 2021 date of final revised submission, july 20, 2021 date of acceptance, july 26, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 131 univ med vol. 40 no 2 introduction the separation of small cell lung cancer and non-small cell lung cancer is no longer based only on morphological differences, but also on i mmu no h is t o c he mi c a l a n d mo l e c u l a r characterization.(1) cell-block cytology using fine-needle aspiration (fna) samples is an immunocytochemical method which plays an important role in the differential diagnosis of lung cancer subtypes. according to the 2015 who c l a s s i f i c at i on , i mmun oc yt o c he mi s t r y i s recommended for many non-small cell lung cancers (nsclc) that cannot be classified as squamous cell carcinoma or adenocarcinoma b a s e d on mor ph ol o gy a l o ne . immunocytochemistry is also used to distinguish primary and metastatic lung tumors that are difficult to differentiate based solely on smear cytology.(2) the role of cell block cytology is in the diagnosis of lung cancer and in determining the administration of immunotherapy, which is currently growing rapidly. cell blocks are similar to conventional tissue paraffin blocks in pathology but consist of cells found in tissue fluids such as bronchial washings or fine needle aspirates. several studies have evaluated the role of cell block cytology as a starting material for the evaluation of programmed death-ligand 1 (pdl1) expression. the study by malapelle et al.(3) looked at the role of cytology in comparison with histology for the evaluation of pd-l1 expression. the results of the study provide evidence of the feasibility of evaluating pd-l1 expression in cell blocks in clinical practice, as well as of the role of cell blocks in evaluating epidermal growth factor receptor (egfr), anaplastic lymphoma kinase (alk) and ros proto-oncogene 1 (ros1). cell blocks of bronchial washings had a higher yield than conventional smears. increased yield was also noted when cell blocks of bronchial washings were combined with biopsy and compared to conventional bronchial washings combined with biopsy.(4) cyt o l ogi c a l t e c hn i qu e s s u c h a s bronchoalveolar lavage or washing (bal), bronchial brushing (bb) and fine ne edle aspiration cytology (fnac) can help diagnose lung malignancies early. for lung cancer diagnosis, fnac is the most effective of the three techniques.(5) the limited material available for adjuvant diagnostic studies is one of the limitations of conventional fine needle aspiration ( fna) . ( 6 ) in t h e c ur r e n t d e ve l op me nt of i mmu no t he r apy, c el l bl ocks c a n pro vid e immunocytochemical material. the present study investigates the importance of the cell block method in the diagnosis and therapy of lung c an ce r. ( 7 -9 ) t h e ai m of thi s st udy w a s t o determine the sensitivity and specificity of transthoracic fna (ttfna) cell block cytology in comparison with bronchial washing smears and ttfna smears in diagnosing lung methods research design t his w a s a c ro s s-s e c t io na l s tu d y of diagnostic tests and was conducted at rsud dr m o e wa rdi ho s pi t a l , sur a ka r t a , f r om september to december 2018. research subjects the research sample consisted of patients with suspected lung cancer who underwent t r e a tme n t . t he s amp l i ng me th o d us e d consecutive sampling with a sample size of 26 patients meeting the inclusion and exclusion criteria. the inclusion criteria were patients aged above 18 years, thorax multi-slice computer tomography (msct) scans showing an image of lung tumors with peripheral lesions to be f o l l ow e d by t t fn a a n d b r on c h os c o py procedures. exclusion criteria were patients with lung tumors with symptoms of acute infection, p r e gna n c y, hi s t o ry o f c he mo t he r a py, r a d i o th e ra py, a n d ma ss i ve he mo p t ys i s. discontinuation criteria were patients refusing bronchoscopy and ttfna and withdrawing. 132 measurements the study subjects’ characteristics were age, gender, smoking history, body mass index (bmi), type of lesion on bronchoscopy, and type of lung cancer histology. bmi was calculated as weight in kilograms divided by height in meters squared. the following categories were used: low bmi group (bmi<18.5 kg/m2), normal bmi group (bmi=18.5-24.0 kg/m2) and high bmi group (bmi>24.0 kg/m2). histology both direct fna smears and cell blocks are useful in the diagnostic work-up of patients. all cytological specimens were prepared according to the standard processing protocol in our laboratory. the papanicolaou (pap) stain was used for wet fixed smears and the diff quik stain for air dried smears. statistical analysis the data were analyzed using spss 21 for windows. the basic data were processed descriptively and presented as a distribution of frequencies and percentages. sensitivity, specificity, positive predictive values and negative predictive values were presented in 2x2 tables. comparative statistical analysis of ttfna cell block versus bronchial washing smear and ttfna smear cytology was carried out using the mcnemar test. a p-value <0.05 was deemed statistically significant. ethical clearance this research received approval for ethical clearance from the faculty of medicine, sebelas maret university, surakarta, under no. 687/ix/ hrec/2018. results the majority of patients were in the age group of over 40 years (80.8%). the proportions of males and females was 14 (53.8%) and 12 (46.2%), respectively. smoking history was obtained in 14 (53.8%) subjects. the majority of subjects with low bmi was 13 (50.0%). the most common type of lesion was compression stenosis, found in 14 (53.8%) subjects. the most common histological types in this study were adenocarcinoma in 11 (64.7%) subjects and high grade neuroendocrine tumors in 5 (29.4%) subjects. the ttfna cell block technique yielded 9 (52.2%) subjects with adenocarcinoma, 5 (29.4%) with high grade neuroendocrine, and 1 (5.9%) patient with adeno-squamous carcinoma (table 1). sensitivity and specificity of ttfna cellblock examination the results showed that 12 (46.15%) subjects had positive results in both ttfna cell block tests and ttfna smears and bronchial washing smears. positive ttfna cell block test results with negative bronchial smears and ttfna smears were found in 3 (11.5%) subjects. negative ttfna-cell block examination with positive bronchial washing smears and ttfna smears was found in 2 (7.7%) subjects. negative ttfna cell block results with negative bronchial washing smears and negative ttfna smears was found in 9 (34.6%) subjects. the sensitivity of the ttfna cell block technique for bronchial washing smears and ttfna smears was 85.7% while the specificity was 75%. the positive predictive value (ppv) was 80.0% and the negative predictive value (npv) was 81.8% (table 2). the value of ttfna cell block positivity for bronchial smear c ytology and ttf na smears the difference in the value of ttfna cell block positivity for bronchial smears and ttfna smears in the diagnosis of lung cancer was calculated using the mcnemar test, which showed no significant difference in the positivity value of the ttfna cell block technique for bronchial washing smears and ttfna smears in lung cancer diagnosis (p>0.05) (table 3). sutanto, santi, wasita, et al cell block technique of ttfna in lung cancer 133 univ med vol. 40 no 2 b c characteristics number of subjects n (%) bronchial washing ttfna total lung cancer patients smear (+) n (%) smear (+) n (%) cell block (+) n (%) total age (years) <40 >40 26 5 (19.2) 21 (80.8) 4 (15.4) 14 (53.8) 15 (57.7) 17 (65.3) 3 (17.6) 14 (82.4) gender male female 14 (53.8) 12 (46.2) 2 (7.7) 2 (7.7) 7 (26.9) 7 (26.9) 9 (34.6) 6 (23.1) 10 (58.8) 7 (41.2) smoking history yes not 14 (53.8) 12 (46.2) 2 (7.7) 2 (7.7) 7 (26.9) 7 (26.9) 9 (34.6) 6 (23.1) 10 (58.8) 7 (41.2) body mass index normal underweight overweight obesity 11 (42.3) 13 (50.0) 1 (3.8) 1 (3.8) 2 (7.7) 1 (3.8) 1 (3.8) 0 5 (19.2) 8 (30.8) 1 (3.8) 0 5 (19.2) 9 (34.6) 1 (3.8) 0 6 (35.2) 10 (58.9) 1 (5.9) 0 types of lesions on bronchoscopy normal compression stenosis infiltrative stenosis endobronchial mass 4 (15.4) 14 (53.8) 1 (3.8) 7 (31.8) 0 1 (3.8) 0 3 (11.5) 2 (7.7) 6 (23.1) 1 (3.8) 5 (19.2) 1 (3.8) 7 (26.9) 1 (3.8) 6 (23.1) 2 (11.8) 8 (47.0) 1 (5.9) 6 (35.3) histological types of lung cancer adenocarcinoma high grade neuroendocrine tumors adenosquamous carcinoma 11 (64.7) 5 (29.4) 1 (5.9) 3 (17.6) 1 (5.9) 0 9 (52.9) 4 (23.5) 1 (5.9) 9 (52.2) 5 (29.4) 1 (5.9) table 1. characteristics of research subjects (n=26) abbreviation: ttfna : transthoracic fine needle aspiration ttfna cell block technique bronchial washing smear technique and ttfna smear technique total positive negative positive 12 3 15 negative 2 9 11 total 14 12 26 sensitivity 85.7% specificity 75% positive predictive value (ppv) 80% negative predictive value (npv) 81.8% table 2. sensitivity and specificity of ttfna cell block technique with bronchial washing smear technique and ttfna smear technique in diagnosing lung cancer abbreviation: ttfna : transthoracic fine needle aspiration discussion lung cancer was found in 57.7% of subjects using the ttfna cell block technique. our study results are almost similar to those of abdulhameed et al.(10) at the rizgary teaching hospital, who reported that the prevalence of lung cancer by means of the ttfna cell block technique was as high as 57.9%. there were more ttfna cell block subjects than subjects undergoing ttfna 134 smear s. this agrees with the study of abdulhameed et al. who also found that the proportions of ttfna cell blocks and smears were 57.9% and 38.8%, respectively. their results also showed that bronchial washing smears had a prevalence of 15.4%. similar results were reported by vadala et al.,(11) in that the diagnosis of lung cancer by bronchial washing smear cytology was found in 36.5% of cases. the study by shivakumarswamy et al.(12) in india showed a lower percentage in lung cancer diagnosis, namely 1.85%. another study by acharya et al. (1 3) gave lower results on cytological examination of bronchial mass washing with 9.2%. most subjects in our study were over 40 years old (80.8%). this is consistent with the risk factors presented by the who or the indonesian lung doctors association (perhimpuan dokter paru indonesia, pdpi) regarding high risk in male subjects, over 40 years of age , a nd smokers.(14) consistent results were shown by li et al.(15) in australia who stated that the most subjects (93%) were in the age group of over 40 years. similar results were found in the studies by raiza et al.(16) and acharya et al.(16) with respect to the subject characteristics, ]this study had a greater proportion of males. lung cancer accounts for 17% and 9% of all cancers in men and women, respectively.(17) however, the incidence of lung cancer in women continues to increase. another study by li et al.(15) also showed that the incidence of males was greater at 15.1%, with an increased incidence in females as well. subjects diagnosed with lung cancer who had a history of smoking accounted for 53.3%. smoking history was higher in males than in females. passive smoking subjects also have risk factors for lung cancer. this is according to the study by charles et al.(18) where subjects with lower or higher bmi comprised 50.0%. lung cancer subjects with low bmi accounted for 53.3%. these results are in line with those of tomita et al.(19) who found that the proportion of lung cancer patients with a low bmi was 46.3%. this may have been due to the fact that the systemic symptoms of lung cancer that often arise are loss of appetite accompanied by weight l os s . t h e c a n c e r c a c he x i a syn d r ome i s characterized by anorexia and weight loss, resulting in impaired immunity, tissue wasting, and decreased performance status.(20) in this study, the types of lesions found on b r o nch os c o p y we r e mos tl y c o mpr e ss i on stenosis at 53.8%, followed by endobronchial mass, normal picture, and infiltrating stenosis at 31.8%, 15.4%, and 3.8%, respectively. lung c a n c e r f ou nd by th e t t fna ce l l b l oc k t e c h ni q u e a c c o un te d f or mo s t of t h e bronchoscopic lesions, namely compression stenosis at 47%, followed by endobronchial mass a t 35 .3% , n or ma l pic tu r e at 11.8% , and infiltrative stenosis at 5.9%. this contrasts with the study by acharya et al.(13) which states that compression stenosis and endobronchial mass have the same prevalence, namely 37.7%. this difference may have been caused by the location and size of the mass, which will affect the type of cancer that is found.(13) ttfna cell block technique bronchial washing smear technique and ttfna smear technique total p value positive negative positive 12 3 15 1.000 negative 2 9 11 total 14 12 26 table 3. mcnemar’s test for comparing ttfna cell block technique versus bronchial washing cytology smear technique and ttfna smear technique in diagnosing lung cancer abbreviation: ttfna : transthoracic fine needle aspiration sutanto, santi, wasita, et al cell block technique of ttfna in lung cancer 135 univ med vol. 40 no 2 the study results showed that the most fre quent histopath ological type was adenocarcinoma, 29.4% from bronchial washing and 52.9% from ttfna. in the research of vivekanand et al.(21) in india, adenocarcinoma was the most diagnosed type of lung cancer. the adenocarcinomas in their study were mostly found in female patients who were passive smokers. in this study, the t tfna cell block examination had a good sensitivity of 85.7% with a sp ec if ic it y of 75% . t he re sult s ar e in accordance with the study by dong et al.(2) who stated that the sensitivity of the ttfna cell block technique to establish the diagnosis of lung cancer was 88.7%. the study by santoshpawar et al.(8) obtained a sensitivity of 89.4% and a specificity of 96%. another study by kakodkar et al.(11) stated that cytological examination with cell blocks increased the positive findings as compared to smears. in their study, the sensitivity and specificity of the ttfna cell block and s me a r t e s ts we r e no t t oo d i ffe r e nt . dr. moewardi hospital has used ttfna smears and bronchial washing smears to diagnose lung cancer and has encountered no problems, so the two tests were used as gold standards in the present study. lung cancer immunotherapy is currently growing rapidly, and the cell block technique becomes inter esting, because one of the immunotherapy r equire ments is immunohistochemi cal examination. immunocytochemistry is a suitable solution for lung cancer immunotherapy, especially in patients who cannot be tested by immunohistochemistry.(3) takigawa et al.(22) stated that it is important to perform a pd-l1 examination using a good cytological sample if a histological sample is not obtained. another study by torous et al. (23) reported no significant difference in the pd-l1 tumor proportion score (tps) in specimens examined by cell block cytology or histology. even though the cell block examination and the smear technique have the same sensitivity, the cell block has the advantage of being able to be used as an immunocytochemical sample. in our study there was no statistically significant difference in the positivity value of the ttfna cell block technique and the positivity value of cytology of bronchus washing smears and ttfna smears in diagnosing lung cancer. there has been no study that tested the difference in the positivity of the ttfna cell block technique and that of the bronchus smear and ttfna smear techniques. the previous study by abdulhameed et al.(10) who performed different tests for fna smear and fna cell block, found no significant difference in positivity. the limitation of the present study is that it used the gold standard for lung cancer diagnosis instead of a histopathological examination. this study suggests that it is possible to determine the primary site of malignancy using cell blocks as well as smears. the cell block technique has the added benefit of allowing multiple sections of the same material to be obtained for special stains and immunohistochemistry.(4) conclusions the ttfna cell block showed good sensitivity and specificity. there was no difference in the positivity value of the ttfna cell block examination versus bronchial washing smears and ttfna smears in lung cancer diagnosis. the cell block technique can be used for immunocytochemical examinations in lung cancer diagnosis and therapy. conflict of interest the author(s) declared no potential conflicts of interest with r esp ect to the research, authorship, and/or publication of this article. acknowledgements the authors thank all patients who were willing to participate and all who supported this study. we also thank the dir ector of dr. moewardi hospital surakarta, for the opportunity to perform this study and collect the data. 136 contributors yss contributed to conceptualization, methodology, and writing the original draft. ns and bw contributed to methodology and data collection and analysis. ar and hr contributed to writing, reviewing, and editing. all authors have read and approved the final manuscript. references 1. kossakowski ca, morresi-hauf a, schnabel pa, eberhardt r, herth fjf, warth a.. preparation of cell blocks for lung cancer diagnosis and prediction: protocol and experience of a highvolume center. respiration 2014;87:432-38.doi: 10.1159/000357068. 2. dong z, li h, zhou j, zhang w, wu c. the value of cell block based on fine needle aspiration for lung cancer diagnosis. j thorac dis 2017;9:2375– 82. doi: 10.21037/jtd.2017.07.912017;9(8):2375–82. 3. malapelle u, iaccarino a, rossi a. cytology versus histology for programmed death-ligand 1 expression evaluation in the landscape of nonsmall cell lung cancer patients selection for immunotherapy. transl lung 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resection in patients with non-small cell lung cancer. asian pacific j cancer prev 2017;18:3353–6. doi: 10.22034/apjcp.2017. 18.12.3353. 20. jusuf a, hudoyo a, andriani r ir. diagnosis of lung cancer. in: jusuf a, editor. basic diagnosis of lung cancer. 1 st ed. jakarta: ui publishing; 2017. p.127–68. sutanto, santi, wasita, et al cell block technique of ttfna in lung cancer 137 univ med vol. 40 no 2 21. giti r, hosseinzadeh m. efficacy of bronchial washing and brushing cytology in the diagnosis of non-neoplastic lung diseases. acta med iran 2017;55:636–41. 22. takigawa n, ochi n, yamane h. histology versus cytology: pd-l1 testing in non-small cell lung cancer. transl lung cancer res 2018;7(suppl 3):s225-7. doi: 10.21037/tlcr.2018.08.08. 23. torous vf, rangachari d, gallant bp, shea m, costa db, vanderlaan pa. pd-l1 testing using the clone 22c3 pharmdx kit for selection of patients with non–small cell lung cancer to receive immune checkpoint inhibitor therapy: are cytology cell blocks a viable option? j am soc cytopathol 2018;7:133–41. doi: 10.1016/j.jasc.2018. 02.003. c:\users\user\documents\41 no 2 139 abstract universa medicina the effect of purple passion fruit juice on superoxide dismutase and malondialdehyde levels in hypercholesterolemic rats alfi muntafiah1*, johanes hasian siahaan1, sofyan hardi1, dody novrial2, and hernayanti3 background hypercholesterolemia due to a high cholesterol diet can increase free radicals resulting in oxidative stress. superoxide dismutase (sod) and malondialdehyde (mda) have been used as the study markers of oxidative stress in cases of hypercholesterolemia. purple passion fruit contains various compounds that may reduce free radicals. this study aimed to determine the effect of purple passion fruit juice on sod and mda levels in hypercholesterolemic rats. methods an experimental analysis with post-test only control group design involving 28 male wistar rats. they were divided into 4 groups: normal control (k1), hypercholesterolemic control (k2), purple passion fruit juice treatment at 4.2 ml/200 gbw/day (k3), and simvastatin treatment at 0.018 mg/200 gbw/ day (k4). the purple passion fruit juice at 4.2 ml/200 gbw/day was administered for 14 days. sod levels were examined by enzymatic colorimetric methods using the ransod kit and mda levels by the tbars method. results the kruskal-wallis test showed a significant difference in sod levels between the tested groups (p<0.05). one-way anova test for mda levels showed a significant difference (p<0.05). post hoc test (mann-whitney for sod and lsd for mda levels) also showed significant differences: k1 vs. k2, k2 vs. k3, k2 vs. k4, and k3 vs. k4 (p<0.05). conclusion this study demonstrated that purple passion fruit juice significantly increases the sod and lowers the mda level in hypercholesterolemic male wistar rats. consumption of purple passion fruit juice may help to modulate oxidative stress caused by hypercholesterolemia in rats. keywords: hypercholesterolemia, malondialdehyde, oxidative stress, passiflora edulis var edulis, superoxide dismutase, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.139-148 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1280 may-august 2022 vol.41no.2 1department of biochemistry, faculty of medicine, universitas jenderal soedirman, purwokerto, indonesia 2department of anatomical pathology, faculty of medicine, universitas jenderal soedirman, purwokerto, indonesia 3department of biology, faculty of biology, universitas jenderal soedirman, purwokerto, indonesia *correspondience: alfi muntafiah department of biochemistry, faculty of medicine, universitas jenderal soedirman, purwokerto, indonesia phone and fax: +6285865137470 email: alfimuntafiah99@gmail.com orcid id: 0000-0002-1424-6111 date of first submission, january 17, 2022 date of final revised submission, june 20, 2022 date of acceptance, june 23, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license ci te t his art icle as: mun tafi ah a, si ahaa n jh, hard i s, novria l d , hernayanti. th e effect of purp le pas sion fru it j uice on superoxi de dismutase and malondialdehyde levels in hypercholesterolemic rats. univ med 2022;41:139-48. doi: 10.18051/ univmed.2022.v41.139-148. 140 muntafiah, siahaan , hardi, et al superoxide dismutase and malondialdehyde levels introduction hypercholesterolemia is a condition where the level of cholesterol in the blood exceeds the normal level due to an increase in cholesterol and low-density lipoprotein (ldl).(1) this condition can be caused by a high-cholesterol diet, increased body weight, age, genetics, and decreased estrogen levels in postmenopausal women.(2) hypercholesterolemia is one of the main risk factors for cardiovascular disease.(3) in 2 01 6 , 1 7. 9 mi l l i o n pe op l e d i e d f r om cardiovascular disease.(4) according to the indonesian ministry of health in 2018 of the indonesian population aged 15 years there were 21.2% with borderline cholesterol (200-239 mg/ dl) and 7.6% with high cholesterol (240 mg/ dl).(5) hypercholesterolemia causes an increase in free radicals, which modify the amino acids in low density lipoprotein (ldl) apolipoproteins so that lipid pe roxida tion occurs in ldl. macrophages engulf the modified ldl causing further free radical formation. this increase in free radicals triggers oxidative stress,(6,7) which is an imbalance between antioxidants and prooxidants, the amount of pro-oxidants being greater than that of antioxidants.(8) oxidative stress can be reduced by incre asing the number of antioxidants. one of the main chain-breaking antioxidants is superoxide dismutase (sod), which acts by scavenging superoxide free radicals.(9) superoxide dismutase catalyzes the conversion of the superoxide free radicals into hydr ogen pe r oxide ( h 2 o 2 ) or mo lecular oxygen.(10,11) according to the american heart association (aha) and the american college of cardiology (acc), hypercholesterolemia therapy is divided into two therapeutic modalities: 1) non-pharmacological therapy through lifestyle changes, and 2) pharmacological therapy with fatlowering drugs.(12) one of these pharmacological therapies are the statins, the most potent drugs in lowering ldl cholesterol.(13) statins act by lowering cholesterol through inhibition of 3hydroxy-3-methyl-glutaryl-coenzyme a (hmgcoa) reductase and increasing the production of high-density lipoprotein (hdl).(14) these statin drugs can cause the side effects of myalgia, rhabdomyolysis, diabetes mellitus, and drug interactions.(13) meanwhile, non-pharmacological ther apies in the management of hypercholesterolemia include increased fruit and vegetable consumption, aerobic physical activity, weight loss, and smoking cessation. (1 2) consumption of fruits and vegetables is very important, since in addition to containing fiber components to lower cholesterol, they also have phytochemical components and vitamins with antioxidant properties.(15) indonesia has a variety of antioxidantcontaining fruits that are beneficial for health.(16) these fruits are a source of natural antioxidants because they contain phytochemical compounds that can reduce pro-oxidants.(17) one of these fruits is the purple passion fruit from the passiflora family.(18) the purple passion fruit (passiflora edulis) is a plant originating from brazil that can live in tropical and sub-tropical areas,(19) and is widely cultivated in indonesia, especially in sulawesi and sumatra.(20) the passion fruit plant is easy to grow but the fruit is often not used because of its sour taste. previous studies have explored the contents of various parts of this fruit (seeds, peel, and juice). purple passion fruit contains flavonoids, alkaloids, phenols, cyanogenic compounds, glycosides, vitamins, minerals, and polyphenol antioxidants.(21) flavonoid antioxidants in fruits are reported to play a role in preventing cell damage caused by free radicals.(22) behind its exotic taste, it turns out that purple passion fruit contains various nutrients and non-nutritive phytochemicals that are beneficial for health. many pharmacological studies have been carried out, where various components of purple passion fruit show antimicrobial, antihypertensive, antidiabetic, antioxidant, and other activities.(23) thi s stud y is a part of our resea rch roadmap. in the last study, we explored the antihypercholesterolemic potential of purple passion fruit juice at various doses.(24) then we studied the effectiveness of this fruit juice against 141 simvastatin in the hypercholesterolemic rat model. research on lipid profiles, which is closely related to this study, has been previously published by muntafiah et al.(25) furthermore, the present study describes the effect of purple passion fruit (passiflora edulis var edulis) juice on the levels of superoxide dismutase (sod) and malondialdehyde (mda) in experimental animal models of hypercholesterolemia. we choose sod as our research parameter because it is the marker enzyme against oxidative stress that plays a key antioxidant role in living cells against the toxicity of superoxide free radicals. in hypercholesterolemia it is known that high cholesterol in cells results in an altered cell me mbr a ne d ue t o lip i d p e r o xi d a t i on . malondialdehyde (mda) is the marker of lipid peroxidation generating free radicals. excess ge ne r a t i on o f fr e e r a d i c a l s i n hypercholesterolemia depletes body antioxidants leading to oxidative stress. several studies have reported the effect of passiflora fruit on antioxidants and oxidative stress, but different results were obtained. the plant materials used in these studies are also different. our study is consistent with a study by silva et al.(26) which states that treatment with a polysaccharide fraction from the peel of passiflora edulis fruit significantly reduced mda concentration and increased glutathione (gsh) levels in mice. this is also consistent with t he s t ud y o n t he a n t i oxi da n t ac ti vi ty of passiflora alata fruit extract by medeiros et al.(18) in contrast, another study by de souza et al.(27) evaluated the effects of passiflora edulis f. flavicarpa degener (yellow passion fruit) juice on the lipid profile and oxidative stress status of wistar rats, showing that the levels of triglycerides and very-low-density lipoproteincholesterol, superoxide dismutase activity (sod), and total glutathione concentration were not statistically different between the two groups. this research is a continuation of a previous study that explored the potential of purple passion fruit juice as an anti-hypercholesterolemic agent, where the fruit juice at a dose of 4.2 ml/200 g bw could significantly reduce total cholesterol levels.(24) methods research design this experimental study used a completely randomized post-test only control group design. t hi s r e s ea r c h wa s c o nd u c te d a t t he biochemistry laboratory and the pharmacology & experimental animal laboratory, faculty of medicine, jenderal sudirman university (fk un soe d) , pu r wo ke r t o, ce n tr a l j a va , indonesia, from october 2016 to november 2016. experimental animals the research subjects were healthy male albino rats (rattus norvegicus) of the wistar strain, 2-3 months old, and weighing 180-220 g, obtained from lppt iii, gadjah mada university (ugm), yogyakarta. the minimum total number of subjects in this study was 28 based on the ‘resource equation’ approach.(28) the animals were maintained at 22"25°c with 12 h/12 h light /dark cycle. all animals were fed rat pellets and provided with tap water ad libitum. establishment of rat hypercholesterolemia model induction of hypercholesterolemia was carried out after the experimental animals underwent a seven-day acclimatization period. induction was carried out in groups k2, k3, and k4 for 10 days with duck egg yolk at a dose of 2 ml/200 g bw/day and pork oil at 3 ml/200 gbw/day orally through a probe. after the induction period, the experimental animals were fasted for 12 hours and 2 ml of blood was drawn from the infra-orbital vein to confirm the hypercholesterolemia.(25) induction was declared successful if the total cholesterol level in the blood exceeded 54 mg/dl. normal rat plasma total cholesterol level is 10-54 mg/dl.(24) univ med vol. 41 no 2 142 muntafiah, siahaan , hardi, et al superoxide dismutase and malondialdehyde levels a b treatment of the hypercholesteremia rats the experimental animals were randomly divided into 4 groups: normal control (k1), hypercholesterolemic control (k2), purple passion fruit juice treatment at a dose of 4.2 ml/200 gbw (k 3), and simvastati n control (k 4). t he experimental animals were put into cages by gr oup. during the tr eatment pe riod, the experimental animals received treatment every morning. the healthy/normal control group (k1) and the hypercholesterolemic control group (k2) were treated with distilled water orally through a probe. the k3 group received purple passion fruit juice at 4.2 ml/200 g bw/day by oral probe. a dose of 4.2 ml/200 g bw is the maximum dose that can be accepted by these animals. based on our previous study, this dose can significantly reduce total blood cholesterol.(24) the k4 group received simvastatin at a dose of 0.018 mg/200 g bw. bef ore the tre atment period, the experimental animals were acclimatized for 7 days, placed in the same environment, in cages of the same size, shape, material, and location, and r eceived food and drinking water ad libitum.(29) the dose of simvastatin used in this study was based on the usual dose given to adult humans, which is 10 mg/day. the conversion factor from humans with a bodyweight of 70 kg to rats (rattus norvegicus) with a body weight of 200 grams is 0.018 mg/200 g bw/day.(25) therefore, the dose of simvastatin for rats equals the conversion factor for rats multiplied by the dose of simvastatin in humans = 0.018 x 10 mg/ day, giving the dose of 0.18 mg/200 g bw/day. plant materials this study used ripe purple passion fruit (with dark purple and smooth or slightly grooved skin). we used fresh fruit juice directly taken from ripe fruit. the fruit was taken in a season with sufficient rainfall and sunshine. the preparation of purple passion fruit juice begins with washing of the fresh fruit, after which the fruit is cut and the fruit flesh is made into juice then filtered through a filter cloth so that the seeds are separated from the fruit juice. the fruit juice is made every day to obtain fresh fruit juice.(30) determination test was carried out at the biology laboratory, jenderal soedirman university. determination of sod and mda posttest blood sampling was carried out at the end of the study period, using a 3 ml hematocrit pipette through the infra-orbital vein. previously, the experimental animals had been fasted for 12 hours. whole blood samples were put into an edta tube, centrifuged at a speed of 1300 rpm for 10 minutes at room temperature, then examined for antioxidant levels of sod and mda.(27) the sod antioxidant levels were measured by the colorimetric enzymatic method using the ransod kit, while mda levels were determined by the thiobarbituric acid reactive substance (tbars) method. the absorbance was read on a spectrophotometer (robert riele gmbh & co kg, germany), at a wavelength of 540 nm. statistical analysis data were processed and analyzed using the spss 10 for windows statistical software. the data distribution was tested for normality with the shapiro-wilk test and continued with the levene’s test for homogeneity. the data on sod and mda levels were found to be normally distributed (p>0.05), but the data variance was n ot homoge neo us ( p< 0. 05 ), s o that da ta t r a n s f o r ma t io n w a s c a r r i e d ou t . af t e r transformation, the data variance of sod was still non-homogeneous (p<0.05) so the kruskalwa l li s n on -p a r a me t r i c te st w a s us e d . meanwhile, the data variance of mda was homogeneous after transformation so it was tested with one-way anova.(31) ethical clearance this research has received ethical approval from the faculty of medicine research ethics committee, jenderal soedirman university under no. ref: 120/kepk/2016. 143 univ med vol. 41 no 2 results as shown in figure 1, there was an increase in body weight in all groups at week 3 of the study. the normal control group (k1) and the hypercholesterolemic group (k2) experienced weight loss at weeks 4 and 5. the treatment group receiving purple passion fruit juice (k3) had relatively stable body weight until the 5th week. the treatment group on simvastatin experienced weight loss. t he da t a of sod a nd m da l e ve l s determined at the end of the study period are presented in table 1. the hypercholesterolemic control group (k2) had the lowest sod and highest mda levels compared to other groups. the purple passion fruit juice group (k3) and the simvastatin group (k4) had higher sod and l ow e r md a le ve l s t h a n t he hypercholesterolemic control group (k2). the sod level in the simvastatin group (k4) was higher than in the hypercholesterolemic control group (k2) but was still lower than in the normal control group (k1). administration of purple passion fruit juice at 4.2 ml/200 g bw to the k3 group could increase sod to levels that were higher than in the normal control group (k1), where the levels increased about 3-fold if compared to the hypercholesterolemic control group (k2). the increase in sod levels was in line with the decrease in mda levels in that group. figure 1. body weight of the experimental animals table 1. distribution of mean sod and mda levels by treatment group statistical test using 95% confidence intervals. *kruskal-wallis test, followed by mann-whitney test shows a significant difference between k1 vs. k2 (p=0.004), k2 vs. k3 (p=0.004), k2 vs. k4 (p=0.020), and k3 vs. k4 (p=0.045 ). **one-way anova. followed by post hoc lsd showed a significant difference between k1 vs. k2 (p=0.00); k2 vs. k3 (p=0.00); k2 vs. k4 (p=0.00); and k3 vs k4 (p=0.00). a,b different notations on the same column indicate significant differences. k1: normal control; k2: hypercholesterolemic control; k3: treatment with purple passion fruit juice at 4.2 ml/200 gbw/day; and k4: treatment with simvastatin at 0.018 mg/200 gbw/day. sod: superoxide dismutase, mda: malondialdehyde, sd: standard deviation data presented as sod: median (min-max) for sod and mda:mean ± sd treatment group p value k1 (n=6) k2 (n=6) k3 (n=6 k4 (n=4) sod (u/ml) mda (µmol/l) 17.10 (11.70 24.00)a 0.61 ± 0.03a 9.15 (7.00 – 10.60)b 2.45 ± 0.12b 22.25 (12.10 -63.90)a,c 0.78 ± 0.07c 12.45 (8.90 – 16.50)a 1.20 ± 0.08d < 0.05* < 0.05** 144 muntafiah, siahaan , hardi, et al superoxide dismutase and malondialdehyde levels mitochondrial electron system or activation of nadph oxidase. these reactive free radicals cause lipid peroxidation in the cell membranes that produce lipid peroxide radicals and other free radicals.(6,33) increased lipid peroxidation (which can be seen from the high levels of mda causes oxidative stress due to an imbalance between peroxides and oxidants.(33) superoxide dismutase is a marker enzyme against oxidative stress in cells. this enzyme degrades superoxide into ordinary oxygen molecules or hydrogen peroxide. it also acts as a key antioxidant in living cells that protects against the toxicity of superoxide free radicals.(6,11) in hype rcholesterolemic conditions, sod antioxidants are at their lowest levels because the increased production of free radicals exceeds the body’s capacity to handle them. the increase in free radicals in the blood occurs because of the increased low-grade systemic inflammatory response, where this systemic inflammation increases the production of ros.(34) these free radicals modify the amino acids in ldl apolipoproteins so that lipid peroxidation occurs in ldl. the macrophages then engulf the modified ldl causing further free radical formation. the continuous increase in free radicals triggers oxidative stress.(6,7) under normal conditions, the body can reduce oxidative stress conditions with e nd ogenous (enzymatic) antioxidants, one of which is sod, but in hypercholesterolemic conditions, the increase in free radicals exceeds the body’s capacity to fight oxidative stress conditions, resulting in a decrease in sod levels in the k2 group.(32) the results of this study showed a significant increase in sod levels in the k3 group, about 3 times higher than in the hypercholesterolemic control (k2). this is in line with the research conducted by kandandapani et al. (35) where administration of purple passion fruit at 500 mg/ kg of leaf, peel, and fruit extract in streptozotocininduced diabetic rats showed a significant increase in sod and a reduction in the tbars levels in the heart, liver, and kidney. in the present study, the increase in sod levels in the k3 group the kruskal-wallis nonparametric test showed a significant difference between the treatment groups (p=0.002). therefore, the post hoc test with mann-whitney was continued to find out which groups had these significant differences. mann-whitney test showed a significant difference in group k1 vs. k2 (p=0.004), group k2 vs. k3 (p=0.004), group k2 vs. k4 (p=0.020), and group k3 vs. k4 (p=0.045). meanwhile, the k1 vs. k3 and k1 vs. k4 groups has no significant differences, with p-values of 0.262 and 0.150, respectively.one-way anova showed significant differences between the treatment groups (p<0.05). post hoc lsd showed significant differences in k1 vs. k2 (p=0.00), k2 vs. k3 (p=0.00), k2 vs. k4 (p=0.00), and k3 vs. k4 (0.00). discussion this study was conducted to determine the effect of purple passion fruit juice (passiflora edulis var edulis) on sod and mda levels in experimental hypercholesterolemic model animals. the results of this study showed that the hypercholesterolemic control group (k2) had the lowest levels of sod antioxidants and highest levels of mda compared to the other groups. this is in line with the research conducted by silva et al.(26) on the effect of the polysaccharide fraction from the peel of passiflora edulis fruit on mda in mice. research by panelli et al.(32) on the benefits of the bark of passiflora edulis under conditions of oxidative stress in obese mice also showed that it was effective in improving antioxidant capa city a nd r educed malondialdehyde levels. hypercholesterolemia is characterized by an increase in total cholesterol and ldl cholesterol. the cell membrane is composed of cholesterol as a structural component that plays a role in the structural and functional integrity of the cell. the presence of high levels of cholesterol in hypercholesterolemia results in changes in the physical properties of the cell membranes, which may facilitate leakage of reactive oxygen spe cie s (ros) from the 145 was made possible by various phytochemical compounds in purple passion fruit juice. the increase in superoxide dismutase levels may be explained by several mechanisms. purple passion fruit juice contains flavonoid compounds that act directly by donating hydrogen ions to neutralize the toxic effects of free radicals. flavonoids also act indirectly by incr easing endogenous antioxidant genes through the activation of nuclear factor erythroid 2 related factor 2 (nrf2). activation of this gene plays a role in the synthesis of antioxidant enzymes such as the sod gene.(36,37) purple passion fruit besides having flavonoid compounds that can reduce free radicals, also contains vitamin c and beta carotene, which act as antioxidants by donating electrons or hydrogen atoms to free radicals to neutralize these free radicals. neutralization of these free radicals can reduce oxidative stress.(38) therefore, the flavonoid compounds, vitamin c, and beta carotene contained in purple passion fruit may increase sod levels in the k3 group.(39) research by doungue et al.(36) showed that the administration of the flavonoid extract fraction to rats with aluminum chloride-induced alzheimer’s disease was able to significantly increase sod levels in the hippocampus and cortex of the rats. the study also stated that flavonoids were the main cause of the increase in sod levels, although other bioactive compounds also played a role. our study is also in line with research conducted by hu et al.(37) which showed that administration of pure purple passion fruit anthocyanin extract, which is a type of flavonoid in purple passion fruit, could significantly increase sod activity at doses of 400 mg/kg and 600 mg/kg in the skeletal muscle of rats with induced muscle fatigue. sod antioxidant levels in the k4 group also showed a significant increase, but the levels were still lower than in the healthy control group (k1). research conducted by eger et al.(40) on the effect of simvastatin administration to wistar rats for 1 month on the increase in free radicals induced by lisdexamfetamine dimesylate (ldx) showed that simvastatin could prevent a decrease in sod activity in the cerebellum of the rat brain. research by yang et al.(41) on sprague dawley rats subjected to smoke inhalation showed that simvastatin in doses of 25 mg/kg to 100 mg/kg could increase sod activity in a dose-dependent manner, but the increase had not yet reached the sod levels in the healthy groups. clinical studies show that statins affect plasma sod levels, while research shows that statins have antioxidant effects through cell antioxidation and increased free radical elimination. statins have antiinflammatory properties by lowering c-reactive protein (crp) and pro-inflammator y cytokines.(42) sod enzymes are important in reducing the effects of oxidative stress by breaking down free radicals into hydrogen peroxide which will then be broken down by catalase or glutathione peroxidase into non-toxic products, namely water, and oxygen. conditions that cause a decrease in the sod enzyme can cause cell damage due to oxidative stress.(4345) purple passion fruit acts as an antioxidant that can r educe hypercholesterolemia-induced oxidative stress by increasing sod activity and neutralizing free radicals. the present study showed that purple passion fruit juice contains antioxidants that are effective in increasing plasma sod and reducing mda levels in wistar rats with induced hypercholesterolemia. one limitation of this research is the short duration of the trial period in which the study has not been able to show the antioxidant or vitamin content of the purple passion fruit extract, so that further research is planned for advanced stages of the study in humans. conclusions administration of purple passion fruit juice (passiflora edulis var edulis) could significantly increase sod antioxidant levels and reduce mda levels in mal e wistar rats (rattus norvegicus) induced by hypercholesterolemia. thus, our result s r einforce the ethnopharmacological use of passiflora edulis in popular medicine. univ med vol. 41 no 2 146 muntafiah, siahaan , hardi, et al superoxide dismutase and malondialdehyde levels conflicts of interest the author(s) declared no potential conflicts of interest concerning this article’s research, authorship, and/or publication. acknowledgment we would like to express our gratitude to the institute for research and community service (lppm) at jenderal sudirman university (unsoed) which has provided a research grant for the blu research scheme for competency improvement so that this research could be carried out. contributors all authors take public responsibility for the content of the 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41 no 2 148 muntafiah, siahaan , hardi, et al superoxide dismutase and malondialdehyde levels 41. yang r, guo p, ma z, et al. effects of simvastatin on inos and caspase 3 levels and oxidative stress following smoke inhalation injury. mol med rep 2020;22: 3405–17. pmid: 32945441. 42. simatupang a. statin (hmg-coa reductase inhibitor): bukti terbaru pengalaman penggunaannya. jakarta: fakultas kedokteran universitas kristen indonesia; 2017. 43. saraswati ra, maharani n, utomo aw. pengaruh ekstrak kulit manggis (garcinia mangostana l.) terhadap kadar enzim superoxide dismutase (sod) tikus yang diinduksi minyak jelantah. j kedokter diponegoro 2018;7:1511–19. doi: https://doi.org/ 10.14710/dmj.v7i2.21470. 44. septembre-malaterre a, stanislas g, douraguia e, gonthier m. evaluation of nutritional and antioxidant properties of the tropical fruits banana, litchi, mango, papaya, passion fruit, and pineapple cultivated in réunion french island. food chem 2016;212:225–33. doi: 10.1016/ j.foodchem.2016.05.147. 45. pretti ir, da luz ac, perdigão tl, araújo ra, batitucci mcp. gene expression and antioxidant enzymatic activity in passion fruit exposed to aluminum. afr j agric res 2018;13:115–20. https:/ /doi.org/10.5897/ajar2017.12834. c:\users\universa medicina\docu 45 abstract universa medicina serum apolipoprotein b increased among tuberculosis patients compared to healthy subjects augustine c. ihim1*, samuel c. meludu1,2, charles c. onyenekwe1, arthur e. anyabolu3 and comfort n.akujobi3 background the burden of tuberculosis (tb) and cardiovascular disease (cvd) is enormous worldwide. public health programs have been challenged with the overlapping of tb and cvd epidemics.mycobacterium tuberculosis (mtb) is also a main driver of atherogenesis, suggesting a potential pathogenic role of tuberculosis in cardiovascular disease. the objective of this study was to compare the serum levels of apolipoprotein b (apo b), apolipoprotein b48 (apo b48) and apolipoprotein b100 (apo b100) between patients with tuberculosis and healthy subjects. methods a cross-sectional study was conducted involving 251 subjects consisting of 120 treatment naïve active tb patients [26 hiv co infected (tb+hiv+) and 82 tb+), 12 malaria parasite co-infected (tb+mp+)], 26 latent tb infected (ltb) and 105 healthy controls. their body mass index (bmi) was calculated. mycobacterium tuberculosis infection was determined by ziehlnelseen (zn) sputum smear microscopy and confirmed positive using genexpert. latent tb was determined by mantoux test, mp was evaluated by microscopy while hiv by immunochromatographic techniques using serial algorithm. apolipoproteins were determined using spectrophotometry. a one-way anova test and lsd’s post hoc multiple comparisons were used for statistical analyses. results significantly lower mean levels of bmi were observed in ltb, tb+, tb+hiv+ and tb+mp+ compared with the controls (p<0.005). the mean serum levels of apo b, apo b48 and b100 were significantly higher in ltb, tb+, hiv+tb+ and tb+mp+ compared with apparently healthy controls (p<0.05). conclusion elevated levels of apolipoproteins among infected tb individuals might predispose them to cardiovascular disease. keywords: apolipoprotein, atherosclerosis, tuberculosis patient, healthy subjects original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.45-51 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1062 january-april, 2021 vol.40no.1 1chemical pathology unit, department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, nigeria 2department of human biochemistry, faculty of basic sciences, nnamdi azikiwe university, nigeria 3faculty of medicine, nnamdi azikiwe university, nigeria *correspondence : augustine c. ihim, ph.d. chemical pathology unit, department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, p.m.b. 5025 awka, anambra state , nigeria email: ac.ihim@unizik.edu.ng phone +2348035985883 orcid id: 0000-0001-9991-0714 date of first submission, october 29 2020 date of final revised submission, march 28, 2021 date of acceptance, april 3, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: ihim ac, meludu sc, onyenekwe cc, anyabolu ae, akujobi cn. serum apolipoprotein b increased among tuberculosis patients compared to healthy subjects. univ med 2021;40: 45-51. doi: 10.18051/univmed.2021. v40.45-51 46 ihim, meludu, onyenekwe, et al serum apolipoprotein b increased among tuberculosis introduction tuberculosis and cardiovascular disease (cvd) pose great public health challenges globally. the world health organization has been challenged with the increasing burden of tuberculosis and cvd epidemics.(1) there is increasing evidence that many infections contribute to the pathogenesis of cvd including mycobacterium tuberculosis. (2) some authors a l s o r e ve al e d t h e a s s o c i a t i on o f la te nt tuberculosis with acute myocardial infarction (ami).(2) the potential mechanism for this association relied on a study that showed continuous activation of the immune system in latent and active tuberculosis.(3) antibodies to mycobacterial hsp65 cross-reacting with selfantigens in human blood vessels leading to autoimmunity may also have an effect on cvd risk. monocytes/macrophages, lymphocytes and cytokines engaged in cell-mediated immune responses against mycobac terium tuberculosis are also mai n driver s of atherogenesis, suggesting a potential pathogenic role of tuberculosis in cvd via mechanisms that have been described for other pathogens that establish chronic infection and latency.(3-5) consequently, apolipoprotein-b (apo b) containing lipoproteins are a suspected cause of atherosclerosis which is the main underlying cause of cvd. apolipoprotein-b enters the wall of the arteries and can transport cholesterol into the arterial wall; if present in increased numbers, ma y b e the ma in i ni t i a t i ng f a c t o r in atherosclerosis.(6) apolipoprotein b100 (apo b100) is the largest of the apob group of proteins, consisting of 4563 amino acids. apolipoprotein b100 is the apolipoprotein found in lipoproteins synthesized by the liver and is found in chylomicrons, very low-density lipoprotein cholesterol (vldl-c), intermediatedensity lipoprotein cholesterol (idl-c), lowde nsi ty li pop ro te in c hol ester ol ( ldl-c) and lipoprotein(a) [lp(a)] particles.(7) all these particles are atherogenic.(8) each of these particles contains a single apo b molecule. several studies have shown that apo b may be a better predictor of cardiovascular disease risk than ldl-c.(9,10) furthermore, it has been shown that apo b may be raised despite normal or low levels of ldl-c. apolipoprotein-b also appears to predict on-treatment risk, when ldlc has been lowered by statin therapy. similarly, a study by ihim et al.,(10) on free fatty acid and lipid profile in active tuberculosis, latent tuberculosis, active tb and hiv, active tb and mal ar ia pa r a si te i n sub je ct s i n anambr a concluded that low level of free fatty acid and hypolipidaemia in tb subjects could be attributed to tuberculosis. this observation is in line with the findings by oyedeji et al.(11) in their study on oxidative stress and lipid profile status in pulmonary tuberculosis patients in south western nigeria. the evaluation of apo b represents the total burden of the main lipoprotein particles involved in the atherosclerotic process. .but another study showed different results, in that there was no significant difference in apo b b e t we e n pa t i e n t s w i th t b a nd h e a l t hy subjects.(12) it has been shown that mycobacterium tuberculosis obtains nutrients and metabolizes host derived lipids mainly fatty acids and cholesterol and uses them to cause and maintain disease.(13,14) despite evidence of hypolipidaemia among individuals with tb infection,(10) cvd is still on the increase among them.(1) consequently, this research objective was to compare the serum levels of apo b, apolipoprotein b48 (apo b48) and apo b100 between patients with tuberculosis and healthy subjects. methods research design t hi s c r os s-se c ti o n a l r e se a r c h w a s conducted at nnamdi azikiwe university teaching hospital (nauth), nnewi, anambra state, nigeria, between may 2015 and january 2018 to compare the serum levels of apo b, apo b48 and apo b100 among individuals with mtb and apparently healthy controls. 47 study population the study population consisted of 5518 suspected individuals with cardinal symptoms of tuberculosis who presented at tuberculosis directly observed therapy (tbdot) clinics of nnamdi azikiwe university teaching hospital (nauth), nnewi, between may, 2015 and january, 2018. sampling technique consecutive non-random sampling techniques was used to select individuals from the tb dot centres before the initiation of therapy. participants were individuals who met the inclusion criteria and consented after the purpose was explained to them and were recruited as they became available until the sample size was attained. study subjects sample size was calculated using g*power software version 3.0.10 (universität düsseldorf, germany). power analysis for one way anova was conducted in g*power to determine a sufficient sample size using an alpha of 0.05, a power of 0.89 and a medium effect size. based on these, the calculated total sample size of 245 has 89% power to detect a difference of 0.25 at a significance level of 0.05. a total of 251 study subjects composed of 120 treatment naïve active tb patients [26 hiv co infected (tb+hiv+) and 82 t b+), 12 malaria parasite c o-infected (tb+mp+)], 26 latent tb infected (ltb) and 105 healthy controls (tb-hiv”tst”) were recruited. at recruitment, all study subjects were interviewed using a standard questionnaire and demographic data were collected. the weight and height of all the subjects were measured and used to determine their body mass index (bmi). mycobacterium tuberculosis infection was determined by ziehl-neelsen (zn) sputum smear microscopy and confirmed positive using genexpert®. newly diagnosed tb positive individuals with or without mp, and or hiv co -infections were recruited. the individuals above were those who did not receive any tuberculosis treatment before they were recruited (category one, first line tb positive individuals), were between 1566 years old, and attending the tb dots clinic, nauth, nnewi, state anambra. individuals infected with tb and on antiretroviral therapy were excluded. patients diagnosed with pulmonary tuberculosis but having diabetes mellitus were also excluded from the study. tobacco smokers, alcohol drinkers and participants who had other clinical problems such as diabetes and cardiovascular diseases were excluded from the study. ethical consideration ethical approval for the study was obtained from nnamdi azikiwe university teaching hospital ethics committee (nauthec) under no. nauth/cs/66/vol.7/79, nnewi, anambra state, nigeria. informed consent the aim, benefits and purpose of the study was explained to the individuals. participation was voluntary and informed consent was obtained from all of the individuals. the individuals were allowed at any time they so desired to discontinue and that would not in any way affect their care. the information obtained from the individuals was kept highly confidential in observance of the privacy act. sample collection sputum collection and processing sputum samples were collected using the directly observed treatment short course (dots) strategy specification and were processed using the ziehl-neelsen staining method and confirmed using the genexpert® by cepheid. blood sample collection blood samples were collected once from individuals with a ctive mycobacterium tuberculosis infection. firstly, immediately the individual was confirmed to be positive for pulmonary tuberculosis by ziehl neelsen’s univ med vol. 40 no 1 48 ihim, meludu, onyenekwe, et al serum apolipoprotein b increased among tuberculosis a b c staining technique and genexpert mtb/rif assay, before the initiation of anti-tuberculosis treatment (att). blood samples were collected once from individuals with latent tb and from apparently healthy individuals (controls). eight milliliters (8mls) of blood was collected from each individual at each period of blood collection, thick and thin blood films were made for microscopic detection of p. falciparum on r e c r u it me n t a n d ma l a r i a pl a s mo di u m falciparum/pan rapid test (carestart tm, access bi o, usa ) wh i c h is a c hr o mat ogr a p h ic immunoassay for the qualitative detection of circulating p. falciparum antigen in whole blood was also used. two milliliters (2ml) of blood was dispensed in ethylene diamine tetra acetic acid (edta) bottles and 6ml of blood was dispensed in plain tubes to separate serum for various biochemical assays.(15) the blood in the plain tubes was allowed to stand for 30 minutes to clot and further centrifuged at 3500 rpm for five minutes using wisperfuge model 1384 centrifuge (samson, holland). serum was separated from the clot with a micropipette into a sterile plain tube for the measurement of biochemical parameters. each individual’s blood sample was stored frozen at -200c in aliquots, in three cryovials to avoid repeated thawing and storing that would affect the result of the analysis. diagnostic assessments the hiv status of the study subjects was determined using the determine hiv-1/2 (abbott laboratories, japan) as the screening test, the capillus hiv-1/2 (trinity biotech, ireland) as the confirmator y test a nd uni-gold hiv-1/2 recombinant (trinity biotech, ireland) as a tie breaker test. diagnosis of malaria p. falciparum malaria was detected using thick and thin blood smears for microscopic detection (employing giemsa staining technique) and malaria plasmodium falciparum rapid test device (carestarttm malaria hrp2 (pf) by access bio,inc. usa). mantoux test tuberculin purified protein derivative (ppd) was utilized for the mantoux test to assist in clinical diagnosis of tuberculosis for diagnosis and differential diagnosis of tuberculosis, early detection of tuberculosis and screening for infection by m. tuberculosis (latent tuberculosis infection). this test is known as the tuberculin skin test. the ppd used was obtained from bb – ncipd ltd, sofia, bulgaria. each vial contained 1ml (10 doses) containing 50tu of ppd = 5tu/0.1ml per dose.(16) estimation of apo b, apo b48, and apo b1 00 apo b, apo b48, and apo b100 were estimated by sandwich enzyme immunoassay technique as described by brodsky and edward.(17) statistical analysis the ibm statistical package for social sciences (spss) version 21 was used for the sta tistical analysis. anova was used to determine if there were any statistical differences between the means of different groups of subjects. lsd’s post hoc multiple comparisons were run to confirm where the differences oc curred in the groups. the results were prese nted as mean ± standard deviation. significant levels were considered at p<0.05. results significantly lower mean levels of bmi were observed in individuals with ltb (25.57 ± 6.53 kg/m2), active tb (24.11 ± 3.84 kg/m2), human immunodeficiency and tuberculosis co-infection (22.48 ± 2.38 kg/m2) and tuberculosis and malaria parasite co-infection (tbmp) (26.69 ± 4.01 kg/ m2) compared with the controls (29.19 ± 7.22 kg/m2) (p<0.05). the mean serum level of apolipoprotein b was significantly higher in individuals with active tb infection, tbhiv and tbmp co-infections (2879.3 ± 1.03 µg/ml, 2759.6 ± 0.37 µg/ml, and 49 univ med vol. 40 no.1 2656 ± 0.462 µg/ml) respectively compared with ltbi (2493.7 ± 0.83 µg/ml) and apparently healthy controls (2033.2 ± 1.10 µg/ml) (p<0.05). fur thermor e, the mean serum level of apolipoprotein b 48 in individuals with active tb infection, tbhiv and tbmp co-infections (364.4 ± 8.5 µg/ml, 358.7 ± 1.98 µg/ml and 386.4 ± 2.36 µg/ml, respectively) is significantly higher than in individuals with ltb (106.3 ± 1.10 µg/ ml) and apparently healthy controls (100.2 ± 5.50 µg/ml)(p<0.05). the mean serum level of apolipoprotein b 100 (µg/ml) was significantly higher in individuals with ltb, active tb infection, tbhiv and tbmp (2489.4 ± 0.84, 2879.5 ± 1.03, 3448.3 ± 0.22 and 2880.8 ± 0.46, respectively) than in apparently healthy controls (2032.3 ± 1.11)(p<0.05). discussion bmi has been used as an indicator of malnutrition (18) or total adiposity,(19) although with many limitations. however, the mean bmi values of the entire study group were within the normal reference range. the lower mean levels of bmi observed in the test groups might be the result of depletion in body lipids and free fatty acids associated with mycobacterium tuberculosis infection.(10) these lower mean levels of bmi observed in the test groups are in agreement with the studies by yen et al.(20) and casha and scarci.(21) the significantly higher mean serum levels of apo b, b48 and b100 in m y c ob ac te ri u m t ub e rcul o s i s i nf e c t e d individuals in this study could be attributed to m y c ob ac te ri u m t u be rc ulo s i s i nf e c t i on . several studies have shown that apo b may be a better predictor of cardiovascular disease risk than ldl-c.(6,9,12) apolipoprotein b is an important component of most atherogenic lipoprotein particles.(11) the apolipoproteins were higher in the active tb and co-morbidity groups than in the controls. apolipoprotein b containing lipoproteins are the ones that are most likely to enter the wall of the arteries. they are capable of trafficking cholesterol into the artery table 1 sociodemographic characteristics parameters of individuals with ltb, active tb infections, hiv&tb, tb & mp and apparently healthy controls (ahc) a-bmeans in a row without a common superscript letter differ (p<0.05), as analyzed by one-way anova. ahc: apparently healthy controls, ltb : latent tuberculosis; tbhiv: tuberculosis and hiv co-infection, tbmp: tuberculosis and malaria parasite co-infection. results are expressed as mean ± sd and are statistically significant at p<0.05 table 2. serum levels of apolipoprotein b, apolipoprotein b48 and b100 among healthy controls, ltb, active tb, tbhiv and tbmp individuals a-b-c means in a row without a common superscript letter differ (p<0.05), as analyzed by one-way anova ahc: apparently healthy controls, ltb : latent tuberculosis, tb+hiv+: tuberculosis and hiv co-infection, tb+mp+: tuberculosis and malaria parasite co-infection. results are expressed as mean ± sd and are statistically significant at p<0.05 ahc (n=105) ltb (n=26) tb (n=82) tb hiv (n=26) tb mp (12) p value apo b (µg/ml) 2033.2 ± 1.10a 2493.7 ± 0.83b 2879.3 ± 1.03c 2759.6 ± 0.37c 2656 ± 0.462c <0.001 apo b 48 (µg/ml) 100.2 ± 5.50a 106.3 ± 1.10a 364.4 ± 8.5b 358.7 ± 1.98b 386.4 ±2 .36b <0.001 apo b 100 (µg/ml) 2032.3 ± 1.11a 2489.4 ± 0.84b 2879.5 ± 1.03c 3448.3 ± 0.22c 2880.8 ± 0.46c <0.001 ahc (n=105) ltb (n=26) tb (n=82) tb hiv (n=26) tb mp (n=12) p value age (yrs) 35.61 ± 9.60 38.31 ± 10.83 36.44 ± 14.20 34.61 ± 11.12 39.33 ± 7.43 0.315 bmi (kg/m2) 29.19 ± 7.22 a 25.57 ± 6.53 b 24.11 ± 3.84b 22.48 ± 2.38b 24.21 ± 3.85b 0.001 50 ihim, meludu, onyenekwe, et al serum apolipoprotein b increased among tuberculosis wall, and if present in increased numbers they ma y b e t he ma i n i n it i a t i n g f a c to r i n atherosclerosis. retention of apo b containing lipoprotein particles within the arterial wall is an essential part of the process. the measurements of apo b represent the total burden of the main l i po p r ot ei n pa r t ic l e s i n vo lve d i n t he atherosclerotic process.(9) apolipoprotein b occurs in two main forms, apob48 and apob100. apob48 is synthesized mainly by the small i nt e st i n e . ( 9 ,1 2 ) a n d is p r i ma r i l y f o un d i n chylomicrons.(13) apo b100 is the largest of the apo b group of proteins, consisting of 4563 amino acids. apolipoprotein b100 is the apo-lipoprotein found in lipoproteins synthesized by the liver and it is found in chylomicrons, vldl, idl, ldl and lp (a) particles.(7) all these particles are atherogenic. each of these particles contains a single apo b molecule. therefore, from the viewpoint of atherosclerosis and cardiovascular risk, apob100 is the important one.(8,18,19) furthermore, the mean serum levels of apo lipoprotein b, apo lipoprotein b48 and b100 were significantly higher in individuals with tbhiv and tbmp coinfections. hence, they might b e a t a h igh e r r i s k of p r e di s po s i ti o n t o cardiovascular disease.(9,12) this finding is in line with previous studies,(8,20) and confirmed that high levels of apo b are indicative of a higher risk to cardiovascular disease in tuberculosis and concluded that concentrations of apo b are superior indicators of vascular/heart disease and cvd risk prediction than standard lipid profile. lack of prior awareness of the participants, on t he i mpor ta nc e o f re se ar c h wa s a ma j or constraint in carrying out this study. monitoring serum apolipoprotein b levels may help reduce the occurrence of cardiovascular disease in patients with tuberculosis. further research is necessary using a longitudinal follow up design t o a s s e s s t he e f f e c t o f m y c ob ac te ri u m tuberculosis (mtb) infection on serum levels of apo b, apo b48, apo b100, atherogenic index and coronary risk index in individuals with mtb infection before, during and after treatment. conclusion it was observed in this research that higher mean serum levels of apo b, apo b48 and apo b100 in tuberculosis patients might predispose them to cardiovascular disease. conflicts of interest the authors declare that they have no conflicts of interest. contributors aci and scm conceived and designed the research proposal. cco and aea performed sample collection, experiments and data analysis. aci and cna contributed to the final version of the manuscript. all authors have read and approved the final manuscript. acknowledgements the authors would like to pay their most profound gratitude to the tertiary education trust fund (tetfund), niger ia for the r e s e a r c h gr a n t s e x t e nd e d f o r t he accomplishment of this research activity. the management and staff of nnamdi azikiwe university teaching hospital, nnewi, and reene m e d ic a l di a gn os t i c la bo r a t or y, awa d a , anambra state, for the laboratory analyses of all biochemical parameters, staff of tbdot laboratory nauth nnewi, anambra state. references 1. huaman ma, henson d, ticona e, sterling tr, garvy ba. tuberculosis and cardiovascular disease: linking the epidemics. trop dis travel med vaccines 2015;1:10. doi: 10.1186/s40794-0150014-5. 2. huaman ma, eduardo t, gustavo m, et al. the relationship between 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doi: 10.21037/jtd.2017.03.47. oktavianus 4 *department of pharmacology, faculty of medicine, indonesian islamic university **professional study program, faculty of medicine, indonesian islamic university correspondence dr. isnatin miladiyah, m.kes. department of pharmacology, faculty of medicine, indonesian islamic university jl. kaliurang km 14.5 sleman, jogjakarta 55564 phone: +62274-898444 ext. 2002 fax: +62274-898444 ext. 2007 email: isnatin@fk.uii.ac.id univ med 2012;31:4-11 abstract universa medicina january-april, 2012january-april, 2012january-april, 2012january-april, 2012january-april, 2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background wound healing is a normal biological process in response to skin injury. anredera cordifolia (ten.) steenis is used traditionally to treat various diseases, including skin disease, hypertension, inflammation and gout. the aim of this study was to evaluate the wound healing activity of the leaves of binahong or anredera cordifolia (ten.) steenis in guinea pigs. methods thirty guinea pigs (1.5-2 kg, 3-4 months old) were randomly divided into 5 groups. group i was given distilled water (negative control), group ii was treated with povidone iodine 10% (positive control), while groups iii-v were treated with ethanolic extract of binahong leaves at concentrations of 10%, 20%, and 40%, respectively. before treatment, a 2 cm long excision wound was made on each animal. all interventions were given by the topical route, twice daily for 15 days. at the end of 15th day, the wound lengths in each group were measured and compared to baseline wound lengths. data were analyzed with one-way anova to compare wound healing activity between groups. results this study showed that groups treated with ethanolic extract of binahong leaves at concentrations of 20% and 40% experienced better wound healing activity than negative and positive controls. there were significant differences (p=0.000) between treatments and negative and positive controls. conclusions this research has succesfully show significance of the binahong leaf extract has a potential for wound healing in guinea pig. keywords : anredera cordifolia (ten.) steenis, wound healing, guinea pigs ethanolic extract of anredera cordifolia (ten.) steenis leaves improved wound healing in guinea pigs isnatin miladiyah* and bayu rizky prabowo** 5 univ med vol. 31 no.1 ekstrak etanol daun anredera cordifolia (ten.) steenis (basellaceae) memperbaiki penyembuhan luka pada marmut latar belakang penyembuhan luka merupakan suatu proses normal sebagai respon adanya cidera pada jaringan kulit. secara tradisional anredera cordifolia (ten.) steenis sering digunakan untuk mengobati berbagai jenis penyakit, termasuk penyakit kulis, hipertensi, peradangan dan gout. penelitian ini bertujuan untuk menilai aktivitas penyembuhan luka daun binahong atau anredera cordifolia (ten.) steenis terhadap luka eksisi buatan pada marmut. metode sebanyak 30 ekor marmut (umur 3-4 bulan, berat 1,5-2 kg dikelompokkan secara acak menjadi 5 kelompok perlakuan yaitu kelompok i diberi olesan akuades (kontrol negatif), kelompok ii diberi povidone iodine 10% (kontrol positif), kelompok iii-v diberi ekstrak etanol daun binahong dengan konsentrasi masing-masing 10%, 20%, dan 40%. pada seluruh hewan uji dibuat luka eksisi sepanjang 2 cm, dan dioleskan perlakuan sesuai kelompoknya, dua kali sehari selama 15 hari. pada akhir hari ke-15, diukur penyembuhan luka dari tiap kelompok (dalam persen, dibandingkan dengan luka awal). data persen penyembuhan luka dianalisis dengan anova satu jalan untuk melihat adanya perbedaan antar kelompok perlakuan. hasil studi menunjukkan bahwa ekstrak etanol daun binahong mampu menyembuhkan luka mulai konsentrasi 20%. semakin tinggi konsentrasi ekstrak, efek penyembuhan luka semakin besar. uji statistik menunjukkan bahwa pada kelompok ekstrak etanol konsentrasi 20% dan 40%, terdapat perbedaan signifikan dengan kontrol negatif (akuades) (p=0,000), maupun kontrol positif (p=0,000). kesimpulan ekstrak etanol daun binahong mampu menyembuhkan luka lebih baik daripada povidone iodine. daun binahong berpotensi pada penyembuhan luka. kata kunci : anredera cordifolia (ten.) steenis., penyembuhan luka, marmut introduction a wound comprises damage to anatomic structures and functions of the skin, thus resulting in loss of epithelial continuity with or without loss of surrounding connective tissue.(1) the effects of a wound may be in the form of p a r t i a l o t c o m p l e t e o rg a n d y s f u n c t i o n , sympathetic activation, bleeding, bacterial contamination and cell death.(2) the wound healing process is a normal biological response to injury, but the underlying biological factors are extremely complex. it is a dynamic process, where the factors involved are regeneration and repair of tissue damage.(3) briefly, the wound healing process may be divided into 4 phases, i.e. (i) hemostasis, (ii) inflammation, (iii) proliferation, and (iv) remodelling .(4) the remodelling phase is associated with the tensile or breaking strength of the skin, where 1 week after wounding the tensile strength is 3% of that of normal dermis, increasing within 3 weeks to 20%, and attaining after 3 months 80% of the breaking strength of normal dermis.(5) the factors playing a role in wound healing include bacterial infection, abstrak 6 n u t r i t i o n a l d e f i c i e n c i e s , u s e o f c e r t a i n medications, obesity, movement of the wound s i t e , a n d l o c a t i o n o f t h e w o u n d . (6 ) t h e surrounding tissues also affect the rate of healing, such as aseptic conditions, disposal of dead tissue, apposition of wound edges, and r e g u l a r a p p l i c a t i o n o f d r e s s i n g s . (7 ) t h e utilization of traditional medicinal plants for wound healing is based on their antiseptic, adstringent, antiinflammatory, and antibacterial properties.(8) the plants increase the rate of w o u n d h e a l i n g b y s u p p l y i n g s u b s t a n c e s r e q u i r e d i n t h e t i s s u e r e g e n e r a t i o n a n d proliferation phases,(7) because they contain saponins, alkaloids, tannins, steroids, and glycosides.(6) among the plant parts used for wound healing, roots account for 27% and leaves 20%, while the remainder is made up of stems, seeds, whole plants, fruits, flowers, tubers, and other parts.(9) one of the plants frequently used by local communities for wound healing is the binahong plant, its scientific name being anredera cordifolia (ten.) steenis.(10,11) the plant belongs to the basellaceae family and is also known as boussingaultia gracilis var. pseudobaselloides or boussingaultia baselloides.(12) it has been used by local communities for a number of healing applications, e.g. following surgery or delivery, and for diabetes and typhoid fever. the parts of the plant used for these purposes are the roots, stems, leaves, and flowers. ( 1 0 ) pharmacological tests conducted on anredera spp. include the following: antibacterial,(13) antiobesity and antihypoglicemic,(14) cytotoxic a n d a n t i m u t a g e n i c , ( 1 5 ) a n t i v i r a l , ( 1 6 , 1 7 ) a n t i d i a b e t i c , ( 1 8 ) a n t i u l c e r, ( 1 9 ) a n d antiinflammatoy.(17,20) in vitro tests of wound healing activity is currently viewed to be inappropriate, since these tests are incapable of demonstrating the efficacy of a substance, such that animal experiments and human clinical trials are being increasingly used.(21) phytochemical analyses of binahong indicate that the leaves contain considerable a m o u n t s o f s a p o n i n s , a l k a l o i d s , a n d flavonoids.(22) thus far, there has been only one study conducted on the wound healing activity of binahong, involving hematomas in rats.(23) in the group receiving binahong there was macroscopic edema, but no infiltration of i n f l a m m a t o r y c e l l s o n h i s t o p a t h o l o g i c a l examination. the present study differs from the previous one in the plant parts used (leaves vs tubers), type of wound (excision wound vs hematoma), test animal (guinea pigs vs rats), and in the type of observation of the effects (macroscopic vs microscopic). based on several preliminary studies, including studies on the phytochemical content of binahong leaves, the present study was performed to determine whether or not an ethanolic extract of the leaves of anredera cordifolia (ten.) steenis showed wound healing activity on excision wounds in guinea pigs. methods research design this was a pure experimental controlled laboratory study to test the effect of binahong leaves on wound healing in guinea pigs. binahong leaves binahong leaves were obtained in april 2011 from the kotagede region of yogyakarta city, consisting of the fresh green leaves of p l a n t s t h a t w e r e a t l e a s t 4 m o n t h s o l d . identification of the leaves was performed at the plant taxonomy laboratory, faculty of biology, gajah mada university. preparation o f l e a f e x t r a c t w a s p e r f o m e d a t t h e pharmaceutical biological laboratory, faculty o f m a t h e m a t i c s a n d n a t u r a l s c i e n c e s , indonesian islamic university. the study was conducted at the integrated research and testing laboratory of gajah mada university. equipment and materials equipment and materials for the study w e r e o b t a i n e d f r o m t h e l a s t m e n t i o n e d laboratory, while povidone iodine for the miladiyah, prabowo activity anredera cordifolia steenis 7 univ med vol. 31 no.1 positive controls was from kimia farma. for anesthesia ketamine (ketalar®) from pfizer was used. preparation of binahong leaf ethanolic extract binahong leaf ethanolic extract was prepared by maceration as follows: binahong leaves in the amount of approximately 1.5 kg was cleansed, cut up into small pieces and dried in a drying cabinet at 38oc for 5 days, then pounded and blenderized into a dry powder. subsequently maceration was performed by putting the dry powder into an erlenmeyer flask containing 500 ml of 70% ethanol and leaving the mixture stand at room temperature for 24 hours. the maceration process was performed up to three times, after which the resulting solution (ethanolic phase) was filtered and concentrated in an evaporator at 50oc, yielding an ethanolic binahong leaf extract of 100%. experimental animals the experimental animals used were 30 adult 3-4 months old guinea pigs weighing from 1.5 to 2 kg. the animals were obtained from the integrated research and testing laboratory of gajah mada university, with the provision of being in healthy condition (active and wellformed). management and care of the animals before, during, and after the intervention was according to the standards of the integrated research and testing laboratory of gadjah mada university. the numbers of test animals were based on previous studies, where with 5 types of intervention the minimum number of animals in each group was 5.(24) the use of guinea pigs in this study was based on the similarity of their characteristics to those of humans, with regard to general physiology of their cells, tissues and organs, thus being an appropriate animal model for several human diseases.(25) t h e t e s t a n i m a l s w e r e a s s i g n e d t o intervention 5 groups, viz. negative controls (distilled water), positive controls (povidone i o d i n e 1 0 % ) , a n d 3 g r o u p s t r e a t e d w i t h b i n a h o n g l e a f e t h a n o l i c e x t r a c t a t c o n c e n t r a t i o n s o f 1 0 % , 2 0 % , a n d 4 0 % , respectively (by dilution of the full extract with distilled water). the treatment consisted of topical application of the agent on the wounds, twice daily (morning and evening) for 15 days. determination of binahong leaf ethanolic extract concentrations t h e d e t e r m i n a t i o n o f b i n a h o n g l e a f ethanolic extract concentrations was based on p r e l i m i n a r y t e s t s , w h e r e a m o n g t h e 5 concentrations used (5%, 10%, 20%, 40%, and 80%), all concentrations from 10% upwards had an effect. preparation of excision wounds b e f o r e e x c i s i o n , t h e a n i m a l s w e r e anesthesized with ketalar. the fur in the dorsal region was shaved, then a 2-cm long excision wound was made. during the study, the wounds were left open and exposed to the environment without topical or systemic antibiotic cover. wound healing was recorded as the percentage of wound closure, calculated according to the formula: [(initial wound length – wound length on a given day)/initial wound length] x 100%.(26,27) statistical analysis the percentages of wound healing on day 15 were analyzed by one-way anova (spss for windows version 15), at significance level of p<0.05. results wound lengths were measured on days 6, 9, and 15, and converted to percentages of wound healing, as shown in table 1. it is apparent that on the last day (day 15), wound healing in the group receiving binahong leaf extract 40% had attained 100%, which was higher than in the negative control group (59.17%), positive control group (68.33%), 8 binahong leaf extract 10% group (81.67%) and binahong leaf extract 20% group (86.67%). results of the anova test indicated that these differences were significant (p=0.000), thus it may be stated that the ethanolic extract of binahong leaves was effective in wound healing. post-hoc anova showed that differences in wound healing expression were significantly differences (p=0.000) between binahong leaf extract 40% with povidone iodine and binahong leaf 10% (table 2). discussion assessment of the effect of a drug on wound healing may be performed by means of various models. one of the models commonly used in experimental studies is the excison wound.(6,26-31) this model is used to obtain information on duration of wound contraction and wound closure after administration of an extract in comparison to controls.(26-27) the use of povidone iodine as positive control was based on previous studies,(31-35) from which it was assumed that the wound healing effect was due to the property of iodine as an effective broadspectrum antimicrobial.(36) the binahong leaf ethanolic extract groups excellent results were found, where increasing the extract concentration led to better wound healing, as compared to the povidone iodine and the distilled water groups. therefore it may be stated that binahong leaf ethanolic extract was effective in wound healing and better than povidone iodine 10%. this study also shows that wound healing i n t h e p o v i d o n e i o d i n e g r o u p g a v e n o satisfactory results, being similar to the results in the negative control group. the use of povidone iodine as a topical drug on wounds is currently subject to debate among clinicians, because iodine may be systemically absorbed and thus cause damage and even toxicity in v a r i o u s t i s s u e s a n d c e l l s , (3 7 ) i n c l u d i n g granulocytes, monocytes, and fibroblasts.(36) povidone iodine is more effective as an antiseptic,(37) and thus more appropriately used as a wound dressing, particularly for infected wounds,(38-40) and not for recent clean uninfected wounds. therefore this study supports previous views that povidone iodine is not effective in wound healing. t h i s s t u d y d e m o n s t r a t e s t h a t administration of binahong leaf ethanolic akuades povidone iodine eeb 10% eeb 20% eeb 40% distilled water 0.225 b 0.005 a 0.0 01 a 0.000 a povidone iodine 0.225 b 0.082 b 0.0 20 a 0.000 a eeb 10%* 0.005 a 0.082 b 0.504 b 0.020 a eeb 20%* 0.001 a 0.020 a 0.504 b 0.082 b eeb 40%* 0.000 a 0.000 a 0.020 a 0.082 table 2. post-hoc anova results on wound healing on day 15 *eeb = ethanolic extract of binahong leaves; asignificant difference; bnon-significant difference miladiyah, prabowo activity anredera cordifolia steenis groups day 6 day 9 day 15 p distilled water 10.83 ± 5.85 25.00 ± 8.94 59.17 ± 18.28 0.000 povidone iodine 12.50 ± 2.74 24.17 ± 6.65 68.33 ± 13.66 eeb 10%* 15.00 ± 4.47 33.33 ± 6.05 81.67 ± 4.08 eeb 20%* 20.83 ± 5.84 45.30 ± 6.65 86.67 ± 16.63 eeb 40%* 27.50 ± 5.24 50.00 ± 6.32 100.00 ± 0.00 table 1. mean percentages of wound healing on days 6, 9, and 15 *eeb = ethanolic extract of binahong leaves 9 univ med vol. 31 no.1 extract was capable of promoting wound healing to a high degree, which may be due to t h e a n t i i n f l a m m a t o r y, a n t i o x i d a n t , antibacterial, and analgesic effects of the extract.(8) these effects may be caused by the saponin, alkaloid, and flavonoid content of binahong leaf ethanolic or aqueous extract.(22) a number of previous studies showed that the presence of saponins, alkaloids, polyphenols, flavonoids, glycosides, and triterpenes in the various parts of a plant may may have a wound healing effect.(41-47) the mechanism of action of saponins in wound healing is to stimulate the production of type i collagen, which has an important role in wound closure and increases epithelialization of tissues.(42) flavonoids act by inhibiting the lipid peroxidation process(48) and are responsible for free radical scavenging,(45) thus preventing and retarding cell necrosis, and increasing vascularization at the wound site. inhibition of lipid peroxidation is believed to enhance the viability of collagen fibrils by increasing collagen fibers and vascularization, preventing c e l l u l a r d a m a g e , a n d p r o m o t i n g d n a synthesis. (48) flavonoids, glycosides, and tannins are known to act as adstringents and a n t i b a c t e r i a l s . (4 3 , 4 8 ) p o l y p h e n o l s , b e i n g compounds with antioxidant properties, also play a role in wound healing, through inhibition o f l i p i d p e r o x i d a t i o n , a s i n t h e c a s e o f flavonoids.(49) the use of antioxidants in wound healing is due to the fact that cell proliferation, suppression of inflammation, and contraction of collageneous tissues are inhibited by the presence of free radicals. it is the presence of saponins, alkaloids, and flavonoids in binahong leaves (22) that presumably plays a role in the wound healing process of the guinea pigs in the present study. however, which of these compounds is the most responsible for wound healing, has to be investigated in future studies, including testing of each of the isolated active compounds. a limitation of the present study is that it only involved macroscopic observation of the wound healing effect, without in-depth investigations into the wound healing process and the cells involved. conclusions binahong leaf ethanolic extract is effective in the healing of excision wounds in guinea pigs. povidone iodine is less effective in wound healing and should preferably not be used as a wound dressing, particularly in infected wounds. acknowledgments thanks are due to dr. farida juliantina rachmawaty, m.kes for corrections to this report and to mr. riyanto (laboratory technician at the faculty of mathematics and natural sciences, indonesian islamic university) and m r. b a y u a n d m r. wa s i n o ( l a b o r a t o r y technicians at integrated research and testing laboratory of gajah mada university) for their assistance before, during and after realization of the study. references 1. nagori bp, solanki r. role of medicinal plants in wound healing. res j med plant 2011;5: 392405. 2. sravanthi c, manthri s, srilaksmi v, ashajyothi v. wound healing herbs: a review. int j pharm technol 2010;2:603-24. 3. jagtap ns, khadabadi ss, farooqi ia, nalamwar vp, sawarkar ha. development and evaluation of herbal wound healing formulations. int j pharm tech res 2009;1:1104-8. 4. diegelmann rf, evans mc. wound healing: an overview of acute, fibrotic, and delayed healing. front biosci 2004;9:283-9. 5. monaco jl, lawrence t. acute wound healing: an overview. clin plast surg 2003;30:1-2. 6. esimone co, ibezim ec, chah kf. the wound healing effect of herbal ointments formulated with napoleona imperialis. j pharmaceutic allied sci 2005;3:294-9. 7. jaiswal s, singh sv, singh b, singh hn. plants used for tissue healing of animals. nat prod radiance 2004;3:284-92. 10 8. gupta n, jain uk. prominent wound healing properties of indigenous medicines. j nat pharmaceutic 2010;1:2-10. 9. chopda mz, mahajan rt. wound healing plants of jalgaon district of maharashtra state india. ethnobotanical leaflets 2009;13:1-32. 10. manoi f. binahong (anredera cordifolia) sebagai obat. warta penelitian pengembangan tanaman obat 2009;15:3-6. 11. barboza ge, cantero jj, nunez, c, pacciaroni a, espinar la. medicinal plants: a general review and a phytochemical and ethnopharmacological screening of the native argentine flora. kurtziana 2009;34:7-365. 12. chuang mt, lin ys, hou wc. ancordin, the major rhizome protein of madeira-vine, with trypsin inhibitory and stimulatory activities in nitric oxide productions. peptides 2007;28:13116. 13. tshikalange te, meyer jjm, hussein aa. antimicrobial activity, toxicity, and the isolation of bioactive compounds from plants used to treat sexually transmitted diseases. j ethnopharmacol 2005;96:515-9. 14. wang l, bang cy, choung sy. anti-obesity and hypolipidemic effects of boussingaultia gracilis miers var pseudobaselloides bailey in obese rats. j med food 2011;14:17-25. 15. yen gc, chen hy, peng hh. evaluation of the citotoxicity, mutagenicity, and antimutagenicity of emerging edible plants. food chem toxicol 2001;39:1045-53. 16. chiang lc, cheng hy, liu mc, chiang w, lin cc. in vitro anti-herpes simplex viruses and antiadenovirus activity of twelve traditionally used medicinal plants in taiwan. biol pharmacetic bull 2003;26:1600-4. 17. li tsc. taiwanese native medicinal plants, phytopharmacology and therapeutic values raton fl. boca raton, florida: taylor and francis group;2006. 18. anh mj, kim jw. identification and qualification of steroidal saponins in polygonatum species by hplc/esi/ms. arch pharm res 2005;28:592-7. 19. lin wc, wu sc, lin yh, hou wc. prevention of ethanol-induced gastric lesions in rats by ethanolic extracts of boussingaultia gracilis. clin pharm j 1997;56:89-93. 20. lin cc, sung tc, yen mh. the antiinflammatory and liver protective effects of boussingaultia gracilis var pseudobaselloides extracts in rats. phytother res 1994;8:201-7. 21. krishnan p. the scientific study of herbal wound healing therapies: current state of play. curr anaesth crit care 2006;17:21–7. 22. cloridina h, nugrohowati n. identifikasi dan isolasi senyawa kimia ekstrak air dan etanol daun anredera cordifolia (ten.) steenis dengan kromatografi lapis tipis. laporan penelitian internal; fakultas kedokteran universitas islam indonesia. 2009. 23. sumartiningsih s. the effect of binahong to hematoma. world acad sci eng technol 2011; 78:743-5. 24. festing mfw, altman dg. guidelines for the design and statistical analysis of experiments using laboratory animals. ilar j 2002;43:24458. 25. foundation for biomedical research. the essential need for animals in medical research. available at: http://www.fbresearch.org. accessed august 28,2011. 26. subhasini s, arunachalam kd. investigation on the phytochemical activities and wound healing properties of adhatoda vasica leave in swiss albino mice. african j plant sci 2011;5:133-45. 27. arunachalam kd, subhasini s. preliminary phytochemical investigation and wound healing activity of myristica andamanica leaves ini swiss albino mice. j med plants res 2011;5:1095-106. 28. suntar i, koca u, keles h, akkol ek. wound healing activity of rubus sanctus schreber (rosaceae): preclinical study in animal models. evidence-based complement alt med 2011. article id 816156, 6 pages. doi:10.1093/ecam/ nep137. 29. karodi r, jadhav m, rub r, bafna a. evaluation of the wound healing activity of a crude extract of rubia cordifolia l (indian madder) in mice. int j appl res nat prod 2009;2:12-8. 30. dalazen p, molon a, biavatti mw, kreuger mro. effects of the topical application of the extract of vernonia scorpioides on excisional wounds in mice. brazilian j pharmacog 2005;15: 82-7. 31. dhumal sv, kulkarni sr. antibacterial and wound healing activity of roots of sesamum indicum. indian drugs 2007;44:937-44. 32. beukelman cj, van den berg ajj, hoekstra mj, uhl r, reimer k, mueller s. anti-inflammatory properties of liposomal hydrogel with povidoneiodine (repithel®) for wound healing in vitro. burns 2008;34:845-55. 33. anand a. comparative efficacy and tolerability of oxum against povidone iodine topical application in post-caesarian sectioun wound management. indian med gazette 2007;498505. 34. roy k, shivakumar h, sarkar s. wound healing potential of leaf extracts of ficus religiosa on miladiyah, prabowo activity anredera cordifolia steenis 11 univ med vol. 31 no.1 wistar albino strain rats. int j pharm tech res 2009;1:506-8. 35. james o, friday et. phytochemical composition, activity, and wound healing potential of euphorbia heterophylla (euphorbiaceae) leaf extract. int j pharm biomed res 2010;1:54-63. 36. boothmann s. the use of iodine in wound therapy;2009. available at: http://www. systagenix.de/cms/uploads/iodine_white_ paper.pdf. accessed august 6, 2011. 37. angel de, morey p, storer jg, mwipatayi bp. the great debate over iodine in wound care continues: a review of the literature. wound pract res 2008;16:6-21. 38. shukrimi a, sulaiman ar, halim ay, azril a. a comparative study between honey and povidone iodine as dressing solution for wagner type ii diabetic foot ulcers. med j malaysia 2008; 63:44-6. 39. yakubu as, abubakar aa, salihu md, jibril a, isah i. comparative analysis of chlorhexidine gluconate, povidone iodine, and chloroxylenol as scrubbing solution. british j pharmacol toxicol 2010;1:93-5. 40. boateng js, matthews kr, stevens hne, eccleston gm. wound healing dressings and drug delivery systems: a review. j pharmaceutic sci 2008;97:2892-923. 41. ekpo m, mbagwu h, jackson c, eno m. antimicrobial and wound healing activities of centroma pubescens (leguminosae). jpcs 2011;1:1-6. 42. mackay d, miller al. nutritional support for wound healing. alt med rev 2003;8:359-77. 43. vinothapooshan g, sundar k. wound healing effect of various extracts of adhatoda vasica. int j pharm bio sci 2010;1:530-6. 44. sasidharan s, nilawaty r, xavier r, latha ly, amala r. wound healing potential of elais guinensis jacq leaves in an infected albino rat model. molecules 2010;15:3186-99. 45. anitha s, suresh gs, ramaiah m, vaidya vp. extraction, isolation, and wound healing activity of flavonoid from coscinium fenestratum. res j pharmaceutic biol chem sci 2011;2:1090-5. 46. ayyanar m, ignacimuthu s. herbal medicines for wound healing among tribal people in southern india: ethnobotanical and scientific evidences. int j appl res nat prod 2009;2:2942. 47. nayak bs, raju ss, ramsubhag a. investigation of wound healing activity of lantana camara l. in sprague dawley rats using a burn wound model. int j appl res nat prod 2008;1:15-9. 48. nayak s, nalabothu p, sandiford s, bhogadi v, adogwa a. evaluation of wound healing activity of allamanda cathartica l. and laurus nobilis l. extract on rats. bmc complement alt med 2006;6:12. doi:10.1186/1472-6882-6-12. 49. neves ala, komesu mc, di matteo ma. effects of green tea on wound healing. int j morphol 2010;28:905-10. c:\users\user\desktop\vol. 41 n 1 universa medicina january-april, 2022 vol.41no.1 tale of the double pandemics, covid-19 and obesity: a never-ending story roslida abdul hamid* *department of biomedical science, faculty of medicine and health sciences, universiti putra malaysia, 43400 serdang, selangor, malaysia it has been almost two years since we have been shocked by the covid-19 pandemic, which initially started in wuhan, china, on december 31, 2019. it was not long before the world health organization (who) finally declared it as a global pandemic on march 11, 2020, after announcing it as a public health emergency of international concern (pheic) on january 30, 2020. since then, we can see the pandemic accelerated in most countries all over the world. globally, as of 4 december 2021, there have been 270,031,622 confirmed cases of covid-19, including 5,310,502 deaths.(1) due to the covid-19 pandemic, a number of non-pharmaceutical interventions colloquially known as lockdowns have been implemented in numerous countries and territories around the world. the results of the pandemic lockdown have instigated another major concern, i.e. obesity. overweight and obesity are defined as excessive or abnormal fat accumulation that may impair health. based on the who classification for adults of both genders and all ages, overweight is defined as a body mass index (bmi) over 25 kg/ m2 and obese as a bmi over 30 kg/m2.(2) covid-19 has ubiquitously proven to be a major challenge for people struggling with obesity as the unfavorable effects of excess body weight in the course of sars-cov-2 infections have been mainly attribut ed to the metabolic perturbations and chronic inflammation of the adipose tissue leading to impaired immunity (blunted macrophage activation, impaired b and t lymphocyte responses) and more severe clinical outcomes.(3) to make the matters worse, the covid-19 pandemic has taken place at a time when there are around 2 billion overweight adults, of whom 650 million are considered to be affected by obesity (bmi ?30 kg/m²). that equates to respectively 39% and 40% of adult men and women aged 18 or over who are overweight, with 13% obese.(4) to top it up, the lockdowns do force us to stay at home to avoid crowds by practising social distancing and locking ourselves at home with limited outdoor activities, which ultimately leads to another undeniable pandemic, obesity. the covid-19 pa ndemic with its lockdowns has caused many to put on pounds. at the same time, obesity has emerged as a major risk for severe disease and death from the virus. in many countries, the interventions required to combat covid-19 have resulted in severe socioeconomic crises.(5) given the well-described relationship between socio-economic status and risk of obesity, a widening societal inequality propelled by the political interventions against covid-19 might translate into an increase in obesity and metabolic diseases in groups with a lower socio-economic status.(6) people of lower socio-economic status with limited income and resources prefer cheap and readily available and palatable foods, which are highly processed and promote high caloric intake beyond energy needs.(7) *email: roslida@upm.edu.my orcid id: 0000-0001-9239-6775 2 universa medicina additionally, we can also closely associate the global pandemic with psychological stress, which is worsened by the socio-economic condition, which thus triggers more anxiety in many individuals who have been cooped up at home, with no social interaction, while embracing loneliness. the deterioration of psychosocial health leads to a negative effect of food-related behavior, such as food hoarding, impulsive eating behavior, and emotional eating. (8) not to mention that psychosocial stress may consequently elevate the energy intake as well as the risk of developing obesity in those who have been confined at home with minimal social interaction. the elimination of social eating practices could encourage a reduction in mindful eating, which might negatively inf luenc e dietary c hoice s and promote overeating.(9) furthermore, the closure of gymnasiums and fitness centers, also the curtailment of various sports in practicing physical distancing makes the maintenance of active lifestyles more troublesome. the disproportionate impact of covid-19 on patients with obesity is now well established. (10-11) patients with obesity and morbid obesity have a known increased susceptibility to viral infections.(5) obesity has been reported to lead to severe forms of covid-19, including acute respiratory distress syndrome (ards), thus is strongly associated with a greater risk of advanced levels of treatment such as admission to intensive or critical care and invasive mechanical ventilation, and with death.(12) the occurrence of obesity may also initiate other metabolic diseases by increasing the risks of developing obesity-linked comorbidities such as diabetes mellitus, and cardiovascular and cerebrovascular diseases.(13) the prevalence of obesity is particularly high in some countries such as the usa where the total number of confirmed covid-19 cases and deaths is one of the highest in the world.(1) people with bmi over 30 in the usa were reported to have increased risks of being admitted to the hospitals due to covid-19 by 113%, of being admitted to intensive care by 74%, and of dying by 48%.(14) similar numbers of mortality were also reported by public health england, with the risk of death from covid-19 increasing by 90% in people with a bmi over 40.(15) sadly, this can also occur in the younger populations who suffer from obesity or overweight, irrespective of their ages. the world obesity federation recently showed further trends that the death rates from covid-19 have been ten times higher in countries where more than half of the population is obese.(16) a total of 191 countries with a complete univariate dataset (168 countr ies with a complete multivariate dataset) found a significant and positive correlation between obesity prevalence and covid-19 case rate.(17) these worrying developments have brought much-needed attention to the world's second pandemic, obesity, especially during the covid19 era. therefore, understanding the mechanisms underlying the link between obesity and disease severity as a result of sars-cov-2 infection is crucial for the development of new therapeutic interventions and preventive measures in this highrisk group. more efforts should be made by the affected individuals who are suffering from obesity, in par ticular, a nd the respective governments, in general, to overcome this "bulky" pandemic. to date, a total of 7,408,870,760 vaccine doses have been administered (1) but there has been no sign of the covid-19 disappearing from this world yet. although fewer cases have been reported after vaccination, but still, we will never know what is waiting for us in the year 2022 and beyond. the only thing that we can do is to live in this new "normal" as we did before covid19, familiarizing ourselves with more incoming lockdowns and staying healthy by restricting ourselves from becoming overweight. with that, we may be able to fight the pandemic, by remembering to always wear a mask when outside, keep a safe distance, clean our hands, and get vaccinated to protect ourselves and stop covid-19 as well as to reduce obesity. 3 january-april, 2022 vol.41no.1 references 1. world health organization. who coronavirus (covid-19) dashboard. geneva: world health organization;2021. 2. dalamaga m, christodoulatos gs, karampela i, vallianou n, apovian cm. understanding the coepidemic of obesity and covid-19: current evidence, comparison with previous epidemics, mechanisms, and preventive and therapeutic perspectives. curr obes rep 2021;10:214-43. doi: 10.1007/s13679-021-00436-y. 3. karampela i, chrysanthopoulou e, christodoulatos gs, dalamaga m. is there an obesity paradox in critical illness? epidemiologic and metabolic considerations. curr obes rep 2020; 9:231-44. doi:10.1007/s13679-020-00394-x. 4. world health organization. obesity and overweight. geneva: world health organization; 2021. 5. woods ja, hutchinson nt, powers sk, et al. the covid-19 pandemic and physical activity. sports med health sci 2020;2:55-64. doi:10.1016/ j.smhs.2020.05.006. 6. clemmensen c, petersen mb, sørensen tia. will the covid-19 pandemic 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obesity and mortality in critically ill covid-19 patients with respiratory failure. int j obes 2021;45:2028-37. doi: 10.1038/s41366-021-00872-9. 13. abdelaal m, le roux cw, docherty ng. morbidity and mortality associated with obesity. ann transl med 2017;5:161. doi: 10.21037/atm.2017.03.107. 14. wadman m. why obesity worsens covid-19. science 2020;369:1280-1. doi: 10.1126/science.369. 6509. 1280. 15. public health england. excess weight can increase risk of serious illness and death from covid-19. london: phe publications;2020. 16. wise j. covid-19: highest death rates seen in countries with most overweight populations. bmj 2021;372: n623. doi: 10.1136/bmj.n623. 17. foo o, hiu s, teare d, syed aa, razvi s. a global country-level analysis of the relationship between obesity and covid-19 cases and mortality. diabetes obes metab 2021;23:2697-706. doi: 10.1111/dom.14523. c:\users\user\documents\39 no 3 199 abstract universa medicina assessment of compliance to standard precautions among nurses using the health belief model noer triyanto rusli*@, muchtaruddin mansyur**, aria kekalih***, astrid sulistomo**, and retno asti werdhani*** background the application of standard precautions (sp) has become a significant challenge for healthcare workers, especially in developing countries, endangering their safety and increasing their exposure to blood-related pathogens. this study was aimed at exploring the factors related to nurses’ compliance with the practice of sp. methods a cross-sectional study was conducted on 120 nurses working at a hospital in palembang. health belief model components of the subjects were recorded through questionnaires. a 12-point observation form assessed the nurses’ sp compliance. multiple logistic regression models were used to explore factors associated with nurses’ compliance. results the results showed that 56.7% of participants had good compliance, although the five moments of hand hygiene still needed to be improved. the study revealed that the proportion of the operating room and emergency room nurses who complied with the sp was larger than the proportion of those who worked at the other wards (or=2.57, 95% ci 1.514.36). the nurses who had received training also showed a larger proportion of compliance with sp than those who had not been trained (or=2.70, 95% ci 1.07-6.79). conclusion nurses’ behavior to sp was significantly associated with the adequacy of the training and work unit. it is suggested that the practice of sp was also influenced by enabling factors and subjective norms. adequate training of nurses, provision of infection prevention equipment, and assessment of occupational exposures need to be introduced. keywords: standard precautions, compliance, health belief model, nurses original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.199-206 september-december 2020 vol.39no.3 cite this article as: rusli nt, mansyur m, kekalih a, sulistomo a, werdhani ra. assessment of compliance to standard precautions among nurses using the health belief model. univ med 2020;39:199-206. doi: 10.18051/ univmed.2020. v39.199-206. *occupational medicine specialist program, faculty of medicine, universitas indonesia **occupational medicine division, department of community medicine, universitas indonesia ***epidemiology division, department of community medicine, universitas indonesia correspondence: @noer triyanto rusli occupational medicine specialist program, faculty of medicine, universitas indonesia, jakarta, indonesia e-mail: noer@u.nus.edu orcid id : 0000-0002-8585-7438 date of first submission, may 18, 2020 date of final revised submission, december 13, 2020 date of acceptance, december 17, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 200 rusli, mansyur, kekalih, et al standard precautions and health belief model introduction standard precautions (sp) are a series of infection control practices that are used to prevent transmission of diseases that can be acquired through contact with body fluids, blood, injured skin (including rashes), and mucous membranes.(1)the components of sp include hand hygiene, injection safety, use of personal protecti ve equipmen t and envir onmental cleanliness, as well as waste management, and respiratory hygiene and cough etiquette.(2) healthcare workers (hcw) are exposed to needle stick injuries, c ontact with blood secretions, hepatitis b or c, and aids / hiv infections through occupational exposure to blood and other body fluids.(3) it is estimated that each year, 35.7 million healthcare workers and related professionals throughout the world are at risk o f c o n tr a c t i ng d is e a s e s c a us e d b y microorganisms in the bloodstream through percutaneous contact.(4) healthcare workers, most specifically nurses, pose the greatest risk of cross-infection among patients and fellow healthcare workers, because of their high visibility and their direct interaction with patients while performing their nursing activities.(5,6) they are at additional risk for a cquiring bloodborne pathogens (bbp) when compared to any other occupational group.(7) although occupational hiv and hepatitis seroconversion is relatively rare, the risks and associated costs of a blood exposure are serious and real. these costs include initial and follow-up treatment of the exposed health care personnel, fear and anxiety about the possible consequences of an exposure, drug toxicities and absence from work.(8) the use of personal protective equipment (ppe) such as gloves and sterile surgical gowns, and sterile equipment, hygiene practices such as washing hands with antiseptic, and the instrument safe waste disposal procedures as descr ibed in the sp guidelines can keep healthcare workers safe from infections caused by blood.(9-11) standard precautions are supposed to be adopted by all healthcare workers. physician compliance with sp is extremely important.(12) in many studies, compliance with sp among healthcare professionals was reported to be inadequate with regard to eye protection, avoidance of needle recapping, glove use when required, washing hands before and after patient contact, use of face masks, and avoidance of a used needle that is disassembled from a syringe and the implementation of precautions for all pa tients.(13) the low adhesion to standard precautions is linked to individual aspects of wo r ke r s , e mp lo ye r s a nd e du c a t i on a l institutions.(14) a study in a tertiary referral center i n no r th -we st er n niger i a fou nd ga ps i n identification of knowledge and practice of infection control among doctors and nurses in the study; hence, it will be beneficial for all hcw to rec e ive f or mal and peri odic r ef resh er trainings.(15). one study in jamaica showed that compliance with sp in the operating department is low (17%).(16) the study conducted by aung (1 7) in m ya n ma r c o n c l ud e d t ha t th e r e wa s inconsistency in some aspects of compliance with sp among myanmar nurses. among health belief model variables, perceived susceptibility, benefits, and perceived barriers were statistically significant for the relation to compliance with sp except perceived seriousness. the health belief model is a most commonly used model to explain and understand the factors that influence someone’s compliance, which may consequently contribute to the adoption of certain behaviors. the difference of the present study with previous studies is found in the fact that nurses’ compliance with sp was objectively assessed by a direct observational approach by an observer. we therefore aimed at exploring the factors related to nurses’ compliance with the practice of sp at a hospital in palembang based on the health belief model. 201 methods research design a cross-sectional study was conducted in a hospital at palembang, indonesia, from september to december 2019. research subjects sample size was determined by total sampling.the study population were all nurses who were on duty in the isolation room, intensive care unit (icu), emergency department, and operating room of the hospital at the time of the study. those who were off-duty were excluded. other medical and nonmedical personnel were also excluded. study participants were recruited by convenience sampling. a total of 120 eligible nurses were included in this study. data collection this study used a questionnaire adapted from aung(18) with content validity (index range 0.306 – 0.756) and reliability (cronbach α range 0.671 – 0.807).the questionnaire was an adaptation of the original questionnaire from a study in myanmar, because it has the similar aim of explaining the predictors of compliance with sp using the health belief model. the following i nf or ma ti o n wa s re c o r d e d : s ub j e c t ’s demographies, hbm components, age, gender, education, nurse group, work unit, infection control training, history of blood exposure, history of needle stick injury, history of hepatitis b vaccination, and history of post-exposure prophylaxis. instruments the he alth belief model consists of perceived threats, perceived benefits, and perceived barriers. the scores of perceived threats (8 items), perceived benefits (8 items), and perceived barriers (8 items) were rated on a likert scale (1=strongly disagree, 2=disagree, 3=agree, 4=strongly agree). the total number of items was 24. in determining the cut-off score for the hbm component, considering that the number of question items is eight and the respondent answers a score of 3 or 4 (agree/ strongly agree) for the good hbm component, the total score is 24. for this reason, the good hbm component was defined as a total score of 24 and poor hbm component as a total score of <24. these categories were determined by mutual agreement between the researchers prior to the start of the study. nu r s e s ’ c o mpl i an c e t o wa r d s t he implementation of sp was measured by using a 12-point observation form. the observer was the head of the work unit of the 120 subjects from the emergency department, intensive care unit (icu), isolation room, and operating room. the observation was made once without the subjects knowing that they were being observed. the categories for compliance with sp were: poor = <12, good = 12 (if participants comply with all 12 points in the observation form). these categories were also determined by mutual agreement between the researchers prior to the start of the study. data analysis statistical analyses were performed using the statistical package for the social science (spss) program, version 20. initial bivariate analyses were done using simple logistic regression to determine the associations between potential variables and nurses’ compliance with sp. variables with p-values of <0.20 in the simple logistic regression were selected for multivariate analysis. multiple logistic regression analysis was used to calculate the odds ratio (or) of each selected independent variable on nurses’ compliance. ethical clearance ethical clearance was obtained from the faculty of medicine universitas indonesia cipto mangunkusumo national hospital (fkuirscm) research ethics committee under no. ket-453/un2.f1/etik/ppm.00.02/2019. univ med vol. 39 no.3 202 rusli, mansyur, kekalih, et al standard precautions and health belief model a b c results in this study, 120 subjects were obtained. data in this study showed that there were more females (82 or 68.3%) than males. the age of the subjects ranged from 26 years to 54 years. the education of the subjects varied with the largest proportion being found at the diploma program level (71.7%). research subjects who had attended infection control training were 80 in number (66.7%). information was also obtained that 81 subjects (67.5%) had been exposed to blood or body fluids and that 52 people (43.3%) had been exposed to a needle stick/ sharp object. detailed information on the basic characteristics of the subjects can be seen in table 1. de t e r m i na nt f a c t or s o f nur s e s ’ sp compliance table 2 shows the results of simple logistic regression and multiple logistic regression a na lysi s o f co mpl ia nc e w ith sp an d the associated independent variables. based on the multiple logistic regression analysis, this study found that work unit and infection control training had a statistical relationship with behavior after being controlled for age, gender, education, nurse group, history of blood/body fluid exposure, history of needle stick injury, history of hepatitis b vaccination, and history of receiving post-exposure prophylaxis (pep). the proportion of the operating room and emergency room nurses who complied with sp was larger than the proportion of those who worked at the other wards (or=2.57, 95% ci 1.51-4.36). the proportion of nurses who had received training also showed a larger proportion in compliance with sp than those who had not been trained (or=2.70, 95% ci 1.07-6.79). discussion in this study, the overall compliance of nurses with sp practices was 56.7%. this figure showed that the compliance was low because characteristics n (%) age (years) ≥30 <30 80 (66.7) 40 (33.3) gender male female 38 (31.7) 82 (68.3) highest education high school diploma bachelor 3 (2.5) 86 (71.7) 31 (25.8) nurses’ group clinical nurse ii clinical nurse iii clinical nurse iv 43 (35.8) 75 (62.5) 2 (1.7) work unit isolation room intensive care unit emergency department operating room 8 (6.7) 39 (32.5) 43 (35.8) 30 (25.0) total duration of working as nurse (years) ≥15 <15 59 (49.2) 61 (50.8) infection control training yes no 80 (66.7) 40 (33.3) exposure to blood/ body fluids yes no 81 (67.5) 39 (32.5) needlestick injury yes no 52 (43.3) 68 (56.7) hepatitis b vaccination yes no 73 (60.8) 47 (39.2) post exposure prophylaxis (pep n=52) yes no compliance good poor 29 (55.7) 23 (44.3) 56.7 43.3 table 1. distribution of demographic characteristics and clinical features of nurses (n=120) * there were no subjects in the group of clinical nurse (pk) i and clinical nurse (pk) ii the indonesian hospital accreditation board requires compliance of more than 85% for sp practices. this finding is almost identical with the study conducted by beyamo et al.(18) which s howe d th a t 65 % of h e a l thc a r e wo r ke r s complied with sp practices. in the present study, when each of the specific components of noncompliance with sp practices was analyzed, the p r o po r t i on of ha n d h ygi e n e c omp l ia nc e 203 univ med vol. 39 no.3 according to the 5 moments was the highest (17.5%). washing hands is one of the most important procedures in preventing nosocomial infections. according to the who, it is necessary t o wa sh h a n ds t o r e d uc e t h e nu mbe r o f nosocomial infections through the 5 moments, namely before touching a patients, before clean/ aseptic procedures, after contact with body fluids table 2. simple and multiple logistic regression of determinants associated with sp compliance *and † in the data analysis group merging was carried out; ‡ variables with p<0.20 were included in the multiple logistic regression; @significant at p<0.05 on multivariate analysis simple logistic regression multiple logistic regression independent variables crude or (95% ci) p-value adjusted or (95% ci) p-value age (years) ≥ 30 2.38 (1.09-5.17) 0.027 3.89 (0.72-20.82) 0.113 < 30 1 gender male 1.26 (0.58-2.76) 0.561 female 1 education bachelor 1 0.055 0.66 (0.18-2.38) 0.657 diploma iii* 0.45 (0.19-1.03) high school* nurses’ classification clinical nurse iv* clinical nurse iii* clinical nurse ii 1.64 1 (0.77-3.49) 0.196 0.52 (1.22-2.22) 0.377 work unit operating room* emergency department* 9.61 (4.10-22.5) <0.001@ 2.57 (1.51-4.36) <0.001@ intensive care unit† 1 isolation room† work duration (years) ≥ 15 2.15 (1.03-4.49) 0.040 1.16 (0.34-4.01) 0.817 < 15 1 infection control training yes 2.79 (1.28-6.09) 0.009‡ 2.70 (1.07-6.79) <0.001@ no 1 exposure to blood/ body fluids yes 1 0.723 no 0.87 (0.40-1.89) needle stick injury yes 1.64 (0.78-3.43) 0.189 1.46 (0.52-4.07) 0.469 no 1 hepatitis b vaccination yes 2.24 (1.06-4.73) 0.034 1.56 (0.58-4.20) 0.381 no post exposure prophylaxis (n=52) yes 1.99 (0.82-4.84) 0.163 1.66 (0.47-5.92) 0.432 no 1 perceived treats yes 1.23 (0.48-3.18) 0.663 no 1 perceived benefits yes 1 no 0.93 (0.45-1.91) 0.853 perceived barriers yes 1 no 0.75 (0.32-1.76) 0.50 204 rusli, mansyur, kekalih, et al standard precautions and health belief model exposure/risk, after touching a patients and after touching patient surroundings.(19) the finding in the present study seems to reflect the indonesian situation which is that hand hygiene has seldom been adopted and encouraged in indonesia. logistic regression analysis was used to identify determinants of compliance with sp. this study found that nurse working unit and previous inf ect ion control tra ini ng a re asso ciat ed significantly with and are determinants of the compliance with sp. this finding is consistent with the study conducted by luo et al.(20) in terms of nurse working unit, the compliance of nurses in the medical departments was lower than that in the surgical departments. this d if f e r e nc e w a s f o u nd to be s t at i st i c a l l y significant, and is probably the result of the greater numbers of chronic internal medicine and elderly patients in the medical departments. also, there is no obvious presence of blood in the medical de partment, which may result in protection being neglected. these results indicate that the administration departments should focus on comprehensive monitoring, especially the monitoring of hospital infections in primary level hospitals. training is also factor that continues to play an important role in the compliance with sp. motaarefi et al.(21) stated that training was the most important factor related to the prevention of needle sticking behavior among nurses. nurses who did not participate in any training session regarding the prevention of needle stick injury in their workplaces faced a higher risk of suffering from these injuries compared to those who participated in several types of training. the suboptima l compliance with sp hightlights the need for interventions to enhance the occupational safety of nurses. to improve compliance of nurses with sp, we suggest to also include other factors, such as enabling factors and subjective norms. enabling factors can be in the form of availability and affordability of facilities such as safety boxes and personal protective equipment (ppe), while subjective norms can be in the form of norms that apply in both work units that encourage subjects to conduct sp. the study of balozi et al.(22) in tanzania showed that subjective norms had a positive influence and a significant mediating effect on knowledge sharing behavior among healthcare workers. their study showed that the higher the subjective norms, the higher the knowledge sharing behavior among healthcare workers. the main strength of the current study is the use of a direct observational approach where nurses are observed objectively by an observer to deter mine their sp complia nce. some limitat ions sho uld be a ckno wled ged and considered. first, the study was of crosss e c t i on a l de s i gn t ha t do e s not a l l o w a n examination of the temporal nature of the association. second, the use of self-filled questionnaires may cause self-reported bias, while observation bias may also have occurred in this study. however, this limitation has been taken into consideration and overcome. the significant implications of this study were for nursing practice and education because it can explore the factors related to nurses’ compliance with the pr actice of standard practices. in addition, to the extent of our knowledge, this is the first study assessing compliance with standard practices which is available in indonesia. this can help to plan and evaluate new interventions in order to increase and ensure stringent compliance with standard practices. further research is needed to better explain the determinants of nurse compliance with standard practices in a better research design namely a cohort study. conclusions nurses’ behavior to sp was significantly correlated with the adequacy of the training and work unit. it is suggested that the practice of sp is also influenced by enabling factors and subjective norms. health authorities in the study area need to improve the training of nurses and provision of infection prevention equipment. 205 conflicts of interest all authors declare that there is no conflict of interest in this research acknowledgements we would like to thank the staff of the department of community medicine, universitas indonesia for the support for this publication. contributors ntr contributed to conceptualization and methodology. ntr and mm contributed to preparing and writing the original draft. mm, ak, as, and raw contributed to supervision. ntr contributed to review and editing. all authors have read and approved the final manuscript. references 1. sadeghi r, hashemi m, khanjani n. the impact of educational intervention based on the health belief model on observing standard precautions among emergency center nurses in sirjan, iran. health educ res 2018;33:327-35. doi: 10.1093/her/ cyy020. 2. ogoina d, pondei k, adetunji b, chima g, isichei c, gidado s. knowledge, attitude and practice of standard precautions of infection control by hospital workers in two tertiary hospitals in nigeria. j infect prev 2015;16:16-22. doi: 10.1177/ 1757177414558957. 3. musa s, peek-asa c, young t, jovanović n. needle stick injuries, sharp injuries and other occupational exposures to blood and body fluids among health care workers in a general hospital in sarajevo, bosnia and herzegovina. int j 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standard precautions and associated factors among healthcare workers in gondar university comprehensive specialized hospital, northwest ethiopia. j environ public health 2017;2017: 2050635. doi: 10.1155/2017/2050635. 14. porto js, marziale mh. reasons and consequences of low adherence to standard precautions by the nursing team. rev gaucha enferm 2016;37:e57395. doi: 10.1590/1983-1447. 2016.02.57395. 15. alice te, akhere ad, ikponwonsa o, grace e. knowledge and practice of infection control among health workers in a tertiary hospital in edo state, nigeria. dir res j health pharmacol 2013;1: 20–7. 16. mcgaw cd, tennant i, harding he, cawich so, crandon iw, walters c. healthcare workers’ 206 rusli, mansyur, kekalih, et al standard precautions and health belief model attitudes to and compliance with infection control guidelines in the operating department at the university hospital of the west indies, jamaica. int j infect control 2012;8. doi: 10.3396/ijic.v8i3. 023.12. 17. aung ss, nursalam, dewi sy. improving compliance with standard precautions among myanmar nurses using health belief model. malaysia j nurs 2016;8:21-8. 18. beyamo a, dodicho t, facha w. compliance with standard precaution practices and associated factors among health care workers in dawuro zone, south west ethiopia, cross sectional study. bmc health services res 2019;19:381.doi: 10.1186/s12913-019-4172-4. 19. world health organization. save lives – clean your hand. geneva: world health organization; 2019. 20. luo y, he gp, zhou jw, luo y. factors impacting compliance with standard precautions in nursing, china. int j infect dis 2010;e1106–14.doi: 10.1016/ j.ijid.2009.03.037. 21. mootarefi h, mahmoudi h, mohammadi e, hasanpour-dehkordi a. factors associated with needlestick injuries in health care occupations: a systematic review. j clin diagn res 2016;10. doi: 10.7860/jcdr/2016/17973.8221. 22. balozi m. mediating effects of subjective norms on the relatioship between career advancement and job characteristics and knowledge sharing behavior among tanzanian healthcare professionals. gadjah mada int j business 2018;20:187-203. doi: 10.22146/gamaijb.23740. c:\users\universa medicina\docu 207 abstract universa medicina upper extremity deep vein thrombosis in an informal porter dewi s. soemarko*and herlinah** background upper extremity deep vein thrombosis (uedvt) is a rare condition, in which job-related arm movements and repetitive, forceful or overhead arm activities have been recognized as the predisposing factor for this condition. uedvt can occur among informal porters. this report describes a case of uedvt in an informal porter due to manual lifting of heavy goods and reviews the literature for occupational reports of this condition. case description a 35-year-old male informal porter presented with marked swelling, pain, and numbness of his right arm 3 days after a prolonged episode of lifting heavy goods. a doppler ultrasound showed thrombosis in the right subclavian and axillary veins and laboratory tests found elevated d-dimer. patient was diagnosed as having axillo-subclavian thrombosis and treated with low molecular-weight heparin. one month after the last follow-up, patient returned to work with modified capacity and after 3 months of return to work, patient was able to resume his regular duties without impairment or disability. conclusion the occupation of informal porter should be considered a risk factor for upper extremity deep vein thrombosis (uedvt), thus the occupational medicine physician should be aware of this condition in the context of manual workers. detailed occupational history to aid the diagnosis and future risk assessment are needed, and education for preventing uedvt should be provided to porters. keywords: informal porter, occupational medicine physician, upper extremity deep vein thrombosis case report pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.207-211 september-december 2020 vol.39no.3 ci te t his art icle as: soemark o ds, herlinah. upper extremity deep vein thrombosis in an informal porter: a case report. univ med 2020;39:207-11. doi: 1 0 . 1 8 0 5 1 / un i vm e d . 2 0 2 0 . v3 9 . 2 0 7 2 1 1 *department of community medicine, faculty of medicine, universitas indonesia, jakarta, indonesia **occupational medicine specialist program, faculty of medicine, universitas indonesia, jakarta, indonesia correspondence: herlinah occupational medicine specialist program, faculty of medicine, universitas indonesia, jakarta, indonesia email: dr.herlina.tan81@gmail.com orcid id: 0000-0001-9774-3270 date of first submission, september 6, 2020 date of final revised submission, october 12, 2020 date of acceptance, october 14, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 208 soemarko, herlinah upper extremity deep vein thrombosis introduction lower limb deep vein thrombosis (dvt) is a common and well-described condition which has recently grown in public awareness. the risk factors for lower limb dvt arise from underlying components of virchow’s triad: venous stasis, hypercoagulability, and injury to the intima of veins.(1,2) upper extremity deep vein thrombosis (uedvt) is a rare disorder, which usually refers to thrombosis of the axillary and/or subclavian veins and may be associated with significant morbidity and complications such as pulmonary embolism.(3) the condition is subject to the same risk factors as the formation of lower limb dvt.(1) upper extremity deep vein thrombosis is caused by problems anterior to the scalene triangle in a space bordered by the bony and ligamentous structures of the costoclavicular junction, as well as the subclavius muscle and tendon. the subclavian vein travels in the proximity of the clavicle, first rib, and anterior s c a l e n e an d s u bc l a vi u s mu sc le s. up p e r extremity deep vein thrombosis is related to the compression and subsequent thrombosis of the subclavian vein due to these structures. with arm abduction, the subclavian vein is compressed at this costoclavicular space, a finding that can often be easily demonstrated, even in patients wi t h ou t e ff or t t hr ombo s i s. in a d d it i o n, hypertrophy of the anterior scalene muscle p os t e r i o rl y or t h e s u bc la viu s mus c l e anteromedially, further narrows the constrained costoclavicular space.(4,5) that uedvt may be due to narrowing of the costoclavicular junction, is demonstrated by the relatively constant finding that uedvt frequently occurs with repetitive and vigorous arm abduction or external rotation of the shoulder d ur ing athletic or oc cupa tional activities.(4) given that uedvt is associated with upper extremity activity, it seems plausible that there may be a preponderance of such c a s e s a mon g t h e wo r kin g p o pu l a t io n s, particularly blue-collar occupations demanding repetitive upper extremity activities on a daily basis throughout one’s working career.(6) in this case report we present a manual worker with uedvt associated with prolonged episodes of lifting heavy goods and review the literature on the work-relatedness with the condition. case presentation a 35-year-old male with the occupation of informal porter presented with a sudden onset of marked swelling, redness, pain, and numbness of his right arm. this occurred 3 days after the most strenuous and prolonged episode of lifting heavy goods (approximately 40 kg) on his shoulder with the weight on both his shoulder and neck, his arm abducted and flexed, his forearm flexed and the hand vertical to help hold goods up. he recalled that it was really painful for the next few days and the pain worsened as he worked, requiring forceful bilateral arm movements with some degree of shoulder elevation. there was no previous medical history of note and in particular no family history of venous thromboembolism or coagulopathy. on physical examination, the right arm was swollen with moderate enlargement compared t o t he l ef t , a nd e r yt h e ma an d ve n o us engorgement was noted. all peripheral pulses were easily palpable. the diagnosis of axillosubclavian thrombosis was made, while doppler ultrasound showed thrombosis in the right subclavian and axillary veins. laboratory tests showed elevated d-dimer. figure 1. clinical appearance of right upper extremity of the patient 209 the patient was put off work and treated with low molecular-weight heparin then warfarin anticoagulation and his signs and symptoms gradually resolved over the next 5 days. a few months later, on subsequent follow-up, he remained well and asymptomatic and repeat venous doppler ultrasound revealed complete resolution of the thrombosis. one month after the last follow-up, patient returned to work with modified capacity. after 3 months of returning to work, patient resumed his regular duties without impairment or disability. discussion upper extremity deep vein thrombosis (uedvt) is a less known and less common phenomenon than lower limb thrombosis, accounting for up to 10% of all deep vein thrombosis (dvt) cases, with an incidence of 11.6 per 10.000 population. the axillary and subclavian veins are the sites most frequently affected. secondary causes account for 80% of cases, most commonly provoked by indwelling central venous catheters.(1,4)the most common primary cause is paget-schroetter syndrome (pss), commonly known as primary effort thr ombosis, which is uedvt caused by strenuous or repetitive upper limb activity, for which it is sometimes referred to as effort-related thrombosis. primary effort thrombosis was first described in the literature by james paget in 1875 and was independently reported again by von schroetter in 1884, giving uedvt the name paget-schoetter syndrome. its incidence is 2/ 100.000 people per year in the united states, equating to approximately 3.000 to 6.000 reported cases yearly.(7,8) this condition is often referred to as effort thrombosis because 60% to 80% of patients with uedvt report repetitive and vigorous overhead upper extremity activity, such as swimming, pitching, weight lifting, or even manual or overhead labor, at the onset of symptoms.(8,9,10) repetitive strain from vigorous physical activity and compr ession of the subclavian vein from adjacent anatomic structures leads to venous injury and subsequent thrombosis. this impedes the venous return from the affected extremity leading to edema. over time, the body forms collaterals t o bypass the venous obstruction. in the chronic phase, the vein becomes fibrotic. surrounding inflammatory changes from the thrombosis lead to scar tissue formation.(5) upper extremity swelling may be present in patients with lymphatic disorders or systemic conditions such as end stage renal disease and congestive heart failure. upper extremity deep venous thrombosis can be seen with indwelling catheters as well.(5,11) a thorough history-taking and an appropriate physical examination are usually sufficient to suggest this diagnosis.(1) the gold standard for diagnosis is contrast venography, but doppler ultrasonography may be an acceptable alternative, because it is cheap and non-invasive with reported specificity of 96% and sensitivity of 97% for uedvt.(7,12) despite being a known cause of uedvt, pss is usua lly undiagnosed or misdiagnosed mainly due to lack of awareness of the syndrome.(13) in the case of this worker, he had been working in his normal capacity as a porter. this included forceful right upper extremity activities. he identifies the onset of pain as occurring while lifting heavy goods. it is likely that these physical factors were significant contributing factors to the subsequent thrombosis. the description of the index event causing his subclavian and axillary vein thrombosis is the repeated compression damage of the subclavian vein intima with subsequent fibrosis and ac tivation of the coagulation cascade by repetitive physical activity and prolonged heavy work in the arm, often in an elevated position.(14) compared with another case in a computer pr ogr ammer, uedv t also occurred in this latter worker when the upper extremity was in particular positions such as the rigid military style of sitting with the back straight and the shoulders placed posteriorly a nd inferiorly.(1) upper extremity deep vein thrombosis is a deep venous thrombosis and as such the first step univ med vol. 39 no.3 210 soemarko, herlinah upper extremity deep vein thrombosis a b c in management is to initiate anticoagulation therapy, typically via an intravenous heparin drip. duration of anticoagulation in patients with underlying hypercoagulability disorders remains unclear. in most patients, anticoagulation for 3 to 6 months following an episode of deep venous thrombosis is reasonable.(5,15) the american college of chest physicians recommends anticoagulation for a minimum of 3 months for all patients identified with uncomplicated primary uedvt.(1) in the present case, the patient was treated with oral anticoagulation for 3 months. while uedvt is a less common phenomenon, much more commonly the condition is reported amongst athletes, but there are surprisingly few repor ts of specific work-r elate d ca ses. pysklywec and cina (6) reported on a case in a millwright who had been doing manual lifting, as did be asl y et al. (3 ) in a tv c ameraman. consideration of these occupational tasks suggests that shoulder posture contributes to the condition. in particular, prolonged or forceful elevation of the arm in overhead work is commonly reported in affected workers. it is difficult to understand the relative paucity of occupational cases reported in the literature.(6) conclusion this case of uedvt is a rare upper extremity condition that may be seen in the working population. occupational medicine physicians should be aware of this condition in the context of manual workers presenting with upper extremity vascular symptoms. detailed occupational history aids diagnosis and future risk assessment. repetitive and forceful overhead shoulder activities may lead to scalene swelling or hypertrophy causing subclavian vascular compromise in predisposed workers. despite a scarcity of occupational cases reported in the literature, effort thrombosis should be recognized as an occupational c ondition in certain circumstances. besi des, employers and employees need to be educated about the risk of prolonged heavy lifting and about work practices. conflict of interest the authors declare no conflict of interest in this study. acknowledgment we would like to thank all residents of internal medicine of universitas indonesia and all the nurses of fatmawati general hospital for the opportunity to complete this case report. contributors h contributed to conceptualization, preparing and writing the original draft. ds and mim contributed to supervision. h contributed to review and editing. all authors have read and approved the final manuscript. references 1. chen aw, oraii yazdani k, candilio l. upper limb deep vein thrombosis: a case report of an increasingly common condition. j teh univ heart ctr 2018;13:73-5. 2. miao j, naik g, muddana s, et al. an uncommon case of lower limb deep vein thrombosis with multiple etiological causes. am j case rep. 2017;18:313–6. doi: 10.12659/ajcr.902391. 3. beasley r, braithwaite i, evans r. upper extremity deep vein thrombosis in a tv cameraman. occup med 2015;65:337-9. doi: 10.1093/occmed/kqu212. 4. bailey cj, illig ka. contemporary management of axillosubclavian vein thrombosis. interv cardiol 2013;5:453-63. 5. saleem t, baril dt. paget schroetter syndrome (paget von schrotter disease). treasure island (fl): stat pearls publishing; 2019. 6. pysklywec m, cina cs. work-related effort thrombosis in a millwright: a case report. am j ind med 2011;54:244–7. http://doi.wiley.com/ 10.1002/ajim.20888. 7. bullock c, johnston amd. upper extremity deep vein thrombosis in a military patient. j r army med corps 2016;162:299–301. doi:10.1136/jramc2015-000418. 8. delisa lc, hensley cp, jackson s. diagnosis of paget-schroetter syndrome/primary effort thrombosis in a recreational weight lifter. phys 211 univ med vol. 39 no.3 t her 2016;97:13–9. https://doi/10.2522/ ptj.20150692. 9. ibrahim r, dashkova i, williams m, et al. pagetschroetter syndrome in the absence of common predisposing factors: a case report. thromb j 2017;15:20. doi: 10.1186/s12959-017-0146-0. 10. keene dj. upper extremity deep vein thrombosis (paget-schroetter syndrome) after surfing: a case report. man ther 2015;20:358–60. doi: 10.1016/ j.math.2014.08.004. 11. delluc a, le mao r, tromeur c, et al. incidence of upper-extremity deep vein thrombosis in western france: a community-based study. haematologica 2019;104:e29–31. doi: 10.3324/ haematol.2018.194951. 12. sayin a, güngör h, bilgin m, ertürk ü. pagetvon schrötter syndrome: upper extremity deep vein thrombosis after heavy exercise. turk kardiyol dern ars 2012;40:354–7. http:// dx.doi.org/10.12687/phleb2256-4-2015. 13. sharma h, tiwari a. recurrent upper extremity thrombosis associated with overactivity: a case of delayed diagnosis of paget-schroetter syndrome. case rep vasc med 2017;2017: 8764903. doi: 10.1155/2017/8764903. 14. noyes am, dickey j. the arm is not the leg: pathophysiology, diagnosis, and management of upper extremity deep vein thrombosis. r i med j 2017 ;100:33-6. 15. engelberger rp, kucher n. management of deep vein thrombosis of the upper extremity. circulation 2012;126:768–73. https://doi.org/10.1161/ circulationaha.111.051276. c:\users\universa medicina\docu 171 abstract universa medicina relationship of occupational health and safety training with health literacy among employees working in the various lines of business nergiz sevinc* and burcu korkut** background health literacy is vital for people’s ability to manage health. it has been known for a long time that the importance of education in increasing health literacy is an undeniable fact. the first aim of this study was to investigate the health literacy levels of employees working in different business lines receiving service from the occupational health and safety unit. the second aim was to reveal how health literacy levels are affected according to the training duration. methods a cross-sectional study was conducted on 202 employees receiving service from the karabuk occupational health and safety unit. the employees were divided into three groups as workers, officers and others. all employees were given the questionnaire consisting of 31 questions including sociodemographic characteristics and the adult health literacy scale. a face-to-face interview was performed with each of the employees. kruskalwallis h and mann-whitney u tests were used to analyze the data. results the range of adult health literacy scale scores of the employees was 4-23. there was no significant difference in adult health literacy scale scores between age groups (p=0.38). the percentage of employees who received more than 16 hours training course was 19.3%, 20.6%, and 46.7% for workers, civil servants and other employees, respectively. adult health literacy scale scores increased in proportion to the amount of training the employees received (p=0.001). conclusion this study reveals that occupational health training has a positive effect on health literacy. keywords: health literacy, employees, various lines of business, occupational health and safety unit, adult health literacy scale original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.171-177 september-december 2020 vol.39no.3 cite this article as: sevinc n, korkut b. relationship of occupational health and safety trai ning with health literacy among employees working in the various lines of business. univ med 2020; 39:171-7 . doi : 10 .18 051/ uni vmed. 2020.v39.171-177 *department of public health, school of medicine, karabuk university, turkey **provincial health directorate, karabuk, turkey correspondence: nergiz sevinc, md karabuk university school of medicine, balhklar kayasi mevkii, demir celik campus karabuk, turkey phone: +905054793362 email: dr.nergizsevinc@gmail.com orcid id: 0000-0003-4763-1902 date of first submission, august 2, 2020 date of final revised submission, october 16, 2020 date of acceptance, october 20, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 172 sevinc, korkut occupational health and safety training introduction even though health literacy (hl) is defined in various forms by many researchers, the world health organization (who) has defined it as cognitive and social skills which determine the motivation and “ability of individuals to gain access to, understand, and use information in wa ys wh ic h p romote and main ta in go od health.”(1) developed countries started to work hard to improve hl when it was determined that low hl was associated with increased hospitalization, decreased vaccination uptake, and low participation in cancer-screening activities in the following years.(2,3) they realized that an individuals’ skills are an important part of improving hl and they increased it by using plain language in both verbal and written communication.(4,5) since hl provides a significant contribution to the health quality of all segments of society, developed and developing countries have prepared health and safety programs to provide occupational health services and increase hl. the content of these programs, which are generally offered by the occupational health and safety (ohs) units and prepared within the framework of laws, vary according to countries. there are a few studies evaluating the hl status of different occupational gr oups such as farmers, students and employees. for example, a study involving 282 workers at a factory who received the ohs training routinely showed that 13.5% had inadequate, 47.5% problematic, 30.9% sufficient, and 8.2% excellent hl scores.(6) there was also a study conducted on 421 farmers working in thailand, which reported that the farmers’ mean hl level was found to be 34.98 (sd=6.87) points over a range of 0-50 points.(7) recently, 686 university students were evaluated for hl status by gamsýzkan et al., who found that 21.1% (n=145) of the students had inadequate hl, 41.8% (n = 287) had limited hl, 30.9% (n=212) sufficient, and 6.1% (n=42) excellent hl.(8) none of these studies were c o nd u c t e d s imu l t ane o u sl y i n va r i o us occupational groups by including a large labor force in the community. the first studies on the state of hl in turkey was initiated by improving the adult health literacy scale (ahls) in 2012.(9) the difference of this study with the previous studies lies in the fact that the subjects were from various occupational groups including civil servants and employees. in the present study, the first objective was to investigate and compare the hl status of civil servants and employees receiving healthcare services from the ohs unit. the second objective was to reveal how hl levels are affected according to the training duration received from the ohs unit. methods design of the study this cross-sectional study was conducted among employees working in different business lines receiving service from the karabuk ohs unit from april to may in 2020. study subjects a total of 213 workers who received the ohs training routinely, were invited to participate in this study. participants enrolled in the study were selected based on the following criteria; (a) aged 18 to 60 years; (b) received training from ohs unit; c) had no significant, untreated chronic disease. those who did not consent or had chronic disease were excluded. after receiving information about the study, 11 refused to take part. a total of 202 workers were analysed. the profession groups involved in the study consisted of the groups of workers (hairdressers, barbers, auto industry workers, cleaning staff), civil servants (railway workers, family health center employees) and ‘others’ (dentists, x-ray technicians, medical secretaries). according to the training duration received from the ohs unit, participants were divided into three subgroups. the first subgroup consisted of 58 participants receiving 8 hours of training or less. the second subgroup consisted of 96 participants receiving 173 between 9-16 hours of training. the third subgroup consisted of 48 participants receiving more than 16 hours of training. instruments and measurements da ta we r e c ol l e c t e d th r o u gh a questionnaire containing 12 questions aimed at evaluating the socio-demographic characteristics of the employees (such as age, gender, marital status, level of education, occupation, and economic status) and the ahls. thirty-one institutions and organizations receiving service f r om k a r a b uk ohs u n it w e r e vi si t e d . occupational health and safety training provided by the workplace doctor and occupational health specialist was mainly aimed at establishing safe working and correct health behaviors in order to prevent occupational accidents and diseases. the training topics provided by ohs unit are shown in table 1. individuals were informed about t he study, and verbal cons ent was obtained. the questionnaire and scale were then applied to individuals who agreed to participate in the study. adult health literacy scale the adult health literacy scale (ahls) was developed in 2012 by sezer (9) who tested its validity and reliability. the adult health literacy scale includes health information to determine the competence of adult individuals in health literacy and drug use and one image showing the organs in the body. thirteen of the items in the scale are yes/no questions, 4 are fill-in-the blank, 4 are multiple choice, and 2 are matching questions. each question type is scored separately. in the yes/no questions, 1 point is given to those who select positive expressions and 0 points is given to those who select negative expressions. in multiple choice questions, 1 point is given to those who mark two or more correct answers, and 0 points to those who do not know at all or who mark the wrong answer together with the true. in matching-type questions, 1 point is given to more than two correct matches and 0 points were given to others. scores that can be obtained from the scale vary between 0 and 23 and the total cronbach alpha value of the scale was determined to be 0.77. the scale has no cut-off point. higher scores indicate a higher level of hl.(9) statistical analysis the data were digitally evaluated by spss software. descriptive statistics were presented as frequency, standard deviation and percentage distribution. the kolmogorov-smirnov test was used to analyze the suitability of quantitative data for normal distribution. the scores of ahls had abnormal data distribution. kruskal-wallis h and mann-whitney u tests were used to compare the ahls scores of the groups formed according to sociodemographic characteristics. a p<0.05 was considered statistically significant in all analyses. ethical clearance univ med vol. 39 no.3 8-hours course 16 -hours course >16 hours course general occupational health and safety rules, causes of work accidents and occupational diseases and risks in the workplace, safe use of work equipment, accident, injury and disease prevention principles and implementation of protection techniques, safe use of work equipment, hygiene legal rights and responsibilities of employees, establishing safe environment and systems in the workplace use of personal protective equipment, working with screened equipment, warning signs, risks arising from chemical, physical and biological substances fire incidence and fire protection, thermal comfort conditions, electricity, dangers, risks and precautions, first aid, rescue. table 1. training topics provided by ohs unit 174 sevinc, korkut occupational health and safety training a b c ethical approval was obtained from the faculty of medicine of karabuk university (2020/ 217) and administrative permission was obtained from the provincial health directorate. results of the 202 employees participating in the study, 25.2% were female, 74.8% were male, while the age range was 18 -51 years. in addition, 16.8% of the employees were at least university graduates, 54.5% were married, and 88.6% had social secur ity. there was no significant difference between male and female participants in terms of marital status, number of children, economic income, and educational status (p>0.05). the characteristics of the employees are shown in the table 2. the range of ahl s sc ores of the employees was 4-23. while there was no difference in ahls score between age groups (p=0.03). ahls score s increased with educational status of the employees (p=0.001). although those with social security and good economic status obtained higher scores from the scale, there was no significant difference in terms of disease history and number of children. the scores of employees according to some variables are shown in table 3. although the ahls scores were higher in the ‘others’ occupational group (dentist, x-ray technician, medical secretary), the difference was not significant (p>0.05). the percentages of employees who received 2 or more training sessions from the ohs unit were 19.3%, 20.6%, and 46.7% for workers, civil servants and other employees, respectively (p=0.001) training duration significantly increased hl levels (p<00.01) (table 3). discussion in the present study carried out in various occupational groups, it was shown that the mean ahls score was 16 (49.0%) out of 23 points, indicating that the employees had moderate hl. in addition, we found that as the number of periodic health training received by the employees from the ohs unit increased, the score obtained from scale also increased. although there are many studies in the literature on hl there is no study evaluating hl in different occupational groups receiving ohs training. in a study conducted by joveini et al.(10) , it was found that 39.4% of individuals between the ages of 18-65 years had adequate health literacy. similarly, güner et al. (6) conducted a study in turkey on 282 factory workers, and reported that 39.1% characteristics n % age groups (years) 18-29 92 45.5 30-39 60 29.7 40+ 50 24.8 gender male 151 74.8 female 51 25.2 educational status primary school 12 5.9 middle school 43 21.3 high school 113 55.9 university and above 34 16.9 social security yes 179 88.6 no 23 11.4 economic status good 46 22.8 moderate 133 65.8 poor 23 11.4 number of children 0 99 49.0 1 40 19.8 2 54 26.7 3 9 4.5 chronic disease yes 77 38.1 no 125 61.9 occupation worker 109 54.0 civil servant 63 31.2 *others 30 14.8 training duration 8 hours and less 58 28.7 9-16 hours 96 47.5 > 16 hours 48 23.8 table 2. distribution of participants according to various characteristics (n=202) *: dentist, x-ray technician, medical secretary 175 univ med vol. 39 no.3 had adequate health literacy. in a recent study, svendsen et al.(11) reported in denmark that 60.9% of adults over the age of 25 had adequate hl. the different results obtained in the present study and other studies may be due to differences of the groups studied, economic income level, and educational status. health literacy is expected to decrease with increasing age, due to reasons such as decrease in using health se rvices and mutual communication, difficulty in accessing health inf ormation, decr ease d internet use, and dependence on family members.(12,13) in contrast, hl increased with age in the present study and in the study of nakayama.(14) on the other hand, jovic et al.(15) investigated 120 people in serbia and found that hl sc ores were lower in individuals over 44 years of age. this difference may be due to the following: the mean age of the sample in the present study was 31 years, there were no elderly employees, the sample consisted of working individuals, and the amount of training received from the ohs unit increased with age. higher level of e duc ation and good economic income affect hl positively in terms variables ahls score (min-max) comparison age 18-29 15 (4-23) 30-39 15.50 (9-21) kw=1.93 p=0.38 40+ 17 (8-22) gender female 16 (8-21) z=0.22 p=0.82 male 16 (4-23) education primary school 10 (4-12) middle school 15 (4-23) kw=31.98 p=0.001 high school 16 (8-21) university 17.50 (9-22) marital status married 17 (4-21) z=1.92 p=0.05 single 15 (4-23) economic status good 18 (8-23) moderate 15 (5-22) kw=8.41 p=0.01 poor 14 (4-21) social security yes 16 (5-23) z=3.34 p=0.001 no 1 2 (4-19) number of children no children 15 (4-23) 1 16 (4-21) kw=1.33 p=0.51 2 or more 17 (8-22) chronic disease yes 16 (4-21) kw=0.74 p=0.38 no 16 (4-23) occupation worker 15 (4-22) civil servant 16 (8-21) kw=4.39 p=0.11 other 17.50 (9-23) training duration 8 hours and less 12 (4-16) 9-16 hours 16 (5-20) kw=118.96 p<0.001 more than 16 hours 20 (15-23) table 3. ahls score of the participants according to sociodemographic characteristics note. ahls : adult health literacy scale; kw: kruskall-wallis test, z= mann-whitney u test 176 sevinc, korkut occupational health and safety training of accessing correct information on health, using health services, follow-up, and communication.(16) in the present study, ahls scores were higher among university graduates and employees who declared that their economic status was good (17.50 and 18.00, respectively). in a study involving 8 european countries, it was found that low education and economic status negatively affected hl.(17) vamos et al.(18) and nutbeam (19) have suggested that receiving any form of health education or training has a positive effect on hl. bayati et al.(20) conducted a study on 256 health representatives in iran, and found a significant relationship between the health literacy scores before and after health education. as mentioned above, in the present study conducted on individuals working in various professions the hl score was found to be higher in participants who received more than 16 hours of ohs training. furthermore, a strong positive correlation was observed between training durations and hl levels. due to lack of related literature, we could not compare the results of the study with any research. given that health and safety training has a significant impact on hl, it can be said that this training encourages employees to access health-related resources and follow current health developments, thereby increasing hl. it is well known that hl is important for every individual of the society and covers the acquisition of health knowledge, its appraisal, and associated decision-making.(17) the results of the present study revealed that providing health literacy training to employees in the various business lines increases their hl levels. hence, as training plays a critical role in raising and promoting hl levels of employees, it is necessary to develop and expand a training program for employees regardless of their literacy or education levels. we think that by achieving this goal, it is possible to create a national action plan with the contributions of public institutions and organizations. the main limitation of this study is that there are numerous different scales on hl and that there is no standard evaluation. secondly, since the present study was conducted only on employees receiving services from the ohs unit, the health literacy scores obtained cannot be generalized to the whole society. in addition to health training, conducting regular examinations for employees receiving services from the ohs unit is important. in this regard, employers should be informed about this issue first and encouraged to receive services from ohs units for their employees. conclusion the hl level of the employees enrolling in this study was found to be moderate, and was related to occupational health and safety training. conflict of interest the authors declare no conflict of interest. acknowledgement we would like to thank the participants for their time and help during the study. contributors ns contributed to writing the original draft. ns and bk contributed to review and editing. bk contributed to data collection and analysis. both authors have read and approved the final manuscript. references 1. world health organization. regional office for south-east asia. 9th global conference on health promotion. health literacy: the mandate for health literacy. shanghai; 2016. 2. rowlands g. health literacy: ways to maximise the impact and effectiveness of vaccination information. hum vaccin immunother 2014;10:2130-35. https://doi: 10.4161/hv.29603. 3. berkman nd, sheridan sl, donahue ke, et al. low health literacy and health outcomes: an updated systematic review. ann int med 177 univ med vol. 39 no.3 2011;155:97-107. https://doi: 10.7326/0003-4819155-2-201107190-00005. 4. harrington kf, valerio ma. a conceptual model of verbal exchange health literacy. patient educ couns 2014;94:403-10. doi: 10.1016/ j.pec.2013.10.024. 5. paige sr, krieger jl, stellefson m, et al. ehealth literacy in chronic disease patients: an item 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in bardaskan, iran. j edu health promot 2019;8:244. https:// doi: 10.4103/jehp.jehp_26_19. 11. svendsen mt, bak ck, sørensen k, et al. associations of health literacy with socioeconomic position, health risk behavior, and health status: a large national population-based survey among danish adults. bmc public health 2020;20:1-12. https://doi: 10.1186/s12889-02008498-8. 12. vogt d, schaeffer d, berens e.m. health literacy in later phases of life: findings from germany and other countries. in: okan o, bauer u, levin-zamir d, pinheiro p, sørensen k, eds. international handbook of health literacy. research, practice and policy across the life span. bristol: policy press; 2019; pp.153-66. 13. okan o, bauer u, levin-zamir d, pinheiro p, sørensen k. international handbook of health literacy: research, practice and policy across the lifespan. bristol: policy press; 2019. 14. nakayama k, osaka w, togari t, et al. comprehensive health literacy in japan is lower than in europe: a validated japanese-language assessment of health literacy. bmc public health 2015;15:505. https:// doi: 10.1186/s12889-015-1835x. 15. jović-vraneš a, bjegović-mikanović v, marinković j, et al. evaluation of a health literacy screening tool in primary care patients: evidence from serbia. health promot int 2014;29:601-7. https://doi: 10.1093/heapro/dat011. 16. kickbusch i, pelikan jm, apfel f, et al. health literacy: the solid facts. copenhagen, denmark: who regional office for europe; 2013. 17. sørensen k, pelikan jm, röthlin f, et al. health literacy in europe: comparative results of the european health literacy survey (hls-eu). eur j public health 2015;25:1053-8. https://doi: 10.1093/ eurpub/ckv043. 18. vamos s, okan o, sentell t, rootman i. making a case for “education for health literacy”: an international perspective. int j environ res public health 2020;17:1436. http://dx.doi.org/10.3390/ ijerph17041436. 19. nutbeam d. health education and health promotion revisited. health educ j 2019;78:705– 9. https://doi.org/10.1177%2f00178969187702120. 20. bayati t, dehghan a, bonyadi f, bazrafkan l. investigating the effect of education on health literacy and its relation to health-promoting behaviors in health center. j educ health promot 2018;7. https://doi: 10.4103/jehp.jehp_65_18. oktavianus 159 universa medicina september-desember, 2015 vol.34 no.3 dietary fat and cardiovascular disease? lie t merijanti s department of occupational medicine, medical faculty, trisakti university dietary saturated fat (sf) intake has been shown to increase low density lipoprotein (ldl) cholesterol and therefore has been associated with increased risk of cardiovascular disease (cvd). this evidence coupled with inferences from epidemiologic studies and clinical trials, h a d l e d t o l o n g s t a n d i n g p u b l i c h e a l t h recommendations for limiting sf intake as a means of preventing cvd. however the relationship between sf and cvd risk remains controversial, due at least in part to the intrinsic limitations of clinical studies that have evaluated this relationship.(1) a recent meta analysis showed that current evidence does not clearly support cardiovascular guidelines that encourage high consumption of polyunsaturated fatty acids (pufa) and low consumption of total sf.(2) they found weak positive associations between circulating palmitic and stearic acids (found largely in palm oil and animal fats, respectively) and cvd, whereas circulating margaric acid (a dairy fat) significantly reduced the risk of cvd.(2,3) saturated fat are not associated with all cause mortality, cvd, chd, ischemic stroke o r t y p e 2 d i a b e t e s , b u t t h e e v i d e n c e i s heterogenous with methodological limitations.(4) dietary lipids are important regulators of cardiac function through their role in membrane phospholipids, as signaling molecules and ligands for nuclear receptors, and as the predominant oxidative substrate for cardiac mitochondria. moreover, long chain n3 pufa decrease very low density lipoprotein assembly a n d s e c r e t i o n r e d u c i n g t r i a c y l g l y c e r o l production. eicosanoids derived from n6 pufa have proinflammatory actions, while eicosanoids from n3 pufa have anti inflammatory ones. previous studies showed that inflammation contributes to both the onset and progression of atherosclerosis actually, atherosclerosis is predominantly a chronic low grade inflammatory disease of the vessel wall.(5) studies in rodents show that in the absence of obesity, replacing refined carbohydrate with fat can attenuate or prevent ventricular expansion and contractile dysfunction in response to hypertension, infarction or genetic cardiomyopathy. clinical studies generally support high intake of n 3 pufa from marine sources to prevent and treat heart failure. this effect is associated with decreased inflammation and improved resistance to mitochondrial permeability transition.(6) dietary guidelines must carefully consider the health effect of recommendations for alternative macronutrient to replace sf.(3) saturated fat intake of around 20 g/day (approximately 10% of total energy) may be optimal, which corresponds to 200 g of milk a day and 150 g of meat every other day.(7) epidemiologic studies and randomized clinical trials have provided consistent evidence that replacing sf with pufa but not carbohydrates is beneficial for chd.(1) in practice, reducing red meat and dairy products in a food supply and increasing intakes of nuts, fish, soy products and non hydrogenated vegetables oils will improve the mix of fa and have a markedly beneficial effects on rates of chd.(4) 160 univ med 2015;34:159-60. doi: http://dx.doi.org/10.18051/univmed.2015.v34.159-160 references 1. siri tarino pw, sun q, hu fb, et al. saturated fatty acid and risk of coronary heart disease: modulation by replacement nutrients. curr atheroscler rep 2010;12:384-90. doi: 10.1007/ s11883-010-0131-6. 2. chowdury r, warnakula s, kunutsor s, et al. association of dietary, circulating, and supplement fatty acids with coronary risk: a systematic review and meta analysis. ann intern med 2014;160:398-406. 3. siri tarino pw, sun q, et al. meta analysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease. am j clin nutr 2010;91:535-46. doi: 10.3945/acjn.2009.27725. 4. de souza rj, mente a, moroleanu a, et al. intake of saturated and trans unsaturated fatty acids and risk of all cause mortality cardiovascular disease and type 2 diabetes: systematic review and metaanalysis of observational studies. bmj 2015;351:3978. doi: 10.1136/bmjh3978. 5. mozaffarian d, katan mb, ascherio a, et al. trans fatty acids and cardiovascular disease. n engl j med 2006;354:1601-13. 6. dessi m, noce a, bertucci p, et al. atherosclerosis, dyslipidemia and inflammation: the significant role of polyunsaturated fatty acids. isrn inflammation: volume 2013. article id 191823, 13 pages. http://dx.doi.org/10.1155/ 2013/191823. 7. stanley wc, dabkowski er, riberio rf, et al. dietary fat and heart failure: moving from lipotoxicity to lipoprotection. circ res 2012;110: 764-76. universa medicina september-desember, 2015 vol.34 no.3 oktavianus 83 *department of parasitology, medical school, atma jaya catholic university of indonesia **medical student of medical school, atma jaya catholic university of indonesia ***department of chemistry, medical school, atma jaya catholic university of indonesia correspondence dr. hanna yolanda, m.kes. department of parasitology medical school, atma jaya catholic university of indonesia pluit raya 2 jakarta 14440 phone: +6221 6693168; +62813 222 85483 email : hanna.yolanda@atmajaya.ac.id univ med 2014;33:83-90 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background to develop new effective antifungals, it is essential to search for antifungal compounds from plants such as nepenthes spp., which have their greatest diversity in indonesia. since chitin-induced liquid (cil) from nepenthes khasiana pitchers has antifungal activity, due to their naphthoquinone content, this study aimed to evaluate antifungal activity of nepenthes rafflesiana pitcher liquids on candida spp. methods collected pitcher liquids were of 3 types: non-induced liquid (nil), prey-induced liquid (pil), and chitin-induced liquid (cil). non-induced liquid (nil) was collected from fresh naturally opened pitchers, pil from opened pitchers after 3 hours of induction with zophobas morio larvae, and cil from closed pitchers after 5 days of chitin solution injection. the antifungal activity of the liquids against c. albicans, c. glabrata, c. krusei, and c. tropicalis were detected by disc diffusion and macrodilution methods. results inhibition zone diameters of nil, pil, and cil against c. albicans were 35.00 (35.00 – 39.33) mm, 26.33 (23.00 – 40.00) mm, and 30.00 ( 28.00 – 32.00) mm, respectively, while for c. glabrata the zone diameters were 22.22 ± 3.66 mm, 29.89 ± 2.79 mm, and 28.89 ± 1.17 mm, respectively. no inhibition zones were found for nil, pil, and cil against c. krusei and c. tropicalis. at concentrations of 80%, almost all samples showed visually apparent inhibition of fungal growth. conclusion the pitcher liquid of n. rafflesiana has antifungal properties, presumably due to the presence of many potentially active substances, such as naphthoquinones, as has been proven in other studies. key words : antifungal, chitin-induced, disc diffusion, nepenthes, candida nepenthes rafflesiana pitcher liquid has antifungal activity against candida spp. hanna yolanda*, ingrid m. makahinda**, maureen aprilia**, nikki sanjaya**, harry gunawan**, and rita dewi*** 84 yolanda, makahinda, aprillia, et al nepenthes rafflesiana against candida spp. aktivitas antijamur cairan kantong nepenthes rafflesiana terhadap candida spp. pendahuluan untuk mengembangkan obat antijamur baru yang efektif, adalah sangat penting untuk mencari senyawa antijamur dari sumber alamiah, seperti nepenthes spp., yang paling aneka ragam di indonesia. karena cairan kantong nepenthes khasiana yang telah diinduksi kitin memiliki aktivitas antijamur akibat kandungan naftokuinon, penelitian ini bertujuan untuk menilai aktivitas antijamur nepenthes rafflesiana terhadap candida spp. metode cairan kantong yang dikumpulkan ada 3 jenis yaitu: cairan yang tidak diinduksi (t), cairan yang diinduksi mangsa (m), dan cairan yang diinduksi kitin (k). t diambil dari kantong yang baru terbuka secara alami, m diambil dari kantong terbuka yang telah diinduksi larva zophobas morio selama 3 jam, dan k diambil dari kantong tertutup yang telah disuntikkan larutan kitin selama 5 hari. aktivitas antijamur ketiga jenis cairan tersebut terhadap c. albicans, c. glabrata, c. krusei, dan c. tropicalis dideteksi dengan menggunakan metode difusi diskus dan makrodilusi. hasil diameter zona inhibisi tampak pada uji t, m, dan k terhadap c. albicans [35,00 (35,00 – 39,33) mm, 26,33 (23,00 – 40,00) mm, dan 30,00 ( 28,00 – 32,00) mm] dan c. glabrata (22.22 ± 3.66 mm, 29.89 ± 2.79 mm, dan 28.89 ± 1.17 mm). pada uji t, m, dan k terhadap c. krusei dan c. tropicalis tidak tampak adanya zona inhibisi. pada konsentrasi 80% didapatkan berkurangnya pertumbuhan jamur secara visual pada hampir semua sampel uji. kesimpulan cairan kantong n. rafflesiana memiliki aktivitas antijamur, karena mengandung beberapa zat potensial, antara lain adalah naftokuinon. kata kunci : antijamur, diinduksi kitin, difusi diskus, nepenthes, candida abstrak introduction t h e e m e r g e n c e o f t h e h u m a n immunodeficiency virus infection, modern patient-management technologies and therapy, s u c h a s b o n e m a r r o w a n d s o l i d o r g a n transplants, and the use of more aggressive chemotherapy have resulted in a rapidly expanding number of immunocompromised patients highly susceptible to fungal infections,(1) the most common being candidiasis. because of increasing use of antifungals and the limited types of antifungals, the development of resistence is an important issue. in addition, adverse effects, drug-drug interactions, and toxixity, are also factors that influence the use of antifungals. therefore, exploration to find new a n d e f f e c t i v e c o m p o u n d s f o r a n t i f u n g a l chemotherapy is of pivotal importance.(2) exploration of natural products that may have antifungal properties has been increasing. naphthoquinones are a group of secondary metabolites with antifungal properties occurring in a number of plant families, such as the nepenthaceae.(5) nepenthes are carnivorous plants that have developed insect capturingtraps, evolved by specific modification of the leaf tips, and capable of utilizing digested insects. nepenthes khasiana, commonly found i n i n d i a , h a s b e e n f o u n d t o c o n t a i n naphthoquinones, especially droserone and 5o-methyldroserone.(3,4) 85 indonesia, as a tropical country, has many species of nepenthes, such as nepenthes rafflesiana, since the distribution of nepenthes is predominantly in indonesia, especially borneo. the liquid inside the pitchers is traditionally used for relieving eye disorders, cough, stomach ache, burn injuries, and skin diseases.(6) several studies have been conducted to detect antifungal activity in nepenthes, but none of them have explored antifungal activity in pitcher fluid of n. rafflesiana.(3,7,8) a study by eilenberg et al.(3) on the antifungal effects of chitin-induced liquid (cil) from n. khasiana to candida spp. concluded that only cil efficiently inhibited fungal pathogens.(3) it has been suggested that either chitin itself or its breakdown products induce the synthesis of antifungal agent/s, that are not produced under natural conditions either in closed or opened pitchers. however, subsequently many enzymes, such as endochitinases, were detected inside closed pitchers.(9) since chitin is one of the major components of fungal cell walls,(10) these results suggested that closed pitchers may have antifungal effects as a result of compounds other than naphthoquinones, e.g. chitinases. in this study, we used 3 types of pitcher liquid of n. rafflesiana, i.e. non-induced liquid (nil), prey-induced liquid (pil), and cil. the objective of this research was to determine if pitcher liquids of n. rafflesiana have antifungal activity on tested candida spp. and have significant differences in activity. n. rafflesiana is a lowland pitcher plant and thus easy to cultivate in jakarta. the pitcher liquid of n. rafflesiana has the unique characteristic of being the most viscoelastic. a 95%-dilution in water still retains enough viscoelasticity to capture all insects dropped into the pitcher.(11) methods research design t h e d e s i g n o f t h i s r e s e a r c h w a s experimental laboratory. this research was done i n m e d i c a l s c h o o l , a t m a j a y a c a t h o l i c university of indonesia, from march 2013 to november 2013. the first six months were used to collect the 3 types of pitcher liquid until we had enough stock. the next months were used to conduct the antifungal activity tests. plant materials n. rafflesiana plants were obtained from borneo island and looked after in the garden of the medical school of atma jaya catholic university of indonesia. we used pitcher liquid from the lower pitchers of n. rafflesiana that were more than 7 cm in height, measured from the base to the spur of the pitcher. we used larvae of zophobas morio as prey for induction. the chitin solution was made from dried prawn exoskeletons. for fungal preparation and disc diffusion test, we used sabouraud dextrose agar (difco, france), mueller hinton agar (difco, france) with the addition of 2% glucose and 0.5 mg/l methylene blue.(12) the discs were 5 mm in diameter, cut out of whatman filter paper no. 42. for the macrodilution method, we used sabouroud dextrose broth (difco, france). as test fungi we used four candida species, viz. c a n d i d a a l b i c a n s , c a n d i d a g l a b r a t a , candida kr usei, and candida tropicalis, obtained from the fungal collection of the parasitology department, medical faculty, university of indonesia. chitin extraction the chitin solution was prepared from ground dried prawn exoskeletons, which were washed by boiling with distilled water for 1 hour. the mixture was centrifuged to precipitate the solid phase, which was separated by filtration through filter paper. the solid phase was stirred in an ethanol : ether : hcl mix (38:38:0.8, v:v:v) for 15 minutes, then the mixture was filtered through filter paper. thereafter, the solid phase was solubilized in 0.2 m naclo for 1 hour at 75oc. after an additional filtering, the solid phase was solubilized in acetone and 32% hcl (1:150, v:v) at 4oc. after centrifugation, the supernatant (solubilized chitin) was collected and the chitin univ med vol. 33 no.2 86 yolanda, makahinda, aprillia, et al nepenthes rafflesiana against candida spp. was precipitated by adding ice-cold water and incubating at 4oc overnight. the acid was removed by subsequent washes with cold water until the ph of the supernatant reached 2.5. the prepared chitin was allowed to dry on a filter paper, then 80 mg of the chitin was homogenized with 10 ml distilled water, and the ph of the colloidal chitin adjusted to 5 by addition of 1 n naoh.(3,4,13) collection of pitcher liquid the nil, pil, and cil were collected as follows.we wrapped target-pitchers with sealed plastic. nil was collected from fresh naturally opened pitchers (opened less than 24 hours). pil was collected from opened pitchers after 3 hours of z. morio induction. cil was collected from closed pitchers after 5 days of 2 ml chitin injection (8 mg/ml chitin solution).(3,4) all of the liquids were stored in dark bottles at 4oc.(14) before being used for the disc diffusion tests, the samples were incubated in a water bath at 50oc for an hour. we collected the liquid from 20 plants of n. rafflesiana. fungal preparation all candida species were subcultured into sabouraud dextrose agar at 37 oc for 24 hours.(15,16) the inocula were prepared from 24hour old cultures of candida spp. the colonies were suspended in sterile saline, then the turbidity of the homogenous suspension was a d j u s t e d b y s p e c t r o p h o t o m e t r y t o ~ 0 . 5 mcfarland standard at 530 nm. this stock suspension was used for the disc diffusion method. for macrodilution, this stock suspension was diluted 1:2000 in sabouroud dextrose broth.(12,15,16) disc diffusion test the 24-hour cultures of candida spp. were cultured onto mueller hinton agar with the addition of 2% glucose and 0.5mg/l methylene blue, by dropping 10 µl fungal suspension onto the agar and spreading it over the entire surface by sterile swab to obtain a confluent growth.(12) the sterile discs were soaked in nil, pil, or cil and placed on the seeded surface.(17) a sterile disc soaked in aquabidest and similarly placed on the seeded medium was used as a control. the plates were incubated at 37oc for 24 hours, then examined for inhibition zones around the paper discs, indicating antifungal activity. the inhibition zone diameters (in millimeters) were measured in triplicate at the point at which there was a prominent reduction in growth, repeating the measurements in different diagonals, and calculating their mean value. all disc diffusion tests were performed in triplicate. macrodilution we used pitcher liquid in concentrations of 20%, 50%, and 80%. a volume of 1000 µl fungal suspension was used as control. the mixtures were incubated at 37oc for 24 hours, then their turbidities were compared with that of the control. these tests were also performed in triplicate. statistical analysis inhibition zone diameters of pitcher liquids were compared using one-way anova test or kruskal-wallis test. for all candida spp. except c. glabrata, the test results are shown as median (minimum – maximum) because they were non-normally distributed and were tested by kruskal-wallis test. the normally distributed c. glabrata data were tested by one-way anova, with the results shown as mean (± standard deviation). results were considered significant if p<0.05. results the color of nil and pil was a cloudy white, with pil being more cloudy than nil. on chitin induction, the color of the pitcher liquid changed to orange red (figure 1). the ph values of nil, pil and cil were 3.1, 3.0, and 2.6, respectively. all samples were slimy. the tested pitcher liquids exhibited different degrees of antifungal activity against candida 87 spp. nil, pil, and cil inhibited the growth of c. albicans and c. glabrata. the diameters of the inhibition zones found on testing of nil, pil, and cil against c. albicans were 35.00 (35.00 – 39.33) mm, 26.33 (23.00 – 40.00) mm, and 30.00 (28.00 – 32.00) mm, respectively. for tests against c. glabrata , the inhibition zone diameters of nil, pil, and cil were 22.22 ± 3.66 mm, 29.89 ± 2.79 mm, and 28.89 ± 1.17 mm, respectively. no inhibition zones were found in nil, pil, and cil tests against c. krusei and c. tropicalis. (table 1 and figure 2). by kruskal-wallis test, there were no significant differences in inhibition zones among the 3 types of pitcher liquid against c. albicans (p=0.298) (table 1). in contrast, for tests against c. glabrata, using one-way anova, significant differences were found between 2 types of pitcher liquid (p=0.027) (table 1). by post-hoc lsd test, there were significant differences in inhibition zones between nil and pil (p=0.014), and between nil and cil (p=0.025), but no significant differences between pil and cil (p=0.671). the concentrations of nil and pil that showed decreased growth of c. albicans, c. glabrata, c. krusei, and c. tropicalis, were observed visually to be 80%, 50%, 80%, and 50%, respectively. on the other hand, the cil concentrations showing decreased growth of c. albicans , c. glabrata, c. krusei, and c . tropicalis were observed visually to be 80%, 20%, 80%, and 80%, respectively (table 2). figure 1. pitcher liquid of n. rafflesiana: non-induced liquid (nil) (a), prey-induced liquid (pil) (b), and chitin-induced liquid (cil) (c) a b c ca b d fe figure 2. results of disc diffusion test of non-induced liquid (nil), prey-induced liquid (pil), and chitininduced liquid (cil) to c. albicans and c. glabrata. inhibition zone of nil to c. albicans (a), pil to c. albicans (b), cil to c. albicans (c), nil to c. glabrata (d), pil to c. glabrata (e), cil to c. glabrata (f) univ med vol. 33 no.2 88 yolanda, makahinda, aprillia, et al nepenthes rafflesiana against candida spp. fungi pitcher liquid diameter of inhibition zone (mm) p value c. albicans nil 35.00 ( 35.00 – 39.3 3)† 0.298* pil 26.33 (23.00 – 40.00) † cil 30.00 ( 28.00 – 32.00) † c. glabrata nil 22.22 ± 3.66‡ 0.027** pil 29.89 ± 2.79‡ cil 28.89 ± 1.17‡ c. krusei nil n o zone pil n o zone cil n o zone c. tropicalis nil n o zone pil n o zone cil n o zone control n o zone table 1. inhibition zone of nil, pil, and cil against candida spp. growth in vitro nil : non-induced liquid; pil : prey-induced liquid;cil : chitin-induced liquid; † data are shown as median (minimum – maximum) because data distribution was not normal; ‡ data are shown as mean ± standar deviation because data distribution was normal; * kruskal-wallis test; ** one-way anova. post-hoc lsd test : nil vs pil p=0.014; nil vs cil p = 0.025; pil vs cil p = 0.671 discussion the cil color change into orange red was similar to that found by eilenberg et al. in cil of nepenthes khasiana.(3) according to these investigators, the color change was associated with the presence of droserone (3,5-dihydroxy2-methyl-1,4-naphthoquinone), which has antifungal properties. the ph of nil, pil, and cil was acidic, as has also been determined by several studies, which found that the ph of most pitcher fluid is acidic, rarely neutral.(18,19) n. rafflesiana has the unique characteristic of its highly viscoelastic fluid, differing in this respect from other nepenthes species. a c c o r d i n g t o a c l i n i c a l l a b o r a t o r y standard institute document, the sensitivity to fluconazole, one of the standard treatments of candidiasis, is defined as an inhibition zone of more than 19 mm.(12,15) since the inhibition zones of nil, pil, and cil towards c. albicans and c. glabrata were more than 19 mm, these r e s u l t s i n d i c a t e t h a t c . a l b i c a n s a n d c . glabrata were susceptible to n. rafflesiana pitcher liquid. in our study, pil and cil showed better inhibition than nil against c. glabrata. types of pitcher liquid c oncentration (%) c. a lbicans c. glabrata c. krusei c. tropicalis + ± + ± + ± + ± nil 20 100 0 0 100 0 0 100 0 0 66.67 33.33 0 50 100 0 0 0 100 0 100 0 0 0 1 00 0 80 0 33.33 66.67 0 100 0 0 100 0 0 1 00 0 pil 20 100 0 0 100 0 0 100 0 0 33.33 66.67 0 50 100 0 0 0 100 0 66.67 33.33 0 0 1 00 0 80 0 100 0 66.67 33.33 0 100 0 0 1 00 0 cil 20 100 0 0 0 100 0 100 0 0 66.67 33.33 0 50 100 0 0 0 100 0 100 0 0 33.33 66.67 0 80 0 100 0 0 0 10 0 0 100 0 0 0 100 nil : non-induced liquid; pil : prey-induced liquid;cil : chitin-induced liquid; + : turbidity equal to control; ± : turbidity less than control; : no turbidity table 2. macrodilution test nil, pil, and cil against candida spp. (in % sample) 89 eilenberg et al.(3) and raj et al.(4) stated that naphthoquinones, which have antifungal properties, are only detected in red-colored c i l . n a p h t h o q u i n o n e s a r e a p p a r e n t l y chromatic pigments.(5) however, in the present study, we found that nil and pil, which are not red in color, also have an antifungal effect. i t i s p o s s i b l e t h a t n i l a n d p i l o f n . rafflesiana contain other substances with antifungal properties, which do not give a red color to pitcher liquid. no inhibition zones were produced in disc diffusion tests of nil, pil, and cil against c. krusei and c. tropicalis, which agrees with the fact that c. kr usei and c. tropicalis are c o m m o n l y m o r e r e s i s t a n t t o a n t i f u n g a l treatment. ( 2 0 ) according to the european committee on antimicrobial susceptibility testing, the drug dosages for mic50 and mic90 of both of fungi, especially c. krusei, are higher than those for the other candida species.(21) the cil concentration that inhibited c. albicans was about 3-fold and 6-fold lower than that required for growth inhibition of c. krusei and c. glabrata, respectively.(3) in this study, we used crude liquids that contain many substances, therefore the concentrations of the active compounds were presumably lower than necessary to obtain inhibition zones against c. krusei and c. tropicalis. however, this may have also been caused by the intrinsic resistance of the fungi. in the macrodilution test, we observed the turbidity of the test samples, compared to control. most of them showed inhibition to tested fungi at 80% pitcher liquid concentration. decreased growth of c. glabrata was shown at 20% cil, compared with 50% nil and pil (table 2). pitcher liquids showed growth inhibition against c. krusei and c. tropicalis at 80%, except for inhibition of nil and pil against c. tropicalis, which were at 50%. these results support the idea of fungal activity of pitcher liquids towards c. krusei and c. tropicalis, although no inhibition zones were detected in disc diffusion test. chitin injection into closed pitchers triggers t h e s y n t h e s i s o f a n t i f u n g a l s a n d c e r t a i n chitinases.(3) as yet, the mechanism underlying the pitcher response to chitin in nepenthes is not clear and may include both the induced synthesis of new compounds and/or the release of existing compounds from the pitcher gland cells to the trap liquid. the presence of naphthoquinones in the pitcher of nepenthes is important for chemical defense.(3) in addition, chitinases hydrolyze chitin which is a major component of fungal cell walls.(7,10,22) chitinase was also identified in the digestion fluid of closed pitchers, not only in chitin-induced pitchers.(9) therefore it may be assumed that chitinase is also potentially antifungal. the difficulties of this study were how to predict the right time to do chitin induction before opening the pitcher and the phenomenon of nonproducing pitchers after several times of taking pitcher liquids. obtaining pitcher liquid at precisely the right time is important to get as much as liquid we can. taking liquids was only done once, and no repetition was performed because of the contamination risk. using special treatments to plants may have to be done to promote the production of pitcher liquid. exploration about potentially active substances in the pitcher liquid of nepenthes should be encouraged. naphthoquinones and chitinase are potential future antifungals. many species of nepenthes in indonesia have not been explored, although most of nepenthes species originate from indonesia. conclusion the pitcher liquid of n. rafflesiana has antifungal properties due to the presence of many potentially active substances, especially against c. albicans and c. glabrata. acknowledgments we thank mr. edwin dwianto from the “pitcher of paradise” nursery and mr. anton univ med vol. 33 no.2 90 yolanda, makahinda, aprillia, et al nepenthes rafflesiana against candida spp. halim for their contribution as providers of nepenthes. we also thank siska tri hapsari, a g u s s i s w a n t o , a n d i d a a f i a h f o r t h e i r contribution in laboratory experimentation. references 1. michallet m, ito jl. approaches to the management of invasive fungal infections in hematologic malignancy and hematopoietic cell transplantation. j clin oncol 2009;27:3398-409. 2. akins ra. an update on antifungal targets and mechanisms of resistance in candida albicans. med mycol 2005;43:285-318. 3. eilenberg h, phini-cohen s, rahamim y, sionov e, segal e, carmeli s, et al. induced production of antifungal naphthoquinones in the pitchers of the carnivorous plant nepenthes khasiana. j exp bot 2009;61:911-22. 4. raj g, kurup r, hussain aa, baby s. distribution of naphthoquinones, plumbagin, droserone, and 5-o-methyl droserone in chitin-induced and uninduced nepenthes khasiana: molecular events in prey capture. j exp bot 2011;62:542936. 5. babula p, mikelova r, potesil d, adam v, kizek r, havel l, et al. simultaneous determination of 1,4naphthoquinone, lawsone, juglone, and plumbagin by liquid chromatography with uv detection. biomed 2005;149:25-7. 6. handayani t, latifah d. diversity and growth behavior of nepenthes (pitcher plants) in tanjung putting national park, central kalimantan province. biodiversitas 2005;6:248-52. 7. buch f, rott m, rottloff s, paetz c, hilke i, raessler m, et al. secreted pitfall-trap fluid of carnivorous nepenthes plants is unsuitable for microbial growth. ann bot 2013;111:375-83. 8. hatano n, hamada t. proteome analysis of pitcher fluid of the carnivorous plant nepenthes alata. j proteome res 2008;7:809-16. 9. rottloff s, stieber r, maischak h, turini fg, heubl g, mithofer a. functional characterization of a class iii acid endochitinase from the traps of the carnivorous pitcher plant genus, nepenthes. j exp bot 2011;62:4639-47. 10. babashpour s, aminzadeh s, farrokhi n, karkhane a, haghbeen k. characterization of a chitinase (chit62) from serratia marcescens b4a and its efficacy as a bioshield against plant fungal pathogens. biochem genet 2012;50:722-35. 11. bonhemme v, pelloux-prayer h, jousselin e, forterre y, labat jj, gaume l. slippery or sticky? functional diversity in the trapping strategy of nepenthes carnivorous plants. new phytol 2011; 191:545-54. 12. canton e, espinel-ingroff a, peman j. trends in antifungal susceptibility testing using clsi reference and commercial methods. expert rev anti infect ther 2009;7:107-19. doi: 10.1586/ 14787210.7.1.107. 13. arbia w, arbia l, ardour l, amrane a. chitin extraction from crustacean shells by biological methods. food technol biotechnol 2013;51:12– 25. 14. gorner h. photoreactions of 1,4 naphthoquinones: effect of substituents and water on the intermediates and reactivity. photochem photobiol 2005;81:376-83. 15. pfaller ma, chaturvedi v, espinel-ingroff a, ghannoum ma, gosey ll, odds fc, et al. reference method for broth dilution antifungal susceptibility testing of yeasts, clsi document m27-a3. 3rd ed. pennsylvania: clsi; 2008. 16. khan s, singhal s, mathur t, rattan a. antifungal susceptibility testing method for resource constrained laboratories. indian j med microbiol 2006;24:171-6. 17. bansod s, rai m. antifungal activity of essential oils from indian medicinal plants against human pathogenic aspergillus fumigatus and a. niger. world j med sci 2008;3:81-8. 18. adlassnig w, peroutka m, lendl t. traps of carnivorous pitcher plants as a habitat: composition of the fluid, biodiversity, and mutualistic activities. ann bot 2011;107:181-94. 19. takeuchi y, salcher mm, ushio m, inatsugi rs, kobayashi mj, diway b, et al. in situ enzyme activity in the dissolved and particulate fraction of the fluid from four pitcher plant species of the genus nepenthes. plos one 2011;6:e25144-52. doi: 10.1371/journal.pone.0025144. 20. fanci r, guidi s, bonolis m, bosi a. candida krusei fungemia in an unrelated allogeneic hematopoietic stem cell transplant patient successfully treated with caspofungin. bone marrow transplant 2005;35:1215-6. 21. cuenca-estrella m, rodriguez-tudela jl. the current role of the reference procedures by clsi and eucast in the detection of resistance to antifungal agents in vitro. expert rev anti infect ther 2010;8:267-73. 22. radjacommare r, ramanathan a, kandan a, sible gv, harish s, samiyappan r. purification and antifungal activity of chitinase against pyricularia grisea in finger millet. world j microbiol biotechnol 2004;20:251-6. higher triglyceride serum level increases atherosclerotic index in subjects 50-70 years of age 205 *department of physiology, faculty of medicine, trisakti university correspondence: dr. martiem mawi, ms department of physiology, faculty of medicine, trisakti university jalan kyai tapa (kampus b), grogol, west jakarta 11440 mobile: +62816 136 3811 email: martiemmawi@ yahoo.com univ med 2015;34:205-12 doi: 10.18051/univmed.2016.v35.205-212 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background atherosclerosis, the underlying cause of heart attack, stroke and peripheral disease, is a main cause of morbidity and mortality worldwide. hypercholesterolemia and hypertriglyceridemia are independent factors in the development and progression of atherosclerosis. the atherosclerotic index (ai) is a strong indicator of cardiovascular heart disease. the objective of this study was to determine the relationship between lipid serum level and ai in subjects 50-70 years of age. methods a study of cross-sectional design was conducted among male and female subjects 50-70 years of age. the inclusion criteria were: healthy, and capable of active communication. the exclusion criteria were: subjects not completing the study, currently consuming antihyperlipidemic drugs. lipid profile comprising total cholesterol, low density lipoprotein (ldl), high density lipoprotein (hdl), triglycerides, and malondialdehyde (mda), was analyzed using commercial kits. systolic and diastolic blood pressure and body mass index was measured in all subjects. atherogenic index was calculated from (total cholesterol – hdl cholesterol) / hdl cholesterol. multiple linear regression was used to analyze the data. results mean age of the subjects was 60.6 ± 3.30 years and there was a significant relationship of ldl cholesterol and triglycerides with ai (β=0.009; p=0.000 and β=0.008; p=0.000, respectively). triglyceride level was the most influencing factor for ai (β=0.008; beta=0.616; p=0.000) conclusions higher triglyceride levels increase ai in subjects 50-70 years of age. subjects with high serum triglyceride level but without symptoms of cardiovascular disease should be examined for the development of coronary artery blockage. keywords : atherosclerotic index, lipid profile, malondialdehyde, 5070 years of age higher triglyceride serum level increases atherosclerotic index in subjects 50-70 years of age martiem mawi*, fransisca chondro*, and juni chudri* doi: http://dx.doi.org/10.18051/univmed.2015.v34.205-212 206 mawi, chondro, chudri triglyceride serum atherosclerotic index kadar trigliseride serum yang tinggi meningkatkan indeks aterosklerotik pada usia 50-75 tahun latar belakang aterosklerosis merupakan penyebab dari stroke, serangan jantung dan penyakit vaskular yang dapat menimbulkan kematian. hiperkolesterolemia dan hipertrigliseridemia merupakan faktor risiko penting terjadinya aterosklerosis. indeks aterosklerotik (ia) merupakan indikator kuat untuk penyakit kardiovaskular. penelitian ini bertujuan untuk menentukan hubungan antara kadar lipid serum dan indeks aterogenik pada usia 50-70 tahun. metode sebuah penelitian dengan rancangan potong silang dilakukan pada 80 subjek laki-laki dan perempuan berusia 50–70 tahun. kriteria inklusi adalah subjek berbadan sehat dan mampu berkomunikasi secara aktif. kriteria ekslusi: tidak mengikuti penelitian secara lengkap, memakan obat anti lipid. pengukuran profil lipid meliputi kadar kolesterol total, low density lipoprotein (ldl), high density lipoprotein (hdl), dan malondialdehid (mda), menggunakan kit komersial. tekanan darah sistolik dan diastolik dan indeks massa tubuh (imt) diukur pada semua subjek. indeks aterogenik diukur berdasarkan (kadar kolesterol total – kolesterol hdl) / kolesterol hdl. analisis regresi linear ganda digunakan untuk analisis data. hasil usia rata-rata subjek penelitian adalah 60,6 ± 3,30 tahun dan terdapat hubungn yang bermakna antara kolesterolldl dan trigliserid dengan ia, masing-masing dengan (β=0.009; p=0,000 dan β=0.008; p=0,000. kadar trigliserid merupakan faktor yang paling besar terhadap ia (β=0.008; beta=0.616;p=0.000). kesimpulan semakin tinggi kadar trigliseride serum semakin tinggi skor ia pada subjek berusia 50-70 tahun. pada subjek dengan kadar trigliserid serum tinggi, walaupun tidak ditemukan gejala penyakit kardiovaskular harus dilakukan pemeriksaan lebih lanjut untuk memastikan ada tidaknya penyumbatan pembuluh darah terutama di arteria coronaria. kata kunci: indeks aterogenik, profil lipid, malondialdehid, usia 50-70 tahun abstrak introduction cardiovascular disease is a worldwide major cause of morbidity and mortality.(1-3) atherosclerosis is considered to be a basic cause of heart attacks, stroke and peripheral vascular d i s e a s e . h y p e r c h o l e s t e r o l e m i a a n d hypertriglyceridemia are important risk factors for the genesis of atherosclerosis and the progressivity of atherosclerotic lesions.(1) the initial event in the induction of atherosclerosis is the accumulation of cells containing excessive amounts of lipids in the arterial wall. it has been demonstrated that increased production of reactive oxygen species (ros) substantially influences the chronic inflammatory response in atherosclerosis.(4) ros reacts with a number of biomolecukes, such as lipids, carbohydrates, proteins, and nucleic acids, resulting in cellular dysfunction. normally there is a balance between the prduction of free oxygen radicals and the antioxidant defense system. antioxidant-oxidant imbalance induces oxidative stress as a result of excessive ros formation. oxidative stress is 207 univ med vol. 34 no.3 known to be a component of the mechanism of cellular and molecular tissue damage in a broad spectrum of human disease.(5) increased ros causes vasoconstriction since it reduces the production of nitric oxide (no), and increases platelet aggregation and neutrophil adhesion to vascular endothelium.(6-8) the risk factors of atherosclerosis, such as hypercholesterolemia, diabetes mellitus, smoking, hypertension, and age, may increase the production of free ros, not only by endothelial cells but also by smooth muscle and adventitial cells. free radicals are extremely reactive chemical compounds, and the majority of molecules in the body may become a target of these free radicals, but lipids are their most frequent target. the cell membrane is a source of polyunsaturated fatty acids and is extremely vulnerable to attack by free radicals, a process known as lipid peroxidation. lipid peroxidation leads to the formation of reactive products, some of which interact with proteins and dna, thus giving rise to toxic and mutagenic components.(9) one of the standards used to evaluate the condition of the vascular wall resulting from the atherosclerotic process is the atherogenic index (ai), which is calculated from the difference between total cholesterol concentration and hdl cholesterol concentration, divided by hdl cholesterol concentration. the atherogenic index is a strong indicator of cardiovascular disease.(1,3) the study of nwagha et al (10) shows a positive correlation of ldl cholesterol and triglycerides with ai in postmenopausal women. a study on women with preeclampsia showed an association between serum mda level and ai.(11) the study of prakash and rao found that hypertriglyceridemia is a risk factor for the atherogenic process, and high ldl cholesterol and triglyceride levels increase the risk of cardiovascular disease.(2) lafta (3) found a positive association between increased triglycerides and incidence of cardiovascular disease. malondialdehydes (mda) are lipid peroxidation products and indicators of oxidationinduced damage. mda have been used for many years as biomarker of lipid peroxidation. the progress of the atherosclerotic process is associated with oxidative stress and can be followed up with measurement of mda.(1,11) the study of yang et al.(1) compared hyperlipidemic and normolipidemic groups and showed a significantly positive association between mda and ai. the investigators found a higher atherogenic index in the groups with increased lipid peroxidation levels. no informatiion could be found in the literature of studies showing an association of lipid and mda levels with ai in healthy adults. the objective of the present study was to determine any association between lipid levels (total cholesterol, hdl cholesterol, ldl cholesterol), and mda on the one hand and atherogenic index on the other in healthy subjects 50-70 years of age. methods study design this study used an experimental crosssectional design and was conducted at the mampang prapatan subdictrict health center, south jakarta from february until may 2014. study subjects males and females aged between 50 and 70 years and residing at 2 kelurahan (villages) in mampang prapatan subdistrict, south jakarta, were recruited into this study. the inclusion criteria were: capable of active communication, literate, physically healthy and not requiring help from others, and agreeing to sign informed consent. the exclusion criteria were: not participating in this study up to its completion, and current consumers of anti-lipid drugs. the sample size was determined with the formula: 3 r)-r)/(1-(11n x 0.5 )z-(z n 2 +       = βα where zá = 1.96, zâ = 0.84, and r = 0.61.(1) 208 mawi, chondro, chudri triglyceride serum atherosclerotic index the minimum sample size required was 18, but in this study it was increased to 80, which is the sampe size for using one commercial kit. anthropometric measurements anthropometric assessment comprised determination of weight, height, and body mass index (bmi). height was measured using a portable microtoise with an accuracy of 0.1 cm. weight was measured using sage portable scales with an accuracy of 0.1 kg. body mass index was calculated from the formula weight (kg) / height (m2) and categorized as follows: normal (< 23.0 kg/m2), overweight or obesity (>23.0 kg/ m2), and obesity according to the criteria determined for asians.(12) laboratory investigations from each subject, a 10-ml blood sample was drawn from the cubital vein using plain tubes after an overnight fast of 12-14 hours and centrifuged at 3000 rpm for 15 minutes. the serum was separated and placed into an automatic analyzer. all determinations of lipid profile components (total cholesterol, ldl cholesterol, hdl cholesterol, and triglyceride concentrations) were performed spectrophotometrically using an advia 1800 system and the respective bayer reagent kits. all procedures produce a complex of a given color, the intensity of which can be spectrophotometrically determined and is proportional to the blood concentration of the lipid component to be assessed. the principles of the lipid profile determinations are as follows. for total cholesterol determination, cholesterol esters in the sample are hydrolyzed by cholesterol esterase into free cholesterol. this is then oxidized by cholesterol oxidase into cholest-4-en-3-on and peroxides. the peroxides are reacted with 4-aminophenazone and phenol to form the colored 4-(p-benzoquinonemonoimino)-phenazone. for ldl cholesterol determination, reagent no. 1 solubilizes chylomicrons, hdl and vldl cholesterol to yield free cholesterol, which is subsequently oxidized into peroxides. the peroxides are then reacted with 4-aminoantipyrine. next, reagent no. 2 releases the ldl from the ldl cholesterol produced in the first reaction so that the ldl is oxidized to form peroxides. these latter are reacted with n,n-bis(4-sulphobutyl)-mtoluidine-disodium (dsbmt) into a blue-violet colored complex. for hdl cholesterol determination, hdl cholesterol is separated from other lipid components and solubilized by a specific detergent. the hdl cholesterol is then oxidized and released from its esters, thus forming peroxides. these are reacted with 4aminoantipyrine and dsbmt using peroxidase catalyst to form a red-colored complex. for triglyceride concentration determination, the triglycerides in the sample are hydrolyzed into glycerol and free fatty acids. the glycerol is then phosphorylated to yield glycerol-3-phosphate, which is then oxidized into dihydroxy-acetonephosphate, forming peroxides. the peroxides are reacted with 4-aminophenazone to form the colored 4-(p-benzoquinone-monoimino)phenazone. the atherogenic index was calculated from the formula: (total cholesterol – hdl cholesterol) /hdl cholesterol.(1) plasma mda concentration was measured using thiobarbituric acid reactive substances (tbars) according to the satoh method. the pink-colored chromogens that are formed by the reaction of barbituric acid with m d a a r e m e a s u r e d a t 5 3 0 n m . ( 1 3 ) t h e d e t e r m i n a t i o n w a s p e r f o r m e d a t p r o d i a laboratories using high-performance liquid chromatography (hplc). ethical clearance ethical clearance was issued by the research ethics commission, faculty of medicine, trisakti university. data analysis the kolmogorov-smirnov test was used to test for normality of the data distribution. since the data were normally distributed, multiple 209 univ med vol. 34 no.3 regression analysis was used to determine the variables which the greatest influence on ai. a pvalue of <0.05 was declared to be statistically significant. result the 80 study subjects who met the inclusion and exclusion criteria were between 50 to 70 years of age, comprising 20 males (25%) and 60 females (75%), with mean age of 60.06 ± 3.30 years. with regard to educational level, 6 subjects (7.5%) had no education, 8 subjects (10%) did not finish primary school, 25 subjects (31.3%) finished primary school, 15 subjects (18.8%) were junior high school graduates, 25 subjects (31.3%) were senior high school graduates, and 1 subject (1.3%) was of academic level. with regard to employment, 38 subjects (47.5%) were employed and 42 subjects (52.5%) were unemployed. mean systolic blood pressure was 137.44 ± 20.93 mmhg and mean diastolic blood pressure 84.33 ± 11.08 mmhg. mean body mass index was 25.94 ± 3.48 kg/m2 (tables 1 and 2). mean total cholesterol was 205.53 ± 36.49 mg/dl, mean ldl cholesterol 140.08 ± 34.31 mg/dl, mean hdl cholesterol was 48.33 ± 8.56 mg/dl, mean triglyceride concentration was 141.90 ± 72.36 mg/dl, mean systolic blood pressure was 137.44 ± 20.93 mmhg, mean table 2. mean values of serum lipid profile, blood pressure and malondialdehyde in subjects (n=80) table 1. distribution of subject characteristics (n=80) diastolic blood pressure 84.34 ± 11.08 mmhg, mean malondialdehyde concentration (mda) was 0.35 ± 0.09 µm, and mean atherogenic index was 3.34 ± 0.92 % (table 2). the results of multiple linear regression analysis showed a significant association between ldl cholesterol (p=0.000), triglycerides (p=0.000) and ai. there was no significant association between ai on the one hand and age (p=0.836), mda, (p=0.867), and bmi (p=0.949), systolic blood pressure (p=0.477) and diastolic blood pressure (p=0.933) on the other. the variable triglyceride concentration had a great influence on ai (β=0.008;beta=0.616; p=0.000) (table 3). discussion among the 80 study subjects a significant association was found between increases in ai and ldl cholesterol (p=0.000) and triglyceride concentration (p=0.000). these results are similar to the study results of nwagha et al.(9) who found that increases in triglyceride and total cholesterol concentrations result a higher ai. atherogenic index is a strong indicator of cardiovascular disease.(3,9) in this study a nonsignificant association was found between ai and mda. similar results were found in the study of jasim,(14) but different from 210 mawi, chondro, chudri triglyceride serum atherosclerotic index the results of the study of yang diperoleh amirkhizi et al.,(15) linna m et al.(16) and emokpae et al.(17) who found a significant association between ai and ma. this may have been caused by the fact that the study subjects came from different populations. our study was conducted on healthy subjects, whereas the study by amirkhizi et al.,(15) linna m et al.(16) and emokpae et al.(17) were carried out on pre-hypertensive women, subjects with hyperlipidemia, and subjects with adult sickle cell nephropathy. no significant association was found between ai and systolic and diastolic blood pressure this differs from the results of the study by amirkhizi f et al.(15) and goyal et al.(18) who found an association between changes in lipid concentrations and hypertension. systolic blood pressure increases progressively with advancing age, in comparison with diastolic blood pressure. oxygen is important and also toxic to human life. within the cell, through enzymatic and nonenzymatic processes, oxygen receives an electron and is transformed into reactive oxygen species (ros) that damages cellular lipids, which are broken up into smaller parts, such as mda, 8-isoprostane, 8isoprostaglandins-f2 and 8-hydroxy-2odeoxyguanosine. oxidative stress occurs when there is an imbalance between ros production and the antioxidant defense system. ros is produced naturally in normal metabolism and plays an important role in biologic processes, such as the killing of bacteria.(19) upon environmental stress, ros levels increases dramatically and may damage the cellular structures, particularly when there is no defense mechanism by antioxidants, such as the enzyme sod or the antioxidant vitamins a, c, e, and polyphenols.(18) vascular changes may be mediated in several ways, therefore oxidative stress may result in the proliferation of vascular smooth muscle cells accompanied by hypertrophy and accumulation of collagen, causing thickening of the tunica media and narrowing of the vascular lumen. in addition, increased oxidative stress may damage the endothelium and disturb vascular relaxation, thus increasing vascular contractile capacity. all of these influences on the blood vessels may lead to hypertension.(20,21) in our study we did not find a significant association between ai and bmi. this agrees with the results of the study by ranjit et al.(22) who found no correlation of bmi and lipid profile with ai. in the present study there was also no significant association between ai and age. advancing age increases the atherosclerotic process and thus ai. the lack of a significant association in this study may have been caused by the small sample size and the narrow age range. these results was in contrast with those of nghawa ut et al.(10) who stated that advancing age increases ai in postmenopausal women. epidemiological studies have shown a positive association between total cholesterol concentration and coronary heart disease. in a number of patients the total cholesterol concentration is not an accurate predictor of the risk of coronary heart disease, because total table 3. multiple linear regression analysis of several independent variables versus atherogenic index (n=80) legend: b=regression coefficient; beta=standardized coefficient; mda=triglycerides, and malondialdehyde; ldl=low density lipoprotein; hdl=high density lipoprotein 211 univ med vol. 34 no.3 cholesterol is a combination of all types of cholesterol, not only atherogenic lipoproteins such as low-density lipoproteins (ldl), very-low density lipoproteins (vldl), and intermediate lipoproteins (idl), but also anti-atherogenic lipoproteins such as high-density lipoproteins. smaller ldl particles are more atherogenic than larger ones. particles that are larger and contain much triglycerides and other smaller particles such as small vldls and idls, known as apo c-iii containing lipoproteins, are extremely atherogenic. a case-control study with prospectively collected samples found that small ldl particles and high triglyceride concentrations are the major independent risk factors for myocardial infarction. several studies have demonstrated an inverse association between lipoprotein size and the ability to penetrate the endothelial barrier and enter the arterial intima. chylomicrons and large vldls cannot enter the arterial wall, but small vldls and idls may do so. therefore triglycerides rich in lipoproteins are termed atherogenic.(23) the design of our study was cross sectional, so that our study cannot explain any cause-andeffect association between triglyceride concentration and ai. the clinical implication of our study results is that increased serum triglyceride concentration is a risk factor for atherogenesis. atherosclerosis is a basic cause of heart attacks, stroke, and peripheral vascular disease.(1) in healthy persons with high serum triglyceride concentrations but no signs of cardiovascular or cerebrovascular disease, further investigations are necessary to determine the presence or absence of vascular obstruction, particularly in the coronary arteries. further studies of longitudinal design are necessary to show a cause-and-effect association between serum triglyceride concentration and ai. conclusions t h e p r e s e n t s t u d y s u c c e e d e d i n demonstrating that higher serum triglyceride concentration increases ai scores in healthy subjects 50–70 years of age. subjects with high serum triglyceride level but without symptoms of cardiovascular disease should be examined for the development of coronary artery blockage. conflict of interest the authors declare that they have no competing interests. acknowledgment the investigators thank the dean and vicedeans of trisakti university for moral and material support in the conduct of this study. thanks are also due to the head of mampang health center (puskesmas mampang) and staff and the study subjects who participated in the study. 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c:\users\universa medicina\docu 178 abstract universa medicina myogenin level tends to decrease in adult male cigarette smokers augustine c. ihim*, patrick o. manafa*, vincent t. ekechukwu* manafa i. vera**, victor n. chukwudi***, and sabina o. ogbuowelu* background the world health organization has warned that cigarette smoking is an avoidable risk factor for endothelial injury. myogenin might play a role in muscle metabolism and energy utilization. electrolytes and minerals are involved in most cellular activities. the objective of this study was to compare myogenin and electrolyte levels between adult male cigarette smokers (cs) and non-smokers (ns). methods a cross-sectional study was conducted involving 90 subjects, consisting of 55 cs and 35 ns. the sandwich enzyme-linked immunosorbent assay was used to determine myogenin levels while the ion-selective electrode method was used to determine electrolyte levels. the levels of sodium, potassium, and chloride and the body mass index (bmi) were measured. mann-whitney and independent t-test were used to analyse the data. results the bmi of cs was significantly lower than that of ns (p < 0.05). the mean serum levels of sodium (145.23 ± 1.87), potassium (4.00 ± 0.31) and chloride (103.95 ± 1.60) were significantly higher in the cs than in the ns (these being 142.38 ± 2.49, 3.83 ± 0.33, and 101.48 ± 2.08, respectively) (p<0.05). myogenin levels (44.24 ± 14.60 pg/ml) tended to decrease in the cs group compared to the ns group (59.66 ± 61.73 pg/ml), but the difference was statistically not significant (p=0.769). conclusion the study demonstrated that higher concentrations of sodium, potassium and chloride with lower concentrations of myogenin in smokers may be associated with higher risk of skeletal muscle tissue injury probably due to the inability of the affected small blood vessels to transport electrolytes to tissues. keywords: tobacco, myogenin, electrolyte, smokers original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.178-183 september-december 2020 vol.39no.3 cite this article as: ihim ac, manafa po, ekechukwu vt, vera mi, chukwudi vn, ogbuowelu so. myogenin level tends to decrease in adult male cigarette smokers. univ med 2020;39:178-83. doi: 10.18051/univmed.2020.v39.178-183 *chemical pathology unit, department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, nnewi campus, anambra state, nigeria **pathology department, east kent hospital university foundation trust, kent ***department of medical laboratory science, faculty of health sciences and technology, owerri, imo state, nigeria correspondence: augustine c. ihim, ph.d. chemical pathology unit, department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, p.m.b. 5025 awka, anambra state, nigeria email:ac.ihim@unizik.edu.ng phone +2348035985883 orcid id : 0000-0001-9991-0714 date of first submission, june 15, 2020 date of final revised submission, october 20, 2020 date of acceptance, october 27, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 179 univ med vol. 39 no.3 introduction cigarette production involves a number of substances and materials other than just tobacco, paper and a filter. tobacco additives include flavorings, enhancers, humectants, sugars, and ammo nium co mpoun ds . ( 1) nic ot in e is a n addictive substance in cigarettes which causes regular intake of the cigarette smoke that leads t o h e a lt h h a r ms a nd de a t h. to da y, t he uncountable nega tive effects of cigarette smoking are widely accepted.(2) the rate of smoking is still rising in some parts of developing nations (3) and close to half of cigarette smokers die of tobacco-related diseases and lose an average 14 years of life.(2) cigarette smoke contains about 7,000 chemical compounds, including not limited to cyanide, lead, nicotine, carbon monoxide, arsenic, formaldehyde, acrolein and other harmful substances and about 70 of these can lead to cancer.(4) smoking, a preventable risk factor for endothelial injury, is said to have become the global number two killer after hypertension.(5) cigarette use is the leading cause of preventable diseases, which could result in pathologies of most organs and tissues in the body, such as the kidneys and oral cavity.(6) cigarette smoking also has great effect on systemic tissues such as skeletal muscle.(7) these tissues are important in the day-today hormonal functions of the body and its metabolism, including electrolyte balance and myo ge ni n s e n s i ti vi t y wh i c h s h ow s t h e remarkable and harmful impact of smoking on metabolism of skeletal muscle and kidney homeostatic function. cigarette smoke contains substances that can stimulate the breakdown of muscle protein and inhibit protein synthesis and this could lead to muscle dysfunction.(8) myogenin is a muscle-specific transcription factor which helps in the co-ordination of the development and repair of skeletal muscle.(9) myogenin as a skeletal muscle biomarker can denote the presence of skeletal muscle dysfunction. even though its role in adult skeletal muscle is still u nc e r t a i n, pr e vi ous s tud ie s s ugge s t tha t myogenin has a vital role as a high-level transcriptional regulator that controls the energy b a l a nc e be tw e e n a e r ob i c a n d a na e r ob i c metabolism of the skeletal muscle of an adult.(10) electrolytes, which are substances that can conduct electric current, are located in the intracellular and extracellular fluid in the body with sodium being the major cation and chloride being the major anion in the extracellular fluid. potassium is the major cation in the intracellular fluid. electrolytes can dissociate in solution and play a major role in homeostasis.(11) the serum electrolytes which include sodium, potassium, chloride and bicarbonate enable the body to maintain and regulate nerve function, water balance, acid-base balance and muscle function and these electrolytes can be increased or decreased in pathological conditions such as organ damage (e.g. of the kidneys) or diseased conditions which lead to imbalance of the serum electrolytes.(12) sodium and potassium are vital for nerve function and nerves have the function of signaling the muscle to contract and relax by passing electric impulses from the brain to the muscle tissues. during muscle relaxation, sodium levels are higher inside the muscle cells than outside. during contraction, which is initiated by a signal from the nerves, sodium flows into the cell while potassium flows out.(13) also research h a s s h ow n t he i mp or t a nc e o f c a l c i u m, magnesium and chloride in the contraction and relaxation of the skeletal muscles.(14,15) nu me r o us s t u di e s h a ve s ho wn t h a t smoking has a very harmful effect on the lungs with an indirect effect on the tissues of the muscles. a recent study shows that smoking decreases the number of small blood vessels that bring oxygen and nutrients to muscles in the legs.(16) it was discovered that smoking makes the muscles weaker and thereby limits the ability of an individual to exercise and this was due to inflammation of the lungs and subsequent destruction by habitual smoking thereby limiting exercise. recent findings suggest that cigarette smoke damages muscle cells directly by reducing the number of blood vessels in leg muscles which 180 leads to oxygen reduction and reduced nutrient intake. this can have an effect on muscle metabolism and its level of activity.(16) myogenin is present in skeletal muscle and is essential to promote terminal differentiation of myocytes. it has been observed that longterm cigarette smoking significantly decreases myogenin expression in the gastrocnemius tissues of rats.(17) besides, salidroside mitigates skeletal muscle atrophy in rats with cigarette smoke-induced chronic obstructive pulmonary disease (copd) by up-regulating myogenin and d ow n-r e gul a t i ng myo st a t i n exp r e s s i on . electrolyte imbalance could greatly affect skeletal muscle function. hypophosphatemia and hypokalemia are the most known causes of myopathy.(18) a study in thirty smokers and thirty non-smokers as matched controls showed a significant increase in sodium level immediately after smoking and no significant change in potassium level in smokers.(19) another study involving 100 male smokers and 40 non-smokers showed a significant effect on the activity of liver enzyme, calcium, phosphate and iron levels in smokers compared to non-smokers but no significant effect on sodium, chloride and potassium levels in smokers.(20) previous studies have focused on the effect of smoking on expression of electrolytes and included myogenin as difference variable. the objective of the present study was to compare the levels of myogenin and electrolytes between adult male cigarette smokers and non-smokers. methods research design this cross-sectional study was designed to compare the levels of myogenin and electrolytes between adult male smokers and non-smokers. this study was carried out between january and august, 2019. study subjects the sample size was calculated using g*power software version 3.0.10 (heinrich heine universität düsseldorf, germany). power analysis for difference between two independent means (groups) was conducted in g*power to determine a sufficient sample size using an alpha of 0.05, a power of 0.80 and a medium effect size. based on these, the calculated total sample size of 84 has a power of 80% to detect a difference of 0.50 at a significance level of 0.05. a total of 90 participants within the age of 1865 years were recruited by convenience sampling for this study, comprising 55 smokers (test) and 35 non-smokers (control). the inclusion criterion was male cs and ns within the age range of 18-65 years. and the exclusion criterion was subjects with underlying disease such as diabetes mellitus, autoimmune disorders and skeletal muscle diseases. participants for this study were recruited from nnamdi azikiwe university teaching hospital, restaurants and drinking pubs within nnew metr opolis, anambra state, nigeria. laboratory analysis determination of the levels of electrolytes was based on a potentiometric method using ion selective electrodes (21) while the evaluation of the levels of myogenin was based on the double antibody sandwich enzyme-linked immunosorbent assay (elisa) using the human myogenin (myog) elisa kit-np_002470.2 (catalog no. mbs765394, mybiosource, san diego (usa)) method according to the ma nufacture r ’s instructions, as described also in a previous protocol.(22) statistical analysis the ibm statistical package for social sciences (spss) version 21 was used for the statistical analysis. in order to decide whether to use parametric or non-parametric tests to calculate significance, the distribution of each group was tested for normality. frequency, duration of cigarette smoking, myogenin, sodium, chloride, and bicarbonate levels were not normally distributed while weight, height, age, potassium and bmi were normally distributed. ihim, manafa, ekechukwu, et al myogenin level tends and cigarette smokers 181 univ med vol. 39 no.3 a b c the majority of the variables were non-normally d is t r i b ute d . t he ma n n -w h it ne y u. a n d independent-t test were used to subject all collected data to statistical analysis. values were deemed significant at p<0.05. ethical approval the ethical approval for this research was obtained from nnamdi azikiwe university teaching hospital ethics committee with reference nauth/cs/66/vol.12/46/2019/ 078. results the mean body mass index (kg/m2) of cs (23.26 ± 1.69) compared with ns (23.92 ± 1.61) was significantly raised (p<0.05). however, mean age was not significantly different between cs and ns (p=0.630) (table 1). discussion the significantly lower body mass index in smokers found in this study corroborated previous studies.(23-25) it has been shown that smoking and nicotine reduce weight by raising the demand for body energy and suppressing a p pe ti t e . ( 2 6 ) smoki ng ha s be e n l i n ke d t o promoting some genetic variants thus causing low bmi value.(27) the low bmi value is also a t t r i b ut e d to a n u mb e r o f ph ys i o lo gi c a l mechanisms associated to the possible role of behavioral pattern common with cigarette s mo ke r s . on e st u dy ( 2 3 ) e xa min e d t h e relationships between sedentary behavior, smoking status, viz. current smokers, nonsmokers and former smokers and weight-related outcomes. sedentary behavior was significantly higher among current smokers compared to nonsmokers and former smokers; former smokers h ad hi ghe r l evel s o f s e de nta ry be ha vior compared to never smokers.(23) it was seen that at low levels of sedentary behavior, current smokers and never smokers did not have significant differences in bmi. however, at moderate and high levels of sedentary behavior, current smokers had a significantly lower bmi compared to non-smokers in those sedentary behavior categories. previous research has demonstrated that nicotine affects the body by increasing its metabolism.(25) thus, nicotineinduced increase in metabolism could have been responsible for lower bmi among smokers.(23) the significantly higher levels of serum sodium, potassium and chloride observed in the present study may be attributed to the effects of cigarette-smoking on small blood vessels leading to reduced blood flow to muscle tissues. the reduced blood flow could lead to reduced flow of electrolytes to the muscles,(16) and could raise the levels of the electrolytes in the blood. the table 1. comparison of age and body mass index between of cigarette smokers and nonsmokers *statistically significant at p<0.05; independent ttest variable smokers (n = 55) non-smokers (n = 35) p-value age (years) 34.70 ± 9.51 38.75 ± 15.97 0.630 bmi (kg/m2) 23.26 ± 1.69 23.92 ± 1.61 *0.024 variable smokers (n = 55) non-smokers (n = 35) p-value myogenin (pg/ml) 44.24 ± 14.60 59.66 ± 61.73 0. 769a sodium (mmol/l) 145.23 ± 1.87 142.38 ± 2.49 0.000*a potassium (mmol/l) 4.00 ± 0.31 3.83 ± 0.33 0.118*b chloride (mmol/l) 103.95 ± 1.60 101.48 ± 2.08 0.000*a bicarbonate (mmol/l) 22.40 ± 1.88 22.68 ± 1.95 0.615a table 2. comparison of myogenin and electrolyte levels between smokers and non-smokers significant at p<0.05;a mann-whitney test;b independent t-test 182 reduction in small blood vessels leads to reduced amounts of oxygen and nutrients they can receive. this can impact on muscle metabolism and activity levels, both of which are risk factors for many chronic diseases including diabetes and c h r o ni c ob st r uc t i ve p ul mon ar y d i se a se (copd).(16) research has shown that with advancement in age of humans and animals, they are less able to regulate sodium and water re ten tio n, uri ne c onc ent ra tion an d thir st compared to their younger counterparts.(28) with increase in duration of smoking, there could be incre ase in sedentary behaviors and also increased physiological effects of nicotine (29) which could lead to an increase in body mass index. the lower values of serum myogenin seen in this study could be due to the presence of muscle stress caused by incre ase in a ge, frequency of smoking and duration of smoking. smoking could damage the muscle cells directly and reduce the number of blood vessels in the leg muscles leading to tissue hypoxia. it is difficult to identify the causes of abnormal serum electrolytes in smokers and non-smokers due to the nature of study design which describes only what is happening at the present time. further research is needed using a better design such as cohort study to evaluate the effect of smoking on myogenin and electrolyte levels. conclusion the observed higher concentrations of sodium, potassium and chloride with lower concentrations of myogenin in smokers may be associated with higher risk of skeletal muscle tissue injury probably due to the inability of the affected small blood vessels to tra nsport electrolytes to tissues. the risk is increased with age, duration of smoking and frequency of smoking. conflicts of interest the authors declare that they have no conflicts of interest. authors’ contributions aci, pom, vte and vnc conceived and designed the research proposal.vte, soo and v n c pe r f or me d s a mpl e c ol l e ct i o n, a nd experiments and aci and vim analyzed the data. discussion was done by vim while aci, vim and pom contributed to the final version of the manuscript. all authors have read and approved the final version. acknowledgements the authors express their gratitude to the management and staff of nnamdi azikiwe university teaching hospital, nnew, anambra state for the permission to analyze the samples in their laboratory. references 1. paumgartten fjr , gomes-carneiro rm, xavier de oliveira aca. the impact of tobacco additives on cigarette smoke toxicity: a critical appraisal of tobacco industry studies. cad. saúde pública 2017;33 sup 3:e00132415. doi: 10.1590/0102311x00132415. 2. di 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518:197-206. 28. ji h, zheng w, wu x, et al. aging and related impairment of urine concentrating mechanisms correlates with dysregulation of adrenocortical angiotensin type 1 receptors in male fischer rats. am j physiol regul integr comp physiol 2016;310:r513–21. doi: 10.1152/ajpregu.00131. 2015. 29. nikoloutsou i, vasileiou v, litsiou e, et al. smoking cessation changes basic metabolism, body weight, leptin and insulin levels, adipose tissue percentage, index of insulin resistance and index of insulin secretion. tob induced dis 2014;12:a24. https://doi.org/10.1186/1617-9625-12-s1-a24. psidium guajava leaves decrease arthritic symptoms in adjuvant-induced arthritic rats 197 *department of pharmacy, faculty of health sciences, jenderal soedirman university, purwokerto, central java, indonesia **medical faculty, universitas brawijaya, malang, east java, indonesia correspondence: hanif nasiatul baroroh, m.sc, apt. department of pharmacy, faculty of health sciences, jenderal soedirman university, jl. dr. soeparno, karangwangkal, purwokerto, central java, indonesia email: h_baroroh@yahoo.co.id univ med 2015;34:197-204 doi: 10.18051/univmed.2016.v35.197-204 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background guava is an herbal with proven antioxidant and anti-inflammatory properties. the aim of this study was to investigate the anti-arthritic activity of the ethanol extract of psidium gujava leaves (eepg) against complete freund’s adjuvant (cfa) induced arthritis in rats. methods an experimental study was conducted on 40 male wistar sprague dawley rats, which were divided into 5 groups. each group was induced with 0.2 ml cfa (1 mg/ml) on day 1 and 0.1 cfa ml booster injection on day 5. group i served as an arthritic control, group ii received dexamethasone (6.75 mg.kg-1 orally), group iii, iv and v received eepg at oral doses of 250, 500, and 750 mg/kg bw, respectively, on days 14 to 28. antiarthritic activity was observed from the arthritis score, the paw circumference was measured on days 0, 1, 4, 8, 12, 16, 20, 24, and 28, the mobility score was determined on days 12 and 28, and the histolopathology of the knee joint was examined on day 29. results ethanol extract of psidium guajava leaves significantly suppressed the swelling of the paws in chronic phase based on increasing of edema (%), while starting on day 20. eepg at 250 mg/kg was most effective in significantly reducing arthritis scores (p<0.05). histopathological examination showed repair of the knee joint synovial membrane and cartilage. conclusions psidium guajava leaf extract is effective in decreasing the inflammatory response and arthritic symptoms in rats with adjuvant-induced arthritis. psidium guajava leaves can be developed into an alternative anti-arthritis treatment. keywords: psidium guajava, antiarthritis, edema, adjuvant induced arthritis, rats psidium guajava leaves decrease arthritic symptoms in adjuvant-induced arthritic rats hanif nasiatul baroroh*, esti dyah utami*, and anisyah achmad** doi: http://dx.doi.org/10.18051/univmed.2015.v34.197-204 198 baroroh, utami, achmad psidium guajava leaves arthritic symptoms daun psidium guajava menurunkan respon inflamasi dan gejala artritis pada tikus terinduksi adjuvant induced arthritis latar belakang daun jambu biji merupakan salah satu tanaman herbal yang telah terbukti sebagai antioksidan dan antiinflamasi. penelitian ini bertujuan untuk menyelidiki aktivitas anti artritis dari efek ekstrak etanol daun psidium guajava (eepg) terhadap artritis yang terinduksi complete freund adjuvant (cfa) pada tikus. metode penelitian eksperimantal dilakukan pada 40 ekor tikus sprague dewley wistar jantan. mereka dibagi menjadi 5 kelompok dan tiap kelompok diinduksi dengan 0,2 ml cfa (1 mg/ml) pada hari ke-1 dan diberikan injeksi booster dengan 0,1 ml cfa (1 mg/ml) pada hari ke-5. kelompok i sebagai control arthritis, ii diberikan dexamethason (6,75 mg.kg-1 p.o.), kelompok iii, iv dan v diberikan eepg dengan dosis 250 mg/kg bb (p.o), 500 mg/kg bb (p.o) dan 750 mg/kg bb (p.o) pada hari ke 14-28. aktivitas antiarthritis diamati melalui skor artritis, lingkar kaki diamati pada hari ke 0, 1, 4, 8, 12, 16, 20, 24, dan 28, skor mobilitas diamati pada hari ke 12 dan 28 serta histopatologi sendi lutut diamati pada hari ke-29. hasil ekstrak etanol daun psidium guajava mampu menurunkan pembengkakan secara signifikan mulai hari ke 20. eepg dosis 250 mg/kg bb paling efektif dalam menurunkan skor artritis secara signifikan (p<0,05). hasil histopatologi sendi lutut menunjukkan perbaikan pada membran sinovial dan kartilago. kesimpulan ekstrak daun psidium guajava efektif menurunkan respon inflamasi dan gejala artritis pada tikus yang terinduksi adjuvant induced arthritis. daun jambu biji dapat dikembangkan menjadi terapi alternatif antiartritis. kata kunci : psidium guajava, antiarthritis, edema, adjuvant induced arthritis, tikus abstrak introduction r h e u m a t o i d a r t h r i t i s ( r a ) i s a n autoimmune disease that attacks the joints of the body and causes chronic inflammation in the synovium. inflammation in ra joints is a very complex process and involves the interaction of a variety of inflammatory cells, autoantibodies and cytokines.(1) conventional treatments that have been used to overcome arthritis and inhibit the development of ra involve the disease modifying anti-rheumatic drugs (dmards).(2) traditional medicine using plant extracts continues to provide health coverage for over 80% of the world’s population, especially in the developing world.(3) recently, the use of plant extracts for arthritis treatment is being promoted in the usa, especially after the withdrawal of fda-approved anti-inflammatory drugs. one of the plants that have the potential to be developed as an antiarthritic drug is the guava plant (psidium guajava). guava contains tannins, phenolic compounds, flavonoids, volatile oils, sesquiterpenes and triterpenoids.(4) flavonoids and phenolic compounds contained in guava have been proven to be antioxidant and antiinflammatory.(5) jahagirdar et al.(6) reported that a hydroalcoholic extract of guava leaves at a dose 199 univ med vol. 34 no.3 of 200 mg/kg/day showed antiarthritic activity. dutta and das (7) observed that an ethanol extract of guava leaves at doses of 250 mg/kg and 500 mg/kg body weight significantly inhibited chronic inflammation and reduced the arthritis index. ojewole (8) suggested that the aqueous extract of guava leaves in the dose range of 50800 mg/kg administered intraperitoneally had analgesic and anti-inflammatory activity in rats with egg albumin-induced arthritis. the aqueous extract of guava leaves at doses of 125, 250, and 500 mg/kg had antiinflammatory activity and decreased edema in rats, with percentage inhibition of 40.81%, 55.45%, and 43.61%, respectively.(9) the freund’s adjuvant model was chosen in this study, as it develops chronic swelling in multiple joints, with influence of inflammatory cells with erosion of joint cartilage and bone destruction. freund’s complete adjuvant-induced arthritis is a well established rat model and has been widely used for many years for evaluation of the anti-inflammatory and anti-arthritic potential of various agents.(10,11) guava leaves has the potential to be developed as an antiarthritic drug. the present study aimed to evaluate the anti-arthritic activity of the ethanol extract of the leaves of psidium guajava (eepg) against complete freund’s adjuvant (cfa) induced arthritis in rats, especially with reference to its anti-inflammatory properties, using dexamethasone as reference drug. methods research design this study used a completely randomized design of undirectional pattern. freund’s adjuvant induced arthritis model was used to assess the anti-arthritic activity of the ethanolic extract of p.guajava in rats. the research was carried out in the laboratory of pharmaceutical biology, department of pharmacy, faculty of medicine and health sciences, universitas jenderal sudirman and the laboratory of pathology and anatomy, medical faculty, gadjah mada university, yogyakarta. the study was conducted from agustus 2013 to february 2014. animals the study was conducted in male sprague dawley (sd) rats weighing 130-150 g obtained from the animal house in the faculty of pharmacy, gadjah mada university. animals were acclimatized to experimental conditions in cages and kept under standard environmental conditions (22 ± 3ºc; 12/12 h light/dark cycle). rats were allowed to feed and water ad libitum. plant material l e a v e s w e r e c o l l e c t e d f r o m karangwangkal, purwokerto. taxonomic identification of the plant was made by the laboratory of taxonomy, faculty of biology. preparation of extract t h e p o w d e r e d l e a v e s ( 1 0 0 0 g ) w e r e successively extracted with ethanol (70-80ºc) for 3x24 h. the macerate was then evaporated in a rotary evaporator for ± 90 minutes in the temperature range of 70-80°c, then evaporated over a water bath until a thick extract was produced, which was stored in the refrigerator until used in the study. experimental animals animals were randomly divided into five groups (n=8). the sample size determination was b a s e d o n f e d e r e r ’s f o r m u l a a n d t h e randomization of the test animals in this research used a completely randomized design (crd) of unidirectional pattern. group i receiving 1 mg/ ml complete freund’s adjuvant (cfa) served as an arthritic control, group ii receiving dexamethasone (6.75 mg.kg-1 p.o.) served as the reference standard, while group iii, iv and v received ethanolic extract at doses of 250 mg/kg bw (p.o), 500 mg/kg bw (p.o) and 750 mg/kg bw (p.o), respectively. each group was induced by 0.2 ml cfa(1 mg/ml) on day 1 and received 200 baroroh, utami, achmad psidium guajava leaves arthritic symptoms a booster injection of 0.1 ml cfa (1 mg/ml) on day 5. to assess the anti-arthritic activity of p. gujava, the extracts were given on days 1428. evaluation of arthritic score each paw was scored on a scale of 0–4 for the degree of swelling, erythema and deformity (maximum score 16 per animal) as follows: 0 = normal, 1 = slight erythema and/or swelling of the ankle or wrist, 2 = moderate erythema and/ or swelling of ankle or wrist, 3 = severe erythema and/or swelling of ankle or wrist and 4 = complete erythema and swelling of toes or fingers and ankle or wrist and inability to bend the ankle or wrist. (12) the arthritic score was measured on days 0, 1, 4, 8, 12, 16, 20, 24, and 28. evaluation of mobility score whole animal mobility was scored between 0 and 4 according to the following definitions: 0 = normal, 1 = slightly impaired, 2 = major impairment, 3 = does not step on paw, and 4 = no movement. the mobility score was measured on days 12 and 28. evaluation of paw edema the paw edema was measured on days 0, 1, 4, 8, 12, 16, 20, 24, and 28. the mean changes in injected paw edema with respect to initial paw volume, were calculated on respective days and the percentage of inhibition of paw edema with respect to the untreated group was calculated on respective days by the following formula: (13) processed for preparation of paraffin-embedded tissue sections (7 ìm thick), which were stained with hematoxylin and eosin. statistical analysis the data were expressed and analyzed using spss software. statistical analysis of difference between groups was evaluated by one-way anova followed by lsd test. the value of p<0.05 was regarded as statistically significant. ethical clearance this study was accorded ethical clearance by the commision on research ethics in medicine and health sciences, faculty of medicine, universitas jenderal soedirman. results injection of cfa increased edema, with a peak increase of 29.46% ± 5.58 on day 12 following the injection of cfa in the arthritic control group (table 1). over the 28-day study, rats treated with extract at doses of 250, 500 and 750 mg/kg showed a significant decrease in edema (p<0.05) starting on day 20 up to day 28, a s c o m p a r e d t o c o n t r o l r a t s ( f i g u r e 1 ) . dexamethasone, the standard anti-arthritic drug, also showed a significant decrease in edema. there was an increase in arthritic scores after induction with cfa that indicated systemic inflammation due to the arthritic condition (figure 1). in the arthritic control group, the arthritis scores remained at their peak level from day 8 to day 24, and started to decrease on day 28. following the cfa injections, the rats developed arthritis beginning on day 4 and reached a peak level on day 8. rats treated with dexamethasone and p.guajava leaf extract showed a significant decrease in arthritis score. throughout the 28-day study, all arthritis groups showed a decrease in mobility scores that was statistically not significant (table 2). rats treated with p. guajava extract with the dose of 250 mg/kg showed the greatest decrease in mobility scores. 100%x vo vo -vt (%) edema of increasing       = vt =paw edema on day t vo = paw edema on day 0 histological processing and assessment of arthritic damage rats were killed by ether anesthesia. knee joints were removed and fixed for 4 days in 10% formalin. after that the specimens were 201 univ med vol. 34 no.3 table 1. the increasing of edema (%) in the control and treated animals groups with p.guajava extracts on freund’s adjuvant complete induced arthritis values expressed in average ± sem of 8 rats; a= significance at the level of p<0.05 compared by control arthritis; b= significance at the level of p<0.05 compared by standard drug histopathologically, after treatment with dexamethasone (figure 2b), there was a reduction in the inflammatory condition of the synovial membranes, accompanied by cartilage repair, compared to the arthritis in the knee joints of the controls. the group treated with guava leaf extract at the dose of 250 mg/kg (figure 2c) also showed a reduction in the i n f l a m m a t o r y c o n d i t i o n o f t h e s y n o v i a l membranes, accompanied by cartilage repair, while treatment with guava leaf extract at the doses of 500 mg/kg (figure 2d) and 750 mg/ kg (figure 2e) showed repair of the synovial membranes. figure 1. arthritis scores in control and experimental groups. as a result of inflammation induced by complete freund’s adjuvant, the arthritis scores were increased (p<0.05) in treated rats when compared with control rats. treatment with dexamethasone and psidium guajava showed significantly decreased (p<0.05) arthritis scores beginning on day 20 202 baroroh, utami, achmad psidium guajava leaves arthritic symptoms table 2. distribution of mean of mobility score by treatment groups after 12th and 28th day on freund’s adjuvant complete induced arthritis values of mobility score expressed in average ± s.e.m of 8 rats figure 2. histopathology of rat knee joint tissues; a (arthritic rat): inflammation of the synovial membrane and cartilage destruction; b (rat on dexamethasone): reduction of synovial membrane inflammation and cartilage repair; c (rat on eepg 250 mg/kgbb): reduction of synovial membrane inflammation and cartilage repair; d (rat on eepg 500 mg/kg): synovial membrane repair; e (rat on eepg 750 mg/kgbb): synovial membrane repair. he staining, magnification 40x. synovial membrane (ms), joint cavity (rs), cartilage (k) 203 univ med vol. 34 no.3 discussion the present study was carried out to evaluate the effect of p. guajava leaf extract in inflammatory disease. the freund’s adjuvant model was chosen as it induces chronic swelling in multiple joints, with influence of inflammatory cells with erosion of joint cartilage and bone destruction. freund’s complete adjuvant-induced arthritis is a well established rat model and has been widely used from many years for evaluation of the anti-inflammatory and anti-arthritic potential of various agents.(14,15) nagakura et al.(10) reported that the induction of arthritis in the freund’s adjuvant model in rats caused joint inflammation, inflammatory cell infiltration, and cartilage and bone destruction. freund’s adjuvant contains mycobacterium tuberculosis h37ra or mycobacterium butyricum, which release endotoxins that trigger the production of nitric oxide (no).(16) nk cells, mast cells, eosinophils, neutrophils, cells and tissues dendritic endothelium are important sources of no production associated with the immune response, because the activation of macrophages results in the production of several cytokines, including il 1, il 6, interferon γ (ifn γ) and tnf α, which have been implicated in immune arthritis.(17) various inflammatory mediators including cytokines (il1b and tnf-α), mcsf, interferons, and platelet derived growth factor (pdgf) are responsible for the swelling joints, bone deformations, and disability of joint function.(1) the progress of the arthritic condition was evident around day 12, indicating systemic inflammation.(18) the swelling was found to be increasing in the initial phase of inflammation and becoming constant within 2 weeks (beginning on day 4). these increases in arthritic and mobility scores were found to be associated with chronic inflamation. psidium guajava extract at doses of 250 mg/kg, 500 mg/kg and 750 mg/kg significantly suppressed the swelling of the paws in chronic inflammation over day 28. the p. guajava leaf extract was found to be effective in decreasing arthritic scores, mobility scores, and inflammatory responses. several studies have shown the antioxidant and anti-inflammatory properties of flavonoids. porwal et al.(4) reported that p. guajava leaf extract contained essential oils, flavonoids, triterpenoids, vitamin c, tannins, and phenolics. although the actual mechanism of suppression of the arthritic condition is not known, it can be correlated with the presence of flavonoids in suppressing the inflammation and antioxidant activity. quercetin in p. guajava suppressed and inhibited nitric oxyde (no) production, catalyzed by inducible nitric oxide synthase (inos).(19) chen et al. (20) reported that quercetin had antioxidant activity by suppressing the nf-κb s i g n a l i n g p a t h w a y. q u e r c e t i n 3 oglucopyranoside and morin in p. guajava leaves have antioxidant activity.(21) vyas et al.(22) reported that p. guajava had antioxidant activity and may have the potential to be developed as an anti-inflammatory drug. in a previous study, aqueous extract of p.guajava leaves at a dose of 250 mg/ml had anti-inflammatory activity in male albino rats with arthritis induced by carrageenan.(9) from the results observed in the current investigation, it may be concluded that p. guajava leaves have a potentially in vivo antiarthritic activity, and warrants more in-depth investigations on the mechanism of action of its anti-arthritic activity. in practice, it can be developed as an alternative anti-arthritis drug. it is suggested that p. guajava leaves may be useful in the teratment of the arthritic condition in rheumatoid arthritis. one limitation of this study was indicated by the animal ethical clearance. the study would have provided better conclusions if validated with a larger sample size. conclusion psidium guajava leaf extract is found to be effective in decreasing the inflammatory response and arthritic symptoms in adjuvant-induced arthritic rats. 204 baroroh, utami, achmad psidium guajava leaves arthritic symptoms conflict of interest all authors declare that there was no actual or potential conflict of interest. acknowledgement the authors thank universitas jenderal soedirman for financial support and gadjah mada university for providing necessary facilities to carry out the research work. references 1. mclnnes ib, schett g. the pathogenesis of rheumatoid arthritis. n engl j med 2011;365: 2205-19. 2. national collaborating centre for chronic conditions. rheumatoid arthritis: national clinical guideline for management and treatment in adults. london: royal college of physicians; 2009. 3. world health organization. traditional medicine: growing needs and potential. geneva: world health organization policy perspectives on medicines; 2002. 4. porwal v, singh p, gurjar d. a comprehensive study on different methods of extraction from guava leaves for curing various health problem. ijera 2012;2:490-6. 5. barbalho sm, machado fmv, goulart rda et al. psidium guajava: a plant of multipurpose medicinal applications. met aromat plant 2012; 4:1-6. 6. jahagirdar k, ghosh p, adil sm, et al. effect of hydroalcoholic extract of psidium guyava linn. on complete freund’s adjuvant induced arthritis in laboratory animals. pharmacologyonline 2010;3:706-19. 7. dutta s, das s. a study of the anti-inflamatory effect of the leaves of psidium guajava linn. on experimental animal models. pharmacognosy res 2010;2:313-7. 8. ojewole ja. antiinflammatory and analgesic effects of psidium guajava linn. (myrtaceae) aqueous extract in rats and mice. methods find exp clin pharmacol 2006;28:441-6. 9. weni l, harliansyah, widayanti. antiinflammatory activity of the extract of guava leaves (psidium guajava l) in the rat (rattus norvegicus l). ijcc 2011;2:169-72. 10. nagakura y, okada m, kohara a, et al. allodynia and hyperalgesia in adjuvant induced arthritis rats: time course of progression and efficacy of analgesics. j pharmacol exp ther 2003;306:490-7. 11. bevaart l, vervoordeldonk mj, tak pp. evaluation of therapeutic in animal models of arthritis, how does it relate to rheumatoid arthritis? review arthritis and rheumatism. arthritis rheum 2010;62:2192-205. 12. brand dd, latham ka, rosloniec ef. collageninduced arthritis. nature protocols 2007;2:126975. 13. panchal ah, patel rk, bhandari a. antiarthritic and synergetic activity of wedelia calendulacea l. with methotrexate in adjuvant induced arthtitis with cardioprotective activity in rat. pharmacologyonline 2012;3:175-87. 14. patil mvk, kandhare ad, bhise sd. antiarthritic and anti-inflammtory activity of xanthium strumarium l. ethanolic extract in freund’s complete adjuvant induced arthritis. biomed aging pathol 2012;2:6-15. 15. snekhalatha u, anburajan m, venkatraman b, et al. evaluation of complete freund’s adjuvantinduced arthritis in a wistar rat model, comparison of thermography and histopathology. rheumatol 2012;72:375-82. 16. stills hf. adjuvants and antibody production: dispelling the myths associated with freund’s complete and other adjuvants. ilar journal 2005;46:280-93. 17. ibiza s. the role of nitric oxide in the regulation of adaptive immune responses. cnic 2008;2: 103-7. 18. venkatesha sh, rajaiah r, berman bm, et al. immunomodulation of autoimmune arthrits by herbal cam. evid based complement alternat med 2011:986797. 19. florez m, gutirez b, sanchez s. quercetin attenuates nuclear factor-b activation and nitric oxide production in interleukin-1 activated rat hepatocytes. j nutr 2005;135:1359-65. 20. chen jc, ho fm, chao pd, et al. inhibition of inos gene expression by quercetin is mediated by the inhibition of iêb kinase, nuclear factorkappa b and stat1, and depends on heme oxygenase-1 induction in mouse bv-2 microglia. eur j pharmacol 2005;521:9–20. 21. tachakittirungrod s, ikegami f, okonogi s. antioxidant active principles isolated from psidium guajava grown in thailand. sci pharm 2007;75:179-93. 22. vyas n, tailang m, gavatia np. antioxidant potential of psidium guajava linn. intern j pharm tech res 2010;2:417-9. c:\users\universa medicina\docu 138 abstract universa medicina the diagnostic value of apparent diffusion coefficient to differentiate benign and malignant meningiomas risa marissa1*, rachmi fauziah rahayu2, hari wujoso3, subandi4, prasetyo sarwono putro2, and widiastuti soewondo2 background meningiomas are the most common primary extra-axial non-glial intracranial tumors. the severe grade of meningioma, according to who, has the highest recurrence rate accompanied by high morbidity and mortality rates. therefore, it is imperative to perform pre-operative assessments so the clinician can give prompt treatment to gain a better prognosis. it is a novel alternative way of predicting meningioma’s malignancy by calculating the tumor’s apparent diffusion coefficient (adc) value. the objective of the study was to determine the value of adc for differentiating benign and malignant meningiomas. methods this cross-sectional study involved 32 subjects with clinically diagnosed or histologically verified meningioma (21 benign and 11 malignant). they underwent a head-magnetic resonance imaging (mri) examination and biopsy. we calculated the adc value by creating regions of interest (rois) on the solid part of the tumor, guided by contrast and fluid-attenuated inversion recovery (flair) sequence. we analyzed the adc value with independent t-test and bland-altman graphs, calculated the average difference, ci 95%, limit of agreement between observers, and roc. results mean adc of malignant meningiomas (0.877 ± 0.167 x 10-3 mm2/s) was significantly lower than that of benign meningiomas (0.990 ± 0.105 x 10-3 mm2/s) (p<0.05). the adc threshold is 0.886 x 10-3 mm2/s with sensitivity 63.6%, specificity 85.7%, positive predictive value 70% and negative predictive value 81.8%. conclusion the adc value measurement provides a discriminative feature to differentiate between benign and malignant meningiomas. however, the clinical applicability still needs to be elucidated, as histopathological confirmation remains the mainstay of definitive diagnosis. keywords: meningioma, apparent diffusion coefficient, magnetic resonance imaging, histopathological grade original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.141-150 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1117 may-august 2021 vol.40no.2 cite this article as: marissa r, rahayu rf, wujoso h, subandi, putro ps, soewondo w. the diagnostic value of apparent diffusion coefficient to differentiate benign and malignant meningiomas. univ med 202 1;40 :13 8 -4 7 . doi: 10. 1805 1/ univmed.2021.v40.141-150. 1resident of radiology, faculty of medicine, sebelas maret university, surakarta 2radiology department, faculty of medicine, sebelas maret university and dr. moewardi hospital, surakarta 3forensic department, faculty of medicine, sebelas maret university and dr. moewardi hospital, surakarta 4neurology department, faculty of medicine, sebelas maret university and dr. moewardi hospital, surakarta *correspondence: dr. r. risa marissa jl. sancang raya no. 14, babakan, bogor tengah, bogor, west java, indonesia, 16128 (+62) 811-135-985 email: rmarissa@gmail.com orcid id: 0000-0002-9306-7399 date of first submission, january 24, 2021 date of final revised submission, july 23, 2021 date of acceptance, july 26, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 139 univ med vol. 40 no.2 introduction m eni ngi o ma s a r e t he mos t c ommon intracranial non-axial primary tumors, ranging from 14 to 20% of all intracranial tumors and are common in middle-aged patients, with a male: female ratio of 1:2.(1) the incidence of meningiomas is 6:100,000 population, of which 2%-3% are asymptomatic, and 8% comprise multiple meningiomas. most meningiomas are benign, slow-growing, and in s ome c a s e s c a n b e c u r e d b y s urge r y. nevertheless, 10-20% show aggressiveness both clinically and histopathologically and cause symptoms that worsen with time.(2,3) the world health organization (who) has divided meningiomas into three groups based on the histopathological findings. these groups are who grade i (typical) that occur in almost 90% of the population, who grade ii (atypical) that have an incidence of 5%-7% in the population, and who grade iii (anaplastic) that account for 1%-3% of the population. it is essential to assess the clinical prognosis to help clinicians determine the next treatment. for example, management of treatment for who grade i meningiomas can be observation or surgery. in contrast, the treatment for meningiomas of who grade ii is total surgery with/without radiotherapy, and for meningiomas of who grade iii is in the form of radical surgery accompanied by radiotherapy. the severest grade of meningioma, according to who, has the highest recurrence rate accompanied by high morbidity and mortality rates and low life expectancy in the next five years.(2) so, it is imperative to preoperatively assess the potential f o r i n va si ve me n i ngi oma t o de t e r mi n e appropriate treatment and improve the patient’s prognosis.(3) magnetic resonance imaging (mri) can be the imaging technique of choice to picture intracranial meningiomas and determine the next treatment. various ways have been taken to assess the grade of meningioma with mri examinations at varying stages before surgery.(2) ho we ve r, c on ve nt i o na l m ri c a n no t distinguish between benign and malignant meningiomas. the picture of heterogeneous lesions and contrast enhancement, margins indistinguishable from surrounding tissue, irregular edges, destruction of the surrounding bone, accompanied by peritumoral edema, leads to an image of atypical/malignant meningioma. still, this is not specific because the picture can also be found in benign meningiomas.(4,5) diffusion-weighted imaging (dwi) in mri examination is a non-invasive technique that can assess free fluid diffusion in tissues. the apparent diffusion coefficient (adc) is a measure of the diffusion of water molecules in tissues and is calculated after determination of dwi in mri. the adc values are automatically computed by the software, resulting in a parametric map showing the rate of diffusion of water molecules, which will vary in each tissue. measurements are made by drawing regions of interest (rois) on the adc map. the adc value is expressed in mm2/s. although there is no clear limit for the normal value, adc values of under 1.0 to 1.1 x 10-3 mm2/s are generally stated to be restricted.(6) moraru and dimitrievici (7) state that the adc value in cortical gray matter is 0.56–0.78 x 10-3 mm2/s, in white matter 0.319–0.686 x 10-3 mm2/s and in cerebrospinal fluid 1.59– 2.43 x 10-3 mm2/s. previous researchers have recognized the adc value obtained from dwi as an effective method for distinguishing benign and malignant meningiomas. however, adc values cannot differentiate between atypical and anaplastic/ malignant meningiomas. fluid diffusion in body tissues depends on the ratio of extracellular to intracellular space. atypical and malignant meningiomas have a high cell proliferation rate and cause reduced extracellular space, resulting in fluid diffusion restriction,(2,3) which shows an increased signal intensity in dwi and decreased signal intensity in adc. in previous studies conducted by surov et al.(8) and azeemuddin et al.,(9) the adc value played an essential role in determining the type of meningioma. 140 for meningiomas with a soft consistency, management to remove the tumor is carried out using a suction probe. in contrast, meningioma with a hard consistency cannot be treated by tumor excision. the adc boundary value between who grade i and ii/iii meningiomas varied in all previous studies. for example, the study conducted by surov et al.(8) used the value of 0.85x10 -3 mm2/s as the thresh old to differentiate who grade i meningiomas, which have low proliferation rates, from the high proliferation potential contained in who grades ii and iii. the adc assessment itself is still rarely done in the mri examination protocol in our hospital, especially in meningioma patients. in some studies, as in the study conducted by yiping et al.,(10) the adc value can distinguish between benign and malignant meningiomas. however, there are several different results from previous studies, one of which is the study of rad et al.,(4) where it said that benign and malignant meningiomas are indistinguishable by adc values. we questioned the difference between adc values in benign and malignant meningiomas because of the previous study results. the present study aimed to determine the value of adc for differentiating benign and malignant meningiomas. methods research design this study was a cross-sectional study conducted in the radiology installation of dr. moewardi hospital, surakarta, from february 2020 to july 2020. study subjects t he t a r get p op ul a t i o n c ons i st e d o f meningioma patients who had undergone headmri and histopathology examinations and were archived at dr. moewardi hospital, surakarta, from march 2016 until april 2020. sample size determination a total of 32 patients with clinically diagnosed or histologically verified meningiomas (21 benign and 11 malignant) were included. all samples were taken using purposive sampling techniques. meanwhile, according to memon et al.,(11) the appropriate sample size in a study is between 30 and 500. even though the sample size between 30 and 500 at 5% confidence level is generally sufficient for many researchers, the decision on the size should reflect the quality of the sample in this wide interval. therefore, the minimum number of samples set in this study was 30 samples. we only included patients that have undergone mri and histopathology examination from march 2016 until april 2020. we e x c l ud e d pa ti en t s wi t h e n-pl a q ue meningiomas and meningiomas related to other tumors as in neurofibromatosis. mri imaging fo r c r a ni a l m ri e x a mi na t i o ns a t dr. moewardi hospital, surakarta, a 1.5 t mri scanner (gemr hdxt) is used, with a standard head coil. pre-contrast t1-weighted spin-echo (se) (tr/te 401/8.5 ms), t2-weighted fast se (fse) (tr/te 5256/73.8 ms), fluid-attenuated inversion recovery (flair) (tr/te/9000/145.2 ms) and dwi sequences (tr/te 5200/81.5 ms) are all taken in axial section. the matrix used is 256×256 with a field of view (fov) of 25 cm, slice thickness of 3 mm, and a 3-mm gap between slices. contrast-enhanced t1-weighted images are obtained in axial section after the patient has been given iv contrast (gadovist, 1 mmol/ml). the adc folders are processed from dwi using functool (ge medical system) softwar e with b-values of 0 and 1000 s/ mm2. simultaneously, for the adc value calculation the radiant dicom viewer (64bit) (medixant company, poznan, poland) is used. data collection and analysis is done using microsoft office 2016 (microsoft corporation, redmond, wa, usa) and ibm® spss statistics version 25 (released 2017, armonk, ny: ibm). in the calculation of adc values, we use radiant dicom viewer software by creating regions of interest (rois) on the solid parts of the tumor, guided by contrast sequence to assess marissa, rahayu, wujoso, et al adc as predictor of meningioma malignancy 141 univ med vol. 40 no.2 b c tumor boundaries and flair sequence to assess the limit of peritumoral edema. the adc value is expressed in units of x10-3 mm2/s. the calcified parts of the tumor are avoided. the assessment is also conducted on the tumor-free contralateral side as control, called normal appearing white matter (nawm). histopathological analysis the pathology preparations of the patients were re-evaluated according to the who 2016 classification. the who categorizes the malignancy grade of meningiomas based on their histopathological features, consisting of who grade i (typical), who grade ii (atypical), and who grade iii (anaplastic). who grade i (typical) meningiomas occur in almost 90% of the population, who grade ii (atypical) meningiomas have an incidence of 5%-7% of the population, and who grade iii (anaplastic) meningiomas a ccount for 1%-3% of the population. each type has many subtypes, namely nine benign subtypes (who grade i), three intermediate subtypes (who grade ii), and three malignant subtypes (who grade iii). this division is based on histopathological appearance.(12) for the more aggressive subtypes, it is unclear what percentage of the histology must be present before being classified as malignant. however, chordoid, clear cell, papillary and rhabdoid subtypes are declared to be categorized as who grade ii and who grade iii if the morphological picture is >50.(12) the level of malignancy referred to in this study are the two groups of benign and malignant meningiomas. benign meningiomas are who grade i (typical) meningiomas, while malignant meningiomas are who grade ii (atypical) and iii (anaplastic) who meningiomas. statistical analysis since the data distribution was normal, we used an independent t-test for the hypothesis. to test the reliability of the measurement results of 2 observers in this study to assess whether it has a good suitability/reliability or not, the researchers made bland altman graphs (13) and calculated the average diff erence, 95% ci and limit of agreement between observers. the receiver operating characteristic (roc) curve was analysed to determine significant cut-off points in distinguishing b enign and malignant meningiomas. ethical clearance this study was approved by the board of health research ethics committee of dr. moewardi general hospital 321/ii/hrec/2020. results the characteristics of the subjects were described in table 1. the study specimens consisted of 11 malignant (34.4%) and 21 benign (65.6%). of these 32 specimens, most were from patients over 30 years old (90.6%), and 24 (75%) were from women. there were no significant differences in age, sex, and peritumoral edema between benign and malignant meningiomas (p>0.05). from the histopathological examination results, only four specimens could not be assigned a known meningioma subtype. in this study, the most common subtypes were meningothelial, clear cell, and fibroblastic meningiomas. in contrast, the least common were lipomatous, transitional, atypical, and rhabdoid meningiomas (data not shown). the reliability/conformity test between the two observers was conducted by assessing the bland-altman scatte r char t of average differences. from the observers’ calculations, the average differenc e be tween the adc assessments was -0.091 and the standard deviation 0.196. the differences between the adc values from the two observers (diff) associated with their average adc value (mmean), produce the bland-alman scatter diagram in figure 1. there was only one observation that was outside the limit of agreement or outside the value of 1.96 times the standard deviation (dotted line), which means there is one observation (3.1%) different from the other observations (outlier). since only 3.1% (α<5%) observations 142 are outside the limit of agreement, we can conclude that the observers have good suitability/ reliability. this study’s data is numerical and of normal distribution based on the shapiro-wilk test. in the independent t-test, there is a statistical difference in the average adc value of benign and malignant meningioma s. the average adc va lue in malignant meningiomas is 0.887 ± 0.167x103 mm2/s, while the average adc value in benign meningiomas is 0.990 ± 0.105 x10-3 mm2/s. thus, the average adc value in malignant meningiomas is lower than in benign meningiomas, and there is a significant difference between the average adc values in malignant and benign meningiomas (p<0.05) (table 1). benign (n =21,%) malignant (n=11,%) p value* age (years) <30 ≥30 gender male female peritumoral edema large size minimum adc 0 (0.0) 21 (72.4) 5 (62.5) 16 (66.7) 15 (65.2) 6 (66.7) 0.990 ± 0.105 x 10-3 mm2/s 3 (100.0) 8 (27.6) 3 (37.5) 8 (33.3) 8 (34.8) 3 (33.3) 0.877 ± 0.167 x 10-3 mm2/s 0.282a 0.829b 0.938b 0.025c table 1. comparison of subject characteristics and clinical features between benign and malignant meningioma (n=32) abbreviation: adc: apparent diffusion coefficient; ap value=fisher exact test; bp value=chi-square-test; cp value=independentt test; *significant at p<0.05 figure 1. bland-altman scatter diagram. diff is the difference between adc values from two observers. mmean is the average adc value from the two observers. the dotted line is the limit of agreement or 1.96 times the standard deviation of the two observers. there is good suitability/reliability if the observation data from the two observers differ from the limit of agreement by less than 5% (α<5%). marissa, rahayu, wujoso, et al adc as predictor of meningioma malignancy 143 univ med vol. 40 no.2 since the average adc value in benign and malignant meningiomas is significantly different from the benign meningiomas adc value, which was higher than in malignant meningiomas, we look for the roc curve’s cutoff point, illustrated in figure 2. from the roc curve, the auc value is 0.732 (p<0.05), with the cutoff point for adc being 0.886 x 10-3 mm2/s with a sensitivity of 0.857 and specificity of 0.636. this can be interpreted to indicate that the strength of the adc’s diagnostic value in determining the malignancy grade of meningiomas is moderate, namely 73.20% (>70%-80%). based on the sensitivity value, it can be interpreted that 85.70% of benign meningiomas can be indicated based on the adc value of >0.886 x 10-3 mm2/s. in contrast, the specificity value means that 63.6% of malignant meningiomas can be indicated by an adc value of <0.886 x 10-3 mm2/s. figure 2. roc curve of mean adc value assessment. larger adc values indicate a benign lesion. the coordinates of the curves are the sensitivity and 1specificity of the variables. the variable is the adc value taken by an observer figure 3. female, 41 years old, complained about headache and weakness of the left side of the body on mri examination, the lesion was hypointense on t1wi (a), which on contrast (b, c, and d) showed contrast enhancement with a dural tail appearance (white arrow) in the convexity of the right parietal lobe. peritumoral edema (blue arrow) was in the right temporal lobe on the t2 flair sequence (g and h). the dwi examination showed fluid restriction (e), and a hypointense lesion appearing on the adc (f) 144 the cutoff points of adc value based on the roc curve are divided into 2 categories for diagnostic test purposes: >0.886x10-3 mm2/s and <0.886 x10-3 mm2/s. the sensitivity of adc value >0.886x10-3 mm2/s as the risk of malignant meningioma incidence is 63.6%, and the specificity value is 85.7%. in contrast, the ppv and npv are 70% and 81.8%, respectively. one of our subjects was a 41-year-old female, who complained of headache and weakness of the left side of the body. there was a tumor in the convexity of the right parietal lobe with peritumoral edema spreading through the right temporal lobe on mri examination. the dwi examination showed fluid restriction, the adc value was 0.882 x 10-3 mm2/s, and the nadc value wa s 0.762 x 10 -3 mm2/s. if categorized based on the cutoff point this tumor leads to the diagnosis of a malignant meningioma (<0.886 x10-3 mm2/s). it is consistent with her histopathological result of chordoid meningioma of who grade ii (figure 3). another subject was a 34-year-old male, with chief complaints of seizures and visual disturbances. the mri examination showed a solid cystic tumor in the left fronto-temporoparietal lobe. the adc value was found to be 0.979 x 10-3 mm2/s; if categorized based on our cutoff point, the diagnosis leads to a benign meningioma (>0.886 x 10-3 mm2/s) that is consistent with the histopathological results of angiomatous meningioma (who grade i) (figure 4). figure 4. male 34 years old, with chief complaints of seizures and visual disturbances. the mri examination showed a heterointense lesion with a solid-cystic component on t1wi (a) and t1 flair (h). when administered contrast (b, c, d, e), it showed contrast enhancement in the left frontotemporoparietal lobe. peritumoral edema (blue arrow) is seen in the left frontotemporoparietal lobe. on dwi examination (f), there was a fluid restriction in the solid area (orange arrow) and a hypointense lesion in the solid area on the adc (g) marissa, rahayu, wujoso, et al adc as predictor of meningioma malignancy 145 univ med vol. 40 no.2 discussion in this study, the prevalence of malignant meningioma was higher than that of benign meningioma and this result was in line with a previous study conducted by surov et al.,(8) which showed that benign meningioma has a higher incidence than malignant meningioma. similar to the previous study of sohu et al.,(1) there were more women among the subjects of the present study. this study also showed a significant difference between adc value in benign meningioma and malignant meningioma with the cutoff point at 0.886 x 10-3 mm2/s, which is close to the adc cutoff point of 0.85×10"3 mm2/s in the studies of sohu et al.(1) and surov et al.(8) the adc measured using diffusion-weighted imaging sequence has been studied previously in predicting the histological nature of brain tumors.(14,15) the adc, which is calculated automatically by software, shows the magnitude of the diffusion of the water molecules. in tumors with a high proliferation of tumor cells, the diffusivity of water molecules is reduced. therefore, an inverse correlation has been reported between adc value and tumor cellularity due to this effect.(16) the sensitivity in this study was 63.6%, which was lower than that in the previous studies conducted by abdel-kerim et al.(17) and neethu et al.,(18) namely 81.2% and 84.0%, respectively. a low sensitivity can be due to the number of benign and malignant subjects, as in the study by abdel-kerim et al.(17) with 36 benign and 11 malignant samples, and in the study by neethu et al.(18) with 53 benign and 13 malignant samples. another source of low sensitivity is the high average malignant adc value close to the cutoff point, because the area of necrosis in malignant meningioma causes increasing fluid diffusion. for example, in one of the samples, where the adc value is 1.067 x 10-3 mm2/s when categorized by cutoff point, the diagnosis leads to a picture of benign meningioma (>0.886 x 10-3 mm2/s). however, histopathology results show a picture of clear cell meningioma with who grade ii. this study also obtained a specificity value of 85.7%, greater than in the previous study of surov et al.,(8) but smaller than in the study by abdelkerim et al.(17) with specificity of 91.7%. the large sensitivity of the study of abdel-kerim (17) may have been influenced by the fairly large average distance of benign adc value from the cutoff point value of the benign meningioma average adc value of 1.02 ± 0.16 x 10-3 mm2/s with a cutoff point value of 0.8 x 10-3 mm2/s. while in the research conducted by surov et al.,(8) the average adc value in benign meningioma was 0.88 ± 0.08 x 10-3 mm2/s with the cutoff point at 0.85 x 10-3 mm2/s. in our study, the adc value of benign meningioma was higher than that of malignant meningioma. these results were in agreement with those of the study of hirunpat et al.,(19) where the average adc value in the benign subgroup (0.83 x 10-3 mm2/s ± 0.37 x 10-3 mm2/ s) was higher than in the malignant subgroup (0.698 x 10-3 mm2/s ± 0.06 x10-3 mm2/s). in the present study, ppv and npv were 70.0% and 81.8%, respectively, and not much different from those of previous studies, with ppv 85.7% and npv 95%. similarly, in the study by abdel-kerim et al.,(17) ppv was 75% and npv 94.3%. our low npv compared to previous studies can be attributed to different study populations and necrotic malignancy images being mistaken for cystic components in the dwi picture of sohu et al.(1) two of the present study’s limitations are inadequately archived data and the observational nature of this study, while the population subtype variants are quite diverse. another limitation is that we did not investigate the diagnostic accur acy of f ir st-orde r histogram (foh) extracted features, which show pixels in an image with similar intensity at a defined intensity level.(20) this study shows that the cutoff value of adc from meningioma may help the clinician predict the diagnosis, but making a prompt diagnosis of meningioma is still based on the histopathological findings. however, the cutoff point obtained in this study may be expected to 146 be useful as raw data for further research. in the radiology field, it could be a guide to be used in the daily protocol in mri examination. conclusions this study demonstrated that the adc is a valid diagnostic tool. further, mean adc can be used as a good test to differentiate benign and malignant meningiomas and help the clinician predict pathological abnormalities due to the malignancy for evaluation before therapy. conflict of interest the authors declare that they have no conflicts of interest. acknowledgements the authors express their gratitude to dr. moewardi general hospital’s management and staf f, surakarta, especia lly the radiology department, for permission to analyze the samples in their mri workstation. contributors rrm conceived, designed, and did data collection, statistical analysis, and manuscript writing. rfr, hw, ss, and psp designed, did the editing, reviewed, and did final approval of the manuscript. ws contributed to revision of the manuscript. all authors have read and approved the final the manuscript. references 1. sohu dm, sohail s, shaikh r. diagnostic accuracy of diffusion weighted mri in differentiating benign and malignant meningiomas. pak j med sci 2019; 35:726-30. doi: 10.12669/pjms.35.3.1011. 2. lu y, liu l, luan s, xiong j, geng d, yin b. the diagnostic value of texture analysis in predicting who grades of meningiomas based on adc maps: an attempt using decision tree and decision forest. eur radiol 2019;29:1318–28. doi: 10.1007/ s00330-018-5632-7. 3. surov a, ginat dt, sanverdi e, et al. use of diffusion weighted imaging in differentiating between malignant and benign meningiomas: a multicenter analysis. world neurosurg 2016;88: 598–602. doi: 10.1016/j.wneu.2015.10.049. 4. saligheh rad h, safari m, kazerooni af, moharamzad y, taheri ms. apparent diffusion coefficient (adc) and first-order histogram statistics in differentiating malignant versus benign meningioma in adults. iran j radiol 2019; 16:e74324. doi: 10.5812/iranjradiol.7432416. 5. baskan o, silav g, bolukbasi fh, canoz o, geyik s, elmaci i. relation of apparent diffusion coefficient with ki-67 proliferation index in meningiomas. br j radiol 2016;89:4–9. doi: 10.1259/ bjr.20140842. 6. madhok r, sachdeva p. measurement of mean adc values in tubercular vertebrae and associated collection. j clin diagn res 2016;10: tc19-tc23. doi: 10.7860/jcdr/2016/20520.8344. 7. moraru l, dimitrievici l. apparent diffusion coefficient of the normal human brain for various experimental conditions aip conference proceedings 1796,2017.040005-1-040005-6. doi: 10.1063/1.4972383. 8. surov a, gottschling s, 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heikal a, mazloum y. differentiation between benign and atypical cranial meningiomas. can adc measurement help? mri findings with hystopathological correlation. egyptian j radiol nuclear med 2018;49:172-5. doi: 10.1016/ j.ejrnm.2017.10.004. 18. neethu pm, saanida mp, subramaniam g, et al. use of mri apparent diffusion coefficient to noninvasively differentiate between the histologic grades of meningioma. j evolution med dent sci 2020;9:1679-83. doi: 10.14260/jemds/2020/ 369. 19. hirunpat s, sanghan n, watcharakul c, kayasut k, ina n, pornrujee h. is apparent diffusion coefficient value measured on picture archiving and communication system workstation helpful in prediction of high-grade meningioma? hong kong j radiol 2016;19:84-90. doi: 10.12809/ hkjr1615346. 20. just n. improving tumour heterogeneity mri assessment with histograms. br j cancer 2014; 111:2205-13. doi: 10.1038/bjc.2014.512. may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 repurposing of fluoxetine for antibacterial activities in catheter-associated urinary tract biofilm infections: an in vitro analysis muhammad musthafa poyil1* , rameesha shafqat2 , and mamoun a. alfaki1 abstract background urinary tract infections are often initiated by indwelling catheters and bring about serious consequences, especially when they are caused by multidrug-resistant bacterial pathogens. the biofilms of uropathogens such as enterococcus faecalis and escherichia coli pose serious challenges. therefore the scientific world is trying to experiment with alternative drugs to replace conventional antibiotics as the latter are more prone to cause the development of antibacterial resistance. here, we evaluate the repurposing of the antidepressant fluoxetine as an antibacterial agent against the mentioned pathogens. methods to repurpose fluoxetine for its antibacterial activity against enterococcus faecalis and escherichia coli, the agar diffusion method was used. the minimal inhibitory concentration was found by the microdilution method. the drug was also analyzed as a coating on catheters to evaluate its efficiency against biofilm formation by pathogens. results the drug fluoxetine showed potential antibacterial and anti-biofilm activities. its minimum inhibitory concentration was found to be 18.75 µg/ ml and 37.5 µg/ml against enterococcus faecalis and escherichia coli respectively. the antibiofilm activity on polystyrene surfaces was also remarkable as it reduced the formation of enterococcus faecalis and escherichia coli biofilms by 70% and 74%, after being treated with 1x mics and 2x mics respectively. conclusions fluoxetine one of the drugs of choice in treating depression, when repurposed, has shown considerable antibacterial and antibiofilm effects against two of the major catheter-associated urinary tract infection-causing bacteria viz. enterococcus faecalis and escherichia coli. therefore, further studies are needed to understand its applicability as an antibacterial agent. keywords: biofilms, catheter-associated urinary tract infections, fluoxetine, repurposing 1department of basic medical sciences, college of medicine, prince sattam bin abdulaziz university, al-kharj 11942, saudi arabia 2dow university of health sciences, karachi 74200, pakistan *correspondence: muhammad musthafa poyil department of basic medical sciences, college of medicine, prince sattam bin abdulaziz university, al-kharj 11942, saudi arabia email: m.poyil@psau.edu.sa orcid: 0000-0003-4826-3603 date of first submission, january 17, 2023 date of final revised submission, june 16, 2023 date of acceptance, june 23, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 128 doi: http://dx.doi.org/10.18051/univmed.2023.v42:128-136 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1421 cite this article as: poyil mm, shafqat r, alfaki ma. repurposing of fluoxetine for antibacterial activities in catheterassociated urinary tract biofilm infections: an in vitro analysis. univ med 2023;42:128-36. doi: 10.18051/ univmed.2023.v42:128-136 mailto:m.poyil@psau.edu.sa https://orcid.org/0000-0002-8415-3893 https://orcid.org/0000-0002-4022-1524 https://orcid.org/0000-0002-1283-8375 https://univmed.org/ejurnal/index.php/medicina/article/view/1421 129 univ med vol. 42 no. 2 introduction in modern medical science, indwelling medical devices have been playing a revolutionary role in the management of various diseases, and urinary catheters which relieve the discomforts of patients with urinary bladder complications are the most significant among them. unfortunately, in hospitalized patients these indwelling catheters significantly increase the risk for iatrogenic infections that are common when the patient is immunocompromised. in the modern age, medical device-associated infections contribute to a major part of nosocomial infections and among these, urinary tract infections (utis) are one of the most significant hospital-acquired infections associated with catheters.(1,2) catheter-associated urinary tract infections (cautis) occur when the presence of uropathogenic bacteria is manifested in the urine of hospitalized patients with catheter usage and are the most common healthcareassociated infections that affect millions of patients globally.(3-5) the use of catheters facilitates the entry of microbial pathogens to the urinary tract and their colonization leads to various complications such as sepsis and bacteriuria, resulting in long hospital stays and in extreme cases, higher mortality rates causing socioeconomic burdens.(6,7) prolonged use of urinary catheters favours polymicrobial infections including those by bacteria and fungi.(8) reports suggest that escherichia coli and enterococcus faecalis are the most prevalent bacterial pathogens that cause cautis.(9,10) these prevalent bacteria form biofilms by adhering to the catheter surfaces and establishing communications among them making their eradication complicated.(11) the biofilmforming ability enables the microorganisms to resist several antibiotics through various resistance mechanisms, such as the physical protective effects of the extracellular polymeric substances, efflux pumps, and the transfer of antibiotic resistance genes between bacterial cells.(12-14) as a result, in most cases, the treatment of cautis has been practically difficult.(15,16) hence, there is an immediate need for alternative therapies that enable the prevention of colonization and biofilm formation on urinary catheters by bacterial pathogens such as enterococcus faecalis and escherich ia coli and their eradication. however, the search for new drugs from the available sources is a time-consuming and expensive process as these drugs under development have to undergo many clinical trials before entering the market. among the sever al ways of dr ug development, repurposing already existing drugs for a new and novel application is one approach that has gained much attention because of the unique nature of this drug development process. the quest of repurposing the existing drug is significant as the drug has already gone through many safety, pharmacological efficacy, and human trials which reduces the cost, time, and risks associated with anti biotic innovation. (1 7) considering all these facts, in the present study, an effort was made to evaluate the antibacterial activity of the antidepressant drug fluoxetine against two of the major cauti biofilm-forming bacterial pathogens, viz, enterococcus faecalis and escherichia coli. methods research design an in vitro analysis was carried out to screen the anti-depression drug fluoxetine against two of the major catheter-associated urinary tract infection-causing bacterial pathogens viz., enterococcus faecalis and escherichia coli. the experiments and the following analyses were conducted a t the ba sic medical science labor atory, prince sattam bin abdulaziz university, al-kharj, saudi arabia, and at biotech research centre, salem636010, tamil nadu, india, during the period between november 4th, 2022 and january 2nd, 2023. reagents and materials for the study, mueller hinton broth, ampicillin, and rifampicin were procured from hi 130 media (usa) and fluoxetine from sigma aldrich. fluoxetine (1 mg/ ml) was prepared in sterile double distilled water. the strains enterococcus faecalis (atcc 29212) and escherichia coli (atcc 25922) were obtained from american type culture collection (atcc). the antibiotics rifampicin (5 µg) and ampicillin (5 µg) were used as controls. determination of antibacterial activity the antibacterial activity of fluoxetine against enterococcus faecalis and escherichia coli was investigated using the well diffusion method as per standard protocols. (18) in brief, overnight cultures of enterococcus faecalis and escherichia coli were grown in mueller-hinton broth (mhb) at densities of 0.5 macfarland units and were used for determining the antibacterial activity of fluoxetine against enterococcus faecalis and escherichia coli. the mentioned cultures were swabbed over the surface of sterile mueller-hinton agar (mha) plates, in which the wells of 6 mm diameters were made to allow the different concentrations of fluoxetine, and incubated at standard conditions overnight. then, the plates were observed for zones of inhibition, which were measured in millimetres (mm), to determine the antibacterial activity of fluoxetine. the exper iments were done in duplicate. ampicillin and rifampicin were used as positive control s for enterococcus faecalis an d escherichia coli respectively. minimum inhibito ry concentr ation determination the microdilution method was adopted, as described by meiyazhagan et al.,(19) to find out the minimum inhibitory concentrations (mics) of fluoxetine against enterococcus faecalis and escherichia coli. here, in 96 well plates, 300 µg/ml of fluoxetine was serially diluted using 200 µl of mhb to achieve a final concentration of 2.3 µg/ml. later, the plates were incubated under standard conditions after adding the overnight cultures of the above-indicated organisms. after incubation, the plates were observed for turbidity, and the optical density was read at 600 nm using a spectrophotometer. the experiments were done in triplicate. effect of fluoxetine on bacterial colony formation to find out the effect of fluoxetine on enterococcus faecalis and escherichia coli colonization, the drug was serially diluted in 96well plates using mhb, then the overnight cultures were added and ncubated for 96 hours. then, the phosphate-buffered saline (pbs) wash was done to each well for the removal of unattached cells. the adherent cells were fixed with methanol and stained with crystal violet for several minutes. the ethanol and acetone (1:9) mixture was added to the stained cells and the plate was r ead a t 570 nm using a spectrophotometer.(19) the experiments were done in triplicate. cells without treatment acted as a negative control. biofilm inhibition efficiency of fluoxetine the biofilm formation assay was performed in polystyrene microlitre plates as illustrated by gowri et al.,(20) to ascertain the effect of fluoxeti ne on enterococcus faecalis an d escherichia coli biofilms. briefly, the biofilm formation was achieved after incubating the enterococcus faecalis and escherichia coli cultures in a 12-well plate for 96 hours. after the incubation, 1x mic and 2x mic concentrations of fluoxetine were added and the plate again incubated for 24 hours. the non-adherent cells were then removed by pbs wash, followed by methanol fixation and crystal violet staining. then, the excess stain was removed by washing followed by air drying. the ethanol and acetone (1:9) mixture was added to the stained cells and the pla te wa s read at 570 nm using a spectrophotometer. cells without treatment were considered negative controls. ampicillin and rifampicin were the positive controls for enterococcus faecalis and escherichia coli respectively. the experiments were done in triplicate. poyil, shafqat, alfaki catheter-associated urinary tract infections 131 univ med vol. 42 no. 2 antibacterial activity of catheter coated with fluoxetine to determine the antibacterial activity of a cathe ter c oated with fluoxetine against enterococcus faecalis and escherichia coli, the in vitro catheter model was adopted as described by goda et al.(21) briefly, small pieces of sterile catheter tubes were dipped into 25 mg/ml of fluoxetine solution for 30 mins followed by air drying. for the assay, the air-dried fluoxetinecoated tubes were placed on the sterile mha plates which were swabbed with enterococcus faecalis and escherichia coli followed by 24 hours of incubation. after incubation, the plates were observed for zones of inhibition. the experiments were done in duplicate. statistical analysis means and standard deviations were calculated for mic determination, colony formation, and biofilm formation assays to analyze the statistical significance. results determination of antibacterial activity of fluoxetine the antibacterial activities of various concentrations of fluoxetine were investigated against enterococcus faecalis and escherichia coli and the zones of inhibition exhibited by different concentrations of fluoxetine against prevalent microbes involved in cauti are presented in figure 1. as seen in the figure, the antibacterial activity was achieved with 75 µg of fluoxetine against enterococcus faecalis and 125 µg of fluoxetine against escherichia coli. it is noted that, when the concentrations increased, the activity of fluoxetine was also higher against both of the microbes. mic determination the mics of fluoxetine were evaluated against enterococcus faecalis and escherichia coli involved in cautis and the lowest concentration of fluoxetine which inhibited the growth of the respective bacteria was calculated, as presented in figur e 2. as shown, the calculated mics of fluoxetine was 18.75 µg/ml against enterococcus faecalis and 37.5 µg/ml against escherichia coli. effect of fluoxetine on bacterial colonization the activities of fluoxetine against colony formation by enterococcus faecalis a nd escherichia coli wer e studi ed and are represented in figure 3. as shown, the presence of fluoxetine up to its mic level in polystyrene figure 1. fluoxetine exhibited zone of inhibition against a) enterococcus faecalis b) escherichia coli 132 plates did not allow any bacterial growth on the surface. surprisingly, even traces of fluoxetine present in the wells were able to decrease the colony-forming ability of enterococcus faecalis and escherichia coli on the surface of the plate when compared with untreated wells which is reflected in the results. effect of fluoxetine on biofilm formation the effects of fluoxetine on enterococcus faecalis and escherichia coli biofilm formation, after treatment with various concentrations of the drug, have been quantified and are shown in figure 4. as seen in the figure, the percentage of effects of fluoxetine on enterococcus faecalis biofilm formation was calculated as 64.4 and 70 for 1x mics and 2x mics respectively. in contrast, escherichia coli biofilm formation was reduced by 69% and 74% after being treated with 1x mics and 2x mics respectively. act ivity of f luoxetine coated cathet ers against the bacteria antibacterial activity was investigated for the fluoxetine-coate d catheter s against enterococcus faecalis and escherichia coli under suitable in vitro conditions as presented in figure 5. as seen in the figure, the drug-coated figure 2. mics of fluoxetine against enterococcus faecalis and escherichia coli values are for mean ± standard deviation (error bar) figure 3. graphical representation of fluoxetine effect on enterococcus faecalis and escherichia coli colony formation poyil, shafqat, alfaki catheter-associated urinary tract infections 133 univ med vol. 42 no. 2 catheter tube exhibited a clear zone of inhibition around the silicone tube which represents the antibacterial activity of fluoxetine. discussion catheter-associated urinary tract infection is one of the most significant hospital-associated infections causing serious clinical complications leading to high morbidity and mortality owing to their polymicrobial nature, which creates treatment challenges because of the antibiotic resistance and biofilm-forming ability of the prevalent organisms.(22,23) considering these facts, there have been novel ways of searching for new antibacterial agents, and one of them is the repurposing of existing drugs for a different scope that has achieved much attention owing to their known pharmaceutical profiles. here, the antidepression drug fluoxetine was repurposed for its antibacterial activity against e. faecalis and e. coli which are prevalent in cauti. the fluoxetine antibacterial activity was explored and the lowest inhibitor y concentr ation was determined. recently, duloxetine had been evaluated against multi-drug resistant e. coli and figure 4. percentage of biofilm inhibition of fluoxetine against a) enterococcus faecalis b) escherichia coli note: pc-positive control figure 5. antibacterial activity of fluoxetine coated catheter tube against a) escherichia coli b) enterococcus faecalis. note: ununcoated, ccoated with drug fluoxetine 134 found to have excellent activity when combined with chloramphenicol.(24) correspondingly, the antibacterial and anti-biofilm activity of the repurposed drug auranofin was investigated and the drug was recorded to possess potential activity against bacteroides fragilis.(25) in the same way, the anti-depression dr ug sertraline was investigated for its antibacterial activity against e. faecalis, acine tobacte r baumanii, pseudomonas aeruginosa, and e. coli. (26) various repurposed drugs such as amodiaquine, curcumin, ibuprofen, ellagic acid, and quercetin were evaluated against two important eskape pathogens and the evaluation revealed the antibacterial activity of curcumin and ellagic acid against staphylococcus aureus a nd pseudomonas aeruginsa.(27) the antiamoebic drug diiodohydroxyquinoline was repurposed for its antibacterial activity against clostridioides difficile. (2 8) likewise , mitomycin-c was repurposed for the antibacterial activity against klebsiella pneumoniae and found to have enhanced antibacterial activity when combined with imipenem.(29) in the present study, fluoxetine was studied for its effect on e. faecalis and e. coli colony formation which is the most important stage in biofilm formation. the insertion of a catheter may provide an entry for bacteria to initiate infection in the urinary tract by adhering to the catheter surface. (30) once the bacteria are attached to the surface area, they can form a complex structure that protects the bacteria from external sources, such as host defence mechanisms resulting in the development of antibiotic resistance, making the management of cauti critical.(31) therefore, the quest is to concentrate on each stage of biofilm formation, from adhesion to mature biofilm, while studying biofilm prevention.(32,33) therefore, the effect of fluoxetine was studied on e. faecalis and e. coli colony formation and it was found that the drug was able to prevent colony formation of e. faecalis and e. coli on surfaces, and thereby inhibiting biofilm formation. similarly, etoposidea was repurposed for its anti-biofilm activity against s. aureus and has shown excellent activity in removing the biofilm which formed on hydroxyapatite.(34) also, the drug penfluridol was repurposed for its antibacterial and antibiofilm activities against e. faecalis,(35)with promising results. besides the antibacterial and antibiofilm activity, fluoxetine was studied for the effect of a coating on the silicone catheter tube which was evaluated against e. faecalis and e. coli. the coating of the catheter tube is an excellent approach to prevent the microbial colonization of uropathogens in the inner and outer surfaces of the tube. hence, our study showed the efficacy of fluoxetine-coated silicone catheter tubes against e. faecalis and e. coli in the invitro bladder model which imitates the general process. the drug fluoxetine was able to suppress the growth of e. faecalis and e. coli which was represented by the clear zone of inhibition. similarly, the antibacterial activity of silver nanocomposite-coated catheter tubes was s t ud i e d a ga i n s t e. co l i a n d s . a u re u s representing biofilm inhibition.(36) in another report by abbott et al.(37) on repurposing fosfomycin, it was said that the drug showed excellent activity against e. faecalis in bladder infection in-vitro model. . in short, fluoxetine can be used as an alternative for cauti when the mechanisms of action will be explored and in vivo efficacy of the drug is analyzed. conclusion this study demon strated tha t the antidepressant drug fluoxetine can make an excellent antibacterial drug and an anti-biofilm coating component. further, in vivo analysis of fluoxetine is needed to determine the efficacy against cauti. conflict of interest the authors declare that the present study was performed in the absence of any conflict of interest. poyil, shafqat, alfaki catheter-associated urinary tract infections 135 univ med vol. 42 no. 2 acknowledgement the authors are grateful to the deanship of scientific research, prince sattam bin abdulaziz university, al-kharj, saudi arabia, for its support and encouragement in conducting the research and publishing this report. author contributions conceptualization mmp; methodology and experiments mmp, maa; result analysis and interpretation mmp, rs, maa; writing of 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10.1002/jbm.a.36561. 31. pelling h, nzakizwanayo j, milo s, et al. bacterial biofilm formation on indwelling urethral catheters. lett appl microbiol 2019;68:277-93. doi: 10.1111/ lam.13144. 32. ghosh a, jayaraman n, chatterji d. smallmolecule inhibition of bacterial biofilm. acs omega 2020;5:3108-15. doi: 10.1021/acsomega. 9b03695. 33. muhammad mh, idris al, fan x, et al. beyond risk: bacterial biofilms and their regulating approaches. front microbiol 2020;11:928. doi: 10.3389/fmicb.2020.00928. 34. ganesan v, meiyazhagan g, devaraj m, et al. repurposing the antibacterial activity of etoposide% a chemotherapeutic drug in combination with eggshell-derived hydroxyapatite. acs biomater sci eng 2022;8: 682-93. doi: 10.1021/acsbiomaterials.1c01481. 35. zeng x, she p, zhou l, et al. drug repurposing: antimicrobial and antibiofilm effects of penfluridol against enterococcus faecalis. microbiologyopen 2021;10:e1148. doi: 10.1002/ mbo3.1148. 36. rahuman hbh, dhandapani r, palanivel v, thangavelu s, paramasivam r, muthupandian s. bioengineered phytomolecules-capped silver nanoparticles using carissa carandas leaf extract to embed on to urinary catheter to combat uti pathogens. plos one.2021;16:e0256748. doi: 10.1371/journal.pone.0256748. 37. abbott ij, van gorp e, van der meijden a, et al. oral fosfomycin treatment for enterococcal urinary tract infections in a dynamic in vitro model. antimicrob agents chemother 2020;64:e0034220. doi: 10.1128/aac.00342-20. poyil, shafqat, alfaki catheter-associated urinary tract infections c:\users\universa medicina\docu 234 abstract universa medicina detection of immunogenic protein from salivary gland of aedes albopictus rike oktarianti1, rochmatul nuryu khasanah1, syubbanul wathon1, and kartika senjarini1* background dengue virus is transmitted by several species of aedes mosquitoes, with aedes albopictus as secondary vector. during blood feeding, these vectors inject saliva into the vertebrate hosts. the saliva contains anticoagulant, anti-inflammatory and immunogenic factors. the objective of this research was to detect immunogenic proteins from ae.albopictus salivary glands reacting with sera of people living in dengue endemic areas. methods the identification of immunogenic proteins of ae. albopictus salivary gland used one-dimensional gel electrophoresis (sodium dodecyl sulfate polyacrylamide gel electrophoresis), and western blot analysis, respectively. to determine the immunogenic nature of the candidate proteins, the antigens from the salivary gland of ae. albopictus were reacted with sera from healthy persons, dengue hemorrhagic fever (dhf) patients, and neonates, each of the groups comprising 10 samples. results the protein profiles of ae. albopictus salivary glands showed 13 bands with molecular weights from 16 kda up to 97 kda, i.e. 16, 17, 26, 28, 31, 32, 45, 55, 60, 67, 73, 76, and 97 kda. according to western blot analysis result, the 31 kda proteins were recognized in all endemic population sera, both in dhf patients and healthy persons. in contrast, protein bands of 47 and 67 kda were only recognized by the sera of dhf patients. conclusion three immunogenic proteins of 31, 47 and 67 kda were detected from ae. albopictus salivary glands. these immunogenic proteins may be developed as candidate biomarkers for bite exposure to ae. albopictus and as vectorbased dhf vaccines. keywords : immunogenic protein, salivary gland, aedes albopictu original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.234-242 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1146 september-december 2021 vol.40no.3 ci te t his article as: ok tariant i r , khasanah rn, wathon s, senjarini k. detection of immunogenic protein from salivary gland of aedes albopictus. univ med 2021;40:234-42. doi: 10.18051/ univmed.2021.v40.234-242. 1department of biology, faculty of mathematics and natural sciences, jember university correspondence : *kartika senjarini department of biology, faculty of mathematics and natural sciences, jember university jl. kalimantan no.37, tegalboto, sumbersari, jember, east java, indonesia phone/fax: +6287857703792 email: senjarini@unej.ac.id orcid id: 0000-0001-7041-1719 date of first submission, march 5, 2021 date of final revised submission, november 8, 2021 date of acceptance, november 20, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 235 univ med vol. 40 no 3 introduction dengue hemorrhagic fever (dhf) often affects people in tropical and subtropical areas.(1) areas at risk of dengue infection comprise 128 countries, including indonesia.(2–4) the latest data show that about two-thirds of the world’s population has the potential to be infected by dengue, with the number of cases reaching 100390 million each year.(5,6) dengue hemorrhagic fever (dhf) is caused by infection with the dengue arbovirus which has four serotypes, i.e. dengue virus (denv)-1, denv-2, denv-3, and denv-4.(7,8) the dengue virus infects the human body through transmission mediated by aedes mosquitoes (aedes ae gypti and aed es albopictus).(9) ae. aegypti is the primary vector because its habitat is around settlements so that it often comes into contact with humans. ae. albopictus acts as a secondary vector because its habitat is in gardens or forests far from human settlements. at present ae. albopictus has adapted to rural and suburban environments.(10) dengue virus transmission to human hosts occurs when the vector is blood feeding.(11) successful transmission of the dengue virus occurs due to the presence of important proteins in aedes salivary glands.(12) aedes saliva has several proteins able to act as antihemostatic, vasodilator, anti-inflammatory, and immunomodulatory substances which play a role in the successful transmission of t he dengue virus. (1 3) antihemostatic substances pla y a role in preventing coagulation of host blood when mosquitoes are blood feeding. vasodilator substances can cause vasodilation of blood capillaries. immunomodulators are substances that can suppress the host immune response so that it can increase the transmission of pathogens into the host by modulating the host immune response so as to facilitate their transmission into the host.(12,14) immunomodulatory proteins in arthropod saliva cause a shift in the immune response towards t helper type 2 (th2), characterized by increased production of th2 cytokines such as interleukin-4 (il-4) and il-10 and decreased production of th1 cytokines such as interferon ga mma ( ifn -γ ) a n d il-12 . ( 1 3 ) re pe a t e d exposure can increase production of specific antibodies in the host against salivary proteins. this is because when the host is repeatedly exposed, there will be an increase in the proliferation of b cells into plasma cells and memory cells which will cause the production of immunoglobulin g (igg). the increase in igg production will trigger the host’s immunity against pathogens. igg production can also be used as a marker for the presence of immunogenic proteins in mosquito saliva.(15,16) pr e vi o us r e s e a r c h i d e n t if i e d t wo immunogenic proteins from ae. aegypti salivary glands of 31 and 56 kda, respectively.(17) both of these proteins were recognized in sera samples of healthy persons and dhf patients living in dhf endemic areas, but not in sera samples of healthy persons in non-endemic areas and in infants (neonates). this shows that persons living in endemic areas develop specific antibodies. because ae. albopictus is also known to be a potential vector of dhf, it is suspected that the salivary glands of ae. albopictus contain immunogenic proteins similar to those in ae. aegypti. therefore, further research is needed to detect the immunogenic protein fraction in the salivary glands of ae. albopictus. the immunogenic protein fraction can be used as a candidate target for the development of vector-based dhf vaccines and as a biomarker of exposure to ae. albopictus. therefore, the aim of this study was to detect the immunogenic proteins from the salivary glands of ae. albopictus. methods research design a laboratory study was conducted from january to november 2020 at the biotechnology laboratory, department of biology, faculty of mathematics and natural sciences (fmipa), jember university. 236 research subjects thirty sera samples were taken from healthy persons, dhf patients, and infants (neonates) living in jember, consisting of 10 samples each. the inclusion criteria of healthy sample sources were persons aged 15-50 years who never had dhf, whereas dhf patients as sample source were persons aged 15-50 years diagnosed as having dhf by a doctor. the exclusion criteria of sample sources were persons under 15 years old or more than 50 years old and non-residents of jember. the study commenced with the rearing of ae. albopictus mosquitoes and was then continued by the identification of ae. albopictus based on morphological characters, isolation of the salivary glands, extraction of salivary gland proteins, protein profile analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis (sds-page), and finally western blot analysis to identify immunogenic proteins. rearing of ae. albopictus rearing was conducted at the animal care unit, university of jember, at room temperature (27°c). rearing was begun with the landing collection of ae. albopictus mosquitoes using aspirators in gardens around the university of jember. male mosquitoes were fed 10% sucrose solution, while female mosquitoes were fed by blood feeding. species identif i cation based on morphological characters identification of spec ies based o n morphological characters was carried out prior to isolation of the salivary glands. ae. albopictus and ae. aegypti are easily distinguished by the mesonotum, mesepimeron, and femur. the mesonotum in ae. aegypti has rows of white hairs forming a white line pattern, namely two curved white lines like a lyre and two parallel lines, while in ae. albopictus it has only one white line. t he mesepimeron of the two mosquitoes is very different: ae. aegypti has a collection of white scales forming two separate white patches, whereas ae. albopictus has a single white patch. ae. aegypti has rows of white hairs that form a long white stripe on the anterior femoral surface of the middle leg, while ae. albopictus does not have such a femoral stripe.(10) isolation and extraction of ae. albopictus salivary gland proteins isolation of the salivary glands was by microdissection using a dissection needle, in which the investigator’s left hand gently presses on the thoracic part and the right hand slowly pulls on the head, so that the 6 clear salivary glands appear. (18) the salivary glands that have been obtained are then separated from the fat body or other tissues until they are clean and then put into a microtube containing 10 µl of 1 mm phenylmethylsulfonyl fluoride (pmsf) in phosphate buffered solution (pbs) in the ratio of 1:1 for the 10 pairs of salivary gland samples and stored at -20oc until used. salivary gland protein extraction was carried out by adding 10 µl of sample buffer into a microtube containing 10 pairs of salivary gland samples + 10 µl of 1 mm pmsf in pbs. the extraction process was followed by boiling using a thermoshaker at a temperature of 95°c for 4 minutes. after boiling, the salivary gland protein extract was centrifuged to obtain the supernatant. blood sera preparation thirty serum samples were taken from healthy persons, dhf patients, and infants (neonates) living in jember, east java, a region within the endemic range for dengue, each group comprising 10 samples. these samples were collected in 4 months from july-october 2020. the serum samples of dhf patients were taken from the dokterku clinic and citra husada hospital, jember. neonatal serum samples were taken from the umi soemarno clinic, jember. blood samples were taken from the brachial veins in the upper arm, except for neonatal sera which were taken from the portion of the umbilical cord that remained with the baby after birth. each blood oktarianti, khasanah, wathon, et al immunogenic protein from salivary gland 237 univ med vol. 40 no 3 a b sample had a volume of 3 ml and was stored in a vacutainer without anti-coagulant. the blood samples were allowed to stand for 30 minutes, then the top clear layer was taken and centrifuged at 3200 rpm for 15 min at 4°c. the resulting supernatant after centrifugation constituted the serum. the serum samples were stored at -20°c until used. protein profile analysis of ae. albopictus salivary glands the salivary gland protein profile of ae. albopictus was analyzed using sds-page. electrophoresis of the protein samples was done using 12% separating gel and 4% stacking gel. each well in the gel contained 20 µl of protein sample (10 pairs of salivary gland samples in 10 µl of pmsf in pbs + 10 µl of sample buffer). electrophoresis was carried out at a constant voltage of 150 v for 100 minutes in a running buffer. then the gel underwent a coloring process using coomassie brilliant blue dye solution (staining) for 60 minutes. the washing process (destaining) was performed 3 times using trisbuffered saline (tbs) every 30 minutes to remove the dye solution that was not bound to protein. identification of the immunogenic protein fraction from ae. albopictus salivary glands identification of immunogenic protein fractions was carried out by western blot analysis, with several modified steps using the semi-dry method. (19) proteins in the polyacrylamide gel from sds-page analysis were transferred to the polyvinylidene fluoride (pvdf) membrane for 1 hour with a constant current of 100 ma. the pvdf membranes were cut according to the size of the polyacrylamide gel then immersed in methanol for 1 minute. the polyacrylamide gel, pvdf membrane, 2 sheets of whatman filter paper, and fiber pad were immersed in the transfer buffer prior to western blot analysis. the pvdf membrane, sds-page acrylamide gel, filter paper, and blotting paper (fiber pad) were arranged sequentially. the immunoblotting process was followed by a reaction between the salivary gland proteins of ae. albopictus on the membranes and primary antibodies derived from sera of healthy persons in dhf endemic areas, as well as dhf patients and neonates, that were dissolved in 5% skim milk in tbs (1: 250) then incubated overnight at 4oc. the membrane was washed with 3 tbs times for 5 minutes each. the secondary antibody used was goat – anti human igg alkaline phosphatase conjugate (rockland, usa) which was dissolved in 5% skim milk in tbs (1: 2500). the membranes that had been treated with secondary antibodies were then incubated for 2 hours. visualization was done by adding the nitro blue tetrazolium-(5-bromo-4-chloro-3-indolylphosphatase) (nbt-bcip) alkaline phosphatase substrate (rockland, usa) to the brackets and incubating in a dark room for 1 minute until immunogenic protein bands appeared. the membranes were washed with distilled water to stop the reaction. positive western blot results were shown by bands on the pvdf membrane, indicating the presence of binding between the antigens from the ae. albopictus salivary gland proteins and primary antibodies. ethical clearance all participants gave written, informed consent to take part in the study. the collecting protocol was approved by the ethical committee of medical research, faculty of dentistry, university of jember under no. 1034/un25.8/ kepk/dl/2020 results the structure of the ae. albopictus salivary glands consists of two parts connected by the salivary duct. each part has three lobes, namely two lateral lobes (l) and one medial lobe (m). each of the lateral lobes consists of two parts, namely the proximal lobe (pl) and distal lobe (dl). the structure of the salivary glands can be seen in figure 1. 238 the protein profile analysis of salivary gland extract of ae. albopictus was conducted by using sds page. the results showed that the salivary gland proteins from ae. albopictus have 13 protein bands with molecular weights of 16, 17, 26, 28, 31, 32, 45, 55, 60, 67, 73, 76, and 97 kda, respectively, as can be seen in figure 2. analysis of the immunogenicity of the salivary gland proteins of ae. albopictus was performed by western blot analysis of the crossreaction between the antigens (salivary gland proteins) and the sera of healthy persons living in endemic areas, dhf patients, and neonates (as negative controls). the 31 kda immunogenic protein can be recognized by all endemic population sera, both the sera of dhf patients and healthy persons. meanwhile, the 47 and 67 kda protein bands were only recognized by the sera of dhf patients (figure 3). discussion the results of sds-page analysis of the salivary gland prot ein extracts fr om ae . albopictus showed 13 protein bands, which are thought to have certain roles such as in anti-host homeostasis and are involved in the metabolism of the mosquito’s body. the role of the 97 kda and 79 kda proteins is unknown. the 60-70 kda proteins are thought to contain apyrase which functions to inhibit platelet aggregation. (16,20–22) the proteins with molecular weights ranging from 40 to 55 kda are serpins which function as figure 1. structure of salivary glands of ae. albopictus (a) and ae.aegypti (b). salivary duct (sd), lateral lobe (l), proximal lobe (pl), distal lobe (dl), medial lobe (m). 50x magnification on the nikon smz735 microscope. camera: xiaomi note 6 pro. marker: prestained blue eye ps-104 (m) figure 2. salivary gland protein profile of ae. albopictus (samsung a50 camera) oktarianti, khasanah, wathon, et al immunogenic protein from salivary gland 239 univ med vol. 40 no 3 protease inhibitor and anticoagulant. approximately 500 serpins are currently known, consisting of between 350 and 400 amino acids. (23) the 31 kda protein is a d7 protein that acts as vasodilator, immunomodulatory and anticoagulant substance. (17) the 27-29 kda bands contain immunogenic proteins and are present in all aedes mosquitoes.(21,22) the 17 kda band has d7 family proteins and lectins, which function in opsonization and as antimicrobials. these proteins control microbial growth on sucrose in the mosquito’s midgut.(24) apyra se (atp-dip hosphohydrolase) contained in the 60-70 kda bands is an enzyme released by ae. albo pictus and other hematophagous arthropods during blood feeding figure 3. results of the immunogenicity analysis of the ae. albopictus salivary gland proteins in individual sera (a, b, c and d) of dhf patients, healthy persons, and neonates (samsung a50 camera) indonesian abbreviations: pasien dbd [demam berdarah dengue] = dengue hemorrhagic fever patients sehat endemik = healthy endemic [sera] 240 that functions as platelet aggregation inhibitor. (25) the 67 kda molecular weight protein in our study is thought to contain apyrase and α-glucosidase. the latter acts as an allergen that will induce ige in humans. this enzyme is also released when mosquitoes suck blood and sugar so that the sugar and blood are not mixed in the mosquito’s mid gut.(26,27) the apyrase of ae. albopictus is in the molecular weight range of 61 kda to 68 kda.(16,20,28,29) western blot analysis of the ae. albopictus salivary gland proteins showed 3 immunogenic proteins of 31, 47 and 67 kda, respectively. the 47 kda band contains the main protein thought to be a serpin which functions as an anticoagulant. (23) in addition, serpins in the body of insects play a role as proteinase inhibitors. serine is a proteinase that breaks down the polypeptide bonds produced by pathogens such as viruses. the action of serine is inhibited by a serineprotease inhibitor, namely serpin.(30) serpins that enter the host are thought to inhibit the action of serine so that the transmission of the dengue virus runs smoothly. dhf patients also activate b cells to produce more antibodies, so the serum of dhf patients are able to recognize more immunogenic proteins. samples taken from each individual presumably reflect the various responses of the individuals to the different antigens carried by the mosquitoes. from the western blot analysis results in figure 3, it is apparent that the immunogenicity analysis of sera of the endemic population shows different bands for each individual. this is presumably because of the differing responses of each individual to exposure to antigens carried by mosquitoes. the intensity of exposure to aedes salivary antigens in endemic areas will influence the human immune system to form specific antibodies (igg) against these antigens.(15,31) exposure to antigens for the first time (primary) will activate the t cells and b cells. the b cells will differentiate and proliferate to produce igg in small amounts. the t cells will be activated by the antigens to become memory cells upon secondary or multiple exposure to the antigens. re-exposure (secondary) to the same antigen will produce more igg.(32) apyrase, serpins, and the d7 family are thought to be the most immunogenic proteins in the salivary glands, so that even low titers can trigger the formation of antibodies in the host body. these three proteins are thought to be immunogenic because they can modulate the immune response of the host. the band of 31 kda is the d7 immunogenic family which facilitates the process of blood feeding. the d7 family functions as a vasodilator, immunomodulator, and anticoagulant.(33) the protein of 47 kda is serpin, tha t acts as anticoagulant and anti-inflammatory,(23,25,26) where as the band of 67 kda is apyrase, that acts as allergen and anticoagulant. the abovementioned three proteins are thought to trigger modulation of the host’s immune response because they comprise proteins that act as allergens and immunomodulators. allergens will react with ige and cause inflammation in the host.(26,27) the immunomodulators function to modulate the host’s immune response so that they can increase the transmission of pathogens into the host.(24) neonate se rum s amples were not recognized as containing immunogenic proteins, presumably because there was no binding between antigens of salivary gland proteins and serum antibodies. this is in accordance with previous r esea rch which sta ted that no immunogenic protein was recognized by neonate sera samples.(33) neonate samples were obtained from the umbilical cord of the baby at birth, thus the neonate samples had never been directly exposed to ae. albopictus salivary gland proteins so that no immunogenic proteins were identified. the three immunogenic proteins of 31, 47 and 67 kda from the salivary glands can be used as biomarker protein candidate for bite exposure to ae. albopictus and can be developed into a vector-based dhf vaccine. therefore, further studies are needed to determine the identities of these proteins and their biological functions in dengue virus transmission and blood feeding using transcriptomic or proteomic analysis. the limitation of this study was the sera samples were oktarianti, khasanah, wathon, et al immunogenic protein from salivary gland 241 univ med vol. 40 no 3 only taken from one dhf endemic region i.e. jember, east java indonesia. therefore, further investigation in the several endemic regions is needed to find out the relevancy of the data”. conclusions this study demonstrated 3 immunogenic proteins that were identified from the salivary glands of ae. albopictus of 31, 47 and 67 kda, respectively. the 31 kda protein fraction could be recognized by all endemic population sera, both the sera of endemic dhf and healthy patients. in contrast, the 67 and 47 kda fractions were only recognized by the sera of dhf patients. these immunogenic proteins can be used as biomarker candidates for bite exposure to ae. albopictus and can be developed into a vectorbased dhf vaccine. conflict of interest the authors declare that there are no conflicts of interest. acknowledgement this study was financially supported by "hibah penelitian pendukung idb" internal financial (pnbp) of jember university. contributors ro conceived and planned the experiments and wrote the manuscript. rnk carried out the experiment and contributed to the interpretation of the results. sw contributed to sample preparation and interpretation of the results. ks conceived the original idea and supervised the project. all authors have read and approved the final manuscript. 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2023;42: 195 -205. doi: 10.18051/univmed. 2023.v42:195-205 may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 attitudes toward fertility and childbearing among female university students derya yüksel koçak1 , nuriye büyükkayaci duman1* , and rukiye hobek akarsu2 abstract background in recent years, there has been a significant decrease in childbearing rates in many parts of the world. young people who delay their marriage plans for various reasons also indirectly delay their childbearing to advanced ages. postponed childbearing may lead to increased health risks for both mother and child. this study aimed to determine attitude towards fertility and childbearing in female university students. methods this cross-sectional study involved 259 female university students. the data were collected with attitudes toward fertility and childbearing scale (afcs). data were analyzed using t test and oneway anova. results in the study 92.7% of the students wanted to become a future mother. the importance of fertility for the future mean score of the students with low income level (21.56±8.14) was found to be lower than the students with medium (25.80±6.51) and high income level (25.29±4.37) (p<0.05). the importance of fertility for the future mean score of only-child students (22.57±7.09) was lower than that of the other students (25.82±6.45). the importance of fertility for the future mean score of the students who did not have a date was also found to be lower than the students who had a date (p<0.05). conclusion in the study, students with a low income level, who are an only child and who do not have a date during the study care less about fertility for the future. also, students with a single-parent family, with siblings, and no previous sexual intercourse identify childbearing more with female identity. keywords: childbearing, attitudes, female university students, turkey 1hitit university, faculty of health sciences, department of nursing, corum, turkey 2bozok university, faculty of health sciences, department of nursing, yozgat, turkey *correspondence: nuriye buyukkayaci duman, hitit university, faculy of health sciences, department of nursing, corum, turkey, tr 19100 phone: +905055015752 email: nurfatihh@hotmail.com orcid id: 0000-0002-4246-3607 date of first submission, march 24, 2023 date of final revised submission, august 8, 2023 date of acceptance, august 22, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2023.v42:195-205 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1435 195 original article https://orcid.org/0000-0001-6890-6755 https://orcid.org/0000-0002-4246-3607 https://orcid.org/0000-0002-3476-0740 https://univmed.org/ejurnal/index.php/medicina/article/view/1435 196 kocak, buyukkayaci duman, hobek akarsu attitudes toward fertility and childbearing introduction in recent years, there has been a significant decrease in childbearing rates in many parts of the world. from 1990 to 2019, the global childbearing rate per woman declined from 3.2 to 2.5. this rate is estimated to fall down to 2.2 in 2050 and 1.9 in 2100.(1) as in the rest of the world, childbearing rates also tend to decrease in turkey, from 2.7 children to 2.3 in the past 25 years.(2) the decline in childbearing rates all over the world is thought to be influenced by factors such as the incr eased education level of women, the widespread use of birth control, and delayed ages of marriage and childbearing.(3) in turkey, childbearing rates differ depending on the region, education level, and income level.(2) the decision to b ear childr en is a multifaceted process that is influenced not only by individual, social, and economic factors but also by socia l policies. (4 ) techno logica l advancements and economic changes have affected people’s lifestyles all over the world. the fact that women are more active in working life and the spread of individualization with globalization has disrupted the traditional family structure.(5) increased education, employment, and career opportunities for women have impacted motherhood and reproductive behaviors. as women took on more active roles in business life and began to move away from home life to achieve their business goals, the number of nuclear families of two has begun to increase. consequently, many european countries have experienced a decrease in the number of births and a rise in dela yed childbearing age.(6) postponing parenting for individual, social, economic, and social reasons is not always a conscious process, but it can also cause an increase in the incidence of infertility.(4) in turkey, the acceptance of childbearing and having an active sexual life is associated with marriage. the median age of first marriage for turkish women in the 25-49 age group is 21.4 years. the age of first marriage stands higher in western countries, urban women, and women with higher education levels.(2) young and educated women delay their plans regarding childbearing to advanced ages to continue their education, achieve their career and business goals, and achieve economic independence, thanks to the developed contraceptive methods.(7) young people who delay their marriage plans for various reasons also indirectly delay their childbearing to advanced ages. (8 ) most young people are considering having children in the future, but cite some reasons for delaying childbearing, such as career goals.(9,10) considering the social status of women in this way, it can be said that the most appropriate age for giving birth is between 25-35 years, when they have completed their education and gained some skills in business life.(11) however, the decreasing number and quality of ovarian follicles with advanced maternal age cause loss of reproductive potential, widespread use of assisted reproductive techniques, and increased obstetric morbidity and perinatal complications.(4,12) on the other hand, studies on the subject show that young women who consider postponing motherhood to later dates do not have enough information about the time of childbearing.(13) in this context, from a cultural and social point of view, it is very important to determine the perspectives of young women on childbearing and having children in conservative societies such as turkey, where the status of women is accepted as equivalent to fertility, in terms of determining fertility and reproductive policies in the future.(14,15) when the national literature on the subject is examined, it is seen that there are a limited number of quantitative studies to determine the fertility and childbearing behaviors and attitudes of young women, mostly qualitative studies.(15,16) regarding the attitudes toward fertility and childbearing scale (afcs) scale developed by söderberg et al.,(8) persian and japanese versions have been published, and turkish validity and reliability studies have been conducted. the information about the attitudes among university students for timing of their parenthood is scanty. a previous study among female students (n=300) 197 visiting a student health centre in sweden, showed that female university students are not very concerned about having children before they get ‘too old’ and had an acceptable understanding about fertility.(17) another study in female university students in cameroon, showed that most of the female students intend to have children in the future, but their fertility awareness knowledge was suboptimal.(18) considering the f act that a deeper understanding of the attitudes towards fertility and motherhood may help clarify the decision to have children among young individuals in turkish young females, there is a great need for further study in this area. therefore the current study aimed to determine the attitude towards fertility and childbearing in female university students methods research design this cross-sectional study was carried out with female university students studying at the undergraduate department of a state university between may and june 2019. research subjects the study population consisted of 450 female students continuing their education in the relevant department of the university as of 2019. since it was impossible to reach the whole population in terms of time, costs, and application, the sample size was calculated using the simple random sampling formula. in the validity and reliability study of the afcs scale, it was calculated as 26.52±5.98 for the future importance subscale, 28.06±8.25 for the present obstacle subscale and 15.68±5.41 for the femininity identity subscale.(19) the sample size required for the future importance subscale was: the sample size for the present obstacle subdimension was: the sample size for the femininity identity subdimension: 𝑛 = 𝑡𝛼 2 ,∞ 2 ∗ 𝑆2 𝑑2 = 1.962 ∗ 5.982 (26.52 ∗ 0.05)2 = 79 𝑛 = 𝑡𝛼 2 ,∞ 2 ∗ 𝑆2 𝑑2 = 1.962 ∗ 8.252 (28.06 ∗ 0.05)2 = 133 𝑛 = 𝑡𝛼 2 ,∞ 2 ∗ 𝑆2 𝑑2 = 1.962 ∗ 5.412 (15.68 ∗ 0.05)2 = 181 the attitudes tow ard fertility and childbearing scale (afcs) used consists of 3 sub-dimensions, and there is only one study in turkey in which the validity and reliability of this scale was tested.(19) since the evaluation of the afcs scale was calculated for each subdimension, the calculation of the sample size in this study was made separately for each subdimension. the effect sizes in the calculations are 5% of the arithmetic mean. as a result of the calculations, the sample sizes of the afcs scale wer e determined to be 79 for the future importance sub-dimension, 133 for the current barriers sub-dimension, and 181 for the identity of women sub-dimension. it was understood that the sample size to be selected in this study should be at least 181. all students who met the inclusion criteria and gave their written consent to participate were included. the study was completed with 259 students. the inclusion criteria were: i). volunteering to participate; ii).being a female university student in the undergraduate program; and iii) single student aged >18 years. the exclusion criteria were: i) having experienced a previous pregnancy or childbirth, and ii) having physical, mental, visual or auditory disabilities that may prevent reading, understanding, and answering the items in the questionnaire to be filled by the participants. data collection female university students who did not meet the inclusion criteria or did not agree to participate after being rationally informed about the study were excluded. although the minimum sample size was 181, the researchers distributed 300 questionnaires, considering possible losses. at the univ med vol. 42 no. 2 198 kocak, buyukkayaci duman, hobek akarsu attitudes toward fertility and childbearing data collection tools during data colle c tion, a per sonal information form was used, consisting of 12 questions about the sociodemogra phic characteristics of the students, namely age, place of birth, place of residence, monthly income, perceived family income, student residence, being an only child, family type (single-parent family), education level of the mother and father, working status of the mother, dating at the time of the study, previous sexual intercourse, and desire to have children in the future. the attitudes tow ards f ertility and childbearing scale (afcs) to determine the attitudes of female university students towards fer tility and childbearing, the attitudes towards fertility and childbearing scale (afcs) was used, which was developed by söderberg et al.(20) and tested for turkish reliability and validity by damar.(19) the afcs was developed for nulliparous women between the ages of 20-30 to determine attitudes towards fertility and childbearing, and the second version of the scale was revised in 2015.(20) it is a 27-item five-point likert-type scale (1-strongly disagree; 2disagree; 3-neither agree nor disagree; 4-agree; 5-strongly agree). lower scores indicate negative attitudes towards fertility and childbearing. the scale consists of 3 subscales on the importance of fertility for the future (8 items), seeing childbearing as a hindrance at present (13 items), and the female identity (6 items). scores range between 8-40 points for the importance of fertility for the future subscale, 13-65 points for the childbearing as a hindrance at present subscale, and 6-30 points for the female identity subscale. the cronbach alpha coefficients of the original scale and the subscales range between 0.862 and 0.945. in the turkish reliability and validity study by damar,(19) the total cronbach alpha coefficient of the scale was found to be 0.82, and the coefficients for the subscales were 0.89 for childbearing as a hindrance at present, 0.92 for importance of fertility for the future, and 0.90 for female identity. characteristics n % age ( years) ≤19 20-22 ≥23 70 160 29 27.0 61.8 11.2 place of birth city district village 68 108 83 26.3 41.7 32.0 place of residence city district village 81 127 51 31.3 49.0 19.7 income level low middle high 25 213 21 9.7 82.8 8.1 student residence dormitory with friends at student house at home with my family 156 63 40 60.2 24.3 15.4 being only child yes no 37 222 14.3 85.7 family type nuclear extended 200 59 77.2 22.8 single-parent family yes no 44 215 17.0 83.0 education level of the mother literate or primary school graduate middle school-high school graduate college-university 196 24 39 75.7 9.3 15.1 education level of the father literate-primary school graduate middle school-high school graduate 84 118 32.4 45.6 table 1. distribution of student’s characteristics (n=259) end of the data collection 285 questionnaires were received. after excluding incomplete and incorrectly filled questionnaires, a total of 259 forms were left to be evaluated. a total of 26 questionnaires were not included in the analysis due to insufficient or blank responses. the students were reached during their breaks. each of the students completed their questionnaire in 15 minutes on average. explanations about the study were made by the researchers and written informed consent was obtained from the students. 199 in our study, cronbach alpha coefficients were 0.83 for the whole scale, 0.91 for the “future importance” subscale, 0.84 for the childbearing as a hindrance at present subscale and 0.81 for the female identity subscale. data analysis statistical analysis was performed using the spss for windows 22.0 software. in statistical evaluation, percentage and mean values were calculated. after e va lua ting the data for conformity to a normal distribution, t test and anova were used. mean scores were given together with standard deviation (sd) (mean± sd) and p<0.05 was accepted as the statistical significance level. ethics permission was obta ined from the researcher who carried out the turkish validity study of the scale. before data collection informed consent was obtained from the students. in order to conduct the study, approval was obtained from the hitit university ethics committee (no. 2019-106). results in the study, 61.8% of the students were aged between 20-22 years. we found that 26.3% of the students were born in the city, 49% lived in a district, and 82.8% had a moderate income level. we also found that 60.2% of the students lived in dormitories, 77.2% had extended families, 14.13% were an only child, and 17% had a single-parent family. in addition, 75.7% of the students had mothers who were literate or primary school graduates, 45.6% had fathers who were middle or high school graduates, and 36.3% had working mothers. finally, 40.9% of the students stated that they were dating at the time of the study, 10.8% stated that they never had sexual intercourse, and 92.7% wanted to become a future mother (table 1). in the study, the students’ afcs total mean score was 73.37±11.49. when the distribution of afcs sub-dimension mean score is examined, the importance of fertility for the future subdimension score is 25.35±6.63, childbearing as a hindrance at present sub-dimension score is 30.73±6.66, and female identity sub-dimension score is 17.29±4.31. in this study, when the distribution of students’ afcs total mean score and afcs subdimension mean score according to some characteristics are examined, the importance of fertility for the future sub-dimension mean score (21.56±8.14) of the students with low income level was found to be lower than for the students with medium (25.80 ±6.51) and high income level (25.29±4.37). this difference was found to be statistically significant (p<0.05). in addition, the importance of fertility for the future sub-dimension mean score of the only-child stude nts (22.57±7.09) is lower than that of the other students (25.82±6.45). during the study, the importance of fertility for the future sub-dimension mean score of the students who did not have a date was also found to be lower than for the students who had a date. this difference was also found to be statistically significant (p<0.05). in other words, according to these findings, it can be said that students with a low income level, who are an only child, and who do not have a date during the study, care less about fertility for the future. the differences between the mean scores of age, place of birth, place of residence, student residence, family type, the state of having single-parent family, education level of the mother, education level of the father, working status of the mother, and having prior sexual intercourse, respectively, in the importance of fertility for the future sub-dimension was found to be statistically not significant ( p>0.05) (table 2). in the study, the childbearing as a hindrance at present sub-dimension mean score of the students whose mothers are working (32.11±5.66) is higher than that of the students whose mothers are not working (29.95±7.06) (p<0.05). in other words, it can be said that students whose mothers are working now see childbearing as an obstacle in planning their lives. the differences between univ med vol. 42 no. 2 200 kocak, buyukkayaci duman, hobek akarsu attitudes toward fertility and childbearing the mean scores of age, place of birth, place of residence, income level, student residence, being an only child, family type, the state of having single-parent family, education level of the mother, education level of the father, dating at the time of the study, and having prior sexual intercourse, respectivley, in the importance of fertility for the future sub-dimension were found to be statistically not significant ( p>0.05) (table 2). in the study, the female identity subdimension mean score (19.09±3.65) of the students with a single-parent family was found to be higher than that of the other students (16.92±4.35). the female identity sub-dimension mean scores of the students with only one child (15.92±4.99) were lower than the students who did not have one child (17.52±4.15). in addition, mean female identity sub-dimension scores of stude nts who have not had prior sexual intercourse are higher (17.52±4.23) than students who have had prior sexual interc ourse (15.39±4.53). this difference was found to be statistically significant (p<0.05). in other words, according to these findings, it can be said that students who have a single-parent family, have siblings, and have no previous sexual intercourse, identify childbearing more with female identity and consider being a mother important in terms of female identity. the differences between mean scores of age, place of birth, place of residence, monthly income, perceived families’ income, student residence, family type, education level of the mother, education level of the father, working status of the mother, dating at the time of the study, respectively, in the desire to have children in the future and female identity sub-dimension mean score was found to be statistically not significant (p>0.05) (table 2). discussion in our study afcs total mean sc ore (73.37±11.49) and afcs sub-dimension total mean scores (the importance of fertility for the future (25.35±6.63), seeing childbearing as a hindrance at present (30.73±6.66), the female identity (17.29±4.31) of female students were found to be higher. in the validity and reliability study of the scale, it was calculated as being 26.52±5.98 for the future importance subscale, 28.06±8.25 for the present hindrance subscale and 15.68±5.41 for the femininity identity subscale.(19) accordingly, it can be said that female students participating in the study generally have a positive attitude towards childbearing and having children in the future. in addition, these findings show that the participants associate childbearing with female identity and consider motherhood as a requirement of being a woman. in addition, the high mean score of seeing childbearing as a hindrance at present indicates that the female students participating in the study currently consider having a child as an obstacle in terms of self-realization and career planning. in our study almost all of the female students in the study reported that they wanted to be mothers in the future (92.7%). in similar studies students thought that childbearing was important and they wanted to have children in the future.(10,21-23) in studies conducted in turkey, the majority of students stated that they would like to have children in the future.(24,25)these finding indicate that young women have a positive attitude and desire towards having children in the future. in our study, the differences in mean scores of age, place of birth, place of residence, student residence, family type, having a single-parent family, education level of the mother, education level of the father, working status of the mother, and having prior sexual intercourse, respectively, were not significant in the importance of fertility for the future sub-domain. it was also determined that students with low income, who are single children and who do not have a date at the moment, care less about having a child in the future than other students. in many studies, parallel to this study, the importance women attribute to childbearing and having children in the future is influenced by many socio-cultural characteristics.(16,23,25) in some studies, similar to the results of our study, it has been determined that the thoughts of young nulliparous women 201 characteristics attitudes to fertility and childbearing scale (afcs) sub-dimensions importance of fertility for the future sub-dimension childbearing as a hindrance at present sub-dimension female identity sub-dimension p value p value p value age (years) ≤19 20-22 ≥23 24.53±6.51 25.43±6.79 26.93±5.89 0.255 30.67±6.09 30.80±7.05 30.52±5.93 0.974 16.83±3.61 17.53±4.54 17.10±4.59 0.515 place of birth city district village 24.81±6.32 25.71±6.77 25.33±6.74 0.680 31.09±6.42 30.83±6.68 30.31±6.87 0.762 16.72±4.38 17.75±4.35 17.16±4.18 0.288 place of residence city district village 24.88±6.28 25.81±6.81 24.96±6.78 0.550 31.01±6.54 30.72±6.77 30.33±6.66 0.850 16.67±4.39 17.60±4.37 17.51±4.00 0.291 monthly income 0-1000 try 1001 try 2600 try ≥ 2601 try 25.04±6.81 26.71±6.54 26.36±4.68 0.323 30.75±6.48 30.77±6.78 30.50±8.34 0.985 17.21±4.46 17.84±3.93 17.27±3.33 0.751 perceived family income low middle high 21.56±8.14 25.80±6.51 25.29±4.37 0.010 28.92±6.84 30.90±6.61 31.24±6.96 0.351 15.76±5.72 17.45±4.18 17.52±3.47 0.175 student residence dormitory with friends at student house at home with my family 25.08±6.65 25.03±7.10 26.93±5.69 0.265 30.28±6.64 31.06±7.07 32.00±6.01 0.312 17.33±4.32 17.11±4.75 17.43±3.57 0.924 being only child yes no 22.57±7.09 25.82±6.45 0.006 32.32±6.40 30.47±6.68 0.117 15.92±4.99 17.52±4.15 0.036 family type nuclear extended 25.23±6.86 25.78±5.84 0.573 30.71±6.87 30.83±5.84 0.899 17.12±4.43 17.86±3.87 0.244 single-parent family yes no 25.07±6.51 25.41±6.67 0.757 30.73±6.25 30.73±6.75 0.995 19.09±3.65 16.92±4.35 0 education level of the mother literate or primary school middle school-high school college-university 25.34±6.62 24.88±7.29 25.69±6.43 0.893 30.84±6.68 30.17±5.10 30.73±6.66 0.885 17.40±4.32 16.67±3.41 17.13±4.81 0.713 education level of the father literate-primary school middle school-high school college-university 25.08±6.85 26.31±6.05 23.77±7.22 0.054 30.32±5.97 31.28±7.15 30.21±6.59 0.482 17.32±4.31 17.63±4.33 16.54±4.25 0.297 working status of the mother working not working 24.54±6.61 25.81±6.62 0.139 32.11±5.66 29.95±7.06 0.012 16.61±4.00 17.68±4.44 0.054 dating at the time of the study dating not dating 26.72±5.89 24.41±6.96 0.006 30.87±7.12 30.64±6.34 0.788 17.87±4.52 16.89±4.13 0.072 prior sexual intercourse yes no 23.82±6.13 25.54±6.68 0.197 29.68±6.06 30.86±6.73 0.376 15.39±4.53 17.52±4.23 0.013 desiring to have children in the future yes no 25.48±6.73 23.79±5.17 0.287 30.63±6.73 32.00±5.70 0.390 17.27±4.37 17.58±3.49 0.762 table 2. distribution of fertility and childbearing scale (afcs) subdimension mean scores by student characteristics note: data presented as mean ± sd; independent-samples t-test or on-way anova; try: turkish lira univ med vol. 42 no. 2 202 kocak, buyukkayaci duman, hobek akarsu attitudes toward fertility and childbearing about childbearing and having children are not affected by some characteristics such as family type, the place where most of their life is spent, parent education and past sexual experience.(24,25) in other studies, it is stated that similar sociodemographic characteristics are determinative in the formation of young nulliparous women’s thoughts on childbearing and having children.(26,27) this is thought to be due to the socio-demographic and cultural characteristics of the groups of subjects in the studies. in our study, the mean score of the subdimension of seeing having children as an obstacle was higher in students whose mothers were employed than in students whose mothers were not employed. hickman et al.(10) reported that the desire to have a good financial status affected women’s childbearing decisions. in another study, having a stable financial situation and good living conditions were also found to be effective in childbearing decisions.(28) considering the care costs of newborn babies, the economic burden of raising children in turkey, where social policies are inade quate in this regard, may cause individuals with low income levels to postpone their future plans for childbearing and parenthood. inadequate social policies and financial resources to cover the cost of childcare, such as childcare services and low wages, make it difficult for women to balance work and family duties. this is another reason for the de cline in childbearing.(29) it is highlighted that having a good financial status, easy access to childcare, and having a job that can be done while raising children are all important in childbea ring decisions.(10,20,30,31) the university students in our sample have not yet completed their education and therefore do not have a regular income, which is indeed very important in childbearing and parenting decisions. in our study, students with siblings scored higher in the future importance of childbearing and the female identity subscales and those grown up in single-parent families scored higher in the female identity subscale. in similar studies it was determined that having siblings affects attitudes towards having children and childbearing in the future (30,32) while in some studies no relationship was found.(16,20) considering the finding that 77.2% of our students had extended families, those who are not a single child may have more positive attitudes towards fertility and childbearing regarding the spiritual influence and social support of having siblings. besides, since having children means gaining status, respect, and social maturity for women in turkey, it is understood that students with siblings attach more importance to fertility and childbearing with regards to their female identity. students who grow up in single-parent families might see it as an advantage to have an extended family and have more children for their female identity. in our study, the mean scores of female identity subscale scores of students who had not had prior sexual intercourse were higher than those of students who were not sexually active. in addition female students who were dating at the time of the study had higher scores from the future importance of the childbearing subscale. chan et al.(9) reported that having a stable partner to share responsibilities with is crucial in childbearing decisions. söderberg et al.(18) found that women with a partner had more positive attitudes in the future importance of childbearing and female identity subscales. in many studies, it was also reported that engaged and married female students had more positive attitudes towards parenthood and childbearing,(19) and that not finding the right partner,(9) having a partner with common characteristics,(28) having a stable relationship, and sharing responsibilities with the partner, were important in the decision to become a parent.(9) this finding was seen as a natural consequence of the dating female university students tending to marriage and dreaming of having children in the future. it was also found that students who had prior sexual intercourse had higher scores in the female identity subscale. this suggests that female university students are aware of the responsibilities of childbearing and 203 that they perceive motherhood and childbearing as important. in our study, 89.2% of the students stated that they had not had prior sexual intercourse. in the study, the term sexual intercourse refers to penile-vaginal penetration. in turkish society, it is not welcomed for women to have sexual intercourse prior to marriage and it is important to protect virginity. for this reason, although the personal information of the students included in the study was not questioned, their concer ns a bout la be ling, exclusion and confidentiality of data may have caused the girls who had previously had sexual intercourse to not answer this question correctly. in fact, it is thought that the number of female students who have had prior sexual intercourse is higher in the sample. there are limitations in this study. the first is that the data is based on self-report. secondly, since our study is cross-sectional, from our results a causal relationship cannot be inferred. it is suggested that the scale use nulliparous women, older individuals, and different populations. conclusion this study demonstrated that female students have a positive attitude towards childbearing and having children. however, the female students participating in the study currently consider having a child as an obstacle in terms of self-realization and career planning. in the future, it can be recommended to conduct a cohort study with female university students and including male students from different regions. acknowledgement we thank the students who participated in this study. fundings the author (s) received no financial support for the research, authorship and/or publication of this article. disclosure statement the authors declare no conflicts of interest associated with this article. author 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10.4274/jpr.47966. univ med vol. 42 no. 2 september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 breast cancer survivors and behavior: cancer care in the time of covid-19 in indonesia widyanti soewoto1* abstract the covid-19 pandemic caused significant disruptions in cancer care, and preliminary research suggests that these disruptions are associated with increased levels of psychosocial distress among cancer survivors. indonesia initially reported positive cases in early march 2020, followed by regular extensive occurrences across 34 provinces. the disease causes acute respiratory failure among people with specific comorbidities, including geriatric disorders, diabetes, cardiovascular and respiratory diseases, and cancer. consequently, the patients’ conditions become more severe, possibly leading to a higher mortality rate. individuals with cancer are at particularly elevated risk of a severe course of covid-19 because they tend to be of older age and are at a greater risk for needing intensive care and for mortality. a reasonably good comprehension of the current covid-19 outbreak, poor awareness of the primary disease and subsequent therapy program, and minimal understanding of the importance of continuing treatment during the pandemic are the probable factors causing the fear of contracting the covid-19 virus that poses a significant threat of aggravating the existing breast cancer conditions in most patients. this anxiety shows a drastic impact in altering the behavioral patterns of survivors undergoing therapy. there is a very good understanding of how it is caused by the sars-cov2 virus. meanwhile, there is a severe lack of understanding of cancer and the therapeutic program. the patients are more afraid of contracting this virus than they are of their cancer. patients prefer development of their cancer to contracting the sarscov2 virus. therefore, massive education and supervision are needed for cancer patients to understand the covid-19 pandemic and ensure that routine checks are appropriately managed. keywords: covid-19, breast cancer, fear, cancer therapy 1 sub division of oncology surgery, faculty of medicine, sebelas maret university, indonesia *correspondence: widyanti soewoto jl slamet riyadi no 419 makamhaji kartasura surakarta jawa tengah indonesia phone: +62817272338, email: widyanti_s@staff.uns.ac.id orcid id: 0000-0003-2513-5801 date of first submission, february 18, 2022 date of final revised submission, august 13, 2022 date of acceptance, august 30, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license original article doi: http://dx.doi.org/10.18051/univmed.2022.v41:277-288 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1302 277 cite this article as: soewoto w. breast cancer survivors and behavior: cancer care in the time of covid-19 in indonesia. univ med 2022;41:27788. doi: 10.18 051/univme d.20 22. v41:277-288. https://orcid.org/0000-0003-2513-5801 http://dx.doi.org/10.18051/univmed.2022.v41:277-288 https://univmed.org/ejurnal/index.php/medicina/article/view/1302 soewoto impact of the covid-19 pandemic in indonesia 278 introduction millions of casualties have been reported globally since the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was first discovered in december 2019 in wuhan, china. this virus is responsible for the rapid spread of the covid-19 pandemic through airborne contact with infected persons or contaminated surf aces. also, the disease causes ac ute respiratory failure among people with specific comorbidities, including geriatric disorders, smoking habits, hype rtension, diabetes, cardiovascular and respiratory diseases, and cancer.(1) as a consequence, the patients’ conditions become more severe, possibly leading to a higher mortality rate. furthermore, every sector of human endeavor has drastically changed, with multiple deaths attributed to the fear of contracting the virus, particularly in persons diagnosed with various comorbidities.(24) the world health organization (who) has declared the covid-19 disease a public health emergency of international concern (pheic). indonesia initially reported positive cases in early march 2020, followed by regular extensive occurrences across the 34 provinces. daily, 223 new patients were observed in less than a year, with an accumulated total of 3,886, the highest rate being in east java. this infection alters diverse societal aspects, specifically in the health sector.(4,5) various methods have been applied to reduce the coronavirus spread and mortality rate. however, the disease appears very complex because the pathophysiology and exact cause are relatively novel. the detection, treatment, prevention, and other related activities are undergoing thorough research. this is because the possibility of several mutations of sars-cov2 poses a severe thr eat to the ef fective management of the growing outbreak.(4-6) community awareness and attitude are significant factors influencing the compliance on preventive measures against the spread of the virus. the fear of contracting the virus is attributed to the increasing rate of daily positive cases and is mainly observed among persons with comorbid diseases such as cancer.(7,8) this pandemic has greatly affected cancer patients who regularly come to health services to undergo cancer therapy, many of whom have postponed their therapy due to fear of infection. only cancer patients with severe conditions are now coming to the health service. likewise with new cases, patients delay treatment as much as possible and only when the complaints become more severe, do they dare to come to the health service. seve ral str ategies to pr event further covid-19 transmission and mortality rates have been initiated at the national and local levels by the indonesian government. one of the significant policies involved strict data collections based on specific locations with high numbers of infectious or suspicious cases, time of occurrence, confirmed persons’ status or condition, and direct or indirect contacts. furthermore, isolation centers were established when a total of 5 or more cases had be en r eported.(9) in a previous study, we distributed the universitas sebelas maret canc er attitude sat isf action k nowledge questionnaire (uns – caskq14) as a google form via the whatsapp application to breast cancer patients throughout indonesia. the uns – caskq14 is an easy-to-understand indonesianlanguage questionnaire, containing an assessment of the quality of and compliance with treatment of cancer survivors in undergoing therapy programs during the covid-19 pandemic. this study was to determine the behavior patterns of breast cancer survivors in indonesia, whether there is anxiety about contracting covid-19 or whether the anxiety about cancer eliminates the fear of contracting covid-19.(10) oncology specialists as well as other providers regularly involved in the diagnosis, active treatment, and longitudinal follow-up of patients with breast cancer must consider how to 1) balance a delay in breast cancer diagnosis or treatment against the risk for a potential covid19 exposure, 2) mitigate the risks for significant care disruptions associated with social distancing 279 behaviors, and 3) manage the appropriate allocation of limited health care resources in this unprecedented time of health care crisis. hopefully, we can see how the behavior patterns of cancer survivors in undergoing their therapy, so as to reduce delays in the enforcement of diagnosis and therapy in cancer patients, prevent cancer patients from being infected with the covid-19 virus and medical personnel in health services who work a little more effectively during the pandemic. management of breast cancer during the covid-19 pandemic the covid-19 pandemic appears not to subside and has recorded approximately 4 million global cases in virtually two years of existence. indonesia tends to experience rapid and increasing mortality rates daily due to human-to-human transmission. in mid-march 2020, the government imposed lockdown measures in the form of cessation of most activities, strict quarantine, and self-isolation.(11-14) all hospitals, from referral to minor hospitals, and other health facilities and medical personnel resources, are of utmost importance during this pandemic. as a significant change in facing this pandemic, most health facilities have been turned into special covid-19 medical centers. some medical services began to be limited from observing the gravity of the disease. patients with life-threatening conditions were to be prioritized, while non-eme rgenc y cases were to be postponed.(15-18) breast cancer is most predominant in women worldwide and has the highest incidence among all cancers diagnosed in indonesian women. over 65,000 new cases were reported in 2020, with the result that breast cancer has become the second malignant cause of death after lung cancer. the covid-19 pandemic is also known to significantly affect breast cancer patients and survivors, particularly in regard to managing schedule and therapy. individuals that have not been diagnosed also experience the impact and are afraid of visiting health facilities.(19,20) from the medical aspect, during this pandemic, medical personnel and health facilities had to make some modifications in treating and handling patients to minimize the risk of exposure and prevent medical personnel from being infected. therefore during this pandemic, there is a challenge in conducting and managing pa tients. (2 1,22 ) sever al new guidelines for cancer patients and survivors were made during this pandemic, with the aim that cancer patients and survivors who have severe comorbidities for covid-19 do not come to health facilities, because these health facilities are a source of covid-19 transmission during the pandemic. in addition, this is also to reduce excessive contact of medical personnel with patients whose infection status is unknown.(23,24) the principles of breast cancer management during the covid-19 pandemic may continue to be instituted as usual according to the applicable guidelines and taking into account the conditions of the hospital and the local area. if the patient’s condition allows, endocrine therapy is safer to administer in the pandemic situation, if it is necessary to postpone treatment that results in the patient being hospitalized.(23,24) according to their treatment, breast cancer patients are broadly grouped into patients not on therapy and patients on therapy. patients with “active disease” should still undergo surgical treatment, chemotherapy and radiotherapy, biologic ther apy, en docrine the rapy, and immunothe rapy (either as an adjuvant or neoadjuvant). this should be done with the application of strict health procedures, namely avoiding crowded places; wearing personal protective equipment when visiting a hospital for visits and treatment; washing hands properly according to who indications; not contacting friends and relatives with covid-19 symptoms or residing in a covid-19 endemic zone.(25-27) patients at high risk for contracting the covid-19 virus are those who are currently receiving or have received chemotherapy in the past three months, patients who have received extensive radiotherapy, patients who have had a bone marrow or stem cell transplant in the last univ med vol. 41 no 3 soewoto impact of the covid-19 pandemic in indonesia 280 a b six months, or who are still taking immunosuppressive drugs, patients with some types of blood or lymphatic cancer that impair the immune system, even if they do not require treatment (for example, chronic leukemia, lymphoma or myeloma). (2 3,2 6) during the pandemic, cancer patients are grouped into three groups: high priority, medium priority, and low priority. this grouping is based on the patients’ condition, both outpatients, patients needing diagnostic and supporting examinations, and patients with planned surgery, radiotherapy, and chemotherapy. (28) high-priority patients are those with lifethreatening, clinically unstable, or intolerable conditions. even if medical personnel are minimal, treatment must be carried out immediately to control the developing disease, relieve symptoms, or preserve the patient’s life. even a short delay will significantly change the prognosis for the worse.(29) a medium priority condition is one that is not immediately life-threatening, but treatment or services should not be postponed indefinitely until the end of the pandemic. most breast cancer patients will be included in the medium priority category. if conditions are unfavorable, treatment for medium priority patients may be delayed for some time during the pandemic. a short delay (6-12 weeks) will not affect the patient’s prognosis. a longer delay may affect outcomes in some medium-priority patients. patients with medium priority status can become top priority if their condition aggravates. (28,29) low priority patients are those in whom treatment or service can be suspended indefinitely until the pandemic ends without adverse ly af fecting c ancer prognosis. some cancer therapies have been revised by adjusting patients’ environmental conditions and covid-19 infection status. several types of surgery have been delaye d, i.v. line chemotherapy was modified to ora l, and radiother apy in some instances was a lso postponed. in addition, the follow-up schedule for cancer monitoring was altered, while several guidelines recommended telemedicine instead of face-to-face consultations. (24) the uns-caskq14 refers to a questionnaire used in a study assessing the quality of and compliance to treatment of cancer survivors who are considering therapy during the present pande mic. this survey was c onducted to determine the coronavirus impact on cancer survivors based on knowledge, attitude, practice, and compliance to clinical therapy. the analysis was expected to determine the behavioral patterns of breast cancer survivors in indonesia, precisely in remote areas with high levels of anxiety of contracting covid-19 or cancer. (30-34) breast cancer dur ing the covid-19 pandemic in indonesia indonesia is an archipelagic country comprising 17,504 large and small islands, with 45% and 55% having named and unnamed status, respectively.(35) however, several limitations have been observed, including the uneven distribution of facilities and infrastructure in terms of transportation, economy, education, information, and social services, with certain regions classified as remote. according to presidential regulation number 63 of 2020 on the designation of underdeveloped regions for 2020-2021, 62 locations in several provinces were designated as underdeveloped (figure 1). (35) based on law number 36 of 2009 on health, health service facilities consist of firstlevel health facilities, including public health centers, primary clinics, private medical/dental practices, and advanced referral health facilities, such as general and special hospitals. also, by december 2019, the total number of public health centers was precisely 10,134, comprising 6,086 inpatients and 4,048 outpatients (figure 2). (36) the fulfillment of immediate health care demands is generally presented from the ratio of public health centers to subdistricts of 1:4 in 2019. this indicates an ideal proportion of at least one public health center in 1 subdistrict and has been accomplished nationally, but with uneven distribution. furthermore, community accessibility 281 is influenced by various factors, including geographical conditions, area size, basic facilities, infrastructure availability, and socio-economic and regional progress. the lowest ratio occurs in west papua province, where community access to primary health care facilities is currently not ideal. a ratio below 1 showed that not all sub-districts have public health centers. at the same time, the complex geographical conditions and the low average socio-economic development in the area demonstrate the need to improve access to medical services. (31,36,37) hospitals are grouped into class a, b, c, and d, based on facilities and service capabilities. sequentially, the maximum number is class c hospitals at 51.6%, followed by class d, b, and a hospitals at 29.4, 14.9, and 2.1%, respectively. according to the who, the standar d for community needs for referral and individual health services in an area is obtained from the ratio of hospital beds to inhabitants of 1:1,000. this ratio was above 1 per 1,000 population between 2014 and 2019, representing the minimum who benchmark. however, the standard has not been fulfilled in 8 provinces, including west nusa tenggara (0.74), east nusa tenggara (0.83), banten (0.87), west java (0.87), lampung (0.90), west sulawesi (0.92), central kalimantan (0.94), and riau (0.98). (36,37) the proportion of medical personnel in all provinces appears to be uneven. approximately 57.2% of these workers are located on the island of java, with the highest number in the provinces of jakarta, east java, and central java. similarly, jakarta and east java record the maximum number of specialist doctors. because the provinces of papua, maluku, sulawesi, and kalimantan have a minor proportion of the population, it is not surprising that the minimum number of specialist doctors is found in west figure 1. map of indonesia with 34 provinces.(35) figure 2. the hospital beds per 1,000 population in indonesia from 2014 – 2019.(36) univ med vol. 41 no 3 soewoto impact of the covid-19 pandemic in indonesia 282 factors affecting attitudes of breast cancer patients and survivors during the covid19 pandemic 1. understanding of the pandemic the who declaration of covid-19 as a global pandemic has compelled the indonesian government to state the condition as a non-natural national disaster. in collaboration with local authorities, the national natural disaster manageme nt agenc y (bnpb) conducts socialization on covid-19 and its development and offers necessary measures to prevent further transmission. during the pandemic, various governmental actions were implemented, with the primary objective of prioritizing policies in health management over other sectors. (39) the central government also initiated health awareness campaigns across remote regions, using electronic media, television, radio, and social networks to complement local governments and health workers’ similar but direct socialization efforts. covid-19 also requires new insights into pathophysiology and management, and therefore, diverse information is generated as experts continue to research the best measures of combating the virus. however, every avenue is currently under research, and no concrete scientific evidence has been obtained, leading to a very vulnerable state of possible misinformation. (5,6) the limitations of facilities and infrastructure in specific remote communities have created complications in comprehending covid-19. this circumstance reduces public awareness in controlling transmission and early detection, leading to excessive panic due to misinformation. the condition also triggers stress and anxiety with a significant impact on the declining immune system of cancer patients and survivors. (40,41) medical care observably changed during the covid-19 pandemic, with adverse effects on susceptible patients, specifically older cancer patients. these alterations in the primary treatment of breast cancer and exposure to sars-cov-2 infection resulted in an extensive stressful situation. a recent survey of increasing covid-19 stress among patients awaiting sulawesi and maluku, although the majority of the doctors are employed in class a and b hospitals. (36,37) indonesia exhibits the optimal frequency of breast cancer in women, with the second-highest mortality rate after lung cancer. health workers and facilities must initiate immediate actions and ensure that these patients and survivors have a high probability of obtaining proper care and therapy. during treatment, adequate protective measures are also necessary to guarantee that the health workers are not infected with covid19 or other viruses.(38) there are challenges in patients as well as medical personnel, so that the professionalism of medical personnel and the priority of breast cancer patients need to adjust to local conditions during this pandemic, in order to minimize the risk of cancer development that must be balanced by reducing the risk of contracting the sars-cov-2 virus, both for the patients and the involved medical personnel. for the priority of breast cancer treatment, the patients are categorized into high risk, moderate risk and low risk patients.(32) high risk patients with are patients with lifethreatening, clinically unstable, or completely intolerable conditions. even a short delay will significantly change the prognosis. this group, requiring immediate treatment to control a developing disease, symptomatic help, or life support, is given top priority, even if resources become scarce. moderate risk patients are patients with conditions that are not directly lifethreatening but treatment or services should not be postponed indefinitely until the end of the pandemic. most patients with breast cancer fall into this group. low-risk patients are breast cancer patients in whom certain treatments or services can be suspended indefinitely until the pandemic ends without any adverse impact on their cancer. with the rearrangement in all lines of health services, especially the handling of breast cancer patients, it is hoped that the covid-19 pandemic will not cause the number of breast events to increase, nor will there be an increase in the mortality rate. 283 surgery detected fears that the pandemic will likely cause delays in their oncological care, leading to higher disease susceptibility, particularly in women with invasive cancer.(25,42) during the outbreak, hospital resources were reorganized into elective and semi-elective procedures to meet the needs of patients in critical conditions. furthermore, scarcity of supplies can delay diagnostic evaluation and breast cancer treatments. in addition, covid-19 anxiety plays a role in the therapeutic timing, as the early pandemic witnessed several patients requesting immediate surgery due to the high risk of suspending treatments. conversely, certain breast cancer patients refused or delayed therapy, including surgery. (43,44) 2. understanding of the required therapy program the relevant information or therapy for breast cancer requires proper delivery, depending on the patients’ education, occupation, and customs. poor communication or the differences in local languages are possible factors causing information deficiency. however, family members believe that patients do not need to be aware of the diseases or therapeutic processes, to reduce unnecessary worries. doctors are prohibited from disclosing sensitive information to external parties, even to certain family members. inadequate or absent understanding among the patients about the treatment possibly leads to failure, therefore the cancer tends to become more progressive, with a high mortality risk.(45-50) the greatest challenge for cancer patients during the pandemic is acquiring the necessary medical services, as a visit to hospitals extends the risk of contracting covid-19. consequently, certain patients are advised not to visit hospitals or leave their homes. at the same time, a number of therapy schedules have been delayed due to several regions implementing the quarantine measures.(51-53) a significant alternative recommended by health services involves the use of telehealth, or telemedicine, where people living with cancer can consult medical personnel, as this process significantly reduces oncology patients’ exposure to covid-19.(54) similar to other technology, accessibility appears to be the primary concern of telemedicine. despite the ease of establishing contacts, the connection in remote areas is typically unstable. another challenge is based on the economic conditions of different patients, as individuals with minimal soc ioeconomic backgrounds have constrained internet access. meanwhile, the category with sufficient socioeconomic and internet facilities prefers to receive treatment remotely, which mainly involves reporting the last complaint, followed by subsequent drug delivery.(55-60) telemedicine is not applied in certain areas because the majority of sick persons prefer direct treatment schedules with the doctor. all the complaints felt at home tend to disappear suddenly when interacting physically with medical personnel. therefore, it is strongly observed that cancer patients prefer visiting health facilities. (61) 3. underst anding the importance of continuity of treatment during the pandemic the unprecedented burden of covid-19 on healthcare systems worldwide generated significant implications, particularly in caring for cancer patients. first, limited data show that cancer patients are more susceptible to an aggravated state of the coronavirus infection, including a greater need for breathing apparatus such as ventilators and a high mortality rate. second, early cancer diagnosis appears to be delayed as screening programs and diagnostic services have been reduced or suspended in various countries. patients who are aware of the high infection risk are more reluctant to visit health facilities. third, the current guidelines for cancer treatment are changing to minimize the potential exposure and infection risk during surgery or radiation therapy. fourth, certain aspects of current clinical care have been prioritized to enable the health system to respond to the covid-19 pandemic, resulting in sub-optimal or delayed medical attention. fifth, cancer research is postponed, leading to a reduction in the current univ med vol. 41 no 3 soewoto impact of the covid-19 pandemic in indonesia 284 therapeutic options that could have been used and jeopardizing the development of long-term therapies.(22,62) gover nments, health facilities, and professional organizations have developed guidelines for managing the continuous welfare of cancer patients in the current pandemic. routine visits are conducted by telephone or rescheduled, and oral medications are delivered to individual homes to prevent further spread. in addition, surgeries, tissue sampling, or other investigations are performed at the local health facility. meanwhile, cancer multidisciplinary team meetings are conducted via virtual platforms, as individual treatment plans are also modified.(6364) due to misleading information about the virus, the confusions and inactions at the advent of the pandemic generated a higher possibility of poorer outcomes for vulnerable populations, including cancer patients. furthermore, the world also faces an ‘infodemic’ condition, where accessibility to selective and quali ty informa tion and understanding appears critical in considering decisions and actions under similar crises. this situation illustrates the need to increase health literacy to enable national health authorities. another obstacle is conducting a precise clinical examination remotely, as this process often reveals early signs that potentially lead to new investigations. the greatest challenge is probably the selection of patients that are asked to report to the clinic and the category that stays at home. furthermore, the patients are divided into three subgroups with different recommendations, including patients in the follow-up phase or patients on oral therapy with newly diagnosed cancer unde r curative care a nd metastatic cases receiving palliative care. (64) a basic priority is maintaining high-quality cancer care for all patients. local factors involving infrastructure, resources, personnel, and social and geographic constraints create differences in access and quality of care, particularly for vulnerable patients, including the patients from rural areas that are culturally, socially, and linguistically diverse. in addition, digital tools provide solution strategies to address logistical challenges contributing to the apparent gaps. overall the pandemic changed the behavior of cancer patients undergoing therapy, due to the lack of information about covid and its effects on cancer, the ignorance of patients as to how they should behave, whether cancer therapy should stop or continue, the lack of education on how patients should continue to undergo therapy for their cancer but also avoid transmission of the sars-cov2 virus. in addition, patients also feel that there is no or ganization that accommodates them so that they cannot ask questions. there is also the factor of limited internet connection that makes it difficult for patients to share news about their illness. conclusion it can be concluded that there is a very good understanding of how the sars-cov2 virus is transmitted. meanwhile, the understanding of cancer and the therapeutic program is very lacking. t he patients a re more af raid of contracting this virus than they are of their cancer. patients prefer the development of their cancer to contracting the sars-cov2 virus. it is very necessary to increase the provision of education by medical personnel so that patients really understand about the disease and the stages of therapy they will undergo. it is very necessary to evaluate periodically the patients’ understanding of their cancer. acknowledgements the author gives thanks to his colleagues dr suprabawati dr spb(k)onk – surabaya, dr yan wisnu dr spb(k)onk – semarang, dr artanto dr spb(k)onk – yogyakarta, dr dedy yulidar dr spb(k)onk – ntt, dr denny rifsal spb(k)onk – medan, dr noer faisal spb(k)onk – nad, dr ramses indrawan spb(k)onk – ntb, dr rudy tabri spb(k)onk – samarinda, dr r. yohana dr spb(k)onk – bandung, dr benny kusuma dr spb(k)onk – palembang, dr 285 albiner simarmata spb(k)onk – medan, dr christian oktavianus manginstar spb(k)onk – manado, dr nilam smaradhania t haufix spb(k)onk– makasar. thanks are also due to his beloved residents dr muhammad david perdana putra, dr galih santoso putra, dr rizka vinkan septiani, dr muhamad muamar, dr muhammad fahmi salafuddin, dr ikhdin radiamin saadhi, dr meirisa ardianti, dr dea alberta setiawati, dr rico alfredo hutabarat, dr dinar kukuh prasetyo. finally, thanks to all breast cancer patients who joined love pink indonesia and teratai pink. competing of interest the authors declare no competing interests. funding this work was supported by the university research group grant with number 261/un/ 27.22/hk.07.00/2021. references 1. abdelhafiz as, mohammed z, ibrahim me, et al. 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2017;389(10071): 847–60. http://dx.doi.org/10.1016/s01406736(16)31392-7 64. momenimovahed z, salehiniya h. epidemiological characteristics of and risk factors for breast cancer in the world. breast cancer (dove med press) 2019;11:151–64. doi: 10.2147/ bctt.s176070. c:\users\universa medicina\docu 200 abstract universa medicina comparison of matrix metalloproteinase-9 and e-cadherin expression in earlyand late-onset preeclampsia tjam diana samara1,2*, heri wibowo1,3, isabella kurnia liem1,4, ani retno prijanti1,5, and andrijono1,6 background preeclampsia (pe) is one of the most common pregnancy complications worldwide. turnover of villous trophoblast is affected by impaired placental perfusion in preeclampsia. among the various factors that influence pro and antiangiogenic factors in trophoblast invasion of pe are e-cadherin and matrix metalloproteinase-9 (mmp-9). the current classification scheme differentiates pe into two variants early-onset (eo) and late-onset (lo) pe. the aim of this study was to compare mmp-9 and e-cadherin expression between early(eo) and late-onset (lo) pe. methods this study used a cross-sectional design involving 26 women with gestational age <34 weeks (eo) and 38 women with gestational age 34 weeks (lo) from pe patients. placentas born to preeclamptic mothers were taken in the form of small pieces of the maternal side to measure the levels of mmp-9 and e-cadherin by the elisa method. statistical analysis was assessed using the mann whitney and independent t-test with a significant p value <0.05. results semiquantitative proteinuria levels were significantly higher in eo-pe group compared to lo-pe group (p=0.000). mean e-cadherin levels were significant lower in the eo-pe group (125.94 ± 54.22 pg/mg) compared to lo-pe group (157.95 ± 54.12 pg/mg) (p = 0.024). however, there was no significance difference in median mmp-9 levels between eo-pe group and lo-pe group (p=0.376). conclusion this study demonstrate that e-cadherin had lower levels in preeclampsia patients who gave birth <34 weeks. this study indicated that lower levels of e-cadherin can lead to early delivery in preeclampsia patients. keywords: mmp-9, e-cadherin, early-onset preeclampsia, late-onset preeclampsia original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.200-206 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1181 september-december 2021 vol.40no.3 cite this article as: samara td, wibowo h, liem ik, prijanti ar, andrijono. comparison of matrix metalloproteinase-9 and e-cadherin expression in earlyand late-onset preeclampsia. univ med 2021;40:200-6. doi: 10.18051/univmed.2021.v40.2002 0 6 1 biomedical science department, faculty of medicine, universitas indonesia, jakarta, indonesia 2 anatomy department, faculty of medicine, universitas trisakti, jakarta, indonesia 3 parasitology department, faculty of medicine, universitas indonesia, jakarta, indonesia 4 anatomy department, faculty of medicine, universitas indonesia, jakarta, indonesia 5 biochemistry and molecular biology department, faculty of medicine, universitas indonesia, jakarta, indonesia 6 obstetric and gynaecologist department, faculty of medicine, universitas indonesia, jakarta, indonesia correspondence : *tjam diana samara anatomy department, faculty of medicine, universitas trisakti, jl. kyai tapa no.260 grogol jakarta email: dianasamara@trisakti.ac.id orcid id: 0000-0003-0610-0638 date of first submission, may 3, 2021 date of final revised submission, september 15, 2021 date of acceptance, september 23, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 201 univ med vol. 40 no 3 background preeclampsia (pe) is a clinical syndrome characterized by the onset of hypertension at more than 20 weeks of gestation or immediately after delivery, and is accompanied by proteinuria or thrombocytopenia, kidney failure, liver failure, pulmonary edema, cerebral disorders, or visual disorders.(1) preeclampsia is a major cause of morbidity and mortality, both in the mother and in the fetus. the worldwide incidence of pe is about 2-8% of all pregnancies, with maternal mortality ranging from 10%-15%.(2) in indonesia, the maternal mortality rate of 225/100,000 deliveries is still high, where pre-eclampsia and eclampsia as most possible cause.(3) many factors that can cause pe, such as obesity, family history of pe, maternal age (>35 years or <17 years), blood vessel disorders such as chronic kidney disease and diabetes mellitus, hypertension, thrombotic disorders such as antiphospholipid syndrome, multiple pregnancie s, nulliparous versus multiparous, and immune maladaptive reaction.(4) in pathogenesis, one of the factors that is considered to be the cause of pe is due to shallow placentation and deficient remodeling of uterine spiral arteries by invasive placental trophoblast cells which results in impaired perfusion of the fetus-maternal unit.(5) the invasion disruption is caused by an imbalance between proangiogenic and antiangiogenic factors. this imbalance is thought to be due to the antiangiogenic protein appearing too early and/or if the production is excessive.(6) among the various factors that influence pro and antiangiogenic factors are ecadherin and matrix metaloproteinase-9 (mmp9).(7,8) e-cadherin is one of the most important molecules in adhesion cells in epithelial tissue.(9) e-cadherin is located on the surface of epithelial cells in areas of contact with cells known as adherent junctions. suppression of e-cadherin expression is the main cause of dysfunction in cell adhesion. one of the important functions of e-cadherin in development is its control of epithelial-mesenchymal transition. e-cadherin, which is involved in wnt signaling, plays a role in regulating migration, proliferation, apoptosis, and differentiation of cells.(10) during the first trimester of pregnancy, the expression of ecadherin in trophoblasts temporarily decreases so that trophoblast cells have the potential to migrate and invade.(11) matrix metaloproteinase-9 is the main mediator for extracellular matrix (ecm) degradation and has a role in placental angiogenesis by tr a nsforming of artery structural.(12) matrix metaloproteinase-9 degrades ecm by activation of major proangiogenic factors such as vascular endothelial growth factor (vegf) and fibroblast growth factor (fgf2).(13) matrix metaloproteinase-9 degrades e-cadherin to soluble e-cadherin, which suppresses ecadherin activity and induces emt.(14) matrix metaloproteinase-9 expression increases from 8 to 11 weeks, becoming the main gelatinase until late in pregnancy.(15) mmp-9 turut berperan dalam invasi sel trofoblas dan ekspresinya banyak didapatkan pada sel stroma desidual.(16) sosa et al.(15) found that mmp-9 has a major role during invasion and contributes to the remodeling of the uterine spiral artery. this is because mmp as a specific proteolytic enzyme plays an important role in the degradation of the extracellular matrix resulting in invasion of proliferative cells into the surrounding tissue. the cur rent classification scheme differentiates pe into two variants.(17) early-onset pe (eo-pe) develops to and requires delivery at 34 weeks of gestation. late-onset pe (lo-pe) appears after 34 weeks of pregnancy as a metabolic syndrome and is not associated with the formation and functioning of the placenta in the second half of pregnancy. a study showed that in eo-pe group the mmp-9 expression in placenta tissues was siginificantly decreased compared to normal pregnancy.(18) another study demonstrated increased expression of e-cadherin in late onset preeclamptic compared to that in gestation normotensive pregnancies.(19) furthermore, there was no difference in the expression of e-cadherin 202 between early ons et and late onset preeclampsia.(19) at present, the role of vascular endothelial injury in eo-pe and lo-pe are concerned, but its regulation mechanism is not clear, and the results of previous studies were inconclusive. therefore, the aim of this study was to compare the mmp-9 and e-cadherin expression between eo-pe and lo-pe patient. methods research design this study was a cross-sectional design with analytic observations. sampling was carried out at cipto mangunkusumo hospital (rscm) and budi kemuliaan hospital, jakarta, indonesia from april 2017 to april 2018. research subject the sample size was determined based of the results of preliminary study of 5 subjects showed that the mean difference of mmp-9 between eo-pe and lo-pe was 5.6, sd=1.66 (data not shown) . using the comparison between two independent group equation with α=0.05 and β=0.2, the sample size for each group was 38 to done to get sample size. the study was divided into two groups: eoand lo-pe. each group needed 38 samples. the samples were collected until the number of samples was met. the subjects were 19 years or more and had agreed to participate in this study by signing an informed consent were included into this study. patients with renal failure and diabetes mellitus were excluded from this study. the placentas were removed at the time of the patient’s delivery either by vaginal or caesarean section. measurements according to the american college of obstetrics and gyna ecologists (acog), preeclampsia is a clinical syndrome characterized by hypertension that appears after 20 weeks of gestation (systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg), and proteinuria (300 mg in 24 hours urine) or with positive protein (1+) taken from mid-stream urine. preeclampsia can be without pr ot einuria but must be accompanied by any of the following symptoms: thrombocytopenia (<100,000/ml), renal failure (serum creatinine >1.1 mg/dl) abnormal liver function (transaminases are twice as high as normal), pulmonary oedema, and cerebral disorders or visual disorders.(1) specimen collection maternal placenta l c otyledons were removed 0.5 x 0.5 x 0.5 cm immediately after vaginal birth or caesarean section. a total of 100 mg of placental tissue was washed with phosphate buffered saline (pbs) and then processed to obtain homogenate in 1 ml pbs and then stored at -800c. measurement of mmp-9 and e-cadherin after all samples were collected, the mmp9 and e-cadherin levels were assessed by the elisa method based on the cloud-clone corp protocol (mmp-9: sea553hu and e-cadherin: sea017hu). the results obtained from the elisa test were then calculated based on the levels in total protein, then the mmp-9 units were ng/mg and e-cadherin pg/mg. ethical clearance this study received ethical permission from the health research ethics committee of the faculty of medicine, university of indonesia no. 34/un2.f1/etik/2017. statistical analysis the distribution of abnormal data was tested based on the kolmogorov-smirnov test, the mann-whitney test to assess the comparative of clinical characteristics and levels of mmp-9 between the two groups. and independent-t test was used to compare levels of e-cadherin between eo-pe and lo-pe. a p<0.05 being considered as statistically significant. samara, wibowo, liem, et al matrix metalloproteinase-9 and e-cadherin expression 203 univ med vol. 40 no 3 a b results demographic inf ormat ion of the study population during the study we could only recruited 26 women in the eo-pe group and 38 in the lope group. the demographic and clinical characteristics of 26 women with eo-pe and 38 women with lo-pe are summarized in table 1. the median maternal age of women with eope [ 29.5 (range 21-42) years old] was not significantly different with median maternal age of women with lo-pe [30.5 (range (19-44) years old] (p=0.545) (table 1). as expected, systolic blood pressure (p=0.607) and diastolic blood pressure (p=0.658) was not significant different between eo-pe and lo-pe gr oup. but semiquantitative proteinuria were significantly higher in the eo-pe group [3 (range (0-4)] compared to lo-pe group [1 range (0-3)] (p=0.000). comparisons of mmp-9 and e-cadherin levels between eo-pe and lo-pe in table 1, it was found that mean ecadherin levels were lower in the eo-pe group (125.94 ± 54.22 pg/mg) compared to lo-pe group (157.95 ± 54.12 pg/mg) (p=0.024). but there was not significant difference of median mmp9 levels between the two groups (p=0.376). . discussion there was no significant difference of systolic and diastolic blood pressure between the eo-pe and lo-pe. this result is different from the result obtained by gomathy et al.(20) that found diastolic blood pressure to be significantly higher in eo-pe than in lo-pe. this may be because there were patients in this study were already being treated with antihypertensives. however, proteinuria was significantly higher at lo-pe compared to eo-pe group. research conducted by dong et al.(21) showed p r o te in u r ia wi t h a l e ve l of 0. 3 g/ l w a s significantly higher in severe pe than in mild pe. in our study, all eope cases were pe cases with severe features. as it’s already known, proteinuria was no longer included as a pe according to the international society of the study of hypertension in pregnancy because it was found that multiple organ dysfunction affecting the kidneys and liver can appear without signs of protein and the amount of proteinuria cannot predict the severity of disease progression.(22) however, although proteinuria is no longer recommended as a criterion for diagnosing pe, in fact clinicians generally use proteinuria levels to inform clinical decisions regarding pe delivery cases. this is because increased levels of proteinuria worsen the development of pe and are associated with poor perinatal outcomes. there was not significance difference levels of mmp-9 between eo-pe and lo-pe in this study. these results differed from the results of a study conducted by timokhina et al.(23) who found mmp-9 levels in eo-pe were lower than in lo-pe. these results showed the e f f e c t o f m m p-9 o n t he sp i ra l a r t e r y characteristics eo-pe (n=26) lo-pe (n=38) p value maternal’s age (years) gestational age systolic blood pressure (mmhg) diastolic blood pressure (mmhg) proteinuria (semiquantitative) mmp-9 (ng/mg) e-cadherin (pg/mg) 29.5 (21-42) 30 (24-33) 167.5 (140-220) 100 (88-150) 3 (0-4) 1.24 (0.18-18.98) 125.94 ± 54.22 30.5 (19-44) 37 (34-41) 170 (130-220) 110 (90-140) 1 (0-3) 1.70 (0.30-6.66) 157.95 ± 54.12 0.545@ 0.000@ 0.607@ 0.658@ 0.000@ 0.376@ 0.024$ table 1. comparison of demographic and clinical characteristics between eo and lo groups of preeclampsia patients @ mann whitney test; $ independent ttest 204 transformation process. research conducted by zhang et al.(12) using immunohistochemical methods on placental samples found that there was a gradual decrease in patients with severe pe c o mp ar e d to mi l d pe and n o r ma l pregnancies. as it’s already known, in early gestation, an increase in mmp-9 is needed to regulate the angiogenesis process for the development of the foetus-maternal interface. while lower levels of mmp-9 in the maternal serum contribute to the abnormal development of blood vessels at the foetus-ma ter nal interface in complicated p r e gna n c i e s of s e ve r e pe . ( 8 ,2 4 ) wi th t h e difference in this study related to mmp-9 levels, further research is needed to find changes in mmp-9 levels based on gestational age both at normal pregnancy and preeclampsia, so that it can be found more clearer regarding changes in mmp-9 levels. so met hin g d if f ere nt happen ed to ecadherin. in this study it was found that ecadherin was significantly lower eo-pe than lo-pe. different results were found by li et al.(7) who found no difference in e-cadherin e x pr e s si o n b e t we en eo -pe a n d lo-pe . however, the expression of e-cadherin in lope was higher than in normal pregnancy, whereas the increase in e-cadherin expression d id n ot c or r e l a t e w i t h t h e se ve r i t y of preeclampsia. li et al.(7) concluded that ecadherin increases cytotrophoblast proliferation and cytokeratin will be highly expressed in proliferative tissue and this causes impaired differentiation to become syncytio-trophoblast. research conducted by osman et al.(25) in sudan using immunohistochemical methods from placental samples found no difference in expression between severe pe and controls. meanwhile, du et al.(26) found an increase in ecadherin levels in pe patients compared to controls. the studies of osman et al.(25) and du et al.(26) did not examine the difference between eo-pe and lo-pe so it cannot be concluded whether there is a difference in levels between the two groups. limitations of this study were there was not enough sample size for eo-pe and the absence of normal gestational controls in c o mp a r a ble ge st a t i o na l a ge a nd t h e measurement of the levels did not follow the course of gestational age from early to term from the same patient. there is a need for a cohort study of gestational age to follow changes in levels in each pregnant woman so that a real change can be obtained. the results of this study indicate that we need to pay attention to the biomolecular changes that occur in eo-pe so that it can reduce the risk of premature birth. conclusion this study demonstrated that e-cadherin expression levels was significantly lower in eope. it shows that lower levels of e-cadherin can lead to early delivery in preeclampsia patients. further studies are nee ded using normal pregnancy and preeclampsia patients in all gestational age to examine the levels of mmp9 and e-cadherin and its relationship. conflict of interest there is no conflict of interest acknowledgment the authors thank the staff of the cipto mangunkusumo hospital, budi kemuliaan hospital, and the integrated laboratory of medical faculty of universitas indonesia for their assistance and support. contributors tds contributed to collecting samples, draft manuscripts, collecting data, and analyse data. hw contributed to analyse data. ikl contributed to manuscript finalization. arp contributed to the research concept. a contributed to the research design and manuscript revision. all authors have read and agreed to the final manuscript. samara, 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pregnancies complicated by severe preeclampsia and/or intrauterine fetal growth restriction. moj women’s health 2017;4:127-31. doi: 10.15406/mojwh.2017.04.00099. 25. osman se, salih m, ahmed em, mohammed aa, adam i. expression of e-cadherin, n-cadherin, and cytokeratin 18 and 19 in placentas of women with severe preeclampsia. open access macedonian j med sci 2020;8:485-9. doi: 10.3889/ oamjms.2020.4004. 26. du l, kuang l, he f, tang w, sun w, chen d. mesenchymal-to-epithelial transition in the placental tissues of patients with preeclampsia. hypertens res 2017;40:67-72. doi: 10.1038/ hr.2016.97. samara, wibowo, liem, et al matrix metalloproteinase-9 and e-cadherin expression cite this article as: m a jnooni a, jamedar sa, azimian a, ghazvini k. association of chlamydia trachomatis and neisseria gonorrhoeae with female infertility: a systematic review and meta-analysis. univ med 2022;41: 302-14. doi: 10.18051/u nivmed 2022.v41.302 -314. september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 association of chlamydia trachomatis and neisseria gonorrhoeae with female infertility: a systematic review and meta-analysis araz majnooni1 , saeed amel jamedar1 , amir azimian2 , and kiarash ghazvini1* abstract background chlamydia trachomatis (ct) and neisseria gonorrhoeae (ng) are commonly diagnosed sexually transmitted infections that have been associated with serious reproductive health outcomes for women. the association of ct and ng infection with female fertility is not completely established yet. this review aimed to determine the association of ct and ng with female infertility. methods this systematic review and meta-analysis was conducted according to the prisma statement. we searched a range of electronic databases, including pubmed, web of science, embase, and scopus, from sept 25, 2017 until february 1, 2021. from the 851 studies screened, 552 that failed to meet our eligibility criteria were excluded. subsequently, we removed 290 studies for not having a possible correlation of ct and ng infections with female infertility. nine studies comprising 1827 infertile patients met our inclusion criteria. two investigators independently extracted a range of data. all analyses were performed using stata (version 13.1, stata corp, college station, tx, usa). results ct infection potentiates female infertility, as 76.47% of the included studies found a positive correlation between them. however, due to the limited number of reported data, we were not able to compare ng infection prevalence in fertile and non-fertile patients. overall prevalences of ct and ng infections among infertile patients were 12 % and 3%, respectively, while ct infection prevalence among the fertile group was 7%. conclusion the prevalences of ct and ng infections were high in infertile women. screening and treatment of c. trachomatis and gonococcal infections during infertility treatment might increase the pregnancy rate. keywords: chlamydia trachomatis, neisseria gonorrhoeae, female infertility, human reproduction 1department of microbiology and virology, school of medicine, mashhad university of medical sciences, mashhad, iran 2department of pathobiology and laboratory sciences, faculty of medicine, north khorasan university of medical sciences, bojnurd, iran *correspondence: kiarash ghazvini, department of microbiology and virology, school of medicine, mashhad university of medical sciences, mashhad, iran email: 30biran@gmail.com orcid id: 0000-0002-8538-1425 date of first submission, august 11, 2022 date of final revised submission, september 26, 2022 date of acceptance, december 19, 2022 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license 302 doi: http://dx.doi.org/10.18051/univmed.2022.v41:302-314 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1358 review article mailto:30biran@gmail.com https://orcid.org/0000-0002-1995-9022 https://orcid.org/0000-0003-4813-6399 https://orcid.org/0000-0002-2532-8198 https://orcid.org/0000-0002-8538-1425 http://dx.doi.org/10.18051/univmed.2022.v41:302-314 https://univmed.org/ejurnal/index.php/medicina/article/view/1358 303 univ med vol. 41 no. 3 introduction sexually transmitted infections (stis) are among the most common acute conditions around the world. (1) it was estimated in 2016 by the world health organization (who) that there were approximately 370 million new cases o f t he thr e e c ur a b le st is wo r l d wi de . ( 1 ) ch la my di a tr a c h om at i s a nd ne i s s e ri a gonorrhoeae are commonly diagnosed sexually transmitted infections (sti) that have been associated with serious reproductive health outcomes for women, and may cause obstetric or perinatal complications,(2-4) including preterm deliveries, preterm rupture of the membranes, low birth weight, or still-birth. tubal factor infertility (tfi) and ectopic pregnancy have been associated with a history of both stis in numerous studies. (5,6) despite these burdensome sequelae, sti control has long languished on health policy agendas. the 2030 agenda on sustainable development (7) aimed to remedy this situation and led to who’s global health sector strategy on st is. (8 ) the st rategy proposes an integrated appr oach for st i prevention and control that addresses core sustainable development goals, mainly through securing universal access to sexual and reproductive health-care services and rights.(7,8) the first strategic direction of this sti strategy i s “t h e nee d to un de r s ta nd t he se xua l l y transmitted infection epidemic and response as a basis for advocacy, political commitment, national planning, resource mobilization and allocation, implementation, and program improvement”.(8) longitudinal studies examining gonorrhea’s adverse health outcomes (a curable infection) are difficult/unethical to conduct. a recent study attempted to overcome this challenge through linking national testing databases to hospital records, but identified too few cases to reach conclusive evidence about gonorrhea’s role in infertility.(9) in the absence of direct evidence, our review aimed to provide indirect evidence for a link between gonorrhea and infertility but strictly did not aim to nor can it establish causality. the underlying hypothesis is that current infection is of unknown duration and persistence and therefore does not allow the establishment of a causal link with infertility, but is often predictive of past exposure.(10-12) this assertion is supported by several lines of evidence. it is established through tens of studies of different designs that gonorrhea as well as chlamydia, being curable infections, carry a high risk of reinfection because of re-exposure to the same sexual partner or to other high-risk partners.(11,13-15) as such, it can be assumed that a curr ent gonorrhea infection is strongly indicative of previous gonorrhea infection;(11,16,17) indeed studies have shown that the strongest predictor of current gonorrhea infection is a histor y of go nor r he a i nf ect ion . ( 10 ,1 8) fo r example, in the uk, a history of gonorrhea infection was found to be the strongest predictor of current gonorrhea infection even after controlling for other demographic and behavioral factors (adjusted or 4.36, 95% ci 1.78 to 10.71).(10) globally, it has been estimated that the prevalence of n. gonorrhoeae is 0.8% and 0.6% among women and men, respectively.(1) for the eastern mediterranean region, the w h o h a s re po r t e d p r e va l e n c e r a t e s o f chlamydia, gonorrhea, and trichomoniasis among women of 3.8%, 0.7%, and 4.7%, respectively.(1) it is estimated that up to 40% of women with untreated c. trachomatis infection will develop pelvic inflammatory disease (pid) which is defined as any combination of endometritis, salpingitis, tubo-ovarian abscess, or pelvic peritonitis.(19) of those with pid from chlamydial or gonococcal sources, 20% will become infertile, 18% will experience debilitating chronic pelvic pain and 9% will have a life-threatening ectopic pregnancy.(19-22) a case-control study did not show an association of active infection due to c. t r ac h omat i s a nd n. g on or r h oe a e wi t h secondary infertility in females.(20) a previous systematic review corroborated the hypothesis that c. trachomatis infection potentiates female infertility, as 76.47% of their included studies 304 found a positive correlation between them.(21) even though this issue is widely recognized amidst the medical community as a secondary effect of female c. trachomatis and gonococcal infection, the level of evidence corroborating the association is relatively weak. an updated systematic review and meta-analysis of these reported data may help to better evaluate the e t i o lo gic r ol e of c. t r ac h o ma ti s a nd n. gonorrhoeae in female infertility. the objective of this review was to verify the scientific investigations related to the association of c. trachomatis and gonococcal infection with female infertility. methods this review was conducted in accordance with the prisma statement.(23) search strategy we s e a r che d a r a nge o f el e c t r o ni c databases, including pubmed, web of science, embase, and scopus, from sept 25, 2017 to february 2021. the search terms used were as follows: [(infertility or sterility or subfertility) and (*genital or vagin*) and (chlamydia trachomatis or neisseria gonorrhoeae) and (microbiota or microbiota or vaginosis or lactobacilli)]. the citation lists of any identified publications were also searched manually to identify any additional references. our searches did not involve any restrictions related to language or country of origin. inclusion and exclusion criteria titles and abstracts were first reviewed by two of the authors and only relevant publications were selected for full review. studies in this meta -analysi s were r equired to meet the following inclusion criteria: (1) observational studies that addressed infertile women and fertile controls; (2) the vaginal microbiota status was characterized by 16s rrna gene amplicon sequencing, nugent score, amsel’s criteria, or spiegel’s criteria; (3) human studies; and (4) original studies that provided clear data relating to vaginal microbiota status. articles were excluded if they were: (1) comments, reviews or conference abstracts; (2) repetitive studies; (3) animal studies; (4) devoid of a control group; (5) linked to a control group that was not made up of fertile women; (6) the status of the vaginal microbiota was based on diagnosis of bacterial vaginosis without any specific methodology; (7) not written in english or chinese; (8) studies related to clinical intervention. data extraction the two above-men tioned r eviewer s independently e xtracted a range of data, including date of publication, authors, study design, study population, sample size, methods used for microbiota characterization/diagnosis, a nd t he s pe ci f ic type of in fe r ti l ity. an y disagreements were resolved through discussion with a third reviewer. quality assessment the two reviewing authors evaluated the quality of the studies included in our analyses based on the newcastle–ottawa scale (nos), which considers three critical aspects: selection, c o mp a r a bil i t y, a nd e xp os u r e. t he t wo investigators scored the studies independently and any discrepancies between reviewers were resolved by reaching a consensus or by a fourth reviewer. statistical analysis associations between the vaginal microbiota and infertility were estimated by pooled odds ratios (ors) and 95% confidence intervals (cis) using the mantel–haenszel method. heterogeneity between studies was tested using cochran’s q two-sided homogeneity test; the i2 statistic was also used as a critical factor to determine which model should be used to pool the effect size (if i2 <50%, then a fixed model was used; otherwise, a random model was used). publication bias was then evaluated by begg’s funnel plots and egger’s regression test, which measures the majnooni, jamedar, azimian female infertility 305 univ med vol. 41 no. 3 a b degree of funnel plot asymmetry. subgroup analyses were performed based on the language used to write the original papers, the different methods used to diagnose vaginal microbiota, different types of infertility, and fertile control gr oups, to reduce he ter ogeneity. sever al sensitivity analyses were performed to evaluate the robustness of our results by excluding some articles that were considered to be of low quality. all analyses were performed using stata (version 13.1, statacorp, college station, tx) and the ‘metan’ command was used to estimate the ors. a two-sided p<0.05 was considered to be statistically significant. results study selection and characteristics in total, we screened 1155 records for eligibility from which 104 were duplicates. out of the remaining 1051 papers , 200 articles excluded based on the irrelevant abstracts, 17 articles were review, 51 have focused on other populations, 7 were letter to editors , 49 did not provide an available full text, and 428 articles did not provide specific data, leaving 229 full texts to be assessed fully. after reviewing these 229 papers, only 9 studies met our inclusion criteria for metaanalysis. and the rest were excluded because of none sufficient data or missing the outcome of interest (figure 1). collectively, these 9 articles reported 1827 cases. because infertility status and vaginal microbiota data were collected at the same time, it was hard to infer a causal correlation between these factors. hence, we considered these articles as cross-sectional rather than case– control studies, despite their original definition. most of the studies (n=8) focused on women who were diagnosed with infertility and seeking clinical treatment; in these studies, the specific type of infertility was ignored. we also found that the control (fertile) groups were quite diverse. for example, some papers used women with a history of pregnancy, antenatal women, and even healthy women without a detailed history of pregnancy. further details of the included studies are shown in table 1. figure 1. the prisma flow diagram: study selection process 306 t ab le 1 . s tu d y ch ar ac te ri st ic s a b b re v ia ti o n s: f : fe m al e; m : m al e; n /a : n o t ap p li ca b le ; p ro s: p ro sp ec ti v e; r et ro : re tr o sp ec ti v e majnooni, jamedar, azimian female infertility 307 univ med vol. 41 no. 3 meta-regression f i nding based on the public ation year and prevalence of chlam ydia trachom atis among inf ertile women the studies’ meta-regression was according to the association between the prevalence of chlamydia trachomatis among infertile women and the publication year of the study. it showed overall prevalence of chlam ydia trachom atis and neisse ria gonorrhoeae among infertile patients the overall prevalence of c. trachomatis and gonococcal infections among infertile patients was 12% and 3%, respectively, while the prevalence of c. trachomatis infection among the fertile group was 7% (figures 2 and 3). abbreviation: es = effect size figure 2. the overall prevalence of chlamydia trachomatis among infertile patients abbreviation: es = effect size figure 3. the overall prevalence of neisseria gonorrhoeae among infertile patients 308 the overall rate of infection was lower in newer studies than in older ones (figure. 4 ). however, there was no statistically significant linear trend in univariate meta-regression to explain effect size variation by publication year of study with coefficient (figure 4). meta-regression finding based on the mean age of patients and prevalence of chlamydia trachomatis among infertile women the studies’ meta-regression was according to the association between mean age of patients and prevalence of chlamydia trachomatis among infertile women. it showed that the overall rate of infection was lower at younger than older ages (figure 5). ho wever, the re was no statistically significant linear trend in univariate meta-regression to explain effect size variation by mean age of patients with coefficient (figure 5). publication bias in figure 6, the funnel plot of publication bias is shown to be symmetrical. the size of the circles shows the weight of the studies (larger circles show more samples and smaller circles show fewer samples). discussion the who estimates that the main curable stis (gonorrhea and chlamydial infection) are commonest in the 15-44-year age groups.(24). they are the most common causes of cervicitis and urethritis, and their sequelae (pelvic inflammatory disease, chronic pelvic pain, tubal factor infertility, and reactive arthritis).(25) chlamydial infections are primarily an issue of women’s health care since the manifestations and consequences are mor e damaging to the reproductive health in women than in men.(26) most urogenital c. trachomatis and gonococcal infections are initially asymptomatic but may subsequently cause considerable long-term morbidity. consequently, accurate diagnosis of both infections requires the use of specific laboratory techniques. the important progress in laboratory diagnosis of chlamydial infection includes the development of non-viabilitydependent tests. (2 7) we provid ed, to our figure 4. meta-regression finding based on the publication year and prevalence of chlamydia trachomatis among infertile women majnooni, jamedar, azimian female infertility 309 univ med vol. 41 no. 3 knowledge, the first systematic review of c. trachomatis and gonorrhea infection in infertile populations. the gonorrhea infection prevalence was several folds higher than that in the general population; the global estimate in infertile populations was 2.2%, compared with only 0.8% in the general population (per who 2016 estimates),(28) but in our study this rate was 12%. these findings should be seen against the expectation that infertile populations should be prone to a lower prevalence than the general population; there is higher frequency of sti testing among them, and therefore earlier detection and higher treatment coverage relative figure 5. meta-regression finding based on the mean age of patients and prevalence of chlamydia trachomatis among infertile women figure 6. begg’s funnel plot 310 to the general population. infertile populations may also unde rgo proph ylactic antibiotic administration, not necessarily with testing, prior to procedures such as in vitro fertilization/embryo transfer.(29,30) it is relatively clear that some sexually transmitted infections (stis) can cause inf ertility. chlamydia. t rachomatis may synthesize a large amount of heat shock protein (hsp60) and thus induce a pro-inflammatory immune response in the human fallopian tube epithelium, resulting in scarring and tubal occlusion.(31) n. gonorrhoeae may attack the epithelial cells of the fallopian tubes, thus exerting an impact on ovum transportation and fertilization.(32) our study revealed a prevalence of 12% for c. trachomatis among infertile women. these findings attest to the potential role of c. trachomatis and gonorrhea in infertility. since early detection and treatment of c. trachomatis and gonococcal infections have been challenged by the asymptomatic nature of the infections (33,34) and growing antimicrobial resistance,(35-40) these findings support the global public health value of developing sti vaccines (41,42) as a fundamental solution to the implications of c. trachomatis and gonorrhea.(43) our results also support the timeliness of a comprehensive prevention approach promoting sexual health to control n. gonorrhoeae and c. trachomatis, mitigate antimicrobial resistance and achieve who global health sector strategy targets. (35) such an approach would focus on the simultaneous implementation of biomedical (rolling-out testing and vaccination), behavioral (promoting healthier sexual lives) and structural prevention interventions (improving access to testing, treatment and care services). indeed, successful and sustainable implementation of biomedical interventions cannot be achieved without adequate levels of public awareness, access to/uptake of services, and adherence/ retention in prevention and treatment cascades. chlamydia trachomatis curr ent infection prevalence was 3% in the middle east and north africa in 2020, similar to who prevalence estimates for this region of about 3% in 2012 and about 3.5% in 2016.(44) the prevalence was also in line with who estimates for the western pacific region (about 4%) and the european region (about 3%), ( 44 ) where broad c. trachomatis control programs, including opportunistic testing, are standard in some highincome countries,(45-47) but higher than that for the south-east asian region (about 1.5%) and lower than that for the african region (about 5%) and the region of the americas (about 5.5%).(44) this high prevalence suggests substantial infection and disease burden that needs to be tackled through sexual health and sti -specific programs, for both women and men. our study has important but unavoidable limitations. data quantity and quality varied across regions and sometimes limited our ability to produce representative summary estimates. it was not possible to conduct full multivariable meta-regression to adjust for potential confounders, with the large number of predictors relative to that of studies. prevalence estimates by infertility diagnosis may have been affected by unavoidable overlap across categories; samples with mixed infertility often included tubal factor infertility (tfi), and those with non-tfi may have included other infertility diagnoses. an analysis by age could not be performed, given the low number of studies reporting patients’ age. quantity and quality of available data varied by country and population, particularly for populations at high risk where most data came from only a few countries. prevalence levels might not have been strictly representative and might have been affected by publication bias, as suggested by the small -study effect observed. the wide array of diagnostics used for ascertainment might have also introduced detection bias. factors that might have contributed to differences in c. trachomatis positivity rates across studies include sampling variation and potential selection bias, spatial or temporal variability in prevalence, and possibly unreported underlying comorbidities. this study did not assess other stis that might have also contributed to infertility, pregnancyrelated morbidity, and other health conditions in women with c. trachomatis or gonorrhea infection. such majnooni, jamedar, azimian female infertility 311 univ med vol. 41 no. 3 potential biases might have contributed to some of the unexplained heterogeneity observed in the prevalence levels. given the potential limitations in the representativeness of the prevalence measures as well as heterogeneity across studies, the calculated pooled prevalence should be interpreted as a pooled average, rather than strictly an estimate of the mean prevalence in the considered population or subpopulation. conclusion the prevalence of c. trachomatis and gonococcal infections were respectively high in infertile women. these infections are strongly implicated in female infertility and are probably an underestimated cause of unexplained infertility. screening and treatment of c. trachomatis and gonococcal infections during the course of infertility treatment might increase pregnancy rate. randomized studies including larger numbers of participants are needed, however, to reach more validated conclusions. moreover, research is strongly recommended on the mechanisms by which c. trachomatis and gonococcal infections impairs fertility and on the link between c. trachomatis and gonococcal infections and elevated androgen levels. conflict of interests all authors declare that they have no conflict of interest. funding no funding was received for conducting this study. author contribution am and kg participated in the conception and design of the study, library searches and assembling relevant literature, critical review of the paper, supervising the writing of the paper, and database mana gement. sj and aa participated in data collection, library searches and assembling relevant literature, writing the paper, and critical review of the paper. all authors have read and approved the final manuscript. references 1. rowley j, vander hoorn s, korenromp e, et al. chlamydia, gonorrhea, trichomoniasis and syphilis: global prevalence and incidence estimates, 2016. bull world health organ 2019;97: 548–62. doi:10.2471/blt.18.228486. 2. menon s, timms p, allan ja, et al. human and pathogen 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2015;5:769-79. doi: 10.4236/aim.2015.512081. majnooni, jamedar, azimian female infertility c:\users\universa medicina\docu 47 abstract universa medicina high glycosylated hemoglobin level as a risk factor of latent tuberculosis infection in patients with uncomplicated type 2 diabetes mellitus yunita arliny1*, dewi behtri yanifitri1, and diennisa mursalin2 background diabetes mellitus (dm) is known to increase the risk of infection including tuberculosis (tb). some studies also showed that 2-15% of latent tb infection (ltbi) will progress to active tb. this study aimed to obtain the prevalence of ltbi and to determine the risk factors of ltbi in patients with type 2 diabetes mellitus (t2dm). methods this was a cross-sectional study on 242 adult t2dm patients. for ltbi screening we performed the interferon gamma release assay (igra) (quantiferon tb gold plus test) and for confirmation of active tb (pulmonary tb) we performed genexpert mtb/rif sputum examination and chest x-ray. glycosylated hemoglobin (hba1c) levels, smoking history and bcg scar were collected. multivariate logistic regression was used to analyze the data. results positive igra results were found in 99 of 242 uncomplicated t2dm patients while ltbi was found in 82 patients (33.8%). there were significant differences between t2dm patients with latent tb and t2dm patients without infection in hba1c and specific ifn-γ levels (tb1 minus nil and tb2 minus nil), i.e. 8.5% and 7.6%, 2.5 iu/ml and 0.06 iu/ml, and 2.6 iu/ml and 0.08 iu/ml, respectively. multivariate analysis showed that the risk factors for ltbi in t2dm patients were smoking history, hba1c >7%, and no bcg scar. conclusions because ltbi is prevalent in t2dm, it is important to screen for it in t2dm patients due to the risk of developing severe active tb. absence of a bcg scar and high hba1c levels are strong predictors of ltbi in t2dm patients. . keywords: diabetes mellitus, latent tuberculosis infection, pulmonary tuberculosis original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.47-55 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1242 january-april, 2022 vol.41no.1 1infection division, department of pulmonology and respiratory medicine, faculty of medicine, syiah kuala university, banda aceh 2department of biomedicine, faculty of medicine, university of indonesia, jakarta *correspondence: dr.dr.yunita arliny,m.kes.sp.p(k), department of pulmonology and respiratory medicine, faculty of medicine, syiah kuala university, banda aceh, indonesia e-mail: nita.arliny@unsyiah.ac.id orcid id: 000-0003-1950-4025 date of first submission, october 16, 2021 date of final revised submission, march 17, 2022 date of acceptance, maret 22, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: arliny y, yanifitri db, mursalin d. high glycosylated hemoglobin level as a risk factor of latent tuberculosis infection in patients with uncomplicated type 2 diabetes mellitus. univ med 2022;41:47-55. doi: 10.18051/ univmed. 2022.v41.47-55 48 arliny, yanifitri, mursalin tuberculosis infection in uncomplicated diabetes mellitus introduction tuberculosis (tb) is a global public health emergency. in 2018, indonesia had an estimated tb incidence of 316 per 100 000 population, or a total of 845,000 cases, and a mortality of 37 per 100 000 population or 98,000 deaths from tb. at the provincial level, in 2018, five provinces contributed to 57% of total new and relapse notifications, i.e. west java (105,794 cases), east j ava (7 1,791), central java (65,014), dki jakarta (41,441) and north su ma tr a ( 3 5 ,035 ) . ( 1 ) t h e p r i ma r y mycobacterium tuberculosis infection will develop into tb disease in 5–10% of individuals and in the remaining 90–95% into latent tuberculosis. the latter is important because individuals with latent infection may progress to active tb at any moment, even years after the primary infection.(2) a previous study reported that diabetes mellitus (dm) is known to have a 3 times greater risk of being infected with tb and that the tb rate in dm is higher than in the general population.(3) in the 2017 who report on estimated dm-tb burden, indonesia ranked second as the country with the highest tb burden and fifth as the country with the highest number of dm patients in the world, so the relationship between dm and tb is important in tb endemic countries as well as countries with an increasing prevalence of dm.(4) although there is a known association between active tb and type 2 dm (t2dm), it is not clear how high the prevalence of ltbi in t2dm is and whether this t2dm increases the risk of developing ltbi. identifying latent tb in t2dm patients is an important measure to prevent reactivation of tb into active tb. a cross-sectional study involving 299 adult t2dm patients showed no significant difference in smoking status, duration of smoking, duration of t2dm, and hba1c level, between ltbi and non-ltbi subjects.(5) in contrast, a different cohort study in egypt involving 102 t2dm patients showed that age and hba1c level are strong predictors of ltbi in t2dm.(6) however, studies investigating the link between dm and ltbi are scarce and heterogeneous.(7) up until now, there have not been a global consensus or specia l re commendation in in done sia on performing latent tb screening in dm patients, making it difficult to find data on prevalence and r i s k f or r e a c t i va ti o n a nd th e r a pe u t i c recommendations for prevention. therefore, we conducted this study to determine the risk factors of ltbi in patients with type 2 diabetes mellitus (t2dm). methods study design this was an analytical observational study that was conducted in 2 steps. the first step was to obtain the prevalence of latent tb infection (ltbi) in dm patients and the second was to obtain the risk factors of ltbi in dm patients. the study was performed at the outpatient clinic for endocrinology, metabolism, and diabetes at the national general hospital dr. cipto mangunkusumo (rscm) and at the integrated diabetic outpatient clinic, pulmonary clinic, persahabatan hospital, from december 2017 to january 2019. research subjects the subjects were adult patients (age >18 years) with type 2 dm (t2dm) and the inclusion criteria were type 2 dm patients without complications who had been diagnosed for >1 year and were willing to participate in the study, as evidenced in writing by signing the informed consent form after receiving the researcher’s explanation and reading the explanation sheet about the study. patients were excluded if they were pregnant and/or lactating, had been diagnosed with c an cer, kidne y and l iver disorders, or were on immune-modulatory medications. sample size determination the sample size was calculated based on the prevalence of 21.6% previously reported in 49 a study on ltbi prevalence among diabetic patients in egypt.(6) using a precision of 5% at the 5% significance level, a total of 248 subjects was found to be required after assuming a nonresponse rate of 10%. the sample size was determined using a formula for sample size for prevalence of ltbi in diabetes patients, with an accuracy and type i error of 5%; we found that the total number of patients with t2dm who were to be screened for latent tb was 248. there were 6 patients who were excluded b e c a u se t he y d i d n ot r e t ur n t o u nd e r go genexpert mtb/rif sputum examination. data collection the subjects were taken by consecutive sampling. subjects who met the inclusion criteria underwent a general physical examination of vital signs and the lungs. after that, the research sub j ects were interviewed usin g a st udy questionnaire that had been prepared to obtain data on persons with t2dm, namely age, gender, length of time with t 2dm, glycosylated hemoglobin (hba1c) levels, levels of fasting plasma glucose (fpg), 2-hour post-prandial plasma glucose (pppg) and data on risk factors for tb infection, namely history of bcg vaccination, presence of bcg scars, bmi, smoking history, alcohol history, history of contact with active tb patients, and symptoms of active tb. assessment of nutritional status was carried out by anthropometric examination to assess body mass index (bmi) by measuring weight and height. bmi assessment is by dividing body weight (kg) by height in meters squared. the duration of diabetes diagnosis (years) in this study is based on the tandem study which divides the duration of diabetes into three categories, namely less than 5 years, 6-15 years a n d mor e th a n 1 5 ye a r s . ta nde m i s a multicenter prospective study with field sites in peru, romania, south africa and indonesia, countries with diverse healthcare systems and population demographics, but all with a relatively high burden of tuberculosis and an increasing prevalence of dm.(8) quantiferon blood test for examination of latent tb with the quantiferon test (qft), from each of the subjects 4 ml of venous blood was taken and 1 ml was put into each of 4 qft tubes. the four tubes were then incubated at 370c for 16"24 hours, then centrifuged, and the second stage was by elisa examination. the qft-plus test is said to be positive if the ifn-γ level is <0.35 iu/ml furthermore, the subjects were asked to have a chest x-ray and each was given two sputum pots to collect sputum. the sputum used was morning sputum. the sputum pot that had been returned by the subjects was then sent in a bag containing a blue ice pack to the laboratory for xpert mtb/rif examination. the chest xray examination was carried out with the patient in the postero-anterior (pa) thoracic position at the radiology installation of the hospital and the resulting radiograph was read and interpreted by the radiology specialist on duty at that time. test for latent tuberculosis the subjects were diagnosed with ltbi if the qft-plus test gave a positive result, the xpert mtb rif sputum examination was negative, and the chest x-ray was normal. if the qft-plus result was positive/negative/indeterminate, the results of the xpert mtb rif sputum examination were positive, and the chest x-ray was positive for tb, the study subject was diagnosed as having active tb. if the subjects obtained negative qftplus results and the xpert mtb/rif sputum was negative, then the subjects were diagnosed as t2dm without tb infection. we performed a risk factor assessment from the results of the qft-plus test. the subjects were divided into 2 groups, namely the positive and negative interferon gamma release assay (igra) groups. then, data were collected regarding risk factors for ltbi including signs and symptoms of tb (cough, coughing up of blood, shortness of breath, chest pain, fever, weight loss, night sweats), history of bcg vaccination, presence of bcg scarring, history of contact with active tb patients, duration of contact, dm univ med vol. 41 no 1 50 arliny, yanifitri, mursalin tuberculosis infection in uncomplicated diabetes mellitus c duration, smoking history, alcohol history and for laboratory analysis measurement of hba1c. statistical analysis the descriptive data in numeric form was presented as mean and standard deviation, whereas the nomina l data was given as percentages. bivariate analyses were performed to compare several variables between the two igra groups. multiple logistic regression analysis was done to obtain adjusted or (aor) and 95% confidence interval. data analysis was performed using statistical program for social sciences (spss) for windows, version 24. ethical clearance the study protocol was approved by the ethics committee, universitas indonesia, under number 17-07-0649 and ethical clearance number 610/un2.f1/etik/2017. results study subjects the subjects of the t2dm study consisted of 242 persons, with the majority being of the female sex, namely 147 subjects (60.7%). the largest age group was the age group of 60 years with 115 subjects (47.5%). the highest number of married subjects was 203 (83.9%) and the most frequent occupation was housewife, at 92 persons (38%). all t2dm subjects without a history of tb had had diabetes for an average of 10 years. the duration of diabetes (years) in this study are based on the tandem study which divides the duration of diabetes into three categories, namely less than 5 years, 6-15 years, and more than 15 years. other variables are shown in table 1. latent tb screening was carried out using igra quantiferon tb gold plus examination, xpert mtb/rif examination, and chest x-ray to eliminate active tb. from the examinations, we found 99 subjects (40.9%) with positive igra. there were 82 subjects with positive igra who met the criteria for latent tb, namely no clinical signs and symptoms of tb, negative mtb sputum characteristics n (%) sex male 95 (39.3) female 147 (60.7) age (years) <40 13 (5.4) 40 ̶ 49 28 (11.6) 50 ̶ 59 86 (35.5) ≥60 115 (47.5) marital status married 203 (83.9) widow/widower 33 (13.6) unmarried 6 (2.5) profession unemployed 13 (5.4) housewife 92 (38.0) employee 77 (31.8) retiree 60 (24.8) history of alcohol consumption yes 3 (1.2) no 228 (94.2) ex-drinker 11 (4.5) history of tb contact yes 28 (11.5) no 133 (55.0) unclear 81 (33.5) history of smoking smoker 24 (9.9) non-smoker 173 (71.5) ex-smoker 45 (18.6) hba1c (%) <7 94 (38.8) 7 ̶ 9.9 108 (44.6) ≥10 40 (16.6) bcg vaccination yes 40 (16.6) no 26 (10.7) unclear 176 (72.7) bcg scar yes 135 (55.8) no 107 (44.2) tb symptoms* yes 27 (11.2) no 215 (88.8) bmi <18.5 5 (2.1) 18.5 ̶22.9 69 (28.5) 23.0 ̶24.9 65 (26.9) ≥25.0 103 (42.5) igra positive 99 (40.9) negative 143 (59.1) ifn-γ tb1 minus nil 0.4 (0.0 ̶ 10.0) tb2 minus nil 0.18 (0.0 ̶ 10.0) xpert mtb/rif* mtb detected 1 (0.4) table 1. sociodemographic characteristics and several risk factors of the subjects (n=242) data presented as n (%) except for ifn-γ [median (min-max); hba1c: glycosylated hemoglobin; bcg: bacille calmetteguérin; bmi: body mass index; igra: interferon-gamma release assay; ifn: interferon gamma; xpert: xpert mtb/rif: mycobacterium tuberculosis and resistance to rifampin 51 univ med vol. 41 no 1 and normal chest x-ray. only 1 subject (0.4%) was identified with active tb from the result of genexpert mtb/rif sputum examination (mtb positive and no rifampicin resistance). none of the latent tb subjects had clinical symptoms and chest x-ray suggestive of tb or a positive genexpert mtb/rif sputum examination. there were 105 subjects (43.4%) without clinical tb symptoms, who had negative igra, negative xpert mtb/rif sputum test, and normal chest x-ray. there was no significant difference in history of tb contact between the two groups (p = 0.535). the presence of a bcg scar was found in different proportions in each group. there was no significant difference in bcg scar presentation between ltbi subjects and subjects without tb infection (p=0.063). the numbers of smokers and ex-smokers in ltbi subjects were greater than in subjects without tb infection. there was a significant difference in history of smoking between the ltbi group and the group without tb infection (p=0.020). the value of hba1c also showed an increasing pattern in the non-tb infection and ltbi groups at 7.6% and 8.5%, respectively. the proportion of subjects with hba1c >10% was found to be significantly greater in latent tb subjects compared to those without tb infection (p=0.002). the subjects with obese bmi but without tb infection had higher bmis compared to the latent tb group, but there was no significant difference between both groups (p=0.165) (table 2). variables latent tb (n=82) n (%) non latent tb (n= 160) (%) p value history of tb contact yes 9 (11.0) 11 (13.4) 0.535a no 46 (56.1) 47 (57.3) unclear 27 (32.9) 24 (29.3) bcg scar yes 41 (50.0) 53 (64.6) 0.063a no 41 (50.0) 29 (35.4) history of smoking smoker 16 (19.5) 9 (10.9) 0.020a ex-smoker 10 (12.1) 5 (6.2) non-smoker 56 (68.2) 68 (82.9) duration of diabetes (years) ≤5 20 (24.3) 22 (26.8) 0.831a 6 ̶ 15 41 (50.1) 35 (42.6) >15 21 (25.6) 25 (30.6) hba1c(%) <7 22 (26.8) 35 (42.6) 0.002a 7 ̶ 9.9 43 (52.5) 37 (45.2) ≥10 17 (20.5) 10 (12.2) bmi (kg/m2) <18.5 5 (6.1) 1 (1.2) 0.165a 18.5 ̶ 22.9 21 (25.6) 23 (28.2) 23.0 ̶ 24.9 21 (25.6) 18 (21.9) ≥25.0 35 (42.7) 40 (48.7) ifn-γ tb1 minus nil 2.5±2.4 0.06±0.1 <0.0001 tb2 minus nil 2.6±2.4 0.08±0.1 <0.0001 table 2. comparison of several risk factors between patients with and without latent tuberculosis data is presented as n (%) except for ifn-γ as mean±sd; a: chi square; b: t-test; hba1c: glycosylated hemoglobin; bcg: bacille calmette-guérin; bmi: body mass index; ifn: interferon gamma 52 arliny, yanifitri, mursalin tuberculosis infection in uncomplicated diabetes mellitus after multivariate analysis, we found only history of smoking, bcg scar, and hba1c value as influencing variables for ltbi in t2dm. table 3 shows that history of smoking (smokers and ex-smokers) had a significant correlation with risk of ltbi in t2dm patients and was 2.5 times higher (aor 2.47; ci 95% 1.151 6 5.295). subjects with hba1c value of >7% had a 2.1 times risk (aor 2.13; ci 95% 1.074-4.225), and those lacking a bcg scar had a 2 times higher risk to become active tb (or1.97; ci 95% 1.029-3.773). discussion of 242 t2dm subjects without tb history, 82 subjects had ltbi (33.9%) and 1 subject active tb (0.4%), based on a positive genexpert mtb/rif sputum examination. the obtained prevalence for ltbi in dm was similar to what k oesoemadinata et al.(9) found. our result was lower than that of a study in mexico (51%) with an almost similar population, but higher than that of studies in singapore (28.2%) and malaysia (28.5%). only 1 active tb patient was found in the present study, which is similar to that of a study by leow et al.,(10) which only found 1 active tb patient among 220 patients screened for tb. a systematic review by workneh et al.(11) mentioned that the tb prevalence in the dm population ranged between 0.38 6 14% with a median of 4.1%. zheng et al.(12) found that the tb prevalence in the dm population of china ranged from 0.3 6 0.8. the lower prevalence in our study compared to koesoemadinata et al.(9) may be because of the different study locations. west java is the province with the highest tb prevalence in indonesia while jakarta is only number four. this surely may have affected the tb exposure. the proportion of ltbi subjects with a smoking history showed that there were more non-smokers than ex-smokers and active smokers, but compared to the non-tb infection group, the ltbi group had more subjects with a history of smoking. we already know that smoking had an obvious relationship with increases in the risk of tb infection, active tb, relapse during tb treatment, and death from tb. smoking affects the important biological defense mechanism against early tb infection and active tb. this includes abnormalities in airway secretion clearance, lowered function of lung macrophages, and decrease in ifnγ and tnf-α production. smoking aggravates tb infection and increases the risk for active tb.(13) some systematic reviews and meta-analyses had reported a correlation of cigarette smoke exposure with tb infection, active tb, and death from tb. nicotine in cigarettes works through α 6nicotinic receptors which will reduce the production of tnf-α by macrophages to prevent protection against tb and increase the progress of tb infection. cigarette smoke also will reduce the secretion of il-12, ifn-γ, and tnf-α to prevent granuloma formation. this will cause an increase in the risk for tb infection which will later on develop into active tb.(13,14) a crosssectional study in china also showed that smoking is a risk factor for ltbi especially in older smokers and that the risk was correlated with the number of cigarettes, while quitting smoking was closely related to successful ltbi therapy.(15) detection of ltbi showing false negative igra results is more frequent in smokers than in nonsmokers. smoking also causes delayed sputum parameter or aor 95 % ci p value smokers hba1c >7% no bcg scar 0.904 0.756 0.678 2.469 2.130 1.970 1.151 – 5.295 1.074 – 4.225 1.029 – 3.773 0.031 0.020 0.041 table 3. multivariable logistic regression analysis of factors associated with ltbi in the t2dm subjects (n=242) or: odds ratio; aor: adjusted odds ratio; ci: confidence interval; hba1c: glycosylated hemoglobin; bcg: bacille calmetteguérin 53 conversion which prolongs therapy.(13,14) exposure to cigarette smoke whether first-hand or secondhand smoking is related to the risk of tb infection and can cause active tb. a study by bai et al.(16) showed that taiwan had a relative risk of 5.61 for positive microscopic sputum examination in tb smokers with dm than in tb non-smokers without dm. therefore tb-dm comorbidity is in general presumably more often found in smokers than in non-smokers. hemoglobin a1c is a product of glucose binding to hemoglobin (hb) in erythrocytes. examination of this parameter is a gold standard examination to evaluate control of blood glucose. generally, the half-life of hb is approximately 34 months so the value of hba1c reflects the control of blood glucose over a long period of time. based on the american diabetes association complete guide to diabetes,(17) hba1c has been approved as the glycemic control measurement parameter in dm patients. the level of diabetic control in our study as measured by hba1c showed uncontrollable diabetes in ltbi subjects. martinez et al.(18) in their study found the mean of hba1c in tb diabetic subjects to be 7.5, which is lower than the 8.5% in our study, which shows that an hba1c value of >7% is correlated with the incidence of ltbi in dm patients. our study is in line with a meta-analysis by chen et al.(19) who concluded that the tb prevalence will increase by 2.05 times in patients with an hba1c of >7% compared to patients with an hba1c of <7%. an epidemiological study in america also showed that patients with poor glycemic control had a 2.2 times higher risk of latent tb infection than had patients without diabetes. the high hba1c level shows poor glycemic control and is correlated with an increased risk of diabetic complications which further increases the risk for tb infection. the effect of the increase in hba1c level on the natural and adaptive immune system has been known from some studies. hba1c is related to a receptor for advanced glycation end product (rage). among the functions of rage is participating in the control of t cell activation and differentiation. one study showed that an elevated hba1c level might be a factor contributing to hb structural modifications in diabetics, which may have deleterious effects linked to the pathological complications of type 2 diabetes mellitus. (20) reaching glycemic control in countries with a high diabetic burden in asia is quite difficult due to the lack of optimum health facilities, poor education, and great economic disparity. a 5-year survey of 11,799 diabetes patients, among whom 5,888 were from asia, showed that only 20–30% had an hba1c of <7%.(21) the diabcare-asia project performed a cross-sectional study and reported that among 24,317 diabetic patients from bangladesh, china, indonesia , malaysia, philippines, singapore, south korea, sri lanka, taiwan, thailand, and vietnam, 55% had an hba1c value of >8%.(11) poor glycemic control in asian diabetes patients, including those from indonesia, is an imp ortant factor in the development of ltbi and also the reactivation of latent tb in patients with dm. gomez et al.(22) in their study showed that attachment to and ingestion of m. tuberculosis by monocytes in dm subjects were lower compared to non-dm subjects. multivariate analysis showed poor glycemic control as measured by hba1c and fbg as predictive factors for the low interaction between m. tuberculosis and monocytes. our study showed that the absence of a bcg scar affected the incidence of latent tb in type 2 dm. this result is in line with the study by lin et al.(23) which reported that the presence of a bcg scar protected t2dm patients against ltbi. the study by chen et al.(24) also showed that bcg vaccination is a protective factor against latent tb. some studies showed similar results and found that the effectivity of bcg vaccine protectivity towards tb infection was 59%.(25) the present study has some limitations including the fact that some misleading factors such as uncontrollable socioeconomic and environmental factors which might be correlated with dm affect the measurement of ltbi risk. however, the present study used the quantiferon tb gold plus test which is the latest generation univ med vol. 41 no 1 54 arliny, yanifitri, mursalin tuberculosis infection in uncomplicated diabetes mellitus of igra for measuring ifn-γ not only from cd4 but also from cd8, so that the reduced sensitivity in t 2dm can be man aged. the clinical implication of our study is the increased awareness of t2dm patients in the future, because they have a high risk of ltbi. a longitudinal study is therefore required to identify the predictive value of hba1c for progression to active tb in patients with dm. conclusion this study demonstrated that a high hba1c level is a risk factor of ltbi in patients with t2dm from a geographic area that has a high incidence of tb. it is important to perform tb screening in dm patients due to the high risk for ltbi and progression to active tb. conflict of interest the authors have no financial or personal conflicts of interest in the present study. acknowledgements the authors thank the participants for their cooperation until the completion of the study. contributors ya contributed to the conception of the study, data collection and final approval of the version to be published. db contributed to data analysis and interpretation. d contributed to drafting the manuscript. all authors have read and approved the final manuscript. references 1. kementrian kesehatan republik indonesia. the republic of indonesia joint external monitoring mission for tuberculosis; 2020. 2. salgame p, geadas c, collins l, jones-lópez e, ellner jj. latent tuberculosis infection revisiting and revising concepts. tuberculosis (edinb) 2015;95:373–84. doi:10.1016/j.tube.2015.04.003. 3. jeon cy, murray mb. diabetes mellitus increases the risk of active tuberculosis: a systematic review of 13 observational studies. plos med 2008;5:1091–101. doi: 10.1371/ journal.pmed.0050152. 4. kementerian kesehatan republik indonesia. indonesia national tb program. current status of integrated community based tb service delivery and the global fund work plan to find missing tb cases; 2018. 5. ping pa, zakaria r, islam ma, et al. prevalence and risk factors of latent 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eastern china of low tb incidence. plos one 2015;109. doi.: 10.1371/journal.pone.0141511. 25. trollfors b, sigurdsson v, dahlgren-aronsson a. prevalence of latent tb and effectiveness of bcg vaccination against latent tuberculosis: an observational study. int j infect dis 2021;109:27982. https://doi.org/10.1016/j.ijid.2021.06.045. univ med vol. 41 no 1 c:\users\user\documents\41 no 2 abstract universa medicina reversible ethambutol-induced optic neuropathy: report of a rare case syntia nusanti1, rasyidia laksmita putri2, and dearaini2 background ethambutol (emb) is one of the first-line anti-tuberculosis therapy. one of its precarious side effects is ethambutol-induced optic neuropathy (eon). the ocular manifestations of eon include painless loss of central vision and cecocentral scotomas in the visual field. case description a 60-year old man presented with gradual and painless visual loss since 3 months prior to visit. the accompanying symptoms were frequent headache without double vision and photophobia. he had been diagnosed with pulmonary tuberculosis (tb) for 9 months and consumed emb for 7 months before being advised to discontinue. there was neither history of systemic diseases nor family history of neuropathy. examination showed reduced visual acuity with positive relative afferent pupillary defect (rapd) on right eye. humphrey test showed bilateral generalized visual loss. ocular computed tomography (oct) showed retinal nerve fiber layer (rnfl) thickness was within normal limit. magnetic resonance imaging (mri) brain indicated unremarkable result for optic neuropathy. based on the examinations listed above, this patient was diagnosed as eon and emb was stopped immediately. patient was given oral citicoline 1000mg and zinc supplementation for one month. after 1 st and 3 rd month follow up, patient’s visual function was gradually improved. the ophthalmic examinations indicated recovery of the visual function. conclusions this case suggested that an early detection and intervention in patient with eon has promising result in visual outcome. eon is a reversible optic neuropathy if the ocular toxicity is monitored closely among the tuberculosis patients who are prescribed emb regiment. keywords: ethambutol, optic neuropathy, toxic optic neuropathy abbreviations: bcva = best-corrected visual acuity, eon = ethambutolinduced optic neuropathy, erg = electroretinogram, iop = intraocular pressure, mferg = multifocal electroretinogram, oct = optical coherence tomography, ou = oculus uterque, rapd = relative afferent pupillary defect, rnfl = retinal nerve fiber layer, vep = visual evoked potential. review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41............... copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1274 may-august 2022 vol.41no.3 cite this article as: nusanti s, putri rl, dea rain i. reversible ethambutol-induced optic neuropathy: report of a rare case. univ med 2022;41: ........... doi: 1 0 . 1 8 0 5 1 / u n i v m e d . 2 0 2 2 . v41................. 1neuro-ophthalmology division, ophthalmology department, faculty of medicine, university of indonesia cipto mangunkusumo hospital, jakarta, indonesia 2ophthalmology department, cipto mangunkusumo hospital, jakarta, indonesia *correspondence: syntia nusanti, md cipto mangunkusumo hospital, jakarta jl kimia no 8, menteng, jakarta email: syntia_nusanti@hotmail.com sidiksyntia@gmail.com orcid id : 0000000273750764 date of first submission, december 24, 2021 date of final revised submission, august 4, 2022 date of acceptance, august 13, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license univ med vol. 41 no 3 introduction tuberculosis (tb) is an infectious disease that is found in every country in the world.(1) indonesia accounts for 44% of global tb cases.(2) one of the first-line medications of tb is ethambutol (emb), which however has been associated with several side effects.(3,4) the most well-documented and precarious ocular side effect is ethambutol-induced optic neuropathy (eon). the worldwide prevalence of eon in 2013 was 100,000.(3) a study in taiwan reported that the incidence of eon was 1.29% in 4803 patients with tb and incidence in male patients was higher than in females.(5) according to a retrospective study in indonesia, eon was recorded in 51 patients with initial presentation of poor visual acuity and mean duration of emb therapy of 6.2 months.(6) the diagnosis of eon is commonly established after the presence of vis u a l s ymp to ms. t h e t yp i c al c l in i c a l manifestations of eon are painless, subacute, and bilateral loss of vision. these manifestations may begin between 1 – 36 months a fter receiving emb, but in general eon may develop at 9 months of treatment.(3) several assessments s uc h a s visu a l a c u i t y a n d vi sua l f i e l ds examination, visual evoked potential (vep), multifocal electroretinogram (mferg) and retinal nerve fiber layer (rnfl) thickness are commonly used to detect early and subclinical toxicity.(3,7) at the beginning, the optic nerve appears normal, but as the disease progresses, optic atrophy might present, which indicates irreversible damage. several risk factors such as hypertension, renal disease and age of more than 65 years are related to a greater risk of eon, although eon is believed to be doseand d ur a t io n -de p e n de nt . ( 3 ,7 ) t he t o xi c it y of ethambutol has been reported to be reversible following early detection of eon. if the patient initially presents with optic disc pallor, the prognosis of eon is considered poor. early detection and immediate emb discontinuation are pivotal in impeding irreversible damage. besides ethambutol cessation, several studies mentioned that citicoline and zinc administration in eon patie nts produce s gives a be tter o ut c o me . h ow e ve r, t h e r e h a s b e e n no established therapy yet to restore ganglion cell loss due to eon. thus, it is necessary to detect eon as soon as possible to prevent severe outcome.(5,7) in the present case report, we aimed to report an eon case that came to our hospital recently, to evaluate the clinical characteristics of eon, and to show that early detection of eon can prevent further damage. case report a 60-year-old man came with bilateral gradual and painless visual loss since 3 months prior to visit. the accompanying symptoms were frequent headache without photophobia and double vision. there was neither history of meta bolic disease s nor f amily history of neuropathy. the patient had been diagnosed with pulmonary tb for 9 months and consumed antituberculosis drugs ever since. the patient took an anti-tuberculosis fixed-drug combination which contains emb 15mg/kg/day. previously, he had seen an ophthalmologist regarding his visual loss complaint, when he was advised to immediately discontinue emb therapy and was given oral corticosteroids for a week. then, the patient was referred to our outpatient department ward for further investigation. the vital signs were within normal limits. initial visual acuity (va) on the right eye was 2/60 and on the left eye 5/60. left and right pupillary light reflexes were normal, but positive relative afferent pupillary defect (rapd) was present on the right eye. there was also increased lens opacity on the left eye, indicating mature cortical cataract. funduscopic examination showed normal optic discs. humphrey visual field testing was done to assess patient’s visual field (vf), showing bilateral generalized visual loss with visual field index (vfi) of 13% and mean deviation (md) of -26.59 db on the right eye, and vfi of 43% and md of -19.9 db on the left eye (as shown in figure 1). testing revealed remarkable improvement, vfi 84% and md -3.98 db for right eye and vfi 87% and md -4.20 db for left eye. relative afferent pupillary defect on right eye became negative, and no recurring symptoms were present. it is possible that left ocular va did not significantly improve due to lens opacity, hence we planned cataract extraction as the next step of treatment. figure 2 and table 1 explain the improvement in visual fields at each given time. written informed consent was obtained from the patient for publication of this article and any accompanying images. discussion the onset of ocular symptoms of eon varies from days to months after emb treatment. the safe dose of emb has not been established, but it has been reported that emb therapy at more than 15mg/kg/day may increase the incidence of eon.(8) in previous studies, the population of individuals aged 22–90 years (mean 66.9 years) color vision and contrast sensitivity test were not performed due to patient’s poor va. optica l c oherenc e tomography (oct) examination found that the retinal nerve fiber layer (rnfl) thickness oculus uterque (ou, both eyes) was within normal limit, indicating no significant degeneration of optic (as shown in figure 2). mri brain scan showed no significant result for acute optic neuropathy, but in relation to patient’s clinical presentation, a subacute phase should be considered. based on history taking and ocular examinations, this patient was diagnosed as eon. since the patient had already stopped taking emb for 2 months prior to visit, we prescribed oral citicoline 1000mg od and zinc supplementation od for 1 month. he was also advised to visit his internist regarding the continuity of his anti-tuberculosis medication. at 1st month follow up, patient’s va was 6/45 for the right eye and 3/60 for the left eye. both va and vf were gradually improved at 3rd month follow up. va became 6/18 and 3/60 for the right and left eye, respectively. humphrey visual field nusanti, putri, dearaini reversible ethambutol-induced optic neuropathy figures 1. humphrey visual field testing at initial visit (a), 1st month follow up (b) and 3rd month follow up (c) a : humphrey visual field testing at initial visit showed generalized visual loss on both eyes b : during 1 st month follow up showed improvement; bitemporal hemianposia c : at 3 rd month follow up showed reversible visual field defect compared to initial visit univ med vol. 41 no 3 a b with history of emb therapy develops eon. the mean emb dosage was 14.5mg/kg/day and duration of treatment was 1 to 21 months.(1,5) this is similar to our patient’s characteristics and history of medication. our patient was 60 years old and received emb at 15mg/kg/day. after a 6-month period of medication, the patient had his first visual symptoms which presented as visual loss. typically, patients with eon complain of subacute, bilateral, painless, and symmetrical central visual loss, which is also in line with our case.(3) humphrey visual field testing in this case showed bilateral generalized visual loss and in the next 3rd months of follow up, the grey-scale images on humphrey visual field testing show a cecocentral scotoma, which is similar to previous studies where visual field examination mostly revealed central or cecocentral scotoma.(3) ocular computed tomography examination in the 1st month of follow-up shows that rnfl thickness was within normal limits. in another longitudinal study performed by han et al.,(9) the rnfl thickness was within the normal range after the patients stopped taking the drug. in a recent study, kim and park (10) reported increase in rnfl thickness among tuberculosis patients receiving ethambutol in the dosage range of 15 to 19 mg/ kg/day. it is important to do oct examination in figure 2. initial ocular computed tomography (oct) showed no significant loss of retinal nerve fiber layer (rnfl) table 1. humphrey visual field testing during initial assessment and follow ups vfi: visual field index, md: mean deviation written informed consent was obtained from the patient for publication of this article and any accompanying images initial assessment 1st follow up 3rd follow up vfi od 13% 48% 84% os 43% 58% 87% md od -26.59 db -17.18 db -3.98 db os -19.9 db -13.8 db -4.20 db eon patient to investigate structural changes to particular retinal layers.(11) our patient showed gradual improvement in both va and vf after emb cessation and start of oral citicoline 1000mg od and zinc supplementation therapy. this can be attributed to a previous taiwanese study which mentioned visual improvement in 50% of the subjec ts af ter discontinuation of emb. (5 ) ethambutol acts by inhibiting mycobacterial cell wall synthesis through its metal chelating properties. the similarity between human mitochondria and bacterial cell wall might lead to accumulation of reactive oxygen species in ocular cells, thus resulting in apoptosis and optic nerve degeneration.(8) previous studies have shown that emb in canines might synthesize a metabolite which significantly decreases the zinc concentrations in ocular tissue.(12) zinc is present in high concentrations in the retina and choroid, therefore zinc deficiency will affect the physiological ocular function, because it plays a role as cofactor in several ocular enzymes.(13) the mechanism of eon is believed to be due to inhibition of lysosomal activity and to zinc chelation.(3) a previous study stated that zinc plays an important role in eon.(14) inhibition of zinc chelation can reduce its cytotoxicity to the retinal ganglion cells.(14) another study stated that the number of retinal ganglion cells decreased in rats which were introduced to emb, hence it is believed that retinal ganglion cell disturbance plays a major role in eon manifestation.(15) our patient was given zinc supplementation in order to suppress the apoptotic rate of the retinal ganglion cells. citicoline 1000 mg was also prescribed to the patient. according to a previous study, retinal ganglion cell damage caused by eon can be suppressed by citicoline administration as shown in the finding that the number of retinal ganglion cells in rats with emb intoxication receiving citicoline was higher than in the other group not receiving citicoline.(6) systemic diseases such as hypertension, diabetes mellitus and renal failure can affect the visual recovery of eon.(3) our patient did not have any underlying diseases, and the final visual outc ome was remarkably improved. currently, there is no effective treatment for ganglion cell damage due to eon. primary prevention is considered the best management. patients with ethambutol therapy should be informed regarding the adverse effect and the need for periodical ocular screening. ocular examination (visual acuity, visual field testing, color vision testing and funduscopy) should be perf ormed prior to treatment with ethambutol.(3) this case report has several limitations. more specific ocular examinations such as mferg and vep should have been performed during initial and follow visit. these examinations could have given information about visual function be fore and after treatment and used as a parameter to evaluate treatment efficacy. conclusion it is still debata ble whether visual disturbance in eon is reversible. however, immediate ethambutol cessation might improve the visual impairment of the patients. this case report shows that early detection of eon in pulmonary tb patients who are taking ethambutol and complain of visual loss may reverse the visual defect. we can conclude that the reversibility of eon relies on early detection and intervention. thus, it is necessary for an ophthalmologist to monitor patients with ethambutol therapy during the treatment period. conflict of interest all the authors declare that there is no conflict of interest. acknowledgments we would like to thank rianti wulandari pratiwi, md and marcia budihardja, md for collecting the data and all the contribution for this paper. we also thank all the doctors, and colleagues at cipto mangunkusumo hospital, jakarta for the contribution. nusanti, putri, dearaini reversible ethambutol-induced optic neuropathy univ med vol. 41 no 3 authors contribution s.n was the ophthalmologist who diagnosed and was responsible for the management of this patient. all authors contributed equally to the presentation of case report and the manuscript. s.n supervised the case report and final manuscript. funding statements there are no financial conflicts or interest to disclose. funding statements there are no financial conflicts or interest to disclose. references 1. kim jh, yim j. achievements in and challenges of tuberculosis control in south korea. emerg infect dis 2015;21:1913–20. doi: 10.3201/ eid2111.141894. 2. world health organization. global tuberculosis report 2013. geneva: world health organization;2013. 3. chamberlain pd, sadaka a, berry s. ethambutol optic neuropathy. curr opin ophthalmol 2017; 28:545-51. doi: 10.1097/icu.0000000000000416. 4. kim yj, lim sh, kim u. natural course and prognostic factors of ethambutol toxic optic neuropathy. res sq 2020;4–11. doi: 10.21203/ rs.3.rs-76098/v1. 5. chen s, lin m, sheu s. incidence and prognostic factor of ethambutol-related optic neuropathy/ : 10-year experience in southern taiwan. kaohsiung j med sci 2015;31:358–62. doi: 10.1016/j.kjms.2015.05.004. 6. sari ri. the effect of citicoline on ethambutol optic neuropathy: histopathology & immunohistochemistry analysis of retina ganglion cell damage level in rat model. [thesis]. jakarta: universitas indonesia; 2016. 7. bandyopadhyay s, banerjee s, bandyopadhyay s, shamantha m, biswas s. a prospective evaluation of ocular toxicity in patients receiving ethambutol as anti tubercular therapy. sudan j ophthalmol 2020;12:10–4. doi: 10.4103/ sjopthal.sjopthal_4_20. 8. tang ra, schiffman j, cruz ra, corsi g. ethambutol: friend or foe?, exp rev ophthalmol 2014;2:59-61. doi: 10.1586/17469899.2014.898564. 9. han j, byun mk, lee j, et al. longitudinal analysis of retinal nerve fiber layer and ganglion cell-inner plexiform layer thickness in ethambutolinduced optic neuropathy. graefe’s arch clin exp ophthalmol 2015;253:2293–9. doi: 10.1007/s00417-015-3150-8. 10. kim kl, park sp. visual function test for early detection of ethambutol induced ocular toxicity at the subclinical level. cutan ocul toxicol 2016; 35:228–32. doi: 10.3109/15569527.2015.1079784. 11. pavan taffner bm, mattos fb, cunha mcd, saraiva fp. the use of optical coherence tomography for the detection of ocular toxicity by ethambutol. plos one 2018;13:e0204655. doi: 10.1371/journal.pone.0204655. 12. sarkar s, ganguly a, sunwoo hh. current overview of anti-tuberculosis drugs: metabolism and toxicities. mycobact dis 2016;6. doi:10.4172/ 2161-1068.1000209. 13. erie jc, good ja, butz ja, pulido js. reduced zinc and copper in the retinal pigment epithelium and choroid in age-related macular degeneration. am j ophthalmol 2009;147:276-82. http:// dx.doi.org/10.1016/j.ajo.2008.08.014. 14. chung h, hee y, jin j, sook k, young j, kim j. ethambutol-induced toxicity is mediated by zinc and lysosomal membrane permeabilization in cultured retinal cells. toxicol appl pharmacol 2009;235:163–70. 15. kinoshita j, iwata n, maejima t, kimotsuki t. retinal function and morphology in monkeys with ethambutol-induced optic neuropathy. invest ophthalmol vis sci 2012;53:7052-62. doi: https:// doi.org/10.1167/iovs.12-103082012;7052–62. cite this article as: ibrahim n, andraini t, yusnidar a, putu indah paramita adi putri pipa, sia nipa r ir. neuroprotective effect of south sulawesi propolis on neurogenesis in primary cultures of rat cerebral cortical neurons. univ med 2023;42 :137-49 . doi: 10. 18051 / u nivm e d. 2023 . v42 : 137 149 may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 neuroprotective effect of south sulawesi propolis on neurogenesis in primary cultures of rat cerebral cortical neurons nurhadi ibrahim1 , trinovita andraini1 , amirah yusnidar2 , putu indah paramita adi putri2 , and imelda rosalyn sianipar1* abstract background neurodegenerative diseases have a significant risk factor, namely aging, which is associated with increased neuronal dysfunction and death. propolis has been widely used as medicine due to its various benefits. this research study investigated the effect of propolis from the stingless bee (tetragonula sapiens) from south sulawesi, indonesia, on neurogenesis in primary cultures of embryonic cerebral cortex of wistar rats at 17-18 days of gestation. methods this research was an experimental study involving 4 female pregnant wistar rats, which were terminated and the cerebral cortex of the embryos collected and grown as primary cultures. the cultures were divided into 3 groups, i.e. control, vehicle, and propolis extract group. the research began with 3-(4,5-dimethylthiazol-2-yl)-5-(3carboxymethoxyphenyl)-2-(4-sulfophenyl)-2h-tetrazolium (mts) to obtain the optimal dose among propolis doses of 0.5 μg/ml, 1 μg/ml, 5 μg/ml, 10 μg/ml, 25 μg/ml, and 100 μg/ml. the study was continued by using the best dose in immunostaining examination using microtubule-associated protein 2 (map2) primary antibody and qrtpcr examination of brain-derived neurotrophic factor (bdnf) mrna expression. one way anova and kruskal-wallis test were used to analyse the data. results the results showed that the propolis doses of 0.5 μg/ml and 1 μg/ml significantly increase cell viability compared to the other doses (p=0.011) and stimulate dendritic growth. the propolis dose group of 1 μg/ml induces a significantly higher expression of bdnf mrna than the control group (p=0.031). conclusion our findings indicate that stingless bee propolis has neuroprotective effects against bdnf mrna in rats. it is shown that propolis can be a candidate inhibitor in neurodegenerative diseases. keywords: propolis, neurodegenerative disease, brain-derived neurotrophic factor, rat 1department of medical physiology and biophysics, faculty of medicine universitas indonesia, jakarta, indonesia 2master program in biomedical sciences, faculty of medicine universitas indonesia, jakarta, indonesia *correspondence: imelda rosalyn sianipar department of medical physiology and biophysics, faculty of medicine universitas indonesia jalan salemba raya no. 6 jakarta pusat, jakarta, indonesia 10430 e-mail: imelda.rosalyn@ui.ac.id orcid id: 0000-0002-1438-096x date of first submission, november 30, 2022 date of final revised submission, june 22, 2023 date of acceptance, july 20, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2023.v42:137-149 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1400 137 original article mailto:imelda.rosalyn@ui.ac.id https://orcid.org/0000-0003-3682-0820 https://orcid.org/0000-0003-4264-7729 https://orcid.org/0009-0008-5636-3376 https://orcid.org/0009-0007-9507-3542 https://orcid.org/0000-0002-1438-096x https://univmed.org/ejurnal/index.php/medicina/article/view/1400 ibrahim, andraini, yusnidar propolis on neurogenesis in primary cultures 138 introduction the global prevalence of elderly is rising, affecting an increasing number of degenerative diseases such as neurodegenerative diseases. neurodegenerative disease is defined as a condition of progressive neuronal loss in the central nervous system (cns), that may cause physical disability, cognitive deficits, or both. alzheimer’s disease and parkinson’s disease are the most common neurodegenerative diseases, characterized by behavioral disturbances and progressive cognitive dysfunction.(1) there are an estimated 6.2 million americans above 65 years of age living with alzheimer’s, and this could potentially grow up to 13.8 million by 2060 if there are no medical developments to prevent, slow down or cure alzheimer’s disease.(2) neurodegeneration is a state where there is a degradation of the cell’s ability to respond to stre ss, resulting in the disruption of cell homeostasis and protein formation.(3) the neurodegenerative diseases are correlated with toxic protein deposition, such as in alzheimer’s disease, where the accumulation of amyloid-β peptides in the human brain trigger s the dysfunction of synapses and neural networks that may cause cognitive deficits.(4) the for mation mechanism of neurodegenerative diseases has been targeted as a therapy and prevention, and several herbal medicines have been investigated that protect cells against free radicals and have antioxidant potential. one of these herbals is propolis, that is collected by honeybees from plants and has a number of important biological functions, including antitumor, immunomodulatory, anti-inflammatory, antioxidant, anticarcinogenic, antiviral, antimicr obial, antiparasitic and antidiabetic properties.(5) the organic compounds that have been identified in propolis are polyphenols, terpenes, esters, amino acids, vitamins, minerals, and sugars.(6) the other common constituents of propolis are organic acids, ketones, aldehydes and hydrocarbons.(7) the bioactive molecular profile of raw propolis is variable depending on geographical and botanical origin, season, bee genetics and environmental factors.(8) propolis contains chrysin, caffeic acid phenethyl ester (cape), and caffeoylquinic acid (cqa) that have neuroprotective effects.(9) previous studies proved that green propolis from brazil can inhibit apoptosis in sh-sy5y neuronal cell culture (a human neuroblastoma cell) exposed to staurosporine by inhibiting caspase-3 and apoptosis through an intrinsic pathway.(10) propolis extract reduced lactate dehydrogenase (ldh) production in cell line cultures of pheochromocytoma pc12 due to h 2 o 2 exposure, thereby showing that propolis extract has a protective effect on mitochondria and neuronal cell membranes.(9) in the neurological and neurodegenerative disease models, namely alzheimer’s disease, parkinson’s disease, and epilepsy, propolis also showed potential therapeutic benefits. propolis was demonstrated to reduce amyloid fibrillation and reduce the i mpa ct of amyloid accumulation.(10,11) several phenol compounds in propolis can increase the expression of neuronal differentiation signalers (gap-43, neurofilament light subunit, synaptophysin, synapsin) that are required for neuronal growth. propolis may boost synapse formation through br ain derived neurotrophic factor (bdnf),(12) which is a neurotrophin that plays a leading role in the development, maintenance, repair and survival of neuronal populations. some evidence shows that decreased function of neurotrophins and their receptors may cause neuronal inj ury and contribute to neurodege nerative diseases. neurotrophins and bioactive compounds with the capability to activate neurotrophic receptors have a great potential in managing neurodegenerative diseases and other neurological disorders.(13) there are various types of propolis growing in indonesia and previous research examined the propolis content of stingless bees (tetragonula sapiens) collected in south sulawesi. this propolis has an antioxidant component from phenol compounds (14) and is observed to be an effective antioxidant and anti-inflammatory. this 139 propolis has been studied based on the isolation conducted by miyata et al.(15) of propolis from tetragonula sapiens from south sulawesi, that contained the following flavonoid compounds: 2',3'dihydro-3'-hydroxypapuanic acid; (–)-papuanic acid, (–)-isocalolongic acid, isopapuanic acid, isocalopolyanic acid, glyasperin a, broussoflavo zero f; (2s)-5,7-dihydroxy-4'-methoxy-8prenylflavanone, and isorhamnetin. however, there is no study regarding the effect of propolis from south sulawesi on neurogenesis of rat cortex primary cultures in the basal condition. therefore this study aims to determine the effect of propolis on neurogenesis, by looking at its effect on cell viability, on bdnf mrna expression, and on dendritic growth in rats. methods research design an in vitro e xper imental study was conducted at the jakarta litbangkes animal laboratory, the imeri-ui molecular biology and proteomics core facilities (mbpcf) laboratory, and the stem cell and tissue engineering (scte) laboratory from may 2022-november 2022. experimental animals female wistar rats were obtained from the animal laboratory of litbangkes jakarta, with a pregnancy of 17-18 days, the initial weight of the rats before pregnancy being 250 grams. the animals were kept on a light/dark cycle for twelve hours on each cycle, at a temperature of ±22oc, while feed and drinking water were served ad libitum. accordingly, the total number of samples in this study was 4 female rats with 2 male rats to fertilize the females. two female rats were used for mts examination and the other two female rats were used for immunostaining and qrt-pcr of bdnf mrna. eac h group treatment was repeate d in triplicate. all experiments were performed in accordance with the standard guidelines for the care and use of laboratory animals. reagents ketamine hcl (pt. guardian phartama indonesia), ilium zylazil-100 ( troy laboratories pty. limited), poly-d lysine (gibco), corning® laminin surface (merck), dimethyl sulfoxide (biomatik), d-(+)glucose solution 45% (gibco), dulbecco’s phosphate buffered saline (dpbs) modified 10x (gibco), neurobasal-a medium (gibco), b-27 supplement serum free (gibco), l-glutamine 100x (gibco), papain lyophilized (sigma), bovine deoxyribonuclease i type ii (sigma), propolis powdered extract (cv. nano biotek indonesia), fetal bovine serum (sigma) , celltiter 96 aqueous solution cell proliferation assay (promega), paraformaldehyde 37% (merck), pipes (sigma), triton x-100 surfact-amps detergent (thermo scientific), map2 antibody (genetex), vectafluor excel amplified antimouse igg (vector laboratories), dylight 488 antibody kit (vector laboratories), vectashield antifade mounting medium with dapi (vector laboratories), quick rna miniprep plus isolation kit (zymo research/california), synthesis kit for cdna revertra ace® qpcr rt master mix with gdna remover (toyobo/osaka, japan), real time pcr kit sensifast sybr® hi-rox (bioline/california), bdnf mrna primer and housekeeping gene gapdh (integrated dna technologies). preparation of propolis extract the propolis preparation used in this study was a product available on the free market that contained propolis extract powder. the extract doses used for the treatment were 0.5 µg/ml, 1 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml and 100 µg/ml. each dose was dissolved in 1% dmso. the primary culture was divided into 8 groups, namely medium (neurobasal growth medium) gr oup a s positive control, ve hicle gr oup, (neurobasal growth medium and 1% dmso) and the groups given propolis extract at doses of 0.5 µg/ml, 1 µg/ml, 5 µg/ml, 10 µg/ml, 25 µg/ml and 100 µg/ml. the best dose in the mts assay univ med vol. 42 no. 2 ibrahim, andraini, yusnidar propolis on neurogenesis in primary cultures 140 was followed by immunostaining examination and measurement of bdnf expression, which was then compared with the control and vehicle groups. the mts assay was carried out 24 hours after treatment, while the immunostaining examination a nd me asurement of bdnf expression were carried out on the 7th day after treatment. each treatment group was tested in triplicate. neuronal cell culture pregnant female wistar rats at 17-18 days gestation were terminated with 90mg/kg ketamine and 10mg/kg xylazine. the abdomen was cleaned with 70% ethanol and an incision made to open the peritoneal space. after the amniotic sac was located, the embryo was removed from the uterus and transferred to a petri dish filled with phosphatebuffered saline (pbs). we used pbs 1x made into 1l and autoclaved at 121oc for 20 minutes. one day before rat dissection, incubation was performed by using poly d-lysine substrate at 0.1 mg/ml in 12 wells that had been placed on a 1ml cover slip of 18 mm diameter (until submerged). incubation was conducted at 4oc overnight. on the next day, the poly d-lysine was aspirated and the well washed three times with pbs. the well was incubated with 600 μl/well of laminin at 5 μg/ml concentration for 4 hours at 4oc. after the rat termination process, the rat embryo cortex isolation process was performed, the rat skull was fixed at the back and the lower part of the head was trimmed. after the brain was released from the meninges, the clean cortex was taken from the brain. the clean cortex was transferred to a 15 ml phial filled with pbs glucose. after the microdissection was completed, the pbs glucose was discarded and papain solution (10 mg papain in 1 ml sterile distilled water) was added and incubated for 15 minutes at 37oc. for papain inactivation, pbs/glucose, b27 supplement, and dnase was added and incubated for 5 minutes at 37oc. the dissociation and centrifugation were conducted at 1000 rpm for 5-10 minutes. the supernatant was discarded and bsa cushion was added (bsa and neurobasal growth medium that had been filtered and homogenized). then recentrifugation was done at 120 g for 6 minutes and the super natant was discar ded and resuspended with 5 ml of neurobasal growth medium. neurobasal growth medium consists of 48.7 ml neurobasal supplemented medium, 50 μl lactic acid, 1 ml b27 supplement, 250 μl lglutamine, 1% amphotericin b and 1% penicillinstreptomycin. cells were grown in each 12-well plate at 300,000 cells per well and in 96-well plates at 10,000 cells per well. before the cells were grown in the wells, the laminin was aspirated before incubation for 4 hours and the wells washed three times with pbs. cell viability assay the 96-well plates were prepared in triplicate at 10,000 cells/well and divided into 8 groups, consisting of medium as the control group, vehicle group, and propolis extract groups at doses of 0.5 µg/ml (p05), 1 µg/ml (p1), 5 µg/ ml (p5), 10 µg/ml (p10), 25 µg/ml (p25) and 100 µg/ml (p100). after being soaked for 24 hours and observed for any changes in cell culture, 100 µl of culture medium was added to the mts solution (10 µl mts stock solution 5 mg/ml). they were incubated for 4 hours at 37°c in an incubator with 5% co 2 . cells were observed after 4 hours to examine the formation of formazan salts and the absorbance at 490 nm was recorded. observations of morphological changes cells were observed until day 7 post treatment which consists of medium, vehicle and the two best doses, namely 0.5 μg/ml and 1 μg/ ml, were se le cte d after the mt s assay procedure. cellular morphology was observed and captured for each day by using an inverted microscope (nikon eclipse ti-s). immunohistochemistry on day in vitro (div) 7, coverslips in wells that consisted of control, vehicle, and best dose propolis groups were subjected to immunostaining procedures. the medium was aspirated from the 141 culture disk and was washed 3 times with pbs 1x. t he culture disk was fixed with 4% paraformaldehyde, incubated for 15 minutes. then washed 3 times with pbs 1x and permeabilized with 0.1% triton-100 for 10 minutes, followed by 3 washings with pbs 1x. afterwards it was incubated with 2-5% normal horse serum for 20 minutes and washed 3 times with pbs 1x. the culture disk was incubated with polymer antibody (map 2) at 1:250 overnight at 4°c and washed 3 times with pbs 1x. the culture disk was incubated with antibody amplifier for 15 minutes and with vectafluor amplified dylight 488 antirabbit igg for 30 minutes, followed by washing 3 times with pbs 1x after each incubation was completed. for preserving fluorescence the slide was mounted with vectashield antifade mounting medium with dapi. the slides were stored in the dark, protected from light at 4°c until imaging. observations were made with a 20x objective on a confocal microscope (zeiss lsm 700). real-time quantitative polymerase chain reaction (qrt-pcr) mrna was isolated from cultured cells of rat embryonic cortical neurons. extracted rna at a concentration of 5 µg/ml was reverse transcribed to cdna using revertra ace® qpcr rt master mix with gdna remover cdna synthesis kit (toyobo/osaka, japan). after polymerase activation was performed at 95°c for 2 minutes, it was followed by 95°c denaturation for 5 seconds with 40 cycles and 60°c annealing for 30 seconds. cdna was amplified by a real time pcr instrument (applied biosystem) with the sensifast sybr® hi-rox real time pcr kit (bioline/california). the primer pair sequences were as follows: bdnf mrna primer 5'tacgagaccaagtgcaatcc-3' (forward); 5'-tcgccagccaattcttt-3' (reverse) and gapdh pr imer 5'accacagtccatgccatcac-3' (forward); 5 ' t c c a c c a c c c t g t t g c t g t a 3 ' (re verse). gapdh primer was used as a housekeeping gene. relative expression was calculated by livak’s formula and each treatment group was processed in triplicate. statistical analysis data were presented as mean ± standard error of the mean. statistical analysis was completed by one way anova with lsd post hoc test as parametric test and kruskal-wallis test followed by multiple comparisons between the groups as nonparametric test using spss (ibm spss statistic). graphpad prism (dotmatics) software was used for making graph. a p<0.05 score was considered to indicate statistical significance. ethical clearance all procedures involving animals were conducted in line with the research protocol approved by the hea lth research ethics commission of the faculty of medicine, universitas indone sia, a nd dr. cipto mangunkusumo national general hospital, under no. ket-518/un2.f1/etik/ppm.00.02/2022 and protocol no. 22-04-0446 on may 30, 2022 results effect of propolis extract on cell viability cell viability in this study was measured by using mts, a calorimetric method to assess cell metabolic activity. the mts assay is applied to assess cell proliferation, cell viability and cytotoxicity. figure 1 shows an overview of the primary culture of rat cerebral cortex neurons figure 1. the overview of neuronal cells before mts treatment contains immature neurons univ med vol. 42 no. 2 ibrahim, andraini, yusnidar propolis on neurogenesis in primary cultures 142 figure 2. the absorbance of cell viability on the first day after treatment data presented as mean ± sem. one way anova with lsd post hoc analysis. n=3 rats/group. (medium: normal control; vehicle: medium with 1% dmso solvent; p1: 0.5μg/ml propolis; p2: 1μg/ml propolis; p3: 5μg/ ml propolis; p4: 10μg/ml propolis; p5: 25μg/ml propolis; p6: 100μg/ml propolis. before the mts solution was given, while the treatment was conducted on day 1 of primary culture. this parameter was assessed on the day after treatment for each group (figure 2). data analysis used one-way anova; the results showed there were differences between groups (p=0.011). from these results, there were two highest absorption values in the dose of propolis given 0.5 μg/ml and 1 μg/ml compared to the other groups. the p0.5 group was significantly different from the medium (p=0.005), vehicle (p=0.032), p100 (p=0.013), p25 (p=0.018) and p10 (p=0.032) groups. on the other hand, the p1 group was significantly different from the medium (p=0.005) and vehicle (p=0.032) groups. thus, the 0.5 μg/ml and 1 μg/ml doses of propolis were found to result in better cell viability compared to the medium and vehicle ground. these two doses were then used as the selected doses for tests on the other parameters, such as immunostaining and bdnf mrna expression test. effect of propolis extract on qualitative analysis of dendrite expression through map2 primary antibody through the mts test, two selected doses were obtained and used for the immunostaining test. the primary cell culture results of the rat cerebral cortical neurons were observed until the seventh day after treatment. figures 3, 4, 5, and 6 give a description of the growth in primary cultures of rat cerebral cortical neurons on day 1 after treatment until day 7 after treatment. the results show neuronal cell growth in all groups, such as medium, vehicle, propolis 0.5 μg/ml, and propolis 1 μg/ml groups. the yellow arrowhead in the figure points to a neuronal dendrite. on div 1-3 the dendrite is still short and immature, and in div 4-7 the dendrite continues to grow, thicken and branch. after 7 days post-treatment, the cells were fixed and treated for immunostaining with map2 primary antibody to see the image of dendrites in the cells. figure 7 shows the results of 143 figure 3. the effect of the propolis extracts on rat cerebral cortex neuron cells after the intervention with medium. arrows indicate the dendrite growth. group (a) day 1 after intervention (b) day 2 after intervention (c) day 3 after intervention (d) day 4 after intervention (e) day 5 after intervention (f) day 6 after intervention (g) day 7 after intervention. on days 1-3 after intervention the dendrites are still short and immature, and on days 4-7 after intervention the dendrites continue to grow, thicken and branch out. figure 4. the effect of the propolis extracts on rat cerebral cortex neuron cells after the intervention with vehicle. arrows indicate the dendrite growth. group (a) day 1 after intervention (b) day 2 after intervention (c) day 3 after intervention (d) day 4 after intervention (e) day 5 after intervention (f) day 6 after intervention (g) day 7 after intervention. on days 1-3 after intervention the dendrites are still short and immature, and on days 4-7 after intervention the dendrites continue to grow, thicken and branch out. univ med vol. 42 no. 2 ibrahim, andraini, yusnidar propolis on neurogenesis in primary cultures 144 figure 6. the effect of the propolis extracts on rat cerebral cortex neuron cells after the intervention with 1μg/ ml dose of propolis. arrows indicate the dendrite growth. group (a) day 1 after intervention (b) day 2 after intervention (c) day 3 after intervention (d) day 4 after intervention (e) day 5 after intervention (f) day 6 after intervention (g) day 7 after intervention. on days i-3 after intervention the dendrites are still short and immature, and on days 4-7 after intervention the dendrites continue to grow, thicken and branch out figure 5. the effect of the propolis extracts on rat cerebral cortex neuron cells after the intervention with 0.5μg/ml dose of. arrows indicate the dendrite growth. group (a) day 1 after intervention (b) day 2 after intervention (c) day 3 after intervention (d) day 4 after intervention (e) day 5 after intervention (f) day 6 after intervention (g) day 7 after intervention. on days 1-3 after intervention the dendrites are still short and immature, and on days 4-7 after intervention the dendrites continue to grow, thicken and branch out. 145 figure 8. brain-derived neurotrophic factor mrna expression data presented as mean ± sem. kruskal-wallis test followed by multiple comparisons between the groups. n=2 rats/group. (medium: normal control; vehicle: medium with 1% dmso solvent; p1: 0.5μg/ml propolis; p2: propolis 1µg/ml) figure 7. overview of cerebral cortex neuronal cultures of day in vitro (div) 7 mouse embryos post-treatment utilizing map2 immunostaining technique. arrows indicate the dendrite growth. group (a) medium and (b) vehicle show fewer cells, shorter dendrites, compared to group (c) 0.5μg/ml dose propolis and (d) 1μg/ml dose of propolis that has thicker and branched dendrites. univ med vol. 42 no. 2 ibrahim, andraini, yusnidar propolis on neurogenesis in primary cultures 146 immunostaining with map2 primary antibody in each group. figure 7 shows that all groups express cell staining by using map2 primary antibody with different characteristics for each treatment, indicating that neuronal cells in the medium, vehicle, p0.5 and p1 groups express map2 protein as dendrite signaling in neuronal cells. effect of propolis extract on bdnf mrna expression in rat cerebral cortex neuron cell cultures the results of bdnf mrna expression analysis in rat cerebral cortical neuron cultures tested by real time pcr are shown in figure 8. the mean value of bdnf mrna relative expression in the medium group was 1, in the vehicle group it was 5.972409, in group p0.5 47.08719 and in group p1 169.6081. according to the normality test, the bdnf mrna relative expression data were not normally distributed and homogeneous, therefore the data were analyzed by using the kruskal wallis test. this study showed that there was a significant difference in the relative expression of bdnf mrna in each group (p=0.031). the data from figure 8 shows that there are significant differences between groups on the relative expression of bdnf mrna, which in group p1 is significantly different (p=0.018) from the medium group. the 1 μg/ml dose of propolis expressed higher bdnf mrna than the medium group. discussion the results in this study showed that 0.5 μg/ ml and 1 μg/ml doses of propolis showed significantly better results in mts examination compared to the medium and vehicle. this implies that the 0.5 μg/ml and 1 μg/ml doses of propolis result in better cell viability compared to the medium and vehicle. propolis contains various neuroprotective agents such as chrysin, caffeic acid phenethyl ester (cape) and caffeoylquinic acid (cqa) that may inhibit apoptotic cells through the inhibition of caspase-3 and intrinsic apoptotic pathways. caffeic acid phenethyl ester can reduce malondialdehyde (mda) as a lipid peroxidation biomarker, therefore it can inhibit harmful agents that cause aging.(5,16,17) in the study by ni et al.,(18) the investigators examined the effect of brazilian green propolis on cell viability in an in-vitro study using shsy5y human neuroblastoma cells which were previously exposed to ros. they found that the viability of cells given propolis was higher than that of cells not given propolis. this showed that propolis has a protective effect on neuronal cells. the propolis used in this study was from stingless bees (tetragonula sapiens) and was collected in south sulawesi, indonesia. this propolis has been known to be rich in polyphenols that are high sources of antioxidants. south sulawesi propolis has the potential as a cox-2 inhibitor that may inhibit inflammation, and has protective effects against oxidative stress.(19) in this study, the optimal doses of propolis were 0.5 μg/ml and 1 μg/ml, while there was a decrease in cell viability at higher doses. flavonoids can prevent the toxic effects of active meta ls such as fe or cu, but in certain circumstances they can also act as pro-oxidants and increase the oxidation of molecules. the role of propolis as antioxidant or pro-oxidant depends on the dose used, therefore propolis doses above the optimal dose can exert pro-oxidant effects and cause cell damage.(20,21) neurodegenerative diseases occur due to alterations in the brain, as a result of agedependent changes that can be stimulated by neuroinflamma tion and ros, c ausing neurodegeneration.(1) in this study, it was found that propolis from south sulawesi has the effect of improving cell proliferation and has a protective effect on neuronal cells compared to the medium and vehicle. thus, propolis can be used as a preventive agent against neurodegenerative diseases. the immunostaining test with map2 primary antibody showed a positive luminescence, indicating the expression of map2 protein in the 147 neuronal cells. it showed the dendrite growth in the propolis group from south sulawesi at doses of 0.5 μg/ml and 1 μg/ml, as compared to medium, and vehicle. the results showed that the groups receiving 0.5 μg/ml and 1 μg/ml doses of propolis had thicker, longer, and branched dendrites compared to the medium and vehicle groups. the measurement of dendrite length in each group was not yet possible, because the images still do not meet the requirements for the calculation of dendrite values, such as the distance between neurons that is still too close, and there are discontinuous lines on the dendrites causing inaccurate calculations. this study used map2 primary antibody, as an outgrowth signaling protein. propolis contains cape which has been shown as an effective modulator of neurogenesis, dendrite formation, elongation and branching as well as inhibiting the deficiency that was induced by neurotoxins. a number of proteins f or neurogenesis are stimulated by propolis, including growth-associated protein 43 (gap-43), microtubule-associated protein (map), tau, synaptophysin, and synapsin.(12) this study showed through qrt-pcr analysis that the group receiving south sulawesi propolis at a dose of 1 μg/ml expressed bdnf mrna significantly higher than did the medium group. in a study by kuswati et al.(22) using rats that had previously experienced induced stress for 14 days, it was found that bdnf expression in the hippocampus was significantly higher in the group given propolis at a dose of 200 mg/kgbw compared to those not given propolis. the latter study is in line with our study which showed that bdnf mrna expression in the group given propolis was higher than that in the medium group as a positive control. propolis is rich in polyphenolic compounds, such as cape, curcumin, and quercetin that are repor ted to elevate bdnf levels. bdnf activation by several polyphenolic compounds incre ases cell viability and antiapoptotic signaling.(12) an increase in bdnf expression was found in the research of ni et al.(18) on the cytotoxicity in sh-sy5y human neuroblastoma cells induced by hydrogen peroxide, with the result that brazilian green propolis significantly increased bdnf mrna expression mediated by pi3k signaling. the limitation of the study is that it cannot calculate dendrite length because there are discontinuous lines on the dendrites, so further optimization of immunostaining using map2 antibodies is needed. the clinical implication of these study results is that propolis may be used to inhibit or prevent neurodegeneration. further studies are needed regarding the effect of propolis on pathological conditions because this study only examined the physiological condition. conclusion this study demonstrated that utilized propolis from south sulawesi can significantly increase cell viability. propolis also expresses dendritic signalers through immunostaining with map 2 primary antibody. meanwhile, in the test of bdnf mrna expression, propolis proved to more effectively increase bdnf mrna expression compared to the medium group. conflict of interests the authors declare no conflict of interests in association with this study. acknowledgements we would like to thank the staff of the jakarta litbangkes animal laboratory, the imeri-ui molecular biology and proteomics core facilities (mbpcf) laboratory, and the stem cell and tissue engineering (scte) laboratory, for their work during this study, the department of physiology, faculty of medicine, universitas indonesia for their support and dr. eng. muhamad sahlan, s.si., m.eng from the faculty of engineering, universitas indonesia to provide propolis for this research. univ med vol. 42 no. 2 ibrahim, andraini, yusnidar propolis on neurogenesis in primary cultures 148 funding this research was funded by a master thesis grant from the ministry of education, culture, research, and technology for the fiscal year 2022 with dr. imelda rosalyn sianipar, m.biomed, phd as the lead researcher. author contributions irs contributed to writing the protocol and revised the manuscript. ni and ta revised the manuscript. ay contributed to the design of the study and wrote the first draft of the manuscript. all authors have read and approved the final manuscript. references 1. azam s, haque me, balakrishnan r, kim is, choi dk. the ageing brain: molecular and cellular basis of neurodegeneration. front cell dev biol 2021;9: 1–22. doi: 10.3389/fcell.2021.683459. 2. alzheimer’s association. 2019 alzheimer ’s disease 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jembrek m. molecular and cellular mechanisms of propolis and its polyphenolic compounds against cancer. int j mol sci 2022;23:10479. doi: 10.3390/ijms231810479. 21. sharifi-rad m, anil kumar n v, zucca p, et al. lifestyle, oxidative stress, and antioxidants: back and forth in the pathophysiology. front physiol 2020;11:694. doi: 10.3389/fphys.2020.00694. 22. kuswati k, nugraha zs, handayani es. pengaruh pemberian propolis terhadap ekspresi bdnf di hippocampus tikus yang diinduksi stres. j kedokt kesehat indones 2021;252-8. doi: 10.20885/ jkki.vol12.iss3.art8. univ med vol. 42 no. 2 c:\users\user\documents\bagus h 90 abstract universa medicina parasite immunomodulatory role in reducing the prevalence of covid-19 in endemic regions bagus hermansyah1*, dini agustina2, siti zulaikha risqiyani3, and ali habibi3 nearly 35 million cases and one million deaths over the nine months of the covid-19 pandemic have been reported worldwide. africa and some countries with endemic parasitic infections had a low incidence of covid19. by contrast, the united states and several european countries, having a non-endemicity of parasitic infections, recorded a high incidence of covid-19. some parasites have an immunomodulatory mechanism that can induce an immune tolerance state in the infected persons by balancing pro-inflammatory and anti-inflammatory responses. emerging reports also stated that covid-19 and helminth co-infections may have more hidden outcomes than predictable ones. hence, the aim of this literature review is to show and identify that an increase in the number of regulatory immune cells due to the immunomodulatory role of a pre-existing parasitic infection could reduce the risk of covid-19. this study explored the existing literature to determine the role of parasitic infections in modulating the immune response and possibly reduce the risk of covid-19 infection in endemic countries. the mechanism of immunomodulation by parasites is the increased numbers of treg cells, m2 macrophages, eosinophils, the th2 cytokines il-4 and il-5, and the pro-inflammatory downregulation of ifn λ, tnf α, and il-6, which play an essential role in inducing cytokine storms in covid19 infection. this condition will probably occur in an individual with parasitic infection in a community with limited facilities and infrastructure to treat parasitic infections, particularly in developing countries. to conclude, in endemic areas, the immunomodulatory effect of parasitic infection to reduce the risk of covid-19 cases/deaths is a possibility if the host is immunocompetent. herein, the current knowledge on the immunomodulatory role of covid-19 and helminth co-infections will be discussed. keywords: covid-19, parasite infection, parasite endemic regions, immunomodulatory review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.90-99 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1212 january-april 2022 vol.41no.1 cite this article as: hermansyah b , agustina d, risqiyani sz, habibi a. parasite immunomodulatory role in reducing the prevalence of covid-19 in endemic regions. univ med 2022;41: 90-99. doi: 10.18051/univmed.2022. v41 . 90 -9 9 1department of parasitology, faculty of medicine, university of jember 2department of microbiology, faculty of medicine, university of jember 3medical science program, faculty of medicine, university of jember *correspondience: bagus hermansyah department of parasitology, faculty of medicine, university of jember jl. kalimantan no.37 kampus bumi tegalboto, jember, indonesia phone: 0331-337877 fax: 0331-337877 email: bagus_hermansyah@unej.ac.id orcid id: 0000-0002-0946-2936 date of first submission, august 12, 2021 date of final revised submission, april 3, 2022 date of acceptance, april 7, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license univ med vol. 41 no 1 91 introduction historically, infectious diseases have caused disasters among communities, have occurred repeatedly, and are now oc curr ing at an unprecedented rate. according to the world health organization (who), the world has been shocked by the emergence of several epidemics over the past decade, caused by more than 20 infectious agents.(1) sars-cov-2 is the virus that causes coronavirus disease 2019 (covid-19) and is a potentially fatal disease agent for global public health. on december 12, 2019, the first case of covid-19 in wuhan, china, was identified as pneumonia. the who declared covid-19 a public health emergency of international concern on january 30 and a pandemic on march 11, 2020.(2,3) as reported to the who on november 21, 2020, there were 57,274,018 confirmed cases of covid-19 globally, including 1,368,000 deaths.(4) parasitic infection is also still considered an extensive global health concern, especially in lowincome developing countries.(5,6) parasitic infections have spread all over the world, with varying prevalence rates in many countries. endemic parasitic infections have also spread worldwide, with varying prevalence rates in those countries.(7,8) some parasites including plasmodium and helminth species such as fasciola hepatica, schistosoma haemat obium, schistosoma mansoni, and necator americanus, have a proficient immunomodulatory mechanism that is currently being defined.(9) they can induce an immunotolerogenic state in infected persons by balancing pro-infl ammator y and antiinflammatory responses.(10,11) parasite infection, especially of helminths, can cause a spike of treg cells, m2 macrophages, eosinophils, and the th2 cytokines il-4 and il-5.(12) these changes in thelper cells represent the host’s response against the invading parasite. the possible beneficial effects that appear are more complex than merely th2 responses.(10,11) africa, one of the endemic parasite regions, had a relatively low number of covid-19 cases, which was lower than in europe, the eastern mediterranean, the americas, and south east asia.(13) africa had numerous reported cases of parasitic infection s such a s malar ia, schistosomiasis, toxoplasmosis, and soiltransmitted helminths, with the highest percentage in 2018, of malaria and schistosomiasis cases of 93.13% and 89.23%, respectively. in 2019, the african region carried a varying number of reported cases of parasitic infections, such as malaria (up to 94%), and the lowest percentage (0.88% or 29,438/3,349,486) of the global covid-19 cases.(9,14) based on these data, the spread of covid-19 infections in africa is relatively slow.(15–17) parasitic infection is still considered a health challenge in the world, especially in southeast asia. the african region holds a poor track record of global parasitic infections, followed by southeast asia. in comparison, according to the who, southeast asia is also known as endemic countries for parasitic infection and instances of third-highest covid-19 cases. by contrast, there are numerous non-endemic countries for parasitic infections with a high prevalence of covid-19 cases. the americas have the highest covid19 cases with non-endemicity of parasitic infection, such as the united states as part of the north american continent, followed by the european region. world health organization data distinctly show that the united states has a substantially high number of covid-19 cases globally.(4,17) as noted above, compared to the developing countries in various parts of the world, the united states as a developed country has a relatively low incidence of parasitic infections but a high prevalence of covid-19 cases. one possible explanation for this situation is that the endemicity of parasitic infecti ons such as malar ia, schistosomiasis, or soil-transmitted helminths can reduce the prevalence of covid-19 cases.(11) therefore, an increase in the number of regulatory 92 immune cells due to the immunomodulatory effects of a pre-existing parasitic infection may reduce the risk of covid-19. the aim of this review is, therefore, to summarize the current understanding of the role of parasitic infections in modulating the immune response and possibly reducing the risk of covid-19 infection in parasite endemic countries. search engines including pubmed central, scopus, and google scholar were used. in the search engines, the keywords or phrases used included but were not limited to covid-19, helminth infections, immunomodula tion, immunopa thology, epidemiology, interactions, and clinical outcome. the search was restricted to english language articles. endemic countries and their parasit ic infections endemic countries are those with persistent diseases generally having a relatively constant incidence rate. parasitic infections caused by organisms such as helminths and protozoa are frequently occurring problems in some endemic countries of the world. helminth infections still affect many people in the world, even up to more than 1 billion. more than 1.5 billion people in the world, almost 20% of the entire human population, are affected by soil-transmitted helminths (sth).(18,19) helminth infections are mainly concentrated in economically disadvantaged or developing tropical countries, including parts of south and central america, most of tropical and subtropical africa, and south asia. simultaneously, one of the caribbean countries, namely haiti, has a high prevalence of sth and lymphatic filariasis.(20,21) not much different from helminthiasis, malaria incidence is also high in low-income developing countries.(15) in 2019, there were more than 200 million malaria cases worldwide, with an annual incidence of 229 per 1,000 persons. most of the cases were reported in africa (213 million cases) with 405,000 deaths (94%).(22) besides africa, india has the greatest burden of malaria cases, accounting for 3% of the global cases with p. falciparum infections, and less commonly with p. vivax malaria.(23,24) toxoplasmosis is an infection caused by the protozoon toxoplasma gondii. in the usa, it is estimated that 11% of the population aged six ye ars and olde r ha s been affe cted by toxoplasma. in various places around the world, toxoplasma has been infecting more than 60% of populations. this parasite infection often occurs in areas with hot and humid climates and lower altitudes, since the oocysts survive better in these types of habitats.(25,26) based on the type of environment, southern brazil (53%), tanzania (46%), and nigeria (40.8%) have a higher prevalence than bangalore (india), korea, or china. the prevalence of toxoplasma also depends on dietary habits, local culture, and the presence of a host. there have been 30-50% of toxoplasma cases reported in western europe, while in france and the us, it has decreased in prevalence.(27,28) helminth and covid-19 co-infections covid-19 is a respiratory disease caused by a coronavirus, of which cases were first found in december 2019 in wuhan, china. who named the coronavirus sars-cov-2. the massive spread and increasing death toll due to covid-19 have diverted all the countries’ resources to deal with the pandemic. meanwhile, other infectious diseases such as parasitic infe ctions continue to attack millions of people.(24,29,30) helminth infections, on the other hand, are known to induce an immune tolerogenic state and modulate responses associated with inflammation. in this case, helminth-induced immunoregulation will help to modulate covid-19-induced inflammation and the interaction of helminth and covid-19 coinfecti ons may perhaps be be neficial for the patient. the beneficial interactions during helminth co-infections have been shown in previous reports. (31)along the same lines, an ecological study by ssebambulidde et al. (9) also stated that the comparatively low number of covid-19 cases/deaths in parasite hermansyah, agustina, risqiyani, et al parasite immunomodulatory and covid-19 univ med vol. 41 no 1 93 a b endemic areas might be due to immunomodulation induced by parasites. epidemiologically, the incidence of covid19 and parasitic infections mostly occurs in lowmiddle income countries (lmics). limited funds and resources in lmics to handle co-infections of covid-19 and parasitic infections, and the immunological reactions of each disease, potentially result in several possibilities, including:(15,29) 1) worse outcomes than mono-infections the incidence of parasitic infections, including malaria , toxoplasmosis, a nd helminthiasis, is high in lmics. their causative agents can alter the immunological response to other infectious agents. malaria, for example, can cause cytokine storms and the pro-coagulant state that also occurs in severe covid-19. thus, coinfection with the two infectious agents can lead to a worse outcome . when two or more epidemics coincide and cause harm or worsen the conditions compared with one or the other, the epidemic is called a syndemic or synergistic epidemic. in addition to poor outcomes, these interactions may shift the age pattern of severe covid-19 to a younger age group. these syndemics occur mainly in acute infection and in immunocompromised persons infected with both parasites and covid-19.(15,29,32,33) 2) increase in cases of parasitic infection endemic countries working to eradicate parasitic infections are very likely to face setbacks due to the covid-19 pandemic. previous progress made to reduce the number of cases and deaths associated with effective vector control, rapid diagnostic tests (rdt), an effective and accessible treatment could be reversed due to erosion of the health system and disruption of control and elimination programs. this has resulted in an increasing number of parasitic infections. this situation is as reflected in iran, which was malaria-free in 2018, and was then threatened with its survival due to the covid19 pandemic, severely impacting the country’s health system.(29,34) endemic countries such as sub-saharan africa and southeast asia are at significantly higher risk of covid-19 and malaria, and the situation may become more difficult over time, due to the weak health systems in these regions, especially in sub-saharan africa.(16,34,35) 3) delayed timing of the epidemic covid-19 epidemic started in wuhan, china, and then spread throughout the world. the timing of the epidemic is different among countries depending on how the disease behaves in each country. several factors play a role in the delayed occurrence of a pandemic in an given area, including the spread of the disease itself (through patterns of population movement between countries or regions), the ability of the area to prevent the disease (usually influenced by the level of a country’s economy), speed and accuracy in tracking and testing, and according to our hypothesis, the existence of an endemic parasitic disease.(2,19,29) there should be delayed timing of the epidemic between endemic parasite countries and parasite-free countries. however, taking the reports into account, covid-19 and helminth coinfections may have detrimental interactions. it is projected that preexisting helminth infections may suppress the efficient immune response against sars-cov-2 in the early stage of the infection, and thereby may increase the morbidity and mortality of covid-19.(36) people living in parasite endemic regions experience changes in the immune system; hence, viruses may not easily infect them. even if infected, they will show milder symptoms. therefore, from the onset of the pandemic, the endemic parasite area experiences a delay in the pandemic’s timing. these theories seem correct in malaria and sth infection, but different results are found in toxoplasmosis. the study findings did not confirm a clear, direct causal association between toxoplasmosis and exposure to the covid 19 pandemic.(19,37–39) 4) low incidence of covid-19 comparing the incidence of covid-19 between lower-middl e-income countries (lmics) and high-income countries, there is the possibility that the incidence of covid-19 in 94 lmics is much lower. the difference in incidence will be seen when we combine incidence maps of covid-19, malaria, toxoplasmosis, and sth. the high number of parasitic infection cases in the world is inversely proportional to the number of covid-19 cases. the dominance of parasitic infection cases is still present in the african region, followed by asia and some in other regions. figure 1. prevalence of covid-19 cases.(40) figure 2. prevalence of toxoplasmosis cases.(39) hermansyah, agustina, risqiyani, et al parasite immunomodulatory and covid-19 univ med vol. 41 no 1 95 meanwhile, cases of parasitic infections in europe and the us are very few or even absent.(23,40) this phenomenon shows a possible link between parasitic infection and covid-19 (see figures 1-4). the relationship involves an immune reaction, as it is known that parasites can modulate the human immune system so that they can survive in the human body.(9) modulation of the immune syst em by infection with parasites in general, the modulation of the immune system by parasites occurs at the stage of chronic infection, and generally, there is a change from the th1 response to the dominant th2. as is well-known, there are five essential stages of covid-19 virus infection, namely: 1) figure 3. prevalence of soil-transmitted helminth infection.(38) figure 4. prevalence of malaria infection.(23) 96 receptor-mediated viral entry, 2) viral replication, 3) immune activation, 4) cytokine release, and 5) immune cell recruitment. different parasites interact with the covid-19 virus at different stages.(41–43) in malaria, it is suspected that the interaction occurs in the early stages of covid19 infection, namely at the stage of entry of the virus into the body. malaria parasites and the covid-19 virus both have membrane proteins, namely glycosylphosphatidylinositol (gpi). glycosylphosphatidylinositol is a phospholipid that can stimulate leukocytes and the expression of adhesion molecules through toll-like receptors (tlrs), especially tlr2 and tlr4 and cause the release of pro-inflammatory cytokines. covid-19 introduces various glycoproteins (gps), namely spike gps, membrane gps, and gps with acetyl esterase and hemagglutination properties.(44) in chronic malaria, the body forms antibodies against the gpi. therefore, in coinf ection of malari a and cov id-19, the antibodies can also recognize the vir al glycoproteins (gps).(45) this indirectly causes people living in malaria-endemic areas to have stronger immunity against covid-19, such that this virus will induce milder symptoms.(42) unlike malaria, toxoplasmosis interacts with covid-19 at the virus replication stage. the dense granule protein-7 (gra-7) is a protein from toxoplasma gondii that is excreted into the host cell and can prevent viral replication. overall, gra-7 can exhibit immunostimulatory and a broad spectrum of antiviral activities. however, although the toxoplasma gondii antivira l activities are limited to the infection’s initial and virulent phase, specific antiviral adaptations are thus expressed by toxoplasma gondii.(37,46) in addition to malaria and toxoplasmosis, sth infection has a more significant interaction wi t h co v id -19 , e sp e c i a l l y in r e d uc in g symptoms arising from the cytokine storm induced by covid-19. helminths, or worms, are invertebrate animals that comprise a whole range of different pathogens capable of affecting human health. parasites are incredibly adept at molding themselves to the environment and, most importantly, evading the host defense syste m.(4 7)in the fight against unicellular pathogens such as bacteria or viruses, the human immune system reacts by the activation of a thelper (th)1 type of immune response that includes the production of pro-inflammatory c yt oki ne s [ t u mo r n e c r os i s f ac t o r -a l p ha (tnfz#), interleukin (il)-1β, type 1 interferons (ifn)γ, interleukin (il)-12, etc.] and the classical activation of m1 macrophages involved in pathogen killing and phagocytosis.(48,49) however, in sth infections, such an immune mechanism does not occur. most helminths find a way, through different molecular tricks, to avoid the fearful th1 immune reaction and, instead, evoke the activation of the th2 network, a completely distinct response of the immune system with often opposite effects to the th1 activation.(18,43,50,51) thus, the cytokine storm that occurs in covid-19 infection characterized by the excessive activation of th1 will not occur or can be suppressed by the helminths to reduce covid-19 severity.(31) most parasites release products with enzymatic activities to modify both parasite and host molecules, leading to either the degradation of antiparasitic molecules or the inactivation of the innate immune system. this usually results in the production of alternative cytokines (il-4, il-5, and il-13) instead of the classical proinflammatory cytokines (tnfz#, il-1β, ifnγ) of the activated th1 response. therefore, the new cytokines trigger the mobilization of a whole array of different cells (th2 lymphocytes, imunoglobulin e (ige) producing b cells, mast c e l l s , b as op hi l s , e os i no p hil s , a n d m 2 mac r oph age s) as we l l a s mole c ul e s t ha t altogether result in a more lively environment for the parasite and manageable damage to the host.(18) thus, chronic infection with parasites is believed to provide some kind of protection against covid-19 for immunocompetent hosts living in endemic areas. such protection can prevent individuals from suffering the lethal complications of this viral disease. hermansyah, agustina, risqiyani, et al parasite immunomodulatory and covid-19 univ med vol. 41 no 1 97 conclusions low-income developing countries that are endemic for parasitic infections might have a smaller number of covid-19 cases than do nonendemic countries. this sheds some light on the possibility that the endemicity of parasitic infections such as malaria, schistosomiasis, or soiltransmitted helminthiasis can reduc e the prevalence of covid-19 cases. in endemic countries, the immunomodulatory effect of the parasitic infection in immunocompetent individuals is postulated to possibly reduce the risk of covid-19 or the severity of the symptoms. conflict of interest no conflicts of interest have been declared. acknowledgement we would like to extend our utmost gratitude to yunita armiyanti and yehuda tri nugroho supranoto for their feedback and comments on the original draft of the manuscript. author contribution bh and da contributed to the design of this study. da, ah, and szr contributed to data collection and analysis. bh, da, ah, and szr contributed to writing the 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response in covid19: a review. j infect public health 2020;13:1619– 29. doi: 10.1016/j.jiph.2020.07.001. 49. mueller sn, rouse bt. immune responses to viruses. clin immunol 2008;421. doi: 10.1016/ b978-0-323-04404-2.10027-2. 50. wait lf, dobson ap, graham al. do parasite infections interfere with immunisation? a review and meta-analysis. vaccine 2020;38:5582–90. doi: 10.1016/j.vaccine.2020.06.064. 51. cruz aa, cooper pj, figueiredo ca, et al. global issues in allergy and immunology: parasitic infections and allergy. j allergy clin immunol 2017;140:1217–28. doi: 10.1016/j.jaci.2017.09. 005. january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 emotional eating in relation to gastrointestinal symptoms and burnout among young women during the pandemic gulay yilmazel1* , emre keles2 , and nur pinar ayaz2 abstract background the emergence of the coronavirus disease (covid-19), created unique constraints in everyday life. emotional eating is a known phenomenon in disasters and is markedly associated with gastrointestinal symptoms. in this study, the aim was to assess the relationship of gastrointestinal symptom severity and covid-19 burnout with emotional eating among young women during the pandemic disaster. methods a cross-sectional study approach was used to allow 462 young women participants in this study. the design of the questionnaires was based on demographics, health behaviors, gastrointestinal symptom severity scale, emotional eating scale and covid-19 burnout scale. data were analyzed using percentages, mean values, independent t-test, chisquared test. the hierarchical multiple regression analysis was performed for predicting risk factors of emotional eating. significance levels were set at the 5% level. results of the women, 73.8% were emotional eaters. the level of covid-19 burnout was moderate with mean score of 29.4±11.1 and emotional eating total score was 21.0±8.1. increased number of meals, increased weight gain and shorter sleep time were significantly associated with emotional eating (p<0.05). participants with more than three meals per day were more likely to be emotional eaters (beta=4.26). the regression model showed that indigestion and covid-19 burnout were strong risk factors of emotional eating (p<0.05). conclusions this study demonstrated that indigestion and covid-19 burnout were strong risk factors of emotional eating. emotional eating could pose an additional health burden to young women in the form of poor food choices. keywords: gastrointestinal symptom, emotional, eating, covid-19, burnout, women 1hitit university, department of public health, faculty of health science, çorum/turkey 2hitit university, department nursing, faculty of health science, çorum/turkey *correspondence: gulay yilmazel assoc. prof. dr. hitit university, faculty of health science, çorum/turkey e-mail: dr.gyilmazel@gmail.com orcid id: 0000-0002-2487-5464 date of first submission, november 19, 2022 date of final revised submission, february 23, 2023 date of acceptance, march 2, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 22 doi: http://dx.doi.org/10.18051/univmed.2023.v42:21-28 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1390 cite this article as: yilmazel g, keles e, ayaz np. emotional eating in rela tion to gastrointestinal symptoms and burnout among young women during the pandemic. univ med 2023;42:21-8. doi: 10.18051/univmed.2023 .v42: 21-28 mailto:dr.gyilmazel@gmail.com https://orcid.org/0000-0002-2487-5464 https://orcid.org/0000-0003-4511-1508 https://orcid.org/0000-0001-7925-1678 22 yilmazel, keles, ayaz emotional eating in covid-19 pandemic introduction emotional eating (ee) is an eating habit that occurs in response t o a se t of emotions characterized by increased food intake or overeating, also often associated with poor diet and obesity.(1-4) it has been shown that impaired eating behavior in the general population can be triggered by feelings of stress experienced after a disaster.(5) the most devastating disaster that humanity has faced at the end of 2019 has been the new coronavirus disease (covid-19).(6) social interactions and daily routines have been limited by government policies to prevent transmission during the pandemic that has exceeded two years.(7,8) on the other hand, increased deaths, the emergence of new variants, fear of being infected, isolation practices, stress, disrupted lives, and negative effects from economic disruption are likely to increase emotional eating.(9) indeed, current research on eating habits during the covid-19 pandemic reveals a shift in self-reported eating habits toward increased appetite and an increase in overall food consumption and between-meal snacking.(10-12) dietary changes caused by emotional eating are often associated with increased consumption of energy-dense foods.(13) these eating habits put extra strain on the digestive system.(14) the gastrointestinal tract and nervous system are closely linked through bidirectional signaling mechanisms characterized by neural, endocrine and immune pathways in a context known as the “gut-brain axis”.(15) specifically, the central stress circuit is the neural network that receives input from the somatic and visceral afferent systems, as well as the visceral motor cortex, and generates the stress response.(16) the changes caused by covid-19 in daily life have the potential to increase levels of stress, anxiety, burnout, fear and frustration.(17,18) all these conditions can increase gastrointestinal motility and visceral sensitivity.(19,20) in general, it is stated that gastrointestinal symptoms (65.6%)(21) and emotional eating (41.4%)(22 ) a re common among women in populations. it is reported that during the covid19 period, the prevalence of emotional eating varies between 52.8%-62.0% in women.(23,24) this high prevalence in women suggests covid19 burnout. burnout is defined as a person’s response to persistent, unresolved stress over a long period of time. it is not a medical condition; but is characterized by physical, emotional or mental exhaustion.(25) the pandemic process can be a source of heavy stress, especially for women, and can trigger emotional eating and accompanying gastric symptoms. a study regarding the most critical phase of the first italian lockdown shows a substantial prevalence of emotional eating in the population, in particular among those that reported feeling depressed or anxious.(26) similarly, a chinese study on emotional eating and gestational weight gain amid the pandemic found that women who worried about covid-19 had higher emotional eating scores.(27) to da te, no publi shed studies have investigated the impact of the covid-19 pandemic on ee, gastrointestinal symptoms, and burnout, especially among women of child-bearing age. compared with men, women are more likely to develop abnormal eating patterns as well as mental health disorders, therefore, women have a higher risk of developing health problems related to eating disorders that may be precipitated or exacerbated by this pandemic.(28) however, in turkey, which is under the obesity epidemic, 29.2% of women aged 19 years and over are overweight and 42% suffer from obesity.(29) the combination of these two factors may aggravate the obesity epidemic in young women and trigger the development of chronic health problems in the future. the aim of this study was to determine the level of emotional eating, gastrointestinal symptom severity and covid19 burnout among young women during the pandemic disaster. 23 methods research design this cross-sectional study was an analysis of outpatients of the family medicine clinic of a university hospital in çorum city, turkey, during may-june 2021. research subjects the size of the female population registered at the family health center was 1170, including 630 women aged 20-49 years. the inclusion criteria were being 20-49 years of age, being women and willing to participate in the study (521 women). patients were excluded if they had a diagnosed covid-19 infection, gastrointestinal disorder or psychiatric disorder. in total, 483 individuals agreed to participate in the study, and among these, 462 completed the questionnaire items relevant to this study. therefore the final sample consisted of 462 women. measurement tools the study data were collected using 4 questionnaires, namely the patient identification form and forms for the gastrointestinal symptom rating scale (gsrs), emotional eating scale (ees) and covid-19 burnout scale (covid19-bs). the design of the patient identification form was based on age, education level, marital status, employment status and some health behaviors. for the purpose of defining their smoking s ta t us , wo men we r e a ske d wh et he r t he y smoked. the answers were as follows: i) i still smoke, ii) i don’t smoke and iii) i quit smoking. in order to determine daily water consumption, the question of “how many glasses of water do you drink per day?” was asked. responses were open-ended and grouped as less than eight glasses and eight glasses and more. t he questions about the number of main meals were also open-ended. when participants’ sleep hygiene was questioned, the responses were grouped as i) <7 hours, ii)7-9 hours, iii)e”9 hours, in line with the recommendations of the american sleep society.(30) in order to determine their weight gain, the participants were asked how much weight they gained during the pandemic, and the answers were i) none, ii) 1-4 kg, and iii) >5 kg. body mass index was calculated by recording the height and weight of the participants according to their own declarations. gas troi ntes tinal sy mpto m r ating sca le (gsrs) the scale was developed to evaluate the symptoms widely reported in gastrointestinal system disorders. the reliability and validity of the gsrs were established by revicki et al. and the turkish version was evaluated by turan et al.(31) the gsrs assesses how the individual felt within the pr evious week with regard to gastrointestinal symptom clusters. the scale consists of 15 items divided into 5 subdimensions: reflux (2 items), indigestion (4 items), diarrhea (3 items), constipation (3 items), and abdominal pain (3 items). a higher score indicates more severe symptoms. turan et al. determined a cronbach alpha value of 0.82.(31) in the present study, the cronbach alpha value of the gsrs was found to be 0.89. emotional eating scale (ees) the ees, which was developed to evaluate the emotional eating behaviors of obese and overweight people,(32) consists of 10 items and three sub-dimensions (disinhibition, food types, guilt). the questions in the scale are answered on a likert-type scale with 4 options (“0” never, “1” sometimes, “2” usually and “3” always). there is no reversed item in the scale. the lowest “0” and the highest “30” points are taken from the scale. high scores indicate a high level of emotional eating behavior. scores were categorized into “non-emotional eater (0-5 points)”, “low emotional eater (6-10 points)”, “emotional eater (11-20 points)” and “very emotional eater (21-30 points)”.(33) univ med vol. 42 no. 1 24 yilmazel, keles, ayaz emotional eating in covid-19 pandemic covid-19 burnout scale (covid-19-bs) the covid-19-bs consists of ten items, adapted from the burnout measure-short version, being a modified scale developed in turkey. a sample question is “when you think about covid-19 overall, how often do you feel hopeless?” participants were asked to respond using a 5-point likert scale ranging from 1 (never) to 5 (always), the total scores ranging from 10 to 50. higher scores indicate higher levels of burnout associated with covid-19.(34) data analysis all analyses were carried out using spss, version 20. skewness and kurtosis values indicated a normal distribution. the data were described and analyzed using percentages, mean values, independent t-test, chi-squared test. the hierarchical multiple regression analysis was performed for predicting preventive behavior. the unstandardized regression coefficients (β) and 95% confidence intervals (cis) were used to quantify the associations between variables. significance levels were set at the 5% level. ethics statement ethics committee approval of this study, which was planned in accordance with the helsinki principles, was obtained from hitit university (2021/143/25). results demographic data in the research group, 51.9% were in the 20-39-year age group, 53.5% were university graduates and 50.6% were married. when their health behaviors were examined, 27.9% were current smokers. daily water consumption was less than eight glasses in 52.6% of the participants. the proportion of three-main-meal consumers was 17.8%. night sleep duration was between 7-9 hours in 52.6% of the respondents, 58% gained weight (5 kg and over) during the pandemic, and 40.7% were overweight/obese. the distributions of demographic characteristics, gsrs, and mental distress are given in table 1. the mean total gsrs score of the patients was 46.5 ± 22.9, indicating a moderate level of symptom severity. the highest mean score was found in the indigestion (12.7 ± 6.1) subdimension, and the lowest score was in the reflux (6.2 ± 4.3) sub-dimension. the level of covid19 burnout was moderate, with mean score of 29.4 ± 11.1, while emotional eating total score was 21.0 ± 8.1. multiple linear regression analysis to predict emotional eating according to socio-demographics and health behaviors is given in table 2. number of meals, weight gain and sleep time were significantly associated with emotional eating (p<0.05). participants with more than three meals per day were more likely to be emotional eaters (beta= 4.26). in table 3 are shown the results of multiple linear regression analysis to predict emotional eating according to gsrs and mental distress. the regression model showed that indigestion and covid-19 burnout were strong predictors of emotinal eating (p<0.05). charactertics n (%) age (years) education ≤ high school university married current smokers sleeping duration gained weight (≥5 kg) gsrs total reflux abdominal pain diarrhea indigestion constipation covid-19-bs emotional eating (emotional eaters) 33.00 ± 9.9 206 (44.5) 256 (53.5) 234 (50.6) 129 (27.9) 7.0 ±1.6 268 (58.0) 46.5 ± 22.9 6.2 ± 4.3 8.2 ± 5.1 7.2 ± 5.5 12.7 ± 6.1 10.1 ± 6.1 29.4 ± 11.1 21.0 ± 8.1 341 (73.8) table 1. demographic characteristics, gsrs, and mental distress in the research subjects note: data presented as mean ± sd and n (%). gsrs = gastrointestinal symptom rating scale 25 discussion unlike natural disasters, the covid-19 pandemic is a disaster that can have advanced layers, which are surrounded by increased mental problems, stress, burnout and worsening nutrition, and which may reveal the invisible part of the iceberg medical and social problems in the future. a strong relationship between emotional eating and disaster has been reported in the literature.(5) the present study was designed to determine the effect of the pandemic on emotinal eating, gastrointestinal symptoms and burnout among young women. in our study, it was found that emotional eating was a common problem among women during the pandemic, with three out of four women being emotional eaters (table 1). this also accords with earlier observations, which showed that emotional eating was prevalent among women in the pandemic period.(24,35). the results of our study indicated that gastrointestinal symptoms had moderate severity among young women during the pandemic, with especially indigestion and constipation being common symptoms (table 1). on the question of covid-19 burnout, this study found that burnout was common among young women (table 1). gastrointestinal problems are widespread and the global prevalence of gastrointestinal disease is about 11.2%.(36) although gastrointestinal complaints are not life-threatening conditions, if they are severe, they are an important public health problem that reduces the quality of life and interferes with daily activities. however, the severity of gastrointestinal symptoms in young women during the pandemic is still unknown and has not previously been described. prior studies that have noted the importance of burnout for healthcare workers,(37,38) report that there is no single reason for covid-19 burnout among young women but that it can be attributed to multiple reasons such as the interaction between social structure, traditional gender roles and working conditions. in our study, a weight gain of >5 kg, number of daily meals of more than three, and sleep time of less than seven hours were the main risk factors of emotional eating based on the simple and multiple regression analysis (table 2). it is clear from the covid-19 literature that the pandemic has altered the behavior of many people. increased number of daily meals, increased food intake, snacking, and homemade meals were verified in a number of studies.(11,39,40) changes in sleeping habits and physical activity reduction were also observed.(12) a study from the usa reported that weight gain was more common among women duri ng the covid-19 pandemic.(41) on the other hand, a chinese study variables β (regression coefficient) beta p value weight gain ≥5 kg -0.857 0.425 0.007 sleep time >7 h/d -0.546 0.579 0.012 number of daily meals >3 1.450 4.263 0.009 table 2. multiple linear regression analysis to predict emotional eating according to socio-demographics and health behaviors *age, education, marital and working status, smoking status, daily water consumption were included the simple linear regression and variables with p<0.25, were included in the model of multivariate regression analysis variables β (regression coefficient) beta p value indigestion -1.677 1.445 0.024 covid-19 bs 1.421 4.137 0.017 table 3. multiple linear regression analyses to predict emotional eating according to gsrs and mental distress note: gsrs = gastrointestinal symptom rating scale; *gsrs sub-dimension and covid-19 bs included in simple linear regression and variables with p < 0.25 were included in the model of multivariate regression analysis univ med vol. 42 no 1 26 yilmazel, keles, ayaz emotional eating in covid-19 pandemic found that emotional eating is associated with excess weight gain among pregnant women.(27) insufficient sleep (less than <7 hours per day), presumed to be one of the unconventional key determinants of excess body weight, interferes with emotion regulation through a range of neurobiological, behavioral, and cognitive processes.(42) short sleep may also be associated with irregular eating behavior and can cause stress and high food consumption in emotional eaters.(43) in this study, the most important clinically releva nt finding was that the severity of gastrointestinal symptoms was higher among women and especially the severity of indigestion was clearly seen. also indigestion and covid19 burnout were more prominent in emotional eaters (tables 1 and 3). digestive function is closely related to the daily diet. it is well known that emotional eating behavior can lead to binge eating or eating disorders.(38,44) however, studies have focused on dietary intake and gastrointestinal reflux disease.(45,46) emotional eating has been shown to be positively associated with laryngopharyngeal symptoms.(14) although the relationship between burnout and eating behaviors has been shown in different studies,(47,48) no study has been found reporting that the burnout caused by the pandemic disaster disrupts emotional eating and accordingly gastrointestinal motility. the scope of this study was limited to young women admitted to a hospital. these findings raise intriguing questions regarding the nature and extent of emotional eating in a pandemic disaster. further studies, which take these variables into account, will need to be undertaken. future research is needed among the understudied to examine the long-term implications of the covid-19 pandemic on emotional eating and disordered eating behaviors. conclusions this study has identified a higher prevalence of emotional eating among young women with moderate severity of gastrointestinal symptoms and covid-19 burnout. overall, this study strengthens the idea that the pandemic disaster increases the pressure on the gastrointestinal system by disrupting the eating behavior. acknowledgements thanks are due to all persons who participated and/or assisted in this study. conflict of interest the authors do not have any conflict of interest. author contribution gy contributed to planning of research, data analysis, writing, and english editing. ek contributed to data collection; npa contributed to literatüre review. all authors have read and approved the final manuscript references 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2012;95:934-43. doi: 10.3945/ ajcn.111.014191. oktavianus 20 *department of medical biology, faculty of medicine, lambung mangkurat university *department of environmental health pskm faculty of medicine, lambung mangkurat university *department of pharmacology and therapeutics, faculty of medicine, lambung mangkurat university correspondence *department of medical biology, faculty of medicine, lambung mangkurat university jl. a yani km.2 no.43 banjarmasin south kalimantan e-mail: roselina_darma@yahoo.co.id univ med 2012;31:20-6 abstract universa medicina january-april ,2012january-april ,2012january-april ,2012january-april ,2012january-april ,2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background dengue hemorrhagic fever (dhf) has long been a serious health problem in indonesia, including kalimantan (borneo), as is evident from the increased case fatality rate in banjarbaru city. synthetic chemical insecticides have frequently been used to eradicate mosquitoes, but are toxic to the body and resistance of adult and larvae mosquito aedes aegypti has been reported. the present study aims to assess the effect of essential oils of turmeric rhizomes (curcuma domestica val) against aedes aegypti larvae methods this was an experimental study of post test one group design, performed in two phases, using aedes aegypti larvae as test organisms. in the first phase, laboratory-reared larvae were used for calculation of the lc50 and lc90, while in the second phase the test organisms were larvae taken from 75 buildings that had been designated based on a preliminary survey in four sub-districts in banjarbaru city with a high incidence of dengue cases. probit analysis of was used to calculate lc50 and lc90, and the kruskal-wallis test to determine the larvicidal potency of turmeric rhizome essential oils. results this study demonstrates that turmeric rhizome essential oils effectively killed laboratory-reared aedes aegypti larvae at an lc50 of 9.239 ppm and an lc90 of 13.565 ppm. the effectiveness of the essential oils of turmeric rhizomes (curcuma domestica val.) for killing aedes aegypti larvae in residential areas was 68%. conclusion turmeric (curcuma domestica val.) rhizome essential oils can kill aedes aegypti larvae, are environment friendly and can be used for the control of mosquitoes. keywords: turmeric rhizome essential oils, larvicidal, aedes aegypti larvae potential of turmeric rhizome essential oils against aedes aegypti larvae roselina panghiyangani*, leni marlinae**, isnaini*** and fauzi rahman** 21 univ med vol. 31 no.1 potensi larvisida minyak atsiri rimpang kunyit terhadap larva aedes aegypti latar belakang demam berdarah dengue (dbd) menjadi salah satu masalah kesehatan di indonesia, termasuk kalimantan, seperti terlihat dari case fatality rate di kota banjarbaru yang semakin meningkat. insektisida kimia sintetis sering digunakan untuk pemberantasan nyamuk, namun bahan kimia ini bersifat toksik bagi tubuh. beberapa laporan menyatakan terjadinya resistensi nyamuk dewasa dan larva aedes aegypti terhadap insektisida tersebut. penelitian ini bertujuan untuk menilai pengaruh minyak atsiri rimpang kunyit (curcuma domestica val.) terhadap larva aedes aegypti. metode rancangan penelitian yang digunakan adalah eksperimental dengan pendekatan post test one group design. obyek penelitian adalah larva aedes aegypti. tahap pertama larva yang digunakan dari hasil kolonisasi di laboratorium untuk menghitung lc50 dan lc90 dan tahap kedua larva diambil dari 75 rumah yang sudah ditetapkan berdasarkan survey pendahuluan di empat kecamatan di wilayah kota banjarbaru yang mengalami kasus dbd tinggi. analisis data yang digunakan adalah uji probit untuk menghitung lc50 dan lc90, dan uji kruskal-wallis untuk menguji daya larvisida minyak atsiri rimpang kunyit. hasil studi ini menunjukkan bahwa minyak atsiri efektif membunuh larva aedes aegypti yang ditangkar di laboratoriun, dengan lc50 sebesar 9,239 ppm dan lc90 sebesar 13,565 ppm. efektivitas minyak atsiri rimpang kunyit (curcuma domestica val.) untuk membunuh larva aedes aegypti di lingkungan perumahan adalah 68%. kesimpulan minyak atsiri rimpang kuyit (curcuma domestica val.) mampu membunuh larva aedes aegypti, ramah terhadap lingkungan sehingga dapat digunakan untuk pengendalian nyamuk. kata kunci : minyak atsiri rimpang kunyit, larvisida, larva aedes aegypti abstrak introduction dengue hemorrhagic fever (dhf) is a disease caused by the dengue virus and transmitted by aedes aegypti and aedes albopictus mosquitoes. in indonesia dhf has been a public health problem for the last 41 years. the number of provinces and districts e n d e m i c f o r d h f h a s i n c r e a s e d f r o m 2 provinces dan 2 cities in 1968 to 32 provinces (97%) and 382 districts (77%) in 2009. in 2007, 2008 and 2009 the number of dhf cases occurring was 158 115, 137.468 and 158.912, respectively.(1) the incidence rates in south kalimantan was 35.59/100,000 inhabitants in 2007, 14.44/100.000 inhabitants in 2008, and 11.26/100,000 in 2009 (january-september). in banjarbaru city the incidence rate in the years 2007–2009 was 45.10/100,000, 34.30/100,000, and 52.09/100,000, respectively, while in 2010 (from january until sptember) the incidence rate was 113.9/100,000 inhabitants. the case fatality rate (cfr) of dhf in banjarbaru city was as follows: 1.9% (2006), 1.8% (2007), 1.9% (2008), 5.11% (2009), and 2% (until september 2010).(2-3) 22 at present there are no drugs and vaccines available for dengue virus eradication in connection with dhf prevention. the disease m a y b e m o s t a p p r o p r i a t e l y m a n a g e d b y eradicating the mosquito vectors. eradication of aedes aegypti may be conducted by killing mosquito larvae by means of larvicides. the most widely used larvicide for control of aedes aegypti larvae is temefos 1% (abate 1sg).(4-6) synthetic insecticides are used by the community because they are practical in use and rapid in action. however, the use of synthetic insecticides has not led to a reduction in dhf rates. on the contrary, there are reports from many countr ies about the occur r ence of insecticide resistance, environmental pollution, and contamination of humans and animals. most of the available synthetic insecticides kill only adult mosquitoes, and only a few kill mosquito larvae.(7-9) previous studies have demonstrated that several plants, such as turmeric (curcuma domestica val.), are a potential alternative source of bioactive phytochemicals for killing aedes a e g y p t i larvae . ( 1 0 1 2 ) t h e s e p l a n t d e r i v e d insecticides may be expected to succeed in replacing conventional insecticides, as they are believed to be target-selective or target-specific. in contrast to synthetic insecticides, these phytochemicals are degradable to nontoxic compounds, thus minimizing the harmful effects on humans and animals, and contributing to a higher degree of environmental safety by minimizing the accumulation of harmful residues in the environment for this reason, they are also potentially suitable for use in the continuation of integrated mosquito control programs.(13-15) b a s e d o n t h e a b o v e m e n t i o n e d considerations, the study and development of alternative larvicides that are environmentfriendly is clearly indicated to decrease the use of synthetic insecticides. these plant-derived larvicides are expected to result in a reduction o f t h e n u m b e r o f d h f c a s e s i n s o u t h kalimantan, especially in banjarbaru city. the alternative candidate larvicides used in the present study are derived from indigenous indonesian plants, such as turmeric, which is readily available, inexpensive, and highly effective. in 2009, panghiyangani et al.(11) concluded that 0.4% ethanolic extract of turmeric rhizome (curcuma domestica val.) was an effective larvicide against aedes aegypti in residential areas of banjarbaru. other studies demonstrated that the essential oils contained in a number of medicinal plant parts, such as the leaves of pandanus spp., sirih (piper betle linn), lemon grass (andropogen nardus), eucalyptus cinerea, and the rhizomes of white turmeric (curcuma zedoaria), were capable of killing aedes aegypti larvae.(8,16,17) the aim of the present study was to evaluate the effects of turmeric rhizome essential oils (curcuma domestica val) on aedes aegypti larvae. methods design of study the study was of experimental design using a post test control group approach and was conducted from may to november 2010. extraction of turmeric rhizome essential oils a p p r o x i m a t e l y 1 0 k g o f t u r m e r i c (curcuma domestica val.) rhizomes was cut into small pieces and placed in the steam distillation apparatus containing 10 liter water, and connected to a condensor. the steam distillation apparatus was then heated, with steps being taken to avoid overheating and to maintain the flow of steam to the condensor. the condensor was kept cool by external packing with ice, to ensure condensation of all essential oils in the water phase. the oil-and-water mixture in the distillate was subsequently separated by means of a separatory funnel. for a complete separation, sodium chloride was added to the distillate. the water phase was collected in an erlenmeyer flask for further separation of remaining traces of oils, and after addition of sodium chloride the water was decanted, then separated in the separatory funnel.(18) panghiyangani, marlinae, isnaini, et al turmeric rhizome against aedes 23 univ med vol. 31 no.1 100 x mortality control % 100 mortality control % -mortality on interventi % ai = larvicide bioassay the study was performed in 2 stages, with the first stage performed in the laboratory using laboratory-reared larvae for determination of lc 50 a n d l c 90, t o s e r v e a s t h e b a s i s f o r calculating the treatment dosage be used in the field. for a mortality of aedes aegypti larvae between 0% and 20%, the number of deaths in the intervention groups was corrected using abbot’s formula:(19,20) statistical analysis probit analysis was performed using spss 17 to calculate lc50 and lc90 values and the kruskal-wallis test for determining larvicidal potency of turmeric rhizome essential oils against aedes aegypti larvae, at a significance level of 5%. results probit analysis found an lc50 value of 9.239 ppm and an lc90 of 13.565 ppm, as shown in table 1. larval mortality in the group given turmeric rhizome essential oils was 65.22%, after abbot’s correction. the results of the larvicidal assay of turmeric rhizome essential oils against aedes aegypti are shown in table 2. the results of the kruskal-wallis test showed a significant difference in larval mortality between the three treatment groups (p=0.021). larval mortality in the group given essential oils was 22% (17/ 75), which was significantly lower than the larval mortality of 33% (25/75) in the group given abate 1%. the effectiveness of turmeric (curcuma domestica val.) rhizome essential oils as larvicide, in comparison with the positive control group (temefos 1%) was 0.68. discussion the first phase of this study found lc50 and lc90 values of 9.239 ppm and 13.565 ppm, respectively, for turmeric (curcuma domestica val.) rhizome essential oils as larvicide (table 1). the lc50 in our study was lower than the 54.5 ppm for the lc50 of white turmeric essential oils.(21) a thai study also found the higher lc50 value of 36.30 ppm for curcuma aromatica essential oils.(15) thus our differing study results show that turmeric essential oils have a lower larvicidal effect than that of curcuma aromatica. the results of kruskall-wallis test showed that turmeric essential oils had the capacity to kill aedes aegypti larvae, although their potency 100 x larvae control ofmortality mean larvaon interventi ofmortality mean esseffectiven = ai: mean corrected persentage of deaths of test larvae the control groups were 2 in number, i.e. positive controls (abate 1%) and negative controls sodium carboxymethylcellulose [cmc-na] 0.5%. there were seven intervention groups, each consisting of 25 larvae, to whom t h e e s s e n t i a l o i l s w e r e a d m i n i s t e r e d a t concentrations of 3.45, 6.9, 13.79, 27.5, 55, 110, and 220 ppm, respectively. the treatment was replicated three times and the larvae were observed for 24 hours. the second phase of the larvicidal potency test used aedes aegypti larvae collected from 75 buildings selected in preliminary surveys, and consisting of 3 groups of 25 larvae, i.e. positive controls, negatiuve controls, and the intervention group receiving turmeric essential oils at a concentration of 13.565 ppm, based on the lc90 obtained in the first stage of this study, on the expectation of 100% larval mortality. these larvae were also observed for 24 hours. the effectiveness of turmeric rhizome (curcuma domestica val.) essential oils in comparison to the positive control groups (temefos 1%) was calculated with the following formula: 24 probability 95% confidence limits esti mate lower bound upper bound probit(a) .010 1.384 -1.0 10 2.943 .020 2.305 .17 0 3.719 .030 2.889 .91 5 4.215 .040 3.328 1.472 4.592 .050 3.685 1.922 4.901 .060 3.989 2.304 5.165 .070 4.256 2.637 5.399 .080 4.495 2.934 5.610 .090 4.712 3.202 5.803 .100 4.912 3.448 5.981 .150 5.739 4.453 6.736 .200 6.397 5.230 7.356 .250 6.961 5.880 7.905 .300 7.468 6.447 8.415 .350 7.938 6.957 8.903 .400 8.383 7.428 9.379 .450 8.814 7.870 9.853 .500 9.239 8.293 1 0.331 .550 9.663 8.706 1 0.820 .600 10.094 9.116 1 1.326 .650 10.539 9.531 1 1.859 .700 11.009 9.959 1 2.428 .750 11.516 10.4 13 1 3.051 .800 12.080 10.9 10 1 3.753 .850 12.738 11.4 81 1 4.580 .900 13.565 12.1 89 1 5.631 .910 13.765 12.3 58 1 5.886 .920 13.982 12.5 42 1 6.164 .930 14.221 12.7 44 1 6.470 .940 14.488 12.9 68 1 6.812 .950 14.792 13.2 23 1 7.204 .960 15.149 13.5 22 1 7.664 .970 15.588 13.8 88 1 8.232 .980 16.172 14.3 73 1 8.988 .990 17.093 15.1 33 2 0.183 table 1. distribution of larvacidal activity of turmeric rhizome essential oils against aedes aegypti panghiyangani, marlinae, isnaini, et al turmeric rhizome against aedes table 2. mortality percentage of aedes aegypti in the treatment groups 25 univ med vol. 31 no.1 was less than that of temefos 1% (abate), as not all larvae were killed. this may be due to the use of a concentration killing 90% of the laboratory-reared larvae (lc90) within 24 hours, whereas the test was done in residential areas, the natural habitat of aedes aegypti. there was a difference in susceptibility between laboratory-reared larvae and wild-type larvae from residential areas with regard to application of turmeric rhizome essential oils. the effectiveness of turmeric ( curcuma domestica val.) rhizome essential oils in killing aedes aegypti larvae from residential areas was two-thirds that of the positive controls. on the basis of the laboratory tests, the results of the effectiveness test should have been more than 68%, because the concentration used was the lc90 (13.565 ppm). there is thus a difference of 22% between laboratory and field test results. since essential oils are volatile compounds, and the field tests were performed in the afternoon hours without practically any control over the temperature of the respective buildings, it may be surmised that a substantial amount of the essential oils was lost through evaporation when pouring the oils into the test containers. this is in contrast with the laboratory tests, which were performed at controlled lower temperatures, thus minimizing loss of oils through evaporation. the rhizomes of turmeric ( curcuma domestica val.) contain the following active s u b s t a n c e s : c u r c u m i n s , s e s q u i t e r p e n e s , turmerones, volatile oils (essential oils), and z i n g i b e r e n s , t u r m e r o l s , p h e l l a n d r e n e s , camphors, curcumons, and various resins with antibacterial properties.(22) according to the studies of heyne,(23) derivatives of oxygenated h y d r o c a r b o n s ( p h e n o l s ) h a v e s t r o n g antibacterial properties. phenolic compounds mainly act by denaturation of cellular proteins and damage to cell membranes. the phenolic content of a substance may result in lysis of larval cells, due to increased permeability of cell membranes, leading to leakage of essential metabolites, while in the cells the phenols disrupt cellular activity. phenolic compounds act as dessicants, and are contact poisons that kill by inducing a continuous leakage of fluids, causing the larvae that come in contact with these poisons to die from dehydration. a contact poison is a larvicide that enters the larvae through the integument and natural orifices (siphons). the larvae die on direct contact with the larvicide. most contact poisons also act as stomach poisons.(24) one limitation in this study was that the stability of turmeric rhizome essential oil preparation could not be maintained over time, due to the volatility of the oils. conclusion turmeric rhizome essential oils (curcuma domestica val.) can kill aedes aegypti larvae from residential areas in several subdistricts in banjarbaru city. we recommend that the larvicidal effect of turmeric rhizome essential oils on aedes aegypti larvae be utilized in dhf vector control. acknowledgements we wish to express our gratitude to direktorat penelitian dan pengabdian kepada masyarakat direktorat jenderal pendidikan tinggi kementerian pendidikan nasional for financial support of this study through a stranas research grant for the fiscal year 2010/2011, and the head and research staff of the entomology laboratory loka penelitian dan pengembangan pemberantasan penyakit bersumber binatang-depkes ri, tanah bumbu, south kalimantan for the use of their laboratory facilities for this study. references 1. sitohang v, brahim r, hasnawati, anggraeni nd, ismandari f. demam berdarah dengue di indonesia tahun 1968-2009. buletin jendela epidemiologi 2010;2:1-14. 2. sukamto. data kasus demam berdarah dengue (dbd) per kabupaten/kota propinsi 26 kalimantan selatan. banjarmasin: dinas kesehatan provinsi kalimantan selatan;2009. 3. arifin z. rekapitulasi jumlah kasus dan kematian demam berdarah dengue (dbd) di wilayah kota banjarbaru menurut puskesmas dan kelurahan tahun 2010 (1 januari s.d 30 september 2010). banjarbaru: dinas kesehatan kota banjarbaru; 2010. 4. lestari k. epidemiologi dan pencegahan demam berdarah dengue (dbd) di indonesia. farmaka 2007;5:12-29. 5. gafur a, mahrina, hardiansyah. kerentanan larva aedes aegypti dari banjarmasin utara terhadap temefos. bioscientiae 2006;3:73-82. 6. kusumawati y, suswardany dl, yuniarno s, darnoto s. upaya pemberantasan nyamuk aedes aegypti dengan pengasapan (fogging) dalam rangka mencegah peningkatan kasus demam berdarah. warta 2007;10:1-9. 7. muhlisin a, arum p. penanggulangan demam berdarah dengue (dbd) di kelurahan singopuran kartasura sukoharjo. warta 2006;9: 123-9. 8. cavalca p, mançano a, gomes ml, de assumpcao l, reis b, bonato cm. homeopathic and larvicide effect of eucalyptus cinerea essential oil against aedes aegypti. braz arch bio technol 2010;53:835-43. 9. suwasono h, mardjan s. uji coba beberapa insektisida golongan pyrethroid sintetik terhadap vektor demam berdarah dengue aedes aegypti di wilayah jakarta utara. j ekol kes 2004;3:437. 10. promsiri s , amara n, maleeya k, usavadee t. evaluations of larvicidal activity of medicinal plant extracts to aedes aegypti (diptera: culicidae) and other effects on a non target fish. insect science 2006;13:179-88. 11. panghiyangani r, rahman f, yuliana. peningkatan kemampuan daya larvasida ekstrak rimpang kunyit (domestica val.) terhadap larva aedes aegypti penyebab dengue hemmorrhargic fever (dhf). laporan hasil penelitian hibah strategis nasional batch i. banjarmasin: universitas lambung mangkurat;2009. 12. marlinae l, lisda h, joharman, vera m. effectiveness of extract rhizome turmeric (curcuma domestica val.) in killing aedes aegypti larva cause of dengue hemmorrhargic fever (dhf). j kes ling 2006;3:22-8. 13. jeyabalan d, arul n, thangamathi p. studies on effects of pelargonium citrosa leaf extracts on malarial vector, anopheles stephensi liston. biores technol 2003;89:185-9. 14. prabakar k, jebanesan a. larvicidal efficacy of some cucurbitacious plant leaf extracts against culexquinquefasciatus (say). biores technol 2004;95:113-4. 15. choochote w, chaiyasit d, kanjanapothi d, rattanachanpichai e, jitpakdi a, tuetun b, et al. chemical composition and anti-mosquito potential of rhizome extract and volatile oil derived from curcuma aromatica against aedes aegypti (diptera: culicidae). j vector ecol 2005; 30:302-9. 16. dewi s, rahman a, eram tp. potensi daun pandan wangi untuk membunuh larva nyamuk aedes aegypti. j ekol kes 2003;2:228-31. 17. parwata im, rita ws, yoga r. isolasi dan uji antiradikal bebas minyak atsiri pada daun sirih (piper betle linn) secara spektroskopi ultra violet-tampak. j kimia 2009;3:7-13. 18. juniarti, yuhernita, susi e. destilasi minyak atsiri daun surian sebagai krim pencegah gigitan nyamuk aedes aegypty l. makara sains 2011; 5:38-42. 19. yasmin y, fitri l. the effect of metharrizium anisopliae fungi on mortality of aedes aegypti larvae. j natural 2010;10:31-5. 20. abbott ws. a method for computing the effectiveness of an insecticide. j econ entomol 1925;18:265-7. 21. panghiyangani r, isnaini, dodo ts. aktivitas larvisida minyak atsiri rimpang kunyit putih (curcuma zedoaria) terhadap larva aedes aegypti. maj kedok fk-uki 2010;xxvii:10813. 22. chattopadhyay i, biswas k, bandyopadhyay u, banerjee rk. turmeric and curcumin: biological actions and medicinal applications. curr sci 2004;87:44-53. 23. heyne. tumbuhan berguna di indonesia. jakarta: badan litbang kehutanan;1987. 24. parwata im, dewi pfs. isolasi dan uji aktivitas antibakteri minyak atsiri dari rimpang lengkuas (alpinia galangal l.). jurnal kimia 2008;2:1004. panghiyangani, marlinae, isnaini, et al turmeric rhizome against aedes c:\users\universa medicina\docu 107 abstract universa medicina poor knowledge of anemia and sexually transmitted disease but better knowledge of hiv/aids among unmarried adolescents aged 15-24 years edhyana sahiratmadja1 *, mugia bayu raharja2, elsa pudji setiawati3, and ramdan panigoro1 background basic knowledge of sexual and reproductive health is important to be introduced during the premarital period, to reduce high-risk sexual behavior among adolescents, as well as to increase the correct attitude in their future marriage. this study explored the knowledge of hiv/aids, sexually transmitted diseases (stds) and anemia among unmarried adolescents. methods this was a cross-sectional analytical study on secondary data from the indonesian demographic and health population survey 2017, initially conducted by the national population and family planning board (bkkbn). knowledge of hiv/aids, stds and anemia among unmarried adolescents aged 15-24 years in west java province and jakarta, the capital city of indonesia, was compared and analyzed using the chi-square test. results in total 5,389 weighted data were retrieved. in general, adolescents in jakarta had better knowledge of all diseases compared to those in west java (p<0.001). knowledge of anemia and stds was poor compared to that of hiv/aids which was moderate to good (p<0.001). interestingly, although the adolescents had a middle-low wealth index (43%), the majority (>90%) had their own mobile phone with almost daily internet access, however most of their information sources on health education were from their school period. conclusion premarital education in schools is imperative to enhance the knowledge of stds and anemia. moreover, poor knowledge of anemia is a challenge for introducing the topic of thalassemia major, an inherited autosomal recessive type of anemia due to hemoglobinopathy. since indonesia is harboring 610% of thalassemia carriers, integrative premarital education is needed for better family planning. keywords: iron deficiency anemia, indonesian demographic and health survey, premarital, thalassemia carrier, thalassemia major original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.110-120 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1163 may-august 2021 vol.40no.2 cite this article as: sahiratmadja e, raharja mb, setiawati ep, panigoro r. poor knowledge of anemia and sexually transmitted disease but better knowledge of hiv/aids among unmarried adolescen ts a ged 1 5-24 yea rs. univ m ed 202 1;40 :1 0 7-1 7 . doi: 10 .18 05 1/ univmed.2021.v40.110-120 1department of biomedical sciences, faculty of medicine, universitas padjadjaran, bandung, indonesia 2bkkbn, national population and family planning board – population research and development unit, jakarta, indonesia 3deparment of public health, faculty of medicine, universitas padjadjaran, bandung, indonesia *correspondence: edhyana sahiratmadja department of biomedical sciences, faculty of medicine, universitas padjadjaran jl. raya sumedang km 21 jatinangor, bandung, indonesia e-mail: e.sahiratmadja@unpad.ac.id, phone: +62-816-996309 orcid id : 0000-0001-9092-5848 date of first submission, march 24, 2021 date of final revised submission, july 9, 2021 date of acceptance, july 13, 2021 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license 108 sahiratmadja, raharja, setiawati, et al anemia and sexually transmitted disease introduction sexual and reproductive health account for a high proportion of global public health problems. the basic knowledge of various sexually transmitted infections and hiv/aids as well as of reproductive health is important to b e in tr od uc ed t o ad ole sc en ts dur ing th e premarital period to reduce high-risk sexual behavior.(1) the incidence of aids cases in indonesia has been reported to be highest at the age of 25-49 years.(2) therefore, adolescents and young adults are the age groups with the highest risk of contracting hiv infection possibly due to poor knowledge regarding hiv/ aids. furthermore, hiv/aids is closely related to sexual transmitted infections. good kno wl e d ge o f h iv / a dis a n d se x u a l l y transmitted diseases (stds) may increase good attitude in their future marriage, as well as eliminate the three infections in mother-to-child transmission, consisting of hiv/aids, syphilis and hepatitis b.(3) another disease that adolescents need to know is anemia, that affects about 40% of children under 5 years of age and 30% of nonpregnant women.(4) the risk factors of anemia are multifactorial, including chronic infections a n d i nf l a mma t i on , mic r on u t r i e n t a nd macronutrient deficiencies, with iron deficiency as the major cause.(5) anemia may lead to i mpa i r e d c o gn i t i ve p e r f or man c e , p oo r educational achievement, resulting in reduced work capacity. especially in young women, anemia may have impacts on their future pregnancies, among others poor pregnancy outcomes and increased maternal and perinatal mortality and morbidity. furthermore, anemic pregnant women have a higher risk during the pregnancy, both for the mother and the fetus, leading to intrauterine growth retardation or preterm delivery.(6) moreover, anemia in children is becoming a public health issue that may contribute to stunted growth and an increased susceptibility to infections. although the prevalence of iron deficiency anemia (ida) due to malnutrition in adolescents is high in the developing countries,(7) ida is not the only major cause in adolescents. since indonesia is located in the thalassemia belt area, hemoglobinopathies such as thalassemia, an inherited autosomal recessive anemia, are also prevalent.(8) good awareness of thalassemia carrier status is important in the premarital phase, as this knowledge is needed for better family planning. adolescence is a unique point of intervention as this age group is more receptive to changes in lifestyle that may determine their life course later.(9) a study in berlin showed that students had heard of hiv, and the majority rated their knowledge as (rather) good and knew that hiv could neither be cured nor vaccinated against.(10) an o th e r s t udy s ho wed d i f f e re n t r e sul t s, knowledge about some aspects of hiv and other stds was inadequate among both science and n on -s c i e n ce h igh sc ho ol a nd u ni ve r s i ty students.(11) this study aimed to assess the knowledge o f hiv /a ids, st ds a n d a n emi a a mo ng unmarried adolescents aged 15-24 years residing in west java and jakarta, the capital city of indonesia, as a reference. methods study design this was a cross-sectional study, using secondary data, pa rtly retrieved from the indonesian demographic and health survey (idhs) for 2017 [sur vei d emog rafi dan kesehatan indonesia (sdki) 2017].(12) research subjects t he id hs 2 01 7 s a mpl e w a s a geographically stratified and clustered sample of households, with a number of respondents representing the population of the provinces. the inclusion criterion was all unmarried men and women aged 15-24 yea rs i n the se lec ted clustered area who agreed to participate. the samples were weighted to inflate the sample 109 data, either from a small province or a large province, to be equivalent to the size of the total national population. in brief, two-stage sampling was designed. the sample size of population ce n su s 20 1 0 w a s c a l c ul a t e d b a s e d o n systematic probability proportional to size, resulting in 1,970 census blocks of urban and rural areas. of each census block, 25 families or households were chosen systematically. the current study had retrieved a total of data (n=5,389; weighted) from two provinces, namely from dki jakarta, the capital city of indonesia, where a premarital health examination a n d e d uc a t i o n pr ogr a m h a d b e e n ma de mandatory,(13) and from west java, the largest province in indonesia, harboring the majority of thalassemia major patients.(8) indonesian demographic and health survey 2017 questionnaires there were two sets of questionnaires namely for reproductive women aged 15-49 years (sdki 17-wus), and for men aged 152 4 ye a r s ( sdk i 1 7 -rp) . t h e s t r u c t ur e d q ue s t i on n ai r e s we r e d i st r i b ut e d t o t he respondents by direct interviews by cadres, conducted from july to september 2017. data of unmarried men and women aged 15 to 24 years were retrieved, and questions related to knowledge of hiv/aids, sexually transmitted diseases (stds) and anemia were explored and compared between the two provinces. the wealth index was computed as a composite index using principal components a na lys es of va r iou s va r i abl es , i nc l ud in g ownership of various assets, access to electricity, and other parameters related to source of drinking water and sanitation facilities and categorized as very poor, poor, middle, rich or very rich classes.(12,14) the questionnaire about hiv/aids consisted of ten closed questions (q1002 to q1008c) and knowledge was categorized as good (score 810), moderate (score 5-7) and poor (score d” 4). the questionnaire about stds consisted of three open questions (q1042 b, c, d), and the questionnaire about anemia consisted of five questions of which two were closed questions (q 1313) about whether the respondents had ever heard of anemia and (q1316) whether anemia was treatable; and three open questions about the definition (q1314), etiology (q1315) and prevention or treatment of anemia (q1317). for the open questions, the respondents were allowe d to answer to the extent of their knowledge. for each correct answer the given score was one. knowledge of anemia and stds was categorized as good (score 10), moderate (score 5-9) and poor score 4). statistical analyses sampling weights were applied to all analyses. the analysis consisted of two steps; first, a descriptive analysis to explore the distribution of variables such as “ever heard of” diseases and definition. the distribution was r e p o r t e d a s p e r c e nt a ge s . sec o nd , t he associations between place of residence and knowledge of hiv/aids, stds or anemia were analyzed using the chi-squared test. results were considered significant when p<0.05. ethical clearance ethical clearance for the initial survey had been granted in the institutional review board findings form (icf irb fwa00000845) for icf project no. 132989.0.000 for the year 2015. for using the se condary idhs 2017 data , no separate ethics approval was required, except the permission from the national population and family planning board, indonesia. results characteristic of unmarried adolescents and young adults in jakarta and west java of 5,389 weighted data retrieved, 19.8% of the respondents was from the capital city of jakarta and 80.2% from west java, the latter residing predominantly in urban areas (71.9%). the respondents comprised 55% men and 45% women, with the proportion of adolescents aged univ med vol. 40 no 2 110 sahiratmadja, raharja, setiawati, et al anemia and sexually transmitted disease a b c 15-19 years being slightly higher (54%) (table 1). the wealth index in urban areas of jakarta and west java was upper middle class, whereas in rural areas of west java the index was lower middle class. interestingly, the mobile phone has b e c o me a n e s s e nt i a l t o ol i n th is e r a of digitalization, with more than 90% of the respondents having a mobile phone. moreover, over 90% had internet access and in 80% the frequency of usage in the previous month was almost daily (table 1). know le dge about anemia com pared to stds and hiv/aids the majority of adolescents in jakarta (82%), west java urban area (70%) and rural area (60%) admitted that they had ‘ever’ heard about anemia. adolescents from jakarta had significantly heard more than had those from west java (p=0.000); moreover, adolescents in urban areas of west java had heard more than had those in rural areas (p 0.000) as depicted in figure 1. jakarta west java province total characteristic n (1065) urban rural n (5389) (%) n (3111) n (1213) gender men 567 1677 742 2986 (55.0) women 498 1434 471 2403 (45.0) age (years) mean ± sd 18.9 ± 2.7 18.6 ± 2.6 18.1 ± 2.6 age category (years) 15-18 501 1651 756 2908 (54.0) 19-24 564 1460 457 2481 (46.0) q1401 age at intended marriage (years) men (median/min-max) 25 (18-38) 25(19-35) 25 (20-37) women (median/min-max) 24 (18-30) 24 (17-30) 23 (17-30) education none 0 15 0 15 (0.3) primary 60 155 107 322 (6.0) junior high 127 440 390 c 957 (18.0) senior high 647 a 1971 a 624 a 3242 (60.0) a academy/university 231 b 530 b 92 853 (16.0) wealth index* very poor 26 160 185 d 371 (7.0) poor 85 375 352 d 812 (15.0) middle 146 e 676 e 308 d 1130 (21.0) rich 305 e 837 e 220 1362 (25.0) very rich 503 e 1063 e 148 1714 (32.0) own mobile phone yes 1000 2873 1070 4943 (92.0) no 65 238 143 446 (8.0) internet usage over last month yes 1033 2962 1098 5093 (95.0) -almost everyday 953 2354 777 4084 (80.0) -at least once a week 66 451 231 748 (15.0) -less than once a week 14 157 91 262 (5.0) no 32 149 114 295 (5.0) table 1. characteristics of unmarried adolescents and young adults from the capital city dki jakarta and west java province based on urban and rural area notes: a senior high school was the most predominant educational level; b academy/university was the second highest educational level in urban areas; c junior high school was the second highest educational level in rural areas; d combined wealth index was lower-middle class; e combined wealth index was upper-middle class. 111 similar to anemia, stds and hiv/aids had been significantly more heard of in jakarta than in west java (p=0.000). evenmore, hiv/aids had been significantly more heard in rural area of west java, compared to the topic of anemia and stds (p=0.000) in jakarta. the question on the definition of anemia was explored and this showed that anemia was generally known as a simple blood deficit. of this knowledge the adolescents in jakarta had the highest proportion among the respondents (60%); as opposed to adolescents in west java rural areas who did not know the definition of anemia (58%) (figure 2a). as for the cause of anemia, the most mentioned causes were lack of vegetables and fruit (31% ) as well as la ck of meat/f ish consumption (27%) as depicted in figure 2b. again, two-thirds of the adolescents in west java rural areas did not know the cause of anemia. as to whether anemia could be treated or prevented, taking a pill was predominantly mentioned by adolescents in jakarta (53%) and in west java urban and rural are as by 37% and 28%, respectively. hence, they did not specifically mention iron tablets in particular. taken together, both definition and cause of anemia were scored significantly better by respondents in jakarta compared to urban and rural areas of west java province (p=0.000). figure 1. distribution of unmarried adolescents and young adults in dki jakarta and west java province who had ‘ever heard’ about anemia, sexual transmitted diseases (stds) and hiv/aids the knowledge of anemia was predominantly poor (69%) in jakarta and even poorer in west java rural area (89%) and the difference was significant (p=0.000). similarly, the knowledge of sexually transmitted diseases (stds) was predominantly poor in jakarta (89%) compared to west java rural areas (98%), the proportions being significantly different (p= 0.000). interestingly, the knowledge about hiv/ aids was moderate (32%) to good (59%) in jakarta. the knowledge distribution in dki jakarta was significantly better than in west java (p<0001), signifying that the knowledge in urban areas was better than in rural areas (figure 3). some of the questions about hiv/aids were incorrectly answered, such as that the infection was spread by mosquitos and sharing of food (data not shown). the most frequent clinical symptoms known for genital infection in men were genital discharge and burning pain on urinating; whereas for genital infection in women the known symptoms we re foul smelling discharge, genital discharge and also burning pain during urination. source of information for health education among adolescents and young adults the most frequent health information source was the school. interestingly, education on hiv/ univ med vol. 40 no 2 112 sahiratmadja, raharja, setiawati, et al anemia and sexually transmitted disease figure 2. distribution of (a) definition and (b) etiology of anemia according to unmarried adolescents aged 15-24 years in dki jakarta and west java province b a aids (63%) was significantly (p 0.000) more given compared to stds (19%). for hiv/aids, radio/tv was the second source of information (53%) followed by information on internet (33%), whereas information about stds was more frequently gathered from friends (11%) and the internet (9%) (figure 4). there was no question in sdki 2017asking for the source of information about anemia that limited the analysis in comparing the source of information. discussion this study has explored the knowledge of hiv/aids, sexually transmitted diseases (stds) and anemia among adolescents aged 15-24 years from two provinces in indonesia, namely west java and jakarta, resulting in poor knowledge of anemia and st ds an d moderate to good knowledge of hiv/aids. interestingly, adolescents in jakarta have heard significantly 113 figure 3. knowledge of anemia, sexually transmitted diseases and hiv/aids among unmarried adolescents aged 15-24 years in dki jakarta and west java province figure 4. sources of information about (a) sexual transmitted diseases and (b) hiv/aids univ med vol. 40 no 2 114 sahiratmadja, raharja, setiawati, et al anemia and sexually transmitted disease more about those diseases compared to those residing in west java, and those in urban areas have heard more than those in rural areas. the disparities in reproductive health education across urban and rural areas may lead to inequities in awareness and utilization of health services.(15) interestingly, even though the wealth index in west java rural area is lower-middle class, over 90% of adolescents have a mobile phone and almost daily internet access. it seems that the mobile phone has become an essential tool in the era of digitalization. the use of mobile phones for example on mobile health or mhealth application via text-messaging information, videos and other social media platforms plays a role, highlighting the significant opportunity to impact health behaviors globally, particularly in lowand middle-income countries.(16) hence, the choice to access information from various sources depends on the individual’s interests and curiosity. even though the internet seems to be essential nowadays in passing the information, radio and tv are also useful sources of information. a study in uganda has shown that the frequency of access to information in traditional mass media such as tv, radio, and print media is more successful in enhancing the knowledge about hiv among adolescents.(17) information about hiv/aids has been regularly broadcast via radio/tv as a campaign to prevent hiv/aids. the hiv/aids campaign program in indonesia may have become a success story, causing knowledge about hiv/ aids to be predominantly moderate and good in j akar ta (91.0%) and west j ava (70.0%). however, the optimal source of reproductive health education is still the school period. for example, the major source of information of stds is the school. yet, the knowledge of stds is very poor. the stds may be seen as a sensitive issue in the community. our result has shown that the information about stds has been gathered from friends and internet. adolescents are prone to stds because of the influence and pressure of their friends and the curiosity to experiment with sexual activity. clearly, health promotion for std prevention needs to be carefully well-designed with a good assessment based on social and behavioral as well as cultural determinants given by teachers and medical professionals in the school curricula.(18) interestingly, educational programs in italy have been designed based on information needs and risk perceptions among adolescents and young adults, resulting in an effective intervention to improve knowledge about hiv and stds.(10) therefore, schools have been proven to be a good place for pr oviding information during the period of adolescence. (19) next to knowledge of stds and hiv/ aids, knowledge of anemia plays a significant role among adolescents. the most common cause of anemia in developing countries is iron deficiency anemia. it seems that the young adults in our study had poor knowledge of anemia , although they have ever heard a lot about anemia. blood deficit in general (48.0%), lack of iron-containing vegetables or fruits (22.0%) as well as lack of meat/fish consumption (18.0%) have been mentioned as the cause of anemia. however, since there was no question in the questionna ire asking about the source of information about anemia, nutrition education at school for the students is imperative. an integrated school-based nutrition program with their mothers have resulted in a significant increase in the knowledge and attitude of the mothers in improving the nutritional status of their children.(20) indeed, in the developing countries, iron deficiency anemia is predominant, as a result of inadequate intake of micronutrientrich foods and the inadequate utilization of available micronutrients in the diet due to i nf e c t i on s, a nd o t he r d i e t a ry f a c t o r s . consumption of vitamin-a rich and iron-rich foods as well as micronutrient fortification in cooking salt are recommended for both women and children.(21) the 1000 days of growth of the fetus into babies is essential. an anemic pregnant woman may deliver low birth weight babies, and children with stunted growth,(22) therefore, knowledge of anemia should be introduced at a young age during lessons in reproductive health education. 115 it is worth noting that next to iron deficiency anemia, hemoglobinopathies such as thalassemia minor or trait are another important etiology of anemia, (23) especially in indonesia and other countries located in the thalassemia belt area.(24) interestingly, thalassemia minor or trait is not well known among adolescents, and none of the respondents acknowledge thalassemia as the cause of anemia in their answer. moreover, knowledge of thalassemia is not wide-spread among general practitioners. (25) thalassemia is inherited in an autosomal recessive manner, thus, when thalassemia carrier is married to another carrier, the probability of having a thalassemia major baby in every pregnancy is 25.0%.(26) with the magnitude of possibly around 6-10.0% thalassemia carriers in west java,(8) the topic of thalassemia needs to be added to the premarital course or education. ideally, early detection for thalassemia carriers should be conducted in adolescents, integrated in the premarital program to plan their future partner.(27) this study, however, has some limitations. unlike the questions on knowledge about hiv/ aids which are true/false questions, the questions on knowledge about anemia and stds are more based on what the respondents know in open questions mode. another limitation is that there are no questions in idhs 2017 about the source of information for anemia. a validated and reliable questionnaire about the knowledge of anemia as well as the source of information needs to be further developed. conclusion the knowledge of anemia and sexually transmitted diseas es among unmarried adolescents from jakarta and west java is poor, whereas knowledge of hiv/aids is moderate to good. poor knowledge of anemia is a challenge in introducing thalassemia, an autosomal recessive inherited hemoglobinopathy. since indonesia is harboring 6-10.0% thalassemia carriers, a holistic approach to premarital education programs in schools is beneficial in enhancing the knowl edge of sexua l and reproductive health for a better family planning to achieve the next golden generation. conflict of interest the authors declare that there are no competing interests. acknowledgement this study has been financially supported by the competitive grant from the national population and family planning board, indonesia (bkkbn) for the year 2020. the authors declare that the results of this study are not affected by the grantor. we are grateful to indra murty surbakti and besral for critical review and fruitful discussion. we thank heru for his support in the study. contributors es, rp conceived the study, developed the idea, participated in the design; mbr retrieved and verified data. es, mbr, ep, rp analyzed the data and reviewed the manuscript. all authors contributed towards drafting and have agreed to be accountable for all aspects of the work; and all the authors read and approved the manuscript. data available statement data is availa ble upon request and permission from the national population and family planning board, indonesia. references 1. snow rc, laski l, mutumba m. sexual and reproductive health: progress and outstanding needs. glob public health 2015;10:149-73. doi: 10.1080/17441692.2014.986178. 2. kementerian kesehatan republik indonesia, pusat data dan informasi. infodatin hiv. jakarta selatan: kementerian kesehatan republik indonesia, pusat data dan informasi;2020. univ med vol. 40 no 2 116 sahiratmadja, raharja, setiawati, et al anemia and sexually transmitted disease 3. world health organization. regional office for the western pacific regional. framework for the triple elimination of mother-to-child transmission of hiv, hepatitis b and syphilis in asia and the pacific, 2018-2030. manila: world health organization regional office for the western pacific;2018. 4. world health organization. anaemia. geneva: world health organization; 2017. 5. chaparro cm, suchdev ps. anemia epidemiology, pathophysiology, and etiology in lowand middle-income countries. ann n y acad sci 2019; 1450:15-31. doi: 10.1111/nyas.14092. 6. di renzo gc, spano f, giardina i, brillo e, clerici g, roura lc. iron deficiency anemia in pregnancy. womens health (lond) 2015;11:891-900. doi: 10.2217/whe.15.35. 7. de andrade cairo rc, rodrigues silva l, carneiro bustani n, ferreira marques cd. iron deficiency anemia in adolescents; a literature review. nutr hosp 2014;29:1240-9. doi: 10.3305/nh.2014.29.6. 7245. 8. kementerian kesehatan republik indonesia. direktorat pencegahan dan pengendalian penyakit tidak menular. pedoman pengendalian penyakit thalassemia di fasilitas kesehatan tingkat pertama. jakarta: kementerian kesehatan republik indonesia; 2017. 9. bakrania s, ghimire a, balvin n. bridging the gap to understand effective interventions for adolescent well-being: an evidence gap map on protection, participation, and financial and material well-being in low and middle-income countries. unicef office of research innocenti, florence. 2018. 10. von rosen ft, von rosen aj, riemenschneider fm, damberg i, tinnemann p. sti knowledge in berlin adolescents. int j environ res public health 2018;15: 110. doi: 10.3390/ijerph15010110. 11. zizza a, guido m, recchia v, grima p, banchelli tinelli a. knowledge, information needs and risk perception about hiv and sexually transmitted diseases after an education intervention on italian high school and university students. int j environ res public health 2021:2069. https://doi.org/ 10.3390/ijerph18042069. 12. badan kependudukan dan keluarga berencana nasional, badan pusat statistik, kementerian kesehatan. survei demografi dan kesehatan indonesia 2017: buku remaja. september 2018. jakarta: badan pusat statistik; 2018. 13. gubernur propinsi daerah khusus ibukota jakarta. peraturan gubernur dki-jakarta no. 185 tahun 2017 tentang konseling dan pemeriksaan kesehatan bagi calon pengantin. jakarta: gubernur propinsi daerah khusus ibukota jakarta; 2017. 14. inner city fund (icf). demographic and health surveys: standard recode manual for dhs 7. the demographic and health surveys program. rockville, maryland, usa: icf; 2018. 15. ilori or, awodutire po, ilori os. awareness and utilization of adolescent reproductive health services among in-school adolescents in urban and rural communities in oyo state. niger med j 2020;61:67-72. doi: 10.4103/nmj. nmj_38_19. 16. källander k, tibenderana jk, akpogheneta oj, et al. mobile health (mhealth) approaches and lessons for increased performance and retention of community health workers in lowand middleincome countries: a review. j med internet res 2013;15:e17. doi: 10.2196/jmir.2130. 17. bago jl, lompo ml. exploringthe linkage between exposure to mass media and hiv awareness among adolescents in uganda. sex reprod health 2019;21:1-8. doi: 10.1016/ j.srhc.2019.04.004. 18. borawski ea, tufts ka, trapl es, hayman ll, yoder ld, lovegreen ld. effectiveness of health education teachers and school nurses teaching sexually transmitted infections/human immunodeficiency virus prevention knowledge and skills in high school. j sch health 2015;85:18996. doi:10.1111/josh.12234. 19. mason-jones aj, sinclair d, mathews c, kagee a, hillman a, lombard c. school-based interventions for preventing hiv, sexually transmitted infections, and pregnancy in adolescents. cochrane database syst rev 2016; 11:cd006417. doi: 10.1002/14651858.cd006417. pub3. 20. angeles-agdeppa i, monville-oro e, gonsalves jf, capanzana mv. integrated school based nutrition programme improved the knowledge of mother and schoolchildren. matern child nutr 2019;15 suppl 3:e12794. doi: 10.1111/mcn.12794. 21. beluska-turkan k, korczak r, hartell b, et. al. nutritional gaps and supplementation in the first 1000 days. nutrients 2019;11:2891. doi: 10.3390/nu11122891. 22. rahman mm, abe sk, rahman ms, et. al. maternal anemia and risk of adverse birth and health outcomes in lowand middle-income countries: systematic review and meta-analysis. am j clin nutr 2016;103:495-504. doi: 10.3945/ ajcn.115.107896. 23. hoffmann jj, urrechaga e, aguirre u. discriminant indices for distinguishing thalassemia and iron deficiency in patients with 117 microcytic anemia: a meta-analysis. clin chem lab med 2015;53:1883-94. doi: 10.1515/cclm-20150179. 24. fucharoen s, winichagoon p. haemoglobinopathies in southeast asia. indian j med res 2011;134:498-506. 25. sahiratmadja e, wijaya ma, widjajakusuma a, maskoen am, dewanto jb, panigoro r. the knowledge about thalassemia among medical students and general practitioners in bandung and the prevalence of -thalassemia carrier. j indon med assoc 2020;70:48-58. doi: 10.47830/ jinma-vol.70.4-2020-232. 26. kohne e. hemoglobinopathies: clinical manifestations, diagnosis, and treatment. dtsch arztebl int 2011;108:532-40. doi: 10.3238/ arztebl.2011.0532. 27. amato a, cappabianca mp, lerone m, et al. carrier screening for inherited haemoglobin disorders among secondary school students and young adults in latium, italy. j community genet 2014;5:265-8. doi: 10.1007/s12687-0130171-z. univ med vol. 40 no 2 c:\users\user\documents\41 no 2 september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 psychological distress and dyslipidemia in adult women: a 6-years follow up study in bogor city, indonesia sulistyowati tuminah1,2* , sudarto ronoatmodjo3 , srilaning driyah4, dewi kristanti4 , and dwi hapsari tjandrarini2 background abstract 1magister program, faculty of public dyslipidemia is of global occurrence, with a prevalence 30% or higher in several countries, including indonesia. one risk factor of dyslipidemia is physical or mental stress, that is more frequent in women. this study aimed at investigating the association between psychological distress and dyslipidemia in adult women. methods this observational longitudinal study involved 1850 women aged 25 years and older at baseline. dyslipidemia was determined from the ratio of low density lipoprotein to high-density lipoprotein. psychological distress was determined using the 20-item self-reporting questionnaire. data analysis was by cox regression for calculating the hazard ratio of the incidence of dyslipidemia as predicted by the psychological distress. results among the 1474 participants without dyslipidemia at baseline, 545 (36.9%) developed dyslipidemia during 6 years of monitoring, while 93 (6.3%) had a history of psychological stress. there was a significant association between psychological distress and dyslipidemia (hr = 3.08; 95% ci: 2.334.07). cox regression revealed that the association was still found to be significant after a further adjustment for the variables of age, bmi, menopause, smoking status, physical activity, and carbohydrate and fat intakes (hr=2.8; 95% ci: 2.10 3.77). conclusions dyslipidemia incidence was high among adult women in bogor. women with psychological distress had higher incidence rates of dyslipidemia than women without psychological stress. psychological distress in women was statistically significantly associated with incidence rate of dyslipidemia. this finding highlights the importance of the need for a dyslipidemiareduction program in women through prevention and control of psychological distress. keywords: psychological distress, ldl/hdl ratio, dyslipidemia, longitudinal study, adult women health universitas indonesia (fkm ui), indonesia 2organization for health research national agency for research and innovation, indonesia 3department of epidemiology, faculty of public health universitas indonesia, indonesia 4center for health management policies agency for health development policies, ministry of health, republic of indonesia, indonesia *correspondence: sulistyowati tuminah badan riset and inovasi nasional (brin) gedung bj. habibie jl. m.h. thamrin no.8, jakarta pusat 10340 email: suli017@brin.go.id / watidarjoko@gmail.com orcid id: 0000-0003-2839-3865 date of first submission, may 25, 2022 date of final revised submission, october 24, 2022 date of acceptance, october 28, 2022 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license cite this article as: tuminah s, ronoatmodjo s, driyah s, kristanti d, tjandrarini dh. psychological distress and dyslipidemia in adult women: a 6 year follow-up study in bogor city, indonesia. univ med 2022;41:236-43. doi: 10.18051/univmed.2022.v41.236245. 236 doi: http://dx.doi.org/10.18051/univmed.2022.v41.236-245 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1348 original article mailto:suli017@brin.go.id mailto:watidarjoko@gmail.com http://dx.doi.org/10.18051/univmed.2022.v41.236-245 https://univmed.org/ejurnal/index.php/medicina/article/view/1348 https://orcid.org/0000-0003-2839-3865 https://orcid.org/0000-0002-2317-0294 https://orcid.org/0000-0002-9694-2064 https://orcid.org/0000-0001-8313-4722 237 univ med vol. 41 no 3 introduction dyslipidemia comprises one or more of the following abnormal lipid concentrations, namely high total cholesterol, high low-density lipoprotein (ldl), low high-density lipoprotein (hdl) or high triglycerides.(1,2) the occurrence and development of dyslipidemia frequently involves a continuous long-term process. various risk factors, including changes in social and economic status, changes in life style, such as westernization of the diet, insufficient physical activity and long-term sedentary behavior, are believed to be contributing to the increased prevalence of dyslipidemia.(3,4) the prevalence of dyslipidemia varies between countries, because of differing ldl cutoffs. the prevalence of dyslipidemia in thailand is 66.5%, using as reference the third adult treatment panels (atp iii) high ldl cutoff of 100 mg/dl and above.(2) the prevalence of dyslipidemia in africa (ldl cutoff = 130 mg/ dl and above) is 25.5% and in iran 83%.(5,6) the prevalence of dyslipidemia in indonesia (ldl cutoff = 160 mg/dl or more) at ages 25 years and above is around 36% (33.1% in men and 38.2% in women).(2) in addition to being affected by nutrition, body weight, physical activity, treatment, and genetic factors, the blood lipid concentration may also be influenced by mental or physical stress.(7) stress is the condition of physical and psychic stress as a result of individual and environmental demands, such as to cause anxiety, which is a ge neral and ear ly s ign of physica l and psychological abnormality.(8) the are 2 types of stress, namely eustress and distress. eustress is a positive response that brings pleasure and health, whereas distress is a negative response of the body that has a deleterious impact on health.(9) psychological distress (psychological stress) is one of the main causes of disability in the global disease burden. the morbidity resulting from psychological distress has reportedly a negative impact on the broader socio-economic health outcomes. the prevalence of psychological distress is especially reported to be higher in lowmiddle income countries (lmics) and is significantly higher in women (as compared to men) and in those with comorbidities.(10,11) because it is associated with different population characteristics and conditions, the prevalence of psychological distress varies widely, being 35.5% in iraq, 16.0% in mexico,(11) 37.6% in br azil, (1 2) and 23.0% in eri trea. (1 3) psychological distress is associated with poor physical health and is predictive of the occurrence of chronic physical conditions.(14) the indonesian national prevalence of psychological distress is 9.8% according to the basic health research (riskesdas) data for 2018. psychological distress tends to increase with increasing age, its proportion being 8.5 15.8%. the prevalence of psychological distress is higher in women (12.1%) than in men (7.6%).(15) the proportion of psychological distress in the cohort study of risk factors of non-communicable disease (studi kohor faktor risiko penyakit tidak menular frptm) in bogor city in 2019 was 28.6%. the proportion of hdl concentration of less than 50 mg/dl in women (or less than 40 mg/dl in men) is around 36 42%.(16) the proportion of subjects with high ldl concentration (100 mg/dl and above) at baseline and in monitoring years 2, 4 and 6, was found to be higher than 80%.(17) multivariate analysis of the study of chen et al.(18) found a significant association between psychological distress and dyslipidemia, with a hazard ratio (hr) of 1.14 (95% ci: 1.04-1.25). on the other hand, the results of the study of zhang et al.(19) showed that the group with mental health disorders had a significant relationship for awareness of dyslipidemia, with odds ratio (or) =1.81, but not for treatment of dyslipidemia (or=1.29) and for controlled dyslipidemia (or=0.93). however, evidence showed that not all people with psychiatric disorders display metabolic impairments due to age and gender, these latter being the most significant factors influencing the relationship between metabolism and mental disorder. for instance, metabolic conditions (abdominal obesity, high triglyceride and glucose levels) are associated with an increased 238 risk of a future depressive episode in middle-aged adults.(20) the present study aimed at investigating the association between psychological distress and dyslipidemia in adult women. methods design of the study this observational longitudinal study was conducted using data from 5 villages (kelurahan) of bogor tengah district, bogor city, west java province from 2011/2012 until 2017/2018. the study was preceded by recruitment of candidate respondents and a baseline study comprising health screening that was conducted in 3 recruitment stages, namely stages 1 (2011), 2 (2012) and 3 (2015), to maintain the minimum sample size. for 6 years, 3 annual routine monitoring studies were performed.(16) new cases of dyslipidemia that occurred in the monitoring period were recorded as the incidence of dyslipidemia. research subjects the study subjects were female respondents of the cohort study of risk factors of noncommunicable disease (frptm) aged 25 years and above (in the baseline study). the reason for selecting female re spondents was that dyslipidemia as well as psychological distress is more frequently found in women.(2,15) the inclusion criteria were: respondents who did not have dyslipidemia at baseline and had been monitored for 6 years. the exclusion criterion was incomplete results of blood lipid examination. from a total of 5258 respondents of the frptm cohort study aged 25 years and above, the data of 3482 female respondents were collected. of the latter, 1850 persons who did not have dyslipidemia at baseline were selected, from whom subsequently wer e collected 1642 respondents who had been monitored up to year 6, i.e. recruited in stages 1 and 2. the data of 168 persons, who had incomplete blood lipid results, were not included in the analysis, such that the number of respondents who met the criteria for analysis was 1474 (figure 1). definition of dyslipidemia dyslipidemia as outcome was determined by the ldl to hdl ratio, because this ratio is a substitute marker for a decrease in lipids.(21) the result of the blood examination is categorized as dyslipidemia if the ldl/hdl ratio is high (2.5 and above) because according to the study of chen et al.(22) this is the optimal cutoff value in women. the ldl and hdl concentrations were obtained from the results of blood lipid examination in the frptm cohort study that are conducted once in 2 years. definition of history of psychological distress the data on exposure to psychological distress were obtained from the results of mental health screening using the 20-item self-reporting questionnaire (srq-20). subjects are categorized as having “psychological distress” if they answer “yes” to at least 6 of the 20 srq-20 questions.(14) subjects are categorized as having “history of psychological distress” if they had been detected as having psychological distress in at least 2 successive monitoring periods prior to the occurrence of the dyslipidemia. covariates seve ral covariate that were seen as potential confounders were included in this analysis. the age variable was divided into 2 groups, namely less than 55 years and 55 years and above, with reference to the study results of sudikno et al.,(23 ) where ldl cholesterol concentration increases 3-fold in the age group of 55 years and above. the values of the menopause variable were obtained from the interview results ba sed on the subj ects’ admissions and were grouped into 2 categories, namely “yes” or “no”. smoking status was estimated using the brinkman index (bi), which is calculated by multiplying the duration of smoking (in years) by the number of cigarettes smoked.(24) smoking status was categorized into 3 groups, namely non-smoker (0), light smoker (1—199), and heavy smoker (200 and above).(25) the physical activity variable is a composite of the type and duration of activity (days per week tuminah, driyah, kristanti psychological distress and dyslipidemia 239 univ med vol. 41 no 3 a b and minutes per day) including the type of sport. vigorous activity as well as vigorous sport has an 8-fold weight, moderate activity or sport has a 4-fold weight, while low activity has a 2-fold weight. subjects were categorized as having insufficient physical activity if they had a total activity of less than 600 metabolic equivalent for task (met) per week.(26) fat and carbohydrate intakes were determined based on the general guideline for balanced nutrition (pedoman umum gizi seimbang, pugs). fat intake was grouped into less than 25% and 25% and above of total energy requirement. carbohydrate intake was grouped into less than 60% and 60% and above of total energy requirement.(27) the nutritional data were obtained through interviews using the food frequency questionnaire (ffq) and food recall. body mass index (bmi) was obtained from weight in kg divided by height in m2, referring to bmi standards for asians. bmi was divided into 2 categories, namely obesity (bmi 25 kg/m2 and above) and no obesity (bmi less than 25 kg/ m2).(28,29) subjects were categorized as having obesity if in at least 2 of 3 monitoring periods (per year) in 6 years they had a bmi of 25 kg/m2 and above. statistical analyses sample characteristics were compared between subjects with and without the onset of dyslipidemia at baseline, using the chi-square test for categorical variables. multivariable analysis was performed with the cox regression test to obtain the hazard ratio (hr) which is the ratio of the incidence dyslipidemia of the group with a history of psychological distress during monitoring to that of the group without such a history in the same period. the results are declared to be statistically significant if the p value is less than 0.05. women respondents (n=3482) no dyslipidemia at baseline (n=1850) monitoring up to 6th year (n=1642) analyzed: complete blood lipid data (n=1474) men respondents (n=1777) dyslipidemia at baseline (n=1632) no monitoring up to 6th year (n=208) incomplete blood lipid data (n=168) respondents of frptm cohort study aged 25 yrs and above (n=5258) figure 1. flow of selection of analyzed sample (n=1474) 240 in a cumulative incidence of dyslipidemia (number of new cases) of 545 (36.9%). the chi-square test results showed that the proportion of dyslipidemia incidence was significantly higher in the group with psychological stress, age less than 55 years, and bmi 25 kg/m2 (p<0.05) (table 1). the results of bivariable analysis showed a higher risk of dyslipidemia incidence in subjects with psychological distress than in subjects without psychological distress (hr=3.08; 95% ci: 2.33-4.07) (table 2). after controlling for the covariate variables of age, bmi, menopausal sta tus, smoking status, physical a ctivity, carbohydrate intake, and lipid intake, the risk of dyslipidemia incidence was higher in the group with psychological distress (hr= 2.8; 95% ci: 2.10 – 3.77 (table 3). ethical clearance the frptm cohort study obtains ethical clearance for each year of conduct, from the national commission of health research ethics (komisi nasional etik penelitian kesehatan, knepk) of the hea lth resear ch and development agency, ministry of health, the current license being no. lb.02.01/2/ke.076/ 2018 dated march 1, 2018. results among the 1474 women without dyslipidemia at baseline, 93 (6.3%) had a history of psychological stress, but the majority (1381 or 93.7%) did not. in the 6-year monitoring period, 57 (61.3%) of the 93 women and 488 (35.3%) of the 1381 women developed dyslipidemia, resulting table 1. comparison of characteristics of adult women with and without dyslipidemia during 6 years of monitoring (n=1474) note: bmi = body mass index risk factor dyslipidemia p value yes (n=545) no (n=929) n (%) n (%) psychological distress <0.001 absent 488 (35.3) 893 (64.7) present 57 (61.3) 36 (38.7) age (years) 0.030 less than 55 412 (38.7) 652 (61.3) 55 and above 133 (32.4) 277 (67.6) bmi (kg/m2) 0.040 less than 25 233 (34.2) 449 (65.8) 25 and above 312 (39.4) 480 (60.6) menopause 0.060 no 340 (39.0) 531 (61.0) yes 205 (34.0) 398 (66.0) smoking status 0.080 non smoker 454 (35.6) 822 (64.4) light smoker 72 (44.4) 90 (55.6) heavy smoker 11 (40.7) 16 (59.3) physical activity 0.05 sufficient 356 (35.3) 653 (64.7) poor 189 (40.6) 276 (59.4) carbohydrate intake (% of total energy) 0.180 less than 60 376 (35.8) 673 (64.2) 60 and above 169 (39.8) 256 (60.2) fat intake (% of total energy) 0.620 less than 25 91 (35.4) 166 (64.6) 25 and above 454 (37.3) 763 (62.7) tuminah, driyah, kristanti psychological distress and dyslipidemia 241 univ med vol. 41 no 3 discussion in this study it was found that the cumulative incidence of dyslipidemia that occurred in the monitoring period of 6 years was 36.9%. this is relatively low as compared with the study of chen et al.(18) who found a cumulative incidence of dyslipidemia of 60.5% in a period of 2.6 years. this is because the definition of dyslipidemia in chen et al. (18) study was at least one abnormal lipid level, whereas in our study dyslipidemia was determined by the combination of a high ldl and a low hdl, resulting in a smaller value. the results of multivariable analysis showed a statistically significant association between psychological distre ss and incidence of dyslipidemia, after controlling for covariate variables, namely age, bmi, menopause, smoking status, physical activity, and carbohydrates and fat intakes. our study results are consistent with the results of other studies that associate stress with dyslipidemia. for the risk of dyslipidemia, the study of catalina-romero et al.(30) had an or=1.11 (95% ci: 1.04 1.19), while the study of assadi (31) had rr=14.54 (95% ci: 3.54 59.65). the study of yu et al.(32) f ound a correlation between psychological distress and high ldl concentration in middle-aged women. the study results of kim et al.(33) showed a significant relationship between chronic mental problems, such as depressive mood, and serum lipid concentration in male adolescents, but not in female adolescents. although psychological distress in the frptm cohort study is determined with the srq-20 that is used to detect mental health in general, the results are consistent with a number of studies that use different and more specific instruments, in that there is a significant table 2. cox regression model of factors associated with the incidence of dyslipidemia in adult women note: bmi = body mass index risk factor crude hazard ratio (chr) 95% ci p value psychological distress absent 1 (ref) present 3.08 2.33—4.07 <0.001 age (years) less than 55 1 (ref) 55 and above 0.79 0.65—0.96 0.020 bmi (kg/m2) less than 25 1 (ref) 25 and above 1.10 0.93—1.30 0.250 menopause no 1 (ref) yes 0.83 0.70—0.99 0.040 smoking status non smoker 1 (ref) light smoker 1.39 1.09—1.79 0.010 heavy smoker 1.14 0.63—2.08 0.660 physical activity sufficient 1 (ref) poor 1.37 1.15—1.64 <0.001 carbohydrate intake (% of total energy) less than 60 1 (ref) 60 and above 1.21 1.01—1.46 0.030 fat intake (% of total energy) less than 25 1 (ref) 25 and above 0.98 0.78—1.35 0.900 242 relationship between psychological distress and the incidence of dyslipidemia. psychological distress may vary by type, number and duration of symptoms that are felt by the subjects, depending on the response of each individual and the way this individual overcome s the pr oblem. (8 ) ac cord ing to marcondes et al.,(34) increases in blood lipids that are triggered by stress are adaptive in nature and will return to normal when the stress disappears. however, if the stress persists for a long time, the dyslipidemia that is triggered by stress will persist, have a damaging effect and contributes to the occurrence of cardiovascular diseases, such as stroke, atherosclerosis, and coronary heart disease. the results of physiological studies show that stress from any source may affect the endocrine, hemopoietic and immune systems.(35) the stress response induces the secretion of cortisol and catecholamine hormones that influence the body metabolism including fat metabolism. there are 2 stress response pathways in the hypothalamus, namely the hypothalamic pituitary adrenal (hpa) and the sympathetic adrenal medullary (sam) axes. both are pathways that play a major role in regulating the balance of cortisol and catecholamine hormones. in a condition of distress, both of these pathways produce cortisol and catecholamines in non-homeostatic concentrations, that result in various types of metabolic changes, both for carbohydrates, proteins and fats.(9) dyslipidemia that is triggered by stress involves a complex interaction between str ess hormones, ins ulin, adipose tissue metabolism and cytokines.(34) table 3. multivariate cox regression analyses on the association between psychological distress and dyslipidemia in adult women *covariates included age, bmi, menopausal status, smoking status, physical activity, carbohydrate intake, lipid intake, and psychological distress. significance was set at p < 0.05. note: bmi = body mass index; ref = reference category risk factor adjusted hazard ratio 95% ci p value psychological distress absent 1 (ref) present 2.81 2.10—3.77 <0.001 age (years) less than 55 1 (ref) 55 and above 0.86 0.65—1.14 0.310 bmi (kg/m2) less than 25 1 (ref) 25 and above 1.10 0.93—1.31 0.240 menopause no 1 (ref) yes 0.92 0.72—1.18 0.540 smoking status non smoker 1 (ref) light smoker 1.29 1.00—1.67 0.040 heavy smoker 1.02 0.56—1.71 0.930 physical activity sufficient 1 (ref) poor 1.38 1.15—1.65 <0.001 carbohydrate intake (% of total energy) less than 60 1 (ref) 60 and above 1.32 1.05—1.66 0.010 fat intake (% of total energy) less than 25 1 (ref) 25 and above 1.26 0.95—1.67 0.100 tuminah, driyah, kristanti psychological distress and dyslipidemia 243 univ med vol. 41 no 3 from the results of the evaluation on each of the covariate variables, no confounder was found. this may have been due to the presence of other confounders that were not analyzed or because previously there had been a restriction in gender, in that only data from female respondents constituted the study sample, on the grounds that 65% of the respondents in the frptm cohort study are women. in addition, based on the literature, both dyslipidemia and psychological distress are more frequently found in women.(2,15) the limitation of this study is that the measurement of dyslipidemia as the outcome is done only once in 2 years. in addition, selfassessment may cause perception bias in the respondents towards the questions in the srq20. our future studies will include more specialized scales on a particular aspect of psychological distress, such as depression. conclusions dyslipidemia incidence was high among adult women in bogor. a psychological distress in women is statistically significantly associated with the incidence of dyslipidemia. therefore, the incidence of dyslipidemia may be reduced if the psychological distress can be prevented or controlled, conflict of interest there was no conflict of interest. funding none. author contributions st contributed to literature search, data analysis, and wr iting of the manuscript (corresponding author); 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( 5) acu te pe i s the r esult of a complicated relationship between different organs; therefore, the clinical symptoms are diverse and non-specific. it is difficult to make an immediate diagnosis, especially in many comorbidities complicating the condition, as d esc ri be d i n thi s c a s e of a p a tie nt wit h pulmonary embolism with signs of pulmonary hypertension (ph) and underlying chronic kidney disease. non-invasive imaging tests enable the clinician to suspect and initiate further workup for pe, with computed tomography pulmonary angiography (ctpa) being the principal tool.(6) unfortunately, the widespread use of multidetector ctpa without selection of patients has introduced new issues, such as overdiagnosis and exposure to radiation.(7) an eff ort to implement the r obust validated strategies in clinical practice could overcome some of these problems. here we report the case of a woman with acute pulmonary embolism who had pulmonary hypertension and dialysis-dependent chronic kidney disease. the purpose of this case report is to highlight the roles of different modalities with different accuracy to help establish the diagnosis. case report a 51-year-old woman came to our hospital with the chief complaint of sudden worsening of shortness of breath with symptoms of right heart failure. the patient had a history of immobilization, uncontrolled hypertension, and chronic kidney disease and was on hemodialysis. on physical examination stable hemodynamics and signs of right heart failure were found, while laboratory examination showed anemia (hb 9.8 g/dl) and lowered kidney function (serum urea 52 mg/dl and creatinine 5.28 mg/dl). the ecg showed a normal sinus rhythm with no sign of a deep s wave in lead i, of a q wave in lead iii, of an inverted t wave in lead iii; or right ventricular strain. echocardiography revealed a normal left ventricular (lv) function, a dilated right ventricle (rv) with impaired function (tricuspid annular plane systolic excursion/ tapse) of 16 mm, longitudinal strain -13.4%), pericardial effusion, and also signs suggesting a high probability of pulmonary hypertension (figure 1). we found no signs of pe, such as mcconnell’s sign and the 60/60 sign. we decided to perform ct pulmonary angiography and found a hypodense lesion within the lateral wall of the main pulmonary artery, right main pulmonary artery, and the superior and inferior right trunk, with central filling defect (polo mint sign) due to thrombus, and dilatation of the main pulmonary artery (figure. 2). after analyzing the resulting ctpa and echocardiography signs suggesting ph, we diagnosed this case as acute pulmonary embolism with stable hemodynamics in a patient with suspected pulmonary hypertension in chronic kidney disease. the patient was treated with parenteral anticoagulation for ten days, overlapping with an oral anticoagulant, and the patient was discharged in a stable condition and 159 figure. 1. echocardiography parameters echocardiography parameters indicate reduced rv function and the probability of ph. (a) free-wall strain of 13.4%; (b) moderate pericardial effusion; (c) reduced tapse value of 16 mm; (d) mean tr velocity of 2.98 m/ s; without signs of pulmonary embolism. rv= right ventricle; ph= pulmonary hypertension; tapse= tricuspid annular plane systolic excursion; tr= tricuspid regurgitation figure. 2. msct of pulmonary embolism. msct images showing (a) hypodense lesion in the lateral wall of mpa, rpa; (b) central filling defect (polo mint sign); (c) dilatation of all cardiac chambers; (d) dilatation of mpa and ratio of mpa to ascending aorta diameter is >1. abbreviations: msct= multi-slice computed tomography; mpa= main pulmonary artery; rpa= right pulmonary artery univ med vol. 40 no 2 160 dewi, hasan, fathini pulmonary embolism in a 51-year-old woman showing improvement in symptoms. further eva luation on pulmonary hypertension is mandatory to conclude the working diagnosis according to the diagnostic algorithm stated in the guidelines, but due to loss to follow-up there are no further data regarding the outcomes or further ph diagnosis. written informed consent was obtained from the patient for publication of this case report. discussion chronic kidney disease can increase the risk of thromboembolism by affecting both procoagulant and anticoagulant factors. chronic kidney disease has significant changes in all important factors of coagulation, as determined by virchow's triad. abnormalities in the coagulation cascade and platelet function may lead to the tendency of thrombosis or bleeding, depending on the defect.(2) one study showed that the occurrence of pulmonary embolism in patients with ckd is 7.6 times higher than in patients with normal kidney function, and also has a higher mortality.(8) acute pulmonary embolism is the most serious c linica l pre sentation of venous thromboembolism (vte). it is often clinically silent or mimics the diagnostic features of other conditions, resulting in under-diagnosis.(6) prediction rules using the wells score help categorize patients with suspected pe into different categories of pre-test probabilities and decide if the diagnostic workup should be initiated. the probability of this patient was likely (intermediate) and needed further evaluation. ddimer has a high negative predictive value (npv), and low positive predictive value (ppv) and is not useful for confirmation of pe. in our case, we did not check the d-dimer due to the low ppv of an in-hospital patient. the ecg features in pe comprise a wide range of abnormalities; 10-25% of patients have a completely normal ecg, such as in our case. the s1q3t3 pattern (mcginn-white sign) is a classic finding found pe cases with a high specificity of 95%, but a low sensitivity of 5%.(9) multivariable analysis of ecg characteristics using the twist score describes right ventricular (rv) strain pattern due to increased pressure and wall tension in the right heart. among these ecg characteristics, the most common finding associated with pulmonary embolism is an s wave in lead i, followed by a t wave inversion (twi) in leads v1-v3, and a tachycardia of ed100 bpm. the higher the twist score, the higher the specificity (97-100%), but the lower the sensitivity (1-7%), with ppv 40100% and npv 44-56%.(9) echocardiography in the diagnosis of acute pe is a favorable alternative, since it is non-invasive, is standardized almost in all cardiology centers, and can be performed at the bedside. right ventricular dysfunction and pressure overload are a common feature in pulmonary embolism, and can be detected by echocardiography. in suspected acute pe, echocardiographic signs are inadequate to rule out pe and only an enlarged rv end-diastolic diameter is 80% sensitive for acute pe. there are also other highly specific signs, such as right heart thrombus with 99% specificity, followed by mcconnell’s sign (rv mid free wall akinesia and apical wall hyper contractility) with 97% specificity, and paradoxical septal movement with 95% specificity.(10) these specificities remain high when performed by a non-cardiologist. however, in our case, we only found a high p r o ba b i lit y o f ph b ut n o sp e c i f ic e c h o c a r d i ogr a p hi c s i gn s su gge s t i ng pe . although echocardiography produces potentially va l ua b l e f i nd i n gs , a c u t e pe c a n n ot be differentiated from other causes of rv failure. echocardiographic findings of rv pressure overload are differentiated into acute and chronic. tricuspid annular plane systolic excursion (tapse) is a widely recognized, obtainable, and validated parameter of global rv systolic function and also has prognostic value for pe.(4) a right heart thrombus is found in 24% of acute pe patients and increases the risk of short-term mortality to 20% in acute pe.(10) co mp ut ed t o mogr a p hy pu lmo na r y angiography is the best modality for showing the 161 pulmonary vascularization in patients with suspected pe. the diagnosis of pe using ctpa has an outstanding accuracy, with a sensitivity of 83% and a specificity of 96%.(4) several studies showed that ctpa is a reliable imaging test for excluding pe. findings for positive ct results include a partial filling defect or central filling defect within the vessel surrounded by contrast material known as the “polo mint sign” on images acquired perpendicular to the long axis and the “railway track sign” on the longitudinal axis of the vessel.(12) computed tomography pulmonary angiography also provides substantial information on vascularity, cardiac anatomy, and t he a n a to my o r a b nor ma l ity o f a d j a ce n t structures. furthermore, ctpa can provide clues to the etiology of ph and other diagnostic information. ph signs in ctpa include dilatation of the main pulmonary artery (mpa) (>33 mm), an mpa to ascending aorta diameter ratio of >1.1, rv enlargement or hypertrophy, an rv to lv diameter ratio of >1, and bending of the ventricular septum toward the lv. the ctpa when used in the diagnosis of chronic ph based on pa diameter has an average accuracy, with a sensitivity of 47-87% and specificity of 41100%.(13) pulmonary hypertension with unclear and/ or multifactorial mechanisms or group 5 ph, is a form of the disease with poorly understood mechanisms, the etiologies of which include pulmona ry vasoconst riction, extr insic compression, intrinsic occlusion, high-output cardiac failure, and left heart failure.(14) one of the disorders that can cause group 5 ph is chronic renal failure. the mechanism of pulmonary hypertension in kidney disease is mostly venous in origin due to left ventricular dysfunction caused by chronic volume overload and pulmonary venous hypertension. comorbid conditions in ckd may contribute to and/or exacerbate pulmonary hypertension. hypertension and diabetes mellitus are two dominant causes of ckd that can increase pulmonary pressure, and trigger lv diastolic dysfunction. in one study, ph in ckd was found in 81% of hemodialysis patients and in 71% of non-dialysis patients.(14) the mechanism is multifactorial; such as anemia and arterio-venous fistula (avf), both of which can cause a high output state. avf has profound hemodynamic effects that lead to decreased systemic vascular resistance, increased venous return, and a significant increase in cardiac output. because the pressure is directly related to flow and resistance, these adaptations eventually will lead to ph.(15) therefore, an increase in pulmonary flow may lead to increased pressure and eventually to pulmonary hypertension.(16) conclusions the risk of either pulmonary embolism or pulmonary hypertension is increased in kidney disease, and several mechanisms certainly play an important role in either manifestation. in general, imaging modalities provide useful information, with var ying sensitivity and specificity, which helps to identify the problem and either rule in or rule out the diagnosis. therefore, the diagnosis of either condition is very challenging, and multiple team evaluation is necessary to provide the best management possible. conflict of interest the authors declare that there are no conflicts of interest. contributors tid contributed to conception or design of the work, and to writing, review, and editing. mh contributed to review and editing. ff contributed to revision of the manuscript. all authors have read and approved the final manuscript. references 1. hill nr, fatoba st, oke jl, et al. global prevalence of chronic kidney disease a systematic review and meta-analysis. plos one 2016; 11: e0158765. doi: 10.1371/journal.pone.0158765. univ med vol. 40 no 2 162 dewi, hasan, fathini pulmonary embolism in a 51-year-old woman 2. kumar g, sakhuja a, taneja a, et al. pulmonary embolism in patients with ckd and esrd. clin j am soc nephrol 2012;7:1584-90. doi: https:// doi.org/10.2215/cjn.00250112. 3. nishiyama kh, saboo ss, tanabe y, jasinowodolinski d, landay mj, kay fu. chronic pulmonary embolism: diagnosis. cardiovasc diagn ther 2018;8:253-71. doi: 10.21037/ cdt.2018.01.09. 4. konstantinides sv, meyer g, bueno h, et al. 2019 esc guidelines for the diagnosis and management of acute pulmonary embolism developed in collaboration with the european respiratory society (ers). eur heart j 2020;41:543603. doi: 10.1093/eurheartj/ehz405. 5. howard l. acute pulmonary embolism published correction appears in clin med (lond) 2019;19:2437. doi: 10.7861/clinmedicine.19-3-247. 6. morrone d, morrone v. acute pulmonary embolism: focus on the clinical picture. korean circ j 2018;48:365-81. doi: 10.4070/kcj.2017.0314. 7. konstantinides s v. trends in incidence versus case fatality rates of pulmonary embolism: good news or bad news? t hromb haemost 2016;115:233-5. doi: 10.1160/th15-10-0832. 8. kumar g, sakhuja a, taneja a, et al. pulmonary embolism in patients with ckd and esrd. clin j am soc nephrol 2012;7:1584-90. doi: 10.2215/ cjn.00250112. 9. hariharan p, dudzinski dm, okechukwu i, takayesu jk, chang y, kabrhel c. association between electrocardiographic findings, right heart strain, and short-term adverse clinical events in patients with acute pulmonary embolism. clin cardiol 2015;38:236-42. doi:10.1002/clc.22383. 10. dabbouseh nm, patel jj, bergl pa. role of echocardiography in managing acute pulmonary embolism. heart 2019;105:1785-92. doi: 10.1136/ heartjnl-2019-314776. 11. ascha m, renapurkar rd, tonelli ar. a review of imaging modalities in pulmonary hypertension. ann thorac med 2017;12:61-73. doi: 10.4103/ 1817-1737.203742. 12. moore aje, wachsmann j, chamarthy mr, panjikaran l, tanabe y, rajiah p. imaging of acute pulmonary embolism: an update. cardiovasc diagn ther 2018;8:225-43. doi:10.21037/ cdt.2017.12.01. 13. hur dj, sugeng l. non-invasive multimodality cardiovascular imaging of the right heart and pulmonary circulation in pulmonary hypertension. front cardiovasc med 2019;6:24. doi: 10.3389/fcvm.2019.00024. 14. pabst s, hammerstingl c, hundt f, et al. pulmonary hypertension in patients with chronic kidney disease on dialysis and without dialysis: results of the pepper-study. plos one 2012;7. doi: 10.1371/journal.pone.0035310. 15. amerling r, ronco c, kuhlman m, winchester jf. arteriovenous fistula toxicity. blood purif 2011;31:113-20. doi: 10.1159/000322695. 16. o'leary jm, assad tr, xu m, et al. pulmonary hypertension in patients with chronic kidney disease: invasive hemodynamic etiology and outcomes. pulm circ 2017;7:674-83. doi: 10.1177/ 2045893217716108. c:\users\universa medicina\docu 57 abstract universa medicina mandatory universal use of cloth mask for prevention of coronavirus disease 2019 transmission yenny*@, elly herwana**, and raditya wratsangka*** since the outbreak in wuhan city, china, in late december 2019, the coronavirus disease 2019 (covid-19) has spread to nearly the whole world, so that it was declared a pandemic by the word health organization. the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), the causative organism of covid-19, is extremely infectious and can adhere to droplet nuclei of < 5 m diameter and become airborne (aerosol). since covid-19 was declared a pandemic, there has been controversy on the use of cloth masks by the public, because of the still inconclusive evidence of the efficacy of cloth masks in protecting against covid-19 transmission. universal masking as a healthcare intervention in the community is currently made mandatory by local governments of most countries, since they follow the recent recommendation by the world health organization. the issuing of the who recommendation on the public use of masks was based on a study demonstrating that covid-19 transmission does not occur only through droplets but also through aerosols. in addition, there was a study showing that covid-19 transmission does not only occur from patients with clinical symptoms but also through asymptomatic and pre-symptomatic subjects, so that universal masking is of benefit in providing protection when used by healthy people and as source control to prevent crosstransmission to other people. this review article aims to discuss the mechanism of covid-19 transmission, the evidence related to the efficacy of cloth masks, and the guidelines related to the selection and use of masks by the general population. keywords: covid-19, prevention, cloth mask, universal masking review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.57-68 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1054 january-april, 2021 vol.40no.1 cite this article as: yenny, herwana e, wratsangka r. mandatory universal use of cloth mask for prevents covid_19 transmission. univ med 2021;40:5768. doi: 10.18051/univmed.2021.v40. 5 7 6 8 . *department of pharmacology & clinical pharmacy, faculty of medicine, trisakti university **department of pharmacology & clinical pharmacy, faculty of medicine, trisakti university ***department of obstetrics & gynecology, faculty of medicine, trisakti university correspondence : @yenny department of pharmacology & clinical pharmacy, faculty of medicine, trisakti university jl. kyai tapa no.260, grogol jakarta email: yennyfarmako@trisakti.ac.id orchid id: 0000-0001-9390-5527 date of first submission, october 18, 2020 date of final revised submission, january 17, 2021 date of acceptance, january 19, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 58 yenny, herwana, wratsangka prevents covid-19 transmission introduction the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), the causative organism of coronavirus disease 2019 (covid19), is an enveloped rna beta-coronavirus that is phylogenetically similar to the first severe acute respiratory syndrome coronavirus (sars-cov1) and the middle east respiratory syndrome coronavirus (mers-cov).(1) the severe acute respiratory syndrome coronavirus-2 is spherical in shape, somewhat pleomorphic, and the virion is 85 nm in diameter,(2) with a size range of 60– 140 nm.(3) coronavirus disease 2019 transmission between humans mainly occurs through exposure to respiratory droplets on exhalation and on close contact with covid-19 patients (within a distance of 1 meter).(4) in addition, transmission can also occur on touching contaminated objects used by covid-19 patients (fomite transmission) and through aerosols (airborne transmission).(4) transmission of covid-19 most frequently occurs via symptomatic patients (those showing symptoms of covid-19) on close contact (±1 meter), but may also occur via presymptomatic patients, who do not yet show symptoms of infection because the disease is still in the incubation period (±14 days) or in asymptomatic patients who do not show clinical symptoms at all but carry the virus that can be transmitted to other people.(5) the prevalence of asymptomatic covid-19 infection is 4.0–45.0%.(6,7) since covid-19 was declared a pandemic by the world health organization (who) on march 11, 2020,(8) the president of the republic of indonesia has formed the action group for accelerated management of coronavirus disease 2019 (covid-19) [gugus tugas percepatan penanganan coronav irus disease 2019 (covid-19)](9) that continually socializes the 3m movement, where 3m is an acronym of the three activities of washing the hands with soap, wearing a mask, and physical distancing with other people in order to break the covid-19 transmission chain. the use of personal protective equipment in the form of masks is one of the essential pillars of covid-19 prevention. to date, the use of masks by the general population is made mandatory by local governments in many countries because they follow the recommendation issued by the who. the use of cloth masks by the general population (universal masking) remains a matter of debate, because of the still inconclusive evidence for the protective efficacy of cloth masks in the preve ntion of cov id-19 transmission.(10,11) in spite of this, several global healthcare organizations have recently issued recommendations in connection with the use of masks by the general population. the united states centers for disease control and prevention (cdc) recommend the use of cloth masks (face cloths) by the community in public places when social distancing is difficult to maintain.(12) similarly, the who that initially did not yet recommend the use of masks by the general population, again revised its guidelines on december 1, 2020, and issued recommendations on the use of masks by the general population in areas of known or suspected cov id-19 infection, accompanied by explanations of the types of mask that may be chosen in certain relevant situations and conditions.(13) the issuing of recommendations on the use of cloth masks by the cdc (12) and who (13) for the general population was due to existence of severa l studies sho wing that cov id-19 transmission can also occur through droplet nuclei (airborne transmission) that are produced upon forceful exhalation such as shouting, singing, or performing physical activities.(14,15) in addition, there are studies indicating that covid-19 transmission may also occur through presymptomatic (16) and asymptomatic individuals.(17) the rationale of mandatory universal masking as a public health intervention when social distancing is impossible is to control the source of disease (mask users infected with covid-19), while universal masking is also of benefit for prevention (protecting healthy users against covid-19 infection).(18) we searched 59 pubmed central and pubmed on 1 october 2020. this review article aims to provide information in connection with the mechanism of transmission of covid-19, evidence associated with cloth masks, and guidelines related to the selection and use of masks by the general population. droplet vs aerosol transmission respiratory droplets are defined as saliva and secretions that are sprayed from the upper respiratory tract through the mouth and nose during exhalation (expelling air from the respiratory tract) such as in coughing, sneezing, laughing, speaking, and even normal breathing.(19) the activity performed determines the diameter of the droplets that are produced. the majority of droplets that are expelled during breathing or clearing the throat are <1 m in diameter, whereas droplets originating during sneezing are larger in size, namely, 74.4 -360.1 m in diameter.(20) the size of the produced droplets determines their extent and penetration in the respiratory tract. based on particle size, several types of droplet are currently recognized: i). small droplet particles (<5–10 m in diameter). droplet particles of <5 m in diameter (droplet nuclei) can penetrate the respiratory tract down to the alveolar spaces, whereas droplet particles >5– 10 m in diameter are designated as respiratory droplets and penetrate below the glottis; ii). large droplet particles (>20 m in diameter) cannot follow the respiratory airflow because they rapidly settle due to gravity; iii). intermediate droplet particles (10–20 m in diameter), have the characteristics of both small and large droplets, but disappear more rapidly than droplet particles of <10 m in diameter and potentially carry a smaller infectious dose than large droplets (>20 m in diameter).(21,22) there ar e three main methods of transmission of viral infection that cause acute respiratory disease, namely contact (direct/ indirect), dr ople t, and aerosol/air borne transmission. direct contact transmission occurs when the virus is transmitted directly by contact from the infected patient to another patient, without the aid of contaminated intermediary objects. indirect contact transmission occurs via the transfer of a virus through contact with intermediary objects contaminated by infected patients. droplet transmission occurs when there is a person-to-person transmission of a virus thr ough the air by droplet sprays. t he characteristic feature of this mode of transmission is the exposure of mucous membranes to viruscontaining droplets. aerosol or air borne transmission occurs when there is a person-toperson transmission of the virus through the air by the formation of aerosols in the size range of respirable particles or smaller.(22) viral respiratory tract infection may occur when infected patients produce droplets by breathing, coughing, or sneezing. transmission between individuals, through large droplets (shortdistance) as well as small droplet nuclei (longdistance), may occur depending on the distance to the patients who are the source of infection. infection may also occur if the droplets deposit onto the surface of objects (fomites) that are subsequently touched and transported by the hands, resulting in self-inoculation into mucosal membranes (ocular, nasal, and oral).(21) in figure 1, infe cted patients are represented by orange-colored heads, while potential recipients have white-colored heads. airborne transmission may occur by close contact (at talking distance) and at longer distances (several meters). both types of heads are potential recipients through self-inoculation when touching the surface of contaminated objects (fomite sources). expiration includes normal breathing exhalation, coughing, and sneezing. airborne droplets may persist on the surface of objects (fomites) that may subsequently be touched by the hands, so causing self-inoculation. transmission through respiratory droplets forms the focus of attention in respiratory infection with droplets of >5–10 m diameter. in general, this has a very low risk of transmission of infection for persons in close contact (1–2 meters), because usually the droplets disappear rapidly from the air. because of gravity, these relatively large univ med vol. 40 no 1 60 yenny, herwana, wratsangka prevents covid-19 transmission a b c droplets deposit onto the floor or other surfaces within a few seconds.(21) environmental factors, in this case the airflow, also play a role in determining the droplet transmission distance. large droplets may persist in the air if the airflow in the environment can maintain the droplet suspension for a long enough time, such as strong cross-flows or ventilation that induces air flows. (21 ) aer osol or airborne transmission may occur through large or small particles that may persist for a considerable time in the air at transmission distances of more than 2 meters.(22) several studies conducted to identify the presence of covid-19 virus in respiratory air or by aerosol transmission yielded inconsistent results. a study conducted by chia et al.(23) showed positive air samples in two out of three isolation rooms of patients with covid-19, with viral particle sizes of around 1-4 m and >4 m (total concentration of sars-cov-2 in the air samples ranging from 1.84 x 103 to 3.38 x 103 rna copies per m3 of sampled air). the study of santarpia et al.(24) showed that 63.2% of air samples were positive, with mean viral load of 2.42 copies/l air. samples taken from around the beds of the patients and those from a distance of more than 2 meters, had covid-19 viral rna copies of 4.07 and 2.48 per l air, respectively. this study supports the opinion that covid-19 may be directly transmitted (droplet and personto-person transmission) as well as indirectly (by contaminated objects and airborne transmission). liu et al.(25) showed that more than half of the viral rna in the air samples had a size of <2.5 m and were therefore associated with aerosol transmission. the above studies show the occurrence of air samples that are positive for the sars-cov2 genome, which supports the notion of airborne covid-19 transmission. in contrast, several studies were showing contradictory results in connection with a irborne covid-19 transmission, such as the singaporean study of figure 1. several possible mechanisms of transmission of respiratory infections. (21) 61 ong et al.,(26) who did not find positive air samples from the isolation rooms of three patients. the study by alsved et al.(27) conducted on 12 choir singers and two patients with confirmed covid19 showed significant differences in particle emission between breathing, talking, and singing, but failed to detect the virus in the respiratory air of patients with c onfirmed covid-19. according to the investigators, the latter may have been caused by several factors, such as low viral concentrations in the air, differences in viral loads in the droplet-producing respiratory tract regions, or the dilution steps in preparing the samples. airborne transmission of covid-19 may occur when a person is exposed to droplet nuclei and is frequently found in healthcare personnel when performing medical procedures in hospital, resulting in the mechanical formation and dispersion of aerosols or triggering an aerosolgenerating procedure, such as when performing bronchoscopy, cardiopulmonary resuscitation, tracheal intubation, mechanical ventilation, oralmaxillofacial surgery, etc.(4) cloth masks cloth masks (cloth/fabric/homemade masks) are defined as masks that are made of any fabric such as cotton, gauze, silk or muslin.(28) the use of cloth or non-medical masks by the community is currently made mandatory by local governments in several countries, including indonesia. up to the present, there are still doubts arising, both from medical personnel and the general population, with regard to the efficacy of cloth masks in protecting against covid-19 transmission. several studies have been conducted to determine the efficacy of cloth masks in reducing the spread of respiratory infection in the community. the study conducted by davies et al.(29) aimed to evaluate the capacity of homemade masks (made of several types of textile) as alternatives to surgical masks as a physical barrier against bacteria and viral aerosols. this study determined the filtration efficiency of various types of cloth, such as cotton t-shirt fabric, linen, silk, and scarfs in comparison with surgical masks against two types of organism, namely bacillus atrophaeus (0.95–1.25 m in diameter) and bacteriophage ms2 (23 nm in diameter). the results showed that the mean filtration efficiency of surgical masks (89.52%) was higher than that of tea towel (72%), cotton mix (70.24%), linen (61.67%), silk (54.32%), cotton t-shirt fabric (50.85%), and scarf (48.87%). these study results also showed that the use of home-made masks was better than not wearing any mask at all.(29) the study conducted by jung et al.(30) aimed to evaluate the filtration capacity of various types of masks, using the protocols of the korean food and drug administration (kfda) and the us national institute for occupational safety and health (niosh). cloth masks made from cotton handkerchiefs consisting of minimally four layers, had a filtration efficiency of only 13%, whereas extremely porous textiles such as gauze, had a filtration efficiency of merely 3%, even though consisting of several layers. the study conducted by jang et al.(31) aimed to evaluate the filtration efficiency, the number of layers, and the effect of washing of commercial cloth masks as compared to respirators. the results of this study showed that the filtration capacity of cloth masks increased 1.7–4.6 times and 2.3–6.8 times when the cloth masks were folded into two and four layers, respectively, as compared with masks consisting only of one layer. after a single washing, the filtration efficiency of the cloth masks decreased 1.04–4.0 times as compared with the products before washing. a cluster randomized controlled trial conducted by macintyre et al.(32) aimed to compare the efficacy of cloth masks to that of medical masks used to protect healthcare personnel from respiratory infection. the study results showed that particle invasion through cloth masks was very high (97.0%) when compared with that of medical masks (44.0%). workers using cloth masks had a 6.64 times higher risk of acquiring an influenza-like infection (rr=6.64; univ med vol. 40 no 1 62 yenny, herwana, wratsangka prevents covid-19 transmission 95%ci=1.45-28.65) and had a 1.72 times higher risk of acquiring laboratory-confirmed viral infection (rr=1.72; 95%ci=1.01–2.94) as compared with those using surgical masks. the investigators concluded that cloth masks did not provide protection equal to that of medical masks so that they should not be recommended for healthcare personnel. the study conducted by neupane et al.(33) aimed to evaluate the filtration efficiency of cloth masks and surgical masks against aerosol-sized particles, and to determine the effects of washing, drying, and stretching on filtration efficiency. the study results showed that the filtration efficiency of cloth masks was variable (63.0–84.0%), while the efficiency of surgical masks was 94.0%. these results also showed that the filtration efficiency of cloth masks decreased by 20.0% after four times washing and drying as a result of changes in pore size and shape, and decreased microfiber in the textile pores due to washing. the study conducted by ma et al.(34) aimed to evaluate the efficiency of three types of mask (home-made, n95, and medical masks) to prevent infection with avian influenza virus. the study results showed that home-made masks consisting of one layer of polyester cloth and four layers of kitchen paper towels were able to inhibit viral aerosol particles by 95.15%, while n95 and surgical masks inhibited by 99.98% and 97.14%, respectively. the investigators stated that cloth masks were more breathable than n95 masks, and that kitchen paper towels could be replaced frequently. the study conducted by konda et al.(35) aimed to evaluate the filtration efficiency of cloth masks made from various types of fabric, namely cotton, silk, chiffon, flannel, synthetics, and combinations of these various types of cloth, and to determine their function in the filtration of aerosol-sized particles (10 mm–10 m). the study results showed that the filtration efficiency of various types of single-layer cloth varied widely for particles of <300nm and >300 nm in diameter, namely 5–80% and 5-95%, respectively. the use of combination cloth (cotton-silk, cotton-chiffon, cotton – flannel) in the manufacture of cloth masks increased the filtration efficiency of the masks by more than 80% (<300 nm) and 90% (>300 nm), respectively. the increased filtration efficiency of combination cloth masks was due to the combined mechanical and electrostatic effects of the cloth. the study results of konda et al.(35) confirm the possibility of using cloth masks for filtration of aerosol-sized particles (100 nm–1000 nm). table 1 may be seen as a summary of studies that determined the efficacy of cloth masks for the prevention of viral infection transmission. a systematic review conducted by jain et al.(36) showed that cloth masks have protective efficacy, but that penetration of cloth masks is higher than that of surgical masks and n95 respirators n95. cloth masks may be used in low risk areas and by the community if there are no surgical masks available. a systematic review and meta-analysis by sharma et al.(37) showed several factors that play a role in the filtration capacity of cloth masks, such as type of cloth used (thread count of the cloth), number of layers, degree of humidity, and compatibility of the masks with the face. cotton cloth masks consisting of three layers or more in combination with silk, chiffon, and flannel, that fit on the face with no or minimal leakage of air around the masks, and are not moist, provide the best protection in preventing viral infection. there are several limitations of cloth masks, namely that the physical features of cloth masks result in a lower filtration capacity in c o mpa r i s o n wi t h medi c a l ma s ks or n9 5 respirators, causing the virus to penetrate and spread through the mask by the capillary diffusion of liquids, particularly because the expelled air frequently causes the masks to become wet. the high humidity and temperature of the expelled air may result in dew precipitation within the mask as a result of the different temperatures within the mask and the outside air. the droplets expelled during speech increase the humidity of the mask, and additionally the 63 table 1. summary of studies that determined the efficacy of cloth versus surgical masks for the prevention of viral respiratory infection transmission investigators aim of the study study design/ subjects type of mask outcome davies et al.(29) to evaluate the potential of home-made mask as surgical mask alternative nonrandomized study on 21 volunteers no mask, homemade mask (100% tshirt cotton) vs surgical mask mean filtration efficiency of surgical mask (89.52%) vs tea towel (72%), cotton combination (70.24%), linen (61.67%), silk (54.32%), t-shirt cotton, and scarf (48.87%). home-made mask better than no mask. jung et al.(30) to evaluate the filtration capacity of various types of mask using protocols of the korean food and drug administration (kfda) and the national institute for occupational safety and health (niosh). laboratory study 44 brands of 4 types of mask and handkerchiefs cloth masks made from cotton handkerchiefs consisting of minimally 4 layers had a filtration efficiency of only 13%, while very porous cloth such as gauze had a filtration efficiency of merely 3% although consisting of several layers. jang et al.(31) to evaluate filtration efficiency, number of layers, and washing of commercial cloth masks laboratory study 5 commercial cloth masks vs respirator the filtration capacity of cloth masks increased 1.7–4.6 times and 2.3–6.8 times, respectively, if cloth masks were folded into two and four layers vs single layer masks. filtration efficiency of cloth masks decreased significantly 1.04–4.0 times after washing. macintyre et al.(32) comparison of efficacy of cloth masks vs medical masks in hospital healthcare personnel cluster randomized clinical trial on 1607 healthcare personnel local medical masks (3 layers of nonwoven material) vs. cloth masks (2 layers of cotton material) particle invasion through cloth masks (97%) vs medical masks (44%). cloth mask user has 6.64 times higher risk of influenza-like infection (rr=6.64; 95%ci=1.45–28.65) and 1.72 times higher risk of laboratoryconfirmed viral infection (rr=1.72; 95%ci= 1.01–2.94) vs surgical mask user univ med vol. 40 no 1 64 yenny, herwana, wratsangka prevents covid-19 transmission breathing process will mechanically increase viral penetration on the surface as well as within the mask.(38) the presence of gaps at the sides of the mask (due to incompatibility of mask and face) may reduce the filtration efficiency of cloth masks by 60.0%.(35) the physical features of cloth masks (poor filtration), reuse, cleaning frequency, and moisture retention, may cause the virus to persist on the surface of the masks, thereby increasing the risk of infection.(32,39) apart from the variable filtration capacity of cloth masks, there are several advantages of cloth masks, namely cloth masks can be made at home by the user and adapted to the user’s face, therefore being able to cover the gaps that are usually found around medical masks, and cloth masks can also be reused after having been washed and subjected to decontamination measures.(32,40) potential benefits and risks of instituting universal masking the recognition of droplet and airborne covid-19 transmission and the discovery of asymptomatic transmission as source of infection has resulted in the wearing of masks becoming one of the methods of preventing covid-19 transmission. the use of masks by the general population has the potential of the occurrence of “variolation”, which was the process in which an individual who was susceptible to smallpox was inoculated with material taken from the vesicles of patients with smallpox, in order to trigger a mild infection that eventually resulted in immunity.(41) the study conducted by sekine et al.(42) shows that cellular immunity results from mild as well as from asymptomatic covid-19 infection. a high prevalence of asymptomatic covid-19 may increase the immunity of the neupane et al.(33) filtration efficiency against aerosolsized particles; effects of washing, drying, and stretching on filtration efficiency laboratory study double-layered cloth masks vs. surgical masks filtration efficiency of cloth masks (63–84%) lower than surgical masks (94%). filtration efficiency of cloth masks decreased by 20% after four times washing and drying due to changes in pore size and shape and reduction in microfiber in pores of the cloth due to washing. ma et al.(34) comparison of filtration efficiency of 3 types of mask on prevention of avian influenza virus laboratory study cloth masks, surgical masks, n95 respirators all three types of masks could effectively inhibit avian influenza virus. home-made masks consisting of one layer of polyester cloth and four layers of kitchen paper towels could inhibit aerosol-sized viral particles (95.15%), n95 masks (99.98%), and surgical masks (97.14%). konda et al.(35) to assess the filtration efficiency of masks made of various materials and/or combinations laboratory study masks made of silk, cotton, flannel, chiffon, various synthetics, and their combinations filtration efficiency of cloth masks increased up to 80% (particles of <300 nm) and 90% (>300 nm) when multiple layers were used in mask fabrication, using combinations of cotton cloth with silk, chiffon, and flannel, respectively. table 1 (continued). 65 community (43) and reduce transmission while waiting for the availability of vaccines, even though the effective antibody concentration and the t cell immune response resulting from various manifestations of covid-19 have not yet been determined.(41) the more individuals are using masks in public places, where they are in close contact with one another, the more the whole community is protected because of the emergence of immunity such as occurs with vaccination.(44) the use of any type of mask can partially filter the viruses present in the droplets (with the filtration capacity depending on the type of mask used).(45) universal masking is of benefit in reducing the “inoculum” or viral dose for mask users.(46) exposure to a lower dose of inoculum may reduce the frequency of severe cases and constitutes the basic principle in vaccine development.(47) the recommendation with regard to the use of masks in the community for the prevention of covid-19 transmission, particularly in indonesia, is acceptable in view of the daily increase in the number of covid-19 cases in indonesia.(48) this shows that physical distancing cannot be sufficiently effective in preventing covid-19 virus transmission. universal masking is of benefit in protecting the individual against crosstransmission and reduces the risk of increasing covid-19 transmission upon relaxation of social distancing.(11) in connection with covid-19 transmission it should be kept in mind that droplet transmission is not the sole factor determining whether or not a person will become infected with covid-19. there are many factors play a role, such as climatic conditions (e.g. temperature and humidity) and population density.(49) other factors to be considered are the virus (viability, infective dose), the infected person (viral load), mode of transmission (droplet vs. airborne), aerosol processes (phase transformation and respiratory deposition), medium (building ventilation), and host (immune system, use of personal protective equipment).(40) apart from the potential benefits of universal masking for the general population, the mandatory use of cloth masks by the general population may engender a spurious feeling of security. because the public feels protected from covid-19 infection due to the use of masks, this may contribute to lowered compliance for maintaining hand hygiene, to poor coughing etiquette, violations in social distancing, and increased risks of touching the face and nose as a result of adjusting the cloth mask on the face.(50) guidelines on the use of masks by the community according to current guidelines, medical masks and respirators should be prioritized for medical personnel and individuals at high risk of infection, in view of their limited availability and higher cost. the who recommends the use of surgical masks for larger-sized droplets (>5 m) and short distances (<2 meters), such as in acute respiratory disease accompanied by fever, in respiratory syncytial virus and adenovirus infections, and influenza. the n95 respirator is used for aerosol infections (<5 m) at longer distances (>2 meters), such as in tuberculosis, measles, sars, and acute respiratory tract infections by new or unknown organisms.(51) on october 5, 2020, the us centers for disease control and prevention (cdc) again revised the guideline related to the potential of airborne transmission of covid-19. according to the cdc, airborne transmission may occur at distances of more than 6 feet (1.8 meters) and persist for several hours after a covid-19 patient or an asymptomatic person has left the room, if the room is closed and inadequately ventilated, or at the moment that the infected patient breathes out forcibly such as when singing or performing physical exercise that results in the production of smaller droplets at relatively high concentrations, thereby spreading the virus to others.(52) in connection with the recommendation on the use of masks by the general population, the cdc recommends the use of cloth masks in public and when being around persons who are not living in our household, particularly when physical distancing is difficult to maintain.(53) univ med vol. 40 no 1 66 yenny, herwana, wratsangka prevents covid-19 transmission on december 1, 2020, the who again revised its guidelines and recommended that the general population use fabric masks when being indoors (such as in shops, workplaces, schools, etc.) or outdoors when physical distancing of minimally 1 meter cannot be maintained. the use of medical masks is recommended for vulnerable subjects who may be at high risk of complications when infected with covid-19 (elderly aged 60 years, persons with comorbidities such as cardiovascular disease, diabetes mellitus, chronic lung disease, cancer, cerebrovascular disease, or on immunosuppressants) and in persons with suspected or confirmed covid-19, irrespective of the presence of symptoms.(13) currently there are guidelines issued by the who in connection with the types of masks that can be used by the general population in areas with known or suspected transmission of covid19.(5) in the selection of non-medical masks it is recommended to pay attention to the following: filtration efficiency, breathability, a combination of materials used, mask shape and coatings, and care of masks. masks should preferably be used by one person only, not shared with others, and should be frequently washed using warm water (60c) and soap or detergent. if no warm water is available, the masks may be washed with soap or detergent using clean water followed by boiling the masks for one minute or immersing the masks in 1% chlorine solution for one minute, then rinsed to remove residual chlorine.(5) conclusions to date, there has been no clinically tested drug that can cure patients with covid-19, so that the best way for breaking the covid-19 transmission chain is by taking measures for preventing viral exposure. based on the currently existing evidence, the filtration efficiency of cloth masks in the prevention of respiratory virus transmission is lower than that of surgical masks. however, there are studies demonstrating that the filtration efficiency of cloth masks may be enhanced by the selection of the textile material and the number of layers used. there is a need for standardization in the fabrication of cloth masks, so that they may give maximal effects to the wearer. universal masking in conjunction with social distancing and hand hygiene are the health interventions that can be instituted at present for the prevention of covid-19 transmission, while waiting for the discovery of definitive treatment for covid-19. conflict of interest all authors declare that they have no competing interests and have no non-financial interests that may be relevant to the submitted work. acknowledgments the authors are grateful to the faculty of medicine, trisakti university, for financial support. contributors y contributed to writing the draft of the manuscript. eh and rw contributed to editing and revising the manuscripts. all authors have read and approved the final manuscript. references 1. guan w, ni z, hu y, et al. clinical characteristics of corona virus disease 2019 in china. n engl j med 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the spread of covid-19. atlanta: centers for disease control and prevention;2020. 228 doi: http://dx.doi.org/10.18051/univmed.2022.v41.228-235 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1339 september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 attitudes towards covid-19 vaccination,vaccine hesitancy and vaccine literacy among unvaccinated young adults gulay yilmazel1 , emre keles1 , ayse calmaz2 , and burcu daysal3 abstract background herd immunity is necessary to control the coronavirus disease 2019 (covid-19) pandemic. unfortunately, attaining herd immunity is a significant challenge for current healthcare systems worldwide, and the prevalence of hesitancy toward covid-19 vaccination remains high. the aim of this study was to determine pandemic vaccine hesitancy, attitudes towards covid-19 vaccination, and covid-19 vaccine literacy level among unvaccinated young adults. methods this cross-sectional study was carried out in the province of çorum in turkey. unvaccinated young adults (n=860) between the ages of 18-30 years studying medicine and non-medical sciences at a university were included in the study. socio-demographic form, and the scales on pandemic vaccine hesitancy, attitudes towards the covid-19 vaccine, and covid-19 vaccine literacy were used to collect the research data. the data were analyzed using percentages, median values, and multiple linear regression analysis. results the study was completed with 860 participants. in the group, 73.8% of participants were 18-24 years old, 67.7% were women and 68.0% were infected with the virus. the median scores from the pandemic vaccine hesitancy scale, vaccine literacy and vaccine attitudes were 33, 27 and 30 respectively. use of social media per day, vaccine literacy and covid-19 vaccine attitude were effective factors on pandemic vaccine hesitancy (p<0.05). negative attitude to vaccines was the most influential risk factor of vaccine hesitancy (beta = 0.248). conclusion pandemic vaccine hesitancy coincides with low vaccine literacy and negative attitudes towards vaccines. covid-19 vaccination information campaigns should promote group strategies, focusing on emphasising the safety of the vaccine and offer reassurance. keywords: vaccine hesitancy, literacy, attitudes, pandemic, young adults 1hitit university, faculty of health science, corum, turkey 2hitit university, iskilip vocational high school, corum, turkey 3hitit university, alaca avni çelik vocational high school, corum, turkey *correspondence: gulay yilmazel hitit university faculty of health sciences, çorum-turkey phone and fax: +903642230730 03642230731 email: gulay-y19@hotmail.com; gulayyilmazel@hitit.edu.tr orcid id: 0000-0002-2487-5464 date of first submission, april 22, 2022 date of final revised submission, october 6, 2022 date of acceptance, october 13, 2022 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article cite this article as: yilmazel g, keles e, calmaz a, daysal b. attitudes towards covid-19 vaccina tion, vaccine hesitancy and vaccine literacy among unvaccinated young adults. univ med 2022;41:228-35. doi: 10.18051/univmed.2022.v41. 228-235. http://dx.doi.org/10.18051/univmed.2022.v41.228-235 mailto:gulayyilmazel@hitit.edu.tr mailto:gulayyilmazel@hitit.edu.tr https://orcid.org/0000-0002-2487-5464 https://orcid.org/0000-0003-4511-1508 https://orcid.org/0000-0002-8253-3956 https://orcid.org/0000-0003-1787-9536 229 introduction the development of vaccines is one of the greatest public health achievements in promoting global health. the burden of communicable diseases and lost years of life have been reduced by vaccination programs that contribute to the strengthening of the health system from a holistic point of view.(1-3) by preventing 2-3 million deaths annually, vaccines are one of the most costeffective ways to avoid disease. despite all these remarkable developments, anti-vaccination attitude is a phenomenon that hinders progress in the fight against vaccine-preventable diseases and is powered by the current of vaccine hesitancy. the reasons for the growing vaccine hesitancy around the world are complex. ignorance, difficulty in accessing vaccines or lack of confidence are the main reasons for not getting vaccinated. vaccine hesitancy is among the top 10 threats to global health according to the world health organization (who),(4) and there is an urgent need to identify the psychological and socio-demographic factors associated with this hesitation.(5) since the middle ages (6) (bubonic plague: 1347/covid-19:2019), there has been an increase in the frequency of pandemics and epidemics. overcoming the pandemic crisis depends on mass immunity and successfully tackling vaccine hesitancy.(7) hesitation about vaccination is theorized to depend on a number of cognitive, social and contextual factors (8) such as subjective risk perception,(9) psychological tendencies, fear of side effects, personal beliefs (10) and established beliefs and prejudices about vaccines. one of the lessons learned from past pandemics has been vaccine hesitancy. as with the swine flu, sars, and mers pandemics, immediately after the announcement of the covid-19 pandemic by the who, discourses about vaccine opposition, vaccine hesitancy or vaccine resistance began to spread.(11,12) in the period of september 2021 to february 2022, ireland comprised 89.8% of people who received at least two doses. western european countries with lower rates include the netherlands (77.2%), germany (73.4%), italy (72.8%) and luxembourg (72.4%).(13) this resistance, hesitation and opposition has accelerated the production and spread of numerous conspiracy theories about vaccines through social media.(14) in the pandemic era, the world is faced with the spread of the “covid19 infodemic” which creates confusion and risktaking behaviors that can be harmful to health, leads to distrust of healthcare, and undermines public health response.(15) another determinant of vaccine hesitancy is vaccine literacy (vl).(16) in parallel with the rapid development of vaccines in the fight against covid-19 globally, reports have emerged characterizing individuals who are vaccine hesitant. in the literature, it has been shown that the intention to vaccinate was higher in those who perceived covid-19 as a serious illness and those who had been infected with coronavirus.(17-19) on the other hand, it has been suggested that the perceived risk of vaccination is influenced by the innovation and rapid development of vaccines, emphasizing fearful cognitions about the speed and new technologies used that are unlike traditional vacc ine development approaches.(19,20) vaccine literacy is an important component not only as a level of knowledge about vaccines, but also for national vaccine coverage in a functioning health system.(21,22) however, limited vl is rarely taken into account when reviewing va ccine hesita ncy factors. (2 2) medi cal countermeasures (mcms) are central to the public health response to mitigate the impact of pandemics.(23) vaccination is the most effective medical countermeasure for contagious disease especially in epidemics and pandemics. this requires developing a well-matched, safe, and effective vaccine in the shortest time possible; deter mining the appr opriate dose; and administering it promptly. giving priority to vaccination to the adult and elderly group has resulted in more than one-third of cases clustering in adolescents and young adults in the covid230 19 pandemic. this situation has made it mandatory to make changes in vaccination schedules. (24) studies have also indicated that the novelty and rapid development of vaccines may have impacted the perceived risk of vaccination, with highlighted fearful cognitions concerning the pace and novel technologies utilized (i.e., mrna vaccines) contrasting with conventional vaccine development approaches.(20) among promotive factors, trust in health officials has been highlighted as a possible factor which may decrease individual perception of vaccination risks.(7) despite these preliminar y advances , several f actors of importance remain uninvestigated. it is accepted that if a certain part of the society is immune to an agent by any means, the chain of transmission will be broken and the spreading will stop. the end of the covid-19 pandemic will be possible with the provision of mass immunity. vaccine hesitancy among young adults may delay the formation of the herd immunity, therefore it should be prevented by scientific evidence, consistent communication, and improving individual vl levels. a study among 4,571 norwegian adults showed that perceived risk of vaccination, belief in the superiority of natural immunity, fear that a significant number of other persons were infected by the virus, and trust in the dissemination of vaccine-related information by health officials were identified as key variables related to vaccine hesitancy.(7) hence, at least a fraction of the remaining unvaccinated population might still be reached and decide to become vaccinated. further research, however, is needed to identify the underlying reasons for vaccine hesitancy of those remaining unvaccinated and to provide guidance on how to design appropriate interventions to reach them specifically. the aim of this study is to determine pandemic vaccine hesitancy, covid-19 vaccine literacy level, and attitudes towards covid-19 vaccination in the vaccination period among unvaccinated young adults. methods research design this cross-sectional study was carried out in the province of çorum in turkey from january to april 2021. study subjects two faculties of medicine and engineering science were included the study (n=2760). we calculated the minimum sample size based on the assumption that covid-19 vaccine acceptance is 50%. participants without cov id-19 vaccination were randomly selected from an online survey. unvaccinated young adults (n=860) between the ages of 18-30 years studying medicine and non-medical sciences at a university were included in the study. primary inclusion criteria for the participating participants were being of turkish nationality and aged between 18-30 years. the single exclusion criterion was having been vaccinated. data collection a questionnaire consisting of a sociodemographic information form and the scales of pandemic vaccine hesitancy, attitudes towards the covid-19 vaccine, and covid-19 vaccine literacy was used to collect the research data. demographic questionnaire a demographic information form was designed by the researchers to gather personal information from participants. participants were asked to report their age, gender, type of education, the presence of chronic illness, use of social media per day, and whether they were infected with covid-19. pandemic vaccine hesitancy scale the “vaccine hesitancy scale in pandemics” was made by modifying the original items of the “vaccine hesitancy scale” developed by larson, (25) and its reliability and validity were determined by çapar and çýnar.(26) this scale is a 5-point yiilmazel g, keles e, calmaz a pandemic vaccine hesitancy and literacy 231 a b likert type measurement tool, where 1=strongly disagree, 2=disagree, 3=neither agree or disagree, 4=agree, 5=strongly agree. high scores from the scale indicate high vaccine hesitancy in pandemics. the scale consists of 10 items contained in two sub-dimensions. the first sub-dimension “lack of confidence” consists of 8 items (m1-r, m2r, m3-r, m4-r, m5-r, m6-r, m7-r, m8-r). items with the letter “r” are reversed items. high scores in this sub-dimension indicate that the mistrust towards vaccines increases in pandemics. the second sub-dimension “risk” consists of 2 items (m9, m10). high scores in this subdimension indicate that the risk of vaccination is high in pandemics.(26) attitudes towards the covid-19 vaccine the sca le has 9 i tems and two subdimensions (positive and negative attitude). the statements in the scale are evaluated as “strongly disagree (1)”, “disagree (2)”, “undecided (3)”, “agree (4)”, “strongly agree (5)”. items in the negative attitude sub-dimension are scored inversely. a value between 1-5 is obtained by dividing the total score obtained by summing the item scores in the scale sub-dimension by the number of items in that sub-dimension. high scores obtained from the positive attitude sub-dimension indicate that the attitude towards the vaccine is positive. the items in the negative attitude subdimension (items 5-9) are calculated after they are reversed, and the high scores in this subdimension indicate that the negative attitude towards the vaccine is less. (27) covid-19 vaccine literacy scale the scale was developed by biasio et al.(28) to assess vaccine literacy (vl) skills and perceptions of covid-19 vaccine candidates, and attitudes and beliefs about available vaccines in the general population. vaccine literacy level had already been validated in terms of content and structure (29) and was evaluated by adapting a 14-item self-report scale for vaccination of adults. (30) from a psychometr ic point of view, functional vl questions are about language, including the semantic system, while interactivecritical questions focus more on cognitive efforts such as problem solving and decision making. each response is on a 4-point likert-type scale (for functional dimension: 4 never, 3rarely, 2 sometimes, 1 – often; for interactive critical dimension: 1 never, 2 rarely, 3 sometimes, 4 frequently). high scores from the scale indicate a high vl level.(28) adaptation of the scale to turkish was made by durmuþ et al.(31) statistical analysis all analyses were carried out using spss, version 21. skewness and kurtosis values indicated non-normal distribution. the data were described using percentages, median values, and analyzed by linear regression analysis. the hierarchical multiple linear regression analysis was performed for pandemic vaccine hesitancy. the unstandardized regression coefficients (β) and standardized regression coefficients (beta) were used to quantify the associations between variables. significance levels were set at the 5% level. ethical clearance ethics committee approval of this study, which was planned in accordance with the helsinki principles, was obtained from hitit university non-invasive ethical committee (2021/152). results the research was completed with 860 participants. of the study group, 73.8% were in the 18-24 age group with a mean age of 22.9±3.3 years, 67.7% were women, 76.3% had medical education, 52.8% used social media for 4 hours or more per day. it was determined that 68.0% of the participants were infected with the covid-19 virus (table 1). the distribution of the scores of the participants from the scales is given in table 2. univ med vol. 41 no. 3 232 discussion this study shows that pandemic vaccine hesitancy in young adults depends on vaccine literacy and attitudes to vaccines. in our study, pandemic vaccine hesitancy and negative attitudes to vaccines were high, whereas vaccine literacy was low among young adults. similarly, in maltese and austrian communities, covid-19 vaccine hesitancy and negative attitudes were found. (32,33) prior studies have reported results on vaccine hesitancy and refusal which differed significantly from country to country.(34-36) globally, there is a wide variation in vaccine hesitancy and refusal, ranging from 20% to 90%.(34,36,37) our citywide research, conducted during the covid-19 vaccination campaigns in turkey, found a relatively high pandemic vaccine hesitancy. our current study also identified that different factors are associated with pandemic vaccine hesitancy. the rapid development of vaccines in the covid-19 pandemic will be effective in the acceptance of vaccines to be developed in current or future pandemics. the dominant factors influencing the acceptance of these new vaccines are concerns about vaccine risk, safety and efficacy. common factors contributing to hesitation towards covid-19 vaccines include fear of side effects and concerns about vaccine safety and effectiveness.(38) previous studies suggested that vl is associated with vaccine perception.(28,39) perceptions regarding future covid-19 vaccines, along with beliefs about vaccination, were mostly positive and significantly associated with functional and interactive-critical vl scale.(22) as a matter of fact, in the present study, lack of confidence in vaccines and the median scores from the pandemic vaccine hesitancy scale was 33, the median of the scores from the lack of confidence sub-dimension was 27, and the median of the scores from the risk sub-dimension was 6. while the median of the scores of the participants from the vaccine literacy scale was 27, the median of the scores from the vaccine attitude scale was 30, the median of the scores from the positive sub-dimension of this scale was 14 and the median of the scores from the negative sub-dimension was 16. similar associations were found in the multivariate linear regression analysis in table 3. in particular, participants with low vaccine literacy and negative attitudes to vaccines were significantly more likely to hesitate (p<0·05). negative attitude to vaccines was the most influential risk factor of vaccine hesitancy (beta = 0.248). table 1. demographic and descriptive data of the subjects (n=860) table 2. the distribution of the scores from the scales yiilmazel g, keles e, calmaz a pandemic vaccine hesitancy and literacy variables n (%) gender male female age (years) (mean ± sd) education type medical non-medical presence of chronic illness yes no use of social media in daily (hour) <4 ≥4 infected with covid-19 yes no 278 (32.3) 582 (67.7) 22.9±3.3 186 (23.7) 674 (76.3) 106 (12.3) 754 (87.7) 406 (47.2) 454 (52.8) 275 (32.0) 585 (68.0) scales mean sd median min max pandemic vaccine hesitancy (total) 32,4 5,6 33 10,0 48,0 lack of confidence sub-dimension 26,2 5,8 27 8,0 40,0 risk sub-dimension 6,2 1,9 6 2,0 10,0 vaccine literacy 27,3 6,5 27 12,0 49,0 attitudes towards the covid-19 vaccine (total) 30,1 6,8 30 9,0 45,0 positive vaccine attitude sub-dimension 13,9 3,8 14 4,0 20,0 negative vaccine attitude sub-dimension 16,3 4,0 16 5,0 25,0 233 univ med vol. 41 no. 3 perception of vaccination risks came to the fore at the height of pandemic vaccine hesitancy. in addition, it was determined that the vl was weak and the negative attitude towards the vaccine was high in the participants. pandemic vaccine hesitancy has incre ased with the lack of confidence in the vaccine. limited vl as a component of vaccine acceptance and adequate herd immunity is rarely considered when revi ewi ng f actors f or vacci ne hesi tancy.(22) health literacy has a great role in the adoption and implementation of preventive health behaviors, especially vaccination. vl is a subset of health literacy and a determinant in examining attitudes towards vaccination.(40) in previous studies, it had been reported that there were limitations in the health literacy levels of students training at medical faculties.(41,42) on the other hand, it has been shown that covid-19 vaccine hesitancy was high in students training in medicine and health sciences.(43) however, the use of social media platforms to seek and share health-related information may influence decision-making about covid-19 va ccination. (4 4) va cc ine misinformation and fake news on social media can increase vaccine hesitancy levels, hinder progress towards mass immunity, and increase the number of new cases of covid-19 variants.(45) all these r esults suggest that strategies to increase vaccine literacy should be considered first in vaccination campaigns, especially among young people. the main limitation of our study is the timing of the survey in the early months of 2021. at that time, vaccination was common in the fragile and in essential working segments of the society (elderly, chronic disease, health personnel, teachers, etc.). in future research studies, the dynamics of vaccine hesitancy in pandemics should be closely monitored and vaccine literacy should be enhanced during school and university periods. conclusions in summary, pandemic vaccine hesitancy is a phenomenon that coincides with low vl and negative attitudes towards vaccines among unvaccinated young adults. the fight against this phenomenon must be included in the pandemic action plans. in addition, pandemic action plans should be updated for young adults as well as at risk groups. conflict of interest compe ting inte re sts: no re levant disclosures. acknowledgement thanks are due to all persons who participated and/or assisted in this study. contributors gy contributed to planning of research, data analysis, writing, a nd english editing; table 3. regression predicting of pandemic vaccine hesitancy note : beta=satndardized regresion coefficient variables β (regression coefficient) beta p value age gender education type presence of chronic illness use of social media in daily infected with covid-19 vaccine literacy covid-19 vaccine attitude -0.028 -0.324 0.798 0.323 -0.827 0.496 -0.131 0.397 0.016 0.027 0.060 0.020 0.073 0.042 0.149 0.284 0.613 0.408 0.065 0.548 0.024 0.199 0.000 0.000 234 ekcontributed to data analysis;aç and bd contributed to data collection. all authors have read and approved the final manuscript. funding: 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the acceptance of the covid-19 vaccine: a cross-sectional study from saudi arabia. patient prefer adherence 2021;15:2673-81. doi:10.2147/ppa.s342535. 45. muric g, wu y, ferrara e. covid-19 vaccine hesitancy on social media: building a public twitter data set of antivaccine content, vaccine misinformation, and conspiracies. jmir public health surveill 2021;7:e30642. doi: 10.2196/30642. univ med vol. 41 no. 3 c:\users\user\documents\39 no 3 212 abstract universa medicina coronavirus disease 2019: a revolution in biological triage in the emergency setting shahram manoochehry*, fatemeh sabouri**, mehrdad faraji*, and mohammad javad behzadnia*@ triage is a familiar concept for all who work at the forefront of the treatment of patients and the emergency medical staff know their duty in different situations including crisis and epidemics. accordingly, the coronavirus outbreak has also a major effect on the emergency department (ed) as it changes the routine ed function. our main question was what are the main triage challenges in the recent pandemic and how we could overcome these challenges? therefore, a search of the main web databases was performed for articles published till may 1st, 2020 using various related keywords. in various steps, the title, summary, methodology, results, and discussion of the selected studies were assessed to find out the recent triage strategies in the covid-19 outbreak. hence, all the available and related english review articles, case series, and experimental studies were evaluated. among 200 studies initially reviewed, 59 met the study criteria for the final assessment. covid-19 puts a significant load on public health services and potential damage to social and psychiatric situations by marked morbidity and mortality. in line with the various presentations and according to the changing of the covid-19 epidemic to a worldwide pandemic problem, the management and treatment protocols changed several times. accordingly, the local and even global hospital protocols were changed as well. the first simple concept of coronavirus triage in an emergency department is the separation of covid-19 infected patients from the others. this approach has been practiced around the world. changing the ed layout from a usual triage or fast track set to an isolated room is necessary for such a pandemic situation. it is very important to consider staff communication and the application of ppe. all the efforts should be taken to protect patients as well as the medical staff from unnecessary exposure and infection; this serves to keep the health facilities working well in the outbreak and diffusion of sars-cov-2. keywords: covid-19, sars-cov-2, triage, emergency medicine, coronavirus review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.212-223 september-december 2020 vol.39no.3 cite this article as: manoochehry s, sabouri f, faraji m, behzadnia mj. coronavirus disease 2019: a revolution in biological triage in the emergency setting. univ med 2020;39:212-23. doi: 10.18051/univmed.2020.v39. 212-223 *trauma research center, baqiyatallah university of medical sciences, tehran, iran **department of pathology, shohada hospital, qom university of medical sciences, qom, iran correspondence: @mohammad javad behzadnia, trauma research center, baqiyatallah university of medical sciences, tehran, ir iran, nosrati alley, sheikh bahaie st, molla sadra st, vanaq sq., tehran, ir iran tel.: +98-2181262012, email: behzadnia@bmsu.ac.ir orcid id: 0000-0002-3062-0839 date of first submission, october 4, 2020 date of final revised submission, november 30, 2020 date of acceptance, december 7, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 213 univ med vol. 39 no.3 introduction triage is a familiar concept for all who work at the forefront of the treatment of patients; almost all of the emergency medical staff know their duty in different situations including crises and epidemics. within the outbreak of coronavirus 2019 and the changing into a pandemic, triage found a new meaning which will be discussed here. the coronavirus outbreak has also a major effect on the emergency department (ed) as it changes the routine ed function. nowadays, triage becomes challenging in various situations during the current covid-19 pandemic; as the clinician should decide which non-covid infection patient should be hospitalized due to the other critical reasons, indeed, they should do this by risk assessment and outcome evaluation. according to different policies, triaging occurs at the entrance into the emergency department and may differ in various hospitals. three, four, or five-level triage systems are commonly used in various parts of the world; the latter seems to be more helpful under normal circumstances, but in this pandemic as a biological attack, it necessitates a change in the routine triage protocol. with the passing of the time and symptom variations, the emphasis on vital signs during triage also changed, given that the purpose of triage in biological crises is to achieve maximum benefits and mortality morbidity reduction. the novel coronavirus (covid-19) which now has spread worldwide was first distinguished in wuhan, china in late december 2019 and was verified by the world health organization as a pandemic on march 1, 2020 while only 118000 cases were globally reported in the whole world.(1,2) this pandemic at a large scale put a burden on the world’s health infrastructures. in line with the various presentations and according to the changing of the covid-19 epidemic to a worldwide pandemic problem, management and treatment protocols changed several times. accordingly, the local and even global hospital protocols were changed as well. because this is a novel topic, to find previously published studies relevant to the current study question, a search of the web of science, google scholar, cochrane, and pubmed databases was performed for articles published t i l l m a y 1 s t, 2 0 20 us i ng t he ke ywor ds “coronavirus”, “corona”,” sars-cov-2", “ sars”,” mers” “emergency medicine”, “emergency”, “triage”, “covid-19”, and “biology*”. a combination search based on mesh terms was also performed. the prisma checklist was used to evaluate the validity of the selected articles.(3) during the previous middle east respiratory syndrome coronavirus (mers-cov) outbreak, saudi arabia used a visual triage scale for evaluating and scoring suspected cases; but further studies showed it had not enough effectiveness. (4) in various steps, the title, summary, methodology, results, and discussion of the selected studies were assessed. initially, the titles and abstracts were evaluated and saved to endnote x8. then, the full-text of english review articles, case series, and experimental studies were evaluated. letters and duplicate studies were excluded. in the next stage, full texts were reviewed by two independent experts, and unrelated studies were excluded. some similar and duplicate studies had no new information. accordingly, some other papers for which the full text was not available were excluded in the second and third stages. among 200 studies initially reviewed, 50 met the study criteria for the final assessment. evidence-based triage changes covid-19 puts a significant load on public health services and causes potential damage to social and psychiatric situations by its marked morbidity and mortality. in this condition, triage has found new meanings in various places such as surgical triage, psychological triage, etc.(5,6) symptom-based triage the first simple concept of coronavirus triage in an emergency department is the 214 separation of covid-19 infected patients from the others. this approach has been practiced around the world since the early days of the pandemic but further evaluation showed no effectiveness as well. in the early days of the problem, triage was mainly based on clinical symptoms such as fever, chills, and sore throat, along with controlling the percentage of blood oxygen saturation (o 2 sat%) and the other vital signs. in this protocol, o 2 sat% (93%) and temperature (37.80c) were more emphasized and even became the criterion for further follow-up evaluations.(7) in previous studies, 28.6% of confirmed coronavirus related respiratory cases had fever, but now, there are different reports from various countries (table 1). in a us healthcare system study; 30.7% of patients were febrile, 17.3% had a respiratory rate of more than 24 breaths/minute and 27.8% received supplementa l oxygen during the triage.(8) smoking and co-infection with two or more viruses were the two significant predictors of fever. fever is less common among adults in viral infection than the children; in a study, more than 75% of adults with viral respiratory infection did not have a fever and this is what we see in the novel covid-19 outbreak. during the first weeks, we have visited many febrile patients who had respiratory symptoms but when the outbreak spread globally most of the patients were afebrile. it seems that the virus symptoms are changing over time. (9) thus, the fever is not considered as an important criterion now. fur thermore, ther e were some confirmed patients who had no symptoms at all. field experiences over the past months have shown that many patients who come to the emergency room accidentally or for other reasons had covid-19 positive tests, so there is a need for a modified evidence-based triage system at the early stages. accordingly, even some routine triage criteria should temporarily change during the pandemic. more exact field investigations showed how medical staff and monitoring systems could change or correct the initial protocols t hrou gh th e c urr ent pa ndemic. according to recent experiences, some triage categories have now developed. consequently, some rare symptoms have been added to the above presentations and some cutaneous manifestations are reported as cov id-1 9 p r e s e n t at i on a nd s ho ul d be considered during the patient’s triage. (10 ) neurological manifestations from mild to severe symptoms are also reported as the new covid19 presentations.(11) triage structure and design in the age of coronavirus-19, it is strongly advised to set up special hospitals, install quarantine wards, and establish preventive and controlled systems for inf ected patients. furthermore, other hospitals could safely treat the other non-covid-19 patients. inpatient and outpatient units have to be separated in such a way that the patients who are in ed are not exposed to the risk of cross-infection at the early stages (figure 1). most common symptoms less common symptoms serious symptoms fever aches and pains difficulty breathing or shortness of breath dry cough sore throat chest pain or pressure tiredness diarrhea loss of speech or movement headache loss of taste or smell a rash on skin, or discoloration of fingers or toes table 1. the most common, less common and serious symptoms of covid -19 manoochehry, sabouri, faraji, et al coronavirus biological triage in the emergency setting 215 univ med vol. 39 no.3 a b c figure 1. emergency department triage in covid-19 outbreak in italy, emergency departments (eds) were reorganized to tackle the covid-19 pandemic, including the introduction of pretriage and different care paths for patients. structurally, the eds we re divi ded into separ ate compartments, termed clean and infected zones, respectively. according to pretriage results, regular non-covid-19 were sent to the clean zones, whereas covid-19 patients were sent to the infected zones. both clean as well as infected patients subsequently underwent the standard triage procedure.(12) designing some field hospitals is necessary to avoid further referral hospital infection; chinese experiences in developing fangcang shelter hospitals are notable. these temporary hospitals have various essential functions such as isolation and triage, basic medical care, screening, and rapid referral parts for public health emergencies.(13) accordingly, appropriate designing for pre triage, diagnosis, and even isolation of doubtful or verified cases is also critical. triage structure design in a hospital is now a multidisciplinary and collaborative task; 216 as it has many different aspects, space definition, resource prioritization, supplies, infection control measures, staff selection, and many other factors need a widespread and multilevel protocol. it should also consider patient and medical staff safety. (14) triage system considerations before admission suspected coronavirus infection triage at the beginning of the pandemic in many parts of the world, workers in healthcare centers continued to visit coronavirus patients in addition to their routine tasks; but gradually the burden of the patients reached such a level that some additional special centers were needed to treat them. thereafter, more challenging issues arose in the triage of the patients. in this way, the traditional triage system could no longer result in the recognition of suspected coronavirus cases. accepting non-corona patients, how to visit and refer them to the other centers, and how to triage and deal with acute presentations of cov id-19 i nf e ct ion b ec ame t he new challenges.(15) triage of high-risk patients against highrisk symptoms changing the specific guidelines has also led to a revolution in emergency triage; the handling of many cases that normally needed to be investigated and treated urgently have been altered to observe more in non-life-threatening circumstances to avoid infection. the first group c o nt a i n s p a t i e nt s w it h a hi s t or y of immunodeficiency for any reason, whether pharmacological or due to an underlying disease; the second group contains the suspected patients with other acute manifestations. besides the lifethreatening situation, we may encounter patients who have high-risk and emergency symptoms; they may need acute surgical, gynecological, or other emergency interventions. furthermore, emergency trauma care is another aspect that needs timelines of interventional effectiveness in the outbreaks, hence elective surgeries and procedures should be postponed for later times.(15,16) a multidisciplinary team approach for triage team work and collaboration in triage during the triage and in the next stages, the number of organ failures, survival prediction, and performance score could help to prioritize the critically ill patients in some situations. however, the proper decision should be made by exper ie nced special teams. ce rta inly, regional, national, and even international collaboration is needed to overcome this pandemic. (17) emergency radiology, as a relatively new subspecialty, plays a critical role in both the diagnosis and triage of acutely ill patients, and provides leadership to coordinate with the radiology department. then they could deal better with novel highly infectious patients.(18) eme r ge n c y a n d r a di o l ogy de pa r t me nt coordination is critical, as the suspected respiratory patients should be referred for a chest x-ray or ct scan evaluation without delay. in this condition, all the patients with respiratory symptoms are considered as probable cases of covid-19 until proven otherwise. risk stratification of covid 19 patients (in terms of clinical condition, patient health, hospital resource availability) and proper decision-making by multidisciplinary teams could decrease the faults and minimize the hospital stay. on the other hand, focusing on restricted protocols may lead to admission of more negative patients and increasing the risk of in-hospital inf e ct ious t ran smis si on. a h igh le ve l of compliance could be achieved only by continuous observation, constant surveillance, and feedback reporting to the medical staff (figure 2.). no wa d a ys, di f f e r e n t pr o toc ol s a nd approaches are needed for different situations and epidemics. accurate triage for surgical procedures is needed not only by telephone at home before hospitalization but also at the time of admission (telemonitoring surveillance).(19) manoochehry, sabouri, faraji, et al coronavirus biological triage in the emergency setting 217 univ med vol. 39 no.3 figure 2. team work and collaboration in triage surgeons need close collaboration with infection control specialists to avoid cross-infection in the hospital and to provide optimized treatment. continued screening and triage have a primary preventive effect. (20) special high-risk groups in triage the coronavirus outbreak requires special considerations on particular groups. as a highrisk population with mor e mortality and morbidities, the elderly with comorbidities require more special consideration.(21) subsequently, remote monitoring of infection risk in people who are on immunosuppressant drugs, during the covid-19 pandemic is a possible and practical approach. pregnant women are a susceptible high-risk group during this pandemic and therefore need special care and management in the emergency department. anatomical feature and immunity alteration make the pregnant women susceptible to viral infections such as covid-19; so all the maternal health settings should have a good management system to minimize their risk during the outbreak.(22) considering minimal contact could significantly decrease the risk. prenatal care, proper triage, and recognition of high-risk cases, admission control, and more supervision are the main things that should be considered in maternal health care during the outbreak of cov id-19.(22) covid-19 has a significant effect on cardiovascular problems, such as increasing the risk of severity, complications, drug-related side effects, and death. having an exaggerated search of covid-19 related symptoms could affect non-coronavirus patients such as those with cardiovascular conditions.(23) any micro aerosol-generating procedure should be stopped during the covid-19 pandemic. in previous outbreaks (2017-18), all age groups had the same mortality rate, but the males were interestingly more affected (74.7%) with an equal mortality rate in both genders (30.5%). it seems that the nove l cov id-19 has a mo re si gni fi ca nt morbidity rate than previous virus outbreaks. therefore, current triage systems should be more a w a r e o f th e i r h a za r do u s a nd i n f e c t i ve features.(24-26) 218 sp eci al c o nsideratio n s f o r health care workers personnel education and safety in triage changing the ed layout from a usual triage or fast track set to an isolated room is necessary for such a pandemic situation. it is very important to consider staff communic ation and the application of personal protective equipment (ppe). all the efforts should be taken to pretest patients as well as the medical staff from unnecessary exposure and infection; this results in keeping the health facilities working well in the outbreak and diffusion of sars-cov-2. the importance of infection control is therefore crucial in limiting the effects of virus diffusion. proper and regular healthcare professions training and education are necessary during the covid-19 outbreak, in which occupational and health safety are the two main components.(27) procedures that produce aerosols such as advanced airway management or cardiopulmonary resuscitation (cpr) have a significant risk. hence, the emergency department pe rsonnel need to be well equipped with appropriate ppe and trained in their use.(28) guidelines of triage should contain a protocol for appropriate use of personal protective equipment and precautions to be taken in every stage of the management and help for allocating scarce resources. the reinforcement of specific infection control training is necessary for medical staff. as in the previous viral infections, the novel coronavirus-19 clinical presentation varies from symptomatic cases to severe fatal disease. healthcare involvement could easily occur among an overcrowded poor compliance infection control setting and unrecognized infected people could easily be missed in a poor triage system.(29) experiences from the outbreaks of the middle east respiratory syndrome coronavirus (mers-cov) showed multiple healthcare association involvement. some studies showed inappropriate donning and doffing of ppe and n95 mask fit testing among the healthcare workers who encountered the patients directly; so the application of special protective protocols could minimize the risk of cross-infection significantly. (30) as their direct contact with covid-19 patients, healthcare providers are at high risk of infection transmission during the triage and treatment procedures; so having a contact distance of at least 2 meters and wearing a special mask and other ppe, could play a strong role. the role of laboratory data in triage in some medical centers, taking a chest ct scan is considered as a screening route for retriage and admission of non-corona patients who have other critical conditions in the ed. problems and critical illnesses such as acute abdomen have a high incidence in the emergency room, requiring selective or emergency surgery. epidemiological and preliminary screening of viral nucleic acids; taking of chest ct examination during the perioperative period to rescreen covid-19 and risk reduction of droplet and aerosol transmission, should be performed to reduce patient’s susceptibility and shorten the length of postoperative hospital stay. in some conditions, the physician could triage the suspected febrile patients with simple laboratory data such as complete blood count (cbc) with a differential count.(31) some other modalities such as lung ultrasonography can also help to find the involved patients and facilitate the triage process. it could determine the workflow of the affected patients to the next step of care. (32) the physician may visit some asymptomatic patients who have taken a laboratory test on their own initiative and are bringing the test results themselves. the diagnostic testing should be simplified and available to healthcare providers to allow them to decide more rapidly and accurately.(33) this categorization could determine the severity and level of infection; subsequently, proper protective equipment is required. merscov patients presented with monocytosis, normal white blood cell (wbc) count, and a lower c reactive protein (crp); but leukopenia and lymphopenia with mild eosinopenia and manoochehry, sabouri, faraji, et al coronavirus biological triage in the emergency setting 219 univ med vol. 39 no.3 elevated crp (4 mg/l) are much more frequent in covid-19 patients than in the controls. these laboratory findings could be helpful for better triage and treatment of febrile symptomatic patients in the ed.(31, 33) evaluation of triage effectiveness is very important for hospital managers for future decision making. in this way, they could recognize the ultra-high risk, high risk, and moderate risk areas according to various risk stratifications.(33) proper clinical parameter definition for triage could help the staff to better manage the positive patients and lead to the best outcomes. furthermore, the effectiveness of an emergency department in pandemic situations such as covid-19 could be measured by a reduction in positive cases, admissions, and control of transinfectivity. so ed function could be improved by identification of the high-risk patients and proper admission.(34) establishing some isolation areas for triage is the first step to counter a pandemic surge such as covid19. exposure to unsuspected patients, lack of triage to use ppe and infection control, lack of awareness and preparedness place the health care providers at a high-risk level.(33,35,36) te l e t r i age and di gi t al t e chno l og y i n covid-19 methods of an effective and applicable triage setting are based on symptom severity and patient comorbidities. in addition, to confirm the virus transmission, all the involved health care systems should use information technologies.(37,38) the role of telemedicine in resource conservation and remote patient care along with management strategies is obviously important. whenever possible, medical staff should shift their visits away from the direct face to face visits to some other kinds such as telemedicine and video screening. (39) web-based monitoring and triage could somewhat help to overcome the limited equipment and prevent unnecessary healthcare exposures.(40) active triaging and screening for symptoms of covid-19 infection could be considered for malignant and palliative care patients who are at their homes to avoi d f urther infection dissemination. telephonic screening could also help to assess their condition.(39, 41) self-triage and self-scheduling plans are suggestive tools that the patients who are not critically ill could carry out themselves, explain their symptoms, and shorten the unnecessary triage times in the hospitals; thereby improving the triage efficacy and postponing all elective visits and surgical procedures.(42) accordingly; digital technologies could help the health organizations to have a valuable role in various stages of the covid-19 outbreak, from screening and triage to diagnosis and monitoring. video consultation in the triage of patients with covid-19 is a way to increase the protection of personnel.(43,44) having a protective policy in triage is helpful for the allocation of the scarce resources when it is necessary to select the patients for urgent visits or telemedicine follow-up.(45) designing a digital triage tool to identify people with a high risk of covid-19 infection is a possible approach for remote monitoring of highrisk patients, therefore, it could limit unnecessary access to healthcare or medical centers and reduce the spread of the infection.(46) delayed diagnosis, inappropriate use of ppe, and ed overcrowding are the main factors that are necessary for a triage setting. the well-being of the health care providers is the first step of every medical care setting; hence all the policies should primarily focus on medical health care providers. workload and psychiatric pressures could eventually lead to caregiver burnout; therefore, it affects medical care and patients’ ma nagement. using telemedicine and social media could partially prevent this workload.(47) in a pandemic such as covid-19, the emergency physicians as the frontline healthcare providers should be aware of ed triage, facilities, and isolation policies, to deal with a surge of suspected cases. the design of appropriate devices and procedures for pre-triage, diagnosis, and isolation of suspected and confirmed cases 220 should be the final goal.(48) elective treatment and procedures should be postponed until the situation become more stable.(49) the covid-19 pandemic leads to delay of many routine treatments; hence it is necessary to have a risk assessment for the patients. developing some new protocols for emergency management among the covid-19 outbreak is essential. all specialties should have their own triage guidelines for their critical patients in the outbreaks. it is also necessary to update the previous protocols. hence, they should design a conceptual framework for emergency and timely triage. (50,51) in order to avoid loss of re sourc es physicians with an overlap in therapies should have well-defined arrangements among each other concerning the treatment spectra. patient triage and risk assessment before any non-urgent procedures, resource allocation and prioritization, regular monitoring of personal protective equipment, infection control measures, and protective device training should be a part of every hospital against the covid-19 infection. eventually, coronavirus outbreaks bring us some excellent lessons in the triage concept. first of all, as a biological threat, covid-19 triage is not as simple as we think. in addition, coronavirus infection symptoms are more complex and various in different peoples. according to new conceptions, many medical policies are changed during these pandemics. using some immune response assay, molecular and rapid igm/igg testing could help to find the suspected patients more rapidly. hence, early screening and triage may improve the response time in clinical and public health work, a lthough their exact importance is still unknown. considering the hospital status, icu, and ventilator capacity or supplies limitations that are likely to evolve over the few hours or days, we saw a significant revolution in medical interrelationship, include some usual medical plans that were not more efficient and in need of modification. this biological threat also has many other lessons such as how to triage different patients with or without covid-19 symptoms and how to guide them to have laboratory and radiological assessment. it teaches our medical sta ff how to protect themselves against a non-visible threat in an emergency setting. conclusion as mentioned, we may encounter some similar biological threats such as covid-19 infections that even change the infrastructure parts of the medical systems. it seems that preparing the medical personnel and wellequipped hospitals is more serious in the future. furthermore, the bioethical points in various parts of triage and diagnosis to do proper treatment are crucial in patient management. finally, we should think about how to conserve hospital critical resources such as icu beds, respirators, transmission capacity as well as protective gear (e.g. ppe for protection of the staff and patients against unnecessary exposure and intrahospital transmission. we hope this information could help the medical staff in all parts of the world. conflict of interest none. acknowledgements the authors would like to thank the “clinical research development unit of baqiyatallah hospital” for all their support, guidance and financial supports during carrying out this study. contributors sm and mjb contributed to conception and design, mf helped in acquisition, analysis and interpretation of data. mjb and fs contributed to drafting the article, critical revision of the article and all of the authors contributed the final approval of the version to be published. manoochehry, sabouri, 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10.3906/sag-2004-132. 51. pulia ms, o’brien tp, hou pc, schuman a, sambursky r. multi-tiered screening and diagnosis strategy for covid-19: a model for sustainable testing capacity in response to pandemic. ann med 2020;52:207-14. doi: 10.1080/ 07853890.2020.1763449. c:\users\universa medicina\docu 56 abstract universa medicina death anxiety and its association with severity of mental illness in patients with depression and schizophrenia hamidreza shahraki mojahed1* and somayeh nakhaei2 background death and the anxiety of it becomes more apparent when confronted with a chronic disease. the aim of this study was to determine the level of death anxiety among patients with depression and schizophrenia, and the relationship between severity of the symptoms and death anxiety in these patients. methods a cross-sectional study was conducted on 29 patients with a depressive disorder, 18 with schizophrenia spectrum disorder and 31 healthy subjects, through face-to-face interviews. the general severity of the psychiatric disorder was measured using the clinical global impression scale (cgi). the severity of depressive symptoms was measured using the beck depression inventory (bdi). psychosocial performance was measured using the personal and social performance scale (psp). to evaluate death anxiety, the bochum questionnaire on attitude to death and death anxiety 2.0 (bofretta 2.0) was used. one-way anova and pearson correlation tests were used to analyze the data. results regarding the bofretta attitude scale, the schizophrenic patients (23.1±9.12) showed significantly higher scores than healthy subjects (18.4 ±4.56) and depressive patients (19.8 ± 5.20) (p<0.050). the healthy controls achieved the least scores of bofretta anxiety scale followed by depressive (30.67±10.33) and schizophrenic patients (31.30±12.18). however, there were no significant between-group differences regarding this scale. we found significant correlations of all bofretta dimensions (attitude, anxiety, sum score) with cgi, psp and bdi. conclusion there was a significant relationship between the severity of symptoms and death anxiety in both depressed and schizophrenic patients. however, the latter showed a more negative attitude towards death. keywords: death anxiety, schizophrenia, depression, severity, mental illness original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.56-63 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1285 january-april 2022 vol.41no.1 cite this article as: shahrakimojahed hr, nakhaei s. death anxiety and its association with severity of mental illness in patients with depression and schizophrenia. univ med 2022;41:5663. doi: 10.18051/univmed.2022.v41. 5 6 6 3 . 1phd program in the clinical psychology, department of psychology, islamic azad university, zabol branch, zabol, iran 2assistant professor of psychology, faculty of human sciences, zabol branch, islamic azad university, zabol, iran *correspondence: hamidreza shahraki mojahed doctor of clinical psychology, department of psychology, islamic azad university, zabol branch, zabol, iran. email:zhn.search@gmail.com orcid id: 0000-0002-9810-1170 date of first submission, january 27, 2022 date of final revised submission, march 20, 2022 date of acceptance, march 28, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 57 univ med vol. 41 no 1 introduction death anxiety is considered one of the basic human characteristics and has been recorded over the past 4000 years. more than three decades of experimental research and hundreds of studies have shown that death anxiety affects a wide range of human behaviors.(1,2) to cope wi t h de a th a n xi e t y, hu ma n be i ngs ha ve developed a number of adaptation strategies, namely seeking for meaningful achievements and relationships, and creation of heritage.(3) while some people may cope with this anxiety effectively, others resort to ineffective coping mechanisms, such as avoidance, which only make their anxiety worse.(1) extensive studies have shown that death anxiety is a “transdiagnostic construct” with the potential to cause maladaptive behaviors, which provides the basis for a range of different mental health states.(4,5) the trans-diagnostic role of death anxiety in mentally ill patients has been confirmed by an increasing number of experimental studies, indicating that death reminders increase the maladaptive behaviors related to specific phobias, anxiety of attending the community,(6) and eating disorders.(7) obsessive-compulsive disorder has been argued to be a major cause of death anxiety. in fact, it has been suggested that death anxiety includes subtypes of this disorder. although it has also been argued that some of these obsessions may not be related to death at all (such as sexual obsessions),(8) death anxiety appears to be the main cause in most forms of obsession. there is increasing evidence supporting the fact that death reminders can stimulate an anxious response among patients with social anxiety and spider phobia. some other studies found significant correlations between death anxiety and hypochondriasis, depression, s c h izo p hr eni a a n d p os t -t r a u mat i c st r e ss disorder.(9-13) several studies suggested a higher rate of death anxiety among depressed patients than in healthy controls.(13,14) soleimani et al.(14) in their systematic review reported a higher rate of death anxiety among patients with breast cancer who developed depression compared to other breast cancer patients. on the other hand sharpe et al.(15) indicated that treatment of depression can significantly reduce death anxiety among patients who suffered from depression. however, the results of studies regarding s c h izo p hr e ni a a r e s o me ho w li mi t e d a nd inconsistent.(16-18) some studies reported a higher rate of death anxiety among these patients compared to patients with depression and healthy cases.(16,17) meanwhile, one study reported a lower rate of death anxiety among schizophrenia patients compared to depressed patients.(18) none of the abovementioned studies evaluated the correlation between the severity of symptoms and death anxiety in these patients. for the above-mentioned reasons, the present study was conducted to evaluate the level of death anxiety among patients with depression and those with schizophrenia, as well as investigating how the severity of the symptoms affects death anxiety in iranian patients. methods research design the present study is a cross-sectional study on patients with depressive disorder and patients with schizophrenia who were referred to the amir-al momenin hospital of zabol from february 1st 2020 to december 1st 2021. research subjects t he s a mpl e s i ze w a s d e t er mi n e d t o compare means between two groups, with effect size of 0.7, α=0.05 and β=0.2, the sample size per group being 34. the inclusion criteria were all patients with the definite diagnosis of depressive disorder or schizophrenia spectrum disorder based on icd-10 criteria,(19) who were referred to the psychiatry department of amiral momenin hospital, zabol, iran. the exclusion criteria were patients with organic disorders based on icd-10, related neurological disorders, or other internal disorders, severe alcohol consumption and substance abuse addiction. 58 subjects with a definite diagnosis of depressive disorder at the psychiatry department of zabol university of medical sciences and patients with schizophrenia spectrum disorder were included in the study. the diagnosis was based on the icd-10 (f2.x) diagnostic criteria.(19) in addition, healthy subjects with no personal or family history of neurological or psychiatric disorders were selected as the control group from an extensive database of healthy subjects in the psychiatry department, zabol university of medical sciences. all subjects underwent a mini international neuropsychiatric interview for diagnostic and statistical manual of mental disorders, and icd-10 disorders-10 (mini international neuropsychiatric interview-plus version),(20,21) and psychometric tests to diagnose signs of depression. healthy subjects and patients were studied through face-to-face interviews. the patients were asked to attend these sessions after the first treatment and establishment of stability in their status. clinical evaluation the general severity of the psychiatric disorder was measured using the clinical global impression scale (cgi).(22) the severity of depressive symptoms was measured using the be c k d e pr e ss i o n in ve n t or y ( bdi) . ( 2 3 ) psychosocial performance was measured using the personal and social performance scale (psp).(24) measure of death anxiety to evaluate death anxiety, the bochum survey for assessment of attitude to death and death anxiety questionnaire was used,(25) the reliability and validity of which were recently confirmed in a sample of about 200 healthy subjects. this questionnaire includes 25 items about attitudes toward death (10 items) and death anxiety (15 items). the criterion was based on the templer ’s death anxiety scale, the le i c e s te r de a t h a nx i e t y sc a le , a n d t he fimest.(26) for a semi-quantitative scale (borfet ta), one c an select one of the following items: “irrelevant” (score 1), “slightly relevant” (score 2), “mostly relevant” (score 3) or “highly relevant” (score 4). a score of 10 to 40 has been considered for the “attitude towards death” subscale (items 2, 3, 10, 16, 17, 19, 21 to 23, 25) , and a score of 15 to 60 has been considered for the “death anxiety” subscale (items 1, 4 to 9, 11 to 15, 18, 20, 24). in the qualitative analysis, participants can express their personal thoughts and anxieties about death, respectively, in two separate columns. statistics statistical analysis of the neuropsychiatric data was performed using statistical analysis s of t wa r e ( ibm spss) f o r t h e wi nd ow s operating system, version 25.0 (ibm, armank, new york, usa). statistical data were analyzed using parametric tests (one-way anova and pearson correlation coefficient). tukey multiple c o mp a r is o ns t e st wa s u se d f or pa i r w i se co mp ar is ons b et we en gr oups. sta ti st ic al significance was defined at a probability value of less than 0.05. ethical clearance all participants submitted their written consent after receiving information about the objectives of this study. the ethics committee of rudehen islamic azad university, iran, approved the study. it was also conducted in accordance with the 1975 helsinki declaration. ethical clearance code: ir.zbmu.rec.1399.172. results a total of 29 patients (13 females and 16 males with mean age of 43.7 ± 15.65 years with a definite diagnosis of depressive disorder at the psychiatry department of zabol university of medical sciences were collected during the study. also, 18 patients (9 females and 9 males with mean age of 41.15 ± 9.68 years with schizophrenia spectrum disorder were included in the study. in addition, 31 healthy subjects (18 shahrakimojahed, nakhaei depression and schizophrenia 59 univ med vol. 41 no 1 a b females and 13 males with mean age of 29.80 ± 16.45 years with no personal or family history of neurological or psychiatric disorders were selected as the control group. table 1 shows the demographic and clinical data of the included cases. as was expected, the patients with schizophrenia showed a s i gn i f i c a nt d i f f e r e n c e i n s oc ia l c l a s s , occupational status and educational level compared to the other two groups (p<0.050). however, the depressed group were mostly categorized as being of middle social class and high-school educational level. meanwhile there was no significant difference in these measures between the depressed group and the healthy individuals. the complete list of characteristics is shown in table 1. the mean ggi value for both study groups and was significantly different from that of the healthy controls (p<0.050). on the other hand, the mean psp score among patients with schizophrenia (47.54 ± 12.30) was significantly lower than those of the healthy (89.41 ± 6.12) and depressed (72.1 ± 12.29)) groups (p<0.050). regarding the bofretta attitude scale, the schizophrenic patients (23.1 ± 9.1) showed significantly higher scores (p<0.050). the healthy controls achieved the least scores (26.3 ± 11.3) in the bofretta anxiety scale followed by depressive (30.6 ± 10.3) and schizophrenic patients (31.3 ± 12.1). however, there was no si gn if ic ant di ff er enc e a mong th e gr oups regarding this scale. the schizophrenic patients also had the highest score of bofretta sum scale (50.1 ± 15.3), but this was not significantly different from those of the other two groups (table 2). we also found significant correlations of all bofretta dimensions (attitude, anxiety, sum score) with cgi, psp and bdi (p<0.050) (data not shown). the post-hoc analysis indicated a significant dif ference between healthy contr ols and schizophrenic patients regarding the bofertta attitude scores (p=0.013). these patients also table 1. demographic and clinical data of healthy subjects, depressive, and schizophrenia patients data presented as n (%), except for age [mean (sd)] variables healthy volunteers (n = 31) depressive patients (n = 29) patients with schizophrenia (n = 18) p value gender female male 15 (48.4) 16 (51.6) 16 (55.2) 13 (44.8) 7 (38.9) 11 (61.1) 0.340 age (years) 29.18 (16.45) 43.7 (15.65) 41.15 (9.68) 0.068 marital status married single 20 (64.5) 11 (35.5) 6 (20.6) 23 (79.4) 2 (11.1) 16 (88.9) 0.036 children yes no 11 (35.5) 20 (64.5) 5 (17.2) 24 (82.8) 1 (5.6) 17 (94.4) 0.051 educational level college high school basic school no school 29 (93.5) 2 (6.5) 0 (0.0) 0 (0.0) 10 (34.5) 15 (51.7) 3 (10.3) 1 (3.5) 3 (16.7) 6 (33.3) 5 (27.8) 4 (22.2) 0.029 occupational status full no activity retired 29 (93.5) 2 (6.5) 0 (0.0) 17 (58.6) 5 (17.2) 7 (24.2) 3 (16.7) 10 (55.6) 5 (27.7) 0.022 social class working class middle class academic 4 (12.9) 5 (16.1) 22 (71.0) 6 (20.7) 19 (65.5) 4 (13.8) 1 0(55.5) 5 (27.8) 3 (116.7) 0.073 60 had significantly higher scores when compared to the depression group (p=0.026). schizophrenic and depressed patients both showed significantly higher cgi scores compared to healthy controls (p=0.010 and 0.034, respectively). however, there was no significant difference between the t wo st u dy gr ou ps r e ga r d i ng cgi s c or e s (p=0.061). regarding the psp scores, although there was no significant difference between depressive and schizophrenic patients, both of these groups obtained significantly higher scores than did the healthy subjects (p=0.018 and p=0.011, respectively) (table 2). discussion according to the results, a significant positive relationship was found between death anxiety and the severity of depression and schizophrenia. also, for both disorders with 29 and 18 participants, a significant relationship was reported between death anxiety and the severity of the symptoms of the disorder. in general, these results support the claim that death anxiety is a trans-diagnostic construct, (4,7) and are consistent with those of previous studies, supporting the relationship between death anxiety and the severity of specific disorders. attitudes toward death were significantly worse in patients with schizophrenia. as shown, a completely negative attitude toward death was found in those patients with significant negative symptoms. patients with recurrent depression disorder, as do patients with schizophrenia, have a higher level of death-related anxiety. death anxiety was significantly high in the two groups, but patients with schizophrenia had higher (in other words, uncontrollable) negative attitude toward death. our results were in line with menzies et al.,(2728) who evaluated 200 mental disorder patients (anxiety, eating and affective disorders, psychotic disorders, obsessive–compulsive disorders) after treatment. in their study, it was reported that the severity of the mental disorder was closely related to the level of the patients’ death anxiety. on the other hand, mavrogiorgou et al.(30) in a more recent study indicated a higher rate of death anxiety among depressed and schizophrenic patients. considering the latest observations, this result is consistent with our results that patients with a chronic period and marked negative symptoms were characterized by a negative and uncontrollable attitude toward death. therefore, it can be stated that these patients have lower death anxiety, and the reason is a disorder in their emotions (due to the disappearance or lack of emotions). in contrast to predictions, although the existence of meaning in life predicted the science of psychopathology, neither the attachment style nor meaning in life moderated the relationship data presented as mean (sd), -one-way anova; bofretta: bochum survey for assessment of attitude to death and death anxiety; cgi: clinical global impression scale; psp, personal and social performance scale; h: healthy volunteers; d: depressive patients; s: patients with schizophrenia table 2. comparison of the psychometric scales applied to the healthy subjects, depressive and schizophrenia patients psychometric scale healthy volunteers (n=31) depressive patients (n=29) patients with schizophrenia (n = 18) p value post hoc analysis h-d h-s d-s p p p bofretta attitude 18.4 (4.56) 19.8 (5.20) 23.1 (9.12) 0.021* 0.056 0.013 0.026 bofretta anxiety 26.3 (11.3) 30.67 (10.33) 31.30 (12.18) 0.812 -- bofretta sum score 43.8 (12.5) 49.9 (12.9) 50.18 (15.3) 0.530 cgi 1.00 (0.0) 5.12 (0.33) 5.15 (0.65) 0.000* 0.034 0.010 0.061 psp 89.41 (6.12) 72.1 (12.29) 47.54 (12.30) 0.000 * 0.018 0.011 0.074 shahrakimojahed, nakhaei depression and schizophrenia 61 univ med vol. 41 no 1 between death anxiety and the science of psychopathology. attachment style can moderate death anxiety among non-clinical participants, (29) and early evidence of an association between death anxiety and understanding of meaning in life among non-clinical participants (30) with mild depression (31) indicates a moderating role of attachment style, which is consistent with previous findings. a recent study conducted by zhang et al.(32) indicated that meaning in life is adversely correlated with death anxiety, which means the higher the meaning in life scores were the lower the rate of death anxiety was. mohammadpour et al.(33) in their study reported that older adults who obtained higher scores regarding meaning in life seemed to completely understand their own values and goals of their life. as a result, they were able to look at death calmly and objectively which resulted in their lower death anxiety. therefore, it is suggested to consider the aspects of meaning in life in the context of death, while trying to treat mental disorders effectively. there are limitations in the interpretation of our data. a realistic study (patients hospitalized in a large university hospital on a daily basis) has its shortcomings. the patient group was more or less homogeneous, while the healthy group was heterogeneous, so they did not match well. the healthy group was far higher than the two patient groups in terms of social and educational status, so the studied groups were not comparable and at the same level. the bofretta scale should be considered critically, since it was previously approved but has not yet been widely used in psychiatry. most of the patients in our relatively small groups were affected by psychotherapy drugs, but we still could show the differences in attitudes of the two groups toward death and the death anxiety. although these results need to be confirmed by using larger sample groups in future studies, it can be suggested that practical interviews must be considered in both depression and schizophrenia cases who seem to be influenced by death anxiety, especially the ones with more severe symptoms. in addition, psychologists must be aware to mention this issue and ask the patient to talk about it in order to see if they have developed death anxiety or not. conclusion there was a significant relationship between the severity of symptoms and death anxiety in both depressed and schizophrenic patients. however, the patients with schizophrenia showed a more negative attitude towards death. acknowledgement we are much thankful to those who supported this project. contributors hsm participated in conception and design of the study, library searches and assembling relevant literature, critical review of the paper, supervising writing of the paper, and database management. sn participated in data collection, library searches and assembling relevant literature, writing the paper, and critical review of the paper. both authors read and approved the final manuscript. conflict of interest we declare that we do not have any conflicts of interests. references 1. rahimah a, khalil s, dang hp, cheng jm. the terror of death and consumers’ sustainability attitudes. j retail consum serv 2020;57:102196. doi: 10.1016/j.jretconser.2020.102196. 2. wong ptp, carreno df, gongora oliver b. death acceptance and the meaning-centered approach to end-of-life care. in: menzies re, menzies rg, iverach l, editors. curing the dread of death: theory, research and practice. samford valley, australia: australian academic press;2017. pp.185-202. 62 3. tradii l, robert m. do we deny death? ii. critiques of the death-denial thesis. mortality 2019;24:377-88. doi: 10.1080/13576275.2017. 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severity of mental illnesses. br j clin psychol 2019;58:452– 67. doi: 10.1111/bjc.12229. 28. mavrogiorgou p, haller k, juckel g. death anxiety and attitude to death in patients with schizophrenia and depression. psychiatry res 2020;290:113148. doi: doi.org/10.1111/bjc.12229. 29. plusnin n, pepping ca, kashima es. the role of close relationships in terror management: a systematic review and research agenda. pers soc psychol rev 2018;22:307-46. doi:10.1177/ 1088868317753505. 30. rogers r, sanders cs, vess m. the terror management of meaning and growth: how mortality salience affects growth-oriented processes and the meaningfulness of life. in: routledge c, vess m,editors. handbook of terror management theory. washington: academic press;2019.pp.325-45. doi: 10.1016/b978-0-12811844-3.00014-7. 31. solomon s, greenberg j. existential meaning and terror management. oxford research encyclopedia;2019. https://doi.org/10.1093/ acrefore/9780190236557.013.241. 32. zhang j, peng j, gao p, et al. relationship between meaning in life and death anxiety in the elderly: self-esteem as a mediator. bmc geriatr 2019;19:18. doi: 10.1186/s12877-019-1316-7. 33. mohammadpour a, sadeghmoghadam l, shareinia h, jahani s, amiri f. investigating the role of perception of aging and associated factors in death anxiety among the elderly. clin interv aging 2018;13:405. doi: 10.2147/cia.s150697. c:\users\universa medicina\docu 79 abstract universa medicina barriers to breast cancer screening in iranian females: a review article nazanin masoudi1, saeed dastgiri2, zohreh sanaat3, zhila abbasi4, and roya dolatkhah3* breast cancer is one of the most common malignancies among females in iran. screening and detection in the early stages can increase the likelihood of breast cancer treatment and prognosis, thereby decreasing the mortality rate and promoting patient survival. however, mass screening programs for breast cancer are still confronted by predominant barriers in iran. we tried to review and recognize the barriers to breast cancer screening and early detection in iranian females as the main research question. we searched all available databases, and screened manuscripts for eligibility according to our inclusion/exclusion criteria. the search strategy was based on the following keywords: breast, malignancy, cancer, screening, early detection, mammography, iran, and barrier. responsible authors performed a literature review and compiled the results into a report. various perceived barriers at different levels (individual, intrapersonal, health systems, and community) play influential and pivotal roles in women’s decisions to participate in breast cancer screening programs to facilitate early detection. lack of awareness, neglect, procrastination, embarrassment, religious beliefs, and lack of primary health workers awareness and recommendation were cited as perceived barriers in screening procedures in iran. there are still barriers in performing mammography at population levels in five major dimensions including availability, accessibility, cost, fears, and acceptability. however, the rate of regular mammography utilization in iran is low compared with other countries and needs increasing through informing women. many perceived barriers can affect women’s willingness and self-efficacy in screening procedures and one of the most effective ways of overcoming these barriers is to increase women’s awareness. keywords: mammography, women; malignancy, early detection, barrier, screening review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41:79-89 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1220 cite this article as: masoudi n, dastgiri s, sanaat z, abbasi z, dolatkhah r. barriers to breast cancer screening in iranian females. univ med 2022;41 :79-89. doi: 10.18051/univmed.2021.v40:79-89 1departmen t o f statistics and epidemiology, faculty of medicine, tabriz university of medical sciences, tabriz, iran 2tabriz health services management research center, tabriz university of medical sciences, tabriz, iran 3hematology and oncology research center, tabriz university of medical sciences, tabriz, iran 4faculty of medicine, tabriz university of medical sciences, tabriz, iran *correspondence: roya dolatkhah hematology and oncology research center, tabriz university of medical sciences, tabriz, iran p.o. box: 5166614731 telephone: +98 4133361358 email: royadolatkhah@yahoo.com/ dolatkhahr@tbzmed.ac.ir orcid id: 0000-0002-6897-7120 date of first submission, august 28, 2021 date of final revised submission, january 22, 2022 date of acceptance, january 26, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license january-april, 2022 vol.41no.1 80 masoudi, dastgiri, sanaat, et al barriers to breast cancer screening introduction b r e a st c an c e r a c c o un ts f o r t he l a r ge st proportion of female cancer-related deaths and is the first common cancer in more than 140 countries worldwide.(1-3) in iran, the international agency for research on cancer (iarc) reported breast cancer as the most common malignancy in females with an age-standardized incidence rate (asr) of 31.0 per 100.000 women.(4,5) recently, breast cancer has had a decreasing pattern in terms of incidence and mortality in highly developed countries. however, in most unde r-d evelo pe d c ountr ies we found increasing incidence rates.(1,6) lack of early detection and screening programs are the predominant causes of low survival rates of patients with breast cancer. these problems lead to an increase in women’s proportions presenting with late-stage disease. (7, 8) based on the studies, screening and detection in the early stages can increase the likelihood of breast cancer treatment and prognosis.(9) the most important way to control and recognize breast cancer in the earlier stages is screening. (10) breast cancer screening methods include: a) breast self-examination (bse) b) clinical breast examination (cbe) and c) mammography. based on the last american cancer society screening recommendations for women with an average breast cancer risk, getting regular mammograms is the only effective method in diagnosing breast cancer at an early stage when the treatment process is the most successful; but, research has not shown a clear benefit of regular bse and cbe.(11,12) however according to national comprehensive cancer network (nccn) guidelines, annual clinical breast examination, mammography and breast awareness have been recommended for asymptomatic women aged 40 years with average risk of breast cancer.(1) it has been established that screening at age 45 or 50 years (as per united states preventive task force/usptf guideline) results in unnecessary loss of life and more severe adverse effects.(13) however, mass screening programs for breast cancer are still confronted by some predominant barriers, especially in developing countries.(14,15) the most important factors affecting the low participation in different breast cancer screening methods include lack of perceived self-efficacy, lack of motivation for health, and different perceived barriers including a belief that the practices are time-consuming, and embarrassing.(15) another reason that has affected the commencement of screening programs in most asian countries, is cultural attitudes. while just less than 20% of at-risk women undergo any provided screening modality, the economic evaluation showed that mammography is the predominant screening method in most asian countries.(16) lack of knowledge on the importance of breast cancer screening and its methodology are other major barriers.(17-19) a significant segment of women remains under-screened with mammography due to the fears of cost and availability o f mammo gra p hy, lack of he a lth insura nce, mammogram-related pain, positive findings, or fear of a poor outcome during screening, which are other factors that play the role of the most commonly reported barriers to breast cancer screening in developed countries.(15,20,21) many barriers prevent women from commencing screening breast cancer and continue their screening according to their lifetime risk of breast cancer, depending on their family history and age as well as environmental factors.(22-25) some psychological factors may lead to defensive avoidance of screening modalities including mammography and may be the result of anxiety, fear of breast cancer and positive findings, and defensive avoidance beliefs about false positive findings.(26) it has also been emphasized that false-positive findings in the screening mammogram lead to psychological distress for up to 3 years, with the result that at least 3% of the women with average risk will be avoiding the next yea r screening appointments.(27) so placement of expert radiologists and breast cancer screening mammogram techniques are important to operate successful breast cancer screening programs. in the present paper, we aimed to review and recognize these barriers in iran. literature search strategy we tried to review and recognize the barriers to screening and early detection of breast cancer in iranian females as the main research question of the study. we searched all available databases including 81 web of science (isi), pubmed, medline (ovid), scopus, embase, google scholar, journal citation reports (jcr), clinical key, cinahl plus with full text (ebsco), as english databases, and scientific information database (sid), magiran, medlib, and irandoc as iranian databases. all manuscripts were screened for eligibility according to our inclusion/ exclusion criteria. we did not include any restriction in time, language and type of manuscripts. the search strategy was based on the following keywords: breast, malignancy, cancer, screening, early detection, mammography, iran, and barrier. responsible authors performed a literature review and compiled the results into a report. challenges of breast cancer screening in iran there are many prominent barriers to screening in iran; for instance, most women are unaware of screening methods and cannot have access to the centers of breast cancer screening.(9) the costs of tests and mammography are also other barriers to breast cancer screening. unfortunately, in iran, 82% of breast cancer cases are diagnosed in the late stages, which result in low survival rates and poor prognosis of breast cancer in women.(9,28,29) since there is still no national breast cancer screening system in iran, there are still many barriers that prevent women from undergoing screening. for instance, there is a lack of mammography screening centers, there are no national breast cancer programs, and insurance comp anies c over only a small p or tion of the costs.(7,19,30-32) the women health volunteers (whvs) program is an important part of iranian health services delivery. the whvs program is interested in local women who are trained to conduct regular home visits to disseminate health messages within their communities. whvs can play an indispensable role in increasing screening awareness and reducing barriers by d isp elling misco nce ptio ns and pr ovid ing comprehensive education to keep women involved in their health.(19) some cross-sectional studies were performed from different provinces of iran based on the women health volunteers program, the bse and mammography. also, standardized questionnairebased surveys were performed using the health belief model to assess women’s beliefs concerning breast cancer screening modalities.(7,17-19) i n a nothe r study in neysha bur city, the investigators checked the validity and reliability of a self-report method, and calculated the effect of awareness and perceived self-efficacy on breast cancer preventive behaviors in women.(33) in another correlational study in 2017, 1509 women in rural and urban areas of isfahan province were selected using a multistage sampling method. data were collected by visiting homes, using a questionnaire, and conducting inte rvie ws. (34 ) i n the per c eive d b ar r ie r s to mammography adoption study, five perceived barriers at different levels (individual, intrapersonal, health systems, and community) were discussed, and all of them have an important role in women’s intention to commence screening programs.(35) the results revealed that most women who participated in abovementioned study have never had mammography and were not planning to have one in the future. lack of any pain or uncomfortable feeling in the breast area is the main reason for not undergoing screening procedures among women. most women do not begin to undergo any screening because they do not see any breast abnormalities and this misconception is a major reason to fail to undergo screening.(35) also, most of the respondents never performed bse and only a few of them regularly performed bse. sometimes, they are not familiar with performing their regular bse correctly, and there are no screening centers available to aid and educate them. the rates of performing clinical breast examinations (cbe) and rates of referral to a doctor, were also low.(7) unfortunately, most women believe that they would only need screening programs when symptoms are detected. in addition, women are not aware of the opportunity of screening programs and their benefits as well as the timeliness of early detection of breast cancer.(36, 37) another substantial factor that has been studied by hosseini et al.,(33) in north khorasan province is self-efficacy on breast cancer preventive behaviors among women. based on the results of this study, the level of preventive behaviors was low. also, perceived self-efficacy and awareness, were significant predictor variables for these behaviors in women, and some univ med vol. 41 no 1 82 masoudi, dastgiri, sanaat, et al barriers to breast cancer screening c women had paid less attention to the aims and benefits of bc screening. they believed that bc is almost familial cancer, and if they do not have any positive family history, they do not need to do the screening.(37) lack of knowledge concerning the objectives and benefits of screening in staff and health-workers among ma mmogra phy ce nter s, and p ro viding misinformation related to mammography and its possible harms, increased the fear and anxiety of participants and in some cases led to their withdrawal. fear or worrying about any screening modality, referral to a hospital or medical center, the finding of a lump or cancer, and positive test results were the most common barriers in most studies.(7,34) in addition, lack of knowledge about curable cancers, fear of mastectomy and of radical treatments were reported.(38) thus, some people prefer not to get informed at all if they have cancer or at least be diagnosed later so that they can live more easily and without worries instead of dealing with cancer treatment side-effects and costs.(39) this shows that primary health workers need in-service training programs. meanwhile, health policymakers should attempt to increase the related knowledge and application success in health personnel that will affect women in their service area.(40) the summary results of included studies are presented in table 1. author, year study period methodology place, population number of women included age mean ± sd/ range (years) main outcome main results salmani et al., (19) june to november 2016 crosssectional, analytic 11 cities of the southern khorasan province 1,410 women health volunteers 20 to 65 36.16 ± 8.81 health beliefs and perceptions about breast cancer and breast cancer screening methods iranian women health volunteers had very low adherence to common bse and mammography practices alizadeh -sabeg et al., (17) randomized controlled trial (rct) 20 rural health centers of abish ahmad district, east azerbaijan 120 iranian rural women 40–69 effect of motivational interviewing on the change of breast cancer screening behaviors among rural iranian women two months after the intervention, a significant difference was found between the two groups in terms of the stages of change for clinical breast examination and mammography shirzadi et al., (35) qualitative component tabriz 24 healthy women 40-50: 15 (62.5%) 50: 9 (37.5%) qualitatively explore the barriers to mammography adoption among iranian women five main themes were extracted, consisting of unawareness of mammography, fear control, priority of mammography needs, inadequate competency of mammography centers, and a sense of losing family support safizade h et al., (18) qualitative study, content analysis four health centers in kerman 45 participants 47 motivational factors for breast cancer screening in iranian women knowledge acquisition, presence of happy-hopeful spirit, positive attitude and self-worth, maternal role, intellectual and financial independence, religious beliefs, motivational fears, and supportive family table 1. summary of the results of related studies 83 hossaini et al., (40) july 2018 to june 2019 qualitative study, content analysis tehran, iran 21 participants (10 health professionals and 11 female patients with breast cancer) 35-65 identifying the barriers to early detection of breast cancer in iranian women individual barriers (limited/lack of knowledge, other life preferences, negative reactions to the disease, and belief in fate), environmental barriers (insufficient social support, inaccurate information sources, and alternative therapy recommendation), and organizational barriers (poor quality of health services, inadequate access to health services, and role of media in informing people). mazarei et al.,(7) crosssectional descriptive health care institutes of bandar abbas 370 healthy women 15-79, 55.35±2. 27 to determine the knowledge and behavior of women regarding screening for breast cancer there was a significant relationship between the knowledge and behavior of the subjects foroozan i et al.,(9) june 2017 to december 2019 hospitalbased crosssectional study shiraz and kermanshah 725 patients with newly diagnosed bc 43.7 ± 8.2 delayed bc diagnosis and late stage presentation of women with bc lack of knowledge about bse, delay at diagnosis kardansouraki et al.,(37) 2016 descriptive crosssectional mazandaran, sari 1,165 healthy women (37.15 ± 8.84) different factors influence the breast cancer screening rate the woman’s age, age at first marriage, age at onset of sexual intercourse, occupation, spouse’s occupation, household income, health status, history of infertility, smoking, and decision-maker on issues of sexual and reproductive health (srh) were the best predictors of participation in screening for breast cancer (p<0.05). hoseini et al.,(33) 2018 crosssectional descriptivecorrelational north khorasan, neyshabur city 248 healthy women 30-69 (37.12± 7.49) to determine the effect of awareness and perceived self-efficacy of women of neyshabur city on the preventive behaviors of breast cancer self-efficacy and awareness variables significantly predicted %67 of the breast cancer preventive behaviors univ med vol. 41 no 1 84 masoudi, dastgiri, sanaat, et al barriers to breast cancer screening badakhs h et al.,(39) 2017 systematic review different regions of iran 10,521 participants 15-79 (33.5) to determining attitudes and practice regarding breast cancer early detection techniques poor performance and low rates for positive attitudes regarding breast cancer early detection techniques in iran bouya et al.,(36) 2017 systematic review different regions of iran 11,756 participants 15 to 75 to determine the knowledge and source of information about early detection techniques of breast cancer among iranian women only one of third of women had sufficient knowledge about bse. the main source of information was healthcare team members salek et al.,(29) from 1980 to 2012 retrospective cross sectional northeastern iran, mashhad 4000 female breast cancer to survey the proportion of stages at the presentation of the disease without a screening program, among a population of breast cancer patients disease is now presented in its earlier stages even in the absence of a screening program ahmadip our et al.,(30) april to september of 2015 cross sectional urban health centers in the city of kerman 240 healthy women 18-64 (31.7± 7) the rate of screening participation the frequency of mammography performance was higher in women with higher education (p=0.01), but there was no statistically significant difference in the frequency of self and clinical breast examinations based on education, household income and employment aminisa ni et al.,(31) 2014 crosssectional six health centers in baneh county, iran 561 healthy women 40-49 (43.64±5 .17) sociodemographic and health related determinants of participation in breast cancer screening the overall uptake of mammography was about 17%, and mostly in older, illiterate and post-menopausal women. the main barrier was lack of access to mammography ghahrem anian et al.,(32) crosssectional 12 health centers of tabriz, east azerbaijan 370 healthy women older than 20 to determine relationships of fear and fatalism with breast cancer screening behavior fatalism and fear had a stimulating effects on breast cancer screening performance 85 from the beginning of october 2016, the integrated health system (samaneyeh iyekparcheye behdashti (sib) system) as an online system has been developed in iran based on technical knowledge and in line with the latest global technologies in order to provide health services by the ministry of health and medical education. all information about households, the type of health care services required in community health centers and databases, and health homes were collected and recorded in this system. however, early detection and screening for breast cancer was the most important of the midwife’s duties, which also included: following the country guides for breast cancer screening using established guidelines, implementation of the classification of breast imagingreporting and data system (bi-rads) and their diagnosis and action, and classification of cases of individual and family history in self-care. with the establishment and development of this program, it is expected that comprehensive screening programs at the population levels, will be launched and integrated in the country soon.(5) barriers to screening of breast cancer in iranian females various perceived barriers at different levels (individual, intrapersonal, health systems, and community) play influential and crucial roles in women’s decisions to participate in breast cancer screening programs (figure 1).(35,41) there are still barriers in performing mammography at population levels in five main dimensions including availability, accessibility, cost, fears, and acceptability.(42-45) i nc r ea sing c a nc e r p re ventio n a nd sc r ee ning knowledge and individual behavior are the main key factors for accessing and adhering to evidence-based guidelines of breast cancer screening in lowand mid d le -inco me c ountr ies like ir a n. (4 6-4 8) multidisciplinary interventions at the health system level are needed to address the barriers that women experience to undergo mammography screening.(49,50) however, the rate of regular mammography in iran is low and should increase by informing the women. while there was not any established and evidence-based screening strategy in the country, we e nc o unter e d maj or b ar r ie r s to p er for ming mammography at average risk at population levels: therefore, cbe and/or bse may play a role as alternative strategies. breast self-exams aid to increase breast cancer awareness and get familiar with how breasts look like and feel; therefore, women can alert their healthcare professional if there are any changes. unfortunately, recently bse has been recommended as grade d strategy for breast cancer screening due to related false-positive results and additional psychological harms and fears.(51) however, bse is so beneficial, inexpensive and simple that it actually can encourage women to take healthy situations into account and aid them to recognize breast cancer in the earlier stages.(19) based on the results of studies, if women perform regular breast examination and manage it at the time, only 95% of breast cancer cases figure 1. various perceived barriers to screening for breast cancer in iranian females (35,41) univ med vol. 41 no 1 86 masoudi, dastgiri, sanaat, et al barriers to breast cancer screening r eac h ad vanc ed stage s. (7 ) the r efo r e, whe n mammography is not available, bse plays an inevitable role in identifying breast cancer in the early stages. studies have indicated barriers consisting of a lack of awareness of the importance and benefits of clinical breast examination and breast self-examination in iran. meanwhile, there is a lack of awareness of bse techniques in individuals and a lack of training by personnel of healthcare centers.(7) results of studies show that the most important barriers to iranian women who do not perform monthly bse include its being time-consuming, feeling embarrassed, and lack of knowledge to perform bse. providing sufficient information regarding early diagnosis and screening programs, curable early stages of cancer, and preventing deaths from breast cancer should be underscored by health policymakers among the populations. increasing awareness and improving women’s beliefs and attitudes toward breast cancer screening are necessary. conclusion many perceived barriers can affect women’s willingness and self-efficacy in screening procedures in iran. lack of awareness, neglect, procrastination, embarrassment, religious beliefs, and lack of primary health workers’ awareness and recommendations were reported. barriers to performing mammography at population levels were discussed in some main dimensions including availability, accessibility, cost, fears, acceptability, and lack of health insurance, mammogram-related pain, positive findings, simply put receiving bad news during screening. since breast cancer incidence, mortality, age pattern and risk factors are completely different in asian countries, it is felt that we need specific diagnostic and prognostic studies for asian breast canc er patients. howeve r, imp le me ntatio n of population-based studies on breast cancer patients from various asian settings is a priority to provide an insight into the effectiveness of different screening modalities in this part of the world. although we should have essential national breast cancer programs for women in the country and try to make them aware of the importance and benefits of early diagnosis and screening of breast cancer, one of the most efficient ways is to increase women’s awareness about breast cancer. conflict of interest the authors have no conflicts of interest. all authors reviewed the drafts of the manuscript and participated in the interpretation of the results and approved the final version. acknowledgements the authors would like to acknowledge mr. ali roshani for scientific native language editing of the text of this manuscript. consent for publication all autho r s c onfir med any c o nse nt for publication. funding this study was supported by hematology and oncology research center, tabriz university of medical sciences, tabriz, iran (id: 58716). contributors the authors confirm contribution to the paper as follows: nm, rd: study conception and design; data collection sd, rd, zs, za: interpretation of 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aghazadeh a, onyeka tc, nabighadim a. beliefs, 89 fear and awareness of women about breast cancer: effects on mammography screening practices. nurs open 2021;8:890-9. doi:10.1002/nop2.696.. 45. rundle a, iles s, matheson k, et al. women’s views about breast cancer prevention at mammography screening units and well women’s clinics. curr oncol 2020;27:e336-42. doi: 10.3747/ co.27.5755. 46. bonsu ab, ncama bp. evidence of promoting prevention and the early detection of breast cancer among women, a hospital-based education and screening interventions in lowand middleincome countries: a systematic review protocol. syst rev 2018;7:234. doi: 10.1186/s13643-0180889-0. 47. hsieh hm, chang wc, shen ct, liu y, chen fm, kang yt. mediation effect of health beliefs in the relationship between health knowledge and uptake of mammography in a national breast cancer screening program in taiwan. j cancer educ 2021;36:832-43. doi: 10.1007/s13187-02001711-7. 48. shen ct, chen fm, hsieh hm. effect of a national population-based breast cancer screening policy on participation in mammography and stage at breast cancer diagnosis in taiwan. health policy 2020;124:478-85. doi: 10.1016/ j.healthpol.2020.02.006. 49. islam rm, billah b, oldroyd j. barriers to cervical cancer and breast cancer screening uptake in lowincome and middle-income countries: a systematic review. asian pac j cancer prev 2017;18:1751-63. doi: 10.22034/apjcp.2017.18.7.1751. 50. wagh b, chaluvarayaswamy r, pal d.. assessment of adaptive breast cancer screening policies for improved mortality reduction in low to middle income countries. asian pac j cancer prev 2017;18:2375-80. doi: 10.22034/ apjcp.2017.18.9.2375. 51. pippin mm, boyd r. breast self examination. statpearls: treasure island (fl);2020. univ med vol. 41 no 1 universa medicina january-april vol.42no.1, 2023 invited editorial placental growth factor as a screening tool of preeclampsia raditya wratsangka department of obstetrics and gynaecology faculty of medicine, universitas trisakti, jakarta, indonesia preeclampsia (pe) is one of the most serious pregnancy-specific multisystem disorders defined by the onset of hypertension accompanied by significant proteinuria after 20 weeks of pregnancy.(1) the prevalence of pe is estimated to be around 2%-8% worldwide but varies greatly depending on racial or ethnic origin and geographic region, and prevalence is higher in some developing countries. in addition, women in low-resource countries are at higher risk of developing pe compared to those in high-resource countries. previous studies have shown that there is a lower frequency of pe among asian women.(2,3) preeclampsia is the leading cause of maternal and perinatal morbidity and mortality and is associated with approximately 80,000 maternal deaths and more than 500,000 infant deaths each year.(4) although a complete understanding of the pathogenesis remains unclear, current theories suggest a two-step process. the first stage is caused by superficial trophoblastic invasion resulting in inadequate spiral artery remodeling. this is thought to lead to the second stage, which involves the maternal response to endothelial dysfunction and the imbalance between angiogenic and antiangiogenic factors, resulting in the clinical picture of the disorder. in late onset disease, the placenta is usually normal; however, fetoplacental demand exceeds supply, resulting in a placental response that triggers the clinical phenotype.(5) although not yet clear, several biochemical, biophysical, and sonographic parameters have been investigated as potential tools for identifying women at high risk of pe that can help us on our long journey.(1) several studies have been conducted by examining first-trimester maternal serum markers to evaluate placental growth factor (plgf) levels in the first trimester to predict the incidence of preeclampsia. placental growth factor is a protein from the family of vascular endothelial growth factors (vegf), which promotes the blood vessels formation. the substance is present in high concentrations in the villous cytotrophoblast and syncytiotrophoblast tissues. placental growth factor has been shown to have a very important role in the prediction of first trimester pe and has a good diagnostic capacity in symptomatic women after 20 weeks of gestation.(6) boutin et al.(7) conducted a study to evaluate the performance of first trimester plgf for predicting pe in nulliparous women. of the 4,652 participants, they observed 232 (4.9%) pe cases including 202 (4.3%) term and 30 (0.6%) preterm pe. placental growth factor was associated with the risk of term pe (auc=0.61, 95% confidence interval [ci] 0.57–0.65) and preterm pe (auc=0.73, 95% ci 0.64–0.83). the model was improved with the addition of maternal characteristics (auc for term pe 0.66, 95% ci 0.62–0.71; auc for premature pe 0.81, 95% ci 0.72–0.91; p<0.01). at a 10% false positive rate, plgf combined with maternal characteristics predicts 26% term pe and 55% preterm pe. agrawal, et al. (8 ) conducted a meta analysis to study the predictive accuracy of plgf *email: raditya@trisakti.ac.id orcid id: 0000-0001-6254-8336 4 mailto:raditya@trisakti.ac.id https://orcid.org/0000-0001-6254-8336 5 universa medicina vol. 42 n0. 1 2023 in asymptomatic women. their analysis included 40 studies with 3189 cases of preeclampsia and 89,498 controls. the overall predictive odds ratio of this test is 9 (6-13). subgroup analysis evaluating different plgf thresholds showed that the highest predictive value for plgf levels was between 80 and 120 pg/ml with a high predictive odds ratio of 25 (7–88), sensitivity of 0.78 (95% ci, 0.67–0.86), specificity 0.88 (95% ci, 0.750.95). the now widely recommended alternative approach to pe screening is based on the fetal medicine foundation’s (fmf) multi-marker prediction algorithm, which is the first trimester combined test for pe, which has been supported by the international federation of gynecology and obstetrics (figo).(1,9) global implementation of an effective first trimester program to screen for and prevent preterm pe will provide an opportunity to reduce the risk of maternal and perinatal morbidity and mortality in the short term. traditional screening methods as recommended in the past have limited predictive performance, so they need to be updated to reflect the latest scientific evidence that the target should be for preterm pe. to achieve optimal screening performance, the key is to establish a standard method for measuring bioma rkers and asse ssing the quality of biomarkers on a regular basis, because each biomarker is prone to inaccurate measurements, thereby affecting screening performance. although the national institute for health and care excellence (nice) recommends plgf as an exclusion test for preeclampsia, it is not currently recommended for routine adoption to exclude or diagnose preeclampsia due to insufficient evidence. further research is needed about repeat plgf-based testing in women with suspected preeclampsia who have previously negative results and about how positive plgfbased tests used to rule out preeclampsia will influence management decisions at the time of delivery and outcome. related to this.(10) references 1. poon lc, shennan a, hyett ja, et al. the international federation of gynecology and obstetrics (figo) initiative on pre-eclampsia: a pragmatic guide for first-trimester screening and prevention. int j gynecol obstet 2019;145(s1):1– 33. doi: 10.1002/ijgo.12802. 2. moungmaithong s, wang x, tai ast, et al. first trimester screening for preeclampsia: an asian perspective. maternal-fetal medicine 2021;3: 116–23. doi: 10.1002/ijgo.12802. 3. bilano vl, ota e, ganchimeg t, mori r, souza jp. risk factors of pre-eclampsia/eclampsia and its adverse outcomes in lowand middle-income countries: a who secondary analysis. plos one 2014;9:e91198. doi: 10.1371/journal.pone.0091198. 4. rana s, lemoine e, granger j, karumanchi sa. preeclampsia: pathophysiology, challenges, and perspectives. circ res 2019;124:1094–112. doi: 10.1161/circresaha.118.313276. 5. mayrink j, costa ml, cecatti jg. preeclampsia in 2018: revisiting concepts, physiopathology, and prediction. scientific world journal 2018;2018: 6268276. doi: 10.1155/2018/6268276. 6. o’gorman n, wright d, syngelaki a, et al. competing risks model in screening for preeclampsia by maternal factors and biomarkers at 11–13 weeks gestation. am j obstet gynecol 2016;214:103.e1–e12. doi: 10.1016/j.ajog.2015. 08.034. 7. boutin a, demers s, gasse c, et al. first-trimester placental growth factor for the prediction of preeclampsia in nulliparous women: the great obstetrical syndromes cohort study. fetal diagn ther 2019;45:69–75. doi: 10.1159/000487301. 8. agrawal s, shinar s, cerdeira as, redman c, vatish m. predictive performance of plgf (placental growth factor) for screening preeclampsia in asymptomatic women: a systematic review and meta-analysis. hypertension 2019;74:1124–35. doi: 10.1161/ hypertensionaha.119.13360. 9. figo working group on good clinical practice in maternal-fetal medicine. good clinical practice advice: first trimester screening and prevention of pre-eclampsia in singleton pregnancy. int j gynaecol obstet 2019;144:325-29. doi: 10.1002/ ijgo.12741. 10. national institute for health and care excellence (nice). plgf-based testing to help diagnose suspected preterm pre-eclampsia. national institute for health and care excellence; 2022. univ med 2023;42:4-5. doi: http://dx.doi.org/10.18051/univmed.2023.v42.4-5 c:\users\universa medicina\docu 69 abstract universa medicina the role of mental health to overcoming the coronavirus disease-19 pandemic ira aini dania1* and nanda novziransyah1 the current coronavirus disease-19 (covid-19) pandemic has changed the order of social life; the efforts made to tackle this pandemic have sometimes raised various obstacles related to norms, ethics and culture. an increased risk of covid-19-associated mental disorders is found in many communities, causing excessive emotional reactions such as feelings of pressure, leading to unhealthy behavior. this situation confirms that health care providers have a key role in dealing with emotional disturbances that occur as a form of pandemic response. public health emergencies can affect the health, safety and well-being of every individual as well as the social stigma in connection with the covid-19 pandemic. various mental health studies related to disasters have determined that emotional distress is widespread and can also be found in populations related to covid-19. there are various kinds of risk factors that have an impact on health workers during the covid-19 pandemic, including social isolation, lack of support, and concerns about safety of family members. we included published, observational and experimental studies that reported the mental health effects on health workers during the covid-19 pandemic. efforts made by policy holders, especially in the health sector, have greatly contributed to the response to the pandemic, but not all of them can run smoothly, several steps and policies in the health sector must involve many stakeholders in order to realize the expected efforts. the main purpose of this review paper was to not only focus on health workers, but include the communities in overcoming the covid-19 pandemic. keywords: pandemic, covid-19, mental health, psychosocial review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.69-76 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1068 january-april, 2021 vol.40no.1 cit e th is article as: da n i a ia, novziransyah n. the role of mental health to overcoming the coronavirus disease-19 pandemic. univ med 2021; 40:6 9 -7 6 . doi: 10 .18 051/ uni vmed. 2021.v40.69 -7 6. 1department of psychiatry faculty of medicine, universitas islam sumatera utara *correspondence : dr. ira aini dania, m.ked (kj), sp. kj. department of psychiatry, faculty of medicine, universitas islam sumatera utara medan mobile: +6285262066240 email :iraaini27@gmail.com orcid id :0000-0002-7534-337x date of first submission, november 18, 2020 date of final revised submission, april 6, 2021 date of acceptance, april 12, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 70 dania, novziransyah mental health and covid-19 pandemic introduction t he c u r r e nt c o r o na vi r u s dis e a se -19 (covid-19) pandemic has changed the order of social life and various financial losses have occurred in various sectors, ranging from the health, educational, social, economic sectors and others.(1) meanwhile, the efforts made to contain this pandemic have sometimes raised various obstacles related to norms, ethics and culture that have been going on and have deep roots. (2) some of the efforts made by policy holders, especially in the health sector, have greatly contributed to the response to the pandemic. however, not all have been able to run smoothly, since several steps and policies in the health sector are considered in some gr oups as restricting personal freedom in carrying out activities.(3) various difficulties and an increased risk of mental disorders associated with covid19 are found in many communities.(4) conditions of mental health in the covid19 pandemic the above conditions emphasize that health care providers have a very important role and are key persons in dealing with emotional disturbances that occur as a form of pandemic response. public health emergencies can affect the health, safety and well-being of each individual, causing such reactions as fear, anxiety, insecurity, confusion, emotional isolation and, most importantly, the social stigma in connection with the covid-19 pandemic. the factors contributing to the burden of emotional distress include economic losses, loss of jobs, problems in the educational sector, resources t ha t a re de eme d i na de q ua t e fo r me di ca l responses and more uneven distribution of basic needs in society.(5,6) conditions that can affect medical staf f, communities, and f amilies involving covid-19 pandemic-related situations, could be the causes of excessive emotional reactions such as feelings of pressure that lead to unhealthy behavioral changes such as increased drug use and non-compliance with public health measures such as self-isolation and direct restrictions in social interactions.(7,8) the public health measures carried out on p e op l e wit h c on f i r me d covi d-1 9 , b o th symptomatic and asymptomatic, create a stigma so that these patients face psychological and social legal conditions of punishment in the community life order, which is not easy for them to deal with. one mental he alth study in hongkong involving 500 respondents found that 19% of the respondents had depression and that 14% had anxiety related to the covid-19 pandemic, indicating that emotional distress is widespread and can be found everywhere in the p op ul at i o n. ( 9) in sp a i n 73 % of t h e 4 1 80 participants had psychological distress.(10) health workers, communities, and family members, should be notified that disaster does not make it more meaningful for them to give up under psychological conditions. they should take lessons from all disasters, such as finding new strengths and trying to be more active and resilient in their striving to fulfill their needs both financially and spiritually.(11,12) however, in a “conventional” study of disasters, accidents, and technological and deliberate mass action, the main concern is post-traumatic stress disorder ( pt sd) th a t a r i s es f r om e x po su r e to trauma.(13,14) medical conditions from natural causes such as life-threatening viral infections do not meet the current criteria for trauma necessary for the diagnosis of ptsd, but other psychopathology such as depression and anxiety disorders may occur.(15,16) age groups including the elderly, pregnant women and toddlers as well as those with comorbidities may be more vulnerable than others to the psychosocial effects of the pandemic.(17,18) the age groups that are at high risk of being infected with covid 19 are especially those in the elderly group, those having a history of chronic diseases such as hypertension, diabetes, heart disease, and people with impaired immune function.(19,20) people living in crowded conditions without possible distancing in social communication, and people with a history of pre-existing medical conditions, 71 psychiatric problems, or drug abuse are at high risk of experiencing adverse psychosocial conditions, while adolescents are also prone to experience distress especially when comorbid with other psychiatric disorders.(21,22) health care providers are also very vulnerable to shocks and emotional changes in the current pandemic, and additionally have a high probability of being exposed to covid 19 concerns about infection that will impact their families plus the cynical views of people who are considered carriers of the virus.(12,23) the activities of caring for their l ove d on e s, l a c k of pe r so n a l p r o t e c t i ve equipment, working longer hours, and being involved in resource allocation decisions as the vanguard, will of course create emotional reactions.(24) preventive measures such as s c r e e ni n g f or me n t a l h e a l t h p r o b le ms , psychoeducation, and psychosocial support should focus on other groups at risk for adverse psychosocial conditions such as the inherent stress of the disease itself, advocating for mass isolation or large-scale social restrictions, including messages to stay put in homes, quarantine, and isolation. all of this is new to society, and of course causes a variety of reactions, where each individual will react in conveying his or her concerns, and people will react individually and collectively.(25,26) several reviews reported the psychological symptoms that were found. a recent review of p syc h ol o gi c a l s ympt o ms i n in di vi du a l s u nd e r goi n g qu a r a n t i ne i nc l u de s s t r e s s , depression, irritability, insomnia, fear, confusion, a n ge r, f r us t r a t i o n, b o r e d om a n d st i gma associated with quarantine, and some of these psychological conditions can persist after quarantine.(27,28) there are many problems that can occur with the existence of large-scale social restrictions and the ongoing quarantine process. some of the individuals do not have adequate supplies or have difficulties in getting medical care and drugs, which are also experienced by people with severe mental disorders. there are limitations for schizophrenics to get mental health services routinely during this pandemic, whereas ideally the mental health service process should continue even though it only uses telemedicine media based on current technology.(29,30) in patients who are chronically stable and sustain financial losses, this will certainly cause significant psychological stress in groups of people who really subsist without savings, while their basic needs must be met immediately.(31) six review articles by kang et al.,(32) cai et al.,(33) liang et al.,(35) xiao et al.,(36) and mohindra et al.(38) showed that current research focused on assessing several aspects of mental health affected in health care workers due to cov id 19 . se ve r a l s oc i o-d e mogr a p h ic variables, such as gender, profession, age, place of work, department of work and psychological variables, such as poor social support and selfefficacy, were associated with increased stress, anxiety, depressive symptoms, and insomnia in h e a l t h c a r e w or ke r s ( ta b l e 1) . t h e r e i s increasing evidence suggesting that covid 19 can be an independent risk factor for stress in health care workers. in the current pandemic the risk factors, such as lack of social support, excessive economic burden, occupation, pressure on medical personnel, has not gone unnoticed; the danger will always lurk at the frontline in the tackling of covid-19.(39) educational and moral messages echoed through electronic and social media by various parties and policy holders, in this case the government through the health service and other related agencies, collaborate with each other to try and break the chain of transmission of covid-19. all of this never escapes attention, but sometimes there is fake news.(40,41) confusion about covid-19 raises various interpretations; some think that covid19 is not a dangerous viral infection and that recommendations that should be adhered to, s uc h a s get t i ng us e d to wa s hin g h a n ds , maintaining social distancing, wearing masks, should be viewed as just ordinary messages that need not be obeyed, because in reality in the field many people who do not comply with these recommendations are doing just fine.(42) univ med vol. 40 no 1 72 dania, novziransyah mental health and covid-19 pandemic a b c authors sample including (n) and place conducted study design instruments used main findings kang et al.(32) 994 medical and nursing staff, wuhan crosssectional study patient health questionnaire-9, generalized anxiety disorder, insomnia seventy index and the impact of event scalerevised 36.9% had subthreshold mental health disturbances, 34.4% had mild disturbances, 22.4% had moderate disturbances, and 6.2% had severe disturbances cai et al. (33) doctors, nurses, and other hospital staff (n543), hubei crosssectional study questionnaire by lee et al.(34) medical staff experienced emotional stress during the covid-19 outbreak. liang et al. (35) 59 doctors and nurses from covid-19 associated department and others, guangdong province crosssectional study zung’s self-rating depression scale (sds), zung’s self-rating anxiety scale (sas). several staff were experiencing clinically significant depressive symptoms. xiao et al. (36) 180 medical staff (doctors or nurses), china crosssectional observational study self-rating anxiety scale, general selfefficacy scale, stanford acute stress reaction questionnaire, pittsburgh sleep quality index, and social support rate scale levels of social support were significantly associated with self efficacy and sleep quality and negatively associated with the degree of anxiety and stress. lai et al. (37) 1257 hcw, 20 hospitals in wuhan, 7 hospitals in other regions of hubei province, 7 hospitals from 7 other provinces crosssectional region stratified study patient health questionnaire-9, generalized anxiety disorder scale, insomnia severity index, and impact of event scale-revised 50.4 % reported symptoms of depression, 44.6 % anxiety, 34.0 % insomnia, and 71.5 % reported distress mohindra et al. (38) frontline health care providers (hp) involved in the care of patients with covid-19 or suspected covid-19 sample size not specified, tertiary hospital in north india qualitative analysis interviews with hp main themes identified for mental health promotion of hp: 1. positive: motivational factors a intellectual b emotional 2. negative: frustrations associated with patient care 3. personal fears and annoyances experienced by doctors table 1. summary of the studies information that is overlapping or different in prevention and management creates confusion for the community so that the impact of p syc h ol o gi c a l p r e s s ur e is fe l t b y t he c o mmuni t y. ( 4 3 ) re s e a r c h c o nd u c t e d i n c o mmu ni t i es s e ve r e ly a f f e c te d by a c u te respiratory syndrome (sars) in the early 2000s revealed that although community members, affected individuals, and health care workers were motivated to comply with quarantine to reduce the risk of infecting others and to protect public health, the emotional distress of some people was the reason why they considered breaking the rules.(44) 73 discussion crisis management personnel, including health workers, are not themselves immune to the psychological consequences of covid-19. health workers facing covid-19 directly in handling these patients are at greater risk than o th er s . ( 4 5 ) t h e r ea s o ns f o r t he a dve r s e psychological outcomes in health workers range f r om e x c e s s i ve wor kl o a d /w or k ho ur s, inadequate personal protective equipment, overenthusiastic media news, and feelings of being inadequately supported. here the psychosocial monitoring that is needed to provide support during in-person patient encounters in clinical practice, is severely limited by this crisis.(46) psychosocial service s are deliver ed via telemedicine, home visits are limited as much as possible, if it is not too emergency, so as not to come to a health service center. in the context of covid-19, psychosocial assessment and monitoring must be carried out including questions about covid-19 and related stressors such as exposure to infected sources, infected family members, and physical distance.(47) other factors include secondary adversity (economic loss, for example), psychosoci al eff ects (such as depression, anxiety, psychosomatics, insomnia, increased drug use, and domestic violence), and indicators of vulnerability (such as preexisting physical or psychological conditions).(35,48) patients with mental health problems, such as excessive anxiety, mild to severe depression, and acute stress reaction, will need referral for evaluation and formal mental health care, while others may benefit from supportive interventions designed to improve health and coping, such as psychoeducation or cognitive behavioral therapy techniques.(49,50) because of the economic crisis and a lot of uncertainty surrounding this pandemic, suicidal ideation may arise, which requires immediate consultation with mental health profe ssionals or ref er ral for e mergenc y psychiatric cases.(51,52) the covid-19 pandemic has changed the basic life order that affects mental health, and various kinds of interventions have been made to reduce emotional distress effectively.(53) the experiences of patients, family members and communities can be normalized by pr oviding the infor mation that the stress experienced during a pandemic is a normal reaction as long as it does not interfere with the function of both individuals and groups.(54,55) one of the supporting efforts is the management of str ess management i n the midst of dire circumstances.(56) conclusions education and training on psychosocial problems is not only for health workers but should be provided to all leaders involved in the health system. mental heal th worke rs and the emergency management community, in this case the covid-19 response acceleration task force, must work together to identify, develop and disseminate evidence-based resources related to mental health disasters, mental health triage and referrals, the needs of special populations, and notification of death and treatment in confirmed covid positive cases. improvement of mental health includes attempts to reduce or eliminate factors with adverse impacts on mental health along with those related to improving the physical health status. it is also important to carry out studies for examining the impact of the pandemic-related mental health on healthcare workers. moreover, it is predicted that implementing the approach for increasing psychological resilience will also protect the mental health of individuals against other risks that may develop. conflict of interest all authors declare no conflict of interest. acknowledgements we would like to thank the department of psychiatry, faculty of medicine, universitas islam sumatera utara. univ med vol. 40 no 1 74 dania, novziransyah mental health and covid-19 pandemic contributors iad and nn conceived idea for this study. nn contributed to interpreting the data. iad wrote the manuscript and made substantial contributions to the revision of the manuscript. all authors have read and approved the final manuscript. references 1. ehrenberg jp, utzinger j, fontes g, et al. efforts to mitigate the economic impact of the covid-19 pandemic: potential entry 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al. changes in mental health symptoms from precovid-19 to covid-19 among participants with systemic sclerosis from four countries: a scleroderma patient-centered intervention network (spin) cohort study. j psychosom res 2020;139. doi: 10.1016/j.jpsychores.2020.110262. 27. thombs bd, bonardi o, rice db, et al. curating evidence on mental health during covid-19: a living systematic review. j psychosom res 2020; 133: 110113. doi: 10.1016/j.jpsychores.2020.110113. 28. spoorthy ms. mental health problems faced by healthcare workers due to the covid-19 pandemic–a review. asian j psychiatr 2020;51: 2018-21. doi: 10.1016/j.ajp.2020.102119. 29. vindegaard n, benros me. covid-19 pandemic and mental health consequences: systematic review of the current evidence. brain behav immun 2020;89:531-42. doi:10.1016/j.bbi.2020. 05.048. 30. lancet. editorial: covid-19: protecting healthcare workers. lancet 2020;395:922. doi: 10.1016/ s0140-6736(20)30644-9. 31. brenner mh, bhugra d. acceleration of anxiety, 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doi: 10.1016/ j.jpsychores.2020.110102. 36. xiao h, zhang y, kong d, li s, yang n. the effects of social support on sleep quality of medical staff treating patients with coronavirus disease 2019 ( covid-19 ) in january and february 2020 in china. med sci monit 2020;26:e923549-1–e-8. doi: 10.12659/msm.923549. 37. lai j, ma s, wang y, et al. factors associated with mental health outcomes among health care workers exposed to coronavirus disease 2019. jama netw open 2020;3: e203976. doi:10.1001/ jamanetworkopen.2020.397.6. 38. mohindra r, suri v, bhalla a, singh sm. issues relevant to mental health promotion in frontline health care providers managing quarantined/ isolated covid19 patients. asian j psychiatr 2020;51: 102084.. doi:10.1016/j.ajp.2020.102084. 39. mahase e. covid-19: mental health consequences need urgent research, paper advises. bmj 2020;369:m1515. doi: 10.1136/ bmj.m1515. 40. international labour organization. managing work-related psychosocial risks during the covid-19 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brain, behavior, and immunity is returning to work during the covid-19 pandemic stressful? a study on immediate mental health status and psychoneuroimmunity prevention measures of chinese workforce. brain behav immun 2020;87:84-92. doi:10.1016/j.bbi.2020.04.055. 47. kusumawardani, arine d, luthfiyana, ulya n. the mental health of medical workers during the covid19 pandemic: how do we manage it? j kes ling 2020;12.21-8. doi: 10.20473/jkl.v12i1si.2020. 48. mann dm, chen j, chunara r, testa p, nov o. covid-19 transforms health care through telemedicine: evidence from the field. j am med inform assoc 2020;27:1132-5. doi: 10.1093/jamia/ ocaa072. 49. kola l, kohrt ba, hanlon c, et al. covid-19 mental health impact and responses in low-income and middle-income countries: reimagining global mental health. lancet psychiatry 2021;0366:1-16. 50. naushad va, bierens jjlm, nishan kp, et al. a systematic review of the impact of disaster on the mental health of medical responders. prehosp disaster 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donnelly r, farina mp. how do state policies shape experiences of household income shocks and mental health during the covid-19 pandemic? soc sci med 2021;269:113-557. doi: 10.1016/ j.socscimed.2020.113557. c:\users\universa medicina\docu 173 abstract universa medicina role of herbs for elevating immunity against sars-cov-2: a concise review aashima anand,1 juhi saraswat1 and rajan patel1* covid-19 has emerged as a dreadful pandemic caused by the virus sarscov-2. though the number of patients recovering from infection is encouraging, sturdy cure is still being researched upon and the effective vaccines are yet to be rigidly resolute. covid-19 has so far instigated a massive loss of lives globally and even the countries with the most advanced healthcare systems have come under its grasp. health experts suggest in such case “prevention is better than cure” which includes an appropriate daily lifestyle and a rugged immune system that could help mankind surmount this peril. this review highlights the importance of influential herbs and their medicinal significance. biomedical articles were explored from pubmed, google scholar, web of science for the period of 2010-2021. guidelines from the ministry of ayush (india) were also considered. the ayurveda does not mention any cure for the novel coronavirus; however, it emphasizes the means of boosting one’s immunity. accordingly, this article highlights some of the role of prevalent ayurvedic herbs in promoting immunity for combating the novel coronavirus. the herbs in combination with drugs could be utilized for the sake of curtailing the side effects as well as the malefic repercussions of strong synthetic drugs used for treating covid-19 patients, which would be a paramount leap in the field of ayurveda as well as western-style medicine. however, the beneficial effects of these traditional medicines and their clinical trials remained to be known. we reviewed the latest updates on traditional medicines proposed for promoting immunity towards covid-19. keywords: covid-19, antiviral agents, ayurveda, herbal plants, immunity, therapeutic uses review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.176-188 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1136 may-august 2021 vol.40no.2 cite this article as: anand a, saraswat j, patel r. role of herbs for elevating immunity against sars-cov-2: a concise review. univ med 2021;40:173-85. doi: 10.18051/univmed.2021.v40.176-188 biophysical chemistry laboratory, centre for interdisciplinary research in basic sciences, jamia millia islamia, new delhi-india. *correspondence: dr. r. patel biophysical chemistry laboratory, centre for interdisciplinary research in basic sciences, jamia millia islamia, new delhi-110025, india tel.: +91 8860634100; fax: +91 11 26983409. email: rpatel@jmi.ac orcid id: 0000-0002-3811-2898 date of first submission, february 12, 2021 date of final revised submission, july 6, 2021 date of acceptance, august 7, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 174 anand, saraswat, patel role of herbs and sars-cov-2 introduction the novel covid-19 pandemic is claiming thousands of human lives worldwide. the world health organization is continuously visualizing the pandemic situation globally regarding the spread, morbidity, and mortality rate. according to the recent who report of 1st may 2021, a total of 150,989,419 conf ir med cases and 3,173,576 deaths have been recorded worldwide which figures ar e e xpec ted to increase exponentially in the coming time.(1) the very high mortality is accounted by its easy human to human transmission from close proximity to an infected person through their respiratory droplets or aerosols via sneezing or getting in contact with any surface presenting these. this virus contains a positively charged single-stranded rna and belongs to the -group of coronaviruses.(2) on the outer surface, the coronavirus exhibits crownlike spikes of glycoprotein, accountable for the attachment of the virus to the host cells’ angiotensin-converting enzyme 2 (ace2) cellular receptors and later through envelope fusion with the cell membrane, the virus detaches the rna into the host cell.(3) this severe acute respiratory syndrome c oronavir us 2 ( sars-cov-2), comprising the contagion, primarily affects the respiratory system.(4,5) initially, cases emerged from wuhan city of china where thousands of patients manifested symptoms of pneumonia that eventually led to the discovery of this virus which apparently originates from bats and which through mutations can infect humans.(6) the initial symptoms include soaring fever, dry cough, shortness of breath, loss of smell (anosmia) besides other uncommon symptoms.(1) several patients are asymptomatic whereas the effects of the virus can be more prominent in patients with a history of diabetes or heart related disorders.(7) pandemics are not an unfamiliar plight and tend to emerge intermittently. the only variance emerging with time is the advancement in medication for treating this peculiar persistant infection in order to shield mankind from its harmful effects. medical therapy employs here in a crucial role for tending to the wellbeing of every suffering individual. the ancient medical system of india, better known as ayurveda, is being emphasized during these momentous days. ayurveda is known not only in india but is also followed across the globe due to its appealing trait being the herbs mentioned there, having no side effects either on humans or on the environment. ayurveda also provides a holistic approach for leading an elementary life. it includes practicing yogasanas and meditation every day to attain a healthy body as well as mind. although the ayurveda does not mention any cure for the novel coronavirus, it emphasizes profoundly on the means of boosting one’s immunity. currently, all countries must team up and collectively fight covid-19 by practicing hand-hygiene and social distancing. accordingly, this article highlights some of the prevalent ayurvedic herbs and their role in promoting immunity for combating the novel coronavirus. virus and antiviral agents viruses are intracellular parasites that do not have any metabolic machinery of their own; hence, they cannot multiply by themselves. to do this, they invade the host cells and employ the biochemical mechanisms of the host cell to promote their own growth by replicating new virus proteins and genetic material and in turn attack the host.(8) for the development of a trailblazing tool against any virus, it is fundamental to understand the mode of viral infection, the intervention of which can prompt its containment. the multiplication of any virus inside the host cells comprises of numer ous steps: attachment (tethering of virus to the host cell membrane), penetration (infiltration into the cell), un-coating (casting its protein coat), multiplication (virus breeding in the host cells), protein synthesis (protein forming post-genome development), assembly (coating of genetic material by protein), release (liberation of the virus) to destroy other cells of the host.(9) the virus can enter the human body anytime; at the earliest a response is initiated 175 by the immune system for combating the invader, the two sub-classes composing the immune system together being intimately associated and functioning in conjugation whenever an immune response is to be ac tivated. the initial classification is referred to as the innate or the non-specific immune system that commences the first ar rangeme nt of response. since the recognition molecules applied by this system are displayed extensively on a wide range of cells, it operates promptly on confronting the virus. the innate immune system allows a generic shield countering detrimental substances and fights utilizing immune cells, in particular, natural killer (nk) cells or the phagocytes that access the body through dermal routes or the digestive system. macrophage activation as part of the innate immune system can be initiated by the release of various cytokines, for instance interferons.(10) interferons strengthen endurance of cells to virus contagion a nd delay the propagation of disease.(11,12) the second classification is named the adaptive, specific, or acquired immune system recognized for producing antibodies to explicitly tackle certain microorganisms, allergens or toxicants the body has hitherto engaged with. since this sort of immune system is unceasingly acquiring knowledge and adapting, the body could also combat viruses that modify in the course of time. this system exhibits distinctiveness for target antigens. accustomed responses are established principally on antigen-specific receptors demonstrated on the surfaces of tand b-lymphocytes. white blood cells (wbcs) are equally essential to the immune system. on discovering any virus es, they initiate an immune response. the wbcs include lymphocytes, in particular b-cells, t-cells and nk cells. an antiviral agent is a substance that kills a virus or inhibits its ability to multiply and produce new virus and narrows the symptoms. the antiviral agent can either be a drug, synthetic or natural, or an antibody-containing vaccine that can produce defensive or curative effects. these drugs can act at varying steps of the virus life cycle such as virus replication by retarding viral genetic matter from replication, penetration and un-coating by hitching to cell-surface receptors or inhibiting protein synthesis and deliverance. antiviral drugs usually possess ‘wide therapeutic window’. however, the fabrication of an antiviral drug or vaccine is never a facile piece of work. the fundame ntal j ustif ica tion for this is the engagement of host cells at each step of the life cycle of the virus in question that endangers the host functions.(13) moreover, the virus changes with time and again undergoing mutations that makes the action of any specific antiviral agent countering it complicated. blocking of enzyme activity is necessary but if it is only partially effective, then resistant mutations can arise, which is more likely to threaten the host. once active inside the host cell, the virus has the tendency of replicating rapidly, hence timely diagnosis and commencing of treatment becomes imperative. concerning the existing covid-19, the screening of patients is largely being done based on the symptoms being apparent in the patients. at the same time, some broad-spectrum antiviral drugs such as remdesivir, ritonavir, lopinavir,(14) azithromycin, hydroxychloroquine (15) and others or treatments in additional forms such as corticosteroids in the form of dexamethasone (16) and plasma therapy have emerged as effective antiviral drugs (17,18) when used in combination with various other drugs or herbs (19) leading to the remarkable reduction in the symptoms of coronavirus to a large extent and eventually resulting in a recovery of the patient. china has made use of its traditional chinese medicine (tcm).(20,21) one of the components of this pathway is the herbal products, in combination with various antiviral drugs, that have shown a high recovery rate amongst coronavirus patients. tcm largely focuses on the overall well-being of the body, rather than emphasizing on treating a particular ailment. the medicines are not a specific herb but comprise of miscellaneous herbal plant products, in formulas selected by and large depending on the individual’s need and ailment, in the form of teas, capsules, or powders. their univ med vol. 40 no 2 176 anand, saraswat, patel role of herbs and sars-cov-2 a b c main focus involves detoxifying preparations and lung cleanser herbal treatments since pneumonia was the highlighted symptom of covid-19 infection in china.(22,23) till date no reliable cures have been developed for this noxious disease. the only way out for saving the mankind from the torment is the vaccination drive being conducted globally. according to world health organization (who), as on 19th march, 2021, at least 7 vaccines have been accepted for emergency use worldwide after successful completion of trials whereas more than 200 auxiliary vaccine contenders are in the development stage, of which more than 60 are in the clinical development phase and a few are in the human trial phase after positive trials.(24) even then, patients with covid-19 are being cured, although there is no certain drug for it yet. taking this into consideration, it is encouraged to follow the best prevention tip against the virus, which is strengthening the body’s natural defense system or in other words the immunity of an individual, rather paying heed to it being baneful. indian herbs and their significance against sars-cov-2 the alarming death rates across countries owing to the novel coronavirus have somehow led to hysteria among the population worldwide since no reliable drug or vaccine is explicitly available for it. the best remedy against this novel virus, which is being laid out in the form of suggestions by numerous nations across the world, is for the individuals to follow a healthy diet in order to improve their immunity to protect themselves from being susceptible to this disease.(25-27) ethnopharmacology becomes a budding concept in these times since diving into the vast pool of knowledge of botanical drugs is called for during this hour of crisis. plant-based medicines are an integral part of the indian medicine system and sculpt a requisite element of our culture. for centuries, this comprehension has been used enormously as a pointer for drug development.(28-30) traditions like ayurvedic, chinese, kampo, unani, iranian, and many more have been inherited by every generation and conceptualized in a distinct manner by each of them. even the who recognizes the gravity of the preservation and acknowledgement of all these ancient medicinal systems.(31) indian ancient and sacred scriptures are laden with investigations involving traditional use of herbal plants and comprehension of their risks and benefits for mankind and report close to 800 plants as medicinal ones. acc or ding to the who, approximately 80% of the population dwelling in developing countries relies on these traditional approaches of he rbal re medies for their therapeutic needs.(32) it is estimated that in india, over 6000 plants are in use as traditional and herbal medicines.(33) the foremost rationality backing it is the cost-effectiveness of these herbal products since these are present in ample quantities and no considerable manipulation from their native manifestation is desired. also, minimum side effects are perceived by employing these herbal plant products in remedial utilization. typical ailments like common cold, flu, fever, cough, microbial or skin infections, hypertension, asthma and others are effortlessly cured using these practices, whereas strenuous maladies comprising diabetes, depression, stress, heart disease, cancer, tuberculosis and many more have the remedies for th eir prevention and management in these practices.(34,35) however, the key hindrance involves the scientific study of the intricate chemical constituents of these herbal products, demanding their extraction and further investigations since there is never an active sole component involved but rather complex modules creating enormous structural complications. every plant consists of pr imar y and secondary metabolites that are involved in numerous functions of the plant. primary metabolites hold significance in the basic obligations of the plant including its cell division, glycolysis, growth and so on and encompass carbohydrates, proteins, amino acids, chlorophyll etc., whereas the commercial importance of these herbal plants lies in their secondary metabolites which are of prime fascination by virtue of their possession of 177 bioactive metabolites such as alkaloids, terpenes, phenolics, lipids and others. (3 2) even the supplementary assets of a plant, such as fragrance, as natural dying agents, flavoring agents or natural insecticides or herbicides, are all associated with these secondary metabolites. the antioxidant, antimicrobial, antidiabetic, anticancer and various other properties are all owing to these secondary metabolites, be it their presence in the roots, seeds, stems, leaves, barks, flowers, or fruits of the concerned plant.(36) these are accumulated in scant quantities in plants and hence the process involving their extraction, purification, separation, identification, and eventual studies is a major root of concern which in a way limits the use of the plant as a therapeutic product. each class of secondary metabolites is responsible for discrete contribution. for example, alkaloids dominate antimicrobial, stimulant properties, flavonoids are accountable for antioxidant, antiinflammatory, anticancer, anti-allergic measures, and so on.(37,38) ayurveda is the indian ancient system of healthcare recommending what practices to comply with for embracing a holistic health as well as accomplishing a vigorous immunity towards procuring a profound life aimed at achieving positive health and longevity. it is identified as the oldest medicinal discipline in the world and is believed to have originated more t ha n 5, 000 ye a r s a go a nd i s e ve n n ow evolving.(39-41) it considers eradicating the root rationale of the malady a pivot strategy about suggested diets and health practices. ayurveda houses varied features of distinct diseases, the ther apeutics incorpor ating detoxification ( pa nc h ka r ma ) a l o ng wi t h yoga a n d pharmacopoeia; it is built entirely on natural mediums and all these therapies being conducted and proposed by a competent vaidya (ayurveda s pe c i a l is t ) . in ay u r v e d a, t h e me d i c i na l preparations entail complex mixing of plant or animal-derived products, metals, and minerals for tending to the ailment. the ancient indian scriptures citing these in specifics include the rig veda, atharva veda, sushruta samhita, charak samhita and ashtanga hridayam sangraha among others. in present times, the ayurvedic practices are persist not only in india but also in developed nations like usa and european countries where these are either taken along the synthetic drugs to reduce the adverse effects done to the body or after successful completion of the course of these drugs so as to harmonize the body.(42) this is attributed to its safety by manifesting minimum side effects, coming across with an extensive therapeutic domain, indicating precise outcomes and being pocket-friendly along with revitalization and nourishment of body tissues or organs in contrast to synthetic drugs that weaken or desecrate them by prolonged use. although, like their synthetic counterparts, these herbal products when taken for a medicinal purpose, must be consumed under an approved guidance. in addition, a variety of antivirals are fabricated from plant herbal sources by replicating the structure of the extracted active ingredient. the aforesaid are accepted as processed drugs with utilization o f c r e a t i ng a nt i vir a l d r u gs o n ma s si ve consignments through steps of semi-synthesis or chemical transformations owing to their inadequacy of interfering with the host cells. acquiring the traits of both herbal medications and synthetic drugs, these are at edge entitling to exigency and are innocuous as well as reliable.(43) these products are persistent for decades for the treatment of critical viral infections such as influenza, herpes, smallpox, hepatitis, and others.(44-47) one quarter of the r o u ti n e me di c i n e s h ol d pl a nt -de r i ve d compounds, the topmost prominent being quinine, procured from cinchona tree bark, and r e f or me d in t o c h lo r o q ui ne b e s i de s hydroxychloroquine. parallel is the account of taxol (periwinkle plant), aspirin (willow bark) and morphine (opium poppy).(48) the immense benefit here is the controlled drug delivery in contrast to their herbal counterparts where it bec omes a pitf all in their use. hence , a c o mb i n a t ion o f t hi s s o r t of syn t he si ze d formulation sets off as a superior preference univ med vol. 40 no 2 178 anand, saraswat, patel role of herbs and sars-cov-2 owing to synergistic sequel and generates a consequential therapeutic effect.(49) a variety of indian herbs have proven to be beneficial against infections and possess properties such as being antiviral, antibacterial and antifungal.(50-52). indian ancient scripts, written thousands of years ago, including charak samhita, mention simple remedies for approaching a fit well being. even drinking lukewarm water throughout the day contributes to the harmony of the body. this alongside following a routine of practicing yogasanas and meditation everyday leads to a blessed immunity. also, relieving and managing stress in the body is an important aspect for reviving the immune system. in this case, even simple deep breathing can prove to be useful. since these exercises primarily focus on breathing, the outcome is a strengthened respiratory system, which is the need of the hour for combating the novel coronavirus, as its main target is the lungs. moreover, if a person is suffering from covid19 infection but his or her immunity is well enough, the recovery can be swift.(53) herbs can be consumed in varied forms by anyone, be it taken as it is, such as tulsi/ basil leaves (ocimum tenuiflorum) , fennel seeds (foeniculum v ulgare), indian gooseberry (phyllanthus emblica), raisin (vitis vinifera), neem (azadirachta indica) or used in cooking, such as garlic (allium sativum), ginger (zingiber officinale), black pepper (piper nigrum), cardamom (elettaria cardamomum), turmeric (curcuma longa), coriander (coriandrum sativum), clove (syzygium aromaticum), cumin (cuminum cyminum), curry leaves (murraya koenigii), bay l eaves/bay la ur el (laurus nobilis).(54) they can also be consumed as an ingredient in herbal tea or in lukewarm water, such as lemon (citrus limon), cin namon (cinnamomum verum), peppermint (mentha piperita), ginger, fennel seeds and tulsi or can be taken with hot milk like turmeric or in the form of extracted juice, such as from the stem of giloy/ heart-shaped moonseed (tinospora cordifolia) or in the form of powder or capsules of giloy or ashwagandha/ indian ginseng (withania somnifera).(54) (figure 1). the immune system is reinforced stronger through the nutrients. inadequate nutrition contributes to a rise in infections whenever provoked by external stimuli and magnifies figure 1. showing the importance of indian herbs against novel cov-2 virus (55-57) 179 vulnerability to manifestations and complexities arising from malfunctioning of the immune system. it is well established that immune function usually lessens with age or may even be hindered if one abandons upholding healthy nutrition. table 1 describes the contribution of these herbs to the immune system. the unparalleled property of these herbs is the numerous forms in which they can be consumed by an individual. most of these can be consumed as they are, through cooking or in a concoction in tea or water or in the form of essential oils. for example, consider the herb tulsi, it can be eaten raw, or concocted with herbal tea, lukewarm water or even as chyawanprash (ancient ayurvedic concoction of various natural herbs), or as an essential oil as well.(63-65) similar is the case with giloy, its powder obtained from the leaves, flower or roots can be consumed in concoction with hot water or ingested in the form of juice from its stems. this can prove to be convenient so as to reap the maximum benefits out of a particular herb. in india, clinical trials are being conducted to comprehend the effect some of the herbs on the body for fighting against covid-19.(66) a few herb main active constituent therapeutic uses turmeric curcuma longa curcumin antiviral (effective against flu, hiv, herpes, hepatitis), antibacterial, anticancer, antioxidant, anti-inflammatory, detoxifier (50,51) neem azadirachta indica azadirachtin antiviral (effective against dengue), anti-diabetic, prevents skin infections, antioxidant, anti-inflammatory, detoxifier (5) peppermint menthapiperita menthol, menthone and menthyl acetate antiviral (effective against influenza and flu), antibacterial, antioxidant, antifungal (52,67) tulsi ocimumtenuiflorum methyl eugenol and methyl chavicol antiviral (effective against flu, common cold), antibacterial, antimalarial, prevents skin infections (56,68) ginger zingiber officinale gingerols, zingerone and shogaols antiviral (effective against flu, common cold), antiinflammatory, antinausea, antipyretic and analgesic (63) indian gooseberry phyllanthus emblica emblicanin a and b, gallic acid, ellagic acid antiviral (effective against influenza and flu), anti-diabetic, anticancer, antioxidant), anti-inflammatory (70) garlic allium sativum allicin and alliinase antiviral (common cold and flu), antifungal, anthelmintic, anticancer, antihypertensive, antioxidant, antidiabetic, natural antibiotic (60) cinnamon cinnamomum verum trans cinnamaldehyde and eugenols antioxidant, antifungal, anti-diabetic, antiinflammatory(72) cumin cuminum cyminum cuminaldehyde antioxidant, anticancer, anti-diabetic, antibacterial (73) lemon citrus limon linanool and geraniol anticancer, antioxidant, detoxifier, anti-nausea, protects against skin diseases (74) giloytinospora cordifolia berberine, tinocordiside and furanolactone antioxidant, anticancer, antibacterial, anti-diabetic, stress relieving, anti-inflammatory, anti-arthritic, antipyretic, analgesic (75,76) ashwagandha withaniasomnifera anaferine, withanone and somniferinine anti-inflammatory, anti-arthritic, stress relieving, anticancer, antioxidant, natural antibiotic, anti-ageing (77) table 1. therapeutic properties of indian herbs for immunity boosting univ med vol. 40 no 2 180 anand, saraswat, patel role of herbs and sars-cov-2 in covid-19 patients is encouraging. some of the herbal formulations which are in the clinical trial phase are listed in table 2. the herbs serve by increasing the production of proteins named interferons and antibodies to trigger an immune response against viruses and therefore accelerate phagocytosis for impairing microorganisms and hence augment immunity by curbing the viral infection.(71) herbs contain of such herbally formulated medications have already been approved for use and more are either in preclinical or clinical trials.(66,67) a protocol has been shared by the ministry of ayush, india regarding the management of covid-19 with ayurvedic approach and yoga that also incorporates the use of ayush 64 and kwatha, both polyherbal medications.(68-70) the recovery shown by the usage of such formulations id title approach remarks 226923 siddha and biomedicine integrative management of novel corona virus disease a case report (78) siddha herbal formulations named nilavembu kudineer & chukku kanji used along with allopathic medications with encouraging results 216624 three case reports of moderate covid19 infection managed through ayurvedic approach (62,63) ayurveda success achieved from kwatha and few others polyherbal formulations used and priority given to mental & physical health of patients 216623 efficacy of ayurveda and yoga in the management of sars-cov-2: two case reports (63) ayurveda & yoga kwatha and few others polyherbal formulations used and focused on personalized holistic treatments for mild to moderate symptoms 206596 a retrospective analysis of the effect of the intervention of arogya kashayam in covid-19 positive cases in madhya pradesh (79) ayurveda a polyherbal formulation arogya kashayam-20 (ak-20) used as a vast case study in mildly symptomatic patients either independently or in combination with hcq 185937 safety and efficacy of herbal extracts to restore respiratory health and improve innate immunity in covid-19 positive patients with mild to moderate severity: a structured summary of a study protocol for a randomised controlled trial (80) ayurveda two polyherbal formulations (ip-1 & ip2) along with calcinated zinc used and their safety and success in restoring respiratory health was accessed 185935 novel coronavirus (covid-19) etiological characteristics, clinical manifestations and unani management (81) unani various unani medicines and syrups tested against covid-19 infection based on the symptoms manifested and history of other diseases in those patients 165568 outcomes of ayurvedic care in a covid19 patient with hypoxiaa case report (82) ayurveda numerous ayurvedic formulations employed along with vitamin c and emphasized on treating hypoxia 164648 ayurvedic treatment of covid19/sars-cov-2: a case report (83) ayurveda treatment carried out entirely based on ayurveda using 4 polyherbal formulations: sudarsana churna, dhanvantara gutika, talisadi churna & deepanam 164467 ayush 64, a polyherbal ayurvedic formulation in influenza-like illness results of a pilot study(84) ayurveda a polyherbal formulation ayush64 effective in treating mild to moderate cases of covid-19 and is currently approved for use. table 2. herbal formulations in the clinical trial phase in india 181 hardly any chemicals that are unfavorable for the human health and seldom show any allergies or other adverse side-effects on the body, a phenomenon common to the synthetic drugs and medicines. in addition, the herbs can strengthen the immune system enough to withstand the infection and provide prompt symptomatic relief. accordingly, an enhanced alternative these days is treating the infection using a combination of modern drugs and herbs. for instance, in the chongqing region of china, doctors resorted to the drug kaletra in combination with tcm to treat the patients and the results came out to be affirmative.(72) in india, the doctors are relying chiefly on the drug hydroxychloroquine for the efficacious treatment of the symptoms of this calamitous disease. on that account, since india has plenteous sources of herbs and all of their properties are well established already, if india goes ahead with the exploration as to how the combination of herbs and drugs can be utilized for the sake of curtailing the side effects as well as the malefic repercussions of strong synthetic drugs used for treating covid-19 patients, it would be a paramount leap in the field of ayurveda as well as medicine. conclusion it is a well-established fact that herbal plants and their derived products have contributed for ages in steering clear of ailments and meanwhile in this pandemic, they are benefitting again. herbal plants have several active ingredients and when used in a complementary manner with synthetic drugs, the therapeutic effects can be collusive. acknowledging that, the world must work towards its success and carry research in the direction of herbs and their products with synthetic drugs and accomplish in the outlook of attaining the utmost from these natural marvels since herbs are genesis of innumerable traits as discussed in this review. until then, in this desperate stretch of sars-cov-2, heed should be at enhancing one’s immunity by virtue of abounding herbs accessible in nature. acknowledgments the authors do acknowledge the efforts of all the doctors, health workers, scientists and researchers currently working against sarscov-2. the authors greatly acknowledges the financial support from science and engineering research board (eeq/2020/000437) new delhi, india. conflict of interest the authors declare no competing financial interest. contributors aa and js wrote the manuscript. rp designed the work and was overall corresponding author. all authors have read and approved the final manuscript. references 1. world health organization. coronavirus disease (covid-19): situation report 182. geneva: world 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https://doi.org/ 10.1016/j.jaim.2020.05.010. univ med vol. 40 no 2 seizure increases electroencephalographic abnormalities in children with tuberculous meningitis 161 department of pediatrics, faculty of medicine, airlangga university, surabaya correspondence: dr. prastiya indra gunawan, spa department of pediatrics, faculty of medicine, airlangga university rsu dr. soetomo jl. mayjen. prof. dr. moestopo 4-6, surabaya 60132 email: prastiya_ig@yahoo.co.id mobile: +62811 342 9476 fax: +6231 5501748 univ med 2015;34:161-7 doi: 10.18051/univmed.2016.v35.161-167 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background tuberculous meningitis (tbm) is a severe intracranial infection with fatal outcomes, permanent disabilities, and electroencephalographic (eeg) abnormalities. seizures may occur in tbm. the eeg findings in tbm vary according to the site of the inflammatory process. there are few studies describing the eeg patterns and clinical manifestations of tbm. the objective of this study was to investigate the correlation between clinical findings and eeg patterns in children with tbm. methods a study of cross-sectional design using medical records was conducted on 12 children with tbm, with their eeg patterns classified as abnormal and normal. clinical manifestations such as seizures, altered consciousness, headache or fever were collected. a positive cerebrospinal fluids mycobacterium tuberculosis culture was considered to indicate definitive tbm. abnormal eeg descriptions were classified into abnormal i, ii or iii. correlation between eeg pattern and clinical manifestation were analyzed with fisher’s exact test. results the study found cases of 12 children with tbm, the majority presenting with seizures, decreased consciousness and fever. abnormal eegs were found in 75% of children and 77% of them showed epileptogenic activities. the eeg results mostly described epileptogenic potentials in the frontotemporal region. there was a significant correlation between eeg abnormality and seizures in children with tbm (p<0.05). conclusions the eeg pattern in children with tbm varies, and eeg abnormalities were more frequently localized in the frontotemporal region. seizures were associated with eeg abnormalities in children with tbm. eeg abnormalities occurring simultaneously with seizures may predict the occurrence of seizures. keywords: tuberculous meningitis, eeg, children seizure increases electroencephalographic abnormalities in children with tuberculous meningitis prastiya indra gunawan* and darto saharso* doi: http://dx.doi.org/10.18051/univmed.2015.v34.161-167 162 gunawan, saharso seizure electroencephalographic tuberculous kejang meningkatkan kelainan elektro ensefalogram pada anak dengan meningitis tuberkulosis latar belakang meningitis tuberkulosis (mt) adalah infeksi intrakranial berat yang dapat berakibat fatal, kecacatan permanen dan menunjukkan keabnormalan pada hasil elektro ensefalogram (eeg). kejang dapat terjadi selama proses mt. hasil eeg pada mt bervariasi tergantung pada daerah setempat dimana proses inflamasi berlangsung. studi yang mendiskripsikan tentang pola eeg dan manifestasi klinis pada mt cukup terbatas. tujuan dari penelitian ini adalah untuk menentukan adanya hubungan antara manifestasi klinis dengan pola eeg pada anak dengan mt. metode studi potong lintang menggunakan catatan medik dilakukan pada anak dengan mt. sebanyak 12 anak dengan mt dilakukan pemeriksaan eeg dan diklasifikasikan sebagai normal dan abnormal. manifestasi klinis berupa kejang, penurunan kesadaran, nyeri kepala dan panas telah dikumpulkan. adanya hasil kultur positif tb dalam cairan serebrospinal dipertimbangkan sebagai meningitis tb definitif. deskripsi eeg yang tidak normal diklasifikasikan sebagai abnormal i, ii, dan iii. hubungan antara pola eeg dan manifestasi klinis dianalisis dengan uji eksak fisher. hasil penelitian ini mendapatkan 12 kasus mt pada anak. kebanyakan kasus bermanifestasi kejang, penurunan kesadaran dan panas. eeg abnormal didapatkan pada 75% anak dimana 77% diantaranya menunjukkan aktifitas epileptik. hasil eeg banyak mendiskripsikan potensial epileptik di daerah frontotemporal. didapatkan hubungan antara eeg yang tidak normal dengan manifestasi kejang pada anak dengan meningitis tb (p<0.05). kesimpulan pola eeg pada anak dengan mt bervariasi. abnormalitas eeg lebih banyak didapatkan pada daerah frontotemporal. kejang berhubungan dengan abnormalitas eeg pada anak dengan mt. abnormalitas eeg yang terjadi bersamaan dengan kejang dapat memprediksi terjadinya kejang. kata kunci: meningitis tb, elektro ensefalogram, anak abstrak introduction tuberculous meningitis (tbm) is one of the common infections of the central nervous system (cns) representing a serious problem, not only in developing countries endemic to tuberculosis, but also worldwide, due to increased immigration. it is characterized by fever, headache, alterations of consciousness, vomiting, focal neurological deficits, and seizures.(1-3) in many areas of africa and asia, the annual incidence of tuberculosis (tb) infection for all ages is approximately 2% and about 15% to 20% of these cases occur in children. tbm complicates about 1 in 300 of untreated cases of tb globally.(1,2) in china, fiftyfour percent of patients aged 0 to 18 years had extrapulmonary tuberculosis (eptb), and 38.8% suffered from tbm.(4) tbm is still one of the common infections of the central nervous system and poses significant diagnostic and management challenges.(1) the most common complications of tbm observed in 81 long-term survivors were epilepsy (11%) and cognitive impairment (12%).(5) seizures are a common clinical feature of tbm, 163 univ med vol. 34 no.3 occurring at any stage of the infection. they cause an additional burden of disability, producing neurological deficits which are a significant predictor of mortality, often requiring long-term antiepileptic therapy.(6) seizures can be the presenting feature of tbm, especially in children.(7) electroencephalography (eeg) has been reported to be better in assessing the gravity of lesions and was recently reported to help in prediction of outcomes.(8) the eeg recordings in tbm depend on the site of the inflammatory process. not much research has been reported on eeg abnormalities in tbm. advanced neuroimaging has yielded valuable data about tbm pathological changes. the relation of clinical findings and eeg pattern may produce better understanding of tbm management and valuable prognostic clues. the objective of this study was to determine the correlation between eeg patterns and clinical manifestations in children with tbm. methods research design a cross-sectional design using medical records was conducted at soetomo hospital, surabaya, indonesia, from january 2012 to june 2013. research subjects a total of 12 children with tbm were investigated. the age of the subjects ranged between 5 to 108 months and eight of them were males and four females. measurements clinical manifestations such as seizures, altered consciousness, headache or fever were recorded. the glasgow coma scale (gcs) was applied to classify consciousness. the tbm diagnosis was based on history, clinical symptoms, and cerebrospinal fluid analysis. the evidence of tuberculosis outside the cns and response to anti tubercular medication were also observed. the presence of a positive csf mycobacterium tuberculosis culture was considered to indicate a case of definite tbm.(8) eeg recordings had been made on hospitalized children with tbm. the eeg descriptions were classified as abnormal eeg i, ii or iii. the level of eeg abnormalities depends on the clinical manifestations and correlates with the severity and specificity of cortical dysfunction of the eeg abnormality. a patient with mild slowing of the background activity associated with a mild diffuse functional cerebral disturbance is classified as abnormal i. if there is intermittent slow activity that is obviously localized or lateralized, then it should be classified as abnormal ii. an eeg that shows spikes but is otherwise unremarkable is classified as abnormal iii.(9) statistical analysis the clinical manifestations and eeg patterns were analyzed by fisher’s exact test. a p value of <0.05 was categorized as statistically significant. ethical clearance the ethical clearance certificate had been issued by the ethics committee of health and research dr. soetomo hospital. results tuberculous meningitis cases were found in 12 children; the median age was 42 (range 5-108) months, eight (66.7%) of them were males and four (33.3%) females. the cases presented with seizures in 10 (83.3%) children, decreased consciousness in 7 (58.3%) children, fever in 9 (75.0) children, spasticity in 2 (16.7%) children, headache in 2 (16.7%) children, and hemiparesis in one child (8.3%) (table 1). approximately two (16.7%) children suffered from hydrocephalus. the eegs were abnormal in 8 (66.7%) children, and 77% was categorized as abnormal iii, 11.5% as abnormal ii and 11.5% as abnormal i while 25% children had normal eegs. the abnormal iii eeg described epileptogenic activity mostly in the frontal (figure 1) and temporal regions as follows: at the left frontal region in 2 children, left and right frontal regions in 1, right frontotemporal region in 1, left frontotemporal 164 gunawan, saharso seizure electroencephalographic tuberculous region in 1, right and left temporal regions in 1 and in both hemispheres in the 6 remaining children. there was a significant relationship between seizures and eeg abnormalities (p<0.05) (table 2). discussion in our research, the eeg recordings were found to be abnormal in 75% patients. the most common abnormalities were abnormal iii in about 77%. the epileptiform activity potentials were mostly in the frontal and temporal regions, at left frontal region in 2 children, left and right frontal region in 1, right frontotemporal region in 1, left frontotemporal region in 1, right and left temporal region in 1 and bilateral cerebral hemisphere region in 1 child. table 1. characteristics of the subjects figure 1. eeg pattern in one tbm patient showing epileptiform waves in left frontal region 165 univ med vol. 34 no.3 table 2. clinical manifestations and eeg patterns in tbm patients tuberculosis of the central nervous system is the most serious complication of tuberculosis in children. many kinds of pathologies may appear simultaneously in tbm, for instance, hydrocephalus, vasculitis, and edema, that result in ischemia, infarction or tuberculoma. the pathological changes in various or isolated combinations may determine eeg abnormality in tbm. eeg descriptions in tbm depend on the site of the inflammatory process. in case of basal meningitis, eegs may be normal and may show mild unspecific changes. mild to severe slowing of eeg background might be caused by the involvement of the cerebral cortex and meninges. the degree of slowing depends on the rate of progression of the disease process, the extent of cortical involvement, associated metabolic factors, medication and level of consciousness. in tbm, severe inflammation with proliferative changes and vasculitis may lead to changes in clinical symptoms such as level of consciousness disturbance ranging from delirium to coma, cranial nerve involvement, hemiplegia, hydrocephalus, and epileptic seizures.(10-12) our study showed that seizures observed clinically were present in 10 patients but epileptogenic activity were present in 8 patients, and the other patients had normal eegs. there were two patients without seizures, one of whom had epileptogenic activity while the other had a normal eeg. this resulted in significant correlation between seizures and eeg abnormalities. the etiology of seizures in tbm is multifactorial and is related to pathophysiological changes occurring during the disease process. cerebral edema, meningeal irritation, and raised intracerebral pressure are thought to represent the most probable causes of seizures in the early phase of tbm. arman et al.(13) reported a case with altered mental status who was diagnosed with non convulsive status epilepticus (ncse). a workup to explain the etiology revealed tbm with increased cerebrospinal fluid protein and positive 166 gunawan, saharso seizure electroencephalographic tuberculous tuberculous dna polymerase chain reaction and interferon-γ assay tests. in a study by kalita el al., (14) eeg descriptions were reported to be commonly abnormal in stage iii tbm patients. out of 21 patients in stage iii, 19 had eeg abnormality whereas 2 out of 6 patients in stage ii and 1 out of 5 in stage i patients had eeg abnormality. epileptiform discharges were also present in stage iii. out of 4 patients with epileptiform discharges, clinical seizures were observed in 3.(14) in tbm, eegs may show at least moderate diffuse slowing with or without focal findings and epileptiform activity, depending on the severity of the clinical symptoms.(6,9) study by kalita et al.(14) showed that the eeg was abnormal in 11 patients which included delta slowing in 5, theta slowing in 6, frontal intermittent rhythmic delta activity (firda) in 3 and epileptiform discharges in 2 patients.(14) any cns infection that affects the brain parenchyma may result in epileptic syndrome as a chronic sequel as well as acute seizures at the time of infection. chronic phase epilepsy as a complication of cns infection in tbm occurs in more than 20% of cases.(1) a novel study focusing on advanced neuroinflammation and cellular injury in the field of pediatric epilepsy surgery has suggested a common pathogenic consequence in childhood epilepsy with different etiologies. the pathophysiologic basis of chronic epilepsy following cns infection is not wellknown. reactive gliosis following severe neuronal injury from changes in the local cellular and physiochemical environments have supported the likelihood of a common final pathway for seizure multiplication in various situations with many causes.(14,15) another study in african children showed that headache, convulsion, motor function, brainstem dysfunction, and cerebral infarction were independently associated with poor outcomes of tbm. (16) a recent study has demonstrated that seizures associated with tuberculomas most often resolve after successful treatment of the underlying cns tuberculosis.(17) in this study, several clinical manifestations such as decreased consciousness, fever, headache, spasticity and hemiparesis had no signification correlation with eeg pattern. head neuroimaging in childhood tbm with epileptiform discharges were abnormal in two and showed hydrocephalus and infarction in both. in the last remaining patients, head ct scans were obviously normal.(3) in the study of kalita et al.,(14) eeg results were associated with three months outcome. the abnormality pattern shown in the eeg descriptions of tbm has been correlated with poor outcome. the relative significance of different types of eeg changes in the prognosis of tbm would need a further study with consecutive long term follow-up. the eeg can often be a reliable predictor of outcome., and is most useful in differentiating organic from psychiatric conditions, in excluding nonconvulsive status epilepticus (ncse), and in providing a rough guide as to the degree of cortical and subcortical dysfunction.(8,11) tuberculous meningitis starts with nonspecific symptoms and is often only diagnosed when brain damage has already occurred. the eeg continues to play a central role in diagnosis and management of patients with seizure disorders, because it is a convenient and relatively inexpensive way to demonstrate the physiological manifestations of abnormal cortical excitability that underlie epilepsy.(8) tuberculous meningitis is associated with a variable degree of encephalitis, which is primarily responsible for the altered sensorium. the other factors contributing to altered sensorium in tbm may include hydrocephalus, infarction, or tuberculoma in a strategic location.(18) the overall prognosis of tbm remains poor, with 10-20% case fatality (worse if treatment is delayed and the patient is comatose) and 20-30% long term morbidity (cognitive and behavioral impairment, epilepsy, cranial nerve palsies and paresis).(19) early diagnosis and treatment correlates with better outcomes. given the difficulties in obtaining a rapid diagnosis, therapy must often be initiated empirically.(20) in the present study, radiograph interpretation should have been performed, and eeg might be useful to monitor tbm patients, if interpreted by professionals in the field of clinicalradiological findings. this study shows the 167 univ med vol. 34 no.3 importance of eegs to detect seizure activity and risk of epilepsy in children with tbm. for further research, it is necessary to conduct studies related to prognosis of children with tbm, especially those with abnormal eegs, and to the quality of life of patients who survive the emergency. conclusion the eeg pattern in children with tbm varies. abnormal iii in frontotemporal regions seems to be more common. seizures were associated with eeg abnormalities in tbm. eegs showing the presence of seizures may assist in localizing the site involved and predict seizure occurrence. conflict of interest the authors declare no conflict of interest evidence related to this article. acknowledgement our highest gratitude to mangihut rumiris for her contribution to data collection. references 1. philip n, william t, vanitha d. diagnosis of tuberculous meningitis: challenges and promises. malaysian j pathol 2015;37:1-9. 2. murthy jm. management of intracranial pressure in tuberculous meningitis. neurocrit care 2005;2:306-12. 3. paganini h, gonzalez f, santander c, et al. tuberculous meningitis in children: clinical features and outcome in 40 cases. scand j infect dis 2000;32:41-5. 4. wu xr, yin qq, jiao ax, et al. pediatric tuberculosis at beijing children’s hospital: 2002–2010. pediatr 2012;130:e1433-40. 5. anderson ne, somaratne j, mason df, et al. neurological and systemic complications of tuberculous meningitis and its treatment at auckland city hospital, new zealand. j clin neurosci 2010;17:1114–8. 6. brigo f, ausserer h, zuccoli g, et al. seizure heralding tuberculous meningitis. epileptic disord 2012;14:329-33. 7. miftode eg, dorneanu os, leca da, et al. tuberculous meningitis in children and adults: a 10-year retrospective comparative analysis. plos one 2015;10:e0133477. 8. smith sjm. eeg in the diagnosis, classification, and management of patient with epilepsy. j neurol neurosurg psychiatry 2005;76 suppl ii:ii2–ii7. 9. luders h, noachtar s. atlas and classification of electroencephalography. philadelphia: saunders;2006. 10. park s, hong yj, lee mk, et al. hippocampal sclerosis and encephalomalacia as prognostic factors of tuberculous meningitis-related and herpes simplex encephalitis-related epilepsy. seizure 2011;20:570-4. 11. kaplan pw. the eeg in metabolic encephalopathy and coma. j clin neurophysiol 2004;21:307-18. 12. ikeda a, klem gh, luders ho. metabolic, infectious and hereditary encephalopathies. in: ebersole js, pedley ta, editors. current practice of clinical electroencephalography. 3rd ed. philadelphia: lippincott williams and wilkins, 2003.p.348-77. 13. arman f, kaya d, akgün y, et al. tuberculous meningitis presenting with nonconvulsive status epilepticus. epilepsy behav 2011;20:111-5. 14. kalita j, misra uk, das bk. spect changes and their correlation with eeg changes in tuberculous meningitis. electromyogr clin neurophysiol 2002;42:39-44. 15. choi j, nordli dr, alden td, et al. cellular injury and neuroinflammation in children with chronic intractable epilepsy. j neuroinflammation 2009;6: 38. 16. van well gtj, paes bf, terwee cb, et al. twenty years of pediatric tuberculous meningitis: a retrospective cohort study in the western cape of south africa. pediatr 2009;123:e1-8. 17. alsemari a, baz s, alrabiah f, et al. natural course of epilepsy concomitant with cns tuberculomas. epilepsy res 2012;99:107-11. 18. misra uk, kalita j, roy ak, et al. role of clinical, radiological, and neurophysiological changes in predicting the outcome of tuberculous meningitis: a multivariable analysis. j neurol neurosurg psychiatry 2000;68:300–3. 19. ginsberg l, desmond k. chronic and recurrent meningitis. practical neurol 2008; 8:348-61. 20. christie l, loeffler am, honarmand s, et al. diagnostic challenges of central nervous system tuberculosis. emerg infect dis 2008;14:1474-5. c:\users\universa medicina\docu 71 abstract universa medicina neutrophil-lymphocyte ratio and fournier gangrene severity index are not prognostic factors of mortality in fournier gangrene patients muhammad achdiar raizandha1,2, furqan hidayatullah1,2, yudhistira pradnyan kloping1,2, and fikri rizaldi1,3* background fournier gangrene (fg) is a life-threatening disease, commonly found in diabetic and immunocompromised patients. recent studies suggested the use of new parameters apart from the commonly used fournier gangrene severity index (fgsi), such as the neutrophil-lymphocyte ratio (nlr), the clinical use of which remains questionable. therefore, we aimed to evaluate the role of the nlr and fgsi as a prognostic factor of mortality in patients with fg. methods this is an analytical study with a retrospective approach involving 109 adult patients diagnosed with fg. data were collected regarding medical history, symptoms, physical examination findings, and laboratory tests. the fgsi score and nlr were determined. bivariate analysis was performed using chi-square test and independent t-test. overall survival between groups was compared using kaplan–meier survival estimates and cox regression test. results of the 109 patients, 90 survived (82.5%, group 1) and 19 died (17.43%, group 2). the cut-off point of nlr among the patients was 10.9, with a 73.7% sensitivity and 60% specificity. the area under curve value was 0.65 (95% ci; 0.524-0.754; p<0.05). the kaplan meier survival analysis showed that nlr was as an independent prognostic factor of mortality in fg patients (hr 5.177; 95% ci; 1.092-8.471; p<0.05), but cox regression analysis showed that nlr and fgsi were not significant prognostic factors of mortality (p=0.09 and p=0.179; respectively). conclusion this study demonstrated that nlr and fgsi are not important as prognostic tools for fg mortality. keywords: fournier gangrene, prognostic factor, neutrophil, lymphocyte, neutrophil-lymphocyte ratio original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.71-78 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1263 january-april, 2022 vol.41no.1 1department of urology, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2department of urology, dr. soetomo general academic hospital, surabaya, east java, indonesia 3department of urology, universitas airlangga teaching hospital, surabaya, east java, indonesia *correspondence: fikri rizaldi department of urology, universitas airlangga teaching hospital, surabaya, east java, indonesia phone/fax: (031)-5916290 email: fikririz@gmail.com orcid id: 0000-0002-4588-6584 date of first submission, december 9, 2021 date of final revised submission, march 30, 2022 date of acceptance, april 9, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cit e th is ar ticle as: ra iza ndh a m a, hidayatullah f, kloping yp, rizald f. neutrophil-lymphocyte rat io a nd fournier gangrene severity index are not prognostic factors of mortality in fournier gangrene patient. univ med 2022;41:7178. doi: 10.18051/univmed.2022.v41. 7178 72 raizandha, hidayatullah, kloping, et al neutrophil-lymphocyte ratio of fg patients introduction fournier gangrene (fg) is a rare type of necrotizing fasciitis that affects the perineal, genital, or anorectal region. it is characterized by widespread soft tissue necrosis and systemic toxicity of the superficial fascia and subcutaneous tissues.(1) even though it is quite uncommon, constituting only 0.02% of all hospital admissions, it is considered a urological emergency as it has a high mortality rate, at 20 to 50% in most reported series.(2,3) in recent years, the incidence of fournier gangrene is increasing with the increase in diabetes prevalence and the number of immunocompromised patients due to various causes.(4) previous studies have determined the possible risk factors for predicting the prognosis of fournier ga ngr ene patie nts, such as comorbidities, fournier gangrene severity index (fgsi) score and disease severity. ( 5– 7 ) parameters to determine the severity and prognosis of the disease have been suggested, one of which is the fgsi, commonly used to assess the severity of the disease by evaluating clinical and laboratory parameters, such as temperature, heart rate, respiratory rate, serum sodium, serum potassium, serum creatinine, serum bicarbonate, hematocrit and white blood cell count.(8) since its introduction, the score has been validated by many studies, but its accuracy remains questionable.(9,10) nevertheless, it is the only well-known tool to assess severity.(10) recent findings have suggested simple and promising parameters by utilizing normal laboratory findings. there is an increasing interest in predicting the prognosis of the patients with a simple blood test since studies began suggesting a correlation between inf lammatory status and disease prognosis. one of the most commonly used parameter is the neutrophil-lymphocyte ratio (nlr).(11) findings regarding its potential use in predicting the prognosis of fournier gangrene patients have been reported. a retrospective study showed that the fgsi scoring system was not associated with determining poor prognosis. however, high nlr and high platelet to lymphocyte ratio (plr) were associated with predictors of mortality in patients with fournier’s gangrene.(10) in contrast, another retrospective observational analytical study of patients diagnosed with fournier fasciitis (ff) showed that ff severity, as measured by nlr and plr, does not correspond to the severity measured by the fgsi.(12) other studies utilized the nlr cutoffvalues of 13.71 (sensitivity 83.3% and specificity 86.6%) (13) and 8 (sensitivity 72.2%, specificity of 52.3%), respectively.(14) the present study included fgsi in the analysis of nlr as mortality predictors. therefore, the aim of this study was to determine the role of nlr and fgsi as prognostic factors of mortality in patients with fg. methods research design thi s wa s an ana lytic al s tudy with a prospective approach utilizing secondary data taken from the medical records of dr. soetomo general-academic hospital from january 2012 to november 2020. research subjects fournier gangrene was defined as an acute necrotic infection involving the scrotum, penis or perineum. a total of 109 adult patients aged 18 and above with fournier gangrene or fasciitis necroticans and complete laboratory examination data including neutrophil and lymphocyte counts were included in the study. patients with a history of malignancy or chemoradiation were excluded, as were also those with incomplete or unclear data in the medical records. data collection the data collected and presented included p a t i e nt a ge , d i a gno s is , l e s i o n l oc a t i o n, comorbidities, fgsi, bacterial culture results, surgical interventions, and survival status. statistical analysis the collected data were grouped and displayed descriptively in the form of tables and 73 narratives. bivariate analysis was performed using chi-square test and independent t-test. the association between the binary marker of nlr and the risk of mortality was evaluated using a survival curve. mortality was defined as disease related death during the hospital stay and survival was measured in days. the separation between the curves of patients with a high nlr and those with a low nlr indicated the prognostic ability o f th e ma r ke r re pr es e nt ed by a r e c ei ve r operating characteristic (roc) curve.(15) the performance of the marker was evaluated by the area under the curve (auc), which is a measure of the ability of a tool to discriminate whether a condition is present or not. an auc value of 0.5 indicates that the test has no discriminating ability, whereas an auc of 1.0 indicates perfect discrimination.(16) overall survival was compared between groups using kapla n–meier survival estimates and the pr oportional-hazards cox regression. the statistical significance was set at p<0.05 for all analyses. ethical clearance the ethical committee of the research and development center of dr. soetomo general academic hospital approved this study under number 0725/109/4/v/2021. results baseline characteristics a total of 109 patients with mean age of 50.31 ± 14.75 years had mean nlr of 15.86 ± 12.75. only 25 patients had an fgsi score of more than 9. the scrotal area was the most commonly af fected a rea ( n= 55, 50. 46%) compared to other areas. most patients also suffered from diabetes mellitus (n=40, 36.7%) l e a d i ng t o i n f e c t i o ns c a us e d ma in l y b y pseudomonas aeruginosa ( n=23, 21.1%), klebsiella pneumoniae (n=21, 19.27%, and acinetobacter baumannii (n=20, 18.35%). most patients were treated with debridement and necrotomy, followed by incision and drainage of the abscess (n=55, 50.46%), after which most p a t i e nt s s u r vi ve d ( n = 9 0, 82.5 7 % ) . t he differences in clinical parameters between the survivors and non-survivors are shown in table 2. the fgsi scores in the two groups did not show significant differences (p=0.248), but the nlr did show significant differences between the two groups (p=0.021) (table 2). nlr and fgsi value as a prognostic marker in this study, the nlr cut-off point among the patients was 10.9, with 73.7 % sensitivity and 60 % specificity, as shown in figure 1. the auc was 0.65 (95% ci; 0.524-0.754; p<0.05). the kaplan-meier curve in figure 2 shows that the nlr cut-off value of 10.9 has a significant impact on the patient’s mortality rate (95% ci; 29.7-19.7; p<0.05). the univariate kaplan meier survival analysis indicated that nlr can be used as an independent predictor for mortality in fournier gangrene patients (hr 5.177; 95% ci; 1.092-8.471; p<0.05). however, the cox regression showed that nlr and fgsi score were not significant as a prognostic factor of mortality in fg patients (the p values of nlr a n d fg si we r e p= 0 . 09 a n d p = 0. 17 9 ; respectively) (table 3). discussion fournier gangrene is a rare and serious c o nd i t i on t h a t c a n be f o un d i n immunocompromised patients.(17) even though fgsi has been validated in numerous studies, it s us e in cl in ica l se tt ings is of te nt imes questionable. there is still a high mortality rate ranging from 20 to 50% among fournier gangrene patients due to sepsis,(17) which is one of the main causes of mortality and extended length of stay in patients with urological infections, including fournier gangrene.(18) to reduce the severity of the disease, the utilization of inexpensive and simple laboratory parameters, such as white blood cell parameters, erythrocyte sedimentation rate, and c-reactive protein, are necessary.(19,20) the use of nlr as a parameter univ med vol. 41 no 1 74 raizandha, hidayatullah, kloping, et al neutrophil-lymphocyte ratio of fg patients c variables n (%) age (years) 50.31 ± 14.79 nlr 15.86 ± 12.75 fgsi > 9 25 (23.0) < 9 84 (70.0) diagnosis fournier gangrene 53 (48.62) fournier gangrene and perianal abscess 33 (30.28) fournier gangrene and scrotal abscess 22 (20.18) fournier gangrene and perianal fistula 1 (0.92) affected region penoscrotal 12 (11.01) penoscrotal and perianal 2 (1.83) penoscrotal and suprapubic 2 (1.83) perianal 27 (24.77) perianal and scrotum 8 (7.34) scrotum 55 (50.46) > 2 regions 3 (2.75) comorbidities diabetes mellitus 40 (36.70) diabetes mellitus and hypertension 21 (19.27) diabetes mellitus and chronic kidney disease 7 (6.42) diabetes mellitus and hepatitis b infection 1 (0.92) hypertension 8 (7.34) chronic kidney disease 4 (3.67) no comorbidities 28 (25.67) bacterial culture pseudomonas aeruginosa 23 (21.10) klebsiella pneumoniae 21 (19.27) staphylococcus epidermidis 12 (11.01) acinetobacter baumannii 20 (18.35) gemella morbillorum 2 (1.83) escherichia coli 13 (11.93) candida spp 8 (7.34) clostridium perfringens 5 (4.59) fusobacterium necrophorum 5 (4.59) survival intervention debridement-necrotomy 48 (44.04) debridement-necrotomy and incision-drainage 55 (50.46) debridement-necrotomy and graft 3 (2.75) debridement-necrotomy and urinary diversion 3 (2.75) survival status survived 90 (82.57) dead 19 (17.43) table 1. characteristics and clinical features of the subjects (n=109) data presented as n (%), except for age and nlr (mean ± sd); nlr: neutrophil-to-lymphocyte ratio; fgsi: fournier gangrene severity index has been suggested by many studies. kaushik et al.(21) recommended its use as a diagnostic marker and predictor in septic patients. its greatest strengths are its efficiency in time, cost, and application compared to other examinations. neutrophils are one of the main immune cells against pathogens and their crucial function is to produce enzymes during the acute inflammatory phase. neutrophils are able to lyse infected cells, produce free radicals, and induce the production 75 univ med vol. 41 no 1 of pro-inflammatory cytokines. (2 2) t he coordination of the transition from innate to adaptive immunity is handled by the lymphocytes. both innate and adaptive immunity are core components of the body’s immune system against pathogens.(23) the ratio of the neutrophil and lymphocyte numbers indicates a transition between innate and adaptive immunity. the relatively low number of lymphocytes could cause a cytokine storm and severe inflammation, leading to a worse prognosis. our study showed the potential role of nlr as a prognostic marker for fournier gangrene patients, since patients with a high nlr had a 5.17-times greater risk of mortality than those with a low nlr. this finding is in line with the study by george et al. in 2020 who discovered a significant difference in nlr among septic patients with multiple organ dysfunction syndrome (mods). they found that most septic patients with mods had a high nlr. the nlr is deemed superior to a white blood cell count.(24) a study with a large sample size conducted by li et al.(25) also showed the predictive capability of nlr in septic patients. however, a study by ni et al.(26) suggested that nlr does not significantly predict septic inpatients with a long hospital stay. t he difference between these findings may have been caused by other factors in these studies which could affect the patients’ nlr. the increased nlr in septic patients is difficult to use as a predictive tool, considering that there are many factors affecting neutrophil and lymphocyte counts. however, it can still be used as a mortality predictor in septic patients. the roc curve, kaplan-meier, and hazard ratio findings in this study are in line with those of the study by yim et al.(10) which suggested that nlr is a useful independent predictor that is associated with increased mortality in fg patients. in our study, cox regression showed that nlr and fgsi score had no significant prognostic value for mortality in fg patients. another study showed that the fgsi scoring system was not found to be valuable in determining prognosis, but that the nlr and plr were valuable.(27) one other study showed similar results, in that the fgsi scoring system was not associated with determining poor prognosis, but that high nlr and high plr were associated with predictors of mortality in patients with fg. (10) in our study, a high fgsi score (>9) was generally associated with the non-surviving group; however, multivariable cox regression analyses found this not to be statistically significant. variables survivors (n=90) non-survivors (n=19) p value age (years) 49 ± 14.9 54 ± 13.8 0.208 fgsi ≤ 9 >9 75 (89.3) 17 (68.0) 9 (10.7) 8 (32.0) 0.248 nlr ≤10.9 >10.9 52 (91.2) 38 (73.1) 5 (8.8) 14 (16.9) 0.021 table 2. differences in clinical parameters between the survivors and non-survivors data presented as n (%), except for age mean ± sd; nlr: neutrophil-to-lymphocyte ratio; fgsi: fournier gangrene severity index variables coefficient hazard ratio p value age (years) fgsi score nlr 0.017 0.081 -0.033 1.017 1.328 2.043 0.356 0.179 0.09 table 3. multivariate cox regression analysis nlr: neutrophil-to-lymphocyte ratio; fgsi: fournier gangrene severity index 76 raizandha, hidayatullah, kloping, et al neutrophil-lymphocyte ratio of fg patients figure 1. receiver operator characteristic curve for neutrophil to lymphocyte ratio (nlr), area under the curve = 0.65 (95% ci; 0.524-0.754; p<0.05, sensitivity = 73.7, specificity = 60.0 figure 2. kaplan-meier survival curves for the overall survival indicating the value of nlr as a survival predictor 77 univ med vol. 41 no 1 t he p r e s e n t st u dy is l i mi t e d b y i t s retrospective design and use of secondary data. most samples included in this study had a high nlr ratio, indicating that most included patients were classified as severe. the inclusion of more patients with different disease severity should be performed in future studies. this study showed the utility of the nlr in fg. the nlr can be examined with high availability and low cost. this marker could be an ideal and simple biomarker to predict the outcome of mortality in patients with fg. conclusions this study demonstrated that a high nlr and fgsi cannot be used as an indicator of poor prognosis of mortality in fg patients. conflict of interest the authors declare no conflicts of interest. acknowledgments the authors thank the medical record staff of dr. soetomo general-academic hospital for their kind cooperation. contributors mar, fh, ypk contributed to concept and design; mar, fh contributed to data collection and analysis. mar and fr contributed to writing manuscript and critical review. all authors have read and approved the final manuscript. references 1. voelzke bb, hagedorn jc. presentation and diagnosis of fournier gangrene. urology 2018;114:8–13. doi: 10.1016/j.urology.2017.10.031. 2. auerbach j, bornstein k, ramzy m, cabrera j, montrief t, long b. fournier gangrene in the emergency department: diagnostic dilemmas, treatments and current perspectives. open access emerg med 2020;12:353. doi: 10.2147/ oaem.s238699. 3. montrief t, long b, koyfman a, auerbach j. fournier gangrene: a review for emergency clinicians. j emerg med 2019;57:488–500. doi: 10.1016/j.jemermed.2019.06.023. 4. chernyadyev sa, ufimtseva ma, vishnevskaya if, et al. fournier’s gangrene: literature review and clinical cases. urol int 2018;101:91–7. https:// doi.org/10.1159/000490108. 5. pehlivanlý f, aydin o. factors affecting mortality in fournier gangrene: a single center experience. surg infect (larchmt) 2019;20:78–82. doi: 10.1089/ sur.2018.208. 6. hahn hm, jeong ks, park dh, park mc, lee ij. analysis of prognostic factors affecting poor outcomes in 41 cases of fournier gangrene. ann surg treat res 2018;95:324-32. doi: 10.4174/ astr.2018.95.6.324. 7. morais h, neves j, ribeiro hm, et al. case series of fournier’s gangrene: affected body surface area–the underestimated prognostic factor. ann med surg (lond) 2017;16:19–22. doi: 10.1016/ j.amsu.2017.02.043. 8. hong ks, yi hj, lee r, kim kh, chung ss. prognostic factors and treatment outcomes for patients with fournier’s gangrene: a retrospective study. int wound j 2017;14:1352–8. doi: 10.1111/ iwj.12812. 9. arora a, rege s, surpam s, gothwal k, narwade a. predicting mortality in fournier gangrene and validating the fournier gangrene severity index: our experience with 50 patients in a tertiary care center in india urol int 2019;102:311–8. doi: 10.1159/000495144. 10. yim su, kim sw, ahn jh, et al. neutrophil to lymphocyte and platelet to lymphocyte ratios are more effective than the fournier’s gangrene severity index for predicting poor prognosis in fournier’s gangrene. surg infect (larchmt) 2016;17:217–23. doi: 10.1089/sur.2015.126. 11. lee js, kim ny, na sh, youn yh, shin cs. reference values of neutrophil-lymphocyte ratio, lymphocyte-monocyte ratio, platelet-lymphocyte ratio, and mean platelet volume in healthy adults in south korea. medicine (baltimore) 2018;97: e11138. doi: 10.1097/md.0000000000011138. 12. guemes-quinto a, godínez-vidal ar, villanuevaherrero ja, et al. usefulness of neutrophil-tolymphocyte ratio and platelet-to-lymphocyte ratio as predictors of severity on fournier fasciitis of the hospital general de méxico "dr. eduardo liceaga." rev med hosp gen mex 2019;82:17 5-8. https://doi.org/10.24875/ hgmx.m19000027. 13. demir cy, yuzkat n, ozsular y, kocak of, soyalp c, demirkiran h. fournier gangrene: association of mortality with the complete blood count 78 raizandha, hidayatullah, kloping, et al neutrophil-lymphocyte ratio of fg patients parameters. plast reconstr surg 2018;142:68e75e. doi: 10.1097/prs.0000000000004516. 14. özlülerden y, ba?er a, çelen s, alki? o. can we predict poor prognosis in fournier gangrene? j surg med 2020;4:1157-60. https://doi.org/ 10.28982/josam.826917. 15. combescure c, perneger t v, weber dc, daurès j-p, foucher y. prognostic roc curves: a method for representing the overall discriminative capacity of binary markers with right-censored time-to-event endpoints. epidemiology 2014;1039. doi: 10.1097/ede.0000000000000004. 16. doluo?lu ög, karagöz ma, k?l?nç mf, et al. overview of different scoring systems in four nie r's ga ngr ene and a sse ssment o f prognostic factors. turk urol derg 2016;42:1906. doi: 10.5152/tud.2016.14194. 17. benjelloun eb, souiki t, yakla n, et al. fournier's gangrene: our experience with 50 patients and analysis of factors affecting mortality. world j emerg surg 2013;8:13. doi: 10.1186/1749-7922-813. 18. abou dagher g, hajjar k, khoury c, et al. outcomes of patients with systolic heart failure pr esenting with se psis to the eme rgency department of a tertiary hospital: a retrospective chart review study from lebanon. bmj open 2018;8:e022185. doi: 10.1136/bmjopen-2018022185. 19. dal-pizzol f, ritter c. à procura do santo graal: aonde vamos com os biomarcadores na sepse? rev bras ter intensiva 2012;24:117-8. ohttps:// doi.org/10.1590/s0103-507x2012000200004. 20. lora-andosilla m, cantillo-garcía k, borrénaranjo d, buelvas-villalba m, ortiz-ruiz g, dueñas-castell c. biomarkers in sepsis. in: ortizruiz g, dueñas-castell c, editors. sepsis. 3rd ed. new york: springer-verlag;2018. p. 39-50. 21. kaushik r, gupta m, sharma m, et al. diagnostic and prognostic role of neutrophil-to-lymphocyte ratio in early and late phase of sepsis. indian j crit care med 2018;22:660-3. doi: 10.4103/ ijccm.ijccm_59_18. 22. wang j, arase h. regulation of immune responses by neutrophils. ann n y acad sci 2014;1319:66-81. doi: 10.1111/nyas.12445. 23. moro-garcía ma, mayo jc, sainz rm, alonsoarias r. influence of inflammation in the process of t lymphocyte differentiation: proliferative, metabolic, and oxidative changes. front immunol 2 01 8 ;9 :33 9 . http s://do i.o r g/1 0 .3 3 89/ fimmu.2018.00339. 24. george aa, thomas tp, gaffoor a. the role of neutrophil/lymphocyte ratio in predicting the severity of sepsis in a tertiary care hospital in south india: a retrospective study. int j res med sci 2020;8:1624-8. doi: http://dx.doi.org/ 10.18203/2320-6012.ijrms20201490. 25. li mx, liu xm, zhang xf, et al. prognostic role of neutrophil-to-lymphocyte ratio in colorectal cancer: a systematic review and meta-analysis. int j cancer 2014;134:2403-13. doi: 10.1002/ ijc.28536. 26. ni j, wang h, li y, shu y, liu y. neutrophil to lymphocyte ratio (nlr) as a prognostic marker for in-hospital mortality of patients with sepsis: a secondary analysis based on a single-center, r etrosp ec tive , c oho rt study. me dic ine (baltimore) 2019;98:e18029. doi: 10.1097/ md.0000000000018029. 27. kahramanca s, kaya o, özgehan g, et al. are neutrophil-lymphocyte ratio and plateletlymphocyte ratio as effective as fournier's gangrene severity index for predicting the number of debridements in fourner's gangrene? ulus travma acil cerrahi derg 2014 ;20:107-12. doi: 10.5505/tjtes.2014.62829. c:\users\universa medicina\docu 88 *department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ rs dr. moewardi hospital, surakarta **department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ rs universitas sebelas maret, surakarta correspondence: dr. nutria widya purna anggraini, sp.og(k), m.kes department of obstetrics and gynecology rsud dr. moewardi jl. kol. sutarto 132 surakarta phone: +62271 665145 e-mail: nutria_dr@staff.uns.ac.id orcid id: 0000-0001-7652-1038 date of first submission, may 31, 2020 date of final revised submission, july 4, 2 02 0 date of acceptance, july 7, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina low neutrophil-to-lmphocyte ratio decreases risk of coronavirus disease in pregnant women nutria widya purna anggraini* and sri sulistyowati** background coronavirus infection 2019 (covid-19), caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), is an emergency condition for global public health. early detection of covid-19 in pregnant women is needed. the neutrophil-to-lymphocyte ratio (nlr), as a marker of viral inflammatory response, is used to determine the presence of a viral or bacterial infection, both acute and chronic. the objective of this study was to determine the relationship between nlr and the polymerase chain reaction (pcr) swab test results in pregnant women with suspected coronavirus disease 2019. methods a cross-sectional study was conducted on 9 pregnant women with suspected covid-19. the subjects were inpatients at moewardi hospital surakarta from 19 april-19 may 2020, who had rapid tests, complete blood examinations, and pcr swab tests. the nlr was categorized based on early warning scores according to research developed at zhejiang university, with cut-off point 5.8. diagnosis of covid-19 was confirmed by pcr swab tests. relation between nlr and pcr swab results was analyzed by the prevalence ratio. results two patients (22.2%) had nlr >5.8, with positive swab results in both (100%). seven patients with nlr <5.8 had positive swabs in only one (33.3%). the relationship between nlr and pcr swab test results showed a prevalence ratio of 0.143 (95% ci 0.023-0.877). conclusion pregnant women with nlr < 5.8 had a decreased risk of covid-19. routine blood examination is more suitable for finding pregnant women with suspected covid-19. keywords: neutrophil-to-lymphocyte ratio, pcr swab, covid-19, pregnancy original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.88-96 cite this article as: anggraini nwp, suli styowa t i s. low n eutrophi l-tolmp hocyte r atio dec reases risk of coronavirus disease in pregnant women. uni v med 2020;3 9:8 8 -9 6 . doi: 10.18051/univmed.2020.v39.88-96 may-august 2020 vol.39no.2 89 may-august, 2020 vol.39no.2 introduction coronavirus disease 2019 (covid-19) is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and emerged initially in wuhan, china, in december 2019. currently the covid-19 infection is a public health emergency, particularly in susceptible populations.(1) pregnant women and their fetuses constitute a susceptible and high risk population, due to the physiological and mechanical factors in pregnancy that increase the susceptibility to inf ections in gener al, particular ly in the cardiorespiratory system, and trigger the occurrence of respiratory failure.(2) during the covid-19 epidemic pregnant women apparently have fewer maternal and neonatal complications than has been reported for sars and mers.(3) there were more than 193.000 deaths (up to 24 april 2020), but no reports of maternal deaths were published. in china a total of 154 pregnant women have been reported with maternal morbidity of 1% and no maternal deaths. in the us, there was 4-10% maternal morbidity and no maternal deaths.(4) pregnant women deve lop spe cial immunological adaptations that are necessary to maintain semi-allograft tolerance to their fetuses.(5) this repressed immunity is modulated by repressed t cell activity, where in pregnancy there is dominance of the t-helper 2 (th2) system that protects the fetus, thereby rendering the mother susceptible to viral infections.(5) coronavirus disease 2019 infection is associated with cytokine storms that are marked by increased concen trations of plasma interleukin-2 (il-2), il-7, il-10, granulocyte colony stimulating factor, interferon--induced protein-10, monocyte chemoattractant protein-1, macrophage inflammatory protein-1 alpha, and tumor necrosis factor alpha (tnf-).(6) pregnant women in the first and third trimesters are in a proinflammatory condition, such that the cytokine storms induced by sars-cov-2 may cause more severe inflammation in these women.(7) based on all of the above, an integrated approach is needed, especially for early detection, prediction and evaluation of covid-19 in pregnant women, both with respect to prevention, breaking of the transmission chain, and establishing the diagnosis as well as management, which are essential for improving the prognosis in pregnant women.(8) the ear ly detection of covid-19 in pregnant women that is recommended by the indonesian obstetr ics and gynecology association (persat uan obstet ri and ginekologi indonesia, pogi), uses the early warning score (ews) developed by the zhejiang university. this co mprises a number of parameters that are rather frequently found in patients with covid-19, namely signs of pneumonia on ct scan, history of contact with covid-19 positive patients, fever, temperature >37.8oc from onset of symptoms, male gender, age >40 years, a number of symptoms of respira tory distre ss and a neutrophil-tolymphocyte ratio (nlr) of 5.8.(9) currently the early warning score is much in use and is a relatively accurate tool for early detection.(10) the neutrophil-to-lymphocyte ratio is a component of the ews, as marker of inflammatory reaction, and is an indicator of the complications associated with pregnancy.(8,11) the inflammatory reaction in pregnant women plays an important role in a number of complications of pregnancy and is a strong contributor to mortality and morbidity in pregnant women. therefore, inflammatory markers may be used both as predictors and prognostic factors. the neutrophil-to-lymphocyte ratio has been used as predictor of the complications of pregnancy, such as preeclampsia, gestational diabetes mellitus, hyperemesis gravidarum, premature labor and ectopic pregnancy.(8) a high neutrophil-tolymphocyte ratio results from an increased number of neutrophils and a decreased number of lymphocytes. the inflammatory response stimulates the production of neutrophils and accelerates apoptosis of lymphocytes. poor immune responses and immunological abnormalities are believed to influence disease severity triggered b y viral infections. (1 2) 90 anggraini, sulistyowati coronavirus disease in pregnant women repression of the immune response it results in an excessive inflammatory reaction and even death. similar to the midlle east respiratory syndrome coronavirus (mers-cov ), the leukocyte diffe renti al count is the most outstanding predictor associated with disease severity.(13) the differential count is characterized by increased neutrophils and monocytes and by decreased lymphocytes, which is found in all deceased patients. studies on covid-19 have shown that cytokine and che mokine concentrations and nlr values are higher in patients with covid-19 and are associated with disease severity.(14,15) in addition, patients with severe viral infection may have bacterial coinfection due to low immunity, resulting in incr ease d nlr, c-re a ctive pr otein and procalcitonin.(16) currently the nlr constitutes a new strong inflammatory marker for the detection of inflammation.(17) the nlr is more superior to the total leukocyte count, the neutrophil count, or the lymphocyte count as an inflammatory marker, because it is less influenced by a number of physiological conditions, such as dehydration or physical activity. moreover, the nlr examination has advantages as compared with other inflammatory markers, such as crp or interleukin, as it is easy to perform and is inexpensive.(14) the neutrophil-to-lymphocyte ratio can be rapidly calculated from routine complete blood examination, so that the physician may identify patients at high risk for covid-19 at an early stage. in covid-19 infection, the numbers of leukocytes, lymphocytes, monocytes and platelets are lower than in the non-covid19 group. the neutrophil-to-lymphocyte ratio is significantly higher in the covid-19 group. the increased nlr in patients with covid-19 is associated with disease severity.(15) the diagnosis of covid-19 depends mainly on the detection of sars-cov-2 nucleic acids through the polymerase chain reaction (pcr) swab test. however, ther e are still many limitations with the occurrence of false negative results caused by a low viral load in the sample and the relative shortage of detection kits, so that many cases remain undetected.(9) there is a need for a method capable of rapid and early detection of covid -19 in pregnant women, that may be used as predictor for preventing maternal morbidity and mortality. a chinese study involving 55 pregnant women (13 pregnant women with covid-19 infection and 42 controls) showed no significant differences in nlr between the two groups, both during hospitalization and post-partum.(18) however, these results differ from those of a spanish study involving 60 pregnant women with covid-19 infection and showing increased nlr in pregnant women with covid-19 infection.(19) these still inconsistent study results point to the need for further studies. in the covid-19 pandemic the prevention and control of covid19 infection in pregnant women is of utmost importance, particularly in the screening of pregnant women who are hospitalized for purposes of delivery. the present study aimed to determine any relationship between nlr and covid-19 pcr swab test results in pregnant women suspected of suffering from covid-19. methods design of the study this study was an observational analytic study of cross-sectional approach that involved as subjects pregnant women hospitalized in dr moewardi hospital, surakarta, which is the central referral hospital for the management of pregnant women with covid-1 9. this study was conducted on nine pregnant women with covid-19 hospitalized in rs dr. moewardi hospital, surakarta, from 19 april 2020 until 19 may 2020. study subjects the study subjects were pregnant women hospitalized with the status of patients under supervision (pasien dalam pengawasan, pdp) for covid-19. the inclusion criteria were pregnant women with signs and symptoms indicative of covid-19, in accordance with the 91 may-august, 2020 vol.39no.2 a b c early warning score (ews) of cough, rhinitis, fever, dyspnea, a picture of pneumonia, having previous contacts, and reactive rapid test results. the exclusion criteria were patients with a history of previous infections and non-reactive rapid test results. diagnostic procedures the diagnosis of covid-19 was based on the guideline for prevention and control of corona virus disease ( covid-19) by the directorate general of disease prevention and control, republic of indonesia.(10) all subjects underwent complete blood examinations, chest x-rays, and pcr swab tests. data measurements the nlr is a systemic inflammatory marker in infectious disease obtained by dividing the absolute neutrophil count by the absolute lymphocyte count. the nlr is categorized into two groups with cut-off point of 5.8, namely group i at 5.8 and group ii at 5.8, in accordance with the early warning score (ews) based on the study developed at the zhejiang university.(9) the cov id-19 pcr swab test is a laboratory test for the detection of the sars cov2 virus using the rapid molecular test based on the pcr. samples were swabbed from the oral and nasal cavities and were used to confirm cases of covid-19 based on the detection of unique viral rna sequences. the real-time reversetranscription polymerase chain reaction (rrtpcr) swab results were confirmed if necessary by nucleic acid sequencing. this examination was performed in the clinical pathology laboratory, dr. moewardi hospital, surakarta. the results were categorized into two groups, namely group 1 with the criterion of positive results and group 2 with the criterion of negative results. statistical analysis the collected data were analyzed using the spss program. subsequently an association was looked for between nlr values and pcr swab results, using the chi square test. the association between nlr and covid-19 in pregnant women was deter min ed by means of the prevalence ratio. ethical clearance ethical clearance was obtained from the health research eth ics committee, dr. moewa rdi hospital/fac ulty of medicine, universitas sebelas maret, surakarta, central java, indonesia under no. 755/vi/hrec/2020. results the results of this study may be seen in table 1 on characteristics of pregnant women with covid-19 infection. table 1 shows that the nine pregnant women were all in the third trimester, with eight patients undergoing cesarean section, and one patient undergoing conservative maintenance of the pregnancy at gestational age of 31 weeks. maintenance of the pregnancy was done because it was still preterm and there were no signs of labor. the age range of the patients was 23-37 years and the gestational age range on admission was 31-42 weeks. two of the eight patients experienced fever but no chills, and none experienced high fever (body temperature >39°c). the body temperature of the patients fluctuated within the range of 36.5–38.8°c. there were no patients with postpartum fever. other symptoms of upper respiratory tract infection in two patients were cough accompanied with dyspnea. one patient was under mechanical ventilation during care. another patient had pulmonar y edema as complication, but not one of the nine patients experienced severe pneumonia or died of covid-19 pneumonia up to may 2020. there was a significant association between nlr and covid-19 infection (prevalence ratio = 0.143; 95% ci = 0.023 – 0.877) (table 2). this signifies that pregnant women with nlr <5.8 have a 0.143 times lower risk for having covid19 infection than do pregnant women with nlr 5.8. 92 variable n (%) mean age (years) 28.44 ± 4.95 20-34 7 (77.8) ≥ 35 2 (22.2) gestational age (weeks) <37 3 (33.3) 37-40 4 (44.4) > 40 2 (22.2) parity primigravida 4 (44.4) multigravida 5 (56.6) contact history yes 1 (11.1) no 8 (88.9) severe preeclampsia yes 1 (11.1) no 8 (88.9) hypertension yes 0 (0.0) no 9 (100.0) diabetes mellitus yes 0 (0.0) no 9 (100.0) fever yes 2 (22.2) no 7 (77.8) cough yes 2 (22.2) no 7 (77.8) dyspnea yes 2 (22.2) no 7 (77.8) myalgia yes 0 (0.0) no 9 (100.0) sore throat yes 0 (0.0) no 9 (100.0) diarrhea yes 0 (0.0) no 9 (100.0) leukocytes (thousands/µl) 10.411 ± 2.02 4.500-11.000 5 (55.6) > 11.000 4 (44.4) lymphocytes (%) 16.43 ± 4.62 < 22 8 (88.9) 22-44 1 (11.1) > 44 0 (0.0) neutrophils (%) 77.08 ± 4.63 55-79 6 (66.7) > 80 3 (33.3) nlr 5.016 ± 1.676 <5.8 7 (77.8) ≥5.8 2 (22.2) table 1. demographic characteristics and clinical manifestations in pregnant women (n= 9) anggraini, sulistyowati coronavirus disease in pregnant women 93 may-august, 2020 vol.39no.2 variable n (%) mean hemoglobin (g/dl) 11.98 ± 1.18 <10 0 (0.0) ≥10 9 (100.0) sgot / sgpt normal 8 (88.9) abnormal 1 (11.1) platelets (thousands/µl) 265.88 ± 50.23 < 150 0 (0.0) > 150 9 (100.0) chest x-ray pneumonia 2 (22.2) no pneumonia 7 (77.8) rapid test positive 8 (88.9) negative 1 (11.1) maternal covid-19 pcr swab positive 3 (33.3) negative 6 (66.7) infant delivery yes 8 (88.9) no 1 (11.1) ventilator yes 1 (11.1) no 8 (88.9) pulmonary edema yes 1 (11.1) no 8 (88.9) renal failure yes 0 (0.0) no 9 (100.0) heart yes 0 (0.0) no 9 (100.0) maternal death death 0 (0.0) no death 9 (100.0) infant birth weight (g) 2825 ± 332.74 < 2500 0 (0.0) ≥ 2500 8 (88.9) newborn apgar score <7 0 (0.0) ≥7 8 (88.9) newborn with severe asphyxia yes 0 (0.0) no 8 (88.9) infant death death 0 (0.0) no death 8 (88.9) newborn covid-19 pcr swab positive 0 (0.0) negative 8 (88.9) nlr: neutrophil-to-lymphocyte ratio table 1 (continued). 94 confirmed group (n=1) control group (n=8) pr (95 % ci) p value nlr < 5.8 ≥ 5.8 1 (14.3) 2 (100.0) 6 (85.7) 0 (0.0) 0.143 (0.0230.877) 0.023 table 2. association between nlr and covid-19 infection in pregnant women notes: nlr: neutrophil-to-lymphocyte ratio; pr: prevalemce ratio; ci: confidence interval; *significant at p < 0.05 discussion our study results showed that the clinical picture of patients with covid-19 infection during pregnancy was similar to that of adult non-pregnant women with covid-19 infection, comprising fever, cough and dyspnea, whereas the symptoms of myalgia, malaise, sore throat, and diarrhea, were seldom f ound, as had previously been reported by chen et al.(16) eight pregnant women in the present study underwent cesarean section, with obstetric indications of severe pr eeclampsia, history of pr evious cesarean sections, and fetal distress, while one case arrived at a gestational age of 31 weeks and was discharged without having undergone delivery and being in good condition without complications. termination of pregnancy by cesarean section is in accordance with the protocol of the indonesian association of obstetrics and gynecology (pogi) thereby avoiding the risk of infection from the mother to the fetus through vaginal delivery.(17) in the present study, fetal outcomes were good for all conditions, and did not require special nursing care. this proves that the postpartum condition of the infant was not affected by the mothers having been infected with covid-19. this agrees with the study of zaigham et al.(20) who showed that the majority of pregnant women were infected with covid-19 in the third trimester and was delivered by cesarean section, with no maternal deaths. in contrast, pregnant women infected with other earlier respiratory viruses, such as h1n1, were reported to be approximately 4–5 times more likely to develop severe disease compared with non-pregnant individuals in the general population.(21) in our study an association was found between nlr values and swab test results in pregnant women with covid-19 infection. however, differing results were obtained in the study of yang et al.(18) which showed that in the prenatal and postpartum periods there were no differences in nlr values. the latter study also states that routine blood tests were more suitable for screening of p regnant women with asymptomatic or mild covid-19 infection, and may be used for screening of covid-19 in pregnant women in areas of epidemic covid19 infection. the increased nlr in our study is in line with the findings in spain by pereira et al.(19) who showed an increase in nlr in pregnant women with covid-19 infection and a decrease in nlr upon improvement of the clinical condition. the study of qin et al.(14) also showed that patients with covid-19 experienced an increase in the neutrophil count and a decrease in the lymphocyte count in the severe phase, suggesting the possibility of a critical condition and serious internal abnormalities in these patients. the study by luo et al.(23) showed that nlr may be used as a sufficiently good inflammatory marker. from the pathophysiological point of view, neutrophils represent the nonspecific immune system that initiate the body responses to inflammation, whereas the lymphocytes represent the protective component against inflammation. the inflammatory condition will trigger hypersecretion of inflammatory cytokines, such as il-6 and tnf-, result ing in a permanently high neutrophil count. in contrast, catecholamines, cortisol, and the increased proinflammatory mediators will bind to lymphocyte surface receptors and subsequently initiate anggraini, sulistyowati coronavirus disease in pregnant women 95 may-august, 2020 vol.39no.2 apoptosis of the lymphocytes, thus causing lymphopenia.(24) similarly, the study of yang et al.(18) on covid-19 also showed that nlr is significantly higher in the covid-19 group, and that the increase in nlr may be considered as an independent marker of poor clinical outcome. there are several limitations in our study. firstly, since this study was limited by its small sample size, therefore a number of considerations have to be taken when interpreting the findings. secondly, for a number of patients in our study, the initial clinical diagnosis of patients under supervision (pdp) was confirmed with the rapid igm specific antibody test. although the reported sensitivity and specificity were relatively good, still the potential for the occurrence of selection bias has to be taken into consideration. this is due to the fact of a shortage of diagnostic equipment, so that not all patients could be directly subjected to the pcr swab test, to the fact that it took a long time for the swab test results to become available, and the fact that the limited supply of test reagents necessitated the inclusion of only those with clinical symptoms or a clinical diagnosis for serologic as well as swab testing, thus producing selection bias. thirdly, samples such as placental tissues, amniotic fluid and cord blood were not collected for the covid-19 test. the results of the present study may be used as input for the early detection of cases of covid-19 in pregnant women, by means of nlr, at a cut-off point of 5.8. there is still a need for further studies on pregnant women with covid-19 and their fetuses. suggestions for further studies consist of increasing the sample size and the study variables to be used in early detection or as predictors. conclusions pregnant women with nlr <5.8 have a 0.143 times lower risk for suffering from covid-19 infection in comparison with pregnant women with nlr 5.8.the nlr may be used as marker for early detection of cases of pregnant women with covid-19 infection. conflicts of interest the authors declare that there were no conflicts of interests in connection with the study, and the writing and publication of this article. acknowledgments the authors thank the director of dr. moewardi hospital surakarta, for the opportunity to perform this study and collect the data. contributors nwpa contributed to drafting of the manuscr ipt, f ormula ting the conceptual framework, and collecting and processing the research data. ss played a role in revising the article. all authors have read and approved the final manuscript. references 1. dashraath p, wong jlj, lim mxk, et al. coronavirus disease 2019 (covid-19) pandemic and pregnancy. am j obstet gynecol 2020;222: 521-31. http://doi.org/10.1016/j.ajog. 2020.03.21. 2. lapinsky se. acute respiratory failure in pregnancy. obstet med 2015;8:126-32. doi: 10.1177/1753495x15589223. 3. qiao j. what are the risks of covid-19 infection in pregnant women? lancet 2020;395:760–2. https://doi.org/10.1016/s0140-6736(20)30365-2. 4. hantoushzadeh s, shamshirsaz aa, aleyasin a, et al. maternal death due to covid-19. am j obstet gynecol 2020;20:30516-20. doi: 10.1016/ j.ajog.2020.04.030. 5. mor g, cardenas i, abrahams v, guller s. inflammation and pregnancy: the role of the immune system at the implantation site. ann n y acad sci 2011;1221:80–7. doi: 10.1111/j.17496632.2010.05938.x. 6. costela-ruiz vj, illescas-montes r, puerta-puerta jm, ruiz c, melguizo-rodríguez l. sars-cov-2 infection: the role of cytokines in covid-19 disease. cytokine growth factor rev 2020;20: 96 30109-x. http://doi.org/10.1016/j.cytogfr.2020.06. 001. 7. liu h, wang ll, zhao sj, kwak-kim j, mor g, liao ah. why are pregnant women susceptible to covid-19? an immunological viewpoint. j reprod immunol 2020;139:103122. doi: 10.1016/ j.jri.2020.103122. 8. hai l, hu zd. the clinical utility of neutrophil to lymphocyte ratio in pregnancy related complications: a mini-review. j lab precis med 2020;5:1. doi: 10.21037/jlpm.2019.10.03. 9. song cy, xu j, he jq, lu yq. covid 19 early warning score: a multiparameter screening tool to identify highly suspected patients. medrxiv 2020;1–22. https://doi.org/10.1101/2020.03.05. 20031906. 10. arif sk, muchtar f. buku pedoman penanganan pasien kritis covid 19. makasar: perhimpunan dokter anestesi dan terapi intensif (perdatin); 2020. 11. liu y, du x, chen j, et al. neutrophil-tolymphocyte ratio as an independent risk factor for mortality in hospitalized patients with covid19. j infect 2020;81:e6-e12. https://doi.org/ 10.1016/jinf.2020.04.002. 12. channappanavar r, perlman s. pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology. semin immunopathol 2017;39: 529–39. https://doi.org/10.1007/s00281-017-0629x 13. min ck, cheon s, ha ny, et al. comparative and kinetic analysis of viral shedding and immunological responses in mers patients representing a broad spectrum of disease severity. sci rep 2016;6:25359. https://doi.org/10.1038/ srep25359. 14. qin c, zhou l, hu z, et al. dysregulation of immune response in patients with coronavirus 2019 (covid-19) in wuhan, china. clin infect dis 2020;ciaa248. https://doi.org/10.1093/cid/ciaa248 15. lou m, luo p, tang r, et al. relationship between neutrophil-lymphocyte ratio and insulin resistance in newly diagnosed type 2 diabetes mellitus patients. bmc endocr disord 2015;15:9. 16. chen h, guo j, wang c, et al. clinical characteristics and intrauterine vertical transmission. lancet 2020;395: 809–15. https:// doi.org/10.1016/s0140-6736(20)30360-3. 17. pokja infeksi saluran reproduksi perkumpulan obstetri dan ginekologi indonesia tahun 2020 . rekomendasi penanganan infeksi virus corona (covid-19) pada maternal (hamil, bersalin dan nifas). surabaya: pogi; 2020. 18. yang h, sun g, tang f, et al. clinical features and outcomes of pregnant women suspected of coronavirus disease 2019. j infection 2020;81:e40– e4. https://doi.org/10.1016/j.jinf.2020.04.003. 19. pereira a, cruz-melguizo s, adrien m, fuentes l, marin e, perez-medina t. clinical course of coronavirus disease-2019 in pregnancy. acta obstet gynecol scand 2020;99:839–47. https:// doi.org/10.1111/aogs.1392119. 20. zaigham m, andersson o. maternal and perinatal outcomes with covid-19: a systematic review of 108 pregnancies. acta obstet gynecol scand 2020;99:823-9. https://doi.org/10.1111/aogs.13867. 21. cérbulo-vázquez a, figueroa-damián r, arriagapizano la, et al. pregnant women infected with pandemic h1n1pdm2009 influenza virus displayed overproduction of peripheral blood cd69+ lymphocytes and increased levels of serum cytokines. plos one 2014;9:e107900. doi: 10.1371/journal.pone.0107900. 22. wang d, hu b, hu c, et al. clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china. jama 2020;323: 1061-69. http://doi.org/ 10.1001/jama.2020.1585. 23. luo y, xie y, zhang w, et al. combination of lymphocyte number and function in evaluating host immunity. aging (albany ny) 2019;11: 12685–707. doi: 10.18632/aging.102595. 24. almeida mq, mendonca bb. adrenal insufficiency and glucocorticoid use during the covid-19 pandemic. clinics. 2020;75:e2022. doi: 10.6061/clinics/2020/e2022. oktavianus 105 *department of pulmonology and respiratory medicine, faculty of medicine, university of indonesia, persahabatan hospital, jakarta **department of clinical pathology, persahabatan hospital/ fatmawati hospital, jakarta ***department of internal medicine, faculty of medicine, trisakti university correspondence dr. rita khairani, msc, sp.p department of internal medicine, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: +6221-5672731 ext.2707 email: ranidarmawan@gmail.com univ med 2012;31:105-12 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background pleural effusions are classified into transudates and exudates based on light’s criteria, but the main disadvantage of light’s criteria is the misclassification of transudates as exudates in about 20% of cases. the aim of this study was to determine the validity of various biochemical parameters to differentiate pleural exudates and transudates. methods an observational study to evaluate diagnostics tests was conducted at the emergency department of persahabatan hospital, jakarta, from september 2010 until december 2011. in total, 119 patients with pleural effusion were evaluated. simultaneous pleural effusion and blood samples were examined for lactate dehydrogenase (ldh), total protein, cholesterol and albumin, with the clinical diagnosis as the gold standard. results there were 104 exudative and 15 transudative pleural effusions. light’s criteria achieved a higher overall accuracy (sensitivity 97%, specificity 80%, accuracy 95%). the optimum cut off values were pleural fluid to serum ratio of ldh 0.4 (sensitivity 95%, specificity 87%, accuracy 94%) and pleural fluid ldh of 178 iu/l (sensitivity 92%, specificity 87%, accuracy 92%). pleural fluid cholesterol was 50 mg/dl (sensitivity 89%, specificity 53%, accuracy 85%), pleural fluid to serum cholesterol ratio 0.41 (sensitivity 75%, specificity 53%, accuracy 72%) and serum-effusion albumin gradient 1.3 g/dl (sensitivity 91%, specificity 73%, accuracy 89%). combination of biochemical tests did not improve sensitivity or accuracy. conclusions light’s criteria remain superior to other biochemical tests, but the new cut off values of ldh pleural fluid to serum ratio of 0.4 and pleural fluid ldh of 178 iu/l appears to yield a slight improvement in diagnostic accuracy. keywords: lactate dehydrogenase, light’s criteria, albumin, pleural effusion validity of pleural lactate dehydrogenase measurements in assessment of pleural effusions elisna syahruddin*, lia g partakusuma**, and rita khairani*** abstract 106 syahruddin, partakusuma, khairani assessment of pleural effusions validitas laktat dehidrogenase pleura sebagai uji tambahan untuk menilai efusi pleura latar belakang efusi pleura diklasifikasikan menjadi eksudat dan transudat berdasarkan kriteria light. kekurangan kriteria light adalah kesalahan mengklasifikasi transudat sebagai eksudat sekitar 20%. tujuan penelitian ini adalah menentukan parameter laboratorium yang valid untuk membedakan eksudat dan transudat pada efusi pleura. metode sebuah studi observasional untuk evaluasi uji diagnostik dilakukan di instalasi gawat darurat rs persahabatan jakarta antara bulan september 2010 – desember 2011. uji diagnostik dilakukan pada 119 pasien efusi pleura. cairan pleura dan serum digunakan untuk memeriksa laktat dehidrogenase (ldh), protein total, kolesterol dan albumin. diagnosis klinik penyebab efusi pleura digunakan sebagai baku emas. hasil terdapat 104 pasien efusi eksudatif dan 15 pasien transudatif. kriteria light mempunyai sensitivitas 97%, spesifisitas 80%, akurasi 95%. titik potong optimum didapatkan pada rasio ldh cairan pleura/serum 0,4 (sensitivitas 95%, spesifisitas 87%, akurasi 94%) dan ldh cairan pleura 178 iu/l (sensitivitas 92%, spesifisitas 87%, akurasi 92%). kolesterol cairan pleura 50 mg/dl (sensitivitas 89%, spesifisitas 53%, akurasi 85%), rasio kolesterol cairan pleura/serum 0,41 (sensitivitas 75%, spesifisitas 53%, akurasi 72%) dan gradien albumin serum-cairan pleura 1,3 gr/dl (sensitivitas 91%, spesifisitas 73%, akurasi 89%). kombinasi ketiga parameter tidak meningkatkan sensitivitas ataupun akurasi diagnostik. kesimpulan kriteria light mempunyai akurasi diagnostik tertinggi tetapi rasio ldh cairan pleura/serum dengan titik potong 0,40 dan ldh cairan pleura dengan titik potong 178 iu/l memiliki akurasi yang lebih tinggi dibandingkan dengan titik potong yang digunakan pada kriteria light. kata kunci: laktat dehidrogenase, kriteria light, albumin, efusi pleura abstrak introduction p l e u r a l e ff u s i o n i s t h e a b n o r m a l accumulation of fluid in the pleural cavities caused by excessive transudation or exudation from the pleural surfaces. pleural effusion may appear in the course of a known disease, or it may present without associated symptoms or previously known cause.(1) however, regardless of presentation, pleural effusion is always abnormal and indicates the presence of an underlying disease.(2) the most frequent causes o f p l e u r a l e ff u s i o n s a r e c a r d i a c f a i l u r e , pneumonia, and malignant neoplasm. the diagnosis of a pleural effusion is based on the clinical history and physical examination, followed by chest radiography, analysis of p l e u r a l f l u i d , (3 ) a n d o p t i o n a l l y b y o t h e r investigations, such as computed tomography ( c t ) o f t h e t h o r a x , p l e u r a l b i o p s y, thoracoscopy, and bronchoscopy. the initial step in the search for the etiology of a pleural effusion is to categorize it as a transudate or an exudate. for this purpose, the criteria formulated by light(4) have been extensively used. according to light’s criteria, 107 univ med vol. 31 no.2 pleural fluid is classified as an exudate if it meets at least one of the following conditions: pleural fluid-serum protein ratio of >0.5; pleural fluid-serum lactate dehydrogenase (ldh) ratio of >0.6; and pleural fluid ldh concentration of minimally 200 u/l. light’s criteria have a high sensitivity (almost 100%) for diagnosing exudates, but have a lower specificity, and may therefore misclassify a transudate as an exudate. in a prospective c o m p a r a t i v e s t u d y o f p l e u r a l e ff u s i o n s involving 172 patients, porcel et al.(5) found that approximately 20% of the patients with heart failure who were taking diuretics also met light criteria for an exudate. exudates require more diagnostic tests to determine their etiology, thus misclassification of a transudate can have s e r i o u s c o n s e q u e n c e s b y u n ne c e s s a r i l y subjecting the patient to invasive procedures and increasing the morbidity of concomitant diseases, such as cardiac, renal or hepatic disorders. a number of investigations have been evaluated for differentiating exudates from transudates, e.g. a pleural fluid cholesterol concentration of >60 mg/dl (1.55 mmol/l) or a pleural fluid protein concentration of >3 g/ dl are considered as indicating an exudate,(6) w h e r e a s a p l e u r a l f l u i d c h o l e s t e r o l concentration of <60 mg/dl, and an serump l e u r a l f l u i d a l b u m i n g r a d i e n t ( i . e . t h e difference between serum and pleural fluid albumin levels) of <1.2 g/dl are deemed to indicate a transudate.(7) the use of light’s criteria for categorizing pleural effusions into exudates and transudates, as an initial step in establishing the etiological diagnosis, suffers from the disadvantage of misclassification, making it necessary to apply additional investigations.(8,9) the aim of the present study was to identify an optimal combination of markers for differentiating between exudates and transudates, namely by measuring ldh and albumin concentrations in the pleural fluid, in addition to the traditional (or standard) light criteria. methods design of the study a n o b s e r v a t i o n a l s t u d y t o e v a l u a t e d i a g n o s t i c s t e s t s w a s c o n d u c t e d a t t h e e m e rg e n c y d e p a r t m e n t o f p e r s a h a b a t a n hospital, jakarta, from september 2010 until december 2011. study subjects as study subjects were taken all patients a t t e n d i n g t h e e m e r g e n c y d e p a r t m e n t a t p e r s a h a b a t a n h o s p i t a l a n d m e e t i n g t h e inclusion and exclusion criteria. the inclusion criteria were patients with pulmonary and nonpulmonary disorders presenting with pleural effusion, who agreed to participate in the study and give written informed consent. patients who were pregnant or in the postpartum period, who h a d a h i s t o r y o f t h o r a c i c o r a b d o m i n a l laparotomy, or had coagulation disorders (a platelet count of < 50,000 per mm3), were excluded from the study. the optimal sample size of 83 subjects was determined from a diagnostic sensitivity of exudates of 0.82, a significance level of 0.05, and a pleural effusion prevalence of 0.75.(10) the sample was selected by non-random consecutive sampling, followed by clinical and radiological examinations. if the posteroanterior chest radiograph showed the presence of fluid in the pleural cavities, the subject underwent pleural puncture. pleural fluid biochemical analysis ten milliliter samples of pleural fluid and 5 milliliter of venous blood without added anticoagulant were collected for analysis at the 24-hour laboratory of persahabatan hospital. pleural fluid was examined by macroscopy (fluid color), biochemical investigations (protein, glucose, ldh, cholesterol, and albumin), and microscopy (number of cells and differential count), while serum biochemistry comprised the same parameters as pleural fluid, viz. protein, ldh, cholesterol, and albumin. 108 syahruddin, partakusuma, khairani assessment of pleural effusions laboratory instruments used were a hitachi 911 and a fuchs rosenthal counting chamber. statistical analysis to determine the cutoff points for the d i a g n o s t i c t e s t s a r e c e i v e r o p e r a t o r characteristic (roc) curve was used based on the results of the pleural fluid and serum analyses. subsequently using these cutoff points, the sensitivity, specificity, positive and negative predictive values, and accuracy (proportion of all correctly diagnosed cases: (tp + tn) / (tp + fp + fn + tn)) of each parameter were calculated. the data analysis was performed by means of the statistical program for social sciences version 17.0. results a total of 119 patients with pleural effusion were recruited into this study, with a mean age of 47.36 ± 16.43 years. the majority (55.5%) of the subjects were males, 40.3% had graduated from senior high school, and 47.1% were employees in the private business sector. among the pleural effusions of the subjects t h e r e w e r e e x u d a t e s i n 1 0 4 ( 8 7 % ) a n d transudates in 15 (13%) patients. tuberculosis was the main cause of the pleural effusions, followed by pulmonary cancers. regarding the latter, 42 patients had adenocarcinoma, 2 patients had atypical carcinoid tumors and 2 patients squamous cell carcinoma. mediastinal tumors were found in 3 patients, viz. lymphomas in 2 patients and teratomas in 1 patient. transudates were mainly caused by heart failure, followed by hepatic cirrhosis and renal failure (table 1). roc curves were constructed from serum and pleural fluid laboratory parameters, comprising protein, ldh, cholesterol and albumin, and used to determine the area under the curve (auc) using a 95% confidence interval (ci), and optimal cutoff points for light’s criteria, cholesterol, and albumin (table 2). the three parameters in light’s criteria had a u c v a l u e s a b o v e 9 0 % . p l e u r a l f l u i d cholesterol (pf chol) and pleural fluid-serum cholesterol ratio (pf/s chol) had an auc of table 1. etiology of pleural effusions (n=119) table 2. area under the curve (auc), auc 95% confidence interval (ci) and optimum cutoff points for each parameter, for differentiation of exudates and transudates pf/s prot=pleural fluid-serum protein ratio; s-pf prot=serum-pleural fluid protein gradient; pf ldh=pleural fluid lactate dehydrogenase; pf/s ldh=pleural fluid-serum lactate dehydrogenase ratio; pf chol=pleural fluid cholesterol; pf/s chol=pleural fluid-serum cholesterol ratio; s-pf alb=serum-pleural fluid albumin gradient; pf/s alb=pleural fluid-serum albumin ratio; ci=confidence interval 109 univ med vol. 31 no.2 66% and 62%, respectively. the serum-pleural fluid protein gradient (s-pf prot), serumpleural fluid albumin gradient (s-pf alb) and pleural fluid-serum albumin ratio (pf/s alb) had auc values of approximately 80%. on the roc curve of each parameter, the cutoff point was selected with the best sensitivity and specificity, and the corresponding sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy calculated (table 3). from the three parameters in the light criteria, the pleural fluid ldh value of >200 u/l had the highest sensitivity (92%), but by combining these parameters, their combined sensitiviy increased to 97%. the pleural fluidserum ldh ratio of >0.6 had the highest specificity (93%), exceeding the 80% of the three light criteria. the highest positive predictive value was found to be 99% for the pleural fluid-serum ldh ratio of >0.6. this was slightly higher than that of light’s criteria (97%). the highest negative predictive value was found to be 80% for pleural fluid ldh, but this was far lower than that of light’s criteria. the diagnostic accuracy of pleural fluid ldh was the highest among light’s criteria but lower than their combined value. the light criteria parameters with their new cutoff point, i.e. the pleural fluid-serum ldh ratio of >0.4, had the highest sensitivity, highest negative predictive value, and highest diagnostic accuracy in comparison with other parameters. on the other hand, the specificity and positive predictive values of the three parameters with t h e n e w c u t o ff p o i n t w e r e t h e s a m e . i n comparison with the standard light criteria parameters, the pleural fluid-serum ldh ratio of 0.4 had a higher sensitivity, negative predictive values, and diagnostic accuracy than other parameters, while the specificity and positive predictive values of the three new parameters were the same. on the roc curve a cutoff point was found with the best sensitivity and specificity for the cholesterol parameter, i.e. a pleural fluid cholesterol value of 50 mg/dl and a pleural fluid-serum cholesterol ratio of 0.4. the results of the roc curve analysis yielded a cutoff point with the best sensitivity and specificity for a pleural fluid-serum albumin gradient parameter value of 1.3 g/dl table 3. diagnostic test results for parameters for classification of exudates, expressed in percentages tp=true positive; fp=false positive; tn=true negative; fn=false negative; ppv=positive predictive value; npv=negative predictive value; pf/s prot=pleural fluid-serum protein ratio; s-pf prot=serum-pleural fluid protein gradient; pf ldh=pleural fluid lactate dehydrogenase; pf/s ldh=pleural fluid-serum lactate dehydrogenase ratio; pf chol=pleural fluid cholesterol; pf/s chol=pleural fluid-serum cholesterol ratio; s-pf alb=serum-pleural fluid albumin gradient; pf/s alb=pleural fluid-serum albumin ratio 110 syahruddin, partakusuma, khairani assessment of pleural effusions and a pleural fluid-serum albumin ratio of 0.6. the specificity and positive predictive values of the pleural fluid-serum albumin ratio parameter were higher than that of the serumpleural albumin gradient parameter. discussion this study found that the majority of pleural effusions (87%) were exudates cansed by local disease of the thoracic cavity, and that the remaining 13 % were caused by systemic diseases. similar results were reported by afful et al.,(11) where 84% of patients had exudative effusions. studies performed in countries with a high tb prevalence found that exudative effusions were more frequent than transudative effusions. a similar result was reported by leers et al.,(12) who found that exudative effusions accounted for approximately 75% of all effusions. in our study 38.7% of the effusions were exudates due to malignancy. essentially the same results were obtained in a us study involving 44 subjects with pleural effusion, with mean age of 46 ± 11.1 years, which showed t h a t t h e p r o p o r t i o n o f e x u d a t e s d u e t o malignancy was 55.0%.(13) the first step in the diagnosis of patients with pleural effusion is to differentiate between exudates and transudates. in this connection, numerous studies have been conducted to evaluate the use of biochemical markers for differentiating exudates and transudates. the markers commonly used are the light criteria, comprising determination of pleural fluid-serum protein ratio, pleural fluid ldh concentration, and pleural fluid-serum ldh ratio. our study found sensitivity and s p e c i f i c i t y v a l u e s o f 9 7 % a n d 8 0 % , respectively, for light’s criteria, while the diagnostic accuracy was 95%. similar results were obtained in the study conducted by gonlugur et al.,(14) who reported a sensitivity of 96% for light’s criteria. however, for the specificity and diagnostic accuracy of these criteria these authors found values of 59% and 88%, respectively. a study conducted with 249 subjects, with mean age of 61 ± 17 years, obtained a value 93% for the accuracy of the light criteria, which is consistent with our results.(15) in addition to the use of the standard light criteria, several studies attempted to find new cutoff points for the light criteria parameters. in these studies the roc curve was used to obtain lower cutoff points for pleural fluid ldh concentration, namely 178 iu/l, with 92% sensitivity, 87% specificity, and 92% diagnostic accuracy. this new cutoff point was similar to the standard cutoff point of 200 iu/l. gonlugur et al.(14) obtained a higher cutoff point for pleural fluid ldh (377 iu/l), with sensitivity of 75%, specificity of 83%, and diagnostic accuracy of 77%. in the present study, at the cutoff point of 0.4 for the pleural fluid ldh ratio, a sensitivity of 95%, specificity of 87% and diagnostic accuracy of 94% were found. in comparison with the cutoff point of 0.6 for the light criteria, our study obtained a higher sensitivity and diagnostic accuracy with the new cutoff point. the light criteria as modified by the new cutoff point did not result in an increased sensitivity, specificity, and diagnostic accuracy. in addition to abovementioned criteria, other frequently used parameters are pleural fluid cholesterol, pleural fluid-serum cholesterol r a t i o , a n d s e r u m p l e u r a l f l u i d a l b u m i n g r a d i e n t . ( 7 ) t h e p l e u r a l f l u i d c h o l e s t e r o l concentration has been used in cases of heart or renal falure treated with diuretics, where a value of >60 mg/dl (1.55 mmol/l) suggests the presence of an exudate.(16) in our study, the pleural fluid cholesterol concentration and the pleural fluid-serum cholesterol ratio had low auc values (66% and 62%, respectively). at a cutoff point for pleural fluid cholesterol of 50 mg/dl we found the sensitivity to be 89%, with the substantially lower specificity of 53%. the pleural fluid-serum cholesterol ratio with a cutoff point of 0.41 had a sensitivity of 75% and a specificity of only 53%. differing results were obtained by leers et al.,(12) who found that pleural fluid cholesterol at a cutoff point of 60 111 univ med vol. 31 no.2 m g / d l h a d a s e n s i t i v i t y o f 7 5 . 7 % a n d a specificity of 98.1%, the latter being higher than that found in our study. a high specificity for pleural fluid cholesterol was also obtained in the study conducted by guleria et al.(17) leers et al.(12) conclude that pleural fluid cholesterol and ldh measurements may be used to differentiate exudates from transudates with a higher diagnostic accuracy. determination of pleural fluid cholesterol and ldh do not require the simultaneous collection of serum samples, thus reducing the number of examinations. several studies found that the serumpleural fluid albumin gradient may also be used i n t h e d i ff e r e n t i a t i o n o f e x u d a t e s a n d t r a n s u d a t e s . i n c a s e s w h e r e t h e c l i n i c a l e v a l u a t i o n i n d i c a t e s t h e p r e s e n c e o f a transudate, whereas pleural fluid analysis indicates an exudate, then it becomes necessary to determine this parameter. in nearly all patients in whom the serum albumin has a value of 1.2 g/dl above the pleural fluid albumin, the effusion will be transudative in nature. our study shows a cutoff point of 1.3 g/dl for the serum-pleural fluid albumin gradient, with 91% sensitivity, 73% specificity, and 89% diagnostic accuracy. these values differ from those obtained in the study conducted by leers et al.,(12) with a cutoff point of 14.5 g/l for the serum-pleural fluid albumin gradient, having a lower sensitivity (44.0%), a higher specificity (88.9%) and a far lower diagnostic accuracy (55.9%). the serum-pleural fluid albumin gradient at the cutoff pont of 1.2 g/dl has been found to be capable of correctly classifying 95% of transudates and exudates.(7) singh et al.(18) also found that the use of the serum-pleural fluid albumin gradient correctly identified all cases of transudative pleural effusion with 97.2% sensitivity and 100% specificity, with only one case from 36 cases of exudative pleural effusion being misclassified. therefore the serum-pleural fluid albumin gradient may be regarded as an effective discriminator between exudates and t r a n s u d a t e s , e x c e p t i n p a t i e n t s w i t h hypoalbuminemia. t h e p l e u r a l f l u i d a l b u m i n f r a c t i o n originates from serum by a process of diffusion. increases in albumin production frequently results in adjustments in the pulmonary microvascular endothelium, which lead to increased fluid leakage, raised protein level, and lowered serum-pleural fluid albumin gradients. the highest serum-pleural fluid albumin gradient occurs in transudative effusions, since there is a low albumin filtration rate through the relatively normal pleural microvasculature. in exudative effusions, the microvasculature is damaged, allowing progressively higher amounts of albumin to enter the pleural cavity, depending on the severity of the lesion. the etiology of exudative effusions involves inflammation, changes in the pulmonary and pleural microvasculature leading to a high fluid leakage rate, high protein levels and a lowered serum-pleural fluid albumin gradient.(7) by combining the pleural fluid ldh parameters of the light criteria with pleural fluid cholesterol values, a sensitivity of 86%, a specificity of 87% and a diagnostic accuracy of 86% was obtained in the present study. by combining a pleural fluid ldh concentration of >207 iu/l or a serum-pleural fluid albumin gradient of <1.3 g/dl, gonlugur et al. (14) obtained a sensitivity of 95%, a specificity of 69% and a diagnostic accuracy of 89%. thus it turns out that combining the three light criteria parameters did not increase either their sensitivity, specificity, or diagnostic accuracy. in most cases of pleural effusion, pleural fluid a n a l y s i s y i e l d s i m p o r t a n t d i a g n o s t i c information, and in certain cases, the use of light’s criteria alone is enough for etiological diagnosis. however, to overcome the limitation of misclassification by using the criteria of light et al.,(4) the new cut-off values of ldh pleural fluid to serum ratio of 0.4 and pleural fluid ldh of 178 iu/l appears should be used, to yield an improvement in diagnostic accuracy i n t h e d i f f e r e n t i a t i o n o f e x u d a t e s a n d transudates in clinical practice. 112 syahruddin, partakusuma, khairani assessment of pleural effusions the cost of performing pleural fluid ldh determinations is affordable and provides essential diagnostic support to the clinicians, enabling them to avoid misclassification of transudates as exudates that may lead to unnecessary and costly investigations, thus obviating the need to order bronchoscopic procedures and computed tomography scans. conclusions the light criteria have a high sensitivity and diagnostic accuracy. determination of pleural fluid ldh concentration is a valid m e a n s f o r d i s t i n g u i s h i n g e x u d a t e s f r o m transudates. further studies of prospective design are necessary to test the validity of various parameters with newer cutoff points. acknowledgements we hereby thank the staff of persahabatan hospital, emergency clinic, laboratory, and medical record for their cooperation. references 1. maskell na, butland rja. bts guidelines for the investigation of unilateral pleural effusion in adults. thorax 2003;58:8-17. 2. rahman nm, chapman sj, davies rj. pleural effusion: a structured approach to care. br med bull 2004;72:31-47. 3. mcgrath ee, paul b. anderson pb. diagnosis of pleural effusion: a systematic approach. am j critical care 2011;20:119-28. 4. light rw. pleural effusion. n engl j med 2002; 346:1971-7. 5. porcel jm, vives m, vicente de vera mc, cao g, rubio m, rivas mc. useful tests on pleural fluid that distinguish transudates from exudates. ann clin biochem 2001;38:671-5. 6. cragun wh. pleural effusion prediction failure. chest 2002;122;1505-6. 7. mangaraj m, kumari s, nanda r, pattnaik mr, mohapatra pc. pleural fluid mda and serumeffusion albumin gradient in pleural effusion. indian j clin biochem 2008;23:81-4. 8. light rw. pleural effusions: the separation of transudates and exudates. ejb 2007;1:8-11. 9. heffner je, highland k, brown lk. a metaanalysis derivation of continuous likelihood ratios for diagnosing pleural fluid exudates. am j respir crit care med 2003;167:1591-9. 10. bachmann lm, puhan ma, riet g, bossuyt pm. sample sizes of studies on diagnostic accuracy: literature survey. bmj 2006;332:1127-9. 11. afful b, murphy s, antunes g, dudzevicius v. the characteristics and causes of pleural effusion in kumasi ghana: a prospective study. tropical doctor 2008;38:219-20. 12. leers mp, kleinveld ha, scharnhorst. differentiating transudative from exudative pleural effusion: should we measure effusion cholesterol dehydrogenase? clin chem lab med 2007;45:1332-8. 13. abouzgheib w, bartter t, dagher h, pratter m, klump w. a prospective study of the volume of pleural fluid required for accurate diagnosis of malignant pleural effusion. chest 2009;135:9991001. 14. gonlugur u, gonlugur te. the distinction between transudates and exudates. j biomed sci 2005;12:985-90. 15. candeira-romero s, hernandez l, romerobrufao s, orts d, fernadex c, martin c. is it meaningful to use biochemical parameters to discriminate between transudative and exudative pleural effusions? chest 2002;122:1524-9. 16. froudarakis me. diagnostic work-up of pleural effusions. respir 2008;75:4-13. doi:10.1159/ 000112221. 17. guleria r, agarwal sr, sinha s, pande jn, ball wc. role of pleural fluid cholesterol in differentiating transudative from exudative pleural effusion. nat med j india 2003;16:64-9. 18. singh g, kajal nc, kaur a, singh m, bhatia as. diagnostic value of serum-effusion albumin gradient in differentiating exudative and transudative pleural effusions. chest 2003; 124:4. c:\users\universa medicina\docu 14 abstract universa medicina life satisfaction is the most significant determinant of quality of life in the elderly thania vinsalia* and yvonne suzy handajani**@ background quality of life tends to decrease as age increases. this study aimed to determine the most significant risk factors (family support, spirituality, and life satisfaction) for the elderly’s quality of life. methods this was a cross-sectional study of 101 subjects aged 60 years in west jakarta. the variables were assessed using the world health organization quality of life–bref (whoqol–bref), family support, daily spiritual experience scale (dses), and the satisfaction with life scale (swls) instruments. multivariate logistic regression was used to analyze the association between quality of life and its determinant factors. results based on sociodemographic characteristics, the respondents were mostly women (66.3%), had more than nine years of education (79.2%), and were married (56.4%). the analysis showed that life satisfaction was significantly associated with overall quality of life (or=9.71; 95% ci:2.04–46.26; p=0.004) and general health (or=7.52; 95% ci:1.70–33.25; p=0.008). life satisfaction was also a risk factor for the environmental domain (or=36.02; 95% ci:5.07– 255.82; p<0.001). furthermore, spirituality was found to be a risk factor for the physical health domain (or=4.18; 95% ci:1.51–11.59; p=0.006), psychological domain (or=6.67; 95% ci:2.4–17.86; p<0.001), and environmental domain (or=11.46; 95% ci:3.10–42.37; p<0.001). conclusion life satisfaction plays a significant role in increasing the environmental domain of quality of life, the overall quality of life, and general health among the elderly. awareness of these factors can assist providers in identifying people at risk and guide new intervention programs to improve care for these invaluable elderly of our communities. keywords : elderly, life satisfaction, spirituality, family support, quality of life original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.14-21 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1037 january-april, 2021 vol.40no.1 cite this article as: vinsalia t, handajani ys. life satisfaction is the most significant determinant of quality of life in the elderly univ med 2021;40:14-21. doi: 10.18051/univmed.2021.v40.14-21 *center of health research, school of medicine and health sciences, atma jaya catholic university of indonesia, jakarta, indonesia **department of public health and nutrition, school of medicine and health sciences, atma jaya catholic university of indonesia, jakarta, indonesia correspondence: @yvonne suzy handajani school of medicine & health sciences, atma jaya catholic university of indonesia, jl. pluit selatan raya no.19, jakarta utara, 14440 phone: +628161353738 email: yvonne.hand@atmajaya.ac.id orcid id: 0000-0002-8245-9354 date of first submission, october 1, 2020 date of final revised submission, january 2, 2021 date of acceptance, january 5, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 15 univ med vol. 40 no 1 introduction the elderly population in indonesia and globally has increased significantly. there are an estimated 962 million people aged 60 years or over worldwide. according to the united nations (un), since 2015, asia and indonesia have entered the era of aging population because the percentage of the population aged 60 or over has exceeded 7%.(1) in 2018, the percentage of the elderly population in indonesia was 9.27% (24.49 million). the indonesian central statistics agency (badan pusat statistik, bps) projected that by 2045, this rate will rise to 20% or approximately 63.31 million people.(2) as age increases, the quality of life tends to decrease. lack of social interactions, poor health condition, decreased physical and mental functions lead to emotional disturbances that affect the quality of life.(3) kaur et al. (4) stated that support from family relations contributes to a higher ability of the elderly to cope with changes in health, social activities, and more. it provides individual emotional, social, and economic support. family support increases not only the quality of life of the elderly, but also the quality of life of the family itself.(5) spiritual needs that are not fulfilled are often associated with a poor quality of life. gonzálescelis et al.(6) compared levels of quality of life in each of the six domains of the whoqol-100 questionnaire and found that spirituality had the highest score. besides that, elderly with high spiritual level tend not to have depression. elderly without depressive symptoms have a better quality of life. high spirituality leads older people to think more positively and therefore to have good health.(7,8) poor et al.(9) also stated that older people with stronger religious beliefs have a greater life expectancy. however, a study by jafaripoor et al.(10) showed a contradictive result. life satisfaction also plays a role as an indicator of the quality of life. physical and mental health significantly influence life satisfaction, which eventually affects the quality of life of the elderly.(11) however, a study by kiarsipour et al. (1 2) in iranian older adults showed a contradictory result where they found a low life satisfaction level due to the negative aging perception. there is a positive association of religiosity and spirituality with quality of life.(13) the empirical evidence concerning the relationship of quality of life with religiosity and spirituality essentially focuses on health, with few studies seeking to analyze this association by incorporating the multidimensional character of quality of life into their empirical analyses. a study conducted in nursing homes has reported a positive correlation between social support received from the family and quality of life.(14) most studies only analyze the relationship between family support or spirituality or life satisfaction with the quality in the elderly. the difference between the present study with previous studies was that in our study, all three risk factors (family support, spirituality, and life satisfaction) were the independent variables on the quality of life, and we assessed the most dominant factors affecting the quality of life. this study aimed to determine the most significant risk factors (family support, spirituality, and life satisfaction) of the elderly’s quality of life. methods research design a study of cross-sectional design was conducted in the pusat sant unan dalam keluarga (pusak a) of m eruya ilir ( a community-based home care center) in west jakarta, indonesia, between october 27th, 2018 until november 24th, 2018. research subjects the sample size determination was done by using the lemeshow formula with  = 0.05. the prevalence of elderly with poor quality of life was 23.6%,(15) which obtained 70 respondents. to anticipate dropouts, 10% of the total sample was added to obtain 77 respondents. simple random sampling was done for the sampling selection. 16 from several community-based home care facilities in 5 areas of jakarta, west jakarta was randomly selected. there are several communitybased home care facilities in west jakarta, and pusaka meruya ilir was randomly selected. all of the 267 elderly living in pusaka meruya ilir were listed and numbered. from these, 101 participants were chosen using spss random sampling. this study’s inclusion criteria were subjects aged 60 years and older residing in pusaka meruya ilir who were willing to become respondents and agreed to sign informed consent. subjects who had hearing and communication impairments were excluded from the study. measurements spirituality was assessed using the present authors’ modification of the daily spiritual experience scale (dses), consisting of 15 items with a 6-scale scoring system, with 1 being almost all day long and 6 being never or seldom. a total score between 15-40 is considered high, and 4188 is considered low. lower scores indicate higher frequency of spiritual experiences. (16) the dses questionnaire has high reliability with a cronbach’s alpha value of  0.9.(17,18) the assessment of family support was done by using the family support scale questionnaire. there are 13 questions which are rated on a fivepoint scale, with 1 being “i disagree a lot” and 5 “i agree a lot”.(19) family support is considered high if the total score is >37.(20) this instrument has proven to be reliable with a cronbach’s alpha value of 0.820.(19) life satisfaction was assessed using the satisfaction with life scale (swls) consisting of 5 questions representing the following five components: the desire to c hange a life, satisfaction with the present life, with life in the past and future, and individual’s appraisal of his life. it uses a 7-scale scoring system, with 1 being ‘strongly disagree’ with the statement and 7 being ‘strongly agree’ with the statement. a total score between 20-35 is considered satisfactory, and a score of 19 is considered unsatisfactory.(21,22) the cronbach’s alpha value for swls ranges between 0.78-0.83.(23) lastly, the quality of life was assessed using the 26-item world health organization quality of lif e – bref (w hoqol – bref) instrument. the four domains were distributed into 24 questions, while the other two questions were about the overall quality of life and general health. each question is assigned a score of 1 to 5, and higher scores represent a better quality of life. a total score of 60 is considered good and a score of <60 is considered moderate-poor.(24) the four quality of life domains have good reliability, with cronbach’s alpha value ranging between 0.41-0.77.(15) data analysis univariate analysis was performed to determine the respondents’ characteristics and summar ize the distri bution of variables. multivariate-adjusted odds ratios (ors) with 95% confidence intervals (cis) were used to determine the association between quality of life and determinant factors. by using multiple logistic regression analysis, the results were automatically adjusted.(25) the data was analyzed using spss version 23. a p-value <0.05 was considered statistically significant. ethical clearance this study obtained ethical clearance with the following reference number: fkik unika atma jaya no.13/12/kep-fkuaj/2018. results the majority of subjects in this study were 65 years old and over (80.2%), female (66.3%), had more than nine years of education (79.2%), and wer e ma rried ( 56.4%). most of the respondents had high family support (73.3%), high spirituality level (75.2%), and high life satisfaction (88.1%) (table 1). as an overview of the quality of life based on the overall quality of life and general health, most subjects had a good overall quality of life (81.2%) and 64.4% had excellent general health. vinsalia, handajani life satisfaction and qol in the elderly 17 univ med vol. 40 no 1 a b c results from the analysis showed that sex was significantly associated with general health (or=5.30; 95% ci=1.72 – 16.36; p=0.004). subjects with low life satisfaction has a risk factor for poor overall quality of life (or=9.71; 95% ci=2.04 – 46.26; p=0.004) and general health (or=7.52; 95% ci=1.70 – 33.25; p=0.008). the elderly with low life satisfaction had a nine times higher risk for low quality of life and seven times higher risk for low general health. low spiritual level was also a risk factor for the overall quality of life (or= 5.84; 95% ci= 1.77 19.23; p= 0.004) (table 2). those with low spiritual level also had a six times higher risk for a low quality of life. those with low spiritual level also had a six times higher risk for a low quality of life. table 3 showed that sex had significant associations with both the social interactions domain (or=4.77; 95% ci=1.87 – 12.16; p= 0.001) and the environmental domain (or= 5.51; 95% ci= 1.20 – 25.24; p= 0.028). spirituality was a risk factor for the physical health domain (or=4.18; 95% ci=1.51 – 11.59; p=0.006, psychological domain (or=6.67; 95% ci=2.49 – 17.86; p<0.001), social interactions domain (or=3.71; 95% ci=1.07 – 12.82; p=0.038), and environmental domain (or=11.46; 95% ci=3.10 – 42.37; p<0.001). furthermore, life satisfaction was also a risk factor of the environmental domain (or=36.02; 95% ci=5.07 – 255.82; p<0.001). discussion sex was significantly associated with general health, social interactions domain, and environmental domain. many studies (26–28) showed similar results. studies conducted in kuala lumpur (26) and the netherlands (28) also showed that sex had a significant association with social interactions and environmental domains. women rated the social domain of quality of life higher variables n (%) age (years) < 65  65 sex male female 20 (19.8) 81 (80.2) 34 (33.7) 67 (66.3) education  9 years > 9 years 21 (20.8) 80 (79.2) marital status single married divorced 7 (6.9) 57 (56.4) 37 (36.6) family support low high spirituality low high life satisfaction not satisfied satisfied overall quality of life good moderate-poor general health good moderate-poor 27 (26.7) 74 (73.3) 25 (24.8) 76 (75.2) 12 (11.9) 89 (88.1) 82 (81.2) 19 (18.8) 65 (64.4) 36 (35.6) factors overall quality of life general health or 95 % c.i. p value or 95 % c.i. p value age sex education marital status family support spirituality life satisfaction 0.95 1.27 0.70 0.37 0.87 5.84 9.71 0.83 1.07 0.33 4.95 0.42 1.18 0.13 1.05 0.23 3.27 1.7719.23 2.04 46.26 0.382 0.730 0.182 0.061 0.837 0.004* 0.004* 1.01 5.30 0.76 0.81 0.48 2.42 7.52 0.92 1.10 1.72 16.36 0.51 1.13 0.38 1.72 0.14 1.59 0.83 1.59 1.70 33.25 0.892 0.004* 0.171 0.582 0.229 0.106 0.008* table 2. multivariate logistic regression for factors related to overall quality of life and general health *significant at <0.05 table 1. distribution of sociodemographic characteristics and variables (n=101) 18 than men. gobbens and van assen (29) showed that social participation was higher in women than in men. in a study from the national health and nutrition examination survey, women also scored higher on social functioning. social interaction was considered to be one of the most important factors of quality of life in older adults.(28) onunkwor et al. (26) stated that women had a significantly lower quality of life in all doma ins than men. this may be because women perceived aging more negatively than men. feelings of unattractiveness among older women could lead to low self-esteem and contribute to the negative perception of aging, resulting in a lower quality of life.(30) family support had no significant association with the four domains of quality of life, and both overall quality of life and general health in this study, which was contrary to previous research done in surakarta, central java, where a better quality of life was found in the elderly with strong family support.(31) a spanish study showed that family support is as important for health as social contacts, which are a true welfare factor, and can be considered an estimate of quality of life.(32) spirituality showed a significant association with the overall quality of life. spirituality was also a risk f actor f or physical domain, psychological domain, and environmental domain. low spiritual level was also a risk factor for poor quality of life. this result was parallel to a research conducted by gonzales-celis et al.,(6) who stated that spirituality had the highest impact and had a significant association with the quality of life. spirituality is associated with better outcomes of quality of life and has been identified as an important dimension of quality of life.(33,34) it was found that people with high spiritual level had a higher quality of life compared to those with low spiritual level.(34) some studies indicated that spirituality has great relationship with an individual’s health. therefore, religion and spirituality are considered to be significant sources for compatibility with life’s stressful events. beside that, it also provides the elderly inscribable equanimity and joy.(35) t ab le 3 . m u lt iv ar ia te lo gi st ic r eg re ss io n f o r fa ct o rs r el at ed to p hy si ca l, p sy ch o lo gi ca l, s o ci al in te ra ct io n a n d e n vi ro n m en ta l d o m ai n s *s ig n if ic an t a t < 0 .0 5 vinsalia, handajani life satisfaction and qol in the elderly 19 univ med vol. 40 no 1 life sa tisfaction showed a positive association with the overall quality of life and general health, as well as with the environmental domain. low life satisfaction was found to be a risk factor for bad overall quality of life, general health, and environmental domain. a similar result was obtained in a study by lucas-carrasco et al.(36) where life satisfaction was found to affect the overall quality of life, general health, and all four domains. life satisfaction is a multidimensional concept that includes physical health, mental health, socio-economic status, social and family re lationships, and the environment, that can be used to predict the mortality and morbidity of the elderly.(11) a research in turkey found a positive relationship between life satisfaction and quality of life. it also stated that life satisfaction will increase along with the improvement of an individual’s quality of life.(37) a correlational analysis done by boylu and günay (22) showed that quality of life tends to decrease as the age increases, however it increases along with high education, good physical health, and high life satisfaction. a previous study by indrayani et al.(38) showed that family support was the most dominant factor related to the quality of life. in our study, we found that family support had no significant association with quality of life. in contrast, spirituality and life satisfaction are significantly associated with the overall quality of life, general health, and the four domains of quality of life. there are three limitations in this study. first, the study was performed in only one communitybased home care center (pusaka), making the generalizability of the findings questionable. second, the respondents were interviewed simultaneously. this could have made them feel uncomfortable to be straightforward about their personal matters. thus, it might have affected the results of the interview. third, there might also be a recall bias due to the use of self-report data. psychological and social functions decrease, and chronic degenerative diseases increase as people age. therefore, knowing the factors that expose the older adults to physical and emotional vulnerability is essential to provide healthy aging and palliative care for geriatric patients. consequently, future research using a more heterogeneous elderly population is advisable. it is better if the research can be developed into a longitudinal study so that related data can be seen more clearly and better results can be obtained. conclusions life satisfaction was the most significant determinant in improving the overall quality of life and general health, as well as environmental domain of quality of life. older people with high spiritual level and life satisfaction had a more excellent quality of life. conflict of interest compe ting inte re sts: no re levant disclosures. acknowledgement the authors would like to thank all the staff of pusaka meruya ilir, who have permitted and helped this research, and all the elderly that were willing to participate in this research. contributors all authors will take public responsibility for the content of the manuscript submitted to universa medicina. ysh and tv contributed to the study concept and design of the study. ysh and tv contributed to drafting the manuscript. ysh contributed to the critical revision of the manuscript. all authors have read and approved the final manuscript. references 1. pusat data dan informasi kementerian kesehatan republik indonesia. analisis lansia di indonesia 2017. jakarta: pusat data dan informasi kementerian kesehatan republik indonesia; 2017. 20 2. badan pusat statistik. statistik penduduk lanjut usia 2018. jakarta: badan pusat statistik; 2018. 3. khaje-bishak y, payahoo l, pourghasem b, asghari jafarabadi m. assessing 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karner-hutuleac a. quality of life and spirituality. eur j sci theol 2012;8:135–41. 14. bakar n, asilar rh. factors affecting depression and quality of life in the elderly. j gerontol geriatr res 2015;04:8. doi:10.4172/2167-7182.1000249. 15. salim oc, sudharma ni, kusumaratna rk, hidayat a. validitas dan reliabilitas whoqolbref untuk mengukur kualitas hidup lanjut usia. univ med 2016;26: 27-38. doi: http://dx.doi.org/ 10.18051/univmed.2007.v26.27-38. 16. soósová ms, mauer b. psychometrics properties of the daily spiritual experience scale in slovak elderly. j relig health 2020;59. doi:10.1007/s10943020-00994-w. 17. underwood lg. the daily spiritual experience scale: overview and results. religions 2011;2:29– 50. doi:10.3390/rel2010029. 18. loustalot f, wyatt sb, sims m, ellison cg, taylor ha, underwood l. psychometric testing of the daily spiritual experiences scale among african americans in the jackson heart study. j relig health 2011;50:675–85. doi:10.1007/s10943-0099278-2. 19. tselebis a, anagnostopoulou t, bratis d, et al. the 13 item family support scale: reliability and validity of the greek translation in a sample of greek health care professionals. asia pac fam med 2011;10:3. https://doi.org/10.1186/1447056x-10-3. 20. bratis d, tselebis a, sikaras c, et al. alexithymia and its association with burnout, depression and family support among greek nursing staff. hum resour health 2009;7:6. doi:10.1186/1478-4491-772. 21. diener e, emmons ra, larsen rj, griffin s. the satisfaction with life scale. j pers assess 2010;49: 71–5. https://doi.org/10.1207/s15327752jpa4901_ 13. 22. boylu aa, günay g. life satisfaction and quality of life among the elderly: moderating effect of activities of daily living. turkish j geriatr 2017;20: 61-9. 23. galanakis m, lakioti a, pezirkianidis c, karakasidou e. reliability and validity of the satisfaction with life scale (swls) in a greek sample. int j humanit soc stud 2017;5:9. 24. silva pab, soares sm, santos jfg, silva lb. cutoff point for whoqol-bref as a measure of quality of life of older adults. rev saúde pública 2014;48:390–7. 25. ranganathan p, pramesh cs, aggarwal r. common pitfalls in statistical analysis: logistic regression. perspect clin res 2017;8:148–51. doi: 10.4103/picr.picr_87_17. 26. onunkwor of, al-dubai sar, george pp, et al. a cross-sectional study on quality of life among the elderly in non-governmental organizations’ elderly homes in kuala lumpur. health qual life outcomes 2016;14:10. doi: 10.1186/s12955-0160408-8. vinsalia, handajani life satisfaction and qol in the elderly 21 univ med vol. 40 no 1 27. campos acv, ferreira ef, vargas amd, albala c. aging, gender and quality of life (ageqol) study: factors associated with good quality of life in older brazilian community-dwelling adults. health qual life outcomes 2014;12:11. doi: 10.1186/s12955-014-0166-4. 28. gobbens rj, remmen r. the effects of sociodemographic factors on quality of life among people aged 50 years or older are not unequivocal: comparing sf-12, whoqol-bref, and whoqol-old. clin interv aging 2019;14: 231–9. doi: 10.2147/cia.s189560. 29. gobbens rj, van assen malm. associations between multidimensional frailty and quality of life among dutch older people. arch gerontol geriatr 2017;73:69–76. doi: 10.1016/j.archger.2017. 07.007. 30. sabik nj. ageism and body esteem: associations with psychological well-being among late middleaged african american and european american women. j gerontol b psychol sci soc sci 2015;70: 189–99. doi: 10.1093/geronb/gbt080. doi: 10.1093/ geronb/gbt080. 31. suwarni s, soemanto rb, sudiyanto a. effect of dementia, family support, peer support, type of residence, and marital status on quality of life of the elderly in surakarta, central java. j epidemiol public health 2018;3:83–94. 32. garcía lmr, navarrro jmr. the impact of quality of life on the health of older people from a multidimensional perspective. j aging res 2018; 2018:7. doi: https://doi.org/10.1155/2018/4086294. 33. counted v, possamai a, meade t. relational spirituality and quality of life 2007 to 2017: an integrative research review. health qual life outcomes 2018;16:18. doi:10.1186/s12955-0180895-x. 34. panzini rg, mosqueiro bp, zimpel rr, bandeira dr, rocha ns, fleck mp. quality-of-life and spirituality. int rev psychiatr 2017;29:263–82. doi: http://dx.doi.org/10.1080/09540261.2017.1285553. 35. seraji m, shojaezade d, rakhshani f. the relationship between spiritual well-being and quality of life among the elderly people residing in zahedan city (south-east of iran). elderly health j 2016;2:84-8. 36. lucas-carrasco r, den oudsten bl, eser e, power mj. using the satisfaction with life scale in people with parkinson’s disease: a validation study in different european countries. sci world j 2014; article id 680659. doi:10.1155/2014/680659. 37. yildirim y, kilic sp, akyol ad. relationship between life satisfaction and quality of life in turkish nursing school students: nursing student quality of life. nursing health sci 2013;15:415– 22. 38. indrayani i, ronoatmodjo s. faktor-faktor yang berhubungan dengan kualitas hidup lansia di desa cipasung kabupaten kuningan tahun 2017. j kesehat reprod 2018;9:1-10. doi: https:// doi.org/10.22435/kespro.v9i1.892.69-78. universa medicina may-august vol.42no.2, 2023 invited editorial more than just a name: "nonalcoholic fatty liver disease (nafld)" versus "metabolic associated fatty liver disease (mafld)" kathryn effendi department of pathology, keio university school of medicine, tokyo, japan the term nonalcoholic fatty liver disease (nafld) has been used for more than three decades, and nafld is known to be a common cause of chronic liver disease.(1) simple steatosis (nafl) with the absence of liver cell damage or inflammation can develop into a condition with more progressive features known as nonalcoholic steatohepatitis (nash), which then can lead to cirrhosis and hepatocellular carcinoma. a working group of liver pathologists in japan has established a consensus regarding the pathological findings of nafl and nash. the presence of hepatocyte ballooning, mallory-denk bodies, and fibrosis are important features for the differential diagnosis of nafl and nash and to predict the progression of nafld.(2) recently, nafld has become a serious public health issue. despite the significant efforts of clinicians and researchers to increase our understanding of nafld, its prevalence is still growing fast and is a cause of concern; indeed, nafld not only affects adults but also children. a cohort study of swedish children and young adults with biopsy-confirmed nafld showed that they have significantly higher overall rates of cancer, cardiometabolic disease, and liver disease.(3) the current definition of nafld requires the exclusion of other causes of liver disease, such as viral infections or autoimmune diseases, and confirmation that the subject has consumed only limited amounts of alcohol. the european association for the study of the liver (easl), the uk national institute for health and care excellence (nice), the italian association for the study of the liver (aisf), and japanese guidelines consider the upper limit of alcohol consumption for a diagnosis of nafld to be 30 g/day in men and 20 g/day in women. (4) however, the level of awareness regarding nafld among the general public is low. it is well known that significant alcohol consumption can lead to liver damage, but it is not widely recognized that such damage also can occur in people who drink only small amounts of alcohol or no alcohol at all. the use of “alcoholic” vs “nonalcoholic” also sparks concern that such diseases result from patient behavior, potentially resulting in social stigmatization and barriers to proper healthcare.(1) in 2020, a panel of international experts published a consensus proposing replacement of the term nafld with metabolic associated fatty liver disease (mafld). (1,5) the use of mafld was considered to more appropriately reflect the fact that metabolic disorders such as overweight or obesity, type 2 diabetes mellitus, dyslipidemia, and hypertension are also key factors associated with the development of liver damage. (6) the updated nomenclature “mafld” was suggested to be advantageous in helping align fatty liver diseases with metabolic risk factors and in providing a path toward precision medicine for fatty liver disease that would not be possible under the guise of nafld. (7) since the new term mafld was proposed, numerous studies have been published in which experts attempted to define the diagnostic criteria for mafld and nafld. the concept of *email: kathryn@a8.keio.jp orcid id: 0000-0001-7844-1170 119 mailto:kathryn@a8.keio.jp https://orcid.org/0000-0001-7844-1170 120 universa medicina vol. 42 n0. 2 2023 mafld and nafld also prompted a wave of debate among experts. the adoption of the new term has been called premature and likely to confuse the existing efforts to promote awareness of liver diseases among patients. one particular concern is that liver diseases also occur and progress in people who do not have metabolic disorders. individuals with normal body mass index may also develop nafld.(8, 9) the percentage of overlap betwe en mafld and nafld is around 80%, the remaining cases comprise combined forms of mafld without nafld (mafld+/nafld– ) and nafld without mafld (mafld–/ nafld+).(6) a meta-analysis of the non-overlap groups comparing epidemiological and clinical features showed that the mafld-only subgroup had higher levels of alanine aminotransferase and aspar tate aminotrans fer ase and a higher prevalence of fibrosis than the nafld-only subgroup in the general population.(6, 10) although these studies suggested that the novel term mafld may be better at identifying patients with long-term risk of progression of liver steatosis, the heterogeneous course of fatty liver disease highlights the need of further studies to assess the pathogenesis of each group in real-world clinical practice. the debate over the use of mafld vs nafld is still ongoing. nonetheless, it would seem appropriate to redefine nafld as our knowledge of the disease also develops. mafld may give us a broader scope to manage the disease more holistically according to its background, but we also need a be tter understanding of mafld subtypes. ultimately, educating patients about their individual health risks and preventing the progression of liver damage is more important than the name of the disease itself. there are still many patients who do not even recognize that fatty liver is actually a disease. therefore, general education about fatty liver disease should be the main focus while the nomenclature question is being resolved. references 1. fouad y, waked i, bollipo s, gomaa a, ajlouni y, attia d. what’s in a name? renaming ‘nafld’ to ‘mafld’. liver int 2020;40:1254-61. doi: 10.1111/liv.14478.. 2. sakamoto m, tsujikawa h, effendi k, et al. pathological findings of nonalcoholic steatohepatitis and nonalcoholic fatty liver disease. pathol int 2017;67:1-7. doi: 10.1111/ pin.12485. 3. simon tg, roelstraete b, hartjes k, et al. nonalcoholic fatty liver disease in children and young adults is associated with increased long-term mortality. j hepatol 2021;75:1034-41. doi: 10.1016/ j.jhep.2021.06.034. 4. leoni s, tovoli f, napoli l, serio i, ferri s, bolondi l. current guidelines for the management of nonalcoholic fatty liver disease: a systematic review with comparative analysis. world j gastroenterol 2018;24:3361-73. doi: 10.3748/wjg.v24.i30.3361. 5. eslam m, sanyal aj, george j; international consensus panel. mafld: a consensus-driven proposed nomenclature for metabolic associated fatty liver disease. gastroenterology 2020;158: 1999-2014.e1. doi: 10.1053/j.gastro.2019.11.312. 6. garcía-compeán d, jiménez-rodríguez ar. nafld vs mafld. the evidence-based debate has come. time to change? ann hepatol 2022;27:100765. doi: 10.1016/j.aohep.2022.100765. 7. eslam m, ratziu v, george j. yet more evidence that mafld is more than a name change. j hepatol 2021;74:977-9. doi: 10.1016/j.jhep.2020. 12.025. 8. singh sp, anirvan p, reddy kr, et al. nonalcoholic fatty liver disease: not time for an obituary just yet! j hepatol 2021;74:972-4. doi: 10.1016/j.jhep.2020.10.015. 9. duseja a, taneja s. changing nomenclature from nonalcoholic fatty liver disease to metabolic dysfunction-associated fatty liver disease – not only premature but also confusing. j clin exp hepatol 2021;11:278-9. doi: 10.1016/j.jceh.2020.08. 002. 10. ayada i, van kleef la, alferink ljm, li p, de knegt rj, pan q. systematically comparing epidemiological and clinical features of mafld and nafld by meta-analysis: focusing on the non-overlap groups. liver int 2022;42:277-87. doi: 10.1111/liv.15139. univ med 2023;42:119-120. doi: http://dx.doi.org/10.18051/univmed.2023.v42.119-120 oktavianus 34 *division of neurology department of child health medical school, airlangga university / soetomo hospital surabaya correspondence dr. prastiya indra gunawan, spa division of neurology department of child health medical school, airlangga university / soetomo hospital surabaya 60286 phone: +6231-5501681 fax: +6231-5501748 email: prastiya_ig@yahoo.co.id univ med 2012;31:34-42 abstract universa medicina january-april, 2012january-april, 2012january-april, 2012january-april, 2012january-april, 2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 background epilepsy remains one of the most frequently occurring pediatric problems. approximately 10-15% patients do not respond to conventional therapy. topiramate as a novel antiepileptic drug has a broad spectrum activity, presumably indicative of multiple anti-seizure mechanisms. previous studies of topiramate as adjunctive and monotherapy in adults have shown beneficial effects. the objective of this research was to evaluate the efficacy and tolerability of topiramate sprinkle monotherapy in pediatric epilepsy. methods this experimental research was conducted in the pediatric neurology outpatient clinic department, soetomo hospital, surabaya, involving 18 consecutive subjects. subjects meeting the inclusion criteria were treated with topiramate sprinkle adjusted dose. seizure frequency and side effects were observed in weeks 1, 4, 8, 12, 16, 20 and 24, respectively. electro encephalogram (eeg) and laboratory examinations were performed prior to and after 6 months of treatment. the t-test for related samples and mcnemar test were utilized for statistical analysis. results a total of 15 subjects completed the study. topiramate-treated patients showed a statistically significant difference of seizure frequency reduction from 2.7 ± 1.16 to 0.13 ± 0.51 (p=0.000) with 93.7% patients being seizure free in 20 weeks. eeg recordings did not differ statistically in decrement of epileptiform activity in 20% subjects. about 7% subjects developed drowsiness and 33.3% subjects suffered from appetite suppression in the initial treatment. laboratory results showed no abnormalities. conclusions there was reduction of seizure frequency and no eeg recording alterations after topiramate sprinkle monotherapy. topiramate as a monotherapy is highly effcicacious in childhood epilepsy. keywords: topiramate sprinkle monotherapy, seizure frequency, side effect, childhood topiramate sprinkle is effective in the treatment of childhood epilepsy prastiya indra gunawan* and darto saharso* 35 univ med vol. 31 no.1 topiramate sprinkle efektif untuk pengobatan epilepsi pada anak-anak latar belakang epilepsi merupakan masalah besar dalam bidang pediatri. masih terdapat 10-15% penderita yang resisten terhadap pengobatan umum. topiramate sebagai obat antiepilepsi baru mempunyai spektrum luas untuk anti kejang. penelitian menunjukkan dan monoterapi topiramate mempunyai potensi yang baik pada orang dewasa. data efektifitas dan efek samping topiramate sebagai monoterapi pada anak-anak masih terbatas. tujuan penelitian ini adalah menilai efikasi dan tolerabilitas topiramate untuk monoterapi penderita anak-anak dengan epilepsi. metode penelitian eksperimental dilakukan di poliklinik neurologi anak rsud dr soetomo surabaya. sebanyak 18 subjek anak-anak diikut sertakan pada penelitian. subyek yang sesuai kriteria inklusi dan ekslusi diberikan terapi topiramate sprinkle dan dilakukan pengukuran frekuensi kejang serta efek samping pada minggu 1, 4, 8, 12, 16, 20 dan 24. gambaran elektro ensefalogram (eeg) dan pemeriksaan laboratorium dilakukan sebelum dan sesudah 6 bulan terapi. analisis statistik menggunakan t-test for related samples dan mcnemar. hasil sebanyak 15 subjek berhasil diikuti sampai akhir penelitian. frekuensi kejang awal sebanyak 2,7 ± 1,16 berkurang menjadi 0,13 ± 0,51 dengan 93,7% penderita bebas kejang pada minggu ke 20 (p=0,000). gambaran eeg awal 100% menunjukkan aktifitas epileptiform menurun menjadi normal pada 20% subyek. sebanyak 30% sampel mengalami penurunan nafsu makan pada saat awal terapi dan 7% subyek mengalami rasa kantuk. setelah pemberian pengobatan topiramate sprinkle hasil pemerikssan laboratorium tidak menunjukkan adanya kelainan. kesimpulan terdapat reduksi frekuensi kejang dan tidak terdapat perubahan eeg pasca pemberian pemberian topiramate sprinkle. topiramate sebagai monoterapi sangat efektif untuk pengobatan epilepsi pada anak-anak. kata kunci: monoterapi, topiramate sprinkle, frekuensi kejang, efek samping, anak-anak abstrak introduction epilepsy is a common chronic neurological disorder that is characterized by recurrent unprovoked seizures. these seizures are transient signs and/or symptoms due to abnormal, excessive or synchronous neuronal activity in t h e b r a i n . m o s t e p i l e p s y c a s e s b e g i n i n childhood. therefore, epilepsy remains one of the biggest pediatric problems.(1,2) antiepileptic drugs (aeds) can control seizures in 70-80% of epileptic children, and advanced studies in pediatric epilepsy have shown that 10-15% of patients are already resistant to conventional treatment.(1-3) the development of new aeds for epilepsy over the last decade has been spurred by the fact that the available aeds did not provide optimal care for patients with epilepsy. many patients “failed” all available options, either because their seizures were not adequately controlled, or they were experiencing side effects. for e t h i c a l a n d m e d i c a l r e a s o n s , n e w a e d s typically are evaluated first as adjunctive t h e r a p i e s i n a d u l t s w i t h a e d r e s i s t a n t seizures.(4) topiramate as a new aed appears to have multiple neurostabilizing activities, including 36 potentiation of ã-aminobutyric acid (gaba) neuroinhibition, state-dependent blockade of voltage-dependent na+ channels, modulation of high voltage-activated ca + channels, and glutamate receptor antagonism at non kainate/ a m pa ( n m e t h y l d a s p a r t a t e / n m d a ) receptors. these activities may account for the broad spectrum of antiseizure effects seen experimentally and clinically. (3-5) recent research has shown that topiramate as a new aed has a better efficacy than other aeds. previous studies of topiramate as adjunctive and monotherapy in adults has shown beneficial effects.(5-6) however, the data on the efficacy a n d t o l e r a b i l i t y o f t o p i r a m a t e s p r i n k l e monotherapy in children are still limited. the aim of the current study was to evaluate the efficacy and tolerability of topiramate sprinkle monotherapy in pediatric epilepsy. methods study design this was an experimental trial with 24week treatment (titration and stabilization) phase conducted at the pediatric neurology outpatient clinic, soetomo hospital, surabaya, from august 2008 to january 2010. patients patients were included if they were above 6 months of age, had a first diagnosis of epilepsy with any type of seizures in accordance with the criteria of the international classification of epileptic seizures, or had been treated with their first aed in monotherapy that failed in efficacy, tolerability, or both. patients were excluded if they had pseudoseizures or the cause of the seizures was treatable, had any clinically relevant progressive or serious illness, which might interfere with the patient completing the trial, had a history or other indications of alcohol or drug abuse, were non-cooperative or were expected to be difficult to follow up, or had hypersensitivity to topiramate or any of its constituents. treatment to p i r a m a t e s p r i n k l e c a p s u l e s w e r e administered by the oral route, either by being swallowed whole or being opened and the contents mixed with a spoonful of soft food. eligible patients selected by consecutive s a m p l i n g r e c e i v e d t o p i r a m a t e s p r i n k l e monotherapy with adjusted dosages, starting from 0.5 mg/kg body weight once daily in the first week, and increasing to 1 mg/kg body weight twice daily in the second week. during the titration period, the dosage increments of topiramate were adjusted to the number of observed seizures. the patients were then followed for a 24-week maintenance period with a maximal tolerated dose of approximately 6 mg/kg body weight daily. if the patient did not tolerate the titration schedule, the rate of titration was either maintained or reduced at t h e d i s c r e t i o n o f t h e i n v e s t i g a t o r. t h e background dose of aed, if any, was tapered during the titration period. evaluation throughout the trial, patients (or their parents or legal guardians) maintained diaries to record the type and frequency of seizures as well as adverse events. at each visit, the investigator reviewed the patient’s seizure diary, classifying each seizure according to the i n t e r n a t i o n a l c l a s s i f i c a t i o n o f e p i l e p t i c seizures, recorded any adverse experiences, and monitored vital signs. patient visits were scheduled for weeks 1, 4, 8,12,16,20, and 24 (final visit), respectively. the primary efficacy analysis outcome was the seizure frequency distribution (i.e. the proportion of patients completing the trial seizure free or experiencing one or two seizures). the secondary efficacy analysis, including electroencephalogram (eeg) examinations, was obtained before and after topiramate treatment. the tolerability was monitored throughout the trial by physical examination, vital signs, clinical laboratory tests including hematology, blood chemistry, and adverse event reports. blood samples for gunawan, suharso topiramate for childhood epilepsy 37 univ med vol. 31 no.1 confirmation of adverse events were collected at weeks 1 and 24. ethical clearance the study protocol was reviewed and approved by the medical research ethics committee of dr. soetomo hospital. the trial was conducted in accordance with international rules of good clinical practice. written informed consent was obtained from the parent or legal guardian of each patient before study-related procedures were initiated. statistical analysis values are expressed as mean ± sd. the ttest for related samples was used for calculation of seizure frequency reduction and mcnemar test for eeg demonstration analysis. results patient characteristics from 18 consecutive patients, 15 subjects completed the study. three patients were excluded from analyses because no safety data were submitted. as shown in figure 1, all subjects with newly or recently diagnosed localization-related epilepsy received topiramate monotherapy with adjusted doses starting from 0.5 mg/kg body weight. most of the subjects were males with no history of familial seizure. three types of epilepsy were recorded, i.e generalized, partial secondarily generalized, and absence. all subjects showed epileptiform activities on eeg before topiramate treatment (table 1). topiramate efficacy on weekly evaluation of the patients, no seizures were observed during the titration period. one patient was recorded as suffering from frequent seizures and hospitalized for a few days. the patient then received additional aed to control the seizure. the percentage of seizure frequency reduction showed 83.7% patients being seizure-free in the 8th week, increasing 93% in the 24th week. one patient did not show improvement up to 20 weeks (table 2). figure 1. percentage of seizure frequency reduction (weeks) table 1. characteristics of the subjects (n=15) * mean ± sd 38 the mean seizure frequency before treatment was 2.71 ± 1.16. in the first week of the study, 100% of subjects did not suffer from seizures. one patient suffered from frequent recurrent seizures in the 4th week of treatment, but the decrement in seizure frequency was statistically significant. the decrements in seizure frequency were not significant in the 8th week of the study, because one patient had intractable frequent seizures. finally, the final observation indicated that the reduction in seizure frequency was statistically significant, despite the continued occurrence of seizures (figure 2). the topiramate dose started from 0.5 mg/ kg body weight once daily at night. the titration dose was then increased to 1 mg/kg body weight daily, divided in two doses. the dose was incremented to 1.3 mg/kg body weight in the 8th week, and finally to 1.4 mg/kg body weight in the 12th week, this dose being sufficient to control frequent seizures. the incremental dose was statistically significant (figure 3). eeg recordings all of subjects showed epileptiform activities on eeg before treatment. after 6 m o n t h s o f t r e a t m e n t , 3 p a t i e n t s s h o w e d normalized eegs. however, the change in eeg recording was not statistically significant with mcnemar test (table 3). table 2. distribution of seizure frequency in each patient g=general, p=partial secondarily generalized, a=absence, ab= abnormal, n=normal, w=week, *p= phenytoin added gunawan, suharso topiramate for childhood epilepsy 39 univ med vol. 31 no.1 adverse effects adverse effects were observed during the first weeks of the study. there were 33.3% of patients who suffered from poor appetite, while 7% of patients became drowsy. in the 8th week there were no more patient who were drowsy, while patients with poor appetite decreased to 7%. in the following weeks, there were no clinically apparent adverse effects. laboratory examination of blood samples collected from the patients at baseline and after 6 months of treatment, revealed no abnormalities. discussion monotherapy trials remain a complex and contentious issue with regard to new aeds. topiramate as a novel aed with broad-spectrum mechanism has been evaluated in previous studies for adjunctive therapy in partial-onset seizures and generalized tonic-clonic seizures. the efficacy results in adults have been very promising, suggesting that topiramate is one the most effective of the newer aeds.(7-9) trials of topiramate as monotherapy have been described in several studies with adults, but only in a few studies with children.(10) most of the studies presented seizure frequency reduction as a primary outcome measure. seizure frequency reduction may be considered a surrogate marker for disease improvement.(4) evaluation of seizure frequency reduction after topiramate monotherapy is shown in figure 1. significant seizure frequency p<0.05 p<0.05p<0.05 p<0.05 p>0.05 p<0.05 p<0.05 figure 2. seizure frequency reduction progress (weeks) figure 3. topiramate adjusted dose progress (mg/kg body weight) (weeks) p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 p<0.05 40 before treatment after treatment p value normal abn ormal 0 15 3 1 2 0,25* table 3. eeg results * statistically not significant with mcnemar reduction was noted in the early titration period to week 4. however, there was one patient suffering from intractable seizures, who had to be titrated to the maximum dose, with the addition of other aeds. this was the reason why the mean seizure frequency in week 8 was not significant, with 86.7% of patients being seizure free, 6.7% of patients with a reduction in seizure frequency of >50% from baseline, and 6.7% patients showing no improvement. the efficacy of topiramate in seizure frequency reduction was 93.7% in week 20. in this study, the types of epilepsy found were generalized epilepsy, simple partial secondarily generalized epilepsy, and absences. the study by valencia on various types of epilepsy found that among 7 patients receiving topiramate monotherapy, there was a seizure frequency reduction of more than 75% in 38.5% subjects.(11) our study showed that topiramate could reduce seizure frequency in absence pediatric epilepsy. this trial is consistent with previous research by cross on the efficacy of topiramate monotherapy in 5 subjects with absence seizures. topiramate seemed to be effective in reducing absence seizures.(12) a c c o r d i n g t o g l a u s e r, t i t r a t i o n i n monotherapy for children should be started from 0.5 mg to 1 mg/kg body weight once daily at night in the first week. the dose is then increased at intervals of 1-2 weeks, from 0.5 to 1 mg/kg body weight daily divided in two doses.(12) glauser’s study achieved a topiramate maintenance dose of 1.43 mg/kg body weight daily in week 20, which remained constant up to week 24. with this maintenance dose there was an increase in topiramate efficacy, with 93.7% of patients being seizure free. kugler has described the topiramate efficacy in babies with partial epilepsy, with dose-related achieved to 7.7 mg/kg body weight, while cross reported topiramate monotherapy efficacy in absence epilepsy to dose-related 5-12 mg/kg body weight.(13,14) a recommended target dose for topiramate monotherapy in children above 2 years is 3-6 mg/kg body weight daily. this research used maximal topiramate dose-related 6 mg/kg body weight daily. patients tend to be highly individual in their responses to aed therapy; therefore the best dose and even the best aed for a specific patient is often a matter of trial and error. the best dose is the lowest possible dose delivering the optimal balance between maximum seizure control, preferably seizure freedom, and minimum adverse effects in early as well as long-term therapy. gradual initiation of aeds, including topiramate, can help clinicians achieve these goals.(14,15) eeg is an essential component in the evaluation of epilepsy. the eeg provides important information about epileptiform discharges and is required for the diagnosis of specific electroclinical syndromes. aidyn reported that 37.1% epileptic patients showed epileptiform activities in their eeg.(16) the present trial demonstrated mild post treatment eeg alterations, which however were not statistically significant. a report on infants 9 to 12 months of age, the eeg recordings had been described in 2 partial epilepsy babies who received topiramate. interestingly, in one baby the eeg normalized in concert with the cessation of clinical seizure, while in the other the interictal eeg remained epileptiform despite the clinical efficacy.(13) the eeg alteration has been reported in 5 absence epilepsy children who received topiramate monotherapy for 6 weeks. it depicted 1 patient with normalized gunawan, suharso topiramate for childhood epilepsy 41 univ med vol. 31 no.1 eeg, 2 patients with confirmed reduction in epilepticform activity and 2 patients without any alterations.(14) the mild and nonspecific eeg abnormality has been found in 15% of the normal population, and approximately 10% epileptic patients have a normal eeg.(17) among the children in the present study there were generally mildly or moderately severe adverse events. most adverse effects were cns r e l a t e d , w i t h a p p e t i t e s u p p r e s s i o n a n d drowsiness being the most common side effects. the incidence of appetite suppresion was 70% on early treatment and tended to disappear without intervention. gurreiro reported that appetite suppresion was observed in 42% of patients with lennox-gastaut syndrome treated with topiramate,(12) and drowsiness in 7%. previous studies had shown that 47% patients with lennox gastaut syndrome suffered from drowsiness after topiramate administration.(18) most adverse effects events occurred early in treatment and related to rapid dose escalation and tended to disappear once patients became acclimatized to topiramate.(19) no acute or longterm idiosyncratic organ toxicity was observed w i t h t o p i r a m a t e m o n o t h e r a p y, w h i c h i s consistent with the laboratory results and the safety profile from earlier studies. there was no discontinuation of therapy due to adverse effects, and no deaths were reported during treatment. a s o u r f i n d i n g s i l l u s t r a t e , g r a d u a l initiation of topiramate sprinkle monotherapy can have a therapeutic effect during titration to effective maintenance dosages and enhance tolerability for pediatric epilepsy. nevertheless, long-term safety and possible adverse sequelae have not been established.(19,20) topiramate may represent a monotherapy option for children with epilepsy. there were several limitations in this study. the experimental methods should have included comparison of subjects to increase the power of the study. this study used patients with several types of seizures. there were some difficulties in determining the type of epilepsy from the eeg results; it would be better to use a defined type of epilepsy that has the highest response to topiramate. conclusions in this study there was a reduction of seizure frequency and no alterations in eeg r e c o r d i n g s a f t e r t o p i r a m a t e s p r i n k l e monotherapy. the most common adverse effects were drowsiness and appetite suppression. to p i r a m a t e a s a m o n o t h e r a p y i s h i g hl y effcicacious in childhood epilepsy. acknowledgement the authors gratefully acknowledge their indebtness to dr windu for contributing to the statistical analysis, and especially to the subjects and their parents who participated in the study. references 1. attumalil tv, sundaram a, varghese vo, vijayakumar k, kunju pa. risk factors of childhood epilepsy in kerala. ann indian acad neurol 2011;14:283–6. 2. saharso d, erny, poerwadi t. pedoman pengobatan epilepsi pada anak. in: permono b, soeparto p, kaspan mf, soegijanto s, soejoso da, narendra mb, editors. surabaya: surabaya intellectual club;2002.p.131-53. 3. das a, balan s, banerjee m, radhakrishnan k. drug resistance in epilepsy and the abcb1 gene. the cilinical perspective. indian j hum genet 2011;17:12-21. 4. french ja, kanner am, bautista j, abou-khalil b, browne t, harden cl, et al. efficacy and tolerability of the new antiepileptic drugs i: treatment of new onset epilepsy. neurology 2004; 62:1252-60. 5. gilliam fg, veloso f, bomhof mam, gazda sk, biton v, ter bruggen jp, et al. a dose-comparison trial of topiramate as monotherapy in recently diagnosed partial epilepsy. neurology 2003;60: 196-202. 6. brigo f. new anti-epileptic drug; overcoming the limits of randomized controlled trial. int j evid based health 2011;9:440-3. 42 7. faught e. topiramate in the treatment of partial and generalized epilepsy. neuropsychiatr dis treat 2007;3:811-21. 8. marson ag, al-kharusi am, alwaidh m, appleton r, baker ga, chadwick dw, et al. the sanad study of effectiveness of valproate, lamotrigine, or topiramate for generalized and unclassifiable epilepsy: an unblended randomized controlled trial. lancet 2007;369: 1016-26. 9. marson ag, al-kharusi am, alwaidh m, appleton r, baker ga, chadwick dw, et al. the sanad study of effectiveness of carbamazepine, gabapentin, lamotrigine, oxcarbazepine, or topiramate for treatment of partial epilepsy: an unblended randomized controlled trial. lancet 2007;369:1000-15. 10. kwon ys, jun yh, hong yj, son bk. topiramate monotherapy in infantile spasm. yonsei med j 2006;47:498-504. 11. valencia i, fons c, kothare sv, khurana ds, yum s, hardison hh, et al. efficacy and tolerability of topiramate in children younger than 2 years old. j child neurol 2005;20:66770. 12. glauser ta. topiramate. epilepsia 1999;40:7180. 13. kugler sl, sachdeo rc. topiramate efficacy in infancy. pediatr neurol 1998;19:320-2. 14. cross jh. topiramate monotherapy for childhood absence seizures: an open label pilot study. seizure 2002;11:406-10. 15. biton v, edwards kr, montouris gd, sackerllares jc, harden cl, kamin m. topiramate titration and tolerability. ann pharmacol 2001;35:173-9. 16. aidyn k. utility of electroencepalography in the evaluation of common neurologic condition in children. j child neurol 2003;18:394-6. 17. panayiotopoulos cp. the epilepsies, seizures, syndromes and management. oxford: bladon medical publishing;2005.p.100-25. 18. crumrine pk.management of seizures in lennox gastaut syndrome. paediatr drugs 2011; 13:107-18. 19. mirza n, marson ag, pirmohamed m. effect of topiramate on acid-base balance: extent, mechanism and effects. br j clin pharmacol 2009;68:655-61. 20. kim jm, kwon s, seo h, choe bh, cho mh, park sp. longterm effectiveness and tolerability of topiramate in children with epilepsy under the age of 2 years: 4 –year follow up. j korean med sci 2009;24:1078-82. gunawan, suharso topiramate for childhood epilepsy september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 predictors of breastfeeding self-efficacy in pregnant adolescents funda özdemir1 , seda karaçay yıkar2 , and evşen nazik2 abstract background breastfeeding is critical for infant health and development globally. current knowledge and attitude of future parents will significantly influence breastfeeding practices. the study was conducted to determine the levels and determinants of breastfeeding self-efficacy among primiparous and secundigravid adolescents. methods a cross-sectional study was performed involving 54 primiparous and secundigravid adolescents in antenatal clinics of hospitals affiliated to the ministry of health in adana, turkey. they were aged between 16 and 19 years, had a single living fetus, did not have pregnancy complications, were in their third trimester of pregnancy, attended an antenatal clinic, and agreed to participate in the study. data were collected using a personal information form and the antenatal breastfeeding self-efficacy short form scale (bses-sf). kruskal-wallis variance analysis, mann whitney u and multiple linear regression analysis test were used to analyze the data. results mean age of the pregnant adolescents was 18.28 ± 0.79 years, and 31.5% had primary education, 96.3% were not working, 55.6% lived in a nuclear family, and 51.9% had not received breastfeeding education. the total bses-sf mean score was 55.37 ± 12.84. breastfeeding education has a statistically significant effect on breastfeeding self-efficacy of pregnant adolescents (p<0.05). regular antenatal care, breastfeeding training status, breastfeeding education source variables and breastfeeding selfefficacy variable of women significantly predicted negatively (p<0.05). conclusions breastfeeding education source was the most influential predictor variable of bses. there is a need for nursing initiatives to enhance the adolescents’ antenatal breastfeeding self-efficacy levels. keywords: breastfeeding, self-efficacy, pregnant, adolescents, nursing 1ankara university, faculty of nursing, obstetrics and gynecology nursing department, ankara/ turkey 2çukurova university, faculty of health sciences, obstetrics and gynecology nursing department, adana/turkey *correspondence: seda karaçay yıkar çukurova university, faculty of health sciences, obstetrics and gynecology nursing department, adana /turkey phone:+903223386484, fax:+903223386970 e-mail: sedakrcyyikar@gmailcom date of first submission, september 26, 2022 date of final revised submission, december 8, 2022 date of acceptance, december 14, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 254 doi: http://dx.doi.org/10.18051/univmed.2022.v41.254-262 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1384 original article cite this article as: özdemir f, yikar sk, nazik e. predictors of breastfeeding selfefficacy in pregnant adolescents. univ med 2022;41:254-62. doi: 10.18051/ univmed.2022.v41:254-262 http://dx.doi.org/10.18051/univmed.2022.v41.254-262 https://univmed.org/ejurnal/index.php/medicina/article/view/1384 https://orcid.org/0000-0002-7483-6690 https://orcid.org/0000-0002-8893-9391 https://orcid.org/0000-0003-1885-5376 255 univ med vol. 41 no. 3 introduction ad o le sc e nc e i s t h e t r a ns it i on f r om childhood to adolescence, and includes ages between 10 to 19 years.(1) every year, an estimated 21 million girls aged between 15 and 19 years, and 2 million girls under the age of 15, become pregnant in developing regions.(2,3) in addition, approximately 16 million girls aged between 15 to 19, and 2.5 million girls under the age of 16, give birth in developing regions.(3) adolescent fertility rate represents children born alive per 1000 women aged between 15 to 19. according to turkish statistical institute’s data from 2017, adolescent fertility rate was 21 in 1000. in other words, every 21 out of 1000 women aged between 15 and 19 years of age had given birth.(4) there are many p hysiologica l and psychosocial changes during adolescence. in addition to these changes, pregnant adolescents are preparing to adapt to new and different situations, and to take new responsibilities during the pregnancy, childbirth and postnatal period.(5) one of the most important responsibilities of adolescents to take during the postpartum period is related to the feeding of the baby. a major sociodemographic factor regarding breastfeeding is age.(6) adolescent mothers are a group that should be considere d as a priority in breastfeeding. a cross-sectional study involving 392 adolescents showed that 290 (74%) had poor knowledge regarding breastfeeding and that there was a statistically significant association between high level of knowledge and positive attitude.(7) özsoy (8) found that adolescent mothers had more ne gative behaviors and attitudes towards breastfeeding and needed more information and help than adult mothers. one of the issues that needs to be addressed first in support of breastfeeding in adolescents is the perception of breastfeeding self-efficacy. according to dennis and faux,(9) breastfeeding self-efficacy perception affects the mother’s ability to breastfeed, the effort she will make for the baby, as well as her emotional thoughts and feelings.(9) yenal et al.(10) found a significant positive relationship between breastfeeding selfefficacy perception and breastfeeding success in their studies. tokat and okumuş (11) found that antenatal education was effective in improving breastfeeding self-efficacy perception and breastfeeding success. in a study developed in china among adult postpartum women during pregnancy and the associated hospitalization, low self-efficacy was found. nevertheless, the selfconfidence to breastfeed among adolescent mothers has been hardly explored.(12) in the present study, the aim was to determine the breastf eeding self-e fficacy of pr egnant adolescents in a province of the mediterranean region. determining the perception of breastfeeding self-efficacy of adolescents is important in shaping the provided healthcare. this study was carried o ut to determine the breastfeeding self-efficacy perceptions in pregnant adolescents. methods research design this study used a cross-sectional design and was performed in antenatal clinics of hospitals affiliated to the ministry of health in adana, turkey, between february and june 2015. in the study, all adolescent pregnant women who came to the outpatient clinic and met the research criteria in the determined time interval were included in the study. research subjects the study was conducted with 54 primiparous and secundigravid adolescents, who were aged between 16 to 19 years, had a single living fetus, did not have pregnancy complications, were in their third trimester of pregnancy, attended an antenatal clinic, spoke turkish, and agreed to participate in the study. data collection the data were collected using a personal i nf or ma tio n f o r m a nd th e a nt e n a t a l 256 özdemir, yikar, nazik breastfeeding self-efficacy in pregnant adolescents breastfeeding selfefficacy scale short form (bses-sf). the personal information form was prepared by the researchers and consisted of questions re lated t o socio-demogr aphic characteristics (age, age of marriage, educational status, occupational status, social security, income level, type of family, place of residence, age of husband, occupational status of husband) and obstetric characteristics (planned status of pregnancy, regular antenatal care, having a health problem during pregnancy, breastfeeding training status). the antenatal breastfeeding self-efficacy short form scale was developed by dennis et al.(13)and is a 14-item self-report instrument to measure breastfeeding confidence. all items are measured on a 5-point likert-type scale, with 1 representing no conf idence at all, and 5 representing greatest confidence. all items are presented positively, and scores are summed to produce scores that range from 14 to 70, with higher scores indicating higher levels of breastfeeding self-efficacy. dennis et al.(13) stated that it is also possible to use it in the antenatal period by using the expression “future time” in the items of bses-sf. the bses-sf turkish reliability and validity study was made by aluş tokat et al.,(14) who found that the cronbach alpha value was 0.86. statistical analysis the statistical analysis were performed using spss for windows version 22.0. descriptive statistics, kruskal-wallis variance analysis, and mann-whitney u test, and multiple linear regression were applied for analyzing the data. significant variables (p<0.05) were included in the multiple linear regressions to investigate the predictors of the breastfeeding self-efficacy. the statistical significance level was accepted as p<0.05. ethical consideration before conductin g the study, the investigators obtained written approval from the ethics board of school of medicine, cukurova university (28.10.2014/24/2), written approvals from the institutions in which the study was to be performed, and verb al consent from the participants. to obtain the adolescents’ verbal consent, all participants were informed of the purpose of the study and were assured that the collected information would be used solely for scientific purposes, be kept confidential and not be shared by others than the researchers. a faceto-face interview method was used by the researchers to administer the questionnaires. the interviews lasted approximately 15 minutes. results the demographic characteristics of the subjects are shown in table 1. the results showed that 83.3 % of the pregnant adolescents were 1819 years old, and 55.6 % were married at the age of 1819 years. furthermore, 50.0% of the adolescents had completed secondary school, 88.9% had socia l se cur ity, 96.3% were unemployed, 68.5% reported that their income equaled their expenditure, 55.6% lived in nuclear families, and 51.9% lived in the city center. the results also showed that 50.0% of the husbands were at least 25 years old, 90.7% of the husbands were employed, and 68.5% of the adolescents chose their partner through love. the average total score of bses-sf was 55.37 ± 12.84 (table 1). no significant difference in mean bses-sf scores between subgroups of age, age of marriage, educational status, having social security, occupational status, economic status, type of family, age of husband, occupational status of husband and type of marriage (p>0.05) (table 2). a statistically significant difference was found in mean bses-sf between subgroups of attending regular antenatal care, attending breastfeeding education, and receiving the education from a healthcare personnel (p<0.05) (table 3). 257 univ med vol. 41 no. 3 characteristics n (%) age (years) mean ± sd 16-17 18-19 age of marriage (years) 15-17 18-19 education primary school secondary school high school having social security yes no occupational status employed unemployed economical level income ≥expenditure income=expenditure income<expenditure family type nuclear large place of residence village town city age of husband (years) mean ± sd 18 – 24 ≥25 occupational status of husband employed unemployed type of marriage arranged love planned status of pregnancy yes no regular antenatal care yes no breastfeeding training status yes no breastfeeding education source (n=27) healthcare personnel family/relative book/magazine/internet did not receive bses-sf (mean ± sd) 18.28 ± 0.78 9 45 24 30 17 27 10 48 6 2 52 10 37 7 30 24 5 21 28 24.56±3.16 27 27 49 5 17 37 45 9 48 6 27 27 22 4 1 27 55.37 ± 12.84 16.7 83.3 44.4 55.6 31.5 50.0 18.5 89.9 11.1 3.7 96.3 18.5 68.5 13 55.6 44.4 9.3 38.9 51.9 50 50 90.7 9.3 31.5 68.5 83.3 16.7 88.9 11.1 50.0 50.0 40.7 7.5 1.8 50.0 table 1. the sociodemographic and obstetrics characteristics of the participant women (n=54) note ; bese-sf : breastfeeding self-efficacy scale short form 258 sociodemographic characteristics mean scores of breastfeeding self-efficacy scale test and p value age kw=1.446 p=0.229 16-17 49.88 ±0.00 18-19 49.00±15.91 age of marriage kw=0.478 p=0.489 15-17 54.00±14.18 18-19 56.46±11.79 education kw=0.542 p=0.763 primary school 57.11±12.75 secondary school 54.77±13.47 high school 54.00±12.19 having social security mw-u= 117.000 p=0.475 yes 55.89±12.84 no 51.16±13.21 occupational status mw-u= 45.000 p=0.771 employed 53.00±12.72 unemployed 55.46±12.96 economical level kw= 2.594 p=0.273 income≥expenditure 57.40±13.58 income=expenditure 55.94±12.62 income<expenditure 49.42±13.18 family type mw-u= 309.00 p=0.372 nuclear 57.06±11.73 large 53.25±14.06 place of residence kw= 6.655 p=003 village 61.00±13.76 town 49.57±14.39 city 58.71±9.93 age of husband mw-u= 282.000 p=0.151 18 – 24 54.18±10.89 25 age and over 56.55±14.65 occupational status of husband mw-u= 110.000 p=0.728 employed 55.08±13.22 unemployed 58.20±8.67 type of marriage mw-u= 299.500 p=0.779 arranged 54.11±13.74 love 55.94±12.56 table 2. comparison of mean scores of breastfeeding self-efficacy scale of pregnant adolescents according to their sociodemographic characteristics note : kw : kruskal-wallis; mw-u : man whitney-u no statistically significant difference was found between place of residence of indigenous wo me n a nd br e a s t f e e d i ng e x tr a c t s . t he variables consisting of regular antenatal care, breastfeeding training status, breastfeeding education source and breastfeeding self-efficacy of women significantly predicted negatively (p<0.05). breastfeeding education source was the most influential predictor of breastfeeding self-efficacy (beta=-0.541) (table 4). discussion in this study, the bses-sf mean scores of pregnant adolescents was 55.37 ± 12.84. a study by alus tokat et al.(14) showed that bses-sf mean scores of pregnant adolescents was 51.72 ± 7.69. in another study conducted in turkey, the mean bses-sf scores of pregnant adolescents was 50.30 ± 9.30.(11) otsuko et al.(15) found the mean bses-sf scores to be 42.4, and brando et özdemir, yikar, nazik breastfeeding self-efficacy in pregnant adolescents 259 univ med vol. 41 no. 3 obstetric characteristics mean scores of breastfeeding self-efficacy scale test and p value planned status of pregnancy mw-u= 191.000 p=0.788 yes 55.80±12.66 no 53.22±14.28 regular antenatal care mw-u= 78.500 p=0.07 yes 56.70±11.88 no 44.66±16.28 breastfeeding training status mw-u= 141.500 p=0.001 yes 61.70±8.62 no 49.03±13.36 breastfeeding education source (n=27) kw= 19.736 p=0.001 healthcare personnel 63.86±6.14 family/relative 53.00±13.85 book/magazine/internet 53.00±0.00 did not receive 48.88±13.33 table 3. comparison of mean scores of breastfeeding self-efficacy scale according to obstetric characteristics of pregnant adolescents note : kw : kruskal-wallis; mw-u : man whitney-u predictors β beta p value place of residence regular antenatal care breastfeeding training status breastfeeding education source 0.072 -0.074 -0.201 -0.824 0.145 -0.297 -0.498 -0.541 0.294 0.029 0.000 0.000 table 4. predictors of breastfeeding self-efficacy among pregnant adolescents (n=54) note: β = regression coefficient; beta = standardized regression coefficient al.(16) obtained the figure of 57.51 ± 8.0 for the bses-sf mean scores of portuguese pregnant adolescents. in another study, the mean bsessf scores of 306 adults and 94 adolescents who were in the early postpartum period were investigated, high self-efficacy levels were found in both groups, and no statistically significant difference was found between the groups.(17) no significant difference was found between age, age of marriage, educational status, having social security, occupational status, economic status, type of family versus bses-sf mean scores of pregnant adolescents. gerçek et al.(18) studied first day postpartum women and found no statistically significant difference in the bsessf mean scores between subgroups of age, educational status, occupational status, economic status, length of marriage. in the study by ince et al.,(19) where self-efficacy levels of mothers and the factors affecting breastfeeding were assessed, no significant difference in the bsessf sc o r e s be t w e e n s ub gr ou p s of a ge , educational level, occupational status, perceived economic status. adolescents with planned pregnancies had higher total bses-sf scores, b ut t h e d if f e r e nc e wa s n ot s t at i st i c a l l y significant. akkoyun and arslan obtained similar results in their study.(20) in a study by aydyn and aba, adolescents with planned pregnancies had higher levels of self-efficacy, but the difference was not statistically significant.(21) pregnant adolescents who attended regular antenatal care, had higher self-efficacy scores. the results of a study by yang et al.(22) were similar to our findings. tucker et al.(23) and wambach et al.(24) both found that women who received antenatal support from healthcare personnel had higher levels of self-efficacy. 260 pregnant adoles cents receiving breastfeeding education had higher mean bsessf scores than adolescents who did not receive any education, and the difference was statistically significant. when investigating the source of the education and the mean bses-sf scores, the highest mean score s were obta ined by adolescents who received the education from healthcare personnel. there are literature studies suggesting that education is effective in improving breastfeeding self-efficacy. myzrak et al.(25) investiga ted the e ffe cts of breastfeeding education given to primipara women during antenatal period, on the mothers’ breastfeeding self-efficacy perceptions and success, and found that the breastfeeding self-efficacy scores were 48.73 during the antenatal period prior to the education, and 66.22 after the education, 8 weeks postpartum. tokat and okumuş found that antenatal education based on strengthening the perception of breastfeeding self-efficacy was effective in improving breastfeeding selfefficacy.(11) in a study by karagöz,(26) where the effects of antenatal breastfeeding education on breastfeeding success and self-efficacy were investigated, repeated tests were conducted to both the experiment and the control groups on the first day and at one month, four, and six months postpartum, and a statistically significant difference was found between the postpartum self-efficacy scores of the experimental and control groups. chan et al.(27) found that education related to self-efficacy given during the antenatal period affected the scores of the second week postpartum, which were significantly higher than the scores prior to education. liu et al.(28) investiga ted the e ffe cts of breastfeeding education on breastfeeding self-efficacy four and eight wee ks postpar tum and found that breastfeeding self-efficacy was significantly higher after this period. the findings of our study are in accordance with the literature, and the results indicate tha t education improves breastfeeding self-efficacy positively. this study has several limitations. first, this study included 54 antenatal care women at a single hospital in southern turkey, limiting the generalizability of the results, since breastfeeding is a long process. the fact that breastfeeding was not evaluated in the postpartum period is one of the limitations of the study. despite its limitations, this study may be helpful in guiding further research. adolescence is one of the importa nt transition processes, which is one of the phases of a wome n’s life. pregnancy is not recommended during this period as it brings risks to the mother and fetus. it is very important to determine the self -efficacy of adolescent pregnant women in or der to continue breastfeeding effectively in the postpartum period, as in every life period. conclusion adolescents who received education regarding breastfeeding had higher levels of breastf eeding self-e fficacy in our study. therefore, it is important that nurses and midwives guide adolescents to pregnancy education classes during the antenatal cares. pregnancy education classes should cover subjects regarding breastfeeding in detail. acknowledgments the authors grateful to all the patients who participated in this study. conflicts of interest nothing to declare. contributors all authors have contributed significantly to this article to design of the work; analysis, or interpretation of data for the work and all authors are in agreement with the content of the manuscript. authors’ contributions sky collected the data. fo and en analyzed the data. fo, en, sky drafted and finalized the manuscript. all özdemir, yikar, nazik breastfeeding self-efficacy in pregnant adolescents 261 univ med vol. 41 no. 3 authors contributed to the design of the study, and reviewed the manuscript. all authors have read and approved the final manuscript. references 1. world health organization. adolescent health. geneva: world health organization;2022. 2. united nations population fund (unfpa). girlhood not motherhood: preventing adolescent pregnancy. new york: united nations population fund (unfpa);2015. 3. neal s, matthews z, frost m, fogstad h, camacho av, laski l. childbearing in adolescents aged 12-15 years in low source countries: a neglected issue. new estimates from demographic and household surveys in 42 countries. acta obstet gynecol scand 2012;91:1114-8. doi: 10.1111/j.1600-0412.2012.01467.x. 4. türkiye ýstatistik kurumu (tüýk) doðum ýstatistikleri;2017. 5. taþkýn l. obstetrics and women’s health nursing. extended 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vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 hepatoprotective activity of averrhoa bilimbi l. fruit extract on carbon tetrachloride-induced acute liver faiure in wistar rats rachellicya ristioni sihaloho1, chodijah1, titiek sumarawati1,3,4, joko wahyu wibowo1, siti thomas zulaikhah1 , agung putra1,2,4* , nurul hidayah4 , shabrina syifa ghaissani4 , and mohammad ariq nazar4 abstract background acute liver failure (alf) is a state of rapid and progressive deterioration of liver function. continuous exposure to chemicals and viruses can increase reactive oxygen species (ros) which leads to prolonged inflammation due to the production of tumor necrosis factor-alpha (tnf-α) thus inhibiting the production of platelet-derived growth factor (pdgf). the objective of this study was to evaluate the effect of administration of averrhoa bilimbi l. fruit extract on pdgf levels and tnf-α levels in carbon tetrachloride (ccl4)-induced alf rats. methods this study used a post-test-only control group design involving 20 wistar rats. they were randomized into 4 groups, namely sham, control, t1, and t2. group t1 was exposed to ccl4 with the administration of a. bilimbi fruit extract at a dose of 500mg/kgbw, while, group t2 was exposed to ccl4 and given a. bilimbi fruit extract of 750 mg/kgbw. on the 15th day, the serum was analyzed to determine the levels of pdgf and tnf-α using elisa. results the highest mean pdgf level in the control group was 146.60±15.36 pg/ml, while the highest mean tnf-α level in group t1 was 40.11±4.44 pg/ml. the one-way anova test showed that there were significant differences in tnf-α (p=0.002) and pdgf (p=0.000) levels between the study groups.. conclusion the administration of a.bilimbi l. fruit extract affected pdgf and tnf-α levels in ccl4-induced alf rats. the present study revealed that a. bilimbi fruits have significant hepatoprotective activity in experimental wistar rats. keywords: acute liver failure, averrhoa bilimbi l., reactive oxygen species, rats 1postgraduate biomedical science study program, faculty of medicine, universitas islam sultan agung (unissula), semarang, indonesia. 2department of pathological anatomy, faculty of medicine, universitas islam sultan agung (unissula), semarang, indonesia. 3department of biochemistry, faculty of medicine, universitas islam sultan agung (unissula), semarang, indonesia. 4stem cell and cancer research laboratory, semarang, indonesia *correspondence: assoc. prof. dr. dr. agung putra, m.si.med postgraduate biomedical science study program, faculty of medicine, unissula jl. raya kaligawe km. 4 semarang email: dr.agungptr@gmail.com orcid id: 0000-0003-4261-9437 date of first submission, september 7, 2022 date of final revised submission, march 26, 2023 date of acceptance, april 10, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article cite this article as: sihaloho rr, chodijah, sumarawati t, wibowo jw, zulaikhah st, putra a, hidayah n, ghaissani ss, nazar ma. hepatoprotective activity of averrhoa bilimbi l. fruit extract on carbon tetrachloride-induced acute liver faiure in wistar rats. univ med 2023;42:61-9. doi: 10.18051/univmed.2023.v42:61-69 doi: http://dx.doi.org/10.18051/univmed.2023.v42:61-69 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1370 61 mailto:dr.agungptr@gmail.com https://orcid.org/0000-0001-5593-9056‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬‬ https://orcid.org/0000-0003-4261-9437 https://orcid.org/0000-0002-0372-0463 https://orcid.org/0000-0003-4677-9139 http://dx.doi.org/10.18051/univmed.2023.v42:61-69 https://univmed.org/ejurnal/index.php/medicina/article/view/1370 62 sihaloho, chodijah, sumarawati , et al averrhoa bilimbi l. fruit extract introduction acute liver failure (alf) is manifested by progressive necrosis of the hepatocytes, followed by impaired liver functions and elevated serum transaminase levels.(1–3) persistent necrosis of hepatocytes induces activation of hepatic stellate cells (hscs) leading to excessive deposition of extracellular matrix (ecm).(4,5) during hepatic fibrogenesis, there is an increase in inflammatory factors such as tumor necrosis factor-alpha (tnf-α) and excessive accumulation of the hepatic extracellular matrix which is regulated by fibrogenic factors, such as platelet-derived growth factor (pdgf).(6,7) the late stage of chronic progressive fibrosis results in cirrhotic hepatic disease (chd), which accounts for approximately 55% of the 1.4 million liverdisease-related deaths reported each year worldwide.(8,9) currently, liver transplantation is the most effective therapy for patients with advanced liver disease, including chd; however, the limited availability of liver donors and the low survival rates among liver transplant patients remain a serious problem.(5,9–11) therefore, novel alternative lf treatments that can be used in place of transplantation must be explored. on the other hand, many previous studies have reported that averrhoa bilimbi l. has oxidative radical scavenging and immunosuppression properties which is mainly due to phenolic substances such as terpenoids, phenols, tannins, and flavonoids.(12-14) tumor necrosis factor-α (tnf-α) is a pleiotropic cytokine associated with a variety of physiological and pathological conditions, including cytotoxicity, differentiation, growth stimulation, immun e modulation, and proinflammatory activity.(5,15,16) tumor necrosis factor-α exacerbates liver damage following exposure to toxic levels of acetaminophen or carbon tetrachloride (ccl4) and plays a role, as well, in liver repair following ccl4 exposure by stimulating hepatocyte growth factors such as interleukin-6 (il-6) and transforming growth factor-α (tgfα) and allowing rapid hepatocyte proliferation.(5,6,17) elevated levels of liver tnfα occur in acute and chronic liver diseases, including fulminant hepatic failure, viral hepatitis, alcohol abuse, metabolic disease, autoimmunity, and biliary obstruction, as well as following chemical-induced hepatotoxicity.(18,19) chronic liver injury can promote fibrosis, characterized by increased hepatic stellate cell proliferation and their transdifferentiation into myofibroblast-like cells, leading to the extracellular matrix and fibrilforming collagen accumulation.(20,21) fibroblast proliferation and collagen production are enhanced by growth factors such as tgf-β1 and pdgf.(22,23) averrhoa bilimbi l. fruits are rich in phytochemical compounds which exert potent antioxidant activity either by scavenging the reactive molecules or by enhancing antioxidant molecules and enzymes. (1 4,24 ) preliminar y phytochemical studies revealed the presence of alkaloids, carbohydrates, phenols, flavonoids, saponins, and tannins.(14,25) a previous study reported that treatment with averrhoa bilimbi l. fruit extract has also optimally maintained the levels of the inflammatory mediators, tnf-α, il6, and il 1β, in an acetic acid-induced ulcerative colon r at model by down-regulating the cyclooxygenase-2 (cox-2) and inducible nitric oxide synthase (i-nos) levels in the colon.(12) another previous report also demonstrated that methanolic extract of a. bilimbi leaves showed significant hepatoprotective and antioxidant activity against ccl4-induced hepatotoxicity by increasing the gsh levels and reducing the thiobarbituric acid reactive substances (tbars) levels.(24) in addition, administration of methanolic extract of averrhoa bilimbi at two different dose levels (250 and 500 mg/kgbw) attenuated the increased levels of the serum enzymes produced by ccl4 and caused a subsequent recovery towards normalization.(24,26) a study to evaluate the hepatoprotective activity of averrhoa bilimbi fruit in wistar albino rats following acetaminophen intoxication, showed that the liver marker enzymes serum glutamate oxaloacetate transaminase (sgot), serum glutamate pyruvate transaminase 63 (sgpt), alkaline phosphatase (alkp) were found to be two fold increased in the control group, while pre-treatment with a. bilimbi inhibited the increase of these enzymes in serum.(27) however, in vivo studies to provide scientific evidence regarding the potential effect of averrhoa bilimbi l. fruit extract in regulating tnf-α and pdgf levels in alf need to be explored, such that they may be useful for improving human health. therefore, this study was designed to investigate the effect of different doses of averrhoa bilimbi l. fruit extract administration on pdgf levels and tnf-α levels in ccl4-induced alf rats. methods research design this experimental study was of completely randomized design to compare the effects of treatment on the experimental and control groups at the end of the treatment. the research was conducted in june–august 2022 at the stem cell research laboratory (laboratorium penelitian sel punca lpsp), faculty of medicine, sultan agung islamic university, semarang. maceration and extraction of averrhoa bilimbi l. fruits the fruits of a. bilimbi l. were collected, air dried, and ground into a fine powder by means of mortar and pestle. extraction from the fruits was done according to the method described by singh et al.(28) the powder (25 g) was extracted with 250 ml of methanol (95% v/v) in a soxhlet appara tus. t he re ma ining methanol was evaporated using a rotary evaporator. the obtained thick semi-solid crude extract was stored at 2–4ºc for further use. ccl4 -induced acute liver failure model the animals were obtained from and certified to be healthy by the agricultural and fishery service of salatiga under no. 524.3/0211/ 421. the calculation of the estimated sample size was performed according to the federer formula: (t-1) (r -1) >15 (t=number of trea tments; r=number of replications); (4-1)(r-1) >15; 3r– 3>18; 3r>18; r>6. therefore, the minimal replication per group is 6 and the total of 4 groups is 24 animals. twenty four male wistar rats (5– 6 weeks old and weighing 250–300 g) were fed ad libitum and housed in plastic cages with mesh wire covers, at a room temperature of 24°c, wi th a 12 -h li gh t-dar k c yc le (l ab ora to ry standard). all rats were intra-peritoneally (ip) injected with 0.1 ml/kg ccl4 (sigma-aldrich), dissolved in olive oil (1:1), twice weekly, for two weeks, to induce alf.(15) to evaluate liver function, an assessment of sgot and sgpt was performed. ext r ac t o f av e r r hoa bi l i m bi l. o r a l administration after a week of acclimatization, rats were randomly divided using a computer into the following five groups and the treatments were administrated by an animal laboratory operator: untreated (sham), ccl-4 treated with olive oil/ vehicle (control group), ccl-4 treated with a low dose (500mg/kgbw) of averrhoa bilimbi l. fruit extract (t1), and ccl-4 treated with a high dose (750mg/kgbw) of averrhoa bilimbi l. fruit extract (t2). each group consisted of f i ve r a t s. t h e i n te r ve nt i o n d os e w a s administered orally daily for up to 14 days. on day 15, all rats were terminated and periorbital blood was collected. pdgf and tnf-α levels using elisa the blood of the rats was harvested via periorbital venous plexus bleeding under general anesthesia on days 0 (pre-treatment), 2, and 7, and the serum was collected by centrifugation a t 4 0 c. we me a su r e d pdg f and t nf -α concentrations by means of enzyme-linked immunosorbent assay (elisa) kits, based on the manufacturer ’s instructions (fine test, wuhan, china) and according to a standard c u r ve c ons t r uc te d f o r e a c h a s s a y. t he colorimetric absorbance was recorded at a wavelength of 450 nm. univ med vol. 42 no. 1 64 sihaloho, chodijah, sumarawati , et al averrhoa bilimbi l. fruit extract sgot and sgpt measurement sgot and sgpt were measure d to determine liver functions at days 0 (pre-treatment) and 7. blood samples were collected from periorbital veins under anesthesia using a solution of ketamine + xylazine (100 mg/kgbw + 5 mg/ kg) (alfasan, the netherlands). the serum was collected by centrifugation at 4ºc. sgot and sgpt serum levels were measured using an automatic analyzer (bt 3000 plus, italy). statistical analysis statistical analyses were accomplished with the ibm spss statistics for windows version 22.0 (ibm corp, armonk, ny). all data were presented as mean ± standard deviation (sd). the data obtained were collected, compiled, and tested for normality with the shapiro-wilk test and for homogeneity with levene’s test. data analysis was performed using one-way anova and c ontinued with the least significant difference (lsd) post hoc test using a p-value <0.05. ethical clearance the ethics committee of sultan agung islamic university approved all procedures performed in this study under number 817/vi/ 2022/ komisi bioetik. results levels of sgpt and sgot the present study revealed that the levels of serum glutamic pyruvic transaminase (sgpt) and serum glutamic-oxaloacetic transaminase (sgot) increased significantly after ccl4 exposure, indicating considerable hepatocellular injury (figure 1). averrhoa bilimbi l. fruit extract decreases tnf-α and pdgf levels in ccl4-induced alf models ccl4 has been shown to induce apoptosis of hepatocytes by increasing the proinflammatory factors, such as il-1, ifn-γ, and tnf-α. the level of tnf-α was measured to examine the effect of averrhoa bilimbi l. fruit extract on the ccl4-induced alf rat liver tissue. the results of the comparative analysis showed that the tnf-α level was increased after ccl4 induction (control group) (36.12±7.11 pg/ml), and increased further after being treated with the low dose averrhoa bilimbi l. fruit extract (group t1) (40.09±4.86 pg/ml), but was significantly reduced with high dose averrhoa bilimbi l. fruit extract (group t2) (27.09±4.46 pg/ml) (figure 2a, tables 1 and 2). these results suggest that the tnf-α level may be regulated by the dosefigure 1. level of sgpt and sgot in rats after two weeks of exposure to ccl-4 (n=6 ± sd); sham=healthy rats; ccl-4=ccl-4 exposure;*p<0.05 65 dependent effect of averrhoa bilimbi l. fruit extract. to determine the role of averrhoa bilimbi l. fruit extract in alf regeneration, the pdgf levels were measured using elisa. the levels of pdgf in averrhoa bilimbi l. fruit extract groups, both low dose and high dose, were significantly decreased (t1=41.07±17.53; t2=30.91±3.74) compared with the control group (146.3±18.26) (p<0.05) (figure 2b, tables 1 and 2). in addition, the level of pdgf expression in the control group was high due to continued inf lammation. the pr esent study had demonstrated the role of hepatoprotective activity of averrhoa bilimbi fr uit extrac t in the experimental animal system. the tnf-α levels and the pdgf levels were decreased in the a. bilimbi treated groups, when compared with the control group. discussion the results of this study showed that averrhoa bilimbi l. fruit extract might reduce tnf-α and pdgf levels in ccl4-induced alf rats. those r esults sugge st that the hepatoprotective effect of averrhoa bilimbi l. may be produced by reducing the tnf-α and pdgf levels in ccl4-induced alf rats. carbon tetrachloride (ccl4) induced toxic liver injury is a well-characterized model of hepatic fibrosis. ccl4 is metabolized by the mitochondrial monooxygenase system (cytochrome p450 2e1) to its active metabolite, an unstable trichloromethyl (ccl3) free radical, and then this active metabolite immediately transforms into trichloromethyl peroxyl (ccl3o2).(17,29,30) these free radicals damage the cell membrane via peroxidative degradation of the fatty acids from phospholipids of the cell membra ne and endoplasmic reticulum.(3,5) subsequently, this process results in the fragmentation of the lipid peroxide radicals, lipid hydroperoxides, and other products, each acting as an active oxidizing a gent. (4 ) consequently, the structure of the cell membrane and intracellular organelles completely deteriorate, and the structural damage expands.(23) these processes are immediately followed by the infiltration of inflammatory cells and the release of various cytokines and growth factors.(31,32) moreover, the level of tnf-α consistently rises during the ongoing cci4-induced liver toxicity.(5) in this study, the decrease in tnf-α level may have been due to the scavenging of free radicals, cytokines experimental groups p value sham (n=6) control (n=6) t1 (n=6) t2 (n=6) tnf-α (pg/ml) pdgf (pg/ml) 25.18±5.01 65.99±5.93 36.12±7.11 146.3±18.26 40.09±4.86 41.07±17.53 27.09±4.46 30.91±3.74 0,002* 0,000* table 1. distribution of tnf-and platelet-derived growth factor by experimental groups note: values are mean ± sd, tnf-α: tumor necrosis factor-α; pdgf: platelet-derived growth factor;* anova test; t1=ccl4 treated with a low dose of averrhoa bilimbi l. fruit extract, t2= ccl-4 treated with a high dose of averrhoa bilimbi l. fruit extract; *p<0.05 group compared group tnf-α (p value) pdgf (p value) sham control 0.007* 0.000* t1 0.001* 0.008* t2 0.611 0.000* control t1 0.349 0.000* t2 0.020* 0.000* t1 t2 0.003* 0.105 table 2. post-hoc lsd result tnf: tumor necrosis factor; pdgf: platelet-derived growth factor; sham: healthy rats; control: ccl-4 treated with olive oil/vehicle, t1=ccl-4 treated with a low dose of averrhoa bilimbi l. fruit extract, t2= ccl-4 treated with a high dose of averrhoa bilimbi l. fruit extract; *p<0.05 univ med vol. 42 no. 1 66 sihaloho, chodijah, sumarawati , et al averrhoa bilimbi l. fruit extract especially nitric oxide (no). nitric oxide is one of the abundant free radicals and is highly formed during inflammation, capable of damaging proteins, lipids, and dna.(33,34) a piece of previous evidence has shown that the antioxidant activity of averrhoa bilimbi l. fruit extract is proven from its high level of no scavenging activity.(12) another previous study also revealed that averrhoa bilimbi l. fruit extract has potential antioxidant activity by reducing the no levels and enhancing the sod enzyme.(13) reducing no levels and increasing sod may inhibit oxidative stress in microenvironment hepatocytes and reduce immune cell recruitment, which sustainably the pro-inflammatory molecules reduction, primarily tnf-α.(12) this finding is also supported by another study that demonstrated averrhoa bilimbi l. fruit extract also optimally maintained the levels of inflammatory mediators, such as tnf-α, il-6, and il-1β in ulcerative colitis rats.(12) acute liver failure is also characterized by impairment of the extracellular matrix (ecm) deposition or fibrosis mediated primarily by pdgf. in this study, the decrease in pdgf level may be suggested due to the reducing inflammatory factor and oxidative stress. platelet-derived growth factor, released and activated after tissue injury, plays an important role in hepatic stellate cell activation and portal fibroblast proliferation, chemotaxis, and migration, thus suppressing its expression is important in fibrosis disease.(22,35) the controlled inflammatory niche may inhibit the activation and differentiation of hepatic stellate cells (hsc) into myofibroblasts, resulting in reducing the hsc stimulator, pdgf.(10) thus, this process will inhibit release of the profibrogenic factor and reduce impaired ecm deposition.(22) this concept is supported by previous reports revealing that the pdgf signaling pathway plays an important role in the development and prognosis of liver fibrosis and that controlling its level is critical for preventing severe liver disease.(2,23) however, this study did not analyze several potent biomarkers such as nf-κb, il-6, tgf-β, no, and sod after administration of averrhoa bilimbi l. fruit extract in ccl4-induced alf rats. overall, the implication of this study is that it may be useful for research on the potential of averrhoa bilimbi l. fruit extract to accelerate and direct target screening through molecular mechanisms to treat alf. future studies will be aimed at identifying the optimum dose and the length of averrhoa bilimbi l. fruit extract administration. conclusion these findings demonstrated that the dosedependent effect of averrhoa bilimbi l. fruit figure 2. effect of averrhoa bilimbi l. fruit extract on tnf-α and pdgf level (n=6)). (a) level of tnf-α in each group, (b) level of pdgf in each group. sham=healthy rats, control=ccl-4 treated with olive oil/vehicle, t1=ccl-4 treated with a low dose of averrhoa bilimbi l. fruit extract, t2= ccl-4 treated with a high dose of averrhoa bilimbi l. fruit extract. *p<0.05. 67 extract administration may reduce the tnf-α and pdgf levels in ccl4-induced alf rats. this study provides new insights into the benefits of averrhoa bilimbi l. administration in most injured tissues, particularly in the proliferation phase of alf. acknowledgment the authors would like to thank the stem cell and cancer research laboratory for facilitating and technically assisting this research. funding none. conflict of interest the authors declare that they have no competing interests. author contribution r.r.s: conception and design, collection and/or assembly of data, data analysis and interpretation, manuscript writing; c.c.: supervising, critical reviewing, final approval of the manuscript; t.s.: 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a, lindarto d, sembiring rj. anti-fibrotic effect of intravenous umbilical cordderived mesenchymal stem cells (uc-mscs) injection in experimental rats induced liver fibrosis. med glas (zenica) 2021;18:62-9. doi: 10.17392/ 1211-21. univ med vol. 42 no. 1 september-december 2022 universa medicina vol.41no.3 pissn: 1907-3062 / eissn: 2407-2230 radiographic lumbosacral angle has a low yield for diagnosing low back pain in workers 20-70 years of age partogi napitupulu1 , gupita nareswari1 , mulia rahmansyah1 , and tandy chintya tanaji1 * abstract background the increasing trend of working from home (wfh) among workers may lead to prolonged sitting time, which is associated with increased complaints of low back pain (lbp). the lumbosacral angle (lsa) is one of the clinically important radiographic angles related to the curves commonly measured to evaluate the biomechanical factors linked with lbp. the purpose of this study was to determine the yield of radiographic lsa for diagnosing lbp among workers aged 20-70 years. methods an analytic cross-sectional study involving 119 participants was carried out in trisakti university. lumbosacral angle was measured using ferguson’s method. data regarding lbp symptoms, sitting duration, and sitting position were collected using a questionnaire. sensitivity and specificity was used to describe the characteristics of lsa as a screening test of lbp. results the subjects consisted of 66 women (55.5%) and 53 men (44.5 %). their ages ranged from 20 to 64 years with a median age of 40.0 years. mean lsa was 37.4 ± 7.3º, while the prevalence of lbp was 75 (63.0%). the optimal cut-off value of lsa for the prediction of lbp was 49.5% (95% ci: 0.385-0.606). the sensitivity, specificity, positive predictive value and negative predictive value of lsa in detecting lbp were 58.7%, 45.5%, 68.8%, and 43.6%. conclusions the radiographic lumbosacral angle has a low yield for the diagnosis of lbp among workers aged 20-70 years. further studies are needed to confirm our results and to test the application of this measurement. keywords: work from home, low back pain, lumbosacral angle, workers 1radiology department, faculty of medicine, universitas trisakti *correspondence: dr. tandy chintya tanaji, sp.rad department of radiology, faculty of medicine, universitas trisakti jl. kyai tapa no.260, grogol jakarta barat, indonesia phone: +6287788834789 email: tandy.chintya@trisakti.ac.id date of first submission, august 15, 2022 date of final revised submission, december 18, 2022 date of acceptance, december 20, 2022 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article doi: http://dx.doi.org/10.18051/univmed.2022.v41:263-270 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1359 263 cite this article as: napitupulu p, nareswari g, rahmansyah m, and tanaji tc. radiographic lumbosacral angle has a low yield for diagnosing low back pain in workers 20-70 years of age. univ med 2022;41:263-70. doi: 10.18051/u nivmed.2022.v41: 263-270. mailto:tandy.chintya@trisakti.ac.id https://orcid.org/0000-0002-1562-7231 https://orcid.org/0000-0002-1652-0495 https://orcid.org/0000-0003-4822-5818 https://orcid.org/0000-0002-2356-3194 http://dx.doi.org/10.18051/univmed.2022.v41:263-270 https://univmed.org/ejurnal/index.php/medicina/article/view/1359 264 napitupulu, nareswari, rahmansyah, et al lsa for diagnosing lbp introduction the implementation of large-scale social restriction in indonesia has given rise to another problem. one of the things that many complain about is that since the introduction of working from home (wfh), the complaints of low back pain (lbp) are increasing among workers. the existence of wfh causes the boundaries and time between work and rest to be unclear.(1) workers unconsciously are constantly sitting at work in front of a computer or laptop. the continuous sitting position, whether ergonomic or non-ergonomic, is one of the causes of the increase in lbp complaints during the pandemic, especially in the 30 to 60 year-olds. in another study, it was said that the lbp prevalence was unchanged or improved in almost 59%, while in more than 41% the lpb became worse.(2) low back pain is the most common musculoskeletal problem in the adult population. low back pain is a pain syndrome that occurs in the lower back region.(3) although the cause in 80-90% of cases of lbp is unknown or nonspecific, it has been reported that workers tend to spend a lot of time working in front of a computer or laptop for hours in a sitting position without the sitting being interspersed with other physical activities, which can trigger lbp that commonly occurs in the lumbosacral region.(4,5) to withstand heavy loads throughout the day, a good lumbosacral curvature is needed so that it can function in supporting the load. the existence of changes or abnormalities of the curvature is what gives rise to mild to severe degrees of lbp. many methods are used to measure this curvature, one that has become the gold standard is the measurement of the lumbosacral angle (lsa) on lateral projection lumbar radiographs.(3) the lumbosacral angle is formed by the intersection of two lines, namely a line drawn across the superior endplate of the s1 vertebra and a horizontal line.(6) aside from other lateral lumbosacral spine mensuration such as lumbar lordosis (ll), lumbosacral disc angle (lsda) and sacral inclination (si), the lsa has been associated with some degree of instability and lbp, therefore knowledge of the lsa range is critical in the management of lbp.(2,7) the risk of lbp was reported to increase in lsa of more than 42 degrees.(3,7) its value is also related to gender, where the lsa value in women is found to be greater than that of men.(8) a previous study reported that changes in lsa may cause lbp, which may guide healthcare providers to include physicians to make decisions clinically, when examining patients with lbp.(9) another research conducted by keskin et al.(10) involving 131 office workers found that lumbar pain was present for less than 6 weeks for those workers who were working in a sitting position for more than 6 hours a day. in conclusion, this study revealed that prolonged sitting at work may be the risk factors for lumbar pain among office workers. the study conducted by cho et al.(4) also reports the effects of standing and different sitting positions but using lumbar lordosis (ll) as its parameters, with more detailed sitting positions being described. their study revealed that sitting position may cause increased or reduced ll, thus showing that sitting in a chair with back support induced minor changes to the ll hence reducing the incidence of lbp. sitting posture can affect the trunk muscle activity and different muscles are predominantly stimulated, depending on the sitting posture (11) previous studies have shown that some sitting postures and behaviours were associated with lbp. bontrup et al.(12) reported that individuals with lbp demonstrated a trend towards more static sitting behaviours compared to non-lbp subjects. jung et al.(13) also showed that prolonged sitting in a non-ergonomic posture could increase the risk of lbp. although there is no firm evidence for the presence or absence of a causal relationship between radiological findings and lbp, many studies have suggested that there should be such a relationship. (1 4,1 5) importantly, since no biomechanical studies have investigated the 265 recovery of spinal biomechanics following sitting in various postures, the aim of this study was to determine the yield of radiographic lsa for diagnosis of lbp among workers aged 20-70 years. methods research design this cross-sectional study was conducted at trisakti university, west jakarta, in the period of september 2020 to february 2021. study subjects this study involved 119 employees of trisakti university who worked from home during the coronavirus disease 2019 (covid-19) pandemic. the sample size was determined based on the following sample size lemeshow’s formula for cross-sectional studies based on a z α value of 1.96 for a 5% level of significance and 35.7% prevalence of low back pain according to patrianingrum et al.,(16) which revealed that a minimum sample size of 89 subjects was required. the inclusion criteria of this study were: (i) men and women aged between 20-70 years (ii) registered employees of trisakti university (iii) willing to fill out an assessment evaluation questionnaire (iv) and willing to undergo lateral lumbosacral spine radiography. the exclusion criteria were: (i) known congenital spinal abnormalities such as spina bifida or congenital scoliosis, (ii) spinal fixation surgery in the lumbosacral region and (iii) suffering from an infectious disease/tumor/malignancy of the spine. data collection a questionnaire was completed by the participants during this study; it included age, sex, body mass index (bmi), and lbp. t he questionnaire also assessed the presence of lbp symptoms in the last 4 weeks and average sitting duration per day. it depicted various sitting positions and the study subject had to choose one of the most frequently used of the sitting positions (figure 1). lumbosacral angle assessment lateral lumbosacral x-ray radiography was performed on each subject. the lumbosacral angle (lsa) was defined as the angle where the line tangential to the upper endplate of the sacral vertebra intersects the lower endplate of the fifth lumbar vertebra. the lsa was measured using the ferguson’s method.(17) the latter method is to first draw the ab line (a line parallel to the superior border of the sacrum), then draw the ac line (horizontal line to the sacrum).(3,8,9) the lumbosacral angle of each subject was measured by experienced radiologists using computerized methods and recorded on a research sheet; then, the editing and coding process was carried out on a computer hard disk and the data cleansed electronically. after having the lumbosacral radiograph taken, each of the subjects was asked to fill the questionnaire. variability of sitting position the questionnaire described various pictures of sitting positions and the research subject had to choose one of the most frequently used pictures figure 1. various sitting positions assessed in this study univ med vol. 41 no. 3 266 napitupulu, nareswari, rahmansyah, et al lsa for diagnosing lbp of the sitting position (figure 1). aside from this, the average duration of sitting during working was also asked to each subj ect within the questionnaire. statistical analysis data analysis was conducted using the spss 25 program. since the data distribution was normal, the intergroup comparisons were performed using the one-way anova test. the cut-off point of lsa in detecting lbp was measured using receiver-operating characteristic (roc) curve analysis. sensitivity and specificity values were used to describe the characteristics of lsa as a screening test of lbp. statistical confidence interval (ci) was set at 95%, and p value <0.05 was considered to indicate statistical significance. ethical clearance the study was approved by the research ethic s committee, fac ulty of medic ine, universitas trisakti under no: 169/ker/fk/xii/ 2020. results characteristics distribution of the study subjects in a sample of 119 participants, there were 66 (55.5%) women and 53 men (44.5%), whose ages ranged from 20 to 64 years with a median age of 40 years, while the prevalence of lbp was 75 (63.0%) (table 1). the median of bmi was 26 kg/m2. the distribution of answers in the study questionnaire is shown in table 2. from all 119 subjects, 49.6% worked from home during the pandemic, of whom 82.4% admitted that they had spent a longer time sitting than in their usual working days. the majority of the respondents admitted to sitting for an average of 3-6 hours (57.1%) without rest, while 26.1% admitted to sitting for an average of 6-9 hours per day (26.1%). in subjects with symptoms of lbp, the majority sat for > 9 hours / day (80%) and most used sitting position d (65.2%). compar ison of m ean lsa betw ee n subgroups of duration and sitting position table 2 shows mean lsa in each average sitting duration and sitting position. according to one-wa y anova ana lysis, there was no significant difference in lsa between subgroups of sitting and duration of sitting position (p=0.099 and 0.188, respectively). accuracy of lsa in identifying workers with lbp in this study, we conducted roc curve analysis to obtain the optimal cut-off point of lsa in patients with low back pain. the analysis was described in table 3. the optimal cut-off point of lsa in this study was 37.21, with area under the curve (auc) of 0.495 (95% ci 0.385–0.606), with sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv) of 58,7%, 45.5%, 68.8%, and 43.6%, respectively. characteristic n (%) age (years), (median, minmax) 40 (20–64) gender man women 53 (44.5) 66 (55.5) body mass index, kg/m2 (median, min-max) 26 (17.9–42.9) average sitting hours per day < 3 3-6 6-9 > 9 15 (12.6) 68 (57.1) 31 (26.1) 5 (4.2) sitting position posture a posture b posture c posture d lsa (mean± sd) lbp yes no 36 (30.3) 15 (12.6) 45 (37.8) 23 (19.3) 37.4 ± 7.3 75 (63.0) 44 (37.0) table 1. baseline characteristics of the research subjects (n=114) data presented as n (%), except for age and body mass index (median, min-max) and lumbosacral angle mean ± sd 267 table 2. comparison of mean lsa between subgroups of duration and sitting position one-way anova discussion our study was unable to identify a significant difference between lsa value and sitting position. with respect to lsa, prior studies have varied greatly in their results.. cho et al. (4) stated that an increase in intradiscal pressure along with flattening of the lumbar curve may result from the sitting position, thus affecting lumbosacral lordosis (ll) rather than lsa. similarly, the study by de baranda et al. (18) which analyzed sitting posture and its effect on sagittal spinal curvature and lbp noted that incorrect posture greatly affects sagittal spinal curvature, which might barely affect lsa. in contrast, caglayan et. al.(9) observed that changes in lsa may cause lbp, as well as the research conducted by melani et al.(19) which also noted a significant but negative correlation between sitting position and lbp. the discrepancy in the findings of these studies and the non-significant correlation between lsa and sitting position may be caused by many factors, including variation in sample size, variation in normal lsa cut-off values, and lack of proper education for the participants with regard to their detailed sitting position in filling out the questionnaire in the present study may contribute to variations in our results. in regard to optimal lsa cut-off value for detecting lbp (37.21º), our study is in line with the study of jha and ravi(3) which noted an optimal cut-off value of 32.42º for detecting lbp. the normal lsa cut-off values in the studies of maduforo et al.(20) and okpala (21) are 36º and 44.5º, respectively. our results emphasize that lsa is a poor predictor of lbp in workers. however, to our knowledge, no previous studies have assessed the diagnostic value (sensitivity and specificity) of lsa in detecting lbp. therefore, we could not compare our results to any previous study regarding the sensitivity and specificity of lsa. we also did not obse rve any significant relationship between lsa and average sitting duration in this study. these findings also support the theory related to biomechanical changes. when a person sits down, the lumbosacral vertebrae carry the largest part of a person’s body weight. because the heavy load from the cervical and thoracic vertebrae is transmitted to the lumbosacral vertebrae, the longer the sitting lumbosacral angle low back pain total positive negative positive negative 44 31 20 24 64 55 total 75 44 119 table 3. accuracy of lsa in identifying workers with lbp sensitivity = 58.7%; specificity = 45.5%; positive predictive value = 68.8%; negative predictive value: 43.6%; optimal cutoff of lsa for predicting lbp: 37.21º univ med vol. 41 no. 3 variable lsa (mean ± sd) p-value average sitting duration (hours) <3 34.3 ± 6.5 3-6 36.9 ± 7.7 0,099 6-9 39.9 ± 6.8 >9 31.7 ± 6.1 sitting position a b c d 38.5 ± 6.9 38.5 ± 7.4 35.6 ± 7.8 38.6 ± 7.4 0.188 268 napitupulu, nareswari, rahmansyah, et al lsa for diagnosing lbp duration, the harder the work of the lumbosacral vertebrae. sitting continuously for a long period of time incre ases t he pressure on the intervertebral discs, thereby stiffening the lumbar vertebrae and weakening the lower back muscles, which subsequently lead to changes in the position of the lumbosacral vertebrae and radiographic lumbosacral spine mensuration, including lsa. it has been reported that more static sitting position may lead to sustained pressure under the buttocks and continuous compression on the intervertebral discs, hence causing lbp.(12) however, it has been established that lumbar lordosis is decreased more in the sitting position than in the standing position. this postural change increases the load and stress on the lumbar spine and surrounding structures, hence increases the risk of low back pain. although proper upright sitting postures involve anterior pelvic tilt, lumbar lordosis and relaxation of the thoracic spine, which reduces the pressure on the intervertebral discs, most people tend to sit in a slouched posture for prolonged time peri ods, which is often accompanied by an abnormal posture of the spine and pelvis. when the posture is maintained with the neck and trunk bent forward, the back extensors are over-activated, and the constant load on these muscles may increase the risk of neck and back pain.(22) another influencing factor is the cut-off lsa value, which varies greatly between studies. this may cause difficulties in confirming the diagnosis of lbp.(23,24) these factors need to be evaluated further and may serve as input for further research. with regard to the fact that the prevalence of lbp was high among workers, for this matter, a multidisciplinary approach involving occupational medicine may be encouraged to educate trisakti employees on how to sit properly and correctly while working from home. the education and counseling may include stretching in between periods of work, and other exercises such as strengthening, endurance, aerobic fitness, walking, yoga, and pilates. all these active interventions may reduce back pain, but no evidence has been found that one form of exercise is superior to the other.(10) according to hayden et al.(25) exercise has been reported to be effective for treatment of chronic low back pain with moderate-certainty evidence, compared to no treatment, usual care, or placebo for pain. figure 2. receiver operating characteristics (roc) curves for low back pain 269 despite the inconsistencies in the literature and great variety in normal lsa cut-off values, several daily activities may also bring about changes in the curvatures that may come in the form of angular changes in the vertebrae affecting the value of lsa. in our process of collecting data, it was challenging to educate participants on how to correctly fill the questionnaire or provide other data about their sitting position and duration; therefore, further studies with larger populations may be warranted. one limitation of our study is the definitions for lbp, this data was obtained from a health interview. such data are subject to self-reporting bias and may have had an effect on the lbp prevalence. conclusion our study did not demonstrate that lsa showed ppv or sensitivities high enough to be effec tive in screening workers for lbp. screening based on lsa would lead to many false positive and negative test results. further studies with a larger number of subj ects may be encouraged to better determine the correlation between lsa and sitting duration. conflict of interests there are no potential conflicts of interest with respect to the research, authorship, and/or publication of this article declared by the author(s). acknowledgement the authors express their gratitude to the faculty of medicine, universitas trisakti, for the funding of this study. contributors all authors will take responsibility for the content of the manuscript submitted to universa medicina publicly. pn, mr, gn and t c contributed to the concept and design of the study and to data acquisition, analysis, and interpretation. gn and tc contributed to the critical revision of the manuscript. all authors have read and approved the final manuscript. references 1. galanti t, guidetti g, mazzei e, zappalà s, toscano f. work from home during the covid-19 outbreak: the impact on employees’ remote work productivity, engagement, and stress. j occup environ med 2021;63:e426-e2. doi:10.1097/ jom.0000000000002236. 2. bailly f, genevay s, foltz v, et al. effects of covid-19 lockdown on low back pain intensity in chronic low back pain patients: results of the multicenter confi-lomb study. eur spine j 2022;31:159-66. doi: 10.1007/s00586-021-07007-8. 3. jha ak, ravi c. a study of normal variation in lumbosacral angle in asymptomatic patients attending orthopaedic out-patient department of a medical college hospital, telangana. indian j orthop surg 2020;4:16–20. https://doi.org/ 10.18231/2395-1362.2018.0005. 4. cho iy, park sy, park jh, kim tk, jung tw, lee hm. the effect of standing and different sitting positions on lumbar lordosis: radiographic study of 30 healthy volunteers. asian spine j 2015;9: 762–9. doi: 10.4184/asj.2015.9.5.762. 5. aminoff mj, boller f, swaab df, barnes mp, good dc. handbook of clinical neurology. vol. 110. oxford: elsevier;2013. 6. kocyigit bf, berk e. comparison of lumbosacral alignment in geriatric and non-geriatric patients suffering low back pain. pakistan j med sci 2018; 34:282–7. doi: 10.12669/pjms.342.13969. 7. shortz sk, haas m. relationship between radiographic lumbosacral spine mensuration and chronic low back pain intensity: a cross-sectional study. j chiropr med 2018;17:1–6. doi: 10.1016/ j.jcm.2017.10.005. 8. mukherjee u, kundu s, gulati r, mandal p. lumbosacral angle variations in middle aged patients with chronic low back pain a retrospective study. j evid based med healthc 2021;8:208–12. doi: 10.18410/jebmh/2021/40. 9. caglayan m, tacar o, demirant a, oktayoglu p, karakoc m, cetin a. effects of lumbosacral angles on development of low back pain. j musculoskelet pain 2014;22:251–5. https:// doi.org/10.3109/10582452.2014.907855. 10. keskin y, ürkmez b, öztürk f, kepekçi m, aydýn t. correlation between sitting duration and position and lumbar pain among office workers. univ med vol. 41 no. 3 270 napitupulu, nareswari, rahmansyah, et al lsa for diagnosing lbp haydarpasa numune med j 2021;61:1–6. doi: 10.14744/hnhj.2019.04909. 11. wong ayl, chan tpm, chau awm, et al. do different sitting postures affect spinal biomechanics of asymptomatic individuals? gait posture 2019;67:230–5. d oi: 10.1016/ j.gaitpost.2018.10.028 12. bontrup c, taylor wr, fliesser m, et al. low back pain and its relationship with sitting behaviour among sedentary office workers. appl ergon 2019;81:102894. doi: 10.1016/j.apergo.2019.102894. 13. jung ks, jung jh, in ts, cho hy. effects of prolonged sitting with slumped posture on trunk muscular fatigue in adolescents with and without chronic lower back pain. med 2021;57:1-8. doi: 10.3390/medicina57010003. 14. cheung km. the relationship between disc degeneration, low back pain, and human pain genetics. spine j 2010;10:958-60. doi: 10.1016/ j.spinee.2010.09.011. 15. maatta jh, wadge s, macgregor a, karppinen j, williams fm. issls prize winner: vertebral endplate (modic) change is an independent risk factor for episodes of severe and disabling low back pain. spine (phila pa 1976) 2015;40:1187-93. doi: 10.1097/brs.0000000000000937. 16. patrianingrum m, oktaliansah e, surahman e. prevalence and risk factors of lower back pain in the anesthesiology workplace in dr. hasan sadikin general hospital bandung. j anestesi perioper 2015;3:47–56. doi: 10.15851/jap.v3n1.379. 17. ferguson ab. roentgen diagnosis of the extremities and spine. 2nd ed. new york: paul b. hoeber, inc.;1949. 18. de baranda ps, cejudo a, martínez-romero mt, et al. sitting posture, sagittal spinal curvatures and back pain in 8 to 12-year-old children from the region of murcia (spain): isquios programme. int j environ res public health 2020;17:1–18. doi: 10.3390/ijerph17072578. 19. melani i, saraswati pas, wahyunin. hubungan posisi duduk dengan nyeri punggung bawah non spesifik pada bagian adminitrasi dan pelayanan di polda bali. maj ilm fisioter indones 2019;7:12– 5. doi: https://doi.org/10.24843/ mifi.2019.v07.i01.p08. 20. maduforo c, west o, nwankwo n, onwuchekwa r, etawo us, ogbulu d. study of the lumbosacral angles of males in port harcourt, south south, nigeria. niger health j 2012;12:22 4. 21. okpala fo. measurement of lumbosacral angle in normal radiographs: a retrospective study in southeast nigeria. ann med health sci res 2014; 4:757-62. 22. hey hwd, wong cg, lau etc, et al. differences in erect sitting and natural sitting spinal alignment—insights into a new paradigm and implications in deformity correction. spine j 2017;17:183-9. doi: 10.1016/j.spinee.2016.08.026. 23. naqvi szg, ali a, siddiqui a, ali sd, qureshi m, aliuddin im. measurement of lumbosacral angle in normal radiographs: a cross-sectional study. j liaquat univ med heal sci 2020;19:238–41. doi: 10.22442/jumhs.201940697. 24. thakur b, prashar r, bhoil r, rana s, gupta u. evaluation of geometrical angles in lumbo-sacral spine on digital radiographs in north indian population. int j health clin res 2021;4:132–5. 25. hayden ja, ellis j, ogilvie r, malmivaara a, van tulder mw. exercise therapy for chronic low back pain. cochrane database syst rev 2021;9: cd009790. doi: 10.1002/14651858.cd009790.pub2. c:\users\universa medicina\docu 87 abstract universa medicina mother's hand tool as a skin-to-skin contact simulation instrument decreases pain levels in newborns yesim ceylantekin1, nuriye buyukkayaci duman2*, dilek ocalan3, senay topuz4, and betul yildiz ucar5 background sensory stimulation activates the gate control mechanism, raises the level of beta endorphins, and the secretion of beta endorphins increases the pain threshold, reducing or eliminating the feeling of pain. it has been reported that skin-to-skin contact or sensual stimulation reduces stress, pain and crying time in newborns. the present study aimed to examine the effect of the mother’s hand tool (mht) developed by the researchers for three purposes: touch, positioning and vibration on pain levels in newborns. methods a quasi-experimental study was conducted involving 52 newborns aged 0-15 days who were being treated in the neonatal intensive care unit. the mht was applied to support the newborn and was applied 8 times in 24 hours for 3 minutes in total. demographic data collection form (ddcf), neonatal evaluation form (nef) to assess the respiratory rate, pulse rate, spo 2 and co 2 level, and neonatal infant pain scale (nips) were used to collect data. the wilcoxon signed rank test was applied to analyse the data. a p value of <0.05 was considered to be statistically significant. results after the mht application, it was revealed that there was a significant difference in pulse rate (p=0.000), s p o 2 level (p= 0.029), co 2 level (p=0.000) and nips pain scores (hour 6,9,12,15,18, and 24) and total nips (p=0.000) pain scores, before and after mht practice. conclusion this study demonstrated that mht application to the newborns had a decreasing effect on pain level, heart rate, co 2 level, and an increasing effect on s p o 2 level. keywords: mother’s hand tool, procedural pain levels, skin-to-skin contact simulation, newborns. original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.90-98 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1147 may-august 2021 vol.40no.2 cite this article as: ceylantekin y, duman nb, ocalan d, topuz s, ucar by. mother's hand tool as a skin-to-skin contact simulation instrument decreases pain levels in newborns. univ med 2021;40:87-95. doi: 10.18051/univmed.2021.v40.90-98 1afyonkarahisar university of health sciences, faculty of health sciences, department of nursing, afyonkarahisar, turkey 2hitit university, faculty of health sciences, department of nursing, çorum, turkey 3tokat gaziosmanpasa university faculty of health sciences, department of nursing, tokat, turkey 4ankara university, faculty of health sciences, department of midwifery, ankara, turkey 5konya numune hospital, third level intensive care unit, konya, turkey correspondence: *nuriye buyukkayaci duman hitit university, faculty of health sciences, department of nursing, corum, turkey email : nurfatihh@hotmail.com orcid id : 0000-0002-4246-3607 date of first submission, march 6, 2021 date of final revised submission, may 24, 2021 date of acceptance, june 2, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 88 ceylantekin, duman, ocalan, et al mother's hand tool and newborn introduction a number of neonates, during the first days of life in the postpartum period, experience painful processes because of drug administrations, heel lancing, venipuncture/arterial puncture, venous interventions, neonatal evaluation and positioning. behavioural or neurological problems may arise in infants if the pain is not treated effectively in this period.(1-3) healthcare professionals are responsible for reducing the pain that neonates experience and helping them to cope with it. the pain has two forms of treatment: pharmacological and non-pharmacologica l. (1 -3) no npharmacological methods are necessary to decrease analgesic drug use in the neonatal intensive care unit (nicu) and hospitalization period. in addition, healthcare professionals may prefer those methods since they are affordable and easily applicable.(4-6)according to the literature, non-pharmacological methods which play a key role in pain management in neonates support them to cope with the pain and reduce the stress level. in addition, these methods are widely preferred since they are easily applicable and affordable. the methods reduce drug use and the risks of side effects as well.(6,7) the distraction methods may be utilized by using the visual and auditory senses or sense of touch and taste in order to reduce the pain in newborns.(6,7)as the distractive stimulant is removed, the pain becomes the main focus.(6,7) skin-to-skin contact (ssc) or kangaroo care (kc), massage, touching and embracing, crooning or avoiding loud talk close to the baby, allowing non-nutritive sucking, giving breast milk, breastfeeding, or giving oral sugared fluids are the distractive methods that reduce pain and stress.(8-10) in addition, such non-pharmacological methods as holding, positioning, individualised care, provision of aromatic and maternal smells, mechanical vibration, acupuncture, and music have been used to control the pain.(8-10) touching and embracing are essential for the newborns with the purpose of maintaining ssc and reinforcing the trust sensation.(11) since preterm newborns are more hypotonic than fullterm newborns, they should be supported and in order to pr eser ve fl exion posture it is recommended that, if necessary, the infant should be swaddled for support. a towel, blanket or roll cloth can be used to maintain stabilisation.(11) similarly, it has been suggested that kc is important in pain management as well. moreover, it has been reported that kc application decreases the pain score in infants and the duration of their crying during heel lancing.(12-14 ) studies on the subject have reported that ssc reduces the emotional stress and shortened crying times in the newborns, as well as improves parent-baby attachment and safe interaction.(1214) in addition, the newborn calms down, the pulse rate decreases and the body temperature is regulated with the sensory stimulation. it has been reported that ssc has a decreasing effect on pain. when the literature is reviewed, there are studies reporting that ssc positively affects blood gas levels in newborns.(12-14) in addition, sensory stimulation activates the gate control mechanism, raises the level of beta endorphins, and the secretion of beta endorphins increases the pain threshold, reducing rather than eliminating the feeling of pain.(15) when studies conducted with newborns on the subject are examined, it is seen that sensory stimulation techniques such as ssc, kc, vibration and massage reduce neonatal stress, resulting in a significant decrease in heart rate, blood pressure and pain scores.(16-18) in the study of herrington and chiodo (16) it was reported that the heart rate and crying time of babies who did not receive the gentle human touch (ght) method as a simulation of ssc during heel lancing in newborns were higher than those in the group receiving ght. in the study of mcginnis et al.(17) it was reported that the newborns to whom vibration was applied during the heel lancing procedure had a more stable heart rate and pain scores were lower than had the group who did not receive vibration. in the systematic review study of ueki et al.(18) it was determined that mechanical vibration was 89 effective in reducing pain from needle-related procedures (nrps) in children. in the study of dolu and karakoc(19) examining the effect of the mechanical vibration technique during heel lancing, the pain scores of the newborns in the intervention group in which mechanical vibration was applied were found to be lower than in the group without mechanical vibration. it was reported by abdallah et al.(20) that the pain scores of premature infants who received massage therapy were lower than those of infants who did not receive massage therapy. when the studies on the subj ect are examined, it is seen that for practices such as reducing neonatal intensive care stress, kangaroo care to relieve pain, sensual contact, and vibration there is definite evidence of improved neurological and behavioural outcomes, although the evidence seems to be insufficient for other applications.(1618) in these studies, it is seen that the relationship of a single technique to neonatal pain management is generally examined.(18-20) in our study unlike other studies, the aim was to examine the effect of the mother’s hand tool (mht) developed by the researchers for three purposes: touch, positioning and vibrationon pain levels in newborns. methods research design a quasi-experimental study was conducted at the intensive care unit in afyonkarahisar university of health sciences, hospital of medicine, faculty of health sciences, between january and march 2018. research subjects fifty two newborns who were 0-15 days old and who met the inclusion criteria of the study, having been treated in incubators for less than a month, were included in the study. the sample size was determined using 90% power to detect a mean of paired differences of 2.1 with a standard deviation of differences of 1.4 and a significance level (alpha) of 0.05, resulting in 52 newborns aged 0-15 days. infants who were aged 0 and 15 days, born between 32 and 40 gestational weeks and whose parents agreed to participate and who met the criteria were included in the study. excluded from the study were infants who had congenital anomalies, haemorrhages, contraindications towards position change, heart disease or severe respiratory distress, had undergone serious operation and infants who received morphine, fentanyl, muscle rel axants, dilantin or phenobarbital. study instruments three instruments were used to collect data. the first instrument was the demographic data collection for m (ddcf) developed by researchers to assess the infant’s gender, birth weight, height, weight, week of birth, delivery type, apgar scores, sleeping position, breastfeeding status, vital signs and the reason for neonatal intensive care admission. the third instrument was the neonatal infant pain scale (nips) that was developed by lawrence et al.(21) to assess behavioural responses and physiologic pain responses of preterm and full-term newborns and was translated into turkish by akdovan and yildirim (22) and validated in 1999 by akdovan. lawrence et al.(21) found the reliability coefficient of nips to be in the range of 0.92-0.97. in addition, akdovan and yyldyrym(22) reported that the cronbach’s value is between 0.83 and 0.86. the scale comprised of five behavioural indicators (facial expressions, cry, state of arousal, arms, legs) and one physiologic indicator (breathing pattern). with the exception of ‘‘cry,’’ which had three possible score descriptors (0, 1, or 2), each behavioural indicator was scored with 0 or 1. the total score ranged between 0 and 7. higher scores represented higher levels of pain. the nips is very easy and quick to use. the data for nips were collected by video recording. in this study, cronbach’s alpha of the scale was between 0.91 and 0.97. study procedure the newborns included in the study were supported in the incubator with a mht instead univ med vol. 40 no 2 90 ceylantekin, duman, ocalan, et al mother's hand tool and newborn a b c of a towel or a rolled cloth. the position of the newborns was changed every 3 hours (prone, supine, right-lateral and left-lateral position) by the mht. the application of mht which included supporting the newborn and providing vibration, was performed 8 times in 24 hours for 3 minutes in total, including interventions. heart rate, s p o 2 value, co 2 rate measurements were taken with a pulse oximeter device before and after the application of the mht. in addition, the respiratory rate of newborns not connected to a mechanical ventilator was measured by the researcher by observing the rib cage movement. the respiratory rate of newborns connected to a mechanical ventilator was measured by means of a device. measurement of the neonate’s vital signs (axillary temperature, heart rate and respiratory rate) was done directly during direct skin to skin contact for 12 times (every 2 hours) to confirm their stability, then their mean values were recorded, and all other routine care was done. the measurement results obtained were recorded in the nef. also, the nips was applied before and after mht application during the interventions. mother’s hand tool (mht) the mht is a hand-like tool, made of rubber and bar of silicone, which vibrates the newborns. non-allergenic components were used in view of the newborn’s skin sensitivity. therefore, more effective results were expected by integrating touching, proper positioning and mechanical vibration techniques without harming the newborn’s skin. it was particularly designed to be like a mother’s hand to induce the feeling that mother is touching. the mht was developed by us and was given its name because it resembles the hand of the mother (figure 1). the mht is considered to be important in creating a sense of trust in the infant who is separated from the mother, since it resembles the mother’s hand, unlike the rolled towel currently used in the clinic (figure 2). the functions of the mht of touching, proper positioning, and mechanical vibration have been integrated with the aim of pain control in newborns. ethical consent this study has been ethically approved on 11/12/2017 by afyon kar ahisar kocatepe university faculty of medicine under no. 2017/ 279. written and oral consent of the respondents’ parents were obtained as well to allow the investigators to evaluate the pain status of the newborns. statistical analysis the data were evaluated using the ibm spss 22.0 package program (ibm corp., armonk, ny, usa). number, percentage, mean, and standar d devia ti on were used in the evaluation of descriptive data. the shapiro-wilk normality test was performed to determine whether the data were normally distributed. in the analysis, it was found that the distribution was figure 1. the maternal mechanism used to position the babies in intensive care 91 not normal. the wilcoxon signed rank test was used to compare two groups of non-parametric data. a p-value of <0.05 was considered to be statistically significant. results a total of 52 newborns participated in this study and were followed up for 24 hours (figure 3). in this study, the median gestation week of the newborns was 35.3 (29-40) weeks, the mean age was 3.4 (0-15) days, and 55.8% of the newborns were girls. the median birth weight of the newborns was 2150 (1100-4000) g, while the median height was 43 (41-46) cm, and 63.5% of the newborns were delivered by caesarean section. the reasons for admission to the nicu were hyperbilirubinaemia (63.5%), hypoxia (25.0%), premature (25.0%), and low birth weight (15.4%). in addition, 96.2% of the newborns received breast milk and all of them slept in the supine position. when the distribution of vital signs of newborns was examined, the median axillary temperature was 36.2 º (35-37) , the median heart rate was 141 (130-160) x/min., and the median respiratory rate was 48 (46-56) x/min. while the median oxygen saturation was 95.5 (94-99) % , and the median carbon dioxide saturation was 36 (35-43) % (table 1). figure 2. the use of mother’s hand tool as a representative in positioning figure 3. flowchart of the participants univ med vol. 40 no 2 92 ceylantekin, duman, ocalan, et al mother's hand tool and newborn a wilcoxon signed rank test showed that there was a significant difference on weight gain on hour 9 (p=0.000), hour 12 (p =0.000), hour 15 (p=0.000), hour 18 (p=0.000), hour 21 (p=0.000) and hour 24 (p=0.000) between infants before the mht practice group and those after the mht practice group. the median co 2 level and heart rate significantly decreased in the post mht practice group compared to the before mht practice group. and the median spo 2 level increased after the mht practice (table 2). discussion in our study, it was determined that the mht application performed during invasive procedures has a significant decreasing effect on the median nips scores of the newborns (at 6, 9, 12, 15, 18, 24 hours) and the median total 24-hour nips score. in other words, it can be said that mht application reduces procedural pain in newborns. *data presented as median (min-max) characteristics n (%) gestational age (weeks) * age (days)* gender male female birth weight (g)* height (cm)* delivery type vaginal birth caesarean section apgar scores 46 79 reason for nicu admission hyperbilirubinaemia hypoxia prematurity low birth weight breastfeeding status taking not taking sleeping position supine vital signs * axillary temperature (ºc) heart rate (x/min) respiratory rate (x/min) spo2 saturation (%) co2 saturation (%) having diagnosed infection status yes no skin contact treatment in incubator 35.3 (29-40) 3.4 (0-15) 23 (44.2) 29 (55.8) 2150 (1100-40000) 43 (41-46) 19 (36.5) 33 (63.5) 27 (52.7) 25 (47.3) (34.6) (25.0) (25.0) (15.4) 50 (96.2) 2 (3.8) 52 (100.0) 36.2 (35-37) 141 (130-160) 48 (46-56) 95.5 (94-99) 36 (35-43) 0 (0.0) 52 (100.0) 8 times and over in 24hour table 1. characteristics of the subjects at base-line (n=52) 93 according to the literature, it is reported that non-pharmacological methods such as skin contact, vibration, massage, and positioning are eff ective in relieving procedural pain in newborns.(1-3) similarly, in our study, it was determined that the application of the mht wh i c h h a s t h r e e f u n c t i on s , s ki n c o nt a c t simulation, vibration and positioning, reduces procedural pain in newborns. in our study, after the mht practice, the median s p o 2 increased and the median co 2 decreased in the newborns. in addition, after the mht application, mean heart rate decreased. studies on the subject show that pain in the newborn increases heart rate, causes ecg changes and negatively affects oxygenation.(1618) on the other hand, with the reduction of pain in the newborn and the elimination of the stress factor of pain, heart rate slows down, oxygenation incr ease s, and carbon dioxide saturation decreases.(16-18) in this context, it can be said that the findings obtained in our study regarding the change in pain scores, vital signs and blood gas values before and after mht application are compatible with the literature. when the literature is reviewed, similar to our study it has been reported that skin-to-skin before mht practice (n=52) after mht practice (n=52) p value nips scores median median 0.05 0.16 0.85 0.00 0.00 1.10 1.24 0.25 time of day (hour) 3: 00 6: 00 9: 00 12:00 15:00 18.00 21:00 24:00 0.06 0.26 1.75 3.50 4.40 2.80 1.50 0.32 3.40 95.5 36 36.2 141 48 0.729 0.285* 0.000 0.000* 0.000* 0.000* 0.000* 0.000* nips total scores 0.22 0.000* spo2 saturation (%) 97 0.029* co2 saturation (%) 31 0.029* axillary temperature (ºc) 36.3 0.652 heart rate (x/min) 139 0.000* respiration rate (x/min) 48 0.445 table 2. comparison of nips scores, total nips score, vital signs in newborns before and after mht practice abbreviation nips, neonatal infant pain scale contact or similar sensual stimulation, vibration and positioning had a decreasing effect on stress, pain and crying time in newborns.(16-18) it has been reported that some of the non-pharmacological studies have found a significant effect on pulse rate, respiration and oxygen saturation, and a decrease in motor activity and arousal states following invasive measures.(16-18) similar to the result of our study, herrington and chiodo (16) investigated the gentle human touch (ght) method during heel lancing in neonates. it has been revealed that infants who did not receive ght had increased heart rate and increased duration of crying during the heel stick. also infants who received ght did not have decreased respiration and elevated heart rate. also, cadlayan and balcy (23) studied the duration of crying and pain scores in preterm neonates both in the usual positions and facilitated tucking during heel lancing. it has been determined that the pain scores of the neonates in facilitated tucking are relatively low and the duration of their crying is shorter as well. chidambaram et al.(24) using a crossover study on the effect of kangaroo care (skin-to-skin contact) in reducing heel lancing pain in preterm infants, found that kangaroo care was effective in reducing pain in univ med vol. 40 no 2 94 ceylantekin, duman, ocalan, et al mother's hand tool and newborn conflict of interest no potential conflict of inter est was reported by the author(s). acknowledgments we would like to thank the participating newborns who took part in this study. we also thank both parents of each of the newborns for their consent. contributors yc, do, nbd designed this study. yc, by, nbd carried out the data analyses and reported the initial findings. nbd, st contributed to the manuscript prepation and submission. all authors have read and approved the final manuscript. references 1. boles j. non-pharmacological strategies for addressing infant pain. pediatr nurs 2017;43:98100. 2. erodlu a, arslan s. perception, evaluation and management of pain in the newborn. duzce university j inst health sci 2018;8:52-60. 3. polkki t, korhonen a, laukkale h. nurses’ perceptions of pain assessment and management practices in neonates: a cross-sectional survey. scand j caring sci 2018;32:725-33. doi: 10.1111/ scs.12503. 4. field t. preterm newborn pain research review. infant behav dev 2017;49:141-50. doi: 10.1016/j.infbeh.2017.09.002. 5. efe e, dikmen s, altas n, boneval c. turkish pediatric surgical nurses’ knowledge and attitudes regarding pain assessment and nonpharmacological and environmental methods in newborns’ pain relief. pain manag nurs 2013;14:343-50. doi: 10.1016/j.pmn.2011.08. 003. 6. bucsea o, riddell rp. non-pharmacological pain management in the neonatal intensive care unit: managing neonatal pain without drugs. semin fetal neonatal med 2019;24:101017. doi: 10.1016/ j.siny.2019.05.009. 7. williams n, mac lean k, guan l, collet jp, holsti l. pilot testing a robot for reducing pain in hospitalized preterm infants. otjr occup preterm infants. similarly, in the study of sen and manav,(25) it was reported that kangaroo care was more effective than oral sucrose in relieving procedural pain in premature babies. in t he s t ud y o f k a r i mi e t a l . , ( 2 6 ) i t wa s determined that massage and kangaroo care had a pain-reducing effect in premature infants.in the randomized controlled trial by mcginnis et al.(17) it was found out that the neonates in the vibration group had significantly lower pain scores and more stable pulse rates during heel stick and 2 minutes following heel lancing compared to those in the non-vibration group. also, dolu and karakoc’s(19) study in which the effect of the mechanical vibration technique during heel stick in order to reduce the pain was observed, mean nips scores in the experimental group during and after the practice was lower than in the control group without the vibration technique. these findings are similar to those of our study. the clinical implication of this study according to the results showed that at least 9 hours of mht was effective in improving pain, heart rate, and s p o 2 and co 2 levels in the newborn . therefore, mothers nee d to be encouraged and supported to provide mht practice in nicus. the limitation of our study was the quasi-experimental design used, which is useful when randomization is not possible. in this study, we chose to use a quasi-experimental design because of the ethical implications of randomising infants to receive or not receive the mht practice. future research using a true experimental design would be more rigorous and assist in controlling confounding variables. conclusions as a result, it was demonstrated that mht practice had a decreasing effect on pain levels, heart rates, and co 2 rates, and an increasing effect on spo 2 values in the newborns. the mht practice is a safe and effective method of pain management in newborn. 95 participation health 2019;39:108-15. https:// doi.org/10.1177/1539449218825436. 8. akcan e, yiðit r. related approaches to pain management of neonatal nurses and physicians in the neonatal clinic in turkey. acu health sci j 2016;3:147-53. 9. de clifford-faugere g, aita m, le may s. nurses’ practices regarding procedural pain management of preterm infants. appl nurs res 2019;45:52-4. https://doi.org/10.1016/j.apnr.2018.11.007. 10. yavuz de, alpar pe. interventional pain and nonpharmacological management in newborns and infants. zeynep kamil med bull 2018;49:169-78. 11. rivas-fernandez m, roque i figuls m, diezizquierdo a, escribano j, balaguer a. infant position in neonates receiving mechanical ventilation. cochrane database syst rev 2016;11: cd003668. doi:10.1002/14651858.cd003668.pub4 12. campbell-yeo m, johnston cc, benoit b, et al. sustained efficacy of kangaroo care for repeated painful procedures over neonatal intensive care unit hospitalization: a single-blind randomized controlled trial. pain 2019;160:2580-8. doi: 10.1097/ j.pain.0000000000001646. 13. pagni am, kellar s, rood m. effects of kangaroo care on procedural pain in preterm infants: a systematic review. honors research projects 2017;441. http://ideaexchange.uakron.edu/ honors_research_projects/441. 14. benoit b, campbell-yeo m, johnston c, latimer m, caddell k, orr t. staff nurse utilization of kangaroo care as an intervention for procedural pain in preterm infants. adv neonatal care 2016; 16:229-38. doi: 10.1097/anc.0000000000000262. 15. zampino c, ficacci r, checcacci m, catacuzzeno l. pain control by proprioceptive and exteroceptive stimulation at the trigeminal level. front physiol 2018;9:1037. doi:10.3389/fphys. 2018.01037. 16. herrington cj, chiodo lm. human touch effectively and safely reduces pain in the newborn intensive care unit. pain manag nurs 2014;15:10715. doi: 10.1016/j.pmn.2012.06.007. 17. mcginnis k, murray e, cherven b, mccracken c, travers c. effect of vibration on pain response to heel lance: a pilot randomized controltrial. adv neonatal care 2016;16:439-48. doi: 10.1097/anc. 0000000000000 315. 18. ueki s, yamagami y, makimoto k. effectiveness of vibratory stimulation on needle-related procedural pain in children: a systematic review. jbi database system rev implement rep 2019; 17:1428-63. doi: 10.11124/jbisrir-2017-003890. 19. dolu kaya fn, karakoc a. efficacy of mechanical vibration of heel stick pain in neonates. clin exp health sci 2018;8:122-7. 20. abdallah b, badr lk, hawwari m. the efficacy of massage on short and long term outcomes in preterm infants. infant behav dev 2013;36:662-9. https://doi.org/10.1016/j.infbeh.2013.06.009. 21. lawrence j, alcock d, mcgrath p, kay j, macmurray sb, dulberg c. the development of a tool to assess neonatal pain. neonatal netw 1993;12:59-66. 22. akdovan t, yyldyrym z. assessment of pain in healthy neonates, investigation of the effects of pacifying and holding in arms. j perinatal med 2001;29:43. 23. çaðlayan n, balcy s. an effective pain reduction in preterm newborns method: fetal position. florence nightingale j nurs 2014;22:63-8. 24. chidambaram ag, manjula s, adhisivam b, bhat bv. effect of kangaroo mothercare in reducing pain due to heel prick among preterm neonates: a cross-over trial. j matern fetal neonatal med 2014;27:488-90. doi: 10.3109/14767058.2013. 818974. 25. sen e, manav g. effect of kangaroo care and oral sucrose on pain in premature infants: a randomized controlled trial. pain manag nurs 2020;21:556-64. doi: 10.1016/j.pmn.2020.05.003. 26. karimi f, abolhassani m, ghasempour z, gholami a, rabiee n. comparing the effect of kangaroo mother care and massage on preterm infant pain score, stress, anxiety, depression, and stress coping strategies of their mothers. int j pediatr 2020. doi:10.22038/ijp.2020.50006.3990. univ med vol. 40 no 2 dark chocolate administration improves working memory in students 229 *department of physiology, faculty of medicine, atma jaya university **department of anatomy, faculty of medicine, atma jaya university correspondence: nawanto agung prastowo department of physiology, faculty of medicine, atma jaya university jalan pluit raya no 2, jakarta utara, jakarta 14444 email: nawanto2005@yahoo.co.id handphone: 081317477849 faksimili: 021-660-6123 univ med 2015;34:229-36 doi: 10.18051/univmed.2016.v35.229-236 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background flavonoids have positive effects on health, including the nervous system. high flavonoid content can be found in chocolate, especially dark chocolate. verbal working memory is important for reasoning, language comprehension, planning, and spatial processing. the purpose of this study was to evaluate the effect of a single dose of dark and white chocolate administration on verbal working memory in medical students. methods a study of experimental pre-post test design with controls was conducted on 60 students. these were simply randomized into two groups: the first group was supplemented with white chocolate as control, and the second group received dark chocolate, at an identical single dose of 100 g. working memory was measured with the digit span forwards (dsf) and the digit span backwards (dsb) tests, before, at 1 hour, and at 3 hours after intervention. independent t and mann-whitney tests were used for data analysis. results scores for dsf and dsb in control and treatment groups were similar at baseline. at 1 hour after dark and white chocolate administration, dsf and dsb scores were not significantly different between the two groups (p=0.832; p=0.683). supplementation of dark chocolate at 3 hours after intervention significantly increased dsb scores compared to white chocolate (p=0.041), but dsf scores were not significantly different between the two groups (p=0.204). conclusions dark chocolate as a single dose is capable of improving verbal working memory in students, 3 hours after its consumption. since cocoa contains multiple bioactive compounds, one approach might be to examine the neurocognitive effects of combinations of potential functional ingredients. keywords: antioxidant, flavonoid, dark chocolate, working memory, students dark chocolate administration improves working memory in students nawanto agung prastowo*, samuel kristanto*, and poppy kristina sasmita** doi: http://dx.doi.org/10.18051/univmed.2015.v34.229-236 230 prastowo, kristanto, sasmita dark chocolate improves working memory pemberian coklat hitam memperbaiki memori kerja pada mahasiswa latar belakang flavonoid memiliki efek positif terhadap kesehatan termasuk sistem saraf. kandungan flavonoid tinggi dapat ditemukan pada coklat terutama coklat hitam. memori kerja penting untuk proses penalaran, pemahaman bahasa, perencanaan, dan pemahaman spasial. tujuan penelitian ini untuk menilai pengaruh pemberian coklat hitam dan coklat putih dosis tunggal terhadap memori kerja pada mahasiswa. metode penelitian eksperimental pre-post test design menggunakan kontrol dilakukan pada 60 mahasiswa. mereka secara acak sederhana dibagi menjadi dua kelompok, yaitu kelompok pertama diberikan coklat putih sebagai kontrol dan kelompok kedua coklat hitam dengan dosis tunggal yang sama sebanyak 100 gr. memori kerja diuji dengan digit span test (dst) forward (dsf) dan backward (dsb) sebelum, 1 dan 3 jam setelah intervensi. uji statistik yang digunakan adalah uji t-independen dan mann-whitney untuk menguji perbandingan skor dst antara kelompok. hasil tidak terdapat perbedaan bermakna skor dsf dan dsb antara kelompok kontrol dan perlakuan pada tahap awal penelitian. setelah 1 jam baik pemberian coklat hitam maupun coklat putih tidak menunjukkan perbedaan yang bermakna skor dsf dan dsb antara kedua kelompok perlakuan (p=0,832;p=0,683). pemberian coklat hitam setelah 3 jam, ternyata skor dsb lebih tinggi secara bermakna dibandingkan coklat putih (p=0,041), dan skor dsf tidak menunjukkan perbedaan yang bermakna (p=0,204) antara kedua kelompok. kesimpulan pemberian coklat hitam dengan dosis tunggal setelah 3 jam mampu meningkatkan memori kerja pada mahasiswa. coklat hitam bersifat antioksidan karena mengandung flavanols bermanfaat untuk meningkat fungsi nerokognitif pada mahasiswa. kata kunci: antioksidan, flavonoid, coklat hitam, memori kerja, mahasiswa abstrak introduction c h o c o l a t e i s p r o d u c e d f r o m c a c a o (theobroma cacao l.) beans and contains flavanols, a class of flavonoids with considerable antioxidant activity, being thus of benefit to human health.(1) in comparison with white chocolate, the antioxidant content of dark chocolate is higher, as a result of fermentation, without the addition of other ingredients, such as powdered milk, sugar, and fat.(2) therefore dark chocolate has a bitter taste and is thus less favored. there are many studies on the effect of flavanols on cognitive function, the majority of which showed enhanced cognitive function after administration of flavanols. however, there are f e w s t u d i e s c o m p a r i n g t h e e ff e c t s o f administration of dark and white chocolate on cognitive function. one study comparing the effects of acute administration of dark and white chocolate was conducted by field et al.(3) using a crossover design, in which dark chocolate with a content of 720 mg cocoa flavanols was given, followed by administration of white chocolate, with an interval of one week between the two 231 univ med vol. 34 no.3 interventions. the results showed that dark chocolate enhanced cognitive function to a higher extent than did white chocolate. working memory plays an important role in cognitive function, particularly in reasoning, language comprehension, planning, and spatial processing.(4) working memory is a combination of attention, concentration, and short-term memory, and refers to the capacity for temporary retention and manipulation of information.(5) working memory is influenced by sleep quality,(6) diet,(7) and age.(8) several disorders, particularly those affecting the brain, such as alzheimer’s disease, stroke, head injury, and brain tumors, result in working memory dysfunction.(5) the digit span test (dst) is a test for measuring working memory function, and is frequently used because of its simplicity and high reliability of up to 0.9.(9) the dst consists of a sequence of digits that must be memorized and verbally repeated by the test subject. dst results are consistently good in normal elderly,(10) in elderly with cognitive dysfunction,(11) and also in children.(12) the objective of this study was to evaluate the effect of administration of dark and white chocolate on working memory in students, and differs from previous studies in the use of a parallel group design. methods design of the study this was an experimental study of preposttest nonblinded design with controls and conducted in jakarta from january to april 2015. subjects the subjects were preclinical medical students (1st – 4th year) who participated in this study and met the inclusion criteria, i.e. male and female students, in the preclinical years, healthy, of normal hearing, and agreeing to participate in the study up to its completion. the exclusion criteria were hypersensitivity to chocolate, insomnia, smoking, drinking, and history of head injury within the last 6 months. the drop-out criteria were consumption of flavonoid-containing foods or drinks 24 hours before the study, and in the 3-hour waiting period before the dst. informed consent was obtained in written form. information on the effect of type of chocolate on dst results was not given to the prospective subjects to avoid bias. sample size was calculated with the formula for comparative numerical analysis of two independent groups: 2 21 )xx( ).sz(z 2n       − + = βα where z á (5%) =1.96, zâ (85%) =1.282, s=0.3, x 1 -x 2 =0.25.(13) anticipating a drop out rate of 10%, the number of subjects for each group was 30, so that the total number of subjects was 60. intervention s u b j e c t s w e r e a l l o c a t e d b y s i m p l e randomization to 2 groups, i.e. the control group and the treatment group, each group consisting of 15 females and 15 males. the control group and the treatment group received white and dark chocolate bars, respectively, at a single oral dose of 100 g, which is the standard dose for determining chocolate contents. (14,15) the flavonoid content of 100 g of dark chocolate is ≈ 28.3 mg, and that of 100 g of white chocolate is ≈ 7.7 mg.(15) the outer wrappers were removed before the bars were given to the subjects, so that it was not apparent what type of chocolate was to be consumed. however, the subjects could still distinguish the type of chocolate by its color, since the white chocolate was of lighter color, whereas the dark chocolate was of darker color. measurement of working memory measurement of working memory was p e r f o r m e d u s i n g t h e d s t. t h e s u b j e c t s underwent a 24-hour wash-out period before the dst test, to remove flavonoids from plasma, by abstaining from consumption of flavonoidcontaining foods and drinks. after the wash-out 232 prastowo, kristanto, sasmita dark chocolate improves working memory period, both groups underwent the dst test, consisting of the dst forwards (dsf) and dst backwards (dsb). after completing the first dst tests (dsf-pre and dsb-pre), the subjects underwent the second and third dst tests, i.e. at 1 hour (dsf1 & dsb1) and 3 hours (dsf3 and dsb3) after chocolate consumption. while waiting for the dst, the subjects were allowed to perform their regular activities, but were not permitted to consume foods or drinks other than those provided by the investigators. in the dsf test, the subjects have to verbally repeat a sequence of digits presented by the investigators in their exact order, whereas in the dsb test the subjects have to repeat the digits in reverse order. the test is started by the investigators calling out a 4-digit number in random order (e.g. 3729) at a rate of 1 digit per second, then the subjects verbally repeating the digits in their given order. if the subjects has repeated the digits in the correct order, the list of digits is increased by 1 digit. if the subjects are unable to repeat the digits in their correct order, the test is followed by the dsb test. the subjects have two opportunities to listen to and repeat the series of digits. the test is discontinued if the subjects on the two occasions are unable to repeat the digits in their correct order. the dst score is the longest list of digits that has been repeated correctly. if the subjects can repeat a 9-digit number, the dst score is 9.(9,16) in the present study there were 3 sets of digit lists for the dsf and 3 sets for the dsb, each set consisting of digit lists in different order. statistical analysis the shapiro-wilk test was used to test for the normality of the data distribution. the independent t-test or mann-whitney test was used to find differences in dst scores between the two groups. the level of significance was set at p<0.05 with a confidence interval of 95%. ethical clearance ethical clearance was obtained from the research ethics commission, faculty of medicine, atma jaya university, jakarta. signed written informed consent was obtained from all subjects. the identity of the subjects was kept secret and was used only for research purposes. results a total of 84 students agreed to participate in the study by filling in an agreement form. a total of 16 students withdrew from the study because of academic reasons. randomization was performed on 68 subjects, consisting of 36 figure 1. flowchart of subject participation 233 univ med vol. 34 no.2 females and 32 males, to determine the type of intervention. in the randomization process the female subjects were separated from the males, to ensure that each intervention group received an identical number of female and males. on completion of the dst tests, the results from 8 subjects (2 males and 6 females) were considered invalid, since they had knowledge of the differing effects of the type of chocolate on the dst test results (figure 1). a total of 60 subjects met the requirements and participated in the study up to its completion. except for the baseline dsf and dsb scores, all dst scores were non-normally distributed (p>0.05), so that a non-parametric test was performed. table 1 presents the characteristics and dst measurement results in both groups at baseline. the independent t-test showed no significant differences for age, dsf scores and dsb scores between both groups (p=0.062; p=0.591; p=0.954). at 1 hour and 3 hours after intervention, mean dsf scores were not significantly different between the two intervention groups (p=0.842; p=0.204). however, at 3 hours after intervention, the dsb scores were significantly different between the two intervention groups (p=0.041) (table 2). administration of dark chocolate resulted in significantly higher dsb scores at 3 hours after intervention than did administration of white chocolate. discussion the present study demonstrates that administration of a single dose of dark chocolate significantly improved working memory of medical students after 3 hours of intervention to a higher extent than did administration of white chocolate. these results are similar to those of the study of field et al.(3) who also compared the effects of acute administration of dark and white chocolate. in the study of field et al. the cognitive function of the treatment group was better than that of the control group. the dose of chocolate in the present study was greater than that used in the study of field (100g vs 35g). for cognitive testing, field et al.(3) used visual spatial working memory, whereas we used dst. the effect of flavanols on cognitive function depends on the dose or concentration. the study by scholey et al.(17) used cocoa flavanols (cf) in differing concentrations (46 mg, 520 mg, and 994 mg). their results showed an increase in cognitive ability and mood in subjects receiving drinks containing cf at concentrations of 520 mg and 994 mg. the study by mastroiacovo et al.(18) also used drinks containing cf at similar concentrations (48 mg, 520 mg, and 993 mg). the results showed that subjects receiving cf at concentrations of 520 mg and 993 mg experienced improvement in the cognitive aspects. in our study, the subjects were given dark and white chocolate at a dose of 100 g. the flavanol content of dark chocolate is around 450 mg per 100 g, whereas that of white chocolate is approximately one-fourth that of *mann-whitney test; dsf1 : digit span forwards 1 hour after intervention; dsf3 : digit span forwards 3 hours after intervention; dsb1 : digit span backwards 1 hour after intervention; dsb3 : digit span backwards 3 hours after intervention table 2. differences in dsf and dsb scores of the control and treatment groups after intervention table 1. mean age and dst scores of the control and treatment groups at baseline dsf=digit span forwards; dsb=digit span backwards 234 prastowo, kristanto, sasmita dark chocolate improves working memory dark chocolate,(15) since white chocolate contains more additives to remove the bitter taste and improve appearance.(2) these additives may reduce the antioxidant content of white chocolate, thus reducing the effectiveness of chocolate on cognitive function.(19) dark chocolate has antioxidant properties because it contains flavanols, which occur as catechin and epicatechin monomers. (19-21) dark c h o c o l a t e i s a l s o n e u r o p r o t e c t i v e a n d neurogenetic. flavanols are lipophilic and can therefore penetrate the blood brain barrier and affect brain function.(1,2) in addition to their antioxidant effect, the neuroprotective and neurogenetic mechanisms of flavanols may occur through the interaction of flavanols with the intracellular signalling pathways, such as the regulation of mitogen-activated protein kinase (mapk) (2), oxidative stress signalling pathways (e.g. nf-κb), and expression of inflammatory genes (il-1β, tnfα).(1,22) the flavonoids used in our study and in previous studies originated from a variety of sources. the study by nurk et al.(7) used grapes, tea, and chocolate, whereas fournier et al.(23) used soy milk and an isoflavone supplement as flavonoid sources. in the present study we used chocolate as flavonoid source, considering that chocolate may be more popular and favored by younger subjects. in addition, we considered the bar form to be more practical for provision and presentation in comparison with fresh fruit, drinks or powders. in the present study, the dst scores at 1 hour after chocolate consumption did not increase significantly. this may have been associated with the peak plasma concentration. the peak plasma concentration of polyphenols is usually reached within 2-3 hours after consumption.(17,19) flavonoids reach a peak plasma concentration within an identical period, so that their effects can be seen after 2-3 hours. t h e r e f o r e i n t h e p e r i o d o f 1 h o u r a f t e r consumption the flavonoid plasma concentration may not have been sufficiently high to yield clinically significant effects. the digit span test (dst) is frequently used in neuropsychology and has been proven to effectively measure cognitive function. the test may be performed as a single test or as part of a method comprising several tests. the dst may be used in children (11) and in the elderly, both in healthy elderly or in elderly with cognitive dysfunction.(12) in the elderly, dst is more acceptable than the mini mental state evaluation (mmse),(12) while in healthy young persons, mmse is apparently too simple so that the test results may not show any differences. working memory is a combination of attention, concentration, and short-term memory that is important for processing and manipulating information that must be retained temporarily.(15) this capacity is important to medical students in following the educational process, especially i n r e c e i v i n g i n s t r u c t i o n s f r o m t u t o r s o r instructors, as well as in listening to patients at anamnesis. a good working memory may help medical students to improve their ability to diagnose a problem or disease, and to decide on the measures associated with their study or patients. the present study has several limitations that may have influenced the results. first, no true blinding could be performed, since the color of the chocolate was visible to the subjects after the removal of the wrappings. the investigators were unable to provide chocolate bars of identical color but of different type. however, blinding could still be maintained, because the subjects had no knowledge of the effects of chocolate type on verbal working memory. the subjects of this study were not informed on the effects of chocolate type on verbal working memory. second, no homogenization of the subjects was performed on the basis of academic achievement, which is an indicator of cognitive function that may affect working memory and dst scores. we propose to conduct further investigations on the effect of chocolate on cognitive facilitation, in addition to substantiation of functional brain changes associated with the components of cocoa. 235 univ med vol. 34 no.2 conclusions dark chocolate as a single dose is capable of improving verbal working memory in students, at 3 hours after its consumption. since cocoa contains multiple bioactive compounds, one approach might be to examine the neurocognitive effects of combinations of potential functional ingredients conflicts of interest this study was not associated with any parties, both financially and non-financially. the cost of the whole study was borne by the investigators themselves without any support of sponsors. the results of this study are solely intended for scientific advancement and not to provide commercial benefits to businesses or certain brands of chocolate. acknowledgements the investigators thank the students of the faculty of medicine, atma jaya university, who agreed to become study subjects. we also thank dr. jimmy barus, sps for useful input for correction of the manuscript. references 1. rendeiro c, guerreiro jdt, williams cm, et al. flavonoids as modulators of memory and learning: molecular interactions resulting in behavioural effects. proc nutr soc 2012;71:24662. 2. mcshea a, ramiro-puig e, munro sb, et al. clinical benefit and preservation of flavonols in dark chocolate manufacturing. nutr rev 2008; 66:630-41. 3. field dt, williams cm, butler lt. consumption of cocoa flavanols results in an acute improvement in visual and cognitive functions. physiol behav 2011;103:255-60. 4. d’esposito m. from cognitive to neural models of working memory. phil trans r soc b 2007;362:761-72. 5. budson ae, price bh. memory dysfunction. n engl j med 2005;352:692-9. 6. alhola p, polo-kantola p. sleep deprivation: impact on cognitive performance. neuropsychiatr dis treat 2007;3:553-67. 7. nurk e, refsum h, drevon ca, et al. intake of flavonoid-rich wine, tea, and chocolate by elderly men and women is associated with better cognitive test performance. j nutr 2009;139:1207. 8. barak o, tsodyks m. working models of working memory. neurobiol 2014;25:20-4. 9. groth-marnat g, baker s. digit span as a measure of everyday attention: a study of ecological validity. percept mot skill 2003;97: 1209-18. 10. choi hj, lee dy, seo eh, et al. a normative study of the digit span in an educationally diverse elderly population. psychiatr investig 2014;11: 39-43 11. leung jlm, lee gth, lam yh, et al. the use of the digit span test in screening for cognitive impairment in acute medical inpatients. int psychogeriatr 2011;23:1569-74. 12. rosenthal en, riccio ca, gsanger km, et al. digit span components as predictors of attention problems and executive functioning in children. arch clin neuropsychol 2006;21:131-9. 13. sastroasmoro s, ismail s. dasar-dasar metodologi penelitian klinis. edisi 4. sagung seto, jakarta. 2011 14. grassi d, lippi c, necozione s, et al. shortterm administration of dark chococolate is followed by a significant increase in insulin sensitivity and decrease in blood pressure in healthy persons. am j clin nutr. 2005;81:6114. 15. meng cc, jalil amm, ismail a. phenolic and theobromine contents of commercial dark, milk and white chocolates on the malaysian market. molecules 2009;14:200-9. 16. conway ara, kane mj, bunting mf, et al. working memory span tasks: a methodological review and user’s guide. psychonom bull rev 2005;12:769-86. 17. scholey ab, french sj, morris pj, et al. consumption of cocoa flavanols results in acute improvements in mood and cognitive performance during sustained mental effort. j psychopharmacol 2010;24:1505-14. 18. mastroiacovo d, kwik-uribe c, grassi d, et al. cocoa flavanol consumption improves cognitive function, blood pressure control, and metabolic profile in elderly subjects: the cocoa, cognition, and aging (cocoa) study-a randomized controlled trial. am j clin nutr 2015;101:53848. 236 prastowo, kristanto, sasmita dark chocolate improves working memory 19. corti r, flammer aj, hollenberg nk, et al. cocoa and cardiovascular health circulation 2009;119:1433-41. 20. agarwal sk. dark chocolate: a therapeutic food. med sci 2013;5:13-5. 21. wan y, vinson ja, etherton td, et al. effects of cocoa powder and dark chocolate on ldl oxidative susceptibility and prostaglandin concentrations in humans. am j clin nutr 2001; 74:596-602. 22. williams rj, spencer jpe. flavonoids, cognition, and dementia: actions, mechanisms, and potential therapeutic utility for alzheimer disease. free radic biol med 2012;52:35-45 23. fournier lr, ryan-borchers ta, robison lm, et al. the effect of soy milk and isoflavone supplements on cognitive performance in healthy postmenopausal women. j nutr health aging 2007;11:155-64. c:\users\universa medicina\docu 162 abstract universa medicina limited health literacy increases the risk of orthorexia nervosa among urban schoolteachers gülay yilmazel* and serpil bozdogan** background orthorexia nervosa (on) describes a pathological obsession with proper nutrition that is characterized by a restrictive diet, ritualized patterns of eating, and rigid avoidance of foods believed to be unhealthy or impure. limited health literacy may play a role in the onset and progression of orthorexia. the aim of this study was to determine the relationship between health literacy and on among urban schoolteachers. methods this cross-sectional study was conducted in central black sea region of turkey with 420 primary and secondary schoolteachers aged between 18 and 51 years. a questionnaire form including socio-demographic characteristics was used. the orthorexia nervosa questionnaire (orto15) was used to assess orthorexia nervosa behavior and the turkey health literacy scale (tsoy-32) to assess health literacy. simple binary and multiple binary logistic regression analyses were carried out to verify the associations between the variables. results of the study group 46.4% were in the 40-49 year age group, 53.8% were male, 78.6% had on and 93.6% had limited health literacy. nearly all of the orthorexics (96.4%) had limited health literacy. female gender, instagram use and limited health literacy was significantly associated with on. limited health literacy increases the risk of on 4.85 times among teachers (aor=4.85;95% c.i. : 2.15-10.94;p=0.000). conclusion the current findings suggest that limited health literacy is the strongest risk factor for on among urban schoolteachers. school health literacy and social media literacy programs can open a new window into revealing on. keywords : urban, schoolteachers, psychological eating disorder, health literacy original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.162-170 september-december 2020 vol.39no.3 ci te t his article as: yi lma zel g, bozdogan s. limited health literacy increases the risk of orthorexia nervosa among urban schoolteachers. univ med 202 0;39 :1 6 2 -7 0 . doi : 10 .18 05 1/ univmed.2020.v39.162-170 *hitit university faculty of health science, public health department, çorum/turkey **hitit university health science institution, çorum, turkey corespondence: assoc. prof. dr. gülay yilmazel hitit university faculty of health sciences, public health department, corum, turkey mobile: +903642230730-31 email : gulcenarin@yahoo.com orcid id: 0000-0002-2487-5464 date of first submission, june 19, 2020 date of final revised submission, october 6, 2020 date of acceptance, october 12, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 163 univ med vol. 39 no.3 introduction noncommunicable diseases are one of the most important health problems of the 21st century and responsible for 41 million of the 57 million deaths in 2016 at the global level. unhealthy diet is one of the key risk factors for noncommunicable diseases.(1) unhealthy diet pattern has increased worldwide.(2) the pattern of nutrition shows a shift towards foods containing high salt, fat and sugar. rapidly growing obesity prevalence due to sedentary lifestyle leads to a dramatic increase in diabetes, cardiovascular diseases, hypertension, osteoarthritis, cancer and many other health problems.(3,4) in developed societies, the main focus of the people is consciousness on healthy nutrition and selectivity towards the quality, quantity and type of food. developing a fixed idea on the amount or type of food often causes an irregular dietary pattern and excessively results in psychological eating disorders.(5) dissatisfaction with one’s body, distorted body image perception and obsession with thinness are central to clinically diagnosed eating disorders (ed) such as anorexia nervosa (an), bulimia nervosa (bn), binge eating disorder (bed) and eating disorders not otherwise specified (ednos).(6) these factors may also contribute to other non-clinical types of disorders including orthorexia nervosa (on).(7) orthorexia nervosa defines a pathological obsession for proper nutrition, characterized by a restrictive diet, ritualized eating pattern, and strict avoidance of unhealthy or supplemented foods.(8) the mean prevalence of orthorexic symptoms is reported to be 6.9% (9) for the general population and 28.3%-74.5% for high risk professions (dietetics, medicine, nursing, physiotherapy, midwifery, optometry, physical education and sport sciences, sociology, and teaching).(7,10) in tur key, on pr evalence is high (approximately 50%) among candidate doctors and nurses.(11, 12) to assess on, the orthorexia nervosa questionnaire (orto-15)(13) is widely used, despite criticism regarding this research tool.(14) a study suggested that the orto-15 questionnaire may not be able to distinguish pathological behaviors and is not clinically relevant.(15) however, a recent review of the studies using orto-15 showed that cronbach’s alpha coefficients were ranging from 0.83 to 0.91.(16) in the original version of orto-15, to distinguish the orthore xic tendency it is recommended to use the cut-off point below 40 (17 ), and so me studies follo wed this recommendation.(18,19) nevertheless, there are also suggestions to lower the cut-off point to 35.(18,20) in recent years, the density and popularity of social media channels have been blamed for the increased incidence of eating disorders worldwide.(21-24) previous studies have revealed that individuals with adequate health literacy consume less sugar-sweetened drinks and fried foods, and better amounts of vegetables and fruits.(25,26) health literacy refers to the competencies related to accessing, understanding, appraising and applying health information in the domains of healthcare, disease prevention and health promotion, respectively.(27) since its importance is increasingly recognized, to date, various frameworks or scales are available for assessing and measuring health literacy based on different subjects, diseases, or theoretical foundations.(2832) to measure health literacy in the general population, there are also different measurement tools such as the european health literacy survey (hls-eu-q47)(33) and turkish health literacy scales.(34) however, no reliable, definitive, and comparable health literacy scale exists for the global population. in turkey, about seven of every ten people has limited health literacy level (34,35) and orthorexia nervosa prevalence reaches 75%.(36) risk factors or characteristics that were listed by professionals to be associated with on were excessive exercise,(37) anxiety, use of social media, thin ideal internalization, and harm avoidance and low self-directedness.(38) however, risk factors that may be particularly relevant to on could include problems with health literacy.(8) 164 teachers are model people in creating healthy lifestyle behaviors and providing health education i n s c h o ol s. t he r e h a s be e n no d e t a i l e d investigation of orthorexia tendency and health literacy among teachers. the aim of this study was to determine the relationship between health literacy and orthorexia nervosa among urban schoolteachers. methods research design this cross-sectional study was conducted between october-december 2019 in corum city located in central black sea region of turkey. study subjects the population consisted of 1914 teachers working in urban primary and secondary schools. based on the prevalence of on of 45% in our country(13), the minimum sample size was calculated as 317 teachers. participants were recruited from nine public schools (four primary and five secondary) covering the urban area. a total of eligible 442 teachers from primary and secondary schools were included to the study with the stratified sampling method. participants with diagnosed diet-related diseases (diabetes mellitus type i and ii, crohn’s disease, celiac d is e a se , ga s t r i t is ) we r e e xc l ud e d ( 2 2 participants). instruments participants were visited at their schools and completed an informed consent form, then received a survey including sociodemographic information, the use of social media, orto-15 and turkey health literacy scale-32 (tsoy32) questionnaire. the survey took 20-30 minutes to answer the questionnaire. three visits were made for each participant who could not be reached. the data was collected with a 64-item form. the first stage of the questionnaire form included the participants’ socio-demographic characteristics (age, gender, institution, branch, chronic condition, health perception, weight satisfaction, height and weight to calculate bmi), and details about the use of social media. social media details participants were asked “which social media channels do you use?” and could select multiple responses out of: instagram, facebook, twitter, pinterest, google+, youtube, snapcheck and linkedin. in the second stage, in order to identify orthorexia tendency and to assess health literacy levels, respectively, the orto-15 scale and tsoy-32 scale were used. orthorexia nervosa (orto-15) scale the scale consists of 15 items and is written in 4-degree format; always (4), often (3), sometimes (2), never (1). items 2, 5, 8 and 9 (items 3, 4, 6, 7, 10, 11, 12, 14, 15) scored on the s c a l e a r e r e ve r se d. t he a n sw e r s wh i c h distinguish criteria for orthorexia, were given as “1”, and those with a tendency to normal eating behavior were given as “4”. a minimum of 15 and a maximum of 60 points can be obtained from the scale. the cut-off point of the scale was considered to be 40 in predicting orthorexic behavior and tendency. those with orto-15 scale score of <40 were considered to be orthorexic and those with a score of 40 were evaluated as normal.(13) an adaptation of the scale into turkish was conducted in 2008.(39) in this study the original cut-off of <40 was considered as indicative of orthorexia nervosa. turkey health literacy scale (tsoy-32) the 32-item scale’s validity and reliability were developed based on the hls-eu study co nc e p tua l fr a me wo r k ( hl s-eu consortium, 2012) in 2016. the scores obtained from the scale vary between 0 and 50. according to the scores, the level of health literacy is classified into four categories: 0-25 = inadequate health literacy; >25-33 = problematic health literacy; >33-42 = adequate health literacy; >42-50 = excellent health literacy.(34) in this study, the inadequate/problematic health yilmazel, bozdogan orthorexia and limited health literacy 165 univ med vol. 39 no.3 a b c literacy of the participants was categorized as “limited” and adequate/excellent health literacy as “sufficient”. statistical analysis data management and analysis were performed using spss software (version 17). simple binary and multiple binary logistic regression analyses were used. to predict significant factors for orthorexia tendency, odds ratio (or) and 95% confidence interval (ci) were calculated. significance levels were set at the 5% level. ethical clearance the research was planned in accordance with helsinki principles. prior to undertaking the investigation ethical clearance was obtained from a local non-interventional clinical research ethics committee (2019-324). results table 1 presents associations of several risk factors and orthorexia nervosa. in general, 78.6% teachers were orthorexics and the mean score of the orto-15 scale was 37.4 (sd=3.5). in the study group, 46.4% were between 40-49 years of age, 53.8% were male, 78.6% were secondary schoolteachers and the mean age was 43.4±7.5 years. a minority of participants (21%) indicated that they had any chronic disease and almost three out of four teachers had good health perception. just over half of those satisfied with body weight and had normal bmi. orthorexia nervosa was significantly more common among variables orthorexia nervosa orthorexic (n=330, 78.6%) normal (n=90, 21.4%) or 95% c.l. p-value age (yrs) 25-39 101 (30.6) 25 (27.8) 0.96 0.52-1.77 0.873 40-49 152 (46.1) 43 (47.8) 0.79 0.37-1.70 50-64 77 (23.3) 22 (24.4) 1 gender male 169 (51.2) 57 (63.3) 0.97 0.37-0..98 0.041 female 161 (48.8) 33 (36.7) 1 institution primary school 70 (21.2) 20 (22.2) 0.94 0.51-1.73 0.836 secondary school 260 (78.8) 70 (77.8) 1 chronic health problem yes 68 (18.2) 20 (31.2) 1 no 262 (81.8) 70 (68.8) 1.06 0.52-2.16 0.698 health perception good 249 (75.5) 66 (73.3) 1 moderate/poor 81 (24.5) 24 (26.7) 1.15 0.60-2.20 0.680 weight satisfaction satisfied 188 (57.0) 55 (61.1) 1 no satisfied 142 (43.0) 35 (38.9) 0.83 0.45-1.53 0.481 bmi normal 177 (53.6) 45 (50.0) 1.39 0.24-2.15 0.513 overweight 123 (37.3) 39 (43.3) 1.24 0.68-2.262 obese 30 (9.1) 6 (6.7) 1 use of social media instagram 196 (59.4) 39 (43.3) 1.73 1.03-2.89 0.036 no instagram user 134 (40.6) 51 (56.7) 1 health literacy limited 318 (96.4) 75 (83.3) 4.95 2.16-11.37 0.000 adequate 12 (3.6) 15 (16.7) 1 table 1. associations of several risk factors and orthorexia nervosa 166 females (p<0.05). there were no significant associations between orthorexia nervosa and age groups, institution, chronic health problem, health perception, weight satisfaction, bmi (p>0.05). more than half of the study group stated that they used instagram and orthorexia nervosa was found to be significantly more common among instagram users (p<0.05). overall, the limited health literacy rate was 93.6% and the sufficient health literacy rate was 6.4%. the mean score was 19.8 (sd=8.9) for the tsoy-32 scale. limited health literacy was significantly more common among orthorexics (p<0.001). multiple binary logistic regression analysis of variables affecting on is given in table 2. on was 4.85 (aor=4.85;95% c.i.=2.15-10.94; p=0.000) times higher in those with limited health literacy, 1.69 (aor=1.69;95% c.i.=1.04-2.75; p=0.033) times higher among instagram users and 0.51 (aor=0.51;95% c.i.=0.29-0.91; p=0.041) times lower among males. discussion al t h ou gh i t d o e s n o t a pp ea r i n t he diagnostic and statistical manual of mental disorders (dsm), on has created a research area. the diagnostic criteria recommended for on include focus on healthy eating, food anxiety and obsession with dietary restrictions, and that these behaviors cause clinical disorders.(6, 40) despite the fact that orthorexic individuals strive for goodness, such behavior often leads to significant negative effects on their quality of life. in extreme cases, orthorexic individuals prefer to be hungry instead of eating the “wrong” food.(41,42) beliefs in maintaining optimal health, the desire to stay healthy and fit and the necessity to manage non-communicable diseases make individuals more sensitive to nutrition. the present study fills a gap in the literature by exploring health literacy in orthorexics and provides an exciting opportunity to advance our knowledge of orthorexia nervosa. in this study, four of every five teachers were determined to be orthorexics. it has been stated that the prevalence of orthorexia nervosa varies according to the communities. a total of 6.9% of the general population in germany (9) and 10.9% of young adults in italy (43) are orthorexic. it was reported that the prevalence in america is less than 1%.(15) in hungarian and lebanese studies, the prevalence of orthorexia has been shown to be 74.2% and 74.5%, respectively.(10,44) on the other hand, in studies conducted in our country, the prevalence of orthorexia was observed to be spread over a wide spectrum (12.0-75.8%).(12, 36, 45). the use of different instruments and cut-off points depending on cultural or religious backgrounds may be explain these differences. however, the results obtained from the present study were higher than those from the other studies conducted abroad and was in line with the hungarian and lebanese studies. in our study such a higher prevalence was anticipated as being due to the educator group and suggests poor knowledge of nutrition among teachers. in the present study, it was determined that almost all of the teachers had limited health literacy. in sri lanka (46) 32.5% of teachers and in turkey (47) 73.8% of teachers were shown to have an insufficient / problematic health literacy level. it has been reported that health literacy is moderate in iranian teachers.(48) contrary to expectations, the present study exhibits an unignorable level of low health literacy among teachers. insufficient health competence of teachers was a thoughtful result. one of the variables adjusted or 95%cl p-value limited health literacy 4.85 2.15-10.94 0.000 instagram use 1.69 1.04-2.75 0.033 male 0.51 0.29-0.91 0.041 table 2. multiple logistic regression analysis of variables affecting orthorexia nervosa yilmazel, bozdogan orthorexia and limited health literacy 167 univ med vol. 39 no.3 issues that emerges from this finding is updating teachers’ knowledge about health protection, development and treatment of diseases. it suggests that school health literacy programs are urgently needed. however, in our study nearly all of the orthorexics had a limited level of health literacy and it has been determined that the strongest risk factor in orthorexia nervosa was limited health literacy. as a matter of fact, a turkish study conducted in university students revealed that the level of health literacy in orthorexics is lower.(45) it can be said that normalization of nutritional behaviors in orthorexic individuals is possible by a sufficient level of health literacy. health literacy can be considered as an important aspect of orthorexia and adequate health literacy may be protective against orthorexia nervosa. gender differences in the occurrence of or thorexia nervosa have not been clearly demonstrated. as a matter of fact, in both national and international studies, there was evidence that the tendency to orthorexia differs between genders.(49,50) on the other hand, a few studies have reported that orthorexic trend did not differ in terms of gender.(39,51) in the present study, females had more orthorexics and higher risk of on than males. this result may be explained by the behaviors to be expected in women with nutrition-sensitive approaches due to their specific life periods such as adolescence, reproductive age, pregnancy, lactation and menopause. instagram and social media use are more broadly related to mental health problems. social media use in young adults has been associated with high levels of depression(23) as well as eating disorders.(24) in the present study, instagram use was another risk factor for orthorexia. indeed, a study conducted in the united kingdom reported that the use of instagram was associated with increased symptoms of orthorexia nervosa.(52) social media and on are interconnected, especially in the field of health promotion.(53) the present study was limited to urban primary and secondary area teachers. other limitations of the current study include the following: (i) the use of the orto-15 tool due to its limited ability to accurately define orthorexia; (ii) the use of the tsoy-32 tool due to lack of a global health literacy scale. orthorexia is a growing public health concern worldwide. despite its poor outcomes, there remains a paucity of evidence on orthorexia and limited health literacy. overall, these findings provide an initial indication of the role that limited health literacy may play in the onset and progression of orthorexia. clinical screening of orthorexia nervosa should be planned for teachers in the preand post-graduation stage. school health literacy and social media literacy programs can open a new window into normalization of nutrition behaviors. future studies on the current topic are therefore recommended. conclusions the current findings suggest that on is framed by limited health literacy among urban primary and secondary schoolteachers. school health literacy programs can open a new window into revealing orthorexic tendencies among teachers. conflict of interest co mp e t i ng i n t e r e s t s : n o r e l e va nt disclosures. contributors gy contributed to the study conception, design, and data acquisition; sb analyzed the data; gy and sb interpreted the data and wrote the manuscript; gy was involved in critically revising the manuscript. all authors have read and agreed to the published version of the 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literacy and factors associated with it among school teachers in an education zone in colombo, sri lanka. bmc public health 2017;17: 631. doi: 10.1186/s12889-017-4543-x 47. yilmazel g, çetinkaya f. health literacy among schoolteachers in çorum, turkey. east mediterr health j 2015;21:598-605. doi:10.26719/ 2015.21. 8.598. 170 48. asadian a, sotoudeh a, zarei venovel m, moosai r. investigating health literacy of teachers in asaluyeh. soc behav res health 2018;2:234-28. doi: 10.18502/sbrh.v2i2.284. 49. parra-fernández ml, rodríguez-cano t, onievazafra md, et al. prevalence of orthorexia nervosa in university students and its relationship with psychopathological aspects of eating behaviour disorders. bmc psychiatry 2018;18:364. doi: 10.1186/s12888-018-1943-0. 50. yeşil e, turhan b, tatan d, şarahman c, saka m. the effect of gender to orthorexia nervosa tendency in adults. j ankara health sci 2018;7:19. 51. brytek-matera a, donini lm, krupa m, poggiogalle e, hay p. orthorexia nervosa and selfattitudinal aspects of body image in female and male university students. j eat disord 2016;16:16. doi:10.1186/s40337-015-0038-2. 52. turner pg, lefevre ce. instagram use is linked to increased symptoms of orthorexia nervosa. eat weight disord 2017;22:277–84. doi: 10.1007/ s40519-017-0364-2. 53. robert m, callahan l, o’leary c. social media: a path to health literacy. stud health technol inform 2017;37:177-87. doi:10.3233/isu-170836. yilmazel, bozdogan orthorexia and limited health literacy january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 covid-19 and the ageing immune system in an elderly patient : a case report ida ayu pradnya paramita1 and i gusti putu suka aryana2* abstract background the severity of covid-19 infection has an increasing trend in the elderly, which contributes to the high morbidity and mortality rates in this population. aging itself is a prominent risk factor for severe disease and death from covid-19. casedescription this case report a 71-year-old woman who complained of shortness of breath for 3 days before being admitted to the hospital. bilateral consolidation and increased bronchovascular pattern were found on chest radiograph, and a positive sars-cov2 nasopharyngeal swab pcr test result was noted. this patient was diagnosed with confirmed severe manifestation of covid-19, community-acquired pneumonia and type 1 respiratory failure, as well as type ii diabetes mellitus and suspicion of acute gastritis. the results of the geriatric status assessment were moderate functional status, risk of malnutrition, and moderate risk of deep vein thrombosis (dvt). this patient underwent treatment in accordance with the covid-19 protocol along with management for geriatric status improvement. the patient was given permission to return home after 14 days of treatment, during which time her health had improved and her functional status had changed to moderate dependency. during follow up, the patient continued to receive therapy. she is still being observed and future evaluations will be conducted. conclusion the increased susceptibility of the elderly to covid-19 infection is caused by various factors. a burden of death and long-term disability brought on by this pandemic may be lessened by new or modified therapies that target aging-associated mechanisms. therefore, covid-19 case management in this population should be done with a comprehensive approach. keywords: covid-19, geriatric, immunity, pneumonia, woman 1medical residency program of internal medicine, faculty of medicine, universitas udayana, sanglah general hospital, denpasar, bali, indonesia 2department of internal medicine, faculty of medicine, universitas udayana, sanglah general hospital, denpasar, bali, indonesia *correspondence: i gusti putu suka department of internal medicine, faculty of medicine, universitas udayana, sanglah general hospital, denpasar, bali, indonesia email: aryanaptsuka_aryana@unud.ac.id orcid id: 0000-0001-8582-2254 date of first submission, january 6, 2022 date of final revised submission, january 12, 2023 date of acceptance, january 18, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2023.v42:101-107 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1276 101 case report cite this article as: para mita iap, aryana igps. ecovid-19 and the ageing immune system in an elderly patient: a case report. univ med 2023 ;42 :101-7 . doi: 10.18051/ univmed.2023.v42:101-107. mailto:aryanaptsuka_aryana@unud.ac.id https://orcid.org/0000-0001-6560-4153 https://orcid.org/0000-0001-8582-2254 http://dx.doi.org/10.18051/univmed.2023.v42:101-107 https://univmed.org/ejurnal/index.php/medicina/article/view/1276 paramita, aryana covid-19 geriatric immunity 102 introduction the covid-19 pandemic that has spread in almost all countries in the world requires clinical and theoretical research related to various populations with di ffer ent demographic characteristics. previous studies have reported that chronic comorbi dities of diabetes, hypertension, and c ardiovascular disease aggravate the infectious condition of the sarscov2 virus.(1) global molecular studies have narrowed down the pathogenesis of this viral infection to the interface between viral surface proteins and ace-ii receptors on pulmonary alveolar epithelial cells.(1,2) these findings are the reasons for several other factors that strengthen the findings regarding epidemiological data, which generally state that the elderly population is the group that is most affected by or most susceptible to this infection, have a history of comorbidities, and decreased numbers or polymorphisms of ace-ii receptors.(2) data from the centers for disease control and prevention (cdc) reports that although the elderly population, or people over the age of 65, only covers 17% of the total population in the united states, this population accounts for up to 31% of total infections, 45% of hospital admission rates, 53% of rates for intensive care, and 80% of deaths due to covid-19 infection.(3) in indonesia until early june 2020 it was shown that the highest proportion of deaths was identified among the elderly (43.8%). the mortality rate indicated a similar trend in which the elderly contributed to the highest rate (17.69%).(4) these data indicate that the severity of covid-19 infection tends to increase, resulting in higher mortality or more severe clinical manifestations in the elderly population. numerous case reports and studies on covid-19 cases that affect elderly people and other vulnerable groups have been published. these reports and studies demonstrate the evidence of an aging immune system and a decline in both its strength and function. a number of changes to the body’s physiological systems occur as a result of the complicated phenomenon of aging. immunosenescence, one of the most significant changes, affects the immune system. given that aging is linked to high rates of morbidity and mortality from numerous diseases, the weakened immune function of the elderly is clinically evident.(5) immunosenescence, or the loss of immune function and diminished immunity to infectious pathogens with aging, is brought on by intricate processes affecting immune cell development and maintenance as well as the origin, maintenance, and termination of appropriately targeted immunological responses.(6) however, the inc idenc e of posthospitalization covid-19 complications, which need additional follow-up and is related to the maturation of the immune system against the covid-19 virus itself, cannot be explained by the pertinent scientific data. throughout this case report, it will be clearly explained how to tackle covid-19 in elderly instances as well as how to treat these patients properly after therapy. the goal of this case report is to learn more about the phenomena that arise in geriatric covid-19 instances and call for a variety of therapeutic stances. based on the data above, the authors are interested in knowing about the differences in clinical symptoms as well as the impact of treatment and therapy needed in the elderly with covid-19 infections. case report a 71-year-old woman came to the hospital complaining of shortness of breath for 3 days before admission. she complained of coughing up yellow phlegm since 3 days ago. she also complained of fever for 3 days (less than 38c), she merely laid in bed as she became weaker and weake r. she had a sore throat and experienced a decrease in appetite accompanied by nausea and heartburn. following questions on secondary infections, cancer, or other related diseases, the patient denied having any illnesses that were related to immunocompromised disorders. 103 the patient had a history of diabetes mellitus since 2016, but she was not undergoing routine treatment. physical examination showed that the body mass index (bmi) was 21.5 kg/m(2) and that the patient was compos mentis. the blood pressure was 130/80 mmhg, pulse rate 108 beats per minute, respiratory rate 28 per minute, temperature 37.8c, and oxygen saturation 90% on room air. there were only additional crackles heard in both lungs on auscultations. there was consolidation in the upper and lower zones of both lungs with increased bronchovascular markings, an impression of pneumonia (figure 1). clinical laboratory findings are shown in table 1. the patient was d ia gn os e d a s h a vi ng se ve r e co v id -19 , community acquired pneumonia (cap) and type1 respiratory failure, type-ii diabetes mellitus, and suspected acute gastritis. a comprehensive geriatric assessment was mandatory, in which the functional status using the activity of daily living (adl) barthel index found the patient to be moderately dependent and without delirium, while from the assessment of nutritional status the patient was found to be at risk of malnutrition. she was in a state of mild cognitive impairment, not depressed and had no incontinence. she was at moderate risk for deep vein thrombosis using the wells score system. she had low risk on the morse fall scale. she was in a pre-frail condition and there was no failure to thrive. based on the fracture risk assessment tool (frax) score, there was a moderate risk of major osteoporosis. there was mild visual and hearing impairment, but no physical disability. the patient was placed in an isolation treatment room and given oxygen therapy, dietary nutrition at 1500 kcal per day and 75 grams of protein per day, levofloxacin 1x750 mg intravenously for 7 days, remdesivir loading dose 1 x 200mg i nt r a ve nous l y on da y 1 a nd 1 x 10 0mg intravenously on days 2-5, dexamethasone 2x5mg intravenously, paracetamol 3x500 mg orally, omeprazole 2 x 40 mg intravenously, vitamin c 2x500 mg, insulin aspart 3x4 units subcutaneously and insulin glargine 14 units every 24 hours subcutaneously and enoxaparin subcutaneously 0.6 ml every 24 hours. th e r e we r e s e ve r al po s t-tr e a t me nt situations that significantly improved in terms of both general health and the findings of patient examinations, including physical examinations and laboratory tests, which also showed a satisfactory improvement. additionally, the figure 1. thorax photo of the patient’s chest when she was first admitted to the hospital univ med vol. 42 no. 1 paramita, aryana covid-19 geriatric immunity 104 patient was monitored more closely while receiving care at home, and her condition improved. the patient was informed of the scientific and clinical interest in her disease as well as of this anonymous publication. she gave informed verbal consent to the anonymous publication. discussion according to a study, elderly people are more frequently affected by sars-cov-2 infection than are people of younger ages, and test parameter values complete blood count wbc (103/µl) neu (103/µl) lym (103/µl) mono(103/µl) eos (103/µl) baso (103/µl) 5.16 4.42 0.49 0.23 0.01 0.01 hb (g/dl) 11.61 hct (%) 34.23 mcv (fl) 83.93 mch (pg) 28.47 mchc (%) 31.29 plt (103/µl) 356.70 blood chemistry sgot (u/l) 53.0 sgpt (u/l) 30.0 bun (mg/dl) 25.90 sc (mg/dl) 1.06 e-gfr 124.04 albumin (g/dl) 3.30 blood glucose (mg/dl) 245 hba1c (%) 13.6 total cholesterol (mg/dl) 178 hdl (mg/dl) 37 ldl (mg/dl) 116 triglycerides (mg/dl) 140 electrolytes k+ (mmol/l) 4.10 na+ (mmol/l) 131 blood gas analysis ph 7.41 pco2 38.7 po2 88.8 base excess (g/dl) 3.3 hco3 24.1 so2 (%) 89 real time-pcr reactive   table 1. clinical laboratory findings of the patient e-gfr: glomerular filtration rate; bun, blood urea nitrogen; hb: hemoglobin; hct: hematocrit; hdl: high-density lipoprotein; ldl: low-density lipoprotein; mcv: mean corpuscular volume; mch: mean corpuscular hemoglobin; mchc: mean corpuscular hemoglobin concentration; plt: platelets; sc: serum creatinine; sgpt: serum glutamic pyruvic transaminase; sgot: serum glutamic oxaloacetic transaminase; wbc: white blood cells this association is more frequently negative.(7) based on a retrospective analysis of 85 patients who had died in wuhan from sars-cov-2 infection, the patients’ median age was 65.8 years.(8) numerous immune system biochemical changes brought on by aging are associated with age-related disorders and increased vulnerability to communicable diseases. senescent immune cell accumulation contributes to the immune system’s decline as it ages, while concurrently increasing inflammatory phenotypes lead to immunological dysfunction. these two processes are known as immunosenescence.(9) 105 atypical symptoms found in elderly patients with cov id-19 are de lirium, low-grade hyperpyrexia, and abdominal pain.(10) a consensus from the infectious disease society of america recommends guidelines for the definition of fever in the elderly, namely the result of measuring an oral temperature of more than 37.8oc.(11) the presence of dna methyltransferase inhibition in treg cells can accelerate the resolution of lung injury in younger persons, whereas in the elderly there is hypermethylation o f tr e g ce l l dn a d u e to mito c h on dr i a l dysfunction caused by the accumulation of toxic metabolites and reactive oxygen species in this population.(12) there are differences in ace-ii levels in lung tissue compared to the young population, which according to several studies showed a decrease in ace-ii expression in the e l d e r l y popu l a t i on . ( 1 0 , 1 3 ) ace -ii ge n e polymorphism was also found in exon 19 of chromosome x p.22.2 in the elderly. one polymorphism that is known to increase mortality in the elderly with covid-19 is the deletion of intron 16 in the ace-ii gene.(2) t cells play a role in the adaptive ability of immunity to increase antigen-specific memory t cells, which can predispose to the development of severe covid-19 infection, increased length of hospitalization and the occurrence of acute respiratory distress syndrome (ards).(12) the population sh ows a continuous production of inflammatory mediators and cytokines which is often known as inflammaging. expression of toll-like receptor (tlr) may increase but the function of the t cells decreases, so that the antigen presenting cell (apc) response in the face of viral infection becomes inappropriate which ultimately triggers excessive cytokine pr oduction. (1 0,1 4) aging-r elated immunosenescence is associated with chronic secretion of pro-inflammatory cytokines known as senescence associated secretory phenotype (sasp). accumulation of sasp cells during aging can lead to persistent withdrawal and activation of effector immune cells, which impairs local communication betwee n proand antiinflammatory systems, leading to tissue damage and incorrect tissue repair.(12) the administration of chloroquine in the elderly must be very closely monitored regarding its lethal effects through increased qt interval and hypoglycemia. administration of tocilizumab is contraindicated in the elderly with a history of diabetes, bacterial infection and the use of corticoste roids, to avoid superimposed infection.(10,15,16) patients with complications of sepsis accompanied by a d dimer level of 3 g/ ml and a coagulation score of >4 can be given anticoagulant thera py with direct or al anticoagulant (doac) to prevent thromboembolic events.(10) in elderly patients infected with covid19, the inflammatory reaction produced on the surface of the lung epithelium makes it difficult to remove the bacteria so that secondary bacterial infections often occur and can worsen the prognosis.(1,3,16) based on the national institute for health and care excellence (nice) guidelines, the administration of antibiotics to the elderly over the age of 75 years with covid-19 infection who have pneumonia due to secondary bacterial infection, should take place at least 4 hours after the onset of infection.(16) the use of corticosteroids is controversial in viral infections due to their immunosuppressant effects.(17) in addition, it should be noted that the use of steroids in the elderly could have metabolic effects that could trigger comorbidities in the elderly such as hypertension, diabetes, bone fractures and cataracts.(10,18) nutrition plays an important role in the management of covid-19 infection in the elderly, where good nutrition will help improve the immune system of the elderly.(19,20) most of the elderly suffer from nutritional disorders, especially malnutrition. the caloric requirement in patients aged over 65 years with multiple comorbidities is 20 to 30 kcal/kgbw/day whereas the target of 30 kcal/kgbw/day should achieved slowly over 3 to 4 days to avoid the risk of refeeding syndrome. protein needs in the elderly with covid-19 infection should be higher due univ med vol. 41 no 2 paramita, aryana covid-19 geriatric immunity 106 to increased protein catabolism due to proinf lammatory media tor s wher e protein requirements are estimated to be 1g/kgbw/day – 1.5g/kgbw/day. the recommended ratio of fat to carbohydrates is 30:70 in patients without respiratory deficiency and 50:50 in patients who use ventilation.(20,21) immunosenescence in elderly patients can be triggered by malnutrition.(14,20) micronutrient intake in the elderly infected with covid-19 must exceed the recommended daily intake because infectious conditions can reduce the body’s mi cronutrient leve ls. micronutrients that must be considered include vitamin c which functions in increasing the immune system which is recommended to be consumed at 24g/day for 7 days. zinc deficiency can increase the risk of infection, where studies examining the effects of zinc administration show that zinc supplementation can increase natural killer cell activity through increased expression of perforins.(22) our patient was observed to be in good health and to be devoid of covid-19-related comorbidities and sequelae at the post-treatment check-up. although complaints including weakness and occasionally dizziness still exist, they are more likely to be caused by the influence of multiple additional co-morbidities than because of covid-19 directly. elderly care at home has an important role in treating covid-19 in the elderly. the related facilities must prepare several pharmacological therapies that can be used for the elderly experiencing covid-19 infection. mortality of the elderly with covid-19 is higher in nursing home facilities than in hospitals so that service providers must be prepared about administering drugs that can help relieve suffering before death..(11) in the case of elderly living in isolation from daily activities without access to communication with their families, this can lead to a worsening of their perception of reality, changes in sleep patterns, and delirium. service providers for the elderly must be ready with psychological support for their patients during this pandemic to minimize the incidence of depression in these elderly.(12) conclusion information on covid-19 immunopathology is still limited and our understanding of the disease is evolving rapidly. thus, the current evidence may soon change with the accumulation of new knowledge of sars-cov-2 biology and host immune responses. assessment of geriatric status must be carried out comprehensively and the complexity of management in elderly patients infected with covid-19 needs to be carefully considered. conflict of interest the authors report no conflicts of interest in this work. acknowledgement the authors would like to thank all colleagues who contributed with the exchange of experiences, information, and discussion to this case report funding statements the authors receive no grants during this study conducted. author contributions igpsa contributed to diagnosis and was responsible for the management of this patient. iapp contributed to revision of the manuscript. both authors contrib uted equally to the presentation of the case report. all authors have read and approved the final manuscript. references 1. zhang x, li s, niu s. ace2 and covid-19 and the resulting ards. postgrad med j 2020;96:403– 7. doi: 10.1136/postgradmedj-2020-137935. 2. sieńko j, kotowski m, bogacz a, et al. covid19: the influence of ace genotype and ace-i and arbs on the course of sars-cov-2 infection in 107 elderly patients. clin interv aging 2020;15:123140. doi: 10.2147/cia.s261516. 3. cdc covid-19 response team, bialek s, boundy e, bowen v, et al. severe outcomes among patients with coronavirus disease 2019 (covid-19) united states, february 12–march 16, 2020. mmwr morb mortal wkly rep 2020;69:343-6. doi: 10.15585/mmwr.mm6912e2. 4. karyono dr, wicaksana al. current prevalence, characteristics, and comorbidities of patients with covid-19 in indonesia. jcemph 2020;3:77-84. doi: 10.22146/jcoemph.57325. 5. fulop t, dupuis g, witkowski jm, larbi a. the role of immunosenescence in the development of age-related diseases. rev invest clin 2016;68:8491. 6. jergović m, coplen cp, uhrlaub jl, nikolichžugich j. immune response to covid-19 in older adults. j heart lung transplant 2021;40:1082-9. doi: 0.1016/j.healun.2021.04.017. 7. wu z, mcgoogan jm. characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention. jama 2020;323:1239–42. doi: 10.1001/jama.2020. 2648. 8. dhama k, patel sk, kumar r, et al. geriatric population during the covid-19 pandemic: problems, considerations, exigencies, and beyond. front public health 2020;8:574198. doi: 10.3389/fpubh.2020.574198. 9. bajaj v, gadi n, spihlman ap, wu sc, choi ch, moulton vr. aging, immunity, and covid-19: how age influences the host immune response to coronavirus infections? front physiol 2021;11: 571416. doi: 10.3389/fphys.2020.571416. 10. perrotta f, corbi g, mazzeo g, et al. covid-19 and the elderly: insights into pathogenesis and clinical decision-making. aging clin exp res 2020; 32:1599-608. doi: 10.1007/s40520-020-01631-y. 11. malone ml, hogan tm, perry a, et al. covid-19 in older adults: key points for emergency department providers. geriatric emergency department collaborative. j geriatr emerg med 2020;1:1-11. 12. torres acosta ma, singer bd. pathogenesis of covid-19-induced ards: implications for an ageing population. eur respir j 2020;56:2002049. doi: 10.1183/13993003.02049-2020. 13. koff wc, williams ma. covid-19 and immunity in aging populations — a new research agenda. n engl j med 2020;383:804–5. doi: 10.1056/ nejmp2006761. 14. bencivenga l, rengo g, varricchi g. elderly at time of corona virus disease 2019 (covid-19): possible role of immunosenescence and malnutrition. geroscience 2020;42:1089-92. doi: 10.1007/s11357-020-00218-9. 15. liu k, chen y, lin r, han k. clinical features of covid-19 in elderly patients: a comparison with young and middle-aged patients. j infect 2020;80:e14-e8. doi: 10.1016/j.jinf.2020.03.005. 16. heneghan c, aronson j, mahtani k. rapidly managing pneumonia in older people during a pandemic. oxford covid-19 centre for evidence based-medicine; 2020. 17. yang jw, yang l, luo rg, xu jf. corticosteroid administration for viral pneumonia: covid-19 and beyond. clin microbiol infect 2020;26:1171-7. doi: 10.1016/j.cmi.2020.06.020. 18. matera mg, rogliani p, calzetta l, cazzola m. pharmacological management of covid-19 patients with ards (cards): a narrative review. respir med 2020;171:106114. doi: 10.1016/j.rmed. 2020.106114. 19. fischer f, raiber l, boscher c, winter mhj. covid-19 and the elderly: who cares? front public health 2020;8:151. doi: 10.3389/fpubh. 2020.00151. 20. azzolino d, saporiti e, proietti m, cesari m. nutritional considerations in frail older patients with covid-19. j nutr health aging 2020;24:696– 8. doi: 10.1007/s12603-020-1400-x. 21. fernández-quintela a, milton-laskibar i, trepiana j, et al. key aspects in nutritional management of covid-19 patients. j clin med 2020;9:2589. doi: 10.3390/jcm9082589. 22. rolles b, maywald m, rink l. influence of zinc deficiency and supplementation on nk cell cytotoxicity. j funct foods 2018;48:322-8. https:/ /doi.org/10.1016/j.jff.2018.07.027. univ med vol. 41 no 2 c:\users\universa medicina\docu 18 abstract universa medicina processed meat consumption increases risk of type 2 diabetes mellitus in adults aged 40 years and older solikhah,1* and asri lestari1 background type 2 diabetes mellitus (t2dm) remains a public health problem in the world, including indonesia. the high mortality of t2dm is triggered by an unhealthy eating pattern and sedentary lifestyle. we aimed to investigate the relationship of food intake pattern and its related factors with t2dm in adults 40 years and older. methods this was a cross-sectional study conducted on 11,022 men and women with t2dm aged 40 years and older. major dietary patterns were collected and multiple logistic regression analysis was used to determine the effect of covariates. statistical significance was set at a p-value of <0.05. results males and individuals aged >40 years comprised 50.17% and 26.19%, respectively, of the 11,022 respondents. individuals aged over 50 years had a higher risk of developing diabetes than those aged less than 50 years (aor =5.67, 95% ci=1.37-21.94, p<0.05). dietary processed meat was associated with a higher risk of t2dm (aor = 4.9; 95% ci=1.08-22.20, p<0.05). carbohydrate and fruit intakes were negatively associated with and protective factors for dm (aor= 0.01; 95% ci=0.01-0.06, p<0.01; aor = 0.35; 95% ci=0.15-0.83, p<0.01). however, physical activity was not a risk factor for t2dm. conclusions processed meat consumption, age over 50 years, and carbohydrate intake may increase the risk of t2dm in adults. conversely, fruit intake may decrease the risk of t2dm in adults. there is a need to control the diet and lifestyle for the early prevention of dm. keywords: diabetes mellitus, dietary processed meat, carbohydrate intake, fruit intake, physical activity, adults original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.18-28 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1203 january-april, 2022 vol.41no.1 1department of public health, faculty of public health, universitas ahmad dahlan, yogyakarta, indonesia correspondence: *solikhah, dr. ph faculty of public health, universitas ahmad dahlan jl prof soepomo, janturan warungboto, yogyakarta 55166, indonesia tel. (+62) (0274) 563515, 511830 fax: (+62) (0274) 564604 e-mail: solikhah@ikm.uad.ac.id orcid id : 0000-0001-6895-6840 date of first submission, july 23, 2021 date of final revised submission, february 8, 2022 date of acceptance, february 15, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: solikhah, lestari a. processed meat consumption increases risk of type 2 diabetes mellitus in adults aged 40 years and older. univ med 2022 ;41 :1 8 -28 . doi: 10. 1805 1/ univmed.2022.v41.18-28 univ med vol. 41 no 1 19 introduction type 2 diabetes mellitus (t2dm) as a degenerative disease is still a serious public health problem in the world. globally, dm ranks seventh as the leading cause of death. (1) data from the international diabetes federation (idf) in 2021 showed that in south east asia the number of adults with diabetes is predicted to rise to 152 million by 2045, an increase of 68%.(2) indonesia, as one of the low-middle income countries, ranks seventh among countries with the highest prevalen ce of dm in the world (3.4%) after china (37.4%), india (24.8%), the united states (9.8%), pakistan (6.2%), brazil (5.4%), and mexico (4.1%).(3) furthermore, this fact is made obvious with the increase in dm prevalence in indonesia from 6.9% in 2013 to 8.5% in 2018 based on data from the indonesian basic health research (ibhr).(4) the increased incidence of dm is triggered by changes in lifestyle that include the lack of awar eness of healthy eating patter ns, ( 5,6 ) increased number of obesity cases,(7) lack of physical activity (8) and prevalence of habitual smoking.(9,10) also, the high prevalence of t2dm in indonesia is due to the fact that nearly all patients with this disease had difficulties in accessing adequate health care services. in addition, the limited ability of health professionals in dm management that encompasses promotive, preventive, curative, and rehabilitative efforts and the unavailability of drugs may raise the incidence rate of t2dm.(11) the high prevalence of dm is also triggered by the food consumption pattern that is high in carbohydrate and saturated fat,(12-15) but low in vitamins and fiber.(16,17) previous studies states that food intake is associated with increased blood sugar level after consuming high-glycemic-index carbohydrates (glucose and sucrose).(18,19) a systematic review of previous studies reported that various fast foods, foods that are high in carbohydrate and calories, or in processed meats, sweetened beverages, and saturated fats, contribute to raising the prevalence of type 2 diabetes.(12) patients with dm are advised not to consume carbohydrates in amounts of more than 45%-65% of the daily total energy need.(20,21) in addition, the amount of protein recommended for these patients is 10-15% of the total daily calorie need. for satur ate d fatty f ood, the recommendation is that it should not exceed 1020 % of the total daily calorie need.(22) if the diet continuously exceeds the recommended amount, the blood glucose level will be beyond control, and the person in question will experience type 2 dm. lack of physical activity also contributes significantly to the high number of type 2 dm cases.(23,24) in contrast, a randomized controlled trial to compare the effects of 3 different modalities of exercise on metabolic control and insulin resistance among patients with type 2 diabetes mellitus, showed that after 12 weeks of training there was no difference across the groups in blood pressure, fasting plasma glucose, postprandial plasma glucose, lipid profile, and high-sensitivity c-reactive protein (hs-crp).(25) in addition, the precise mechanism of how physical activity acts to reduce the risk of type 2 diabetes mellitus, such as through altered insulin sensitivity or altered insulin production, is still unknown.(26) if the amount of energy consumed exceeds the amount of energy expended, this gives rise to a positive balance of power in adipose tissue (27) which then triggers insulin resistance that will lead to type 2 dm.(28,29) previous studies have reported that unhealthy eating patterns and low physical activity have been recognized as the risk factors for the high incidence of dm; 30) therefore, it is necessary to encourage and promote the shift to a healthy lifestyle in the community. a study in saudi arabia showed that most of the respondents aged at least 40 years old had diabetes mellitus (44.6%).(31) a systematic review and meta-analysis showed that after adjusting for the potential factor of gender, total carbohydrate was no longer significantly associated with type 2 diabetes risk (rr=1.11, 95% ci: 0.97 to 1.26, p=0.12). fructose, glucose, lactose, maltose, a nd sucr ose were not significantly associated with type 2 diabetes risk.(32) in their systematic review, hemmingsen 20 et al.(33) concluded that there was no effect of diet or physical activity alone on the risk of type 2 dm in people at high risk of developing t2dm. in indonesia as one of the asian countries that have a high rate of urbanization and modernization triggered by rapid development, the changes into an unhealthy lifestyle marked by the consumption of unhealthy food and adoption of the sedentary lifestyle (34, 35) may impact on non-communicable diseases such as t2dm. the results of previous studies show that the relationship between carbohydrate diet and physical activity with t2dm is inconsistent and that further studies are needed to confirm this relationship. the aim of the present study wa s to de ter mine the relationship of food intake pattern and its related factors with t2dm in adults aged 40 years and older. methods research design a cross-sectional study was conducted in 13 provinces in indonesia, namely north sumatra, south sumatra, west sumatra, lampung, jakarta, west java, central java, yogyakarta, east java, bali, west nusa tenggara, south kalimantan, and south sulawesi. we used secondary data from the fifth wave of the indonesia family life survey (ifls-5). our secondary data collection was performed from october 2014 to august 2015. research subjects a total of 11,022 respondents aged 40 years consisting of males (5,530; 50.17%) and females (5,492; 49.83%) from the fifth wave of the indonesia family life survey (ifls-5). the latter is a longitudinal survey that collects panel data on socioeconomics and health at the individual, household and community levels in the abovenamed 13 provinces. the inclusion criteria applied to the sample in this study were individuals aged 40 years, having completed the questionnaire, and having been diagnosed with t2dm. respondents who relocated to another village or passed away during data collection and those who did not complete the questionnaire were excluded. measurements a questionnaire was used comprising closed questions on the variables of age, gender, occupation, educational level, and smoking habit (yes/no). psychosocial stress was measured using the centres for epidemiologic studies depression scale (ces-d) instrument to assess the symptoms of stress. (3 6) based on the symptoms, stress was classified into “stress” and “no stress”. physical activity variables were assessed using the international physical activity questionnaire (ipaq) (37) and were categorized into 3 categories, namely “high,” “moderate,” and “low” activity. a respondent was stated to have a “high” physical activity when he or she met one of the following criteria: 1) physical activity of high intensity for at least 3 days and reaching a total physical activity of at least 1500 metminutes/week; or 2) combination of walking activity for at least 7 days with moderate to high intensity physical activity and reaching a total activity of at least 3000 met-minutes/week. meanwhile, a respondent that met the following 3 criteria was considered to have a “moderate” physical activity: 1) high-intensity physical activity for at least 3 days with at least 20 minutes per day, 2) moderate-intensity physical activity for at least 5 days and/or at least 30 minutes of walking activity per day, 3) combination of moderate-high intensity walking activities and reaching a total of 600 met-minutes/week of physical activity. in addition, respondents were categorized as having a “low” physical activity if they did physical activities that were not included in the “high” and “moderate” categories. laboratory analysis glycosylated hemoglobin (hba1c) levels of the respondents were collected by the ifls in collaboration with the laboratory of the clinical pathology department, gadjah mada university. the measurement of hba1c was performed by a physician or paramedic (yes = hba1c  6.5% and no = hba1c <6.5%).(38) solikhah, lestari food intake pattern and diabetes mellitus univ med vol. 41 no 1 21 c assessment of dietary intake eating patterns were obtained from the participants’ answers to the question about their average frequency of daily intake per week.(39) those patterns were measured by counting the frequency of consumption of carbohydratecontaining foods (sweet potatoes/rice); fruits; green leafy vegetables; dairy products; fish; processed meats (fresh processed beef/chicken/ pork/eggs, etc.) and instant noodles, which was divided into 3 categories once/week, 1-3 times/ week, and >3 times/week).(39) ethical clearance written ethical approval for the study was obtained from the research ethics committee of universitas ahmad d ahlan, yogyakarta, indonesia (no: 011905053). written informed consent was obtained from all patients before being included in the study. statistical analysis participant characteristics were analyzed descriptively and presented as mean and standard deviation for continuous variables and as frequency and pe rcentage for categorical variables. the demographic characteristics analyzed in this study included age, level of education (elementary school/junior high school/ senior high school/higher education/did not attend formal sc hool), and occupational sta tus (unemployed/working). a logistic regression model was constructed to obtain adjusted bivariate and adjusted multivariate effects for outcomes as well as estimated adjustment of association between age, gender, education level, occupational status, smoking habit, physical activity, stress, diet, and dm. statistical significance was set at a p-value of <0.05, and all data analyses were performed using stata 15.0 software. results males and individuals aged >40 years comprised 50.17% and 26.19%, respectively, of the 11,022 respondents. more than half of the respondents did not smoke (58.15%) and were not under stress (84.83%). meanwhile, a higher percentage of the respondents had a low educational level (elementary school) (51.38%), were working (74.46%), and were less active (55.03%). further details on the participants’ characteristics in this study are presented in table 1. in summar izing the frequency of food consumption among participants (table 2), the most common foods consumed (3 times/week) were carbohydrates (99.58%) and processed meats (60.99%). table 3 provides the results of multivariable binary logistic regression analyses, which revealed that carbohydrate intake of more than 3 t i me s per w e e k wa s si gni f ic a nt l y a n d negatively associated with 1% and a protective factor for t2dm (aor=0.01; 95% ci=0.010.06; p<0.001). subjects who consumed fruit >3 times/week were shown to be significantly associated with t2dm, with the odds of the prevalence of t2dm decreasing by 65% (aor =0.35, 95% ci=0.15 to 0.83, p<0.01). meanwhile, the odds of subjects with processed meat intake (>3 times per week) was 4.90 times higher for the prevalence of t2dm (aor=4.90, 95% ci=1.08 to 22.20, p<0.05). regarding the patients’ age, our study showed that in the age groups of 50-55 years, 55-59 years, and 60 years, respectively, age was significantly associated with t2dm, (aor=5.67, 95% ci=1.37-21.94, p<0.05; aor=5.46, 95% ci = 1.16-24.00, p<0.05; aor = 6.15, 95% ci = 6.15, 95% ci=1.32-22.56, p<0.05). in addition, there were no significant associations of t2dm with physical activity and consumption of green leafy vegetables, dairy products, and instant noodles. discussion the results of this study indicated that frequent excessive consumption of processed meat incr eased the risk for dm among respondents. this is in line with other studies which state that consuming red meat and 22 processed meat increases the risk of insulin resistance and type 2 dm.(40, 41) meanwhile, the results of this study are inversely proportional to those of a study in korea which stated that consuming processe d red meat was not associated with the incidence of t2dm.(42) in addition, saturated fat can cause obesity and result in glucose intolerance, insulin resistance and diabetes.(43) furthermore, the results of our study showed that freque nt consumption of carbohydrates is a protective factor against developing t2dm. this research is in line with a study in korea which showed that very high carbohydrate intake was associated with an increased risk of t2dm in men and women.(44) however, these results are in contrast to a study in europe which stated that consuming easily digested carbohydrates was not associated with the incidence of diabetes. (4 5) controlling carbohydrate consumption is the primary key to controlling dm by lowering the glycemic index (gi) through regulating dietary patterns,(46) such as through consumption of brown rice, beans, bananas, corn, breadfruit, and various types of tubers.(47) dm is known as one of the silent killers because patients often do not realize that they have dm due to nonspecific symptoms of this disease. this is aggravated by the fact that most dm patients suffer from complications, especially those who live in low-middle income countries such as indonesia. some of the complications of this disease are cardiovascular disease (cvd), blindness, heart failure, and even amputation of the lower limbs. in pregnancy, uncontrolled diabetes increases the risk of characteristics diabetic (%) non-diabetic (%) total participants (%) prevalence 95% ci overall 28 (0.25) 10,994 (99.75) 11,022 (100.00) 0.001 – 0.003 gender male 19 (67.86) 5,511(50.13) 5,530 (50.17) 0.002 0.005 female 9 (32.14) 5,483 (49.87) 5.492 (49.83) 0.001 – 0.003 age (years) 40-44 3 (10.71) 2,884 (26.23) 2,887 (26.19) 0.001 – 0.002 45-49 6 (21.43) 2,368 (21.47) 2,366 (21.47) 0.001 – 0.005 50-54 7 (25.00) 1,918 (17.45) 1,925 (17.47) 0.001 – 0.006 55-59 5 (17.86) 1,475 (13.42) 1,480 (13.43) 0.004 – 0.006 60 7 (25.00) 2.357 (21.44) 2,364 (21.45) 0.007 – 0.005 education level elementary school 11 (39.29) 5,678 (51.65) 5,689 (51.61) 0.001 – 0.003 junior high school 3 (10.71) 1,702 (15.48) 1,705 (15.47) 0.001 – 0.004 senior high school 11 (39.29) 2,428 (22.08) 2.439 (22.13) 0.002 – 0.007 higher education 3 (10.71) 1,186 (10.79 1,189 (10.79) 0.001 0.005 smoking habit no 14 (50.00) 6,395 (58.17) 6,409 (58.15) 0.001 – 0.003 yes 14 (50.00) 4,599 (41.83) 4,613 (41.85) 0.002 – 0.005 stress condition no stress 22 (78.57) 9,328 (84.85) 9,350 (84.83) 0.001 – 0.003 stress 6 (21.43) 1,666 (15.15) 1,672 (15.17) 0.001 – 0.006 occupational status unemployed 7 (25.00) 2,808 (25.54) 2,815 (25.54) 0.001 – 0.004 working 21 (75.00) 8,186 (74.46) 8,207 (74.46) 0.001 – 0.003 physical activity high 12 (42.86) 4,506 (40.99) 4,518 (40.99) 0.001 – 0.004 moderate 1 (3.57) 438 (3,98) 439 (3.98) 0.002 – 0.007 low 15 (53.57) 6,050 (55.03) 6,065 (55.03) 0.001 – 0.004 table 1. characteristics of participants with and without t2dm (n=11,022) note: t2dm: type 2 diabetes mellitus solikhah, lestari food intake pattern and diabetes mellitus univ med vol. 41 no 1 23 however, the findings in this study indicated that physical activity did not significantly influence t2dm in the adult population. this study contradicts 2 studies which state that adults who have moderate physical activity are associated with the incidence of dm (50) and that decreased physical activity increases the risk of t2dm. (51) this is due to the fact that the intensity and duration of physical activities performed by most participants were still low, despite the fact that high-intensity physical activity provides benefits in reducing the glycemic level in individuals who are at risk of developing dm. (52) low physic al acti vity affects the suboptimal metabolism of blood and body cells that use glucose as the source of fuel for food consumption diabetic (%) non-diabetic (%) total participants (%) carbohydrate (sweet potatoes and rice)  once/week 2 (7.14) 24 (0.22) 26 (0.24) 1-3 times/week 2 (7.14) 18 (0.16) 20 (0.18)  3 times/week 24 (85.71) 10,952 (99.62) 10,976 (99.58) fruits  once/week 14 (50.00) 3,938 (35.82) 3,952 (35.86) 1-3 times/week 4 (14.29) 1,623 (14.76) 1,627 (35.86)  3 times/week 10 (35.71) 5,433 (49.42) 5,443 (49.38) green leafy vegetables (include carrot)  once/week 2 (7.14) 1,819 (16.55) 1,821 (16.52) 1-3 times/week 3 (10.71) 1,468 (13.35) 1,471 (13.35)  3 times/week 23 (82.14) 7,707 (70.10) 7,730 (70.13) dairy products (milk, yogurt, cheese, etc.)  once/week 22 (78.57) 8,475 (77.09) 8,497 (77.09) 1-3 times/week 2 (7.14) 640 (5.82) 642 (5.82)  3 times/week 4 (14.29) 1,879 (17.09) 1,883 (17.08) fish  once/week 11 (39.29) 3,800 (34.56) 3,811 (34.58) 1-3 times/week 3 (10.71) 1,953 (17.76) 1,956 (17.75)  3 times/week 14 (50.00) 5,241 (47.67) 5,255 (47.68) processed meats (fresh beef/chicken/ pork, egg, etc.)  once/week 2 (7.14) 2,523 (22.95) 2,525 (22.91) 1-3 times/week 4 (14.29) 1,771 (16.11) 1,775 (16.10)  3 times/week 22 (78.57) 6,700 (60.94) 6,722 (60.99) instant noodle  once/week 16 (57.14) 7,156 (65.09) 7,172 (65.07) 1-3 times/week 5 (17.86) 1,791 (16.29) 1,796 (16.29)  3 times/week 7 (25.00) 2,047 (18.62) 2,054 (18.64) table 2. the frequency of food consumption among participants with and without t2dm note: t2dm : type 2 diabetes mellitus maternal and fetal complications.(2) therefore, to reduce mortality due to dm, efforts are needed to improve dm management by, among others, increasing knowledge on self-management among dm patients, increasing physical activities, and implementing nutritional therapy to control blood sugar levels and complications.(48) it is essential to change the patient’s lifestyle into a healthier one, mainly by reducing the consumption of food that has high calories as well as food high in unsaturated fat, sugar, salt, or sodium and with minimal fiber content. in addition, shifting the behavior from less active with a minimum amount of exercise into a more active one will have a very significant impact in reducing the incidence of dm.(30,49) 24 items unadjusted or (95%ci) p-value adjusted or (95%ci) p-value age (ref: 40-44) 45-49 2.44 (0.61 9.78) 0.207 2.71 (0.66 11.09) 0.165 50-54 3.51 (0.91 13.58) 0.069 5.67 (1.37 21.94) 0.014 55-59 3.26 (0.78 13.65) 0.106 5.46 (1.16 24.00) 0.029 60 2.86 (0.74 11.05) 0.129 6.15 (1.32 22.56) 0.015 education level (ref: elementary school) junior high school 0.91 (0.25 3.26) 0.885 1.05 (0.28 3.89) 0.955 senior high school 2.33 (1.01 5.40) 0.047 3.32 (1.32 8.32) 0.014 higher education 1.30 (0.36 4.68) 0.683 1.57 (0.41 6.02) 0.517 smoking habit (ref: no smoking) yes 1.39 (0.66 2.92) 0.384 1.18 (0.52 – 2.64) 0.683 stress condition (ref: no stress) stress 1.52 (0.62 3.77) 0.359 1.64 (0.65 – 4.17) 0.293 occupational status (ref: unemployed) working 1.02 (0.44 2.42) 0.948 1.23 (0.47 – 3.22) 0.672 physical activity (ref: high) moderate 0.86 (0.11 6.61) 0.883 0.63 (0.08 – 5.07) 0.663 low 0.93 (0.43 – 1.99) 0.854 0.78 (0.35 – 1.72) 0.542 carbohydrate (sweet potatoes and rice) (ref:  once/week) 1-3 times/week 1.33 (0.17 10.38) 0.784 0.81 (0.08 – 7.60) 0.763  3 times/week 0.03 (0.01 – 0.11) 0.001 0.01 (0.01– 0.06) 0.002 fruits (ref:  once/week) 1-3 times/week 0.69 (0.22 2.10) 0.519 0.55 (0.17 – 1.74) 0.339  3 times/week 0.51 (0.22 – 1.17) 0.112 0.35 (0.15 – 0.83) 0.024 green leafy vegetables (ref:  once/week) 1-3 times/week 1.85 (0.31 11.13) 0.497 1.98 (0.32 12.40) 0.443  3 times/week 2.71 (0.64 11.52) 0.176 2.96 (0.66 13.22) 0.137 dairy products (milk, yogurt, cheese, etc.) (ref:  once/week) 1-3 times/week 1.20 (0.28 – 5.13) 0.802 1.25 (0.29 – 5.48) 0.797  3 times/week 0.82 (0.28 – 2.38) 0.715 0.58 (0.19 – 1.76) 0.331 fish (ref:  once/week) 1-3 times/week 0.53 (0.14 – 1.90) 0.331 0.56 (0.15 – 2.06) 0.377  3 times/week 0.92 (0.42 – 2.03) 0.842 0.87 (0.38 – 2.00) 0.750 processed meats (beef/chicken/pork, egg, etc.) (ref:  once/week) 1-3 times/week 2.84 (0.52 – 15.57) 0.227 2.81 (0.49 15.88) 0.237  3 times/week 4.14 (0.97 – 17.62) 0.054 4.90 (1.08 – 22.20) 0.035 instant noodle (ref:  once/week) 1-3 times/week 1.24 (0.45 – 3.41) 0.665 1.06 (0.37 – 3.05) 0.993  3 times/week 1.52 (0.62 – 3.72) 0.349 1.73 (0.68 – 4.41) 0.283 table 3. multivariable adjusted or and 95% ci for t2dm note: ref: reference; or: odds ratio; 95%ci: 95% confidence interval; t2dm: type 2 diabetes mellitus, ci: confidence interval the body so that it will not significantly affect the blood glucose level.(53). routine and regular physical activities are strongly recommended for diabetic patie nts to improve insulin sensitivity, control the gi, and control the metabolic profile, which was also true for individuals who were at risk of developing diabetes.(54) mild physical activity with high solikhah, lestari food intake pattern and diabetes mellitus univ med vol. 41 no 1 25 intensity and a minimum duration of 30 minutes was sufficient for preventing t2dm.(55) however, this study also had several limitations. first, it used secondary data from data collected using a structured questionnaire that allowed for biased information, especially for details regarding eating frequency and physical activity frequency that entirely relied on the respondents’ memory about their eating habits and activities in the past week. also, missing data was found in this study that requires the researchers to only analyze data from respondents that met the established research inclusion criteria to prevent selection bias. the cross-sectional design of this study also makes causal inferences between exposure and disease impossible, because it cannot determine whether the exposure preceded the disease or vice versa. there is a need for further welldesigned prospective cohort studies to examine the potential association between macronutrient intake and t2dm. our findings provided valuable information for the primary prevention of t2dm through dietary modifications in a middle-aged indonesian population. conclusion dietary processed meat increased the risk of t2dm in adults. while physical activity did not affect the prevalence of t2dm, nevertheless, all parties need to continue to exercise and to exert control over dietary intake through early preventive measures as a way to minimize the development of t2dm. conflict of interest none. funding none. acknowledgment this study was supported by the fifth wave of the indonesian family life survey (ifls-5) conducted by rand and survey meter (https:// www.rand.org/well-being/social-and-behavioralpolicy/data/fls/ifls/ifls5.html). we wish to express our gratitude to rand for their permission to use the survey data and to the study participants who provided the survey data. 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https://doi.org/10.2337/dc19-s005. 55. colberg sr, sigal rj, yardley je, et al. physical activity/exercise and diabetes: a position statement of the american diabetes association. diabetes care 2016;39:2065–79. doi: 10.2337/dc161728. solikhah, lestari food intake pattern and diabetes mellitus c:\users\user\documents\41 no 2 129 abstract universa medicina highest economic status increases risk of cesarean section in women of childbearing age haerawati idris1*and rini anggraini1 background cesarean section (cs) rates that are higher than the who recommendation may pose morbidity and mortality risks for both mother and child. in recent years, the number of cs deliveries has been increasing in developed and developing countries. the aim of the present study was to determine the rate of cs delivery and socioeconomic and demographic factors as risk factors of cesarean delivery in women of childbearing age. methods this cross-sectional study used data from the 2017 indonesian health demographic survey (idhs). the research subjects were 14,724 women of childbearing age aged 15-49 years who had given birth and met the inclusion criteria. the rate of cs was determined and the associations between independent and dependent variables were explored using logistic regression. results the cs rate was 17.9%. variables that had a significant relationship with cesarean delivery were geographic region, economic status, occupation, education, birth attendant, insurance ownership and antenatal care visits. the most dominant influencing variable was the highest economic status (or 3.566; 95% ci: 2.857-4.452). respondents with the highest economic status had a 3.5 times greater risk of having a cesarean delivery than subjects with the lowest economic status after controlling for the other variables. conclusion this study demonstrated that the highest economic status increased the risk of cs delivery in women of childbearing age. the current epidemiological findings and evidence suggest adopting and implementing some strict guidelines in the health system to avoid unnecessary delivery by cs. keywords: cesarean section, demographic health survey, economic status, women of childbearing age original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.129-138 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1306 may-august 2022 vol.41no.2 1faculty of public health, sriwijaya university, palembang correspondence: haerawati idris faculty of public health, sriwijaya university, raya palembang-unsri km 32 indralaya street, ogan ilir, south sumatra, indonesia, 30862 email: haera@fkm.unsri.ac.id orcid id: 0000-0002-3483-6717 date of first submission, february 27, 2022 date of final revised submission, june 6, 2022 date of acceptance, june 10, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: idris h, anggraini r. highest economic status increases risk of cesarean section in women of childbearing age. univ med 2022;41: 1 2 9-3 8 . doi: 10 .18 051/ univmed. 2022.v41.12 9-13 8. 130 idris, anggraini cesarean section in women of childbearing age introduction the world health organization (who) recommends that the average number of cesarean sections should range from 10% to 15% as the maximum -target limit in an effort to avoid the risks of this mode of delivery to mother and baby.(1) however, it turns out that the cesarean section rate (csr) continues to increase and to exceed the who recommended average. betran et al.(2) reported that based on their studies from 1990 to 2014 in 121 countries, mean csr increased by 12.4% (from 6.7% to 19.1%). some countries with higher csr than the who standards are the latin american and caribbean countries with the highest csr of 40.5%, europe with 25%, and asia 19.2%, whereas africa with 7.3% is still below the recommended average.(2) on the other hand, the csr in some areas of pakistan ranges from 47% to 65%. the number of mothers who are giving birth by cesarean section also continues to increase in indonesia; acc ording to the indonesian demographic and health survey (idhs) data, the csr in indonesia was 5%, then it rose to 12% in 2012 and increased again to 17.0% in 2017.(4) as the proportion of births increased in health facilities in indonesia, in the last 30 years indonesia’s csr has also increased considerably, from 1.6% in 1991 to 17.6% in 2017.(5) even the study of oktarina et al.(6) in government and private hospitals in jakarta, indonesia, reported that the trend of cesarean section (cs) deliveries is very high, reaching 70%, especially in private hospitals. currently cs is a life-saving intervention for mothers and children during childbirth, but this procedure can also have short-term and long-term health effects for women and children. the prevalence of maternal morbidity and mortality is higher after cs compared to vaginal delivery. cesarean section has been shown to increase the risk of uterine rupture, abnormal placentation, ectopic pregnancy, stillbirth and premature delivery.(7) cesarean delivery also affects the child’s sensory perception, sensory integration ability, and neuropsychiatry, and the relationship between mother and child.(8) exclusive breastfeeding is especially important for the baby’s growth and development, but cs can reduce the chances of continued breastfeeding.(9) breast milk production is strongly influenced by the release of the hormones oxytocin and prolactin.(10) in cesarean delivery, maternal anxiety due to postoperative pain and also the effects of anesthetics/drugs may delay the onset of breastfeeding.(11) based on previous studies, it was reported that the majority of mothers who gave birth by the cesarean method stopped breastfeedin g after 12 weeks of delivery.(12) research in niger by hitachi et al. (13) also reported that many mothers who had cesarean section did not give exclusive breastfeeding, and even stopped breastfeeding after 1 week of giving birth. medically, the most common indication for cs is cephalopelvic disproportion (cpd) or narrow pelvis. (1 4.1 5) other indications are obstructed labor, multiple pregnancies, nonreassuring fetal heart rate pattern (nrfhrp), failed induction and augmentation of labor, malpresentation and malposition, and antepartum hemorrhage.(16) sungkar and basrowi (17) also stated that sociodemographic factors, namely advanced maternal age, high socioeconomic status, higher education, living in urban areas and ownership of health insurance were also found to be associated with the incidence of cesarean delivery. cesarean section procedures aim to save lives when there are medical indications for delivery, but cs can pose a higher risk of morbidity and mortality when performed without medical indications.(5) ironically, according to sandall et al.(7) the rate of cesarean delivery continues to increase, especially in cases without medical indications, even though this procedure can have short-term and long-term health effects on women and children. in addition, it turns out that a high csr of more than 10% is also not associated with a decrease in maternal and neonatal mortality.(17.18) 131 based on this description, it can be concluded that there is a large risk that threatens the mother and child due to cs without medical indications which contributes greatly to the high csr but is proven not to be associated with a decrease in maternal and neonatal mortality. a study in ghana involving 4948 research participants revealed that cs delivery is associated with maternal age, level of education, occupation, parity and antenatal care (anc) visits.(19) women’s socioeconomic status and health system factors were associated with the increased use of cs.(20) in contrast, a bangla desh study found that the logistic regression analysis has traced no significant variation in cs rate among mothers of varying educational levels as compared to the reference group (mothers with no education) (p>0.05). similarly, mothers’ religious status, age at first childbirth, age at first marriage, working status, exposure to media, including wanted indexed child and husband’s educational level, resulted in no significant difference in cs rate among various groups as compared to their corresponding reference groups.(21) because the results of previous studies that found an association of socioeconomic and demographic factors, working status, and age with cs, are still inconsistent, therefore the association of socioeconomic and demographic factors, working status, and age with cs delivery are still to be determined. this study aimed to raise the importance of investigating the causes of high csr, especially in indonesia, which is useful in developing strategies for optimizing the use of appropriate cs. the objective of this study was to determine the socioeconomic and demographic factor as risk factors of cs in indonesia. methods research design this was a cross-sectional study using secondary data from the 2017 indonesian health demographic survey (idhs), which is a survey data jointly carried out by the central statistics agency (bps), the national population and family planning agency (bkkbn), and the ministry of health (kemenkes) from 24 july to 30 se ptember 2017. survey funding was provided by the government of indonesia, assisted by the united states agency for international development (usaid). study subjects the population of this study comprised all respondents in 34 provinces of indonesia who were successfully interviewed by the idhs t e a m. t he s a mpl e f o r t he p r es e n t s t ud y amounted to 14,724 respondents who were selected based on one inclusion criterion, namely women of childbearing age in the range of 1549 years who had given birth in the last 5 years at the time of the interview and two exclusion criteria consisting of missing data and invalid data. main outcomes the main outcome was cesarean delivery a n d wa s ca t e go r i ze d i nt o s ub j e c t s wh o underwent cesarean delivery (coded as 1) and otherwise (coded 0). main exposure variables the main exposure variables in this study were education, occupation, economic status, region, insurance ownership, birth attendant, and anc visits. educational level was divided into three categories, namely low if respondent had no education or had attended elementary school, intermediate if respondent had attended junior high school or senior high school, and high if respondent had studied for diploma degree or attended college. economic status was based on the economic quintile owned by a household. households were scored based on the number and type of items they had, from television sets t o bi c yc l e s o r c ar s, a n d on h ou s in g characteristics, such as drinking water sources, toilet facilities, and main building materials for the floor of the house. this score was calculated using principal component analysis. national economic quintiles were arranged based on univ med vol. 41 no 2 132 idris, anggraini cesarean section in women of childbearing age a b hou sehold scor es for each pers on in the household and then divided into five quintiles, namely lowest, low, middle, high and highest. statistical methods data were analyzed by univariate analysis and by bivariate analysis using the simple logistic regression test to determine the relationship between the independent variable and the dependent variable. multivariate analysis was by means of logistic regression test with predictive model to analyze the independent variable with the most dominant effect on the dependent variable. the significance level set at p<0.05. ethical clearance ethical clearance was obtained for the 2017 idhs from the national ethics committee. the respondents’ identities have all been deleted from the dataset. respondents provided written consent for their involvement in the study. the researchers have obtained permission to use the data for the purposes of this study through the following website: https://dhsprogram.com/data/ new-user-registration.cfm. this study was appro ved by the health research e thics committee of the faculty of public health, sriwijaya university, under no. 217/un9.fkm/ tu.kke/2022. results from table 1 it can be seen that around 17.9% of respondents gave birth by cesarean section. the majority of respondents (58.6%) had secondary education, 58.8% had insurance, 57.6% resided on java/bali, 51.5% were e mpl oyed , 4 1.4% h ad mi ddl e t o h igh es t economic status, 86.3% gave birth assisted by hea lth wor ke rs a nd 91.7% had c ompl ete antenatal care visits. based on the results of the simple logistic regression analysis in table 2, it was found that all independent variables namely education, occupation, economic status, geographic region, insurance ownership, birth attendants, and anc visits have a significant relationship with incidence of cesarean delivery. all of the independent variables studied also have an or of >1, which means that there is an association in the form of increasing the risk of an outcome (i.e. a risk factor for cesarean delivery). based on the or value, respondents with the highest economic status have a 6.3 times gre ater probability of having a cesarean delivery compared to respondents with the lowest ec onomic s ta tu s ( 95% ci: 5 .2 43 -7. 733) . respondents with complete anc visits have a 3 times greater probability of having a cesarean d e l i ve r y c o mpa r e d t o r e s po nde n t s wi t h incomplete anc visits (95% ci: 2.413-3,926). table 1. socio-demographic characteristics of the study participants (n= 14,274) *education: low: no education or elementary school; intermediate: junior high school or senior high school; high: diploma or college; anc : antenatal care variable n % education high 2,188 14.9 middle 8,634 58.6 low 3,902 26.5 insurance policy ownership yes 8,661 58.8 no 6,063 41.2 region sumatra 3,282 22.3 java/bali 8,477 57.6 east indonesia 2,965 20.1 employment status employed 7,585 51.5 unemployed 7,139 48.5 economic status highest 2,829 19.3 high 3,054 20.7 middle 3,053 20.7 low 2,980 20.2 lowest 2,808 19.1 childbirth attendant health workers 12,702 86.3 non-health workers 2,022 13.7 anc visits complete 13,501 91.7 incomplete 1,223 8.3 cesarean section delivery yes no 2,632 12,092 17.9 82.1 133 univ med vol. 41 no 2 respondents with a higher educational level have a 4.6 times greater probability of having a cesarean delivery compared to respondents with a low level of education (95%ci: 3.944-5.562). the results of the simple logistic regression selection analysis show that the independent variables have a p-value <0.25. based on the results, it can be concluded that the variables of education, geographic region, insurance policy ownership, occupation, economic status, birth attendant, and anc visits can be included in the multivariate analysis modeling. based on the results of the multivariate analysis in table 3, which shows the most influential variable as indicated by the adjusted odds ratio is the highest economic status variable (aor= 3.566; 95% ci: 2.857-4.452). this means that re spondents with t he highest economic status have a 3.5 times gre ater probability of having a cesarean delivery when compared to respondents with the lowest economic status after controlling for the variables of education, geographic region, insurance ownership, birth attendant and anc visits. discussion the cesarean delivery rate in indonesia has continually increased, from 1.6% in 1991 to 17.6% in 2017.(5) the present study, which used idhs 2017 data, also reported that indonesia’s csr was 17.9%. this figure exceeds the range of csr recommendations from who, which is 10%-15%.(1) medically, the study by aprina and puri reported on the factors that could influence the occurrence of cesarean delivery, namely severe preeclampsia, placenta previa, fetal table 2. crude odds ratios of determinants of cs delivery notes: anc: antenatal care; education: low: no education or elementary school; intermediate: junior high school to senior high school; high: diploma or college; or: odds ratio variable cs delivery or(95% ci) p value yes (n,%) no (n,%) region sumatra 649 19.8 2633 80.2 1.655 (1.418 -1.930) 0.000 java/bali 1899 18.9 6878 81.1 1.561 (1.346 -1.809) 0.000 east indonesia 384 13.0 2581 87.0 1 economic status highest 911 32.2 1917 67.8 6.367 (5.243-7.733) 0.000 high 691 22.6 2363 77.4 3.918 (3.215-4.775) 0.000 middle 483 15.9 2569 84.1 2.524 (2.072-3.074) 0.000 low 351 11.8 2629 88.2 1.788 (1.457-2.195) 0.000 lowest 195 6.9 2613 93.1 1 childbirth attendant health workers 2508 19.7 10194 80.3 3.746 (2.940-4.772) 0.000 non-health workers 125 6.2 1897 93.8 1 anc visits complete 2547 18.9 10954 81.1 3.078 (2.413-3.926) 0.000 incomplete 86 7.0 1137 93.0 1 education high 735 33.6 1453 22.4 4.684 (3.944-5.562) 0.001 intermediate 1517 17.6 7117 82.4 1.974 (1.703-2.287) 0.003 low 380 9.7 3522 90.3 1 insurance policy ownership yes 1854 21.4 6807 78.6 1.850 (1.651-2.071) 0.000 no 778 12.8 5285 87.2 1 employment status employed 1455 19.2 6130 80.8 1.201 (1.080-1.336) 0.000 unemployed 1178 16.5 5961 83.5 1 134 idris, anggraini cesarean section in women of childbearing age sub-saharan africa shows the diversity of csr in public health facilities that ranges from 3% in burkina faso to 15.6% in ghana. the results of the present study show that the highest csr in women of high educational status is around 33.6% and in working women is about 19.2%, which is greater than the 16.5% of non-working women. this finding is also in line with previous research and the global trend of csr in general. the factors of urban residence and high educational status influence the occurrence of cs in other developing countries as well, such as in south and southeast asia.(25) mumtaz et al.(26) reported that the prevalence of cs in pakistan among working mothers tended to increase in the following three survey periods, namely 16.6% (dhs 1990), 24.7% (dhs 2006) and 25% (dhs 2012). women with high educational status tend to choose to work, so there is a similar pattern of relationship of educational factors and occupational factors with the incidence of cs. hea lt h pre f e re n c es of wome n wi th h igh position abnormalities, and delayed parturition.(22) several predictors of cs in pakistan according to murtaza et al.(23) were multiparity, breech position, fetal distr ess, oligohydramnios, preeclampsia, and previous uterine scars. in indonesia, the variety of csr in each ge ogr a p h ic r e gio n i s c a us e d b y sociodemographic factors. the disparity in csr between one region and another, which is influenced by the diversity of the regions, education, and occupation in indonesia, is similar to the world trend.(5) sumatra, which is the western part of indonesia, has a csr of 19.8%, followed by java/bali with 18.9% and east indonesia with 13%, all of which are greater than the 10% recommended by the who. in addition, pregnant women residing in sumatra are 1.6 times more likely to have a cs procedure than those in east indonesia. the csr in pakistan varies by province, being 40% in khyber pakhtunkhwa and punjab, 43% in islamabad, 4 9% i n sin dh a n d 51 % i n aza d j a mmu kashmir.(23) the study of yaya et al.(24) on cs in table 3. adjusted odds ratios of determinants of cs delivery note : anc: antenatal care; education: low: no education or elementary school; intermediate: junior high school to senior high school; high: diploma or college; aor: adjusted odds ratio variable aor 95 % ci p value education 1.646 2.433 1.095 – 1.507 0.000 0.002 high 2.001 intermediate 1.285 low 1 region 1.257 1.694 0.990 1.314 0.000 0.068 sumatra 1.459 java/bali 1.140 east indonesia 1 insurance policy ownership 1.349 1.710 0.000 yes 1.519 no 1 economic status 2.857 4.452 2.195 3.366 1.575 2.393 1.216 1.860 0.000 0.000 0.000 0.000 highest 3.566 high 2.718 middle 1.941 low 1.504 lowest 1 childbirth attendant 1.701 2.799 0.000 health worker 2.182 non-health worker 1 anc visits 1.497 2.431 0.000 complete 1.907 incomplete 1 135 educational status are generally higher than those of women without formal education.(27) high formal education has a strong relationship with women’s decision-making autonomy in making choices.(28) in addition, women with high formal education may believe that cs is safer and does not interfere with their workload.(29) the who recommends anc to provide a positive pregnancy experience, the minimum recommendation in 2006 being 4 anc visits, followed by the minimum of 8 anc contacts (in 2016) with a trained anc service provider. t he s e e f f or ts a r e t h e ma n a ge me nt of complications during pregnancy that are likely to lead to cs procedures.(30) however, the results of the present study showed a different finding, in that 18.9% of mothers had cesarean delivery and 91.7% had complete anc visits. the latter percentage is much higher than in the group of mothers who did not receive complete anc services, which is around 7%. a secondary analytical study in pakistan using dhs 20122013 data reported that mothers who received anc care more than four times were more likely to give birth to children through cs procedures. in women who reported complications of pregnancy, gynecological risk avoidance was the reason for the choice of cesarean section.(31) pregnant women who attend antenatal care are more likely to give birth in health facilities and follow postnatal care,(32) such that they have safe deliveries by health workers. the high prevalence of anc of 91.7% is also followed by a high trend of birth attendance with health personnel, namely 86.3% (table 1). in addition to the fact that the prevalence of cesarean delivery is still relatively high among mothers who underwent complete anc, it is note-worthy that the prevalence of cesarean delivery is also quite high in the group of mothers whose birth attendants are health workers, namely 19.7%. even though the csr in government-owned hospitals is around 30%-40%, there are private hospitals whose csr can reach 70%.(6) the percentage of women with cesarean delivery in pakistan is high at 13.6%. according to amj ad et al. (3 1) pakistani wome n who experience pregnancy complications, have high anc utilization and deliver in private hospitals have a higher probability of obtaining a cs procedure. therefore decisive action is needed such as detailed medical indications from doctors for cs and awareness of pregnant women to reduce the chances of pregnancy complications. cesarean section delivery costs many times more than vaginal delivery, such that the f a mi l y’s e c on omi c a bi l i t y b e c o me s a consideration in choosing the cs method of delivery.(35) this can be seen from the results of the study which showed that the economic s t a t us va ri a bl e w a s t h e mo s t d o mi n a n t. respondents with the highest economic status have a 3.5 times greater probability of having a cesarean delivery than do respondents with the lowest economic status after controlling for other variables. economic factors are also seen in csr patterns in other countries, where the percentage of cs births in ghana ranges from 5% in very poor women to 27.5% in very rich women.(34) the csr in brazil is particularly high among wealthier women or those in maternity hospitals in the private sector.(35) in developing countries, economic inequality factors influence the pattern of cesarean delivery rates, which may be due to inadequate access to emergency obstetric care in the very poor and the poor, in co ntra st w ith the high ra tes of ce sare an deliveries without medical indication in the very rich group.(36) the data show that insurance ownership is related to the incidence of cesarean delivery. the percentage of cesarean deliveries in the group of women who have insurance is relatively high, namely 21.4% (table 2). these results are in line with the research of jenabi et al.(37) who reported that the factors of educational level, socioeconomic status, place of residence and insurance ownership were significantly related to the mother’s choice of cesarean delivery. the link between insurance and cesarean delivery rates in indonesia can be seen from the national health insurancehealthy indonesian card univ med vol. 41 no 2 136 idris, anggraini cesarean section in women of childbearing age quality control and cost control team report, that in the period of 2014-2018 around 57% of j a mi n an ke se ha t an nas i o na l kar t u indonesia sehat (jkn-kis) participants who gave birth chose the cs method. according to widjayanti,(38) the csr for jkn participants even reached 79.21%. se ve r a l f a c t or s o ut s i de o f sociodemographic and midwifery variables such as women’s choice, women’s fear, psychological factors and doctor’s preference also contribute to high csr.(23,39) severe fear of childbirth in women causes them to be at higher risk for emergency cs and to be more likely to choose elective cs, mostly due to non-obstetr ic indications.(40) solanki et al.(41) examined 6,542 births in south africa, of which 4,815 were cesarean births, causing the csr to reach 73.6%. most of the cs were emergency cs (39.7%), followed by elective cs (39.5%). weak regulations concerning hospitals that offer cs service packages (profit-oriented) as well as regulations that encourage moral hazard in doctors, thus allowing cs requests without adequate medical indications, also contribute to high csr.(6) therefore, adequate awareness, appropriate prenatal and perinatal counseling, fetomaternal monitoring, monitoring of vaginal bir th a fter ces ar ean (vbac) eff ort s and promotion of institutional delivery (in health facilities with trained birth attendants) can minimize obstetric complications and csr.(23,39) the present study has its limitations, such as the use of cross-sectional data that cannot determine a causal relationship, and the fact that there are several variables that have not been explored due to limited data regarding age, parity, maternal physical activity during pregnancy, delivery complications and the demand for cesarean delivery. on the other hand, it becomes difficult to limit the autonomy of women, especially those with higher income to demand or to suggest cesarean delivery. the government should consider this factor for controlling the cesarean delivery rate in indonesia. further study should expand this study by adding other variables related to cesarean delivery with a better design like cohort study. conclusions the current investigation based on the indonesian health demographic survey-2017 data has revealed that the prevalence of cs delive ry in indonesia is higher than that recommended by the who. economic status is the dominant variable in the prevalence of cesarean delivery in indonesia. conflict of interest the authors declare no competing interest acknowledgement we gratefully acknowledge the use of secondary data from the 2017 idhs. contributors hi contributed to conceptualizing the study design and acquiring the raw data for analysis; ra contributed to writing the manuscript then prepared the original draft of the manuscript. all authors have read and approved the final manuscript. references 1. betran ap, torloni mr, zhang jj, gülmezoglu am. who statement on caesarean section rates: a commentary. bjog 2016;123:667-70. doi: 10.1111/1471-0528.13526. 2. betrán ap, ye j, moller ab, zhang j, gülmezoglu am, torloni mr. the increasing trend in caesarean section rates: global, regional and national estimates: 1990-2014. plos one 2016; 11:1–12. https://doi.org/10.1371/journal.pone. 0148343. 3. tahir n, adil m, fatima s, khan s. cesarian sections: frequency and indications at peripheral tertiary care hospital. pak armed forces med j 2018;68:273-9. 4. badan kependudukan dan keluarga berencana nasional badan pusat statistik. laporan survei 137 demografi dan kesehatan indonesia, 2017. jakarta: badan pusat statistik;2020. 5. zahroh ri, disney g, betrán ap, bohren ma. trends and sociodemographic inequalities in the use of caesarean section in indonesia, 1987-2017. bmj global health 2020;5:e003844. doi:10.1136/ bmjgh-2020-003844. 6. ayuningtyas d, oktarina r, misnaniarti m, sutrisnawati nnd. bioethics in childbirth through sectio caesaria without medical indication. media kesehatan masyarakat indonesia universitas hasanuddin 2018;14:9-16. doi: http://dx.doi.org/ 10.30597/mkmi.v14i1.2110. 7. sandall j, tribe rm, avery l, et al. short-term and long-term effects of caesarean section on the health of women and children. lancet 2018; 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(2016). faktor-faktor yang berhubungan dengan persalinan sectio caesarea di rsud dr. h. abdul moeloek provinsi lampung. j kesehatan 2016;7:90-6. doi: http:// dx.doi.org/10.26630/jk.v7i1.124. 23. murtaza k, chaudhry m, nazeer s, malik s. prevalence-pattern and risk factors of cesarean section in a multiethnic cohort. pak j med sci 2021;37:711-5. doi: 10.12669/pjms.37.3.3186. 24. yaya s, uthman oa, amouzou a, bishwajit g. disparities in caesarean section prevalence and determinants across sub-saharan africa countries. global health res pol 2018;3:1-9. https:/ /doi.org/10.1186/s41256-018-0074-y. 25. verma v, vishwakarma rk, nath dc, khan hta, prakash r, abid o. prevalence and determinants of caesarean section in south and south-east asian women. plos one 2020;15:e0229906. doi: 10.1371/journal.pone.0229906. 138 idris, anggraini cesarean section in women of childbearing age 26. mumtaz s, bahk j, khang yh. rising trends and inequalities in cesarean section rates in pakistan: evidence from pakistan demographic and health surveys, 1990-2013. plos one 2017;12:e0186563. https://doi.org/10.1371/journal.pone.0186563. 27. ushie ba, udoh ee, ajayi ai. examining inequalities in access to delivery by caesarean section in nigeria. plos one 2019;14:e0221778. https://doi.org/10.1371/journal.pone.0221778. 28. epstein rm, gramling re. what is shared in shared decision making? complex decisions when the evidence is unclear. med care res rev 2013;70(1 suppl):94s–112s. https://doi.org/10. 1177/1077558712459216. 29. anwar i, nababan hy, mostari s, rahman a, khan ja. trends and inequities in use of maternal health care services in bangladesh, 1991–2011. plos one 2015;10:e0120309. https://doi.org/ 10.1371/ journal.pone.0120309. 30. tunçalp æ, pena rosas jp, lawrie t, et al. who recommendations on antenatal care for a positive pregnancy experience—going beyond survival. bjog 2017;124:860-2. doi: 10.1111/14710528.14599. 31. amjad a, amjad u, zakar r, usman a, zakar mz, fischer f. factors associated with caesarean deliveries among child-bearing women in pakistan: secondary analysis of data from the demographic and health survey, 2012–13. bmc pregnancy childbirth 2018;18:113. https://doi.org/ 10.1186/s12884-018-1743-z. 32. fekadu ga, kassa gm, berhe ak, muche aa, katiso na. the effect of antenatal care on use of institutional delivery service and postnatal care in ethiopia: a systematic review and metaanalysis. bmc health serv res 2018;18:577. https://doi.org/10.1186/s12913-018-3370-9. 33. sihombing nm, saptarini i, putri ds. determinan persalinan sectio caesarea di indonesia (analisis lanjut data riskesdas 2013). indonesian j reprod health 2017;8:63-73. doi: 10.22435/ kespro.v8i1.6641.63-75. 34. dankwah e, kirychuk s, zeng w, feng c, farag m. socioeconomic inequalities in the use of caesarean section delivery in ghana: a crosssectional study using nationally representative data. int j equity health 2019;18:162. https:// doi.org/10.1186/s12939-019-1063-6. 35. barros aj, victora cg, horta bl, et al. antenatal care and caesarean sections: trends and inequalities in four population-based birth cohorts in pelotas, brazil, 1982–2015. int j epidmiol 2019;48(supplement_1):i37-i45. https://doi.org/ 10.1093/ije/dyy211. 36. boatin aa, schlotheuber a, betran ap, et al. within country inequalities in caesarean section rates: observational study of 72 low and middle income countries. bmj 2018;360:k55. doi: https:/ /doi.org/10.1136/bmj.k55. 37. jenabi e, khazaei s, bashirian s, aghababaei s, matinnia n. reasons for elective cesarean section on maternal request: a systematic review. j matern fetal neonatal med 2020;33:3867-72. https:// doi.org/10.1080/14767058.2019.1587407. 38. widjayanti tb. analysis of sectio caesaria characteristics at profit hospital x around jakarta under national health insurance. j epidemiol kesehatan indonesia 2020;4:23–8. doi: 10.7454/ epidkes.v4i1.3699. 39. gonda a, bukhari s, karim mt, karim s. frequency of caesarean section at a tertiary care hospital. j sheikh zayed med coll 2017;8:124850. 40. ryding el, lukasse m, parys av, et al. fear of childbirth and risk of cesarean delivery: a cohort study in six european countries. birth 2015;42:4855. https://doi.org/10.1111/birt.12147. 41. solanki g, fawcus s, daviaud e. a cross sectional analytic study of modes of delivery and caesarean section rates in a private health insured south african population. plos one 2019;14:e0219020. https://doi.org/10.1371/journal.pone.0219020. c:\users\user\documents\41 no 2 169 abstract universa medicina long-covid neurological symptoms are associated with d-dimer levels in covid-19 patients diah kurnia mirawati1*, pepi budianto1, subandi1, rivan danuaji1, hanindia riani prabaningtyas1, ira ristinawati1, shabrina nur zidny1, and novian anindito santosa1 background coronavirus disease 2019 (covid-19) is a disease designated as a global pandemic by the who that can manifest clinically as neurological disorders that can occur in the acute phase or after the acute phase (long covid-19), such as headache, myalgia, anosmia, and cognitive impairment. these neurological disorders as symptoms of long covid-19 are presumably caused by hypercoagulable conditions characterized by an increase in ddimer level. this study aims to determine the correlation of long covid-19 neurological symptoms with hypercoagulable conditions and the role of d-dimer as a biomarker of long covid-19 neurological symptoms. methods this was a cross-sectional study involving 31 patients with long covid19 symptoms. admitted long covid-19 cases with recorded d-dimer levels and definitive outcomes were included consecutively. long covid-19 neurological symptoms were collected. d-dimer level was measured using immunofluorescence assay and reported in fibrinogen equivalent units (μg/ml). the correlation between d-dimer levels and neurological clinical manifestations was assessed by using ordinal regression analysis. the pvalue of <0.05 was considered statistically significant. results the mean age of the subjects was 38.81 ± 11.58 years and 18 (58.06%) were female. long covid neurological symptoms comprised myalgia, anosmia and cephalgia, and most subjects complained of myalgia (80.65%). on multivariable analysis, long-covid-19 neurological symptoms were significantly correlated with d-dimer [odds ratio (or) = 1.05; p=0.020]. conclusion the number of neurological long covid symptoms were significantly correlated with level of d-dimer. ultimately, more clarity is needed on the neurological impact of covid-19, its diagnosis, and its treatment. keywords: covid-19, d-dimer, long covid neurological symptoms original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.169-175 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1246 cite this article as: mirawati dk, bud iant o p, suba ndi, da nuaj i r , pra bani ngtyas hr , r isti nawa ti i, zidny sn, santosa na. long-covid neurological symptoms are associated with d-dimer levels in covid-19 patients. univ med 2022;41:169 -7 5. doi: 10.18051/univmed.2022 .v41 . 1 69 -1 75 . 1department of neurology, faculty of medicine, universitas sebelas maret, surakarta, indonesia correspondence: *diah kurnia mirawati jl. ir. sutami no. 36, jebres, surakarta, central java, indonesia phone and fax: (+62)271-664-178 email: diahkm@staff.uns.ac.id orcid id: 0000-0002-0558-5680 date of first submission, november 5, 2021 date of final revised submission, august 6, 2022 date of acceptance, august 13, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license may-august 2022 vol.41 no.2 170 mirawati, budianto, subandi, et al long-covid neurological symptoms introduction on march 11, 2020, the world health organization (who) declared covid-19 a pandemic. the disease is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2).(1) as of january 25, 2021, there were 99,851,223 confirmed cases and 2,140,684 de aths from this infection worldwide. in indonesia, there were 999,256 confirmed cases with a death toll of 28,132.(2) seve re acute resp iratory syndr ome coronavirus 2 enters target cells through the angiotensin-converting enzyme 2 (ace2) receptor with an incubation period of 2-14 days.(3,4) common symptoms include fever, dry cough, fatigue, dyspnea, pneumonia, and even respiratory failure.(5) other manifestations of neurological disorders also frequently appear, such as headache, myalgia, or anosmia. these manifestations can be considered as a direct effect of the virus on the central nervous system (cns), or as a neurological complication in the para-infectious or post-covid-19 phase.(6,7) similar to sars-cov, the sars-cov-2 virus uses the angiotensin 2 (ace2) receptors to enter the cells.(3,8) angiotensin-converting enzyme 2 receptors in the central nervous system are located on the cell surface, namely on neurons and on endothelial and smooth muscle cells in cerebral blood vessels. the sars-cov virus will stimulate the emergence of a cytokine storm which causes disruption of tight junctions in the blood-brain barrier resulting in its impaired permeability. through this scheme, the sarscov virus is able to enter the central nervous system and cause neurological symptoms.(9,10) long covid-19 symptoms are symptoms that appear persistently for more than 3 weeks after a patient is infected with covid-19.(11) it can appear in the form of neurological or nonneurological symptoms. the most common symptoms are fatigue (87%) and shortness of breath (71%) that appear within 79 days after infection with covid-19.(12) other symptoms include cognitive and memory disorders, headache, myalgia, pain in the chest and joints, disturbances of smell and taste, coughing, hair loss, sleep disturbances, wheezing, rhinorrhea, excess mucus production and disturbances in the heart and digestive system. the symptoms may persist for 6 months after the patient is discharged from the hospital (13) and can interfere with the patient’s activities of daily living and quality of life. the coagulopathy in covid-19 is caused by the induction of proinflammatory cytokines known as cytokine storms which can cause vasoconstriction resulting in increased deposition of fibrin and d-dimer.(9,14) the increase in ddimer levels can last up to 2 months in patients who have been infected with covid -19.(15) this is in line with other studies which claim that long covid-19 symptoms can last up to 2-3 months.(12) interestingly, in long-covid-19 patients, it is possible to detect a prolonged e l e va t i on of d-d i me r, r e ga r d le s s o f t h e inflammatory indices and the severity of the acute phase.(15) one of the indicators of a condition induced by hypercoagulative infection is the increase in d-d i me r t ha t c a n c a us e va r i o us c li n i c a l manifestations. the commonest lung function abnormality (16.36%) is a decline in diffusion capacity for carbon monoxide (dlco) in patients after 30 days discharge for acute covid-19. a higher level of d-dimer at admission is significantly associated with dlco%<80% suggesting that d-dimer might be a potential biomarker for the prediction of dlco decline in patients with covid-19.(15) a study involving 30 long covid-19 patients and 20 non-covid-19 subjects, showed that l on g co v id -19 i s i n d e pe n de nt l y a nd s i gn i f i c an t ly c or r e l a t e d wit h d-d i me r (standardized coefficient =0.259; p=0.047). however, these long-covid-19 patients had no e vi de n c e o f hypo f i b r i n oge ne mi a o r thrombocytopenia.(16) because of the high incidence of covid19 in the pandemic and inconsistent results of previous studies, the researchers were interested 171 in conducting a study to determine the role of ddimer as a biomarker of long neurological covid symptoms in covid-19 patients. methods research design this cross-sectional study was conducted at the neurology outpatient clinic and clinical pathology laboratory of dr. moewardi general hospital of surakarta from january to august 2021. study subjects a total of 31 patients aged at least 18 years with long covid symptoms who came to the neurology outpatient clinic of dr. moewardi general hospital of surakarta from january to august 2021 were included as subjects of the study. exclusion criteria were (1) having no data on d-dimer examination when initially infected with covid, (2) pregnant patients, and (3) possessing a history of symptoms and disorders related to hypercoagulation such as stroke infarct, deep vein thrombosis (dvt), and pulmonary embolism (pe). consecutive non-random sampling was used to select the subjects. data collection demographic characteristics of patients (age, sex, comorbidity), d-dimer on admission, neurological symptoms, length of stay, and ddimer outcome were recorded for each patient. all data were recorded on a standardized data collection f orm usin g standard units of measurement and were verified by the principal investigator. laboratory testing blood samples for d-dimer assessment were collected within 24 hours of admission and sent to the clinical pathology laboratory of dr. moewardi general hospital of surakarta. all measurements were done in the laboratory within 2 hours of sample collection. d-dimer was measured by immunofluorescence using the mispa-i2 analyzer (agappe diagnostics ltd., india). the kit used had a biological reference range of<0.5 μg/ml and the results were reported in fibrinogen equivalent units [feu (μg/ml)]. the results of laboratory testing were verified by a certified clinical pathologist. statistical analysis by using the normality test, the age data for the initial and final d-dimer levels in this study were not normally distributed. a multivariate ordinal logistic regression was used to assess the correlation between the d-dimer levels and the long covid-19 neurological symptoms. the software used for statistical analysis was statistical package for social sciences (spss) 22.0 version. a p-value of less than 0.05 was considered statistically significant. ethical clearance the subjects who met the inclusion and exclusion criteria and agreed to participate in this study were asked to fill out an informed consent form. this research has been declared ethically compliant by the health resea rch ethics committee of universitas sebelas maret through the ethics eligibility certificate no. 118/un27.06/ kepk/2019. results there were more female than male patients, with a mean age of 35 ± 11.58 years. the most common initial symptom complained of by the patients was fever, followed by anosmia, headache, myalgia, cough, and shortness of breath, while the least common symptom was diarrhea. the length of stay in hospital ranged from 8 to 21 days. there were 7 subjects with a history of comorbid diseases, the most common of which was hypertension followed by diabetes mellitus (dm), obesity, and asthma. the highest initial d-dimer levels of the patients (the d-dimer levels obtained when they were initially infected with covid and undergoing hospitalization) was 2740 μg/ml, while the lowest was 245 μg/ml. univ med vol. 41 no. 2 172 mirawati, budianto, subandi, et al long-covid neurological symptoms a b the final d-dimer levels (d-dimer levels checked at the polyclinic) ranged from 147 μg/ml to 871 μg/ml. there were 8 subjects whose final ddimer levels were higher than the initial. the lowest increase in d-dimer levels was 20 μg/ml, while the highest was 272 μg/ml (table 1). based on the multivariate ordinal regression analysis, there was a significant correlation between the d-dimer levels and the number of long covid-19 neur ological symptoms (or=1.05; p=0.020) (table 2). discussion covid-19 infection has a very wide range of clinical manifestations. the most frequent symptoms complained of by the subjects of this study were fever (67.74%), followed by anosmia (67.74%), and cough (38.71%). this result was in line with the findings of other researchers. based on several studies summarized by iser et al.(17) symptoms such as fever, cough, and dyspnea are the most frequent indicators/ table 2. ordinal logistic regression analysis of d-dimer level in covid-19 patients or: odds ratio, aor: adjusted odds ratio; ci: confidence interval *statistically significant variable or p-value aor 95 % ci early model age 0.787 0.040* 1.53 0.89-1.97 neurological symptoms 0.637 0.288 1.19 0.91-1.74 final model neurological symptoms 0.841 0.020* 1.05 0.96-1.15 table 1. characteristics of study subjects characteristics mean  sd median (min – max) n (%) sex male 13 (41.94%) female 18 (58.06%) age 38.81  11.58 initial covid symptoms fever 21 (67.74%) anosmia 16 (51.61%) cough 12 (38.71%) myalgia 7 (22.58%) shortness of breath 4 (12.90%) diarrhea 3 (9.68%) long covid neurological symptoms myalgia 25 (80.65%) anosmia 13 (41.94%) cephalgia 6 (19.35%) number of long covid neurological symptoms 0 1 (3.23%) 1 16 (51.61%) 2 14 (45.16%) comorbidity diabetes mellitus 2 (6.45%) hypertension 5 (16.13%) obesity 1 (3.23%) asthma 1 (3.23%) d-dimer level (μg/ml) initial d-dimer 460 (245 – 2740) final d-dimer 283 (147 – 871) difference d-dimer 198 (-2540 – 272) length of stay (days) 12.32  2.91 173 symptoms of covid-19. gastrointestinal symptoms and loss of taste (ageusia) or loss of smell (anosmia/hyposmia) are frequently reported in mild cases, while shortness of breath or dyspnea is common in severe and fatal cases. according to lechien et al.,(18) anosmia is a specific symptom of covid-19 infection, so it can serve as a diagnostic tool for developing countries in the current pandemic. the mechanism of anosmia in covid-19 does not appear to directly involve nasal obstruction but is more associated with the damage to the olfactory neuroepithelium. the long covid neurological symptoms complained by our study subjects were myalgia (80.65%), anosmia (41.9%) and cephalgia (19.35%). this result is in accordance with the study conducted by stefanou et al.(9) who found that after 6 months following acute covid-19 infection, the patients complained of at least one of the symptoms of fatigue or muscle weakness, sleep disturbances, smell or taste impairment, myalgia and headache. in this study, the subjects’ d-dimer levels during admission were 245 μg/ml at the lowest and 2740 μg/ml at the highest. meanwhile, the d-dimer levels examined after covid-19 infection were 147 μg/ml at the lowest and 871 μg/ml at the highest. this finding is in accordance with those of the studies conducted by yao et al.(19) and poudel et al.(20) who showed d-dimer to be a biomarker of the severity of covid-19 cases. numer ous studies have shown the correlation between the d-dimer levels and the severity of covid-19 cases, but no studies have presented the correlation between the d-dimer levels and the type of clinical manifestations in patients. the higher the final d-dimer levels were, the higher would be the number of long covid symptoms experienced by the patient. in our study, the long covid symptoms consisted of cephalgia, anosmia, and myalgia. this may be because the subjects in this study were limited to patients with mild to moderate covid-19 symptoms, such that the clinical manifestations of covid-19 and long covid symptoms were less diverse. camargo-martínez et al.(21) concluded that the common cephalgia symptoms experienced by covid-19 patients tend to occur together with gastrointestinal symptoms related to the braingut axis. activation of the brain-gut axis is mediated by the release of inflammatory cytokines such as tnfα, interleukins, and peptides linked to the calcitonin and neuropeptides genes that are significantly associated with trigeminovascular activation leading to cephalgia. osawa et al.(22) revealed that covid-19 infection is usually associated with coagulopathy resulting in elevated d-dimer levels. the increase in d-dimer levels during admission is associated with thromboembolic events.(15) the increase also indicates an increase in fibrinolysis, the occurrence of coagulation in blood vessels, the incidence of vascular thrombus, and tissue damage which can be the cause of the appearance of severe clinical symptoms in covid-19 patients. at this moment, there is no consensus regarding the limit for d-dimer levels to predict the prognosis of covid-19 patients. however, other studies stated that covid-19 is associated with an increased incidence of thrombosis, although its correlation with an increase in ddimer is still unclear.(23) there is significant heterogeneity among studies on d-dimer and covid-19. different laboratories use different kits for measurement (such as standard f200 analyzer from sd biosensor,korea; poct axceed p200 from bi oscience (tianjin) diagnostic technology co., ltd; getein 1100 immunofluorescence quantitative analyzer from getein biotech inc., china; and mispa-i2 from agappe diagnostics ltd., india) and the accuracy and reliability of measurement can vary according to the kit manufacturer. furthermore, there is variation in reporting units. favaloro and thachil (24) analyzed 20 papers on covid-19 and ddimer and found that most papers did not report which manufacturer and reagent kit was used and whether d-dimer values were reported in d-dimer units (ddu) or fibrinogen equivalent units (feu). they also found that nearly half of the studies did not report normal cutoff values. this lack of standardization leads to chances of univ med vol. 41 no. 2 174 mirawati, budianto, subandi, et al long-covid neurological symptoms pitfalls in the analysis and interpretation of ddimer values in covid-19. a major limitation of our study is selection bias because only patients admitted to the hospital were included, which meant that asymptomatic patients with high oxygen saturation, who were not admitted according to hospital guidelines, were not included in the study. in addition, the time from the onset of the illness to hospital presentation may affect the d-dimer values. the d-dimer assay is a widely available, relatively inexpensive, and easy to perform laboratory test. finally, the findings of this study supported the potential benefit of measuring d-dimer as biomarkers in the prevention of long-covid neurological symptoms and provided an indication for further research. apart from the need of covid-19 treatment options, there is actually an urgent need for the discovery of new biomarkers of early longcovid neurological symptoms as well as biomarkers for monitoring the severity of covid19 which may also be useful as new treatment targets. conclusion this study demonstrated that there was a significant correlation between the final d-dimer levels a nd the numbe r of long covid neurological symptoms in covd-19 subjects. it can be concluded that the neurological long covid symptoms were significantly correlated with the hypercoagulable condition, and that the d-dimer level could be a predictor of this condition. conflict of interest compe ting inte re sts: no re levant disclosures. acknowledgment we would like to express our gratitude to the management staff of the clinical pathology laboratory of dr. moewardi general hospital for facilitating this research. contributors conceptualization, manuscript writer, and guarantor: dkm. data collecting, analysis, and manuscript writer: pb, s,rd, nas. writing, review and editing the final manuscript: hrp, ir, snz. all authors have read and approved the final manuscript. references 1. world health organization. coronavirus cases. geneva: world health organization;2021. 2. kementerian kesehatan republik indonesia. coronavirus. jakarta: kementerian kesehatan republik indonesia;2021. 3. zhang h, penninger jm, li y, zhong n, slutsky as. angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic 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mechanisms. acs chem neurosci 2020;11:995–8. doi: 10.1021/acschemneuro. 0c00122. 9. stefanou ml, palaiodimou l, bakola e, et al. neurological manifestations of long-covid syndrome: a narrative review. ther adv chronic dis 2022;13:20406223221076890. doi: 10.1177/ 20406223221076890. 10. jesuthasan a, massey f, manji h, zandi ms, wiethoff s. emerging potential mechanisms and 175 univ med vol. 41 no. 2 predispositions to the neurological manifestations of covid-19. j neurol sci 2021; 428:117608. doi: 10.1016/j.jns.2021.117608. 11. akbarialiabad h, taghrir mh, abdollahi a, et al. long covid, a comprehensive systematic scoping review. infection 2021;49:1163–86. doi: 10.1007/s15010-021-01666-x. 12. garg m, maralakunte m, garg s, et al. the conundrum of ‘long-covid-19¹: narrative review. int j gen med 2021;14:2491–506. doi: 10.2147/ ijgm.s316708. 13. yong sj. long covid or post-covid-19 syndrome: putative pathophysiology, risk factors, and treatments. infect dis (lond) 2021; 53:737-54. doi: 10.1080/23744235.2021.192439. 14. ortega-paz l, capodanno d, montalescot g, angiolillo dj. coronavirus disease 2019– associated thrombosis and coagulopathy: review of the pathophysiological characteristics and implications for antithrombotic management. j am heart assoc 2021;10:1–24. https://doi.org/ 10.1161/jaha.120.019650. 15. townsend l, fogarty h, dyer a, et al. prolonged elevation of d-dimer levels in convalescent covid-19 patients is independent of the acute phase response. j thromb haemost 2021;19:1064– 70. doi: 10.1111/jth.15267. 16. connors jm, levy jh. covid-19 and its implications for thrombosis and anticoagulation. blood 2020;135:2033-40. doi: 10.1182/blood. 2020006000. 17. iser bpm, sliva i, raymundo vt, poleto mb, schuelter-trevisol f, bobinski f. suspected covid-19 case definition: a narrative review of the most frequent signs and symptoms among confirmed cases. epidemiol serv saude 2020;29: e2019354. doi: 10.5123/s1679-49742020000300018. 18. lechien jr, barillari mb, jouffe l, saussez s. anosmia is a key symptom of covid-19 infection and should be used as a diagnostic tool. ear nose t hroat j 2020;99:577-8. doi: 10.1177/ 0145561320925191. 19. yao y, cao j, wang q, et al. d-dimer as a biomarker for disease severity and mortality in covid-19 patients: a case control study. j intensive care 2020;8:49. https://doi.org/10.1186/s40560-02000466-z. 20. poudel a, poudel y, adhikari a, et al. d-dimer as a biomarker for assessment of covid-19 prognosis: d-dimer levels on admission and its role in predicting disease outcome in hospitalized patients with covid-19. plos one 2021;16: e0256744. https://doi.org/10.1371/journal.pone. 0256744. 21. camargo-martínez w, lozada-martínez i, escobar-collazos a, et al. post-covid 19 neurological syndrome: implications for sequelae’s treatment. j clin neurosci 2021;88:21925. doi: 10.1016/j.jocn.2021.04.001. 22. osawa i, okamoto k, ikeda m, otani a, wakimoto y, yamashita t. dynamic changes in fibrinogen and d-dimer levels in covid-19 patients on nafamostat mesylate. j thromb thrombolysis 2021;51:649–56. doi: 10.1007/s11239-020-02275-5. 23. simadibrata dm, lubis am. d-dimer levels on admission and all-cause mortality risk in covid19 patients: a meta-analysis. epidemiol infect 2020;148:e202. doi: 10.1017/s0950268820002022. 24. favaloro ej, thachil j. reporting of d-dimer data in covid-19: some confusion and potential for misinformation. clin chem lab med 2020;58: 1191–9. doi: 10.1515/cclm-2020-0573. cite this article as: surasmiati nma, suryathi nma, andayani a. serum microrna-126 expression as a biomarker of diabetic retinopathy. univ med 2023;42:121-7. doi: 10.18051/ univmed.2023.v42:121-127 may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 serum microrna-126 expression as a biomarker of diabetic retinopathy ni made ayu surasmiati1* , ni made ari suryathi1 , and ari andayani1 abstract background diabetic retinopathy (dr) is a microvascular complication of diabetes mellitus (dm). diabetic retinopathy causes permanent blindness in the productive age group and has a multifactorial pathogenesis. microrna 126 (mirna-126) regulates the expression of the vascular endothelial growth factor (vegf) gene at the post-transcriptional level, vegf being an important angiogenic protein regulating inflammation in dr development. this study aimed to determine serum mirna-126 expression as a biomarker in dm patients with dr. methods this was a cross-sectional study involving 4 healthy persons and 21 type 2 dm patients. subjects consisted of 4 groups: i) healthy controls, ii) dm patients without diabetic retinopathy (ndr), iii) dm patients with non-proliferative dr (npdr) and iv) dm patients with proliferative dr (pdr). venous blood was collected from subjects for mirna-126 examination by real-time polymerase chain reaction (pcr). mirna-126 in each group was analyzed using the one-way anova test and p<0.05 was considered to be statistically significant. results mean mirna-126 expression was significantly decreased in pdr (1.86±1.03) and npdr (1.01±0.43) groups when compared to healthy control (2.44±1.29) and ndr groups (2.15± 0.48) (p=0.027). mirna126 values of less than 1.81 can differentiate ndr from the control group (sensitivity 83%, specificity 75%) and mirna-126 of less than 1.56 can be used to predict npdr when compared to the control group (sensitivity 86%, specificity 75%). conclusion serum mirna-126 is a potential biomarker for screening of npdr and ndr in type 2 dm patients, and could be considered a non-invasive diagnostic parameter. keywords: microrna-126, diabetes mellitus, diabetic retinopathy, biomarker 1ophthalmology department, faculty of medicine, udayana university, bali, indonesia *correspondence: ni made ayu surasmiati sekretariat ilmu kesehatan mata rsup prof i.g.n.g ngoerah gedung cempaka lantai iv jalan diponegoro denpasar bali indonesia 80113 email: surasmiati@unud.ac.id orcid: 0000-0001-7835-224x date of first submission, march 2, 2023 date of final revised submission, june 13, 2023 date of acceptance, june 19, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2023.v42:121-127 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1445 121 original article https://orcid.org/0000-0001-7835-224x https://orcid.org/0000-0003-2181-9480 https://orcid.org/0009-0006-2431-5439 122 surasmiati , suryathi , andayani serum microrna-126 expression introduction diabetic retinopathy (dr) is a form of microvascular complication of diabetes mellitus (dm) caused by prolonged hyperglycemia. the prevalence of dr is predicted to increase along with the increase in dm patients. diabetic retinopathy is the leading cause of vision loss in adults aged 20–74 years.(1) according to the wisconsin epidemiologic study of diabetic retinopathy (wesdr), in patients with a dm duration of 20 years, the incidence of dr reaches 99% in type 1 dm and 60% in type 2 dm.(2) diabetic retinopathy can be clinically classified into non-proliferative dr (npdr) and proliferative dr (pdr). retinal microvascular changes in the npdr stage do not cross the internal limiting membrane (ilm). clinical features of the r etina may include microaneurysms, non-perfused capillary areas, nerve fiber layer (nfl) infarction, intraretinal microvascular abnormalities (irmas), intraretinal dot and blot hemorrhages, retinal edema, hard exudates, arterial abnormalities, retinal vein dilation and beading. proliferative dr consists of extraretinal fibrovascular tissue proliferation, such as the formation of new blood vessels, with minimal fibrous tissue crossing the ilm, with the blood vessels changing in size to become larger in diameter and thinner, accompanied by an increase in fibrous tissue. when the new vessels regress, the fibrovascular tissue remnants will proliferate along the posterior hyaloids. the proliferation of new blood vessels around the optic nerve is called neovascularization at the disc (nvd), whereas proliferation of new vessels in other places is called neovascularization elsewhere (nve). decreased visual acuity and blindness occur in severe cases of dr with complications such as vitreous hemorrhage, rhegmatogenous retinal detachment, and fibrovascular proliferation.(3,4) one of the main factors which plays an essential role in angiogenesis in the hyperglycemic condition is vascular endothelial growth factor (vegf). intravitreal anti-vegf injection has been used for dr treatment but the clinical responses have been very diverse.(5,6) research in microrna (mirna) is currently being developed. microrna is a short chain noncoding rna segment that inhibits gene expression in the post-transcriptional stage. microrna-126 binds to the 3' untranslated region (utr) of the vegf messenger rna and also plays a role in suppressing two negative regulators of vegf, i.e. sprout-related evh1 domain-containing protein 1 (spred1) and phosphoinositide-3-kinase regulatory subunit 2 (pik3r2). it has been found that in the hyperglycemic state mirna-126 expression is low in endothelial cell culture,(7) plasma,(8) and serum.(9–12) the low expression of mirna-126 in both hyperglycemic endothelial cells (representing intracellular mirna) and in serum could make mirna-126 a biomarker for the screening of retinal endothelial cell damage and the early diagnosis of the proliferative phase of dr.(11,12) many studies about the lower level of mirna-126, both in vitro and in vivo, have been done in the hyperglycemic state. microrna-126 is a stable molecule in the circulation and its serum level is equivalent to its level in the retinal endothelium, making it a good marker for tissue damage in hyperglycemia. most of the studies in human subjects aimed to identify markers for the late stage of dr, such as pdr.(13–16) the ability to distinguish npdr as the early stage of dr from non-dr (ndr) in type 2 dm based on the level of unregulated mirnas, remains unknown. many studies showed that mirnas have key roles in the development of many diseases including dr. therefore, assessment of mirna levels in plasma may be an important tool for diagnosing disea ses and following their progression.(17) the present study was conducted to obtain data on serum mirna-126 expression as a biomarker for the discrimination of ndr, npdr and pdr in type 2 dm patients. methods research design this study was an observational crosssectional study to determine serum mirna-126 expression in dm patients with dr divided into 123 npdr and pdr. the research was conducted at the sanglah general hospital, bali, from march to august 2021. research subjects the study involved 25 subjects consisting of 4 healthy non-dm patients and 21 patients with type 2 dm , namely 8 type 2 dm patients without dr (ndr), and 13 dm patients with dr which were subdivided into npdr and pdr groups of 7 and 6 patients, respectively. the inclusion criterion for dm subjects was type 2 dm patients who were willing to have their venous blood collected and in whom retinal evaluation under dilated pupil was feasible. exclusion criteria were patients with a history of malignancy, under corticosteroid treatment in the last 3 months, having undergone vitrectomy, and patients with a history of bevacizumab injections in the last 3 months. data collection history taking was done by a research assistant collecting age, length of dm diagnosis, and dm treatment. the ophthalmological examination included visual acuity, intraocular pressure, and fundus examination under dilated pupil (with instillation of 1% tropicamide). the fundus examination was done using a slit lamp biomicroscope and 78 d condensing lens by an external vitreoretinal consultant. some patients underwent optical coherence tomography for macular edema. laboratory analysis all subjects were instructed to fast for 8-10 hours. six milliliter of venous blood was drawn from the antecubital vein of the fasting subjects under aseptic conditions. the blood samples were then divided into two parts that were placed into an ethylenediaminetetraacetic acid (edta) tube for hba1c examination and a plain red tube for mirna examination. serum mirna-126 examination a total of 3 ml venous blood in a plain red tube was centrifuged at 3000 rpm for 15 minutes to obtain the serum, which was then stored at minus 80 degrees celsius. from the serum samples, 400 mirnas were isolated from rna by quantitative real-time polymerase chain reaction (qrt-pcr). total rna including mirna was extracted using mirva nat m paristm rna reagent. reverse transcription was done with the taqman microrna reverse transcription kit on a takara thermal cycler pcr instrument. expression was detected by real time pcr with taqman fast advanced master mix and taqman microrna assay reagents. gene expression was calculated by relative quantification (comparative delta ct=2-(ct)(18) and required comparator genes in the form of endogenous control and reference/calibrator gene samples. the endogenous control used was mirna-328-3p, which normalizes the number of target mirnas to the total number of mirnas in the sample.(19) the calibrator sample was a comparison sample from healthy individuals. relative quantification (rq) is a value that represents the ratio of mirna expression in the target sample to that in the calibrator sample. this result is then made the cut-off point, where the values below the cut-off point are classified as low. the cut-off point is obtained from the calculation of the statistical magnitude using the roc curve. statistical analysis data was analyzed using the computer program statistical package for the social sciences (spss) version 22.0 from ibm corp., armonk, ny, usa. numerical data were tested by the shapiro-wilk test. comparison of variables between groups was done using the kruskalwallis and one way anova tests. post-hoc lsd analysis was used for further analysis of betweengroup differences. a p-value of <0.05 was considered statistically significant. diagnostic accuracy was presen te d using the terms sensitivity and specificity. receiver operator characteristics (roc) analysis was used to determine the optimum cut-off value for mirna126 in differentiating ndr, npdr, and pdr from the healthy controls. univ med vol. 42 no. 2 124 surasmiati , suryathi , andayani serum microrna-126 expression ethical clearance ethical clearance for the study was obtained from the research ethics committee, faculty of medicine, udayana university, under registration number 752/un14.2.2.vii.14/lt/2021. results most of the subj ects were women, consisting of 17 persons (68%). the mean age of the subjects was 58.10±7.29 years. most of the 21 patients with type 2 dm received oral antidiabetic therapy (61.9%) and had dm for less than 10 years (57.1%). mean hba1c was 7.89 ± 1.48% (table 1). mean mirna-126 expression was lower in the npdr group than in the pdr and ndr groups and was highest in the healthy controls (p=0.027) (table 1). post-hoc analysis using lsd showed that there were significant differences between the healthy control group and the npdr group (p=0.009) and between the npdr and ndr groups (p=0.011) (table 2). the cut-off point for ndr compared to the healthy group was 1.81 (sensitivity 83%, specificity 75%). npdr can be distinguished from the healthy group by a mirna-126 level of less than 1.56 (sensitivity 86%, specificity 75%) (table 3). the best cut-off point of mirna-126 was 1.81 for ndr and 1.56 for npdr. (table 3, figure 1) based on the roc analysis, mirna-126 level in the pdr group showed a tendency to cluster around and above the diagonal. thus, mirna-126 cannot be used as a biomarker to differentiate between the pdr group and the healthy control group. discussion in this study, it was found that 17 (68%) of the 25 study subjects consisted of women. based on the 2018 riskesdas report, the prevalence of dm in people aged more than 15 years in bali parameter healthy controls (n=4) ndr (n=8) npdr (n=7) pdr (n=6) p value sex male 2 (25.0) 1 (12.5) 2 (25.0) 3 (37.5) 0.422* female 2 (11.8) 7 (41.2) 5 (29.4) 3 (17.6) age (years) 58.50 ± 9.68 56.88 ± 9.13 61.14 ± 5.24 54.67 ± 4.84 0.462** dm treatment insulin 0 1 (12.5) 2 (25.0) 5 (62.5) 0.001* oad 0 7 (53.8) 5 (38.5) 1 (7.7) no other treatment 4 (100) 0 0 0 duration of dm (years) < 10 0 8 (66.7) 2 (16.7) 2 (16.7) 0.001* ≥ 10 0 0 5 (55.6) 4 (44.4) hba1c (%) 7.38 ± 1.54 7.91 ± 1.40 8.53 ± 1.48 0.539** mirna-126 2.44 ± 1.29 2.15 ± 0.48 1.01 ± 0.43 1.86 ± 1.03 0.027** notes: data presented as n (%), except for age, hba1c and mirna-126 (mean ± sd); oad= oral antidiabetic drugs; ndr= non diabetic retinopathy; npdr= non-proliferative diabetic retinopathy; pdr= proliferative diabetic retinopathy; dm= diabetes mellitus; hba1c= hemoglobin a1c; mirna= micro rna; *analyzed using kruskal-wallis test; **one way anova analysis was done to compare means between groups table 1. characteristics of research subjects mean difference p value healthy ndr 0.288 0.558 npdr 1.429* 0.009 pdr 0.585 0.263 ndr npdr 1.141* 0.011 pdr 0.297 0.492 npdr pdr -.844 0.068 note : ndr: non diabetic retinopathy; npdr : non-proliferative diabetic retinopathy; pdr : proliferative diabetic retinopathy table 2. post-hoc analysis of mirna-126 using lsd 125 auc cut-off point sensitivity specificity healthy vs ndr healthy vs npdr healthy vs pdr 0.250 0.036 0.563 1.81 1.56 1.83 0.83 0.86 0.75 0.75 0.75 0.50 table 3. roc curve analysis of mirna-126 note: ndr= non diabetic retinopathy; npdr= non-proliferative diabetic retinopathy; pdr= proliferative diabetic retinopathy province was 1.7%. the prevalence of dm in indonesia based on gender was found to be higher in women (2.4%) than in men (1.7%).(20) the present study found that most of the subjects with dm used oral antidiabetic agents rather than insulin injections. this is in line with the 2018 riskesdas data where the proportions of dm treatments in the province of bali were 64.6% for oral antidiabetic drugs, 15.7% for insulin injections, and 15.3% for combinations of oral and injected drugs, with 4.4% for the untreated.(20) microrna (mirna) is an endogenous, single-stranded rna sequence of about 19-24 nucleotides that interacts with the target rna and regulates gene expression at the posttranscriptional level. microrna-126 is the most widely expressed mirna in endothelial cells and plays an important role in angiogenesis and vascular integrity. under conditions of hypoxia and hyperglycemia, mirna-126 plays a role in regulating the process of angiogenesis. mirna126 inhibits vegf expression by binding to the 3' untranslated region of the vegf-a messenger rna (mrna).(21-25) microrna-126 is one of the mirnas which plays a role in the dr angiogenesis and inflammation pathways.(8,10–12) (a) (c)(b) figure 1. roc curve analysis of plasma mirna-126 for discriminating: a. healthy controls from ndr; b. healthy controls from npdr; and c. healthy controls from pdr diabetic retinopathy (dr) is one of the microvascular complications of dm causing changes in the retinal vascular endothelium. the early phase of these changes is marked by the loss of endothelial pericytes resulting in disruption of vascular permeability. disruption of endothelial cells is manifested as microaneurysms as a marker of the npdr phase, whereas retinal neovascularization is a sign of the pdr phase.(3,4) the retinal vegf molecule is an important molecule as a marker of pdr, making anti-vegf a major treatment for this condition. as the circulating vegf molecule cannot be used as a biomarker for dr and pdr, other biomarkers are needed for diagnostic purposes. research conducted in type 2 dm patients found lower expression of mirna-126 compared to non-dm controls,(9,11) similar to that found in type 1 dm patients. patients with pre-diabetes also showed lower mirna-126 levels than did healthy controls.(9,12) low mirna-126 is also seen in patients with other vascular diseases such as coronary artery disease (26) and intracerebral hemorrhage.(27) our study also found a similar condition, in which the mean mirna-126 levels in the healthy controls was higher compared to the dm groups. we also found lower mirnauniv med vol. 42 no. 2 126 surasmiati , suryathi , andayani serum microrna-126 expression 126 expression in the dr group than in the nondr group. the present study found that the mean mirna-126 was higher in the pdr group and therefore does not reflect dr severity. however, if we compare the npdr and ndr groups with the healthy control group, the roc area under the curve is below the diagonal curve. this shows that mirna-126 can be used as predictor for ndr and npdr but not for pdr. an endogenous control functions to normalize the number of target mirnas to the total number of mirnas. the requirement for an endogenous control is stable expression and not being affected by the target mirna expression, the environment, or the treatment given to the study subjects. the endogenous control used in this study was mirna-328-3p, although a great number of other studies use mir-39 and u6 snrna.(11,19) in this study, we conclude that mirna-126 is expressed in serum, for which the endogenous control mirna-328-3p can be used in dr subjects. there are differences in mean mirna126 expression in the healthy control, type 2 dm without dr, and type 2 dm with dr groups. in the pdr group there was a decrease in mirna126 expression compared to the healthy group such that mirna-126 can be used to predict dr in type 2 dm patients. this study did not consider the other microvascular complications of dm such as neuropathy and nephropathy. a cohort study on diabetes patients needs to be done to further evaluate the role of serum mirna-126 on dr progressivity and microvascular improvement with dm treatment. conclusion this study showed serum mirna-126 to be a biomarker for identifying npdr and ndr in type 2 dm patients. conflict of interest competing interests: no relevant disclosure. acknowledgement the author would like to thank the biomolecular laboratory, faculty of medicine and health sciences, warmadewa university, and the integrated biomedical laboratory, faculty of medicine, udayana university, for assisting in the molecular testing for this research. funding this research was funded by dipa pnbp udayana university ta-2021 in accordance with the research implementation assignment agreement no. b/5168/un14.2.2.v ii.1/ pt.01.03/2021 of june 7, 2021. author contribution nmasm 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10.3389/fnins.2019.00866. univ med vol. 42 no. 2 c:\users\universa medicina\docu 226 abstract universa medicina outcomes of ureteral stent placement for hydronephrosis in patients with ureteral stone dyah ratih widyokirono1,2, yudhistira pradnyan kloping1,2, zakaria aulia rahman1,2, and lukman hakim1,3* background routine ureteral stent placement after ureteroscopy (urs) for ureteral stone treatment is arguable due to the possible stent-related symptoms. several studies claimed that its use is necessary, while others reported that its use is excessive. hydronephrosis occurs when urine cannot drain out from the kidney to the bladder due to blockage or obstruction. we aimed to evaluate the role of ureteral stents in hydronephrosis resolution in ureteral stone patients following urs lithotripsy. methods this was a prospective observational study using secondary data involving 130 ureteral stone patients undergoing urs lithotripsy [99 patients (76.2%) with stent placement and 31 patients (23.8%) without stent]. data consisting of baseline characteristics, pre-operative status, intraoperative characteristics, and postoperative complications were collected from the medical record database and presented descriptively. the patients were divided into two groups based on stent placement. comparison of hydronephrosis resolution between the groups was analyzed with chisquare. results ureteral lesions were the most common indication of ureteral stent placement following urs lithotripsy (28.3%). the most bothersome symptoms were dysuria in 18 patients (18.2%); followed by frequency in eight patients (8.1%) and low back pain in six patients (6.1%). all symptoms were successfully treated with oral medications. there were 41 patients (91.1%) with pre-operative hydronephrosis significantly resolved after stent placement compared to 5 (62.5%) patients without stent placement (p=0.027). conclusion ureteral stenting significantly resolves pre-operative hydronephrosis after urs lithotripsy in patients with ureteral stone. ureteral stent placement is the preferred method for the treatment of pre-operative hydronephrosis. keywords: ureteral stent, ureteral stenting, ureteroscopic lithotripsy, ureteral stone original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.226-233 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1214 september-december 2021 vol.40no.3 cite this article as: widyokirono dr, kloping yp, rahman za, hakim l. outcomes of ureteral stent placement for hydronephrosis in patients with ureteral stone. univ med 2021;40:226-33. doi : 10. 1805 1/un ivmed.20 21.v40. 2 26 -2 33 . 1department of urology, faculty of medicine, universitas airlangga, surabaya, east java, indonesia 2dr. soetomo general-academic hospital, surabaya, east java, indonesia 3universitas airlangga teaching hospital, surabaya, east java, indonesia correspondence: *lukman hakim department of urology, faculty of medicine, universitas airlangga, universitas airlangga teaching hospital, surabaya, east java, indonesia email: lukman-h@fk.unair.ac.id orcid id : 0000-0003-1518-8010 date of first submission, august 14, 2021 date of final revised submission, november 2, 2021 date of acceptance, november 4, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 227 univ med vol. 40 no 3 introduction urinary tract stones are the third most common disease of the urinary tract, apart from urinary tract infections and pathological conditions of the prostate.(1) the prevalence of urinary tract stones is estimated to be between 1% and 15% depending on age, gender, race, and geography.(2) ureteral stones may cause several complications such as renal colic, ureteric obstruction, hydronephrosis and infection. the management of ureteral stones va r i e s f ro m e x t r a c o r p or e a l s ho c k wa ve lithotripsy (eswl), ureteroscopy (urs), me di c a l ex pu l si ve th e r a py (m e t ) , a nd laparoscopic surgery.(3) urs has been used to treat lower urinary tract stones as well as those in the upper tract.(1,2) it is a method of treating ureteric stones besides eswl. compared to eswl, urs has a greater stone free rate in four weeks and is a safe option for obese patients.(3) the technological advancements of urs lithotripters, such as the development of smaller and more flexible urs and the use of laser lithotripsy have made this procedure more effective and safer for urinary stone treatment. indications for ureteric stone removal include persistent pain or obstruction, renal insufficiency, and low chance of spontaneous discharge, comorbidity profile, social situation and patient preferences.(3) treatment after stone treatment varies depending on patient characteristics and comorbidities, residual stones, complications, and the decision of the surgeon. stent placement after urs is recommended by the latest european association of urology (eau) guidelines if there is an increase in postoperative complications; in case of ureteral lesions, bleeding, residual fragments, perforation, urinary tract infection and pregnancy; and in patients who are at increased risk of complications.(3) ureteral stent insertion after urs reduces the risk of ureteral stricture, which can lead to ureteral obstruction and obstructive nephropathy.(4) the stents sustain the patency of the ureter for drainage of stone fragments to prevent steinstrasse formation and s i mu l t a ne ou sl y r e s ol ve hydr on e p hr o s i s. howeve r, ureteral stent insertion for the treatment of ureteral stone is still debatable, considering the stent-associated symptoms i nc l ud i ng h e ma t ur i a , d ys u r i a , a nd o t h e r discomforts such as stent fragmentation and encrustation.(4,5) symptoms associated with stenting cannot be ignored and it is still controversial whether the patient should have a stent inserted after surgery.(6,7) the european association of urology (eau) guidelines state that routine stenting after an uncomplicated urs procedure with clean stone removal is unnecessary.(3) a meta-analysis showed that stenting failed to improve the stonefree rate, and instead, it generated complications such as hematuria, irritative urinary symptoms, urinary infection, and dysuria, which were significantly higher in the stented group.(8) these symptoms may reduce the quality of life of affected patients. despite the recommendations made by the eau and american urological association (aua) guidelines, recent studies reported that there is an excessive use of ureteral s t e nt s a mo ng p a t i e n t s un d e rgoi n g urs lithotripsy.(9) stent placement is indicated in patients with symptomatic hydronephrosis and patients with renal dysfunction. one study reported that 85.2% of patients with symptomatic hydronephrosis showed symptom improvement after stent placement, (10) while the failure rate of ureteral stent placement was reported to be 16–58%.(11) in view of this, we aimed to evaluate the role of ureteral stents in hydronephrosis resolution following urs lithotripsy in ureteral stone patients. methods research design this was an ana lyt ic al study with a prospective approach utilizing secondary data taken from the medical records of dr. soetomo general-academic hospital from january 2018 to december 2019. 228 study subjects total sampling was performed in this study; thus, the sample size represents the whole population of all eligible patients admitted to the hospital. a total of 130 patients who had ureteral stones and underwent urs lithotripsy were included in this study. the inclusion criteria of this study were male and female patients with ureteral stones who underwent urs lithotripsy, and were aged 18 years or above. the exclusion cri teri a of this stud y we r e p atien ts with comorbidities, history of malignancy, abnormal laboratory results, and patients with incomplete data. patient assessment all patients were admitted to the hospital and assessed preoperatively by history and physical examination. laboratory data collected included full blood counts; kidney function tests including serum creatinine, urea, sodium, and potassium; urine analyses; and urine cultures. st o ne s i ze a nd l o c a t i o n we r e a ss e s s e d preoperatively by plain abdominal radiographs of the kidneys, ureters, and bladder (kub) and by non-enhanced computed tomography (ct). upper ureteric stones were defined as those located above the super ior border of the sacroiliac joint. mid-ureteric stones were defined as those located between the superior and inferior borders of the sacroiliac joint, and distal ureteric stones as those located below the inferior border of the sacroiliac joint. statistical analysis all statistical values were presented as frequency and percentage. comparison of variables between groups with and without stent placement was analyzed using the chi-square test. a p value of <0.05 was c onsi dered statistically significant. all statistical analyses in this study were performed using spss ver. 21 software. ethical clearance this study was approved by the health research ethical committee, medical faculty of airlangga university (1895/kepk/iii/2020). all subjects participating in this study were informed about the aims and benefits of the study and signed informed consent during admission. results there were 130 patients with ureteric stones who underwent urs lithotripsy. table 1 displays the baseline characteristics of subjects, as well as their pre-operati ve status and stone characteristics. many patients who used stents after ureteroscopy (urs) were above 40 years old (85.9%) with a mean age of 51.12 years. in the patient group with a body mass index (bmi) <25 kg/m2 there were 29 patients (29.3%) using stents and 16 patients (51.6%) not using stents. there was a significant difference in patient’s bmi among the stent users (p=0.039). a total of 47 patients (47.5%) with stones in the proximal ureter, 14 patients (14.1%) with stones in the middle ureter and 38 patients (38.4%) with stones in the distal ureter underwent stent insertion posturs. stone location did not have a significant correlation with stent placement post urs (p=0.760). there were 68 patients (68.7%) with one stone and 31 patients (15.2%) with more than one stone that had stent placement; however, the analysis results showed that there was no significant relationship between the number of stones and stent placement post-urs (p=0.351). there were 61 patients (61.6%) with ureteral stones less than 1 cm in diameter, 23 patients (23.2%) with ureteral stones of 1-2 cm in diameter, and 15 patients (12.1%) with stones of more than 2 cm. the analysis results showed that the size of the stone had a significant effect on ste nt placement af ter urs ( p=0.021). preoperative hydronephrosis was present in 53 patients, with 45 patients undergoing stent insertion. table 2 displays the details regarding the intraoperative characteristics of the subjects, in which there were 13 out of 130 patients (13.1%) who experienced intraoperative complications and widyokirono, kloping, rahman, et al ureteral stenting following ureteroscopic lithotripsy 229 univ med vol. 40 no 3 a b underwent ste nt placement. there was a significant difference in complications in the stent group and the non-stent group during surgery (p=0.037). patients that underwent stent insertion had longer duration of surgery and there was a statistically significant difference when compared with the group without stents (p=0.001). there were four patients with remaining stones and eleven patients with retropulsion of stones during the urs procedure and all of these patients had stent insertion. the stone-free rate in the stent and non-stent groups was 84.8% and 100%, respec tive ly, and there was a significant difference between the two groups (p=0.022) (table 2). table 3 shows the postoperative characteristics of the subjects. most of the indications for stent placement in this study were the presence of lesions in the ureter, which occurred in 28 patients (28.3%), followed by impacted stones in 20 patients (20.2%). there were also 16 patients (16.2%) with residual stones, 14 patients (14.1%) with a duration of surgery of more than 60 minutes, and 14 patients (14.1%) in whom stent placement was the surgeon’s preference. characteristics stent (n,%) non stent (n,%) p value age (years) < 40 ≥ 40 14 (14.1) 85 (85.9) 7 (22.6) 24 (77.4) 0.494 gender male female 70 (70.7) 29 (29.3) 23 (74.2) 8 (25.8) 0.883 bmi (kg/m2) * < 25 ≥ 25 29 (29.3) 70 (70.7) 16 (51.6) 15 (48.4) 0.039 * stone size < 1 cm 1-2 cm > 2 cm 61 (61.6) 23 (23.2) 15 (12.1) 18 (58.1) 13 (41.9) 0 (0.0) 0.021* stone quantity 1 ≥ 68 (68.7) 31 (15.2) 18 (58.1) 13 (41.9) 0.351 pre-operative hydronephrosis no yes 54 (54.5) 45 (45.5) 23 (74.2) 8 (25.8) 0.052 table 1. patients demographic, stone characteristics and pre-operative status *bmi : body mass index characteristics stent (n,%) non-stent (n,%) p value intraoperative complications none 86 (86.9) 31 (100.0) 0.037* present 13 (13.1)) 0 (0) duration of surgery (min)‡ > 60 82 (82.8) 7 (22.6) 0.001* < 60 17 (17.2) 24 (77.4) stone free rate free of stones 84 (84.8) 31 (100.0) 0.022* residual stone 4 (4.0) 0 (0.0) retropulsion 11 (11.1) 0 (0.0) table 2. intraoperative characteristics during ureteroscopy procedure ‡min : minutes 230 on post-operative evaluation, the most frequent symptom that occurred in patients undergoing stent insertion was dysuria with a total of 18 patients and 4 patients without stents had the major complaint of low back pain. there was a significant difference in postoperative complications between the stent and non-stent groups (p=0.016). the majority of patients did not require further follow-up after urs, but four patients were planned f or pe rcutaneous nephrostolithotomy (pnl) and three patients needed eswl afterwards. all patients requiring eswl and pnl after urs had stent placement. in this study all patients used a 6 french stent. a total of 91 patients (91.9%) had a stent indwelling time of less than 90 days, whereas in 8 patients (8.1%) the stent indwelling time was more than 90 days, as shown in table 3. after stent placement, 41 patients (91.1%) had their hydronephrosis resolved significantly compared with 5 (62.5%) patients with non-stent placement (p=0.027). however, the analysis showed that stent placement had a significant effect on the incidence of postoperative hydronephrosis in patients who had preoperative hydronephrosis. discussion in this study, there were 130 patients with ureteric stones who underwent urs procedure at dr. soetomo hospital, surabaya for the period 2018 to 2019. the patients comprised 99 (76.2%) with stents and 31 (23.8%) without stents. most patients who used stent post-ureterorenoscopy (urs) were over 40 years old (85.9%) with a mean age of 51.12 years. t here were no significant differences in age group as well as gender regarding the use of post-urs stents. this result is similar to that of a study conducted by muslumanoglu et al.(12) who found that about 60% characteristic stent (n,%) non-stent (n,%) p value post-operative complications none hematuria dysuria urinary retention low back pain frequency indications ureteral lesions impacted stones residual stones duration >60 mins surgeon preferences ureteral edema ureteral stricture postoperative follow up none pnl * eswl ** stent size 6 french stent indwelling time (days) <90 >90 hydronephrosis resolution after stenting persisted resolved 61 (61.6) 5 (5.1) 18 (18.2) 1 (1.0) 6 (6.1) 8 (8.1) 28 (28.3) 20 (20.2) 16 (16.2) 14 (14.1) 14 (14.1) 4 (4) 3 (3) 92 (92.9) 4 (4.0) 3 (3.0) 99 (100.0) 91 (91.9) 8 (8.1) n=45 4 (8.9) 41 (91.1) 25 (80.6) 1 (3.2) 0 (0.0) 0 (0.0) 4 (12.9) 1 (3.2) 31 (100.0) 0 (0.0) 0 (0.0) n=8 3 (37.5) 5 (62.5) .016* 0.130 0.027* table 3. characteristics of stent placement postureteroscopy * pnl: percutaneous nephrostolithotomy; eswl: extracorporeal shock wave lithotripsy widyokirono, kloping, rahman, et al ureteral stenting following ureteroscopic lithotripsy 231 univ med vol. 40 no 3 of patients with ureteral stones had a stent attached post urs. however, there are many factors influencing the success of urs and whether or not stent placement is necessary after urs.(5,12) various parameters have been used, such as gender, age, bmi, and medical history of congenital disorders, solitary kidney, previous stone surgery histo ry, and the use of anticoagulants. in addition, stone location, number of stones, and pr esence/absence of hydronephrosis before surgery have also been used as basic patient characteristics. this data is important for predicting postoperative stent placement after urs. one study stated that patients with a solitary kidney or on anticoagulant use and with preoperative double-j stent were less likely to have a postoperative double-j stent place d. (1 2,13 ) t he operative duration wa s calculated from ureteral opening until the stone was removed or crushed. the duration of urs surgery increased when stent insertion was performed; in this case there was a statistically significant difference in the duration as compared with no stent insertion. this result is similar to that of research conducted by netto et al.,(14) but contrary to a study conducted by jeong et al.(15) which stated that there was no significant difference in the duration of urs with and without stents. another study also stated that if the duration of urs with lithotripsy is more than 45 minutes, it can potentially cause postoperative complications in the stent-free group.(16) however, stent placement after urs may also cause complications. in our study, the most frequent indication for stent placement was ureteral lesions in 28 patients (28.3%) and the most frequent complications after stent placement were dysuria in 18 patients and frequency in 8 patients. symptoms that arise after stent insertion include hematuria, dysuria, urinary retention, low back pain and frequency.(17,18) a different outcome was found in patients who did not use stents, where the main complaint was low back pain in four patients. a similar result was found in the study by abdelaziz et al.,(17) who reported that all patients who had stent insertion experienced dysuria, while a few of them had hematuria and recurrent fever. however, there was no significant difference in the complication rate between the stent and non-stent groups after urs. several randomized, prospective trials also reported that ther e was no signif icant dif ference in complications and postoperative pain between the stent and non-stent groups.(17–19) however, there is a significant difference in the cost of caring for patients with a stent attached which is more expensive than caring for patients without a stent.(14,15,18) in the present study, among the patients who used stents, there were 8 patients (8.1%) whose stent indwelling time was more than 90 days. this may increase morbidity because the longer stent indwelling time is associated with increased stent encrustation frequency, urinary infection, secondary stone formation, obstruction, and hematuria. the study conducted by abdelaziz et al.(20) has also found that post-operative imaging showed that forgotten stents can cause an upper tract obstruction, and fragmentation and encrustation of the stent. this condition occurred as a result of various risk factors such as decreased acidification of urine which may lead to risk for infection and nonacidified urine, which was associated significantly with stent fragmentation. therefore urine acidification is important to prevent stent fragmentation. this condition may require further investigation regarding different types of stent biofilms. there was also a significant difference in the incidence of resolved hydronephrosis. in the group of patients who had a stent inserted, among the 45 out of 130 patients who had preoperative hydronephrosis, 41 (91.1%) patients had their hydronephrosis resolved compared to the 5 (62.5%) patients without stent placement, the difference being statistically significant. the possible benefits of stent placement may be seen from several studies stating that stents reduce the risk of urethral stricture, edema, or mucosal inflammation at the site of the stone.(21-24) the limitation of our study lies mainly in the use of secondary data, in which there is limited information including stone density, the use of 232 urs lasers or pneumatics and the biomaterial of the stent. these data can help further analyze the predicting factors for stent placement and the symptoms that occur after stent insertion. the study implies that ureteral stents are necessary in preventing and resolving hydronephrosis. future studies are needed, using an experimental design which may contribute to evaluate the stent placement indications and to reduce complications due to stent insertion. however, as to the conduct of this study, these findings are beneficial toward adding data regarding ureteral stent use in the indonesian population. conclusions in this study, ureteral stenting significantly resolved pre-operative hydronephrosis after urs lithotripsy in patients with ureteral stone. the stenting procedure is necessary and safe, especially in patients with pre-operative hydronephrosis, as it shows significant resolution and has only treatable mild associated symptoms. conflict of interest compe ting inte re sts: no re levant disclosures. acknowledgement we would like to thank the subjects who participated in this study and our colleagues in the soetomo general hospital. special thanks are also due to my supervisor for the advice to improve the manuscript. contributors this study was conceived and designed by drw, ypk, zar, and lh. drw and ypk implemented the study and analyzed the results. lh drafted the manuscript. drw and lh 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2012;61:764-72. doi: 10.1016/j.eururo.2012.01.009. c:\users\user\documents\hotma.p 243 abstract universa medicina viral load as a risk factor of reverse transcriptase inhibitor drug resistance mutation in antiretroviraltreated people living with hiv/aids hotma martogi lorensi hutapea1, tri nury kridaningsih1, khoirul huda prasetyo1, and milton boaheng antwi2 background the human immunodeficiency virus type 1 (hiv-1) is a major contagion faced by the population of indonesia. the success of antiretroviral treatment (art) is threatened by the emergence of drug resistance mutations (drm). the aim of this study was to determine the association between cd4 count, cd4 count changes, viral load, adherence to therapy, and therapy history in the presence of drm in people living with hiv/aids (plwha). methods this was a cross-sectional study involving 269 adults who underwent antiretroviral (arv) therapy for at least 6 months. the frequencies of drm and polymorphisms were measured by partial amplification of the reverse transcriptase (rt) gene using rt-nested pcr on samples with viral loads of >1000 copies/ml. sequencing was performed using the sanger method, and edited by bioedit. the edited sequences were submitted to http:// hivdb.stanford.edu for drm determination. respondents’ medical data, cd4 count, viral load, and drm were analyzed by simple and multiple logistic regression. results the multiple logistic regression analysis showed a significant association of cd4 count (aor=12.47; 95% ci: 1.45 -107.39; p=0.023) and viral load at the time of study (aor=29.56; 95% ci: 3.47-251.52; p=0.002) with the presence of drm in respondents. arv substitution history was not associated with the presence of drm. there were 17 respondents (6.3%) carrying hiv-1 drm, with m184v/i (11 sequences) as the most frequent pattern of nrti resistance, and k103 (9 sequences) as that of nnrti resistance. conclusion this study demonstrated that viral load at the time of the study was the most influential determinant factor for the presence of drm in plwha. keywords: antiretroviral, drug resistance mutation, papua barat province original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.243-253 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1193 september-december 2021 vol.40no.3 cite this ar ticle a s: hu ta p ea hm l, kridaningsih tn, prasetyo kh, antwi m b. viral load as a risk fact or of reverse tra nscript ase inh ibitor d rug resistance mutation in antiretroviraltreated people living with hiv/aids. uni v med 20 21;4 0:2 4 3-5 1 . doi: 1 0 . 1 8 0 5 1 / u n i v m ed . 2 0 2 1 . v 4 0 . 2 4 3 253. 1center of health research and development papua, indonesia 2vib center for medical biotechnology, department of biomolecular medicine, ghent university, ghent, belgium correspondence: hotma martogi lorensi hutapea jl. a. yani no.48, jayapura phone (0967) 534389 fax: (0967) 534697 email: hotmahutapea@litbang.kemkes.go.id orcid id: 0000-0002-7099-3891 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license date of first submission, june 14, 2021 date of final revised submission, november 22, 2021 date of acceptance, november 30, 2021 244 hutapea, kridaningsih, prasetyo, et al rti drug resistance mutation introduction human immunodeficiency virus type 1 (hiv-1) infection remains one of many major health challenges in indonesia, including papua barat province.(1,2) human immunodeficiency virus type 1 is a retrovirus with lack of proofreading activity of reverse transcriptase (rt). as a result, hiv-1 has high genetic diversity due to its high mutation rate. the manifestation of this characteristic is the presence of mutation which is caused by spec ific selection by antiretrovirals.(3–6) another impact of this dynamic diversity is the presence of natural polymorphisms in hiv-1. these polymorphisms by themselves are not significantly affecting viral susceptibility changes to antiretrovirals (arv); however, in combination with drug resistance mutations (drm) they might increase or decrease the effect of arv on the virus.(7) antiretroviral therapy has been used to decrease the infection and increase the health quality of people l iving with hiv /aids (plwha). the major target of arv therapy is hiv-1 rt. there are two classes of arv, namely nucleotide reverse transcriptase inhibitors (nrti), and non-nucleotide reverse transcriptase inhibitors (nnrti). an effective life-long drug adherence is required to achieve successful treatment.(4,8) higher risk in developing drm has been associated with low drug adherence.(9–12) drug resistance mutation is one of the major problems in arv therapy management, especially in upscaling the arv coverage program. in 2017 in papua province, there were 5.16% drm cases reported with a dominant mutation in hiv-1 rt amino acid 184 which changed from methionine to valine or isoleucine (m184v/i) in nrti drm patients.(13) m184v/i reduces the susceptibility of hiv-1 to lamivudine (3tc) and emtricitabine (ftc), but increases its susceptibility to zidovudine (azt) and tenofovir (tdf). another major mutation called k103n is prevalent in nnrti drm patients. this mutation decreases the susceptibility of hiv-1 to nevirapine (npv) and efavirenz (efv). combinations of m184v/i and k103n in individual patients are commonly identified. several important mutations have been studied that affect viral susceptibility to arv.(14– 1 6 ) monitoring of the cd4 cell count is important to predict the severity of hiv-1 infection during arv therapy. low cd4 cell counts are frequently associated with the presence of opportunistic infections, advanced disease, and death.(17) during arv therapy, the viral load decreases, which improves the immune response, as shown by the increased cd4 cell count.(9,17) the success of arv therapy is measured by the clinical appearance of the patients, the reduction in viral load to an undetectable level, and cd4 cell counts above 200 cells/ml. papua barat province originally consisted of the 3 districts manokwari, sorong, and fakfak, and by 2010 was administratively divided into 10 districts.(18) the total population of papua barat has grown by approximately 32.94% from 2010 to 2020. the hiv cases in papua barat province in 2019 cumulatively comprised 697 cases with aids case rate of 176.32. based on the aids case rate, papua barat has the third highest rate in indonesia, while the national aids case rate is 38.93.(19) the majority of hiv-1 cases in papua barat are found in manokwari, sorong, and fakfak. particularly, plwha in manokwari, sorong, and fakfak are treated with a fixed dose combination (fdc) containing tenofovir (tdf), emtricitabine (ftc), and efavirenz (efv) in a single tablet. the ministry of health reported that crf01_ae is the most predominant hiv-1 variant in most regions in indonesia and that subtype b is common in papua and papua barat.(20) a study showed that detection of resistance and of certain drm during virological failure of art has a small but significant favorable effect on cd4 decline. (17) by contrast, an european study involving 6180 individuals with a resistance test prior to starting antiretroviral treatment accessing care in hiv clinics showed that there was no definitive evidence that drm influenced cd4+ cell decline.(21) viral loads and cd4 counts 245 were found to be associated with hiv-1 drug resistance.(22) it is important to understand the interplay of factors associated with hiv drug resistance, especially in low to middle-income countries (lmics) where there is limited access to viral load testing.(23) therefore, to understand the relationship of cd4 cell count and viral load with the presence of drm, the aim of this study was to determine the association of clinical and arv therapy related variables (cd4 cell count collected at the time of the study, changes in cd4 cell count, viral load, drug adherence, and regimen substitution history) with the presence of drm in arv-treated plwha in papua barat province. we also identified dominant drms and important polymorphisms in these drm sequences to determine the potential resistance to the fdc according to the hiv-1 arv resistance database. methods research design this was a c ross-sec tional study on plwha in the care, service, and therapy (cst) clinics of government hospitals in fakfak, manokwari, and sorong city, papua barat province. this study was conducted over a 10month period from february to december 2019 in fakfak, manokwari, and sorong, papua barat province. study subjects the population in this study consisted of plwha registered in the cst clinics, who were older than 18 years, and had received therapy for more than 6 months. an important inclusion criterion was that the respondents should be healthy at the time of data collection. sample size determination this study noted from previous research that hiv-1 subtyping is successfully identified in 98% of patients by sequencing.(24) the sample size determination for each study area was performed with the lemeshow formula: n = sample size z2α = 95% level of confidence: 1.96 p = population proportion estimation (0.98); q=0.02 d = permissible error (0.01) based on the calculations, at least 84 subjects were required in each studied area. the total number of subjects obtained was 269 plwha selected by consecutive sampling. laboratory analysis data and specime n collection was conducted in 2 rounds (of 14 days each) in the clinic. therapy initiation cd4 cell count was copied from the patient’s medical record by authorized health workers in the clinic. blood specimen collection was performed by competent and qualified health workers assigned by the head of the cst clinic. the blood specimens were divided into 3 aliquots to obtain whole blood, serum, and plasma. the whole blood was used to measure cd4 cell count, performed on the same day using pimatm-alere (germany). serum and plasma were stored in a temperaturecontrolled freezer before shipment to jayapura for viral load measur ement and mutation identification. sample shipment was conducted according to the 3-layer packaging and cold chain standard. the serum was used to measure viral load b y us i n g a vi r a l l o a d a n a l yze r wi t h t he accupower® hiv-1 quantitative rt-pcr kit ( bio ne er, repu bl ic of k or ea ) . mu t a ti o n identification was performed on selected samples with a viral load of >1000 copies/ml. the reverse transcription-polymerase chain reaction (rt-pcr) was nested by using who hiv-dr primers (25) that partially amplified the rt genes using the high fidelity enzyme mix superscript iii one-step rt-pcr system with platinum univ med vol. 40 no 3 246 hutapea, kridaningsih, prasetyo, et al rti drug resistance mutation a b taq® (invitrogen). pcr for sequencing was performed using the inner primer pair, and reading of the product was performed using the sanger method. each sample produced 2 sequences which were complementary to each other. bioedit sequence alignment editor was used for trimming, editing, and creating the consensus sequences. the consensus sequence for each sample was submitted to the website http:// hiv.db.stanford.edu for drm analysis. the output from the website was used as the reference of mutation identified in the patients and its clinical implication regarding arv therapy. statistical analysis patients’ data, cd4 cell count, viral load measurement and mutation status were added to a spreadsheet and descriptively analyzed using spss version 21. respondents’ medical data, cd4 counts, viral loads, and drm were analyzed using simple and multiple logistic regression. a statistical result was considered significant when p < 0.05. ethical clearance this study was ethically approved by the health research ethics committee, national institute of health research and development (hrec – nihrd) under number lb.02.01/2/ ke.008/2019. results demographic characteristics of respondents the majority of the respondents was female (54.3%), papuan (53.0%), and older than 35 years (80.5%). most of the respondents were married (78.6%), graduated from higher education (74.0%), and employed (66.9%) (table 1). the cd4 cell count measured in this study showed that most of the respondents (81.4 %) had cd4 t-cell counts of more than 200 cells/ml and viral loads below 1000 copies/ml was 210 (78.1%) ((table 1). variables n (%) sex female male ethnicity papuan non-papuan age (years) ≤ 35 > 35 marital status married single formal education elementary to junior high senior high to higher education occupation employed unemployed cd4 count (cells/ml) ≤ 200 > 200 cd4 count before therapy (cells/ml) ≤ 200 > 200 not available cd4 count changes after therapy (cells/ml) decreased to ≤ 200 or maintained ≤200 increased to > 200 or maintained > 200 viral load (copies/ml) ≤ 1,000 > 1,000 mutation associated to drug resistance yes no art-naïve art failure therapy adherence moderate – poor good antiretroviral therapy fdc non-fdc na length of therapy ≤ 12 months > 12 months na regimen substitution yes no 146 (54.3) 123 (45.7) 123 (45.7) 146 (54.3) 143 (53.2) 126 (46.8) 209 (77.7) 60 (22.3) 69 (26.0) 196 (74.0) 88 (33.1) 178 (66.9) 50 (18.6) 219 (81.4) 74 (27.5) 80 (29.7) 115 (42.8) 22 (14.2) 132 (85.8) 210 (78.1) 59 (21.9) 17 (6.3) 252 (93.7) 0 (0.0) 43 (16.0) 39 (14.5) 230 (85.5) 221 (82.2) 46 (17.1) 2 (0.7) 197 (73.2) 45 (16.7) 27 (10.1) 32 (11.9) 237 (88.1) table 1. characterization of the study population (n=269) na = not available; fdc : fixed dose combination 247 according to the arv therapy adherence history, the majority (85.5%) of the respondents in this study was categorized as having good adherence. most of the respondents (82.2%) was receiving fdc for treatment and 73.2% had undergone therapy for more than 12 months. the majority of respondents (88.1%) never had regimen substitution for their therapy (table 1). based on their drm status, the majority of respondents of nnrti classes was papuan (4.1%), female (3.7%), and 4.1% had a cd4 cell count below 200 cells/ml (table 2). associat ion of cli nical and therapy characteristics of respondents with drm presence in rt nucleotide sequence simple logistic regression analysis on variables regarding respondents’ clinical data showed that there was a significant association between cd4 cell count and drm (p< 0.001). respondents with cd4 cell counts of less than 200 cells/ml had a higher probability of carrying hiv-1 with drm compared to respondents with cd4 cell counts of more than 200 cells/ml (or=.38.45;95% ci:5.19-285.24; p=<0.001). in this study, a significant association was also f ound between viral load and drm (p<0.001). this study showed that respondents with viral loads of more than 1000 copies/ml had higher chances o f de veloping drm (or=56.95;95% ci: 7.71-420.61); p<0.001) than did respondents with viral loads below 1000 copies/ml. however, there was no significant association between therapy adherence and drm, and between regimen substitution and drm. the multiple logistic regression analysis showed the viral load to be the most influential determinant of drm (aor=29.56;95% ci: 3.47251.52; p=0.002) (table 3). identification of subtype, drm pattern, and polymorphisms among 59 respondents with a viral load of more than 1000 copies/ml, 30 rt partial gene sequences (50.8%) were successfully amplified. however, only 21 sequences (70.0%) were of sufficiently good quality to be analyzed further. subtype b was dominant in this study (10 out of 21 sequences, 47.6%), followed by circulating recombinant form (crf) 01_ae (7 out of 21 variables drug resistant drug sensitive to both nnrti and nrti nnrti nrti nnrti or nrti n % n % n % n % papuan 11 4.1 6 2.2 6 2.2 117 43.5 non-papuan 5 1.9 5 1.9 4 1.5 135 50.2 gender male 6 2.2 4 1.5 3 1.1 117 43.5 female 10 3.7 7 2.6 7 2.6 135 50.2 adherence yes 13 4.8 9 3.3 9 3.3 216 80.3 no 3 1.1 2 0.7 1 0.4 36 13.4 cd4 <200 cells/ml 11 4.1 8 3.0 8 3.0 63 23.4 cd4 <200 cells/ml before treatment and maintained at <200 cells/ml after treatment 5 3.2 4 2.6 4 2.6 14 9.1 viral load >1000 copies/ml 14 5.2 9 3.3 8 3.0 43 16.0 table 2. profile of patients with drm univ med vol. 40 no 3 note : drm = drug resistance mutations; nnrti : non-nucleotide reverse transcriptase inhibitors; nrti : nucleotide reverse transcriptase inhibitors 248 hutapea, kridaningsih, prasetyo, et al rti drug resistance mutation sequences, 30%) (table 4). the drm patterns were identified in 17 sequences. m184v/i was the most frequent pattern identified for nrti resistance (11 respondents, 64.7%). mutation k103n was the most frequent pattern (9 respondents, 52.9%) identified for nnrti resistance (figure 1). of the important natural polymorphisms, k122e was identified in almost all sequences (14 respondents, 70.0%), and g335d in one sequence (table 4). discussion based on viral load and cd4 cell count data, the majority of respondents in this study were successfully treated by arv therapy. the significant association between cd4 cell count and the presence of drm was in accordance with previous studies.(9,17,26) our study showed that respondents with cd4 cell counts below 200 cells/ml had a 38.45 times higher probability of carrying hiv-1 with drm than did those with cd4 cell counts above 200 cells/ml. other studies have shown that cd4 cell count may be a strong predictor of drm present in plwha undergoing arv therapy.(13) monitoring the cd4 cell count regularly may help in predicting the presence of drm in plwha in papua barat, s i nc e d e te c t i ng d rm i s i nt ri c a t e a n d challenging. another benefit of measuring cd4 cell counts regularly is to detect reductions in cd4 cell count and to anticipate the progression of infection into acquired immunodeficiency syndrome (aids) diseases.(27) factors drm rate (n,%) crude or (95% ci) p value adjusted or (95% ci) p value cd4 count (cells/µl) < 200 ≥ 200 viral load (copies/ml) ≤ 1,000 > 1,000 therapy adherence moderate – poor good regimen substitution yes no 16 (5.94) 1 (0,003) 1 (0.003) 16 (5.94) 3 (1.1) 14 (5.2) 1 (0.003) 16 (5.94) 38.48 (5.19 – 285.24) 56.95 (7.71– 420.61) 1.26 (0.38 – 4.19) 0.46 (0.06 – 3.37) <0.001 < 0.001 0.70 0.45 12.47 (1.45 -107.39) 29.56 (3.47 – 251.52) 0.56 (0.13 – 2.48) 0.28 (0.03 – 2.56) 0.023 0.002 0.52 0.26 table 3. factors associated with rtidrm among participants (using simple and multiple logistic regression) figure 1. the distribution of drm pattern in papua barat note : drm : drug resistance mutations; or = odds ratio 249 sample code subtype nrti mutation nrti resistance nnrti mutation nnrti resistance polymorphism 910101 b e138ea etr, rpv k122e, i135v, i142im, s162c, i195t, t200a, l228m, v245e, i293v, p313t 910114 crf01_ae m1841, t215tfis, k219q abc, azt, ftc, 3tc v1081, y181c dor, efv etr, nvp, rpv k122e, i135v, s162c, i195t, l228m, v245e, e297ea, p313t 910143 a k122e, d123g, i135t, r172k, k173kr, q174k, d177e, i178m, l193f, e194p, q207d, v245e, s251n, a272p, t286a, e291k 910166 b e138ek dor, efv etr, nvp, rpv d121y, k122e, i135v, i142imt, s162c, g196e, q207qhr, r211g, l228m, i293v 910167 b k122e, i135v, s162c, i195t, l228m, v245e, e297ea, p313t 910179 a l1001, k103n dor, efv etr, nvp, rpv k122e, d123s, r172k, k173i, q174k, d177deg, e194p, t200i, q207a, r211n, v245e, a272g, k281r, t286a, e291d, v292i, i293v, l295x, t296k, e297k, e305v, n306s, i309t, l310f, e312s 910508 b t107s, k122e, s162c, t200i, r211k, v292i, i293v, l301i, y319f 910516 b m184v abc, azt, ftc, 3tc k103r, i135v, s162c, i178m, q207e, r211k, l228m, i293v 910520 b m184v abc, azt, ftc, 3tc k103n, p225h dor, efv etr, nvp k102q, d123e, i135l, s162c, t200i, r211k, l228q, a272p, v276i, l283i, v292i, i293v 910528 b l100f, k103n dor, efv etr, nvp k122e, d123n, t128ts, s162c, i178l, t200i, h208r, r211k, l228f, v245e, e248d, a272p, v292i, i293v, v317a 910530 b k103n, v108vi dor, efv etr, nvp k102q, d123e 910532 crf01_ae m184v abc, azt, ftc, 3tc p225h, k103n dor, efv etr, nvp k122e, d123n, k173v, q174k, v179i, i202v, q207a, r211s, k238r, v245e, a272p, e291d, v292i, i293v, e312n, i329v. 910701 crf01_ae m1841 abc, azt, ftc, 3tc y318f, l1001, k103n dor, efv etr, nvp k122e, d123s, t139k, k173i, q174k, d177e, t200e, q207a, r211k, k238r, v245e, t286a, i293v, e312a, i326v, i329v, k331n 910764 a k173i, q174k, q197qe, q207a, r211s, v245e, t286a, i293v, e312t, s322a, l325v, i329y, q330r, g335d. 910749 b k103n dor, efv etr, nvp g112a, k122e, d123s 910765 a m184v, t215f, m41l, d67n, abc, azt, ftc, 3tc v179d, m230l, v106l dor, efv etr, nvp, rvp v106l, k122e, d123n, i135k, k173t, q174k, g196e, i202v, q207a, r211k, l228h, v245e, l283i, e291d, v292i, i293v, k311r, e312a, i329t 910781 crf01_ae m184i abc, azt, ftc, 3tc k103n, y318f dor, efv etr, nvp k122e, d123s, t139k, k173i, q174k, d177e, t200e, q207a, r211k, k238r, v245e, t286a, i293v, e312a, i326v 9105105 crf01_ae m184v abc, azt, ftc, 3tc y181c, g190a dor, efv etr, nvp, rvp i142m, s163c, k173i, q174k, q207a, r211s, v245e, a272p, v276i, t286a, e291d, i293v, e312n, i326v, i329r 910149 crf01_ae m184v, k219kr abc, azt, ftc, 3tc p225h dor, efv etr, nvp d123e, i135m, s162c, t200ta, q207e, l228km, i257l, a272p, i293v, k311n, e312q, h315q, v317a 910150 crf01_ae m184 i abc, azt, ftc, 3tc y188l dor, efv etr, nvp, rvp k122e, d123n, k173i, q174k, d177e, v189i, e194p, t200i, e203d, q207a, r2 11s, k238r, v245e, a272g, k281r, t28 6a, e291d, v292i, i293v, e297ek, e312 n, i326v, i329v 910714 b m184v abc, azt, ftc, 3tc k103n, p225h dor, efv etr, nvp i142m, s163c, k173i, q174k, q207a, r211s, v245e, a272p, v276i, t286a, e291d, i293v, e312n, i326v, i329r table 4. subtype, polymorphisms, and drm identified from patients and clinical implication univ med vol. 40 no 3 250 hutapea, kridaningsih, prasetyo, et al rti drug resistance mutation the cd4 cell count of patients with good virological control will increase by approximately 50-150 cells/mm3 per year.(26) a study conducted by schultze et al.,(17) showed that drm at a particular site in rt might contribute to the stable cd4 count during therapy. in our study, there was a significant association between the difficulty of increasing the cd4 cell count to more than 200 cells/ml and the presence of drm. the group of respondents with difficulties of increasing their cd4 cell count had an approximately 48 times higher probability of carrying hiv-1 with drm compared to the group who had their cd4 cell count increased to or maintained above 200 cells/ml. the mutations m184v, d67n and t215y were associated with reduced cd4 count, whereas mutations v179d and l74v were associated with a steeper decline in cd4 count. in our study, m184v was the most frequent mutation in the nrti class of arv, and one respondent carried hiv-1 with mutation v179d (table 4). another strong predictor of hiv-1 infection progression is viral load. the gold standard is to measure the number of rna copies in the patient’s sample.(28,29) a patient with a viral load above 1000 copies/ml in two consecutive tests within 3 months after at least 6 months of therapy and with a cd4 cell count below 200 cells/ml is considered to be experiencing treatment failure.(30) in this study, the viral load was measured only once during the study, due to the lack of resources and certain equipment in papua barat province. however, there was a significant association between viral load and the presence of drm. respondents with viral loads above 1,000 copies/ml had a 56 times higher probability of carrying hiv-1 with drm than those with less than 1,000 copies/ml, regardless of the number or pattern of the mutations. the immediate objective of arv therapy is to inhibit viral replication and reduce the number of virus particles to undetectable levels. in this condition, arv therapy is adequate and drm will not develop. this can only be achieved by the patients’ excellent adherence to arv therapy.(7) patients’ adherence to therapy is important to optimize the benefits of arv therapy. studies h a ve r e vea le d t ha t go od a dhe r e nc e w a s associated with an increase in cd4 t cells and a reduction in viral load.(12,31) in our study, adherence was measured by the numbers of pills left after each month of therapy as counted by the health workers in the clinic. in our study, the moderate to poor adherence to therapy was not significantly associated with the presence of drm. this result was inconsistent with a previous study which showed that adherence to arv therapy was one of the important factors that affected the outcome of the therapy. the non-adherence behavior in taking therapy might cause viral rebound and was significantly associated with the presence of drm. nonadherence to taking arv might also be a predictor of the severity of aids diseases.(32) this inconsistent re sult might be due to difficulties in tracking the accurate numbers of pills taken by the patients daily that were faced by the physicians or assigned health workers. the percentage of rti-associated drm was 6.3% and the most frequent pattern was m184v / i f or nrt i-as soci ate d drm a nd k103n for nnrti. this was higher compared to the rti-associated drm percentage in papua province in 2017 (5.16%).(13) several studies reported that mutations at m184 were the most frequently observed.(29–31) mutation m184v/i confers high level resistance to lamivudine (3tc) and emtricitabine (ftc). based on the guideline for arv therapy for adults and adolescents, there are several regimen substitutions to be considered when m184v/i is detected in patients based on their clinical scenario.(33) another major drm identified in the present study was k103n which confers high level resistance to nnrti classes of arv and was the most frequent pattern identified in this study. this result is similar to the finding in mexico.(34) 251 in our study, we a lso found natural polymorphisms associated with changes in the characteristic of the virus, for example k122e, i135v/i, and g335d.(14,34) k122e was identified in almost all patients with or without any nrti or nnrti mutations. this was in line with another study reporting that k122e was common in hiv1 infected patients with nrti mutations. the accumulation of k122e with other polymorphisms or mutations might increase the numbers of nrti and nnrti mutations and contribute to higher levels of nrti resistance.(4) another polymorphism identified in our study was g335d. a study conducted by tanuma et al.(35) showed that the pre sence of g335d polymorphism in hiv-1 crf01_ae made the virus less susceptible to azt in the background of m41l, l210w and t215y (8.2 – to 23.2-fold) except for 3tc. the g335d polymorphisms increased viral susceptibility to azt, abc, and tdf (52.7, 4.2, and 2.4-fold respectively) in the background of d67n, k70r and t215f drm. our study highlighted that g335d was identified in patients without nrti. how this polymorphism works without any mutation as the background is not clear. one limitation of this study was the inability to identify polymorphism g335 in all of the sequences due to the differences in length as a result of sequence 3’-end trimming. due to the limitation of continuity of cd4 cartridge supply, not all respondents were provided with a complete data set of cd4 cell counts. however, the drm and polymorphism data obtained in this study is important in monitoring the usage of arv in papua barat. this study might be useful in making recommendations that could contribute to reviewing treatment monitoring policy in papua barat. conclusions this study highlighted the necessity of monitoring patients’ cd4 cell count and viral load to reduce the possibility of drm development. the presence of polymorphisms should be brought to attention with regard to the possibility that their combination with mutations might affect virus susceptibility to arv. conflict of interest compe ting inte re sts: no re levant disclosures. acknowledgement we gratefully acknowledge the cooperation of the health workers and patients involved in this study. we also thank th e labora tory technicians who helped with the collection of laboratory data for this study. contributors hmlh contributed to conception of the study, data acquisition, analysis and interpretation, drafting the manuscript, and revising the manuscript. tnk contributed to conception of the study, data acqui sition, ana lysis and interpretation, and revising the manuscript. khp contributed to data acquisition, analysis and interpretation. mba contributed to revising the manuscript critically for important intellectual content. all authors have read and approved the final manuscript. references 1. menteri kesehatan ri. rencana aksi nasional pengendalian hiv-aids tahun 2015-2019. jakarta: kementerian kesehatan ri; 2015. 2. pemerintah provinsi papua barat dinas kesehatan. profil kesehatan papua barat; 2018. 3. abecasis ab, wensing amj, paraskevis d, et al. hiv-1 subtype distribution and its demographic determinants in newly diagnosed patients in europe suggest highly compartmentalized 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antiretroviral therapy. in: consolidated guidelines on the use of antiretroviral drugs for treating and preventing hiv infection. geneva: world health organization ;2016. p. 72–190. 31. memiah p, shumba c, etienne-mesubi m, et al. the effect of depressive symptoms and cd4 count on adherence to highly active antiretroviral therapy in sub-saharan africa. jiapac 2014;13:346–52. doi: 10.1177/2325957413503368. 32. chen y, chen k, kalichman sc. barriers to hiv medication adherence as a function of regimen simplification. ann behav med 2017;176:139–48. doi: 10.1007/s12160\-016-9827-3. 33. dhhs panel on antiretroviral guidelines for adults and adolescents – a working group of the office of aids research advisory council (oarac). guidelines for the use of antiretroviral agents in adults and aadolescents. department of health and human services; 2018. 34. arguello jr, noyola de, centro c. prevalence of drug resistance mutations in protease, reverse transcriptase and integrase genes of northcentral mexico hiv isolates. aids res hum retroviruses 2017;52:1–29. doi: 10.1089/aid. 2017.0257. 35. tanuma j, hachiya a, ishigaki k, et al. impact of crf01_ae-specific polymorphic mutations g335d and a371v in the connection subdomain of human immunodeficiency virus type 1 (hiv-1) reverse transcriptase (rt) on susceptibility to nucleoside rt inhibitors. microbes infect 2010;12:1170–7. doi: 10.1016/j.micinf.2010.08.003. univ med vol. 40 no 3 c:\users\universa medicina\docu 22 abstract universa medicina body mass index as the most influential factor of highsensitivity c-reactive protein in non-diabetic adults winda mulyamin*, liong boy kurniawan**@, endy adnan***, yuyun widaningsih**, irfan idris†, arif santoso‡, and ika yustisia†† background high-sensitivity c-reactive protein (hscrp) has been widely accepted as a predictor of future cardiovascular risk that reflects a microinflammatory state. obesity linked to microinflammation increases the prevalence of metabolic disorders and cardiovascular diseases. this study aimed to determine the association between several obesity indices namely body mass index (bmi), waist circumference (wc), body fat percentage (fat), and visceral fat (vf) with hscrp in non-diabetic adults. methods this was a cross-sectional study performed on 80 non-diabetic adults with ages ranging from 20-40 years. the obesity indices bmi, wc, body fat percentage, and vf were measured. we then measured the hscrp levels using an immunoturbidimetric method. simple and multiple linear regression tests were used to analyze the association between obesity indices and hscrp levels. results mean of log bmi, log wc, and log vf was 1.41 ± 0.08 kg/m2, 1.93 ± 0.06 cm, and 0.95 ± 0.27 units, respectively. simple linear regression tests showed that log bmi (β=3.506; p<0.001), log wc (β=3.672; p<0.001), log vf (β=0.833; p<0.001), and log systolic blood pressure (β=3.739; p=0.024) had a significant positive correlation with log hscrp levels. further multiple linear regression test showed that log bmi (β=3.772; beta=0.674; p<0.001) had the greater effect on log hscrp levels compared to other indices. conclusions bmi had a greater influence on hscrp levels compared to other obesity indices in non-diabetic adults. body mass index can be used as a better index in predicting hscrp levels compared to other indices. keywords: body mass index, waist circumference, visceral fat, body fat, hscrp, non-diabetic adults original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.22-28 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1045 january-april, 2021 vol.40no.1 cite this article as: mulyamin w, kurniawan lb, adnan e, et al. body mass index as the most influential factor of high-sensitivity c-reactive protein in non-diabetic adults. univ med 2021;40:22-8. doi: 10.18051/univmed.2021.v40.22-28 *aging and regenerative medicine concentration, biomedical magister programme, hasanuddin university, makassar, indonesia **department of clinical pathology, facu lty of medicine, hasanu ddin university, makassar, indonesia ***department of internal medicine, facu lty of medicine, hasanu ddin university, makassar, indonesia †department of physiology, faculty of medicine, hasanuddin university, makassar, indonesia ‡department of pulmonology and respiratory medicine, faculty of medicine, hasanuddin university, makassar, indonesia ††department of biochemistry, faculty of medicine, hasanuddin university, makassar, indonesia correspondence: @liong boy kurniawan medical faculty, hasanuddin university, perintis kemerdekaan street km 10, makassar, indonesia phone: +6281241738007 email: liongboykurniawan@yahoo.com; liongboykurniawan@med.unhas.ac.id orcid id: 0000-0002-2633-0441 date of first submission, october 5, 2020 date of final revised submission, january 20, 2021 date of acceptance, january 21, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 23 univ med vol. 40 no 1 introduction c-reactive protein (crp) is commonly associated with infl ammatory reactions, particularly during infection. high-sensitivity crp (hscrp), which can be detected at levels of <1 mg/l, has been linked to several chronic lowgrade inflammator y processes leading to cardiovascular disease (cvd).(1) high-sensitivity crp has been used to predict future cvd risk worldwide in both developed and developing countries in asia, africa, europe, and north/ central/south america.(2) persons with hscrp levels of >3mg/l are considered to have high hscrp levels because they are at high risk of developing cvd in the future.(3) obesity marked by excess of body fat is associated with insulin resistance and systemic low-grade inflammation leading to cvd.(4) obesity is commonly measured by body mass index (bmi) and waist circumference (wc). visceral obesity is linked to higher cvd risk compared to other types of fat deposition.(5) high-sensitivity crp levels are reported to have a significant correlation with obesity indices. in the indian adult population, hscrp showed a significant correlation with bmi and wc.(6,7) in the swiss caucasian population, several obesity indices including bmi, body fat percentage, and wc also had a significant correlation with hscrp levels.(8) a significant correlation of hscrp with bmi, wc, hip circumference, and waist-hip ratio was also reported in the young saudi population.(9) the body adiposity index is correlated with hscrp in hypertensive and normotensive obese adult subjects.(10) on the other hand, it was reported that waist-to-hip ratio (whr) and waist-to-height ratio (whtr) had a significant association with hscrp, but that bmi and wc showed no significant association with hscrp.(11) the present study was focused on the association of several obesity indices with hscrp levels in the indonesian non-diabetic population, especially young adults (age range 20-40 years). a previous study reported that hscrp had a better association with bmi compared to wc, but its association with other newly proposed obesity indices including body fat percentage (fat), and visceral fat (vf) had not been revealed.(12) the present study aimed to determine the relationship between traditional obesity indices, such as bmi, wc, and the newly proposed obesity indices including body fat percentage, and visceral vf with hscrp levels, and to determine which index had the best value in predicting high hscrp levels as the novelty of this study. methods study design this was an analytical cross-sectional study, conducted from august to september 2020 among non-diabetic indonesian adults at hasanuddin university hospital, makassar, indonesia. study subjects the study subjects were non-diabetic adults in the age range of 20-40 years who voluntarily joined this research by signing informed consent according to the declaration of helsinki. the inclusion criteria were male and female adults aged 18 years who had no histor y of cerebrovascular disease (cvd) and diabetes and had never experienced other cardiovascular symptoms including cardiac chest pain and dyspnea. the exclusion criteria were subjects with fasting plasma glucose levels of 126 mg/ dl, smokers, and those suffering from infectious disease in the last month. the sample size was determined based on the correlation coefficient of 0.359 between bmi and hscrp levels as reported in previous research.(8) by using α=0.05, β=0.20, and drop-out rate of 20%, the minimal sample size required was 59. a total of 80 adults joined the study, consisting of 39 male and 41 female subjects. measurement of obesity indices and blood pressure obesity indices were measured right before blood sampling. body weight and height of the 24 subjects were measured and bmi was calculated using the formula: body weight (kilograms)/height2 (meters, squared). waist circumference was then measured by using tape at midway between the lower border of the 12th rib and the iliac crest. the bioelectrical impedance analysis (bia) method was used to measure body fat and vf using a tanita bc-541 body composition monitor (tokyo, japan). blood pressure was measured using a riester sphygmomanometer (germany) after the subject had rested for 15 minutes in the seated position. systolic and diastolic blood pressures were measured on the subject’s right arm. all measurements were taken twice, and the average was calculated. laboratory analysis blood sampling was performed after 8-12 hours of overnight fasting. a 3 ml venous blood sample was taken from each subject, separated to obtain the serum, and directly tested for fasting plasma glucose levels by the hexokinase method (abx pentra 400, horiba, usa) for excluding those with diabetes. the remaining serum was kept in 200 µl aliquots and kept refrigerated at 20o celcius until the time for the hscrp test (immunoturbidimetry, cobas c 311 analyzer, roche diagnostics, mannheim). statistical analysis the normality of data was tested with the kolmogorov-smirnov test. all parameters including hscrp, bw, height, bmi, wc, vf, systolic and diastolic blood pressures which were not normally distributed, were transformed into logarithmic (log) format to normalize them. simple and multiple linear regression were performed to determine which obesity index had the largest effect on log hscrp levels. statistical analyses were performed by using the ibm statistical package for the social sciences statistics program (ibm, usa), version 21.0. statistical analysis was considered significant if p-value <0.05. ethical clearance this study was approved by the komite etik penelitian kesehatan (committee of health research ethics), medical faculty, hasanuddin university, makassar, indonesia, under number 395/un4.6.4.5.31/pp36/2020, and protocol number uh20060245. mulyamin, kurniawan, adnan, et al bmi as the most influential factor of hscrp notes: normally distributed parameters are expressed as mean ± standard deviation, non-normally distributed parameters are expressed as median (minimum-maximum). bw: body weight, bmi: body mass index, wc: waist circumference, vf: visceral fat, hscrp: high sensitivity c-reactive protein table 1 distribution of age, anthropometrics, blood pressure and hscrp of subjects (n=80) variable mean ± sd/ median (min-max) age, years 29.96 ± 5.00 bw, kg 64.55 (48.70-137.60) log bw, kg 1.82 ± 0.09 height, m 1.60 (1.49-1.84) log height, m 0.21 ± 0.02 bmi, kg/m2 24.43 (19.73-47.61) log bmi, kg/m2 1.41 ± 0.08 wc, cm 83 (68-136,50) log wc, cm 1.93 ± 0.06 fat, % 31.35 ± 7.39 vf, unit 9 (3-30) log vf, unit 0.95 ± 0.27 systolic blood pressure, mmhg 120 (90-140) log systolic blood pressure, mmhg 2.06 ± 0.03 diastolic blood pressure, mmhg 80 (60-80) log diastolic blood pressure, mmhg 1.88 ± 0.04 hscrp, mg/l 1.65 (0.20-11.0) log hscrp, mg/l 0.15 ± 0.45 25 univ med vol. 40 no 1 c results the characteristics of the research subjects are shown in table 1. mean of age was 29.96 ± 5.00 years, while mean of log bmi, log wc, and log vf were 1.41 ± 0.08 kg/m2, 1.93 ± 0.06 cm, and 0.95 ± 0.27 units, respectively. mean of body fat percentage was 31.35 ± 7.39%, while the mean of log hscrp was 0.15 ± 0.45 mg/l. simple linear regression analysis results are shown in table 2. log bmi (β=3.506; p<0.001), log wc (β=3.672; p<0.001), log vf (β=0.833; p<0.001), and log systolic blood pressure (β=3.739; p=0.024) correlated with log hscrp, but body fat (β=0.013; p=0.067), age (β=0.013; p=0.215), and log diastolic blood pressure (β=1.693; p=0.218) had no significant correlation with log hscrp. all variables with p-value <0.200 were subsequently entered for analysis into a multiple linear regression model. as presented in table 3, multiple linear regression analysis showed that log bmi was the most effective marker for increased levels of log hscrp (beta=0.674, p<0.001) compared to other obesity indices. discussion in this study, we found that the obesity indices had a significant correlation with hscrp levels in non-diabetic adults, with bmi as the most effective on hscrp levels compared to the other obesity indices. several other reports have supported our findings. sadanand et al.(6) reported that bmi (r=0.51), wc (r=0.42), and waist-tohip ratio (whr) (r=0.32) had a significant correlation with hscrp levels in the indian population. the same finding was reported by lavanya et al.(7) who performed a study on the indian adult population aged 20-70 years and by farooq et al.(9) in the saudi population. marquesvidal et al.(8) reported that bmi, wc, and percent body fat had a significant correlation with hscrp levels both in the male and female caucasian swiss population. kawamoto et al.(13) reported that the overweight japanese population (bmi 25 kg/m2) aged<75 years had a higher prevalence of elevated hscrp concentration compared to those with bmi <22 kg/m2, but that the association did not occur in persons aged 75 years. a significant correlation between bmi and hscrp levels was also found in obese children and adolescents.(14) on the other hand, mahwati et al.,(11) after performing logistic regression, reported a significant association of whr and whtr with hscrp, but no association was observed of bmi and wc with hscrp. the different results compared with those of the present study might be caused by different research models. mahwati et al.(11) adjusted the effect of social variables including marital status, education, and residence in the analysis while the present study measured the effect of several variables β beta p-value log bmi 3.772 0.674 <0.001 log wc -1.382 -0.172 0.329 fat -0.010 -0.161 0.179 log vf 0.278 0.165 0.417 log systolic blood pressure 1.506 0.102 0.295 table 3. multiple linear regression of obesity indices and systolic blood pressure with log hscrp a b variables β p-value log bmi 3.506 <0.001 log wc 3.672 <0.001 fat 0.013 0.067 log vf 0.833 <0.001 age 0.013 0.215 log systolic blood pressure 3,739 0.024 log diastolic blood pressure 1.693 0.218 table 2. simple linear regression of obesity indices and other parameters with log hscrp notes: β: regression coefficient; bmi: body mass index; wc: waist circumference; vf: visceral fat notes: β: regression coefficient; beta: standardized regression coefficient; bmi: body mass index, wc: waist circumference, vf: visceral fat 26 obesity indices, age, and blood pressure on hscrp levels. in our study, age and diastolic blood pressure showed no significant association with hscrp levels, and after multiple linear regression analysis, bmi was a consistent indicator associated with hscrp. obesity is a condition marked by an excess of body-fat mass that may impair health by generating a low-grade inflammatory state.(15,16) obesity occurs when energy intake exceeds energy expenditure.(17) several indices have been used to measure obesity including bmi, wc, and body adiposity index.(18) obesity induces chronic inflammatory conditions through the role of adipose tissue. the adipocytes secrete proinflammatory adipokines such as interleukin-6 (il6) and tumor necrosis factor-α (tnf-α) while the secretion of adiponectin which has an antiinflammatory role is decreased.(19,20) this results in the development of endothelial dysfunction and a hypercoagulability state, therefore triggering the atherosclerotic process.(21) high -sensitive crp is a protein produced mainly in the liver in response to inflammation and has been identified as a cvd risk predictor marker. c-reactive protein is synthesized in response to cytokine expression including il-6 and has an important role in the atherogenic process. it acts by binding oxidized low-density lipoprotein (oxldl) which has a high atherogenic property. c-reactive protein might induce the adhesion and movement of monocytes to the arterial wall as the early step of the atherosclerotic process. monomer ic crp a lso has prothrombogenic and inflammatory properties which increase its atherogenic role.(1,22) our findings reveal that obesity indices particularly bmi, wc, and vf may be used to predict the high hscrp levels which reflect the high cvd risk in the indonesian non-diabetic adult population, while body fat percentage has no significant correlation with hscrp. this may be explained by the fact that body fat percentage not only measures white fat which has an adverse inflammatory function but also brown fat which has a beneficial catabolic function. multiple linear regression analysis reveals that bmi has the largest effect on hscrp levels compared to other obesity indices. therefore, we propose that bmi might be used as the chosen obesity index to predict cvd risk, particularly in the indonesian population. this study to our knowledge is among the few studies that compare and evaluate the ability of several obese indices in predicting hscrp levels and stratify the ability of each index as a predictor in the indonesian adult population. there are several limitations to this study. first, the cross-sectional design cannot explain a causal association between obesity indices and hscrp levels. second, we did not use magnetic resonance imaging (mri) or computed tomography (ct) as the gold standard method in measuring obesity especially the body fat percentage and vf. third, our findings can only be applied to adult subjects with ages ranging from 20-40 years. the clinical implication of this study is that bmi can be used as a simple traditional obesity index to better predict hscrp levels than do wc and other non-traditional obesity indices including body fat and vf. further research must be done using a better design like cohort study to explain the causal association between obesity indices and hscrp levels in the elderly. conclusion this study demonstrated that bmi had the most consistent effect on hscrp levels compared to other obesity indices. bmi may be used to predict hscrp levels as a cvd risk factor for early intervention. conflict of interest statement the authors state that they have no conflicts of interest associated with this manuscript. acknowledgment we would like to thank all study participants who voluntarily joined this research and to all mulyamin, kurniawan, adnan, et al bmi as the most influential factor of hscrp 27 univ med vol. 40 no 1 laboratory staff who helped us in collecting the samples. contributors wm contributed to designing the study, collecting samples, performing the experiment, and writing the manuscript draft; lbk analyzed and interpreted the data, and also wrote the manuscript draft; ea, yw, ii, as, and iy contributed to the revision and finalization of the manuscript. all authors have read and approved the final manuscript. references 1. salazar j, martínez ms, chávez-castillo m, et al. c-reactive protein: an in-depth look into structure, function, and regulation. int sch res notices 2014; article id 653045. doi: 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2019;11:74. doi: 10.1186/s13098-019-0468-0. 16. muller mj, geisler c. defining obesity as a disease. eur j clinl nutr 2017;71:1256-8. doi: 10.1038/ejcn.2017.155. 17. oussaada sm, van galen ka, cooiman mi, et al. the pathogenesis of obesity. metabolism 2019;92:26-36. doi: 10.1016/j.metabol.2018.12.012. 18. gadde km, martin ck, berthoud hr, heymsfield sb. obesity: pathophysiology and management. j am coll cardiol 2018;71:69-84. doi: 10.1016/ j.jacc.2017. 11.011. 19. frasca d, blomberg bb, paganelli r. aging, obesity, and inflammatory age-related diseases. front immunol 2017;8:article 1745. doi: 10.3389/ fimmu.2017.01745. 20. jura m, kozak lp. obesity and related consequences to ageing. age 2016;38:23. doi: 10.1007/s11357-016-9884-3. 28 mulyamin, kurniawan, adnan, et al bmi as the most influential factor of hscrp 21. csige i, ujvarosy d, szabo z, et al. the impact of obesity on the cardiovascular system. j diabetes res 2018; article id 3407306. doi: 10.1155/2018/ 3407306. 22. yousuf o, mohanty bd, martin ss, et al. highsensitivity c-reactive protein and cardiovascular disease: a resolute belief or an elusive link?. j am coll cardiol 2013;62:397-408. doi: 10.1016/j.jacc. 2013.05.016. honey improves lipid profile of diet-induced hypercholesterolemic rats 177 *department of physiology, faculty of medicine, islamic university of indonesia correspondence: dr. titis nurmasitoh, m.sc. department of physiology, faculty of medicine, islamic university of indonesia, jln. kaliurang km 14,5 yogyakarta 55584 phone: +62815 685 7437 email: titisnurmasitoh@gmail.com univ med 2015;34:177-86 doi: 10.18051/univmed.2016.v35.177-186 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background coronary heart disease (chd) is a major cause of morbidity and mortality throughout the world, including indonesia. one of the risk factors for chd is hypercholesterolemia. one of the natural products that has been developed for the treatment of hypercholesterolemia is honey. honey contains fructooligosaccharides, various vitamins, minerals, and enzymes which are supposedly able to lower blood cholesterol levels. this research aimed to study the influence of honey on the levels of blood total cholesterol, triglyceride, and low density lipoprotein (ldl) levels in wistar rats. methods this study was of experimental post test control group design. twentyfour male wistar rats (rattus norvegicus) were randomly divided into 4 groups. k1 was the negative control group (with normal diet), k2 was the positive control group (with high-fat diet), p1 was fed a high-fat diet for 7 days, followed by high-fat diet plus honey for the next 7 days. p2 was fed a high-fat diet for 7 days, followed by regular diet plus honey for the next 7 days. after completion of this treatment, total cholesterol, triglycerides, and ldl levels were measured by the cholesterol oxidase phenol+aminophenazone (chod-pap) method using enzymatic spectrophotometry principles. results there were significant differences in total cholesterol, triglyceride, and ldl levels between all groups after day 15 (p<0.05). conclusion honey supplementation was able to reduce the blood levels of total cholesterol, triglycerides, and ldl. honey supplementation accompanied by non-cholesterol feeds could more effectively lower total cholesterol, triglycerides, and ldl serum levels in wistar rats. keywords: cholesterol, triglycerides, ldl, honey, hypercholesterolemia, rats honey improves lipid profile of diet-induced hypercholesterolemic rats titis nurmasitoh* and miranti dewi pramaningtyas* doi: http://dx.doi.org/10.18051/univmed.2015.v34.177-186 178 nurmasitoh, pramaningtyas honey improves hypercholesterolemic madu memperbaiki profil lipid pada tikus yang diinduksi diet hiperkolesterolemia latar belakang penyakit jantung koroner (pjk) merupakan penyebab utama kesakitan dan kematian di seluruh dunia, termasuk indonesia. salah satu faktor risiko pjk adalah hiperkolesterolemia. salah satu bahan alami yang banyak dikembangkan dalam pengobatan adalah madu. madu mengandung fruktooligoakarida, berbagai vitamin, mineral, dan enzim yang diduga dapat menurunkan kadar kolesterol dalam darah. penelitian ini bertujuan untuk mempelajari pengaruh madu terhadap kadar kolesterol total, trigliserida, dan ldl darah. metode penelitian ini merupakan penelitian eksperimental menggunakan post test control group design. dua puluh empat ekor tikus wistar (rattus norvegicus) jantan dibagi secara acak dalam 4 kelompok. kelompok k1 adalah kontrol negatif (diberi pakan biasa), k2 adalah kontrol positif (diberi pakan tinggi lemak), p1adalah kelompok yang diberi pakan tinggi lemak selama 7 hari dilanjutkan pakan tinggi lemak ditambah madu selama 7 hari berikutnya, dan p2 adalah kelompok yang diberi pakan tinggi lemak selama 7 hari dilanjutkan pakan biasa ditambah madu selama 7 hari berikutnya. setelah selesai perlakuan, darah diambil untuk diperiksa kadar kolesterol total, trigliserid, dan ldl menggunakan metode chod-pap dengan prinsip spektrofotometri enzimatis. hasil terdapat perbedaan bermakna kadar kolesterol, trigliserida, dan ldl setelah hari ke-15 antara keempat kelompok (p<0,05). kesimpulan pemberian suplementasi madu dapat menurunkan kadar kolesterol total, trigliserida, dan ldl dalam darah pada tikus. pemberian suplementasi madu disertai dengan pemberian pakan non-kolesterol dapat menurunkan kadar kolesterol total, trigliserida, dan ldl dalam darah secara lebih efektif. kata kunci: kolesterol, trigliserida, ldl, madu, hiperkolesterolemia,tikus abstrak introduction coronary heart disease (chd) is a major cause of morbidity and mortality throughout the world, including indonesia. during the last fifty years, the number of chd patients has continued t o i n c r e a s e , w i t h s u d d e n d e a t h a s f i n a l outcome.(1-4) the increased incidence of chd is associated with increased chd risk factors such as dyslipidemia, obesity, hypertension, smoking habit, diabetes mellitus, and other risk factors related to lifestyle. in order to decrease chd cases, the risk factors should be controlled. as the increased levels of blood cholesterol is also a risk factor for chd, a decrease in total cholesterol levels, especially reduced levels of low-density lipoprotein (ldl), is one of the therapeutic targets to prevent and decrease the incidence of chd.(1,3,5) the american college of cardiology and the american heart association (acc/aha) have issued guidelines for cholesterol therapy (also known as cholesterol treatment guidelines, ctg). the main treatment for hypercholesterolemia is by the use of statins. statins act to lower blood cholesterol levels by 179 univ med vol. 34 no.3 inhibiting 3-hydroxy-3-methyl-glutaryl-coenzyme a (hmg-coa) reductase.(6) several other substances used to lower cholesterol, are among others bile-acid sequestrants (inhibiting the absorption of bile acids), vitamin e, and gemfibrozil. so far, the use of statins is the most effective strategy to lower blood ldl levels.(1) statins can lower blood cholesterol in more than 30% of patients with hypercholesterolemia. however, the use of statins and other anticholesterol drugs, especially in the long term, might cause permanent side effects, such as those associated with gastrointestinal symptoms, skin rash, anxiety disorders, and hepatic dysfunction.(7,8) therefore, controlling risk factors by improving lifestyle and performing early monitoring is the best measure to prevent chd caused by hypercholesterolemia. various studies on the benefits of honey have been performed. honey is widely used empirically for its anti-inflammatory, antibacterial, antifungal, antiviral, antihypertensive, antioxidant, cardioprotective, hypoglycemic and other actions.(3,9,10) however, scientific evidence supporting honey as a medicine still needs to be developed to optimize its utilization. various literature reports mention that honey contains a complete array of nutrients and has the potential to be developed into a medication. various vitamins, minerals, fructo-oligosaccharides, polyphenols, amino acids, and enzymes contained in honey are expected to be useful for lowering blood cholesterol levels. this research aimed to study the influence of honey on the levels of total cholesterol, triglycerides, and ldl in the blood of diet-induced hypercholesterolemic wistar rats (rattus norvegicus). methods research design this was an experimental laboratory research using post test control group design. the study took place in the physiology laboratory, faculty of medicine, islamic university of indonesia. the study was conducted from january to may 2010. animals a total of twenty four male wistar rats (rattus norvegicus) aged 15 weeks, weighing 200250 grams, were obtained from the animal house at the integrated research and testing laboraory (laboratorium penelitian dan pengujian terpadu, lppt), gadjah mada university, yogyakarta. the number of rats needed was determined based on the 3rs principle (replacement, refinement, and reduction) and was calculated using the following equation: e = n-t where: e = a constant between 10-20, n = planned number of animals per group multiplied by number of treatment groups, t = number of treatment groups. this study used 4 groups. if n or the number of animals per group = 6, then e = (6x4) -4, therefore e=20, which is still included in the specified range of values between 10-20. thus, the total number of rats was 24 and each group contained 6 rats.(11) before the treatment was performed, the rats were isolated for an adaptation stage of 7 days. they were placed in cages with regulated indoor light intensity, consisting of 12 hours light and 12 hours dark periods. during this stage, feed and water were given ad libitum. treatment rats were randomly assigned into four groups, namely the negative control group (k1), the positive control group (k2), treatment group 1 (p1), and treatment group 2 (p2). each group of six rats was assigned randomly. group k1 was given regular feed from the beginning to the end of the study. group k2 was given a high-fat diet for 14 days. group p1 was given high-fat feeds for 7 days, followed by high-fat feeds plus honey for the next 7 days. group p2 was given high-fat feeds for 7 days, followed by regular feed plus honey for the next 7 days. all rats were weighed daily to determine changes in body weight from day to day. 180 nurmasitoh, pramaningtyas honey improves hypercholesterolemic preparation of honey honey from one local honey store was chosen to be used in the study. based on the printed label of the packaging, the honey used for this study contained glucose, fructose, sucrose, amino acids, zinc, anti-oxidant, potassium, phosporus, iron, vitamins (b1, b2), and some enzymes. the honey was given daily by gavage at a dose of 0.8 ml/day. the dose of honey for the rats was obtained by multiplying the usual dose of honey for daily human consumption by a conversion factor. the usual dose for human consumption as printed on the packaging and empirically considered adequate to reduce cholesterol was 2 tablespoons (30 ml) each day. after multiplying the dose for humans by the conversion factor of man to rat (0.018), a dose of 0.8 ml/day was obtained. each dose of honey was dissolved in water to a volume of 2 ml. the honey used in this experiment belonged to the kapok tree type of honey, which had been commercialized with a specific brand. preparation of high-fat diet high-fat feed was prepared by mixing the regular feed with white butter at a ratio of 5:1, i.e. 5 parts of feed (20 g/day/rat) was mixed with 1 part white butter (4 grams/day). the dose of white butter was obtained from a preliminary study. white butter was chosen in this study on the basis of economic and accessability aspects. the feed and butter were evenly mixed and then placed in a feed container in each cage, to be consumed by the rats. the amount of feed consumed was determined daily by measuring the feed left in the container. biochemical analyses blood sampling of the rats was performed three times, i.e. before treatment, on day 7 of treatment, and on day 15, after completion of the treatment. blood was taken at the retro-orbital plexus using 2 ml hematocrit pipettes. the blood samples were taken to the laboratory of nutrition, central inter-university laboratory, gadjah mada university, to check the levels of total cholesterol, triglycerides, and low density cholesterol (ldl) using the cholesterol oxidase phenol + aminophenazone (chod-pap) method, based on the principles of enzymatic spectrophotometry. the results were expressed in mg/dl. statistical analysis the data on total cholesterol, triglycerides, ldl, and hdl obtained from laboratory tests were analyzed using statistical software and presented as mean ± sd. the distribution of the obtained numerical data were also examined using shapiro wilk test followed by levene test in order to determine the homogeneity of the data. if the data were distibuted normally, then analysis of variance (anova) test was used, and kruskall wallis test was used if the data were not normally distributed. differences were considered significant when the probability value was less than <0.05 (p<0.05). ethical clearance this study was approved by the ethical commision of the islamic university of indonesia. results before treatment, blood sampling was performed for all groups in order to measure the initial levels of total cholesterol, triglycerides, and ldl. the mean levels of total cholesterol, triglycerides, and ldl are presented in table 1. the data analysis began with the shapiro wilk normality test and homogenity test using levene test. the shapiro wilk test for cholesterol, triglycerides, and ldl indicated that the data had a normal distribution. therefore, one way anova test for cholesterol, triglyceride, and ldl was applied. there were no signifficant differences between groups before treatment (p>0.05). the statistical analysis indicated that the subjects were in similar condition before treatment. during the first seven days, k1 rats were given standard feed and water ad libitum, while 181 k2, p1, and p2 rats were treated with high-fat feed and water ad libitum. on day 7, blood sampling was again performed. mean total cholesterol, triglyceride and ldl levels on day 7 are shown in table 2. one way anova test results for cholesterol on day 7 showed significant differences between groups (p=0.000). lsd post hoc test showed significant differences (p <0.05) between groups k1 in comparison with groups k2, p1 and p2. the results of one way anova test for triglyceride levels on day 7 showed significant differences between groups (p=0.000). lsd post hoc test showed significant differences (p<0.05) between group k1 in comparison with groups k2, p1 and p2. levels of ldl on day 7 also showed significant differences between groups (p=0.008) using kruskal wallis test (table 2). thus, the administration of high-fat feeds in groups k2, p1 and p2 led to significantly increased levels of cholesterol, triglycerides, and ldl compared with the negative control group (group k1). the results for day 14 indicated that the levels of cholesterol, and triglycerides, and ldl showed significant differences between groups (p=0.000; p=0.000; and p=0.001) (table 3). in this study, total cholesterol, triglycerides, and ldl levels were highest in group k2 (the group receiving high-fat feeds without honey). this showed that the induction with high-fat feeding in rats led to an increase in the levels of total cholesterol, triglycerides, and ldl. discussion the induction with high-fat feeds causes an increase in cholesterol synthesis and eventual a c c u m u l a t i o n o f b l o o d c h o l e s t e r o l (hypercholesterolemia). increase in cholesterol synthesis is facilitated by the enzyme hmgcoareductase. tomkin and owens (12) state that the decrease in cholesterol synthesis resulting from inhibition of hmgcoa-reductase enzyme activity reduces ldl levels and vice versa. hmgcoa-reductase inhibitory activity is shown b y s t a t i n s u s e d i n t h e t r e a t m e n t o f hypercholesterolemia. if the cholesterol level in the blood increases, ldl as a carrier of cholesterol to cells throughout the body will also increase. furthermore, the accumulation of cholesterol followed by free radical activity causes oxidative damage to various tissues. in addition, ldl is a substance that is readily oxidized. oxidized ldl binds to macrophages and subsequently forms foam cells to cause atherosclerotic lesions. the hypercholesterolemic table 1. mean total cholesterol, triglyceride, and ldl levels by treatment groups at base line k1: standard feed control group, k2: high fat feed control group, p1: high fat feeds for 7 days followed by high-fat feed plus honey for the next 7 days, p2: high-fat feeds for 7 days followed by regular feed plus honey for the next 7 days. a anova test k1: standard feed control group, k2: high fat feed control group, p1: high fat feeds for 7 days followed by high-fat feed plus honey for the next 7 days, p2: high-fat feeds for 7 days followed by regular feed plus honey for the next 7 days; a anova test; bkruskall wallis test table 2. mean total cholesterol, triglyceride, and ldl levels by treatment groups after 7 days of treatment univ med vol. 34 no.3 182 nurmasitoh, pramaningtyas honey improves hypercholesterolemic table 3. mean total cholesterol, triglyceride, and ldl levels by treatment groups after 14 days of treatment k1: standard feed control group, k2: high fat feed control group, p1: high fat feeds for 7 days followed by high-fat feed plus honey for the next 7 days, p2: high-fat feeds for 7 days followed by regular feed plus honey for the next 7 days; a anova test; bkruskall wallis test condition can lead to atherosclerosis, resulting in complications such as myocardial infarction and stroke.(12,13) uncontrolled hypercholesterolemia can d e v e l o p i n t o c a r d i o v a s c u l a r d i s e a s e . hypercholesterolemia is strongly associated with high total cholesterol and ldl levels in the blood. high levels of total cholesterol and ldl in the blood attract attention because they are correlated with the incidence of coronary heart disease.(1,3) cardiovascular disease, such as coronary heart disease, is one of the leading causes of worldwide death. mortality and morbidity due to cardiovascular disease are aggravated by a variety of risk factors, such as: minimal physical activity and exercise, smoking, poor dietary habits (diets high in fat and low in fiber), alcoholism, and others.(14,15) several studies were developed to address the conditions of hypercholesterolemia and maintain cholesterol homeostasis. cholesterol homeostasis is maintained via a complex arrangement in terms of absorption, anabolism, catabolism, and excretion.(3,7,14) according to previous studies, cholesterol levels in the blood may be reduced through several mechanisms. first, through competition with intestinal niemann pick c1-like 1 (npc1l1). around 1200-1700 mg of cholesterol enters the lumen of the small intestine every day. approximately 300-500 mg of the cholesterol comes from the diet and the rest comes from bile acids. cholesterol absorption in the small intestine is mediated by npc1l1. once absorbed, cholesterol is transported by npc1l1 from the lumen of the small intestine into the enterocytes. cholesterol is then converted into cholesteryl ester (ce) by intestinal acyl-coa cholesterol acyltransferase 2 (acat2). in addition, the ce will be packaged into chylomicrons by microsomal triacylglycerol transport protein (mtp). eventually, chylomicrons head into the circulatory system in the form of very-low density lipoprotein (vldl). next, vldl is degraded forming ldl to deliver cholesterol to cells throughout the body. the addition of substances that are structurally similar to the npc1l1 may reduce the levels of cholesterol and ldl.(7,12,16) the second mechanism is inhibition of intestinal acyl-coa cholesterol acyltransferase 2 (acat2). in mammals, acat2 is important for lipid esterification in enterocytes and the liver to produce cholesteryl ester which is useful for the formation of chylomicrons and vldl. inhibition of acat2 may lower total cholesterol and ldl. the third mechanism is inhibition of 3-hydroxy3-methylglutaryl (hmg-coa) reductase, which is required in the biosynthesis of cholesterol. thus, inhibition of hmg-coa reductase may inhibit the cholesterol synthesis pathway so that the levels of cholesterol and ldl in the blood are reduced.(7,12,16) the fourth mechanism is activation of ldl receptors. increasing the number and activity (upregulation) of ldl receptors may improve the cholesterol-ldl clearance from the circulation. therefore, activation of the receptors may reduce the levels of blood cholesterol and ldl. the fifth mechanism is inhibition of bile acid reabsorption. this is done through the binding of bile acids in the intestine by certain substances to form insoluble complex compounds that can not be 183 reabsorbed into the liver and are eventually excreted. inhibition of bile acid reabsorption lowers cholesterol levels and increases the hepatic synthesis of bile acids from cholesterol. the increase in hepatic bile acid synthesis triggers blood cholesterol influx into the liver. this is also caused by ldl receptor activation due to decreased hepatic cholesterol levels. thus, cholesterol levels and ldl in the blood will decrease.(7,12,16) the sixth mechanism is activation of cytochrome p450 7a1 (cyp7a1) or cholesterol7á -hydroxylase. activation (up-regulation) of cyp7a1 is theoretically able to lower the cholesterol in the liver, causing blood cholesterol influx, and ultimately lowering cholesterol levels in the blood.(7,12,16) the last mechanism is inhibition of plasma cholesteryl ester transporting protein (cetp). cetp inhibition could be expected to lower blood cholesterol and ldl levels because the transportation of cholesteryl ester as a raw material for making chylomicrons is inhibited.(7,12,16) research on the mechanisms of cholesterollowering therapy is important to determine the substances and the working points which are expected to lower total cholesterol and ldl levels in the blood. one substance that is widely studied as cholesterol-lowering agent is honey. studies on honey as an anti-hypercholesterolemia agent has been widely developed, in both humans and experimental animals with varying results. in the present study, administration of honey in group p1 and p2 resulted in significantly lower cholesterol, triglycerides, and ldl levels in comparison with those in group k2. group p2 (the group receiving high-fat feeds followed by normal feed plus honey) showed lower results compared with group p1 (the group receiving high-fat feeds followed by high-fat feeds plus honey). in fact, total cholesterol, triglycerides, and ldl levels of rats in group p2 were almost identical with those in group k1 (the negative control group receiving regular feed up to the end of treatment). based on the data analysis perfomed in this study, honey was proven to be able to reduce the levels of cholesterol, triglycerides, and ldl. this is in line with previous studies conducted on various experimental animals. nemoseck et al.(17) stated that honey has many advantages that would be promising in the world of health. one of the benefits of honey that is described in their study was to lose weight and lower blood triglyceride levels in sd strain rats. in contrast, the other lipid profile parameters were increased or were not significantly different from those of the positive control group or the group given additional sucrose diet. in addition, alagwu et al.(13) suggest that the administration of pure nigerian honey can reduce levels of blood ldl and total cholesterol in rats. however, alagwu’s research showed elevated levels of triglycerides and vldl in the blood. adnan et al.(1) also suggest that honey from the acacia tree (desi kikar) may lower the levels of triglycerides, total cholesterol, and ldl in dietinduced hypercholesterolemic albino rats, as well as in the group treated with simvastatin. the group treated with a combination of acacia honey and simvastatin showed greater reductions in lipid profile, approaching the values in the negative control group. research on the effects of different variants of honey on the lipid profile has also been conducted in humans. a research study by mushtaq et al.(18) mentions that the consumption of 40 grams of honey was shown to improve lipid profiles in obese adult subjects. yaghoobi et al.(19) also found that 70 grams of pure honey daily for 30 days was proven to reduce cardiovascular disease risk factors and did not cause weight gain in overweight or obese subjects. in addition, studies on healthy young subjects with similar dose and duration (70 grams daily for 4 weeks) also showed a decrease in total cholesterol, triglycerides, and ldl compared with the control group.(20) honey is a nutrient-rich food that is produced mainly by the honeybee (apis mellifera l.). honey has been used by humans since univ med vol. 34 no.3 184 nurmasitoh, pramaningtyas honey improves hypercholesterolemic ancient times. various literature reports mention that honey has antibacterial, antifungal, antiviral, anti-inflammatory, antihypertensive, antioxidant, antitumor, cardioprotective, hepatoprotective, and hypoglycemic effects. honey contains much carbohydrate (glucose and fructose—85% content of honey, sucrose, maltose, isomaltose, maltulose, and others), vitamins (thiamine, riboflavin, pyridoxine, vitamin a, niacin, pantothenic acid, vitamin e, vitamin c), trace elements and minerals (aluminum, arsenic, lithium, sulfur, iodine, cobalt, sodium, calcium, potassium, magnesium, phosphorus, zinc, copper, iron, selenium, manganese, and others), polyphenols, organic acids, amino acids (proline, glutamic acid, alanine, phenylalanine, tyrosine, leucine, isoleucine, and others), enzymes (glucose oxidase, invertase, amylase, catalase, and acid phosphatase), polyphenols (phenolic acids, flavonoids, phenolic acid derivatives [quercetin, chrysin, galangin, luteolin, kaempferol, apigenin, and others]), nitric oxide (no), and other substances. the composition of the substances contained in honey is influenced by the type of crop, climate, and environmental conditions.(10,15, 21) the hypotriglyceridemic effects of honey may be caused by fermented nondigestibleo l i g o s a c c h a r i d e s ( n d o s ) , s u c h a s fructooligosaccharides (fos) or other similar carbohydrate isomaltulose which are contained in honey. nondigestible-oligosaccarides (ndos) are not hydrolyzed in the small intestine, but are degraded by microflora in the colon. in the colon, the degradation process will produce short chain fatty acids that will affect the normal flora in the colon. the normal flora of the colon is important and has a beneficial effect on lipid metabolism.(17) polysaccharides that can not be digested and short chain fatty acids resulting from the fermentation process by the normal flora will be able to reduce the levels of ldl cholesterol in the blood by inhibiting hmg-coa reductase (7) and bile acid reabsorption into the liver.(3,7,9,22) in addition, the normal flora species which are beneficial and present in sufficient numbers in the colon, such as lactobacillus, b i f i d o b a c t e r i u m , e n t e ro c o c c u s , a n d streptococcus, will be able to help lower blood cholesterol levels by inhibiting the reabsorption of bile acids into the liver.(7,22) bogdanov et al.(9) mention that honey can increase lactobacillus in the small intestine and colon. the content of antioxidants in honey is also predicted to have a beneficial effect on lipid metabolism. nevertheless, the content of antioxidants in honey varies greatly depending on the type of plant and its environment. honey with darker colors have higher antioxidant content than the honey with a lighter color.(1,17) according to chen et al.,(7) antioxidant compounds, such as quercetin, kaempferol, galangin, and various other flavonoid compounds, are able to help lowering ldl and cholesterol levels of blood. the antioxidant compounds work by inhibiting acat2, activating (up-regulating) ldl receptors, and activating cyp7a1. chepulis and starkey ( 2 3 ) found that although there was an increase in hdlcholesterol in honey-fed rats compared with rats fed sucrose or a sugar free diet, but there were no other differences in lipid profiles. nemoseck et al.,(13) alagwu et al.(17) and majid et al.(20) also suggest that the supplementation of honey will increase the excretion of cholesterol through bile acids thereby lowering blood cholesterol levels. in addition to antioxidants, honey also contains niacin. the content of niacin in honey could be expected to lower blood cholesterol by inhibiting the mobilization of deposits of triglycerides from a d i p o s e c e l l s a n d t h r o u g h i n h i b i t i o n o f diacylglycerol acyltransferase enzymes in liver cells that are involved in cholesterol synthesis in the hmg-coa reductase pathway. (13,18,20) yaghoobi et al.(19) suggest that antioxidants in honey, in addition to their role in lowering blood c h o l e s t e r o l a n d l d l l e v e l s , a r e a l s o advantageous by inhibiting the formation of atherogenic plaques. inhibition of atherogenic plaque formation is also determined by a variety of trace elements, minerals, vitamins, and no contained in honey. some trace elements that prevent the formation of atherogenic plaques are 185 copper and zinc. meanwhile, some of the vitamins contained in honey playing a role in preventing oxidative stress and atherosclerotic plaque formation are vitamin e (niacin), vitamin c, vitamin b, and vitamin a.(20,24) honey is a food supplement with a complete array of substances, of which some are potentially of use as antihypercholesterolemic agents. honey contains several amino acid components.(15) according to chen et al.,(7) protein has the ability to lower of blood cholesterol and ldl cholesterol levels by inhibiting the reabsorption of bile acids, inhibiting hmg-coa reductase and activating ldl receptors. in addition, honey also contains calcium. supplementation of calcium in the diet is beneficial to lower cholesterol levels by inhibiting intestinal npc1l1, activating cyp7a1, and increasing bile acid excretion via feces.(7,25) meanwhile, there are still many substances in honey which have not been scientifically proven to be beneficial in lowering blood cholesterol levels. in our study, the group treated with honey followed by a normal diet had the lowest levels of cholesterol, triglycerides, and ldl among all treatment groups. moreover, the level was almost identical with that in the negative control group. this indicates that in addition to adequate therapy with antihypercholesterolemia agents, dietary modification also has a positive effect on the prevention of cardiovascular disease.(7,14,22) this study had a limitation in the dose of honey that should be given to rats. further studies should use a number of honey doses. the results of this study may be useful to researchers and clinicians as a reference in the use of honey supplementation for those who are dyslipidemic. conclusions honey supplementation can reduce levels of total cholesterol, triglycerides, and ldl in the blood. supplementation of honey accompanied by a non-cholesterol diet can more effectively lower total cholesterol, triglycerides, and ldl in the blood. conflict of interest there was no conflict of interest with other institutions. acknowledgement this study was funded by the faculty of medicine, islamic university of indonesia. references 1. adnan f, sadiq m, jehangir a. antihyperlipidemic effect of acacia honey (desi kirar) in cholesterol-diet induced hyperlipidemia in rats. biomedica 2011;27:62-7. 2. chapman mj, ginsberg hn, amarenco p, et al. triglyceride-rich lipoproteins and high-density lipoprotein cholesterol in patients at high risk of cardiovasculer disease: evidence and guidance for management. eur heart j 2011;32:1345-61. 3. nijjar ps, burke fm, bloesch a, et al. role of dietary supplements in lowering low-density lipoprotein cholesterol: a review. j clin lipidol 2010;4:248-58. 4. zahrawardani d, herlambang ks, anggraheny hd. analisis faktor risiko kejadian penyakit jantung koroner di rsup dr kariadi semarang. jurnal kedokteran muhammadiyah 2013;1:1320. 5. siri-tarino pw, sun q, hu fb, et al. saturated fat, carbohydrate, and cardiovascular disease. am j clin nutr 2015;91:502-9. 6. martin ss, abd tt, jones sr, et al. acc/aha cholesterol treatment guideline, what was done well and what could be done better. j am coll cardiol 2014;63:2674-8. 7. chen z, ma ky, liang y, et al. role and classification of cholesterol-lowering functional foods. j funct foods 2011;3:61-9. 8. djide mn. efek hipokolesterolemia kultur bakteri asam laktat dalam soyghurt terhadap tikus putih. j sains teknol 2006;6:13-8. 9. bogdanov s, jurendic t, sieber r, gellmann p. honey for nutrition and health: a review. am j coll nutr 2008;27:677-89. 10. kumar kps, bhowmik d, chiranjib, et al. medicinal uses and health benefits of honey: an overview. j chem pharm res 2010;2:385-95. 11. festing mfw, altman dg. guidelines for the design and statistical analysis of experiments using laboratory animals. ilar 2002;43:24458. univ med vol. 34 no.3 186 nurmasitoh, pramaningtyas honey improves hypercholesterolemic 12. tomkin gh, owens d. ldl as a cause of atherosclerosis. open atheroscl thromb j 2012; 5:13-21. 13. alagwu ea, okwara je, nneli ro, et al. effect of honey intake on serum cholesterol, triglycerides, and lipoprotein levels in albino rats and potential benefits on risks of coronary heart disease. nig j physiol sci 2011;26:161-5. 14. wang s, melnyk jp, tsao r, et al. how natural dietary antioxidants in fruits, vegetables, and legumes promote vascular health. food res int 2011;44:14-22. 15. al-waili n, salom k, al-ghamdi a, et al. honey and cardiovascular risk factors, in normal individuals and in patients with diabetes mellitus or dyslipidemia. j med food 2013;16:1063-78. 16. lagor wl, millar js. overview of the ldl receptor: relevance to cholesterol metabolism and future approaches for the treatment of coronary heart disease. j receptor ligand channel res 2010;3:1-14. 17. nemoseck tm, carmody eg, furchner-evanson a, et al. honey promotes lower weight gain, adiposity, and triglycerides than sucrose in rats. nutr res 2011;31:55-60. 18. mushtaq r, mushtaq r, khan zt. effects of natural honey on lipid profile and body weight in normal weight and obese adults: a randomized clinical trial. pakistan j zool 2011;43:161-9. 19. yaghoobi n, al-waili n, ghayour-mobarhan m, et al. natural honey and cardiovascular risk factors; effects on blood glucose, crp, and body weight compared with sucrose. scientific world j 2008;8:463-9. 20. majid m, younis ma, naveed ak, et al. effects of natural honey on blood glucose and lipid profile in young healthy pakistani males. j ayub med coll abbottabad 2013;25:44-7. 21. perez-perez e, vit p, huq f. flavonoid and polyphenols in studies of honey antioxidant activity. int j med plant altern med 2013;1:6372. 22. izadi z, nasirpour a, izadi m, et al. mini review: reducing blood cholesterol by a healthy diet. international food res j 2012;19:29-37. 23. chepulis, starkey n. the long-term effects of feeding honey compared with sucrose and a sugar free diet on weight gain, lipid profiles, and deaa measurements in rats. j food scie 2008; 73:1-7. 24. kshitiz kk, sinha rb, bhattacharjee j. a study of effects of smoking on lipid and vitamin c metabolism, a pilot study in central bihar. int j pharma bio sci 2010;1:106-12. 25. ma ky, yang n, jiao r, et al. dietary calcium decreases plasma cholesterol by down regulation of intestinal npc1l1 and mtp and up regulation of cyp7a1 and abcg 5/8 in hamster. mol food res 2011;55:247-58. blood viscosity increases the degree of coronary stenosis in coronary heart disease 168 *department of clinical pathology, faculty of medicine, diponegoro university/dr.kariadi hospital **department of biochemistry, faculty of medicine, diponegoro university/dr.kariadi hospital correspondence: dr. antonius ferika indrianto, sppk jl. diponegoro 213 ungaran kab. semarang mobile: +62812 293 0293 email: f3rry_ind@yahoo.co.id univ med 2015;34:168-76 doi: 10.18051/univmed.2016.v35.168-176 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2015september-december, 2015september-december, 2015september-december, 2015september-december, 2015 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 vol.34 no.3 background endothelial dysfunction and associated increased blood viscosity (bv) play an important role in the formation of atherosclerosis during inflammatory processes. c-reactive protein (crp) is a biomarker of inflammation with an active role in endothelial dysfunction and development of atherosclerosis that is marked by degree of coronary stenosis (cs). the purpose of this study was to determine the relationship of bv and crp with varying degrees of cs among coronary heart disease (chd) patients. methods this cross-sectional study involved 24 subjects taken consecutively among patients with chd who underwent angiography. blood viscosity levels were determined using an ostwald viscometer, with reference limits of 1.5 to 1.72 (cp). c-reactive protein was determined by turbidimetric immunoassay, with a normal reference value of <3 mg/l. degree of cs was examined with angiography, where 0% = no stenosis; <50% = nonsignificant stenosis; > 50% = significant stenosis. data was analyzed using non-parametric spearman correlation test. results there were 17 male and 7 female subjects, with mean age of 55.96 ± 7.29 years. the angiography results were: non-significant stenosis in 2 subjects (8.3%), significant stenosis in 22 subjects (91.7%) and none with normal stenosis. statistical analysis of the relationship of bv with degree of cs found a moderate significant positive relationship (r=0.549; p=0.005). a poor significant positive relationship was found between crp and degree of cs (r=0.481; p=0.017). conclusion blood viscosity increased the degree of cs in chd patients. therapy should target both bv-associated risk and angiographically evident stenosis. keywords: c-reactive protein, blood viscosity, degree of coronary stenosis, coronary heart disease blood viscosity increases the degree of coronary stenosis in coronary heart disease antonius ferika indrianto*, indranila kustarini samsuria*, and kusmiyati deddy kurniawan** doi: http://dx.doi.org/10.18051/univmed.2015.v34.168-176 169 univ med vol. 34 no.3 viskositas darah meningkatkan derajat stenosis koroner pada penderita jantung koroner latar belakang peran penting dalam pembentukan aterosklerosis selama proses inflamasi berlangsung adalah terjadinya disfungsi endotel dan sangat berhubungan erat dengan peningkatan viskositas darah (vd). c-reactive protein (crp) adalah biomarker inflamasi yang berperan aktif dalam disfungsi endotel, dan perkembangan ateroskeloris yg ditandai derajad stenosis koroner (dsk). tujuan penelitian ini untuk menentukan adanya hubungan antara vd dan crp dengan berbagai derajat stenosis koroner (dsk) pada penderita jantung koroner. metode penelitian ini dilakukan dengan pendekatan belah lintang. dua puluh empat penderita penyakit jantung koroner yang menjalani angiografi yang diambil secara konsekutif. kadar vd diperiksa menggunakan viskometer ostwald dengan batas nilai rujukan 1,5-1,72 (cp).c-reactive protein diperiksa dengan turbidimetric imunoassay dengan acuan normal <3 mg/l. derajad stenosis koroner diperiksa dengan angiografi, 0% : tidak ada stenosis ; <50% non significant stenosis; >50% significant stenosis. dilakukan uji korelasi non parametrik spearman. hasil terdapat 17 subyek laki-laki dan 7 subyek wanita, yang rata-rata umurnya 55,96±7,29 tahun. pemeriksaan angiografi didapatkan pasien yang non signifikan stenosis 2 subyek (8,3%), signifikan stenosis 22 subyek (91,7%) dan tidak didapatkan yang normal / tidak ada stenosis. analisis hubungan vd dengan dsk didapatkan adanya hubungan yang bermakna dengan kekuatan sedang (r=0,549; p=0,005). terdapat pula hubungan yang bermakna antara crp dan dsk dengan kekuatan lemah (r=0,481; p=0,017). kesimpulan viskositas darah meningkatkan derajat derajat stenosis jantung koroner pada penderita jantung koroner. pengobatan harus ditujukan pada risiko yang terkait vd maupun dsk yang terlihat pada pemerikssan angiografi. kata kunci : c-reactive protein, viskositas darah, derajad stenosis koroner, penderita jantung koroner abstrak introduction coronary heart disease (chd) is a terrifying disease that is still a problem in both developed and developing countries. according to data of the world health organization (who), heart disease is the number one cause of mortality in the world. sixty percent of all causes of deaths from heart disease is accounted for by ischemic heart disease. at least 17.5 million or 30% of global deaths are caused by heart disease, and this number is projected to be 23.6 million in the year 2030.(1) coronary heart disease in indonesia causes the highest number of deaths, approaching 26% or 53.5 per 100.000 inhabitants. in addition to being the top-ranking cause of death, chd also lowers the quality of life of the patients.(2) chd starts with endothelial dysfunction and ends in atherosclerosis. endothelial cells have an integral role in the vascular structure, hemodynamics, and hemostasis. abnormalities of vascular hemostasis that occur in endothelial dysfunction result in abnormalities of vascular motor function, inflammation, and thrombosis.(3,4) blood viscosity is a parameter used in hemorheology. hyperviscosity of the blood injures 170 the endothelial wall and causes an inflammatory response in the large arteries, resulting in plaque formation and subsequent rupture. all of the above shows that blood viscosity is plays an important role in the holistic approach for prevention and early detection in connection with cardiovascular health.(5) many studies have been conducted on blood viscosity in connection with atherosclerosis or chd. a number of these studies have indicated that hemorheological parameters play an important role in the pathogenesis and development of chd(6) and that there is a relationship between hemorheological abnormalities and chd severity. these studies have also found that therapeutic interventions for normalizing blood rheology may reduce the incidence and/or progressivity of chd, and that changes in hemorheological parameters that lead to hyperviscosity may result in occlusive thrombotic events.(7,8) atherosclerosis is not merely a disease connected with lipid deposition, since systemic inflammation plays an essential role in the initiation, progression, and destabilization of atheromas.(9) a large number of inflammatory markers are known, such as c-reactive protein (crp). this acute phase protein is measured by the hs-crp (high sensitivity c-reactive protein) method, which is sensitive and practical, with known cut off values. crp is a better cardiovascular predictor than ldl cholesterol and several other inflammatory markers. the crp concentration is raised in infections, inflammation, or trauma.(10,11) the role of crp in evaluating the risk of cardiovascular disease is understandable, since it is a reflection of all pathologic mechanisms of chd, being increased in chronic infections, inflammation, and atherosclerosis.(12) there have been many studies conducted on the relationship of crp and atherosclerosis or chd. several of these studies found a significant positive correlation between coronary artery stenosis and crp concentration, where increased crp concentration was accompanied by degree of stenosis.(13) crp concentration is also associated with severity of coronary heart disease as detected on angiography and supports the hypothesis of a cause-and-effect relationship between the acute phase reaction and the p a t h o g e n e s i s o f c o r o n a r y a r t e r y atherosclerosis.(14,15) blood viscosity is a basic biological p a r a m e t e r a ff e c t i n g t h e m a c r o a n d microcirculation. increased blood viscosity causes injury to the endothelial cell wall, thus interfering with endothelial function, and playing a role in the inflammatory process.(16) crp is an acute phase protein and an inflammatory marker t h a t i s a c t i v e l y i n v o l v e d i n e n d o t h e l i a l dysfunction and the formation and development of atherosclerotic plaques.(17) the study of lee et al. ( 8 ) s h o w e d a n a s s o c i a t i o n b e t w e e n hemorheological abnormalities (blood viscosity, erythrocyte aggregation) and severity of coronary artery disease. chatzizisis et al.(18) showed a role for endothelial stress wounds in atherosclerosis and vascular remodelling. toth et al.(19) found changes in hemorheological parameters in chd. hasnat et al.(20) concluded that there is an association in patients with chd between the concentration of highly sensitive c reactive p r o t e i n ( h s c r p ) a n d c h d s e v e r i t y i n comparison to controls.(20) atherosclerotic plaques, in addition to being a disease of lipid deposition, also have a role in systemic inflammation as their main key, and may be expected to predict the early stages of coronary stenosis. in view of the above, the objective of the present study was to determine the relationship between blood viscosity as h e m o r h e o l o g i c a l p a r a m e t e r a n d c r p a s inflammatory marker in patients with coronary artery stenosis. methods design of the study this study used a cross-sectional approach and was conducted from january until march 2014 in the outpatient and inpatient departments of the cardiac disease unit, dr. kariadi central hospital, semarang. indrianto, samsuria, kurniawan blood viscosity degree coronary stenosis 171 univ med vol. 34 no.3 study subjects the study subjects were recruited from the cardiac outpatient and inpatient units of dr. kariadi central hospital, semarang. the subjects were selected by consecutive nonrandom sampling among patients with chd who had undergone angiography and were screened by the inclusion and exclusion criteria. a total of 24 subjects were included, with the sample size n calculated according to the formula for comparison of 2 proportions: by gravitation between two marks on the ostwald viscometer. the flow time of the tested liquid was compared with the time needed by a liquid with known viscosity (usually water) to pass by gravitation between the two marks on the ostwald viscometer. a clean viscometer was filled by pipetting the test liquid and aspirating with the pushball past the two marks. the pushball was released to allow the liquid to drop from the first to the second mark, the resulting time being recorded by stopwatch. the viscosity coefficient was then expressed in si units (systeme international) as ns/m2 = pa.s (pascal second). the unit centimeter gram second (cgs) for the si viscosity coefficient is dyn.s/cm2 = poise (p). viscosity is also frequently expressed in centipoise (cp). 1 cp = 1/100 p.(23,24) the crp concentration was determined by the hscrp method, using the particle enhanced turbidimetric immunoassay (petia). the serum sample was reacted in a cuvette with anti-crp antibody (abcrp), antimicrobial buffer and polyethylene glycol buffer, the reactants becoming turbid at 37° c. the resulting turbidity is a measure of the crp concentration, as determined by the bichromatic rate technique (340 and 700 nm). the results of the test are shown on a monitor, and the test takes 20 minutes to perform. other methods for crp determination are based on nephelometry and turbidimetry, which are applied in automated analysis. the reference value of crp is <3 mg/l.(25) angiography is the gold standard for determining the degree of coronary stenosis. the finding of minimal stenosis in one coronary artery, expressed as 0% stenosis, indicates a normal coronary without stenosis. if the stenosis is <50% of the luminal diameter, this indicates a non significant stenosis, while a stenosis of >50% of the luminal diameter indicates significant stenosis.(26) data analysis the non parametric spearman test was used to determine the relationship between blood viscosity and coronary stenosis and the relationship between crp and coronary stenosis. ( ) { }      ++ + 3r)] (1 / r) [(1n 51.0 zβ zα 2 2 where zα = 1.96 (level of significance), zβ = 1.28 (power of the study = 90%), r=0.64,(26) giving the total sample size of 21.3 or 22. to anticipate possible drop outs, the sample size was increased by 10%, so that the final sample size was 24. the inclusion criteria for the study subjects were males or females, with normal body temperature, agreeing to participate in the study by signed informed consent, having normal serum sgpt (for determining normal liver function) and normal hemoglobin (hb) (>13 g/ dl for males and >12 g/dl for females). the exclusion criteria were patients with a history of trauma or surgery; lipemia, jaundice, and hemolysis; pregnancy, dehydration, smokers, and crp >10 mg/l. laboratory analysis from the patients meeting the inclusion and exclusion criteria, fasting blood samples were drawn from the median cubital vein, using vacutainers filled with 5 ml edta anticoagulant, for determination of blood viscosity and crp within 3 hours of collection.(21) determination of blood viscosity was by means of an ostwald viscometer, in which the blood sample in the ostwald tube descends by gravitation. the time of descent was recorded with a stopwatch.(22) the viscosity of the liquid was determined by measuring the time needed by the liquid to pass 172 significance was set at p<0.05. the test indicates a very weak correlation, if r=0.00-0.25; a weak correlation if r=0.25-0.50; a moderate correlation if r=0.50-0.75; a strong correlation if r>0.75 and a very strong correlation if r=0.80-1.00.(27) ethical clearance this study obtained ethical clearance under no. 462/ec/fk/rsdk/ 2013. results with regard to subject characteristics, the youngest subject was 37 years old and the oldest 70 years (table 1). mean age of the subjects was 55.96 ± 7.29 years. the number of study subjects was 24, consisting of 17 males (70.8%) and 7 females (29.2%). when the age was grouped according to gender, the youngest age for males was 37 years and the oldest 65 years. among the female subjects, the youngest age was 49 years and the oldest 70 years. on the basis of degree of stenosis, there were 2 subjects (8.3%) with nonsignificant stenosis. significant stenosis was found in 22 subjects (91.7%) and no study subjects were found with normal stenosis (0%). on statistical analysis, the correlation between blood viscosity and degree of stenosis was positively significant and of medium strength (r=0.549; p=0.005). this signifies that increases in blood viscosity will be accompanied by increasingly severe stenosis. from the scatter plot (figure 1), an increasing trend may be seen between degree of stenosis and blood viscosity. a significant positive correlation was also found after statistical analysis of the relationship between crp and degree of coronary stenosis, the correlation being of low strength (r=0.481; p=0.017). this indicates that increases in crp table 1. characteristics of study subjects (n=24) figure 2. scatter plot of relationship between degree of stenosis and crp figure 1. scatter plot of relationship between degree of stenosis and blood viscosity (bv) indrianto, samsuria, kurniawan blood viscosity degree coronary stenosis 173 univ med vol. 34 no.3 concentration are accompanied by severe degrees of coronary stenosis. from the scatter plot (figure 2), an increasing trend may be seen between degree of stenosis and blood viscosity. discussion the positive correlation between ds and bv agrees with the study by kesmarky et al.,(6) w h o i n v e s t i g a t e d t h e r e l a t i o n s h i p o f hemorheological parameters with severity of coronary artery disease, which was divided into 3 groups, i.e. no significant disease, single vessel d i s e a s e , a n d m u l t i v e s s e l d i s e a s e . t h e investigators found that all hemorheological parameters increased significantly in comparison with controls. hematocrit and viscosity increased significantly in the group with multivessel disease, in comparison with the groups with no significant disease and single vessel disease. blood viscosity plays an important role in the regulation of vascular flow and changes in h e m o r h e o l o g i c a l p a r a m e t e r s m a y c a u s e h y p e r v i s c o s i t y, t h e r e b y s u p p o r t i n g t h e occurrence of occlusive thrombotic events.(7) blood viscosity as the material determinant of viscoelasticity in direct contact with endothelium may modulate the shape, elasticity, and gene expression of the endothelial cells, and the distribution of adhesion molecules in these cells, and directly affects endothelial cells through shear stress.(18) increased bv increases the traumatic forces on the endothelial cell walls, thus having a poor impact on endothelial function a n d p l a y i n g a n i m p o r t a n t r o l e i n t h e inflammatory process. this condition will also inhibit the production of nitric oxide (no) through inhibition of the synthesis of endothelial nitric oxide synthase (enos) and increases the production of reactive oxigen species (ros) in endothelial cells and vascular smooth muscle cells. on the other hand, no has an important role in the regulation of vascular tone, in addition to having strong anti-inflammatory, antiapoptotic, anti-mitogenic, and anti-thrombotic actions. increased blood viscosity may directly influence wall or endothelial shear stress (ess), resulting in altered vasomotor tone and decreased ess as a result of lowered blood flow.(17,18) increased bv may promote the development of atherosclerosis by increasing platelet adhesion to the subendothelium, increasing protein infiltration into the arterial wall, and changing the shear stress at the site of atherogenesis.(17) in a r t e r i a l r e g i o n s w i t h u n d i s t u r b e d f l o w, physiologically in ess, the endothelial cells express various atheroprotective genes and repress several pro-atherogenic genes, thus finally leading to stability and undisturbed flow in those regions. by contrast, in regions with low and disturbed flows, at sites of low ess, the expression of endothelial genes will be modulated by a complex process of mechanoreception and m e c h a n o t r a n s d u c t i o n , t h u s i n d u c i n g t h e atherogenic endothelial phenotype and the early formation of atherosclerotic plaques.(18) similar results were found in the study by l e e e t a l . (8) w h o i n v e s t i g a t e d s e v e r a l hemorheological parameters in subjects with stable angina (sa), unstable angina (ua) and acute myocardial infarction (ami). on all subjects angiography was performed to establish the diagnosis. the resulting findings as compared to controls showed significant increases in all groups of study subjects, in the order of ami>ua>sa>controls. the conclusion was t h a t t h e r e w a s a r e l a t i o n s h i p b e t w e e n hemorheological abnormalities and severity of coronary artery disease. the investigators showed in their evaluation of intervention therapy to normalize blood rheology, that it could reduce the incidence or progressivity of coronary artery disease.(8) similarly, in the study conducted by toth et al.,(19) 121 subjects with coronary artery disease who underwent ct scans of the coronary arteries, were assigned to 4 groups, i.e. those with negative stenosis, with non significant stenosis, with single vessel stenosis and with multi vessel stenosis. the results showed a significant relationship between hematocrit and blood viscosity, which were higher in all groups, in comparison with the negative group.(18) 174 the above mentioned correlation between degree of stenosis and crp is in agreement with the results of the study by hasnat et al. in which crp showed a positive significant correlation in 90 subjects with chd, as determined by vessel score, stenosis score, and extent score. it was concluded from above mentioned study that increases in crp concentration were substantially correlated with severity and degree of stenosis of chd. the increase in crp concentration was associated with cardiovascular disease incidence in the future and may predict coronary atherosclerotic disease.(20) nyandak et al.(13) also investigated the relationship between crp and coronary stenosis by angiography and found a positive correlation; whereas if crp was correlated with angiographic extent score. the crp concentration was higher in patients with this angiographic degree of stenosis, showing that crp concentration was correlated with degree of disease severity in patients with chd. momiyama et al., (21) piranfar,(22) masood et al.,(23) and lai et al.,(24) arrived at similar conclusions, namely that crp is an index of chd severity and may reflect plaque activity in patients with chd, and is therefore important for clinical diagnosis and evaluation chd risk.(21-24) another study by ciccone et al.(25) found that thickening of the carotid intima media was correlated with crp, thus indicating that inflammatory markers affect the progressivity of atherosclerosis in subjects with obstructive sleep apnea.(25) c-reactive protein plays an active role in endothelial dysfunction and the formation and development of atherosclerotic plaques, since it has been found in atherosclerotic plaques. creactive protein down-regulates the transcription of enos protein and messenger ribonucleic acid (mrna) in endothelial cells, thus resulting in a reduction of no release. the inhibition of no facilitates endothelial cell apoptosis and inhibits angiogenesis. among other proatherogenic effects, crp regulates intercellular adhesion molecule 1 (icam-1), vascular cell adhesion molecule-1 (vcam-1), and e-selectin. this occurs through up-regulation of the genes by nuclear factor κ-b (nf-κb), which is involved in nuclear transcription of several proatherosclerotic genes and may facilitate leukocyte migration by stimulating the release of monocyte chemotactic protein-1 (mcp-1). in addition, nf-κb also upregulates angiotensin type-1 receptor (at-1) to stimulate the migration and proliferation of vascular smooth muscle cells, formation of neointima, and production of ros. crp also inhibits the survival and differentiation of bone marrow-derived endothelial progenitor cells and interferes with the maintenance of vascular integrity. another mechanism in which crp actively participates is in atheroma formation, i.e. facilitating the opsonization of ldl cholesterol by macrophages to form foam cells. furthermore, crp also activates complement, particularly c3, which is found in atherosclerotic lesions and is involved in initiation and progressivity of atherosclerotic plaques. c-reactive protein also induces the production of endothelin-1 as endogenous vasoconstrictor and mediator of endothelial dysfunction, activator of leukocytes and platelets and cellular proliferation. finally, crp stimulates the production of il-6 (interleukin 6) in blood vessels, which is very important since il-6 is involved in positive feedback for stimulating crp production in the liver.(26) a limitation of this study was that we did not measure other influencing components such as lipid profile and markers of endothelial dysfunction. further studies are necessary on the dominant factors affecting endothelial dysfunction, as an extension of the present study. the present study may viewed as preliminary research for the prediction of coronary stenosis events in the community. conclusions there was a moderate positive correlation of bv and crp with degree of coronary stenosis. increases in bv and crp levels will be accompanied by increased severity of coronary stenosis. indrianto, samsuria, kurniawan blood viscosity degree coronary stenosis 175 univ med vol. 34 no.3 conflict of interest the authors had no conflict interest with any companies in this study. all authors participated in data collection, study design, statistical analysis, and manuscript preparation. all authors gave final consent for the publication. acknowledgment we wish to express our gratitude to the staff and personnel of the outpatient and inpatient departments of the cardiac disease unit, dr. kariadi central hospital, semarang, who participated from the start of the study up to the preparation of this paper. references 1. world health organization. cardiovascular diseases. geneva: world health organization; 2015. 2. dinas kesehatan jawa tengah. profil kesehatan provinsi jawa tengah. dinas kesehatan. jawa tengah. available at: http://www.dinkes jatengprov.go.id/dokumen/profil/profile2004/ bab5.htm. accessed march 21, 2014. 3. suwaida ja, hamasaki s, higano st, et al. long-term follow-up of patients with mild coronary artery disease and endothelial dysfunction. circulation 2000;101:948-54. 4. ganz p, hsue py. endothelial dysfunction in coronary heart disease is more than a systemic process. eur heart j 2013;34:2025-7. 5. froom p. blood viscosity and the risk of death from coronary heart disease. eur heart j 2000; 21:513-4. 6. kesmarky g, feher g, koltai k, et al. viscosity, hemostasis, and inflammation in atheroscerotic heart diseases. clin hemorheol microcirc 2006; 35:67-73. 7. cecchi e, mannini l, abbate r. role of hyperviscosity in cardiovascular and microvascular diseases. g ital nefrol 2009;26: 20-9. 8. lee bk, durairaj a, mehra a, et al. hemorheological abnormalities in stable angina and acute coronary 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sensitivity c-reactive protein with presence and severity of coronary artery disease. pak j med sci 2013;29:1425-9. 16. ergun ck, gurel ei, ozeke o, et al. blood viscosity changes in slow coronary flow patients. clin hemorheol microcirc 2011;47:27-35. 17. ridker pm. high-sensitivity c-reactive protein potential adjunct for global risk assessment in the primary prevention of cardiovascular disease. circulation 2001;103:1813-8. 18. chatzizisis ys, coskun au, jonas m, et al. role of endothelial shear stress in the natural history of coronary atherosclerosis and vascular remodeling. jacc 2007;49:2379–93. 19. toth a, szukits s, varady e, et al. hemorheology parameters in coronary artery disease detected by multi-slice ct. korea-australia rheology j 2014;26:229-35. 20. hasnat ma, islam aemm, chowdhury aw, et al. high sensitive c-reactive protein (hs-crp) and its correlation with angiographic severity of patient with coronary artery disease (cad). j dhaka med coll 2010;19:91-7. 21. momiyama y, ohmori r, fayad za, et al. association between plasma c-reactive protein levels and the severities of coronary and aortic atherosclerosis. j atheroscler thromb 2010;17: 460-7. 22. piranfar ma. the correlation between highsensitivity c-reactive protein (hscrp) serum levels and severity of coronary atherosclerosis. int cardiovasc res j 2014;8:6-8. 23. masood a, jafar ss, akram z. serum high sensitivity c-reactive protein levels and the 176 severity of coronary atherosclerosis assessed by angiographic gensini score. j pak med assoc 2011;61:325-7. 24. lai cl, ji yr, liu xh, et al. relationship between coronary atherosclerosis plaque characteristics and high sensitivity c-reactive proteins, interleukin-6. chin med j 2011;124: 2452-6. 25. ciccone mm, scicchitano p, zito a, et al. correlation between inflammatory markers of atherosclerosis and carotid intima-media thickness in obstructive sleep apnea. molecules 2014;19:1651-62. 26. osman rl, allier pl, elgharib n, et al. critical appraisal of c-reactive protein throughout the spectrum of cardiovascular disease. vasc health risk manag 2006:2:221–37. 27. dawson b, trapp rg. basic & clinical biostatistics. 4th ed. new york: lange medical books/mcgraw-hill;2004. indrianto, samsuria, kurniawan blood viscosity degree coronary stenosis c:\users\universa medicina\docu 36 abstract universa medicina protective effect of ethanolic feronia elephantum correa fruit extract on high-fat diet induced steatohepatitis in rats maruni wiwin diarti1, khoviya yuwina selinada harmi2, and dwi nur ahsani3* background a high-fat diet can lead to hyperlipidemia which will end up as liver damage (steatohepatitis). ethanolic feronia elephantum correa fruit extract (efec) has an antioxidant activity which is expected to overcome hyperlipidemia in the liver. the objective of this study was to determine the effects of efec on liver function and morphological changes in rats. methods this was an experimental study with a post-test only group design. a total of 20 male wistar rats aged 2-4 months were randomized into 5 groups, a= negative control, b= positive control (high fat diet + 10 mg/kgbw simvastatin), c = high fat diet + 500 mg/kgbw efec fruit extract, d = high fat diet + 600 mg/kgbw efec, and e = high fat diet + 700 mg/kgbw efec). high-fat diet was given for 4 weeks (quail egg yolks, 10ml/200gbw). efec was administered for 4 weeks after induction of hypercholesterolemia. examination of liver serum glutamic oxaloacetic transaminase (sgot) and serum glutamic pyruvic transaminase (sgpt) was performed on rat blood serum and histopathological examination was carried out using steatohepatitis grading. one way anova test and post-hoc tamhane’s test were used to analyze the data. results administration of efec at 700 mg/kgbw improved the liver enzymes (p=0.00 for sgpt and sgot) and steatohepatitis grading in high-fat diet induced rats (mild condition, e = 75% vs a = 100% mild) conclusion ethanolic feronia elephantum correa fruit extract at 700 mg/kgbw was able to improve steatohepatitis in high-fat diet induced rats. keywords: feronia elephantum correa, liver enzyme, steatohepatitis, highfat diet, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.36-44 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1055 january-april, 2021 vol.40no.1 1health analyst, poltekkes kemenkes mataram, indonesia 2undergraduate student, faculty of medicine, universitas islam indonesia 3department of histology, faculty of medicine, universitas islam indonesia correspondence: *dwi nur ahsani department of histology faculty of medicine, universitas islam indonesia jl. kaliurang km. 14.5 sleman yogyakarta. indonesia phone: 085229030982 email : dwi.nurahsani@uii.ac.id orcid id : 0000-0003-1344-3997 date of first submission, october 19, 2020 date of final revised submission, february 21, 2021 date of acceptance, february 24, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: diarti mw, harmi kys, ahsani dn. protective effect of ethanolic feronia elephantum correa fruit extract on high-fat diet induced steatohepatitis in rats. univ med 2021; 40:3 6 -4 4 . doi: 10 .18 051/ uni vmed. 2021.v40.36-44 37 univ med vol. 40 no 1 introduction a high-fat diet can induce hyperlipidemia and disrupt organ functions. in the liver, hyperlipidemia is frequently associated with nonalcoholic fatty liver disease (nafld),(1–4) which is histologically characterized by features of steatosis (lipid accumulation in hepatocytes), inflammation, and cell degeneration.(5) this disorder is characterized by elevated liver enzyme levels, including highe r serum gl utamic oxaloacetic transaminase/sgot (also known as alanine aminotransferase/alt), serum glutamic pyruvic transaminase/sgpt (also known as aspartate aminotransferase/ast), and alkaline phosphatase (alp), as well as increased triglyceride and cholesterol levels.(6,7) higher levels of hepatic enzymes and progressive morphological changes in the liver can lead to irreversible liver disease, such as fibrosis and hepatic cirrhosis.(5,8) statins a re dr ugs used for hypercholesterolemia that reduce steatohepatitis, but not liver fibrosis.(9) the use of statin therapy for hypercholesterolemia is also inseparable from its side effects. statins can cause muscle-related problems, such as myopa thy (including cardiomyopathy) and rhabdomyolysis, as well as damage to the kidneys and liver. hepatic disorders caused by statins can be indicated by elevated levels of transaminase enzymes (sgot and sgpt).(10) the side effects of statins can incr ease the risk of progression of such comorbidities as hepatic disorders in patients who also experience hyperlipidemia. exogenous antioxidant supplements emerge as an alternative therapy to substitute statins as standard drugs for hyperlipidemia, particularly in hyperlipidemia with coexisting liver disease. consumption of a diet containing antioxidants can reduce the risk of nafld.(11) many studies have examined the presence of antioxidants in plants, one of which is related to their effect on the liver.(12) feronia elephantum correa (also known as kawista; wood apple; limonia acidissima linn; feronia lemonia swingl e) a nd subsequently in this article referred to as fec, are a potential herbal medication for conditions that are high in reactive oxygen species (ros). an in-vitro study found that methanolic feronia elephantum correa fruit extract (mfec) can also reduce the proliferation of breast cancer cells.(13) fec fruit at doses of 500 mg/kgbw, 600 mg/ kgbw, and 700 mg/kgbw dissolved in 1 ml distilled water (for each administration) can prevent increased mda levels in the blood serum of rats exposed to cigarette smoke.(14) with regard to hyperlipidemia, fec is able to improve choleste rol profiles in animal models of hyperlipidemia, such as triton wr-1339 induced hyperlipidemia in rats.(15) in addition, fec is also able to improve carbohydrate metabolism in the liver (by improving the activity of the hepatic enzyme glucose-6phosphatase and increasing liver glycogen). the antihyperlipidemic effects of fec can be explained with different mechanisms. saponins and phytosterols can inhibit pancreatic lipase and reduce intestinal absorption of lipids. fibers of the extract can lower ldl level. in addition, flavonoids and ascorbate increase hdl and decrease ldl.(16) the studies of pandit et al.(15) and vasant et al.(16) have demonstrated the antihyperlipidemic effect of methanolic fec fruit extract and fec fruit powder, respectively. in contrast, the present study did not aim to examine the effect of fec on lipid profiles in hyperlipidemic conditions, but rather its effect on the liver. the objective of this study was to determine the effects of efec fruit extract on liver function and morphological changes in rats. methods research design this research involved an experimental design with a posttest-only control group approach. the experiment lasted for four months (january-april 2020) and was conducted in several laboratories. the preparation of efec 38 fruit extract, animal care, group treatment, and termination of experimental animals were carried out in the pharmacology laboratory, faculty of medicine, universitas mataram. the o bs e r va t i on a n d ima ge r e tr i e va l ( ti s s ue micrographs) were done in the laboratory of histology and anatomical pathology, faculty of medicine, universitas islam indonesia. collection of plant material and extraction ripe kawista (fec) fruits were obtained from bima regency, west nusa tenggara (ntb), indonesia. to prepare the extract, the fruit pulp was first dried under the sun and crushed to obtain a coarse powder, which was then sieved to yield the finest particles. the extraction process used maceration in 70% ethanol (1:3) at room temperature and was repeated for 3 da ys. the viscous extr act obtained after evaporation was stored in a closed container at room temperature and given to the experimental animals as per predetermined doses. animals and treatment t he r a t s we r e ob t a i ne d f r om t he ph a r ma c o logy la bo r a t o r y, u ni ve r s i t a s mataram. the inclusion criteria were male wistar rats aged 2-4 months, weighing 150-200 grams, and apparently healthy without any physical disabilities. a total of 20 rats (4 per group) were used according to the festing research equation.(17) based on this formula, the minimum total number of subjects required in this study was 15-25 (3-5 in each group). therefore, this study used four animals per group. the research subjects were selected through a random sampling technique. the rats were randomized into five research groups in this study, namely a = healthy controls (regular feed), b = positive controls (high-fat diet + 10 mg/kg bw simvastatin), c = high-fat diet + 500 mg/kgbw efec fruit extract, d = high-fat diet + 600 mg/kgbw efec fruit extract, and e = high-fat diet + 700 mg/kgbw efec fruit extract. efec fruit extract was administered for 4 weeks as soon as the rats were confirmed to have hypercholesterolemia. the efec fruit extract was dissolved in 0.5% carboxymethyl cellulose (cmc). the high-fat diet and therapy (simvastatin or efec fruit extract) were given via a nasogastric tube, and the rats received feed and water ad libitum during the study. the three different intervention groups c to e were each treated with a different dose of efec fruit extract (500 mg /kgbw= c, 600 mg/kgbw= d and 700 mg/kgbw= e). all treatments were performed by the researchers. high fat diet induced hyperlipidemia a high-fat diet was provided by feeding 10 ml/200 g bw quail egg yolk for 4 weeks. the intervention involving efec fruit extract was p r o vi d e d i f t he r a t s e x p er i e n c e d a hypercholesterolemic condition which was confirmed if the total cholesterol level was >200 mg/dl (using the easy-touch® rapid test on peripheral/tail blood). if the rats showed no hypercholesterolemia after four weeks of induction through feeding on quail egg yolk, they were excluded from this study. steatohepatitis grading the livers obtained after termination (after 4 weeks of hypercholesterolemia induction and 4 weeks of fec therapy) were stored in a 10% formalin phosphate-buffered saline fixative for 24 hours. the liver tissues were then placed in 70% ethanol for further processing. the liver samples were transversely sliced, blocked in paraffin, and stained with hematoxylin eosin (he). all of the liver tissue processing was p e r f o r me d i n t he an a t omi c a l pa t ho l o gy laboratory, faculty of medicine, universitas mataram (unram). the histological changes were observed wi t h t h e br u nt sys t e m ( gr a d i ng of steatohepatitis). according to this system, changes in the liver are categorized into mild, modera te, and se vere. (1 8) t he parame ters observed in these three categories include the degree of steatosis, ballooning cells, and diarti, harmi, ahsani feronia elephantum correa and steatohepatitis 39 univ med vol. 40 no 1 a b c inflammation. liver damage is deemed mild if steatosis (to a maximum of 2/3 of the liver) and ballooning cells appear to be minimal, and inflammation seems diffuse. the moderate degree is marked by visible steatosis and ballooning cells (in zone 3, i.e. the area of hepatic lobules near the central vein), and inflammation appears as a result of ballooning cells. severe liver damage is signified by a high degree of steatosis (>2/3), and ballooning cells are clearly visible in zone 3. the histological observation was performed wit h si ngl e bl in ding by a n ex per ie nc ed , independent anatomical pathologist from the labor atory of histology and anatomical pathology, faculty of medicine, universitas islam indonesia, using a light microscope. imaging was conducted using a light microscope wi t h a c ame r a ( op t i la b ) a t ta c h e d a n d connected to a computer. observations were made in all fields of view on each sample. a low magnification (100x) was used when observing the location of the damage, while high magnification (400x) was employed to observe morphological changes in detail. laboratory analysis the liver protection of high-fat diet induced rats was observed from the liver enzyme (sgot and sgpt) levels, which were examined from the rat blood serum obtained during termination. the blood samples were collected via retroorbital blood sampling. both examinations were carried out in the pharmacology laboratory, faculty of medicine, universitas mataram using spectrophotometric analysis. statistical analysis the histological changes in the liver were stated as a percentage (the number of positive samples compared to the total samples). the levels of liver enzymes were analyzed using the shapiro-wilk normality test, one way analysis of variance/anova (significance test), and tamhane’s t2 test (post-hoc test). the data were declared as significantly different if p <0.05. ethical clearance the ethical research permit was obtained from the ethics committee, faculty of medicine, universitas mataram prior to the study (number 354/un18.f7/etik/2019). results all of the rats survived until the end of the study and met the inclusion and exclusion criteria. from the examination of liver tissue, no significant histopathological changes were found in the liver parenchyma of the normal healthy (negative control) group (a). however, changes in the liver were observed in the positive control group (b = 50% severe) and the groups c – e receiving efec fruit extract. therapy using 700mg/kgbw efec fruit extract showed the ability to reduce the steatohepatitis induced by a high-fat diet (e = 75% mild, table 1). figure 1. histopathological changes in liver structure. a: mild grade, b: moderate grade, c: severe grade. yellow arrows: cell steatosis, red arrows: lymphocytes, blue arrow: ballooning cell (400x magnification, he staining). 40 there were significant differences in the levels of sgot and sgpt from the five study groups (p<0.001). the administration of efec fruit extract at 500 mg/kgbw (c), 600 mg/kgbw (d), and 700mg/kgbw (e) resulted in reduced sgot and sgpt levels compared to the group receiving standard therapy (table 2). in the posthoc tamhane’s test, significant differences in sgpt levels were seen in all of the treatment groups with efec fruit extract therapy as opposed to the positive control group (p<0.001, table 3). similar results were found in the sgot levels with the p values for sgot levels in groups c, d, and e reaching 0.008, 0.000, and 0.002, respectively (table 3). there was no significant difference in the sgpt levels between the group receiving 700 mg/kgbw efec fruit extract and the normal healthy group a (p=0.535). similarly, the sgot levels of the groups given 500 mg/kgbw and 600 mg/kgbw efec fruit extract did not seem significantly different from group a. meanwhile, group n grade n % a 4 mild 4 100 moderate 0 0 severe 0 0 b 4 mild 2 50 moderate 0 0 severe 2 50 c 4 mild 0 0 moderate 2 50 severe 2 50 d 4 mild 0 0 moderate 1 25 severe 3 75 e 4 mild 3 75 severe 0 0 moderate 1 25 note: a = healthy control (normal feed, no therapy), b = positive control (high-fat diet + 10 mg/kgbw simvastatin), c = high-fat diet + 500 mg/kgbw efec fruit extract, d = high-fat diet + 600 mg/kgbw efec fruit extract, e = highfat diet + 700 mg/kgbw efec fruit extract). #percentage obtained from the ratio of n to n table 1. grading system for steatohepatitis in the study groups variable group n mean ± sd p-value sgpt a 4 57.25 ± 3.04 0.00* b 4 122.05 ± 9.52 c 4 92.90 ± 4.96 d 4 85.0 ± 3.71 e 4 48.44 ± 6.19 sgot a 4 159.85 ± 14.8 0.00* b 4 221.3 ± 13.2 c 4 158.1 ± 5.85 d 4 120.3 ± 10.6 e 4 77.62 ± 2.1 table 2. levels of sgot and sgpt by treatment groups note: * p<0.05. a = healthy control (normal feed, no therapy), b = positive control (high-fat diet + 10 mg/kgbw simvastatin), c = high-fat diet + 500 mg/kgbw efec fruit extract, d = high-fat diet + 600 mg/kgbw efec fruit extract, e = high-fat diet + 700 mg/kgbw efec fruit extract) the sgot levels of rats in the group with 700 mg/kgbw efec fruit extract were lower than those in group a (p = 0.013) (table 3). discussion this study showed that the administration of efec fruit extract can improve the functional and morphological features of a high-fat diet induced steatohepatitis in rats. compared with the group receiving simvastatin therapy, the efec fruit extract therapy at doses of 500 mg/ kgbw and 600 mg/kgbw was able to improve only the features of liver function without improving the hepatic morphological changes. the therapeutic effect of efec fruit extract can be found at 700 mg/kgbw (sgot and sgpt levels and steatohepatitis grades were similar to those of the normal healthy group). the protective effect of fec extract can be observed in a number of induced tissuedamage models. in animal models of skin wounds (incision, excision, and dead space), the administration of methanolic fec fruit extract (mfec) at doses of 200mg/kgbw and 400mg/ kgbw can accelerate wound healing. the potential dose in that study is 400 mg/kgbw.(19) diarti, harmi, ahsani feronia elephantum correa and steatohepatitis 41 univ med vol. 40 no 1 although the prepared extract in that study is different from this present research (ethanol), the therapeutic effect of fec extract on tissue damage is similar to that of the present study. at a dose of 2000mg/kgbw, no side effects of fec treatment were found.(15,19) therefore, the dose of 700 mg/kgbw in this study remains potential as it is still far below the toxic dose. f. elephantum correa extract also has a protective effect in numerous tissue-damage mod e l s . m fe c f r u i t e xt r ac t ha s a n e ur op r o t ec t i ve e f f e c t on s t ro ke a n i ma l mod e l s. ( 2 0 ) re se a r c h b y sh a rma e t a l . ( 2 1 ) showed that premedication using aqueous f. elephantum correa (aqfec) leaf extract at doses of 400 mg/kgbw and 800 mg/kgbw can reduce the percentage of deaths and prevent the increase in weight and volume of rat liver induced by alloxan (induction of type-1 dm) and thioacetamide (a hepatotoxic substance). with regard to the functional parameters (liver enzymes), premedication involving aqfec leaf extract can also prevent elevated hepatic enzymes (sgot and sgpt).(21) similarly, the administration of efec fruit extract at doses of 500mg/kgbw, 600mg/kgbw, and 700mg/kgbw in the present study can improve the features of hepatic function (sgot and sgpt) compared to the group receiving a standard drug. ad mini ster ing fec ext rac t ca n al so improve the morphological features of the liver. aqueous f. elephantum correa leaf extract has t he po t e nt ia l t o r e du c e t he de gr e e of hepatocellular necrosis in alloxan-thioacetamidei nd u c e d ra t s ( mi l d i nj u r y) . ho we ve r, premedication using aqfec leaf extract is unable to make a significant difference to the degree of inflammation, congestion (lobular as we ll a s va sc ul a r ) , a n d he pa t o c yte regeneration.(21) the increasing protective effect o f fec c or r e sp o nd s to t he hi gh e r d o se administered. in line with the studies by sharma et al.(21), the present study also showed that administration of efec fruit extract at a dose of 700 mg/kgbw results in hepatic features that are close to the normal liver architecture (75% mild). the presence of moderate damage of up to 25% in the high-fat diet-induced group treated with efec fruit extract at a dose of 700 mg/ kgbw indicates that the ability of the liver to regenerate at such dose remains imperfect. meanwhile, the liver regeneration ability has yet to be shown at lower doses (500 mg/kgbw and 600 mg/kgbw). the varied results of liver enzyme and steatohepatitis grading improvement in the present study indicates that the effect of efec fruit extract is dose-dependent. efec fruit extract therapy at doses of 500mg/kgbw and 600mg/kgbw is only able to improve the liver e nzyme le ve l s al o ne wi t hout si gni fi c an t improvement in the steatohepatitis grading. liver en zyme s an d st ea tohe pa ti ti s gr ad in g ar e concurrently observed at a dose of 700mg/ kgbw. in line with the therapeutic and protective dose of fec fruit extract, the higher the dose group p-value a b c d e sgpt a 0.000* 0.000* 0.000* 0.535 b 0.000* 0.000* 0.000* c 0.798 0.000* d 0.000* sgot a 0.008* 1.00 0.060 0.013* b 0.008* 0.000* 0.002* c 0.019* 0.000* d 0.032* table 3. post-hoc test of liver enzyme levels in rat blood serum note: post hoc test using tamhane’s t2 test; a = healthy control (normal feed, no therapy), b = positive control (high-fat diet + 10 mg/kgbw simvastatin), c = high-fat diet + 500 mg/kgbw efec fruit extract, d = high-fat diet + 600 mg/kgbw efec fruit extract, e = high-fat diet + 700 mg/kgbw efec fruit extract) 42 used, the more significant the effects that can b e o bs e r ved . in l i n e w it h t ha t , a mo r e considerable improvement in steatohepatitis condition in this study can be found at a higher dose (700 mg/kgbw efec fruit extract). in addition, fec fruit extract also has a therapeutic effect on metabolic disorders. administration of mfec fruit extract can reduce the serum hyperlipidemia parameters (in hyperlipidemia animal models).(15) with regard to such findings, the therapeutic effect on liver enzymes and steatohepatitis grading in this study is made possible by the ability of fec fruit extract to reduce cholesterol levels. high ros levels will induce oxidative stress conditions which will damage the cell membrane and the dna.(22) the liver is the main target of high ros levels which induce changes in cell metabolism. the increased metabolism of ethanol by hepatocytes in oxidative stress conditions will result in steatosis. in addition, ac ti va te d ku pff er c el ls will a ls o induce inf lammation, apoptosis (thr ough t nf-α induction) and fibrosis (occurrence of collagen p r o du c t i on) . in f l a mma t i o n w ill f ur th e r exacerbate oxidative stress conditions.(23) reducing the ros levels is one of the potential strategies to prevent worsening of steatosis conditions in oxidative stress. steatosis is characterized by an increase in cytochrome p4 50 fa mil y 2 su bf a mi l y e m e mbe r 1 (cyp2e1).(24) studies show that flavonoids can suppress inflammation (reduce tnf-α), and reduce liver enzyme and mda levels and also reduce cyp2e1. this shows that flavonoids have an antioxidant activity and are also h e pa to p r ot e c t ive i n o xi d a t ive s t r e s s conditions.(25) the fec used in this study also contain flavonoids, alkaloids and phytosterols.(15) in line with xiang et al.(25) it seems that the flavonoids in fec play an important role in preventing the worsening of steatosis in rats with oxidative stress induced by high fat diet. the protective effects of efec fruit extract in this study are strongly associated with the antioxidant content. mfec fruit extract has proved to be able to increase the level of antioxidants (sod and catalase/cat) in the granulation tissue that forms during incisions, excisions, or dead space wounds in animal models. (19) reduced nitrite and lipid peroxidation levels as well as elevated catalase levels can also be observed in stroke animal models.(20) a study by darsini et al (26) shows that long-term fec supplementation (30 and 60 days in fish) results in increased levels of antioxidants (sod, glutathione s-transferase/gst, and glutathione peroxidase/gpx) in the blood serum as well as in liver tissue and muscle. the highest sod level is found in the liver. with fec administration, gpx shows activities in the liver similar to those in the blood serum. in contrast, gst shows the lowest activity in the liver as opposed to the blood serum and muscle. meanwhile, the mda free ra dica l l eve ls i n hepa tic tis sue dec re a se s i gn i f i c an t ly a f t e r 60 d ays of fe c supplementation. these studies suggest that the activation of antioxidant pathways is associated with the type of damage models and the kind of organs affected. in line with darsini et al.,(26) the protective effect of efec fruit extract in the present study is likely to occur through increased antioxidant activities of sod and gpx in liver tissue. however, the present study has some limitations in that the researchers did not involve a fec negative control group (high-fat dietinduced, unhealthy group). in addition, the researchers did not further investigate the antioxidant content in efec fruit extract and the response of the observed tissue to ros. the researchers did not reexamine as well the total c h ol e s t e r ol l e ve l s i n t h e r a t s a f t e r t he administration of efec fruit extract, thereby leaving unexplained the correlations among the changes in cholesterol levels, antioxidant levels, and ros as well as changes in liver morphology and function. despite such limitations, this study has proved that the hepatic functional and morphological changes in the group induced by a high-fat diet and given efec fruit extract at a therapeutic dose of 700mg/kgbw are nearly the diarti, harmi, ahsani feronia elephantum correa and steatohepatitis 43 univ med vol. 40 no 1 same as in normal liver. further research is recommended to determine the ranges of therapeutic and toxic doses as well as the mechanism of action of efec fruit extract in hyperlipidemia. conclusion efec fruit extract at 700 mg/kgbw was able to improve steatohepatitis in high-fat diet induced rats. conflict of interest the authors declare no conflicts of interest. acknowledgement part of this study was funded by a 2019 dipa re s ea r c h gr a nt f r om po l te kke s kemenkes mataram. contributors dna interpreted the data and prepared the manuscript for publication, mwd and kys collected the data, conducted the study and prepared the manuscript. all of the authors have read and approved this manuscript. references 1. ferramosca a, di giacomo m, zara v. antioxidant dietary approach in treatment of fatty liver: new insights and updates. world j gastroenterol 2017;23:4146–57. doi: 10.3748/wjg.v23.i23.4146. 2. bril f, 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antioxidants in alcoholic liver disease. med phoenix 2018;3:75– 88. 23. li s, tan hy, wang n, et al. the role of oxidative stress and antioxidants in liver diseases. int j mol sci 2015;16:26087–124. doi: 10.3390/ ijms161125942. 24. appak-baskoy s, cengiz m, teksoy o, ayhanci a. dietary antioxidants in experimental models of liver diseases. in: strawberry preand postharvest management tech high fruit quality. boston: intechopen; 2019;pp.1–22. doi: 10.5772/ intechopen.83485. 25. xiang c, teng y, yao c, et al. antioxidant properties of flavonoid derivatives and their hepatoprotective effects on ccl4 induced acute liver injury in mice. rsc adv 2018;8:15366–71. 26. darsini dtp, maheshu v, srinivasan p, nishaa s, castro j. dietary supplementation of limonia acidissima l. fruit on in vivo antioxidant activity and lipid peroxidation of cyprinus carpio l. ipcbee 2013;57:73–9. diarti, harmi, ahsani feronia elephantum correa and steatohepatitis restu 91 *anatomy department, faculty of medicine, yarsi university **physiology department, faculty of medicine, yarsi university ***biochemistry departement, faculty of medicine, yarsi university correspondence dr. indra kusuma, m.biomed physiology department, faculty of medicine, yarsi university cempaka putih, jakarta, 10510 6221-4206674 ext. 3091 email: indra.kusuma@yarsi.ac.id univ med 2014;33:91-9 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background transplanted allogeneic dermal fibroblasts retain stem cell subpopulations, and are easily isolated, expanded and stored using standard techniques. their potential for regenerative therapy of chronic wounds should be evaluated. the aim of this study was to determine allogeneic fibroblast viability in the presence of peripheral blood mononuclear cells (pbmc). methods in this experimental study, fibroblasts were isolated from foreskin explants, expanded in the presence of serum, and stored using slow-freezing. we used one intervention group of allogeneic fibroblasts co-cultured with pbmc and 2 control groups of separate fibroblast and pbmc cultures.fibroblasts were characterized by their collagen secretion and octamer-binding transcription factor 4 (oct4) expression. viability was evaluated using water soluble tetrazolium-1 (wst-1) proliferation assay. absorbances were measured at 450 nm. data analysis was performed by student’s paired t-test. results dermal fibroblasts were shown to secrete collagen, express oct4, be recoverable after cryopreservation, and become attached to the culture dish in a co-culture with pbmc. co-cultured and control fibroblasts had no significantly different cell viabilities (p>0.05). calculated viable cell numbers increased 1.8 and 5.1fold, respectively, at days 2 and 4 in vitro. both groups showed comparable doubling times at days 2 and 4 in vitro. pbmc did not interfere with allogeneic fibroblast viability and proliferative capacity conclusions allogeneic fibroblasts remain viable and proliferate in the presence of host pbmc. future research should evaluate allogeneic human dermal fibroblast competency in clinical settings. dermal fibroblasts are a potential source for cell therapy in chronic wound management. key words: fibroblast, pbmc, co-culture, foreskin allogeneic human dermal fibroblasts are viable in peripheral blood mononuclear co-culture restu syamsul hadi*, indra kusuma**, and yurika sandra*** 92 hadi, kusuma, sandra allogeneic human dermal fibroblasts fibroblas dermis manusia alogenik viabel dalam sistem ko-kultur bersama sel mononuklear darah tepi latar belakang dermis pada kulit memiliki subpopulasi sel punca. fibroblas asal dermis dapat diperoleh, diperbanyak, dan disimpan dengan mudah. potensi fibroblas sebagai salah satu alternatif strategi regeneratif untuk penyakit luka kronik perlu dievaluasi lebih lanjut. studi ini bertujuan untuk menentukan viabilitas fibroblas asal dermis yang di kultur bersama dengan sel mononuklear asal darah tepi individu yang berbeda. metode studi ini menggunakan desain eksperimental. fibroblas diisolasi menggunakan teknik eksplan. kultur perbanyakan menggunakan serum serta disimpan dengan metode slow-freezing. studi ini menggunakan satu kelompok perlakuan berupa ko-kultur antara fibroblas dan sel mononuklear darah tepi dan dua kelompok kontrol terdiri atas kultur tersendiri fibroblas dan sel mononuklear darah tepi. fibroblas dikarakterisasi dengan kemampuan sekresi kolagen dan ekspresi octamer-binding transcription factor 4 (oct4). viabilitas dievaluasi menggunakan uji proliferasi kolorimetrik water soluble tetrazolium-1 (wst-1). absorbansi diukur pada panjang gelombang 450 nm. analisis data dilakukan menggunakan paired student t-test. hasil fibroblas mensekresikan kolagen, mengekspresikan oct4 serta dapat dipulihkan dari kriopreservasi. tidak ada perbedaan signifikan viabilitas antar kelompok kontrol dan kelompok perlakuan (p>0,05). jumlah sel mengalami peningkatan 1,8 dan 5,1 kali lipat pada hari kedua dan keempat eksperimen. kedua kelompok memperlihatkan doubling time yang sebanding pada hari kedua dan keempat in vitro. sel mononuklear darah tepi tidak mengganggu viabilitas dan proliferasi fibroblas dari individu yang berbeda. kesimpulan fibroblas dapat mempertahankan viabilitas dan proliferasinya dalam ko-kultur bersama sel mononuklear darah tepi dari individu yang berbeda. penelitian selanjutnya dapat menilai kompetensi fibroblas pada situasi klinis. fibroblas asal dermis adalah sumber potensial untuk terapi sel pada luka kronis. kata kunci: fibroblas, pbmc, ko-kultur, preputium abstrak introduction recently, adult dermal fibroblasts were found to retain stem cells subpopulations. these cells expressed stage-specific embryonic antigen 3 (ssea-3) and octamer-binding transcription factor 4 (oct4), and co-expressed with cd105, a w e l l k n o w n m a r k e r f o r m a r r o w a n d adiposederived mesenchymal stem cells. human dermal fibroblasts (hdfs) can differentiate into epidermal melanocytes, neurons, adipocytes, hepatocytes and islet-like cell clusters.(1–3) this ability suggests that human dermal fibroblasts have multipotent stem cell subpopulations. human dermal fibroblasts are easily isolated from foreskin. the compulsory moslem practice of male circumcision in indonesia constitutes an abundant and accessible source of adult stem cells for cell therapy. human dermal stem cells secrete high levels of growth factors, such as basic fibroblast growth factor (bfgf), insulin-like growth factor-1 (igf-1), vascular 93 e n d o t h e l i a l g r o w t h f a c t o r ( v e g f ) , a n d keratinocyte growth factor (kgf), in addition to extracelullar proteins, such as collagen, which i s i m p o r t a n t i n d e r m a l a n d e p i d e r m a l regeneration.(3–5) potential applications of these cells include regenerative therapy for chronic wounds, such as diabetic foot, decubitus, and burns. diabetic foot is reported to cause one million amputations annually.(6) this report has led to an interest to develop novel rehabilitation efforts using regenerative technologies. r e g e n e r a t i v e s t r a t e g i e s f o r w o u n d treatment using cell therapy requires evaluation of cell functionality, storage ability, and compatibility, particularly in allogeneic settings. the risk of host tissue rejection needs to be evaluated before starting clinical trialson humans. tissue rejection is an immune reaction caused by cellular immunity in the form of skin graft-versus-host disease (gvhd), marked by increased c-x-c motif chemokine 10 (cxcl10) and langerhans cells as antigen presenting cells (apcs). receptors for cxcl10 are present in t cells,(7) and donor t cells presented to skin with intact langerhans cells will trigger gvhd. a recent study on mouse dermal fibroblasts found similarities in multipotent capacity between dermal fibroblasts and mesenchymal stem cells. further investigations revealed that allogeneic mouse dermal fibroblasts retain imunosupressive properties at days 3-6 in co-culture with mononuclear cells in a mixed lymphocyte r e a c t i o n ( m l r ) e x p e r i m e n t a l s e t u p . (8) however, another study using induced pluripotent stem cells (ipscs) derived from pigs showed that autologous major histocompatibility complex (mhc)-matched ipscs were still susceptible to innate immunity, natural killer (nk) cells, and serum complement cytotoxic reactions.(9) the present study was designed to evaluate the viability of allogeneic human dermal fibroblasts in the presence of blood mononuclear cells. this study did not use heat-inactivated fetal bovine serum that could inhibit serum complement reactions. methods research design the present study using an experimental design was conducted from april 2013 to march 2014 at the integrated laboratory of yarsi university. cell culture cell cultures and proliferation assays were carried out at the cell culture laboratory, yarsi university, jakarta. one healthy human foreskin sample (from an 8-year old boy) was obtained after a circumcision procedure conducted at the indonesia house of circumcision (ihc) in accordance with a manual provided by who, with informed consent given by the parents.(10) fibroblasts were cultured in a modification of minimal essential medium (α-mem, sigma) supplemented with fetal bovine serum (fbs, sigma) and glutamine (sigma). antibiotics and antimycotics used were penicillin-streptomycin (sigma) and amphotericin-b (sigma). the same medium was used for co-culture with peripheral blood mononuclear cells (pbmc). sample preparation t h e c o l l e c t e d f o r e s k i n s a m p l e w a s r e f r i g e r a t e d f o r 6 0 m i n u t e s b e f o r e decontamination using povidone iodine and 70% ethyl alcohol (etoh). the technique for separation of the dermis from the epidermis has been described elsewhere.(11) the skin sample was minced and placed in a 35-mm diameter culture dish with α -mem and incubated overnight at 37o c with 5% co2. the medium was increased to working volume the next day and supplemented with 20% fbs for expansion culture. fibroblast out growths appeared within 7 days (figure 1a) and were subcultured twice before being used for the experiment. excess cells were cryopreserved using a slow-freezing method. approximatelly one million viable cells were resuspended in cryomedium [dulbecco’s modified eagle’s medium (dmem) 20% fbs, univ med vol. 33 no.2 94 hadi, kusuma, sandra allogeneic human dermal fibroblasts fbs and dimethyl sulfoxide(dmso) in a 7:2:1 ratio], incubated overnight at -80 oc and transferred directly to liquid nitrogen cryofreezer the next morning.(12) fibroblast characterization cultured fibroblasts were characterized functionally by quantifying collagen 1 and 3 secretion. conditioned medium from cultured fibroblasts at 1-3 days in vitro were collected, centrifuged at 1500 rpm for 10 minutes, and the resulting supernatant used to quantify collagen 1 and 3 using col1a1 and col3a1 elisa kits (uscn). to find out whether the dermal fibroblasts retained a multipotent subpopulation, we calculated relative oct4 mrna expression between passages 4-6. a total of one million viable cells were collected for total rna isolation using high pure rna isolation kit (roche), quantified using a qubit fluorometer (invitrogen). real-time polymerase chain reaction (rt-pcr, roche) was performed using lightcycler rna master sybr green i (roche). forward and reverse primers for oct4 were 5'-aggtgttcagcc aaacgacc-3' and 5'-tgatcgtttgccc ttctggc-3', respectively. beta-actin was used as house keeping gene with the following primer sequences: forward 5’-agagctacga gctgcctgac-3’ and reverse 5’-agcac t g t g t t g g c g ta c a g 3 ’ . b o t h p r i m e r sequences had been confirmed to be specific using the nucleotide blast facility from ncbi. peripheral blood monocyte isolation the peripheral blood samples collected from one healthy human volunteer was treated with edta and cooled in a refrigerator for 60 minutes. pbmc isolation was conducted by centrifugation for 30 minutes at 2000 rpm using ficoll hypaque (1.077 g/dl; sigma) with a 1:1:1 ratio of blood, phosphate buffer saline (pbs, gibco) and ficoll.(12) the thin buffy coat layer visible between serum and ficoll layer was collected and washed three times with pbs at 1500 rpm for 10 minutes. co-culture the experiments were conducted on 96well culture plates. a total of 3 x 103 viable fibroblasts were seeded with 1.5 x 105 pbmc in 100 µl á-mem supplemented with 10% fbs per well. the experimental groups used in this study were the intervention group consisting of a co-culture of fibroblast and pbmc, and two control groups consisting of fibroblast control group and pbmc control group. this set up was arranged on two plates and repeated in triplicate per plate. both plate were incubated at 37o c with 5% co2. and assessed at two time point, one for each plate at 2 and 4 day in vitro, respectively. we used separate fibroblast and pbmc cultures, each in their own 35 mm culture dishes,seeded at the same density as used for the experiment. this separate setup was for evaluating fibroblast confluence and pbmc attachment in standard culture. mitochondrial activity was assessed by colorimetric method using water soluble tetrazolium-1 (wst-1) proliferation assay (roche) on a 96-well culture plate. a microplate reader with 450 nm filter was used to read the absorbance. data analysis the collected data were processed on an excel spreadsheet (version 2007; microsoft). blank absorbance values were subtracted from the original data. absorbance values were used to calculate viable cells with an equation derived from the linear chart shown in figure 1. calculated viable cell numbers were further analyzed to derive population doubling, doubling time, and simulated growth curve. oct4 expression data were presented as ratios, using data from passage 6 for comparison. statistical significance was determined using paired student’s t-test. results the separate dermal fibroblast control culture in the 35-mm culture dish at the same 95 figure 1a. outgrowth of primary human dermal fibroblasts and keratinocytes from skin explants seen as spindle-shaped and cobble-stone cells, respectivelly. div 6, 20x nikon (div = day in vitro) figure 1b. fibroblast confluence >80% ( div4, 20x nikon) figure 1c. plastic-attached, spindle-shaped, fibroblast-like cells from pbmcs known as mesenchymal progenitor cells (mpcs) or fibrocytes (div18, 10x axiocam; div = day in vitro) figure 1b. fibroblast confluence >80% ( div4, 20x nikon) univ med vol. 33 no.2 96 hadi, kusuma, sandra allogeneic human dermal fibroblasts s e e d d e n s i t y a s t h e c o c u l t u r e , r e a c h e d confluence in 4 days in vitro (figure 1b), while the pbmc control culture in their own 35 mm culture dish showed plastic-attached, spindleshaped cell colonies after 5 days in vitro (figure 1c). to evaluate the results we subtracted absorbance values of the co-culture group with those of the pbmc control group and compared the resulting data with the fibroblast control group. mitochondrial celullar dehydrogenase activity was used to quantify viable cells after incubation. this study did not reveal any significant differences in viable cell numbers between the co-culture group and the fibroblast control group (p>0.05) (figure 2). the numbers of viable cells in both coculture and fibroblast control groups after four days in vitro were correspondingly greater than the numbers of viable cells after two days in vitro. calculated viable cell numbers increased figure 2. no significant difference in calculated viable cell number after 2 days (div2) and 4 days (div4) of culture (p>0.05) (ratio of viable cells calculated from absorbance reading/initial seed number) 1.8 and 5.1 times, respectively, at two and four days in vitro. from the population doubling and doubling time values it was apparent that in the first two days of culture, the fibroblasts were in the lag phase (long doubling time, 56 hours) and entered the proliferative phase (shorter doubling time, 32 hours) in the next two days. however, the simulated growth curve derived from the doubling time differs with the actual cell numbers by more than 40% (table 1). these data reveal that co-culture of fibroblasts with pbmc did not reduce fibroblast proliferation capacity. the fibroblasts were confirmed to secrete collagen 1 and 3 into the culture media; collagen 1 secretion was however significantly lower than collagen 3 secretion (p<0.05) (figure 3). oct4 mrna expression levels show a steady decline from passage 4 to 6 (figure 4). cryopreserved fibroblasts were thawed after 2 days in liquid nitrogen and evaluated using the trypan-blue exclusion method. fibroblast hours actual co-culture actual fibroblasts simulated co-culture simulated fibroblasts 0 3 ,00 3,00 3,00 3,00 40 5 ,43 5,49 5,49 5,62 80 15,38 15,23 26,91 26,04 table 1. comparison of cell numbers between simulated cell numbers derived from fibroblast doubling time and actual cell numbers calculated using linear wst-1 curve 97 recovery was over 90%; however, we recorded a loss of about 40% of the initial cell population under going cryopreservation. discussion our findings reveal that co-culture of allogeneic fibroblasts with pbmc is feasible. a mixed lymphocyte reaction study will show different results, because in that case both fibroblasts and pbmc will contain immunereactive cells, such as t lymphocytes and apcs. classic study of epidermal cells in a mixed skin cell lymphocyte reaction study (mlsr) requires the presence of langerhans cell as apcs to stimulate the blood mononuclear cells. our study results indicate that the fibroblast culture was not contaminated by langerhans cells or t lymphocytes, both of them able to act as apcs. the results also show no adverse effects from innate immunity, nk cell activity, and serum complement cytotoxic reactions. the fibroblasts used in this study were s u b c u l t u r e d t w i c e b e f o r e e n t e r i n g t h e experiment. the cells were considered to be functional because they were able to secrete collagen 1 and 3. the different amounts of collagen secreted by both types is considered figure 3. secreted collagen 1 and 3 on div1, div2 and div3, significantly different (p<0.05); highest difference at div2 (div: day invitro) figure 4. oct4 mrna relative expression level compared between passages, 1.0e+06 viable cells used for total mrna extraction univ med vol. 33 no.2 98 hadi, kusuma, sandra allogeneic human dermal fibroblasts acceptable because collagen 3 is secreted in larger amounts by younger children.(13) passagetwo fibroblasts were used because further passaging showed significantly lower oct4 expression levels. this finding suggested a down regulation of oct4 which could be attributed to spontaneous differentiation, frequently occurring in stem cells cultured in the presence of serum. passaging also sufficiently removed potential contaminants from the cell suspension, such as epidermal langerhans cells and t-cells, which are common contaminants in fibroblasts isolated from skin explants. the actual fibroblast growth curve in our study shows that both control and intervention fibroblasts remained for the same amount of time in the lag phase and entered the proliferative phase at about the same time. the difference between simulated and actual growth curve may be explained by assuming that space restriction in the culture wells initiated contact inhibition, thus lowering the cell numbers observed. the results indicate that after allogeneic transplantation, the fibroblasts should be able to integrate with the host tissue. dermal substitute products such as transcytetm and dermagrafttm, that are derived from allogeneic living fibroblasts, are known to have been successfully applied empirically for various types of wound.(14,15) the r e s u l t s o f o u r e x p e r i m e n t p r o m i s e t h e achievement of similar results using locally collected sources of fibroblasts. our study should be followed up by other experiments, such as the generation of induced pluripotent stem cells from fibroblasts (16–18) or clinical trials that require large numbers of cells demanding a s t o r a g e s o l u t i o n . o u r s l o w f r e e z i n g cryopreservation method at low (10%) dmso concentration showed a high recovery rate (90%), but with a technical loss of 40% of the initial cell numbers. this study was limited by the lack of autologous co-culture because of difficulties in obtaining samples. the incubation period was restricted to 4 days, because on the 5th day pbmc subpopulations known as mesenchymal progenitor cells (mpcs) or fibrocytes exhibiting plastic-attachment ability, started to interfere with the fibroblast viability assessment.(19,20) this study demonstrated the potential application of locally collected foreskin-derived dermal fibroblasts for cell therapy, particularly the regenerative approach, for chronic wound h e a l i n g . s u c c e s s f u l i s o l a t i o n o f d e r m a l fibroblasts that retain multipotent stem cell subpopulations also offers patient-specific fibroblast populations for regenerative and reprogramming purposes. conclusions allogeneic human dermal fibroblasts retain their viability and proliferative capacity in pbmc co-culture system. future research should be directed to evaluate allogeneic human dermal fibroblast competency in clinical settings. research could also be directed to construct dermal-epidermal composites for the production of bilayered human skin equivalents that can be used in wound therapy, cosmetic toxicological protocols, and drug delivery studies. acknowledgements f u n d i n g w a s p r o v i d e d b y t h e r i s e t unggulan universitas 2013 grant scheme. we thank intan razari for laboratory asisstance, ratih rinendyaputri and prof. drh. arief b o e d i o n o , p h . d f o r c o n s u l t a t i o n o n cryopreservation methods, dr. linda julianti wijayadi, sp.kk for the elisa procedure, dr m a h d i a n n u r n a s u t i o n , s p . b s f o r t h e circumcision procedure and viko and wiza for their enthusiasm in research. references 1. li l, fukunaga-kalabis m, yu h, xu x, kong j, lee jt, et al. human dermal stem cells differentiate into functional epidermal melanocytes. j cell sci 2010;123:853–60. 99 2. bi d, chen fg, zhang wj, zhou gd, cui l, liu w, et al. differentiation of human multipotent dermal fibroblasts into islet-like cell clusters. bmc cell biol 2010;11:46. 3. crigler l, kazhanie a, yoon tj, zakhari j, anders j, taylor b, et al. isolation of a mesenchymal cell population from murine dermis that contains progenitors of multiple cell lineages. faseb j 2007;21:2050–63. 4. shim jh, park jy, lee mg, kang hh, lee tr, shin dw. human dermal stem/progenitor cell-derived conditioned medium ameliorates ultraviolet ainduced damage of normal human dermal fibroblasts. plos one 2013;8:e67604. 5. crespo av, awe jp, reijo pera r, byrne ja. human skin cells that express stage-specific embryonic antigen 3 associate with dermal tissue regeneration. bio res 2012;1:25–33. 6. bakker k, schaper n. the development of global consensus guidelines on the management and prevention of the diabetic foot 2011. diabetes met res rev 2012;28:116–8. 7. ding w, stohl ll, wagner ja, granstein rd. calcitonin gene-related peptide biases langerhans cells toward th2-type immunity. j immunol 2008;181:6020–6. 8. mukonoweshuro b, brown cj, fisher j, ingham e. immunogenicity of undifferentiated and differentiated allogeneic mouse mesenchymal stem cells. j tissue eng 2014;5:2041731414534255. doi: 10.1177/2041731414534255. 9. mizukami y, abe t, shibata h, makimura y, fujishiro sh, yanase k, et al. mhc-matched induced pluripotent stem cells can attenuate cellular and humoral immune responses but are still susceptible to innate immunity inpigs. plos one 2014;9:e98319. 10. hargreave t. male circumcision: towards a world health organisation normative practice in resource limited settings. asian j androl 2010;12: 628–38. 11. kusuma i, hadi rs. geraniin supplementation increases human keratinocyte proliferation in serum-free culture. univ med 2013;32:3-10. 12. djuwantono t, wirakusumah ff, achmad th, sandra f, halim d, faried a. a comparison of cryopreservation methods: slow-cooling vs. rapid-cooling based on cell viability, oxidative stress, apoptosis, and cd34+ enumeration of human umbilical cord blood mononucleated cells. bmc research notes 2011;4:371. 13. coolen na, schouten kc, middelkoop e, ulrich mm. comparison between human fetal and adult skin. arch dermatol res 2010;302:47–55. 14. pereira c, gold w, herndon d. review paper: burn coverage technologies: current concepts and future directions. j biomater applic 2007;22:101– 21. 15. auger f, lacroix d, germain l. skin substitutes and wound healing. skin pharmacol physiol 2009; 22:94–102. 16. pereyra-bonnet f, gimeno ml, argumedo nr, ielpi m, cardozo ja, gimenez, et al. skin fibroblasts from patients with type 1 diabetes (t1d) can be chemically transdifferentiated into insulin-expressing clusters: atransgene-free approach. plos one 2014;9:e100369. 17. simeonov kp, uppal h. direct reprogramming of human fibroblasts to hepatocyte-like cells by synthetic modified mrnas. plos one 2014;9: e100134. 18. durruthy-durruthy j, briggs sf, awe j, ramathal cy, kayumbaran s, lee pc, et al. rapid and efficient conversion of integration-free human induced pluripotent stem cells to gmp-grade culture conditions. plos one 2014;9:e94231. 19. curnow sj, fairclough m, schmutz c, kissane s, denniston ako, nash k, et al. distinct types of fibrocyte can differentiate from mononuclear cells in the presence and absence of serum. plos one 2010;5:e9730. 20. pilling d, vakil v, gomer rh. improved serum-free culture conditions for the differentiation of human and murine fibrocytes. j immunol methods 2009; 351:62–70. univ med vol. 33 no.2 may-august 2023 universa medicina vol.42 no.2 pissn: 1907-3062 / eissn: 2407-2230 research trends in brain imaging of mild cognitive impairment in 25 years: a bibliometric analysis daniella satyasari1* and agnes tineke waney rorong1 abstract mild cognitive impairment (mci) is a condition that is experienced by most elderly in the world. although there has been a huge rise in research on developing brain imaging tests that can identify and evaluate mci early on, a bibliometric analysis of this issue is still lacking. the purpose of this review is to determine the pattern and growth of research trends related to mci and brain imaging using bibliometric analysis, based on scopus data from 1996 to 2021. the data was converted to comma separated values (csv) and exported to vosviewer to bibliometrically analyze the origin by country, keywords, frequently cited articles, author, and journals. over a 25-year period, 5081 articles were discovered, with the number rising, particularly in the past four years, and significantly in 2022 when 561 articles (11.04%) were found. the journal of alzheimer's disease (19.22%) and neuroimage clinical (10.22%) published the largest number of articles on this subject. the united states (24.31%) led all other countries in the number of publications, followed by china (14.84%) and uk (6.5%). the most cited article was by petersen rc in 1999 (41 citations) about mci and its clinical characterization. the keywords that appeared the most frequently were mild cognitive impairment (984 occurrences) associated with biomarkers, brain scanning procedures, brain part, age, and human subject. the most frequently cited authors were petersen rc (1365 citations) and jack cr (1103 citations). neuroimage (4164 citations), and neurology (3268 citations) are the most repeatedly cited journals. this bibliometric study displays the trend in the last 25 years for mci and brain imaging. keywords: mild cognitive impairment, brain imaging, bibliometric analysis, vos viewer 1department of psychiatry, faculty of medicine, universitas trisakti, jakarta, indonesia *correspondence: daniella satyasari department of psychiatry, faculty of medicine universitas trisakti, jl. kyai tapa no.1, jakarta, indonesia 11440 email: daniella.satyasari@trisakti.ac.id orcid id: 0000-0002-8415-3893 date of first submission, november 21, 2022 date of final revised submission, may 9, 2023 date of acceptance, may 29, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license review article doi: http://dx.doi.org/10.18051/univmed.2023.v42.xxxx copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1393 cite this article as: satyasari d, rorong atw. research trends in brain imaging of mild cognitive impairment in 25 years: a bibliometric analysis. univ med 2023;42: xxxxx. doi: 10.18051/univmed. 2023.v42.xxxxx. mailto:daniella.satyasari@trisakti.ac.id https://orcid.org/0000-0002-8415-3893 https://orcid.org/0009-0009-7681-7440 http://dx.doi.org/10.18051/univmed.2023.v42.xxxx https://univmed.org/ejurnal/index.php/medicina/article/view/1393 univ med vol. 42 no. 2 introduction the proportion of the world’s elderly population will double from 12% to 22% by 2050 according to the world health organization (who). physical and mental abilities may gradually deteriorate as people age and there is also a reduction in cognitive function, manifested as mild cognitive impairment (mci).(1) mild cognitive impairment is defined as a neurological disorder in the elderly which is characterized by decreased cognitive functioning, especially in memory, but has no significant impact on daily life and has not met the criteria for dementia.(2) the prevalence rate of mci in the elderly population varies greatly in various studies around the world ranging from 3% to 42%.(3) based on indonesian national socio-economic survey (susenas) data in 2022, the proportion of the elderly population is 10.48%.(4) according to data from the directorate general of medical services of the indonesian ministry of health in 2010, the prevalence of mci in the elderly in indonesia was around 32.4%.(5) to both identify and protect patients from cognitive deterioration, it is necessary to examine biomarkers that can predict individuals who are predisposed to developing alzheimer’s disease (ad). various diagnostic and screening techniques are used to detect these biomarkers and evaluate mci, such as magnetic resonance imaging (mri), positron emission tomography (pet), and cerebrospinal fluid (csf) sampling.(6) brain imaging technique s such as mri are recommended by various guidelines in europe and the united states because these techniques can assess structural damage, such as in blood vessels, and changes in parts of the brain. the presence of biomarkers allows pre-symptomatic diagnosis and evaluation of disease progression.(7) however, to determine how well these screening techniques and biomarkers can predict the risk in mci individuals, further studies are needed in individuals with different levels of risk.(8) this review was conducted because of the rise in the incidence of mci and the need to prevent its potentially worsening effect in the elder ly. (6) t hr ough bibliometric a nalysis, researchers can understand the focus of current research, identify widely cited publications, and predict the direction of future research.(9) however, this approach is still lacking in the case of mci. therefore, the aim of this review is to conduct a bibliometric analysis of mci and brain imaging using scopus and vosviewer to ascertain the scientific framework (year of publication, articles, authors, countries, keywords, and journal). the findings of this bibliometric analysis are expected to provide an overview of the connections between the two topics and offer recommendations regarding ways to conduct examination for mci screening, diagnosis and evaluation. methods this bibliometr ic analysis has thre e methodological phases (figure 1), namely data collection (phase 1), data visualization (phase 2), and data analysis with data interpretation (phase 3).(10) data collection data collection in the first phase begins by using a search for journals according to keywords and research criteria. the search for bibliometric journals using basic data from the scopus database with keywords was carried out on april 13, 2023. the keywords used were mci, brain, imaging, and brain imaging. keyword intake was associated with boolean logical functions such as or and and which led to the following search topics: [title-abs-key (“mci”) and title-abskey (“brain” and “imaging” or “brain imaging”) and (exclude (pubyear, 2023))]. in phase 1, there are no set criteria for the year of publication to show the trend of these topics, but we omit 2023 because it is still in progress. the types of data are not limited to articles, but also include books, book chapters, reviews, and conference papers. based on these keywords, 5081 articles were obtained from 1996 to 2021. figure 1. the methodological phases and analytical criteria applied in this study.(10) data visualization in the second phase, the data found in scopus was subsequently converted to a comma separated value (csv) file using microsoft excel. the csv file was then extracted using the openrefine application so that the number of keywords in the author and index keywords section can be streamlined. after data conversion, the csv file was exported by vosviewer software for bibliometric analysis of countries, authors, journals, articles, and keywords.(10) in this study, the bibliometric method was used to visualize mci and brain imaging through vosviewer by means of bibliographic coupling (independent articles citing the same article), cocitation analysis (showing linked journals and authors from stored citations) and co-occurrence of keywords (showing frequently repeated author keyword maps).(11) data analysis in the third phase, bibliometric analysis was carried out using the performance analysis approach and scientific structure analysis. performance analysis is carried out by evaluating trends in year of publication, country, journal, number of articles and most frequent citations. scientific structure analysis uses scientific mapping of keywords, authors, most frequently cited articles, and co-citation networks of authors and journals. the results will be displayed in the form of interconnecte d circles. (1 2) da ta interpretation is done by looking at the relationship between circles, color, and size. the closer the distance between the two circles, the greater the strength of the relationship between the terms in these circles. in addition, the larger circle size is correlated with a higher frequency of occurrence of the terms represented. in the end, bibliometric analysis enables the achievement of our research objectives, namely the mapping of scientific structures, patterns and developments in research trends related to m ci themes and brain imaging.(13) ethics statement ethical approval is not required for this study because it is based on secondary data. results analysis of annual publications and trend it was found that there were 5081 articles that matched the criteria. figure 2 shows that articles on this topic just began to appear in 1996, with yearly increases and decreases until 2005. the increase in articles continues to occur every year from 2006, but there is a sharp increase in satyasari, rorong bibliometric analysis of mild cognitive impairment univ med vol. 42 no. 2 a b figure 2. development of articles on published study topics figure 3. visualization of country distribution analysis by year of publication the 4 years from 2019 until 2022, with 387 (7.61%), 442 (8.69%), 506 (9.95%), and 561 articles (11.04%), respectively. this indicates the increasing interest of researchers in this topic and the possibility of future advances in imaging examination capabilities. sources of scientific publications when viewed from the number of articles published annually on scopus, it was found that there were five journals with the highest numbers of articles. the journal of alzheimer’s disease publishes the largest number of articles on this topic with 94 articles (19.22%), followed by neuroimage: clinical with 50 articles (10.22%). the other three journals that publish articles on this topic are frontiers in aging neuroscience (9.61%), alzheimer’s and dementia (6.74%), and alzheimer’s research and therapy (5.72%). country contribution the more countries that contribute to creating articles and publishing them, the more likely it is to link knowledge on the topic. bibliographic coupling helps provide a consistent picture, as well as differences in research from different countries over different timescales.(13,14) using 5 as the minimal number of articles from a country, we found 34 countries that are divided into six clusters. a visualization of the country distribution analysis based on the year of publication regarding mci and brain imaging is shown in figure 3. the country with the largest number of publications in this category is the united states, with 452 articles (24.31%), followed by asia, with china having 276 articles (14.84%). the next highest contributors are the uk with 121 articles (6.5%), south korea with 105 articles (5.64%), table 1. the ten most frequently cited articles regarding mci and brain imaging.(15–24) rank author year article citations 1 petersen et al.(15) 1999 mild cognitive impairment: clinical characterization and outcome 41 2 petersen (16) 2004 mild cognitive impairment as a diagnostic entity 40 3 tzourio-mazoyer et al.(17) 2011 automated anatomical labeling of activations in spm using a macroscopic anatomical parcellation of the mni mri single-subject brain 25 4 albert et al.(18) 2011 the diagnosis of mild cognitive impairment due to alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease 23 5 fischl (19) 2012 freesurfer 15 6 rathore et al.(20) 2017 a review on neuroimaging-based classification studies and associated feature extraction methods for alzheimer’s disease and its prodromal stages 13 7 sperling et al.(21) 2011 toward defining the preclinical stages of alzheimer's disease: recommendations from the national institute on aging-alzheimer's association workgroups on diagnostic guidelines for alzheimer's disease 13 8 belleville et al.(22) 2014 predicting decline in mild cognitive impairment: a prospective cognitive study 11 9 dubois et al.(23) 2014 advancing research diagnostic criteria for alzheimer's disease: the iwg-2 criteria 9 10 besson et al.(24) 2015 cognitive and brain profiles associated with current neuroimaging biomarkers of preclinical alzheimer’s disease 9 and canada with 102 articles (5.48%). there are also asian and european countries in this distribution. most studies on this subject were conducted between 2017 and 2020. articles from the united states and uk are from 2018 (bluish green). those from europe are largely from 2017 to 2019 (purple, bluish green and green), and those from china and other asian nations are the newest ones, from 2020 (yellow). most frequently cited articles the search for the most frequently cited articles aims to see the importance of an article and its discussion.(14) the analysis was carried out by selecting 5 as the minimum number of citations, with a total of 201 articles being found. the results showing the most frequently cited articles can be seen in table 1. the 10 most frequently cited articles are the following, sorted in descending order. the first 2 are both on mci, both were authored by petersen et al.(15,16) and were published in 1999 and 2004, with 41 and 40 citations, respectively. the next 2 articles with respectively 25 and 21 citations are by tzourio-mazoyer et al.(17) and by albert et al.,(18) both published in 2011. then come 3 articles, the first of which is by fischl (19) about freesurfer and published in 2012, with 15 citations, while the next 2 articles are about ad, authored by rathore et al.(20) and sperling et al.,(21) and published in 2017 and 2011, respectively, both with 13 citations. the next article with 11 citations is by belleville et al.(22) about mci and published in 2014. the last 2 articles both have ad as topic and are by dubois et al.(23) and besson et al.,(24) both having 9 citations. co-occurrences map by author keyword keyword analysis can provide an overview of the concepts that most frequently arise from the research area under investigation.(25) with a minimum of 29 keywords that appear in each satyasari, rorong bibliometric analysis of mild cognitive impairment univ med vol. 42 no. 2 figure 4. visualization of keywords used from year to year in scopus indexed journals article, out of 7,468 keywords, 236 were found. in figure 4, these keywords are mapped by frequency of occurrence. keywords that appeared the most frequently wer e mild cognitive impairment (984 occurrences), male (822 occurrences), female (815 occurrences), aged (789 occurrences), cognitive defect (704 occurrences), nuclear magnetic resonance imaging (731 occurrences), cognitive dysfunction (579 occurr ences), magnetic resonance imaging (547 occurrences), neuroimaging (547 occurrences), and diagnostic imaging (535 occurrences). there are four clusters in the search for publications based on keywords. the red colored cluster mostly highlights human subjects and parts of the brain. the blue cluster shows mild cognitive impairment associated with the procedures of brain scanning, whereas the green cluster is associated with biomarkers and the yellow cluster with age. co-citation map by most frequently cited author this kind of analysis emphasizes outstanding authors which are linked using citation records. there are 69.231 cited authors who are the references of this study. by using the criterion of 20 as the number of authors, the study found 2250 authors who met the criterion. six clusters are created here using the default setting of 1000 authors. shen d (758 citations) is the most frequently referenced author in the red cluster, and many other chinese authors are listed as being associated with studies on imaging of brain anatomy and medical image analysis. morris jc (538 citations) and klunk we (346 citations) in the green cluster, whose research focuses on the role of biomarkers in aging and ad as well as brain amyloid imaging. the author with the most citations overall in the blue cluster is petersen rc (1365 citations) with numerous studies about mci and dementia, from guidelines up to biomarkers to detect the disease and it’s progression. the blue cluster linking up with other authors whose research focuses mostly on aging and dementia. the authors who ar e most frequently cited in the yellow cluster are jack cr (1103 citations), knopman ds (665 citations) and scheltens p (626 citations) who are associated with other authors whose research focuses on csf and inflammatory biomarkers in ad. the author with the most citations in the purple cluster is blennow k (674 citations), associated with authors whose studies are about risk of ad from plasma biomarkers and genetic factors. aarsland d (320 items) is the most prominent author in the light blue cluster, related with other authors whose studies are about the diagnosis, risk factors, and clinical manifestations of ad and other related dementia. the top 10 most co-cited authors on mci and brain imaging are shown in table 2. co-citation map of scientific journals a map of the co-citation journal was conducted to emphasize direct observation of source that have been cited repeatedly by a particular discipline.(26) this analysis shows scientific journals that are repeatedly cited in studies on mci and brain imaging. it was found that there were 10.283 scientific journals and with a minimum criterion of 20 citations, there were 437 journals that met the criterion. table 3 shows the top 10 scientific journal sources co-cited on the subject of mci and brain imaging. the cluster analysis of journal sources is divided into four groups with high similar properties. the red cluster consists of the journal alzheimer’s & dementia (1308 citations) and other journals on geriatrics and psychiatry in this network. neuroimage (4164 citations) is the most prominent journal in the green cluster and is a s s oc i a t e d wi t h a dd i t io n a l ima gi ng a nd ranking author citations links total link strength 1 2 petersen rc jack cr 1363 1103 999 999 170.174 141.981 3 4 5 6 7 8 9 10 shen d blennow k knopman ds scheltens p morris jc fox nc fischl b weiner mw 758 674 665 626 538 528 520 495 970 997 997 999 998 996 990 999 78.147 91.941 91.941 92.606 82.523 70.542 74.320 69.126 table 2. top 10 authors co-cited in references on mci and brain imaging ranking journal source citations links total link strength 1 2 neuroimage neurology 4164 3268 435 435 159.938 155.258 3 4 5 6 7 8 9 10 brain alzheimer’s & dementia plos one journal of alzheimer’s disease neurobiology of aging archives of neurology the lancet neurology annals of neurology 1410 1308 942 903 829 690 652 605 435 424 435 339 337 321 398 292 73.700 55.796 41.556 40.880 46.145 37.015 31.954 31.471 table 3. top 10 scientific journals co-cited on mci and brain imaging satyasari, rorong bibliometric analysis of mild cognitive impairment univ med vol. 42 no. 2 neurobiology journals. the blue cluster is dominated by the journal neurology (3268 c i t a t i on s) , wh ic h i s l i nke d to o th e r inte r dis ci pl inar y j our na ls t ha t de a l wit h neurological disorders. the yellow cluster has plos one (946 citations) as the most well-known publication associated with journals broadly discussing science and medicine. discussion according to the analysis of scientific production, there has been an increase in research output on mci and brain imaging since 2006, but there has been a strong increase particularly since 2019, with production hitting a peak of 561 articles (11.04%) in 2022. it is indisputable that over the past four years, it has become crucial that the topic of mci and brain imaging has generated a great deal from both research and clinical perspectives. the growing knowledge about this topic can also signal a greater need for newer alternative approaches to imaging tools and techniques that are developed to detect, treat, prevent, and evaluate mci globally. the country with the greatest number of publications in this category is the united states, followed by china, the uk, south korea, and canada. when sorted by year, articles from the united states are largely from 2018, those from europe are from 2017 to 2019, and those from china and other asian nations are the newest ones from 2020.(27–29) the growing number of studies on this subject coming from various countries demonstrates the need for the ability to deal with cognitive issues in both developing and developed countries, especially with the use of brain imaging technique that can then be taken into consideration.(30,31) due to asia’s prominence as the world’s most populous region and its fast expanding societies, there has been a rise in research on this subject recently in the asian region.(32,33) according to recent studies, the prevalence of mci is almost the same in the east and the west, at 3–42%, but asia has a very dense population, and particularly the number of people who are aging and at risk of developing mci would rise in this region.(34,35) the rapid rise in the prevalence of mci causes significant issues for the healthcare system that can be influenced by factors such as culture, economics, education, social conceptions of aging, and geographic location.(36,37) according to the source of scientific publications as well as the co-citation map of scientific journals, the majority of mci and brain imaging journals are multidisciplinary, including journals about geriatrics, psychiatry, imaging, neurobiology, neuropsychology, neurology, science, behavior, and medicine. there are various ways to look at this issue, therefore it is possible that all parties involved will need to work together to find a solution. (38,39) with a multidisciplinary team, broad approaches to mci are required, encompassing pharmaceutical and non-pharmacological interventions from prevention to rehabilitation.(40–42) to improve patient-centered care and present a viable strategy for providing patients and families with integrated health an d medica l care, the collaborative care model should be established and put into practice concurrently.(43–45) the foundation of a successful collaboration is built on open communication among team members, teamwork, trust and respect for each team member ’s knowledge, collegiality, and understanding of the area of medical practice.(46) according to the research that has received the most citations on this subject, petersen rc’s 1999 publication emphasizes that clinical traits that fit the requirements for mci can be distinguished from mild ad and from cognitive domain deficits in more severe ad.(15) petersen rc’s research from 2004 that also featured a similar topic on improving the mci criteria and making the therapy target more apparent was the secondmost-cited study.(16) articles from 2011 to 2017 have also received many citations in other studies. based on the discussion of prior research, studies from before 2015 continue to focus more on diagnostic criteria that are clinically evaluated using tests of memory and cognition.(15–19) it appears that numerous studies from 2015 and later have started to talk about brain profiles and bioma rker methods using neuroimaging techniques to detect mci and other cognitive issues. to forecast cognitive decline, early detection, diagnostic guidelines, and the goal of an extensive early treatment, neuroimaging techniques using mri and pet scans for the detection of a 1-42 and tau have started to be used.(20–24) the keyword that appears the most frequently in each article is mild cognitive impairment which is connected to the procedures of brain scanning. the other keywords that tend to come up frequently are male and female, which is related to human subjects, brain structure, and function. moreover, positron emission tomography is frequently used and connected to biomarkers. the keywords age and those connected to it are the most prevalent. the correlation between keywords demonstrates a connection between mci, imaging methods, and biomarkers as aging progresses. according to the visual analytical findings, there has been considerable research done on the topic of brain scanning techniques because keywords related to these techniques are frequently discovered. this highlights the fact that more studies are examining the use of neuroimaging as a test to diagnose and evaluate cognitive impairment by looking at specific biomarkers, potentially because it is easier and provides data more rapidly.(47,48) in the co-citation map by most frequently cited author, petersen rc is the most prominent cited author, followed by jack cr, shen d, blennow k, knopman ds, scheltens p, klunk we, and aarsland d. petersen rc, a professor in neurology(49) has began to write articles in 1974 and since 2002 has produced numerous articles related to mci, dementia, and alzheimer’s disease.(50–52) he produces a lot of research and interestingly, starting in 2013, he began to concentrate more on bioma rker s and neuroimaging methods.(53–57) it is not surprising that he is the author who is most commonly cited considering the approximately 1000 publications on this subj ect and the continual yearly development of those numbers.(49) this is in contrast with shen d, a professor of radiology,(58) who began writing articles in 2002 on a variety of subjects, including genetics, anatomical brain networks, medical image analysis, and others on topics ranging from infants to elderly people who suffer from dementia.(59–61) because of the broadness of his writing, although not as a first author, shen d has received numerous citations for his work. the writers who are commonly ref erenc ed here come f rom a var ie ty of institutions, nations, and fields of study, indicating that the distribution of authors on this subject is quite diverse.(62–66) this 25-year trend can be seen by looking bibliometrically at the top productive authors, countries, keywords, most cited articles, and journals. according to the trend of the discussions, this subject started to be popular around 2015, its popularity increased until 2022, and this mapping supports the premise that brain imaging approaches to look for distinctive biomarkers seem promising for future directions in building guidelines for diagnosis and treatment of mci. arguably it cannot be the only test used to diagnose cognitive impair ments, but its significance is obviously important given the increased discussion about this topic. the weakness of this research is that the data were limited to the scopus database, therefore it is possible that some overview from a different angle was not included in the study. in addition, bibliometric analysis also cannot be used to measure the validity and quality of scientific publications, and the number of citations to an article is highly dependent on various factors. although the results of this bibliometric analysis can be used for further study and for developing the topic of mci and brain imaging, these limitations need to be considered in interpreting the results of this study. conclusions this review provides a research trend related to mci and brain imaging through satyasari, rorong bibliometric analysis of mild cognitive impairment univ med vol. 42 no. 2 bibliometric analysis over a period of 25 years. this theme is getting more popular by looking at the growing articles, particularly in the past four years. since 1996, there has been research on mild cognitive impairment alone. however, since 2015 and beyond, there has been a rise in interest in the topic in relation to brain imaging techniques and biomarkers, with asia in 2020 receiving particular emphasis. the mapping’s findings provide more evidence for the relationship between aging, mci, and numerous biomarkers and brain imaging procedures. the primary method used in the research is brain imaging to identify a 1-42 and tau as biomarkers for mci and other cognitive diseases, as well as for diagnostic guidance and treatment. the results of journals that are frequently cited and clearly shown suggest that there is a relationship between various multidisciplinary fields, necessitating team collaboration with a patient focus to achieve better results in preventing mci and its deterioration. however, until now, brain imaging cannot be used as a single examination in diagnosis and therapy for mci. hence, more thorough analysis and research are needed to determine how to apply brain imaging in clinical practice. conflict of interest we know of no conflicts of inte rest associated with this publication. acknowledgements we thank dewan riset dan pengabdian kepada masyarakat (drpmf) universitas trisakti for 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cincau leave infusion at dosages of 2.5 mg/kg bw, 5 mg/kg bw, and 10 mg/kg bw, respectively, while group iv (positive control) received antacid at a dosage of 20 mg/kg bw, and group v (negative control) one milliliter of distilled water. all interventions were given by the oral route, once daily for seven days. on day 7, the mice were given aspirin (600 mg/kg bw) to induce gastric ulcer. after 30 minutes, all mice were sacrified, and their stomachs examined macroscopically for gastric ulcer, characterized by the presence of ulcer(s) and bleeding. total ulcer scores were analyzed by one-way anova to compare between-group protective effect of interventions against aspirin-induced gastric ulcer. results showed that groups treated with cincau leaf infusion at all dosages experienced a gastric ulcer protective effect. there were significant differences (p=0.002) between treatments, compared to the negative control, but no significant differences (p>0.05) when compared to the positive control. thus cincau leaves (cyclea barbata miers) at dosages of 2.5 mg/kg bw, 5 mg/kg bw, and 10 mg/ kg bw, had a protective effect against aspirin-induced gastric ulcer in mice. higher dosages of cincau leaf infusion have a correspondingly higher gastric ulcer protective power. keywords : cyclea barbata miers, aspirin-induced gastric ulcer, mice *department of pharmacology, medical faculty, islam indonesia university yogyakarta correspondence dr. isnatin miladiyah, m.kes. department of pharmacology, medical faculty, islam indonesia university jl. kaliurang km 14.5 sleman yogyakarta 55564 phone: 0274-898444 ext. 2002 email: isnatin@fk.uii.ac.id univ med 2011;30:88-94 protective effects of cyclea barbata miers leaves against aspirin-induced gastric ulcer in mice iskandar muda siregar* and isnatin miladiyah* may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina introduction gastric ulcer is one of the intestinal disorders that occur frequently due to an imbalance between offensive factors (gastric acid secretion) and defensive factors (gastric mucosal defense).(1) aspirin and other nonsteroidal anti-inflammatory drugs (nsaids) may induce these ulcers through inhibition of 89 univ med vol.30 no.2 prostaglandin biosynthesis, reduced gastric blood flow, local irriation, and disruption of local tissue restitution and repair.(2) the cincau plant (cyclea barbata miers) belongs to the menispermaceae family and is traditionally utilized by many indonesian communities as a refreshing beverage in the form of a gel or infusion. some communities in indonesia also use the infusion as medication against cold. cold is frequently felt to be a digestive disorder, with the complaints of sprue, diarrhea, epigastric fullness, and reflux of gastric acid into the pharynx.(3) it is the tannin content of cincau leaves that is believed to be beneficial for abdominal pain. in addition, this plant contains various b i s b e n z y l i s o q u i n o l i n e a l k a l o i d s , i . e . berbamine, chondocurine, alpha and beta cyclanoline, fangchinoline, homoaromoline, isochondocurine, isotetrandrine, lemacine, and tetrandrine,(4) as well as the minerals calcium and phosphorus, and vitamins a and b.(5) cincau leaves contain mucin, thus cincau leaf extract readily turns into a gel, resembling agar.(5) the major component of cincau gel is the polysaccharide pectin, consisting of watersoluble vegetable fibers that are readily fermented by the colonic microflora.(6) because of the pectin content, cincau leaf extract is considered to be a good source of vegetable fiber, the more so as its caloric content is sufficiently low. the high fiber content of cincau leaves leads to ease of defecation in those consuming cincau gel.(5). the effect of the pectin fibers in cincau leaves may be enhanced by the addition of citric acid, which also increases their antioxidant effect.(7) several studies have been conducted on the medicinal properties of cincau leaves, such as antitumor,(3) antipyretic, antimalarial (8) and a n t i h y p e r t e n s i v e . ( 9 ) t h e p h a r m a c o l o g i c activities of cincau leaves are thought to be c a u s e d b y t h e b i s b e n z y l i s o q u i n o l i n e alkaloids.(8-9) thus far, the effect of cincau leaves on gastric functions has been investigated in a study on gastric hydrochloric acid (hcl) concentration and gastric histopathology after simultaneous administration of an aqueous extract of cincau leaves and aspirin to wistar rats. the study results indicated that cincau leaves protected the gastric mucosa from the effects of aspirin, as demonstrated by gastric histopathology, but did not affect the gastric hcl concentration.(10) the present study differs from the abovementioned one in the method of preparation of the cincau leaves (infusion vs gel), and in the observed effects (macroscopic examination and measurement of ulcer index vs microscopic examination and measurement of hcl concentration). on the basis of several studies on the content of active substances in cincau leaves, the present study aims to evaluate the protective effect of cincau leaves against gastric ulcer after administration of aspirin in mice. methods research design the study was designed as a purely experimental laboratory investigation using controls to evaluate effect of cincau leaves on gastric ulcer. test animals this study used 25 balb/c mice aged 2-3 m o n t h s a n d w e i g h i n g 2 0 3 0 g r a m s . t h e experimental animals were healthy mice (active and without any physical defects), whose condition was monitored before and during the study, and who were handled with reasonable care during the experiments. according to the previous study, with 5 intervention groups, each consisting of 5 test animals (minimum size), the size of the sample was 25, selected by simple random sampling.(11) cyclea barbata miers cincau leaves (cyclea barbata miers) were obtained during the period of februarymarch 2009 in and around the sleman district. the leaves selected were clean and shiny ones, 90 lesions score ulcer length > 10 cm 4 length 2-10 cm 2 length 1-2 cm 1 length < 1 cm 0.5 bleeding 2 table 1. ulcer score (13)as such leaves have a substantially higher content of active substances. the leave picking was started from the lower stem upwards. the leaves were identified at the pharmacognosyphytochemistry laboratory, pharmacy program, faculty of mathematic and natural sciences (fmipa), uii, yogyakarta, from where the chemicals used in the preparation of the cincau leaf infusion were also obtained. a liquid antacid preparation (bayer) was used as positive control, and aspirin (bayer) as inducer of gastric ulcer in the laboratory animals. preparation of cincau leaf infusion cincau leaves were washed, after which 50 grams were weighed off, and 300 ml water was added to cover the leaves. the material was then placed in a waterbath at 80-90oc for 30 minutes. the solution was filtered with filter paper, replaced in the waterbath, and left to evaporate, until only 50 ml water was left. induction of gastric ulcer as inducer of gastric ulcer, aspirin was administered orally to the mice. the animals had been fasted previously for 12 hours to empty the stomach of food, and to increase the gastric acid level, thereby facilitating the induction of gastric ulcer upon administration of aspirin. the dosage of aspirin was 600 mg/kg bw, which was modified from previous studies. (12) determination of the dosage of cincau leaf infusion the basic dosage of cincau leaf infusion was taken from previous studies to be 2.5 mg/ kgbw, but modified for the higher dosages to two and four times the basic dosage, becoming 5 and 10 mg/kgbw, respectively. determination of antacid dosage the antacid dosage was calculated from the regular dosage in humans of 50 kg, thus obtaining a dosage of 20 mg/kgbw for the animals weighing 20-30 grams. calculation of gastric ulcer protective power on macroscopic examination, the signs of gastric ulcer are the presence of an ulcer (or u l c e r s ) a n d t h e o c c u r r e n c e o f g a s t r i c hemorrhage, which were scored as shown in table 1.(13) data analysis the total scores of ulcer(s) and hemorrhage in the various treatment groups were statistically analyzed using one-way anova (spss version 15 for windows) for significant differences, with p<0.05 being considered significant. results among the 25 mice (n=25) used in the study, one mouse in group i did not live to completion of the study, as it died through an error in using the stomach tube, thereby leaving only 24 animals for analysis. mean total ulcer scores of the experimental animals are shown in table 2 and figure 1. discussion apirin is an anti-inflammatory drug known for its gastric toxicity, which is frequently c h a r a c t e r i z e d b y g a s t r i c u l c e r s a n d hemorrhage.(14) therefore this drug is frequently u s e d a s a m o d e l i n s t u d i e s o n i n v i v o cytoprotective activity of new substances or compounds.(1-2,15-18) administration of nsaids, including aspirin and indomethacine, inhibits the biosynthesis of prostaglandins, particularly of pge2 and pgi2, which are protective factors siregar, miladiyah cyclea barbata against gastric ulcer 91 univ med vol.30 no.2 (mucosal resistance factors) against irritation of the stomach by gastric acid.(18, 19) inhibition of prostaglandins results in early damage to the mucosal, parietal, and endothelial cells,(15) thus leading to the formation of an ulcer. moreover, the occurrence of an ulcer is also mediated by f r e e r a d i c a l s f r o m t h e c o n v e r s i o n o f hydroxyperoxyl into hydroxyl fatty acids, c a u s i n g d e s t r u c t i o n o f t h e c e l l s . t h e s e h y d r o x y p e r o x y l c o m p o u n d s i n t u r n a r e produced by the degranulation of mast cells and the complete lipid peroxidation accompanying cellular damage.(20) figure 1 shows that the mice in the groups receiving cincau leaf infusion and antacid had lower total ulcer scores, in comparison with the negative controls receiving distilled water. this indicates that cincau leaf infusion at the three dosage levels administered simultaneously with antacids, was capable of reducing the occurrence of gastric ulcer due to oral aspirin administration. from these data it is also apparaent that with increasing dosage of cincau leaf infusion, there is a proportional increase in gastric ulcer protective effect, with the highest dosage of 10 mg/kg bw giving the highest protective power. treatment group total ulcer score of mice no. mean ± sd p 1 2 3 4 5 infusion of cincau leaves at dose 2,5 m g/kgbw 3 4 2 4 3.25 ± 0.96 0.002 infusion of cincau leaves at dose 5 mg/kgbw 3 1 4 2 2.5 2.5 ± 1.12 infusion of cincau leaves at dose 10 mg/kgbw 0.5 0 1 1 1 0.7 ± 0.45 antacid at dose 20 mg/kgbw 0 1 0.5 1 0.5 0.6 ± 0.42 aquadest 25 6 9 8 6.5 10.9 ± 7.97 table 2. mean total ulcer scores based on treatment groups in mice figure 1. mean of total ulcer score by treatment groups in mice. group i: infusion of cincau leaves at dose 2,5 mg/kgbw; group ii: infusion of cincau leaves at dose 5 mg/ kgbw; group iii: infusion of cincau leaves at dose 10 mg/kgbw; group iv: antacid at dose 20 mg/kgbw; group v: aquadest 92 the results of one-way anova indicated that there were significant differences in ulcer protection at all dosage levels of cincau leaf infusion, compared with negative controls (p=0.002). however, the three dosages of cincau leaf infusion did not show significant differences with antacid (p>0.05). from the lack of significant differences with antacid it may be concluded that cincau leaf infusion at the dosages of 2.5 mg/kg bw; 5 mg/kg bw; and 10 mg/kg bw, has a gastric ulcer protective power equivalent to an antacid dosage of 20 mg/kg bw. the results of the present study supports those of a previous study, in which cincau leaf press liquid was protective against gastric u l c e r, b e i n g c a p a b l e o f i m p r o v i n g t h e histopathological picture of aspirin-induced gastric ulcer.(10) the present study supports the previous study results, as with a different method of preparation, cincau leaves are still capable of exerting gastroprotective activity against ulcers. although tis study was able to provide evidence that the cincau plant (cyclea barbata miers) possesses protective activity against gastric ulcer, the active compounds involved in exerting this protective effect are not known with certainty. one of the suggestions relate to the antioxidant effects of the flavonoids, saponins, and tannins, that are known to be present in the cincau leaves.(3,5) antioxidant compounds have been demonstrated to have cytoprotective effects, thus being capable of protecting cells from various kinds of damage and possibly of functioning as anti-ulcer drugs.(21-23) antioxidants act by inhibiting lipid peroxidation and by scavenging free radicals, thereby preventing the occurrence of gastric ulcer.(24) s e v e r a l s t u d i e s d e m o n s t r a t e d t h a t flavonoids from various plants are reportedly capable of preventing the occurrence of gastric ulcer. this may take place through an increase in the amounts of neutral glycoproteins and in prostaglandin concentrations, and inhibition of h i s t a m i n e s e c r e t i o n f r o m m a s t c e l l s b y inhibition of histidine decarboxylase,(25) thus reducing stimulation of h2 receptors,(26) or by secretion of prostaglandin-like compounds.(27) another possible mechanism of action for inhibiting ulcer occurrences is by decreasing pepsin secretion and activity,(28) in view of the f a c t t h a t o n e o f t h e m a j o r p a t h o g e n e t i c m e c h a n i s m s o f u l c e r f o r m a t i o n i s b y hypersecretion of gastric juice and by pepsin hyperactivity.(29) however, elucidation of the exact mechanism underlying the protective effect of cincau leaves awaits further studies. one of the flavonoids in the cincau plant are the bisbenzylisoquinolines (including tetrandrine), which is thought to play a role in t h e m e c h a n i s m o f u l c e r p r e v e n t i o n . b i s b e n z y l i s o q u i n o l i n e c o m p o u n d s f r o m various plants possess anti-inflammatory, antiallergic, antioxidant, antifibrogenetic, and immunomodulating effects. the alkaloid tetrandrine from various plants also has antiinflammatory properties against a number of lung infections.(30) however, whether or not the bisbenzylisoquinolines (including tetrandrine) from the cincau plant possess these effects, is a subject for further study. i n a d d i t i o n t o f l a v o n o i d s , o t h e r compounds playing a role in ulcer protection are the saponins, which have hemolytic, expectorant, immunostimulant, and antiinflammatory properties.(31) another member of the menispermaceae is cyclea peltata, which also has a high saponin content.(32) it is thought that the anti-inflammatory effects of s a p o n i n s r e d u c e s t h e r i s k o f u l c e r s , b y increasing defensive factors of gastric mucosa a n d s t o p p i n g t h e i n f l a m m a t o r y p r o c e s s resulting from induction by aspirin (indicated by absence of edema in the gastric mucosa of mice receiving cincau leaf infusion). the protective effect of saponins against gastric ulcer may also be mediated by the formation of a protective mucus layer on the gastric m u c o s a a n d b y s e l e c t i v e i n h i b i t i o n o f pgf2α.(25) siregar, miladiyah cyclea barbata against gastric ulcer 93 univ med vol.30 no.2 one study reported that cincau leaves are safe for consumption by the community, as it was demonstrated that cincau leaf ethanolic extract at a dosage of up to 30,000 mg/kg bw did not induce significant toxic effects, in other words, the ld50 of cincau leaf extract (cyclea barbata miers) in mice is greater than 30,000 mg/kg bw.(33) there are several limitations to this study, as there were no observations on the effect of cincau leaf infusion in ulcer healing, active compounds playing a role in prevention of gastric ulcer were not isolated, and the specific mechanism of action of the gastroprotective effect was not studied. conclusions i n f u s i o n s o f c i n c a u l e a v e s ( c y c l e a barbata miers) have protective activity against the occurrence of gastric ulcers in mice induced by aspirin at a dosage of 20 mg/kg bw, from an initial dosage of 2.5 mg/kg bw. higher dosages of cincau leaf infusion are correlated with a correspondingly higher gastric ulcer protective power. further studies are needed on the active compounds in cincau leaves responasible for protective activity against gastric ulcer. acknowledgement thanks are due dr noviana nugrohowati as her assistance in constructing the study report and to mr. sumarno as laboratory technician at the pharmacology laboratory, pharmacy program, faculty of mathematics and natural s c i e n c e s , u n i v e r s i t a s i s l a m i n d o n e s i a , yogyakarta for their valuable support before, during, and after the execution of this study. daftar pustaka 1. nilesh m, dinesh s, dhirendra s. evaluation of anti-ulcer potential of leaves of jasminum grandiflorum l. int j ph sci 2009;1:247-9. 2. al-howiriny t, al-sohaibani, el-tahir k, rafatullah s. prevention of experimentallyinduced gastric ulcers in rats by an ethanolic extract of “parsley” petroselinum crispum. am j chinese med 2003;31:699-711. 3. chalid sy. effect of green cincau leaves (cyclea barbata miers and premna oblongiofolia merr) extracts on antioxidant activity and tumor growth of mammary gland of transplantable mice (thesis). bogor:institut pertanian bogor;2003. 4. tantisewie b, ruchirawat s. alkaloids from the plants of thailand. in: brossie a, cordell ga, editors. the alkaloids. vol. 41st. california: academic press, inc.;1992.p.1-7. 5. ekasaputra b, setyawan h, eko ip, humar r, bayu s. application of cincau leaves for controlling viscosity and gel strength of drilling mud (skripsi). (pemanfaatan daun cincau dalam mengontrol viskositas dan gel strength lumpur pemboran) yogyakarta: teknik perminyakan upn;2007. 6. gallagher d. dietary fiber and its physiological effect in essential of functional food. maryland: aspen publication;2000. 7. nurdin su, zuidar as, suharyono. dried extract from green cincau leaves as potential fibre sources for food enrichment. afr crop sci conf proc 2005;7:655-8. 8. saxena s, pant n, jain dc, bhakuni rs. antimalarial agents from plant sources. curr sci 2003;85:1314-29. 9. jia-qing q. cardiovascular pharmacological effects of bisbenzylisoquinoline alkaloid derivatives. acta pharmacol sin 2002;23:108692. 10. djam’an q. effect of press liquid from leaves of cyclea barbata miers on gastric acid concentration and histopathological picture of gastric ulcers induced by acetylsalicylic acid (thesis). (pengaruh air perasan daun cyclea barbata miers (cincau hijau) terhadap konsentrasi hcl lambung dan gambaran histopatologik lambung tikus galur wistar yang diinduksi acetylsalicylic acid.) semarang: fk universitas diponegoro;2008. 11. festing mfw, altman dg. guidelines for the design and statistical analysis of experiments using laboratory animals. ilar j 2002;43:24458. 12. farah aj, aslam asm, javed k, jafri ma. antiulcerogenic activity of elettaria cardamomum maton and amomum subulatum roxb seeds. indian j trad knowledge 2005;4: 298-302. 13. best r, lewis da, nasser n. the antiulcerogenic activity of the unripe plantain banana (musa species). british j pharm 1984;82:41-5. 94 14. toruner m. aspirin and gastrointestinal toxicity. anatol j cardiol 2007;(7suppl 2):27-30. 15. rajkapoor b, anandan r, jayakar b. anti-ulcer effect of nigella sativa linn against gastric ulcer in rats. curr sci 2002;82:177-9. 16. khalil j, akhter s, bhatti sa, bukhari mh. gastric ulcer healing effects of nigella sativa: a comparative experimental study with cimetidine. biomedica 2010;26:61-6. 17. vinothapooshan g, sundar k. anti-ulcer activity of imosa pudica leaves against gastric ulcer in rats. rjpbcs 2010;1:606-14. 18. wang z, hasegawa j, wang x, matsuda a, tokuda t, miura n, et al. protective effects of ginger against aspirin-induced gastric ulcer in rats. yonago acta medica 2011;54:11-9. 19. sharma rk, mandal s, rajani gp, gupta n, srivastava dp. antiulcer and antiinflammatory activity of fresh leave extracts of polyalthia longifolia in rats. int j drug dev res 2011;3:3519. 20. umamaheswari m, asokkumar k, rathidevi r, sivashanmugam at, subhadradevi v, ravi tk. antiulcer and in vitro antioxidant activities of jasminum grandiflorum l. j ethnopharmacol 2007;110:464-70. 21. okokon je, nwafor pa. antiulcer and anticonvulsant activity of croton zambesicus. pak j pharm sci 2009;22:384-90. 22. dekanski d, janicijevic-hudomal s, tadic v, markovic g, arsic i, mitrovic dm. phytochemical analysis and gastroprotective activity of an olive leaf extract. j serb chem soc 2009;74:367–77. 23. gregory m, vithalrao kp, franklin g, kalaichelavan v. anti-ulcer (ulcer-preventive) activity of ficus arnottiana miq. (moraceae) leaf methanolic extract. am j pharmacol toxicol 2009;4:89-93. 24. naseri mkg, mard sa. gastroprotective effect of alhagi maurorum on experimental gastric ulcer in rats. pak j med sci 2007;23:570-3. 25. okokon je, antia bs, umoh ee. antiulcerogenic activity of ethanolic leaf extract of lasianthera africana. afr j trad cam 2009;6:150-4. 26. kishore dv, jennifer p, mini kv. anti ulcer activity of methanolic and aqueous extracts of leaves of sapindus trifoliatus. linn. int j pharmaceutic sci rev res 2011;6:25-7. 27. nguelefack tb, watcho p, wansi s, mbonuh n, ngamga d, tane p, et al. the antiulcer effects of the methanolic extract of the leaves of aspilia africana (asteraceae) in rats. afr j trad cam 2005;2:233-7. 28. borikar vi, jangde cr, philip p, rekhe ds. study of antiulcer activity of bauhinia racemosa lam in rats. vet world 2009;2:215-6. 29. khandare ra, gulechal vs, mahajani ms, mundadal as, gangurdel hh. evaluation of antiulcer activity polyherbal formulation. ijprd 2006;1:1-6. available at: http:// www.ijprd.com. accessed june 16, 2011. 30. qiang-min x, hui-fang t, ji-qiang c, ru-lian b. pharmacological actions of tetrandrine in inflammatory pulmonary diseases. acta pharmacol sin 2002;23:1107-13. 31. sahelian rmd. antispasmodic saponins from bulbs of red onion (allium cepa l. var tropea). j agric food chem 2005;23:935-40. 32. hullatti kk, sharada ms. comparative phytochemical investigation of the sources of ayurvedic drug patha: chromatographic fingerprinting analysis. indian j pharm sci 2010; 72:39-45. 33. angelina m, hartati s, dewijanti id, banjarnahor sds, meilawati l. determination of cincau leaf (cyclea barbata miers) ld50 in mice (penentuan ld50 daun cincau (cyclea barbata miers) pada mencit). makara sains 2008;12:236. siregar, miladiyah cyclea barbata against gastric ulcer c:\users\universa medicina\docu 118 abstract universa medicina extracoporeal shockwave treatment decreases pain, functional limitations and medial collateral ligament thickness in subjects aged 50-70 years with knee osteoarthritis andwi setiawan kokok1*, tirza z. tamin1, nyoman murdana1, and indah suci widyahening2 background pain from knee and hip osteoarthritis (oa) can have a significant impact on the physical function and quality of life of affected individuals worldwide. the objective of this study was to evaluate the effect of extracorporeal shockwave therapy (eswt) on pain, flexibility, function, and medial collateral ligament (mcl) thickness in knee osteoarthritis (koa). methods a study of quasi experimental design was performed involving 15 subjects aged 50 – 70 years with kellgren-lawrence grade 2-3 koa. all subjects were evaluated regarding baseline -pain using visual analogue scale (vas), range of motion (rom), functional outcome using western ontario and mcmaster universities arthritis index (womac), and mcl size. extracorporeal shock wave therapy was given 3 times, at baseline, and 4 and 8 weeks after intervention. all subjects were given 4000 shocks at intensities of 1.5 – 4 bar (raised gradually) per session. the shocks were given in the supine position, knee flexed 90o, without topical anesthetic. statistical analyses were conducted using a dependent t-test. results after 8 weeks of intervention, eswt significantly improved pain score (p<0.01), womac (p<0.01) and mcl thickness (p<0.01) in patients with oa of the knee. however, there was no significant difference in knee rom, both for degree of flexion and extension (p>0.05). conclusion the use of eswt for treatment of knee oa had a beneficial effect on pain relief, function outcome and mcl thickness. however, there remains a lack of clarity regarding the frequency and dosage levels of eswt required to achieve maximum improvement. keyword: knee osteoarthritis, quasi-experimental, extracorporeal shockwave therapy, medial collateral ligament original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2021.v40.121-132 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1164 may-august 2021 vol.40no.2 cite this article as: kokok as, tamin tz, m u r d a n a n, wi d ya h eni n g is. extracoporeal shockwave treatment decreases pain, functional limitations and medial collateral ligament thickness in subjects aged 50-70 years with knee osteoarthritis. univ med 2021;40:118-29. doi: 10.18051/univmed.2021.v40.121132. 1medical rehabilitation department, musculoskeletal division, faculty of medicine, universitas indonesia, jakarta 2department of community medicine, faculty of medicine, universitas indonesia, jakarta *correspondence: andwi setiawan kokok medical rehabilitation department, faculty of medicine, universitas indonesia, jakarta phone: +6281905954798 email: andwisetiawan@yahoo.com orcid id: 0000-0001-6708-9984 date of first submission, march 25, 2021 date of final revised submission, july 18, 2021 date of acceptance, july 30, 2021 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 119 univ med vol. 40 no 2 introduction osteoarthritis (oa) is the most common form of arthritis (inflammation of the joints) and the most common cause of disability worldwide, mainly due to pain, as the main symptom of this disease. osteoarthritis occurs most often in the weight bearing joints such as the knees and hips. the diagnosis of oa of the knee can be confirmed based on clinical and/or radiological findings. the economic burden caused by oa is quite impactful, including the costs that are related to management, environmental and lifestyle modifications, and also decreased work productivity.(1) the number of patients with knee oa (koa) in the us has multiplied twice as much c o mp a r e d t o t he mi d -20 t h c e n t ur y. ( 2 ) approximately 13% of women and 10% of men aged 60 years or older have symptomatic oa. in indonesia, the prevalence of oa that has been diagnosed radiologically reaches 15.5% in men and 12.7% in women aged 40-60 years.(3) also a t t h e ou tpa ti e nt c l i n ic of the m e d i c a l re h a bi l it a t i on de p a r t me nt , ci p to mangunkusumo hospital, indonesia, the number of patients with koa in 2016 reached 1,018 patients (15.2% of the total number of outpatients i n t h e mus c u l os kel e t a l di visi o n) . ( 4 ) t he proportion of people with symptomatic koa increases with age and their body mass index (bmi).(2) osteoarthritis is a disease of multifactorial etiology, including local and systemic factors. increasing age, female, overweight and obesity, history of knee injury, muscle weakness, and joint hyperlaxity are risk factors for oa.(5) to date, ther e is no therapy that can cure oa. management is primarily aimed at controlling/ relieving pain, improving motion and joint function and improving quality of life. optimal management of oa includes pharmacological and nonpharmacological management. joint replacement surgery is reserved for patients with severe degrees of oa that do not respond to conservative treatment.(6) extracorporeal shock wave therapy (eswt) has been widely used for the management of musculoskeletal abnormalities and pain. the curative effect of eswt has been demonstrated in plantar fasciitis, calcified tendinitis of the shoulder, epicondylitis, patellar and achilles tendinopathy, non-union and malunion fractures of long bones, and avascular necrosis of the femoral head.(7) extracorporeal shock wave therapy is a non-invasive treatment modality that has a low complication rate, can be performed in the outpatient setting, and has a relatively low cost compared to other conservative therapies and surgery.(8) the principle of eswt is the production of pressure waves by electrohydraulic, electromagnetic, piezoelectric, or pneumatic sources. these waves work on both liquids and solid matter. the shock wave energy focused on one area is defined as energy flux density (efd) which is recorded in mj per unit area (mm2).(5) based on the energy flux density, eswt is classified into low dose (<0.08 mj/mm2), moderate dose (0.08-0.28 mj/mm2), and high dose (>0.28-0.60 mj/mm2).(9) seve ral animal st udies have shown improvement in motor dysfunction and decrease in pain in oa.(10) extracorporeal shockwave therapy has been increasingly used in patients suffering from koa.(11-13) observational research by li et al.(11) demonstrated12better13improvement in koa after 6 and 12 weeks of eswt compared to laser therapy, measured using the numeric rating scale and the western ontario and mcmaster universiti es arthritis index (womac) as outcome parameters. imamura et al.(12) did a clinical test on radial eswt in patients with grade 2 – 4 knee oa. their study compared radial eswt at doses of 0.1 – 0.16 mj/mm2 with sham radial eswt. the study showed that the radial eswt group had a significant difference only in the womac pain component, therefore the authors considered that this dose is less effective to treat knee oa.(12) several studies reported varying results, but generally, the improvement in pain score and 120 functional score after being given eswt is due to its regenerative effect on soft tissue and its pain reduction effect. structural abnormalities in knee oa can occur not only in the cartilage but also in the ligaments that function as passive stabilizers of the knee. the medial collateral ligament (mcl) is a ligamentous structure that serves as the knee’s main stabilizer against valgus stress. the mcl is also the most commonly injured knee ligament. injuries to the mcl are most commonly the result of trauma; however, mcl injuries can also result from non-traumatic causes, e.g. degenerative diseases of the knee joint, particularly those involving the medial tibiofemoral compartment. grade 1 mcl injury is characterized by edema around the mcl structure and involves disruption of a few fibers resulting in localized tenderness but no instability.(14) the difference of this study with the previous study lies in the changes in mcl features in knee oa before and after receiving eswt management. therefore, the aim of this study was to evaluate the effect of eswt on pain, flexibility, functional outcome, and mcl thickness in koa subjects. methods research design a study of quasi experimental design was conducted in cipto mangunkusumo hospital, jakarta from october 2019 – february 2020. research subjects a total of 15 koa male and female subjects aged 50-70 years were recruited into the study. the inclusion criteria of this study were: i) knee pain with vas 31 69 mm; ii) diagnosed with primary knee oa of kellgren-lawrence (kl) grade 2 and 3 with mcl size > 5.6 mm that was confirmed by ultrasound examination; iii) sedentary level of physical activity; iv) could understand and follow instructions and v) willing to participate in this study and had signed informed consent after given information regarding the study. the exclusion criteria of this study were: i) had a history of total knee replacement (tkr) surgery; ii) genu varus / valgus >15°; iii) had existing tumor or infection or signs of acute inflammation in the knee and the surrounding tissue; iv) had conditions that cause a tendency to bleed (including taking anticoagulants; v) requiring invasive management; and vi) received injection therapy in the knee in the last 6 months. the sample size was calculated using mean difference formula within paired group of differences in womac scores of 15 points with standard deviation of 20 points (data from a preliminary study). assuming a significance level of 5% and a test power of 80%, we calculated that we needed a minimum of 15 participants. data collection all subjects fulfilling the inclusion criteria would be given complete oral and written information about the study by the principal researcher, who was also to collect the basic data. these consisted of age, gender, occupation, body weight, height, diagnosis of knee osteoarthritis, baseline vas score, baseline rom using a goniometer, baseline functional score using the womac questionnaire, and baseline mcl thickness using knee ultrasonography examination. measurements body mass index was calculated from the subject’s weight in kilograms and the height in meters squared. the diagnosis of knee oa was established using clinical criteria and radiological findings according to the american college of rheumatology (acr) criteria. intervention the eswt used was the btl® radial eswt type swt-6000. the patient would be given eswt in the supine position with the knee flexed at 90o, without topical anesthetics. the eswt would be given at 2 of the most painful locations according to the patient. the dose was started from the lowest dose (1.5 bars) and kokok, tamin, murdana, et al extracoporeal shockwave treatment decreases pain 121 univ med vol. 40 no 2 a b c increased gradually to 4 bars in 8 levels, each consisting of 500 shocks for a total of 4.000 shocks. the eswt probe was held stationary on a trigger point around the knee or at the patellofemoral and tibiofemoral borders of the target knee (if the patient was unable to point out the 2 most painful locations), we avoided a direct placement on the peroneal nerve or blood vessel. in order to limit the loss of shockwave energy at the interface, we used an aqueous gel, not containing any phar macologically active substance as a coupling medium between the probe of the device and the skin, and it was applied in circular motions. each treatment session did not exceed 30 minutes and was given once per week. before receiving eswt therapy, the patients were informed that there would be pain/ discomfort during therapy. the patients received the first eswt therapy according to the protocol and were instructed to come back and undergo the second and third eswt therapy respectively 7 and 14 days after the first to complete the therapy. patients were also directed to come in weeks 1, 2, 4 and 8 for evaluation. outcome measures one researc her per formed all the evaluations at baseline (week 0) and after 1 (week 1), 2 (week 2), 4 (week 4), and 8 weeks (week 8). the following parameters were assessed during the evaluations: pain using visual analogue scale (vas): scores wer e re corded by making a handwritten mark on a 10-cm line that represents a continuum between “no pain” and “worst pain.(15) knee extension and flexion using range of motion (rom) – the knee extension and flexion were measured in the prone starting position with lower limb extension in the hip and knee joint. the lateral femoral condyle was used a s a l andma rk for the measurement of the knee extension and flexion. the central pivot of a goniometer was placed over the midpoint of the lateral joint margin, with the stationary arm of the goniometer aligned with the great trochanter. the moving arm of the goniometer was then aligned with the lateral malleolus. during the measurement we stabilized the joint at half the length of the thigh by a girdle. each knee (in extension or flexion) was measured twice with an accuracy of 1 degree, and the higher angle was recorded for the statistical analysis. knee flexion and extension rom in degrees were measured bilaterally in the prone position. for this purpose, the lateral femoral condyle was used as a landmark for the measurement of knee flexion and extension. the central pivot of a universal goniometer was placed over the midpoint of the lateral joint margin, with the stationary arm aligned with the great trochanter. the moving arm was then aligned with the lateral malleolus, with the neutral position taken as zero. to measure knee flexion, the hip was initially at 0 degree of extension, abduction, and adduction. in order to avoid knee flexion, the examiner held the lower limb and stabilized the femur to prevent hip rotation, abduction or adduction. to measure knee extension, the lower limb was extended. the previous precautions were taken to prevent compensations (i.e. adduction, abduction, and rotation). functional outcome using the womac questionnaire, a disease-specific index of disability, that was used as a subjective measure of perceived health and physical function. the womac questionnaire is a thr ee-par t questio nnaire tha t can be completed by the subjects in approximately 10 minutes. it consists of 24 questions and probes clinically important symptoms in the areas of pain (5 questions), stiffness (2 questions), and physical function (17 questions) for patients with oa of the hip and/or knee. the patients answer the questions to describe their symptoms and difficulties from the past 3 days.(16) medial collateral ligament (mcl) thickness was measured using knee ultrasonography. 122 for examination of the medial side of knee, the patient is in the supine position and is asked to do hip external rotation with knee f l exi on of 20o – 30o. the transducer is placed obliquely in the long-axis of the mcl. mean thickness of normal mcl is 4.3mm (3.3mm – 5.6 mm).(17) data analysis all data were analyzed using the statistical package for the social sciences software v.20.0 (ibm corp). univariate analysis was done to produce descriptive data. numeric data were expressed as mean/median, and categoric data as proportion (%). the paired t test (for normal distribution) or wilcoxon signed rank test (for non-normal distribution) was used to analyze the data. two-sided p values of <.05 were regarded as the statistical significance levels. ethical clearance this study protocol was approved by the eth ics commi ttee, fac ulty of me dici ne, universitas indonesia under no. 19-08-0947. all the patients gave their written informed consent to participate in the study. results during the study, 3 subjects resigned, 1 of them claimed to have increased pain in the knee, and 2 subjects were unable to follow up due to the covid-19 pandemic. a total of 12 female subjects were analyzed (figure 1). figure 1. recruitment and flow of participants through the trial kokok, tamin, murdana, et al extracoporeal shockwave treatment decreases pain 123 univ med vol. 40 no 2 table 1. characteristics of the participants abbreviations: bmi: body mass index; kl: kellgren-lawrence; vas: visual analog scale; mcl: medial collateral ligament; womac: western ontario and mcmaster universities arthritis index characteristics mean ± sd n (%) age (years) 59.33 ± 4.33 age category (years) < 60 7 (58.33) > 60 5 (41.67) gender female 12 (100.0) male 0 (0.0) education elementary-middle school 3 (25.0) senior high school 6 (50.0) diploma 1 (8.34) bachelor 2 (16.67) profession housewives 11 (91.66) government employees 1 (8.34) bmi 28.34 ± 2.30 kl grade 2 4 (33.33) 3 8 (66.67) vas 51.09 ± 13.81 flexion degree 124.29 (120.1 – 130.2) extension degree -1.43 (-10.0 0.2) mcl 0.7 ± 0.16 womac total 37.50 ± 16.61 womac pain 9.0 ± 2.49 womac stiffness 2.75 ± 2.45 womac function 24.25 ± 11.83 comorbidity hypertension 5 (41.67) diabetes 2 (16.67) hypercholesterolemia 5 (41.67) mean age of subjects was 59.33 ± 4.33 years and mean bmi 28.58 ±2.30 kg/m2, while knee oa had kl 2 on 5 knees (35.7%) and kl 3 on 9 knees (64.3%). the characteristics of the subjects are presented in table 1. after the subjects received the intervention, at the eighth week of follow up, there was a significant improvement in pain score (p<0.001), functional abilities (p=0.007), and mcl thickness (p=0.001). however, there was no significant difference in the stiffness (p=0.317), nor the womac subscore for stiffness (p>0.05). paracetamol use was also evaluated in this study. there were 2 subjects (16.67%) who used paracetamol 3x1000 mg for 2 days in the first week, but then discontinued the drug because the pain had decreased. discussion several studies have been done previously to observe the changes in patients with knee oa after being given the eswt intervention, such as the observational study by li et al. (11) who showed improvement in subjects with knee oa after receiving eswt for 6 and 12 weeks compared with laser treatment, the changes being measured with the numeric rating scale p a r a me t e r a n d t h e we s te r n ont a r io a n d mcmaster universities index (womac). 124 t ab le 2 . m ea n ( ± s d ) o f w it h in -g ro u p d if fe re n ce s fo r p ai n , w o m a c , r o m a n d m c l o u tc o m e af te r in te rv en ti o n a b b re v ia ti o n s: b m i: b o d y m as s in d ex ; k l : k el lg re n -l aw re n ce ; v a s : v is u al a n al o g s ca le ; m c l : m ed ia l co ll at er al l ig am en t; w o m a c : w es te rn o n ta ri o a n d m cm as te r u n iv er si ti es a rt h ri ti s in d ex kokok, tamin, murdana, et al extracoporeal shockwave treatment decreases pain 125 univ med vol. 40 no 2 several other studies also compared the use of eswt with another modality. lee et al.(7) compared eswt with hyaluronic acid injection and said that eswt significantly improved the va s a nd w om ac, bu t d i d not d i ff e r significantly compared to hyaluronic acid injection in the first and third months. the study by lizis e t a l. ( 13 ) c ompa r ed esw t w it h kinesiotherapy and showed that there was a significant difference in the womac score of the eswt group compared to the kinesiotherapy group. in this study, the eswt dose used was the highest dose (0.4 mj/mm2) and no adverse effects were reported immediately from all subjects. as noted previously, imamura et al.(12) studied the administration of eswt at doses of 0.1 – 0.16 mj/mm2 to patients with grade 2–4 knee oa as compared to sham eswt and found that eswt had only a significant effect on womac. t herefore the investigators considered these doses to be less effective for treatment of knee oa. the clinical dose of eswt is usually not more than 0.5 mj/mm2. there is as yet no fixed recommended dose of eswt for treating knee oa. the intervention give n in ou r s t ud y f ol l ow e d t h e ped r o systematic recommendation, in which the treatment protocol of the optimum eswt for a musculoskeletal problem consists of 3 sessions at one week intervals, with 2000 shocks per session with the highest efd that the patients can tolerate.(1) however, because this research uses the interval method, the researchers used a larger number of total shocks to prevent the lack of shocks needed to affect knee oa. this is similar to the study by zhao et al.(10) who compared the effect of eswt to sham eswt and showed that administration of 4000 shocks in one session with 0.25 mj/mm2 efd did not show any adverse effect. because imamura et al.(12) found that doses of 0.1 – 0.16 mj/mm2 did not significantly improve knee oa, the initial efd dose (0.15 mj/mm2) was considered to be less effective in producing the eswt effects, and was only used as a warm-up. the main symptoms of knee oa, such as pain and stiffness, will hinder the patient’s daily activities and functional abilities. eventually there is decreased use of the knee as a result of this pain in an attempt to avoid pain due to activity. the evaluation of the subject’s function used the womac questionnaire, which not only assesses the oa symptoms, such as pain and stiffness, but also evaluates the daily activities, such as climbing up and down stairs, squatting and walking. with the reduction in pain, the functional limitations are also expected to decrease. our study did not use any local anesthesia in the treatment area. this is in accordance with the pedro systematic recommendation that reported decreased effectivity of the eswt if the treatment area was given local anesthesia.(18) the molecular mechanism that causes the decreased effect has not been understood yet; however, there is ample evidence leading to the central role of the peripheral nervous system in mediating the cellular and molecular effects of the shockwave whe n applied to the musculoskeletal system. these effects might be inhibited by local anesthesia. therefore, generally the shockwave application for the musculoskeletal system is recommended to be given without local anesthesia. in the present study, vas measurement was done in weeks 1, 2, 4, and 8, to observe the duration needed to effect a clinically significant de crea se in th e pa in scor e. t here was a statistically significant decrease in vas in weeks 1, 2, 4 and 8, but a decrease in vas that was considered clinically significant was only found in week 4, namely a reduction of 16.64 mm and a further reduction was seen in week 8 (21.76 mm). according to the study by concoff et al.,(2) the decrease in moderate pain that is considered clinically significant is a reduction of 14–15 mm. this is similar to the study by lee et al.(11) who reported a decrease in vas score of 22.4 mm in week 12. in several other studies, a higher reduction in vas score was reported, such as by zhao et al.(8) and li et al.,(11) who reported a 126 decrease in vas score of 37.3 mm and 47 mm, respectively, in week 12. the differences between these studies could be caused by a different basic vas score, which in the two previous studies was reported to be >70 mm. in addition, both studies did the follow up in week 12, therefore further studies with longer follow up periods might be needed to evaluate these differences. the decreasing vas pattern for 8 weeks of follow up showed that the vas score at week 8 is the lowest vas score. this agrees with the study by xu et al.,(20) which showed that‘the maximum ef fe ct of red ucing pain in the treatment of knee oa with eswt appeared in the eighth week. ge ne ra ll y, th e eff e ct ivene ss of pa in reduction after eswt application can be divided into two, namely the short-term and the longterm analgesic effects. the short-term analgesic effect can be explained by the hyperstimulation theory, in which hyperstimulation of axons by pain stimuli can produce analgesic effects.(21) the pain sensations can be affected by the stimuli to the nervous system as mechanism to block the neuronal path against the pain signals. the increasing nociceptive impulse transmission at h igh f r e qu e n c y c o ul d p r e ve nt t h e p a i n transmission according to the ‘gate control theory’ and is started by the activation of the adelta and c nerve fibers. the signals are transmitted through the type c nerve fibers to the posterior horn up to the periaqueductal gray (pag) region and retransmitted to the posterior horn as inhibitors, causing pain signals to be ignored.(22) the myelin of a-delta nerve fibers inhibit the signal transmission from the c fibers. the memory of pain can be eliminated by this mechanism, so that normal movement can be restored, the neural and muscular compensation being no longer required. thus, eswt is able to break the vicious cycle of pain. for long-term analgesia, several studies have been done to explain the biological effects of eswt on pain and concluded that some molecular and cellular changes, for example a reduction in the number of neurons that are responsible for substance p in the dorsal root ganglia, reduction in the calcitonin-gene related peptide (cgrp) of the dorsal root ganglia, and reducing the amount of u nmye li n at e d n e r ve f i be r s af t e r e sw t application.(21) substance p is concentrated in the a-delta and c nerve fibers, then released to the nociceptive neurons in the central and peripheral sensory system after stimulation. calcitonin-gene related peptide is the marker of that is typically related to the pain perception and appear simultaneously with p substance. in the activation of small diameter nerve fibers through local depolarization, axonal reflexes or dorsal root reflexes release cgrp and substance p.(23) then both of the substances will act on the peripheral target cells such as mast cells, immune cells and vascular smooth muscle cells, which will lead to inflammation. this phenomenon is known as neurogenic inflammation. this chronic inflammation might affect the chronic pain of knee oa. the reduction in substance p has been proven to be able to reduce the inflammation of the experimental animal. therefore, it can be hypothesized that the reduction in substance p of dorsal root ganglia plays an important role in mediating the long-term analgesic effects after eswt application. local selective unmyelinated neuronal damage also might play a role in the long-term analgesic effects. in this study, there were no significant changes in the degree of knee flexion as well as knee extension after intervention, whether in the first, second, fourth, or eighth weeks. there was one subject who experienced limited knee flexion (90p) and two subjects who experienced limited knee extension (-10p ), while the rest of the study subjects achieved the full rom of flexion and extension. because in most subjects the rom of knee flexion and extension was already full, the changes seen after intervention could not exceed the full knee rom. the subjects who experienced limited knee flexion and extension, also did not get improvement after intervention. all of the study subjects who has limited knee rom had grade 3 knee oa. kokok, tamin, murdana, et al extracoporeal shockwave treatment decreases pain 127 univ med vol. 40 no 2 th e limi ta tio n of rom in kne e oa generally appears in advanced oa and might be caused by narrowing of the knee joint space caused by cartilage damage and changes in the knee alignment. in other pathological conditions, such as calcified rotator cuff tendinopathy that has rom limitation due to soft tissue pathology, eswt treatment has been shown to improve the rom, such as in the study by wijayanti et al.(24) according to this study, eswt therapy has not been proven to affect the rom of knee oa patients. in the present study, it was found that the difference in womac score in weeks 1, 4 and 8 of follow up was statistically significant with a decrease in week 4 by 6.16 points. this is similar to a study by lee et al.(7) who reported a decrease in womac score of about 7.14 points in the first month. however, there was a difference in the results of the eighth week of follow up between the present study and the others. in this study, the reduction in womac score was only 8.34 points, while several previous studies reported a higher reduction in womac score. the study by lee et al.(7) reported a reduction of about 15.4 points during the twelfth week of follow up, with increasing reduction trends since the sixth week. the bmi score is a factor that plays a role in oa formation and progression and could be explained by the increased excessive load which might affect the degeneration of the cartilage. meanwhile, the adipose tissue increases the release of proinflammatory cytokines, such as il-6, il-1, il8, tnf-alpha and il-18, and decreases the regulator cytokines, such as il-10. therefore, very likely the subjects’ nutrition status affected the eswt treatment results. however, the researchers realize that more research is required to prove it. the collateral ligament edema in one subject could be caused by trauma or secondary abnormalities of meniscus extrusion and osteoarthritis. medial collateral ligament injury is marked by increased thickness, which can be examined by using musculoskeletal usg.(22) in this study, there was a significant change in mcl thickness in the fourth and eighth weeks, with the greatest reduction in the eighth week (reduced by 0.93 mm). this might be related to a regenerative effect after eswt treatment. the eswt has been proven to be able to reduce the expression of inflammatory mediators (metalloproteinases and interleukin matrix). therefore, eswt can be considered to produce a r e ge n e ra t i o n e f f e c t a n d impr ove t he mus c u l os kele t a l t i ss u e , a n d no t o n ly a disintegration effect as expected before. another study by liao et al.(23) reported that eswt has given mechanical stimuli conducted by wave sound, through mechanotransduction stimuli that are converted to sequences of biochemical signals that can enhance tissue regeneration. they also reported that there was increased production of protein, nitric oxide and growth factors, which increased neoangiogenesis, tenocyte and fibroblast proliferation, and collagen synthesis, which further increa sed tissue catabolism, healing and remodeling.(3) acoustic cavitation is also formed due to the tensile phase of the shock wave. this cavitation effect is the second effect of eswt, which is also increasing t i ss u e r e ge n e r a t io n b y in c r e ase d ti s s ue permeability, and efficiently destroying the calcification deposits of the soft tissue.(22) the series of biological events mentioned before can support the use of eswt to reduce pain, increase blood flow to the ischemic tissue, soften the calcified tissue, and release the adhesions, which will improve physical function and performance. a study by cho et al.(6) has proven that there is increased vascularization in the medial knee area that is confirmed by musculoskeletal usg evaluation at the knee immediately after eswt treatment. the eswt adverse effects are related to high doses. the adverse effects reported in previous studies were local bleeding (petechiae, hematoma) , increased pain, arr hythmias, hypertension, and peripheral nerve paresthesia. in general, researchers have concluded that eswt is a safe modality to treat knee oa. 128 however, studies with larger samples and more specific soft tissue involvement might be required to know about the increased pain after eswt application in knee osteoarthritis patients. the limitations of this study are its pre-post design and there were no control subjects to minimize other variables that may or may not interfere with the outcome measurement. regardless, the results of this study may be used as baseline data and further study implementing randomized controlled trial design with larger sample size can be done to produce higher levels of evidence supporting the use of eswt for knee osteoarthritis. conclusion there was a significant difference in pain score, functional capacity, and mcl thickness after eswt therapy, but there was no significant difference in flexibility after eswt therapy. in general, eswt is a safe modality to treat patients with knee oa. conflict of interest this study was partially funded by btl and the rest of the total cost was self-funded by the authors. acknowledgement this study was supported by the faculty of medicine, university of indonesia, by btl, a n d by a l l r e s e a r c h s ub j e c t s t h a t h a ve participated. references 1. neogi t. the epidemiology and impact of pain in osteoarthritis. osteoarthritis cartilage 2013;21: 1145-53. doi: 10.1016/j.joca.2013.03.018. 2. wallace ij, worthington s, felson dt, et al. knee osteoarthritis has doubled in prevalence since the mid-20th century. proc natl acad sci usa 2017;114:9332–6. doi: 10.1073/pnas.1703856114. 3. ahmad iw, rahmawati ld, wardhana th. demographic profile, clinical and analysis of osteoarthritis patients in surabaya. biomol health sci j 2018;1:34. doi:10.20473/bhsj.v1i1.8208. 4. medical rehabilitation department rscm outpatient data 2016. jakarta: medical rehabilitation department rscm;2016. 5. palazzo c, nguyen c, lefevre-colau mm, rannou f, poiraudeau s. risk factors and burden of osteoarthritis. ann physic rehab med 2016;59: 134-8. doi:10.1016/j.rehab.2016.01.006. 6. cho sj, yang jr, yang hs, yang he. effects of extracorporeal shockwave therapy in chronic stroke patients with knee osteoarthritis: a pilot study. ann rehabil med 2016;40:862-70. doi: 10.5535/arm.2016.40.5.862. 7. lee jk, lee by, shin wy, an mj, jung ki, yoon sr. effect of extracorporeal shockwave therapy versus intra-articular injections of hyaluronic acid for the treatment of knee osteoarthritis. ann rehabil med 2017;41:828-35. doi: 10.5535/arm. 2017.41.5.828. 8. zhao z, jing r, shi z, zhao b, ai q, xing g. efficacy of extracorporeal shockwave therapy for knee osteoarthritis: a randomized controlled trial. j surg res 2013;185:661–6. doi: 10.1016/j.jss.2013. 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knee: a pilot randomized controlled trial. j back musculoskelet rehabil 2017;30:1121-8. doi: 10.3233/bmr-169781. 14. andrews k, lu a, mckean l, ebraheim n. review: medial collateral ligament injuries. j orthop 2017; 14:550–4. doi: 10.1016/j.jor.2017.07.017. 15. delgado da, lambert bs, boutris n, et al. validation of digital visual analog scale pain kokok, tamin, murdana, et al extracoporeal shockwave treatment decreases pain 129 univ med vol. 40 no 2 scoring with a traditional paper-based visual analog scale in adults. j am acad orthop surg glob res rev 2018;2:e088. doi: 10.5435/ jaaosglobal-d-17-00088. 16. mascarin nc, vancini rl, andrade mds, magalhaes edp, lira cab, coimbra ib. effects of kinesiotherapy, ultrasound and electrotherapy in management of bilateral knee osteoarthritis: prospective clinical trial. bmc musculoskelet disord 2012;13:182. doi: 10.1186/1471-2474-13-182. 17. madani h, tincey s, tavare an, drumm o, chan o. chronic mcl syndrome: anatomy, pathology, clinical 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orthop division rev 2012;25:1-8. doi: 10.1186/1749-799x-7-11. 22. mokhtar m, singh p, editors. neuroanatomy, periaqueductal gray. treasure island (fl): statpearls publishing; 2021. 23. iyengar s, ossipov mh, johnson kw. the role of calcitonin gene–related peptide in peripheral and central pain mechanisms including migraine. pain 2017;158:543–59. doi: 10.1097/j.pain. 0000000000000831. 24. wijayanti i, murdana in, tamin tz, kekalih ak. perbandingan efektivitas extracorporeal shockwave therapy dosis menengah dengan extracorporeal shockwave therapy dosis tinggi terhadap tendinitis kalsifikasi rotator cuff (tesis). jakarta: program pendidikan dokter spesialis ikfr fkui; 2017. 25. alves ti, girish g, brigido mk, jacobson mja. us of the knee: scanning techniques, pitfalls, and pathologic conditions. radio graphics 2016;36:1759–75. doi: 10.1148/rg.2016160019. 26. liao cg, xie gm, tsauo jy, chen hc, liou th. efficacy of extracorporeal shock wave therapy for knee tendinopathies and other soft tissue disorders: a meta-analysis of randomized controlled trials. bmc musculoskelet disord 2018;19:278. doi: 10.1186/s12891-018-2204-6. january-april 2023 universa medicina vol.42no.1 pissn: 1907-3062 / eissn: 2407-2230 effect of aerobic and resistance exercises on body composition and quality of life in overweight and obese women: a randomized control trial guner cicek1* and rabia hurrem ozdurak singin1 abstract background obesity has become a major health issue in the world and directly reduces the quality of life. the aim of this study was to compare the effect of aerobic and resistance exercises on body composition and quality of life (qol) in overweight and obese women. methods an experimental study involving 90 overweight/obese women aged between 20-45 years participated in the study voluntarily and were randomized into control (n=30), aerobic exercise (ae) (n=30), and resistance exercise (re) (n=30) groups. either ae or re groups performed exercise for 60 minutes per day for 10 weeks, whereas the control group did not perform any exercise. the body composition and qol of the women were measured before and after the exercise. results body weight, bmi, fat mass, and waist and hip circumferences decreased significantly in both ae and re groups after the exercise intervention compared to the control group (p<0.05). in addition, quality of life sub dimensions including physical health, psychological health, social relationship, and environment sub-dimensions improved significantly in the ae and re groups compared to the control group (p<0.001). post hoc tukey test showed that ae was as effective as re for improving body composition parameters and qol sub dimension compared to control group. conclusion this study demonstrated that both ae and re were effective in improving body composition and thereby increasing the qol in overweight and obese women. both ae and re programs are effective and should be considered within any multicomponent therapy program in overweight and obese women. keywords: obese women, aerobic exercise, resistance exercise, quality of life 1physical education and sports department, faculty of sports sciences, hitit university, çorum, turkey *correspondence: dr. guner cicek physical education and sports department, faculty of sports sciences, hitit university, çorum, turkey tel: +900533385398 email: gunercicek@hitit.edu.tr orcid id: 0000-0002-6909-3028 date of first submission, january 18, 2023 date of final revised submission, april 6, 2023 date of acceptance, april 10, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article doi: http://dx.doi.org/10.18051/univmed.2023.v42:70-83 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1423 70 cite this article as: cicek g, ozdurak singin rh. effect of aerobic and resis tance exercises on body composition and quality of life in overweight and obese women: a randomized control trial. univ med 2023;42:70-83. doi: 10.18051/ univmed.2023.v42:70-83 mailto:gunercicek@hitit.edu.tr https://orcid.org/0000-0002-6909-3028 https://orcid.org/0000-0003-3729-5028 http://dx.doi.org/10.18051/univmed.2023.v42:70-83 https://univmed.org/ejurnal/index.php/medicina/article/view/1423 71 univ med vol. 42 no. 1 introduction obesity is defined as abnormal or excessive fat accumulation due to excessive food intake and might impair health because of the high increase in adipose tissue.(1) weight gain causes an increase in body mass index (bmi) which is calculated by dividing weight in kg by the square of height in meters.(2) while bmi between 25 to 30 kg/m2 is classified as overweight, bmi above 30 kg/m² is classified as obese.(1) obesity is a complex, multifactorial, chronic disease affected by genetic, metabolic, behavioral, physiological, and psychological components, and social determinants.(3) obesity has been accepted as a serious problem for public health all over the world due to the related complications.(4) the gradual increase in the prevalence of obesity has prompted more research into the risks associated with excess fat gain and/or being overweight.(5) overweight or obese individuals are at increased risk for serious diseases such as type 2 diabetes, cardiovascular disease, metabolic disease, and cancer.(6,7) moreover, studies have shown that obesity has been associated with a variety of negative psychological disorders, including depression, motivational disorders, low selfesteem, emotional and behavioral disorders and poor quality of life.(8,9) quality of life (qol) focuses on the physical and mental health and functional performance of individuals, thus it is an important component in assessing people’s overall health.(10) on the other hand, bmi is an indicator of health (11) and the association with qol has been studied in detail in previous research.(12,13) findings of previous studies showed contradictory results for the relationship between bmi and qol. although some studies stated that obesity has been associated with reduced qol, even if the comorbid disease has been taken under control, other studies argue that being overweight has been associated with improved qol.(14,15) overall health-related qol decreases among obese individuals but appears to increase in overweight individuals,(16) thus there is no consistent explanation for the association between high or low bmi and health-related qol.(17) physical inactivity is one of the main factors associated with obesity, whereas low physical fitness is an independent risk factor for premature death.(18) in contrast, exercise is an important strategy for managing the health risks of obesity and is considered as a milestone of the weight loss program.(19,20) therefore, various types of exercise at different intensities might be used as complementary therapies in the treatment of obesity.(21) several studies have suggested that aerobic exercise (ae) is probably the most effective exercise type for the treatment and prevention of excessive weight gain and obesityrelated morbidities, as it can be easily administered to obese individuals and provides high energy expenditure.(22,23) resista nce exercise (re) is the gold standard exercise mode for accrual of lean muscle mass, but the isolated effect of resistance training on body fat is unknown.(24) the american college of sports medicine position stand on physical activity for weight loss and prevention of weight regain (25) states that re will not promote clinically significant weight loss, and may increase loss of body fat mass only when combined with aerobic exercise.(25) a systematic review and meta-analysis showed that reductions in regional adiposity and body weight measures were also observed following combined re and ae programs including caloric restriction.(26) on the other hand, some studies have highlighted that re is also safe and effective for the treatment of overweight and obesity, especially in increasing lean body mass and physical fitness components related to muscle strength.(20,22,24) epidemiologic studies noted specific relationships between physical activity and health outcomes that include increased cardiorespiratory and muscle fitness (27) as well as decreased risk of all-cause mortality and cardiovascular disease.(28) physical training is an effective strategy for improving quality of life and emotional health status(29) and can diminish morbidities associated with obesity, such as depression and anxiety.(15) 72 studies have shown that physically active individuals have higher scores for certain subdimensions of qol domains compared to physically inactive individuals.(30,31) at the same time, physical activity helps to improve one’s physical, psychological and emotional health (32) and might positively affect the personal perception of qol and well-being.(33) in this context, the aim of this study was to evaluate the effect of a 10week re and/or ae program on body composition and qol in overweight and obese women. methods research design this study was carried out on the hitit university campus in the province of çorum in turkey as a randomized control trial with parallel group design according to consort guidelines. the study was conducted between september and december 2022 in turkey. study subjects subjects recruited for this study were selected through advertising flyers displayed on supermarket doors and around the university campus. a total of 214 participants responded to the advertisement and were invited to the campus for face-to-face interviews. after the interview for inclusion and exclusion criteria, 87 participants were not eligible for the study due to having menopause, smoking, alcohol consumption, exercising regularly for the last 6 month, having bmi below 25 and over 35 kg/m2, leaving 127 potential participants, who were asked to sign a written informed consent statement. inclusion criteria were age 20 to 45 years, sedentary (exercising >1–2 times/week), and overweight or moderately obese (body mass index 25–35 kg/ m2). subjects were nonsmokers without a history of diabetes, hypertension, or coronary artery disease. before the initial clinical evaluation, they underwent health examinations to ensure that they were not taking any medications for heart disease, and respiratory, metabolic or inflammatory disorders, and not suffering from chronic disease related to carbohydrate and lipid metabolism. sample size determination in order to generalize the research results, the size of the sample was determined using gpower 3.9.1. in order to determine the effect of aerobic and resistance exercises on body composition and quality of life in overweight and obese women, the total number required for the expectation of an effect size (f=0.60) to be statistically significant (α=0.05;1-β=0.80) was 30 for each group. randomization of subjects after written informed consent was obtained and baseline tests were completed, all subjects were asked to maintain their current lifestyle for a ten-week run-in period followed by pre-exercise testing, with subsequent randomization. both stratified and block randomization strategies were combined for grouping participants. initially, participants were stratified into overweight or obese according to their bmi and subsequently grouped in blocks of six which were determined using randomly generated numbers. ninety eight subjects started the run-in period and were then randomized into three groups: control group (n=30), ae group (n=33), and re group (n=35) while the assignment of a greater number of participants to the intervention gr oups was due to th e r isk of dropouts. participants not joining the exercise for 4 times in total and 2 times consecutively were excluded from the study, therefore the final numbers of participants were equal in all three groups, each having 30 participants for analysis. both ae and re groups participated in their respective exercise intervention programs for 10 weeks, whereas the control group did not participate in any exercise program. the exercise groups performed 10 weeks of physical training. aside from the training, all subjects were instructed to maintain the physical activity that they had been practicing previously without any caloric restriction. at baseline and cicek, ozdurak-singin exercises on body composition in overweight women 73 univ med vol. 42 no. 1 after the 10 weeks o f physical training, anthropometric and body composition measurements as well as quality of life parameters were determined in all of the groups. exercise training protocols the study groups were 1) ae; 2) re; and 3) control. the 10-week intervention consisted of a physical exercise program, including 3 sessions of training per week (on mondays, wednesdays, and fridays). a total of 30 training sessions were carried out for groups ae and re. all exercise sessions were supervised by the same trained physical education professional. training was performed under the supervision of a qualified and certified fitness instructor. women participated three times per week at an intensity of 60% of maximal heart rate for 60 minutes for 10 weeks in either aerobic or resistance exercises according to their groups. all participants performed aerobic exercises during the warm-up for 10 minutes before the main part of the exercise protocol at each exercise session, whereas both target heart rate and exercise intensity was calculated for each participant separately. the main part of the ae group was a 40-minute walk at an intensity of 60% of maximal heart rate. for the re group, the 10-week program was performed three times per week at nonconsecutive days, totaling 30 sessions with duration of 40 min each. resistance loads were 60% of one repetition maximum testing (1rm) that a person can lift only for 1 repetition for all major muscle groups. the ten different types of resistance exercises were leg press, leg curl, leg extension, lat pulldown, chest press, bench press, shoulder press, biceps curl, triceps extension and basic crunch. movements were performed in 10 repetitions and for 2 sets with 1-minute rest intervals. each exercise session ended with a 10-minute cool-down period including stretching for both exercise groups. anthropometrics and body composition height was measured to the nearest 0.1cm on a stadiometer when the participants were shoeless. the body weights of participants in bare feet, t-shirts, and tights were measured by the weighing instrument in kilograms ±0.01 kg. bmi was calculated as weight/height2 (kg/m2). the participants were asked to breathe out for measurement of their waist circumference, which was measured to the nearest 0.1cm at the iliac crest. when viewed from the sagittal plane, hip circumference was evaluated at the level of the maximum extension of the thigh, and the waisthip ratio equals the waist circumference divided by the hip circumference. the “tanita body composition analyzer bc-418” bioelectrical impedance analyzer was used to determine the body fat percentage of the subjects participating in the study. in order to determine the body compositions of the subjects, after their height, age, gender, and clothing weight were recorded and entered as data on the analyzer screen, the subjects were asked to stand on the platform with bare feet. the assessment was performed after overnight fasting, early in the morning on an empty stomach and urinary bladder. quality of life (qol) the qol was assessed using the validated turkish version of whoqol-bref that had been translated and adapted into turkish by fidaner et al.(34) the whoqol-bref contains 26 items, with each item representing one facet. the four main domains and their respective facets are as follows: (1) physical health domain: to be free of any pain, sleep and rest, mobility, having energy, mobility, activities of daily living, to be fre e of dependence on medication and treatments, and work capacity; (2) psychological health domain: happiness and enjoyment of life, to be able to concentrate, feeling positive about yourself, body image and appearance, to be free of negative feelings, and religion/spirituality/ personal beliefs; (3) social relationships: sexual activity, personal relationships, and social support; (4) environment: feeling physical safety and security, home environment, financial resources, to be able to access adequate health care, chances of ge tting new inf ormation and 74 knowledge, participation in recreation/leisure, adequate transport, and physical environment. all items on the whoqol-bref are scored on a 5-point likert scale with total scores ranging from 25 to 125 points and higher scores representing a greater qol.(34) statistical analysis statistical analysis was performed by using spss 23.0 software while the normality of data distribution was checked by using kolmogorov– smirnov test and homogeneity of variances were deter mined with levene’s test. one-way anova was used to compare group differences for body composition in terms of weight, bmi, fat mass, waist and hip circumferences, waistto-hip ratio, and for the qol sub-dimensions of physical and psychological health, social relationship, and environment. significant differences among groups were further tested by tukey post hoc analysis (honestly significantly difference, hsd). all statistical analysis tests were performed at the significance level of p<0.05 and p<0.01. ethical clearance ethics committee approval of this study, which was planned in accordance with the helsinki principles, was obtained from hitit university non-invasive ethics committee (acceptance number: 2022-goaek-0072). results of the 127 subjects who entered the 10weeks run-in phase of the study, 98 (77.1%) were randomized to one of three exercise groups. there was a 8.2% dropout rate from the exercise intervention across all groups, leaving 90 to complete the study intervention and testing. the flow diagram showing the patients participating in the study is shown in figure 1. a total of 90 obese women aged between 20 and 45 years were included in the controlled randomized experimental study, with a mean age of 35.49 ± 4.21 years for all participants. mean age for the groups were 32.80 ± 0.75 years (95% ci: 31.26-34.34) for control, 37.53 ± 0.34 years (95% ci:36.84-38.22) for ae, and 36.13 ± 0.85 years (95% ci:34.39-37.88) for re groups. the mean height of all participants was 160.21± 5.85 cm, while the mean height for groups were 161.17 ± 1.13 cm (95% ci:158.85-163.48) for control, 159.03±0.99cm (95% ci:156.99-161.07) for aerobic exercise, and 160.43 ± 1.07 cm (95% ci:158.24-162.62) for resistance exercise respectively. baseline demographic variables in terms of body composition measured as weight, bmi, fat mass, waist circumf erenc e, hip circumference, waist-to-hip ratio, and the qol sub-dimension physical health, psychological health, social relationship and environment scores for ae, re and the control groups were analyzed by anova and shown in table 1. none of these variables showed a significant difference, indicating that the randomization of participants into groups had been successfully performed (p >0.05) (table 1). at the end of the 10-week exer cise intervention program, differences among ae, re and control groups in terms of body composition parameters were analyzed with anova, the results of which are shown in table 2. according to the results, weight (p=0.003), bmi (p=0.009), fat mass (p=0.016) , waist circumfe rence (p=0.005), hip circumference (p=0.008) variables were significantly different among the groups, whereas waist-to-hip ratio (p=0.535) did not show any significant difference among the groups (table 2). variables that showed significant differences among groups were further analyzed with tukey’s hsd test for post hoc analysis and pairwise comparisons revealed statistically significant differences between control and ae (p=0.002) and between control and re (p=0.045) groups for weight, whereas the difference between ae and re was not statistically significant (p=0.563). moreover, fat mass was found to be significantly different between control and ae (p=0.024) and between control and re (p=0.048), but not between ae and re (p=0.961). cicek, ozdurak-singin exercises on body composition in overweight women 75 univ med vol. 42 no. 1 f ig u re 1 . f lo w ch ar t o f th e p ar ti ci p an ts . 76 variables treatment groups p value ae (n=30) re (n=30) c (n=30) weight (kg) 72.20 ± 11.54 74.79 ± 8.08 80.72 ± 9.42 0.003** bmi (kg/m2) 28.45 ± 3.58 29.16 ± 3.79 31.12 ± 2.81 0.009** fat mass (kg) 32.52 ± 6.48 33.87 ± 3.24 35.02 ± 4.96 0.016* waist (cm) 89.98 ± 7.97 89.30 ± 8.27 94.27 ± 8.71 0.005** hip (cm) 109.07 ± 8.01 108.67 ± 7.94 113.20 ± 8.14 0.008** waist/hip 0.83 ± 0.07 0.82 ± 0.62 0.83 ± 0.05 0.535 table 2. comparison of body composition parameters weight, bmi, fat mass, waist and hip circumferences, and waist-to-hip ratio by treatment groups after 10 weeks intervention *significance level is at p<0.05, **significance level is at p<0.01 note: data presented as mean ± sd; ae: aerobic exercise; re: resistance exercise; c: control; bmi: body mass index table 1. baseline demographics and exercise prescription by treatment groups *significance level is at p<0.05 note: data presented as mean ± sd; ae: aerobic exercise; re: resistance exercise; c: control; bmi: body mass index table 3. pairwise comparisons of post hoc analysis for control and exercise intervention groups for body composition parameters bmi, fat mass, waist and hip circumferences *significance level is at p<0.05, **significance level is at p<0.01 note: ae: aerobic exercise; re: resistance exercise; c : control; bmi: body mass index variables ae (n=30) re (n=30) c (n=30) p value body composition weight (kg) 77.52 ± 11.59 79.42 ± 7.91 81.20 ± 9.20 0.343 bmi (kg/m2) 30.55 ± 3.55 30.96 ± 3.76 31.24 ± 2.82 0.734 fat mass (kg) 36.75 ± 6.80 38.27 ± 4.28 34.76 ± 5.16 0.052 waist (cm) 94.17 ± 8.82 93.10 ± 8.47 94.0 7 ± 8.59 0.870 hip (cm) 112.67 ± 7.90 112.13 ± 8.11 112.8 3 ± 7.97 0.939 waist/hip ratio 0.83 ± 0.71 0.83 ± 0.60 0.83 ± 0.05 0.918 whoqol-bref physical health 19.67 ± 2.56 18.80 ± 2.63 19.73 ± 2.99 0.342 psychological health 15.87 ± 3.04 16.83 ± 3.91 14.93 ± 2.91 0.091 social relationship 7.03 ± 2.24 7.97 ± 1.77 6.97 ± 1.75 0.087 environment 19.07 ± 3.88 20.30 ± 4.16 19.07 ± 3.54 0.366 variables treatment groups mean difference p value weight bmi fat mass waist hip ae re ae re ae re ae re ae re re c c re c c re c c re c c re c c 2.590 8.70 6.14 0.626 2.65 3.27 0.350 3.50 3.15 0.633 6.60 5.97 0.067 5.60 5.53 0.563 0.002** 0.045* 0.699 0.009** 0.001** 0.961 0.024* 0.048* 0.953 0.008** 0.018* 0.999 0.018* 0.020* cicek, ozdurak-singin exercises on body composition in overweight women 77 univ med vol. 42 no. 1 also, bmi was significantly different between control and ae (p=0.009) and between control and re (p=0.001) but not between ae and re (p=0.699). according to the results of tukey’s post hoc analysis, waist and hip circumferences were significantly different between control and ae (p=0.008) and between control and re (p=0.018) groups, but not between ae and re groups (p=0.953) (table 3). according to anova results of qol after the 10-week exercise intervention programs, physical health (p<0.001), psychological health (p<0.001), social relationship (p<0.001) and environment (p<0.001) sub-dimensions showed statistically significant differences among the groups (table 4). pairwise comparison according to tukey’s hsd test performed for post hoc analysis revealed statistically significant differences between control and ae (p<0.001) and between control and re (p<0.001) groups for physical health, psychological health, social relationship and environment, whereas the difference between ae and re was not statistically significant for physical health (p=0.499), psychological health (p=0.931), social relationship (p=0.961) and environment (p=0.999) (table 5). discussion the findings of this study showed that re intervention for 10 weeks was as effective as ae intervention for improvement not only in body composition parameters such as weight, bmi, fat mass and waist and hip circumferences but also for qol subdimensions in overweight and obese women. interestingly, waist-to-hip ratio did not show any significant differences among groups, variables treatment group mean difference p value physical health psychological health social relationship environment ae re ae re ae re ae re re c c re c c re c c re c c 0.433 -6.07 -5,63 0.333 -9.87 -953 0.133 -3.73 -3.60 0.033 -9.53 -9.57 0.499 <0.001** <0.001** 0.931 <0.001** <0.001** 0.961 <0.001** <0.001** 0.999 <0.001** <0.001** table 5. pairwise comparisons of post hoc analysis for control and exercise intervention groups for qol subdimensions **significance level is at p<0.001 note: ae: aerobic exercise; re: resistance exercise; c: control variables treatment group p value ae (n=30) c (n=30) re (n=30) whoqol-bref physical health 26.33 ± 3.14 25.23 ± 4.93 20.17 ± 2.93 <0.001** psychological health 24.77 ± 3.47 24.43 ± 4.05 14.90 ± 3.16 <0.001** social relationship 11.10 ± 1.99 10.97 ± 1.97 7.37 ± 1.77 <0.001** environment 28.77 ± 3.40 28.80 ± 3.97 19.23 ± 3.29 <0.001** table 4. comparison of qol sub-scales physical, psychological, social, and environmental health by treatment groups after 10 weeks intervention **significance level is at p<0.01 note: ae: aerobic exercise; re: resistance exercise; c: control 78 although waist and hip circumference showed significantly differences according to the anova results after the 10-week exercise intervention. the literature states that ae is the preferred exercise type for weight loss and improving body composition, whereas re is more effective in gaining lean mass without weight loss.(24,25) resistance exercise was as effective as ae such that bmi, fat mass, waist and hip circumferences of women were similar after the exercise intervention and did not show any statistically significant difference. in the light of our findings, re can be an alternative exercise to ae for weight loss, improving bmi and reducing fat mass in overweight and obese women (tables 2 and 3). previous studies support these findings in that regular aerobic and strength exercises lead to significant reductions in body composition parameters such as body weight, bmi, fat mass, and waist and hip circumference.(35-37) skrypnik et al.(38) found a decline in body weight, bmi, waist and hip circumference values in women with abdominal obesity after three months in both groups who performed either only endurance exercise or a combined exercise program with endurance and strength training. in addition, marandi et al.(39) found that both light and moderate aerobic exercises improved body composition parameters in obese and overweight women. aerobic exercises are low-impact physical activities recommended for obese individuals with weak physical strength(40) and are recognized as the most effective strategy for improving body we ight and fat mass parameters.(41) a combined exercise program might be effective in the treatment of obesity since cardiovascular exercises burn fat stored in adipose tissue, while resistance exercises increase basal metabolism by increasing muscle mass.(42) additionally, resistance training leads to a stable resting metabolic rate, resulting in an increased likelihood of weight loss in obese individuals.(8) reviews clearly st ate that exer cise programs improve body weight and composition in overweight or obese adults due to a decline in body weight, total body fat and visceral adipose tissue. while the effect on weight and fat loss is relatively small and limited to only a few kilograms, reduction of visceral fat is likely to improve cardiometabolic health in these patients.(43) according to the european clinical practice guidelines of the european society for the study of obesity (easo), moderate but gradually increasing intensity aerobic endurance exercise should be performed for 30-60 minutes a day and most days of the week in all ages, tailored to the patient’s health status and ability.(44) on the other hand, recent research shows that strength training might be an alternative in the treatment of obesity due to numerous potential benefits of resistance exercises including increased muscle strength, prevention of sarcopenia with aging, preservation of bone mineral density and reduction of body fat.(38,45) the findings of the present study are in accordance with the literature supporting that re have a positive effect and show improvements in body composition in overweight and obese females. quality of life is a multidimensional construct that includes psychological, physical, social and environmental domains. it has been stated that physical activity is associated with an improved perception of the physical aspect of qol, as well as other domains such as the social, emotional or psychological aspects of qol.(46) a recent randomized clinical trial showed that moderate or high-intensity exercise training programs help improve the qol among obese adults.(47) in contrast, in a meta-analysis that focused on postintervention outcomes, but not on the mean change in physical, psychological, mental, social and environment qol domains, improvement was reported only in the physical health subdimension due to exercise, but not in any other qol subdimension.(48,49) the findings of the present study in terms of qol sub-dimensions including physical health, psychological health, social relationship and environment show that exercise intervention, either aerobic or resistance exercise, for 10 weeks causes an increase in qol in overweight and obese females. at the beginning of the study, cicek, ozdurak-singin exercises on body composition in overweight women 79 univ med vol. 42 no. 1 physical health, psychological health, social relationship, and environment were similar for each group, while all sub-dimensions showed significant differences according to comparisons of groups after 10 weeks of exercise intervention (table 4). pairwise comparisons with the tukey post hoc test showed that the control group had significantly lower scores compared to both aerobic and resistance exercise groups for physical health, psychological health, social relationship and environment sub-dimensions of qol. however, physical health, psychological health, social relationship and environment, were not significantly different between ae and re (table 5). the findings of the study are in accordance with the literature which shows that physical exercise has a positive effect on qol in obese individuals.(50) as a crucial therapeutic strategy for improving physical and mental health, exercise training that includes aerobics, strength, stretching and balance exercises improves qol in the general population and is consistently among the top health and wellness trends.(51,52) however, psychological problems can lead to weight gain,(53) while weight loss can lead to better psychological outcomes, which may contribute to maintaining successful weight loss.(54) the detrimental effect of obesity on physical and psychological qol domains has already been demonstrated by normal-weight adults showing be tt er qol compar ed to overweight and morbidly obese individuals.(55) longitudinal and cross-sectional studies have shown a positive relationship between physical activity and perception of better mental health and qol.(56) findings of the present study showed that exercise interventions, both aerobic and resistance exercise for 10 weeks, had a positive effect on all qol sub-dimensions and resulted in an increase in psychological health, social relationship and even in environment, whereas control group scor e s re mained the same throughout the study for all sub-dimensions. the current findings provide evidence of the effectiveness of both aerobic and resistance exercise components which might be used in combination in overweight and/or obese females in the context of weight loss. it seems that inclusion in an aerobic and resistance exercise program can result in improvements in healthrelated qol independent of changes in weight.(8) as a result, different types of physical activity can contribute to improving qol. in addition, the mental and emotional benefits may be motivating factors in people’s perception of physical activity programs, especially for overweight/obese individuals.(15) obesity is also associated with reduced life expectancy.(16) pimenta et al. (57) found an inverse relationship between bmi and all sub-dimensions of qol. in addition to physical and psychological scores, also total scores for qol showed a linear decrease with increasing bmi. global scores demonstrated in the literature found a negative linear relationship between qol and high bmi.(16,58) based on previous studies, scores of psychological sub-dimension including selfesteem, body image and emotions showed that qol decreases with weight gain.(59,60) pimenta et al.(57) stated that higher bmi is associated with lower general qol in brazilian males and females, based on lower scores in the physical health, psychological health, social relationship and environment sub-dimensions. most studies show that physical activity has a positive effect on subjective well-being in terms of happiness and life satisfaction.(61,62) the findings of our study are in accordance with the literature showing that physical activity improves body composition and increases qol, which might have positive outcomes throughout life span.(61-63) the main goal of weight loss interventions in overweight individuals or obesity treatment is to lose weight and improve physical, physiological, and psychological health, often defined as the indicator of success.(53) a few limitations of this study need to be acknowledged such as dietary intake and life habits. participants were advised not to change their daily physical activity level and eating habits, however, they were not strictly controlled for their diet and might have reduced their caloric intake 80 due to positive effect of exercise on behavioral eating. in addition to the drop-outs, the selection of participants who were particularly motivated to increase physical activity before undergoing a conventional weight loss may reduce the generalizability of study results. another limitation is the single gender and the single obesity type 1 included in the study. from a practical point of view, it might be appropriate to inform patients with overweight and/or obesity that resistance training improves not only bmi and body composition, but also qol as effectively as does aerobic exercise. since overweight and obese women get tired in a shorter time period and might be shy due to their apperance, resistance training might be a possible choice to exercise at home with a resistance band and for shorter times than walking and running outdoors. future studies should include mixed methods such as resistance training at different intensities and caloric restriction, and a complete program should be evaluated for the most effective intensity and duration according to different obesity types for both gender. conclusion it can be concluded that ae and re improve body composition and qol in overweight and obese women. exercise intervention for 10 weeks might cause a decline in body weight, bmi, fat mass, and waist and hip ratios in overweight and obese females. also, qol components such as physical health, psychological 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van rooij fja, et al. physical activity types and health-related quality of life among middle-aged and elderly adults: the rotterdam study. j nutr health aging 2018;22: 246-53. doi:10.1007/s12603-017-0902-7. 63. legey s, aquino f, lamego mk, et al. relationship among physical activity level, mood and anxiety states and quality of life in physical education students. clin pract epidemiol ment health 2017;13:82. doi: 10.2174/ 1745017901713010082. c:\users\universa medicina\down 121 abstract universa medicina microbiological profile of diabetic foot infections and the detection of meca gene in predominant staphylococcus aureus ponmurugan karuppiah1, 2, suresh s. s. raja3, and muhammad musthafa poyil4* background diabetes mellitus (dm) is a serious health problem that is rapidly expanding worldwide. staphylococcus aureus is a pathogenic bacterium which has a number of drug resistant strains. different variants of this pathogen have been isolated from patients with diabetic foot ulcers in persons having uncontrolled blood sugar level all over the world, resulting in high rates of morbidity and mortality. the objective of this study was to determine the prevalence of drug resistant staphylococcus aureus in diabetic foot infections (dfis). methods an epidemiological survey was conducted and 300 pus samples were collected from wounds, abscesses, skin and soft tissue lesions of patients having type ii diabetes with foot ulcer infections at a tertiary care hospital. further, the antibacterial susceptibility patterns of all the isolated staphylococcus aureus were determined against methicillin, oxacillin, vancomycin and novobiocin. results pathogenic bacterial species including coagulase positive and coagulase negative staphylococcus aureus, escherichia coli, klebsiella sp., proteus sp., pseudomonas sp. and citrobacter sp. were identified, among which staphylococcus was the main genus identified. a total of 13 (4.3%) isolates of coagulase positive staphylococcus aureus were resistant to methicillin. using pcr, 7 (53.8%) staphylococcal isolates were detected with the meca gene. conclusion staphylococcus aureus is the most common cause of dfis. this study demonstrates that about 53.8% of all methicillin resistant staphylococcus aureus isolates have meca genes. such a finding is the primary step in understanding and tackling the resistance mechanism. keywords: diabetic foot ulcer, staphylococcus aureus, meca, methicillin original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.121-128 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1291 may-august 2022 vol.41no.2 1department of botany and microbiology, college of science, king saud university, riyadh, saudi arabia 2department of microbiology, k.s. rangasamy college of arts and science, tiruchengode, namakkal, tamil nadu, india 3department of microbiology, government arts and science college, preambular, tamil nadu, india. 4department of basic medical sciences, college of medicine, prince sattam bin abdulaziz university, al-kharj, saudi arabia correspondence: muhammad musthafa poyil department of basic medical sciences, college of medicine, prince sattam bin abdulaziz university, al-kharj, 11942, saudi arabia email: pmusthu@gmail.com orcid id: 0000-0003-4826-3603 date of first submission, februari 2, 2022 date of final revised submission, may 20, 2022 date of acceptance, may 26, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: karuppiah p, raja sss, poyil mp. microbiological profile of diabetic foot infections and the detection of meca gene in predominant staphylococcus aureus. univ med 2022;41:121-8. doi: 10.18051/ univmed.2022.v41.121-128. 122 karuppiah, raja, poyil diabetic foot ulcer and staphylococcus aureus introduction di a b e t e s me ll i tu s ( d m ) wh i c h i s encountered when the blood glucose level is increased, poses a serious threat to the public health with high rates of morbidity and mortality worldwide.(1) unfortunately, it affects 463 million adults globally, indicating that dm is the seventh major cause of death (2,3) and this estimation is likely to be increased 1.5-fold in 2045.(4) diabetes mellitus introduces many complications such as diabetic foot ulcers (dfu), pressure ulcers and other types of venous leg ulcers. among them, dfu is affecting 15-25% of the diabetic population every year across the world,(5,6) which increases the number of limb amputations by 524% within a period of 6–18 months after the first evaluation. by infecting soft tiss ues and bony str uctu res . (7 -9 ) th e pathogenesis of dfu is unknown (10) and its incidence and severity are based on the effect of host-associated conditions such as neuropathy and also pathogen-linked factors such as colonization, microbial resistance and virulence factors.(11) initially, dfu would be a single microbial infection, but later it may turn into polymicrobial infections with both aerobic and anaerobic microbes.(12,13) bacterial virulence factors and the host resistance level play a major role in the diagnosis and management of dfu infections.(14) the treatment of dfu remains a changing one because of the overuse of antibiotics as the clinical symptoms of dfu mimic the ulcer infections. unfortunately, this phenomenon has an impact on the ecology of t he h uma n mic r obi ome, re s ul ti ng i n th e occurrence of drug-resistant organisms. many reports indicate that dfu is caused by a variety of drug-resistant organisms including methicillinresistant staphylococcus aureus (mrsa) (15,16) and hence the incidences are higher. among the mic roorganisms, staphylococcus aureus is one of the most frequently isolated dfu bacterium which is a commensal as well as human pathogen (17,18) causing approximately 30% of human infections such as sepsis, bacteraemia, pneumonia and skin inf ections. (1 9) methicilli n-resistant staphylococcus aureus has been acquired through a variety of risk factors such as prior amputation, previous hospitalization, prior antibiotic usage and stay in chronic care facilities.(20,21) methicillin-resistant staphylococcus aureus has the ability to resist all the currently used antibiotics which makes the treatment procedures costly, while the side effects related to antibiotics create difficulty in the management of dfu infections.(22) moreover, staphylococcus aureus genomes consist of variety of genes which are responsible for antibiotic resistance and other virulence factors. a study on diabetic foot infections showed that polymicrobial cultures were obtained from 83.7% of patients with a rate of isolation of 3.0 ± 1.4 bacteria per patient.(23) a meta-analysis clearly identified a high prevalence of bacterial species/ genera classically associated with diabetic foot infection, e.g. s. aureus.(24) the prevalence of methicillin-resistant staphylococcus aureus (mrsa) identified by this meta-analysis (18.0%) matches closely that of a previous metaanalysis.(25) studies indicate that the withincountry and between-country prevalence of mrsa are heterogeneous.(26,27) in addition, there is a need for evidence-based guidance to prescribe an appropriate drug of choice to concerned patients based on local data. hence, this study aimed to isolate staphylococcus aureus strains and antibiotic sensitivity profiles from diabetic foot ulcer infections and to investigate for the presence of the meca gene that codes for methicillin resistance in staphylococcus aureus. methods research design an epidemiological study was conducted in both male and female in-patients from the wards of the government district hospital, erode, india, in the period of 07-06-2021 to 23-12-2021. 123 sam ple collection and isolat ion and identification of s. aureus stains a total of 300 pus samples were collected using sterile swabs from wounds, abscesses, skin and soft tissue lesions of patients having type ii diabetes with foot ulcer infections. the swabs were transported to the laboratory without any further delay. the samples were swabbed on to macconkey, blood and mannitol salt agar plates and incubated overnight at 37o c. then, the plates were observed for colony formation. the isolated colonies were used for morphological, gram stain, biochemical analyses (coagulase, catalase, oxidase, indole, methyl red, voges-proskauer, citrate utilization, triple sugar iron agar, urease, dnase and gelatinase tests) and carbohydrate fermentation tests (glucose, sucrose, mannitol and lactose).(28) determination of antibacterial activity the isolated and identified s. aureus strains were diluted and adjusted to form cell suspensions of 0.5 mcfarland units. these suspensions were used for the disc diffusion method as described by gowri et al.(29) mueller hinton agar (mha) plates were used to study the antibacterial activity of isolated s. aureus strains against commercially available antibiotics such as methicillin (5 g), oxacillin (1 g), vancomycin (30 g) and novobiocin (30 g), which were purchased from hi media (india). briefly, the prepared mha plate surfaces were swabbed with diluted inocula of s. aureus and left for five min. after that, the antibiotic discs were placed individually and the plates were incubated for 24 hrs at 37o c. after incubation, the plates were observed for zones of inhibition. det ection of meca f r om s. aureus by polymerase chain reaction the presence of meca from all isolates of s. aureus was determined as described by akhi et al. (2 8) using the f or ward (f5’ctcaggtactgctataccacc-3’) and reverse (r 5’-cacttggtatatcttcacc-3) primers. briefly, a single bacterial colony was obtained from a fr esh subculture and resuspended in 100 μl of sterile water and 1 ml of suspension was added to each pcr ready mix. the pcr was programmed as follows: bacterial lysis and dna denaturation step of 5 min at 95o c; 30 cycles with a 30-s denaturation step at 94o c; a 30-s annealing step at 42o c; a 30-s extension at 72o c; and final 10-min extension step at 72o c. after 30 cycles, the final pcr product was detected by gel electrophoresis. gel electrophoresis for the gel electrophoresis, the resulting product was loaded onto the 1.5 % agarose gel with ethidium bromide along with standard dna 100 bp marker and the electrophoresis was performed at 50 volts using 1 x tris-borate edta (t be) as the r unning buffer. then the electrophoresis was stopped by turning off the power supply when the product had migrated a distance sufficient for separation of the dna fragments. next, the gel was observed for bands on an uv trans-illuminator. statistical analysis the microbiological experiments were performed in triplicate and all the data were statistically analysed by using ibm spss software, version 22.0 (armonk, ny, usa) and qualita tive variable s were expressed as percentages. ethical clearance this study was approved by the departmental ethical committee (ksrcas/ deciii-2021/03), at the postgraduate and research department of microbiology, k.s. rangasamy college of arts and sciences, tiruchengode, india and written informed consent was obtained from every participating patient. results isolation and identification out of 300 samples collected from patients having type ii diabetes with foot ulcer infections, univ med vol. 41 no 2 124 karuppiah, raja, poyil diabetic foot ulcer and staphylococcus aureus a b table 1. biochemical analysis of bacteria isolated from dfu infections notes: + indicates positive, indicates negative, k/kalkali slant and butt, a/aacid slant and butt, k/aalkali slant and acid butt and v-variable 172 samples were from males (58%) and 128 from females (42%) of the age groups between 45 and 85 years. from the samples, 300 distinct c o l on y mor p ho l ogi e s we r e obs e r ve d o n macconkey, blood and mannitol salt agar plates and the individual colonies were subjected to gram’s staining reaction, showing that 104 isolates were gram positive (34.4%) and 196 were gram negative (65.3%). the results of various biochemical analyses such as coagulase, catalase, oxidase, indole, methyl red, vogesproskauer, citrate utilization, triple sugar iron agar, urease, dnase and gelatinase tests, and carbohydrate fermentation tests (glucose, sucrose, mannitol and lactose) to which the isolates were subjected are mentioned in table 1. based on the microscopy, biochemical reactions and cultural characteristics, the isolates were identified as s. aureus, escherichia coli, klebsiella sp., proteus sp., pseudomonas sp. and citrobacter sp. and the results are displayed in table 2. the results also showed that, among isolated microbes n (%) coagulase positive s. aureus (cps) 31 (10.3) coagulase negative s. aureus (cns) 73 (24.3) e. coli (ec) 30 (10.0) proteus sp. (pr) 66 (22.0) pseudomonas sp. (ps) 45 (15.0) klebsiella sp. (kl) 15 (5.0) citrobacter sp. 40 (13.3) table 2. distribution of the isolates from diabetic patients with foot ulcers (n=300) 104 gram positive isolates, 31 were coagulase positive s. aureus (10.3 %) and 73 we re coagulase negative s. aureus (24.3%). in the remaining isolates, 30 were e. coli (10.0%), 66 isolates were proteus sp. (22.0%), 45 isolates were pseudomonas sp. (15.0%), 15 isolates klebsiella sp. (5.0%) and 40 isolates were citrobacter sp. (13.3%). the coagulate positive and coagulase negative s. aureus isolates were used for further analyses. name of the biochemical tests s. aureus e. coli klebsiella sp. proteus sp. pseudomonas sp. citrobacter sp. gram stain +ve cocci -ve rod -ve rod -ve rod -ve rod -ve rod coagulase + catalase + + + + + + oxidase + indole + methyl red + + + vogesproskauer + + citrate utilization + + + + + triple sugar iron agar k/a, gas +ve, no h2s a/a gas +ve, no h2s a/a, no gas, no h2s k/a gas +ve, h2s +ve k/k no gas, no h2s a/a gas +ve, h2s +ve urease, + + + v dnase + + gelatinase + + + glucose + + + + + sucrose + + + + mannitol + + + + + lactose + + + + xylose + + + + 125 univ med vol. 41 no 2 s. aureus strains antibiotics sensitivity profile$ methicillin oxacillin vancomycin novobiocin r s r s r s i r s coagulase positive (n-31) 13 (41.9) 18 (58.1) 2 (6.4) 29 (93.6) 9 (29.0) 21 (67.7) 1 (3.3) 0 (0.0) 31 (100.0) coagulase negative (n=73) 0 (0.0) 73 (100.0) 28 (38.3) 45 (61.7) 22 (30.1) 51 (69.9) 0 (0.0) 17 (23.3) 56 (76.7) table 3. antibiotics sensitive pattern of s. aureus isolates note: r=resistant, s=sensitive, i=intermediate; data presented as n (%) antibiotic sensitivity patterns the antibiotic sensitivity patterns of all isolated coagulase-positive and coagulasenegative s. aureus were determined against above mentioned commer cially available antibiotics. the results showed that, among the 31 coagulase positive s. aureus, 13 (41.9%) isolates were resistant and 18 (58.1%) isolates were susceptible to methicillin. two (6.4 %) of the isolates were resistant to oxacillin whereas 29 (93.6%) of the isolates showed susceptibility to the same anti bioti c. to th e ant ibio tic va nco myc i n , 9 ( 2 9 .0 % ) i s ola t e s show e d resistance, 1 (3.2%) isolate was of intermediate resistance and 21 (67.8%) isolates showed susceptibility. all 31 coagulase-positive s. aureus were susceptible to novobiocin and at the same time, all 73 coagulase-negative s. aureus were susceptible to methicillin (table 3). detection of meca from s. aureus the presence of meca in the isolated s. aureus was detected using the pcr technique and the result is presented in figure 1. among the 13 isolates which were susceptible to methicillin, 7 (53.8%) were shown to have the p r e s e n c e of t h e me c a ge n e a f te r pcr amplification. figure 1. detection of meca gene from coagulase positive s. aureus. lane 1: dna marker, lane 2-14: 13 methicillin susceptible isolates shows meca 126 karuppiah, raja, poyil diabetic foot ulcer and staphylococcus aureus discussion diabetic foot ulcer infections (dfuis) are serious complications of diabetic mellitus and are caused by a var iety of micr oorganisms particularly s. aureus which have strains that are resistant to many of the antibiotics in common use, making the treatment procedures complicated and costly.(30) the findings of the present study also underline the seriousness of the dfu inf ections as the y r eveal the natur e and characteristics of the bacterial isolates from a total of 300 pus samples collected from dfuis of type ii diabetes patients. based on detailed analyses, the bacterial isolates from the dfu pus samples were identified and belonged to s. aureus, escherichia coli, klebsiella sp., proteus sp., pseudomonas sp., and citrobacter sp., with 65.3% gram negative and 34.4% gram positive bacteria, a clear indication that the dfuis are polymicrobial. similarly, tiwari et al. (3 1) investigated 62 cases of dfuis and isolated 82 bacteria wherein they found that the percentages of gram negative and gram-positive isolates were 68% and 32%, respectively. the current study was correlated with a previous report of mutonga et al.(32) in whose study 80 swabs were collected and who found that the percentages of the gram negative and gram-positive populations were 65% and 29%, respectively. among them, 16% were s. aureus, 15% e. coli, 11% proteus mirabilis, 7% klebsiella p neumoniae and 7% pseudomonas aeruginosa, indicating that s. aureus is the most frequently isolated organism in dfus. many other studies have also reported that s. aureus is an important agent causing dfu, in line with the present study which reports its presence in 34.4% of patients.(33,34) the antibacterial susceptibility patterns are helpful for recommending suitable antibiotics for the treatment of dfuis. in this study, all 31 coagulase-positive s. aureus were susceptible to novobiocin and all 73 coagulase-negative s. aureus were susceptible to methicillin. an investigation by mergenhagen et al.(35) determined that the prevalence of staphylococcus aureus isolated from patients with dfuis was 89.2%, with 7.5% of mrsa and 24.8% of methicillinsusceptible staphylococcus aureus. recently, anafo et al.(36) investigated the variety of bacteria in 100 patients with dfuis in ghana and found that s. aureus was the most prevalent bacterium showing resistance to pe nicillin (100%), tetracycline (47.4%), cotrimoxazole (42.1%) and so on. nowadays, the genotypic method such as pcr plays a vital role in the detection of genes involved in the resistance mechanism. in the current study, the meca genes which are responsible for methicillin resistance were detected in s. aureus using pcr. among the 13 isolates which were resistant to methicillin, 7 isolates showed the presence of meca genes after pcr amplification and the remaining mrsa may have other genes such as mecc. our findings were correlated with an earlier report by anwar et al.(37) wherein they investigated 46 samples for the detection mrsa and predicted that 45.8% were mrsa. pcr showed the presence of meca in 41.6% of mrsa. there are many limitations for the present study, essentially to be examined at the time of interpreting its findings. firstly, the specimen collection using swabs has a demerit that it cannot isolate the bacterial pathogens from the inner parts of the ulcers such as the bones. secondly, the antibiotics that were being received by the patients in the hospital facility were also not considered. still, the present investigation report affords important basic information for future studies as a thorou gh knowledge of the microbiology of dfuis is important in monitoring the treatment and managing the adverse effect of antimicrobial resistance among high risk diabetic patients. further studies are recommended which should include samples from different levels of skin lesions in a larger number of patients and analyse the virulence factors. conclusion this study demonstrated that the 13 methicillin-resistant isolates were analysed for the presence of the meca gene using pcr indicating 127 univ med vol. 41 no 2 that 7 (53.8%) isolates had the meca gene. overall, the results suggest that dfu infections are polymicrobial in nature and comprise s. aureus as the dominant bacterial pathogen. conflict of interest the authors declare that the present study was performed in the absence of any conflict of interest. acknowledgement the authors are grateful to the deanship of scientific research, prince sattam bin abdulaziz university, al-kharj, saudi arabia for the support and encouragement in conducting the research and publishing this report. contributors pk and mmp: concept development; pk, sssr and mmp: work design and supervision; pk and mmp: sampl ing, processing, identification, antibiotic susceptibility tests; pk, sssr, and mmp: pcr, gel electrophoresis, data analysis and 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bm, oncul o, tulek n, et al. a prospective, multi-center study: factors related to the management of diabetic foot infections. eur j clin microbiol infect dis 2012;31:2345-52. doi: 10.1007/s10096-012-1574-1. 21. lee a, de lencastre h, garau j, et al. methicillinresistant staphylococcus aureus. nat rev dis primers 2018;4:18033 . https://doi.org/10.1038/ nrdp.2018.33. 22. cervantes-garcía e, garcía-gonzález r, reséndizalbor a, salazar-schettino pm. infections of diabetic foot ulcers with methicillin-resistant staphylococcus aureus. int j low extrem wounds 2015;14:44-9. doi: 10.1177/ 1534734614564053. 23. mendes jj, marques-costa a, vilela c, et al. clinical and bacteriological survey of diabetic foot infections in lisbon. diabetes res clin pract 2012;95:153-61. doi:10.1016/j.diabres.2011.10.001. 24. macdonald ke, boeckh s, stacey hj, jones jd. the microbiology of diabetic foot infections: a meta-analysis. bmc infect dis 2021;21:770. https://doi.org/10.1186/s12879-021-06516-7. 25. stacey 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three of diabetic foot ulcer. iraqi j sci 2014;55:1232–5. 34. jouhar l, jaafar rf, nasreddine r, et al. microbiological profile and antimicrobial resistance among diabetic foot infections in lebanon. int wound j 2020;17:1764-73. doi: 10.1111/iwj.13465. 35. mergenhagen ka, croix m, starr ke, sellick ja, lesse aj. utility of methicillin-resistant staphylococcus aureus nares screening for patients with a diabetic foot infection. antimicrob agents chemother 2020;24;64:e02213-19. doi: 10.1128/aac.02213-19. 36. anafo rb, atiase y, dayie ntkd, et al. m e t h i c i l l i n r e s i s t a n t s t a p h y l o c o c c u s aureus (mrsa) infection of diabetic foot ulcers at a tertiary care hospital in accra, ghana. pathogens 2021;24;10:937. doi: 10.3390/ pathogens10080937. 37. anwar k, hussein d, salih j. antimicrobial susceptibility testing and phenotypic detection of mrsa isolated from diabetic foot infection. int j gen med 2020;13:1349-57. doi: 10.2147/ ijgm.s278574. alvina 138 abstract *departement of public health, **departement of pediatrics, faculty of medicine airlangga university correspondence dr. pudji lestari, m.kes., phd. departement of public health faculty of medicine airlangga university jl. prof. moestopo no. 47 surabaya phone: 031-5020251 fax: 031-5022472 email: pules30@yahoo.com univ med 2011;30:138-45. home humidity increased risk of tuberculosis in children living with adult active tuberculosis cases pudji lestari*, florentina sustini*, anang endaryanto**, and retno asih** september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina indonesia is one of the countries with the largest number of tuberculosis (tb) cases in the world. environmental factors play significant roles in infection and disease development in children living with adult active tb cases. the aim of this study was to explore the environmental factors affecting tb risk in children, including humidity and number of people living in the same house with the children. using a cross-sectional study design, children living with active tb adults for at least 8 weeks were recruited. the subjects underwent clinical examination, tuberculin skin test (tst) and radiological investigations. home visits were conducted three times daily, namely in the morning, at midday, and in the evening, to measure humidity by digital hygrometer and to observe home conditions. of 56 index cases living with active tb adults in an urban low socioeconomic setting, 64 contact tb children were recruited. these children were classified as class i with negative tst and no clinical signs, class ii with positive tst and no clinical signs, and class iii with both positive tst and clinical signs. there were 32 (50%) class i, 10 (15.6%) class ii and 22 (34.3%) class iii children. however, a sub-sample of 43 showed positive results for mtb 16s rrna, indicating that all children were infected. the humidity cut-off point was set at 75%, p=0.04 and ep 2.09 (ci= 1.32-3.29), signifying that children living in houses with a humidity higher than 75%, were twice more likely to be tuberculin positive. environmental conditions playing a role in disease development were humidity and number of people living in the house. key words: tuberculosis, home condition, humidity, children introduction indonesia is one of the countries with the largest number of tuberculosis (tb) cases in the world, affecting people in the productive age range of 15-65 years. in endemic countries, tb in children accounts for 12-20% of all tb cases.(1) children living with active tb parents, termed contact children, are infected by m y c o b a c t e r i u m t u b e rc u l o s i s ( m t b ) , t h e causative pathogen of tb. these contact children are at higher risk for infection and d i s e a s e d e v e l o p m e n t , a n d a s a d u l t s w i l l constitute a reservoir of m.tuberculosis as well 139 univ med vol.30 no.3 a s a s o u r c e o f i n f e c t i o n f o r t b i n t h e community.(2) adverse environmental conditions in the c o n t a c t c h i l d r e n ’s h o m e s , s u c h a s h i g h humidity and poor ventilation can increase the risk of tb. moreover, a high population density in the house and a high number of household members further increase the infection rate from adult active tb cases, termed index cases, to children.(3) according to epidemiological data, areas with low socioeconomic status have a high prevalence of tb. whether children living with tuberculous adults will develop active tb depends both on the closeness of contact and the concentration of the mtb agent in the sputum of index cases. the aim of this study was to identify the home environmental conditions affecting the risk of tb infection in children living with adult active tb patients. methods research design an observational cross-sectional study w a s c o n d u c t e d f r o m s e p t e m b e r 2 0 0 9 t o november 2009 at dr. sutomo hospital, surabaya. research subjects children under 15 years of age who lived with adult open tb cases for at least 8 weeks consecutively were recruited into the study. adult tb patients with positive smears who came to the pulmonology clinic at dr sutomo hospital were asked whether or not they were living with children under 15 years. patients giving an affirmative answer were suggested t o b r i n g t h e i r c h i l d r e n t o t h e c l i n i c f o r screening. sample size determination s a m p l e s i z e w a s d e t e r m i n e d b y t h e following formula: n= {n. z2 1-α/2 p (1-p)}/ {(n-1) d 2+ z2 1-α/2 p (1-p)} where n = 140 (estimated monthly number of new sputum positive cases at the pulmonology clinic); α = 5%, therefore z2 1-α/2 = (1.96)2 = 3.84; p = proportion of affected children among contact children; d = deviation from actual value = 12.5%. the minimum sample was 50 tb patients, with all children living with them being included in the study sample. the subjects were selected by systematic random sampling. data collection the children of the tb patients were asked to visit the clinic twice, the first visit being for blood collection, tuberculin tests and chest radiographs, while the second visit was for reading the tuberculin skin test (tst) results and collection of the stool samples b r o u g h t b y t h e c h i l d r e n . c l i n i c a l a n d radiological examinations were conducted at the pediatric clinic, while tst results were examined by a physician. the homes of the c h i l d r e n w e r e v i s i t e d t h r e e t i m e s . a l l observations, interviews and measurements were performed by trained field workers. diagnosis of tuberculosis the diagnosis of tuberculosis was according to the who system as follows: table 1(4) class class type description tb-0 no tb exposure. not infected: no history of exposure, negative reaction to tst or negative in-vitro laboratory diagnostic tests. tb-1 tb exposure. no evidence of infection: history of exposure and negative reaction to tst or negative in-vitro latent tb infection (ltbi) laboratory diagnostic tests. tb-2 ltbi, no disease. positive tst or positive invitro ltbi laboratory diagnostic tests, negative bacteriologic studies (if done) and no clinical and/or radiographic evidence of active tb. tb-3 tb, clinically active. laboratory, clinical, bacteriological, and/or radiographic evidence of current disease. note: our study subjects were contact children, therefore we did not have tb class 0. table 1. description of tuberculosis class (4) 140 tuberculin test purified protein derivative (biofarma, bandung) was used for tst. results were examined after 72 hours, the presence of an induration of >10 mm in diameter indicating a positive tst, regardless of age and bcg status. measurements each adult patient’s house was visited and observed, the humidity of the house being measured by means of a digital hygrometer, and expressed as the percentage of water in the air. a dwelling is considered to be healthy at a humidity of 40-60%.(5) the cut-off point in our study was set at a humidity of 75%, which was higher than recommended in the reference,(5) because all dwellings housed tb patients. the numbers of bacilli found in index cases is very important, because it is a measure of both the degree of infectivity of the patient and the severity of the disease. the international union a g a i n s t tu b e r c u l o s i s a n d l u n g d i s e a s e (iuatld) recommends the following grading scale of smear microscopy results (table 2).(6) m. tuberculosis 16s rrna was measured on pbmc by means of a commercial kit (cobas amplicor roche), exactly as written in the m a n u f a c t u r e ’s p r o t o c o l . s e n s i t i v i t y a n d specificity of the kit, as compared to bacterial culture, was 80.2 % and 96.6%, respectively. ethical clearance ethical clearance of the study protocol was granted by the ethical committee of dr sutomo hospital. statistical analysis data were analyzed using spss version 13. the prevalence exposure rates of several e n v i r o n m e n t a l f a c t o r s , v i z . h u m i d i t y, ventilation, number of people living in the s a m e h o u s e , a n d g e n e t i c p r o x i m i t y (relatedness) of index case, were compared between tuberculin skin test (tst) results. for ordinal data, mann-whitney and kruskalwallis tests were used for comparison between groups. the level of significance was set at 0.05. results a total of 73 children under 15 years of age who were living with 56 adult smearpositive cases were examined. most of them came from households of low economic status. the mean age of the children was 6.8 ± 3.5 years (range 1 year to 15 years), with a slight excess of males (39 or 53%) over females (34 or 47%). most of the homes were crowded, with 5 ± 1 persons (range 3–9) per dwelling and calculated mean density per 10 m2 of 2 ± 1 persons (range 0.15 – 10) (table 3). acid fast bacilli (afb) counts recording/reporting no afb in at least 1 00 fields 1 to 9 afb in 100 fields* 10 to 99 afb in 100 fields‡ 1 to 10 afb per field in at least 50 fields† > 10 afb per field in at least 20 fields‡ 0/negative actual afb counts† + ++ +++ table 2. iuatld-recommended grading of sputum smear microscopy results(6) *a finding of 1 to 3 bacilli in 100 fields does not correlate well with culture positivity. the interpretation of the significance of this result should be left to the national tuberculosis program (ntp) and not to the microscopist. it is recommended that a new smear be prepared from the same sputum specimen and be re-examined. †the reporting of actual acid fast bacilli (afb) counts is recommended to allow a competent authority to determine whether the number fits the tb case definition of the ntp. ‡in practice most microscopists read a few fields and confirm the finding by a quick visual scan of the remaining fields. lestari, sustini, endaryanto, et al home humidity and tuberculosis in children 141 univ med vol.30 no.3 although 9 children had clinical tb, their t b c l a s s w a s u n d e t e r m i n e d , b e c a u s e o f negative tst results. based on the igra/ elispot results, only 71 children could be tested. in addition, there were 8 children with clinical tb but negative igra/elispot test r e s u l t s , w h o w e r e t h e r e f o r e a l s o n o t categorized as to tb class. regarding the genetic proximity of the children to the index cases, in 54 children (78%) the index cases were their parents (first degree relatives), while 19 children (22%) had non-parents as index cases. most of the children (57 or 67%) shared bedrooms with their index case and only 16 (23%) did not. the majority of the children had also longstanding contact with their index case, with a variable indicator n ( %) housing density * number of people living in the sam e house density per 10 m2 proximity to index cases genetic proximity: parents non-parents sharing the sam e room: yes no duration of contacts (weeks) * afb** index case : positive 1 positive 2 positive 3 smoking and chr onic cough sm oking household member : one more th an one none chronic cough in other household mem bers : yes no 5 ± 1 2 ± 1 54 (78) 19 (22) 57 (67) 16 (23) 40 ± 46.9 37 (51) 27 (37) 9 (12) 36 (49) 5 (6.8) 32 (42.2) 9 (12) 64 (88) table 3. distribution of housing density, proximity to index cases, smoking and chronic cough of household members (n= 73) * mean ± sd; **afb=acid fast bacilli mean duration of 40 weeks (range 8–208 weeks). as to the concentration of acid-fast bacilli (afb) in the sputum of index cases, 37 adults (51%) were positive 1(+), 27 (37%) were positive 2 (++), and 9 adults (12%) positive 3 (+++). a total of 36 children (49%) h a d o n e s m o k i n g h o u s e h o l d m e m b e r, 5 children (6.8%) had more than one smoker in the household and 32 (43%) had none (table 4). children with parents as index case had significantly different tst results and tb class compared to those with non-parents as index case. this difference was not apparent for geographic proximity to index case, bacterial density and presence of bcg scar. (table 5). in our study, mtb 16s rrna were found in all 43 sub-samples. table 4. social status and housing conditions of respondents (n = 73) var iables n (%) social st atus house ownersh ip : own ed b y respon dent own ed b y family of spouse ren ted productive person in h ouseh ol d : parents othe rs food con sum ption p er day (in th ousand rup iah) * housing conditions and en vir onm en t : size of dw ellin g* bu ilding mater ial : con crete wall board special room for children : yes no humid ity (%)* ventilation : good poor ill umin ation : dark light 31 (42) 27 (37) 15 (20) 63 (86) 10 (14) 20.8 ± 8 55.4 ± 54.6 6 7 (91.8) 6 (8.2) 2 0 (27.4) 5 3 (72.6) 73.0 ± 10.0 14 (19.2 %) 59 (80.8 %) 30 (40) 43 (60) * mean ± sd 142 the environmental variables yielding significant differences were the number of people living in the same house and the humidity, both according to tb class and tuberculin test results, while size of the house and illumination were not significantly different. the humidity cut-off point was set at 75%, p=0.04 and exposure prevalence (ep) was 2.09 (ci: 1.32-3.29), indicating that children living in dwellings with a humidity more than 75%, were twice more likely to be tuberculin positive. the high cut-off point of 60% as minimum criterion of a healthy dwelling was chosen considering that all dwellings housed tb patients (table 6). variables tb class p class 1 (n=32) class 2 (n=1 0) class 3 (n=22) humidity (%) illum ination light dark ventilation ≤ 20% > 20% size of house# number of people living in the same house genetic proximity: parent non-paren t proximity to index case sharing of bed room: yes no duration of contact (weeks) bcg scar present absent bact erial density of index case positive 1 positive 2 positive 3 66.7 ± 12.1 17 15 24 8 52.5 (9-30) 47.7 ± 1.0 19 13 22 10 33.8 (41.5) 19 13 15 11 6 76.1 ± 18.3 8 2 9 1 24.5 (6-90) 5.0 ± 0.8 10 0 9 1 40 (50.1) 6 4 6 4 0 74.5 ± 11.9 16 6 17 5 46.0 (9-216) 5.6 ± 1.4 20 2 18 4 43.7 (56.1) 15 7 10 9 3 0.03*† 0.172 0.558 0.397 0.018* 0.011* 0.275 0.754 0.791 0.441 table 5. distribution of risk factors according to tb class (n=64) * significant; #median (min-max) continuous data tested by t test/anova and mann-whitney/kruskal-wallis when not normally distributed. nominal data tested by x2 variable tuberculin skin test exposure prevalence (95% c.i.) negative (n=32) positive (n=32) humidity <75% >75% index case parents non-parents 34 7 19 13 14 18 30 2 2.09 (1.32-3.29) 4.59 (1.241 7.01) table 6. exposure prevalence for risk factors associated with tuberculin skin test lestari, sustini, endaryanto, et al home humidity and tuberculosis in children 143 univ med vol.30 no.3 discussion t b w a s d i a g n o s e d b y d e t e c t i n g t h e mycobacterium in sputum, gastric lavage, or blood. since infection is defined as colonization of the host by the agent,(7) and the 16s rrna subsample was taken randomly, we may conclude that all children were infected. this study showed for the first time that living in the same house with active tb patients for at least 8 weeks, was sufficient for children to get infected. most adult cases are secondarily infected, either through reinfection or reactivation. children may get primary infection from adults and the disease manifestations are more severe. as adults they in turn may become a source of infection to others.(8) in general, this may be the reason why tuberculosis in children is more apparent, systemic, and has broad clinical manifestations, whereas in adults with their mature immune response, there are already memory t cells against mtb, resulting in a protective immune response and a more contained pulmonary tb. unfortunately, the tb bacilli, however contained, are still living in the h o s t m a c r o p h a g e s , w a i t i n g f o r t h e h o s t immunity to wane. therefore, in an endemic country setting like indonesia there will always be the threat of a new upsurge of pulmonary tb cases, originating from the reservoir of adults who contracted tb in childhood. in addition, the increasing number of patients with hiv/aids and/or diabetes mellitus may result in a tb pandemic as new emerging disease. prospective studies have shown the tb prevalence in hhc children in colombia and senegal to be 1.6%(9) and 1.5%,(10) respectively, in contrast with a non-endemic area in the uk, where the morbidity risk in school contacts was only 1.5%.(11) the discrepancy with our study might be due to the diagnostic criteria used. diagnosis of tb in these studies was based on the results of bacterial culture and smear examination, while our study was based on mtb 16s rrna and clinical signs and symptoms. our study results support the prevailing opinion that bacterial cultures or smears are capable of detecting only a small proportion of child tb cases. the children in this study came from the same social class. we found that there were significantly more positive tsts in children with parents as index cases, as compared to children with other persons as index cases (p=0.031). however, sharing the same bedroom and index case bacterial density did not lead to significantly different tst results. these findings are similar to those of other studies, where it was found that first degree genetic proximity (parents) had an odds ratio of 3.15 times that of third/fifth degree proximity, which was similar to our threeor four-fold exposure prevalence.(3) the number of people living in the household showed a significant difference between tb class 1 and tb class 3. in this study, groups who were developing disease tended to have larger households than those with negative tst results. in gambia, on the contrary, among children under five years old, a higher risk of tst positivity was found in the smaller households, which was caused by a more intensive contact between tb parents and their childen.(3) in laos, however, no difference was found between the number of persons living with children with latent tb infection (ltbi children) and non-ltbi children.(13) the risk of tb infection is presumably influenced by the pattern of child care and local endemicity. in indonesia, where the society is communal, there are more people in one house and more people attending the children, so the probability for contacts is higher. the situation is different in laos, where the study was conducted in a rural area with a low density of people, thus no variability was found in the number of people living in a house between tst result groups. the presence of a bcg scar in the present study was essentially similar between tstpositive and tst-negative groups, being consistent with the results in gambia and 144 l a o s . ( 3 , 1 2 ) a p p a r e n t l y, a s u c c e s s f u l b c g vaccination, as indicated by the presence of a scar, is not correlated with cellular immune response to mtb infection. a low effectiveness of bcg vaccination could be due to co-infection with helminths,(13) although in all 50 fecal samples examined, no evidence of helminths was found. before the bcg era, tb disease in children was mostly non-pulmonary.(14) along with the fact that infection is systemic, bcg vaccine seems to protect the body from disease developing in other organs, but to be of no influence on the spreading process itself. that tuberculosis is correlated with humidity and other environmental factors, was shown in a russian study finding an increased incidence rate associated with high relative humidity, low ambient temperature and severe air pollution.(15) in indonesia, one case control study on children showed an eight-fold higher odds ratio (or) in cases from homes with a higher humidity,(16) in comparison to an or of 1.7 in adult cases, with a humidity cut-off of 70%,(17) suggesting that humidity has a higher impact on children than adults. this research used different approach than nurhidayah did; by given the exposure index case clear. the humidity factor here represents the condition where successful transmission happened. this was also the reason why we chose the higher cut-off point of 75%, instead of the standard 60% humidity for a healthy environment,(5) considering that all houses already had tb patients. an in vitro study on cytokine production by human cell lines found that the fungal species stachybotrys chantarum stimulated il-6 production.(18) in this connection, it should be emphasized that mycobacteria are also found in proportional numbers with fungi in moisturedamaged building materials.(19) the present study found that home humidity differed significantly between tb classes as well as between tst groups, so that humidity might a play role in host responses to tb infection and development of disease. as suggested by sun et al., humidity shifts the th1-th2 balance, resulting in disease development.(20) another environmental factor, namely altitude, was shown in a peruvian study to affect tst positivity, where the prevalence of positive tst results was lower at higher altitudes (+/6%), in comparison to tst results at sea level (+/30%).(21) similar reults were also found in vietnam.(22) our study, which was conducted in surabaya, just 5 meters above sea level, found a proportion of 50% positive tst results. these findings support the opinion that the humid and warm air near sea level may be important for mycobacterial transmission. since the indonesian national guidelines for children with tb prescribe mandatory preventive drugs for children living with tb patients,(22) the observations in this study could only made before diagnosis. for this reason, we chose a cross-sectional design for this study. however, an inherent weakness of this design is that it cannot determine cause-and-effect relationships, all variables being observed at the same time. a one-way relationship between independent and dependent variable cannot be determined with certainty. that was why, we s u b s t i t u t e d o d d s r a t i o , w i t h e x p o s u r e prevalence. conclusions housing conditions that play a significant role in the host reaction to tuberculosis are humidity and household size, i.e. the number of persons living in a household. on the other hand, presence of bcg scars, sharing bedrooms, and mycobacterial density of index cases, presumably play less important roles. in the risk approach to child tb those factors should be included on the scoring system available. appropriate housing conditions and the optimal number of persons living in a household should be taken into consideration for a long term tb national program. lestari, sustini, endaryanto, et al home humidity and tuberculosis in children 145 univ med vol.30 no.3 acknowledgment this study was performed in part on a grant from the minister of education. references 1. donald pr. childhood tuberculosis: the hidden epidemic. int j tuberc lung dis 2004; 8:627-9. 2. brent aj, anderson bst, kampmanna b. childhood tuberculosis: out of sight, out of mind? trans r soc trop med hyg 2008;102: 217–8. 3. lienhardt c, sillah j, fielding k, donkor s, mameh k, warndoff, et al. risk factor for tuberculosis infection in children in contact with tuberculosis cases in the gambia, west africa. pediatrics 2003;111:e608-14. 4. world health organization. global tuberculosis control: surveillance, planning and financing. who report 2007. geneva: world health organization;2007. 5. keman s. kesehatan perumahan dan lingkungan pemukiman. j kesehatan lingkungan 2005;2;2942 6. laszlo a. for the international union against tuberculosis and lung disease. technical guide: sputum examination for tuberculosis by direct microscopy in low income countries. 5th ed. paris: international union against tuberculosis and lung disease;2000. 7. ryan kj. sherris medical microbiology. 4th ed. new york. mcgraw hill;2004. 8. lillebaek t, dirksen a, baess i, strunge b, thomsen vo, andersen ab, et al. molecular evidence of endogenous reactivation of mycobacterium tuberculosis after 33 years of latent infection. j infect dis 2002;185:401–4. 9. del coral h, paris sc, marin nd, marin dm, lopez l, henao hm, et al. ifn gamma response to mycobacterium tuberculosis, risk of infection and disease in house hold contacts of tuberculosis patiens in colombia. plos one 2009;4:e 8257. 10. lienhardt c, fielding k, hane aa, niang a, ndao ct, karam f, et al. evaluation of the prognostic value of ifn gamma release assays and tuberculin skin test in house hold contacs of infectious tuberculosis cases in senegal. plos one. 2010;5:e 10508. 11. caley m, flower t, welch s. risk of developing tuberculosis from a school contact: retrospective cohort study, united kingdom, 2009. euro surveill 2010;15:19510. 12. nguyen th, odermatt p, slesak g, barennes h. risk of latent tuberculosis in children living in household with tuberculosis patiens: cross sectional survey in northern lao people’s democratic republic. bmj inf dis 2009;9:96. 13. elias d, akuffo h, britton s. helminthes could influence the outcome of vaccines against tb in the tropic. parasite immunol 2006;28:507-13. 14. alcais a, fieschi c, abel l, casanova jl. tuberculosis in children and adults: two distinct genetic diseases. j exp med 2005;19: 1617-21. 15. shilova mv, glumnaia tv. influence of seasonal and environmental factors on the incidence of tuberculosis. prob tuberk bolezn legk 2004;2: 17-22. 16. nurhidayah i, lukman m, rakhmawati. hubungan antara karakteristik lingkungan rumah dengan kejadian tuberculosis pada anak di kecamatan paseh kabupaten sumedang. makalah tuberculosis bandung;2007. 17. nursanti i. faktor -faktor risiko terjadinya tb paru pada umur 15 tahun ke atas di kota jambi prop nsi jambi [thesis]. semarang: program pasca sarjana universitas diponegoro;2002. 18. huttunen k, hyvarinen a, nevalainen a, komulainen h, hirvonen mr. production of proinflamatory mediators by indoor bacterial and fungal spores in mouse and human cell line. environ health perspec 2003;111:85-92. 19. torvinen e, meklin t, torkko p, suomalainen s, reiman m, katila ml, et al. mycobacteria and fungi in moisture-damaged building materials. appl environ microbiol 2006;72: 6822–4. 20. sun y, zhang y, sundell j, fan z, bao l. dampness in dorm rooms and its association with allergy and airways iinfection among college students in china: a cross sectional study. indoor air 2009;19:348-56. 21. olender s, saito m, apgar j, gillenwater k, bautista ct, lescano ag, et al. low prevalence and increased household clustering of mycobacterium tuberculosis infection in high altitude villages in peru. am j trop med hyg 2003;68:721-7. 22. vree m, hoa nb, sy dn, co nv, cobelen fg, borgdoff mw. low tuberculosis notification in mountainous vietnam is not due to low case detection: a cross-sectional survey. bmc infect dis 2007;19:109. 23. rahajoe nn, basir d, ms makmuri, kartasasmita cb, editors. pedoman nasional tuberkulosis anak. edisi kedua. jakarta: unit kerja koordinasi respirologi ikatan dokter anak indonesia;2008. high mmp-9 and tnf-a expression increase in preterm premature rupture of membranes 33 *department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ rsud dr. moewardi surakarta corresponding author: dr. sri sulistyowati, spog *department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ rsud dr. moewardi surakarta jl. ir sutami 36a surakarta, jawa tengah 57126 phone: +62812 2968 215 email: elis_spog@yahoo.co.id univ med 2016;35:33-9 doi: 10.18051/univmed.2016.v35.33-39 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 high mmp-9 and tnf-ααααα expression increase in preterm premature rupture of membranes sri sulistyowati*, yuniarsih zakia*, and soetrisno* background preterm delivery is one of the causes of high perinatal morbidity and mortality. matrix metalloproteinase 9 (mmp-9) is important for extracellular matrix (ecm) remodeling and may cause preterm labor and premature rupture of membranes (prom). tumor necrosis factorá (tnf-α) as a pro-inflammatory cytokine plays a role in stimulating uterine activity and cervical ripening by degrading the ecm of the amniotic membranes through mmp-9. this study aimed to determine differences between mmp-9 and tnf-α expression of the membranes in preterm delivery with premature rupture of membranes (pprom) and without prom. methods an analytic observational study with cross-sectional approach was conducted in 24 subjects, who were divided into 2 groups, with 12 subjects in the preterm delivery group with prom and 12 subjects in the preterm delivery group without prom. the expression of mmp-9 and tnf-α in the amniotic membrane was determined by immunohistochemistry. data were analyzed using the t test. results mmp-9 expression in the amniotic membrane of preterm delivery subjects with prom (8.6 ± 3.1%/field) differed significantly with that of preterm delivery subjects without prom (5.5 ± 2.3%/ field) (p=0.001). tnf-α expression in the amniotic membrane of preterm delivery subjects with prom (8.0 ± 3.0%/field) also differed significantly with that of preterm delivery subjects without prom (3,3 ± 1.5%/field) (p=0.000). conclusion expression of mmp-9 and tnf-α was higher in the amniotic membrane of preterm delivery subjects with prom than in preterm delivery subjects without prom and can thus be used as predictor to avoid pprom. keywords: mmp-9, tnf-α, preterm delivery, premature rupture of membranes doi: http://dx.doi.org/10.18051/univmed.2016.v35.33-39 34 sulistyowati, zakia, soetrisno mmp-9 and tnf-α expression in premature rupture introduction p r e t e r m p r e m a t u r e r u p t u r e o f t h e membranes (pprom) is the spontaneous rupture of the fetal membranes during pregnancy before 37 weeks in the absence of regular uterine contractions. preterm prom occurs in 3–8% of pregnancies and in one-third of cases results in preterm delivery. pprom is an important cause of perinatal morbidity, since it is associated with infection and compression of the umbilical c o r d c a u s e d b y l a c k o f a m n i o t i c f l u i d (oligohydramnion).(1) the prevalence rate of preterm delivery differs by country. in developed countries such as europe, the rate ranges between 5 and 11%.(2) in the usa around one of nine infants is born preterm (11.9%).(3) in indonesia the preterm prevalence rate is roughly 47.35%.(4) preterm delivery increases the perinatal and neonatal complications, with a fetal mortality risk of 1–2% and increased perinatal morbidity such as the respiratory distress syndrome and neonatal sepsis.(1) there are many factors that may cause preterm delivery, such as infection, uterine overdistension, uteroplacental ischemia, e n d o c r i n e f a c t o r s , a n d c e r v i c a l a n d immunological disorders that may trigger preterm delivery. (5) one of the causes of infections in pregnancy is premature rupture of the membranes (prom).(6) the incidence of prom in indonesia ranges from 4.5 to 7.6%.(7) prom contributes to 30–40% of preterm deliveries. the prevalence rate of prom in preterm pregnancies is around 2–3%.(6) prom is an important problem in obstetrics, as it is associated with preterm delivery.(8) matrix metalloproteinase-9 (mmp-9) is the main mmp involved in normal delivery and also plays an important role in pathological delivery. matrix metalloproteinase-9 is the most active mmp in the amnion, and is found to increase significantly in the amniotic membranes after the onset of contractions. matrix metalloproteinase, also known as matrixin, plays an important role in the breakdown and remodeling of the extracellular matrix (ecm), finally causing both p r e t e r m d e l i v e r y a n d p r e t e r m p r o m . manipulation of mmp may play a role in preventing spontaneous preterm delivery.(9) preterm prom increases mmp-9 concentrations in the amniotic fluid, causes amniotic fluid infection, threatened preterm delivery and poor neonatal outcome.(10) tumor necrosis factor-α (tnf-α) is a proinflammatory cytokine that is mainly produced in monocytes and/or macrophages. excessive secretion of tnf-α may cause deleterious effects, such as abortion and preterm delivery. increase or non-increase of the tnf-α proinflammatory cytokine presumably plays an important role in preventing preterm delivery.(11) in preterm delivery, tnf-α stimulates uterine activity and the cervical ripening process by producing prostaglandin and cortisol, and degrading the ecm of the membranes through the mmp-2 and mmp-9 pathways.(12) the tnf-α concentration in the amniotic fluid in preterm delivery increases significantly in comparison with term delivery. the tnf-α (and/or il-6) concentration in amniotic fluid at mid-trimester may identifiy pregnant mothers at risk for chorioamnionitis, which may cause preterm delivery.(13) the purpose of this study was to compare the expression of mmp-9 and tnf-α in the membranes in preterm delivery with prom and that in preterm delivery without prom. methods design of the study an analytical-observational study with crosssectional approach was conducted at the department of obstetrics and gynecology, rsud dr. moewardi surakarta, from december 2014 until may 2015. subjects of the study the study subjects were pregnant mothers who had undergone preterm delivery with prom and without prom. the sample size was 12 per 35 group, based on the difference between the prevalence of preterm delivery without prom and the prevalencs of preterm delivery with prom, the difference being 0.3, with α=1.96 and β= 0.2.(14) there were thus 12 preterm delivery subjects with prom and 12 preterm delivery subjects without prom. the inclusion criteria were pregnant mothers delivering at rsud dr. moewardi surakarta, who had undergone preterm delivery with prom and preterm delivery without prom, and whose infants were without major congenital abnormalities. the exclusion criteria were pregnant mothers with chronic diseases such as diabetes mellitus, kidney and heart diseases, and hypertension; pregnant mothers with systemic inflammation and infection, such as pneumonia, typhoid, malaria, hepatitis; pregnant mothers who were smokers, drinkers, and drug abusers, on antibiotic treatment, with trauma in pregnancy, twin pregnancies, intrauterine fetal death, and abnormally shaped uteri. immunohistochemistry for all subjects, immunohistochemical examination of mmp-9 and tnf-α expression in the membranes was performed at the pathological anatomy laboratory, universitas sebelas maret, surakarta. matrix metalloproteinase 9 was determined immunohistochemically by the avidin biotin complex method, using bioss mmp-9 antibody reagent bs-0397r. mmp-9 expression in the membranes was examined under the microscope at 400x magnification. mmp-9 expression for a strong positive result was shown by a reddish-brown color in the cell nucleus and the cytoplasm, for a moderate positive result by a dark brown color, for a weak positive result by a light brown color, and for a negative result by a bluish color. the histological score was then calculated as percentages per field of view, the data being of continuous scale. similarly, expression of tumor necrosis factor α was determined immunohistochemically by the avidin biotin complex method, using bioss tnf-α antibody reagent bs-0078r. expression of tnf-α in the membranes was examined under the microscope at 400x magnification. tnf-α expression for a strong positive result was shown by a reddish-brown color in the cell nucleus and the cytoplasm, for a moderate positive result by a dark brown color, for a weak positive result by a light brown color, and for a negative result by a bluish color. the histological score was then calculated as percentages per field of view, the data being of continuous scale. data analysis the data were analyzed with the t-test using spss, with p<0.05 being considered significant. ethical clearance ethical clearance wa obtained from the commission for medical research ethics, rsud dr. moewardi/ faculty of medicine, universitas sebelas maret, under no. 642/xi/hrec/2014, dated 11 november 2014. results there were 50% working mothers in the preterm delivery group with prom and 25% in the preterm delivery group without prom. the majority of mothers (75%) in the preterm delivery group with prom as well as in the preterm delivery group without prom, had a junior high school education. the percentage of primigravida in the preterm delivery group with prom and the preterm delivery group without prom was 75% and 50%, respectively (table 1). the majority of pregnant mothers were 21– 35 years old, accounting for 83% of the preterm delivery group with prom, and for 75% of the p r e t e r m d e l i v e r y g r o u p w i t h o u t p r o m . regarding the age of pregnancy of 34–36 weeks, the percentage in both the preterm delivery group with prom and the preterm delivery group without prom was identical, namely 58% (table 1). from table 2 it may be seen that the mean mmp 9 expression in the membranes was significantly higher in the preterm delivery group with prom (8.6 ± 3.1%/field), in comparison univ med vol. 35 no.1 36 sulistyowati, zakia, soetrisno mmp-9 and tnf-α expression in premature rupture table 1. distribution of characteristics of study subjects table 2. mean expression of mmp-9 and tnf-α in the membranes in the preterm delivery group with prom and the preterm delivery group without prom *significant at p<0.05 with the preterm delivery group without prom (5.5 ± 2.3%/field) (p=0.001). the mean expression of tnf-α in the membranes was significantly higher in the preterm delivery group with prom (8.0 ± 3.0%/field) than in the preterm delivery group without prom (3.3 ± 1.5%/field) (p=0.000). expression of mmp-9 and tnf-α in the membrane of the pretern delivery groups with and without prom were showed in figure 1 and figure 2. discussion in our present study we found that the mean mmp-9 expression in the membranes was significantly higher in the preterm delivery group with prom (8.6 ± 3.1%/field) in comparison with the preterm delivery group without prom (5.5 ± 2.3%/field). prom is one of the factors for the initiation of delivery and is an important p r o b l e m , s i n c e i t i s a s s o c i a t e d w i t h t h e complications of pregnancy, one of them being preterm delivery, which results in both infant morbidity and mortality.(8) the pathogenesis of preterm delivery is associated with cytokines, matrix metalloproteinases, and prostaglandins. the amniotic membrane is the inner layer of the fetal membranes that line the amniotic cavity. the amnion consists of an epithelial cell layer above the thicker basement membrane and a spongy collagen layer containing mesenchymal cells. the amnion is part of the fetal development and protects the fetus against mechanical injury by surrounding it with amniotic fluid. the amnion receives its strength from collagen in the basement membrane, especially type iv collagen. collagen in the basement membrane and collagen 37 mmp-9 expression in preterm delivery group with prom mmp-9 expression in preterm delivery group without prom figure 2. expression of tnf-α in the membranes of the preterm delivery group with and without prom, determined by immunohistochemistry and evaluated using an olympus cx21 microscope at 400x magnification. reddish-brown color in the cell nucleus and the cytoplasm indicates a strongly positive result; dark brown = moderately positive result; light brown = weakly positive result; bluish color = negative result. tnf-α expression was stronger/higher in the preterm delivery group with prom in comparison with the preterm delivery group without prom. tnf-α expression in preterm delivery with prom tnf-α expression in preterm delivery without prom figure 1. expression of mmp-9 in the membranes of the preterm delivery groups with and without prom, determined by immunohistochemistry and evaluated using an olympus cx21 microscope at 400x magnification. reddish-brown color in the cell nucleus and the cytoplasm indicates a strongly positive result; dark brown = moderately positive result; light brown = weakly positive result; bluish color = negative result. mmp-9 expression was stronger/higher in the preterm delivery group with prom in comparison with the preterm delivery group without prom univ med vol. 35 no.1 38 sulistyowati, zakia, soetrisno mmp-9 and tnf-α expression in premature rupture degradation in the chorioamnion is controlled by matrix metalloproteinases (mmps). matrix metalloproteinase-1 (mmp-1) degrades type i, ii and iii collagens, while mmp-2 and mmp-9 (gelatinase b) degrade type iv collagen. in human chorionic cells, tumor necrotic factor alpha (tnf-α) has been found to promote the production of mmp and prostaglandin e2 (pge2), and to repress tissue inhibitors of metalloproteinase [timps]). therefore, tnf-α tends to cause the membranes to weaken and rupture through collagen degradation in the ecm.(15) increased concentrations of interleukin-6 (il-6), c-reactive protein (crp), and matrix metalloproteinase 9 (mmp-9) in the fetal and neonatal compartments have been shown to be associated with increased risk of preterm delivery.(16) mmp-9 expression, apoptosis index (ia) of cervical cells, and mmp-9 gene polymorphism (c-1562t) in pregnancies of 21– 36 weeks without and with prom have been demonstrated by sabarudin et al.,(17) who found no significant differences in mmp-9 expression and cervical cell ia between preterm delivery with prom and preterm delivery without prom. in the present study, mean tnf-α in the membranes was found to be higher in the preterm delivery group with prom than in the preterm delivery group without prom. in preterm d e l i v e r y c a s e s , t n f α a n d o t h e r p r o inflammatory cytokines play a role in stimulating uterine activity and cervical ripening by p r o d u c i n g p r o s t a g l a n d i n s , c o r t i s o l , a n d degrading the ecm of the amniotic membranes through the mmp-2 and mmp-9 pathways.(12) tnf-α is a predictor of prom, since increased tnf-α levels in the patient’s serum may trigger mmp-9 expression, thus causing increased degradation of type iv collagen in the membranes of pregnant women and possibly causing prom.(8) tu m o r n e c r o s i s f a c t o r, c a c h e x i n o r cachectin, previously known as tumor necrosis factor-α, is a pleiotropic inflammatory cytokine that is mainly produced in monocytes and/or macrophages. excessive secretion of the proinflmmatory cytokines tnf or il-1, such as occur in infections of the amnion, has been known to be detrimental to intra-uterine tissues such as in abortion and preterm delivery.(11) cytokines can increase the production of prostaglandins in the amniotic membranes and decidua. various factors may be involved in the release of tnf-α and the activation of mmp-9, such as psychological stress, which causes the release of corticotropin releasing hormone (crh), infection, and inflammation, that directly stimulates the release of inflammatory mediators, such as tnf-α. the concentration of bioactive tnf, the availability of tnf receptors (tnfr1 and tnfr2), and the affinity of tnf for its receptors, and also its signaling pathway, may display different kinerja of tnf-α. tnf-α in most frequently involved in the activation of mmp-9.(13) mmp-9 increases significantly upon exposure of the amnion to tnf-α or il-1 beta, whereas its secreted concentration in the chorion is not altered. each of the inflammatory cytokines (il-1, il-6, and tnf-α) have been found in higher concentrations in the amniotic fluid of women with prom. the cytokine that influences the mmp family in pregnancy is tnf-α, which is the most characterized cytokine. this member of the proinflammatory cytokines has been shown to be able to trigger mmp expression in various tissues, thus causing the release of active mmp-9 from the amniotic membranes and also triggering apoptosis in the amniotic membranes and activation of mmp-9, which finally causes prom.(9) advanced understanding of the biochemical causes of fetal membrane weakening and rupture indicates the possibility of inhibiting the process and ultimately preventing pprom.(18) in this study, we did not discuss in detail the causes of prom in the study groups, e.g. we did not examine the occurrence of urinary tract infection (uti). we also did not differentiate the duration of prom or the degree of infection. another limitation of this study was that we did not equalize the leukocyte concentrations in the 39 preterm delivery groups, despite the fact that this can influence the mmp-9 and tnf-α concentrations in preterm deliveries with prom. further studies are required that control for confounding variables such as the causes of prom and the degree of infection. it is expected that pregnant mothers with prom may be detected earlier so that they can be better managed to prevent preterm delivery resulting in morbidity and mortality of the mother and her fetus. conclusions the expression of mmp-9 and tnf-α were higher in the membranes of preterm deliveries with prom, in comparison with preterm deliveries without prom. mmp-9 and tnf-α may be strong predictors of prom, particularly in preterm deliveries. conflict of interest the authors declare that there is no conflict of interest associated with this study, the authors, and / or the publication of this article. references 1. okeke tc, enwereji jo, okoro os, et al. the incidence and management outcome of preterm premature rupture of membranes (pprom) in a tertiary hospital in nigeria. am j clin med res 2014;2:14–7. 2. martin ja, hamilton be, sutton pd, et al. births: final data for 2005. natl vital stat rep 2007;5;1-103. 3. spong cy. prediction and prevention of recurrent spontaneous preterm birth. obstet gynecol 2007; 110 2 pt 1:405–15. 4. sudarmi. hubungan ketuban pecah dini > 12 jam dengan gawat janin di ruang bersalin rsup ntb tahun 2012. media bina ilmiah 2013;7:31–4. 5. razzak msa, al-sa’adi mak, hussainy taia. the role of tumor necrosis factor-alpha (tnfα) in the induction of preterm labor. karbala j med 2010;3:779–83. 6. veleminsky m, sak p. management of pregnancy with premature rupture of membranes (prom). j health sci management public health 2006; 192–7. 7. wiradharma, kardana i, wyn dai. risiko asfiksia pada ketuban pecah dini di rsup sanglah. sari pediatri 2013;14:316-9. 8. wibowo ap, sulistyowati s, respati sh. difference of serum mmp-9 and tnf-α level in preterm and term premature rupture of membranes. ijog 2015;3:15–8. 9. weiss a, goldman s, shalev e. the matrix metalloproteinases (mmps) in the decidua and fetal membranes. front biosci 2007;1:649–59. 10. yoon bh, oh sy, romero r, et al. an elevated amniotic fluid matrix metalloproteinase-8 level at the time of mid-trimester genetic amniocentesis is a risk factor for spontaneous preterm delivery. am j obstet gynecol 2001;185: 1162-7. 11. haider sm, knofler. human tumour necrosis factor: physiological and pathological roles in placenta and endometrium. placenta 2009; 30:111–23. 12. calleja-agius j, muttukrishna s, jauniaux e. the role of tumor necrosis factor-receptors in pregnancy with normal and adverse outcome. int j interferon, cytokine mediator res 2012;4: 1–15. 13. thomakos n, daskalakis g, papapanagiotou a, et al. amniotic fluid interleukin-6 and tumor necrosis factor-a at mid-trimester genetic amniocentesis: relationship to intra-amniotic microbial invasion and preterm delivery. eur j obstet gynecol reprod biol 2010;148:147–51. 14. hulley sb, cummings sr, browner ws, et al. designing clinical research. 4th ed. new york: lippincot williams & wilkins, wolters kluwer; 2013. 15. izumi-yoned n, toda a, okabe m, et al. alpha 1 antitrypsin activity is decreased in human amnion in premature rupture of the fetal membranes. mol hum reprod 2009;15:49-57. 16. sorokin y, romero r, mele l, et al. maternal serum interleukin-6, c-reactive protein, and matrix metalloproteinase-9 concentrations as risk factors for preterm birth <32 weeks and adverse neonatal outcomes. am j perinatol 2010; 27:631–40. 17. sabarudin u, mose jc, krisnadi sr. polimorfisme gen mmp-9, ekspresi mmp-9, dan indeks apoptosis sel serviks pada kehamilan 21 – 36 minggu. mkb 2011;43:199–206. 18. kumar d, schatz f, moore rm, et al. the effects of thrombin and cytokines upon the biomechanics and remodeling of isolated amnion. in vitro placenta 2011;32:206-13. univ med vol. 35 no.1 alvina 197 abstract as older persons are steadily increasing in number and there are no specialized comprehensive healthcare services for older persons in indonesia, including east kalimantan, the aim of the present study was to determine the extent of the problems facing healthcare staff and officials in tarakan city, east kalimantan, in providing comprehensive healthcare services attuned to the needs of older persons.this study was a qualitative interview-based survey with focus group discussions, involving heads and healthcare officials of seven puskesmas in tarakan city, east kalimantan district, with the addition of a number of district health planning officials. the results revealed a difference between daily hospital referral rate of older persons by puskesmas staff and actual daily hospital admission rate for the referrals. none of the consulted healthcare staff had any speciality education in geriatrics or older persons health. the older persons most frequently presented to the hospital with hypertension, diabetes, and myocardial insufficiency. on the other hand, at the health centers the presenting conditions were hypertension, gastroenteritis, rheumatism, sleep disorders, and upper respiratory tract infections. improved access to healthcare for older persons should be achieved by improving knowledge and skills of human resources, including communication skills, and of supporting healthcare service infrastructure at puskesmas, specifically oriented towards the needs of and designed for use by older persons, such as ramps for wheelchairs, and handrails in corridors. local governments should strengthen the appropriate service delivery to older persons, and provide support for the maintenance, sustainability and strengthening of community-based care for older persons. keywords: health services, community based-care, older persons, tarakan city *department of public health, faculty of medicine, trisakti university **dicipline of public health, university of adelaide, south australia ***district health office, tarakan city, east kalimantan correspondence dr. rina k. kusumaratna, dr, m.kes. department of public health, faculty of medicine, trisakti university jln. kyai tapa no. 260, grogol jakarta barat – 11440 phone: 021-5672731 ext.2504 email: rkusumaratna@gmail.com univ med 2011;30:197-204 health services needs of older persons: emerging findings from tarakan city, east kalimantan september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina rina kusumaratna*, mohammad afzal mahmood**, maskito asmadi soerjoasmoro*, and khairul*** introduction currently the ideal of old age does not only focus on the added number of years, but has the additional requirement of quality of life (qol), with emphasis on healthy lifestyle as determinant of qol. this shift in concept n e c e s s i t a t e s t h e i m p l e m e n t a t i o n o f a 198 kusumaratna, mahmood, soerjoasmoro, et al physical activity reduced hypertension in elderly comprehensive health care program from health center up to hospital level. populations in developing countries are aging more rapidly than those in the developed world, and this has implications for medical training in these health institutions. by knowing older persons’ health care needs it should be possible to build a strong health program for older persons. improvements in life expectancy have resulted in incremental growth of the older p o p u l a t i o n g r o u p , a c c o m p a n i e d b y a correspondingly smaller increase in the younger population groups, thus altering the balance between the younger and older population g r o u p s . t h e a v e r a g e l i f e e x p e c t a n c y i s predicted to rise continuously from 67 to 74 years by the year 2025. the increasing age and size of the older population group lead to public health issues, the solution of which should be implemented through appropriate policies and programs that stress the right of older persons to stay healthy.(1-4) up to the present time, the health centers in south jakarta have not yet i m p l e m e n t e d c o m m u n i t y b a s e d p r i m a r y healthcare services for older persons, who still receive the same kind of treatment as the other age groups, while there are only few hospitals with geriatric wards and specialists in geriatric medicine.(5-7) primary healthcare services for older people can play an integral part in healthy aging a n d t h e l i f e c o u r s e a p p r o a c h . p r i m a r y h e a l t h c a r e s e r v i c e s c a n s u p p o r t h e a l t h promotion, disease reduction and prevention, early diagnosis, and effective management of chronic illnesses. the reality of primary healthcare services in the developing countries is that healthy aging and the life-course approach is not well-understood or applied by the healthcare providers at the primary level. in the area of health promotion, health care providers typically do not spend time with older persons to encourage the development of behavioral changes (for example, healthy diets and routine exercise) that can reduce the risks of chronic illnesses. in indonesia there has so far been little research conducted to identify the preparedness of the health services in the public sector for providing specialized care to older persons, particularly in places outside j a k a r t a . l i k e o t h e r p r o v i n c e s , t h e e a s t k a l i m a n t a n h e a l t h s e r v i c e s y s t e m i s decentralized at the district level. district health services are striving to improve access to care and quality of care, particularly for the socioeconomically disadvantaged, including older persons. the east kalimantan government has launched initiatives to strengthen its health system. because of the need to build access for older persons due to their increased needs for health services, the east kalimantan government is keen to increase the health services access for every resident of east kalimantan, including older persons. the objective of the present study was to determine the extent of the problems facing healthcare staff and officials with regard to health services f o r o l d e r p e r s o n s i n ta r a k a n c i t y, e a s t kalimantan. in particular, the study aimed to obtain input from these officials on challenges and opportunities of a decentralized district health service system to improve access to comprehensive and integrated care for older persons. methods research design qualitative methods were used through ind e p t h i n t e r v i e w s i n v o l v i n g d i f f e r e n t stakeholders, such as health care providers, officials at puskesmas and district health officers. the research project was completed in 4 months, from september to december 2010. study subjects the study interviewed 14 respondents f r o m 7 p u s k e s m a s , c o n s i s t i n g o f t w o healthcare staff from each puskesmas, namely the head of puskesmas and the healthcare provider or official for older persons. in 199 addition, the respondents included 3 district government officials responsible for older persons’ healthcare planning at district level. data collection after obtaining informed consent from the respondents for the qualitative interviews, initial guided conversations were held, for determining the main topics for the interviews, which were conducted by personal face-to-face contacts. during the interviews, all of the conversation were recorded using an audio recorder, which w e r e s u b s e q u e n t l y c o n f i r m e d b y t h e respondents. however, due to difficulties in the field, most of the data were recorded by handwritten notes only, with the important points of the interviews being transcribed afterwards. the qualitative data were converted into electronic format and stored in secure passwordprotected computer for the duration of the project. any data not in electronic format (e.g. completed copies of surveys) were stored in a locked filing cabinet at the faculty of medicine, trisakti university, jakarta. statistical analysis the data analysis provided a description of a narrative explaining the main issues, marking the underlying ideas in the data, grouping similar information together, and relating different ideas and themes to one another. in the final stages of the analysis, the data and links were organized to create a clear description of an issue topic. analysis of the issues of discussion with healthcare providers and officials identified their perspectives on factors determining the quality of care, and the need of comprehensiveness of integrated care for older persons. ethical clearance the study was approved by the medical research ethics committee of the faculty of medicine, trisakti university, jakarta and the human research ethics committee of the university of adelaide. results i n f o c u s g r o u p d i s c u s s i o n s w i t h 1 7 healthcare officials at health center and health district level, the issues included their knowledge on geriatric medicine and gerontology, and older persons’ health, and their training in these areas. additionally, they were asked about the main type of illness of older persons and the readiness of the healthcare services for older persons. table 1 shows the characteristics of officials responsible for the healthcare of older persons. extent of service provision and health center-to-hospital referrals the healthcare staff at the health centers reported that every day on average between 5 and 15 older patients visited each puskesmas, and that on average they referred about four patients to hospitals every day. however, the daily hospital admittance rate was reported to be only about one older person. according to puskesmas staff, they provided the history of the disease, symptoms, and information on use of medicines with their referral letters. most of these referrals were for hypertension, diabetes mellitus complications, heart disease a n d s t r o k e . i t w a s m e n t i o n e d t h a t t h e s e puskesmas have access to 24-hour ambulance service, and if needed the patient could be transferred from the puskesmas to the hospital using the puskesmas ambulance. table 1. characteristics of officials responsible for older persons’ healthcare variables n (%) age (mean,sd) yr 35 ± 7 gender male 9 (53) female 8 (47) location puskesmas 14 (82) district health office 3 (18) specialty physician / dentist 11 (65) nurse / midwife 6 (35) univ med vol.30 no.3 200 kusumaratna, mahmood, soerjoasmoro, et al physical activity reduced hypertension in elderly education, training, and experience in older persons’ health none of the consulted healthcare staff had any speciality education in geriatrics or older persons’ health, and only one had received some training about healthcare for older people. this training focussed on nutrition, mental health, and health promotion. one of the healthcare staff had attended a seminar two years ago, on disease prevention and maintenance of the health of the eldery. the healthcare staff mentioned that their experience was based on their examining older persons on a daily basis. they also pointed to the availability of some manuals on management of diseases among older persons. however, these healthcare providers were not familiar with various screening instruments, except the geriatrics depression scale, that was used in hospitals, and helped identify mental health issues among older persons. signs and symptoms the older persons in this study most frequently presented to the hospital with h y p e r t e n s i o n , d i a b e t e s , a n d m y o c a r d i a l insufficiency. on the other hand, at the health c e n t e r s t h e p r e s e n t i n g c o n d i t i o n s w e r e hypertension, gastroenteritis, rheumatism, sleep disorders, and upper respiratory tract infections. challenges to provision of care to older persons in addition to the lack of training and education in geriatrics, the staff pointed to the issue that many older persons presented with multiple and complex problems. diagnosis and treatment of a combination of diseases is quite difficult and challenging for the local staff. the s t a f f a l s o p o i n t e d t o t h e d i f f i c u l t y i n communication with older patients. suggestions by the healthcare staff to improve care for older persons it was suggested that there is a need for training in geriatrics and in communication skills while providing care to older persons, as only a small percentage (24%) of healthcare officials had attended workshops or seminars in geriatrics a n d h a d s o m e k n o w l e d g e o f g e r i a t r i c assessment tools (table 2). additionally, more than 50% of the respondents had problems related to diagnosis and therapy. it was also highlighted that medicines needed for treating multiple complex issues were at times not available, as is the case with the needed laboratory investigations. therefore, the staff also suggested improving the availability of medicines and providing a comprehensive range o f l a b o r a t o r y t e s t s f o r o l d e r p e r s o n s . furthermore, the healthcare staff at puskesmas pointed to the need for a special emphasis on older persons care by providing specific areas within the health center for interacting with older persons and administering prompt treatment. the interviewed staff mentioned that improving t h e q u a l i t y o f h e a l t h c a r e n e c e s s i t a t e s improvement of the skills and knowledge of the staff, and of the available infrastructure, including allied health. discussion the findings revealed several issues that could be listed for improving and strengthening t h e e x i s t i n g p r o g r a m s t h a t h a v e b e e n implemented in the field. the point of view of the healthcare staff indicated that education and training skills in older persons’ healthcare system n e e d s t o b e i m p r o v e d , i n c l u d i n g t h e infrastructure, so that the quality of services for older persons thereby will also improve. these table 2. experiences in older persons’ healthcare variables n (%) geriatric medicine / gerontology conference 4 (24) geriatric assessmen t tools 4 (24) problem s in diagnosis 10 (5 9) problem s in therapy 11 (6 5) 201 needs are equivalent to the factors affecting the c h a n c e s o f o l d e r p e r s o n s f o r a c c e s s i n g comprehensive healthcare at the health center. improvement of communication skills between medical practitioners and older persons or their caregivers is essential for improving the quality of care. service providers could directly support medical management, especially adherence to medication (compliance) and provide patients with accurate information related to health education, that they might need for understanding and managing their illness. healthcare service providers should have sufficient knowledge and skills to provide services of high quality and give satisfaction to their clients. in addition, healthcare workers should also have an awareness of their negative attitudes to older people and the lack of involvement of older people in decision-making about their care and treatment. as stated by the who,(8) the community health care (chc) framework has proposed the primary health care (phc) approach to better address the needs of older persons. the approach provides a framework for integrating health and welfare systems and strengthening of older peoples’ relations with their family and communities, regardless of abilities or disabilities. current impediments to holistic patientcentered care are to be found in the short time allocated for consultations and the single-disease approach in consultations. older persons frequently suffer from co-morbidities and therefore need a multidisciplinary approach that is focused on their individual needs, instead of the medically oriented singular approach, which leads to fragmentation of care and treatment. o t h e r a r g u m e n t s f o r i m p l e m e n t i n g a multidisciplinary approach include both raised community expectations for health care as well a s e c o n o m i c c o n s i d e r a t i o n s o f c o s t effectiveness. in indonesia, accessibility of health services is relatively good, while accessibility of public services is still limited. since 2001, the indonesian ministry of health has launched the “puskesmas santun usia lanjut” program for older persons, consisting of five levels of preventive activities, but the results were limited.(5,8) thus it is important to maintain the accessibility of older persons’ health services at the highest level and improve the existing public health services to support older persons’ accessibility to public facilities. the need is for a comprehensive and holistic approach(9-11) with an overview of all important health problems, in order to arrange and reduce these into a unified treatment scheme. this approach should be divided into t w o s t a g e s , i . e . a c c o m p l i s h m e n t o f a comprehensive health overview, and selection of important health problems to develop a holistic care plan. health services could play an important role in this regard by provision of comprehensive curative, preventive, and promotive care. however, many of the health services lack resources and structures to prioritise and provide better quality and comprehensive services for older persons. barriers to delivery of services need to be removed by prioritising older persons’ needs, assessing the current situation and introducing needed changes in provision of care for older persons and inter-service cooperation for achieving integrated care. an innovative and comprehensive approach to improving the health and well-being of older people by promoting healthy living at home needs to be developed, especially in the health centers for first level of prevention and also in the hospitals for second and third levels of prevention. however, all levels should have a better referral system to make the system optimally functional and relevant to the complex needs of older persons. the spectrum of older persons’ care often require both outpatient and inpatient care due to chronic disease affecting them. (4,5,12) the community setting should encourage healthy living, and healthcare services should be able to cater to complex needs. the health services can do this by reorienting towards comprehensive care, care that is cost effective and affordable, univ med vol.30 no.3 202 kusumaratna, mahmood, soerjoasmoro, et al physical activity reduced hypertension in elderly and where referral pathways are well established and easy to follow and maintain. working together requires good discharge planning as well. service access needs to be flexible and responsive to short-term care needs, as the number and types of health services used by older persons are influenced by many factors. frequency and intensity of service use are related to health status, level of impairment and access factors. many older persons suffer from co-morbidities. a study in malaysia involving older persons with knee joint osteoarthritis showed that more than 90% of those older persons had more than one disease. even in the early 1990s, older persons in indonesia had a high incidence of cvd and hypertension.(4,12) with increasing population aging, these issues have become a highly important public health issue, requiring immediate attention. older people become more vulnerable as they grow older because of the gradual onset of physical and mental disabilities. family assistance given to older persons may be limited, particularly among poor families, and the support for older persons’ welfare provided by the government and the community is very limited and practically inaccessible to many older persons.(4) the incidence of disability is known to increase with old age, and the first most likely causal factor of disability is mobility. impaired mobility increases dependence on caregivers. in a number of cases the disability may be permanent, which makes older persons person dependent until death. as such, the required intensity of care for older persons increases with disability.(10,11) thus, older persons with a disability, particularly those who are bed-ridden, need special care. current trends in the aging population of east and south-east asia imply that there will be a growing need for providing such special care facilities for an increasing proportion of the population of older persons.(13) economic considerations may also have an impact on the behavior of providers and patients, as both parties have to consider the economics of the provided health care. financial incentives created by healthcare systems to providers and caregivers may affect the implementation of health care services to older persons, both qualitatively and quantitatively. t h e r e f o r e , t o i m p r o v e t h e c o s t effectiveness of healthcare for older persons requires a thorough understanding of the intricacies of the healthcare system. priority s e t t i n g i n v o l v e s c h a n g e s i n o r g a n i z i n g consultations, by initially obtaining health overviews for the majority of older patients, followed by systematic processing of the various health problems affecting a patient, to be completed within one consultation. finally, to reduce the number of treatable problems to an ideal minimum through shared priority setting, a n e s s e n t i a l t o o l i s p a t i e n t c e n t e r e d communication. person-centered holistic health care requires not only medical competency of caregivers, but also empathy for older persons as patients, ensuring a mutually respectful relationship and cooperation between caregivers, patients and their family, and thus transcending traditional ideas of individualised care.(14) in this connection, to arrive at optimal health care decisions, the health care workers must also take into consideration the personal history of the older patients and their perceptions, aspirations and concerns, frequently provided by the patients’ family.(15,16) one limitation of this study was the small number of healthcare officials as respondents, due to the scope of this study, which was restricted to governmental health offices and therefore did not include private institutions or clinics. conclusions improvements are needed in providing access to health care, especially for older persons. some aspects should be developed, s u c h a s k n o w l e d g e a n d s k i l l s o f h u m a n resources, and the supporting healthcare service infrastructure at puskesmas for implementing 203 primary healthcare for older persons. local governments should make plans for strengthening the appropriate service delivery to the growing numbers of older persons, and should provide support for the maintenance, sustainability and strengthening of community-based care for older persons. acknowledgement we g r a t e f u l l y a c k n o w l e d g e t h e collaboration and support provided by the staff a t d i n a s k e s e h a t a n k o t a ta r a k a n , e a s t kalimantan, and by our colleague dr. novia sudarma, as member of the aging research center (arc) team. funding this study was financially supported by the dikti scheme research fund with co-contribution from the university of adelaide and dinas kesehatan kota tarakan, east kalimantan. the sponsors of the study had no role in the design and conduct of the study, and in data collection, analysis and interpretation. the authors are financially independent from the funders. author contributions all authors (rk, mam, mas and kh) contributed equally to the conduct of the field work and data management, including data analysis. rk and mam were also responsible for conceptualization of the study and preparation o f t h e r e s e a r c h r e p o r t , a n d t a k e f u l l responsibility for the content of this report. declaration of conflicting interest the authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article. references 1. united nations population funds (unfpa). population ageing in east and south-east asia: current situation and emerging challenges. population ageing series no.1, thailand;2006. 2. united nations population funds (unfpa). population ageing in east and south-east asia: current situation and emerging challenges. population ageing series no.3, thailand;2007. 3. department of health. basic health research : national report 2007. jakarta: health research and development board department of health;2008. 4. mujahid g. population ageing in east and southeast asia, 1950-2050: implication for elderly care. asia pac pop j 2006;19:25-43. 5. trihandini i. the role of medical check-up utilization on basic physical activities in older persons: study panel for older persons 1993-2000. makara kesehatan 2007;11:90-6. 6. kusumaratna rk. effects of daily multimicronutrient supplementation on nutritional and health status in the elderly: a community based randomized controlled trial [dissertation]. jakarta: university of indonesia;2009. 7. abikusno n, kusumaratna rk. characteristic of elderly club participants of tebet health center south jakarta. asia pac j clin nutr 1998;7:320324. 8. world health organization. aging and health: a health promotion approach for developing countries. manila: who regional office for the western pacific;2002. 9. van eeuwijk p. old-age vulnerability, ill-health and care support in urban areas of indonesia. ageing society 2006;26:61-80. 10. kusumaratna rk. factors influencing frailty among community-dwelling elderly in manggarai sub-district, south jakarta. proceeding of 8th national conference of emerging researchers in ageing, monash university on 22-23 october 2009. 11. beaglehole r, epping-jordan j, patel v, chopra m, ebrahim s, kidd m, et al. improving the prevention and management of chronic disease in low-income and middle-income countries: a priority for primary health care. lancet 2008;372: 940-949. 12. zakaria zf, bakar aa, hasmoni hm, rani fa, kadir sa. health-related quality of life in patients with knee osteoarthritis attending two primary care clinics in malaysia: a cross-sectional study. asia pac fam med 2009;8:10. 13. witoelar f, strauss j, sikoki b. socioeconomic success and health in later life: evidence from the indonesia family life survey. rand labor and population working paper series, 2009. 14. mccormack b. a conceptual framework for person-centred practice with older people. int j nurs pract 2003;9:202-9. univ med vol.30 no.3 204 15. clarke a, hanson e, ross h. seeing the person behind the patient: enhancing the care of older people using biographical approach. j clin nurs 2003;12: 697-706. kusumaratna, mahmood, soerjoasmoro, et al physical activity reduced hypertension in elderly 16. hummers-pradier e, beyer m, chevallier p, eilattsanani s, lionis c, peremans l, et al. research agenda for general practice/family medicine and primary health care in europe. maastricht (holland): european general practice research network egprn; 2009. alvina 3 abstract cassava (manihot esculenta crantz) leaves have long been used as a vegetable in many countries and empirically as a febrifuge. the aim of the present study was to evaluate the analgesic activity of an ethanolic extract of cassava leaves in mice. thirty balb/c mice (20-30 g, 2-3 months old) were randomly divided into 6 groups. group i was given distilled water 1 ml as negative control, group ii paracetamol 65 mg/kgbw as positive control, and group iii-vi received an ethanolic extract of cassava leaves in 4 doses, i.e. 12.8 mg/kgbw, 25.6 mg/ kgbw, 51.3 mg/kgbw, and 102.6 mg/kgbw, respectively. all interventions were administered as a single dose by oral route on a given day. acetic acid 0.6% (w/v) was used as a pain inductor. analgesic activity was measured by counting the percentage of writhing movements as a measure of the analgesic effect produced by each intervention. data were analyzed with one-way anova to compare analgesic activity between treatment groups. the results showed that groups treated with ethanolic extract of cassava leaves at dosages of 12.8 mg/kgbw, 25.6 mg/kgbw, 51.3 mg/kgbw, and 102.6 mg/kgbw had an analgesic activity of 59.2%; 73.9%; 62.1%; and 55.9%, respectively. on statistical analysis there were significant differences (p=0.00) between these treatments compared to the negative control, but no significant differences (p>0.05) with the positive control (paracetamol). it may be concluded that the analgesic effect of an ethanolic extract of cassava (manihot esculenta crantz) leaves in mice was of similar potency as paracetamol. keywords: manihot esculenta crantz, analgesic activity, flavonoid, mice *department of pharmacology, faculty of medicine, islam indonesia university yogyakarta correspondence dr. isnatin miladiyah, m.kes. department of pharmacology, faculty of medicine, islam indonesia university yogyakarta jl. kaliurang km 14.5 sleman yogyakarta 55564 phone: 0274-898444 ext. 2002 email: isnatin@fk.uii.ac.id univ med 2011;30:3-10 analgesic activity of ethanolic extract of manihot esculenta crantz leaves in mice isnatin miladiyah*, ferdiyanto dayi*, and sufi desrini* january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction the cassava plant (manihot esculenta crantz, family euphorbiaceae) is one of the staple food crops in most regions of africa, asia, and latin america.(1) the parts of the plant that are commonly utilized are the roots and leaves. in indonesia cassava roots are used as an alternative staple food and cassava meal (tapioca flour) as a wheat flour substitute in the preparation of bread and cookies.(2) cassava leaves have also been used against many disorders, such as rheumatism, fever, headache, diarrhea, and loss of appetite.(3,4) cassava 4 miladiyah, dayi, desrini analgesic activity of manihot esculenta crantz leaves reportedly also possess antihemorrhoid, a n t i i n f l a m m a t o r y, ( 5 ) a n d a n t i m i c r o b i a l activity.(6) in nigeria, cassava is used for the treatment of ringworm, tumor, conjuctivitis, sores and abscesses. to date there are very few reports of studies on the analgesic activity of the cassava plant. a study in nigeria showed that oral administration of an aqueous cassava leaf extract to rats induced anti-inflammatory and analgesic effects.(8) the cassava plant is rich in macroand micronutrients(1,9) and is therefore commonly used as a staple food. in addition the plant contains a number of antioxidant compounds, namely â-carotene (around 23-86 mg/100g),(7) vitamin c (1.7-419 mg/100g),(1) vitamin a, anthocyanins (flavonoids), saponins, steroids, and glycosides.(7,10) previous studies have reported that several saponinand flavonoidcontaining plants possess analgesic, antiinflammatory, antipyretic, and antimicrobial activities. these plants include mahkota dewa ( p h a l e r i a m a c ro c a r p a ) , ( 11 ) k a y u u l i n (eusideroxylon zwageri) t et b,(12) star fruit ( b e l i m b i n g m a n i s , av e r rh o a c a r a m b o l a l i n n . ) , ( 1 3 ) d a u n b a n g u n b a n g u n ( c o l e u s amboinicus l.,(14) and curcuma alismatofolia gagnep.(15) the flavonoid fraction and volatile flavonoid compounds of these plants are t h o u g h t t o h a v e a n t i i n f l a m m a t o r y a n d analgesic effects.(16) i n a d d i t i o n t o b e n e f i c i a l c h e m i c a l compounds, cassava leaves also contain toxic substances, which are associated with the high concentration of cyanogenic glycosides. these are carbohydrate derivatives that release h y d r o c y a n i c a c i d u p o n h y d r o l y s i s . hydrocyanic acid inhibits the reduction of oxygen in the (mammalian) respiratory system, the electron transport in photosynthesis, and the activity of enzymes such as catalase and oxidase, which play a role in transport of iodine in cases of goiter, although one study did not find evidence for a relationship between cassava consumption and the prevalence of goiter.(17) the cyanogenic glycoside content depends to a great extent on the age and cultivar of cassava and on environmental factors.(18) younger plants tend to have a higher cyanogenic glycoside content and the mature leaves on the lower part of the plant have a lower cyanogenic glycoside content.(2) therefore in the present study only mature leaves were collected for preparation of the extract. since cassava leaves have a high level of antioxidants, such as flavonoids, saponins, vitamin c, and â-carotenes, the cassava plant is presumably capable of counteracting the effects of oxidants (free radicals), suggesting the possibility that the leaves affect the inflammatory process, the pathophysiology of which also involves free radicals. the aim of the present study was to evaluate the analgesic effect of an ethanolic extract of cassava leaves (manihot esculenta crantz) in mice. methods research design this was a pure experimental controlled l a b o r a t o r y s t u d y w i t h t h e o b j e c t i v e o f evaluating the analgesic activity of an ethanolic extract of cassava (manihot esculenta crantz) leaves. plant material cassava (manihot esculenta crantz) leaves were collected in january 2010 in the hamlet (dusun) of ngalangan in jetisbaran village, ngaglik subdistrict, sleman district, yogyakarta province. the collection procedure selected the ripe leaves growing near the ground level, in order to obtain leaves with a minimal content of cyanogenic glycosides. identification of the c a s s a v a l e a v e s w a s p e r f o r m e d a t t h e pharmacognosy-phytochemistry laboratory, pharmacy program, faculty of mathematics and natural sciences (fmipa), uii, yogyakarta, which also supplied the chemicals used in the preparation of the ethanolic extract. material for assessment of analgesic activity was obtained 5 f r o m t h e p h a r m a c o l o g y l a b o r a t o r y o f abovementioned faculty. extract preparation c a s s a v a l e a f e t h a n o l i c e x t r a c t w a s prepared by maceration as follows: one kilogram of cassava leaves was rinsed in water, cut up into small pieces, then left to dry in a drying cabinet at 38oc for 5 days, before undergoing maceration. subsequently the leaves were pulverized by means of a pestleand-mortar and an electric blender to yield a dry powder of around 200 g in weight. for maceration, 100 g dry cassava leaf powder was put in an erlenmeyer flask, then 500 ml 70% ethanol was added, after which the mixture was left standing at room temperature for 24 hours. the leaf powder was subjected three times to the maceration process. the resulting solution (ethanol phase) was filtered and concentrated in an evaporator at 50oc to yield a concentrated ethanolic cassava leaf extract of about 10 g in weight. experimental animals this study used thirty balb/c mice aged 23 m o n t h s a n d w e i g h i n g 2 0 3 0 g . t h e experimental animals were healthy and in excellent general condition, as was evident from their active movements and their black and round fecal pellets without signs of diarrhea. oral administration of manihot esculenta crantz thirty mice were randomly allocated to six intervention groups comprising 5 mice each. group i animals were given distilled water 1 ml and served as negative controls, while group ii mice received paracetamol 65 mg/kgbw and served as positive controls. the dosages of paracetamol were based on dosages for human adults, using a conversion factor of 0.026. groups iii-vi were given 1 ml of ethanolic cassava leaf extract in dosages of 12.8 mg/ kgbw, 25.6 mg/kgbw, 51.3 mg/kgbw, and 102.6 mg/kgbw, respectively. the lower dose of 12.8 mg/kgbw was equivalent to a dose of dried cassava leave powder of 1 g/kgbw. this dosage was determined in preliminary studies from consideration of effective doses of previous studies. all interventions were administered on a given day as a single dose by gavage. acetic acid induced writhing in mice as pain inducer 0.6% (w/v) acetic acid was given by intraperitoneal injection in dosages of 10 ml/kgbw.(19) the resulting pain was inferred from the writhing movements of the mice, which were monitored at intervals of five minutes, for up to one hour for each experimental animal, yielding the cumulative total of writhing movements per hour. calculation of analgesic power analgesic power was calculated on the b a s i s o f t h e i n h i b i t o r y c a p a c i t y o f t h e interventions on the writhing movements of the experimental animals,(20) as follows: % writhing protection = 100 – [(e/c) x 100] where e = cumulative total of writhing in the experimental animals after intervention, and c = cumulative total of writhing in the negative controls. a medication is said to have analgesic activity if it is capable of effecting a reduction of > 50% in the number of writhings of the experimental animals as compared to the control group. (20) to determine analgesic power, the percentage reduction in cumulative total number of writhings is calculated against the negative control group. the calculated percentage is termed the analgesic power of cassava leaf ethanolic extract against the pain induced by acetic acid stimulation of the experimental animals. statistical analysis percentages of writhing protection in the intervention groups were analyzed by means of a one-way anova, at significance level of p<0.05. univ med vol.30 no.1 6 miladiyah, dayi, desrini analgesic activity of manihot esculenta crantz results i n p r e l i m i n a r y a c u t e t o x i c i t y t e s t s , monitoring was performed on clinical signs considered to be representative of autonomic, b e h a v i o r a l , s e n s o r y, n e u r o m u s c u l a r, cardiovasculer, and respiratory systems. the tests involved four test animals, each of them receiving one of the four different dosages of the extract to be used in the present study. in these acute toxicity tests, all test animals failed to display clinical signs of toxicity, from which it was concluded that the four administered dosages could safely be used for the actual analgesic tests. in the present study all test animals (n = 30) completed the study, as none of them died or suffered from toxic effects. the number of w r i t h i n g s o f a l l t e s t a n i m a l s a n d m e a n cumulative total of writhings in the intervention groups are presented in table 1 and figure 1, respectively. from table 1 and figure 1 it may be seen that there was a reduction in the cumulative total of writhings in the ethanolic extract g r o u p s a n d t h e p a r a c e t a m o l g r o u p , i n c o m p a r i s o n w i t h t h e n e g a t i v e c o n t r o l s receiving distilled water. this indicates that paracetamol and cassava leaf ethanolic extract at the four dosages were capable of reducing intervention group cumulative number of writhings in mice mean ± sd 15’ 210’ 315’ 420’ 525’ distilled water 1 ml (n egative control) 40 78 71 127 133 89.8 ± 39.44 paracetamol 65 mg/kgbw (positive control) 17 24 35 46 44 33.2 ± 12.56 cassava leaf ethanolic extract 12.8 mg/kgbw 27 36 13 46 61 36.6 ± 18.26 cassava leaf ethanolic extract 25.6 mg/kgbw 7 11 20 46 33 23.4 ± 16.10 cassava leaf ethanolic extract 51.3 mg/kgbw 28 31 24 39 48 34 ± 9.56 cassava leaf ethanolic extract 102.6 mg/kgbw 23 33 26 34 82 39.6 ± 24.15 table 1. number of writhings in mice after intervention and induction of pain by 0.6% acetic acid   89.8 33.2 36.6 23.4 34  39.6 0 20  40 60  80  100 mean cummulative total of writhings  aquadest paracetamol extract 12.8 mg/kgbw extract 25.6 mg/kgbw extract 51.3 mg/kgbw extract 102.6 mg/kgbw figure 1. mean cumulative total of writhings in intervention groups 7 the occurrence of writhing movements as a response to the pain induced by intraperitoneal injection of 0.6% acetic acid. the analgesic power (%) of cassava leaf ethanolic extract at several dosages on the writhing reflex induced by intraperitoneal acetic acid is presented in table 2, while the percentage of writhing protection in the intervention groups is shown graphically in figure 2. it is apparent from both table 2 and figure 2 that of the original six groups there are now only five, because the analgesic power of the negative control is not calculated, being used as the divisor in the calculation of analgesic power. the one-way anova found a significant d i f f e r e n c e b e t w e e n c u m u l a t i v e t o t a l o f writhings in the paracetamol group and the ethanolic extract groups at all dosages as c o m p a r e d t o t h e n e g a t i v e c o n t r o l g r o u p (p<0.05). post hoc testing demonstrated that the cumulative totals of writhings differed significantly between the negative control group and the five other intervention groups figure 2. percentage of writhing protection in intervention groups table 2. percentage of writhing protection of paracetamol and cassava leaf ethanolic extract on pain in mice induced by 0.6% acetic acid note: negative control group: writhing protection = 0% univ med vol.30 no.1 8 miladiyah, dayi, desrini analgesic activity of manihot esculenta crantz (p<0.05), but did not differ significantly b e t w e e n t h e f i v e i n t e r v e n t i o n g r o u p s themselves (p>0.05). calculation of analgesic power with percentage of writhings in the negative control group as divisor showed that paracetamol and ethanolic extract of cassava leaves at all dosages did not differ significantly in analgesic power (p>0.05). discussion from table 2 and figure 2 it is clear that there was an increase in percentage of writhing protection at dosages of 12.8 mg/kgbw and 25.6 mg/kgbw, but a decrease at dosages of 51.3 mg/kgbw and 102.6 mg/kgbw. the graph shows that the analgesic power was highest at the dosage of 25.6 mg/kgbw, whereas at the h i g h e s t d o s a g e o f 1 0 2 . 6 m g / k g b w, t h e analgesic power was lowest. it is still unclear in the present study why the increase in dosage of the extract was not accompanied by an increase in analgesic power. the results of the one-way anova shows that the analgesic power of cassava leaf e t h a n o l i c e x t r a c t a t a l l d o s a g e s w a s significantly different compared with the n e g a t i v e c o n t r o l s ( p < 0 . 0 5 ) , b u t w a s n o t significantly different from that of paracetamol (p>0.05). therefore it may be concluded that cassava leaf ethanolic extract at dosages of 12.8 mg/kgbw, 25.6 mg/kgbw, 51.3 mg/ kgbw, and 102.6 mg/kgbw has an analgesic power equivalent to that of paracetamol at a dosage of 65mg/kgbw. an anti-inflammatory study in rats yielded similar results; the analgesic effect produced by an aqueous cassava leaf extract was significantly higher than that of indomethacin (10 mg/kg, s.c.).(8) from the foregoing it can be concluded that m a n i h o t e s c u l e n t a c r a n t z l e a f e x t r a c t s presumably contain orally safe, topically and o r a l l y e ff e c t i v e a n t i i n f l a m m a t o r y a n d analgesic principles. although the present study demonstrated that the cassava plant has an analgesic effect, it is still unclear what active principles are involved in this effect. one hypothesis is based o n t h e f a c t t h a t c a s s a v a l e a v e s c o n t a i n flavonoids(10) and that nearly all flavonoid compounds possess analgesic activity, due to inhibition of prostaglandin synthesis and c o n s e q u e n t d e c r e a s e d s t i m u l a t i o n o f nociceptors. flavonoids are thought to interact with the cyclooxigenase system, so as to interfere with arachidonic acid synthesis and inhibit the production of prostaglandins(21). in addition to the flavonoids, the analgesic effect may also be due to the presence of other antioxidants in the cassava plant, e.g. âcarotenes and vitamin c.(1,9) antioxidants are capable of neutralizing the free radicals released by phagocytes in response to cellular injury, so as to suppress the inflammatory response caused by these radicals, leading to a decreased pain response.(22) this idea is supported by the f i n d i n g t h a t c a s s a v a l e a v e s c o n t a i n anthocyanins, which play an important role in the antioxidant effect(23), while antioxidants in the cassava plant have a demonstrated ability to prevent alcohol-induced hepatotoxicity.(24) other substances to be suspected of playing a role in the analgesic activity are the saponins, as previous studies on different plant species indicate that saponins have analgesic and antiinflmmatory effects, and eliminate pain without affecting cell viability.(25) which of the possible active compounds in the cassava plant is the most responsible for its analgesic activity is still unclear. the alcoholic solvent used in this study extracts semipolar active compounds, thus it is probable that the analgesic substances are soluble in polar solvents, but to test this suggestion requires futher study. as a preliminary study, the present one has several limitations, among which may be mentioned the simple assessment method of analgesic effect with its high subjectivity level, the relatively narrow dosage range (thus requiring extension), and the limited sample size (five animals for each intervention). 9 conclusions ethanolic extracts of cassava (manihot esculenta crantz) leaves exert analgesic activity on pain in mice induced by 0.6% acetic acid, but increasing the dosage of the extract beyond a certain level does not result in an increase in analgesic power. further studies are necessary on the active constituents of cassava leaves that are responsible for their analgesic properties. acknowledgement thanks are due to mr sumarno, technician at the laboratory of pharmacology, pharmacy program, fmipa uii yogyakarta, for his assistance before, during and after the study. references 1. fasuyi ao. nutrient composition and processing effects on cassava leaf (manihot esculenta crantz) antinutrients. pakistan j nutr 2005;4:3742. 2. hidayat a, zuraida n, hanarida i. the cyanogenic potential of roots and leaves of ninety nine cassava cultivars. indonesian j agric sci 2002;3:25-32. 3. sastroamidjojo s. obat asli indonesia. jakarta: dian rakyat;2001. 4. yuniarti t. ensiklopedia tanaman obat tradisional. yogyakarta: media pressindo, yogyakarta;2008. 5. okpuzor j, oloyede am. anti-inflammatory, antipyretic and anti-diarrhoeal properties of an antihaemorrhoid tri-herbal pill. nature sci 2009;7:8994. 6. popoola, yangomodou, od, akintokun ak. antimicrobial activity of cassava seed oil on skin pathogenic microorganism. res j med plant 2007;1:60-4. 7. okeke cu, iweala e. antioxidant profile of dioscorea rotundata, manihot esculenta, ipoemea batatas, vernonia amygdalina and aloe vera. j med res technol 2007;4:4-10. 8. afolabi l , adeyemi oo, yemitan ok. cassava leaves have anti-inflammatory and analgesic principles, which justify its use in traditional african medicine. j ethnopharmacol 2008;119:611. 9. chavez al, bedoya jm, sanchez, iglesias, ceballos, roca w. iron, carotene, and ascorbic acid in cassava roots and leaves. food nutr bull 2000;21:410-3. 10. departemen kesehatan . inventaris tanaman obat indonesia. jilid i. jakarta: departemen kesehatan republik indonesia;2000. 11. soeksmanto a. pengaruh ekstrak butanol buah tua mahkota dewa (phaleria macrocarpa) terhadap jaringan ginjal mencit. biodiversitas 2006;7:278-81. 12. ajizah a, thihana, mirhanuddin. potensi ekstrak kayu ulin (eusideroxylon zwageri t et b) dalam menghambat pertumbuhan bakteri staphylococcus aureus secara in vitro. bioscientiae 2007;4:3742. 13. sukadana im. senyawa antibakteri golongan flavonoid dari buah belimbing manis (averrhoa carambola linn.l). j kimia 2009;3:109-16. 14. santosa cm, hertiani t. kandungan senyawa kimia dan efek ekstrak air daun bangun-bangun (coleus amboinicus l.) pada aktivitas fagositosis neutrofil tikus putih. maj farmasi indonesia 2005;16:141-8. 15. akter r, hasan smr, siddiqua sa, majumder mm, hossain m, alam a. evaluation of analgesic and antioxidant potential of the leaves of curcuma alismatofolia gagnep. stamford j pharm sci 2008;1:3-9. 16. hashemi vah, ganhadi s, mosavi d. analgesic and antiinflammatory effects of total extract, flavonoid fraction, and volatile. j res med sci 2000;5:17-27. 17. kittivachra r. effects of cassava on thyoroid gland in rats. thai j pharm sci 2006:57-62. 18. fransisco ia, pinotti mhp. cyanogenic glycosides in plants. braz arch biol techno 2000;43:487-92. 19. somchit mn, shukriyah, bustaman aa, zuraini a. anti-pyretic and analgesic activity of zingiber zerumbet. int j pharmacol 2005;1:277-80. 20. hendershot lc, forsaith j. antagonism of the frequency of phenylquinone induced writhing in the mouse by weak analgesics and non-analgesics. j pharmac exp therap 1959;125:237-40. 21. purwantini i, purwantiningsih, puspita oe. efek analgesik fraksi etanol dari ekstrak etanol daun mindi (melia azedarach l.) pada mencit jantan. j ilmiah farmasi 2007;4:88-103. 22. raji y, udoh us, oluwadara, akinsomiyose, awobajo, adeshoga k. anti-inflammatory and analgesic properties of the rhizome extract of zingiber officinale. afr j biomed res 2002;5: 121-4. 23. byamukama r, namukobe j, kiremire b. anthocyanins from leaf stalks of cassava (manihot esculenta crantz). afr j pure appl chem 2009;3:20-5. univ med vol.30 no.1 10 miladiyah, dayi, desrini analgesic activity of manihot esculenta crantz 24. boby rg, indira m. effect of co-administration of cassava (manihot esculenta crantz) rich diet and alcohol in rats. indian j physiol pharmacol 2004:48:41-50. 25. borgi w, recio mc, rios jl, chouchane. antiinflammatory and analgesic activities of flavonoid and saponin fractions from ziyzyphus lotus (l.) lam. south afr j bot 2008;7:320-4. alvina 65 abstract dengue is a disease that is caused by dengue virus and transmitted to humans through the bite of infected aedes mosquitoes, especially aedes aegypti. the disease is hyper-endemic in southeast asia, where a more severe form, dengue hemorrhagic fever (dhf) and dengue shock syndrome (dss), is a major public health concern. the purpose of the present study was to find evidence of dengue virus transovarial transmision in local vectors in jakarta. fifteen aedes larvae were collected in 2009 from two areas in tebet subdistrict in south jakarta, namely one area with the highest and one with the lowest dhf prevalence. all mosquitoes were reared inside two cages in the laboratory, eight mosquitoes in one cage and seven mosquitoes in another cage and given only sucrose solution as their food. the results showed that 20% of the mosquitoes were positive for dengue virus. dengue virus detection with an immunohistochemical method demonstrated the occurrence of transovarial transmission in local dhf vectors in tebet subdistrict. transovarial dengue infection in ae.aegypti larvae appeared to maintain or enhance epidemics. further research is needed to investigate the relation of dengue virus transovarial transmission with dhf endemicity in jakarta. keywords: dengue hemorrhagic fever, aedes mosquitoes, transovarial transmission *department of biology, **department of parasitology, medical faculty, trisakti university correspondence dr. monica dwi hartanti, mbiomed department of biology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2312 email: mdhartanti@gmail.com univ med 2010;29:65-70 dengue virus transovarial transmission by aedes aegypti monica dwi hartanti*, suryani**, and ingrid a.tirtadjaja** may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction dengue is an acute disease in humans caused by antigenically distinct serotypes of dengue virus (den) and transmitted by the bite of infected mosquitoes, especially ae. aegypti as a prime vector and ae. albopictus as a covector. the disease is endemic in most tropical and subtropical areas of the world, with an estimated annual occurrence of 100 million cases of dengue fever (df) and 250,000 cases of dengue hemorrhagic fever (dhf).(1) dengue viruses are plus-sense, ssrna viruses belonging to the genus flavivirus. it is believed that they once were mosquito viruses that b e c a m e a d a p t e d t o l o w e r p r i m a t e s a n d humans.(3) there are four serotypes of dengue virus, den-1, den-2, den-3, and den-4. den-3 virus is the dominant serotype in indonesia, capable of causing fatal disease, 66 hartanti, suryani, tirtadjaja transovarial transmission by aedes aegypti especially in children.(2) demographic and societal changes such as population growth, urbanization, and modern transportation greatly contributed to the increased incidence and geographic spread of dengue activity. the world health organization (who) classifies dengue as a major international public health concern because of the expanding geographic distribution of the virus and its mosquito vector, the increased frequency of epidemics, the cocirculation of multiple viral serotypes, and the emergence of dhf in new areas.(4) to date, dhf prevention still mostly depends on vector control because there is no effective treatment for dhf and no vaccine for dhf prevention. control of aedes mosquitoes is the most important measure in dhf prevention, because these mosquitoes tend to act as a reservoir or amplifier host for dengue viruses and the virus can be transmitted horizontally a s w e l l a s v e r t i c a l l y ( t r a n s o v a r i a l transmission). most remarkably, the virus can b e m a i n t a i n e d i n n a t u r e b y t r a n s o v a r i a l transmission in mosquitoes and can replicate in potentially vulnerable tissues of mosquito ovaries and embryos without deleterious effects.(5) transovarial transmission of dengue virus i s a c r u c i a l e t i o l o g i c a l p h e n o m e n o n responsible for persistence of virus during inter-epidemic periods.(6) the occurrence of dengue virus transovarial transmission in aedes mosquitoes was demonstrated in a study conducted in 2005 by rohani et al., who found infected larvae from 16 different locations in malaysia, with higher virus infection rates (vir) in ae. aegypti than in ae. albopictus.(7) there is increasing evidence for dengue transovarial transmission, which tends to potentially become a factor that can initiate dhf outbreaks in endemic areas.(4) for this reason, it was considered imperative to conduct a research study on the ability of local vectors i n j a k a r t a t o t r a n s m i t d e n g u e v i r u s transovarially, with the goal of supporting dengue virus and aedes mosquito control measures for prevention of dhf outbreaks in jakarta. methods research design this preliminary study of transovarial t r a n s m i s s i o n o f d e n g u e v i r u s i n a e d e s mosquitoes as local vectors was conducted as a survey and used a cross-sectional design. mosquito collection a e . a e g y p t i l a r v a e w e r e c o l l e c t e d randomly from two different areas in tebet subdistrict, south jakarta, in the year 2009, consisting of 8 larvae from an area with the highest dhf prevalence (designated rw 01) and 7 larvae from an area with the lowest dhf prevalence (rw 02). tebet subdistrict was selected as the research location because it was endemic for dhf in 2009. fifteen aedes mosquitoes were grown from the collected larvae and reared inside two cages in the laboratory for about 1 week to allow replication of any dengue virus present. the mosquitoes were only given sugar water as food and no blood, to rule out cross-infection by dengue virus in the blood meal. detection of dengue virus after identification of the fifteen aedes mosquitoes by light microscopy, the presence o f d e n g u e v i r u s i n e a c h m o s q u i t o w a s d e t e r m i n e d b y m e a n s o f h e a d s q u a s h preparations. any dengue virus present in the tissues was detected by monoclonal dengue antibodies using the immunocytochemical s t r e p t a v i d i n b i o t i n p e r o x i d a s e c o m p l e x (isbpc) assay. the head squash preparation, after fixation in cold methanol (-20°) for 2-3 minutes, was first incubated with peroxidase blocking solution to quench endogenous peroxidase activity. the specimen was then incubated with serum blocking solution to 67 univ med vol.29 no.2 suppress non-specific binding of subsequent reagents which would interfere with the results, followed by incubation with primary antibody. the specimen was then washed in phosphate buffer solution, and sequentially incubated with biotin-labelled secondary antibody and peroxidase-streptavidin conjugate to bind the biotin residue. the presence of peroxidase was visualized by substrate-chromogen solution. peroxidase catalyzes its substrate, hydrogen p e r o x i d e , a n d c h a n g e s t h e c h r o m o g e n substrate, diaminobenzidine tetrachloride (dab), into a brown precipitate at the antigen site. positive results were characterized by a brown coloring of the tissues, the color intensity depending on the total amount of chromogens reacting with peroxidase. denguenegative tissue specimens were blue in color. the study also included dengue virus-positive a n d n e g a t i v e h e a d s q u a s h s p e c i m e n s a s controls.(8) statistical analysis the chi-square test was done to compare the proportion of dengue virus in the two areas, rw 01 and rw 02, which had the highest and t h e l o w e s t p r e v a l e n c e o f d h f i n 2 0 0 9 , repectively. a p value less than 0.05 was c o n s i d e r e d a s s t a t i s t i c a l l y s i g n i f i c a n t . statistical analysis was performed with spss 15.0 software. results there were two (25%) positive results among the eight head squash specimens from the area with the highest dhf prevalence in tebet subdistrict, south jakarta. in contrast, there was only one (14.28%) positive result among the seven head squash specimens from the area with the lowest dhf prevalence. overall, 20% of the mosquitoes were positive for dengue virus. table 1 lists the number of larvae collected in the two study areas. table 2 presents the dengue virus distribution of the two areas. there was no significant difference in positive results between the two areas (p > 0.05). figure 1 shows a dengue-positive ae. aegypti head squash, where the brownish precipitation (black arrow) indicates the location of dengue antigen in the infected mosquito. location number of aedes mosquitoes dengue positive area of h ighest dhf prevalen ce (rw 01)* 8 2 area of lowest dhf prevalence (rw 02)* 7 1   table 1. numbers of aedes mosquitoes from areas with highest and lowest dhf prevalence in tebet subdistrict *rw = rukun warga table 2. dengue virus distribution of two areas in tebet subdistrict *chi-square test; **rw = rukun warga 68 hartanti, suryani, tirtadjaja transovarial transmission by aedes aegypti discussion t h e p r e s e n t s t u d y s h o w e d t h a t t h e precentage of positive results among the head squash specimens was 20%, consisting of 25% for the area with the highest dhf prevalence in 2009, and 14.28% for the area with the lowest dhf prevalence in 2009. the results s h o w e d t h a t t h e r e w a s d e n g u e v i r u s transovarial transmission in local dhf vectors in tebet subdistrict, south jakarta. although the percentage of positive results was higher in the area of highest dhf prevalence in 2009, there was no significant difference between the areas with the highest and the lowest dhf p r e v a l e n c e i n 2 0 0 9 i n te b e t s u b d i s t r i c t (p>0.05). similar results were obtained in the 2005 study of rohani et al., who demonstrated the occurrence of transovarial transmission of dengue virus in ae. aegypti and ae. albopictus mosquitoes at 13% and 4%, respectively.(7) there are many reports of transovarial transmission of dengue viruses.(9-11) in a recent s t u d y, m o u r y a e t a l . h a v e s h o w n t h a t transovarially infected mosquitoes can orally transmit the virus. this study also found that when eggs obtained from infected females were hatched after several weeks of incubation at r o o m t e m p e r a t u r e , t h e r a t e o f v e r t i c a l transmission increased. it has been suggested that at room temperature the virus has an opportunity to multiply and increase its copy number in the quiescent embryos. occurrence o f t h i s p h e n o m e n o n i n n a t u r e m a y b e advantageous for maintenance of this virus.(12) in their experimental study, joshi et al. reported on the persistent transovarial infection of s u c c e s s i v e g e n e r a t i o n s o f a e . a e g y p t i mosquitoes with dengue-3 virus.(3) several workers have also reported the transovarial transmission of the dengue virus in both ae. aegypti and ae. albopictus experimentally and/ figure 1. ae. aegypti head squash preparation with positive streptavidin-biotin peroxidase complex staining (40x magnification) 69 univ med vol.29 no.2 or from field collected mosquito larvae.(11,13,14) it is important to point out that both of the principal dengue vectors have now been demonstrated to be capable of sustained transovarial maintenance of virus in the absence of any horizontal transmission from vertebrate blood meals. the transovarial transmission of the dengue virus in the aedes vectors is now a well-documented phenomenon reported from many parts of the endemic areas in the world. this observation has further emphasized the importance of larval control since the immature stages may become the reservoir of the virus during the interepidemic periods. there are three types of mechanisms of d e n g u e t r a n s o v a r i a l t r a n s m i s s i o n : ( 1 5 ) ( i ) uninfected female mosquitoes bite and suck the host’s viremic blood, allowing the virus to replicate within the mosquitoes, the infected e g g s g i v i n g r i s e t o i n f e c t e d l a r v a e ; ( i i ) uninfected female mosquitoes have sexual activity with transovarially infected male mosquitoes, resulting in sexual transmission of infection to the female mosquitoes; and (iii) the ovarial tissues of female mosquitoes become infected and the virus is genetically transmitted to the next generation. transovarial transmission of the dengue virus potentially increases the probability of dengue outbreaks or at least contributes to persistence of dhf cases in endemic areas.(15) infected female mosquitoes lay their eggs in microniches, where most of the eggs survive through the inter-epidemic season, to emerge as adult mosquitoes that may enter the humanm o s q u i t o h u m a n c y c l e . t h e s u c c e s s i v e generations obtained from infected parental mosquitoes showed that the virus could persist in mosquitoes into succesive generations through transovarian passage.(4) transovarially infected mosquitoes have the ability to transmit the dengue virus orally, through the salivary glands. (7) multigenerational, transovarial passage and horizontal transmission of dengue from vertically infected progeny has been observed with ae. aegypti.(3,12) in indonesia, on average dengue outbreaks take place every three to four years, while in some regions the outbreaks occur annually or biannually. without proper intervention, dhf cases in indonesia could amount to 125.000 cases in 2007, an increase of almost 11% compared to 2006.(16) according to jakarta health official profile for the year 2009, about 2300 persons among the inhabitants suffered from dhf, of whom 65 died.(12) in jakarta, there are 174 areas of dengue outbreaks in 10 subdistricts and it is highly probable that these figures will increase.(17) modern dengue control should focus on early detection of dengue virus infection in the mosquito vectors prior to its introduction into the human population, so that remedial control action can be taken immediately to curb the impending outbreak. akbar et al. showed that detection of dengue virus in adult mosquitoes using reverse transciptase-polymerase chain reaction (rt-pcr) was successful in predicting an outbreak in bandung, indonesia.(18) our investigation has several limitations. the aedes larvae were collected only from households that were willing to participate, thus the sample was not representative of current dengue infection in tebet subdistrict. time limitation was also a problem, because the dengue virus was probably not allowed enough time for replication, which may have resulted in false negative results. further research studies are needed to provide more data about dengue transovarial transmission in local vectors in the tebet area, to be followed up with studies in the whole jakarta area, to support an early warning system on the development of dengue outbreaks, so that in the future the number of dhf cases might be reduced. conclusion transovarial transmission of dengue virus by available vector species in a dengue endemic area could explain the specific emergence of dengue in this region. 70 hartanti, suryani, tirtadjaja transovarial transmission by aedes aegypti acknowledgements the authors are grateful to all persons for supporting the present study, especially to prof. dr. dr. adi hidayat, ms for his endless support and to mr. rasimin for supporting the laboratory work. references 1. clarke t. dengue virus: break-bone fever. nature 2002;416:672–4. 2. world health organization. dengue fever in indonesia – update 3. communicable disease surveillance & response (csr). geneva : world health organization; 2004. 3. joshi v, mourya dt, sharma rc. persistence of dengue-3 virus through transovarial transmission passage in successive generations of aedes aegypti mosquitoes. am j trop med hyg 2002; 67:158–61. 4. gubler dj. the global emergence/resurgence of arboviral diseases as public health problems. arch med res 2002;33:330–42. 5. borucki mk, kempf bj, blitvich bj, blair cd, beaty bj. la crosse virus: replication in vertebrate and invertebrate hosts. microbes infect 2002;4:341–50. 6. angel b, joshi v. distribution and seasonality of vertically transmitted dengue viruses in aedes mosquitoes in arid and semi-arid areas of rajasthan, india. j vector borne dis 2008;45:56– 9. 7. rohani a, zamree i, lee hl, mustafakamal i. detection of transovarian dengue virus for field caught aedes aegypti and aedes albopictus mosquito using c6/36 cell line and rt-pcr. trop biomed 2005;22:149–54. 8. umniyati rs. teknik imunositokimia dengan menggunakan antibodi monoklonal dssc7 untuk kajian patogenesis infeksi dan penularan transovarial virus dengueserta surveilansi vektor dengue [dissertation]. fakultas kedokteran, universitas gadjah mada; 2009. 9. arunachalam n, tewari sc, thenmozhi v, rajendran r, paramasivan r, manavalan r, et al. natural vertical transmission of dengue viruses by aedes aegypti in chennai, tamil nadu, india. indian j med res 2008;127:395-7. 10. günther j, martínez-muñoz jp, pérez-ishiwara dg, salas-benito j. evidence of vertical transmission of dengue virus in two endemic localities in the state of oaxaca, mexico. intervirology 2007;50:347-52. 11. lee hl, rohani a. transovarial transmission of dengue virus in aedes aegypti and aedes albopictus in relation to dengue outbreak in an urban area in malaysia. dengue bull 2005;29: 106-11. 12. mourya dt, gokhale, basu a, barde pv, sapkal gn, padbidri vs, et al. horizontal and vertical transmission of dengue-2 virus in highly and lowly susceptible strains of aedes aegypti mosquitoes. acta virol 2001;45: 67–71. 13. perich mj, davila g, turner a, garcia a, nelson m. behavior of resting aedes aegypti (culicidae: diptera) and its relation to ultra-low volume adulticide efficacy in panama city, panama. j med entomol 2000;37:541-6. 14. xu g, dong h., shi n, liu s, zhou a, cheng z, et al. an outbreak of dengue virus serotype 1 infection in cixi, ningbo, people’s republic of china, 2004. associated with a traveler from thailand and high density of aedes albopictus. am j trop med hyg 2007;76:1182–8. 15. thavara u, siriyasatien p, tawatsin a, asavadachanukorn p, anantapreecha s, wongwanich r, et al. double infection of heteroserotypes of dengue viruses in field populations of aedes aegypti and aedes albopictus (diptera: culicidae) and serological features of dengue viruses found in patients in southern thailand. southeast asian j trop med public health 2006;37:468-76. 16. lembaga penelitian dan pengembangan departemen kesehatan. demam berdarah dengue. bagian jaringan informasi iptek dan promosi penelitian, sekretariat badan litbang kesehatan, c2007. available at: http// www.litbang.depkes.go.id . accessed june 10, 2008. 17. dinas kesehatan dki. demam berdarah dengue, makin mengecoh makin mengganas. dinas kesehatan profinsi dki jakarta, c2007. available at: http//www.dinkes-dki.go.id. accessed june 10, 2008. 18. akbar mr, agoes r, djatie t, kodyat s. pcr detection of dengue transovarial transmissibility in aedes aegypti in bandung, indonesia. proc asean congr trop med parasitol 2008;3:849. alvina 111 abstract *department of anatomy and rehabilitative medicine, medical faculty, trisakti university jakarta correspondence dr. maria regina rachmawati, sprm department of anatomy and rehabilitative medicine, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone. 021-5672731 ext.2101 email: ati_dr@yahoo.com univ med 2011;30:111-9. high erector spinae endurance reduced low back pain in postmenopausal women maria regina rachmawati* may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina introduction back pain, particularly low back pain (lbp), is the most common complaint in all age groups, including postmenopausal women. inappropriately managed, lbp may become chronic and carries the risk of disability.(1) failure of management of lbp may increase economic loss, both direct costs for treatment and indirect costs due to decreased work productivity and early retirement.(2) the lower back region is the area of the vertebral column low back pain (lbp) is the most frequent health problem in all age groups, including postmenopausal women, who are at risk of disability, due to degenerative processes affecting all organ systems, including the erector spinae muscles. in addition, postmenopausal women also experience a decrease in esterogen levels, which stimulates osteoclastic activity and initiates a reduction in bone mineral density (bmd). the aims of the present study were to determine the relationship between lbp prevalence and erector spinae endurance, bmd reduction, and body mass index (bmi) respectively. this was a cross-sectional study, conducted on 213 postmenopausal women, selected by simple random sampling. assessment of pain, expressed on a visual analog scale (vas), was performed by interviews and measurement of erector spinae muscle endurance time using a modified biering-sorensen technique, while bmd was assessed by bone densitometry. the results of the study showed that the postmenopausal women had a mean age of 53.5 ± 3.6 years. the prevalence of lbp and osteoporosis in the postmenopausal women was 58.2% and 21.6%, respectively. a significant positive linear correlation was found between bmi and vas (r=0.165; p=0.016). in addition, the t-test demonstrated a significant difference in mean vas scores between good and poor erector spinae endurance (p=0.008). however, the chi-square test showed no significant relationship between reduced bmd and lbp (p>0.05). it is recommended that low back pain in postmenopausal women be managed by strengthening exercises of the erector spinae. key words: low back pain, erector spinae, bone mineral density, postmenopause 112 rachmawati spinae endurance reduced low back pain from below the twelfth ribs down to the inferior gluteal folds. anatomically, the lower back region is composed of the lumbar vertebrae, which are held in their vertical position by the action of the enveloping muscles.(3,4) in general, t h e p r e v a l e n c e o f l b p i s 2 3 . 7 % i n postmenopausal women,(5) 42.1% in workers(6) and 62.3%(7) in young adults. muscular activity is required for stabilizing the vertebral column when supporting heavy loads, and is directed upwards, opposing gravity. according to electromyographic (emg) studies conducted by noorbaksh,(8) there is postural muscular activity in each body segment in the standing position, to maintain the erect posture. in the relaxed standing posture, muscular activity is minimal in the sacrospinal region or the abdomen, but varies according to the position of the vertebral column relative to the line of gravitation. a change in the position of the center of gravity results in increased activity in the dorsal region, in comparison with the abdominal region.(9) the muscles that maintain the position of the lumbar vertebrae in the erect position are the erector spinae muscles. the erector spinae are type 1 muscles, which are designed for sustained low-level activity. they comprise the spinal, iliocostal, and longissimus muscles, which have the main function of t h o r a c o l u m b a r e x t e n s i o n ( f i g ure 1 ) . ( 1 0 ) electromyographic studies have shown that the erector spinae are more rapidly fatigued in patients with lbp than in healthy subjects. the study by hides found that the multifidus muscle in subjects with lbp have a 31% smaller diameter than in healthy subjects.(11) figure 1. mm. extensor spinae.(10) 113 the function of the erector spinae muscles is to support the vertebral column, control its movements, and protect it, particularly in truncal flexion. protection of the vertebral column may be disturbed if the erector spinae are in fatigued condition, as found in patients with nonspecific lbp. biering-sorensen emphasized that the time during which an individual can maintain horizontal thoracolumbar extension without support is a predictor of back pain.(12) a deficiency in isometric lower back muscle endurance is supposed to be a major risk for non-specific lbp.(13) these studies also found decreased proprioceptive input to the erector spinae, causing the patient to be unable to maintain the erect position, leading to decreased overall physical activity, and ultimately to lowered productivity. several previous studies have demonstrated that management of back pain by thoracolumbar motor control exercises resulted in significant relief of pain, compared with other methods, such as manual therapy and surgical methods.(14) in postmenopausal women, the organ systems start to experience a decrease in functioning, such as decreased estrogen synthesis, which causes a reduction in bone mineral density (bmd) by activation of osteoclasts, leading to a more rapid bone r e s o r p t i o n , i n c o m p a r i s o n w i t h b o n e mineralization.(15) bmd is assessed by means of standard bone densitometry and expressed as t-score, according to the following criteria: t score values of >-1 indicate normal bmd, t scores of <-1 or >-2.5 signify osteopenia (start of osteoporosis), and t scores of <-2.5 indicate osteoporosis.(16,17) in postmenopausal women, the degenerative process also results in a decreased work capacity of the muscles, i n c l u d i n g t h e e r e c t o r s p i n a e m u s c l e s . assessment of biering-sorensen muscle endurance (bsme) for the erector spinae is performed with the patient lying in the prone position, the pelvis, thighs and ankles being fixed by straps, and the arms crossed on the chest. the patient is then asked to perform and maintain horizontal truncal extension, without support to the upper body. the time elapsed in m a i n t a i n i n g t h e u p p e r b o d y i n t h e abovementioned position is recorded as the bsme score of the erector spinae. ( 1 8 ) in connection with the above, the present study was conducted to find correlations of lbp in postmenopausal women with erector spinae endurance and bmd, respectively. methods study design this was a cross-sectional study conducted in mampang prapatan district, south jakarta, from february to april 2010. subjects of study p o s t m e n o p a u s a l w o m e n r e s i d i n g i n mampang prapatan district were selected for this study on the basis of inclusion and exclusion criteria. the inclusion criteria used in this study were: women in the age range of 47–60 years, in menopause for at least one year, mobile, capable of communication, in good health, without acute disease, and willing to give written informed consent. exclusion criteria were: hysterectomy, diabetes mellitus, renal d i s o r d e r s , h e p a t i c d i s o r d e r s , p u l m o n a r y disorders, and hormonal therapy during the previous three years. calculation of sample size according to the formula for cross-sectional studies, with confidence interval of 95% and lbp prevalence of 23.7%, resulted in a minimal sample size of 231. the subjects were selected by simple random sampling in the catchment area of the mampang primary health center, south jakarta. data were collected by interview and physical examination, performed by physiotherapists who had received preliminary training. questionnaire the questionnaire used for the interviews comprised items on the following subject characteristics: age, education, employment, univ med vol.30 no.2 114 rachmawati spinae endurance reduced low back pain n u m b e r o f p r e g n a n c i e s , a n d d u r a t i o n o f menopause. visual analogue scale a visual analogue scale (vas) was used to express the intensity of lbp, as it is a commonly used, quick, reliable, and valid means to measure subjective pain. this scale is a 100mm horizontal line, where 0 mm indicates “no pain” and 100 mm indicates “unbearable pain”. bone mineral density assessment of bmd was done by means of dual-energy x-ray absorptiometry, using a lunar dpx bravo nosuma densitometer (ge medical systems) at budi jaya hospital, jakarta. the results were expressed as t-scores of the lumbar spine (l1–l4). categorization of b m d w a s a c c o r d i n g t o t h e d e f i n i t i o n r e c o m m e n d e d b y t h e wo r l d h e a l t h organization, as follows: (i) normal: a value of bmd greater or less than one standard deviation (sd) below the peak bone mass of healthy adults; (ii) osteopenia: a value of bmd between 1.0 sd and 2.5 sd below the average peak bone mass of healthy adults; (iii) osteoporosis: a value of bmd more than 2.5 sd below the average peak bone mass of healthy adults.(17) anthropometric measurements height, weight, and body mass index (bmi) were measured on all subjects. height was recorded to the nearest 0.1 cm using a portable microtoise, and weight was measured using sage portable scales to the nearest 0.1 kg. bmi was calculated as weight (kg) divided by the square of height (m2). bmi is classified i n t o t h e f o l l o w i n g c a t e g o r i e s f o r a s i a n populations: underweight (<18.5 kg/m 2), normal (18.5-22.9 kg/m2), overweight (23.027.5 kg/m2), and obese (>27.6 kg/m2).(18) biering-sorensen test erector spinae endurance was assessed by the biering-sorensen test, which yields a measure of static endurance of the erector spinae. the bsme test measures the elapsed time during which the participant is still capable of maintaining the unsupported upper part of the body in a horizontal position (figure 2). figure 2. erector spinae endurance testing 115 in this test, the load is equal to the weight of the upper part of the body, with torque determined by the lever arm from the pubic symphysis to the upper-body centre of gravity.(19) the patient was positioned prone on a treatment couch with the lower half of the body-below the level of the anterior superior iliac spinesstrapped to the couch at the ankles and at the level of the greater trochanter of the femur. the straps were pulled as tight possible without causing undue discomfort to the patient. before beginning the test patient was allowed to rest the top half of the body on a chair. then the trunk was raised to the horizontal position with hands crossed over the chest. the test was continued until the participant could no longer control the horizontal posture, or until he or she reached the limit for fatigue or pain. since preliminary tests on 10 postmenopausal subjects yielded a mean endurance time of 28.5 (±2.3) s e c o n d s , n o r m a l b s m e t i m e f o r postmenopausal women in the present study was set at 30 seconds (unpublished data). erector spinae endurance was categorized as good for bsme times of >30 seconds, and poor for bsme times of <30 seconds. ethical clearance the study was explained in detail to the subjects and any questions or concerns were addressed and the subjects were informed that they could withdraw from the study at any time. a consent form was read and signed prior to participation in the study, which was approved by the research ethical committee, faculty of medicine, trisakti university. data analysis data analysis was performed by means of the statistical package for social science (spss) program, version 15. all variables related to lbp were compared by means of student’s t test, while pearson’s correlation coefficients were also analyzed. a 5% level of probability was used to indicate statistical significance. results t h e p r e s e n t s t u d y i n v o l v e d 2 1 3 postmenopausal women with mean age of 53.5 ± 3.6 years. most subjects had attended primary school (50.7%) and were unemployed (67.1%). on average the subjects had menopause for 4.4 ± 2.2 years (range 1-10 years) (table 1). the majority of the subjects was obese (41.3%), 58.0% complained of lbp within the previous 3 months, and mean vas score was 2.0 ± 1.9. the biering-sorensen test showed that the majority of subjects (57.7%) had bsme t i m e s o f < 3 0 s e c o n d s . m o s t o f t h e p o s t m e n o p a u s a l w o m e n s u ff e r e d f r o m osteopenia (49.8%), while 46 (21.6%) had osteoporosis (table 2). with advancing age, the proportion of postmenopausal women with osteopororsis tended to increase, with 12.9% in the age range of 46 49 years and 22.2% between 55 and 60 years of age (table 3). *mean ± sd characteristic n (%) age (years)* range (years) educational level no form al education primary school (not finished) primary school junior high school senior high school university/ college employment entrepreneur government offi cial trader unemployed age at menarche (years)* duration of menopause (years)* range (years) number of pregnancies* range 53.5 ± 3.6 46 60 21 (9.8) 20 (9.3) 109 (50.7) 40 (18.6) 19 (8.8) 4 (1.9) 19 (8.9) 2 (0.0 1) 49 (23.0) 142 (67.1) 14.3 ± 1.7 4.4 ± 2.2 1 10 5.0 ± 2.5 0 13 table 1. characteristics of study subjects comprising 213 postmenopausal women univ med vol.30 no.2 116 rachmawati spinae endurance reduced low back pain variable n (%) height (cm)* weight (kg)* body mass index (kg/m2)* underweight normal overweight obese bone mineral density lumbar t-scor e* normal osteopen ia osteoporosis vas score* back pain yes no bsme time good (= 30 seconds) poor (< 30 seconds) 148.8 ± 5.1 59.2 ± 11.1 26.6 ± 4.6 10 (4.7) 30 (14.1) 85 (39.9) 88 (41.3) -1.60 ± 1.03 61 (28.6) 106 (49.8) 46 (21.6) 2.0 ± 1.9 124 (58.2) 89 (41.8) 90 (42.3) 123 (57.7) table 2. key variables of subjects (n=213) variable vas p r body mass index lumbar t-score 0.165 -0.039 0.016 0.575 table 4. correlation between body mass index and lumbar t-score with vas in postmenopausal women table 5. mean vas score by erector spinae endurance group table 3. age-specific prevalence of osteopenia and osteoporosis in postmenopausal women age range (years) lumbar spine l1-4 osteoporosis (%) osteopenia (%) 46 – 49 50 – 54 55 – 60 12.9 23.9 22.2 48.4 45.7 54.4 this study found a significant positive linear correlation of bmi with vas, where s u b j e c t s w i t h h i g h e r b m i s c o r e s h a d c o r r e s p o n d i n g l y h i g h e r va s s c o r e s (r=0.165;p=0.016). in contrast, lumbar t-scores were not significantly correlated with vas scores (table 4). in postmenopausal women with good erector spinae endurance, the mean vas score was 1.60 ± 1.74, which was significantly lower than mean vas score of 2.32 ± 2.10 in postmenopausal women with poor erector spinae endurance (p=0.008) (table 5). this signifies that a high endurance capacity of the erector spinae muscles was capable of reducing the intensity of lbp in postmenopausal women. discussion in the present study the prevalence of lbp in postmenopausal women was 58.2 %, which differed from the value of 20.8% in a study involving 2.127 women between 35 and 85 years of age, including women in menopause.(20) t h i s d i ff e r e n c e i n l b p p r e v a l e n c e w a s presumably due to the higher age of the postmenopausal women in the present study, ranging from 46 to 60 years. a cohort study on 438 postmenopausal women in the age range of 72-96 years indicated that 37% of women with incident vertebral deformity reported back symptoms at follow-up, as compared with 23.6% of women without incident vertebral deformity.(21) the present study showed the prevalence of osteoporosis in postmenopausal women in the age groups of 46-49 years, 50-54 years, and 55-60 years to be 12.9%, 23.9%, and 22.2%. respectively. the results of a study in thailand, involving 1935 women at age 40*mean ± sd variable erector spinae enduran good (n=90) p vas 1.60 ± 1.74 117 8 0 y e a r s s h o w e d t h a t t h e p r e v a l e n c e o f osteoporosis in those aged 45-49 years and 5054 years (12.0% and 23.7%, respectively) was similar to the present study results, whereas for the age group of 55-59 years, osteoporosis prevalence was considerably higher (42.7%).(22) a german study found a significantly higher prevalence of osteoporosis of 39% among women. up to 6 out of 10 women over the age of 75 were estimated to have osteoporosis.(23) the differences in osteoporosis prevalence rates clearly indicates the diversity of factors a ff e c t i n g o c c u r r e n c e o f o s t e o p o r o s i s i n postmenopausal women, such as life style, climate, culture, and dietary intake. many authors report changes in the control of the trunk muscles in people with low back p a i n . a l t h o u g h t h e r e i s c o n s i d e r a b l e disagreement regarding the nature of these changes, we have consistently found differential effects on the deep intrinsic and superficial muscles of the lumbopelvic region. two issues require consideration; first, the potential mechanisms for these changes in control, and secondly, the effect or outcome of changes in control for lumbopelvic function. recent data indicate that experimentally induced pain may replicate some of the changes identified in people with lbp. while this does not exclude the possibility that changes in control of the trunk muscles may lead to pain, it does argue that, at least in some cases, pain may cause the changes in control. there are many possible mechanisms, including changes in excitability in the motor pathway, changes in the sensory system, and factors associated with the attention demanding, stressful and fearful aspects of pain. a new hypothesis is presented regarding the outcome from differential effects of pain on the elements of the motor system. taken together these data argue for strategies of prevention and rehabilitation of lbp.(24) the result of this study agree with previous study confirmed a muscle deficiency model was supported chronic low back pain, and a muscle spasm model was not. after 50 years, the research evidence is suggesting the hypothesis of the hyperactivity-causality model for musculoskeletal pain. the lack of convincing evidence to support the belief in hyperactivity as an etiological factor in musculoskeletal conditions has been pointed out in many of the research reviews contained in this compendium. these data indicate that the body’s reaction to injury and pain is not primarily increased muscular tension and stiffness, rather muscle inhibition is often more significant. monitoring maximum surface integrated electromyography of lumbar muscles during isometric effort facilitates classification of chronic low-back pain patient. the fact is emg not a common diagnostic tool in indonesia, and many health centre don’t have one. otherwise we need to determined trunk stabilization, particularly erector spinae muscle endurence on low back pain patient. many physician has to make lumbar muscle endurance classification without emg. this study proved that biering sorensen muscle endurence can be done to a n a l y z e l u m b a r m u s c l e e n d u r a n c e . i n postmenopausal women that degenerative process already happen in all body system, definitely has different erector spinae muscle endurance classification compare with young age. the result of this study suggested that postmenopausal women can do as long as 30 seconds or more bsme had lower low back pain incidence. this result supported the previous study that proved that trunk muscle weakness is the low back pain risk factor.(8) the present study demonstrated that bmi had a significant positive correlation with vas scores, indicating that postmenopausal women with higher bmi scores experienced higher i n t e n s i t i e s o f l b p. a s t u d y o n 1 3 4 postmenopausal women showed essentially similar results, in which lbp was associated with menopausal symptoms, bmd, duration of menopause, hormonal therapy, obesity, and inactivity.(25) in the post menopausal years, all women experience the physical effects of aging univ med vol.30 no.2 118 rachmawati spinae endurance reduced low back pain and may also be affected by the hormonal changes responsible for menopause. these changes can include serious health conditions. the present study revealed that the static erector spinae endurance times were lower in the lbp group compared to the normal group, which was consistent with a study among rice farmers(26) reduced endurance capacity of erector spinae muscles has been associated with chronic lbp.(27) the decreased erector spinae endurance could be explained as a result of prolonged lbp. repetitive and accumulative trauma to erector spinae muscles may cause fibrosis and vascular change in the muscles, resulting in decreased erector spinae endurance. however, these explanations may need to be proven by future research. poor back muscle fitness is believed to be associated with back pain and dysfunction regardless of sex and age.(28) longitudinal research suggests that a shorter bsme time denotes an increased risk of developing chronic non-specific lbp. presence of back pain increases the probability of a new vertebral fracture in postmenopausal women with osteoporosis.(20) since the present study is cross-sectional and cannot reflect a causal relationship between decreased erector spinae endurance and lbp, future research should focus on a cohort study to investigate this relationship. in addition, future research should emphasize evaluation of the effectiveness of specific back exercise programs for lbp in postmenopausal women. conclusions the prevalence of lbp and osteoporosis in the postmenopausal women in this study was 58,2% and 21,6% respectively. subjects with poor erector spinae muscle endurance had more pain intensity according to vas. prevention of decreased quality of life in postmenopausal women due to chronic low back pain, requires instructional programs for improving body posture and erector spinae muscle endurance, as well as the program to reduce body weight. acknowledgements the author thanks the postmenopausal women who volunteered for participation this study. thanks are also due to the dean and vice deans of the faculty of medicine, trisakti 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deformity, back symptoms, and functional limitations among older women: the framingham study. osteoporos int 2005;16: 1086–95. 22. limpaphayom kk, taechakraichana n, jaisamrarn u, bunyavejchevin s, chaikittisilpa s, poshyachinda m, et al. prevalence of osteopenia and osteoporosis in thai women. menopause 2001;8:65–9. 23. haussler b, gothe h, gol d, glaeske g, pientka l, felsenberg d. epidemiology, treatment and costs of osteoporosis in germany—the bone eva study. osteoporos int 2007;18:77–84. 24. hodges pw, moseley gl. pain and motor control of the lumbopelvic region: effect and possible mechanisms. j electromyogr kinesiol 2003;13: 361-70. 25. ahn s, song r. bone mineral density and perceived menopausal symptoms: factors influencing low back pain in postmenopausal women. j adv nurs 2009;65:1228-36. 26. taechasubamorn p, nopkesorn t, pannarunothai s. comparison of physical fitness between rice farmers with and without chronic low back pain: a cross-sectional study. j med assoc thai 2010; 93:1415-21. 27. ebadi s, ansari nn, henschke n, naghdi s, van tulder mw. the effect of continuous ultrasound on chronic low back pain: protocol of a randomized controlled trial. bmc musculoskel disord 2011;12:59. 28. payne n, gledhill n, katzmarzyk pt. healthrelated fitness, physical activity and history of back pain. can j appl physiol 2000;24:236–49. univ med vol.30 no.2 c:\users\universa medicina\docu 184 abstract universa medicina gynecological and breast cancer risk factors and screenings in lesbian and bisexual women nuriye buyukkayaci duman1* and fatma yildirim1 it is estimated that individuals with lesbian, gay, bisexual, transsexual, intersexual, and other sexual tendencies make up approximately 3-12% of the world population. the proportion of lesbian and bisexual women (lbw) varies between 1-20% of all women. studies on the subject show that risk factors for gynecological malignancies and breast cancer are more common in lesbian and bisexual women than in heterosexual women. in lbw, it is reported that obesity or high body mass index, inactivity, smoking, alcohol and substance abuse, stress, anxiety disorders, depression, and sexually transmitted diseases are more common than in heterosexual women. in addition, sexually transmitted infections (stis) such as genital herpes, human papilloma virus (hpv) infections, bacterial vaginosis, trichomoniasis, hepatitis a, syphilis, and hiv disease, that can be transmitted by homosexual relationships, are also common in these women. it is known that genital herpes and hpv are important risk factors especially in the development of cervical cancer. in addition, breastfeeding rates and lactation periods are known to have protective effects against gynecological malignancies and lactation periods are known to have protective effects against breast cancer. this review aims to discuss gynecological malignancy and breast cancer incidence in lbw and the risk factors for these cancers according to current literature. in this context, a total of 340 articles were accessed by entering keywords related to the subject in the pubmed database and analyzed according to their titles and abstracts, while duplicates were removed. however, reviews, qualitative studies, and summaries were not included in the study. a total of 23 research articles were examined, published between 2012-2022, whose full text can be accessed through the pubmed database, investigating gynecological cancer and breast cancer risk factors and screenings in lbw. keywords: lesbians, bi-sexual women, gynecological cancers, breast cancer, screening review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2022.v41.184-193 copyright@author(s) available online at https://univmed.org/ejurnal/index.php/medicina/article/view/1224 may-august, 2022 vol.41no.2 1departmen t of obstetrics and gynecology nursing, faculty of health sciences, hitit university, çorum, turkey correspondence: *nuriye büyükkayaci duman associate professor, hitit university, faculty of health sciences, department of obstetrics and gynecology nursing, çorum, turkey. orcid-id: 0000-0002-4246-3607 email: nurfatihh@hotmail.com telephone: +90 5055015752 orcid id : 0000-0002-4246-3607 date of first submission, october 30, 2021 date of final revised submission, april 25, 2022 date of acceptance, may 18, 2022 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: buyukkayaci duman n, yildirim f. gynecological and breast cancer risk factors and screenings in lesbian and bisexual women. univ med 202 2;41 :1 8 4 -9 3 . doi : 10 .18 05 1/ univmed.2022.v41.184-193 univ med vol. 41 no 2 185 introduction the abbreviation “lgbti +” is used for lesbian, gay, bisexual, transsexual, intersex, and other sexual trends that ref lect differ ent dimensions of homosexuality worldwide.(1,2) while men with a sexual orientation towards their sex are defined as gay and women as lesbian, sexual tendency towards both sexes is defined as bisexual.(1,3) when the literature is examined, it will be seen that lgbti + individuals make u p a p pr ox i ma te ly 3-1 2 % of t h e t ot a l population.(4,5) it is estimated that the proportion of lesbian and female bisexuality in the world varies between 1-20% of all women.(2,6) this finding clea rly r eveals that the problems of lesbian and bisexual women (lbw) who have to live in society invisibly, should be addressed separately. according to a study conducted in the united states (usa), almost 50% of “lgbti +” individuals were diagnosed with cancer.(7) studies on the subject show that smoking, alcohol use, excess weight/obesity, inactivity, stress, anxiety disorders, depression, and sexually transmitted diseases that are seen more in lesbian and bisexual women (lbw) than in heterosexual women, are considered to be risk factors for gynecological malignancies and breast cancer.(810) in addition, the use of oral contraceptives is known to protect against endometrial cancer while birth and breastfeeding rates are known to protect against ovarian cancer and breast cancer, all of which are lower in this group of women than in heterosexual wo me n. ( 11 ,-1 3 ) al s o , s e xu a l l y t r a n s mi t t e d infections (stis) such as genital herpes, human papilloma virus (hpv) infections, bacterial vaginosis, trichomoniasis, hepatitis a, syphilis, and hiv disease, which can be transmitted by homosexual relationships, are also common in these women.(9,14) it is known that genital herpes and hpv are important risk factors, especially in the development of cervical cancer.(9,14) although they have many risk factors, it is known that lbw avoid gynecological examination and gynecological cancer screenings and receive inadequate health care services related to women’s health.(11-15) although the rates are only estimates, the increase in gynecological cancer prevalence in lbw today may be associated with the insufficiency of protective gynecological health services despite the increased cancer risk in this group of women. in this review paper, a total of 340 articles were reached by entering keywords in the pubmed database. however, reviews, qualitative studies, and summaries were not included in the study. the flow of review information is shown in figure 1. this review aims to discuss gynecological malignancies and breast cancers in lbw according to the current literature. a total of 23 research articles, published between 2012-2022, were examined, whose full text can be accessed through the pubmed database, and which investigated gynecological cancer and breast cancer risk factors and screenings in lbw (table 1). gy n ec o logi cal and b r east cancer risk factors in lesbian and bisexual women cervical cancer cervical cancer is the third most common type of gynecological cancer in the world.(16,17) the prevalence of cervical cancer is estimated to be over 400,000. it is the second most common gynecological cancer type among the top 10 cancer types seen in women in the usa,(17) where the incidence of cervical cancer is 7.7/100.000, and the mortality rate is 2.3/ 100.000.(16) the literature on cervical cancer and screening studies in lbw seems to be quite insufficient.(9,15) in turkey, a study on this group of women in the reproductive health and health care services has not been demonstrated outside of the limited number of studies.(6,11) according to the california health interview questionnaire, lesbians (16.5%) were reported to have a higher prevalence of cervical cancer than heterosexual women (14.0%). according to the same study results, the prevalence of cervical cancer . 186 (41.2%) was found to be higher in bisexual wo me n c o mp a r e d t o he te r o se x u a l a n d lesbians.(18) these findings require that risk factors related to cervical cancer be examined in lbw. it is known that cervical cancer risk factors such as smoking, alcohol use, substance abuse, and obesity are common in lbw. (8,19) in addition, the main risk factors for cervical cancer, namely early sexual intercourse before the age of 16, polygamous sexual life, and stis such as hpv-16 and hpv-18, [especially hpv-11 and hpv-16], are common in lbw and are reported to increase the risk of getting caught.(1,9) it is kno wn t ha t h pv-16 a n d h pv-1 8 a r e responsible for almost 70% of all cervical cancer cases. when the literature is examined, different study results related to the subject were found. although previous studies indicate that stis are rare among lesbians, studies in recent years are drawing attention.(14,15) this has been linked to the fact that lesbians or their spouses have past or current heterosexual relationship histories. in addition, stis such as hpv, genital herpes, bacterial vaginosis, trichomoniasis, hepatitis a, syphilis, and hiv disease have been reported t o be t r an smi t t e d t hr o u gh homos e x u a l intercourse in women.(14,15) according to the literature, hpv transmission can also occur through skin contact with infected body fluids, genital mucosa, penis, scrotum, vagina, vulva, or anus. for this reason, it is known that lesbians who do not have vaginal/anal intercourse are also at risk of hpv infection and thus cervical cancer.(14,15) endometrial cancer endometrial cancer is the most common type of gynecological cancer. one in every 40 women is diagnosed with endometrial cancer in her life.(20,21) in the usa, there are an average of 54,870 new cases of uterine corpus cancer each year, and it is predicted that endometrial cancer wil l develop mostly a mong these cases.(17, 22) when the literature is examined, it is seen that, like other types of gynecological cancer, there is a limited number of studies buyukkayaci duman, yildirim breast cancer risk factors in lbw figure 1. flow of review information univ med vol. 41 no 2 187 authors article title type of study sample size results united states preventive services task force (42) the guide to clinical preventive services: screening for cervical cancer. baltimore descriptive 9581 women pap smear screening rates in lesbian women were found to be significantly lower than in heterosexual women. cochran & mays (46) risk of breast cancer mortality among women cohabiting with same sex partners: findings from the national health interview survey descriptive 693 women lesbian women are less likely to undergo clinical breast examination (cbe) in 2 years than heterosexual women, but mammography rates do not differ. sexual orientation has been found to be at risk of fatal breast cancer. gooren et al.(34) breast cancer development in transsexual subjects receiving cross-sex hormone treatment descriptive 15,974 people cross-sex hormone therapy of transgender subjects does not appear to be associated with an increased risk of developing malignant breast cancer. bernat et al.(40) characteristics associated with initiation of the hpv vaccine among a national sample of male and female young adults descriptive 3,448 people the rate of lbw (44.9%) receiving at least one dose of hpv vaccine was lower than that of heterosexual women (51.1%) in their web-based studies involving women between the ages of 18 and 24 years. however, the relationship between sexual orientation and hpv vaccination was statistically insignificant. fredriksengoldsen(15) hpv vaccination among lesbian and bisexual women: findings from a national survey of young adults descriptive 543 people many lbw were not vaccinated against hpv. 45% of lesbian and bisexual women received one dose of hpv vaccine, but 70% completed all three doses of the vaccine. clavelle et al.(36) breast cancer risk in sexual minority women during routine screening at an urban lgbt health center descriptive 423 women the nullipara rate and the lifetime risk of breast cancer in lbw compared with the gail model were found to be higher than in heterosexual women lacombeduncan & logie (48) correlates of clinical breast examination among lesbian, gay, bisexual, and queer women descriptive 22410 women the rate of participation in the clinical breast examination of lbw in the last two years, knowledge of stds, sexual risk practices, the last two years of pap test, regular health checkups and the state of health personnel's knowledge of sexual orientation were positive. the thought of stigmatization and the belief that healthcare personnel are uncomfortable with their sexual orientation were negatively correlated. table 1. data on the studies conducted between 2000-2020 on gynecological cancer and breast cancer risk factors and screening in lesbian and bisexual women 188 related to endometrial cancer incidence in lbw. ( 1 8 ,2 1 ) in s o me s t ud ie s, t h e r i sk o f endometrial cancer in lbw has been reported to be higher than in heterosexual women,(3) while in other studies it has been reported to be lower.(1) it is known that the most important factors that increase the risk of endometrial cancer in lbw c authors article title type of study sample size results liu &yeo (47) breast health, risk factors, and cancer screening among lesbian, bisexual, and queer/questioning women in china descriptive 310 women the rates of breast selfexamination and clinical breast examination of lbw were low. the study concluded that lbw are vulnerable to breast cancer. greene et al. (44) association of pregnancy history and cervical cancer screening in a community sample of sexual minority women descriptive 430 women a positive correlation was found between pregnancy history and having a pap test. in addition, disclosure of one's bisexual identity and sexual orientation to healthcare providers also positively affects the pap test. milner & mcnally(20) non adherence to breast and cervical cancer screening among sexual minority women descriptive 1115 women among women who had a general physical in the last year, concealment, stigma consciousness, rejection sensitivity, and fear of negative evaluation were all positively associated with lower rates of timely pap tests. among all women, these psychological variables were positively associated with never obtaining a pap test and concealment was also negatively associated with clinical breast exam adherence. kim, lee & choi-kwon (43) cervical cancer screening and human papillomavirus vaccination among korean sexual minority women by sex of their sexual partners descriptive 671 women lbw with only female partners had lower rates of pap testing and completion of hpv vaccine than women with male and female partners. herriges et al. (1) the association of sexual orientation with prostate, breast, and cervical cancer screening and diagnosis descriptive overall, 4,441 and 6,333 heterosexual men and 225 and 213 lgb men and women, a higher proportion of heterosexual women than lbw were screened for cervical cancer with pap smears (95.36% vs. 90.48% and 86.11%, p=<0.001). legend: lbw = lesbian women and bisexual women are obesity, nulliparity, medications suppressing the ovaries, and anovulation. risk factors such as polycystic ovarian syndrome, tamoxifen use, hypertension and diabetes mellitus, and lynch syndrome are thought to affect the prevalence at the same rate as in heterosexual women.(1,10) according to the american cancer society (3) buyukkayaci duman, yildirim breast cancer risk factors in lbw univ med vol. 41 no 2 189 comparing gynecological cancer risk factors over 40 years of age, cancer risk factors such as nulligravida, high body mass index, and immobility in lbw were found to be higher in homosexual than in heterosexual women. moreover, the number of pregnancies, breastfeeding, and oral contraceptive use rates are lower in lbw than in heterosexual women. in the study, it was concluded that gynecological malignancy risk factors were higher than in heterosexual women.(3) according to the women’s health initiative (whi), older lbw who have never had sex with men have been reported to have a lower prevalence of endometrial cancer than heterosexual women.(1) however, the limited literature on the subject reveals the need for further study. ovarian cancer although ovarian cancer is the second most common type of gynecological cancer, it is also seen as the most common cause of death among other gynecological cancer types.(22,23) risk factors include nulligravida, early menopause, unsatisfactory use of estrogen, breast cancer gene 1 (brca1) or breast cancer gene 2 (brca2) gene mutation, overweight, and obesity.(22,23) the factors with a protective effect are pregnancy, high parity, oral contraceptive use, lactation, and ovarian cancers. in lbw, o be s it y, l ow use o f or a l co ntr a ce pti ves, nulligravida, or decreased number and duration of pregnancy, are associated with ovarian cancer.(19,24,25) in addition, some studies on the subject reported that the health beliefs and behaviors of this group of women towards ovarian cancer genetic risk tests (brca1 or brca2) are inadequate.(13-15) in the studies of azagba et al.(26) and semlyen et al.(27) lesbians had a higher body mass index while in the studies of waterman and voss (14) and barefoot et al.(28) the number of pregnancies, number of children, number of abortions, and use of birth control pills were higher. also, in the study of fallin at al.(19) smoking rates are higher in lesbians than in heterosexual women. breast cancer breast cancer is the most common type of cancer in women after skin cancer and is observed in 18% of women worldwide. while one out of every nine women gets breast cancer at some time in their lives, one out of thirty women die due to breast cancer.(17,22) the estimated incidence of breast cancer in the usa is 124.8/ 100,000 per year.(29) breast cancer risk factors include early menarche, late menopause, pregnancies over 35, hormone replacement therapy, family history, brca1-2 gene mutation, alcohol use, obesity, and nulliparity.(30,31) studies on breast cancer in lbw have shown that these women have a higher risk of breast cancer risk factors such as alcohol use, obesity, and nulliparity compared to heterosexual women. it is reported that the rate of breastfeeding, known to be protective against breast cancer, is also lower in lbw.(10) in the studies of austin et al.(32) the incidence rate ratio of breast cancer in lbw were found to be higher than in heterosexuals. in this same study one year incidence rates per 100,000 person-years were 131.6 lesbian, 131.7 bisexual women and 122.6 heterosexual women.(32) in addition, it is known that the risk of developing breast cancer in lesbians who have not had mastectomy a nd who received testosterone treatment is not clear, but treatment taken does not affect estradiol levels.(31,33) according to the whi, lbw have a higher pr evalence of br east cancer compar ed to heterosexual women.(34,35) when the literature is examined, different study results related to the subject were found. the study of boehmer et al.(10) reported that in the 2001, 2003, 2005, and 2007 california health interview surveys, the lesbian population density was associated with higher breast cancer incidence, and the executive bisexual population density was associated with lower breast cancer incidence. clavelle et al. (36) in their study investigating breast cancer risk factors in lbw, found that the nullipara rate and the lifetime risk of breast cancer in these women compared with the gail model were higher than in heterosexual women. 190 a short training session with a lesbian doctor, it was found that one in four women (58-66 years) who did not have a pap test had a pap test within 3 years.(1) similarly, in the studies of kim et al.(43) it was found that lbw with only female partners had lower rates of pap testing and completion of hpv vaccine than women with male and female partners. as can be seen, studies on the subject show that the health behaviors of lbw regarding gynecological examination and pap test are not at a sufficient level.(1,14,40) on the other hand, the frequency of gynecological examination and pap test in women with heterosexual relationships is higher than in women with only homosexual relationships. similarly, in the study conducted by greene et al.(44) on lbw, it was reported that previous pregnancy history positively affected the rates of having pap tests. another factor that positively affects the rates of lbw having pap tests is the disclosure of sexual orientation to healthcare providers. in the studies of milner and mcnally,(20) fear of stigmatization, tendency to hide, and fear of exclusion negatively affect the pap test in lbw. psychological barriers and concealment of sexual identity may negatively affect compliance with gynecological screening behaviors among some sexual minority women. strategies that facilitate positive disclosure experiences to healthcare providers and address psychological factors related to minority stress can improve compliance with gynecological cancer screening.(20,44) breast cancer there are also different results in studies examining the effect of sexual orientation on awareness of breast cancer and screening. while some studies reported that lbw are less interested in breast self-examination (bse) and mammography applications than heterosexual women,(15) sexual orientation does not affect awareness of these scans.(34,45) in the studies of cochran and mays,(46) it was found that lesbians had a lower probability of performing clinical breast examinations (cbe) within 2 years gynecological and breast cancer screening in lesbian and bisexual women gynecological cancers the american academy of pediatrics (apa) recommends routine administration of 3 doses of hpv vaccine between the ages of 1112. it has been reported that those who have not had immunity or who have not completed their doses should be vaccinated by completing their doses between the ages of 13 and 26.(37) the american food and drug administration (fda) approved two types of hpv vaccines, namely the bivalent (hpv-16, hpv-18) and quadrivalent (hpv-6, hpv-11, hpv-16, hpv-18) vaccines. (38) the american college of obstetrics and gynecology (acog) recommends performing an annual pelvic examination and pap test for all sexually active women aged 21 and over.(39) these recommendations also include lbw. however, although limited data are available, including cervical cancer screenings and hpv vaccination for lbw, studies show that hpv vaccination and cervical screening are insufficient in this group of women.(14,29) bernat et al.(40) reported that the rate of lbw (44.9%) receiving at least one dose of hpv vaccine was lower than heterosexual women (51.1%) in their web-based studies involving women between the ages of 18 and 24. however, the relationship between sexual orientation and hpv vaccination was found to be statistically insignificant. in addition, in the studies of mcree et al.,(41) which included 543 lbw between the ages of 18-26, it was found that 45% of women received a dose of the hpv vaccine, but that 70% completed three doses of the vaccine. in the studies of the united states preventive services task force (uspstf), it was found that the papanicolaou (pap) smear screening rates in lesbians were quite low compared to that in heterosexual women.(42) similarly, according to the whi, compared to heterosexual women, older lbw have been reported to be significantly less likely to undergo a pap test at any time.(1) in the same study, after buyukkayaci duman, yildirim breast cancer risk factors in lbw univ med vol. 41 no 2 191 compared to heterosexual women, but their mammography rates did not differ. similarly, in the studies of liu and yeo,(47) lbw were reported to have low bse and low clinical breast examination (cbe) rates. in the studies of fredriksen-goldsen et al., (1 5) l bw mammography rates were f ound to be significantly lower compared to those of heterosexual women. in lacombe-duncan and logie’s (48) studies the rate of participation of lbws in cbe in the last two years, knowledge of stds, sexual risk practices, the last two years of pap test, regular health checkups and the state of health personnel’s knowledge of sexual orientation were positive. the thought of stigmatization and the belief that healthcare personnel are uncomfortable with their sexual orientation was negatively correlated. the contradictory findings may be due to the small sample sizes in the studies to represent the universe, the demographic determinants that prevent access to breast cancer screenings, homogeneous distribution in the studied groups, how sexual orientation is classified, and differences in methodologies. for this reason, it is thought that more studies are needed to generalize the results of the studies. conclusion although lbw carry many risk factors in terms of gynecological malignancies and breast cancer, their awareness and healthcare rates are insufficient compared to heterosexual women. while lbw should receive reproductive health and sexual health services based on their risk factors specific to their sexual orientation, the fact that this group of women tends to hide their sexual orientation makes them invisible to the health system. however, the results of this study on the subject show that lbw need to be handled specially in the provision of health services and the preparation of guidelines when developing preventive health policies for gynecological malignancies and breast cancer. when the literature is examined, it is seen that studies examining gynecological malignancies and breast cancer incidence and risk factors in lbw are insufficient. therefore, more studies are needed to produce good and generalizable evidence. in addition, healthcare providers should question sexual orientation and evaluate lbw-specific health risk factors, not only in the provision of preventive healthcare, but also in therapeutic services. training and counseling services to increase awareness of gynecological malignancies and breast cancer and to encourage screening in this group of women should be made 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health 2013; 53:630-6. doi: 10.1016/j.jadohealth.2013. 07.035. 41. mcree al, katz ml, paskett ed, reiter pl. hpv vaccination among lesbian and bisexual women: findings from a national survey of young adults. vaccine 2014;32:4736-4742. doi: 10.1016/ j.vaccine.2014.07.001. 42. united states preventive services task force (uspstf). the guide to clinical preventive services 2012: screening for cervical cancer. rockville (md): agency for healthcare research and quality (us); 2012. 43. kim s, lee sy, choi-kwon s. cervical cancer screening and human papillomavirus vaccination among korean sexual minority women by sex of their sexual partners. international j environ res public health 2020;17:8924. doi: 10.3390/ ijerph17238924. 44. greene mz, hughes tl, sommers ms, et al. association of pregnancy history and cervical cancer screening in a community sample of sexual minority women. j women’s health 2019;28:526– 34. doi: 10.1089/jwh.2018.6960. 45. cathcart-rake ej. cancer in sexual and gender minority patients: are we addressing their needs? current oncol reports 2018;20:1–8. doi: 10.1007/ s11912-018-0737-3. 46. cochran sd, mays vm. risk of breast cancer mortality among women cohabiting with same sex partners: findings from the national health interview survey, 1997–2003. j women’s health 2012;21:528–33. doi: 10.1089/jwh.2011.3134. 47. liu pl, yeo ted. breast health, risk factors, and cancer screening among lesbian, bisexual, and queer/questioning women in china. health care women int 2019;42:947–61. doi: 10.1080/ 07399332.2019.1571062. 48. lacombe-duncan a, logie ch. correlates of clinical breast examination among lesbian, gay, bisexual, and queer women. can j public health 2016;107:467–72. doi: 10.17269/cjph.107.5351. alvina 162 abstract immune dysfunction in postmenopausal women tends to decrease health-related quality of life (hrqol). the present study’s objective was to evaluate the effect of daily supplementation of 100 mg soy isoflavones on specific immune responses among healthy postmenopausal women. the study design was a communitybased double blind randomized controlled trial involving 60 healthy postmenopausal women, aged between 48–60 years, in the mampang prapatan district of south jakarta. participants were randomized to receive either 100 mg soy-isoflavone + 500 mg calcium (intervention group) or 500 mg calcium only (control group). both supplements were taken daily for 12 weeks, from january to april 2010. specific immune responses (measured by serum ig g and cd4+) were assessed at baseline and after supplementation. statistical analysis using independent t-test was performed to evaluate the effect of soy isoflavone supplementation on specific immune responses. fifty-six (93.3%) participants completed the study without any significant side-effects or adverse events. daily supplementation of 100 mg soy isoflavones for 12 weeks did not significantly increase the humoral specific immune response (p=0.242), but tended to improve the cellular specific immune response (p= 0.850). other findings of this study were that soy isoflavone supplementation tends to improve specific immune responses in postmenopausal women with normal body mass index and adequate daily dietary isoflavone intakes. short-term soy isoflavone supplementation is unable to improve the humoral and cellular specific immune responses in postmenopausal women aged 48 to 60 years. keywords : soy-isoflavone, specific immune response, postmenopausal women *department of obstetrics and gynecology, medical faculty, trisakti university correspondence dr. raditya wratsangka, dr., sp.og(k) department of obstetrics and gynecology, medical faculty, trisakti university jalan kyai tapa no. 260, grogol jakarta barat – 11440 phone: 021-5672731 ext. 2706 email: raditya@trisakti.ac.id univ med 2011;30:162-72 soy isoflavone supplementation tends to improve specific immune responses in postmenopausal women raditya wratsangka* september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina introduction menopause, according to the definition of the world health organization and american association of clinical endocrinologists, is the last natural menstruation, which is the time of permanent cessation of menstruation due to loss of activity or nonfunctioning of ovarian follicles caused by structural and functional changes, thereby causing decreased secretion of ovarian steroidal sex hormones, particularly estrogens and progesterones.(1,2) the decrease 163 univ med vol.30 no.3 r subjects o1 o2 x o3 o4 figure 1. diagram of research design notes : r = randomization; o = observation; x = intervention; o1 and o2 = pre-supplementation; o3 and o4 = post-supplementation in steroidal sex hormone concentrations in the body, particularly estrogens, does not change the menstrual pattern, but impacts on general health, particularly as somatic/urogenital and vasomotor abnormalities, known as menopausal symptoms.(3) in addition to risk of coronary heart disease, osteoporosis and several malignancies, as a result of the aging process, postmenopausal women also undergo reduction of the immunity of the body, such that menopausal women become more susceptible to infectious diseases, and commonly also to various degenerative disorders.(4) several studies have shown that postmenopausal women have decreased total numbers of lymphocytes, in comparison to women of reproductive age, as a result of decrease in antibody-producing b lymphocytes (which produce antibodies) and cytokineproducing t helper (th) cells, although the existence of other mechanisms involved in the control of lymphocyte numbers in women cannot be eliminated.(5) the results of the study conducted by the wo m e n ’s h e a l t h i n i t i a t i v e , s h o w i n g a n increased risk of breast cancer, coronary heart disease, stroke, and venous thromboembolism (vte) in users of hormonal therapy, has caused some menopausal women to refuse hormone t h e r a p y ( h t ) a n d t o s e e k a l t e r n a t i v e treatments capable of filling their need for managing the menopausal abnormalities.(6,7) p h y t o e s t r o g e n s a l s o k n o w n a s “ h e r b a l estrogens” are natural substances from plants with chemical structures and effects that are similar to those of estrogens. these compounds bind to estrogen receptors (er-á and er-â), and have both estrogenic and weakly antiestrogenic activities, depending on endogenous estrogen and estrogen receptor concentrations, such that phytoestrogens are also known as s e l e c t i v e e s t r o g e n r e c e p t o r m o d u l a t o r s (serms) with both agonist and antagonist effects.(8,9) i s o f l a v o n e s a r e a m o n g t h e b e s t phytoestrogens, being found in soy beans, which a r e a r e m u c h c o n s u m e d b y i n d o n e s i a n communities and other asian populations. since the human immune system is partially influenced by steroidal hormones, it may be assumed that soy isoflavones are capable of affecting the immune system through estrogen-receptormediated mechanisms. epidemiological data indicate that the prevalence of chronic and degenerative disorders is significantly lower in asia populations consuming soy-rich foods, in c o m p a r i s o n t o a m e r i c a n o r e u r o p e a n populations.(10) the present study had as objective to determine the increase in humoral and cellular specific immune responses in postmenopausal women after soy isoflavone supplementation. methods research design t h i s w a s a n e x p e r i m e n t a l s t u d y o f community-based double blind randomized controlled trial design to investigate the effect of supplementation of soy isoflavones 100 mg for 12 weeks on humoral and cellular specific immune responses in postmenopausal women. this study was conducted from january to midapril 2010. 164 research subjects t h e s t u d y p o p u l a t i o n c o m p r i s e d postmenopausal women (minimally one year without menstruation). the inclusion criteria were: postmenopausal women aged 48-60 years; healthy (capable of independently performing activities of daily living and capable of communication, not consuming menopauserelated hormonal medications within the last three months, and agreeing not to consume other vitamins/minerals during the 12 weeks of s u p p l e m e n t a t i o n , n e v e r h a v i n g h a d hysterectomy and/or oophorectomy. the exclusion criteria for the subjects were: past history of diabetes mellitus, renal or hepatic disorders, heart disease, and hypertension. sample size the study sample was divided into two independent groups, namely the intervention group and the control group. the number of s u b j e c t s i n e a c h g r o u p w a s c a l c u l a t e d a c c o r d i n g t o t h e f o l l o w i n g f o r m u l a f o r hypothesis testing of means difference of two independent groups(11) the above calculation and a design effect of 2, a total of 30 subjects was required for each group, therefore the total sample size required was 60 postmenopausal women. sample selection this experimental study was conducted in the catchment area of the mampang prapatan subdistrict health center, south jakarta. the mampang prapatan subdistrict was chosen as the study area by purposive sampling. selection of kelurahan (villages), rw (hamlets) and rt (neighborhoods) to be involved in this study was performed by cluster random sampling, whereas selection of subjects in rts was by proportional simple random sampling. intervention the study subjects were divided into two groups, as the isoflavone and the control group, respectively. random allocation was performed with a block size of 4 on 60 subjects by random number generation. the intervention was conducted by administering the supplement at a dose of one tablet per day for 12 consecutive weeks. the supplement tablets were prepared by pt ikapharmindo putramas with a special license from badan pengawasan obat dan makanan republik indonesia (bpom-ri). the tablets were labelled a and b by pt ikapharmindo putramas, where the tablets labelled a contained 250 mg of 40% soybean isoflavone extract (equivalent to 100 mg soy isoflavones) + 500 mg calcium, while the other type of tablet only contained 500 mg calcium. to minimize the effect of bias on the study results, the tablets were administered in a doubleblind manner. soy isoflavone intake daily dietary intakes (carbohydrates, lipids, proteins, and isoflavones) were assessed and calculated by trained nutritionists, based on the quality and quantities of the foods consumed. assessment was done by using the food record method, in which the subjects 21 χ−χ effectdesignx )( sd)zz( 2n 2 21 βα ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ χ−χ + = sd = standard deviation of the two groups = expected clinical difference (ä) á = 0.05 z á = 1.96 â = 20% z â = 0.84 based on the results of a previous study on the effect of soy isoflavone administration on immune function in healthy postmenopausal women conducted by ryan-borchers et al.,(10) who measured cd4+ concentrations in blood plasma, a standard deviation (sd) of 6.9 was obtained, and the expected effect size (ä) after administration of soy isoflavones for 3 months being 15%, the calculated sample size n for each group was 12 subjects. assuming a drop-out rate of 20% during the study, the required sample size for each group was 15 subjects. based on wratsangka isoflavone and specific immune responses 165 univ med vol.30 no.3 t h e m s e l v e s r e c o r d e d t h e i r d a i l y f o o d consumpton three times weekly, on 2 working days and 1 holiday (saturday or sunday). assessment of the quantity and quality of dietary isoflavone intakes was based on the c o m p l e t e n e s s o f f o o d s c o n t a i n i n g s o y isoflavones and their weekly frequency of c o n s u m p t i o n , b y m e a n s o f t h e s e m i quantitative food frequency questionnaire (sq-ffq), which contains a list of isoflavonecontaining foods. the subjects were grouped into quintiles (q 1 to q 5 ), according to their dietary isoflavone intakes, as follows: q 1 were the subjects considered to have poor dietary isoflavone intakes, while subjects in q 2 –q 5 were considered to have adequate dietary isoflavone intakes. determination of body mass index (bmi) the subjects were measured as to height and body weight. height was measured using a portable microtoise to the nearest 0.1 cm and weight was measured using sage portable scales to the nearest 0.1 kg. the bmi criteria used w e r e d e t e r m i n e d b y t h e wo r l d h e a l t h organization. according to these criteria, bmi is classified as normal (18.5 25.0 kg/m2), mildly overweight (25.1 – 27.0 kg/m2), and severely overweight (> 27.0 kg/m2).(12) laboratory measurements at baseline and after supplementation, from each study subject in both groups (control and intervention), a 10 ml venous blood sample was collected by a trained laboratory technician from prodia clinical laboratories. the plasma isoflavone, cd4+ and immunoglobulin g (igg) concentrations were determined at baseline ( b e f o r e s u p p l e m e n t a t i o n ) a n d a f t e r 1 2 consecutive weeks of supplementation. isoflavone concentrations plasma isoflavone concentrations were m e a s u r e d b y h i g h p e r f o r m a n c e l i q u i d chromatography (hplc) at the dki jakarta provincial health laboratory (laboratorium kesehatan daerah, labkesda, provinsi dki j a k a r t a ) . h e l i x p o m a t i a g l u c u r o n i d a s e sulfatase h-2 was added to serum (1 ml), and the mixture was incubated at 37 °c for 15 to 18 h to hydrolyse genistein and daidzein. to the sample was added trichloroacetic acid and the isoflavones extracted with 7.5 ml ethyl acetate. extracted isoflavones were dissolved in 80% methanol in water for hplc. imunoglobulin g and cd4+ concentrations l a b o r a t o r y d e t e r m i n a t i o n o f i g g concentrations was performed at prodia clinical laboratories, by means of turbidimetry ( w i t h a d v i a 1 8 0 0 r e a g e n t f r o m a d v i a chemistry systems – bayer health care), with a coefficient of variation (cv) of 2.7%. determination of cd4+ concentrations was p e r f o r m e d a t d h a r m a i s c a n c e r h o s p i t a l laboratory, using flow cytometry (with bd tritesttm reagent from becton dickinson – usa), with a coefficient of variation (cv) of 2.58%. the abovementioned laboratories had passed iso 9001:2000 and iso 9001:2008 certification. ethical clearance this study received ethical clearance from the commission for research ethics, faculty of public health, university of indonesia. statistical analysis the study data were tested for normality of distribution using the kolmogorov-smirnov test. effect differences between the isoflavone group and the control group were assessed by independent t-test . all analyses used the statistical package for social sciences (spss) for windows version 17. p-values <0.05 were considered statistically significant. results characteristics of subjects screening of postmenopausal women selected for participation as study subjects 166 yielded 80 postmenopausal women, among whom 18 women did not meet the inclusion criteria and 2 women declined to participate. the remaining 60 subjects were assigned to two groups. after 12 weeks of supplementation, 3 subjects from the isoflavone group and 1 subject from control group dropped out from the study, because they had to move outside the study area. therefore 56 subjects were available for analysis per protocol and at the completion of the study, namely 27 subjects in the isoflavone group and 29 subjects in the control group (figure 2). among subjects participating in this study, the youngest subject was 48 years old and the oldest 60 years. as shown in table 1 (below) the results of comparability testing between the isoflavone and control groups showed that several important characteristics (such as age, marital status, educational level, employment, physical exercise, smoking, coffee consumption) as well as a number of clinical characteristics ( s u c h a s b o d y m a s s i n d e x , d u r a t i o n o f menopause, estradiol concentration, dietary intake) at the start of the study (baseline data) were not significantly different in both groups. mean igg and cd4+ concentrations at baseline were also not significantly different in both intervention groups. this indicates that the random allocation with a block size of 4 succeeded in uniformly distributing all nonintervention variables. figure 2. flowchart for screening & recruitment of study subjects, allocation of intervention, follow-up, and data analysis assessed for eligibility (n=80) randomized (n=60) excluded (n=20) not m eeting inclusion criteria (n=18) declined to p arti cipate (n=2) isoflavone (n=30) control (n= 30) lost to follow-up moved (n= 3) analyzed (n=29) lost to follow-up moved (n= 3) analyzed (n=27) wratsangka isoflavone and specific immune responses 167 univ med vol.30 no.3 effect of isoflavone supplementation on specific immune responses i n t h i s s t u d y, t h e s p e c i f i c i m m u n e responses assessed were the humoral immune response with igg concentration as parameter and the cellular immune response with cd4+ concentration as parameter. i n ta b l e 2 i t i s a p p a r e n t t h a t a f t e r supplementation the isoflavone group had higher mean igg and cd4+ concentrations, in comparison with the control group, although the difference was not statistically significant. in table 3, the mean igg concentration after supplementation decreased in both the isoflavone and the control subgroups based on duration of menopause, with a larger decrease in the isoflavone group. in subgroups based on body mass index, mean igg concentration in the isoflavone group with normal bmi was slightly increased after supplementation (1%), while mean igg concentration in the control group decreased by 3.6%. in subgroups based o n d i e t a r y i s o f l a v o n e i n t a k e , m e a n i g g concentration after supplementation in the variable intervention group p isoflavone (n=27) control (n=29) age (years) * 53.0 (3.2) 54.0 (3.4) 0.261 a) marital status ** married (w/ living husban d) 59.2 75.9 0.254b) unmarried/widowed 40.8 24.1 educational level ** low (< sd) 62.9 68.9 0.635b) interm ediate (smp/sma) 33.3 31.1 high (> d1) 3.8 0 employment ** emplo yed 37.0 37.9 0.945b) unemployed 63.0 62.1 physical exercise ** yes, r egular 29.6 20.7 0.151 b) yes, irregular 40.8 24.1 never 29.6 55.2 smoking ** no 92.6 93.1 1.000 c) yes 7.4 6.9 coffee drinking ** no 85.2 79.3 0.731 c) yes 14.2 20.7 body mass ind ex (kg/m2)* normal ** overweight ** duration of men opause (years) * early (1-4 years) ** late (> 5 year s)** hormones/”hormone like substance” estradiol conc. (pg/ml) * isoflavone conc. (ng/l) * daily dietary intake pr oteins (g) * 27.4 (4.8) 40.7 59.3 4.3 (2.5) 59.2 40.8 7.7 (4.1) 4.8 (3.7) 41.9 (13.7) 26.5 (3.7) 31.0 69.0 5.0 (2.4) 44.8 55.2 7.0 (3.6) 4.1 (2.7) 49.5 (18.7) 0.438 0.143 0.238 0.280 0.519 0.363 0.093 table 1. distribution of demographic,habitual, and clinical characteristics of subjects at baseline, by intervention group * mean (sd) ; ** number of subjects (n) in %; a independent t-test; b chi-square test; c fisher’s exact test 168 isoflavone group with poor isoflavone intake increased by 4.6%, as compared with baseline concentrations, while in the control group the concentration decreased by 4.4%. in the subgroup of subjects with “overweight bmi” and “adequate isoflavone intake”, mean igg after supplementation of 100mg soy isoflavones for 12 weeks decreased by 10.7% and 8.3%, respectively, while the decrease in mean igg concentration in the isoflavone was larger than that in the control group. in general, these results of subgroup analyses indicate that isoflavone supplementation for 12 weeks had no positive effect, in other words, it was incapable of increasing the humoral response, with the exception of the subgroup with normal body mass index and poor isoflavone intake. from table 3 it may been seen that in g e n e r a l t h e s u b g r o u p a n a l y s e s a l s o demonstrated increased cd4+ concentrations table 3. relative difference percentage (ä) of mean igg concentration before and after supplementation, by intervention group, for all subjects and selected subgroups *results of independent t-test on mean igg and cd4+ concentration postsupplementation between isoflavone group and control group; **dietary isoflavone intake: poor<0.13 g/d; adequate: 0.13 0.44 g/d ä: relative difference percentage of mean igg concentration before and after supplementation, calculated according to the formula : igg concentration after supplementation – igg concentration before supplementation ——————————————————————————————————— x 100% igg concentration before supplementation ä: relative difference percentage of mean cd4+ concentration before and after supplementation, calculated according to the formula : cd4+ concentration after supplementation – cd4+ concentration before supplementation ———————————-————————————————————————— x 100% cd4+ concentration before supplementation subgroups mean igg mean cd4+ isoflavone (n=27) control (n=29) p* isoflavone (n=27) control (n=29) p* duration of menopause early menopasue late m enopause body mass index normal overweigh t dietary isofl avone intake** poor adequate 6.9 5.1 1.0 -10.7 4.6 8.3 2.8 2.9 3.6 4.4 4.4 4.0 0.319 0.381 0.073 0.925 0.951 0.158 2.8 6.0 14.2 1.4 -0.1 5.1 -1.5 1.3 -1.2 0.5 -5.7 1.6 0.886 0.626 0.559 0.945 0.938 0.955 intervention group specific immune response isoflavone (n = 27) control (n = 29) p b) igg (mg/dl) a) 1516.1 (228.0) 1441.5 (242.6) 0.242 cd4+ (cells/µl)a ) 773.5 (264.3) 759.3 (292.4) 0.850 table 2. mean igg and cd4+ concentration after supplementation of soy isoflavones for 12 weeks, by intervention group legend: a) mean (sd); b)p value from independent t-test wratsangka isoflavone and specific immune responses 169 univ med vol.30 no.3 postsupplementation in the isoflavone subgroups, both in those in early and late menopause. although the increase in cd4+ concentrations after 12 weeks of isoflavone supplementation was statistically not significant, in the late menopausal subgroup this increase was 4 times that in the control group, revealing a tendency for a raised cd4+ concentration in the late menopausal subgroup postsupplementation. among the subgroups with normal bmi, the mean cd4+ concentration increased by 14.2% in the isoflavone group, but decreased by 1.2% in the control group. in addition, there was also a reduction in mean cd4+of 1.4% in the isoflavone subgroup with above normal bmi. similarly, in the subgroups with adequate d i e t a r y i s o f l a v o n e i n t a k e , t h e c d 4 + concentrations in both the intervention and the control group showed an increase, with a higher increase in the former (5.1% vs. 1.6%). thus it may be concluded that supplementation of soy isoflavones 100mg/day for 12 weeks tends to raise cd4+ concentrations in postmenopausal women, particularly in the subgroups with normal bmi and those with adequate dietary isoflavone intakes. side effects and adverse events none of the subjects in the control group experienced adverse effects after consumption of their supplementation tablets for 12 weeks, whereas in the isoflavone group there were 3 subjects (11.1%) who reported having mild symptoms of nausea in the first week of supplementation, with the symptoms decreasing in the second week and disappearing thereafter. discussion in this study, the results indicate that there were no significant differences in igg and cd4+ concentrations after supplementation of soy isoflavones 100 mg per day for 12 weeks in postmenopausal women. supplementation of soy isoflavones 100 mg per day for 12 weeks tended to increase cd4+ concentrations, but had an immunosuppressant effect on igg. the i n c r e a s e i n c d 4 + c o n c e n t r a t i o n s a f t e r supplementation of soy isoflavones tended to be more pronounced in the subgroup of women in late menopause, with normal bmi and adequate dietary isoflavone intake. studies on the effects of soy isoflavone supplementation on specific immune responses in postmenopausal women are still very limited in number. the concentrations of estradiol (which is also an immunomodulator) in postmenopausal women are extremely low (<20 mg/dl). in this connection it should be noted t h a t t h e s o y i s o f l a v o n e s ( g e n i s t e i n , a n d daidzein), whose structures are similar to those of estrogens (17 â-estradiols), will bind to estrogen receptors (er-á and er-â), and therefore are administered as a supplement and expected to be capable of affecting immune functions in humans, as they exert agonist and weakly antagonist effects on estrogens.(13) estradiol has equal affinities to both type of receptors, whereas isoflavones bind more strongly to er-â. (14,15) in postmenopausal women both types of estrogen receptor also have reduced functionalities and affinities, e s p e c i a l l y f o r i s o f l a v o n e s w i t h t h e i r considerably lower potency in comparison to estradiol (e2).(16-18) to date it is still unclear what immune mechanisms are involved, whether through binding of estradiol to er-á or to er-â receptors. several investigators have concluded that lymphocyte potentiation probably does not involve estrogen receptor ( e r ) d e p e n d e n t m e c h a n i s m s , b e c a u s e daidzein, but not genistein, is capable of potentiating lymphocytes, while both types of isoflavone have equal affinities for estrogen receptors in er-sensitive tissues.(19,20) the subgroups of subjects with normal bmi in this study showed an increase in cd4+ c o n c e n t r a t i o n s o f 1 4 . 2 % , w h e r e a s t h e subgroup with above normal bmi tended to h a v e r e d u c e d c d 4 + c o n c e n t r a t i o n s a f t e r supplementation. these data indicate that c i r c u l a t i n g e s t r a d i o l c o n c e n t r a t i o n s i n 170 postmenopausal women did not influence the effects of isoflavone supplementation. data f r o m s e v e r a l i n v i t r o i n a n i m a l s t u d i e s demonstrate that isoflavones may either e n h a n c e o r i m p a i r i m m u n o c o m p e t e n c e , d e p e n d i n g o n d i e t a r y i s o f l a v o n e i n t a k e , isoflavone concentration, and most importantly, sensitivity of the target tissue and route of administration of the isoflavones.(21) the results of the present study indicate that the subjects i n t h e s u b g r o u p w i t h a d e q u a t e d i e t a r y isoflavone intakes showed a better response to supplementation of 100 mg soy isoflavones for 12 weeks, in the form of increased cd4+ concentrations after supplementation. several investigators have cited the estimated daily dietary isoflavone intakes in asians to be approximately 50–200 mg.(22) a study among postmenopausal women showed that the dietary isoflavone intake was 69.5 mg/d.(23) in-vitro studies using high doses of genistein (as a type of flavonoid) have shown that genistein is capable of inducing apoptosis and inhibiting angiogenesis, both of which are involved in cell proliferation and consequently also in immune responses. in the studies by wang and colleagues on cultures of mitogen-activated murine lymphocytes, addition of daidzein in physiological concentrations to the culture medium was capable of potentiating the lymphocytes and increasing the production of interleukins (il-2 and il-3).(24) the study conducted by paes on administration of soy isoflavones 100 mg/day for 4 weeks, found that the supplementation did not induce significant changes in lymphocyte proliferation and production of the cytokines il2 and interferon (ifn), and was incapable of increasing immune responses in postmenopausal women.(25) ryan-borchers et al. administered a daily supplementation of 70 mg soy isoflavones to healthy postmenopausal women for 16 weeks, and succeeded in increasing the population of b lymphocytes by 12.2% as compared to the control group, but there were no significant differences in the numbers of t lymphocytes and natural killer (nk) cells.(10) these conflicting study results may be due to several differences regarding the study subjects, types of isoflavone used, route of administration, and duration of supplementation. yellayi administered genistein injections directly to oophorectomized mice, resulting in a reduction in the proportions of cd4+ and cd8+ cells.(26) the results of this study indicate that supplementation of 100 mg soy isoflavones was c a p a b l e o f i n c r e a s i n g c e l l u l a r i m m u n e responses in postmenopausal women, but only in those with certain characteristics. this study also demonstrates that supplementation of 100 mg soy isoflavones was incapable of increasing humoral immune responses, and even tended t o h a v e i m m u n o s u p p r e s s a n t e f f e c t s o n p o s t m e n o p a u s a l w o m e n w i t h c e r t a i n characteristics. therefore it may be concluded t h a t t h e e f f e c t s o f s o y i s o f l a v o n e s u p p l e m e n t a t i o n a r e a s s o c i a t e d w i t h characteristics of postmenopausal women, thus preventing generalization of the results to all. i n o t h e r w o r d s , t h e m a n a g e m e n t o f postmenopausal women still requires an individual approach and cannot be generalized. there are many questions emerging around the optimal dose and duration of supplementation required for effecting the expected clinical response. some authors question the length of administration of supplements in several studies, considereing them to be too short for obtaining a clinical response. this study has several limitations, one of the limitations being that the outcome variables used only one immune response indicator, namely igg concentration for assessing humoral specific immune responses, and cd4+ concentration for assessing cellular specific immune responses. other immune response indicators were not assessed in this study, as a consequence of limited funding. another limitation was that the subjects remained in their homes, making it difficult to e n s u r e t h a t t h e y d i d n o t c o n s u m e o t h e r supplements in addition to the supplements administered in this study. wratsangka isoflavone and specific immune responses 171 univ med vol.30 no.3 conclusions s u p p l e m e n t a t i o n w i t h 1 0 0 m g s o y i s o f l a v o n e s f o r 1 2 w e e k s i n h e a l t h y postmenopausal women did not result in i n c r e a s e d h u m o r a l a n d c e l l u l a r i m m u n e r e s p o n s e s . p o s t m e n o p a u s a l w o m e n w i t h normal bmi tended to have increased igg concentrations, while those with adequate daily dietary intakes of isoflavones tended to have increased cd4+ concentrations. further studies are required to explore the effects of soy isoflavones in immunologically challenged subjects. acknowledgements thanks are due to all study subjects and the staffs at the mampang prapatan subdistrict health center (puskesmas kecamatan) and t h e vi l l a g e h e a l t h c e n t e r s ( p u s k e s m a s kelurahan), and all others who assisted in the successful completion of this study, particularly to the faculty of medicine, trisakti university, for the funding and research facilities. references 1. american association of clinical endocrinologist. aace medical guidelines for clinical practice for the prevention and treatment of postmenopausal osteoporosis. endocrinol pract 2003;9:544-64. 2. nelson hd. menopause. lancet 2008;371:760-70. 3. wu jm, zelinski mb, ingram dk, ottenger ma. ovarian aging and menopause: current theories, hypotheses, and research models. experiment biol 2005;230:818-28. 4. biben a. pemberdayaan perempuan menopause alami menuju menua sehat dengan memanfaatkan panca daya alam. pidato pengukuhan guru besar tetap dalam ilmu obstetri dan ginekologi pada fakutas kedokteran universitas padjadjaran, bandung 2003. 5. yang jh, chen cd, wu my, chao kh, yang ys, ho hn. hormone replacement therapy reverses the decrease in natural killer cytotoxicity but does not reverese the decrease in the t-cell subpopulation or interferon-gamma production in post-menopausal women. fertil steril 2000;74: 261-7. 6. allen j, teutsch s. hormone replacement therapy and risk of venous thromboembolism. systematic evidence review. rockville, md: agency for healthcare research and quality;2002. available at: http://www.ahrg.gov/clinic/serfiles.htm. accessed november 12, 2010. 7. anderson gl, judd hw, kaunitz am, barad dh, beresford saa, mary pettinger m, et al. effects of estrogen plus progestin on gynecologic cancers and associated diagnostic procedures, the women’s health initiative randomized trial, jama 2003;290:1739-48. 8. ryan-borchers ta, park js, chew bp, mcguire mk, fournier lr, beerman ka. soy-isoflavones modulate immune function in healthy postmenopausal women, am j clin nutr 2006;83:1118-25. 9. rosett, jw. menopause, micronutrients, and hormone therapy, am j clin nutr 2005;8:1223-31. 10. nahas eap, nahas-neto j. the effects of soy isoflavones in postmenopausal women: clinical review. current drug therapy 2006;1:31-6. 11. dawson b, trapp rg, editors. research questions about two seperate or independent groups. in : basic & clinical biostatistics. 4th ed. new york : mcgraw-hill companies;2004.p.134-61. 12. world health organization. bmi clasification; 2006. available at: http://apps.who. int/bmi/ index.jsp?intro page=intro_3.html. accessed november 14, 2010. 13. cooke ps, selvaraj v, yellayi s. genistein, estrogen receptors, and the acquired immune response. j nutr 2006;136:704–8. 14. beato m, klug j. steroid hormone receptors: an update. hum reprod update 2000;6:225-36. 15. bouman a, heineman mj, faas mm. sex hormones and the immune response in humans. hum reprod update 2005;11:411-23 16. faas m, bouman a, moesa h, heineman mj, de leij l, schuiling g. the immune response during the luteal phase of the ovarian cycle: th2-type response? fertil steril 2000;74:1008-13. 17. bouman a, moes h, heinemann mj, de leij lf, faas mm. the immune response during the luteal phase of the ovarian cycle: increasing sensitivity of human monocyes to endotoxin. fertil steril 2001;76:555-9. 18. giltay ej, fonk jc, von bloomberg bm, drexhage ha, schalkwijk c, gooren lj. in vivo effects on sex steroids on lymphocyte responsiveness and immunoglobulin levels in human. j clin endocrinol metabol 2000;85:1648-57. 172 19. girmes-grieco nk. soy isoflavone supplementation does not alter distribution of circulating lymphocytes or natural killer cell activity in postmenopausal women [thesis]. blacksburg va: faculty of the virginia polytechnic institute and state university;2001. 20. guo tl, mccay a, zhang lx, brown rd, you l, karrow na, et al. genistein modulates immune response and increases host resistance to b16f10 tumor in adult female b6c3f1 mice. j nutr 2001; 131:3251-58. 21. jenkins dja, kendal cwc, connelly pw. effects of highand lowisoflavone (phytoestrogen) soy foods on inflammatory biomarkers and proinflammatory cytokines in middle-aged men and women. metabolism 2002;51:919-24. 22. taku k, melby mk, takebayashi j, mizuno s, ishimi y, omori t, et al. effect of soy isoflavone extract supplements on bone mineral density in menopausal women: meta-analysis of randomized controlled trials. asia pac j clin nutr 2010;19:3342. 23. nurmillah, yolantha is, livinus v. soyfood consumption and risk of glycosuria in postmenopausal women. univ med 2010;29:162-8. 24. wang w, higuchi cm, zhang r. individual and combinatory effects of soy isoflavones on the in vitro potentiation of lymphocyte activation. nutr canc 1997;29:29-34. 25. paes cm. soy iisoflavone supplementation does not alter lymphocyte proliferation and cytokine production in postmenopausal women [thesis]. blacksburg va: faculty of virginia polytechnic institute and state university;2001. 26. yellayi s, naaz a, tomomi s, szecwzykowski m, cooke p. genistein causes thymic atrophy and changes in lymphocyte subpopulation: potential health implications? faseb j 2001;15:498-510. wratsangka isoflavone and specific immune responses alvina 63 abstract polymyalgia rheumatica (pmr) is commonly found in northern europe and in persons of scandinavian extraction in the us, with an annual incidence of around 50 per 100,000 population over 50 years of age, in whom it should be considered in the differential diagnosis of musculoskeletal disorders. the disorder is twice more common in women than in men. pmr is closely related to giant cell arteritis and both disorders are considered to have a common pathogenesis, associated with genetic and environmental factors (viral and bacterial infections). there is no gold diagnostic standard for pmr and the diagnosis rests on a high index of suspicion in persons older than 50 years with musculoskeletal symptoms. as an aid in diagnosis, several sets of diagnostic criteria have been used, usually related to age at onset, duration, symptoms, inflammatory markers, and response to corticosteroids. patients with pmr usually present with acute onset of stiffness and pain in the shoulder and pelvic musculature, which may be accompanied by fever, malaise, and weight loss. the symptoms of pmr seem to be related to synovitis of proximal joints and extra-articular synovial structures. pmr may occur as an isolated syndrome or accompany other diseases, mainly giant cell arteritis. it usually responds quickly to once-daily, low-dose prednisone, but some patients require treatment for several years. monitoring for corticosteroid-associated side effects such as osteoporosis and diabetes, as well as for relapses and flare-ups, is key to chronic management. keywords: pain, stiffness, polymyalgia rheumatica, corticosteroids *department of anatomy medical faculty, trisakti university correspondence dr. diana samara, mkk department of anatomy medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2101 email: dianasamara2@gmail.com univ med 2011;30:63-70 prognosis and management of polymyalgia rheumatica diana samara* january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction polymyalgia rheumatica (pmr) is a v a s c u l a r i n f l a m m a t o r y d i s o r d e r w i t h musculoskeletal manifestations and is closely related to giant cell arteritis (gca), as both disorders may coexist in the same individual. of the two conditions, pmr is more common and is characterized by bilateral pain and stiffness, typically affecting the shoulders, arms, neck, and hips. pmr affects caucasian women and men over age 50 in a 2:1 ratio.(1-3) pmr and gca are most frequent at northern latitudes, the incidence being highest in scandinavian countries and in parts of the us w i t h l a r g e p o p u l a t i o n o f s c a n d i n a v i a n extraction. in general, the prevalence of pmr in individuals older than 50 years is 0.5-0.7% 64 samara polymyalgia rheumatica and the estimated annual incidence in the us is 12-50 per 100,000. in northern europe the reported incidence is between 20.4 and 68.3 per 100,000 population older than 50 years, while in the united kingdom (uk) between 1990 and 2001 the incidence of pmr has increased by 35%.(4) a retrospective cohort study on pmr in primary health care patients in the uk found an overall annual incidence of 11.3 per 10,000 patients aged 50 or over.(5) gca is less common, with an average annual incidence in the us of 18 per 100,000 population among persons over 50 years of age.(2) the lowest prevalence of gca of 1.47 per 100,000 population older than 50 years was reported from japan.(6) gca is found in 10-15% of pmr patients and approximately 50% of persons with gca also have pmr. the incidence of pmr and gca increases after the age of 50 years and peaks between 70 and 80 years of age.(2) there is no specific test for pmr, thus the diagnosis should be considered in a p a t i e n t o l d e r t h a n 5 0 y e a r s w i t h t h e characteristic symptoms and history.(5) pathogenesis the exact cause of pmr is unknown, but the disorder is believed to be influenced by infectious and genetic factors. the condition is associated with hla-dr antigens, which seem to induce immunological responses to certain proteins.(3,9) some theories have postulated viral infection of the immune system in genetically susceptible persons, such as the respiratory syncytial virus (rsv), which may induce the production of anti-rsv antibodies. other viral etiologies may be associated with an increased i n c i d e n c e o f p m r a n d e p i d e m i c s o f mycoplasma pneumoniae, parvovirus b19, and chlamydophila pneumoniae.(10-12) patients with pmr frequently have increased levels of interleukin-2 (il-2) and interleukin-6 (il-6).(13) pain and stiffness is due to the activity of inflammatory cells and proteins that normally are part of the immune system for combating disease. in pmr, it appears that inflammatory activity is concentrated in the tissues with effects on joints. the associated muscle pain is due to referred pain.(14) signs and symptoms pmr is a disorder of rapid onset, initiated by severe, deep-seated rheumatic pains in the shoulders (most frequent), neck, lower back, buttock, hip, and thigh. the pain usually has a symmetrical pattern and may be aggravated by movement of the adjacent joints. the pain may also appear at rest, and frequently the patient wakes up at night on account of the pain. the patient has difficulty buttoning his/her clothes, putting on a jacket, or standing up from a sitting position. the joint pain is felt upon active as well as passive movement (shoulder pain in 7094% of cases, hip and neck pain in 50–70%). the presence of bilateral upper arm muscle weakness is not a feature of the disorder, but may be due to disuse atrophy. all above symptoms constitute the most evident features of pmr.(15) the patient also suffers from stiffness (of more than 1 hour duration) in the involved parts, particularly in the morning or after a long period of inactivity, such as after long-distance car driving. some patients have joint swelling, usually in the knee, wrist, and sternoclavicular joints. in addition, there may be systemic symptoms, such as lethargy, mild fever, malaise, fatigue, lack of appetite, loss of weight, and occasionally depression.(5) diagnostic criteria in the absence of a gold standard diagnostic test, the diagnosis of pmr is based on some recommended criteria, such advanced by bird, hunder, chuang, healy, hazelman and others. most criteria include age over 50 years, although bird differed in using age over 65 years. minimum duration of illness is usually taken as one month, but here bird again used a different period, namely two weeks. the various sets of diagnostic criteria usually have the following four in common: age over 50, bilateral shoulder or hip pain, morning stiffness, and erythrocyte sedimentation rate (esr) >40 mm/ 65 univ med vol.30 no.1 h. in addition, the response to a standard dose of corticosteroids may also used to aid in establishing the diagnosis.(2) e v a l u a t i o n o f t h e v a l i d i t y o f p m r specificity and sensitivity data is hampered by the fact that there is no gold standard diagnostic investigation for pmr.(5) physical examination on physical examination the patient is commonly found to be fatigued, subfebrile, with swelling of distal extremities and pitting edema. on musculoskeletal evaluation, the muscles are of normal strength, and there is shoulder pain and hip pain on movement, without clinically significant swelling. in addition, the patient has synovitis of the knee, wrist and sternoclavicular joints. the muscles are flaccid, with impaired movement of the hips and legs, and/or shoulders and arms. in advanced stages there is disuse atrophy of muscles and weakness of proximal muscles and even contracture of the shoulder c a p s u l e , l i m i t i n g a c t i v e a n d p a s s i v e movements.(17) pmr accompanies gca in approximately 50% of cases, therefore the diagnosis of gca may be made if the prominent musculoskeletal discomfort is presumably due to pmr.(18) musculoskeletal pain of abrupt onset occurring in the early morning hours, and responding dramatically to glucocorticoids, is suggestive of pmr.(19) laboratory evaluation the following investigations are required for establishing the diagnosis of pmr:(20) (i) tests for inflammatory markers (esr, plasma viscosity, and/or crp); (ii) routine blood tests; (iii) ultrasonography of hips and shoulders; and (iv) other diagnostic tests, such as magnetic resonance imaging (mri), bone scan, and radionuclide scan. esr values of >40 mm/hr are characteristic of pmr, even though in normal persons the esr may be raised by more than 20%. in this connection, crp level is more sensitive than esr. mri, bone and radionucleide scans are of lesser import in establishing the diagnosis of pmr. ultrasonography shows the characteristic pathological findings at the shoulder and hip joints that may differentiate pmr from other disorders. the pathological findings consist of subdeltoid bursitis, tenosynovitis of the long head of the biceps, and synovitis of the glenohumeral joint. at the hips synovitis and trochanteric bursitis are frequently found. although knee joint synovitis with effusions is common in patients with pmr, there is rarely a need for arthrocentesis.(21) the diagnosis of pmr is based on the typical history of persistent muscle and joint pain and stiffness, accompanied by increased levels of inflammatory markers, such as esr. slight increases in liver function tests are also not uncommon.(13) high esr values are a sensitive but nonspecific indicator of pmr. the esr is on average over 40 mm/hr, but may be up to 100 mm/hr. in 20% of cases, the esr is not unduly raised and may even be normal. in these cases the diagnosis is established by a rapid response on administration of oral prednisone 10-15 mg/ day.(22) the differential count shows mild normocytic normochromic anemia. the white blood cell (wbc) count may be normal or slightly raised, but platelet counts are usually high.(2,18) of the liver function tests, serum glutamic oxaloacetic transaminase (sgot) and serum glutamic pyruvic transaminase (sgpt) may be normal, while alkaline phosphatase may be slighly increased. serum albumin may be slightly reduced. creatinine kinase is usually normal, and this feature may be used for differentiating pmr from polymyositis and other myopathies. antinuclear antibody and rheumatoid factor are usually normal. serum il6 is raised and frequently parallels with the course of inflammation.(23) mri of the shoulders shows subacromial and subdeltoid bursitis and glenohumeral synovitis in the majority of patients (figure 1).(2) mri of the hands and feet reveals inflammation of the tendon sheaths in many patients.(24) 66 samara polymyalgia rheumatica on ultrasonography there is effusion in the bursa of the shoulder joint. (figure 2).(2) the f i n d i n g s o n m r i a r e c o r r e l a t e d w i t h ultrasonographic findings, thus ultrasonography is indicated when the diagnosis is uncertain.(25) differential diagnosis the differential diagnosis may be divided into inflammatory and non-inflammatory disorders.(28) inflammatory disorders to be differentiated from pmr comprise rheumatoid arthritis; advanced spondyloarthropathy, including ankylosing spondylitis and psoriatic arthritis; systemic lupus erythematosus; scleroderma, sjögren’s syndrome, and vasculitis; dermatomyositis and polymyositis. of the noninflammatory disorders may be mentioned osteoarthritis; spinal spondylosis; rotator cuff disease, adhesive capsulitis (frozen shoulder); figure 1. magnetic resonance imaging of the shoulder of a patient with untreated polymyalgia rheumatica. an axial t2-weighted section shows subacromial and subdeltoid bursitis (arrowheads), joint effusion (arrow), and tenosynovitis of the long head of the biceps (curved arrow). [reproduced with permission from dr. carlo salvarani].(2) drug-induced myalgia; infections, including viral infections, osteomyelitis, bacterial endocarditis, tuberculosis; malignancies (such as lymphoma, leukemia, myeloma, prostatic carcinoma); amyloidosis; parkinson syndrome; chronic pain syndrome, fibromyalgia, depression; and endocrinopathies and metabolic bone diseases, such as hyperthyroidism, hypothyroidism, hyperparathyroidism, hypoparathyroidism, osteomalacia, and pseudogout with calcium pyrophosphate deposits. associated disorders t h e d i a g n o s i s o f g c a s h o u l d b e c o n s i d e r e d i n a l l p m r p a t i e n t s . ( 2 9 ) t h e symptoms of gca are headache, claudication of the muscles of the jaw, and visual loss. the temporal artery is abnormal on palpation and vasculitis is detectable on biopsy. 67 univ med vol.30 no.1 figure 2. ultrasonography of the shoulder of a patient with untreated polymyalgia rheumatica ultrasonography reveals the presence of fluid within the subacromial bursa (arrows) and surrounding the long biceps tendon groove (arrowheads). [reproduced with permission from dr. carlo salvarani].(2) complications pmr usually persists from several months to 5 years. untreated patients frequently suffer from discomfort and decreased quality of life. the discomfort arises because the patients frequently experience difficulties in (i) getting up from their beds, standing up after sitting, getting out of a car; (ii) washing their hair (shampooing), or performing acts of personal hygiene; (iii) putting on clothes (shirts).(27) generally pmr is not accompanied by serious complications. patients receiving long-term corticosteroid therapy are at risk of osteoporosis, corticosteroid myopathy, bruising, emotional s y m p t o m s ( e . g . , i n s o m n i a , r e s t l e s s n e s s , hypomania, depression), hypertension, diabetes, elevated cholesterol, and fluid retention.(27) these complications have an impact on their health, social interaction, physical activity, and daily living. management management of pmr is oriented toward reduction of inflammation, mostly using lowdosage oral corticosteroids, such as prednisone or prednisolone, which characteristically produce a rapid and satisfactory response, not infrequently with a single one-day course. as a rule, however, the symptoms of pmr disappear within a few days of oral low-dosage prednisone (10 to 20 mg per day), although optimal responses may take more than two weeks to develop. tapering of corticosteroid dosage should be started after the patient has been stabilized for two to four weeks, but the should be individualized. typically, corticosteroids can be tapered to a low dosage (7.5 to 10 mg per day) after six months, with complete tapering to discontinuation within two to three years.(27,30) a l t h o u g h s o m e p a t i e n t s w i t h m i l d symptoms may recover on non-steroidal anti68 samara polymyalgia rheumatica i n f l a m m a t o r y d r u g s , s u c h a s a s p i r i n o r ibuprofen (motrin, advil), corticosteroids are currently considered the drug of choice for pmr, because they are capable of effecting a complete recovery and returning the esr to normal levels. the addition of nsaids does not confer any additional advantages in duration of therapy or daily or cumulative prednisone doses, while producing more adverse events. however, some patients with pmr may achieve sustained remission with nsaids.(20, 31) st u d i e s t e s t i n g c o m b i n a t i o n s o f methotrexate and prednisone for a supposed corticosteroid-sparing effect have produced inconclusive results, although in one study the a b o v e m e n t i o n e d c o m b i n a t i o n w a s m o r e effective than prednisone alone in preserving remission and reducing overall corticosteroid exposure.(32) for prevention of corticosteroidinduced osteoporosis vitamin d and calcium have been found to be effective. the american college of rheumatology recommends calcium supplementation (1,200 mg per day), vitamin d supplementation (800 u per day), lifestyle modification, regular weight-bearing exercise, and bisphosphonate therapy for preventing glucocorticoid-induced osteoporosis, and suggests bone-density assessment for patients receiving long-term glucocorticoid therapy.(33) no limitation on movements is necessary. in connection with the foregoing, the management principles of pmr may be taken to be as follows: (18) (i) glucocorticosteroids constitute the most effective treatment and non-steroidal anti-inflammatory drugs offer very few benefits; (ii) in case of lack of response to prednisone (younger persons, muscle weakness, peripheral joint disease, and pain with or without stiffness), alternative diagnoses should be considered; (iii) there is scant evidence for the efficacy of steroid substitutes such as methotrexate or anti tumor necrosis factor; (iv) retraining of the remaining physical and psychosocial capabilities in c o l l a b o r a t i o n w i t h p h y s i o t h e r a p i s t s a n d o c c u p a t i o n a l t h e r a p i s t s . m o n i t o r i n g o f responses to treatment should be done by managing morning stiffness, pain and disability of proximal hip muscles, and the possibility of osteoporosis that might result in fractures. occasionally, patients may suffer from relapses of pmr several years after the symptoms have d i s a p p e a r e d . s e v e r a l s t u d i e s h a v e demonstrated that relapses of pmr occur in 23% to 29% of patients during the entire follow-up period and, according to other studies, in 33% of patients during the first year. (13) a higher relapse rate (55%) was r e p o r t e d b y a r e t r o s p e c t i v e s t u d y u s i n g prednisone at wide dose ranges (1-100 mg/d; median dose, 15 mg/d).(22) relapses of pmr may be treated by returning to previous high doses of prednisolone. duration of therapy and occurrence of relapses have been related to high baseline inflammatory markers,(34,35) older age, female gender, low initial steroid dose, f a s t e r t a p e r i n g , l o n g e r d u r a t i o n o f symptoms,(36) and human leukocyte antigen (hla) alleles.(37) prognosis pmr is usually a self-limited disease, but the progress and prognosis of pmr is extremely variable, although with prompt diagnosis and adequate treatment the prognosis is frequently very good. for recalcitrant cases, response to systemic corticosteroids is rapid and dramatic, but the treatment should be constinued for 1-2 years and some patients need low doses for a relatively long period. although relapses frequently occur within 1-3 years, the patients still respond to higher doses of systemic corticosteroids. pmr is not associated with increased mortality, but morbidity and mortality may occur as a result of immunosuppression or side effects of steroids.(38) conclusions there is an urgent need for standardized diagnostic and classification criteria for pmr. 69 univ med vol.30 no.1 i n g e n e r a l , p m r r e m i s s i o n o c c u r s o n a p r e d n i s o n e d o s e o f 1 5 m g / d , a n d d o s e reductions to less than 10 mg/d should be done by tapering at a rate of less than 1 mg/mo. treatment produces relief of pain and other symptoms and a return to previous function. the prognosis depends on the underlying c o n d i t i o n a n d c o n t r o l o f c o r t i c o s t e r o i d associated complications. references 1. gonzalez-gay ma, vazquez-rodriguez tr, lopez-diaz mj, miranda-filloy ja, gonzalezjuanatey c, martin j. epidemiology of giant cell arteritis and polymyalgia rheumatica. arthritis rheum 2009;61:1454-61. 2. salvarani c, cantini f, boiardi l, hunder gg. polymyalgia rheumatica and giant-cell arteritis. n engl j med 2002;347:261-71. 3. mandell bf. polymyalgia rheumatica: clinical presentation is key to diagnosis and treatment. clev clin j med 2004;71:489-95. 4. smeeth l, cook c, hall aj. incidence of diagnosed polymyalgia rheumatica and temporal arteritis in the united kingdom,1990–2001. ann rheum dis 2006;65:1093–8. 5. barraclough k, liddell wg, du toit j, foy c, dasgupta b, thomas m, et al. polymyalgia rheumatica in primary care: a cohort study of the diagnostic criteria and outcome. family practice 2008;25:328–3. 6. kobayashi s, yano t, matsumoto y. clinical and epidemiologic analysis of giant cell (temporal) arteritis from a nationwide survey in 1998 in japan: the first government-supported nationwide survey. arthritis rheum 2003;48:1532-7. 7. catanoso mg, macchioni p, boiardi l, pipitone n, salvarani c. treatment of refractory polymyalgia rheumatica with etanercept: an open pilot study. arthritis rheum 2007;57:1514-9. 8. chatelain d, duhaut p, loire r, bosshard s, pellet h, piette jc, et al. small-vessel vasculitis surrounding an uninflamed temporal artery: a new diagnostic criterion for polymyalgia rheumatica? arthritis rheum 2008;58:2565-73. 9. gonzalez-gay ma, hajeer ah, dababneh a. seronegative rheumatoid arthritis in elderly and polymyalgia rheumatica have similar patterns of hla association. j rheumatol 2001;28:122–5. 10. elling p, olsson at, elling h. synchronous variations of the incidence of temporal arteritis and polymyalgia rheumatica in different regions of denmark: association with epidemics of mycoplasma pneumoniae infection. j rheumatol 1996;23:112-9. 11. elling h, olsson at, elling p. human parvovirus and giant cell arteritis: a selective arteritic impact? clin exp rheumatol 2000;18:suppl 20:s12-4. 12. mitchell bm, font rl. detection of varicella zoster virus dna in some patient s with giant cell arteritis. invest ophthalmol vis sci 2001;42:2572-7. 13. krupa wm, dewan m, jeon ms. trapping of misdirected dendritic cells in the granulomatous lesions of giant cell arteritis. am j pathol 2002; 161:1815-23. 14. hutchings a, hollywood j, lamping dl, pease ct, chakravarty k, silverman b, et al. clinical outcomes, quality of life, and diagnostic uncertainty in the first year of polymyalgia rheumatica. arthritis rheum 2007;57:803-9. 15. frearson r, cassidy t, newton j. polymyalgia rheumatica and temporal arteritis: evidence and guidelines for diagnosis and management in older people. age ageing 2003;32:370–4. 16. dasgupta b, borg fa, hassan n, barraclough k, bourke b, fulcher j, et al. on behalf of the bsr and bhpr standards, guidelines and audit working group. bsr and bhpr guidelines for the management of polymyalgia rheumatica. rheumatology 2010;49:186–90. 17. hunder gg. the early history of giant cell arteritis and polymyalgia rheumatica: first descriptions to 1970. mayo clin proc 2006;81:1071-83. 18. salvarani c, cantini f, hunder gg. polymyalgia rheumatica and giant-cell arteritis. lancet 2008; 372:234-45. 19. stone jh, papaliodis gn, dunbar mr, stone jr. a 53-year-old man with arthralgias, oral ulcers, vision loss, and vocal-cord paralysis. n engl j med 2010;362:537-46. 20. dasgupta b, salvaran ic, schirmer m, crowson cs, maradit-kremers h, matteson el and members of the american college of rheumatology work group for development of classification criteria for pmr. development of classification criteria for polymyalgia rheumatica (pmr): results from an expert work group meeting and a wider survey. j rheumatol 2008;35: 270–7. 21. ceccato f, roverano sg, papasidero s, barrionuevo a, rillo ol, paira so. peripheral musculoskeletal manifestations in polymyalgia rheumatica. j clin rheumatol 2006;12:167-71. 22. kremers hm, reinalda ms, crowson cs, zinsmeister ar, hunder gg, gabriel se. relapse in a population based cohort of patients with 70 samara polymyalgia rheumatica polymyalgia rheumatica. j rheumatol 2005;32: 65-73. 23. weyand cm, fulbright jw, hunder gg, evans jm, goronzy jj. treatment of giant cell arteritis: interleukin-6 as a biologic marker of disease activity. arthritis rheum 2000;43:1041-8. 24. mcgonagle d, pease c, marzo-ortega h, o’connor p, gibbon w, emery p. comparison of extracapsular changes by magnetic resonance imaging in patients with rheumatoid arthritis and polymyalgia rheumatica. j rheumatol 2001;28:1837–41. 25. macchioni p, catanoso mg, pipitone n, boiardi l, salvarani c. longitudinal examination with shoulder ultrasonography of patients with polymyalgia rheumatica. rheumatology 2009;48:1566–9. 26. weyand cm, goronzy jj. giant-cell arteritis and polymyalgia rheumatica. ann intern med 2003;139:505-15. 27. unwin b, williams cm, gilliland w. polymyalgia rheumatica and giant cell arteritis. am fam physician 2006;74:1547-54. 28. michet cj, matteson el. polymyalgia rheumatica. bmj 2008;336:765-9. 29. smetana gw, shmerling rh. does this patient have temporal arteritis? jama 2002;287:92-101. 30. knights g. polymyalgia rheumatica and giant cell arteritis. innovait 2009;2:580–4. 31. hernandez-rodriguez j, cid mc, md, lopezsoto a, espigol-frigole g, bosch x. treatment of polymyalgia rheumatica: a systematic review. arch intern med 2009;169:1839-50. 32. jover ja, hernandez-garcia c, morado ic, vargas e, banares a, fernandez-gutierrez b. combined treatment of giant-cell arteritis with methotrexate and prednisone. a randomized, double-blind, placebo-controlled trial. ann intern med 2001;134:106-14. 33. american college of rheumatology ad hoc committee on glucocorticoid-induced osteoporosis. recommendations for the prevention and treatment of glucocorticoidinduced osteoporosis: 2001 update. arthritis rheum 2001;44:1496-503. 34. myklebust g, gran jt. prednisolone maintenance dose in relation to starting dose in the treatment of polymyalgia rheumatica and temporal arteritis. a prospective two-year study in 273 patients. scand j rheumatol 2001;30:260–7. 35. pulsatelli l, boiardi l, pignotti. serum interleukin-6 receptor in polymyalgia rheumatica: a potential marker of relapse/recurrence risk. arthritis rheum 2008;59:1147–54. 36. salvarani c, cantini f, niccoli l. acute-phase reactants and the risk of relapse/recurrence in polymyalgia rheumatica: a prospective follow up study. arthritis rheum 2005;53:33–8. 37. martinez-taboda vm, bartolome mj, lopezhoyos m. hla-drb1 allele distribution in polymyalgia rheumatica and giant cell arteritis: influence on clinical subgroups and prognosis. semin arthritis rheum 2004;34:454–64. 38. salvarani c, macchioni p, manzini c, paolazzi g, trotta a, manganelli p, et al. infliximab plus prednisone or placebo plus prednisone for the initial treatment of polymyalgia rheumatica: a randomized trial. ann intern med 2007;146:6319. oktavianus 52 *department of biology, faculty of medicine, trisakti university, jakarta correspondence dr. ml. edy parwanto, m.biomed department of biology, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: +6221-5672731 ext.2302 email: edy.parwanto@gmail.com univ med 2012;31:52-62. universa medicina january-april, 2012january-april, 2012january-april, 2012january-april, 2012january-april, 2012 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 vol.31 no.1 introduction the bone loss that occurs with ageng in postmenopausal women is related to a decrease in serum levels of bioavailable estrogen and testosterone, which are mainly bound to sex hormone-binding globulin (shbg) and albumin. phytoestrogens are thought to exert hormonal effects in the body due to their structural resemblance to 17â-estradiol. the objective of this study was to evaluate the effect of isoflavone supplementation on levels of shbg in postmenopausal women aged 4760 years. methods a study of pre and post test design with controls was conducted in 70 women aged 4760 years. subjects were randomly divided into 2 groups, the isoflavone group received 100 mg isoflavones/day + calcium 500 mg/day and the control group calcium 500 mg/day for 6 months. measurement of bone mineral density was performed prior to supplementation, and serum shbg levels before and after supplementation. results supplementation of isoflavones for 6 months reduced the shbg levels by 31.1% in the isoflavone group (p=0.000), whereas supplementation of calcium for 6 months did not affect the levels of shbg in the control group (p=0.359). supplementation of isoflavones for 6 months reduced shbg levels of postmenopausal women in the isoflavone group with either osteopenia (p=0.028) or osteoporosis (p=0.008). conclusion supplementation of isoflavones for 6 months decreased the shbg levels of postmenopausal women in the isoflavone group with osteopenia and osteoporosis. our findings suggest that phytoestrogens may significantly decreased shbg levels in postmenopausal women. keywords: isoflavones, calcium, sex hormone-binding globulin, postmenopausal women isoflavone supplementation reduced serum sex hormonebinding globulin concentration in postmenopausal women ml. edy parwanto*, yanti indrawati* and herman setiawan* abstract 53 suplementasi isoflavon menurunkan kadar sex hormonebinding globulin pada perempuan pascamenopause latar belakang osteoporosis pada perempuan pasca menopause umumnya terjadi akibat kekurangan estrogen dan progesterone. sex hormone-binding globulin (shbg) berperan dalam aksi estrogen maupun progesteron terhadap sel sasaran. phytoestrogens – isoflavon berefek serupa dengan 17â – estradiol. tujuan penelitian ini adalah untuk menilai pengaruh suplementasi isoflavon terhadap kadar shbg pada perempuan pascamenopause yang berusia 47–60 tahun. metode sebuah rancangan eksperimental secara buta ganda menggunakan kontrol mengikutsertakan sebanyak 70 perempuan berusia 47–60 tahun. subjek penelitian secara acak menggunakan blok sebesar 4 dibagi dalam 2 kelompok perlakuan, yaitu diberikan isoflavon 100 mg/hari + kalsium 500 mg/hari (kelompok isoflavon) dan diberikan kalsium 500 mg/hari (kelompok kontrol). pemberian suplemen dilakukan selama 6 bulan. pengukuran densitas massa tulang (bmd) terhadap subjek dilakukan sebelum pemberian suplemen. pengukuran kadar shbg serum dilakukan sebelum dan sesudah pemberian suplemen. untuk menguji pengaruh pemberian suplemen terhadap kadar shbg digunakan uji t-independen. hasil suplementasi isoflavon 100 mg/hari + kalsium 500 mg/hari selama 6 bulan menurunkan kadar shbg pada perempuan pascamenopause (p=0,000), sedangkan suplementasi kalsium 500 mg/hari selama 6 bulan tidak mempengaruhi kadar shbg (p=0,359). suplementasi isoflavon 100 mg/hari + kalsium 500 mg/hari selama 6 bulan menurunkan kadar shbg baik pada perempuan pascamenopause penderita osteopenia (p=0,028) maupun osteoporosis (p=0,008). kesimpulan suplementasi isoflavon 100 mg/hari + kalsium 500 mg/hari selama 6 bulan mampu menurunkan kadar shbg pada perempuan pascamenopause yang mengalami osteopenia dan osteoporosis. implikasi klinik dari hasil studi ini memerlukan konfirmasi lebih lanjut. kata kunci: isoflavon, kalsium, sex hormone-binding globulin, perempuan pascamenopause introduction in post menopausal women, estrogen deficiency causes reduced bone mineral density (bmd), resulting in osteoporosis.(1) in addition to reduced bmd, patients with osteoporosis also have bone marrow degeneration.(2) according to t h e wo r l d h e a l t h o rg a n i z a t i o n ( w h o ) , individuals have osteoporosis if their bmd value expressed as a t score is < –2.5 and osteopenia if the t score is within the range of – 1 up to – 2.5, while those with a t score from t –1 to 0 or more have normal bone mineral density.(3) estrogens and progesterones are frequently associated with osteoporosis. estrogens are steroid hormones produced by women for their menstrual cycle. estrogens are also produced by men, but at a low level. menopausal women experience a reduction in estrogen levels that subsequently stabilize during postmenopause. high levels of estrogens indicate an increased risk of endometrial and mammary cancers, abstrak univ med vol. 31 no.1 54 whereas low estrogen levels increase the risk of bone fracture. women with low levels of estradiol have a twofold higher risk of fracture, which is independent of the bmd.(4) progesterone is a steroid hormone with androgenic properties and is produced by both men and women. progesterone is synthesized from cholesterol as pregnenolone, which is subsequently converted into progesterone. progesterone synthesis needs steroid hormone precursors, including cortisol, estrogen and testosterone. in women, progesterone is synthesized by the corpus luteum, adrenal glands, and placenta during pregnancy. in adults, progesterone levels decrease with age. in the menopausal phase, progesterone levels decrease drastically to nearly undetectable levels. as with estrogen, low progesterone levels reduce bone density and cause osteoporosis.(5) in women, estrogen and progesterone levels decrease at menopause, and subsequently both hormones decrease progressively in postmenopause. at menopause, estrogen concentrations decrease by approximately 60%, whereas estrone concentrations decrease by approximately 40%, leading to increased bone resorption. as is the case with estrogen, progesterone and progesterone receptors are decreased in menopausal women, thus reducing new bone formation. progesterone deficiency is the major early cause of osteoporosis, while a r e d u c t i o n i n e s t r o g e n i n c r e a s e s b o n e resorption by osteoclasts. in postmenopausal women serum estradiol concentrations are lower than before menopause and serum estradiol concentrations in postmenopausal women with osteoporosis are lower than in those with osteopenia.(6) sex hormone-binding globulin (shbg) is produced by hepatocytes and is then secreted into the circulatory system. besides by hepatocytes, shbg is also produced by target cells. within the circulatory system, shbg binds to estrogen or testosterone. free shbg functions as a regulator of steroid activity on target cells. in healthy adult women, both estrogen and progesterone are positively correlated with shbg.(7,8) isoflavones are phytoestrogens similar to 17â–estradiol and are also called estrogen-like molecules or non-steroidal estrogens. phytoestrogens are categorized as isoflavones (genistein, daidzein, biochanin a a n d f o r m o n e t i n ) , l i g n a n s ( m a t a i r e s i n o l , secoisolariciresinol diglucosides), coumestans (coumestrol, 4–metoxy coumestrol) and stilbens ( r e s v e r a t r o l ) . c u r r e n t l y m o r e t h a n 1 0 0 molecules have been found to belong to the phytoestrogen family.(9) in view of this, it is not surprising that isoflavone supplementation in postmenopausal women, for the purpose of increasing bmd, have shown variable results. in contrast, calcium and vitamin supplementation to postmenopausal women has been found to be beneficial for preventing loss of bone mass and decreased bone turnover, and for reducing nonvertebral fractures.(10) isoflavone supplementation have also been found to have variable effects on shbg concentrations. isoflavones do not affect shbg l e v e l s i n b o t h p r e m e n o p a u s a l ( 11 ) a n d p o s t m e n o p a u s a l w o m e n . ( 11 , 1 2 ) a l t h o u g h isoflavone supplementation for a period of 6 months does not affect shbg levels, isoflavone supplementation for 1 year increases shbg concentrations.(13) the increase in shbg concentrations by isoflavones has been shown to be inconsistent.(14) another study showed that isoflavone supplementation decreases shbg concentrations.(15) the purpose of the present study was to evaluate the effect of isoflavone supplementation on shbg concentrations in postmenopausal women in the age range of 47– 60 years. methods design of the study t h i s s t u d y w a s a p r e a n d p o s t t e s t e x p e r i m e n t a l d e s i g n w i t h c o n t r o l s , a s diagrammed in figure 1. parwanto, indrawati, setiawan isoflavone and human binding globulin 55 study subjects the subjects of the study were women with minimally one year of menopause but not exceeding 10 years, who resided at mampang subdistrict, south jakarta. recruitment of subjects was by cluster random sampling at the villages (kelurahan) of kuningan barat, mampang prapatan, tegal parang and pela m a m p a n g . i n c l u s i o n c r i t e r i a w e r e : postmenopausal women, aged 47–60 years, p r o n o u n c e d h e a l t h y b y a p h y s i c i a n , comunicative, not taking hormonal drugs in the last 3 months and agreeing not to consume vitamins and minerals during the study period, never having undergone hysterectomy and/or oophorectomy. exclusion criteria were: patients with diabetes, renal, hepatic and cardiovascular diseases, and hypertension. sample size the number of subjects in each of the two groups was calculated using the formula: figure 1. diagram of study design legend: r = randomization, x1 and x2 = presupplementation observations, x3 and x4 = postsupplementation observations, t1 = supplementation of isoflavones + calcium (isoflavone group), t2 = calcium supplementation (control group). subjects r x1 x2 t1 x3 x4 t2 intervention there were two treatments administered to the subjects. the first used a combination of isoflavones 100 mg + calcium (ca) 500 mg, g i v e n d a i l y i n o n e c a p l e t f o r 6 m o n t h s (isoflavone group). the second treatment used ca 500 mg 1 caplet daily for 6 months (control group). the supplements were given to the s u b j e c t s b y s p e c i a l l y t r a i n e d p e r s o n n e l . supplementation was administered in a double blind manner. to determine subject compliance in consuming the supplements, the remaining caplets were counted every other day by the personnel entrusted with this task. analysis of serum blood samples were drawn from the left cubital vein of each subject. serum was obtained from the blood by centrifugation at 2000 x g for 20 minutes, then stored at -20o c. measurement of serum shbg concentrations by immunoradiometric assay was performed in the laboratory for immunoendocrinology ( m a k m a l te r p a d u i m u n o e n d o k r i n o l o g i ) , faculty of medicine, university of indonesia, jakarta. the reagents for the assay irmanotes: n = sample size; ×1 = mean shbg concentration (72.2 nmol/l) from a preliminary study on 5 indonesian postmenopausal women given a supplement of isoflavone + calcium; ×2 = mean shbg concentration (58.6 nmol/l) from a preliminary study on 5 indonesian postmenopausal women given a supplement of calcium. sd = pooled standard deviation = 13.3 nmol/l. α = 5%; zα = 1.96 (two-sided test); β = 10%; power of the test 1 β = 0.90, zβ = 1.645. univ med vol. 31 no.1 56 c o u n t s h b g f r o m d i a g n o s t i c p r o d u c t s c o r p o r a t i o n ( d p c ) . s e r u m s h b g concentrations were expressed in nmol/l. the normal value of serum shbg is 55 nmol/l. measurement of bmd bmd was measured by dual-energy x-ray absorptiometry (dxa), using a lunar dpx bravo nomusa densitometer (ge medical s y s t e m s ) . m e a s u r e m e n t o f b m d w a s performed on the lumbar vertebrae, femoral neck and distal radius. the results of bmd measurement were expressed as the t score. t scores between -1 and 0 signify normal bone mineral density, t scores from -1 up to -2.5 signify osteopenia and t scores < 2.5 indicate osteoporosis.(3) measurement of bmd was performed at budi jaya hospital, south jakarta. data analysis comparison of demographic, physical and laboratory variables, including mean shbg concentratiions, between the isoflavone group a n d t h e c o n t r o l g r o u p w a s b y m e a n s o f independent sample t-tests. comparison of mean shbg concentrations before and after supplementation in the isoflavone group and the control group was done with the paired t-test. the level of significance used was 0.05. ethical clearance t h i s s t u d y w a s a p p r o v e d b y t h e commission on research ethics, faculty of m e d i c i n e , tr i s a k t i u n i v e r s i t y. b e f o r e participating in the study, all subjects gave written informed consent. results subject characteristics postmenopausal women meeting the inclusion criteria were 96 in number, among whom 20 subjects did not attend meetings and 6 subjects withdrew from the study. the remaining seventy subjects were divided into two groups of 35 subjects each. the first group was the isoflavone group, who were given isoflavone + ca supplementation, and the second group was the control group, who were given ca supplemention (figure 2). figure 2. diagram of screening, recruitment, follow-up and data analysis of study subjects parwanto, indrawati, setiawan isoflavone and human binding globulin 57 the youngest subjects participating in this study were 47 years old and the oldest subjects were 60 years old. in table 1 are shown the results of comparing subject characteristics between the isoflavone group and the control group, indicating no differences in age, bmi, bmd and laboratory test results. in the group with shbg concentrations of > 55 nmol/l, the results of data analysis showed that mean shbg concentrations between the isoflavone g r o u p a n d t h e c o n t r o l g r o u p w e r e n o t significantly different (p=0.481). these results indicated that randomization had successfully achieved a uniform distribution of the nontreatment variables between both groups. effect of isoflavone supplementation on shbg concentrations t h e r e s u l t s o f c o m p a r i n g s h b g concentrations between the isoflavone group and the control group after supplementation for 6 months are presented in table 2. there were no significant differences between shbg concentrations in the isoflavone a n d c o n t r o l g r o u p s a f t e r 6 m o n t h s o f supplementation (p=0.177). effect of isoflavone supplementation on shbg levels in each group t h e e ff e c t o f s u p p l e m e n t a t i o n o f i s o f l a v o n e s a n d c a l c i u m o n s h b g concentrations in each intervention group of postmenopausal women is presented in table 3. isoflavone supplementation for 6 months significantly reduced shbg concentrations by 31.1% in the isoflavone group (p=0.000), whereas ca supplementation for 6 months did not affect shbg concentrations in the ca group (p=0.059). table 1. demographic, physical, and laboratory characteristics of subjects at baseline *(mean ± sd); p = significance level of independent sample t-test; bmi = body mass index; bmd = bone mineral density; hdl = high density lipoprotein; ldl = low density lipoprotein; shbg = sex hormone-binding globulin table 2. comparison of shbg concentrations between isoflavone group (isoflavones 90 mg + ca 500 mg/day) and control group (ca 500 mg/day) after 6 months of supplementation shbg = sex hormone-binding globulin; nmol/l = nano mol per liter; p = level of significance univ med vol. 31 no.1 58 parwanto, indrawati, setiawan isoflavone and human binding globulin discussion . in the present study it was found that after 6 months isoflavone supplementation there was no significant difference in shbg concentrations between the isoflavone group and the control group. isoflavone supplementation for 6 months did not affect shbg concentrations. similar results were obtained in a meta-analytic study involving 35 studies of the effects of isoflavone supplementation for 4 weeks or longer on shbg concentrations, indicating that isoflavone supplementation had no significant influence on shbg concentrations. (11) the results of a randomized, placebo-controlled, double-blinded, crossover trial showed that administration of a supplementation of red clover-derived isolated isoflavones of 84 mg/day for 2 months did not affect serum shbg concentrations.(16) other studies also found that isoflavones did not result in differences in shbg and t e s t o s t e r o n e l e v e l s i n p o s t m e n o p a u s a l women.(12) in postmenopausal women, estrogens a n d a n d r o g e n s a r e b i o s y n t h e s i z e d f r o m dehydroepiandrosterone (dhea) and dhea sulfate (dhea-s). dhea and dhea-s are steroidal precursors of estrogens and androgens p r o d u c e d b y t h e a d r e n a l g l a n d s . ( 1 7 ) t h e abovementioned biosynthesis of estrogens and androgens takes place in peripheral intracrine tissues.(18) our study results showed that isoflavone supplementation for 6 months reduced shbg concentrations, both in patients with osteopenia and in those with osteoporosis, but that calcium supplementation over the same period had no effect on shbg concentrations. supplementation for 6 months in this study did not lead to differences in shbg concentrations between the isoflavone group and the control group. these results differed those of other studies, in whom it was shown that although supplemenation of isoflavones 70 mg/day for 6 months did not affect shbg concentrations, supplementation for 1 y e a r d i d e ff e c t a n i n c r e a s e i n s h b g concentrations.(13) the increase in shbg concentrations caused by isoflavones in postmenopausal women is inconsistent.(14) o u r s t u d y s h o w e d t h a t i s o f l a v o n e supplementation for 6 months was capable of significantly reducing shbg concentrations by 31.1% only in the isoflavone group. essentially similar results were shown by a study on postmenopausal women below 65 years of age, stating that daily consumption of isoflavones in the form of 25 g protein from soy products for one year decreased shbg concentrations by 14.5% in the isoflavone group, but did not affect the control group.(15) several factors, such as age, body weight, sex steroids, and insulin, regulate plasma shbg levels to a varying degree. shbg levels have been associated with a number of diseases, including osteoporosis. in several studies, an inverse correlation was found between serum shbg levels and bmd in both males and females. shbg levels may be predictive of various macro-architectural features of cortical bone.(19) high shbg levels predict the occurrence of osteoporotic fractures of the vertebrae and peripheral bones, particularly the proximal femur. estradiol and shbg play an important role in bone loss and osteoporotic fractures. as table 3. effect of supplementation of isoflavones and calcium on shbg concentrations in postmenopausal women shbg=sex hormone-binding globulin; ca=calcium; p=level of significance 59 increased serum shbg concentrations are associated with multiple fractures, measurement o f s e r u m s h b g l e v e l i n t h e g e n e r a l p r a c t i t i o n e r ’s o ff i c e m a y b e u s e f u l f o r predicting the severity of osteoporosis.(20) the variable effects of isoflavones on shbg levels may be due to genetic factors, dosage differences, duration of supplementation, and type of isoflavone. the influence of genetic factors on shbg concentrations is apparent from the positive correlation of isoflavone supplementation with shbg concentrations found in women with the genetic shbg n variant (p=0.006), but not in those with the d variant (p=0.999).(21) isoflavones are steroids, and they exert their actions on target cells in two ways, i.e. activation of both intracellular and membrane erâ, and interaction with steroidal hormone metabolism.(9) regarding the reduction in shbg concentrations by isoflavones, there are two possibilities. the first possibility is that isoflavones are bound by shbg to form isoflavone-shbg complexes, causing the isoflavones to become unavailable to the target cells. the second possibility is that isoflavones are bound by shbg receptor-shbg complexes (rshbg – shbg) on the surface of the cell membrane, subsequently activating their genes according to the signal tranduction theory.(22) these cell surface-bound isoflavonershbg shbg complexes are then used for activating bone formation by osteoblasts, causing them to undergo proliferation and differentiation, and form mineral deposits (mineralization).(23) studies on femoral-metaphyseal tissues of aged female rats demonstrated that genistein and genistin increased the osteoblastic calcium content, the activity of alkaline phosphatase as marker enzyme in osteoblasts, and their dna content as an index of bone cell numbers.(24) phosphogenistein and phosphodaidzein increased b o n e c o m p o n e n t s i n t i s s u e c u l t u r e . phosphoisoflavones containing low levels of genistein and daidzein had no effect on bone components, such that it was assumed that the oh group at position 7 in genistein and daidzein plays a role in the anabolic effect of isoflavones on bone components.(25) a study on postmenopausal women showed that the combination of genistein and daidzein increased lumbal bmd scores and decreased bone resorption.(26) other phytoestrogens, such as resveratrol, may also be beneficial in osteoporosis.(27) isoflavones also have direct effects on protein synthesis. genistein enhances the capacity of osteoblasts for producing collagen a n d a l k a l i n e p h o s p h a t a s e , a n d e v e n i n nonosteogenic media mineralized bone nodules are formed.(28) genistein, but not daidzein, is bound to estrogen receptors (á and â). both genistein and daidzein are bound to transcription proteins within osteoblastic cells. there have been no studies demonstrating any effects of genistein or daidzein on various protein kinases a n d p r o t e i n p h o s p h a t a s e s t h a t s h o w a relationship between osteoblastic proliferation and dna synthesis.(29) it has been demonstrated that genistein increases production of estrogens by increasing the activity of aromatases and mrna. in estrogen production, genistein also a c t i v a t e s s e v e r a l p r o t e i n k i n a s e s a n d transcription factors, such as cyclic amp response element (cre)-binding protein (creb) in gene regulation.(30) shbg concentrations can be affected by several factors, such as genetic factors, by pregnancy, exogenous steroidal sex hormones, and body weight. the genetic factor that has been most investigated is polymorphism of the shbg gene located on chromosme 17. recent studies have reported that several shbg genotypic variants are strongly associated with shbg concentrations in postmenopausal women, but have no effect on bmd.(31) however, several studies have shown that shbg levels are negatively correlated with bmd.(32,33) shbg genetic variants also influence estrogenic activity in normal as well as neoplastic tissues.(34,35) soy isoflavones are effective in raising lumbar bmd scores(26) and reducing bone univ med vol. 31 no.1 60 parwanto, indrawati, setiawan isoflavone and human binding globulin resorption in postmenopausal women.(36) bone resorption is performed by osteoclasts, while osteoblasts are responsive to bone synthesis and m i n e r a l i z a t i o n , e s p e c i a l l y d u r i n g b o n e formation and remodeling.(37) the isoflavones genistein and daidzein stimulate osteoblasts and i n h i b i t o s t e o c l a s t s . ( 3 8 ) t h e s e i s o f l a v o n e s decrease the number of osteoclasts, thus reducing bone resorption. in this connection, with a stable number of osteoblasts and an increase in bone components, bone bmd will of necessity also be increased. in addition, genistein also enhances mineralization of the extracellular matrix.(39) o t h e r s t u d i e s s h o w t h a t c a l c i u m supplementation effected some increase in b m d , b u t d i d n o t r e d u c e f r a c t u r e s a n d increased the risk of renal calculus.(40) low calcium intakes result in increased bone resorption, decreased bone mass, and increased osteoporosis. in japanese postmenopuasal women, low calcium intakes of less than 400 mg/day was associated with increased bone resorption but not with bone formation.(41) one limitation in this study is that no investigations were conducted on shbg polymorphism of the subjects, although shbg polymorphism has been found to affect shbg levels. investigations of shbg polymorphism should be done for accurate evaluation of the effect of isoflavone supplementation on shbg concentrations in postmenopausal women. another limitation is that the supplementation was conducted in the homes of the subjects and thus it was difficult to monitor whether or not the subjects took other supplements. conclusions isoflavone supplemenatation for 6 months decreased shbg concentrations in osteopenic and osteoporotic postmenopausal women. soy supplementation may have increased the availability of estrogens without affecting actual concentrations. acknowledgements thanks are due to the study subjects and t h e s t a ff o f p u s k e s m a s k e c a m a t a n m a m p a n g p r a p a t a n , s o u t h j a k a r t a , f o r participation in this study. we also thank the d e a n a n d vi c e d e a n s o f t h e f a c u l t y o f medicine, trisakti university for providing the funds for this study. references 1. venkat k, desai m, arora mm, singh p. age – related changes in 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lk. the soy isoflavone genistein induces estrogen synthesis in an extragonadal pathway. mol cell endocrinol 2009;302:73-80. univ med vol. 31 no.1 62 31. riancho ja, valero c, zarrabeitia mt, garcía – unzueta mt, amado ja, gonzalez – marcías j. genetic polymorphisms are associated with serum levels of sex hormone binding globulin in postmenopausal women. bmc med genet 2008;9:112–18. 32. goderie-plomp hw, van der km, de ronde w, hofman a, de jong fh, pols ha. endogenous sex hormones, sex hormone-binding globulin, and the risk of incident vertebral fractures in elderly men and women: the rotterdam study. j clin endocrinol metab 2004;89:3261–9. 33. rapuri pb, gallagher jc, haynatzki g. endogenous levels of serum estradiol and sex hormone binding globulin determine bone mineral density, bone remodeling, the rate of bone loss, and response to treatment with estrogen in elderly women. j clin endocrinol metab 2004;89:4954–62. 34. abrahamson pe, tworoger ss, aiello ej, bernstein l, ulrich cm, gilliland fd, et al. associations between the cyp17, cypib1, comt and shbg polymorphisms and serum sex hormones in post-menopausal breast cancer survivors. breast cancer res treat 2007;105: 45–54. 35. cui y, shu xo, cai q, jin f, cheng jr, cai h, et al. association of breast cancer risk with a common functional polymorphism (asp327asn) in the sex hormone-binding globulin gene. cancer epidemiol biomark prev 2005;14:1096– 101. 36. harkness ls, fiedler k, sehgal ar, oravec d, lerner e. decreased bone resorpstion with soy isoflavone supplementation in postmenopausal women. j womens health 2004;13:1000–7. 37. prapan s, udomrat s, puntheeranurak t. characterization of osteoblast and biomineral formation by atomic force microscopy study. j microsc soc thailand 2009;23:7–11. 38. de wilde a, lieberherr m, colin c, pointillart a. a low dose of daidzein acts as an er beta – selective agonist in trabecular osteoblasts of young female piglets. j cell physiol 2004;200: 253–62. 39. kukuljan s, nowson ca, sanders km, nicholson gc, seibel mj, salmon j, et al. independent and combined effects of calciumvitamin d3 and exercise on bone structure and strength in older men: an 18-month factorial design randomized controlled trial. j clin endocrinol metab 2011;96:955–63. 40. jackson rd, lacroix az, gass m, wallace rb, robbins j, lewis ce, et al. calcium plus vitamin d supplementation and the risk of fractures. n engl j med 2006;354:669–83. 41. nakamura k, saito t, yoshihara a, ishikawa m, tsuchiya y, oshiki r, et al. low calcium intake is associated with increased bone resorption in postmenopausal japanese women: yokogoshi study. public health nutr 2009;2: 2366–70. parwanto, indrawati, setiawan isoflavone and human binding globulin alvina 137 abstract exercise training can improve blood pressure in normotensive, prehypertensive, and hypertensive subjects. one of the mechanisms of blood pressure reduction in hypertensive patients with obesity is through weight loss. this study aimed to examine the effect of exercise training on bodyweight and the relationship between weight loss and reduction of blood pressure. an experimental pre-post test design without controls was used to evaluate the effect of exercise training on weight loss. the study involved 89 elderly aged 50 years or more, consisting of 40 men and 49 women, who were members of senayan sport fitness club and had been exercising for at least three months. exercise training was programmed and performed three times a week, consisting of aerobic (walking, jogging, static cycling), and resistance exercise. all exercise was performed for one to two hours with mild to moderate intensity. blood pressure and body weight were obtained from medical records. paired t-test showed that systolic blood pressure (sbp), diastolic blood pressure (dbp), mean arterial pressure (map), pulse pressure (pp), and body mass index (bmi) were significantly lower after training [(systolic, 126.3 ± 2.9 vs 122.3 ± 2.7, p=0.02), (diastolic, 80.2 ± 3.1 vs 77.2 ± 2.4, p=0.00), (map, 95.6 ± 4.6 vs 92.2 ± 3.4, p=0.00), (pp, 46.1 ± 4.2 vs 45.1 ± 3.6, p=0.04), (bmi, 24.5 ± 2.9 vs 23.6 ± 2.9, p=0.04)]. duration of training was the most influential factor affecting bmi, (beta = 0.38; p=0.00). exercise training could lower bmi and the reduction in diastolic blood pressure was higher for the subjects aged 70 years and over. keywords: exercise, body mass index, blood pressure, pulse pressure, elderly *department of physiology, school of medicine, atma jaya catholic university correspondence dr. ignatio rika haryono, spko department of physiology, school of medicine, atma jaya catholic university jl. pluit raya no.2 jakarta 14440 phone: 021-5469177 email: rika_haryono@yahoo.com univ med 2010;29:137-43. exercise training decreases body mass index in subjects aged 50 years and over ignatio rika haryono* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction the effect of exercise training on blood pressure is obvious. this is the reason why exercise has been be a part of protocol based therapy in people with prehypertension and hypertension. a study by pinto et al. showed that fast walking reduced ambulatory systolic blood pressure (sbp) and diastolic blood pressure (dbp) by 7.6 and 6.3 mmhg in hypertensive subjects.(1) hayashi et al. found that the decrease in sbp could even reach up to 10 mmhg.(2) the decrease in blood pressure is usually greater in hypertensive than in normotensive persons.(3) the decrease in blood pressure due to 138 haryono training decreases body mass index exercise training can occur in several ways. one of the mechanisms of blood pressure reduction in hypertensive patients with obesity is through weight loss. sbp and dbp will decrease about 3 mmhg for a 3-9% decrease in body weight.(4) a study by engeli et al. showed that in obese women 5% weight loss reduced levels of angiotensinogen by 27%, renin by 43%, aldosterone by 31%, and angiotensin-converting enzyme (ace) by 12%.(5) sbp and dbp, mean arterial pressure (map), and pulse pressure (pp) are risk factors of cardiovascular diseases. the study by sesso showed that increased sbp, dbp, map, and pp in men increases the risk of cardiovascular disease by 1.43 to 2.52 times.(6) systolic pressure tends to increase, while diastolic pressure rises until 60 years of age and then declines or stabilizes as increasing age.(7) the difference between the pattern of increase in systolic pressure and that in diastolic pressure with increasing age causes the map and pp to be important factors for assessment of the risk of cardiovascular disease.(8,9) a study in japan on 246 healthy middle-aged and older adults (4478 years) showed that after moderate and highintensity interval walking training body weight a n d b o d y m a s s i n d e x ( b m i ) d e c r e a s e d significantly in women.(10) the objective of the present study was to investigate the effect of exercise training on weight loss, sbp, dbp, map, and pp. methods research design an experimental pre-post test design without controls was used to evaluate the effect of exercise training on weight loss, sbp, dbp, map, and pp.this study was carried out in senayan sport fitness club (ssfc), from august to october 2010. subjects t h e s t u d y s u b j e c t s c o n s i s t e d o f 8 9 participants, namely 40 men and 49 women, who were members of the senayan sport fitness club (ssfc), jakarta. the sample size was calculated for numeric in paired sample (á=0.05%, β=0.20%, sd=10, significant mean difference=4). inclusion criteria were members aged 50 years or more, with normal or above normal blood pressure, who had practiced three times a week continuously in the last three months (checked from the daily presence list) a n d u n d e r w e n t r e g u l a r b l o o d p r e s s u r e examination at the ssfc. the sole exclusion criterion was members who did not practice for the last three months or more. exercise training the intensity of the training was in a c c o r d a n c e w i t h t h e i n d i v i d u a l t r a i n i n g programs that had been designed based on the member’s level of fitness and health (50-60% vo2max) for exercise for at least 30 minutes. aerobic training was done either outdoors (walking, jogging) or indoors (static cycling), whereas weight training was done indoors. exercise was performed three times a week. data collection data of the subjects included age, gender, and exercise test variables (sbp, dbp, bmi, and weight, map), which were obtained from patient fitness and medical records. pre-exercise values of blood pressure, height, and body weight were mean values taken before exercise bouts in the first 10 days, whereas post-exercise values were mean values taken before exercise bouts in the last 10 days of exercise training. blood pressure was measured by trained nurses using a sphygmomanometer (nova). body weight was measured using camry scales to the nearest 0.1 kg, and height was measured using a stature meter to the nearest 0.1 cm. bmi was calculated as the weight (kg) divided by the square of the height (m2). ethical clearance ethical clearance was approved by the ethical commission of atma jaya catholic 139 university. the participants were requested to give written consent, with attached signature. i d e n t i f y o f a l l p a r t i c i p a n t s w a s k e p t confidential and used for research purposes only. statistical analysis the data obtained were processed and a n a l y z e d u s i n g s p s s v e r s i o n 1 6 . 0 , a n d normality of distribution of the data was checked using kolmogorov-smirnov test. paired t-test was performed to confirm the difference of mean sbp, dbp, map, and pp before and after training, whereas anova was used to compare the same variables between a g e g r o u p s . t h e p e a r s o n o r s p e a r m a n correlation test was conducted to determine the relationship between the above variables. significance of the results was set at p <0.05. results of the participants enrolled in the study, 40 were male (44.9%) and 49 (55.1%) were female. mean age of the subjects was 61.3 ± 11.5 years and most of the subjects, both male and female, were aged 60 to 69 years (47.2%) (table 1). there were more female than male subjects in all age groups. exercise test variables consisting of duration of training, bmi, sbp, dbp, map, and pp, are shown by age group in table 2, for comparison of the between-group means. the mean duration of training was 6.3 years. the longest duration of training was 7.1 years, occurring the age group of 70-79 years, and differed significantly from the duration of training in the age group of >80 years. the mean body weight above normal was found in the age group of 70-79 years, but there was no difference in bmi between groups. the mean sbp and mean dbp of all groups were higher than normal. mean sbp of age groups 60-69, 70-79, and >80 years were significantly higher than that of the group of 50-59 years, whereas for mean dbp no significant between-group difference was found. the mean map of all groups were within the normal range (70-110 mm hg). mean map in the age group of 7079 years was higher than that in the age group of 50-59 years. similarly, the mean pp of all groups were above normal (30-40 mmhg), and mean pp of the age group of 60-69 years was also significantly higher than that of age group of 50-59 years.   age group g ender total (% ) male female 50 -59 y 6 8 14 (15.7) 60 -69 y 19 23 42 (47.2) 70 -79 y 8 10 18 (20.2) >8 0 y 7 8 15 (16.9) total 40 49 89 (100) table 1. subject distribution according to the gender and age group age group traini ng durati on (years) bmi (kg/m2) sbp (mmhg) dbp (mmhg) map (mmhg) pp (mmhg) 50-59 y 5.3 24.1 120.7 78.4 92.5 42.3 60-69 y 6.7 25.0 127.6* 79.9 95.8 47.7* 70-79 y 7.1* 25.9 128.7* 81.9 97.5* 46.8 >80 y 4.2 23.0 128.2* 80.6 96.5 47.6 overall 6.3 24.5 126.3 80.2 95.6 46.1   table 2. exercise test variables by age group at baseline one-way anova test *significant at p<0.05, compared to lowest mean value univ med vol.29 no.3 140 haryono training decreases body mass index ∆ bmi ∆ sbp ∆ dbp ∆ map ∆ pp p p p p p age -0.14 0.18 -0.01 0.94 -0.04 0.75 -0.03 0.82 0.02 0.90 gender -0.24 0.03 -0.06 0.60 -0.03 0.78 -0.05 0.66 -0.06 0.63 training durati on 0.38 0.00 0.07 0.58 0.12 0.32 0.10 0.39 0.01 0.97   table 4. multiple regression analysis of age, gender and training duration on exercise variables β= standardized coefficients * significance at p<0.05 a comparison of preand post-training values of the exercise test variables are shown in table 3, where it is apparent that the values for body mass index and all other parameters d e c r e a s e d s i g n i f i c a n t l y i n t o t h e n o r m a l category after exercise training. mean post exercise values of bmi in the age groups of 60-69 and 70-79 years, post exercise sbp the age group of 50-59 years, and post exercise dbp of age groups 70-79 and >80 years, fell into the normal category. however, post exercise pp of all groups were still above normal. table 4 shows the results of multiple regression analysis demonstrating that training duration was the most influential factor affecting bmi (beta = 0.38; p=0.00). however, both age, gender, and training duration did not affect sbp, dbp, map dan pp. discussion this study was conducted in the elderly to confirm decrease in body weight after exercise training and its relationship to changes in cardiovascular parameters. the results showed there was significant reduction in bmi and all exercise test paramenters. overall, bmi, sbp, dbp decreased about 3.7%, 4 mmhg, and 3 mmhg, respectively. this could be interpreted as meaning that a decrease in bmi by 1% will decrease sbp and dbp by about 1 mmhg. this result was quite similar to that of other studies. the study by steven et al. showed that the decrease in blood pressure after exercise training can occur even with a slight decrease in bodyweight. (12) mulrow et al. found a decrease in sbp and dbp of 3 mmhg for a 39% decrease in body weight.(4) age group bmi sbp dbp map pp pre pos p pre post p pre po p pre post p pre post p 50-59 y 24.1 22.7 0.01 120.7 118.1 0.04 78.4 75.9 0.01 92.5 89.9 0.05 42.3 42.2 0.68 60-69 y 25.0 24.0 0.04 127.6 120.8 0.00 79.9 76.0 0.00 95.8 90.9 0.29 47.7 44.8 0.00 70-79 y 25.9 24.8 0.04 128.7 122.5 0.01 81.9 78.1 0.03 97.5 92.9 0.02 46.8 44.4 0.02 >80 y 23.0 22.9 0.74 128.2 127.8 0.62 80.6 78.8 0.06 96.5 95.1 0.62 47.6 49.0 0.07 overall 24.5 23.6 0.04 126.3 122.3 0.02 80.2 77.2 0.00 95.6 92.2 0.00 46.1 45.1 0.04   table 3. exercise variables before and after training, by age group one-way anova test 141 a meta-analysis by cornelissen and fagard showed that exercise can lower sbp and dbp in the normotensive by 3.3 and 3.5 mmhg, respectively, whereas in patients with hypertension sbp and dbp decreased by 6.9 and 4.9 mmhg.(13) our subjects consisted of b o t h n o r m o t e n s i v e a n d h y p e r t e n s i v e individuals, who were however not divided into normotensive and hypertensive groups, because our study emphasized the association b e t w e e n w e i g h t l o s s a n d r e d u c e d b l o o d pressure. therefore, the overall mean decrease in blood pressure was the combined result of the decreases in both types of subjects, so that the mean reduction in sbp and dbp was less, being only 4 and 3 mmhg, respectively. in the present study the grouping of the subjects according to age was consistent with m a c a u l e y ’s s t u d y w h i c h f o u n d t h a t t h e relationship between blood pressure and physical activity was influenced more by age.(14) however, although in the present study post-exercise values of sbp, dbp, map, and pp of the elderly were higher, age had no influence on the increase in the values of these exercise variables. this difference may be caused by the wider age range of the subjects (16-74 years) in the macauley study which impacted significantly on blood pressure.(14) a t h o u g h t h e d e c r e a s e i n s b p w a s statistically significant, mean sbp of all age groups after training, except in the age group of 50-59 years, was clinically less significant, because it was still in the prehypertension category.(15) in contrast, the mean dbp of all a g e g r o u p s a f t e r t r a i n i n g w a s c l i n i c a l l y significant, as it reflected a beneficial change i n c a t e g o r y, f r o m p r e h y p e r t e n s i o n t o normotension. the clinically non-significant decrease in sbp may be the result of the considerably higher sbp values in a number o f s u b j e c t s , l e a d i n g t o n o c h a n g e i n hypertensive category. the decrease in blood pressure due to weight loss may occur by several mechanisms. o b e s i t y, e s p e c i a l l y c e n t r a l o b e s i t y, i s associated with an increase in renin angiotensin aldosteron (raa) system activity, sympathetic activity, endothelial dysfunction, and insulin resistance.(16,17) engeli et al. found that weight loss could reduce angiotensinogen, renin, a l d o s t e r o n e , a n d a n g i o t e n s i n c o n v e r t i n g enzyme.(5) the study by dengel et al. of obese people showed that weight loss increased i n s u l i n s e n s i t i v i t y a n d b r a c h i a l a r t e r y compliance. (18) furthermore, the study by straznicky et al. found that weight loss in obese p e o p l e l o w e r e d p l a s m a n o r e p i n e p h r i n e spillover rate, muscle sympathetic activity, and increased baroreflex cardiac sensitivity.(19) in our study, the decrease in blood pressure by exercise training-induced weight loss may have been caused by a similar effect on the raa s y s t e m , i n s u l i n s e n s i t i v i t y, s y m p a t h e t i c activity, and endothelial function. map and pp, in addition to sbp and dbp, are also risk factors for cardiovascular disease, especially in the elderly.(6) the map value is determined by the value of sbp and dbp. an increase in sbp and dbp would increase map, while a decrease in sbp and dbp would lower map. mean arterial pressure is proportional t o c a r d i a c o u t p u t ( c o ) a n d p e r i p h e r a l resistance. increased co and peripheral resistance will increase map and vice versa.(10) in this study, mean preand post exercise map values were within normal range and decreased significantly due to reduction in sbp and dbp. the study by seals et al. also found decreased sbp and dbp, with consequently decreased map, after three months of aerobic exercise.(20) the study by garcia-palmieria et al. e x a m i n e d t h e r e l a t i o n s h i p b e t w e e n p p, stratified by quartiles, and cardiovascular mortality in men. they found that the highest quartile of pp (>57 mmhg) had a 1.4 times higher cardiovascular mortality than the lowest quartile (<38 mmhg).(9) mean pre-and post exercise pp of all age groups and overall in the present study were substantially below 57 m m h g a n d t h e r e f o r e d i d n o t l e a d t o a n increased risk of cardiovascular mortality. univ med vol.29 no.3 142 haryono training decreases body mass index exercise has been proven to be beneficial in lowering the blood pressure, especially in patients with hypertension. regularity of exercise training also reduces the risk of d e v e l o p i n g c a r d i o v a s c u l a r d i s e a s e . ( 2 1 ) programmed and supervised physical activities are more efficient in sustaining blood pressure i n t h e e l d e r l y w i t h h y p e r t e n s i o n . ( 2 2 ) t h e american college of sports medicine (acsm) recommends an optimal exercise program to optimize blood pressure improvement in hypertension. exercise should be performed on most, preferably all days of the week, with moderate intensity (40-60% vo2max), for 30 minutes of continuous or accumulated activity. aerobic or endurance exercise should be the primary activity, supplemented by weight training or resistance exercise.(23) the exercise p r o g r a m s i n t h e p r e s e n t s t u d y m e t t h e recommended acsm criteria of exercise for hypertension. this study has several limitations that led to bias and influenced the results. in the first place, the study did not record the amount of food consumed by the subjects, which is important for determining the actual cause of the loss in bodyweight and the decrease in blood pressure. another limitation is that a n u m b e r o f s u b j e c t s a t t i m e s t o o k antihypertensive medication before blood pressure examination. therefore the mean blood pressure value did not purely reflect the effect of exercise training; however, this may be eliminated by taking the mean from 10 examinations. conclusions exercise training leads to a lower body mass index that is also influenced by gender. moreover, the reduction in diastolic blood pressure is higher for persons aged 70 years and over. it is recommended to examine the effect of exercise on blood pressure in subjects on a controlled diet and taking no antihypertensive medication. acknowledgments the author would like to thank sadoso, md and all staff of senayan sport fitness club for permission for this study and support in collecting the data. references 1. pinto a, di raimondo d, tuttolomondo a, frenandez p, arnao v, licata g. twenty-four hour ambulatory blood pressure monitoring to evaluate effects on blood pressure of physical activity in hypertensive patients. clin j sport med 2006;16:238-43. 2. hayashi t, ohshige k, tochikubo o. exclusion of influence of physical activity on ambulatory blood pressure. clin exp hypertens 2007;29:2330. 3. whelton sp, chin a, xin x, he j. effect of aerobic exercise on blood pressure. a metaanalysis of randomized controlled trials. ann intern med 2002;136:493-503. 4. mulrow cd, chiquette e, angel l, grimm r, cornell j, summerbell cd, et al. withdrawn: dieting to reduce body weight for controlling hypertension in adults. cochrane database syst rev 2008;8:cd000484. 5. engeli s, böhnke j, gorzelniak k, janke j, schling p, bader m, et al. weight loss and the renin angiotensin aldosterone system. hypertension 2005;45:356-61. 6. sesso hd, stampfer mj, rosner b, hennekens ch, gaziano jm; manson jae, et al. systolic and diastolic blood pressure, pulse pressure, and mean arterial pressure as predictors of cardiovascular disease risk in men. hypertension 2000;36:801-7. 7. franklin ss, gustin w, wong nd, larson mg, weber ma, kannel wb, et al. hemodynamic patterns of age-related changes in blood pressure. the framingham heart study. circulation 1997;96:308-15. 8. miura k, dyer ar, greenland p, daviglus ml, hill ma, liu k, et al. pulse pressure compared with other blood pressure indexes in the prediction of 25-year cardiovascular and all-cause mortality rates. the chicago heart association detection project in industry study. hypertension 2001;38:232-7. 9. garcia-palmieria mr, crespob cj, geec dm, semposb c, smithb e, sorlied pd. wide pulse pressure is an independent predictor of 143 cardiovascular mortality in puerto rican men. nutr metab cardio diseases 2005;15:71-8. 10. nemoto k, gen-no h, masuki s, okazaki k, nose h. effects of high-intensity interval walking training on physical fitness and blood pressure in middle-aged and older people. mayo clin proc 2007;82:803-11. 11. tortora gj, derrickson bh. principles of anatomy and physiology. 12th ed. danvers:wiley & sons;2009. 12. steven vj, obarzanek e, cook nr, lee im, appel lj, west ds, et al. long-term weight loss and changes in blood pressure: results of the trials of hypertension prevention, phase ii. ann intern med 2001;134:1-11. 13. cornelissen va, fagard rh. effect of endurance training on blood pressure, blood pressureregulating mechanisms, and cardiovascular risk factors. hypertension 2005;46:667-73. 14. macauley d, mccrum ee, stott g, evans ae, mcroberts b, boreham ca, et al. physical activity, physical fitness, blood pressure, and fibrinogen in the northern ireland health and activity survey. j epidemiol community health 1996;50:258-63. 15. chobanian av, bakris gl, black hr, cushmanwc, green la, izzo jl jr, et al. the national high blood pressure education program coordinating committee. jnc 7: complete report. seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. hypertension 2003;42:1206-52. 16. mathieu p, poirier p, pibarot p, lemieux i, després jp. visceral obesity: the link among inflammation, hypertension, and cardiovascular disease. hypertension 2009;53:577-84. 17. wolk r, shamsuzzaman asm, somers vk. obesity, sleep apnea, and hypertension. hypertension 2003;42:1067-72. 18. dengel dr, kelly as, olson tp, kaiser dr, dengel jl, bank aj. effects of weight loss on insulin sensitivity and arterial stiffness in overweight adults. metabol 2006;55:907-11. 19. straznicky ne, lambert ea, lambert gw, masuo k, esler md, nestel pj. effects of dietary weight loss on sympathetic activity and cardiac risk factors associated with the metabolic syndrome. j clin endocrinol metabol 2005;90:5998-6005. 20. seals dr, tanaka h, clevenger cm, monahan kd, reiling mj, hiatt wr, et al. blood pressure reductions with exercise and sodium restriction in postmenopausal women with elevated systolic pressure: role of arterial stiffness. j am coll cardiol 2001;38:506-13. 21. djousse l, driver ja, gaziano jm. relation between modifiable lifestyle factors and lifetime risk of heart failure. jama 2009;302:394-400. 22. barroso wk, jardim pc, vitorino pv, bittencourt a, miquetichuc f. the influence of programmed physical activity on blood pressure of hypertensive elderly patients on nonpharmacologic treatment. rev assoc med bras 2008;54:328-33. 23. pescatello ls, franklin ba, fagard r, farquhar wb, kelley ga, ray ca. american college of sports medicine position stand. exercise and hypertension. med sci sports exerc 2004;36:533-53. univ med vol.29 no.3 editorial i univ med novel swine origin influenza a (h1n1) virus in humans lie t merijanti s department of occupational medicine, medical faculty, trisakti university in march and early april 2009, while much of the world was focusing on the threat of avian influenza originating in asia, intelligence gathering systems were also extracting evidence of an epidemic of acute respiratory infections in mexico and southern california. although the exact sequence of events is uncertain, the centers for disease control and prevention (cdc) had confirmed the presence of a novel reassortment of influenza a strain h1n1 from avian, swine, and human strains.(1,2,3) on 25 april 2009, the world health organization (who) declared the swine flu outbreak in north america a public health emergency of international concern. on 29 april 2009, the international health regulations emergency committee recommended a change from who pandemic influenza phase 4 to phase 5. this means that who views a pandemic as imminent.(3) who and the cdc have confirmed that the new swine flu virus is transmitted between humans.(4) a novel swine origin influenza a virus (s-oiv) was identified as the cause of outbreaks of febrile respiratory infection ranging from self limited to severe illness. the symptoms including fever, cough, sore throat, rhinorrhea, myalgia, headache, chills, fatigue, vomiting or diarrhea until respiratory failure and death. it is likely that the number of confirmed cases underestimates the number of cases that have occurred. the cdc issued recommendations to clinicians, asking that they consider the diagnosis of s-oiv infection in patients with an acute febrile respiratory illness who met the following criteria: residence in an area where confirmed cases of human infection with s-oiv had been identified, a history of travel to such areas, or contact with ill persons from these areas in the seven days before the onset of illness. if s-oiv infection was suspected in a patient, clinicians were asked to obtain a nasopharyngeal swab from the patient and to contact their local health departments in order to facilitate initial testing of the specimen by reverse transcriptase polymerase chain reaction (rt-pcr) assay.(5) the modes of transmission of influenza viruses in humans, including s-oiv, are not known but are thought to occur mainly through the dissemination of large droplets and possibly small particle droplet nuclei expelled when an infected person coughs.(6) there is also potential for transmission through contact with vomites that are contaminated with respiratory or gastrointestinal material.(7) since many patients with s-oiv infection have had diarrhea, the potential for fecal viral shedding and subsequent fecal oral transmission should be considered and investigated. until further data are available, all potential routes of transmission and sources of viral shedding should be considered. january-april 2009, vol. 28 no.1 ii the incubation period for s-oiv infection appears to range from 2 to 7 days. most patient with s-oiv infection might shed virus from one day before the onset of symptoms through 5 to 7 days after the onset of symptoms or until symptoms resolve. in young children and in immunocompromised or severely ill patients, the infectious period might be longer.(8) the potential for persons with asymptomatic infection to be the sources of infection to others is unknown but should be investigated. patients who are at highest risk for severe complications of s-oiv infection are children under the age of 5 years, adults 65 years of age or older, children and adults of any age with underlying chronic medical conditions, and pregnant woman.(9) as of may 5, 2009, the cdc has recommended that given the severity of illness observed among some patients with s-oiv infection, therapy with neuraminidase inhibitors (oseltamivir and zanamivir) should be prioritized for hospitalized patients with suspected or confirmed s-oiv infection and for patients who are at high risk for complications from seasonal influenza. genetic and phenotypic analyses indicated that s-oiv is susceptible to oseltamivir and zanamivir but resistant to the adamantanes.(10) early 2008-2009 influenza season in the united states surveillance data suggest that human influenza a (h1n1) viruses were resistant to oseltamivir.(11,12) the food and drug administration (fda) has issued an emergency use authorization that approves the use of oseltamivir to treat influenza in infants under the age of 1 year (treatment that is normally approved for those 1 year of age or older) and for chemoprophylaxis in infants older than 3 months of age (chemoprophylaxis that is normally approved for children 1 year of age or older).(13) the cdc has recommended that health care workers who provide direct care for patients with known or suspected s-oiv infection should observe contact and droplet precautions, including the use of gowns, gloves, eye protection, face masks, fit tested, and disposable n95 respirators.(14) there is no vaccine available right now to protect against swine flu. you can help prevent the spread of germs that cause respiratory illnesses like influenza by covering your nose and mouth with a tissue when you cough or sneeze, washing your hands often with soap and water, especially after you cough or sneeze, trying to avoid close contact with sick people and staying home from work or school if you are sick. references 1. brownstein js, freifeld cc, madoff lc. influenza a (h1n1) virus, 2009 – online monitoring. n engl j med 2009; 360: 2155-6. 2. baden lr, drazen jm, kritek pa, curfman gd, morrisey s, campion ew. h1n1 influenza a disease information for health professionals (editorial). n engl j med 2009; 360: 2666-7. 3. coker r. swine flu. bmj 2009; 338: b1791. 4. komaroff al. the sudden birth of h1n1 “swine” flu: what does the future hold? j watch 2009. 5. novel swine origin influenza a (h1n1) virus investigation team. emergence of a novel swine origin influenza a (h1n1) virus in humans. n engl j med 2009; 361: 1-10. 6. blachere fm, lindsley wg, pearce ta, stacey e. anderson se, melanie fisher m, rashida khakoo r, et al. measurement of airborne influenza virus in a hospital emergency department. clin infect dis 2009; 48: 438– 40. 7. boone sa, gerba cp. the occurrence of influenza a virus on household and day care center fomites. j infect 2005; 51: 103-9. 8. carrat f, vergu e, ferguson nm, lemaitre m, cauchemez s, steve leach s, et al. time lines of infection and disease in human influenza: a review of volunteer challenge studies. am j epidemiol 2008; 167: 775-85. iii univ med 9. centers for disease control and prevention. 2008-09 influenza prevention & control recommendations: influenza vaccination coverage levels. available at: http://www.cdc.gov/flu/professionals/acip/ coveragelevels.htm. acessed may 7, 2009. 10. centers for disease control and prevention. update: drug, susceptibility of swine origin influenza a (h1n1) viruses, april 2009. morb mortal wkly rep 2009; 58: 433-5. 11. centers for disease control and prevention. update: influenza activity—united states, september 28, 2008 – april 4, 2009, and composition of the 2009-10 influenza vaccine. morb mortal wkly rep 2009; 58: 369-74. 12. centers for disease control and prevention. update: infections with a swine origin influenza a (h1n1) virus united states and other countries, april 28, 2009. morb mortal wkly rep 2009; 58: 431-3. 13. dharan nj, gubavera lv, meyer jj, okomo-adhiambo m, mcclinton rc, marshall sa, et al. infections with oseltamivir resistant influenza a (h1n1) virus in the united states. jama 2009; 301: 1034-41. 14. centers for disease control and prevention. interim recommendations for facemask and respirator use to reduce novel influenza a (h1n1) virus transmission. available at: http://www.cdc.gov/h1n1flu/masks.htm. accessed may 7, 2009. january-april 2009, vol. 28 no.1 iv not an hiv cure, but encouraging new directions the history of infectious diseases frequently i n c l u d e s p e o p l e w h o w e r e r e s i s t a n t t o a pathogen. such a phenomenon helped the spanish, who had resistance to smallpox, in their conquest of south america, but not the aztecs or the incas, who had no resistance to smallpox and were decimated by the virus.(1) microbial resistance involves adaptive (acquired) immunity (e.g., the hla subtype) or innate (natural) immunity resulting from the genetic makeup of the host.(2) with the human immunodeficiency virus (hiv) and its known destruction of the immune system, resistance to infection and disease was not initially expected. however, certain people — long-term survivors — have been infected with hiv for more than 10 years (and sometimes 30 years) and received no treatment yet remain without disease.(2) in addition, some people who have been exposed to hiv on many occasions do not become infected. (3) both long-term survivors and those who have been exposed to hiv but remain seronegative offer a great o p p o r t u n i t y t o s t u d y t h e m e c h a n i s m s o f resistance to hiv infection and disease. hiv enters cells primarily through attachment to the cd4 molecule and subsequent binding to coreceptors, of which two chemokine receptors, ccr5 and cxcr4, are the most common. r5 hiv types bind to ccr5; x4 hiv types use cxcr4.(4) people whose cells lack expression of the ccr5 gene are markedly resistant to hiv infection despite multiple exposures to r5 hiv, which is the most prominent virus detected after transmission.(2,4) this mutation is found in 1 to 3% of the western population. among people with hiv who have only one copy of the wildtype ccr5 gene, progression to disease appears to be slower than among those who have two.(4,5) obviously, such information is of value in efforts to develop new approaches for therapy. in 2007, an estimated 2 million people died from aids and 2.7 million contracted the virus. currently, infected patients can benefit from antiretroviral therapies that effectively delay or prevent progression to aids.(6) these people are in many cases healthy but continue to carry hiv. if the antiretroviral therapy is stopped, however, a rebound in virus production occurs that can lead to aids.7 moreover, the virus can develop resistance to antiretroviral therapy and reemerge in the host. long-term treatment with these drugs is also costly and can cause toxicities that are often lifethreatening, including disorders of the cardiovascular system, pancreas, kidney, and liver.(8) and only a fraction of the people who are infected and need antiretroviral therapy are receiving therapy, particularly in countries with limited resources. for these reasons, a search for better, longterm treatment for hiv infection continues. in this issue of the journal, hütter et al. highlight an innovative approach that could prove beneficial for the long-term control of hiv without v univ med antiretroviral therapy.(9) these investigators selected an hla-compatible person whose cells lacked expression of ccr5 as the donor for stem-cell transplantation from bone marrow to a patient with acute myelogenous leukemia who w a s i n f e c t e d w i t h h i v. a f t e r t w o transplantations, there has been no recurrence of leukemia or detectible hiv in the bloodstream, as determined by analyses of viral rna and cellular proviral dna. in addition, after nearly 2 years, the cd4+ t cells in this patient have returned to a normal range, all carrying the donor’s homozygote-deleted ccr5 gene. although some observers may consider the p a t i e n t c u r e d o f h i v, t h i s c o n c l u s i o n i s premature. much evidence has shown that hiv can be lurking in cells found in the lymph nodes and other parts of the body, including the brain, gut, liver, kidneys, and heart.(2) eventually, the virus could induce disease in these tissues. nevertheless, the results of this study and others(10) provide further encouragement for those examining approaches to treatment that reduce ccr5 expression in persons with hiv infection. bone marrow transplantations requiring ablation of host immune cells, as in this case, are risky; m a n y p a t i e n t s d i e f r o m t h e p r o c e d u r e . autologous stem-cell administration after manipulation to eliminate ccr5(10) carries a similar risk. consequently, an approach designed to modify hiv target cells without eliminating the host’s own bone marrow could be helpful. an example would be to inject into the patient with hiv a ccr5-inactivating biochemical compound or genetic vector that would enter white cells and eventually make them resistant to hiv. a compound that could enter the stem cells of the patient would be the most effective for long-lasting protection. development of such technologies could include injecting into the bloodstream vectors carrying small interfering rna (sirna), antisense rna, or ribozymes, a l l o f w h i c h r e d u c e c c r 5 c e l l u l a r expression.(10,11) another approach could involve s m a l l , i n j e c t a b l e , a r g i n i n e r i c h p a r t i c l e s containing rna that down-regulates ccr5 e x p r e s s i o n b y i n t e r r u p t i n g n o r m a l g e n e splicing.(12) although such techniques need to be perfected, they point in directions that may serve as stimuli for other innovative gene therapies to help those infected with hiv. certain issues, however, need to be appreciated. an x4 type of hiv that was detected at low levels in the blood of the patient studied by hütter et al. could eventually emerge. this virus grows in cells lacking ccr5 expression.(2-4) moreover, after transplantation, the patient’s remaining ccr5-expressing macrophages — major cells for r5 virus infection — had no evidence of hiv. what protected these cells? perhaps the ccr5 protein was present at low density on these cells, since the patient was heterozygous for the mutated allele. or, since hiv-specific t cells were not prominent, innate immune responses could be suppressing both the r5 and x4 viruses.(13) one caveat is that people lacking the ccr5 gene can be more susceptible to serious effects from certain infections, such as west nile virus.(14) therapeutic targeting of ccr5 could delay the onset of disease and reduce the cost and toxicity of antiretroviral therapy, as it has in this patient for nearly 2 years. this case places further emphasis on gene therapies and treatments directed at blocking the ccr5 receptor with decoy drugs. maraviroc, a recently approved ccr5 inhibitor, has had some success,(15) but it m u s t b e a d m i n i s t e r e d a l o n g w i t h o t h e r antiretroviral medications. it is probable that hiv resistance to maraviroc occurs because the ccr5 molecule remains expressed on cells. in january-april 2009, vol. 28 no.1 vi summary, the case reported by hütter et al.(9) could pave the way for innovative approaches that provide long-lasting viral control with limited toxicities for persons with hiv infection. references 1. prescott wh. history of the conquest of mexico and the history of the conquest of peru. new york: rowan & littlefield, 2000. 2. levy ja. hiv and the pathogenesis of aids. 3rd ed. washington, dc: asm press, 2007. 3. shearer gm, clerici m. protective immunity against hiv infection: has nature done the experiment for us? immunol today 1996; 17: 214. 4. berger ea, murphy pm, farber jm. chemokine receptors as hiv-1 coreceptors: roles in viral entry, tropism, and disease. annu rev immunol 1999; 17: 657-700. 5. de roda husman am, koot m, cornelissen m, keet ip, brouwer m, broersen sm, et al. association between ccr5 genotype and the clinical course of hiv infection. ann intern med 1997; 127: 882-90. 6. palella fj jr, delaney km, moorman ac, loveless mo, fuhrer j, satten ga, et al. declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. n engl j med 1998; 338: 853-60. 7. jubault v, burgard m, le corfec e, costagliola d, rouzioux c, viard jp. high rebound of plasma and cellular hiv load after discontinuation of triple combination therapy. aids 1998; 12: 2358-9. 8. nolan d, reiss p, mallal s. adverse effects of antiretroviral therapy for hiv infection: a review of selected topics. expert opin drug saf 2005; 4: 201-18. 9. hütter g, nowak d, mossner m, ganepola s, müssig a, allers k, et al. long-term control of hiv by ccr5 delta32/delta32 stem-cell transplantation. n engl j med 2009; 360: 692-8. 10. cohen j. building an hiv-proof immune system. science 2007; 317: 612-4. 11. an ds, donahue re, kamata m, poon b, metzger m, mao sh, et al. stable reduction of ccr5 by rnai through hematopoietic stem cell transplant in non-human primates. proc natl acad sci u s a 2007; 104: 13110-5. 12. moulton hm, nelson mh, hatlevig sa, reddy mt, iversen pl. cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. bioconjug chem 2004; 15: 290-9. 13. levy ja. the importance of the innate immune system in controlling hiv infection and disease. trends immunol 2001; 22: 312-6. 14. glass wg, mcdermott dh, lim jk, lekhong s, yu sf, frank wa, et al. ccr5 deficiency increases risk of symptomatic west nile virus infection. j exp med 2006; 203: 35-40. 15. deeks sg. challenges of developing r5 inhibitors in antiretroviral naive hiv-infected patients. lancet 2006; 367: 711-3. alvina 14 abstract aeromonas species are gram-negative, motile, facultative anaerobic, rod shaped, oxidase positive bacteria of the recently assigned family aeromonadaceae. the significance of aeromonas species as causative agent of human diarrhoea has recently been established. the aim of the present study was to investigate the distribution, and antibiotic sensitivity of aeromonas in nonhospitalized children with diarrhea.one hundred and seventeen rectal swabs from children with diarhhea were cultured for isolation of aeromonas organisms as the etiological agents. in addition to aeromonas, other enteric pathogens were also isolated. overall, the isolates of enteric pathogens amounted to 36.8%, consisting of salmonella, shigella, aeromonas, and vibrio. aeromonas was only found in 5.1% of cultures, with a ratio of a. caviae and a. hydrophila of 2:1, while salmonella made up the majority of causative organisms with an isolation frequency of 18.8%, followed by shigella with 11.1%. in this study no isolates of vibrio cholerae o1 were found as etiological agents of diarrhea; however, v. cholerae non-o1 and v. parahaemolyticus were found in small numbers (<1%). all isolates of aeromonas were resistant to ampicillin and tetracycline, but sensitive to ciprofloxacin and ceftriaxone, as were the other enteric pathogens. although the frequency of isolation of these enteric pathogens was higher than for vibrio spp., their role in infective diarrhea was less clearcut in comparison with salmonella and shigella. keywords: diarrhea, aeromonas, resistance, children *department of pharmacy, **department of public health, ***department of microbiology, medical faculty, trisakti university correspondence a dr. meiyanti, spfk department of pharmacy medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2805 univ med 2010;29:14-20 isolation and antibiotic sensitivity of aeromonas from children with diarrhea meiyanti*a, oktavianus ch. salim**, julius e. surjawidjaja***, and murad lesmana*** january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina introduction there are approximately 40 species of diarrhea-causing pathogens, and around half of them were only described within the last t h r e e d e c a d e s . a l l t h e s e p a t h o g e n s a r e relatively important in some age groups and/or geographical localities, both because of the chronicity of the involved pathogens as well as the duration of the disease, or because of progress of the disease from the acute to the 15 univ med vol.29 no.1 chronic form.(1) to date many cases of diarrhea a r e c o n s i d e r e d t o b e c a u s e d b y v i r u s e s , because of the difficulties and failures in isolation and identification of these pathogenic bacteria.(1) among these bacteria is the species aeromonas, which is one of the important groups of enteropathogenic microorganisms whose etiological role in diarrhea is subject to controversy, such that it has received scant attention and is frequently ignored.(2) the s p e c i e s a e ro m o n a s c o n s i s t s o f m o t i l e mesophilic gram-negative rods. as a result of various studies(3-6) it is recognized that the species aeromonas is universally distributed in freshwater and other environments and may be isolated from clinical material, potable w a t e r, f o o d s a n d b e v e r a g e s . i n t h e s e environments the bacteria are capable of reproduction even at low temperatures.(7) among the bacteria causing diarrhea the role of a e ro m o n a s a s a n e n t e r i c p a t h o g e n i s i n c r e a s i n g l y a c k n o w l e d g e d a n d t h e r e i s d e f i n i t e e v i d e n c e t h a t t h e o r g a n i s m i s associated with gastroenteritis in children, adults, and the elderly(6,7) and with cases of ’traveler ’s diarrhea’. (7) although healthy individuals may become carriers of this bacterium, it is reported that aeromonas is a significant cause of diarrhea.(8,9) the three phenotypes of aeromonas that are considered to be the main pathogens and to be associated with gastrointestinal infectious disease are a.hydrophila, a.caviae, and a. veronii biovar sobria, but the most frequently associated with diarrheal infection is a. hydrophila,(5) as reported by juan et al.(10) in addition these organisms produce virulence f a c t o r s t h a t a r e u s e f u l a s m a r k e r s f o r d i ff e r e n t i a t i n g b e t w e e n p a t h o g e n i c a n d nonpathogenic strains.(11) in indonesia, data on the presence of aeromona s in connection with diarrheal d i s e a s e a r e d i ff i c u l t t o o b t a i n , b e c a u s e aeromonas is not among the bacteria that are routinely reported as etiological agents of d i a r r h e a , a s i s t h e c a s e w i t h s h i g e l l a , salmonella or vibrio cholerae. actually this bacterium may cause various disorders, either intestinal (diarrhea) or extraintestinal, (11) making it necessary to pay attention to its presence. the aim of the present study was to investigate the distribution, and antibiotic sensitivity of aeromonas in nonhospitalized children with diarrhea. methods research subjects and location the study was performed in infants and children aged <14 years with diarrhea, who were visiting the tebet and mampang health centers in south jakarta. the study was c o n d u c t e d f r o m d e c e m b e r 2 0 0 8 u n t i l november 2009. media x y l o s e l y s i n e d e o x y c h o l a t e ( x l d ) , macconkey (mac), salmonella-shigella (ss), and thiosulfate citrate bile salts sucrose agar (tcbs) (difco, becton dickinson, s p a r k s , m d ) , w e r e u s e d a n d p r e p a r e d according to standard methods. sampling and culture rectal swabs were collected from the study participants after informed consent had been obtained from their parents or caregivers. personal data and data related to the diarrhea were obtained from the parents or caregivers and recorded on standard forms by special personnel. collection of rectal swabs was done irrespective of degree of diarrhea (mild, moderate or severe). rectal swabs were taken at the time of admission to the health center and before administration of antibiotics. the swabs were placed in cary blair transport medium and stored in a refrigerator, pending transportation to the laboratory. the samples were transported in thermos flasks to the microbiological laboratory, medical faculty, trisakti university. the rectal swabs were plated on mac, ss, xld and tcbs agar and 16 the agar plates were incubated under aerobic conditions at 370c for 18-20 hours. non-lactose fermenting colonies on mac, ss and xld agar, and suspected colonies of vibrio on tcbs agar, were selected and characterized in biochemical test media, namely kligler’s iron agar (kia), mannitol motility ornithine (mio), and sucrose semi-solid (sss).(12) preliminary screening for colonies of aeromonas was performed by means of the oxidase test. oxidase negative, ornithine positive (mio positive) colonies were considered to be aeromonas and were subjected to further testing for species determination. confirmatory serological testing was performed using specific antisera (difco laboratories, detroit, mi). the bacterial isolates were subsequently tested for susceptibility to a variety of antibiotics by the disk diffusion method as described in the manual of the national committee for clinical laboratory standards (nccls).(13,14) the antibiotics used in these sensitivity tests were ampicillin, chloramphenicol, tetracycline, cotrimoxazole, ciprofloxacin, and ceftriaxone (becton dickinson and company, cockeysville, ma, usa). escherichia coli atcc 25922 was included in the tests as control strain. data analysis percentage analysis was done to describe the distribution and antibiotic sensitivity of aeromonas. results bacterial culture of 117 rectal swab samples collected from infants and children aged <14 years with diarrhea resulted in the isolation from 43 (36.8%) subjects of the f o l l o w i n g e n t e r o p a t h o g e n i c b a c t e r i a i n descending order of frequency: salmonella 22 (18.8%), shigella 13 (11.1%), aeromonas 6 (5.1%) and vibrio 2 (1.7%). the number of aeromonas caviae isolates was higher than that of a.hydrophila (3.4% vs 1.7% or 2:1). isolates o f s a l m o n e l l a b e l o n g e d t o s e r o g r o u p b ( 3 6 . 5 % ) , c ( 2 2 . 7 % ) , d ( 1 8 . 1 % ) , a n d e (22.7%), while isolates of shigella comprised s.flexneri (53.8%) and s.sonnei (46.2%). isolates of aeromonas ranked third among the causative organisms in this study, consisting o f a . h y d ro p h i l a ( 3 3 . 3 % ) a n d a . c a v i a e (66.7%). vibrio cholerae was not found among the children with diarrhea in the locality of the present study, but v.cholerae non-o1 and v. age of patient/number of isolates (%) organism number of positives 0-1 yr >1-4 yr >4-14 yr salmonella spp. 22 salmonella b 8 3 (37.5) 4 (50) 1 (12.5) salmonella c 5 2 (40) 3 (60) 0 salmonella d 4 1 (25) 3 (75) 0 salmonella e 5 2 (40) 3 (75) 0 shigella spp. 13 s. flexneri 7 1 (14.3) 6 (85.7) 0 s. sonnei 6 4 (66.6) 1 (16.7) 1 (16.7) aeromonas spp. 6 a. hydrophila 2 0 2 (100) 0 a. caviae 4 1 (25) 3 (75) 0 vibrio spp 2 v. cholerae non-o1. 1 0 0 1 (100) v.parahaemolyticus 1 0 0 1 (100) table 1. distribution of enteric pathogens isolated from children with diarrhea meiyanti, salim, surjawidjaja, et al isolation of aeromonas 17 univ med vol.29 no.1 parahaemolyticus were obtained in small numbers (<1%) (table 1). the results of the antibiotic sensitivity testing indicated that both a.hydrophila and a. c a v i a e w e r e r e s i s t a n t t o a m p i c i l l i n a n d tetracycline, but that a.hydrophila was also resistant to co-trimoxazole (table 2). shigella flexneri was resistant to various antibiotics, such as ampicillin, chloramphenicol, tetracycline and c o t r i m o x a z o l e , f o l l o w e d b y s a l m o n e l l a serogroup b, which was resistant to ampicillin, c h l o r a m p h e n i c o l a n d t e t r a c y c l i n e . a l l enteropathogenic organisms were still sensitive to ceftriaxone and ciprofloxacin. discussion there are still doubts as to the role of a e ro m o n a s i n t h e e t i o l o g y o f d i a r r h e a l infections in humans, which is presumably because aeromonas is a heterogenous group of microorganisms, with only some subgroups being pathogenic, while there are still problems in subgrouping of these microorganisms.(11,15) however, within the last two or three decades the role of aeromonas in various human diseases has been proven, particularly as a cause of gastroenteritis.(8,10,11,15) although clinically it may be obvious that aeromonas causes diarrhea, this organism is not looked for routinely by means of fecal culture or rectal swab, such that very little is known about the clinical evaluation of this b a c t e r i u m . t h e i s o l a t i o n o f a e ro m o n a s hydrophila as a cause of diarrhea has been reported from taiwan,(10) india,(11)and other localities. (15-17) juan et al. (10) reported that aeromonas hydrophila is more prevalent in the summer, around august and september, with an isolation frequency of 24-30%. the number of affected males and females is almost equal, and is highest between the age of 1-3 years. from various localities it has been reported that the main causative organisms of diarrhea are salmonella and shigella,(18,19) followed by others such as campylobacter and aeromonas table 2. distribution of antibiotic sensitivity of pathogenic enteric bacteria isolated from children with diarrhea (n=43) antibiotic / proportion of resistant bacteria organism total number of isolates am c te sxt cro cip na salmonella spp. salmonella b 8 5 (62.5) 1 (12.5) 5 (62.5) 0 0 0 0 salmonella c 5 1 (20) 0 1 (20) 1 (20) 0 0 1 (20) salmonella d 4 1 (25) 0 1 (25) 0 0 0 0 salmonella e 5 1 (20) 0 1 (20) 0 0 0 0 shigella spp. s. flexneri 7 4 (57.1) 4 (57.1) 4 (57.1) 6 (85.7) 0 0 0 s. sonnei 6 0 0 6 (100) 6 (100) 0 0 0 aeromonas spp. a. hydrophila 2 2 (100) 0 2 (100) 2 (100) 0 0 0 a. caviae 4 4 (100) 0 4 (100) 0 0 0 0 vibrio spp v. cholerae non-o1. 1 1 (100) 0 0 0 0 0 0 v.parahaemolyticus 1 1 (100) 0 0 0 0 0 0 am = ampicillin; c = chloramphenicol; te = tetracycline; sxt = cotrimoxazole; cro = ceftriaxone; cip = ciprofloxacin; na = nalidixic acid 18 hydrophila. (10, 20) in the present study the isolation of a. hydrophila ranks after that of salmonella and shigella in order of frequency. their numbers are much smaller than those reported by juan et al.,(10) but their order of frequency is similar to that reported by yates(18) in his discussion on the etiologic agents of traveler’s diarrhea. the order of presentation of the most frequently isolated organisms may differ from one locality to another, due to s e v e r a l f a c t o r s , s u c h a s d i f f e r e n c e s i n a p p l i c a t i o n o f m i c r o b i a l i d e n t i f i c a t i o n techniques, seasonal variation and geographic location.(21) the frequency of isolation of aeromonas obtained in the present study is less than the 9.7% reported by subashkumar et al.(21) and the 17.7% reported by sinha et al.,(11) but almost identical to the frequency reported by other investigators.(7, 23,24) the isolation frequency of aeromonas might have been higher, had the study been conducted in a hospital, because patients with severe diarrhea are generally hospitalized. it has been reported that gastroenteritis due to aeromonas, particularly a.hydrophila, is more commonly found in children and the e l d e r l y ( 1 0 , 1 7 ) a n d i n t o u r i s t s ( t r a v e l e r ’s diarrhea).(6,7,18,25) according to vila(7) a.caviae is isolated with the highest frequency in traveler’s diarrhea, whereas a.hydrophila is predominant in the local population.(9-11,26) although diarrhea due to aeromonas is a self-limiting disease,(5) occasionally it may become severe and around 50% of patients with diarrhea due to this organism may develop persistent diarrhea(7) such that antibiotic therapy is indicated. the drugs of choice for diarrhea d u e t o a e ro m o n a s a r e t h e q u i n o l o n e s (ciprofloxacin). in our study, all aeromonas species, both a.hidrophila and a.caviae, were still sensitive to ciprofloxacin. however, resistance to quinolones should receive more attention, because within the last years the emergence of quinolone-resistant aeromonas strains have been reported in industrialized countries.(25) as to the â -lactam antibiotics, the majority of aeromonas strains have been reported to be resistant to ampicillin, but to be still sensitive to third-generation cephalosporins such as ceftriaxone,(7,10) as is evident from the results of the present study. resistance to chloramphenicol, tetracycline and cotrimoxazole is due to extensive usage of these antibiotics, particularly in developing countries.(7) the limitation of this study lies in the relatively small sample size that is inadequate for ascertaining that aeromonas spp. plays an important role as an etiological agent of diarhhea in children. however, the presence of aeromonas itself in feces is indeed less recognized and has received less attention, such that the organism is seldom looked for in patients with diarrhea.(10) although the isolation frequency of aeromonas is higher than that of vibrio spp., its role in diarrheal infection is less obvious in comparison to salmonella and shigella. from the aspect of antibiotic therapy, the emergence of multiresistant aeromonas strains should receive attention and antibiotic administration should be performed with care. conclusions the main etiological agents of diarrhea are salmonella, shigella, aeromonas and vibrio, although the role of aeromonas spp has to be put under scrutiny. all aeromonas isolates are resistant to ampicillin and tetracycline, but are still sensitive to ciprofloxacin and ceftriaxone. acknowledgements the investigators wish to express their gratitude to the dean and vice-deans of the medical faculty, trisakti university, for the funding of the present study. we also wish to extend our thanks to the staff of the mampang and tebet health centers in south jakarta, for their support and cooperation in this study. we also thank ms rosma oppusunggu for her tireless efforts in the laboratory. meiyanti, salim, 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infect immun 2002;70:192435. 17. ghenghesh ks, ahmed sf, el-khalek ra, algendy a, klena j. aeromonas-associated infections in developing countries. j infect developing countries 2008;2:81-98. 18. lesmana m, surjawidjaja je, herwana e, salim och, bukitwetan p. distribusi serotipe dan pola resistensi antibiotika dari isolat salmonella nontifoid di jakarta. univ med 2006;25:7-14. 19. boekitwetan p, surjawidjaja je, salim oc, aildilfit m, lesmana m. bacterial diarrhea: the aetiology and the antibiotika sensitivity in two community health centres in jakarta. j kedokter trisakti 2001;20:57-65. 20. yates j. traveler’s diarrhea. am fam physician 2005;71:2095-100. 21. gascon j. epidemiology, etiology and pathophysiology of traveler’s diarrhea. digestion 2006;73 suppl l:s102-8. 22. subashkumar r, thayumanavan t, vivekanandhan g, lakshmanaperumalsamy p. occurrence of aeromonas hydrophila in acute gastroenteritis among children. indian j med res 2004;123:61-6. 23. vasalkar s, saraswathy k, de a, varaiya a, gogate a. aeromonas species isolated from cases of acute gastroenteritis. indian j med microbiol 2002;20:107-9. 24. alavandi sv, ananthan s. biochemical characteristics, serogroups, and virulence factors of aeromonas species isolated from cases of diarrhea and domestic water samples in chennai. indian j med microbiol 2003;21:233-8. 25. vila j, marco f, soler l, chacon m, figueras mj. in vitro antimicrobial susceptibility of clinical 20 isolates of aeromonas caviae, aeromonas hydrophila and aeromonas veronii biotype sobria. j antimicrob chemother 2002;49:701-2. 26. kandakai-olukemi yt, mawak jd, olukemi ma, ojumah so. aeromonas-related diarrhea in nasarawa, nigeria. ann afr med 2009;6:76-9. meiyanti, salim, surjawidjaja, et al isolation of aeromonas oktavianus 100 *veterinary pharmacy subdivision, department of veterinary basic science, faculty of veterinary medicine, airlangga university. ** pharmacology department, medical faculty, airlangga university correspondence prof. dr. mochamad lazuardi, drh.msi veterinary pharmacy subdivision department of veterinary basic science faculty of veterinary medicine airlangga university “c” campus airlangga university, mulyorejo rd. faculty of veterinary medicine surabaya 60115 phone : +62 31 8705165 email: lazuardi@fkh.unair.ac.id univ med 2014;32:100-8 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background human infertiliy cases in indonesia have tended to increase at about 2-5% annually since 2000. many tropical plants in indonesia are potential sources of novel anti-infertility compounds, e.g. dendrophthoe pentandra l. miq. (benalu duku), a parasitic plant growing on lansium domesticum. the objective of this study was to identify the effect of crude methanolic dendrophthoe pentandra leaf extract on follicle stimulating hormone (fsh) and progesterone levels in female rats. methods fourteen rattus norvegicus wistar strain female rats were divided into an intervention group and a control group, and synchronized to estrus via the pheromone synchronizing method. the intervention group was given daily single intramuscular injections of crude methanolic dendrophthoe pentandra leaf extract at 100 mg/kgbw for 4 days, while the control group was given daily single intramuscular injections of 1 ml of distilled water. determination of fsh and progesterone levels in whole blood was done using the randox evidence investigator analyzer. the data were subjected to the t test for two independent samples. results mean fsh in the intervention group was 9.28 ± 6.72 miu/ml, which was lower than mean fsh of 24.80 ±16.35 miu/ml in controls (p<0.05). mean progesterone level in the intervention group was 33.55±13.96 nmol/l, twice as high as that in the control group, which was 18.47± 06.47 nmol/l (p<0.05). conclusions crude methanolic dendrophthoe pentandra leaf extract reduces fsh and increases progesterone levels in female rats, but cannot yet be recommended for use as fertility hormone inhibitor or stimulator in rats. key words: dendrophthoe pentandra, estrus synchronization, cuboidal cells, progesterone, female rats dendrophthoe pentandra methanolic leaf extract increases progesterone levels in female rats lazuardi mochamad* and bambang hermanto** 101 univ med vol. 33 no.2 ekstrak methanol daun dendrophthoe pentandra meningkatkan kadar progesteron pada tikus betina latar belakang kasus ketidaksuburan pada manusia di indonesia cenderung naik 2-5% setiap tahun sejak tahun 2000. disisi lain diketahui banyak tumbuhan tropis di indonesia yang berpotensi sebagai sumber komponen baru anti infertilitas (contoh benalu duku atau dendrophthoe pentandra l. miq., tumbuh di lancium demesticum). tujuan penelitian ini adalah mengidentifikasikan kinerja ekstrak kasar metanol daun benalu duku pada tikus betina terhadap induksi siklus oestrous dan pengaruhnya terhadap kinerja follicle stimulating hormone (fsh) dan progesteron. metode sebanyak empat belas tikus betina (rattus norvegicus wistar strain) dibagi dua kelompok (kelompok perlakuan dan kelompok kontrol) dan diatur menjadi oestrous melalui metode sinkronisasi feromon. kelompok perlakuan selanjutnya diberi ekstrak kasar daun benalu duku 100 mg/kg berat badan (satu kali sehari) selama 4 hari melalui intra muskular. kelompok kontrol diberi 1 ml aqua secara intra muskular (satu kali sehari) selama 4 hari. pengukuran kadar fsh dan progesteron, sampel darah diproses menggunakan metode analisis evidence investigator dari randox. data yang diperoleh dilakukan uji t dua sampel saling tak bergantung dengan tingkat siknifikansi sebesar 0,05. hasil rata-rata kadar fsh pada kelompok perlakuan besarnya 9,28 ± 06,72 miu/ml lebih rendah secara bermakna dari kelompok kontrol sebesar 24,80 ± 16,35 miu/ml, (p<0,05). pada kelompok perlakuan, rata-rata kadar progesteron besanya 33,55 ± 13,96 nmol/l dua kali lebih tinggi secara bermakna dari kelompok kontrol sebesar 18,47 ± 6,47 nmol/l (p<0,05). kesimpulan ekstrak kasar metanol daun benalu duku mampu menurunkan kadar fsh dan meningkatkan kadar progesteron pada tikus betina. pada penelitian ini masih belum dianjurkan untuk menggunakan hormone fertilitas lainnya sebagai penghambat atau peningkat hormon pada tikus betina. kata kunci: benalu duku, penyeragaman birahi, sel kubus, progesteron, tikus betina abstrak introduction the follicle stimulating hormone (fsh) and progesterone are fertility hormones in the human female and essential for control of the menstrual cycle. fertility hormones are not only used for pregnancy testing in women but are also important in the diagnosis of cases of early onset of menopause and gonadal abnormalities. in males, fertility hormones can also be used as accurate indicators of cases of liver cirrhosis and testicular cancer.(1) fertility hormones can be produced by medicinal plants such as j a t ro p h a c u rc a s , w h i c h p r o d u c e s testosterone,(2) and dioscorea macrostachya, which produces diosgenine (intermediate product of cortisone), and compounds with antiestrogenic activity.(3) dendrophthoe pentandra (l.) miq (local name: benalu duku), a parasitic plant of the loranthaceae family and growing on lansium domesticum, has been known to possess 102 medicinal properties since 1990 and is still being investigated for its content of local and systemic anticancer compounds.(4) the plant is also known to contain compounds with anti-myeloma cell and antibiotic activity.(5,6) several investigators have r e p o r t e d t h a t d e n d ro p h t h o e p e n t a n d r a contains components that are useful for preventing disease, such as compounds with antiproliferative activity. among its known organic compounds are essential amino acids, alkaloids, flavonoids, polyphenols, terpenoids, and free steroids.(4) a more recent report states that members of the genus dendrophthoe may contain immune hormones that prevent the formation of internal free radicals as a result of stimulation by external substances.(7) however, the use of dendrophthoe pentandra l. miq for treatment of menstrual cycle disorders has not yet been reported. on the basis of the above mentioned background, we attempted to determine the levels of the steroid hormones follicle stimulating hormone (fsh) and progesterone in adult female rats receiving a methanolic extract of d e n d ro p h t h o e p e n t a n d r a l e a v e s . t h e objective of this study was to evaluate the influence of a methanolic dendrophthoe p e n t a n d r a l e a f e x t r a c t o n f s h a n d progesterone levels in adult estrous female rats. methods research design this study was of experimental design with posttest only control group using experimental animals as research subjects. this study was conducted from july to october 2013 in the l a b o r a t o r y o f t h e ve t e r i n a r y p h a r m a c y subdivision, department of veterinary basic science, faculty of veterinary medicine, airlangga university. sample size the sample size was calculated according to rumke’s table with the assumption of a 100% s u c c e s s r a t e a f t e r a d m i n i s t r a t i o n o f dendrophthoe pentandra leaf extract and 40% failure rate without administration of dendrophthoe pentandra leaf extract (table 1).(4,8) therefore the sample size required was 7 female rats for each treatment group, giving a total of 14 female rats (7 in the intervention group and 7 in the control group). plant material the leaves of dendrophthoe pentandra l., miq growing on lansium domesticum were collected from the plant’s natural habitats in the palembang district, south sumatera, and table 1. determination of sample size analyte availability on administration of dendrophthoe pentandra leaf extract (% ) analyte availability without administration of dendrophthoe pentandra leaf extract (%) 10 20 30 40 50 60 70 80 90 10 0 53 28 17 12 9 7 0 270 83 42 26 18 13 9 7 10 402 111 53 31 20 14 9 20 294 128 58 32 20 13 30 539 134 58 31 18 7 40 539 128 53 26 9 50 494 111 42 12 60 402 83 17 70 270 26 80 53 90 lazuardi, hermanto dendrophthoe pentandra increases progesterone 103 univ med vol. 33 no.2 authenticated by mrs. yayah, biological research center – indonesian science institute (l e m b a g a i l m u p e n g e t a h u a n i n d o n e s i a , lipi), bogor. extraction t h e f r e s h d e n d ro p h t h o e p e n t a n d r a leaves were cleaned, air dried, and powdered using an electric homogenizer. then 450 g of the powdered sample was extracted with 2 l of analytical grade methanol for 72 h by using the r o t a t i n g p e r c o l a t i o n m e t h o d . t h e c r u d e methanolic extract was dried by flushing with nitrogen gas to reduce the methanol level. from the crude methanolic extract subsequently a injectable preparation was made that was pyrogen-free, sterile, stable, isotonic, iso-ionic, isohydric, with a ph of 7.2-7.5. the last step in the preparation of the extract was filtration at 0.20 µm and dispensing to disposable sterile vials at 40c. experimental animals fourteen healthy adult female rats 3-4 months of age (rattus norwegicus wistar strain) were obtained from rachmad priyadi dvm in trosobo, sidoarjo. the rats were assigned by simple randomization to one control group (n=7) and one intervention group (n=7). all animals were ear-marked according to the following code: o : no ear markings; ka : markings on right ear, ki : markings on left ear, kk : markings on both ears. ka3: code for cage no. 3; code for cage no. 4. ear markings were notches on the ear lobes. in this study, cages nos. 1 (ka1) and 2 (ka2) were used by male rats for estrus synchronization purposes only. synchronization of estrus all rats were synchronized for estrus by the whitten effect [pheromonally induced estrus synchronization in female rats by the presence of a male] or pheromone effect technique for two cycles at intervals of 10 days as follows: all fourteen female rats of the two groups were kept in cages no. 3 (8 females) and no. 4 (6 females). cages no. 1 and 2 contained the male rats.(9,10) giemsa stained vaginal smears of the f e m a l e r a t s w e r e e x a m i n e d a t 1 0 0 0 x magnification for cuboidal and cornified cells during the study period.(10) experimental procedures the treatments in this study were applied after all rats were in estrus. during the fourday treatment period, the rats in the intervention group were given once daily single intramuscular injections of dendrophthoe pentandra leaf extract diluted with aqua pro injection (w/v) at a dose of 100 mg/kg body weight. control rats were given a single daily intramuscular injection of 1 ml aqua pro injection during the four days. at the end of the treatment period, the rats were sacrificed and cardiac blood samples were collected, ranging from 1.5 to 2 ml. plasma was s e p a r a t e d f r o m t h e b l o o d s a m p l e s b y centrifugation at 8000 g for 15 minutes and stored at 2-80c until required for determination of fsh and progesterone concentrations. measurement of fsh and progesterone the levels of fsh and progesterone were measured using the evidence investigatortm semi-automated analyzer based on a proprietary b i o c h i p a r r a y t e c h n o l o g y. t h e c o m p l e t e evidence investigator package includes the biochip imaging module, nine-biochip carrier, biochip carrier handling tray, thermoshaker, as well as a personal computer, imaging software, and barcode scanner. the core technology is the randox biochip, which is a 9-mm2 solid substrate containing an array of discrete test regions for different markers. the biochip allows the simultaneous quantitative detection of multiple analytes from a single subject sample. for chemiluminescent immunoassay d e t e r m i n a t i o n o f f s h a n d p r o g e s t e r o n e concentrations, the present study used the randox fertility hormone array package (cat no. ev3610) consisting of assay diluent, conjugate, biochip, calibrator, luminance or px, and wash 104 buffer. the light signal generated from each of the test regions on the biochip was detected using a ccd camera and digital imaging technology, then compared to that from a stored calibration curve, from which the concentration was calculated.(11) procedural details after adding 150 µl of assay diluent into the appropriate biochip wells and a volume of 75 µl each of calibrators, samples and controls into the respective wells, the reagents were mixed by gently tapping all edges of the handling tray. then the handling tray containing the biochip carriers was fixed to the base plate of the thermoshaker, and incubated for 30 minutes at +37 0c and 350 rpm. after removal of the handling tray from the thermoshaker, volumes of 75 µl of conjugate were added to the biochip wells and handled using the same sequence of tapping/mixing, fixing to thermoshaker and incubating under the same conditions as described above. at the end of the procedure, the reagents were discarded using a sharp flicking action of the handling tray, and immediately subjected to 2 quick wash cycles by adding approximately 350 µl wash buffer to each well, using the wash bottle containing diluted wash buffer. the edges of the handling tray were then tapped to release any reagents trapped below the biochip, then the reagents were flicked to waste with a sharp action. to reduce potential well-to-well contamination, care was taken not to overfill the wells. after a further 4 wash cycles, in which the edges of the handling tray were tapped for approximately 10 to 15 seconds, the tray was then left to stand for 2 minutes to allow the biochips to soak in wash buffer. after the final wash, the wells were filled with wash buffer and left to soak until directly prior to imaging. for imaging, the carrier was removed from the handling tray and 250 µl of working signal reagent added to each of the well, which were then covered to protect from light. directly before addition of signal, the wash buffer was removed by flicking and tapping the carrier onto lint free tissue. after exactly 2 minutes (±10 seconds) the carrier was then placed in the evidence investigator machine. image capture was automatically initiated by the dedicated software. all blood samples of the intervention and control groups were assayed in triplicate, the results being coded n1, n2, and n3. statistical analysis data analysis was performed using the minitab statistical software version 17.0. i n d e p e n d e n t t t e s t w a s u s e d t o t e s t t h e differences between control and intervention groups at a significance level of 0.05. animal ethical clearance the rats were handled according to the principles for animal experiments under control of the commission on animal research ethics from the faculty of veterinary medicine, airlangga university, and the minimum standard r e q u i r e m e n t s f o r a n i m a l h a n d l i n g f o r experiments from the experimental animal ethics unit of the indonesian veterinary pharmacy and pharmacology association (www.affaveti.org). results from the study results it is apparent that estrus synchronization was achieved in all animals in around 10 days. for a view of cuboidal cells at 1000 x magnification in vaginal smears prepared by the giemsa staining method, see figures 1 and 2 (at 1000x magnification). the coefficient of variation interval for the fsh assays from 36 replicates was 10.01% to 16.68%, while for the progesterone assay it was 10.00% to 16.66%. the assay results for fsh and progesterone in both intervention and control groups performed in triplicate are presented in table 2. lazuardi, hermanto dendrophthoe pentandra increases progesterone 105 univ med vol. 33 no.2 treatment group p value dendrophthoe pentandra control fsh (miu/ml) ka3ka ka3ki ka3kk ka3o ka4o ka4ka ka4ki overall progesterone (nmol/l) ka3ka ka3ki ka3kk ka3o ka4o ka4ka ka4ki overall 16.98 ± 2 7.85 4.74 ± 6.26 14.54 ± 1 2.30 16.15 ± 1 3.80 2.04 ± 0.33 9.27 ±10.47 1.27 ± 1.05 9.28 ± 6.72 46.04 ± 3 0.85 38.56 ± 3 7.87 16.30 ± 1 8.86 12.0 3 ± 8.36 35.6 6 ± 3.97 38.7 7 ± 0.00 47.52 ± 2 5.33 33.55 ± 1 3.96 15.24 ± 9.16 17.55 ± 14.02 5.74 ± 4.72 10.80 ± 0.00 46.92 ± 37.54 35.14 ± 10.68 42.23 ± 4.98 24.80 ± 16.35 13.45 ± 5.76 15.40 ± 9.58 27.36 ± 23.61 28.67 ± 28.10 16.91 ± 13.34 12.02 ± 8.76 -- 18.47 ± 6.47 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 <0.05 -- <0.05 table 2. effect of dendrophthoe pentandra l. on fsh and progesterone levels in female rats each pair of fsh and progesterone values is from a single blood sample —lyzed blood sample; ka3: cage no. 3, ka4: cage no. 4; o: no ear markings; ka: markings on right ear, ki : markings on left ear, kk: markings on both ears, significance level at p <0.05 using independent t test figure 1. cuboidal cells in vaginal smear (giemsa stain, 1000x) figure 2. cornified cells in vaginal smear (giemsa stain, 1000x) discussion the present study achieved estrus in rats more rapidly than is possible by the bruce effect [pheromonally induced pregnancy block and return to estrus by exposing recently inseminated females with an unfamiliar male], as described by other investigators in mice.(12,13) thus our method is applicable for laboratory studies using rats for assays of short-acting hormones, such as luteinizing hormone. this study demonstrated that the fsh concentrations in the intervention group was threefold lower than those in the control group, whereas the progesterone concentrations in the intervention group were twice those in the 106 control group. these facts indicate that the analytes contain some unknown substance(s) with two pharmacodynamic actions, viz. as an fsh suppressor and progesterone stimulator. these unknown substances from the crude m e t h a n o l i c e x t r a c t o f d e n d ro p h t h o e pentandra leaves have been identified as having “suppressor fsh-like effects” and “stimulant progesterone-like effects”. several other substances with “suppressor fsh-like effects” are human seminal plasma components with 92amino acid polypeptides and alpha-inhibin-92 (alpha-ib-92).(14) synthetic compounds with “progesterone-like effects” that have been known as early as 2000 are dydrogesterone, 17α -hydroxyprogesterone caproate, medroxy progesterone acetate, and 17-megestrol acetate. the fsh suppression phenomenon is analogous to the concept of “receptor downregulation of gonadotrophin releasing hormone ( g n r h ) ” . a s d o w n r e g u l a t i o n o c c u r s , production of gonadotrophins by the pituitary i.e fsh ceases, effectively shutting down control hormones for cyclic ovarian functioning in the female. this effect was well known as early as 1989 and has been suggested as a potential estrus suppressor hormone.(15) the analytes from the crude methanolic extract of dendrophthoe pentandra leaves may act by occupying gnrh receptors in the pituitary and after a short period of stimulation cause the cells to reduce or stop the synthesis of receptor proteins, making the cells insensitive to gnrh.(15) the progesterone level increases after the cells receive a “calling signal” from the anterior pituitary, impacting on the corpus luteum in the form of inhibition of graafian follicle development in the ovary. the relationship b e t w e e n f s h i n h i b i t i o n a n d i n c r e a s e d progesterone is not a direct one, but has an influence on other fertility hormones, such as luteinizing hormone (lh), while estrogen may have an important role in strengthening the relationship between fsh inhibition and progesterone stimulation.(16) hormones are generally administered to patients for one of three purposes. first, when patients fail to produce sufficient quantities of hormones, therapy is directed at correcting the d e f i c i e n c y. s e c o n d , w h e n n o p e r m a n e n t hormonal deficiency exists, hormones are used to obtain more physiological effects. for example, synthetic progesterone, used as an estrus control agent, may be administered in cases of normal estrus. third, when hormone production is excessive, therapy is directed to the target by administration of antagonist hormones to inhibit excessive production. our study results probably indicate that the analytes act by stimulating the corpus luteum so as to produce a larger than two-fold increase in progesterone as compared with the normal production. this will activate a feedback mechanism for decreasing fsh production by gnrh signaling to inhibit fsh, as referred to by konishi et al.(17) our study cannot clarify the mechanism whereby the analytes induce low fsh levels and excessive production of progesterone after administration of the crude m e t h a n o l i c e x t r a c t o f d e n d ro p h t h o e pentandra leaves. there is a possibility that through further exploration of the “progesteronlike” effects of the extract, the mechanism of a b o v e m e n t i o n e d a n a l y t e a c t i o n m a y b e explained. a limitation of our study is that we did not study the expression of enzymes encoded by base-paired microsomal dna subunits acting as “control messenger” to inhibit fsh and stimulate progesterone, which may provide the answer to the analyte mechanism of action. in women with fertility disorders, the unique phenomena of fsh inhibition and progesterone stimulation by crude methanolic extract of dendrophthoe pentandra leaves may be used to treat pregnant woman with low progesterone levels, particularly in early pregnancy with a risk of miscarriage. in early pregnancy, abdominal pain and vaginal bleeding may be signs of a m i s c a r r i a g e , b u t c o n s u m p t i o n o f c r u d e lazuardi, hermanto dendrophthoe pentandra increases progesterone 107 univ med vol. 33 no.2 m e t h a n o l i c e x t r a c t o f d e n d ro p h t h o e pentandra leaves probably will be of benefit in increasing progesterone levels. several natural products from medicinal or other plants used to stimulate progesterone in early pregnancy are oils in yam and soy plants, compounds from curcuma comosa especially its diaryl-heptanoid compounds, plants frequently growing in waste water (wwtps) in beijing, china, and alkaloids from digitalis lanata leaves known as 5-â-cardenolides.(17-19) as explained earlier, some alkaloids of the dendrophthoe family are very beneficial for the treatment of steroid disorders in experimental animals.(20) other researchers even mention that dendrophthoe species are potential muscle relaxants. (21) however, through future studies to explore the leaf alkaloids of dendrophthoe pentandra as a member of the dendrophthoe family, we may presumably find new compounds for the treatment of cases of low progesterone levels in early pregnancy or for management of infertile uterine conditions. conclusions t h e c r u d e m e t h a n o l i c e x t r a c t o f dendrophthoe pentandra leaves is capable of increasing progesterone concentrations and of decreasing fsh production in healthy female rats. further studies are required on the components in crude methanolic extract of dendrophthoe pentandra leaves that have progesterone stimulating properties. other fertility hormones such as prolactin, testosterone, estrogen, and luteinizing hormone are not recommended yet to be used in the treatment of steroid disorders in healthy female rats. acknowledgements the investigations were financed by the directorate general of higher education, ministry of education and culture by contract no. 8714/un3/kr/2013. the authors wish to thank the director of the livestock products test station, directorate general of livestock services and animal health, bogor, indonesia, for providing research facilities for assays of fsh and progesterone by means of the evidence investigator analyzer. the research team is grateful to pt rafa topaz utama as distributors of randox products in indonesia, for their technical assistance in fsh and progesterone assays using the evidence investigator analyzer in bogor, indonesia. references 1. bashin s, cunningham gr, hayes fj, matsumoto am, snyder pj, swerdloff rs, et al. testosterone therapy in adult men with androgen deficiency syndromes: an endocrine society clinical practice guideline. j clin endocrinol metab 2010;95:253659. 2. onyeka ca, aligwekwe au, olawuyi ts, nwakanma aa, kalu ec, oyeyemi aw. antifertility effects of ethanolic root bark extract of chrysophyllum albidum in male albino rats. int j appl res nat prod 2012;5:12-7. 3. pathak ak, 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salivary concentration of progesterone and cortisol significantly differs across individuals after correcting for blood hormone values. am j phys anthropol 2012;149:231-41. 18. chang h1, wan y, wu s, fan z, hu j. occurrence of androgens and progestogens in wastewater treatment plants and receiving river waters: comparison to estrogens. water res 2011;45:73240. 19. ernst m1, de padua rm, herl v, muller-uri f, kreis w. expression of 3beta-hsd and p5betar, genes respectively coding for delta5-3betahydroxysteroid dehydrogenase and progesterone 5beta-reductase, in leaves and cell cultures of digitalis lanata ehrh. planta med 2010;76:9237. 20. gupta rs1, kachhawa jb. evaluation of contraceptive activity of methanol extract of dendrophthoe falcata stem in male albino rats. j ethnopharmacol 2007;30:215-8. 21. pooja s, irchhaiya r, bhawna s, gayatri s, santosh k. anticonvulsant and muscle relaxant activity of the ethanolic extract of stems of dendrophthoe falcata (linn. f.) in mice. indian j pharmacol 2011;43:710-3. lazuardi, hermanto dendrophthoe pentandra increases progesterone alvina 90 abstract postmenopausal women are at high risk of disease, such as coronary heart disease, stroke, malignancies, dementia and osteoporosis. this is due to decreased levels of estrogen/estradiol, produced mainly in the ovaries, leading to reduced bone mineral density (bmd), which is the gold standard for diagnosis of osteoporosis. the purpose of the present study was to determine the relationship between serum estradiol levels and bmd in postmenopausal women. the study, which was of cross-sectional design, involved 184 postmenopausal women meeting the inclusion criteria, viz. healthy postmenopausal women aged between 47 and 60 years having taken no hormonal medications in the previous 3 years. the subjects were assessed for anthropometric and biochemical characteristics, including bmd and serum estradiol levels. bmd was measured at the lumbar spine, right femoral neck and at the distal radius by the dual-energy x-ray absorptiometry (dxa) instrument. the mean serum estradiol concentration was 7.54 ± 4.65 pg/ml, while in 49.5% of the subjects the estradiol concentration was ≤ 5 pg/ml. in postmenopausal women with estradiol concentrations of > 5 pg/ml, a significant positive relationship was found between bmd and the tscores for the femoral neck. thus the higher the serum estradiol levels, the higher the bmd values for femoral neck region. in conclusion, the results of this study point to estradiol levels as a major factor in determining the bmd values in postmenopausal women. keywords: postmenopausal, estradiol, bone mineral density, femoral neck *department of physiology medical faculty, trisakti university correspondence dr. martiem mawi, ms department of physiology medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2804 univ med 2010;29:905. serum estradiol levels and bone mineral density in postmenopausal women martiem mawi* may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction with the increased longevity there is a concomitant increase in the numbers of the elderly, particularly postmenopausal women, in developed as well as developing countries. according to world health organization ( w h o ) e s t i m a t e s t h e n u m b e r o f postmenopausal women in the year 2025 will be around 60–70 million.(1) postmenopausal women are at high risk of disease, such as osteoporosis, hypertension, coronary heart disease, and stroke.(2) the menopause is the 12month period after cessation of menstruation, while the postmenopausal is the period after menopause onwards.(1) in the postmenopausal period the ovaries as main producers of estradiol are no longer functional, leading to 91 univ med vol.29 no.2 d e c r e a s e d l e v e l s o f e s t r a d i o l . ( 3 , 4 ) i n t h e postmenopausal period there is degeneration of the ovaries, resulting in cessation of estradiol production by these organs. the remaining sources of estradiol are the adrenals, a d i p o s e t i s s u e , a n d m u s c l e s t h r o u g h aromatization of androstenedione.(5-7) estradiol is synthesized in osteoblasts and chondrocytes in osseous tissue from circulatory androgens, p a r t i c u l a r l y t e s t o s t e r o n e f o l l o w e d b y dehydroepiandrosterone and androtenedione.(5) the reduction in bone mass is significantly associated with decreased androgen levels in perimenopausal and postmenopausal women.(8) testosterone level is positively associated with bmd, and circulatory androgens play an important role in maintaining estrogen levels in osseous tissue.(9) in young adults the processes of bone f o r m a t i o n a n d a b s o r p t i o n a r e c l o s e l y associated to one another. after the age of 30 years there is a gradual diminution of bone mass, which is most clearly seen in women, who experience a significant decrease in bone mass. this is associated with lowered estrogen production several years prior to the onset of the menopause and continues for a period of up to 5 years, followed by a slow decrease in bone mass for the remaining life span of the women.(10) osteoporosis is a condition of diminished bone mass and changes in bone architecture up to the fracture threshold without clinical signs or symptoms.(12) in this d i s o r d e r t h e r a t e o f b o n e f o r m a t i o n i s f r e q u e n t l y n o r m a l , b u t t h e r a t e o f b o n e absorption is increased. bone loss occurs more frequently in trabecular bone, such as in the vertebrae, femoral neck and distal radius.(11) the rate of bone loss is 2-2.5% in the first 5 post-menopausal years, 39% in the age range of 70-79 years and 70% at age 80+ years.(12) the who has estimated the prevalence of osteoporosis among white postmenopausal women in the us to be 14% at age 50-59 years, 22% at 60-69 years, 39% at age 70-79 years and 70% at age 80+ years.(13) unmodifiable risk factors for osteoporosis are gender (women are of lesser weight and have smaller bones than men), advanced age, family history of osteoporosis, race (asian and c a u c a s i a n w o m e n a r e a t g r e a t e r r i s k o f osteoporosis than african women), body build (thin and small women are at higher risk of osteoporosis), and a number of disorders (anorexia, diabetes, chronic diarrhea, renal and hepatic disorders). modifiable risk factors are smoking, alcohol consumption, low calcium intake, lack of exercise, underweight and use of medications (steroids, phenobarbital and phenytoin).(10) the objective of this study was to determine serum estradiol levels and their r e l a t i o n s h i p w i t h b m d v a l u e s i n postmenopausal women. methods research design the present study was of cross-sectional design in order to determine serum estradiol levels and their relationship with bmd values in postmenopausal women. study subjects the study subjects were randomly selected postmenopausal women aged 47 up to 60 years from four villages (kelurahan) in the mampang prapatan subdistrict, south jakarta, namely the villages of kuningan barat, mampang prapatan, tegal parang and pela mampang. inclusion criteria for this study were: p o s t m e n o p a u s a l w o m e n , d u r a t i o n o f menopause of more than one year, age 47 up to 60 years, willing to participate in the study and sign informed consent, able to actively communicate, and actively mobile (requiring no walking aids). the exclusion criteria were women with hysterectomy or bilateral oophorectomy, acute infections, diabetes mellitus, diseases of the kidneys, lungs and liver, malignancies, and those consuming hormonal medications in the last 3 years. the respondents were interviewed 92 from february to april 2010 by 14 health center cadres, using a questionnaire that i n c l u d e d i t e m s o n a g e a n d d u r a t i o n o f menopause. all participating women signed an informed consent form. assessment of physical characteristics the physical characteristics assessed were height, weight, and body mass index (bmi). height was measured to the nearest 0.1 cm with the subjects in the upright position without shoes. body weight was determined to the nearest 0.1 kg with the subjects wearing i n d o o r c l o t h e s b u t n o s h o e s . b m i w a s calculated as weight (kg)/ height (m)2. bmi is classified into the following categories: underweight (< 18.5 kg/m2), normal (18.5-22.9 kg/m2), overweight (23.0-27.5 kg/m2), and obese (> 27.6 kg/m2).(14) biochemical measurements b l o o d s a m p l e s f o r b i o c h e m i c a l measurements were drawn by venipuncture after a fast of 12-14 hours. subsequently the serum was separated and stored at –700c until needed for examination. d e t e r m i n a t i o n o f s e r u m e s t r a d i o l concentration (coefficient of variation = 3.2%) was performed at prodia laboratories, jakarta, u s i n g r o c h e r e a g e n t s ( c a t a l o g n o . 03000079122, lot no.154701-04 ed). the d e t e c t i o n r a n g e o f t h e s e r u m e s t r a d i o l determinations was 5.00 4300.00 pg/ml. bmd measurements bmd of the lumbar spine (first to fourth lumbar vertebrae), the right femoral neck, and the distal left radius was determined by dualenergy x-ray absorptiometry (dxa), using a lunar dpx bravo nomusa densitometer (ge medical systems) at budi jaya hospital, jakarta. according to definitions of diagnostic categories suggested by a who study group in 1994, the normal category has a bmd within 1 sd of the reference mean (young adults). in osteopenia the bmd value is more than 1 sd b e l o w t h e r e f e r e n c e m e a n , w h e r e a s i n osteoporosis the bmd is 2.5 sd or more below the reference mean.(16) expressed as t-scores: normal if t-score > -1; osteopenia if -2.5 < tscore < -1; osteoporosis if t-score < -2.5.(15) ethics ethical clearance was provided by the research ethics commission of the medical faculty, trisakti university. data analysis subsequent to coding of the collected data, data entry was performed using statistical package for social science (spss) version 15 for windows. initially a test of normality was done by means of the komogorov-smirnov t e s t . s i n c e t h e d a t a w e r e n o n n o r m a l l y distributed, correlation analysis was performed by calculating spearman’s rho in order to determine correlations between bmi and serum e s t r a d i o l l e v e l s . t h e l e v e l o f s t a t i s t i c a l significance was set at p < 0.05. results among 215 subjects aged 47-60 years, 31 (31/125 = 0.25%) did not meet the inclusion c r i t e r i a o f w h o m 2 0 s u b j e c t s w e r e o n biochemical testing revealed to have diabetes mellitus and 11 did not show up for laboratory tests. a total of 184 women meeting the inclusion criteria participated in this study. mean age of the subjects was 53.58 ± 3.53 years, with age range of 47 – 60 years. mean bmi was 26.75 ± 4.68 kg/m2. the majority of p o s t m e n o p a u s a l w o m e n ( 6 5 . 3 % ) w e r e overweight and 30.4% were in the normal category. mean estradiol concentration was 7.54 ± 4.65 pg/ml, while 46.5% of women were in the low estradiol category (≤ 5 pg/ml). mean lumbar t-score was –1.63 ± 1.04, mean femoral neck t-score was –0.99 ± 0.96, and mean distal radial t-score was –1.95 ± 1.17. mean duration of menopause was 4.47 ± 2.24 years, with a range of 2 to 11 years (table 1). mawi serum estradiol levels and bone mineral density 93 univ med vol.29 no.2 in this study, the highest prevalence of osteopenia was for the lumbar vertebrae (50%), while the prevalence of osteoporosis was highest in the left distal radius (35.3%) (table 2). t h e k o l m o g o r o v s m i r n o v t e s t o f normality showed that serum estradiol was not normally distributed. nonparametric correlation according to spearman rho showed a positive correlation between serum estradiol and bmd of the femoral neck (r=0.021;p=0.042) (table 3). thus the higher the serum estradiol levels, the higher the bmd values for the femoral neck region. discussion i n t h e p r e s e n t s t u d y 4 9 . 5 % o f p o s t m e n o p a u s a l w o m e n h a d e s t r a d i o l concentrations of < 5 pg/ml. in postmenopausal women aged between 47 and 60 years the estradiol concentration was > 5 pg/ml, this study showed a significant positive relationship between estradiol levels and t-scores for the femoral neck region. essentially similar results were found by bagur et al. in postmenopausal women < 65 years of age, who had estradiol levels of > 10 pg/ml and higher bmd values in all skeletal sites examined, in comparison with women whose estradiol levels were < 10 pg/ ml.(5) a study involving 370 postmenopausal women aged between 55 and 85 years found a significant positive relationship between serum estradiol levels and bmd values for the lumbar spine and the femoral neck.(16) in postmenopausal women, the ovaries as the main producers of estradiol undergo degeneration resulting in diminished estradiol levels, which may lead to osteopenia and table 1. mean values for age, bmi, lumbar, femoral and radial t-scores, estradiol level and duration of menopause in postmenopausal women (n=184) general characteristics mean ± sd age (years) 53.58 ± 3.53 body mass in dex (kg/m2) underweight normal overweight 26.74 ± 4.68 8 (4.3%) 56 (30.4%) 120 ( 65.3%) lum bar t-score -1.63 ± 1.04 right fem oral t-score -0.99 ± 0.96 left radial t-score 1.9 5 ± 1.17 estradiol (pg/ml) ≤ 5.0 > 5.0 36.14 ± 16.14 91 (46.5%) 93 (50.5% duration of menopause (years) 4.47 ± 2.24 table 2. distribution of osteopenia and osteoporosis by bmd site in postmenopausal women (n=184) bmd site o steopenia (n,%) osteoporosis (n,%) lum bar vertebrae 1-4 righ t fem oral neck left distal radius 92 (50.0) 91 (49.5) 77 (41.8) 43 (23.4) 9 (2.2 ) 63 (35.3) table 3. correlation between serum estradiol and bone mineral density in postmenopausal women with estradiol concentrations of > 5 pg/ml (n=93) bmd lumbar spine femoral neck distal radius estradiol (pg/ml) r=0.080 r= 0.211* r= 0.063 *p=0.042 94 osteoporosis. extragonadal sources of estradiol are the adrenals, adipose tissue, liver, kidney and muscle, where estradiol is synthesized through aromatization of andrestenedione.(7) t h e c r i t i c a l r o l e o f e s t r o g e n s i n b o n e homeostasis is widely recognized, and one of its consequences is the loss of bone occurring a f t e r t h e m e n o p a u s e , w h e n t h e g o n a d a l function ceases.(17) extragonadal aromatization is affected by age, gender, and body weight. overweight women have higher estrogen c o n v e r s i o n a n d c i r c u l a t i o n r a t e s t h a n underweight women.(1,2) lack of estrogen also causes osteoporosis, as in the case of women athletes in whom menstruation is delayed, resulting in less than normal estrogen levels. one in three women and one in twelve men will suffer from o s t e o p o r o s i s t h r o u g h o u t t h e i r l i v e s , p a r t i c u l a r l y a f t e r t h e a g e o f 5 0 y e a r s . osteoporosis may occur at age under 45 years in women who have undergone hysterectomy and oophorectomy.(1,2) osteoporosis is a common disorder of old age, creating a worldwide health problem, as the aged tend to suffer from bone fractures from mild injury or even without injury. these fractures increase the morbidity and mortality rates, and health funding, and reduce the quality of life of the patients.(10,18) in the study by morton et al. it was found that bmd decreased with advancing age, whereas bmd was proportional to bmi.(19) in the present study, it is unclear whether the extremely low estradiol level (<5 pg/ml) in the majority of the respondents was caused by lack of dietary cholesterol (fat), since estrogen is synthesized in the liver and some is of dietary origin. osteoporosis may also be caused by low calcium, low vitamin d and lack of exercise, which affects bone density. it is reported that osteoporosis in asia is intimately associated with low calcium and lack of vitamin d. around 35% of postmenopausal women in asia have osteoporosis and 50% osteopenia. in postmenopausal women, the most frequently affected by osteoporosis are the trabecular bones (spongious bones).(1) as is well-known, two risk factors for osteoporosis are age and menopause. with advancing age there is increased resorption of old bone in comparison to new bone formation, so that the bones weaken and fracture easily at the slightest i n j u r y o r e v e n s p o n t a n e o u s l y. t h e m o s t c o m m o n f r a c t u r e s a r e t h o s e o f t h e h i p , vertebrae and wrist.(20) the reduction in bone mineral mass of the radius in men occurs after the age of 60 years. however, in women bone loss commences around the age of 50 years, is most rapid 3 or 4 years after menopause, and on average continues at a greater rate from the age of 67 years upwards.(19,20) in approximately half of white postmenopausal women the radial mineral mass is below normal. in the study by rogers et al. a positive association was found between estradiol and bone density at all body sites examined.(21) in another study, it was found that increased age was associated with decreased estradiol and b m d , b e c a u s e r e d u c e d s e r u m e s t r a d i o l concentrations increased bone turnover, which is a risk factor for fractures.(16) zarrabeitia et al. revealed a significant correlation of serum estradiol concentration with bmd of the spine in postmenopausal women. in addition, the investigators also found in subjects with high b m i t h a t b o d y w e i g h t a n d e s t r a d i o l concentration were the principal factors determining bmd.(22) t h i s s t u d y w a s s u b j e c t t o s o m e limitations. first, as this was a cross-sectional community-based study, no conclusions can be drawn as regards the cause-effect relationship b e t w e e n e s t r a d i o l l e v e l s a n d b m d , a n d longitudinal studies will be necessary to confirm our conclusions. secondly, the women in this study were aged between 47 and 60 years, for whom no bmd data were available. thirdly, no measured values of free estrogen were available. mawi serum estradiol levels and bone mineral density 95 univ med vol.29 no.2 conclusions postmenopausal women aged 47 to 60 years 49.5% had serum estradiol levels of ≤ 5 pg/ml. there was a significant correlation between estradiol and femoral neck bmd v a l u e s i n p o s t m e n o p a u s a l w o m e n w i t h estradiol levels of > 5 pg/ml. acknowledgements the investigator expresses her gratitude to the medical faculty, trisakti university, for the funding of this study. and also thanks the respondents who were willing to participate in this study. references 1. baziad a. menopause and andropause. jakarta: yayasan bina pustaka sarwono prawirohardjo; 2003. 2. molina pe. endocrine physiology. new york: the mcgraw-hill companies; 2004. 3. sacco sm, ward we. revisiting estrogen: efficacy and safety for postmenopausal bone health. j osteoporos 2010;2010:1-7. 4. shanafelt td, barton dl, adjei aa, loprinzi cl. pathophysiology and treatment of hot flushes. mayo clin proc 2002;77:1207-18. 5. bagur a, oliveri b, mautalen c, beotti m, mastaglia s, yankelevich d, et al. low level of endogenous estradiol protects bone mineral density in young postmenopausal women. climacteric 2004;7:181-8. 6. simpson er. aromatization of androgens in women: current concepts and findings. fertil steril 2002;77:6-10. 7. matsumine h, hirato k, yanaihara t, tamada y, yoshida m. aromatization by skeletal muscle. j steroid biochem mol biol 2003;84:485-92. 8. guthrie jr, lehert p, dennerstein l, burger hg, ebeling pr, wark jd. the relative effect of endogenous estradiol and androgens on menopausal bone loss: a longitudinal study. osteoporos int 2004;15:881-6. 9. larionov aa, vasyliev da, mason jl, howie af, berstein lm, miller wr. aromatase in skeletal muscle. j clin endocrinol metab 2002;3: 1327-36. 10. nurhonni sa. osteoporosis and pencegahannya. maj kedokt indon 2000;50:565-8. 11. tana l. pencegahan osteoporosis. media litbang kesehatan 2005;15:50-7. 12. sowers mr, jannausch m, mc connell d, little r, greendale ga, filkelstein js, et al. hormone predictors of bone mineral density changes during the menopausal transition. j clin endocrinol metab 2006;91:1261-7. 13. ninghua l, pinzhong o, harmin z, dingzhuo y, pinru z. prevalence rate of osteoporosis in the mid-aged and elderly in selected parts of china. clind med j 2002;115:773-5. 14. world health organization. appropiate body mass index for asian populations and its implications for policy and intervention strategies. the lancet 2004;363:157-63. 15. world health organization. assessment of fracture risk and its application to screening for postmenopausal osteoporosis. who technical report series 843. geneva: world health organization;1994. 16. van geel tacm, geusens pp, winkens b, sels jpje, dinant gj. measures of bioavailable testosterone and estradiol and their relationships with muscle mass, muscle strength and bone mineral density in postmenopausal women: a cross-sectional study. eur j endocrinol 2009;160: 681-7. 17. riggs bl, khosla s, melton lj iii. sex steroids and the construction and conservation of adult skeleton. endocr rev 2002;23:279–302. 18. lea ck, ebrahim h, tennant s, flanagan am. aromatase cytochrome p450 transcripts are detected in fractured human bone but not in normal skeletal tissue. j gerontol a biol sci med sci 2003;3:266-70. 19. morton dj, connor eb, silverstein dk, wingard dl, schneider dl. bone mineral density in postmenopausal caucasian, filipina, and hispanic women. int j epidemiol 2003;32:1506. 20. miller pd, hochberg mc, wehren le, ross pd, and wasnich rd. how useful are measures of bmd and bone turnover? curr med res opin 2005;21:545. 21. rogers a, saleh g, hannon ra, greenfield d, eastell a. circulating estradiol and osteoprotegerin as determinants of bone turnover and bone density in postmenopausal women. j clin endocrinol metab 2002;87:4470-5. 22. zarrabeitia mt, hernandez jl, valero c, ziarrabeitia a, amado ja, macias jg, et al. adiposity, estradiol, and genetic variants of steroid-metabolizing enzymes as determinants of bone mineral density. eur j endocrinol 2007; 156:117-22. alvina 36 abstract worldwide, the proportion of people aged 60 and over is growing faster than any other age group. it has been well-established that the aging process can be associated with increased susceptibility to chronic conditions, disability, and co-morbidity, which however may be minimized or even partially reversed by physical activity. the assessment of physical activity is becoming an increasingly important component in the evaluation of elderly persons. nutritional intake and status play an essential role in determining the physical activity level potentially capable of minimizing the health burden of older persons. the objective of this study was to find out whether nutritional intake and status were correlated with physical activity in community-dwelling older persons. the study population included 219 aged 60 to 69 years, of whom complete measures of socio-demographic characteristics, nutritional status, nutritional intake and physical activity were obtained. serum total protein, albumin, globulin and hemoglobin concentration were measured as nutritional indicators (biomarkers). the nutrient content of food intakes was analyzed and calculated by “nutrisurvey” software. analysis indicated that there was a significant correlation between nutritional biomarkers [total protein (r=-0.211; p=0.002) and globulin (r=-0.247; p=0.000)] and physical activity. compared to other food intakes, intakes of carbohydrate (r=0.241; p=0.001) and animal protein (r=0.149; p=0.027) were significantly correlated with physical activity. our findings lend support to the existence among healthy older persons of a relationship between nutrional intake and status and physical activity. key words: nutritional status, nutritional intake, physical activity, older persons *department of community medicine medical faculty, trisakti university correspondence dr. dr. rina k. kusumaratna, m.kes. department of community medicine medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2504 email: rkusumaratna@gmail.com univ med 2011;30:36-44. protein intake as a determinant factor of physical activity in older persons rina k kusumaratna* and adi hidayat* january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction relative to the younger age groups, the group of older persons is steadily growing worldwide, as a result of increased longevity. in 2002, the global population of older persons was around 600 millions and is expected to double by the year 2025. around 80% of the e l d e r l y w i l l b e l i v i n g i n d e v e l o p i n g countries.(1,2) indonesia is one of the nations with a large population of older persons and ranks as number five in the world. thus, the 37 univ med vol.30 no.1 maintenance of a healthy physical and mental state in the elderly population is an important public health challenge now and in the future.(3) a number of clinical and epidemiological studies have produced confirmatory evidence for a relationship between health (physical and mental), nutrition and quality of life. many studies have strived to find specific nutrients for older persons that could link nutrition with chronic diseases and could impact on health in later life. other studies have focused on the preventive effects of physical activity on physical health, while others have highlighted the relationship between nutritional intake and status. one of the factors believed to be important for performance of physical activity is the composition of the diet. there are as yet no established criteria for optimal nutritional intakes and requirements in older persons. nutritional status plays an essential role in determining health status and is potentially capable of minimizing the health burden of older persons. currently a nutritional transition is occurring on a global scale, due to unhealthy lifestyles of younger people, such as declining physical activity, non-nutritious diets, and smoking. the current trend in developing countries is to eat more fat and more refined diets, which has contributed to increased risk of chronic disease. social and demographic changes have an impact on older persons, in that the aged population has a great risk of food insecurity and suffers from malnutrition. in facing the expanding older population segment, the who and the jean mayer usda human nutrition research center on aging at tufts university in 1998 jointly set up a list of priority concerns comprising documentation of “the nutritional status of older persons, better definition of their nutrient requirements, and identification of factors affecting dietary intake and nutrient absorption in different cultural and environmental settings”.(4) the normal aging process causes multiple physiological changes that could influence the n u t r i e n t i n t a k e a n d n u t r i t i o n a l s t a t u s o f individuals. optimal nutrition ensures that the body has sufficient nutrients to maintain its functions, prevent nutritional deficiencies, and delay the onset of degenerative diseases. recently, there has been a shift in the concept of healthy and successful aging, from an emphasis on number of years to quality of life (qol) during those years. this shift has led to the view that a healthy lifestyle, specifically optimal food intake and physical activity, are important determinants of qol and wellbeing. exercise is most commonly used for h e a l t h p r o m o t i o n o f o l d e r p e r s o n s , a n d adequate nutritional intake may be expected to produce an improvement in physical activity. a previous study reported that physical activity is not only of benefit in preventing lifestylerelated disease and functional decline in the older persons, but also in decreasing their mortality.(5) these considerations led to the formulation of the aim of the present study, namely to investigate whether nutritional intake and status have a relationship with physical activity among community-dwelling older persons. methods study design a cross-sectional study was carried out in the mampang prapatan district, south jakarta municipality, dki jakarta province from august to october 2007. study subjects the study enrolled 219 communitydwelling older persons aged 60 to 69 years, who were residents of five sub-districts in the mampang prapatan district. five kelurahan w e r e r a n d o m l y c h o s e n a n d f r o m t h e s e kelurahan were selected 8 hamlets (rw) with high numbers of resident older persons. subjects were listed and recruited by simple random sampling from the older persons living in the hamlets, especially those who were nonsupervised. the inclusion criteria were: male 38 or female persons 60-69 years of age, mobile, independent, able to verbally communicate, willing to join the study and prepared to sign the informed consent form. exclusion criteria were subjects with dementia or uncontrolled hypertension, and bedridden or physically handicapped persons. data collection the general characteristics of the subjects i.e. identity of respondents, age, date of birth, address, formal education level, history of occupation, financial support (income and expenses), ethnicity and religion, history of health, housing condition, and life style were recorded by interview using a standardized questionnaire. anthropometric measurements the subjects were measured as to height, body weight, and waist circumference. height was measured using a portable microtoise to the nearest 0.1 cm and weight was measured using sage portable scales to the nearest 0.1 kg. body mass index (bmi) was calculated as the weight (kg) divided by the square of the height (m2). for asian populations, bmi is classified into the following categories: underweight (<18.5 kg/m2), normal (18.5–22.9 kg/m2), overweight (23.0–27.5 kg/m2), and obese (>27.6 kg/m2).(6) waist circumference was measured with the subject wearing light clothing, at a level midway between the lower rib margin and iliac crest, to the nearest cm using a plastic measuring tape. nutritional evaluation blood was collected from the antecubital vein in the fasting state in early morning. serum total protein, albumin, globulin and hemoglobin concentration were measured as nutritional indicators. dietary intake dietary intake was assessed using semiquantitative-food frequency questionnaire (sq-ffq), 2 x 24 hours non-consecutive food r e c a l l a n d w e i g h t f o o d . f o r m i n i m i z i n g memory bias and assist the elderly in recalling the foods consumed, the trial used threedimensional food models in standard portion sizes. the sq-ffq and 24-hour dietary recall assessments were done through interviews administered by the nutritionist on home visits. the nutrient content of food intakes was analyzed and calculated by “nutrisurvey” software.(7) physical activity habitual physical activity was calculated using the short-form [short version] of the international physical activity questionnaire ( s f i pa q ) . t h e i pa q s h o r t f o r m i s a n instrument designed primarily for population s u r v e i l l a n c e o f p h y s i c a l a c t i v i t y a m o n g adults.(8) the ipaq was developed as an instrument for cross-national monitoring of physical activity. the sf-ipaq has been extensively used for measurement of physical activity in the age range of 15 to 69 years, which was the reason for the age limit of 69 years used is the present study. total physical activity was calculated as met-minutes/week. statistical analysis the data analysis was by descriptive statistical methods and bivariate analysis by chi square and independent t tests to compare several variables between males and females. pearson correlation coefficient was used to determine the relationship between nutrional intake and status and physical activity. a p value of <0.05 was regarded as statistically s i g n i f i c a n t . t h e s t a t i s t i c a l a n a l y s i s w a s performed with the spss software package for windows, version 15 (spss inc., chicago). ethical clearance permission and administrative clearance for conducting the study were provided by the district health office and the district health center. ethical clearance was given by the kusumaratna, hidayat protein intake of physical activity 39 univ med vol.30 no.1 demographic profile and nutritiona l status g ender pa males n=80 (%) females n=139 (%) ag e (yr) * education (%) educated illiterate marital status (%) married wid owed unmarried nu tritional status und erweight normal overweight obese bmi (kg/m2) * weight (kg) * height (cm) * waist circumference (cm) * 64.2 ± 2.9 74 (43.0) 6 (12.8) 67 (68.4) 13 (10.9) 0 (0.0) 13 (26.0) 37 (43.0) 23 (41.1) 7 (25.9) 22.1 ± 3.4 57.2 ± 9.4 160.7 ± 4.3 81.3 ± 10.3 63.7 ± 2.5 98 (57.0) 41 (87.2) 31 (31.6) 106 (89.1) 2 (100.0) 37 (74.0) 49 (57.0) 33 (58.9) 20 (74.1) 21.7 ± 4.4 49.5 ± 11.1 150.8 ± 3.8 77.7 ± 12.2 0.241 0.000 0.000 0.124 0.480 0.000 0.000 0.026 table 1. demographic profile and nutritional status by gender (n=219) note : * mean ± sd; a p values were derived from χ2 and independent t test nutritional biomarkers gender pa males (n=80) females (n=139) hemoglobin (g/dl) total protein (g/dl) albumin (g/ dl) globulin (g/ dl) 14.1 ± 1.5 7.1 ± 0.9 4.0 ± 0.6 3.0 ± 0.5 12.7 ± 1.4 7.1 ± 1.2 3.9± 0.6 3.3 ± 0.7 0.000 0.614 0.121 0.018 table 2. mean values of nutritional biomarkers by gender note: values are mean ± sd; a p values were derived from independent t test human ethics committee of the faculty of medicine, university of indonesia. results demographic profile and nutritional status as shown in table 1, the majority of participants were females (63.5%) with mean age of 63.7 ± 2.5 years. the underweight category was higher in females (74.0%) compared to males (26.0%). similarly, there were more overweight females (74.1%) than males (25.9%), although the difference was not statistically significant (p=0.124). most female older persons (87.2%) were illiterate and 31.6% were married. in male older persons, weight, height and waist circumference were significantly higher compared to females. nutritional biomarkers ta b l e 2 c o m p a r e s t h e n u t r i t i o n a l biomarkers between males and females. the hemoglobin concentration was significantly higher among males (14.1 ± 1.5 d/dl) than females (12.7 ± 1.4 g/dl) (p=0.000). however, the globulin concentration was significantly higher in females (3.3 ± 0.7 g/dl) compared to males (3.0 ± 0.5 g/dl) (p=0.018). total protein, albumin and globulin levels were within the normal ranges in both groups. 40 table 3. mean nutritional intakes based on gender note: values are mean ± sd; a p values were derived from independent t test nutritional intakes d a i l y i n t a k e s o f m a c r o n u t r i e n t a n d micronutrients are presented in table 3. estimated intakes of energy and nutrients were calculated on the basis of the average value of two non-consecutive 24-hour food recall sessions. from these values the distribution of usual intakes in the population was estimated. micronutrient intakes were calculated on the basis of the sq-ffq. t h e f o l l o w i n g u s u a l i n t a k e s w e r e obtained: mean intake of total energy per day was 1251.1 ± 437.2 kcal/day in males, or 6 1 . 3 % o f t o t a l e n e r g y i n t a k e b a s e d o n indonesian recommended daily allowances [irda] for males. in females, mean intake of total energy per day was 1056.8 ± 355.8 kcal/ day or 66.1% of irda, carbohydrate 153.3 ± 55.6 g per day, protein 36.2 ± 16.8 g per day and fat 36.3 ±15.6 g per day. iron, zinc, vitamin c and â-carotene intakes per person per day were 29.4 ± 26.4 mg, 5.4 ± 2.7, 70.0 ± 54.0 mg, and 0.66 ± 0.88 mg, respectively. there were significance differences of total energy, carbohydrate, total protein and animal protein intakes between males and females (<0.05) (table 3), with higher intakes of carbohydrate, total protein, and animal protein in males. t h e p e r c e n t a g e s o f t h e n u t r i e n t composition in females indicate that average carbohydrate intake accounted for 58.2% of total energy intake, while 10.5% of total energy per day was from protein and 20.4% of total energy per day from fats. in addition, the n u t r i e n t c o m p o s i t i o n o f t h e e l d e r l y w a s moderate for protein (normal range 10-20%), w i t h i n t h e n o r m a l r a n g e o f 5 0 6 0 % f o r carbohydrate, and was high in fat (> 25%) in both groups. the vitamin and mineral intakes in both groups were lower than estimated intakes based on irda, especially intakes of vitamin e and zinc, which were considerably below irda. from these data it is apparent that there were significant differences in nutritional intakes by gender, except for fat and several micronutrients (table 3). physical activity physical activity score in males was 731.5 ± 383.8 met-minutes/week and in females 590.0 ± 259.7 met-minutes/week (p=0.001) (data not presented). kusumaratna, hidayat protein intake of physical activity 41 univ med vol.30 no.1 nutritional status, nutritional intakes and physical activity table 4 shows the relationship between nutrional intake and status and physical activity. weight and height were correlated significantly with physical activity (r=0.175; p=0.010 and r=0.169; p=0.010), but bmi and waist circumferences were not significantly correlated with physical activity. the significant correlation between nutritional biomarkers and physical activity comprised serum total protein (r=-0.211; p=0.002) and globulin (r=-0.247; p=0.000). in contrast to other food intakes, intakes of carbohydrate (r=0.241; p=0.001) and animal protein (r=0.149; p=0.027) were significantly correlated with physical activity. discussion the ideal outcome of longevity is to live through the added years as a healthy individual free of disability and disease, and enjoy a good qol. however, older persons may expect to experience disability and poor qol, even if they are apparently healthy. as a rule, older persons s u ff e r f r o m n u t r i t i o n a l d e f i c i e n c e s , ( 9 , 1 0 ) decreased physical activity,(11) decline in health status,(9) decline in cognitive functions,(11) and impaired functional status,(12) all of which ultimately affect their qol.(13) hemoglobin, total protein, albumin, and globulin levels are widely used as indicators for screening of nutritional status. the older persons in our study had normal values of hemoglobin, total protein, albumin, and globulin. a study among italian older persons aged 75 to 85 years and japanese older women aged 60 to 80 years, showed normal hemoglobin and albumin levels in all subjects.(14,15) our study results showed a significant relationship between blood albumin concentration and physical activity, which was consistent with the results of a previous study indicating that albumin levels in blood were correlated with walking ability and physical fitness.(16) the study conducted by chin et al.(17) demonstrated that decreased physical activity was the best indicator for frailty and described its relation to nutritional intake and nutritional status. in contradistinction to the albuminphysical activity relationship, there was no significant correlation between energy intake and physical activity in our study. although blood biomarkers are important for determining nutritional status, a more important point to consider is dietary or nutritional guidance to older persons for ensuring maintenance of proper dietary intakes, especially intakes of protein. it is known that both exercise and nutrition are important in promoting health in elderly people. in health promotion, the relationship between nutritional intake and/or status and physical activity has to date not been adequately clarified. the present study showed that total energy intakes of both females and males were lower than estimated energy requirements based on irda, with moderate protein and normal carbohydrate intakes but high intakes of fat, relative to irda, for persons aged 60 years and over. vitamin and mineral intakes in both groups were also lower than irda, especially for intakes of vitamin e and zinc which were considerably below irda. our study results table 4. relationship between nutritional status and intake and physical activity in all subjects (n=219) a p values were derived from pearson correlation test 42 differed from a study in japan on female subjects aged 60 to 82 years, which showed that intakes of macronutrients, micronutrients and v i t a m i n s s a t i s f i e d e s t i m a t e d e n e rg y requirements based on the japanese dietary intake reference 2005.(15) intake of antioxidants (vitamin c, â-carotene, vitamin e, and trace m i n e r a l s s u c h a s z i n c ) i s e s s e n t i a l f o r maintaining health and physical activity in old age. according to findings from one of the studies of the italian inchianti (invecchiare i n c h i a n t i ) p r o j e c t , p l a s m a a n t i o x i d a n t concentration significantly correlates with physical performance and leg extension strength in older persons.(18) socio-economic factors are also important as underlying determinants of morbidity, mortality and malnutrition, including m i c r o n u t r i e n t d e f i c i e n c y. e d u c a t i o n a l background can be used to assess the potential vulnerability of a population with poor dietary patterns. in this study only 21.5% of illiteracy was found, and therefore other factors may have affected the dietary pattern. in urban areas, such as mampang prapatan, the population depends heavily on cash income, and employment can be a useful indicator of standard of living. in general, older people might be at risk of malnutrition due to impaired energy intake, social isolation or decreased income. in the present study, socio-economic factors, including source of income and employment, could be also used as indicators to identify those older persons at high risk for micronutrient deficiency. overall, the diet of the subjects conformed to that of other developing nations, being largely composed of foods of plant origin. their diet includes few animal protein sources, such as meat or poultry, since these are expensive and older persons may be unable to afford them on a regular basis. the association between poverty and nutrition has long been recognized, because p o o r p o p u l a t i o n s o f t e n r e l y h e a v i l y o n monotonous plant-based diets low in animal products. in addition, some of the older persons may have difficulties in mastication due to dental problems, such that most of them substitute the softer tofu or tempeh for relatively tough animal protein. malnutrition in older p e o p l e i s a n i m p o r t a n t p r o b l e m a n d i s encountered in both community-dwelling and institutionalized older persons. the risk and prevalence of malnutrition rise proportionally w i t h a g e , m a k i n g t h e m a n a g e m e n t o f malnutrition among older people an urgent public health need, that is also beneficial from an economic viewpoint. in general, nutritional requirements change with increasing age. older people are less efficient in processing and metabolizing their foods, and require lesser amounts of energy. they therefore should consume nutrient-dense f o o d s , i n w h i c h s m a l l s e r v i n g s p r o v i d e substantial amounts of macroand micronutrients. inadequate food intake leads to undernutrition among older persons that is difficult to reverse, because low food intakes are likely to increase their risk of energy and nutritional deficiency. ideally, older persons should have balanced and varied intakes of macroand micronutrients for optimum health. however, most subjects in this study had a total energy intake of 63.7%, which is lower than the minimal requirement (80%) of irda for persons aged 60+ years. although in absolute terms the total energy intake of both genders were significantly d i ff e r e n t , i t w a s m o s t l y i n a d e q u a t e a n d unbalanced. most macroand micronutrient intakes were lower than irda for age 60+ years, especially intakes of vitamin c, vitamin e and zinc, which were far below irda. therefore it appears that the diet of the study subjects was relatively poor in macronutrients and energy, but definitely deficient in vitamins and minerals. these results are consistent with those of several studies reporting that 50% of older persons have a micronutrient intake of less than the daily recommended intake, while 10–30% has marginal levels of micronutrients.(9,19) therefore it should be emphasized to older persons to kusumaratna, hidayat protein intake of physical activity 43 univ med vol.30 no.1 consume sufficient amounts of animal protein, b o t h f o r i t s r e a d y a b s o r b a b i l i t y a n d micronutrient content. an adequate protein intake results in a positive nitrogen balance, thus accelerating protein synthesis. our study did not find a correlation between bmi and physical activity. this agrees with a swedish study on subjects 60–84 years of age, where no significant difference was found between bmi and leisure time physical activity, both among subjects born in sweden as well as among swedish residents born in iran.(21) furthermore, in our study a significant c o r r e l a t i o n w a s o b s e r v e d b e t w e e n carbohydrate and animal protein intake with physical activity. similar results were found in elderly japanese women,(15) which may be explained by the fact that among ordinary older people nutritional intake may relate to physical activity, regardless of the subject’s body composition. muscle mass gradually declines after age 50 years, and muscle loss leads to muscle weakness; greater risks of falls, fractures, disability, and loss of independence.(22) some of the age-related muscle weakness and muscle loss is due to physical inactivity, and as such can be reversed or at least minimized through increased physical activity. strength training can be an effective way for older persons to increase muscular strength and improve balance, and may assist the elderly to cope with a variety of chronic conditions; thus physical activity may have a preventive effect against disease and immobility.(23) as the world population is growing older and older, the increasing number of older people will present economic and social problems over the next decade. an important goal of research today is to identify possible strategies for ensuring the health of the elderly people in the near future in order to reduce social and economic expenditure. our data showed that nutrition is correlated with physical activity. although links between diet, physical activity, and risk of chronic disease have been well documented, more needs to be known about the p o s s i b l y r e c i p r o c a l e ff e c t s o f r e g u l a r l y performed physical activity and improved diet in older persons.(24,25) several limitations of our study should be mentioned. the cross-sectional design of the study did not permit us to evaluate the causality of daily nutrient intakes on physical activity. further longitudinal studies are needed to evaluate the role that nutrition status and intake may have on physical performance in older persons. the study population tended to be overweight, which prevented us from assessing the effect of very poor nutrition and may have influenced our findings. to better identify the effects of nutritional intake on physical activity, it may be useful to compare one group of persons with undernutrition with a control group of normal nutritional status. finally, another l i m i t a t i o n o f o u r s t u d y w a s t h e l i m i t e d assessment of biomarkers. the evaluation of both dietary intake estimates and biomarkers would have been the ideal combination. conclusions the present study found differences between actual and recommended dietary patterns of older persons in indonesia, with both genders having nutrient and energy intakes below irda. the protein intake have a relationship with physical activity in healthy elderly people. albumin blood levels as a targeted marker of nutrition status in blood significantly correlated with physical activity. acknowledgement the authors wish to thank all the elderly subjects participating in the study, and the mampang prapatan health center, south jakarta for their cooperation. the present study was supported by the faculty of medicine, trisakti university. 44 references 1. unfpa. population ageing in east and southeast asia: current situation and emerging challenges. population ageing series no.1, thailand, 2006. 2. meydani a, ahmed t, meydani sn. aging, nutritional status and infection in the developing world. nutr rev 2005;63:233-46. 3. bappenas, bps. projeksi penduduk indonesia (indonesia population projection) 2000-2025. jakarta: bappenas;2005. 4. tucker kl, buranap s. nutrition and aging in developing countries. j nutr 2001;131:s2417-23. 5. burke gl, arnold am, bild de, cushman m, fried lp, newman a, et al. factors associated with healthy aging: the cardiovascular health study. j am geriatr soc 2001;49:254-62. 6. world health organization. appropriate body mass index for asian populations and its implications for policy and intervention 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2001;56:m146-56. 12. inouye sk, studenski s, tinetti me, kuchel ga. geriatric syndromes clinical, research and policy implications of a core geriatric concept. j am geriatr soc 2007;55:780-91. 13. cella d, nowinski cj. measuring quality of life in chronic illness; the functional assessment of chronic illness therapy measurement system. arch phys med rehabil 2002;83:s10-7. 14. pasini e, corsetti g, bandera f, tolomin s, salvetti m, pedreti e, et al. nutritional status and physical activity of a selected sample of elderly healthy italian people. mediterr j nutr metab 2008;1:43-8. 15. sakato y, tanabe k, nishijima t, fukunaga t, kuno s. relationship between nutrition intake status, nutrition condition and physical fitness in elderly women. int j sport haeth sci 2006;4:54454. 16. kwon j, suzuki t, kim h, yoshida h, kumagai s, yoshida t, et al. health status and physical fitness of undernourished community-dwelling elderly people. jap jour phys fit sport med 2005;54:99-106. 17. chin a, paw mj, de groot lc, van gend sv, schoterman mh, schouten eg, et al. inactivity and weight loss: effective criteria to indentify frailty. j nutr health aging 2003;72:55-60. 18. cesari m, pahor m, bartali b, cherubini a, penninx bwjh, williams gr, et al. antioxidants and physical performance in elderly persons: the invecchiari in chianti (inchianti) study. am j clin nutr 2004;79:289-94. 19. thomas dr. in: morley je, thomas dr, eds. nutritional requirements in older adults. in: geriatric nutrition, crc press, boca raton, 2007.p.103-21. 20. schunemann hj, sperati f, barba m, santesso n, melegari c, elie aa, et al. an instrument to assess quality of life in relation to nutrition: item generation, item reduction and initial validation. health qual life outcomes 2010;8:26:1-13. 21. koochek a, johansson se, kocturk to, sundquist j, sundquist k. physical activity and body mass index in elderly iranians in sweden: a population-based study. eur j clin nutr 2008;62: 1326–32. 22. chapman im. obesity in old age. obes metabol 2008;36:97–106. 23. drewnowski a, evans wj. nutrition, physical activity, and quality of life in older adults. j gerontol 2001;56a:89-94. 24. kubota a, ishikawa-takata k, ohta t. effect of daily physical activity on mobility maintenance in the elderly. int j j sport health sci 2005;3:8390. 25. evans wj. protein nutrition, exercise and aging. j am coll nutr 2004;23:s601–9. kusumaratna, hidayat protein intake of physical activity paul1 25 multimicronutrient supplementation and asymptomatic urinary tract infections in the elderly january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina paul boekitwetana*, julius e. suryawidjaja*, mahyunis aidilfit*, and murad lesmana* *department of microbiology, medical faculty, trisakti university correspondence adr. paul bukitwetan, mars department of microbiology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone:021-5672731 ext.2401 email : paulbw@gmail.com univ med 2009; 28: 25-33 abstract as human life expectancy continues to increase, developing countries are reporting higher percentages of elderly in their respective populations. the defense mechanisms of the elderly are reduced due to several factors, such as increased susceptibility to infection, specifically urinary tract infection (uti). a randomised, placebo-controlled, double-blind trial was conducted to assess whether multimicronutrient supplementation is effective in reducing utis in older people in the community. a total of 261 elderly who lived in mampang prapatan district, south jakarta, were randomised to daily multimicronutrient supplementation or control groups. the primary outcomes were the incidence of asymptomatic uti, the organisms responsible for utis and the results of sensitivity tests on uti microorganisms. uti was defined as culture-positive urine yielding a single species of organisms in numbers greater than 104 cfu/ml urine specimen. at base-line 19.5% of the elderly had uti, namely 23.7% patients in the mmn group and 16.7% in the control group, but the difference was not statistically significant (p=0.158). escherichia coli was the most common microorganism, isolated in 20.7% of the mmn group and 17.5% of the control group. after six months of supplementation, uti in the mmn group decreased by 40.6% compared with only 14.4% in the control group. the numbers of e. coli also declined by 64.3% in the mmn group compared to 37.8% in the control group. this study has confirmed the beneficial effect of multimicronutrient supplementation on uti in the elderly. keywords: multimicronutrients, urinary tract infection, resistance, elderly introduction in 2004 the number of elderly in indonesia was 5.8% of the general population and will continue to increase.(1) from the year 2000 up to 2050, the elderly population throughout the w o r l d w i l l e x p e r i e n c e a t w o t o f o u r f o l d increase.(2) infection is among the most common of disorders in older americans, and elderly individuals are 2–10 fold more likely to die of a variety of infections than are young adults.(3) urinary tract infection (uti) is very common 26 boekitwetan, surjawidjaja, aidilfit, et al asymptomatic urinary tract infections i n o l d e r p e o p l e , b e i n g t h e m o s t f r e q u e n t bacterial infection recorded in older people, followed by pneumonia and skin/soft tissue infections.(4) the majority of uti in elderly over 65 years of age in the urology department of rscm hospital occurred in men (95.0%), in whom the proportion of uti was 14.4%. the most frequent etiological organism was e.coli (38.6%).(5) a s t u d y i n t h e u k s h o w e d a u t i prevalence of 5.6% in the elderly.(6) the causes of the increased susceptibility to uti in older people are multiple: decline in cell-mediated immunity, altered bladder defenses due to o b s t r u c t i v e u r o p a t h y, n e u r o g e n i c b l a d d e r dysfunction, increased bacterial receptivity of u r o e p i t h e l i a l c e l l s , i n c r e a s e d r i s k o f c o n t a m i n a t i o n d u e t o f a e c a l a n d u r i n a r y incontinence as well as urethral instrumentation and catheterization, and decrease in prostatic and vaginal antibacterial factors associated with changes in zinc levels, urinary and vaginal ph, and hormones, especially lack of estrogens. men become more susceptible to utis after 50 years of age, when they begin to develop prostate problems. benign prostatic hyperplasia (bph) can produce obstruction in the urinary tract and increase the risk for infection. in men, r e c u r r e n t u t i s a r e a l s o a s s o c i a t e d w i t h prostatitis caused by e. coli.(7) although only a b o u t 2 0 % o f u t i s o c c u r i n m e n , t h e s e infections can cause more serious problems than they do in women. men with utis are far more l i k e l y t o b e h o s p i t a l i z e d t h a n w o m e n . ( 8 ) bacterial utis can be classified according to localization as urethritis (urethra), cystitis (bladder), or pyelonephritis (kidney). in men, prostatitis may mimic or complicate uti. alternatively, uti can be classified by the p r e s e n c e o r a b s e n c e o f s y m p t o m s ( a s symptomatic or asymptomatic), the frequency of its occurrence, the presence or absence of complications, and especially important in the elderly whether uti is associated with catheter use. older people are more likely to have adverse reactions to drugs. up to the present time older women with uncomplicated uti were treated longer than younger patients without any scientific evidence and with an increased risk of adverse drug reactions.(9) older adults are at risk for malnutrition, which may contribute to their increased risk of infection. uti is defined when the number of bacteria on urinary culture is more than 100,000 colony forming units per ml.(10) if not promptly and appropriately managed, uti may lead to infection of the kidneys and renal failure. urine was the most common specimen of e.coli inactive isolated.(11) the etiology of uti in the elderly is mainly e.coli (50%).(12) studies on the prevalence of bacteriuria in indonesian elderly are few in number. the effects of m u l t i m i c r o n u t r i e n t s u p p l e m e n t a t i o n o n bacteriuria and microbial antibiotic resistance in the elderly are still not clearly understood, and will be the subject of the current study. methods research design a randomized, placebo-controlled, doubleblind trial was conducted to assess whether multimicronutrient supplementation is effective in reducing utis in older people. study population the target population were the elderly residents of mampang prapatan district, south jakarta. the inclusion criteria were: male and female elderly aged 60 years and over, mobile, healthy, able to communicate and willing to participate in the study. terminally ill persons and those currently receiving antibiotic therapy for infections were excluded. written informed 27 consent was obtained from the participants and t h e s t u d y w a s a p p r o v e d b y t h e e t h i c s c o m m i t t e e , m e d i c a l f a c u l t y, tr i s a k t i university. a total of 261 subjects were enrolled using cluster and simple random sampling. randomization participants were randomized to receive multi-micronutrients (mmn) consisting of 40 mg elemental zinc (as zinc gluconate), 120 mg ascorbic acid, 6 mg â-carotene, 15 mg átocopherol (d-á-tocopheryl acid succinate) and 400 µg folic acid. the control group received daily oral tablets containing 400 mg calcium carbonate. randomization was performed using a blocks-of-six design in a numbered sequence prepared from computer-generated random numbers (microsoft excel) by an individual not otherwise involved in the study. intervention the supplementation was given every day for 6 months by the field workers at their home visits. participants in the treatment group received a daily oral tablet containing the amounts of vitamins and minerals stated above, except that on saturdays they received two t a b l e t s . p a r t i c i p a n t s i n t h e c o n t r o l g r o u p received a daily oral tablet containing calcium. both supplements were identical in color, size and taste, and would cause similar changes in t h e c o l o r o f t h e p a r t i c i p a n t s ’ u r i n e . p t ikapharmindo pharmaceutical prepared the supplement and control tablets specifically for this study, according to the specifications of the investigators. all participants were required to take the tablets daily for 6 months. at the home visits, the field workers recorded all occurring side effects and at the end of the month noted the number of supplements taken. the subjects were designated as withdrawals when they had n o t t a k e n t h e s u p p l e m e n t s f o r t w o w e e k s consecutively. collection of specimens after the elderly stated their willingness for participation in the study by signing the i n f o r m e d c o n s e n t f o r m , t h e e x a m i n i n g physician filled out the forms on personal data and clinical information for each participant. collection of early morning mid-stream urine was done on the fasting subject to avoid dilution of the urine. for female elderly, before voiding urine the external genital area was cleansed with soap and water and rinsed with clean water. the first portion of the urine was discarded, and only 20 ml of the mid-stream portion was collected in a sterile container. collection of urine samples was conducted before and after the 6 months of mmn supplementation. transport of urine samples the containers with the urine samples labeled with the subject’s identification number were put in an ice-box for transport to the microbiological laboratory, medical faculty, tr i s a k t i u n i v e r s i t y w i t h i n 4 h o u r s a f t e r collection. the urine samples that could not be sent on time were put in refrigerators at the study location and were sent to the laboratory within 24 hours. on arrival at the microbiological laboratory the specimens were recorded in a logbook. bacteriological examination on arrival at the laboratory, the urine specimens were promptly processed according to standard methods.(4) direct microscopy was performed on a gram smear of uncentrifuged urine. the number of bacteria per high-power f i e l d w a s c o u n t e d a n d t h e p r e s e n c e o f leucocytes was recorded. a volume of 10 ml u r i n e w a s c e n t r i f u g e d a t 2 5 0 0 r p m f o r 5 minutes. the supernatant was discarded and one drop of the sediment was placed on a glass slide, a cover slip was put on top, and the preparation was examined under a microscope. the mean univ med vol.28 no.1 28 boekitwetan, surjawidjaja, aidilfit, et al asymptomatic urinary tract infections of three high-power field readings was taken as the outcome. pyuria was diagnosed when more than 5 leucocytes per high-power field were found. urine culture methods the urine sample of elderly with pyuria was cultured for assessment of bacterial colony count. volumes of 0.01 ml and 0.001 ml of u n c e n t r i f u g e d u r i n e w e r e t a k e n f r o m t h e container by means of a calibrated loop, which was inserted vertically into the urine sample. the urine sample was streaked evenly on blood agar and mcconkey agar plates, using the same l o o p . t h e a g a r p l a t e s w e r e s u b s e q u e n t l y incubated aerobically at 350-370 c for 24-48 hours, after which the colonies were counted. from the results the mean was taken to obtain the number of colonies per milliliter of urine. i d e n t i f i c a t i o n o f i s o l a t e s w a s p e r f o r m e d according to standard methods.(4) a subject with a colony count of < 10,000 cfu/ml on blood agar plate was not considered to be suffering from bacteriuria. a count of 10,000-100,000 cfu/ml might be due to contamination, but a count of >100,000 cfu/ml was considered indicative of bacteriuria.(4) microbial susceptibility to antibiotics antibiotic sensitivity tests were conducted on antimicrobial drugs commonly used for treatment of bacteriuria, such as ciprofloxacin, norfloxacin, nalidixate, tetracycline, ampicillin, and chloramphenicol. the sensitivity tests were performed according to the kirby-bauer disk diffusion method and the minimal inhibitory concentration (mic) were assessed using the national committee for clinical laboratory standards. (8,9) statistical methods sample size based on a study by achmad(6) on uti, it was predicted that a final sample of 260 participants would be required to have a power of 80% at p<0.05 for detecting a 50% reduction in the i n c i d e n c e o f s u b j e c t s h a v i n g a t l e a s t o n e episode of uti in the mmn group. figure 1. participant recruitment and follow-up 29 statistical analysis data were entered onto excel database and analyzed using spss version 15.0. full statistical analysis was performed prior to breaking the treatment code. between-group comparisons were made using the unpaired t-test for normally distributed variables. categorical variables were compared using the chi squared test. results figure 1 outlines the results of the trial selection process. a total of 261 elderly were enrolled and randomized into mmn (n=135) and control (n=126) groups. participant characteristics table 1 summarizes the characteristics o f t h e s t u d y p a r t i c i p a n t s a t b a s e l i n e . participants were similar at baseline with no s i g n i f i c a n t d i ff e r e n c e s b e t w e e n t h e t w o groups (table 1). the overall sample was predominately female (62.8%), and between the ages of 60 and 78 years. a total of 19.5% (53/261) had at least one uti, consisting of 32/135 (23.7%) subjects in the mmn group and 21/126 (16.7%) in the control group, but the numbers were not significantly different ( p = 0 . 1 5 8 ) . e . c o l i w a s t h e m o s t c o m m o n microorganism, being isolated in 20.7% of the mmn group and in 17.5% of the control group, b u t t h e d i f f e r e n c e i n f r e q u e n c i e s w a s n o t s t a t i s t i c a l l y s i g n i f i c a n t ( p = 0 . 1 8 1 ) . a n o t h e r isolated microorganism was klebsiella, found only in the mmn group (2.2%). antimicrobial sensitivity tests at baseline t h e r e s u l t s o f s e v e r a l a n t i m i c r o b i a l sensitivity tests on 50 e.coli isolates indicated that there were no significant differences in sensitivity tests between the mmn and control g r o u p s f o r c i p r o f l o x a c i n , n o r f l o x a c i n , tetracycline, ampicillin and chloramphenicol (p > 0.05). however, antimicrobial sensitivity tests f o r n a l i d i x a t e o n e . c o l i i s o l a t e s s h o w e d a significant difference between the mmn and control groups, antimicrobial resistance being higher in the control group compared with the mmn group (p = 0.037) (table 2). urinary tract infections after supplementation a f t e r 6 m o n t h s o f m u l t i m i c r o n u t r i e n t supplementation, the proportions of the elderly with uti were 12.6% in the intervention group and 14.3% in the control group, the difference table 1. baseline demographic characteristics and urinary tract infections in the elderly according to study group at baseline univ med vol.28 no.1 30 boekitwetan, surjawidjaja, aidilfit, et al asymptomatic urinary tract infections being statistically not significant (p=0.719). however, when compared with baseline values, uti in the mmn group after supplementation was reduced by 40.6% (11.1%/27.3%), whilst in the control group the reduction in uti was only 14.4% (2.4%/16.7%). the proportions of e . c o l i a n d k l e b s i e l l a a f t e r 6 m o n t h s o f supplementation were respectively 7.4% and 4.4% in the intervention group and respectively 12.7% and 0.8% in the control group, these figures being statistically also not significant (p=0.078). the proportion of e.coli was also decreased by 64.3%% {(20.7% 7.4%)/20.7%} in the intervention group after 6 months of supplementation, compared with the proportion at baseline. in the control group the proprtion of e.coli decreased by 37.8%{(17.5% 12.7%)/ 17.5%}. tabel 2. antimicrobial sensitivity tests on 50 e.coli isolates in the elderly by study group at baseline table 3. urinary tract infection in the elderly after 6 months of supplementation by study group 31 antimicrobial sensitivity tests on e.coli after supplementation sensitivity tests for several antimicrobials o n e . c o l i a f t e r 6 m o n t h s o f m m n supplementation indicated that e. coli was sensitive (100%) to ciprofloxacin, norfloxacin a n d n a l i d i x a t e i n t h e i n t e r v e n t i o n g r o u p , compared with the control group, where 87.5% 93.8% of the bacteria was sensitive. however, the difference was statistically not significant (p> 0.05) (table 4). discussion infectious diseases are a common cause of increased morbidity and mortality in the elderly people. a number of health factors contribute to genitourinary infections. comorbid diseases, functional status, and living environments each play a role in a patient’s susceptibility to i n f e c t i o n , a n d e a c h m a y c o m p l i c a t e a n otherwise simple urinary infection. among healthy elderly patients, these infections are usually benign; however, in patients with significant comorbidities, utis can ultimately l e a d t o m o r e s e r i o u s c o m p l i c a t i o n s . ( 1 2 , 1 3 ) infections in the elderly are quite different from infections in a younger population. these differences are due to age-related alterations in immunity. there are multiple reasons for higher rates of infection when compared with younger patients. anatomic variations during the aging process (such as changes in prostatic function in men and changes in vaginal flora associated with menopause in women) increase the risk of utis.(14) incidence and bacterial spectrum depends on the site of infection and whether the patient is hospitalized, living in a nursing home or in the community. in this study, of 261 elderly in the community 19.5% had uti. our results showed a similar uti prevalence when compared with a study in 520 elderly people in table 4. antimicrobial sensitivity tests on 25 e.coli isolates in elderly by study group after supplementation univ med vol.28 no.1 32 boekitwetan, surjawidjaja, aidilfit, et al asymptomatic urinary tract infections nursing home at iran, where the uti prevalence was 15.5%.(1) on the other hand, our study showed a higher uti rate than that found in a study of 262 nursing home residents, where the uti rate was 3.4%.(15) the reason for these differing figures may lie in the fact that in the present study the number of females was higher than that of males, and that males are less prone to uti, possibly because of their longer urethra and the presence of antimicrobial substances in prostatic fluids.(16) most uti are asymptomatic, which is remarkable in view of the prevalence of bacteriuria in 15% 50% of all residents of long-term care facilities.(17) diagnosis of uti in confused elderly patients can be difficult because of the nonspecific and frequently misleading symptoms and signs, and is therefore primarily based on microscopic examination of urine.(18) a study o f i n s t i t u t i o n a l i z e d e l d e r l y p e r s o n s demonstrating the benefits by means of clinical end points suggested that trace minerals, rather than vitamins, may be the key nutritional factor for preventing infection in older adults.(18) e s c h e r i c h i a c o l i w a s t h e c o m m o n e s t o r g a n i s m i s o l a t e d i n o u r s t u d y, a n d i s recognised as the most common urinary tract p a t h o g e n i n e l d e r l y p e o p l e . ( 1 0 ) we f o u n d significantly fewer infections with e. coli in the multi-micronutrient group than in the control group. an important reason for evaluating the effectiveness of multimicronutrients is because of their potential to improve immune responses to infection and thus prevent illness. multimicronutrient supplementation for older people increased their in-vitro lymphocyte response to mitogens.(20) the use of antimicrobials for treatment of urinary infection is a part of the larger concern about appropriate antimicrobial use in the elderly and the impacts of the selective pressure of antimicrobials on colonization and infection with resistant organisms. treatment of urinary i n f e c t i o n s i n e l d e r l y p a t i e n t s s h o u l d b e conservative when compared with younger, healthier individuals. higher rates of failure and relapse are more often associated with advanced age. fluoroquinolones are the preferred firstline treatment when the pathogen is not known, as they have a wider spectrum of coverage and g r e a t e r p e n e t r a t i o n o f t h e p r o s t a t e g l a n d compared with other antibiotics.(11) in our study, a f t e r s i x m o n t h s o f m u l t i m i c r o n u t r i e n t supplementation antimicrobial sensitivity tests showed that e. coli was 100% sensitive to ciprofloxacin and norfloxacin compared with the control group (sensitivity 87.5% – 93.8%), which figures were however not significantly d i ff e r e n t . t h e o c c u r r e n c e o f i n t e r a c t i o n s between host and numerous potential pathogens should be further investigated using a larger number of e. coli isolates. it should be of interest to explore whether multi-micronutrient supplementation might not be able to affect the h o s t p a t h o g e n b a l a n c e , i m p a c t i n g o n antimicrobial sensitivity of the pathogens.(21) numerous micronutrients play a special role in human immune function. should escherichia coli after micronutrient supplementation of the h o s t b e c o m e m o r e s e n s i t i v e t o t h e fluoroquinolones, by what amount, and how effective would the supplementation be. we p r o p o s e a m o r e i n t e g r a t e d a p p r o a c h t o investigate the actions of micronutrients in human beings on the interaction between host and pathogen biology and genomics. conclusions a multi-micronutrient supplement reduced the incidence of uti by 40.6% in the mmn group and only by 14.4% in the control group. infections with e. coli were less frequent in the multimicronutrient group than in the control group after six months of supplementation. 33 references 1. badan koordinasi keluarga berencana nasional [national coordinating board of family planning]. data keluarga penduduk indonesia. jakarta: badan koordinasi keluarga berencana nasional. 2004. available at http://www.bkkbn.go.id. accessed august 10, 2008. 2. united nations. world population aging 1950-2050. new york: united nations; 2001. available at http:/ / w w w. u n . o rg / e s a / p o p u l a t i o n / p u b l i c a t i o n s / worldageing19502050/pdf/preface_web.pdf. accessed august 12, 2008. 3. high kp. nutritional strategies to boost immunity and prevent infection in elderly individuals. clin infect dis 2001; 33: 1892-900. 4. castle sc. clinical relevance of age-related immune dysfunction. clin infect dis 2000; 31: 578–85. 5. achmad ia, birowo p. profil pasien infeksi saluran kemih usia lanjut di rumah sakit cipto mangunkusumo jakarta [profile of urinary tract infection in elderly patients]. maj kedokter indon 2006; 56: 114-7. 6. mcmurdo et, bissett ly, price rjg, phillips g, crombie ik. does ingestion of cranberry juice reduce symptomatic urinary tract infections in older people in hospital? a double-blind, placebocontrolled trial. age ageing 2005; 34: 256–61. 7. williams me. treatment of uti in an elderly male. medscape fam med 2002; 4: 2-6. 8. trotter cl, stuart jm, george r. increasing hospital admissions for pneumonia, england. emerg infect dis 2008; 14: 727–33. 9. lutters m, vogt-ferrier nb. antibiotic duration for treating uncomplicated, symptomatic lower urinary tract infections in elderly women. cochrane database of systematic reviews 2008, issue 3. art. no.: cd001535. doi: 10.1002/14651858. cd001535. pub2. 10. thomson rb, miller jm. specimen collection, transport, and processing: bacteriology in: murray p, baron ej, jorgensen jh, pfaller ma, yolken rh, editors. manual of clinical microbiology. 8th ed. washington dc: asm press; 2003. p. 286-330. 11. noviana h. antibiotic susceptibility of escherichia coli isolated from various clinical specimens. j kedokter trisakti 2004; 23: 122-6. 12. adedipe a, lowenstein r. infectious emergencies in the elderly. emerg med clin n am 2006; 24: 433-48. 13. ginde aa, rhee sh, katz ed. predictors in outcome in geriatric patients with urinary tract infections. j emerg med 2004; 27: 101–8. 14. shortliffe ldm, mccue jd. urinary tract infections at the age extremes: pediatrics and geriatrics. am j med 2002; 113: 55s–66s. 15. bucher a, sorkness n, lundqvist k, ronning k. infection and use of antibiotics in nursing home. tidsskr nor laegeforen 2001; 121: 827–30. 16. nowroozi j, mirgalili as, bagheri kp. study on nutrition status and urinary tract infection in elderly people at nursing home. iranian j publ health 2004; 33: 36-9. 17. nicole le. urinary tract infection in long-term facility residents. clin infect dis 2000; 31: 757-61. 18. rao gg, patel m. urinary tract infection in hospitalized elderly patients in the united kingdom: the importance of making an accurate diagnosis in the post broad-spectrum antibiotic era. j antimicrob chemother 2009; 63: 5–6. 19. high kp. nutritional strategies to boost immunity and prevent infection in elderly individuals. clin infect dis 2001; 33: 1892–900. 20. barringer ta, kirk jk, santaniello ac, foley kl, michielutte r. effect of a multivitamin and mineral supplement on infection and quality of life. ann int med 2003; 138: 365-71. 21. prentice am, ghattas h, cox se. host-pathogen interactions: can micronutrient tip the balance?. j nutr 2007; 137: 1334-7. univ med vol.28 no.1 waist1 77 waist circumference as a predictor for blood glucose levels in adults may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina shinta l hardiman*a, intan nevita bernanthus*, puspa k rustati* and eva susiyanti** *medical profession study program, medical faculty, trisakti university **distric health center, cipete selatan, south jakarta correspondence adr. shinta l hardiman medical profession study program, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta univ med 2009;28:77-82 abstract anthropometric indexes such as body mass index (bmi), waist circumference (wc), hip ciucumference (hc), and waist–hip ratio (whr), are all useful anthropometric measurements to provide important information on blood glucose concentrations. the aim of this study was to determine different anthropometric measurements, in particular bmi, waist circumference, hip circumference and waist-to-hip ratio, in their ability to predict the blood glucose levels in men and women 40 to 60. a cross-sectional study was conducted on a sample of 44 men and 127 women aged 40 to 50 who lived in cipete selatan subdistrict, south jakarta. blood glucose levels was assessed and anthropometric measurements comprising bmi, wc, hc, whr were collected. multiple linear regression analysis was used to determine the best predictor for blood glucose levels. the study showed that the prevalence of dm type 2 was 25.7% and the prevalence was higher in men (40.9%) compared to women (23.5%). the significant predictive variables in the simple regression analysis were age and waist circumference. multiple linear regression showed that after adjustment for age, wc was positively associated with blood glucose levels. standardized a value was 0.172 (p=0.026). wc predict blood glucose levels, beyond that explained by traditional diabetic risk factors and bmi. these findings provide support for the recommendation that wc be a routine measure for identification of diabetes mellitus type 2 in men and women aged 40 to 60 years. keywords: anthropometric indexes, blood glucose levels, diabetes mellitus type 2 introduction the increase in the number of diabetes mellitus (dm) type 2 patients all over the world have been predicted by the experts. (1) the prevalence rate of dm in the developing world, e s p e c i a l l y i n t h e a s i a p a c i f i c r e g i o n , increasingly compared with other countries. in thailand the increase in the prevalence of dm type 2 among the population aged ≤35 was 9.6%, 78 hardiman, bernanthus, rustati, et al waist circumference for blood glucose an increase of 20% over a period of 5 years.(2) according to the survey that was carried out by the world health organization (who) in the year 2000, indonesia occupied the 4th place in the number of dm patients in the world after india, china and the united states. during 2000, the prevalence of the dm was 8.4 million and in 2030 will be increased to 21.3 million.(1) whereas data from the indonesian department of health showed that the number of dm inpatients and outpatients treated in hospitals occupied the first place from all over endocrine disorders.(3) in developing countries the highest prevalence of dm type 2 was in people 45 to 65 years of age.(4) the complications that could result from dm, i n c l u d i n g c o r o n a r y h e a r t d i s e a s e ( c h d ) , necessitate larger expenditures for medical treatment. therefore, early diagnosis and prevention of dm are very important to prevent chd as a complication after the onset of diabetes. several factors that could increase the incidence of dm type 2 are sedentary habits, obesity, excessive consumption of polyunsaturated fats and refined sugars, and smoking. several studies showed that obesity was correlated with the increase in the p r e v a l e n c e o f h y p e r t e n s i o n , d m a n d dyslipidemia.(5) the importance of obesity as a risk factor for type 2 diabetes and hypertension has been well recognized, but its role as a coronary heart disease (chd) risk factor in nondiabetic, normotensive individuals has been less well established.(6) anthropometric indices, s u c h a s b o d y m a s s i n d e x ( b m i ) , w a i s t circumference (wc), and waist-hip ratio (whr), are anthropometric measurements that could give risk information on dm and chd.(7) the who and the national institute of health have defined bmi, wc, and whr cut-off levels for white, black and hispanic american adults; however, these definitions cannot be readily applied to other populations.(8,9) it is beneficial to healthcare to assess which anthropometric measurements are associated with the presence of dm in different populations. although it is argued that there is no justification for general population screening, early detection of individuals at risk of diabetes could be beneficial because early intervention has the potential to prevent the development of diabetes and its complications. the identification of individuals at risk of diabetes and treatment of risk factors is therefore relevant to prevent cardiovascular disease and mortality in addition to diabetes.(10) the aim of this study was to determine which anthropometric measurements, in particular bmi, wc, and whr, were able to predict the risk of type 2 diabetes in people 40 to 60 years of age. methods research design we carried out a cross-sectional study during october to november 2007 among people 40 to 60 years of age. study subjects the subjects of the study were men and women 40 to 60 years of age who lived in cipete selatan subdistrict. the inclusion criteria were age 40 to 60 years and no history of diabetes. after exclusion of participants with any history of severe diseases (cancer, liver cirrhosis), mental retardation and lack of communication, data were collected on subject characteristics, such as age, gender, level of education, exercise and occupation. measurements the anthropometric measurements used to calculate bmi, using standardized procedures, were height and weight. height was measured using a portable microtoise to the nearest 0.1 cm and weight was measured using sage portable scales to the nearest 0.1 kg. bmi was calculated as the weight (kg) divided by the square of the 79 univ med vol.28 no.2 height (m). wc was taken as the minimum circumference between the umbilicus and xiphoid process and measured to the nearest 0.5 cm. hip circumference (hc) was measured as the maximum circumference around the buttocks posteriorly and the symphysis pubis anteriorly and measured to the nearest 0.5 cm. then whr was calculated from wc and hc. blood was taken at any time for measurement of plasma glucose (pg). blood glucose level was measured using glucotest and glucostrip. the american diabetes association criteria were used to diagnose diabetes.(11) diabetes was defined by the presence of classic dm symptoms and a casual plasma glucose level of > 200 mg/dl (11.1 mmol/l). statistical analysis differences between men and women were e x a m i n e d b y st u d e n t ’s t t e s t . to t e s t t h e contribution of age, wc, hp, bmi, whr on c a s u a l b l o o d g l u c o s e c o n c e n t r a t i o n s a s dependent variable we used a single regression analysis. furthermore a multiple step-wise regression analysis, including all variables that were significantly (p<0.05) associated with c a s u a l b l o o d g l u c o s e l e v e l s , w a s u s e d t o determine the best predictor of casual blood glucose concentrations. all analyses were performed using the spss/pc statistical program (version 11.0 for windows; spss, inc.chicago, il). results the subjects consisted of 127 (74.3%) women and 44 (25.7%) men. the prevalence of dm type 2 was 25.7%. the age, anthropometric indices, blood glucose levels and prevalence of type 2 dm of the subjects are summarized in table 1. the means of height and weight were significantly higher in men compared to women. in men, wc (88.3 ± 10.8 cm) was statistically significantly higher compared with women (83.5 ± 9.4 cm) (p=0.005). hc and whr differences were also statistically significant between men and women, but the difference in blood glucose levels was not statistically significant (p=0.197). table 2 presents the results of the simple linear regression analysis in which several anthropometric indices were used to predict the blood glucose levels. the analysis showed that age and wc had a statistically significant positive association with casual blood glucose levels, whereas bmi, hc, and whr were statistically not significantly associated with anthropometry index men (n=44) women (n=127) p value age (yr) 51.3 (6.3) 47.6 (6.1) 0.001 height (cm) 162.8 (7.0) 151.8 (5.1) 0.000 weight (kg) 77.6 (10.3) 59.1 (10.1) 0.000 bmi * 25.2 (3.3) 25.7 (4.1) 0.393 wc (cm) ** 88.3 (10.8) 83.5 (9.4) 0.005 wh ratio *** 0.98 (0.06) 0.93 (0.04) 0.000 hc (cm) **** 92.6 (9.4) 96.2 (9.8) 0.041 blood glucose level (mg/dl) 136.6 (70.4) 120.8 (69.6) 0.197 dm § 40.9% 23.5% 0.081 table 1. anthropometry and prevalence of type 2 dm in the studied population *bmi = body mass index; **wc = waist circumference; ***whr = waist to hip ratio; **** hc = hip circumference; §dm = diabetes mellitus type 2; values are expressed as mean and standard deviation (in parentheses). 80 hardiman, bernanthus, rustati, et al waist circumference for blood glucose predictors b p age 1.684 0.048 bmi* 0.383 0.827 wc** 1.351 0.011 hc*** 0.491 0.303 whr**** 103.371 0.345 table 2. simple regression analysis for predictors of blood glucose levels in men and women *bmi = body mass index; **wc = waist circumverence; ****whr = waist to hip ratio; *** hc = hip circumference table 3. multiple linear regression model for age and wc on blood glucose levels model â standardized â p aged 1.348 0.121 0.115 wc* 1.202 0.152 0.026 *wc = waist circumference casual blood glucose levels. multiple linear regression models showed that wc was the best predictor for casual blood glucose levels among men and women 40 to 60 years of age. wc had a statistically significant positive association with casual blood glucose levels (p=0.026) (table 3). discussion in the present study the prevalence of dm type 2 was 25.7% among men and women 40 to 60 years of age. this result was similar with that of a survey in mexico, where the prevalence of dm type 2 among men and women over 20 was 26.6%.(12) however, the prevalence of dm in our study was higher than that found in the american survey (dm 7.8% in both genders), probably because in the latter the data were analyzed based on age groups and not on gender, whereas in our study the analysis was on gender and not on age groups.(13) the primary finding of our study is that wc significantly predicts the levels of blood glucose. the study by janiszewski et al(14) of approximately 3,000 men and 2,800 women showed similar results. after controlling for age, sex, race, smoking status, cardiometabolic risk factors, and bmi, patients with wc in the medium and high categories had a twofold and fivefold increased risk of diabetes, respectively. schulze e t a l . s h o w e d t h a t w c h a d t h e s t r o n g e s t association of single anthropometric measures for type 2 dm.(15) several previous cohort studies(1619) that compared different anthropometric measurements with regard to diabetes risk p r e d i c t i o n s u g g e s t t h a t a n t h r o p o m e t r i c m e a s u r e m e n t s t h a t d e s c r i b e c e n t r a l f a t distribution, in particular wc, may be superior to measurements of general adiposity. a study in central jakarta on men and women 35 to 55 years of age, showed a similar result, namely that wc was the best predictor for screening dm type 2, compared with bmi, waist to height ratio and whr.(20) wc by itself was also strongly related to risk of myocardial infarction, and this association remained significant after adjustment for other risk factors.(21) in the present study, wc significantly predicted the levels of blood glucose, but bmi did not predict diabetes after consideration of common diabetic risk factors and wc. the recent consensus statement of the american diabetes association, the obesity society, and the american society for nutrition questions the sequence of clinical measures of diabetes mellitus and more importantly, the relevance of wc measurement in clinical practice.(22) one of the limitations of this study was that we were unable to include the fasting and 2 hour glucose concentration to determine a better diagnosis for dm type 2. the simplest parameter for identifying those at risk of diabetes is wc, beyond that explained by commonly evaluated 81 univ med vol.28 no.2 diabetic risk factors such as weight, bmi and whr. conclusions we found that among men and women, wc appeared to be the best predictor than any other single direct measure. wc was a better predictor of diabetes occurrence than bmi among men and w o m e n . g e n e r a l l y, m e a s u r e m e n t o f anthropometric characteristics beyond wc had little predictive information. wc can be used for early intervention to delay or prevent type 2 dm. acknowledgement we hereby wish to extend our heartfelt gratitude to all men and women who graciously participated in this study, to drg. eva susiyanti as head of cipete selatan health center and to all staff of the health center, for their support of this study. and last but not least, we thank prof. dr. dr. adi hidayat, ms for editing and revising the manuscript. references 1. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care 2004;27: 1047-53. 2 aekplakorn w, stolk rp, neal b, suriyawongpaisal p, chongsuvivawong v, cheepudomwit s, et al. the prevalence and management of diabetes in thai adults: the international collaborative study of cardiovascular disease in asia. diabetes care 2003;26:2758–63. 3. departemen kesehatan republik indonesia. jumlah penderita diabetes indonesia ranking ke-4 di dunia. 5 september 2005. available at: http://www. depkes.go.id/index.php?option=news&task= viewarticle&sid=1183. accessed august 12, 2007. 4. wild s, roglic g., green a. global prevalence of diabetes. diabetes care 2004;27:1047-53. 5. griffin sj, little ps, hales cn, kinmonth al, wareham nj. diabetes risk score: towards earlier detection of type 2 diabetes in general practice. diabetes metab res rev 2000;16:164-71. 6. abbasi f, brown jr bw, lamendola c, mclaughlin t, reaven gm. relationship between obesity, insulin resistance, and coronary heart disease risk. j am coll cardiol 2002;40:937–43. 7. us national institutes of health. clinical guidelines for the identification, evaluation, and treatment of overweight and obesity in adults. national institutes of health: bethesda, md;1998. 8. world health organization. obesity. preventing and managing the global epidemic. report of a who consultation on obesity, geneva, 3 – 5 june. world health organization: geneva;1998. 9 okosun is, liao y, rotimi cn, choi s, cooper rs. predictive values of waist circumference for dyslipidaemia, type 2 diabetes and hypertension in overweight white, black, and hispanic american adults. j clin epidemiol 2000;53:401-8. 10. nijhof n, ter hoeven cl, de jong mdt. determinants of the use of a diabetes risk-screening test. j comm health 2008;33:313-7. 11. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2005;28 (suppl 1):s37-s42. 12. berber a , go´mez-santos r, fanghänel g, sanchezreyes l. anthropometric indexes in the prediction of type 2 diabetes mellitus, hypertension and dyslipidaemia in a mexican population. int j obes 2001;25:1794–9. 13. harris mi, flegal km, cowie cc, eberhardt ms, goldstein de, little rr, et al. prevalence of diabetes, impaired fasting glucose, and impaired glucose tolerance in u.s. adults. third national health and nutrition examination survey, 1988 – 1994. diabetes care 1998;21:518-24. 14. janiszewski pm, janssen i, ross r. does waist circumference predict diabetes and cardiovascular disease beyond commonly evaluated cardiometabolic risk factors? diabetes care 2007;30:3105-9. 15. schulze mb, heidemann c, schienkiewtz a, bergmann mm, hoffmann k, boeing h. comparison of anthropometric characteristics in predicting the incidence of type 2 diabetes in the epic-potsdam study. diabetes care 2006;29:19213. 16. stevens j, couper d, pankow j, folsom ar, duncan bb, nieto fj, et al. sensitivity and specificity of anthropometrics for the prediction of diabetes in a biracial cohort. obes res 2001;9:696-705. 82 hardiman, bernanthus, rustati, et al waist circumference for blood glucose 17. snijder mb, dekker jm, visser m, bouter lm, stehouwer cd, kostense pj, et al. associations of hip and thigh circumferences independent of waist circumference with the incidence of type 2 diabetes: the hoorn study. am j clin nutr 2003;77:1192–7. 18. sargeant la, bennett fi, forrester te, cooper rs, wilks rj. predicting incident diabetes in jamaica: the role of anthropometry. obes res 2002;10:792– 8. 19. castillo s, monroy v. anthropometric cut off points for predicting chronic diseases in the mexican national health survey 2000. obes res 2003;11: 442-51. 20. delima. indeks antropometri sebagai uji diagnostic diabetes mellitus tipe 2. avaiable at: http:// digilib.litbang.depkes.go.id/go.php?id=jkpkbppkgdl-res-2002-delima-1134-diabetes&q=indeks+ antropometri. accessed september 1, 2008. 21. walling ad. waist-to-hip ratio better predictor of disease than bmi. am fam physician 2006; 73: 2046-7. 22. klein s, allison db, heymsfield sb, kelley de, leibel rl, nonas c, et al. waist circumference and cardiometabolic risk: a consensus statement from shaping america’s health: association for weight management and obesity prevention; naaso, the obesity society; the american society for nutrition; and the american diabetes association. diabetes care 2007;30:1647–52. alvina 114 abstract currently nonalcoholic fatty liver disease (nafld) and nonalcoholic steatohepatitis (nash) are medical problems associated with the increasing prevalence of diabetes mellitus, obesity, hypertension and hypertriglyceridemia, usually designated as the metabolic syndrome associated with insulin resistance. one study demonstrated an increase in nafld prevalence of around 17-33% and in nash prevalence of 5.7-16.5%. nafld comprises a range of mild to severe conditions, from simple steatosis to steatohepatitis, hepatic fibrosis and cirrhosis. the diagnosis of hepatic fibrosis is important for prognosis, stratification for treatment, and monitoring of treatment efficacy. ultrasonography (usg) is a simple method for detecting fatty infiltrates in the liver. usg has a sensitivity of 82-89% and a specificity of 93%, but cannot differentiate between hepatic steatosis and fibrosis. the gold standard for evaluation of hepatic fibrosis is liver biopsy, which however is a painful and invasive procedure. currently determination of serum type iv collagen has been suggested as an alternative to liver biopsy among the non-invasive methods for evaluation of hepatic fibrosis, as its serum concentration is closely correlated with advanced hepatic fibrosis in nash. type iv collagen is one of the components of basement membrane and its serum concentration is indicative of degradation of the extracellular matrix. keywords: nafld, nash, type iv collagen, hepatic fibrosis *department of clinical pathology, medical faculty, trisakti university correspondence dr. alvina, sppk department of clinical pathology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2404 email: vina_march_dr@yahoo.com univ med 2010;29:114-24. type iv collagen as marker of fibrosis in nonalcoholic liver disease alvina* may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction nonalcoholic fatty liver disease (nafld) a n d i t s a d v a n c e d s t a g e , n o n a l c o h o l i c steatohepatitis (nash), currently are the subject of heated discussion in the field of m e d i c i n e . t h e i n c r e a s i n g p r e v a l e n c e o f d i a b e t e s , o b e s i t y, h y p e r t e n s i o n , a n d hypertriglyceridemia is considered to be an important cause of nafld.(1) the world health organization (who) has estimated that there were 200 million patients with obesity worldwide in 1995 and 300 million in 2002.(2) on the basis of data published by the nafld/ nash 2006 concensus in cebu, indonesia has the highest percentage of nafld patients in southeast asia, namely 30%, in contrast with 17% for malaysia and 5% for singapore.(3) nafld comprises a range of conditions, from simple steatosis to steatohepatitis, hepatic fibrosis and cirrhosis(4) and is histologically differentiated into four stages, where stages iii 115 univ med vol.29 no.2 and iv are also known as nash.(1) ultrasonographic imaging (usg) is a simple method for diagnosis of fatty liver, based on visualization of fatty infiltrates in the liver.(5,6) it has a sensitivity of 82%-89% and a s p e c i f i c i t y o f 9 3 % , b u t c a n n o t p r o p e r l y differentiate between steatosis and hepatic fibrosis. for detecting hepatic fibrosis, the gold standard is liver biopsy, which however is an invasive procedure and has its limitations.(5-7) currently several studies are in progress for detecting hepatic fibrosis by means of noninvasive procedures. it has been reported that the serum concentration of type iv collagen may be used as a non-invasive alternative to liver biopsy for evaluation of hepatic fibrosis. epidemiology nafld, a clinical condition that may advance to terminal hepatic disease, is found in 10-24% of the general population of various countries.(4,8) according to population studies based on ultrasound and liver function tests, the prevalence of nafld tends to increase by 17-33%, while that of nash increases with 5.7-16.5%.(9) n a f l d i s a s s o c i a t e d w i t h i n s u l i n resistance and the metabolic syndrome.(10-12) the prevalence of nafld is 50% in diabetic patients and 76% in obese patients.(10) the risk factors associated with nafld are obesity, diabetes mellitus type 2, and hyperlipidemia.(4) approximately 67% of patients with body mass index (bmi) >30 kg/m2 and more than 90% of patients with bmi >39 kg/m2 tend to have s t e a t o s i s . ( 5 ) m a n y n a f l d p a t i e n t s a r e asymptomatic, although a number of patients reportedly suffer from fatigue, malaise, and a s e n s a t i o n o f f u l l n e s s i n t h e r i g h t u p p e r abdominal quadrant. hepatomegaly may be f o u n d i n 5 0 % o f p a t i e n t s o n i n i t i a l examination.(4,5,9) etiology there are several causative factors of fatty liver, such as drugs and metabolic disorders. among the causes of macrovesicular steatosis, which is associated with an imbalance of hepatic nutritional drugs* metabolic or genetic other protein-calorie malnutrition† starvation† total parenteral nutrition† rapid weight loss† gastrointestinal surgery for obesity† glucocorticoids† synthestic estrogens† aspirin‡ calcium-channel blockers† amiodarone§ tamoxifen† tetracycline‡ methotrexate† perhexiline maleate§ valproic acid‡ cocaine‡ antiviral agents zidovudine† didanosine‡ fialuridine‡ lipodystrophy† dysbetalipoproteinemia† weber-christian disease† wolman’s disease§ cholesterol ester storage§ acute fatty liver of pregnancy‡ inflammatory bowel disease† small-bowel diverticulosis with bacterial overgrowth† human immunodeficiency virus infection† environmental hepatotoxins phosphorus ‡ petrochemicals†‡ toxic mushrooms† organic solvents bacillus circus toxins‡  * this is a partial list of agents that produce fatty liver. some drugs produce inflammation as well. the association of fatty liver with calcium-channel blockers and valproic acid is weak, whereas the association with amiodarone is strong. druginduced fatty liver may have no sequelae (e.g., cases caused by glucocorticoids) or can result in cirrhosis (e.g., cases caused by methotrexate and amiodarone). † this factor predorminantly causes macrovesicular steatosis (mostly owing to imbalance in the hepatic synthesis and export of lipids). ‡ this factor predominantly causes microvesicular steatosis (mostly owing to defects in mitochondrial function). § this factor causes hepatic phospholipidosis (mostly owing to the accumulation of phospholipids in lysosomes). table 1. causes of fatty liver disease(4) 116 l i p i d s y n t h e s i s , a r e s u c h a g e n t s a s glucocorticoids, calcium channel blockers, tamoxifen, and methotrexate, and disorders such as lipodystrophy and dysbetalipoproteinemia. in contrast, microvesicular steatosis is mostly the result of mitochondrial functional defects, e.g. t h o s e c a u s e d b y v a l p r o i c a c i d , a s p i r i n , tetracycline, and acute fatty liver of pregnancy. wo l m a n ’s d i s e a s e c a u s e s h e p a t i c phospholipidosis due to accumulation of phospholipids in lysosomes. (table 1)(4) the metabolic syndrome, associated with insulin resistance (ir), is a collection of symptoms comprising diabetes, central obesity, h y p e r t e n s i o n a n d d y s l i p i d e m i a . ( 1 3 ) i r i s associated with increased production of free fatty acids in the liver from glucose; in addition adipose tissue is also a source of tnf-α which is a regulator of insulin sensitivity. tnf-α presumably increases insulin resistance by d e c r e a s i n g r e g u l a t i o n o f p e r o x i s o m e p r o l i f e r a t o ra c t i v a t e d r e c e p t o r g a m m a (pparγ), which is the most important receptor for maintaining insulin sensitivity within normal limits.(10,14) in obese patients there is an increased hepatic uptake of free fatty acids, which s u b s e q u e n t l y u n d e rg o β o x i d a t i o n o r e s t e r i f i c a t i o n w i t h g l y c e r o l , f o r m i n g t r i g l y c e r i d e s t h a t p l a y a r o l e i n t h e accumulation of fat in the liver.(15) nafld classification nafld may be classified into four histopathological stages, with stage i being characterized by fatty infiltration of the liver, table 2. grading and staging the histopathological lesions of nonalcoholic fatty liver disease(4) grading for steatosis grade 1: <33% of hepatocytes affected grade 2: 33% to 66% of hepatocytes affected grade 3: >66% of hepatocytes affected grading for steatohepatitis grade 1, mild steatosis: predominantly macrovesicular, involves up to 66% of lobules ballooning: occasionally observed; zone 3 hepatocytes lobular inflammation: scattered and mild acute inflammation (polymorphonuclear cells) and occasional chronic inflammation (mononuclear cells) portal inflammation: none or mild grade 2, moderate steatosis: any degree; usually mixed macrovesicular and microvesicular ballooning: obvious and present in zone 3 lobular inflammation: polymorphonuclear cells may be noted in association with ballooned hepatocytes; pericellular fibrosis; mild chronic inflammation may be seen portal inflammation: mild to moderate grade 3, severe steatosis: typically involves >66% of lobules (panacinar); commonly mixed steatosis ballooning: predominantly zone 3; marked lobular inflammation: scattered acute and chronic inflammation; polymorphonuclear cells may be concentrated in zone 3 areas of ballooning and perisinusoidal fibrosis portal inflammation: mild to moderate staging for fibrosis stage 1: zone 3 perivenular, perisinusoidal, or pericellular fibrosis; focal or extensive stage 2: as above, with focal or extensive periportal fibrosis stage 3: bridging fibrosis, focal or extensive stage 4: cirrhosis alvina type iv collagen in liver disease 117 univ med vol.29 no.2 stage ii by fatty infiltration with inflammation, stage iii by fatty infiltration with ballooning degeneration, and stage iv by fatty infiltration with lesions similar to alcoholic hepatitis and sinusoidal fibrosis and by polymorphonuclear infiltrates with or without mallory’s hyaline. nafld stages iii and iv are known as nash.(1) the term nash was first introduced by ludwig to describe the lesions of alcoholic steatohepatitis (ash) in patients who had no history of regular alcohol consumption and did not have other liver diseases.(14) the grading and staging for steatosis, steatohepatitis, and hepatic fibrosis are based on histopathological criteria. (table 2)(4) pathogenesis of nafld fatty acids in the liver are normally esterified into triglycerides, some of which are excreted by the hepatocytes in the form of very low density lipoproteins (vldl). the elevated level of lipids, particularly triglycerides, in the hepatocytes of patients with nafld is the r e s u l t o f a n i m b a l a n c e b e t w e e n e n z y m e systems that promote the uptake and synthesis of fatty acids and those that promote the oxidation and export of fatty acids. (figure 1)(4) ir also plays an important role in the accumulation of lipids in hepatocytes, via two pathways, i.e. lipolysis (which increases circulating fatty acids), and hyperinsulinemia. high fatty acid uptake results in mitochondrial β-oxidation overload leading to fatty acid accumulation within the hepatocytes. fatty acids are substrates and inducers of the microsomal lipoxygenases cytochrome p-450 2e1 and 4a. cytochrome p-450 2e1, the concentration of which is elevated in patients with steatohepatitis, produces free oxygen radicals that are capable of inducing lipid figure 1. pathogenesis of nonalcoholic fatty liver disease through imbalance between enzyme systems promoting uptake and synthesis of fatty acids and those promoting oxidation and export of fatty acids.(4) 118 figure 2. pathogenesis of nonalcoholic fatty liver disease through insulin resistance.(4) figure 3. pathogenesis of development of steatosis into steatohepatitis and fibrosis through lipid peroxidation, cytokine induction, and fas ligand induction.4 alvina type iv collagen in liver disease 119 univ med vol.29 no.2 p e r o x i d a t i o n o f h e p a t o c y t e m e m b r a n e s . h y p e r i n s u l i n e m i a d u e t o i r i n c r e a s e s glycolysis which subsequently increases fatty acid synthesis in hepatocytes and favors the a c c u m u l a t i o n o f t r i g l y c e r i d e s w i t h i n hepatocytes through decreased production of hepatic apolipoprotein b-100. (figure 2)(4) mitochondrial reactive oxygen species p r o m o t e p r o g r e s s o f s t e a t o s i s i n t o s t e a t o h e p a t i t i s a n d f i b r o s i s b y t h r e e mechanisms, viz. lipid peroxidation, cytokine i n d u c t i o n , a n d i n d u c t i o n o f f a s l i g a n d . reactive oxygen species (ros) trigger lipid p e r o x i d a t i o n , l e a d i n g t o c e l l d e a t h a n d production of malondialdehyde (mda) and 4hydroxynonenal (hne). mda and hne cause cell death, are involved in the synthesis of mallory’s hyaline, and activate stellate cells, thus increasing collagen synthesis. ros also induce the production of the cytokines tnfα, transforming growth factor β (tgf-β) and interleukin-8. tgf-β activates stellate cells i n t o s y n t h e s i z i n g c o l l a g e n , w h e r e a s i n t e r l e u k i n 8 i s a c h e m o a t t r a c t a n t f o r neutrophils. mitochondrial reactive oxygen species can activate fas ligand in hepatocytes, which normally express the fas receptor. fas l i g a n d s m a y b i n d t o f a s r e c e p t o r s o n neighboring hepatocytes, thus killing these cells. (figure 3)(4) hepatic fibrosis h e p a t i c f i b r o s i s i s t h e e x c e s s i v e accumulation of extracellular matrix proteins in response to stimuli in acute or chronic liver disease.(16,17) around 15%-40% of patients with nash progress to hepatic fibrosis,(18) in which there occur changes in the number and quality of extracellular matrix proteins,(19) leading to s t r u c t u r a l c h a n g e s i n t h e l i v e r t h r o u g h formation of fibrous scars.(16) pathogenesis of hepatic fibrosis fatty acid accumulation in the cytosol increases oxidation of the fatty acids in peroxisomes and endoplasmic reticulum. peroxisomal oxidation reactions are catalyzed by acyl-coa oxidase (aox) with the formation o f h y d r o g e n p e r o x i d e . o x i d a t i o n i n t h e e n d o p l a s m i c r e t i c u l u m i s c a t a l y z e d b y cytochromes p450 (cyp) 2e1, 4a10, and 4a14. polyunsaturated fatty acids (pufa) are the products of lipid peroxidation by reactive o x y g e n s p e c i e s ( r o s ) , a n d t h e p u fa peroxidation products are trans-4-hydroxy-2nonenal (hne) and malondialdehyde (mda). ros and aldehydes can induce oxidative stress and cause cell death through depletion of atp and nad, damage to dna and protein, and depletion of glutathione. ros and lipid peroxidation products play a role in the formation of fibrosis through activation of collagen-synthesizing hepatic stellate cells. (figure 4)(20) type iv collagen as noninvasive marker of hepatic fibrosis liver biopsy is the gold standard for determination of the severity of hepatic inflammation and fibrosis, while possessing limitations such as being painful and invasive to the patient.(19,21) in large studies, pain is a significant issue in 20% of cases and severe complications have been reported to occur in 0.57%.(22) the biopsy may represent only 1/50 000th of the liver, and sampling error has been shown to be an issue in a variety of liver diseases, including nafld.(23) all staging systems in widespread use share common failings that have been discussed at length.(24,25) currently evaluation of hepatic fibrosis may be made through noninvasive methods, such as serology, imaging and genetics.(19,26) noninvasive markers of liver fibrosis have been most extensively studied in the context of hepatitis c. there has been considerable interest in extending this work into the field o f n a f l d b e c a u s e o f t h e i n c r e a s i n g p r e v a l e n c e o f d i s e a s e . t h e p r e s u m p t i v e diagnosis of nafld is rapidly becoming the commonest cause for referral to hepatology outpatient clinics.(27) markers of fibrosis in the 120 blood may be divided into two types, i.e. direct markers that represent transformation of extracellular matrix and indirect markers that represent changes in hepatic functions. direct serologic markers of fibrosis include those a s s o c i a t e d w i t h m a t r i x d e p o s i t i o n — e .g . procollagen type iii amino-terminal peptide (p3np), type i and iv collagens, laminin, hyaluronic acid, and chondrex.(28) type iv collagen is a useful marker of hepatic fibrosis because its concentration is a s s o c i a t e d w i t h t h e o c c u r r e n c e o f hepatocellular decline and dysfunction, it plays a role in hepatocellular regeneration and r e p a i r a n d i t i s t h e f i r s t c o l l a g e n t o b e synthesized by hepatocytes in experimental hepatic injury.(29) ty p e i v c o l l a g e n i s c o m p o s e d o f polypeptide α chains and is divided into 3 domains, i.e. the amino terminal 7s domain, the triple helical domain and the carboxy terminal globular non-collagenous (nc)-1 domain. the nc-1 domain is thought to play a role in the formation of the trimeric structure of type iv collagen.(30) type iv collagen is one of the main components of the basement membrane in addition to laminin (ln), heparan sulfate proteoglycan and nidogen/entactin, and is the principal component of all basement membrane proteins. type iv collagen is capable of forming networks with itself and is thus also called network forming collagen; this property differentiates type iv collagen from other collagen types.(30) type iv collagen is one of the essential components of the extracellular matrix, as it does not undergo proteolysis and i s d e p o s i t e d i n t a c t i n t h e m a t r i x . t h e concentration of type iv collagen is indicative of matrix degradation. type iv collagen is positively correlated with the stage of fibrosis in patients with chronic hepatitis. patients with advanced hepatic fibrosis have elevated type iv collagen concentrations.(19,31) the study by yoneda et al. found that 7s collagen concentrations were increased in nash patients with advanced fibrosis in comparison to nash patients with moderate fibrosis.(31) kubo et al. reported that in cases of hepatocellular carcinoma preoperative s e r u m 7 s c o l l a g e n c o n c e n t r a t i o n s w e r e correlated with post-operative hepatic failure and that a collagen concentration of > 12 ng/ m l w a s a r e l a t i v e c o n t r a i n d i c a t i o n f o r figure 4. mechanism of lipid-induced hepatic cell damage in nafld.(20) alvina type iv collagen in liver disease 121 univ med vol.29 no.2 resection of the liver.(32) the study conducted by sakugawa et al. also revealed that 7s collagen as well as hyaluronic acid (ha) may be used for differentiating nash from simple fatty liver or patients with advanced fibrosis from those without advanced fibrosis.(33) in other study, serum levels of ha, ln, and type iv collagen increased significantly in hepatic fibrosis, suggesting that detection of ha, ln, and type iv collagen level is an optimal choice.(34) although various types of collagen like type i, iii, iv, v and vi increase proportionally in the liver with the progression of fibrosis, type iv collagen, a constituent of t h e b a s e m e n t m e m b r a n e , i s p a r t i c u l a r l y important and noteworthy for the following r e a s o n s : t o r e l a t e t h e a g g r a v a t i o n o f hepatocellular damage and hepatocellular dysfunction, to play an important role in h e p a t o c e l l u l a r r e g e n e r a t i o n a n d t h e rearrangement of the lobular architecture and for being the earliest type of collagen to be synthesized by hepatocytes in experimental liver injuries.(35) similar reports of elevated type iv collagen levels in serum were reported by dos santos et al.(36) one of the reasons why the field of non-invasive markers for nafld may lag behind hepatitis c is because of the uncertainty of which end point to measure. the current field is divided into studies attempting to distinguish between stages of fibrosis, fibrosis from nash, and nash from simple steatosis. recently, investigators have published a panel test of noninvasive serum markers to diagnose steatosis alone within nafld.(37) the arguments of whether this is superior to ultrasound aside it highlights the uncertainty and open debate of which stages of nafld require diagnosis. conclusions nafld and its advanced stage nash can s t i l l p r o g r e s s t o f i b r o s i s a n d c i r r h o s i s . determination of the stage of hepatic fibrosis is performed by means of liver biopsy as the gold standard. type iv collagen level is highly correlated with stage of hepatic fibrosis and its determination may presumably be utilized for evaluation of hepatic fibrosis in patients with nash. references 1. das sk, mukherjee s, vasudevan d. nonalcoholic fatty liver disease: an underrecognized cause with emerging importance. current science 2006;90:659-65. 2. basaranoglu m, neuschwander-tetri ba. nonalcoholic fatty liver disease: clinical features and pathogenesis. gastroenterol hepatol 2006;2: 282-9. 3. amarapurkar d. how common is nafld in asia pacific and is there local differences. the consensus on nafld/nash. cebu; 2006. 4. angulo p. nonalcoholic fatty liver disease. n engl j med 2002;346:1221-31. 5. bayard m, holt j, boroughs e. nonalcoholic fatty liver disease. am fam physician 2006;73:19618. 6. lupsor m, badea r. imaging diagnosis and quantification of hepatic steatosis: is it an accepted alternative to needle biopsy? rom j gastroenterol 2005;14:419-25. 7. farrell gc, larter cz. nonalcoholic fatty liver disease: from steatosis to cirrhosis. hepatology 2006;43:s99-112. 8. gani ra. manifestasi klinik dan penatalaksanaan nonalcoholic fatty liver disease (nafld). jakarta: ilmu penyakit dalam fkui; 2002. 9. lesmana la. penyakit perlemakan hati nonalkoholik. dalam: sulaiman a, akbar n, lesmana la, noer s, editors. buku ajar ilmu penyakit hati. ed 1. jakarta: jayabadi; 2007.p. 301-5. 10. adams la, angulo p, lindor kd. nonalcoholic fatty liver disease. jamc 2005;172:899-905. 11. mcavoy n, ferguson j, campbell i, hayes p. nonalcoholic fatty liver disease: natural history, pathogenesis, and treatment. br j diabetes vasc dis 2006;6:251-60. 12. mcavoy n, lockman a, hayes p. nonalcoholic fatty liver disease (nafld): an overview. j r coll physicians edinb 2007;37:9-17. 13. olufadi r, byrne c. clinical and laboratory diagnosis of the metabolic syndrome j clin pathol 2008;61:697-706. 14. oneta cm, dufour jf. nonalcoholic fatty liver disease: treatment options based on pathogenic considerations. swiss med wkly 2002;132:493505. 122 15. feldstein a, werneburg n, canbay a, guicciardi m, bronk s, rydzewski r. free fatty acids promote hepatic lipotoxicity by stimulating tnfalpha expression via a lysosomal pathway. hepatology 2004;40:185-94. 16. bataller r, brenner da. liver fibrosis. j clin invest 2005;115:209-18. 17. tiniakos d, kittas c. pathology of nonalcoholic fatty liver disease. ann gastroenterol 2005;18: 148-59. 18. tetri ban. fatty liver and nonalcoholic steatohepatitis. liver dis 2001;3:47-54. 19. grigorescu m. noninvasive biochemical markers of liver fibrosis. j gastrointestin liver dis 2006;15:149-59. 20. browning jd, horton jd. molecular mediators of hepatic steatosis and liver injury. j clin invest 2004;114:147-52. 21. afdhal nh. biopsy or biomarkers: is there a gold standard for diagnosis of liver fibrosis? clin chem 2004;50:1299-300. 22. cadranel jf, rufat p, degos f. practices of liver biopsy in france: results of a prospective nationwide survey. for the group of epidemiology of the french association for the study of the liver (afef). hepatology 2000;32: 477–81. 23. ratziu v, charlotte f, heurtier a. sampling variability of liver biopsy in nonalcoholic fatty liver disease. gastroenterology 2005;128:1898– 906. 24. scheuer pj. assessment of liver biopsies in chronic hepatitis: how is it best done? j hepatol 2003;38:240–2. 25. rosenberg wm. rating fibrosis progression in chronic liver diseases. j hepatol 2003;38:357– 60. 26. king mw. the extracellular matrix. available at: http://www.making@medicine.indstate.edu. accessed april 2, 2010. 27. guha in, parkes j, roderick pr, harris s, rosenberg wm. non-invasive markers associated with liver fibrosis in non-alcoholic fatty liver disease. gut 2006;55:1650–60. 28. carey e, carey wd. noninvasive tests for liver disease, fibrosis, and cirrhosis: is liver biopsy obsolete? cleveland clinic j medicine 2010;77: 519-27. 29. insert package for determination of human type iv collagen in serum panassay iv c (latex). daichi. 2008 30. kalluri r. basement membranes: structure, assembly, and role in tumour angiogenesis. nat rev cancer 2003;3:422-33. 31. yoneda m, mawatari h, fujita k, yonemitsu k, kato s, takahashi h. type iv collagen 7s domain is an independent clinical marker of the severity of fibrosis in patients with nonalcoholic steatohepatitis before the cirrhotic stage. j gastroenterol 2007:375-81. 32. kubo s, tsukamoto t, hirohashi k, tanaka h, shuto t, takemura s. correlation between preoperative serum concentration of type iv collagen 7s domain and hepatic failure following resection of hepatocellular carcinoma. ann surg 2004;239:186-93. 33. sakugawa h, nakayoshi t, kobashigawa k, yamashiro t, maeshiro t, miyagi s, et al. clinical usefulness of biochemical markers of liver fibrosis in patients with nonalcoholic fatty liver disease. world j gastroenterol 2005;11:255-9. 34. li ch, piao dm, xu wx, yin zr, jin js, shen zs. morphological and serum hyaluronic acid, laminin and type iv collagen changes in dimethylnitrosamine-induced hepatic fibrosis of rats. world j gastroenterol 2005;11:7620-4 35. surapaneni km, saraswathi p, shyama s, subramaniam s. type iv collagen: a non invasive bio marker to detect non-alcoholic steato hepatitis (nash), among non-alcoholic fatty liver disease (nafld) patients. j clin diagnos res 2010;4:2483-8. 36. dos santos vm, leite-mór mmb, kondo m, martins jr, nader h, lanzoni vp, et al. serum laminin, type iv collagen and hyaluronan as fibrosis markers in non–alcoholic fatty liver disease. brazilian j med biol res 2005;38:747–53. 37. poynard t, ratziu v, naveau s. the diagnostic value of biomarkers (steatotest) for the prediction of liver steatosis. comp hepatol 2005;4:10. alvina type iv collagen in liver disease non-exposure parenting increases risk of bullying 56 *department of psychiatry, faculty of medicine, atma jaya catholic university **medical profession program, faculty of medicine, atma jaya catholic university of indonesia correspondence : dr. dr. surilena, spkj(k) department of psychiatry, faculty of medicine, atma jaya catholic university of indonesia, jl. pluit raya no. 2, north jakarta, indonesia phone: +6221-6693168 fax: +6221-6606123 email: surilenahasan@yahoo.co.id / surilena@atmajaya.ac.id univ med 2016;35:56-64 doi: 10.18051/univmed.2016.v35.56-64 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 non-exposure parenting increases risk of bullying behavior in junior high school students surilena* and jessica** background bullying behavior is one of many behavioral and disciplinary problems among school students, which has a wide impact on youth, families, schools, and communities. parenting and the role of parents as good educators (exposure) can prevent mental, emotional and behavioral disorders caused by bullying. the aim of this study was to determine the role of self-esteem and family factors on bullying behavior in junior high schools students. methods a cross-sectional study was conducted including 1324 junior high school students of penjaringan village, north jakarta. respondents filled out questionnaires on demographics (age, gender, economic status, educational level), rosenberg self-esteem questionnaire, strength and difficulties questionnaire, olweus bullying questionnaire, hamilton scale for depression, parenting style, and family adaptability and cohesion scales iii. simple and multivariate logistic regression tests were used to analyze the data. results respondents consisted of 53.5% females and 46.5% males, with an age range of 13-16 years. a total of 45% was involved in bullying (victims, perpetrators, and victims and perpetrators). gender, self-esteem, family relationships, and parenting were significantly associated with bullying role (p<0.05). multiple logistic regression analysis showed that self-esteem (odds ratio=23.89; 95% ci:7.899-12.990) and non-exposure parenting (odds ratio=39.11; 95% ci:2.455-8.210) were significantly associated with bullying behavior. conclusions non-exposure parenting was the most relevant risk factor of bullying behavior. low self-esteem increases the risk of bullying behavior. these findings suggest the need of timely bullying prevention and intervention programs that should have a special focus on families of primary high school students. keywords: bullying behavior, family factors, self-esteem, junior high school students doi: http://dx.doi.org/10.18051/univmed.2016.v35.56-64 57 univ med vol. 35 no.1 introduction bullying is a phenomenon that is widespread in the whole world. bullying is a term that sounds foreign to most indonesians, although this phenomenon has been occurring for a long time and in various aspects of life, including the educational world.(1) bullying is a phenomenon characterized by negative acts towards peers, with the intention to hurt, that is performed repeatedly and is accompanied by physical or verbal aggression, or relational harassment by social manipulation and ugly rumors. during the last decades alarming conditions have been found, where more bullying is performed by adolescents, particularly in schools.(2) the considerable number of cases of violence that occur at present among school children is of much concern to educators and parents.(2,3) the school that should be a place for the children to acquire knowledge and to help persons in forming a positive character, turns out to be a place for the fertile growth of bullying practices, thus inspiring fear to children and adolescents.(2,3) the prevalence of bullying is estimated to be up to 50% in several countries in asia, the us and europe. in the phillippines the prevalence is as large as 21.2% and in thailand as large as 10.3% in children aged 11-16 years.(4) the prevalence of bullying in junior high school students varies between countries, being 11% in the us, 14.7% in italy and 27% in the uk.(5,6) a survey in china states that 20% of adolescents experience bullying behavior, while in venezuela around 31.5% of adolescents experience bullying behavior.(4) the prevalence of bullying in state primary school pupils is 89.5%.(7) accurate data on the prevalence of bullying in junior high school students in indonesia are lacking. bullying is behavior that is deleterious to the development of mental and physical health of children or adolescents. bullying behavior should not be tolerated, since it has serious physical, psychological, and psychosomatic effects for all concerned, i.e. the perpetrators (bullies), victims, and bully-victims (aggressive victims, persons who are both victims and bullies), that can persist into adult life. some of the effects that may occur are difficulties in socializing, self-withdrawal, anxiety, depression, suicidal ideation [thinking about or attempting suicide], eating problems, somatic symptoms (decreased appetite, headaches, sleeping problems, abdominal pain, and easy fatiguability, substance abuse, and other mental disturbances.(8,9) van der wal et al.(10) state that with regard to bullying in schools, particularly those who are involved as victim or bully-victim, may feel excluced, have decreased academic achievement, become drop-outs, and be lacking in self-esteem. the factors affecting the occurrence of bullying behavior, particularly in schools, are internal and external. internal factors are age, gender, physical condition, psychological condition, and others.(11) the phenomenon of bullying behavior is like an iceberg that is apparently “small” at the surface, but may have many problems in store that are mostly not easy to discover or to be aware of, by teachers or even parents.(1,2) the community (especially parents and teachers) are frequently misled by the apparent “insignificance” of bullying behavior, so that they ignore the future impact and extraordinary danger towards both victims and bullies, as well as bully-victims, and the widespread impact towards the community.(1,3) there is a need to detect at an early stage the presence of bullying behavior and to know the role of family factors, so as to be able to take early preventive measures against bullying behavior, particularly in schools, and to perform at an early stage interventions on the resulting effects, in their physical as well as mental aspects. therefore this study aimed to determine the role of family factors and self-esteem in bullying behavior among junior high school students. methods study design the design of the study used was crosssectional and the study was conducted from april – september 2015 at a junior high school in the 58 kelurahan (village) of penjaringan, north jakarta. study subjects there were 1336 students of state and private junior high schools in kelurahan penjaringan, north jakarta. the study sample was recruited by consecutive sampling. all male and female students of the i, ii, and iii grades of state and private junior high schools in kelurahan penjaringan, north jakarta, were selected as study subjects. instruments the instruments used in this study were as follows. first, the demographic questionnaire comprising age, gender, educational level, and economic status of the family. second, the rosenberg self-esteem questionnaire comprising 10 questions with 4 categories. self-esteem is high if the total score is >15, while self-esteem is low if the total score is <15. third, the strength and difficulties questionnaire (sdq) comprising 28 questions used to measure behavioral and emotional problems; behavioral problems are positive if the total score is <15 and negative if the total score is >16. fourth, the olweus bullying questionnaire (obq) comprising 39 questions and consisting of 2 parts, i.e. a q u e s t i o n n a i r e a b o u t v i c t i m s o f b u l l y i n g (questions nos. 1-23) and a questionnaire about perpetrators of bullying (questions nos. 24-39); bullying behavior is positive if the respondent selects option c (only 2 or 3 times per month), or d (around once weekly), or e (more than once weekly) and bullying behavior is negative if the respondent selects option a (i did not do that in the last 6 months), or b (once or twice in the last 6 months). fifth, the hamilton rating scale for depresssion (hdrs) comprising 17 questions used to measure depression. the interpretation of the total score is: 0-7= no depression, 8-13= mild depression, 14-18= moderate depression, 19-22= severe depression, and >23= very severe depression. next, the questionnaire on parental nurturing pattern (parenting type) consisting of 26 questions in group a and 6 questions in group b. the parenting pattern involves non-exposure if the total score is <27 and exposure if the total score is >28. finally, the questionnaire on family relations (family adaptibility and cohesion scales iii) consisting of 16 questions with their interpretations. there are 4 types of family cohesion and 4 types of family adaptibility, which are subsequently summarized into 3 types of family relation, i.e. balanced (high), midrange and extreme (low) family relation. statistical analysis the data of this study were analyzed using binary logistic regression analysis for an association of bullying behavior with self-esteem, gender, type of family relation, and parenting pattern. multiple logictic regression analysis was used for finding an association of bullying behavior with self-esteem and parenting pattern. ethical clearance this study obtained ethical clearance from the ethics commission of the faculty of medicine, atma jaya catholic university of indonesia in march 2015. results there were 12 subjects who were not present during the course of the study, so that they were excluded. the participating subjects comprised 1324 persons, among whom 53.5% females, 46.5% males, with an age range of 1316 years, while 91.3% subjects had a middle economic status. at junior high schools, the subjects were mostly in grade i (35.9%), grade ii (32.8%) and grade iii (31.3%). this study also showed that 45.1% of the subjects were involved in bullying behavior, i.e. as victims (18.6%), bullies (13.4%) and bully-victims (13.1%). in contrast, the proportion of subjects who were not involved in bullying behavior was 54.9%. the types of bullying behavior that were encountered were verbal bullying (37.6%), psychological and emotional bullying (29.9%), surilena, jessica non-exposure parenting and bullying behavior 59 univ med vol. 35 no.1 physical bullying (24.7%) and cyber bullying (7.8%) (table 1). the subjects who were involved as victim and bully-victim stated that most of the bullies were classmates (58.5%), bullying behavior occurred more frequently in the classroom (37.2%) and during school recess (43.4%). a total of 24.1% victims and bullyvictims reported the occurrence of bullying to their friends, 12.8% reported to their older or younger siblings, 12.2% to their parents and 6.4% to their teachers (table 1). the results of simple logistic regression analysis showed that the variables of self-esteem, type of family relation, and parenting pattern were significantly associated with bullying role (p<0.050). evaluation of the results of the rosenberg self-esteem questionnaire indicated that 67.9% of subjects with low self-esteem were involved in bullying i.e. 38.3% as victims, 11.1% as bullies and 18.5% as bully-victims. on the other hand, subjects with low self-esteem who were not involved in bullying accounted for 32.1%. simple logistic regression showed that the proportion of self-esteem was significantly different between subjects who were involved and those who were not involved in bullying (victims p=0.011; bullies p=0.021; bully-victims p=0.012). depression in subjects who were involved in bullying was higher (54.7%) than in subjects who were not involved in bullying (45.3%). subjects with depression who were involved in bullying were encountered in the groups of victims (22.5%), bullies (16.3%) and bully-victims (15.9%). depression was not a risk factor of bullying behavior (victims p=0.664; bullies p=0.583; bully-victims p=0.783) (table 2). evaluation of the total score for the strength and difficulties questionnaire (sdq) indicated that 47.5% of subjects involved in bullying had emotional and behavioral problems, of which 18.1% as bullies, 17.6% as victims and 12.1% as bully-victims. simple logistic regression showed that emotion and behavior did not constitute a risk for bullying behavior (victims p=0.293; bullies p=0.272; bully-victims p=0.274). table 1. distribution of characteristics and bullying behavior of study subjects (n=1324) 60 table 2. relationship of psychological and family factors with bullying behavior the majority (56.6%) of study subjects had a midrange type of family relation (adequate cohesion and adaptation), while 41.4% of subjects involved in bullying had an extreme type of family relation (low cohesion and adaptation), with 17.1% victims, 10.8% bullies, and 13.55% bullyvictims. simple logistic regression showed that the proportion of the extreme type of family relation was significantly different between the subjects who were involved and those who were not involved in bullying (victims p=0.022; bullies p=0.020; bully-victims p=0.031). the majority of subjects who were involved in bullying (57.1%) had a non-exposure pattern of parenting (unexpected, inconsistent, permissive, overprotective), while of those who were not involved in bullying, there were more (79.1%) who had an exposure parenting pattern (expected, democratic). simple logistic regression showed that the proportion of non-exposure parenting was significantly different between subjects who were involved and those who were not involved in bullying (victims p=0.020; bullies p=0.011; bullyvictims p=0.010). simple logistic regression found three variables with p<0.25, i.e. self-esteem, family relation, and parenting pattern, that played a role in the occurrence of bullying behavior. these three variables were further analyzed using multiple logistic regression. the results of the latter analysis showed that there were two variables affecting bullying behavior, i.e. selfesteem and parenting pattern. the variable of self-esteem had an odds ratio of 23.89 (95% tabel 3. factors with a role in the occurrence of bullying behavior note: ci= confidence interval to mean 95%; p*= significant association surilena, jessica non-exposure parenting and bullying behavior 61 univ med vol. 35 no.1 ci:7.899-12.990), signifying that subjects with low self-esteem had a 23.89 times greater probability of being involved in bullying behavior, as compared with subjects with high self-esteem. the variable of parenting pattern had an odds ratio of 39.11 (95% ci: 2.4558.210), signifying that subjects with the nonexposure type of parenting (permissive, authoritarian, inconsistent) had a 39.11 times greater probability of being involved in bullying behavior, as compared with the exposure type of parenting (democratic). discussion the prevalence of bullying in this study was found to be 45.1%, with 53.5% females and 41.4% males. females were more involved as victims of bullying (20.3%), while males were more involved in bullying as perpetrators (23.5%). bullying behavior is a worldwide health problem and its prevalence is increasing from year to year.(1) flemming et al.(12) stated that the prevalence of bullying in asia, america and europe was estimated to be 40-80%. the incidence rate of bullying in the present study was essentially similar to that of the study by flemming. data from the national mental health and education center in the us indicate that bullying is a form of violence that generally occurs in the school environment, where 15% of students are the perpetrators of bullying and 30% of students are the victims of bullying.(13) the frequently encountered type of bullying in this study was the verbal type, followed by psychological and emotional, physical, and cyber bullying. the verbal, psychological and emotional types of bullying and cyber bullying were more frequently experienced by female than male subjects. on the other hand, the physical type of bullying was more frequently experienced by male than by female subjects. the most frequently found form of bullying in schools were taunting, gossiping or rumor-spreading, followed by hitting and threatening.(12,13) the study of stein et al.(14) showed that the most frequent type of bullying was the verbal type (taunting, name calling), followed by psychological and emotional bullying (spreading of rumors, social isolation), physical bullying (threatening, hitting), and cyber bullying. the results of this study found that bullying occurred more frequently in the classroom. this finding is in agreement with the study by nordhagen et al.(15) where the classroom, when the teacher was absent, was most frequently used as the location of bullying behavior. interactions between students were most frequent in the playgrounds, hallways, and classrooms, so that bullying was frequently experienced by the victims in those locations. bullying may occur anywhere, particularly in locations with minimal supervision by teachers and school personnel, due to the high teacher-student ratio. this study showed that only around 55.5% of victims and bully-victims reported the bullying episodes that they had experienced to their friends and family members (parents, siblings) and that only 6.4% of both victims and bully-victims reported to their teachers. the unwillingness of the students to report the bullying that they experienced to their teachers may be caused by the lack of confidence of the students in their teachers. incorrect perception, attitude, and acts of teachers towards bullying behavior may result in the low confidence of students in the educational institution and encourage bullying behavior in schools. actually the ability of teachers to encourage student confidence and remove the gap between teachers and students constitutes the foundation for fighting bullying in schools.(15,16) low self-esteem was most frequently found among subjects who were involved in bullying (67.9%) compared with those who were not involved in bullying (32.1%). subjects with low self-esteem who were involved in bullying were most numerous among the victims and bullyvictims. the victim of bullying is a weak person without peer group support. the majority of victims had low self-esteem and were taciturn and shy. our study results agree with those of the study by spade (17) who also used the 62 rosenberg self-esteem questionnaire, stating that adolescents who were involved in bullying behavior, both as victims and bully-victims, had a significant correlation with low self-esteem. spade also stated that adolescents with low selfesteem were at risk both as victims and bullyvictims. in this study the prevalence of depression was most frequently found among victims (22.5%), followed by bullies (16.3%) and bullyvictims (15.9%). the incidence rate of depression in this study among subjects who were involved in bullying differed with those of the study of kaltiala-heino et al.(18) who stated that the prevalence of depression was higher in the bullyvictims (44% females and 23% males) than in the victims (26% females and 16% males). depression may be one of the psychological impacts of bullying behavior or an influential risk factor for the occurrence of bullying behavior. our study did not perform initial screening for depression as impact or risk factor of bullying behavior. this study also showed that the proportion of emotional and behavioral problems among subjects who were involved in bullying as perpetrators (bullies) was 18.1%, as victims 17.6% and as bully-victims 12.1%. our study results differed with those of the study by gini (19) who also used the sdq instrument and found that the proportion of emotional and behavioral problems among subjects involved in bullying as victims was 19.2%, as bullies 14.3%, and as bully-victims 27.1%. victims of bullying and bully-victims were at higher risk for experiencing mentalemotional problems than were the perpetrators.(18) victims and bully-victims frequently showed symptoms such as anxiety, depression, always feeling insecure, being extremely cautious, having low self-esteem and tending to have limited social interaction and to be isolated by their peers. p e r p e t r a t o r s o f b u l l y i n g b e h a v i o r m o r e frequently showed behavioral problems and abnormalities such as aggressive behavior, impulsivenes, lack of empathy, challenging and destructive attitude.(19,20) around 17.1% of subjects who were involved in bullying had extreme family relations (rigid family relation, low emotional bonding). victims were most frequently found to have extreme family relations. in families with rigid and poor family relations, lack of communication, lack of emotional bonding (closeness) and lack of h a r m o n y b e t w e e n f a m i l y m e m b e r s , t h e adolescent members may lack guidance and care in their moral and emotional development, so that they grow up with negative perceptions and views towards themselves and their environment, and are at risk of mental emotional problems (depression, anxiety, bipolarity) and behavioral problems (conduct disorder, bullying).(21) the n o n e x p o s u r e t y p e o f p a r e n t i n g p a t t e r n (unexpected, inconsistent, overprotective or overindulgent) were more frequently found in subjects who were involved in bullying (57.1%). achenbach and edelbrock (22) stated that overindulgent, too rigid and harsh (authoritarian) or inconsistent parenting in disciplining, i n f l u e n c e d t h e p e r s o n a l i t y f o r m a t i o n o f adolescents, who will be at high risk of mentalemotional problems (depression, anxiety, bipolarity) and behavioral problems (conduct disorder, bullying). our study showed that the non-exposure type of parenting pattern constituted the strongest risk factor for bullying behavior among adolescents. this is in accordance with the study of bacikovasleskova et al.(23) who found a significant association between parenting pattern and bullying behavior. their study also stated that the role of parents was essential to instill good behavior and emotions in their adolescent children. permissive and inconsistent (nonexposure) parenting is the nurturing pattern of parents who give too much freedom to their a d o l e s c e n t c h i l d r e n w i t h o u t a d e q u a t e supervision and tends to form adolescents who are agressive, impulsive, and egoistic. in contrast, children with authoritarian parenting may acquire a too rigid character, become headstrong and easily anxious and depressed. parenting pattern and the role of parents as good surilena, jessica non-exposure parenting and bullying behavior 63 univ med vol. 35 no.1 educators (exposure, democratic) can prevent the occurrence of behavioral and emotional problems, mental-emotional disorders and bullying behavior. parents are expexted to be capable of giving basic counseling and guidance about morals and religion, and build effective c o m m u n i c a t i o n w i t h t h e i r a d o l e s c e n t children.(22,23) self-esteem is a risk factor of bullying behavior. adolescents with low self-esteem tended to be 23.89 times more involved in bullying as compared with adolescents with high self-esteem. tritt and duncan (24) stated that adolescents with low self-esteem tended to be 18.90 times more involved in bullying than those with high selfesteem. adolescents with low self-esteem were at risk for the occurrence of mental-emotional health problems, substance abuse and addiction, and bullying behavior. salmon (25) stated that students who had been involved in bullying, either as bullies, victims or bully-victims, had a significant correlation with low self-esteem; the worst bullying behavior was associated with the lowest self-esteem. a limitation of this study lies in its crosssectional design, so that it cannot explain any causal relationship among junior high school students between parenting pattern and self-esteem on the one hand and bullying behavior on the other. further studies should be conducted using a longitudinal study design, to be able to explain any such causal relationship. this study was carried out among junior high school students, so that our study results are not representative of bullying behavior in adolescents outside the school environment. these study results are expected to lead to comprehensive management of adolescents who are involved in bullying behavior at school, i.e. both the physical and psychological aspects, by involving the family (particularly the parents) and school teachers. conclusions non-exposure parenting is the largest risk factor of bullying behavior. low self-esteem i n c r e a s e s t h e r i s k o f m e n t a l e m o t i o n a l disturbances (depression, anxiety), conduct disorder, and bullying behavior . conflict of interest there are no conflicts of interest. acknowledgment we thank all students of grades i, ii, and iii of state junior high school 23 (smp negeri 21) and of the private junior high schools (pluit r a y a , g e n e s a r e t h , wi j a y a k u s u m a ) i n kelurahan penjaringan who agreed to become respondents in this study. we also thank the heads and teachers of the schools, who permitted this study and helped in the course of this study. references 1. novianti i. fenomena kekerasan di lingkungan pendidikan. j insania 2010;13:1-10. 2. harris s. bullying at school among older adolescents. school nurs rev 2010;22:18-21. 3. nation m, vieno a, perkins d. bullying in school and adolescent sense of empowerment: an analysis of relationship with parents, friends, and teachers. j community applied soc psychol 2011;10:115-27. 4. wang j, iannotti rj, nansel tr. school bullying among adolescents in the united states: physical, verbal, relational, and cyber. j adolesc health 2009;45:368–75. 5. olweus d. bullying at school: basic fact and effect of a school based intervention program. j child psychol psychiatry 2009;35:171-90. 6. cook c, williams kr, guerra ng, et al. predictors of bullying and victimization in childhood and adolescence: a meta-analytic investigation. sch psychol 2010;25:65–8 7. nurhamzah w, maureen a, wiguna t. gambaran bullying dan hubungannya dengan masalah emosi dan perilaku pada anak sekolah dasar. sari pediatri 2013;15:174-80. 8. weir e. the health impact of bullying. can med assoc j 2011;1:1245-9. 9. arseneault l, bowes l, shakoor s. bullying victimization in youths and mental health problems: ‘much ado about nothing’? psychol med 2010;40:717-29. 64 10. van der wal mf, de wit cam, hirasing ra. psychosocial health among young victims and offenders of direct and indirect bullying. pediatrics 2003;111:1312-7. 11. brixval cs, rayce sl, rasmussen m. overweight, body image and bullying–an epidemiological study of 11-15 years olds. eur j public health 2011;3:123-5. 12. flemming lc, jacobsen kh. bullying among middle school students in low and middle income countries. j health promotion 2010;25:73-82. 13. nansel tr, overpeck m, pilla rs, et al. bullying behaviors among u.s. youth: prevalence and association with psychosocial adjustment. jama 2001;285:2094-100. 14. stein ja, dukes rl, warren ji. adolescent male bullies, victims, and bully-victims: a comparison of psychosocial and behavioral characteristics. j pediatric psychol 2007; 32:273-82. 15. nordhagen r, nielsen a, stigum h, et al. parental reported bullying among nordic children: a population-based study. child care health dev 2009;31:693-701. 16. smokowski, kopasz. bullying in school: an overview of types, effects, family characteristics, and intervention strategies. j pediatric psychol 2010;27:101-9. 17. spade ja. the relationship between student bullying behaviours and self-esteem [dissertation]. new york: college of bowling green state university;2010. 18. kaltiala-heino r, rimpela m, marttunen m, et al. bullying, depression, and suicidal ideation in finnish adolescents: school survey. bmj 2011;3:348-51. 19. gini g. association between bullying behaviour, psychosomatic complaints, emotional and behavioural problems. j paed chlid health 2011; 44:492-7. 20. garner pw, hinton st. emotional display rules and emotional self-regulation: associations with bullying and victimization in community-based after school programs. j community appl psychol 2010;20:480–9. 21. holt mk, espelage dl. perceived social support among bullies, victims, and bully-victims. j youth adolesc 2007;36:984–9. 22. achenbach tm, edelbrock cs. behavioral problems and competencies reported by parents of normal and disturbed children aged four through sixteen. monogr soc res child dev 2010;2:123-9. 23. bacikova-sleskova m, madarasova ga, van dijk jp, et al. parental support and adolescents’ health in the context of parental employment status. j adolesc 2011;34:141-9. 24. tritt c, duncan rd. the relationship between childhood bullying and young adult self-esteem and loneliness. j humanistic educ develop 2009;36:35–44. 25. salmon g. bullying in school: self-reported anxiety and self-esteem in secondary school children. bmj 2008;3:924–5. surilena, jessica non-exposure parenting and bullying behavior editorial 71 dietary salt reduction for control of hypertension richard tjan editor univ med vol. 30 no.2 in developed as well as developing countries, the four main factors affecting blood pressure are high salt intake, low potassium intake, overweight, and low physical activity level. this is also true for the increase in blood pressure with advancing age, occurring in all societies. it is now accepted that excess dietary salt raises blood pressure levels, whereas dietary salt reduction reduces blood pressure and prevents vascular complications.(1) the effect of salt on blood pressure is presumably due to the inability of the kidneys to excrete large amounts of salt, as humans are evolutionary adapted to ingest and excrete less than 1 gram of salt per day.(2) in this connection it should be noted that the more important element in common salt (sodium chloride) is the sodium ion, and any restrictions applying to common salt also apply to all food items that contain sodium ions, such as sodium glutamate and baking soda. worldwide, dietary salt intakes tend to be high. surveys in european countries (switzerland, united kingdom, italy, finland) based on 24-h urine collections found salt intakes in the range of 10-11 g/day in men and 8-9 g/day in women.(3) similarly, in period of 2005-2006 the average salt consumption in the united states was 10.4 g/day in men and 7.3 g/day in women.(4) in comparison, the total salt intake of 15 javanese women was 5.8 g/day, as determined by the urinary lithiummarker technique.(5) with regard to dietary salt (sodium) reduction, the who and fao have recommended a maximum daily intake of 5 g salt per day for blood pressure control and reduction of hypertension prevalence and related health risks in populations.(6) several european countries, namely united kingdom, germany, austria, switzerland, and finland, have set the recommended salt consumption at 6 g/day,(1) while in the united states a daily salt intake of less than 5.8 g (2300 mg of sodium) has been recommended by the department of health and human services and the department of agriculture. certain population groups, such as hypertensives and blacks should limit their sodium intake to 1500 mg/day.(4) on 20 april 2010, the institute of medicine (iom) issued a report on “strategies to reduce sodium intake in the united states,” the recommendations of which has since been under careful review and evaluation by the food and drugs administration (fda). recently the american public health association has recommended a daily sodium intake of less than 1500 mg. in developed countries about 80% of dietary salt comes from commercially prepared foods. the united kingdom was notably the first country in the world where a salt reduction of more than 30% in some food products was achieved. since 2005 a consensus was reached with the food industry to voluntarily reduce the salt content of their products gradually within a few years, thus preventing rejection by the consumer. actually, the acquired taste for highly salted foods, especially in children, was due to habituation to these commercial products. these efforts to 72 univ med vol. 30 no.2 reach the recommended salt consumption of 6 g/day are still continuing.(1) other countries that have achieved population-wide dietary salt reduction are japan, finland, and portugal.(4) the finnish national nutrition council in 2005 recommended an absolute salt intake level of 7 g/day for men and 6 g/day for women.(7) after a nation-wide dietary sodium reduction campaign since 1978, after 30 years the average sodium intake in finland has decreased more than 40%. the cost-effectiveness of dietary salt reduction has been the subject of many studies, such as the study by bibbins-domingo et al. this study used the coronary heart disease (chd) policy model to determine the cost-effectiveness of daily reduction of up to 3 g of salt (1200 mg of sodium), as compared with the treatment of hypertension with medications. according to this study, dietary salt reduction of 3 g per day is projected to effect an annual reduction of 60,000 to 120,000 new chd cases, 32,000 to 66,000 stroke cases, 54,000 to 99,000 cases of myocardial infarction, and 44,000 to 92,000 deaths from any cause.(4) in the united states, the center for science in the public interest (cspi), which is supported by subscribers to their nutrition action health newsletter, won a legal action against the fda, resulting in the 1990 nutrition labeling and education act. unfortunately, in 2006 cspi found that sodium consumption had actually increased, with a 50% annual decrease in new low-sodium foods and a concomitant steady decrease in consumer concern about sodium. it is to be hoped that in the near future the fda will take a more active role in salt reduction efforts, in cooperation with such organizations as cspi, iom, and apha. in developing countries, the main source of excess dietary salt is foods prepared at home.(1) other sources are the products of national and international food and beverage industries available in supermarkets and foods prepared by informal sidewalk food vendors. assuming that the branded products could be regulated as to salt content, there still remains the problem of persuading the housewives and the sidewalk vendors to use less salt in preparing their products. in the absence of an official policy on salt reduction, there remains the option of health education at primary and secondary school level, and also at individual and community level, possibly taking several decades before showing results. in addition to promoting dietary salt reduction, countries with endemic iodine deficiency should also increase the level of iodine fortification in salt to 80-100 ppm of kio3 to provide a daily iodine intake of 150µg, as recommended by mustafa. (5) references 1. world health organization. reducing salt intake in populations: report of a who forum and technical meeting, 5–7 october 2006 paris, france. geneva: world health organization;2007. 2. meneton p, jeunemaitre x, de wardener he, macgregor ga. links between dietary salt intake, renal salt handling, blood pressure, and cardiovascular diseases. physiol rev 2005;85:679–715. 3. beer-borst s, costanza mc, pechère-bertschi a, morabia a. twelve-year trends and correlates of dietary salt intakes for the general adult population of geneva, switzerland. euro j clin nutr 2009;63:155–64. 4. bibbins-domingo k, chertow gm, coxson pg, moran a, lightwood jm, pletcher mj, et al. projected effect of dietary salt reductions on future cardiovascular disease. n engl j med 2010;362:590-9. 5. mustafa a, muslimatun s, untoro j, lan mcpj, kristianto y. determination of discretionary salt intake in an iodine deficient area of east java-indonesia using three different methods. asia pac j clin nutr 2006;15:362-7. 6. world health organization. diet, nutrition and the prevention of chronic diseases. report of a joint who/fao expert consultation. who technical report series no. 916. geneva: world health organization;2003. 7. karppanen h, mervaala e. sodium intake and mortality. lancet 1998;351:1509. alvina 46 abstract *department of microbiology medical faculty, trisakti university correspondence prof. dr. murad lesmana department of microbiology medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2611 email: jmurad@cbn.net.id univ med 2010;29:46-55 helicobacter pylori and gastro-esophageal reflux disease (gerd) murad lesmana* january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina helicobacter pylori is the major cause of gastritis and duodenitis, and is responsible for the majority of gastric and duodenal ulcers. it is thought that h.pylori may have a benign influence in the prevention of reflux esophagitis and gastro-esophageal reflux disease (gerd). gerd is due to failure of the gastro-esophageal anti-reflux mechanism, thus allowing gastric acid to damage the esophageal mucosa, but the main abnormalities may also be found in the stomach and duodenum. the recently decreased prevalence of h.pylori has led to a lower incidence of duodenal ulcer and gastric carcinoma, but increasing acid secretion resulted in an increase in gerd. eradication of h.pylori does not aggravate the symptoms of gerd and also does not induce the development of gerd in the normal population or in patients with duodenal ulcer. pharmacological suppression of acid secretion is more effective in patients infected with h.pylori in comparison to non-infected individuals. the evidence on the efficacy of acid suppression therapy and its association with h.pylori infection is still contradictory. the sharply differing opinions regarding the role or association of h.pylori infection with gerd have not been reconciled and contradictory study results are still widespread. keywords: helicobacter pylori, gastro-esophageal reflux, eradication introduction t h e d i s c o v e r y o f t h e b a c t e r i u m helicobacter pylori (h.pylori) in 1982 was the starting point of a revolution in the concept and management of gastroduodenal disorders. h.pylori was known to be asscociated with inflammation of the gastric mucosa, and it was agreed that the common gastric disease known as peptic ulcer was an infectious disease, and that the causal organism was h.pylori. the isolation of this bacterium opened a new era in gastric microbiology.(1,2) ample evidence showed that once an infection of h.pylori has o c c u r r e d , t h i s b a c t e r i u m p e r s i s t s i n t h e patient’s body for life and causes chronic active 47 univ med vol.29 no.1 gastritis.(2) the national institute of health (nih) consensus conference of 1994 concluded that h.pylori was the main cause of gastric ulcer and recommended a therapy of eradication of the causative organism.(3) based on reports indicating an association between h.pylori infection and gastric carcinoma, the who also reviewed the existing data and stated that there was sufficient epidemiological and histological e v i d e n c e d e m o n s t r a t i n g t h a t h . p y l o r i i s carcinogenic for humans.(4) the most effective approach to reduce the pravalence of gatric carcinoma is to prevent childhood h.pylori infection.(5) t h e p r i n c i p a l a b n o r m a l i t y i n t h e e s o p h a g u s , s t o m a c h a n d d u o d e n u m i s associated with h.pylori infection or with gastro-esophageal reflux disease (gerd). h.pylori causes chronic infection and has a severe impact on the gastric mucosa, whereas gerd occurs as a result of abnormal exposure of the esophagus to gastric acid. in developed countries such as the united states and europe, gerd is a controversial problem because it is associated with h.pylori infection.(6,7) gerd has since long considered to be independent of colonization or infection with h.pylori. gerd may occur with the same frequency and grade of severity both in h.pylori positive as well as in h.pylori-negative patients. however, this concept was revised in connection with the results of several studies indicating that h.pylori protects against the development of gerd.(6) some additional aspects such as the reduction in the prevalence of h.pylori in gerd patients and the increase in gerd following measures for eradication of h.pylori, led to the widespread opinion that h.pylori has a protective effect for the esophagus: h.pylori can prevent the occurrence of gerd with its various complications. however, the existing d a t a a n d t h e i r i n t e r p r e t a t i o n a r e s t i l l controversial such as to cause the relationship between gerd and h.pylori to be a problem without a definitive solution. microbiological characteristics of h.pylori h.pylori is a helical gram-negative microaerophilic organism with rounded ends. the bacterium has 4-6 sheathed unipolar flagella w i t h s w o l l e n e n d s . t h e f l a g e l l a h a v e a propulsive function, allowing the bacterium to move at great speed within viscous liquids such as the mucus on the surface of gastric epithelial cells.(1,2) there is ample evidence linking h.pylori with chronic superficial gastritis. eradication of h.pylori through antimicrobial therapy cures the patient of gastritis and the titer of specific a n t i b o d y d e c r e a s e s a f t e r e r a d i c a t i o n ; i n contrast, nearly all patients infected with h.pylori show a persistent immunological response against this organism.(7) h . p y l o r i h a s a c h a r a c t e r i s t i c t i s s u e specificity with an exclusive affinity to gastric mucosal cells but not to intestinal epithelial cells .(8,9) after ingestion by the host, the organism has to be able to protect itself against the bactericidal activity of the gastric luminal contents and subsequently enter the mucus layer (figure 1). within the gastric lumen, h.pylori is capable of spatial orientation in the mucus layer, adhere to the epitelial cell, colonize it and settle in the gastric lumen.(10) t h e p a t h o g e n i c d e t e r m i n a n t s o f t h e microorganism may be divided into two main groups, i.e. (i) virulence factors that play a role in the pathogenic effect of the microorganism, and (ii) maintenance factors that allow the microorganism to colonize and settle in the body of the host.(9) the two important factors here are urease production and motility of the microorganism. urease hydrolyzes urea into carbon dioxide and ammonia, thus enabling h.pylori to persist in an acid environment, w h i l s t m o t i l i t y i s e s s e n t i a l f o r t h e m i c r o o rg a n i s m i n o r d e r t o c o l o n i z e t h e stomach. urease is a strong stimulus for activation of mononuclear phagocytes and production of inflammatory cytokines. in vitro, 48 figure 1. mechanism of h.pylori colonization of the gastric mucosa(10) urease activity is also toxic for human gastric cells. therefore, urease probably functions s i m u l t a n e o u s l y a s v i r u l e n c e f a c t o r a n d m a i n t e n a n c e f a c t o r. ( 1 , 9 , 1 0 ) p a t i e n t s w h o s e gastritis is situated predominantly in the antral region of the stomach (which is the most common form of gastritis) tend to suffer from duodenal ulcers, whereas patients whose gastritis is predominantly in the gastric corpus and is multifocal and atrophic, tend to have gastric ulcers, gastric atrophy, intestinal m e t a p l a s i a , a n d u l t i m a t e l y g a s t r i c carcinoma.(4,7,11) gastric or duodenal ulcers are called peptic ulcers, which are mucosal defects > 0 . 5 c m i n d i a m e t e r, t h a t p e n e t r a t e t h e muscularis mucosae. gastric ulcers commonly o c c u r a l o n g t h e m i n o r c u r v a t u r e a t t h e transition between the mucosa of the body and the gastric antrum. duodenal ulcers generally affect the duodenal bulb, which is the site most exposed to acid. some experts are of the opinion that certain strains of h.pylori are more virulent than others. preliminary studies indicated that the pathogenicity was associated with the activity of a protein called caga (cytotoxigenic -associated gene a).(1,2) caga is an extremely immunogenic protein encoded by the caga gene, which is possessed by around 50-70% of h.pylori strains, where caga is a marker of bacterial virulence. patients infected with caga-positive h.pylori experience a more severe inflammatory response. infection with these caga-positive strains increases the risk for the occurrence of duodenal ulcer and adenocarcinoma of the distal part of the stomach.(11,12) in the united states and europe around 60% of h.pylori strains reportedly possess the caga gene. another virulence factor also considered t o p l a y a r o l e i n h . p y l o r i i n f e c t i o n s i s vacuolating cytotoxin (vaca), that is a strongly immunogenic protein capable of inducing v a c u o l i z a t i o n o f e p i t h e l i a l c e l l s . va c a accumulates in the mitochondria and causes tissue necrosis.(7) lesmana gastro-esophageal reflux disease 49 univ med vol.29 no.1 epidemiology and transmission of the bacterium h . p y l o r i i n f e c t i o n m a y b e f o u n d w o r l d w i d e , b u t i t s p r e v a l e n c e v a r i e s considerably by country and by population g r o u p i n t h e s a m e c o u n t r y. t h e o v e r a l l prevalence is intimately associated with the socio-economic conditions of the involved region, as the infection predominantly affects t h e l o w e r s o c i o e c o n o m i c g r o u p s . ( 7 ) in developing countries 70-90% of the population is h.pylori-positive, approximately 80% are elderly people and nearly all acquired the infection under the age of 10 years. in contrast, in industrial countries the prevalence of the infection is only around 25-50%.(6) to date there is no evidence for a zoonotic transmission, while a reservoir other than humans has not been found for h.pylori.(1,2) therefore it is thought that there are 3 routes of transmission from one individual to another, namely (i) by means of endoscopic apparatus (an extremely rare event); (ii) fecal-oral transmission; (iii) oral-oral transmission, e.g. in africa where mothers masticate food before giving them to their infants. in general, infection is acquired by oral i n g e s t i o n o f h . p y l o r i o rg a n i s m s a n d i s t r a n s m i t t e d a m o n g f a m i l y m e m b e r s . transmission may be effected through saliva, utensils, fecal matter, and water. unlike other regions,western countries have an approximately fourfold frequency of duodenal ulcer compared with gastric ulcer. duodenal ulcer primarily affects the age range of 20-50 years, while gastric ulcer is more frequent in patients older than 40 years.(7) h.pylori infection in adults is usually chronic and does not heal without specific treatment. on the other hand, spontaneous elimination of the bacteria may occur in children, particularly if these children receive antibiotic treatment for other infections. in developed countries the prevalence of h.pylori has substantially decreased in the last decades in connection with improved nutrition, living standards, and sanitation in these countries, but it is to be feared that without intervention h.pylori will still be endemic.(12) detection of h. pylori infection currently there are several tests available f o r t h e d i a g n o s i s o f h . p y l o r i i n f e c t i o n , comprising noninvasive and invasive methods. the latter require endoscopic biopsy of the gastric mucosa. the endoscopic specimen may be subjected to histological examination, bacterial culture, rapid urease test, dna p r o b i n g a n d p c r a n a l y s i s . n o n i n v a s i v e methods are the urea breath test, serological tests, and fecal antigen assays. the choice of test depends mainly on the required clinical information, test availability and cost. testing endoscopic biopsy specimens the best specimens for detection of h.pylori organisms are endoscopic biopsy specimens. the specimens are collected before adminstration to the patients of antibiotics or antisecretoric agents, particularly proton pump i n h i b i t o r s ( p p i ) , r e s u l t i n g i n a b n o r m a l distribution of the organisms. two weeks before endoscopic specimen collection the patients should not be given the abovementioned drugs. t h e d i s t r i b u t i o n o f h . p y l o r i a n d associated inflammation is patchy, such that it frequently leads to endoscopic sampling errors and thus yielding negative results. therefore at least two biopsy specimens should be taken from the antrum at a distance of around 2-5 cm from the pylorus. the biopsy specimens may be subjected to (i) culture and (ii) rapid urease test. it is r e c o m m e n d e d t h a t t h e c o l l e c t e d b i o p s y specimens first be used for culture, before being used for histopathological examination. rapid urease test the clotest (delta west ltd, bentley, australia) was the first commercial biopsy urease test developed by marshal.(1) the test 50 consists of gall agar containing urea and phenol red. the biopsy specimen is placed on the agar and in the presence of urease (produced by h.pylori), the urea undergoes hydrolysis, which effects a change in the ph of the agar (the latter becoming alkaline) such that there is a color change of the phenol red. this test can be read up to 24 hours after placing the biopsy tissue on the agar gel. i n a d d i t i o n t o t h e c l o t e s t , o t h e r commercially available biopsy urease tests are “hpfast” and “pyloritek” (as a strip test). all three tests have almost identical sensitivities a n d s p e c i f i c i t i e s ( s e n s i t i v i t y 8 8 9 3 % ; specificity 99-100%).(7,8) the results of the “pyloritek” may be read in one hour, but the test has a lower sensitivity, namely 66-71%. culture h.pylori is a slow-growing organism that requires complex growth factors. culture of h.pylori has two principal advantages (i) antibiotic sensitivity testing can be performed; (ii) the culture isolate can be subjected to further detailed characterization. culture may be performed on biopsy specimens, gastric juice and feces, but the most common specimens yielding high sensitivity are gastric biopsies. transport of biopsy specimens is an important factor in the successful culture of h.pylori. the problem in this case is that gastroenterologists have a negative view of bacteriological culture systems, as errors in specimen transport lead to negative results. it should be noted that h.pylori is an extremely fragile organism that has to be protected from drying and from contact with oxygen and room temperature. after the biopsy specimen has been collected, it is placed in normal saline (0.1 ml nacl-0.85%) and stored at a low temperature (in a coolbox, 40c) prior to and during transport to the laboratory. normal saline is a simple transport medium used for transport times of <6 hours. for longer tranport times a more complex medium is utilized (transport medium), usualy in the form of semisolid agar. a commercially available transport medium for this purpose is portagerm pylori (biomerieux, france).(13) in the transport medium the culture specimen can survive for 24 hours at the temperature of 40c used for its transport. if this condition cannot be met or if long-term storage is required, the biopsy specimen can be frozen and stored in sterile conditions at a temperature of -700c.(14) culture is performed on a nonselective medium (blood agar 5-10%), such as columbia agar or brucella agar with the addition of 5-10% blood, and also on a selective medium for increasing the sensitivity of the culture. useful selective media are (i) blood agar + vancomycin, trimethoprim, amphotericin b and polymyxin b (skirrow); or (ii) blood agar + vancomycin, trimethoprim, amphotericin b and cefsulodin. h.pylori requires microaerophilic culture conditions (n280%; co2-5-10%) (campypak microaerophilic system, columbia diagnostics), a temperature of 35-370c, high humidity and an incubation time of 7-10 days. in positive cultures growth occurs within 3-5 days as translucent and clear colonies on the agar plates, with the microscopic picture of gram-negative curved rods that are positive for urease, catalase, and oxidase. culture of biopsies is the gold standard for detecting h.pylori infection. in the case of a negative culture, a combination of the rapid urease test and histological results may be used. culture has a sensitivity of 95%.(1,2,15) urea breath test (ubt) this test is based on the substantial urease activity of h.pylori in the stomach and its sensitivity depends on the bacterial load. the urea breath test qualitatively detects active infection with a sensitivity and specificity of over 90%. the principle of ubt is identical to that of other urease tests. urea as the substrate in ubt is administered as ingested [13c]urea. or [14c]urea. in the presence of h.pylori, urea is hydrolyzed by the bacterial urease, leading to absorption of labeled co2 by the blood and elimination of the co2 from lesmana gastro-esophageal reflux disease 51 univ med vol.29 no.1 the respiratory tract by exhalation. the test accuracy depends on several factors, i.e. collection time of the breath, gastric emptying time, and distribution pattern of urea (tablets vs liquids). a commercially available test is the ubt kit (meretek diagnostics). ubt is indicated for initial diagnosis of h.pylori infection and for follow-up of eradication therapy. in the latter case, the urea breath test should not be performed until after a time interval of 4 weeks has elapsed, to prevent false negative test results. ubt is valid for children over 6 years old, while for younger children the test has not been definitively validated. serum antibody test h.pylori infections of the gastric mucosa result in local and systemic immune responses, including raised specific serum igg and iga and gastric iga and igm levels. this allows the detection of bacteria by serological tests. in general, serologic testing is performed by means of enzyme-linked immunosorbent assay (elisa) which has a reported sensitivity and specificity of 85% and 79%, respectively. without therapeutic intervention the antibody levels are persistently high, perhaps for the lifetime of the patient, and are used as a measure of the duration of infection. following eradication of h.pylori, the specific igg and i g a l e v e l s t e n d t o d e c r e a s e w i t h i n approximately 6 months. these serologic tests are of considerable benefit in the screening of large numbers of individuals such as in epidemiological studies. a commercially available test kit for assessing serum anti-h.pylori antibodies is the hm-cap elisa (ez-em inc, new york). stool antigen test tests for fecal h.pylori antigens have the advantages of being non-invasive, of using easily obtainable test materials and of being capable of detection of bacteria or bacterial components (dna, antigen). fecal h.pylori antigen tests in general use are elisa with polyclonal antibody reagents such as the hpsa test (meridian diagnostics, cincinnati, oh) or the amplified-idea hpstar (dako, glostrup, denmark) with monoclonal antibodies. amplified-idea hpstar reportedly has a higher accuracy(2) and sensitivity than hpsa. st o o l a n t i g e n t e s t s h a v e s e v e r a l limitations; e.g. rapid transit time maintains the integrity of the antigens, whereas constipation may increase their degradation.(16) pcr this technique has great potential for detection of h.pylori as it has high sensitivity and specificity. it can be applied to biopsy specimens as well as specimens obtained by non-invasive means, such as gastric juice, blood, saliva, urine and fecal specimens. h o w e v e r, s e v e r a l f a c t o r s m a y a ff e c t i t s accuracy, including selection of dna primer and target, specimen preparation, and technical procedures. gerd and h.pylori infection: is there any association? t h e m a i n l e s i o n s i n t h e e s o p h a g u s , stomach and duodenum are associated with h. pylori infection or gastro-esophageal reflux d i s e a s e ( g e r d ) . w h e n t h e n u m b e r s o f h.pylori decrease and the organisms disappear from the human stomach, the incidence of gastro-esophageal reflux disease or gerd i n c r e a s e s d r a m a t i c a l l y, p a r t i c u l a r l y i n developed countries such as the united states and europe. gerd has become a problem associated with infection by h.pylori.(6,17) h. pylori causes a chronic infection and has a profound impact on the gastric mucosa, while gerd occurs as a result of abnormal exposure of the esophagus to gastric acid. it was hypothesized that h.pylori has a beneficial effect by regulating the acidity of 52 gastric contents and reducing the impact of regurgitation of gastric acid into the esophagus. this hypothesis has not been universally accepted because a number of studies have been unable to demonstrate aggravation of the r e f l u x d i s e a s e f o l l o w i n g e r a d i c a t i o n o f h.pylori. in patients with chronic atrophic gastritis as a result of h.pylori infection, gastric acid is suppressed, and thus is not produced in the critical quantities necessary for the induction of gerd.(6) is there an actual increase in prevalence of gerd in connection with the decreased prevalence of h.pylori infection, or is it the result of more accurate observations of endoscopists in detecting erosive esophagitis? g e r d w a s l o n g c o n s i d e r e d t o b e independent of h.pylori colonization or infection . both in h.pylori positive and negative patients, gerd may occur at similar frequencies and severity. however, this concept was subsequently modified by a group of investigators in c o n n e c t i o n w i t h n u m e r o u s s t u d y r e s u l t s indicating that h.pylori may be protective against development of gerd such that it confers benefits on the subject in question. the hypothesis put forward by this group is that inflammation of the gastric corpus caused by h.pylori has a suppressive effect on acid production such that the individual is prevented from developing gerd. reports from japan mention that patients with atrophic gastritis have an increased production of gastric acid f o l l o w i n g e r a d i c a t i o n o f h .p y l o r i , t h u s inducing the development of gerd in a subset of the patients. h.pylori infection presumably has a protective effect against gerd.(6,17) conversely it has been suggested that longterm suppression of gastric acid may aggravate gastritis of the gastric corpus due to h.pylori and increase the risk of gastric carcinoma.(6,17) however, european studies have reported that e r a d i c a t i o n o f h . p y l o r i d i d n o t m o d i f y duodenogastro-esophageal reflux and h.pylori status in patients with gerd did not affect the degree of exposure of the esophagus to acid. from these data it was concluded that the risk of gerd after h.pylori eradication was not high. currently this conclusion is still the subject of controversy. many populations of patients have h.pylori infection concomitantly w i t h g e r d a n d t h e r e i s n o c o n c l u s i v e evidence that severe gerd is associated with low or decreased prevalence of h.pylori infection, or that low infection rates are associated with virulent strains of h.pylori. the sharply differing opinions regarding the role or association of h.pylori infection with gerd have not been reconciled and contradictory study results are still widespread. the opinion that the association between gerd and h.pylori is a negative one is suggested by studies on endoscopically proven reflux esophagitis rather than symptomatic gerd.(18,19) the majority of the study results f r o m e a s t a s i a n c o u n t r i e s r e v e a l e d a considerably greater negative association compared with that from west european studies.(18) this negative association is greater in patients infected with caga-positive strains of h.pylori.(20) these data support the opinion that patients with gerd have a low probability of experiencing infection with h.pylori, b e c a u s e h . p y l o r i i n f e c t i o n p r o t e c t s t h e esophagus against reflux of gastric acid. however, there are still other possibilities to account for the fact that patients with gerd are protected against h.pylori, such as the influence of social status in the community. one of the opininons advanced in the initial debates about the role of h.pylori in connection with gerd is the problem of treatment for eradication of h.pylori. cases of duodenal ulcer reflux esophagitis occur more frequently in patients receiving eradication therapy for h.pylori compared with those receiving placebo.(18-20) nakajima and hattori(21) recently reviewed the data on development of gerd following eradication of h.pylori in patients with peptic ulcer and concluded that there were no results supporting the opinion lesmana gastro-esophageal reflux disease 53 univ med vol.29 no.1 that eradication of h.pylori results in the development of gerd or symptoms of gerd. apparently the opinions advanced in various study results are paradoxical in nature. the present situation is therefore such that on one hand there is a negative association between prevalence of h.pylori infection and gerd, but on the other hand eradication of h.pylori does not induce or aggravate gerd. what lesson is to be learned from these contradictory opinions? in reality, the incidence of gerd has increased drastically since 1995, whereas in the same time period duodenal ulcers have s h a r p l y d e c r e a s e d . st a t i s t i c a l d a t a f r o m developed countries on gastric carcinoma and duodenal ulcer reveal that the decreased p r e v a l e n c e o f h . p y l o r i i s t h e c a u s e o f abovementioned changes. is it possible that the decrease in h.pylori also causes an increase in gerd? gerd is an abnormality due to failure of the gastro-esophageal anti-reflux mechanism, thus allowing gastric acid to damage the esophageal mucosa. koike et al.(22) have confirmed that there is a high level of g a s t r i c a c i d s e c r e t i o n i n p a t i e n t s w i t h esophagitis. this is not surprising since it is the acidity of the refluxate that determines the extent of damage to the esophageal mucosa. according to axon(18) gastric acid secretion in the japanese community, both in the elderly and the young, increased substantially in the last decades, indicating that there had been a gradual rise in gastric acid secretion over a number of years. however, it is of interest that the increase in gastric acid secretion did not occur only in h.pylori-negative individuals, but also in those with h.pylori-positive status. t h u s t h e o v e r a l l i n c r e a s e d g a s t r i c a c i d secretion is not a direct result of decreased incidence of h.pylori. it is possible that this increased gastric acid secretion is the cause of the increase in gerd in patients with an abnormal anti-reflux mechanism. to date there has not been any direct evidence supporting the hypothesis that a high level of gastric acid secretion protects against h.pylori infection. the mode of transmission of h.pylori is still unclear. however, this organism apparently prefers a hypochloric environment to an achlorhydric one or one with high acid secretion. high acid secretion a p p a r e n t l y p r o v i d e s p r o t e c t i o n a g a i n s t colonization by h.pylori, possibly by inhibiting or reducing the colonizing capacity of the microorganism. the effect of h.pylori on acid secretion is complex. in individuals with an high natural acid secretion, h.pylori gastritis is limited to the antrum, similar to the pattern of gastritis encountered in patients with duodenal ulcer.(18) in these patients the post-prandial secretion of gastric acid is raised and this abnormality may be partially corrected through treatment of the infection. in contrast, individuals with an inherently low acid secretion tend to suffer from pangastritis, which subsequently results in impairment of the parietal cells in the gastric c o r p u s a n d f u r t h e r m o r e c a u s e s f u r t h e r r e d u c t i o n i n a c i d s e c r e t i o n . s u c c e s s f u l treatment of the infection in these patients may increase their gastric acid secretion.(18) t h e e p i d e m i o l o g y o f t h e d i s e a s e s a s s o c i a t e d w i t h h . p y l o r i h a s c h a n g e d dramatically over the last two centuries. gastric ulcer was prevalent in europe in the 19th century, being replaced by duodenal ulcer in the 20th century. this condition was followed by a decreased prevalence of h.pylori, leading to a lower incidence of duodenal ulcer and gastric carcinoma, but the still increasing acid secretion resulting in an increase in gerd. several reports have drawn attention to the presence of a strong negative association between gastritis of the gastric corpus and g e r d . ( 23,2 4) t h i s o b s e r v a t i o n l e d t o t h e hypothesis that patients with pangastritis have damaged parietal cells, which secrete lesser amounts of acid, thus there is a low probability of the patients suffering from reflux and therefore they are protected from developing gerd. actually this is a circular argument as 54 individuals who develop gastritis of the gastric corpus have at the moment of infection a naturally lower acid secretion anyway and thus a lower probability of developing gerd, whether they are infected or not. from the reported data it is apparent that eradication of h.pylori does not aggravate the symptoms of gerd and also does not induce the development of gerd in the normal population or in patients with duodenal ulcer.(18) thus arises the question why patients with gerd are not given treatment in the form of h.pylori eradication? pharmacological suppression of acid secretion is reportedly more effective in patients infected with h . p y l o r i i n c o m p a r i s o n t o n o n i n f e c t e d individuals. therefore eradication of h.pylori theoretically makes pharmacologic control of the symptoms more difficult.(18,24) however, as in other aspects of the connection between gerd and h.pylori, the evidence on the efficacy of acid suppression therapy and its association with h.pylori infection is still contradictory. conclusions apart from the diversity of studies on the relationship between gerd and h.pylori, the prevalence of h.pylori is significantly lower in patients with gerd compared with those without. however, there a still a great number of mutually contradictory study results on h.pylori infection in connection with gerd. references 1. kusters jg, van vliet ahm, kuipers ej. pathogenesis of helicobacter pylori infections. clin microbiol rev 2006;19:449-90. 2. megraud f, lehours p. helicobacter pylori detection and antimicrobial susceptibility testing. clin microbiol rev 2007;20:280-32. 3. nih consensus conference. helicobacter pylori in peptic ulcer disease. nih consensus development panel on helicobacter pylori in peptic ulcer disease. jama 1994;272:65-9. 4. uemura n, okamoto s, yamamoto s, matsumura n, yamaguchi s, yamakido m, et al. helicobacter pylori infection and the development of gastric cancer. n engl j med 2001;345:784-9. 5. tehuteru es. management of helicobacter pylori infection in children. univ med 2004;23:110-4. 6. malfertheiner p, peitz u. the interplay between helicobacter pylori, gastro-oesophageal reflux disease, and intestinal metaplasia. gut 2005;54:1320. 7. suerbaum s, michetti p. helicobacter pylori infection. n engl j med 2002;347:1175-86. 8. ottemann km, lowenthal ac. helicobacter pylori uses motility for initial colonization and to attain robust infection. infect immun 2002;70: 1984-90. 9. schreiber s, konradt m, groll c, scheid p, hanauer g, werling ho, et al. the spatial orientation of helicobacter pylori in the gastric mucosa. proc natl acad sci 2004;101:5024-9. 10. petersen am, krogfelt ka. helicobacter pylori: an invading microorganism? a review. femas immunol med microbiol 2003;36:117-26. 11. peek rm, crabtree je. helicobacter pylori and gastric neoplasia. j pathol 2006;208:233-48. 12. rupnow mf, shachter rd, owens dk, parsonet ja. a dynamic transmission model for predicting trends in helicobacter pylori and associated diseases in the united states. emerg infect dis 2000;6:228-37. 13. dunn be, cohen h, blaser mj. helicobacter pylori. clin microbiol rev 1997;10:720-61. 14. heep m, scheibl a, degrell a, lehn m. transport and storage of fresh and frozen gastric biopsy specimens for optimal recovery of helicobacter pylori. j clin microbiol 1999;37:3764-6. 15. frenck rw jr, fathy hm, sherif m, mohran z, el mohammedy, francis w, et al. sensitivity and specificity of various tests for the diagnosis of helicobacter pylori in egyptian children. pediatrics 2006;118:el1195-202. 16. matsuda m, noda y, takemori y. utility and limitations of a method for detecting helicobacter pylori-specific antigens in the stool. j gastroenterol 2003;38:222-8. 17. richter je. effect of helicobacter pylori eradication on the treatment of gastro-oesophageal reflux disease. gut 2004;53:310-1. 18. axon atr. personal view: to treat or not to treat? helicobacter pylori and gastro-oesophageal reflux disease – an alternative hypothesis. aliment pharmacol ther 2004;19:253-61. 19. ragunath a, hungin aps, wooff d, childs s. prevalence of helicobacter pylori in patients with gastro-oesophageal reflux disease: systematic review. br med j 2003;326:737-9. lesmana gastro-esophageal reflux disease 55 univ med vol.29 no.1 20. loffeld rjlf, werdmuller bfm, kuster jg, perez gl, blaser mj, kuipers ej. colonization with caga-positive helicobacter pylori strains inversely associated with reflux oesophagitis and barret’s oesophagus. digestion 2000;62:95-9. 21. nakajima s, hattori t. active and inactive gastroeophageal reflux disease related to helicobacter pylori therapy. helicobacter 2003; 8:279-93. 22. koike t, ohara s, sekine h, iijima k, abe y, kato k, et al. helicobacter pylori prevents erosive reflux oesophagitis by by decreasing gastric acid secretion. gut 2001;49:330-4. 23. richter je. helicobacter pylori: the bug is not all bad. gut 2001;49:319-21. 24. haruma k, hamada h, mihara m, kamada t, yoshihara m, sumi k, et al. negative association between helicobacter pylori infection and reflux esophagitis in older patients: case-control study in japan. helicobacter 2000;5:24-9. oktavianus 27 *anatomy department faculty of medicine and health sciences jenderal soedirman university purwokerto **physiology department faculty of medicine gadjah mada university ***education programme reproduction and development c/o monash institute of medical research australia correspondence dr. fitranto arjadi, m.kes anatomy department faculty of medicine and health sciences jenderal soedirman university kampus berkoh purwokerto 53146 phone : +62281-622022 email: f.arjadi@yahoo.com univ med 2014;33:27-35 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background chronic stress increases glucocorticoid levels and accelerates reduction in leydig cells functions and numbers. chronic stress models in the working place comprise sleep deprivation, sedentary stress, and physical stress. the aim of this study was to evaluate the effect of various work stress models, such as stress from paradoxical sleep deprivation (psd), immobilization, and footshock, on serum testosterone levels and number of leydig cells in male albino rats. methods this study was of experimental randomized post-test only with control group design using 24 male wistar albino rats (rattus norvegicus). the sample was divided into 4 groups: k1 (control), k2 (psd), k3 (immobilization) and k4 (footshock), receiving treatment for 25 days. measured parameters were serum testosterone level and leydig cell number. analysis of variance (anova) was used for statistical analysis, followed by post hoc lsd. results mean serum testosterone levels (0.07 ± 0.08 ng/ml) and leydig cell numbers (4.22 ± l0.96) were lowest in the psd stress model. serum testosterone levels differed significantly between controls and psd group (p=0.014), while there was a significant difference in numbers of leydig cells between footshock stress and psd (p=0.011) and between the three stress groups and controls (p=0.006). conclusion this study demonstrated that psd, immobilization and footshock stress significantly decreased serum testosterone levels and number of leydig cells in male albino rats (rattus norvegicus). the mechanism by which psd affects serum testosterone is still unclear. keywords: leydig cells, serum testosterone, work stress, male albino rats paradoxical sleep deprivation decreases serum testosterone and leydig cells in male rats fitranto arjadi*, sri kadarsih soejono**, and mulyono pangestu*** 28 arjadi, soejono, pangestu paradoxical sleep deprivation and testosterone paradoxical sleep deprivation menurunkan kadar testosteron dan sel leydig pada tikus jantan latar belakang stres kronik meningkatkan glukokortikoid dan mempercepat penurunan fungsi dan jumlah sel leydig. model stres kronik di dunia kerja meliputi pengurangan waktu tidur, pola sedentarian dan stres fisik. tujuan penelitian adalah untuk menilai pengaruh berbagai model stress kerja, yaitu paradoxical sleep deprivation (psd), imobilisasi, dan footshock kronik terhadap jumlah sel leydig dan kadar testosteron pada tikus putih (rattus norvegicus) jantan. metode sebuah penelitian eksperimental post test only dengan menggunakan kontrol digunakan pada 24 ekor tikus putih jantan galur wistar. tikus ini dibagi menjadi 4 kelompok: ki (kontrol), kii (psd), kiii (imobilisasi), dan kiv (footshock) dan diberikan perlakuan selama 25 hari. parameter yang diperiksa adalah kadar testosteron serum dan jumlah sel leydig. analisis statistik menggunakan analysis of variance (anova) dengan post hoc-lsd. hasil rata-rata kadar testosteron besarnya 0,07 ± 0,08 ng/ml dan rata-rata jumlah sel leydig 4,22 ± 0,96 dengan jumlah terendah pada stres jenis psd. ada perbedaan rata-rata kadar testosteron serum yang bermakna antara kontrol dan psd (p=0,014) dan stres footshock dengan psd (p=0,011) dan ada perbedaan jumlah sel leydig yang bermakna pada kelompok perlakuan dibanding kontrol (p=0,006). kesimpulan model stres psd menurunkan kadar testosteron dan jumlah sel leydig pada tikus putih jantan. mekanisme kerja psd terhadap kadar serum testoesteron masih belum jelas. kata kunci : sel leydig, stres kerja, testosteron serum, tikus putih jantan abstrak introduction in the projected numbers of indonesian w o r k e r s f o r t h e y e a r 2 0 1 3 , t h e r e i s a predominance of males, accounting for around 66.8 million out of 107.7 workers. therefore the impact of stress in male workers has the potential to cause huge business losses in the form of decreased performance, efficiency and work productivity.(1) work stress connected with male reproduction follows the psychogenic hypothesis that work stress is part of the cause of infertility problems, where work stress levels are higher in infertile men than in fertile men.(2) chronic stress accelerates the reduction in functions and numbers of leydig cells(3) through the hormonal system and oxidative stress by increasing the levels of glucocorticoids, which trigger an increase in reactive oxygen species (ros) and initiate leydig cell apoptosis, resulting in decreased levels of testosterone hormones.(4) the plasma membrane of leydig cells has a high phospholipid content and is therefore susceptible to ros, which is a lipid-, proteinand dna-oxidizer,(5) causing damage to leydig cells and decreased testosterone levels.(6) glucocorticoid can directly inhibit testosterone b i o s y n t h e s i s b y s u p p r e s s i o n o f 11 b e t a 29 hydroxysteroid dehydrogenase-1 (11â hsd1), a unique enzyme in leydig cells that controls intracellular active glucocorticoid concentration, thus protecting leydig cells from the negative effects of glucocorticoids.(7) the decreased level of the 11â hsd1 enzyme increases intracellular glucocorticoid levels, subsequently inhibiting the intracellular steroidogenic function of leydig cells by suppressing testosterone enzyme biosynthesis.(4) stress in experimental animals comprises several models, namely paradoxical sleep deprivation (psd), representing the physical and psychological stress of sleep deprivation;(8) i m m o b i l i z a t i o n , r e p r e s e n t i n g p h y s i c a l , psychological and social stress, which form the sedentary stress pattern;(9) and footshock stress, which is a reflection of physical stress.(10) these stress models were shown to be able to decrease t e s t o s t e r o n e l e v e l s . p a r a d o x i c a l s l e e p deprivation and footshock stress led to lowered testosterone levels of 13.7 ng/dl and 31.7 ng/ dl, respectively, as compared with 371.4 ng/dl in the control group,(11) while immobilization stress decreased testosterone levels.(12) however, there have been no studies comparing the effect of these stress models on serum testosterone levels and numbers of leydig cells, which is therefore the objective of our present study conducted on male rats. figure 1. psd models using the modified multiple platform method univ med vol. 33 no.1 30 arjadi, soejono, pangestu paradoxical sleep deprivation and testosterone methods research design this experimental study used a completely randomized post-test only design with control group and was conducted from october 2012 to january 2013 at the animal laboratory, medical and health sciences faculty, jenderal soedirman university, and the medico labora clinical laboratory. experimental animals the experimental animals were 24 male wistar albino rats (rattus norvegicus), aged 34 months and weighing 200-300 grams. the sample size was determined by means of federer ’s formula (t-1)(r-1) >15 where t= n u m b e r o f t r e a t m e n t s a n d r = n u m b e r o f replications. intervention experimental animals were divided equally into 4 treatment groups. k1 was the control group, in which the rats were left free in the cage and had normal sleep patterns; k2 was the stress model of psd, where the rats were treated using the modified multiple platform method (mmpd). in this method the animals were put in a tank measuring 123 x 44 x 44 cm and filled with water to a height of 1 cm. filled with water to a level 1 cm below the surface of the platforms. the tank had two rows of 14 platforms located at 10-cm distances from each other, and muscle atonia tools that were switched on automatically every 10 minutes to cause the sleeping animals to fall into the water and thus re-awaken. two days prior to the experiments, the rats underwent a social adaptation and interaction process with each other. the mmpd test was carried out for 18 hours from 04.00-22.00, after which the animals were returned to their cages and allowed to sleep for 6 hours from 22.00-04.00.(13) k3 was the group subjected to immobilization stress, by placing the animals in a 21-cm long transparent cylinder with a diameter of 6 cm for 1 hour/day. k4 was the footshock stress model group, in which the rats were placed on an acrylic box of 14x25x28 cm with an electrified base. the animals were subjected to 0.1 sec shocks of 5 ma for 1 hour/day at variable intervals every 5 minutes to avoid anticipation by the rats.(11) the shock treatment was given 4-6 times per day. all stress treatments were given at 09.00 am every day for 25 days to produce chronic stress.(14) hormonal assays blood samples were collected from the orbital plexus by means of hematocrit pipettes, put into individual test tubes and centrifuged at 4000 rpm for about 20 minutes to separate the blood serum, after which the serum-containing tubes were kept at -20°c until required for analysis. measurement of serum testosterone levels was by means of an enzyme-linked immunosorbent assay (elisa) reagent kit from rodent testosterone endocrine technologies inc. isolation of leydig cells the number of leydig cells was counted in 50 fields of view of the interstitial tissue in the left testis of the male albino rats (rattus norvegicus) at a magnification of 400 x. observations were made using a nikon eclipse e100® microscope equipped with an optilab® digital camera and image raster software. data analysis results of univariate analysis of the frequency distribution were given as mean ± s t a n d a r d d e v i a t i o n . n o r m a l i t y o f d a t a distribution was tested with the shapiro wilk test, while homogeneity of variance was tested using the levene test. the analysis was continued with one-way analysis of variance (anova), and for determining different of means in each treatment the post hoc lsd test was used for testosterone levels and number of leydig cells.(15) ethical clearance all experimental procedures were approved by the medical and health research ethics 31 committee, faculty of medicine, gadjah mada university. results table 1 shows that the mean serum testosterone level (0.07 ± 0.08) ng/ml) and the mean number of leydig cells (4.22 ± 0.96) were lowest in the psd stress model. the anova statistical test on serum testosterone levels (p=0.032) and the number of leydig cells (p=0.001) showed a significant difference in at least two treatment groups. figure 2 shows that the numbers of leydig cells decreased to their lowest value in the psd stress group. the results of post hoc lsd (p<0.05) showed a significant difference in the mean serum testosterone level between control and footshock groups on the one hand, and the psd group on the other. there was no significant difference figure 2. leydig cells of rattus norvegicus, he stain, 400 x. leydig cells (yellow arrows) with large round nuclei and eosinophilic cytoplasm. a) k1 (control); b) k2 (paradoxical sleep deprivation); c) k3 (immobilization); d) k4 (footshock) table 1. mean serum testosterone level and number of leydig cells by treatment groups values are mean ± sd; psd = paradoxical sleep deprivation univ med vol. 33 no.1 32 arjadi, soejono, pangestu paradoxical sleep deprivation and testosterone table 2. result post hoc test lsd of testosterone level and number of leydig cells psd = paradoxical sleep deprivation; * significantly different (p<0.05) (p>0.05) between control and immobilization versus footshock, psd versus immobilization, and immobilization versus footshock. the posthoc lsd test showed that the mean number of leydig cells in the three treatment groups was lower than in the control group, with the lowest number being in the psd group (table 2). discussion the effect of psd on number of leydig cells has never been studied, although the effect on t e s t o s t e r o n e l e v e l s h a s f r e q u e n t l y b e e n investigated. previous studies showed significant differences in plasma testosterone levels in rats treated with a variety of work stress models, such as psd, footshock, cold, and swimming, with the lowest plasma testosterone level being found in the psd stress test.(11) this was presumably due to the duration of the applied stress, since the psd test was applied for around 18 hours/ day, whereas the duration of the immobilization and footshock tests was only 1 hour/day.(16) other studies also showed that serum testosterone levels in rats given psd stress were lower than in those given food limitation stress.(17) the mean decrease in serum testosterone levels in the stress groups presumably led to suppressed testosterone production through several mechanisms, viz. suppression of the hypothalamus-pituitary-testis axis (hpt) by the hypothalamus-pituitary-adrenal axis (hpa) through inhibition of gonadotropin releasing hormone (gnrh) secretion and inhibition of enzyme activity that plays a role in testosterone biosynthesis and the leydig cell apoptosis induction process.(18) the initiation of leydig cell apoptosis by increased glucocorticoid levels occurs through several mechanisms, namely fas, procaspase-3 cleavage, loss of mitochondrial membrane potential (äøm) and increased ros. high g l u c o c o r t i c o i d l e v e l s c a u s e c h a n g e s i n mitochondrial membrane permeability through the intrinsic pathway as a result of activation of pro-apoptotic molecules (bax, bak, bad) and suppression of anti-apoptotic molecules (bcl-2, bcl-xl, bcl-w), causing cytochrome c release and activation of caspase-9, active caspase-8 and caspase-9 activate caspase-3. activation of this cascade leads to apoptosis of mature leydig cells due to the high number and sensitivity of their glucocorticoid receptors.(4) high glucocorticoid levels also increase r o s b y s u p p r e s s i n g t h e p r o d u c t i o n o f nicotinamide adenine dinucleotide phosphate (nadph) which is important in the regeneration of the antioxidant glutathione (gsh) from o x i d i z e d g l u t a t h i o n e . t h e d e c r e a s e i n antioxidants and increase in free radicals lead to oxidative stress,(19) which induces an increase in lipid peroxidation. the latter results in changes i n c e l l m e m b r a n e p e r m e a b i l i t y, s o t h a t cytochrome c releases (2) and oxidizes dna bases that produce 8-hydroxy-2-deoxiguanosine ( c a u s i n g m u t a t i o n s a n d d e l e t i o n s o f mitochondrial dna), while cytochrome c also leads to apoptosis through p53 activation.(20) the increase in glucocorticoids inhibits steroidogenic enzyme secretion due to the excessive oxidative 33 capacity of 11âhsd enzymes. another result of increased intracellular glucocorticoids is that the leydig cells cannot be protected from the adverse effects of glucocorticoids.(18) the lowest mean serum testosterone level in the psd stress group signifies that oxidative stress caused by high glucocorticoid levels leads to an increase in ros and also a decrease in the antioxidant glutathione (gsh).(21) in footshock stress, the increase in ros is caused by the electric shocks given to the animals, and reduces the effectiveness of antioxidant utilization in the body.(22) physical sleep deprivation is analogous to sleep deprivation in humans that increases the melatonin hormone levels, especially at night after sleep deprivation.(23) melatonin acts directly on neuronal gonadotropin-releasing hormone (gnrh) receptors to induce gonadotropin inhibiting hormone (gnih) expression in the hypothalamic paraventricular nuclei and the testis.(24) at central nervous system level, increases in gnih is effected by inducing gnih e x p r e s s i o n i n g n i h n e u r o n s o f t h e paraventricular nuclei through its melatonin subtype ic (mel1c) receptors. increased gnih inhibits gnrh and decreases the sensitivity of the pituitary to gnrh, resulting in lowered luteinizing hormone (lh) and follicle stimulating hormone (fsh). in the testis, binding of melatonin to its binding site on leydig cells suppresses steroidogenic acute regulatory (star) e x p r e s s i o n ( 2 5 ) t h u s d e c r e a s i n g t h e phosphorylation and activity of p450 side chain cleavage enzyme that converts cholesterol to pregnenolone, and ultimately to testosterone. the reduction in star protein expression is the key to steroidogenesis and decreases testosterone l e v e l s a f t e r 2 4 a n d 4 8 h o u r s o f s l e e p deprivation.(26) in this study, the numbers of leydig cells in the psd, immobilization, and footshock stress groups were lower than in the control group, the lowest being in the psd stress group. the duration of the applied psd stress was 18 hours, while the duration of the immobilization and footshock stresses was 1 hour per day, showing that stress is influenced by the duration of s t r e s s o r e x p o s u r e . ( 1 6 ) t h e i n c r e a s e d glucocorticoid levels decrease the number of leydig cells through the inhibition of mitosis and differentiation of leydig cell precursors or through an increase in the rate of leydig cell apoptosis, starting with the inhibition of hypothalamic gnrh synthesis and secretion. the decrease in lh and fsh secretion(27) further i n f l u e n c e s l e y d i g c e l l f u n c t i o n a n d differentiation. ( 2 8 ) st r e s s a l s o c a u s e s macrophages to produce hydrogen peroxide, resulting in oxidative stress in leydig cells,(29) causes leydig cell apoptosis through the extrinsic and intrinsic pathways, and increases p53 protein as a marker of dna damage.(30) a limitation of this study was that several b l o o d s a m p l e s u n d e r w e n t h e m o l y s i s o n aspiration, and could not be read with the microtiter well reader (elisa reader), thus necessitating repeat aspiration to obtain validated data. another limitation was that this study did not evaluate other types of stress in rats, such as starvation and social stress. the results of this study may be of use to company doctors, psychiatrists and andrologists in promoting health status, particularly fertility problems in male workers. in future, we will investigate the effect of a traditional medicine known as purwoceng (pimpinella pruatjan molk) as an aphrodisiac and antioxidant to improve the fertility status of rats with psd. conclusion after psd the serum testosterone levels and the leydig cells decreased in male rats. the mechanism by which psd affects serum testosterone is still unclear. acknowledgements the authors thank the directorate general of higher education, ministry of education and culture, for research funding through the hibah univ med vol. 33 no.1 34 arjadi, soejono, pangestu paradoxical sleep deprivation and testosterone fundamental scheme in 2011. thanks are also due to the head of the pathology anatomy laboratory, medical faculty, gadjah mada university, and the head of the experimental animal laboratory, medical faculty, jenderal soedirman university, for the use of their research facilities. references 1. tjiptoherijanto p. proyeksi penduduk, angkatan kerja, tenaga kerja, dan peran serikat pekerja dalam peningkatan kesejahteraan. majalah perencanaan pembangunan 2001;23:1-10. 2. sheiner ek, eyal s, refael c, gad p, ilana sv. potential association between male infertility and occupational psychological stress. j occup env med 2002;44:1093-9. 3. wang ff, wang q, chen y, lin q, gao hb, zhang p. chronic stress induces ageingassociated degeneration in rat leydig cells. asian j androl 2012;14:643-8. 4. gao hb, ming-han t, yan-qin h, hai-yan y, qiang-su g, ren-sha g, et al. mechanisms of glucocorticoid-induced leydig cell apoptosis. mol cell endocrinol 2003;199:153-63. 5. agarwal a, sekhon lh. antioxidant therapy on male fertility. human fertility 2010;13:217-25. 6. chigurupati s, tae gs, dong hh, justin dl, mohamed rm, jason s, et al. lifelong running reduces oxidative stress and degenerative changes in the testes of mice. j endocrinol 2008; 199:333-41. 7. hu g, lian q, lin h, latif sa, morris dj, hardy mp, et al. rapid mechanisms of glucocorticoid signaling in the leydig cell. j steroids 2008;73: 1018-24. 8. hipolide dc, suchecki d, pimentel cp, chiconelli f, sergio t, luz j. paradoxical sleep deprivation and sleep recovery: effects on the hypothalamic-pituitary-adrenal axis activity, energy balance and body composition of rats. j neuroendocrinol 2006;18:231-8. 9. kvetnansky r, mccarty r. stress immobilization. endocrinol 2007;2:503-6. 10. cui r, li b, suemaru k, araki h. differential effects of psychological and physical stress on the sleep pattern in rats. act medic okayama 2007;61:319-27. 11. andersen ml, bignotto m, machado rb, tufik s. different stress modalities result in distinct steroid hormone responses by male rats. brazilian j med biol res 2004;37:791-7. 12. al-damegh ma. stress-induced changes in testosterone secretion in male rats: role of oxidative stress and modulation by antioxidants. open j animal sci 2014;4:70-8. 13. machado rb, suchecki d, sergio t. comparison of the sleep pattern throughout a protocol of chronic sleep restriction induced by two methods of paradoxical sleep deprivation. brain res bull 2006;70:213–20. 14. joels m, henk k, harmen jk, paul jl. chronic stress: implications for neuronal morphology, function and neurogenesis. neuroendocrinol 2007;28:72-96. 15. dahlan ms. statistik untuk kedokteran dan kesehatan. jakarta: salemba medika;2008. 16. chen y, qian w, fei fw, hui bg, ping z. stress induces glucocorticoid-mediated apoptosis of rat leydig cells in vivo. stress 2012;15:74-84. 17. alvarenga ta, monica la, javier vm, sergio t. food restriction or sleep deprivation: which exerts a greater influence on the sexual behaviour of male rats? behavioral brain res 2009;202: 266-71. 18. hardy pm, hui-bao g, qiang d, renshan g, qiang w, wei rc, et al. stress hormone and male reproductive function. cell tissue res 2005;322:147-53. 19. patel r, mcintosh l, mclaughlin j, brooke s, nimon v, sapolsky r. disruptive effects of glucocorticoids on glutathione peroxidase biochemistry in hippocampal cultures. j neurochem 2002;82:118-25. 20. kregel kc, zhang hj. an integrated view of oxidative stress in aging: basic mechanisms, functional effects, and pathological considerations. am j physiol regul integr comp physiol 2007;292:r18-36. 21. tufik s, andersen ml, bittencourt lra, mello mt. paradoxical sleep deprivation: neurochemical, hormonal and behavioral alterations. evidence from 30 years of research. an acad bras sci 2009;81:521-38. 22. maslachah l, rahmi s, rahma k. hambatan produksi reactive oxygen species radikal superoksida (o 2 ) oleh antioksidan vitamin e pada tikus putih (rattus norvegicus) yang menerima stressor renjatan listrik. media kedokteran hewan 2008;24:21-6. 23. galvão mol, rita sc, suzi ek, sergio t, deborah s. paradoxical sleep deprivation activates hypothalamic nuclei that regulate food intake and stress response. psychoneuroendocrinol 2009;34:1176-83. 24. tsutsui k, george eb, takayoshi u, etsuko s, hong y, tomohiro o, et al. the general and 35 comparative biology of gonadotropin-inhibitory hormone (gnih). endocrinology 2007;153:36570. 25. frungieri mb, mayerhofer a, zitta k, pignataro op, calandra rs, calvar sig. direct effect of melatonin on syrian hamster testes: melatonin subtype 1a receptors, inhibition of androgen production, and interaction with the local corticotropin-releasing hormone system. endocrinology 2005;146:1541-52. 26. wu jl, wu rsc, yang jg, huang cc, chen kb, fang kh, et al. effects of sleep deprivation on serum testosterone concentrations in the rat. neuroscience lett 2011;494:124-9. 27. whirledge s, cidlowski ja. glucocorticoids, stress, and fertility. minerva endocrinol 2010;35: 109-25. 28. sriraman v, sairam mr, rao aj. evaluation of relative roles of lh and fsh in regulation of differentiation of leydig cells using an ethane 1,2-dimethylsulfonate-treated adult rat model. j endocrinol 2003;176:151-61. 29. gautam dk, misro mm, chaki sp, sehgal n. h 2 o 2 at physiological concentrations modulates leydig cell function inducing oxidative stress and apoptosis. apopt 2006;11:39-46. 30. maheshwari a, man mm, archana a, rajnesh s, deoki n. pathway involved in testicular germ cell apoptosis induced by h 2 o 2 in vitro. febs j 2009;870-81. univ med vol. 33 no.1 albendazole therapy reduces serum zinc in elementary school children with ascaris lumbricoides infections 206 *department of parasitology, faculty of medicine, tarumanagara university, jakarta **department of parasitology, faculty of medicine, trisakti university, jakarta ***department of parasitology, faculty of medicine, university of indonesia, jakarta correspondence: dr. ria buana, m.biomed department of parasitology, faculty of medicine, tarumanagara university jl. s. parman no. 1 jakarta barat 11440 +6221 5671781, +62812 9657 508 riabuana@ymail.com univ med 2016;35:206-12 doi: 10.18051/univmed.2016.v35.206-212 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 albendazole therapy reduces serum zinc in elementary school children with ascaris lumbricoides infections ria buana*, jane florida kalumpiu**, yenny djuardi***, and taniawati supali*** background soil-transmitted helminth (sth) infection can cause malnutrition and vice versa, and decrease serum zinc concentrations. no previous studies were found on the effect of sth treatment on serum zinc concentrations in elementary school children. the objective of this study was to evalaute the effect of sth treatment on serum zinc concentration in elementary school children in jakarta. methods a pre-experimental study (one group pre and post) was conducted on 3rd 5th grade students in one elementary school in north jakarta. flotac examination was used to determine infection status and sth intensity, while serum zinc concentrations were measured by atomic absorption spectrometry. all children were given albendazole 400 mg for three consecutive days. stool and blood samples were collected before and three weeks after treatment with albendazole. a paired t test was used to analyze the data. results the sth prevalence was 63.4%; these were mainly ascaris lumbricoides and trichuris trichiura infections. before treatment, children infected with a.lumbricoides and t.trichiura had significantly higher serum zinc concentrations (p=0.028; p=0.014). after treatment, children with a.lumbricoides had significantly lower serum zinc concentrations (p=0.039), while children with t.trichiura had slightly lower serum zinc concentrations (p=0.659) than before treatment. conclusions albendazole treatment produces lower serum zinc concentration in elementary school children with a.lumbricoides infections. a profound understanding of the interaction between helminth infections and zinc may assist in guiding integrated and sustainable intervention strategies among affected children throughout the world. keywords: serum zinc, albendazole, sth, children doi: http://dx.doi.org/10.18051/univmed.2016.v35.206-212 207 univ med vol. 35 no.3 introduction soil-transmitted helminth (sth) infection is the most frequent helminth infection in humans, particularly inhabitants of tropical or subtropical countries. the who estimates that more than 2 million people are infected with sth. helminth infection occurs more frequently in school-age children.(1) soil humidity, climate, environmental sanitation, lack of sources of clean water and habits of the inhabitants are factors that influence the high prevalence of sth infection.(1,2) in 2006, 195 million persons in indonesia resided in endemic areas and 50 million among them were school-age children.(3) in jakarta, the prevalence of sth infection is still high, particularly in crowded slum areas.(4) helminths can evade the host immune system, resulting in chronic infection. helminth infection stimulates the production of the cytokines interleukin-4 (il-4), il-5, il-10, il-13 and immunoglobulin e (ige).(5) the diagnosis of sth infection is established by finding helminth eggs in the stool by microscopy with the flotac technique, because of its higher sensitivity.(6) albendazole is an anthelminthic that effectively manages sth infection with substantially high cure rates for ascaris lumbricoides and hookworm infections, but is less effective in trichuris trichiura infections.(7) helminth infections and micronutrient deficiencies are highly prevalent in developing countries and often occur in the same individuals.(8) zinc is an essential element that is required for cell growth, development and differentiation, and plays a role in the synthesis of proteins, enzymes and transcription factors. zinc deficiency can occur as a result of malabsorption syndrome in chronic gastrointestinal disturbances.(9) zinc cannot be stored in the body and is excreted by the kidneys, the skin and the intestine, while its concentration is maintained through tissue catabolism and bone resorption.(10) helminths require zinc for their biology, such as the production of the zincdependent metalloproteinases, invasion of host tissues, digestion of food, development of their eggs, and for neutralizing the host immune system.(11) zinc deficiency in children can cause abnormalities of growth and cognitive development, and render them more susceptible to disease.(12) a number of previous studies found that serum zinc in children with helminth infections did not undergo significant changes.(13,14) since sth infections still occur frequently in indonesia, particularly in communities of low economic status with poor sanitation, and may have a negative impact on serum zinc concentration that can affect immunity, growth and cognitive function in children, it was considered necessary to carry out a study to determine serum zinc concentration in school-age children before and after treatment for sth infection. methods study design this was a pre-experimental study of one group preand post-test design that was conducted at kalibaru state elementary school 07 (sdn 07), cilincing, north jakarta from may to october 2014. study subjects the population comprised students of elementary school grades 3-5, and the inclusion criteria were: not consuming anthelminthic drugs in the last 6 months, healthy and without fever, agreeing to participate in this study with written informed consent, with their parents allowing them to participate in this study after signing informed consent. the exclusion criterion was refusal to consume albendazole. the estimated sample size was calculated using the formula for hypothesis testing of two paired populations by taking power of 80% and significance level of 5%, resulting in the required sample size of 32 children. in anticipation of drop outs (10%), the required sample size was taken as 35 children.(15) based on a preliminary survey performed in the sdn 07 kalibaru complex, yielding an sth infection 208 prevalence of 30%, the required sample was 117 children for screening. parasitological examination and treatment the stool samples were examined in august 2014 in the parasitology laboratory, faculty of medicine, university of indonesia, while the blood examination was performed in september 2014 in the seameo recfon laboratory, faculty of medicine, university of indonesia. the stool samples were examined by the flotac method, which is based on a centrifugal flotation technique. the weight of the stool samples was 1 gram per sample. the apparatus used in the flotac technique was a cylinder made of polycarbonate amorphous thermoplastic and consisting of 3 physical components (core components), i.e. base, translation disc and reading disc, in which two flotation chambers are located for holding the samples, each chamber having a volume of 5 ml (total volume = 10 ml). there are five accessories, i.e. screw, key, bottom, centrifuge adapter and microscope adapter, which together with the core components make up the assembled apparatus for use in the centrifugation process and at microscopic examination. the present study used the dual technique with two types of flotac solution, namely nacl and sucrose.(16) infection intensity was determined by counting the number of helminth eggs in 1 gram of feces (table 1).(17) albendazole 400 mg chewable tablets were administered orally and ingested on the spot by the respondents as witnessed by the investigators assisted by the form teacher. serum zinc serum zinc concentrations were determined in the seameo recfon laboratory, faculty of medicine, university of indonesia, using atomic absorption spectrometry (aas) to determine the quantities of chemical substances by measuring the radiation absorbed by the chemical substance in question. this is accomplished by reading the spectrum produced by the sample upon stimulation by radiation. the atoms will absorb ultraviolet or other rays and convert them into certain levels of energy. this method measures the amount of energy produced in the form of photons absorbed by the sample. a detector is used to measure the wave length of the transmitted radiation, while a processor integrates the absorbed wave length into the result. this method is based on the beer-lambert law. (18) data analysis the difference of serum zinc concentration before and after treatment was analyzed using the paired t test. statistical significance was defined as a p value below 0.05, ethical clearance this study was approved by the ethics commission, faculty of medicine, university of indonesia and cipto mangunkusumo hospital under no. 500/h2.f1/etik/2014. results a total of 127 children agreed to participate in this study, but only 115 stool samples and 88 blood samples were successfully collected. after treatment with albendazole 400 mg for 3 consecutive days, stool and blood samples were again collected, resulting in 82 stool samples and 87 blood samples. a total of 78 paired blood samples (before and after treatment) were successfully collected (figure 1). the majority of respondents were 10 years of age, of female gender, while their parents had low educational level (junior high school graduates), and most were employed as laborers table 1. infection intensity based on number of eggs per gram (epg).(17) buana, kalumpiu, djuardi albendazole therapy reduces serum zinc 209 univ med vol. 35 no.3 (table 2). upon examination of the 115 stool samples before treatment, 72 (63.4%) children were found to be infected with sth, consisting of 30 (26.0%) children with a.lumbricoides, 20 (17.4%) children with t.trichiura, and 23 (20.0%) children with mixed infection (table 2). in the 82 samples after treatment, a decrease in the prevalence of sth infection was found, in which 14 (17.1%) children still had helminth infection, consisting of 3 (3.6%) children with a.lumbricoides, 10 (12.2%) children with t. trichiura, and 1 (1.2%) child with mixed infection (a.lumbricoides and t.trichiura). before treatment, sth infection was more prevalent in female children than in male children, but after treatment the prevalence of sth infection in children of both genders was reduced. before treatment, the majority of children with a.lumbricoides infection had mild infection (1-4999 epg), while the remaining children had moderate infection (5.000 – 49.000 epg). in the mixed infections, the intensity of a.lumbricoides infection was mild to moderate, whereas t.trichiura infection was of mild intensity only (1-999 epg). after treatment, the prevalences of sth infection, both moderate and mild, were substantially reduced in each type of sth infection (data not shown). table 2. distribution of sosciodemographic characteristics, nutritional and sth infection status (n=115) *al : ascaris lumbricoides; tt : trichuris trichiura 210 after treatment, mean serum zinc concentration decreased by 0.15 ± 1.95 µmol/l in children with sth infection and by 0.36 ± 1.86 µmol/l in children without sth infection. using the paired t-test, no significant differences were found in mean serum zinc concentration before and after treatment, both in children with sth infection (p=0.583) and in those without sth infection (p=0.302) (table 3). to test for differences in mean serum zinc concentration for each type of helminth infection before and after treatment, an analysis was made with the paired t-test, which found a significant decrease in mean serum zinc concentration after treatment in children with a.lumbricoides infection (p=0.039), but no significant decrease in children with t.trichiura infection and in those without infection (p=0.659). on the other hand, in children with mixed infection a nonsignificant increase in mean serum zinc concentration was found after treatment (p=0.082) (table 3). discussion before treatment, serum zinc concentration in children with sth infection was higher than in children without infection, but there were no significant differences. this finding agrees with that of a study conducted by osei et al.(19) in elementary school children in india, in that helminth infection of the intestine does not influence zinc concentration. however, this study did not differentiate between helminth species. in contrast, in our study serum zinc concentration was analyzed in relation to the infecting helminth species. our results differ from those of a study carried out by kongsbak et al.(20) in bangladesh, where it was found that children with sth infection had significantly lower serum zinc concentrations than those without infection. there are several possible mechanisms that may clarify the differences between our study results and those of the aforementioned study. firstly, these differences may be caused by differing tissue catabolism as a result of the helminth infection, thus increasing the serum zinc concentration in our study. secondly, sth infection may cause abnormal absorption of zinc, resulting in compensatory changes in the form of increased synthesis of zinc transporters, such as zip4 that transports zinc from the intestinal lumen into enterocytes and znt1 that functions to transport zinc out of the enterocytes into the blood, leading to increases in serum zinc concentrations.(21) after treatment, serum zinc concentration was significantly decreased in children with a.lumbricoides infection, but not significantly in children with t.trichiura infection. furthermore, in children with mixed infection or without infection, there was a nonsignificant increase in serum zinc concentration. these results may be due to the fact that the small intestine is undergoing mucosal repair after helminth infection, thus causing a decrease in zinc transporter molecules in the enterocytes (zip4 and znt1),(22) and also decreasing the amount of zinc table 3. relationship between sth infection and serum zinc concentration before and after treatment apaired t-test; * significant; ** mean zinc concentration after treatment subtracted by mean zinc concentration before treatment buana, kalumpiu, djuardi albendazole therapy reduces serum zinc 211 univ med vol. 35 no.3 that is transported into the blood. in addition, the body may be attempting to remove the helminths that had died after treatment, through the cytokines il-4, il-5, il-9, il-10, and il-13, that require more zinc for their production and activity.(23) another factor may be the possibility of other infections that were not investigated in this study, that can cause decreased zinc concentration, such as infection with the intestinal protozoa giardia lamblia.(24. 25) the present study has several limitations, the first being that no investigation was done on possible infection with intestinal protozoa, such as g.lamblia. the second limitation is the possibility that several blood samples underwent hemolysis, thus increasing serum zinc concentration. further studies are required, with longer surveillance periods and higher infection intensities, including the possibility of other infections that were not investigated in this study apart from sth infection. conclusions treatment with the maximal dose of albendazole causes decreased serum zinc in children with sth infection. a profound understanding of the interaction between helminth infections and zinc may assist in guiding integrated and sustainable intervention strategies among affected children throughout the world. conflict of interest the authors declare that there is no conflict of interest in this study. acknowledgment the investigators are greatly indebted to all parties without whose participation this study would not have been conducted. references 1. world health organization. eliminating soil transmitted helminthiases as a public health problem in children. progress report 2001-2010 and strategic plan 2011-2020. geneva: world health organization; 2012. 2. brooker s, clements ac, bundy da. global epidemiology, ecology and control of soiltransmitted helminth infections. adv parasitol 2006;62:221-61. 3. tan m, kusriastuti r, savioli l, et al. indonesia: an emerging market economy beset by neglected tropical diseases (ntds). plos negl trop dis 2014;8:e2449. 4. mardiana, djarismawati. prevalensi cacing usus pada murid sekolah dasar wajib belajar pelayanan gerakan terpadu pengentasan kemiskinan di wilayah kumuh di daerah dki jakarta. jurnal ekologi kesehatan 2008;72:76974. 5. bethony j, brooker s, albonico m, et al. soiltransmitted helminth infections: ascariasis, trichuriasis, and hookworm. the lancet 2006;367:1521-32. 6. utzinger j, rinaldi l, lohourignon lk, et al. flotac: a new sensitive technique for the diagnosis of hookworm infections in humans. trans roy soc trop med 2007;102:84-90. 7. samuel a, degarege ab, erko b. efficacy and side effects of albendazole currently in use against ascaris, trichuris and hookworm among school children in wondo genet, southern ethiopia. parasitol int 2014;63:450-5. 8. hurst rj, else kj. retinoic acid signalling in gastrointestinal parasite infections: lessons from mouse models. parasite immunol 2012;34:351– 9. 9. john e, laskow tc, buchser wj, et al. zinc in innate and adaptive tumor immunity. j transl med 2010;8:118. 10. de benoist b, darnton-hill i, davidsson l, et al. conclusions of the joint who/unicef/ iaea/izincg interagency meeting on zinc status indicators. food nutr bull 2007;28 suppl 3:s480-4. 11. dzik jm. molecules released by helminth parasites involved in host colonization. acta biochim pol 2006;53:33-64. 12. cole cr, grant fk, ellis ed, et al. zinc and iron deficiency and their interrelations in lowincome african american and hispanic children in atlanta. am j clin nutr 2010: 91:1027-34. 13. osei a, houser r, bulusu s, et al. nutritional status of primary schoolchildren in garhwali himalayan villages of india. food nutr bull 2010;31:221-33. 14. orden ab, apezteguía mc, ciarmela ml, et al. nutritional status in parasitized and nonparasitized children from two districts of 212 buenos aires, argentina. am j hum biol 2014; 26:73-9. 15. madiyono b, moeslichan s, sastroasmoro s, et al. perkiraan besar sampel. in: sastroasmoro s, ismail s (editors). dasar-dasar metodologi penelitian klinis, 4th ed. sagung seto: jakarta; 2011.p.348-82. 16. cringoli g, rinaldi l, maurelli mp, et al. flotac: new multivalent techniques for qualitative and quantitative copromicroscopic diagnosis of parasites in animals and humans. nat protoc 2010;5:503-5. 17. world health organization. prevention and control of schistosomiasis and soil-transmitted helminthiasis, who technical report series, no. 912. geneva: who;2002. 18. garcía r, báez ap. atomic absorption spectrometry (aas). in: farrukh am, editor. atomic absorption spectroscopy. croatia: intech open;2012. 19. osei a, houser r, bulusu s, et al. nutritional status of primary school children in garhwali himalayan villages of india. food nutr bull 2010;31:221-33. 20. kongsbak k, wahed ma, friis h, et al. acute phase protein levels, t. trichiura, and maternal education are predictors of serum zinc in a crosssectional study in bangladeshi children. j nutr 2006;136:2262-8. 21. roohani n, hurrell r, kelishadi r, et al. zinc and its importance for human health: an integrative review. j res med sci 2013;18:144– 57. 22. geiser j, de lisle rc, andrews gk. the zinc transporter zip5 (slc39a5) regulates intestinal zinc excretion and protects the pancreas against toxicity. plos one 2013;8:e82149. 23. allen je, maizels rm. diversity and dialogue in immunity to helminths. nature reviews immunol 2011;11:375-88. 24. zarebavani m, dargahi d, einollahi n, et al. serum levels of zinc, copper, vitamin b12, folate and immunoglobulins in individuals with giardiasis. iranian j publ health 2012;41:47-53. 25. blackwell ad, martin m, kaplan h, et al. antagonism between two intestinal parasites in humans: the importance of co-infection for infection risk and recovery dynamics. proc r soc b 2013;280:1671-8. buana, kalumpiu, djuardi albendazole therapy reduces serum zinc puspa1 6 *medical profession study program, medical faculty trisakti university **cipete selatan health center correspondence shinta larasati h medical profession study program, medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 universa medicina 2008; 27: 6-11. anthropometric indexes such as body mass index (bmi), waist circumference (wc), hip circumference (hc), and waist hip ratio (whr), are useful measurements to provide important information on blood glucose concentration. the aim of this study was to determine several anthropometric measurements, in particular bmi, wc, hc and whr, in their ability to predict the blood glucose levels in men and women 40 to 60 years of age. a cross-sectional study was conducted on 44 men and 127 women aged 40 to 60 who lived in cipete selatan subdistrict, south jakarta. blood glucose levels were assessed and anthropometric indexes such as bmi, wc, hc, whr were measured. multiple linear regression analysis was used to determine the best predictor for blood glucose levels. the study showed that the prevalence of diabetes mellitus (dm) type 2 was 12.9% and higher in men (20.5%) than in women (10.2 %). the significant predictive variables in the simple regression analysis were age and wc. multiple linear regression showed that after adjustment for age, wc was positively associated with blood glucose levels. standardized β value was 0.172 (p=0.026). wc predicts blood glucose levels, beyond that explained by traditional diabetic risk factors and bmi. the findings support the recommendation that wc be a routine measure for identification of dm type 2 in men and women aged 40 to 60. keywords: anthropometric indexes, blood glucose levels, diabetes mellitus type 2, forty to sixty years introduction the increase in the number of diabetes mellitus (dm) type 2 patients all over the world h a s b e e n p r e d i c t e d b y t h e e x p e r t s . ( 1 ) t h e prevalence rate of dm in the developing world, e s p e c i a l l y i n t h e a s i a p a c i f i c r e g i o n w a s increasing compared to that in other regions. in thailand, during 2001 the prevalence of dm type 2 among the population aged ≤35 was 9.6%, an increase of 20% over a period of 5 years.(2) according to the survey that was conducted by waist circumference as a predictor for blood glucose levels among men and women 40 to 60 years of age january-march 2008january-march 2008january-march 2008january-march 2008january-march 2008 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 abstract shinta larasati h*, intan nevita ob*, puspa krisna r*, and eva susiyanti** universa medicina 7 universa medicina vol.27 no.1 t h e wo r l d h e a l t h o r g a n i z a t i o n ( w h o ) , indonesia occupied the 4th place in the number of dm patients in the world after india, china a n d t h e u n i t e d st a t e s . d u r i n g 2 0 0 0 , t h e prevalence of the dm was 8.4 million and in 2030 it is expected to increase 21.3 million.(1) data from the department of health showed that the number of dm inpatients and outpatients treated in the hospital occupied the highest rate among all endocrine disorders.(3) in developing countries the highest prevalence of dm type 2 was in people 45 to 65 years of age.(4) the complications that could result from dm, including coronary heart disease (chd), need a larger expenditure for medical treatment. therefore, early diagnosis and treatment of dm are very important in preventing the chd complication after the onset of diabetes. several factors that could increase incidence of dm type 2 are sedentary habits, obesity, excessive consumption of polyunsaturated fats and refined sugars and smoking. several studies showed that obesity was correlated with the increase of the p r e v a l e n c e o f h y p e r t e n s i o n , d m a n d dyslipidemia.(5) anthropometric indexes such as body mass index (bmi), waist circumference (wc), hip circumference (hc) and waist-hip ratio (whr) are anthropometric measurements that could give risk information on dm and chd.(6) the who and the national institute of health have defined bmi, wc, hc, whr cutoff l e v e l s f o r w h i t e , b l a c k a n d h i s p a n i c american adults; however, these definitions are not applicable yet to other populations.(7,8) it is b e n e f i c i a l t o h e a l t h c a r e t o a s s e s s w h i c h anthropometric measurement are associated with the presence of dm in different populations. although it is argued that there is no justification for general population screening, early detection of individuals at risk of diabetes could be beneficial because early intervention has the potential to prevent the development of diabetes and its complications. the identification of individuals at risk of diabetes and treatment of risk factors are therefore relevant to prevent c a r d i o v a s c u l a r d i s e a s e a n d m o r t a l i t y i n d i a b e t e s . ( 9 ) t h e a i m o f t h i s s t u d y w a s t o d e t e r m i n e t h e a b i l i t y o f a n t h r o p o m e t r i c measurements, in particular bmi, wc, hc, and whr in predicting risk of type 2 diabetes in people 40 to 60 years of age. methods research design a cross-sectional study was conducted during october to november 2007 among people 40 to 60 years of age. study subjects study subjects were men and women 40 to 60 years of age who lived in cipete selatan subdistrict south jakarta. a simple randomized sampling design was used to select the subjects. the inclusion criteria were subjects aged 40 to 60 years and no history of diabetes. after exclusion of participants with any history of severe diseases (cancer, liver cirrhosis), mental r e t a r d a t i o n a n d l a c k o f c o m m u n i c a t i o n , information on subject characteristics such as age, g e n d e r, l e v e l o f e d u c a t i o n , e x e r c i s e a n d occupation were collected. measurements the anthropometric measurements used to calculate bmi, by standard procedures, included both height and weight. height was measured u s i n g m i c r o t o i s p o r t a b l e a n d w e i g h t w a s measured using sage portable scales to the nearest 0.1 kg. bmi was calculated as the weight (kg) divided by the square of the height (m). wc was taken as the minimum circumference between the umbilicus and xiphoid process and m e a s u r e d t o t h e n e a r e s t 0 . 5 c m . h c w a s measured as the maximum circumference around the buttocks posteriorly and the symphysis pubis 8 anteriorly and measured to the nearest 0.5 cm. whr was then calculated. blood was taken for measurement of casual plasma glucose (pg). the american diabetes association (ada) criteria were used to diagnose diabetes.(10) diabetes was defined as a casual plasma glucose > 200 mg/dl (11.1 mmol/l). statistical methods differences in age, height, weight, bmi, w c , h c , w h r a n d b l o o d g l u c o s e l e v e l between men and women were examined by student’s t test. to test the contribution of age, wc, hp, bmi, whr on casual blood glucose concentration as dependent variable a single r e g e s s i o n a n a l y s i s w a s u s e d . a m u l t i p l e step-wise regression analysis, including all variables that were significantly (p < 0.05) associated with blood glucose levels, was used t o d e t e r m i n e t h e b e s t p r e d i c t o r f o r b l o o d glucose concentrations. all analyses were performed using spss/pc statistical program ( v e r s i o n 1 1 . 0 f o r wi n d o w s ; s p s s , inc.chicago,il). results the subjects consisted of 127 (74.3%) women and 44 (25.7%) men. among the 171 participants without any signs and symptoms of diabetes, the prevalence of dm type 2 was 12.9%. their age, anthropometric indexes, blood glucose levels and prevalence of type 2 dm are summarized in table 1. the means of height, weight and wc were significantly higher in men compared to women. in men, wc (88.3 ± 10.8 c m ) w a s s t a t i s t i c a l l y s i g n i f i c a n t l y h i g h e r compared to women (83.5 ± 9.4 cm) (p=0.005). in women, hc (92.6 ± 9.8 cm) was statistically significantly higher compared to men (92.6 ± 9.4 cm) (p=0041). whr was also statistically significant higher in men (098 ± 0.06) compared to women (0.93 ± 0.04) (0.000), but blood glucose level was not significantly different between men and women (p=0.197). table 2 presents the results of the simple linear regression analysis in which several anthropometric indices were used to predict b l o o d g l u c o s e l e v e l s . a g e a n d w c h a d significantly positive association with casual blood glucose levels. bmi, hc, and whr were not significantly associated with casual blood glucose level. multiple linear regression models showed that wc was the best predictor for blood glucose levels among men and women 40 to 60 years of age. wc was statistically significantly associated with blood glucose levels (p=0.026) (table 3). table 1. anthropometry and prevalence of type 2 dm in the studied population *bmi = body mass index; **wc = waist circumference; ***whr = waist to hip ratio; **** hc = hip circumference; §dm = diabetes mellitus type 2; ( ) = standard deviation larasati, nevita, krisna, et al waist circumference for blood glucose 9 universa medicina vol.27 no.1 discussion in the present study, the prevalence of dm type 2 was 12.9% among men and women 40 to 60 years of age. this result was lower compared to a survey in mexico, where the prevalence of dm type 2 among men and women over 20 was 26.6%.(11) the prevalence of dm in both genders was higher than that found in the american survey (dm 7.8%), probably because different age groups were not considered.(12) the primary finding of this study was that wc significantly predicted the levels of blood glucose, beyond that explained by commonly evaluated diabetic risk factors such as weight, bmi and whr. conversely, bmi did not predict diabetes after consideration of common diabetic risk factors and wc. a study in jakarta on men and women of 35 to 55 years, a showed similar result. wc was the best predictor for screening dm type 2 compared to bmi and whr.(13) the recent consensus statement of the ada, the obesity society, and the american society for nutrition questioned the sequence of clinical measures of dm and, more importantly, the relevance of wc measurement in clinical practice.(14) schulze et al showed that wc was the strongest associations of single anthropometric measures for dm type 2.(15) several previous cohort studies(16-19) that compared different anthropometric measurements with regard to d i a b e t e s r i s k p r e d i c t i o n s s u g g e s t e d t h a t anthropometric measurements that described central fat distribution, in particular wc, may be superior to measurements of general adiposity. a cohort study of 721 mexican americans aged 2 5 6 4 y e a r s w h o w e r e f r e e o f n o n i n s u l i n dependent diabetes mellitus (niddm) at baseline and were followed for an average of 7.2 years, showed that wc was the only significant predictor o f n i d d m i n m o d e l s t h a t i n c l u d e d o t h e r anthropometric variables.(20) but a meta analysis based on published studies from 1996 to 2004 retrieved from pubmed search showed that bmi, wc and whr have similar associations with diabetes.(21) waist circumference was also more c l o s e l y r e l a t e d t o p o t e n t i a l l y a t h e r o g e n i c metabolic disturbances associated with abdominal obesity. distribution of body fat, especially abdominal localization, is a more important determinant than the total amount of body fat for the development of dm type 2. increased wc was more consistently associated with the table 2. simple regression analysis of predictors for blood glucose levels in men and women *bmi = body mass index, ** wc = waist circumference, *** hs = hip circumference, *** whr = waist to hip ratio table 3. multiple linear regression model for age and wc on blood glucose levels *wc = waist circumference 10 increased plasma insulin levels.(22) wc alone was strongly related to risk of myocardial infarction and this association remained significant after adjustment for other risk factors.(23) from the c l i n i c a l p e r s p e c t i v e , c e n t r a l o b e s i t y (approximated by wc or whr) is known to generate diabetogenic substances and should therefore be more informative than general obesity (bmi). lifestyle modification has been proven to effectively prevent and delay the development of diabetes.(24,25) therefore, early recognition of and intervention for the condition will be beneficial, particularly as cardiovascular complications set in early after the onset of diabetes.(26) knowledge of the risk of diabetes could enhance people’s awareness, leading to lifestyle modification. among the modifiable risk factors that played a substantial role on dm was obesity, measured by bmi or wc. in the present study, wc was found to increase blood glucose levels. wc was better than whr and bmi for identifying the blood glucose levels among subjects 40 to 60 years of age. conclusion waist circumference appeared to be a better predictor than any other anthropometric indices among men and women. generally, measurement of anthropometric indices other than wc had little predictive information on blood glucose levels. acknoledgement we thank the respondents who took part in this study, and to prof.dr.dr.adi hidayat ,ms for revision of this paper. references 1. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care 2004; 27: 1047–53. 2 aekplakorn w, stolk rp, neal b, suriyawongpaisal p, chongsuvivawong v, et al . the prevalence and management of diabetes in thai adults: the international collaborative study of cardiovascular disease in asia. diabetes care 2003; 26: 2758–63. 3. departemen kesehatan republik indonesia. jumlah penderita diabetes indonesia ranking ke-4 di dunia. available at: http://www.depkes.go.id/ index.php?option=news&task=viewarticle&sid=1183. accessed august 12, 2007. 4. king h, aubert re, herman wh. global burden of diabetes, 1995–2025: prevalence, numerical estimates, and projections. diabetes care 1998; 21: 1414–31. 5. must a, spadano j, coakley eh, field ae, colditz g, dietz wh. the disease burden associated with overweight and obesity. jama 1999; 282: 15239. 6. us national institutes of health. clinical guidelines for the identification, evaluation, and treatment of overweight and obesity in adults. national institutes of health: bethesda, md; 1998. 7. world health organization. obesity. preventing and managing the global epidemic. report of a who consultation on obesity, geneva, 3–5 june. world health organization: geneva; 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naaso, the obesity society; the american society for nutrition; and the american diabetes association. diabetes care 2007; 30: 1647–52. 15. schulze mb, heidemann c, schienkiewtz a, bergmann mm, hoffmann k, boeing h. comparison of anthropometric characteristics in predicting the incidence of type 2 diabetes in the epic-potsdam study. diabetes care 2006; 29: 1921-3. 16. stevens j, couper d, pankow j, folsom ar, duncan bb, nieto fj, et al. sensitivity and specificity of anthropometrics for the prediction of diabetes in a biracial cohort. obes res 2001; 9: 696-705. 17. snijder mb, dekker jm, visser m, bouter lm, stehouwer cd, kostense pj, et al. associations of hip and thigh circumferences independent of waist circumference with the incidence of type 2 diabetes: the hoorn study. am j clin nutr 2003; 77: 1192– 7. 18. sargeant la, bennett fi, forrester te, cooper rs, wilks rj. predicting incident diabetes in jamaica: the role of anthropometry. obes res 2002; 10: 7928. 19. janiszewiski pm, janssen i, ross r. does waist circumference predict diabetes and cardiovascvular disease beyond commonly evaluated cardiometabolic risk factors? diabetes care 2007; 30: 3105-9. 20. wei m, gaskill sp, haffner sm, stern mp. waist circumference as the best predictor for noninsulin dependent diabetes mellitus (nidmm) compared to body mass index, waist/hip ratio and other anthropometric measurements in mexican american – a 7 year prospective study. obes res 1997; 5: 16-23. 21. vazquez g, duval s, jacobs dr jr, silventoinen k. comparison of body mass index, waist circumference, and waist/hip ratio in predicting incident diabetes: a meta-analysis. epid rev 2007; 29: 115-28. 22. gibson rs. anthropometric assessment of body size. in: principles of nutritional assessment. 2nd ed. new york: oxford university press; 2005. pp. 245-98. 23. walling ad.waist to hip ratio better predictor of disease than bmi. am fam physician 2006; 73: 2046-7. 24. tuomilehto j, lindstrom j, eriksson jg, valle tt, hamalainen h, ilanne-parikka, et al. the finnis diabetes prevention study group: prevention of type 2 diabetes mellitus by changes in lifestyle among subjects with impaired glucose tolerance. n engl j med 2001; 344:1343-50. 25. lindstrom j, tuomilehto j. the diabetes risk score: a pratical tool to predict type 2 diabetes risk. diabetes care 2003; 26: 725-31. merijanti1 65 department of occupational medicine, medical faculty, trisakti university correspondence adr. lie t. merijanti, mkk department of occupational medicine, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2802 email : lie_mery@hotmail.com univ med 2008; 27: 65-71. the role of night shift work on blood pressure among healthy female nurses april-june 2008april-june 2008april-june 2008april-june 2008april-june 2008 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 abstract universa medicina lie t. merijantia, diana samara, reza tandean, and ridwan harrianto although hypertension is probably the most common disease on earth, little is known about the association between shift work and hypertension. the aim of this study was to determine the relationship of night shift work and blood pressure among healthy nurses. a cross-sectional study was performed from march to april 2007 among 152 healthy female nurses from one hospital in central jakarta. historical data were obtained from a self-administered detailed questionnaire, while anthropometric and blood pressure variables were measured directly. shift workers had a significantly lower age (29.2 ± 5.6 years) and shorter job experience (6.9 ± 5.9 years) than day workers (age = 36.5 ± 8.7 years; job experience 15.4 ± 8.4 years). there was a higher prevalence of overweight individuals among shift workers (31/43 = 72.1%) in comparison with day workers (12/43 = 27.9%), even though the difference in prevalence did not reach statistical significance (p=0.121). analysis of covariance yielded a significant relationship between shift work and systolic blood pressure (p=0.046), even after taking into account age and job experience. systolic blood pressure was significantly influenced by shift working, while diastolic blood pressure was not (p=0.283). working in the hospital on a 24-hour shift leads to abnormally high systolic blood pressure in female nurses, thus suggesting that this type of work may be a risk factor for cardiovascular disease. keywords : shift work, systolic blood pressure, female nurses introduction according to the most recent european union (eu) survey on working conditions,(1) carried out in the year 2000 in the 15 eu countries, only 24% of the working population (27% of employed and 8% of self-employed workers) are now engaged in the so-called ‘normal’ or ‘standard’ daywork, that is between 07.30–8.00 and 17.00–18.00 h from monday to friday. this means that the vast majority of workers are engaged on ‘non-standard’ working hours, including shift and night work, part-time work, weekend work, compressed work week, 66 merijanti, samara, tandean, et al shift work and blood pressure varying working hours, split shifts, seasonal work, on-call work, etc. various types of work schedules have been adopted by organizations and companies in which working hours are extended to evening and night hours. shift work is one of work hour systems in which a relay of employees extends the period of service beyond the conventional eight hour working day.(2) numerous psychological and physiological variables have been found to have a demonstrable 24-hour, circadian rhythm (latin: circa = about, and dies = a day), e.g. body temperature, the sleep-wake cycle, cardiovascular parameters, c o g n i t i v e p e r f o r m a n c e , e n d o c r i n e a n d immunological factors, therapeutic response to certain medications, and psychological variables of mood and anxiety. b i o l o g i c a l r h y t h m s a r e a n e s s e n t i a l component of homoeostasis: ‘everything is rhythmic unless proved otherwise’. most rhythms are driven by an internal biological clock located in the hypothalamic suprachiasmatic nucleus.(3) blood pressure (bp) is also modulated in a circadian rhythm over a 24-h cycle, where the dipper pattern shows high bp in the daytime and low bp at night. it has been suggested that the rhythm closely follows the sleep-wakefulness cycle and changes in bp are merely a result of differences in physical activity; thus, the circadian bp rhythm is largely independent of the internal circadian rhythm.(4) however, some abnormal conditions, such as autonomic failure or small lacunar infarcts, show non-dipper bp patterns independent of the sleep-wakefulness cycle. it is quite appropriate that attention is paid to shift work as a very important feature of sociotechnical systems, which may adversely affect mental and physical health, social life and safety of shiftworkers. in the medical domain, physicians, nurses and other ancillary staff are expected to be performing their jobs or to be on-call around-the-clock. studies have shown that a greater percentage of health service workers are engaged in a shift system than in any other employment sector. hospitals, the biggest employers in the health care field, employ more night shiftworkers than any other industry.(5) shift work is also usually associated with hypertension, but positive(6-8) and negative(9,10) relationships have been reported. the aim of this study was to evaluate the relationship of night shift work and blood pressure among healthy female nurses working in a hospital. methods study design from march to april 2007 a cross-sectional study was carried out among healthy female nurses in hospital x, central jakarta. study subjects the study sample consisted of female nurses actively employed at hospital x in central jakarta and meeting the inclusion c r i t e r i a , i e . f e m a l e n u r s e s w h o h a d b e e n employed for more than 1 year and were willing to participate in the study by signing an informed consent form. excluded from the study were nurses with a history of coronary heart disease, stroke, renal disease, or hypertension. the study protocol has been approved by the research ethics committee at the medical faculty of trisakti university data collection all participants in the study filled out a questionnaire relating to age, level of education, u s e o f h o r m o n a l c o n t r a c e p t i v e s , j o b characteristics, including length of employment and work unit. additionally, there were questions on factors external to the job, such as sports, smoking habits, coffee drinking, and periodical medical examinations. shift workers were defined as those nurses answering yes to the 67 question: “do you have shift work, or do you work during weekends?” body weight was recorded to the nearest kilogram on barefoot subjects using health scale instruments, while body height was measured with a mikrotoise to the nearest centimeter without shoes. body mass index (bmi) was calculated as the weight (kg) divided by the height squared (m2), and defined b y t h e f o l l o w i n g t h r e s h o l d s : u n d e r w e i g h t (<18.5), normal (18.5 – 25.0) and overweight (>25.0) kg/m2. blood pressure was measured after five minutes’ rest in a relaxed condition. systolic (sbp) and diastolic blood pressure (dbp) readings were recorded as the mean of two measurements with the subjects seated and using a mercury sphygmomanometer with an appropriate cuff size. statistical analysis for the statistical analysis of the collected data the spss program version 11.5 was used. c o m p a r i s o n o f t h e m e a n s o f c o n t i n u o u s variables between shift workers and day workers was performed by independent student’s t test, while the chi-squared test was used to evaluate the differences between the two groups for the categorical variables. evaluation of the influence of independent continuous and categorical variables on dependent variables (sbp and dbp) was by analysis of covariance. the minimal statistical significance level was defined at p<0.05. results general characteristics of the study subjects the subjects consisted of 152 female nurses with a mean age of 39.9 ± 7.2 years and a mean body mass index of 21.7 ± 7.1 kg/m2. among these subjects, 116 (76.3%) were shift workers and 36 (23.7%) day workers, while five persons (3.3 %) were smokers and 33 (21.7%) coffee drinkers (table 1). the mean job experience of the nurses was 8.9 ± 2.6 years, while mean dbp was 73.8 ± 8.4 mmhg and mean sbp was 111.0 ± 10.2 mmhg. table 1. characteristics of female nurses univ med vol.27 no.2 68 merijanti, samara, tandean, et al shift work and blood pressure table 3. comparison of several cathegorical variables between day workers and shift workers among female nurses (n=152) shift workers had a significantly lower age (29.2 ± 5.6 years) and shorter job experience (6.9 ± 5.9 years) than day workers (age 36.5 ± 8.7 years; job experience 15.4 ± 8.4 years). (table 2) o v e r w e i g h t i n d i v i d u a l s w e r e m o r e prevalent in shift workers (31/43 = 72.1%) than in day workers (12/43 = 27.9%), even though t h i s d i f f e r e n c e d i d n o t r e a c h s t a t i s t i c a l significance (x2 test= 4.229; p=0.121). day table 2. comparison of several continuous variables between day workers and shift workers among female nurses (n=152) table 4. analysis of covariance in the 152 female nurses 69 workers were more frequently smokers (3/ 5=60%) than were shift workers (2/5=40%), and this difference also did not reach statistical significance (÷2 test = 3.772; p=0.052). (table 3). table 4 shows the results of the analysis of covariance, which in particular indicated a significant influence of shift work on sbp, even after adjustment for age and job experience, while the influence on dbp was insignificant. discussion this study shows that, while dbp was not significantly influenced by shift working, shift workers have significantly higher sbps than day workers, independent of age and job experience. this is an important finding since a higher sbp is associated with an increased risk for morbidity and mortality from cardiovascular disease. these results are consistent with previous studies showing that shift work is a significant and independent risk factor for the progression of hypertension in male japanese workers.(7) a study in korea revealed that shift work was significantly associated with sbp in male workers aged 30 or more, while among female nurses younger than 30 years, shift work was inversely associated with dbp.(11) a study among male factory workers in malaysia showed that prevalence of hypertension was significantly h i g h e r i n s h i f t w o r k e r s c o m p a r e d t o d a y workers.(12) karlsson et al.(13) and bøggild et al.(14) found no difference in the prevalence of hypertension when comparing shift workers and day workers, but ohira et al(15) demonstrated a rise in either dbp or sbp in shift workers. the exact mechanisms that link shift work and greater cardiovascular mortality are not fully understood, although modifications in blood pressure (bp) are possibly an important factor.(16) a study showed that the prevalence of hypertension increasing with age.(17) recent data h a v e s h o w n t h a t a n i n c r e a s e d r i s k o f cardiovascular disease is present in persons with bp levels as low as 115/70 mmhg and that this risk increases steadily with rising bp.(18) the activities of the human body undergo a 24-hourly rhythmical change, being at their lowest at 4 o’clock in the morning, reaching a peak at noon, and then gradually decreasing again. this rhythmical change is known as the c i r c a d i a n r h y t h m , r e g u l a t e d b y t h e suprachiasmatic nucleus in the brain, and i n f l u e n c i n g b o d i l y f u n c t i o n s s u c h a s temperature, metabolism, heart beat, respiratory volume, adrenaline production, blood pressure, and mental abilities. workers employed in the shift system, especially on night shifts, will s u f f e r f r o m d i s s y n c h r o n i z a t i o n w i t h t h e circadian rhythm of their bodies.(4) the increased susceptibility of shift working female nurses for developing higher sbps can be explained by the fact that shift work triggers the effects of other life-style factors such as disruption of the c i r c a d i a n r h y t h m , s t r e s s a n d b e h a v i o u r modification (which includes lack of physical exercise and unhealthy diet). in this study the proportion of overweight persons was higher among the shift workers (72.1%) compared with day workers (27.9%), although this difference was not statistically significant. a limitation in the present study is the definition of exposure to shift work. the female nurses were defined as shift or day workers after answering yes or no to one single question in the questionnaire, viz.”do you have shift work or weekend work?” this question is rather weak a n d i m p r e c i s e , a s i t d o e s n o t e l i c i t a n y information about day to day patterns of shift work, frequency of night work, type of rotation, or duration of shift work. another limitation of this study is the use of a cross-sectional design, which yields inconclusive results, as it does not r e f l e c t t h e t r u e c a u s a l e ff e c t o r t e m p o r a l relationship between shiftwork and blood univ med vol.27 no.2 70 merijanti, samara, tandean, et al shift work and blood pressure p r e s s u r e . h o w e v e r, w h a t o u r s t u d y d o e s demonstrate is that a higher sbp is more common in shift workers than in day workers, which may be an indication that shift work is associated with a risk of systolic hypertension. conclusion the present study has strengthened the existing evidence for a relationship between shift work and systolic blood pressure among female nurses, independently of age and job experience. this further underlines the need for routine bp measurements in nurses to identify subjects with hypertension who should be the target of lifestyle modification. acknowledgements f i n a n c i a l s u p p o r t f o r t h i s s t u d y w a s provided by the medical faculty, trisakti university. we thank the nursing administrator for supporting this study and all nurses whose participation made this study possible. references 1. boisard p, cartron d, gollac m, valeyre a. temps et travail: la durée du travail. dublin: european foundation for the improvement of living and working conditions, 2002. 2. harrington jm. health effects of shift work and extended hours of work. occup environ med 2001; 58: 68-72. 3. nagai k, nagai n, sugahara k, niijima a, nakagawa h. circadian rhythms and energy metabolism and special reference to the suprachiasmatic nucleus. neurosci biobehav rev 1994; 18: 579-84. 4. t kitamura, k onishi, k dohi, t okinaka, m ito, n isaka, et al. circadian rhythm of blood pressure is transformed from a dipper to a non-dipper pattern in shift workers with hypertension. j hum hypertens 2002; 16: 193-7. 5. a choobineh, a rajaeefard, m neghab. problems related to shiftwork for health care workers at shiraz university of medical sciences. east mediterr health j 2006; 12: 341-6. 6. sakata k, suwazono y, harada h, okubuyu y, kobayashi e, nogawa k. the relationship between shift work and the onset of hypertension in male japanese workers. j occup environ med 2003; 45: 1002-6. 7. oishi m, suwazono y, sakata k, okubu y, harada h, kobayashi e, et al. a longitudinal study on the relationship between shift work and the progression of hypertension in male japanese workers. j hypertens 2005; 23: 2173-8. 8. inoue m, morita h, inagaki j, harada n. influence of differences in their jobs on cardiovascular risk factors in male blue-collar shift workers in their fifties. int j occup environ health 2004; 10: 3138. 9. murata k, yano e, shinuzaki t. impact of shift work on cardiovascular function in a 10-year follow-up study. scan j work environ health 1999; 25: 272-7. 10. virkkunen h, harma m, kauppinen t, tenkanen l. shift work, occupational noise and physical workload with ensuing development of blood pressure and their joint effect on the risk of coronary heart disease. scan j work environ health 2007; 33: 425-34. 11. ha m, park j. shift work and metabolic risk factors of cardiovascular disease. j occup health 2005; 47: 89-95. 12. nazri sm, tengku ma, winn t. the association of shift work and hypertension among male factory workers in kota bharu kelantan, malaysia. southeast asian j trop med public health 2008; 39: 176-83. 13. b karlsson, a knutsson, b lindah. is there an association between shift work and having a metabolic syndrome? results from a population based study of 27 485 people. occup environ med 2001; 58: 747-52. 14. bøggild h, knutsson a. shiftwork, risk factors and cardiovascular disease. scand j work environ health 1999; 25: 85-99. 15. ohira t, tanigawa t, iso h, odagiri y, takamiya t, shimomitsu t, et al. effects of shift work on 24-h ambulatory bp and its variability among japanese workers. scand j work environ health 2000; 26: 421-6. 16. hoshide s, kario k, hoshide y, umeda y, hashimoto t, kunii o, et al. association between nondipping of nocturnal blood pressure decrease 71 and cardiovascular target organ damage in strictly selected community-dwelling normotensives. am j hypertens 2003; 16: 434-8. 17. budijanto d, astuti wd, anggaeini r. analisis kecenderungan hipertensi dalam hubungannya dengan usia dan body mass index. j kedokter trisakti 2000; 19: 47-54. 18. lewington s, clarke r, qizilbash n, peto r, collins r. age-specific relevance of usual blood pressure to vascular mortality: a meta-analysis of individual data for one million adults in 61 prospective studies. lancet 2002; 360: 1903-13. univ med vol.27 no.2 nurman1 59 imaging of common bile duct stones january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina a nurman *department of internal medicine, medical faculty, trisakti university correspondence adr. rizaldy pinzon department of internal medicine, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440. tel 021-5672731 ext.2707 e-mail: nurman _achmad@yahoo.com univ med 2009; 28: 59-68 abstract the gallbladder serves as the repository for bile produced in the liver. however, bile within the gallbladder may become supersaturated with cholesterol, leading to crystal precipitation and subsequent gallstone formation. gallstone is one of the most common gastrointestinal diseases in clinical practice. common bile duct stone may be silent and symptomless; alternatively the stone can cause acute cholangitis with jaundice, pain and fever and acute pancreatitis. imaging of the gallbladder is typically requested for evaluation of right upper quadrant pain in patients with or without fever and jaundice. hence,imaging is central to the investigation and diagnoses of choledocholithiasis. there are many options in the field of imaging of choledocholithiasis from a simple to more sophisticated examinations. ultrasonography (us) has been the traditional modality for evaluating gallbladder disease, primarily owing to its high sensitivity and specificity for both stone disease and gallbladder inflammation. however, us is limited by patient body habitus, with degradation of image quality and anatomic detail in obese individuals. with the advent of faster and more efficient imaging techniques, magnetic resonance (mr) imaging has assumed an increasing role as an adjunct modality for gallbladder imaging. mr imaging allows simultaneous anatomic and physiologic assessment of the gallbladder and biliary tract. magnetic resonance cholangiopancreatography is excellent for identifying the presence and the level of biliary obstruction. with newer diagnostic imaging technologies emerging, endoscopic retrograde cholangiopancreatography is evolving into a predominantly therapeutic procedure. keywords : bile duct stone, ultrasound, cholangiography, magnetic resonance imaging introduction gallstones is one of the most common gastro intestinal diseases in clinical practice. gallstones are found in about 10% of the general population, are twice as common in women as in men, and become more prevalent w i t h i n c r e a s i n g a g e . m o s t p a t i e n t s w i t h gallstones are asymptomatic and discovered after routine general check up or for evaluation 60 nurman common bile duct of patients with upper abdominal pain or with disturbance of liver function test. laboratory test are helpful but results are not specific for t h e d i a g n o s i s o f c h o l e d o c h o l i t h i a s i s . ( 1 , 2 ) ultrasonography (us) is the technique of choice to detect gall bladder stone having a s e n s i t i v i t y o f a p p r o x i m a t e l y 9 9 % a n d a s p e c i f i c i t y o f 9 9 % . ( 3 ) t h e y a r e s e e n a s echogenic foci within the gall bladder and cast accoustic shadows. choledocholithiasis is the present of stone in the common bile duct (cbd); the cbd stones are missed frequently by us (sensitivity 1540%). unlike the gallbladder stone, cbd stone only produce accoustic shadow in about 20% of cases. (3) the detection of cbd stone is i m p e d e d b y t h e p r e s e n c e o f g a s i n t h e duodenum, possible reflection and retraction of the sound beam of the duct and the location of t h e d u c t b e y o n d t h e o p t i m a l f o c i o f t h e transducer. on the other hand, cbd dilatation i s i d e n t i f i e d a c c u r a t e l y w i t h u p t o 9 0 % accuracy.(4) it is therefore another options are needed to detect and confirm cbd stone. clinical syndrome choledocholithiasis may be silent and symptomless, discovered only by imaging at the time of a routine cholecystectomy for chronic calculous cholecystitis. alternatively, the stone may cause an acute cholangitis with jaundice, pain and fever.(5) a history of cholelithiasis is n o t e s s e n t i a l f o r t h e d i a g n o s i s o f choledocholithiasis because gall bladder stone c a n b e a s y m p t o m a t i c . p a i n i s t h e m o s t presenting symptom. the pain is colicky in nature, moderate in severity and located in in right upper quadrant of the abdomen. jaundice occur when the cbd becomes obstructred and conjugated bilirubin enters the blood stream. a history of clay colored stools and tea colored urine, the jaundice can be episodic.(6) specific findings upon physical examination are few and are principally abdominal tenderness found the r i g h t u p p e r q u a d r a n t o f t h e a b d o m e n a n d jaundice. laboratory test are helpful, but the results a r e n o t s p e c i f i c f o r t h e d i a g n o s e s o f o f choledocholithiasis because some patients are often asymptomatic and laboratory test results can be completely normal. the laboratory examination may revealed disturbance of the liver function test ie the elevation of serum bilirubin, gamma-glutamyl transpeptidase/ alkaline phosphatase and transaminase. patients with cholangitis and pancreatitis have abnormal laboratory values: i) white cell blood count elevation indicate the presence of infection or inflammation; ii) serum bilirubin level elevation indicates obstruction of the cbd; iii) alkaline phosphatase and gammaglutamyl transpeptidase levels are elevated. these tests results have a good predictive value for the presence of cbd stone and iv) liver t r a n s a m i n a s e ( s e r u m g l u t a m i c p y r u v i c transaminase and oxaloacetic transaminase) levels are elevated. multiple modalities permit depiction of the normal anatomy as well as disease processes of the cystic duct, including i) plain photo of the abdomen; ii) abdominal us; iii) endoscopic u l t r a s o u n d ; i v ) a b d o m i n a l c o m p u t e d tomography scan (ct scan); v) endoscopic retrograde cholangio pancreography (ercp); vi) percutaneous transhepatic cholangiography (ptc); vii) intra operative cholangiography (ioc); and viii) magnetic resonance cholangio pancreatogarphy (mrcp). plain photo of the abdomen abdominal plain photos often play an important role in the evaluations of patients with abdominal pain.(7) for detecting cbd stones, however, the diagnostic yield is low and this test is tending to be omitted. it may reveal gall bladder stones, a calcified gall bladder, 61 univ med vol.28 no.1 pancreatic calcification or rarely outline of a distended gall bladder.(6) the wall of the gall bladder may also calcified (porcelain gall bladder). gas in the biliary tree (aerobilia) may be seen after endoscopic sphincterotomy or surgical bile duct of bowel anastomosis. bile duct dilatation and bile duct stones however, c o u l d n o t b e s e e n b y p l a i n p h o t o o f t h e abdomen. ultrasonography this is a non invasive, inexpensive, and readily modality for assessment of the bilary tree. it is usually the first modality used in the d i a g n o s e s o f p a t i e n t s w i t h b i l i a r y r e l a t e d symptoms.(6) usg findings are accurate in the diagnoses of gallbladder stones (97%) in elective situations (figure 1) and 80% in the presence of acute cholecystitis), but cbd stones are missed frequently (sensitivity 15-40%). right upper quadrant pain and/or jaundice and disturbance of liver function test are the most common indications for imaging of the biliary tract. us is the recommended initial imaging test in the evaluation of these patients. based on clinical circumsrances, the differential diagnosis includes choledocholithiasis, biliary stricture or tumor. this is a non invasive, inexpensive, and readily modality for assessment of the bilary tree. us is very sensitive in the detection of biliary conctriction and stone diseases, allhough less sensitive for the detection of obstructing tumors, i n c l u d i n g p a n c r e a t i c c a r c i n o m a a n d cholangiocarcinoma. it is usually the first modality used in the d i a g n o s e s o f p a t i e n t s w i t h b i l i a r y r e l a t e d symptoms us is the most commonly used modality in the evaluation of gallstone disease, with a high specificity (95%) and sensitivity (95%) for stones larger than 2 mm.(8,9) the a c c u r a c y o f s o n o g r a p h y i n d e t e c t i n g choledocholithiasis approximates 70% although detection of intrahepatic biliary ductal calculi by sonography is more problematic. the biliary ductal system and gallbladder is best imaged with the patient fasted for eight to twelve hours.the fasting state achieves 2 objects, first, an empty stomach and duodenum to enble better imaging of the distal cbd and a relatively distended gall bladder to enable adequate study of the gall bladder wall and inraluminal contents. recognition of dilated bile ducts is central to sonographic imaging of the biliary system. dilated intrahepatic bile ducts are greater than 2 mm in diameter and run parallel to the portal v e i n s i n t h e p o r t a l t r i a d s r e s u l t i n g i n t h e appearance “parallel channel sign”. the normal extra hepatic bile duct is 7 to 8 mm in diameter and usally wider in the subsegment than at the portal hepatic (figure 2). conditions that may mask biliary dilatation include pneumobilia of intrahapatic ducts when the posterior margin of a d i l a t e d d u c t i s o b s c u r e d b y a c o u s t i c shadowing and the presence of biliary sludge or infected bile which may make the dilated ducts relatively invisible on sonography. figure 1. gall bladder stone (arrow) with associated accoustic shodows (arrow head) 62 nurman common bile duct f a l s e n e g a t i v e s t u d i e s f o r b i l i a r y dilatation and/or small choledocholithiasis are usually because of overlying bowel gas obscuring the subhepatic common bile duct or to the absence of biliary dilatation in the p r e s e n c e o f s m a l l n o n o b s t r u c t i n g calculi.common duct dilatation may not o c c u r f o r 2 4 h o u r s a f t e r i n i t i a l s t o n e impaction and intrahepatic duct may not dilate for up 72 hours after impaction. post cholecystectomy patients may have a relatively distended cbd of greater than 8 m m i n d i a m e t e r w i t h o u t m e c h a n i c a l obstruction.(10) in such patients presenting with acute right upper quadrant pain who have obstructive liver chemistry or develop jaundice, a negative sonographic study should b e f o l l o w e d b y a c h o l a n g i o g r a p h i c e x a m i n a t i o n , e i t h e r m a g n e t i c r e s o n a n c e c h o l a n g i o p a n c r e a t o g r a p h y ( m r c p ) o r e n d o s c o p i c r e t r o g r a d e p a n c r e a t o g r a p h y ( e r c p, t h e l a t t e r s u p p l e m e n t e d b y endoscopic ultrasound (eus), to evaluate ductal calculi. endosonography tr a n s c u t a n e o u s u l t r a s o n o g r a p h y i s frequently hampered by interfering intestinal or pulmonal gas, bone or thick layers of fat tissue t h e d e f i c i e n c e s i n c o n v e n t i o n a l i m a g i n g t e c h n i q u e s p r o m p t e d t h e d e v e l o p m e n t o f e n d o s c o p y c o m b i n e d w i t h u l t r a s o n o g r a p h y (endoscopic ultrasound/eus) in the early of 1980s in an effort to obtain more information about diseases of the gastrointestinal and its surroundings.(11) transcutaneous ultrasonography detects more than 95% of gallbladder stone in the bile duct by imaging from the descending bulb or duodenal bulb.(12) cbd stones can be identified when us is used to evaluate the patients with a dilated bile duct or jaundice of unknown cause. i f a c o m m o n d u c t s t o n e i s s u s p e c t e d f r o m transcutaneous imaging procedure or on clinical grounds, ercp should be undertaken rather than eus because it allows removal of the stone; however, eus appears to be more accurate with less invasiveness.(13,14) the ability to place an ultrasonographic probe in close proximity to suspected abnormalities, obviating the effects of gas, fat, and bone, enables eus to yield a highresolution view of the gastrointestinal tract and a d j a c e n t s t r u c t u r e s . s e v e r a l s t u d i e s h a v e compared the accuracy of eus in detection of cbd stones with ercp, and ct.(13,14) in these reports, the sensitivity of eus ranged from 88% to 97%, with specificity of 96% to 100%, which is comparable with ercp and much better than ct. moreover, the accuracy of eus is better than ercp for the detection of small stones. small stones play an important role in acute pancreatitis and biliary-type pain.(15) conventional us is routinely used in the detection of cholelithiasis but it may not be able to detect small stones. there has been an interest in the role of eus in detecting microlithiasis and in evaluating patients with idiopathic pancreatitis. microlithiasis, occult common bile duct stones, small pancreatic neoplasm and pancreas divisum figure 2. ultra sound scan showing dilated intra-hepatic (arrowed) and common bile duct (marked ++) 63 univ med vol.28 no.1 can be found in 75% of this population.(16,17) due to the lower risk of complication, eus should be considered as an alternative to ercp in this group of patients. computed tomogram (ct) scan u s i n g c u r r e n t c t t e c h n o l o g y a n d intravenous contrast enhanced (figure 3), n o r m a l i n t r a h e p a t i c b i l e d u c t s m a y b e visualized in over 40% of normal patients; these should not be confused with ductal dilatation.(18) the extra hepatic bile ducts are identified normally in 80% of patients at ct and usually have diameters 4-10 mm.(19) the extra hepatic bile tree may be up to 10 mm in diameter in patients who have undergone cholecystectomy. the presence or abscence of bile duct stones can be determined on the basis of the four criteria peviously described by baron.(20) with the target sign, the stone is seen as a central density surrounded by hypoattenuating bile or ampullary soft tissue. with the the rim sign one can see a faint rim of increased density along the margin of a low-density area. a c a l c u l o u s w i t h i n c r e a s e d d e n s i t y t h a t i s surrounded by a crescent of hypoattenuating bile is suggestive of the crescent sign. indirect signs, such as the abrupt termination of the duct or ductal dilatation, can be helpful. ct is comparable to us in its ability to detect biliary obstruction and exceeds us in the ability to p r e d i c t t h e l e v e l a n d c a u s e o f b i l i a r y obstruction, although some controversies still e x i s t s . ( 2 1 ) m o s t p a t i e n t s w i t h o b s t r u c t i v e jaundice will undergo us with ct used as a problem solving modality. the reported ct sensitivity for cbd stone detection varies from 45% to 90%.(22) the ability to thin collimation and overlapping image reconstruction available with volume data acquisition of helical ct scanners improves stone detection rate. non contrast ct may improve the recognition of choledocholithiasis to more than 90%.(23) in dilated ducts it is easier to visualized stones. the ct appearance of cbd stones parallels that of gall stones and depend on the chemical composition for each stone. densely calcified stones can be seen as high attenuation structures within the duct lumen, but most duct stones are is attenuating with soft tissue or bile on ct scan. several processes can simulate duct stones on ct and care must be taken to avoid misdiagnoses. pancreatic calculi, oral contrast in an adjacent duodenal diverticulum, residual contras material from prior cholangiogram, and p a p i l l a r y d u c t a l n e o p l a s m c a n s i m u l a t e choledocholithiasis. e n d o s c o p i c r e t r o g r a d e c h o l a n g i o pancreatography (ercp) e n d o s c o p i c r e t r o g r a d e c h o l a n g i o p a n creaticography is the standard therapy for the therapy of biliary obstruction. however, the success rate is not 100%, depending on various patient and physician related factors.(24) the c b d w a s c a n u l a t e d t h r o u g h t h e a m p u l l a , contrast material is injected and films are obtained. complications are hyperamilassemia and cholangitis. in most patients, ercp is the modality of choice when choledocholithiasis is suggested. figure 3. common bile duct stone on contrast-enhanced ct scan.(straight arrow) 64 nurman common bile duct e r c p i s a d i a g n o s t i c p r o c e d u r e t o diagnose problem in the liver, gall bladder, bile ducts and the pancreas in which x-ray is combined with the use flexible scope. with this p r o c e d u r e p a p i l l a o f va t e r i s s e a r c h e d endoscopically, followed by canulation of the catheter and contrast is injected (figure 4). this method needs a high skill ability. from ercp, the blockade and stone in the bile duct, leakage o f b i l e o r p a n c r e a t i c d u c t , p a n c r e a t i c obstruction or stenosis, the existence of tumor may be found. in most patients, ercp is the modality of c h o i c e f o r d i a g n o s i n g a n d t r e a t i n g choledocholithiasis when choledocholithiasis is s u s p e c t e d b u t c a r r i e s p o t e n t i a l r i s k s o f complications (figure 5). complications that occur were morbidity in 1-3%, mortality in 0,2%, and these were directly in connection with skills and experiences of the operator and t o t h e u n d e r l y i n g b i l i a r y a n d p a n c r e a t i c diseases. the complications might occur are acute pancreatitis, cholangitis and bleeding particularly from sphincterotomy sites and d u o d e n a l p e r f o r a t i o n . ( 6 , 2 5 ) p r o p h y l a c t i c antibiotics are often recommended, especially in patients with cbd obstruction. ercp should be reserved for therapeutic purposes for those patients with a high likelihood of stones being present despite negative findings on prior studies. percutaneous transhepatic cholangiography (ptc) in ptc intrahepatic biliary duct is directly punctured with fine flexible chiba needle with the diameter 0.7 mm (22 gauge), so that the patient still can normally breath during the procedure (figure 6).(6) us is used as a guidance in which the puncture was performed into the dilated bile duct. this method is easy and will 100% success if the bile duct is dilated. ptc may be the modality of choice in whom ercp is difficult and failed due to the technical difficulties or when ercp is not possible to p e r f o r m i . e . i n p a t i e n t s w i t h h e p a t i c o e n terostomy, post billroth ii or in klatskin tumor. complication occured less than 5% and consisted of bleeding, bile peritonitis and sepsis.(6) less common were pneumothorax and puncture of viscera. these complications can be reduced by ensuring fastidious technique, figure 4. ercp, normal appearance pd= pancreatic duct, g=gall bladder, c= common bile duct figure 5. ercp showing common bile duct stone. asphincterotome hasbeen passed into the lower end of the bile duct 65 univ med vol.28 no.1 with a single puncture of the liver capsule, avoid over distention of ducts which may allow reflux of infected bile to the circulation through hepatic sinusoid, and ensuring that maneuver are carried out in suspended respiration.(26) u n c o r r e c t e d c o a g u l o p a t h y i s a contraindication for ptc, and the normal size of the intrahepatic duct makes the procedure d i f f i c u l t . p r o p h y l a c t i c a n t i b i o t i c s a r e recommended to reduce the risk of cholangitis. intraoperative cholangiography (ioc) ioc is cholangiography that performs during surgical procedure. the most common indication of ioc is determination the need for e x p l o r a t i o n o f c b d a t t h e t i m e o f cholecystectomy. (6,26) ioc is performed by inserting a catheter intra operatively into the cystic duct, followed by injecton of diluted (50%) contrast material to outline biliary tree.(1) films are taken and are assessed for the precence of filling defects, the anatomy and the calibre tree, and the flow of contrast into the duodenum. this procedure can be performed at open or laparoscopic cholecystectomy (figure 7).(27) ioc findings have a positive predictive value of 60-75% for the detection of cbd stones. the procedure can fail due to i) inability to cannulate the cystic duct, ii) the leakage of contrast during the injection, iii) air bubbles mimicking stones, iv) contrast flowing too quickly into the duodenum, preventing proper filling of the biliary tree, and v) spasm of the sphincter of oddi. postoperative cholangiography through a t-tube is indicated to ensure that all stones have been removed following exploration of the bile duct.(26) i n p a t i e n t s w h o h a v e n o h i s t o r y o f pancreatitis, normal liver function test and cbd stone less than 10 mm diameter, laparoscopic c h o l e c y s t e c t o m y p e r f o r m e d w i t h o u t i n t r a operative chlolangiography does not result in an increased incidence of retained stone.(28) figure 6. diagnosis percutaneous transhepatic cholangiogram showing normal right and life intrahepatic ducts and common bile duct and free flow of contrast into duodenum. the gail bladder is beginning to fill figure 7. normal intraoperative cholangiogram (1) right hepatic duct; (2) left hepatic duct; (3) cystic duct; (4) two surgical clips holding the cholangio catheter in the cystic duct; (5) common hepatic duct; (6) common bile duct; (7) pancreatic duct; (8) ampulla of vater; (9) duodenum 66 nurman common bile duct magnetic resonance cholangio pancreatography (mrcp) mrcp was first described by walner et al in 1991(29) is a noninvasive method of imaging the biliary and pancreatic ducts. the basic principle underlying mrcp is that body fluids such as bile and pancreatic secretions, have high s i g n a l i n t e n s i f y i n h e a v i l y t 2 w e i g h t e d magnetic resonance (ie they appear white), w h e r e a s b a c k g r o u n d t i s s u e s s u c h a s t h e surrounding liver and flowing blood generated little signal (ie they appear dark). as a result of this combination of imaging characteristics, mrcp provides optimal contrast between the hyperintense signal of the bile and pancreatic secretions and the hypointense signal of the background tissue (solid organs), and blood vessels have no measurable signal. on these images the intrinsic fluids of the biliary and pancreatic ducts give the cholangiogram and pancreatogram. no special patient preparation is required but the usual contraindication to mr scanning a p p l y. p a t i e n t s w i t h c a r d i a c p a c e m a k e r, neurostimulator, ferromagnetic aneurism clips a r e e x c l u d e d . t h e e x a m i n a t i o n i s u s u a l l y performed after fasting for several hours. no sedation is required, no hospital admittance is needed.(30-32) in some patients claustrophobia might be a problem. oral contrast agent are not administered, no intravenous contrast agents are needed, and there is no radiation exposure. it is an ideal imaging method for patients with allergies to iodine based contrast or those with a general history of atopy, and prevent the occurrence of contrast nephropathy.(31) mrcp is an alternative to diagnostic ercp f o r t h e i m a g i n g o f t h e b i l e t r e e a n d t h e pancreatic ducts (figure 8). it is almost as good as ercp in the diagnoses of cbd stones, though the ability of mrcp to detect small stones in a non dilated ducts may be limited.(33) it is highly sensitive (97,9%) and specific (84,4%) for choledocholithiasis and avoids the need for invasive imaging in most patients with suspected choledocholithiasis (figure 9).(34) mrcp can be considered as a new gold standard for the investigation of cbd stones and permits reservation of ercp to patients w i t h a h i g h p r o b a b i l i t y o f t h e r a p e u t i c intervention.(35) the european association of endoscopic surgeons consider mrcp to be a standard test for patients with an intermediate probability of cbd stones.(36) a major feature of mrcp is that is not a therapeutic procedure, w h i l e i n c o n t r a s t e r c p i s u s e d f o r b o t h diagnoses and treatment.(30) figure 8. normal mr cholangiography large arrow = common bile duct, g = gall bladder, d = duodenum, small arrow = pancreatic duct figure 9. mrcp in patient with distal common bile duct stone 67 i n m r c p t h e r e i s n o m o r b i d i t y n o r mortality like in ercp/ptc. more over mrcp is an important choice in patients where ercp failed.(37) mrcp is a non invasive tool that is s u i t a b l e i n p a t i e n t s s u s p e c t e d t o h a v e pancreatico biliary diseases where there is no likelyhood or little possibility to perform therapeutic intervention e.g. patients with asymptomatic cholelithiasis without clinical evidence of clinical bile duct disease like the presence of jaundice and abnormalities of liver function test. conclusion cbd stone may be silent and symptomless; alternatively the stone can cause right upper quadrant abdominal pain with jaundice, pain and fever and acute pancreatitis. imaging is central to the investigation and diagnoses of choledocholithiasis. sonography is recommended as initial imaging and from the result further imaging techniques can be chosen. ct may b e c o m e m o r e i m p o r t a n t a n d a c o m p e t i t o r against mrcp in the near future because of improvements in the detectors and the software. for imaging of the biliary system, mrcp has r e p l a c e d i n v a s i v e e r c a n d p e r c u t a n e o u s transhepatic cholangiography as the method of c h o i c e . wi t h n e w e r d i a g n o s t i c i m a g i n g technologies emerging, ercpis evolving into a predominantly therapeutic procedure. references 1. dandan is, soweid am, abiad f. choledocholithiasis: overview. emedicine gastroenterology; 2008. available at: http:// emedicine.medscape.com/article/172216-overview. accessed november 8, 2008. 2. catalana oa, sahani dv, kalva sp, cushing ms, hahn pf, brown jj, et al. mr imaging of the gallbladder: a pictorial essay. radio graphics 2008; 28: 135–55. 3. yeh bm, breiman rs, taouli b. biliary tract depiction in living potential liver donors: comparison of conventional mr, mangafodipir trisodium-enhanced excretory mr, and multidetector row ct cholangiography-initial experience. radiology 2004; 230: 645–1. 4. turner ma, fulcher as. the cystic duct: normal anatomy and disease processes. radio graphics 2001; 21: 3–22. 5. eisen gm, dominitz ja, faigel do, goldstein jl, kalloo an, petersen bt, et al. an annotated algorithm for the evaluation of choledocholithiasis. gastrointest endosc 2001; 53: 864–66. 6. sherlock s, dooley j. gallstones and inflammatory gallbladder diseases. in: sherlock s, dooley j, editors. diseases of the liver and biliary system. 11th ed. malden, mass: blackwell; 2002. p. 597– 628. 7. gupta k, bhandari rk, chander r. comparative study of plain film abdomen and ultrasound in nontraumatic acute abdomen. int j radiol imaging 2005; 15: 109-15. 8. fowley wd, quiroz fa. the role of sonography in imaging of the biliary tract. ultrasound q 2007; 23: 123-5. 9. stroszcynski c, hunerbein m. malignant biliary obstruction: value of imaging findings. abdom imaging 2005; 30: 314-23. 10. khalid tr, casillas vj, montalvo bm, centeno r, levi ju. using mr cholangiopancreatographyto evaluate iatrogenic bile duct injury. am j roentgenol 2001; 177: 1347–52. 11. rosch t, lorenz r, suchy r. colonic endoscopic ultrasonography: first results of a new technique. gastrointest endosc 1990; 36: 382-6. 12. yoshitsugu k, makoto t, shin y, nobuyuki s, massaki s, shoichiroh t, et al. diagnosis of common bile duct calculi with intraductal ultrasonography during endoscopic biliary cannulation. j gastroenterol hepatol 2002; 17: 70812. 13. kohut m, nowakowska-dulawa e, marek t, kaczor r, nowak a. accuracy of linear endoscopic ultrasonography in the evaluation of patients with suspected common bile duct stones. endoscopy 2002; 34: 299–303. 14. buscarini e, tansini p, vallisa d, zambelli a, buscarini l. eus for suspected choledocholithiasis: do benefits outweigh costs? a prospective, controlled study. gastrointest endosc 2003; 57: 510–8. 68 nurman common bile duct 15. venneman ng, renooij w, rehfeld jf, vanbergehenegouwen gp. small gallstones, preserved gallbladder motility, and fast crystallization are associated with pancreatitis. hepatology 2005; 41: 738–6. 16. tandon m, topazian m. endoscopic ultrasound in idiopathic acute pancreatitis. j gastroenterol 2001; 96: 705-9. 17. frossard jl, soca-valencia, amouyal g, marty o, hadenque a, aouyal j. usefulness of endoscopic ultrasonography in patients with “idiopathic” acute pancreatitis. am j med 2000; 109: 196-200. 18. liddell rm, baron rl, ekstrom je. normal intrahepatic bile duct: ct depiction. radiology 1990; 176: 633-5. 19. schulte sj, baron rl, teefy sa. ct of the extrahepatic bile ducts: wall thickness and contrast enhancement in normal and abnormal ducts. am j roentonolog 1990; 154: 79-85. 20. baron rl. common bile duct stones: reassessment of criteria for ct diagnoses. radiology 1987; 162: 419-24. 21. upadhyaya v, upadhyaya dn, ansari ma, shilka vk. comparative assessment of imaging modalities in biliary obstruction. in j radiol imag 2006; 16: 577-82. 22. knowlton jq, tayloraj, reichelderfer m, stang j. imaging of biliary tract inflammation: an update. am j roentonolog 2008; 190: 984–92. 23. soto ja, alvarez o, múnera f, velez sm, valencia j, ramírez n. diagnosing bile duct stones: comparison of unenhanced helical ct, oral contrast–enhanced ct cholangiography, and mr cholangiography. am j roentgenol 2000; 175: 1127–34. 24. puspok a. biliary therapy: are we ready for eusguidance? minerva med 2007; 98: 379-84. 25. topal b, van de moortel m, fieuws s, vanbeckevoort d. the value of magnetic resonance cholangio-pancreatography in predicting common bile duct stones in patients with gall stones diseases. br j surg 2003; 90: 42-7. 26. karani j. the biliary tract. in: sutton d, editor. texbook of radiology imaging vol.i. london: churchill livingstone; 2003. p. 711-36. 27. massarweh nn, flum dr. role of intraoperative cholangiography in avoiding bile duct injury. j am coll surg 2007; 10: 656-64. 28. mcfarlane mec, thomas cal, mccartney t. selective operative cholangiography in the performance of laparoscopic cholecystectomy. int j clin pract 2005; 59: 1301-3. 29. wallner wk, schumacher ka, weidemaier w, friedrich jm. dilated biliary tract: evaluation with magnetic resonance cholangio pancreatography with a t2-weighted contrast-enhanced fast sequence. radiology 1991; 18: 805-8. 30. mceneaney p, mitchell mt, mcdermoth r. update on magnetic resonance cholangio pancreatography. gastroenterol clin n am 2002; 31: 731-46. 31. calco mm, bufanda l, calderon a, heras i, cabriada jl, bernal a, et al. role of magnetic resonance cholangio pancreatography in patients with suspected choledocholithiasis. mayo clin proc 2002; 77: 422-8. 32. romagnuolo j, bardon m, rahme e, joseph l, reinhold c, barkun an. magnetic resonance cholangio pancreatography: a meta analysis of test performance in suspected biliary disease. ann inter med 2003; 139-7: 547-57. 33. williams ej, green j, beckingham i, park r, martin d, lombard m. guidelines of the management of common bile duct stones (cbds). gut 2008; 57: 1004-21. 34. shanmugan v, beattie gc, jule sr, reid w, loudon ma. is magnetic resonance cholangio pancreatography the new gold standard in biliary imaging ?. br j radiol 2005; 78: 888-93. 35. gore rm, yaghmai v, newmark gm, berlin jw, miller fh. imaging benign and malignant disease of the gallbladder. radiol clin north am 2002; 40: 1307–23. 36. neugebauer e, sauerland s, fingerhut a. european association endoscopic surgery guidelines for endoscopic surgery. berlin: springer; 2006. 37. leung j. fundamentals in ercp in advanced digestive endoscopy: endoscopic retrograde cholangio pancreatography. in: cotton pb, leung j, editors. 1st ed. massachusets: blackwell publishing; 2006. p. 17-80. editorial i warfarin: do we need genotype-based dose prediction? yenny department of pharmacology and therapeutics, medical faculty, trisakti university univ med vol. 29 no.3 for the treatment and prevention of thrombo-embolic disease, the most frequently used anticoagulant drug worldwide is warfarin, an oral coumarin derivative, with more than 30 million prescriptions written for this drug in the united states in 2004.(1) the drug has a narrow therapeutic index and its metabolism varies by as much as a factor of 10 among individual patients, making warfarin therapy difficult to manage. hemorrhagic complication rates of warfarin are estimated to be 5-7.9% for major (life threatening) hemorrhage and 14-36% for minor hemorrhage (e.g. nosebleeds, microscopic hematuria).(2) this condition makes it difficult to establish the appropriate dose of warfarin. warfarin is administered as a racemic mixture of sand r-enantiomers (mirror-image isomers), which differ in metabolism and potency, the s-enantiomer being the more potent form with a variable metabolism, resulting in the large variability in warfarin dose requirements. of the overall anticoagulation response, 60-70% is due to the s-enantiomer, while the r-enantiomer is responsible for the remaining 30-40%. metabolism of the enantiomers occurs in the liver via distinct cytochrome p450 (cyp) enzymes. in s-warfarin metabolism, cyp2c9 serves as the principal enzyme and cyp2c8, cyp2c18, cyp2c19 as minor metabolic pathways. r-warfarin is mainly metabolized by cyp1a2 and cyp3a4, with cyp1a1, cyp2c8, cyp2c18, cyp2c19, and cyp3a4/5 serving as minor pathways to inactive metabolites.(3) the pharmacological effect of warfarin is produced by the drug interfering with the synthesis of vitamin k-dependent clotting factors via inhibition of vitamin k 1 2,3-epoxide reductase complex, subunit 1 (vkorc1). this interferes with the post-translational gamma-carboxylation of glutamic acid residues on coagulation factors ii, vii, ix, and x, as well as the anticoagulant proteins c and s. depletion of reduced vitamin k leads to the production of nonfunctional coagulation factors, resulting in anticoagulation.(3) resistance to warfarin has been described as the inability to prolong the prothrombin time or raise the international normalized ratio (inr) into the therapeutic range when the drug is given at normally prescribed doses. however, a higher warfarin requirement does not itself establish the diagnosis of warfarin resistance. the range of normally recommended daily or weekly warfarin doses to maintain a therapeutic prothrombin time or inr depends on the study population. patients who need more than 105 mg per week (15 mg/day) (4) should be considered warfarin resistant. it should be noted that another reference paper gives the lower dose of 70 mg per week.(3) an important characteristic of warfarin resistance is that patients need much smaller doses of vitamin k to reverse the effect of warfarin. ii univ med vol. 29 no.3 warfarin resistance can be classified in practical terms as acquired and hereditary. acquired resistance to warfarin is most frequently the result of poor patient compliance, but may also be due to other causes, such as high consumption of vitamin k, decreased absorption or increased clearance of warfarin, and drug interactions. hereditary resistance is presumably governed by genetic factors that result in faster metabolism (pharmacokinetic resistance) or in lower activity of the drug (pharmacodynamic resistance).(4) genetic variability are important in causing warfarin resistance or warfarin sensitivity, the latter being associated with some vkorc1 and cyp2c9 variant alleles.(5) the variant alleles cyp2c9*2 and *3 are more prevalent in european americans than in african americans, while in asian populations, cyp2c9*2 has a frequency of 2-4%, and *3 alleles are not commonly found.(3) in a meta-analysis of nine studies involving almost 3000 warfarin patients it was shown that carriers of cyp2c9*2 and *3 had lower mean daily doses and a significantly higher risk of hemorrhage.(6) the relative bleeding risk for cyp2c9*2 and * 3 was 1.91 (1.16-3.17) and 1.77 (1.07-2.91), respectively. inter-individual variability in warfarin dose among patients of european, asian and african descent has been associated with the prevalence of variant vkorci 1173 c>t and 1693 g/a.(3) several rare vkorci missense mutations have been found in patients with warfarin-resistance, requiring the higher doses of >70 mg/week.(5,7) although the frequency and clinical impact of these mutations at the population level is poorly documented, they may be clinically relevant in compliant patients requiring higher warfarin doses.(3) warfarin resistance is diagnosed through patient history and laboratory studies, and the search for potential causes requires a full drug and dietary history. subtherapeutic plasma warfarin levels should alert the pharmacologist to the possibility of intestinal malabsorbtion or poor compliance. the range of therapeutic total plasma warfarin levels is 0.5 – 3.0 µg/ml, depending on the laboratory and patient population. to determine the type of warfarin resistance, drug absorption and clearance is evaluated by determination of plasma levels at specific intervals after administration, commonly every 60 to 180 minutes. the clearance rate of the s-enantiomer is normally twice that of the r-enantiomer (5.2 vs 2.5 ml/min/70 kg). a normal clearance rate precludes the possibility that the resistance is caused by enhanced drug elimination. in addition, bentley et al.(8) have devised an algorithm for using the plasma warfarin levels to determine the type of resistance. a clotting assay of factors ii, vii, ix, and x has been used for determining warfarin dose. in patients with an unreliable or prolonged baseline prothrombin time and inr, some studies have targeted a factor ii and x activity level of 10% 30% of normal biologic activity for achieving a therapeutic warfarin effect. plasma warfarin levels are typically measured with a turn-around time of 2 to 7 days, as opposed to 24 hours for factor ii and x activity.(4) treatment of warfarin-resistant cases should be based on the cause. patient education is necessary to improve compliance and to reduce adverse effects of warfarin therapy, regardless of the dose. in true hereditary warfarin resistance, treatment may be effected by increasing warfarin dosage (possibly attaining 100 mg/day or more), or by switching to other types of anticoagulant. there are several alternative anticoagulants, such as subcutaneous heparins (unfractionated and low-molecular-weight heparins), fondaparinux (arixtra, a subcutaneous factor xa inhibitor), dabigatran (an oral direct thrombin inhibitor, currently undergoing phase 3 studies for use in long-term anticoagulation), rivaroxaban (a direct factor xa inhibitor), and vitamin k antagonists (bishydroxycoumarin, phenprocoumon, acenocoumarol, phenindione).(4) iii in 2007, the food and drug administration (fda) added pharmacogenetic information to the warfarin package insert, presumably in recognition of the fact that genetic variations in the cyp2c9 and vkorc1 genes contribute significantly to the variability in dose requirements for warfarin. however, the fda did not propose a specific method for using genetic information to predict the dose required in individual patients.(9) one study on the use of a pharmacogenetic algorithm for estimating the appropriate initial dose of warfarin yielded recommendations that were significantly closer to the required stable therapeutic dose.(10) on the other hand, the review by limdi et al.(3) did not find supporting evidence to suggest that genotype-guided therapy will improve anticoagulant control and prevent or reduce the risk of hemorrhagic or thromboembolic complications. in any case, it would be both reasonable and prudent to use cyp2c9 and vkorc1 genotypes as part of diagnostic efforts to understand unusual responses to standard medical care. references 1. wysowski dk, nourjah p, swartz l. bleeding complications with warfarin use: a prevalent adverse effect resulting in regulatory action. arch intern med 2007;167:1414-9. 2. hylek em, evans-molina c, shea c, henault le, regan s. major hemorrhage and tolerability of warfarin in the firs years of therapy among elderly patients with atrial fibrillation. circulation 2007;115:2689-96. 3. limdi na, veenstra dl. warfarin pharmacogenetics. pharmacotherapy 2009;28:1084-97. 4. osinbowale o, malki ma, schade a, bartholomew jr. an algorithm for managing warfarin resistance. cleve clin j med 2009;76:724-30. 5. scott sa, edelmann l, kornreich r, desnick rj. warfarin pharmacogenetics: cyp2c9 and vkorc1 genotypes predict different sensitivity and resistance frequencies in the ashkenazi and sephardic jewish population. am j hum genet 2008;82:495-500. 6. sanderson s, emery j, higgins. cyp2c9 gene variants, drug dose, and bleeding risk in warfarin-treated patients: a hugenet systematic review and meta-analysis. genet med 2005;7:97-104. 7. d’ambrosio rl, d’andrea g, cafolla a, faillace f, margaglione m. a new vitamin k epoxide reductase complex subunit-1 (vkorc1) mutation in a patient with decreased stability of cyp2c9 enzyme. j thrombo haemost 2007;5:191-3. 8. bentley dp, backhouse g, hutching a, haddon rl, spragg b, routledge pa. investigation of patients with abnormal response to warfarin. br j clin pharmacol 1986;22:37-41. 9. food and drug administration (fda) approves updated warfarin (coumadin) prescribing information. new genetic information may help providers improve initial dosing estimates of the anticoagulant for individual patients. available at: http://www.fda.gov/cder/drug/infopage/warfarin/default.htm. accessed july 7, 2010. 10. the international warfarin pharmacogenetic consortium. estimation of the warfarin dose with clinical and pharmacogenetic data. n engl j med 2009;360:753-64. alvina 152 anemia decreases quality of life of the elderly in jakarta september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina meiyanti,*a joice viladelvia kalumpiu,** julius i.mulia* *department of pharmacy, **department of pharmacology, medical faculty, trisakti university correspondence adr. meiyanti, spfk department of pharmacy, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2805 univ med 2009;28:152-60 abstract anemia is a very common disorder both in clinical practice and in the community. the recent rise in the population of the elderly has become the focus of attention in developing countries, because of the increasing longevity of the elderly, whilst the prevalence of anemia increases with age. the objective of this study was to determine the prevalence of anemia and its association with the quality of life in the elderly. a cross-sectional study was conducted from april to june 2008, located in the mampang prapatan district, south jakarta. a total of 298 elderly persons participated in this study, comprising 109 (36.6%) males and 189 (63.4%) females. anemia was measured by the parameters of hemoglobin and quality of life was assessed by the whoqol-bref instrument, a short version of the whoqol-100 instrument. the results of the study showed the prevalence of anemia to be 26.2%, with a higher prevalence rate in females compared with males. the quality of life in the physical, psychological, social and environmental domains differed significantly with decreasing hemoglobin levels, whereas the physical and mental quality of life declined significantly with age. anemia is thus indicative of a reduction in the quality of life of the elderly. keywords: anemia, elderly, quality of life introduction anemia is a very common disorder in clinical practice as well as in the community. scientific advances and improved health services have resulted in an increased life span and a rise in the elderly population. the us census bureau has estimated that more than 36.3 million americans are over the age of 65 years and that this number will increase up to 85 million in the y e a r 2 0 5 0 . ( 1 ) i n 1 9 7 1 t h e t o t a l n u m b e r o f indonesian elderly was 5.3 million or 4.48% of the total population of indonesia, in the year 2000 the number increased to 14.4 million (7.18%) and in 2020 is projected become 28.8 million (11.34%).(2) anemia may occur at all ages, but its prevalence rises with advancing age.(3) according to epidemiological data, the 153 univ med vol.28 no.3 prevalence of anemia increases sharply at ages over 60 years. the us nhanes iii (national health & nutrition examination survey) has reported that the overall prevalence of anemia in the elderly is estimated to be 10% (3.5 million), with a prevalence in males of 11% and in females of 10.2%.(4) in asia, the prevalence of anemia in korean elderly is 13.6% and the prevalence in females (14.7%) is higher than that in males (9.9%).(5) anemia shows abnormal laboratory values, the most common parameters for defining anemia being hemoglobin levels, hematocrit and red cell count. the world health organization (who) defines anemia as a hemoglobin (hb) concentration of <12 g/dl in females and of <13 g/dl in males.(6) a number of causative factors for anemia in the elderly are i) anemia due to hemorrhage resulting from nsaid usage, malnutrition (e.g. deficiencies of iron, folic acid, vitamin b12), alcoholism, and atrophic gastritis; ii) anemia associated with chronic disease; iii) anemia of unknown origin.(4,5) the who definition of q u a l i t y o f l i f e ( q o l ) a s ‘ a n i n d i v i d u a l ’s perception of their position in life in the context of the culture and value systems in which they lives, and in relation to their goals, expectations, standards and concerns’. the quality of life in the elderly is assessed by the whoqol-100 and the whoqol-bref instruments, where the last-mentioned instument is a short version of the first.(7) several cross-sectional studies on the e l d e r l y h a v e d e m o n s t r a t e d a n a s s o c i a t i o n between anemia and functional disability, r e d u c e d p h y s i c a l p e r f o r m a n c e , d e c r e a s e d cognitive function and a lowered qol as a result of declining daily activities of life.(4,8) although it can be hypothesized that anemia is a predictor of quality of life in the elderly, this hypothesis h a s t o b e t e s t e d b y f u r t h e r r e s e a r c h . t h e objective of the present study was to determine the prevalence of anemia and its association with qol in the elderly. methods research design in this study a cross-sectional research design was used. the study was conducted in the catchment area of the mampang prapatan health center, south jakarta, from april to june 2008. study subjects there are several definitions of the elderly. i n d e v e l o p i n g c o u n t r i e s , a n i n d i v i d u a l i s considered to be elderly if he or she is 60 years of age or older, whereas in developed countries an elderly is a person aged 65 years of age and older.(9) the subjects of the present study were healthy elderly aged >60 years, meeting the inclusion criteria and chosen randomly from each rukun tetangga (rt). there were 298 elderly who participated in this study, meeting the following inclusion criteria: male or female, a c t i v e , h e a l t h y, i n d e p e n d e n t , a b l e t o communicate verbally and willing to participate in this study. data collection and assessment information was obtained through interviews using structured questionnaires, followed by physical examination and measurement of blood pressure and anthropometric variables. quality of life was assessed using the indonesian version of t h e w h o q o l b r e f q u e s t i o n n a i r e . t h i s questionnaire consists of 24 items to assess 4 d o m a i n s : ( i ) t h e p h y s i c a l h e a l t h d o m a i n , consisting of 7 items, (ii) the psychological domain, consisting of 6 items, (iii) the social relations domain, consisting of 3 items, (iv) the environment domain, consisting of 8 items. the w h o q o l b r e f a l s o c o n t a i n s 2 i t e m s 154 meiyanti anemia and quality of life measuring overall quality of life and general health, bringing the total number of items to 26. a domain is not given a score if >20% of the items remain unanswered by the study subject. the assessment of quality of life by means of the whoqol-bref had been validated and was of high validity.(10) the interviewer had also gone through a standardized training course for conducting interviews and the questionnaires had been tested in a pilot study. measurements blood pressure was measured by means of a calibrated mercury sphygmomanometer. in accordance with the criteria of the joint national committee 7 (jnc 7) the study subjects were considered to have hypertension if the systolic pressure was >140 mmhg or the diastolic pressure >90 mm hg(11) or if the subjects were under treatment with antihypertensive drugs. assessment of hemoglobin (hb), fasting glucose, total protein, and creatinine was performed in a c l i n i c a l l a b o r a t o r y ( p r o d i a ) a c c o r d i n g t o p r o c e d u r a l s t a n d a r d s . a n t h r o p o m e t r i c measurements comprised calculation of body mass index (bmi) by standard procedures, including measurement of height and weight. height was measured using a portable microtoise accurate up to 0.1 cm and weight was measured by means of portable scales accurate up to 0.1 kg. from the study subjects meeting the inclusion and exclusion criteria, a 5 ml blood sample was taken by venipuncture from the cubital fossa. from each blood sample 2 ml was put into a v a c u t a i n e r t u b e c o n t a i n i n g e d ta a s anticoagulant. hemoglobin concentration was assessed by the cyanmethemoglobin method, using a standard photometer. the remaining blood sample was centrifuged at 300 rpm for 10 minutes in order to obtain serum for the initial determinations of total protein, albumin and globulin, using a trx 7010 automated clinical analyzer. ethical clearance ethical clearance was approved by the research ethics committee of the medical faculty, trisakti university. each study subject was asked to fill in an informed consent form, legalized with affixed signature for the literate or a thumbprint for the illiterate. the identity of all study subjects was kept confidential and only used for research purposes. statistical analysis analysis of variance was performed on background characteristics, and normality of d a t a d i s t r i b u t i o n w a s a s s e s s e d b y t h e k o l m o g o r o v s m i r n o v t e s t . a n o r m a l d a t a distribution was expressed as the mean, standard d e v i a t i o n a n d p e r c e n t a g e . s i m p l e l i n e a r regression analysis was used to determine the association between age, hemoglobin level and quality of life of the elderly. for the statistical analysis spss for windows version 15.0 was used, and p< 0.05 was considered statistically significant. results a total of 298 elderly participated in this study, consisting of 109 (36.6 %) males and 189 (63.4%) females. there were 233 subjects (78.2%) in the age group of 60-69 years and 65 (21.8%) in the age group of > 70 years. the mean systolic pressure was 145.0 ± 22.9 mmhg and the mean diastolic pressure was 86.6 ± 12.4 mmhg. according to the jnc 7 criteria grade i hypertension is defined by a systolic pressure of 140-159 mmhg or a diastolic pressure of 90-99 mmhg. as judged from the mean systolic p r e s s u r e , t h e s t u d y s u b j e c t s h a d g r a d e i hypertension. the bmi values were normal, with a mean of 21.7 ± 4.0 kg/m2. most laboratory parameters were within normal limits, with mean hb of 13.2 ±1.6 g/dl, mean fasting glucose of 117 g/dl, and mean total protein of 7.2 ± 1.1 g/ 155 univ med vol.28 no.3 dl. however, albumin concentrations were reduced, whereas those of globulin were raised. regarding formal education, the majority of the elderly did not finish primary school (74.4%). as to marital status, most of the elderly were widowed (54.7%), whilst 44.6% was married and 0.7% single. the overall prevalence of anemia in this study was 26.2% (78/298), with a prevalence in females of 30.2% and in males of 19.3% (table 1). the results of simple linear regression analysis between hb level and the four qol domains, viz. physical, psychological, social and environmental, showed that each rise of 1 g/dl in hb concentration resulted in an increase in all four qol domains in the elderly. there was a significant difference in each qol domain due to decreased hb. the four qol domains, namely p h y s i c a l , p s y c h o l o g i c a l , s o c i a l , a n d environmental, had p=0.001, p=0.001, p=0.000 and p=0.001, respectively (table 2). table 3, listing the results of simple linear regression analysis between age and the four qol domains, indicates that with advancing age there was a decrease in the four qol domains, although a significant reduction occurred only in 2 domains, i.e. the physical (p=0.000) and psychological domains (p=0.029). for the other two domains (social and environmental) no significant difference was apparent. simple linear regression analysis between albumin and hb concentrations revealed that hb concentration was proportional to albumin domain physical psychological social environmental β* β β β hb 0.333 0.279 0.285 0.425 p 0.001 0.001 0.000 0.001 * β: regression coefficient table 2. simple linear regression analysis between hb level and qol domains concentrations, with â = 0.867 and significant association (p=0.000). on the other hand, hb concentrations were inversely proportional to globulin concentrations, with â = -0.812 and significant association (p=0.000) (table 4). characteristic n (%) age (years) 60 – 69 233 (78.2) > 70 65 (21.8) blood pressure (mm hg) systolic (mean ± sd) 145.0 ± 22.9 diastolic (mean ± sd) 86.8 ± 12.4 body weight (kg) (mean ± sd) 51.9 ± 10.9 height (cm) (mean ± sd) 154.3 ± 6.4 bmi (kg/m2) (mean ± sd) 21.7 ± 4.0 hb (g/dl) (mean ± sd) 13.2 ± 1.6 fasting glucose (g/dl) (mean ± sd) 117.9 ± 61.1 albumin (g/dl) (mean ± sd) 3.9 ± 0.6 globulin (g/dl) (mean ± sd) 3.2 ± 0.7 total protein (mean ± sd) 7.2 ± 1.1 length of education < 6 years 222 (74.4) 7 – 9 years 37 (12.4) > 9 years 40 (13.2) marital status married 133 (44.6) widow/widower 163 (54.7) single 2 (0.7) prevalence of anemia males 21 (19.3) females 57 (30.2) table 1. distribution of subject characteristics (n=298) 156 discussion based on the who criteria, the prevalence of anemia in the present study was found to be 26.2%, which was higher than in the us (10%)(4) and korea (13%). the prevalence of anemia in this study was higher in females (30.6%) than in males (19.3%), these results being not noticeably different from those of a korean study.(5) previously, anemia of the elderly was viewed as a normal physiological process, but currently it is considered to be a pathological condition due to chronic disease or other comorbidities. in healthy elderly the hb concentrations are similar to those of young adults,(12) so that the who criteria for anemia can be used for defining anemia in the elderly. anemia in the elderly is caused by various factors, such as malnutrition, nutritional deficiencies (iron, folate or vitamin b12), and acute or chronic hemorrhage (e.g. gastrointestinal hemorrhage, hemorrhage from side effects of medications). other causes of anemia are chronic disease or inflammation, such as renal insufficiency, congestive heart disease, hepatitis c, diabetes mellitus, rheumatoid arthritis, osteoarthritis, stroke, chronic obstructive pulmonary disease, and cancer. anemia of unknown origin in the elderly is usually associated w i t h a b n o r m a l i t i e s o f t h e b o n e m a r r o w, presumably because advanced age leads to decreased renal function and decreased response o f t h e b o n e m a r r o w a s a s o u r c e o f erythropoietin.(3,4,12) advanced age increases the prevalence of anemia, guralnik et al.(4) reporting the prevalence of anemia to rise sharply at age 75 years and above, estimated to be 42.9%, which is higher than the prevalence of anemia at age 6574 years, estimated at 25% . the bmi values in the present study were normal (mean 21.7 kg/m2), but as bmi is only indicative of the total amount of body fat, it cannot be used by itself for evaluating the general nutritional status of the elderly.(13) the general nutrional status is commonly assessed from body weight. an individual is said to be malnourished if there is a reduction in body weight of >10% from the initial weight within a period of 6 months or a reduction in body weight of >5% from the initial weight within a period of 3 months. a reduction in body weight of >40% from the initial weight may cause irreversible organ damage or even death.(14) mean systolic and diastolic pressure in the elderly was 145.0 mmhg and 86.8 mmhg, respectively, indicating that the elderly in the p r e s e n t s t u d y p r e s u m a b l y h a d g r a d e i meiyanti anemia and quality of life table 3. simple linear regression analysis between age and qol domains * β: regression coefficient; † p< 0.05: significant tabel 4. results of simple linear regression analysis between albumin/globulin and hb concentrations 157 univ med vol.28 no.3 h y p e r t e n s i o n o r e v e n i s o l a t e d s y s t o l i c hypertension. the fasting blood glucose level of the elderly was normal, but there remains the possibility of an abnormal glucose tolerance. it is estimated that 50% of the elderly have an abnormal glucose tolerance with normal fasting blood glucose,(13) which may be associated with obesity and decreased activity of the elderly. meneilly et al.(15) reported that at age 75 years and over an estimated 20% of the population have diabetes mellitus. mean albumin concentration in this study was 3.9 g/dl, indicating a low albumin level, which in turn indicates malnutrition. serum albumin is a practical indicator for assessing malnutrition in young adults as well as in the elderly. low serum albumin is associated with a variety of conditions, such as malnutrition, h e p a t i c d i s e a s e , p r o t e i n u r i a , d e h y d r a t i o n , inflammation, and disorders of testosterone.(13) with advancing age there is an increase in chronic disorders. based on literature reports, 50-80% of the elderly aged over 65 years have on average more than one chronic disorder, whilst yenny et al.(16) found a figure of 68.8% for the elderly with more than one chronic disorder. the chronic disorders most frequently suffered by elderly males as well as females are m u s c u l o s k e l e t a l d i s o r d e r s . i n a d d i t i o n , cardiovascular disease is a frequent cause of death and disability in the elderly. advanced age is said to be one of the most important risk factors for musculoskeletal disorders in the elderly population. other risk factors that also play a role are hypertension, hyperlipidemia, diabetes and obesity.(17) in the elderly with d e g e n e r a t i v e d i s e a s e t h e r e i s a d e c r e a s e d production of erythropoetin. in patients with heart disease there is a rise in the levels of the proinflammatory cytokine il-6 as a result of a s t r e s s r e s p o n s e g e n e e x p r e s s i o n , c a u s i n g decreased erythropoietin production and response, resulting in anemia. male hypogonadism may induce a decreased hb level, where administration of testosterone may raise hb levels, because t e s t o s t e r o n e d e p r e s s e s i l 6 p r o d u c t i o n . testosterone increases the production of stem cells for satellite muscle cells and the hemopoetic system.(18,19) the testosterone level also decreases with advancing age in females and plays an important role in the pathogenesis of anemia in elderly males as well as females.(19) the majority of the elderly in the present study had minimal education, as over 50% of them merely had six years of education or less. this low educational level may have been responsible for their low economic status, which is usually associated with malnutrition (due to reduced intakes of meat, fish, eggs, vegetables, and fruit) and with poor health status, such as chronic disease, dental and periodontal disease, decreased gustation and olfaction. the use of medications such as reserpine and digoxin may be a causative factor for malnutrition in the elderly. many elderly in the present study were also widowed (54.7%), and it is well-known that the loss of a life partner is a predisposing factor for depression in the elderly, leading to loss of appetite and subsequently malnutrition.(12) in the present study there are several possible causes for the high prevalence of anemia in the elderly, viz. (i) advanced age in association with declining organ functions and decreased reponse of the bone marrow as a source of erythropoietin; (ii) chronic disease such as hypertension and glucose intolerance; (iii) malnutrition as assessed from the low albumin levels. choi et al.(5) reported that the most frequent causes of anemia in the elderly were chronic disease and iron deficiency d u e t o g a s t r o i n t e s t i n a l h e m o r r h a g e . t h e investigators also reported that gender, advanced age, low albumin, and low bmi were among the independent factors for anemia. s i m p l e l i n e a r r e g r e s s i o n a n a l y s i s demonstrated an association between hb level and the four qol domains, where a rise of 1 g/ 158 meiyanti anemia and quality of life dl hb caused an increase in the four qol domains. a low hb level has a negative impact o n a p p e a r a n c e , p h y s i o l o g y a n d p h y s i c a l limitations. in the elderly aged >65 years anemia is associated with physical weakness, decreased activity, declining cognitive functions, increased risk of dementia, reduction in muscle and bone mass, increased risks of falling and increased risk of a major depressive illness.(20,21) similarly lucca et al.(22) stated that hypoxia due to anemia affected physical functions, cognition and qol in the elderly. denny et al.(23) reported that anemia was a risk factor for loss of physical appearance and cognitive function. chronic disease accompanied by anemia may also induce depression in the elderly, as reduced activity due to fatigue causes the elderly to become more i s o l a t e d a n d t o h a v e f e w e r s o c i a l a n d e n v i r o n m e n t a l i n t e r a c t i o n s , w h i c h a r e a l l responsible for the decrease in the four qol domains of the elderly. the positive clinical outcome for treating anemia, such as inproved quality of life, decreased hospitalization, and decreased mortality, demand that a hemoglobin concentration of less than 12 g/al showed be investigated and treated wherever possible.(24) the results of simple linear regression analysis between age and qol showed that advanced age causes a significant decrease in the physical and mental domains of qol. as reported by canbaz et al.(25) in a study on the quality of life in the elderly, there was also a significant reduction in qol with advancing years. with advancing age, the body of the e l d e r l y e x p e r i e n c e s a n u m b e r o f c h a n g e s associated with the sensory organs (hearing, vision) and with the functioning of vital organs, such as the cardiovascular system, the central nervous system and the respiratory system. advanced age is associated with decreases in anabolic hormones, which causes reduced igf1 levels, and also contributes to reduced lean body mass, such that the body becomes thinner. advanced age is accompanied by a reduction in the levels of testosterone, which plays an important role in physiological activities and affects the muscles and bone marrow.(18) these conditions result in a decreased physical qol in the elderly. t h e c h a n g e s o c c u r r i n g i n t h e c e n t r a l nervous system with age are in the form of changes in neurotransmitter concentrations, p a r t i c u l a r l y c a t e c h o l a m i n e rg i c n e u r o transmitters, which play a role in the occurrence of depression in the elderly.(12) depression is also closely associated with loss of memory and dementia. decreased functioning of the senses of vision and hearing, dependency on others, loss of work, and reduced income are also causative factors for the occurrence of depression in the elderly. loss of a life partner, such as is apparent from the large number of widowed elderly, may also cause depression. the ensuing depression and dementia lead to decreased psychological qol in the elderly. the social and environmental qol also decreased with age, but the decrease was non-significant. increased albumin level was correlated with increased hb, whilst increased globulin levels caused decreased hb levels. a high globulin level may be associated with hepatic disease, chronic i n f e c t i o n s , a u t o i m m u n e d i s e a s e s u c h a s rheumatoid arthritis, decreasesd renal functions. the results of the present study is similar to the study conducted by alfons et al.,(26) stating that hb level was correlated with prealbumin and albumin levels, but not with age. several other studies also demonstrated increased albumin levels upon an increase in hb level in patients on erythropoetin therapy.(27) the limitations of the present study are the relatively small sample size, limitations in laboratory ancillary examinations, and limitations in tracing of comorbidities of the elderly. 159 univ med vol.28 no.3 conclusions anemia is identical with decreased qol in all four domains. further research is needed on the common types of anemia, and on preventive measures and management of anemia in order to enhance the quality of life of the elderly. acknowledgements the investigator expresses her gratitude to the dean and vice-deans of the medical faculty, trisakti university, for the funding of the present study, and to the staff of mampang prapatan health center and all persons who made this study possible. references 1. steensma, teffani a. anemia in elderly: how should we define it, when does it matter, and what can be done? mayo clin proc 2007;82:958-66. 2. badan pusat statistik. proyeksi penduduk indonesia per propinsi 1995-2005. jakarta: badan pusat statistik;1998. 3. smith dl. anemia in elderly. am fam physician 2000;62:1565-72. 4. guralnik jm, eisentaedt rs, ferruci l, klein hg, woodman rc. prevalence of anemia in persons 65 year and older in the united states: evidence for a high role of unexplained anemia. blood 2004;104:2263-68. 5. choi cw, lee j, park kh, yoon sy, choi ik, oh sc, et al. prevalence and characteristics of anemia in the elderly: cross sectional study of three urban korean population samples. am j hematol 2004;77:26-30. 6. spivak jl. anemia in elderly: a growing health concern. available at: http//www.medscape.com/ viewarticles. accessed nov 10, 2009. 7. the whoqol group. the world health organization quality of life assesment (whoqol) development and general psychometric properties. soc sci med 1998;46: 1569-85. 8. riva e, tettamanti m, masconi p, apolone g, gandini f, nobili a, et al. association of mild anemia with hospitalization and mortality in elderly: the health and anemia population-based study. hematologica 2009;94:22-8. 9. departemen sosial republik indonesia. pedoman rencana aksi nasional untuk kesejahteraan lanjut usia. jakarta: depsos-ri-yel-unfpa-help age international;2003. 10. salim oc, sudharma ni, rina k. kusumaratna rk, hidayat a. validity and reliability of world health organization quality of life-bref to assess the quality of life in the elderly. univ med 2007;26:2738 11. chobanian av, bakris gl, black hr, cushman wc, green la, izzo jl, et al. the seventh report of the joint national committee on prevention, detection, evaluation and treatment of high blood pressure: the jnc 7 report. jama 2003;289:246072. 12. thomas dr. anemia: it’s all about quality of life. jamda 2007:80-2. 13. kane rtl, ouslander jg, abras ib, editors. essentials of clinical geriatrics. new york: mc graw-hill;2004.p.305-33. 14. wilson mmg, morley je. aging and energy balance. j appl physiol 2003;95:1728-36. 15. meneilly gs, tessier d. diabetes in elderly adults. j gerontol a biol sci med sci 2001;56a:5-13. 16. yenny, herwana e. chronic disease dan quality of life elderly. universa medicina 2006;25:164-70. 17. nauman vj, byrne gj. whoqol-bref as a measure of quality of life in older patients with depression. int psychogeriatr 2004;16:159-73. 18. wittert ga, chapman jm, haren rt, mackintosh s, coates p, morley je. oral testosterone supplementation increase muscle and decrease fat mass in healthy elderly males with low-normal gonadal status. j gerontol sci 2003;58a:618-25. 19. morley je, perry hm. androgens and women at the menopause and beyond. j gerontol med sci 2003;58a:409-16. 20. penninx b, pahor m, caseri m, corsi am, woodman rc, bandinelli s, et al. anemia is associated with disability and decreased physical performance and muscle strength in the elderly. j am ger soc 2004;52:719-24. 21. penninx b, pahor m, woodman r, guralnik jm. anemia in old age associated with increased mortality and hospitalization. j gerontol med sci 2006;61a:474-9. 22. lucca u, tettamanti m, mosconi p, apolone g, gandini f, nobili a, et al. association of mild 160 anemia with cognitive, functional, mood and quality of life outcome in elderly: the “health and anemia” study. available at: http//www.plosone.org. accessed nov 17, 2009. 23. denny sd, kuchibhatia mn, cohen hj. impact of anemia on mortality, cognition, and function in community-dwelling elderly. am j med 2006; 119:327-34. 24. thomas dr. anemia and quality of life: unrecognized and undertreated. j gerontol med sci 2004;59a:238-41. meiyanti anemia and quality of life 25. canbaz s, sunter at, dabak s, peksen y. the prevalence of chronic disease and quality of life in elderly people in samsun. turk j med sci 2002; 33:335-40. 26. alfons r, palmi j, sigurbjom b, inga t. anemia, nutritional status and inflammation in hospitalized elderly. nutrition 2008;24:1116-22. 27. agarwal r, davis jl, smith l. serum albumin is strongly associated with erythropoietin sensitivity in hemodialysis patients. clin j am soc nephrol 2008;3:9801-4. alvina 95 abstract microbiological procedures for v. cholerae isolation from clinical specimens are important factors in clinical and epidemiological management of cholera. the standard preservation medium for enteric pathogenic bacteria, including v. cholerae, is cary-blair medium (cb), a semisolid medium for preservation and transport of specimens containing intestinal bacteria. a special medium for vibrio organisms is alkaline peptone water (apw), which is both a transport and an enrichment medium. the purpose of this study was to ascertain the suitability of apw supplemented with 0.5% agar (apw-0.5) as a sensititive preservationtransport medium for rectal swab specimens for isolation of v. cholerae. a total of 144 paired rectal swab specimens were collected from children and adults with acute diarrhea. of each specimen pair, one was placed in cb and the other in apw-0.5, from which they were plated out to thiosulfate citrate bile sucrose (tcbs) agar. altogether, from both cb and apw-0.5 transported specimens, v. cholerae non-o1 was present in 29 (20.1%) specimens, while only 2 (1.4%) specimens were positive in cb and 9 (6.3%) positive in apw-0.5 transported specimens. the number of v. cholerae non-o1 isolates from apw-0.5 transported specimens was significantly higher (p=0.000) as compared to that from caryblair transported specimens. it may be concluded that for isolation of v. cholerae, specimen transport in apw-0.5 medium was more effective than transport in cary-blair medium. keywords: alkaline peptone water, agar, vibrio cholerae, rectal, swab *department of pharmacology, **department of public health, ***department of microbiology, medical faculty, trisakti university jakarta correspondence dr. meiyanti, spfk department of pharmacology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone. 021-5672731 ext.2805 univ med 2011;30:95-101. alkaline peptone water plus 0.5% agar suitable for transport of vibrio cholerae meiyanti*, oktavianus ch. salim**, julius e. surjawidjaja***, and murad lesmana*** may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina introduction cholera is an acute diarrheal disease caused by serogroups o1 and o139 of v. cholerae. in its severest form the disease is characterized by profuse diarrhea with rice water stools, rapidly leading to dehydration. cholera has two special epidemiological f e a t u r e s , n a m e l y i t s t e n d e n c y t o c a u s e explosive epidemics, frequently at several foci simultaneously, and its capability for causing p a n d e m i c s t h a t p r o g r e s s i v e l y i n v o l v e numerous parts of the world, as has been the case up to the present.(1-3) 96 meiyanti, salim, surjawidjaja, et al alkaline peptone water for vibrio cholerae in several countries this disease has become endemic, from time to time causing epidemics.(1,4-6) it is estimated that around 5.5 million cholera cases occur annually in asia and africa, 8% of these being of such severity as to need hospitalization, with 20% of these hospitalized cases terminating fatally, giving rise to a total annual mortality of 120,000.(7) consequently cholera is still considered to be a serious health problem in many developing c o u n t r i e s w i t h p o o r s o c i o e c o n o m i c c o n d i t i o n s . (8,9) c h o l e r a s u r v e y s a r e s t i l l i m p o r t a n t i n s t r u m e n t s f o r d e t e r m i n i n g behavioral trends of the disease in many areas around the globe and within a given country, in view of the fact that the majority of the reported numbers of cases are underestimates.(8,9) among the enteric pathogenic bacteria v. cholerae is probably the easiest organism to identify, for which however a laboratory infrastructure is required. the accuracy of laboratory investigations, including the efficient use of material such as culture media,(10,11) has a considerable impact on the exactness of bacterial identification and the ensuing accuracy of the report. although treatment of cholera cases is not based on prior identification of the causal organism, the microbiological procedures for the isolation of v. cholerae organisms from clinical specimens are important factors determining the clinical and epidemiological management of cholera.(7) the specimens, either from stools or rectal swabs, are collected at the earliest opportunity or at onset of the disease, prior to administration of antibiotics. if the specimens cannot be processed within two hours, they are placed in preservation media.(7,12) the most common preservation medium used for enteric pathogenic bacteria, including v. cholerae, is cary-blair medium, which is a semisolid medium for preservation and transport of specimens containing intestinal bacteria. a special medium for vibrio organisms is alkaline peptone water (apw), which is both a transport and an enrichment medium.(7,12) however, apw is almost never used for transport of vibrio organisms, due to concern that longtime storage of specimens in apw at room temperature encourages overgrowth by organisms such as pseudomonas, proteus and vibrio non-o1, which are capable of inhibiting growth of v. cholerae.(7,11) in contrast, there are reports stating that specimens enriched in apw and incubated for more than 20 hours did not show suppression of vibrio isolation rates by growth of commensals.(7) as a consequence, apw may presumably be used for transport of v. cholerae within a period of 20-24 hours, yielding a substantially high sensitivity of isolation, due to the enrichment features of apw. concern that apw in its liquid form is subject to spillage, resulting in contamination and infection by specimens containing v. cholerae, may be countered by the addition of 0.5% agar, thus transforming apw into a semi-solid medium. the purpose of this study was to ascertain the suitability of apw supplemented with 0.5% agar (apw-0.5) as a safe and sensititive preservation-transport medium for rectal swab specimens for isolation of v. cholerae. methods study population subjects recruited for this study were patients with diarrhea attending the mampang prapatan primary health center in south jakarta. the subjects were categorized as having diarrhea on the basis of a self-reported frequency of defecation of 3 or more times within 24 hours, with watery/liquid/soft stools.(8,10) before collection of the fecal specimens, the subjects were informed on the purpose of the study and were requested to sign a consent form for participation in the study. in the case of children, the consent form would be signed by their caregivers. subsequently a clinical questionnaire was to be filled out by research personnel with data obtained by interviewing the subjects, comprising data on age, gender, duration of diarrhea, type of diarrhea, and clinical symptoms. 97 preservation and transport medium standard cary-blair medium was prepared according to the manufacturer’s instructions, while apw supplemented with 0.5% agar (apw-0.5) was prepared by adding 10 g peptone, 10 g sodium chloride, and 5 g agar to 1 l of distilled water (ph 8.4). specimen collection and transport the stool or rectal swab specimens were collected prior to administration of antibiotics. rectal swabs were obtained by means of cotton swabs mounted on sticks. for subject comfort, the cotton swabs were wetted in transport medium before rectal insertion. to obtain a rectal specimen, the cotton swab was inserted past the anal sphincter (around 2-3 cm), slowly rotated and withdrawn, and immediately placed in transport medium. from each patient one pair of rectal swabs was taken, one of the pair being placed in cary-blair medium and the other in apw-0.5 medium. the specimens were then stored at room temperature prior to being sent to the microbiology laboratory of the medical faculty, trisakti university. in the case of patients with prior antibiotic therapy, this fact w a s r e c o r d e d i n t h e q u e s t i o n n a i r e f o r consideration in the subsequent data analysis. bacteriological procedure on arrival at the laboratory the rectal swab specimens in cary-blair and apw-0.5 were immediately plated out on thiosulfate citrate bile salts sucrose (tcbs) agar, a selective medium for vibrio organisms. the tcbs plates were then incubated aerobically at 370c for 18-20 hours. bacterial growth resembling vibrio colonies were picked and processed according to the following standard procedures for identification of v. cholerae,(8,12) viz. oxidase t e s t , b i o c h e m i c a l r e a c t i o n s ( c o m p r i s i n g kligler’s iron agar, sucrose, semisolid, motility, i n d o l e , o r n i t h i n e , l y s i n e , a rg i n i n e ) , a n d serological tests with specific antiserum for determination of v. cholerae serotypes. statistical analysis the significance of the difference in transport media was assessed by comparing the proportions of positive specimens. a p value of <0.05 was considered to indicate statistical significance. computations were performed by means of epi info version 3.5.1. results within a period of 10 months a total of 144 paired rectal swab specimens were collected from children and adults with acute diarrhea. from these paired specimens, vibrio species were isolated from 44 (30.6%) rectal swab specimen pairs, comprising 40 isolates (90.9%) of v. cholerae non-o1 and 4 isolates (9.1%) of v. parahaemolyticus, without any isolates of v. cholerae o1 organisms being found in this study. there were therefore 100 (69.4%) paired rectal swab specimens with negative results, yielding no isolates of vibrio species (table 1). among the rectal swab specimens in caryblair medium, v. cholerae non-o1 organisms were present in 21.5% (31/144) specimens and v. parahaemolyticus in 2.8% (4/144) specimens. in comparison, the rectal swab specimens transported in apw-0.5 yielded the following results: v. cholerae non-o1 was present in 26.4% (38/144) specimens, while the frequency table 1. number of isolates of v. cholera non-o1 and v. parahaemolyticus recovered from 144 patients with diarrhea number of isolates (%) n bacterial isolate v. cholerae non-o1 v. parahaemolyticus positive 44 40 (90.9%) 4 (9.1%) negative 100 univ med vol.30 no.2 98 meiyanti, salim, surjawidjaja, et al alkaline peptone water for vibrio cholerae of v. parahaemolyticus isolates was identical to the cary-blair isolates (table 2). table 2 also s h o w s t h a t 2 9 ( 2 0 . 1 % ) s p e c i m e n p a i r s transported in cary-blair and apw-0.5 yielded positive results for v. cholerae non-o1, while only 2 (1.4%) rectal swab specimens were positive in cary-blair and 9 (6.3%) positive in apw-0.5 transported specimens. results of m c n e m a r ’s t e s t i n d i c a t e d a s i g n i f i c a n t difference between number of isolates of v. cholerae in cary-blair and that in alkaline p e p t o n e w a t e r ( p < 0 . 0 0 5 ) . i s o l a t e s o f v. c h o l e r a e i n a p w ( 9 / 4 0 = 2 2 . 5 % ) w e r e substantially higher in number than isolates in cb (2/40 = 5%) (table 3). discussion the specimens used for isolation of v. cholerae are stools or rectal swabs, collected at the earliest opportunity or at onset of the disease, prior to administration of antibiotics, as the numbers of v. cholerae organisms in stools decline immediately upon antibiotic a d m i n i s t r a t i o n . ( 8 ) t h e s p e c i m e n s s h o u l d preferably not be collected from bedpans, because they may have been contaminated by p r e v i o u s u s e o r m a y c o n t a i n t r a c e s o f disinfectants after cleaning, leading to false positive or false negative results. for collecting stools, sterile stool cups may be used. rectal swab specimens give as good results as stools in the isolation of v. cholerae, particularly in the acute phase of the disease.(5) in addition, rectal swabs specimens are superior to stools for infants and field surveys, but are inadequate for convalescents or contacts.(7) incubation for 6-8 hours is the method recommended for apw,(5) and is used in the majority of laboratories.(8,13,14) in the initial 68 hours of incubation, vibrio organisms show a rapid growth, but more than 8 hours of incubation is considered to result in growth of competing bacteria and inhibition of vibrio organisms, impeding isolation of the latter. h o w e v e r, r e c e n t r e s e a r c h p o i n t s t o t h e contrary, in that enrichment in apw for 24 hours, although promoting the growth of nonvibrio organisms such as proteus to greater numbers than vibrio organisms, yet did not decrease the growth of v. cholerae.(4) in the present study it was found that after 24 hours there was a large number of both non-vibrio and vibrio colonies on the culture plates, the latter thus being easily picked and identified. enrichment in apw for 6-8 hours does not yield significantly better results in comparison with enrichment in apw for 24 hours, with r e g a r d t o c a p a b i l i t y f o r i s o l a t i o n o f v. cholerae.(15) it may be concluded that apw is superior as to frequency of isolation of vibrio species, when compared to cary-blair medium. cb apw + + 29 2 9 104 table 3. number of isolates of v. cholerae non-o1 in cary-blair (cb) and alkaline peptone water (apw) + 0.5% agar culture results v. cholerae non-o1 v. parahaemoluticus cb apw +0.5% number % number % positive positive 29 20.1 4 2.8 positive negative 2 1.4 0 0 negative positive 9 6.3 0 0 negative negative 104 72.2 1 40 97.2 table 2. distribution of culture results of v. cholerae non-o1 and v. parahaemolyticus from 144 rectal swab specimens in cary-blair (cb) vs. alkaline peptone water (apw) + 0.5% agar 99 this is presumably because apw is both an enrichment and a transport medium. in its original form, apw is a liquid medium. supplementation with 0.5% agar minimizes s p i l l a g e o r l e a k a g e , t h u s p r e v e n t i n g e n v i r o n m e n t a l c o n t a m i n a t i o n b y e n t e r i c pathogens. however, the results of transport of v. cholerae in agar-supplemented apw for a period of more than 24 hours cannot as yet be predicted. there is a possibilty that after 48 hours the commensal organisms start to reproduce rapidly, thus inhibiting v. cholerae, ultimately leading to negative isolation results for v. cholerae.(12) in that case, specimen transit times in apw for days or even for more than o n e w e e k , a s i s c o m m o n w i t h c l i n i c a l specimens in cary-blair medium, will cause apw to be ineffective as transport medium for v. cholerae. in cary-blair medium vibrio organisms remain viable for 4 weeks, without any possibility of inhibition by excessive growth of commensal organisms.(12) according to this line of reasoning, cary-blair medium is to be preferred for transport of stool or rectal s w a b s p e c i m e n s f o r i s o l a t i o n o f e n t e r i c pathogenic bacteria, including v. cholerae, whereas enrichment-transport media are to be used only when the field-collected specimens can be cultured within 12-24 hours after collection.(7,12) if it can be demonstrated that specimen transit times in enrichment-transport media of more than 48 hours or even more than one week does not impair the viability of enteric pathogenic bacteria such as vibrio organisms, use of such media for preservation and transport of vibrio organisms may be taken into consideration. the present study succeeded only in isolating v. cholerae non-o1 at a frequency of 27.8%, whereas no v. cholerae o1 were found. this may be due to the drastic decline in the v. cholerae o1 population in indonesia since 2000, from 10.5% 18.3% in the period of 1993-1998(16) to < 2% after the year 2000,(17) for unknown reasons. v. cholerae o1 could probably be isolated with a larger sample. however, because v. cholerae o1 and v. c h o l e r a e n o n o 1 h a v e i d e n t i c a l c u l t u r e characterisitics, the results on the viability of v. cholerae non-o1 in cary-blair and apw may be assumed to refer also to v. cholerae o1. both strains may be said to differ only in serology, clinical picture, and epidemiology. serologically, v. cholerae non-o1 does not show agglutination with anti-o1 antiserum.(12) with respect to clinical manifestations, vibrio o1 causes profuse diarrhea, while v. cholerae non-o1 causes a milder diarrhea, but tends to r e s u l t i n e x t r a i n t e s t i n a l i n f e c t i o n s a n d bacteriemia.(7,19) epidemiologically, v. cholerae o1 frequently causes epidemics, whereas v. cholerae non-o1 is sporadic and non-epidemic in nature.(18) reports on evaluations of apw as an enrichment and transport medium for v. cholerae have never been encountered after the year 1997,(15) making it difficult to obtain descriptions of more recent studies on apw. in spite of this, there have presumably been few changes since the last published reports on this topic. however, the discovery of a new variant of v. cholerae o1 that is a hybrid of the classic and el tor biotypes(20-23) poses a new threat for the occurrence of epidemics of pandemic proportions. therefore, there should be increased efforts at maximal isolation of v. cholerae, particularly v. cholerae o1, using various culture and transport systems. the efficacy of enrichment in apw is reportedly higher for v.cholerae non-o1 as compared with o1 strains. enrichment in apw r e s u l t s i n s i g n i f i c a n t l y h i g h e r y i e l d s o f v.cholerae non-o1 isolates, in comparison with direct plating.(15) our study results are similar to those of previous studies on the evaluation of enrichment in apw for isolation of v. cholerae, demonstrating the superiority of enrichment to direct plating. consequently, enrichment media with features of transport media, such as apw, have a higher effectivity a n d s e n s i t i v i t y f o r i s o l a t i o n o f vi b r i o organisms. univ med vol.30 no.2 100 meiyanti, salim, surjawidjaja, et al alkaline peptone water for vibrio cholerae one limitation of the present study is the relatively short transport time of the specimens in apw (less than 48 hours), which cannot describe the ultimate fate of commensal organisms in the apw. further long-term studies are necessary to resolve this question. conclusion for isolation of v. cholerae, specimen transport in apw-0.5 medium is significantly more effective than transport in cary-blair medium. acknowledgements the investigators thank the dean and viced e a n s o f t h e m e d i c a l f a c u l t y, tr i s a k t i university, for the funding of this study. thanks are also due to the doctors and staff of the mampang prapatan primary health center, and to all assisting parties for the successful completion of this study. references 1. simanjuntak ch, larasati w, arjoso s. cholera in indonesia in 1993-1999. am j trop med hyg 2001;65:788-97. 2. lesmana m. recent developments of cholera infections. j kedokter trisakti 2004;23:101-9. 3. salim a, lan r, reeves pr. vibrio cholerae pathogenic clones. emerg infect dis 2005;11: 1758-60. 4. nguyen bm, lee jh, cuong nt, choi sy, hien nt, anh dd, et al. cholera outbreaks caused by an altered vibrio cholerae o1 el tor biotype strain producing classical cholera toxin b in vietnam in 2007 to 2008. j clin microbiol 2009; 47:1568-71. 5. krishna bv, patil ab, chandrasekhar mr. fluoroquinolone-resistant vibrio cholerae isolated during a cholera outbreak in india. trans r soc trop med hyg 2006;100:224-6. 6. theophilo gn, rodrigues dp, leal nc, hofer e. distribution of virulence markers in clinical and environmental vibrio cholerae non-o1, nono139 strains isolated in brazil from 1991-2000. rev inst med trop sao paolo 2006;48:65-70. 7. sack da, sack rb, nair gb, siddique ak. cholera. lancet 2004;363:223-33. 8. agtini md, soeharno r, lesmana m, punjabi nh, simanjuntak c, wangsasaputra f, et al. the burden of diarrhea, shigellosis, and cholera in north jakarta, indonesia: findings from 24 months surveillance. bmc infect dis 2005;17: 341-50. 9. kosek m, bern c, guerrant rl. the global burden of diarrhoeal disease, as estimated from studies published between 1992-2000. bull world health organ 2003;81:197-204. 10. thielman nm, guerrant rl.acute infectious diarrhea. n engl j med 2004;350:38-47. 11. world health organization. cholera 2001. wkly epidemiol rec 2002;77:257-68. 12. farmer jj iii, janda jm, birkhead k. vibrio. in: murray pr, baron ej, jorgensen jh, pfaller ma, yolken rh, editors. manual of clinical microbiology. 8th ed. washington dc: american society for microbiology;2003.p.706-18. 13. bhuiyan na, qadri f, faruque asg, malek ma, salam ma, nato f, et al. use of dipstick for rapid diagnosis of cholera caused by vibrio cholerae o1 and o139 from rectal swabs. j clin microbiol 2003;41:3939-41. 14. tamrakar ak, goel ak, kamboj dv, singh l. surveillance methodology for vibrio cholerae in environmental samples. int environ health res 2006;16:305-12. 15. lesmana m, richie e, subekti d, simanjuntak c, walz se. comparison of direct plating and enrichment methods for isolation of vibrio cholerae from diarrhea patients. j clin microbiol 1997;35:1856-8. 16. lesmana m, subekti ds, tjaniadi p, simanjuntak ch, punjabi nh, campbell jr, et al. spectrum of vibrio species associated with acute diarrhea in north jakarta, indonesia. diagn microbiol infect dis 2002;43:91-7. 17. oyofo ba, lesmana m, subekti d, tjaniadi p, larasati w, putri m, et al. surveillance of bacterial pathogens of diarrhea disease in indonesia. diagn microbiol infect dis 2002;44:227-34. 18. cariri famo, costa apr, melo cc, theophilo gnd, hofer e, de melo neto op, et al. characterization of potentially virulent non-o1/ non-o139 vibrio cholerae strains isolated from human patients. clin microbiol infect 2010;16: 62-7. 19. dalsgaard a, forslund a, hesselbjerg a, bruun b. clinical manifestations and characterization of extra-intestinal vibrio cholerae non-o1, nono139 infections in denmark. clin microbiol infect 2000;6:626-8. 101 20. pourshafie m, grimont f, kohestani f, grimont pad. a molecular and genotypic study of vibrio cholerae in iran. j med microbiol 2002;51:3928. 21. nair gb, faruque sm, bhuiyan na, kamruzzaman m, siddique ak, sack da. new variants of vibrio cholerae o1 biotype el tor with attributes of the classical biotype from hospitalized patients with acute diarrhea in bangladesh. j clin microbiol 2002;40:3296-9. 22. lizarraga-partida ml, quilici ml. molecular analyses of vibrio cholerae o1 clinical strains, including new nontoxigenic variants isolated in mexico during the cholera epidemic years between 1991-2000. j clin microbiol 2009;47: 1362-71. 23. nair gb, qadri f, holmgren j, svennerholm am, safa a, bhuiyan na, et al. cholera due to altered el tor strains of vibrio cholerae o1 in bangladesh. j clin microbiol 2006;44:4211-3. univ med vol.30 no.2 oktavianus 11 *health analyst faculty of nursing and health muhammadiyah university semarang **department of nutrition consultant of dr. kariadi hospital/ diponegoro university semarang ***department of pathology consultant at dr. kariadi hospital/ diponegoro university semarang ****department of pharmacy dr. kariadi hospital/ diponegoro university semarang correspondence dr. budi santosa, s.km, m.si.med health analyst faculty of nursing and health muhammadiyah university jl. kedungmundu raya 18 semarang email: budi_unimus@yahoo.co.id univ med 2014;33:11-8 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background lead acetate inhibits pyrimidine-5’-nucleotidase resulting in ribonucleic acid and ribosome accumulation in erythrocytes (rbc), visible as basophilic stippling (bs). lead exposure disrupts rbc membrane, shortens the rbc life span and decreases hematocrit. zinc supplementation increases lead-binding proteins (metallothioneins). the study objective was to determine whether zinc supplementation prior to lead exposure decreases bs and increases the hematocrit in rats. methods the study was a randomized post-test only control-group trial, using 28 rats allocated to one control and 3 intervention groups (zinc i, zinc ii, zinc iii) receiving 0.2 mg, 0.4 mg, and 0.8 mg of zinc by oral gavage daily for 3 weeks. all groups were then exposed to lead at 0.5 g/kg bw/day by gavage for 10 weeks. on the last day of the 10 weeks bs was determined from giemsastained blood smears and hematocrit by hematology analyzer. between-group differences were tested with one-way anova, followed by bonferroni’s test. results mean bs was significantly decreasing 7.93 ± 0.99% in controls, 6.91 ± 1.04%, 4.66 ± 0.79%, and 1.73 ± 0.88%, respectively, in intervention groups (p=0.000). mean hematocrit was significantly increasing 26.16 ± 3.60% in controls, and 30.33 ± 6.12%, 36.83 ± 3.31%, and 40.00 ± 4.77%, respectively, in intervention groups (p=0.000). one-way anova and bonferroni’s test showed significant differences in bs and hematocrit between controls and intervention groups receiving zinc supplementation of 0.4 and 0.8 mg (p=0.000). conclusion zinc supplementation before lead exposure significantly decreases basophilic stippling and increases hematocrit level in rats exposed to lead. keywords: zinc, basophilic stippling, hematocrit, rats zinc supplementation decreases basophilic stippling in rats exposed to lead budi santosa*, hertanto wahyu subagio**, lisyani suromo***, and henna rya sunoko**** 12 santosa, subagio, suromo, et al basophilic stippling in exposed to lead suplementasi seng menurunkan jumlah basophilic stippling pada tikus yang terpajan plumbum latar belakang plumbul (pb) asetat menghambat enzim pirimidin-5’-nukleotidase yang menimbulkan akumulasi ribonulcleic acid serta ribosom eritrosit ditandai adanya basophilic stippling (bs). pajanan pb menyebabkan kerusakan membran eritrosit, umur eritrosit pendek dan penurunan hematokrit. suplementasi seng meningkatkan protein metallothionein yang mengikat pb. tujuan penelitian ini adalah untuk membuktikan suplementasi seng sebelum pajanan pb menurunkan bs dan meningkatkan hematokrit pada tikus. metode randomized post test only control-group design digunakan pada 28 tikus yang dibagi menjadi 4 kelompok yaitu 1 kelompok kontrol dan 3 kelompok perlakuan yang disuplementasi seng 0,2; 0,4; 0,8 mg diberikan setiap hari melalui sonde sampai minggu ke-3. kemudian semua kelompok diberi pajanan pb 0,5 gr/kg bb/hari melalui sonde sampai minggu ke-13. hari terakhir minggu ke-13 diperiksa jumlah bs menggunakan pengecatan giemsa dan kadar hematokrit menggunakan haematology analyzer. perbedaan antar kelompok dilakukan menggunakan one way anova, dilanjutkan dengan uji bonferroni. hasil rata-rata bs menurun secara bermakna mulai kelompok kontrol sampai perlakuan ke-3 (7,93 ± 0,99%; 6,91 ± 1,04; 4,66 ± 0,79%; 1,73 ± 0,88%) (p=0,000). rata-rata hematokrit meningkat secara bermakna mulai kelompok kontrol sampai perlakuan ke-3 (26,16 ± 3,60%;30,33 ± 6,12%; 36,83 ± 3,31%; 40,00 ± 4,77%) (p=0,000). uji anova dilanjutkan dengan uji bonferroni menunjukkan bs dan hematokrit berbeda bermakna antara kelompok kontrol dengan kelompok yang disuplementasi seng 0,4 dan 0,8 mg (p=0,000). kesimpulan suplementasi seng sebelum pajanan pb mampu menurunkan bs dan meningkatkan hematokrit secara signifikan pada tikus terpajan pb. kata kunci: seng, basophilic stippling, hematokrit, tikus abstrak introduction one of the hazardous air pollutants is lead that may cause health problems and may be lifethreatening to humans. the level of lead utilization in indonesia is still considerable, although its use in gasoline has largely been abandoned. several large cities such as jakarta, bandung, semarang, surabaya, medan and others are at potential risk of lead poisoning.(1) in west and central java the use of phosphate fertilizers, pesticides, and herbicides has increased, thus potentially increasing lead exposure. the main sources of lead poisoning are vegetables, batteries, paints, cosmetics, jewelry, toys, gasoline, and others. exposure to lead in humans may occur by way of lead-contaminated air, foods, and drinks. the highest risk of lead poisoning is in children, pregnant women, and industrial workers.(2) the main signs of lead poisoning are anemia,(2) decreased hematocrit and the presence of inclusions in the red blood cell (rbc) called basophilic stippling (bs).(3,4) 13 the lead tocicity threshold of 20-40 µg/dl is already capable of causing anemia in children.(5) a previous study conducted in india on children found that 38% of the children had a blood lead content 10-19.9 µl/dl, and 9% had a lead level of >20 µl/ dl. children with a lead level of >10 µl/dl have a 1.7 times higher risk of anemia that those with a lead level of <10 µl/dl.(6) the study conducted by hariono (7) showed that administration to rats of oral lead acetate at 0.5 g/kg/bw/day for 16 weeks caused anemia, weight loss, and increased numbers of reticulocytes since the 10th week, accompanied by increased activity of ä-aminolevulinic acid dehydratase (alad). another study showed that blood lead levels of >7 µg/dl in children can inhibit the activity of enzymes involved in hemoglobin synthesis, resulting in subclinical effects characterized by elevated levels of ä-ala and protoporphyrins.(8) in heme biosynthesis, lead poisoning depresses enzyme alad activities at the starting point, at midpoint (coproporphyrinogen oxidase), and at the endpoint (ferrochelatase) of heme biosynthesis, thus leading to anemia. approximately 90% of the lead introduced into the circulation will enter the rbc, where it acts as a prooxidant, causing oxidative stress that results in damage to the membranes of rbc and shortening of their lifespan.(3,4) membrane damage and shortened lifespan causes a decrease in rbc number and volume, as indicated by the hematocrit. lead also causes glucose-6 phosphate dehydrogenase (g6pd) deficiency and inhibition of pyrimidine-5’nucleotidase. the latter results in accumulation of ribonucleid acid (rna) and ribosomes in rbc, characterized by bs of rbc,(3,4) which can be used as a marker of lead poisoning. the management of lead poisoning by means of chelating agents has been studied previously. the chelating agents act to bind lead with the formation of polar (hydrophilic) complexes which are excreted by the kidneys. according to the study by suaniti,(9) the success rate of reducing lead toxicity by intravenous administration of the chelating agent edta is only 4.91%. the use of edta is only for therapeutic or curative purposes and thus not maximal. therefore preventive measures to combat lead toxixity are necessary. lead in the tissues is bound to the metalbinding proteins called metallothioneins at their sulfhydryl groups.(10) metallothioneins can be synthesized in the liver as well as the gastrointestinal wall through absorption of optimal amounts of zinc.(11) zinc supplementation at stepwise increasing daily doses of 0.2 mg, 0.4 mg, and 0.8 mg to rats (rattus norvegicus) may increase metallothionein levels.(12) the administration of zinc supplements as an alternative preventive measure against lead poisoning needs to be subjected to further study, the results of which can be measured by the amount of bs and the hematocrit. a meta-analysis of zinc supplementation in humans stated that doses of 1.5-50 mg zinc per day, by considering the influence of various food materials such as fibers, phosphates, and phytates, which are capable of inhibiting the level of zinc absorption.(13) to observe the effectivity of certain doses in increasing metallothioneins in humans, stepwise increasing doses of zinc supplementation should be considered, e.g. 10 mg per day, 20 mg per day and 40 mg per day. conversion of these doses to experimental animals yields the following doses of 0.2 mg, 0.4 mg, and 0.8 mg, respectively.(12) previous sttudies on lead exposure impacting on bs and hematocrit have yielded consistent results, i.e. considerable increases in bs and decreases in hematocrit. the present study differs from the previous ones, since it does not aim to merely observe the increase in bs nor the decrease in hematocrit after lead exposure. on the contrary, this study aims to determine whether zinc supplementation to rats before exposure to lead may decrease bs and increase the hematocrit. methods research design this study was of randomized post-test only control-group design. the experimental animals were kept and subjected to the intervention at the integrated research and testing laboratory, gadjah mada university, yogyakarta. the animals univ med vol. 33 no.1 14 santosa, subagio, suromo, et al basophilic stippling in exposed to lead were kept for 100 days, starting from the selection period up to the end of the intervention period, from december 2012 to march 2013. experimental animals the sample size was determined by means of the formula: s = (t–1) (r–1) >15, where t = number of groups, r = number of animals per group. this study uses 3 intervention groups and one control group, therefore t=4, (4-1) (r-1) >15, yielding r >6. the number of rats per group was 6, thus giving a sample size of 24 animals. to each group was added one animal in anticipation of death of the animals, so that the total number of animals used in this study was 28 male rats (rattus norvegicus) aged 15 weeks. zinc supplementation by simple random assignment, the rats were allocated to 4 groups, viz. one control group and 3 intervention groups. from the first until the third week, the intervention groups received zinc supplementation by oral gavage at stepwise increasing doses of 0.2, 0.4, 0.8 mg/day, respectively, whereas the control group did not receive zinc supplementation. lead exposure from the third week until the 13th week all groups of animals were exposed to lead at 0.5 g/kg bw/day, in the form of oral lead acetate solution given by oral gavage (gastric tube). measurement of blood parameters on the last day of the 13th week the control group and the intervention groups were examined for amount of bs and hematocrit level. basophilic stippling was measured using methanol-fixed blood smears with giemsa staining. the results were expressed as percentages (%), the normal values being 0-1%. the hematocrit was determined by means of a hematology analyzer, based on the principle of electrical impedance, using lyse, rinse, and diluent solutions. the specimens used were whole blood samples of 3 ml with added edta as anticoagulant collected in vacutainer tubes. determination of blood parameters was done at the health analyst clinical pathology laboratory, faculty of medicine and health science, muhammadiyah university, semarang. data analysis for testing of significant between-group differences in amounts of bs and hematocrit levels, one-way anova test was used, followed by bonferroni’s test. ethical clearance the present study obtained ethical clearance no.339/ec/fk/rsdk/2012 from the ethics commission, faculty of medicine, diponegoro university /dr. kariadi hospital, semarang. after the head of the laboratory for integrated research and testing, gadjah mada university, yogyakarta, was informed of the decision, the study was allowed to be conducted. results there was weight loss in the control animals between the third week until the 13th week after exposure to the lead acetate. in contrast, there was a continued increase in weight of the animals in all three intervention groups, in spite of exposure to the lead acetate at identical doses. in table 1 is shown the weight of the animals of the control group and the groups receiving stepped doses of zinc, at baseline and at the end of the third and 14th weeks. basophilic stippling occurs from accumulation of ribonucleic acid (rna) and ribosomes in rbc as a result of inhibition of pyrimidine-5’-nucleotidase upon lead exposure. as shown in figure 1, the non-supplemented control animals had more bs than the animals in the zinc iii group receiving zinc supplementation of 0.8 mg. the mean amount of bs in the control group differed from that in the intervention groups. the highest mean value (7.93 ± 0.99%) was in the control group receiving no zinc supplementation, while the lowest (1.73 ± 0.88%) was in the intervention group receiving zinc supplementation of 0.8 mg. according to the anova test results, 15 there was a significant difference between control and intervention groups (p=0.000) (table 2). on the basis of the bonferroni test results (table 3), the difference was between the control group versus the zinc-ii and zinc-iii intervention groups (both at p=0.000). there was a decrease in bs from the control group to the stepped-dose zinc supplementation groups. the mean hematocrit values in the control and intervention groups are shown in table 2. there was an increase in hematocrit values starting from the control group up to the third intervention group. the lowest mean was in the control group (26.16 ± 3.60%), and the highest in the third intervention group receiving zinc supplementation of 0.8 mg (40.00 ± 4.77%). the anova results showed a significant difference between the control and intervention group (p=0.000). according to the bonferroni test results (table 3), the significant difference was between the control group versus the second and third intervention groups (p=0.000). increased zinc supplementation was accompanied by an increase in hematocrit level in rats exposed to lead. table 1. mean weights of the animals control zinc-iii discussion in this study there was a decrease in bs after zinc supplementation. the highest mean value for bs was in the control group who did not receive zinc supplementation, while the lowest mean was in the intervention group receiving zinc supplementation at the highest dose of 0.8 mg. statistically, there was a significant difference in bs between the control group and all intervention groups, where the higher zinc supplementation dose resulted in the lowest amounts of bs in leadexposed rattus norvegicus. our findings are supported by those of hariono,(7) who found evidence that exposure of rats to lead at 0.5 g/kg/bw increased the amount of bs in the 10th week. basophilic stippling is visible as blurred blue points in rbc and frequently occurs in cases of lead exposure. lead inhibits pyrimidine-5'-nucleotidase and alters the functions of other nucleotides. lead can interfere with heme synthesis, so as to alter the concentrations of enzymes and intermediates involved in heme synthesis or their derivatives. figure 1. basophilic stippling in control and zinc-iii groups after intervention univ med vol. 33 no.1 16 santosa, subagio, suromo, et al basophilic stippling in exposed to lead table 2. mean basophilic stippling and mean hematocrit after the 13th week, by intervention group table 3. bonferroni test results for basophilic stippling and hematocrit after the 13th week, by intervention group lead poisoning can also lead to increased amounts of bs in rbc.(14) lead exposure causes membrane damage in rbc, resulting in a shortened rbc lifespan and a decreased rbc count. the bone marrow as the site of erythropoiesis compensates for the decreased rbc numbers in the circulation by increased erythropoietic activity, leading to increased numbers of immature rbc in the circulation.(4,15) in the present study lead exposure without zinc supplementation results in high mean percentages of bs, whereas lead exposure with zinc supplementation results in reduction in mean percentages of bs. this shows that zinc supplementation is capable of decreasing the amount of bs in rats exposed to lead. the hematocrit is the percentage volume of rbc in 100 ml of blood and is largely influenced by the rbc count. in our study, the hematocrit level of rats exposed to lead without zinc supplementation was lower than that of rats receiving preventive zinc supplementation. there was a significant difference in hematocrit level between the control group receiving no zinc supplementation and the intervention groups receiving stepped-dose supplementation. most of the lead in the blood is localized to the rbc, with 90% in the rbc cytoplasm and 10% in the rbc membrane, mainly bound to lipids and lipoproteins.(5) the distribution of lead in rbc cytoplasm and membrane is due to binding of lead to cytoplasmic elements, its transportation is through the rbc membrane, and its excretion via the calcium pump. the toxic effect of lead on rbc is caused by its ability to form complexes with negatively charged ligands, particularly sulfhydryl and carboxyl groups, and with imidazole enzymes and other proteins.(16) many investigators have reported that the most important mechanism of lead toxicity is the production of free radicals. reactive oxygen species react with cellular macromolecules, namely dna, proteins, and lipids.(17) red blood cells are very sensitive to induction of oxidative stress by high lead exposure.(18,19) of the amounts of lead entering the circulation, around 90% goes to the rbc. in the rbc membrane, there are compounds or chemical reactions capable of producing potentially toxic oxygen species called pro-oxidants. increased amounts of pro-oxidants may cause oxidative stress. lead may also cause g-6pd deficiency that can inhibit rbc maturation in the bone marrow.(3,4) gugliotta et al.(19) in their investigations on the effect of lead on rbc membrane proteins, found a decrease in rbc count, in which membrane permeability plays an important role. this finding is supported by the study of patit et al.(20) on lead exposure in battery manufacturing workers as compared to a normal group without lead exposure. 17 these investigators found a significant decrease in rbc count in the lead-exposed group as compared with the control group. in addition, the findings of gugliotta et al.(19) was also supported by a laboratory experimental study on the addition of lead chloride to heparinized blood with a hematocrit concentration of 3%, which was incubated for 1 hour at 25°c in a medium containing stepped concentrations of lead chloride (0, 0.3, 0.5 and 1 µm). this procedure increased rbc permeability through cell damage and cell death, particularly at high lead concentrations, caused morphological and structural alterations in the rbc membrane, and decreased atp concentrations in rbc.(19) exposure to lead causes reduced rbc counts, ultimately impacting on decreased hematocrit levels. in the present study the zinc supplementation prior to lead exposure was presumably capable of increasing the amounts of metallothioneins, which function to bind lead, so that the hematocrit levels increased in the zinc supplementation groups, and increased significantly at a doses of 0.4 mg and 0.8 mg. one limitation of this study was that the hematology analyzer used was not capable of counting bs, which was therefore counted manually in giemsa stained slides. the clinical implication of the study is that zinc supplementation is capable of decreasing bs in lead-exposed rats, indicating that zinc is capable of reducing the impact of lead exposure. therefore further studies are needed to apply this finding to humans. conclusion zinc supplementation prior to lead exposure significantly decreases bs and increases hematocrit level in the blood of rats exposed to lead. references 1. suherni. lead poisoning in indonesia. in: robert a, o’brien e, editors. the global lead advice and support (glass) provides information and referels on lead poisoning and lead contamination prevention and management. the lead group incorporated. sydney: australia; 2010.p.1-16. 2. hegazy aa, zaher mm, abd el-hafez ma, morsy aa, saleh ra. relation between anemia and blood levels of lead, copper, zinc and iron among children. bmc 2010;3:133-8. 3. murray rk. porphyrin and bile pigments. in: murray rk, granner dk, rodwell vw. harper’s illustrated biochemistry. 27th ed. new york: mcgraw-hill;2006.p.279-93. 4. patrick l. lead toxicity, a review of the literature. part i: exposure, evaluation, and treatment. altern med rev 2006;11:2-22. 5. united states environmental protection agency (epa). air quality criteria for lead. epa 2006;2: 136-41. 6. jain nb, laden f, guller u, shankar a, kazani s, garshick e. relation between blood lead levels and childhood anemia in india. am j epidemiol 2005; 161:968-73. 7. hariono b. efek pemberian plumbum (timah hitam) pada tikus putih anorganik (rattus norvegicus). j sain vet 2006;24:125-33. 8. sunoko hr. peran gen polimorfik á asam aminolevulinic acid dehidratase (alad) pada intoksikasi pb. mmi 2008;43:1-10. 9. suaniti nm. pengaruh edta dalam penentuan kandungan timbal dan tembaga pada kerang hijau (mytilus viridis). ecotrophic 2007;2:1-7. 10. cheung rck, chan mhm, ho cs, lam cwk, law elk, lit ccw, et al. heavy metal poisoning clinical significance and laboratory investigation. asia pacific analyte note 2001;7:2-8. 11. hijova e. metallothionein and zinc: their functions and interactions. bratislavia med j 2004;105:2304. 12. santosa b, subagio ws, suromo l, sunoko hr. zinc supplementation dosage variations to metallothionein protein level of rattus norvegicus. int j sci eng 2013;5:15-7. 13. nissenson m, villegas as, lugo df, sanches ph, alonzo jd, lowe nm, et al. effect of zinc intake on serum/plasma zinc status in infants: a metaanalysis. matern child nutr 2013;9:285-98. 14. jun l, christopher jk. reticulocyte counts in sports medicine. n z j med lab sci 2012;66:36-8. 15. fry mm, mcgavin md. bone marrow, blood cell, and the lymphatic system. in: zachary jf m, mcgavin md, editors. pathologic basis of veterinary disease. 7th ed. st. louis, missouri mosby elsevier;2012.p.699-702. 16. bosiacka b, hlynczak aj, wiszniewska b, marchlewicz m. disorders of purine metabolism in human erythrocytes in the state of lead univ med vol. 33 no.1 18 santosa, subagio, suromo, et al basophilic stippling in exposed to lead contamination. pol j environ study 2004;13:46776. 17. oktem f, arslan mk, dundar b, delibas n, gultepe m, ergürhan ii. renal effects and erythrocyte oxidative stress in long-term low-level lead-exposed adolescent workers in auto repair workshops. arch toxicol 2004;78:681-7. 18. slobozhanina ei, kozlova nm, lukyanenko lm, oleksiuk ob, gabbianelli r, fedeli d, et al. leadinduced changes in human erythrocytes and lymphocytes. j app toxicol 2005;25:109-14. 19. gugliotta t, luca gd, romano p, rigano c, scuter a, romano p. effects of lead chloride on human erythrocyte membranes and on kinetic anion sulphate and glutathione concentrations. cel mol biol lett 2012;17:586-97. 20. patit aj, baghwar vr, patil ja, dongre nn, ambekor je, jaikhani r, et al. effect of lead (pb) exposure on the activity of superoxide dismutase and catalase in battery manufacturing workers of western maharashtra (india) with reference to heme biosynthesis. int j environ res public health 2006;3:329-37. puspa1 106 homocysteine and cognitive function in the elderly may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina pusparini* *department of clinical pathology, medical faculty, trisakti university correspondence dr. pusparini, sppk department of clinical pathology medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11410 telp. 021-5672731 ex.2404 email: pusparini_sppk@yahoo.com univ med 2009;28:106-16 abstract dementia is prevalent among elderly people, and projections show that the number of affected might triple over the next 50 years, because of a large increase in the oldest segment of the population. the objective of this study was to investigate the relationship between cognitive function and serum homocysteine level. this study was a cross-sectional study carried out in mampang district, south jakarta. a total of 94 elderly people was recruited for this study consisting of 44 females and 50 males. in this study serum homocysteine level was assessed by fluorescent polarization immunoassay and cognitive function with the mini mental state examination (mmse). in elderly women mmse scores for plasma homocysteine concentrations of <11.7 µmol/l, 11.7-14.9 µmol/l and >14.9 µmol/l were 24.00 ± 3.68, 23.80 ± 6.51, and 20.80 ± 9.00 respectively, with p=0.000. in elderly men the mmse scores for plasma homocysteine concentrations of <11.7 µmol/l, 11.714.9 µmol/l and >14.9 µmol/l were respectively 27.66 ± 2.06, 26.33 ± 3.79 and 24.03 ± 5.52 with p=0.008. homocysteine concentrations of >14.9 µmol/l were more commonly found in males (66%) than in females (22.70%). the results of a regression analysis indicated that the factors influencing cognitive function were level of education (p=0.001) and age (p=0.035), whereas gender and homocysteine concentration did not affect cognitive function (p=0.554 and p=0.714, respectively). plasma homocysteine concentration was inversely related to cognitive function. the most important factors affecting cognitive function were level of education and age. keywords : plasma homocysteine, cognitive function, elderly introduction no other organ system in the human body is as dependent on nutritional intake as the central nervous system, while conversely this system also affects nutritional intake. several theories have been put forward regarding the functions of brain receptors for cholecystokinin, opioid-like endorphins and serotonin that play a role in determining human food habits.(1,2) 107 univ med vol.28 no.2 studies on experimental animals indicate that with advancing age there is a decline in the functioning of the brain and in the number of brain receptors. in the elderly there is also a diminished sense of taste and a decline in olfactory nerve functions that affect appetite.(2,3) the nervous system requires a constant intake of glucose for adequate functioning of the brain, which is particularly dependent on intake of essential nutrients. deficiencies of vitamins and minerals, such as vitamin b12, folate, vitamin b6 and trace minerals, may result in disturbances of brain functions such as cognitive functions.(4,5) vitamin deficiencies commonly occur in the elderly, especially of the vitamin b group, caused by various factors, such as gastric atrophy with achlorhydria or hypochlorhydria, which in the elderly may account for up to 20-50% of cases. studies have yielded evidence for an association in the elderly between decreased levels of vitamin b12, folate, and vitamin b6 on the one hand and impaired cognitive function on the other.(1,5) one of the manifestations of impaired c o g n i t i v e f u n c t i o n i s d e m e n t i a , w h i c h i s c h a r a c t e r i z e d b y a p r o g r e s s i v e d e c l i n e i n cognitive function, resulting in a decreased ability for conducting activities of daily living. dementia affects 8% of the elderly population over 65 years of age, and in canada it gives rise to more than 60.000 new cases annually. another disease, alzheimer’s, is the cause of 50% of all cases of dementia in canada,(6) whereas other studies indicate that dementia due to alzheimer’s disease may account for up to 70% of all dementia cases.(7) it is estimated that in the following 50 years the number of dementia cases may undergo a threefold rise due to a increase in the elderly population.(8) the prevalence of this disorder is extremely high and as the disorder may lower the quality of life of the elderly, prevention and early detection are important.(6) factors that have been associated with the risk of dementia are age and level of education, which are of the highest relevance for dementia. recently high homocysteine levels have also been associated with an increased risk of dementia.(6,7,9) homocysteine is an amino acid resulting from the metabolism of methionine that is dependent on cobalamine, vitamin b 6 , and folic acid. (8,10) plasma homocysteine level is an indicator for vitamin b status, including that of f o l i c a c i d , w h e r e a h i g h h o m o c y s t e i n e concentration implies an inadequate vitamin b status. data from several laboratories have shown that high homocysteine level is extremely common in old age and does not depend on v i t a m i n s t a t u s . ( 11 1 3 ) h i g h t o t a l p l a s m a homocysteine level is a major vascular risk factor that is associated with an increased risk of atherosclerotic sequelae, including death due to cardiovascular disease, coronary heart disease, carotid atherosclerosis and stroke.(7,14,15) c o g n i t i v e f u n c t i o n m a y b e a s s e s s e d through various instruments, such as the mini mental state examination (mmse). this test comprises five categories of criteria, viz. criteria on orientation with a maximum score of 10, registration criteria with maximum score of 3, attention and calculation with maximum score of 5, recall criteria with maximum score of 3, and language criteria with maximum score of 9. in addition to these five criteria the subjects are also asked to write a sentence and copy a drawing.(8) r e s e a r c h r e s u l t s o n t h e i n f l u e n c e o f homocysteine on cognitive function in the elderly are still subject to controversy. a number of studies have demonstrated a significant assciation,(7,15-17) whereas other studies have shown contradictory results.(8,18-19) an italian study by ravaglia et al.(9) on dementia-free 108 elderly found an increased risk of low mmse scores with increasing plasma homocysteine levels, although homocysteine was not directly associated with cognitive disorders. the objectives of the present study were t o f i n d a n a s s o c i a t i o n b e t w e e n p l a s m a homocysteine concentration and cognitive function in the elderly and to determine the factors that might play a role in the occurrence of dementia. methods subjects the study subjects were randomly selected residents aged 60 years and over from the catchment area of the mampang prapatan district health center in south jakarta. the inclusion criteria were as follows: age 60 years and over, mobile, not having terminal diseases or being comatous, and not suffering from severe psychotic disorders. the study subjects had also expressed their willingness to participate in the present study by signing a letter of informed consent. exclusion criteria were acute infection, intake of vitamins, cod liver oil or other supplements within the previous month, and abnormal renal function. assessment of renal function and cognitive function as renal function affects the metabolism of homocysteine, (15) subjects with a creatinine c o n c e n t r a t i o n a b o v e r e f e r e n c e l e v e l w e r e considered to have abnormal renal function. the reference level for females is 0.5-1.3 mg/dl and for males 0.5-1.4 mg/dl.(14) cognitive function was assessed by the mmse. an mmse score of 26-30 is considered questionably significant/no cognitive impairment, a score of 21-25 implies mild cognitive impairment, a score of 10-20 suggests moderate cognitive impairment and 0-9 indicates severe cognitive impairment.(13) data collection the study participants were interviewed by field workers using a questionnaire that had been tested in a preliminary trial. the filling in of q u e s t i o n n a i r e s w a s p e r f o r m e d d a i l y f r o m monday to friday, and the weekly number of elderly who completed their interviews was around 25. these later were invited for a visit to the mampang prapatan district health center on the following saturday. for the purpose of collection of a blood sample for laboratory examination, the elderly were asked to conduct a 10 – 12 hour fast prior to their visit to the health center. the following saturday morning the participants underwent measurement of blood pressure, pulse rate, weight, height, waist circumference, and hip circumference. the m e a s u r e m e n t s w e r e t a k e n b y t w o n u r s e s previously trained in the appropriate techniques. data collection was performed four times within the period of one month between november 2006 and december 2006. assessments height (cm), waist circumference (cm) and hip circumference (cm) of the subjects were measured using a standard tape measure accurate to 1 mm. weight (kg) was measured on a calibrated scale accurate to 0.5 kg with normal indoor clothing. body mass index (bmi) was calculated as weight in kilograms divided by the square of the height in meters). blood pressure measurements were performed with the subject sitting, using a standard sphygmomanometer accurate to 5 mmhg. hypertension was defined as a systolic blood pressure of >140 mmhg and/ or a diastolic blood pressure of >90 mmhg. laboratory examinations from each study subject 5 ml of venous blood was collected using a vacutainer without anticoagulant. the blood sample was centrifuged at 300 rpm for 10 minutes in order to obtain pusparini homocysteine and cognitive function 109 univ med vol.28 no.2 serum for assessment of renal function and h o m o c y s t e i n e l e v e l . d e t e r m i n a t i o n o f homocysteine level was performed only on the serum of subjects with a renal function consistent with reference values. creatinine concentration was determined by the jaffe method on a trx, whereas homocysteine was a s s e s s e d b y f l u o r e s c e n t p o l a r i z a t i o n i m m u n o a s s a y o n a n a b b o t t i m x . t h e coefficient of variation (cv) of creatinine was 2.1% for normal concentrations and 1.6% for high concentrations, whilst the corresponding cv values for homocysteine were 3.4% and 2.3%, respectively. data analysis for data analysis the statistical program f o r s o c i a l s c i e n c e s ( s p s s ) w a s u s e d . normality of data distribution was determined by the kolmogorov – smirnov (ks) test. for n o r m a l l y d i s t r i b u t e d d a t a t h e m e a n a n d standard deviation was calculated and for nonnormally distributed data the median and standard deviation. to determine the presence of a statistically significant difference between groups for normally distributed data the analysis of variance (anova) was used, whilst for non-normally distributed data the kruskal – wa l l i s t e s t w a s a p p l i e d . f o r d e t e r m i n i n g correlation between homocysteine level and cognitive function, for normally distributed data the pearson’s product moment correlation was used and for non-normally distributed data the spearman rho test. a multiple linear regression was done to examine the cross-sectional association between homosysteine blood levels and mean mmse scores adjusted for sociodemographic factors. statistical significance was defined for all analyses as p <0.05. results a total of 94 elderly consisting of 44 (47%) females and 50 (53%) males meeting the inclusion and exclusion criteria participated in this study. the age of the subjects ranged from 60 80 years, with mean age of 64.5±8.4 years for females and 66.3±5.2 years for males. the characteristics of the study subjects are presented in table 1. there was no appreciable difference in age between female and male subjects, whereas the body mass index (bmi) was higher in the male group compared with the female group, which was consistent with the greater weight and stature of table 1. mean values of main characteristics of female and male subjects 110 males. the educational level of male subjects w a s h i g h e r t h a n t h a t o f f e m a l e s . wa i s t circumference of males was greater than that of females, but in both groups it did not exceed the r e c o m m e n d e d w a i s t c i r c u m f e r e n c e v a l u e s according to asia-pacific criteria, i.e. 80 cm for females and 90 cm for males. hip circumference was similar in males and females. both systolic and diastolic blood pressures were higher in females than in males, with p=0.03 for systolic bp and p= 0.30 for diastolic bp, whilst mean systolic bp was > 140 mmhg in both groups. creatinine and homocysteine levels were both higher in males than in females, with p=0.000, indicating a significant difference between both genders. mmse scores tended to be higher in males compared with females, but did not show a significant difference. in table 2 homocysteine levels were divided into three categories, i.e. <11.7 µmol/l, 11.7 14.6 µmol/l and >14.6 µmol/l, both for females and males. among the study subjects with homocysteine levels above 14.6 µmol/l, there were 10 females (22.70%) with mean homocysteine level of 19.2 ± 2.4 µmol/l and 32 males (66%) with mean homocysteine level of 22.1 ± 7.3 µmol/l. homocysteine levels above 14.6 µmol/l were more frequent in males compared with females. table 3 presents mmse scores in females and males by category of homocysteine level, s h o w i n g a s i g n i f i c a n t d i ff e r e n c e b e t w e e n categories in females with p=0.000 and in males w i t h p = 0 . 0 0 8 . f r o m t h e t a b l e i t m a y b e concluded that higher plasma homocysteine levels are associated with lower mmse scores. in table 4 are shown homocysteine levels and mmse scores by age and level of education. homocysteine levels in males for the categories of no education, primary school, junior high school and senior high school were greater than 14.6 µmol/l, whereas in females for primary school, senior high school and university education homocysteine levels were <14.6 µmol/ l. there was no significant difference in homocysteine concentrations of females and males at various levels of education. mmse scores were proportional to level of education, with higher mmse scores being found at higher levels of education (p=0.000). from table 4 it is also apparent that in females there was no significant difference in homocysteine levels between various age groups, whereas in males the homocysteine levels did show a significant difference between various age groups. table 2. plasma homocysteine levels of study subjects table 3. mmse scores by categories of homocysteine level pusparini homocysteine and cognitive function 111 univ med vol.28 no.2 according to the results of data analysis (table 5), several factors were shown to play a role in cognitive function, i.e. age, level of education, and plasma homocysteine level. the results of the multiple linear regression analysis indicated that mmse is affected by level of education and age, whilst homocysteine level did not influence mmse score in males as well as in females. table 6 presents mmse scores for the subitems of temporal orientation, registration, attention and calculation, recall, and language, both in females and in males at several plasma homocysteine levels. only female mmse scores for attention and calculation and for language show a significant difference (p=0.041 and p=0.024). discussion in the present study homocysteine levels in females were lower compared with those in males, with means of 13.50 ± 3.69 µmol/l and 18.96 ± 7.56 µmol/l, respectively. homocysteine levels showed a significant difference between females and males with p=0.000, therefore data analysis was done based on gender. the study conducted by haan4 showed comparable results, where mean homocysteine level in females (10.07 ± 4.13) was significantly different from that in males (11.80 ± 8.76), with the age of the subjects in the range o f 6 0 8 0 y e a r s . i n c o n t r a s t , t h e s t u d y b y seshasdri(7) found no difference in homocysteine levels between females and males, the subjects being in the age range of 60 – 94 years. table 5. factors affecting mmse score table 4. mean homocysteine concentration and mmse score by age and level of education 112 table 6. mmse scores category at several plasma homocysteine levels in females and males homocysteine is a sulfur-containing amino acid closely associated with the metabolism of methionine and cysteine. there is no dna that codes for homocysteine synthesis. homocysteine is not synthesized in nature but results from the metabolism of the amino acid methionine through a methylation cycle as a unique source of homocysteine. methionine is obtained through intake of protein in the daily diet.(20,21) homocysteine occurs in several forms in plasma. the sulfhydryl or reduced form is designated homocysteine and the disulfide or oxidized form is designated homocystine. the disulfide form may occur together with cysteine and with proteins having reactive cysteine residues (protein-bound homocysteine), and this form is called mixed disulfide. the oxidized forms account for most of the homocysteine in plasma (98-99%), whilst the reduced forms make u p o n l y 1 % o f t o t a l h o m o c y s t e i n e . to t a l h o m o c y s t e i n e ( t h c y ) i s t h e s u m o f a l l homocysteine forms present in plasma.(20,21) homocysteine is converted by the enzyme cystathionine β synthase (cbs) into cysteine with vitamin b6 as cofactor, in a process called d e m e t h y l a t i o n . i n t h e r e v e r s e p r o c e s s o f remethylation, homocysteine is reconverted into m e t h i o n i n e b y t h e e n z y m e s m e t h y l e n e tetrahydrofolate (mthfr) and methionine synthase (ms), with folic acid, and vitamin b12 as substrates and cofactors. thus a deficiency of one of these vitamins leads to hyperhomocysteinemia.(21-23) u n d e r n o r m a l c o n d i t i o n s b l o o d homocysteine levels are relatively low, ranging from 5-15 µmol/l. the homocysteine level in the extracellular compartment is determined by its intracellular synthesis, metabolism and e x c r e t i o n . i f i n t r a c e l l u l a r h o m o c y s t e i n e synthesis exceeds the metabolic capacity, homocysteine is released into the extracellular compartment; in contrast, decreased synthesis leads to a fall in release of homocysteine from the cell. this mechanism maintains intracellular homocysteine at a low level. the homocysteine equilibrium may be upset by abnormal enzyme activity or as a result of a reduction in the number o f c o f a c t o r s t h a t p l a y a r o l e i n i t s metabolism.(22,24) in table 2 homocysteine levels were subdivided into several categories, i.e. <11.7 µmol/l, 11.7-14.6 µmol/l and >14.6 µmol/l. this was based on the results of a study conducted by quadri(15) with the objective of finding a reference value for hyperhomocysteinemia, as currently there are no standard criteria for defining hyperhomocysteinemia. a number of investigators have used variable values for hyperhomopusparini homocysteine and cognitive function 113 univ med vol.28 no.2 cysteinemia, e.g. ravaglia(9) used a homocysteine level of >15 µmol/l, whilst seshasdri(7) and dufouil(2) used levels of >14 mol/l and >15 µmol/ l, respectively. in the present study, homocysteine levels o v e r 1 4 . 6 µm o l / l w e r e f o u n d i n a l a rg e proportion of males (32 subjects or 66%) with a mean level of 22.06 ± 7.25, but only in 10 females (22.70%). in comparison, in the study by quadri(15) homocysteine levels of >14.6 µmol/ l were found in 51.85% of subjects with vascular dementia and in 56.36% of control subjects. mean homocysteine level was 14.6 ± 6.1 µmol/ l in control subjects with mean age of 75.6 ± 8.5 years, whereas subjects with vascular dementia with mean age of 80.5 ± 5.7 years had a mean homocysteine level of 18.9 ± 7.9 µmol/ l and those with alzheimer’s disease and mean age of 79.1 ± 7.7 years showed a homocysteine level of 16.8 ± 7.0 µmol/l. in contrast, the present study with a lower mean age showed a higher mean homocysteine level in males that was almost equal to the mean homocysteine level in patients with vascular dementia, although the study subjects had signs of mild cognitive impairment only. the mean mmse score in male subjects in the present study was 25.11 ± 5.03, indicating that they had mild cognitive impairment, whereas in quadri’s study the mean mmse score in vascular dementia was 18.6 ± 5.6, and in alzheimer ’s disease 18.6 ± 5.1, signifying moderate cognitive impairment. the mean mmse score in females was 23.18 ± 6.4, indicating mild cognitive impairment. according to several studies, the contribution of sex is controversial with regard to the risk of cognitive impairment. however, the results obtained by huadong et al indicate that the incidence of cognitive impairment was 10.7% in men and 13.2% in women and that there was a significant association of sex and cognitive impairment.(30) i n t h e p r e s e n t s t u d y a s s e s s m e n t o f homocysteine levels was performed after the subjects had fasted for a minimum of 12 hours. measurement of homocysteine on non-fasting subjects results in an increase in homocysteine levels of around 20% compared with those in fasting subjects. comparison of mmse scores in the three categories of plasma homocysteine levels (table 3) indicated that mmse scores in females were also lower than those in males for each category of plasma homocysteine level. higher homocysteine levels were associated with lower mmse scores in both female and males, with p=0.000 and p=0.008, respectively. this i n d i c a t e s t h a t h o m o c y s t e i n e l e v e l m a y presumably affect cognitive function. seshasdri’s study7 revealed a tendency for increased plasma homocysteine levels to precede the onset of dementia. therefore it is probable that the increased homocysteine levels in the p r e s e n t s t u d y m a r k t h e e a r l y s t a g e s o f a developing dementia, although the mmse scores were categorized as mild cognitive impairment only. in general, level of education can be said to affect cognitive function. according to the results of the present study cognitive function in elderly females and males was affected by l e v e l o f e d u c a t i o n b u t n o t b y p l a s m a homocysteine levels. these results were similar with those of huadong’s study, where level of e d u c a t i o n w a s a s s o c i a t e d w i t h c o g n i t i v e function.(30) besides level of education, age may also affect mmse scores and homocysteine levels. tabel 4 shows that older subjects, both females and males, tended to have higher homocysteine levels, although these levels showed a significant difference in males only. mmse scores showed no significant difference between age groups, both in females with p=0.864 as well as in males with p=0.734. 114 multiple linear regression analysis revealed that the influencing factors on mmse scores w e r e l e v e l o f e d u c a t i o n a n d a g e , w h i l s t homocysteine levels were not significantly different in females and in males. this is consistent with the results of the study by joosten(25) where no significant correlation was f o u n d b e t w e e n h o m o c y s t e i n e l e v e l s a n d cognitive function. in the present study mean homocysteine level in males was >14.9 µmol/l but the decline in cognitive function was only slight. the study conducted by bell(26) indicated that in normal subjects there was no significant correlation between plasma homocysteine level and mmse score. a correlation was found only in subjects with depression. borroni(27) and miller(28) concluded from the results of their respective cross-sectional studies that raised plasma homocysteine concentration was not a causative factor in dementia and alzheimer’s disease, but was only a marker for concomitant vascular disease, independently of cognitive status. their results are contrary to those of seshasdri (7) where a correlation was found between increased plasma homocysteine levels and decreased mmse scores, but the correlation was found only after 4 years of follow-up. thus only long-term intervention trials can yield definitive evidence for a causal relation between hyperhomocysteinemia and cognitive impairment. as already stated above, in the multiple linear regression analysis the factors affecting m m s e s c o r e s w e r e f o u n d t o b e l e v e l o f e d u c a t i o n a n d a g e . m m s e s c o r e w a s proportional to level of education, indicating better cognitive function in the better educated. several investigators used subjects with a similar educational background to find a correlation between homocysteine levels and mmse scores. the study by ravaglia(9) indicates that in subjects w i t h a s i m i l a r e d u c a t i o n a l b a c k g r o u n d hyperhomocysteinemia was an independent predictor for the occurrence of dementia or a l z h e i m e r d i s e a s e . i n t h e p r e s e n t s t u d y, arrangement of the homocysteine levels into the categories <11.7 µmol/l, 11.7-14.6 µmol/l and >14.6 µmol/l revealed a significant difference in mmse scores. thus although with the correlation regression analysis no significant c o r r e l a t i o n w a s t o b e f o u n d b e t w e e n homocysteine levels and mmse scores, after categorization homocysteine levels appeared to also affect mmse scores. when the mmse scores were categorized by component or sub-item, it became apparent that in female and male subjects the sub-items “attention and calculation” and “language” lead to the conclusion that homocysteine levels were inversely related to mmse scores. the study by levitt(29) showed similar results in that the significant mmse scores were in the “attention and calculation” category. the associations between homocysteine and cognitive function in community-dwelling elderly are nonsignificant, whereas demographic variables, particularly age and education, were found to be much stronger determinants of cognitive function scores. conclusion although there is suggestive evidence that plasma homocysteine levels and mmse are inversely associated, the most important factors affecting mmse score are education and age. therefore an interventional study is called for t o p r o v i d e d e f i n i t e d a t a t o a s c e r t a i n t h e relationship between homocysteine levels and mmse. acknowledgements the authors wish to express their gratitude to the medical faculty of trisakti university, to the subjects of this study for their willingess to participate in this study, and to all who made this study possible. pusparini homocysteine and cognitive function 115 univ med vol.28 no.2 references 1. weir dg, molloy am. microvascular disease and dementia in the elderly: are they related to hyperhomocysteinemia? am j clin nutr 2000;71:859-60 2 dufouil c, alperovitch a, ducros v, tzourio c. homocysteine, white matter hyperintensities, and cognition in healthy elderly people. ann neurol 2003;53:214-21. 3 roberts s, sternberg s. do nutritional supplements improve cognitive function in the elderly? nutr 2003;19:976-80. 4 haan mn, miller jw, aiello ae, whitmer ra, jagust wj, mungas dm, et al. homocysteine, b vitamins, and the incidence of dementia and cognitive impairment: result from the sacramento area latino study on aging. am j clin nutr 2007;85:511-7. 5 refsum h. total homocysteine. guidelines for determination in the clinical laboratory. clinical laboratory news. aacc 2002;28:12-14. 6 duel pb, malinow mr. homocysteine: an important risk factor for atherosclerotic vascular disease. curr opin lipidol. 1997;8:28-34. 7 seshadri s, beiser a, selhub j, jacques pf, rosenberg ih, a’agostiono rb, et al. plasma homocysteine as a risk factor for dementia and alzheimer’s disease. n eng j med 2002;346: 47683. 8 mooijaart sp, gussekloo j, frolich m, jolles j, stott dj, westendorp rgj. homocysteine, vitamin b12 and folic acid and the risk of cognitive decline in old age: the leiden 85-plus study. am j clin nutr 2005;82:866-71. 9 ravaglia g, forti p, maioli f, martelli m, servadei l, brunetti n, et al. homocysteine and folate as risk factors for dementia and alzheimer disease. am j clin nutr 2005;82:636-43. 10 ueland pm, refsum h, stabler sp, malinow mr, anderson a, allen rh. total homocysteine in plasma or serum: methods and clinical applications. clin chem 1993;39:1764-78. 11 garcia a, haron y, pulman k, hua l, freedman m. increases in homocysteine are related to worsening of stroop scores in healthy elderly persons: a prospective follow-up study. j of gerontol 2003;59a:1323-27. 12 tucker kl, qiao n, scott t, rosenberg i, spiro a. high homocysteine and low b vitamins predict cognitive decline in aging men: the veterans affairs normative aging study. am j clin nutr 2005;82:62735. 13 garcia a, zanibbi k. homocysteine and cognitive function in elderly people. cmaj 2004; 171:897904. 14 selhub j, bagle lc, miller j, rosenberg ih. b vitamins, homocysteine, and neurocognitive function in the elderly. am j clin nutr 2000;71(suppl): 614s-20s. 15 quadri p, fragiacomo c, pezzati r, zanda e, forlani g, tettamanti m, et al. homocysteine, folate, and vitamin b-12 in mild cognitive impairment, alzheimer disease, and vascular dementia. am j clin nutr 2004;80:114-22. 16 miller jw, green r, ramos mi, allen lh, mungas dm, jagust wj, et al. homocysteine and cognitive function in the sacramento area latino study on aging. am j clin nutr 2003;78:441-7. 17 andres e, loukili nh, noel e, kaltenbach g, abdelgheni mb, perrin ae, et al. vitamin b12 (cobalamin) deficiency in elderly patients. cmaj 2004;171:1503-55. 18 stott dj, macintoch g, lowe gdo, rumley a, mcmahon ad, langhorne p, et al. randomized controlled trial of homocysteine lowering vitamin treatment in elderly patients with vascular disease. am j clin nutr 2005;82:1320-6. 19 lei f, ng tp, chuah l, niti m, e hk. homocysteine, folate, and vitamin b12 and cognitive performance in older chinese adults: findings from singapore longitudinal ageing study. am j clin nutr 2006;84:1506-12. 20 kruman ii. homocysteine elicits a dna damage response in neurons that promotes apoptosis and hypersensitivity to excitoxicity. j neuroscience 2000;20:6920-6. 21 hsu kk, sorond fa, jen hc, hashmi a, milberg wp, lipsitz la. the role of homocysteine in multisystem age-related problems: a systematic review. j of gerontol.2005;60a:1190-1201. 22 massachusetts medical society. a controlled trial of homocysteine lowering and cognitive performance. n eng j med 2006;354:2764-72. 23 miller jw. assessing the association between vitamin b12 status and cognitive function in older adults. am j clin nutr 2006;84:1259-60. 24 krumdieck cl, prince cw. mechanisms of homocysteine toxicity on connective tissue: implications for the morbidity of aging. j nutr 2000;130:365s-8s. 116 25. joosten e, lesaffre e, riezler r. is metabolic evidence for vitamin b12 and folate deficiency more frequent in elderly patients with alzheimer’s disease? gerontol biol sci med sci 1997;52:76-9. 26. bell ir, edman js, selhub j. plasma homocysteine in vascular disease and in nonvascular dementia of depressed elderly people. acta psychiatr scand 1992;86:386-90. 27. borroni b, agosti c, panzali af, di luca m, padovani a. homocysteine, vitamin b6, and vascular disease in patients with alzheimer disease. neurology 2002;59:1475-6. 28. miller jw, green r, mungas dm, reed br, jagust wj. homocysteine, vitamin b6, and vascular disease in alzheimer disease. neurology 2002;58:1471-5. 29. levitt aj, karlinsky h. folate, vitamin b12 and cognitive impairment in patients with alzheimer’s disease. acta psychiatr scand 1992;86:301-5 30. huadong z, juan d, li jc, wang yj, zhang m, he h. study of the relationship between cigartette smoking, alcohol drinking and cognitive impairment among elderly people in china. age and ageing 2003;32:205-10. pusparini homocysteine and cognitive function elly 98 increased irrational use of antibiotics promotes bacterial resistance to these drugs. among the resistance mechanisms developed by bacteria is the production of âlactamase which can destroy the â-lactam ring and cause resistance to the other âlactam antibiotics, such as amoxycillin. extended spectrum â-lactamase (esbl), an enzyme found in bacterial plasmids, is capable of hydrolyzing third-generation cephalosporins, namely cefotaxime, ceftazidime, ceftriaxone, and the monobactams. esbl is predominantly found in klebsiellas spp., escherichia coli and other bacteria of the enterobacteriaceae family. this study was conducted to determine the prevalence of klebsiella resistant to cephalosporins and the prevalence of those producing esbl. a total of 65 clinical isolates of klebsiella were tested by the disk diffusion method according to kirby-bauer to determine their antibiotic susceptiblity and by the double-disk synergy method to detect the presence of esbl. the results show that 18.5% of klebsiella isolates tested were resistant to ceftazidime and cefixime, 13.9% to ceftriaxone, and 23.1% to aztreonam. testing for esbl revealed that the prevalence of esbl producers in clinical klebsiella isolates ranged from 10.8% to 12.3%. the presence of esbl, plus the potential for plasmidmediated quinolone and carbapenem resistance, undoubtedly will create significant therapeutic problems in the future. keywords: extended spectrum â-lactamase, klebsiella, antibiotics *department of pharmacology, **department of microbiology medical faculty, trisakti university correspondence adr. elly herwana, m.biomed department of pharmacology, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2801 email : elly_herwana@cbn.net.id elly_herwana@yahoo.com univ med 2008; 27: 98-105 prevalence of extended spectrum beta-lactamase in klebsiella pneumoniae july-september, 2008july-september, 2008july-september, 2008july-september, 2008july-september, 2008 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 abstract universa medicina elly herwana*a, yenny*, laurentia pudjiadi*, julius e. surjawidjaja**, and murad lesmana** introduction a n t i b i o t i c s h a v e l o n g b e e n u s e d f o r managing infectious disease and lately have tended to be used excessively and irrationally. this tendency has increased the prevalence of antibiotic resistance in previously sensitive bacteria. bacteria synthesize the enzyme βlactamase that is capable of opening the βlactam ring and inducing resistance to β-lactam antibiotics such as amoxicillin. the emergence of extended spectrum β-lactamase in these bacteria subsequently has resulted in their resistance to third-generation cephalosporins. 99 univ med vol.27 no.3 this situation may complicate antibiotic therapy in patients with infections caused by bacteria of the family enterobacteriaceae, especially klebsiella spp. and e. coli, as has been reported in many countries.(1-5) extended spectrum β-lactamase (esbl) is a group of enzymes found in plasmids that are c a p a b l e o f h y d r o l y z i n g t h i r d g e n e r a t i o n cephalosporins, such as cefotaxime, ceftazidime, ceftriaxone, and the monobactam group of antibiotics, such as aztreonam, thus causing resistance to these antibiotics in esbl-producing bacteria. esbl is predominantly found in klebsiella spp.(4) and escherichia coli,(5) and also in other members of the enterobacteriaceae.(6) most of these esbl-producing organisms are still sensitive to carbapenem, whilst their action against ciprofloxacin, cefepim, and combinations of beta-lactam antibiotics with a beta-lactamase inhibitor, is variable. the prevalence in microorganisms of betalactamase induced resistance to beta-lactam antibiotics is steadily increasing. bacterial resistance due to beta-lactamase was first encountered in staphylococcus aureus, and has subsequently spread to neisseria gonorrhoeae and haemophilus influenzae, and later also to e n t e ro c o c c u s f a e c a l i s . e x t e n s i o n o f t h e s p e c t r u m o f b e t a l a c t a m a s e s p r o d u c e d b y v a r i o u s b a c t e r i a l h o s t s h a s n o t b e e n accompanied by a change of enzyme substrate s p e c t r u m . t h u s t h e b a c t e r i a h a v e r e a d i l y become resistant to beta-lactam antibiotics, such as the penicillins and cephalosporins, which at the time of emergence of extendedspectrum beta-lactamases possessed only a small number of enzyme substrates. a number of pathogenic bacteria have become resistant through mutation, making them capable of inactivating the substrate. isolates of klebsiella may become resistant at cephalosporins through acquisition of plasmidmediated resistance.(3) there are more than 30 esbl plasmids, based on substrate profile, response to inhibitors, and isoelectric point. in indonesia there have been only a small number of reports on esbl-producing bacteria, but this problem will clearly complicate antibiotic therapy, particularly in patients with infections caused by members of the enterobacteriaceae, especially klebsiella, as has been reported in many countries.(4-6) studies conducted in several countries have demonstrated the presence of klebsiella pneumoniae in cases of severe sepsis, 38% of which were fatal. the aim of the present study was to determine the prevalence of klebsiella isolates resistant to third-generation cephalosporins and the prevalence of esblinduced resistance in these isolates. materials and methods bacteria the bacteria used were clinical isolates of klebsiella pneumoniae, which had been stored i n t r y p t i c s o y b ro t h + 1 5 % g l y c e ro l a t a temperature of -70°c. antibiotics antibiotic disks were obtained from bbl (becton dickinson, sparks, md), comprising ceftriaxone (cro; 30 µg), ceftazidime (caz; 30 µg), aztreonam (atm; 30 µg), cefixime (cfm; 30 µg), and amoxicillin-clavulanic acid (amc; 30 µg and 15 µg). a n t i b i o t i c s u s c e p t i b i l i t y a s s a y b y d i s k diffusion method (kirby-bauer) the test isolate was suspended in brainheart infusion broth (bhi), the suspension was incubated at 370c, and subsequently diluted with saline to a concentration equivalent to mcfarland standard 0.5. the broth culture was taken with a cotton swab and plated on muellerhinton ii agar (mh-ii). the antibiotic disks (cro, caz, atm, cfm, and amc) were then 100 placed on the inoculated agar surface and the agar plate incubated at 370c for 20-24 hours. interpretation of disk diffusion assay results t h e r e s u l t s o f t h e k i r b y b a u e r d i s k diffusion antibiotic susceptibility test was interpreted according to the criteria defined by the national committee for clinical laboratory standards (nccls).(7-8) for ceftriaxone (cro), ceftazidime (caz), and cefixime (cfm), the bacteria were classified as resistant if the zone of inhibition was <16 mm and as susceptible (sensitive) if the zone of inhibition was >20 mm. f o r a z t r e o n a m ( at m ) t h e b a c t e r i a w e r e classified as resistant if the zone of inhibition was <19 mm and sensitive if the zone of inhibition was >23 mm. double-disk synergy esbl assay an mh-ii agar plate was inoculated with the isolate to be tested as in the disk diffusion assay and a cro disk placed at the center. then an amc disk was placed on the agar surface at a distance of 20 mm from the cro test disk and the agar plate was incubated for 20-24 hours at 370c. the procedure was repeated for caz, atm, and cfm, using a separate mh-ii plate for each antibiotic.(2) interpretation of double-disk synergy esbl assay results an extension of the inhibition zone of the t e s t e d a n t i b i o t i c t o w a r d s t h e a m c d i s k containing clavulanic acid signifies a positive result, indicating that the test organism is an esbl producer. data analysis percentage analysis was performed for demonstrating the prevalence of klebsiella strains resistant to the test antibiotics and the prevalence of esbl producing microorganisms. results isolation of bacteria from a total of 78 klebsiella pneumoniae isolates recovered from the blood of febrile patients, thirteen did not grow on repeat culture. thus only 65 klebsiella pneumoniae isolates were available for testing by the kirby-bauer and esbl assays. the results of the disk diffusion assay according to kirby-bauer on 65 isolates of klebsiella pneumoniae revealed that bacterial resistance was highest against aztreonam with a bacterial resistance pattern of 15 (23.1%) resistant and 50 (76.9%) sensitive isolates. the patterns of resistance to ceftazidime and cefixime were similar, namely 53 (81.5%) vs. 52 (80%) sensitive and 12 (18.5%) vs. 13 (20%) resistant isolates (including 1 or 1.5% of intermediate r e s i s t a n c e ) . d i m i n i s h e d s u s c e p t i b l i t y w a s demonstrated in the kirby-bauer results for c e f t r i a x o n e , n a m e l y 5 5 ( 8 4 . 6 % ) s e n s i t i v e i s o l a t e s , 1 ( 1 . 5 % ) i s o l a t e o f i n t e r m e d i a t e resistance and 9 (13.9%) completely resistant isolates (table 1, figure 1). herwana, yenny, pudjiadi, et al extended spectrum beta-lactamase sensitivity test result antibiotic sensitive (%) intermediate (%) resistant (%) ceftazidime (caz) 53 (81.5) 0 (0) 12 (18.5 ) cefixime (cfm) 52 (80) 1 (1.5) 12 (18.5) ceftriaxone (cro) 55 (84.6) 1 (1.5) 9 (13.9) aztreonam (atm) 50 (76.9) 0 (0) 15 (23.1) table 1. kirby-bauer assay on 65 isolates of klebsiella pneumoniae 101 univ med vol.27 no.3 esbl assay (n/%) antibiotic n esbl + esbl ceftazidime (caz) 65 8 (12.3%) 57 (87.3) cefixime (cfm) 65 8 (12.3%) 57 (87.3) ceftriaxone (cro) 65 7 (10.8%) 58 (89.2) aztreonam (atm) 65 8 (12.3%) 57 (87.3) table 2. esbl assay on isolates of klebsiella pneumoniae resistant to 4 cephalosporins isolates yielding kirby-bauer results of intermediate resistance and complete resistance were subsequently tested by the esbl assay. the results of the esbl assay showed that among the 65 klebsiella pneumoniae isolates tested, 8 (12.3%) produced esbl against ceftazidime, cefixime, and aztreonam. similar results were found for ceftriaxone, where 7 isolates (10.8%) were esbl positive (table2, figure 2). figure 1. kirby bauer test results the test bacterium shows sensitivity to amoxicillin+clavulanic acid (amc) [left], ceftriaxone (cro) [center], and ceftazidim (caz) [right], as indicated by the substantially wide zones of inhibition around the disks. the bacterium is resistant to aztreonam (atm) [top] and ceftazidime (cfm) [bottom] as there is no zone of inhibition around the respective disks amc cro caz atm cfm 102 discussion i n t h e p r e s e n t s t u d y, e s b l m e d i a t e d resistance was found in 10.8% to 12.3% of our isolates. this prevalence rate is much higher than those obtained in india(9) and the united states,(10) which were reported to be 6.6% and 5%, respectively, but lower than those in france (14%) and the united kingdom (16%). ( 11 ) studies performed in malaysia in 2001 reported that esbl production by klebsiella spp. in the asia-pacific region amounted to 27-38% and in indonesia was 33.3%. ( 1 2 ) another study conducted in 2005 at dr. soetomo hospital in surabaya, reported that out of 85 klebsiella pneumoniae isolates recovered from urine and blood, 73 (86%) were esbl-positive.(13) s i n c e t h e d i s c o v e r y o f t h e â -l a c t a m antibiotics, inactivation of the drugs by â lactamase has become a principal feature of the r e s i s t a n c e m e c h a n i s m s o f g r a m n e g a t i v e bacteria. in the enterobacteriaceae, esbl production has become an important resistance mechanism to â-lactam antibiotics, affecting 5 0 % o f a n t i b i o t i c s w i d e l y u s e d a g a i n s t i n f e c t i o u s d i s e a s e . ( 1 4 ) e s b l i s c a p a b l e o f hydrolyzing a large number of â-lactam drugs, including the newer cephalosporins, but is inactive against cephamycin and carbapenem. in the family enterobacteriaceae, esbl is found particularly in klebsiella spp. and e. coli, but to a lesser degree also in other members of the family.(4-6) in general, this resistance is coded for by a plasmid-borne gene that is readily transmissible among the enterobacteriaceae, particularly in klebsiella spp. and e. coli. increased broads p e c t r u m c e p h a l o s p o r i n u s a g e h a s b e e n herwana, yenny, pudjiadi, et al extended spectrum beta-lactamase figure 2. esbl positive results by the double disc synergy test the disk at center contains ceftriaxone and the disk at left amoxicillin + clavulanic acid. the zone of inhibition around the ceftriaxone disk has enlarged in the direction of the disk containing clavulanic acid 103 univ med vol.27 no.3 associated with nosocomial infections in both hospitalized and ambulatory patients.(4-6) esbl, first discovered in germany, was subsequently widely reported in many european countries, and has now been found in nearly all parts of the world.(2-5) infections caused by esbl-producing microorganisms have resulted in serious antibiotic resistance problems. several investigators reported that esbl produced by bacteria have frequently induced bacterial r e s i s t a n c e t o a n u m b e r o f c e p h a l o s p o r i n derivatives, such as ceftazidime, cefotaxime, ceftriaxone, and aztreonam. a brooklyn survey has revealed that esbl is produced by 17.2% of k. pneumoniae, e. coli, and p. mirabilis.(15) however, a larger study conducted in france found an esbl prevalence of only 3.2% in enterobacteriaceae.(16) several recent studies in the united staes and europe have reported that e s b l p r o d u c i n g s t r a i n s i n t h e f a m i l y enterobacteriaceae have increased dramatically and that this trend is having a significant impact on mortality rate and hospitalization costs.(17-20) i n m a n y h o s p i t a l s , e s b l p r o d u c i n g organisms are already endemic. is there any hope of controling esbl producers in such a setting? a number of authors have shown that ceftazidime restriction alone is insufficient to c o n t r o l c o n t i n u e d i n f e c t i o n s w i t h e s b l producing organisms.(21,22) rahal et al.(21) were forced to withdraw cephalosporins as an entire class in order to exact control over endemic esbl producers. some authors have suggested t h a t u s e o f l a c t a m / l a c t a m a s e i n h i b i t o r combinations, rather than cephalosporins, as workhorse empirical therapy for infections suspected as being due to gram-negative bacilli, may facilitate control of esbl producers.(23,24) the mechanism by which these drugs may reduce infections with esbl producers is not certain. it should be noted, however, that many organisms now produce multiple-lactamases, which may reduce the effectiveness of lactam/ l a c t a m a s e i n h i b i t o r c o m b i n a t i o n s . ( 2 5 , 2 6 ) a p p a r e n t l y t h e p r o b l e m o f r e s i s t a n c e i n klebsiella spp. due to esbl-production by t h e s e b a c t e r i a h a s b e c o m e i n c r e a s i n g l y common, such that it may become a serious problem in the future management of infectious diseases. conclusions klebsiella organisms still susceptible to c e f t a z i d i m e , c e f i x i m e , c e f t r i a x o n e , a n d aztreonam show a sensitivity rate of 80% or more. testing for esbl revealed that the prevalence of esbl producers in clinical klebsiella isolates ranged from 10.8% to 12.3%. acknowledgements the authors wish to express their gratitude to the dean and vice-deans of the medical faculty, trisakti university for their financial support and to the department of microbiology, medical faculty, trisakti university for the use of laboratory facilities. references 1. bradford pa. extended broad-spectrum ßlactamases in the 21st century: characterization, epidemiology, and detection of this important resistance threat. clin microbiol rev 2001; 49: 3351. 2. hoge cw, gambel jm, srijan a, pitarangsi c, echeverira p. trends in antibiotic resistance among diarrheal pathogens isolated in thailand over 15 years. clin infect dis 1998; 26: 341-5. 3. vercauteren e, deschemaeker p, ieven m, sanders cc, goossens h. comparison of screening methods for detection of extended spectrum ß-lactamases and their prevalence among blood isolates of escherichia coli and klebsiella spp. in a belgian teaching hospital. j clin microbiol 1997; 219: 17. 4. shehabi aa, mahafzah a, baadran i, qadar fa, dajani n. high incidence of resistance of klebsiella 104 pneumoniae clinical isolates to extended spectrum ß-lactam drugs in intensive care units. diagn microbiol infect dis 2000; 36:53-6. 5. quinteros m, radice m, gardella n, rodriguez m, costa n, korbenfeld d, et al. extended spectrum ß-lactamases in enterobacteriaceae in buenos aires, argentina, public hospitals. antimicrob agents chemother 2003; 47: 2864-7. 6. kang ci, kim sh, park wb, lee kd, kim hb, kim ec, et al. bloodstream infection due to extended-spectrum ß-lactamase-producing escherichia coli and klebsiella pneumoniae: risk factors for mortality and treatment outcome, with special emphasis on antimicrobial therapy. antimicrob agents chemother 2004; 48: 4574-81. 7. national committee for clinical laboratory standards. performance standards for antimicrobial disk susceptibility testing sixth edition. m2-a6. villanova (pa): nccls; 1997 8. national committee for clinical laboratory standards. performance standards for antimicrobial disk susceptibility testing tenth informational supplement. m100-s11 wayne (pa): nccls: 2001. 9. subha a, ananthan s. extended spectrum beta lactamase (esbl) mediated resistance to third generation cephalosporins among klebsiella pneumoniae in chennai, indian j med microbiol 2002; 20: 92-5. 10. jacoby ga. antimicrobial-resistant pathogens in the 1990s. annu rev med 1996; 47: 169-79. 11. sirot d. extended-spectrum plasmid-mediated beta lactamases. antimicrob agents chemother 1995; 36: 19-34. 12. ministry of health malaysia, academy of medicine of malaysia, malaysian society of infectious diseases and chemotherapy. consensus guidelines for the management of infections by esblproducing bacteria; 2001. 13. severin ja, mertaniasih nm, kuntaman k, lemmens n, verbrugh ha, goessens whf. evaluation of the vitek 2r esbl test in detecting esbl-positive clinical escherichia coli and klebsiella pneumoniae isolates. in: proceedings of the 16th european congress of clinical microbiology and infectious diseases; 2006 apr 1-4; nice, france. oxford: blackwell publishing; 2006. p. 451. 14. spanu t, luzzaro f, perilli m, amicosante g, toniolo a, fadda g, et al. occurrence of extendedspectrum â-lactamases in members of the family enterobacteriaceae in italy: implications for resistance to â-lactams and other antimicrobial drugs. antimicrob agents chemother 2002; 46: 196-202. 15. saurina g, quale jm, manikal vm, oydna e, landman d. antimicrobial resistance in enterobacteriaceae in brooklyn, ny: epidemiology and relation to antibiotic usage patterns. j antimicrob chemother 2000; 45:895-8. 16. de champs c, sirot d, chanal c, bonnet r, sirot j, the french study group. a 1998 survey of extended-spectrum â -lactamases in enterobacteriaceae in france. antimicrob agents chemother 2000; 44: 3177-9. 17. bisson g, fishman no, patel jb, edelstein ph, lautenbach e. extended-spectrum â -lactamase producing escherichia coli and klebsiella species: risk factor for colonization and impact of antimicrobial formulary interventions on colonization prevalence. infect control hosp epidemiol 2002; 23: 254-60. 18. du b, long y, liu h, chen d, liu d, xu y, et al. extended-spectrum â -lactamase producing escherichia coli and klebsiella pneumoniae bloodstream infection: risk factors and clinical outcome. intensive care med 2002; 28: 1718-23. 19. tumbarello m, spanu t, sanguinetti m, citton r, montuori e, leone f, et al. bloodstream infection caused by extended-spectrum â -lactamase producing klebsiella pneumoniae: risk factors, molecular epidemiology, and clinical outcome. antimicrob agents chemother 2006; 50: 498-504. 20. schwaber mj, navon-venezia s, kaye ks, benami r, schwartz d, carmeli y. clinical and economic impact of bacteremia with extended spectrum â-lactamase producing enterobacteriaceae. antimicrob agents chemother 2006; 50: 1257-62. 21. rahal jj, urban c, horn d, freeman k, segalmaurer s. maurer j, et al. class restriction of cephalosporin use to control total cephalosporin resistance in nosocomial klebsiella. jama 1998; 280: 1233-7. 22. szabo d, filetoth z, szentandrassy j, nemedi m, toth e, jeney c, et al. molecular epidemiology of a cluster of cases due to klebsiella pneumoniae producing shv-5 extended-spectrum betalactamase in the premature intensive care unit of a hungarian hospital. j clin microbiol 1999; 37: 4167-9. 23. patterson je, hardin tc, kelly ca, garcia rc, jorgensen jh. association of antibiotic utilization measures and control of multiple-drug resistance herwana, yenny, pudjiadi, et al extended spectrum beta-lactamase 105 univ med vol.27 no.3 in klebsiella pneumoniae. infect control hosp epidemiol 2000; 21: 455-8. 24. paterson dl, bonomo ra. extended-spectrumlactamases: a clinical update. clin microbiol rev 2005; 18: 657-86. 25. baraniak a, sadowy e, hryniewicz w, gniadkowski m. two different extended spectrum beta-lactamases (esbls) in one of the first esblproducing salmonella isolates in poland. j clin microbiol 2002; 40: 1095-7. 26. shen d, winokur p, jones rn. characterization of extended spectrum beta-lactamase-producing klebsiella pneumoniae from beijing, china. int j antimicrob agents 2001; 18: 185-8. alvina i blood transfusion: is it safe for users? pusparini clinical pathology department, medical faculty, trisakti university blood transfusion is a long-known life-saving procedure for patients in need of blood, as its history can be traced to about 200 years ago. the first successful blood transfusion performed from one human to another was pioneered by james blundell, an obstetrician who in 1818 succeeded in transfusing 227 ml of blood to a patient with postpartum hemorrhage.(1) the modern practice of blood transfusion dates from the discovery of the abo blood groups by karl landsteiner in 1901 and of the rhesus blood group in 1940, in association with wiener. the collection, storage, and banking of blood and the use of anticoagulants became a recognized procedure immediately after the end of world war i.(2) currently blood is packaged in sterile plastic bags with the addition of anticoagulants. it may be distributed as whole blood or in the form of blood products, such as packed red cells, frozen fresh plasma, platelet concentrates, and cryoprecipitates.(3) every minute there is one person in the world who needs blood and blood products. in all countries, surgery, trauma, severe anemia and complications of pregnancy are the clinical conditions where blood transfusions is necessary. the problem arising is that there are many patients who do not have access to blood when needed. it is estimated that worldwide only 80 million units of blood are donated each year. this particularly has its impact on women with complications of pregnancy, trauma victims, and children with life-threatening anemia. blood transfusion may be likened to a two-edged sword, because on the one hand blood transfusion is needed to save a person’s life while on the other hand it can lead to side effects and complications ranging from the most mild to the most severe, i.e. death. therefore the issue of safe blood transfusion is an urgent one. the world health organization (who) has issued guidelines for performing safe blood transfusion in every country. the who strategy for blood safety comprises: (i) a well-organized, nationally coordinated blood transfusion service that can provide adequate and timely supplies of safe blood for all patients in need; (ii) the collection of blood only from voluntary non-remunerated blood donors from low risk populations; (iii) testing of all donated blood for transfusion transmissible infections, blood grouping and compatibility testing; (iv) the appropriate clinical use of blood, including the use of alternatives to transfusion whenever possible and the safe administration of blood and blood products; (v) a quality control system covering all stages of the transfusion process. with adequate donor selection among volunteers and screening of the blood for transfusion transmissible infections (tti,) the incidence of tti can at present be reduced to extremely low rates. the transmission rate of hepatitis b has reportedly declined substantially, as has the transmission rate of syphilis. the transmission rate of hepatitis c has decreased by 90%, from ii 1 : 200 in the year 1990 to 1 : 3000 in 1992. it is currently predicted that the transmission rate of hepatitis c through blood transfusion will decrease to 1:103.000. in the past, the transmission of hiv as a result of transfusion was estimated to be between 5% and 10%, but in 1996 all branches of the pmi started to perform screening tests for hiv (human immunodeficiency virus). the current estimate of hiv transmission by transfusion is 1 : 676.000.(4) in addition to the risk of tti there are several noninfectious hazards of blood transfusion, resulting in considerable morbidity and mortality rates. the noninfectious hazards include hemolytic transfusion reactions, reactions associated with leukocytes and leukocyte antibodies, graft versus host disease, transfusion related acute lung injury dan mistransfusions.(6) the majority of deaths are caused by acute hemolysis due to errors in identification of blood samples, blood components and the blood of recipients. in the last decade one in 38,000 units of red cells transfused in new york was the result of abo incompatibility due to errors. half of these errors took place outside the blood banks. in an international survey involving 62 hospitals, among 690,000 blood samples one in 165 was mislabelled or miscollected. it is said that in the united states the incidence of mislabeled and miscollected samples was 1000 to 10,000 times higher than the risk of tti.(5) transfusion of blood fractions rich in leukocytes causes severe febrile reactions, whereas in transfusions with an identical number of units containing low levels of buffy coat cells no febrile reactions develop. the minimum numbers of leukocytes capable of inducing transfusion reactions vary from 0.25 x 109 to 25 x 109 and the degree of temperature increase is correlated with incompatible numbers of leukocytes and transfusion speeds. transfusion reactions associated with leukocyte antibodies exhibit variable symptoms and signs, such as fever, dyspnea, hypotension, hypertension, and stiffness.the pathophysiology of leukocyte antibody reactions may be explained by the hypothesis that antibody binding of transfused leukocytes produces a monocyte-activating complex resulting in release of cytokines and their pyrogens. these transfusion reactions may be minimized by using leuko-depleted blood.(5) tr a n s f u s i o n a s s o c i a t e d g r a f t v e r s u s h o s t d i s e a s e ( ta g v h d ) o c c u r s w h e n immunocompetent allogeneic lymphocytes in the transfused blood bind to the blood of the recipient, proliferate and attack host tissues. recent reports mention rare but fatal events involving fetuses receiving exchange transfusions and children with immune disorders such as wiskottaldridge syndrome and thymic aplasia. to date no ta-gvhd cases have been reported in patients with acquired immune deficiency syndrome (aids). ta-gvhd develops 4 to 30 days after transfusion of blood and blood products. the treatments of ta-gvhd range from difficult to useless. with the development of a full blown syndrome the mortality rate approaches 90%. what is needed for effecting a decline in ta-gvhd incidence is a pathogen reduction technology involving nucleic acids, to be applied in the production of future blood components.(7) another severe transfusion reaction is termed transfusion related acute lung injury (trali), previously called noncardiogenic pulmonary edema. trali is currently the most frequent cause of death due to transfusions as reported by the food and drug administration (fda). trali is estimated to occur in 1:5000 blood transfusions, with a reported mortality of up to 15%. several strategies have been formulated to prevent the development of trali, including avoding the use of plasma containing human leukocyte antigens (hla), minimizing the use of blood from female donors to reduce exposures to hla and leukocyte antibodies that might have been induced in pregnancy.(8) in conclusion, although blood transfusion cannot be said to be completely safe, the risk of transfusion reactions may be minimized by improvements in the blood bank management system, involving donor recruitment, tti screening, and use of sophisticated technology. iii references 1. blundell j. experiment on the transfusion of blood by a syringe. med chir trans 1818;9-56. cited by pj schmidt, ag leacock. forgotten transfusion history: john leacock of barbados. bmj 2002;325:1485–7. 2. blundell j. a successful case of transfusion. lancet 1829;1:431. cited by en i. hajdu si. a note from history: blood transfusion from antiquity to the discovery of the rh factor. annals clinical laboratory science 2003;33:471-3. 3. mahmud z. are the blood transfusions safe! jafmc 2009;5:1-2. 4. improving blood safety worlwide (editorial). the lancet 2007;370:361. 5. alter hj, klein hg. the hazards of blood transfusion in historical perspective. blood 2008;112:2617-25. 6. dzik wh, murphy mf, andreu g. an international study of the performance of sample collection from patients. vox sang 2003;85:40-47. 7. klein hg. transfusion associated graft versus host disease: less fresh blood and more gray for an aging population. transfusion 2006;46:878-80. 8. kopko pm, marshall cs, mackenzie mr, holland pv, popovsky ma. transfusion related acute lung injury:report of clinical look back investigation. jama 2002;287:1968-71. alvina 21 abstract with increasing maternal age hopefully the mother knows more about raising a baby and supervising their growth and development. with normal birth weight and adequate duration of exclusive breastfeeding the baby can be expected to have optimal growth and development. the goal of this historical-cohort prospective study was to analyze the relation between maternal age, infant birth weight and duration of breastfeeding on the one hand with infant weight gain on the other. the population under study were six-month-old infants in the catchment area of pagar gading community health center, pino raya region, south bengkulu regency. the total sample consisted of 31 six-month-old infants, selected by consecutive nonrandom sampling of subjects meeting the inclusion criteria. statistical analysis was performed by means of the correlation-regression test. there was a significant correlation between duration of breastfeeding and infant weight gain using the correlation test (p<0.05), whilst there was no correlation of infant weight gain with maternal age (p>0.05). furthermore, with the multiple linear regression test, duration of breastfeeding affected infant weight gain (p<0.05), but infant birth weight and maternal age had no effect (p>0.05). the conclusion of this study is that duration of exclusive breastfeeding affects infant weight gain. breastfeeding of infants should be prolonged beyond the age of 6 months. keywords: breastfeeding, maternal age, infant weight gain *pagar gading community health center pino raya region, south bengkulu regency correspondence a dr. dedi kurniawan saputra pagar gading community health center pino raya region, south bengkulu regency email : dedi_phantom@yahoo.com univ med 2010;29:21-26 introduction the growth patterns of healthy infants during the first year of life and the standards for their assessment have been important subjects of research among nutritionists and child health workers in recent decades.(1) it is well-established that human milk is t h e o p t i m a l f o r m o f i n f a n t n u t r i t i o n . b r e a s t f e e d i n g c o n f e r s i m m u n o l o g i c , psychological, and developmental benefits to the infant.(2) breastfed infants grow differently during the first year of life than do those fed infant formula. a growing body of evidence duration of breastfeeding has a positive effect on infant weight gain dedi kurniawan saputra*a, carolina*, and riko rusli* january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina 22 saputra, carolina, rusli breastfeeding on infant weight gain shows that breastfeeding is associated with longterm benefits for the infant, such as a reduced risk of developing overweight and obesity during childhood.(3,4) the 2001 national household health survey (nhhs) showed that only 47% children aged 0-3 months were exclusive breastfed, and 6% children 6-7 months old still had exclusive breastfeeding without food supplement.(5) in rural areas the mother commonly breastfeeds her child, but study results indicate the influence of bad habits, such as giving the infant food/ drink as breast milk substitute in the initial postpartum days before mother ’s milk is produced. the substitute foods are among others rice water, a kind of rice preparation (nasi makmak), honey, and coconut water, which may endanger the infant’s health and reduce the opportunities for stimulating breast milk production as early as possible through the infant’s nursing at the mother’s breasts. in addition, there are many mothers who do not utilize the colostrum (breast milk secreted in the initial postpartum days), because it is viewed as unfit for infant consumption, as soured milk, etc. furthermore, breast milk substitutes (bms) are given at inappropriate times (too early or too late) and are qualitatively as well as quantitatively inadequate. it is recommended that bms be given from the age of 4-5 months, so that at age 6 months and above all infants have been given bms.(5) formula-fed infants are often introduced to complementary foods earlier than are their breastfed counterparts; these infants are exposed to a different feeding pattern than are infants who continue to breastfeed.(6,7) this combination of short periods of breastfeeding and early introduction of complementary food may contribute to the altered growth patterns seen in nonbreastfed infants. the age range of 0-24 months is a period of intense growth and development, such that it is frequently designated the golden period and also the critical period. the golden period may be realized if at this time period the infant and child receives nutritional intakes that are a p p r o p r i a t e f o r o p t i m u m g r o w t h a n d development. on the other hand, if in this period the infant and child do not receive food appropriate with the nutritional requirements, the golden period will turn into a critical period that disturbs their growth and development, either at the present stage or in subsequent stages. in indonesian infants, birth weight and length have also been found to be important determinants of infant growth.(8) infant birth weight and length are determinants of future nutritional status as was shown in bangladeshi infants who experienced very little catch-up growth after birth.(9) in order to achieve optimal growth and development, in the global strategy for infant and young child feeding, the who/unicef recommend 4 important measures: firstly, breastfeeding newborn babies within the initial 30 minutes of birth; secondly, exclusive breastfeeding from birth up to the age of 6 months; thirdly, giving complementary foods at 6-24 months; and fourthly, continuing breastfeeding until the age of 24 moths and o v e r. t h e s e r e c o m m e n d a t i o n s s t r e s s sociocultural norms; complementary foods should be prepared from inexpensive and indigenous food.(10) this study was conducted to test the hypothesis that breastfeeding has a positive effect on the weight gain in infants 0 to 6 months of age. methods research design a historical-cohort observational study was conducted to assess the relation between breastfeeding and the growth performance of infants 0–6 months of age from november 2008 until april 2009. subjects all infants recruited were born in a m a t e r n a l a n d c h i l d c a r e f a c i l i t y a t t h e puskesmas pagar gading, pino raya district, 23 s o u t h b e n g k u l u r e g e n c y. i n f a n t s w e r e enrolled consecutively at birth if they were term, singleton, had a birth weight of between 2000 to 4000 g inclusive, and whose mothers agreed to participate in the study. infants with congenital malformations or neonatal diseases were excluded from the study. sample size an a priori calculation for sample size for multiple regression was performed, indicating that a sample of 31 subjects or more would achieve a power level of 0.80 with a level of p< 0.05, using as many as 3 predictor variables.(11) data collection the anthropometric data collected was infant birth weight, performed monthly by means of a beam balance (dacin) calibrated in kilograms, up to one digit after the decimal p o i n t . t h e w e i g h i n g o f t h e i n f a n t s w a s performed at the health center. information was obtained on maternal age, socioeconomic characteristics, and duration of breastfeeding. data analysis independent t-test was performed for comparing maternal age, infant birth weight, and gender between the group of infants breastfed for 4 months and those breastfed for 6 months. to adjust for additional explanatory variables while exploring the association between the main exposure (duration of breastfeeding) and the main outcome (infant weight gain), a multiple linear regression model was constructed that included the potential confounding variables maternal age and infant birth weight. all statistical analyses were performed by means of spss version 11.0 at level of significance of 0.05. results data for analysis were available for 31 infants participating in the study, consisting of 13 male and 18 female infants, whose mothers had a mean age of 26.1 ± 4.2 years with a range of 18-38 years. the mean infant birth weight was 2.9 ± 0.5 kg, with a range of 2.0 4.0 kg. infants exclusively breastfed for 6 months amounted to 51.6% and those breastfed for 4 months 48.4%. there was no difference in maternal age, infant birth weight and infant gender between infants breastfed for 4 months and those breastfed for 6 months (table 1). figure 1 shows that after breastfeeding for more than 4 months there was a difference in infant weight gain. in infants breastfed for 6 months, the mean weights at 5 and 6 months were 7.1 kg and 7.6 kg higher than infants breastfed for 4 months, with mean weights at 5 and 6 months of respectively 6.7 kg and 7.1 kg. from figure 2 it is apparent that mean weight gain of infants breastfed for 6 months was greater than that of infants breastfed for 4 months. in month i there was no significant difference in mean infant weight gain. in month ii mean infant weight gain of infants breastfed for 0-4 months was greater than that of infants breastfed for 0-6 months, but thereafter breastfeeding for 0-6 months led to a higher mean infant weight gain compared with breastfeeding for 0-4 months. duration of breastfeeding variables 6 months (n=16) 4 months (n=15) p maternal age (yrs) 25.5 ± 4.1 26.6 ± 4.4 0.451 birth weight (kg) 3.1 ± 0.5 2.9 ± 0.6 0.268 gender male female 5 (38.5%) 10 (55.6%) 8 (61.5%) 8 (44.4%) 0.268 table 1. maternal age, infant birth weight, infant gender by duration of breastfeeding univ med vol.29 no.1 24 saputra, carolina, rusli breastfeeding on infant weight gain maternal age was inversely associated with infant weight gain, but this association was statistically not significant (â=-0.024, p=0.981). infant birth weight had a statistically non-significant negative association with i n f a n t w e i g h t g a i n ( â = 1 . 6 9 3 , p = 0 . 1 0 2 ) . duration of breastfeeding had a statistically p o s i t i v e e ff e c t o n i n f a n t w e i g h t g a i n . a d d i t i o n a l b r e a s t f e e d i n g f o r o n e m o n t h resulted in an increase in infant birth weight of 0.415 kg (â = 0,415, p=0.048) (table 2). 0 0.2 0.4 0.6 0.8 1 1.2 1 2 3 4 5 6 months in fa nt w ei gh t breastfeed 0-4 months breastfeed 0-6 months figure 2. mean monthly infant weight gain by duration of breastfeeding figure 1. mean monthly infant weight by duration of breastfeeding 1.00 2.00 3.00 4.00 5.00 6.00 7.00 8.00 bbl 1 2 3 4 5 6 infant age (months ) in fa nt w ei gh t (k g) breastfeed 0-4 months breastfeed 0-6 months 25 discussion the results of this study indicated that there was no correlation between maternal age and infant weight gain in the age range of 0-6 months. this finding was consistent with that obtained in a study in mexico city on infants between 0 and 6 months of age, showing no significant association between maternal and household characteristics on the one hand, comprising maternal age, education, marital status, crowding, sewage disposal, and use of piped water, and weight gain of infants between 0 and 6 months of age.(11) similar results were obtained in a danish study indicating that maternal age had no significant association with infant weight gain.(12) in addition, infant birth weight did not affect infant weight gain up to the age of 6 months, which finding was also obtained by taveras et al.(13) our study results differed from those obtained in a study in bangladesh on 1654 infants followed from birth up to the age of 12 months, showing that infant birth weight was significantly associated with infant weight gain from 0 to 12 months.(9) the studies conducted by villalpando et al.(11) and taveras et al.(13) obtained results similar to ours, viz. that infant birth weight was not significantly a s s o c i a t e d w i t h i n f a n t w e i g h t g a i n . a longitudinal study of maternal feeding style throughout infancy showed that infant birth weight was not associated with infant weight gain from 6 to 12 months.(14) the latter study revealed that duration of exclusive breastfeeding for 6 months affected infant weight gain, which was also seen in infants from an afro-columbian community,(15) thus once again demonstrating the superiority of exclusive breastfeeding in influencing infant weight gain. consistent results were also found in a multicenter study involving 7 localities (chengdu, china; new delhi; guatemala city; sagamu, nigeria; santiago, chile; uppsala, sweden; and westmead, australia), where 1252 infants were followed up from birth up to the age of 32 weeks.(16) in contrast to our results, in the latter study it was found that infants who were breastfed gained less weight at 1 year than did formula or mixed-fed infants.(12) children exclusively breastfed for less than 6 months had a greater risk of elevated weight gain at the age of 2 years than children breastfed for 6 months and more.(17) during the first 6 months of life the growth of exclusively breastfed infants was also similar to that of the infants regularly receiving formula at 12–16 weeks of age, mostly in addition to breast milk. the fact that exclusively breastfed infants showed the same growth as infants who were not exclusively breastfed may be partly due to the fact that there was also a high breastfeeding rate in the latter group. many studies have shown more rapid growth during the second half of the first year in infants fed with formula during the first 6 months.(1) complementary food in addition to breastfeeding turned out to have no great effect on infant growth and development.(18) there were several limitations to this study. firstly, diseases suffered by infants as confounding variables were not available and thus could not be analyzed. secondly, data on infant feeding patterns were not collected and infant weight should preferably be collected minimally up to the age of one year. conclusions this study demonstrates that exclusive breastfeeding is an ideal nutrition that can adequately support optimal growth in the first 6 m o n t h s o f l i f e . d u r a t i o n o f e x c l u s i v e variables β p maternal age infant birth weight -0.024 -1.693 0.981 0.102 duration of breastfeeding 0.415 0.048 table 2. adjusted multiple linear regression analysis of factors that contributed to the model univ med vol.29 no.1 26 saputra, carolina, rusli breastfeeding on infant weight gain breastfeeding enhances infant weight gain optimally. acknowledgements the investigators gratefully acknowledge the support of the head of the health service of south bengkulu regency and the head of the pagar gading health center and their r e s p e c t i v e s t a ff m e m b e r s f o r t h e d a t a collection. references 1. aarts c, kylberg e, hofvander y, gebre-medhin m. growth under privileged conditions of healthy swedish infants exclusively breastfed from birth to 4–6 months: a longitudinal prospective study based on daily records of feeding. acta pediatr 2003;92:145-51. 2. mortensen el, michaelsen kf, sanders sa, reinisch jm. the association between duration of breastfeeding and adult intelligence. jama 2002;287:2365-71. 3. dietz wh. breastfeeding may help prevent childhood overweight. jama 2001;285:2506-7. 4. toschke am, vignerova j, lhotska l, osancova k, koletzko b, von kries r. overweight and obesity in 6 to 14-year-old czech children in 1991: protective effect of breast-feeding. j pediatr 2002;141:764-9. 5. supraptini, lubis a, lrianto j. the coverage of immunization and exclusive breastfeeding in indonesia: result of national health survey 2001. j ekologi kesehatan 2003;2:249-54. 6. norris fj, larkin ms, williams cm, hampton sm, morgan jb. factors affecting the introduction of complementary foods in the preterm infant. eur j clin nutr 2002;56:448-54. 7. barton sj, howard pk, rayens mk. the effects of infant feeding decisions on infant growth. j spec pediatr nurs 2002;7:64-70. 8. kolsteren p w, kusin j a, kardjati s. pattern of linear growth velocities of infants from birth to 12 months in madura, indonesia. trop med int health 1997;2:291-301. 9. arifeen s e, black re, caulfield le, antelman g, baqui ah, nahar q, et al. infant growth patterns in the slums of dhaka in relation to birth weight, intrauterine growth retardation, and prematurity. am j clin nutr 2000;72:1010-7. 10. kresnawan, ranida ia, zainab s, zainal e, djasmidar, sianturi g, et al. pedoman umum pemberian makanan pendamping air susu ibu (mp-asi) lokal. jakarta: departemen kesehatan ri;2006. 11. villalpando s, lo´pez-alarco´ m. growth faltering is prevented by breast-feeding in underprivileged infants from mexico city. j nutr 2000;130:546-52. 12. baker jl, michaelsen kf, rasmussen km, sørensen tia. maternal prepregnant body mass index, duration of breastfeeding, and timing of complementary food introduction are associated with infant weight gain. am j clin nutr 2004;80: 1579-88. 13. taveras em, scanlon ks, birch l, rifas-shiman sl, rich-edwards jw, gillman mw. association of breastfeeding with maternal control of infant feeding at age 1 year. pediatrics 2004;114:57783. 14. worobey j, coper mi, hoffman rs. maternal behavior and infant weight gain in the first year. j nutr educ behav 2009;41:169-72. 15. beatriz ea, zunzunegui mv, delisle h, osorno j. growth trajectories are influenced by breastfeeding and infant healthy in an afro-colombian community. j nutr 2005;135:2171-8. 16. who working group on the growth reference protocol and the who task force on methods for the natural regulation of fertility. growth of healthy infants and the timing, type, and frequency of complementary foods. am j clin nutr 2002; 76:620-7. 17. kalies h, heinrich j, borte m, schaaf b, von berg a, von kries r, et al. for the lisa study group. the effect of breastfeeding on weight gain in infants: results of a birth cohort study. eur j med res 2005;60:36-42. 18. fronggilo e, de onis m, garza c. growth of healthy infants and the timing, type and frequency of complementary foods. am j clin nutr 2002; 76:620-7. alvina 71 abstract the tyrosine kinase receptor ret, which is the protein product of the ret gene, is involved in the development of the mammalian nervous system that causes hirschsprung’s disease (hscr). rets are cell surface molecules that are expressed in cells derived from the neural crest. the purpose of this study was to investigate the polymorphism of the ret gene in hscr in the yogyakarta population. genomic dna was extracted from surgically removed bowel tissues of 54 unrelated hscr patients. exon 2 of the ret gene was amplified by polymerase chain reaction (pcr) and analyzed by restriction fragment length polymorphism (rflp). molecular results were compared with clinical performance of hirschsprung patients. ret polymorphism was detected in exon 2 in all of the 54 indonesian hscr patients. the allelic distribution of the c135g a polymorphism in the ret exon 2 indicated that the a allele was more frequent in patients than in control individuals (chisquare test, p= 0.001). thus the ret variant allele a is over-represented in patients affected with the hscr phenotype. polymorphism of exon 2 of the ret gene was found in sporadic hirschsprung’s disease in the yogyakarta population, which suggests that the ret gene plays important roles in the pathogenesis of hscr. keywords: hirschsprung’s disease, ret gene, c135g a, exon 2 *department of biochemistry, school of medicine, soedirman university, purwokerto **department of paediatric surgery, ***department of biochemistry, school of medicine, gadjah mada university, yogyakarta ****department of biochemistry, school of veterinary, gadjah mada university, yogyakarta correspondence saryono, skp, mkes, ph.d department of biochemistry, school of medicine, soedirman university, purwokerto jl. gumbreg no.1 purwokerto jawa tengah 53416 email: sarbiokim@gmail.com univ med 2010;29:71-7 ret single nucleotide polymorphism in indonesians with sporadic hirschsprung’s disease saryono*, rochadi**, wiryatun lestariana***, wayan t artama****, and ahmad hamim sadewa*** may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction hirschsprung’s disease (hscr) is a congenital malformation of the colonic region c a u s e d b y a g a n g l i o n o s i s . m e i s s n e r aganglionosis in submucosal plexuses and auerbach aganglionosis in muscularis plexuses lead to a lower peristaltic coordination and f u n c t i o n a l i n t e s t i n a l o b s t r u c t i o n . ( 1 ) t h e phenotypes in hscr have aganglionosis of variable length and can be classified into two groups: short segment aganglionosis (about 80% of cases) and long segment aganglionosis (about 20% of cases), each having different characteristics.(2) clinically, hscr shows failure to defecate, abdominal distension, vomiting, and enterocolitis.(3) the disease has a complex genetic etiology and is likely to be of multifactorial inheritance with a variety of susceptibility genes, including ret. in 10-40% 72 saryono, rochadi, lestariana, et al sporadic hirschsrung’s disease of hscr cases, mutations of the ret receptor tyrosine kinase have been found. ret proteins are cell-surface molecules expressed on human intestinal neural crest cells and the ret gene is known as supportive gene of several congenital autosomal disorders, such as hscr.(4) the ret gene belongs to the cadherin superfamily and encodes one of the tyrosine kinase receptors on the surface of cells t h a t a r e i n v o l v e d i n s i g n a l t r a n s d u c t i o n required for cell growth and differentiation. the ret gene plays a crucial role in intestinal neural crest cells. the ret gene has been described as the most frequently mutated gene in hscr patients, with 50% of cases being familial and 7-35% sporadic hscr.(5) the gene exhibits variable polymorphism, is overor under-represented in hscr patients and results in visible phenotypic modification.(6) however, there have been no r e p o r t s o n r e t g e n e p o l y m o r p h i s m i n indonesians, in particular in the yogyakarta population. in order to investigate the role of ret polymorphism in the hscr pathogenesis we examined in indonesians with sporadic h s c r t h e o c c u r r e n c e o f t h e c 1 3 5 g a p o l y m o r p h i s m i n r e t e x o n 2 , w h i c h i s considered to be a genetic marker for hscr. methods research design this was a case control study conducted on patients with hscr admitted to dr sardjito hospital, yogyakarta in 2007. study subjects the dna of the hscr cases was taken from an aganglionic part of the tissues obtained from patients with hscr who underwent surgery in 2007 and the dna of the control group was taken from blood samples of healthy people without past history of congenital disorders in 2008. the research was conducted after obtaining ethical clearance and informed consent. measurements dna extraction f i f t y f o u r s p o r a d i c h s c r p a t i e n t s admitted to sardjito hospital yogyakarta who had undergone surgery were included in this study, after informed consent was obtained. hscr was diagnosed based on histological examination of either biopsy or surgical resection material. histopathological criteria for hscr were absence of enteric plexuses on histological examination of the aganglionic t r a c t a n d i n c r e a s e d a c e t y l c h o l i n e s t e r a s e staining in the nerve fibers. normal controls group were unselected, unrelated, race matched subjects from yogyakarta without a diagnosis of hscr. genomic dna was extracted from b o w e l s p e c i m e n s o f p a t i e n t s w h o h a d undergone surgery and from whole blood of control individuals by use of qiaamp dna kit® (qiagene) according to standard methods, and stored at -800c. p o l y m e r a s e c h a i n re a c t i o n re s t r i c t i o n f r a g m e n t l e n g t h p o l y m o r p h i s m ( p c r rflp) pcr amplification of exon 2 of ret was carried out on a pe 9600® pcr machine (perkin elmer pe biosystems) in twenty five µl of reaction mixture containing 200 ng of dna template, 1x pcr buffer, 3.5 mm mgcl2, 200 µm dntps, 0.2 µm of each primer and 1u of taq dna polymerase. the sequence of the forward primer was 5´ tca ctc act tcc cta ctt cc 3´ and that of the reverse primer 5´ ctt atg cgg aca ctg agc 3´. the conditions for pcr included an initial denaturation step at 96°c for 5 minutes, followed by 35 cycles of denaturation at 94°c for 30 seconds, annealing at 60°c for 45 seconds, extension at 72°c for 1 minute, and an additional extension step at 72°c for 5 m i n u t e s . t h e a m p l i f i e d f r a g m e n t s w e r e e l e c t r o p h o r e s e d o n 2 % a g a r o s e g e l a n d visualized by ethidium bromide under uv transillumination. the expected pcr product was 294 bp in length. 73 univ med vol.29 no.2 figure 1. the digestion product of c135g a, polymorphism ret gene with eagi on hscr in yogyakarta. lane 10 is homozygosity (gg), lane 2, 5 and 7 are heterozygosity (ga), lane 6 and 12 are undigested product (aa). ga aa gg aa ga ga gg the c135g a polymorphism of ret was genotyped by use of the eagi restriction enzyme (new england biolabs inc., usa) according to the manufacturer’s instructions. the digestion products were electrophoresed on 3% agarose gel and visualized by ethidium b r o m i d e u n d e r u v t r a n s i l l u m i n a t i o n . individuals with gg (wild homozygosity) showed two bands of 87 bp and 207 bp, those with ga (heterozygosity) showed three bands of 87 bp, 207 bp and 294 bp, and those with aa (mutated homozygosity) showed a single undigested band of 294 bp (figure 1). statistical analysis allelic frequency was calculated from the genotype frequencies and compared between the patient and control groups by chi-square test. the significance level of each statistical test was taken to be 0.05. hardy-weinberg equilibrium was tested by chi-square test in both control and patient sets. results o f 5 4 s p o r a d i c h s c r p a t i e n t s participating in this study, 36 were male and 18 female, giving a male:female ratio of 2:1. t h e g e n o t y p i c d i s t r i b u t i o n s o f t h e r e t c135g a polymorphism in indonesians with sporadic hscr were gg 9.25%, ga 42.59% a n d a a 4 8 . 1 4 % , w h i l e t h o s e o f c o n t r o l individuals were gg 21.73%, ga 62.21% and aa 13.04%. there was a significant difference in the genotype frequencies between the case and the control groups (chi-square = 14.54; p = 0 . 0 0 1 ) . i n d i v i d u a l s c a r r y i n g t h e a a phenotype were the most frequent in hscr patients (48.14%), whereas those carrying the ga genotype were the most frequent in controls (62.21%) (table 1). in this study there w a s a n o v e r r e p r e s e n t a t i o n o f t h e r e t c135g a polymorphism in hscr cases compared with controls. a m o n g a t o t a l o f 1 0 8 h s c r c h r o m o s o m e s , 6 9 . 4 4 % h a r b o r e d t h e polymorphic variant a and 30.55% the wild type g at nucleotide 135 (codon 45). in contrast, among 92 control chromosomes, 45.64% had the variant a and 54.34% the wild type g. the allele associated (p<0.05) with hirschsprung’s disease in our study sample was allele g of c135g a. allele 135a of the exon 2 polymorphism has been shown to be overrepresented in patients with hscr. it is table 1. genotype distribution of the ret gene polymorphism in hscr patients and controls in yogyakarta * chi-square test 74 saryono, rochadi, lestariana, et al sporadic hirschsrung’s disease a p p a r e n t t h a t a l l e l e d i s t r i b u t i o n d i ff e r s significantly between patient and control groups (chi-square test = 11.584; p=0.001) (table 2). hardy-weinberg equilibrium (hwe) analysis showed that genotype distribution for t h e r e t c 1 3 5 g a p o l y m o r p h i s m i n indonesians with sporadic hscr was in a c c o r d a n c e w i t h t h e h a r d y we i n b e rg equilibrium. among male and female patients with h s c r , t h e a a g e n o t y p e f r e q u e n c y o f c135g a was significantly higher than in controls (p<0.05) (table 3). this indicates that the aa genotype is one risk factor for hscr in male and female patients. the difference in frequency based on gender indicates that there are some modificator gene or other factors contributing to the incidence of hscr. discussion the present study is the first to show a genetic analysis of hscr disease in indonesia. the ret gene encodes a tyrosine kinase receptor on the surface of intestinal neural crest cells and is used to ligand several receptors.(10) ret is the most frequently mutated gene in hscr patients, but its surgical implications h a v e n o t b e e n e l u c i d a t e d . r e t g e n e polymorphism in the coding regions (exons) frequently contribute to hscr, although hscr has been shown to be associated with multiple genes.(11-14) our data show that there are differences in genetic distributions between patient and control groups. the results of our study showed that ret c135g a polymorphism in the indonesian population differed significantly between patient and control groups. it may be concluded that ret c135g a is correlated with hscr. there is over-representation of the a allele in the patient group as compared with the control group in indonesian populations. the results are similar to studies in chinese populations, although having different amounts of allele frequencies. the a allele on c135a represents the risk of hscr in indonesians, which is c o n s i s t e n t w i t h o t h e r s t u d i e s i n s e v e r a l p o p u l a t i o n s . ( 2 , 8 , 9 ) t h e r e s u l t s o f a s t u d y conducted in a chinese population showing over-representation in sporadic hscr patients of the variant allele of snp2 (a45a) are consistent with an ancient founding locus. our data indicate that ret polymorphism plays a table 3. sex distribution of ret polymorphism in exon 2 pursuant to genotype in yogyakarta *chi-square test table 2. the frequencies of ret polymorphism in hscr patients and controls *chi-square test 75 univ med vol.29 no.2 role in the etiology of some sporadically occurring hscr. the ethnic differences o b s e r v e d i n t h e s i n g l e n u c l e o t i d e p o l y m o r p h i s m ( s n p ) f r e q u e n c i e s w e r e r e f l e c t e d i n t h e f r e q u e n c i e s o f t h e r e t genotypes associated with hscr. r e c e n t s t u d i e s r e v e a l e d t h a t polymorphism of allele c135a on exon 2 was over-represented in patients with hscr(2) and that the a45a polymorphic allele was strongly associated with the occurrence of hscr. interestingly, the frequency of allele c135a, which was previously found to be overrepresented in hscr patients, was almost equal to the control group in our study. our d a t a s u g g e s t t h a t e x o n 2 p o l y m o r p h i s m c o n t r i b u t e s t o h s c r . t h i s a s s o c i a t i o n demonstrated in the present study may be confidently assumed to be true, given the high statistical probability with which the null hypothesis was rejected, and in view of the fact that the allele frequencies encountered in the controls were almost similar to those obtained in other studies. (2) the frequency of the polymorphic alleles at the a45a locus in our study appeared to be higher than that found in french, italian, spanish, polish and taiwanese populations.(2,7-9) if the current molecular epidemiological observation is valid, then several mechanisms are possible. first, it is possible that a new cryptic splice site (donor, acceptor or enhancer site) had been created. this would certainly be plausible for the a45a-hscr association, since the g to a substitution could result in the creation of a new alternative splice acceptor site 4 bp downstream of nucleotide 135. this could lead to the formation of a truncated protein or a receptor that did not strongly bind to its ligand. unfortunately, rna from a proper tissue source is not available to test this hypothesis, while ret is not expressed in peripheral lymphocytes.(6) the second possible mechanism is that the sequence variant may predispose to decreased expression of the variant-bearing allele, thus l e a d i n g t o l o w l e v e l f u n c t i o n a l haploinsufficiency.(15) another possibility is that the loci that are over-represented in the hscr cases compared to controls may lie in linkage disequilibrium with other sequences t h a t m a y d i r e c t l y c o n f e r a l o w l e v e l predisposition to or protection against hscr. the fourth possibility is that preferential usage of trna molecules may be involved, although this has yet to be shown in humans, but is well described among prokaryotes. if this were true, the wild type would be the favored sequence, with the variant being less favored, thus resulting in slightly decreased efficiency of r e t t r a n s l a t i o n i n t h e l a t t e r. t h e l a s t possibility is that when an amino acid is a l t e r e d , o n e m a y p o s t u l a t e t h a t s u c h a n apparently conservative change could subtly alter structure or function or both if located in a critical region. these postulated mechanisms are not mutually exclusive, and may account for the ret sequence variants acting as common low penetrance alleles in hscr predisposition.(6) these observations, taken t o g e t h e r, a rg u e f o r t h e v a l i d i t y o f t h e association of the a45a variant with the development of hscr, perhaps in a low penetrance fashion. in addition, the data also suggest that other polymorphic alleles might protect against the development of hscr in a low penetrance manner. these reasons suggest t h a t h s c r i s a c o m p l e x m u l t i g e n i c o r oligogenic disorder, in which the cumulative e f f e c t o f m u t a t i o n s i n m u l t i p l e g e n e s contributes to individual phenotype, suggesting genetic heterogeneity.(12,13) some polymorphic variants of the ret gene, including those found in the noncoding ret sequence, are believed to modify the effect of mutations in ret genes.(16-19) the sex ratio in this study was found to be 2:1 (36 males : 18 females), indicating that hscr predominantly occurs in males, as compared to females. the ratio was lower than those found in studies from other geographical areas, this observation having never been 76 saryono, rochadi, lestariana, et al sporadic hirschsrung’s disease reported before.(1,2,20) gender bias indicates the p r e s e n c e o f a d d i t i o n a l g e n d e rs p e c i f i c s u s c e p t i b i l i t y o r m o d i f y i n g l o c i , o r o f influencing environmental factors. there is a specific locus that plays a role in sex regulation in hscr, whilst there is another factor that codes for sex distribution in hscr patients. the present study has the limitation that it was c o n d u c t e d o n o n e r a c i a l t y p e o n l y i n yogyakarta, precluding comparative analysis with patients of other racial types. conclusions the results of this study showed overr e p r e s e n t a t i o n o f r e t c 1 3 5 g a polymorphism in hscr in one indonesian population. the findings are indicative of a correlation between codon 45 polymorphism of the ret gene and hirschsprung’s disease. ret polymorphism has been described in association with disease, suggesting a role for this gene in hscr predisposition. acknowledgements we extend our gratitude to all subjects who participated in this study. we are grateful to dewajani purnomosari, ph.d., for technical assistance. this study was supported by a doctoral scholarship granted by gadjah mada university, yogyakarta, indonesia. references 1. sangkhathat s, kusafuka t, chengkriwate p, patrapinyokul s, sangthong b, fukuzawa m. mutations and polymorphisms of hirschsprung disease candidate genes in thai patients. j hum genet 2006;51:1126–32. 2. garcia-barcelo mm, sham mh, lui vc, chen bl, song yq, lee ws, et al. chinese patients with sporadic hirschsprung’s disease are predominantly represented by a single ret haplotype. j med genet 2003;40:e122. 3. amiel j, lyonnet s. hirschsprung disease, associated syndromes, and genetics: a review. j med genet 2001;38:729–39. 4. zhang xn, zhou mn, qiu yq, ding sp, qi m, li jc. genetic analysis of ret, ednrb, and edn3 genes and three snps in mcs + 9.7 in chinese patients with isolated hirschsprung disease. biochem genet 2007;doi 10.1007/ s10528-007-9093-y. 5. sakai t, nirasawa y, itoh y, wakizaka a. japanese patients with sporadic hirschsprung: mutation analysis of the receptor tyrosine kinase proto-oncogene, endothelin-b receptor, endothelin-3, glial cell line-derived neurotrophic factor and neurturin genes: a comparison with similar studies. eur j pediatr 2000;159:160–7. 6. borrego s, ruiz a, saez me, gimm o, gao x, lopez-alonso m, et al. ret genotypes comprising specific haplotypes of polymorphic variants predispose to isolated hirschsprung disease. j med genet 2000;37:572–8. 7. smigiel r, lebioda a, patkowski d, czernik j, dobosz t, pesz k, et al. single nucleotide polymorphisms in the ret gene and their correlations with hirschsprung disease phenotype. j appl genet 2006;47:261–7. 8. lantieri f, griseri p, puppo f, campus r, martucciello g, ravazzolo r, et al. haplotypes of the human ret proto-oncogene associated with hirschsprung disease in the italian population derive from a single ancestral combination of alleles. ann hum genet 2005;70: 12–26. 9. wu tt, tsai tw, chu ct, lee zf, hung cm, su cc, et al. low ret mutation frequency and polymorphism analysis of the ret and ednrb genes in patients with hirschsprung disease in taiwan. j hum genet 2005;50:168–74. 10. heanue ta, pachnis v. enteric nervous system development and hirschsprung’s disease: advances in genetic and stem cell studies. nature 2007;8:466-79. 11. passarge e. dissecting hirschsprung disease. nat genet 2002;31:11-2. 12. brooks, as. a novel susceptibility locus for hirschsprung’s disease maps to 4q31.s3-q32.3. j med genet 2006;43:e35. 13. bolk s, pelet a, hofstra rm, angrist m, salomon r, croaker d, et al. a human model for multigenic inheritance: phenotypic expression in hirschsprung disease requires both the ret gene and a new 9q31 locus. proc natl acad sci usa 2000;97:268–73. 14. gath r, goessling a, keller km, koletzko s, coerdt w, muntefering h, et al. analysis of the ret, gdnf, edn3, and ednrb genes in patients with intestinal neuronal dysplasia and hirschsprung disease. gut 2001;48:671–5. 77 15. fitze g, cramer j, ziegler a, schierz m, schreiber m, kuhlisch e, et al. association between c135g/ a genotype and ret proto-oncogene germline mutations and phenotype of hirschsprung’s disease. lancet 2002;359:1200–5. 16. griseri p, pesce b, patrone g, osinga j, puppo f, sancandi m, et al. a rare haplotype of the ret proto-oncogene is a risk-modifying allele in hirschsprung disease. am j hum genet 2002;71: 969–74. 17. griseri p, sancandi m, patrone g, bocciardi r, hofstra r, ravazzolo r, et al. a single-nucleotide polymorphic variant of the ret protooncogene is underrepresented in sporadic hirschsprung disease. eur j hum genet 2000;8:721–4. 18. lesueur f, corbex m, mckay jd, lima j, soares p, griseri p, et al. specific haplotypes of the ret proto-oncogene are over-represented in patients with sporadic papillary thyroid carcinoma. j med genet 2002;39;260-5. 19. sancandi m, griseri p, pesce b, patrone g, puppo f, lerone m, et al. single nucleotide polymorphic aleles in the 5’ region of the ret proto-oncogene define a risk haplotype in hirschsprung’s disease. j med genet 2003;40:714–8. 20. li jc, ding sp, song y, li mj. mutation of ret gene in chinese patients with hirschsprung’s disease. world j gastroenterol 2006;8:1108-11. univ med vol.29 no.2 yenny1 29 the role of polyphenols in causing cardiovascular disease department of pharmacology, medical faculty, trisakti university correspondence dr. yenny department of pharmacology, medical faculty, trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 ext. 2801 email : stasia_mk@yahoo.com universa medicina 2008; 27: 29-38. january-march, 2008january-march, 2008january-march, 2008january-march, 2008january-march, 2008 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 abstract yenny and elly herwana research on flavonoids has increased since the discovery of the french paradox, the low cardiovascular mortality rate observed in mediterranean population in association with red wine consumption and a high saturated fat intake. plant polyphenol (flavonoid) occurs naturally in fruits, vegetables, and beverages such as tea and wine. epidemiologic studies suggest that higher polyphenol intake from fruits and vegetables is associated with decreased risk for cardiovascular disease. the mechanisms explaining this observation remain unclear. the vascular endothelium is a critical regulator of vascular homeostasis, and endothelial dysfunction contributes to the pathogenesis and clinical expression of coronary artery disease. platelet aggregation is a central mechanism in the pathogenesis of acute coronary syndromes, including myocardial infarction and unstable angina. there are numerous reports suggesting that plant polyphenols improve endothelial function and inhibit platelet aggregation in humans. keywords: polyphenol, flavonoid, cardiovascular universa medicina introduction c a r d i o v a s c u l a r d i s e a s e , e s p e c i a l l y ischemic heart disease, is the main cause of morbidity and mortality in many countries.(1) this disease is a multifactorial of complex etiology. a number of risk factors have been extensively studied and documented, as in the case of serum lipids and lipoproteins,(2) which are the cause of at least one-half the incidence of ischemic heart disease in europe. subsequent research has shown that cases of atherosclerosis may occur in spite of normal serum lipid and lipoprotein concentrations.(3) other factors of interest to be discussed as causes of ischemic heart disease are endothelial dysfunction and platelet aggregation. the relationship between flavonoid intake and the risk for a number of chronic diseases has been investigated by knekt et al.(4) this study shows that flavonoid intake is beneficial in decreasing the risk of chronic d i s e a s e , s u c h a s i s c h e m i c h e a r t d i s e a s e . f l a v o n o i d s r e p u t e d l y h a v e a n u m b e r o f beneficial effects in controlling endothelial t h r o m b o s i s , i n f l a m m a t o r y p r o c e s s e s , a n d vascular tone, i.e. factors which may cause occlusion of the arterial lumen, thus leading to acute coronary syndromes. 30 yenny, herwana polyphenols in cardiovascular pathogenesis of cardiovascular disease atherosclerosis is a chronic inflammatory disorder occurring at prone regions in mediumsized arteries. atherosclerotic lesions may develop asymptomatically for a number of decades before becoming active and causing clinical conditions such as acute myocardial infarction, unstable angina, or sudden death. a b o v e m e n t i o n e d e v e n t s a r e i n g e n e r a l frequently caused by acute rupture or erosion of a susceptible plaque causing exposure of the strongly thrombogenic subendothelium to the blood. the resulting disorder is an acute one, w h e r e p l a t e l e t a c c u m u l a t i o n c a u s e m u r a l thrombus formation, leading to a total or partial o c c l u s i o n o f t h e a r t e r i a l l u m e n , a n d t h u s influencing the occurrence of infarction or ischemia. to date the mechanisms explaining plaque susceptibility and rupture, are still incompletely understood, but available data point to local inflammation within the plaque and thinning of fibrotic peaks, while accumulation of fatty plaques may be a contributing factor. once rupture of a plaque takes place, the extent of thrombus formation and the occurrence of acute changes in vascular tone will play a role i n d e t e r m i n i n g t h e e x t e n t o f i s c h e m i a o r infarction.(5) the xanthine oxidase pathway also is implicated as an important pathway in the process of injury due to oxidation within the tissues. both types of xanthine, i.e. xanthine d e h y d r o g e n a s e a n d x a n t h i n e o x i d a s e , a r e involved in xanthine metabolism into uric acid. xanthine dehydrogenase is the enzymic form present in physiological conditions, but in ischemic conditions the dehydrogenase will undergo configurational change into the oxidase. xanthine oxidase is a source of oxygen free radicals. in the reperfusion phase, e.g. after reoxygenation, xanthine oxidase will react with molecular oxygen, releasing free radicals in the form of superoxide anions,(5) which may attract various mediators of inflammation, leading to inflammation and tissue damage. leukocyte immobilization and adhesion to the endothelial cell wall is another major m e c h a n i s m r e s p o n s i b l e n o t o n l y f o r t h e generation of reactive oxygen from free radicals, but also for the release of cytotoxic oxidants and mediators of inflammation, and the subsequent activation of the complement system. in normal conditions the leucocytes can freely pass through the endothelial cell wall. however, in ischemia a n d i n f l a m m a t i o n , v a r i o u s m e d i a t o r s , p a r t i c u l a r l y t h o s e o r i g i n a t i n g f r o m t h e endothelium, and complement factors, may cause adhesion of leucocytes to the endothelial wall, thereby causing immobilisation and stimulation of neutrophil degranulation, resulting in tissue injury.(5) numerous studies undertaken recently, with a focus on vascular biology, provide the basis for a productive approach to developing the latest management programs and prevalence strategies for cardiovascular diseases. this strong interest is particularly aimed at antithrombotic and other therapies for improving endothelial cell fucntion as the key factor in cell regulation within the vascular wall. the vascular endothelium is an important k e y f a c t o r i n t h e r e g u l a t i o n o f v a s c u l a r homeostasis. there is evidence indicating that functional changes of the endothelium contribute to the pathogenesis and expression of clinical cardiovascular disease.(6) the endothelial cells regulate vascular homeostasis by producing factors acting locally in the vascular lumen and vascular wall, through formation of nitric oxide (no). originally no was known as endotheliumderived vasodilator, but at present it is apparent that no also regulates other important aspects of vascular homeostasis. for example, no prevents leucocyte adhesion to the endothelial surface and inhibits expression of adherent leukocytes. no also prevents platelet adhesion 31 and aggregation. additionally, no inhibits proliferation of vascular smooth muscle fibers and alters expression of noncellular components of the vascular wall matrix. these features of no result in a correlation between no and lesion formation, vascular wall hypertrophy, and vascular compliance. in other words, there is an interrelationship between endothelium-derived no and all stages of the atherosclerotic process, due to its important properties as vasodilator, anti-inflammatory and anti-thrombotic agent, a n d s u p r e s s o r o f v a s c u l a r s m o o t h m u s c l e proliferation.(7) being endothelium-derived is not only a feature of no, but also that of other products having the same characteristics in regulating v a s c u l a r h o m e o s t a s i s , s u c h a s s u b s t a n c e s influencing the vascular tone (prostacyclins and e n d o t h e l i n s ) , f i b r i n o l y t i c f a c t o r s ( t i s s u e p l a s m i n o g e n a c t i v a t o r a n d p l a s m i n o g e n activator inhibitor-1), coagulation factors (tissue factors, heparan, and von willebrand factor), and pro-inflammation factors (adhesion molecules and inflammatory cytokines).(8) in general, loss of no in parallel with changes in the regulatory system mechanism, results in the development of a pathological endothelial p h e n o t y p e . t h i s o b s e r v a t i o n s h o w s t h a t endothelial condition may be an indicator of v a s c u l a r h e a l t h , a n d t h a t e x a m i n a t i o n o f endothelial vasomotor function may yield clinical benefits. cardiovascular risk factors which may adversely affect the endothelium are such as dyslipidemias, hypertension, diabetes mellitus, smoking, ageing, physical inactivity, s y s t e m i c i n f l a m m a t i o n , i n f e c t i o n , a n d hyperchromocysteinemia; the postmenopausal c o n d i t i o n i s a l s o l i n k e d t o e n d o t h e l i a l dysfunction. genetic and environmental factors may influence the effect of risk factors on endothelial function. here genetic variation influences the synthesis of no and the activity of antioxidant enzymes, such as superoxide d i s m u t a s e , c a t a l a s e , a n d g l u t a t h i o n e peroxidase.(10) food may also influence the effect of risk factors on endothelial function, thus polyphenol intake may be important in d e t e r m i n i n g t h e r i s k f o r c a r d i o v a s c u l a r events.(5) polyphenols polyphenols are a major constituent of v e g e t a b l e s a n d c o m p r i s e t h e m a j o r i t y o f antioxidants found in our food. polyphenols have various properties, depending on the molecular structure. these substances are abundant in fruits, vegetables, nuts, barks, roots, stems, flowers, tea, and wine.(10) most polyphenols are important in imparting attractive colors and flavors to plant foods and beverages, which is termed their organoleptic qualities. (11) the m a j o r p i g m e n t i n p l a n t p o l y p h e n o l s i s anthocyanin, detectable by its red, violet, or blue color, while the less numerous yellow colors are due to flavonols and flavones. some polyphenols, such as vanillin and eugenol (imparting the typical fragrance to cloves), h a v e v o l a t i l e p r o p e r t i e s , b u t t h e m a i n characteristic of polyphenols is their bitter taste.(11) plant polyphenols are broadly divided into t w o m a i n g r o u p s , i . e . f l a v o n o i d s a n d nonflavonoids. nonflavonoids have a simpler s t r u c t u r e ; e x a m p l e s o f n o n f l a v o n o i d s a r e phenolic acids (subdivided into benzoic acid and hydroxycinnamic acid, based on the c1-c6 and c 3 c 6 c h a i n s , r e s p e c t i v e l y ) , s t i l b e n e a n d d e r i v a t i v e s , ( s u c h a s s t i l b e n e o l i g o m e r s , gallotannins, ellagitannins, and lignins). the flavonoids possess a core consisting of two phenolic rings and an oxygenated heterocyclic pyrane ring. they are the most important group, and are further subdivided into 13 classes (figure 1), based on differences in oxidation state of the heterocyclic pyrane ring.(12) universa medicina vol.27 no.1 32 yenny, herwana polyphenols in cardiovascular q u e r c e t i n i s o n e o f t h e f l a v o n o i d s f o u n d abundantly in onions, apples, broccoli, and berries. the second group are the flavanones, mainly found in citrus fruits. an example of flavonoids in this group is narigin. catechincontaining flavonoids are mainly found in green and black tea, and in red wine. anthocyanins are encountered in strawberries and other kinds of berry, grapes, red wine, and tea.(9) flavonoids are thought to contribute to the potential protective effect against cardiovascular disease through their antioxidant,(14) anti-thrombogenic,(15) and antiinflammatory properties. the most recent research results indicate that flavonoids may also improve vascular function.(16) numerous epidemiological studies are in existence,(17,18) indicating a decreased risk for cardiovascular disease in subjects consuming large amounts of flavonoids in tea or other food sources. one of these is the meta-analysis study by peter at al,(17) showing a decrease in risk for cardiovascular disease of approximately 11% on daily consumption of three cups of tea. the results are supported by the study of geleijnse et al,(15) indicating a lower relative risk (rr) for incidence of myocardial infarction in the group of subjects ingesting more than 375 ml of tea d a i l y ( r r : 0 . 5 7 ; 9 5 % c i : 0 . 3 3 0 . 9 8 ) , a s compared to non-users. mechanism of action of flavonoids the cells of the body and various tissues are continually subject to injury due to free radicals and reactive oxygen produced during oxygen metabolism. the mechanisms and events by which free radicals disrupt cellular function are not fully understood. one of the events which appears to be most important in membrane damage is lipid peroxidation. the membrane damage caused may induce a shift in net electric charge of the cells and changes in osmotic pressure, leading to swelling of the cells and even cell death. the formation of free radicals may to date, more than 4000 flavonoid variants have been successfully identified from plants.(13) the four main flavonoid groups together with their constituents and food sources are listed in table 1. flavones are described as planar structures marked by the presence of flavonoid double bonds at the center of the aromatic ring. figure 1. group classes of flavonoid 33 table 1. main flavonoid groups, components, and food source (9) also attract various mediators of inflammation, which play a role in systemic inflammatory r e s p o n s e s a n d t i s s u e d a m a g e . t h e l i v i n g organism is capable of protecting itself from reactive oxygen through a number of defense m e c h a n i s m s . o n e o f t h e s e i s a n t i o x i d a n t formation, either through enzymatic activity, such as of superoxide dismutase, catalase, and g l u t a t h i o n e p e r o x i d a s e , o r t h r o u g h t h e n o n e n z y m a t i c a c t i v i t y o f s u p p l e m e n t a r y glutathione, ascorbic acid, and α-tocopherol.(9) nearly all flavonoid groups have the capability of functioning as antioxidants. flavones and catechins appear to be the flavonoids with the strongest effect in protecting the body against reactive oxygen.(9) flavonoids are also able to prevent injury caused by a number of free radicals. oxidation of flavonoids by free radicals will result in more stable and less reactive radicals. in other words, flavonoids stabilize reactive oxygen by reacting with this component. the high hydroxyl group reactivity of flavonoids inactivates free radicals t h r o u g h t h e f o l l o w i n g r e a c t i o n e q u a t i o n : flavonoid (oh) + r* > flavonoid (o*) +rh, where r* is a free radical and o* is an oxygen free radical. some flavonoids can scavenge highly reactive oxygen originating from radicals called peroxynitrites. some flavonoid types such as epicatechin and rutin are potent radical scavengers. this scavenging ability is due to inhibition of xanthine oxidase activity. the universa medicina vol.27 no.1 34 yenny, herwana polyphenols in cardiovascular ability of flavonoids for scavenging free radicals causes flavonoids to be able to inhibit oxidation of low density lipoproteins (ldl). oxidation of ldl by oxygen radicals will cause endothelial wall injury, thus increasing atherosclerotic changes. the ability of flavonoids for protecting the endothelial wall against injury due to ldl particles, has led to the statement that they have a preventive effect against atherosclerosis.(9) this was proven in a study conducted by arai e t a l . ( 1 9 ) i n j a p a n , r e p o r t i n g a n i n v e r s e relationship between flavonoid intake and total plasma cholesterol concentration. the anti-atherosclerotic effect of flavonoids takes place through synthesis of no, which functions in maintaining vascular dilatation. the higher no concentration, as a result of its synthesis in macrophages, apparently will cause oxidative damage. this is presumably linked to simultaneous activation of macrophages, which increases no production concurrently with enhanced large-scale synthesis of superoxide anions. no is produced by various cell types, including endothelial cells and macrophages. no reacting with free radicals may possibly produce the strongly deleterious peroxynitrites. injury arising from no through peroxynitrites plays the main role, as peroxynitrites can directly react with oxidized ldl, resulting in irreversible membrane damage. the use of flavonoids as antioxidants leads to scavenging of free radicals, because these indirectly react with no, such that the resulting damage is reduced. interestingly, no itself can behave as a radical, and it is reported that the no molecule is directly scavenged by flavonoids.(9) cyclo-oxygenase and 5-lipoxygenase play an important role as mediators of inflammation. involvement of both substances is preceded by arachidonic acid release, as the starting point f o r a s y s t e m i c i n f l a m m a t o r y r e s p o n s e . neutrophils produced by the lipoxygenase pathway synthesize chemotactic components f r o m a r a c h i d o n i c a c i d , a n d a l s o p r o v o k e cytokine release. some phenolic components, particularly quercetin, may down-regulate both cyclooxygenase and 5-lipoxygenase pathways, thus inhibiting the formation of inflammatory metabolites, with resultant arachidonic acid release. the precise mechanism of enzyme inhibition by flavonoids is still unclear. the anti-inflammatory effects are also caused by the ability of flavonoids for inhibiting the biosynthesis of eicosanoids. eicosanoids, such as prostaglandins, are involved in various immunological responses and are the end p r o d u c t s o f c y c l o o x y g e n a s e a n d 5 lipoxygenase. flavonoids also inhibit tyrosine kinases in the cytosol and cell membrane. integral membrane proteins, such as tyrosine 3-monooxygenase kinase, are involved in various functions, such as enzyme catalysis, cross-membrane transport, signal transduction for hormone receptors and growth factors, and energy transfer in adenosine triphosphate (atp) synthesis. this protein-to-protein inhibition causes down-regulation of uncontrolled cell growth and proliferation. tyrosine kinase is an important key factor in the signal pathway r e g u l a t i n g c e l l p r o l i f e r a t i o n . t h e a n t i i n f l a m m a t o r y e f f e c t s o f f l a v o n o i d s a r e demonstrated by the capability of flavonoids for inhibiting neutrophil degranulation. this is a direct effect of flavonoids in decreasing arachidonic acid release by neutrophils and other immune cells.(9) some flavonoids are actively involved in inhibiting platelet aggregation. certain flavonols have an antithrombotic effect because they directly scavenge free radicals and thereby maintain endothelial prostacyclin and no concentrations. flavonoids show antithrombotic effects in vitro and in vivo, due to their capability in inhibiting cyclooxygenase and 5-lipoxygenase activation. currently it is well known that arachidonic acid released in inflammation is 35 metabolized by platelets to form prostaglandins, endoperoxides, and thromboxane a2, causing platelet activation and aggregation. the antiaggregation effect of flavonoids is thought to take place through inhibition of thromboxane a2 formation. the effects of flavonoids on other e n z y m e s y s t e m s i s t h r o u g h i n h i b i t i o n o f arachidonic acid metabolism, thus conferring a n t i i n f l a m m a t o r y a n d a n t i t h r o m b o g e n i c properties on flavonoids.(9) effects of polyphenols on endothelial function e n d o t h e l i a l d y s f u n c t i o n i s a n e a r l y pathophysiologic abnormality occurring in atherosclerosis and is an independent predictor of adverse prognosis in nearly all cardiovascular diseases. most studies on the effects of tea (camellia sinensis l) on endothelial function utilize the noninvasive ultrasound method, which tests endothelium-dependent flow-mediated dilation in the brachial artery. a study conducted by modena et al.(20) succeeded in proving that impaired flow-mediated dilation in the brachial artery can identify patients at high risk for cardiovascular disease. preliminary studies on the effects of the water-soluble antioxidant, ascorbic acid, and its r e l a t i o n s h i p t o i n c r e a s e d e n d o t h e l i a l n o synthesis, has promoted research on tea in connection with endothelial function, as tea also contains water-soluble flavonoid antioxidants.(21) te a i s v e r y r i c h i n p o l y p h e n o l s ; w i t h a concentration in dry tea leaves of 30%. tea c o n t a i n s v a r i o u s w a t e rs o l u b l e f l a v o n o i d antioxidants, including catechin, quercetin, kaempferol, and other polyphenols. the majority of flavanols are subsequently converted into a number of products, including thearubigin and theaflavin, which impart a black or dark brown color to tea, and a smaller number is converted into theaflavic acid and bisflavanol, also known as theasinensis.(11) a n u m b e r o f s t u d i e s ( 1 6 , 2 2 ) h a v e b e e n undertaken to search for a correlation between tea consumption and endothelial function. a cross-sectional placebo-controlled study on 50 subjects with coronary artery disease (confirmed by angiography), was conducted by duffy et al.(16) with the objective of detecting an effect of shortand long-term tea consumption on flowmediated dilation of the brachial artery. the results of this study indicate that both short-term and long-term tea consumption has been proven to be able to enhance endothelial function. these results are also corroborated by the finding that tea consumption has no effect on nitroglycerinemediated vascular dilatation, basal arterial diameter, or extent of reactive hyperemia. this f u r t h e r s t r e n g t h e n s t h e b e l i e f t h a t t e a consumption influences endothelial function . a study done by hodgson et al.(22) has investigated the effect of consumption of 5 cups of black tea daily for 5 weeks in healthy subjects with moderate hypercholesterolemia, and this also showed improvement in flow-mediated dilation o f t h e b r a c h i a l a r t e r y. i n t e r e s t i n g l y, t e a consumption was also associated with enhanced nitroglycerine-mediated vascular dilation, indicating that tea improves the bioactivity of endothelium-derived no or that tea also has an effect on vascular smooth muscle function. other flavonoid-containing beverages are mainly derived from wine products. products derived from purple grapes, including red wine and purple grape juice, contain flavonoids. the flavonoid content of red wine is approximately 10 times greater than that of white wine. consumption of moderate amounts of red wine (5ml/kg or 1-2 glasses a day), or of 5-10 ml of purple grape juice, may reduce the incidence of c a r d i o v a s c u l a r d i s e a s e . t h i s d e c r e a s e i n cardiovascular disease incidence is due to the ability of this substance to protect ldl against oxidative processes. ( 2 3 ) red wine contains flavonoids in the form of resveratrol. because universa medicina vol.27 no.1 36 yenny, herwana polyphenols in cardiovascular red wine also contains alcohol, there is some question as to whether its cardioprotective effect might be due to interaction of alcohol and resveratrol. a study comparing the effects of red wine, dealcoholized red wine, and resveratrol, indicated that atherosclerotic plaque size and thickening of the tunica intima decrease on administration of those three substances. the results of this study show that dealcoholized red wine has a cardioprotective effect comparable to that in subjects given resveratrol only.(24) the study by guarda et al.(25) attempted to find an association between red wine consumption in moderate doses with endothelial function and oxidative stress in patients with acute coronary s y n d r o m e . tw e n t y i n d i v i d u a l s w h o h a d undergone percutaneous coronary intervention subsequent to acute coronary syndrome, were divided at random into two groups. for two months, the group on red wine (n=9) ingested cabernet sauvignon 250 ml daily, while the control group (n=11) was free from alcoholic beverages. the results of the study indicate that red wine is effective in improving endothelial function and in reducing oxidative stress in patients with acute coronary syndromes. numerous studies have also been conducted on chocolate, one of the food sources rich in flavonoids, in relation to endothelial function. one of such studies is that undertaken by perezviscaino et al. ( 2 6 ) intake of chocolate and chocolate-based foodstuffs in high doses (176 mg/dl) for two weeks may improve endothelial function.(27) other studies were conducted on p o s t m e n o p a u s a l w o m e n a t h i g h r i s k f o r cardiovascular disease, yielding a correlation with endothelial dysfunction. the subjects in this study were 32 postmenopausal women with hypercholesterolemia, who were randomly assigned to a group on high-dose chocolate (440 mg total flavanols) and a group on low-dose chocolate (43 mg total flavanols) for six weeks. in both groups there was a significant increase in flow-mediated dilation of the brachial artery, amounting to 76% in the high-dose group and 32% in the group on low-dose chocolate. the r e s u l t s o f t h i s s t u d y s h o w t h a t c h r o n i c c o n s u m p t i o n o f c h o c o l a t e m a y i m p r o v e endothelial function and decrease vascular cell adhesion in postmenopausal women with hypercholesterolemia.(28) polyphenols and platelet function platelet aggregation contributes to formation of atherosclerosis and acute platelet thrombi, followed by embolization of the stenotic arteries. activation of platelets adhering to the vascular endothelium induces the formation of lipid peroxidation and oxygen free radicals, which will inhibit synthesis of endothelial prostacyclins and no. it is apparent here that platelet aggregation plays a critical role in the pathogenesis of acute coronary syndromes. this is supported by extensive evidence that antiplatelet therapy can reduce the risk of cardiovascular disease.(29) the effect of polyphenols in decreasing platelet activity has a strong impact on cardiovascular disease and can explain the epidemiological data on polyphenol function in cardiovascular disease. based on the potential mechanism of flavonoids o n p l a t e l e t f u n c t i o n , f r e e d m a n e t a l ( 3 0 ) investigated the effect of purple grape juice on platelet function. the observational results indicate that the addition of purple grape juice to platelets ex vivo causes a decrease in platelet aggregation and superoxide anion generation, and an increase in nitric oxide production by platelets. these beneficial effects appear to be related to the ability of purple grape juice for inhibiting protein-c kinase activity. this study is important due to the reproducibility of its results, when repeated with platelets isolated from healthy volunteers consuming purple grape juice for two weeks.(5) there are numerous data on the effect of tea consumption on platelet function. among 37 these are those from a study undertaken by duffy et al,(31) on the effect of short-term and longterm tea consumption on platelet aggregation ex vivo in response to adp (adenosine diphosphate) or thrombin-related activated peptides in patients with coronary artery disease. this study did not show any effects of tea consumption on platelet function, although when the tea was administered concurrently with aspirin therapy, it did have an influence on the research results. other studies indicate that tea consumption may decrease the plasma concentration of p-selectin, an in vivo platelet aggregation marker.(32) although polyphenols show many beneficial effects, especially in improving endothelial function and inhibiting platelet aggregation, further study is required on the ability of polyphenols in preventing cardiovascular disease. conclusions consumption of food rich in polyphenols, such as fruits and vegetables, and beverages derived from plants, such as tea, red wine and chocolate, has a protective effect on the risks for cardiovascular disease. most acute coronary syndromes are due to adhesion, aggregation, and thrombus formation in ruptured atheromatous plaques. here flavonoids will demonstrate their a b i l i t y f o r r e d u c i n g p l a t e l e t a g g r e g a t i o n , enhancing platelet no release and decreasing superoxide anion production. although these f i n d i n g s s h o u l d n o t b e i n t e r p r e t e d a s recommending flavonoid consumption in the prevention or treatment of cardiovascular disease, nevertheless the beneficial effects of f l a v o n o i d c o n s u m p t i o n a r e a p p a r e n t . furthermore, the results of these studies are in accord with the american heart association recommendation for increased consumption of vegetables and fruits, including plant-derived b e v e r a g e s , s u c h a s t e a , f o r p r e v e n t i o n o f cardiovascular disease. references 1. american heart association. heart and stroke statistical update. dallas: american heart association, 2000. 2. expert panel. executive summary of the third report of the national cholesterol education program (ncep). expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii). jama 2001; 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275: 399406. 22. hodgson jm, puddey ib, burke v, watts gf, beilin lj. regular ingestion of black tea improves brachial artery vasodilator function. clin sci 2002; 102: 195-201. 23. folts d. potential health benefits from the flavonoids in grape products on vascular disease. adv exp med bio 2002; 505: 95-111. 24. wang z, zou j, cao k, hsieh tc, huang y, wu jm. dealcoholized red wine containing known amounts of resveratrol suppresses atherosclerosis in hypercholesterolemic rabbits without affecting plasma lipid level. int j mol med 2005; 16: 53340. 25. guarda e, godoy i, foncea r, perez dd, romero c, venegas r, et al. red wine reduces oxidative stress in patients with acute coronary syndrome. int j cardiol 2005; 104: 35-8. 26. perez-vizcaino f, duerte j, andriantsitohaina r. endothelial function and cardiovascular disease: effects of quercetin and wine polyphenols. free radic res 2006; 40: 1054-65. 27. heiss c, dejam a, kleinbongard p, schewe t, sies h, kelm m. vascular effects of cocoa rich in flavan3-ols. jama 2003; 290: 1030-1. 28. wang-polagruto jf, villablanca ac, polagruto ja, lee l, holt rr, schrader hr, et al. chronic consumption of flavanol-rich cocoa improves endothelial function and decreases vascular cell adhesion molecules in hypercholesterolemic postmenopausal women. j cardiovasc pharmacol 2006; 46: 177-86. 29. awtry eh, loscalzp j. aspirin. circulation 2000; 101: 1206-18. 30. freedman je, parker iii c, li l, perlman ja, frei b, ivanav v, et al. selected flavonoids and whole juice from purple grapes inhibit platelet function and enhance nitric oxide release. circulation 2001; 103: 2792-8. 31. duffy sj, vita ja, holbrook m, swerdloff pl, keaney jf jr. effect of acute and chronic tea consumption on platelet aggregation in patients with coronary artery disease. arterioscler thromb vasc biol 2001; 21: 1084-9. 32. hodgson jm, puddey jb, mori ta, burke v, barker ri, beilin lj. effects of regular ingestion of black tea on haemostasis and cell adhesion molecules in humans. eur j clin nutr 2001; 55: 881-6. fajar 125 *department of histology, medical faculty, trisakti university correspondence dr. fajar arifin gunawijaya, ms department of histology, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2701 univ med 2008; 27: 125-31 the role of tea in cardiovascular disease july-september, 2008july-september, 2008july-september, 2008july-september, 2008july-september, 2008 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 abstract universa medicina fajar arifin gunawijaya* tea is an infusion of the leaves of the camellia sinensis plant, which is not to be confused with so-called ‘herbal’ teas. some biologically active chemicals in tea include flavonoids, caffeine and fluoride. for as long as people have been drinking tea, there have been health benefits associated with it. however, it has only been within the past 20 to 30 years that scientific studies have been conducted to investigate the validity of these claims. many animal studies, and recently an increasing number of human studies, including epidemiological studies and clinical trials, have examined the relationship between tea drinking and health. overall, observational studies in humans suggest that daily consumption of at least 3 cups of tea may be associated with a risk reduction of mortality (44%) after myocardial infarction compared with non-tea drinkers. the possibility that tea consumption reduces the risk of cardiovascular events remains open to the need for further clinical trials to clarify the effects of tea polyphenols in humans in order to recommend their use against cardiovascular diseases. keywords : tea, coronary heart disease, endothelium introduction the indonesian national health survey 2001 showed that the main cause of death a m o n g t h e i n d o n e s i a n p o p u l a t i o n w a s cardiovascular disease (diseases of the heart and great vessels), amounting to 26.3% of all deaths. when compared with the mortality rates due to cardiovascular disease in 1975, 1986 and 1995 of 5.9%, 9.1%, and 19%, respectively, these values indicate an increasing trend in the mortality rate of cardiovascular disease. (1) epidemiological observations and clinical trials have established an association between regular t e a c o n s u m p t i o n a n d h e a l t h , p a r t i c u l a r l y p r e v e n t i o n o f c a r d i o v a s c u l a r d i s e a s e a n d stroke.(2,3) d i e t a r y f a c t o r s m a y b o t h r e d u c e a n d contribute to risk of cardiovascular disease (cvd) in a number of ways. antioxidants, such as vitamin c, vitamin e and a range of other plant compounds, such as the flavonoids, are considered beneficial.(4) it is widely known that t h e i n c i d e n c e o f c o r o n a r y e v e n t s ( d e a t h definitely or probably due to coronary heart disease or non-fatal myocardial infarction) is 126 gunawijaya tea in cardiovascular substantially lower in the japanese than in western populations, although this is still open to question.(5) in japan, as well as in other asian countries, tea consumption is very high, and green tea, in particular, is favored by the japanese. chemical composition of tea tea is an infusion of the leaves of the tea plant (camellia sinensis l.) and its consumption worldwide is second only to that of water. the t e a p l a n t i s f o u n d i n 3 0 c o u n t r i e s , b u t i t originally came from china, where it was cultivated about 4000 years ago. however, recent archeological evidence has revealed that the plant was already utilized about 500,000 years ago.(6) although the belief in the beneficial qualities of tea had been closely associated with this beverage since its discovery, scientific studies on tea and the substances contained therein were initiated only around 30 years ago. there are three kinds of tea, namely green tea, black tea and oolong tea, which differ in the processing methods used in the curing of the tea leaves. green tea is prepared from unfermented tea leaves and has been demonstrated to have the highest catechin content. black tea undergoes a fermentation process, such that most of its catechins are converted into theaflavins and thearubigins. on the other hand, oolong tea is processed by partial fermentation. black tea is mainly consumed in europe, north america and north africa (except marocco), whereas green tea is consumed in asia and oolong tea is renowned in china and taiwan. most of the tea produced is black tea, amounting to 76-78%, which is consumed throughout the world. production of green tea is around 20-22% of the global tea production, whereas oolong tea production is only 2%.(7) tea contains a number of polyphenols, particularly flavonoids. the classification of polyphenols and the origins of the chemical substances contained in the tea plant are listed in table 1.(8) flavonoids are phenol derivatives that are found in a variety of plants and exhibit an enormous diversity, as more than 40,000 kinds have been recognized.(9) the principal flavonoids present in green tea are the catechins, such as epicatechin (ec), epicatechin-3-gallate (ecg), epigallocatechin (egc) and epigallocatechin-3gallate (egcg). the catechins of black tea undergo polymerization (fermentation/enzymatic oxidation) to form mainly theaflavins and thearubigins. the amount of catechin found in tea depends on the curing process, geographic location and cultivation methods.(8) epidemiological studies mukamal kj, et al. carried out a cohort s t u d y o n p a t i e n t s w i t h a c u t e m y o c a r d i a l infarction, who were asked about their tea drinking habits in the last year before the occurrence of the attack, and subsequently observed for two to four years. those patients who consumed less than 14 cups per week (moderate tea drinkers) had a hazard ratio of 0.69 (95% ci 0.53 to 0.89), whereas those consuming 14 or more cups per week (heavy tea drinkers), had a hazard ratio of 0.61; 95% ci 0.42 to 0.86). both groups had a mortality lower risk when compared with persons who did not drink tea.(2) sesso and colleagues used a case-control design to examine the relation between tea and c o f f e e c o n s u m p t i o n a n d m y c o c a r d i a l infarction.(9) they examined tea and coffee intake by questionnaire in 340 subjects and 340 matched controls from the boston health study and observed a 44% reduction in cardiovascular risk in individuals drinking more than one cup of tea per day. no significant relationship was f o u n d b e t w e e n c o f f e e c o n s u m p t i o n a n d cardiovascular disease. 127 a d u t c h s t u d y d e m o n s t r a t e d t h a t t e a drinking may reduce the risk of fatal heart attacks. the sudy was performed on 4,807 men and women aged 55 years and older, who had suffered no heart attacks during the past five years or more. persons drinking more than 375 ml of tea daily had a lowered risk of myocardial i n f a r c t i o n , i n c o m p a r i s o n w i t h n o n t e a drinkers.(10) in contrast, a 7.7 year follow-up on participants of the scottish heart study showed n o r e l a t i o n s h i p b e t w e e n t e a d r i n k i n g a n d cardiovascular disease.(11) peters et al. have recently pooled data from the usa and europe into a meta-analysis and reported a protective effect of tea on myocardial infarction (11% per three cups per day).(12) this analysis specifically excluded data from the uk and thus has not clarified t h e i s s u e . m e t a a n a l y s i s i s a p o w e r f u l s t a t i s t i c a l t o o l , b u t i t c a n n o t a c c o u n t f o r exclusion of major confounding factors, such as dietary intake of other antioxidants (eg. vitamin c and e), or social factors. crossculturally this is a major drawback, since the social patterns of tea consumption and its relationship to cvd in the uk are the opposite of those in other countries. even controlling for a confounding factor, such as social class, table 1. classification of polyphenols and origin of chemicals in tea plants(8) category classes major food sources phenolic acids ferulic acid caffeic acid: chlorogenis acid condensed tannins hydrolyzable tannins: gallotannins: ellagitannins dietary fibre – hemicelluloses many fruits and vegetables, coffee mango fruit blackberries, raspberries, strawberries, wine, brandy aged in oak barrels flavonoids flavones flavonols: quercetin flavanols: catechins : epicatechin (ec) epicatechin-3-gallate (ecg) epigallocatechin (egc) epigallocatechin-3-gallate (egcg) flavanones : hesperetin isoflavones : genistein daidzein anthocyanins : cyanidin proanthocyanidins sweet red pepper, celery tea, onions, apples, many other fruits and vegetables tea, especially green tea, chocolate, cocoa oranges, citrus fruits soybeans, soy protein-containing foods red fruits: cherries, plums, strawberries, raspberries, blackberries, grapes, red and black currants apples, pears, grapes, red wine, tea lignans enterodiol flaxseed, flaxeed oil stilbenes resveratrol red wine univ med vol.27 no.3 128 gunawijaya tea in cardiovascular is often not adequate to remove the whole effect of interest.(13) d r e g e r e t a l . h a v e d e m o n s t r a t e d t h e protective effect of egcg and theaflavin-3,3’digallate (tf3) against oxidative stress on rat cardiac muscle, when the substances were administered one hour before induction of oxidative stress by means of hydrogen peroxide. thus green tea and black tea extracts, containing egcg and tf3 respectively, may protect the heart muscle against oxidative stress.(14) several possible mechanisms could explain an association between tea consumption and survival among patients with acute myocardial infarction. a recent randomized trial found that a c u t e a n d c h r o n i c b l a c k t e a c o n s u m p t i o n improved endothelial function in patients with coronary heart disease in an additive fashion.(15) this provides a suggestive mechanism for a beneficial impact of tea intake on survivors of acute myocardial infarction, given the adverse prognosis associated with coronary endothelial dysfunction in patients with coronary heart d i s e a s e . ( 1 6 ) f l a v o n o i d s a l s o i n h i b i t l d l oxidation, perhaps by reducing macrophage superoxide production.(17) oxidized ldl may promote atheroma formation by increasing macrophage uptake, monocyte recruitment, and direct endothelial cell damage, thus antioxidant use may prevent myocardial infarction, at least in some patients.(2) platelet aggregation is a precipitating event in cardiovascular disease, and tea contains antioxidant flavonoids that are known to decrease platelet aggregation in vitro. plasma flavonoids increase with acute and chronic tea consumption, indicating adequate absorption of tea flavonoids. a randomized cross-over design demonstrated that acute and chronic black tea consumption does not affect ex vivo platelet aggregation in patients with coronary artery disease. this finding suggests that an effect of tea flavonoids on platelet aggregation is unlikely to be the explanation for the reduction in risk of cardiovascular events noted in epidemiological studies.(18) antioxidant capacity of tea experimentally it has been shown that tea flavonoids may increase the number of gapjunctions, stimulate b cell proliferation, and inhibit cytochrome p430 enzymes in hepatic cells in vitro. ( 19) b y oxy g e n ra d i c a l ab s o r b a n c e capacity (orac) assessment it was found that both green and black tea had a higher antioxidant capacity against peroxyl radicals than vegetables such as garlic, kale, spinach and brussels sprout. additionally, by determination of ferric reducing antioxidant power (frap) it turned out that green tea had a higher antioxidant capacity.(20) the in-vitro antioxidant capacity of tea depends on the test used and does not reflect factors such as bioavailability and metabolism. thus, for determining the benefits of tea, an invivo antioxidant capacity test would be more appropriate. recently, several clinical trials have shown that drinking tea increases plasma antioxidant levels in adults after 30 to 60 minutes.(21) the statistically significant increase in plasma antioxidant levels (p<0.001) was determined by t h e f r a p a s s a y, a f t e r t h e s u b j e c t s h a d consumed 300 ml of tea infusion (prepared from 20 g tea leaves in 500 ml water) or 2 g of green or black tea extract (equivalent to 3 cups). (21,22) on average, plasma antioxidant levels were raised about one to two hours after d r i n k i n g t e a a n d s u b s e q u e n t l y d e c r e a s e d rapidly.(7) drinking tea or tea extract repeatedly within one to four weeks appeared to decrease biomarkers of oxidative stress. a study on 40 c h i n e s e m a l e c i g a r e t t e s m o k e r s a n d 2 7 american males and females in the usa, found that drinking 6 cups of green tea daily for one week reduced the levels of dna oxidation damage, lipid peroxidation, and free radical 129 synthesis.(23) similarly, among ten patients with type 2 diabetes who consumed high doses of flavonoids for two weeks, including 6 cups of black tea, there was a reduction in oxidative stress against lymphocyte dna. (24) plasma malondialdehyde level, another indicator of lipid peroxidation, was reduced in 20 healthy women aged 23 to 50 years, who ingested capsules containing linoleic acid and tea extract (equivalent to 10 cups of green tea). however, administration of placebo did not change the concentration of 8-isoprostaglandin f2α and oxidized glutathione in blood.(25) the results of the latter study are somewhat misleading, b e c a u s e s o m e i n d i v i d u a l s i n b o t h t h e intervention and the control groups had also consumed black tea up to 560 ml per day. tea and endothelial cell function i t i s n o w g e n e r a l l y a c c e p t e d t h a t endothelial dysfunction plays a role in the early d e v e l o p m e n t o f a t h e r o s c l e r o s i s . ( 1 6 , 2 6) t h e endothelium is made up of squamous cells l i n i n g t h e b l o o d v e s s e l s , a n d p r o d u c e s vasoactive factors, such as nitric oxide (no) and prostacyclins. these substances are active i n v a s o d i l a t i o n d a n p l a t e l e t i n h i b i t i o n . i m p a i r m e n t o f n i t r i c o x i d e a c t i v i t y o f e n d o t h e l i a l c e l l s m a y c o n t r i b u t e t o t h e pathogenesis of atherosclerosis and disorders of coronary blood flow, which subsequenly may cause coronary heart disease.(27) recent research findings indicate that black tea may reduce the risk of heart disease b y c o n t r i b u t i n g t o t h e m a i n t e n a n c e o f endothelial cell functions. presumably there are chemical substances in tea that enhance the dilatory capacity of the arteries. a study by sudjarwo sa on 30 rabbits has proved that catechin administration may decrease plasma m a l o n d i a l d e h y d e p r o d u c t i o n a n d e n h a n c e acetylcholine-induced endothelium-dependent r e l a x a t i o n o f t h e a o r t a i n r a b b i t s o n a cholesterol-rich diet.(28) in a cross-sectional study by duffy sj et al., freshly infused black tea and plain water were randomly assigned to subjects, in whom brachial artery dilation was monitored using doppler ultrasound. both short-term (2 hours after drinking 450 ml black tea) and long-term ( 9 0 0 m l b l a c k t e a d a i l y f o r 4 w e e k s ) consumption may enhance vasodilation (p < 0.001). this phenomenon is associated with increased plasma levels of tea polyphenols. on a d m i n i s t r a t i o n o f 2 0 0 m g c a f f e i n e o r n i t r o g l y c e r i n n o v a s o d i l a t o r y e f f e c t w a s found.(15) n a g a y a n e t a l . a l s o r e p o r t e d t h a t administration of 400 ml green tea to cigarette smokers may increase the blood circulation in the arms.(29) it is a well-known fact that free r a d i c a l s i n c i g a r e t t e s m o k e m a y i m p a i r endothelial function. therefore further research is needed to prove that antioxidants in green tea can lower the risk of cardiovascular events. vasodilation in patients with coronary heart disease is impaired, in comparison with healthy individuals, thus this study proved that tea can enhance endothelial cell function of the blood vessels. te a d r i n k i n g i s i n v e r s e l y r e l a t e d t o atherosclerosis formation and progressivity. in the rotterdam study involving 3,454 adults aged 55 years or older, who were followed-up for two to three years, geleijnse et al. reported on radiological assessment of aortic atherosclerosis by measuring calcification in the abdominal aorta. the results showed that individuals drinking 1-2 cups of tea per day had an odds ratio of 0.54, which was reduced to 0.31 in those drinking 4 cups or more.(10) sasazuki et al. a s s e s s e d a t h e r o s c l e r o s i s b y c o r o n a r y arteriography in 512 japanese over the age of 3 0 y e a r s , a n d r e p o r t e d t h a t t e a p r o v i d e s protection against atherosclerosis.(30) univ med vol.27 no.3 130 gunawijaya tea in cardiovascular debette s et al. observed 6,597 french men and women aged >65 years, and reported that t h e f r e q u e n c y o f p l a q u e f o r m a t i o n i n t h e common carotid artery was in 44% women who did not drink tea, 42.5% in those drinking 1–2 c u p s o f t e a p e r d a y, a n d 3 3 . 7 % i n t h o s e consuming >3 cups daily. thus there was a reduction in the frequency of common carotid artery plaque formation when drinking >3 cups of tea daily. (p<0,0001). in this study, tea consumption in men had no effect on common carotid artery plaque formation.(31) drinking 5-6 cups of green tea daily or 200-300 mg egcg, as reported by wolfram s, may maintain cardiac health and metabolic f u n c t i o n i n g o f t h e b o d y. ( 32) t h e s e s t u d i e s indicate that tea antioxidants may help prevent atherosclerosis, particularly coronary heart disease. conclusions te a i s a e x c e l l e n t s o u r c e o f d i e t a r y a n t i o x i d a n t f l a v o n o i d s , w h o s e a c t i o n s o n endothelial function may help explain the cardioprotective effects of tea drinking that has been observed in several countries. both green and black tea have strong in-vitro and in-vivo antioxidant capacities. tea plays a role in lowering the risk of cardiovascular disease and stroke. tea is a drink of global renown, thus f u r t h e r r e s e a r c h s h o u l d b e o f a p p r o p r i a t e design, by measuring total daily consumption of tea and its polyphenol content, with an emphasis on its role in primary and secondary prevention of cardiovascular disease. references 1. department of health republic of indonesia. laporan studi mortalitas 2001: pola penyakit penyebab kematian di indonesia. jakarta: national institute of health, research and development; 2002. 2. mukamal kj, maclure m, muller je, sherwood jb, mittleman ma. tea consumption and mortality after acute myocardial infarct. circulation 2002; 105: 2474-79. 3. weisburger jh. tea and health; the underlying mechanism. proc soc exp biol 1999; 220: 271-5. 4. nijveldt rj, van nood e, van hoorn de, boelens pg, van norren k, van leeuwen pa. flavonoids: areview o probable mechanismsof action and potential applications. am j clin nutr 2001; 74: 418-25. 5. egusa g, yamane k. lifestyle, serum lipids and coronary artery disease: comparison of japan with the united state. j atheroscler thromb 2004; 11: 304-12. 6. green tea. available at: http://www.umm.edu/ altmed/articles/green-tea-000255.htm. accessed may 26, 2007. 7. mckay dl, blumberg jb. the role of tea in human health: an update. j am coll nutr 2002; 21: 1-13. 8. vinson j, dabbagh y, serry m, jang j. plant flavonoids, especially tea flavonols, are powerful antioxidant using an in vitro oxidation model for heart disease. j agri food chem 1995; 43: 28002. 9. sesso h, gaziano j, buring j, hennekens c. coffee and tea intake and the risk of myocardial infarction. am j epidemiol 1999; 149: 162-7. 10. geleijnse jm, launer lj, van der kuip dam, hofman a, witteman jcm. inverse association of tea and flavonoid intakes with incident myocardial infarction, the rotterdam study. am j clin nut 2002; 75: 880-6. 11. woodward m, tunstall-pedoe h. coffee and tea consumption in the scottish heart health study follow up: conflicting relations with coronary risk factors, coronary disease and all cause mortality. j epidemiol commun health 1999; 53: 481-7. 12. peters u, poole c, arab l. does tea affect cardiovascular disease? a meta-analysis. am j epidemiol 2001; 154: 495-503. 13. poulter j, bolton-smith c, rietveld a. is there any evidence that tea drinking impacts on cardiovascular health in the uk? br j cardiol 2003; 10: 281-6. 14. dreger h, lorenz m, kehrer a, baumann g, stangl k, stangl v. characteristics of catechin and theaflavin mediated cardioprotection. experimental biology and medicine 2008; 233: 427-33. 15. duffy sj, keaney jr jf, holbrook m, gokce n, swerdloff p, frei b, et al. short and long-term black 131 tea consumption reverses endothelial dysfunction in patients with coronary artery disease. circulation 2001; 104: 151-6. 16. schachinger v, britten mb, zeiher am. prognostic impact of coronary vasodilator dysunction on adverse long-term outcome of coronary heart disease. circulation 2000; 101: 1899-906. 17. yoshida h, ishikawa t, hosoai h, michio suzukawa, makoto ayaori, tetsuya hisada, et al. inhibitory effect of tea flavonoids on the ability of cells to oxidize low density lipoprotein. biochem pharmacol 1999; 58: 1695-703. 18. duffy sj, vita ja, holbrook m, swerdloff pl, keaney jr jf. effect of acute and chronic tea consumption on platelet aggregation in patients with coronary artery disease. arterioscler hromb vasc biol 2001; 21: 1084-9. 19. yang c, chung j, yang g, chhabra s, lee m. tea and tea polyphenols in cancer prevention. j nut 2000; 130: 472s-478s. 20. langley-evans sc. antioxidant potential of green and black tea determined using the ferric reducing antioxidant power (frap) assay. int j food sci nutr 2000; 51: 181-8. 21. leenen r, roodenburg a, tijburg l, wiseman s. a single dose of tea with or without milk increases plasma antioxidant activity in humans. eur j clin nutr 2000; 54: 87-92. 22. langley-evans sc. consumption of black tea elicits an increase in plasma antioxidant potential in humans. int j food sci nutr 2000; 51: 309-15. 23. klaunig j, xu y, han c, kamendulis l, chen j, heiser c, et al. the effect of tea consumption on oxidative stress in smokers and non smokers. proc soc exp biol med 1999; 220: 249-54. 24. lean m, noroozi m, kelly i, burns j, talwar d, sattar n, et al. dietary flavanols protect diabetic human lymphocytes against oxidative damage to dna. diabetes 1999; 48: 176-81. 25. freese r, basu s, hietanen e, nair j, nakachi k, bartsch h, et al. green tea extract decreases plasma malondialdehyde concentration but does not affect other indicators of oxidative stress, nitric oxide production, or hemostatic factors during a highlinoleic acid diet in healthy females. eur j nutr 1999; 38:149-57. 26. zhang x, zhao sp, li xp, gao m, zhou qc. endothelium-dependent and independent functions are impaired in patients with coronary heart disease. atherosclerosis 2000; 149: 19-24. 27. sargowo d. peran endotel pada patogenesis penyakit kardiovaskular dan program pencegahannya. medika 1999; 10: 643-54. 28. sudjarwo sa. protective effect of catechin on endothelial cell in hypercholesterolemia. j kedokt trisakti 2004; 23: 1-5. 29. nagaya n, yamamoto h, uematsu m, itoh t, nakagawa k, miyazawa t, et al. green tea reverses endothelial dysfunction in healthy smokers. heart 2004; 90: 1485-6. 30. sasazuki s, kodama h, yoshimasu k, liu y, washio m, tanaka k, et al. relation between green tea consumption and the severity of coronary atherosclerosis among japanese men and women. ann epidemiol 2000; 10: 401-8. 31. debette s, courbon d, leone n, gariepy j, tzourio c, dartigues jf, et al. tea consumption is inversely associated with carotid plaques in women. arteriosclerosis, thrombosis and vascular. biology 2008; 28: 353-59. 32. wolfram s. effects of green tea and egcg on cardiovascular and metabolic health. j am coll nutr 2007; 26: 373s-388s. univ med vol.27 no.3 rizaldy1 1 risk factors of intracranial stenosis among older adults with acute ischemic stroke january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina rizaldy pinzon*, laksmi asanti*, sugianto*, and kriswanto widyo* *neurology department, bethesda hospital, yogyakarta correspondence adr. rizaldy pinzon neurology department, bethesda hospital, yogyakarta email: medidoc2002@yahoo.com univ med 2009; 28: 1-7 abstract intracranial large artery atherosclerosis is an important cause of stroke worldwide. previous studies have shown that it is found more commonly in asians. however, studies of intracranial stenosis in indonesian stroke patients have been very few in number. the aim of this cross-sectional study was to determine the frequency and risk factors of intracranial stenosis in acute ischemic stroke. the data were obtained from 234 consecutive patients in the transcranial doppler (tcd) registry. documentation of risk factors was performed systematically and for tcd sonography td-dop 9000 equipment with a 2-mhz probe was used for the examination of the intracranial circulation. the criteria of middle cerebral artery (mca) stenosis used in this study were a peak systolic velocity (psv) >140 cm/s or mean systolic velocity (msv) >80 cm/s. for stenosis of the posterior circulation the criteria were psv > 90 cm/s or msv >60 cm/s. the data were obtained from 234 patients, and complete examinations were performed in 182 patients (77.7%). ischemic stroke is the most common indication for performing tcd sonography. stenosis was present in 38% of cases, with stenosis iof the anterior circulation being the more common. the most frequent risk factors were hypertension, dyslipidemia, and diabetes. intracranial stenosis is common in stroke patients, in whom the risk factors are hypertension and diabetes. keywords: stenosis, intracranial, risk factor, acute ischemic stroke introduction stroke is the leading cause of mortality and d i s a b i l i t y, a n d m a y b e c a u s e d b y e i t h e r modifiable or non-modifiable risk factors. it is the third leading cause of death and disability among adults. the incidence of stroke has continued to increase since the mid-1960s, with up to 700,000 new cases being reported in the united states each year.(1,2) intracranial arterial stenosis is a significant risk factor for vascular events, having been implicated as a causal factor in 8% to 10% of all ischemic strokes(3) and as a major factor in recurrent stroke and 2 pinzon, asanti, sugianto, et al acute ischemic stroke vascular mortality.(4,5) an important cause of intracranial arterial stenosis is intracranial a t h e r o s c l e r o s i s . ( 6 , 7 ) a t h e r o s c l e r o s i s i s a systemic, multifocal disease with various signs a n d s y m p t o m s , c a u s e d b y a t h e r o s c l e r o t i c lesions in the arterial tree. in the brain the lesions may be located in the extracranial carotid or intracranial arteries.(8-10) effective preventive measures are needed for preventing stroke and its recurrence.(6,11) symptomatic atherosclerotic intracranial stenosis accounts for approximately 10% of ischemic strokes. risk of stroke in patients with middle cerebral artery (mca) stenoses was reported to be around 24% during a 6-year follow-up study. the cerebral hemodynamic study and its alterations may be a good indicator for long-term outcome.(12) previous studies have caused some controversies about the risk factors of intracranial stenosis. (8,9) increasing lood pressure, increasing blood cholesterol, and atrial fibrillation are definite causal risk factors f o r i s c h e m i c s t r o k e b e c a u s e r a n d o m i z e d c o n t r o l l e d t r i a l s ( r c ts ) h a v e s h o w n t h a t treating them reduces the incidence of ischemic s t r o k e . ( 1 3 1 5 ) c i g a r e t t e s m o k i n g , d i a b e t e s mellitus, ischemic heart disease, and valvular heart disease are probably also causal risk f a c t o r s f o r i s c h e m i c s t r o k e b e c a u s e epidemiological case-control and cohort studies h a v e s h o w n t h a t t h e s e c h a r a c t e r i s t i c s a r e significantly associated with an increased risk of stroke; moreover, the association is strong, c o n s i s t e n t a m o n g s t u d i e s , b i o l o g i c a l l y plausible, and independent of other factors that were measured and analyzed. however, it is possible that these associations are confounded by other factors that have not been measured a n d a n a l y z e d i n e p i d e m i o l o g i c a l s t u d i e s uncontrolled confounding. (16-17) a study in s u r a b a y a s h o w e d t h a t o u t o f t h e 6 7 5 intracranial arterial segments examined, 118 (17%) segments had stenosis of more than 29% o r h a d o c c l u s i o n o n d i g i t a l s u b t r a c t i o n angiography (dsa).(18) there are only a few studies about intracranial stenosis in indonesia. the aim of this study was to determine the frequency and risk factors of intracranial stenosis in acute ischemic stroke. methods research design in this cross-sectional study, we examined 2 3 4 c o n s e c u t i v e p a t i e n t s u n d e r g o i n g transcranial doppler (tcd) studies. further evaluation was performed for acute stroke p a t i e n t s w i t h s y m p t o m s o f o b s t r u c t i o n i n anterior or posterior cerebral artery territories. patients with a tcd diagnosis of intracranial stenosis were further evaluated. data collecting the recorded data consisted of demographic characteristics, risk factors and tests results. hypertension was defined as a history of elevated blood pressure (systolic blood pressure >140 mmhg or diastolic blood pressure >90 mmhg) in two separate recordings or elevated blood pressure before admission that required antihypertensive medications. diabetes mellitus was defined as a history of elevated blood glucose in two separate tests (fasting level >125 mg/dl) or diabetes treated with medication before admission. hyperlipidemia was defined as history of an abnormally high lipid level that r e q u i r e d e i t h e r d i e t a r y o r p h a r m a c o l o g i c intervention. smoking was documented by quantity-frequency assessment at the time of admission. measurements tcd sonography is a non-invasive, noni o n i z i n g , i n e x p e n s i v e , p o r t a b l e a n d s a f e technique that uses a pulsed doppler transducer for assessment of intracerebral blood flow. the 3 purpose of this test is to detect any narrowing or blockage in these arteries that may decrease or stop the flow of blood to the brain. a small hand-held, wand-like probe covered with a conductive gel will be placed and held at various areas on the head of the subject, usually at each temple, over each eye, and at the base of the skull. this enables the examiner to image the blood flow in the arteries and to record the flow in each artery. the procedure should take 20 to 60 minutes and is painless.(19,20) tcd studies of intracranial vessels were performed by tdop-tc9000p unit with a 2mhz probe for the examination of intracranial circulation. mca was designated as stenotic if its peak systolic velocity was greater than 140 cm/s or its mean velocity exceeded 80 cm/s.(21) a diagnosis of vertebral segment and proximal basilar artery stenosis was made if there were focal increases of the peak systolic and mean velocities to 90 cm/s and 60 cm/s or more.(22) data analysis we used spss to save and analyze the data. independent t-test and chi square test were used to compare the variables. results we a n a l y z e d t h e t c d r e s u l t s o f 2 3 4 patients from our tcd registry, with mean age of 58.35 years. complete data were collected for 182 patients (77.7%). the most common reason for incomplete data was poor temporal window and bedridden-ventilated patients. the study sample consisted of 52% males and 48% females. the most common indication for tcd e x a m i n a t i o n s i n t h i s s t u d y w a s n o n hemorrhagic stroke, which was found in around 5 8 % o f t h e s t u d y s a m p l e ( 1 3 6 p a t i e n t s ) , f o l l o w e d b y v e r t e b r o b a s i l a r i n s u ff i c i e n c y (figure 1). the results of the tcd studies in ischemic stroke patients is presented in figure 2. the proportions of abnormalities detected were as follows: stenosis (38%), atherosclerosis (30%), and hypoperfusion (21%). figure 3 shows stenosis in the right middle cerebral artery. in comparison to the posterior circulation, stenosis in the anterior circulation is more common (figure 4), and the most frequent sites are the middle cerebral artery and the internal carotid artery siphon. univ med vol.28 no.1 figure 1. indications for tcd studies 4 pinzon, asanti, sugianto, et al acute ischemic stroke figure 4. distribution of sites of the stenosis in patients with intracranial stenosis (52 patients) figure 3. stenosis in the right middle cerebral artery figure 2. distribution of abnormalities of tcd studies in patients with acute ischemic stroke 5 we analysed the distribution risk factors in patients with intracranial stenosis, and compared it with ischemic stroke patients without stenosis. the most frequent risk factor for atherosclerosis was hypertension with a frequency of 72%, whilst diabetes mellitus, hyperlipidemia, and smoking had frequencies of 24%, 38%, and 22% respectively. table 1 presents the frequency of vascular risk factors in intracranial stenosis patients. t h e r e w a s a s i g n i f i c a n t r e l a t i o n s h i p b e t w e e n h y p e r t e n s i o n a n d d i a b e t e s w i t h intracranial stenosis. both hypertension and diabetes increase the risk of stenosis (or=2.97, 95% ci=1.76-4.98, p<0.05 for hypertension, and or=1.48, 95% ci=1.12-2.43, p<0.05 for diabetes). discussion t h e f i n d i n g s o f t h i s s t u d y a r e i n agreement with those of previous studies, s u g g e s t i n g t h a t a t h e r o s c l e r o t i c o c c l u s i v e lesions are common in asians. (23-25) in our studies intracranial lesions appeared to be the cause of atherosclerotic occlusive disease in more than around 30% cases. however, this figure may be an overestimation because we excluded patients with poor temporal windows (mostly elderly women), and patients who were moribund with severe stroke (commonly from cardioembolic stroke). multiple intracranial stenoses were found frequently, a finding that is in agreement with some other studies.(26,27) our data showed that m c a w a s t h e m o s t f r e q u e n t s t e n o t i c intracranial artery. despite some previous suggestions, we found no statistical difference i n t h e f r e q u e n c y o f i n t r a c r a n i a l l e s i o n s between men and women. however, due to the better trans-temporal windows, we suggest that tcd is a better device for studying intracranial arteries in men. in contrast, results from the warfarin-aspirin symptomatic intracranial disease (wasid) trial showed that women have a higher risk for recurrent ischemic stroke and for the combined end point of stroke and vascular death. (28) the explanation for this finding is complex. women have a greater clustering of risk factors that are indicative of metabolic abnormalities (e.g. high body mass indexes, hypertension, and diabetes) and those t h a t p o r t e n d i n c r e a s e d r i s k b a s e d o n s o c i o d e m o g r a p h i c f e a t u r e s , l i f e s t y l e , a n d family history of stroke. women may have smaller intracranial arteries than men, which could pose a greater risk for stroke in the territory of a stenotic intracranial artery.(29) an i n t e r e s t i n g f i n d i n g i n o u r s t u d y i s t h a t i n t r a c r a n i a l s t e n o s i s i s m o r e f r e q u e n t l y table 1. frequency of vascular risk factors in stroke patients with intracranial stenosis (n = 52) table 2. the risk factors of intracranial stenosis in acute ischemic stroke univ med vol.28 no.1 6 pinzon, asanti, sugianto, et al acute ischemic stroke detected in patients with a history of either diabetes mellitus or hypertension. this finding is in agreement with some studies,(12,30) and in d i s a g r e e m e n t w i t h s o m e o t h e r s . ( 8 , 3 1 ) t h e wa s i d t r i a l i n n o r t h a m e r i c a s h o w e d consistent results, in which elevated mean sbp and dbp were associated with increased risk of stroke after adjustment for a number of factors. these were age, type of qualifying event (transient ischemic attack versus stroke), gender, body mass index, time from qualifying event, history of diabetes mellitus, history of hypertension, race, percentage of intracranial a r t e r y s t e n o s i s a t s t u d y e n t r y, a n d h y p e r l i p i d e m i a . ( 3 2 ) a l t h o u g h w e f o u n d n o association between the site of the lesion and a history of known lipid disorder, this finding should be interpreted with caution, as blood lipids and lipoprotein concentrations were not systematically measured in our study. because we can now detect intracranial occlusive disease safely and reliably with tcd and magnetic resonance angiography (mra), studies of the prevalence of intracranial stenosis in asymptomatic patients with multiple risk factors is feasible.(33) identifying patients with asymptomatic intracranial stenosis may enable us to implement preventive measures early in this high-risk population. this is particularly i m p o r t a n t b e c a u s e l i m i t e d s u c c e s s o f pharmacological interventions in preventing stroke has now been replaced by technological developments offering improved methods for e n d o v a s c u l a r t h e r a p y o f c e r e b r a l a r t e r y stenosis. conclusions intracranial stenosis is present in more than one third of ischemic stroke patients. hypertension and diabetes are significantly more prevalent in intracranial stenosis. acknowledgement the authors would like to express their appreciation to associate professor tan kay sin for the constructive comments and guidance during the asna biennial meeting in kuala lumpur 2009. references 1. quilliam bj, lapane kl. clinical correlates and drug treatment of residents with stroke in long-term care. stroke 2001; 32: 1385–93. 2. american heart association. heart disease and stroke statistics—2003 update. available at: http:/ /www.americanheart.org. accessed sept 1, 2008. 3. the warfarin aspirin symptomatic intracranial disease (wasid) trial investigators. design, progress and challenges of a double-blind trial of warfarin versus aspirin for symptomatic intracranial arterial stenosis. neuroepidemiology 2003; 22: 106 –17. 4. klijn cjm, kappelle lj, algra a, van gijn j. outcome in patients with symptomatic occlusion of the internal carotid artery or intracranial arterial lesions: a meta-analysis of the role of baseline characteristics and type of antithrombotic treatment. cerebrovasc dis 2001; 12: 228–34. 5. wong ks, li h. long-term mortality and recurrent stroke risk among chinese stroke patients with predominant intracranial atherosclerosis. stroke 2003; 34: 2361–6. 6. bang oy. intracranial atherosclerotic stroke: specific focus on the metabolic syndrome and inflammation. curr atheroscler rep 2006; 8: 3306. 7. elmore em, mosquera a, weinberger j. the prevalence of asymptomatic intracranial largevessel occlusive disease: the role of diabetes. j neuroimaging 2003; 13: 224-7. 8. suwanwela nc, chutinet a, phanthumchinda k. inflammatory markers and conventional atherosclerotic risk factors in acute ischemic stroke: comparative study between vascular disease subtypes. j med assoc thai 2006; 89: 2021-7. 9. shin dh, lee ph, bang oy. mechanisms of recurrence in subtypes of ischemic stroke: a hospital-based follow-up study. arch neurol 2005; 62: 1232-7. 7 10. meyers pm, schumacher hc, tanji k, higashida rt, caplan lr. use of stents to treat intracranial cerebrovascular disease. annu rev med 2007; 58: 107-22. 11. aboyans v, lacroix p, criqui mh. large and small vessel atherosclerosis: similarities and differences. prog cardiovasc dis 2007; 50: 112-25. 12. hermes p. the importance of doppler studies in asymptomatic intracranial and extracranial arterial disease. keio j med 2002; 51: 189-92. 13. lawes cmm, bennett da, feigin vl, rodgers a. blood pressure and stroke: an overview of published reviews. stroke 2004; 35: 776-85. 14. cholesterol treatment trialists’ (ctt) collaborators. efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90,056 participants in 14 randomised trials of statins. lancet 2005; 366: 1267–78. 15. hankey gj. potential new risk factors for ischemic stroke. what is their potential? stroke 2006; 37: 2181-8. 16. kizer jr, wiebers do, 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alexandrov av. screening for intracranial stenosis with transcranial doppler: the accuracy of mean flow velocity thresholds. j neuroimaging 2002; 12: 9-14. 22. li h, lam wwm, wong ks. distribution of intracranial vascular lesion in the posterior circulation among chinese stroke patients. neurol asia 2002; 7: 65-9. 23. wong ks, huang yn, gao s, lam ww, chan yl, kay r. intracranial stenosis in chinese patients with acute stroke. neurology 1998; 50: 812-3. 24. hsu hy, yang fy, chao ac, chen yy, chung cp, sheng wy et al. distribution of carotid arterial lesions in chinese patients with transient monocular blindness. stroke 2006; 37: 531-3. 25. tan ty, chang kc, liou cw, schminke u. prevalence of carotid artery stenosis in taiwanese patients with one ischemic stroke. j clin ultrasound 2005; 33: 1-4. 26. lee sj, cho sj, moon hs, shon ym, lee kh, kim di. combined extracranial and intracranial atherosclerosis in korean patients. arch neurol 2003; 60: 1561-4. 27. suh dc, lee sh, kim kr, park st, lim sm, kim sj, et al. pattern of atherosclerotic carotid stenosis in korean patients with stroke: different involvement of intracranial versus extracranial vessels. am j neuroradiol 2003; 24: 239-44. 28. williams je, chimowitz mi, cotsonis ga, lynn mj, waddy sp for the wasid investigators. gender differences in outcomes among patients with symptomatic intracranial arterial stenosis. stroke 2007; 38: 2055-62. 29. liebeskind ds, laughton al, kim d, starkman s, salamon n, villablanca p, et al. vessel size explains gender differences in response to intravenous thrombolysis for acute stroke. stroke 2006; 37: 6229. 30. leung sy, ng th, yuen st, lauder ij, ho fc. pattern of cerebral atherosclerosis in hong kong chinese severity in intracranial and extracranial vessels. stroke 1993; 24: 779-86. 31. mendes i, baptista p, soares f, oliveira v, ferro jm. diabetes mellitus and intracranial stenosis. rev neurol 1999; 28: 1030-3. 32. turan tn, cotsonis g, lynn mj, chaturvedi s, chimowitz m (for the warfarin-aspirin symptomatic intracranial disease (wasid) trial investigators relationship between blood pressure and stroke recurrence in patients with intracranial arterial stenosis. circulation 2007; 115: 2969-75. 33. zarei h, ebrahimi h, shafiee k, yazdani m, aghill k. intracranial stenosis in patients with acute cerebrovascular accidents. arya atherosclerosis j 2008; 3: 206-10. univ med vol.28 no.1 alvina 11 abstract the fragile x syndrome is the most common form of inherited mental retardation in humans, caused by an expansion of the cytosine-guanine-guanine (cgg) repeat in the fragile x mental retardation 1 (fmr1) gene located on the x chromosome. antibody tests have been developed to identify fragile x patients, based on the presence or absence of fragile mental retardation protein (fmrp) in both lymphocytes and hair roots. the objective of this study was to compare correlations of hair root and lymphocyte fmrp expression with cognitive functioning in female rural area probands carrying the full mutation. thirty females (normal, premutation, or full mutation) were selected from indonesian fragile x families and were tested for fmrp expression in lymphocytes and hair roots using the fmrp antibody test. subject genotype was determined by southern blot analysis, and iq equivalent by raven’s standard progressive matrices. statistical analysis was by pearson correlation. fmrp expression in blood lymphocytes was relatively higher than that in hair roots, but hair root fmrp expression was strongly correlated with cognitive functioning in female full mutation carriers (r=0.64, p=0.015), whereas no significant correlation between lymphocyte fmrp and cognitive functioning was found (r=0.31, p= 0.281). around 14% of subjects had a normal and 7% a borderline iq level, while 79% had mild mental impairment. in conclusion, hair root fmrp expression may be a useful marker for identification of fragile x full mutation females. keywords: fragile x syndrome, full mutation females, cognitive functioning *department of biotechnology, medical faculty and health sciences, jenderal soedirman university **center for biomedical research, faculty of medicine, diponegoro university ***erasmus medical center, erasmus university correspondence dr. lantip rujito, msi.med. department of biotechnology, medical faculty and health sciences, jenderal soedirman university jl gumbreg no 1, purwokerto 53146 email: l.rujito@unsoed.ac.id univ med 2011;30:11-21 hair root fmrp expression for screening of fragile x full mutation females lantip rujito*, dwi kustiani** , lies anne severijnen***, peter hanzon***, sultana mh faradz** and rob willemsen*** january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction the fragile x syndrome (fxs) is the most prevalent inherited form of mental retardation (mr) in humans. the prevalence of this disease is approximately 1/4000 for males and 1/6000 for females in western countries.(1) in i n d o n e s i a t h e p o p u l a t i o n o f f x s i s approximately 2% of the mr population, and in central java more than 50 patients have thus far been identified.(2,3) in the same study 53% of institutionalized males and females with fragile x of an isolated village in central java could be traced back to a common ancestor.(4) 12 rujito, kustiani, severijnen, et al fragile x full mutation another study in a special school for mentally retarded individuals revealed that 3.9% of these were fxs probands.(5) the causative mutation of fxs resides in the fragile x mental retardation 1 (fmr1) gene, which is located on the long arm tip of the x chromosome. the vast majority of mutations c o m p r i s e s a m p l i f i c a t i o n o f t h e c y t o s i n e guanine guanine (cgg) repeat in the 5' noncoding region of the fmr1 gene. in normal people the cgg repeats vary from 6 to 54, while premutation carriers have between 55 and 200 repeats, and full mutation subjects have more than 200 cgg repeats in their fmr1 gene6. full mutation expansion causes hypermethylation of the fmr1 promoter and the expanded repeat itself, resulting in absence of fmr1 protein (fmrp) in the neurons, which is responsible for the observed mr in fragile x patients.(7) males carrying the full mutation are usually cognitively affected, whereas female full mutation carriers show mild to moderate mental impairment in only approximately 60% of cases. f e m a l e s c a r r y i n g t h e f u l l m u t a t i o n a r e characterized by cells with and without fmrp expression, which can be explained by the random inactivation of one of the two x c h r o m o s o m e s ( l y o n i z a t i o n ) . i t h a s b e e n suggested that an insufficient number of fmrpexpressing neurons in the brain of affected females causes the learning deficits as a result of the proportion of mutant fmr1 alleles on the active x chromosome.(8) willemsen et al. have described alternative diagnostic tests to identify patients with fragile x syndrome on the basis of the absence of fmrp in lymphocytes and hair roots. this antibody test has advantages when compared with the dna test, including low cost and short (one day) work period; absence of radioactivity; and capability to detect all loss-of-function mutations, including the prevalent cgg repeat amplification.(9) the ectodermal origin of hair roots may explain the strong correlation existing between lack of fmrp expression in hair roots from full mutation females and cognitive functioning, c o m p a r e d t o f m r p e x p r e s s i o n i n lymphocytes. ( 1 0 ) i t i s l i k e l y t h a t t h e x inactivation pattern within the ectoderm during early development give rise to similar xinactivation patterns in both brain and hair roots. therefore, hair roots might be of value for predicting the mental capacities of females with a full mutation. methods research design this was an observational analytic research study with cross sectional design, conducted in senin district, gunung kudul regency, from september 2007 up to august 2008. subjects of study fxs subjects were selected according to the following criteria: female, coming from a rural fxs family, age range 4 to 65 years, and without other disabilities. purposive nonrandom sampling was used for recruiting the subjects. thirty females from seven families, both affected and nonaffected, were included in this study. three mentally retarded persons as non-fxs controls were also taken to find out whether fmrp also influenced their cognitive functioning. measurements after obtaining written informed consent, from each subject we collected 20 to 30 hair roots and a ten ml venous blood sample, from which smears were immediately made. the remaining blood sample was subsequently stored in edta tubes for dna extraction in the laboratory. twenty to thirty hair roots were plucked smoothly from different areas behind the ears, either manually or by means of a special pincette, and were then coded. fmrp expression in hair roots fmrp expression was determined by the willemsen method: hair roots were fixed in 3% 13 paraformaldehyde at room temperature (rt) for 10 min. cells were permeabilized by treatment with 100% methanol at rt for 20 min. hair roots were washed with pbs + (phosphate-buffered saline with added 0.5% bovine serum albumin and 0.15% glycine), and i n c u b a t e d a s w h o l e m o u n t w i t h m o u s e monoclonal anti-fmrp antibodies at 4 c° overnight. visualization of antibody-antigen complexes was achieved by an indirect alkaline phosphatase technique, using á m-powervision polymeric alkaline phosphatase (poly ap) for 1 hr, followed by incubation in new fuchsin substrate system (dako) for 10-15 min. levamisole was added to the substrate solution a c c o r d i n g t o t h e g u i d e l i n e s o f t h e manufacturer, to block endogenous alkaline phosphatase activity. immunolabeled hair roots were examined with a stereo zoom microscope at a final magnification of 70x. the number of fmrp-positive hair roots showing red color was expressed as a percentage of the total number of hair roots examined. fmrp expression in blood smears b l o o d s m e a r s w e r e f i x e d i n 3 % paraformaldehyde for 10 min followed by a p e r m e a b i l i z a t i o n t r e a t m e n t w i t h 1 0 0 % methanol for 20 min at rt. after washing in phosphate-buffered saline (pbs) for 5 min, the endogenous peroxidase activity was blocked with pbs-blocked (100 ml 0.1m pbs, 2 ml 30% h2o2, 1 ml 12.5% sodium azide) for 30 min. the smears were washed in pbs+ and incubated with mouse monoclonal anti-fmrp antibodies at 4o c overnight. smears were rinsed in pbs + for 3x5 min followed by biotinylated secondary antibody treatment for 10 min (zymed-kit reagent b). subsequently, s m e a r s w e r e i n c u b a t e d w i t h p e r o x i d a s e conjugated streptavidin for 10 min after rinsing in pbs+ for 3x5 min. finally, smears were rinsed in pbs+ for 4x5 min and pbs for 5 min, respectively. as a final step, smears were incubated with dab substrate (dako liquid dab substrate-chromogen system) for 2x20 min and 1x10 min followed by nuclear fast red counterstaining. the smears were serially dehydrated in ethanol and xylene, mounted in e n t e l l a n ( m e r c k # 1 . 0 7 9 6 0 ) a n d d r i e d o v e r n i g h t a t 3 7 oc i n a n i n c u b a t o r. immunolabeled smears were examined under a l i g h t m i c r o s c o p e a t 1 0 0 0 x f i n a l magnification. a brown precipitate in the cytoplasm of lymphocytes signified positive fmrp expression. the percentage of fmrp expression was obtained by counting one hundred lymphocytes in different areas of the smears and scoring them for fmrp expression. the test results were evaluated by three different investigators who were unaware of the identity of the probands and the counting results of each other. southern blot analysis using digoxigenin labeled probe pp2 seven mg of genomic dna extracted by the miller method (35ìl of 200 ng/ìl) was mixed well with 4.75 ìl aquadest, 5 ì l 10x surecut buffer h (roche), 3 ì l 50 mm spermidine, 1.25 ì l hind iii (recognition site = aagctt – roche cat. #10656313001), 1 ì l eag i, a methylation sensitive enzyme (recognition = site cggccg biolabs cat. #r0505l). the mix was incubated overnight at 370c to complete digestion. five ì l 10x ficoll loading buffer was added to 45 ì l of e a c h d i g e s t i o n p r o d u c t , w h i c h w a s t h e n electrophoresed on 0.7% agarose gel overnight at 45v. dna was transferred to a nylon m e m b r a n e ( h y b o n d n 1 , a m e r s h a m ) i n s o u t h e r n b l o t t i n g a p p a r a t u s o v e r n i g h t , followed by baking the membrane for 20 min at 120°c. prehybridization was performed in roller bottles with pre-heated dig easy hyb h y b r i d i z a t i o n s o l u t i o n ( r o c h e c a t . #11603558001) for at least 1 hr at 42°c and 10 rpm. the prehybridization solution was r e p l a c e d w i t h a h y b r i d i z a t i o n s o l u t i o n containing denatured digoxigenin labeled probe pp2 (courtesy prof ben oostra, erasmus medical center) and left at 42°c overnight to univ med vol.30 no.1 14 rujito, kustiani, severijnen, et al fragile x full mutation complete hybridization. the membrane was washed for 2x5 min with 50 ml 2xssc/0.1 % sds at room temperature. detection was achieved in clean trays with the following procedure: equilibration of membrane in 10 ml w a s h i n g b u f f e r f o r 5 m i n f o l l o w e d b y incubation in blocking solution for 60 min. the membrane was put in a piece of saranwrap. 10 ì l anti-digoxigenin-ap-conjugate was a d d e d t o t h e b l o c k i n g s o l u t i o n a n d t h e membrane was incubated in this solution for 30 min. after incubation, the antibody solution was discarded and the membrane washed in washing buffer for 2x15 min and in detection buffer for 5 min. in the meantime 15 cdp star solutions (roche cat. no. 1685627) were diluted in 1500 ì l detection buffer. after the washing procedure, the membrane was placed between the two sides of a plastic bag and cdp star solution was pipetted on top of the membrane. bubbles present under the sheets w e r e r e m o v e d a n d t h e m e m b r a n e w a s incubated for 5 min. after incubation, liquid excess was removed and the plastic bag was sealed. the membrane was exposed to film for 1 0 a n d 3 0 m i n . a n o r m a l b a n d w a s characterized by the presence of a 2.8 kb band indicating a normal x chromosome, while a 5 . 2 k b b a n d r e f l e c t e d a m e t h y l a t e d x chromosome. determination of iq equivalents raven’s standard progressive matrices were used to measure iq equivalents of the subjects. testing and analysis were performed b y a c e r t i f i e d p s y c h o l o g i s t . c o g n i t i v e functioning was measured by the non-verbal raven test based on non-verbal intervention.(11) statistical analysis using spss 16.0 for windows, pearson c o r r e l a t i o n a n a l y s i s w a s p e r f o r m e d t o determine a possible correlation of fmrp expression in hair roots and lymphocytes with cognitive functioning (p<0.05). results fmrp expression fmrp expression was demonstrated in both blood smears and hair roots by using an immunohistochemical test introduced by willemsen (figure 1). positive specimens have a clear red staining in bulb and hair shaft respectively, whereas negative specimens exhibit no red color in either bulb or hair shaft. fmrp expression in lymphocytes is shown by brown staining of the cytoplasm, while no staining indicates a negative result. the distribution of fmrp expression in all probands is depicted in table 1. normal samples and premutation carriers showed high expression of fmrp both in blood smears and hairs roots, with an average of 91.78% and 8 2 . 8 9 % , r e s p e c t i v e l y. t h r e e s a m p l e s o f mentally retarded people from non-fragile x families also showed a high fmrp expression (over 80%), whereas full mutation subjects showed on average 65.85% expression in blood and 53.98 % in hair roots. these findings show that in full mutation individuals the average fmrp expression in blood smears was higher than in hair roots. fifteen samples show a different result for fmrp expression in blood smears and hairs of over 10%, which were mostly in the full mutation group. a comparison of fmrp expression in lymphocytes vs. hair roots is shown in figure 2. to determine whether a relationship exists between the percentages of fmrp expression in lymphocytes and hair roots, a correlation test was applied for these samples. the pearson c o r r e l a t i o n o f t o t a l s a m p l e w a s h i g h l y significant (r=0.82, p<0.001), while in the full mutation group correlation was lower, although still having statistical significance (r=0.58, p=0.028). this finding shows that in cells of normal individuals and premutation or full mutation carriers, fmrp expression was presumably similar in lymphocytes as well as 15 hair roots, although more variation was seen in the full mutation group. iq equivalents of probands distribution of iq levels among these groups is depicted in figure 3. thirty percent of the sample shows iq levels of >80 whereas the iq levels of the other subjects were below 80. the mean value was 77.88 in the normal group, 83.42 in premutation carriers, and 63.07 in full mutation carriers. in the full mutation group about 14% of subjects was in the normal range (>85), 7% in the borderline range (7084) and 79% showed mild mental impairment (50-69). genotypes of probands to confirm genotyping of the probands, dna testing was performed by southern blot analysis, using a methylation sensitive enzyme, allowing discrimination between methylated and unmethylated fmr1 alleles. this analysis showed the classification of the alleles, whether normal, premutation, or full mutation alleles. results are shown in figure 4. correlation between fmrp expression and iq equivalents of full mutation females in the full mutation group, pearson correlation test was carried out to determine whether a relationship exists between fmrp expression and iq equivalent. the p value of the pearson correlation test for fmrp in hair roots and iq equivalent parameter showed high significance (r=0.635, p= 0.015). in contrast, fmrp expression in lymphocytes showed no statistically significant relationship with iq equivalents (r = 0.31, p = 0.281) (figure 5). figure 1. fragile x mental retardation protein (fmrp) expression in both blood smears and hair roots. a: positive appearance in follicle hair roots from normal probands, b: negative appearance in follicle hair roots from full mutation probands, c: positive appearance in lymphocytes cell from normal probands, d: negative appearance in lymphocytes cell from full mutation probands   c  d  a b univ med vol.30 no.1 16 rujito, kustiani, severijnen, et al fragile x full mutation discussion studies on fmrp expression in hair roots have shown that this may serve as a predictor of cognitive functioning in females carrying the fragile x full mutation,(9) while fmrp expression in blood smears can be used to identify male fragile x patients. however, number of probans iq equivalent % fmrp in blood smear % fmrp in hair roots genotypes 1 60 98 85 non f 2 65 97 90 non f 3 70 95 86 non f 4 95 97 89 n 5 75 88 85 n 6 75 90 84 n 7 85 87 82 n 8 77 97 70 n 9 75 93 87 n 10 73 92 86 n 11 66 75 76 n 12 80 95 83 n 13 83 80 73 p 14 75 94 85 p 15 77 94 77 p 16 75 97 80 p 17 88 86 90 p 18 101 94 85 p 19 85 95 82 p 20 60 65 50 f 21 65 65 50 f 22 60 44 64 f 23 60 76 40 f 24 63 63 41 f 25 70 55 60 f 26 65 61 54 f 27 50 97 75 f 28 85 88 87 f 29 50 45 31 f 30 50 58 25 f 31 55 62 50 f 32 85 76 69 f 33 65 67 60 f table 1. distribution of percentage of fmrp expression in hair roots and blood smear, iq equivalent, and genotypes of individual samples in indonesia note: n = normal; p = premutation; f = full mutation; non f= mr non fragile x there were overlapping results in the female group (full mutation versus controls). in this study, all samples from normal and premutation female carriers expressed normal levels of fmrp (75 -100%), both in blood smears and hair roots, whereas full mutation female carriers showed a large variance in percentage of fmrp expression (figure 2). 17 non fraxa normal sample permutation fullmutation not educated and live in a simple environment. also the diet may play a role because they eat more cassava than rice, as they live in a relatively arid area. as a consequence their protein intake is low, which may be harmful to the body due to the cyanide content of cassava. this finding represents the condition that they may have low micronutrient intake l e a d i n g t o i n h i b i t i o n o f n o r m a l b r a i n development. this finding is in line with previous literature mentioning that dietary intake may affect development of cognitive functioning.(12) hypothyroidism is also one of the problems in this rural area.(13) during this finding is in line with the current h y p o t h e s i s t h a t n o r m a l a n d p r e m u t a t i o n carriers show a normal translation of the fmr1 mrna, in contrast to carriers of the full m u t a t i o n a l l e l e s , w h i c h u s u a l l y s h o w a methylation of the fmr1 gene, resulting in lack of fmrp. a random lyonization process in the cells causes the variance of fmrp expression in full mutation females. normal and premutation subjects in this study show relatively low iq equivalents. a possible explanation for this observation is that these subjects live in an isolated area where modern facilities are limited. most of them are percentages figure 2. diagram of percentage of fmrp expression in lymphocytes and hair roots in individual samples. non-full mutation groups show high expression of above 75% except two samples, whereas the full mutation group varies from 25% to 97%. the hair roots fmrp is lower than in lymphocytes univ med vol.30 no.1 18 rujito, kustiani, severijnen, et al fragile x full mutation figure 3. iq equivalents (converted from raven test) distribution in individual samples. normals and premutation carriers have an iq range between 66 and 101, whereas full mutation carriers have iq levels between 50 and 85. the average iq value in normals and premutation carriers is 77.88 and 83.42 respectively, whereas full mutation carriers have 63.07. the highest iq value is 101 in a premutation carrier and the lowest value is 50 in a full mutation carrier        5,2 kb      2,8 kb      2,8 kb      5,2 kb       5,2 kb       2,8 kb  a: lanes 4,14,21 were normal samples. lanes 1,3,13,20,23 correspondent to premutation samples whereas full mutation samples were shown in lanes 2,5,7,8,9,10,11,12,15,18,19, and 22. lanes 6 and 17 indicated full mutation and premutation control, respectively. b: lanes 1,2,5,6,7 were normal samples. lane 3 correspondents to premutation sample and line 4 were full mutation sample. dna ladder was indicated in lane 8. c: lane 1 was full mutation and lane 2 was premutation figure 4. figure of southern blotting test from individual samples using double digestion with hindiii and eagi. 19 figure 5. correlation between fmrp expression and iq equivalents in blood smear (a) and hair roots (b) in full mutation carriers. the chart indicated a significant correlation between percentage of fmrp expression in hair roots and iq equivalents in our group of full mutation carriers a b application of the raven test; we had to explain the test to the probands more frequently, as they seem did not care about it. in contrast, they finished their test very fast. this case relates with a previous study that mentioned an alternative hypothesis to general ability (g factor) as to the reasons why non-western p o p u l a t i o n s s c o r e l o w e r t h a n we s t e r n populations, such as being test-wise, less i n t e r e s t e d , m o r e a n x i o u s , w o r k i n g l e s s efficiently, or giving up sooner on items they find difficult.(14) distributions of iq equivalents in the full mutation group were as follows: about 14% in the normal range (>85), 7% in the borderline range (70-84%) and 79% showing mild mental impairment (50-69). these percentages are higher than those found in previous research studies, (15) namely 60% with mild mental retardation. however, in our study the number of subjects was limited and should be increased in the near future to allow drawing of definite/ final conclusions. the differences in iq equivalents of the full mutation, premutation, and normal groups are shown in figure 3. in the full mutation group we found subjects with relatively high iq levels compared t o o t h e r g r o u p s s h o w i n g h i g h e r f m r p expression in their cells (compared with other samples). this may be due to the fact that f e m a l e s c a r r y i n g a f u l l m u t a t i o n a r e characterized by cells with fmrp expression as well as those without, which can be explained b y t h e p r e s e n c e i n f e m a l e s o f t w o x chromosomes and random inactivation of one x chromosome (lyonization). this finding also noticed that although human intelligence is influenced by many genetic factors, a single mutation in the fmr1 gene resulting in lack of f m r p s i g n i f i c a n t l y r e d u c e s c o g n i t i v e functioning in female full mutation carriers.(16,17) fmrp expression in blood smears and hair roots are also correlated with cognition, although fmrp expression in blood smears seemed to be higher than in hair roots. this may be explained by differences in origin of the tissues. blood originates from mesoderm, whereas both hair roots and brain tissue are derived from ectoderm. lymphocytes originate from a common lymphoid progenitor cell before univ med vol.30 no.1 20 rujito, kustiani, severijnen, et al fragile x full mutation differentiating into their distinct lymphocyte types. the formation of lymphocytes, known as lymphopoiesis, has a daily turnover and the lifespan of lymphocytes ranges from several weeks to several years.(18,19) it has also been suggested that there might be a selection against cells with a full mutation in dividing lymphocytes or that there is a bias toward inactivation of the x-chromosome in women during aging.(20,21) this may explain why there seems to be a better correlation between lack of fmrp expression in hair roots and mental retardation. in addition, lymphocytes may poorly reflect the conditions in brain tissue, because the rapid turnover may lead to high variation in clones that either do or do not carry t h e f m r 1 m u t a t i o n o n t h e a c t i v e x chromosome. earlier studies using the fmrp test on lymphocytes did show a weak statistical correlation; however, the significance was not high enough to use this method as a reliable diagnostic or predictive test. the benefit of hair roots as a diagnostic tool is shown in this study because we found, although in a small-sized study, a highly significant correlation between the percentage of fmrp expression in hair roots and iq equivalent in female full mutation carriers. the statistical analysis showed that level of cognitive functioning in female full mutation carriers typically reflected by fmrp in hair roots rather than in blood smears. the origin of the tissue is important, but methodological aspects may play a role as well and cannot be excluded from this research. some of the limitation aspects were the limited number of full mutation carriers tested, problems with sample delivery by courier, and climatic differences. conclusion fmrp expression in hair roots is probably more useful to predict cognitive functioning in female full mutation carriers than is fmrp expression in blood lymphocytes. ackowledgments we would like to express our gratitude to ben oostra (clinical genetics department of erasmus medical center rotterdam), ben hamel, erick sisterman and arie smith (clinical genetics department of radboud university nijmegen) for providing us with the facilities and opportunity to do the genetic tests in the netherlands. references 1. acog committee opinion no. 469. carrier screening for fragile x syndrome. obstet gynecol 2010;116:1008-10. 2. hussein sm. fragile x mental retardation and fragile x chromosome in the indonesian population (dissertation). sydney: faculty of medicine, university of new south wales;1998. 3. faradz smh, buckley m, tang l, liegh d, holden jja. molecular screening for fragile x syndrome among indonesian children with developmental disability. am j med genet 1999; 83:350-1. 4. faradz smh, gasem mh, nillesen wm, sistermans e, hamel bcj, hagerman r. a high rate of fragile x in a small district of indonesia can be traced back to one common ancestor. the 8th international fragile x conference; 2002 july 17-21; chicago, usa. 5. faradz smh, setyawati an, winarni t, mundhofir fe, nillesen wm, zamhari m, et al. molecular screening for fragile x syndrome in institutionalized mild mentally retarded individuals at bbfsbg temanggung, central java (a preliminary study). the 12th international workshop on fragile x and x-linked mental retardation; 2005 august 26-29;williamsburg, va, usa. 6. hagerman rj, hagerman pj. testing for fragile x gene mutations throughout the life span. jama 2008;300:2419-21. 7. penagarikano o, mulle jg, warren st. the pathophysiology of fragile x syndrome. annu rev genomics hum genet 2007;8:109-29. 8. luo xf, zhong jm, zhang xz, zou y, chen y, wu hp, et al. hair root fragile x mental retardation protein assay for the diagnosis of fragile x syndrome. chinese j contemp pediatr 2009;11:817-20. 9. willemsen r, mohkamsing s, de vries b, devys d, van den ouweland a, mandel jl, et al. rapid 21 antibody test for fragile x syndrome. lancet 1995;345:1147–8. 10. willemsen r, smits a, severijnen la, jansen m, jacobs a, de bruyn e, et al. predictive testing for cognitive functioning in female carriers of the fragile x syndrome using hair root analysis. j med genet 2003;40:377–9. 11. raven j. the raven’s progressive matrices: change and stability over culture and time. cognit psychol 2000;41:1-48. 12. the british nutrition foundation. the influence of diet on cognitive function, appetite and mood. sainsbury, uk;2007. 13. gunungkidul regency, in cooperation with regional development and poverty reduction program – rdprp, atlas gunung kidul regency, final main report, march 2005. available at: http://siteresources.worldbank.org/intindo nesia/resources/projects/288973-8033888998 /1218077-1150284192230/2654894-115028466 8244/atlas.pdf. accessed mei 12, 2009. 14. nell v. cross-cultural neuropsychological assessment: theory and practice. london: erlbaum;2000. 15. barlow-stewart k, emery j, metcalfe s. genetic in family medicine: the australian handbook for general practitioners. canberra: biotechnology australia;2007. 16. kenneson a, zhang f, hagedorn ch, warren st. reduced fmrp and increased fmr1 transcription is proportionally associated with cgg repeat number in intermediate-length and premutation carriers. hum mol genet 2001;10: 1449-54. 17. zhou y, lum jms, yeo g-h, kiing j, tay skh, chong ss. molecular diagnostics and genetics simplified molecular diagnosis of fragile x syndrome by fluorescent methylation-specific pcr and gene scan analysis. clin chem 2006; 52:1492-500. 18. janeway c, travers p, walport m, shlomchik m, immunobiolog. 5th ed. new york: garland science;2001. 19. claycombe k, king le , fraker pj. a role for leptin in sustaining lymphopoiesis and myelopoiesis. pnas 2008;105:2017-21. 20. rousseau f, heitz d, oberlé i, mandel jl. selection in blood cells from female carriers of the fragile x syndrome: inverse correlation between age and proportion of active x chromosomes carrying the full mutation. j med genet 1991;28:830–36. 21. nossa gjv. the double helix and immunology. nature 2003;421:440-4. univ med vol.30 no.1 alvina 146 abstract *department of clinical pathology **department of pharmacology, faculty of medicine, trisakti university correspondence dr. pusparini, dr, sppk department of clinical pathology, faculty of medicine, trisakti university jl. kyai tapa no. 260 grogol jakarta 11440 phone: 021-5672731 ext.2403 email: pusparini_sppk@yahoo.com univ med 2011;30:146-54. low density lipoprotein cholesterol decreases vascular cell adhesion molecule-1 in postmenopausal women pusparini*, danny wiradharma*, and elly herwana** september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina in premenopausal women cardiovascular disease is rarely encountered, but after menopause the prevalence of cardiovascular disease increases drastically. there are several risk factors for cardiovascular disease, known as traditional risk factors, among others body fat concentration, age, duration of menopause, body mass index (bmi), and estradiol concentration. cardiovascular disease is considered as an inflammatory disorder, in which adhesion molecules play an important role. vascular cell adhesion molecule-1 (vcam-1) is one of the adhesion molecules with an important role in the atherosclerotic process. the aim of this study was to determine the relationship of risk factors affecting the expression of vcam-1 in postmenopausal women. this study was a cross-sectional study involving 182 postmenopausal women in the age range of 4760 years, who were residents of mampang prapatan subdistrict, south jakarta. venous blood samples were obtained for laboratory investigations, viz. fasting blood glucose, total cholesterol, high density lipoprotein (hdl) cholesterol, low density lipoprotein (ldl) cholesterol, triglycerides, creatinine, serum glutamic oxaloacetic transamirase (sgot), serum glutamic pyruvic transmirasi (sgpt), bilirubin, total protein, albumin, estradiol and vascular cell adhesion molecule-1 (vcam-1).a multiple regression analysis was performed on traditional risk factors and their relationship with vcam-1 concentration. the results showed there were five traditional risk factors influencing vcam-1 concentration, viz. duration of menopause, bmi, estradiol concentration, hdl cholesterol, and ldl cholesterol. among these five factors, ldl cholesterol had the greatest influence on vcam-1 expression (beta coefficient = -0.253 and p=0.001). in conclusion, ldl cholesterol concentration decreased vcam-1 expression in postmenopausal women. key words: vcam-1, ldl cholesterol, postmenopausal women introduction cardiovascular disease (cvd) is rarely f o u n d i n p r e m e n o p a u s a l w o m e n , b u t cardiovascular and cerebrovascular disease are the main causes of mortality in postmenopausal women, particularly in developed countries. the prevalence of these two diseases is up to 147 univ med vol.30 no.3 75%, which is larger than the prevalence of 68% for breast cancer.(1,2) in postmenopausal women decreased estrogen levels may result in changes in the distribution of body fat from the gynoid into the android type, decreased glucose tolerance, dyslipidemias, high blood p r e s s u r e , i n c r e a s e d s y m p a t h e t i c t o n e , e n d o t h e l i a l d y s f u n c t i o n , a n d v a s c u l a r inflammation.(3-5) the abnormalities caused by decreased estrogens in postmenopausal women comprise vasomotor changes, i.e. hot flushes on the c h e s t , n e c k a n d f a c e , w h i c h m a y b e accompanied by anxiety and palpitations. other manifestations include reproductive tract symptoms, psychological symptoms, cognitive d i s o r d e r s , a n d i n c r e a s e s i n c h r o n i c degenerative disorders, such as osteoporosis, c a r d i o v a s c u l a r d i s e a s e , s t r o k e , a n d alzheimer’s disease.(6-8) cvd is considered to be an inflammatory disorder.(9-11) the results of several studies indicate that atherosclerosis is associated with inflammatory processes, formation of reactive o x y g e n s p e c i e s ( r o s ) a n d e n d o t h e l i a l dysfunction. several conditions that are regarded as risk factors for inflammation in the development of cardiovascular disease, include dyslipidemias, comprising increased total cholesterol, low density lipoprotein (ldl) cholesterol, triglycerides, and reduced high density lipoprotein (hdl) cholesterol.(9,10,12) cvd in postmenopausal women is associated with endothelial dysfunction, in which there is a defect in the vascular mechanism that protects the vascular endothelium against physical and chemical stimuli, such as inflammation, procoagulation and vasoconstriction.(3,10) one of the markers of endothelial dysfunction is the vascular cell adhesion molecule-1 (vcam-1), which plays a role in the development of endothelial dysfunction, along with other adhesion molecules. vcam-1 plays its role at the time of entry of inflammatory cells into the s u b e n d o t h e l i a l t i s s u e s . ( 1 3 1 6 ) l e u k o c y t e s normally circulate within the blood stream, but when there is tissue damage or inflammation, these leukocytes adhere to the endothelium of blood vessels and migrate across the vessel wall into the affected tissues. leukocyte adherence to blood vessel walls comprises a series of events, namely selectin-mediated rolling, activation mediated by chemoattractants, and adhesion to the vascular endothelium mediated by integrins, which are an immunoglobulin superfamily of counter receptors such as vcam-1 and intercellular cell adhesion molecule-1 (icam-1). subsequently the leukocytes move between the endothelial cells and migrate into the subendothelial tisues. the aim of the present study was to determine the factors influencing vcam-1 expression in postmenopausal women.(13-16) methods design of the study this study was of cross-sectional design and was conducted in the area of mampang p r a p a t a n s u b d i s t r i c t h e a l t h c e n t e r, f r o m january until march 2010. study subjects the subjects of this study were postmenopausal women between 47 and 60 years of age, with as inclusion criteria healthy postmenopausal women, with a natural cessation of menstruation of minimally 1 year and less than 10 years, not caused by total hysterectomy, b i l a t e r a l o o p h o r e c t o m y, i r r a d i a t i o n , a n d chemotherapy, with body mass index (bmi) < 35 kg/m2, not taking medications within the last 6 months (steroids, oral antidiabetics, antihypertensives, antihyperlipidemic drugs, hormonal therapy); laboratory tests: bilirubin <2 m g / d l , s e r u m g l u t a m i c o x a l o a c e t i c transaminase (sgot) and serum glutamic pyruvic transminase (sgpt) within reference range (10–36 u/l and sgpt 7–35 u/l, respectively), no abnormalities on physical e x a m i n a t i o n , s u c h a s h e p a t o m e g a l y, hypertension, disorders of cardiac rhythm, 148 ( ) ( )( ) 3 r1r15,0 ζβα n 2 +⎥ ⎦ ⎤ ⎢ ⎣ ⎡ −+ +ζ = nl creatinine <1.5 mg/dl, capable of actively walking without aids, capable of communication (able to answer questions by themselves or with a s s i s t a n c e f r o m o t h e r s ) a n d a g r e e i n g t o participate in the study by signing informed consent after receiving information on the aim of the study. the exclusion criteria were: women with chronic or terminal diseases, such as mammary carcinoma, carcinoma of the cervix, endometrial carcinoma, renal failure, cardiac failure, diabetes mellitus, stroke and myocardial infarction / heart attack, severe psychosis (such as schizophrenia), and mastectomy. the study subjects were selected by multistage cluster r a n d o m s a m p l i n g f r o m f o u r k e l u r a h a n (villages), namely kuningan barat, mampang prapatan, tegal parang dan pela mampang, ressorting under mampang prapatan subdistrict, south jakarta. sample size the sample size was calculated using the formula : (17) c i r c u m f e r e n c e t a k e n w a s t h e m i n i m a l circumference of the abdomen between the midpoint of the lower rib and the superior anterior iliac spine. hip circumference was the largest circumference between the gluteal muscle dorsally and the pubic symphysis ventrally. bmi was calculated as the weight in kg divided by the square of the height and using the standard for asians. blood pressure was determined on the right arm of the sitting subject after a rest of at least 15 minutes, using a riester mercury sphygmomanometer. blood pressure was taken from the mean of two measurements performed by different nurses. systolic pressure was based on the first korotkoff sound, whilst diastolic pressure was based on the fifth korotkoff sound. in case the determinations differed by more than 10 mm hg, the blood pressure measurement was repeated by a third nurse, and only those measurements were used that differed by <10 mm hg. pulse rate was determined after blood pressure measurements, by counting the pulsation of the radial artery for 1 minute, after the subject had rested for at least 15 minutes. the pulse rate was counted twice and the mean of the results was taken. analytic procedures after the subjects had fasted for 12 hours, a 10 ml venous blood sample was collected from each subject for laboratory investigations, viz. fasting blood glucose, total cholesterol, h d l c h o l e s t e r o l , l d l c h o l e s t e r o l , t r i g l y c e r i d e s , c r e a t i n i n e , s g o t, s g p t, bilirubin, total protein, albumin, estradiol and vcam-1. ldl cholesterol and estradiol were d e t e r m i n e d u s i n g r e a g e n t s f r o m r o c h e d i a g n o s t i c s . a s s e s s m e n t o f v c a m 1 concentration was by means of sandwich enzyme linked immunosorbent assay (elisa), using reagents produced by r&d systems, catalog no. dvcoo, lot no. 279922, date of expiry 20 april 2011. the coefficient of variation (cv) of ldl cholesterol assessment was 1.2% between day and 1.7% within run, where: r = coefficient of correlation between hdl cholesterol and vcam-1 = 0.798.(15) z α = 1.96 z β = 0.842 n= 68 the minimum sample size required was 68 subjects. a n t h r o p o m e t r i c a n d b l o o d p re s s u r e measurements height and weight were determined on lightly clothed subjects without foot wear. height was measured by means of a portable microtoise with a precision of 0.1 cm. body weight was determined using sage portable scales with a precision of 0.1 kg. circumference measurements were by means of a measuring tape to the nearest 0.1 cm. the abdominal pusparini, wiradharma, herwana ascular cell adhesion molecule-1 in women 149 univ med vol.30 no.3 charac te ristic n (%) age (yea rs) # 53.5 ± 3.5 < 50 27 (15) 50 –54 81 (44) 55 – 60 74 (41) age at menarche (ye ars) # 14.3 ± 1.7 < 15 146 (80) =15 36 (20) age at menopau se (year s) # 49.1 ± 3.3 < 50 97 (53) 50 –54 79 (43) 55 –60 6 (6) duration of menopa use (yea rs)* 4 (3 – 6 ) < 5 101 (55) 5– 10 81 (45) education n o forma l ed ucation 17 (9) d id not finish primary sc hool 17 (9) primary school 90 (50) junior high sch ool 36 (20) senior high school 18 (10) tertiary educ ation 4 (2) employmen t status employed 84 (46) un employed 98 (54) abdominal circumference (cm)# 83.9 ± 10.4 hip circumferen ce (cm)# 96.9 ± 8.3 weigh t (kg)# 59.4 ± 11.3 height (cm)# 148.9 ± 5.2 body mass index (k g/m2)# 26.7 ± 4.7 < 25 65 (35.5 ) 25 – 29.99 79 (43.2 ) > 30 38 (20.8 ) systolic pressure (mmhg) 124.8 ± 2 1.3 diastolic pre ssure (m mhg) 78.6 ± 12.6 pulse rate (p er minute) 77.9 ± 6.5 la boratory tests estradiol (pg/ ml)* 5 .1 (5 – 8.9) sgot (u/l) 20.7 ± 5.2 sgpt(u/ l) 13.8 ± 7.3 c reatinine (g /dl) 0.65 ± 0.15 total bilirubin (m g/d l) 0.47 ± 0.20 protein (g/dl) 7.5 ± 0.5 a lbum in (g/dl) 4.4 ± 0.3 fa stin g g lucose (mg/dl) 87.8 ± 12.7 total choleste rol (mg/dl) 207.0 ± 3 6.6 hdl choleste rol (mg/dl) 57.2 ± 12.2 ldl ch ol esterol (mg /dl) 129.7 ± 3 3.6 triglyc erides (mg/dl) 115.2 ± 6 5.5 v cam -1 (n g/ml) 847.2 ± 275.4 table. 1 demographic, physical, and laboratoratory characteristics of the study subjects (n=182) # mean ± sd, * values are median (lowest and highest quartiles). sgot: serum glutamic oxaloacetic acid transaminase, sgpt: serum glutamic pyruvic transaminase, hdl cholesterol: high density lipoprotein cholesterol, ldl cholesterol: low density lipoprotein cholesterol, vcam-1: vascular cell adhesion molecule-1 for estradiol determination, the cv was 1.7% between day and 1.8% within run, while the cv for vcam-1 was 2.1% between day and 2.1% within run. statistical analysis the normality of data distribution was determined by using the kolmogorov-smirnov test. data on demographic characteristics, physical measurements, and laboratory tests were presented as descriptive statistics, using the mean and standard deviation for normally distributed data or normalized data after logarithmic transformation. age of menarche and estradiol concentration were presented as median and interquartile range, because the data were non-normally distributed even after transformation. pearson correlation tests were used to determine the association between vcam-1 and several traditional factors such as age, duration of menopause, systolic and diastolic blood pressure, bmi, total cholesterol concentration, hdl and ldl cholesterol, triglycerides, fasting glucose, and estradiol concentration a multiple linear multiple regression analysis was used to determine the m o s t i n f l u e n t i a l f a c t o r o n v c a m 1 concentration. the level of significance used was 0.05. ethical clearance the study protocol was approved by the r e s e a r c h e t h i c s c o m m i t t e e , f a c u l t y o f medicine, trisakti university. all candidate study subjects willing to participate were given information on the aim and benefits of the study, before signing informed consent. results at the start of the study there were 200 subjects who agreed to participate. eighteen subjects did not meet the inclusion and exclusion criteria, among whom 14 subjects had fasting glucose concentrations of more than 126 mg/dl and 4 subjects were hypertensive, thus resulting in 182 subjects meeting the criteria. 150 tabel 3. factors assocaiated with vcam-1 concentration *p <0.05 significant the mean age of the subjects was 53.5 ± 3.5 years, with the majority (44%) between 50 and 54 years of age. mean age at menarche was 14.3 ± 1.7 years, and 146 subjects (80%) had menarche at the age of less than 15 years. mean age at menopause was 49.1 ± 3.3 years and 97 subjects (53%) had menopause at the a g e o f l e s s t h a n 5 0 y e a r s . d u r a t i o n o f menopause had a median value of 4 years, with the lowest quartile at 3 years and the highest quartile at 6 years. ninety subjects (50%) had attended primary school, while a similar p e r c e n t a g e h a d n o e m p l o y m e n t . t h e demographic and physical characteristics are presented in table 1. in addition, table 1 lists the laboratory p a r a m e t e r s o f t h e s u b j e c t s . e s t r a d i o l concentrations had a median of 5.1 pg/ml with the lowest quartile being 5 pg/ml and the highest quartile 8.9 pg/ml. the concentrations of sgot, sgpt, creatinine, bilirubin, total protein, albumin, fasting glucose, and the lipid profile were within normal limits. vcam-1 concentration was found to be 847.2 ± 275.4 ng/ml. table 2 presents several factors that could possibly affect vcam-1 expression, namely fasting glucose, estradiol, total cholesterol, hdl cholesterol, ldl cholesterol, triglycerides, and other factors, such as age at menopause, duration of menopause, bmi, and blood pressure. among the various factors in table 2 above, duration of menopause, estradiol, bmi, total cholesterol, hdl cholesterol, and ldl cholesterol were theorectically associated with vcam-1 concentration. these five factors were entered into a multiple regression model. backward model analysis showed that the factor influencing vcam-1 expression was l d l c h o l e s t e r o l c o n c e n t r a t i o n (beta coefficient=0.253; p=0.001) (table 3). table 2. correlation test for several factors affecting vcam-1 concentration bmi=body mass index; hdl=high density lipoprotein; ldl=low density lipoprotein pusparini, wiradharma, herwana vascular cell adhesion molecule-1 in women 151 univ med vol.30 no.3 discussion among the five factors analyzed in the present study, only ldl cholesterol had an influence on vcam-1 expression, with a beta coefficient of –0.253 and p=0.001. the role of ldl cholesterol in the atherosclerotic process lies among others in the formation of foam cells. ldl cholesterol has five subtypes on the basis of molecular size. small ldl cholesterol molecules are more atherogenic, because they can easily enter the vascular endothelium. the subtypes that are considered to be the most atherogenic are subtypes 4 and 5.(18,19) ldl cholesterol entering the vascular endothelium easily become oxidized ldl cholesterol, which are ultimately phagocytosed by macrophages, which turn into foam cells. oxidized ldl cholesterol is also one of the factors affecting the decrease in vascular smooth muscle cells through apoptosis in atherosclerotic plaques, secretion of metalloproteinases and other connective tissue enzymes, resulting in a reduction in collagenous tissue, thinning of the atherosclerotic capsule, ultimately making the atherosclerotic plaques vulnerable to rupture.(2022) the results of the present study are similar with those of the study by bossowska et al,(23) indicating that the concentration of adhesion molecules (icam-1, vcam-1, se-selectin) is correlated with traditional risk factors for cvd, such as systolic blood pressure, diastolic blood pressure, and concentration of lipids in the blood.(23) the mean hdl cholesterol concentration in the present study was 57.2 ±12.2 mg/dl, falling within the reference range of 35–80 mg/ dl.(24) hdl cholesterol is essential for lipid transport from the peripheral circulation to the liver, thus decreasing peripheral accumulation of lipids. hdl and ldl cholesterols function synergistically in maintaining cholesterol balance. reduction of hdl cholesterol is an atherogenic risk factor, whereas an increase in hdl cholesterol concentration acts as a protective factor. multiple regression analysis indicates that hdl cholesterol is not an influencing factor in vcam-1 expression, with a beta coeffcient of –0.113 and p=0.157. this differs from the results of a cross-sectional study by demerath et al.(25) on 592 healthy male and female subjects 18-82 years old, demonstrating a n i n d e p e n d e n t a s s o c i a t i o n b e t w e e n t h e concentration of adhesion molecules and hdl c h o l e s t e r o l , p a r t i c u l a r l y i n s m o k e r s . ( 2 5 ) demerath’s study subjects were in the age range of 18-82 years and were differentiated into smokers and nonsmokers, whereas in the present study all subjects between 47 and 60 years old and did not include any smokers. in our study, hdl cholesterol was not correlated with vcam-1. our result is different from that of a study in chechia involving both obese and lean postmenopausal women with mean age of 49.9 ± 2.9 years, indicating that serum soluble vcam-1 concentration is positively correlated with hdl-cholesterol levels.(26) estradiol concentration in this study had a median of 5.1 pg/ml with the lowest quartile = 5 pg/ml and the highest quartile = 8 . 9 p g / m l . e s t r a d i o l c o n c e n t r a t i o n i n postmenopausal women had a reference value of <130 pg/ml.(27) all subjects in this study had estradiol concentrations that were below r e f e r e n c e v a l u e . a s t u d y c o n d u c t e d b y ausmanas et al.(27) on 1020 postmenopausal women, comprising 9 asian ethnic groups, reported that estradiol concentrations varied with ethnic group, the lowest being 13.6 pg/ml in chinese and the highest 29.1 pg/ml in vietnamese. the estradiol concentrations in these asian postmenopausal women had a mean of 20.2 pg/ml (sd = 34.0 pg/ml) and a median of 12.26 pg/ml. in abovementioned study, the estradiol concentration in indonesian women was 19.6 pg/ml. ausmanas et al. (28) also confirmed menopausal status by determination of follicle stimulating hormone (fsh) and luteinizing hormone (lh) concentrations. in this connection, fsh concentration was better than estradiol concentration for confirmation of menopause, since fsh is not influenced by 152 a g e ( p = 0 . 8 5 2 ) , i n c o n t r a s t t o e s t r a d i o l ( p = 0 . 0 0 1 ) . (2 8 ) t h e i n c r e a s e d r i s k f o r cardiovascular disease in postmenopausal women, such as the subjects of this study, may be attributed to the reduction in estrogen concentrations as one of the causal factors.(6,29) the american heart association has formulated strategies for early detection of cvd in high risk groups, including the use of inflammatory markers,(20,21) as atherosclerosis is thought to be an inflammatory process. among the various inflammatory markers for primary prevention of cvd,(2,21,28) the one used in this study is vcam-1, which is both an i n f l a m m a t o r y m a rk e r a n d a n a d h e s i o n molecule.(13,30) inflammation in atherosclerosis is marked by an infiltration of leukocytes in the vascular endothelium, which is a dynamic o rg a n t h a t i n n o r m a l c i r c u m s t a n c e s h a s anticoagulant properties. injury to the vascular endothelium leads to platelet adhesion and aggregation, ultimately resulting in thrombus f o r m a t i o n . o t h e r r e s p o n s e s i n c l u d e t h e mediation of leukocyte migration into the endothelium, and it is this pathological process that is thought to be the initiating event of atherosclerosis.(31-33) in leukocyte migration there are several useful markers, such as intercellular adhesion molecule-1 (icam-1), endothelial leukocyte adhesion molecule-1 (elam-1), and vcam-1. vcam-1 is an adhesion molecule of the igg superfamily and interacts with its counterreceptor very late appearing antigen 4 (vla-4), a â1 integrin expressed on the surface of monocytes and lymphocytes.(25,32,34) in this study estradiol concentration was not significantly correlated with vcam-1 concentration. the results of r e g r e s s i o n a n a l y s i s a l s o s h o w e d a b e t a coefficient of 0.071 and a p value of 0.349. t h e r e f o r e i n t h i s s t u d y t h e e s t r a d i o l concentration did not play a role in vcam-1 expression. this is in contrast with an in vivo study in rabbits conducted by nathan et al.(34) to determine the role of estradiol in vcam-1 expression. the results of the study by nathan et al.(34) indicated that there were gender differences in monocyte adhesion to vascular endothelial cells and transendothelial migration after induction of hypercholesterolemia in male and female rabbits. the investigators concluded that estradiol presumably inhibits monocyte adhesion and ultimately vcam-1 expression.(34) the differences in the results of the present study and those of nathan et al. (34) may be due to the fact that inhibition of vcam-1 expression presumably constitutes a multifactorial process involving other factors in addition to estrogens. at present the precise mechanisms by which estrogens may protect vascular endothelium from atherosclerosis are as yet not known with certainty.(13,33,34) another presumed risk factor for cvd in postmenopausal women is the duration of menopause. in the present study the median duration of menopause in the study subjects was 4 years, with the lowest and highest quartiles being 3 and 6 years, respectively. the majority of the subjects had a duration of menopause of less than 5 years (55%). bivariate analysis showed that duration of menopause was not correlated with vcam-1 concentration, while similarly regression analysis showed that duration of menopause was not a factor playing a role in vcam-1 expression, with a beta coefficient of 0.100 and a p value of 0.175. these results were consistent with those of women health initiative (whi) study,(6) which i n d i c a t e d t h a t c v d p r e v a l e n c e w a s n o t increased with duration of menopause. the whi study showed a peak prevalence of less than two years and hazard ratio of 1.12, while for durations of 2-5 years and >5 years, the hazard ratios were 1.05 and 0.83, respectively.(6) this whi study are similar to those of a study conducted by rossouw et al,(35) showing that the risk of cvd was not influenced by age and duration of menopause.(35) bmi is another factor presumed to effect a rise in cvd prevalence in postmenopausal w o m e n , i n w h o m d e c r e a s e d e s t r o g e n c o n c e n t r a t i o n s r e s u l t i n a l t e r ed l i p i d pusparini, wiradharma, herwana vascular cell adhesion molecule-1 in women 153 univ med vol.30 no.3 metabolism and distribution, from a gynoid type into an android type. with advancing age, the metabolism of the body decreases, such that body weight tends to increase, leading to increased bmi. in the present study, mean bmi was 26.7 ± 4.7 kg/m2, thus falling into the obese category, and bmi was not correlated with vcam-1 concentration. the results of a linear regression analysis also showed that bmi did not influence expression of vcam-1, with a beta coefficient of –0.010 and p=0.898. these results are contrary to those of a study by bossowska et al,(23) demonstrating a correlation of icam-1 and vcam-1 concentrations with bmi. these conflicting results may have been due to different characteristics of the study subjects. the study by bossowska et al (23) used male and female subjects with mean age of 60 years with past myocardial infarction, whereas t h e p r e s e n t s t u d y i n v o l v e d h e a l t h y p o s t m e n o p a u s a l w o m e n a s s u b j e c t s . i n addition, 65% of the subjects of the present study had bmi values of more than 25 kg/m2, thus resulting in inadequate variation in bmi among the study subjects to represent the normal, obese i and obese ii categories.(36) one of the limitations of the present study is that menopause was confirmed by past history (anamnesis) and estradiol concentrations in the blood, and not by follicle-stimulating hormone (fsh) concentration. another limitation was that the study used a cross-sectional design, such that it yielded only the factors affecting vcam1 concentration, but could not demonstrate a casuse-and-effect relationship. therefore it is necessary to conduct experimental studies to determine whether or not ldl cholesterol and vcam-1 concentrations are involved in a cause-and-effect relationship. conclusion th e t r a d i t i o n a l r i s k f a c t o r w i t h t h e greatest influence on vcam-1 expression in postmenopausal women is ldl cholesterol concentrration. acknowledgements the authors thank the dean and vice deans of the faculty of medicine, trisakti university, for sponsoring this study. thanks are also due to the postmenopausal women who agreed to participate in this study, and to the head and staff of mampang prapatan health center, south jakarta, who facilitated this study. references 1. rosano gmc, vitale c, marazzi g, volterrani m. menopause and cardiovascular disease : the evidence. climacteric 2007;10:19-24. 2. ross r. atherosclerosis an inflammatory disease. n engl j med 1999;340:115-26. 3. ungvari z, kaley g, de cabo r, sonntag we, csiszar a. mechanisms of vascular aging: new perspective. j gerontol a biol sci med sci 2010;65:1028-41. 4. wu jm, zelinski m, ingram dk, ottinger ma. ovarian aging and menopause: current theories, hypotheses, and research models. exp biol med 2005;230:818-28. 5. ferrari a, radaelli a, centola m. aging and the cardiovascular system. j appl physiol 2003;95: 2591-7. 6. maturana ma, irigoyen mc, spritzer pm. menopause, estrogens and endothelial dysfunction: current concepts. clinics 2007;62: 77-86. 7. nelson hd. menopause. lancet 2008;371:7609. 8. shapiro s. recent epidemiological evidence relevant to the clinical management of 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2004;110:3068-74. 20. pearson ta, mensah ga, alexander rw, anderson jl, cannon ro, criqui m, et al. markers of inflammation and cardiovascular disease. application to clinical and public health practice. a statement for healthcare professionals from the centers for disease control and prevention and the american heart association. circulation 2003;107499-511. 21. vasan rs. biomarkers of cardiovascular disease. circulation 2006;113:2335-62. 22. verna l, ganda c, stemerman mb. in vivo low density lipoprotein exposure induces intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 correlated with activator protein-1 expression. arterioscler thromb vasc biol 2006; 26:1344-9. 23. bossowska a, kiersnowska r, bssowski a. assessment of serum levels of adhesion molecules (sicam-1, svcam-1, se-selectin) in stable and unstable angina and acute myocardial infarction. przegl lek 2003;60:445-50. 24. fiscbach f, dunning mb, editors. chemistry studies. in: fiscbach f, dunning mb, editors. a manual of laboratory and diagnostic tests. 7th edition. philadelphia: lippincott, williams & wilkins;2004.p.316-455. 25. demerath e, towne b, blangero j. the relationship of soluble icam-1, vcam-1, pselectin and e-selectin to cardiovascular disease risk factors in healthy men and women. ann hum biol 2001;28:664-78. 26. bošanska l, michalský d, lacinová z, dostálová i, bártlová m, haluzíková d, et al. the influence of obesity and different fat depots on adipose tissue gene expression and protein levels of cell adhesion molecules. physiol res 2010;59:79-88. 27. fiscbach f, dunning mb, editors. urine studies. in: fiscbach f, dunning mb, editors. a manual of laboratory and diagnostic tests. 7th ed. philadelphia: lippincott,williams&wilkins; 2004.p.163-263. 28. ausmanas mk, tan da, jaisamrarn u, tian xw, holinka cf. estradiol, fsh, and lh profiles in nine ethnic groups of postmenopausal asian women:tte pan asia menopause (pam) study. climateric 2007;10:427-37. 29. mosca l, appel lj, benjamin ej, berra k, strobos nc, fabunmi rp, et al. evidence based guidelines for cardiovascular disease prevention in women. arterioscler thromb vasc biol 2004; 24:e29-50. 30. sbarouni e, kroupis c, kyriakides zs, koniavitou kk, kremastinos dt. cell adhesion molecules in relation to simvastatin and hormone replacement therapy in coronary artery disease. eur heart j 2000;21:975-80. 31. libby p. inflammation in atherosclerosis. nature 2002;420:868-74. 32. galkina e, ley k. vascular adhesion molecules in atherosclerosis. arterioscler thromb vasc biol 2007;27:2292-301. 33. galkina e, ley k. immune and inflammatory mechanism of atherosclerosis. annu rev immunol 2009;27:165-97. 34. nathan l, pervin s, singh r, rosenfeld m, chaudhuri g. estradiol inhibits leukocyte adhesion and transendothelial migration in rabbits in vivo. circ res 1999;85:377-85. 35. rossouw je, prentice ri, manson je, wu ll, barad d, barnabei vm, et al. postmenopausal hormone therapy and risk of cardiovascular disease by age and years since menopause. jama 2007;297:1465-76. 36. world health organization. appropriate body mass index for asian populations and its implication for policy and intervention strategies. lancet 2004;363:157-63. pusparini, wiradharma, herwana vascular cell adhesion molecule-1 in women alvina 126 abstract *department of clinical pathology, medical faculty, trisakti university jakarta correspondence dr. alvina, sppk department of clinical pathology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone. 021-5672731 ext.2403 email : vina_march_dr@yahoo.com univ med 2011;30:126-34. idiopathic thrombocytopenic purpura: laboratory diagnosis and management alvina* may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina idiopathic thrombocytopenic purpura (itp) or immune thrombocytopenic purpura is a disease characterized by low platelet count (<150,000/ìl) caused by autoantibody-mediated platelet destruction and the absence of other causes of thrombocytopenia. acute primary itp is more common in children 2-6 years of age, with similar incidence between males and females, while the chronic form is usually encountered in adults with median age of 40-45 years. the clinical signs of itp are purpura, ecchymosis, petechiae and gastrointestinal tract bleeding, gingival bleeding, epistaxis, and urinary tract bleeding. spontaneous mucosal, intracranial, and gastrointestinal hemorrhage may occur at platelet counts of <10000/ìl. to date, the diagnosis of itp is still arrived at by exclusion, i.e. by elimination of other causes of thrombocytopenia. the diagnosis of itp also requires a medical history (anamnesis), physical examination, platelet count, and examination of a peripheral blood smear. the latter examination in itp shows low numbers of normal-sized platelets, occasionally also giant platelets, while erythrocytes and leukocytes have a normal morphology. the bone marrow is usually normal or shows increased megakaryocytes. assessment of antithrombocyte antibody may assist in establishing the diagnosis of itp. management of itp is based on platelet count and severity of bleeding. treatment is aimed at interfering with antibodies that damage the platelets, by inhibiting the functions of macrophage fcã receptors and decreasing the production of antiplatelet antibodies. thrombopoietin (tpo) receptor agonists including eltrombopag and romiplostim have offered an important new option in treating itp. key words: idiopathic thrombocytopenic purpura, antiplatelet, antibodies, splenectomy, thrombopoietin introduction idiopathic thrombocytopenic purpura (itp) is an acquired disorder characterized by decreased numbers of circulating platelets, known as thrombocytopenia,(1) and resulting in subnormal platelet counts. thrombocytopenia may be subdivided into four grades as follows: 127 univ med vol.30 no.2 grade 1 with a platelet count of 75,000-150,000/ µl; grade 2 with a platelet count of 50,000<75,000/µl; grade 3 with a platelet count of 25,000-<50,000/µl; and grade 4 with a platelet c o u n t o f < 2 5 , 0 0 0 / µl (2) t h e c a u s a t i v e mechanisms of itp are varied, making itp a heterogeneous disorder.(3,4) thrombocytopenia may be caused by failure of or reduction in platelet production or by increased platelet destruction (table 1).(2) in 1735 paul gottlieb werlhof described a disorder, morbus maculosus hemorrhagicus, which became a recognized diagnostic entity at the turn of the twentieth century and is currently called idiopathic thrombocytopenic purpura (itp) or immune thrombocytopenic purpura.(5) this disorder is characterized by a decrease in platelet count down to <150,000/µl, due to autoantibody-mediated platelet destruction, purpuric rash, normal bone marrow, in the absence of other causes of thrombocytopenia.(6,7) although itp has been known for a long time, there are still many unresolved issues regarding the pathogenetic mechanisms, epidemiology, diagnosis and management. itp is distinguished into primary and secondary types. primary itp comprises two forms, acute and chronic. the acute form is more common in children 2-6 years of age, with similar incidences between males and females, while the chronic form is usually encountered in adults with median age of 40-45 years, among whom the prevalence is higher in women, with a female to male ratio of 3:1. the chronic form i s c h a r a c t e r i z e d b y a t h r o m b o c y t o p e n i a persisting for more than 6 months,(8,9) typically has an insidious onset and often requires medical intervention to prevent bleeding.(10) the t r u e i n c i d e n c e o f i t p i n a d u l t s r e m a i n s unknown, but based on one study, the age adjusted prevalence of itp was estimated to be 9.5 per 100,000 persons.(11) thrombopoietin (tpo) is a naturally occurring hormonal growth factor that is primarily produced in the liver. it regulates megakaryocytopoiesis and stimulates platelet production in the bone marrow.(12) pathophysiology i t p i s c a u s e d b y s p e c i f i c p l a t e l e t autoantibodies binding to the platelets. the igg a u t o a n t i b o d y c o a t e d p l a t e l e t s u n d e rg o accelerated clearing in the spleen and liver after binding fc receptors expressed on tissue macrophages. platelet destruction is presumably triggered by antibody, leading to the formation of neoantigens, thus resulting in the production of autoantibodies in amounts sufficient to cause thrombocytopenia. figure 1 gives an explanation about the trigger factors for autoantibody production, which to this date are still unknown.(13) in the i n i t i a l s t a g e , g l y c o p r o t e i n s i i b / i i i a a r e recognized by autoantibodies, while antibodies recognizing glycoproteins ib/ix have not been formed at this stage. autoantibody-coated platelets will bind to antigen presenting cells (apcs), i.e. macrophages, through fc receptors, and are then internalized and degraded. the apcs process not only glycoproteins iib/iiia, but also other glycoproteins. the activated decreased production increased destruction hem atologic malignancies aplastic anemia mds (myelod ysplasia) drugs: chemotherapy hiv her editary thrombocytopen ia cancer metastases in bone marrow immune: itp hiv posttransfusion purpura non-i mmune: dic sepsis ttp-hus table 1. causes of thrombocytopenia(2) 128 figure 1. pathogenesis of autoantibody production in itp.(13) apcs express new peptides on their surface, aided by costimulation (shown by interaction between cd154 and cd40). the new peptides s u b s e q u e n t l y a r e p r e s e n t e d t o t c e l l s , whereupon these activated t cells produce cytokines and activate b cells to produce a n t i b o d y a g a i n s t s p e c i f i c p l a t e l e t glycoproteins.(14-16) in addition to autoantibodymediated platelet destruction, itp-associated thrombocytopenia may also be caused by suboptimal platelet production.(17) clinical manifestations bleeding in itp is manifested by purpura, ecchymoses and petechiae, and mucosal hemorrhages. hemorrhagic vesicles or bullae may be seen in the oral cavity and other mucosal surfaces. gingival bleeding and epistaxis are the most frequent types of hemorrhage. other types of bleeding may occur in the gastrointestinal tract as melena, and in the genitourinary tract as hematuria and menorrhagia.(9) spontaneous mucosal, intracranial and gastrointestinal bleeding occur at a platelet count of <10,000/ ì l .(2) diagnosis to date the diagnosis of itp is still arrived at by a process of exclusion, by eliminating other causes of thrombocytopenia, such as thrombocytopenia associated with autoimmune diseases, exposure to drugs such as heparin or quinine, and hiv or hepatitis c infection.(13,18) thrombocytopenia associated with hiv or h e p a t i t i s c i n f e c t i o n m a y b e c l i n i c a l l y i n d i s t i n g u i s h a b l e f r o m p r i m a r y thrombocytopenia and may occur several years before the development of other symptoms.(19) the diagnosis of itp also requires a medical history (anamnesis), physical examination, platelet count, and examination of a peripheral blood smear. the medical history may eliminate other causes of thrombocytopenia such as drugs or alcohol. on physical examination usually alvina idiopathic thrombocytopenic purpura 129 univ med vol.30 no.2 figure 3. peripheral blood smear with 2 giant platelets.(20) figure 4. bone marrow picture showing increased numbers of mega-karyocytes.(20) figure 2. petechiae and purpura in male patient with itp.(13) signs of bleeding are encountered, such as petechiae, purpura, and conjunctival and mucosal hemorrhages (figure 2). (13) mild splenomegaly may also be found in young patients.(19) laboratory investigations laboratory examination of peripheral blood smears for itp show low numbers of normal-sized platelets, occasionally also giant platelets, while erythrocytes and leukocytes have a normal morphology (figure 3). (20) anemia and iron deficiency from blood loss may also occur.(19) the bone marrow is usually normal or shows increased megakaryocytes (figure 4).(20) in itp patients with hiv infection the bone marrow shows pycnotic and dyspoetic megakaryocytes devoid of cytoplasm, termed naked megakaryocyte nuclei, of unknown etiology.(21,22) bone marrow examination is performed in patients with poor therapeutic outcomes, ( 1 3 ) and may provide relatively significant information in patients aged over 60 years with systemic symptoms or abnormal signs. details of platelet morphology may also b e o b t a i n e d b y m e a n s o f c y t o g e n e t i c examination or flow cytometry.(19,23) the direct antiglobulin test (dat) may also be used for itp diagnosis. according to aledort et al., dat was positive in 22% of 205 patients with itp, but its clinical significance is unclear.(19,24) assessment of antiplatelet antibodies may assist in establishing the diagnosis of itp, although the results may not be positive in all patients with itp, and negative results cannot always be taken as excluding itp. assays for detection of antiplatelet antibodies have been d e v e l o p e d s i n c e 1 9 5 0 , i n v o l v i n g t h r e e phases.(25,26) initially indirect methods were d e v e l o p e d t o a s s e s s s e r u m a n t i p l a t e l e t antibodies. patients’ sera were mixed with normal platelets, and the final outcome could be in the form of either platelet aggregation or lysis. this method is now obsolete, having low sensitivity and specificity due to the presence of many substances in serum, such as immune 130 figure 5. detection of antiplatelet antibody by the monoclonal antigen capture assay.(13) complexes and complement, that are capable of inducing platelet activation, thus leading to false positive results. in the next phase assays were developed for detection of platelet associated igg (pa-igg) and the most commonly used method was the enzyme linked immunosorbent assay (elisa), for detecting platelet-bound immunoglobulin. this assay is sensitive as it is capable of detecting immunoglobulins bound to platelets but is less specific, because platelets from patients with immune as well as nonimmune thrombocytopenia may increase paigg levels. in the third phase specific antibodies against platelet glycoproteins were detected by means of a modified antigen capture enzyme linked immunosorbent assay (mace), as illustrated in figure 5,(13) one example of a mace assay being the monoclonal antibody specific immobilization of platelet antigen assay (maipa). i n t h e m a i pa a s s a y, m o u s e antiglycoprotein antibodies (anti-gpab) bound to platelet glycoproteins (gp) initially form mouse antigpab-gp complexes, which upon addition of auto-antibody (autoab) bearing platelets will form mouse antigpab-gpautoab complexes. these mouse anti-gpabgp-autoab complexes are put into goat antim o u s e a n t i b o d y c o a t e d w e l l s , t o w h i c h enzyme-labeled anti-human ig is added, which binds to the complexes (figure 6).(26) the maipa assay has the advantage of preserving the platelet antigen (gp), as the antigen is p r e s e n s i t i z e d w i t h a n t i b o d y b e f o r e solubilization. other antigen capture assays are the microtiter plate assay and the immunobead a s s a y. i n t h e m i c r o t i t e r p l a t e a s s a y, glycoprotein is added to wells coated with a n t i p l a t e l e t m o n o c l o n a l a n t i b o d y, t h e n antibody-bearing platelets from the patient are added, followed by enzyme-labeled antihuman antibody (figure 6).(26) in the immunobead assay, the following reagents are added to the wells, namely antiplatelet monoclonal antibody attached to beads, glycoprotein complexed with autoantibody-bearing platelets from the p a t i e n t , a n d e n z y m e l a b e l e d a n t i h u m a n antibody (figure 6).(26) alvina idiopathic thrombocytopenic purpura 131 univ med vol.30 no.2 figure 6 . illustration of antigen capture assays.(26) management of itp s e v e r a l f a c t o r s p l a y i n g a r o l e i n management of itp are profuse bleeding, hemorrhagic comorbidity predisposition, complications of specific treatment, activities of living and life style, and tolerance to adverse effects.(19) treatment is rarely indicated in patients with platelet counts above 50,000 per ìl, without bleeding associated with platelet dysfunction or other hemostatic defects, trauma, and surgery.(19,27) the management of itp depends on platelet count and degree of bleeding. treatment is aimed at interfering with antibodies capable of platelet destruction, by inhibiting the function of macrophage fcã receptors and decreasing the production of antiplatelet antibodies.(28) first line treatment includes observation, corticosteroids, ivig, or anti-d.(29) corticosteroid therapy in patients with itp aims at increasing the platelet count through several mechanisms, i.e. reducing the plateletdestroying capability of macrophages, reducing autoantibody production, inhibiting plateletautoantibody binding, and increasing levels of thrombopoeitin for stimulating megakaryocyte progenitors. treatment with corticosteroids, such as prednisone at a dosage of 1-2 mg/kg/ day may increase the platelet count by about 75%. prednisone as initial firstline standard t h e r a p y f o r p a t i e n t s w i t h i t p i s u s u a l l y administered at a dosage of 0.5-2 mg/kg/day, until the platelet count increases by >30,00050,000/ìl, which takes several days to several w e e k s . to p r e v e n t c o m p l i c a t i o n s f r o m c o r t i c o s t e r o i d u s e , p r e d n i s o n e m a y b e administered by tapering off and subsequently stopping, if there is no response after four weeks of therapy.(19) parenteral administration of highdose methylprednisone is indicated for treating p a t i e n t s w h o f a i l t o r e s p o n d t o f i r s t l i n e treatment. (19) there are several studies on corticosteroid therapy in adult patients with itp.(30,31) these studies found that 51.9%-80% o f p a t i e n t s h a d a g o o d r e s p o n s e t o d e x a m e t h a s o n e , w i t h t h e p l a t e l e t c o u n t i n c r e a s i n g a f t e r t r e a t m e n t . anti-d immunoglobulin is one of the treatments for itp, after the food and drug administration (fda) l i c e n s e d t h e u s e o f i n t r a v e n o u s r h ( d ) immunoglobulin (anti-d ivig) in march 1995. ( 3 2 ) anti-d immunoglobulin is only effective in non-splenectomized rh (d) positive patients, where the antibodies are bound to the d a n t i g e n o n r e d b l o o d c e l l s . ( 3 3 ) anti-d immunoglobulin acts by clearance of anti-d coated red blood cells through macrophage fc receptors in the reticuloendothelial system, thus 132 minimizing the clearance of antibody-coated platelets and increasing the platelet count.(32-34) the standard dose of intravenous anti-d immunoglobulin is 50 mg/kg/day and the preparation takes 72 hours to produce a s i g n i f i c a n t i n c r e a s e i n p l a t e l e t c o u n t . treatment with anti-d immunoglobulin is not recommended as a firstline treatment to increase the platelet count in patients with advanced thrombocytopenia.(33) the goal of secondline treatment, such as splenectomy in patients with itp is to attain increased platelet counts. splenectomy is indicated in patients failing to respond to corticosteroid therapy or requiring continuous p l a t e l e t t h e r a p y. s p l e n e c t o m y r e d u c e s interactions between t and b cells involved in a n t i b o d y s y n t h e s i s . ( 1 3 ) i n d i c a t i o n s f o r splenectomy after corticosteroid therapy are the following: a) platelet counts of less than 50,000 per ìl after 4 weeks of therapy; b) platelet counts remaining below normal after 6-8 weeks; and c) normal platelet counts, but decreasing upon reduction of corticosteroid dosage.(14) around 80% of patients with itp respond to splenectomy, with a five-year continuous response being encountered in 66% of patients without the need for additional therapy. around 14% of patients are unresponsive to treatment and about 20% of patients relapse within weeks, months or years afterwards. splenectomy also gives rise to complications, such as bleeding, infections, and thrombosis. since itp and splenectomy are both associated with a risk of thromboembolism, patients with itp should receive appropriate thromboprophylaxis after surgery, and additionally long-term antibiotic prophylaxis, such as phenoxymethypenicillin 250-500 mg or erythromycin 500 mg twice daily.(19,29) in secondline treatment, cyclosporine a may be administered, at a dosage of 2.5 3 mg/ kg/day. the drug is effective as a single agent in patients with itp, but its side effects may make some patients uncomfortable, particularly patients of advanced age.(19,35) i m m u n o s u p p r e s s i o n w i t h o r a l o r intravenous cyclophosphamide is beneficial in p a t i e n t s w h o a r e d i ff i c u l t t o t r e a t w i t h c o r t i c o s t e r o i d s a n d / o r s p l e n e c t o m y. ( 1 9 ) m y c o p h e n o l a t e m o f e t i l ( m m f ) i s a n antiproliferative immunosuppressant useful in certain patients with itp. it is administered at increasing dosages (250 mg up to the optimal dosage of 1,000 mg/day twice weekly), and increases platelet production in 39% of patients with refractory itp.(19,36) however, according to 2011 clinical guidelines there are insufficient data for specific recommendations to use immunosuppressant therapy.(29) a m o r e r e c e n t d e v e l o p m e n t i n i t p management is the use of tpo receptor agonists, such as romiplostim and eltrombopag, for s t i m u l a t i n g p l a t e l e t p r o d u c t i o n t h r o u g h activation of tpo receptors. these preparations are second generation tpo receptor agonists which have been approved by the us food and drug administration (fda) since 2008. the first generation tpo to be studied for clinical application was recombinant human tpo ( r h t p o ) , b u t t h i s p r e p a r a t i o n h a d t h e disadvantage of inducing tpo autoantibody f o r m a t i o n i n h e a l t h y v o l u n t e e r s . ( 3 7 , 3 8 ) romiplostim is injected subcutaneously once weekly at doses of 1–10 ìg/kg.(39) eltrombopag is to be used at an initial oral dose of 50 mg once daily, but not exceeding 75 mg/day,(40) to achieve and maintain a platelet count >50 x 109/l necessary to reduce or prevent the risk of bleeding.(40,41) the drug has to be given on an empty stomach one hour before or two hours after a meal. nplate and promacta are brand names of romiplostim and eltrombopag, r e s p e c t i v e l y, w h i l e r e v o l a d e i s a n eltrombopag-containing preparation that has been eu-approved since 2010 for adult chronic itp.(41) in emergency cases, such as patients requiring immediate surgery, or patients with uncontrolled gastrointestinal or urinary tract hemorrhages, where an increased platelet count is necessary, firstline combination therapy may alvina idiopathic thrombocytopenic purpura 133 univ med vol.30 no.2 be given, such as prednisone and ivig. highdose methylprednisolone is also beneficial for emergencies, and platelet transfusion with or without ivig will increase the platelet count by more than 20,000/ìl in 42% of itp patients with hemorrhage and may possibly attenuate the hemorrhage. administration of antifibrinolytics, such as oral or intravenous tranexamic acid and e p s i l o n a m i n o c a p r o i c a c i d , m a y a l s o b e b e n e f i c i a l f o r p r e v e n t i o n o f r e c u r r e n t h e m o r r h a g e i n p a t i e n t s w i t h s e v e r e thrombocytopenia, although their effectiveness has not been evaluated in patients with itp.(19) conclusions idiopathic thrombocytopenic purpura is a disorder characterized by decreased platelet c o u n t s d u e t o p l a t e l e t b o u n d s p e c i f i c autoantibodies, accompanied by clinical signs of hemorrhage. management of itp depends on the platelet count and degree of bleeding. tpo receptor agonists including eltrombopag and romiplostim are an important new option in treating itp. references 1. 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review of eltrombopag (revolade) for the treatment of adult chronic immune (idiopathic) thrombocytopenic purpura (itp): summary of the scientific assessment of the committee for medicinal products for human use (chmp). haematologica. 2011; 96 doi:10.3324/haematol. 2011.048819. may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 physicians' awareness and practice of home blood pressure measurement in indonesia: asia home blood pressure monitoring survey 2020 yuda turana1* , noriko matsushita2 , ebtehal salman3 , arieska ann soenarta4 , vidya gani wijaya1, bambang widyantoro4 , eka harmeiwaty5, rarsari soerarso4 , and tunggul diapari situmorang6 background abstract 1school of medicine and health hypertension is a significant mortality risk factor. the knowledge and practice among physicians of hypertension management and home blood pressure monitoring (hbpm) in blood pressure control is important. we aimed to investigate the awareness and practice of hbpm among physicians in indonesia after publishing of the local 2019 hbpm guidelines. methods this was a cross-sectional study involving 611 physicians in indonesia that was conducted between february and october 2020. the questionnaire covered awareness, knowledge, and practice of hbpm. results a total of 330 male physicians (54.0%) aged 20-49 years participated in the survey. more than half were specialists (51.6%) and recommended hbpm to their patients with hypertension (89.0%). the awareness of hbpm benefits was substantial among the physicians; however, the knowledge of the home blood pressure (hbp) reference values of was low (7.9%). around 54% of the respondents thought that the barriers to low recognition of hbpm are lack of hbpm guidelines or lack of understanding of hbpm among physicians. a considerable percentage provided instruction on hbpm that aligned with the local guidelines, but between 7.5-29.5% gave no instruction on hbpm. conclusion most physicians recommend hbpm, but there is still a lack of knowledge and attitude toward hbpm. in indonesia, local hbpm guidelines were published in 2019, but have not yet sufficiently penetrated the country, therefore robust dissemination of the published hbpm guidelines is still needed. for efficient utilization of hbpm by physicians in clinical practice, developing user-friendly educational tools such as physicians’ pocket guide on hbpm instructions is essential. keywords: attitude, hypertension, hbpm, knowledge, physicians sciences, atma jaya catholic university of indonesia, jakarta, indonesia 2global medical affairs, asia pacific region, omron healthcare singapore, singapore 3technology development hq, strategic clinical r&d department, omron healthcare co., kyoto, japan 4department of cardiology and vascular medicine, faculty of medicine, universitas indonesia national cardiovascular center harapan kita, jakarta, indonesia 5department of neurology, national cardiovascular centre, harapan kita hospital, jakarta, indonesia 6division of nephrology and hypertension, department of internal medicine, siloam hospital jakarta, indonesia *correspondence: prof. yuda turana, md, phd school of medicine and health sciences, atma jaya catholic university of indonesia, jl. pluit raya no. 2, jakarta 14440, indonesia phone: +62 21 5885125 email: yuda.turana@atmajaya.ac.id orcid id: 0000-0003-4527-0285 date of first submission, may 18, 2023 date of final revised submission, august 15, 2023 date of acceptance, august 21, 2023 original article doi: http://dx.doi.org/10.18051/univmed.2023.v42:182-194 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1472 182 mailto:yuda.turana@atmajaya.ac.id https://orcid.org/0000-0003-4527-0285 https://orcid.org/0009-0002-5878-7523 https://orcid.org/0000-0002-9572-0814 https://orcid.org/0000-0003-0907-3858 https://orcid.org/0000-0001-5325-4125 https://orcid.org/0000-0002-4992-6686 https://univmed.org/ejurnal/index.php/medicina/article/view/1472 183 univ med vol. 42 no. 2 introduction in indonesia, 37% of total deaths are caused by cardiovascular diseases (cvds). especially stroke is a serious problem in the country, causing 21% of deaths, whereas 9% of deaths is due to coronary heart disease (chd).(1) high blood pressure (bp) is a known prominent risk factor for cvds. the indonesia family life survey (ifls) conducted in 2016 estimated high bp as an attributable factor to 37% of strokes in males and 39% in females, while accounting for 20% of chd in males and 25% in females.(2) the prevalence of hypertension in indonesia is estimated at around 34%, which makes it one of the he alth burdens affecting the c ountry economically and socially.(3) measured bp is the indicator of an individual’s bp value and diagnosis of hypertension. however, according to the ifls national survey, 70% of participants answered to have measured their own bp for the very first time making the screening for hypertension in the country still limited.(4) one key aspect in preventing cvds is keeping bp under control; however, the indonesia family life survey (ifls) national study revealed that the awareness of hypertension was 37% and the bp control rate was only 25% among hypertensive patients in indonesia. another study conducted in indonesia by the asiabp@home study group showed that the bp control rate among 202 hypertensive patients was 23% and sustained uncontrolled bp was 54%, while white-coat hypertension was 13% and masked hypertension was 9%, using both clinic bp (cbp) and 7-day home bp (hbp).(5) the available evidence on bp control is based on routine cbp; nevertheless, this index has the limitation of assessing bp only on one occasion. additionally, recent evidence shows the cite this article as: turana y, matsushita n, salman e, soenarta aa, wijaya vg, widyantoro b, harmeiwaty e, soerarso r, situmorang td. physicians' awareness and practice of home blood pressure measurement in indonesia: asia home blood pressure monitoring survey 2020. univ med 2023;42:182-94. doi: 10.18051/univmed.2023.v42:182-194. this open access article is distributed under a creative commons attribution-non commercialshare alike 4.0 international license superiority of out-of-office bp measurement, more specifically home blood pressure monitoring (hbpm), in identifying white-coat and masked hyper te nsion, impr oving the medications adherence and patient treatment compliance, and hypertension management.(6-9) the indonesian hypertension guidelines recommend hbpm for its affordability and practicality in comparison to ambulatory blood pressure monitoring (abpm), which allows for multiple blood pressure measurements of bp at the convenience of the patient’s own environment and as confirmatory measurement of cbp in the diagnosis and treatment of hypertension. a national survey in 2019 targeting general practitioners and specialists revealed that most healthcare practitioners recommend hbpm in hype rtension manage me nt; however, the knowledge of the hbpm technique among physicians is still lacking.(10) to help implement hbpm in clinical practice in indonesia, the indonesian society of hypertension (inash) established hbpm guidelines for physicians in 2019, which focused on key aspects of hbpm including clear hbpm technique and clinical benefits.(11) the physicians’ awareness and the usability of the hbpm guidelines in their practice are important features of managing hypertension. thus far, several studies have investigated the knowledge, attitude, and practice of hbpm among physicians in relation to hypertension guidelines. a paper by xavier et al.(12) reported that among french general practitioners (gp) 94.5% use hbpm for diagnosis and 92.5% for monitor ing hypertension. a recent survey conducted in 2020 among 11 asia pacific countries, including indonesia, showed that the recommendation of hbpm by physicians in asia 184 data collection the survey was distributed using an online panel through surveymonkey and an offline platform during a national annual scientific meeting (indonesian society of hypertension conference). the questionnaire was composed of 37 questions and sub-questions which covered the respondents’ awareness, knowledge, and practice of hbpm, including questions on the recommendations of hbpm and device type, barriers to hbpm recognition, benefits of hbpm, instructions regarding hbp measurement, and knowledge of diagnostic bp reference value for hypertension (supplementary material 1). the key elements of hbpm from the survey were compared with the recommendations in the 2019 local hbpm guidelines. additionally, the physicians’ perceptions of their patients’ ownership of hbpm device, measuring of hbp, and recognition of hbpm and the barriers to recognition among their patients were explored through the survey questions. statistical analysis the statistical analysis was done as descriptive statistics for the proportions of respondents per que stion expressed as percentages only. all analyses were performed on jmp statistical software, version 15.2.1 (sas institute, cary, nc). ethical clearance informed consent was obtained directly from the participants. this study was approved by and received ethical clearance from the school of medicine and health sciences, atma jaya catholic university of indonesia (no.12/05/kepfkuaj/2017). results a total of 611 respondents participated in this study. there were more males than females among the participants (54.0% males vs 41.9% females), and the age proportion fell gradually is high (95.9%) nevertheless, the knowledge and instruction of hbpm were low (at 22.4% and 54.1%, respectively).(13) in the survey, almost half the physicians in asia attributed the barriers to the dissemination of hbpm to la ck of understanding of hbpm and lack of hbpm guidelines. indonesia published its local hbpm guidelines in 2019; however, the results of the survey show low understanding of hbpm, which could be related to low dissemination of the local guidelines. as part of the same survey, we aimed to scale the dissemination of hbpm guidelines and assess the knowledge, attitude, and practice of hbpm after the dissemination of the local hbpm guidelines and to explore the barriers to dissemination of hbpm among physicians and patients in indonesia. methods research design this explorator y survey was an observational cross-sectional study conducted in jakarta, indonesia, between february and october 2020 as a part of the asia hbpm survey 2020. the overall results of the asia hbpm 2020 survey which was conducted in 11 asian countries (china, india, indonesia, japan, malaysia, the philippines, singapore, south korea, taiwan, thailand, and vietnam) were recently published.(13) research subjects the participants eligible for the survey were: 1) healthcare professionals (hcp) and 2) living in indonesia. the primary sample size target was 1000 hcp; nevertheless, due to covid-19 pandemic, the recruited sample size was 792. to focus on the awareness of hbpm by physicians only (including gener al practitioners a nd specialist), nurses and other hcp were excluded. physicians with mis sing data from the characteristics section of the questionnaire (questions 1-5) were excluded from the analyses leading to the final sample size being 611. turana, matsushita, salman, et al hbp awareness among physicians in indonesia 185 univ med vol. 42 no. 2 (figure 1). moreover, only 34.9% of physicians utilize hbpm to evaluate their patients’ cvd risk. in respect of the knowledge of hypertension diagnostic reference values of 140/90 mmhg for cbp and 135/85 mmhg for hbp, 45.5% of the physicians knew the reference value for cbp, while only 7.9% answered the correct reference value for hbp (table 2). a considerable 53.9% of the physicians answered that systolic hbp and systolic cbp are of the same value, whereas less than a quarter of the respondents (24.4%) answered that the value of hbp is less than that of cbp. among the respondents, 89.0% recommend hbpm to their hypertensive patients, which shows high acceptance of hbpm among the responding physicians. however, out of 498 physicians who manage hypertension patients, the perceived percentage of patients that own a hbpm device was 22.3%, whereas only 23.4% of patients measure their hbp (figure 2). the attitude of the other physicians in the country was investigated by asking the respondents about the recognition level of hbpm among physicians in indonesia and the barriers to the dissemination of hbpm. among the respondents, 36.5% think hbpm is well known by other physicians in the from the participants aged 20 years to those 70 years of age (table 1). nearly 52% of the physicians were specialists. the majority of the physicians worked at hospitals (76.3%) and on average they managed 46 hypertension patients per week. regarding the physicians’ knowledge, they were asked about their awareness of hbpm benefits and their knowledge of hypertension reference values evaluated by cbp and hbp. the physicians’ knowledge of hbpm benefits was substantial for hypertension management (69.6%), the diagnosis of white-coat hypertension (59.6%), and masked hypertension (55.3%), but less than 50% of the physicians knew hbpm as a tool to evaluate anti-hypertensive medication efficacy characteristics n (%) gender male 330 (54.0) female 256 (41.9) age (years) 20-29 154 (25.2) 30-39 137 (22.4) 40-49 114 (18.7) 50-59 106 (17.3) 60-69 56 (9.2) ≥70 41 (6.7) no answer 3 (0.5) specialty general practitioner (gp) 280 (45.8) specialist 315 (51.6) other specialty 6 (1.0) no answer 10 (1.6) specialist cardiologist 67 (21.3) internist 6 (1.9) nephrologist 13 (4.1) neurologist 79 (25.1) other 0 (0.0) no answer 150 (47.6) workplace hospital 466 (76.3) clinic 114 (18.6) other 22 (3.6) no answer 9 (1.5) number of hypertension patients/week, n 45.6 table 1. distribution of physicians’ characteristics (n=611) values are expressed as n (%) unless otherwise stated n (%) cbp (mm/hg) 130/80 57 (9.3) 135/85 7 (1.2) 140/90 278 (45.5) other 182 (29.8) no answer 87 (14.2) hbp (mm hg) 130/80 66 (10.8) 135/85 48 (7.9) 140/90 211 (34.5) other 202 (33.1) no answer 84 (13.7) home systolic bp < clinic systolic bp 149 (24.4) home systolic bp = clinic systolic bp 329 (53.9) home systolic bp > clinic systolic bp 39 (6.4) table 2. awareness of hypertension reference value among physicians in indonesia (n=611) values are expressed as n (%); cbp: clinic blood pressure; hbp: home blood pressure; bp: blood pressure 186 figure 1. the physicians’ knowledge of hbpm benefits (n= 611) hbpm: home blood pressure monitoring; hbp: home blood pressure; cvd: cardiovascular disease; bp: blood pressure and accuracy of hbpm device are a barrier to the dissemination of hbpm in the country (figure 3b). apart from the physicians’ point of view on hbpm, the participants were asked questions about their patients’ attitude toward hbpm. the knowledge of hbpm among the patients was perceived to be high among 31.1%, moderate among 26.2%, and poor among 33.1% (figure country, while 41.4% thought it is moderately known, and 12.8% believe it is poorly known (figure 3a). the barriers causing the moderate and poor knowledge of hbpm among physicians in indonesia were lack of guidelines for hbpm (47.1%), lack of understanding of hbpm (43.5%), and low recommendation of hbpm in the guidelines (35.1%), whereas around one-third of the physicians think that concerns of reliability figure 2. physicians’ recommendations of hbpm to patients and perceived percentage of patient ownership of bp device and hbp measurement in indonesia hbpm: home blood pressure monitoring; hbp: home blood pressure; bp: blood pressure turana, matsushita, salman, et al hbp awareness among physicians in indonesia 187 univ med vol. 42 no. 2 b a figure 3. physicians’ views on other physicians’ knowledge of hbpm in indonesia. a) answer to “do you think that the significance of hbpm is well known by physicians in your country?”. b) the barriers of moderate or poor knowledge of hbpm by other physicians in indonesia hbpm: home blood pressure monitoring 188 a b figure 4. physicians’ views on patients’ knowledge of hbpm in indonesia. a) answer to “do you think that significance of hbpm is well known by patients with hypertension in your country?”. b) the barriers of moderately or poorly recognition of hbpm by patients in indonesia hbpm: home blood pressure monitoring turana, matsushita, salman, et al hbp awareness among physicians in indonesia 189 univ med vol. 42 no. 2 4a). the main barriers to adequate knowledge of hbpm among the patients were their lack of understanding of hbpm (66.9%), high cost of the bp monitoring devices (61.6%), low educational level of the patients (56.1%), and lack of recommendation to patients by physicians (45.0%) (figure 4b). with respect to th e pr actic e of the physicians, a large percentage of the respondents recommends hbpm, in line with the recommendations in the local hbpm guidelines regarding the type of device being the automatic digital device (75.3%) and the upper-arm cuff device (88.2%) (table 3). on the instructions to measure hbp, a considerable proportion of the participants’ answers were in line with the hbpm guidelines on morning hbp measurement one hour after waking up (84.1%), after micturition (48.9%), in the sitting position (62.7%), before breakfast (47.3%), and in the evening before bedtime (62.9%). a noticeable fraction of the participants, ranging between 7.5-29.5% , provided no instructions on the preceding guided steps of hbp measurement. in regard to taking antihypertensive medications in the morning, the local guidelines specified measuring hbp prior to taking the medications. however, only 36.5% of the respondents answered “prior to taking the medication”, 35.8% answered “after taking the medication”, and 18.3% gave no instruction on taking the medication. discussion this survey was conducted to obtain the indonesian physicians’ knowledge, attitude, and practices on hbpm after the dissemination of the local hbpm guidelines. with respect to the physicians’ knowledge of the reference value of hypertension diagnosis, less than half the physicians knew the cbp reference value, whereas under 10% knew the hbp reference value. the main barriers to the physicians’ awareness of hbpm in the country were anticipated to be due to the lack of hbpm guidelines and low recommendations of hbpm in the guidelines, in addition to the physicians’ lack of understanding of hbpm. on the practice of hbpm that derived fr om the physicians’ instructions on hbpm to their patients, the majority of the physicians was in line with the recommendations. in comparison with local hbpm guidelines, however, a substantial percentage of the physicians gave no instructions on hbpm. the findings from the survey highlighted the still poor dissemination of the indonesian hbpm guidelines among the physicians in the country and the fact that robust and effective dissemination and implementation of the local hbpm guidelines in clinical practice is still needed in indonesia. we found that 89.0% of the physicians recommended hbpm to their patients, which percentage was higher than in the other surveys conducted in spain (67%), and us (32%).(14,15) this finding in comparison to that of the other surveys shows that indonesian physicians accept and recommend hbpm to a greater extent than do physicians in other countries. even though the recommendation is high among the physicians, around 7.5 29.5% gave no instructions on measuring hbp to their patients, and 18.3% gave no instructions on the timing of taking antihypertensive medications, whereas an equal number of physicians instructed their patients to measure hbp before and after taking antihypertensive medications. additionally, only 47.3% of the physicians instructed their patients to measure hbp before breakfast, whereas around 23% gave no instructions. a similar survey in japan among physicians exploring the same aspects of hbpm instructions showed that a higher proportion of physicians instructed their patients to measure hbp before taking antihypertensive medications (74.7%) and before breakfast (79.2%).(16) since japan was the first country in the world to establish guidelines on selfmonitoring blood pressure at home in 2003,(17) the results of the japanese survey show that efficient dissemination of the hbpm guidelines in clinical practice leads to proper instructions on hbpm 190 values are expressed as n (%); hbpm: home blood pressure monitoring table 3. the instructions the physicians recommended to their patients on practicing hbpm (n=611) hbpm practice n (%) type of hbpm device recommended to patients automatic digital device 460 (75.3) aneroid device 24 (3.9) mercury device 70 (11.5) no answer 57 (9.3) type of automatic device recommended to patients upper-arm-cuff device 539 (88.2) wrist device 18 (3.0) finger device 2 (0.3) none specified 6 (1.0) no answer 46 (7.5) instructions on hbpm measurement morning timing of measurement after waking up just after 218 (35.7) within 30min 218 (35.7) within an hour 78 (12.8) no instruction 46 (7.5) no answer 51 (8.3) micturition before 77 (12.6) after 299 (48.9) no instruction 180 (29.5) no answer 55 (9.0) body position sitting position 383 (62.7) recumbent position 109 (17.8) no instruction 64 (10.5) no answer 55 (9.0) time of rest before measurement (min) none 20 (3.3) 1–2 50 (8.2) 3–4 38 (6.2) ≥ 5 390 (63.8) no instruction 59 (9.7) no answer 54 (8.8) taking antihypertensive drug before 223 (36.5) after 219 (35.9) no instruction 112 (18.3) no answer 57 (9.3) breakfast before 289 (47.3) after 125 (20.5) no instruction 142 (23.2) no answer 55 (9.0) evening before dinner 58 (9.5) after dinner 57 (9.3) before bedtime 384 (62.9) other 2 (0.3) no instruction 48 (7.9) no answer 62 (10.1) turana, matsushita, salman, et al hbp awareness among physicians in indonesia 191 univ med vol. 42 no. 2 by physicians to their hypertensive patients. japan could be taken as an example that can also be locally emulated, since indonesia already has local hbpm guidelines. the inconsistent instructions on the timing of hbpm before anti-hypertensive medications to hypertensive patients in indonesia as seen in this survey is troubling, since this point is mentioned clearly in both local and regional hypertension and hbpm guidelines.(17-21) proper education on the instructions to measure hbpm, especially before taking any hypertension medications, is vital to accurate measurement of hbp and efficient utilization of its value in hypertension management. accurate diagnosis of hypertension stands at the top of appr opriate hyper te nsion management, in which the key lies in the physicians’ knowledge of the standard reference values to diagnose hypertension. a moderate proportion of physicians in this survey understands the use of hbp to diagnose white-coat (59.6%) and masked hypertension (55.3%). however, the physicians’ knowledge of the reference value to diagnose hypertension by hbp is considerably low (7.9%). this low understanding of the standard reference value of hbp to accurately diagnose white-coat and masked hypertension may lead to the misdiagnosis of these phenotypes of hypertension. the local hbpm guidelines, as well as global guidelines, indicate the clear definition of correct diagnosis of hypertension by hbp to be 135/85 mmhg.(22,23) nevertheless, regardless of the source of information, the indonesian physicians’ utilization of the guidelines for hbp reference values still seems to fall short and attention should be focused on providing userfriendly, clear, and concise standard education to the physicians on the basic knowledge of hypertension diagnosis and management, especially using hbpm. the benefits and applicability of hbpm in clinical practice range from the accurate diagnosis of hypertension phenotypes to decisions on therapy, titration of bp-lowering medications, and monitoring of bp control in patients.(17,24,25) the physicians and healthcare providers are the primary source of information for the patients in recommending hbpm and instructing the patients appropriately on the method of bp measurement at home. a study by wake et al.(26) showed that the tendency of patients to selfmonitor bp is six times greater when they receive recommendations from health professionals to self-monitoring of bp. another study revealed that the healthca re provider ’s a dvice was the prominent contributor to hbpm device ownership and weekly practice of hbpm by the patients, with the odds ratio being at 13.50 and 8.97, respectively.(27) this available evidence further magnifies the importance of physicians’ proper education and knowledge of hbpm. from our study, even though a high proportion of the physicians recommend hbpm, a substantial percentage think that the main barrier to hbpm recognition among other physicians in the country is lack of guidelines, low recommendations of hbpm in the guidel ines, and lack of understanding of hbpm by the physicians. the findings from this survey reveal that thus far the available local hbpm guidelines have not yet penetrated greatly among the physicians and that their awareness of the instructions in the guidelines is still low. more robust and practical methods to disseminate the local hbpm guidelines are essential and finding alternative platforms for this dissemination may help in reaching a wider spectrum of physicians and healthcare professionals to efficiently bring about the awareness of and proper instruction on hbpm. furthermore, developing user-friendly educational tools, such as a physicians’ pocket guide for clinical practice, may play an important role as a reachable source of knowledge of hbpm instructions. the survey also studied the knowledge of the patients on hbpm in indonesia. around 59% of the physicians answered that hbpm is moderately or poorly known among their patients, the main reason being a lack of understanding o f hbpm a mo ng t he p a t i e nt s . bo t h t he ownership of the hbpm device and hbp measurement were considerably low among the 192 patients as perceived by the respondents (22.3% and 23.4%, respectively). a survey study in singapore among hypertensive patients showed that even though the awareness rate of hbpm among the patients was 61.7%, only 24.0% actually used hbpm.(28) the results from our survey were comparably low as perceived by physicians in indonesia. the measurement rate of hbpm seems still to be low in this country, and raising the awareness of hbpm among patients through education and through active recommendation of hbpm by physicians are the key factors to improving the practice of hbpm among patients in indonesia. the strength of this study is that it is a part of the asia hbpm survey 2020 that was conducted in 11 asia pacific countries, and was published recently by wang et al.,(13) making it possible to compare the results with neighboring countries. additionally, the study was conducted using both online and offline platforms which increased the participation rate, hence the high number of respondents. nevertheless, the study has the limitation of being susceptible to selection bias, as a larger proportion of the respondents was recruited during a local annual scientific meeting and almost a quarter of the specialists who responded were neurologists, a specialty that does not primarily manage hypertensive patients. additionally, the study results of the patients are perceived from the physicians’ responses, therefore a comparative survey from the patient’s point of view is needed. lastly, although the sample size is moderately sufficient, the survey needs to be conducted on a larger sample size to be representative of the indonesian physicians’ population and at a nationwide level, as the current survey was conducted mainly in jakarta. the survey results revealed that even though the majority of physicians recommend hbpm to their patients, the knowledge, attitude, and practice of hbpm among the physicians were suboptimal. these findings after the establishment of the local hbpm guidelines show that much work is needed to disseminate the guidelines properly and effectively among physicians and to provide the educational tools to implement hbpm in clinical practice. moreover, another survey a couple of years after the initiative of educational tools and guidance of hbpm is needed to evaluate the effectiveness of the activities and to assess the knowledge and application of hbpm in practice and understanding of the importance of hbpm in hypertension management. conclusion in conclusion, the majority of the physicians in indonesia recommends hbpm to their patients; however, the knowledge, attitude, and practice of the physicians were suboptimal. the awareness and dissemination of the local hbpm guideline published in 2019 in indonesia are still low and robust efforts should be dedicated to disseminating the local guidelines among the physicians and healthcare providers in the country, in addition to developing more user-friendly educational tools such as a physicians’ pocket guide of hbpm instructions to be utilized in clinical practice. conflict of interest es is an employee of omron healthcare co. ltd. nm is an e mployee of omron healthcare singapore pte. ltd. the other authors declare that there is no conflict of interest regarding the publication of this paper. acknowledgments we appreciate the contribution of physicians, medical technicians, and the expert committees for orga nizing the indone sian society of hypertension conference which was one of the main platforms for conducting this survey. contributors yt, es, and nm c ontr ibuted to the conception, study design, interpretation, and writeup; yt, es, nm, vgw, aas, bw, eh, rs, and turana, matsushita, salman, et al hbp awareness among physicians in indonesia 193 univ med vol. 42 no. 2 tds contributed to data collection for analysis and writeup of the draft and final manuscript. yt and vgw oversaw the data collection process and overall research work including interpretation of results, and critically reviewing and revising the manuscript. all authors read and approved the final version to be submitted for publication. data availability statement the data used to support the findings of this study is available from the corresponding author upon request. fundings this survey was funded by omron healthcare co., ltd., japan. award/grant number: n/a. patient and public involvement no patients were involved in the study. the participants were physicians and they were not involved in the design, or conduct, or reporting, or dissemination of the plans of this research. provenance and peer review not commissioned; internally peer-reviewed. references 1. cardiovascular division & health services research centre. reducing the burden of cvd in indonesia. newtown: the george 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jp, stevens r, et al. selfmonitoring of blood pressure in hypertension: a systematic review and individual patient data meta-analysis. plos med 2017;14:e1002389. doi: 10.1371/journal.pmed.1002389. 25. george j, macdonald t. home blood pressure monitoring. eur cardiol 2015;10:95-101. doi: 10.15420/ecr.2015.10.2.95. 26. wake ad, bekele dm, tuji ts. knowledge and attitude of self-monitoring of blood pressure among adult hypertensive patients on follow-up at selected public hospitals in arsi zone, oromia regional state, ethiopia: a cross-sectional study. integr blood press control 2020;13:113. doi: 10.2147/ibpc.s242123. 27. zuo hj, ma jx, wang jw, chen xr. assessing the routine-practice gap for home blood pressure monitoring among chinese adults with hypertension. bmc public health 2020;20: 1770. doi: 10.1186/s12889-020-09901-0. 28. tan nc, khin lw, pagi r. home blood-pressure monitoring among hypertensive patients in an asian population. j hum hypertens 2005;19:55964. doi: 10.1038/sj.jhh.1001865. turana, matsushita, salman, et al hbp awareness among physicians in indonesia alvina 173 abstract sedentary lifestyle gives rise to important health problems in the world, such as cardiovascular disorders and hypertension. hypertension is an important modifiable risk factor for cardiovascular disease and is the most significant risk factor for stroke. there is mounting evidence that physical activity is beneficial for the elderly, and may have positive effects on chronic disorders, such as hypertension. the purpose of the present study was to assess the effectiveness of physical activity for reducing hypertension and the healthcare costs of hypertension in the elderly. this cross-sectional study, conducted from august to october 2009, involved 237 community-dwelling elderly in one district of south jakarta, comprising 86 males and 151 females aged 66.4 ± 6.3 years. data on background characteristics were collected using a validated questionnaire, while physical activity was assessed using the short version of the international physical activity questionnaire. results showed that 32.9% elderly was physically active and 67.1 % less physically active. the overall prevalence of hypertension was 18.6%, while there was no significant difference of hypertension between male and female elderly (p=0.737). the risk for hypertension in the physically active group was 0.40 times lower than that in the less physically active group (or = 0.40; 95% c.i. 0.16 – 0.97), leading to annual healthcare cost savings in the physically active group of 60%. in conclusion, physical activity reduces hypertension and the associated healthcare expenditure among community-dwelling elderly. key words : physical activity, hypertension, cost-effectiveness analysis, elderly *department of anatomy pathology, faculty of medicine. trisakti university **department of community medicine, faculty of medicine, trisakti university correspondence dr. robert d. gandasentana, dr, ms department of anatomy pathology, medical faculty, trisakti university jalan kyai tapa no. 260, grogol jakarta barat – 11440 phone: 021-5672731 ext.2703 email: robsentana@yahoo.com univ med 2011;30:173-81 physical activity reduced hypertension in the elderly and cost-effective september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina robert d. gandasentana* and rina k. kusumaratna** introduction the aging individual is subject to a reduction in musculoskeletal mass, which is largely due to physical inactivity, as most elderly are commonly unemployed or retired and generally pay less attention to their physical health. in addition, the elderly frequently suffer f r o m c h r o n i c d i s e a s e s , p a r t i c u l a r l y h y p e r t e n s i o n a n d o t h e r c a r d i o v a s c u l a r disorders. a world health organization (who) report estimated that 80% of adults aged 65 174 gandasentana, kusumaratna physical activity reduced hypertension in elderly years and older has more than one chronic health condition and that 50% has more than two chronic health conditions.(1) hypertension is an independent and powerful prognostic indicator for cardiovascular and renal disease, whereas it is significantly associated with the increased morbidity and mortality from cerebrovascular disease, myocardial infarction, congestive heart failure and renal insufficiency.(2,3) arterial hypertension is highly prevalent in the elderly, and according to the national health and nutrition examination survey iii study, its prevalence rate for subjects >60 years old (white non-spanish speaking americans) is estimated to be >60%. (4) h y p e r t e n s i o n p r e v a l e n c e increases with advancing age and is higher in men than in women until the age of 55 years, but is slightly higher in postmenopausal women.(5) hypertension is the major risk factor for stroke, heart failure, and coronary artery disease in older adults, while all of these disorders are important contributors to mortality and functional disability. hypertension is defined as a systolic blood pressure of 140 mmhg or greater and/or a diastolic blood pressure of 90 mmhg or greater. isolated systolic hypertension is defined as a systolic pressure of 140 mmhg or greater with a diastolic pressure of less than 90 mmhg.(6) according to the most recent national american guidelines for hypertension, values of 120-139 mm hg and 80-89 mmhg, for systolic and diastolic blood pressure respectively, are characterized as a precursor stage of hypertension, since these values are b e i n g a s s o c i a t e d w i t h i n c r e a s e d r i s k o f hypertension development compared to lower values of arterial pressure.(7) sedentary lifestyle is a major risk factor for cardiovascular disease and being physically active reduces risk by 40%.(8) several studies support the important role of physical activity for both primary and secondary prevention of cardiovascular diseases,(9) and according to epidemiological evidence there exist positive associations between regular physical activity and health benefits. (10) the who recommends at least 30 minutes of regular, moderate-intensity physical activity on most days of the week to reduce the risk of diseases.(10) to change physical activity behavior and promote regular physical exercise among community-dwelling elderly, there is a need for cost-effectiveness analyses using standardized methods to determine the costs and benefits of participation in physical activity.(11) outside of indonesia, a large number of interventions have been conducted to promote physical activity, ranging from individual counseling by general practitioners to massmedia campaigns. the cost-effectiveness of several interventions have been evaluated using v a r i o u s m e t h o d o l o g i e s . c o b i a c e t a l . (8) performed cost-effectiveness analyses on six intervention programs, and concluded that all six could lead to substantial improvement in population health at a cost saving to the health sector. cobiac and colleagues modelled the costs a n d h e a l t h o u t c o m e s a s s o c i a t e d w i t h interventions to improve physical activity in the population, and identify specific interventions that are likely to be cost-saving. rubinstein et al. (12) used generalized cost-effectiveness (gce) analysis, a who-designed methodology, to evaluate a cvd reduction intervention package in buenos aires. d e s p i t e t h e h i g h p r e v a l e n c e o f hypertension in older adults, it should not be considered a normal consequence of aging. primary hypertension is the result of multiple c o n d i t i o n s , s u c h a s g e n e t i c , n u t r i t i o n a l , psychosocial, and life style factors. one of the life style factors leading to hypertension is obesity, which is usually related to a sedentary life style and inadequate physical activity. therefore one way to prevent hypertension or at least decrease the risk of hypertension is to perform habitual physical activity (exercise), including work-related activity and activities of daily living. in general, cost-effectiveness analysis has been used by governments and international 175 organizations for analyzing the efficiency of alternative programs, including healthcare intervention programs, such as promotion of physical activity among population groups. h o w e v e r, t h e p r i n c i p l e s o f c o s t effectiveness analysis are presumably universal, and may therefore be applied at the level of the individual, especially where large healthcare expenditures are to be borne by the individual concerned. this is particularly true for most of the middleto low-income groups of indonesian elderly, as many, if not all, of them have no health insurance cover, apart from free but extremely restricted health insurance for the poorest, provided by the provincial governments. in view of the foregoing, the aim of the p r e s e n t s t u d y w a s t o c o n d u c t a c o s t effectiveness analysis of physical activity in relation to primary hypertension in communitydwelling elderly, in order to provide evidence of the economic benefits of physical activity in hypertensive individuals. methods research design the study was an observational crosss e c t i o n a l s t u d y t o d e t e r m i n e t h e c o s t effectiveness of physical activity among community-dwelling elderly with hypertension, conducted from august to october 2009. study subjects the population under study comprised community-dwelling elderly residing in the vicinity of a primary community health center in south jakarta. inclusion criteria were: age 60 years and older, male or female gender, mobile and independent, able to verbally communicate, and apparently healthy. the participants’ health status was determined by self-reported medical history and by physical examination during recruitment, and cross-checked with their medical record at the heath center. the sample size was determined to be around 229 subjects, based on a power of 95% and expected dropout of 20%. the subjects were recruited from a district in south jakarta with a relatively high number of elderly persons of low to medium socioeconomic status among the permanent residents, as indicated by official data from the district health office (sudinkes). cluster random sampling was used to determine the participating villages in the district and the numbers of hamlets (rw) and rt required to yield the calculated sample size. in indonesia, rts are the smallest administrative units comprising around 20 households, similar to neighborhood associations. the study sample consisted of community dwelling elderly selected from each rt in five villages by proportionate simple random sampling. the subjects meeting the inclusion and exclusion criteria gave written informed consent, confirmed by signature for the literate and by thumbprint for the illiterate. physical activity assessment physical activity among the participants was evaluated by a modification of the short f o r m a t i n t e r n a t i o n a l p h y s i c a l a c t i v i t y questionnaire (sf-ipaq), recommended by the who to measure physical activity among adults from 15–69 years.(13) as a consequence, this study could assess only the physical activity status of elderly aged 60–69 years. the 9-item short form assesses time spent on different activities. the short form of ipaq records four types of physical activity: vigorous activity such as aerobics; moderate-intensity activity such as leisure cycling; walking; and sitting. the ipaq-sf contains questions on the frequency (number of days per week), duration (total time spent in physical activity per day), and intensity of physical activity over the previous week ( l i g h t , m o d e r a t e , a n d v i g o r o u s ) . a f t e r assessment of physical activity, the subjects were assigned to two groups, i.e. one group with moderate to high levels of physical activity, and one group with low levels of physical activity, designated in the present study as the physically active group and the less physically active group, respectively. univ med vol.30 no.3 176 gandasentana, kusumaratna physical activity reduced hypertension in elderly blood pressure measurement tw o s i t t i n g b l o o d p r e s s u r e ( b p ) measurements were taken for all subjects and the mean value was used to determine bp status: hypertensive (systolic bp >140 mm hg, diastolic bp >90 mm hg, or not. body mass index height was measured with microtoise with a precision of 0.1 cm. weight was measured with sage portable scales with a precision of 0.1 kg. body mass index (bmi) was calculated as the weight (kg) divided by the square of the height (m2). for asian populations, bmi is classified into the following categories: underweight (<18.5 kg/m2), normal (18.5–22.9 kg/m2), overweight (23.0–27.5 kg/m2), and obese (> 27.6 kg/m2).(14) cost-effectiveness analysis c o s t e f f e c t i v e n e s s a n a l y s i s ( c e a ) generally considers the costs and effects of adding new interventions to current practice or the cost of replacing an existing intervention with another targeting the same condition. the cea in this study indicated that for hypertensive patients attending a health center (puskesmas), t h e h e a l t h c a r e e x p e n d i t u r e i n c l u d i n g medications was approximately rp. 12,000 at the time of the study. patients with hypertension are usually advised to visit their physician once weekly for follow-up, until their bp is adequately controlled, with a systolic bp of <140 mmhg and a diastolic bp of <90 mmhg.(15) therefore the annual healthcare costs for regular followup at a health center is approximately rp. 624,000. ethical clearance ethical clearance was issued by the r e s e a r c h e t h i c s c o m m i t t e e , f a c u l t y o f medicine, trisakti university. statistical analysis the background characteristics were analyzed using descriptive statistics, after c h e c k i n g f o r n o r m a l d i s t r i b u t i o n b y t h e kolmogorov-smirnov test of normality. costeffectiveness of physical activity was analyzed using the odds ratio as a measure of the risk of hypertension among the physically active s u b j e c t s a s c o m p a r e d w i t h t h e r i s k o f hypertension in the less physically active subjects. the level of significance was set at p<0.05. . results background characteristics of the elderly a total of 237 elderly people aged 60–69 years from eight hamlets in the five sub-districts participated in the present study. the socioeconomic status of the subjects consisted of education, history of employment, income sources, health services, history of occupation as well as their housing status. regarding the distribution of socio-economic status of the study subjects, among the 237 elderly there were 138 (58.2%) in the age range of 60-64 years and 99 (41.8%) in the age range of 65-69 years. overall mean age was 63.9 ± 2.6 years. with regard to gender, the subjects comprised 86 (36.3%) males and 151 (63.7%) females. the majority (77.2%) of the elderly had been educated and most of them in their youth had received their formal education at religious elementary schools. with regard to marital status, approximately half of them (55.7%) was widowed or unmarried. a total of 60.8% of the elderly did not have regular income for supporting their daily life and most of the income came from their children. only 13.5% of the elderly had insurance and for access to health services, most of them had the kartu miskin (health services card for the poor). around 80.6% of the elderly lived in their own house (table 1). the prevalence of hypertension was 18.6%, while 32.9% was physical active (table 2). there was no significant difference in the prevalence of hypertension between males and females. similar results were obtained for nutritional status and smoking habits. the results 177 indicated that for elderly with adequate physical activity, the risk of hypertension was 0.4 times lower, as compared with the less physically active elderly (o.r.=0.4; 95% confidence interval 0.16 – 0.97) (table 3). cost-effectiveness of physical activity cost-effectiveness analysis was used to evaluate the impact of physical activity on h y p e r t e n s i o n , b y a s s e s s i n g t h e r i s k o f hypertension in the physically active group as compared to the risk of hypertension in the less physically active group. t h e p r e v a l e n c e o f e l d e r l y w i t h hypertension was 10.3% in the physically active group and 22.0 % in the less physically active group. these data indicate that there w e r e s i g n i f i c a n t l y m o r e e l d e r l y w i t h hypertension in the less physically active group than in the physically active group (p=0.027). the risk for hypertension in the physically active group was 0.40 times lower than that of the less physically active group as shown in table 3. this signifies that the risk of suffering from h y p e r t e n s i o n a m o n g e l d e r l y w h o w e r e physically active was significantly lower than in elderly who were less physically active. from table 4 it is apparent that the cost of a patient in the health center is rp.12,000 per ambulatory visit, amounting to 52 x rp.12,000 = rp.624,000 per year. in subjects who are physically active, the risk of hypertension was 0.40 lower, such that the total cost per year amounts to rp.249,600, yielding a 60% reduction in healthcare expenditure, compared to subjects who are less physical active. discussion for the 237 subjects of the present study, the prevalence of hypertension was 18.6%. a study in costa rica on elderly aged 60 to 80 years (mean age = 76 years) yielded differing results, the prevalence of hypertension being 65.0%.(16) this may have been due to the higher variables n (%) age 60-64 138 (58.2) 65-69 99 (41.8) gender male 86 (36.3) female 151 (63.7) education illiterate 54 (22.8) educated 183 (77.2) marital status married 105 (44.3) widowed/unmarried 132 (55.7) income sources regular 93 (39.2) non-regular 144 (60.8) health services insurance 32 (13.5) non-insurance 205 (86.5) history of occupation enterpreneur 25 (10.5) laborer 19 (8.0) goverment employee 19 (8.0) merchant 35 (14.8) unemployed 139 (58.6) housing status own 191 (80.6) not own 46 (19.4) table 1. socio-economic and demographic profile of the participants (n=273) variables n (/%) nutritional status bmi* kg/m2 (mean ± sd) 21.80 ± 4.04 underweight 55 (23.2) normal 95 (40.1) overweight 87 (36.7) health status chronic 186 (78.1) non-chronic hypertension yes no physical activity active les active 51 (21.5) 44 (18.6) 193 (81.4) 78 (32.9) 159 (67.1) table 2. nutritional, health status, and physical activity of the elderly (n=273) *bmi = body mass index univ med vol.30 no.3 178 gandasentana, kusumaratna physical activity reduced hypertension in elderly age of the subjects, whereas the elderly in our study were relatively younger, with a mean age of 63.9 years. o u r s t u d y s h o w e d t h a t t h e r i s k f o r hypertension in the physically active group was 0.40 times lower than that of the less physically active group as shown in table 3. similar results were obtained in a prospective study of 27,055 apparently healthy women, where vigorous physical activity (1500 kcal/week) led to a reduction in the incidence of hypertension (r.r. = 0.62;95% c.i. (0.51–0.74;p< 0.001).(17) a study in australia showed similar results, in that the prevalence of poor physical activity was higher among the hypertensive elderly.(18) many epidemiologic observational studies have reported a consistent inverse association between physical activity and the risk of incident cardiovascular diseases.(19) even at an advanced age, becoming more active still confers a reduction in coronary heart disease mortality.(20) the cardio-protective effect of physical activity may be related to its beneficial effects on body weight, blood pressure, serum cholesterol and glucose tolerance.(21) cobiac et al.(8) analysed cost-effectiveness of six physical activity intervention programs in community-dwelling frail elderly taken from a larger number of meta-analyses. their analysis indicated that these interventions would be costhypertension risk factors yes (n= 44,/%) no (n=193,/%) p gender male female age (years) 60 – 64 65 – 69 > 70 smoking yes no nutritional status underweight normal overweight physical activity active less active 15 (17.4) 29 (19.2) 25 (18.1) 18 (18.9) 1 (25.0) 8 (17.0) 36 (18.9) 9 (16.4) 24 (18.0) 11 (22.4) 8 (10.3) 35 (22.0) 71 (82.6) 122 (80.8) 113 (81.9) 77 (81.1) 3 (75.0) 39 (83.) 154 (81.1) 46 (83.6) 109 (82.0) 38 (77.6) 70 (89.7) 124 (78.0) 0.737 0.934 0.761 0.709 0.027 o.r.* = 0.40 (95% c.i.=0.16-0.97) table 3. risk factors of hypertension in the elderly (n=237) * o.r.=odds ratio c.i.= confidence interval cost per ambulatory visit cost-effectiveness per year reduction iitervention hc* hc* hc* physical activity 12,000,374,400,60.0% table 4. costs, effects and cost-effectiveness of physical activity on hypertension * hc : health center 179 saving to the health sector, leading to substantial improvements in health for the australian population. although the studies analyzed by cobiac et al.(8) differed from the present study in south jakarta in several respects, one of them being that their subjects were frail elderly, their findings were similar to the results of costeffectiveness analysis of the present study regarding the cost-savings of physical activity in the elderly. the pragmatic, single-blind randomized clinical trial conducted by mccarthy et al.,(22) showed that a home-based exercise program supplemented with an 8-week class-based exercise program yielded significantly better results even 12 months after cessation of the intervention, while their economic evaluation suggested that structured physical activity programs are likely to be cost-effective. the findings of the mccarthy study that elderly conducting regular physical activity have costsavings in health care expenditure, therefore are consistent with the results of abovementioned study, although differing in study design. the beneficial effect of regular exercise in hypertension is not limited to reduction of blood pressure only. it has also been shown to reduce left ventricular hypertrophy,(23) and improve exercise capacity and quality of life.(24) when combined with dietary alterations, regular exercise causes reduction of oxidative stress, increases nitric oxide availability and improves the overall metabolic profile. (25) recently published large prospective epidemiological studies have reported physical inactivity as a predictor of not only cardiovascular but also total mortality in middle-aged men and women.(26,27) this association is strong, and independent of other major risk factors, and illustrates the enormous preventive potential of regular exercise, especially given the high prevalence of a sedentary lifestyle in most communities. it also implies that physical activity also has a protective effect on noncardiovascular mortality, although the beneficial mechanisms are not clearly understood. in general, blood pressure rises with age, certainly up to the seventh decade. the rise is more marked for systolic pressure and is more pronounced in men. hypertension needs life-long medical control. once the subject has been diagnosed as suffering from hypertension, he or s h e i s o b l i g e d t o t a k e a n t i h y p e r t e n s i v e medications for life, thus becoming a burden to the family and society. ideally, the hypertension patient has to visit his or her physician for control of the disorder at regular intervals of one week. for the calculation of cost of healthcare in health centers, the present study used the standard healthcare costs as determined by the department of health.(28) the cost savings for the physically active subjects in comparison to the less physically active subjects was 60%. it is apparent that regular physical activity in older persons results in healthcare cost savings due to reduced risk of hypertension. the costeffectiveness measured in the present study is seen from the viewpoint of the study subjects (on a micro scale), as most did not have health insurance and had to pay for health services out of their own pocket. several limitations of the present study should be taken into consideration. as physical activity was assessed by self-report, it is possible that more precise assessment of this factors may have resulted in a different contribution of this variable to the reduction in hypertension. we also did not have information on other variables that are favorably influenced by physical activity, such as those related to heart rate or autonomic balance, or insulin sensitivity, so we could not evaluate their contributions to the inverse relation between physical activity and hypertension risk. conclusions the prevalence of hypertension in the elderly was 18.6%, while 67.1 % of the subjects w a s l e s s p h y s i c a l l y a c t i v e . t h e r i s k o f hypertension among the elderly who were physically active was 0.4 lower compared to less physically active elderly. in the economic univ med vol.30 no.3 180 gandasentana, kusumaratna physical activity reduced hypertension in elderly evaluation the physically active group had higher annual healthcare cost savings of approximately 60%. future randomized controlled studies will be needed to confirm the potential beneficial effect of physical activity on health status of the elderly and the attendant reduction in healthcare expenditure. acknowledgement we wish to express our gratitude to the d e a n a n d vi c e d e a n s o f t h e f a c u l t y o f medicine, trisakti university, for the funding of this study, to the staff of mampang prapatan district health center and sudinkes of south jakarta for the use of the health facilities, and also to the participants of this study. references 1. world health organization. diet, nutrition and the prevention of chronic disease: joint who/ fao expert consultation. geneva: world health organization;2003. 2. mason pj, manson je, sesso hd, albert cm, chown mj, cook nr, et al. blood pressure and risk of secondary cardiovascular events in women: the women’s antioxidant cardiovascular study (wacs). circulation 2004;109:1623-9. 3. babatsikou f, zavitsanou a. epidemiology of hypertension in the elderly. health sci j 2010;4:2430. 4. ong kl, cheung bm, man yb, lau cp, lam ks. prevalence, awareness, treatment and control of hypertension among united states adults 19992004. hypertension 2007;49:69-75. 5. kannel wb. prevalence and implications of uncontrolled systolic hypertension. drugs aging 2003; 20:277-86. 6. world health organization-international society of hypertension (ish). guidelines for the management of hypertension. j hypertens 1999; 17:151-83. 7. u.s. department of health and human service. the seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. bethesda, md: nhlbi health information center;2004. 8. cobiac lj, vos t, barendregt jt. costeffectiveness of interventions to promote physical activity: a modelling study. plos med 2009;6:e1000110. doi:10.1371/journal.pmed. 1000110. 9. kahn eb, ramsey lt, brownson rc, heath gw, howze eh, powell ke, et al. the effectiveness of interventions to increase physical activity: a systematic review. am j prev med 2002;22:73– 107. 10. world health organization. global strategy on diet, physical activity and health. geneva: world health organization;2004. 11. ministry of health. basic health research 2007. jakarta: health research and development board department of health;2008. 12. rubinstein a, martí sg, souto a, ferrante1 d, augustovski f. generalized cost-effectiveness analysis of a package of interventions to reduce cardiovascular disease in buenos aires, argentina. cost eff resour alloc 2009;7:10 doi:10. 1186/1478-7547-7-10. 13. international physical activity questionnaire (ipaq) research committee. guidelines for data processing and analysis of the international physical activity questionaire (ipaq) : short and long forms. available at: http://www.ipaq.ki.se. accessed october 10, 2009. 14. world health organization. appropriate body mass index for asian populations and its implications for policy and intervention strategies. the lancet 2004;363:157-63. 15. cushman wc, ford ce, cutler ja, margolis kl, davis br, grimm rh, et al. success and predictors of blood pressure control in diverse north american settings: the antihypertensive and lipid-lowering treatment to prevent heart attack trial (allhat). j clin hypertens 2002;4:393-404. 16. méndez-chacón e, santamaría-ulloa c, roserobixby l. factors associated with hypertension prevalence, unawareness and treatment among costa rican elderly. bmc public health 2008;8:275 doi:10.1186/1471-2458-8-275. 17. mora s, cook n, buring je, ridker pm, lee im. physical activity and reduced risk of cardiovascular events: potential mediating mechanisms. circulation 2007;116:2110-8. 18. nelson mr, alkhateeb am, ryan p, willson k, gartlan jg, reid cm on behalf of the second australian national blood pressure management committee. physical activity, alcohol and tobacco use and associated cardiovascular morbidity and mortality in the second australian national blood pressure study cohort. age ageing 2010;39:112– 39. 19. mcneill am, katz r, girman cj, rosamond wd, wagenknecht le, barzilay ji, et al. metabolic syndrome and cardiovascular disease in older 181 people: the cardiovascular health study. j am geriatr soc 2006;54:1317–24. 20. pitsavos c, panagiotakos db, chrysohoou c, kavouras s, stefanadis c. the associations between physical activity,inflammation, and coagulation markers, in people with metabolic syndrome: the attica study. eur j cardiovasc prev rehabil 2005;12:151–8. 21. bianchi g, rossi v, muscari a, magalotti d, zoli m and the pianoro study group. physical activity is negatively associated with the metabolic syndrome in the elderly. q j med 2008; 101:713– 21. 22. mccarthy cj, mills pm, pullen r, richardson g, hawkins n, roberts cr, et al. supplementation of home-based exercise programmme with a classbased programme for people with osteoarthritis of the knees: a randomized controlled trial and health economic analysis. health tech assess 2004;8:46. 23. sherwood a, gullette ecd, babyak m, waugh r, georgiades a. reduction of left ventricular hypertrophy after exercise and weight loss in overweight patients with mild hypertension. arch intern med 2002;162:1333–9. 24. tsai jc, yang hy, wang wh, hsieh mh, chen pt, kao cc, et al. the beneficial effect of regular endurance exercise training on blood pressure and quality of life in patients with hypertension. clin exp hypertens 2004;26:255–65. 25. roberts ck, vaziri nd, barnard rj. effect of diet and exercise intervention on blood pressure, insulin, oxidative stress, and nitric oxide availability. circulation 2002;106:2530–2. 26. barengo nc, hu g, lakka ta, pekkarinen h, nissinen a, tuomilehto j. low physical activity as a predictor for total and cardiovascular disease mortality in middle-aged men and women in finland. eur heart j 2004;25:2204–11. 27. hu g, tuomilehtoa j, silventoinenb k, barengoc n, jousilahtia p. joint effects of physical activity, body mass index, waist circumference and waistto-hip ratio with the risk of cardiovascular disease among middle aged finnish men and women. eur heart j 2004;25:2212–9. 28. department of health. indonesian diagnosis related group fees for general and special c & d type hospitals. jakarta: department of health directorate general for medical services upbuilding;2007. univ med vol.30 no.3 alvina 96 abstract arthritis is a disease of the joints that causes pain and musculoskeletal stiffness, and may cause limitation of joint movement. age, obesity, smoking behavior, and occupation are risk factors for rheumatic diseases. the objective of the present study was to evaluate the role of body mass index (bmi), smoking behavior, and occupation on rheumatic disease among persons of productive age in indonesia. a cross-sectional study was conducted using national basic health research data. the inclusion criterion was age 15-64 years. rheumatic disease diagnosis was based on interview results and was defined as the presence of a history of rheumatic disease diagnosed by health professionals and/or rheumatic symptoms in the past 12 months. the study sample consisted of 609.097 persons who fulfilled the inclusion criterion. compared to persons with normal bmi, rheumatic disease was more prevalent in the overweight (or 1.25; 95%ci 1.21—1.29) and the obese (or 1.52; 95%ci 1.47—1.56), but less prevalent in the underweight (or 0.91; 95%ci 0.88—0.93). compared to nonsmokers, rheumatic disease was more prevalent in every day smokers (or 1.65; 95%ci 1.60—1.70), occasional smokers (or 1.41; 95%ci 1.35—1.47), and ex-smokers (or 1.85; 95%ci 1.76—1.95). measures for prevention of rheumatic disease are needed, e.g. through education to increase knowledge about the impact of smoking and obesity on rheumatic disease. keywords: rheumatic disease, smoking, body mass index, productive age *center of biomedical and pharmaceutical research and development national institute of health research and development, moh ri correspondence dr. lusianawaty tana, ms, spok center of biomedical and pharmaceutical research and development national institute of health research and development, moh ri jl. percetakan negara no.23a jakarta phone: 021-6500266 email: lusianawaty@litbang.depkes.go.id univ med 2010;29:96-103 impact of smoking and obesity on rheumatic disease in persons of productive age lusianawaty tana* and daroham mutiatikum* may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction rheumatic disease comprises more than 100 disorders and conditions, constituting a health problem frequently experienced by the community. the most frequently encountered rheumatic diseases are osteoarthritis (oa) and rheumatoid arthritis (ra).(1) in 2001 there was an estimated 43 million people in america who reportedly had rheumatic disease.(2) one study stated that rheumatic disease was the cause of impaired mobility and agility in more than 30% of the elderly population.(3) the reported prevalence of rheumatic diseases or disorders vary considerably in a number of countries, being 33% in australia, 26.3% in bangladesh, 18.2% in india, and 14.9% in vietnam.(4-7) the varying prevalence between countries indicates differences in the definition of rheumatic disease. 97 univ med vol.29 no.2 oa is the most frequently encountered t y p e o f a r t h r i t i s t h r o u g h o u t t h e w o r l d , p a r t i c u l a r l y a m o n g t h e e l d e r l y, a n d t h e principal cause of disability both in developed as well as in developing countries.(8) the symptoms of oa initially appear around the age of 40 years and by the age of 50 years female oa patients outnumber males.(9) the etiology of oa is still undetermined, but the risk factors of oa are: (i) ethnicity; (ii) overweight; iii) joint injury; (iv) overuse of joints; (v) infrequent excercise; (vi) nerve injury; and vii) advanced age. there is a dearth of data on oa prevalence, due to p r o b l e m s o f d e f i n i t i o n a n d m e t h o d s o f d e t e r m i n i n g t h e o n s e t o f t h e d i s e a s e . worldwide, the proportions of male and female oa patients have been estimated at 9.6% and 18.0%, respectively.(10) in australia, the incidence of oa in females is higher than in males of all age groups (2.95 per 1000 vs 1.71 per 1000 population). in females, the peak incidence is at age 65–74 years, while in males it is at age 75 years and over.(10) in malaysia the prevalence of oa is around 10– 20%.(11) the incidence and prevalence of ra increase with age up to the age of 70 years and subsequently decrease. females suffer from ra twice as frequent as males. the prevalence of ra in industrial countries is around 0.3-1%, while in developing countries it is estimated to be less than 0.3%.(10) although rheumatic disease is rarely fatal, the resulting pain may be extremely uncomfortable/incapacitating and may lead to limitations in activity and thus affect quality of life. the course of the disease is chronic, and if not properly treated may result in mild disability (joint damage) to severe disability (paralysis). the data collected by the national basic h e a l t h r e s e a r c h 2 0 0 7 ( r i s k e s d a s 2 0 0 7 ) include data on rheumatic disease and various variables such as individual characteristics, b m i , a n d s m o k i n g b e h a v i o r. ( 1 2 ) i n t h i s connection it was desired to analyze the riskesdas 2007 data to determine the role of obesity and smoking on rheumatic disease in persons of productive age in indonesia. this s t u d y i s p a r t o f t h e r e s e a r c h p r o j e c t o n “ f a c t o r s a s s o c i a t e d w i t h r h e u m a t i c a n d inherited diseases in indonesia according to riskesdas data”.(13) methods design of the study the study was of cross-sectional design, and attempted to attain the research objectives using the riskesdas 2007 data. subjects of the study the inclusion criterion of the study was age 15-64 years, resulting in a sample size of 609.097. data collection the variables came from 3 types of questionnaire, i.e. the rkd07.rt (riskesdas 2 0 0 7 h o u s e h o l d q u e s t i o n n a i r e ) , t h e rkd07.ind (riskesdas 2007 individual q u e s t i o n n a i r e ) , a n d t h e s u s e n a s 2 0 0 7 vsen2007.k questionnaire (national socioeconomic survey of the 2007 census). data on individual characteristics were obtained from the rkd07.rt questionnaire and data on rheumatic disease, smoking and bmi from the rkd07.ind questionnaire. data on per capita household expenditure were from the susenas 2007 vsen2007.k questionnaire.(17) the diagnosis of rheumatic fever was based on two questions, viz. (i) had respondent in the past 12 months been diagnosed as having rheumatic joint disease/rheumatism by health personnel and (ii) did respondent in the past 1 2 m o n t h s e v e r s u ff e r f r o m a c h e s / p a i n / stiffness/swelling around the joints and/or joint stiffness on waking up or after a long rest, that did not result from accidents.(14) 98 data analysis data analysis was by means of the spss program version 15.0. univariate, bivariate and m u l t i v a r i a t e a n a l y s i s w a s p e r f o r m e d o n variables with p<0.25 or on variables with p>0.25 that were theoretically influential. the level of significance was set at < 0.05 and the confidence interval at 95%. results a total of 609.097 respondents aged 1564 years were recruited in this study. only the data on the variables age, gender and type of residence were collected for an identical total of 609.097 (100%). data on education, per capita expenditure, occupation, smoking, and bmi were obtained in differing percentages, i.e. respectively 99.63%, 99.61%, 99.59%, 99.54% and 97.04%. the proportion of respondents aged 15-44 years was 71.9% while those between 45-64 years of age accounted for 28.1 %. the sample comprised 52% females and 48% males, among whom 55% were rural residents and 45% were urban residents. a total of 71.8% respondents had an educational level of junior high school (smp) a n d l o w e r, w h i l e 2 8 . 2 % h a d a n educational level of senior high school (sma) and above. among the respondents, 56.8% had a monthly per capita household expenditure in the 1-3 quintile range and 43.2% in the 4-5 quintile range. on the aspect of occupation, 32.6% of the respondents were farmers/ fishermen/laborers, 21.4% housewives, 15.4% entrepreneurs, 11% employees, 17.2% without occupation and without education, and 2.4% “other”. t h e p r o p o r t i o n o f r e s p o n d e n t s w i t h normal bmi was 67.5%, overweight 9.2%, characteristics n rheumatic disease (%) or* 95% ci** p yes no age ( year s) 45-64 164.607 50.7 49.3 3.88 3.80 —3.95 0.0001 15-44 444.490 21.0 79.0 gender female 315.061 31.0 69.0 1.19 1.17 —1.21 0.0001 male 294.036 27.4 72.6 education < junior high (smp) 435.168 33.7 66.3 2.27 2.21 —2.32 0.0001 > senior high (sma) 171.683 18.3 81.7 occupation housewife 127.782 34.3 65.7 1.99 1.92 —2.07 0.0001 employee 62.156 19.9 80.1 0.95 0.90 —0.99 entrepreneur 85.103 30.3 69.7 1.66 1.59 —1.72 farmer/fisherman/laborer 210.866 37.3 62.7 2.27 2.19 —2.35 other 15.621 26.4 73.6 1.37 1.29 —1.46 no occupation and no educati on 53.769 13.6 86.4 type of residence rural 378.712 32.3 67.7 1.38 1.34 —1.42 0.0001 urban 230.385 25.7 74.3 per capita expen ditur e quintile 1-3 344.551 30.4 69.6 1.13 1.11 —1.16 0.0001 quintile 4-5 262.178 27.9 72.1 table 1. bivariate relationship between individual characteristics and rheumatic disease *or = odds ratio; **95%ci = 95% confidence interval tana, mutiatikum smoking and obesity on rheumatic disease 99 univ med vol.29 no.2 obese 10.5% and underweight 12.8%. the respondents who smoked daily accounted for 27.6%, those who smoked occasionally 6.3%, ex-smokers 3%, and those who never smoked (non-smokers) 63.2%. the proportion of respondents in the age range of 15-64 years with rheumatic disease was 29.3% and those without rheumatic disease 70.7%. the results of the bivariate analysis on the relationship between individual characteristics and type of occupation on the one hand and rheumatic disease on the other hand are shown in table 1, while the bivariate analysis results o n b m i a n d s m o k i n g b e h a v i o r v e r s u s rheumatic disease are shown in table 2. from the bivariate relationship in table 1 it is apparent that the proportion of rheumatic diseases increases significantly with advancing age, lower educational level, rural residence and lower per capita household expenditure. i n c o m p a r i s o n w i t h t h e g r o u p w i t h o u t occupation, the proportion of rheumatic disease was higher in farmers/fishermen/ laborers and housewives. table 2 shows the bivariate relationship between bmi and smoking behavior on the one hand and rheumatic disease on the other. from table 2 it can be seen that in comparison to t h e p r o p o r t i o n o f r h e u m a t i c d i s e a s e i n respondents with normal bmi, the proportion of rheumatic disease was higher in overweight a n d o b e s e r e s p o n d e n t s a n d l o w e r i n underweight respondents. the proportion of rheumatic disease in respondents who smoked daily and in ex-smokers was higher when compared with the non-smokers. the multivariate relationship between several factors and rheumatic disease is shown in table 3. the variables fulfilling the criteria for multivariate analysis were age, gender, education, type of residence, occupation, monthly per capita household expenditure, bmi, and smoking behavior. from the multivariate relationship shown in table 3, it appears that all eight factors included in the analysis were significantly a s s o c i a t e d w i t h r h e u m a t i c d i s e a s e . i n comparison to the respondents in the age group of 15-44 years, the proportion of rheumatic disease was 3.2-fold higher in the age group of 45-64 years. compared with the respondents who never smoked, the proportion of rheumatic disease in the respondents who smoked daily was 1.6-fold, in ex-smokers 1.8-fold, and in non-smokers 1.4-fold higher. the proportion of rheumatic disease in respondents with an educational level of smp and below was 1.6fold higher than in those who had an sma certificate or higher. in comparison with the group without occupation, the proportion of table 2. relationship of bmi and smoking with rheumatic disease *or = odds ratio; **95%ci = 95% confidence interval 100 rheumatic disease was higher in farmers/ f i s h e r m e n / l a b o r e r s a n d h o u s e w i v e s . i n comparison with the respondents with normal bmi, the proportion of rheumatic disease in the overweight and obese were 1.2and 1.5fold, respectively. discussion in the present study the proportion of rheumatic disease was 21.0% in indonesians of productive age between 15-44 years and 50.7% in the age group of 45-64 years. the r e s u l t s o f t h e 1 9 9 7 u s n a t i o n a l h e a l t h interview study (nhis) showed a prevalence of arthritis of 30% in the age range of 55-64 years and of 45-46% in the age range of 65-84 years, thus differing from the present study results.(15) the results of the present study indicate that the prevalence of rheumatic disease is higher females than in males and increases with age. this is consistent with the results of the us national health interview survey (nhis) for 2003–2005, showing a higher prevalence of rheumatic disease for females (25.4%) as table 3. multivariate relationship between several factors and rheumatic disease *or = odds ratio; **95%ci = 95% confidence interval tana, mutiatikum smoking and obesity on rheumatic disease 101 univ med vol.29 no.2 compared to males (17.6%). in this survey the prevalence of rheumatic was also higher in the older age groups, namely 29.3% in the age range of 45-64 years and 50% in the 65+ group, as compared to 7.9% in the age group of 1844 years.(16) similarly, the present study results showed that the risk of rheumatic disease in the age group of 45-64 years is 3 times that in the age group of 15-44 years. one of the rheumatic diseases frequently encountered is oa, which is a degenerative disorder. age is the principal determinant of oa, as the incidence and prevalence of oa is proportional to age, and oa is the main cause of rheumatic disease in the 65+ population group.(17) in respect to occupation, the risk of rheumatic disease was higher in the group of farmers/fishermen/laborers and in housewives. in the occupations of farmer/fisherman/laborer, the persons involved commonly must perform muscular work, such as lifting of loads and working in non-ergonomical positions, which in the long run may lead to complaints of pain in the involved joints. similarly, the occupation of housewife is connected with lifting and stooping movements or working in nonergonomical positions. in addition, as the occupation of housewife is predominantly a female occupation and the female gender is one of the risk factors of rheumatic disease, this c o m b i n a t i o n o f f a c t o r s i s p r e s u m a b l y a s s o c i a t e d w i t h t h e h i g h p r e v a l e n c e o f rheumatic disease detected in the present study. similar results were obtained in male japanese workers aged > 45 years, comprising factory laborers, construction workers, farmers or fishermen, who had a significantly higher risk of oa of the joints (or = 2,54;95% ci 1.34 . 8 2 ) . ( 1 8 ) h o w e v e r, t h e s t u d y f o u n d n o association of activities such as standing, climbing stairs, kneeling, driving, and sitting with a higher risk of oa in male japanese workers. similar results were found in female japanese workers.(19) compared with the respondents with normal bmi, the risk of rheumatic disease was respectively 1.2 and 1.5 times higher in overweight and obese respondents. with respect to bmi, it was found that overweight a n d o b e s i t y w e r e a t h i g h e r r i s k f o r t h e o c c u r r e n c e o f r h e u m a t i c d i s e a s e , i n comparison to normal body weight. the results obtained in the present study are consistent with those in the literature, in that one of the risk factors of rheumatic disease is overweight. a person with oa of the knee joint has in general a higher body weight as compared to persons without oa of the knee joint. a number of studies similarly showed that obesity increases the risk of oa. these studies also showed a positive correlation between bmi and severity of oa of the knee.(20-22) oa is due to the aging process, resulting in damage to joint cartilage. the involved joints are the frequently moved and weight-bearing joints, such as the carpal, elbow, lumbar, knee and tarsal joints. there were several limitations in this study, such as lack of specific data on the type of rheumatic disease and on the location, n u m b e r a n d s i z e o f t h e i n v o l v e d j o i n t s . therefore the present study was unable to p r o j e c t t h e p r e v a l e n c e o f s p e c i f i c j o i n t disorders. as arthritis may cause functional impairment, disability, and impaired quality of life, this may lead to future socio-economic problems. attention should be focussed on prevention of oa in order to reduce the problems associated with this disease. obesity is a remediable risk factor for several types of joint disorder, thus epidemics of obesity s h o u l d b e c o n t a i n e d b y w e i g h t c o n t r o l , changes in dietary patterns and increased physical activity, in order to impact on the prevalence of arthritis. every patient with rheumatic disease should receive counseling regarding this disorder. changes in lifestyle are necessary to ensure that these patients r e m a i n p h y s i c a l l y f i t a n d b e c a p a b l e o f controlling their disease. in the case of severe disease the patient should be referred to a physiotherapist. 102 conclusion in indonesians of productive age, bmi, s m o k i n g , a n d o c c u p a t i o n a r e t h e m o s t influential among the various factors affecting rheumatic disease, indicating the importance of obesity prevention and the urgent need of a behavioral approach to weight control. acknowledgement we hereby express our deepest gratitude to dr.triono soendoro, phd, dr. dr. trihono, msc, dr emiliana tjitra msc, phd, and riskesdas 2007 data managers, enabling us to conduct this study using the riskesdas 2007 data from the national institute of health research and development, moh ri. references 1. feinglass j, nelson c, lawther t, chang rw. chronic joint symptoms and prior arthritis diagnosis in community surveys: implications for arthritis prevalence estimates. public health rep 2003;118:230-9. 2. centers for disease control and prevention. prevalence of self-reported arthritis or chronic joint symptoms among adults-united states, 2001. mmwr 2002; 51:948–50. 3. leveille s, ling s, hochberg mc, resnick h, bandeen-roche kj, won a, et al. widespread musculoskeletal pain and the progression of disability in older disabled women. ann intern med 2001;135:1038-46. 4. minaur n, sawyers s, parker j, darmawan j. rheumatic disease in an australian aboriginal community in north queensland, australia. a who-ilar copcord survey. j rheumatol 2004;31:965-72. 5. haq sa, darmawan j, islam mn, uddin mz, das bb, rahman f, et al. prevalence of rheumatic diseases and associated outcomes in rural and urban communities in 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2006;55:1089-92. 17. cicutini fm, teichtahl aj, wluka ae, davis s, strauss bj, ebeling pr. the relationship between body composition and knee cartilage volume in healthy middle-aged subjects. arthritis rheum 2005;52:461-7. 18. yoshimura n, kinoshita h, hori n, nishioka t, ryujin m, mantani y, et al. risk factors for knee osteoarthritis in japanese men: a case-control study. mod rheumatol 2006;16:24–9. 19. yoshimura n, nishioka s, kinoshita h, hori n, nishioka t, ryujin m, et al. risk factors for knee osteoarthritis in japanese women: heavy weight, past joint injuries and occupational activities. j rheumatol 2004;31:157–62. tana, mutiatikum smoking and obesity on rheumatic disease 103 univ med vol.29 no.2 20. felson dt, goggins j, niu j, zhang y, hunter d. the effect of body weight on progression of knee osteoarthritis is dependent on alignment. arthritis rheum 2004;50:3904-9. 21. jadelis k, miller m, ettinger wh, messier sp. strength, balance, and the modifying effects of obesity and knee pain: results from the observational arthritis study in seniors (oasis). j am geriatric society 2001;884-91. 22. sharma l, lou c, cahue s, dunlop d. the mechanism of the effect of obesity in knee osteoarthritis. arthritis rheum 2000;43:568-75. paradoxical sleep deprivation changes testicular malondialdehyde and caspase-3 expression in male rats 87 *anatomy department, faculty of medicine and health sciences, jenderal soedirman university, purwokerto **physiology department, faculty of medicine, gadjah mada university, yogyakarta ***department of occupational health, faculty of medicine, gadjah mada university, yogyakarta ****education programme reproduction and development c/o monash institute of medical research, australia correspondence: dr. fitranto arjadi, mkes. anatomy department, faculty of medicine and health sciences, jenderal soedirman university kampus berkoh, purwokerto 53146 phone : +62281-622022 / +628156996806 email: f.arjadi@yahoo.com univ med 2015;34:87-95 doi: 10.18051/univmed.2016.v35.87-95 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background sleep deprivation is a significant problem among adult men and is considered as a risk factor for several diseases. paradoxical sleep deprivation (psd) induces leydig cell apoptosis through elevation of corticosterone, with testicular malondialdehyde (mda) and leydig cell caspase-3 expression as parameters. the aim of this study was to observe testicular mda level and caspase-3 expression treated with paradoxical sleep deprivation (psd), immobilization, and footshock stress and to determine the stress model with a significant effect in white male rats (rattus norvegicus) . methods this experimental randomized study of posttest only with control group design was conducted on 24 white male wistar strain rats, randomly allocated into four treatment groups, i.e. control (k1) without any stress treatment, psd (kii), immobilization (kiii), and footshock stress (kiv). treatments were given for 25 days to produce chronic stress. testicular mda concentration was examined by the elisa method while caspase3 was examined by the tunel method. results mean testicular mda concentration with one-way anova test showed differences in means between the groups (p=0.000) and post hoc tukeyhsd test showed significant results between psd stress group versus control, immobilization and footshock stress groups. one-way anova test showed a significant difference in caspase-3 expression in at least two treatment groups (p=0.008) and post-hoc tuckey-lsd test showed significant differences between controls and all stress groups. conclusion sleep deprivation is a type of stress inducing changes in testicular mda concentration and caspase-3 expression in male rat testes. keywords: caspase-3 expression, stress, testicular malondialdehyde, male rat paradoxical sleep deprivation changes testicular malondialdehyde and caspase-3 expression in male rats fitranto arjadi*, ginus partadireja**, lientje setyawan maurits***, and mulyono pangestu**** doi: http://dx.doi.org/10.18051/univmed.2015.v34.87-95 88 arjadi, partadireja, maurits, et al stress changes testicular malondialdehyde paradoxical sleep deprivation mengubah kadar malondialdehid testis dan ekspresi caspase-3 pada tikus jantan latar belakang sleep deprivation merupakan masalah penting bagi laki-laki dewasa dan merupakan faktor risiko bagi beberapa penyakit. paradoxical sleep deprivation (psd) stres menyebabkan apoptosis sel leydig akibat peningkatan kadar kortikosteron dengan parameter kadar mda testis dan caspase-3. tujuan penelitian adalah menyelidiki kadar mda testis dan ekpresi caspase-3 (rattus norvegicus) jantan yang diberi perlakuan stres psd, stres imobilisasi dan stress footshock pada tikus putih jantan. metode metode penelitian adalah eksperimental dengan posttest only with control group design. tikus putih jantan strain wistar sebanyak 24 ekor dibagi 4 kelompok perlakuan; kontrol (ki) yaitu kelompok yang tidak diberi perlakuan stres, kelompok stres psd (kii), kelompok stres imobilisasi (kelompok iii), dan kelompok stres footshock (kiv). perlakuan diberikan selama 25 hari untuk menghasilkan stres kronik. kadar mda testis diperiksa menggunakan metode elisa sedangkan caspase-3 menggunakan metode tunel. hasil hasil rerata pengukuran kadar mda testis dengan one way anova menunjukkan perbedaan rata-rata antara kelompok (p=0,000) dan uji post hoc tukey hsd menunjukkan rata-rata kadar mda pada stres psd berbeda signifikan dengan kelompok kontrol, imobilisasi dan footshock. hasil uji anova menunjukkan ada perbedaan yang bermakna rata-rata ekspresi caspase-3 sel leydig antara tiap kelompok data (p=0,008) dan dilanjutkan dengan uji post-hoc tuckey-lsd ada perbedaan rerata jumlah caspase-3 antara kelompok kontrol dengan semua perlakuan. kesimpulan paradoxical sleep deprivation merupakan salah satu jenis stress yang mampu mengubah kadar mda pada testis dan ekspresi caspase-3 pada testis tikus jantan kata kunci : stres, mda testis, ekspresi caspase-3, tikus jantan abstrak introduction at the molecular level, male infertility occurs because of abnormal mitochondrial deoxyribonucleic acid (dna) and apoptosis mechanism.(1) when apoptosis is improperly activated or regulated in the testis, infertility can result. pinpointing how environmental conditions can affect apoptosis is important for the advancement of preventive medicine and behavioral science, especially as there are potentially harmful environments in workplaces. moreover, familiarity with stressful conditions in workplaces and with testicular processes, especially the induction of apoptosis, is essential for promoting male fertility. in male patients with idiopathic infertility, reactive oxygen species (ros)-induced sperm damage is associated with increased apoptosis.(2) st r e s s e x p o s u r e c a n i n c r e a s e t h e concentration of cortisol which can disturb the testis by reduction of leydig cell numbers and 89 univ med vol. 34 no.2 inducing apoptosis by glucocorticoid receptors (3) and also by reducing the quality of sertoli cells which can decrease the production of germinal cells, depress spermatogenesis, and finally can c a u s e i n f e r t i l i t y. ( 4 ) h i g h g l u c o c o r t i c o i d concentrations during the stress period can induce leydig cell apoptosis by various mechanisms, such as activation of the fas system, cleavage of procapase-3, removal of mitochondrial membrane potential (deltapsi) and elevation of ros concentration.(3) one of the apoptosis markers is the elevated expression of caspase-3 (the active form of procaspase-3).(5) work stress induces higher oxidative stress and stimulates the formation of high levels of ros.(6) the main target of ros are lipids, such as in testiscular tissues which have high amounts of unsaturated fats which in the end can influence the capacity of the germinal cells to produce spermatozoa and to initiate the process of leydig c e l l s t e r o i d o g e n e s i s . ( 7 ) t h e h i g h r o s concentrations activate lipid peroxidation, which ultimately produces hydrogen peroxide (h 2 o 2 ), exactly in the form of malondialdehyde (mda), a cytotoxic oxidant in the body.(8) testicular mda level has been used as biomarker to measure the degree of oxidative stress (9) and to describe testicular tissue damage.(10) the present research objective was to evaluate the effect of work environment stress on the concentrations of testicular mda and caspase3 in male rats. in the experimental animals, sleep deprivation stress was implemented with the paradoxical sleep deprivation (psd), immobilization and footshock tests. in the experimental animals, an analogous form of working stress is the lack of sleep which is induced with the psd test as a form of physical and psychological stressor. the sedentarian pattern in office workers was modeled with the immobilization test as a form of social, physical, and psychological stressor, and high risk work with the footshock test as a form of physical stressor. the electric foot stress is usually used as a stressor in animal models such as conditioned fear stress and learned helplessness.(11) methods research design this research used an experimental post test only design with completely randomized sampling repeated five times. this research was conducted from january to december 2013 at the animal house of the medical faculty of jenderal soedirman university, and at the biochemistry laboratory and anatomical pathology laboratory, medical faculty, gadjah mada university. animals a total number of 24 male wistar albino rats (six rats for each treatment), aged 3-4 months and weighing 200-300 grams, were obtained from the animal house of the faculty of pharmacy, gadjah mada university. the sample size was determined by federer’s formula (t-1) (r-1) >15 where t = number of treatments and r = number of replications. intervention the rats were placed in stainless steel cages with the dimensions 23 x 13 x 16 cm for 1 week with controlled lighting (12 hours light/12 hours dark cycle) and temperature (21-24°c) and received food and water ad libitum.(12) the rats were placed in cages of the same size, form, and material. the experimental animals were divided into four treatment groups: ki as the control group, in which the rats were kept in a wide cage with their normal sleep pattern, and the groups kii, kiii, and kiv which were subjected to the psd, immobilization and footshock stress tests, repectively. in group kii the rats were treated with psd stress using the modified multiple platform method (mmpd), by placing the rats in a 123 x 44 x 44 cm tank containing water to a height of 1 cm and having 14 platforms, where each platform was separated by a distance of 10 cm, so the rats could move freely inside the cage, jump from platform to platform, and interact with the other rats in the cage. a muscle atonia-inducing device was turned on automatically every 10 minutes, 90 arjadi, partadireja, maurits, et al stress changes testicular malondialdehyde so that when the rats begin to sleep, they will fall down into the water and awaken from sleep. this cycle was done in 18 hours from 04.00 until 22.00 o’clock and after that the rats were returned to their regular cages, in which they sleep for 6 hours.(13) in group kiii, in which the rats were treated with immobilization stress, the rats were placed for 1 hour, from 09.00 until 10.00, in a transparent cylinder, 21 cm in length and 6 cm in diameter, with ventilation holes at the sides.(13) in group kiv, the footshock stress group, the rats were placed in an 14 x 25 x 28 cm acrylic box, in which an 0.1 second electric current of 5 ma was applied to the base for 1 hour/day from 09:00 until 10:00. each rat was shocked 4-6 times at different intervals, which were changed every 5 minutes to avoid anticipation by the rats.(14) each treatment (kii, kiii and kiv) was given for 25 days to induce a chronic stress effect.(3) the rats were then anesthetized with ether, decapitated, and incised to harvest the left testicle, which was cleaned in nacl solution, weighed, and preserved in bouin solution. measurement of testicular mda measurement of testicular mda was performed by means of a kit (catalog number: 201-11-0157, shanghai sunred biological technology, china). the isolated testis was weighed, pbs solution was added at the ratio of 50-100 mg testis to 1 ml pbs, and the testis was mashed in a mortar until homogenous, placed in a microtube and centrifuged at 4000 rpm for 5 minutes, after which the supernatant solution was stored at -80 oc. the testicular tissue mda concentration was measured by the elisa method with the double-antibody sandwich technique. caspase – 3 expression measurement of tunel caspase-3 expression was done in 50 fields of view of testis interstitial tissue at 400x magnification. the methods used were tunel and staining with anti caspase-3. a positive expression was shown by a brown color of the cytoplasm. observations were done using a nikon eclipse e100® microscope with optilab® and image raster software version 2.1. data analysis univariate analysis was done on the mean testicular mda concentration and caspase-3 expression data, data normality and homogeneity were analyzed with shapiro wilk and levene’s test. comparisons of mean values was analyzed by one-way anova and each treatment compared with post-hoc tukey test, and if there was a difference between treatments, the analysis was to be continued with duncan’s multiple range test (dmrt) at a value of p<0.05. ethical clearance all experimental procedures were approved by the research ethics committee of medical and health sciences of jenderal soedirman university. results the data was collected without drop outs and the caspase-3 counts were conducted by the investigator and two other observers to yield valid values. normality tests on the results of caspase-3 expression and testicular mda using the saphiro-wilk test showed the data to be normally distributed. the variance test using levene’s test showed equal data variances (p=0.146), so that a parametric test using oneway anova could be done. the mean mda concentrations analyzed with parametric oneway anova showed significant differences in mda concentrations between groups (p=0.000) (table 1). p o s t h o c tu k e y h s d t e s t s h o w e d significant differences between the psd group on the one hand and the control, immobilization stress and footshock stress groups on the other (table 2). the results of anova test for leydig cell caspase-3 expression showed significant differences between all treatment groups (p=0.008). post-hoc tuckey-lsd showed that mean leydig cell caspase-3 expression in three of the treatment groups was significantly 91 univ med vol. 34 no.2 different from the control group, with the lowest mean in the kii group as compared with the control group (p<0.05) (table 2). compared with the controls, the number of caspase-3 expression decreased in all stress models, with the lowest number in the psd stress group (figure 1). discussion the highest mean testicular mda concentration and lowest caspase-3 expression was in the psd stress group, showing that psd stress increases oxidative stress which can induce lipid peroxidation reaction and damage the antioxidant mechanism of the body. paradoxical sleep deprivation increases in mda level and may enhance the metabolic rate and in turn increase free radical generation and induce cell damage. oxidative stress caused by psd induction decreases the damage of endogenous antioxidant mechanism by inhibition of g6dp (glucose-6 diphosphatase) and stimulation of a larger lipid peroxidation reaction. the lipid peroxidation reaction is a cascade of reactions that can produce free radicals and continuous peroxidation, with mda as the end product. these results suggest that sleep has an antioxidative role. free radicals which accumulate during wakefulness are removed during sleep and this is accomplished by decreasing the rate of free radical formation and increasing the efficiency of endogenous antioxidant mechanisms. (15) paradoxical deprivation increases early morning pineal melatonin, which induces apoptosis through activation of caspase-3, caspase-6, caspase-7, and caspase-9, in association with up-regulation of the pro-apoptotic factor bax and also with the release of cytochrome c from mitochondria.(16) stress disturbs the reproductive system and decreases the numbers of leydig cell because of the increase in glucocorticoids induced by psd stress.(17) glucocorticoids induce the apoptotic process in mature and immature leydig cells because of the high numbers and high sensitivity of the glucocorticoid receptors. glucocorticoids cause leydig cell apoptosis by several mechanisms such as fas system activation, procaspase-3 activation, reduction of mitochondrial membrane potential and elevation of ros concentration. caspase-3 is a protein in the caspase cascade pathway that is important in apoptosis and is activated by caspase 8 and 9. procaspase-3, which is in the form of an active enzyme, can cleave into a new subunit, caspase3, which can activate caspase 6 and 7. caspase3 with caspase 6 and 7 is proteolytic and can induce apoptosis.(18) table 1. mean testicular mda and number of caspase-3 expression by treatment groups note: values are mean ± sd; mda = malondialdehyde; psd = paradoxical sleep deprivation note *= p<0.05 means significantly different; mda = malondialdehyde; psd = paradoxical sleep deprivation table 2. post hoc test lsd for mda and caspase-3 by treatment groups 92 arjadi, partadireja, maurits, et al stress changes testicular malondialdehyde in immobilization stress, the study used tunel and active caspase-3 staining methods. t h e r e s u l t s s h o w e d t h a t a p o p t o s i s w a s significantly higher in the stress groups than in the control group, with the markers being the number of tunel (+) and caspase 3 (+) cells, which was increased significantly in the stress groups, in comparison with the cells in the control group. the study demonstrated the occurrence of testicular damage on the basis of elevated mda levels. the results showed that there is a relationship between immobilization stress on the one hand and induction of apoptotosis and lipid peroxidation on the other.(19) l e y d i g c e l l a p o p t o s i s i s n o t e l e v a t e d i f endogenous corticosterone is low, proving that leydig cell apoptosis occurs if corticosterone concentration is elevated, which can reduce t e s t o s t e r o n e c o n c e n t r a t i o n . (3) r e d u c e d testosterone in immobilization stress also can elevate spermatocyte apoptosis by activation of caspase-3 so the normal hormonal concentration is still needed for steroidogenesis but in this research the effect was stronger in the psd stress model.(2) footshock stress as physical stress showed a significant increase in mda levels on the 30th day of the experiment compared with the 1st day, but no significant difference was observed at any time between mda levels of the control and physical stress groups.(20) the effect of footshock stress on cell leydig apoptosis remains unclear, and studies and references about the mechanism are also still limited. all well-known stress models may elevate glucocorticoid levels, therefore causing leydig cell apoptosis by intrinsic and extrinsic pathways. apoptosis by the extrinsic pathway (death receptor pathway) is initiated by the elevation of glucocorticoids which can continue to induce release of death signal proteins such as fas-ligand (fas-l).(3) the release of fas-l causes interaction figure 1. leydig cell caspase-3 of rattus norvegivus interstitial testis tissue. red arrows indicate caspase-3 expression with positive expression shown by brown cytoplasm. stain: ihc. magnification: 400 x. note: a) control; b) psd stress; c) immobilization stress; d) footshock stress 93 univ med vol. 34 no.2 between fas and fas-l, which can form fasassociated death domain (fadd). fadd formation cause formation of death-inducingsignaling-complex (disc) which can cause activation of caspase-8.(21) apoptosis by the intrinsic pathway (stress-induced, mitochondrion pathway) is marked by the reduction in mitochondrial membrane potential and the formation of superoxide anions (part of ros).(3) the intrinsic pathway is initiated by the cleavage of bid (part of bcl-2) and can cause the loss of mitochondrial membrane potential so that cytochrome c can be released. released cytochrome c can form apoptosomes which can activate caspase-9. active caspase-8 and 9 can cause activation of procaspase-3 into caspase-3, which forms two subunits that complex into a tetramer with a number of cellular substrates, leading to leydig cell apoptosis. leydig cell apoptosis is marked by abnormal morphologic changes, such as reduction of cellular volume, chromatin condensation, mitochondrial disintegration, swelling of the cell membrane, and cytoplasm vacuolization and biochemical changes, i.e. exposure of phosphatidylserine in the plasma membrane, activating caspase, and resulting in dna cleavage.(2) reduction in sleeptime can elevate the concentration of melatonine, which is known to influence testicular activity and gonadotropin secretion. melatonin has a direct effect on gnrh neuron receptors to induce expression of gnih, which can inhibit lh release and cause a reduction in testicular function.(22) elevation of melatonin concentration can reduce leydig cell number and function by two mechanism, i.e. by reduction of calcium ion from the cytosol and by inhibition of camp activity in steroidogenesis.(23) the elevation of glucocorticoid concentration because of oxidative stress can suppress the antiapoptotic group and can the activate proapoptotic group. these can cause mitochondrial membrane permeability change and reduction of membrane potential so that leakage occurs in cytochrome c protein. cytochrome c protein release into the cytosol can activate the mitochondrial pathway by activation of caspase 9. caspase 8 and caspase 9 can activate caspase3 so that the cell will enter the degradation phase and finally, cell death can occur. the morphologic changes in the cell that undergoes apoptosis consist of reduction in cellular volume, chromatin condensation, apoptotic body formation and mitochondrial disintegration as a result of caspase3 activation. caspase-3 is a protease with proteolytic abilities and can cause cleavage of many intracellular substrates.(18) caspase-3 expression is also elevated in the penis and the prostate upon reduction of testosterone because of leydig cell damage.(24) one the limitation of the study is that no stereological methods were used, of which the advantages are that the sampling is uniform to ensure that all cells in the testis have equal probabilities of being sampled and no assumptions about shape, size or orientation of the cells or shrinkage of the testis during histological processing are required. the conventional counting method is morphometric and may severely bias the results in unpredictable ways (e.g. the results are influenced by differential tissue shrinkage in hormone-treated rats and control rats).(25) the results of this study could be useful to company doctors, psychiatrists and andrologists in promoting health status, particularly for fertility problems in male workers. in future, we will investigate the effect of stress models in infertile human males and the use of traditional antioxidants to improve the fertility status. the study of the various workstress types in humans is very limited because of inadequacy of samples, low participancy, selection and recall bias, ethic problems and no exact clues to the source of the stressors. conclusion this study reveal that sleep deprivation caused alterations in testicular mda concentration and caspase-3 expression in male rats. 94 arjadi, partadireja, maurits, et al stress changes testicular malondialdehyde conflicts of interest none declared. acknowledments the authors would like to express their thanks to the directorate general of higher education, ministry of education and culture for funding through the hibah disertasi doktor scheme for 2013 and to the department of biochemisty, medical and health sciences faculty, jenderal soedirman university, and the pathological anatomy laboratory, medical faculty, gadjah mada university, for research facilities. references 1. nakada k, sato a, yoshida k, et al. mitochondria-related male infertility. pnas 2006;103:15148-53. 2. premendu p, huang l, kashou a, et al. environmental toxicants and testicular apoptosis. reprod sci j 2011;3:114-24. 3. arjadi f, soejono sk, pangestu m. paradoxical sleep deprivation decreases serum testosterone and leydig cells in male rats. univ med 2014;33: 27-35. 4. pfaff tj, rhodes mb, weinbauer gf. inhibin b as a marker of sertoli cell damage and spermatogenic disturbance in the rat. birth defects res 2013;98:91-103. 5. ghavami s, hashemi m, ande sr, et al. apoptosis and cancer: mutations within caspase genes. j med genet 2009;46:497-510. 6. lima ama, de bruin vms, rios erv, et al. differential effects of paradoxial sleep deprivation on memory and oxidative stress. naunyn-schmiedeberg’s arch pharmacol 2014; 378:399-406. 7. aitken rj, roman sd. antioxidant systems and oxidative stress in testes. oxid med cell longev 2008;1:15-24. 8. pusparini cw, aulanni’am, oktavianie da. kadar malondialdehyde (mda) dan gambaran histopatologi organ paru pada hewan model tikus (rattus norvegicus) asma dengan induksi lipopolisakarida. ubj chem 2012;1:1-7. 9. sakr sa, somaya ys. carbendazim-induced testicular damage and oxidative stress in albino rats: ameliorative effect of licorice aqueous extract. toxicol ind health 2014;30;259-67. 10. duru fio, noronha cc, akinwande ai, et al. effects of torsion, detorsion and melatonin on testicular malondialdehyde level. west african j med 2007;26:312-5, 11. hipolide dc, suchecki d, pimentel dcp, et al. paradoxical sleep deprivation and sleep recovery: effects on the hypothalamic-pituitary-adrenal axis activity, energy balance and body composition of rats. j neuroendocrinol 2006;18: 231-8. 12. willems ra. regulatory issues regarding the use of food and water restriction in laboratory animals. lab anim 2009;38:325-8. 13. andersen ml, silva a, kawakami r, et al. the effects of sleep deprivation and sleep recovery on pain thresholds of rats with chronic pain. sleep sci 2009;2:82-7. 14. dagyte g, van der zee ea, postema f, et al. chronic but not acute foot-shock stress leads to temporary suppression of cell proliferation in rat hippocampus. neuroscience. 2009;162:904-13. 15. thamaraiselvi k, mathangi dc, subhashini as. effect of increase in duration of rem sleep deprivation on lipid peroxidation. int j biol med res 2012;3:1754-9. 16. perdomo j, cabrera j, estévez f, et al. melatonin induces apoptosis through a caspase-dependent but reactive oxygen species-independent mechanism in human leukemia molt-3 cells. j pineal res 2013;55:195-206. 17. alvarenga ta, monica la, javier vm, at al. food restriction or sleep deprivation: which exerts a greater inûuence on the sexual behaviour of male rats? behav brain res 2009;202:26671. 18. shaha c, tripati r, mishra dp. male germ cell apoptosis: regulation and biology. phil trans r soc b 2010;365:1501-15. 19. bitgul g, tekmen i, keles d, et al. protective effects of resveratrol against chronic immobilization stress on testis. isrn urol 2013; 1-10. 20. jafari m, salehi m, zardooz h, et al. response of liver antioxidant defense system to acute and chronic physical and psychological stresses in male rats. xcli j 2014;13:161-71. 21. thorburn a. tumor necrosis factor-related apoptosis-inducing ligand (trail) pathway signaling. j thorac oncol 2007;2:461-5. 22. tsutsui k, ukena k. hypothalamic lpxrfamide peptides in vertebrates: identification, localization and hypophysiotropic activity. peptides 2006;27:1121-9. 95 univ med vol. 34 no.2 23. ahmed na. effect of short photoperiod on the leydig cells of rat’s testis: ultrastructural and immunohistochemical study. egyptian j histol 2008;31:354-1. 24. taufiqurrachman. decreased expression of caspase-3 in penis and prostat tissue of rat after the treatment of buceng (pimpinella alpina molk and euricoma lungifolia jack). med j indones 2013;22:2-8. husnun1 83 effectiveness of piper betle leaf infusion as a palpebral skin antiseptic may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina introduction the indonesian basic health survey of 2007 (riset kesehatan dasar tahun 2007) indicates that the prevalence of cataracts in indonesia is up to 1.8%.(1) the management of cataract is by cataract surgery. good surgical results are dependent on preand intra-operative procedures as a whole, including treatment with antiseptic solutions. the use of antiseptics before surgery is a standard procedure. in 1867, joseph l i s t e r f o r t h e f i r s t t i m e i n t r o d u c e d t h e husnun amalia*a, ratna sitompul**, johan hutauruk**, andrianjah*** and abdul mun’im**** *department of ophthalmology, medical faculty, trisakti university **department of ophthalmology, *** department of microbiology, ****department of matemathics and natural science, faculty of medicine, university of indonesia correspondence adr. husnun amalia, spa department of ophthalmology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11410 telp. 021-5672731 ex. 2614 univ med 2009;28:83-91 abstract the goal of an antiseptic is to eliminate or greatly reduce the number of microorganisms in the surgical field at the time of the surgery. the objective of this study was to verify the effectiveness of 20% piper betle leaf infusion as an antiseptic solution in pre-surgery cataract patients. a clinical trial with partner-matching design was conducted on 31 pairs of eyelids. from each pair of eyelids, one eyelid was asigned to the piper betle infusion group and the opposite one to the povidoneiodine group. the microorganisms were collected by swab from the patient’s palpebral skin, inoculated on nutrient agar, and incubated at 37oc for 20 hours. the antiseptic effectiveness was measured by counting the microbial colonies before and after administration of the antiseptic solutions. this study demonstrates that the mean colony counts after application of 20% piper betle leaf infusion showed a significant reduction of 27-100% compared with those before administration (p=0.001). mean colony counts after 10% povidone-iodine administration showed a significant reduction of 88-100% compared with the mean counts before the solution was applied (p=0.000). the 20% piper betle infusion has an antiseptic potential. nevertheless, the 10% povidone-iodine solution has more effective antiseptic capability. . keywords: antiseptic, palpebral skin, piper betle leaf infusion, povidone-iodine 84 amalia, sitompul, hutauruk, et al piper betle leaf infusion as antiseptic preoperative use of antiseptics.(2) preoperative antisepsis is performed by cleansing the periocular skin with an antiseptic solution in order to prevent contamination by normal palpebral microbial flora at the time of surgery.(3) povidone-iodine is an antiseptic that has been known for more than four decades and is extensively used to date.(4) povidone-iodine is a broad-spectrum antimicrobial agent that is effective against both gram-positive and gramnegative bacteria, fungi, bacterial spores, viruses, protozoa, mycobacteria and yeasts. this solution contains iodine as the active principle, which has an excellent and rapid antimicrobial action.(4) the concentration used for periocular s k i n c l e a n s i n g i s 5 1 0 % . ( 5 , 6 ) t h e t o p i c a l application of povidone-iodine in a significantly high concentration can result in irritation and s e n s i t i v i t y r e a c t i o n s i n 0 . 7 2 0 % o f t h e population.(4,7) although the incidence of allergic reactions to povidone-iodine is not high, if these reactions occur preoperatively, they may become an impediment to the course of surgery and the patient will suffer. the betel vine (piper betle linn., sirih), the leaves of which are extensively consumed as betel quid in the indian subcontinent, has been used as a medicinal plant by southeast asian peoples for more than 3000 years. betel leaf has broad antiseptic properties and to date there have been no reports in the literature on the occurrence of cases of allergy to the plant. the use of betel leaf as an antiseptic is increasing.(9) the active substances in betel leaf are phenol and its derivatives. the phenolic derivatives contained in betel leaf have a five-fold greater antibacterial potency than phenol itself.(10) hydroxychavicol, a major phenolic compound present in the aqueous extract of the piper betle leaf, is known for its antioxidant and anticancer properties.(11,12) the betel leaf has been demonstrated to be e f f e c t i v e a g a i n s t st re p t o c o c c u s s p . , staphylococcus sp., escherichia coli, vibrio eltor, salmonella typhosa, shigella shigae, m i c ro c o c c u s l u t e u s , b a c i l l u s s u b t i l i s , pseudomonas aeruginosa, and others.(13,14) c a s e s o f a l l e rg y t o p o v i d o n e i o d i n e encountered in the population need an alternative antiseptic solution. an infusion of betel leaves have the potential for becoming an alternative antiseptic because of its bactericidal action that has been demonstrated in a number of studies. this is the reason for the present study, which aims to compare the effectiveness of the antiseptic action of a betel leaf infusion to that of povidoneiodine as a control in the antisepsis of the palpebral skin in pre-surgery cataract patients. methods study design the present study was an experimental trial c o n d u c t e d i n t h e o p e r a t i n g r o o m o f t h e department of ophthalmology of a government hospital in central jakarta in november 2007 with matching approach. the laboratory part of t h e i n v e s t i g a t i o n s w e r e p e r f o r m e d a t t h e university of indonesia, respectively in the laboratory of the department of microbiology, faculty of medicine, and the phytochemical laboratory of the department of pharmacy, faculty of mathematics and natural sciences, both of which are located in depok. study subjects t h e s a m p l i n g i n t h i s s t u d y w a s d o n e consecutively. the prospective samples for this study were patients who were planned to undergo cataract surgery in the operating room of the department of ophthalmology. the eyelids of selected patients were assigned by matching into the s group comprised eyelids of non-surgical patients, which underwent antisepsis with betel leaf infusion and the b group comprising eyelids of surgical patients, which underwent antisepsis with povidone-iodine solution. each matched 85 univ med vol.28 no.2 pair in the s and b groups came from the same patient, namely the left and right eyelids, that were subjected to different treatments. t h e i n c l u s i o n c r i t e r i a w e r e p a t i e n t s presenting to the self-supported opthalmology operating room of a government hospital in central jakarta who were planned to undergo cataract surgery and consenting to participate in the study; all eyes and eyelids that belonged to surgery and non surgery candidates and were healthy, without previous surgery, and free of tumors, infections, or wounds; and the selected patients had to be willing to fill out an informed consent form for this study. extraction method for p. betle leaves all stages of the study were performed in conditions of antisepsis. the test substances in this study were betel leaf infusion and povidoneiodine solution. the betel leaves were from piper betle linn., as identified by the biological research center of the indonesian scientific institue (lembaga ilmu pengetahuan indonesia lipi) in cibinong. the betel leaves were washed, dried and comminuted by rubbing them between the hands. of the reduced simplicia, 10and 20-twenty gram portions were weighed on an analytical balance (shimadzu, type aw120) and subsequently made into infusions in sterile glass beakers. each of the beakers was filled with 100 ml distilled water, which was supplemented with 20 and 40 ml of distilled water (twice the dry weight of the simplicia). the contents of the beakers were subsequently heated for 15 minutes at 90oc, with occasional stirring. the betel leaf preparations were left to cool and strained through sterile filter paper. hot distilled water was poured through the strained leaves and this was added to the infusions up to a volume of 1 0 0 m l . t h e r e s u l t i n g i n f u s i o n s h a d a concentration of 20% (w/v). the infusions were filtered by means of bacterial filters of 0.22 mm pore size (millipore filters) and kept in sterile bottles at room temperature in a dry place and protected from direct sunlight. the povidoneiodine solution used as test substance was a commercially available solution under the brand name of betadine. the clinical specimens were collected by using cotton swabs before and after antiseptic treatment of the palpebral skin on both eyes of the patients. a preliminary study was performed to determine the concentrations of the betel leaf infusions (10% atau 20%), the type of transfer medium (normal saline [nacl 0.9%] or nutrient broth), the transfer volume (1 ml, 2 ml, or 3ml), and the appropriate dilution. the collected clinical specimens were then serially diluted in normal saline and nutrient broth (nb), to obtain inoculum concentrations of 1, 1:10, 1:100, 1:1000, and 1:10.000. volumes of 100 ml of the clinical specimen suspensions w e r e t a k e n w i t h s t e r i l e m i c r o p i p e t t e s f o r inoculation on nutrient agar (na) plates. the inoculation was performed in duplicate for both treatment groups by spreading sterile glass beads onto the plates, following which the inoculated plates were incubated for 20 hours at 370c. the effectiveness was assessed by the reduction in colony counts and the percentage of reduction. the number of colony-forming units (cfus) was determined manual the agar culture. data analysis the primary data were statistically analyzed using spss version 12, by means of the t-test. the primary data were transformed into their log10 to obtain a normal distribution of the data. the transformed data were tested for normality, if p >0.05 indicated that the obtained data were distributed normally. paired t-test was used for analyzing within-group data, whereas unpaired t-test was for between-group data. the data in the preliminary study was tested by anova. a p value of < 0.05 (2-tailed) was considerd to be significant. 86 amalia, sitompul, hutauruk, et al piper betle leaf infusion as antiseptic figure 1. flow of study results the infusions made in this study had concentrations of 10% and 20% (w/v), were brownish in color, had the characteristic betel leaf odor, with ph = 6 for the 10% and ph = 5.2 for the 20% infusions. the control solution used in this study was a 10% povidone-iodine solution under the brand name of betadine. the latter was an opague reddish-brown solution with ph = 4. 87 univ med vol.28 no.2 tests using the 20% betel leaf infusion s h o w e d a h i g h e r p e r c e n t a g e r e d u c t i o n i n microbial colony count in comparison with the 10% betel leaf infusion. therefore, in this study the 20% infusion was subsequently used. the preliminary study with nutrient broth yielded substantial numbers of microbial colonies. in contrast, normal saline yielded smaller colony counts, which were however adequate for quantitative analysis. thus in this study normal saline was used as the transfer medium. the tested transfer volumes in the preliminary study were 3 ml, 2 ml, and 1 ml, respectively. the colony counts in the preliminary study were inversely related to the transfer volumes, where the smallest transfer volumes yielded the highest counts; thus the best transfer volume was 1 ml. the serial dilution tests in the preliminary study indicated that the microbial counts could be easily determined from undiluted samples. therefore the samples contained in the transfer medium could be directly inoculated on nutrient agar plates. effectiveness of the antiseptics the present study involved 31 pre-surgery cataract patients meeting the inclusion criteria. table 1 shows that the majority of patients were elderly persons with a mean age of > 60 years (64.5%), and that there were 54.8% female and 45.2% male elderly. both age and gender were not correlated with the initial colony count (before antiseptic treatment, respectively with b e t e l l e a f i n f u s i o n a n d p o v i d o n e i o d i n e solution). there was no significant difference for colony counts in both groups before antiseptic treatment with betel leaf infusion and povidoneiodine solution (p > 0.05), and thus the antiseptic power of both types of antiseptic could be compared between groups (table 2). mean colony count after antisepsis with 20% b e t e l l e a f i n f u s i o n w a s s l i g h t l y l o w e r i n comparison with mean colony count before antisepsis (p=0.001) (figure 2). this indicates that the 20% betel leaf infusion had a potential antiseptic capability. mean colony count after antisepsis with povidone-iodine solution was always within the range of 0-7, irrespective of the colony count before antisepsis (p=0.000) (figure 3), indicating that antiseptic effectiveness of povidone-iodine solution was extremely high for all types of microorganisms present on palpebral skin. table 2. mean colony counts before and after treatment, by treatment group table 1. distribution of demographic characteristics (n=31) 88 amalia, sitompul, hutauruk, et al piper betle leaf infusion as antiseptic figure 4 shows that the effectiveness of povidone-iodine solution was in most cases around 100%, implicating that this solution has a maximal antiseptic capability that is superior to that of 20% betel leaf infusion. discussion antiseptic solutions play an important role in preventing sepsis of the skin and mucous membranes by significantly inhibiting and killing the microorganisms present on these tissues. (15) the human skin has a normal microbial flora that is extremely varied in species and numbers, therefore an antiseptic substance needs to have considerable antiseptic capability for use in preoperative procedures of antisepsis.(16) the present study demonstrates that there was a significant reduction in colony count after antiseptic treatment with povidone-iodine as well figure 2. mean colony counts with 20% betel leaf infusion figure 3. mean colony counts with 10% povidone-iodine solution 89 univ med vol.28 no.2 as with 20% betel leaf infusion. this indicates that both test substances are effective antiseptics and possess antimicrobial properties against the normal palpebral flora. twenty-percent betel leaf infusion has a potentially strong antibacterial capability but is less effective. the difference in effectiveness of betel leaf infusion between the in vitro study by murnikusumawatie(17) and the results of the preliminary investigation for the present study may possibly be the result of differences in species of microorganisms and in the antiseptic power of the infusions against certain microbial species. betel leaf infusions are capable of inhibiting the growth of two microbial species in minimal inhibitory concentrations (mic) of 5% and 20%, but these concentratons are inadequate for inhibiting other microorganisms.(13) povidone-iodine as a 10% solution exhibits an extremely effective antibacterial capability against all species in the normal human microbial flora, irrespective of their numbers on the palpebral skin. this solution kills all normal flora within 2-3 minutes, whether present in large or small numbers. ferguson et al.(4) reported that 5% povidone-iodine solution has an effective antiseptic capability as pre-operative antiseptic solution on the palpebrae and adnexa. irrigation of the fornices with 5% povidone-iodine was associated with significantly fewer positive conjunctival cultures at the time of surgery. (18) the present study demonstrates that 20% betel leaf infusion and 10% povidone-iodine had effective antibacterial capability in reducing the colony count. however, 10% povidone-iodine was capable of killing greater numbers of bacteria after antiseptic treatment (p=0.001), indicating that the antibacterial capability of 10% povidone-iodine was higher than that of 20% betel leaf infusion. povidone-iodine has been shown to be bactericidal against a wide range of bacteria, and is also effective against fungi, protozoa, and viruses.(19) povidone-iodine is an antiseptic solution with a concentration of 10%, which is equivalent to 1% iodine. houang et al. ( 2 0 ) figure 4. percentages in reduction of colony counts with 20% betel leaf infusion and 10% povidone-iodine 90 amalia, sitompul, hutauruk, et al piper betle leaf infusion as antiseptic reported the minimal inhibitory concentration (mic) and minimum bactericidal concentration (mbc) of 1% povidone-iodine solution against four species of test bacteria to be in the range of 1:256 – 1:8192. the content of active substance in povidone-iodine is accurately known and has been determined in numerous studies. the betel leaf infusion used in the present study had a concentration of 20% (w/v), but its content of active substances is as yet undetermined. s u r i a t m i n i r e p o r t e d t h a t t h e t o t a l concentration of phenolic derivatives present in a 5-10% betel leaf infusion was 0.72-2.28%, while the concentration of phenolic derivatives used as antibacterial solution was 0.5-2%. (21) betel leaf infusions contain phenolic derivatives with a six-fold greater antibacterial power than phenol itself. however, their concentrations are not known with certainty and there have been n o s t u d i e s o n t h e m i n i m u m b a c t e r i c i d a l c o n c e n t r a t i o n ( m b c ) o f t h e s e p h e n o l i c derivatives. the reduction in microbial colony count after antiseptic treatment with 20% betel leaf infusion varied between 27 and 100%, but a value of <50% was found in only four patients. this may be due to variation in the normal flora of individual patients and is evidenced by the variation in colonial morphology in each sample, even though no identification of the colonies was performed. i n t h i s s t u d y, a s s e s s m e n t o f a l l e rg i c reactions was done at 10 minutes, 60 minutes and 24 hours after application of the antiseptic. the signs and symptoms used for assessment were redness of the skin, feelings of warmth, and itching. none of the participants showed any of the above signs and symptoms after application of the antiseptic to the palpebral skin, indicating that 10%and 20%betel leaf infusions did not induce allergic reactions on palpebral skin. conclusion although 20% piper betle leaf infusion is potentially antiseptic, 10% povidone-iodine solution has more effective antiseptic capability. thus 20% betel leaf infusion has less effective a n t i s e p t i c c a p a b i l i t y c o m p a r e d w i t h 1 0 % povidone-iodine solution. references 1. department of health, republic of indonesia. results of the basic health survey 2007. jakarta: board for research and development, department of health, republic of indonesia;2008. 2. lister j. on the antiseptic principle in the practice of surgery. the lancet 1867 cited by connor jj, connor jth. being lister: ethos and victorian medical discourse. j med ethics 2008;34:3–10. 3. hefny c, edelhauser h. surgical pharmacology: intraocular solutions and drugs used for cataract surgery. in: buratto l, liliana w, zanini m, apple d, editors. pacoemulsification principles and techniques. 10th ed. thorofare: slack;2003.p.21920. 4. fergsuon a, scott j, mcgavigan j, elton r, mclean j, schmidt u, et al. comparison of 5% povidoneiodine solution against 1% povidon-iodine solution in preoperative cataract surgery antisepsis: a prospective randomised double blind study. br j opthalmol 2003;87:163-7. 5. surgery for cataract. in: ophthalmology aa, editor. lens and cataract. san francisco: american academy of ophthalmology;2005.p.89-97. 6. bausz m, fodor e, resch md, kristof k. bacterial contamination in the anterior chamber after povidone-iodine application and the effect of the lens implantation device. j cataract refract surg 2006;32:1691-5. 7. de kaspar hm, chang rt, singh k, egbert pr, blumenkranz ms, ta cn. prospective randomized comparison of 2 different methods of 5% povidoneiodine applications for anterior segment intraocular surgery. arch ophthalmol 2005;123:161-5. 8. yoshida k, sakurai y, kawahara s, takeda t, ishikawa t, murakami t, et al. anaphylaxis to polyvinylpyrrolidone in povidone-iodine for 91 univ med vol.28 no.2 impetigo contagiosum in a boy with atopic dermatitis. int arch allergy immunol 2008;146: 169–73. 9. moeljanto rd, mulyono. khasiat dan manfaat daun sirih. 1st ed. depok: agromedia pustaka; 2003. 10. nalina t, rahim zha. 2007. the crude aqueous extract of piper betle l. and its anti bacterial effect towards streptococcus mutans. am j biochem biotechnol 2007;3:10–5. 11. chang mc, uang bj, wu hl, lee jj, hahn lj, jeng jh. a major phenolic compound in piper betle leaves. br j pharmacol 2002;135:619–30. 12. jeng jh, wang yj, chang wh. reactive oxygen species are crucial for hydroxychavicol toxicity toward kb epithelial cells. cell mol life sci 2004;61:83–96. 13. razak fa, rahim zh. the anti-adherence effect of piper betle and psidium guajava extracts on the adhesion of early settlers in dental plaque to salivacoated glass surfaces. j oral sci 2003;45:201–6. 14. nasagewa m, hayashi h, nakayoshi t. in vitro evaluation of skin sensitivity of povidone-iodine and other antiseptics using a three-dimensional human skin model. dermatology 2002;204:109-13. 15. mcgreal gt, joy a, manning b, kelly jl, o’donnell ja, kirwan wwo, et al. antiseptic wick: does it reduce the incidence of wound infection following appendectomy? world j surg 2002;26:631-4. 16. tortora gj, funke br, case cl microbiology. 6th ed. california: addison wesley longman; 1998. 17. murnikusumawatie r. perbandingan daya antibakteri rebusan dan minyak atsiri daun sirih (piper betle l.) terhadap staphylococcus aureus dan pseudomonas aeruginosa yang diisolasi dari ulkus kornea [spm thesis]. jakarta: fkui; 2007. 18. de kaspar hm, chang rt, singh k, egbert pr, blumenkranz ms, ta cn. prospective randomized comparison of 2 different methods of 5% povidoneiodine applications for anterior segment intraocular surgery. arch ophthalmol 2005;123:161-5. 19. ciulla ta, starr mb, masket s. bacterial endophthalmitis prophylaxis for cataract surgery. an evidence-based update. ophthalmology 2002;109:13-24. 20. houang e, gilmore o, reid c, shaw e. absence of bacterial resistance to povidone-iodine. j clin path 1976;29:752-5. 21. suriatmini s. pengaruh cara sterilisasi terhadap stabilitas rebusan daun sirih [s.si skripsi]. depok: universitas indonesia; 2005. alvina 73 abstract bone remodeling is a physiological process of cortical and trabecular bone reconstruction, with initial bone resorption, by osteoclasts and concurrent bone formation by osteoblasts. oxidative stress due to coal dust exposure is not only found in the lungs, but also in the circulation or systemically. the aim of this study was to determine the effect of oxidative stress from coal dust exposure on the number of osteoblasts and osteoclasts in rats. in this experimental study, four groups were evaluated: control; coal dust exposure at 6.25 mg/m3 for 28 days; coal dust exposure at 12.5 mg/m3 for 28 days; coal dust exposure at 25 mg/m3 for 28 days (all exposures were given daily for one hour). circulatory oxidative stress was measured by malondialdehyde level. osteoblast and osteoclast numbers were counted by light microscopic examination of distal femoral cross-sections stained with hematoxylin eosin. this study showed that malondialdehyde levels were significantly increased in coal dust exposure groups, in comparison with the control group (p<0.05). there were also significantly decreased numbers of osteoblasts (p<0.05) and significantly increased numbers of osteoclasts (p<0.05) numbers in coal dust exposure groups, as compared with the control group. no correlations were found between malondialdehyde levels (oxidative stress) and respective numbers of osteoblasts and osteoclasts in all coal dust exposure groups (p>0.05). coal dust exposure increased malondialdehyde level and osteoclast numbers, and decreased osteoblast numbers, but no correlation was found between oxidative stress (caused by coal dust exposure) and osteoblast and osteoclast numbers. key words: coal dust, subchronic, oxidative stress, osteoblast, osteoclast, rat *department of orthopedic surgery, **department of pathology anatomy, ulin general hospital medical faculty, lambung mangkurat university, banjarmasin ***department of medical chemistry, medical faculty, lambung mangkurat university, banjarbaru ****department of pharmacology, medical faculty, brawijaya university, malang east java correspondence dr. izaak zoelkarnain akbar, spot(k) department of orthopedic surgery, ulin general hospital medical faculty, lambung mangkurat university, jl. a yani km.2 no.43 banjarmasin south kalimantan email : izaakakbar@yahoo.co.id univ med 2011;30:73-9. decreased osteoblasts and increased osteoclasts in rats after coal dust exposure izaak zoelkarnain akbar*, nia kania**, bambang setiawan***, nurdiana****, and m. aris widodo**** may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina introduction o s t e o b l a s t s a r e b o n e f o r m i n g c e l l s d e r i v e d f r o m m u l t i p o t e n t m e s e n c h y m a l precursor stem cells, which are also stem cells for the bone marrow stroma, chondrocytes, muscle cells, and adipocytes, with mean life span of around 3 weeks.(1,2) osteoclasts are multinuclear cells of large size (50 to 100 µm i n d i a m e t e r ) , o r i g i n a t i n g f r o m t h e 74 akbar, kania, setiawan, et al osteoblasts and osteoclasts in rats hematopoietic system,(1,3) with a life span of up to ∼2 weeks.(4) the precursors of osteoclasts a r e h e m a t o p o i e t i c c e l l s o f m o n o c y t e / macrophage lineage,(1,2) and the osteoclasts reach the bones through cell migration of progenitors from adjacent connective tissue.(1) b o n e r e m o d e l i n g i s a p h y s i o l o g i c a l p r o c e s s o f c o r t i c a l a n d t r a b e c u l a r b o n e reconstruction, with initial bone resorption by osteoclasts and concurrent bone formation by osteoblasts. this process is coordinated throughout life and is the dominant process in a d u l t s k e l e t o n s . i f t h e r e s o r p t i o n a n d absorption are quantitatively proportional, the remodeling proceeds in a balanced manner.(5,6) the remodeling process may be disrupted by various toxic substances from the environment, resulting in disease. exposure to metals, such as cadmium, aluminum, and lead is a risk factor for osteoporosis.(7) south kalimantan is the largest coal producer in indonesia, with mines located throughout the area (banjar, tanah laut, kotabaru, tanah bumbu, hulu sungai tengah, hulu sungai utara, hulu sungai selatan, tapin, and tabalong). coal dust is a complex admixture of several minerals, trace metals, a n d o r g a n i c s u b s t a n c e s o t h e r t h a n c o a l particles.(8) coal dust particles deposited in the alveolar epithelium are phagocytosed by alveolar macrophages, subsequently increasing release of h2o2 and •o2, triggering oxidative stress.(9,10) oxidative stress from coal dust exposure is not only found in the lungs, but also in the circulation or systemically.(10) high levels of oxidative stress causes apoptotic necrosis.(11) the numbers of osteoclasts and osteoblasts depend on two processes, i.e. osteoclastogenesis or osteoblastogenesis, and apoptosis. (4) the study by parhami et al. demonstrated that lipid oxidation inhibits osteoblast differentiation, thus disturbing bone formation.(12) in vitro and in vivo it was also demonstrated that free radicals are involved in osteoclastogenesis and bone resorption.(13) therefore the aim of our study was to evaluate the effect of oxidative stress, induced by coal dust exposure, on the numbers of osteoblasts and osteoclasts in rats. methods research design this was a controlled experimental study conducted in the period of october-december 2010. experimental animals the experimental subjects were 2-3 month old male wistar rats weighing 200-250 grams, subdivided into 4 groups: group 1 was the control group (p1) without exposure to coal dust; group 2 was exposed to coal dust at a dose of 6.25 mg/m3 for 1 hour per day for 28 days (p2); group 3 was exposed to coal dust at a dose of 12.5 mg/m3 for 1 hour per day for 28 days (p3); and group 4 was exposed to coal dust at a dose of 25 mg/m3 for 1 hour per day f o r 2 8 d a y s ( p 4 ) . t h e s a m p l e s i z e w a s calculated from the formula of stell et al.(14) to be 6 rats per group. all male wistar rats were obtained from the pharmacology laboratory, faculty of medicine, brawijaya university, malang. preparation of coal dust coal dust was prepared by pulverizing coal particles by means of equipment consisting of ball mills, ring mills and raymond mills at pt. carsurin coal laboratories, banjarmasin the resulting coal dust was <75 µm in diameter, and subsequently further screened, using microsieve (biodesign, usa), producing coal dust of <10 µm diameter as respirable particulate matter. exposure to coal dust exposure to coal dust was performed by means of a coal dust exposure device of 0.5 m3 c a p a c i t y, d e s i g n e d a n d p r o v i d e d b y t h e pharmacology laboratory, faculty of medicine, 75 brawijaya university. this device works on the principle of providing an ambient environment containing coal dust as particulate matter, which c a n b e i n h a l e d i n t o t h e a i r w a y s o f t h e experimental animals. the air flow through the blower is 1.5-2 liter/minute, which corresponds to the air flow in collieries. the dose of coal d u s t e x p o s u r e h a d b e e n d e t e r m i n e d b y preliminary studies to be 12.5 mg/m3 for a high dose.(13) parameter measurements after undergoing coal dust exposure for 28 days, the experimental animals were sectioned. the rats were anesthesized by means of ether on cotton wool in a plastic container. sectioning was performed on rats with beating hearts by opening the abdomen, cutting the ribs, and opening the thoracic cavity to find the heart. blood was obtained from the heart to be used for parameter measurements. the p a r a m e t e r s m e a s u r e d i n t h e b l o o d w e r e m a l o n d i a l d e h y d e ( m d a ) c o n c e n t r a t i o n , performed in the pharmacology laboratory, faculty of medicine, brawijaya university, malang. in addition to the blood samples, the posterior extremities were also taken for histological sections of the femoral bones with hematoxylin-eosin staining. subsequently the numbers of osteoblasts and osteoclasts were counted in the pathology laboratory, faculty of medicine, brawijaya university, malang, by means of image analyzer software. for each p r e p a r a t i o n , 1 0 h i g h p o w e r f i e l d s w e r e examined at 400 x magnification. ethical clearance e t h i c a l c l e a r a n c e f o r w o r k o n experimental animals for this study was issued by the research ethics committee, faculty of medicine, lambung mangkurat university. statistical analysis the collected data were analyzed using anova, and a p value of p<0.05 indicates a significant differences between intervention groups. significant results of anova were followed up by a post hoc test. in addition correlation tests were performed. all statistical analyses were performed using spss software (15.0) for windows. minimal significance was assumed at p<0.05. figure 1. mda levels according to coal dust exposure groups in rats. p1=control; p2=coal dust exposure at 6.25 mg/m3 for 28 days; p3=coal dust exposure at 12.5 mg/m3 for 28 days; p4= coal dust exposure at 25 mg/m3 for 28 days univ med vol.30 no.2 76 akbar, kania, setiawan, et al osteoblasts and osteoclasts in rats results according to anova results, subchronic e x p o s u r e t o c o a l d u s t a t v a r i o u s d o s e s significantly increased malondialdehyde levels in comparison with the control group (p=0.000). using post hoc testing, there were significant differences between intervention groups, except between group p2 and control (p1) (p=0.192) (figure 1). u s i n g a n o va , c o a l d u s t e x p o s u r e significantly reduced the mean number of osteoblasts, as compared to the control group (p=0.000). on post hoc testing significant differences were found between intervention groups (p<0.05) (figure 2). based on anova, coal dust exposure significantly increased the mean number of osteoclasts, as compared to the control group (p=0.000). using post hoc testing significant differences were found between intervention groups, except between p2 and p3 (p= 0.064) (figure 3). figure 2. mean number of osteoblasts based on coal dust exposure groups in rats p1=control; p2=coal dust exposure at 6.25 mg/m3 for 28 days; p3=coal dust exposure at 12.5 mg/m3 for 28 days; p4= coal dust exposure at 25 mg/m3 for 28 days figure 3. mean number of osteoclasts based on group in rats p1=control; p2=coal dust exposure at 6.25 mg/m3 for 28 days; p3=coal dust exposure at 12.5 mg/m3 for 28 days; p4= coal dust exposure at 25 mg/m3 for 28 days 77 according to the results of the pearson correlation test, there was no significant correlation between mda levels and osteoblast and osteoclast numbers in the intervention groups (p>0.05). discussion inhalation of coal dust leads to formation of reactive oxygen species (ros) through d i r e c t a n d i n d i r e c t m e c h a n i s m s . d i r e c t mechanisms involve bioactive components c o n t a i n e d i n c o a l d u s t , w h i l e i n d i r e c t mechanisms form ros through respiratory bursts during phagocytosis by macrophages.(10) the content of transitional metals, comprising fe, cr, mn, co, ni, cu, zn, and silicon, may catalyze fenton reactions to produce reactive oxygen compounds.(15) during phagocytosis of inhaled particles, superoxide radicals are f o r m e d , t h a t a r e s u b j e c t t o s p o n t a n e o u s dismutation to produce hydrogen peroxide. in t h e p r e s e n c e o f t r a n s i t i o n a l m e t a l s , t h e hydrogen peroxide is converted into hydroxyl radicals.(16) in the present study, increased levels of mda are the result of oxidative stress, due to coal dust exposure. formation of mda is by lipid peroxidation, which is significantly different between intervention groups. increased lipid peroxidation is consistent with the study by armutcu et al.(10) in addition, according to the hypothesis of parhami,(12) lipid peroxidatiom may affect the numbers of osteoblasts and osteoclasts in the aged condition (figure 4). in our study, the number of osteoblasts decreased concurrently with increased dose of coal dust exposure, although no significant difference was found between p2 and p3. in addition, the number of osteoclasts increased concurrently with increased doses of coal dust exposure, although no significant difference was found between p2 and p3. this indicates an imbalance between bone formation and resorption. interestingly, there was no correlation between mda levels and the number of osteoblasts, or between mda levels and the n u m b e r s o f o s t e o c l a s t s , w h i c h w a s n o t according to parhami’s hypothesis.(12) this is presumably due to the presence of other figure 4. mechanisms of osteoporosis triggered by coal dust. oxidation products are formed and accumulate in bone or are transformed into more reactive molecules, thus triggering a) direct effects via mechanisms of 1) inhibition of osteoblast differentiation and increased apoptosis of osteoblasts, thus decreasing bone formation; 2) induction of osteoclasts differentiation, thus increasing bone resorption; b) indirect effects via production of proinflammatory cytokines. modified after parhami.(12)   lipids & lipoproteins  lipid oxidation and  accumulation in bone  inflammation and cytokine  production inflamasi  effects on bone cells  inhibition of osteoblast differentiation  and increased apoptosis  induction of osteoclast  differentiation  transformation of molecules  osteoporosis  coal dust   univ med vol.30 no.2 78 akbar, kania, setiawan, et al osteoblasts and osteoclasts in rats molecules capable of affecting the numbers of osteoblasts and osteoclasts. mda molecules are a c t i v e m o l e c u l e s t h a t m a y u n d e rg o transformation into other molecules, e.g. via e n z y m a t i c g l y c o s y l a t i o n . t h e a v a i l a b l e aldehyde groups may react with amino groups of proteins, giving rise to advanced glycation end products (ages), which subsequently may affect osteoblasts and osteoclasts.(17-19) another mechanism of coal dust is through triggering of increases in nitrous oxide levels, which may trigger apoptosis of osteoblasts.(10,20) one limitation of this study is the failure to explore the various parameters that are increased as a result of coal dust exposure, in order to demonstrate the markers that are the cause of the reduction in the numbers of osteoblasts and the increased numbers of osteoclasts. conclusion exposure to coal dust increases mda levels and the numbers of osteoclasts, while reducing the numbers of osteoblasts, although there was no significant correlation between oxidative stress due to coal dust exposure and the numbers of osteoblasts and osteoclasts. acknowledgement we thank pt. carsurin coal laboratories for the supply of coal dust of <75 µm diameter. references 1. manolagas sc. birth and death of bone cells: basic regulatory mechanisms and implications for the pathogenesis and treatment of osteoporosis. endocr rev 2000;21:115-37. 2. okamoto m, murai j, yoshikawa h, tsumaki n. bone morphogenetic proteins in bone stimulate osteoclasts and osteoblasts during bone development. j bone min res 2006;21:1022-33. 3. saltel f, chabadel a, zhao y, lafage-proust m, clezardin p, jurdic p, et al. transmigration: a new property of mature multinucleated osteoclasts. j bone min res 2006;21:1913-23. 4. wu x, pan g, mckenna ma, zayzafoon m, xiong w, mcdonald jm. rankl regulates fas expression and fas-mediated apoptosis in osteoclasts. j bone min res 2005;20:107-16. 5. lerner uh. bone remodeling in postmenopausal osteoporosis. j dent res 2006;85:584. 6. epstein s. the roles of bone mineral density, bone turnover, and other properties in reducing fracture risk during antiresorptive therapy. mayo clin proc 2005;80:379-88. 7. alfven t, elinder c, carlsson md, grubb a, hellstrom l, persson b, et al. low-level cadmium exposure and osteoporosis. j bone min res 2000;15:1579-86. 8. dalal ns, newman j, pack d, leonard s, valyathan v. hydroxyl radical generation by coal mine dust: possible implication to coal workers’ pneumoconiosis. free radic biol med 1995;18: 1-20. 9. huang x, finkelman rb. understanding the chemical properties of macerals and minerals in coal and its potential application for occupational lung disease prevention. j toxicol environ health part b 2008;1:45-67. 10. armutcu f, gun bd, altin r, gurel a. examination of lung toxicity, oxidant/antioxidant status and effect of erdosteine in rats kept in coal mine ambience. environ toxicol pharmacol 2007;24:106-13. 11. glorie g, legrand-poels s, piette j. nf-κb activation by reactive oxygen species: fifteen years later. biochem pharmacol 2006;72:1493505. 12. parhami f. possible role of oxidized lipids in osteoporosis: could hyperlipidemia be a risk factor? prostaglandins leukot essent fatty acids 2003;68:373-8. 13. ghanem mm, porter d, batteli la, valyathan v, kashon ml, ma jy, et al. respirable coal dust particle modify cytochrome p4501a1 expression in rat alveolar cells. am j resp cell mol biol 2004;31:171-87. 14. steel rgd, torrie jh, dickey da. principles and procedures of statistics, a biometrical approach. 3rd ed. singapore: mcgraw hill book company; 1997. 15. nadif r, mintz m, jedlicka a, bertrand j, kleeberger sr, kauffmann f. association of cat polymorphisms with catalase activity and exposure to environmental oxidative stimuli. free rad res 2005;39:1345–50. 16. altin r, kart l, tekin i, armutcu f, tor m, ornek t. the presence of promatrix mettaloproteinase3 and its relation with different categories of coal worker’s pneumoconiosis. mediators inflamm 2004;13:105–9. 79 17. hein g, wiegand r, lehmann g, stein g, franke s. advanced glycation end-products pentosidine and nε-carboxymethyllysine are elevated in serum of patients with osteoporosis. rheumatol 2003; 42:1242-6. 18. franke s, siggelkow h, wolf g, hein g. advanced glycation endproducts influence the mrna expression of rage, rankl and various osteoblastic genes in human osteoblasts. arch physiol biochem 2007;113:154-61. 19. alikhani m, alikhani z, boyd c, maclellan cm, raptis m, liu r, et al. advanced glycation end products stimulate osteoblast apoptosis via the map kinase and cytosolic apoptotic pathways. bone 2007;40:345-53. 20. chen rm, chen tl, chiu wt, chang cc. molecular mechanism of nitric oxide-induced osteoblast apoptosis. j orthop res 2005;23:4628. univ med vol.30 no.2 rina1 34 body mass index and quality of life among the elderly january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina rina k. kusumaratna,*a and adi hidayat* *department of community medicine, faculty of medicine trisakti university correspondence adr. dr. rina k.kusumaratna, m.kes. department of community medicine, faculty of medicine trisakti university jl. kyai tapa no.260, grogol jakarta 11440 email: rkusumartna@yahoo.com univ med 2009; 28: 34-41 abstract obesity in many countries is a major public health problem. the health problems associated with an increase in the proportion of elderly in the population are further compounded by the increasing prevalence of obesity and overweight in older people. the aimed of this study was to explore any difference of quality of life (qol) attributable body mass index (bmi) categories using the whoqol-bref instruments. an observational cross-sectional study was conducted in mampang prapatan district, south jakarta. a total of 296 free-living elderly aged 60 years and above were participated in this study. bmi was calculated as the weight (kg) divided by the square of the height (m2). quality of life was measured by whoqolbref questionnaire in the indonesian language version. the results showed that the percentage of overweight subjects was 27.5% in male and 22.2% in female elderly, while the percentage of obese subjects was respectively 8.3 and 13.2.1% respectively. in males, the mean scores on each domain of the qol were higher compared with females. the significant differences were in social relationship (p=0.000) and environment (p=0018) domains. among male and female elderly the highest mean scores of each domain of qol was observed in the environment domain, and mean scores of the environment domain was significantly higher in males compare with females (p=0.018). the results have practical implications for the health of older persons because they show that the overweight have a worse score in two qol domains, namely psychological health and environment. key words: body mass index, quality of life, elderly introduction most developed and developing countries i n t h e w o r l d a r e e x p e r i e n c i n g a n o b e s i t y epidemic.(1,2) the high and growing frequency of excess body weight and its association with n u m e r o u s c h r o n i c d i s e a s e s , s u c h a s cardiovascular disease, diabetes, certain tumors and osteomuscular disorders, signifies that overweight and obesity are an important public health problem.(3) the increased life expectancy will lead to growing numbers of frail, disabled, and institutionalized older persons with a decreased quality of life and increased costs for h e a l t h c a r e . (4 ) i n t h e u n i t e d s t a t e s , t h e prevalence of overweight and obesity among 35 univ med vol.28 no.1 adults has increased markedly during the past few decades.(5) the impact of excess weight on physical health is well documented, as obesity is associated with higher morbidity risk and shorter life expectancy.(6) obesity could be measured by using height and body weight indices, both of which are anthropometric indices usually used as a body mass index ( b m i ) o f a n i n d i v i d u a l ; w h e r e b m i i s calculated as body weight in kilograms divided by the square of height in meters.(5) in old age, the elderly are expected to have a good quality of life to conduct independent daily activities. various definitions of quality of life have been proposed by the experts. the world health organization (who) has defined qol as “the individual’s perception of his/her position in life in the context of the culture and value systems in which he/she lives and in relation to his/her goals, expectations, standards and concerns”.(6) this international body has recently developed a generic profile subjective qol instrument, the whoqol-100, and its brief version, the whoqol-bref.(7) this instrument is to be used for measuring the quality of life among the elderly. although it could be hypothesized that obesity is a predictor o f t h e q u a l i t y o f l i f e o f t h e e l d e r l y, t h e hypothesis needs further investigation. the objective of this study was to explore any difference of quality of life (qol) attributable to bmi categories using the whoqol-bref instrument in the 60+ age group. methods research design an observational cross-sectional study was conducted in mampang prapatan district, south jakarta. the study was carried out from april to september 2008. research subjects s u b j e c t s o f t h e s t u d y, i . e . a p p a r e n t l y h e a l t h y f r e e l i v i n g e l d e r l y w h o m e t t h e inclusion criteria were randomly selected from a list in each rukun tetangga (rt). a total of 296 free-living elderly aged 60 years and above w e r e p a r t i c i p a t e d i n t h i s s t u d y w i t h t h e following inclusion criteria: individuals of both g e n d e r s , m o b i l e , a p p a r e n t l y h e a l t h y, independent, able to verbally communicate and willing to join the study. data collecting and measurements information was collected through homebased personal interview using a structured q u e s t i o n n a i r e , f o l l o w e d b y a p h y s i c a l examination to measure blood pressure and anthropometric variables. blood pressure was m e a s u r e d w i t h a c a l i b r a t e d m e r c u r y sphygmomanometer. subjects were deemed to be hypertensive where their systolic blood pressure (sbp) was > 140mmhg, their diastolic blood pressure (dbp) was >90mmhg or they w e r e o n c u r r e n t a n t i h y p e r t e n s i v e d r u g treatment.(9) the anthropometric measurements used to calculate bmi, by standard procedures, included both height and weight. height was measured using a portable microtoise to the nearest 0.1 cm and weight was measured using sage portable scales to the nearest 0.1 kg. bmi was calculated as the weight (kg) divided by t h e s q u a r e o f t h e h e i g h t ( m 2) . f o r a s i a n population, bmi is classified into the following categories: underweight (<18.5 kg/m2), normal (18.5–22.9 kg/m2), overweight (23.0–27.5 kg/ m2), and obese (> 27.6 kg/m2).(10) q u a l i t y o f l i f e w a s m e a s u r e d b y w h o q o l b r e f q u e s t i o n n a i r e i n i t s indonesian translation. this questionnaire is made up of 26 items which assess the following four domains: i) physical domain, consisting of 36 kusumaratna, hidayat quality of life among the elderly s e v e n i t e m s , i i ) p s y c h o l o g i c a l d o m a i n , consisting of six items, iii) social relationship domain, consisting of three items, and iv) environmental domain, consisting of eight items. the whoqol-bref also measures two facets of quality of life as general outcomes, namely: i) overall quality of life, and ii) general health. each question was assigned scores b e t w e e n 1 a n d 5 , w i t h t h e h i g h e r s c o r e s indicating a better quality of life. the score f r o m e a c h d o m a i n w a s c a l c u l a t e d b y multiplying each facet by a factor of four. a domain would not be assigned a score if more t h a n 2 0 % o f t h e q u e s t i o n s h a d n o t b e e n answered by the subjects. the whoqolbref instrument was validated and indicated high validity and reliability for measuring the quality of life among the elderly.(11) interviewers underwent standardized training to administer the questionnaire and take blood pressure readings and anthropometric measurements. ethical clearance ethical clearance was approved by the research ethics committee of the faculty of medicine, trisakti university. the participants were requested to give a written consent, with a t t a c h e d s i g n a t u r e f o r t h e i l l i t e r a t e o r thumbprint for the illiterate. identify of all participants was kept confidential and used for research purposes only. statistical analysis univariate analyses were performed for background characteristics, and normality of distribution of the data was checked using the kolmogorov-smirnov test. normally distributed data were expressed as mean and standard d e v i a t i o n , a n d p e r c e n t a g e . a n o va w a s conducted to compare each qol domain by bmi c a t e g o r i e s . t h e s t a t i s t i c a l a n a l y s i s w a s performed with the spss software package (windows version 15.0 spss chicago), and all p-values less than 0.05 were considered to be statistically significant. results the subject characteristics are shown in table 1. one hundred and eight (36.5%) were male and 188 (63.5%) were female elderly. mean age of the elderly was 65.8 ± 4.6 years (range 60-85). the majority (81.2%) of the elderly were aged 60 to 70 years and 18.2% were aged 70+ years. from the anthropometric indices it may be concluded that male elderly were taller and heavier than females, and also had a greater knee-height. overall, the body mass index was in the normal category for both genders. most of the elderly (74.4%) had six years of education, while male elderly had a higher educational background. as to marital status of the elderly, there were more widowed f e m a l e s t h a n m a l e s . t h e p e r c e n t a g e o f overweight subjects was 27.5% in male and 22.2% in female elderly, and that of obese subjects, 8.3 and 13.2%, respectively (table 1). table 2 shows the mean scores for the qol questionnaire scales based on gender. in males, the mean scores for each qol domain were higher compared with females. the significant difference was in social relationship (p=0.000) and environment (p=0018) domains. among male and female elderly the highest mean scores of each domain of qol were observed in the environment domain and were significantly h i g h e r i n m a l e s c o m p a r e d w i t h f e m a l e s (p=0.018). t h e h i g h e s t m e a n s c o r e s f o r t h e psychological health and environment domains were observed in overweight subjects. as c o m p a r e d w i t h u n d e r w e i g h t , n o r m a l , overweight and obesity the mean scores of psychological (p=0.019) and environment (p=0.005) domains showed significantly higher values (table 3). 37 univ med vol.28 no.1 table 2 mean scores for quality of life domain based on gender table 1 subject characteristics based on gender (n=296) table 3. adjusted mean scores for quality of life domains according to bmi categories 38 after adjusting for mean scores of qol d o m a i n s ( ta b l e 3 ) , t h e f o l l o w i n g f o u r domains of qol remained higher in the o v e r w e i g h t c a t e g o r y, n a m e l y t h e psychological health domain (p=0.019), and environment domain (p=0.005). on average the qol as measured in psychological and environment domains increased significantly with level of the body mass index, especially among overweight category. the tukey hsd multiple comparisons illustrated that the scores for the physical h e a l t h a n d e n v i r o n m e n t d o m a i n s w e r e significantly lower in the underweight group when compared to the overweight group (p=0.013 and p=0.008) (table 4). discussion a s m o r e p e o p l e a r e b e c o m i n g i n c r e a s i n g l y o l d e r, t h e y m a y e x p e c t t o experience disability and poor qol, even if they are apparently healthy. apart from morbidity, these issues should receive due consideration. the ideal outcome of added years of life is to live through those years as a healthy individual free of disability and disease. the results of the present study indicate that among persons aged 60 years and over in jakarta, overweight is associated with worse scores in the psychological health and environment domains. these results were observed across the genders, regardless of age or type of anthropometric measure used. in contrast, obesity was not associated with a worse hrql, but the percentage of overweight and obese subjects in this study was substantial, and was comparable with that reported by a recent nationally representative study in indonesia (8.8% overweight, 10.3% obesity), especially in jakarta province (11.9% overweight, 15% obesity) and other provinces. the proportion of obese is greater than that of overweight, although this has also been observed in other populations around the world that have a very high prevalence of obesity. in this study there were more overweight subjects (24.3%) compared to obese (11.5%). the percentage of overweight subjects was 27.5% in male and 22.2% in female, and that of obese subjects, 8.3 and 13.2.1%, respectively. this results were difference compared to the study in the population aged 60 y and over in spain, the percentage of overweight subjects was 48.5% in men and 39.8% in women, and of obese subjects, 31.9 and 41.1% respectively.(13) the mean bmi in both genders were similar, but elderly men were taller and heavier compared to women. compared with the normal weight and obese categories, overweight elderly had higher v a l u e s f o r p s y c h o l o g i c a l h e a l t h , s o c i a l relationships and environment scores. similar results were found in the study by daviglus et al,(5) in which the bmi for men and women had significant inverse-graded associations with all health status questionnaire. the proportion of overweight was higher than that of obese in our study, but the study in spain showed a greater table 4 multiple comparisons of qol domains between bmi groups among the elderly kusumaratna, hidayat quality of life among the elderly 39 univ med vol.28 no.1 proportion of obese compare to overweight.(13) although these results have also been observed in other populations around the world which a very high prevalence of obesity.(14,15) overweight and obesity are associated w i t h h i g h e r m o r b i d i t y a n d s h o r t e r l i f e expectancy, but the effect of body mass index (bmi) assessed in middle age on subsequent quality of life among older survivors is as yet undetermined. while even this has not been conclusively proved, it has been reported that i n d i v i d u a l s w i t h f a v o r a b l e h e a l t h r i s k s (determined by smoking status, bmi, and exercise patterns) have half the cumulative disability of those at high risk after 32 years of follow-up. in addition, onset of disability was postponed by approximately 5 years in the lowrisk compared with the high-risk group. (16) these findings suggest that healthy habits and traits may lead to postponement of initial disability and decreased lifetime disability. a l o t o f a t t e n t i o n h a s b e e n f o c u s e d o n t h e prevention of chronic diseases by prevention a n d c o n t r o l o f m a j o r r i s k f a c t o r s , w i t h a resultant reduction in mortality and an increase in longevity. however, risk factors for chronic disease, which can be largely asymptomatic, can also cause subclinical disease and affect quality of life.(17) overweight (bmi, 23.0-27.5) and obesity (bmi, >27.6) are important determinants of type 2 diabetes mellitus, insulin resistance, hypertension, and dyslipidemia, (18) and are independently associated with future morbidity and mortality from coronary heart disease and cardiovascular disease. in addition, excess weight has is strongly correlated with a higher risk of some types of cancers (endometrial, prostate, colon, and breast), gallbladder disease, o s t e o a r t h r i t i s , s l e e p a p n e a , r e s p i r a t o r y problems, and other conditions. furthermore, high bmi levels have been associated with increased health care costs. it is well known that the obese suffer social discrimination and, in particular, that those who come to clinics in search of treatment tend to have lower selfesteem, more anxiety and a poorer body image. nonetheless, a number of studies have found that this has affected the mental less than it has the physical components of hrql. (20) the influence of age on the relation between bmi and mortality has not been widely studied, especially in the elderly. a population-based california cohort study showed the association between increased weight and higher mortality is primarily relevant for the younger elderly.(21) one limitation of this study was that it did not determine the influence of other factors related to overweight and obesity, such as physical inactivity or diet, on physical, mental, and social health among the elderly. the findings of this study demonstrate the adverse impact of high bmi in the elderly on future health-related quality of life. with much o f t h e o l d e r p o p u l a t i o n f a c i n g t r e n d s o f increasing overweight and obesity, preventive measures are urgently required to lessen future individual and societal burdens of disease, disability, cost of care, and impaired quality of life associated with excess weight. strategies that emphasize primary prevention of excess weight from an early age, with the potential of ending the obesity epidemic and leading to improved quality of life, should also become an ongoing component of national public health policy. conclusions the results have practical implications for the health of older persons because they show that the overweight have a worse score in the qol domains of psychological health and environment. thus, the study emphasizes the need for appropriate approaches in primary care to break the vicious circle of overweight, 40 obesity and decreasing qol. not only does this shed new light on the enormous impact that excess weight could exert upon the health of the elderly population, but should serve as a s p u r t o i m p l e m e n t t h e n e c e s s a r y c o n t r o l measures. acknowledgements this study was supported by the medical faculty of trisakti university as a part of big study of “supplementation among the elderly”. we thank to those who was involved in this s t u d y a n d a l s o t o m y c o l l e a g u e s w h o s e invaluable cooperation and assistance made this study possible. references 1. gutie´rrez-fisac jl, banegas jr, rodrý´guez af, regidor e. increasing prevalence of overweight and obesity among spanish adults, 1987–1997. int j obes relat metab disord 2000; 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152: 324-33. 20. ford es, moriarty dg, zack mm, mokdad ah, chapman dp. selfreported body mass index and health related quality of life: findings from the behavioral risk factor surveillance system. obes res 2001; 9: 21–31. 21. corrada mm, kawas ch, mozaffar f, paganinihill a. association of body mass index and weight change with all-cause mortality in the elderly. am j epidemiol 2006; 163: 938–49. alvina 133 duration of rhegmatogenous retinal detachment predicts recovery of retinal sensitivity september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina rose* *department of ophthalmology medical faculty, trisakti university correspondence dr. rose, spm department of ophthalmology medical faculty, trisakti university jl. kyai tapa no. 260, grogol jakarta 11440 phone: 021-5672731 ext.2614 email: rose_149@yahoo.com univ med 2009;28:133-8 abstract the decision to treat a disease is often based on the presence or absence of symptoms, one prototype case being rhegmatogenous retinal detachment. detachment of the neural retina from the pigment epithelium is a major cause of anatomical and functional dysfunction of the retina, where retinal recovery is inversely related to duration of detachment. the purpose of retinal reattachment is to effect recovery of the photoreceptors and pigment epithelium from degeneration. the aim of this study was to determine the critical duration of rhegmatogenous retinal detachment resulting in optimal retinal recovery after reattachment. a prospective study was conducted at a private hospital in yogyakarta. thirty five eyes were involved in this study. three months after reattachment, central retinal recovery was measured by means of a goldmann manual kinetic perimeter. the results showed that retinal recovery developed three months after surgery if the onset of rhegmatogenous retinal detachment was less than 28 days before surgery. the results were not significant if the onset of rhegmatogenous retinal detachment was more than 35 days. although the goldmann manual kinetic perimeter can efficiently detect central retinal sensitivity, it should be supported by more sensitive tools to evaluate the anatomy and function of the retina. keywords: duration, rhegmatogenous retinal detachment, reattachment surgery, retinal sensitivity introduction rhegmatogenous retinal detachment (rrd) is characterized by separation of the neural retina from the retinal pigment epithelium (rpe) with collection of fluid derived from liquefied vitreous gel through the retinal defect into the subretinal space.(1-3) this condition can lead to loss of vision especially when it involves the m a c u l a . ( 4 6 ) u n t r e a t e d , r r d c a n l e a d t o irreversible damages of the rods and cones, frequently ending in blindness.(7) rrd leads to damage of the photoreceptors and retinal pigment epithelium (rpe),(8,9) where 134 rose rhegmatogenous retinal detachment the death of the photoreceptors is mediated by a p o p t o s i s . ( 6 , 1 0 , 11 ) a l l o f t h e s e w i l l l e a d t o development of reactive changes in the inner retinal neurons and macroand microglial cells, and prolonged retinal circulation times.(6,12,13) therefore, retinal reattachment should be performed to reduce the effect of the metabolic and cellular changes.(8,14,15) successful anatomic reattachment is usually s t a b l e a t t h e t h i r d m o n t h a f t e r r e t i n a l reattachment.(1,6) unfortunately, it is not always followed by functional recovery. although anatomic reattachment is successful, visual dysfunction and color vision defects may persist.(4,16,17) the visual dysfunction is affected by the duration of symptoms, the preoperative visual acuity, the type and extent of retinal d e t a c h m e n t , a n d t h e i n v o l v e m e n t o f t h e macula.(1,11,14) therefore, there is a need for the detection of functional alterations in the retina after reattachment of the retina.(11,17) there is no general consensus in identifying a critical period beyond which the prognosis for recovery dims.(6,18) in this study, we set out to determine the period of rrd before surgery which would still result in recovery of central retinal sensitivity after retinal reattachment. methods research design a prospective study was conducted at a private hospital in yogyakarta from june 2007 to june 2008. the study was designed to evaluate whether the duration of rhegmatogenous retinal detachment before reattachment surgery would impact on the retinal sensitivity 3 months after successful anatomical retinal reattachment. research subjects thirty five eyes of 35 patients with primary rrd were involved in this study. all of them underwent retinal reattachment surgery between june 2007 to march 2008 at a private hospital in yogyakarta. the inclusion criteria used to select patients for this study were: patients with primary rhegmatogenous retinal detachment with macular involvement, agreeing to have retinal reattachment surgery, and showing successful retinal reattachment 3 months after surgery. exclusion criteria included retinal redetachment within 3 months after surgery, hereditary retinal disorders (retinitis pigmentosa), vascular retinal disorders (proliferative diabetic retinopathy), chorioretinal inflammation, and opacity of the refractive media. drop-out was indicated if the patient did not return for follow-up at 3 months after surgery. data collection and measurements the data were collected by anamnesis and ophthalmological examination. they included age, gender, duration of symptoms, location and extent of the retinal detachment, presence of proliferative vitreoretinopathy (pvr), type of retinal surgery, and central retinal sensitivity before and 3 months after retinal reattachment surgery. all of the subjects underwent retinal examination by indirect ophthalmoscopy. rrd was defined by the finding of a separation of the retina with associated hole or tear. the duration of the retinal detachment was calculated from the first day of visual complaints until the day of the retinal reattachment surgery. central retinal sensitivity, expressed in isopters, was quantified by an ophthalmologist using the goldmann manual kinetic perimeter. recovery of retinal sensitivity was defined as increased r e t i n a l s e n s i t i v i t y 3 m o n t h s a f t e r r e t i n a l reattachment. ethical clearance ethical clearance was approved by the research ethics committee of the dr. yap eye hospital. all patients participating in this study gave their informed consent before surgery. 135 statistical analysis regression analysis by means of spss 15.0 f o r wi n d o w s w a s u s e d t o e v a l u a t e t h e relationship between the duration of rrd before surgery and recovery of retinal sensitivity after successful anatomical reattachment. results t h i r t y f i v e e y e s o f 3 5 p a t i e n t s w e r e involved in this study. the patients consisted of 23 (65.7%) men and 12 (34.3%) women between the age of 20 and 60 years (mean age 44.4 ± 11.6 years). the duration of detachment ranged from 5 to 60 days with mean duration of 25.8 ± 9.2 days. twenty five eyes had 2 quadrants of retinal detachment and the majority of the detachments was temporally located. all of the detachments involved the macula. only 4 eyes demonstrated pvr, showing of scars of long-standing rrd. the subject characteristics are shown in table 1. table 2 lists the types of retinal surgery performed in the subjects. the type of retinal surgery chosen depended on the condition of the retinal detachment before surgery. twenty nine e y e s ( 8 2 . 9 % ) u n d e r w e n t v i t r e c t o m y i n combination with scleral buckling and gas tamponade, whilst the other eyes were treated with either scleral buckling alone or vitrectomy combined with gas tamponade, with or without another procedure. all of the surgery was conducted by a single retinal surgeon. no retinal redetachment was found during follow-up. three months after successful anatomical reattachment, the subjects were examined for retinal sensitivity. the mean sensitivity of the central retina 3 months after surgery (28.5 ± 3.7 db) was higher than before surgery (19.8 ± 5.7 db). table 3 reveals that the recovery of central retinal sensitivity was not significantly different in subjects who suffered from rrd for 35 days or less (p=0.465) compared with the groups of 14 days (p=0.021), 21 days (p=0.011), and 28 days (p=0.047). discussion the present study provides an evaluation of macular rrd. it has been suggested that retinal detachment involving the macula has m o r e f u n c t i o n a l i m p a c t t h a n p e r i p h e r a l detachment.(1,6,18,19) the metabolic rate in the cells univ med vol.28 no.3 retinal surgery n (%) sb sb + vitrectomy + gas sb + vitrectomy + so sb + vitrectomy + gas + retinotomy sb + vitrectomy + gas + ilm peeling 2 (5.7) 29 (82.9) 1 (2.9) 2 (5.7) 1 (2.9) table 2. types of retinal surger note: sb = scleral buckling; so = silicone oil; ilm = internal limiting membrane characteristics n gender men women mean age ± sd (years) mean duration of detachment ± sd (days) extent of detachment 2 quadrants 3 quadrants 4 quadrants location of detachment temporal inferior superior total pvr no yes 23 (65.7%) 12 (34.3%) 44.4 ± 11.6 25.8 ± 9.2 25 (71.4%) 6 (17.1%) 4 (11.4%) 16 (45.7%) 10 (28.5%) 5 (14.3%) 4 (11.4%) 31 (88.6%) 4 (11.4%) table 1. characteristics of subjects note: sd = standard deviation; pvr = proliferative vitreoretinopathy 136 rose rhegmatogenous retinal detachment of the macula is the highest compared to any other cell in the body.(6,9,11) detachment of this area will result in nutritional deprivation of the photoreceptors and may cause hypoxia and/or ischemia, leading to necrosis and apoptosis of retinal cells. in addition, retinal detachment causes changes in retinal blood supply, resulting in a prolonged retinal circulation time.(8,9,12) the l o c a l i m m u n e a n d i n f l a m m a t o r y r e s p o n s e represents a major causative factor for reactive changes in the retina after detachment.(6,13,14) hypoxia of the photoreceptors is proportional to the duration of detachment, therefore retinal reattachment should be performed promptly to alleviate the hypoxia and prevent photoreceptor degeneration. the recovery of photoreceptors after macular detachment is limited and in how far the process is different from extra-macular detachment is still unclear.(1,6,11) t h e r e s u l t s o f t h i s s t u d y s h o w a n improvement of retinal anatomy and central retinal sensitivity three months after surgery. some factors contributing to retinal recovery in rrd are duration of retinal detachment, the height of macular detachment, the patient’s age, previous macular disease or previously reduced visual acuity.(6,19-21) the mean duration of retinal detachment in this study was 25.8 ± 9.2 days. the duration of the retinal detachment before surgery was estimated by the symptoms of vision. the presence of macular detachment is known to produce disturbances in visual acuity, metamorphopsia, and color vision. (17,19) because all our subjects had retinal detachments that i n v o l v e d t h e m a c u l a , t h e s i g n s o f v i s u a l dysfunction could be used as estimator when the macular detachment occurred for the first time, e v e n t h o u g h i t c o u l d l e a d t o b i a s d u e t o subjective symptoms and difficulties of recall. in the present study, most of the patients had retinal surgery after 2 weeks of detachment, so the retinal recovery in this study was indicative of long-term rhegmatogenous retinal detachment ( 7 d a y s o r m o r e ) . d e g e n e r a t i o n o f t h e photoreceptors in the outer segment generally occurs within 12 hours after retinal detachment, and the cones and rods are damaged within 2472 hours. the degeneration and the peak of proliferation in the inner segments develop within 1-3 days after retinal detachment and decline slowly within several weeks and müller cells grow 3 days after detachment.(6,13,14 ) in longterm retinal detachment (7 days or more), the proliferative response may induce the formation of multiple layers of cells whose polarity does not match that of the original monolayer.(6,12,14) naturally, the results of this study would have been different with detachment of recent onset. several studies had reported that functional recovery of the retina was best evaluated in rrd of recent onset (less than 10 days).(13,21-23) the duration of detachment and the age of patients did not influence the anatomical success of subjects with recent onset rrd, but the height of the macular detachment did.(13,24) patients were advised to undergo scleral buckling within 7 days n β p rrd ≤ 14 days rrd ≤ 21 days rrd ≤ 28 days rrd ≤ 35 days 10 18 22 30 -1.355 -0.581 -0.333 0.092 0.021 0.011 0.047 0.465 table 3. regression analysis for predicting changes in central retinal sensitivity by duration of rrd note: rrd = rhegmatogenous retinal detachment 137 after rrd.(22,25) the present study demonstrates minimal retinal recovery if the onset of rrd was more than 35 days (5 weeks). this result is supported by others, in that the duration of macular detachment was not of prognostic value after 30 days, (19,21,23,26) which may be explained as follows. in experimental detachments of owls, monkeys, and cats, proliferation of the rpe occurs at very low levels after long-term d e t a c h m e n t ( 1 2 2 4 m o n t h s ) . i n l o n g t e r m detachments, disorganized lamellar debris is f o u n d i n t h e s u b r e t i n a l s p a c e , a ff e c t i n g photoreceptor recovery. hypertrophy of the müller cells in the outer plexiform layer will produce glia scars in the subretinal space, leading to failure of photoreceptor regeneration after retinal reattachment.(6,8,10) however, there is still a chance for retinal recovery if there are a number of remaining and retained ciliary stalks along with a few rudimentary discs or membrane evaginations. this is a crucial point because the connecting cilium is essential for production of the outer segment. (6) the goldmann manual kinetic perimetry used to measure the central retinal sensitivity is very efficient in quantifying visual field defects and detecting the location of neurologic lesions but not optimal in quantifying the central retinal sensitivity compared to automated perimetry.(27,28) some studies have demonstrated that multifocal electroretinography (mferg) could detect functional recovery of the retina.(14,25,28) in longterm retinal detachment, the retinal function will decline not only in the detached areas of the retina, but also in the attached areas.(6,28,29) however, this was not found in studies of recent onset rrd; it seems that central retinal function can remain fairly unaffected in peripheral rrd for some time.(1,6,11) in the present study, as optical coherence tomography was not performed, either before or after reattachment surgery, such data as the height of detachment, the presence of edema, and the condition of the borderline areas between d e t a c h e d a n d a t t a c h e d r e t i n a , w e r e n o t available.(20,30) conclusion duration of rrd of less than 28 days leads to significant improvement in retinal sensitivity three months after reattachment. further studies o f r e t i n a l f u n c t i o n u s i n g m o r e s e n s i t i v e instruments are necessary to evaluate any variables that may affect the retinal sensitivity after reattachment. acknowledgments this study was funded by the faculty of medicine of trisakti university. the author wishes to thank all study participants and those persons who supported this study. references 1. kanski jj. retinal detachment. in: kanski jj, editor. clinical ophthalmology a systematic approach. 5th ed. england: butterworth heinemann;2003.p.34888. 2. liesegang tj, skuta gl, cantor lb. retinal detachment. in: liesegang tj, editor. retina and vitreous 2004-2005. section 12. san francisco: american academy of ophthalmology (aao); 2004.p.268-77. 3. sharma a, tolentino mj, maguire am. retinal detachment. top emerg med 2000;22:20-7. 4. polkinghorne pj, craig jp. northern new zealand rhegmatogenous retinal detachment study: epidemiology and risk factors. clin exp ophthalmol 2004;32:159-63. 5. gariano r, kim ch. evaluation and management of suspected retinal detachment. am fam physician 2004;69:1691-8. 6. fisher sk, anderson dh. cellular effects of detachment on the neural retina and the retinal pigment epithelium. in: ryan sj, editor. retina. 3rd ed. singapore: mosby; 2001.p.1961-86. 7. go sl, hoyng cb, klaver cw. genetic risk of rhegmatogenous retinal detachment: a familial univ med vol.28 no.3 138 rose rhegmatogenous retinal detachment aggregation study. arch ophthalmol 2005;123: 1237-41. 8. sakai t, calderone jb, lewis gp, linberg ka, fisher sk, jacobs gh. cone photoreceptor recovery after experimental detachment and reattachment: an immunocytochemical, morphological, and electrophysiological study. invest ophthalmol vis sci 2003;44:416-25. 9. zacks dn, hänninen v, pantcheva m, ezra e, grosskreutz c, miller jw. caspase activation in an experimental model of retinal detachment. invest ophthalmol vis sci 2003;44:1262-67. 10. guimaraes ca, benchimol m, amarante-mendes gp, linden r. alternative program of cell death in developing retinal tissue. jbc 2003;278:41938-46. 11. hollborn m, francke m, iandiev i, buehner e, foja c, kohen l. early activation of inflammation and immune response-related genes after experimental detachment of the porcine retina. invest ophthalmol vis sci 2008;49:1262-73. 12. heij ec, blaauwgeers hgt, de vente j, markerink m, liem ata, kessels ag, et al. decreased level of cgmp in vitreous and subretinal fluid from eyes with retinal detachment. br j ophthalmol 2003;87: 1409-12. 13. hassan ts, sarrafizadeh r, ruby aj, garretson br, kuczynski b, williams ga. the effect of duration of macular detachment on results after the scleral buckle repair of primary, macula-off retinal detachments. ophthalmology 2002;109:146-52. 14. sakai t, iida k, tanaka y, kohzaki k, kitahara k. evaluation of s-cone sensitivity in reattached macula following macula-off retinal detachment surgery. jpn j ophthalmol 2005;49:301-5. 15. jin m, chen y, he s, ryan s, hinton dr. hepatocyte growth factor and its role in the pathogenesis of retinal detachment. invest ophthalmol vis sci 2004;45:323-9. 16. liu f, meyer ch, mennel s, hoerle s, kroll p. visual recovery after scleral buckling surgery in macula-off rhegmatogenous retinal detachment. ophthalmologica 2006;220:174-80. 17. hartono, agni an, rose. changes of retinal sensitivity and central fixation after rhegmatogenous retinal detachment surgery. pertemuan ilmiah tahunan (perdami) batam; 9-12 juni 2005. 18. mowatt l, shun-shin ga, arora s, price n. macula-off retinal detachments. how long can they wait before it is too late? eye j ophthalmol 2005; 15:109-17. 19. doyle e, herbert en, bunce c, williamson th, laidlaw da. how effective is macula-off retinal detachment surgery. might good outcome be predicted? eye 2007;21:534-40. 20. benzerroug m, genevois o, siahmed k, nasser z, muraine m, brasseur g. results of surgery on macular holes that develop after rhegmatogenous retinal detachment. br j ophthalmol 2008;92:2179. 21. yang ch, lin hy, huang js, ho tc, lin cp, chen ms, et al. visual outcome in primary macula-off rhegmatogenous retinal detachment treated with scleral buckling. j formos med assoc 2004;103: 212-7. 22. diederen rm, la heij hc, kessels ag, goezinne f, liem at, hendrikse f. scleral buckling surgery after macula-off retinal detachment: worse visual outcome after more than 6 days. ophthalmology 2007;114:705-9. 23. ozgür s, esgin h. macular function of successfully repaired macula-off retinal detachments. retina 2007;27:358-64. 24. ross w, lavina a, russell m, maberley d. the correlation between height of macular detachment and visual outcome in macula-off retinal detachment of seven days duration or less. ophthalmology 2005; 112:1213-7. 25. schatz p, holm k, andreasson s. retinal function after scleral buckling for recent onset rhegmatogenous retinal detachment: assessment with electroretinography and optical coherence tomography. retina 2007;27:30-6. 26. salicone a, smiddy we, venkatraman a, feuer w. visual recovery after scleral buckling procedure for retinal detachment. ophthalmology 2006;113:73442. 27. hong s, narkiewicz, kardon rh. comparison of pupil perimetry and visual perimetry in normal eyes: decibel sensitivity and variability. invest ophthalmol vis sci 2001;42:957-65. 28. riemann cd, hanson s, foster ja. a comparison of manual kinetic and automated static perimetry in obtaining ptosis fields. arch ophthalmol 2000;118:65-9. 29. wu d, gao r, zhang g, wu l. comparison of preand post-operational multifocal electroretinograms of retinal detachment. chin med j 2002;115:15603. 30. ajtony c, balla z, somoskeoy s, kovacs b. relationship between visual field sensitivity and retinal nerve fiber layer thickness as measured by optical coherence tomography. invest ophthalmol vis sci 2007;48:258-63. alvina 144 abstract aging is associated with a gradual impairment in cognitive function. the elderly also show a high prevalence of undernutrition, whereas nutrition plays an important role in the metabolism of neuronal cells and enzymes. homocysteine is an amino acid resulting from methionine metabolism and is dependent on intake of vitamin b12, vitamin b6 and folic acid. homocysteine is said to play a role in cognitive function. the objective of this study was to evaluate the effect of micronutrient supplementation for 6 months on serum homocysteine levels and cognitive function in older people. this study was an experimental study of pre-post test design, carried out in mampang subdistrict, south jakarta. a total of 94 elderly people was recruited for this study, consisting of 44 females and 50 males. serum homocysteine level was assessed by fluorescent polarization immunoassay and cognitive function by means of the mini mental state examination (mmse) before and after micronutrient supplementation. mean serum homocysteine concentration after supplementation decreased significantly to 14.8 ± 5.8 µmol/l, compared with mean serum homocysteine level of 15.9 ± 5.9 µmol/l before supplementation (p=0.000). multiple regression analysis indicated that the factors influencing post-supplementation mmse scores were gender (â=-0.350; p=0.000), education (â=0.510; p=0.000) and postsupplementation homocysteine levels (â=-0.201; p=0.000), while age, presupplementation homocysteine levels and bmi did not affect mmse scores. homocysteine concentration decreased significantly after 6 months of supplementation. the factors affecting post-supplementation mmse scores were gender, level of education, and post-supplementation homocysteine level. keywords: micronutrient, mmse, serum homocysteine, cognitive function, elderly *department of clinical pathology, medical faculty, trisakti university correspondence dr. pusparini, sppk department of clinical pathology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2404 email: pusparini_sppk@yahoo.com univ med 2010;29:144-52. decreased serum homocysteine levels after micronutrient supplementation in older people pusparini* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction no other organ system in the human body is as dependent on nutritional intake as the central nervous system, while conversely this system also affects nutritional intake. several theories have elucidated the functions of brain receptors for cholecystokinin, opioid-like endorphins and serotonin that play a role in determining human dietary habits.(1,2) studies on experimental animals indicate that with advancing age there is a decline in 145 univ med vol.29 no.3 the functioning of the brain and in the number of brain receptors. in the elderly there is also a diminished sense of taste and a decline in o l f a c t o r y n e r v e f u n c t i o n s t h a t a f f e c t appetite.(2,3) the nervous system requires a constant intake of glucose for adequate functioning of the brain, which is particularly dependent on intake of essential nutrients. deficiencies of vitamins and minerals, such as vitamin b12, folate, vitamin b6 and trace minerals, may result in disturbances of brain functions such as cognitive functions.(4,5) in the elderly, vitamin deficiencies are common, especially of the vitamin b group, caused by various factors, such a s g a s t r i c a t r o p h y w i t h a c h l o r h y d r i a o r hypochlorhydria, which in the elderly may account for up to 20-50% of cases. studies have yielded evidence for an association in the elderly between decreased levels of vitamin b12, folate, and vitamin b6 on the one hand and impaired cognitive function on the other.(1,5) one of the manifestations of impaired cognitive function is dementia, which is characterized by a progressive decline in cognitive function, resulting in a decreased ability for conducting activities of daily living. in canada dementia affects 8% of the elderly population over 65 years of age, and gives rise to more than 60.000 new cases annually. another disease, alzheimer’s, is the cause of 50% of all cases of dementia,(6) whereas other s t u d i e s i n d i c a t e t h a t d e m e n t i a d u e t o alzheimer’s disease may account for up to 70% of all dementia cases.(7) it is estimated that in the following 50 years the number of dementia cases may undergo a threefold rise.(8) the prevalence of this disorder is extremely high and as the disorder may lower the quality of life of the elderly, prevention and early detection are important.(6) factors that have been associated with the risk of dementia are age and education, both of which are of the highest relevance for dementia. recently the role of homocysteine in dementia has been elucidated, where high homocysteine levels have been associated with an increased risk of dementia.(6,7,9) homocysteine is an amino acid resulting from the metabolism of methionine that is d e p e n d e n t o n b 12, v i t a m i n b 6, a n d f o l i c acid.(8,10) plasma homocysteine level is an indicator for vitamin b status, including that of folic acid, where a high homocysteine concentration indicates an inadequate vitamin b status. data from several laboratories have s h o w n t h a t h i g h h o m o c y s t e i n e l e v e l i s extremely common in old age and does not depend on vitamin status.(11-13) total plasma homocysteine level is a major vascular risk factor, and raised plasma homocysteine level i s a s s o c i a t e d w i t h a n i n c r e a s e d r i s k o f atherosclerotic sequelae, including death due to cardiovascular disease, coronary heart d i s e a s e , c a r o t i d a t h e r o s c l e r o s i s a n d stroke.(7,14,15) research results on the influence of homocysteine on cognitive function in the elderly are still subject to controversy. a number of studies have demonstrated a significant association,(7,15-17) whereas other studies have shown contradictory results.(8,18,19) preliminary studies with small samples in a population of healthy elderly in italy showed no significant c o r r e l a t i o n b e t w e e n m m s e s c o r e s a n d homocysteine levels. however, the same investigator found in larger samples that older persons with low mmse scores had a higher risk of suffering from abnormal cognitive function with increasing plasma homocysteine levels. the study also found that homocysteine level was not directly associated with cognitive disorders.(9) the present study had as objectives to find a decrease in homocysteine concentrations after s i x m o n t h s o f s u p p l e m e n t a t i o n w i t h a combination of micronutrients and vitamins, and to determine whether decreased postsupplementation homocysteine levels can improve cognitive function in older persons. 146 methods design of the study this study used an experimental pre-post test design to investigate the occurrence of a decrease in homocysteine concentrations after administration of a supplement comprising a combination of micronutrients and vitamins for a period of six months. the study was carried out from november 2008 to april 2009. subjects the study subjects were residents aged 60+ years from the catchment area of the mampang prapatan subdistrict health center in south jakarta who met the following inclusion criteria: healthy males and females, m o b i l e , i n d e p e n d e n t , a b l e t o v e r b a l l y communicate and willing to join the study by giving written informed consent. subjects with acute infections, intake of vitamins, cod liver oil or other supplements within the previous month, and abnormal renal function, were excluded from the study. selection of the subjects was by cluster and simple random sampling. supplementation the supplements for the subjects were in the form of oral tablets, each containing 280 mg zinc gluconate (equivalent to 40 mg zinc), 120 mg ascorbic acid, 6 mg β-carotene, 15 mg α-tocopherol, and 400 µg folic acid. the tablets were administered to the study subjects by field workers who assisted in this study. to each study subject was allocated one bottle containing 30 tablets to be taken once daily. c o m p l i a n c e w a s m o n i t o r e d b y t h e f i e l d workers and the investigator by counting the remaining tablets returned with the bottles each month. data collection the field workers conducted interviews using a questionnaire that had been tested in a preliminary trial on elderly who were willing to participate in this study. the filling in of questionnaires was performed daily from monday to friday, and those subjects who had been interviewed were invited to come to the mampang prapatan subdistrict health center on saturday. the weekly number of elderly who completed their interviews was around 25. the subjects were asked to fast overnight on friday for 10–12 hours prior to the collection of a blood sample for laboratory examination. t h e f o l l o w i n g s a t u r d a y m o r n i n g t h e participants visited the health center for measurement of blood pressure, pulse rate, weight, height, waist circumference, and hip circumference. the measurements were taken by two previously trained nurses. data were collected within six months, before and after the supplementation period. in addition to anthropometric assessment and laboratory examination, diet recall was performed 2x24 h o u r s o n w o r k d a y s a n d 1 x 2 4 h o u r s o n holidays. assessment of dietary recall was done twice, namely at the start and at the end of the study. measurements height was measured to the nearest 0.1 cm using a portable microtoise and weight to the nearest 0.1 kg using sage portable scales. body mass index (bmi) was calculated as the weight (kg) divided by the square of the height (m 2) . wa i s t c i r c u m f e r e n c e ( c m ) a n d h i p circumference (cm) of the subjects were measured using a standard tape measure a c c u r a t e t o 1 m m . b l o o d p r e s s u r e measurements were performed with the subject sitting, after a rest period of 15 minutes, by means of a standard sphygmomanometer accurate to 5 mmhg. hypertension was defined as a systolic blood pressure of >140 mmhg and/or a diastolic blood pressure of >90 mmhg. assessment of renal function from each subject meeting the inclusion and exclusion criteria a 5 ml blood sample was c o l l e c t e d u s i n g a v a c u t a i n e r w i t h o u t pusparini homocysteine levels after micronutrient 147 univ med vol.29 no.3 a n t i c o a g u l a n t . t h e b l o o d s a m p l e s w e r e centrifuged at 300 rpm for 10 minutes in order to obtain serum for assessment of renal function and homocysteine level. since renal f u n c t i o n a ff e c t s t h e m e t a b o l i s m o f homocysteine,(15) subjects with a creatinine concentration above reference level were considered to have abnormal renal function. the reference level for females is 0.5-1.3 mg/ d l a n d f o r m a l e s 0 . 5 1 . 4 m g / d l . ( 1 4 ) determination of homocysteine level was performed only on the serum of subjects with a renal function consistent with reference values. homocysteine concentrations of all study subjects were divided into 3 categories, i.e. <11.7 µmol/l, 11.7-14.6 µmol/l, and >14.6 µmol/l. ( 1 5 ) creatinine concentration was determined by the jaffe method on a trx, w h e r e a s h o m o c y s t e i n e w a s a s s e s s e d b y fluorescent polarization immunoassay on an abbott imx. the coefficient of variation (cv) o f c r e a t i n i n e w a s 2 . 1 % f o r n o r m a l c o n c e n t r a t i o n s a n d 1 . 6 % f o r h i g h concentrations, whilst the corresponding cv values for homocysteine were 3.4% and 2.3%, r e s p e c t i v e l y. s e r u m f o r h o m o c y s t e i n e assessment was stored at –700c and examined after collection of the last blood sample. assessment of cognitive function cognitive function was assessed by means of the mmse instrument. this comprises five c a t e g o r i e s o f c r i t e r i a , v i z . c r i t e r i a o n orientation with a maximum score of 10, registration criteria with maximum score of 3, attention and calculation with maximum score of 5, recall criteria with maximum score of 3, and language criteria with maximum score of 9. in addition to these five criteria the subjects are also asked to write a sentence and copy a d r a w i n g . ( 8 ) m m s e s c o r e s o f 2 6 3 0 a r e d e s i g n a t e d q u e s t i o n a b l y s i g n i f i c a n t / n o cognitive impairment, while scores of 21-25, 10-20 and 0-9 respectively indicate mild, moderate and severe cognitive impairment.(13) data analysis data processing was performed by means of the statistical program for social sciences (spss) software version 15. normality of data d i s t r i b u t i o n w a s d e t e r m i n e d b y t h e kolmogorov – smirnov (ks) test. for normally d i s t r i b u t e d d a t a t h e m e a n a n d s t a n d a r d deviation (sd) was calculated and for nonnormally distributed data the median. to determine the presence of a statistically significant difference between groups for   assessed for eligibility (n=137) enrollment (n=107) supplementation for 6 months completed trial (n=94) not meeting inclusion criteria (n=25) declined to participate (n=5) withdrawn: death (n=3) illness (n=2) emigration (n=3) unable to cope withtrial (n=5) figure 1. flow chart describing the progress of the subjects during the trial 148 normally distributed data the analysis of variance (anova) was used, whilst for nonnormally distributed data the kruskal – wallis test was applied. multiple linear regression was used to identify the factors affecting mmse levels. a value of p <0.05 was taken to indicate a significant difference. results at the start of the study 137 respondents were recruited, among whom 107 were found to meet the inclusion and exclusion criteria. during the study period of six months 13 respondents dropped out, due to various reasons, viz. 3 persons moved to another area, 3 persons died, 5 persons withdrew from the study, and 2 persons were taken ill and thus unable to take the supplements for more than 7 days. at the end of the study the remaining subjects totaled 94 persons, comprising 44 females and 50 males (figure 1). supplementation to respondents who p a r t i c i p a t e d u p t o t h e e n d o f t h e s t u d y proceeded satisfactorily, since 98% of the respondents received the full supplementation for six months and 2% skipped or missed 1-2 days of supplementation. monitoring of subject c o m p l i a n c e w a s d o n e b y c o u n t i n g t h e remaining tablets returned with the bottles. no subject was dropped for non-compliance. at the completion of the study there were 94 elderly, consisting of 44 (47 %) females and 50 (53%) males. the characteristics of the study subjects are listed in table 1. the age of the subjects ranged from 60 to 79 years, with a mean age of 65.9 ± 4.7 years. there were 46 (48.9%) study subjects with homocysteine concentrations above 14.6 µmol/ l, having at the start of the study homocysteine levels of 20.1± 5.9 µmol/l, which decreased to 18.9 ± 5.9 µmol/l at the completion of the study. homocysteine concentrations before and after supplementation showed a significant difference (p=0.000), with decreased post-supplementation levels in all groups (table 2). the factors that were thought to be of influence on cognitive function were among others age, bmi, education, gender and blood homocysteine levels. from table 3 it may been seen that mmse scores were influenced by gender, post-supplementation homocysteine levels, and education. age, bmi, and prehomocystei ne conc. (µmol/l) before supplementation after supplementation p* n (%) x + sd n (% ) x + sd < 11.7 22 (23.4 %) 10.2 ± 1.4 28 (29.8%) 9.9 ± 1.7 0.000 11.7 – 14.6 26 (27.7%) 13.2 ± 0.6 22 (23.4%) 12.9 ± 0.9 0.000 > 14.6 46 (48.9%) 20.1 ± 5.9 44 (46.8%) 18.9 ± 5.9 0.000   table 2. preand post-supplementation serum homocysteine concentrations of subjects *paired t-test characteristics n* gender female 44 (47%) male 50 (53%) age (years) 65.9 ± 4.7 body mass index (bmi) (kg/m2) 22.4 ± 3.4 level of education (years) 5.1 ± 4.4 weight (kg) 59.9 ± 10.9 height (cm) 155.7 ± 4.6 waist circumference (cm) 83.3 ± 11.2 hip circumference (cm) 91.1 ± 7.8 systolic pressure (mmhg) 143 ± 18 diastolic pressure (mmhg) 87 ± 10 creatinine (mg/dl) 0.84 ± 0.2 homocysteine (µmol/l) 15.9 ± 5.9 mmse** 24.1 ± 5.9 table 1. characteristics of study subjects at start of study (n=94) *values expressed as mean ± sd, except for gender ** mmse: mini mental state examination pusparini homocysteine levels after micronutrient 149 univ med vol.29 no.3 supplementation homocysteine levels did not affect mmse scores. discussion the aging process is related to a reduction in various physiological functions. it is well known that physiological changes are the result o f i n a d e q u a t e i n t a k e o f v i t a m i n s , t r a c e elements, minerals and other nutrients.(20) even nutritional intakes that are considered to be of high quality for health cannot comprise all nutritional elements required for promotion of health and prevention of acute as well as chronic diseases. the results of the present study showed that the pre-supplementation homocysteine concentration of 15.9 ± 5.9 µmol/l decreased significantly to 14.8 ± 5.8 µmol/l after micronutrient supplementation for six months. in a chinese study on 381 subjects with mean age of 46.9 ± 8. 3 years, who received supplementation of vitamin c 250 mg, vitamin e 100 mg and selenium 37.5 µg twice daily for 88 months, the plasma homocysteine concentration tended to decrease 6 . 4 % , t h e d e c r e a s e h o w e v e r b e i n g n o t statistically significant (95% ci: -13.5 to 1.3%) ( p = 0 . 1 0 ) . ( 2 1 ) i n c o n t r a s t , i n a s t u d y o n g u a t e m a l a n e l d e r l y, w h o w e r e g i v e n a supplement containing zinc and folic acid, results similar to the present study were obtained, which may be due to the fact that in the chinese study the supplement did not include these two micronutrients, which are capable of reducing serum homocysteine levels.(22) homocysteine is a sulfur-containing a m i n o a c i d c l o s e l y a s s o c i a t e d w i t h t h e metabolism of methionine and cysteine. there is no dna that codes for homocysteine synthesis. homocysteine is not synthesized in nature but results from the metabolism of the amino acid methionine through a methylation cycle as a unique source of homocysteine. methionine is obtained through intake of protein in the daily diet.(23,24) homocysteine occurs in several forms in plasma. the sulfhydryl or reduced form is designated homocysteine and the disulfide or oxidized form is designated homocystine. the disulfide form may occur together with cysteine and with proteins having reactive cysteine residues (protein-bound homocysteine), and this form is called mixed disulfide. the oxidized forms account for most of the homocysteine in plasma (98-99%), whilst the reduced forms make up only 1% of total homocysteine. total homocysteine (thcy) is the sum total of all homocysteine forms present in plasma.(23,24) homocysteine is converted by the enzyme cystathionine β synthase (cbs) into cysteine with vitamin b6 as cofactor, in a process called demethylation. in the reverse process of remethylation, homocysteine is reconverted into m e t h i o n i n e b y t h e e n z y m e s m e t h y l e n e tetrahydrofolate (mthfr) and methionine synthase (ms), with folic acid and vitamin b12 as substrates and cofactors. folic acid, vitamin b12 and vitamin b6 are substrates of the cofactors i n t h e m e t a b o l i s m o f m e t h i o n i n e a n d homocysteine, thus a deficiency of one of these vitamins leads to hyperhomo-cysteinemia.(24-26) factor beta coefficient p age 0.115 0.234 gender -0 .350 0.000* pre-supplem entation homocysteine -0 .154 0.583 post-supplementation homocysteine -0 .201 0.000* education 0.510 0.001* body mass index -0 .072 0.098   table 3. factors affecting mmse scores * p< 0.05 indicates a significant difference 150 u n d e r n o r m a l c o n d i t i o n s b l o o d homocysteine levels are relatively low, ranging from 5-15 µmol/l. the homocysteine level in the extracellular compartment is determined by its intracellular synthesis, metabolism and e x c r e t i o n . i f i n t r a c e l l u l a r h o m o c y s t e i n e synthesis exceeds the metabolic capacity, homocysteine is released into the extracellular compartment. in contrast, decreased synthesis leads to a fall in release of homocysteine from t h e c e l l . t h i s m e c h a n i s m m a i n t a i n s intracellular homocysteine at a low level. the homocysteine equilibrium may be upset by abnormal enzyme activity or as a result of a reduction in the number of cofactors that play a role in its metabolism. (25,27) in table 2 homocysteine levels were subdivided into three categories, i.e. <11.7 µmol/l, 11.7-14.6 µmol/ l and >14.6 µmol/l. this was based on the results of a study conducted by quadri(15) with the objective of finding a reference value for hyperhomocysteinemia, as currently there are n o s t a n d a r d c r i t e r i a f o r d e f i n i n g h y p e r h o m o c y s t e i n e m i a . a n u m b e r o f investigators have used variable values for hyperhomocysteinemia, e.g. ravaglia(9) used a homocysteine level of >15 µmol/l, whilst seshasdri(7) and dufouil(2) used levels of >14 mol/l and >15 µmol/l, respectively. in the present study homocysteine levels of >14.6 ± 6.1 µmol/l were found in 46 subjects (48.9%) with mean level of 20.06 ± 5.9 µmol/l at the start of the study, decreasing to 44 subjects (46.8%) with mean level of 18.9 ± 5.9 µmol/ l. in comparison, in the study by quadri(15) homocysteine levels of >14.6 µmol/l were found in 51.85% of subjects with vascular dementia and in 56.36% of control subjects. in quadri’s study, mean homocysteine level was 14.6 ± 6.1 µmol/l in control subjects with mean age of 75.6 ± 8.5 years, whereas subjects with vascular dementia with mean age of 80.5 ± 5.7 years had a mean homocysteine level of 18.9 ± 7.9 µmol/l and those with alzheimer’s disease and mean age of 79.1 ± 7.7 years showed a homocysteine level of 16.8 ± 7.0 µmol/l. the different results may be due to the lower mean age of the subjects (65.9 ± 4.7 years) in the present study. in the present study determination of homocysteine concentration was performed after the subjects had fasted at least 10 hours. assessment of homocysteine levels without fasting results in a rise in homocysteine levels of around 20%, compared with those in fasting subjects.(7) seshasdri’s study(7) revealed a tendency for increased plasma homocysteine levels to precede the onset of dementia. therefore it is probable that the increased homocysteine levels in the present study mark the early stages of a developing dementia, although the mmse scores were categorized as mild cognitive impairment only. seshasdri’s results are also consistent with the results of the present study, w h e r e h o m o c y s t e i n e l e v e l s s h o w e d a s i g n i f i c a n t p o s t s u p p l e m e n t a l d e c r e a s e , although the increases in mmse scores did not reach statistical significance. t h e r e a r e s e v e r a l f a c t o r s t h a t a r e considered to be of influence on cognitive function, namely age, gender, education, bmi a n d p r e a n d p o s t s u p p l e m e n t a t i o n homocysteine levels. the results of the multiple regression analysis in this study revealed that gender, education, and post-supplementation homocysteine levels constituted the factors affecting post-supplementation mmse scores, whereas age, bmi, and pre-supplementation homocysteine levels did not affect postsupplementation mmse scores. these results are similar to the results of quadri’s study,(15) i n d i c a t i n g t h a t e d u c a t i o n a l s o d i ff e r r e d significantly between the vascular dementia group and the control group. with higher educational levels the mmse scores are found to increase, indicating better cognitive functioning. several investigators u s e d s u b j e c t s o f s i m i l a r e d u c a t i o n a l backgrounds to determine an association between homocysteine levels and mmse scores. the study by ravaglia(9) indicated that pusparini homocysteine levels after micronutrient 151 univ med vol.29 no.3 i n s u b j e c t s w i t h a s i m i l a r e d u c a t i o n a l background hyperhomocysteinemia was an independent predictor for the occurrence of dementia or alzheimer’s disease. the present study results showed that post-supplementation mmse scores were not influenced by age, w h e r e a s i n t h e c r o s s s e c t i o n a l s t u d y b y pusparini(28) age was found to be an influencing factor in males (p=0.006) and in females (p=0.000). both studies, the one of crosssectional design as well as the study of preand-post design, yielded similar results, in that education affected mmse scores. in the study by pusparini(28) initial homocysteine levels had n o e ff e c t o n m m s e s c o r e s , w h i c h w a s consistent with the results of the present study and those of the study by joosten,(29) where no significant difference was found between homocysteine levels and cognitive function. the study conducted by bell (30) found an association between plasma homocysteine levels and mmse scores only in subjects with depression and not in normal subjects. in the cross-sectional studies conducted by borroni(31) and miller(32) it was found that increased plasma homocysteine concentration was not a causative factor in dementia and alzheimer’s disease, but only an accompanying marker of vascular disorders and was independent of individual cognitive status. intervention studies are n e c e s s a r y f o r p r o v i d i n g e v i d e n c e o f a n association between hyperhomocysteinemia and cognitive function. the study by seshasdri(7) revealed a correlation between raised plasma homocysteine levels and decreased mmse scores, but the correlation was found only after 4 years of monitoring. one limitation of this study is the lack of a control group for comparative purposes, thus it cannot prove that the decrease in postsupplementation homocysteine levels was the result of the supplementation factor itself rather than other causes. in addition, in this study there was no assessment of folic acid and vitamin b concentrations, which affect the metabolism of homocysteine, both initially and at the completion of the study, so that the investigator could not ascertain whether the administered supplementation was capable of i n c r e a s i n g h o m o c y s t e i n e l e v e l s t h r o u g h increases in folic acid and vitamin b levels or whether there were other causes. conclusions from the results of this study it may be concluded that homocysteine levels decreased a f t e r s i x m o n t h s o f m i c r o n u t i e n t supplementation. the main factors influencing post-supplementation mmse scores were gender, post-supplementation homocysteine levels, and education. acknowledgements the author wishes to express her gratitude to the medical faculty of trisakti university, to the study subjects for their willingness to participate in this study, and to all parties who facilitated this study. references 1. weir dg, molloy am. microvascular disease and dementia in the elderly: are they related to hyperhomocysteinemia? am j clin nutr 2000;71: 859-60 2. dufouil c, alperovitch a, ducros v, tzourio c. homocysteine, white matter hyperintensities, and cognition in healthy elderly people. ann neurol 2003;53:214-21. 3. roberts s, sternberg s. do nutritional supplements improve cognitive function in the elderly? nutr 2003;19:976-80. 4. haan mn, miller jw, aiello ae, whitmer ra, jagust wj, mungas dm, et al. homocysteine, b vitamins, and the incidence of dementia and cognitive impairment: result from the sacramento area latino study on aging. am j clin nutr 2007; 85:511-7. 5. refsum h. total homocystein. guideline for determination on the clinical laboratory. clinical laboratory news. aacc 2002;28:12-4. 6. duel pb, malinow mr. homocystein: an important risk factor for atherosclerotic vascular disease. curr opin lipidol 1997;8:28-34. 152 7. seshadri s, beiser a, selhub j, jacques pf, rosenberg ih, a’agostiono rb, et al. plasma homocysteine as a risk factor for dementia and alzheimer’s disease. n engl j med 2002;346: 476-83. 8. mooijaart sp, gussekloo j, frolich m, jolles j, stott dj, westendorp rgj. homocysteine, vitamin b12 and folic acid and the risk of cognitive decline in old age: the leiden 85-plus study. am j clin nutr 2005;82:866-71. 9. ravaglia g, forti p, maioli f, martelli m, servadei l, brunetti n, et al. homocyteine and folate as risk factors for dementia and alzheimer disease. am j clin nutr 2005;82:636-43. 10. ueland pm, refsum h, stabler sp, malinow mr, anderson a, allen rh. total homocysteine in plasma or serum methods and clinical applications. clin chem 1993;39:1764-78. 11. garcia a, haron y, pulman k, hua l, freedman m. increases in homocysteine are related to worsening of stroop scores in healthy elderly persons: a prospective follows up study. j gerontol 2003;59a:1323-27. 12. tucker kl, qiao n, scott t, rosenberg i, spiro a. high hmocysteine and low b vitamins predict cognitive decline in aging men: the veterans affairs normative aging study. am j clin nutr 2005;82:627-35. 13. garcia a, zanibbi k. homocysteine and cognitivie function in elderly people. cmaj 2004;171:897-904. 14. selhub j, bagle lc, miller j, rosenberg ih. b vitamins, homocysteine, and neurocognitive function in the elderly. am j clin nutr 2000; 71(suppl):4s-20s. 15. quadri p, fragiacomo c, pezzati r, zanda e, forlani g, tettamanti m, et al. homocysteine, folate, and vitamin b-12 in mild cognitive impairment, alzheimer disease, and vascular dementia. am j clin nutr 2004;80:114-22. 16. miller jw, green r, ramos mi, allen lh, mungas dm, jagust wj, et al. homocysteine and cognitive function in the sacramento area latino study on aging. am j clin nutr 2003;78:441-7. 17. andres e, loukili nh, noel e, kaltenbach g, abdelgheni mb, perrin ae, et al. vitamin b12 (cobalamin) deficiency in elderly patients. cmaj 2004;171:1503-55. 18. stott dj, macintoch g, lowe gdo, rumley a, mcmahon ad, langhorne p, et al. randomized controlled trial of homocysteine lowering vitamin treatment in elderly patients with vascular disease. am j clin nutr 2005;82:1320-6. 19. lei f, ng tp, chuah l, niti m, e hk. homocysteine, folate, and vitamin b12 and cognitive performance in older chinese adult: findings from singapore longitudinal ageing study. am j clin nutr 2006;84:1506-12. 20. chandra rk. effect of vitamin and trace element supplementation on cognitive function in elderly subjects. nutr 2001;17:709-12. 21. wang y, zhang l, moslehi r, ma j, pan k, zhou t, et al. long term garlic or micronutrient supplementation, but not anti-helicobacter pylori therapy, increases serum folate or glutathione without affecting serum vitamin b 12, or homocysteine in rural chinese population. j nutr 2009;139:106-12. 22. nguyen p, grajeda r, melgar p, markinkevage j, flores r, martorell r. weekly may be as efficacious as daily folic acid supplementation in improving folate status and lowering serum homocysteine concentration in guatemalan women. j nutr 2008;138:1491-8. 23. kruman ii. homocystein in elicits a dna damage response in neuron that promotes apoptosis abd hypersensitivity to excitoxicity. j neuroscience 2000;20:6920-6. 24. hsu kk, sorond fa, jen hc, hashmi a, milberg wp, lipsitz la. the role of homocysteine in multisystem age related problems: a systematic review. j gerontol 2005;60a:1190-201. 25. massachusetts medical society. a controlled trial of homocysteine lowering and cognitive performance. n engl j med 2006;354:2764-72. 26. miller jw. assessing the association between vitamin b12 status and cognitive function in older adults. am j clin nutr 2006;84:1259-60. 27. krumdieck cl, prince cw. mechanisms of homocysteine toxicity on connective tissue: implications for the morbidity of aging. j nutr 2000;130:365s-8s. 28. pusparini. homocysteine and cognitive function in the elderly. univ med 2009;28:106-16. 29. joosten e, lesaffre e, riezler r. is metabolic evidence for vitamin b12 and folate deficiency more frequent in elderly patients with alzheimer’s disease? j gerontol a biol sci med sci 1997;52: m76-9. 30. bell ir, edman js, selhub j. plasma homocysteine in vascular disease and in nonvascular dementia of depressed elderly people. acta psychiatr scand 1992;86:386-90. 31. borroni b, agosti c, panzali af, di luca m, padovani a. homocysteine, vitamin b6, and vascular disease in patient with alzheimer disease. neurology 2002;59:1475-6. 32. miller jw, green r, mungas dm, reed br, jagust wj. homocysteine, vitamin b6, and vascular disease in alzheimer disease. neurology 2002;58:1471-5. pusparini homocysteine levels after micronutrient atik 12 effect of clean intermittent self-catheterization on urinary tract infection in subject with spinal cord injury *department of anatomy, medical faculty, trisakti university **rehabilitation medicine unit, fatmawati general hospital, jakarta correspondence dr. maria regina rachmawati, sp.rm department of anatomy medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 ext.2101 email:rachmawati@trisakti.ac.id universa medicina 2008; 27: 12-7. january-march, 2008january-march, 2008january-march, 2008january-march, 2008january-march, 2008 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 patients with spinal cord injury are often unable to empty their bladders sufficiently. if this condition is left untreated, urinary tract infection (uti) is unavoidable, with potentially lethal consequences. the objective of this study was to evaluate the effect of clean intermittent self-catheterization (cic) on urinary tract infection in subject with spinal cord injury. a randomized controlled design was conducted in subjects who lived at two dormitories were included in this study. they were randomized to clean intermittent selfcatheterization or control group. all the subjects were followed for one months. primary outcome measure was symptomatic urinary tract infection as diagnosed by, pyuria (leukocytes of 100,000 or more per high power field.). twenty-six patients between 22 to 62 years with cervical spinal cord injuries and urine residue more than 50 cc were recruited. of the 26 subjects, 18 (69.2%) developed urinary tract infection. the incidence rate of uti in the cic group was 5/13 (38.5%); significantly lower than the control group 11/13 (84.6%) (p=0.016). clean intermittent self catheterization in patient with spinal cord injury decreased the risk of developing urinary tract infection. keywords : spinal cord injury, clean intermittent self-catheterization, urinary tract infection abstract maria regina rachmawati*, suleiman sutanto*, and rosiana p wirawan** universa medicina introduction one of the most problematic consequences of a spinal cord injury (sci) is alterations in lower urinary tract function. although mortality related to urological complications has been reduced during the last decades, urinary tract problems continue to be a prominent cause of morbidity after sci.(1) urinary tract infection ( u t i ) i s a v e r y c o m m o n c o m p l i c a t i o n i n persons with sci.(2,3) after sci, subjects may not feel the urge to urinate when their bladder is full. they also may not have a voluntary control of their bladder and sphincter muscles. 13 universa medicina vol.27 no.1 this condition was named neurogenic bladder.(4) patients with neurogenic bladder dysfunction a r e o f t e n u n a b l e t o e m p t y t h e i r b l a d d e r s sufficiently. if this condition is left untreated, uti is unavoidable, with potentially lethal consequences. the use of indwelling urinary c a t h e t e r s m a k e s i t p o s s i b l e t o e m p t y t h e bladder, but the risk for complicating uti is s t i l l h i g h . f o l l o w i n g t h e i n t r o d u c t i o n o f i n t e r m i t t e n t c a t h e t e r i z a t i o n w i t h s t e r i l e technique, the prognosis for these patients improved.(5) s e v e r a l s t u d i e s i n d i c a t e d t h a t c l e a n intermittent catheterization (cic) is a safe and e ff e c t i v e m e a n s o f m a n a g i n g n e u r o g e n i c bladder dysfunction and to decrease the risk for r e n a l c o m p l i c a t i o n s . ( 6 , 7 ) n o w a d a y s c i c i s widely accepted as the first line in management of patients with neurogenic bladder dysfunction a n d u n b a l a n c e d v o i d i n g . s o f a r, v a r i o u s urethral catheters have been introduced to the markets.(8) disposable catheters either with self-lubrication or in connection with jelly injected into the urethra, are used to minimize urethral trauma and infection. they increase ease of use during catheterization.(9) clean intermittent self-catheterization (cisc) has proven to be a most satisfying form of bladder drainage as stated by many authors.(10) a randomized controlled trials suggest that cic technique does not jeopardize patient health, but this studies are limited by small sample size, heterogeneous groups, and inconsistent measures of symptomatic uti.(11) the purpose of this study was to evaluate the effect of cic on uti in patients with chronic sci. methods research design an experimental controlled parallel group study was performed from january to march 2007. research subjects a total of 37 subjects 22 years and older who lived at two disabled dormitories, had chronic sci and agreed to participate were enrolled. subjects were included if they had no urinary tract symptom (fever) and had urinary residue more than 50 cc. exclusion criteria was using pharmacologic agent within the previous 3 months. subjects gave written informed consent for their participation. catheterization subjects were randomized into two groups: group 1, subjects under cic and group 2, were controls. as preparation cic group were trained how to do cic using python catheter. they were instructed to do the cic once a day for one month. the two groups were informed about urinary tract complication as a consequence of neurogenic bladder. laboratory analysis the primary outcome was the incidence of uti. a uti is defined as a leukocytes in urine of 100,000 or more per high power field.(12) urinary analysis was done at base-line and one month after cic intervention. measurement u r i n e r e s i d u e w a s m e a s u r e d u s i n g ultrasound sonography (usg). levels of injuries were classified according to the american spinal injury association (asia) classification, as asia a, b, c, or d.(13) asia a = complete: no motor or sensory function is preserved in the sacral segments s4-s5; b = incomplete: sensory but not motor function is preserved below the neurological level and includes the sacral segments s4-s5; c= incomplete : motor function is preserved below the neurological level, and more than half of key muscles below the neurological level have a muscle grade less than 3; d=incomplete : motor function is preserved 14 total number of patients that potentially could have been recruited: n= 37 exclusion n= 11 reasons: using indwelling catheter rest urine < 50 ml total number of patients registered n= 26 a = clean intermittent catheterization (cic) b = control one month follow-up n = 13 outcome data developed uti group a n = 13 figure 1. flow diagram for clean intermittent catheterization study in patients with spinal cord injury below the neurological level and at least half of key muscles below the neurological level have a muscle of grade 3 or more; e = normal: motor and sensory function are normal. statistical analysis the results were analyzed using chi-square or fisher ’s exact tests to compare categorical variables between the two groups and student’s t -tests to compare continuous variables. results over 37 potential subjects with cervical, thoracic or lumbar injuries were approached. reasons for non-participation included using indwelling catheter (1 subject) and rest urine < 50 ml (10 subjects). the final sample was 26 subjects between 22 to 62 years, 13 as the cic group and 13 as control group (figure 1). rachmawati, sutanto, wirawan clean intermittent self-catherization 15 universa medicina vol.27 no.1 characteristics cic (n= 13) age (years) gender male female onset of injury (years) level of sci* c5-c7 t1-t12 l1-l5 completeness of lesion ** asia a (complete) asia b (incomplete) asia c (incomplete) urinary residue 50 – 100 cc 100 – 500 cc > 500 cc urinary tract infections 40.7 ± 10.8 9 (69.2) 4 (30.8) 18.7 ± 6.5 0 8 5 8 4 1 2 10 1 8 table 1. the baseline patient characteristics in the cic § and control groups * sci : spinal cord injury; ** asia : american spinal cord injury association ; §cic : clean intermittent catheterization urinary tact infections cic (n-13) positive negative 4 9 table 2. incidence of urinary tract infections after one month intervention in the cic and control groups group 1 was composed of 13 patients (nine males and four females), mean age 40.7 years, and mean disease duration 18.7 years. group 2 was made of 13 patients (11 males and two females), mean age 41.6 years, and mean disease duration 15.5 years. the clinical features of the two groups are described in table 1. spinal cord injuries ranged from c5 to c8, t1 to t12 and l1-l5. causes included hitting a moose on an isolated highway, falling asleep at the wheel and vehicle rollover, falling from a height at work, a n d s p i n a l c o r d t u m o r . d a t a c o n c e r n i n g completeness and level of the lesion were available from 26 subjects. in total, 19 (73.1%) h a d a c o m p l e t e l e s i o n , a n d 7 ( 2 6 . 9 % ) a n incomplete lesion. after one month, of the 26 subjects, 15 (57.7%) developed uti. the incidence rate of u t i i n t h e c i c g r o u p w a s 4 / 1 3 ( 3 0 . 8 % ) ; significantly lower than the control group 11/13 (84.6%) (p=0.016). (table 2) spinal cord injuries ranged from c5-c7 to l1-l5 with a t1-t12 injury being the most common (61.5%). among subjects whose spinal cord injuries ranged from t1-t12 to l1-l5, the incidence rate of uti in the cic group (30.8%) was significantly lower compared to the control group (91.7%) (p= 0.016). (table 3) 16 urinary tract infections cic (n=11) yes no 5 6 table 3. incidence of urinary tract infection after one month intervention in the cic and control groups in patients injury t7-t12 and l1-l5 discussion in this study there was a clear on outcome measure of uti (the presence of leukocytes in urine of 100,000 or more per ≥/10 per high power field). urinary tract abnormalities are quite common after spinal cord injuries and represent a cause of morbidity and mortality.(14) of the 26 subjects, 15 (57.7%) developed uti: 4/13 (30.8%) in the cic group; 11/13 (84.6%) in the control group (p=0.016). this result was consistent with others who suggest that cic in a rehabilitation setting in patient with sci decreased the risk of uti.(15,16) three randomized controlled trials ranging from 1992 to 1997 were found similar results that cic in an institutional setting decreased the risk of uti.(17-19) our data also confirms the main role of cic for the prevention of uti. another study in italy showed that cic would prevent the vesicoureteral reflux a n d u r i n a r y t r a c t a b n o r m a l i t i e s i n s c i patients. (20) cisc has proved to be a most satisfying form of bladder drainage. it is a useful solution to the major problems of neuropathic bladder dysfunction.(21) what is the best method of intermittent c a t h e t e r i z a t i o n i n p a t i e n t s w i t h b l a d d e r dysfunction? in the hospital setting, strict sterile technique is used by healthcare professionals. this technique is not only a time-consuming method for healthcare providers but also costly.’ in the community, patients are instructed to perform intermittent catheterization using a clean technique, which is less time consuming a n d d e c r e a s e s t h e c o s t o f i n t e r m i t t e n t catheterization. people living with a neurogenic bladder are at risk of uti, including when intermittent catheterization is the method of b l a d d e r m a n a g e m e n t . c o n s i s t e n t t e a c h i n g methods and modeling of desired behaviors will increase patient and family skill with proper technique and satisfaction. this study has indicated that cic is a safe and effective means of managing neurogenic bladder dysfunction and to decrease the risk for renal complications. limitations of the study recruitment was the major obstacle in achieving a larger sample size and proved extremely challenging in the population under study. despite subjects having a major disability which we expected would mean a nurse-managed continence protocol, many patients started catheterizing or had a caregiver provide care several developed non-urinary tract infections which required antibiotics. conclusion cic appears safe and effective for sci patients with urinary residue more than 50 cc who lived at disabled dormitories. simplification of the catheterization procedure may enhance the transition from rehabilitation to home. acknowledgements thanks to all subjects in the two dormitories for supporting this research study. the study was f u n d e d b y t h e m e d i c a l f a c u l t y tr i s a k t i university. mr. reindert reintje chief of sasana bina daksa budi bhakti pondok bambu, and rachmawati, sutanto, wirawan clean intermittent self-catherization 17 universa medicina vol.27 no.1 british woman association in jakarta for recruiting the subjects. this grants do not carry any restrictions as to publication of the study or use of the data and none of the authors had a financial or product interest in the study. references 1. devivo mj, krause js, lammertse dp. recent trends in mortality and cause of death among persons with spinal cord injury. arch phys med rehabil 1999; 80: 1411-9. 2. siroky mb. pathogenesis of bacteruria and infection in the spinal cord injured patient. am j med 2002; 113 (suppl 1a): s67-s79. 3. garcia leoni me, esclarin de ruz a. management of urinary tract infection in patients with spinal cord injuries. clin microbiol infect 2003; 9: 7805. 4. lindsey l, klebine p. bladder care and management. 2001. available at: http://images. main.uab.edu/spinalcord/pdffiles/info-11.pdf. accessed december 12, 2006. 5. svensson e, ertzgaard p, forsum u. bacteriuria in spinal cord injured patients with neurogenic bladder dysfunction. upsala j med sci 2004; 109: 25-32. 6. barton r. intermittent self-catheterization. nurs stand 2000; 15: 47-52. 7. duffin h. intermittent self-catheterization. j comm nurs 2000; 14: 29-32. 8. kovindha a, mai w nc, madersbacher h. reused silicone catheter for clean intermittent catheterization (cic): is it safe for spinal cordinjured (sci) men? spinal cord 2004; 42: 638-42. 9. wyndaele jj, de ridder d, everaert k, heilporn a, congard-chassol b. evaluation of the use of urocath gel catheters for intermittent selfcatheterization by male patients using conventional catheters for a long time. spinal cord 2000; 38: 97-9. 10. parmar s, baltej s, vaidyanathan s. teaching the procedure of clean intermittent catheterization. paraplegia 1993; 31: 298-302. 11. prieto-fingerhut t, banovac k, lynne cm. a study comparing sterile and nonsterile urethral catheterization in patients with spinal cord injury. rehabil nurs 1997; 22: 299-302. 12. biering-sorensen f, bagi p, hoiby n. urinary tract infections in patients with spinal cord injuries. drugs aging 2001; 61: 1275-87. 13. young w, w.m. keck center for collaborative neuroscience rutgers university, piscataway, nj. spinal cord injury levels & classification. 2002. available at: http://www.sci-info-pages.com/ levels.html. accessed december 18, 2006. 14. winn c, thompson j. urinary catheters for intermittent use. prof nurse 1999; 14: 859-65. 15. moore kn, burt j. intermittent catheterization in the rehabilitation setting: a comparison of clean and sterile technique. clin rehab 2006; 20: 4618. 16. lemke jr, kasprowicz k, worral ps. intermittent catheterization for patients with a neurogenic bladder: sterile versus clean. j nurs care quat 2005; 20: 302-6. 17. biering-sorensen f, bagi p, hoiby n. urinary tract infections in patients with spinal cord injuries. drugs aging 2001; 61: 1275-87. 18. de ridder djmk, everaert k, garcia fernandez l. intermittent catheterization with hydrophiliccoated catheters (speedicath) reduces the risk of clinical urinary tract infection in spinal cord injured patients: a prospective randomized parallel comparative trial. eur urol 2005; 48: 991-5. 19. svensson e, ertzgaard p, forsum u. bacteriuria in spinal cord injured patients with neurogenic bladder dysfunction. upsala j med sci 2004; 109: 25-32. 20. giannantoni a, scivoletto g, di stasi sm, silecchia1 a, finazzi-agroá e, micali i, et al. clean intermittent catheterization and prevention of renal disease in spinal cord injury patients. spinal cord 1998; 36: 29-32. 21. wyndaele jj, de ridder d, everaert k, heilporn a, congard-chassol b. evaluation of the use of urocath-gel1 catheters for intermittent selfcatheterization by male patients using conventional catheters. spinal cord 2000; 38: 97-9. alvina 169 abstract osteoporosis is a disorder having the characteristic features of low bone mass and structural degeneration. there are several factors affecting the prevalence of osteoporosis in postmenopausal women, such as age, age at menarche, duration of menopause, dietary or nutritional intakes, life style and level formal education. in connection with the increasing global prevalence of osteoporotic fractures, the purpose of the present study was to determine the prevalence and the influencing factors of osteoporosis in postmenopausal women. a cross-sectional study, involving 203 postmenopausal women aged 47-60 years, was conducted in 4 villages of mampang prapatan subdistrict, from february to april 2010. bone mineral density (bmd) of lumbar spine 1-4, femoral neck, and left radius was measured by dual-energy x-ray absorptiometry (lunar dpx bravo nomusa densitometer, ge medical systems) at budi jaya hospital, jakarta. the t-score threshold, defined as ≤ -2.5 was used to identify subjects with osteoporosis the results of the study showed that the highest prevalence of osteopenia of 45.8% was found in the femoral neck, while the highest prevalence of osteoporosis of 30% was found in the distal radius. age, duration of menopause, and number of pregnancies yielded a significant correlation with bmd of the lumbar vertebrae and the distal radius. body mass index (bmi) was also significantly correlated with bmd of lumbar vertebrae, femoral neck and distal radius. effective strategies for the prevention and management of osteopenia and osteoporosis are needed. keywords: menopause, osteoporosis, bmd, postmenopausal women *department of pharmacy, medical faculty, trisakti university correspondence dr. meiyanti, spfk department of pharmacy, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5672731 ext.2805 univ med 2010;29:169-76. epidemiology of osteoporosis in postmenopausal women aged 47 to 60 years meiyanti* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction osteoporosis is a disorder having the characteristic features of low bone mass and s t r u c t u r a l d e g e n e r a t i o n , p r o m o t i n g t h e development of brittleness of the bones and increasing the risk of fractures of the bones of the pelvis, vertebral column, and wrist. to date, the global prevalence of osteoporotic fractures is increasing.(1,2) osteoporosis is a “silent” disorder, because the loss of minerals occurs without discernable symptoms, and is also agerelated. at the age of around 30 years the bone mass ceases to increase and the function of healthy bone is to maintain the existing bone mass as long as possible.(3) in women the loss of bone mass increases several years after menopause, then decreases 170 meiyanti epidemiology of osteoporosis in women again, while in men the loss of bone mass occurs more slowly, until at the age of 60-70 years the loss of bone mass in men and women is comparable. it has been estimated that in the united states around 13-18% women over the age of 50 years suffer from osteoporosis, while a r o u n d 3 7 5 0 % h a v e a l o w b o n e m a s s (osteopenia).(4) the occurrence of fractures in old age is related to the fragility of bone. with the introduction of the bone densitometer, the measurement of bone fragility has been standardized. in postmenopausal women aged 55 years and over the possiblity of fractures should be kept in mind, in connection with the process of osteoporosis. the risk of fractures increases 1.5-3 times or more for a decrease of bone mineral density (bmd) by 1 standard deviation (sd). according to the criteria of the wo r l d h e a l t h o r g a n i z a t i o n ( w h o ) , osteoporosis is defined by a bone mineral density value (t score) of –2.5 or lower, while osteopenia is present if the t score is >-2.5 up to -1, and the bones are normal if the t score is >-1.(5) several risk factors are correlated with the occurrence of osteoporosis and contribute to the probability of an individual suffering from osteoporosis. there are several risk factors that a r e c o n t r o l l a b l e , s u c h a s s e x h o r m o n e s , anorexia nervosa, dietary intake of calcium and vitamin d, consumption of medications such as longterm corticosteroids, life style, smoking, consumption of alcohol and coffee, and hyperparathyroidism. on the other hand, gender, age, body size, ethnicity, and family history are uncontrollable risk factors for osteoporosis.(6) according to a study by pietschmann et al., the prevalence of fractures of the vertebrae, pelvic bones, and distal bones of the forearm in white american women aged 50 years or more is estimated at around 40%, while in white american men it is around 13%. from this study it was also apparent that the risk of fractures increased in women as compared to men. osteoporotic fractures is related to pain and disability, and also increases the mortality rate. the mortality rate after hip fracture in women is 4 times higher than in men. (7) vertebral fractures are the cause of longterm morbidity in women and mortality in women as well as men. costs of treatment of fractures are higher in women than in men. the objective of this study was to determine the prevalence of osteoporosis and several of its risk factors in postmenopausal women aged 47 to 60 years. methods design of the study a cross-sectional design was used in this study, conducted in the catchment area of m a m p a n g p r a p a t a n h e a l t h c e n t e r, f r o m february to april 2010. subjects of the study the study subjects were postmenopausal women aged 47 to 60 years, selected according to the following inclusion criteria: having e n t e r e d n a t u r a l m e n o p a u s e , d u r a t i o n o f menopause >1 year and <10 year, not consuming longterm steroids, not suffering from chronic diseases, capable of active communication, and willing to participate in this study. the exclusion criteria were: acute infectious diseases, diabetes mellitus, functional abnormalities of the kidneys a n d l i v e r, h i s t o r y o f c a n c e r, h i s t o r y o f hysterectomia, hormonal therapy. the selection of the study subjects was performed using cluster random sampling from four villages (kelurahan), i.e. kuningan barat, mampang prapatan, tegal parang, and pela mampang, in the area of mampang prapatan subdistrict, south jakarta. measurements weight and height were measured with the subjects wearing light clothing and no shoes. height was determined by means of a portable microtoise accurate to 0.1 cm and weight was measured with sage portable scales accurate 171 t o 0 . 1 k g . b o d y m a s s i n d e x ( b m i ) w a s calculated by dividing weight (in kilograms) by the square of the height (in meters). the obtained bmi values were independent of age and gender. bmi classification for the asiapacific region were as follows: underweight if b m i < 1 8 . 5 , n o r m a l i f b m i 1 8 . 5 2 2 . 9 , overweight if bmi between 23-24.9, obese i if bmi between 25-29.9, and obese ii if bmi > 30.(8) t h e i n s t r u m e n t s a n d m e t h o d s f o r e v a l u a t i n g b m d w e r e s t a n d a r d i z e d t o minimize interobserver variation. bmd (mg/ cm2) was measured by dual-energy x-ray absorptiometry (lunar dpx bravo nomusa densitometer, ge medical systems) at budi jaya hospital, jakarta. the results given were those for the mean bmd values of l1–l4 at the spine, femoral neck, and left radius. demographic data were collected through interviews using a structured questionnaire, f o l l o w e d b y p h y s i c a l e x a m i n a t i o n f o r measurement of blood pressure. statistical analysis univariate analysis was conducted on background characteristics, and normality of the data distribution was evaluated by means of the kolmogorov-smirnov test. normal data distributions were expressed as mean, standard deviation, and percentage. pearson correlation analysis was used to determine correlations between age, duration of menopause, number of pregnancies, and bmi on the one hand and bmd on the other. for nonnormally distributed data, spearman’s rho was used. statistical analyses was done using spss software for windows version 15.0 (spss inc., chicago), while statistical significance was set at p< 0.05. ethical clearance ethical clearance was issued by the research ethics committee, medical faculty, trisakti university. written informed consent was obtained from all subjects in this study, duly legalized by attached signature or thumb print. identity of all study subjects was kept c o n f i d e n t i a l a n d o n l y u s e d f o r r e s e a r c h purposes. results in this study there were 203 women m e e t i n g t h e i n c l u s i o n c r i t e r i a , w i t h t h e following characteristics: age between 47 and 60 years, mean age 53.3 ± 5.15 years, mean age at menarche 14.35 ± 1.74 years; mean duration of menopause 4.45 ± 2.21 years, with a r a n g e o f 2 7 y e a r s ; m e a n n u m b e r o f pregnancies 4.96 ± 2.49 , with a range of 2-7 pregnancies. mean bmi was 28.8 ± 4.55 kg/m2, with most of postmenopausal women (61.58%) in this study being overweight, while 7.08% was u n d e r w e i g h t a n d 3 0 . 5 4 % i n t h e n o r m a l category. mean lumbar vertebral t-score was -1.62 ± 1.03 g/cm2, mean femoral neck t-score -1.00 ± 0.93 g/cm2, and mean distal radius tscore was -1.91 ± 1.16 g/cm2. with regard to formal education of the study subjects, 50.7% f i n i s h e d p r i m a r y s c h o o l , b u t o n l y 9 . 4 % attended senior high school (sma) or higher institutions of learning. the majority of subjects (52.7%) was unemployed at the time of the study (table 1). the presence of osteroporosis is inferred from the bmd value, expressed as the t score, which compares bmd to mean bone density of young adults. in the present study, the p r e v a l e n c e o f o s t e o p e n i a o f t h e l u m b a r vertebrae was found to be 28.1%, that of the distal radius was 23.2% and the highest prevalence of osteopenia was in the femoral neck, accounting for 45.8% of subjects. regarding the prevalence of osteoporosis, in the lumbar vertebrae it was 20.2%, and in the femoral neck 4.9%, with the highest prevalence of 30% in the distal radius (table 2). the results of a simple linear regression analysis between age, duration of menopause, number of pregnancies on the one hand and bmd on the other, showed that advanced age 172 meiyanti epidemiology of osteoporosis in women leads to a reduction in bmd. a significant reduction in bmd was seen in the lumbar vertebrae and distal radius. for lumbar bmd a value of p=0.012 was found, for the distal radius p=0.039, while for the femoral neck p=0.367. simple linear regression analyis between bmi and bmd revealed that bmi positively affected bmd, in the sense that higher bmi values were related to higher bmd. bmd scores of the lumbar vertebrae were significant at p=0.000, while bmd scores of the femoral neck and distal radius were both significant at p=0.000 (table 3). discussion t h e p r e v a l e n c e o f o s t e o p o r o s i s i n postmenopausal women aged 47 to 60 years, based who criteria using t scores, was 20.2% in the lumbar vertebrae, while the highest prevalence of osteoporosis of 30% was found in the distal radius. similar results were obtained in postmenopausal women aged 50 to 64 years in germany, with a prevalence of osteoporosis of 23.3%.(1) in comparison, a thai s t u d y r e p o r t e d t h a t t h e p r e v a l e n c e o f osteoporosis in the lumbar spine of thai postmenopausal women aged 50 to 54 years and 55 to 59 years, was 9.4% and 22.6%, r e s p e c t i v e l y. i n t h e f e m o r a l n e c k t h e prevalence of osteoporosis in the age groups 50-54 years and 55-59 years was 4.9% and 10.3%, respectively.(9) however, differring results were encountered in a study conducted at the nutritional research and development center of the indonesian department of health (pusat penelitian dan pengembangan gizi [puslitbang] depkes), yielding a prevalence of osteoporosis of 10.3%.(10) the type of osteoporosis found in the present study was primary osteoporosis due to estrogen deficiency. as is commonly accepted, table 1. characteristics of study subjects (n=203) characteristic n (%) age (years) 53.30 ± 5.15 age at menarche (year s) 14.35 ± 1.74 duration of men opause (years) 4.4 5 ± 2.21 num ber of pregnancies 4.9 6 ± 2.49 blo od pressure (mm hg) syst olic 124.7 ± 20.49 diastolic 78.55 ± 12.02 body mass index underweight 16 (7.88) normal 62 (30.54) over weight 125 (61.58) weight (kg) 59.45 ± 10.97 length (cm) 148.83 ± 5.17 bmd (t score) right femur -1.00 ± 0.93 lumbar vertebrae 1.62 ± 1.03 left radius 1.19 ± 1.16 education non e 21 (10.3) did not finish primary school 20 (9.9) finished primar y sch ool 10 3 (50.7) junior high school (smp) 40 (19.7) seni or high school (sma) 17 (8.4) academ y / university 2 (1.0) employment entrepreneur 18 (8.9) public servant (pns) 1 (0.5) businessman 46 (22.7) unemployed 10 7 (52.7) other 31 (15.3)  abbreviation bmd, bone mass density table 2. distribution of osteopenia and osteoporosis by bmd site in postmenopausal women (n=203) bmd site osteopenia (n,%) osteoporosi s (n,%) lumbar ver tebr ae 57 (28.1) 41 (20.2) rig ht femoral neck 93 ( 45.8) 10 (4.9) left distal radius 47 ( 23.2) 61 (30.0)  abbreviation bmd, bone mass density 173 # pearson correlation coefficient; **: spearman’s rho; *significant (p < 0.05). the values in parentheses are p value; abbreviation: bmi, body mass index estrogen plays an important role in osteoblasts and mineral homeostasis. in addition, estrogen also has extraskeletal effects in the form of d e c r e a s e d a b s o r p t i o n o f c a l c i u m i n t h e intestines, which leads to increased parathyroid hormone levels and increased bone degradation in postmenopausal women. according to mawie et al.(11) up to 49.5% of postmenopausal women have estradiol levels of <5 pg/ml, while a positive correlation was found between estradiol levels and t score in the femoral neck region. estradiol inhibits the generation and activity of osteoclasts, the inhibition being mediated by upregulation of osteoprotegerin.(7) the results of the correlation test showed a correlation between age and bmd, with increasing age leading to decreasing bmd in the lumbar vertebrae and the distal radius. in old age (>60 years), the type of osteoporosis encountered is senile osteoporosis as a result of the aging process, with the occurrence of osteoblastic dysfunction in trabecular and cortical bone, thereby increasing the risk of fractures of the vertebrae and pelvic bones.(7,12) in their lifetime women will lose 50% of their trabecular bone mass and 30% of their cortical bone mass, and half of the loss occurs within 10 years after menopause.(13) sakondhavat et al. ( 1 4 ) have stated that the prevalence of osteoporosis increases proportionally with advancing age and duration of menopause in the lumbar vertebrae as well as the femoral neck. zhai et al.(2) reported that with increasing age a significant loss of bone mass occurs in t h e v e r t e b r a e a n d f e m o r a l n e c k o f postmenopausal women, the relationship between loss of bone mass and age not being linear, but quadratic. in contrast, liu-ambrose et al.(15) found no relationship between loss of bone mass and age. in the present study the duration of menopause appears to have a significant impact on the reduction in bmd of the lumbar vertebrae and distal radius. in women entering menopause, there is a loss of bone mass of 12% annually for a period of 5-10 years. the loss of bone mass is particularly rapid in trabecular bone, with fractures of the vertebrae and distal radius comprising the frequently e n c o u n t e r e d c l i n i c a l m a n i f e s t a t i o n s . trabecular bone is affected by several factors, such as bmi, age, estradiol levels and physical activity..(16,17) in postmenopausal women there is a decrease in trabecular bone mass due to age and diminished estradiol levels. at the start of menopause the mean loss in trabecular bone mass is reportedly between 1.8% and 2.3% in the veterbral column and 1.0-1.4% in the pelvic bones. after 5 years of menopause the mean decrease in bmd is 7-10% in the vertebral column and 5-7% in the pelvic bones, thus increasing the risk of fractures.(18) finkelstein et al. (13) showed that the decline in bmd occurs significantly in late perimenopause and is extremely rapid in the first postmenopausal year. li et al. (19) also found a significant decrease in bmd occurring with increasing age and duration of menopause. mean bmi in the present study was 28.8 k g / m 2, a n d o n t h e b a s i s o f t h e b m i table 3. correlation between several risk factors and bmd scores lumbar vertebrae femoral neck distal radius age** -0.175 (0.012)* -0.065 (0.357) -0.145 (0.039)* duration of menopause# -0.215 (0.002)* -0.129 (0.066) -0.182 (0.009)* no. of pregnancies** -0.210 (0.003)* -0.138 (0.050) -0.179 (0.011)* bmi# 0.3 11 (0.000)* 0.305 (0.000)* 0.366 (0.00 0)*   174 meiyanti epidemiology of osteoporosis in women classification used, most subjects were in the overweight category (obese i = 25-29.9 kg/m2). the simple linear regression results indicate the presence of a positive correlation between bmi and bmd. bmi is representative of total body fat. several studies demonstrated that body weight and adipose tissue may influence bmd in several ways, such as the following: an overweight individual has a greater weight bearing load, body weight in adolescents affects peak bone mass, and in postmenopause adipose tissue is converted into active estrogens (estrones), so that obese postmenopausal women have higher endogenous estrogen levels.(20) adipose tissue synthesizes leptins that are associated with bone mass, body weight and reproduction. leptins are associated with osteoblasts, which stimulate mineralization of the bones, regulate bone formation and inhibit the generation of osteoclasts. circulating leptins are related to bmi and size and geometry of hand bones, while in regulating body weight leptins are linked to eating habits and energy utilization.(21,22) sukumar et al.(23) reported similar results, in that bmi values were closely correlated with bmd scores in trabecular and cortical bone, with serum parathyroid hormone levels, and with decreased serum 25-hydroxy vitamin d levels. similarly, felson (20) found that bmi values were more closely correlated with bmd in older women than in men. genetic factors are strongly correlated with b o n e m i n e r a l c o m p o s i t i o n a n d b o d y composition, and in 70-80% of cases are of influence in determining peak bone mass. the mean number of past pregnancies was 5 and correlation analysis indicated that f r e q u e n c y o f p r e g n a n c i e s s i g n i f i c a n t l y decreased bmd. pregnancy and lactation are a s s o c i a t e d w i t h c h a n g e s i n c a l c i u m homeostasis, which lead to decreases in bmd. it is common knowledge that during pregnancy maternal calcium is transferred to the fetus, and the amounts transferred increase in the second and third trimesters, when calcium is required for the development of fetal bones. as a compensatory change, during pregnancy there is a 50% increase in the absorption of calcium from intestines, as a result of raised levels of 1,25 dihydroxy-vitamin d and estrogen, thus protecting the body against the effects of calcium loss from the bones. during pregnancy parathyroid hormone levels are also raised, leading to mobilization of calcium from the b o n e s . ( 2 4 , 2 5 ) o n t h e o t h e r h a n d , a h i g h e r frequency of pregnancies may protect against postmenopausal loss of bone mass, because pregnancy is associated with increased body weight, increased absorption of calcium from the intestines, and cumulative exposure to estrogens, and older age at menopause.(26) several studies found supportive evidence for a positive correlation between number of p r e g n a n c i e s , b m d , a n d r e d u c t i o n i n prevalence of femoral fractures. streeten et al.(27) showed that the number of pregnancies was associated with increased bmd values in the hip, which was related to increased bmi during pregnancy. lenora et al.(28) did not find any influence of the number of pregnancies and duration of lactation on bmd in middle-class women, which was related to nutrional intake during pregnancy. the present study found three factors possibly associated with the high prevalence o f o s t e o p o r o s i s , v i z . a g e , d u r a t i o n o f menopause, and number of pregnancies. in addition, genetic factors should receive due consideration. deng et al.(29) reported that genetic factors play an important role in bmi and bmd of the spine and hip in ethnic han chinese. lazcano-ponce et al.(30) stated that genetic factors impact on bmd and peak bone mass at the ages of 20-30 years, and on p o s t m e n o p a u s a l l o s s o f b o n e m a s s . furthermore, girls and young adult women whose mother had a history of osteopenia or osteoporosis, would have a similar experience. in the present study the majority of subjects (70.9%) had a low level of formal education (primary school or lower), while ho et al.(31) in china found that 50.5% of their subjects 175 were in this category. they also found that a higher level of education was associated with a l o w e r p r e v a l e n c e o f o s t e o p o r o s i s . i n developing countries, educational level is an essential marker of socio-economic status. education is closely linked with life style, dietary intakes (especially of high-calcium foods, vegetables, and nutrients with an impact on bone mass), and intakes of nutrients and calcium during pregnancy and lactation. socioe c o n o m i c s t a t u s i s a l s o a s s o c i a t e d w i t h nutrition in childhood and adolescence, which is reflected in body height and age at menarche. in addition, life style activities, such as consumption of alcohol, smoking, and habitual participation in sports and physical activities, are of influence on bmd. (31) the present study was not exempt from several limitations. firstly, the study design was of cross-sectional type and thus was unable to prove that the osteoporosis was the outcome of several risk factors. secondly, the study did not collect data on life style of postmenopausal w o m e n , a s a n i n f l u e n c i n g f a c t o r o n t h e prevalence of osteoporosis. conclusions t h e p r e v a l e n c e o f o s t e o p o r o s i s i n postmenopausal women aged 47 to 60 years was 20.2% and 30% respectively for the lumbar spine and distal radius. several factors, such as age, duration of menopause, number of pregnancies, and bmi, affected the risk for o s t e o p o r o s i s . p u b l i c h e a l t h p r o m o t i o n a l activities should be implemented in view of the relatively high prevalence of osteoporosis. futher studies are also needed on the factors associated with osteoporosis and on prevention and management of primary osteoporosis. acknowledgements the author thanks the dean and viced e a n s o f t h e m e d i c a l f a c u l t y, tr i s a k t i university, for the funding of this study. thanks are also due to the postmenopausal women for their participation and to the doctors and staff of the mampang prapatan primary health center for their support in this study. references 1. häussler b, gothe h, göl d, glaeske g, pientka l, felsenberg d. epidemiology, treatment and costs of osteoporosis in germany-the boneeva study.osteoporos int 2006;18:77-84. 2. zhai g, hart dj, valdes am, kato bs, richards hb, hakim a, et al. natural history and risk factors for bone loss in postmenopausal caucasian women: a 15-year follow up population based study. osteoporos int 2008;19:1211-7. 3. national institutes of health consensus development panel on osteoporosis prevention, diagnosis, and therapy. osteoporosis prevention, diagnosis, and therapy. jama 2001;285:785-95. 4. brown se. osteoporosis: snap, crackle and pop some pills. northeast florida med 2006;57:1420. 5. world health organization. assessment of fracture risk and its application to screening for postmenopausal osteoporosis. geneva: who technical report series 843;1994. 6. johnson nk, clifford t, smith km. understanding risk factors, screening and treatment of postmenopausal osteoporosis. orthopedics 2008;31:676-80. 7. pietschmann p, rauner m, sipos w, schindl kk. osteoporosis: an age related and gender specific disease a mini review. gerontology 2009;55:312. 8. world health organization. appropiate body mass index for asian populations and its implication for policy and intervention strategies. the lancet 2004;363:157-63. 9. limpaphayom kk, taechakraichana n, jaisamrarn u, bunyavejchevin s, chaikittisilpa s, poshyachinda m, et al. prevalence of osteopenia and osteoporosis in thai women. menopause 2001;8:65-9. 10. puslitbang gizi depkes. osteoporosis. available at: http://www.pppl.depkes.go.id. accessed september 2, 2010. 11. mawie m. serum estradiol level and bone mineral density in postmenopausal women. univ med 2010;29:90-5. 176 meiyanti epidemiology of osteoporosis in women 12. finkelstein js. osteoporosis. in: goldman l, auseillo n, editors. cecil textbook of medicine. 22nd ed. philadelphia: saunders;2004. p.1547-55. 13. finkelstein js, brockwell se, mehta v, greengale ga, sowers mr, ettinger b. bone mineral density changes during the menopause transition in a multiethnic cohort of women. j clin endocrinol metab 2008;93:861-8. 14. sakondhavat c, thangwijitra s, soontrapa s, kaewrudee s, somboonporn w. prevalence of osteoporosis in postmenopausal women at srinagarind hospital, khon kaen university. srinagarind med j 2009;24:113-0. 15. liu-ambrose t, kravetsky l, bailey d. change in lean body mass is a major determinant of change in areal bone mineral density of proximal femur: a 12 year observational study. calcif tissue int 2006;79:145-51. 16. uusi k, sievanen h, pasanen m, kannus p. agerelated decline in trabecular and cortical density: a 5 year peripheral quantitative computed tomography follow up study of preand postmenopausal women. calcif tissue int 2007; 81:249-53. 17. khosla s, melton lj, achenbach sj, oberg al, riggs bl. hormonal and biochemical determinants of trabecular microstructure at the ultradistal radius in women and men. j clin endocrinol metab 2006;91:885-91. 18. finkelstein js, lee ml, sowers m, ettinger b, neer rm, kelsey jl, et al. ethnic varation in bone density in premenopausal and early perimenopausal women: effects of anthropometric and life style factors. j clin endocrinol metab 2002;87:3057-67. 19. li hl, han mz. menarche and menopause, age, factors such a postmenopausal osteoporosis and the incidence of relationship. chinese j gynaecol obstet 2005;12;796-98. 20. felson dt, zhang y, hannan mt, anderson jj. effects of weight and body mass index on bone mineral density in men and women: the framingham study. j bone miner res 2003;8: 567-73. 21. livshits g, pantsulaia ia, trofimov s, kobyliansky e. genetic variation of circulating leptin is involved in genetic variation of hand bone size and geometry. osteoporos int 2003;14:47683. 22. thomas t, burguera b. is leptin the link between fat and bone mass? j bone miner res 2002;17: 1563-69. 23. sukumar d, schlussel y, riedt s, gordon c, stahl t, shapses a. obesity alters cortical and trabecular bone density and geometry in women. osteoporos int 2010;22:634-45. 24. o’brien ko, nathanson ms, macini j, witter fr. calsium absorption is significantly higher in adolescents during pregnancy than in the early postpartum period. am j clin nutr 2003;78:118893. 25. o’brien ko, donangelo cm, zapata cl, abrams sa, spencer em, king j. bone calcium turnover during pregnancy and lactation in womwn with low calcium diets is associated with calcium intake and circulating insulin-like growth factor i concentrations. am j clin nutr 2006;83:31723. 26. cure-cure c, cure-ramirez p, teran e, lopezjaramillo p. bone mass peak in multiparity and reduced risk of bone fractures in menopause. int j gynaecol obstet 2002;76:285-91. 27. streeten ea, ryan ka, mc bride dj, pollin ti, shuldiner ar, mitchell bd. the relationship between parity and bone mineral density in women characterized by a homogeneous lifestyle and high parity. j clin endocrinol metab 2005; 90:4536-41. 28. lenora j, lekamwasan s, karlsson m. effects of multiparity and prolonged breast-feeding on maternal bone mineral density: a communitybased cross-sectional study. bmc womens health 2009;9:19. 29. deng fy, lei sf, li mx, jiang c, dvornyk v, deng hw. genetic determination and correlation of body mass index and bone mineral density at the spine and hip in chinese han ethnicity. osteoporos int 2006;17:119-24. 30. lazcano-ponce e, tamayo j, diaz r, burguete ai, salmeron j. correlation trends for bone mineral density in mexican women: evidence of familiar predisposition. salud publica de mexico 2009;51suppl 1:s93-9. 31. ho sc, chen ym, woo jlf. educational level and osteoporosis risk in postmenopausal women. am j epidemiol 2005;161:680-90. alvina 188 pharmacokinetic interactions between rifampicin and efavirenz in hiv-tb coinfections september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina yenny* *department of pharmacology and therapeutics medical faculty, trisakti university correspondence dr. yenny, spfk department of pharmacology and therapeutics medical faculty, trisakti university jl. kyai tapa no. 260, grogol jakarta 11440 phone: 021-5672731 ext.2801 email: stasia_mk@yahoo.com univ med 2009;28:188-201 abstract the increased percentage of patients with hiv-tb coinfection leads to inevitable interactions between rifampicin and efavirenz. efavirenz is a potent non-nucleoside reverse transcriptase inhibitor (nnrti) for the treatment of hiv infection. the use of this drug combination with rifampicin causes problems in determination of the optimal dosage of efavirenz when administered concomitantly with rifampicin. efavirenz is metabolized by the enzyme cytochrome p-450 (cyp), i.e. the cyp2b6 and cyp3a4 isozymes, of which rifampicin is an inducer. the induction of cytochrome p-450 by rifampicin is mediated by pregnane x (pxr) and constitutive androstane receptors (car) in the cell nucleus, resulting in a wide variation in the plasma efavirenz concentrations, such that a therapeutic failure or the occurrence of toxic effects are to be expected. the optimal dosage of efavirenz is commonly determined through pharmacokinetic studies, but this is problematic in the combined use of the drug with rifampicin, due to the wide variation in study design, method, and sample size of each study. ethnic factors and genetic polymorphism of the enzymes that metabolize efavirenz contribute to the problem of determining the optimal dose of this drug. pharmacokinetic studies with good measurement parameters and methods are still necessary as the basis for determining the optimal dose of efavirenz in the indonesian population. keywords: drug interaction, efavirenz, rifampicin, hiv, tb introduction tu b e r c u l o s i s ( t b ) i s o n e o f t h e opportunistic infections frequently encountered in patients with human immunodeficiency virus ( h i v ) i n f e c t i o n . t h e r e s u l t s o f a s u r v e y conducted by the department of pulmonology and respiratory medicine of the faculty of medicine, university of indonesia and the persahabatan hospital in the years 2000-2005 revealed an sharp increase in the percentage of tb in patients with hiv from 5.88% in 2000 to 189 univ med vol.28 no.3 33% in 2005.(1) hiv infection is the main risk factor of tb reactivation. forty percent of patients with hiv against only 5% of those without hiv are said to be potentially liable to tb reactivation.(1) the mortality rate of patients with hiv accompanied by tb exceeds 50%.(2) in the therapeutic regimen for tb the use of rifampicin is absolutely necessary for the successful outcome of the treatment of patients with hiv who are coinfected with tb. clinical t r i a l s h a v e d e m o n s t r a t e d t h a t t h e r a p e u t i c regimens for tb that do not use rifampicin or only in the initial two months of treatment show an increase in the therapeutic failure or relapse rates.(3) efavirenz is a non-nucleoside reverse transcriptase inhibitor (nnrti) recommended by the who as one of the components for the initial first-line treatment of patients with hiv.(4) the combined use of efavirenz and rifampicin gives rise to the problem of drug interaction.(5) rifampicin is a potent inducer of the enzyme cytochrome p450 which metabolizes a number of drugs, including efavirenz, thus significantly decreasing plasma levels of this nnrti. the problem concerns the determination of t h e o p t i m a l d o s a g e o f e f a v i r e n z w h e n administered concomitantly with rifampicin. longitudinal studies conducted by friedland et al.(6) showed a wide variation in the plasma drug concentrations of efavirenz when administered in combination with rifampicin. this causes the patients who are exposed to subtherapeutic doses to be at risk of therapeutic failure and the development of drug resistance, whereas patients exposed to high doses of the drug are at high risk of experiencing central nervous system side effects. the purpose of this literature review is to discuss opportunistic tb infections in patients with hiv, the interactions of efavirenz and rifampicin (in relation to nuclear receptors), and to trace the extent to which the interactions between these two drugs may be applied to the determination of the optimal dose of efavirenz. opportunistic tb infections in patients with hiv hiv causes difficulties in the management of tuberculosis, and vice versa. hiv infection increases the risk of reactivation of latent tb, while individuals already infected with hiv and only afterwards become infected with tb have a higher progressivity of the disease.(7) cd4+ t lymphoctes and macrophages play an important role in the immune response to infection with mycobacterium tuberculosis. infection with tb leads to the release by cd4+ lymphocytes of proinflammatory cytokines, such as interferon-γ (ifn-γ), interleukin (il)-1, il-2, and tumor necrosis factor-α (tnf-α). these cytokines subsequently activate macrophages a n d k i l l i n t r a c e l l u l a r t b o rg a n i s m s . t h e production of cytotoxic t cells is an additional protective mechanism in the response to tb infection.(8) hiv infection results in dysfunction of cd4+ cells and a reduction in their absolute numbers,(8) causing patients with hiv to be at high risk of experiencing reactivation of latent tb, this risk being similar to the risk of acquiring a new tb infection. conversely the tb infection itself may increase hiv progressivity. the presence of the tb organism and its major cell wall antigen (lipoarabinomannan) within the body leads to upregulation of the genes coding for the proinflammatory cytokines il-1, il-2, il6, and tnf-α. the induced cytokines increase hiv replication by activation of the transcription factors nuclear factor kb (nf-kb) and il-6, thus increasing the transcription of the long terminal repeats (ltrs) of hiv. this process is directly and indirectly stimulated by il-1 and tnf-α, cytokines which in normal conditions play a role in the defense mechanisms against tb infection. in contrast, in hiv-tb coinfection the above 190 process accelerates the progressivity of hiv to the advanced stages.(8) in principle, adequate chemotherapy of tuberculosis should be administered to all i n d i v i d u a l s r e c e i v i n g t r e a t m e n t f o r t b , irrespective of the presence of hiv infection in the individual in question. this therapeutic principle for tb includes the use of standard combinations of antituberculosis drugs, of which the first-line drugs are isoniazid, rifampicin, pyrazinamide, ethambutol, and streptomycin. in the standard therapeutic regimen for tb, these drugs should always be used in combinations of two or more agents. the standard regimen of 69 months consists of 2 months treatment with i s o n i a z i d , r i f a m p i c i n , p y r a z i n a m i d e , a n d ethambutol (base-line), followed by 4-7 months of treatment using isoniazid and rifampicin (endline). for all responsive tb cases, this standard therapeutic regimen is presumably adequate both in hiv-infected and in non-infected patients.(9) drug interactions drug interaction occurs if the effect of a drug is modified by the presence of another.(3) d r u g i n t e r a c t i o n s a r e d i v i d e d i n t o p h a r m a c o d y n a m i c a n d p h a r m a c o k i n e t i c interactions. pharmacokinetic interactions occur if the presence of a drug is capable of modifying t h e a b s o r p t i o n , d i s t r i b u t i o n ( t r a n s p o r t ) , metabolism, and excretion of another drug given concomitantly, so as to affect the bioavailability of the latter. pharmacokinetic interactions i n c l u d e c h a n g e s i n g a s t r i c p h a n d d r u g absorption, altered drug metabolism mediated by c y p 4 5 0 , m o d u l a t i o n o f p g l y c o p r o t e i n s (cellular transport proteins), and alterations in renal drug elimination. pharmacodynamic interactions are interactions between drugs acting on the same receptor system, site of action or physiological system. the induced responses may be additive, synergistic, or antagonistic. pharmacodynamic interactions do not alter the plasma concentrations of the interacting drugs.(10) pharmacokinetic interactions frequently occur in metabolic processes located in the liver (and less frequently in the intestines). these metabolic interactions take place when two drugs compete for being metabolized by the same enzyme, where one of the most important metabolic interactions occurring is through the enzyme cyp450. drugs may affect cyp450 in two ways, namely by induction or inhibition. inhibition of cyp450 results in the slower metabolism of the drug that is the substrate of the enzyme, leading to accumulation of the drug in the blood, thus p o t e n t i a l l y c a u s i n g t o x i c i t y. i n d u c t i o n o f cyp450 leads to increased metabolism of the drug that is the substrate of the enzyme, thus reducing the efficacy of the treatment. drug interactions are the main problem in optimization of dosage. all drugs of the rifamycin group are cyp450 inducers of varying potency.(11) drug metabolism and cytochrome p-450 the process of biotransformation plays an important role in discontinuing the action of a drug and the main organ for biotransformation is the liver. the biotransformation process consists of several phases. phase i comprises oxidation, reduction, and hydrolysis, commonly m e d i a t e d b y c y p 4 5 0 . phase ii comprises conjugation, mediated by uridine diphosphateglucuronosyl-transferase (ugt), sulfotransferase (sult), and glutathione-s-transferase (gst). in general, this process gives rise to metabolites that are more polar, more easly ionized (more easily eliminated by the kidneys), or more fat-soluble (excreted into the bile and subsequently into the feces).(12) cyp450 is particularly found in the smooth endoplasmic reticulum of the hepatic cells and the epithelial enterocytes of the small intestine. in general, cyp450 plays a role in 80% of the oxidative metabolism and in nearly 50% of the yenny rifampicin and efavirenz in hib-tb 191 univ med vol.28 no.3 elimination of a drug.(12) currently 57 human genes for cyp450 have been identified, but only a small number code for proteins (particularly the cyp1, cyp2 and cyp3 families), and contribute to drug metabolism. cyp450 is classified on the basis of amino acid homology, designated by a digit indicating the family, a letter standing for the subfamily, and a final number referring to the individual enzyme, e.g. cyp3a4, 2d6, 2c19.(12) n u c l e a r re c e p t o r s a n d i n d u c t i o n o f cytochrome p-450 xenobiotics may affect the rate of drug metabolism by activating the transcription process and induce the expression of genes coding for the enzymes that metabolize the drug t h r o u g h i n t e r a c t i o n s b e t w e e n t h e n u c l e a r receptor superfamily and their ligands.(12) t h e n u c l e a r r e c e p t o r s u p e r f a m i l y a r e transcription factors functioning as sensors of lipophilic xenobiotics, including drugs. ( 1 3 ) activation of certain nuclear receptors by xenobiotics that are the ligands of the nuclear receptors induce the transcription of a series of t a rg e t g e n e s ( a m o n g t h e m c y p a n d d r u g transporters), which function as an adaptive defense mechanism against toxic substances or xenobiotics. ( 1 3 ) the structure of a nuclear receptor is depicted in figure 1. o n e o f t h e m o s t i m p o r t a n t i n d u c t i o n mechanisms is effected through a type 2 nuclear receptor, belonging to the same superfamily as the steroid hormones. type 2 nuclear receptors play an essential role in drug metabolism and therapy. the group of receptors and ligands belonging to the type 2 nuclear receptors are pregnane x receptor (pxr) with rifampicin as ligand, constitutive androstane receptor (car) with its ligand phenobarbital, and peroxisome proliferators activated receptor (ppar) whose ligand is fibrate.(11) figure 1. structure of a nuclear receptor.(13) a. the nuclear receptor consists of 4 domains, viz. the n-terminal region containing the activation function 1 (af-1), dna binding domain (dbd), hinge domain, and ligand binding domain (lbd) containing the activation function 2 (af-2). b. dna binding domain of the nuclear receptor. pxr and car are members of the nuclear receptor superfamily functioning as sensors for the presence of lipophilic xenobiotics, including drugs. when the nuclear receptor senses the presence of a xenobiotic, it binds to rxr (heterodimer) of the responsive element of the target gene and triggers the transcription of nucleotide hexamers. 192 pxr is activated by a number of drugs, one of them being rifampicin. pxr is expressed in the same tissue where cyp3a4 is expressed. a c t i v a t i o n o f p x r i n d u c e s e x p r e s s i o n o f c y p 3 a 4 , a n d o f g e n e s c o d i n g f o r d r u g transporters (e.g. p-glycoprotein), and phase ii enzymes including ugt and sult.(13) the genes induced by car include those encoding several cyp enzymes (cyp2b6, cyp2c9, and cyp3a4), a number of phase ii gst, ugt and slut enzymes, and endobiotic transporters. cyp3a4 is induced by pxr and car, such that the level of this enzyme is c o n s i d e r a b l y a ff e c t e d b y m a n y d r u g s a n d xenobiotics.(13) induction of cytochrome p-450 by rifampicin the mechanism by which rifampicin induces the enzyme cyp3a4 is mediated by activation of the nuclear receptor pxr (also known as “steroid x receptor”) and car. rifampicin functions as a ligand that activates the transcription factor when bound to pxr. this process is followed by the formation of a heterodimer with the retinoid x receptor (rxr), the heterodimer ultimately binding to the responsive elements of dna in the regulator region of the cyp3a gene. the outcome is upregulation of dna transcription resulting in increased synthesis of the cyp3a4 enzyme and increased oxidative metabolism of the substrate of the enzyme.(14) drug interaction at molecular level is illustrated in figure 2. another nuclear receptor, car, is also involved in the activation of transcription factor cyp3a4. car exhibits cross-talk(16) (functional i n t e r a c t i o n b e t w e e n 2 o r m o r e s i g n a l i n g pathways) with pxr, in the sense that both nuclear receptors bind to the same responsive element in the promoter region of the gene for cyp3a4 as well as cyp2b6 (figure 3). cross-talk enables the two nuclear receptors to induce activation of the transcription factor of the cyp3a4 and cyp2b6 genes as a result of rifampicin administration. in addition, the occurrence of cross-talk causes car and pxr to jointly compensate for loss or malfunction of each nuclear receptor.(17) the effect of rifampicin on car is smaller than on pxr.(17) efavirenz and nevirapin induce the genes for cyp2b6 and cyp3a4 principally through activation of car.(16) figure 2. drug interaction at molecular level(15) nuclear receptor pxr is a transcription factor regulating the expression of the cyp3a gene (yellow) in the liver and small intestine. in order to function pxr binds to the nuclear receptor rxr forming a heterodimer. binding of drug a to pxr and induction of expression of enzyme cyp3a (pink) accelerates the metabolism of drug b, which is the substrate of cyp3a. cyp = cytochrome p450; oh = hydroxyl group; pxr= pregnane x receptor; rxr= retinoid x receptor. yenny rifampicin and efavirenz in hib-tb 193 univ med vol.28 no.3 rifampicin currently there are 3 types of rifamycin that are used in clinical practice, namely rifampicin (rifampin), rifapentin, and rifabutin. the use of rifapentin is not recommended for treatment of tb in hiv-positive patients, due to the high microbiological failure rate. (18) rifabutin is recommended more for use in hiv patients with concomitant tb infection, as the risk of its p o t e n t i a l i n t e r a c t i o n w i t h a rv ( p r o t e a s e inhibitor and nnrti) is the lowest among the existing rifamycins. rifamycin acts to inhibit deoxyribonucleic acid (dna)-dependent ribonucleic acid (rna) p o l y m e r a s e o f m y c o b a c t e r i a a n d o t h e r microorganisms (figure 4). inhibition of dnadependent rna polymerase leads to suppression of the initiation of dna chain formation (but n o t o f c h a i n e x t e n s i o n ) r e q u i r e d f o r microbacterial replication.(19) rifampicin is well-absorbed following oral administration, the peak concentration being reached within 2-4 hours. after being absorbed, the drug is rapidly eliminated into the bile and enters the enterohepatic circulation. the half-life of rifampicin is from 25 hours. the half-life is progressively shortened within 14 days of therapy, due to the ability of rifampicin to induce hepatic m i c r o s o m a l e n z y m e s a n d a c c e l e r a t e t h e deacetylation process that plays an important role in drug elimination. elimination of rifampicin in the urine is only 30%, and in the feces 60-65%, less than half in intact form. therefore its dose does not need to be adjusted with impaired renal function.(18) the pharmacokinetic profile of rifampicin is shown in table 1. rifampicin is in general well-tolerated. less than 4% of patients experience side effects, the most common in treatment of tb being skin rashes, fever, nausea, and vomiting. rarely hepatitis and hepatic failure may occur.(19) the most important interactions of the members of the rifamycin class occur mainly with the cyp3a4 and cyp2c8/9 isozymes. in addition to these two cyp isozymes, rifampicin also induces the activity of isozymes cyp2c19 and cypb6 to a smaller extent.(5) the members of the rifamycin class differ in cyp induction potential, and in order of decreasing potency these are rifampicin > rifapentin > rifabutin, with a d i ff e r e n c e i n i n d u c t i o n p o t e n t i a l o f approximately 3:2:1.(20) in addition to being a potent cyp inducer, rifampicin also upregulates the synthesis of cytosolic enzymes involved in the metabolic reactions of phase ii drugs, e.g. uridine diphosphate-glucuronosyltransferase and sulfotransferase.(12) figure 3. cross-talk between pxr and car signaling pathways.(16) both nuclear receptors pxr and car are modulated by xenobiotics and steroids. to become active, pxr and car bind to rxr to form a heterodimer at the responsive element of the regulator region of the genes involved in the metabolism of xenobiotics and steroids, including those coding for phase i and phase ii enzymes and transporters. 194 efavirenz efavirenz is one of four nnrti drugs licensed by the food and drug administration (fda) for treatment of hiv. the other three agents are nevirapin, delaverdin, and etravirdin. among these, delavirdin is rarely used in clinical practice because of its relatively lower potency and higher frequency of administration when compared to the others. etravirdin is reserved for cases resistant to the other nnrti. all four drugs have differing structures, but a common mechanism of action. nnrti act as noncompetitive inhibitors of t h e r e v e r s e t r a n s c r i p t a s e o f t h e h u m a n immunodeficiency virus type-1 (hiv-1), that transcribes viral rna into dna. binding of nnrti to the hydrophobic pocket in the hiv-1 r e v e r s e t r a n s c r i p t a s e s u b u n i t p 6 6 i n d u c e s c o n f o r m a t i o n a l c h a n g e s a n d d i s t u r b s t h e catalytic activity of the enzyme, thus resulting in inhibition of viral dna synthesis.(21) orally administered efavirenz is wellabsorbed in the digestive tract, with a peak concentration within 5 hours. the bioavailability of the drug is increased by 22% on simultaneous intake of foods high in fat. over 99% of the drug is bound to plasma proteins. the metabolism of efavirenz is mainly mediated by cyp2b6 to p r o d u c e 8 h y d r o x y e f a v i r e n z , f o l l o w e d b y oxidation into 8,14-dihydroxyefavirenz. other isozymes (cyp3a4, 3a5, and 1a2) contribute only minimally to the metabolism of efavirenz.(22) efavirenz is an inducer of cyp3a4, thus being able to induce its own metabolism. (21) the autoinduction of efavirenz results in a difference in the half-times of the drug on administration of a single dose (52-76 hours) as compared with administration of multiple doses (after reaching a stable level) with half-life of 40-55 hours. elimination of the unmodified drug by way of the kidneys is insignificant (< 1%), thus no dose adjustment is necessary in renal dysfunction.(21) the pharmacokinetic profile of efavirenz is shown in table 2. the commonly encountered side effects of efavirenz are skin rashes, disorders of the central n e r v o u s s y s t e m ( d i z z i n e s s , d i s t u r b e d concentration, dysphoria, nightmares, and i n s o m n i a ) i n c l u d i n g p s y c h o t i c e p i s o d e s (depression, hallucinations, and/or mania). the central nervous system side effects are generally tolerated and disappear within the initial 4 weeks of therapy. to reduce the side effects, the drug is taken on an empty stomach at bedtime. other reported side effects are headaches and increased levels of hepatic transaminases and serum cholesterol. administration of the drug to pregnant women results in malformations of the fetal brain and spinal cord.(21) rifampicin bioavailability (%) 68 effect of food auc* 6%, cmax** 36% main metabolic pathway deacetylation, hydrolysis to formal derivatives effect on cyp3a4 induction excellent autoinduction yes half life 2-5 bound protein (%) 85 table 1. pharmacokinetic profile of rifampicin(19) * auc = area under the time-concentration curve ** cmax = maximal drug concentration yenny rifampicin and efavirenz in hib-tb 195 univ med vol.28 no.3 all nnrti drugs are metabolized by cyp450, but by different isozymes. nevirapin is the substrate of cyp3a4 whilst efavirenz is mainly metabolized by cyp2b6, such that its concentration is less affected by administration of other drugs modifying the activity of the c y p 3 a 4 e n z y m e s , e . g . r i f a m p i c i n , w h e n compared with other members of the nnrti group.(21) studies on the interactions of rifampicin and efavirenz t h e o p t i m a l d o s e o f e f a v i r e n z w h e n administered concomitantly with rifampicin is s t i l l t h e s u b j e c t o f c o n t r o v e r s y. s e v e r a l pharmacokinetic studies that have been conducted will be discussed here. the pharmacokinetic study by lopez-cortez et al.(23) in spain on 24 patients w i t h c o n c o m i t a n t i n f e c t i o n s o f h i v a n d tuberculosis was of non-blinded randomized design. group a (n=16) received antituberculosis drugs without rifampicin (comprising isoniazid, pyrazinamide, and streptomycin), but with added antiretroviral therapy containing efavirenz 600 mg/day, on days 1–7. the patients were then switched to a standard antituberculosis regimen c o n t a i n i n g r i f a m p i c i n ( p l u s i s o n i a z i d a n d pyrazinamide) with an antiretroviral regimen containing efavirenz 600 mg/day (group a1; n=8) or efavirenz 800 mg/day (group a2; n=8). the second treatment group, i.e. group b (n=8), r e c e i v e d s t a n d a r d t b t h e r a p y c o n t a i n i n g rifampicin on days 1-7, with the addition of an antiretroviral regimen containing efavirenz 800 mg/day on day 8. the plasma concentration of efavirenz was determined on days 7 and 14. the levels of auc, c max , and c min of efavirenz decreased respectively 24%, 25%, and 22%, after addition of rifampicin to the efavirenz of 600 mg/day (treatment a1 vs a). the exposure to efavirenz in group a2 (efavirenz 800 mg/day with rifampicin) and group a (efavirenz 600 mg/ day without rifampicin) yielded similar results. the investigators stated that there was a positive association between body weight, dose of efavirenz (dose/kg), and the pharmacokinetic parameters of efavirenz. however, when the data were analyzed by body weight (<50 kg vs >50 kg), it was apparent that the concentration of efavirenz with 800 mg daily dose administered concomitantly with rifampicin was identical to that seen with the dose of 600 mg/day without rifampicin, independent of body weight. because of the uncertainty regarding the minimum plasma c o n c e n t r a t i o n o f e f a v i r e n z r e q u i r e d f o r v i r o l o g i c a l s u p p r e s s i o n , t h e i n v e s t i g a t o r s suggested increasing the dose of efavirenz from 600 mg/day to 800 mg/day as a single dose, irrespective of body weight of the patients on concomitant rifampicin. efavirenz bioavailability (%) unknown effect of food taken on empty stomach. admistration with food increases auc by 20% & cmax by 40-50%. main metabolic pathway cyp2b6, cyp3a4 (minimal) effect on cyp3a4 induction induction autoinduction yes (no need for dose adjustment) half life 40-55 bound protein (%) >99 table 2. pharmacokinetic profile of efavirenz(21,23) 196 manosuthi et al.(24,25) conducted an nonblinded randomized clinical trial for respectively 24 and 48 weeks on 84 patients with hiv-tb coinfection in thailand. patients receiving r i f a m p i c i n f o r m o r e t h a n 1 m o n t h w e r e randomized into 2 groups of identical size, to w h o m w a s g i v e n a t h e r a p e u t i c r e g i m e n containing efavirenz 600 mg/day (n=42), or efavirenz 800 mg/day (n=42). the plasma concentrations of efavirenz were determined respectively 12 hours after drug administration and on day 14. the numbers of cd4+ cells and the levels of hiv rna were also assessed on weeks 16, 24, and 48. the study results indicated similar means of body weight in the two groups (50.6 kg vs 52.9). both groups had 12-hr e f a v i r e n z c o n c e n t r a t i o n s ( c 1 2 ) o f s i m i l a r magnitude (median 3.02 mg/l vs 3.39 mg/l); but the upper c 12 levels were higher in the group receiving efavirenz at the dosage of 800 mg/day (21.3 mg/l) in comparison with the group on 600 mg/day (12.2 mg/l). in general, auc and c min were important predictor parameters used for finding a correlation between antiretroviral exposure and virological response.(24) other data indicate that the 12-hr efavirenz concentration was also useful for predicting virological failure, similar to the central nervous system side effects o c c u r r i n g a s a r e s u l t o f e f a v i r e n z administration.(26) follow up studies were done up to 48 weeks in order to assess the virological and imunological responses. on the 48th week, 31 of 34 patients (91.2%) in the group on 600 mg/day, and 27 of 31 patients (87.1%) in the group on 800 mg/day attained hiv rna levels of <50 copies/ml. in addition, there were no differences in cd4+ levels between the two groups on the 24th and 48th weeks. the conclusion of this study was that efavirenz with a dose of 600 mg/day is adequate for the majority of hivinfected patients in thailand (with a body weight o f a r o u n d 5 0 k g ) r e c e i v i n g r i f a m p i c i n concomitantly. the investigators also state that the recommended dosages should not be applied to other ethnic groups, or to populations with a higher body weight (>50 kg). the study conducted by brennan-benson et al.(28) was aimed at determining the effects of higher body weights and differing ethnicities on exposure to efavirenz. this observational study involved 9 patients with body weights of over 50 kg (mean 66.4 kg; range 52-107 kg). the patients received an antiretroviral regimen c o n t a i n i n g e f a v i r e n z 8 0 0 m g / d a y a n d antituberculosis therapy containing rifampicin. during a time period of 2 years the clinical toxicity and plasma concentrations of efavirenz were monitored. seven out of the 9 patients with h i v / t b c o i n f e c t i o n e x p e r i e n c e d c l i n i c a l toxicity, namely central nervous system toxicity in 6 persons, and hepatitis in 1 person. the induced toxicity necessitated a reduction of the doses or cessation of therapy. all patients experiencing toxicity had supratherapeutic trough concentrations of efavirenz (median 11.7 m g / l ; r a n g e 5 . 3 7 1 9 . 6 m g / l ) . t h e t a rg e t therapeutic range of trough concentrations of efavirenz were between 1.2 and 4.0 mg/l. among the 7 patients experiencing toxicity, 6 of them were ethnic black africans, and one was a caucasian. the investigators were of the opinion that cyp2b6 polymorphism contributes heavily to the high rate of central nervous system toxicity. around 20% of ethnic black africans possess homologous g516t alleles versus 3% of caucasians.(28) this cyp2b6 polymorphism i s a s s o c i a t e d w i t h e x p o s u r e t o h i g h e r concentrations of efavirenz and with central nervous system toxicity due to slower clearance of efavirenz.(28) friedland et al.(6) revealed the existence of a wide variability in efavirenz concentrations among 19 ethnic black africans with hiv/tb coinfection (mean body weight 59.4 kg; range 45-97 kg) who received efavirenz 600 mg/day concomitantly with rifampicin. the investigators yenny rifampicin and efavirenz in hib-tb 197 univ med vol.28 no.3 monitored the minimal plasma concentrations of efavirenz (c min ) intermittently during a 6-month c o m b i n a t i o n t h e r a p e u t i c r e g i m e n f o r t b containing rifampicin and antiretroviral drugs, and 15 months after cessation of tb therapy. the s t u d y r e s u l t s i n d i c a t e d t h a t i n t e rs u b j e c t variability in efavirenz concentrations was greater if the drug was administered with rifampicin (coefficient of variation [cv] 157%) compared with the inter-subject variability after discontinuation of rifampicin (58%), with relatively consistent intra-subject variation over t i m e ( c v 2 4 % ) . c m i n , h o w e v e r, w a s n o t significantly different during versus post tb t h e r a p y. e f a v i r e n z c o n c e n t r a t i o n w a s n o t correlated with body weight, irrespective of concomitant rifampicin administration. of the 17 patients who completed the hiv/tb therapy, 16 had an hiv rna level of <50 copies/ml. seven persons (35%) reportedly had dizziness and poor concentration during the initial weeks of efavirenz therapy, but no association was found between plasma efavirenz concentrations a n d n e u r o p s y c h i a t r i c s y m p t o m s . t h e investigators were of the opinion that the optimal dosage of efavirenz should be mainly determined o n t h e b a s i s o f s t u d i e s a s s e s s i n g c l i n i c a l outcomes and supported by pharmacokinetic data for informing their design. notwithstanding the variability in plasma efavirenz concentrations during rifampicin administration, excellent hiv clinical treatment outcomes were obtained. the authors support the use of a routine dose of e f a v i r e n z 6 0 0 m g / d a y o n c o n c o m i t a n t administration of rifampicin for african patients with hiv/tb coinfection. a summary of studies assessing the interactions between efavirenz and rifampicin in patients with hiv/tb may be seen in table 3. the determination of the optimal dosage is generally based on pharmacokinetic studies. however, assessing the optimal dosage of efavirenz for use in combination with rifampicin is not a simple question. this is due to the wide variation in the following aspects of the studies: the study design used (randomized (24-26) vs observational);(6,27) the duration of follow-up; the time of administration of efavirenz. (in some studies the drug was given with meals, whereas in others it was given to fasting patients). the food factor contributes to the difficulties in determining the optimal dose of efavirenz, and is connected with the occurrence of side effects. administration of efavirenz with meals increased the auc and c max of the drug by 20% and 4050%, respectively.(21) the studies did not provide important information in connection with the collection of pharmacokinetic data, such as time of sample collection or the methods used for a s s e s s i n g t h e r e l e v a n t p h a r m a c o k i n e t i c parameters. most studies used an inadequate p h a r m a c o k i n e t i c s a m p l e s i z e b e c a u s e t h e investigators believed that the study subjects would not consent to repeated blood sampling for an extended period of time. in studies providing pharmacokinetic parameters, the available pharmacokinetic data for assessing exposure to efavirenz were extremely variable (c 12h , c min , auc). the relationship between c 12h and c min is still unclear. in addition, assessment of the efficacy and safety of efavirenz based on c 12h concentration has still to be validated. the majority of patients in the study had a body weight of <50 kg; the data showed that e f a v i r e n z 6 0 0 m g / d a y w a s a d e q u a t e f o r maintaining virological and immunological control up to the 48th week on concomitant a d m i n i n s t r a t i o n o f r i f a m p i c i n . h o w e v e r, considerable amounts of data are still required on patients with body weights of >50 kg, because some studies have demonstrated toxicity of efavirenz necessitating reduction of dosages or cessation of treatment in patients with higher body weights.(24,25,27) 198 t ab le 3 . s u m m ar y o f st u d ie s o n c o n co m it an t ef av ir en z an d r if am p ic in a d m in is tr at io n i n p at ie n ts w it h h iv a n d t b c o in fe ct io n e f v = e fa vi re nz ; r f p = r if am pi ci n; c n s = c en tr al n er vo us s ys te m @ s in gl e ce nt er ; ex ce pt s tu dy b y m an os ut hi e t al . ( t w oce nt er s tu dy ) s tu d y d es ig n @ n u m b er of su b je ct s b od y w ei gh t b ad an ( k g) d os e of e fa vi re nz (s in gl e d os e) p h ar m ac ok in et ic s & v ir ol og y s id e ef fe ct s l op ez -c or te z et a l. (2 4) (s pa in ) r an do m iz ed , un bl in de d, c on tr ol le d, pr os pe ct iv e; f ol lo w up 2 w ee ks 24 m ed ia n 54 ; r an ge 38 -8 0 60 0 m g (n = 8 ) 80 0 m g (n = 1 6) a dd it io n o f r f p t o e f v 6 00 m g ca us ed c m in e f v 25 % , a u c 22 % . e xp os ur e to e f v 8 00 m g + r f p i de nt ic al to e f p 60 0 m g on ly , i rr es pe ct iv e of b od y w ei gh t (< 50 k g vs ≥ 5 0 kg ) 60 0 m g: s s p ( 4) , r as h (1 ), li ve r fu nc ti on s (1 ). 80 0 m g: s s p ( 1) , li ve r fu nc ti on s (2 ) m an os ut hi e t a l. (2 5, 26 ) (t ha il an d) r an do m iz ed , un bl in de d, co nt ro ll ed ; fo ll ow u p 48 w ee ks . 84 m ea n 50 .6 ( 60 0 m g gr ou p) m ea n 52 .9 ( 80 0 m g gr ou p) 60 0 m g (n = 4 2) 80 0 m g (n = 4 2) m ed ia n e f v c 12 h id en ti ca l in b ot h gr ou ps , bu t up pe r ra ng e of c 1 2h h ig h er in 8 00 m g (2 1, 3 m g/ l ) gr ou p vs 6 00 m g (1 2, 2 m g/ l ) gr ou p. v ir ol og ic al a nd i m m un ol og ic al o ut co m es at w ee k 48 id en ti ca l i n bo th g ro up s. 60 0 m g: r as h (1 ), he ad ac he ( 1) 80 0 m g: r as h (1 ), he pa ti ti s (1 ) b re nn an -b en so n et a l.( 28 ) (e ng la n d) o be rv at io na l, un bl in de d, pr os pe ct iv e; f ol lo w up 2 y ea rs 9 m ea n 66 .4 ; r an ge 52 -1 07 80 0 m g m ed ia n c m in 1 1. 7 m g/ l ( ra ng e 5. 419 .6 m g/ l ) in 7 p at ie n ts ( th er ap eu ti c ra ng e 1. 24 m g/ l ) s ev en o ut o f 9 pa ti en ts h ad e fa vi re n z to xi ci ty n ec es si ta ti ng do se r ed uc ti on o r ce ss at io n o f th er ap y. c n s ( 6) , h ep at it is ( 1) f ri ed la nd e t a l. (6 ) (s ou th a fr ik a) o bs er va ti on al , un bl in de d, pr os pe ct iv e; f ol lo w up 2 1 m on th s 20 m ea n 59 .4 ; r an ge 45 -9 7 60 0 m g m ea n c m in e f v i de nt ic al , w it h or w it h ou t co n co m it an t r f p ; in te rs ub je ct va ri ab il it y of e f v o n co n co m it an t r f p . s ix te en o ut o f 17 p at ie nt s co m pl et in g tr ea tm en t ha d h iv r n a < 50 c op ie s/ m l in m on th 2 1 c n s ( 7) yenny rifampicin and efavirenz in hib-tb 199 univ med vol.28 no.3 ethnicity and pharmacogenetics provide i m p o r t a n t c o n t r i b u t i o n s a n d s h o u l d b e c o n s i d e r e d i n c o n n e c t i o n w i t h t h e w i d e variability among the patients to efavirenz exposure. the majority of patients in the study were asians or ethnic black africans, whilst caucasians were underrepresented. ethnicity plays an important role because ethnic black africans are frequently of the homologous cyp2b6 516-t/t genotype in comparison to other ethnic groups.(28) this fact is supported by the study conducted by friedland et al.(6) where the study results indicated that black african patients suffered more frequently from central nervous system side effects although receiving a dose of 600 mg with rifampicin. the significant toxicity of efavirenz leading to a dose reduction was also reported in predominantly ethnic black africans receiving 800 mg efavirenz with rifampicin.(27) a study performed in japan on patients with hiv infection had the goal of determining the possibility of a dose reduction of efavirenz on the basis of genotype. among 456 patients of whom the genotype was determined, 16 were found to be carriers of 516-t/t. in all 16 patients a concentration of efavirenz of >6000 ng/ml was found when given a standard dose of 600 mg/ day. for 12 patients a dose reduction was effected to 400 mg/day, and among 11 subjects the dose was reduced further to 200 mg/day in 7 subjects. among 4 persons receiving an initial dose of 400 mg, 2 of them had persistently high efavirenz concentrations, thus necessitating a dose reduction to 200 mg/day. the ascertained dosages were continued for >6 months, in which period hiv levels were still undetectable. these study results demonstrate the successful outcome of a dose reduction of efavirenz to 400 mg/day or 200 mg/day in japanese 516-t/t carriers with hiv (without tb infection). dose reduction according to genotype resulted in reduction of central nervous system side effects and was capable of maintaining virological control. these findings indicate that an increase in dosage of up to 800 mg/day on concomitant administration of rifampicin may not be necessary in patients with the homologous allele variant 516t/t.(29) o t h e r s t u d i e s d e m o n s t r a t e d i n t e r s u b j e c t variability in pharmacokinetics of efavirenz due to another cyp2b6 polymorphism, namely c1459t.(30) clinical application to date several guidelines have been prepared by clinicians on the use of efavirenz with rifampicin. the recommendation issued by the british hiv association (bhiva)(31) in the therapeutic guidelines for hiv/tb in 2009 currently favors a dose of efavirenz of 600 mg/ day for patients with a body weight of < 50 kg, and recommends consideration of increasing the dosage to 800 mg/day for patients with a body w e i g h t o f > 5 0 k g ( 3 1 ) o r > 6 0 k g . ( 9 ) c i p t o m a n g u n k u s u m o h o s p i t a l u s e s a d o s e o f efavirenz 600 mg for patients with hiv/tb, irrespective of body weight, in accordance with the guidelines issued by the world health organization (who)(4) which only recommend a dose of 600 mg, without regard to body weight. the food and drug administration(32) states that the available data are inadequate for supporting a recommendation of the dosages of efavirenz when used concomitantly with rifampicin. conclusions c o n c o m i t a n t u s e o f r i f a m p i c i n w i t h efavirenz leads to unavoidable drug interactions. interactions between the two drugs result in a w i d e v a r i a t i o n i n t h e p l a s m a e f a v i r e n z concentrations, thus being capable of affecting therapeutic outcomes or the occurrence of toxic effects. this leads to problems in determining the optimal dosage of efavirenz in patients with hiv/tb coinfection. before assessing the 200 optimal dosage, it is necessary to study the p a t t e r n s o f i n t e r a c t i o n o f t h e t w o d r u g s . pharmacokinetic studies with good measurement parameters and methods are still needed as the basis for determining the optimal dosage of efavirenz in the indonesian population. references 1. manuhutu ej. peran riset dalam bidang tuberkulosis paru untuk menunjang pembangunan nasional. pidato pengukuhan guru besar fk ui, april 2006. 2. clain kp, anekthananon t, burapat c, akkslip s, mankhatitham w, srinak c, et al. causes of death in hiv-infected persons who have tuberculosis. thailand emerg infect dis 2009;15:258-64. 3. jindani a, nunn aj, enarson da. two 8-month regimens of chemotherapy for treatment of newly diagnosed pulmonary tuberculosis: international multicenter randomized trial. lancet 2004;364: 1244-51. 4. world health organization. antiretroviral therapy for hiv infection in adults and adolescents: recommendations for a public health approach. geneva, switzerland: world health organization; 2006 revision. available at: http://www.who.int/hiv/ pub/guidelines/artadultsguidelines.pdf. accessed may 19, 2009. 5. center for disease control and prevention. managing drug interactions in the treatment of hivrelated tuberculosis [online].2007. available at: http://www.cdc.gov/tb/tb_hiv_drugs/default. htm. accessed february 18, 2009. 6. friedland g, khoo s, jack c, lallo u. administration of efavirenz (600 mg/day) with rifampicin results in highly variable level but excellent clinical outcomes in patients treated for tuberculosis and hiv. antimicrob agents chemother 2006;58:1299-302. 7. idemyor v. hiv and tuberculosis coinfection: inextricably linked liaison. j natt med assoc 2007; 99:1414-9. 8. burzynski jn, schluger nw, garay sm. tuberculosis and the human immunodeficiency virus infection. in: rom wn, garay sm, editors. tuberculosis. 2nd ed. philadelphia: lippincott williams & wilkin;2004.p.663-8. 9. dhhs panel of antiretroviral guidelines for adults and adolescent. guidelines for the use of antiretroviral agents in hiv-1 infected adults and adolescents. department of health and human services. available at: http://www.aidsinfo.nih. gov/ contentfiles/adultsandadolescentgl.pdf. accessed june 25, 2009. 10. drug interaction. in: grahame-smith dg, aronson jk, editors. oxford textbook of clinical pharmacology and drug therapy. 3rd ed. new york: oxford university press;2002.p.104-17. 11. piscitelli sc, gallicano kd. interaction among drugs for hiv and opportunistic infections. n engl j med 2001;344:984-96. 12. gonzalez fj, tukey rh. drug metabolism. in: brunton ll, lazo js, parker kl, editors. goodman & gilman’s. the pharmacological basis of therapeutics. 11th ed. new york: mcgraw-hill; 2006.p.71-91. 13. handschin c, meyer ua. induction of drug metabolism: the role of nuclear receptors. pharmacol rev 2003;55:649-73. 14. chen j, raymond k. roles of rifampicin in drugdrug interactions: underlying molecular mechanisms involving the nuclear pregnane x receptor. ann clin microbiol antimicrob 2006;5:111. 15. wilson tm, kliewer sa. pxr, car, and drug metabolism. nat rev drug discov 2002;1:259-66. 16. kliewer sa, goodwin b, willson tm. the nuclear receptor pregnane x receptor: a key regulator of xenobiotic metabolism. endocr rev 2002:23:687702. 17. chen j, raymond k. roles of rifampicin in drugdrug interactions: underlying molecular mechanisms involving the nuclear pregnane x receptor. ann clin microbiol antimicrob 2006;5:111. 18. mcllleron h, meintjes g, burman wj, maartens g. complications of antiretroviral therapy in patients with tuberculosis: drug interactions, toxicity, and immune reconstitution inflammatory syndrome. j infect dis 2007;196:s63-s75. 19. petri wa. chemotherapy of tuberculosis, mycobacterium avium complex disease, and leprosy. in: brunton ll, lazo js, parker kl, editors. goodman & gilman’s. the pharmacological basis of therapeutics. 11th ed. new york: mcgraw-hill;2006.p.1207-10. 20. vernon aa. rifamycin antibiotics, with a focus on newer agents. in: rom wn, garay sm, editors. tuberculosis. 2nd ed. philadelphia: lippincott williams & wilkin;2004.p.760-70. yenny rifampicin and efavirenz in hib-tb 201 univ med vol.28 no.3 21. flexner c. antiretroviral agents and treatment of hiv infection. in: brunton ll, lazo js, parker kl, editors. goodman & gilman’s the pharmacological basis of therapeutics, 11th ed. united state of america: mcgraw-hill;2006.p.1292-6. 22. ward ba, gorski jc, jones dr, hall sd, flockhart da, desta z. the cytochrome p450 2b6 (cyp2b6) is the main catalyst of efavirenz primary and secondary metabolism: implication for hiv/aids therapy and utility of efavirenz as a substrate marker of cyp2b6 catalytic activity. pharmacol exp ther 2003;306:287-300. 23. lopez-cortez lf, ruiz-valderas r, viciana p, alarcon-gonzalez a, gomez-mateos j, leonjimenez e, et al. pharmacokinetic interactions between efavirenz and rifampicin in hiv-infected patients with tuberculosis. clin pharmacokinet 2002;41:681-90. 24. manosuthi w, sungkanuparph s, thakkinstian a, vibhagool a, kiertiburanakul s, rattanasiri s, et al. efavirenz levels and 24-weeks efficacy in hivinfected patients with tuberculosis receiving highly active antiretroviral therapy and rifampicin. aids 2005;19:1481-6. 25. manosuthi w, kiertiburanakul s, sungkanuparph s, ruxrungtham k, vibhagool a, rattanasiri s, et al. efavirenz 600 mg/day versus efavirenz 800 mg/ day in hiv-infected patients with tuberculosis receiving rifampicin: 48 weeks results. aids 2006; 20:131-2. 26. marzolini c, talenti a, decostered la, greub g, biollaz j, buclin t. efavirenz plasma level can predict treatment failure and central nervous system side effect in hiv-1 infected patients. aids 2001; 15:71-5. 27. brennan-bonson p, lyus r, harrison t, pakianathan m, macallan d. pharmacokinetic interactions between efavirenz and rifampicin in the treatment of hiv and tuberculosis: one size does not fit all. aids 2005;19:1541-3. 28. haas dw, ribaudo hj, kim rb, tierney c, wilkinson gr, gulick rm, et al. pharmacogenetics of efavirenz and central nervous system side effects: an adults aids clinical trial group study. aids 2004;18:2391-400. 29. gatanaga h, hayashida t, tsuchiya k. successful efavirenz dose reduction in hiv type-1-infected individuals with cytochrome p450 2b6*6 and *26. clin infect dis 2007;45:1230-7. 30. burger d, van der heiden i, la porte c, van der ende m, groeneveld p, richter pk, et al. interpatient variability in the pharmacokinetics of the hiv non-nucleoside reverse transcriptase inhibitor efavirenz: the effect of gender, race, and cyp2b6 polymorphism. br j clin pharmacol 2005; 61:148-54. 31. pozniak al, collins s, coyne km, freedman ar, johnson ma, lipman mci, et al. british hiv association guidelines for the treatment of tb/hiv co-infection (2009). available at: http://www.bhiva. org/cms1223707.asp. accessed september 11, 2009. 32. digiacinto jl, chan-tack km, robertson sm, reynolds ks, struble ka. are literature references sufficient for dose recomendations? an fda case study of efavirenz and rifampin. j clin pharmacol 2008;48:519-23. age decreases quality of life in adolescents with intractable epilepsy 104 *division of neurology, department of child health, medical school, airlangga university, dr. soetomo hospital, surabaya correspondence: dr prastiya indra gunawan spa division of pediatric neurology, department of child health, faculty of medicine, airlangga university hp: 08113429476 email: prastiya_ig@yahoo.co.id univ med 2015;34:104-11 doi: 10.18051/univmed.2016.v35.104-111 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background intractable epilepsy considerably affects both the private and social life of the patient. the objective of this study was to determine the quality of life of intractable epileptic adolescents and its correlated factors. methods a cross sectional study was conducted in the pediatric neurology outpatient clinic of dr. soetomo hospital. all intractable epileptic adolescents aged between 10 to 16 years were asked to complete a questionnaire on quality of life in epilepsy for adolescents (qolie-ad-48). a multiple linear regression was used to analyze the data through spss v17.0. results thirty one patients with mean age of 12.41 ± 1.40 years were enrolled in the study. the mean duration of diagnosed intractable epilepsy was 6.12 ± 4.30 years. mean score for impact of epilepsy was 57.11 ± 24.50, for memory and concentration 53.54 ± 26.66, physical functioning 65.56 ± 23.67, social stigma 52.23 ± 17.48, social support 52.64 ± 22.69, behavior at school 57.51 ± 26.50, attitude 53.40 ± 16.70 and health perception 61.51 ± 11.30. multiple linear regression results showed that quality of life (qol) was not significantly correlated with duration of epilepsy, sex and nutritional status (p>0.05), but increasing age was significantly decreases quality of life (p<0.05). conclusions intractable epileptic adolescents have higher scores for physical functioning and health perception, but lower scores for social stigmatization. social support has extremely low scores. increasing age decreases quality of life in adolescents with intractable epilepsy. keywords: intractable epileptic adolescents, quality of life age decreases quality of life in adolescents with intractable epilepsy prastiya indra gunawan*, theresa laura limanto*, and darto saharso* doi: http://dx.doi.org/10.18051/univmed.2015.v34.104-111 105 univ med vol. 34 no.2 semakin tinggi usia remaja menurunkan kualitas hidup pada epilepsi intraktabel latar belakang epilepsi intraktabel sangat mempengaruhi kehidupan pasien, baik kehidupan pribadi maupun sosialnya. tujuan penelitian ini adalah untuk menentukan adanya hubungan antara faktor-faktor dan kualitas hidup pasien epilepsi intraktabel remaja. metode sebuah penelitian potong lintang dilakukan di poli neurologi anak rs dr. soetomo. semua penderita epilepsi intraktabel remaja berusia 10–16 tahun diminta untuk mengisi kuesioner kualitas hidup epilepsi remaja (quality of life in epilepsy for adolescents/qolie-ad-48). uji regresi linear ganda diguanakan untuk analisis data dengan piranti lunak spss versi 17.0. hasil tiga puluh satu pasien dengan rata-rata usia 12,41 ± 1,40 tahun diikut sertakan pada penelitian. lama terdiagnosis mengalami epilepsi intraktabel rata-rata 6,12 ± 4,30 tahun. nilai rata-rata dampak epilepsi 57,11 ± 24,50; ingatan dan konsentrasi 53,54 ± 26,66; kemampuan fisik 65,56 ± 23,67; stigma masyarakat 52,23 ± 17,48; dukungan sosial 52,64 ± 22,69; perilaku di sekolah 57,51 ± 26,50; tingkah laku 53,40 ± 16,70 dan persepsi kesehatan 61,51 ± 11,30. analisis regresi linear ganda menunjukkan kualitas hidup tidak berhubungan secara bermakna dengan dengan lama menderita epilepsi, jenis kelamin dan status gizi (p>0,05), tetapi semakin tinggi usia menurunkan kualitas hidup secara bermakna pada remaja (p<0,05). kesimpulan penderita epilepsi intraktabel remaja memiliki nilai yang lebih tinggi pada kemampuan fisik dan persepsi akan kesehatan namun lebih rendah pada stigma masyarakat. dukungan sosial memiliki nilai yang sangat rendah. semakin tinggi usia menurunkan kualitas hidup pada remaja dengan epilepsi intraktabel. kata kunci: epilepsi intraktabel remaja, kualitas hidup abstrak introduction intractable epilepsy affects the life of the patient and society as a whole in a profound way. the patients suffer in terms of schooling and employment and are stigmatized. in addition, they frequently suffer from psychiatric complications, especially depression and anxiety. mortality is increased secondary to seizure related accidents, higher suicide rates, and sudden unexpected death in epilepsy. also, there is mounting evidence that uncontrolled seizures can lead to deterioration in cognition and developmental function.(1) the goal of management and care of adolescents with epilepsy should enable them and their families to lead a life as free as possible from adverse medical and psychosocial complexities of epilepsy.(2) adolescents have different activities, levels of responsibility, different interests and areas of concern from those of younger adolescents and adults. pediatric epilepsy is a very complex neurological condition primarily characterized by the unexpected, episodic, and chronic nature of a variety of seizures, but also by different developmental, psychological, behavioral, 106 educational, and social difficulties. as such, pediatric epilepsy has pervasive impacts on all aspects of a child’s life. health-related quality of life (hrqol) is an important outcome in studies of pediatric patients with chronic physical illnesses such as asthma, diabetes or epilepsy.(3) several quality of life instruments for children with epilepsy are the questionnaire on quality of life for children with epilepsy (qolce); brazillian quality of life questionnaire for children with epilepsy (quce); health related quality of life for childhood epilepsy (cheqch-25) and quality of life in epilepsy for adolescents (qoliead-48).(4) the quality of life in epilepsy for adolescents (qolie-ad-48) questionnaire is a health-related quality of life (hrqol) instrument that has been developed to assess psychometric properties of specific areas for adolescents with epilepsy.(5) a study showed that duration of treatment was correlated with quality of life of adolescents with epilepsy.(6) we aimed to determine the hrqol in adolescents with intractable epilepsy and its correlated factors. methods research design this was a cross sectional study held in march 2014 in the pediatric neurology outpatient clinic of dr.soetomo hospital, surabaya. research subjects we included adolescents with intractable epilepsy (partial or generalized), aged 10 to 16 years, who had their typical seizure recorded by electroencephalography (eeg). in this study, intractable epilepsy was defined according to the international league against epilepsy (ilae) as seizures which occur despite the adequate administration of two tolerated, appropriately chosen and adequately used anti-epileptic drugs (aeds).(4) subjects were excluded if they had a history of brain surgery, another psychiatric or progressive neurologic illness, and motoric deficits (spastic diplegia, spastic quadriplegia, hemiplegia, dyskinesia, ataxia and hypotonia). a total of 31 subjects were included in this study. questionnaires all eligible subjects and their parents who agreed to participate in the study were interviewed to obtain demographic and personal information. age was calculated from the birth date. gender (sex) was assessed by physical examination. nutritional status was based on the center for disease control 2000 chart. duration of epilepsy was determined from chronological age minus age at first diagnosis of epilepsy. quality of life in epilepsy for adolescents the subjects were asked to complete a selfassessment of qolie-ad-48 with the assistance of an english interpreter. the qolie-ad-48 questionnaire contained 48 items in 8 subscales: epilepsy impact (12 items), memory-concentration (10 items), attitudes toward epilepsy (4 items), physical functioning (5 items), stigma (6 items), social support (4 items), school behavior (4 items), health perceptions (3 items), and total summary score. total summary score was calculated and transformed linearly into scales of 1-100 points, using the scoring manual for qolie-ad-48. a higher value represents better functioning and wellbeing. score 0 indicates very poor and score 100 indicates highest hrqol.(3) the health related quality of life scores are presented as mean and sd. data analysis descriptive statistics (means, sds, ranges, minimum and maximum scores) were calculated using spss version 17.0 for each scale and for the summary score. multiple linear regression test was used to analyze correlation between hrqol and the variables duration of epilepsy, age, sex and nutritional status. ethical clearance the ethical clearance certificate had been issued by the committee of human health research and ethics of dr. soetomo hospital. gunawan, limanto, saharso quality of life in intractable epilepsy 107 univ med vol. 34 no.2 results thirty one patients with mean age of 12.41 ± 1.40 years were enrolled, among whom twenty four (77.5%) had moderate malnutriton. the mean duration of intractable epilepsy was 6.12 ± 4.30 years. thirty subjects (96.8%) used two antiepileptic drugs (aeds) and one (3.2%) used three aeds. among the adolescents who used two aeds, valproic acid and phenytoin were prescribed for twenty two (70.9%) subjects. the only subject (3.2%) using three aeds was prescribed valproic acid, carbamazepine and topiramate. twenty four (77.4%) adolescents attended public schools, and nine (37.5%) of them had to repeat their school grades twice or more (table 1). among the subscales of qolie-ad 48, the physical functioning scales of daily activities (such as walking to school, running, bicycle riding, carrying bags or taking a bath alone) was found to have a mean score of 65.48 ± 23.68. stigmatization scales (considering themselves to be less happy, mentally unstable, rejected by opposite sex) had a mean score of 52.17 ± 17.49. one subject to whom was prescribed three aeds had scores below the mean for all subscales, which consisted of epilepsy impact score 4.5, memory and concentration 7.5, physical functioning 25, stigma 33.3, social support 100, school behavior 12.5, attitude toward epilepsy 25 and health perception score 33.3. overall mean score of 8 aeds : antiepileptic drugs table 1. distribution of characteristics of patients (n=31) figure 1. qolie-ad 48 scores for each subscale in adolescents with intractable epilepsy 108 subscales from 31 subjects was 51.86 ± 19.32. figure 1 presents the scores of each subscale in adolescents with intractable epilepsy. there was no correlation between hrqol and duration of epilepsy, sex and nutritional status variables. there was a significant correlation between hrqol and age of intractable epileptic adolescents. the subjects’ quality of life decreased with increasing age (â= -3.81; p=0.03) (table 2). discussion a full assessment of hrqol issues in adolescents is complex because of the wide range of maturity within this age group, differences in independence and experience, and potential volatility of emotions. to consider adolescents as adults is problematic because of these developmental issues.(5) our study recruited 31 adolescents with intractable epilepsy according to the inclusion criteria, aged 10-16 years, with equal sex distribution. the mean duration of epilepsy was 6.12 ± 4.30 years. overall mean score of 8 subscales from 31 subjects was 51.86 ± 19.32. sabaz et al.(8) used the child behaviour checklist (cbcl) on adolescents with refractory epilepsy and found that intellectually normal adolescents with refractory epilepsy were more likely to have emotional, behavioral, and cognitive problems and to be less competent in socializing and school performance. the total problem score was 48.82 ± 11.12 in adolescents with refractory epilepsy without seizures during the past 4 weeks and 54.01 ± 11.17 in adolescents with refractory epilepsy with seizure fequency of more than 10 seizures per month. the correlation between seizure frequency and total problem score was variable s β p duration of epilepsy sex age nutritiona l status -0.06 -1.33 -3.81 0.02 0.75 0.47 0.03 0.99 table 2. multiple linear regression of several variables with total mean score of hrqol β = regression coeficient significant (p<0.01).(8) sabaz et al.(8) mentioned as the limitations of previous studies using cbcl the diagnostic criteria in the classification of epilepsy syndrome, the evaluation of epilepsy severity, and the fact that they did not accurately established the cognitive normality of the subjects. cramer et al.(5) used the same qolie-ad 48 instrument as in our study, and found a higher overall mean score. in epileptic adolescents without seizure, the overall mean score was 77.3 (sd 12.6) and in epileptic adolescents with high severity seizure, the overall mean score was 62.7 (sd 16.5). the different results were due to the dissimilarity of the inclusion criteria. cramer et al.(5) included all adolescents with epilepsy, including those who were not intractable and used aed monotherapy. furthermore, the types of epilepsy were not mentioned in their inclusion criteria for the study. monir et al.(9) stated that a lower mean score of all domains of quality of life was correlated with generalized epilepsy, frequent episodes of seizure, use of aed polytherapy, younger age of seizure onset, and longer duration of illness. in our study, valproic acid and phenytoin were prescribed for most of the subjects, while phenytoin and carbamazepine were used in eight subjects. modi et al.(10) described that adolescents to whom carbamazepine monotherapy was prescribed had declining emotional functioning in seven months of follow up, compared with those to whom valproic acid was prescribed, who demonstrated steady or improving emotional functioning regardless of initial side-effect severity. eddy et al.(11) stated that phenytoin has been implicated in decline in consentration, memory, visuomotor functions and mental speed. however, the decline in attention and motor performance were said to be improved after withdrawal of therapy. a randomized, doubleblinded, placebo-controlled study involving 150 epilepsy subjects on aed monotherapy (mainly carbamazepine or valproic acid) found that drug discontinuation significantly improved performance requiring complex cognitive processing.(12) these previous studies were not gunawan, limanto, saharso quality of life in intractable epilepsy 109 univ med vol. 34 no.2 designed to evaluate overall hrqol, but only the emotional, attention and cognitive aspects, related to type and side effects of the aeds prescribed to the subjects. the type of epilepsy and the frequency of seizures were not mentioned. nevertheless, a routine assessment of aed side effects whenever used as a polytherapy and its implication on hrqol may be useful for further longitudinal, prospective studies in adolescents with intractable epilepsy. stigmatization scales (considering themselves to be less happy, mentally unstable, rejected by opposite sex) had a mean score of 52.17 ±17.49. social support scales had a mean score of 52.62 ± 22.70. aydemir et al.(13) conducted a study on 70 subjects with epilepsy and noted that the impact of stigma in epileptic subjects was greater and extended beyond the neurological condition itself, and lowered the overall hrqol score. siqueira et al.(14) stated that a high prevalence of social support and knowledge about epilepsy may influence the way adolescents relate to the disease and thus provide a better adaptation to and acceptance of the limits imposed by epilepsy. both values will promote a valuable way to adaptation, acceptance, increased self-esteem, overcoming of stigma, resulting in a higher hrqol. in our study, stigma and social support were two of the lowest scales found. parents and close family were the only main source of social support for our subjects. limited parental education and family income may contribute to poor overall hrqol scales. level of knowledge about epilepsy and its impact were not assessed in this study. better social support, apart from that of the family, is needed in order to obtain higher self esteem and hrqol scores. in our study there was no correlation between duration of epilepsy and hrqol. a similar study using qolie-ad 48 conducted by taylor et al.(15) found that adolescents with new-onset epilepsy (less than 3 years of onset), particularly those with comorbid conditions, are at risk of reduced qol at the time of diagnosis. it is stated furthermore that psychosocial problems are the comorbid conditions that could lower qol scores and need to be assessed earlier to improve the qol scores.(15) shakir and al-asadi stated that the association between duration of epilepsy (less than 5 years of onset versus more than 5 years) and qol was not significant. a possible explanation might be related to age as a potential confounder. a specific age sampling of epileptic patients is needed in further studies.(16) another study also stated that the correlation between duration of illness and qol was not significant. however, the duration of illness was significantly correlated with caregiver rating of the patient as having higher qol.(17) the correlation of hrqol and age was significant in the present study. few previous studies noted that there was a fair correlation between epilepsy impact and memory or concentration, and that hrqol was significantly associated with age. our overall scores of qoliead-48 show that adolescents with epilepsy have moderate levels of qol.(18-20) the other nonsignificant correlation in this study was with the variable of sex. similar results were found in a few prior studies.(21,22) other factors that correlated with hrqol and needed to be approached in order to achieve a higher hrqol were psychosocial consultation, family support programs and health education for parents, teachers and the public about different aspects of epilepsy.(23) seizure control and early behavioral problem identification also play a role in obtaining better hrqol scores.(24) the results of this study reflect the hrqol of a limited population of adolescents due to the relatively small and homogeneous study sample. the cross-sectional design of the study precluded conclusions about the direction of association between predictors and hrqol variables. further studies with greater numbers of subjects and longer periods of follow up are needed to better assess the risk factors for higher hrqol in adolescents with intractable epilepsy. a routine hrqol assessment may be useful in daily practice, especially in intractable epilepsy patients who need aed polytherapy. 110 conclusions intractable epileptic adolescents in the pediatric neurology outpatient clinic of dr. soetomo hospital had higher scores for physical functioning and health perception, but lower scores for stigmatization of epilepsy. social support had the lowest score. increasing age was significantly decreases quality of life in adolecents with intractable epileptic. conflict of interests the authors declare to have no conflict of interest. acknowledgement the authors would like to thank ms mahmudah for her enthusiasm in supporting us with statistical analysis. references 1. sinha s, siddiqui ka. definition of intractable epilepsy. neurosciences 2011;16:3-6. 2. lach lm, ronen gm, rosenbaum pl, et al. health-related quality of life in youth with epilepsy: theoretical model for clinicians and researchers. part i: the role of epilepsy and comorbidity. qual life res 2006;15:1161-71. 3. zashikhina a, hagglof b. health-related quality of life in adolescents with chronic physical illness in northern russia: a cross-sectional study. health qual life outcomes 2014;12:12. 4. waters e, davis e, ronen gm, et al. quality of life instruments for children and adolescents with neurodisabilities: how to choose the appropriate instrument. dev med child neurol 2009;51:6609. doi: 10.1111/j.1469-8749.2009.03324.x. 5. cramer j, westbrook le, perrine k, et al. development of the quality of life in epilepsy inventory for adolescents: the qolie-ad-48. epilepsia 1999;40:1114-21. 6. malhi p, singhi p. correlates of quality of life with epilepsy. indian j pediatr 2005;72:131-5. 7. kwan p, arzimanoglou a, berg at, et al. definition of drug resistant epilepsy: consensus proposal by the ad hoc task force of the ilae commission on therapeutic strategies. epilepsia 2010;51:1069-77. 8. sabaz m, cairns dr, lawson ja, et al. the health-related quality of life of children with refractory epilepsy: a comparison of those with and without intellectual disability. epilepsia 2001;42:621-8. 9. monir zm, el-alameey ir, eltahlawy e. health related quality of life of children with epilepsy in egypt. j arab soc med res 2013;8:53-66. 10. modi ac, ingerski lm, rausch jr, et al. treatment factors affecting longitudinal quality of life in new onset pediatric epilepsy. j pediatr psychol 2011;36:466-75. 11. eddy cm, rickards he, cavanna ae. the cognitive impact of antiepileptic drugs. ther adv neurol disard 2011;4:385-407. 12. hessen e, lossius mi, reinvang i, et al. influence of major antiepileptic drugs on attention, reaction time and speed of information processing: result from a randomized, doubleblind, placebo-controlled withdrawal study of seizure-free epilepsy patients receiving monotherapy. epilepsia 2006;47:2038-45. 13. aydemir n, ozkara c, unsal p. a comparative study of health related quality of life, psychological well-being, impact of illness and stigma in epilepsy and migraine. seizure 2011; 20:679-85. 14. siqueira nf, guerreiro mm, pedroso de souza ea. self-esteem, social support perception and seizure controllability perception in adolescents with epilepsy. arq neuropsiquiatr 2011;69:7704. 15. taylor j, jacoby a, baker ga, et al. self-reported and parent-reported quality of life of children and adolescents with new-onset epilepsy. epilepsia 2011;52:1498-98. 16. shakir m, al-asadi j. quality of life and its determinants in people with epilepsy in basrah, iraq. squ medical journal 2012;12:449-57. 17. ohaeri ju, awadalla aw, farah aa. quality of life in people with epilepsy and their family caregivers: an arab experience using the short version of who quality of life instrument. sustech 2009;30:1-22. 18. kerr c, nixon a, angalakuditi m. the impact of epilepsy on children and adult patients’ lives: development of a conceptual model from qualitative literature. seizure 2011;20:764-74. 19. sharoni sk, rahman ja, jamaludin ma, radzi nm. hrqol among children with epilepsy. international j undergraduates study 2013;2:1925. 20. aggarwal a, datta v, thakur lc. quality of life in children with epilepsy. indian pediatrics 2011; 48:893-6. gunawan, limanto, saharso quality of life in intractable epilepsy 111 univ med vol. 34 no.2 21. norsa’adah b, zainab j, knight a. the quality of life of people with epilepsy at a tertiary referral centre in malaysia. health qual life outcomes 2013;11:1-6. 22. karakis i, cole aj, luciano ms, meador kj, piperidous c. caregiver burden in epilepsy: determinants and impact. epilepsy res treat 2014;1:1-9. 23. abbas z, elseed ma, mohammed in. the quality of life among sudanese children with epilepsy and their care givers. sudanese j pediatr 2014;14:51-8. 24. kanemura h, aihara m. behavioural consequences in children with epilepsy. j child adolesc behav 2013;1:1. alvina 123 abstract the kelakai fern (stenochlaena palustris) contains bioactive substances, such as flavonoids, steroids, and alkaloids. the kelakai is used by the kenyah dayak communities for the treatment of anemia, fever, and cutaneous disorders. fever is increased temperature regulation of the body, which process indirectly increases the production of peroxides, leading to peroxidative stress. the aim of this research study was to evaluate the influence of an aqueous extract of the kelakai fern on peroxidative stress in the hoary marmot (marmota caligata) with experimentally-induced fever. this was a true experimental study, with post test-only and control group design, involving 6 groups, each made up of 4 animals. peroxide concentration was measured by a modification of the fox2 method and peroxidase activity by the kanehira method. the kelakai extract was administered to the animals by gavage at weight-adjusted dosages between 7.75 x 10-4 mg/kg body weight to 1.24 x 10-2 mg/kg body weight. data were analyzed using singe linear regression test. the results showed that kelakai extract dose significantly reduced the peroxide levels, regression equation with the y = 2.513-106.03 x (r2 = 0.8057), indicating that 80.5% variation of peroxide levels is predicted by the kelakai extract dose. from the results we conclude that kelakai aqueous extract decreases peroxide levels in hoary marmots with experimentally-induced fever. keywords: fever, peroxidative stress, stenochlaena palustris, hoary marmots *department of public health, medical faculty, lambung mangkurat university **department of chemistry/ biochemistry, medical faculty, lambung mangkurat university ***study group on free radicals and utilization of natural substances correspondence drs. eko suhartono, msi department of chemistry/ biochemistry, medical faculty, lambung mangkurat university jl. a yani km 36 banjarbaru 70714 email: ekoantioksidan@yahoo.com univ med 2010;29:123-8. stenochlaena palustris aqueous extract reduces hepatic peroxidative stress in marmota caligata with induced fever adenan* and eko suhartono**,*** september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction the potential of indonesian wetlands, including those of south kalimantan, is still u n d e r e x p l o i t e d . t h e p r o v i n c e o f s o u t h kalimantan has relatively extensive areas of marshland (both freshwater and peat swamps) of 287,000 hectares. it is estimated that peat swamps account for 8-11% of the total area of this province, occupying extensive lowland areas of kalimantan. the peat swamps have a distinctive type of forest formation with relatively limited flora, in which the kelakai fern [stenochlaena palustris (burm.f) bedd] constitutes one of its species.(1) in south kalimantan, the kelakai has few 124 adenan, suhartono stenochlaena palustris reduces peroxidative uses. however, on the basis of empirical studies, the kelakai is used by the kenyah dayak communities for the treatment of anemia, fever, and cutaneous disorders. in spite of this, few scientific studies have confirmed this empirical evidence. previous studies state that the plant contains bioactive substances, such as flavonoids, steroids, and alkaloids.(2) the study by suhartono demonstrated that the kelakai fern may act as an analgesic as well as antiphlogistic; the mechanism of synthesis involves oxidative reactions by peroxide molecules and the enzyme peroxidase acting holistically.(3) when the peroxide concentration exceeds the activity of peroxidases by which it is catalyzed, peroxidative stress occurs, w h i c h i s m a r k e d b y a l o w p e r o x i d e t o peroxidase ratio. the results of the study conducted by suhartono et al. revealed that hoary marmots with experimentally-induced fever have raised plasma peroxide levels, presumably as an immunological response of the body.(4) since peroxides possess oxidant properties and are thus capable of forming hydroxyl radicals t h r o u g h f e n t o n a s w e l l a s h a b e rwe i s s reactions, administration of kelakai extract to hoary marmots with experimentally-induced fever may be expected to reduce peroxidative stress. thus the objective of the present study was to explore the influence of kelakai aqueous extract on hepatic peroxidative stress in hoary marmot. methods design of the study the present study was a true experimental study with post-test-only and control group design to examine the impact of kelakai extract on peroxidative stress in hoary marmots subjects the study involved 6 groups of 4 male hoary marmots (marmota caligata), where one group was the control group, while the other five constituted intervention groups. the test substance was an aqueous extract of kelakai ferns. preparation of kelakai aqueous extract kelakai ferns were collected in june 2009 from gambut subdistrict, south kalimantan. species identification was performed by the department of biology, pharmacy study program, faculty of mathematics and natural s c i e n c e s (fmipa), l a m b u n g m a n g k u r a t university. the active principles in the kelakai plants were extracted by maceration. animal experiments the experimental animals used in this study were 3-4 months’ old hoary marmots weighing approximately 300-400 g, obtained from the veterinary research and investigation station (balai penelitian dan penyidikan veteriner, bppv) at banjarbaru. the animals were kept for one week prior to the experimental treatment to bring them to approximately equal physical and psychological conditions. in the adaptation period, the animals received animal feed and water ad libitum, the water being tap w a t e r s u p p l i e d b y t h e p r o v i n c i a l w a t e r purification plant. complete random sampling was used to assign the animals into 6 groups, each consisting of 4 animals. each group was kept in one small cage for adaptation. one group, the control group, was designated p0, while the remaining five intervention groups were designated p1-p5. the animals were subsequently weighed and their rectal temperature taken, after which all animals received 0.16 ml dpt vaccine by intramuscular injection in the right thigh, the dose having been determined in preliminary tests. the rectal temperature of the animals was again taken at the onset of fever (the time of onset of fever was determined in preliminary t e s t s t o b e 9 0 m i n u t e s p o s t i n j e c t i o n ) . afterwards kelakai extract was administered to the animals by gavage (stomach tube) at weight-adjusted dosages as follows: 125 22 bu bs oh mm 1x aa aa ionconcentrat peroxide − − = 1-10 min 5 aa activity peroxidase − = experimental group kelakai aqueous extract (mg/kg body weight) distilled water p0 1 ml p1 7.75 x 10 -4 p2 1.55 x 10 -3 p3 3.10 x 10-3 p4 6.20 x 10-3 p5 1.24 x 10-2 at the peak of fever (determined in preliminary tests to be 2 hours after onset), the animals were sacrificed by decapitation and their livers surgically removed. subsequently the livers were washed in phosphate buffer at ph 7, then minced into a liquid, of which 5 ml was centrifuged at 3500 rpm for 10 minutes, and 200 µl of the supernatant was taken for determination of peroxide concentration by the modified fox2 method, as described below. the standard and test solutions consisted of 1 m h2o2 200 µl and 200 µl plasma, respectively, with the addition of 160 µl pbs p h 7 . 4 , 1 6 0 µl f e c l 3 ( 2 5 1 . 5 m g f e c l 3 dissolved in 250 ml distilled water) and 160 µl o-fenantroline (120 mg o-phenantroline dissolved in 100 ml distilled water) for both solutions. the composition of the blank solution was identical to that of the test solution, except for absence of fecl3 in the blank. subsequent to preparation, all solutions w e r e i n c u b a t e d f o r 3 0 m i n u t e s a t r o o m temperature, then centrifuged at 12,000 rpm for 10 minutes, and the absorbance of the standard (as), test (au) and blank (ab) solutions measured at λ=505 nm, using the supernatant of each solution. peroxide concentration was calculated from the following formula, ab= absorbance of blank solution; and au = absorbance of test solution. peroxidase activity was determined by the kanehira method.(5) one ml of the supernatant layer of the test solution was mixed with 45 µl fecl3 , 45 µl phosphate buffer (ph 7) and 45 µl o-phenantroline, then the absorbance was measured at ë=505 nm (a0). subsequently the mixture was incubated for one minute at room temperature and its absorbance (a1) measured at the same wavelength. peroxidative stress is the ratio of peroxide concentration and peroxidase activity. statistical analysis the relationship between kelakai aqueous extract dose and levels of peroxidative stress was analyzed by single linear regression. data processing was done using microsoft excel. p values < 0.05 were considered statistically significant. results as shown in figure 1, marmot hepatic peroxidative stress due to fever was inversely proportional to test substance concentration, decreasing with increasing concentration of the test substance. from figure 1, it may be concluded that administration of kelakai extract is capable of inhibiting peroxidative stress. the correlation of the administered dose of kelakai extract with peroxidative stress is given in the equation: ln y = 2 . 5 1 3 1 0 6 . 0 3 x w i t h r 2 = 0 . 8 0 5 7 , indicating that 80.5% variation of peroxidative stress level is predicted by the kelakai extract dose. this indicates that there is a strong correlation between fever-induced hepatic univ med vol.29 no.3 where as = absorbance of standard solution; 126 adenan, suhartono stenochlaena palustris reduces peroxidative figure 1. correlation of hepatic peroxidative stress in marmots with experimentally-induced fever peroxidative stress and the administered dose of kelakai extract. discussion the febrile process indirectly leads to an increase in free radicals such as superoxide anions (•o2) which afterwards are converted t o p e r o x i d e s b y t h e e n z y m e s u p e r o x i d e dismutase (sod). the peroxides are then catalyzed by the enzyme peroxidase into water and oxygen. administration of distilled water (p0) to dpt-injected control animals resulted in very high peroxide concentrations in the liver, since administration of dpt vaccine induces a r e s p i r a t o r y b u r s t b y n e u t r o p h i l s a n d macrophages through concurrent formation of h2o2. at this stage there are a considerable number of phagocytosed bacteria, necessitating high h2o2 levels. in addition, arachidonic acids are ultimately transformed to prostaglandins, w h i c h a r e m e d i a t o r s o f p a i n a s w e l l a s inflammation,(6,7) the prostaglandin synthesis i n v o l v i n g o x y g e n a t i o n c a t a l y z e d b y cyclooxygenase to form peroxides.(8) the peroxides formed from these sources will in appropriate amounts be beneficial to the body that is being subjected to infection, acting as a line of defense, since peroxides possess microbicidal properties that aid in eradication of pathogenic bacteria. on the other hand, peroxides may also have a negative impact on the host, which may occur if the p r o d u c t i o n o f p e r o x i d e s e x c e e d s t h e c o u n t e r a c t i n g a n t i o x i d a n t a c t i v i t y. t h i s condition is called peroxidative stress, which may cause biomolecular damage comprising a m o n g o t h e r s c e l l u l a r d a m a g e , e n z y m e inactivation, and mutation. additionally, peroxidative stress may lead to oxidation of dna and proteins followed by membrane damage due to lipid peroxidation, causing m e m b r a n e p e r m e a b i l i t y c h a n g e s a n d alterations in protein structure and functions. oxidative damage also occurs in mitochondrial m e m b r a n e s , t h u s c a u s i n g m e m b r a n e depolarization, disruption of the oxidative 127 phosphorylation chain, and altered cellular respiration. this finally causes damage to the m i t o c h o n d r i a , r e s u l t i n g i n s y n t h e s i s o f cytochrome c and activation of apoptosis (programmed cell death).(9,10) the results of the present study indicate that kelakai aqueous extract is capable of significantly decreasing hepatic peroxidative stress. this is presumably due to bioactive s u b s t a n c e s i n k e l a k a i f e r n s , n a m e l y flavonoids, steroids, and alkaloids, which are useful as antipyretics, antioxidants, and antiphlogistics.(11-13) the capture mechanism of free radicals b y f l a v o n o i d s i s i n i t i a t e d b y r e l e a s e o f hydrogen, forming reactive flavonoid radicals. the latter will bind to free radicals (•o2), causing the reactivity of the latter to diminish or even disappear,(14-16) leading to decreased formation of peroxides. other bioactive substances contained in kelakai are alkaloids and steroids. in addition to being antipyretic, the alkaloids and steroids possess anti-inflammatory properties. the study by sudjarwo revealed that the alkaloid piperine contained in kelakai can function as antipyretic through inhibition of prostaglandin synthesis,(1719) while the steroids inhibit phospholipase activity and arachidonic acid transformation to prostaglandins, reduce microvascular leakage, prevent direct migration of pyretic cells, and inhibit cytokine production.(17,20) conclusion administration of kelakai (stenochlaena palustris) aqueous extract reduces the hepatic peroxidative stress in febrile hoary marmots (marmota caligata). references 1. suhartono e, yasmina a. kajian farmakologis ekstrak air tumbuhan kelakai sebagai antiyeri dan antiradang dengan pendekatan mekanisme oksidatif. laporan penelitian universitas lambung mangkurat; 2007. 2. maharani, haidah, hainiyah. studi potensi kelakai sebagai pangan fungsional. kumpulan makalah pimnas xix, 26-29 juli 2006;universitas muhamadiyah malang; 2006. 3. suhartono e. potention of aquaeus extract kelakai as antiinflammation by oxidative mechanism, congress international korean medicine; 2008 october 22-23;sangji university korea; 2008. 4. suhartono e, bakhriansyah m, sandi da, prabandari k. hydrogen peroxide concentration and catalase activity in the plasma of fever marmota caligata after giving extracts of stenochlaena palustris. proceedings of the 38th meeting of national working group for indonesian medicinal plant; 2010 july 23-24; widya mandala university; 2010. 5. kanehira t, shibata k, kashiwazaki h, inoue n, morita m. comparison of antioxidant enzymes in saliva of elderly smokers and non-smokers. gerodontology 2006;23:38-42. 6. devaux y, seguin c, grosjean s, talance n, camaeti v, burlet a, et al. lipopolysaccharideinduced increase of prostaglandin e2 is mediated by inducible nitric oxide synthase activation of the constitutive and induction of membraneassociated prostaglandin e synthase. j immunol 2001;167:3962-71. 7. warner td, vojnovic i, giuliano f, jime’nez r, bailey db, mitchell ja. cyclooxygenase 1, 2, and 3 and the production of prostaglandin i2: investigating the activities of acetaminophen and cyclooxygenase-2-selective inhibitors in rat tissues. jpet 2004;310: 642-47. 8. lobanova em, tahanovich ad. hydrogen peroxide metabolism in alveolar macrophages after exposure to hypoxia and heat. physiol. res. 2006;55:569-75. 9. guzik tj, korbut r, adamek-guzik t. nitric oxide and superoxide in inflammation and immune regulation. j physiol pharmacol 2003;54:469-87. 10. matsumura ki, matsumura k, terao a, watanabe y. attenuated fever in pregnant rats is associated with blunted synthesies of brain cyclooxygenase2 and pge2. am j physiol regul integr comp physiol 2002;283:r1346-53. 11. byrne, djo., devaraj. s., grundy, sm., jialal i. comparison of the antioxidant effects of concord grape juice flavonoids and á -tocopherol on markers of oxidative stress in healthy adults. am j clin nutr 2002;76:1367–74. 12. pignatelli p, pulcinelli fm, celestini a, lenti l, ghiselli a, gazzaniga pp, et al. the flavonoids quercetin and catechin synergistically inhibit platelet function by antagonizing the intracellular univ med vol.29 no.3 128 adenan, suhartono stenochlaena palustris reduces peroxidative production of hydrogen peroxide. am j clin nutr 2000;72:1150-5. 13. patral a, jha s, murthy pn, chattopadhyay p, panigrahi g, roy d, et al. anti-inflammatory and antipyretic activities of hygrophila spinosa t. anders leaves (acanthaceae). trop j pharm res 2009;8:133-7. 14. ranju sp, ariharasivakumar g, kundlik g, ashutosh u. in-vitro antioxidative activity of phenolic and flavonoid compounds extracted from seeds of abrus precatorius. int j pharmacy pharmaceutical sciences 2009:1:136-40 15. o’byrne dj, devaraj s, grundy sm, jialal i. comparison of the antioxidant effects of concord grape juice flavanoid and á-tocopherol on markers of oxidative stress in healthy adults. am j clin nutr 2002;76:1367-74. 16. konyahoglu s, karamenderes c. screening of total flavanoid, phenol contents and antioxidant capacities of some achillea l. species growing in turkey. acta pharmaceutica turcica 2004;46:163-70. 17. sudjarwo sa. the potency of piperine as antiinflammatory and analgesic in rats and mice. folia medica indonesiana 2006;41:190-4. 18. teh bs, seow wk, li sy, thong yh. inhibition of prostaglandin and leukotrien generation by the plant alkaloids tetradine and barbamine. j immunopharmacol 2000;12:321-6. 19. moura dj, marc fr, jane mb, joao aph, jenifer s. antioxidant properties of β-carboline alkaloids are related to their antimutagenic and antigenotoxic activities. mutagenesis 2007;22: 293–302. 20. runtti pp, lakari e, raivio ko, kinnula vl. expression of antioxidant enzymes in human inflammatory cells. am j physiol cell physiol 2000;278:118–25. cite this article as: ekasari dp, basuki s, kurniasih w, brahmanti h, rofiq a. effect of physalis angulata leaf extract cream on interleukin-4, interleukin-6, and immunoglobulin-e in mice with in duced atopic dermatitis. univ med 2023; 42:150 -9. doi: 10.18051/univmed. 2023.v42:150-159 may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 effect of physalis angulata leaf extract cream on interleukin-4, interleukin-6, and immunoglobulin-e in mice with induced atopic dermatitis dhany prafita ekasari1 , santosa basuki1, wuriandaru kurniasih1* , herwinda brahmanti1 , and aunur rofiq1 abstract background the prevalence of atopic dermatitis (ad) and allergic or irritant contact dermatitis has been increasing significantly in the general population. interleukin4 (il-4), interleukin-6 (il-6), and immunoglobulin e (ige) play a key role in the pathogenesis of ad. physalis angulata (pa) leaves reportedly have anti-inflammatory effects by impeding il-4, il6, and ige. this study aimed to evaluate the effect of pa leaf extract cream on il-4, il-6, and ige using 2,4-dinitrochlorobenzene (dncb) to induce ad-like skin inflammation in a mice model. methods this study used an experimental design involving 30 balb/c mice, that were randomized into 3 groups: 1) control group receiving no treatment; 2) vehicle treatment group receiving vehicle cream preparation; 3) pa treatment group receiving 10% pa leaf extract cream after induction of ad-like skin inflammation by dncb. after 30 days, tissue samples were extracted from the skin lesions to measure il-4 and il-6 levels, and serum to measure ige using elisa. oneway anova, kruskal-wallis and mann-whitney tests were used to analyze the data. results group 3 (pa treatment) had significantly lower il-4 (281.15 ± 43.14 pg/ml) than group 2 (vehicle cream treatment) (388.89±135.88 pg/ml) (p=0.001). however, although il-6 and ige levels were lower in group 3 than in group 2, the differences were statistically not significant (p=0.096 and p=0.479 respectively). conclusion there were lower levels of il-4, il-6, and ige in the group receiving pa leaf extract cream than in the group receiving vehicle cream preparation. therefore, pa leaf extract cream may have therapeutic potential in ad. keywords: atopic dermatitis, physalis angulata, balb/c mice, dinitrochlorobenzene 1department of dermatology and venereology, faculty of medicine, universitas brawijaya, malang, indonesia *correspondence: wuriandaru kurniasih jl. percetakan negara iva no. 9a. jakarta pusat 10560 email: swuriandaru@gmail.com orcid: 0000-0001-8790-5918 date of first submission, march 17, 2023 date of final revised submission, july 13, 2023 date of acceptance, july 24, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license 150 doi: http://dx.doi.org/10.18051/univmed.2023.v42:150-159 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1451 original article mailto:swuriandaru@gmail.com https://orcid.org/0000-0002-2469-7127 https://orcid.org/0000-0001-8790-5918 https://orcid.org/0000-0002-1970-6551 https://univmed.org/ejurnal/index.php/medicina/article/view/1451 151 univ med vol. 42 no. 2 introduction atopic dermatitis (ad) is a chronic inflammatory skin disease with characteristic symptoms and clinical signs, such as itching, erythema, oozing/cru sting, excor iations, lichenification, and dry skin. this condition is caused by inflammation of the skin as a result of increased histamine and inflammatory cytokine levels.(1-3) according to the world health organization global burden of diseases at least 230 million people worldwide suffer from ad.(4) the prevalence of ad in children is about 10% to 20% whereas in adults it is about 5-10%.(5) atopic dermatitis is marked by infiltration of the inflammatory cytokine interleukin-4 (il-4) producing cells in the skin and high levels of il-4 in peripheral blood. the high il-4 levels stimulate th2 cells and provide positive feedback causing these cells to synthesize more il-4.(6) enhanced regulation of th2 immunity can also cause maturation of b cells and differentiation of plasma cells, resulting in ige hypersecretion and release of mast cells that will aggravate ad.(7) the presence of damaged keratinocytes disturbing the skin barrier, as well as an increased number of macrophages, cause increased secretion of il6, which then triggers th2 differentiation and inhibits th1 polarization.(8) topical immunosuppressant steroids or calcineurin inhibitors are the most frequent medications used because of their rapid effect in repairing ad lesions. however, the prolonged use of steroids can cause various side effects such as skin atrophy, telangiectasia, acne, and hypertrichosis. therefore, the development of new pharmacological agents as a safe alternative therapy is very much needed, considering that ad is chronic and recurrent.(9,10) currently, there have been many developments in various types of therapy that can target the specific mediators involved in the pathogenesis of ad. these targets include il-4, il-13, ige, il-6, b cells, il-5, il31, janus kinase-signal transducer and activator of transcription (jak-stat), phosphodiesterase -4 (pde-4), il-12, il-17, il-23, il-22, histamine h4 receptor (h4r), thymic stromal lymphopoietin (tslp) and peroxisome proliferator-activated receptor-gamma (ppar-).(11) physalis angulata (pa) reportedly contains flavonoids such as quercetin and steroids such as physalin and angulatin-a.(12) quercetin has been developed for ad treatment resulting in lower levels of nuclear factor kappa b (nf-κb), tslp, il-4, il-6, and ige.(10) the steroid content of pa such as physalin has also been much studied as an anti-inflammatories agent that can inhibit various types of proinflammatory cytokines such as nf-κb and il-6.(13) physalis angulata was reported to have a mechanism of action on the skin as an immunomodulator and antiinflammatory agent with effects similar to corticosteroids and was suggested to be able to increase wound healing factors but not to reduce tumor growth factor β1 (tgf-β1) production in normal human keratinocytes, in contrast to corticosteroids which can inhibit the wound healing process and cause skin atrophy. topically applied physalis angulata is also reported as a new and innovative phytopharmaceutical with va rious pharmacologi cal effects that are potentially useful to protect human skin, especially against inflammation.(14) another study also showed that pa cream containing physalin-e conferred benefits as an anti-inflammatory agent in acute and chronic dermatitis in experimental animals, which has therapeutic potential for the chronic and recurrent inflammatory disease ad.(15) our study might give suggestions about whether physalis angulata is a potential antiinflammatory drug. the objective of this study was to evaluate the effect of pa leaf extract cream against levels of il-4, il-6, and ige in the development of a balb/c mouse model that resembles ad with 2,4-dinitrochlorobenzene (dncb) induction. methods study design this was an experimental study using an animal model with a post-test only control group 152 design. this study wa s conducted at the integrated biomedical laboratory unit, faculty of medicine, universitas brawijaya, dr saiful anwar general hospital, malang from october until november 2021. preparation of pa leaf extract and phytochemical tests physalis angulata leaves were obtained from malang, east java. the leaves were ovendried at 40 0c and made into a powder. subsequently, the leaves were extracted by macerating in 70% ethanol. the maceration process consisted of soaking 200 grams of powdered pa leaves in 1 liter of 70% ethanol. after five days the solution was filtered to separate the residue from the filtrate, from which the ethanol was evaporated using a rotary evaporator, leaving only the pure extract without the ethanol. to analyze the plant extract, phytochemical tests were carried out at the malang medicinal plant station, consisting of quantitative analysis of quercetin and qualitative analysis of flavonoids and steroids. preparation of pa leaf extract cream the formulation of the cream was identical to that used in the study of herdiana et al., (16), except for the green tea used as perfume, while the 10% w/w concentration of the pa leaf extract was identical to the study of abdul-nasir-deen et al.(17) t he vehicle cream had the same composition except for the pa leaf extract. the materials used in the cream were glycerin 10%, triethanolamine 2%, corn oil 20%, stearic acid 7%, cetyl alcohol 2%, methylparaben 0.1%, and distilled water 100%. the cream was prepared using tano’s method, commencing by warming up the stearic acid, corn oil, and cetyl alcohol to a temperature of 70°c. the temperature was then lowered to 65°c, and triethanolamine was slowly added (mixture 1). glycerin and water in separate containers were heated to 80°c, stirred, and cooled down to 35°c (mixture 2). mixtures 1 and 2 were combined, while being stirred manually until they increased in volume and formed a smooth cream emul sion ( mixture 3). methylparaben and pa leaf extract were added while stirr ing until a smooth cream was formed.(16) experimental animals the research animals were 6-week-old male balb/c mice weighing 20-30 grams, obtained from the bioscience laboratory, universitas brawijaya (malang, indonesia). all mice were acclima tized f or one week at controlled temperature (23±3°c) and humidity (55±15%) in a cycle of 12 hours light and 12 hours darkness. body weight and food intake were measured once a week. the sample size was determined by the federer formula: (t-1) (r-1) >15 (t=number of treatments; r=number of replications); (3-1)(r1) >15; 2(r-1)>15; r>8.5. in anticipation of a dropout rate of 10%, a total sample of 30 was obtained for the three groups. procedure of ad i nduction in m ice development and treatment model this study used 30 mice that were randomly assigned to three groups: group 1 was the control group consisting of mice that were not given any treatment; group 2 was the vehicle cream group consisting of mice with dncb-induced ad that were given the vehicle cream preparation; and group 3 was the ad treatment group consisting of mice with dncb-induced ad that were given 10% pa leaf extract cream. the ad in the balb/c mice was induced with dncb using the procedure of lim et al.,(3) as follows: a 2x2 cm area of mouse dorsal skin was shaved on day 0 and cleaned with sterile gauze and 0.9% nacl. sensitization with dncb 0.5% was done for 3 consecutive days, namely on days 1, 2, and 3 using 0.15 ml of 0.5% dncb on the shaved dorsal skin area. subsequently the sensitized skin was challenged on days 14, 17, 20, 23, 26, and 29 (every 3 days) with 0.15 ml of 1% dncb in groups 2 (vehicle cream) and 3 (pa leaf extract cream) at 8:00 a.m.(3) in groups 2 and 3 on day 4 of sensitization, skin lesions resembling ad were ekasari , basuki , kurniasih, et al physalis angulata leaf extract cream 153 univ med vol. 42 no. 2 found, such as erythema, erosion, excoriation, scaling, and dry skin.(18) on days 14 to 29, group 2 balb/c mice were given vehicle cream topically on the dorsal skin every day, while group 3 was given 10% pa leaf extract cream. the respective creams were given in amounts of 0.1 g for the 2x2 cm areas on the dorsal skin of the mice with dncb-induced ad.(3,17,19) the vehicle and pa creams were applied every day at 2:00 p.m. on the respective animals. on day 30th, the mice were euthanized by cervical dislocation. the mechanism of ad induction in the mice is depicted in the figure 1. analysis of mouse serum and skin after being left to stand at room temperature for 2 hours, each of the blood samples was centrifuged at 3000 g for 15 minutes and the supernatant was collected for determining ige levels (elisa kit thermo scientific, usa). examination of levels of il-4 (elisa kit cusabio, usa) and il-6 (elisa kit, mybiosource, usa) was performed on 200 mg of skin tissue that was put in a 1.5ml eppendorf tube, homogenized in 1 ml pbs using mortar and pestle, and stored overnight at -20 °c. the levels of each cytokine were then measured using the elisa kits. figure 1. induction of atopic dermatitis-like inflammation in balb mice with 2,4-dinitrochlorobenzene (dncb) figure 2. atopic dermatitis-like lesion in 2,4-dinitrochlorobenzene (dncb) treated balb/c mice 154 statistical analysis data obtaine d fr om the study was quantitative data, comprising mouse skin il-4 and il-6 levels, and mouse serum ige in all three groups. quantitative data was then processed with the shapiro-wilk normality test using spss software (v21.0). if the data was normally distributed, it was next analyzed with one-way anova to find differences between the three groups. if the data distribution was not normal, the kruskal-wallis test was used for analysis. a pva lue <0.05 was cons ider ed sta tistically significant. ethical clearance ethical approval of the study on balb/c mice was obtained from the ethics research commission, brawijaya university (animal care and use committee) with the ethical clearance letter no. 037-kep-ub-2021. results phytochemical results phytochemical testing of 70% ethanolic pa leaf extract found the presence of flavonoids, but did not find steroid compounds. on quantitative analysis, a mean quercetin level of 7.61% was obtained. effect of pa leaves on ad-like skin lesions induced by dncb in mice a total of 30 skin lesions on day four had met the criteria for lesions resembling atopic dermatitis, namely erythema, erosion, excoriation, or crusting, while repeated dncb application induced skin lesions similar to marked ad with clearly visible erythema, excoriation, and lichenification on the dorsal skin. healing of the observable skin lesions resulted in better looking skin, especially in the pa leaf extract cream group (group 3) compared to the vehicle cream group (group 2). administration of pa leaf extract cream resulted in visible repair of proven lesions, accompanied by decreases in erythema, edema, lichenification, dry skin, and excoriations, although of insufficient potential compared to the mice that did not receive any treatment (group 1). effect of pa cream on il-4, il-6, and ige levels based on the results of anova analysis, it was found that there was a significant difference in the mean il-4 level in the three treatment groups (p= 0.001). the mean difference between il-4 levels in group 1 and group 2 was statistically significant (p<0.001). however, the mean difference between il-4 levels in group 1 and group 3 was not significant (p=0.226), whereas the mean il-4 level in group 2 was significantly different from the mean in group 3 (p=0.024) (table 1). it was found that the p value for ige was 0.479 (p>0.05), signifying that there was a nonsignificant difference between the groups at an error rate of 5% (table 1). in contrast, mean ige in group 2 was higher than in groups 1 and 3, and mean ige levels in group 3 that was given pa cream was lower than in group 2 that was given vehicle cream preparation. the il-6 normality test showed that the kolmogorov-smirnov test value in group 3 had a p=0.007 (p<0.05), which means that it had an abnormal distribution. then, kruskal-wallis and mann-whitney tests were performed. analysis treatment groups p value control (n=10) dncb + vehicle cream (n=10) dncb + pa cream (n=10) interleukin-4 (pg/ml)@ interleukin-6 (pg/ml)$ immunoglobulin e (ng/ml)@ 216.09 ± 42,64 4.05 (3.93-4.10) 125.04 ± 4.76 388.89 ± 135.97 4.17 (4.09-4.27) 128.51 ± 7.53 281.15 ± 43.14 4.13 (4.05-4.14) 126 ± 6.75 0.000 0.011 0.479 table 1. the effect of pa leaves on il-4, il-6 and ige by treatment groups note: data presented as mean ± sd, except for interleukin-6 [median (q1-q3)]; @ one-way anova test; $ kruskal-wallis test; pa: physalis angulata; dncb: 2,4-dinitrochlorobenzene; significant at p<0.05 ekasari , basuki , kurniasih, et al physalis angulata leaf extract cream 155 univ med vol. 42 no. 2 results of the kruskal-wallis test obtained a pvalue of 0.011 (p>0.05), signifying that although il-6 was slightly lower in group 3 than in group 2, there was no significant between-group difference in the effect. after post-hoc analysis, only the mean difference in il-6 between group 1 and group 2 was statistically significant (p = 0.008 <0.05). on the other hand, the mean il-6 levels in groups 1 and 3 were not significantly different (p=0.053), while the mean il-6 level in group 2 also did not differ significantly from that in group 3 (p=0.096) (table 2). discussion the preliminary phytochemical tests of the ethanolic pa leaf extract revealed the presence of flavonoids, but not of steroids were. in this study flavonoids such as quercetin accounted for 7.16% while the ster oid content was not detectable. the mice in the pa leaf extract cream group had significantly lower il-4 levels than the mice in the vehicle cream group. however, although the il-6 and ige levels in the pa leaf extract cream were lower than in the vehicle cream group, the between-group difference was statistically not significant for both parameters. our study results are similar to those of abdul-nasir-deen et al.,(17) where the methanolic pa leaf extract, which also contained flavonoids but no steroids, reportedly still had antiinflammatory properties and reduced paw edema in a mouse model of dermatitis. hou et al.(20) concluded that a cream made of quercetin, which is a flavonoid, is useful in reducing ad symptoms. quercetin cream was applied on the dorsal side of the left ears of the a d mice (c57bl/6 mice treated with topical mc903, a low-calcemic vitamin d3 analog) and reportedly reduced the expression of ccl17, ccl22, il-4, il-6, ifnγ, and tnf-α. possible cause why our pa leaves do not contain steroids is because in our study as well as in previous studies, the fresh pa leaves came from different locations, e.g. from bogor in the study by herdiana et al.(16) and from kalimantan in the study by iswahyudi et al.(21) the place of origin of fresh pa leaves can influence the contained metabolites. the diversity of secondary metabolites is determined by various factors, such as genetics, age, season at harvest time, environmental fluctuations, soil conditions, and biochemical interactions between competing plants, that may regulate the synthesis of various secondary metabolites.(22,23) in addition, the solvents used for extracting the processed materials and the drying method (sun-, air-, or oven-drying) can also cause quantitative changes in the composition of the phytochemical compounds.(21) the quantity of the metabolites is also affected by the location or origin of the pa. akomolafe et al.(24) from nigeria reported that the most abundant type of flavonoid found in pa leaf extract is quercetin at 56.74 mg/g. in contrast, nguyen and kim (12) in vietnam reported that quercetin is found in pa leaves at much lower concentration, in the range of 40.12-66.10 mg/ kg. atopic dermatitis is characterized by three main features, namely abnormalities of the th2 immune response, disruption of the skin barrier, and chronic pruritus.(25) th2 cells produce il-4 and il-13, which can increase vascular adhesion, parameter compared groups p-value il-4a 1 2 0.000* 3 0.226 2 3 0.024* il-6b 1 2 0.008* 3 0.053 2 3 0.096 igec 1 2 0.462 3 0.925 2 3 0.691 table 2. results of tukey multiple comparison test notes: group 1 = no treatment, group 2 = dncb induced mice treated with vehicle cream, group 3 = dncb induced mice treated with pa cream. * significant at p<0.05; aone-way anova, followed by tukey test showed significant differences between group 1 and 2 (p=0,000) and group 2 vs. 3 (p=0.024); bkruskal-wallis test, followed by mann-whitney test showed a significant difference between group 1 and 2 (p=0.008); cone-way anova, followed by tukey test showed no significant between-group difference 156 which is molecularly related to the adhesion of eosinophils in dermatitis lesions. th2 cells induce proliferation of b cells, which can produce ige, which then binds to the surface of mast cells, and subsequently, mast cells release inflammatory mediators and cytokines, such as histamine, il6, il-1β, or tnf-α.(3) lesions in ad skin are marked by an excessive expression of il-4, il-13, and tslp secreted by keratinocytes.(26) the il-4 cytokine interferes with the skin barrier and inhibits the flg (profilaggrin) gene expression, as well as the synthesis of other proinflammatory cytokines that cause inflammation in ad. interleukin-4 and/ or il-13 are strongly associated with three of the abovementioned ad characteristics which are key elements in the th2 response. these cytokines could trigger and control the immune response in ad so that specific antagonism against these cytokines has developed as a therapeutic target in ad.(25,27) interleukin-6 that is especially produced by activated macrophages, and il-4 that is mainly released by t cells, are key mediators in the initiation and development of ad. interleukin-6 also can influence the enhancement of t cells and b cells. the enhanced release of proinflammatory cytokines could be detected at different ad phases which shows that cytokines are closely involved in the development of ad.(28) immunoglobulin e plays a role in ad via binding to mast cells and basophils that have high affinity ige receptors (fceri), whereupon these cells will then release a signal for degr a nulation and cause inflammation.(29) atopic dermatitis affects the human immune system and causes clinical symptoms such as erythema, skin edema, excoriation, lichenification, and abnormal epidermal thickening that are observed in mice with dncb-induced ad.(28) the immune response in mice with dncbinduced ad can be observed from the increased serum levels of ige, il-4, and il-6 in the study, and this condition is correlated with enhanced infiltration of inflammatory cells and mast cells in the dorsal skin. however, ad symptoms appear to be reduced after treatment with pa leaf extract cream for two weeks. our study shows that pa leaf extract cream can significantly lower il-4 levels in the skin of the dncb-sensitized mice. the pa leaf extract cream can also lower il-6 levels in the mouse skin and ige levels in mouse serum compared with mice that were only given the vehicle cream preparation. the mechanism of action of the pa leaf extract cream in ad is due to the existence of the flavonoid quercetin, as well as steroids such as physalin that have been reported by many to be able to suppress tslp or nf-kb which is a path in the formation of il-4 or to directly inhibit il-4, il-6, and ige.(10) abnormalities of the epidermal barrier in ad increases the enzyme kallikrein-5, which then binds to par2 and triggers nf-κb, which in turn can increase the release of tslp cytokines by keratinocytes.(30,31) thymic stromal lymphopoietin induces dendritic cells and activated langerhans cells to migrate to the lymph nodes as antigen-presenting cells (apc) and convert naive t cells into th2 cells.(32,33) the th2 cells could return to the dermis and release il-4 and il-13 which can trigger the signs and symptoms of ad. in addition, th2 causes a change in the ige class produced by the b cells.(31) the immunoglobulin e that is produced then stimulates mast cells, which degranulate when bound to specific allergens and release various types of molecules including il-4. increasing il4 will also aggravate ad by reducing epidermal diff erentiation and downregulating amp expression thereby increasing the risk of infection.(34) quercetin is a pleiotropic molecule that can be used against various molecular targets to control proinflammatory cytokines and therefore has potential for ad therapy.(35) several studies have demonstrated that quercetin constitutes a potent anti-ad agent that can be given orally or topically. karuppagounder et al. (36) used quercetin orally (at 50 mg/kg) for two weeks in the nc/ nga ad mouse model and reported that quercetin could lower serum il-4 levels and modulate hmgb1/rage/ nf-κb signaling and nrf2 ekasari , basuki , kurniasih, et al physalis angulata leaf extract cream 157 univ med vol. 42 no. 2 protein induction. the study of hou et al.(20) concludes that administration of 1% quercetin cream for seven days can reduce the expression of il-4, ccl17, ccl22, il-6, ifn-γ, and tnfα in the ears of c57bl/6 mice with mc903induced ad. the quercetin derivative quercetin3-o-(2”-gallate)-α-l-rhamnopyranoside (qgr) has also been reported to lower the expression of il-4, il-5, and il-13 when 1% qgr was given topically to nc/nga mice for four weeks.(37) treatment with quercetin derivatives could push the expression of proinflammatory cytokines through modulation of nf-κb, il-4, il-5, and il13, serum ige, and levels of eosinophils, inos, and cox2, hmgb1, rage which play an essential role as inflammatory mediators. quercetin has also been reported to inhibit the hmgb1, nf-κb, j ak/stat, and tslp pathways in the nc/nga ad mouse model. in addition, it has been shown that quercetin inhibits cytokines and other pro-inflammatory agents, such as il-1β, il-6, and tnf, and increases il-10 levels in liposaccharide-induced inflammation in mice.(10,36) according to the literature, quercetin can inhibit nf-κb, tslp, and il-4 pathways which are unique inflammatory pathways in ad. however, this does not preclude the possibility that the quercetin contained in the pa leaf extract cream can inhibit il-4 through other pathways, in view of the fact that the pathophysiology of ad is very complex, because pa could inhibit a variety of signaling pathways or cytokines. one of the limitations of this study is that the pa leaf extract contained no steroids, which are considered important for suppressing the inflammation of atopic dermatitis. therefore it is necessary to search for pa leaves that contain both flavonoids and steroids, in the expectation that they can provide a more synergistic effect against the ad-like inflammation in the mice. another limitation is that there was no comparison of the pa cream with other standard topical agents for ad, such as topical corticosteroids and calcineurin inhibitors. it is still unclear whether the improvement in il-4, il-6, and ige levels due to pa leaf extract cream equals or surpasses that of topical steroids or calcineurin inhibitors. even so, the il-4, il-6, and ige levels in mice given pa cream at a concentration of 10% proved to be lower than in mice given only the vehicle cream. further studies are needed on the proportion of the extract in different creams, on experiments with other preparations, such as ointments or gels, and on the use of solvents and other extraction methods to obtain a maximal yield of flavonoids and steroids, thus providing adequate treatment of ad lesions. conclusions physalis angulata leaf extract cream at 10% concentration can inhibit the inflammatory response in mice dncb-induced ad lesions by lower ing il-4, il-6, and ige levels. the therapeutic use of pa leaf extract cream is exceedingly promising and has the potential to be developed into standard ad therapy. conflict of interest the authors declare no conflicts of interest. acknowledgement the authors would like to tha nk the biomedical labora to ry unit, brawij aya university. funding non-tax state revenue fund, faculty of medicine , brawij aya unive rsity ( dana penerimaan negara bukan paj ak/pnpb, fakultas kedokteran universitas brawijaya). author contributions dpe: basic concept, design, writing the manuscript and performing the experiment. sb: supervising, critical reviewing and final approval of the manuscript. wk: analyzing, interpreting data, and helping draft the manuscript. hb and 158 ar: critical reviewing and final approval of the manuscript. all authors have read and approved the final manuscript. references 1. peng w, novak n. pathogenesis of atopic dermatitis. clin exp allergy 2015;45:566-74. doi: 10.1111/cea.12495. 2. hur ss, choi sw, lee dr, park jh, chung th. advanced effect of moringa oleifera bioconversion by rhizopus oligosporus on the treatment of atopic dermatitis: preliminary study. evid based complement alternat med 2018;2018: 7827565. doi: 10.1155/2018/7827565. 3. lim jm, lee b, min jh, et al. effect of peiminine on dncb-induced atopic dermatitis by inhibiting inflammatory cytokine expression in vivo and in vitro. int immunopharmacol 2018;56:135-42. doi: 10.1016/j.intimp.2018.01.025. 4. rodrigues ma, torres t. jak/stat inhibitors for the treatment of atopic dermatitis. j dermatolog treat 2019;31:33-40. doi: 10.1080/09546634. 2019.1577549. 5. simpson el, leung dym, eichenfield lf, boguniewicz m. atopic dermatitis: in: kang s, amagai m, bruckner al et al., editors. fitzpatrick’s dermatology in general medicine, 9th ed. new york: mcgraw hill; 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kim jy, jeong ms, et al. effect of topical application of quercetin-3-o-(2 3 -gallate)-α-lrhamnopyranoside on atopic dermatitis in nc/ nga mice. j dermatol sci 2015;77:166-72. doi: 10.1016/j.jdermsci.2014.12.005. soursop leaf extract increases neuroglia and hepatic degeneration in female rats 17 *anatomy department, faculty of medicine, islamic university of indonesia, yogyakarta **histology department, faculty of medicine, islamic university of indonesia, yogyakarta ***medical profession program, faculty of medicine, islamic university of indonesia, yogyakarta correspondence dr. ety sari handayani, m.kes anatomy department, faculty of medicine, islamic university of indonesia jl. kaliurang km14,5 yogyakarta phone: +62274-898470 ext. 2033 fax: +62274-898470 ext. 2007 email: eshyasser@yahoo.co.id univ med 2015;34:17-24 doi: 10.18051/univmed.2016.v35.17-24 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background soursop leaf contains annonaceous acetogenins and alkaloids. the acetogenins act as inhibitors of mitochondrial complex i, suppress atp production and cause cell degeneration, whereas the alkaloids act as neurotoxins. neuronal degeneration will be followed by an increase in neuroglia (gliosis). hepatic clear cell foci represent the morphology of liver degeneration. the purpose of this study was to evaluate the effect of soursop leaf extract on number of neuroglia brain gliosis and hepatic clear cells in female rats. methods this study was an experimental study with a post-test only control group design. ten female sprague-dawley strain rats were divided into one control and one treatment group. the control group was gavaged with distilled water, while the treatment group was gavaged with aqueous soursop leaf extract at a dose of 1000 mg/kgbw/day for 90 days. rat brain tissue samples were taken at day 91 with a transcardial perfusion technique. the number of neuroglia in rat cerebral cortex, hippocampus, substantia nigra, and nucleus accumbens and the number of hepatic clear cells were determined. independent t-test was used to examine the differences in the numbers of neuroglia and hepatic clear cells between control and treatment groups. results the results of independent t-test analysis found a significant difference in the number of neuroglia in the cerebral cortex (p=0.015) and nucleus accumbens of the rats (p=0.030), and significant differences in the number of hepatic clear cells (p=0.029). conclusions aqueous soursop leaf extract orally increases neuroglia of the cerebral cortex and nucleus accumbens, and hepatic degeneration in female rats. keywords: soursop leaf, degeneration, brain, liver, female rats soursop leaf extract increases neuroglia and hepatic degeneration in female rats ety sari handayani*, zainuri sabta nugraha*, ika fidianingsih**, and prilly raleka pahlevawati*** doi: http://dx.doi.org/10.18051/univmed.2015.v34.17-24 18 handayani, nugraha, fidianingsih, et al soursop leaf increases gliosis ekstrak air daun sirsak meningkatkan neuroglia dan degenerasi hepar pada tikus betina latar belakang daun sirsak mengandung senyawa aktif annonaceous acetogenins dan alkaloid. acetogenin sebagai inhibitor kompleks i mitokondrial menurunkan produksi atp menyebabkan degenerasi sel. senyawa alkaloid bersifat neurotoksin. degenerasi neuron ditandai dengan peningkatan neuroglia (gliosis). clear cell focus hepatosit adalah gambaran morfologi degenerasi hepar. tujuan dari penelitian ini adalah untuk menilai efek ekstraks air daun sirsak terhadap jumlah neuroglia dan jumlah clear cell focus hepatosit pada tikus betina. metode rancangan penelitian eksperimental ini adalah post test only control group design, menggunakan 10 ekor tikus betina galur sprague-dawley, terbagi menjadi dua kelompok yaitu kelompok kontrol dan perlakuan. kelompok kontrol mendapatkan sondase aquades, dan kelompok perlakuan mendapatkan ekstrak air daun sirsak dosis 1000 mg/kgbb/hari selama 90 hari. pada hari ke 91, jaringan otak tikus diambil dengan menggunakan transcardial perfusion technique. jumlah neuroglia diamati pada cortex cerebri, hippocampus, substansia nigra dan nucleus accumbens tikus. uji t-independen digunakan untuk menguji adanya perbedaan jumlah neroglia dan haposit hepar antara kelompok kontrol dan perlakuan. hasil analisis dengan uji t independen menunjukkan adanya perbedaan bermakna antara kedua kelompok perlakuan, jumlah neuroglia di cortex cerebri (p=0,015) dan nucleus accumbens tikus (p=0,030), serta terdapat perbedaan bermakna clear cell focus hepatosit (p=0,029). kesimpulan pemberian ekstrak air daun sirsak secara oral meningkatkan neuroglia di cortex cerebri dan nucleus accumbens otak serta degenerasi hepar pada tikus betina. kata kunci : daun annona muricata, degenerasi, otak, hepar, tikus betina abstrak introduction annona muricata has been used for the prevention and treatment of cancer.(1) the phytochemicals present in the plant possess antioxidant activities, which can be used in the prevention or treatment of many diseases, including cancer. however, several species of the annonaceae family, including a. muricata, have also been targeted for investigation due to appurtenant substances among the acetogenins.(2) annonaceous acetogenins have cytotoxic properties that can inhibit the mitochondrial complex i enzyme. inhibition of this enzyme significantly reduces the production of energy (atp), cause cancer cell death.(1,3) however, the inhibitory effect may also occur in normal cells, particularly those with high atp requirements, such as the brain, liver, and kidney. various studies show that the soursop plant exerts side effects on healthy cells. administration for 28 days of purified acetogenins derived from soursop plant roots, was capable of causing degeneration of substantia nigra neurons in the r a t b r a i n , w i t h s y m p t o m s s i m i l a r t o parkinsonism. annonacin can cause cortical 19 neuronal death in in-vitro experiments.(4) inhibition of mitochondrial complex i causes a reduction in atp associated with increased tau protein.(5) administration of annonacin does not always result in toxicity. mutant r406w mice had a decreased number of cortical, parietal and hippocampal neurons in comparison with nonmutant mice.(6) the liver is composed of hepatocytes that are arranged radially around the central vein. the cytoplasm of hepatocytes contain 2000 mitochondria. the large number of mitochondria in hepatocytes is associated with the high performance of the liver in the body. in rat hepatocytes receiving ethanolic leaf extract of the sugar apple (annona squamosa), another member of the annonaceae family, there was an increase in their apoptotic index in he stained preparations.(7) the brain and the liver are vital organs in the body. the performance of the brain and the liver is highly dependent on the availability of atp in the cells. physiologically, if the decrease in atp production occurs in the long run, the cells may degenerate and die by necrosis or apoptosis.(8-10) damage to the neurons will be followed by an increase in the number of neuroglia (gliosis). liver damage can result in hepatic clear cell foci. (11) based on these facts, the objective of this study was to evaluate the effect of soursop leaves extract on brain gliosis and hepatic clear cell foci in female rats. methods research design t h e d e s i g n o f t h i s r e s e a r c h w a s a n experimental posttest only control group. the study was conducted from april until october 2013 at the pharmacy laboratory and histology laboratory, islamic university of indonesia, the integrated research and testing laboratory, gadjah mada university (lppt ugm) and the microanatomy laboratory, faculty of veterinary medicine, gadjah mada university (fkh ugm). animals and experimental procedure the animals used in this study were female rats (rattus norvegicus of the sprague-dawley strain) that had met the inclusion and exclusion c r i t e r i a . t h e r a t s w e r e r e a r e d i n t h e pharmaceutical laboratory, islamic university of indonesia. inclusion criteria for this study were healthy 3-month old female rats without any defects, of 175-250 g body weight, and normal hemoglobin and sgot levels. exclusion criteria were pain and disability in the rats during the course of the study. the samples used were 10 rats that were divided into 2 groups: the control and the treatment group, each group consisting of 5 rats. sample size determination was according to the guidelines of lwanga and lemeshow. the treatment group was gavaged with aqueous soursop leaf extract at 1000 mg/ kgbw/day given in volumes of 2 ml/day. the control group was gavaged with distilled water at a dosage of 2 ml/day. the interventions were performed for 90 days. preparation of soursop extract soursop leaves were selected from the 4th to 6th level branches of the trees. preparation of the aqueous soursop leaf extract was done in the integrated research and testing laboratory, gadjah mada university. preparation of brain tissue samples rat brain tissue samples were taken at day 91 with a transcardial perfusion technique. the samples were then made into paraffin blocks and he stained sections. the parts of the rat brain that were studied comprised the cerebral cortex, hippocampus, nucleus accumbens and substantia nigra. observations on vacuolization and neuroglial number were made using an olympus cx21 light microscope with an optilab camera and linked to a computer containing the optilab viewer software program. in each sample, all fields of view were taken. in one field of view univ med vol. 34 no.1 20 handayani, nugraha, fidianingsih, et al soursop leaf increases gliosis were taken all neuroglia located to the right of the left border of the photo frame and above the lower limit of the photo frame. the results were read by a single person. the neuroglia were studied morphologically. oligondendrocytes have dark-colored round or oval nuclei with darkcolored cytoplasms. microglia have dark-colored bean-shaped or irregular nuclei and dark-colored cytoplasms that are lighter in color than those of oligodendrocytes. astrocytes have pale nuclei. rat liver tissue samples were taken on day 91 prior to the transcardial perfusion technique. the microscopic features of focal hepatocyte lesions were observed in their cytoplasms. the number of hepatic clear cells was determined on he stained sections, in all fields of view in each section. the main microcopic feature was the nearly white color of the unstained cytoplasm of the hepatocytes. the observation were made using an olympus cx21 light microscope with an optilab camera and linked to a computer containing the optilab viewer software program. in each sample, all fields of view were taken. the cytoplasmic vacuolization was observed at 1000 x total magnification (using a 100x objective lens and a 10 x ocular lens). after microscopic observation, the morphology was recorded as images of 640x480 pixels using the optilab viewer. statistical analysis independent t-test was used to determine the differences in the numbers of neuroglia and hepatic clear cells between control and treatment groups. ethical clearance this study was reviewed by the ethical clearance committee for preclinical research, integrated research and testing laboratory, gadjah mada university (lppt ugm) and obtained ethical clearance under no.139/keclppt/iii/2014. figure 1. vacuolization (black arrows) in substantia nigra neurons (a, b), cytoplasmic vacuolization of neurons (black arrows), neuroglia (red arrows) in the hippocampus (c), necrosis of neurons (pycnotic nuclei) (yellow arrows) in the hippocampus (d) (he staining at 1000x magnification). a b c d 21 table 1. mean number of hepatic clear cells and brain neuroglia, by treatment group results the present study used 10 rats assigned randomly to the control and treatment groups. the rats were in healthy condition and during the study no rats were disabled or died. overall, the administration of the aqueous extract of soursop (annona muricata) leaves o r a l l y f o r 9 0 d a y s r e s u l t e d i n n e u r o n a l degeneration in the rat substantia nigra and neuronal vacuolization in the hippocampus (figure 1). in the rat liver, there were cytoplasmic vacuolization, clear cell foci, and eosinophilic hepatocytes (figure 2). the number of neuroglia and hepatic clear cells were counted in the entire field of view of each preparation. administration of soursop leaf extract for 90 days resulted in a significant mean increase in hepatic clear cells and brain neuroglia in the treatment group in comparison with the control group, except in the hippocampus and substantia nigra (table 1). discussion the interesting thing about this study is the increase in neuroglia in the nucleus accumbens and cerebral cortex. in contrast, there was no increase in neuroglia in the substantia nigra and the hippocampus of the rats. increased neuroglia (gliosis) occurs when neurons undergo necrosis. the necrotic neurons will be surrounded by neuroglia. this relates to the function of neuroglia as supporter cells. the results of this study showed that aqueous soursop leaf extract given orally for 90 days had not been able to cause necrosis in the rat substantia nigra. the damage in the substantia nigra neurons only reached the stage of degeneration. this is supported by microscopic findings in substantia nigra neurons in the form of cytoplasmic vacuolization. similarly there was neuronal cytoplasmic vacuolization in the hippocampus. this is understandable given the strength of acetogenin compounds in soursop plants. the leaves of annona muricata contain neurotoxic figure 2. a. acidophilic hepatocyte (red arrow), b. hepatic clear cells (black arrows), c. cytoplasmic vacuolization (black arrow) (he staining at 1000x magnification) ba c univ med vol. 34 no.1 22 handayani, nugraha, fidianingsih, et al soursop leaf increases gliosis isoquinolinic alkaloids and acetogenins. isoquinolinic alkaloids can damage dopaminergic neurons and non-dopaminergic neurons (gabaergic). annonacin, an acetogenin compound that is present in soursop leaves, is able to passively diffuse into the cell. this compound is 50 times more toxic to dopaminergic neurons and 2000 times more toxic to non-dopaminergic neurons, w h e n c o m p a r e d w i t h 1 m e t h y l 4 phenylpyridinium (mpp).(12,13) the substantia nigra is a brain structure that is rich in dopaminergic neuronal cell bodies. these findings raise the possibility that the acetogenins in aqueous soursop leaf extract preferentially affect non-dopaminergic neurons as compared to dopaminergic neurons. this supports the findings in this study where the nucleus accumbens and cerebral cortex have more severe damage resulting in gliosis compared with the substantia nigra and the hippocampus, which merely undergo a process of degeneration characterized by cytoplasmic vacuolization of rat neurons. the results are consistent with research f i n d i n g s b y c h a m p y e t a l . ( 1 3 ) o f s e v e r e degeneration of dopaminergic neurons in the substantia nigra in the form of cytoplasmic vacuolization upon administration of purified annonacin for 28 days, giving rise to symptoms of parkinson’s disease in rats. degenerating neurons showed a picture of dystrophy and enlargement of the nerve fibers, and swelling of the nucleus.acetogenins and alkaloids contained in the plant soursop are able to damage dopaminergic neurons. various studies at the cellular as well as community level have demonstrated the neuronal damage after administration of this compound.(14,15) neuronal necrosis occurs after repeated exposure to neurotoxins. the term necrosis can be used to describe the state of ischemic neurons. the cell body will shrink due to reduced neuronal cell cytoplasm. in he staining, the cell body will a p p e a r e o s i n o p h i l i c . t h e f i n a l s t a g e o f degeneration, neuronal necrosis, will appear in t h e f o r m o f c h a n g e s t o t h e n u c l e u s a s karyorrhexis or karyolysis. these changes can be observed by using a light microscope. necrotic neurons will be surrounded by glia (gliosis). cellular mechanisms underlying neurodegeneration involve the accumulation of tau protein in the cytoplasm of dopaminergic neurons. tau is a microtubule-associated protein (map) located in the axon. the presence of microtubular damage stimulated by atp upon the administration of annonacin causes tau protein to accumulate in the cytoplasm of dopaminergic neurons.(12) the ability of acetogenins to damage dopaminergic neuronal cell bodies in the area ventralis tegmenti and the substantia nigra will indirectly affect the neurons in the nucleus accumbens. dopaminergic neurons project to the nucleus accumbens so that in the event of damage or cell death of dopaminergic neurons in the area ventralis tegmenti or substantia nigra, it will affect the neurons in the nucleus accumbens.(16,17) the toxic effect of soursop leaves may also be due to its alkaloid content. mohanty found that the imino sugar alkaloid swainsonine has n e u r o t o x i c p r o p e r t i e s . s w a i n s o n i n e i s a mannosidase inhibitor that exert neurotoxic effects by inhibition of lysosomal acidic alpha mannosidase. the swainsonine content of soursop leaves is 0.0004% of their dry weight. if consumed in amounts of 0.001% of the diet, t h e c o m p o u n d m a y c a u s e n e u r o l o g i c a l disorders.(17) other soursop alkaloids are 1 benzyl-1,2,3,4-tetrahydroisoquinoline (1bntiq) and annonamine. the compound 1bnt1q can demonstrably induce symptoms of parkinsonism in experimental animals. annonamine is neurotoxic for cathecholaminergic neurons, thus inducing parkinsonism.(18) the toxic effects of s o u r s o p i n h u m a n s h a v e b e e n r e p o r t e d previously. it is said that there is a relationship between consumption of soursop products and brain atrophy with enlargement of the cerebral ventricles.(19) the microscopic picture of hepatic focal lesions can be seen in the cytoplasm of hepatocytes. on the basis of hematoxylin-eosin 23 staining, hepatic focal lesions can be divided into the following types: basophilic, eosinophilic, clear cell, or mixed. the results of this study indicate the presence of clear and acidophilic cell foci. a clear cell focus is characterized by swelling of hepatocytes, followed by an increase in the size of the nucleus. all drugs of any kind either herbal, natural, or synthetic can give side effects to various organs, among which the liver is the center of d r u g m e t a b o l i s m a n d d e t o x i f i c a t i o n . ( 2 0 ) nascimento et al.(7) in their study on the effects of annona coriacea (araticum) found that the e t h a n o l e x t r a c t o f t h e p l a n t c a n c a u s e hepatotoxicity. hepatotoxicity is determined by a decrease in the number of cells per area, changes in the morphology of hepatocyte nuclei cytoplasmic vacuolization, and decreased appetite. there are two theories regarding the m e c h a n i s m o f l i v e r d a m a g e i n d u c e d b y acetogenin. the first theory is through the mechanism of adaptation of the liver to the burden of acetogenin metabolism. the liver is the organ most frequently damaged. around 80% of the liver blood supply comes from the hepatic p o r t a l v e i n t h a t d r a i n s b l o o d f r o m t h e gastrointestinal system. toxic substances including those from plants, fungi, and bacteria, and also metals, minerals and other chemical substances that are absorbed into the portal blood system are carried toward the liver as the center of drug metabolism. the second theory is through a direct mechanism of acetogenin on hepatocytes, because hepatocytes have mitochondria. acetogenin which is the active compound contained in soursop inhibits mitochondrial complex i activity and is likely to cause side effects. but the complete explanation is not known with certainty.(21) subacute treatment with a. muricata aqueous extract for 14 days had no effect on the weights of the liver, kidneys, heart and stomach, while the uterus increased in weight to 1000 mg/ kg and beyond. hematological parameters, alt, ast, alp, urea and albumin were unaffected while creatinine levels were increased at 2500 mg.(22) a limitation of this study was the use of a single dose of aqueous soursop leaf extract, thus precluding the determination of the minimal dose that can cause damage to healthy cells. the study results demonstrate that soursop leaves are not completely safe when used as herbal medication, since it can cause injury to healthy cells. consumers of soursop leaves will hopefully pay a t t e n t i o n t o t h e d o s e a n d d u r a t i o n o f administration, in order to prevent the side effects of degeneration and necrosis of healthy cells. further studies are indicated to determine the minimal dose and duration of administration that can induce degeneration and necrosis of healthy cells. conclusion an aqueous extract of soursop (annona muricata) leaves given orally increases the numbers of neuroglia in the cerebral cortex and nucleus accumbens of the brain and the number of hepatic clear cells in rats. acknowledgements the authors are indebted to uppm medical faculty, islamic university of indonesia, for supporting this study. references 1. moghadamtousi sz, kadir ha, paydar m, et al. annona muricata leaves induced apoptosis in a549 cells through mitochondrial-mediated parhway and involvement of nf-kb. complement alt med 2014;14:299. 2. mishra s, ahmad s, kumar n, et al. annona muricata (the cancer killer): a review. global j pharm res 2013;2:1613-8. 3. liaw cc, wu ty, chang fr, et al. historic perspectives on annonaceous acetogenins from the chemical bench to preclinical trials. planta med 2010;76:1390-404. 4. pott lf, luzzio fa, smith sc, et al. annonacin in asimina triloba fruit: implication for univ med vol. 34 no.1 24 handayani, nugraha, fidianingsih, et al soursop leaf increases gliosis neurotoxicity. neurotoxicol 2012;33:53-8. doi: 10.1016/j.neuro.2011.10.009. 5. hollerhage m, matusch a, champy p, et al. natural lipophilic inhibitors of mitochondrial complex i are candidate toxins for sporadic neurodegenerative tau pathologies. exp neurol 2009;220:133-42. 6. yamada es, respondek g, mussner s, et al. annonacin, a natural lipophilic mitochondrial complex i inhibitor, increases phosphorylation of tau in the brain of ftdp-17 transgenic mice. exp neurol 2014;253:113-25. 7. nascimento gnl, valadares mc, nishijo h, et al. investigation of the toxic potential of crude ethanol extract of annona coriacea (araticum) seeds in acute exposed mice. brazilian j pharmacog 2012;22:580-6. 8. alvarez co, neske a, chahboune n, et al. ucupentol, a novel mono-tetrahydrofuranic acetogenin from annona montana, as a potent inhibitor of mitochondrial complex i. chem biodivers 2009;6:335-40. 9. dayeef ay, karyono s, sujuti h. the influence of annona muricata leaves extract in damaging kidney cell and inducing caspase-9 activity. j pharm biol sci 2013;8:48-52. 10. li k, li q, li j, et al. alkaloid from angelicae daharaicae inhibits hela cell growth by inducing apoptosis and increasing cascape–3 activity. lab med 2008;399:540-6. 11. mcmartin dn, o’donoghue jl, morrissey r, et al, editors. non-proliverative lesions of the nervous system in rats, ns-1. in: guides for toxicologic pathology. washington, dc: stp/ arp/afip;1997.p.51-76. 12. escobar-khondiker m, hollerhage m, paulemuriel m, et al. annonacin, a natural mitochondrial complex i inhibitor, causes tau pathology in cultured neurons. j neurosci 2007; 27:7827-37. 13. champy p, melot a, guérineau v, et al. quantification of acetogenins in annona muricata linked to atypical parkinsonism in guadeloupe. mov disord 2005;20:1629-33. 14. shaw ca, hoglinger gu. neurodegenerative disease: neurotoxins as sufficient etiologic agent? neuromol med 2008;10:1-9. 15. arif ia, khan ha. environmental toxins and parkinson’s disease: putative roles of impaired electron transport chain and oxidative stress. toxicol ind health 2010;26:121-8. doi: 10.1177/ 0748233710362382. 16. richard jm, berridge kc. nucleus accumbens dopamine/glutamate interaction switches modes to generate desire versus dread: d1 alone for appetitive eating but d1 and d2 together for fear. j neurosci 2011;31:12866-79. 17. mohanty s, hollinshead j, jones l, et al. annona muricata (graviola): toxic or therapeutic. natural product comm 2008;3:31-3. 18. matsushige a, kotake y, matsunami k, et al. annonamine, a new aporphine alkaloid from the leaves of annona muricata. chem pharm bull 2012;60:257-9. 19. lannuzel a, hoglinger gu, verhaeghe s, et al. atypical parkinsonism in guadeloupe: a common risk factor for two closely related phetotypes? brain 2007;130:816-27. 20. warisno dk. daun sirsak: langkah alternatif menggempur penyakit. jakarta: gramedia pustaka utama;2012. 21. lim tk. edibel medicinal and non-medicinal plants. fruits 2012;1. doi: 10.1007/978-90-4818662-7_1. 22. arthur fkn, woode e, terlabi eo, et al. evaluation of acute and subchronic toxicity of annona muricata (linn.) aqueous extract in animal. eur j 2011;4:115-24. alvina 56 abstract cancer-associated retinopathy (car) is a paraneoplastic syndrome most commonly associated with small-cell carcinoma of the lung, but also less frequently reported in patients with breast, endometrial, and other cancers. a paraneoplastic syndrome (pns) is a secondary organ dysfunction occurring in a cancer patient at a site that is anatomically remote from the tumor. pns is not due to a direct effect of the tumor itself or its metastases but caused by other mechanisms, commonly autoimmune mechanisms develop when malignant tumors express proteins, paraneoplastic antigens (pna), which are normally present only in neurons. one retinal antigen implicated in the autoimmune mechanism of car is recoverin, a 23 kda photoreceptor-specific calciumbinding protein modulating the activity of photoreceptor guanylyl cyclase. the anti-recoverin antibodies induced by the primary tumor may on contact with intraretinal recoverin initiate a photoreceptor degeneration and trigger photoreceptor death by apoptosis, thus causing blindness. other circulating antibodies directed against a 46 kda protein identified as retinol enolase and a 60 kda retinal protein have been demonstrated in patients with clinically diagnosed car syndrome. in certain patients no specific antibody has been identified. this suggests that the car syndrome includes an heterogenous group of autoimmune conditions directed against various retinal proteins. keywords: cancer-associated retinopathy, paraneoplastic, recoverin, autoimmune *department of ophthalmology, gatot subroto central military hospital correspondence dr. nurbuanto tradjutrisno, spm department of ophthalmology, gatot subroto central military hospital jl. abdul rahman saleh no.24, jakarta pusat phone: 021-3441008 email: ntradjutrisno@gmail.com univ med 2010;29:56-64 cancer-associated retinopathy: an autoimmune retinopathy nurbuanto tradjutrisno* january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina introduction a paraneoplastic syndrome is a secondary organ dysfunction occurring in a cancer patient at a site that is anatomically remote from the tumor. paraneoplastic syndromes are a complex of symptoms not due to a direct effect of the tumor itself, its metastases, opportunistic infections, metabolic abnormalities, vascular diseases, coagulopathies, complications of therapies, or malnutrition, but caused by i m m u n e o r o t h e r m e c h a n i s m s . m o s t paraneoplastic syndromes are associated with small-cell carcinoma of the lung (sccl), 57 univ med vol.29 no.1 g y n e c o l o g i c m a l i g n a n c i e s , a n d b r e a s t neoplasms (ductal adenocarcinoma), while the symptoms frequently are in the form of a b n o r m a l i t i e s o f t h e n e r v o u s s y s t e m [paraneoplastic neurologic syndrome (pns)]. pns are rare neurological disorders that are related to cancer, even though the primary tumor and its metastases have not invaded the n e r v o u s s y s t e m . p n s a r e t h o u g h t t o b e autoimmune diseases that develop when m a l i g n a n t t u m o r s e x p r e s s p r o t e i n s , paraneoplastic antigens (pna), which are normally present only in neurons.(1) in one case pns occurred in a patient with occult breast cancer(2) and in a child with langerhans cell histocytosis.(3,4) the abnormalities of pns are frequently the result of autoimmune disorders, but may also be due to other mechanisms. in pns associated with autoantibodies, these antibodies are targeted against antigens shared by tumor cells and nervous tissue (onconeural antigens). these immune responses subsequently will recognize the same antigens or epitopes occurring in other organs, e.g. in the nervous system. a second group of autoimmune pns are monoclonal g a m m o p a t h y s y n d r o m e s a s s o c i a t e d w i t h secretion of an antibody by the neoplasm.(3) that some pns are based on autoimmune reactions was first demonstrated for the lambert-eaton syndrome, which is a myasthenia-like disorder o f t h e p e r i p h e r a l n e r v o u s s y s t e m w i t h autoantibodies that bind to presynaptic calcium channels at the neuromuscular junction. this binding disturbs the release of acetylcholine, thus leading to weakness of the proximal muscles and associated abnormalities. in another paraneoplastic syndrome, namely p a r a n e o p l a s t i c c e r e b e l l a r d e g e n e r a t i o n , autoantibodies were found against purkinje cells in the cerebellum of a patient. the researchers subsequently also found purkinje cell antigens in gynecologic tumors in the affected patient, but not in patients with similar t u m o r s w h o d i d n o t h a v e n e u r o l o g i c a l abnormalities. paraneoplastic syndromes affecting the visual system have also been reported, mainly consisting of cancer-associated retinopathy (car) and melanoma-associated retinopathy (mar). car was first described by sawyer et al.,(5) while mar was first described in 1988 by berson et al.(6) the prevalence of car is unknown, with less than 100 reported cases worldwide, but car is still more common than mar. the primary tumor most commonly associated with car (90%) is small sclc, but it has also been less frequently reported in patients with breast, endometrial, and other cancers. mar has been described only in a s s o c i a t i o n w i t h c u t a n e o u s m a l i g n a n t melanoma, and not with melanomas affecting other tissues. in car, the visual loss frequently precedes the discovery of sclc, while in mar the retinopathy commonly presents after the melanoma is diagnosed, often at the metastatic stage.(7) in this review the characteristics and probable pathogenesis of the car syndrome will be discussed, with some notes on apoptosis due to antirecoverin antibodies. clinical picture car is characterized by a functional and progressive abnormality of the rods and cones, without any other neurological dysfunction. car should be suspected in patients who present with visual symptoms such as rapid unexplained visual loss and seeing shimmering lights, a c c o m p a n i e d b y a m a r k e d l y a t t e n u a t e d electroretinogram (erg). the chief complaint in car is unilateral or bilateral, symmetric or asymmetric, total or partial loss of vision, but night blindness may also be the presenting symptom. the visual loss occurs either gradually or in a stepwise pattern over weeks to months. other symptoms may include visual shimmering, sparkling, or distortions. examination shows poor visual acuity, impaired color vision, constriction of visual fields, and an afferent pupillary defect. visual field defects most commonly consist of 58 central and/or ring scotomas. histologically, there is severe loss of the inner and outer s e g m e n t s o f t h e r o d s a n d c o n e s , w i t h widespread degeneration of the outer nuclear layer. the electroretinogram (erg) is usually flat due to loss of the photoreceptors. the symptoms of car are in the majority of cases bilateral, although occasionally a time interval o f s e v e r a l w e e k s m a y s e p a r a t e t h e symptomatology between the two eyes, which in turn commonly precedes the discovery of the primary tumor by several weeks to several months.(7,8) the clinical presentation of car and mar is rather similar, but the erg pattern is different. in car, scotopic and photopic aand b-waves are either absent or severely abnormal, whereas in mar maximal combined rod-cone erg responses are electronegative. as an apparent exception goetgebuer et al.(7) reported a case of car with electronegative erg, leading the investigators to initially suspect mar. however, the underlying malignancy turned out to be an oat-cell carcinoma in the right lung. the patient died of pneumonia 2 years after presentation. treatment of car with corticosteroids (methylprednisolone iv), immunoglobulins a n d p l a s m a p h e r e s i s h a v e p r e s u m a b l y demonstrated their efficacy in reducing the titer of circulating antibodies and in maintaining effective vision until demise of the patient. however, spontaneous recovery of vision in car has not been reported. the prognosis ad vitam depends on the primary tumor.(8,9) an old man with lung adenocarcinoma presenting with car at the age of 65 years was reported by oohira.(10) the patient had been followed-up for the last 15 years after the resection of the lung tumor in 1991. igg of 23 kda and 65 kda, identified as antirecoverin and anti-heat shock cognate protein antibodies, respectively, were detected in his serum preoperatively, and the patient was treated with corticosteroids. the fundi and visual fields of both eyes gradually deteriorated over 13 years, but they had become stable in the last 2 years. espandar et al.(11) reported on a 66-year old women who had lumpectomy and chemotherapy for breast cancer in 1978 and serologically p r o v e n c a r a n d p a r a n e o p l a s t i c o p t i c neuropathy in 1988. over an 8-year follow-up period after the initial diagnosis of car, the p a t i e n t w a s s u c c e s s f u l l y t r e a t e d w i t h alemtuzumab after therapeutic failures of respectively prednisone, plasmapheresis and cyclosporin. alemtuzumab therapy allowed her to maintain fairly good visual function during the entire follow-up period, although she experienced some permanent decline in her visual fields and visual acuity after each of three episodes of car. however, it should be noted that her serum contained antibodies against the 40 kda car antigen but not the 23 kda car antigen. alemtuzumab is a recombinant dnaderived humanized monoclonal antibody that is directed against the 21-28 kda cell surface glycoprotein cd52. cd52 is expressed on the surface of normal and malignant b and t lymphocytes, natural killer cells, monocytes, m a c r o p h a g e s , a n d t i s s u e s o f t h e m a l e reproductive system. pathogenesis as in other autoimmune pns, car is a p p a r e n t l y t h e r e s u l t o f a n a u t o i m m u n e process. in the serum of one patient with carcinoma of the cervix, antibodies against human retinal photore-ceptors were found, whereas in two others their serum reacted with ex-tracts of normal human, murine and bovine retinas, also with human and bovine choroid, but not with extracts of the lens or iris. there were only low background readings by sera of normal individuals or of cancer patients without disturbances of vision. these findings suggested that the antigen for this pns was situated in the retina-choroidal complex.(12) one of the first retinal antigens to be implicated in the autoimmune mechanism of the car syndrome was a 23 kda protein tradjutrisno cancer-associated retinopathy 59 univ med vol.29 no.1 identified as recoverin, a photoreceptorspecific calcium-binding protein modulating t h e a c t i v i t y o f p h o t o r e c e p t o r g u a n y l y l cyclase.(13,14) recoverin is aberrantly expressed in more than 50% of 33 cancer cell lines.(15) the anti-recoverin antibodies induced by the primary tumor may on contact with intraretinal recoverin initiate a photoreceptor degeneration and trigger photoreceptor death by apoptosis, t h u s c a u s i n g b l i n d n e s s . ( 1 6 ) c i r c u l a t i n g antibodies directed against a 46 kda protein i d e n t i f i e d a s r e t i n o l e n o l a s e h a v e b e e n demons tr ated in patients with clinically diagnosed car syndrome. other autoantigens include the s-antigen (arrestin) and tubby-like p r o t e i n 1 ( t u l p 1 ) w h i c h i s a m o l e c u l e e x p r e s s e d i n s y n a p t i c t e r m i n a l s o f photoreceptor cells.(17) some reports have also implicated heat shock proteins (specifically h e a t s h o c k c o g n a t e p r o t e i n 7 0 ) i n c a r autoimmunity. however, because both enolases and heat shock proteins are expressed in every tissue, it is difficult to attribute an organspecific autoimmune disease to immunological activity with these ubiquitous antigens. in certain patients no specific antibody has yet been identified, suggesting that the car syndrome includes an heterogenous group of a u t o i m m u n e c o n d i t i o n s d i r e c t e d a g a i n s t various retinal proteins.(18,19) neuronal calcium sensors calcium as a second messenger controls many biological processes and one of the mechanisms is by interaction with calciumbinding proteins. among these proteins there is one class, the ef-hand superfamily, that shares a common calcium binding motif, namely the ef-hand. ef-hand proteins with regulatory roles are often termed calcium s e n s o r p r o t e i n s ( c s p ) , c o m p r i s i n g a heterogeneous class of proteins that includes calmodulin (cam), neuronal calcium sensors (ncs), visinlike protein (vilip), neurocalcin, hippocalcin, and the recently identified s100 family members. ncs are neuron-specific calcium sensor proteins, of which recoverin is the best-known example. recoverin (also called s-modulin in the frog) is a myristoylated csp e x p r e s s e d p r e d o m i n a n t l y i n v e r t e b r a t e photoreceptor cells. calcium ion plays a critical role in the recovery phase of visual excitation and in adaptation to background light. the lightinduced lowering of the calcium level in retinal rod outer segments restores the dark state by stimulating guanylate cyclase, decreasing the photosensitivity of the cgmp phosphodiesterase and switching the cgmp-gated channel to the high affinity form. calcium-bound recoverin prolongs the photoresponse most likely by blocking the phosphorylation of photoexcited rhodopsin. this effect of recoverin is reversed by the light-induced lowering of the calcium level. the shortened lifetime of photoexcited rhodopsin, and hence of the phosphodiesterase, at low calcium concentrations promotes recovery of the dark state and contributes to adaptation to background light.(20) characteristics and effects of recoverin recoverin, a relatively new member of the ef hand superfamily, was discovered in the early 1990s in the search for a soluble calciumsensitive activator of guanylate cyclase. it is a 23 kda calcium-binding protein present only in vertebrate photoreceptors, in certain other retinal neurons, and in pineal glands. the threed i m e n s i o n a l s t r u c t u r e o f r e c o m b i n a n t unmyristoylated recoverin was elucidated and shown to be a compact protein made up of two domains separated by a narrow cleft. each domain contains a pair of ef hands, which are the 29 residue helix-loop-helix motifs found in parvalbumin, troponin c, calmodulin, and other members of the superfamily. thus there are four potential ef hand calcium-binding sites in the recoverin molecule, but only two (the second and the third ef hands) are capable of binding calcium ions. in the crystal form of recombinant unmyristoylated recoverin, a calcium ion is bound to ef hand 3, while ef 60 hand 2 can bind samarium but not calcium. the other two ef hands have novel structural f e a t u r e s t h a t p r e v e n t o r i m p a i r c a l c i u m binding. recoverin can form a complex with rhodopsin kinase (grk-1) in vitro, thus inhibiting phosphorylation of the visual receptor rhodopsin in a calcium-dependent manner. it has been confirmed recently that r e c o v e r i n s e r v e s a s a c a l c i u m s e n s o r o f rhodopsin phosphorylation under physiological conditions. recoverin is also found as an a n t i g e n i n v a r i o u s n e o p l a s m s t h a t a r e accompanied by car.(17,20,21) the recombinant equivalent of this protein is now routinely used a s t h e t e s t a n t i g e n i n t h e s e r o l o g i c a l identification of recoverin hypersensitivity.(12) investigations into the mechanism of cell damage caused by antirecoverin antibodies revealed an apoptosis-inducing activity on in vitro cultivated monolayers of rat retinal cells, and antibody-mediated retinal degeneration in vivo following intraocular injection of these immunoglobulins into the lewis rat. this animal is genetically prone to the induction of autoimmune reactions in the eye and provides a useful model to demonstrate the pathological processes involved in immune-mediated retinal degeneration and the significance of inherited susceptibility.(12) to find out whether recoverin was able to induce the immunological mechanism leading to photoreceptor degeneration, lewis rats were immunized with recoverin and subsequently showed a high titer of serum a n t i b o d i e s a g a i n s t r e c o v e r i n , a n d a l s o degeneration of immunocompetent t cells and photorecep-tors.(12,17) polans et al.(17) proved that recoverin was expressed in the lung tumor of one patient with car, but not in similar lung tumors in patients without car. the immunodominant portion o f r e c o v e r i n w a s f o u n d b y s o l i d p h a s e immunoassay using overlapping heptapeptides t h a t i n c l u d e d t h e c o m p l e t e s e q u e n c e o f recoverin. the major determinant is formed by two linear strands of amino acids, namely residues 64-70 (lys-ala-tyr-ala-gln-his-val) and 48-52 (gln-phe-gln-ser-ile). residues 6180 are also pathogenic, causing photoreceptor degeneration in lewis rats immunized with these peptides. interestingly, amino acid residues 64-70 promote both autoantibody binding and pathogenicity. thus it is clear that recoverin as a nervous system antigen is in all probability responsible for the loss of vision in some cancer patients. while autoimmune diseases are widespread, car is one of the few autoimmune disorders where the specific selfantigen is known.(17) tumor tissues in car patients selectively express recoverin, which will react with the patients’ autoantibodies. thus there is the possiblity that the expressed recoverin, when released, will be able to trigger an autoimmune response resulting in retinal degeneration. in the study by adamus et al. it was proven that antirecoverin antibodies enter the cell and subsequently cause cell death by apop-tosis.(24) other circulating antibodies directed against a 46 kda protein identified as retinol enolase and a 60 kda retinal protein have been demonstrated in patients with clinically diagnosed car syndrome. however, in certain p a t i e n t s n o s p e c i f i c a n t i b o d y h a s b e e n identified. therefore, this suggests that the car syndrome includes an heterogenous group of autoimmune conditions directed against various retinal proteins. apoptosis as a cause of photoreceptor damage to evaluate the pathogenic effect of antirecoverin antibodies on retinal cells, in vitro experiments were performed using the immortalized rat retinal cell line e1a.nr3. this cell line contains cells expressing antigens specific for photoreceptors, bipolar cells and ganglion cells, including recoverin. it has been demonstrated that normal as well as specific antibodies may enter retinal cells. to find out the influence of antibodies on cell growth, the tradjutrisno cancer-associated retinopathy 61 univ med vol.29 no.1 following autoantibodies were used: five types of antirecoverin autoantibodies from car patients, antirecoverin antibodies of rats with experimental autoimmune uveoretinitis, and rabbit antirecoverin antibodies.(22) all of the above antirecoverin antibodies had a similar cytotoxic effect on e1a.nr3 cells, which effect depends on the number of antirecoverin antibodies administered and on time of exposure. the toxic effect was greater with high doses of antirecoverin antibodies and t h e n u m b e r o f v i a b l e c e l l s d e c r e a s e d s i g n i f i c a n t l y a f t e r 4 8 h o u r s . n o r m a l nonspecific antibodies had no effect on cell viability with identical doses and time of exposure. in a subsequent experiment e1a.nr3 cells were cultured in combination with antibody and complement. human antirecoverin antibodies are of the igg1 class and posses complement fixing ability. however, compared with cultures incubated with antirecoverin antibodies only, those with added complement showed no further cellular damage after 48 hours. the possible expression of fc receptors on the surface of e1a.nr3 cells was determined by an fc rosetting assay using igg-coated sheep erythrocytes. the assay results showed that the sheep erythrocytes did not form rosettes with the surface of e1a.nr3 cells. to test whether fc receptors were induced by antibodies, the e1a.nr3 cells were initially incubated with antibody for 4 hours, then the rosette test was performed. in this case also no rosette formation was seen. these results indicate that e1a.nr3 cells do not express fc-binding activity on their surface. to a s c e r t a i n w h e t h e r a n t i r e c o v e r i n antibodies are capable of influencing the growth o f c e l l s t h a t d o n o t e x p r e s s r e c o v e r i n , antirecoverin antibodies were cultured together with three types of cells, ie. human retinoblastoma cells y79, rat pheochromocytoma cells pc12, and rat hyphophyseal tumor cells gh3. the proteins extracted from these cells were examined for the presence of recoverin by western blot analysis using antirecoverin antibodies. none of these cell types expressed recoverin in culture, although retinoblastoma y79 cells expressed recoverin mrna. the cells h a d b e e n c u l t u r e d w i t h a h i g h d o s e o f antirecoverin (300 µg/ml) for 24 to 48 hours. by the immunoperoxidase method normal antibodies and antirecoverin antibodies were found within the cytoplasm of y79, pc12 and gh3 cells. however, the results of mtt cytotoxic assays showed that antirecoverin antibodies did not affect the growth and viability of cells not expressing recoverin. i n a s u b s e q u e n t e x p e r i m e n t , t o t h e e 1 a . n r 3 c e l l c u l t u r e w e r e a d d e d photoreceptor-specific monoclonal antibodies against another protein, i.e. arrestin, and incubated for 48 hours. arres-tin is expressed by e1a.nr3 cells. the results of culture with monoclonal antiarrestin antibody were similar with those of culture with antirecoverin antibody. after incubation with the highest dose of antibody, only 40 percent of the cells were still viable, compared with 100 percent viable cells in cultures with normal antibody. in the car syndrome there are occasional inflammatory cell infiltrates in the diseased tissues, but inflammation of the retina is rarely reported. as there is no inflammation in car, it may be surmised that retinal degeneration i n c a r d e v e l o p s a s a r e s u l t o f a n o n inflammatory process involving humoral i m m u n i t y, i . e . t h e p r o c e s s o f a p o p t o s i s . comparative analysis on e1a.nr3 cells c u l t u r e d w i t h s p e c i f i c a n t i b o d y a g a i n s t recoverin, irrespective of cellular origin (human, rat, rabbit), showed changes in cell morphology, including shrinking of cell bodies, blebs, retraction of cell processes, and release of cells from the tissue culture plates. cells cultured with control antibody on identical doses showed normal morphology. because specific as well as normal antibody may enter the cell, the effect of antirecoverin antibodies must be due to specific antibody activity. incubation of cells with antirecoverin antibodies 62 caused fragmentation of dna into 200-bp integers and condensation of nuclear chromatin. dna fragmentation was observed with all antirecoverin antibodies, both from patients’ and animal sera. however, dna fragmentation was not seen when the cells were incubated with normal antibody. condensation of chromatin was analyzed using the fluorochromes hoechst 33342 and propidium iodide. addition of antirecoverin antibodies to the cells caused apoptosis, which was apparent from the numerous brightly-colored cell nuclei, most of which belonged to dead (pink-colored) cells. the number of apoptotic cells was on average 20 percent of the cell population, whilst in the control culture without added antibodies or with normal antibodies, only 1-2 percent of the cells were apoptotic, which may have been due to the natural cell cycle process.(16) a possible mechanism of antirecoverininduced apoptosis of the photoreceptor cells is passage of the antibodies through leaks in the blood-retina barrier as a consequence of biochemical influences of the tumor, allowing internalization of the antirecoverin antibodies into the photoreceptor cells and access to the intracellular car antigen(s). the major result is that autoantibodies specific to recoverin penetrate into living retinal cells and trigger photoreceptor cell death through apoptotic mechanisms.(12,16) recoverin as a paraneoplastic antigen without paraneoplastic syndrome it might be assumed that autoantibodies against recoverin should be detected only in car patients. however, many paraneoplastic antigens have the capacity to induce low titer a u t o a n t i b o d i e s w i t h o u t a n y s i g n s o f paraneoplastic syndromes. in the process of raising antibodies against recoverin, bazhin et al. found that immunized rabbits developed an immune response to recoverin at variable titers. fundus examination and light microscopy of retinal sections detected retinal degeneration in rabbits with high titers of the antibody, while the eyes of rabbits with low titers did not differ from those of control animals. these findings led the researchers to screen serum samples of patients with lung cancers irrespective of the presence of car. their search revealed 15 patients with sclc among 99 individuals investigated (15%) and 9 of 44 patients with n o n s m a l l c e l l l u n g c a n c e r s ( 2 0 % ) w i t h relatively low titers of the autoantibodies in their sera, but without manifestations of car at the time of the serum sampling. therefore they concluded that autoantibodies against recoverin can be detected in sera of patients with lung cancer without manifestation of paraneoplastic syndromes. for these findings the investigators suggested the following explanations: (i) to overcome the blood-retinal barrier, the titer of the autoantibodies in the blood stream of p a t i e n t s s h o u l d e x c e e d a c e r t a i n l e v e l . otherwise the autoantibodies cannot penetrate into the cells, bind to the corresponding intracellular antigen and initiate apoptosis of the cells; (ii) the development of paraneoplastic syndromes might depend on the actual epitopes recognized by the autoantibodies. in the case o f r e c o v e r i n , a u t o a n t i b o d i e s f r o m c a r p a t i e n t s m o s t l y b i n d t o r e s i d u e s 6 4 7 0 representing the ef-hand 2 domain and initiate apoptosis by blocking calcium-binding; (iii) the manifestation of car might well depend on a second event resulting in transmissibility of the blood–retinal barrier.(23) i n c a r p a t i e n t s , a n t i e n o l a s e autoantibodies may induce the apoptotic death of other retinal cells such as ganglion cells. autoantibodies against retinal proteins from retinopathy patients with and without cancer had similar cytotoxic effects on retinal cells.(25) conclusions in car, certain tumors may express antigens that are normally present in the retina, tradjutrisno cancer-associated retinopathy 63 univ med vol.29 no.1 thus inducing autoantibodies against these r e t i n a l p r o t e i n s . t h e i d e n t i f i c a t i o n o f antiretinal antibodies is neither specific nor sensitive for the diagnosis of car. analysis of immune-mediated vision loss is in its i n f a n c y, a n d a c a r e f u l a n a l y s i s a n d c h a r a c t e r i z a t i o n o f a n t i r e t i n a l a n t i b o d y specificities will help in our understanding of the mechanisms and the diagnosis of patients with this form of vision loss. however, the pathogenic mechanisms of retinopathies are complex and our understanding of car is still incomplete. references 1. gure ao, stockert e, scanlan mj, keresztes rs, jager d, altorki nk, et al. serological identification of embryonic neural proteins as highly immunogenic tumor antigens in small cell lung cancer. proc natl acad sci usa 2000;97:4198–203. 2. altaha r, abraham j. paraneoplastic neurologic syndrome associated with occult breast cancer: a case report and review of literature. breast j 2003;9:417–41. 3. minisini am, pauletto g, bergonzi p, fasola g. paraneoplastic neurological syndromes and breast cancer. regression of paraneoplastic neurological sensorimotor neuropathy in a patient with metastatic breast cancer treated with capecitabine: a case study and mini-review of the literature. breast cancer res treat 2007;105:133–8. 4. hayashi m, hatsukawa y, yasui m, yanagihara i, ohguro h, fujikado t. cancer-associated retinopathy in a child with langerhans cell histiocytosis. jpn j ophthalmol 2007;51:390–8. 5. sawyer ra., selhorst jb, zimmerman le, hoyt wf. blindness caused by photoreceptor degeneration as a remote effect of cancer. am j ophthalmol 1976;81:606–13. cited by hooks jj, tso mom, detrick b. retinopathies associated with antiretinal antibodies. clin diagn lab immunol 2001;8:853–8. 6. berson el, lessell s. paraneoplastic night blindness with malignant melanoma. am j ophthalmol 1988;106:307–11. cited by hooks jj, tso mom, detrick b. retinopathies associated with antiretinal antibodies. clin diagn lab immunol 2001;8:853–8. 7. goetgebuer g, kestelyn-stevens am, de laey jj, kestelyn p, leroy bp. cancer-associated retinopathy (car) with electronegative erg: a case report. doc ophthalmol 2008;116:49–55. 8. de potter p, disneur d, levecq l, snyers b. manifestations oculaires des cancers. j fr ophtalmol 2002;25:194-202. 9. hooks jj, tso mom, detrick b. retinopathies associated with antiretinal antibodies. clin diagn lab immunol 2001;8:853-8. 10. oohira a. fifteen-year follow-up of patient with cancer-associated retinopathy. jpn j ophthalmol 2007;51:68–81. 11. espandar l, o’brien s, thirkill c, lubecki la, esmaeli b. successful treatment of cancerassociated retinopathy with alemtuzumab. j neurooncol 2007;83:295–302. 12. thirkill ce. immune-mediated paraneoplasia. br j biomed sci 2006;63:185-95. 13. ohguro h and nakazawa m. pathological roles of recoverin in cancer-associated retinopathy. adv exp med biol 2002;514:109–24. 14. heckenlively jr, ferreyra ha. autoimmune retinopathy: a review and summary. semin immunopathol 2008;30:127–34. 15. maeda a, ohguro h, maeda t, wada i, sato n, kuroki y, nakagawa t. aberrant expression of photoreceptor-specific calcium-binding protein (recoverin) in cancer cell lines. cancer research 2000;60:1914-20. 16. adamus g, machnicki m, elerding h, sugden b. blocker ys, fox da. antibodies to recoverin induced apoptosis of photoreceptor and bipolar cells in vivo. j autoimmunity 1998;11:523–33. 17. polans as, witkowska d, haley tl, amundson d, baiz-er l, adamus g. recoverin, a photoreceptor-specific calcium binding protein is expressed by the tumor of a patient with cancerassociated retinopathy. proc nat acad sci usa 1995;92:9176-80. 18. magrys a, anekonda t, ren g, adamus g. the role of anti-á -enolase autoantibodies in pathogenicity of autoimmune-mediated retinopathy. journal of clinical immunology 2007;27:181-92. 19. misiuk-hojlo m, ejma ma. gorczyca wa, szymaniec s, witkowska d, fortuna w, et al. cancer-associated retinopathy in patients with breast carcinoma. arch immunol ther exp 2007; 55:261–5. 20. iacovelli l, sallese m, mariggio s, de blasi a. regulation of g-protein-coupled receptor kinase subtypes by calcium sensor proteins. faseb j 1999;13:1–8. 64 21. bazhin av, schadendorf d, philippov pp, eichmuller sb. recoverin as a cancer-retina antigen. cancer immunol immunother 2007;56: 110–6. 22. adamus g, sugden b, seigal gm. cytotoxic and apoptotic effect of anti-retinal autoantibodies of autoimmune retinopathy. faseb j 2000;14: a1107. 23. adamus g, ren g, weleber rg. autoantibodies against retinal proteins in paraneoplastic and autoimmune retinopathy. bmc ophthalmology 2004;4:5. tradjutrisno cancer-associated retinopathy eveline1 78 department of physiology, medical faculty, trisakti university correspondence adr. eveline margo department of physiology medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2803 email : eveline_margo@yahoo.com univ med 2008; 27: 78-86. management of anemia among patients in intensive care units april-june 2008april-june 2008april-june 2008april-june 2008april-june 2008 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 abstract universa medicina eveline margo anemia is frequently encountered in critically-ill patients in the intensive care unit (icu). anemia may occur both at the time of admission, during treatment and after discharge from the icu. the causes are multifactorial and include acute blood loss, blood loss from diagnostic testing and blunted red blood cell production. blood transfusions are frequently given to patients in the icu to treat low hemoglobin levels due to either acute blood loss or subacute anemia associated with critical illness. although blood transfusion is a life-saving therapy, evidence suggests that it may be associated with an increased risk of morbidity and mortality. a feature of anemia of critical illness is lack of appropriate elevation of circulating erythropoietin concentrations in response to physiological stimuli. one important concern is that anemia may not be well tolerated by a critically ill patient. a number of blood conservation strategies exist that may mitigate anemia in hospital patients and limit the need for transfusion. these strategies include the use of hemostatic agents, hemoglobin substitutes and blood salvage techniques, the reduction of blood loss associated with diagnostic testing, the use of erythropoietin and the use of restrictive blood transfusion triggers. in this article we review the prevalence of anemia during critical illness specifically among patients in the icu, and discuss the various factors that contribute to its development, the prevention and treatment of anemia by appropriate red cell transfusion and the place of erythropoietin in treatment. keywords : anemia, blood transfusion, critically ill, hemoglobin, hematocrit introduction anemia frequently accompanies criticallyill patients under treatment in the intensive care unit (icu). (1,2) more than 85% patients or approximately five in six patients treated in the icu for more than 1 week requires daily blood t r a n s f u s i o n b e c a u s e o f a n e m i a . ( 2 ) tw o observational studies reported that 35 to 45% o f p a t i e n t s a d m i t t e d t o t h e i c u r e c e i v e transfusions of almost 5 red-cell units while in the icu.(3,4) however, the view that red-cell 79 univ med vol.27 no.2 transfusion is beneficial for critically ill patients has been questioned because of data suggesting that red-cell transfusion may decrease the likelihood of survival in critically ill adults.(5,6) the anemia in critically-ill patients is not exclusively due to loss of blood, as there are a number of reciprocally interacting factors playing a causative role.(1-3) impaired production of red cells contributes to the development and persistence of anemia. anemia is defined by a hemoglobin (hb) concentration below normal, when factors such as age, gender, pregnancy and environment (elevation above sea level) have been taken into account. (1) according to the world health organization (who), anemia is defined by a hb level of <13 g/dl (hematocrit/hct <39%) in adult men and <12 g/dl (hct <36%) in adult non-pregnant women. (7) anemia is usually assessed by measurement of hb or hct levels, which indicate the relationship between red cell count and blood plasma. in critically-ill patients there are a large number of factors influencing both components, so that the prevalence of anemia in these patients requires a more precise interpretation in connection with therapy and pathophysiology.(1,3) factors affecting the prevalence of anemia a m o n g c r i t i c a l l y i l l p a t i e n t s i n c l u d e t h e underlying disease, its severity, and aggravating factors. based on the time of occurrence of the anemia, these patients may be subdivided into t h r e e g r o u p s , i . e . a n e m i a a t t h e t i m e o f admission, during icu treatment and after treatment. anemia in critical lilnes characteristics production of rbcs by the bone marrow is impaired in critically ill patients and this phenomenon contributes to both the development and, more importantly, the persistence of anaemia. critically ill patients are anemic early in their icu course and hemoglobin levels fall during the icu stay.(3) the prevalence of anemia on admission into the icu turns out to be relatively high, as about 20-30% of patients experiences moderate to severe anemia with hb levels of <9 g/dl.(1,3) there are three sufficiently large studies on the prevalence of anemia in patients at the time of admission into the icu, as follows: (i) vincent et al. in their cohort study obtained a mean hb level of approximately 11.3 g/dl, while around 29% of patients had a hb level of <10 g/dl on admission to the icu;(3) (ii) corwin et al. found a mean hb level of approximately 11 g/dl in patients admitted to the icu, and almost twothirds of patients had a hb level of <12 g/dl;(4) (iii) walsh et al., reported in a cohort study on patients admitted to the icu that the patients showed a mean hb level of around 10.5 g/dl, while about 25% of those patients had a hb level of <9 g/dl.(8) according to nguyen et al. among icu patients who didn’t get transfusions, there was a mean reduction in hb levels of 0.52 g/dl/day.(9) a study showed that on average there was a reduction in hb levels of 0.66 g/dl/day in the first three days and 0.12 g/dl in subsequent days. a notable reduction in hb levels in the first days of icu treatment was also seen in the crit study, where the mean hb level of patients decreased from 12 g/dl at the time of admission to 11 g/dl at day 3-4, while afterwards the decrease would slow down.(10) walsh et al. estimated that moderate to severe anemia (hb level <9 g/dl) within the icu treatment period occurred in about 40-50% of the population. when anemia did not arise at the time of admission to the icu, then in some patients it would soon occur within the first 2-3 days. in addition, they found that anemia occurring during icu treatment remained constant until the patients were discharged from 80 the icu, except when the patients received blood transfusion during icu treatment.(1) the prevalence of anemia in patients convalescing from critical illness is still little known. a study in england on 283 patients receiving conditional transfusion during icu treatment (transfusion if hb = 7 g/dl) and after discharge from the icu, showed that around 77.4% of patients suffered from anemia, with hb levels below 10 g/dl in 32.5%, while 11.3% had levels of less than 9 g/dl. above data indicate that the probability of anemia remains constant for a period of time after the patients have been discharged from the icu.(1) causes critically ill patients with anemia may be placed into two categories, i.e. patients suffering from anemia prior to admission to the icu, and patients developing anemia during treatment in the icu. critically-ill patients often experience hypovolemia requiring fluid resuscitation in the initial days of treatment. currently the fluids given for resuscitation are crystalloids or colloids, while transfusion is restricted, except in patients with severe hemorrhage and hb <7 g/dl.(1-3) consequently there will be a change in blood plasma volume relative to red cell volume (rcv), effecting a reduction in hb level, especially during fluid resuscitation. (figure 1) loss of blood loss of blood is one of the main causes of anemia in critically ill patients. according to von ahsen et al., based on their study on hct level, blood volume and criteria for transfusion, it is estimated that on average patients treated in the icu experience a blood loss of >100 ml/day,(10) where the primary sources of blood loss in particular are multiple collections of blood for diagnostic samples, and hemorrhage elsewhere in the body.(1,2,10) however, critically ill patients often have elevated levels of factor viii and von willebrand factor, both induced bleeding.(11) figure 1. relationship between red blood cell volume, plasma volume, hematocrit and hemoglobin concentration during hemorrhage, normovolemia and fluid resuscitation.(1) *pv = plasma volume; rcv = red cell volume; hct = hematocrit; hb = hemoglobin concentration margo anemia in intensive care units 81 univ med vol.27 no.2 collection of blood for diagnostic samples plays a role in the development of anemia in patients treated in the icu. a study showed about 30% of total blood transfusions is due to collection of blood for diagnostic purposes, with an average blood loss of around 40-70 ml/ day. (12) loss of blood by this route occurs m a i n l y w i t h i n t h e f i r s t 2 4 h o u r s o f i c u treatment and will decline in the following days, in proportion to the need for routine blood examinations.(3,12) a p o t e n t i a l s o u r c e o f h e m o r r h a g e i n critically-ill patients is bleeding in the digestive tract, particularly in patients on ventilators, and in those with coagulation disorders and renal f a i l u r e . ( 1 , 2 ) a c c o r d i n g t o wa l s h e t a l . , approximately 12% of patients under treatment in the icu experience at least one significant episode of gastrointestinal tract hemorrhage (blood loss = 300 ml) and around 18% of patients require blood transfusion.(1) reduction of red blood cells life span accurate data on the reduction in red cell life span in critically-ill patients, particularly those with sepsis, are still lacking, but there are a number of experimental data indicating that mediators of inflammation, such as tumor necrosis factor (tnf-α) and interleukin-1 (il1), and also free radicals, can cause premature red cell damage and programmed cell death (apoptosis).(1,2,12,13) decreased production of red blood cells there are a number of factors leading to a decreased in red cell production, inter alia: disordered iron metabolism, bone marrow depression, low erythropoietin levels and a b n o r m a l r e d c e l l m a t u r a t i o n . t h e b o d y normally responds to anemia by increasing red cell production, but in critically-ill patients this mechanism is inoperative. the pathophysiology of anemia in critically-ill patients is similar to that in chronically-ill patients, especially the mechanism of inhibition of red cell formation (figure 2).(1,2,12,13) disorders of iron metabolism the inflammatory process reduces the a m o u n t o f s e r u m i r o n a v a i l a b l e f o r erythropoiesis. in inflammation, most of the iron is located in the macrophages in the form of ferritin, resulting in a decrease in the total serum iron available to the erythropoietic process. this condition is known as functional iron deficiency.(1,8,13) according to andrew et al, in patients with anemia due to chronic disease, inflammatory cytokines stimulate synthesis of hepcidin, a peptide produced by liver cells in response to interleukin-6 exposure. hepcidin plays an i m p o r t a n t r o l e i n t h e r e g u l a t i o n o f i r o n metabolism by increasing the retention of iron in macrophages, hepatocytes and enterocytes t h r o u g h i n h i b i t i o n o f i r o n i o n e ff l u x v i a ferroportin (see figure 2). in addition, hepcidin may also inhibit absorption of iron in the digestive tract.(8,10,12) although an identical mechanism has still to be demonstrated in c r i t i c a l l y i l l p a t i e n t s , a d m i n i s t r a t i o n o f supplementary iron by the intravenous and oral routes does not lead to increased red cell production.(8,10,12) low erythropoietin levels in the circulation the normal body response to anemia is to i n c r e a s e e r y t h r o p o i e t i n r e l e a s e f r o m t h e kidneys. however, in critically-ill patients e r y t h r o p o i e t i n r e s p o n s e t o a n e m i a i s inadequate, due to inhibition by inflammatory cytokines.(1,2,13) low erythropoietin levels in the circulation will cause decreased red cell production. it is this factor that causes the occurrence of anemia o r t h e a g g r a v a t i o n o f e x i s t i n g a n e m i a i n critically-ill patients. 82 figure 2. pathophysiology of anemia of chronic disease. panel a. invasion of microorganisms, malignancies or autoimmune disorders activate t cells (cd3+) and monocytes. these cells produce cytokines such as tumor necrosis factor (tnf-α), interleukin-1, interleukin-6 and interleukin-10. panel b. interleukin-6 and iipopolysaccharide stimulate expression by hepatic cells of hepcidin, an acute-phase protein which inhibits absorption of iron in the duodenum. panel c. interferon-γ and liposaccharide increase expression of divalent metal transporter 1 on macrophages. the anti-inflammatory cytokine interleukin-10 increases ferrous ion uptake of transferrin-bound iron into monocytes. these mechanisms result in a decreased iron concentration in the circulation and lead to accumulation of iron in macrophages in the form of ferritin. panel d. tnf-α and interferon-γ inhibit the production of erythropoietin in the kidneys. panel e. tnf-α, interferon-γ and interleukin-1 directly inhibit differentiation and proliferation of erythroid progenitor cells. all of the above mechanisms in concert cause the development of anemia.(13) margo anemia in intensive care units 83 univ med vol.27 no.2 abnormal red cell maturation mediators of inflammation, such as tumor necrosis factor-α (tnf-α), interleukin-1 and interleukin-6, will increase in critically-ill patients, especially those with sepsis. these mediators have been proven to inhibit maturation of red blood cells. moreover, in experimental studies the existence of interferon-γ capable of stimulating apoptosis of erythroid progenitor cells has successfully been demonstrated.(1,2,13) t h e s e f a c t o r s , i n c o m b i n a t i o n w i t h erythropoietin deficiency and functional iron deficiency, lead to an inadequate erythroid response to anemia. management reduction of blood loss in critical ill patients below is a list of measures for blood loss reduction in patients treated in the icu:(1,2,4) (i) avoidance of daily routine blood examination, blood chemistry and blood gas analysis, when unnecessary and without clinical indications; (ii) conversion of laboratory procedures by using microtubes to minimize the necessary blood volumes collected; (iii) use of diagnostic equipment requiring minimal blood samples, such as use of sticks in blood sugar determinations, etc.; (iv) use of monitoring equipment for alternative screening, such as pulse oxymetry for estimating oxygen saturation and arterial oxygen p a r t i a l p r e s s u r e , u s e o f c a p n o g r a p h s f o r determination of co 2 partial pressure, etc.; and (v) prevention of stress ulcers in patients at high risk for gastro-intestinal hemorrhage, such as patients on mechanical ventilation, and those with renal failure and disorders of coagulation. blood transfusion red cell transfusion is one of the most rapid methods of anemia management for overcoming anemia and the most frequently applied in clinical practice. although at present screening tests for diseases transmitted through blood transfusions are increasingly being perfected, yet t h e r e a r e m a n y h a r m f u l s i d e e f f e c t s o f transfusions, particularly if inappropriately a d m i n i s t e r e d . c o n s e q u e n t l y, f o r b l o o d transfusions to be right on target, it is necessary to pay attention to the criteria for starting blood transfusions and the targeted hb levels in individual patients.(1) red blood cells are often transfused to increase oxygen delivery and mitigate tissue ischemia; however, the ability of red blood cell transfusions to increase oxygen consumption has not been clearly demonstrated. critical hb concentrations critical hb concentrations are usually interpreted as hb concentrations below the level where oxygen consumption will be dependent on supply, assuming that normovolemia is being maintained. this level has no absolute values, but differs in various organs and depends on the tissue metabolic activity and the capacity for oxygen extraction by the tissues.(1) a number of studies on human volunteers and on patients undergoing operations have acquired data showing that the critical hb concentration in healthy and normovolemic individuals is around 4-5 g/dl, while hypoxia will be apparent at lower hb levels. tolerable hb concentrations in critically-ill patients the hb limits that is still tolerated by critically-ill patients depend on the balance between the risks of blood transfusions and the risks of low hb levels themselves. there have been two relatively large-scale studies large showing the limits of hb concentration still tolerated by patients with critical illness. the transfusion requirements in critical care (tricc) study divided the patients into two groups. the first group had as transfusion criterion a hb level of <7 g/dl and a target hb 84 level of 7-9 g/dl (restricted group), while the second group had a more liberal transfusion criterion, i.e. hb level of <10 g/dl and target hb level of 10-12 g/dl (liberal group). between both groups there was no significant difference i n m o r t a l i t y r a t e a f t e r 3 0 a n d 6 0 d a y s o f treatment. even in the restricted group, patients <50 years old with the acute physiological and chronic health evaluation (apache ii) scores <20 showed significantly lower mortality rates.(14) other study had used tissue hypoxia as a c r i t e r i o n f o r b l o o d t r a n s f u s i o n . t h e m o s t frequently used parameters were tissue oxygen consumption, lactate concentration, mixed vein saturation and mixed vein partial pressure. the study showed that if the hb level was >8 g/dl, administration of blood transfusion will not improve the above-mentioned indicators, as long as the patients were of normovolemic status.(15) the above studies also confirm the results of the tricc study that hb levels of 7-9 g/dl were safe for the majority of patients with critical illness. criteria for transfusion in special patients although according to the tricc study a hb level between 7-9 g/dl is considered safe in the majority of critically-ill patients, there are some groups of patients that require a higher hb level. although still controversial, a list of such groups of patients is as follows:(1,2,10,13) (i) patients with ischemic heart disease, most authorities suggest a hb level of 7-8 g/dl as condition for starting blood transfusion with a target hb of around 7-9 g/dl, except in cases of acute myocardial ischemia, when the criteria for starting blood transfusion will be higher, viz. blood transfusion is administered at a hb level of 8 g/dl, with a target hb level of >9 g/ dl;(1,13,15) (ii) for patients with sepsis, early goaldirected therapy (egdt) carried out in the early stages has been proven to significantly reduce the mortality rate. one of its algorithms uses a central vein oxygen saturation of <70% as indication for taking interventional measures, e.g. by maintaining the hct = 30% and hb = 10 g/dl.(1,14,15) although it is still unclear which of the two components is responsible for the reduction of mortality rate in these patients, in general patients on egdt will receive more blood transfusions. to date there is a consensus of opinion that in patients with sepsis the target hb level should be 10 g/dl (hct = 30%), if the central venous oxygen saturation < 70%.(10,15,16) erythropoietin the low endogenous erythropoietin response t o a n e m i a i n c r i t i c a l l y i l l p a t i e n t s m a k e s administration of exogenous erythropoietin one of the therapeutic alternatives. in critically-ill patients with multiple organ damage, recombinant human erythropoietin (rhuepo) is capable of stimulating erythropoiesis.(1,2,13,14) erythropoietin acts as a cytokine with antiapoptotic activity.(17) in this role, erythropoietin has been shown in preclinical and small clinical studies to protect cells from hypoxemia and ischemia. multiple t i s s u e s e x p r e s s e r y t h r o p o i e t i n a n d t h e erythropoietin receptor in response to stress and also to mediate local stress responses. these nonhematopoietic activities of erythropoietin in t h e p r o t e c t i o n o f c e l l s s u g g e s t a r o l e f o r erythropoietin in critically ill patients.(17) there have been two clinical studies showing the success of erythropoietin use in critically-ill patients. corwin et al. in their most recent study using recumbent human erythropoeutin (rhuepo) in doses of 40,000 units weekly for 4 weeks combined with oral iron supplementation, obtained a lower percentage of patients requiring red cell transfusion during the first 28 days in the group on rhuepo, in comparison with the placebo group (50.5% vs 60.4%, p<0.001).(18) corwin et al in their study showed that the use of margo anemia in intensive care units 85 univ med vol.27 no.2 40,000 u epoetin alfa weekly, for a maximum of 3 weeks does not reduce the incidence of red-cell transfusion among critically ill patients, but may reduce mortality in patients with trauma.(19) the above-mentioned studies have proven that administration of erythropoietin is effective in increasing hb levels in critically-ill patients; however, its wide-spread use is still constrained by the high cost. consequently some authorities suggest the use of erythropoeitin according to the following strategy:(2) (i) administration of rhuepo in doses of 30000 40000 u weekly; (ii) weekly administration of rhuepo is started on admission for all critically-ill patients s u ff e r i n g f r o m a n e m i a , w h o h a v e a g o o d prognosis and estimated icu treatment of >7 days; (iii) avoid using rhuepo in patients with minimal survival chances, non-anemic patients and those with icu treatment estimate of <7 days, such as in asthma, copd, drug overdose, d i a b e t i c k e t o a c i d o s i s , e t c . ; a n d ( i v ) r e assessment at day 4-7 for deciding whether or not to continue rhuepo administration. it is necessary to determine whether there are clinical o u t c o m e b e n e f i t s i n c r i t i c a l l y i l l p a t i e n t s admitted to either the icu and/or long-term acute care facilities associated with the reduction in t h e e x p o s u r e t o r b c t r a n s f u s i o n w i t h erythropoietin administration.(20) conclusions management of anemia in critically-ill patients may take the form of prevention of excessive blood loss due to diagnostic blood sampling, prevention of stress ulcers in patients a t h i g h r i s k , r e d c e l l t r a n s f u s i o n a n d administration of erythropoietin. the benefits and costs of blood transfusions should be taken into account, considering that on the basis of a number of clinical studies it has been proven that c r i t i c a l l y i l l p a t i e n t s a r e w e l l c a p a b l e o f tolerating a hb level of around 7-9 g/dl. although the supporting data are not fully accurate, certain groups of patients, for example those with acute ischemic heart disease or sepsis, possibly require a higher hb concentration. administration of erythropoietin may indeed increase hb levels and reduce the need for blood transfusion, but currently its use is still constrained and requires a special strategy because of the relatively high price of the drug. references 1. walsh ts, saleh eed. anemia during critical illness. br j anaesth 2006; 97: 278-91 2. marini jj, wheeler ap. transfusion and blood component therapy. in: critical care medicine. 3rd ed. philadelphia : lippincott williams & wilkins, 2006. p. 247-58. 3. vincent jl, baron jf, reinhart k, gattinoni l, thijs l, webb a, et al. anemia and blood transfusion in critically ill patients. jama 2002; 288: 1499-507. 4. corwin hl, gettinger a, pearl rg, fink mp, levy mm, abraham e, et al. the crit study: anemia and blood transfusion in the critically ill current clinical practice in the united stated. crit care med 2004; 32: 39-52. 5. napolitano lm, corwin hl. efficacy of red blood cell transfusion in the critically ill. crit care clin 2004; 20: 255-68. 6. taylor rw, o’brien j, trottier sj, manganaro l, cytron m, lesko mf, et al. red blood cell transfusions and nosocomial infections in critically ill patients. crit care med 2006; 34: 2302-8. 7. emmanuel je, mcclelland b, page r, editors. the clinical use of blood in medicine, obstetrics, paediatrics, surgery anaesthesia, trauma & burns. geneva: world health organisation; 1997. 8. walsh ts, lee rj, maciver c, garrioch m, mackirdy f, binning ar, et al. anemia during and at discharge from intensive care: the impact of restrictive blood transfusion practice. intensive care med 2005; 32: 100-9. 9. nguyen bv, bota dp, melot c, vincent jl. time course of hemoglobin concentrations in nonbleeding intensive care unit patients. crit care med 2003; 31: 406-10. 10. von ahsen n, muller c, serke s, frei u, eckardt ku. important role of nondiagnostic blood loss and 86 blunted erytropoietic response in the anemia of medical intensive care patients. crit care med 1999; 27: 2630-9. 11. pusparini. peran faktor von willebrand dalam sistem hemostasis. j kedokter trisakti 2000; 20: 130-8. 12. scharte m, fink mp. red blood cell physiology in critical illness. crit care med 2003; 31: 5651-7. 13. weiss g, goodnough lt. anemia of chronic disease. n engl j med 2005; 352: 1011-23. 14. hebert pc, wells g, blajchman ma, marshall j, martin c, paqliarello g, et al. a multicenter, randomized, controlled clinical trial of transfusion requirements in critical care. transfusion requirements in critical care investigators, canadian critical care trials group. n engl j med 1999; 340: 409-17. 15. hebert pc, wells g, martin c, tweeddale m, marshall j, blajchman m, et al. canadian survey of transfusion practices in critically ill patients. transfusion requirements in critical care investigator and the canadian critical care trials group. crit care med 1998; 26: 482-7. 16. brines m, cerani a. discovering erythropoietin’s extra-hematopoietic functions: biology and clinical promise. kidney int 2006; 70: 246-50. 17. coleman t, brines m. recombinant human erythropoietin in critical illness: a role beyond anemia? crit care 2004; 8: 337-41. 18. corwin hl, gettinger a, pearl rg, fink mp, levy mm, shapiro mj, et al. efficacy of recombinant human erythropoietin in critically ill patients: a randomized controlled trial. jama 2002; 288: 2827-35. 19. corwin hl, gettinger a, fabian tc, may a, pearl rg, heard s, et al. for the epo critical care trials group. efficacy and safety of epoetin alfa in critically ill patients. n engl j med 2007; 357: 965-76. 20. corwin hl, eckardt k-u. erythropoietin in the critically ill: what is the evidence? nephrol dial transplant 2005; 20: 2605-8. margo anemia in intensive care units centella asiatica increases b-cell lymphoma 2 expression in rat prefrontal cortex 10 *department of anatomy, faculty of medicine, islamic university of indonesia **department of anatomy, embryology and anthropology, faculty of medicine gadjah mada university. ***department of pathological anatomy, faculty of medicine, sebelas maret university ****department of anatomy, faculty of medicine, sebelas maret university correspondence dr. kuswati, m.sc department of anatomy, faculty of medicine, islamic university of indonesia yogyakarta 55281 phone: +6285228478487 email: kuswati2005@yahoo.com univ med 2015;34:10-6 doi: 10.18051/univmed.2016.v35.10-16 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background stress is one of the factors that cause apoptosis in neuronal cells. centella asiatica has a neuroprotective effect that can inhibit apoptosis. this study aimed to examine the effect of centella asiatica ethanol extract on bcell lymphoma 2 (bcl-2) protein expression in the prefrontal cortex of rats. methods an experimental study was conducted on 34 brain tissue samples from male sprague dawley rats exposed to chronic restraint stress for 21 days. the samples were taken from following groups: non-stress group k, negative control group p1 (stress + arabic gum powder), p2 (stress + c.asiatica at 150 mg/kgbw), p3 (stress + c.asiatica at 300 mg/kg bw), p4 (stress + c.asiatica at 600 mg/kg body weight) and positive control group p5 (stress + fluoxetine at 10 mg/kgbw). the samples were made into sections that were stained immunohistochemically using bcl-2 antibody to determine the percentage of cells expressing bcl-2. data were analyzed using one way anova test followed by a post hoc test. results there were significant differences in mean bcl-2 expression between the groups receiving centella asiatica compared with the non-stress group and stress-only group (negative control group) (p<0.05). the results were comparable to those of the fluoxetine treatment group. conclusion the centella asiatica ethanol extract was able to increase bcl-2 expression in the prefrontal cortex of sprague dawley rats exposed to restraint stress. this study suggests that centella asiatica may be useful in the treatment of cerebral stress. keywords : centella asiatica, b-cell lymphoma 2, prefrontal cortex, stress, rat centella asiatica increases b-cell lymphoma 2 expression in rat prefrontal cortex kuswati*, djoko prakosa**, brian wasita***, and nanang wiyono**** doi: http://dx.doi.org/10.18051/univmed.2015.v34.10-16 11 univ med vol. 34 no.1 centella asiatica meningkatkan ekspresi protein b-cell lymphoma 2 pada korteks prefrontalis tikus latar belakang stres merupakan salah satu faktor yang dapat menyebabkan apoptosis pada sel saraf. centella asiatica memiliki efek neuroprotektif yang dapat menghambat apoptosis. penelitian ini bertujuan untuk menilai peningkatan ekspresi protein b-cell lymphoma 2 (bcl-2) setelah pemberian ekstrak etanol centella asiatica pada tikus yang diberi perlakuan stres restraint kronik. metode sebuah penelitian eksperimental dilakukan pada 34 jaringan otak tikus sprague dawley yang diberi perlakuan stres restraint kronik selama 21 hari. jaringan otak tikus berasal dari 6 kelompok berikut: kelompok non stres (k), kelompok kontrol negatif stres + pulvis gumma arabicum (p1), kelompok stres + c.asiatica dosis 150 mg/kgbb (p2), kelompok stres + c.asiatica dosis 300 mg/kgbb (p3), kelompok stres + c.asiatica dosis 600 mg/kgbb (p4) dan kelompok stres + fluoksetin dosis 10 mg/kgbb sebagai kontrol positif (p5). jaringan otak tersebut dibuat preparat yang dicat secara imunohistokimia untuk penghitungan prosentase sel yang mengekspresikan bcl-2. data yang diperoleh dianalisis menggunakan uji one way anova dilanjutkan analisis post-hoc test. hasil terdapat perbedaan bermakna pada rerata prosentase ekspresi bcl-2 antara kelompok yang mendapat terapi centella asiatica dan fluoksetin dibandingkan dengan stres (p<0,05). ekspresi bcl-2 pada kelompok yang mendapat terapi centella asiatica dan kelompok fluoksetin lebih tinggi dibanding kelompok stres. kesimpulan ekstrak etanol centella asiatica dapat dapat meningkatkan ekspresi protein antiapoptosis bcl-2 di cortex prefrontalis tikus sprague dawley yang diberi perlakuan stres restraint kronik. centella asiatica dapat berperan sebagai pengobatan pada restraint stress yang dialami tikus. kata kunci : centella asiatica, b-cell lymphoma 2, cortex prefrontalis, stres, tikus abstrak introduction st r e s s c a n c a u s e d i s o r d e r s o f c e l l homeostasis, because it increases the secretion of glucocorticoids. in the central nervous system, an increase in glucocorticoids will trigger release of glutamate. the latter binds to n-methyl-daspartate (nmda) receptors, resulting in the opening of calcium channels, with subsequent calcium entry into the cytoplasm and increase in cytoplasmic free calcium. the entry of calcium and water into the mitochondria results in mitochondrial edema, impaired balance between bcl-2-associated x protein (bax) and b-cell lymphoma 2 (bcl-2) expression, increase in mitochondrial membrane permeability and release of cytochrome c, triggering apoptosis.(1) stress affects several areas of the prefrontal cortex, hippocampus, amygdala and brainstem. in the prefrontal cortex, stress results in a decrease in the activity of neurons, a reduction in the number of dendrites and shortening of dendrites in the medial prefrontal cortex.(2) chronic stress results in retraction of the apical dendrites of pyramidal cells in layer ii / iii and v of the medial prefrontal cortex.(3) therefore, 12 individuals who experience stress should be given therapy to prevent apoptosis and morphological changes in the prefrontal cortex. drugs that can be used to inhibit apoptosis in the central nervous system caused by stress belong to the class of antidepressant drugs. olanzapine, imipramine, amitriptyline, citalopram and fluoxetine can inhibit apoptosis by increasing the expression of bcl-2 and b-cell lymphomaextra large (bcl-xl) and decreasing the expression of bax.(4,5) although they may inhibit apoptosis, antidepressant drugs have many side effects. side effects of tricyclic antidepressant drugs include dry mouth, dizziness, decreased vision, constipation, sedation, orthostatic hypotension and tachycardia. one class of antidepressant drug, the selective serotonin reuptake inhibitors (ssris), have side effects such as nausea, sleep disturbances, sexual dysfunction, changes in appetite, headache and dry mouth.(6) therefore, it is necessary to find an alternative drug to inhibit apoptosis, one of which can be found in the class of herbs such as pegagan (centella asiatica). centella asiatica has a wide therapeutic dose range. centella asiatica extract at a dose of 100-1000 mg/kg body weight did not cause any side effects, and had a median lethal dose (ld50) of more than 4000 mg/kg body weight. centella asiatica extract up to a dose of 4000 mg/kg did not result in impaired liver and kidney function as well as changes in the macroscopic picture of the liver, lung and kidney.(7) centella asiatica contains madecassoside, asiaticoside, asiatic acid and madecassic acid which have antioxidant and neuroprotectant effects.(8) administration of c. asiatica extract in mice with stress can improve spatial memory, increase hippocampal cornu ammonis (ca) 1 pyramidal layer thickness, and increase the number of neuroglia in the hippocampus.(9,10) the objective of this study was to evaluate the anti-apoptotic effects of c. asiatica ethanol extract, in particular on the expression of bcl-2 in the prefrontal cortex of rats exposed to chronic restraint stress. methods research design this study is part of an umbrella research using an experimental design to evaluate the effect of centella asiatica ethanol extract on memory, apoptosis and blood biochemistry (corticosteroid hormone levels and interleukins). the present study was limited to the examination of bcl-2 protein expression in the prefrontal cortex of sprague dawley rats. the study period was from august 2012 until september 2013. research subjects the sample size was determined from a mean difference in bcl-2 expression between groups of 9.0%, with σ=0.05 and β=0.2, yielding 6 subjects per group. therefore the total sample size for 6 groups was 36.(11) the subjects were 34 brain tissue samples from male sprague dawley rats exposed to chronic restraint stress for 21 days. the samples were taken from 6 experimental groups consisting of a non-stress group (k), a negative control group exposed to stress + arabic gum powder (p1), 3 groups exposed to stress + c. asiatica at dosages of 150 mg/kg body weight (p2), 300 mg/kg body weight (p3), and 600 mg/kg body weight (p4), respectively, and the positive control group exposed to stress + fluoxetine at a dosage of 10 mg/kg body weight (p5). in the present study chronic restraint stress was used, by placing the rats into plastic transparent acrylic restraint tubes of 15 cm length and 5.5 cm diameter, with a number of 3 mm diameter holes at their closed ends. this treatment caused the rats to be immobilized, resulting in psychological stress. starting from day 1, the stress groups were exposed to restraint stress by placing them into the restraint tubes daily for 6 hours, from 09.00 to 15.00, for 21 days. in the stress groups receiving c. asiatica or fluoxetine, the treatments were given by gavage at predetermined dosages, 30 minutes before being stressed. the non-stress and stress control groups were given arabic gum powder for 21 days. on day 23 the rats were kuswati, prakosa, wasita, et al centella asiatica and b-cell lymphoma 2 13 univ med vol. 34 no.1 terminated and perfused, followed by taking brain tissue samples. subsequently the samples were made into paraffin blocks, sliced on a microtome, and stained immunohistochemically with bcl-2 antibody. preparation of centella asiatica extract c. asiatica was obtained in simplicia form from cv. merapi farma herbal (a commercial herbal manufacturer). c. asiatica identification was performed in the plant systematics laboratory, faculty of biology, gadjah mada university. the centella asiatica extract was standardized nonspecifically by examination of o f i t s w a t e r c o n t e n t a n d s p e c i f i c a l l y b y determining its asiaticoside content by thin layer chromatography. the c. asiatica extract contained 4.16 ± 0.51% asiaticoside. the c. asiatica extract was diluted to give three concentrations of 30 mg/ml, 60 mg/ml and 120 mg/ml, respectively. these concentrations were used to prepare the three treatment dosages of 150 mg/kg, 300 mg/kg body weight and 600 mg/ kg, respectively. dilution of the extract was performed once in three days at the laboratory of pharmacology, medical faculty, gadjah mada university. the dilutions were stored in the refrigerator. to calculate the volume of the c. asiatica extract for each of the rats, the following formula was used: volume = dose (mg/kgbw) x weight (kg) / concentration (mg/ml). immunohistochemical staining paraffin blocks were made from the brain tissue and then cut into 4 µm-thick sections. the cut brain areas comprised the prelimbic, infralimbic and cyngulate cortex at bregma 3.72 mm to 2.52 mm.(12) five sections were made from each rat brain at 50 µm spacings. the first step in immunohistochemical staining consisted of deparaffinizing the sections using xylol and descending alcohol series. the tissue sections were then incubated in 3% h 2 o 2 in 10% methanol for 20 minutes and rinsed 3 times with phosphate buffered saline (pbs). the tissue sections were blocked by background sniper blocking reagent for 10 minutes. without rinsing, the tissue sections were incubated overnight at 40 c with anti-bcl-2 primary antibody (1:400) (bioworld no. bs1511). the following day, the sections were rinsed using pbs 2 times, 0.2% tween 2 times and pbs 2 times. the sections were incubated in trekki universal link, followed by incubation w i t h h o r s e r a d i s h p e r o x i d a s e c o n j u g a t e d streptavidin for 10 minutes. the tissue sections were again rinsed using pbs 2 times, 0.2% tween 2 times and pbs 2 times. the final color product for single labeling was typically visualized by immersion in 3,3’-diaminobenzidin (1:100) solution for 5 minutes, rinsed 5 times with distilled water, counterstained with meyer’s hematoxylin for 1 minute, and rinsed with tap water for 2 minutes. the sections were then dehydrated in an ascending alcohol series (70%, 80%, 90%, 95% and 100%) for 1 minute each, cleared with xylene, and then covered with canada balsam and cover slip. measurements for each slide, cell counts were performed on 500 or more cells. the percentage of cells expressing bcl-2 was calculated as the number of cells expressing bcl-2 divided by the total number of cells multiplied by 100%. ehical clearance this study has received ethical clearance from the ethics committee, faculty of medicine, gadjah mada university, under number ke/fk/ 399/ec. statistical analysis te s t s o f n o r m a l i t y s h o w e d t h a t t h e percentage data were normally distributed, therefore one-way anova was performed to compare the mean number of cells between groups, followed by a post-hoc test to determine differences between specific groups. the critical level for rejection of the null hypothesis was considered to be a p value of 0.05. 14 results a total of 34 rats were analyzed, because two rats died during the study. the mean expression of the anti-apoptotic protein bcl-2 in the non-stress group was 1.41 ± 0.32%. mean bcl-2 expression in this group was the lowest compared to the other groups. mean bcl-2 expression in the stress-only group (p1) was 5.00 ± 1.12%, which was higher than that in the nonstress group, but lower than in the groups receiving therapy (group p2, p3, p4, p5). groups p2, p3, and p4, receiving c. asiatica ethanol extract at a dose of 150 mg, 300 mg, and 600 mg, respectively, had mean bcl-2 expression values of 24.89 ± 5.11%, 21.91 ± 4.52% and 2 5 . 3 1 ± 5 . 8 4 % , r e s p e c t i v e l y. t h e m e a n expression of bcl-2 in the group receiving fluoxetine (p5) was 21.84 ± 4.37%. thus, the groups that received c. asiatica ethanol extract and fluoxetine showed higher bcl-2 expression than the non-stress group k and the stress-only group p1. the results of the one-way anova test showed significant differences in the expression of bcl-2. to find out between which groups there was a significant difference, followup analysis using a post-hoc test was performed. the latter showed significant differences between the stress-only group p1 on the one hand, and the groups treated with c.asiatica ethanol extract and fluoxetine (p2, p3, p4, p5 ) on the other hand, with p-values of 0.000 to 0.002 (p<0.05) (table1). results of multiple comparisons analysis s h o w e d t h a t t h e c o n t r o l g r o u p ( k ) w a s significantly different from the groups exposed to respectively stress + c. asiatica dosage of 150 mg/kg body weight (p2), stress + c.asiatica dosage of 300 mg/kg body weight (p3), stress + c. asiatica dosage of 600 mg/kg body weight (p4) and stress + fluoxetine dosage of 10 mg/kg body weight (p5). group p1 was significantly different from the groups exposed to respectively stress + c. asiatica dosage of 150 mg/kg body weight (p2) , stress + c. asiatica dosage of 300 mg/kg body weight (p3), stress + c. asiatica dosage of 600 mg/kg body weight (p4) and stress + fluoxetine dosage of 10 mg/kg body weight (p5). there were no significant differences between the groups exposed to stress + c. asiatica dosage of 150 mg/kg body weight (p2), stress + c.asiatica dosage of 300 mg/kg body weight (p3) , stress + c. asiatica dosage of 600 mg/kg body weight (p4) and stress + fluoxetine dosage of 10 mg/kg body weight (p5) (table 2). table 1. mean bcl-2 expression by treatment groups k : control; p1: stress only; p2: stress + c. asiatica dosage of 150 mg/kg body weight; p3: stress + c. asiatica dosage of 300 mg/kg body weight; p4: stress + c. asiatica dosage of 600 mg/kg body weight; p5: stress + fluoxetine dosage of 10 mg/ kg body weight table 2. post-hoc analysis of bcl-2 expression percentages between groups *the mean difference is significant at the 0.05 level kuswati, prakosa, wasita, et al centella asiatica and b-cell lymphoma 2 15 univ med vol. 34 no.1 discussion in this study, stress-exposed rats showed higher expression of bcl-2 than did rats not e x p o s e d t o s t r e s s . d i ff e r e n c e s i n b c l 2 expression between both groups were not significant. research conducted by kim et al.(13) found that rats exposed to restraint stress, noise stress and cold stress for 10 days showed impaired memory and higher expression of bcl2, bax and caspase-3 than non-stressed rats. increased bcl-2 expression was presumably a c o m p e n s a t o r y m e c h a n i s m f o r n e u r o n a l protection from exposure to stress.(14) in the present study, the expression of bcl2 in the stress-only group (p1) was lower than in the groups treated with c. asiatica and fluoxetine (group p2, p3, p4 and p5). treatment with ethanol extract of c. asiatica at a dose of 150 mg/kg body weight, 300 mg/kg body weight and 600 mg/kg body weight could increase the expression of bcl-2 protein. dose differences did not result in significantly different expression of bcl-2 protein. this result shows that the ethanol e x t r a c t o f c . a s i a t i c a c a n p r o v i d e neuroprotective effects by inhibiting apoptosis through increased expression of bcl-2 protein. this result is consistent with research conducted by uvarajan et al.(15) and omar et al.(16) who reported that the administration of asiaticoside at a dose of 50 mg/kg for 21 days can increase the expression of bcl-2 protein and decrease the expression of bax protein in the striatum and midbrain in rats with induced 1-methyl-4-phenyl1 , 2 , 3 , 6 t e t r a h y d r o p y r i d i n e ( m p t p ) neurotoxicity. in our study, there were no significant differences in the mean expression of the bcl-2 protein between the groups that received ethanol extract of c.asiatica and the group on fluoxetine. administration of fluoxetine at a dose of 10 mg/kg body weight could increase the expression of bcl-2 protein in the prefrontal cortex of rats exposed to chronic restraint stress. the results are consistent with research by k o s t e n e t a l . ( 1 7 ) w h i c h s t a t e s t h a t t h e administration of fluoxetine at a dose of 5 mg/ kg for 21 days in rats exposed to repeated unpredictable stress increases bcl-2 mrna in the cingulate, frontal, and lateral parietal cortex. the study by guirado et al.(18) showed that in middle-aged rats chronic fluoxetine treatment induces changes in the expression of molecules related to neuronal structural plasticity. a p r e v i o u s s t u d y h a s c l e a r l y s h o w n t h a t intraperitoneal injections per se of fluoxetine in rats have effects on the structure of neurons after chronic stress.(19) in the present study some of the rats died from gavaging trauma and some paraffin blocks were damaged due to blunted microtome knifes and fatigue of the investigators. we also experienced difficulties in determining the limits of the medial prefrontal cortex, although we were guided by the atlas of the rat brain by paxinos and watson.(12) further researches are required to determine the expression of the bax pro-apoptotic protein, and the bax/bcl-2 ratio. in addition, further r e s e a r c h e s a r e n e e d e d t o e x a m i n e o t h e r parameters such as the expression of active caspase-3 and tunel assay. conclusion ethanol extract of c. asiatica could increase the expression of bcl-2 protein in the prefrontal cortex of sprague dawley rats exposed to chronic restraint stress. this study suggests that centella asiatica may be useful in the treatment of cerebral stress. acknowledgments the authors would like to thank the faculty of medicine, islamic university of indonesia, prof. dr. mustofa, m.kes. apt and dr. dwi cahyani ratnasari, m.kes. pa(k) for all advice given. we also thank rina susilowati m.d. ph.d as laboratory supervisor for her support. thanks are also due to anggraini janar wulan, rizal adi kusnomo, and suparno of the department of physiology, faculty of medicine, gadjah 16 mada university, and to sumaryati of the department of histology and cellular biology, faculty of medicine, gadjah mada university, for their technical assistance. references 1. chen sd, yang di, lin tk, et al. roles of oxidative stress, apoptosis, pgc-1α and mitochondrial biogenesis in cerebral ischemia. int j mol sci 2011;12:7199-215. 2. wilber aa, walker ag, southwood cj, et al. chronic stress alters neural activity in medial prefrontal cortex during retrieval of extinction. neuroscience 2011;174:115–31. 3. goldwater ds, pavlides cp, hunter rg, et al. structural and functional alterations to rat medial prefrontal cortex following chronic restraint stress and recovery. neuroscience 2009;164:798– 808. 4. murray f, hutson ph. hippocampal bcl-2 expression is selectively increased following chronic but not acute treatment with antidepressants, 5-ht1a or 5-ht2c/2b receptor antagonists. eur j pharmacol 2007;569: 41–7. 5. shiskina gt, kalinina ts, berezova iv, et al. stress-induced activation of the brainstem bclxl gene expression in rats treated with fluoxetine: correlation with serotonin metabolism and depressive-like behavior. neuropharmacology 2012;62:177-83. 6. ferguson jm. ssri antidepressant medications: adverse effects and tolerability. j clin psychiatry 2001;3:22-7. 7. chauhan pk, singh v. acute and subacute toxicity study of the aceton leaf extract of centella asiatica in experimental animal models. asian pac j trop bio 2012;2:511-3. 8. kumar a, dogra s, prakash a. neuroprotective effects of centella asiatica against intracerebroventricular colchicine-induced cognitive impairment and oxidative stress. int j alzheimer dis 2009. article id 972178, 8 pages, doi:10.4061/2009/972178. 9. sari dcr. the relationship between memory, ca1 hippocampus and neuroglia after centella asiatica leaf extract’s administration in stressinduced rat (rattus norvegicus), proceeding of 2nd international joint symposium frontier in biomedical sciences: from genes to applications faculty of medicine gadjah mada university. yogyakarta;2011. 10. sari dcr, aswin s, suharmi s, et al. the effect of centella asiatica ethanolic extract’s administration duration on spatial memory in rat (rattus norvegicus) after electric-stress induced. mutiara medika jurnal kedokteran kesehatan 2011;3:1-10. 11. charan j, kantharia nd. how to calculate sample size in animal studies? j pharmacol pharmacother 2013;4:303–6. doi: 10.4103/0976500x.119726 12. paxinos g, watson c. the rat brain. 6th ed. philadelphia: elsevier inc.;2007.p.11-8. 13. kim bk, ko ig, kim se, et al. impact of several types of stresses on short-term memory and apoptosis in the hippocampus of rats. int neurourol j 2013;17:114-20. 14. tan ml, ooi jp, ismail n, et al. programmed cell death pathways and current antitumor targets. pharm res 2009;26;7:1547-56. 15. uvarajan s, janardhanam va, gopinath g. neuro-shielding effect of asiaticoside against mptp induced variations in experimental mice. ijest 2012;4:960-5. 16. orhan ie. centella asiatica (l.) urban: from traditional medicine to modern medicine with neuroprotective potential. evid based complement alternat med 2012;10:1-8. 17. kosten, ta, galloway mp, duman rs, et al. repeated unpredictable stress and antidepressants differentially regulate expression of the bcl-2 family of apoptotic genes in rat cortical, hippocampal and limbic brain structures. neuropsychopharmacology 2008;33: 1545-58. 18. guirado r, matarredona ds, varea e, et al. chronic fluoxetine treatment in middle aged rats induces changes in the expression of plasticity related molecules and in neurogenesis. bmc neurosci 2012;5:1-13. 19. huang gj, herbert j. stimulation of neurogenesis in the hippocampus of the adult rat by fluoxetine requires rhythmic change in corticosterone. biological psychiatry 2006;59:619-24. kuswati, prakosa, wasita, et al centella asiatica and b-cell lymphoma 2 red fruit oil increases trophoblast cells and decreases caspase-9 expression in placenta of lead exposed mice 110 *department of veterinary anatomy veterinary faculty, airlangga university **department of veterinary anatomy, veterinary faculty, airlangga university ***department of veterinary reproduction, veterinary faculty, airlangga university correspondence : portia sumarsono veterinary faculty, airlangga university kampus c unair, jl. mulyorejo, surabaya 60115 phone : +62857 4654 4651 email: portia.sumarsono@gmail.com univ med 2016;35:110-8 doi: http://dx.doi.org/10.18051/ univmed.2016.v35.110-118 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 red fruit oil increases trophoblast cells and decreases caspase-9 expression in placenta of lead exposed mice portia sumarsono*, widjiati**, and suherni susilowati*** background lead is able to pass through the placental barrier and interfere with fetal development. red fruit has high antioxidant activity, due to carotenoids (pro-vitamin a), tocopherols (vitamin e) and unsaturated fatty acids. this study aims to examine the effect of red fruit oil towards placenta in pregnant mice before lead exposure. methods this was a laboratory experimental post test only study, using 20 pregnant mice (mus musculus) that were randomized into five groups. the negative control group was not exposed to lead; intervention group 0 (p0) was exposed to lead at 0.011 mg/20 g bw/day on days 6-15 of gestation. the intervention groups p1-p3 were given red fruit orally at 0.3 ml/20 g bw/day, 0.8 ml/20 g bw/day, 0.9 ml/20 g bw/day, respectively, before lead exposure. normal trophoblast cell count and caspase-9 expression of trophoblasts were calculated. one-way anova, kruskal-wallis and mann whitney tests were used to analyze the data. results the intervention groups p2 and p3 given red fruit at 0.8 ml/20 g bw/ day and 0.9 ml/20 g bw/day showed a significant increase in normal trophoblast cell count of 88.2% (p=0.024) and a decrease in caspase-9 expression of 70.8% compared with p0 to which was not given red fruit (p=0.020). conclusion red fruit increased normal trophoblast cell count by 88.2% and decreased caspase-9 expression by 70.8% in pregnant mice before lead exposure. it should be noted that red fuit may prevent oxidative damage to cells. keywords: lead, red fruit, caspase-9, trophoblasts, pregnant mice doi: http://dx.doi.org/10.18051/univmed.2016.v35.110-118 111 univ med vol. 35 no.2 introduction one of the heavy metals that is capable of decreasing the quality of life of humans is lead (pb). if lead is present in water, it will be c o n s u m e d b y l i v i n g o rg a n i s m s a n d w i l l ultimately cause disease and other negative impacts. lead is capable of crossing the p l a c e n t a l b a r r i e r o f p r e g n a n t m i c e a n d accumulate in fetal tissues, so that it constitutes a potential hazard to the fetus. lead crosses the placental membrane early in the 12th to 14th week of gestation, i.e. the period of organogenesis, and accumulates in the fetal tissues, with more than 90% being stored in bone and released into the circulation.(1) lead is an element that may increase the formation of free radicals and decrease antioxidants in the body, thus causing organ damage. the mechanism of lead toxicity is oxidative stress, which is defined as an imbalance between the amount of reactive oxygen species (ros) produced and that eliminated by the antioxidant system.(2) the reactive oxygen species that are formed cause hypoxia, which mediates a proapoptotic effect that is presumably involved in the mitochondrial pathway. hypoxia may cause apoptosis of trophoblasts by decreasing bcl-2 expression, increasing p53 and bax expression, and activating caspase.(3) one of the native indonesian plants with a high antioxidant content is red fruit (buah merah, pandanus conoideus lam.). from this plant, red fruit oil may be extracted, which is highly beneficial since it has high antioxidant activity, in the form of carotenoids (provitamin a), tocopherol (vitamin e) and unsaturated fatty acids.(4) in the study by dearth et al.(5) it was demonstrated that lead concentrations in the blood of females receiving lead during pregnancy and lactation was equal to lead concentrations in the blood and tissues of their offspring, thereby also affecting hormonal levels and onset of puberty. lead may depress insulin-like growth factor-1 (igf-1), luteinizing hormone (lh), and estradiol (e2), thus delaying puberty. in addition, isradji (6) proved in his study that lead acetate affects the fertility of male mice through a reduction in the number of pregnancies and litter size of their mates. sharma et al.(7) reported that administration of the antioxidant vitamins e and c to mice exposed to lead was capable of improving fertility by increasing implantation rate, fetal weight and by protecting the ovaries. red fruit can be used as a source of natural antioxidants to prevent diseases associated with free radicals.(8) thus there is a need to prove the protective efficiency of red fruit (pandanus conoideus lam.) antioxidants in pregnant mice (mus musculus) when administered before exposing them to lead, thereby revealing the potency of the plant against placental disorders during pregnancy. the objective of the present study was to evaluate the effect of red fruit oil antioxidants on the placenta of mice exposed to lead. methods research design the present study was an experimental study of completely randomized design conducted from october to december 2015 at the experimental animal and the veterinary pathology laboratories, veterinary faculty, airlangga university. experimental animals the subjects of this study were 20 pregnant balb/c mice (mus musculus) selected from among 35 three-month-old females weighing around 25-30 grams, who had been acclimatized for one week. the sample size was determined using federer’s formula, resulting in 4 pregnant mice per group. the optimal total sample size was 20. the mice were assigned by simple random sampling to five groups. the negative control group (k) consisted of pregnant mice that were not exposed to lead on days 6 to 15 of gestation. the intervention group 0 (p0) consisted of pregnant mice exposed to lead acetate at an oral dose of 0.011 mg/20 g bw/ 112 day, dissolved in 0.1 ml distilled water, on days 6 to 15 of gestation. the intervention groups (p1, p2, and p3) consisted of pregnant mice receiving red fruit oil at a dose of 0.3 ml/20 g bw, 0.8 ml/20 g bw, 0.9 ml/20 g bw, and being exposed to lead acetate one hour afterwards. extraction of red fruit oil selected ripe fruits were split lengthwise to remove the pith, then the fruit pulp was cut up into slices and rinsed. the pulp was steamed for 1-1.50 hours until well-done or soft, after which it was taken out to cool. a small quantity of water was added to the cooked slices, which were subsequently mashed into a paste. the fruit paste was screened to remove seed residues, then boiled for 4-5 hours. it was allowed to remain on the fire for 10 minutes until a black oil appeared on the surface. the boiled paste was removed from the fire and left standing for 24 hours, then the oil was carefully ladled to a transparent vessel, and left standing for 2 hours to separate the oil from the water and fruit paste. this last step was repeated several times until no more water remained under the layer of red fruit oil, which was then cooled and packaged. estrus synchronization procedure induction of estrus was effected by the use of pregnant mare serum gonadotropin (pmsg), followed by human chorionic gonadotropin (hcg) for estrus synchronization. the pmsg h o r m o n a l p r e p a r a t i o n w a s a d m i n i s t e r e d intraperitoneally at a dose of 5 iu, while 5 iu of hcg was given 48 hours afterwards.(9) intervention the hormonally-induced females were subsequently mated with the stud male by monomating. mated females were identified by the formation of a vaginal plug 17 hours after mating. the presence of a vaginal plug is considered indicative of copulation and day 0 of gestation. the dose of 4 mg/kg bw of lead for rats was converted to a dose of 0.011 mg/20 g bw for mice. the intervention groups p1, p2, and p3 received red fruit antioxidants one hour before lead exposure. the lead exposure dose of 0.011 mg/20 g bw for the mice was based on the study of aprioku and siminialayi (10) who proved in wistar rats that at a dose of 4 mg/kg bw, lead exposure was capable of decreasing the number of births and of causing teratogenic effects in the form of abnormally-formed skulls, short forelimbs, and absence of hindlimbs in the fetus. the three doses of red fruit antioxidants were obtained from the study of udju et al.(11) who demonstrated that red fruit antioxidants at a dose of 1199.25 mg/kg bw could reduce total cholesterol concentrations in male swiss albino mice. there are no previous data regarding the effect of red fruit administration on caspase expression in lead exposed mice. the interval between red fruit antioxidant administration and lead exposure is known from study results showing that the concomitant administration of vitamin c and vitamin e with lead may protect cells from damage by free radicals during reproduction and fertilization.(7) the one-hour interval between administration of antioxidants and lead is presumably effective in protecting the body against free radicals. measurements the trophoblast cell count was performed in five fields of view. trophoblast cells are located in the floating villi of the placenta, so that the five fields of view comprise 5 floating villi. floating villi are characterized by the presence of trophoblast cells, hofbauer cells, and blood vessels. the scoring of caspase-9 expressing placental trophoblast cells was by means of a modified semiquantitative irs scale of remmele, in which the scores are obtained by multiplying the percentage of positive cells and the intensity of the color reaction.(12) statistical analysis caspase-9 expression scores were analyzed by means of the kruskal-wallis test, followed by the mann whitney test. trophoblast cell counts sumarsono, widjiati, susilowati red fruit oil caspase-9 expression in mice 113 univ med vol. 35 no.2 were analyzed by one-way anova, followed by duncan’s test, at a significance level of 0.05 ethical clearance ethical clearance for the present study was obtained from the research ethics commission, veterinary faculty, airlangga university, surabaya. results from the results shown in tables 1 and 2 it is apparent that administration of red fruit antioxidants to pregnant mice of intervention groups 2 and 3, before they were exposed to lead, was capable of substantially decreasing caspase9 expression in placental trophoblast cells. in general the normal trophoblast cell counts differed considerably. according to one-way anova, there were significant differences in mean trophoblast cell counts between groups (p<0.05) (table 1). this was followed by duncan’s test to find differences between intervention groups. the results of duncan’s test for mean normal trophoblast cell count and sd showed that in the negative control group, i.e. the group of pregnant mice without lead exposure or red fruit administration, there were significant differences with the intervention group zero, i.e. the group of pregnant mice receiving only lead exposure. in contrast, there were no significant differences between the negative control group and intervention groups 1, 2, and 3, i.e. the groups of pregnant mice receiving red fruit antioxidants at different doses, and one hour afterwards being exposed to lead. however, there were significant table 2 . results of multiple comparisons test k= pregnant mice not exposed to lead on days 6-15 of gestation; p0= pregnant mice exposed to lead at 0.011 mg/ 20 g bw/day dissolved in 0.1 ml of distilled water on days 6-15 of gestation; p1, p2, and p3= pregnant mice given red fruit at 0.3 ml/20 g bw, 0.8 ml/20 g bw, and 0.9 ml/20 g bw and then exposed to lead differences between the intervention group 0 and the three intervention groups 1, 2, and 3 (table 2). in the intervention group given red fruit at 0.9 ml/20g bw and lead exposure one hour afterwards (p3) there was a significant increase of 88.2% in trophoblast cell count, in comparison with the group exposed to lead at 0.011 mg/20g bw/day (p0) (p=0.024). there was also a significant reduction of 70.8% in caspase-9 expression of group p3 (p=0.020) (table 2). table 1. mean distribution of normal trophoblasts and caspase-9 expression, by intervention groups in pregnant mice (mus musculus) k= pregnant mice not exposed to lead on days 6-15 of gestation; p0= pregnant mice exposed to lead at 0.011 mg/20 g bw/day dissolved in 0.1 ml of distilled water on days 6-15 of gestation; p1, p2, and p3 = pregnant mice given red fruit at 0.3 ml/20 g bw, 0.8 ml/20 g bw, and 0.9 ml/20 g bw and then exposed to lead; significant at p<0.05 114 in immunohisto chemical and he stained placental slide preparations, the intervention group 0 receiving lead exposure only showed a reduction in normal trophoblast cell count in comparison with the negative control group (figure 1 and 2). with regard to red fruit doses, intervention group 1 given red fruit antioxidants at 0.3 ml/20 g bw, yielded no significant differences with intervention group 0, since the normal trophoblast cell counts approximated those of intervention group 0. in contrast, intervention groups 2 and 3 yielded significant differences with intervention group 0, since the normal trophoblast cell counts approximated those of the negative control group. discussion this study showed that there was increased caspase-9 expression in intervention group 0 (lead exposure only). the three intervention groups showed low caspase-9 expression, when compared with intervention group 0. this is because lead as a toxicant causes damage to both the plasma membranes and the membranes of internal organelles, and also causes lipid peroxidation. the membranes of mitochondria and microsomes are very sensitive to lipid peroxidation, because of the high polyunsaturated fatty acid (pufa) content of the figure 1. caspase-9 expression in placenta of pregnant mice (mus musculus) for each intervention. (a) not showing caspase-9 expression and (b) showing caspase-9 expression. (immunohistochemical staining; 400x). sumarsono, widjiati, susilowati red fruit oil caspase-9 expression in mice 115 univ med vol. 35 no.2 phospholipid membranes.(13) lead exposure during organogenesis causes stress in the placental cells and in the mitochondria of these cells, resulting in the expression of several proteins by these cells, such as bax, a protein carrier of apoptosis signals from within the cells. excessive bax expression in the cytoplasm may damage the mitochondrial membrane so that the cell loses energy. the damaged mitochondrial membrane releases one of the most important proteins, i.e. cytochrome-c, into the cytoplasm. the expression of bcl-2 protein increases, represses bax and inhibits apoptosis. this causes release of mitochondrial cytochrome-c into the cytosol, where it promotes oligomerization of apoptotic protease activating factor-1 (apaf-1), procaspase-9 and atp, forming a complex called the apoptosome. the apoptosome is recruited and activates caspase-9, which in turn promotes caspase-3 activation. this mechanism constututes the intrinsic pathway of apoptosis. the high level of apoptosis in this study is reflected in the brownish colored caspase-9 antibody-antigen complexes. this proves that caspase-9 plays a role in the apoptosis of placental cells at the final stages of pregnancy. the study results show that caspase-9 is expressed by placental cells, i.e. trophoblast cells, decidual cells, fibroblasts, and placental blood cells. caspase-9 induces cell death through the intrinsic pathway, which is activated through the stress induced pathway (mitochondria–mediated pathway).(14) caspase-9 expression was also seen in the negative control group, because apoptosis of placental cells is a physiological process at the approach of parturition. therefore the negative control group still exhibited a brown color, but figure 2. histopathology of normal trophoblasts in pregnant mice (mus musculus) before lead exposure. yellow arrows = normal trophoblasts; red arrows = necrosis of trophoblasts (hematoxylin eosin stain, 400x) 116 not more than did group p0 receiving lead exposure. apoptosis of decidual cells and trophoblast cells increases with age of gestation in normal pregnancies.(15) however, qumsiyeh et al.(16) state that apoptosis of decidual cells and chorionic villi in cases of spontaneous abortion is more than in normal pregnancies. physiologically, apoptosis in trophoblast cells occurs in the third trimester. with increasing age of gestation, the rate of apoptosis is normally increased, so that the number of living cells decreases. placental trophoblast cells differentiate into cytotrophoblasts, then the cytotrophoblasts fuse with one another, forming the syncytiotrophoblast. in addition, the cytotrophoblasts also differentiate into spongiotrophoblasts. in humans and mice, the trophoblasts at the end of gestation differentiate into glycogen cells that play a role in energy storage. furthermore, at the end of gestation the trophoblasts normally also undergo apoptosis at the approach of parturition. lead exposure influences the morphology and physiological reponse of the rat placenta, particularly in the placental labyrinth, i.e. the confluence of the maternal and fetal circulation. the presence of lead causes high ros levels in placental cells, including vascular endothelial cells, resulting in necrosis and apoptosis of these endothelial cells. damage to the blood vessels triggers a physiological response to form new blood vessels (angiogenesis).(17) lead results in a high rate of cell death and affects the physiological response and morphology of the rat placenta. lead exposure during organogenesis in pregnant mice may cause accelerated apoptosis, thus resulting in abortion. increased placental cell death and high levels of inflammation induces secretion of inflammatory cytokines in the blood, while within the cells there is oxidative stress. remodelling of placental vascularization does not occur because the majority of placental cells have undergone normal apoptosis, with extremely high cell death rates and decreased supply of nutrients and vascularization, resulting in abortion. one method to find abnormalities of pregnancy due to lead is to determine the level of apoptosis of placental cells, and one of its indicators is expression of caspase-9 protein in placental trophoblasts. the presence of caspase9 demonstrates the occurrence of apoptosis in the placenta, and may be determined by immunohistochemical techniques. in group p3 receiving the highest dose of antioxidants there was a substantial difference with group p0, but none with group p2. this was because the dose range between groups p2 and p3 was not too great. however, if compared with group p1 it may be seen that the doses of groups p2 and p3 were more influential in decreasing caspase-9 expression, showing that at these doses the antioxidants were capable of reducing caspase9 expression. the antioxidants contained in red fruit function as immunosuppressor, thus acting to decrease caspase-9 expression in trophoblasts as a result of induction by lead. therefore red fruit antioxidants decrease apoptosis of the cells and increase the number of normal trophoblasts. group p0 showed a substantial difference from the other intervention groups, whether the negative control group or the intervention groups. the lead exposure presumably induced many of the placental trophoblasts to undergo necrosis due to the presence of the inflammatory cytokine tnfα, that is capable of causing apoptosis of trophoblasts. in contrast, among the intervention groups receiving red fruit antioxidants before lead exposure, it was found that groups p2 and p3 did not substantially differ from the negative control group, but did substantially differ from group p0. this was due to the betacarotene and tocopherol content of red fruit as a source of natural antioxidants capable of protecting the body against ros. antioxidants are not only important for preventing oxidative stress and tissue damage, but also for preventing increased production of proinflammatory cytokines, that are the result of continuous activation of the defense system. the benefit of red fruit antioxidants is to disrupt the free radical chain reaction, such as is done by vitamin e (alpha tocopherol), vitamin c (ascorbic acid), and vitamin a (betacarotene). sumarsono, widjiati, susilowati red fruit oil caspase-9 expression in mice 117 univ med vol. 35 no.2 tocopherol and betacarotene are antioxidants that can terminate the free radical chain reaction.(18) tocopherol has the ability to terminate lipid peroxidation by donating one hydrogen atom from the oh group to the lipid peroxyl radical, so that it becomes less reactive and does not cause damage. when there are free radicals, lipid peroxides increase due to reactions between lipids and free radicals. the doses of 0.8 ml/20g bw and 0.9 ml/20 g bw of red fruit antioxidants in groups p2 and p3 showed an increase in normal trophoblast cell counts, which was greater than the increase produced by the dose of 0.3 ml/20 g bw. primary antioxidants are capable of cleaving free radicals by initiating a chain reaction through chain-breaking, i.e. by donating a free electron to ros and lipid radicals that are produced in biological systems and converting them to more stable molecules.(19) tocopherol when given to lead-exposed rats can neutralize lead toxicity by cleaving free radicals and preventing oxidative stress.(20) tocopherol as an antioxidant acts by competitive inhibition of lead and prevents the formation of lipid peroxides as a result of lead exposure. the carotenoids contained in red fruit antioxidants may induce metabolic enzymes so as to prevent cell damage or death caused by lead.(21) all carotenoids, whether as provitamin a or not, may act as antioxidant. carotenoids also prevent the formation of lipid peroxides and bind to free radicals. the antioxidant mechanism of carotenoids is through quenching of singlet oxygen and subsequently interacting with free radicals.(22) the limitations of this study are firstly, that the sampling was done in stages, since not all of the mice were found to be pregnant. secondly, that several oxidative stress parameters were not measured, e.g. malondialdehyde (mda) and superoxide dismutase (sod). in accordance with the results of this study, its clinical implication is that the use of antioxidants as a preventive measure, both in humans and animals, is urgently required, in view of the occurrence of heavy metal pollution that may affect reproductive health. further studies are needed to evaluate red fruit administration at various time points before lead exposure. conclusions administration of red fruit to pregnant mice before lead exposure has the potential to increase the normal trophoblast cell count by 88.2% and decrease caspase-9 expression by 70.8%. red fruit is a potential protective agent against rosmediated disorders. conflict of interests none declared. acknowledgement we thank the staff of the embryology and veterinary pathology laboratories. veterinary faculty, airlangga university for support in the completion and publication of this study. references 1. barness eg. teratogenic causes of malformations. ann clin lab sci 2010;40:99114. 2. garcia-nìno wr, chaverri jp. protective effect of curcumin against heavy metals-induced liver damage. food chem toxicol 2014;69:182–201. 3. levy r, smith sd, yusuf k, et al. trophoblast apoptosis from pregnancies complicated by fetal growth restriction is associated with enhanced p53 expression. am j obstet gynecol 2002;186: 1056-61. 4. sarungallo zl, hariyadi p, andarwulan n, et al. analysis of α-cryptoxanthin, β-cryptoxanthin, α-carotene, and β-carotene of pandanus conoideus oil by high-performance liquid chromatography (hplc). procedia food sci 2015;3:231–3. 5. dearth rk, hiney jk, srivastava v, et al. effects of lead (pb) exposure during gestation and lactation on female pubertal development in the rat. reprod toxicol 2002;16:343–53. 6. isradji i. pengaruh pemberian pb-asetat terhadap fertilitas mencit jantan, dimonitor melalui jumlah kebuntingan dan jumlah anak sekelahiran. sains medika 2010;2:2:170-177. 118 7. sharma rn, qureshi sm, panwar k. lead induced infertility in swiss mice and role of antioxidants. universal j environ res technol 2012;2:72-82. 8. rohman a, riyanto s, yuniarti n, et al. antioxidant activity, total phenolic, and total flavaonoid of extracts and fractions of red fruit (pandanus conoideus lam). int food res j 2010; 17:97-106. 9. widjiati awr, arifin mz. pengaruh berbagai konsentrasi krioprotektan propanediol pada proses vitrifikasi terhadap viabilitas embrio mencit pasca thawing. veterinaria medika 2011; 4:2. 10. aprioku js, siminialayi im. maternal lead exposure and pregnancy outcome in wistar albino rats. j toxicol environ health sci 2013; 5:185-93. 11. udju ist, martono y, soetjipto h. ekstrak enzimatis antioksidan buah merah (pandanus conoideus lam.) sebagai antikolesterol terhadap mencit putih jantan galur swiss. prosiding seminar nasional sains dan pendidikan sains vii uksw. salatiga;2012.p.117-26. 12. nowak m, madej ja, dziegeil p. intensity of cox2 expression in cells of soft tissue fibrosarcomas in dogs as related to grade of tumour malignancy. bull vet inst pulawy 2007; 51:275-9. 13. suryohudoyo p. kapita selekta ilmu kedokteran molekuler. jakarta: sagung seto;2007. 14. mcilwain dr, berger t, mak tw. caspase functions in cell death and disease. cold spring harb perspect biol 2013;5:a008656. 15. halperin r, peller s, sandbank j, et al. expression of the p53 gene and apoptosis in gestational tropoblastic disease. placenta 2000; 21:58–62. 16. qumsiyeh mb, kim kr, ahmed mn, et al. cytogenetics and mechanisms of spontaneous abortions: increased apoptosis and decreased cell proliferation in chromosomally abnormal villi. cytogenet cell genet 2000;88:230-5. 17. veras mm, damaceno-rodrigues nr, caldini eg, et al. particulate urban air pollution affects the functional morphology of mouse placenta. biol reprod 2008;79:578-84. 18. christijanti w, utami nr, iswara a. pemberian antioksidan vitamin c dan e terhadap kualitas spermatozoa tikus putih terpapar allethrin. biosaintifika 2010;2:18-26. 19. vaya j, aviram m. nutritional antioxidants: mechanisms of action, analyses of activities and medical applications. curr med chem imm endo metab agents 2001;1. 20. sajitha gr, jose r, andrews a, et al. garlic oil and vitamin e prevent the adverse effects of lead acetate and ethanol separately as well as in combination in the drinking water of rats. indian clin biochem 2010;25:280-8. 21. sharma b, singh s, siddiqi nj. biomedical implications of heavy metals induced imbalances in redox systems. bio med res int 2014, article id 640754, 26 pages http://dx.doi.org/10.1155/ 2014/640754. 22. polyakov ne, foscan al, bowman mk, et al. free radical formation in novel carotenoid metal ion complexes with astaxanthin. j phys chem b 2010;114:16968–77. sumarsono, widjiati, susilowati red fruit oil caspase-9 expression in mice david 106 *department of histology, medical faculty, trisakti university correspondence adr. david department of histology, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2702 email : davesaboch@yahoo.com univ med 2008; 27: 106-112 evaluation of the oral toxicity of formaldehyde in rats july-september, 2008july-september, 2008july-september, 2008july-september, 2008july-september, 2008 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 abstract universa medicina david*a, and hanslavina arkeman* formaldehyde is used in the production of plywood, particleboard, a wide variety of molded or extruded plastic items, and for preserving cadavers. experimental posttest only control group design was used to determine the histopathological changes of rat stomach tissue exposed to formaldehyde for 12 weeks in thirty adult female sprague-dawley rats weighing 150–200 g. the animals were randomized into three groups, namely the control group, the treatment group given 4 mg/l of formaldehyde, and the group treated with 6 mg/l of formaldehyde. the results showed that there was a significant difference in the thickness of the mucous layer of the gastric fundus between the control group and the group treated with 6 mg/l (p = 0.011), and also a significant difference in the thickness of the mucous layer of the gastric fundus between both treatment groups (4 mg/l vs. 6 mg/l) (p = 0.003), indicating that there was necrosis of the superficial layers of the gastric mucosa due to high concentrations of formaldehyde. in histopathology sections from all groups, similar changes were observed in both treatment groups, in the form of vacuolar (hydropic) degeneration of gastric fundus glands. in conclusion, administration of formaldehyde in drinking-water for 12 weeks caused histopathologic effects on the gastric mucosa in rats. keywords : formaldehyde, oral, drinking water, toxicity introduction formaldehyde is used in many industries, in the production of fertilizer, paper, plywood, and polyacetal, phenolic, urea, and melamine resins. the latter three kinds of resins are widely u s e d a s a d h e s i v e s a n d b i n d e r s f o r w o o d products, pulp and paper, synthetic fibers, and i n t h e f i n i s h i n g o f t e x t i l e s . f u r t h e r m o r e , formaldehyde may be converted into a number of chemicals, such as 1,4-butanediol, 4,4’diphenylmethane diisocyanate, pentaerythritol and hexamethylene-tetramine. formaldehyde is also used in many hospitals and laboratories to preserve tissue specimens,(1-3) and in mortuaries a s a n e m b a l m i n g f l u i d . f o r m a l d e h y d e i n drinking water commonly originates from the oxidation of natural organic matter during the o z o n i z a t i o n a n d c h l o r i n a t i o n p r o c e s s . formaldehyde in drinking water may also come from deterioration of the protective finish on polyacetal plastics.(4,5) 107 univ med vol.27 no.3 formaldehyde is a gas widely encountered in natural and man-made environments and is readily soluble in water to form a 40% solution (formalin®). the hazards of exposure to this gas by inhalation have been the subject of many studies, leading to the reclassification by the international agency for research on cancer (iarc) of formaldehyde from group 2a to g r o u p 1 ( c a r c i n o g e n i c t o h u m a n s ) , w h i c h e x p r e s s e s t h e c u r r e n t c o n c e r n a b o u t t h e c a r c i n o g e n i c e f f e c t s o f i n h a l a t i o n o f formaldehyde.(2,6) however, in indonesia the most recent issue was about the indiscriminate use of formaldehyde as a food preservative, especially for sources of protein, such as beef, poultry, fish, as well as tofu and tempeh. formaldehyde is a colorless, flammable gas at room temperature. it has a pungent, distinct odor and at high concentrations may cause a burning sensation to the eyes, nose, and lungs. formaldehyde is also known as methanal, methylene oxide, oxymethylene, methylaldehyde, and oxomethane. formaldehyde can react with m a n y o t h e r c h e m i c a l s , a n d a t v e r y h i g h temperatures it will break down into methanol (wood alcohol) and carbon monoxide. a number o f e x p e r i m e n t a l s t u d i e s o n t h e e ff e c t s o f formaldehyde by ingestion have been conducted in rodents, particularly in rats. owing to formaldehyde’s high reactivity, effects in the tissue on first contact following ingestion are more likely to be related to the concentration of the formaldehyde consumed than to its total intake.(7,8) the aim of the present s t u d y w a s t o a s s e s s t h e e ff e c t s o f o r a l l y administered formaldehyde on the thickness and histopathology of the gastric fundal mucosa in rats. methods design of the study a posttest only control group design was used to attain the objectives of the study. sample thirty adult female sprague dawley rats weighing 150–200 g. the sample size was calculated using the formula of federer (9) : (n1) (t-1) >15. when using three groups (t = 3), the optimal sample size required will be 10 adult female rats per group. intervention the experimental animals were randomly assigned to three groups, namely group i comprising the control group which was not g i v e n f o r m a l d e h y d e c o n t a i n i n g d r i n k i n g water, group ii as the treatment group that was given drinking water containing formaldehyde in a concentration of 4 mg/l daily for 12 weeks, and group iii as the treatment group t h a t w a s g i v e n d r i n k i n g w a t e r c o n t a i n i n g formaldehyde in a concentration of 6 mg/l daily for 12 weeks. methods all three groups of experimental animals were given food pellets and plain drinking water, and it was only in the intervention groups (groups ii and iii) that formaldehyde was added to the drinking water. the formaldehyde used was formalin® diluted with distilled water to obtain the required dose. after 12 weeks the experimental animals were sacrificed and the organs taken for histological sections stained with hematoxylin-eosin (he). data collection the weight of the rats in each group was measured at the beginning and at the end of the experiment to monitor increases in the weights of the rats in each group. measurements were also taken of the thickness of the fundal mucosa in the rats of each group using a micrometer in a double-blind manner. 108 data analysis continuous data were expressed as mean ± sd. one-way analysis of variance (anova) was used for comparing the difference in the body weights of the rats before and after exposure to formalin® between the three groups, at a significant david, arkerman oral toxicity of formaldehyde mean control (n=10) formaldehyde 4 mg/l (n=10) formaldehyde 6 mg/l (n=10) p difference in body weight (g) 32.9 ± 12.5 33.0 ± 15.6 31.7 ± 7.6 0.953 tunica mucosa of gastric fundus (µm) 1051.24 ± 132.07 1075.00 ± 65.04 919.00 ± 138.91* 0.004 table 1. mean difference in body weight and between thickness of the fundal mucosa the three groups * significantly different from control group and group on 4 mg formaldehyde level of 0.05. the same method was used for comparing the thickness of the fundal mucosa of the rats in each group at a significant level of 0.05. when significant differences were found between the three groups, the analysis was continued with the tukey multiple comparison method. tunica mucosa figure 1c. tunica mucosa of rat gastric fundus of 6 mg formaldehyde group stained with he, at 100x magnification figure 1b. tunica mucosa of rat gastric fundus of 4 mg formaldehyde group stained with he, at 100x magnification tunica mucosa figure 1a. tunica mucosa of rat gastric fundus of control group stained with he, at 100x magnification 109 univ med vol.27 no.3 results after 12 weeks’ exposure to formaldehyde, the mean difference (± sd) in body weight before and after exposure was 32.9 ± 12.5 g in the control group and 33.0 ± 15.6 g in the group treated with a formaldehyde concentration of 4 mg/l, whereas in the group treated with 6 mg/ l formaldehyde a mean difference in body weight of 31.7 ± 7.6 was found (table 1). the results of the anova revealed that there was no significant difference in the body weights o f t h e r a t s b e f o r e a n d a f t e r e x p o s u r e t o formaldehyde between the three groups (p = 0 . 9 5 3 ) . t h e a n o va r e s u l t s f o r t h e m e a n thickness of the tunica mucosa of the rat gastric fundus showed a significant difference between t h e t h r e e g r o u p s ( p = 0 . 0 0 4 ) . m u l t i p l e comparison analysis according to tukey showed that the mean thickness of the tunica mucosa of the gastric fundus in the group on 6 mg/l formaldehyde was significantly different from that of the control group (p = 0.011) and that of the group on 4 mg/l formaldehyde (p = 0.003). in the histological sections structural changes may be seen in the cells of the fundal mucosa of rats exposed to formaldehyde in c o n c e n t r a t i o n s o f 4 m g / l a n d 6 m g / l , respectively. necrosis of fundal mucosal cells o c c u r r e d e s p e c i a l l y i n t h e c y l i n d r i c a l epithelium, affecting a higher percentage of c e l l s i n t h e g r o u p e x p o s e d t o 6 m g / l formaldehyde (figures 1a, 1b, and 1c). t h i s g r o u p a l s o s h o w e d h y d r o p i c d e g e n e r a t i o n o f t h e l a m i n a p r o p r i a a n d associated glands, which percentage was more severe than that in the group on 4 mg/l of formaldehyde (figures 2a, 2b, and 2c). figure 2a. cells of the glands in the fundal mucosa stained with he, at 400x magnification figure 2b. hydropic degeneration in the fundal mucosa stained with he, at 400x magnification figure 2c. hydropic degeneration in the fundal mucosa stained with he, at 400x magnification hydropic degeneration 110 discussion this study shows that administration of drinking water containing formaldehyde in a concentration of 6 mg/l to experimental rats for 12 weeks, resulted in a mean thickness of the fundal mucosa of 919.00 ± 138.91 ì m. the fundal mucosa in the group given drinking water containing formaldehyde in a concentration of 6 mg/l was thinner, in comparison with that in t h e c o n t r o l g r o u p a n d i n t h e g r o u p g i v e n drinking water containing formaldehyde in a concentration of 4 mg/l. this indicates the occurrence of necrosis in the superficial cells that were directly exposed to irritation by formaldehyde. for purposes of comparison, a n u m b e r o f p r e v i o u s s t u d i e s b y v a r i o u s investigators may be mentioned. in one study, wistar rats (10 per sex per dose) were given formaldehyde in drinkingwater at 0, 10, 50, or 300 mg/kg of body weight per day. at the end of 12 months, all rats of both sexes in the high-dose group were observed to have gastric erosions, ulcers, squamous cell hyperplasia, hyperkeratosis, and basal cell hyperplasia. however, only one male and one f e m a l e f r o m t h e m i d d o s e g r o u p s h o w e d hyperkeratosis.(6) in a carcinogenicity study, a group of 10 rats was given drinking-water containing 0.5% formalin® (0.2% formaldehyde) for 32 weeks. histopathological changes were observed in the stomach, as well neoplastic changes in the forestomach and papillomas. takahashi et al. reported that formaldehyde enhanced gastric two-stage carcinogenesis in rat stomach induced by n-methyl-n’-nitro-nguanidine. (10) subsequently, furihata et al. administered formaldehyde at doses of 11 to 110 mg/kg body weight by gastric intubation to male f 3 4 4 r a t s , u s i n g o r n i t h i n e d e c a r b o x y l a s e activity and dna synthesis as markers of tumor-promoting activity. their study showed an increase in ornithine decarboxylase activity (up to 100-fold) and in dna synthesis (up to 49-fold) in the pyloric mucosa of the glandular stomach, both with a maximum after 16 hr. these results suggest that formaldehyde has tumor-promoting activity in carcinogenesis in the glandular stomach.(11) a number of other long-term studies by the oral route have been conducted, and these have been reviewed in detail by the international p r o g r a m m e o n c h e m i c a l s a f e t y. ( 6 ) t h e c o n c l u s i o n o f t h e s e r e v i e w s w a s t h a t f o r m a l d e h y d e i s a n o r m a l m a m m a l i a n metabolite and is not carcinogenic at low levels of exposure. similarly, bosetti et al reviewed c o h o r t s t u d i e s o n f o r m a l d e h y d e r i s k o f nasopharyngeal and selected other cancers, and found a pooled rr of 0.63 for nasopharyngeal cancer. they concluded that occupational exposure to formaldehyde shows no appreciable excess risk for oral and pharyngeal, sinonasal or lung cancers, and that the non-significantly increased rr for nasopharyngeal cancer among industry workers was attributable to a cluster of deaths in a single plant.(12) these views have still to be reconciled with the reclassification of formaldehyde by the iarc. the histological changes observed in the current study showed that the group given drinking water with a high concentration of formaldehyde experienced hydropic degeneration in a larger percentage of the cells of the fundal glands, compared with the group given drinking water with a low concentration of formaldehyde. t h i s i n d i c a t e s t h a t t h e e x t e n t o f t h e histopathological changes was proportional to the formaldehyde concentration to which the rats had been exposed. this is in accord with the view that, owing to the high reactivity of formaldehyde, its effects in the tissue of first contact following ingestion are more likely to be related to the concentration of the formaldehyde consumed than to its total intake.(7,8) david, arkerman oral toxicity of formaldehyde 111 univ med vol.27 no.3 the current study also found that even s h o r t e x p o s u r e t i m e s w e r e s u f f i c i e n t f o r inducing histological changes in the rat gastric fundus. the observed histopathological changes c o n f i r m t h e s t u d y c o n d u c t e d b y m a z i m khan,(11) which clearly demonstrated that one of the effects of formaldehyde is induction of cell vacuolization. however, the study by til et al. revealed that long-term administration of formaldehyde in drinking water (2 years) may induce the development of gastric papillomas.(6) the hydropic degeneration induced after 12 weeks in our study could represent the initial stage of such a development. one study in male rats showed an increased incidence of forestomach papillomas, while another study in male and female rats found that t h e i n c i d e n c e o f g a s t r o i n t e s t i n a l l e i o myosarcomas was increased in females and in males and females combined. in a third study, also using male and female rats, the number of m a l e s b e a r i n g m a l i g n a n t t u m o r s a n d t h e incidences of hemolymphoreticular tumors (lymphomas and leukemias) and testicular i n t e r s t i t i a l c e l l a d e n o m a s i n m a l e s w e r e i n c r e a s e d . h o w e v e r, a f o u r t h s t u d y g a v e negative results.(13) a health-based value can be derived on the b a s i s o f a t o l e r a b l e c o n c e n t r a t i o n , s i n c e irritation by formaldehyde is a concentrationdependent outcome and is not directly related to the total intake. the international programme on chemical safety has established a tolerable c o n c e n t r a t i o n o f 2 . 6 m g / l f o r i n g e s t e d formaldehyde based on the no-observed-effect level (noel) of 260 mg/l for histopathological effects in the oral and gastric mucosa of rats.(14) the environmental protection agency (epa) recommends that an adult should not drink water containing more than 1 milligram of formaldehyde per liter of water (1 mg/l) for a lifetime exposure, and a child should not drink water containing more than 10 mg/l for 1 day or 5 mg/l for 10 days. the occupational safety and health administration (osha) has set a permissible exposure limit for formaldehyde of 0.75 parts per million (ppm) for an 8-hour workday, 40-hour workweek. the national institute for occupational safety and health (niosh) recommends an exposure limit of 0.016 ppm.(15) the weight of evidence indicates that ingested formaldehyde is not carcinogenic in humans. there are good grounds for confidence that the present understanding of the two step n a t u r e o f f o r m a l d e h y d e c a r c i n o g e n e s i s r e q u i r i n g a h i g h l e v e l o f e x p o s u r e t o formaldehyde in the first instance remains valid. the world health organisation (who) in its concise international chemical assessment document outlined that “the weight of evidence indicates that formaldehyde is carcinogenic only at concentrations that induce the obligatory precursor lesion of proliferative regenerative response associated with cytotoxicity, although interaction with the dna must be taken into account”.(6) conclusions short-term administration of formaldehyde in drinking water was capable of effecting histopathological changes in the cells of the gastric fundal glands in rats. the severity and extent of the histopathological changes were proportional to the formaldehyde concentration to which the rats had been exposed. references 1. dutch expert comittee on occupational standards (decos). formaldehyde health-based recommended occupational exposure limit. health council of the netherlands (gezondheidsraad) 2003; 02 osh: 124. 2. international agency for research on cancer (iarc) monographs on the evaluation of the 112 carcinogenic risk of chemicals to humans vol: 88; 2004. 3. thomsen gk. health effects of selected chemicals. nord 1995; 28: 211–37. 4. organization for economic co-operation and development (oced). sids initial assessment report for siam 14. formaldehyde. paris: organization for economic co-operation and development; 2002. 5. world health organization. guidelines for drinking water quality. formaldehyde in drinking water. geneva: world health organization; 2005. 6. world health organization. concise international chemical assessment document: formaldehyde. geneva: world health organization; 2002. 7. cogliano vj, yann g, baan rs, kurt s, secretan mb, el ghissassi f, et al. advice on formaldehyde and glycol ethers. lancet oncol 2004; 5: 528. 8. pitcher m. lca treatment of human health exemplified by formaldehyde within the furniture industry. presented at the 4th australian lca conference, sydney. february 2005. 9. federer w. statistics and society: data collection and interpretation. 2nd ed. new york: marcel dekker, 1991. 10. takahashi m, hasegawa r, furukawa f, toyoda k, sato h, hayashi y. effects of ethanol, potassium metabisulfite, formaldehyde and hydrogen peroxide on gastric carcinogenesis in rats after initiation with n-methyl-n’-nitro-n-guanidine. jpn j cancer res 1986; 77: 118-24. 11. furihata c, yamakoshi a, matsushima t. inductions of ornithine decarboxylase and dna synthesis in rat stomach mucosa by formaldehyde. jpn j cancer res 1988: 79: 917-20. 12. bosetti c, mclaughlin jk, tarone re, pira e, la vecchia c. formaldehyde and cancer risk: a quantitative review of cohort studies through 2006. ann oncol 2008: 19: 29-43. 13. international agency for research on cancer. formaldehyde. available at: http://www. m o n o g r a p h s . i a r c . f r / e n g / m e e t i n g s / 8 8 formaldehyde.pdf. accessed june 2, 2008. 14. international programme on chemical safety (ipcs). formaldehyde. geneva: world health organization (concise international chemical assessment document 40); 2002. 15. agency for toxic substances and disease registry (atsdr). 1999. toxicological profile for formaldehyde. atlanta, ga: u.s. department of health and human services, public health services. available at: www.atsdr.cdc.gov/ toxprofiles/tp111-c1.pdf. accessed march 3, 2007. david, arkerman oral toxicity of formaldehyde alvina 78 abstract to date the use of computers is widely distributed throughout the world and the associated ocular complaints are found in 75-90% of the population of computer users. symptoms frequently reported by computer users were eyestrain, tired eyes, irritation, redness, blurred vision, diplopia, burning of the eyes, and asthenopia (visual fatigue of the eyes). a cross-sectional study was conducted to determine the etiology of asthenopia in computer-using students. a questionnaire consisting of 15 items was used to assess symptoms experienced by the computer users. the ophthalmological examination comprised visual acuity, the hirschberg test, near point accommodation, amplitude accommodation, near point convergence, the cover test, and the alternate cover test. a total of 99 computer science students, of whom 69.7% had asthenopia, participated in the study. the symptoms that were significantly associated with asthenopia were visual fatigue (p=0.031), heaviness in the eye (p=0.002), blurred vision (p=0.001), and headache at the temples or the back of the head (p=0.000). refractive asthenopia was found in 95.7% of all asthenopia patients with accommodative insufficiency (ai), constituting the most frequent cause at 50.7%. the duration of computer use per day was not significantly associated with the prevalence of asthenopia (p=0.700). there was a high prevalence of asthenopia among computer science students, mostly caused by refractive asthenopia. accommodation measurements should be performed more routinely and regularly, maybe as screening, especially in computer users. keywords : asthenopia, acomodative insuffieciency, computer users, students *department of ophthalmology, medical faculty, trisakti university **department of ophthalmology, faculty of medicine, university of indonesia correspondence dr. husnun amalia, spm department of ophthalmology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2614 univ med 2010;29:78-83 accommodative insufficiency as cause of asthenopia in computer-using students husnun amalia*, gusti g. suardana**, and widya artini** may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction the number of computer users has rapidly increased throughout the world and is expected to reach 1 billion by the year 2010 as a result of an increasing number of new computer users in developing countries such as china, india and russia.(1) there is a growing body of evidence that the use of computers can adversely affect visual health. working at a computer terminal is invariably associated with ocular disorders that may affect visual acuity. initially research was focused on the ocular effects of radiation hazards, but subsequently came to include symptoms due to exposure of the eyes to video d i s p l a y t e r m i n a l s ( v d t ) , k n o w n a s t h e 79 univ med vol.29 no.2 computer vision syndrome.(2) several studies showed that around 75% of computer users had visual problems. (3,4) symptoms frequently reported by computer users were eyestrain, tired eyes, irritation, redness, blurred vision, diplopia, burning of the eyes, and asthenopia.(5-7) the main cause of asthenopia is thought to be fatigue of the ciliary and extraocular muscles due to the prolonged accommodation and vergence required by near vision work. a n o t h e r c a u s a t i v e f a c t o r t h a t h a s b e e n implicated in asthenopia is dryness of the eyes resulting from an increased exposed surface area of the cornea when focusing straight ahead (rather than down at written text) and a d e c r e a s e d b l i n k r a t e d u e t o m e n t a l concentration.(8,9) etiologically, asthenopia may be classified a s m u s c u l a r a s t h e n o p i a a n d r e f r a c t i v e asthenopia. muscular asthenopia may be caused by heterophoria, intermittent heterotropia, and convergence insufficiency (ci), whereas refractive asthenopia is due to ammetropia, presbyopia, accommodative insufficiency (ai), and a combination of accommodative and convergence insufficiency. anomalies of convergence may be detected by means of the near point convergence (npc) test, while accommodative abnormalities are detected by the near point accommodation (npa) test.(10) symptoms occurring in asthenopia are redness, heaviness of the eye, dry eye, frontal headache with periocular pain and occipital headache, blurred vision, intermittent diplopia at near fixation, and difficulty of focus, especially when reading and writing. (11) computer-related headache and eyestrain are reported during as m a n y a s 1 0 % t o 1 5 % o f r o u t i n e e y e examinations, and several investigators state that nearly 50% of vdt workers experience some eye discomfort . the world health organization has initiated a program to eradicate blindness in the year 2020, known as vision 2020.(12) asthenopia is a reversible condition and does not result in a reduction of performance o r e f f i c i e n c y o f t h e p a t i e n t . r e f r a c t i v e asthenopia can be corrected by glasses, whilst muscular asthenopia may be corrected by accommodative and convergence training. current technological advances can prevent asthenopia symptoms through appropriate ergonomic design of the vdt.(13) available data show that there are large numbers of computer and internet users. consequently, the risk of asthenopia in computer users may reach epidemic proportions in the foreseeable future. the purpose of the present study was to determine the prevalence of asthenopia and its etiology in computer users. methods design of the study this study was an analytical study with cross-sectional approach, with the objective of determining the prevalence of asthenopia, in order to ascertain the extent of ocular health hazards resulting from computer use. the study was conducted in february 2007 at the faculty of medicine, university of indonesia, jakarta. subjects of study the subjects of this study comprised 99 s t u d e n t s f r o m t h e f a c u l t y o f c o m p u t e r science, university of indonesia. the inclusion criteria in this study were strata 1 and strata 2 students with visual acuity of 6/6 with or without correction who were willing to fill in t h e q u e s t i o n n a i r e a n d t o r e c e i v e a n ophthalmological examination. the exclusion criteria were presence of manifest heterotropia, amblyopia, and ocular disorders capable of obstructing the visual axis. data collection data collection was carried out by means of a questionnaire and several ophthalmological tests. the data collected by questionnaire comprised demographic status, total hours of near-vision work and 15 items on subjective complaints due to asthenopia. responses to the 80 table 1. distribution of etiologies of asthenopia 15 items in the questionnaire were scored as follows: 0, never; 1, infrequent; 2, occasionally; 3, relatively frequent; 4, frequent; and 5, always. the total score for the 15 questions given to each subject ranged from 0 to 75. subjects were considered to be at risk of suffering from convergence insufficiency if their total score was ≥9. subjects having a total score of <9 were assigned to the group with negative symptoms, while those with a total score of ≥9 were placed in the group with positive symptoms.(14) subsequently the subjects received an ophthalmological examination, comprising determination of visual acuity, the hirschberg test, tests for npa, amplitude accommodation (aa), and npc, the cover test (ct), and the alternate cover test (act). subjects were diagnosed as suffering from ai if the obtained aa score was less than the normal score for age according to donder’s table.(11) the aa score for the age range of 17-19 years is ≥11 d, 2024 years 8 d, 25-29 years ≥6 d, 30-34 years ≥5 d, and 35-39 years ≥4 d (d=dioptries). npc is the convergence ability of the patient’s eye in maintaining fusion, and the test result is expressed in cm. a score of <10 cm is said to be normal and a score of >10 cm means that the subject has ci.(8) data analysis the study data were divided into two groups, i.e. data on the group of subjects with asthenopia and data on the group of normal subjects. the relationships between variables of the asthenopia group and the normal group were analyzed using the chi square tests. a pvalue of <0.05 was considered significant. the data analysis used the spss program version 15.0. results the total number of subjects who were willing to participate in this study was 133, of whom 34 did not meet the inclusion criteria, for t h e f o l l o w i n g r e a s o n s : o n e s u b j e c t h a d congenital cataract, four subjects suffered from manifest heterotropia, and 29 subjects did not complete the questionnaire. the total number of subjects who participated until completion of the study was 99, consisting of 61 (61.4%) males and 38 (38.4%) females. mean age of the subjects (± sd) was 20.2 ± 1.8 years, with a range of 18 up to 26 years. the majority of the subjects (68 persons or 68.3%) used a computer from 3-6 hours per day, and only 7 (7.1%) used a computer for <2 hours per day. the prevalence of asthenopia in the present study was 69 (69.7%), while that of normal subjects was 30 (30.3%). ta b l e 1 s h o w s t h e d i s t r i b u t i o n o f etiologies of the asthenopic subjects. among the two principal causes of asthenopia, i.e. refractive anomalies and muscular anomalies, refractive anomalies accounted for the majority of asthenopia causes (95.7%), where ai ranked highest at 50.7%. the results of the analysis (table 2) indicated that there was a significant difference between the two groups for the symptoms of visual fatigue (p= 0.031), heaviness (p=0.002), difficulty of recall of recent reading (p=0.001), parietal or occipital headaches (p=0.000), blurred vision (p=0.001), confusion during reading (p=0.014), and failing focus or loss of concentration during reading (p=0.003). most of the subjects in the asthenopia group were active at the computer for 2-4 hours per day. amalia, suardana, artini accommodative insufficiency and asthenopia 81 univ med vol.29 no.2 complaints p visual fatigue 0.03 1* redness 0.10 6 heaviness in eye 0.00 2* dry eye 0.08 8 drowsiness 0.18 4 difficulty of recall of recent reading 0.00 1* per iocular pain 0.22 9 parietal/occipital headaches 0.00 0* blurred vision 0.00 1* occasional diplopia 0.10 3 line skipping during readin g 0.20 7 confusion during readin g 0.01 4* difficulty of focus/loss of concentration 0.00 3* need for momentary rest for the eyes 0.05 2 floating tex t 0.69 5 table 2. analysis of subjective complaints between asthenopia and normal groups * significance table 3. distribution of asthenopia in relation to duration of computer use (hours/day) duration of computer use (hours/day)* asthenopia p present (%) absent (% ) < 2 3-4 5-6 > 7 4 (57.1) 27 (73.0) 20 (64.5) 18 (75.0) 3 (42.9) 10 (27.0) 11 (35.5) 6 (25.0) 0.700 * chi-square test from table 3 it is apparent that duration of daily computer use was not significantly associated with occurrence of asthenopia (p=0.700). discussion to date the use of computers has spread e x t e n s i v e l y t h r o u g h o u t t h e w o r l d a n d accompanying ocular complaints occur in 7590% of the population of computer users.(2) the present study showed a high prevalence of asthenopia of 69.7% in computer users. essentially similar results were obtained in an indian study on computer users aged 18-55 years, of whom 46.3% suff er ed from asthenopia.(1) a study in italy on bank employees f o u n d t h a t 3 9 . 1 % o f t h e s u b j e c t s h a d asthenopia.(5) a total of 92.9% of computer science students in our study had ocular complaints, of w h o m 6 9 . 7 % s u ff e r e d f r o m r e f r a c t i v e anomalies. blehm et al. obtained similar results, where 75–90% of computer users had ocular complaints.(2) in all probability their subjects h a d o c u l a r s u r f a c e a b n o r m a l i t i e s a n d photophobia, which were not investigated in their study. ocular surface abnormalities are caused by dry eye and problems with contact lenses. dry eye may be due to reduced blinking r e f l e x e s a n d e n v i r o n m e n t a l f a c t o r s . ( 1 , 2 ) photophobia is sensitivity of the eye to light, and computer users are commonly exposed to intense illumination and monitor flicker.(16) the usual cause of intense illumination is a window facing the operator and a less than adequate ambient lighting. ai is the major cause of asthenopia, because computer-related activities overburden t h e a c c o m m o d a t i o n m e c h a n i s m . t h e concurrence of refractive errors leads to an increased probability of subjective complaints. the occasional occurrence of blurred vision is due to failure of the accommodative mechanism to maintain focus on near objects. blurred vision may be remedied by fitting a positive spherical lens or by accommodative training, in which case the subject with ai is fitted with a spherical lens of +0.75d up to +1.25d for near vision.(17) accommodative training may be accomplished by means of the flip lens technique,(3,4) which uses 2 negative spherical lenses and 2 positive spherical lenses, each differing 0.5d in power. 82 the patient is asked to focus on an object located at a distance of 40 cm in front of her, then the lenses are placed in turn, with the patient attempting to maintain focus on the object. the procedure is repeated 5 times daily, each time for a period of 3 minutes. ci is a condition where a person lacks the ability for maintaining binocular convergence. this condition is the most frequent cause of muscular asthenopia, being found in 3-5% of the general population.(5) and in 4.3% of the asthenopia group of the present study. ci may be managed by convergence training, also known as pencil push up,(19) where the patient focuses on her thumb or on a pencil held in her hand. the object is held vertically at arm’s length in front of the eye, then the object is slowly moved towards the eye, while the p a t i e n t a t t e m p t s t o m a i n t a i n b i n o c u l a r convergence. this procedure is performed 3-4 times daily for 5 minutes and in general the patient will recover within 5-15 weeks. r e f r a c t i v e e r r o r s m o s t f r e q u e n t l y encountered are myopia (21.7%), astigmatism (2.9%), and compound myopic astigmatism ( 4 . 3 % ) , w h i c h m a y b e c o r r e c t e d w i t h glasses.(18) on the other hand, the combination of ai and ci is managed by accommodative and convergence training.(6) in the asthenopia group of the present s t u d y, c o m p u t e r r e l a t e d a c t i v i t i e s w e r e apparently more frequent than other types of near work. there are still conflicting opinions on the relationship of duration of computer use to occurrence of asthenopia. a total of 73.0% o f s u b j e c t s w i t h a s t h e n o p i a p e r f o r m e d computer-related activities for periods of 3–4 hours per day, but no significant association was found between duration of computer use and the occurrence of asthenopia. in contrast, a study in india found that mean computer use of 78 minutes duration was associated with occurrence of asthenopia.(5) seven subjective complaints were proven to be significant in the asthenopia group, i.e. visual fatigue (p=0.031), heaviness (p=0.002), difficulty of recall (p=0.001), blurred vision (p=0.001), parietal or occipital headaches (p=0.000), confusion during reading (p=0.014), and failing focus or loss of concentration during reading (p=0.003). visual fatigue and heaviness in the eye may be caused by inability to maintain binocular fixation, occurring in subjects with ci.(3) blurred vision and failing focus are due to inability of the accommodation mechanism to maintain focus on near objects, as found in s u b j e c t s w i t h a i . i n s u b j e c t s w i t h p o o r accommodation there also occur headaches when reading.(19) the above symptoms may diminish and disappear with management of the asthenopia. improving the ergonomic design of workstations and modifying the work habits of computer users (with supplementary breaks) have been shown to have positive effects on visual fatigue in these workers.(20,21) asthenopia has been extensively studied in the computer literature, and the most commonly used assessment method is selfr e p o r t i n g , b e c a u s e t h i s i s a r a p i d a n d inexpensive method that can be applied to general populations. however, the method carries the risk of self-reporting bias, whereby the personal experiences of the respondent may affect the recall, interpretation, and reporting of symptoms. conclusions this study suggests that asthenopia is a common problem among students who are computer users and is mostly caused by accommodative insufficiency. acknowledgements we hereby wish to express our graditude to the dean of the faculty of computer science, university of indonesia, for the support and facilities accorded to us in conducting this study. we also thank the students of above faculty as the participants of the study for their willingness and cooperation. amalia, suardana, artini accommodative insufficiency and asthenopia 83 univ med vol.29 no.2 references 1. bhanderi dj, choudhary s, vikas g, doshi vg. a community-based study of asthenopia in computer operators. indian j ophthalmol 2008; 56:51-5. 2. blehm c, vishnu s, khattak a, mitra s, yee rw. computer vision syndrome: a review. surv ophthalmol 2005;50:253-62. 3. anshel j. visual ergonomics in the workplace. aaohn j 2007;55:414-20. 4. rajeev a, gupta a, sharma m. visual fatigue and computer use among college students. indian j comm med 2006;31:192-3. 5. mocci f, sera s, corrias ga. psychological factors and visual fatigue in working with video display terminals. occup environ med 2001;58: 267-71. 6. cole bl. do video display units cause visual problems?-a bedside story about the processes of public health decision-making. clin exp optom 2003;86:205-20. 7. sheedy je, hayes jn, engle j. is all asthenopia the same? optom vis sci 2003;80:732–9. 8. verma sb. computers and vision. j postgrad med 2001;47:119–20. 9. vertinsky t, forster b. prevalence of eye strain among radiologists: influence of viewing variables on symptoms. ajr 2005;184:681–6. 10. carlson nb, kurtz d. ocular examination. 3rd ed. new york: mcgraw-hill; 2004. 11. abdi s, rydberg a. asthenopia in schoolchildren, orthoptic and ophthalmological findings and treatment. doc ophthalmol 2005;111:65-72. 12. gilbert c, foster a. childhood blindness in the context of vision 2020 the right to sight. bull world health organ 2001;79:227–32. 13. tracy bd. computer vision care: clear path to productivity. compensation benefits management 2001;17:49-51. 14. borsting ej, rouse mw, mitchell gl, scheiman m, cotter sa, cooper j, et al. validity and reability of the resived convergence insufficiency symptom survey in children aged 9 to 18 years. optom vis sci 2003;80:832-8. 15. clark c. end user computing ergonomics facts or fads? j organiz end user comput 2006;18:6676. 16. zhang m, bi lf, ai yd, yang lp, wang hb, liu zy, et al. effect of taurine supplementation on vdt work induced visual stress. amino acids 2004;26:59-63. 17. sterner b, abrahamsson m, sjostrom a. accommodative facility training with long term follow up in a sample of school aged children showing accommodative dysfunction. doc ophthalmol 1999;99:93-101. 18. jenkins rh. characteristics and diagnosis of convergence insufficiency. am orthopt j 1999; 49:7-11. 19. adler p. efficacy of treatment for convergence insufficiency using vision therapy. ophthal physiol opt 2002;22:565-71. 20. van den heuvel sg, de looze mp, hildebrandt vh, the kh. effects of software programs stimulating regular breaks and exercises on workrelated neck and upper-limb disorders. scand j work environ health 2003;29:106–16. 21. ketola r, toivonen r, hakkanen m, luukonen r, takala e, viikari-juntura e. effects of ergonomic intervention in work with video display units. scand j work environ health 2002;28:18–24. alvina 1 abstract asthma is a common chronic disease and information on its management practices at the community level is helpful in identifying problems and improving asthma care. the prevalence of asthma in children below 18 years of age is around 9.3% and is on the increase. the aim of the present study was to determine the relationship between pulmonary function and duration of asthma in children. this was a cross-sectional study conducted at the outpatient clinic of rsupn dr. cipto mangunkusumo in central jakarta. the study subjects were children aged 6-18 years with frequent episodic or persistent asthma. among the 31 subjects there were 28 children with frequent episodic asthma and 3 children with persistent asthma. the duration of frequent episodic asthma ranged from 4 to 84 months, with a mean duration of 28 months. the fev1 and v50 values decreased in proportion to the duration of asthma (p=0.003 and p=0.012, respectively). mean fev1 in persistent asthma was lower than that in frequent episodic asthma (82.7% vs. 61.2% at p=0.005). similarly v50 and v25 were lower in persistent asthma, but the decrease was not statistically significant. the decrease in fev1 and v50 values was proportional to the duration of asthma. the severity of asthma is indicative of inadequate asthma control, resulting in a proportional decrease in pulmonary function. therefore prevention of asthmatic attacks is an essential feature of asthma management in children in order to enhance their quality of life. keywords: pulmonary, function, severity, asthma, children *department of pediatrics, medical faculty, trisakti university correspondence dr. ellen p. gandaputra, spa department of pediatrics, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2705 email: e1entity@yahoo.com univ med 2010;29:1-7 duration of asthma affects pulmonary function in asthmatic children ellen p. gandaputra* january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina introduction asthma is one of the major chronic health problems in children. worldwide, approximately 40% of all young children have at least one episode of asthmatic symptoms like wheezing, coughing, and dyspnea.(1) although asthmatic symptoms are common in preschool children, only 30% will have asthma at the age of 6 years and over. the rest of the children with recurrent respiratory symptoms is symptom-free at 6 years and does not have asthma but transient, viral 2 gandaputra pulmonary function in asthma associated wheeze.(2) according to the indonesian national guidelines for asthma in children (pedoman nasional asma anak) the diagnosis of asthma should be based on the presence of wheezing and/or cough, and the following characteristics: episodic and/or chronic; occurring at night or i n t h e e a r l y m o r n i n g h o u r s ( n o c t u r n a l ) ; seasonal; involvement of precipitating factors such as physical activity; reversible (either spontaneously or upon treatment); positive past history of asthma or other atopic disorders in the patient or the family, after exclusion of other causes.(3) a common precipitating factor in the development of asthmatic symptoms are housedust mites, animal hair, and pollen. in addition, several pollutants such as cigarette s m o k e a n d e x h a u s t g a s e s o f a u t o m o t i v e vehicles, may also precipitate an attack of asthma, while among asthma precipitating drugs may be mentioned aspirin and nonsteroidal anti-inflammatory drugs.(4) the prevalence of asthma in preschool children may be up to 32% in the united states and europe, whilst that of children below the age of 18 years is around 9.3% and is still increasing.(5) the prevalence of self-reported wheezing in the previous 12 months in 13 to 14 year old children varied from 1.6% to 36.7% i n d i f f e r e n t c e n t e r s t h e c o r r e s p o n d i n g prevalence for parent-reported wheezing for the 6 to 7 year old age group was 0.8% to 3 2 . 1 % . ( 6 ) wi t h i n c e r t a i n r e g i o n s a s t h m a prevalence is generally lower in developing countries than in more affluent countries. for example, in southeast asia, the centers with the lowest prevalence of asthma symptoms were in indonesia (2.1%) and china (3.3– 5.1%), and the centers with the highest rates were in japan (13.4%), thailand (12.6– 13.5%), and hong kong.(7) based on the degree of severity, asthma i s c o m m o n l y c a t e g o r i z e d a s i n f r e q u e n t episodic asthma, frequent episodic asthma, and persistent asthma. (table 1).(8) an asthmatic attack develops upon acute and extensive obstruction of the airways. the degree of a s t h m a i s d e t e r m i n e d b y t h e f r e q u e n c y, duration, and intensity of the asthmatic attack, activity and symptoms outside attacks, and the results of pulmonary function tests. asthma is a chronic inflammatory disease and several studies have suggested that the remodelling process in asthma occurs since its onset and increases in proportion to the f r e q u e n c y o f a s t h m a t i c a t t a c k s . ( 9 , 1 0 ) t h e continuing inflammatory process affects the pulmonary function of the asthmatic child and ultimately its quality of life. atopic sensitization has long been known to be related to childhood asthma.(11) the available evidence suggested that usually only less than half of the asthma cases were attributable to atopic sensitization. in addition, studies showing a strong relation between asthma and atopy come mainly from affluent western countries.(12) thus, the link between asthma and atopic sensitization differs between countries.(13,14) prevention of asthmatic attacks is the goal of long-term management of a s t h m a , w h i c h i s e x p e c t e d t o i m p r o v e pulmonary function and comprises avoidance of allergens and drug therapy with inhalatory corticosteroids, leukotriene antireceptors, slowrelease theophylline, and long-acting beta-2agonists.(8) pulmonary obstruction is a characteristic finding in acute exacerbation of asthma; however, there is a scarcity of data comparing the pulmonary function of children with degree of asthma. the aim of the present study was to compare the pulmonary function of children with degree of asthma and to clarify the relationships between duration of asthma and pulmonary function. methods research design the present study is of cross-sectional d e s i g n , c o n d u c t e d a t r s u p n d r. c i p t o mangunkusumo from mei-december 2008. 3 subjects of study the study subjects were children with frequent episodic asthma or persistent asthma who were visiting the outpatient allergy or respirology clinics at rsupn dr. ciptomangunkusumo. the children were selected as study subjects when meeting the following inclusion criteria: (i) age between 6-18 years; (ii) diagnosed as mild or severe intermittent or persistent allergic rhinitis with frequent episodic or persistent asthma outside of attacks; (iii) subjects or their parents willing to sign informed consent and agreeing to pulmonary function tests for their children. exclusion criteria were (i) other pulmonary disorders or abnormalities; (ii) other disorders affecting pulmonary functions; (iii) currently on long-term intranasal, inhalatory, or systemic corticosteroid therapy (>5 consecutive days) by; (iv) unfit for pulmonary function tests. data collection and assessment data were collected by means of interviews f o l l o w e d b y p h y s i c a l e x a m i n a t i o n a n d pulmonary function tests. the degree of asthma was categorized as infrequent episodic asthma, frequent episodic asthma, and persistent asthma. pulmonary function tests were performed by means of a spirometer with forced vital capacity maneuver for measuring forced vital capacity (fvc), forced expiratory volume in 1 second (fev1), 50% fvc expiratory flow volume (v50), and 25% fvc expiratory flow volume (v25). these tests were performed outside of asthmatic attacks. the evaluation of spirometric results comprises fev1, v50, and v25. abnormal pulmonary function is designated obstruction if fev1/fvc are less than 70%, and fev1 is less than 80% of standard value. if fev1 is less than 80%, there is borderline obstruction, if fev 1 is less than 60% there is moderate obstruction, and if fev1 is less than 40% there is severe obstruction. the v50 and v25 values are parameters for determining the presence of an obstruction in the smaller airways.(15) ethical clearance ethical clearance was issued by the research ethics committee of the faculty of medicine, university of indonesia. statistical analysis all data was analyzed by means of the spss 16 software program. descriptive data parameter infrequent episodic asthma frequent episodic asthma persistent asthma frequency of attacks <1x/month >1x/months frequent duration of attacks < 1x/week >1x/week almost throughout the year intensity of attacks usually mild usually moderate usually severe between attacks symptoms none symptoms frequent symptoms day and night sleep and activities not disturbed frequently disturbed extremely disturbed physical examination outside attacks normal some abnormality may be found never normal controling drugs unnecessary necessary necessary lung function tests (outside attacks) pef/fev1 >80% pef/fev1 60-80% pef/fev1<60%, variability 20-30% variability of lung function (during attacks) > 15% > 30% > 50% table 1. classification of asthma by degree of severity(8) notes: pef = peak expiratory flow; fev1= forced expiratory volume in 1 second univ med vol.29 no.1 4 gandaputra pulmonary function in asthma were presented in textual and tabular form, and analyzed using the t-test. analysis of variance was used to assess differences in lung function between children grouped according to the classifications described previously in this paper, with p< 0.05 considered as statistically significant. results o v e r a l l t h e r e w e r e 3 1 s u b j e c t s participating in the present study, consisting of 21 males and 10 females, with mean age of 9.5 ± 2.3 years and most of them (87.1%) being in the age range of 6-12 years. the youngest study subject was 6 years old and the eldest 15.9 years. a total of 28 (90.3%) subjects had frequent episodic asthma and 3 (9.7%) subjects had persistent asthma. the duration of frequent episodic asthma ranged from 4 to 84 months, with mean duration of 28.3 ± 4.1 months. there was no significant difference between males and females, age group and degree of asthma. mean duration of frequent episodic asthma (28.3 ± 4.1 months) was not significantly different from that of persistent asthma (44.0 ± 18.3 months) (p=0.185) (table 2). mean fev1 in persistent asthma (61.2 ± 7.3) was significantly lower than that of f r e q u e n t e p i s o d i c a s t h m a ( 8 2 . 7 ± 1 2 . 2 ) (p=0.005). this was also the case with the values for v50 and v25 of respectively 83.2 ± 28.9 in frequent episodic asthma and 54.3 ± 16.3 in persistent asthma (p=0.102), and the values for v25 of 85.3 ± 29.4 in frequent episodic asthma and 50.4 ± 21.1 in persistent asthma, respectively (p=0.056) (table 3). duration of frequent episodic asthma in the study subjects ranged from 4 up to 84 months, with mean duration of 28 months. the fev1 (p=0.003) and v50 values (p=0.012) decreased in proportion to duration of asthma. the v25 values decreased also, but the decrease was not statistically significant (p=0.71). pulmonary function as measured by fev1 decreased significantly with duration of asthma ( r = 0 . 5 2 2 ; p = 0 . 0 0 3 ) . s i m i l a r l y v 5 0 a l s o decreased significantly with duration of asthma (r=-0.448; p=0.012). however, the decrease in degree of asthma pulmonary function frequent episodic (n=31) persistent (n=3) p fev1 (%) v50 (%) v25 (%) 82.7 ± 12.2 83.2 ± 28.9 85.3 ± 29.4 61.2 ± 7.3 54.3 ± 16.3 50.4 ± 21.1 0.005 0.102 0.056 table 3. comparison of pulmonary function by severity of asthma degree of asthma variables frequent episodic (n=28) persistent (n=3) p gender male female 19 (90.5%) 9 (90.0%) 2 (9.5%) 1 (10.0%) 0.967 age group (years) 6 12 >12 24 (88.9%) 4 (100.0%) 3 (11.1%) 0 (0%) 0.123 duration of asthma (months) (mean ± sd) 28.3 ± 4.1 44.0 ± 18.3 0.185 table 2. gender, age, and duration of asthma by degree of asthma 5 v25 values with duration of asthma was not significant (r=-0.329; p=0.071) (table 4). age, duration of asthma, and degree of a s t h m a h a d a s i g n i f i c a n t i n f l u e n c e o n pulmonary function as measured by fev1. the regression analysis revealed that duration of asthma had the highest impact on fev1 in asthmatic children (table 5). duration of asthma also affected v50 but not v25. discussion in this study the majority of children in the age range of 6-18 years had frequent episodic asthma (90.3%) and only 9.7% had p e r s i s t e n t a s t h m a . s i m i l a r r e s u l t s w e r e obtained in 10-year old children in hong kong and guang zou, where 83% of the children had intermittent asthma and 27% persistent asthma (mild and moderate).(16) asthma is a chronic inflammatory disease that commonly affects pulmonary function tests. a descriptive study in children 10-19 years old, showed that the lung function test in children with asthma can be obstructive, restrictive or combination.(17) the longer the duration of asthma and the more frequent the asthmatic attacks, the greater the d e c l i n e i n p u l m o n a r y f u n c t i o n s d u e t o remodelling of the bronchial wall.(9,10) this may be seen from the increasingly lower fev1 and v50 values in proportion to the duration of asthma. the lowered fev1 and v50 values indicate the presence of obstruction in the large and small airways that may occur in asthma. in addition to asthma, there are several factors influencing the pulmonary functions, namely height, birth weight, and the occurrence of w h e e z i n g u n d e r t h e a g e o f o n e y e a r. ( 1 8 ) however, these data were not collected in the present study and therefore this constitutes one limitation of this study. the severity of asthma also affects the pulmonary functions. the study conducted by bacharier et al.(19) demonstrated that fev1 did not differ substantially with various degrees of asthma, while apparently fev1/fvc was reduced in more severe degrees of asthma. these findings do not support the results of the present study, where the decrease in fev1 and v50 was greater in persistent asthma than in frequent episodic asthma. the lower values of these parameters indicate a more severe obstruction occurring in the airways. in pulmonary function duration of asthma p fev1 v50 v25 r*=-0.522 r=-0.448 r=-0.329 0.003 0.012 0.071 table 4. relationship between duration of asthma and pulmonary function *r : pearson correlation variables age asma duration asma severity fev1 β 2.801 -0.318 -17.706 beta 0.351 -0.521 -0.400 95% c.i. β 0.411 3.751 -30.142 5.270 -0.495 0.141 v50 β 3.691 -0.631 -20.883 beta 0.287 -0.474 -0.218 95% c.i. β -0.692 8.074 -1.096 -0.166 -53.255 11.758 v25 β 0.347 -0.370 -28.663 beta 0.026 -0.266 -0.287 95% c.i. β -4.649 5.334 -0.849 0.159 -65.838 8.512 table 5. multiple linear regression of several main variables by pulmonary function univ med vol.29 no.1 6 gandaputra pulmonary function in asthma persistent asthma the fev1 and v50 values were in the range of 50-60%, even though the c h i l d r e n h a d n o a s t h m a t i c a t t a c k s . t h i s indicates that the obstruction has become permanent and may affect the capacity of the children for activities. the duration of asthma is inversely and significantly related to pulmonary functions. consistent findings were obtained in children with mild and moderate asthma. zeiger and colleagues(20) using the baseline data from the childhood asthma management program (camp) of the national heart, lung, and blood institute, reported a change in prebronchodilator fev1 of almost 1% per year of asthma duration in children with mild to moderate asthma. the decline in fev1 indicates the presence of obstruction in the large airways. whereas v25 and v50 indicate the presence of obstruction in the smaller airways. in children with asthma there is also obstruction in the smaller airways, which is aggravated by attacks. the occurring inflammation also becomes permanent.(10) the concern in childhood asthma is that the disease adversely impacts the growth of a child’s airways such that maximal lung growth is not achieved. lower lung function in young adults w i t h d i a g n o s e d o r u n d i a g n o s e d a s t h m a compared with healthy control subjects is seen in various studies(21) in addition, childhood fev1% predicts adult lung function level. (22) in children with asthma, it turns out that the variable with the greatest influence on fev1 and v50 is not the degree of frequent episodic asthma and persistent asthma, but the duration of asthma. thus there is a need for long term drug therapy capable of preventing future attacks. such a therapy is expected to be able to improve the decreased pulmonary functions. the inflammatory process in asthma may be reduced by long-term administration of inhalatory corticosteroids,(23) as was also demonstrated in the study by reddel et al.,(24) where inhalatory fluticasone improved fev1. similarly the study conducted by ramsdell et al.(25) showed significant improvement in fev1 t h r o u g h i n h a l a t o r y a d m i n i s t r a t i o n o f methasone furoate dry powder, compared with placebo (20.7% vs 5.1%). repeated objective m e a s u r e m e n t s o f l u n g f u n c t i o n m a y immediately detect the occurrence of airway o b s t r u c t i o n , w h i c h m a y b e a m e n a b l e t o adequate treatment. another factor capable of affecting the degree of asthma severity and the frequency of asthma attacks is comorbidity, viz. allergic rhinitis and sinusitis. this indicates that with the recovery from sinusitis, waning of asthma symptoms and improvement of pulmonary functions may be expected. in the present study the issue of comorbidity was not evaluated. conclusions most children in this study had frequent episodic asthma and the duration of asthma had the most impact on the pulmonary function of asthmatic children, ultimately affecting their quality of life. prevention of asthmatic attacks is essential, comprising avoidance of allergens and administration of controling drugs in the long term. acknowledgements the investigators wish to acknowledge the support of the dean and vice-deans of the medical faculty, trisakti university, for the funding of the present study. we also wish to thank the doctors and staff of rsupn dr. cipto mangunkusumo, and all persons who provided assistance and support until the completion of this study. references 1. bisgaard h, szefler s. prevalence of asthma-like symptoms in young children. pediatr pulmonol 2007;42:723-8. 2. kurukulaaratchy rj, fenn mh, waterhouse lm, matthews sm, holgate st, arshad sh. characterization of wheezing phenotypes in the first 10 years of life. clin exp allergy 2003;33: 573-8. 7 3. kling s, gie r, goussard p. inhaled corticosteroids in childhood asthma. curr allergy clin immunol 2003;16:8-10. 4. terr ai. the atopic diseases. in: parslow tg, stites dp, terr ai, imboden jb, editors. medical immunology. 10th ed. boston: mcgraw hill;2003. p.349-69. 5. savichi g, dovey m. chronic childhood asthma: definition, epidemiology, and pathophysiology. available at : http://www.uptodate.com. accessed november 11,2009. 6. beasley r, keil u, von mutius e on behalf of the isaac steering committee. worldwide variation in the prevalence of symptoms of asthma, allergic rhinoconjunctivitis and atopic eczema: the international study of asthma and allergies in childhood (isaac). lancet 1998;351:1225–32. 7. beasley r, ellwood p, asher i. international patterns of the prevalence of pediatric asthma: the isaac program. pediatr clin n am 2003; 50:539–53. 8. ukk pulmonologi. pedoman nasional asma anak. dalam: rahajoe n, supriyatno b, setyanto db, penyunting. jakarta: pp idai;2004.h.1-44. 9. bousquet j, jeffery pk, busse ww, johnson m, vignola am. asthma, from bronchoconstriction to airways inflammation and remodeling. am j respir cri care med 2000;161:1720-45. 10. payne dnr, rogers av, adelroth e, bandi v, guntupalli kk, bush a, et al. early thickening of the reticular membrane in children with difficult asthma. am j respir crit care med 2003;167: 78-82. 11. von mutius e. environmental factors influencing the development and progression of pediatric asthma. j allergy clin immunol 2002;109:s525– 32. 12. weinmay g, weiland sk, bjo¨rkste´n b, brunekreef b, bu¨chele g, cookson woc, et al. atopic sensitization and the international variation of asthma symptom prevalence in children. am j respir crit care med 2007; 176:565–74. 13. scrivener s, yemaneberhan h, zebenigus m, tilahun d, girma s, ali s, et al. independent effects of intestinal parasite infection and domestic allergen exposure on risk of wheeze in ethiopia: a nested case-control study. lancet 2001;358:1493–9. 14. palmer lj, celedon jc, weiss st, wang b, fang z, xu x. ascaris lumbricoides infection is associated with increased risk of childhood asthma and atopy in rural china. am j respir crit care med 2002;165:1489–93. 15. kaswandani n. uji fungsi paru pada batuk kronik. dalam: trihono pp, kurniati n, penyunting. strategi pendekatan klinis secara professional batuk pada anak. jakarta: departemen ilmu kesehatan anak fkui-rscm;2006. h.26-39. 16. ko fws, wang hy, wong gw, leungz tf, hui dsc, chan dps, et al. wheezing in chinese schoolchildren: disease severity distribution and management practices, a community-based study in hong kong and guangzhou. clin exp allergy 2005;35:1449–56. 17. tehuteru es. profile of lung function test in pediatric asthma patients. univ med 2003;22:14. 18. joseph-bowen j, de klerk nh, firth mj, kendall ge, holt pg, sly pd. lung function, bronchial responsiveness, and asthma in a community cohort of 6-year-old children. am j respir crit care 2004;169:850-4. 19. bacharier lb, strunk rc, mauger d, white d, lemanske rf, sorkness ca. classifying asthma severity in children. am j respir crit care med 2004;170:426-32. 20. zeiger rs, dawson c, weiss s. relationships between duration of asthma and asthma severity among children in the childhood asthma management program (camp). j allergy clin immuno 1999;103:376-87. 21. apostol gg, jacobs dr, tsai aw, crow rs, williams od, townsend md, et al. early life factors contribute to the decrease in lung function between ages 18 and 40: the coronary artery risk development in young dults study. am j respir crit care med 2002;166:166–72. 22. covar ra, spahn jd, murphy jr, szefler sj, for the childhood asthma management program research group. progression of asthma measured by lung function in the childhood asthma management program. am j respir crit care med 2004;170:234–41. 23. mauad t, bel eh, sterk pj. asthma therapy and airway remodeling. j allergy clin immunol 2007;120:997-1009. 24. reddel hk, belousova eg, marks gb, jenkins c. does continuous use of inhaled corticosteroids improve outcomes in mild asthma? a doubleblind randomized controlled trial. primary care respiratory j 2008;17:39-45. 25. ramsdell jq, nayak as, bensch gw. efficacy of treatment with once-daily evening dosing of mometasone furoate dry powder inhaler 200 micrograms in asthma stratified by baseline severity. am j respir crit care med 2009;179: 19-24. univ med vol.29 no.1 alvina 27 abstract cardiovascular disease (cvd) risk factors, such as diabetes, hypertension, hypercholesterolemia, smoking, and obesity tend to occur together in the general population. increasing prevalence of multiple cvd risk factors has been related to increased risk of death from coronary heart disease and stroke. studies have suggested that people with several risk factors of cvd may have impaired healthrelated quality of life. the objective of this study was to assess the association of cvd risk factors with quality of life (qol) among adults aged 40 to 65 years. a cross-sectional study was conducted involving 220 subjects 40 65 years of age at a health center. the cvd risk factors were assessed using the framingham risk score that is the standard instrument for assessment of the risk of a first cardiac event. the risk factors assessed were age, smoking, blood pressure, total cholesterol and high density lipoprotein cholesterol concentrations. qol was assessed by means of the whoqol-bref instrument that had been prevalidated. the results of the study showed that 28.2% of subjects were smokers, 56.4% had stage 1 hypertension, 42.8% high total cholesterol and 13.6% low hdl cholesterol. the high risk group amounted to 45.5% and 42.3% constitued an intermediate risk group. high cvd risk scores were significantly associated with a low qol for all domains (physical, psychological, social and environment) (p=0.000). preventing or reducing the multiple cvd risk factors to improve qol is necessary among adults. keywords: framingham risk score, quality of life, adult *medical profession study program, medical faculty, trisakti university correspondence achristian yosaputra medical profession study program, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 univ med 2010;29:27-33 high framingham risk score decreases quality of life in adults christian yosaputra*a, erica kholinne*, and erick susanto taufik* january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina introduction chronic diseases are considered major threats to the quality of life (qol). prevalence rates of most chronic diseases increase with age and a substantial part of the elderly population suffers from more than one chronic disease. since chronic diseases and conditions a r e p a r t o f t h e e v e r y d a y l i f e o f a l a rg e proportion of the population, it is considered important to do research on their qol. one of the frequently encountered chronic diseases is cardiovascular disease (cvd). cvd has become the leading cause of morbidity and 28 yosaputra, kholinne, taufiq m o r t a l i t y f o r m e n a n d w o m e n , b o t h f o r developed and developing countries and rates are expected to rise further over the next few decades.(1-3) the prevalence and incidence of cardiovascular disease increases exponentially with age.(4) despite the fact that over the lifespan, approximately the same proportion of the female population as the male population dies of complications resulting from cvd, it has been traditionally considered as a middleage disease. the increasing burden of cvd has important economic implications. cvd occurs typically at a younger age in developing as c o m p a r e d t o d e v e l o p e d c o u n t r i e s , w i t h i m p o r t a n t c o n s e q u e n c e s s u c h a s l o s s o f r e v e n u e a t h o u s e h o l d l e v e l a n d l o s s o f productivity at macroeconomic level.(5) in indonesia, the results of the basic health research survey (riset kesehatan d a s a r ) o f t h e y e a r 2 0 0 7 , s h o w e d t h e prevalence of cvd to be 7.2%.(6) cvd is inseparable from an individual’s unhealthy lifestyle in consequence of changes in patterns of living. the framingham heart study has d e v e l o p e d m a t h e m a t i c a l f u n c t i o n s f o r predicting risk of clinical coronary heart disease (chd) events, viz. multivariable mathematical functions that assign weights to major chd risk factors such as sex, age, blood pressure (bp), total cholesterol (tc), lowdensity lipoprotein cholesterol (ldlc), highdensity lipoprotein cholesterol (hdl-c), smoking behavior, and diabetes status.(7) these mathematical functions are known as the framingham risk score (frs), on the basis of which the risk of cvd for a given individual may be predicted. physical conditions such as high bp or body mass index (bmi) as well as risk behaviors (such as smoking, alcohol drinking and poor nutritional habits) have been recognized as risk factors for cardiovascular diseases. in addition, psychosocial factors have been suggested as risk factors for atherosclerotic disease.(8) it is known that health is one of the significant factors that influence a person’s qol. chronic diseases are considered major threats to the qol of western populations.(9) prevalence rates of most chronic diseases increase with age and a substantial part of the elderly population suffers from more than one chronic disease. the goal of a health service is not only viewed from the aspect of an individual’s survival rate, but also from his/her qol. the world health organization (who) generally evaluates an individual’s qol by means of multi-dimensional parameters, comprising physical, psychological, environmental and social functions, which affect the well being of an individual’s life. (10) the who has recently developed a generic profile subjective qol instrument, the whoqol-100, and its brief version, the whoqol-bref.(11) this instrument may be used for assessing an individual’s quality of life. if an individual has many risk factors for cvd, his/her qol will decline. in the physical dimension the lowered health status will be clearly seen, the more so if the parameters of lipid and bp levels are at least borderline (threshold). (12) it may be hypothesized that a high frs may lower an individual’s qol, which naturally has to be proven. the aim of this study was to investigate the associations between the cardiovascular risk factors as measured by frs and the level of qol in adult subjects in primary care. methods study setting and design t h e p r e s e n t s t u d y w a s a n a l y t i c a l observational with cross-sectional approach performed at one of the south jakarta districts in november 2007. subjects of study the study subjects were patients visiting the district health center selected according to the following inclusion criteria: aged between 40 and 65 years, able to communicate verbally a n d w i l l i n g t o p a r t i c i p a t e i n t h e s t u d y. 29 exclusion criteria were past history of stroke and coronary heart disease based on the information provided by their physician. the age range of 45 to 65 years constitutes the r e p r o d u c t i v e a g e a n d i s a s s o c i a t e d w i t h substantially better health-related qol and less illness in older age.(13) demographic variables and history of cvd the collected variables were age, sex, occupation, and educational attainment. the educational level was categorized as no education, not finishing elementary school, elemenrary school graduate, junior high school graduate, senior high school graduate, college or university graduate. a history of cvd was determined by an affirmative answer to the question, “has a doctor, nurse, or other health professional ever told you that you had any of the following: (a) a heart attack, also called a myocardial infarction; (b) angina or coronary heart disease; or (c) a stroke?” cardiovascular risk factors the framingham risk score (frs) is the standard instrument used to assess the risk of a first cardiac event and to guide the institution of primary preventive therapy in individuals at risk of cvd.(8,14) on the basis of the frs, the risk of cvd is categorized as (i) low: score = 0; (ii) intermediate: score = 1-10; (iii) high: score =11-20;and (iv) very high: score ≥ 20. in this study, the cvd risk factors collected comprised total and hdl-c, hypertension and smoking. total and hdl-c after the subjects had fasted overnight, venous blood samples were drawn to measure t c a n d h d l c i n w h o l e p l a s m a . hiperlipidemia is the condition of high blood lipid levels. the normal tc concentration is 140-200 mg/dl or less. the normal hdl-c concentration is more than 40 mg/dl. thus a hdl-c concentration of < 40 mg/dl is a risk factor for cvd. the ratio of tc to hdl-c should be < 4.6 in males and < 4.0 in females. the risk for chd increases proportionally with tc: hdl-c ratio. according to the frs, the lipid profile affecting chd risk comprises total a n d h d l c . ( 7 , 1 5 ) t h e l i p i d p r o f i l e s w e r e determined at the pramita utama laboratory. systolic and diastolic blood pressure single systolic bp measurements were performed with a mercury sphygmomanometer, with the cuff placed on the right brachial artery at the upper arm 2 cm proximal to the cubital fossa in seated non-smoking subjects. bp categories were defined on the basis of the s e v e n t h r e p o r t o f t h e j o i n t n a t i o n a l committee (jnc vii).(16) smoking all participants were interviewed about present and past smoking habits. current and past smokers of cigarettes and cigars were judged to have a history of smoking. qol assessment qol was assessed using the indonesian version of the whoqol-bref questionnaire. this questionnaire is made up of 26 items which assess the following four domains: (i) physical domain, consisting of 7 items, (ii) psychological domain, with 6 items, (iii) social r e l a t i o n s c o m p r i s e s 3 i t e m s , a n d ( i v ) environment, with 8 items. the whoqolbref also measures 2 facets of general qol: (i) overall qol and (ii) general health. each item is given a score of 1–5, and a higher score indicates a better qol. scores for each domain are calculated by multiplying the average score of each facet by 4. a domain is not given a score if ≥ 20% items have been left unanswered by the respondent. the whoqol-bref instrument has been validated and has shown a high validity and reliability for assessment of the qol of the elderly.(17) calculations of high and low qol categories in each domain are based on the highest and lowest scores for the respective domains according to the univ med vol.29 no.1 30 yosaputra, kholinne, taufiq responses of the respondents. the highest and lowest scores are totaled and then divided by 2. the value obtained by this division is taken as the cut-off point for separating the group with a high qol and that with a low qol. data analysis for every participant, we calculated the frs using the classic framingham equation, and participants were assigned to high risk, intermediate risk, and low risk groups. the distributions of several major factors of cvd and the qol domains were analyzed by means of percentage analysis. the pearson correlation analysis was used for determining a correlation between cvd risk factors (according to frs) and qol. the analyses were done using spss for windows version 15. a p-value < 0.05 was considered statistically significant. results a total of 220 study subjects participated in this study with an age range of 40 65 years. more than half of the subjects (58.6%) were females, while males accounted for 41.4%, eighty percent of the subjects had a low educational level (uneducated and not passing e l e m e n t a r y s c h o o l ) a n d 5 5 . 9 % w e r e housewifes. the distribution of cardiovascular risk is presented in table 2. some 28.2% of subjects were smokers, 56.4% had stage 1 hypertension, 42.8% high tc and 13.6% low hdl-c. of all of the individual risk factors included in the frs calculation, approximately 45.5% was in the high risk category, 42.3% was categorized as intermediate risk and 8.6% as low risk (table 3). from table 4, it is apparent that in the physical domain subjects with a good qol a m o u n t e d t o 4 0 . 5 % , w h e r e a s i n t h e psychological domain good qol subjects totaled 51.8%. in the social domain good qol s u b j e c t s a c c o u n t e d f o r 5 5 % a n d i n t h e environmental domain 55.5% of subjects had a good qol. table 2. distribution of cardiovascular risk for all subjects (n=220) table 1. classification of blood pressure for adults(16) note: bp= blood pressure; sbp= systolic blood pressure; dbp= diastolic blood pressure table 3. cardiovascular risk for all subjects based on framingham risk score 31 the results of the pearson correlation test demonstrated the presence of a statistically significant inverse correlation between frs and qol in the physical (r = -0.491; p = 0.000), psychological (r = -0.52; p = 0.000), social (r = -0.496; p = 0.000), and environmental domains (r = -0.512; p = 0.000) (table 5). high frs values indicate a poor qol in each domain. discussion t h e p r e s e n t s t u d y e x a m i n e d t h e association between cvd risk and qol. according to frs values, 45.5% of subjects in the age range of 40 to 65 years were at high risk of cvd. subjects with a high frs had a correspondingly high risk of cvd. frs is used for predicting the occurrence of cvd after 10 years (10-year risk prediction) in individuals aged 30 to 74 years who have not suffered from cvd. a prospective study in subjects aged >45 years without any complaints of cvd and were followed-up for approximately 8.5 year, indicated that the risk of chd death or nonfatal mi for participants in the highest frs group was 4.3 higher that of participants in the lower frs group (p=0.009).(18) our study determined that only 8.6% of subjects were at low risk of cvd. similar results were obtained in a cohort study of subjects in the age range of 36-64 years, indicating that only a small minority of t h e c o h o r t ( < 1 0 % o f t h e e n t i r e 3 9 5 2 2 participants) met the criteria for low risk at the baseline examination in 1967-1973. of note, participants with 0 risk factors also had better outcomes than those with 1 or more high risk factors.(19) subjects at high risk of cvd should attempt to reduce their risk factors, as most of them are amenable to modification. smokers should stop this habit. our study showed that the prevalence of main risk factors was: 56.4% for stage i hypertension (140-159 mmhg); 42.8% for high tc (≥240 mg/dl); 13.6% for low hdl-c (<40 mg/dl); and 28.2% for s m o k i n g . a d d i n g a l r e a d y r e c o g n i z e d cardiovascular risk factors in this group may put them in a challenged life situation. they are a vulnerable population subgroup that needs puhlic health attention. the three major coronary risk factors, viz. serum cholesterol, bp, and cigarette smoking, are important because of their high prevalence and impact on risk, and their prevention could have a great influence on longevity, health care costs and qol. assessing effects of cardiovascular risk profile, rather than individual risk factors, is also of public health importance. table 5. correlation between framingham risk score and respective qol domains * r = pearson correlation coeficient univ med vol.29 no.1 table 4. distrbution of each domain of qol 32 yosaputra, kholinne, taufiq our study indicates that high cvd risk tend to significantly lower the qol in subjects aged 40-65 years for all four domains (physical, psychological, social and environmental). several previous studies obtained results consistent with ours, in that higher cvd risk (a combination of hypertension, hyperlipidemia, and smoking) had an adverse impact on hrqol.(20-22) the influence of increasing numbers of risk factors, was generally additive. thus accumulation of more cvd risk factors may be associated with a more substantial impairment of qol.(23) however, no causality can be drawn from the presented results and therefore prospective studies are needed to allow causal inferences to be drawn. actually the three major coronary risk factors (serum cholesterol, bp, and cigarette s m o k i n g ) a r e v a r i a b l e s a m e n a b l e t o intervention. a study has demonstrated that p r i m a r y p r e v e n t i o n t h r o u g h l i f e s t y l e i n t e r v e n t i o n i n p r i m a r y h e a l t h c a r e h a s favourable effects on several cardiovascular risk factors in moderate to high risk patients, and the improvements can persist for up to one year.(24) subjects in the high cvd risk group should be motivated to complete such an intervention program. a weakness of our study would be the limited number of cvd biomarkers selected for investigation. other biomarkers such as diabetes, low density lipoprotein cholesterol, and obesity should also have been included. a second limitation of our study is that the majority of subjects were female with a low level of education, so that the study results cannot be generalized to populations with differing characteristics. conclusions a high prevalence of major risks factors was found in our study. the majority of subjects were in the groups of intemediate and high risk cvd, as assessed by the frs. the data in this study support the hypothesis that an individual with a high risk of cvd at 10years prediction will experience a lowered qol. these findings stress the need for affordable, cost-effective treatment strategies and the critical importance of public health strategies aimed at reducing risk factors in the entire population. acknowledgements we are deeply indebted to all respondents who participated in this study. we are also g r a t e f u l f o r h e l p f u l c o m m e n t s f r o m t h e reviewers of this paper. references 1. mckay j, mensah ga. the atlas of heart disease and stroke. geneva: world health organization; 2004. 2. ezzati m, lopez ad, rodgers a, vander hoorn s, murray cjl. selected major risk factors and global and regional burden of disease. lancet 2002;360:1347-60. 3. yusuf s, reddy s, ounpuu s, anand s. global burden of cardiovascular diseases: part i: general considerations, the epidemiological transition, risk factors, and impact of urbanization. circulation 2001;104:2746-53. 4. mcdermott mm. the international pandemic of chronic cardiovascular disease. jama 2007;297: 1253-5. 5. bovet p, shamlaye c, gabriel a, riesen w, paccaud f. prevalence of cardiovascular risk factors in a middle-income country and estimated cost of a treatment strategy. bmc public health 2006;6:9. 6. badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia. laporan nasional 2007: riset kesehatan dasar (riskesdas). jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia;2008. 7. d’agostino rb, grundy s, sullivan lm, wilson p. validation of the framingham coronary heart disease prediction scores: results of a multiple ethnic groups investigation. jama 2001; 286:180-7. 8. wilson pwf, d’agustino rb, levy d, balenger am, silbershatz h, kannel wb. prediction of coronary heart disease using risk factor categories. circulation 2005;97:1837-47. 33 9. benjak t, mavrinac gv. subjective quality of life and cadiovascular risk factors in a croatian adult population. coll antropol 2009;33:159-63. 10. coelho r, amorin i, prata j. coping styles and quality of life in patients with non-insulindependent diabetes mellitus. psychosomatics 2003;44:312-8. 11. the whoqol group development of the world health organisation. whoqol-bref quality of life assessment. psychol med 1998;28:51–8. 12. stamler j, daviglus m, garside m. relationship of baseline serum cholesterol levels in 3 large cohorts of younger men to long-term coronary, cardiovascular, and all-cause mortality and to longevity. jama 2000;284:311-8. 13. stampfer mj, hu fb, manson je, rimm eb, willett wc. primary prevention of coronary heart disease in women through diet and lifestyle. n engl j med. 2000;343:16-22. 14. lauer ms, fontanarosa pb. updated guidelines for cholesterol management. jama 2001;285: 2508-09. 15. ramachandran s, french jm, vanderpump mpj, croft p, neary rh. using the framingham model to predict heart disease in the united kingdom: a retrospective study. br med j 2000;320:676-7. 16. chobanian av, bakris gl, black hr, cushman wc, green la, izzo jl jr, et al. and the national high blood pressure education program coordinating committee. seventh report of the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure. hypertension;2003;42:1206–52. 17. salim oc, sudharma ni, kusumaratna rk, hidayat a. validity and reliability of world health organization quality of life-bref to assess the quality of life in the elderly. univ med 2007;26:27-38. 18. greenland p, labree c, azen sp, doherty tm, detrano rc. coronary artery calcium score combined with framingham score for risk reduction in asymptomatic individuals. jama 2004;291:210-5. 19. daviglus ml, liu k, pirzada a, yan ll, garside db, feinglass j, et al. favorable cardiovascular risk profile in middle age and health-related quality of life in older age. arch intern med 2003; 163:2460-8. 20. chambers ba, guo ss, siervogel r. cumulative effects of cardiovascular disease risk factors on quality of life. j nutr health aging 2002;6:179– 84. 21. oldridge nb, stump te, nothwehr fk, clark do. prevalence and outcomes of comorbid metabolic and cardiovascular conditions in middle and older age adults. j clin epidemiol 2001;54:928–34. 22. wee hl, cheung yb, li sc. the impact of diabetes mellitus and other chronic medical conditions on health-related quality of life: is the whole greater than the sum of its parts? health qual life outcomes 2005;3:2. 23. li c, ford es, mokdad ah, balluz ls, brown dw, giles wh. clustering of cardiovascular disease risk factors and health-related quality of life among us adults. value health 2008;11:689– 99. 24. erikssoni km, westborg cj, eliasson mce. a randomized trial of lifestyle intervention in primary healthcare for the modification of cardiovascular risk factors : the bjo¨rkna¨ s study. scand j public health 2006;34:453–61. univ med vol.29 no.1 lusi 161 cataract surgical coverage rate among adults aged 40 years and above september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina lusianawaty tana* *center of research and development biomedical and pharmaceutical national institute of health research and development, moh ri correspondence dr. lusianawaty tana, ms, sp.ok center of research and development biomedical and pharmaceutical national institute of health research and development, moh ri jl. percetakan negara no.23a jakarta phone: 021-6500266 email: lusianawaty@litbang.depkes.go.id univ med 2009;28:161-9 abstract cataract is a leading cause of curable blindness. hence, in its global declaration of ‘vision 2020 right to sight’, the world health organization (who) encouraged its member countries to address the problem of incident cataract. many factors are related to the cataract surgical coverage rate, such as gender and diabetes mellitus. the objective of this study was to determine the cataract surgical coverage rate and investigate the determinants factors of cataract surgical coverage rate among adults 40 years old and above with cataract. a cross sectional study was conducted using national basic health research (riskesdas) 2007 data. cataract surgery was defined as surgery conducted within the last 12 months before the survey was performed. there were 6939 subjects (3105 male, 3834 female) who fulfilled the study criteria. the cataract surgical coverage rate was 19.3%. the cataract surgical coverage rate was lower in subjects with low education, in the group of farmers/ fishermen/laborers, in the 40-49 years age group, in rural areas, and in subjects of low socioeconomic status (p<0.05). the cataract surgical coverage rate was not different between gender and diabetes mellitus (p>0.05). determinants that were related to cataract surgical coverage rate were age, type of area of residence, socioeconomic status, and region of residence (p<0.001). the implementation of educational programs and reforms to local ophthalmic health services may improve the cataract surgical coverage rate. keywords: coverage rate, cataract surgery, health services accessibility introduction cataract is a leading cause of curable blindness. hence, in its global declaration of ‘vision 2020 right to sight’, the world health organization (who) encouraged its member countries to address the problem of incident c a t a r a c t . ( 1 , 2 ) t h e e x i s t i n g c a t a r a c t s u rg i c a l coverage rate is much lower than expected in most developing countries. the cataract surgical 162 tana cataract surgical coverage coverage rate is defined as the number of cataract who have been operated divided by the total number of cataract in the population. knowledge of factors affecting the coverage of cataract services is crucial for increasing the cataract surgical coverage rate in these countries.(3) in indonesia the prevalence of blindness due to cataract is 0.78% (based on survey on vision and hearing 1993-1996).(4) surgery is a safe and effective option for treatment of cataract-induced visual impairment. t h e b a s i c h e a l t h r e s e a r c h r e p o r t (laporan riset kesehatan dasar, riskesdas) for the year 2007 showed the prevalence of cataract in indonesians aged 30 years and above, based on medical diagnosis and/or symptoms of cataract, for the previous 12 months to be 17.3%. the prevalence was higher in females than in males, being 17.8% in farmers/fishermen/ l a b o r e r s a n d 1 6 . 1 % i n h o u s e w i v e s . t h e proportion of cataract cases diagnosed by medical personnel and having undergone cataract surgery in the last 12 months was only 18%.(5) the cataract surgical coverage rate in indonesia v a r i e s b e t w e e n 5 . 2 % a n d 3 1 . 5 % . t h e s i x provinces with a cataract surgical coverage rate o f m o r e t h a n 2 3 % w e r e n o r t h s u l a w e s i , gorontalo, nusa tenggara barat, dki jakarta, bali, and east kalimantan, while those with a p r o p o r t i o n o f l e s s t h a n 1 0 % w e r e s o u t h sumatera, jambi, lampung, and west papua.(5) cataract surgery has been reported to be associated with several factors, among others age, gender, and diabetes mellitus.(2,6,7) lewallen et al.(2) reported on the results of a meta-analysis of 21 community-based surveys conducted up to the year 2000, where there was a difference in cataract surgical coverage rate by gender. cataract surgical coverage rate in low and middle-income countries was reported to be lower in females than in males. cataract surgical coverage rate in males was 1.7-fold higher than in females (95 % ci: 1.48 –1.97). similar results were reported by nirmalan et al.(6) in south india, in that cataract surgical coverage rate in females was lower, although cataract blindness in females aged 50 years and older was significantly higher than in males (adjusted or 0.71, 95 % ci: 0.570.87). advanced age, male gender and diabetes mellitus were the factors associated with cataract surgical coverage rate.(7) in indonesia cataract surgery has been performed in order to overcome the problem of impaired vision due to cataract, but the backlog of cataract cases is fairly high. this has been correlated with several factors, such as low c a t a r a c t s u rg i c a l c o v e r a g e r a t e , l a c k o f knowledge, high cost of surgery, limited numbers of ophthalmic surgeons and surgical facilities.(8) many measures have been taken to identify the factors that affect the cataract surgical coverage rate in the indonesian community. information on the role of these factors is expected to assist measures for prevention of cataract blindness and to determine the appropriate strategies for managing the problem. in connection with the above, this study was performed with the aim to determine the cataract surgical coverage rate and the relationship between individual characteristics, occupation, and type of area of residence on the one hand and cataract on the other. methods research design the research design was analytical with a cross-sectional approach, using secondary data from the riskesdas 2007. inclusion criteria for the study subjects were respondents with cataract diagnosed by medical personnel and aged 40 years and over. 163 data collection t h e v a r i a b l e s u s e d o r i g i n a t e d f r o m questionnaires from the riskesdas 2007 and the survei sosio-ekonomi nasional (susenas) 2007. the riskesdas 2007 questionnaires used were the rumah tangga (rkd07.rt) and individu (rkd07.ind) questionnaires, whereas the susenas 2007 questionnaire used was the vsen2007.k questionnaire. data on individual c h a r a c t e r i s t i c s c o m p r i s i n g a g e , g e n d e r, occupation, education, type of area of residence and region of residence were obtained from the rkd07.rt questionnaire. data on cataract, previous cataract operations, and diabetes mellitus (dm) came from the rkd07.ind questionnaire. data on rt per capita expenditure w e r e f r o m t h e s u s e n a s v s e n 2 0 0 7 . k questionnaire. (9) socioeconomic status was a s s e s s e d f r o m t h e rt p e r c a p i t a m o n t h l y household expenditures categorized into 5 quintiles. in the present study, cataract surgical events were obtained by submitting the following question to the subjects diagnosed as suffering from cataract by medical personnel: have you ever undergone cataract surgery in the last 12 months? regions of residence were subdivided into the regions of sumatera, java-bali, and east indonesia. data analysis the odds ratios (or) of the cataract surgical coverage rate were calculated for a comparison between gender, age groups, occupation, income, type of area of residence, and region of residence. the strength of association was expressed as or and their 95% ci. a final multivariate logistic regression model included all of the above variables meeting the preconditions (p<0.25 or p>0.25 but theoretically of influence). a p-value of 0.05 was taken as significant for this analysis. spss version 15.0 (spss inc., chicago, il, usa) was used for data analysis. results the total number of subjects meeting the inclusion criteria was 6939. data on the variables of age, gender, type of area of residence, and region of residence amounted to an identical total of 6939 (100%). the total numbers of subjects f o r t h e v a r i a b l e s e d u c a t i o n , p e r c a p i t a expenditure, and occupation were respectively 6915 (99.7%), 6913 (99.6%), and 6935 (99.9%). for the variables past history of dm and cataract surgery the data obtained were 6929 (99.9%) and 6896 (99.4%), respectively. the proportion of subjects aged 50 years and over was 84.4% and of those in the age range of 40-49 years 15.6%. the study subjects consisted of 54.6% females and 45.4% males. a total of 75.1% of the subjects had an education lower than junior high school and 24.9% had an education of junior high school and higher. about 30.3% of the subjects were unemployed, 25.6% were farmers/fishermen/laborers, and the proportion of housewives was 18.0%, selfemployed 13.0%, employees 7.6%, teachers 7.6% and others 5.0%. around 51.5% resided in rural areas and 48.5% in urban areas, 58.8% had a l o w rt m o n t h l y p e r c a p i t a e x p e n d i t u r e (quintiles 1-3) and 41.2% had a high per capita expenditure (quintiles 4-5). fifty-nine percent of the subjects originated from the java-bali region, 24% from the sumatera region, and 16.5% from the region of east indonesia. the proportion of respondents who had previously undergone cataract surgery was 19.3% and those who did not have prior cataract surgery was 80.7%. table 1 presents the results of simple logistic analysis between a number of important variables and cataract surgery. the proportion of cataract surgeries was significantly lower among subjects in the age group of 40–49 years, the less educated, residents of rural areas, those with low per capita expenditure per rt, and those from the sumatera region. in comparison to other univ med vol.28 no.3 164 tana cataract surgical coverage types of occupation, the group of farmers/ fishermen/laborers had the lowest rate of cataract s u rg e r y. t h e r e w a s n o d i ff e r e n c e i n t h e proportions of cataract surgeries by gender and past history of dm. the determinants associated with cataract surgery were obtained through multiple logistic regression analysis by entering several variables into a model. variables meeting the preconditions for inclusion into the multivariate model were age, gender, education, type of area of residence, rt per capita expenditure, occupation, region of residence, and past history of dm. table 2 shows a significant association between age, type of area of residence, region of residence, and monthly rt per capita expenditure and cataract surgery. discussion the present study showed a low cataract surgical coverage rate of 19.3%, which is similar surgery (%) variables n yes no or 95 %ci p age group (years) 40-49 1105 14.0 86.0 0.64 0.50 0.83 0.001 >50 5834 20.3 79.7 ref gender male 3105 19.5 80.5 1.03 0.87 1.21 0.7329 female 3834 19.1 80.9 ref education < junior high school 5198 18.1 81.9 0.74 0.61 0.89 0.001 junior high school + higher 1717 23.1 76.9 ref occupation unemployed 2003 18.9 81.1 0.71 0.52 0.96 0.0001 teachers 46 29.9 70.1 1.29 0.51 3.26 housewives 1252 19.0 81.0 0.71 0.51 1.00 self-employed 799 22.4 77.6 0.87 0.62 1.24 farmers/fishermen/laborers 2012 15.3 84.7 0.55 0.40 0.75 employees 321 24.8 75.2 ref others 502 26.1 73,9 1.07 0.69 1.66 type of area of residence rural 3943 15.2 84.8 0.59 0.50 0.70 0.0001 urban 2996 23.2 76.8 ref region of residence sumatera 2570 14.4 85.6 0.63 0.52 0.76 0.0001 east indoneisa 2058 20.1 79.9 0.94 0.78 1.14 java-bali 2311 21.1 78.9 ref rt per capita expenditure quintiles 1 3 4015 16.9 83.1 0.70 0.59 0.82 0.0001 quintiles 4 5 2898 22.6 77.4 ref dm yes 580 21.9 78.1 1.19 0.91 1.55 0.2168 no 6349 19.0 81.0 ref table 1. relationships between a number important individual characteristics, dm and cataract surgery legend: rt: rumah tangga; dm: diabetes mellitus; or: odds ratio; ci: confidence interval; ref: reference group 165 to the results of previous studies conducted in tanzania,(10) ethiopia,(11) malawi(12) and south africa.(13) even among urban respondents, the present study revealed that the proportion of respondents with previous cataract surgery was o n l y 2 3 . 2 % . t h e r e a r e s e v e r a l p o s s i b l e explanations for these study results. first, there may have been a tendency among medical personnel or ophthalmologists to refrain from performing cataract surgery before the cataract has become mature, resulting in extending the period of blindness of the respondents. ( 1 0 ) second, a program that uses visual acuity cuttoffs (blind or severe visual impairment) to determine on whom to perform cataract surgery may exclude the majority of people who should receive cataract surgery. in this study, age had a significantly positive association with cataract surgery, the rate of cataract surgery being proportional to age. this finding is in accord with the results of a study by lavanya et al.(7) where advanced age was a factor associated with cataract surgery. the explanation may be that there will be a higher number of mature cataracts in the older age groups, which is consistent with reports in the l i t e r a t u r e i n d i c a t i n g t h a t a m o n g t h e approximately 40% of lens opacities in the age group of 55-64 years, 5% were found to be mature cataracts. similarly, from 70% of lens opacities at ages 65-74 years, 18% of cataracts become mature, and in more than 90% of lens opacities at ages 79-84 years almost half were mature cataracts.(14) it may be concluded that with advancing age, in addition to an increased percentage of cataracts, there is also an increased percentage of mature cataracts. in other words it may be stated that the higher percentage of mature cataracts in the older age groups causes the cataract patients to undergo cataract surgery in order to improve their visual acuity. in the present study, from the results of bivariate as well as multivariate analysis, no association was found between gender and cataract surgery. these findings are at variance w i t h t h o s e r e p o r t e d b y l e w a l l e n e t a l . ( 2 ) suggesting that females in low and middle-income countries had a lower coverage of cataract surgery, when compared with males. the coverage of cataract surgery in males was 1.7-fold higher than univ med vol.28 no.3 cataract surgery (%) determinant yes no or adjusted 95 %ci p age group (years) 40-49 14.0 86.0 0.64 0.49 0.82 0.0001 >50 20.3 79.7 ref type of area of residence rural 15.2 84.8 0.63 0.53 0.75 0.0001 urban 23.2 76.8 ref rt per capita expenditure quintiles 1 3 16.9 83.1 0.72 0.61 0.85 0.0001 quintiles 4 5 22.6 77.4 ref region of residence sumatera 14.4 85.6 0.69 0.57 0.84 0.0001 east indonesia 20.1 79.9 1.02 0.84 1.24 java bali 21.1 78.9 ref table 2. multivariate logistic regression analysis between age, type of area of residence, household expenditure, and region of residence and cataract surgery legend: rt= rumah tangga; or= odds ratio; ci= confidence interval; ref= reference group 166 tana cataract surgical coverage in females. lavanya et al.(7) reported a similar finding, i.e. that male gender was associated with cataract surgery. in addition, nirmalan et al.(6) also reported that although blindness due to cataract i n f e m a l e s 5 0 y e a r s o f a g e o r o l d e r w a s significantly higher than that in males, the number of cataract surgeries in females was lower (adjusted or 0.71, 95% ci: 0.57 0.87). it may be concluded from the results of various studies conducted in urban populations of asia and south india and a meta-analysis on 21 population-based surveys conducted up to the year 2000, that the number of cataract surgeries in females was lower than in males. in respect to region of residence, the present study differs from the literature and found that overall there was no significant difference in coverage of cataract surgery between males and females in the regions of java-bali, sumatera, and east indonesia, although the proportion of cataracts was higher in females. the strategies for increasing the cataract surgical coverage rate in females should receive due attention. the lower cataract surgical coverage rate in women as compared to men in many countries results in significantly higher numbers of cataract blindness and impaired vision due to cataract.(15,16) in the present study a significant association was found between type of area of residence of the respondents and cataract surgery, where the proportion of rural respondents with previous cataract surgery was lower than that of urban respondents. results consistent with this finding were obtained in india, demonstrating a higher cataract surgical coverage rate in urban areas as compared to rural areas.(17) these results may be explained from several aspects such as level of education, socioeconomic status, and access to healthcare facilities. from the results of an analysis performed by tana(18) it was found that the proportion of respondents with a low level of education and residing in rural areas was higher than that of r e s p o n d e n t s i n u r b a n a r e a s . t h e l e v e l o f education may possibly affect the knowledge and understanding of the respondents with regard to cataract, the importance of cataract surgery for treatment of cataract, and the success of cataract surgery in restoring vision. another possibility connected with cataract surgical coverage rate is ease of access to the relevant healthcare facilities for the respondents. access to healthcare facilities capable of performing cataract surgery is not as simple for respondents in rural areas as it is for urban r e s p o n d e n t s . f r o m t h e n a t i o n a l r e s u l t s o f riskesdas 2007, for rural rts with access to health services outside a radius of 5 km the cataract surgical coverage rate was 8.9%, whilst at a distance of 1-5 km the rate was 50.9%. a total of 4% of rural rts had to spend 1 hour to reach the health services.(4) thus distance to health services and travel time are still a problem for a proportion of rts in indonesia, including the respondents in the present study. still another possible explanation for the l o w c a t a r a c t s u rg i c a l c o v e r a g e r a t e i s socioeconomic status. low socioeconomic status may affect the respondent’s decision to undergo cataract surgery. a study in myanmar showed that the most commonly reported barrier to cataract surgery was its cost. the impact of cost varies between different communities, but it is still one of the most commonly reported barriers to obtaining care.(19,20) in indonesia, however, due to the availability of governmental health insurance for the indigent and the multitude of free cataract surgical services offered in the community, the low socioeconomic status of the r e s p o n d e n t s m a y b e e x p e c t e d t o o ff e r n o impediments to the respondents in their decision for undergoing cataract surgery. however, there remains the question whether these free services are 100% without cost, for the surgery itself as 167 well as the surgical instruments used for the operations. this should be subject to further investigation in future studies. in the present study there was no difference in cataract surgical coverage rates between respondents with and without dm, both in bivariate and multivariate associations. the results reported by investigators was that there is an association between dm and cataract surgery.(7) dm is one of the factors associated with cataract, as it has been reported that cataract in patients with dm is 1.6 times higher than in patients without dm.(21) the cataract surgical coverage rate in respondents with a low level of education was l o w e r t h a n i n t h o s e w i t h a h i g h l e v e l o f e d u c a t i o n . t h i s m a y b e b e c a u s e l e v e l o f e d u c a t i o n o f t h e r e s p o n d e n t s i s p r o b a b l y associated with level of knowlegde of diseases, particularly of cataract. the reports in the literature indicate that only 29.2% of the respondents had some knowledge of cataract, although 46.5% of them had heard of the term cataract.(22) the low level of knowledge of cataract resulted in the respondents being ignorant of the fact that their impaired vision was caused by cataract and that it was curable. in addition, lack of knowledge may impact on ignorance of the successful outcome of cataract surgery, which is consistent with literature reports on the existence of an association between educational level and cataract surgical coverage rate. (23) the association between rt per capita expenditure and cataract surgical coverage rate obtained in the present study was proportional. t h i s m a y b e e x p l a i n e d a s f o l l o w s : l o w socioeconomic status of the respondents may be related to their low financial capacity for paying for cataract surgery. however, because of the availability of governmental health insurance for indigent community members, it is expected that this may eliminate these financial limitations. similarly, several studies have also found an association between lower socioeconomic status and cataract surgical coverage rate where access to ophthalmic services is limited,(24,25) but even if these are available the uptake of surgery can s t i l l b e l o w. ( 2 6 , 2 7 ) r e g a r d i n g t h e b i v a r i a t e association between occupation and cataract s u rg i c a l c o v e r a g e r a t e i t w a s f o u n d t h a t respondents who were farmers/fishermen/ l a b o r e r s h a d t h e l o w e s t c a t a r a c t s u rg i c a l coverage rate among all occupations extracted from the riskesdas 2007 data, especially in comparison with employees. this may be explained from the aspect of the different needs for visual acuity of the respondents in performing their job. the group of employees need good visual acuity at work, such that the need for optimal visual acuity tends to motivate the employee to seek treatment when suffering from visual disorders, including cataract. the group of farmers/fishermen/laborers perform their work in the open air and do not need to write or read for a living, thus their need for optimal vision is relatively less pronounced. therefore, even though they may already have poor vision, they may be not aware of the fact, as it does not affect their work. another possibility is that respondents who are farmers/fishermen/laborers reside more in rural areas rather than in urban areas, so that there is a lower possibility of their a c c e s s t o h e a l t h c a r e f a c i l i t i e s c a p a b l e o f performing cataract surgery, unlike urban respondents. in addition, respondents from the group of farmers/fishermen/laborers have a lower per capita expenditure, which may affect their access to adequate treatment. the low level of education in farmers/fishermen/laborers may also influence their perception of the need for cataract treatment. tana(18) conducted a study on the role of occupation on cataract surgical coverage rate in indonesian communities and found that among farmers/fishermen/laborers aged 30 years and older, 72.3% had a per capita univ med vol.28 no.3 168 tana cataract surgical coverage extensive rural area in the sumatera region compared with other regions may possibly be a factor associated with the low rate of cataract surgery in that region. conclusions this study has helped to determine the cataract surgical coverage rate and several of its determinant factors. it is suggested to implement a health program which could identify and address the barriers to cataract surgery among identified high-risk groups. acknowledgements for the support provided in conducting the present study we gratefully acknowledge our indebtedness, especially to dr triono soendoro, phd, dr trihono, msc, dr atmarita, dr emiliana tjitra, msc, phd, and dr lutfah rif’ati spm, for enabling the present study to be carried out on the basis of data from the riskesdas 2007 of the health research and development center, department of health, republic of indonesia (badan penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia). references 1. resnikoff s, pararajasegaram r. blindness prevention programmes: past, present and future. bull who 2001;79:222–6. 2. lewallen s, mousa a, bassett k, courtright p. cataract surgical coverage remains lower in women. br j ophthalmol 2009;93:295-8. 3. lewallen s, courtright p. gender and use of cataract surgical services in developing countries. bull who 2002;80:300–3. 4. departemen kesehatan republik indonesia. gangguan kesehatan indera penglihatan dan pendengaran. analisis data morbiditas-disabilitas, skrt-surkesnas 2001. direktorat jenderal bina kesehatan masyarakat direktorat kesehatan khusus dan badan penelitian dan pengembangan expenditure in the quintiles 1-3, 79.7% resided in rural areas, and 83.2% had a low educational level (lower than junior high school). t h i s w a s i n c o n t r a s t t o t h e g r o u p o f employees, who had the highest proportion of c a t a r a c t s u rg e r y c o m p a r e d w i t h a l l o t h e r occupations. among employees aged 30 years and over, only 38.3% had a monthly rt per capita expenditure in the quintiles 1-3, 27.6% lived in rural areas, and 11.2% had a low educational level. besides the group of farmers/fishermen/ laborers, the group of housewives also had a low rate of cataract surgery. on further scrutiny of individual characteristics (education, type of area of residence, access to health services, per capita expenditure), tana et al.(28) reported that the proportion of housewives aged 30 years and over, according to riskesdas 2007 data was 99.5%; 62.6% had an rt per capita expenditure in the quintiles 1-3, half of them lived in rural areas, and 63.7% had a low level of education. regarding the association between region of residence and cataract surgery, it was found that the lowest rate of cataract surgery was in respondents residing in the sumatera region, in comparison with the regions of java-bali and east indonesia. when viewed from the aspect of cataract surgical coverage rate in all provinces of indonesia, it was found that the lowest coverage rate was in the province of west papua (5.2%) and the highest in the province of southeast sulawesi (31.5%).(4) still another fact that may explain the differences in cataract surgical coverage rate based on region is that from the aspect of type of area of residence (urban and rural), in the sumatera region the rural area accounts for 64.1% and the urban area 35.9%. in contrast, in the region of java-bali only 39.6% of the area is rural, while the urban area amounts to 60.4%, and lastly in east indonesia the rural area is 58.1% and the urban area 41.9%.(4) the more 169 kesehatan, sekretariat surkesnas. jakarta; 2004. 5. badan penelitian dan pengembangan kesehatan departemen kesehatan ri. laporan nasional riset kesehatan dasar 2007. jakarta;2008. 6. nirmalan p k, padmavathi a, thulasiraj r d. sex inequalities in cataract blindness burden and surgical services in south india. br j ophthalmol 2003;87:847-9. 7. lavanya r, wong ty, aung t, tan dth, saw sm, tay wt, et al. prevalence of cataract surgery and post-surgical visual outcomes in an urban asian population: the singapore malay eye study. br j ophthalmol 2009;93:299-304. 8. departemen kesehatan republik indonesia. ditjen bina kesehatan masyarakat. rencana strategi nasional penanggulangan gangguan penglihatan dan kebutaan untuk mencapai vision 2020. keputusan menteri kesehatan republik indonesia no. 1437/ menkes/sk/x/2005. jakarta;2006. 9. badan penelitian dan pengembangan kesehatan departemen kesehatan ri. riset kesehatan dasar 2007. pedoman pengisian kuesioner. jakarta; 2007. 10. chibuga e, massae p, genau r, mahonde h, lewallen s, cortright p. acceptance of cataract surgery in a cohort tanzanian with operable cataract. eye 2008;22:839-43. 11. melese m, alemayehi w, friedlander e. courtright p indirect cost associated with asseccessing eye care services as a barrier to service use in ehiopia. trop med int health 2004;9:426-31. 12. courtright p, metcalfe n, hoechsmann a, chirambo m, lewallen s, barrows j, et al. the chikawa survey team. cataract surgical coverage and outcome of cataract surgery in a rural district in malawi. canad j ophthalmol 2004;39:25-30. 13. rotchford ap, rotchford km, methwa cp, johnson cj. reasons for post cataract surgery intake: a qualitative study in rural southern africa. trop med int health 2002;7:288-92. 14. medline plus. cataract. available at: http:// www.nlm.nih.gov/medlineplud/ency/article/ 001001.htm. accessed august 20,2009. 15. venkata g, murthy s, gupta sk, bachani d, jose r, john n. current estimates of blindness in india. br j ophthalmol 2005;89:257-60. 16. abou-gareeb j, lewallen s, bassett k, courtright p. gender and blindness: a meta-analysis of population-base survey. ophthalmic epidemiol 2001;8:39-56 17. nirmalan pk, padmavathi a, thulasiraj rd. sex inequalities in cataract blindness burden and surgical services in south india. br j ophthalmol 2003;87:847-9. 18. tana l. determinan kejadian cataract di indonesia. riset kesehatan dasar 2007. laporan penelitian. jakarta. bul penelit kesehat 2009;37:114-25. 19. sapkota yd, pokharel gp, dulal s, byanju rn, maharjan im. barriers to up take cataract surgery in gandaki zone, nepal. kathmandu univ med j 2004;2:103–12. 20. rabiu mm. cataract blindness and barriers to uptake of cataract surgery in a rural community of northern nigeria. br j ophthalmol 2001;85:776– 80. 21. mukesh bn, le a, dimitrov pn, ahmed s, taylor hr, mccarty ca. development of cataract and associated risk factors: the visual impairment project. arch ophthalmol 2006;124:79-85. 22. rumah sakit mata cicendo, fakultas kedokteran universitas padjadjaran, helen keller internasional indonesia. laporan penelitian: survei kebutaan dan kesehatan mata di jawa barat tahun 2005. bandung; 2006. 23. tana l, delima, hastuti e, gondhowiardjo t. katarak pada petani dan keluarganya di kecamatan teluk jambe barat. media penelit pengembang kesehat 2006;xvi:43-51. 24. klein be, klein r, lee ke, meuer sm. socioeconomic and lifestyle factors and the 10-year incidence of age-related cataracts. am j ophthalmol 2003;136:506–12. 25. navaro esteban jj, gutierrez leiva ja, valero caracena n, buendia bermejo j, calle puron me, martinez vizcaino vj. prevalence and risk factors of lens opacities in the elderly in cuenca, spain. eur j ophthalmol 2007;17:29–37. 26. rotchford ap, rotchford km, mthethwa lp, johnson gj. reasons for poor cataract surgery uptake – a qualitative study in rural south africa. trop med int health 2002;7:288–92. 27. de lima dm, ventura lo, brandt ct. barriers in the access to senile cataract treatment at altino ventura foundation. arq bras oftalmol 2005;68: 357–62. 28. tana l, rif’ati l, ghani l. peranan pekerjaan terhadap kejadian cataract pada masyarakat indonesia riset kesehatan dasar 2007. bul penelit kesehat 2009; suplemen bulletin. univ med vol.28 no.3 yenny1 117 pleiotropic effects of statins in stroke prevention may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina yenny* *department of pharmacology medical faculty, trisakti university, jakarta correspondence dr. yenny department of pharmacology medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11410 telp. 021-5672731 ex.2801 email: stasia_mk@yahoo.com univ med 2009;28:117-26 abstract cardiovascular disease is the leading cause of death and disability, and contributes substantially to healthcare budgets. the lipid-lowering drugs, 3-hydroxy-3methylgulutaryl-coenzyme a (hmg-coa) reductase inhibitor or statins, reducing mortality and cardiovascular morbidity in patients with established cardiovascular disease. statins therefore have a place in the secondary prevention of cardiovascular disease. recent experimental and clinical studies suggest that statins may exert vascular protective effect beyond cholesterol reduction. the cholesterol-independet or “pleiotropic” effects of statin include the upregulation and activation of endothelial nitric acid synthase (enos) that can increase nitric oxide (no) production. augmentation of no production increases cerebral blood flow, which can lead to neuroprotection during brain ischaemia. by inhibiting mevalonate synthesis, statins prevent the formation of several isoprenoids (including farnesylpyrophosphate and geranylgeranylpyrophosphate). inhibiting geranylgeranylation of rhoa small g proteins increases the stability of enos mrna through the remodeling of endothelial actin microfilamens. moreover, statins directly increase enos activity within minutes by activating the pathway involving phosphoinositide 3-kinase and protein kinase b. in the secondary prevention of stroke, the use of statins reduces the incidence of either recurrent stroke or other major vascular events and treatment should be initiated soon after the event. the use of statins does not increase hemorrhagic stroke or cancer and may also favor atherosclerotic plaque regression. key word: statin, endothelial nitric synthase stroke ischemic introduction according to data from heart disease and stoke statistic update in 2009, each year 795.000 stroke cases are reported in the united states, consisting of 610.000 cases of first stroke events and 185.000 cases of repeat stroke. stroke is also reported to be the third major cause of death after heart disease and cancer. total mortality in 2005 in the united states is reported to amount to 242.000 cases, and in 143.579 cases the cause of death is stroke.(1) 118 yenny pleiotropic effects of statins to d a t e t h e r e i s n o e ff e c t i v e pharmacological treatment for stroke. the development of drugs capable of preventing stroke events will play an important role because of the high mortality and disability resulting from t h i s d i s o r d e r, w h i c h n o t o n l y i m p o s e s a n economic burden but also has an impact by lowering the quality of life of the patient. the discovery of statins in 1976 by akira endo occurred fortuitously in the course of a search for new antibiotic compounds.(2) the discovery of statins was a major advance in prevention and treatment of ischemic stroke, similar to the discovery of antiplatelet and antithrombotic drugs, with the objective of maintaining and restoring cerebral blood flow. to date it is debatable whether increased serum cholesterol level is a risk factor for stroke events, and whether statins have a preventive e f f e c t o n s t r o k e e v e n t s . t h e c h o l e s t e r o l treatment triallists collaborators(3) in their m e t a a n a l y s i s c o m p r i s i n g 9 0 . 0 5 6 p a t i e n t s showed that the use of statins significantly resulted in a proportional reduction of first stroke events of 17% for each 1 mmol/l reduction of low-density lipoprotein (ldl) concentration. however, it is unclear whether the results of this meta-analysis are caused by the effects of statins in lowering ldl cholesterol levels or by the (greek: “pleio” or many, and “tropos” manner) p l e i o t r o p i c e ff e c t s o f s t a t i n s . t h e s t r o k e p r e v e n t i o n b y a g g r e s s i v e r e d u c t i o n i n cholesterol levels (sparcl) study,(4) which had the objective of evaluating the secondary p r e v e n t i v e e ff e c t s o f s t a t i n s o n s t r o k e , demonstrated that atorvastatin reduced the risk of repeated cerebrovascular events in patients without a history of coronary heart disease who had recently experienced a stroke or transient ischemic attack (tia). the effects of statins that are independent of their blood cholesterol-reducing effects are also called their pleiotropic effects. these are apparently related to the ability of statins to improve plaque stability, reduce the number of i n f l a m m a t o r y c e l l s i n p l a q u e s , r e s t o r e endothelial function, inhibit platelet function, increase fibrinolytic activity, cerebral blood flow and nitric oxide (no) levels. due to their pleiotropic effects, statins have been claimed to possess neuroprotective effects in the prevention of ischemic stroke.(5,6) the biosynthesis of no is mainly performed by an isoform of enos that is dependent on the presence of ca2+. this process is triggered by binding of agonists or by fluid shear stress (1) and is facilitated by several cofactors and the hsp90 heat-shock protein. conversion by enos of l-arginine (l-arg) to no (2) gives rise to l-citrulline (l-cit) as a by-product. subsequently no diffuses into the neighboring smooth muscle cells (3) where it activates the effector enzyme guanylate cyclase (gc). gc (4) then converts guanosine triphosphate (gtp) into the second messenger cyclic guanosine monophosphate (cgmp), which in turn activates protein kinase g (pkg) (5) resulting in modulation of myosin light chain kinase and smooth muscle relaxation. pkg also modulates the activity of potassium channels (hyperpolarization) and causes relaxation. no itself can also directly modulate potassium channels (independently of cgmp). figure 1. biosynthesis of no and effects of no on blood vessels(8) 119 the present review discusses the pleiotropic effects of statins in the prevention of ischemic stroke in connection with endothelial nitric oxide synthase (enos). in addition, this paper aims to find supporting evidence for a rationalized u t i l i z a t i o n o f s t a t i n s i n i s c h e m i c s t r o k e prevention. no, enos and vascular disorders enos (endothelial nitric oxide synthase) is an enzyme for the production of nitric oxide (no) by vascular endothelium.(7) no is produced by enos through oxidative conversion of larginine to l-citrulline. activation of enos occurs through specific phosphorylation at ser1177 by protein kinase b (pkb/akt), which also mediates no synthesis induced by fluid shear stress. after synthesis by the vascular endothelium, no diffuses into the neighboring cells and activates soluble guanylate cyclase. this process subsequently mediates various beneficial effects of no.(7) in vascular smooth muscle, no is a potent vasodilator and regulates regional blood flow. figure 1 presents a diagram of biosynthesis of no and its effects on blood vessels.(8) in addition to the vasodilator effects of no, o t h e r b e n e f i c i a l e ff e c t s o f n o a r e i t s a n t i t h r o m b o t i c , a n t i i n f l a m m a t o r y, a n d antiproliferative effects. on the other hand, loss of no leads to impaired vascular relaxation, platelet aggregation, increased proliferation of vascular smooth muscle, increased leukocyte adhesion to the endothelium, and raised blood p r e s s u r e . t h e r e f o r e i t m a y b e s t a t e d t h a t endothelial no acts as a protector of the vascular wall.(7) currently three isoforms of nitric oxide s y n t h a s e ( n o s ) h a v e b e e n i d e n t i f i e d i n mammals,(9) each of these nos isoforms being encoded by a different gene and showing differences in location and function, as presented in table 1. in the brain, besides enos, other nos isoforms, namely neuronal nos (nnos) and inducible nos (inos), also play a role during c e r e b r a l i s c h e m i a . l i k e e n o s , n n o s i s constitutively expressed and is ca2+/calmodulindependent. macrophage nos (‘immunologic’ n o s o r i n o s ) i s i n d u c e d b y s e l e c t i v e immunologic stimuli and is ca2+-independent.(7) the no produced by enos and nnos is essential for the regulation of cerebral blood flow,(7,9) and enos is also said to play a role in r e d u c i n g t h e e x t e n t o f a n i n f a r c t i o n . ( 7 ) furthermore, no from nnos also functions as a neurotransmitter and is involved in synaptic p l a s t i c i t y, m o d u l a t i o n o f n e u r o e n d o c r i n e f u n c t i o n , a n d b e h a v i o r a l a c t i v i t y. ( 9 ) i n p a t h o l o g i c a l c o n d i t i o n s , s u c h a s c e r e b r a l ischemia, no is produced in large quantities in the brain as a result of induced expression of inos. induction of inos expression occurs as a result of increased transcription of the inos univ med vol.28 no.2 table 1. differences of nos isoforms(10) 120 yenny pleiotropic effects of statins g e n e i n r e s p o n s e t o l o c a l l y p r o d u c e d inflammatory cytokines. the ischemia induced b y o v e r p r o d u c t i o n o f n o i s r e l a t e d t o glutamatergic activation mediated by increased intracellular ca2+ concentrations, which causes calmodulin-dependent upregulation of nnos and enos activity.(9) the augmented no production in the early ischemic phases occurs as a result of increased enos and nnos activity, which is of short duration (~1 hour) and rapidly decreases within a few hours, both in transient and permanent ischemia. this process is subsequently followed by the second stage of large-scale no production as a result of induced expression of inos, which is started several hours after the initial ischemic phase and persists up to 4-7 days. no production by inos apparently contributes secondarily to cerebral damage that occurs in the final stages.(9) depending on the cellular source and evolutionaty stage of the ischemic process, no may be protective or destructive. the double role of no in cerebral ischemia forms the basis for a s e l e c t i v e t h e r a p e u t i c a p p r o a c h a i m e d a t inhibiting nnos and inos, while increasing enos.(7) statins and enos statins are a class of drugs that are known to be capable of increasing enos levels. the principal mechanism of action of statins is reduction of cholesterol levels. this effect is m e d i a t e d b y 3 h y d r o x y 3 m e t h y l g l u t a r y l coenzyme a (hmg-coa) reductase inhibitors in the liver, which are necessary for cholesterol biosynthesis. around 60-70% of serum cholesterol is s y n t h e s i z e d i n t h e l i v e r, w i t h h m g c o a reductase playing an essential role in the cholesterol biosynthetic pathway. oxidized lowdensity lipoprotein (ldl) has been known to be capable of inhibiting the expression of enos that is bound to caveolin-1 (an integral protein c o m p o n e n t o f t h e c e l l m e m b r a n e ) w i t h i n caveolae.(7) inhibition of hmg-coa reductase by statins causes a dramatic reduction in circulating l d l c h o l e s t e r o l l e v e l s ( f i g u r e 2 ) . ( 11 ) additionally, reduction of ldl cholesterol leads to upregulation of ldl receptors, thus effecting i n c r e a s e d l d l c l e a r a n c e . t h i s e f f e c t i s presumably due to the ability of statins to reduce the abundant amounts of caveolin-1.(7) besides their cholesterol-lowering effects, statins also exert cholesterol-independent or pleiotropic effects, as a result of the ability of statins in inhibiting the conversion of hmg-coa to l-mevalonic acid. inhibition of l-mevalonic acid synthesis by statin leads to inhibition of the synthesis of ‘isoprenoid’ intermediates such as farnesylpyrophosphate (fpp) and geranylgeranylpyrophosphate (ggpp). isoprenoids function as lipid attachment sites for intracellular signal m o l e c u l e s . st a t i n c a u s e s i n h i b i t i o n o f isoprenilation required for modification of small g proteins (gtpases), one of which being the rho a protein, to enable them to occupy the appropriate locations at the membrane and thus perform their functions. isoprenilation inhibition by statin prevents downregulation of enos expression, and maintains enos activity in conditions where downregulation of enos e x p r e s s i o n o c c u r s , s u c h a s h y p o x i a ( e . g . ischemic stroke) and the presence of oxidized ldl in the blood. this process occurs through inhibition of the rho kinase activator (rho a), leading to post-translation stabilization of enos mrna and prevention of downregulation of enos expression.(11,12) c u r r e n t l y t h e r e a r e 8 s u b f a m i l i e s o f gtpases that have been identified in mammals. in performing its function, gtpases undergoes cyclical changes from the gdp-bound (inactive) form to the gtp-bound (active) form, and vice v e r s a . ( 11 ) o f t h e e x i s t i n g 8 s u b f a m i l i e s o f gtpases, only the ras and rho gtpases have 121 m e r i t e d s p e c i a l a t t e n t i o n , a s t h e y e ff e c t transduction of extracellular stimuli into multiple intracellular signal transmission.(11) the ras family plays an important role in signal transduction, and in cellular proliferation and malignant transformation. representatives of the rho gtpases comprising rhoa, rac and cdc42, have specific functions in cellular shape, motility, secretion, and proliferation. the biological effects of the rho gtpase family is presented in figure 2. inhibition of geranylgeranylation of rhoa gtpase by statin enhances stability of enos mrna through remodelling of endothelial actin microfilaments.(11) in addition to inhibition of isoprenoid synthesis, statin directly enhances enos activity b y a c t i v a t i o n o f t h e p a t h w a y i n v o l v i n g phosphoinositide 3-kinase (pi3k) and pkb/ akt.(13) e v i d e n c e f r o m a n i m a l m o d e l studies animal model studies with the objective of finding evidence for the neuroprotective effects of statins and their underlying mechanisms have been conducted. for inducing cerebral infarction in the experimental animals (rats), permanent occlusion of the middle cerebral artery was performed. the temporal progression of the resulting infarction was monitored by magnetic resonance imaging (mri). subsequently the figure 2. isoprenoids and statins(11) diagram of cholesterol biosynthetic pathway and inhibition of hmg-coa-reductase by statins. inhibition of isoprenilation by statin results in modulation of various cellular functions univ med vol.28 no.2 122 yenny pleiotropic effects of statins experimental animals were given simvastatin (20 mg/kg) on the third hour after occlusion. the study results showed that administration of simvastatin prevented increases in the volume of infarction within 24 hours and decreased the size of the infarction up to 46.6% within 48 hours after the intervention. on the basis of immunoreactivity a s s a y s o f e x p e r i m e n t a l a n i m a l s r e c e i v i n g simvastatin, the neuroprotective effects of simvastatin paralleled the increase in enos.(14) a study with the objective of determining the physiological relevance of regulation of enos expression by rho kinase (rock) has been conducted. this study was performed by administration of fasudil (rock inhibitor) to rats in the 48-hour period prior to occlusion of t h e m i d d l e c e r e b r a l a r t e r y, f o l l o w e d b y assessment of cerebral blood flow, extent of cerebral infarction, and neurological deficits. the results indicated that administration of rock inhibitors led to increased blood flow in the ischemic as well as the non-ischemic areas of the brain, reduction in size of infarction of up to 33%, and enhancement of neurological deficit s c o r e o f u p t o 3 7 % . t h e s e s t u d y r e s u l t s confirmed the premise that the neuroprotective effects due to rock inhibition was mediated by enos.(15) s tat i n s a n d s e c o n d a ry prevention of stroke the stroke prevention by aggressive reduction of cholesterol level (sparcl) is a study that for the first time investigates the effects of statins on cerebrovascular risk in patients without a history of coronary heart disease. this study is a multicenter, placebocontrolled double-blind randomized clinical trial. the atorvastatin dose used is 80 mg/day. inclusion criteria in this study are patients with previous tia or stroke, ldl levels between 100 mg/dl and 190 mg/dl, and without evidence of coronary heart disease. the primary endpoint is the first occurrence of fatal and non-fatal (cerebrovascular) stroke, while the secondary e n d p o i n t i s t h e f i r s t o c c u r r e n c e o f a cardiovascular event.(16) the number of subjects for this study was 4.731 persons with a history of stroke or tia within the previous six months. after a 6-year follow up, there were 265 patients with fatal or n o n f a t a l s t r o k e i n t h e g r o u p r e c e i v i n g atorvastatin, whereas in the control group there were 311 of these patients. there was a risk reduction of 16% of first occurrence of stroke in the group on atorvastatin (adjusted hazard ratio: 0.85, 95% ci: 0.71-0.99; number needed to treat: 46). for the secondary endpoint of this study, namely occurrence of stroke or tia, there was a risk reduction of 23% (hazard ratio:0.77, 95%ci: 0.67-0.88) with 375 events in the atorvastatin group and 476 events in the control group. furthermore, there was a risk reduction of 35% in coronary events (hazard ratio: 0.65, 95% ci: 0.49-0.87).(4) the heart protection study(17) investigated a total of 20.536 patients with coronary disease, other occlusive arterial disorders, or diabetes. among the study subjects were 3280 patients with stroke prior to randomization, of which 1822 had stroke without evidence of chd. the subjects were randomized for simvastatin 40 mg/ day or placebo for 5 years. the results of this study showed a significantly reduced all-cause mortality, particularly mortality due to coronary disease. the number of stroke events was also reduced significantly up to 25% [444 stroke events in the simvastatin group (4.3%) versus 585 stroke events in the placebo group (5.5%); or:0.75; 95%ci: 0.66-0.79; p<0.0001]. the reduction in stroke events was mainly due to i s c h e m i c s t r o k e , w h i l e t h e n u m b e r o f hemorrhagic stroke events was similar in both groups. 123 analysis of the hps study results indicated that there was a reduction in major vascular events of 23%, but the interpretation of these results has been inconclusive. the reduction in the endpoint of this study included major coronary events, stroke, or revascularization, but the reduction was mainly for major coronary events and revascularization, as the number of events of repeat stroke was almost identical in both groups (10.4% simvastatin versus 10.5% placebo).(18) the lack of an apparent effect of simvastatin in preventing repeat stroke may be due to the subgroup analysis performed or due to the small numbers of patients with repeat stroke, resulting in a decreased power for detection of differences.(19) the mini meta-analysis conducted on the sparcl and hps studies of patients with a history of cerebrovascular disorders without chd and with major vascular events and repeat stroke events is presented in figures 3 and 4.(20) according to the results of the mini metaanalysis on the sparcl and hps studies, when compared to placebo, statin shows a nearly nonsignificant difference for repeat stroke events (or: 0.87, 95% ci: 0.75-1.01; p: 0.07), but a significant difference for major vascular events (or: 0.78, 95% ci: 0.68-0.88; p: 0.0001).(20) clinical practice the american heart association/american stroke association(21) has issued recommendations on stroke prevention in patients with a history of ischemic stroke or transient ischemic attack (tia). the recommendations comprise two important points. firstly, patients with ischemic stroke or tia, having a high cholesterol level, coronary heart disease comorbidity, or evidence of atherosclerosis, should be managed according to the guidelines issued by the national cholesterol education program (ncep iii),(22) involving lifestyle changes, dietary regulation, and use of medications (class ia, evidence level a). univ med vol.28 no.2 figure 3. effects of statin on major vascular events in patients with history of cardiovascular disease without chd.(20) comparison : statins versus placebo outcome : major vascular events study treatment control or (random), 95% ci or (random), 95% ci or subcategory n/n* n/n* hps 172/920 212/900 0.75; 0.60-0.94 sparcl 334/2,365 407/2,366 0.79; 0.68-0.93 total 3,285 3,266 0.78;0.68-0.88 total events: 506 (treatment), 619 (control) test for heterogeneity: χ2 = 0.18, d.f. = 1 (p = 0.68), i2 = 0% test for overall effect: z = 3.84 (p = 0.0001) 0.2 0.5 1 2 5 favors treatment favors control *n/n= number of subjects per event per total number of subjects 124 yenny pleiotropic effects of statins administration of statin is recommended, where the target of cholesterol reduction in patients with symptomatic chd or atherosclerosis is ldl cholesterol <100 mg/dl, whilst for high risk patients with multiple risk factors (class i, evidence level a) the target is ldl<70 mg/dl. secondly, patients with ischemic stroke or tia s u s p e c t e d o f h a v i n g b e e n c a u s e d b y atherosclerosis but without indications for statin u s a g e ( n o r m a l c h o l e s t e r o l l e v e l , w i t h o u t coronary heart disease comorbidity, or without e v i d e n c e o f a t h e r o s c l e r o s i s ) a r e s u i t a b l e candidates for statin therapy to reduce the risk of vascular events (class iia, evidence level b). based on the sparcl study results, the sparcl investigators suggest a modification of a b o v e m e n t i o n e d c l i n i c a l g u i d e l i n e s , b y specifically stating that stroke and tia shall be categorized as ‘coronary heart disease risk equivalent’ in connection with the indications for statin therapy.(4,20) c e s s at i o n o f p o s t i s c h e m i c s t r o k e s tat i n t h e r a p y a n d clinical outcomes the majority of post ischemic stroke patients are expected to derive benefits of longterm statin therapy. in clinical practice, however, the patient frequently discontinues the use of statins. a study conducted by colivicchi et al(23) had the objective of assessing the impact of cessation of post ischemic stroke statin therapy, with a follow-up period of 12 months. the study subjects comprised 631 stroke patients without clinical evidence of coronary heart disease. the results of this study indicated that within a period of 12 months a total of 246 patients (38.9%) stopped statin therapy, with a mean period of statin therapy cessation of 48.6±54.9 days. during the follow-up period 116 patients died f r o m c a r d i o v a s c u l a r ( 8 0 . 1 % ) , a n d n o n cardiovascular (6.9%) causes. this study also figure 4. effects of statin on repet stroke events in patients with history of cardiovascular disease without chd.(20) comparison : statins versus placebo outcome : recurrent study treatment control or (random), 95% ci or (random), 95% ci or subcategory n/n* n/n* hps 265/920 311/2,366 0.83; 070-0.99 sparcl 96/920 95/900 0.99; 0.73-1.33 total 3,285 3,266 0.87; 0.75-1.01 total events: 361 (treatment), 406 (control) test for heterogeneity: χ2 = 0.91, d.f. = 1 (p = 0.34), i2 = 0% test for overall effect: z = 1.80 (p = 0.07) 0.2 0.5 1 2 5 favors treatment favors control * n/n= number of subjects per event per total number of subjects 125 showed that the patients dying during follow-up were commonly those who were elderly, had d i a b e t e s m e l l i t u s , a n d w e r e o b e s e . discontinuation of statin therapy was said to be an independent predictor of all-cause mortality in the first year after cessation of statin therapy (hazard ratio: 2.78; 95% ci: 1.96-3.72; p: 0.003). a s t u d y w i t h s i m i l a r o b j e c t i v e s w a s performed by liao et al(24) in italy on 631 post ischemic stroke patients. inclusion criteria were patients without clinical and laboratory evidence of coronary heart disease, or other cardiac disorders, who had stopped using statin. in the f o l l o w u p p e r i o d 2 4 6 p a t i e n t s ( 3 8 . 9 % ) discontinued statin therapy, and 116 patients died within the first year after cessation of therapy. as in the previous study cessation of statin therapy was said to be an independent predictor of all-cause mortality within 12 months. it is to be regretted that there was no clear description of the causes of death, whether stroke or cardiovascular disease, as had been done in the study by colivicchi et al.(23) the study findings mention the occurrence of a rebound effect due to statin withdrawal, where the clinical effects were more severe than the clinical effects of patients not on statin therapy. on the basis of the hypothesis that statin has pleiotropic effects through upregulation of enos, it is believed that cessation of statin therapy causes a reduction in no release, due to rebound in the form of increased rho gtpase levels, which inhibit enos expression.(25) conclusions apart from the ability of statins to lower cholesterol levels, the pleiotropic effects of statins in enhancing the expression of enos result in the beneficial effects of these drugs when used in post acute ischemic stroke patients. on the basis of the results of a mini-metaanalysis on the sparcl and hps studies, statins showed an almost non-significant difference for repeat stroke events, but a significant difference for major vascular events. the studies indicate that the discontinuation of statin therapy in post acute ischemic stroke patients leads to an almost threefold increase in mortality in the first year. it has even been stated that cessation of statin treatment is an independent predictor of all-cause mortality in the first year. apparently the clinical recommendations issued by aha/asa regarding statin usage for stroke prevention in patients with a history of ischemic stroke or tia are still applicable. the results of the sparcl trial support the necessity of modifying the clinical guidelines, i.e. by specifically stating that stroke and tia shall be categorized as ‘coronary heart disease risk equivalent’ in connection with indications for statin treatment. references 1. lloyd-jones d, adam r, carnethon m, de simone g, ferguson b, flegal k, et al. heart disease and stroke statistics 2009 update: a report from the american heart association statistics committee and stroke statistics subcommitte. circulation 2009;119:e21-e181. 2. endo a, kuroda m, tsujita y. ml-236a, ml-236b, and ml-236c, new inhibitor of cholesterogenesis produced by penicillium citrinium. j antibiot 1976;29:1346-8. 3. cholesterol treatment triallist collaborators: efficacy and safety of cholesterol-lowering treatment: prospective meta-analysis of data from 90.056 participants in 14 randomized trials of statins. lancet 2005;366:1267-78. 4. sparcl investigators. high-dose atorvastatin after stroke or transient ischemic attack. n engl j med 2006;355:549-59. 5. takemoto m, liao jk. pleiotropic effects of 3hydroxy-3-methylglutaryl coenzyme a reductase inhibitors. arterioscler thromb vasc biol 2001;21:1721-9. 6. amarenco p, moskowitz ma. the dynamics of statins: from event prevention to neuroprotection. stroke 2006;37:294-6. univ med vol.28 no.2 126 yenny pleiotropic effects of statins 7. endres m, laufs u, liao jk, moskowitz ma. targeting enos for stroke protection. trends neurosci 2004;27:283-9. 8. khurana vg, meyer fb. translational paradigms in cerebrovascular gene transfer. j cereb blood flow metab 2003;23:1251-62. 9. pacher p, beckman js, liaudet l. nitric oxide and peroxynitrite in health disease. physiol rev 2007;87:315-424. 10. griffith o, stuehr d. no synthases: properties and catalytic mechanism. annu rev physiol 1995;57:707-36. 11. wang cy, liu py, liao jk. pleiotropic effects of statin therapy. trends mol med 2008;14:37-44. 12. rikitake y, liao jk. rho gtpases, statins, and nitric oxide. circ res 2005;97:1232-5. 13. zhang l, zhang zg, liu xs, hozeska-solgot a, chopp m. the pi3k/akt pathway mediates the neuroprotective effect of atorvastatin in extending trombolytic therapy after embolic stroke in the rat. arterioscler thromb vasc biol 2007;27:2470-5. 14. sironi l, cimino m, guerrini u, calcio am, lodetti b, asdente m, et al. treatment with statins after induction of focal ischemia in rats reduces the extent of brain damage. arterioscler thromb vasc biol 2003;23:322-7. 15. rikitake y, kim hh, huang z, seto m, yano k, asano t, et al. inhibition of rho kinase (rock) leads to increased cerebral blood flow and stroke protection. stroke 2005;36:2251-7. 16. sparcl investigators. design and baseline characteristics of the stroke prevention by aggressive reduction in cholesterol levels (sparcl). cerebrovasc dis 2003;16:389-95. 17. heart protection study collaborative group. mrc/ bhf heart protection study of cholesterol lowering with simvastatin in 20.536 high-risk individuals: a randomized placebo-controlled trial. lancet 2002;360:7-22. 18. heart protection study collaboration group. effect of cholesterol-lowering with simvastatin on stroke and other major vascular event in 20.536 people with cerebrovaskuler disease or other high-risk condition. lancet 2004;363:757-67. 19. coull bm. statin therapy after acute ischemic stroke in the heart protection study. stroke 2004;35:22334. 20. paciaroni m, hennerici m, agnelli g, bogousslavsky j. statins and stroke prevention. cerevrovasc dis 2007;24:170-82. 21. sacco rl, adam r, albers g, alberts mj, benavente o, furie k, et al. guideline for prevention of stroke in patients with ischemic stroke or transient ischemic attack: a statement for healthcare professionals from the american heart association/american stroke association council on stroke. circulation 2006;113:e409-49. 22. grundy sm, cleemon ji, merz nb, brewer hb, clark lt, hunninghake db, et al. iimplications of recent clinical trials for the national cholesterol education program adult treatment panel iii guidelines. circulation 2004;110:227-39. 23. colivicchi f, bassi a, santini m. caltagirone c. discontinuation of statin therapy and outcome clinical after ischemic stroke. stroke 2007;38:26527. 24. liao jk. is statin discontinuation an option in patients who have had a stroke? nat clin pract neurol 2008;4:18-9. 25. laufs u. suppression of endothelial nitric oxide production after withdrawal of statin treatment is mediated by negative feedback regulation of rho gtpase gene transcription. circulation 2000;102:3104-10. alvina 120 abstract *department of parasitology, medical faculty, trisakti university jakarta **department of parasitology, medical faculty, university of indonesia jakarta correspondence dr. hj. suriptiastuti, ms department of parasitology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone. 021-5672731 ext.2311 univ med 2011;30:120-5. intestinal parasites from fingernails of sidewalk food vendors suriptiastuti* and widiastuti s. manan** may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina intestinal infections with soil-transmitted helminths and protozoa are still prevalent in indonesia, particularly in urban communities. transmission of parasitic infections is effected directly or indirectly through objects contaminated with feces, including food, water, fingers and fingernails, indicating the importance of fecal-oral human-to-human transmission. sidewalk food vendors (sfvs) preparing food for their customers are a potential source of infections with many intestinal helminths and protozoa. compared to other parts of the hand, the area beneath fingernails harbors the most microorganisms and is most difficult to clean. the objective of this cross-sectional study was to determine the prevalence of intestinal parasites in fingernail dirt of sfvs and to identify the associated factors. this study involved 112 sfvs in the vicinity of hospital x in central jakarta, and used microscopic examination of sfv fingernail dirt for determining species prevalence of intestinal parasites. this study showed that 94 samples out of 112 (83.9%) were positive for intestinal parasites; 60 samples (63.8%) represented single infections and 34 (36.2%) mixed infections. ascaris lumbricoides eggs were found in 30 (26.8%) samples and giardia lamblia cysts in 12 (17.89%). the highest prevalence was found in subjects with primary school education, among whom 20 (30.8%) had single infections of a. lumbricoides and 16 (24.6%) mixed infections with a. lumbricoides and trichuris trichiura. in conclusion, prevalence of intestinal parasites in sfv fingernail dirt is extremely high, with the highest prevalence among less educated sfvs. it is recommended to provide health education and training to all sfvs. key words: parasites, intestinal, fingernails, food vendors introduction in tropical countries, parasitic infections remain a serious public health problem, in view of the high prevalence of protozoan and helminth infections. (1) the world health organization has estimated that helminth infections affect around 2 billion people, among whom 5-10% are children under 24 months of age. (2,3) helminth infections may lead to 121 univ med vol.30 no.2 malnutrition, anemia, and disturbed appetite.(4,5) and may ultimately result in retarded physical and cognitive development in children.(6) a study in north sumatra reported a prevalence of ascariasis of 76.7% among primary school pupils.(7) in jakarta, a study conducted among f e m a l e w o r k e r s s h o w e d a p r e v a l e n c e o f intestinal parasitic infections of 70.47%, comprising ascaris lumbricoides (38.13%), tr i c h u r i s t r i c h i u r a ( 2 8 . 1 3 % ) , n e c a t o r americanus and ancylostoma duodenale mixed i n f e c t i o n ( 1 3 . 5 9 % ) , a n d e n t e ro b i u s vermicularis (4.84%).(8) the factors associated with intestinal parasitic infections in tropical countries include poverty, illiteracy, poor hygiene, poorly organized clean water supply, and hot and humid e n v i r o n m e n t a l c o n d i t i o n s . p r o t o z o a a n d helminthic parasites are responsible for the prevalence of diseases capable of affecting an i n d i v i d u a l ’s h e a l t h . i n t e s t i n a l p a r a s i t i c infections (ipi) are global health problems c a u s i n g c l i n i c a l i l l n e s s i n 4 5 0 m i l l i o n inhabitants, particularly children and women of reproductive age in developing countries.(9) ipi, and helminths in particular, are associated with increased risks for nutritional anemia, proteinenergy malnutrition, growth retardation in children, poor increase in body weight in pregnancy, intrauterine growth retardation, and low birth weight.(10,11) children infected with soil-transmitted helminths (sths) have poor educational level and performance at school and a high level of truancy, thus impacting on their future earnings and productivity.(12,13) the most important sths are the common roundworm (a s c a r i s l u m b r i c o i d e s ), t h e w h i p w o r m (trichuris trichiura) and the hookworms (n e c a t o r a m e r i c a n u s a n d a n c y l o s t o m a duodenale), which currently are a group of neglected tropical diseases.(14) food vendors with poor personal hygiene could be potential sources of infections of many intestinal helminths and protozoa. food vendors who harbor and excrete intestinal parasites may cause fecal contamination of foods via their fingers and their preparation of foods, and finally to healthy individuals. compared to other parts of the hand, the area beneath fingernails harbors the most microorganisms and is most difficult to clean.(15) the presence of sidewalk food vendors (sfvs) is essential for the middleclass society to satisfy their daily food needs away from home. the types of food sold by sfvs include meatballs, chicken noodles, fried noodles, gado-gado (jakarta salad), dumplings, and chicken porridge. these traders of foods and beverages are much in demand at lunchtime. however, sfvs are a source of infectious parasitic diseases if hygiene is not maintained. intestinal parasites are found everywhere, such as in fingernail dirt, in areas abounding in flies and cockroaches, and in contaminated foods. the aim of the present study was to determine the prevalence of intestinal parasites from fingernail dirt and to identify the associated factors among sfvs. methods research design this study used a cross-sectional design and was conducted from 15 june to 15 august 2010. study subjects a total of 112 sfvs in the vicinity of a large hospital in central jakarta participated in this study. the inclusion criteria were: male and female sfvs selling foods to employees and patients of the hospital, and having dirty (blackish) fingernails. informed consent was obtained verbally after informing the sfvs of the purpose of the study. the study location was around abovementioned hospital in central jakarta. data collecting a structured questionnaire was used for collecting information on age, sex, education, types of food sold, and hygienic status of each sfv. fingernail clippings were collected from 122 both hands of each subject using clean nail clippers and placed in labeled containers. laboratory investigation the following procedure was used for preparing wet mounts of the nail clippings: initially the clippings were immersed in 10% potassium hydroxide solution for 24 hours, and subsequently centrifuged at 2500 rpm. the supernatant and sediment of each specimen were then stained with eosin and lugol, and examined by light microscopy at low to medium magnifications (100-400x) for species identification. helminths were identified from their characteristic egg morphology, and protozoa from their cysts and/or vegetative f o r m s . tr i c h r o m e s t a i n i n g w a s u s e d f o r preserving positive preparations for future reference. the microscopic work was done in the department of parasitology, faculty of medicine, university of indonesia, jakarta. data analysis the collected data were analyzed using spss version 15.0. the prevalence of parasites was presented as descriptive statistics, while the relationship between several variables (age, gender, educational level) and presence of parasites was determined by the chi-square test. the level of significance used was 0.05. results a total of 112 sfvs were recruited for this study, consisting of 62 (55.4%) male and 50 (44.6%) female sfvs. most sfvs (65.2%) went to primary school, and only 1.8% had tertiary education. the subjects’ age ranged from 12 to 40 years, with mean age of 23.7 ± 8.6 years (table 1). with regard to the types of foods for sale, 20.5% of sfvs prepared meatballs, 15.2% sold chiken noodles, and only 3.6% of sfvs had fried foods for sale. gado-gado (a kind of salad with peanut sauce) was sold by 7.1% of sfvs and drinks were sold by 5.4%. a total of 94 n a i l s a m p l e s ( 8 3 . 9 % ) w e r e p o s i t i v e f o r intestinal parasites, while 18 samples (16.1%) were negative. of the positive samples, 60 (63.8%) had a single parasite species, while 34 (36.2%) had two or more species. ascaris lumbricoides eggs were found in 30 (26.8%) samples and giardia lamblia cysts in 12 (17.89%). (table 2) table 1. distribution of respondent characteristics (n=112) * mean ± sd suriptiastuti, manan intestinal parasites from fingernails table 2. prevalence of intestinal parasites in fingernail samples of food vendors (n=94) 123 univ med vol.30 no.2 gender and age of the subjects did not show any differences in parasite prevalence between males and females or between age groups (p=0.116 and p=0.398). subjects with primary school education significantly had the highest prevalence of parasites in fingernail dirt, as compared with subjects at other educational levels (junior high school, senior high school, academy) with p=0.005 (table 3). in the subjects with primary school education, the highest prevalence was 20 (30.8%) for a single parasite species, namely a. lumbricoides, while for the combined presence of a.lumbricoides and t. trichiura it was 16 (24.6%) (data not shown). discussion in the present study 94 (83.9%) of the 112 subjects was positive for enteropathogenic parasites, indicating the poor health status and hygiene of the sfvs. similar results were found in a study in abeokuta (nigeria) where the prevalence of parasitic infections among street food vendors was 98.7%.(16) in contrast, a study on fingernails from 127 ethiopian food-handlers showed a prevalence of intestinal parasites of 29.1%.(17) sfvs commonly do not have access to any toilet facilities, even when established semi-permanently at their locations. in addition, public toilets commonly have an inadequate supply of clean water, and therefore sfvs c a n n o t w a s h t h e i r h a n d s p r o p e r l y a f t e r defecation, assuming they were highly aware of the importance of personal hygiene. the importance of food-handlers in the transmission of parasitic diseases has been stressed by many investigators worldwide.(18-20) the control and prevention of intestinal infections, either by parasites or bacteria, requires understanding of the epidemiological aspects of the problem, for guidance in the design of practical and economic control and prevention measures. such a high prevalence of intestinal parasites is largely due to poor personal hygiene and environmental sanitation, lack of supply of safe water, poverty, ignorance of health promotion practices, and impoverished health services. most of the common intestinal parasitic infections of man are fecal borne infections and the transmission occurs either directly hand-tomouth or indirectly through food and water. their transmission within the community is predominantly related to human habits with regards to eating, defecation, personal hygiene, cleanliness and level of education. their prevalence in the community can be used as an i n d i c a t o r o f t h e l i v i n g c o n d i t i o n s , a n d environmental sanitation, as well as the socioeconomic status of the community. the table 3. presence of parasites by gender, age, and educational level of subjects (n=112) chi-square test results; * significant 124 ingestion of parasitic cysts frequently occurs through contaminated fingers and there is an a s s o c i a t i o n b e t w e e n t h e p r e s e n c e o f enteroparasites under the fingernails of sfvs and poor personal hygiene. our study showed a high prevalence of intestinal parasites in fingernail dirt specimens of sfvs. to effectively prevent the transmission of pathogens from sfvs to consumers via the foods prepared by these sfvs requires good personal hygiene and hygienic food-handling practices. therefore it is recommended that education and training in good hygienic practices should be provided to all sfvs. d e s p i t e t h e i r h i g h p r e v a l e n c e i n developing countries, persons with intestinal parasites have very low morbidity and mortality rates, thus intestinal parasites are commonly viewed as low-priority health problems. h o w e v e r, a s c a r i a s i s c a u s e d i n t e s t i n a l obstruction in 5-35% of pediatric cases in a comparison of studies conducted throughout the tropics. the intestinal obstruction by ascaris worms is often fatal. in addition, hookworm infection can cause iron deficiency anemia; and t r i c h u r i a s i s i s a s s o c i a t e d w i t h c h r o n i c dysentery and rectal prolapse. amebiasis can r e s u l t i n d y s e n t e r y a n d e x t r a i n t e s t i n a l complications, while giardiasis is associated with acute diarrhea, steatorrhea and lactose intolerance.(21,22) our study indicates that there were no s i g n i f i c a n t d i ff e r e n c e s i n p r e v a l e n c e o f intestinal parasites in sfv fingernail dirt between male and female sfvs. however, studies from iran and the philippines obtained different results. in iran the prevalence of intestinal parasites was higher in men than in women,(23) while in the philippines the reverse was true.(24) these different results may possibly be explained by differences in environmental factors, where philippine women do more farm work than their iranian counterparts, while our study pertains only to sfvs and not the whole population as in iran and the philippines. the results of our study showed that sfvs with low educational level had a significantly higher prevalence of helminths under their fingernails, as compared to the better educated sfvs. similar results were obtained in an nigerian study on prevalence of helminths in stools, which showed that the highest prevalence was in subjects with low education.(25) in this connection it should be conceded that an individual’s educational level greatly influences his/her health related behavior, where poor education prevents a person from realizing the importance of personal hygiene. one limitation of the study was the relatively small sample size of sfvs, thus precluding the use of advanced analysis to make associations. conclusions in this study there was a high prevalence o f i n t e s t i n a l p a r a s i t e s i n f i n g e r n a i l d i r t specimens of sidewalk food vendors. low educational level was associated with high prevalence of intestinal parasites. an effective means of preventing the transmission of pathogens from sidewalk food vendors as foodhandlers via food to consumers is strict adherence to good personal hygiene and to hygienic food-handling practices. acknowledgments the investigators thank the sidewalk food vendors who were willing to participate in this study. last but not least, we express our gratitude to colleagues at the department of parasitology, faculty of medicine, trisakti university and the department of parasitology, faculty of medicine, university of indonesia for their support and assistance. references 1. sehgal r, reddya gv, verweijb jj, atluri v, rao avs. prevalence of intestinal parasitic infections among school children and pregnant women in a suriptiastuti, manan intestinal parasites from fingernails 125 univ med vol.30 no.2 low socio-economic area, chandigarh, north india. rif 2010;1:100-3. 2. world health organization. the millenium development goals. the evidence is in: deworming helps meet the millenium development goals. geneva: world health organization;2005. 3. world health organization. report of the who: informal consultation on the use of praziquantel during pregnancy/lactation and albendazole/ mebendazole in children under 24 months. geneva: world health organization;2002. 4. world health organization. action against worms: deworming young children. geneva: world health organization;2007. 5. stoltzfus rj, chway hm, montresor a, tielsch jm, jape jk, albonico m, et al. low dose daily iron supplementation improves iron status and appetite but not anemia, whereas quarterly anthelminthic treatment improves growth, appetite and anemia in zanzibari preschool children. j nutr 2004;134:348–56. 6. stoltzfus rj, kvalsvig jd, chwaya hm, montresor a, albonico m, tielsch jm, et al. effects of iron supplementation and anthelmintic treatment on motor and language development of preschool children in zanzibar: double blind, placebo controlled study. bmj 2001;323:1389– 93. 7. putra ds, dalimunthe w, lubis m, pasaribu s, lubis c. the efficacy of single dose albendazole for the treatment of ascariasis. paediatr indones 2005;45:118-22. 8. suriptiastuti. some epidemiological aspects of intestinal parasites in women workers before going abroad. trop biomed 2006;23:103–8. 9. quihui l, valencia me, crompton dwt, phillips s, hagan p, morales g, et al. role of the employment status and education of mothers in the prevalence of intestinal parasitic infections in mexican rural schoolchildren. bmc public health 2006;6:225. doi.10.1187/1471-2458-6225. 10. sackey me, weigel mm, armijos rx. predictors and nutritional consequences of intestinal parasitic infections in rural ecuadorian children. j trop pediatr 2003;49:17-23. 11. rodriguez-morales aj, barbella ra, case c, arria m, ravelo m, perez h, et al. intestinal parasitic infections among pregnant women in venezuela. infect dis obstet gynecol 2006; 23125. doi: 10.1155/idog/2006/23125. 12. miguel e, kremer m. worms: identifying impacts on education and health in the presence of treatment externalities. econometrica 2004;72: 159–217. 13. hotez pj, fenwick a, savioli l, molyneux dh. rescuing the bottom billion through control of neglected tropical diseases. lancet 2009;373: 1570–5. 14. feasey n, wansbrough-jones m, mabey dcw, solomon aw. neglected tropical diseases. br med bull 2010;93:179–200. 15. lin cm, wu fm, kim hk, doyle mp, michael bs, williams lk. a comparison of hand washing techniques to remove escherichia coli and caliciviruses under natural or artificial fingernails. j food prot 2003;66:2296-301. 16. idowu oa, rowland sa. oral fecal parasites and personal hygiene of food handlers in abeokuta, nigeria. afr health sci 2006;6:160-4. 17. andargie g, kassu a, moges f, tiruneh m, huruy k. prevalence of bacteria and intestinal parasites among food-handlers in gondar town, northwest ethiopia. j health popul nutr 2008;26: 451-5. 18. wiwanitkit v, assawawitoontip s. high prevalence of positive culture and parasites in stool samples of food handlers in a thai hospital setting. med gen med 2002;4:8. 19. hundy rl, cameron s. an outbreak of infections with a new salmonella phage type linked to a symptomatic food handler. commun dis intell 2002;26:562-7. 20. feglo pk, frimpong eh, essel-ahun m. salmonellae carrier status of food vendors in kumasi, ghana. east afr med j 2004;81:35861. 21. bethony j, brooker s, albonico m, geiger sm, loukas a, diemert d, et al. soil-transmitted helminth infections: ascariasis,trichuriasis, and hookworm. lancet 2006;367:1521–32. 22. world health organization. world health report 2003 annex: burden of disease in dalys by cause, sex and mortality stratum in who regions, estimates for 2002. geneva: world health organization;2003. 23. nasiri v, esmailnia k, karimi g, nasiri m, akhavan o. intestinal parasitic infections among inhabitants of karaj city, tehran province, iran in 2006-2008. korean j parasitol 2009;47:2658. 24. kim bj, ock ms, chung di, yong ts, lee kj. the intestinal parasite infection status of inhabitants in the roxas city, the philippines. korean j parasitol 2003;41:113-5. 25. amuta eu, houmsou rs, mker sd. impact of socio-demographic and economouc factors on the prevalence of intestinal parasites among female gender in makurdi, benue state-nigeria. int j acad res 2010;1:56-60. atik1 92 musculoskeletal pain and posture decrease step length in young adults may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina maria regina rachmawati*a, diana samara*, purnamawati tjhin*, magdalena wartono*, and yefta bastian* *department of anatomy/ physical and rehabilitation medicine mecical faculty, trisakti university, jakarta correspondence adr. maria regina rachmawati, sprm department of anatomy/ physical and rehabilitation medicine, mecical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11410 telp. 021-5672731 ex.2101 email: rachmawati@trisakti.ac.id univ med 2009;28:92-9 abstract pain of the musculoskeletal system, especially low back pain, is one of the most frequent problems with a high risk of disability. the aim of this research study was to determine the existence of an association between low back pain on one hand, posture and step length on the other. this cross-sectional study was conducted on 77 healthy young adult subjects. step length was measured with the biodex gait trainer 2 (230 vac). the study results indicate that 62.3% of the young adult subjects had suffered from low back pain. there was no significant association between gender and pain. in male subjects no significant association was found between pain on one hand and mean difference in step length and posture on the other. however, in female subjects with abnormal posture, there was a highly significant difference in left step length between subjects with back pain and those without (p=0.007). the results of a multiple regression analysis indicate that posture has the greatest influence on left step length (b=4.135; 95% confidence interval 0.292-7.977). it is recommended that in the examination of low back pain an assessment be made of posture, step length and difference in step lengths. keywords: musculoskeletal pain, posture, step length, young adults introduction pain is one of the most frequent health problems that causes an individual to consult a physician. the most prevalent pain suffered by an individual is low back pain (lbp). inadequate management of lbp may result in disability and impact on socio-economic aspects, both due to high costs of medical care and to indirect costs, such as loss of working hours and forced early retirement.(1,2) the lower back is that area of the spinal column from below the twelfth rib to the inferior gluteal fold, anatomically composed of the lumbal vertebrae associated with the inferior 93 univ med vol.28 no.2 extremities, comprising the pelvis and the bones of the extremities. based on this structure, the biomechanical system of the spinal column forms the axis of the biomechanical system of the inferior extremities, and a disorder of one of these systems may affect the other as well.(3-6) walking is an activity of the inferior extremities that is essential to human activities, as a result of complex actions of the neuromuscular and musculoskeletal systems on the spinal column and inferior extremities. the assessment of gait pattern may be performed by measuring several components of gait, namely step length, cycle length, and walking speed, which may also be termed the vital signs of gait.(7) a disturbance of gait may ultimately lead to disability. the spinal column and inferior extremities constitute one interrelated biomechanical system. thus a disorder of the spinal column, such as postural abnormalities (scoliosis, s posture, kyphosis, hyperlordosis), may presumably affect the gait pattern, due to a shift of the gravitational axis in relation to the spinal column, causing the body to find a new equilibrium, e.g. by modifying the gait pattern. pain in the region of the spinal column as well as in the inferior extremities leads t o a c o m p e n s a t o r y m o v e m e n t t h a t m a y presumably also affect gait pattern, among others by asymmetry of right and left step lengths. asymmetrical step lengths are thought to result i n d i s e q u i l i b r i u m o f t h e a c t i o n s o f t h e musculoskeletal biomechanical system of the spinal column and inferior extremities, such that one component is overworked, leading to the c o m p l a i n t o f p a i n . ( 8 11 ) o n e s t u d y h a s demonstrated that in patients with chronic low back pain a decrease in lumbar curvature and anteroposterior movement occurs on prolonged s t a n d i n g . t h i s m a y p o s s i b l y b e d u e t o a disturbance of the sensorimotor system causing a decreased adaptibility on prolonged standing, in comparison with normal individuals, thus increasing the risk of recurrence of low back pain.(12,13) walking is frequently promoted as a safe exercise treatment for back pain patients. the purpose of this investigation was to determine the existence in young adults of an association between musculoskeletal pain on one hand posture and step length on the other. methods research design this cross-sectional study was used to answer the research question and attain the goal of the study. research subjects the subjects for this study were students of productive age enrolled at a private medical faculty in jakarta. at the start of the study, the students were informed about the background and goal of the study, and those willing to participate in the study by signing an informed consent form were recruited for this study. assessment complaints of musculoskeletal pain in the back and/or lower extremities were assessed t h r o u g h i n t e r v i e w s . t h e q u a l i t y o f musculoskeletal pain was scored according to the visual analogue scale (vas). the subjects graded their symptom of low back pain on a 10-cm scale, anchored with the descriptors ‘no pain’ at one end and ‘pain as bad as it could possibly be’ at the other end. severe pain was defined as vas 75–100.(14) assessment of posture was performed by physical examination, in which the subjects were asked to remove clothing and footwear and stand upright with their backs to the examiner. a plumb line was hung from the level of the spinous process of the seventh cervical vertebra without touching the subject’s skin, and used for assessment of the alignment of the subject’s spinal column. the examination also involved a 94 rachmawati, samara, tjhin, et al musculoskeletal pain and step determination of a difference in length between the right and left legs, which is deemed capable of causing postural abnormalities. right and left step lengths (in centimeters) were measured using the biodex gait trainer 2 (230 vac). measurement of step length was performed while the subjects were walking for 5 minutes at their normal daily walking speed (figure 1). statistical analysis the study data were analyzed using the statistical package for social sciences (spss) version 10.0 for windows (spss inc., chicago, il, usa). descriptive statistical methods were u s e d t o e v a l u a t e s o c i o d e m o g r a p h i c characteristics. a p-value below 0.05 was considered to be statistically significant. the independent samples t-test and chi-square test were used to compare groups for the parameter of pain. results of the 126 subjects examined at the start of the study, comprising 68 (54%) females and 58 (46%) males, only 77 (64%) completed the study. this was due to the fact that the step length measuring instrument was kept in a hospital figure 1. biodex gait trainer 2 (230 vac) 95 univ med vol.28 no.2 table 1. distribution of relevant subject characteristics table 2. distribution of back pain by posture on examination of posture 40 (51.9%) subjects had a normal posture, whilst the remaining 37 (48.1%) had postural abnormalities, consisting of postural kyphosis in 2 (5.4%) subjects, hyperlordosis in 1 (2.7%), postural scoliosis in 28 (75.6%), and structural scoliosis in 6 (16.3%) subjects. the results of step length measurement using the biodex gait trainer were as follows: mean right step length = 42.8 cm (sd 6.1); mean left step length = 40.0 cm (sd 8.5); mean variation in right step length = 23% (sd 12); and mean variation in left step length = 25% (sd 16). subjects with equal right and left step lengths were only 6 (7.8%) in number, whereas 92.2% had unequal right and left step lengths. mean difference in right and left step lengths was 4.3 cm (sd 3.2). there was no significant difference between subjects with normal or abnormal posture and the occurrence of pain (p=0.331) (table 2). table 2 shows that the proportion of back pain sufferers was higher among subjects with normal posture (67.5), compared with subjects with a b n o r m a l p o s t u r e ( 3 2 . 5 % ) , a l t h o u g h t h e difference in proportions was not statistically significant. in male subjects there was no significant difference in right and left step lengths among subjects with back pain and among those without back pain (table 3). in male subjects with back pain the difference in right and left step lengths was smaller than that in male subjects without back pain, although the difference was not statistically significant. situated at a considerable distance from the location of the basic data collection. the analyzed study subjects consisted of 28 (36.4%) males and 49 (63.6%) females, with mean age of 22.2 years (sd=0.7). (table 1) from the medical history a total of 48 (62.3%) subjects was found to have had back pain with the most frequent duration of onset of < 1 month (45.4%). 96 in female subjects no significant difference was found in right or left step lengths between those with or without back pain (table 4). female subjects with back pain had a larger difference in right and left step lengths compared with those without back pain, but the difference was not statistically significant. the results of stratification analysis of back pain and step length by gender and posture is presented in tables 5 and 6. table 5 indicates that, both in male subjects with normal posture and those with abnormal posture, no significant difference was found in right or left step lenghts between subjects with or without back pain. in female subjects with normal posture there was no significant difference in right or left step lengths between subjects with or without back pain. however, in female subjects with abnormal posture a considerably significant difference was found in left step lengths between subjects with back pain and those without (table 6). left step length in female subjects with abnormal posture was significantly shorter in those with back pain, c o m p a r e d w i t h t h o s e w i t h o u t b a c k p a i n (p=0.007). rachmawati, samara, tjhin, et al musculoskeletal pain and step table 3. differences in step lengths by back pain in male subjects table 4. differences in step lenghts by back pain in female subjects table 5. differences in step lenghts by posture and back pain in male subjects 97 univ med vol.28 no.2 the results of a multiple regression analysis showed that in subjects with abnormal posture left step length was significantly shorter by 4.135 cm, compared with those subjects with normal posture (b = 4.135; 95% confidence interval 0.292 -7.977) (table 7). gender and back pain had no significant influence on left step length. in subjects with abnormal posture the right step length was considerably shorter by 1.469 cm compared with those subjects with normal posture, but the difference was not statistically significant (b = 1.469; 95% confidence interval – 1.322 – 4.259) (table 8). gender and back pain had no significant influence on right step length. discussion the results of this study demonstrate that the prevalence of low back pain in young adults was 62.3%. this figure is at variance with that from a propspective study in young danish twins s h o w i n g a p r e v a l e n c e o f l b p o f 3 5 % . ( 1 5 ) however, a finnish study comprising 7,333 male c o n s c r i p t s ( m e d i a n a g e 1 9 ) , y i e l d e d a c o n s i d e r a b l y l o w e r p r e v a l e n c e o f l b p o f 12.7%.(16) there are several factors associated with lbp in young adults, such as smoking, which has been shown to be significantly associated with the occurrence of lbp in young adults.(17) in the present study no association was f o u n d b e t w e e n b a c k p a i n a n d p o s t u r a l abnormalities. a number of causal mechanisms have been proposed for adolescent back pain, including rapid bony growth, inadequate fit of furniture to body size, poor muscle strength, poor motor control, balance and coordination, and poor posture.(18-20) it is imperative that a study be conducted on the occurrence of a step length difference table 6. differences in step length by. posture and back pain in female subjects table 8. multiple regression analysis of right step length justified by gender, posture and back pain table 7. multiple regression analysis of left step length justified by gender, posture and back pain 98 rachmawati, samara, tjhin, et al musculoskeletal pain and step between a study group with back pain and one without, and that a stratification analysis be performed based on posture and gender. the present study shows that in female subjects with abormal posture, a significant difference in left step length was found between subjects with and w i t h o u t b a c k p a i n . f e m a l e s u b j e c t s w i t h abnormal posture had a shorter left step length when also having back pain. previous studies have yielded evidence that posture and pelvic shape play a significant role in determining the gait pattern of an individual. this is the result of a shift in the center of gravity in persons with abnormal posture, which affects the mechanism of action of the muscles and their surrounding supporting tissues.(3,4) according to previous studies, in the elderly age group a significant association has been found between back pain and step length of the inferior extremities, which is due to abnormal posture.(5) however, the present study shows that left step length is significantly shorter in female subjects with abnormal posture and back pain. currently, whether abnormal postural coordination can cause or contribute to lbp is not known. some authors have hypothesized that p o o r c o o r d i n a t i o n c o u l d l e a d t o p a i n , f o r instance, by producing greater loading within the trunk.(21,22) in support of this hypothesis, people with lbp have greater muscular activity and greater spinal loading than people without l b p. ( 2 3 ) a l t e r n a t i v e l y, a b n o r m a l p o s t u r a l coordination could result from the pain itself (eg, splinting to avoid pain). the results of a multiple linear regression analysis showed that posture had the greatest influence on mean left step length (b=4.135: 95% ik 0.292-7.977), which is consistent with previous study results indicating that posture and p e l v i c s h a p e p l a y a n i m p o r t a n t r o l e i n determining an individual’s gait pattern, due to a shift in the gravitational center of persons with abnormal posture, thus affecting the mechanism of action of the muscles and surrounding supporting tissues.(6,7) the present study did not find a significant association between abnormal posture and back pain, which is at variance with previous study results in the elderly, namely the finding of a significant difference between back pain and/or pain in inferior extremities on one hand, and abnormal posture on the other.(8,24) the differing results may be due to a difference in age of the s u b j e c t s , a s i n y o u n g s u b j e c t s t h e musculoskeletal system is still flexible and has not undergone degenerative processes, thus facilitating adjustment of the biomechanical system to the existing structural abnormalities. conclusions the mayority of young adult subjects had suffered from back pain. right and left step lengths did not differ in subjects with or without back pain. female subjects with abnormal posture and back pain had a shorter left step length. however, it is unclear whether postural abnormalities are caused by asymmetry in step lengths or whether asymmetrical step lengths are caused by abnormal posture. in the management of back pain, attention should be paid to improvement of posture and gait pattern. young adults should be screened for postural abnormalities and gait pattern in order to lower the risk of occurrence of back pain in old age. acknowledgements the investigators wish to thank our students who were willing to participate in this study. we also express our gratitude to the dean and vicedeans of the medical faculty, trisakti university for the funding of this study. 99 univ med vol.28 no.2 references 1. ehrlich ge. low back pain. bull world health organ 2003;81:671-6. 2. woolf ad, pfleger b. burden of major musculoskeletal conditions. bull world health organ 2003;81:646-56. 3. mitchell t, o’sullivan pb, burnett af, straker l, smith a. regional differences in lumbar spinal posture and the influence of low back pain. bmc musculoskelet disord 2008;9:152. 4. scannell j, mcgill s. lumbar posture – should it, and can it, be modified? a study of passive tissue stiffness and lumbar position during activities of daily living. phys ther 2003;83:907-17. 5. smith a, o’sullivan p, straker l. classification of sagittal thoraco-lumbo-pelvic alignment of the adolescent spine in standing and its relationship to low back pain. spine 2008;33:2101-7. 6. cholewicki j, vanvliet jj. relative contribution of trunk muscles to the stability of the lumbar spine during isometric exertions. clin biomech 2002;17: 99-105. 7. al-obaidi s, wall jc, al-yaqoub a, al-ghanim m. basic gait parameters: a comparison of reference data for normal subjects 20 to 29 years of age from kuwait and scandinavia. j rehabilit res develop 2003;40:361–6. 8. weiner dk, sakamoto s, perera s, breuer p. chronic low back pain in older adults: prevalence, reliability, and validity of physical examination findings. j am geriatr soc 2006;54:11–20. 9. sweeting k, mock m. gait and posture : assessment in general practice. aust fam physician 2007;36: 398-405. 10. adam ma, dolan p. spine biomechanics. j biomech 2005;38:1972-83. 11. curran sa, dananberg hj. future of gait analysis: a podiatric medical perspective. j am pediatr med assoc 2005;95:130–42. 12. mok nw, brauer sg, hodges pw. hip strategy for balance control in quiet standing is reduced in people with low back pain. spine 2008;29:107–12. 13. lafond d, champagne a, descarreaux m, dubois jd, prado jm, duarte m. postural control during prolonged standing in persons with chronic low back pain. gait & posture 2009;29:421-7. 14. löfvander m, taloyan m. pain intensity and severe pain in young immigrant patients with long-standing back pain. eur spine j 2008;17:89–96. 15. hestbaek l, leboeuf-y c, kyvik ko. are lifestylefactors in adolescence predictors for adult low back pain? a cross-sectional and prospective study of young twins. bmc musculoskelet disord 2006;7: 27-34. 16. mattila, vm, sahi t, jormanainen v, pihlajamäki h. low back pain and its risk indicators: a survey of 7,040 finnish male conscripts. eur spine j 2008; 17:64-9. 17. power c, frank j, hertzman c, schierhout g, li l. predictors of low back pain onset in a prospective british study. am j public health 2001;91:1671-8. 18. louw qa, morris ld, grimmer-somers ka. the prevalence of low back pain in africa: a systematic review. bmc musculoskelet disord 2007;8:105. 19. bakker ew, verhagen ap, lucas c, koning hj, koes bw. spinal mechanical load: a predictor of persistent low back pain? a prospective cohort study. eur spine j 2007;16:933-41. 20. el-metwally a, salminen jj, auvinen a, macfarlane g, mikkelsson m. risk factors for development of non-specific musculoskeletal pain in preteens and early adolescents: a prospective 1-year follow-up study. bmc musculoskelet disord 2007;8:46. 21. hodges pw. the role of the motor system in spinal pain: implications for rehabilitation of the athlete following lower back pain. j sci med sport 2000; 3:243–53. 22. newcomer kl, jacobson td, gabriel da. muscle activation patterns in subjects with and without low back pain. arch phys med rehabil 2002;83:816– 21. 23. marras ws, davis kg, ferguson sa. spine loading characteristics of patients with low back pain compared with asymptomatic individuals. spine 2001;26:2566–74. 24. rachmawati mr, tjhin p, wartono p. musculoskeletal pain and its relationship with functional physical ability in the elderly. univ med 2006;25:179-86. andi1 8 relationship between noise and job stress at a private thread spinning company january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina andi mursali,*a endang basuki,* and suryo dharmono** *department of community medicine, faculty of medicine, university of indonesia, jakarta **department of psychiatric, faculty of medicine, university of indonesia/ central national general hospital dr. cipto mangunkusumo, jakarta correspondence adr. andi mursali, m.kes department of community medicine, faculty of medicine, university of indonesia, jakarta email: dr_andimursali@yahoo.com univ med 2009; 28: 8-16 abstract noise exposure is one of the major occupational hazards in many places, and has several health effects, including hearing loss and psychological effects such as sleep disturbances and mental stress. the objective of this research was to determine the relationship between noise and stress and other risk factors that affect job stress. a cross-sectional comparative design involved a total of 326 workers consisting of 220 workers in high noise conditions and 106 workers in low noise conditions. the data were collected directly from the respondents by filling out the questionnaire on worker characteristics, the stress diagnostic questionnaire, and the symptom checklist 90, through discussions with company management, and by measurement of noise levels. the job stress prevalence in workers in high noise conditions was 55% and in those in low noise conditions 24.5%. the results showed that risk factors such as gender of workers, noise conditions, habitual use of ear plugs, shift work, and work stressors had a significant relationship with job stress. multivariate analysis indicated that the habit of using earplugs was a dominant risk factor causing job stress. the prevalence of job stress in high noise conditions was higher than that in workers in low noise conditions. noise conditions have a significant relationship with job stress (or=2.46; 95% ci 1.33 4.55). workers in high noise conditions who did not always use or never had used ear plugs had a higher chance of experiencing stress than those in low noise conditions who did not need ear plugs (or=21.76; 95% ci 8.09 58.52). this study supported that noise exerts its health effect via stress, since noise often raise stress in various ways. keywords: noise, job stress, ear plugs, workers, thread spinning introduction stress can be defined in several ways, and it is important to use an approach that covers the different concepts. one definition of stress at work (job stress, work-related stress) is “the harmful physical and emotional responses that occur when the requirements of the job do not match the capabilities, resources, or needs of the worker”.(1,2) normally the worker should 9 univ med vol.28 no.1 make a successful adaptation to the work environment. the purpose of this adaptation is to minimize or eliminate discomfort caused by environmental stressors. successful adaptation to a stressor will not cause problems, thus the resulting stress is an agreeable stress called eustress or positive stress. if the adaptation is incomplete or if the stressor is stronger than the defense mechanism of the individual, then the result will be disagreeable or uncomfortable stress. this condition is frequently called distress or negative stress. (3) negative stress (in the following discussion to be referred to simply as ‘ s t r e s s ’ ) m a y i n f l u e n c e h e a l t h a n d c a u s e numerous health problems, including anxiety, arthritis, cancer, depression, heart disease, high blood pressure, and insomnia. in severe cases of job stress occurring over a prolonged period of time, psychological problems and psychiatric disorders may develop.(1) stress may occur in various types of occupation, and european studies have found that the prevalence of stress v a r i e s b e t w e e n d i ff e r e n t o c c u p a t i o n s . professionals reported the highest level of stress (40%), as compared with 17% in elementary occupations.(4) two british studies, one of head teachers and one of police officers, found the prevalence of self-reported work-related stress t o b e 4 3 % a n d 4 1 % , r e s p e c t i v e l y. ( 5 , 6 ) t h e prevalence of job stress in the production section of a japanese machinery factory was 57.7%.(7) noise is one of the intrinsic occupational factors that may cause job stress. several studies indicate that occupational noise exposure is correlated with blood pressure changes and cardiovascular disease.(8,9) a cross-sectional study showed that there was no significant a s s o c i a t i o n b e t w e e n n o i s e i n t e n s i t y a n d increased noise induced heaving loss.(10) noise plays a substantial role in stress causation in workers. this is due to the fact that certain individuals are unduly sensitive to noise in comparison to others. ivancevich is of the opinion that repeated excessive noise (around 80db) for a prolonged period of time may induce s t r e s s . ( 11 ) h i g h i n t e n s i t y n o i s e m a y c a u s e headaches, drowsiness, high blood pressure, occupational stress, muscle fatigue, and lack of concentration. exposure to excessive noise may reduce the work ethos and result in increased a b s e n t e e i s m , t h u s d e c r e a s i n g w o r k productivity.(12) japanese studies in male workers aged 40 to 59 years indicate that around 35% experience noise-related stress.(13) exposure to moderate levels of noise may also result in psychological disorders (gastrointestinal, sleep and psychosomatic disorders) and physical disorders (high blood pressure and raised levels of stress hormones).(14) among a total of 2,368 company workers, female workers exposed to n o i s e a p p a r e n t l y s u ff e r e d f r o m s o m a t i c symptoms, anxiety, depression and irritability, whereas male workers presumably suffered only from irritability.(15) the use of ear mufflers is one of the means for reducing the intensity of perceived noise. if early stress symptoms are found in workers and the etiologic factors of these s y m p t o m s h a v e b e e n d e t e r m i n e d , t h e n immediate steps have to be taken for managing early stress symptoms in those workers. (15) stress is caused by multiple factors, thus no simple solution is available for application. different cases of stress call for individualized solutions in stress management. one of the most ideal solutions is preventing the occurrence of early stress symptoms in workers. this may be attained by taking care of the core of the stress problem, this being the cause of stress itself. studies on the relationship between noise caused by spinning machinery and job stress have been few in number. the aims of the study was to identify the risk factors of job stress at a private thread spinning company, where noise is presumably the principal risk factor of job stress. 10 methods research design the present study uses a comparative cross-sectional design for determination of a relationship between noise and the occurrence of job stress, conducted from december 2007 until january 2008. population and study subjects t h e t a r g e t p o p u l a t i o n u n d e r s t u d y comprised the rank and file workers at a private spinning company in citeureup, totalling 601 individuals. the minimal sample size needed was 326 persons, calculated using the following formula:(17) variables, where the dependent variable was job stress. the independent variables comprised personal characteristics (age, educational level, gender, marital status, length of employment, u s e o f e a r p l u g s ) , e n v i r o n m e n t a l f a c t o r s (occupational stressors in the form of role conflicts, ambiguity of roles, work load, career development, work shift and the availablity of ear plugs), and agent factor (noise). data were c o l l e c t e d b y f i l l i n g o u t o f t h r e e t y p e s o f questionnaire, namely the questionnaire on worker characteristics, the stress diagnostic s u r v e y q u e s t i o n n a i r e , a n d t h e s y m p t o m s checklist-90 (scl 90). the questionnaire on w o r k e r c h a r a c t e r i s t i c s c o l l e c t e d d a t a o n workers, namely age, educational level, gender, marital status, length of employment, work shift, and the use of ear plugs. the stress diagnostic survey questionnaire consisted of 30 q u e s t i o n s f o r m e a s u r i n g w o r k s t r e s s o r variables, namely ambiguity of roles, role conflicts, qualitatively excessive work load, quantitatively excessive work load, and career development. stressors were negative (-) if the total score was 0–9, while stressors were positive (+) if the score was > 10. the scl 90 questionnaire was used for measuring emotional mental disorders in workers, and consisted of 90 questions.(18) mental or psychopathological disorders were positive (+) if the total score was > 61 and negative (-) if the total score was <60. data on the use of ear plugs were obtained by discussions with the facory management. measurements noise intensity data were obtained from the results of measurements performed by the investigators in the production section (high noise conditions) and in the non-production sections (low noise conditions). in both cases, noise level was determined using a lutron sound level meter. n 1 = z∝2 x p x (1-p) d2 = 1.962 x 0.26 x 0.74 0.052 = 296 n 2 = n 1 + 10% n 1 = 296 + 30 = 326 mursali, basuki, dharmono noise and job stress the inclusion criteria in this study were: workers who had worked for at least 6 months with the company, and who were willing to participate in the study and sign a written consent, the exclusion criterion in this study was workers with severe hearing disorders. the study participants were recruited by proportional purposive sampling among workers in high and low noise conditions. for workers in high noise conditions (> 80 db) the sample size was 220, whilst for workers in low noise conditions (< 80 db) a sample size of 106 was obtained. data collection the variables investigated in this study c o n s i s t e d o f d e p e n d e n t a n d i n d e p e n d e n t 11 univ med vol.28 no.1 data analysis percentage analysis was performed to obtain stress prevalence, while simple logistic regression analysis was used to test various risk factors and stress events. to determine the dominant risk factor affecting the development of job stress, a multiple logistic regression analysis was conducted, and the risk factor affecting stress in the simple logistic regression analysis was included in the model. the level of significance of the bivariate analysis was p<0.05. for the data analysis the spss 13 program was used. results a total of 326 workers participated in this study, consisting of 55.8% (182/326) males and 44.2% (144/326) females. the mean age of the workers was 30.5 years. the number of workers in sections with low noise conditions was 32.5% (106/326), whilst the number of those employed in high noise conditions was 67.5% (220/326). the prevalence of job stress in high n o i s e c o n d i t i o n s w a s 5 5 . 0 % w h e r e a s t h e prevalence of job stress in those in low noise conditions was 24.5%. relationship between risk factors and job stress the results of the relationship between several risk factors and job stress is shown in table 1. gender, use of earplugs and noise condition were risk factors of job stress among workers. the risk of job stress among workers who worked in a high noise condition were 3.76 higher than workers who worked in low noise condition (or=3.76; 95% ci = 2.24-6.30). dominant risk factor on occurrence of job stress to o b t a i n t h e d o m i n a n t r i s k f a c t o r influencing the occurrence of job stress, a m u l t i p l e l o g i s t i c r e g r e s s i o n a n a l y s i s w a s performed. all significant variables in the s i m p l e l o g i s t i c r e g r e s s i o n a n a l y s i s w e r e included in the model. from table 2 it is apparent that there were two significant risk factors, namely noise conditions and use of ear plugs. a correlation m a t r i x , d e m o n s t r a t i n g a h i g h c o r r e l a t i o n between noise conditions and use of ear plugs. this indicated the presence of colinearity between noise conditions and use of ear plugs. from table 3 it may be seen that noise condition was the dominant risk factor for occurrence of job stress (or=2.46; 95% ci 1.33 4.55). from table 4 it is apparent that the risk factor ear plug was a dominant risk factor for the occurrence of job stress, with (or=21.76; 95% ci 8.09 58.52). discussion in high noise conditions the prevalence of job stress was 55.0%, whereas the prevalence of job stress in low noise conditions was 24.5%. the prevalence of job stress of the production section in this study was lower than the value of 57.7% obtained in the study by yoshio et al. in japanese workers in the machinery section.(7) the difference may be due to differences in research design. the study by yoshio used the cohort method based on observations made during a period of 2 years, whereas in the present study the cross-sectional method was employed, where study results were based on observations made at a given point in time. i n t h e p r e s e n t s t u d y g e n d e r h a d a significant correlation with occurrence of job stress. female workers had a 1.57 fold greater chance for stress than their male counterparts. the available literature states that working females experience a higher level of stress in comparison with males workers.(3) a 6-year french cohort study on 8,847 male and 2,886 12 mursali, basuki, dharmono noise and job stress table 1. relationship between risk factors and job stress 13 univ med vol.28 no.1 tabel 4. multivariate analysis with inclusion of ear plug risk factor table 2. dominant risk factor on occurrence of job stress table 3. multivariate analysis with inclusion of noise condition risk factor 14 female workers reported that female workers experienced higher stress levels when compared with males.(19) evans randomly assigned 40 experienced clerical workers (all female with a mean age of 37 years) to either a quiet office or one with low-intensity office noise (including speech) for three hours. the study showed that the workers in the noisy office experienced s i g n i f i c a n t l y h i g h e r l e v e l s o f s t r e s s ( a s measured by urinary epinephrine).(20) a study of nurses yielded essentially similar results. urinary cortisol, adrenaline and noradrenaline levels were all increased with age in both g e n d e r s , w i t h a g r e a t e r d i ff e r e n c e i n t h e younger age group compared with the older g r o u p . t h e s e h o r m o n e s w e r e l o w e r i n premenopausal women compared with male nurses of similar age.(21) a survey conducted in japan found psychosomatic disorders in 88% of female workers and in only 80% of male workers.(22) the habit of using ear plugs in the present s t u d y w a s s i g n i f i c a n t l y c o r r e l a t e d w i t h occurrence of job stress. workers in high noise conditions who always used ear plugs had a 1.79 fold greater chance of stress than workers in low noise conditions who did not need ear plugs. on the other hand, workers in high noise conditions who never or not always had used ear plugs had a 26.15 fold greater chance of stress than workers in low noise conditions who did not need ear plugs. use of ear plugs is one of the measures for reducing perceived noise intensity. workers who always use ear plugs may still have a greater chance of experiencing stress compared with those not needing ear plugs. this may be due to the fact that use of ear plugs constitutes a stressor for the workers. the worker may not feel comfortable using the supplied ear plugs or they feel compelled to use ear plugs out of feelings of fear for the company. another reason may be that although the workers always use ear plugs, they do not use them correctly. the results of this study indicate that all w o r k s t r e s s o r v a r i a b l e s w e r e s i g i f i c a n t l y correlated with occurrence of job stress. if the risk factors of ambiguity of roles, role conflicts, q u a l i t a t i v e l y e x c e s s i v e w o r k l o a d , quantitatively excessive work load and career development are present in the workers, these w o r k e r s w i l l h a v e a g r e a t e r c h a n c e o f e x p e r i e n c i n g s t r e s s c o m p a r e d w i t h t h o s e workers without above risk factors. work shift a l s o h a s a s i g n i f i c a n t c o r r e l a t i o n w i t h occurrence of job stress. this is consistent with studies conducted in japan indicating that work shift is a main source of stress in factory workers.(23) according to spurgeon, 10 out of 11 studies show that work shift is correlated with mental health disorders.(24) the results of the present study reveal two significant risk factors for job stress, namely noise conditions and habitual use of ear plugs. this indicates the presence of colinearity between noise conditions and habitual use of ear plugs. after performing a multivariate analysis by exclusion of one of those factors, it was found that habitual use of ear plugs was the dominant risk factor of job stress, with or=21.76 (95% ci 8.09-58.52). this signifies that workers in high noise conditions who did not always use or never had used ear plugs had a 21.76 fold greater chance of stress compared with workers in low noise conditions who did not need ear plugs. use of ear plugs is one of the measures for reduction of perceived noise i n t e n s i t y l e v e l s . t h e s t u d y c o n d u c t e d b y melamed et al. showed that workers exposed to noise levels of >80 db had substantially elevated cortisol levels.(25) after the use of earmuffs, which decreased the noise levels to 30db, the cortisol levels were drastically lowered.(26) studies in the usa have indicated that the use of hearing protectors is increasing, but there is still much to be improved in this respect. mursali, basuki, dharmono noise and job stress 15 univ med vol.28 no.1 many factors have been reported as 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melchior m, krieger n, kawachi i, berkman lf, niedhammer i, goldberg m. work factors and occupational class disparieties in sickness absence: findings from the gazel cohort study. am j publ health 2005; 95: 1206-12. 20. evans gw, johnson d. stress and open-office noise. j applied psychology 2000; 85: 779-83. 21. deane r, chummun h, prashad d. differences in urinary stress hormones in male and female nurses at different ages. j adv nurs 2002; 37: 304-10. 22. reika k, teruyo k, ayumi m, setsuko k. work related reproductive, musculoskeletal and mental 16 disorders among working women-history, current issues and future research directions. ind health 2002; 40: 101-12. 23. fujino y, mizoue t, izumi h, kumashiro m, hasegawa t, yoshimura t. job stress and mental health among permanent night workers. j occup health 2001; 43: 301-6. 24. spurgeon a. working time: its impact on safety and health. international labour office occupational safety & health research institute korea occupational safety & health agency. international labour organization; 2003. 25. melamed s, bruhis s. the effects of chronic industrial noise exposure on urinary cortisol, fatigue and irritability: a controlled field experiment. j occup environ med 1996; 38: 252-6. 26. clemens k, dirk h. noise and stress salivary as a noun invasive measure of allostatic load. noise health int j 1999; 1: 57-69. 27. patel ds, witte k, zuckerman c, murray-johnson l, orrego v, maxfield am, et al. understanding barriers to preventive health actions for occupational noise induced hearing loss. j health comm 2001; 6: 155-68. 28. kerr mj, lusk sl, ronis dl. explaining mexican american workers’ hearing protection use with the health promotion model. nursing res 2002; 51: 100-9. mursali, basuki, dharmono noise and job stress alvina 155 abstract *department of occupational medicine, faculty of medicine, trisakti university correspondence lie t. merijanti susanto, dr, mkk department of occupational medicine, faculty of medicine, trisakti university jl. kyai tapa no. 260 grogol jakarta 11440 phone: 021-5672731 ext.2802 email: lie_mery@hotmail.com univ med 2011;30:155-61. serum osteocalcin and bone mineral density in postmenopausal women lie t. merijanti susanto* september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina since high bone turnover is associated with decreased bone mass, biochemical markers of bone remodeling, such as serum osteocalcin, may be used to assess osteoporosis and to predict fractures in elderly women, particulary those involving trabecular bone, and use of a combination of bone mineral density (bmd) and biochemical markers may improve fracture prediction. the serum levels of osteocalcin constitute a specific biochemical parameter of bone formation. compared to imaging techniques, assays for osteocalcin are safe, noninvasive and easily performed. the aim of this study was to determine the relationship of serum osteocalcin and bmd in postmenopausal women. a cross sectional study was performed on 53 postmenopausal women in south jakarta from february to april 2010. the subjects were assessed for anthropometric characteristics, serum osteocalcin levels and bmd. bmd was measured at the lumbar spine, right femoral neck and at the left distal radius by dual energy x-ray absorptiometry (dxa). mean serum osteocalcin was 28.99 ± 10.02 ng/ml. the pearson correlation test on all subjects indicated a significant inverse correlation between serum osteocalcin and femoral neck bmd (r = 0.29; p=0.034). by arranging the data into tertiles, a significant association was found in non-obese subjects between mean femoral neck bmd and serum osteocalcin (p=0.036). the tukey posthoc multiple comparison test showed a significant mean difference in femoral neck bmd between the lowest and the highest tertiles of osteocalcin serum concentrations (p=0.028). maintenance of body weight is important for maintaining bmd in postmenopausal women. key words: serum osteocalcin, bmd, body weight, postmenopausal women introduction the increased knowledge of society on healthy living has resulted in an increase in the numbers of older persons worldwide, including indonesia. the 2010 population census indicates that older persons constitute approximately 10 percent of the indonesian population, whilst the 15.5 million menopausal women in the year 2000 are predicted to 156 susanto osteocalcin and bone mineral density increase to 24 million in 2015.(1) osteoporosis is one of the health problems arising in women entering menopausal age, and will presumably seriously affect the lives of postmenopausal women. osteoporosis and the potentially serious consequences of osteoporotic fracture increase with advancing age of the population, making skeletal health assessment an important component of routine care in postmenopausal women.(2) osteoporosis-related fracture risk is usually assessed by bone mineral density (bmd) as a measure of bone mass and a predictor of fracture, since bone mass affects bone strength or the ability of a bone to withstand trauma. it is well established that 7590% of the variance in bone strength is related to bmd. the risk of fracture is known to be higher in women with low bmd, with the risk d o u b l i n g f o r a r e d u c t i o n o f 1 s t a n d a r d deviation in bmd.(3) however, since bone strength is also determined by other factors, such as bone geometry, microarchitecture, and size, bmd by itself is not a good predictor of fracture risk. epidemiological data indicate that high body weight or high body mass index (bmi) are related to high bone mass, while a reduction in body weight may result in loss of bone mass.(46) on the other hand, some are of the opinion that fat mass is negatively related to bmd.(7,8) hsu et al. showed that postmenopausal women with a higher percentage of body fat may have a higher risk for osteoporosis, osteopenia, and nonspine fractures.(9) decreased estrogen concentrations at m e n o p a u s e a g e l e a d t o l o w e r i n t e s t i n a l absorption of calcium, resulting in low serum c a l c i u m c o n c e n t r a t i o n s a n d i n c r e a s e d osteoclastic resorption of bone. both increase bone turnover and constitute risk factors for the development of osteoporosis.(9) menopause and aging are associated with accelerated loss of cortical bone. bone loss is the result a negative remodeling balance due to impaired b o n e f o r m a t i o n a n d / o r i n c r e a s e d b o n e resorption.(10,11) fractures may be predicted from bone turnover markers either via bmd, because low bmd is associated with high bone turnover,(12) or independently of bmd, since increased b o n e t u r n o v e r n e g a t i v e l y a ff e c t s b o n e microarchitecture and fragility.(13) currently biochemical markers of bone turnover are being used for predicting the rate of bone loss and for assessing the risk of f r a c t u r e s i n p o s t m e n o p a u s a l w o m e n . estimation of bone turnover rates may be obtained through determination of the serum or urinary concentrations of certain proteins that are representative of the bone remodeling process. these proteins may be divided into bone formation markers and bone resorption markers. the most specific and sensitive bone formation markers include osteocalcin and b o n e a l k a l i n e p h o s p h a t a s e , w h i c h a r e indicative of osteoblastic activity, whereas bone resorption markers, such as tartrate resistant acid phosphatase and cross-linked telopeptides, reflect osteoclastic activity.(14) osteocalcin (oc), also termed gla (ã carboxyglutamic acid) protein, is a bone matrix protein synthesized by mature osteoblasts, and c o n s t i t u t e s a p p r o x i m a t e l y 1 5 % o f noncollagenous bone matrix proteins.(15) most of the osteocalcin (80-90%) is adsorbed to bone hydroxyapatite, with a minor percentage leaking into the circulation.(16) serum oc may be detected by various tests, such as assays using monoclonal antibodies against oc n-mid and n terminal oc fragments. osteoporotic females have significantly higher levels of oc, bone alkaline phosphatase, and crosslinked telopeptide-c (ctx ).(17) in general, serum oc concentrations increase with age, such that women aged over 65 years of age have an approximately 2 fold higher oc concentration in comparison with women under 44 years of age.(18) the aim of the present study was to investigate the relationship between serum o s t e o c a l c i n a n d b m d v a l u e s i n postmenopausal women. 157 methods study design from february to april 2010 a crosssectional study was carried out at the mampang prapatan community health center, south jakarta. study subjects the study sample was selected by cluster a n d s i m p l e r a n d o m s a m p l i n g f r o m postmenopausal women in the mampang prapatan area meeting the following inclusion criteria: postmenopausal women in the age range of 47-60 years, duration of menopause of more than one year, not consuming steroids and bisphosphonates, actively mobile, able to communicate, and willing to participate in this study. the exclusion criteria were: a past h i s t o r y o f h y s t e r e c t o m y, m a l i g n a n c i e s , h o r m o n a l t h e r a p y, d i a b e t e s m e l l i t u s , abnormality of liver and kidney functions, and acute infections. a total of 53 female subjects were included in this study. all participants gave written informed consent. questionnaire the respondents filled out a questionnaire relating to demographic data, including age, education, employment, number of pregnancies, and duration of menopause. anthropometric assessments height was measured to the nearest 0.1 cm using a portable microtoise and weight to the nearest 0.1 kg using sage portable scales. body mass index (bmi) was calculated as the weight (kg) divided by the square of the height (m). for asian populations, bmi classified into the following categories: underweight (<18.5 kg/m2), normal ( 18.5-22.9 kg/m2), overweight (23.0 -25 kg/m2), and obese (>25 kg/m2).(19) bmd measurements b m d m e a s u r e m e n t s ( g / c m 2) for the lumbar spine (l1-l4), femoral neck and distal radius were obtained by dual energy x ray absorptiometry (dxa) using a lunar dpx bravo nomusa densitometer (ge medical systems). the bmd measurements were performed at budi jaya hospital, south jakarta. osteocalcin measurements measurement of oc was performed on fasting serum samples collected before 10 a.m. and stored at a temperature below –70 ºc before a n a l y s i s . d e t e r m i n a t i o n o f s e r u m o c c o n c e n t r a t i o n w a s p e r f o r m e d a t p r o d i a laboratories, jakarta, using the elecsys n mid o s t e o c a l c i n a s s a y r e a g e n t k i t ( r o c h e diagnostics, cat no. 12 149 133). the elecsys n mid osteocalcin assay utilizes two monoclonal antibodies, one specific for epitopes on the n mid oc fragment and the other specific for the n terminal oc fragment, and therefore detects the intact oc as well as the n-mid fragment. the detection range of the kit was 0.5 – 300 ng/ ml, while the coefficient of variation was 2.28%. serum oc concentrations were divided into tertiles, the lowest tertile ranging from 12 up to 24.39 ng/ml, while the middle and highest tertiles were 24.4 – 32.45 ng/ml and 32.46 – 70.59 ng/ml, respectively. ethical clearance the protocol for this study was approved by the research ethics committee, medical faculty, trisakti university. statistical analysis data were entered to spss format using version 17.0. categorical variables were s u m m a r i z e d u s i n g f r e q u e n c i e s a n d percentages, while continuous variables were s u m m a r i z e d u s i n g m e a n a n d s t a n d a r d deviation. one-way analysis of variance (anova) was used to compare the mean d i ff e r e n c e i n b m d i n t h e t e r t i l e s e r u m osteocalcin levels. a significant anova test was followed by a tukey post hoc multiple comparison test to determine which specific univ med vol.30 no.3 158 susanto osteocalcin and bone mineral density groups were significantly different. statistical significance was set at p < 0.05. results i n t h i s s t u d y t h e r e w e r e 5 3 w o m e n meeting the inclusion criteria, for whom the major characteristics are shown in table 1. the mean age of the respondents was 53.49 ± 3.36 years, while the duration of menopause was 2 7 years. a total of 41 subjects (77.4 %) was non-obese and the mean of femoral neck bmd was 0.78 ± 0.12 g/cm2. in non-obese postmenopausal women, femoral bone mass was found to be significantly higher (0.82 ± 0.89 g/cm2) in the lowest serum oc tertile, in comparison with that in the mid (0.77 ± 0.11 g/cm2) and higher tertiles and (0.71 ± 0.13 g/cm2) respectively (table 2). however, for the lumbar and radial bones, the mass was not significantly different between lower serum oc tertile and the other two tertiles. chara cteristic n (%) age (yrs) 53.49 ± 3.36 a duration of menopause (yrs) 4.55 ± 2.3 8 a education did not finish senior high school (sma) 4 5 (84.9) senior high school graduate 7 (13.2) academy/university 1 (1.9) employment unemployed 3 1 (58.5) self-em ployed (informal sector) 2 2 (41.5) number of pregnancies 0-3 pregn ancies 1 9 (35.8) > 3 pregnancies 3 4 (64.2) osteocalcin (n g/ml) 28.99 ± 10.02 a tertiles of serum osteocalcin (ng/m l) lowest 1 7 (32.1) mid 18 (34) highest 18 (34) nutritional status non obese 4 1 (77.4) obese 1 2 (22.6) bmd values lumbar bmd (g/cm2) 0.90 0.12 a femoral neck bmd (g/cm2) 0.78 ± 0.1 2 a radial bmd (g/cm2) 0.53 ± 0.0 8 a table 1 characteristics of the subjects (n=53) a mean ± sd a mean ± sd tertiles of serum osteoca lcin (ng/ml ) p lowest (n=13) mid (n=14) h ighest (n=14) 12 – 24.39 24.4 – 32.45 32.46 – 70.59 bmd (g/cm 2)a lumbar 0.91 ± 0.11 a 0.86 ± 0.10 0.8 5 ± 0.11 0.364 femoral neck 0.82 ± 0.89 0.77 ± 0.11 0.7 1 ± 0.13 0.036 radial 0.56 ± 0.09 0.51 ± 0.08 0.5 1 ± 0.05 0.271 table 2. mean of bone marrow density by tertiles of serum osteocalcin in non-obese subjects 159 the results of the tukey post hoc multiple comparison test indicated a significant mean difference in femoral neck bmd between the lowest and highest teriles of serum osteocalcin concentrations (p=0.028) (table 3). the results of the pearson correlation test showed a significant inverse correlation between serum osteocalcin concentrations and femoral neck bmd (r = -0.29; p = 0.034) (table 4). discussion in the present study, femoral neck bmd had a significant negative correlation with serum osteocalcin. since there is a complex relationship between bone turnover and bone m a s s , s u c h t h a t h i g h b o n e t u r n o v e r i s associated with decreased bone mass,(21) it has been suggested that bone markers can predict fractures in elderly women, particularly those involving trabecular bone,(22) and that the use of a combination of bmd and bone markers can improve fracture prediction.(23) a case control study of 90 postmenopausal women with mean age of 66 ± 8 years showed results that were consistent with the results of the present study, in that there is a significant inverse correlation of proximal femur bmd with osteocalcin. (24) however, a study in pakistan, involving 50 postmenopausal women with mean age of 54.36 ± 0.81, obtained inconclusive results indicating lack of a significant association between bmd and s e r u m o s t e o c a l c i n c o n c e n t r a t i o n s . ( 2 5 ) biochemical markers of bone turnover, such as osteocalcin, may also be used for early e v a l u a t i o n o f t h e e f f e c t s o f t r e a t m e n t . additionally, serial measurements of bone markers may assist in evaluating the patients’ responses to specific antiresorptive therapy.(26) serum oc is considered a specific marker of osteoblast function, as its levels have been shown to correlate with bone formation rates. however, since it is also released from bone matrix during bone resorption, it reflects the overall turnover of bone and is considered as a bone turnover marker. oc has a high affinity for calcium and has a compact á helical conformation that is calcium dependent. the ã carboxyglutamic acid (gla) residues of oc a r e c a p a b l e o f b i n d i n g t o b o n e m a t r i x h y d r o x y a p a t i t e , t h u s l e a d i n g t o b o n e mineralization. calciumand phosphorusdeficient osteoporotic women may have a decreased rate of bone mineralization due to a reduction in hydroxyapatite crystal formation. in this condition, free oc may be present in the circulation, thus explaining the increased serum oc concentration in osteoporotic postmenopausal women.(2,12,18) tertile of serum osteocalcin femoral neck bmda p lowest mid 0.051 ± 0.041 0.444 highest 0.112 ± 0.041 0.028 mid lowest -0.051 ± 0.041 0.444 highest 0.061 ± 0.041 0.305 table 3. mean differences and ses of femoral neck bmd in non-obese subjects (tukey multiple comparisons procedures) a mean ± sd bmd serum osteocalcin p r values lum bar 0.079 0.572 femoral neck -.0.290 0.034 radi al -.0.093 0.502 table 4. correlation between serum osteocalcin and bmd in all subjects (n=53) univ med vol.30 no.3 160 susanto osteocalcin and bone mineral density the current study showed that non-obese postmenopausal women in the high osteocalcin tertile had the lowest mean femoral neck bmd values. these results are similar to those of a population survey that included women in the age range of 20–69 years, indicating that women with high oc concentrations had the lowest mean bmd values.(27) other studies on elderly women have shown low body weight to be a major predictor of bmd and bone loss, and this association may be mediated by increased bone turnover.(28,29) in the study by zhao et al.(8) it was found that fat mass is negatively related to bone mass when adjusted for body weight.(8) in another study, there was a significant risk of osteoporosis, osteopenia, and non spine fractures in subjects with high weight-adjusted body fat percentages.(12) bmd in postmenopausal women is determined by peak bone mass and the amount of bone lost since menopause. in several studies, body weight or bmi was shown to be inversely associated with bone turnover markers, where persons with a low body weight have high bone turnover, whereas overweight and obese persons had a lower serum oc than those of normal weight.(30-32) apparently, the effect of o b e s i t y o n f r a c t u r e r i s k d e p e n d s o n i t s definition. if defined on the basis of bmi or body weight, obesity may be protective against b o n e m i n e r a l l o s s o r v e r t e b r a l f r a c t u r e . however, if obesity is based on the percentage of body fat, obesity may be a risk factor for o s t e o p o r o s i s . i n t h i s c o n n e c t i o n , b o t h metabolic syndrome and hyperglycemia could be risk factors for osteoporosis. high hdl protects against osteoporosis, increasing bmd and lowering fracture risk.(33) while biochemical markers of bone turnover may be able to predict bone loss and thus fracture risk, they may also predict fracture risk independently of bmd. high bone turnover can disrupt the trabecular architecture by i n c r e a s i n g t h e i n c i d e n c e o f t r a b e c u l a r perforation and buckling, thus reducing bone strength, without necessarily appreciably affecting bmd. because of the overlap in bmd distribution of normal and fracture populations, the bmd criteria used are useful in populationbased measurements but less useful in assessing i n d i v i d u a l r i s k . ( 3 4 ) s i m i l a r l y, a s i n g l e measurement of a biochemical marker of bone turnover may be unable to predict even shortterm individual fracture risk. however, there is increasing evidence that a combination of a biochemical marker and bmd may be a better p r e d i c t o r o f f r a c t u r e t h a n b m d a l o n e . biochemical markers of bone turnover cannot substitute for serial bmd measurements, but may be useful when considered in conjuction with bmd measurement. the present study admittedly has several limitations. firstly, this study performed only investigations on bone formation without investigating bone resorption. secondly, the study design was cross-sectional and therefore cannot depict a causal relationship between oc concentrations and bmd. lastly, the age of the respondents was relatively homogenous in the 50–60 year range, while the duration of menopause was 2-7 years, such that this study can only describe bone changes occurring during early menopause. conclusions this study demonstrates a significant inverse correlation between serum osteocalcin and femoral neck bmd. in non-obese subjects there is a significant difference in femoral neck bmd values between the lowest and the h i g h e s t t e r t i l e s o f o s t e o c a l c i n s e r u m concentrations. acknowledgements the author wishes to thank the faculty of medicine, trisakti university, for funding this study and the respondents who were willing to participate in this study. special thanks are notably due to prof.dr.dr. adi hidayat, ms for his helpful comments and suggestions on the manuscript. 161 references 1. badan perencanaan pembangunan nasional. proyeksi penduduk indonesia 2000-2025. jakarta: bps, bappeneas, dan unfpa indonesia; 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21:751–5. doi 10.1007/s00198-009-1001-3. univ med vol.30 no.3 alvina 22 abstract cataract is a cause of avoidable blindness and its prevalence increases with age. cataract surgery is the only possible therapeutic modality for improving visual function in cataract patients. the rising numbers of cataract patients require a type of cataract surgery with minimal side effects and without hospitalization. in this connection the day care cataract surgery has been introduced, with excellent results. therefore the aim of the present study was to compare the visual acuity of day-care patients and inpatients after extracapsular cataract extraction (ecce). a comparative observational study was carried out on 30 mature senile cataract patients with regard to type of care after ecce surgery. the study subjects were allocated to two groups of 15 subjects each, i.e. the day-care group and the in-patient group. the patients were observed postoperatively on the first day, first week, fourth week, and eighth week. they subsequently were corrected for best visual acuity. hypothesis testing was done using independent t-test and the mann-whitney test. the study results indicated that the visual acuity of the subjects after the eighth postoperative week did not differ significantly between both groups (p>0.05). there was no significant difference in outcome or risk of postoperative complications between day care and in-patient cataract surgery.this study therefore presents evidence that both post-ecce day-care as well as post-ecce inpatients had similar visual acuity levels. keywords: cataract, in-patient, day-care, visual acuity, postoperative *department of ophthalmology medical faculty, trisakti university correspondence dr. erlani kartadinata, spm department of ophthalmology medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2614 email: univ med 2011;30:22-8. day-care patients and inpatients have similar visual acuity erlani kartadinata* january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction m o r e t h a n 4 0 % o f c a t a r a c t c a s e s ultimately suffer from blindness, with the majority of blind patients being found in the developing countries of asia.(1,2) the results of i n d o n e s i a n s t u d i e s s h o w e d t h a t 6 3 % o f bilateral blindness was due to cataract.(2) because of the high prevalence of cataracts, coupled to the fact that cataract is the most frequently encountered cause of blindness, cataracts constitute a considerable community health problem requiring special attention. the prevalence of all-cause blindness is three to four times higher in low-income countries than in industrialized countries, and more than 75% 23 univ med vol.30 no.1 of global blindness is either preventable or treatable.(3) s e v e r a l n o n s u r g i c a l t h e r a p e u t i c modalities for cataract can effectively improve vision, but only temporarily. for permanent improvement of visual functions, cataract s u rg e r y i s t h e o n l y t r e a t m e n t o p t i o n . ( 4 ) currently three types of surgical operations for cataract are in existence, viz. extra-capsular extraction, phaco-emulsification, and intracapsular extraction.(5) one study demonstrated that both extracapsular cataract extraction (ecce) and manual small incision cataract surgery (msics) are both safe and effective techniques for treatment of cataract patients in community eye care settings. msics needs similar equipment to ecce, but gives better uncorrected vision.(6) cataract surgery has increased 400% during the last 10 years (8) to become the most common surgical procedure in the developed world, exceeding 1.6 million operations in the united states alone because of an aging p o p u l a t i o n a n d d r a m a t i c e x p a n s i o n o f i n d i c a t i o n s f o l l o w i n g i m p r o v e m e n t s i n technology.(7) in indonesia, the cataract surgical coverage rate among adults aged 40 years and over was 19.3%.(8) the prevalence of blindness was significantly higher in poor households and poor households were also more likely to have had less than optimal cataract surgery; this was also reflected in the higher prevalence of blindness due to uncorrected aphakia.(9) cataract s u rg e r y r e q u i r e s s u b s t a n t i a l f i n a n c i a l expenditures, which are increased by additional by administrative costs such as hospitalization costs, such that they become a heavy economic burden to the patient. the economic advantage of the day surgery has been emphasized in a recent randomized clinical study, which reported lower costs for outpatients than for inpatients. this difference in cost was largely due to the higher cost associated with an overnight stay in hospital, while the costs of surgical intervention and of follow-up were similar in both patient groups.(10,11) this is the reason most frequently given by patients for declining to undergo cataract surgery and the associated follow-up treatment for returning vision to the patients. on the other hand, with the remarkable advances in the techniques of cataract surgery and the skill of the ophthalmologists, there is a corresponding improvement in surgical outcomes. these considerations led to the abandonment of postoperative hospitalization procedures by several countries and to the application of one day care (odc) procedures, where the patient receives postoperative care on the same day only, instead of inpatient care, so that the care may be safely administered. the result is to keep the costs to a minimum, while the patient is more comfortable living at home instead of in a hospital. one of the indicators of a successful outcome of cataract surgery is visual acuity. the majority of the indonesian people belongs to the middle and low social classes, with unequal distribution of educational levels and relatively inaccessible health facilities, which is in contrast with the situation in developed countries that have instituted the n o n h o s p i t a l i z a t i o n p r o c e d u r e , w h e r e educational level and health facilities are superior. in view of the fact that most of the i n d o n e s i a n c o m m u n i t i e s h a v e a l o w educational level and many people are still uneducated, while health facilities are still limited, there is a need for a study that compares the visual acuity of post-ecce inpatients and day-care patients at “prof. dr. r. d. kandou” hospital (pdrdk hospital), manado. methods study design the present study was a comparative observational study conducted on two groups of post-ecce patients, i.e. the inpatient group and the day-care group, at the department of ophthalmology of pdrdk hospital from june 2007 until march 2008. 24 study subjects the subjects of the present study were all p a t i e n t s a t t e n d i n g t h e a b o v e m e n t i o n e d department, who were diagnosed as having senile cataract and fulfilled the inclusion and exclusion criteria. the sample size was calculated with the following formula: (12) ( ) 2 µ2-µ1 s zβ-zα 2n2n1 ⎥ ⎦ ⎤ ⎢ ⎣ ⎡ == [ ] 142.14 3.2 2.2(-0.84)-1.96 2n2n1 2 ==⎥⎦ ⎤ ⎢⎣ ⎡== where á = 0.05; â = 0.20; s = 2.2; µ1-ì 2 = 2.3, thus giving: thus each group consisted of >14 eyes. the inclusion criteria used in this study were the following: i) age 50-70 years; ii) mature senile cataract; iii) intraocular pressure (iop) <22 mmhg; iv) blood pressure <140/90 mmhg ; v) fasting blood glucose <150 mg/dl, random blood glucose <200 mg/dl; and vi) cooperative and willing to participate in the study by giving informed consent. exclusion criteria were: i) traumatic cataract; ii) other o c u l a r d i s e a s e , a p a r t f r o m c a t a r a c t ; i i i ) previous intra-ocular surgery in the eye to be operated; iv) intra-operative complications of ecce (hemorrhage, vitreous loss, capsular tear; and v) posterior segment abnormalities. operative procedure from every patient a signed letter of consent was obtained for surgery/research, then the past history was elicited in connection with the inclusion and exclusion criteria, and the identity of the patient was recorded. ophthalmological examinations performed on the subjects were among others visual acuity testing, assessment of intra-ocular pressure, and biomicroscopic examination. in addition, measurement of blood pressure and laboratory tests were done (routine blood tests, blood glucose, clotting time, bleeding time), ekg, and keratometry and biometry for determining strength of the intra-ocular lenses to be implanted. before the patient was brought into the operating room, the blood pressure and iop was rechecked. all cataract surgery was done under an operating microscope by residents in o p t h a l m o l o g y u s i n g s t a n d a r d e c c e t e c h n i q u e s . t h e e c c e w a s i n i t i a t e d b y standard incision and was completed by the insertion of an intra-ocular lens (iol), in the following sequence: superior limbal incision of 140-160º, anterior capsulotomy by the “can opener” method, removal of lens nucleus, removal through a cannula of cortical matter by aspiration with ringer lactate solution, implantation of a posterior chamber iol, and suturing of the corneal incision with five 10.0 nylon sutures. after the operation the patient received eye drops containing an antibiotic and a corticosteroid (tobroson), analgetic eye drops (noncort) for one to two weeks, a systemic antibiotic (cefadroxil 500 mg) and analgesic (mefenamic acid 500 mg) for five days. the inpatient group was hospitalized for three days, starting from the time of cataract surgery up to two days after the operation. the day-care group received care for one hour after surgery only, after which the patients were allowed to return home. postoperative examination all subjects were monitored for one day, one week, four weeks and eight weeks after surgery. subsequently there was an assessment of visual acuity, which is the capacity of the eye to discern the smallest letters on the snellen optotype at a given distance. this was followed by correction of refractive errors for best attainable visual acuity. in addition the postoperative iop level was also measured. statitical analysis for hypothesis testing, the collected data kartadinata day-care and inpatients visual acuity 25 univ med vol.30 no.1 was statistically analyzed using the computer software program statistical package for social sciences (spss) for windows version 15.0. the statistical tests used were independent ttest for normally distributed data and mannwhitney test for non-normally distributed data. the level of significance for this study was set at p<0.05. results t h e p u r p o s e o f t h e p r e s e n t s t u d y, conducted from june 2007 until march 2008, was to compare the visual acuity of day-care patients and inpatients after extracapsular cataract extraction (ecce). among 30 patients with mature senile cataract who attended the ophthalmology clinic at pdrdk hospital, only 27 were found to meet the criteria of the study, two of whom required bilateral surgery. during the study peruiod, three patients were dropped out for the following reasons: one patient came only twice for follow-up, one had diabetic retinopathy, and the last had vitreous loss. the distribution of males and females was not significantly different (p>0.05) (table 1). mean age of the subjects in the inpatient group was 61.1 years (sd = 7.49 years), while in the daycare group mean age was 63.3 years (sd = 5.74 years). the results of the t-test showed that age was not significantly different in both groups (p>0.05). median iop was 14.6 mmhg in the inpatient group and 17.3 mmhg in the day-care group. the iop in both groups was shown by the mann-whitney u-test to be similar. mean visual acuity in the inpatient group on the first day after surgery was 0.3 (6/20) with sd = 0.20, while in the day-care group mean visual acuity was 0.3 (6/20) with sd = 0.19. according to the t-test the visual acuity on the first day after surgery was not significantly different in both groups (p>0.05) (table 2). median visual acuity one week after surgery in the inpatient group vs. the day-care g r o u p w a s 0 . 4 ( 6 / 1 5 ) a n d 0 . 5 ( 6 / 1 2 ) , respectively. these values were shown by the mann-whitney u-test to be not significantly different (p>0.05). identical results were obtained at four and eight weeks after surgery, where median visual acuity values in both groups of subjects were identical (all amounted to 0.6 or 6/9), and mann-whitney u-test results were also not significantly different (p>0.05). table 2. differences in visual acuity values on first day, one week, four weeks and eight weeks after surgery between inpatient and day-care subjects *mean (sd); **median; aindependent t-test postoperative period inpatient (n=15) day-care (n=15) pa first day* 0.3 (0.20) 0.3 (0 .19) 0.402 first week** 0.4 0.5 0.407 fourth week** 0.6 0.6 0.352 eighth week** 0.6 0.6 0.334 table 1. distribution of important characteristics of respondents in the inpatient group (n=15) and the day-care group (n=15) *independent t-test; **chi-square test; *** mann-whitney u-test variable inpatient (n=15) day-care (n=15) p age (years) 61.1 (7.49) 63.0 (5.74) 0.434* gen der male female 9 (60.0%) 6 (40.0%) 11 (73.3%) 4 (26.7%) 0.600** iop 14.6 17.3 0.230 *** 26 as seen in table 3, 100% of subjects in both groups had postoperative corneal edema as a complication of cataract surgery. in the inpatient group at one week after surgery, cortical rests were found in one patient (6.6%), whereas in the day-care group dislocation of the haptic occurred in one patient (6.6%) one week after surgery. in the eighth week after surgery both groups had a corrected visual acuity of 1.0 (6/6). astigmatism was found in five patients (33.3%) in the day-care group and in six patients (40%) in the inpatient group. the chi-square test found no significant difference in complication rate between the i n p a t i e n t g r o u p a n d t h e d a y c a r e g r o u p (p=0.065). discussion the present study involved 30 patients with mature senile cataract, of whom three patients were dropped out of the study due to death of one patient (after only 2 follow-ups), while one patient was found on funduscopy to have diabetic retinopathy, and the last one had vitreous loss upon insertion of the iol. among the remaining 27 patients, 3 had bilateral ecce. of the initial 30 eyes, 15 were assigned to the inpatient group and 15 to the day-care group. both groups were monitored on the first day, first week, fourth week, and eighth week after surgery, and were corrected for best attainable visual acuity. postoperative visual acuity on the first day was identical in both groups (0.3 or 6/20), presumably because of postoperative corneal edema and inflammation in all patients of both groups. there are several factors that may affect the occurrence of corneal edema, namely mechanical trauma and prolonged irrigationa s p i r a t i o n w i t h s u b s e q u e n t e n d o t h e l i a l damage.(13) visual acuity of the subjects on the first postoperative day was not significantly different between groups. in comparison with the visual acuity value of 0.3 on the first postoperative day in both groups, the visual acuity scores were slightly better in the first week after surgery, being respectively 0.4 (6/15) and 0.5 (6/12) for the inpatient group and day-care group. this improvement in visual acuity was due to clearing of the corneal edema. as a rule corneal edema disappears spontaneously within 4-6 weeks,(13) when the inflammatory process is considerably reduced. in the first postoperative week, in one inpatient cortical rests were found t h a t h a d n o t b e e n a p p a r e n t o n t h e f i r s t postoperative day due to corneal edema. in this patient no further invasive measures were taken, since the cortical rests were relatively few and small and thus likely to be reabsorbed. the finding of cortical rests may be due to their unobtrusive location behind the iris or to inadequate irrigation-aspiration. in the day-care group one patient experienced blunt trauma resulting in dislocation of the haptic, which was immediately repositioned. as was the case in the first postoperative day, the visual acuity of the patients in the first postoperative week also did not show statistically significant differences between both groups. t h e v i s u a l a c u i t y i n t h e f o u r t h postoperative week was identical in both groups, having the score of 0.6 (6/9), which was an improvement over the vidual acuity of 0.4 in the first week. this may have been table 3. postoperative complications in the inpatient group and the day-care group postoperative complication inpatient (n=1 5) day-care (n=15) pa corneal edema cortical rests dislocation of haptic astigmatism 15 1 0 6 15 0 1 5 0.0648 a chi-square test kartadinata day-care and inpatients visual acuity 27 univ med vol.30 no.1 caused by stabilization of the wound repair process, by completion of corneal clearing, or by the absence of inflammation. to minimize the chances of astigmatism, the sutures were removed in this postoperative period. in this fourth postoperative week also the visual acuity did not differ significantly between the two groups. th e v i s u a l a c u i t y i n t h e e i g h t h postoperative week was identical in both groups and also identical to the visual acuity in the fourth week (0.6 or 6/9). in general, the refractive power of the patients commence to stabilize between the sixth and the eighth week after surgery. all patients of both groups in the present study received best correction of visual acuity, which was 1.0 (6/6) in all patients. astigmatism was found in 5 patients of the daycare group and in 6 of the inpatient group, possibly due to refractive variability, a normal postoperative finding resulting from the wound repair process. another cause of decreased visual acuity in these postoperative patients was inaccuracy of keratometric and biometric m e a s u r e m e n t s , r e s u l t i n g i n i n c o r r e c t p r e o p e r a t i v e c o m p u t a t i o n o f t h e i o l dimensions. in this postoperative period visual acuity was also not significantly different between both groups (p>0.05). these findings were consistent with a review by fedorowicz et al., where no difference was found in visual acuity and risk in post-ecce inpatients and day-care patients.(1) similar results were found in the study by castells in 2001, where visual a c u i t y, s a f e t y, e f f e c t i v e n e s s a n d c o s t effectiveness did not differ between ecce with postoperative day care and inpatient care. (14) one retrospective study in india showed that the number of patients wishing to undergo cataract surgery increased after the introduction of day care cataract surgery,(15) suggesting that day care cataract surgery is a viable health service in developing countries. a prospective studied 851cataract surgery extraction at a tertiary centre in vancouver showed after 3 months postoperatively, the v i s u a l a c u i t y h a d i m p r o v e d i n 7 8 6 e y e s (92.4%), remained the same in 42 (4.9%) and had worsened in 23 (2.7%).(16) cataract is the leading cause of visual impairment and is associated with lower levels of self-reported quality of life.(17) a study showed a low cataract surgical coverage rate (19.3%) and even among urban subjects the rate was only 23.2%.(18) cataract surgery visual outcome can be used as an indicator by ophthalmologists to monitor the quality of their services. the outcome can be assessed with full spectacle correction (‘best vision’) or with available correction (‘functioning vision’). good outcome is defined as 6/6-6/18 (available and best correction grades >85% and >90% respectively), borderline outcome as <6/18-6/ 60 (available and best correction <15% and <5% respectively), and poor outcome as <6/ 60 (available and best correction <5% for each type). these broad categories can further be subdivided into: 6/6 excellent, 6/9 very good and 6/12 good.(19) with advances in technology, it has been found that phaco-emulsification and manual small incision cataract surgeries achieve excellent visual outcome with lower complication rates.(19) the vision 2020 initiative aims to reduce the number of people with blindness or impaired vision as a result of cataract, especially in lowand middle-income countries. however, in spite of vision 2020 programs, cataract surgery coverage in women of these countries still remains lower than in men. although previous research showed the need to increase utilization of cataract services by women, only a few programs report efforts to meet women’s needs,(20,21) and some blindness surveys still do not report important indicators by gender. conclusions no significant difference in visual acuity was found among inpatients and day-care postecce patients at pdrdk hospital. there was no significant difference in outcome or risk of 28 postoperative complications between day care and in-patient cataract surgery there is a need for studies on larger samples for extended p e r i o d s o f t i m e t o d e t e c t l o n g t e r m c o m p l i c a t i o n s , w h i c h p r e s u m a b l y m a y substantially influence the visual acuity of inpatients and day-care post-ecce patients. references 1. fedorowicz z, lawrence d, gutierrez p. day care versus in-patient surgery for age-related cataract. the cochrane database of systematic reviews 2005, issue 1. art. no.: cd004242. pub3.doi: 10.1002/14651858.cd004242.pub3. 2. ang cl, tan th, wong ty. overview of visual impairment, blindness and major eye diseases in asia. in: ang cl, chee sp, yap he, editors. clinical ophthalmology. philadelphia: w.b. saunders;2005.p.1-5. 3. resnikoff s, pascolini d, etya’ale d, kocur i, pararajasegaram r, pokharel gp, et al. global data on visual impairment in the year 2002. bull world health organ 2004;82:844-51. 4. who. magnitude and causes of visual impairment. fact sheet no 282, november 2004. available at: http://www.who.int/mediacentre/ factsheets/fs282/en/index.html. accessed august 12, 2010. 5. bollinger k, langston rhs. what can patients expect from cataract surgery? clev clin j med 2008;75:193-200. 6. gogate pm, deshpande m, wormald rp, deshpande r, kulkarni sr. extracapsular cataract surgery compared with manual small incision cataract surgery in community eye care setting in western india: a randomised controlled trial. br j ophthalmol 2003;87:667-72. 7. westcott mc, tuft sj, minassian dc. effect of age on visual outcome following cataract extraction. br j ophthalmol 2000;84:1380-2. 8. tana l. cataract surgical coverage rate among adults aged 40 years and older. univ med 2009; 28:161-9. 9. gilbert ce, shah sp, jadoon mz, bourne rb, dineen b, khan ma, et al. on behalf of the pakistan national eye survey study group. poverty and blindness in pakistan: results from the pakistan national blindness and visual impairment survey. bmj 2008;336:29 doi 10 1136/bmj 39395 500046 ae. 10. kroneman mw, westert gp, groenewegen pp, delnoij dm. international variations in the availability and diffusion of alternatives to inpatient care in europe: the case of day surgery. ambul surg 2001;9:147-54. 11. nghiem-buffet mh, de pouvourville g, renard g, ullern m, boureau c, chaine g. cost of managing cataracts. evaluation of traditional hospitalization and ambulatory surgery. presse med 2001;30:1924-6. 12. dawson b, trapp rg, editors. research questions about two separate or independent groups. in: basic & clinical biostatistics. 3rd ed. new york: lange medical books/mcgraw-hill;2001.p. 132-60. 13. thomas jl, gregory ls, louis bc. international ophthalmology. usa: american academy of ophthalmology 2005;5:161-76. 14. castells x, alonso j, castilla m, ribo c, cots f, anto j. outcomes and costs of outpatient and inpatient cataract surgery: a randomized clinical trial. j clin epidemiol 2001;54:23-9. 15. prakash g, sharma n, jhanji v, agarwal t, titiyal js. impact of day care cataract surgery on inpatient services in a tertiary care ophthalmic setup. tropical doctor 2009;3:141-4. 16. noertjojo k, mildon d, rollins d, law f, blicker j, courtright p, et al. cataract surgical outcome at the vancouver eye care centre: can it be predicted using current data? can j ophthalmol 2004;39:38-47. 17. broman at, munoz b, rodriguez j. the impact of visual impairment and eye disease on visionrelated quality of life in a mexican-american population: proyecto ver. invest ophthalmol vis sci 2002;43:3393-8. 18. isawumi ma, soetan eo, adeoye ao, adeoti co. operative complications especially posterior capsular opacity led to significantly poor visual outcome. ghana med j 2009;43:169-74. 19. tabin g, chen m, espander l. cataract surgery for the developing world. curr opin ophthalmol 2008;19:55-9. 20. jefferis jm, bowman rjc, hassan gh, hall ab, lewallen s. use of cataract services in eastern africa a study from tanzania. ophthalmic epidemiol 2008;15:62–5. 21. lewallen s, mousa a, bassett k, courtright p. cataract surgical coverage remains lower in women. br j ophthalmol 2009;93;295-8. kartadinata day-care and inpatients visual acuity riany 39 ischemic stroke in a young adult male as a manifestation of takayasu’s arteritis neurology department medical faculty trisakti university correspondence dr. riani indiyarti, sp.s neurology department medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 ext. 2806 universa medicina 2008; 27: 39-50. january-march, 2008january-march, 2008january-march, 2008january-march, 2008january-march, 2008 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 takayasu arteritis is a chronic, inflammatory large vessel arteriopathy that primarily affects the aorta, its main branches, and the pulmonary arteries. fibrosis and thickening of the arterial wall often occur in later stages, resulting in a cerebrovascular accident. a 20-year old man presented with ischemic stroke of young adults due to takayasu’s arteritis, from which he had been suffering since childhood (age 10). physical examination showed an abdominal aortic aneurysm, renal artery stenosis, leading to renovascular hypertension, and asymmetrical blood pressure. aneurysm of abdominal aorta was diagnosed by panaorto-arteriography. subsequently ischemic stroke of young adults developed, because the arteritis had led to stenosis of the left anterior and middle cerebral arteries, with renovascular hypertension as additional risk factor. on this patient a percutaneous transluminal angioplasty operation of the right renal artery was performed, and it is intended to do an extra-intracranial arterial bypass. preventive treatment is necessary to stem the extension of the arteritis to other blood vessels. keywords: ischemic stroke, young adults, takayasu’s arteritis, renovascular hypertension abstract riani indiyarti introduction in indonesia, stroke is the number three killer after heart disease and cancer. moreover, according to a survey in the year 2004, stroke is number one killer in governmental hospitals throughout indonesia. formerly stroke only affected the elderly, but at present there is a tendency for stroke to endanger people at productive age or even under the age of 45 years. according to the indonesian stroke foundation (yayasan stroke indonesia, yastroki), the number of stroke patients in indonesia has tended to increase in the last decades. the disease tends to affect the younger members of the productive generation. this has the effect of lowering the level of productivity and can lead to disruption of the social-economic situation of the family.(1) the causes of ischemic stroke of young adults in spain are non-atherosclerotic disorders of the blood vessels, atherosclerosis of the large vessels, cardiac embolism, and hematological universa medicina 40 indiyarti stroke and takayasu’s arteritis disorders.(2) risk factors for ischemic stroke of young adults in india are metabolic syndromes and smoking.(3) in contrast, the most common risk factors causing ischemic stroke of young adults i n ta i w a n a r e h y p e r l i p i d e m i a , s m o k i n g , hypertension and a family history of stroke. i n t r a c r a n i a l s t e n o s i s o f t h e c a r o t i d a n d vertebrobasilar systems is more common than e x t r a c r a n i a l s t e n o s i s , w h i l e p r e m a t u r e atherosclerosis is the most frequent cause of intracranial stenosis.(4) stroke in young adults may be a manifestation of narrowing of large and medium-sized arteries, in particular the aorta and its branches. one of the causes of this narrowing is takayasu’s arteritis.(5) in this case report a discussion is presented of stroke of young adults caused by takayasu’s arteritis, a very uncommon disease. case report a 20-year old male was addmitted to a hospital, at karawaci, tangerang, with the chief complaint of weakness in the right arm and leg since the day before admission. headache, nausea, vomiting, convulsions or lowering of consciousness were absent. the patient had a past history of renovascular hypertension since 10 years ago. at 10 years of age (december 1997), he underwent preparation of the renal artery and abdominal aorta, fibromuscular excision and prostethic graft placement on the diagnosis of hypertension and stenosis of the abdominal aorta and right renal artery. the pathologic examination revealed fibrosis of the muscularis of the blood vessels. five years later, at the age of 15 (december 2002 ), the patient had a laparatomic resection of the abdominal aortic aneurysm. abdominal ultrasound at that time detected a suspected aneurysm of the proximal abdominal aorta, while renal and aortic arteriography indicated the presence of a fusiform aneurysm of the distal abdominal aorta, and severe bilateral renal artery stenosis. pathology results were consistent with above findings. on examination the patient was found to be conscious, blood pressure in the right arm was 170/100 mmhg, in the left arm 150/100 mmhg, in the right leg 160/100 mmhg, and in the left leg 150/100 mmhg. the pulse rate was 80 per minute, the breathing rate 20 per minute, and the patient was afebrile. there was no bruit at the location of the carotid artery. pulsation of the right and left dorsalis pedis arteries was +/+. chest: heart and lungs were within normal limits. abdomen: liver and spleen not palpably enlarged, normal intestinal sounds. on neurological examination hemiparesis of the right arm was found, arm muscle strength 3, leg strength 4. physiological reflexes were +/+, pathological reflexes -/-. there was left hemihypesthesia. laboratory results: hemoglobin was 16.8 g per deciliter, white-blood-cell count 8,000 per cubic millimeter, with 0 percents eosinophils, 0 percent basophils, 0 percents stab or band neutrophils, 63 percent segmented neutrophils, 33 percent lymphocytes, and 4 percent monocytes, r e d b l o o d c e l l c o u n t w a s 5 , 8 0 0 c u b i c p e r millimeter, hematocrit was 47 percent, platelet count was 208,000, erythrocyte sedimentation rate was 17 mm per hour. the ureum level was 17 mg per deciliter, the creatinine level was1.0 mg per deciliter, the uric acid level was 4.8 mg per deciliter, and the blood sugar level was 95 mg per deciliter, sgot level was 30, sgpt level 27, and total cholesterol level was 210 mg per deciliter. the sodium level was 133 nmol per liter, the potassium level was 4.1 nmol per liter, and the chloride level was 100 nmol per liter. the clotting time, bleeding time, prothrombin time and activated partial thromboplastn time (aptt) were ormal. the d-dimer was 0.2 (< 0.3), fibrinogen level was 394 (200-400), amd anti dsdna igg (eia) level was 0.28. cranial c o m p u t e r i z e d t o m o g r a p h y ( c t ) s c a n o n admission was within normal limits (figure 1). 41 figure 1. cranial ct scan performed prior to 2 x 24 hours from onset; still within normal limits result of abdominal ct and multi slides (ms) ct angiography showed severe stenosis of approximately 70-90% at the origin of the right renal artery along a length of about 2.5 cm, as a result of extension of a mural thrombus of the abdominal aortic aneurysm. in addition there was mild stenosis of the aorta of about 30 50 % at the origin of the left renal artery, along a length of around 0.5 cm (figure 2). the abdominal aortic aneurysm was located at the level of the l1-l3 disks (figure 3), starting at the inferior part of the origin of the superior mesentric artery, extending past the origin of the renal artery, and ending approximately 4.5 cm above the aortic bifurcation (figure 4). this aneurysm had a caliber of around 3 cm and a length of approximately 9.5 cm (figure 5). the proximal portion of the aneurysm (at the level of l1-l2, along a length of about 4.5 cm) was ulcerated and accompanied by a very large nodular mural thrombus (measuring around 7,2 by 5 cm), probably post rupture. the left and right suprarenal arteries appeared to be in good condition (figure 6). universa medicina vol.27 no.1 42 indiyarti stroke and takayasu’s arteritis figure 2. abdominal ct angiography showing severe stenosis of ± 70 –90% at origin of right renal artery ± 2.5 cm in length and mild stenosis of ± 30–50% at origin of left renal artery ± 0.5 long figure 3. abdominal ct angiography showing abdominal aortic aneurysm at level of l1 –l3 disks 43 figure 5. abdominal ct angiography showing abdominal aortic aneurysm 3 cm in caliber, ± 9.5 cm long, with calcification of the tunica intima figure 4. msct angiography shows presence of abdominal aortic aneurysm, starting at origin of superior mesenteric artery, passing origin of renal artery and ending ± 4.5 cm above aortic bifurcation. proximal part of aneurysm is ulcerated and accompanied by very large nodular mural thrombus, presumably post rupture universa medicina vol.27 no.1 44 indiyarti stroke and takayasu’s arteritis the celiac trunk, common hepatic artery, right and left hepatic arteries, and superior mesenteric artery were of normal form and caliber. no stenosis was seen. the right and left common iliac arteries were within normal limits, while the inferior vena cava was of normal caliber. intra-abdominal organs were within normal limits. results of panaorto-arteriography showed aneurysm of abdominal aorta, beginning from 0.58 cm below the left renal artery, with a maximal diameter of 3.82 cm, and length of 9 cm. stenosis of left renal artery was 50 % at the origin. (figure 7 and figure 8). the right renal artery was small, without any stenosis. the carotid and vertebral arteries were bilaterally normal. the left anterior cerebral artery had a stenosis of 100 % at the origin, collaterals were present (+) distally is but were inadequate. the left middle cerebral artery had a stenosis of 70% (figure 9). the right and left coronary arteries were normal. figure 7. arteriography showing abdominal aortic aneurysm, starting 0.58 cm below left renal artery, with maximal diameter 3.82 cm, length 9 cm. left renal artery stenosis of 50 % at origin and small right renal artery figure 6. abdominal ct angiography shows left and right suprarenal arteries to be in good condition 45 figure 8. abdominal ct angiography revealing renal artery stenosis figure 9. intracranial arteriography showing 100% stenosis at origin of anterior cerebral artery with inadequate collaterals distally, and 70% stenosis of left middle cerebral artery t h e p a t i e n t w a s g i v e n t h e f o l l o w i n g treatment: antihipertensive therapy: amlodipine 1x5 mg in the morning. anti-platelet aggregation therapy: thrombo aspilet 1x1 tablet, clopidogrel 1 x 1 t a b l e t , pe t o x y f i l l i n 2 x 4 0 0 m g . a n t i hyperlipidemic therapy: simvastatin 1x5 mg in the evening. neurotropic therapy: citicholin 2x500 mg, piracetam 4x1200 mg. the patient underwent stenting/percutaneous transluminal angioplasty (pta) of the right renal artery and an extra-intracranial bypass operation was planned (pending availability of funding). discussion takayasu’s arteritis, also called ‘pulseless disease’, ‘martorell syndrome’, or ‘occlusive thromboarthropathy’, is an vascular inflammatory disease (granulomatous vasculitis), in particular universa medicina vol.27 no.1 46 indiyarti stroke and takayasu’s arteritis of the large to medium-sized arteries, with a predilection for the aortic arch and its main b r a n c h e s , a n d m a r k e d b y a p r o g r e s s i v e i n f l a m m a t o r y p r o c e s s o f t h e v e s s e l w a l l , involving the tunica media and adventitia, and subsequently causing wall thickening, dilatation, aneurysm, stenosis, occlusion and thrombosis. the disease is more common in asians, and occurs more frequently in females than males, affecting all age groups, most commonly in the second and third decades. the etiology is thought to be an autoimmune process, because there is an association with the human leucocyte antigens (hla), eg. b51, b52, drb1 1502 etc. there is also a rise in the concentrations of il-1, il-6 and rantes. tuberculosis and virus infections are suspected to be a trigger of the vascular inflammation. clinical manifestations vary from non-specific symptoms to severe sequelae due to involvement of the carotid arteries or other large vessels. there are two stages in takayasu’s arteritis, i.e. the ‘ pre-pulseless’ phase with a non-specific type of inflammation, followed by a chronic phase with vascular insufficiency. the onset is preceded by non-specific systemic symptoms, such as fever, malaise, night sweats, joint pains, anemia, anorexia and loss of weight. after a couple of months or years, symptoms of s t e n o s i s a n d o rg a n i s c h e m i a w i l l b e c o m e apparent, such as diminished pulsation of the artery, vascular bruits, blood pressure difference between the right and left brachial arteries, c l a u d i c a t i o n , r e n o v a s c u l a r h y p e r t e n s i o n , retinopathy, aortic regurgitation, coronary vessel disease up to congestive heart failure, neurologic m a n i f e s t a t i o n s a n d p u l m o n a r y a r t e r y involvement. clinical symptoms are caused by target organ damage due to involvement of the carotid artery, hypertension due to renal artery stenosis or aortic regurgitation due to aortitis. m o r e t h a n 5 0 % o f p a t i e n t s e x p e r i e n c e neurological symptoms, such as headache, disturbances of vision, convulsions, transient ischemic attacks (tia), cerebral infarction, i n t r a c e r e b r a l h e m o r r h a g e a n d a t t a c k s o f orthostatic syncope. approximately 10% of takayasu’s arteritis patients will have ischemic stroke or tia due to vasculitis or embolism. c e r e b r o v a s c u l a r d i s e a s e i s a c o m m o n l y occurring complication. the thyrocervical trunk, the anterior spinal artery or the circle of willis may also be involved. ischemic stroke occurs frequently in the early phase of takayasu’s arteritis (1-2 years after the onset of arteritis), possibly because of inadequate collaterals. cerebral hemodynamics may possibly also be chronically disrupted, and a mild reduction in blood pressure may lead to cerebral ischemia in the chronic phase of this disease. hemorrhagic stroke may also occur, including subarachnoid hemorrhage, although less frequently than ischemic stroke. subarachnoid hemorrhage in takayasu’s arteritis is caused by rupture of an a n e u r y s m . t h e a n e u r y s m i s f o r m e d b y hemodynamic changes at the circle of willis, due to agenesis of the internal carotid artery; alternative causative mechanisms of aneurysm formation are immunologic reactions damaging the elastic lamina; necrosis of the vessel wall may also occur, resulting in aneurysmal rupture and hemorrhage. bruits and diminished pulsation of the artery are the most common symptoms. most patients experience bilateral stenosis, accompanied by aneurysm in the same segment of the involved vessel. there is a predilection for the aortic arch, but there may also be involvement of the renal artery (28-75%), coronary artery < 10%), and pulmonary artery (14-100%). the subclavian artery is the vessel most commonly affected (90%), while two-thirds of cases involve the aorta, particularly just above and below the diaphragm. more than half of c a s e s i n v o l v e t h e c o m m o n c a r o t i d a r t e r y, especially on the left side. the incidence of specific organ involvement varies with ethnicity; in the japanese the disease mainly affects the 47 aortic arch and its branches, while in indian people the abdominal aorta and renal arteries are more commonly involved. based on the v e s s e l s i n v o l v e d , ta k a y a s u ’s a r t e r i t i s i s classified as type i (aortic arch), type ii (aortic arch and descending thoracic aorta), type iii (descending thoracic aorta and abdominal aorta), type iv (abdominal aorta), and type v (aortic arch, descending thoracic aorta and abdominal aorta).(6-13) takayasu’s arteritis is associated with focal mural inflammation causing sclerosis of the tunica media and intima. on macroscopic e x a m i n a t i o n o f t h e i n t i m a l s u r f a c e , i t s appearance is irregular, with a network of scar tissue in the form of horizontal lines. when the sclerosis involves the coronary artery, three forms of abnormalities will be apparent, ie. stenosis or occlusion of the coronary ostia or the proximal coronary artery; focal coronary arteritis, which may extend to all epicardial branches; or coronary aneurysm. when the coronary ostia are affected by sclerosis, the result will be luminal narrowing leading to m y o c a r d i a l i n f a r c t i o n . t h e p u l m o n a r y circulation may also be affected, usually by s y s t e m i c a r t e r y d i s e a s e . h i s t o l o g i c a l examination reveals fibrosis of the tunica adventitia, rupture of elastic fibers in the tunica media, proliferation of fibroblasts in the tunica media, luminal narrowing and the presence of m u l t i n u c l e a t e h i s t i o c y t e s . i n ta k a y a s u ’s arteritis, the artery undergoing partial occlusion will acquire a new large and organized lumen, called the ‘vessel-in-vessel’ phenomenon, which i s n o t p r e s e n t i n s y s t e m i c a r t e r i t i s . t h i s p h e n o m e n o n d i f f e r s f r o m o r g a n i z e d thromboembolism involving blood clots with ingrowth of endothelial cells, hemosiderin, and a myxoid fibrous network with smaller and m o r e n u m e r o u s v a s c u l a r t u n n e l s . ( 1 2 ) t h e diagnosis of ischemic stroke of young adults in the present case was based on the clinical symptom of right hemiparesis, even though the results of a head ct scan were within normal limits. the ct scan was made prior to 2 x 24 hours after admission; thus the picture of infarction was absent. the ischemic stroke had its onset in the childhood period, at age 10, starting with an aneurysm of the abdominal aorta and stenosis of the renal artery, the latter resulting in renovascular hypertension. the renovascular hypertension and takayasu’s arteritis were the causes of ischemic stroke in this patient. renovascular hypertension is secondary hypertension caused by narrowing of blood vessels supplying the kidneys. the most common cause is renal artery stenosis. r e n o v a s c u l a r h y p e r t e n s i o n m a y l e a d t o complications: hypertensive heart disease, heart attacks, congestive heart failure, vascular damage, renal damage, renal failure, stroke, and blindness. the management of takayasu’s a r t e r i t i s c o n s i s t s o f m e d i c a l t h e r a p y, endovascular therapy/angioplasty with stenting to maintain a patent lumen, or a renal artery bypass (revascularization).(14-16) the differential diagnosis of takayasu’s a r t e r i t i s i s s y p h i l i t i c a o r t i t i s , t u b e r c u l o u s aortitis, lupus and other vascular collagen diseases, ‘giant cell arteritis’, and kawasaki disease. there are no diagnostic pathological signs, and the diagnosis is established through a combination of the clinical picture and results o f i m a g i n g ( r a d i o l o g i c ) . to e s t a b l i s h t h e diagnosis of takayasu’s arteritis, the ishikawa c r i t e r i a ( 1 7 ) ( ta b l e 1 ) o r t h e c r i t e r i a o f t h e american college of rheumatology may be applied (18) (table 2). the american college of rheumatology criteria (1990) consist of: (a) age < 40 years, (b) claudication of extremities, (c) decreased pulsation of the brachial artery, (d) right-left brachial systolic pressure difference > 10 mmhg (e) subclavian or aortic bruit, (f) narrowing or occlusion of the aorta and its proximal branches at angiography. universa medicina vol.27 no.1 48 indiyarti stroke and takayasu’s arteritis the diagnosis of takayasu’s arteritis is established when any 3 from above 6 criteria are present. the gold standard for examination is angiography. inflammation and dilatation of blood vessels may be revealed by doppler ultrasound and magnetic resonance imaging (mri). ultrasound, ct and magnetic resonance angiography (mra) may be used for diagnosis of takayasu’s arteritis. mra can reveal wall thickening and luminal configuration of blood vessels. helical ct angiography may detect mural thickening of vessel walls, apart from v a s c u l a r s t e n o s i s a n d o c c l u s i o n . t h e erythrocyte sedimentation rate is raised in the acute phase. although correlation with some hla types supports an autoimmune etiology, this hypothesis remains unproven.(11,12,19,20) the etiological diagnosis of ischemic stroke for this particular patient is considered to be takayasu’s arteritis, based on the clinical picture and imaging test results indicating the p r e s e n c e o f a b d o m i n a l a o r t i c a n e u r y s m , bilateral stenosis of the renal arteries and stenosis of the left anterior and middle cerebral arteries. the patient fulfills both the criteria of ishikawa and those of the american college of rheumatology for the diagnosis of takayasu’s arteritis. the course of takayasu’s arteritis is variable and may lead to spontaneous remission. tr e a t m e n t m a i n l y c o n s i s t s o f s t e r o i d administration with a response rate of 50%. patients unresponsive to steroids may be given cytotoxic drugs, such as cyclophosphamide, a z a t h i o p r i n e , m y c o p h e n o l a t e m o f e t i l a n d methotrexate. treatment is aimed at controlling d i s e a s e a c t i v i t y a n d m a i n t a i n i n g v a s c u l a r function, with minimal long-term effects. s u r g i c a l t r e a t m e n t a n d p e r c u t a n e o u s intervention therapy, yielding variable results, are recommended in cases not responding to medical therapy or where life-threatening ischemia is present. approximately 10-15% of patients show monophasic clinical symptoms w i t h f i b r o s i s , s t e n o s i s , l o w e r e d b l o o d sedimenation rate and absence of inflammatory markers, indicating a subclinical inflammatory phase. in this condition the management of arterial insufficiency by angioplasty, stenting table 2. criteria of the american college of rheumatology, takayasu arteritis if meeting 3 criteria. sensitivity is 90.5% table 1. ishikawa criteria, takayasu arteritis if meeting 2 major criteria, or 1 major and 2 minor criteria, or 4 minor criteria. sensitivity is 84% 49 or surgery is more useful than medical therapy. indications for surgery are severe renal artery srtenosis, claudication of the extremities, stenosis of three or more cerebral vessels or coronary artery involvement. the five-year survival rate is reported to be 83–91%, while the ten-year survival rate is 84%.(6,11,12) laboratory hematological results in this p a t i e n t d i d n o t s h o w a n i n c r e a s e d b l o o d sedimention rate as acute phase marker, thus leading to the question of whether a course of steroids is warranted and of the duration of such a course. at present management is aimed at improving the blood circulation with anti-platelet aggregation drugs, control of hypertension and other risk factors, and placement of stenting/pta in the renal artery to prevent subsequent renal complications. additionally it is intended to perform an extra-intacranial artery bypass to further improve circulation in the left cerebral hemisphere, thus preventing the occurrence of right hemiplegia and aphasia. the carotid arteries, cardiac and other vessels are still in good condition. it is imperative to implement preventive measures to stem the extension of the arteritic process to other vessels, considering that the patient is still young. conclusion a c a s e o f ta k a y a s u ’s a r t e r i t i s w i t h childhood onset was discussed, which had caused an abdominal aortic aneurysm and renal artery s t e n o s i s r e s u l t i n g i n t h e o c c u r r e n c e o f renovascular hypertension. ischemic stroke of young adults occurred with the picture of left anterior and middle cerebral artery stenosis due t o ta k a y a s u ’s a r t e r i t i s , w i t h r e n o v a s c u l a r hypertension as additional risk factor. a stenting operation was performed on the right renal artery and an extra-intracranial artery bypass has been planned. prevention is imperative to stem extension of the arteritic process to other vessels. references 1. stroke mengancam usia produktif. available at: http://www.medicastore.com/stroke. accessed october 4, 2007. 2. verona jf, guerra jm, bermejo f, molina ja, gomez a. causes of ischemic stroke in young adults, and evolution of the etiological diagnosis over the long term. europ neurol 2007; 57: 2128. 3. lipska k, sylaja pn, sarma ps, thankappan kr, kutty vr, vasan rs, et al. risk factors for acute ischaemic stroke in young adults in south india. j neurol, neurosurg psychiatry 2007: 78: 959-63. 4. lee th, hsu wc, chen cj, chen st. etiologic study of young ischemic stroke in taiwan. stroke 2002: 33: 1950. 5. nomano f, okawara m, inomata h, kobayashi y. takayasu’s arteritis. lancet 2000; 356: 1023-5. 6. ahmed w, ahmad z. takayasu’s arteritis: a case report with global arterial involvement. rawal med j 2005: 30: 7. brey rl, moore m. infammatory arteritis and stroke. in: batjer hh, caplan lr, friberg l, greenlee rg, kopitnik ta, young wl. cerebrovascular disease. lippincott-raven. 1997: 506. 8. adam rd, victor m, ropper ah. principles of neurology. 7th ed. new york: mc graw-hill; 2001. 9. ringleb pa, strittmatter ei, loewer m, hartmann m, fiebach jb, lichy c, et al. cerebrovascular manifestations of takayasu’s arteritis in europe. rheumatology 2005; 44: 1012-5. 10. cantú c, pineda c, barinagarrementeria f, salgado p, gurza a, de pablo p, et al. non-invasive cerebrovascular assessment of takayasu’s arteritis. stroke 2000; 31: 2197-202. 11. krishna mv, rudresh, namratha s. takayasu’s arteritis stroke as an initial presentation. j indian acad clin med 2004; 5: 274-6. 12. harris nl, mcneely wf, shephard jo, ebeling sh, ellender sm, peters cc. case records of the massachusetts general hospital. n engl j med 2002; 347: 2057-65. 13. kim ds, kim jk, yoods, huh pw, cho ks, kang jk. takayasu’s arteritis presented with subarachnoid hemorrhage: report of two cases. j korean med sci 2002; 17: 695-8. 14. renovascular hypertension. available at: http:// www.nlm.nih.gov/medlineplus/ency/article/ 000204.htm. accessed october 3, 2007. universa medicina vol.27 no.1 50 indiyarti stroke and takayasu’s arteritis 15. renovascular hypertension. available at: http:// www.merck.com/mmpe/sec07/ch071/ch071b. html. accessed oktober 4, 2007. 16. renovascular hypertension: what is it ? available at: http://www.vdf.org/diseaseinfo/ras/. accessed oktober 4, 2007. 17. ishikawa k. diagnosis approach and proposed criteria for the clinical diagnosis of takayasu’s arteriopathy. j am coll cardiol 1988; 12: 964-72. 18. arend wp, michell ba, bloch da. the american college of rheumatology 1990 criteria for the classification of takayasu’s arteritis. arthritis rheum 1990; 33: 1129-34. 19. neidhart b, kosek r, bachmannl, stey c. exertional dyspnea as initial manifestation of takayasu’s arteritis a case report and literature review. bmc pulmonary medicine. available at: http://www.biomedcentral.com/1471-2466/1/3. accessed december 30, 2007. 20. pinheiro lw, leblang sd, romano j, corteza a. the acute diagnosis of takayasu’s arteritis based on helical ct angiography of the chest and neck in the emergency room. ajnr am j neuroradiol 1999; 20: 1983-5. editorial i the polypill: the solution for prevention of coronary heart disease? hendarto natadidjaja department of internal medicine medical faculty, trisakti university in western countries, cardiovascular disease is the most common cause of death and it is expected that it will continue to be so in the near future.(1) if the resulting physical impairment and psychosocial disturbances are also taken into account, clearly this is a serious problem from the viewpoint of productivity, quality of life, as well as community health level. therefore the institution of preventive measures is an important issue. unfortunately, however, currently preventive measures that are effective, safe, and at the same time practical and economical, are almost nonexistent. changes in lifestyle and control of dietary patterns in themselves do not always produce the desired results. in addition, these measures can potentially reduce or eliminate only some of the risk factors, as changing personal lifestyle and dietary habits of an individual is not always easily accomplished. the lifestyle and dietary habits of an individual are commonly influenced by environmental and other factors, in addition to the individual himself or herself, particularly if the individual’s motivation and willpower is not strong enough and his or her self-discipline is weak. the addition of anti-platelet aggregation and anti-hyperlipidemic agents to changes in lifestyle and dietary pattern does not per se provide significant support. there are indeed reports suggesting that post-myocardial infarction patients who received four kinds of preventive drugs simultaneously, i.e. aspirin, beta-blockers, angiotensin-converting-enzyme (ace) inhibitors, and statins, had a better survival rate in comparison with those who received no medication or only one or two drugs. other studies have emphasized that patients with acute myocardial infarction to whom three preventive drugs were administered simultaneously, had a better one-year survival rate compared with those who received only one drug, either an anti-platelet aggregation, anti-hyperlipidemic, or antihypertensive agent. the preliminary conclusion that may be drawn(2) is that by adding as many kinds of drugs as possible, in addition to changes in lifestyle and dietary pattern, a higher level of prevention may be attained. this is easily understandable since preventive measures that involve more risk factors should logically be more effective; however, this kind of approach is not without problems. consuming too many drugs simultaneously may lower the effectiveness of the preventive measures, because of decreased compliance in taking the drugs and because of the high cost. univ med vol. 28 no.2 ii the concept of a “polypill”, where a number of drugs for prevention of coronary heart disease, such as aspirin, beta-blockers, ace inhibitors, and statins, are packaged as a single “pill”, is neither a new idea nor an original one. with the polypill, prevention is expected to be more effective and efficient, due to better patient compliance. on the basis of the results of a meta-analytical study by wald and law(3) published in 2003, a hypothetical polypill containing a beta-blocker, a thiazide antidiuretic, and an ace inhibitor, all at half the standard dose, with the addition of folic acid, a statin and aspirin, if consumed daily by all individuals over 50 years of age, could be expected to reduce the incidence of coronary heart disease by more than 80%. additionally, one third of the population would be free from heart attacks or stroke for the next 11-12 years of their life.(4) the objective of the hypothetical polypill was to improve four key risk factors of coronary heart disease simultaneously, namely hypertension, ldl cholesterol, serum homocysteine levels and platelet function. the adverse effects of the antihypertensive drugs contained in the polypill would presumably be reduced by using only half-doses. wald and law estimated the probability of discontinuation of the pill due to unwanted effects to be only around 1%-2%, whilst fatal side effects would occur in just less than 1:10,000 cases. sleight et al support the polypill concept, but are of the opinion that a sufficiently long period of time is needed for educating healthcare workers so that they would be prepared to accept this concept. however, besides the promises of the polypill, there are still several doubts about its feasibility. not every patient needs all of the drugs in the polypill with the same dosages. there may be individuals who have to avoid taking beta-blockers or aspirin, such as patients with asthma or peptic ulcer. furthermore, not a few patients are unwilling to take such a large-sized pill, presumably leading to a high drop-out rate. it is also unclear if the total cost of taking polypills will be considerably lower than that of taking only the needed drugs individually. discontinuation of treatment because of adverse effects is also much easier in the case of drugs that are administered separately. apart from the validity of the meta-analysis of wald and law, because the costs of production and promotion of the polypill has not yet been taken into consideration and its effectivity has still to be demonstrated in practice, compared to the currently accepted methods of preventing coronary heart disease, at present the polypill is still not more than a concept. it is to be hoped that research on the polypill be intensified, taking into consideration also the morbidity rates, epidemiological characteristics and risk factors prevalent in various communities with differing psychosocial and cultural conditions. references 1. ezzati m, hoorn sv, rodgers a, lopez ad, mathers cd, murray cj. estimates of global and regional potential health gains from reducing multiple major risk factors. lancet 2003:362:271-80. 2. panchin n, cambon jp, hanama g, kadri z, genes n, cablanche jm, et al. usic 2000 investigators. impact of combined secondary prevention therapy after acute myocardial infarction: data from a nationwide french registry. am heart j 2005;150:1147-53. 3. wald nj, law mr. a strategy to reduce cardiovascular disease by more than 80%. bmj 2003;326:1419. 4. sleight p, pouleur h, zannad f. benefits, challenges, and registerability of the polypill. european heart journal 2006;27:1651–6. univ med alvina 182 abstract * professional study program, faculty of medicine, trisakti university **cilandak subdistrict health center correspondence kirana asmara, sked. professional study program, faculty of medicine, trisakti university jl. kyai tapa no. 260 grogol jakarta 11440 univ med 2011;30:182-8. erectile dysfunction and health-related quality of life in elderly males kirana asmara*, meitty marisha*, hengky*, fitria agustanti* and hotma p tampubolon** september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina one important aspect that will determine the quality of human life is sex life. therefore sexual activity may be used in the assessment of the quality of life. erectile dysfunction (ed) is the inability to continuously achieve or maintain a penile erection quality so as to achieve a satisfying sexual relationship. the aim of this study was to determine the relationship between ed and quality of life in the male elderly. a cross-sectional study was carried out on males aged 60 years and over in west cilandak village, south jakarta. the presence of ed was evaluated by means of the international index of erectile function (iief) and quality of life with the short form health survey (sf-36). one-way analysis of variance (anova) was used to compare the mean difference of qol by erectile dysfunction category. the correlation between the total ed score and qol was analyzed using the pearson correlation analysis. subjects included 199 elderly male patients, with mean age of 66.7 years (age range 60-81 years), among whom 80.5% presented with ed. the results of anova analysis showed that ed affects quality of life total score (p=0.000), with a significant difference in the vitality (p=0.029), physical function (p=0.048), and role emotional subscales (p=0.011). ed is a highly prevalent among elderly males and impairs the overall quality of life, the sf-36 is applicable and suitable for evaluation of quality of life. keywords: erectile dysfunction, health-related quality of life, elderly males introduction owing to advances in public health, human life expectancy continues to increase, with the result that people aged 65 years and older comprise the fastest growing segment of the global population. increasing life expectancy h a s c h a n g e d t h e s t r u c t u r e o f t h e w o r l d population and the numbers of the aging population increase continuously. in 2005, there were 673 million (10%) elderly people over 60 years old out of a world population of 6.6 billion.(1) indonesia has experienced population aging since the last few 183 univ med vol.30 no.3 decades. the proportion of elderly males was 7.8% in 2010, and is predicted to reach 12.6% in 2025.(2) as people age, the incidences of a great number of chronic diseases dramatically i n c r e a s e , a n d a s t h e p o p u l a t i o n a g e s , expectations for their health-related quality of life (hrqol) increase. hrqol is a subdivision of quality of life (qol), and most commonly refers to people’s experience of their global health. it may also refer to health-related subjective well-being, functional status, or selfperceived health. a representative definition of hrqol is “a multi-dimensional concept that encompasses the physical, emotional, and social components associated with an illness or treatment”.(3) qol measures may help construct a comprehensive health profile of older people after they experience an illness.(4) age-related p h y s i o l o g i c c h a n g e s i n c l u d e t h e s l o w e r progression of the sexual response cycle. medical illnesses and medications, as well as cultural, societal, and psychological factors, also impact on sexual function. one important aspect that also determines qol in humans is their sexual life. an enjoyable sexual life has a positive influence on qol. conversely, poor sexual life may impair qol therefore sexual activity may be used to assess the qol in elderly males.(5) in 1995, the number of asian men with erectile dysfunction (ed) ranked first at 86.9 million, followed by approximately 30 million in europe and an equal number in united states, while the estimated prevalence in the rest of north america, africa, south america, and oceania were estimated at 11.9, 11.5, 10.5 and 1.0 m i l l i o n , r e s p e c t i v e l y. (6) e d m a y b e a manifestation of a serious disorder, such as cardiovascular disease, diabetes, or depression, and commonly has a negative influence on qol and interpersonal relationships.(7) with the increasing numbers of the elderly population, ed may possibly become a public health problem. according to the national institute of health consensus development panel on impotence ed is defined as ‘the consistent inability to sustain an erection sufficient for sexual performance”.(8) the international index of erectile function (iief-15), a screening instrument developed by rosen et al. and validated in more than 63 languages, comprises 15 items, while an abridged version, the iief5, contains just 5 items.(9,10) some studies have demonstrated that ed deteriorates the hrqol. hrqol among men in the general population with ed is poorer in those with comorbid illnesses and improves with age.(11) in the general population there is commonly a reduction in hrqol, with an impairment in physical functioning, but minimal changes in mental health.(12) a man’s inability to achieve or maintain an erection is inevitably linked to troublesome feelings of disappointment, frustration, and loss of confidence, which may spill over into other areas of his life. the purpose of this cross-sectional study was to determine the relationship between ed and hrqol in older men. methods research design an analytical cross-sectional study was conducted to determine the presence or absence of an association between ed and health-related qol in elderly males attending a primary health center at the village (kelurahan) of west c i l a n d a k , s o u t h j a k a r t a . t h e s t u d y w a s conducted from july to august 2011. research subjects subjects of the study were men aged 60 years and older, residing in the village of west cilandak; who did not suffer from severe disorders, such as stroke, coma, or cancer; were capable of good communication; and willing to participate in the study. the sample size required for this study was determined according to the formula for sample size of a cross-sectional study, with the prevalence of elderly males with poor qol of 0.79 , alpha level of 0.05 and acceptable margin of error for proportion being 184 estimated = 0.05, giving an optimal sample size of 137.(13) selection of the subjects was by cluster and simple random sampling. in each rw (rukun warga or hamlet, a subunit of kelurahan), the number of subjects were selected by proportional sampling. data collection personal interviews were conducted to obtain information on age, smoking habits, and comorbidities, namely hypertension and diabetes mellitus. assessment of hr-qol hr-qol (subsequently called qol) was assessed by means of the short form health survey (sf-36) questionnaire, which is a reliable and valid instrument for assessment of qol in older people. the sf-36 contains 36 questions that have been translated into indonesian.(14) the sf-36 includes 8 subscales namely: physical functioning (pf), role physical (rp), bodily pain (bp), general health (gh), vitality (vt), social functioning (sf), role emotional (re), and mental health (mh). it also p r o v i d e s t w o s u m m a r y s c a l e s , p h y s i c a l c o m p o n e n t s u m m a r y ( p c s ) a n d m e n t a l component summary (mcs). scores range from 0 to 100 for each subscale with higher scores indicating a better condition. assessment of erectile dysfunction ed was assessed using the international index of erectile function (iief) questionnaire, a multi-dimensional self-report instrument for the assessment of male sexual function, and recommended as a primary endpoint for ed. the questionnaire comprises 15 items that are divided into five domains of sexual function: erectile function (six items), orgasmic function ( t w o i t e m s ) , s e x u a l d e s i r e ( t w o i t e m s ) , intercourse satisfaction (three items), and overall satisfaction (two items). a scoring key for each of the sexual function domains was developed and validated. the subscale scores range as follows: a) erectile function: 1-30; b) orgasmic function: 0-10; c) sexual desire: 2-10; d) intercourse satisfaction: 0-15; and e) overall satisfaction: 2-10. the subjects’ ed was measured and categorized according to severity using a five-level ordinal scale based on their iief score: i) severe dysfunction (score 0-6); ii) moderate dysfunction (score 7-12); iii) mild to moderate dysfunction (score 13-18); iv) mild d y s f u n c t i o n ( s c o r e 1 9 2 4 ) ; a n d v ) n o dysfunction (score 25-30).(15) data analysis to describe the subjects’ characteristics, observed frequencies were used for qualitative variables. one-way analysis of variance (anova) was used to compare the mean difference of total qol and the 8 domains of sf-36 by ed category. the correlation between the total score of ed and qol was analyzed using the pearson correlation test. all statistical calculations were performed by means of spss version 17.0. a p-value of 0.05 was considered statistically significant. results a total of 137 elderly males participated in this study, with mean age of 66.7 ± 8.2 years (age range: 60 81 years), of whom 23 men (16.8%) were smokers, 38.7% had a past history of hypertension, and 59 men (43.1%) had a past history of diabetes mellitus. among these subjects, 36 men (26.3%) had severe ed, 26 men (19.0 %) had mild ed, while 13 men (9.5%) had no ed (had normal erection) (table 1). the results of one-way anova showed that total qol was significantly greater in elderly males with normal erectile function (p = 0.000). similar results were obtained for p h y s i c a l f u n c t i o n i n g ( p = 0 . 0 4 8 ) , v i t a l i t y ( p = 0 . 0 2 9 ) a n d r o l e e m o t i o n a l ( p = 0 . 0 11 ) subscales. higher mean scores from these three domains were found in elderly males with normal erectile function, in comparison to elderly males with severe ed (table 2). asmara, marisha, hengky, et al erectile dyfunction in older men 185 univ med vol.30 no.3 pearson correlation analysis indicated a significant correlation between ed and qol (r = 0.376; p = 0.000). better ed in elderly males result in better qol (table 3). discussion our study demonstrated that 80.5% of elderly males had ed, among whom 26.3% had severe ed according to the iief instrument. vari able n (%) age (years) 60– 74 >75 smoking yes ex-sm oker no history of hypertension yes no history of diabetes m ellitus yes no erectile dysfunction severe moderate mild to moderate mild no 1 15 (83.9) 22 (16.1) 23 (16.8) 56 (40.9) 58 (42.3) 53 (38.7) 84 (61.3) 59 (43.1) 78 (56.9) 36 (26.3) 28 (21.2) 33 (24.1) 26 (19.0) 13 (9.5) table 1. distribution of several key features of the respondents (n=137) qol dimension erectile dysfunction p severe moderate mild to moderate mild normal total sf-36 sf 36 physical functioning sf 36 role physical sf 36 bodily pain sf 36 general health sf 36 vitality sf 36 social functioning sf 36 role emotional sf 36 mental health 49.9±11.9 50.2±28.1 43.8±43.7 46.8±24.0 52.4±15.9 51.1±14.2 57.2±24.7 42.6±39.5 55.3±16.0 56.2±11.9 64.3±27.5 50.0±43.8 50.4±24.9 54.2±14.0 46.4±13.3 57.2±26.2 53.5±36.8 56.7±16.3 53.3±12.7 63.9±26.1 42.4±35.1 47.7±26.5 51.7±15.0 48.8±18.2 61.8±30.1 60.3±38.4 57.0±17.4 63.2±12.2 65.9±30.9 65.4±37.5 53.4±25.1 59.6±16.5 56.1±19.2 62.3±19.5 83.4±30.5 56.6±21.8 65.5±10.1 73.1±24.7 71.2±39.3 47.3±25.3 65.6±17.7 58.3±14.4 67.6±29.1 86.3±19.2 67.3±15.4 0.000 0.048 0.061 0.864 0.115 0.029 0.672 0.011 0.066 table 2. mean total and 8 subscales of qol by erectile dysfunction category * one-way anova comparable results were found in a crosssectional taiwanese study on ed in communitydwelling males aged 65 years and older, using the iief-5, the abridged version of iief.(16) the ed prevalence was 57.6% among the 125 males completing the study, of whom 39.6% were mild cases of ed; 20.8% were moderate to mild; 9.4% were moderate; and 30.2% were severe ed. voluntary health examinations of men between 20 and 80 years revealed that 32% had some degree of ed when screened using iief-5. ed was present in 37.5% (51–60 years), and increased to 71.2% in between 71 and 80 years.(17) in contrast, the study conducted by nicolosi et al.(18) found an overall ed prevalence of 11% among indonesian men. the subjects in nicolosi’s study were men and women aged 40-80 years, and the instrument used to assess ed differred from the instrument used in our study. the presence of sexual dysfunction was assessed using two sequential questions; the first was “ d u r i n g t h e l a s t 1 2 m o n t h s h a v e y o u experienced any of the following for a period of 2 months or more?”: ‘lacked interest in having sex’, ‘was unable to reach climax (experience orgasm)’; ‘reached climax (experienced orgasm) too quickly’; ‘experienced physical pain during intercourse’; ‘did not find sex pleasurable’; (men only) ‘had trouble achieving or maintaining an erection’; (women only) ‘had trouble becoming adequately lubricated’. those who answered positively to the first question 186 were asked a second question: “for each of these experiences, how often would you say this has occurred during the last 12 months?” and the o ff e r e d a n s w e r s w e r e ‘ o c c a s i o n a l l y ’ , ‘sometimes’ and ‘frequently’. the prevalence of ed in men between 60 and 69 years of age in japan, singapore and china are 70%, 77% and 65%, respectively.(1921) these differing prevalence rates may have been caused by the fact that ed is subject to numerous influences. age has been shown to be one of the most, if not the most, powerful and consistent predictor of ed. given the differences in the age distribution of the study populations in different studies, and hence the differences in age-related ed, it is almost impossible to compare the prevalence rates of ed between different studies.(22) our study results demonstrate a significant association between ed and qol. male subjects without ed had a higher qol score than the group of subjects with ed. we observed that average values of the physical functioning, vitality, and role emotional domains decreased when subjects had moderate or severe ed. in addition, several studies showed that clinically and statistically significant improvements in some qol domains (mental and social health, and self-esteem) have been documented after ed treatment, but that other general parameters (physical and cognitive functioning, and global health perceptions) are unaffected.(23) these r e s u l t s d e m o n s t r a t e t h a t g e n e r a l q o l parameters (mental health, depression, and psychological well-being) are profoundly affected by ed. however, the physical capacity and environmental domains did not reveal an impact due to ed; the reasons for this are unclear. further study is needed to explore the possible cause. our study found the role emotional subscale of the sf-36, a multipurpose shortform health survey with 36 questions, to be more profoundly associated with ed than the physical domains. similar results were found in a study on men aged 18 years or older in metropolitan philadelphia who were treated for chronic ed among hemodialysis patients for at least six months.(25) sexuality is an important component of emotional and physical intimacy that men experience through their lives. cardiovascular risk factors and vascular disease result in endothelial dysfunction and early development o f e r e c t i l e f a i l u r e . s m o k i n g , o b e s i t y, hypertension, diabetes and dyslipidemias contribute to ed in elderly males.(26-28) several instruments have been developed to be selfadministered in the context of qol and ed a s s e s s m e n t . ( 2 9 , 3 0 ) t h e u s e o f v a l i d a t e d instruments in clinical work has been suggested in order to improve the diagnostic rate of ed and create an increased opportunity for therapy, but just asking one general question as opposed to none is the first need in clinical practice. two potential limitations of our study must be discussed. first, the presence of ed was based on subjects’ self-report, without any attempt to clinically confirm the diagnosis. second, the cross-sectional design of our study does not allow us to draw conclusions about the causal link between ed and qol. these aspects must be further investigated using a cohort longitudinal study. conclusions ed is a highly prevalent disease among elderly males, with the decline in ed that possibly affecting the qol in elderly males. the qol of many elderly males, especially in the physical functioning, vitality, and role emotional subscales, are already impaired by the presence of ed. the sf-36 can be used for quality of life p erectile dysfunction r = 0.376* 0.000 table 3. correlation between erectile dysfunction and quality of life in elderly males (n=137) * pearson correlation coefficient asmara, marisha, hengky, et al erectile dyfunction in older men 187 univ med vol.30 no.3 clinical research and evaluating treatment outcomes for ed. longitudinal population s u r v e y s s h o u l d b e d o n e t o i m p r o v e t h e understanding of sexuality in the older old. acknowledgements we thank the head and staff of the west c i l a n d a k vi l l a g e h e a l t h c e n t e r a n d t h e participating elderly males for their assistance and cooperation in the conduct of this study. thanks are also due to prof. dr. dr adi hidayat mph for valuable comments and suggestions in the preparation of this paper. references 1. united nations. world population prospects: the 2006 revision. new york: united nations;2006. 2. badan perencanaan pembangunan nasional, badan pusat statistik, united nations population 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older men diana1 42 restless legs syndrome and impact on work performance january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina diana samara* *department of occupational medicine, faculty of medicine, trisakti university correspondence dr. diana samara, mkk department of occupational medicine, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2101 email: davidwp@cbn.net.id univ med 2009; 28:34-40 abstract restless legs syndrome (rls) is a neurological sensorimotor disorder characterized by unpleasant sensations in the legs and an uncontrollable urge to move them for relief. the rsl prevalence in the general population is 0.1% 11.5%, and increases with age, with the highest effect of producing a primary sleep disorder (70%-80%). women appear to be at increased risk, as do individuals with certain chronic conditions, including renal failure and anemia. the pathophysiology of rls is incompletely understood, but it probably results from derangements in dopamine and iron metabolism, and has a genetic component. rsl could be idiopathic or secondary (usually related with iron deficiency, terminal renal failure, pregnancy, and spinal cord lesions). rls patients usually have sleep disorders, so the disease can cause difficulties and problems in occupational and social life. subjects with rls symptoms appear to experience significantly more daytime problems, including being late for work, making errors at work, or missing work because of sleepiness. the diagnosis of rls is made by following the criteria of the international restless legs syndrome study group (irlssg). pharmacologic rls therapy, in which dopaminergic drugs constitute the first line, is effective and may have a dramatic effect on symptoms and quality of life. identifying and treating rls may improve sleep quality, daytime function and work performance. keywords: restless legs syndrome, sleep disorder, workers introduction r e s t l e s s l e g s s y n d r o m e ( r l s ) i s a n e u r o l o g i c a l s e n s o r i m o t o r d i s o r d e r characterized by by unpleasant sensations in the legs and an uncontrollable urge to move them for relief. the patients complain of a burning sensation, of worms moving, of a sudden pulling movement of the leg, or of ants crawling within the legs. the sensations range from uncomfortable to irritating and painful.(1,2) the urge to move the legs has a circadian pattern most occur in the evening or at night when the p a t i e n t i s n o t a c t i v e , a t w h i c h t i m e r l s symptoms are manifested as sudden movements of the legs, such that this condition is frequently 43 univ med vol.28 no.1 accompanied by sleep disturbances.(2) rsl may have a negative impact on the quality of life because it causes unpleasant waking up, chronic sleep disorders, and stress. this condition has an effect on various aspects of life, such as occupational and social problems and difficulties in family life. disturbed sleep and intolerance to activities where the individual has to remain without moving, may cause loss of work, inability to enjoy life and relational problems.(3) epidemiology in the general population, the prevalence of rsl ranges from 0.1% to 11.5%, with the occurrence of a primary sleep disorder as the highest effect (70%-80%).(4) studies conducted by the rls epidemiology, symptoms and treatment (rest) have shown that among the majority of patients seeking relief, only 6.2% was diagnosed as rsl. the symptoms are more frequently associated with conditions such as back pain, arthritis, or peripheral neuropathy.(5) the ratio of females to males with rls is 2:1, but this ratio does not differ significantly from the female to male ratio of respondents in all surveys6. a survey in japan found that 5% of respondents had symptoms of rsl, whilst clinically significant rsl amounted to 1.5%.(7) in the rls epidemiology, symptoms, and treatment (rest) survey of 16,202 individuals in the united states and 5 european countries, which is perhaps the largest study conducted to date, 7% of participants screened positively for rls.(8) a telephone interview of a random sample of 1803 adults in predominantly rural kentucky found an age-adjusted rls prevalence of 10%.(9) results were similar in the wisconsin sleep cohort, a prospective community-based epidemiologic study, in which 10.6% of 2821 participants reported rls symptoms occurring at least weekly.(10) rangarajan et al reported their f i n d i n g s o f a d o o r t o d o o r s u r v e y o n a l l individuals in bangalore, india. they found a prevalence of 2.1% based on the international restless legs syndrome study group (irlssg) criteria, and 1.2% of rsl patients had sleep disturbances.(11) a high prevalence of rls symptoms was observed in one idaho primary care population. b a s e d o n t h e r e s t l e s s l e g s s y n d r o m e questionnaire (rlsq), this study found that 504 (24.0%) of the 2099 patients surveyed reported all 4 basic diagnostic symptoms of rls.(12) etiology the main cause of rsl is not known. rsl m a y b e i d i o p a t h i c o r s e c o n d a r y ( u s u a l l y associated with iron deficiency, terminal renal failure, pregnancy, and spinal cord lesions). a d d i t i o n a l s t u d i e s i n d i c a t e t h a t r s l i s associated with type 2 diabetes mellitus and m u l t i p l e s c l e r o s i s . h a l f o f p a t i e n t s w i t h idiopathic rls have a positive family history of rls.(2,4) several studies also found that caffeine, alcohol, and tobacco may induce symptoms in individuals with a predisposition for developing rls. a number of studies have shown that using a reduced amount of the substances, or not using them at all, may relieve the symptoms, although it is unclear whether elimination of these s u b s t a n c e s c o u l d a l t o g e t h e r p r e v e n t t h e occurrence of rls symptoms.(1) pathophysiology the basic pathophysiologic mechanism of rls is incompletely understood. pharmacologists, physiologists, pathologists, and neuroradiologists have found an association of rls with dopamine transmission. dopamine is a messenger molecule responsible for transmission of signals between one area of the brain, i.e. the substantia nigra, and the next relay station in the brain, which is the corpus striatum, resulting in smoothly coordinated activity of muscles as intended. it 44 is this dopamine transmission that is believed to play a role in the development of rls.(1,13) additionally, the development of rsl is also thought to be associated with a low iron store in the neurons of the substantia nigra and with spinal cord dysfunction.(2,13) symptomatology patients with rsl have uncomfortable sensations in the legs, particularly when sitting or lying down, accompanied with an urge to move the legs. these sensations are usually felt in the legs between the knee and the ankle, but occasionally also in the feet, thighs, arms and hands. the sensations mostly appear on both sides of the body (bilaterally), but may also be on one side only (unilateral). most of the symptoms are ignored in daytime settings and a r e o n l y f e l t i n t h e e v e n i n g o r a t n i g h t , particularly when asleep. most of the symptoms disappear by morning, when the patient is at last able to get some sleep. the symptoms may occur at any stage of life, but commonly become increasingly frequent with advancing age.(1) r e s t l e s s l e g s s y n d r o m e a n d w o r k performance most patients with rls suffer from a sleep disorder called periodic limb movements in sleep (plms). individuals with both rls and plms have difficulty in falling asleep and s t a y i n g a s l e e p , a n d u s u a l l y f a l l a s l e e p a t daytime because of difficulty in falling asleep in the early morning hours. the national sleep foundation 2005 poll on 1,506 adults found associations for persons at risk for rls and sleep behaviors, who had shorter durations of sleep (less than six hours nightly) and had symptoms of insomnia. in comparison to persons not at risk for rsl, they also stayed up longer than intended (several nights per week), took more than 30 minutes to fall asleep, and had more daytime fatigue (p < 0.05).(14) the rls epidemiology, symptoms, and treatment (rest) study in which 16.202 adults aged > 18 years participated, showed that more than 75% of the rls sufferers reported at least one sleep-related symptom. disrupted sleep, an inability to fall asleep, and insufficient hours of s l e e p a r e e x p e c t e d c o n s e q u e n c e s o f t h e sensorimotor abnormalities of rls, which are worse at night and while at rest.(15) consequently persons suffering from rls have problems at w o r k , i n s o c i a l l i f e a n d i n r e c r e a t i o n a l activities.(16) they frequently come late to work and make errors at work, due to sleepiness.(14) the study by ulfberg et al in 200 women between 18-64 years of age, using multivariate logistic regression, found that 11.4% of the women suffered from rls. these women with rls had more sleep disturbances than those without rls. the sleep disorder had an impact on their daily life, where rsl sufferers were f i v e t i m e s m o r e l i k e l y t o c o m p l a i n o f headaches, and tended to have diminished performance at work. in consequence, these conditions caused 9 times more problems at work among women with rsl.(17) a n u m b e r o f s t u d i e s , i n c l u d i n g t h e national sleep foundation 2005 poll, have consistently reported the presence of a strong association between mental and physical health problems on the one hand and rls symptoms on the other.(14,18,19) diagnostic criteria because of a lack of objective biological diagnostic signs, the diagnosis is based on the clinical criteria of the international restless legs syndrome study group (irlssg), consisting of four essential and four supportive criteria. the four essential criteria are (i) an urge to move the legs, (ii) worse at rest or when inactive, (iii) relief by activities such as walking, and (iv) worse in the evening or at night. supportive samara restless legs syndrome 45 univ med vol.28 no.1 clinical conditions include (i) family history of rsl, (ii) response to dopaminergic therapy, and (iii) periodic movement of the extremities at sleep or when awake but relaxed, and (iv) chronic symptoms occurring with varying degrees of sleep disturbance and generally w i t h o u t n e u r o l o g i c a l a b n o r m a l i t i e s o n examination (table 1). ( 2 0 ) the movements usually occur periodically, on average at 20second intervals. the most common movement is dorsoflexion of the ankle and flexion of the knee or hip.(3) in a number of dubious cases, neurophysiological examinations, such as polysomnography and/or immobilization tests, may be needed for confirmation of rsl.(4) management n o t a l l p a t i e n t s w i t h r l s n e e d pharmacological treatment. confirmation of diagnosis and reassurance is sufficient for most patients with rls, and only approximately 20% of all rls patients may have symptoms severe enough to merit pharmacological treatment.(21) rsl can be cured, although determining the most suitable treatment and dosage may take time, and treatment may occasionally have to be individually modified. treatment should begin by examining the patient’s lifestyle and looking for opportunities to initiate lifestyle changes, especially with regard to substances known to exacerbate symptoms (table 2).(22) table 1. diagnostic criteria for restless legs syndrome(20) abbreviation: rls, restless legs syndrome table 2. factors influencing treatment of rls(22) 46 t h e p r i n c i p a l d r u g s u s e d i n pharmacological management of rsl are ldopa, dopamine agonists, anticonvulsants and benzodiazepines. significant recovery with therapy occurs after a sufficiently long period of time.(2) t h e s t u d y c o n d u c t e d b y m o n t a g n a indicated that dopaminergic drugs constituted the first line in pharmacology for treatment of rsl, at least for a short period of time (table 3).(23) a l t h o u g h m o s t p a t i e n t s r e q u i r e pharmacologic treatments, non-pharmacologic treatments are available from which patients m a y a l s o b e n e f i t . ( 2 4 ) m o s t o f t h e n o n pharmacologic treatments for rls are from case series or case reports; however, one small randomized study assessed the effects of e x e r c i s e o n r l s i n 2 8 i n d i v i d u a l s . ( 2 5 ) participants were assigned to no exercise (n=17) or thrice weekly lower body resistance training and treadmill walking (n=11). severity as measured by the international restless legs syndrome (irls) scale decreased by 39% during the first 6 weeks of the intervention. this improvement was maintained for the remaining 6 weeks of the study period. in addition to moderate exercise, good sleep hygiene, including rising and going to bed at the same times e a c h d a y, s h o u l d f o r m t h e b a s i s f o r n o n pharmacologic treatment of rls. (26,27) little information is available about the effects of lifestyle on the symptoms of rls. limiting caffeine, tobacco, and alcohol use may improve s y m p t o m s . a c t i v i t i e s t h a t p r o v i d e m e n t a l stimulation may also provide relief. one survey showed a higher prevalence of rls in persons who were sedentary and overweight.(28) many experts recommend abstaining from alcohol, caffeine, and nicotine. activities that improve mental alertness (crossword puzzles, video games) may also reduce r l s s y m p t o m s ( 2 9 ) h o t b a t h s , m a s s a g e , a n d stretching may also improve symptoms.(30) prognosis rls has a variable course, but symptoms tend to progress with advancing age. some i n d i v i d u a l s m a y e x p e r i e n c e s p o n t a n e o u s improvement in their symptoms for a period of time, but symptoms tend to recur.(31) individuals with rls secondary to an underlying condition m a y h a v e i m p r o v e m e n t o f s y m p t o m s i f t h e underlying condition is treated. medications, when needed, may provide relief of symptoms. table 3 recommendations for primary rsl therapy(23) abbreviation: rls, restless legs syndrome; plms : periodic limb movements in sleep samara restless legs syndrome 47 univ med vol.28 no.1 conclusions rls is a disorder that frequently causes sleep disturbances, which if sufficiently severe will result in impairment of daily activities of life and thus may cause work-related problems. therefore an accurate diagnosis of rls needs to be made to prevent increasing the numbers of patients with sleep disturbances, which will have an impact on their work. the physician should have an increased understanding and a w a r e n e s s o f t h e c o n d i t i o n , t o p r e v e n t m i s d i a g n o s i s a n d c o n s e q u e n t i n e ff e c t i v e treatment. identifying and treating rls may improve sleep quality and daytime function. references 1. restless legs syndrome. national institute of neurological disorder and stroke available at: http://www.ninds.nih.gov/disorders/restless_legs/ restless_legs.htm. accessed december 3, 2008. 2. sonka k, kemlink d. restless legs syndrome in 2004. prague med rep 2004; 105: 337-56. 3. national heart lung and blood institute working group on restless legs syndrome. restless legs syndrome: detection and management in primary care. am fam physician 2000; 62: 108-14. 4. merlino g, valente m, serafini a, gigli gl. restless legs syndrome: diagnosis, epidemiology, classification, and consequences. neurol sci 2007; 28: (suppl 1): s37-46. 5. agarwal p, griffith a. restless legs syndrome: a unique case and essentials of diagnosis and treatment. medscape j med 2008; 1012: 296. 6. hening w, walters as, allen rp, montplaisir j, myers a, ferini-strambi l. impact, diagnosis and treatment of restless legs syndrome (rls) in a primary care population: the rest (rls epidemiology, symptoms, and treatment) primary care study. sleep med 2004; 5: 237–46. 7. kageyama t, kabuto m, nitta h, kurokawa y, taira k, suzuki s, et al. prevalences of periodic limb movement-like and restless legs-like symptoms among japanese adults. psychiatry clin neurosci 2000; 54: 296–8. 8. mccrink l, allen rp, wolowacz s, sherrill b, connolly m, kirsch j. predictors of health-related quality of life in sufferers with restless legs syndrome: a multi-national study. sleep med 2007; 8: 73-83. 9. phillips b, young t, finn l, asher k, hening wa, purvis c. epidemiology of restless legs symptoms in adults. arch int med 2000; 160: 2137-41. 10. winkelman jw, finn l, young t. prevalence and correlates of restless legs syndrome symptoms in the wisconsin sleep cohort. sleep med 2006; 7: 545-52. 11. rangarajan s, rangarajan s, d’souza ga. restless legs syndrome in an indian urban population. sleep med 2007; 8: 247-51. 12. nichols da, allen rp, grauke jh, brown jb, rice ml, hyde pr, et al. restless legs syndrome symptoms in primary care: a prevalence study. arch intern med 2003; 163: 2323-9. 13. paulus w, dowling p, rijsman r, stiasny-kolster k, trenkwalder c, de weerd a, et al. pathophysiological concepts of restless legs syndrome. mov disord 2007; 22: 1451-6. 14. phillips b, hening w, britz p, mannino d. prevalence and correlates of restless legs syndrome: results from the 2005 national sleep foundation poll. chest 2006; 129: 76–80. 15. allen rp, walters as, montplaisir j, hening h, myers a, bell tj, ferini-strambi l. restless legs syndrome prevalence and impact. arch intern med 2005; 165: 1286-92. 16. the foundation for better health care. restless legs syndrome. available at: http://www.fbhc.org/ patients/modules/rls.cfm. accessed december 17, 2008. 17. nyström jub, carter n, edling c. restless legs syndrome among working-age women. eur neurol 2001; 46: 17-9. 18. rothdach aj, trenkwalder c, haberstock j. prevalence and risk factors of rls in an elderly population: the memo study; memory and morbidity in augsburg elderly. neurology 2000; 54: 1064–8. 19. berger k, luedemann j, trenkwalder c. sex and the risk of restless legs syndrome in the general population. arch intern med 2004; 164: 196–202. 20. allen rp, picchietti d, hening wa, trenkwalder c, walters as, montplaisir j. restless legs syndrome: diagnostic criteria, special considerations, and epidemiology. a report from the restless legs syndrome diagnosis and epidemiology workshop at the national institutes of health. sleep med 2003; 4: 101-19. 48 21. chaudhuri kr. the restless legs syndrome: time to recognize a very common movement disorder. prac neurol 2003; 3: 204–13. 22. dekokker a, whitehead h, chaudhuri kr. therapeutic strategies in restless legs syndrome. future prescriber 2005; 6: 5–10. 23. montagna p. the treatment of restless legs syndrome. neurol sci 2007; 28: s61-s6. 24. ryan m, slevin jt. restless legs syndrome. j phar pract 2007; 20; 6: 430–48. 25. aukerman m, aukerman d. exercise and restless legs syndrome: a randomized controlled trial. j am board fam med 2006; 19: 487-93. 26. parker kp, rye db. restless legs syndrome and periodic limb movement disorder. nurs clin n am 2002; 37: 655-73. 27. hening w, allen r, earley c, kushida c, picchietti d, silber m. the treatment of restless legs syndrome and periodic limb movement disorder. sleep 1999; 22: 970-99. 28. phillips b, young t, finn l, asher k, hening wa, purvis c. epidemiology of restless legs symptoms in adults. arch intern med 2000; 160: 2137-41. 29. silber mh, ehrenberg bl, allen rp. an algorithm for the management of restless legs syndrome. mayo clin proc 2004; 79: 916-2. 30. parker kp, rye db. restless legs syndrome and periodic limb movement disorder. nurs clin n am. 2002; 37: 655-73. 31. bayard m, avonda t, wadzinski j. restless legs syndrome. am fam physician 2008; 78: 235-40. samara restless legs syndrome may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 evaluation of clinical abdominal scoring system for predicting outcomes of blunt abdominal trauma nico odolf yordanius1* , i ketut wiargitha2 , nyoman golden3 , i wayan periadijaya2 , i wayan sudarsa4 , and i wayan niryana3 abstract background trauma has been called the neglected disease of modern society and the most common cause of death under 45 years. determining the optimal prospective course of action may be aided by the adoption of a scoring system to evaluate urgent laparotomy intervention. a quick and easy technique to identify whether there are any intra-abdominal injuries is to use the clinical abdominal scoring system (cass). the objective of this study was to evaluate cass in predicting the outcomes in patients with blunt abdominal trauma (bat). methods a retrospective observational study was conducted involving 80 patients with suspected bat that arrived at the emergency department. all patients with suspected bat were scored using cass and radiological investigations that were done in the ed. the decision to proceed with the surgery would be made if the patient had cass >12 and/or if the radiological investigation showed features of bat such as air under the diaphragm. results mean cass score was 10.28 ± 1.340. the majority of the subjects (75 or 93.5%) had successful laparotomies, whereas only five (6.3%) had unsuccessful ones. injuries to the spleen (42.6%), and liver (32%), combined injuries to the spleen and liver (2.6%), intestine (16%), pancreas (1.3%), bladder (4%), and kidneys (1.5%) were all found in positive laparotomies. the cass has specificity of 60%, sensitivity of 80%, ppv 96.7%, and npv 16.6%. conclusions according to our data results, the cass has a poor ability to predict the need for laparotomy in cases of blunt abdominal injuries as it shows low specificity. keywords: blunt abdominal trauma, exploratory laparotomy, scoring system, emergency, trauma management, mortality cite this article as: yordanius no, wiargitha ik, golden n, periadijaya iw, sudarsa iw, niryana iw. evaluation of clinical abdominal scoring system for predicting outcomes of blunt abdominal trauma. univ med 2023;42:173-81. doi: 10.18051/ univmed.2023.v42:173-181 1general surgery training programme, faculty of medicine, universitas udayana/central general hospital professor i.g.n.g. ngoerah, denpasar, bali, indonesia 2division of trauma, critical care and acute care surgery, department of surgery, faculty of medicine, universitas udayana/central general hospital professor i.g.n.g. ngoerah, denpasar, bali, indonesia 3division of neurosurgery, department of surgery, faculty of medicine, universitas udayana/ central general hospital professor i.g.n.g. ngoerah, denpasar, bali, indonesia 4division of surgical oncology, department of surgery, faculty of medicine, universitas udayana/ central general hospital professor i.g.n.g. ngoerah, denpasar, bali, indonesia *correspondence: nico odolf yordanius universitas udayana/central general hospital professor i.g.n.g. ngoerah, denpasar, bali, indonesia email:nicoodolf@gmail.com orcid id: 0009-0008-4140-3005 date of first submission, june 6, 2023 date of final revised submission, august 4, 2023 date of acceptance, august 10, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2023.v42:173-181 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1475 173 original article mailto:nicoodolf@gmail.com https://univmed.org/ejurnal/index.php/medicina/article/view/1475 https://orcid.org/0009-0008-4140-3005 https://orcid.org/0000-0002-3421-8409 https://orcid.org/0000-0001-9700-9025 https://orcid.org/0009-0001-8470-6021 https://orcid.org/0000-0002-6581-7244 https://orcid.org/0000-0003-2626-5931 yordanius, wiargitha, golden, et al cass in abdominal trauma 174 introduction abdominal blunt trauma continues to be a serious issue that needs more studies, as 15% of all trauma cases seen in emergency rooms throughout asia, including qatar, were blunt abdominal trauma cases, according to a previous study.(1) a total of 44 new cases of blunt abdominal trauma were recorded, of which 75% were males, and 79.5% of the ct scans revealed intraabdominal organ rupture.(2) determining the optimal choice for the patient’s care requires weighing whether or not to do a laparotomy in the event of blunt abdominal injuries. screening techniques, such the clinical abdominal scoring system (cass), have been demonstrated to have great validity for patients with suspected intraabdominal organ injury.(3) the validity of cass was examined in a number of studies. the cass is highly effective at determining laparotomy patients with abdominal trauma, according to research comprising 749 abdominal trauma patients. (4) another study that included 46 patients with only blunt abdominal trauma (bat) at the emergency unit of the department of general surgery at zagazig university hospitals revealed that cass has higher sensitivity and specificity in bat patients than do imaging modalities such as usg of the abdomen and ct scan.(5) this score has a minimal measurement cost and a high degree of accuracy. with the aid of this score, patients who have suffered blunt abdominal injuries can cut down on the number of follow-up exams and consequently, on the expense of their care. the cass score is calculated from the glasgow coma scale (gcs), blood pressure, pulse rate, post-trauma status, and the findings of a clinical abdominal exam.(3) the clinical abdominal scoring system was found to result in considerably higher scores in patients with a positive need for laparotomy than in patients with a negative need, indicating that the cass score can be used as a reliable index in identifying patients who need laparotomy following acute abdominal injuries. the clinical abdominal scoring system (cass) uses a different approach than the blunt abdominal trauma severity score (batss), has gained popularity recently, and is thought to be more effective. additionally, it is considered to be a reliable scoring system because it is affordable and rapid detection results, allowing clinicians to manage more emergency cases. the cass score system also makes it easier for clinicians to decide which patients require additional procedures like laparotomies by allowing them to make this determination based solely on the total cass score without the need for additional examinations.(6) this can be explained as cass was designed for predicting the need for laparotomy in trauma rather than to predict mortality. however, in our study, cass was not only used for predicting the need for laparotomy in bat, but also for predicting the mortality rate. there are various components to our study that could potentially be seen as being more up to date than several other studies. however, the total cass score acquired can be used to determine whether surgery, particularly exploratory laparotomy, can be performed immediately without anticipating the findings of complementary examinations. however, there were no conclusions in prior investigations about the usage of this cass score in the therapeutic setting. furthermore, by initiating prompt action, the cass score can reduce postoperative problems caused by delayed therapy, as well as the patient’s longer length of stay (los), which can be avoided, reducing the incidence of in-hospital mortality (ihm). the objective of the present study was to evaluate cass in predicting the outcomes in patients with bat. method research design a study of observational design, using a retrospective study approach was carried out in the emergency department of central general 175 hospital professor i.g.n.g. ngoerah, denpasar, bali, indonesia, between february 2022 and july 2022. research subjects a total of 80 subjects was included in the study. the inclusion criteria in this study were patients with blunt abdominal trauma and aged more than 18 years, who were available from medical records. the exclusion criteria were incomplete medical records, pregnant women with acute abdominal trauma, patients with blunt abdominal trauma who did not undergo a laparotomy, patients with blunt abdominal trauma and laparotomy who were entered in the medical record outside the period of the study, and patients with glasgow coma scale (gcs) = 3. for each participant, the investigators examined the cass score recorded in the medical record at the moment the patient entered triage. data collection data c olle cted from medical rec ords included age, sex, pulse rate, systolic blood pressure, glasgow coma scale, clinical complaints on the abdomen , and laparotomy. all patients who were suspected to have bat were scored using cass and radiological investigations that were done in the emergency department of central general hospital professor i.g.n.g. ngoerah, denpasar, bali by the junior resident under the guidance of a consultant. the decision to proceed with the surgery would be done if the patient had cass of more than 12 and/or if the radiological investigation showed features of bat. the cass is a scoring system that yields a maximum of 15 points ba sed on clinical parameters. total score range: 5-15 classified into 3 categories: priority one with a score of 12, in which patients underwent immediate lifesaving laparotomy following an initial phase of resuscitation; priority two with a score of 9-11, in which patients underwent auxiliary investigations in the form of abdomino-pelvic usg, ct scan, and x-ray. final management was decided according to the observed findings; and priority three with a score of 8, in which patients were kept under observation with no auxiliary investigations for an average of 24 hr for the suspected abdominal injury. the score was reevaluated 6 hr after admission and before discharge to avoid any missed injuries. statistical analysis data analysis was performed with the stata se 12.1 program. t he receiver operating characteristic (roc) curve was used to determine the cut-off point of cass in assessing the need for a laparotomy. based on the area under the curve (auc), the ability of cass to determine the need for a laparotomy was good if the auc score 0.7. the best intersection point of the cass score to determine whether or not a laparotomy is needed is based on the coordinate farthest from the curve to the diagonal line. ethical clearance under license number 508/ un14.2.2.vii.14/lt/2022, the ethics committee of the faculty of medicine at udayana university in denpasar granted approval for this research. result the mean age of the subjects was 33 (±15.0) years. the 80 subjects consisted of 59 (73.8%) men and 21 (26.3%) women. there are three categories of trauma onset or time of presentation to the emergency room: 2 hours, 2–6 hours, and >6 hours. trauma onset was later than 6 hours in 53.8% of participants overall, and followed by 2–6 hours in 28 (35%). the characteristics of the study participants are displayed in table 1. after determining the patients’ pulse rate, systolic blood pressure, and glasgow coma scale (gcs) score, it was found that their vital signs were generally stable. there were 42 (52.5%) 28 participants with a pulse rate of >110. forty eight participants (60.0%) had a systolic blood pressure between 90-120 and. 69 (86.3%) of the univ med vol. 42 no. 2 yordanius, wiargitha, golden, et al cass in abdominal trauma 176 (1.5%) were all observed. for evaluating the cass capacity to predict the necessity of a laparotomy in cases of blunt abdominal injuries, the roc curve was developed (figure 1). the cass score has a poor ability to predict the need for laparotomy in cases of blunt abdominal injuries, as shown by the area under the curve (auc) value, which was reached at 0.687 (0.395 0.978). the roc curve is shown in figure 1. the sample was then divided into two groups according to their cass scores, which were moderate-high (eh10) and low (<10). the cass score sensitivity, specificity, positive predictive value (npp), and negative predictive value (npn) as a predictor of the need for laparotomy in patients with blunt abdominal trauma were measured as part of the validity test. these values were 80.0%, 60.0%, 96.7%, and 16.6%, respectively. table 2 displays the outcomes of the cass score validity test. discussion one of the leading causes of death worldwide is trauma. after head and chest trauma, abdominal trauma has the third highest prevalence, with the majority of cases being blunt abdominal trauma.(6) the mean age of the study participants was 33 (± 15.0) years. there were 21 ( 26.3%) female and 59 (73.8%) male participants. according to research by kumar et al.,(7) men may sustain blunt abdominal injuries in as muc h as 81% of cases, while women comprise the remaining 19%. a startling 38.1% of cases of blunt abdominal injuries occurs in variable age (years) 33 ± 15.00 gender male female trauma onset <2 hours 2-6 hours >6 hours pulse rate (beats/minute) < 90 90-110 >110 systolic blood pressure (mmhg) <90 90-120 >120 glasgow coma scale (gcs) <9 9-12 13-15 clinical complaints on the abdomen painful tenderness abdominal guarding cass score laparotomy positive negative 59 (73.8) 21 (26.3) 9 (11.3) 28 (35) 43 (53.8) 28 (35.0) 10 (12.5) 42 (52.5) 19 (23.8) 48 (60.0) 13 (16.3) 2 (2.5) 9 (11.3) 69 (86.3) 8 (10.0) 28 (35.0) 44 (55.0) 10.28 ± 1.34 75 (93.8) 5 (6.3) table 1. distribution of characteristics of the research subjects (n=80) data presented as n (%), except for age and cass score (mean ± sd);cas : clincal abdominal scoring system cass laparotomy total yes no score ≥10 score <10 60 15 2 3 62 18 total 75 5 80 table 2. cass score validity test results as predictors of the need for laparotomy in blunt abdominal trauma note: sensitivity = 80% (95 % ci : 69.1-88.3); specificity = 60% (95% c.i. 14.4-94.7); positive predictive value (ppv) = 96.7% (85% c.i. 91.0-98.8); negative predicitve value (npv) =16.7% (95% c.i. 7.9-31.8); cass: clincal abdominal scoring system individuals had gcs 13 to 15. in this study, a cass score of 10.28 ± 1.340 was considered average. among those who had a successful laparotomy, injuries to the spleen (42.6%), liver (32%), spleen and liver combined (2.6%), intestine (16%), pancreas (1.3%), bladder (4%), and kidney 177 people between the ages of 21 and 30 years. furthermore, up to 57.1% of cases presented after 6 hours of trauma, 26.2% after 2–6 hours, and just 16.7% within 2 hours.( in india, research on 100 patients with abdominal trauma demonstrated that 48% of the patients were in the 21–30 year age group, followed by the 31–40 year age group with 25% of patients. the remaining 12 cases involved women, providing a total of 88 cases for men.(8) the majority of blunt abdominal injuries occurred in women (62.5%), with a mean age of 33.52 (13.84) years. however these findings were not statistically significant. in general, men in the productive age range have a higher frequency of blunt abdominal trauma. men prefer to be employed in professions that involve more mobility due to culture and traditions, and men additionally suffer more substance and alcohol abuse compared to women.(7) this seems congruent with da ta showing that 61% of cases of blunt abdominal trauma are from road accidents, while up to 25% are caused by falls from heights, and the other cases are due to other causes.(9) in order to determine whether patients with acute abdominal injuries require surgical treatments such as laparotomies, the clinical abdominal scoring system (cass) has started to be utilized widely.(6,10) this evaluation is essential in helping clinicians choose a course of treatment that is appropriate for the patient’s medical condition. the glasgow coma scale (gcs), clinical abdominal signs, pulse rate, systolic blood pressure, and presentation following an accident or trauma contribute to part of the 15-point total cass score.(3) among the elements that significantly impact a patient’s prognosis is the time of the patient’s emergency room visit following a traumatic event.(7) pursuant to our study, the time of presentation or the commencement of trauma was >6 hours in 43 (53.8%) of the subjects, followed by 2–6 hours in 28 (35%), and 2 hours in 9 (11.3) of the individuals. these findings are univ med vol. 42 no. 2 figure 1. receiver operating characteristics (roc) curve of clincal abdominal scoring system as a predictor of the need for laparotomy in patients with blunt abdominal trauma yordanius, wiargitha, golden, et al cass in abdominal trauma 178 reinforced by a study by kumar et al.,(7) which revealed that only 16.7% of instances occurred within two hours of the trauma, whereas 26.2% occurred between two and six hours after the trauma, and 57.1% occurred within six hours. the group with the shortest presentation time after trauma (less than 1 hour) had the highest percentage of cases (57.5%), followed by the group with a longer presentation time (1-4 hours), which had the second-highest percentage (36.2% ), and the group with the longest presentation time (>4 hours) had the third-highest percentage (6.2%). patients with negative laparotomy outcomes tended to present to health services more quickly than those with positive laparotomy results. this difference in presentation time after trauma was statistically significant between patients with negative and positive laparotomy results. the potential of the patient’s condition and complications due to progressively severe trauma is increased the longer the time elapsing between the beginning of the trauma and arrival at the health center. (11) a more severe hypovolemic condition is indicated by a greater pulse rate, which is divided into three groups. stage i hypovolemic shock is a pulse rate of less than 100 beats per minute; stage ii is 100 to 120 beats per minute; stage iii is 120 to 140 beats per minute; and stage iv is greater than 140 beats per minute.(12) a pulse rate that is less than 90 beats per minute receives a score of 1, a rate between 90 and 110 beats per minute receives a score of 2, and a greater rate than 110 beats per minute receives a score of 3.(6) the results of this study showed that 12.5% of subjects had a pulse rate that was below 90 beats per minute, followed by 52.5% of patients who had a pulse rate between 90 and 110 beats per minute, and 35% of subjects who had a pulse rate that was over 110 beats per minute. similar findings were also obtained which showed that the majority of the subjects (16/30) had a pulse rate between 90 and 110 times per minute, followed by a pulse rate below 90 times per minute (10/30 samples), and a pulse rate of over 110 times per minute (4/30 samples).(6) systolic blood pressure is another indicator of hypovolemia in a patient. significant bleeding in intra-abdominal solid organs can lead to hemodynamic imbalances, including reduced cardiac output and contractions. hypovolemic shock and lowered blood pressure are also effects of this condition. an individual has reached the shock phase when his or her systolic blood pressure is reduced to 90 mmhg.(12) a blood pressure below 90 mmhg in patients with suspected abdominal trauma receives the highest score of 3, while a blood pressure between 90 and 120 mmhg receives a score of 2, and a blood pressure above 120 mmhg receives a score of 1.(6) in our investigation, systolic blood pressure was 90-12 mmhg, >120 mmhg, and< 90 mmhg in 48 (60.0%), 13 (16.3%), and 19 (23.8%) patients, respectively. this is similar to the results of kumar et al. (7), where the majority of subjects (22/30) had a systolic blood pressure between 90 and 120 mmhg, followed by >120 mmhg (6/30 subjects), and finally 90 mmhg (2/30 subjects). the glasgow coma scale (gcs), which assesses the patient’s consciousness, is the fourth criterion tested by the cass. reduced gcs may arise from the body’s hemodynamic instability, and one of the causes is hypovolemic shock. the degree of trauma or bleeding experienced is correlated with the gcs score, which is lower and implies a lesser level of awareness in the patient.(12) a clinical examination needs to be performed because tests solely based on changes in systolic blood pressure and pulse frequency carry the risk of leading to incorr ect diagnose s or inef ficie ncies in treatment. (1 3,1 4) clin ical examination of the abdomen will result in a score of 3 for findings of abdominal guarding, a score of 2 for tenderness, and a score of 1 for complaints of pain.(6) although the remainder of 8 (10.0%) and 28 (35.0) patients complained of discomfort and soreness, a total of 44 (55.0%) subjects displayed clinical abdominal guarding. although soreness and guarding were more frequent with positive laparotomy findings, abdominal discomfort was more frequent with 179 univ med vol. 42 no. 2 negative laparotomy findings (75.5% vs. 64.2%). once irritation due to peritonitis occurs, the muscles of the abdominal wall will become rigid, which is a sign of abdominal guarding. the presence of organs that have been traumatized or of peritonitis may be indicated by the presence of tenderness and by the location of the tenderness. the presence of peritonitis is one of the signs that a laparotomy is necessary in cases of blunt abdominal injuries.(15) the severity of the patient’s trauma is indicated by the cass score; the higher the score in this abdominal clinical examination, the more serious the trauma.(6) regarding the predictor of whether or not a laparotomy needed to be performed in cases of blunt abdominal trauma, our study results showed that cass had a sensitivity of 80%, a specificity of 60%, a positive predictive value (npv) of 96.77% and a negative predictive value (npv) of 16.67%. the mean cass score was 10.28 (±1.34) was obtained from our study. a higher mean cass score was found in a different study that was conducted by vanitha et al. (3) on 100 subjects, where the mean cass score of 11.56 (±2.02) points was found in the group that had surgery. (3) the study of sivarajan et al. (8) also found that the mean cass score was 11.56 with a standard deviation of 2.02 for the operative group and that a cass value of more than 12 had a specificity of 100%, sensitivity of 83.5%, positive predictive value of 100%, and negative predictive value of 91.3%. according to both studies, in blunt abdominal trauma the calculated cass score with a cut-off of 12 has a specificity of 100%, a sensitivity of 54%, a positive predictive value of 100%, and a negative predictive value of 78.7% for predicting the necessity for laparotomy. (3,8) the findings of the present study contrast with those of other studies done in the past. during research conducted by kumar et al.,(7) specificity, sensitivity, ppv, and npv were found to be 84.62%, 99.2%, 33.3%, and 100%, respectively. another study showed the specificity, sensitivity, ppv, and npv to be 88%, 100%, 90%, and 100%, respectively.(16) although in the two preceding investigations of vanitha et al. and srivarajan et al. the cass score had a high specificity of 100%, the current study discovered that it only had a poor specificity of 60%. the sensitivity value has not changed very significantly and remains at 80% . (2) it was discovered in our study that the proportion of subjects with trauma onset of more than six hours was 53.8% and achieved hemodynamic stability (systolic bp > 120 mmhg). this is due to the fact that the study patients had received fluid resuscitation while they were being transported to the hospital.(17–19) the cass predicted the need for operative intervention with good accuracy. (4,20) the limitations of the present study were, firstly, that the subjectivity of operators in conducting clinical examinations was very high, resulting in measurement bias. secondly, the number of subjects in this study was based on the research period, not based on statistical calculations. finally, this study was based on secondary data (medical records). the cass score is based on clinical assessment, which is an advantage of this study since it enables measures to be promptly carried out without the need for supporting tests. conclusion the cass is not re commended f or assessing the need for laparotomy in cases of blunt abdominal trauma. further studies in this regard ne ed to be performed on a larger population and in multiple centers to validate the results of the study. conflict of interest none declared. funding none. yordanius, wiargitha, golden, et al cass in abdominal trauma 180 acknowledgment the authors would like to extend their gratitude to everyone who took part in this study, and particularly to the staff at the department of surgery at universitas udayana in bali, for their assistance throughout the entirety of the study. author contributions noy: conceptualization, design, writing and reviewing the manuscript, ikw: conceptualization, design, writing, and manuscript revision. ng: manuscript writing and revision. iws: conceived, designed, wrote, and revised the manuscript. iwn: contributed to the study’s conception and design, and critically revised it for key intellectual content. all authors have read and approved the final manuscript . references 1. arumugam s, al-hassani a, el-menyar a, et al. frequency, causes and pattern of abdominal trauma: a 4-year descriptive analysis. j emerg trauma shock 2015;8: 193–8. https://doi.org/10.4103%2f09742700.1665902. 2. karjosukarso as, wiargitha ik, bagus mahadewa tg. validitas diagnostik blunt abdominal trauma scoring system (batss) pada trauma tumpul abdomen di rsup sanglah denpasar, bali. medicina 2019;50: 377-40. https://doi.org/10.15562/medicina. v50i1.181 3. vanitha dt. prospective study comparing the clinical abdominal scoring system (cass) with blunt abdominal trauma severity scoring (batss) in predicting the necessity of laparotomy. j dent med sci 2018;17:25-33. https://doi.org/10.9790/0853-1703162533 . 4. jang y. analysis of trauma scoring system for patients with abdominal trauma. ulus travma acil cerrahi derg 2022;29:68-72. doi: 10.14744/tjtes.2022.94475. 5. el arini amh, abdellatif t, sallam a, ebrahim a. clinical abdominal scoring system in predicting the necessity of laparotomy in blunt trauma abdomen. zagazig univ med j 2021;29:56-63. doi: 10.21608/zumj.2021.63767.2141. 6. dave d, bansal n, astik h. “clinical abdominal scoring system”: role of clinical judgement in patients of solid organ injury in blunt abdominal trauma in age of emerging technology. j dent med sci 2019;18:10–4. https://doi.org/10.9790/0853-1804011014 . 7. kumar d, kumar mk. effectiveness of clinical abdominal scoring system in the management of patients with blunt trauma abdomen. int surg j 2021;8:1175-80. https:/ /doi.org/10.18203/2349-2902.isj2021129.3. 8. siva raj an n, mo hamed wasim sk , kamalakannan c, bhardwaj a, grover h, sugandhi. prospective study comparing clinical abdominal scoring system (cass) with blunt abdominal trauma severity scoring (batss). medpulse int j surg 2021;19:53– 7. https://doi.org/10.26611/1061925. 9. abebe k, bekele m, tsehaye a, lemmu b, abebe e. laparotomy for abdominal injury: indication & outcome of patients at a teaching hospital in addis ababa, ethiopia. ethiopian j health sci 2019;29:503–12. https://doi.org/10.4314/ejhs.v29i4.12. 10. adelgais km, kuppermann n, kooistra j, et al. accur acy of the abdominal examination for identifying children with blunt intra-abdominal injuries. j pediatr 2014; 165:1230-5.e5. https://doi.org/10.1016/j. jpeds.2014.08.014. 11. faget c, taourel p, charbit j, et al. value of ct to predict surgically important bowel and/ or mesenteric injury in blunt trauma: performance of a preliminary scoring system. eur radiol 2015;25:3620–8. https:/ /doi.org/10.1007/s00330-015-3771-7. 12. hardisman. memahami patofisiologi dan aspek klinis syok hipovolemik: update dan penyegar. j kes andalas 2013;2:178–82. https://doi.org/10.25077/jka.v2i3.167. 181 univ med vol. 42 no. 2 13. wiargitha ik, karjosukarso as. validitas diagnostik skor blunt abdominal trauma scoring system (batss) pada trauma tumpul abdomen di rsup sanglah denpasar, bali [diagnostic validity of blunt abdominal trauma scoring system (batss) in blunt abdominal trauma at sanglah central general hospital, denpasar, bali] [master’s thesis]. denpasar (bali): universitas udayana; 2017. 14. weledji p. perspectives on the management of abdominal trauma. j univ surg 2018;6: 17. doi: 10.21767/2254-6758.100106. 15. umboh i, sapan h, lampus h. hubungan penatalaksanaan operatif trauma abdomen dan kejadian laparotomi negatif di rsup prof. dr. r. d. k andou manado. j biomedik 2016;8:s52–7. https://doi.org/ 10.35790/jbm.8.2.2016.12702. 16. saber a, shams m, farrag s, ellabban g, gad m. incidence, patterns, and factors predicting mortality of abdominal injuries in trauma patients. north am j med sci 2012; 4:129. https://doi.org/10.4103/1947-2714. 93889. 17. haghbeen m, rezaabbasi a, kalani n. epidemiology of causes of intra-abdominal hemorrhage in blunt trauma over the last decade: a cross-sectional study. int j sci stud 2017;5:353-6. doi: 10.17354/ijssi/2017/ 47. 18. behboodi f, mohtasham-amiri z, masjedi n, shojaie r, sadri p. outcome of blunt abdominal traumas with stable hemodynamic and positive fast findings. emerg (tehran) 2016;4:136-9. 19. bhatia k, cra nme r hh. tra uma in pregnancy. in: marx ja, hockberger rs, walls rm, editors. rosen’s emergency medicine: concepts and clinical practice. 8th ed. philadelphia (fa): elsevier health sciences;2014.p.296–304. 20. alam a, gupta ak, gupta n, yelamanchi r, bansal lk, durga ck. evaluation of iss, rts, cass and triss scoring systems for pr edicting outcome s of blunt trauma abdomen. evaluation of iss, rts, cass and triss scoring systems for predicting outcomes of blunt trauma abdomen. pol przegl chir 2021;93:9-14. doi: 10.5604/ 01.3001.0014.7394. alvina 80 abstract coal dust and cigarette smoke are pollutants found in coal mines that are capable of inducing oxidative stress, the effects of which on blood malondialdehyde (mda) level and serum superoxide dismutase (sod) level are still unknown. the purpose of the present study was to evaluate the effect of coal dust and cigarette smoke on levels of mda and sod in rats. an experimental study was done on wistar male rats divided into the following groups: control (c), coal dust exposure (14 days) (cde), cigarette smoke exposure (14 days) (cse), coal dust exposure (7 days) followed by cigarette smoke exposure (7 days) (cde+cse), cigarette smoke exposure (7 days) followed by coal dust exposure (7 days) (cse+cde). all exposures increased mda levels and decreased sod activity significantly between groups (p=0.000). all exposure groups had significantly increased blood mda levels, compared to the control group, although there was no difference between cse + cde and cde + cse. for sod levels, all exposure groups had significantly decreased the sod levels compared to control. but there were no significant differences between cse vs cde and cde + cse vs cse + cde. we conclude that exposure to cigarette smoke significantly increases blood mda level and decreases serum sod activity, which was not found in exposure to coal dust. combined exposures also increase blood mda level and decrease serum sod activity significantly. key words: coal dust, cigarette smoke, malondialdehyde, superoxide dismutase, rats *department of pathology anatomy, ulin general hospital, medical faculty, lambung mangkurat university, banjarmasin **department of medical chemistry, medical faculty, lambung mangkurat university, banjarbaru ***department of paediatric, saiful anwar general hospital, medical faculty, brawijaya university, malang correspondence dr. nia kania, sppa(k) department of pathology anatomy, ulin general hospital, medical faculty, lambung mangkurat university jl. a yani km.2 no.43 banjarmasin-south kalimantan email: kaniazairin@yahoo.com univ med 2011;30:80-7. oxydative stress in rats caused by coal dust plus cigarette smoke nia kania*, bambang setiawan**, and h.m.s. chandra kusuma*** may-august, 2011may-august, 2011may-august, 2011may-august, 2011may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina introduction coal mine dust is a heterogenous and complex mixture of more than 50 organic and inorganic compounds and their oxides. minerals contained in coal dust include silicates, oxides, carbonates, sulfites, and sulfates. a number of particles are respirable, and therefore increase the risks of disease in coal miners.(1) the diseases among the coal miners do not only c o m p r i s e p u l m o n a r y d i s o r d e r s , s u c h a s pneumoconiosis, progressive fibrosis, chronic bronchitis, and accelerated loss of pulmonary function,(2) but also cardiovascular disease 81 univ med vol.30 no.2 ( c v d ) . a n e p i d e m i o l o g i c a l s t u d y i n appallachian mines found a increased risk of cvd (or=1.22; 95%ci=1.14-1.30), coronary heart disease (chd) (or=1.29; 95%ci=1.191.39) and heart attacks (or=1.19; 95%ci=1.101.30).(3) oxidative stres is defined as a condition marked by an imbalance between free radicals and antioxidants on the cellular or individual level. oxidative damage is one of the results of this imbalance, comprising oxidative modification of cellular macromolecules.(4) several transcription factors that are sensitive to changes in redox status, are activated and coordinate certain biological responses. lowgrade oxidative stress induces transcription of nuclear factor-erythroid related factor 2 (nrf2), for gene transactivation of the enzymatic antioxidant superoxide dismutase (sod) as a frontline antioxidant in the defense to oxidative stress.(5,6) habitual cigarette smokers are frequently found among coal miners, causing these individuals to be burdened by oxidative stress from coal dust and cigarette smoke. coal dust particles deposited in the alveolar epithelium are phagocytosed by alveolar macrophages that subsequently increase release of h2o2 and o2 -.(7) cigarette smoke contains free radicals in the tar and gas phases. the tar phase contains 1017 radicals per gram, which are detectable by electron spin resonance (esr), are very stable, and persist for hours. the gas phase contains 1015 radicals per exhalation, consist a shorter half life radicals than the tar phase radicals.(8) the study by armutcu et al. demonstrated increased serum malondialdehyde (mda) levels in rats exposed to coal dust.(9) the study by junior et al. showed an increase in plasma t h i o b a r b i t u r i c a c i d r e a c t i v e s u b s t a n c e (tbars) level and a decrease in sod activity in residents of coal districts and in coal miners, compared to controls.(10) ulker et al. found increased serum sod in active coal miners and those on leave, in comparison to controls.(11) the study by murarescu et al. evaluated increases in serum mda levels in rats exposed to cigarette smoke.(12) on the basis of above mentioned studies, there no studies have been conducted on the combined effect of exposures to coal dust and cigarette smoke. the meeting of two free radical molecules results in termination of their radical status and the formation of neutral compounds. in addition, the reaction velocity of free radicals determines the triggering effect on oxidative damage in biomolecules or reactions with enzymatic antioxidants. this study examined the effect of coal dust and cigarette smoke on levels of mda and sod in rats. methods research design this was an experimental study, using male wistar rats weighing 200-250 grams, obtained from the pharmacology laboratory, faculty of medicine, brawijaya university, malang. experimental animals the experimental animals were assigned to the following groups: control (c); coal dust exposure (14 days) (cde); cigarette smoke exposure (14 days) (cse); coal dust exposure (7 days), followed by cigarette smoke exposure (7 days) (cde + cse); and cigarette smoke exposure (7 days), continued with cigarette smoke exposure (7 days) (cse + cde). there were five rats in each group.(13) preparation of coal dust coal dust was prepared by pulverizing gross coal with ball mill, ring mill and raymond mill at carsurin coal laboratories, banjarmasin. the resulting coal dust was <75 µm in diameter, and subsequently further screened using microsieve (biodesign, usa), producing coal dust of <10 µm diameter as respirable particulate matter. the coal dust 82 characteristics were analyzed by scanning electron microscopy and x-ray fluorometry at t h e p h y s i c s l a b o r a t o r y, m a l a n g s t a t e university. exposure to coal dust exposure to coal dust was performed by means of a coal dust exposure device of 0.5 m3 c a p a c i t y, d e s i g n e d a n d p r o v i d e d b y t h e pharmacology laboratory, faculty of medicine, brawijaya university. this device works on the principle of providing an ambient environment containing coal dust as particulate matter, which c a n b e i n h a l e d i n t o t h e a i r w a y s o f t h e experimental animals. the air flow through the blower is 1.5-2 liter/minute, corresponding to the air flow in collieries. the dose of coal dust exposure had been determined by preliminary studies to be 12.5 mg/m3 for a high dose.(14) cigarette smoke exposure was performed by means of a smoking pump apparatus, designed and provided by the pharmacology laboratory, faculty of medicine, brawijaya university. five rats were placed into the exposure box, then the cigarettes were lighted and the cigarette smoke was introduced into the box, the exposure lasting five minutes. parameter measurements following a 14-day exposure, the rats were anesthesized in plastic jars containing cotton wool drenched in ether. section was performed on rats with beating hearts by opening the abdomen, cutting the ribs, and opening the thoracic cavity to find the heart. blood was obtained from the heart to be used for parameter measurements. the parameters measured were blood malondialdehyde (mda) level and serum superoxide dismutase (sod) activity, by means of a colorimetric method developed in the pharmacology laboratory, faculty of medicine, brawijaya university, malang. ethics this study was approved by ethics local committe in faculty of medicine lambung mangkurat university, banjarmasin, south kalimantan. kania, setiawan, kusuma coal dust plus cigarette smoke figure 1. scanning electron microscopy of coal dust (magnification 5000 x) 83 univ med vol.30 no.2 statistical analysis t h e c o l l e c t e d d a t a w e r e t e s t e d f o r n o r m a l i t y o f d i s t r i b u t i o n a n d v a r i a n c e . normally distributed data of identical variance w e r e a n a l y z e d u s i n g a n o v a . s i g n i f i c a n t differences between intervention groups ( p < 0 . 0 5 ) w e r e f o l l o w e d u p b y m u l t i p l e comparison test. non-normally distributed data of differing variances were tested by nonparametric tests. results the technique for preparation of coal d u s t , a s s t a t e d i n t h e m e t h o d s s e c t i o n , produced coal dust particles with a maximum diameter of <10 µm, as shown in the scanning electron microscopy results in figure 1. the coal dust particles of <10 µm had a variety of shapes and tended to form aggregates. x-ray fluorescence yielded information on the mineral content, comprising iron (fe) 36.9%; silicon (si) 17.9%; molybdenum (mo) 15%; aluminum (al) 10%; calcium (ca) 8.67%; sulphur (s) 4.7%; and titanium (ti) 3.65%. other minerals of <1% included vanadium (v), chromium (cr), manganese (mn), nickel (ni), copper (cu), and yterbium (yb). mean mda levels of the treatment groups are shown in figure 2. the collected data were not suitable for parametric tests, and were therefore analyzed using non-parametric tests. in the single exposure groups for cigarette s m o k e a n d c o a l d u s t , a n d t h e c o m b i n e d exposures to cigarette smoke, followed by coal dust, or coal dust exposure plus cigarette s m o k e , k r u s k a l wa l l i s t e s t i n g f o u n d significantly different increases in mda levels b e t w e e n t r e a t m e n t g r o u p s ( p = 0 . 0 0 0 ) . futhermore, using the mann whitney test, significant differences were found between c vs cse (p=0.009); c vs cde + cse (p=0.009); c vs cse + cde (p=0.009); cde vs cse + cde (p=0.028). no significant differences were found between c vs cde (p=0.117); cde vs cde + cse (p=0.113); cse vs cde + cse (p=0.463); cse vs cse + cde (p=0.347); and cde + cse vs cse + cde (p=0.116). figure 2. mean mda concentrations in the treatment groups. c=control; cse= cigarette smoke exposure; cde= coal dust exposure; cse+cde= cigarette smoke exposure followed by exposure to coal dust; cde + cse= coal dust exposure followed by cigarette smoke exposure 84 mean sod activities in the treatment groups are shown in figure 3. the collected data were not suitable for parametric tests, and were therefore analyzed using non-parametric t e s t s . i n t h e s i n g l e e x p o s u r e g r o u p s f o r c i g a r e t t e s m o k e a n d c o a l d u s t , a n d t h e combined exposures to cigarette smoke, followed by coal dust, or coal dust exposure plus cigarette smoke, kruskal wallis testing found significantly different decreases in sod activity between treatment groups (p=0.000). the mann whitney test found significant differences between c vs cse (p=0.009); c v s c d e ( p = 0 . 0 2 8 ) ; c v s c s e + c d e (p=0.009); c vs cde + cse (p=0.009); cde vs cse + cde (p=0.009); cde vs cde + cse (p=0.009); cse vs cse + cde (p=0.0059); cse vs cde + cse (p=0.016). no significant differences were found between cse vs cde ( p = 0 . 11 7 ) ; c d e + c s e v s c s e + c d e (p=0.917). discussion the technique for preparation of coal dust produced coal dust particles with a maximum diameter of <10 mm, having a variety of shapes a n d t e n d i n g t o f o r m a g g r e g a t e s . t h i s aggregative tendency is presumably due to attraction between charged atoms of the coal dust particles, in the form of cations or oxoions. the diameter of the coal dust particles used in this study was less than the diameter of dust partilces used in the study by pinho et al.,(7) but larger than in the study by ghanem et al. (14) and armutcu et al.(9) the unique physical characteristics (shape, size, surface area) and the metal constituents of these particles are major predictors for vascular oxidative stress. nanoparticle are an important constituent part of pm10. nanoparticle are c l o s e l y a s s o c i a t e d w i t h r e s p i r a t o r y, cardiovascular diseases and even the death rate figure 3. sod activity in the treatment groups. c=control; cse= cigarette smoke exposure; cde= coal dust exposure; cse+cde= cigarette smoke exposure follwed by exposure to coal dust; cde + cse= coal dust exposure follwed by cigarette smoke exposure kania, setiawan, kusuma coal dust plus cigarette smoke 85 univ med vol.30 no.2 due to their nanosize effects, high specific s u r f a c e a r e a a n d c o m p l i c a t e d c h e m i c a l composition allowing redox reaction take place.(15-17) the main finding in this study is that exposure to cigarette smoke and coal dust triggers systemic oxidative stress. this is consistent with the findings of brook et al.(18) malondialdehyde is a lipid peroxidation product involving a three-stage reaction, i.e. initiation, propagation, and termination. the overall reaction is: phagocytosis is a cellular process requiring molecular and cellular communication, thus i n v o l v i n g m o r e t i m e f o r i n i t i a t i n g l i p i d p e r o x i d a t i o n r e a c t i o n s . d e t a i l s s t e p s o f phagocytosis consist of recognition, response, and removal.(19) although coal dust contains catalytic minerals for redox reactions, such as fe and ti, these reactions also require the p r e s e n c e o f h 2o 2 f o r h y d r o x y l r a d i c a l formation. for the combined exposures, all exposure groups had significantly increased blood mda levels, compared to the control group, although there was no difference between cse + cde and cde + cse. the free radicals in the gaseous phase, the products of coal dust phagocytosis, and the redox catalyzation p r o d u c t s w i l l i n i t i a t e l i p i d p e r o x i d a t i o n reactions, although it cannot be determined which of these was the most dominant. there is a variety of reactive compounds produced by coal dust, including •o2 -, h2o2, and no. cigarette smoke contains •o2 and no.4 one of the catalytic redox reactions is the fenton or haber-weiss reaction:(8) ti(iii) + h2o2 → ti(iv) + oh + •oh in comparing the single exposures with the combined exposures, a significant increase was found for cde vs cse + cde. this is due to the diffusive capacity of gaseous reactive compounds derived from cigarette smoke, leading to their appearance in the b l o o d , t o b e a u g m e n t e d w i t h r e a c t i v e compounds from coal dust. in contrast, for cde vs cde + cse; cse vs cde + cse; and cse vs cse + cde, no significant differences w e r e f o u n d . t h i s w a s o n t h e o n e h a n d presumably due to the high level of reactive compounds in cigarette smoke, and on the other hand to reactions between radicals neutralizing one another (termination reactions). superoxide dismutase is an enzymatic antioxidant that catalyzes the conversion of •o2 into h2o2. the function of sod is to convert radicals into oxidants, or reduce the reactivity of radical molecules. the effect of for single exposures, significant increases in blood mda levels versus controls were found in the cigarette smoke exposure group. t h i s f i n d i n g i s s i m i l a r t o t h e s t u d y b y murarescu et al., where increased serum mda levels were found in rats exposed to cigarette smoke.(12) coal dust exposure at a dosage of 12.5 mg/m3 for 14 days failed to significantly increase blood mda levels, compared with the controls. the results of the present study were at variance with the study of armutcu et al., which demonstrated increased serum mda levels in rats exposed to coal dust for 14 days (24 hours per day in underground mines).(9) this discrepancy in results is presumably due to the prolonged daily exposures in the study by armutcu et al. the underlying reason for the different effects of single cigarette smoke exposure (cse) and single coal dust expsosure (cde) is the gaseous form of the active substances in cigarette smoke, leading to diffusion directly of the free radicals to all parts of the lungs. the active substances can also diffuse via the alveolar membrane into the blood stream, where they immediately initiate lipid peroxidation reactions. this is in contrast with coal dust exposure, where the coal dust is initially p h a g o c y t o s e d t o p r o d u c e f r e e r a d i c a l s . lh + oxidant → l• + oxidant-h (initiation) l• + o2 → loo• (propagation) loo• + lh → l• + looh (propagation) l• + l• → non radical products (termination) l• + loo• → non radical products (termination) 86 single cigarette smoke exposures on sod was to significantly reduce sod activity, in comparison to controls, which was also the c a s e w i t h c o a l d u s t e x p o s u r e . t h i s i s presumably caused by the gaseous phase free radicals in cigarette smoke (•o2 -) triggering spontaneous dismutation of sod in the blood, through the diffusion of cigarette smoke gases into the circulation. in the case of single exposures to coal dust, the reduced sod a c t i v i t y w a s d u e t o n o n p h a g o c y t i c mechanisms, in the form of reactivity of i n o rg a n i c c o m p o n e n t s , o r t o p h a g o c y t i c mechanisms resulting in superoxide radical production. the effect of coal dust exposure on blood sod activity in this study was contrary to the study by armutcu et al.,(9) who found no significantly increased sod activity even with exposure times of 24 hours per day. this difference in study results was due to a differences in exposure dose, namely 1.17 mg/ m3 in the study of armutcu et al. and 12.5 mg/ m3 in the present study. in the case of combined exposures, there was a significant decrease in sod activity, as compared to controls or to single exposures. this may be because cigarette smoke and coal dust are sources of •o2 as the substrate of s o d . t h e p o i n t o f i n t e r e s t i s t h e nonsignificancy of single coal dust exposures in comparison with single cigarette smoke exposures, or combined exposures of both, indicating that the formation of •o2 in the circulation, as a result of cigarette smoke gases, has a similar effect as coal dust exposures. beside that, tar phase of cigarettes smoke is an effective metal chelator wherein iron is chelated to produce tar-semiquinone + tar-fe2+, which can inhibit redox properties of metal.(20) a limitation of this study is that we were unable to determine the types of the free radicals produced, in order to determine their respective roles in coal dust and cigarette s m o k e e x p o s u r e s . t h i s i s d u e t o t h e i r considerably high reactivity, requiring electron paramagnetic resonance or electron spin resonance apparatus for identification of their types. conclusions s i n g l e e x p o s u r e t o c i g a r e t t e s m o k e triggered significant increases in blood mda levels and significant decreases ini serum sod activity. single coal dust exposure failed to trigger increases in blood mda levels and only induced decreases in serum sod activity. combined exposures to cigarette smoke and coal dust triggered significant increases in blood mda levels and significant decreases in serum sod activity. acknowledgement the authors thank pt. carsurin coal laboratories for their donation of coal dust with particle size of <75 µm. author also thank to mr. moch. abuhari dan mrs. ferida for technical assistance. references 1. huang x, finkelman rb. understanding the chemical properties of macerals and minerals in coal and its potential application for occupational lung disease prevention. j toxicold environ health part b 2008;11:45-67. 2. huang x, li w, attfield md, nadas a, frenkel c, finkelman rb. mapping and prediction of coal workers’ pneumoconiosis with bioavailable iron content in the bituminous coals. environ health perspect 2005;113:964–8. 3. hendryx m, zullig kj. higher coronary artery disease and heart attack morbidity in appalachian coal mining regions. prev med 2009;49:355-9. 4. lykkesfeldt j, svendsen o. oxidant and antioxidants in disease: oxidative stress in farm animals. vet j 2007;173:502-11. 5. glorie g, legrand-poels s, piette j. nf-κb activation by reactive oxygen species: fifteen years later. biochem pharmacol 2006;72:1493505. 6. romieu i, castro-giner f, kunzli n, sunyer j. air pollution, oxidative stress and dietary supplementation: a review. europ respir j 2008; 31:179-96. kania, setiawan, kusuma coal dust plus cigarette smoke 87 univ med vol.30 no.2 7. pinho ra, silveira pcl, silva la, steck el, dalpizzol f, moreira jcf. n-acetylsisteine and deferoxamine reduce pulmonary oxidative stress and inflammation in rats after coal dust exposure. environ res 2005;99:355-60. 8. halliwell b, whiteman m. measuring reactive species and oxidative damage in vivo and in cell culture: how should you do it and what do the results mean? br j pharmacol 2004;142:231-55. 9. armutcu f, gun bd, altin r, gurel a. examination of lung toxicity, oxidant/antioxidant status and effect of erdosteine in rats kept in coal mine ambience. environ toxicol pharmacol 2007;24:106-13. 10. junior sa, possamai fp, budni p, backes p, parisotto eb, rizelio vm, et al. occupational airborne contamination in south brazil: oxidative stress detected in blood of coal miners. ecotoxicology 2009;18:1150-7. 11. ulker oc, yucesoy b, demir o, tekin io, karakaya a. serum and bal cytokine and antioxidant enzyme level at different stages of pneumoconiosis in coal workers. human exper toxicol 2008;27:871-7. 12. murarescu ed, iancu r, mihailovici ms. morphological changes positive correlates with oxidative stress in copd. preliminary data of an experimental rat model-study literature review. romanian j morphol embryol 2007;48:59-65. 13. stell, rgd, torrie h. principles and procedures of statistics, a biometrical approach 2nd ed. singapore: mcgraw hill book company;1991. 14. ghanem mm, porter d, batteli la, valyathan v, kashon ml, ma jy, et al. respirable coal dust particle modify cytochrome p4501a1 expression in rat alveolar cells. am j respir cell molecular biol 2004;31:171-87. 15. xi yd, hou xm, hong y, wei lx, ke z, chang w, et al. physicochemical properties and potential helath effects of nanoparticles from pulverized coal combustion. chine sci bull 2009;54:124350. 16. donaldson k, tran l, jimenezi la, duffin r, newby de, mills n, et al. combustion-deroved nanoparticles: a review of their toxicology following inhalation exposure. part fibre toxicol 2005;2:10. 17. simeonova pp. nanoparticle exposure and systemic/cardiovascular effects-experimental data. nanotechnol toxicol issues environ safety 2007:53-64. 18. brook rd, rajagopalan s, pope a, brook jr, bhatnagar a, diez-roux av, et al. particulatte matter air pollution and cardiovascular disease: an update to the scientific statement from the american heart association. circulation 2010; 121:2331-78. 19. gregory cd, pound jd. microenvironmental influences of apoptosis in vivo and in vitro. apoptosis 2010;15:1029-49. 20. biswas sk, rahman i. environmental toxicity, redox signalling, and lung inflammation. mol aspect med 2009;30:60-76. editorial 135 high dose simvastatin and adverse muscle effects yenny department of pharmacology & therapeutics medical faculty, trisakti university univ med vol. 30 no.3 high blood cholesterol, or hyperlipidemia, is a cardiovascular risk factor. surveys have demonstrated that 35.6% of adults in the united states suffer from hyperlipidemia.(1) currently there are various therapeutic regimens available for hyperlipidemia. the use of lipid-lowering drugs, patient education, dietary modification, and exercise have all been recommended for the management of hyperlipidemia.(2) among the lipid-lowering drugs, the most effective and best-tolerated agents are the statins, which decrease ldl-cholesterol production in hepatoctyes by competitive inhibition of 3-hydroxy3-methylglutaryl coenzyme a (hmg-coa) reductase. because of their ldl cholesterol–lowering potency, the statins are the most effective in reducing cardiovascular risks, with the highest decrease in serum ldl-cholesterol being due to rosuvastatin 40 mg (63%), atorvastatin 80 mg (57%), and simvastatin 80 mg (46%).(3) however, statin therapy also has a high incidence of adverse effects, particularly affecting skeletal muscle and the liver, with the statin-induced myopathies being the most recognized. statin myopathies range from asymptomatic increases in creatine kinase concentration to muscle aches or weakness to fatal rhabdomyolysis (in ascending order of severity). the risk of statin-induced myopathy increases with the lipophilicity, cholesterol-lowering potency, and dosage of the drugs. with the exception of cervastatin, all lipophilic statins (simvastatin, fluvastatin, lovastatin, atorvastatin) are metabolized by cytochrome p450 3a4 (cyp3a4) enzymes via first-pass metabolism in the gastrointestinal tract and liver. inhibition of first-pass metabolism by competing substances using the same pathway may increase statin toxicity from 5% to 100%.(4) damage to skeletal muscle is commonly assessed by determining the concentration of creatine kinase (ck), an enzyme essential for maintaining atp stores in skeletal muscle.(4) most definitions of myopathy involve a higher than tenfold rise in serum creatine kinase concentration.(5) in the study of the effectiveness of additional reductions in cholesterol and homocysteine (search),(6) involving approximately 12,000 subjects with past myocardial infarction and randomly assigned in approximately equal numbers to receive 80 mg and 20 mg simvastatin, myopathy was defined as a serum creatine kinase level exceeding 10 times the upper limit of normal, accompanied by unexplained muscle weakness or pain. the most severe form of statin myopathy is rhabdomyolysis, with muscle cell destruction or enzyme leakage. rhabdomyolysis is defined as unexplained muscle pain or weakness with a serum creatine kinase level of more than 40 times the upper limit of normal.(7) the search trial found 98 (1.6%) cases of definite or incipient myopathy among the 6,000 subjects on 80 mg simvastatin, during approximately 6 years of follow-up, but only 2 definite and 6 incipient cases of myopathy in the 20 mg group (0.1%). although the search 136 univ med and other clinical trials have generally found a relatively percentage of statin-induced myopathy (<5%), in clinical practice, myalgias occur in 5–10% of patients receiving statins.(6) the statin myopathies are presumably the result of inhibition of mevalonate synthesis, causing depletion of its metabolites, such as cholesterol, isoprenoids, and coenzyme q19. lack of these substances causes abnormal membrane behaviors, impaired intracellular signaling, and decreased mitochondrial respiratory function, respectively. in a genomewide association study, as part of the search trial, genetic predisposition to statin myopathy was demonstrated to be associated with a variant of the slco1b1 gene that is common among individuals of european ancestry.(6) the slco1b1 gene encodes an organic anion transporter that regulates the hepatic uptake of statins and other drugs. single nucleotide polymorphism (snp) analysis of slco1b1 found a strong association of myopathy with the rs4363657 and rs4363656 snps within the slco1b1 gene. the rs4363656 c allele has a prevalence of 15% in the population. the cumulative risk for myopathy in individuals taking 80 mg daily of simvastatin was 18.0% for cc homozygotes, 3.0% for the ct genotype, and 0.6% for the tt genotype. collective evidence indicates that individuals with the c allele of the rs4149056 slco1b1 genotype have higher statin blood concentrations, suggesting that this allele is a high-risk allele for statin-induced myopathy. the percentage of simvastatin-induced myopathy attributable to variant slco1b1 is approximately 60%. therefore, the incidence of simvastatin myopathy could be reduced by 60% if simvastatin were not prescribed to individuals homozygous or heterozygous for the variant allele. on the other hand, prescribing low-dose simvastatin to heterozygous individuals only, and none to homozygous individuals, would result in a 25% reduction in myopathy incidence. this approach merits to be more fully evaluated.(8) based on the search trial and other data on the risks of high-dose (80 mg) simvastatin, the us food and drug administration (fda) has recently instituted safety-labeling changes and related measures to prevent the prescription of high-dose simvastatin to new patients. if during the year 2012 these measures prove to be ineffective, high-dose simvastatin may ultimately be withdrawn from the market.(9) currently, high-dose simvastatin should be prescribed only to patients who have been using the drug for at least one year without clinically significant muscle toxicity. in addition, because simvastatin is extensively metabolized by the cyp3a4 enzyme system, the drug should not be used concomitantly with other drugs metabolized by cyp3a4, such as antifungal azoles, macrolide antibiotics, hiv protease inhibitors, and nefazone. moreover, simvastatin at a dosage of more than 10 mg should not be used concomitantly with gemfibrozil, cyclosporine, and danazol. amiodarone and verapamil should not be used concomitantly with a simvastatin dose of more than 20 mg, while diltiazem should not be used concomitantly with a simvastatin dose of more than 40 mg. references 1. rosamond w, flegal k, furie k, go a, greenlund k, haase n, et al. heart disease and stroke statistics, 2008 update: a report from the american heart association statistics committee and stroke statistics subcommittee. circulation 2008;117:e25–146. 2. third report of the national cholesterol education program (ncep). expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii). available at: http:// www.nhlbi.nih.gov/guidelines/cholesterol/atp3full.pdf. accessed august 1, 2011. 3. smith meb, lee nj, haney e, carson s. drug class review: hmg-coa reductase inhibitors (statins) and fixed-dose combination products containing a statin. final report. update 5. portland: oregon health & science university;2009. available at: http://www.ncbi.nlm.nih.gov/books/nbk47273/pdf/toc.pdf. accessed august 1, 2011. 137 4. di stasi sl, macleod td, winters jd, binder-macleod sa. effects of statins on skeletal muscle: a perspective for physical therapists. phys ther 2010;90:1530–42. 5. eckel rh. approach to the patient who is intolerant of statin therapy. clin endocrinol metab 2010;95:2015– 22. 6. the study of the effectiveness of additional reductions in cholesterol and homocysteine (search) collaborative group. slco1b1 variants and statin-induced myopathy a genomewide study. n engl j med 2008;359:789-99. 7. the study of the effectiveness of additional reductions in cholesterol and homocysteine (search) collaborative group. intensive lowering of ldl cholesterol with 80 mg versus 20 mg simvastatin daily in 12.064 survivors of myocardial infarction: a double-blind randomised trial. lancet 2010;376:1658– 69. 8. nakamura y. pharmacogenomics and drug toxicity. n engl j med 2008;359:856-8. 9. egan a, colman e. weighing the benefits of high-dose simvastatin against the risk of myopathy. n engl j med 2011;365:285-7. vol. 30 no.3 alvina 45 abstract the prevalence of obesity in indonesia is rapidly increasing, particularly in older people. obesity is characterized by increased percentage of body fat in the form of visceral fat and non-visceral or subcutaneous fat. the aim of this study was to analyze body fat percentage (bfp) and associated risk factors, i.e. type of residence (rural or urban), physical activity, gender, age, intakes of energy and fat, and socio-economic background (educational level and occupational status). this cross-sectional study was conducted on 812 older persons (517 females and 295 males) from december 2007february 2008 in the cities of surabaya, semarang, yogyakarta, wonogiri, gunung kidul, and magetan subdistricts. bfp was assessed using an omron fat analyzer. nutritional intakes were collected through interviews using semi-quantitative food frequency questionnaire (ffq). to obtain overall total energy expenditure for physical activity (pa), the energy expenditures for exercise (sports) pa, daily activities, and leisure time pa were added together. the study results indicated that urban residence and light pa at age 55 years constituted risk factors for high bfp. light pa at 55 years of age was the most influential risk factor, since it was 4.3 times greater than vigorous pa at the same age (or=4.3; 95% confidence interval 2.6-7.1) it is recommended to implement nutritional counseling about adequate intakes for increased pa in all age groups (adolescents, adults, older persons), particularly in urban areas with their greater risk of high bfp. keywords: body fat percentage, older persons, rural, urban *department of public health nutrition, faculty of public health, university of indonesia correspondence dr. fatmah, skm, msc department of public health nutrition, faculty of public health, university of indonesia kampus ui depok 16424 phone: 021-7863501 email: ffatmah@yahoo.com univ med 2011;30:45-53 light physical activity increased body fat percentage in elderly javanese fatmah* and yusran nasution* january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction the prevalence of obesity is rapidly increasing in both developed and developing countries. this disorder is steadily increasing in older persons, leading to increased mortality due to degenerative diseases, such as diabetes, hypertension, stroke, and coronary heart disease (chd). obesity is characterized by increased total body fat in the form of visceral fat and non-visceral or subcutaneous fat. both forms of fat increase in quantity with advancing age up to the age of 40 years in males and 50 years in females, stabilize up to the age of 70 years 46 fatmah, nasution physical activity body fat percentage and subsequently decline due to loss of adipose tissue. in addition, more of the body fat in older persons is intramuscular and intra-abdominal.(1) concurrently, lean mass [muscle mass] declines steadily with age, resulting in an initial increase of body fat percentage (bfp) with age (with peak level in middle age), followed by a decrease and finally a leveling-off in old age.(1) body fat acts as a reservoir of energy, protects the body in times of illness, protects vital organs against injury (mechanical shock protection), and maintains body temperature (thermal protection). however, excessive accumulation of body fat leads to medical complications in o l d e r p e r s o n s , i . e . t h e e m e rg e n c e o f degenerative diseases. obesity has become a public health problem, as it is associated with metabolic diseases, among others hypertension, dyslipidemia, and atherosclerosis, all of which may increase the risk of chd. accumulation of adipose tissue, particularly in the abdominal or visceral region, plays an essential role in the development of metabolic syndrome, chd, or both.(2) data from the indonesian basic health research survey for 2007 (riskesdas 2007) indicate that the national prevalence of obesity at age 14 years and over is 19.1%, with waisthip circumference (whc) as indicator.(3) the prevalence of obesity is higher in urban areas as compared to rural. the prevalence of obesity is proportional to monthly per capita household expenditure. it is to be regretted that no national data are available on the prevalence of obesity in older persons in indonesia, but only data from local studies. a study of 296 older persons in jakarta in the year 2008 found that 27.5% of the males and 22.2% of the females were overweight.(4) there are also e x t r e m e l y f e w ( a n d r e l a t i v e l y o b s c u r e ) e p i d e m i o l o g i c a l d a t a o n b f p i n o l d e r i n d o n e s i a n s . d e m o g r a p h i c c h a n g e s i n indonesia in the last decade have resulted in increased longevity, such that the numbers of older persons are expected to increase. the availability of information on bfp is an urgent need in the prevention of obesity in older persons. these data may be used as input for governmental, private, and other interested parties in designing health programs for older persons, particularly for prevention of obesity in indonesia. there is thus a need for a study on bfp as one of the indicators for assessing the risk of obesity in older persons, in order to obtain data on prevalence of obesity in older persons. ethnic javanese were selected as study subjects, as they constitute the largest ethnic group in indonesia.(5) the aim of the present study was to determine bfp level and associated risk factors in javanese older persons aged 55 years and older. methods design of the study this cross-sectional study was conducted from december 2007 up to february 2008 to determine the risk factors of bfp. study subjects a total of 812 older persons (517 females and 295 males) participated in this study. sample size was determined on the basis of calculations for multiple regression analysis.(6) inclusion criteria of the respondents were: ethnic javanese males and females aged over 55 years, apparently healthy, and with good verbal communication. respondents who were ill, suffering from loss of memory, incapable of adequate speech, paralyzed or unable to stand, and unable to completely extend both arms anteriorly, were excluded from the study. twostage stratified random sampling was used to select the study area, comprising 2 stages of s e l e c t i o n , i . e . a t s u b d i s t r i c t a n d v i l l a g e (kelurahan) level. the subdistricts and villages were randomly selected from a list of all subdistricts and villages in the province. the study sample was taken by simple random sampling from 54 villages in 18 subdistricts. 47 assessment of dietary intake and physical activity data on daily energy and protein intakes were collected through interviews using semiquantitative food frequency questionnaire (ffq), while data on pa at age 55 were collected by means of a structured questionnaire. the respondents were questioned about their history of food consumption and physical activities at 55 years. the purpose of collecting data at this point in time was to find possible associations of nutritional intakes and physical activities in old age with bfp at the time of interview. bfp was measured by a trained nutritionist using an omron body fat analyzer hbf 352. bfps were grouped into the following categories: i) normal 20-30% (women) and 10-20% (men); ii) near high 30-35% (women) and 20-25% (men); and iii) high > 35% (women) and 25% (men).(7) daily dietary intakes of a selection of foods (in grams) were calculated from the frequency of consumption of the foods, as follows. depending on the food frequency time units ( d a i l y, w e e k l y, m o n t h l y, a n n u a l l y ) , t h e frequencies were divided by the appropriate number of days, as illustrated for carbohydrate in table 1. the columns marked with a cross represent the reported food frequencies, while the rightmost column shows the calculated daily food intakes in grams. total energy and protein intakes were obtained from analysis of nutritional data using the nutrisoft software program from the center for nutrition research and development (puslitbang gizi) in bogor for the year 2005. inputs for the nutrisoft program were the quantities of each food type consumed (in grams), while the outputs were the daily intakes of all nutrients making up each type of food. a d e q u a c y o f n u t r i t i o n a l i n t a k e w a s assessed by comparison with indonesian recommended dietary allowances (indonesian rda, irda) based on age and gender.(8) nutritional intake was deemed adequate if meeting the minimum of 80% irda and inadequate if below 80% irda. data on physical activity (pa) comprised daily activities (da), daily leisure time pa, and exercise (sports) pa at age 55 years. types of da were adjusted to the type of occupation at age 55 years. in sports pa, three items were evaluated, i.e. type of exercise, duration per week, and frequency per week. type of exercise was divided into 3 categories, i.e. light (weight = 3.5), moderate (5.5) and vigorous (7.5), on the basis of durnin and passmore categories.(9) duration of exercise per week in hourly units was scored on a scale of 1-5 as follows: i) score = 1 for a duration/week of <1 hour; ii) score = 2 for a duration of 1-2 hours/week; iii) score = 3 for a duration of 2-3 hours/week; iv) score = 4 for a duration of 3-4 hours/week; and v) score = 5 for a duration of >4 hours/week. frequency of exercise per week was also scored o n a s c a l e o f 1 5 , i n w h i c h s c o r e s 1 – 4 corresponded to the actual weekly frequency, while a score of 5 was given for a weekly frequency of >4 times. food type frequency of consumption household measures (gram) >1x per day 1x per day 3 –5 x per week 1-2 x per week 2 –3 x per month 1x per month 1-2x per year never carbohydrate rice x 2/7 x 100 biscuits or crackers x 2/365 x 50 table 1. frequencies of food consumption reported by the subjects univ med vol.30 no.1 48 fatmah, nasution physical activity body fat percentage to find the energy expenditure per minute for a given type of exercise, the score for weekly duration of exercise was multiplied by weekly f r e q u e n c y s c o r e , t h e n d i v i d e d b y 7 a n d multiplied by 60, and finally multiplied by the weight of exercise type. weighting was applied to all das at 55 years.(9,10) for more than one type of da performed, the weight of each a c t i v i t y w a s c a l c u l a t e d s e p a r a t e l y, t h e n multiplied by duration/day and by 60. duration of da was scored similarly to weekly scores, and calculation of daily energy expenditure per minute was also similar to that for weekly scores. for more than one type of da, the energy expenditures for each type were added to yield the total daily energy expenditure. leisure time activity was weighted according to severity of activity,(8,9) and the weights were multiplied by the score for duration per day, which was identical to abovementioned da scores. calculation of daily energy expenditure for leisure time activity was similar to that for das. to obtain overall total energy expenditure for pa, the energy expenditures for exercise (sports) pa, da, and leisure time were added together. level of pa was categorized as light, moderate, and vigorous, on the basis of overall total energy expenditure. a high energy score corresponds to a high level of pa. body mass index (bmi) was calculated as body weight (kg) divided by the square of height (m). statistical analysis to compare means and standard deviations b e t w e e n i n d e p e n d e n t v a r i a b l e s ( t y p e o f residence, gender, age, educational level, physical activity at age 55, and intakes of energy and fat at age 55) and bfp as dependent variable, the data were analyzed using paired-t tests. multiple logistic regression analysis was used to determine the relationship between bfp status (high and normal) and associated risk factors. all statistical analyses were performed by means of the spss program version 13. values of p<0.01 and p<0.05 were considered significant. variables bfp status p high (n,%) near high (n,%) normal (n,% ) type of residence urban rur al gender male female age gr oup (yrs) 55-65 66-85 educational level low high employment unemployed informal sector formal sector physical activity light moderate vig orous 28 8 (63.9) 13 7 (38.0) 12 8 (43.4) 29 7 (57.4) 31 5 (51.0) 11 0 (56.7) 21 4 (44.5) 21 1 (73.7) 20 9 (65.5) 15 1 (38.1) 65 (67.0) 15 5 (57.2) 15 7 (57.9) 11 3 (41.9) 119 (26.4) 111 (30.7) 89 (30.2) 141 (27.3) 178 (28.8) 52 (26.8) 149 (31.0) 81 ( 24.5) 85 (26.6) 124 (31.3) 21 (21.6) 80 (29.5) 76 (28.0) 74 (27.4) 44 (9.8) 113 (31.3) 78 (26.4) 79 (15.3) 125 (20.2) 32 (16.5) 118 (24.5) 39 (11.8) 25 (7.8) 121 (30.6) 11 (11.3) 36 (13.3) 38 (14.0) 83 (30.7) 0.001 0.001 0.001 0.001 0.001 0.001 table 2. distribution of bfp status by type of residence, gender, age group,educational level, employment, and physical activity level at age 55 years 49 results half of the respondents (52.3%) had a high bfp, while 28.3% had near high and 1 9 . 3 % n o r m a l b f p. b a s e d o n t h e b m i categories, 56.7% of the study subjects was in the normal bmi category and 33.1% was o v e r w e i g h t o r o b e s e . t h e f r e q u e n c y distribution of bfp by type of residence, gender, age, educational level, employment, and level of pa at age 55 years is presented in table 2. overall, total proportions of bfp status were significantly different for pa levels at the age of 55 years. the percentage of high bfp status was smallest and normal bfp status was greatest in older persons with high pa level. high pa level was proportional to normal bfp status and inversely proportional to high bfp status. based on type of residence, the proportion of high bfp in urban areas was greater than in rural areas, while normal bfp was more frequent in rural areas. there were differences in high, near high, and normal bfp between rural and urban areas. the proportions of bfp at all levels (high, near high, normal) were higher in females in comparison with males, while normal bfp was more frequent in m a l e s . t h e p r o p o r t i o n o f h i g h b f p w a s variable coefficient s.e. p wald or 95.0% c.i. or urban type of residence 0.68 60 0.2229 0.0021 1.9858 1.2829 3.0737 ligh t ph ysical activity at 55 years 1.46 39 0.2545 0.0000 4.3230 2.6251 7.1189 constant -0.0457 0.1434 0.7501 0.9554 table 4. multiple logistic regression analysis of bfp risk factors significantly greater in the age group of 66-85 years, compared with the age group of 55-65 years, whereas for normal bfp the converse was true (more frequent at 55-65 years). thus it may be concluded that bfp at 55-65 years was significantly lower than bfp at 66-85 years. regarding pa at age 55 years, older persons with light pa had slightly higher bfps than those with heavy pa. da was significantly different from bfp proportion. the proportion of high bfp status was higher in older persons with light pa compared with those with vigorous pa, which may be due to the fact that the latter expended more energy for performing pa. table 3 shows mean bfp level by intakes of energy and fat at age 55 years. there was a significant difference in high and normal bfp status for an adequate level of energy intake at age 55 years. multiple logistic regression analysis of bfp risk factors is presented in table 4. the most influential risk factor on bfp status was light pa (or=4.3; 95% confidence interval= 2.6-7.1), indicating that older persons with light pa had a probability of 4.3 times that of older persons with vigorous pa in possessing a high bfp status, after adjustment for urban type of residence. table 3 mean bfp level by energy and fat intakes in respondents at age 55 years variable mean bfp level p high mean ± sd near high mean ± sd normal mean ± sd adequacy of energy intake inadequate (< 80% irda) 36.3 ± 5.4 29.0 ± 5.2 21.3 ± 5.5 < 0.05 adequate (>=80% irda) 35.8 ± 5.6 28.6 ± 5.1 21.2 ± 6.4 < 0.05 fat intake 29.7 ± 23.6 27.5 ± 16.4 30.4 ± 24.2 > 0.05 univ med vol.30 no.1 50 fatmah, nasution physical activity body fat percentage f i g u r e 1 s h o w s t h e e x i s t e n c e o f a correlation between bmi and bfp (r = 0.641), while table 5 describes the pattern of increases and/or decreases in bfp by increases in body height and weight. in both older men and women, higher body weight was proportional to mean bfp, whereas older women had higher mean bfp than men. figure 1. correlation between bmi and bfp table 5 mean bfp by height, body weight, and gender discussion the differences in bfp status are caused by life style and dietary habits of urban older persons, who consume more high-fat foods in comparison to rural older persons. this is presumably because urban older persons have high intakes of cholesterol and energy as a result of different dietary patterns. these findings are at variance with those of a study on the dietary intakes of rural and urban older persons in poland. the energy and macronutrient content of the foods consumed by both groups was similar, except for intakes of several vitamins and minerals, particularly vitamins a and c, which were lower in rural older persons.(11) the proportion of normal bfp was higher in males than in females, as the latter have a higher bfp than the former. females have lower pa levels and energy expenditures, and higher energy intakes, while males have a 51 larger lean mass and higher pa levels, and most of their body is composed of muscle.(12) the differing proportions of the three bfp levels based on age are due to increases in body fat and loss of muscle mass (lean mass) with age in both genders. older females have entered the menopause, when there is a decreased functioning of the ovaries and estrogens, resulting in increased absorption of calcium. menopause is a significant predictor of decrease in or loss of muscle mass as a result of estrogen deficiency. the results of the present study are consistent with a study in elderly aged 70–79 years in pittsburgh, pa, and memphis, tn, both in the us. these data showed that, in older men and women, later cohorts had a greater bfp than did earlier cohorts. the increase in bfp was due to a larger increase in fat mass and a moderate increase in lean mass. lean mass was found to decline with age, while fat mass initially increased, but subsequently decreased around the age of 80 years. these changes caused an initial increase in bfp with age, followed by a leveling-off in both older men and women.(13) on the basis of educational level, the present study results indicate that the high educational level group had a greater proportion of high bfp status than the low education group, whereas in the low education group there was a greater proportion of normal bfp status. this is because educational level affects income and ultimately impacts of purchasing power with regard to foods. an age-related decline in physical activity was observed among loweducation individuals. for low-education individuals, unemployment and job losses were associated with reduced pa, whereas for highly educated individuals the reverse was true.(14) the differences in bfp status based on pa level are due to differences in energy expenditure in older working and non-working persons. the lack of a significant relationship between bfp status on the one hand and intakes of energy and fat at age 55 years on the other may be due to the high level of pa of the repondents. when consumption of foods rich in energy and fat is balanced by high levels of pa, then the input calories will be converted into energy, thus there is no accumulation of calories. the lack of a significant relationship between high fat intakes and bfp is similar to the results of a norwegian study on the influence of dietary fat intake on body fat in middle-aged (47-49 years) and older (71-74 years) persons, in men as well as in women.(15) h o w e v e r, b o t h s t u d i e s f a i l e d t o f i n d a relationship between energy intakes and bfp. a high-fat diet may cause increased body fat, since it has a high energy density, while total energy may also be affected by intakes of other macronutrients low in energy, namely proteins and carbohydrates. older persons who do not perform active work have a higher bfp, as inactivity affects the distribution of body fat, thus increasing the number of cases of obesity. this is consistent with the results of a study on aging, body composition, and life style in older persons,(16) where physical work was associated with a decrease in bfp in male and female older persons. a somewhat different result was found in new england, where bfp was also inversely related to pa (r= 0.37, p=0.007), but in elderly men only.(17) the changes in bfp in overweight and obese older persons were due to increased pa. these findings were contrary to the results of a study on variations in bfp and bmi in spanish older persons.(18) bfp in older persons depends on age, menopausal status, and body weight. increased body weight due to body fat may increase bfp. with normal aging there is a progressive increase in fat and decrease in fat-free mass, the latter being mainly due to loss of skeletal muscle. consequently, at any given weight, older people, on average, have substantially more body fat than young adults. the increase in body fat with aging is multifactorial in origin, with decreased pa as a major cause, a n d c o n t r i b u t i o n s f r o m r e d u c e d g r o w t h hormone secretion, declining sex hormone univ med vol.30 no.1 52 fatmah, nasution physical activity body fat percentage action and reduced resting metabolic rate, and t h e r m i c e ff e c t o f f o o d . t h e r e w a s a proportional relationship between increased body weight and bfp in both genders. body weight consists of fat mass and lean body mass, a n d w i t h i n c r e a s i n g b o d y w e i g h t t h e percentage of body fat and visceral fat also increases. (19) in older persons there is an increase in body weight as a result of increased e n e r g y i n t a k e s a n d d e c r e a s e d e n e rg y expenditures through pa. female older persons have a higher bfp than males, due to the fact that accumulation of fat in males takes place in the upper body (waist circumference, waist/ hip ratio, subscapular/triceps). the body of a woman is naturally built for protecting itself and the fetus in pregnancy, therefore it has numerous enzymes for accumulating and burning fats. estrogen promotes replication of the enzymes involved in the building of fat reserves. (20) there were several limitations in this study. first, the cross-sectional design of the study, assessing all independent and dependent variables at a given point in time, has the disadvantage of not being able to find causeand-effect relationships between bfp and associated risk factors. there is also the possibility of recall bias when the respondents have to recall their sports activities, adls, leisure time pa, type and mean level of consumption of carbohydrate and protein sources, as well as their frequencies, at age 55 years. the sq-ffq instrument has several advantages in collecting data on longterm dietary consumption, as it is capable of determining the types of foods commonly consumed by older persons and does not affect their dietary habits. the validity of the ffq method is relatively high, in comparison with other methods. one study on post-menopausal w o m e n i n c h i n a ( 2 1) f o u n d t h a t f f q i s sufficiently valid in describing the dietary habits of the respondents, since ffq measures t h e v o l u m e o f f o o d s c o n s u m e d b y t h e respondents and not the weight of the foods. h o w e v e r, t h i s m e t h o d h a s s e v e r a l disadvantages, as it requires recall of past dietary patterns, where current intakes may influence reports of past intakes. the method a l s o n e e d s a c c u r a c y i n e s t i m a t i n g f o o d frequency and servings, and there is limited possibility for inserting food specifications into the questionnaire, particularly of local foods not listed in the nutrisoft software program for analysis of nutritional content. in addition, the relatively long interview period of 30 minutes per respondent results in a high level of inattention, such that the respondent m a y u n t h i n k i n g l y m e n t i o n f o o d t y p e s , frequency of consumption and food servings. the cross-sectional study design and the inherent recall bias of the sq-ffq method may be the cause of the lack of association between energy and protein intakes and bfp. acknowledgements the investigators wish to express their gratitude to all parties who assisted in this study and enabled the study to be conducted, namely the health services of the participating cities and districts, the local primary health centers, the cadres of the comprehensive health service programs for older persons (posyandu lansia) and all older persons as respondents in this study. references 1. ghosh a. age and sex variation in measures of body composition among the elderly bengalee hindus of calcutta. coll anthropol 2004;2:553– 61. 2. hughes va, frontera wr, roubenoff r, evans wj, singh maf. longitudinal changes in body composition in older men and women: role of body weight change and physical activity. am j clin nutr 2002;2:473-81. 3. badan litbang depkes ri. laporan nasional riset kesehatan dasar (riskesdas) 2007. jakarta: badan litbangkes depkes ri; 2008. 4. kusumaratna r, hidayat a. body mass index and quality of life among the elderly. univ med 2009; 28:34-41. 53 5. aris a, arifin en. report on ageing at provinces and districts in indonesia. singapore: institute of southeast asian studies singapore;2005. 6. dawson b, trapp rg. basic & clinical biostatistics. 4th ed. new york: mcgrawhill; 2004. 7. omron. health management by checking the body fat (use of fat analyzer scale). 2006. 8. biro pusat statistik. prosiding widyakarya nasional pangan dan gizi dan gizi viii ketahanan pangan dan gizi di era otonomi daerah dan globalisasi. jakarta;2008. 9. passmore r, durnin jvga. human energy expenditure. department of physiology, university of edinburgh, edinburgh, scotland and the institute of physiology, university of glasgow, glasgow, scotland;1995. 10. fao/who/unu. energy and protein requirements. geneva: who;1991. 11. szponar r. dietary intake elderly subjects in rural and urban area in poland. pol mekur lekarski 2002;78:490-6. 12. odenigbo um, odenigbo cu, oguejiofor oc. nutritional status of elderly in asaba, delta state, nigeria. pak j nutr 2010;5:416-8. 13. ding j, kritchevsky sb, newman ab, taaffe dr, nicklas bj, visser m, et al. for the health abc study. effects of birth cohort and age on body composition in a sample of community-based elderly. am j clin nutr 2007;85:405–10. 14. shaw ba. examining the association between education level and physical activity changes during early old age. j aging health 2008;20: 767-87. 15. vinknes kj, de vogel s, elshorbagy ak, nurk e, drevon ca, gjesdal cg, et al. dietary intake of protein is positively associated with percent body fat in middle-aged and older adults. j nutr 2011;141:440-6. 16. kromhout d, bloemberg b, seidell jc, nissinen a, menotti a. physical activity and dietary fiber determine population body fat levels: the seven countries study. int j obes relat metab disord 2001;25:301–6. 17. hughes va, roubenoff r, wood m, frontera wr, evans wj, singh maf. anthropometric assessment of 10-y changes in body composition in the elderly. am j clin nutr 2004;80:475–82. 18. gutierrez-fisac jl, lopez e, banegas jr, graciani a, rodriguez-artalejo f. prevalence of overweight and obesity in elderly people in spain. obes res 2004;12:710–5. 19. jackson as, stanforth pr, gagnon j. the effect of sex, age and race on estimating percentage body fat from body mass index: the heritage family study. int j obes relat metab disord 2002; 6:789-96. 20. bedogni g, pietrobelli a, heymsfield sb, borghi a, manzieri am, morini p, et al. is body mass index a measure of adiposity in elderly women? obes res 2001;1:17-20. 21. xua1 l, dibley mj, d'este c. reliability and validity of a food-frequency questionnaire for chinese postmenopausal women. public health nutr 2003;7:91–8. univ med vol.30 no.1 alvina 139 hepatic enzyme concentrations as indicators of nonalcoholic fatty liver disease september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina alvina* *department of clinical pathology, medical faculty, trisakti university correspondence dr. alvina, sppk department of clinical pathology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2404 email: vina_march_dr@yahoo.com univ med 2009;28:139-45 abstract nonalcoholic fatty liver disease (nafld) has emerged as a world-wide problem because it runs an asymptomatic course, ultimately leading to cirrhosis of the liver and portal hypertension, resulting in death. the prevalence of the disease accounts for 3-24% of the population in several countries. generally there are increased concentrations of hepatic enzymes as markers of liver damage, such as serum glutamic oxaloacetic transaminase (sgot), serum glutamic pyruvic transaminase (sgpt) and gamma glutamyl transferase (ggt). the aim of the present study was to determine the concentrations of hepatic enzymes as markers of nafld. the study design was cross-sectional, involving 90 subjects meeting the inclusion and exclusion criteria. the degree of severity nafld was determined by ultrasonography and the concentrations of sgot, sgpt and ggt by automated clinical chemistry analyzer. the results indicated that there were 32 subjects with mild nafld (35.6%), 35 subjects with moderate nafld (38.9%) and 23 subjects with severe nafld (25.6%). there was a significant difference in degree of nafld by gender (p<0.05), where severe nafld was more frequent in males than in females. concentrations of sgot, sgpt and ggt were significantly different between degrees of nafld (p<0.05). the conclusion is that sgot, sgpt and ggt concentrations are indicators of degree of nafld. keywords: nonalcoholic fatty liver disease, liver enzyme concentrations, ultrasonography introduction nonalcoholic fatty liver disease (nafld) is a pathological clinical condition with a wide spectrum, including steatosis and steatohepatitis, with a potential for cirrhosis and hepatoma. the incidence of nafld varies with ethnicity or culture and with gender.(1) according to the american association for the study of liver disease, nafld is a condition characterized by excessive accumulation of fat in the liver of 510% of the weight of the liver and the presence 140 alvina nonalcoholic fatty lever disease of fat-containing hepatocytes on microscopic examination.(2) nafld is a sufficiently important problem because it runs an insidious asymptomatic course and ultimately causes cirrhosis of the liver and portal hypertension, culminating in death.(3) the prevalence of nafld has been reported to account for 3-24% of the population of several countries, in parallel with the concomitant increase in the prevalence of diabetes and obesity, while the disease is the leading cause of liver disease in western countries.(4-6) nafld is also associated with type 2 diabetes, as 70% of patients with type 2 diabetes has steatosis, as determined by ultrasonography.(7) in addition, visceral obesity in adults is considered to be the main risk factor for developing steatosis.(1) the majority of patients with nafld are asymptomatic, although commonly having elevated circulatory concentrations of markers of hepatic damage, such as serum glutamic o x a l o a c e t i c t r a n s a m i n a s e ( s g o t ) , s e r u m glutamic pyruvic transaminase (sgpt) and gamma glutamyl transferase (ggt).(4,8) sgpt is closely associated with accumulation of fat in the liver, such that in epidemiological studies sgpt is commonly used as surrogate marker of nafld. elevated concentrations of sgpt are frequently associated with obesity and the metabolic syndrome.(4,9,10) ggt is considered to be a sensitive but nonspecific indicator of hepatic d a m a g e a n d i s t h e m a i n c o m p o n e n t o f i n t r a c e l l u l a r a n t i o x i d a n t d e f e n s e s . t h e association of ggt with diabetes is due to h e p a t i c d y s f u n c t i o n a s a r e s u l t o f f a t t y i n f i l t r a t i o n o f t h e l i v e r a n d t h i s e ff e c t i s considered to be capable of increasing oxidative stress.(11) o n t h e b a s i s o f t h e a b o v e m e n t i o n e d discussion, the purpose of the present study was to determine the concentrations of hepatic enzymes as markers of nafld. methods research design the study was designed as a cross-sectional study, conducted from april to september 2008 in hospital x in jakarta. study subjects the study subjects were patients with nafld, chosen consecutively from outpatients visiting the hepatology clinic of hospital x and meeting the following inclusion criteria: male and females aged 20-66 years, suffering from nafld as proven by ultrasonography (usg). the exclusion criteria were alcoholics and/or patients with hepatitis b and/or hepatitis c. on usg examination, nafld may be graded into t h r e e s t a g e s , n a m e l y m i l d , m o d e r a t e , a n d severe.(12) in mild nafld there is hepatomegaly with increased echogenicity of the hepatic parenchyma, designated as bright liver, where the diaphragmatic dome and all hepatic vascular structures are clearly visualized. in moderate nafld, in addition to increasing echogenicity of the hepatic surface, there is decreased e c h o g e n i c i t y o f t h e p a r e n c h y m a o f t h e subsurface areas, the diaphragm is somewhat hypoechogenic and the peripheral hepatic vascular structures become increasingly difficult to recognize. in severe nafld, the echogenicity of the hepatic surface is enhanced and most of t h e s u b s u r f a c e h e p a t i c p a r e n c h y m a i s hypoechogenic whilst the diaphragm and the hepatic vascular structures are difficult to identify. measurements the specimens to be examined, consisting of 5 ml venous blood taken from the cubital vein were collected in tubes without anticoagulant. t h e s e r u m w a s s e p a r a t e d f r o m t h e b l o o d maximally one hour after collection. the blood 141 was centrifuged at 3000 rpm for 10 minutes, then t h e s e r u m w a s p u t i n a s a m p l e c u p f o r determination of the sgot, sgpt and ggt a c t i v i t i e s . s g o t a n d s g p t l e v e l s w e r e d e t e r m i n e d b y t h e i f c c m e t h o d w i t h o u t p y r i d o x a l p h o s p h a t e a c t i v a t i o n , w h e r e a s determination of ggt was by the szasz method using 66-3’ carboxy-4-nitranilide at 37ºc. the determinations were performed with a hitachi 912 analyzer. data analysis the collected data were analyzed by means of spss version 11.5. chi-square tests were done to analyze the difference of degree of nafld between genders. to compare the hepatic enzyme concentration between degrees of nafld, analysis of variance (anova) was performed. all the reported p values were 2tailed, and values of p<0.05 were considered to be statistically significant. results the study involved 90 persons, consisting of 28 males (31.1%) and 62 females (68.9%), within the age range of 20-66 years. on usg examination, there were 32 subjects with mild nafld (35.6%), 35 subjects with moderate nafld (38.9%) and 23 subjects with severe nafld (25.6%). findings for risk factors for nafld were as follows: 53 subjects with one risk factor (58.9%), 31 subjects with two risk factors (34.4%) and 6 subjects with three risk factors (6.7%). in subjects with one risk factor, the most frequently encountered risk factor was hypercholesterolemia (15.5%), followed in descending order of frequency by diabetes mellitus (14.4%) and obesity (10.0%). in subjects with two risk factors, there were combinations of risk factors, such as obesity and hypercholesterolemia, obesity and d i a b e t e s m e l l i t u s , d i a b e t e s m e l l i t u s a n d hypertension. in subjects with three risk factors, t h e c o m b i n a t i o n s w e r e d i a b e t e s m e l l i t u s hypertension-dyslipidemia and diabetes mellitushypertension-obesity (table 1). among the subjects with mild nafld there were 9 males (28.1%) and 23 females (71.9%). subjects with moderate nafld comprised 7 males (20%) and 28 females (80%) and those with severe nafld consisted of 12 males (52.2%) and 11 females (47.8%). the chi-square t e s t b y g e n d e r a n d d e g r e e o f n a f l d demonstrated a significant difference in degree of nafld between males and females (p<0.05). the proportion of severe nafld in males (42.9%) was significantly higher than in females (17.7%), as may be seen from table 2. a subtantial proportion of the subjects (36.0%) was in the age group of > 50 years. however, the variable n (%) degree of nafld mild 32 (35.6) moderate 35 (38.9) severe 23 (25.6) nafld risk factors dyslipidemia 11 (12.2) dm 13 (14.4) hypercholesterolemia 14 (15.5) hypertriglyceridemia 4 (4.4) hypertension 2 (2.2) obesity 9 (10) dyslipidemia + hypertension 3 (3.3) dyslipidemia + dm 5 (5.5) dm + hypercholesterolemia 3 (3.3) dm + hypertriglyceridemia 1 (1.1) dm + hypertension 10 (11.6) hypercholesterolemia + hypertension 1 (1.1) obesity + dyslipidemia 2 (2.2) obesity + dm 1 (1.1) obesity + hypercholesterolemia 5 (5.5) three risk factors* 6 (6.6) table 1. distribution of degrees and risk factors of nafld *dm-hypertension-dyslipidemia and obesity-dm-hypertension univ med vol.28 no.3 142 alvina nonalcoholic fatty lever disease m a j o r i t y o f s u b j e c t s ( 6 2 . 5 % ) w i t h s e v e r e nafld was in the age group of 20–29 years. in mild nafld the mean concentrations of sgot, sgpt and ggt were 19.1 u/l, 18.1 u/ l, and 23.5 u/l, respectively. in moderate nafld mean sgot concentration was 26.2 u/ l, while mean sgpt was 27.1 u/l and mean ggt 45.2 u/l. in severe nafld mean sgot, sgpt and ggt activities were respectively 58.0 u/l, 64.1 u/l, and 60.7 u/l. anova results showed sgot, sgpt and ggt activities to be significantly elevated in proportion to degree of nafld (p < 0.05) as indicated in table 3. discussion in the present study, among the 90 study subjects there were a higher number of persons with nafld of mild and moderate degree (35.6% and 38.9%, respectively), compared with those with nafld of severe degree. these findings may have been due to the fact that in the present study no distinction was made between subjects with newly acquired nafld and those who had recovered from nafld of severe degree. thus the possibility exists that subjects with nafld of severe degree made a recovery after treatment and thus were entered into the study as nafld of moderate or mild degree. in this study the number of female subjects was higher than that of males, which is supported by the study of de lusong et al.(14) in the philippines in 2005 where it was also found that female nafld patients amounted to 71% of the 134 study subjects.(13) in contrast, amarapurkar et al. in india found more males with nafld than females (24.6% vs 13.6%). the reason why nafld is found more frequently in males than in females may be due to an excess of intraabdominal fat in males, and possibly also to the fact that the distribution of body fat is influenced enzyme mild nafld moderate nafld severe nafld p sgot (u/l) 19.1 ± 7.3 26.2 ± 10.2 58.0 ± 41.5 0.00* sgpt (u/l) 18.1 ± 6.9 27.1 ± 13.1 64.1 ± 48.6 0.00* ggt (u/l) 23.5 ± 10.1 45.2 ± 27.2 60.7 ± 35.3 0.00* table 3. sgot, sgpt and ggt activities in nafld * significant (p<0.05) table 2. characteristics of the study participants by degree of nafld degree of nafld characteristics mild (n=32) moderate (n=35) severe (23) p age (years) 20 – 29 30 – 39 40 – 49 > 50 1 (12.5%) 5 (29.4%) 12 (40.0%) 14 (40.0%) 2 (25.0%) 9 (52.9%) 12 (40.0%) 12 (34.3%) 5 (62.5%) 3 (17.6%) 6 (20.0%) 9 (25.7%) 0.715 gender male female 9(32.1%) 23(37.1%) 7(25.0%) 28(45.2%) 12(42.9%) 11(17.7%) 0.037* * significant (p<0.05) 143 by sex hormones. sex hormone binding globulin (shbg) synthesized in the liver is strongly correlated with insulin sensitivity.(15) the age range of the subjects in the present study was 20-66 years. in another study in pakistan the age was between 47 and 54 years.(16) another investigator also found that nafld was more common in the younger age groups, which may have been due to changes in dietary pattern and the habit of consuming soft drinks, as r e v e a l e d b y a n o t h e r s t u d y i n d i c a t i n g t h a t consumption of soft drinks was associated with an elevated risk of nafld, although the precise mechanism is unclear.(17) according to shibata, the rapid rise of nafld in the younger age groups is the result of a westernized life style, such as is reflected by a high-fat and high-calorie diet and a decline in physical activity.(18) these results provide confirmatory evidence for our study, where severe nafld was most frequent in the age group of 20-29 years. the most frequent risk factors of nafld in the present study were hypercholesterolemia, diabetes mellitus, dyslipidemia, and obesity. the study found the cause of nafld to be the m e t a b o l i c s y n d r o m e , i . e . h y p e r l i p i d e m i a (80.6%), obesity (37%) and type 2 diabetes mellitus (18%).(19) the metabolic syndrome is associated with insulin resistance (ir), which in turn is related to elevated synthesis of free fatty acids from glucose in the liver. in addition, adipose tissue is a source of tumor necrosis factor-á (tnf-á ), which is a regulator of insulin s e n s i t i v i t y. t n f á m a y i n c r e a s e i n s u l i n resistance by depressing peroxisome proliferator activated receptor gamma (pparã), which is an important receptor for maintaining insulin sensitivity within normal limits.(20,21) generally the number of nafld patients increase in proportion to the course of the metabolic syndrome and insulin resistance. the metabolic syndrome is a collection of signs and symptoms c o n s i s t i n g o f d i a b e t e s , c e n t r a l o b e s i t y, hypertension and dyslipidemia.(22,23) in obese patients there is an increased uptake of free fatty acids by the liver, whereafter the acids are subjected to â -oxidation or esterification with glycerol to form triglycerides that play a role in the accumulation of fat in the liver.(24) the study by chang et al.(25) showed an association between sgpt and the incidence of nafld as determined by usg. therefore it may b e c o n c l u d e d t h a t e l e v a t e d s g p t i s a n i n d e p e n d e n t p r e d i c t o r o f n a f l d e v e n t s . because sgpt is a glucogenic enzyme, elevated s g p t i s a l s o a n i n d i c a t o r o f i n s u l i n abnormalities.(26) nafld patients have higher sgpt concentrations in comparison with sgot, and is mainly synthesized in the liver, making sgpt more specific for liver disease.(27,28) an elevated sgpt concentration in asymptomatic individuals is considered of value as a screening test for nafld patients, as sgpt concentrations are correlated with abdominal fat and as higher sgpt concentrations are associated with the degree of steatosis.(1,29,30) the present study also found elevated ggt concentrations proportional to the degree of nafld, thus it may be assumed that ggt is correlated with nafld. the precise mechanisms of the correlation of ggt with nafld is still unclear. however, the explanation suggested by grundy et al.(31) is that accumulation of fat in the liver probably increases oxidative stress, leading to increased consumption of glutathione (gsh). this is compensated for by increased ggt synthesis, as ggt plays a role in glutathione c a t a b o l i s m . g g t i s a l s o i n v o l v e d i n t h e p a t h o g e n e s i s o f d i a b e t e s t h r o u g h a n o t h e r mechanism associated with oxidative stress. the a s s o c i a t i o n b e t w e e n g g t a n d d i a b e t e s i s indicated by continued exposure to organic pollutants, where these latter may interact with obesity, thus resulting in diabetes.(11) univ med vol.28 no.3 144 alvina nonalcoholic fatty lever disease elevated hepatic enzyme concentrations may indicate the presence of excessive fat deposits in the liver, which are indirectly associated with obesity and deposits of visceral fat, now considered as part of the metabolic syndrome.(11) according to tiikkainen et al., elevated liver enzymes are correlated with i n c r e a s e s i n h e p a t i c f a t a n d n a f l d . ( 3 2 ) according to thamer et al. sgot and ggt concentrations are also correlated with hepatic fat independently of obesity, although sgot is less sensitive than sgpt.(33) the present study has several limitations. f i r s t , t h e s t e a t o s i s w a s d e t e r m i n e d b y ultrasonography instead of liver biopsy as the gold standard. however, ultrasonography is s e n s i t i v e , n o n i n v a s i v e a n d p r a c t i c a l f o r determining nafld. but ultrasonography cannot accurately differentiate steatosis from fibrosis.(34) second, the study participants were mostly persons on medical checkup and thus less representative of the general population. third, the sample size was too small. nafld is generally asymptomatic and it is only the development of cirrhosis that has clinical consequence. at present, therapy in nafld concentrates on managing risk factors but in the future clinical trials may provide robust evidence for the use of insulin sensitising agents and other potential therapies.(35) conclusions in patients with nafld the increase in sgot, sgpt and ggt concentrations were independent indicators of the degree of nafld. references 1. rocha r, cotrim hp, bitencourt agv, barbosa dbv, santos as, almeida adm, et al. nonalcoholic fatty liver disease in asymptomatic brazilian adolescents. world j gastroenterol 2009;15:473-7. 2. kotronen a, yki-jarvinem h. fatty liver: a novel component of the metabolic syndrome. arterioscler thromb vasc biol 2008;28:27-38. 3. ahmed mh. the road map for the diagnosis of nonalcoholic fatty liver disease. am j clin pathol 2007;127:20-2. 4. chen zw, chen ly, dai hi, chen jh, fang lz. relationship between alanine aminotransferase levels and metabolic syndrome in nonalcoholic fatty liver disease. j zhejiang univ sci b 2008;9:61622. 5. clark jm. the epidemiology of nonalcoholic fatty liver disease in adults. j clin gastroenterol 2006;40 suppl:s5-10. 6. ong j, younossi z. epidemiology and natural history of nafld and nash. clin liver dis 2007;11:1-16. 7. targher g, bertolini l, padovani r, rodella s, tessari r, zenari l. prevalence of nonalcoholic fatty liver disease and its association with cardiovascular disease amongtype 2 diabetic patients. diabetes care 2007;30:1212-8. 8. clark j, diehl a. defining nonalcoholic fatty liver disease: implications for epidemiologic studies. gastroenterology 2003;124:248-50. 9. marchesini g, avagnina s, barantani e, ciccarone a, corica f, dalle g, et al. aminotransferase and gamma glutamyltranspeptidase levels in obesity are associated with insulin resistance and the metabolic syndrome. j endocrinol invest 2005;28:333-9. 10. schindhelm r, diamant m, dekker j, tushuizen m, teerlink t, heine r. alanine aminotransferase as a marker of nonalcoholic fatty liver disease in relation to type 2 diabetes mellitus and cardiovascular disease. diabetes metab res rev 2006;22:437-43. 11. kim ch, park jy, lee ku, kim jh, kim hk. association of serum gamma-glutamyltransferase and alanine aminotransferase activities with risk of type 2 diabetes mellitus independent of fatty liver. diabetes metab res rev 2009;25:64-9. 12. kim hc, choi sh, shin hw, cheong jy, lee kw, lee hc, et al. severity of ultrasonographic liver steatosis and metabolic syndrome in korean men and women. world j gastroenterol 2005;11:531421. 13. lusong mad, labio e, daez l, gloria v. nonalcoholic fatty liver disease in the philippines: 145 comparable with other nations? world j gastroenterol 2008;14:913-7. 14. amarapurkar d, kamani p, patel n, gupte p, kumar p, agal s, et al. prevalence of nonalcoholic fatty liver disease: population based study. annals of hepatology 2007;6:161-3. 15. schwimmer j, mcgreal n, deutsch r, finegold mj, lavine je. influence of gender, race and ethnicity on suspected fatty liver in obese adolescents. pediatrics 2005;115:e561-5. 16. khurram m, shakoor a, arshad mm, khaar hb, hasan z. characteristic features of 50 nafld patients. 2004: dhq teaching hospital rawalpindi. 17. alwis nd, day c. non alcoholic fatty liver disease: the mist gradually clears. j hepatol 2008;48 suppl 1:s104-12. 18. shibata m, kihara y, taguchi m, tashiro m, otsuki m. nonalcoholic fatty liver disease is a risk factor for type 2 diabetes in middle-aged japanese men. diabetes care 2007;30:2940-4. 19. bahcecioglu ih, koruk m, yilmaz o, bolukbas c, bolukbas f, tuncer i, et al. demographic and clinicopathological characteristics of nonalcoholic fatty liver disease in the east southeastern anatolia regions in turkey. med princ pract 2006;15:62-8. 20. adams la, angulo p, lindor kd. nonalcoholic fatty liver disease. jamc 2005;172:899-905. 21. oneta cm, dufour jf. nonalcoholic fatty liver disease: treatment options based on pathogenic considerations. swiss med wkly 2002;132:493505. 22. olufadi r, byrne c. clinical and laboratory diagnosis of the metabolic syndrome. j clin pathol 2008;61:697-706. 23. reynolds k, he j. epidemiology of the metabolic syndrome. am j med sci 2005;330:273-9. 24. feldstein a, werneburg n, canbay a, guicciardi m, bronk s, rydzewski r. free fatty acids promote hepatic lipotoxicity by stimulating tnf-α expression via a lysosomal pathway. hepatology 2004;40:185-94. 25. chang y, ryu s, sung e, jang y. higher concentrations of alanine aminotransferase within the reference interval predict nonalcoholic fatty liver disease. clin chem 2007;53:686-92. 26. giboney pt. mildly elevated liver transaminase levels in the asymptomatic patient. am fam physician 2005;71:1105-10. 27. harrison sa, tetri bn. clinical manifestations and diagnosis of nafld. in: farrell gc, george j, hall pdlm, mccullough aj, editors. fatty liver disease: nash and related disorders. 1st ed. massachusetts: blackwell publishing;2005. p.159-67. 28. fraser a, longnecker m, lawlor d. prevalence of elevated alanine aminotransferase among us adolescents and associated factors: nhanes 19992004. gastroenterology 2007;133:1814-20. 29. stranges s, dorn j, muti p, freudenheim j, farinaro e, russell m, et al. body fat distribution, relative weight and liver enzyme level: a population-based study. hepatology 2004;39:754-63. 30. nurman a. non alcoholic fatty liver disease. univ med 2007;26:205-15. 31. grundy sm. gamma glutamyl transferase, another biomarker for metabolic syndrome and cardiovascular risk. arterioscler thromb vasc biol 2007;27:4-7. 32. tiikkainen m, bergholm r, vehkavaara s, aila rissanen a, häkkinen am, tamminen m, et al. effects of identical weight loss on body composition and features of insulin resistance in obese women with high and low liver fat content. diabetes 2003;52:701-7. 33. thamer c, tschritter o, haap m, shirkavand f, machann j, fritsche a, et al. elevated serum ggt concentrations predict reduced insulin sensitivity and increased intrahepatic lipids. horm metab res 2005;37:246-51. 34. siegelman es, rosen ma. imaging of hepatic steatosis. semin liver dis 2001;21:71-80. 35. mcavoy nc, ferguson jw, campbell anw, hayes pc. non-alcoholic fatty liver disease: natural history, pathogenesis and treatment. br j diabetes vasc dis 2006;6:251-60. univ med vol.28 no.3 typhonium flagelliforme decreases telomerase expression in hela cervical cancer cells 3 *department of anatomy, faculty of medicine, yarsi university, jakarta **department of biochemistry, faculty of medicine, yarsi university, jakarta correspondence prof. dr. endang purwaningsih, ms, pa department of anatomy, faculty of medicine, yarsi university, jl. letjend. suprapto cempaka putih, jakarta 10510 phone: +6221-4206674/76 email: endpurwaning@gmail.com univ med 2015;35:3-9 doi: 10.18051/univmed.2016.v35.3-9 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 typhonium flagelliforme decreases telomerase expression in hela cervical cancer cells endang purwaningsih*, yulia suciati**, and etty widayanti* background cancer cells have a relatively high telomerase activity compared to normal cells, so that cancer cells have the ability for continued proliferation and uncontrolled mitosis. telomerase is an enzyme responsible for the length of telomeres, dna segments located at the ends of eukaryotic chromosomes. natural materials such as rodent tuber (typhonium flagelliforme) have anticancer potential. the purpose of the present study was to determine the effects of typhonium flagelliforme extract on telomerase expression in hela cervical cancer and t47d breast cancer cells. methods this experimental laboratory study was conducted on cultured hela and t47d cancer cell lines, with normal vero cells as controls, and using rpmi and m199 culture media. the study comprised three groups, i.e. controls, and groups receiving typhonium flagelliforme extract at doses of ½ ic50 and ic50. telomerase expression was measured by immunohistochemistry (ihc). analysis of variance and lsd multiple comparison test were used to analyze the data. results telomerase expression in cancer cells showed significantly higher values compared to normal vero cells. typhonium flagelliforme extract was capable of significantly decreasing telomerase expression in cancer cells receiving the extract. conclusion typhonium flagelliforme extract at different doses is capable of decreasing telomerase expression more effectively in cervical cancer cells than in breast cancer cells. this study shows that typhonium flagelliforme may have anti-cancer activity, necessitating further investigations. keywords: telomerase, ihc, typhonium flagelliforme, hela and t47d cancer cells doi: http://dx.doi.org/10.18051/univmed.2016.v35.3-9 4 purwaningsih, suciati, widayanti typhonium flagelliforme in hela cancer cells introduction the use of traditional medications or natural substances is at present steadily increasing. although modern therapeutics has advanced considerably, it is relatively expensive a n d m a y c a u s e m a j o r a d v e r s e e ff e c t s , particularly in the treatment of cancers, while its active compounds are not or less selective in killing cancer cells. in addition, the management of tumors or cancers has many constraints, since t u m o r s o r c a n c e r s a r e i n s e n s i t i v e t o antiproliferative signals and are capable of avoiding apoptosis.(1,2) one type of therapy that is attracting attention is chemotherapy using bioactive substances obtained by synthesis or isolation of natural materials, one of these being the rodent tuber (typhonium flagelliforme lodd.).(3,4) the dna segments located at both ends of the chromosomes, known as telomeres, are one of the factors associated with the development of cancers. telomere length is maintained by the telomerase enzyme. the actual shape of this enzyme may differ from species to species, but each of these versions have an rnaspecific template for the formation of new telomere subunits. telomerase activity in normal cells is mainly found in reproductive cells (germl i n e c e l l s ) , s i n c e t h e s e h a v e u n l i m i t e d proliferative capacity.(5) indonesia is known as one of the countries with the greatest biological diversity, so that it has the potential for developmental efforts in medicine to produce drugs that are derived from m e d i c i n a l p l a n t s . t h e s e p l a n t s p r o d u c e secondary metabolites of the alkaloid, terpenoid and flavonoid categories with anticancer a c t i v i t i e s . o n e o f t h e p l a n t s c o n t a i n i n g compounds with potential anticancer activity is rodent tuber (typhonium flagelliforme lodd.),(6) which also may counteract the adverse effects of chemotherapy. several previous studies using cultured t4 lymphoblastoid and wehi-3 leukemia cells have reported that typhonium flagelliforme may inhibit cancer cell proliferation and induce apoptosis.(7-9) according to a study conducted in malaysia, rodent tuber may decrease pain, metastases or dissemination of cancer cells, comprising mammary, nasopharyngeal, cervical, prostatic, pancreatic, and pulmonary cancers.(7) in addition, rodent tuber grows abundantly in indonesia and malaysia, and has been used for the treatment of hemorrhoids and skin diseases, for neutralizing narcotic drugs, and has antibacterial and antioxidant properties.(8,9) regarding the chemical compounds of rodent tuber, there is a scarcity of information. p h y t o c h e m i c a l a n a l y s e s o f r o d e n t t u b e r performed at the research station for medicinal and aromatic plants (balai penelitian tanaman obat dan aromatik) showed the presence of alkaloids, saponins, steroids, and glycosides, but the specific active substances in rodent tuber that play a role in the healing of cancers are as yet unknown.(10) the saline extract of typhonium flagelliforme contains ribosome inactivating proteins (rips), which are proteins with the ability to cut cancer cell dna or rna, so that they inactivate cancer cell development without damage to the surrounding tissues. in addition, rips can also block cancer cell growth.(10, 11) typhonium flagelliforme extract contains antineoplastic or anticancer compounds. (12) it has also been reported that typhonium flagelliforme extract at a dose of 200 mg/kgbw may decrease protein kinase and ki67 protein expression in rats.(13) furthermore, typhonium flagelliforme extract may have antiproliferative properties and significantly induce apoptosis of nci-h23 pulmonary cancer cells and nontumoral balb/ c3t3 rat fibroblasts.(14,15) since rodent tuber has anticancer potential and cancer cells in general show high telomerase expression, we wished to determine the potency o f ty p h o n i u m f l a g e l l i f o r m e e x t r a c t o n telomerase expression. no information is a v a i l a b l e o n t h e e ff e c t o f ty p h o n i u m flagelliforme extract on telomerase expression in t47d breast cancer and hela cervical cancer cells. typhonium flagelliforme extract has cytotoxic effects, with differing ic 50 doses in 5 different cancer cell types. the purpose of this study was to evaluate telomerase expression in cultured hela and t47d cancer cells after administration of typhonium flagelliforme extract. methods study design this was an experimental laboratory study conducted at the parasitology laboratory, faculty of medicine, gadjah mada university from april until november 2013. the study used one control group (of normal vero cells) and two experimental groups (of hela and t47d cancer cells, respectively), where each cancer cell group received three doses (control dose, ½ ic50 and 1 ic50). study subjects the study subjects were cultured t47d breast cancer, hela cervical cancer and normal vero cell lines, obtained from the parasitology laboratory, faculty of medicine, gadjah mada university. the cells were cultured in 24-well microplates using rosswell park memorial institute (rpmi) medium, with two replications per group. the cultures were incubated for 72 hours or until the cultures became confluent, at a density of 1 x 10 4 cells per well. after confluence, the cultures were treated with their previously determined doses of typhonium flagelliforme extract. plant materials rodent tuber plants were obtained from medicinal herb gardens in the jagakarsa area in south jakarta. preparation of cell culture and cell growth media the cell culture media for hela and t47d cells were prepared by dissolving one sachet each of rpmi and m199 powders, respectively, in around 800 ml twice-distilled water, with the addition of 2.0 gram sodium bicarbonate and 2.0 gram hepes, and adding twice-distilled water to obtain a volume of one liter. the solutions were mixed with a magnetic stirrer for around 10 minutes until homogenous, then neutralized with 1 n hcl to a ph of 7.2 – 7.4. the cell growth medium was prepared by mixing 19 ml fetal bovine serum (fbs), 2 ml penicillinstreptomycin, and 0.5 ml fungizone, then diluting with the respective culture media to a volume of 100 ml. subsequently the solutions were aseptically filtered using sterile 0.2 µm polyethylene sulfone filters. the filtered solutions were stored in the refrigerator in stoppered bottles. preparation of typhonium flagelliforme extract typhonium flagelliforme whole plant extract was prepared as follows: all fresh and cleansed plant parts were cut into thin strips and air-dried. then the strips were blenderized into a powder. the powder was macerated in 80% ethanol in an erlenmeyer flask, under continued shaking. subsequently the ethanolic extract was poured into a rotary flask and concentrated using a rotary evaporator. the rodent tuber concentrate was dissolved in dimethyl silfoxide (dmso), diluted into several concentrations, and filtered through 0.2 µm filters before being placed on the 24-well plates. from the preliminary c y t o t o x i c i t y a s s a y o f t h e ty p h o n i u m flagelliforme extract on the three cell lines (vero, t47d and hela cells) were obtained the respective ic50 values of 60.50 µg/ml, 66.90 µg/ml, and 30.198 µg/ml. the interventions were performed after the cell cultures attained a concentration of 5 x 104 cells per well. immunohistochemical telomerase expression assay the hela, t47d, and vero cell cultures at a density of 104 cells per well, that were attached to coverslips, were immunohistochemically stained at the parasitology laboratory, faculty of medicine, gadjah mada university, using the following procedure. the cell cultures on univ med vol. 35 no.1 6 purwaningsih, suciati, widayanti typhonium flagelliforme in hela cancer cells coverslips that had been given interventions, were incubated in a 5% co 2 incubator at 37oc for 24 hours, and were then removed and placed on poly-l-lysine slides, and fixed with acetone or methanol for 10 minutes at -200c. the slides were then washed in phosphate-buffered solution (pbs) for 3 x 5 minutes (3 times for 5 minutes each), and 0.3% h 2 o 2 was dripped onto the slides for 20 minutes. the slides were dripped with normal mouse serum (1:50) for 15 minutes, and the liquid was discarded without washing. then the slides were dripped with primary (telomerase) antibody for 60 minutes. after washing in pbs for 3 x 5 minutes, the slides were incubated in biotinylated secondary antibody for 5-10 minutes, and washed in pbs for 3 x 5 minutes. after incubation in streptavidin-peroxidase for 5-10 minutes and washing in pbs for 3 x 5 minutes, the slides were incubated in the chromogen diaminobenzidine tetrahydrochloride (dab) for 5-10 minutes, at a ratio of chromogen to substrate of 1:20, then washed in distilled water. the slides were then immersed in hematoxylin for 3-5 minutes for counterstaining, washed in distilled water and dehydrated using 95% ethanol followed by xylene, each for 10 minutes. subsequently the slides were given a drop of mounting medium (canada balsam) and covered with a coverslip. telomerase expression was observed using the light microscope. cells with telomerase expression are brown in color, whereas cells without telomerase expression are bluish purple in color. observation of telomerase expression by the ihc method is performed by counting the number of expression-positive (brown-colored) cells among 100 cells examined. the results are expressed as percentages. data analysis data were analyzed using the anova test, followed by the least significant difference (lsd) multiple comparison test using spss version 18. results the telomerase test results for each cancer cell type after administration of typhonium f l a g e l l i f o r m e e x t r a c t i n t h e f o r m o f t h e percentages of telomerase expression-positive cells in all three cell groups at their respective doses are presented in table 1. telomerase expression was significantly higher in t47d breast cancer and hela cervical c a n c e r c e l l s t h a n i n n o r m a l ve r o c e l l s . administration of typhonium flagelliforme extract showed decreased telomerase expression at doses both of one-half and one hela cell ic50 and at doses of one-half and one t47d cell ic50 (table 1). according to the results of the multiple comparison test with lsd on telomerase expression, there were significant differences between the groups (data not shown). administration of typhonium flagelliforme extract was capable of decreasing telomerase expression in both cancer cell types, both at doses of ½ ic50 and of 1 ic50. typhonium flagelliforme extract at a dose of ½ ic50 and 1 ic50 was capable of producing a highly significant decrease in telomerase expression (p<0.01) in hela cells. on the other hand, in t47d cells, a far greater dose was of ½ ic50 and 1 ic50 required, to be able to cause a highly significant reduction in telomerase expression (p<0.001). table 1. mean telomerase expression of each type of cancer cells by experimental group 7 discussion our study results showed that telomerase expression in both types of cancer cells was greater than in normal vero cells. after administration of rodent tuber (typhonium flagelliforme lodd.) extract, telomerase expression in both cancer cell types showed decrease, and telomerase expression was lower in hela cells than in t47d cells. at a lower dose, telomerase expression in hela cells showed a greater decrease in expression in comparison with the dose of 1 ic50 in t47d cells. this shows that the compound in typhonium flagelliforme extract was more effective or more potent as an anticancer agent in hela cells than in t47d cells. it has been reported that there are 4 active fractions contained in rodent tuber, i.e. pheophorbide-a, pheophorbide-a’, pyropheophorbide-a and pyropheophorbide-a’. other compounds that have been identified are oleic acid, linoleic acid, stigmasterol, and beta sitosterol. (15) it is postulated that the active substances in typhonium flagelliforme extract are more potent in decreasing telomerase expression in hela cells than in t47d cells. it is known that telomerase has an important role in the maintenance and stabilization of telomere length, which may affect cancer cell proliferation.(16). in this case the active compounds in typhonium flagelliforme extract are probably more effective in affecting telomere length of hela cells, so that the inhibition of proliferation of these cells is greater. however, this necessitates further studies. in this connection, previous investigators have reported that cancer cells have differing resistance or sensitivity to different chemotherapeutic or antineoplastic compounds, and it is possible that telomerase plays a role in this case. several tumor cell cultures show decreased telomerase activity after treatment with antineoplastic drugs.(17) the use of rodent tuber (typhonium flagelliforme lodd.) as an anticancer agent has demonstrated that this plant has an anticancer effect against pulmonary cancer cells (nci-h23 cells) by inhibiting their proliferation and stimulating their apoptosis. (14) dichloromethane and acetylacetic extracts of typhonium flagelliforme lodd. also have an anticancer effect against leukemia cells. it has been reported that administration of rodent tuber leaf extract (at doses of 19.8 and 5.8 µg/ml) and rodent tuber rhizomes (at doses of 6.5 and 8.2 µg/ml) may be antiproliferative against cemss leukemia cells.(8) telomerase is a ribonucleoprotein enzyme that serves to maintain the protective structures at the ends of eukaryotic chromosomes, called the telomeres. telomerase is the structure responsible for the maintenance of the telomere, which consists of repeats of the nucleotides (ttaggg)n. in most human somatic cells such as fibroblasts, telomerase expression is repressed and the telomere shortens progressively at each cell divison. on the other hand, the majority of human tumor or cancer cells express telomerase, so that the telomere length is stable. this observation shows that telomere maintenance is essential for tumor or cancer cell proliferation. this telomerase activity determines cell proliferation, both of cancer cells and normal cells in in-vitro and in-vivo conditions.(18) in normal human cells such as fibroblasts, the aging process is not only determined by telomere length but also by htert (human telomerase reverse transcriptase) expression and telomerase activity. abnormalities of telomerase activity in normal human cells slow down cell proliferation, limit the lifespan of the cells, and alter the maintenance of the single-strand telomeric overhang at the 3' end of the chromosome without altering the overall telomere shortening. normal and cancer stem cells have different telomerase activities.(19,20) in this study, in addition to the two cancer cell lines (hela and t47d), we also used normal vero cells, which are somatic cells, namely fibroblasts. it is well-known that normal cells have a limited capacity for cell division, in general 40 to 50 generations, after which the cells stop dividing. normal somatic cells, including stem cells, experience telomere shortening, that is univ med vol. 35 no.1 8 purwaningsih, suciati, widayanti typhonium flagelliforme in hela cancer cells meant for cell renewal. therefore somatic cells have programmed aging and low telomerase expression.(21) previous studies on gastrointestinal (gastric and colonic) cancer cells have reported higher telomerase expression of the sub-units htert, htr (human telomerase rna), and telomeraseassociated protein 1 (tp1/tep1) in these cells than in normal cells. similarly, telomerase activity in the tested cancer cells was higher than in normal cells. therefore telomerase may be a satisfactory marker for cancer incidence.(22) studies have been conducted by previous investigators on hela cells using triethylene tetraamine (teta) by the modified telomeric repeat amplification (trap) method and flow cytometry. it is reported that triethylene tetraamine (teta) in micromolar concentrations may inhibit proliferation of hela cells at the g1 phase. telomerase activity in hela cells is considerable and the cells divide rapidly, in other words their proliferative level is high. this leads to the idea of telomerase inhibitors playing a role in the treatment of cancers through molecular mechanisms.(23) other studies reported that t47d cells also have high telomerase activity. administration of curcumin and silibinin can inhibit the growth or proliferation of t47d cells and reduce the expression of the human telomerase reverse transcriptase (htert) gene.(24) in addition, telomerase activity in pancreatic cancer cells is also high. telomerase activity was determined in pancreatic cancer specimens as well as in metastatic lesions and was found to be higher in the metastatic lesions than in the primary tumor. telomerase activity is low in non-malignant pancreatic lesions, such as benign adenomas, and acute and chronic lesions.(25) this study had one limitation, in that it did n o t d e t e r m i n e t h e e ff e c t o f ty p h o n i u m flagelliforme extract on telomere length in hela and t47d cancer cells as compared with vero cells. further studies are necessary to determine the effect of typhonium flagelliforme extract on telomere length in hela and t47d cells. conclusions administration of typhonium flagelliforme e x t r a c t i s c a p a b l e r e d u c i n g t e l o m e r a s e expression, both in hela and t47d cells, although at differing doses. the reduction of telomerase expression in hela cells was more effective than that in t47d cells. conflict of interest the investigators had no conflicting interests in this study. acknowledgements the authors wish to express their gratitude to the directorate general of higher education, w h o t h r o u g h t h e r e s e a r c h f u n d f o r decentralization of outstanding research in higher education, provided additional funding for the conduct of this study. references 1. farida y, rahayu l, faizatun. aktivitas antioksidan serbuk n-heksana dan 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jw, wright we. role of telomer and telomerase. biology 2011;21:349-53. 22. nowak j, januszzkiewicz, lewandowski k, et al. activity and expression of human telomerase in normal and malignant cells in gastric and colon cancer patient. eur j gastroenterol 2003; 15:75–80. 23. ivankovic m, cukusic a, gotic i. telomerase activity in hela cervical carcinoma cell line proliferation. biogerontology 2007;8:163-72. 24. nasiri m, zhargami n, koshki kn, et al. curcumin silibinin inhibit telomerase expression in t47d human breast cancer cells. asian pasific j cancer prev 2013;14:3449–53. 25. zisuh av, han tq. expression of telomerase and its significance in the diagnosis of pancreatic cancer. indian j med res 2012;135:26-30. univ med vol. 35 no.1 alvina 104 abstract amebiasis is caused by e. histolytica, which is the only species pathogenic in humans, while the pathogeneicity of e. dispar and e. moshkovskii is still unclear. the disease is endemic in the developing countries, mainly due to poor sanitation and lack of clean water supplies. infection occurs by ingestion of e. histolytica cysts in fecally contaminated food or water. excystation in the small intestine releases motile invasive trophozoites which migrate to the large intestine, adhere to the colonic epithelium by means of galactose and an amebic surface antigen, n-acetyl-d-galactosamine-specific lectin. this results in killing of epithelial cells, neutrophils, and lymphocytes by the trophozoites, presumably through secretion of the pore-forming proteins called amebapores and activation of caspase 3. the trophozoite virulence factor, cysteine proteinase, induces an inflammatory response, resulting in neutrophil-mediated damage. hematogenous spread of trophozoites causes extraintestinal amebiasis, particularly amebic liver abscess (ala), in the formation of which caspase 3 presumably also plays a role. the trophozoites in the liver induce tissue destruction, cellular necrosis and formation of microabcesses that coalesce into a large solitary abscess in 65-75% of cases. results from pediatric studies reveal that partial immunity is acquired after infection with e.histolytica, the immunity however declining with age. keywords: amebiasis, host-parasite interactions, host defenses *department of parasitology, medical faculty, trisakti university correspondence dr. hj. suriptiastuti, ms department of parasitology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2311 univ med 2010;29:104-13 host-parasite interactions and mechanisms of infection in amebiasis suriptiastuti* may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina introduction diarrhea still constitutes a public health problem in many developing countries, being associated with the morbidity rate and the impact on children in the form of retarded growth and sequelae. it has been reported that around 2.5 million deaths occur annually in developing countries as a result of diarrheal diseases.(1) the annual prevalence of dysenteric diarrhea caused by the shigella bacterium worldwide is around 164.7 million, with 163.2 million in the developing countries.(1) another disease besides shigellosis that also induces symptoms of dysentery is amebiasis, which globally is a parasitic infection with a high mortality rate. the world health organization (who) jointly with the pan american health organization (paho) have defined amebiasis a s a n i n f e c t i o n c a u s e d b y e n t a m o e b a histolytica, irrespective of clinical symptoms.(2) the parasite has a worldwide distribution, but 105 univ med vol.29 no.2 it has been reported that prevalences exceeding 10% are frequently encountered in developing countries. in developed countries the parasite i n f e c t s m a i n l y t r a v e l e r s t o d e v e l o p i n g countries and immigrants. there are reports from bangladesh that dysenteric diarrhea due to e. histolytica kills 1 in 30 children under 5 years of age.(3) the presumed diagnosis of e. histolytica infection is mainly based on stool examination against the parasite, but this method is not sensitive because e. histolytica is morphologically indistinguishable from nonpathogenic entamoeba species. the genus entamoeba consists of many species, six among them being e. histolytica, e. dispar, e. moshkovskii, e. polecki, e. coli and e. hartmanni, located in the human intestinal lumen. e. histolytica is the sole species that is definitely pathogenic in humans. although several recent studies found e. dispar a n d e . m o s h k o v s k i i i n p a t i e n t s w i t h g a s t r o i n t e s t i n a l s y m p t o m s , t h e r e i s n o u n e q u i v o c a l e v i d e n c e o f a n a s s o c i a t i o n between both species and the pathology and symptoms exhibited by the patients.(4-6) the clinical picture of e. histolytica a m e b i a s i s r a n g e s f r o m a s y m p t o m a t i c c o l o n i z a t i o n t o d y s e n t e r i c d i a r r h e a a n d invasive extraintestinal amebiasis, commonly in the form of liver abscess. although effective t h e r a p y i s a v a i l a b l e , t h e m o r b i d i t y a n d mortality of amebic infections is still high, a mortality rate of 100,000 per 50 million patients with liver abscess per year having been reported.(7) from hue city, vietnam,(8) it was reported that the annual incidence of amebic l i v e r a b s c e s s i s a r o u n d 2 5 p e r 1 0 0 , 0 0 0 population, whereas in the united states oralfecal transmission rarely occurs. amebiasis is more severe in very young and old patients,(9) indicating the ineffectiveness of interventions performed for elimination of this disease. man is the sole host for this parasite, such that there is a need for an effective control program designed for eradication of amebiasis. one of the most important clinical questions arising is with regard to the existence of protective immunity against amebiasis. there is still a s c a r c i t y o f d a t a f r o m h u m a n s t u d i e s o n protective immunity and most of them are difficult to interpret, because in the majority of cases infection by e. histolytica is almost indistinguishable from colonization by e. dispar.(10) epidemiology the epidemiology of the e. histolytica parasite is still not known with certainty, because of the morphological similarities between the three species of entamoeba, n a m e l y e . h i s t o l y t i c a , e . d i s p a r a n d e . moshkovskii. several reports state that e. moshkovskii is frequently found in areas endemic for e. histolytica, thus hampering d e t e r m i n a t i o n o f t h e p r e v a l e n c e o f amebiasis.(11,12) the worldwide prevalence of e. histolytica and e. dispar as distinct species has not been investigated extensively and the prevalence of e. moshkovskii is practically unknown. on subcontinents such as india, africa, asia, and south and central america, the prevalence is fairly high. in developing countries the prevalence depends on cultural practices, socioeconomic conditions, age, inadequate supply of clean water, population density, and poor sanitation, facilitating fecaloral transmission of the parasites from one person to another. in developed countries, infections are mostly caused by e. dispar and in the majority of cases are limited to certain c o m m u n i t y g r o u p s . i n f e c t i o n s b y e . m o s h k o v s k i i i n h u m a n s a r e r e p o r t e d l y asymptomatic, but the parasite is considered potentially capable of causing disease in humans. most of the data come from studies u s i n g m e t h o d s t h a t a r e i n c a p a b l e o f differentiating the three aforementioned species. the difficulty of distinguishing the three entamoeba species led the world health o rg a n i z a t i o n ( w h o ) t o r e c o m m e n d t h e development and application of improved 106 methods for specific diagnosis of e. histolytica. i n f e c t i o n . ( 2 ) e p i d e m i o l o g i c a l s u r v e y s o n amebiasis should involve the utilization of test instruments capable of differentiating between e. histolytica, e. dispar and e. moshkovskii, individually, simultaneously, and accurately. single-round pcr assay is reported to be an accurate, rapid, and effective diagnostic method for the detection and discrimination of the three aforementioned entamoeba species as an alternative tool in both routine diagnosis of amebiasis and epidemiological surveys. however, to date no inexpensive laboratory tests are available for use in endemic areas of amebiasis. the identification of e. histolytica remains the principal target of the clinical parasitology laboratory and accurate and definite diagnosis constitutes an important step in the management of patients infected with e. histolytica. it has been known for a long time that i n f e c t i o n w i t h e . h i s t o l y t i c a r e s u l t s i n pathology in the host, although not in all of those infected. however, recently it became clear that 1 in 4 infections with e. histolytica lead to clinical symptoms.(7,12) thus the parasite remains a significant cause of morbidity and mortality for the populations of developing countries. in bangladesh the annual incidence of amebic dysentery in pre-school children is reported to be 2.2%, in comparison with the a n n u a l i n c i d e n c e o f 5 . 3 % f o r s h i g e l l a dysentery, and 1 in 30 children die of diarrhea or dysentery before the age of 5 years.(15) acuna-soto et al.(16) in their summary of cases from various reports between 1929 and 1997 found the male-to-female ratio for invasive intestinal amebiasis and asymptomatic carriers to be respectively 3.2:1 and 1:1, where many asymptomatic carriers were due to e. dispar. some studies reported that the prevalence rate of acute amebic diarrhea was around 1.5% in persons recently returning from travels to southeast asia and 3.6% in persons recently returning from central america, while the overall prevalence rate was 2.7%. (17) the prevalence rate of amebiasis in the united states is around 4%, with asymptomatic e. dispar infections being tenfold more frequent than e. histolytica infections. in addition, only 10% of the latter are invasive, so that only 1% of individuals with entamoeba-positive stools on microscopic examination exhibit symptoms of amebiasis. invasive amebiasis, including amebic liver abscess, is more frequently found in males than in females, while in prepubertal children amebic liver abscess is equally distributed in both genders. however, amebic liver abscess is tenfold more frequent in adults than in children. it is suggested that the higher proportion of amebic liver abscess in males is d u e t o t h e i r s u s c e p t i b i l i t y t o i n v a s i v e amebiasis.(18) pathogenesis of amebiasis the asymptomatic passage or entry of cysts is the most common manifestation of e. histolytica, but this conclusion comes from studies using microscopic examination of fecal samples.(6) in the stool, cysts are commonly seen but trophozoites are rarely encountered. individuals with asymptomatic e. histolytica infection may form antibodies, even though there are no invasive abnormalities. however, untreated asymptomatic colonization with e. histolyticca may lead to amebic dysentery and various invasive disorders.(9) haque et al.(18) reported that after a one-year follow-up, 4-10% of individuals with asymptomatic e. histolytica colonization had colitis or extraintestinal disorders. intestinal amebiasis ingestion of quadrinucleate cysts of e. histolytica in food or water contaminated with fecal matter initiates infection. this event is a daily occurrence for inhabitants in developing countries and constitutes a threat to inhabitants of developed countries.(18,19) the infective form of the cyst survives in the stomach and suriptiastuti host-parasite interactions in amebiasis 107 univ med vol.29 no.2 intestines. excystation takes place within the lumen of the small intestine, whereafter motile invasive trophozoites are released to migrate i n t o t h e l u m e n o f t h e l a r g e i n t e s t i n e . trophozoite adherence occurs by means of galactose and n-acetyl-d-galactosamine (gal/ galnac)-specific lectin, which is a surface molecule of the ameba. the trophozoite adheres to the colonic mucin layer, thereby c o l o n i z i n g t h e l a r g e i n t e s t i n e . ( 2 0 , 2 1 ) t h e reproduction of the trophozoites lacks a sexual c y c l e a n d t h e o v e r a l l p o p u l a t i o n o f e . histolytica is presumably clonal. in most infections, the trophozoites aggregate in the mucin layer and form new cysts by binary fission, thus resulting in a self-limited and asymptomatic infection. the cysts that are excreted in the stool maintain the life cycle by further fecal-oral spread. being protected by their thick walls the cysts are capable of surviving for days to weeks in the external environment and play a role in the transmission of the infection. in contrast, the trophozoites that are excreted in the stool and come to be o u t s i d e t h e h u m a n b o d y a r e r a p i d l y incapacitated and die. ingested trophozoites do n o t s u r v i v e w i t h i n t h e h u m a n s t o m a c h . commonly the trophozoites remain within the figure 1. life cycle of entamoeba histolytica(22) 108 intestinal lumen as a noninvasive infection in asymptomatic individuals, who thus become carriers releasing new cysts. cysts are typically found in formed stools whereas trophozoites are found in liquid stools. i n a n u m b e r o f c a s e s , a d h e r e n c e o f trophozoites to colonic epithelium results in epithelial lysis, initiating invasion of the colon by trophozoites or hematogenous spread of trophozoites to extraintestinal locations, with their various pathological manifestations. in view of the fact that purified lectin has no cytotoxic effect even in high concentrations, it is assumed that cytolysis is effected by a d h e s i n s t h r o u g h s t i m u l a t i o n o f a c t i n polymerization. neutrophils react to the invasion and cause cellular injury locally. invasion of intestinal epithelium is followed by extraintestinal spread to the peritoneum, liver, and other locations.(9,18) invasive intestinal disease may occur days to years after initial infection and is characterized classically by abdominal pain and bloody diarrhea.watery or mucus-containing diarrhea, constipation, and tenesmus may also occur. this clinical picture corresponds histologically with trophozoites invading and laterally undermining the intestinal surface to form the so-called flask-shaped ulcers (figure 2).(19) colitis occurs when the trophozoites p e n e t r a t e t h e i n t e s t i n a l m u c o s a , w h i c h functions as a barrier to invasion by preventing adhesion of the amebae to the epithelium and b y d e c r e a s i n g t r o p h o z o i t e m o t i l i t y. ( 9 , 1 8 ) trophozoite invasion is mediated by the killing o f e p i t h e l i a l c e l l s , n e u t r o p h i l s , a n d lymphocytes by the trophozoites. these events do not occur until lectins of the parasite bind to host n-acetyl-d-galactosamine groups on ofigure 2. “flask-shaped” ulcer of invasive intestinal amebiasis (hematoxylin-eosin, original magnification ×50). note that the apex of the ulcer at the bowel lumen is narrower than the base, accounting for the flask shape. this is formed as trophozoites invade through the mucosa and move laterally into the submucosa (direction of ulcer expansion is marked by arrows). microscopically, trophozoites are localized to the advancing edges of the submucosal ulcer. image courtesy of john williams, cbiol, mibiol, london school of hygiene and tropical medicine(19) suriptiastuti host-parasite interactions in amebiasis 109 univ med vol.29 no.2 linked cell surface oligosaccharides. the l e c t i n g l y c o c o n j u g a t e i n t e r a c t i o n i s stereospecific and multivalent. the identity of t h e h i g h a ff i n i t y r e c e p t o r s o n i n t e s t i n a l epithelial cells is as yet unclear. the secretion by the amebae of amebapores, which are 5-kd proteins capable of forming pores in lipid b i l a y e r s , p r e s u m a b l y p l a y s a r o l e i n aforementioned killing. activation of caspase 3, a distal effector molecule in the apoptotic pathway, occurs soon after contact with the amebae, and caspases are required for killing of cells in vitro and for the formation of amebic liver abscess in vivo.(24) patients with amebic colitis typically show symptoms of several weeks duration, such as abdominal cramping, weight loss, and watery or bloody diarhhea. the insidious onset and variability of the symptoms and signs make t h e d i a g n o s i s d i ff i c u l t t o e s t a b l i s h . t h e occurrence of diarrhea with bloody stools should lead to consideration of bacterial i n f e c t i o n s w i t h s h i g e l l a , s a l m o n e l l a, c a m p y l o b a c t e r a n d e n t e r o i n v a s i v e a n d e n t e r o h e m o r r h a g i c e s c h e r i c h i a c o l i. uncommon manifestations of amebic colitis may also be found, such as acute necrotizing c o l i t i s , t o x i c m e g a c o l o n , a m e b o m a a n d perianal ulceration, the latter potentially l e a d i n g t o f i s t u l a f o r m a t i o n . a l t h o u g h extremely rare, these manifestations should be properly managed. i n d e v e l o p i n g c o u n t r i e s , i n t e s t i n a l amebiasis may frequently be diagnosed by finding and identifying entamoeba cysts or m o t i l e t r o p h o z o i t e s o n m i c r o s c o p i c preparations. a drawback of this method is its p o o r s e n s i t i v i t y, a s i t i s i n c a p a b l e o f differentiating e. histolytica infection from e. dispar and e. moshkovskii infection.(25) for definitive diagnosis, specific tests for fecal e. h i s t o y i t i c a a n t i g e n s , s e r u m a n t i a m e b i c antibodies, or amebic dna, should be utilized. an important aid to fecal antigen tests is detection of serum antibodies against amebae, which may be found in more than 70% of p a t i e n t s w i t h s y m p t o m a t i c a m e b i a s i s . ( 1 6 ) however, these serologic test results remain positive for years after amebic infection, thus making it difficult to differentiate between recent and past infections. extraintestinal amebiasis t h e m o s t c o m m o n e x t r a i n t e s t i n a l manifestation of amebiasis is amebic liver abscess (ala). ala is a progressive disorder caused by hematogenous spread of invasive trophozoites from the colon through the portal vein into the liver. the abscess is four times more common in the right lobe, as this part receives most of its blood supply from the cecum and ascending colon. although amebic liver abscess may affect all ages and both genders, it occcurs with greater frequency in males between 20 and 40 years of age. in c e r t a i n i n d i v i d u a l s a l a i s e n c o u n t e r e d c o n c o m i t a n t l y w i t h a m e b i c c o l i t i s , b u t f r e q u e n t l y w i t h o u t s y m p t o m s , w h i l e microscopic examination of the stool for trophozoites and cysts of e. histolitica yields negative results. the possibility of amebic liver abscess should be suspected in individuals from endemic areas with fever, pain in the right upper quadrant and tenderness in the hepatic region, but rarely with jaundice. the most serious complications of amebic liver abscess are rupture of the abscess and bacterial superinfection. the trophozoites may have infected the patient’s liver for months or years before an abscess develops. in the intervening period hopefully an immune response is induced in the patient’s body, resulting in the formation of circulating immunoglobulins. as mentioned above, the dominant surface antigen of the parasite is the gal/galnac-specific lectin. a r o u n d 8 0 % p a t i e n t s w i t h a l a h a v e circulating igg against the antigens of the parasite, but this does not result in recovery from infection caused by the parasite, thus suggesting the presence of other defense mechanisms.(9,18,25) 110 during liver invasion by e. histolytica, the host will both sequentially and simultaneously deploy a number of mechanisms to kill the parasite. the first line of tissue defense is composed of innate immune system cells that recognize pathogen-associated molecular patterns (pamps) and trigger an inflammatory response. it was previously reported that female and male mice differ in their early responses to e. histolytica liver invasion.(26) in in vitro models, the effects of gamma interferon (ifn ã) can be bypassed by the recognition of ppgs of e. histolytica by toll-like receptor 2 (tlr2) and tlr4, which results in direct tumor necrosis factor (tnf), interleukin-12p40 (il-12p40), and il-8 production. (27) this proinflammatory cytokine profile underlines the importance of the early recognition of pamps (such as proteophosphoglycans/ ppgs) and appropriate inflammatory cell recruitment and activation during ala onset.(28) although the body of the host mounts a massive inflammatory response against e. histolytica, the parasites are capable of survival within their niche. virulence of the parasites in their invasion of the host confers survival and invasive potential. the clonal origin of the t r o p h o z o i t e p o p u l a t i o n c a n n o t e x p l a i n adequately why some of the trophozoites are able to survive within the host body and cause infection, whereas other trophozoites are unable to survive. this suggests the presence of local and individual adaptation mechanisms of the parasites, leading to the hypothesis of specialization of subpopulations, in which the trophozoites are capable of defense against the c o m p l e m e n t s y s t e m o f t h e h o s t . t h e s e trophozoite subpopulations subsequently invade the host tissues, causing cell death through parasite cytotoxicity.(23) hepatic invasion by amebic trophozoites induce tissue destruction, cellular necrosis and formation of microabcesses that gradually coalesce. in 65-75% of cases a solitary abscess is found, although multiple abcesses may also be formed.(29) the abcesses consist of soft, necrotic, acellular yellow-brown debris, called “anchovy paste”.(30) the occurrence of severe jaundice indicates the possibility of multiple abcesses. the parasites themselves are rarely i d e n t i f i a b l e , a s t h e y a r e l o c a t e d i n t h e peripheral parts of the lesions. in general, the symptoms of amebic liver abscess are pain in the right hypochondrium radiating to the right s h o u l d e r a n d s c a p u l a r r e g i o n . t h e p a i n increases on deep inspiration, on coughing, and on stamping the right foot when walking. host immunty and inflammatory response the interaction of the parasites with the intestinal epithelium results in an inflammatory response marked by activation of nuclear factor ê b a n d s e c r e t i o n o f l y m p h o k i n e s . t h e epithelial response depends on the trophozoite virulence factor cysteine proteinase, causing various intestinal abnormalities through neutrophil-mediated damage.(25) however, the neutrophils may also be protective, such as in the activation of neutrophils or macrophages b y t u m o r n e c r o s i s f a c t o r á ( t n f á ) o r interferon ã, which in vitro are capable of killing amebae and of limiting the size of the liver abscess. in contrast with the severe inflammatory response that is typical of early invasive amebiasis, the inflammation occurring after the formation of colonic ulcers and liver abscess is of minimal intensity only. in the case of the infection becoming chronic, e. histolytica maintains itself against the host immune response in several ways. the gal/galnac-specific lectin exhibits sequence homology and antigenic cross-reaction with cd59, a leukocyte antigen that prevents the formation of the c5b-c9 membrane attack complex. amebic cysteine proteinase rapidly degrades complement anaphylatoxins c3a and c5a, and also degrades secretory iga and serum igg, presumably protecting the amebas from opsonization.(18) the immunity against e. histolytica infection is associated with the mucosal ig a reponse to the gal/galnac-specific lectin. suriptiastuti host-parasite interactions in amebiasis 111 univ med vol.29 no.2 after a period of around one year, children with the above response experience less reinfection than do children without the response. the cellmediated response is reportedly found in p a t i e n t s w i t h a m e b i c l i v e r a b s c e s s , characterized by lymphocyte proliferation and lymphokine secretion that is amebicidal in vitro. it should be realized that the pandemic of acquired immunodeficiency syndrome (aids) has not shown increased amebic invasion, although asymptomatic intestinal c o l o n i z a t i o n i s f r e q u e n t l y e n c o u n t e r e d . however, this statement is still controversial, because there are reports stating that both in endemic and non-endemic areas amebic liver abscess is an infectious disease that threatens the individual with hiv-aids. among 31 hospitalized patients with amebic liver abscess at the seoul national university hospital in 1990-2005, ten of them (32%) were hiv-aids patients.(31) r e c e n t l y a p e d i a t r i c c o h o r t s t u d y c o n d u c t e d b y h a q u e e t a l . ( 1 2 ) i n c l u d e d determination of fecal iga antilectin antibodies and found that children with iga antibodies against amebic adherence lectins showed r e s i s t a n c e a g a i s n t r e i n f e c t i o n w i t h e . histolytica. however, the immunity was shortlived, and around 20% of the children in the cohort study suffered from a second episode of e. histolytica infection. these findings demonstrate that immunity to amebiasis may occur in a number of children after a prior infection, but that the immunity is of temporary character. at the time of invasion of the liver by e. histolytica, a number of responses take place in the host, in the form of defense mechanisms for killing the parasites, consisting of immune cells that trigger an inflammatory response. e x p e r i m e n t a l l y, f e m a l e m i c e h a v e b e e n reported to mount a distinct response against invasion of the liver by e. histolytica. these animals rapidly clear the parasites from the liver, release natural killer t cells (nktc) in larger numbers into the infected areas and produce higher levels of gamma interferon (ifn-ã).(32) deficiencies of nktc as well as of ifn-ã result in a higher survival rate of the parasites. ifn-ã is an important regulator in the initial inflammatory process, because it activates the synthesis of tumor necrosis factor (tnf), which increases no synthesis by neutrophils and macrophages. the role of monocytes in hepatic e. histolytica infection is not known in detail, but it is certain that the parasite strongly activates p e r i t o n e a l m a c r o p h a g e s a n d c i r c u l a t i n g monocytes, leading to synthesis of cytokines that subsequently cause the destruction of the parasite. conclusions entamoeba histolytica is the causative organism of the globally distributed human amebiasis. most infections are asymptomatic, but invasion of the tissues by the parasite may result in amebic colitis and liver abscess, which is the most common extraintestinal amebic i n f e c t i o n . t h e o c c u r r e n c e o f i n v a s i v e amebiasis in some individuals undergoing colonization by e. histolytica and of secondary infection indicates that acquired immunity against the parasite is only partial. however, it is clear that amoebic factors potently activate p e r i t o n e a l m a c r o p h a g e s a n d c i r c u l a t i n g monocytes, leading to cytokine and reactive oxygen species (ros) production and then parasite and tissue destruction. references 1. ravdin jl, stauffer wm. entamoeba histolytica (amebiasis). in: mandell gl, bennett je, dolin r, editors. mandell, douglas, and bennett’s priciple and practice of infectious diseases. 6th ed. philadelphia, pa: churchill livingstone; 2005:3097-111. 2. who/paho/unesco report: a consultation with experts on amoebiasis: mexico city, mexico 28-29 january 1997. epidemiol bull 1997;18:134. 112 3. petri wa jr, haque r, lyerly d, vines rr. estimating the impact of amebiasis on health. parasitol today 2006;16:320-1. 4. parija sc, khairnar k. entamoeba moshkovskii and entamoeba dispar-associated infections in pondicherry, india. j health pop nutr 2005;23: 292-5. 5. tanyuksel m, ulukanligil m, guclu z, araz e, koru u, petri wa jr. two cases of rarely recognized infection with entamoeba moshkovskii. am j trop med hyg 2007;76:7234. 6. fotedar r, stark d, beebe n, marriott d, ellis j, harkness j. laboratory diagnosis techniques for entamoeba species. clin microbiol rev 2007;20:511-32. 7. haque r, petri wa. diagnosis of amoebiasis in bagladesh. arch med res 2006;37:273-7. 8. blessmann j, van linh p, nu pa, thi hd, mullermihsok b, buss h, et al. epidemiology of amebiasis in a region of high incidence of amebic liver abscess in central vietnam. am j trop med hyg 2002;66:578-83. 9. stanley sl jr. amoebiasis. lancet 2003;361: 1025-34. 10. stanley sl jr. protective immunity to amebiasis: new insight and new challanges. j infect dis 2001;184:504-6. 11. ali ikm, hossain mb, roy s, ayeh-kumi pf, petri wa jr, haque r, et al. entamoeba moshkovskii infections in children, bangladesh. emerg infect dis 2003;9:580-4. 12. fotedar r, stark d, marriott d, ellis j, harkness j. entamoeba moshkovskii infections in sydney, australia. eur j clin microbiol infect dis 2008; 27:133-7. 13. hamzah z, petmitr s, mungthin m, leelayoova s, chavalitshewinkoon-petmitr p. differential detection of entamoeba histolytica, entamoeba dispar, and entamoeba moshkovskii by a singleround pcr assay. j clin microbiol 2006;44: 3196–200. 14. blessmann j, ali ikm, nu pa, dinh bt, viet tq, van al, et al. longitudinal study of intestinal entamoeba histolytica infections in asymptomatic adult carriers. j clin microbiol 2003;41:474550. 15. haque r, mondal d, kirkpatrick bd, akhter s, farr bm, sack rb, et al. epidemiologic and clinical characteristics of acute diarrhea with emphasis on entamoeba histolytica infections in preschool children in urban slums of dhaka, bangladesh. am j trop med hyg 2003;69:398405. 16. acuno-soto r, maguire jh, wirth df. gender distribution in asymptomatic and invasive amebiasis. am j gastrointerol 2000;95:1277-83. 17. freedman do, weld lh, kozarsky pe, fisk t, robins r, von sonnenburg f, et al. spectrum of disease and relation to place of exposure among ill returned travelers. n engl j med 2006;11930. 18. haque r, huston cd, hughes m, houpt e, petri wa jr. current concept: amebiasis. n engl j med 2003;346:1565-73. 19. pritt bs, graham c. amebiasis. mayo clin proc 2008;83:1154-60. 20. blessmann j, van al, tannich e. epidemiology and treatment of amebiasis in hue, vietnam. arch med reas 2006;37:270-2. 21. haque r, ali im, sack rb, farr bm, ramakrishnan g, petri wa. amebiasis and mucosal iga antibody against the entamoeba histolytica adherence lectin in bangladesh children. j infect dis 2001;183:1787-93. 22. petri wa jr, mann bj, haque r. the bittersweet interface of parasite and host: lectin-carbohydrate interactions during human invasion by the parasite entamoeba histolytica. annu rev microbiol 2001;56:39-64. 23. cdc.amebiasis. available at: http://www.dpd. cdc.gov/dpdx/html/amebiasis.htm. accessed june 22, 2010. 24. yan l, stanley sl jr. blockade of caspase inhibits amebic liver abscess formation in a mouse model of disease. infect immun 2001;69:7911-4. 25. santi-rocca j, rigothier m-c, guillen n. hostmicrobe interactions and defense mechanisms in the development of amoebic liver abscesses. clin microbiol rev 2009;22:65-75. 26. lotter h, jacobs t, gaworski i, tannich e. sexual dimorphism in the control of amebic liver abscess in a mouse model of disease. infect immun 2006;74:118–24. 27. maldonado-bernal c, kirschning cj, rosenstein y, rocha lm, rios-sarabia n, espinosacantellano m, et al. the innate immune response to entamoeba histolytica lipopeptidophosphoglycan is mediated by toll-like receptors 2 and 4. parasite immunol 2005;27:127–37. 28. ivory cp, chadee k. activation of dendritic cells by the gal-lectin of entamoeba histolytica drives th1 responses in vitro and in vivo. eur j immunol 2007; 37:385–94. 29. salles jm, moraes la, salles mc. hepatic amebiasis. braz j infect dis. 2003;7:96-110. 30. gill gv, beeching nj. tropical medicine. 5th ed. oxford, uk: blackwell publishing; 2004. suriptiastuti host-parasite interactions in amebiasis 113 univ med vol.29 no.2 31. muzzafar j, madan k, sharma mp, kar p. randomized, single-blind, placebo-controlled multicenter trial to compare the efficacy and safety of metronidazole and satranidazole in patients with amebic liver abscess. dig dis sci 2006;51:2270-3. 32. lotter h, jacobs t, gaworski i, tannich e. sexual dimorphism in the control of amebic liver abscess in a mouse model of disease. infect immun 2006; 74:118-24. editorial 1 does vitamin e play a role in the primary prevention of stroke? adi hidayat department of community medicine, medical faculty, trisakti university univ med vol. 30 no.1 a few decades ago it was generally accepted from experimental studies, animal experiments, and epidemiological cohort studies that atherogenesis is linked to oxidative stress and that antioxidant supplementation, notably vitamin e supplementation, is associated with a low incidence of cardiovascular disease (cvd). this view led to the widespread use of vitamin e and other antioxidants for reducing cvd morbidity and mortality. in vitro and animal studies on antioxidant and anti-inflammatory properties of alphaand gamma-tocopherols also found that these antioxidants had anti-inflammatory properties.(1-3) however, many large-scale prospective randomized trials failed to find beneficial effects of high dose vitamin e supplementation either to healthy persons or to cvd patients.(4,5) recent meta-analytical studies concluded that vitamin e supplementation results in increased mortality.(5) although this conclusion has been much criticized, a decision analysis using markov modeling on the same data also found that indiscriminate supplementation of high doses of vitamin e is not beneficial in preventing cvd.(6) in contrast, available evidence indicates that vitamin e may have beneficial effects on stroke. one study reported a significant inverse association between stroke mortality and dietary vitamin e intake (rr from lowest to highest fifth of intake, 1.0, 0.80, 0.93, 0.67, and 0.40; p for trend < 0.008) in women with a mean age of 61 years.(7) a meta-analysis of 9 randomized controlled trials on the effect of vitamin e on incident stroke found a differential effect on stroke subtypes, as vitamin e supplementation resulted in a 10% decrease in risk of ischemic stroke, but increased the risk of hemorrhagic stroke by 22%. this differential effect may be due the different mechanisms underlying ischemic and hemorrhagic events.(8) these opposing effects of vitamin e on stroke subtypes should be clarified previous to issuing public health recommendations on the use of vitamin e in potential stroke patients.(7) in absolute terms, the reduction in ischemic stroke events by vitamin e is relatively small. actually, considerably lower ischemic stroke risks may be produced by other preventive measures, such as healthy lifestyle, low body mass index, moderate alcohol consumption, abstinence from smoking, regular physical activity, and healthy diet. in this setting, vitamin e supplements may not lead to further risk reduction. the use of vitamin e for primary prevention of hemorrhagic stroke should ideally be under regular medical supervision, since this subtype of stroke carries a higher morbidity and mortality, while vitamin e in high doses may increase all cause mortality.(8) the best alternative to vitamin e as a primary preventive measure against stroke, especially ischemic stroke, seems to be the adoption a low-risk lifestyle by healthy individuals, who at the same time will be achieving a state of long-term well-being. 2 univ med vol. 30 no.1 references 1. jiang q, ames bn. gamma-tocopherol, but not alpha-tocopherol, decreases proinflammatory eicosanoids and inflammation damage in rats. faseb j 2003;17:816–22. 2. singh u, jialal i. anti-inflammatory effects of alpha-tocopherol. ann n y acad sci 2004;1031:195–203. 3. young is, woodside jv. antioxidants in health and disease. j clin pathol 2001;54:176–86. 4. cook nr, albert cm, gaziano jm, zaharris e, macfadyen j, danielson e, et al. a randomized factorial trial of vitamins c and e and beta carotene in the secondary prevention of cardiovascular events in women: results from the women’s antioxidant cardiovascular study. arch intern med 2007;167:1610-8. 5. miller er, pastor-barriuso r, dalal d, riemersma ra, appel lj, guallar e. meta-analysis: high-dosage vitamin e supplementation may increase all-cause mortality. ann intern med 2005;142:37-46. 6. dotan y, pinchuk i, lichtenberg d, leshno m. decision analysis supports the paradigm that indiscriminate supplementation of vitamin e does more harm than good. arterioscler thromb vasc biol 2009;29:13049. 7. donnan ga, fisher m, macleod m, davis sm. stroke. lancet 2008;371:1612-23. 8. schürks m, glynn rj, rist pm, tzourio c, kurth t.effects of vitamin e on stroke subtypes: metaanalysis of randomised controlled trials. bmj 2010;341:c5702 doi:10.1136/bmj.c5702. alvina 129 abstract to date there is increasing pressure on surgeons to minimize the time that the patient stays in hospital. patients with acute appendicitis without perforation or peritonitis are not discharged early due to concern on the part of the surgeon that it would increase surgical wound infection rates. the aim of this study was to compare surgical wound infection rates after open appendectomy (oa) of patients with acute appendicitis without perforation or peritonitis in an early discharge (ed) group and a control group without early discharge (late discharge [ld] group. the study was also to evaluate patient acceptance of ed in comparison to the ld group. a cross sectional study was carried out on adult patients (age >14 years) with acute appendicitis without perforation or peritonitis, who underwent open appendectomy. the results showed that there was no significant difference (p>0.05) in surgical wound infection rates in both the intervention (ed) and control (ld) groups. in addition, there was a significant difference (p<0.05) in patient acceptance (satisfaction) in both groups, where patients in the ed group were more satisfied than the patients who had not been discharged early. the conclusions of this study are that early discharge from hospital after open appendectomy does not increase surgical wound infection rate and has good patient acceptance. keywords : early discharge, open appendectomy, infection, patient acceptance *department of surgery, medical faculty, trisakti university correspondence dr. hari krismanuel, spb department of surgery, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2808 email: hr_kris@yahoo.com univ med 2010;29:129-36. early versus late discharge from hospital after open appendectomy hari krismanuel* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction at present, many operations have only a one-day length of stay.(1,2) although length of s t a y f o r c o m m o n o p e r a t i o n s i n v a r i o u s c o u n t r i e s t e n d s t o d e c r e a s e , p a t i e n t s undergoing open appendectomy often remain in hospital for 3-5 days after operation.(3,4) in saudi arabia, the mean length of stay in open appendectomy was 3.02 ± 1.27 days.(5) to date there is increasing pressure on surgeons to minimize the time that the patients stay in hospital. therefore, there is a need to establish guidelines for reasonable length of stay standards for common operations.(3) patients with acute appendicitis without perforation or peritonitis are not discharged early, due to concern on the part of the surgeon that it would increase surgical wound infection rates(3) and that postoperative pain would inhibit 130 krismanuel early versus late discharge after appendectomy mobilization of the patient.(6-8) surgical wound infection is commonly due to contamination of the wound at the time of appendix removal.(9-11) with minimal contamination, prophylactic antibiotic administration would bring the incidence of wound infection to a low level.(12,13) p a i n a f t e r o p e n a p p e n d e c t o m y c o u l d b e managed with analgesics. after the pain d i s a p p e a r s , t h e p a t i e n t s w o u l d b e m o r e comfortable, could be mobilized immediately and discharged earlier.(6,8) surgical wound infection is affected by s e v e r a l r i s k f a c t o r s , i . e . p a t i e n t , l o c a l , environmental, procedural, surgeon/operator, and care factors. patient factors consist of age, nutritional status (malnutrition, obesity), the p r e s e n c e o f o t h e r d i s e a s e s ( m a l i g n a n c y, chronic diseases [diabetes mellitus, hepatic cirrhosis], associated infection), the treatment received (corticosteroids, immunosuppresants, radiation) and psychological state of the patient (anxiety, fright, and sleeping difficulty).(10,12) local factors that participate in surgical wound infection are necrotic tissue, avascular tissue, hematoma, poor hemostasis, foreign material in the wound, suture material and suturing technique, skin infection in the surgical area.(12) environmental factors consist of sterility of the operating room, number of personnel in operating room, air circulation of operating room, and sterility of instruments. procedural factors of influence are length of hospital stay before operation, preoperative preparation such as cleansing of the incision site, hair shaving, aseptic and antiseptic techniques for preparation, antibiotics administration, and degree of contamination of the type of surgical procedure, whether categorized as a clean, clean-contaminated, contaminated or dirty operation. emergency or elective operation and duration of the operation are also included in procedural factors. (10,12) surgeon/operator factors are knowledge, skill and experience of t h e s u rg e o n / o p e r a t o r, t i s s u e h a n d l i n g , prevention of the spread of contamination, selection of needles, suture material and suturing technique. (10,12) care factors that i n f l u e n c e s u rg i c a l w o u n d i n f e c t i o n a r e misevaluation (inexperience/lack of attention), lack of asepsis in wound care, less careful evaluation about progress/healing/symptoms of the patients, less mobilization, incorrect nutritional support.(10,12) open appendectomy (oa) patients with early discharge (ed) from hospital may be expected to differ in risk factors from those who are not discharged early (late discharged/ld), presumably due to care factors after the operation. the aim of this study was to compare the surgical wound infection rate and patient acceptance after oa in ed and ld patients. methods research design a cross sectional study was carried out from january 1999 to december 2001 at the irna a2 and irna a3 (3rd class) wards of the surgery division of dr. kariadi hospital, semarang. participants participants were adult patients (more than 14 years old) with acute appendicitis on whom open appendectomy was performed. the inclusion criteria, determined at oa, were phlegmonous, suppurative or gangrenous appendicitis. exclusion criteria were: i) patients with malnutrition, obesity (body mass i n d e x > 2 5 . 0 ) , ( 1 4 ) a n e m i a ( h e m o g l o b i n concentration <11.5 g/dl for women and < 12.5 g/dl for men),(15) malignancy, chronic diseases (diabetes mellitus, hepatic cirrhosis), skin infection on surgical area before operation, patients receiving corticosteroids and/or chemotherapy, patients on radiotherapy; ii) patients who declined to participate in this study and iii) patients with intraoperative findings such as appendiceal mass, perforated appendicitis or normal appendix.(16,17) 131 surgery g e n t a m y c i n i n j e c t i o n w a s g i v e n a s prophylactic antibiotic (80 mg for adults or 2.5 mg/kg body weight). the choice of antibiotic was based on pattern and sensitivity tests of aerobic microorganisms in intraperitoneal fluid o f p a t i e n t s w i t h n o n p e r f o r a t e d a c u t e appendicitis in the surgery division of dr kariadi hospital, semarang.(18) the patients were also given one 500 mg metronidazole s u p p o s i t o r y ( 1 5 m g / k g b o d y w e i g h t ) a s prophylaxis against gram negative anaerobic bacteria (including bacteroides fragilis).(19,20) on all of the patients standard appendectomy was performed via a muscle splitting incision under general anesthesia. closing of muscle layers was done with absorbable material. skin was closed by interrupted sutures with 4-0 silk. postoperative management a f t e r t h e o p e r a t i o n , a n a n a l g e s i c s suppository was administered. in addition the patients received mefenamic acid. antibiotic administration was continued up to 24 hours post operation. (21,22) no intravenous fluid replacement was done after the operation. the patients were supported to early mobilization. they were permitted to drink and eat after being free from anesthetic influence. the patients were evaluated on the next day and discharged when their condition was good. b e f o r e d i s c h a rg e , t h e p a t i e n t s a n d t h e i r families received oral and written instructions on the procedure of wound care. surgical wound care for patients in the ed group was performed by the patients and their families at home. surgical wound care for patients in the control (ld) group was performed by the attending physician in the ward. outcomes the patients with ed (discharged in 24 hours) and ld (more than 24 hours) were followed up one week later. the surgical wound was examined and all problems such as discharge, bleeding or infection were noted. the definition of surgical wound infection is based on center for disease control and prevention (cdc) criteria.(10,23) the surgical wound infection that were evaluated were superficial incisional surgical site incision (ssi) and deep incisional ssi. the patients w e r e a s k e d t o e x p r e s s t h e i r a c c e p t a n c e (satisfied or not satisfied) of their discharge. then the patients were requested to visit the hospital one week later for reevaluation. evaluation of the patients discharged after more than 24 hours was performed by the attending physician in the ward. sample size the approximate sample size (n) needed for this study, involving two groups with the outcome is expressed in terms of proportions, was calculated from the following formula: 21 2 22111 )-(1)-(1z)2z n ππ πππππ βα − ++ = the appropriate sample size for our study was calculated before the beginning of the trial, based on an analysis of sample sizes required for each of the main parameters (wound infection and patient acceptance) for an á of 0.05 and a power of 90%. nandi et al. stated that surgical wound infection rate for cleancontaminated operations (ð1) was 7.7%. (24) s u rg i c a l w o u n d i n f e c t i o n r a t e f o r e a r l y discharge after open appendectomy in a study in royal berkshire hospital, london, (ð2) was 6.12%.(3) we determined that we would need at least 89 patients per group for fulfilling the requirements to perform statistical analysis for surgical wound infection rate with pearson chi square.(25-27) the total number of patients who were included in this study was 208, consisting of 102 patients in the ed group and 106 patients in the ld group, thus meeting the sample size requirement. univ med vol.29 no.3 132 krismanuel early versus late discharge after appendectomy statistical analysis nominal or ordinal data were tested by pearson chi square. data on a ratio scale were tested for normality of distribution. the analysis was continued with independent t-test for data with normal distribution (skewness approaching 0 and kurtosis approaching 3).(25,28) the proportions of risk factors in both groups were tested for equality. all p values reported are 2-sided, and p< 0.05 denotes statistical significance. results twenty two patients were excluded from 2 3 7 a c u t e a p p e n d i c i t i s p a t i e n t s . o f t h e remaining 232 patients 215 (92.68 %) agreed to participate after open appendectomy, among whom there were five patients with perforated appendicitis and two patients with appendiceal mass. the latter were not included in the study, bringing the total number of study subjects to 208. one hundred and two patients were assigned to the ed group and 106 patients to the ld group (figure 1). the values (expressed as mean ± standard deviation) of age, duration of operation, stage of acute appendicitis, and class of operator (e.g. resident) in the ed group and ld group and the probability of each risk factor in both groups is shown in table 1. both groups were figure 1. flow of participants through trial   acute appendicitis (n=237) excluded: anemia (n=1) malignancy (n=1) chronic disease (n=1) skin infection around surgical area (n=1) chemotherapy + radiation (n=1) declined to participate (n=17) open appendectomy (n=215) late discharge (n=106) early discharge (n=102) 102 included in analysis control at 1 week 106 included in analysis simple randomization excluded: -appendiceal mass (n=2) -perforated appendicitis (n=5) -normal appendix (n=0) open appendectomy (n=208) 133 comparable in terms of age and duration of operation (p>0.05). the mean operative duration in the ed group was 65.54 ±21.95 minutes compared to 68.2 ±29.62 minutes in the ld group, yielding a statistically nonsignificant difference (p=0.463). similarly, no significant differences were found between both groups regarding stage of appendicitis and class of operator (p>0.05) t h e p e a r s o n c h i s q u a r e t e s t f o r differences in postoperative surgical wound infection rate between ed group and ld group found no significant difference between both g r o u p s ( p = 0 . 4 3 1 ) , a s s h o w n i n ta b l e 2 . however, the pearson chi square did find a significant difference in patients acceptance (satisfaction) between ed group and ld group (table 3). there was a significant difference (p < 0.05) of patient acceptance (satisfaction) between ed group and ld group, where patients in the ed group were more satisfied with their discharge than the ld patiens. discussion the present study proved that there was no difference in wound infection rates after oa between ed and ld patients. early discharge was also acceptable to both groups. the results of this study were similar to those of a previous study in another country, stating that early discharge of patients with acute appendicitis without perforation after oa was safe and well accepted by the patients. surgical wound infection rate was 4.90% in the ed group and 7.41% in the ld group. statistically there was early discharge (n=102) late discharge (n=106) p value age (years) 26.28 ± 8.45 26.45 ± 8.83 0.888 duration of operation (min) 65.54 ± 21.95 68.21 ± 29.62 0.463 stage of acu te appendicitis : phlegmonous appendicitis 70 66 suppurative appendicitis 19 19 gangrenous appendicitis 13 21 0.382 class of operator : second degree 59 60 third degree 43 46 0.857   table 1. demographic and operative data of the study groups wound infection early discharge (n=102) late discharge (n=107) p value no 97 9 8 0.431 yes 5 8   table 2. surgical wound infection in ed group and ld group patient acceptance early discharge (n=102) late discharge (n=106) p value satisfied 78 4 3 0.000 dissatisfi ed 24 6 3   table 3. patients acceptance in ed group and ld group univ med vol.29 no.3 134 krismanuel early versus late discharge after appendectomy no significant difference (p>0.05) in surgical wound infection rates between the ed and ld groups.(3) the present results also showed similar surgical wound infection rates as compared to the reference values. according to gottrup et al. the surgical wound infection rate for clean-contaminated operations was between 6–9%.(13) nandi et al. declared that surgical wound infection rate for cleancontaminated operation was 7.7%,(24) while surgical wound infection rate for oa in the royal berkshire hospital study in london was 6 . 1 2 % . ( 3 ) e a r l y d i s c h a rg e w a s a l s o w e l l accepted by the patients. it was found that 76.47% of patients in the ed group were satisfied with their discharge, compared with 3 9 . 8 1 % o f p a t i e n t s i n t h e l d g r o u p . statistically, there was a significant difference ( p < 0 . 0 5 ) i n p a t i e n t a c c e p t a n c e o f t h e i r discharge between the ed and ld groups.(25,27) thus more patients were satisfied with early discharge compared to patients satisfied with late discharge. the study in royal berkshire hospital, london, on 147 post appendectomy patients (adults and children) without perforation or peritonitis, who were discharged in 24 hours after operation, showed that among patients who consulted general practitioners there were 2 patients (1.36%) with pain at the incision site, 8 p a t i e n t s ( 5 . 4 4 % ) w i t h s e r o s a n g u i n o u s discharge, 1 patient (0.68%) with wound abscess and 1 patient (0.68%) with pyrexia and malaise caused by mesenteric adenitis. the patients had a normal appendix, as determined in a previous operation. all of the wound p r o b l e m s a p p e a r e d i n 7 – 1 0 d a y s a f t e r o p e r a t i o n , a f t e r t h e p a t i e n t s h a d b e e n discharged from hospital. therefore, a 3–5 day discharge after operation, as was done in the past, was no guarantee that the patients would be free from surgical wound infection.(3) on the contrary, a longer length of hospital stay i n c r e a s e d t h e r i s k o f n o s o c o m i a l infection.(9,10,11) early discharge was also well accepted by the patients and their families, as i t a l l o w e d t h e m t o r e s u m e t h e i r n o r m a l activities. (3) santacroce et al. from state university at bari, italy gave early discharge to patients with acute appendicitis without complications, on whom had been performed o p e n a p p e n d e c t o m y o r l a p a r o s c o p i c appendectomy, with good results.(29) there is a general tendency in health care for decreasing length of stay in hospitals. l e n g t h o f s t a y i s o n e o f t h e c l i n i c a l performance indicators or the main measure of efficiency.(2) early discharge from hospital after open appendectomy results in the patients returning more quickly to normal activities. early discharge also reduces hospital costs compared with late discharge.(2) ramesh and galland showed that there was no morbidity caused by early discharge from hospital. most patients and their families showed good acceptance of early discharge.(3) currently patient acceptance (satisfaction) has become a p a r t o f t h e c l i n i c a l d a t a b a s e . p a t i e n t s a t i s f a c t i o n i s c o n s i d e r e d i n c r e a s i n g l y important in the medical field.(30) the most common problem faced by the patients after oa was surgical wound infection. there is little question that surgical wound infection contributes significantly to the cost, morbidity, and possible long-term consequences of a surgical procedure.(10,12) surgical wound infection was more often due to contamination with endogenous microorganisms, rather than e x o g e n o u s m i c r o o rg a n i s m s . ( 9 , 1 0 ) t h e polymicrobial aerobic and anaerobic flora, closely resembling the normal endogenous microflora of the surgically resected organ, are the most frequently isolated pathogens in clean-contaminated operations.(10) in oa, surgical wound infection is mainly due to contamination of the wound at the time of a p p e n d i x r e m o v a l . ( 9 , 1 0 , 11 ) i n m i n i m a l c o n t a m i n a t i o n , p r o p h y l a c t i c a n t i b i o t i c a d m i n i s t r a t i o n r e d u c e d t h e i n c i d e n c e o f complications to a low level.(11-13) even with a length of stay of 3-5 days in hospital, most surgical wound infection occurred after the 135 patients were discharged from hospital (7–10 days after the operation).(3) wound infections may not constitute serious complications per se but represent a major inconvenience to the patient, impacting on convalescence time and quality of life.(31) this study supports previous studies that declared early discharge from hospital after oa to be safe and well accepted by the patients.(3) although statistically there was no significant difference in surgical wound infection rate between both groups, there still was an obvious c l i n i c a l d i ff e r e n c e , t h e s u r g i c a l w o u n d infection rate in the ed versus ld groups being 4.90% and 7.41%, respectively. the theoretical implication of this study is that late discharge will increase nosocomial infection rates.(12,32,33) the present study has s o m e l i m i t a t i o n s . t h e s t u d y p o p u l a t i o n consisted of patients with acute appendicitis without perforation in 2nd and 3rd class in the surgery division of dr. kariadi hospital, semarang, having different socio-economic status, ward conditions and hospital care. therefore, this study may not be representative of the general population in semarang. our f o l l o w u p w a s l i m i t e d t o t h e f i r s t w e e k postoperatively. conclusions late discharge after open appendectomy is not superior to early discharge. early d i s c h a rg e f r o m h o s p i t a l a f t e r o p e n appendectomy does not increase surgical wound infection rate and is also well accepted by the patients. acknowledgements i gratefully acknowledge my indebtness for the support provided in conducting the present study, especially to dr. andy maleachi, sp.b, sp.bd and prof.dr.dr. i. riwanto, sp.b, sp.bd. references 1. wasowicz dk, schmitz rf, go pm. assessment of one day surgery in a district training hospital: safety, efficacy and patient’s satisfaction. ned tijdschr geneeskd 2000;144:1919-23. 2. gilliard n, eggli y, halfon p. a methodology to estimate the potential to move inpatient to one day surgery. bmc heath services research 2006;6:78. 3. ramesh s, galland rb. early discharge from hospital after open appendicectomy. br j surg 1993;80:1192-3. 4. oludiran oo, ohanaka ce. early discharge following open appendicectomy – a nigerian experience. j med biomed res 2002;1:51-4. 5. yagnik vd, rathord jb, phatak ag. a retrospective study of two-port appendectomy and its comparison with open appendectomy and three-port appendectomy. saudi j gastroenterol 2010;16:268-71. 6. carr ecj. exploring the effect of post operative pain on patient outcomes following surgery. acte pain 2000;3:183-93. 7. pyati s, gan tj. perioperative pain management. cns drugs. 2007;21:185-211. 8. key rw. a preemptive strike against surgical pain. contemporary ob/gyn 2001;46:65. 9. kavic ms, kavic sm, kavic sm. laparoscopic appendectomy. in: wetter pa, kavic ms, levinson cj, kelley we jr, mcdougall em, nezhat c, editors. prevention and management of laparoendoscopic surgical complications. 2nd ed. miami, fl: society of laparoendoscopic surgeons; 2005.p.151-7. 10. nichols rl. preventing surgical site infections: surgeon’s perspective. emerg infect dis 2001;7:220-4. 11. nichols rl. current strategies for prevention of surgical site infections. curr infect dis rep 2004;6:434-42. 12. rubin rh. surgical wound infection: epidemiology, pathogenesis, diagnosis and management. bmc infect dis 2006;6:171. 13. gottrup f, melling a, hollander da. an overview of surgical site infections: aetiology, incidence and risk factors. emwa j 2005;5:11-5. 14. world health organization. bmi classification. in: global database on body mass index.geneva: who; 2004. 15. chavez p, pregler j, buckler d. study challenges who anemia definition: current criteria for healthy adults may not always apply to older women. amnews; 2001. univ med vol.29 no.3 136 krismanuel early versus late discharge after appendectomy 16. carr nj. the pathology of acute appendicitis. ann diagn pathol 2000;4:46-58. 17. nabipour f. histopathological feature of acute appendicitis in kerman – iran from 1997 to 2003. am j environ sci 2005;1:130-2. 18. sugiarto d, riwanto i. pola dan uji kepekaan kuman cairan intra peritoneal pada apendisitis akuta non perforata. muktamar ahli bedah indonesia xi medan;1993. p.1-15. 19. kasatpibal n, norgaard m, sorensen ht, schonheyder hc, jamulitrat s, chongsuvivatwong v. risk of surgical site infection and efficacy of antibiotic prophy-laxis: a cohort study of appendectomy patients in thailand. bmc infectious diseases 2006;6:1117. 20. munckhof w. antibiotic for surgical prophylaxis. austr prescr 2005;28:38-40. 21. dettenkofer m, forster dh, ebner w, gastmeier p, ruden h, daschner fd. the practice of perioperative antibiotic prophylaxis in eight german hospitals. infection 2002;30:164-7. 22. nichols rl. preventing surgical site infections. clin med res 2004;2:115-8. 23. petherick es, dalton je, moore pj, cullum n. methods for identifying surgical wound infection after discharge from hospital: a systematic review. bmc infectious diseases 2006;6:170. 24. nandi pl, soundara rajan s, mak kc, chan sc, so yp. surgical wound infection. hkmj 1999;5:82-6. 25. dawson b, trapp rg. research questions about two separate or independent groups. in: basic and clinical biostatistics. 3rd ed. new york: lange medical books/ mcgraw-hill; 2001.p.132-60. 26. dahlan ms. besar sampel dan cara pengambilan sampel dalam penelitian kedokteran dan kesehatan. jakarta: salemba medika; 2009. 27. mcdonald jh. chi square test for goodness-offit. in: handbook of biological statistics. 2nd ed. baltimore, maryland: sparky house publishing; 2009.p.57-63. 28. knoke d, bohrnstedt gw, mee ap. statistics for social data analysis. 4th ed. itasca, il : f. e. peacock publisher;2002. 29. santacroce l, ocha jb. appendicitis. emedicine 2010. available at: http://emedicine.medscape. com/article/195778. accessed june 25, 2010. 30. torpy jm. changes in anesthesiology practice are explicated. jama 2002;287:1924-6. 31. katkhouda n, mason rj, towfigh s, gevorgyan a, essani r. laparoscopic versus open appendectomy: a prospective randomized doubleblind study. ann surg 2005;242:439-50. 32. shah mh, gandhi md, mehta vp, udani dl, mundra mp, swadia mm. nosocomial infections in surgical wards. internet j surg 2010;24:1. 33. safdar n, maki dg. the commonality of risk factors for nosocomial colonization and infection with antimicrobial-resistant staphylococcus aureus, enterococcus, gram-negative bacilli, clostridium difficile, and candida. ann intern med 2002;136:834-44. obesity contributes toward hypertension 96 *centre for public health research and development, nihrd, republic of indonesia **centre for health resources and services, nihrd, republic of indonesia correspondence: julianty pradono centre for public health research and development, nihrd, republic of indonesia phone: +62812 1004 523 email: jpradono@yahoo.com univ med 2016;35:96-104 doi: 10.18051/univmed.2016.v35.96-104 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 obesity contributes toward hypertension in young and older adult julianty pradono* and sintawati** background obesity and hypertension are independent risk factors in the increasing prevalence of non-communicable diseases. the proportion of obesity in indonesia has increased in the last 5 years according to the national health survey. the purpose of this research was to obtain the contribution of obesity toward hypertension in the age group of 18 years and above. methods an observational study of cross-sectional design was conducted using the national basic health research 2013 population data. the total sample of subjects was 2,741,297 from 33 provinces in indonesia. data were collected through interviews, blood pressure measurement, and anthropometry. analysis of categorical data was by means of the chisquare statistical test, followed by calculation of the attributable fraction in the population, and analysis of factors influencing the impact fraction. results the prevalence of general obesity (bmi>25 kg/m2) was 31.5% in males and 48.0% in females, while central obesity was 20.9% in males and 42.5% in females. in females with general obesity the risk of hypertension was 1.84 (95% ci: 1.82-1.86) times higher than in those with normal bmi. if general obesity and central obesity can be reduced, the prevalence of hypertension may be reduced by 2.9% in males and by 12.2% in females. conclusion the prevalence of hypertension in the age group of 18 years or more can be reduced by 2.9% in males and by 12.2% in females, by overcoming the contribution of obesity. therefore efforts should be geared towards promoting healthy eating habits and maintaining a healthy weight through health education. keywords: obesity, hypertension, age >18 years doi: http://dx.doi.org/10.18051/univmed.2016.v35.96-104 97 univ med vol. 35 no.2 introduction obesity is one of the independent risk factors for the increased incidence of coronary heart disease and diabetes mellitus in the population.(1) according to the national health survey, the proportion of general obesity in indonesian males increased from 13.9% in 2007 to 19.7% in 2013. in indonesian females, the proportion of general obesity also increased, from 14.8% to 32.9%.(2) similarly, the proportion of central obesity increased from 18.8% in 2007 to 26.6% in 2013, both in males and females. the two most common measurements used in population surveys are body mass index (bmi) to indicate general obesity and abdominal circumference to indicate central obesity. during an observation period of 46 years on 508 young adults, a longitudinal cohort study in the us found that bmi scores of >30 kg/m2 were substantially associated with the prevalence of hypertension (hazard ratio, 4.17; 95% confidence interval 2.34-7.42). similarly, bmi scores of 25-29 kg/ m2 increased the risk of hypertension (hazard ratio 1.58; 95% confidence interval 1.28-1.96).(3) another study showed that the risk of hypertension is much greater in men than in women at any given level of bmi. for men, the risk of hypertension is strongly positively associated with bmi only at bmi levels above 25, but for women a positive relationship is observed at all bmi levels. this indicates that using a bmi cut off point of 25 to define overweight is more appropriate for men than for women.(4) hypertension is an increase in blood pressure that remains above normal limits. in general, hypertension does not show any symptoms and is usually detected at the time of consultation for a different illness. the results of the indonesian basic health research (riskesdas) indicate that the prevalence of hypertension in 2007 was 31.7% in the age group of 18 years or above. among the reported prevalences, only 23% of subjects had ever been diagnosed by health personnel and were taking antihypertensive drugs, whereas 77% were found upon blood pressure measurement during the survey. of the subjects who had ever been diagnosed as having hypertension by health personnel, only 24.0% had their blood pressure controlled and regularly consulted their physician.(5) obesity in the long term causes injury to t a rg e t o rg a n s , p a r t i c u l a r l y t h e k i d n e y s . hypertension caused by obesityis more difficult to control, thus requiring several antihypertensive drugs and treatment of the risk factors, including dyslipidemia, insulin resistence, and diabetes mellitus.(6) the objective of the present study was to determine the level of the contribution of obesity toward the prevalence of hypertension in subjects aged 18 years and over. methods study design this observational study was of crosssectional design and was conducted from august until october 2013 in 33 provinces in indonesia. study subjects the subjects were individuals aged >18 years, who resided in urban and rural areas, and were members of house holds selected as samples in riskesdas 2013. the inclusion criteria were residents aged >18 years, males and female, and agreeing to be interviewed and to under go measurements. the number of subjects meeting the inclusion criteria was 2,918,858 from 33 provinces in indonesia. the exclusion criteria were persons with severe illness, who could therefore not be interviewed and were unable to stand or sit for measurements. interviews interviews using a questionnaire were p e r f o r m e d b y t r a i n e d i n t e r v i e w e r s . t h e questionnaire comprised socio demographic characteristics, history of illness, (whether ever diagnosed by health personnel as having h y p e r t e n s i o n a n d c u r r e n t l y t a k i n g a n t i hypertensive drugs), and behavioral risk factors. 98 measurements all subjects under went measurements by trained data collectors. anthropometric measures comprised body weight, which was measured with a personal balance and expressed in kg, and height, which was measured using a statometer and expressed in cm, on subjects without footwear or belts. abdominal circumference was measured using a measuring tape and expressed in cm. measurement of abdominal circumference was carried out by politely asking the subject to open or lift the outer garments, then with the subject standing upright and breathing no rmally, determining the midpoints between the lower margin of the last palpable rib and the top of the iliac crest on both sides of the body and marking them with a marking pen. the measuring tape was placed around the subject’s abdomen through both midpoints. central obesity was defined as an abdominal circumference of >90 cm for males and >80 cm for females.(7) the body mass index was calculated as follows: bmi (kg/m2) = weight (kg)/[height (m)2]. standard bmi scores for asians are: underweight <18.5; normal weight >18.5 <25.0; overweight >25 <27; and obesity >27.0.(7) the who showed that the relationships between bmi, percentage of body fat, and health risks in asian populations differ from those of european populations. the proportion of asians at high risk of non-communicable disease has a bmi score of >25 kg/m 2. ( 7 ) in the analysis for application in preventive programs, general obesity was defined as a bmi score of >25 kg/ m2. blood pressure was measured using a digital sphygnomanometer. the subjects were asked not to perform physical activity such as sports, at least 30 minutes before undergoing blood pressure measurements. the subjects were asked to sit and rest for at least 5-10 minutes before the measurements. the measurements were performed twice with an interval of 3-5 minutes between two successive measurements, during which interval the cuff around the arm w a s l o o s e n e d . i f t h e f i r s t a n d s e c o n d measurements differed by >10 mmhg, a third measurement was taken after a rest of 10 minutes, with loosening of the cuff around the arm. the recorded systolic and diastolic blood pressure was taken as the mean of 2 of the closest measurements. hypertension was defined if the systolic blood pressure was >140 mmhg, or the diastolic blood pressure >90 mmhg, or if the subject was taking antihypertensive drugs.(8) data analysis the chi-square test was used to compare the prevalence of obesity between age groups in the population under study. multiple logistic regression analysis was used to obtaina risk factor model of hypertension, while the odds ratio was used to test for an association between hypertension and several other variables. furthermore, to determine the level of the contribution of each of these variables, the attributable fraction in the population (afp) was determined by calculating the adjusted or value of the respective risk factor variables. in astudy of cross-sectional designand using logistic regression analysis, if the frequency is >10% and or > 2.5, the calculated or value is frequently overestimated. therefore, to obtain a better estimate of the effect of an association, the adjusted or should be corrected to estimate the relative risk, which will subsequently be termed the corrected relative risk (rrcor).(9) the rrcor is calculated from the following formula: ( ) ( )xorpp1 or rrcor 00 +− = where p0=out come of non-exposed group, or= odds ratio, rrcor=corrected relative risk. after obtaining the value of the relationhip of several risk factors and the prevalence of hypertension with corrected relative risk, an analysis is performed on the impact fraction againts these influencing factors. thus will be known the level of the contribution of each of pradono, sintawati obesity and hypertension in adult 99 univ med vol. 35 no.2 the risk factors toward the dependent variables, yielding a priority scale for intervention programs against these variables. if the corrected rr is >1, the calculated impact fraction is the value of attributable fraction of exposure in the exposed population (afe),(10) which is calculated using the formula rrcor 1rrcor afe − = the attributable fraction in the whole population (afp) is then ( ) afe* ca a afp + = where a = number of subjects at risk of hypertension, and (a+c) = all subjects with hypertension. the level of contribution of the risk factors is calculated as follows: level of contribution = afp * proportion of exposed outcome. the analysis was performed using the spss version 15.0® software. ethical clearance the basic health research (riskesdas) for the year 2013 had already been granted ethical clearance from the ethics commission, national institute for health research and deveopment, ministry of health, republic of indonesia, under number lb.02.01/5.2/ke.006/2013. results among the 2,918,858 subjects in the age group of 18 years and over who participated in the interviews, a total of 2,741,297 (93.9%) subjects had complete data and anthropometric measures. t h e n u m b e r o f m a l e s u b j e c t s w a s 1,284,299 (46.9%), and that of the females 1,456,998 (53.1%). mean age of the subjects was 43.18 ± 14.23 years, with no significant difference in mean age between males and females. mean height and weight were higher in males than in females. the converse was true for the means of bmi, abdominal circumference, and diastolic blood pressure (table 1). the prevalences of general obesity (bmi >25 kg/m2), central obesity, hypertension, and smoking behavior were 30.4%, 29.5%, 29.8% and 32.6%, respectively. these prevalences, except that for smoking behavior, were higher in females than in males. the educational level of the female subjects was lower than that of the males. more than half of the females had only primary school education, did not finish primary school, or never went to school (table 1). table 1.comparison of several characteristics between genders 100 general obesity = bmi >25 kg/m2 table 2. prevalences of general obesity, central obesity and hypertension by age group and gender the prevalences of general obesity in the males of the age groups of 18 up to 44 years were on average lower than those in the females, but the opposite was found in the age groups of 45 years and over (table 2). after controlling for age, educational level, and smoking behavior, females with general obesity had a 1.84-fold higher risk (95% ci: 1.821.86) for hypertension than females with normal bmi, while males with general obesity had a 1.69table 3. relationship of bmi and abdominal circumference with hypertension, by gender bmi=body mass index; ac=abdominal circumference; *controlled for age, educational level, smoking fold higher risk (95% ci: 1.68-1.71) for hypertension than males with normal bmi. after controlling for age, educational level, and smoking behavior, males with central obesity (>90 cm) had a 1.94-fold higher risk (95% ci: 1.91-1.96) than those with an abdominal circumference of <90 cm. on the other hand, females with central obesity (>80 cm) had a 1.53-fold higher risk (95% ci: 1.51-1.54) than those with an abdominal circumference of <80 cm (table 3). pradono, sintawati obesity and hypertension in adult 101 univ med vol. 35 no.2 table 4. reduction in prevalence of hypertension if risk factors of obesityare eliminated if both risk factors, namely general obesity (bmi >25 kg/m2) and central obesity, are eliminated, the contribution in males will be 2.9%, the prevalence of hypertension may be reduced from 28.7% to 25.8%. in contrast, the contribution in females will be 12.2%, the prevalence of hypertension may be reduced from 36.3% to 24.1% (table 4). discussion the prevalence ofobesity in residents aged 18 years and above was 30.4% (21.7% in males and 38.0% in females), with the prevalence in females being significantly higher than in males. these results are consistent with the findings in the study by wang et al.(11) in china.the prevalence of central obesity was 12.0% in males and 45.0% in females, showing significant results. these values are lower than those of wang et al.(11) in china. this may have been due to the use of different age groups in both studies. the study by wang used subjects aged 50 years and over from urban areas, where as the present study used subjects aged 18 years and older from both urban and rural areas. the prevalence of general obesity increased with age, in males as well as females. the highest prevalence of general obesity was in the age group of 35-44 years. central obesity showed significant differences by age group, both in males and females. in males the highest prevalence occurred at a 10-year younger age, i.e. in the age group of 35-44 years. these results show that the third and fourth decades are very important in the development of central obesity, particularly in females.(12) from the prevalence of general and central obesity in our study, it is apparent that if these can be prevented or controlled, the prevalence of hypertension in both males and females in the age group of 18 years and over in indonesia may be lowered and with due attention to other risk factors, may impede the development of noncommunicable disease. numerous studies have shown that the risk of hypertension is increased in persons with obesity, and that the relationship between obesity and hypertension varies with age, gender, geographic area, and race.(13) in the analysisof t h e r e l a t i o n s h i p b e t w e e n o b e s i t y a n d hypertension in residents aged 18 years and above in indonesia, it was shown that height, weight, and the prevalences of general obesity (bmi), central obesity, blood pressure, smoking and educational level differed significantly between males and females. this indicates that gender is a strong confounding variable, although the sample sizes of the males and females were almost balanced. according to the who, the definition for overweight and general obesity in caucasian populations are 25 and 30 kg/m2, respectively.(14) on the other hand, studies in china and many o t h e r a s i a n c o u n t r i e s s h o w e d t h a t t h e i r population shad lower bmi scores but higher percentages of body fat in comparison with european countries.(15) the relationship between obesity and hypertension may be explained as being due to 102 the role of leptin-releasing adipose tissue.(16) leptin isan adipose tissue-derived hormone that plays a rolein interactions involving increased blood pressure, in association with metabolic, inflammatory, hemostatic, and other factors. this was demonstrated in the us national health and nutritional examination survey in the age group of 20 years or above (n=5,599; females 54.7%). the plasma leptin concentration was categorized in quartiles (females: <7.68, 7.68-13.18, 13.1921.70, >21.70 fg/l; males: <2.64, 2.64-4.36, 4.37-7.12, >7.12 fg/l), showing that plasma leptin concentration was higher and positively associated with hypertension, after having been controlled for age, gender, ethnicity, education, smoking, alcohol intake, bmi, diabetes mellitus, serum cholesterol and c-reactive protein. in comparison with the first quartile leptin (reference), the odds ratio for hypertension associated with the fourth quartile leptin was 1.89 (95% ci: 1.24-2.09). subgroup analysis by gender and bmi also showed a consistently positive association.(17) shidfar et al.(18) in their study stress the importance of attention to changes in body fat distribution that lead to increased risks for cardiac and metabolic disease, particularly in menopause. a cross-sectional study on 138 females in the age group of 26-35 years showed significantly increase in blood pressures (systolic and diastolic) in subjects within creased bmi and central obesity, in comparison with subjects who had normal bmi and no central obesity.bmi and abdominal circumference showed a significant positive correlation with systolic and diastolic blood pressure, and had several independent effects on therisk of high blood pressure in females.(19) central obesity is a better predictor than general obesity for the risk of cardiovascular disease in females.(20) hypertension is strongly correlated with obesity,(21) which is consistent with our study results confirming a correlation between hypertension, general obesity and central obesity. there is an agreement between countries, which is known as the 25×25 target, to reduce premature mortality as a result of the four main non-communicable diseases (cardiovascular disease, chronic respiratory disease, cancer, and diabetes) by 25% from 2010 to 2025, between the ages of 30 to 70 years. international studies of various countries have shown that, upon achievement of the risk factor target, the probablity of death from the four main noncommunicable diseases will decrease by 22% in malesand by 19% in females between the years 2010 and 2025. achieving the risk factor target will delay the death of more than 37 million persons, which will be mostly felt inlow-to middleincome countries.(22) the sample in this study has a considerably high power in the national population, thus allowing for a correct estimate of the contribution of obesity to ward the prevalence of hypertension among the study subjects. one limitation of this study is that 6.1% of respondents had incomplete data. in addition, errors in blood pressure measurement are more freqnent in obese persons, if a cuff of the wrong size is used. although an analysis of the risk factors of hypertension was performed, including smoking and educational level, there are presumably other risk factors that may affect the contribution of obesity toward hypertension, but were not included in the analysis. the study design used was crosssectional, which cannot explain any cause-andeffect relationship. the nationally-based study results reveal that a reduction in general obesity and central o b e s i t y m a y r e d u c e t h e p r e v a l e n c e o f hypertension, particularly in females of the third or fourth decades. further studies are needed to identify increased obesity as an impediment in the management of hypertension, which continuously threatens to increase the number of persons with non-communicable disease. on the other hand, decreases in weight may be associated with reductions in blood pressureand risk of non-communicable disease, particularly cardiovascular disease.(23) strategies should be developed for optimal increases in interventions or interventional therapies for hypertension due to obesity. pradono, sintawati obesity and hypertension in adult 103 univ med vol. 35 no.2 conclusion the prevalence of hypertension in the age group of 18 years or more can be reduced by 2.9% in males and by 12.2% in females, by over coming the contribution of obesity. therefore efforts should be geared towards promoting healthy eating habits and maintaining a healthy weight through health education. conflict of interests no relevant conflict of interest present. acknowledgement we extend our thanks to the head and s e c r e t a r i a t o f t h e h e a l t h r e s e a r c h a n d development agency, ministry of health, republic of indonesia, who gave us the opportunity for further analysis of the riskesdas 2013 data. we 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pradono, sintawati obesity and hypertension in adult alvina 153 abstract one of the impacts of exposure to noise is stress. natural killer (nk) cells are one of the leukocyte subsets that are responsive to physiological and psychological stress. the objective of the present research was to determine the relationship between cortisol levels and nk cell activity among women with aircraft noise stress in the area of adi sumarmo airport, solo. this study was an analytical survey with a cross sectional design. the number of subjects was 39, who were divided into 3 groups of 13 subjects each. groups 1 to 3 were exposed to noise levels of 92.29 db, 71.79 db and 52.17 db, respectively. the sample was taken using simple random sampling. the data were analyzed by pearson correlation test and anova followed by post hoc test using lsd test. the anova test showed that there were significant differences in circulating cortisol levels among all groups (p = 0.018). the pearson correlation test showed that there was a positive association between circulating cortisol levels and the number of nk cells (r = 0.547; p< 0.05) and a negative association between circulating cortisol levels and nk cell activity (r = 0.578; p < 0.05). this study indicated that cortisol levels decreased nk cell activity among women with exposure to aircraft noise. women who experienced aircraft noise stress showed increased cortisol levels and decreased nk cells activity. keywords : aircraft noise, cortisol, natural killer cells, women *department of physiology, sebelas maret university school of medicine, solo correspondence dr. dr. hartono, m.si department of physiology, sebelas maret university school of medicine jln. ir. sutami 36 a, surakarta 57126 phone: 0271-664178 email: hartonofkuns@yahoo.co.uk univ med 2010;29:153-61. cortisol level decreases natural killer cell activity among women exposed to aircraft noise hartono* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction there is sufficient scientific evidence that noise has an influence on ischemic heart disease, hypertension, hearing impairment, sleep disturbance, and duodenal and gastric disorders. however, for other effects such as changes in the immune system, the evidence is limited.(1) one of the impacts of exposure to noise is stress. (1-3) in response to a stressor, physiological changes are set into motion to help an individual cope with the stressor. however, a chronic activation of these stress responses, which include the hypothalamic-pituitary-adrenal (hpa) axis and the sympathetic-adrenalmedullary (sam) axis, results in chronic production of glucocorticoid hormones and catecholamines.(3,4) glucocorticoid receptors expressed on a variety of immune cells bind cortisol and interfere with the function of nfkappa beta (nf-kβ), which regulates the 154 hartono cortisol decreases natural killer activity of cytokine producing immune cells. adrenergic receptors bind epinephrine and norepinephrine and activated camp response e l e m e n t b i n d i n g p r o t e i n , i n d u c i n g t h e transcription of genes encoding for a variety of cytokines. the changes in gene expression mediated by glucocorticoid hormones and catecholamines can dysregulate the immune system. lymphocytes, natural killer cells, m a c r o p h a g e s a n d g r a n u l o c y t e s e x h i b i t receptors for many neuroendocrine products of the hpa and sam axes, such as cortisol and catecholamines which can cause changes in cellular trafficking, proliferation, cytokine secretion, antibody production and cytolytic activity.(5-7) high intensity noise is more annoying than noise of low intensity, whilst intermittent noise is more annoying than continuous noise.(8) women are more sensitive in response to noise than are men.(9) in the category of intermittent noise, aircraft noise is significantly more annoying. chronic exposure to noise is thought to bring about significant impacts if it happens for more than a year.(1) natural killer (nk) cells are important components of the innate immune systems, o w i n g t o t h e i r c y t o k i n e a n d c h e m o k i n e production and ability to lyse target cells without prior sensitization. human nk cells comprise ±15% of all lymphocytes and are defined phenotypically by their expression of cd56 and cd16, and lack of expression of cd3 (cd56+cd16+cd3-).(10) among the many indices of immune function, nk activity and nk cell subsets have been of interest to researchers because nk cells are known to be important in host defense against viral diseases a n d a p p e a r t o p l a y a s i g n i f i c a n t r o l e i n protection against neoplastic growth.(11-13) nk cells are also one of the leukocyte subsets that are responsive to physiological stress and psychological stress.(14-16) based on the above considerations, the aim of this research study was to find out the presence of a correlation of circulating cortisol levels with nk cell numbers and nk cell activity among women with aircraft noise stress in the area of adi sumarmo airport of solo, indonesia. methods research design this research study was an observational analytical study with cross sectional design. it was conducted from july 2008 to june 2009 among residents in the neighborhood of the runway of adi sumarmo international airport of solo, indonesia, namely in the villages of dibal and gagak sipat, ngemplak subdistrict, boyolali regency. subjects t h e s t u d y p o p u l a t i o n c o m p r i s e d a l l residents of dibal village and gagak sipat v i l l a g e , n g e m p l a k s u b d i s t r i c t , b o y o l a l i regency who fulfilled the following inclusion criteria: female, married, housewife, aged 2040 years old (age influences the number of nk cells),(10) and having lived in the area for at least 1 year. exclusion criteria were consumption of non-herbal or herbal medicines, pregnancy, hearing loss, infectious disease (colds, flu, and diarrhea), and diabetes mellitus. the respondents who fulfilled the criteria were selected by means of simple random sampling. the sample size was calculated using the formula of snedecor and cochran or by u s i n g wi n e p i s c o p e 2 . 0 w i t h e s t i m a t e d difference between means (ä = 0.05), for a confidence level of 95% and significance level of 5%. based on the results of previous studies in the area,(17) the number of lymphocytes sd was 700; m1 was 3.6 x 10 3/µl; m2 was 2.5 x 103/µl, and the number of subjects per group was 13. the total number of subjects was 39, who were divided into 3 groups, on the basis of the distance of their residential area from the runway. group 1 respondents lived at a distance of less than 500 meters from the tip of the runway; group 2 respondents were 155 subjects whose residential area was between 500 and 1,000 meters from the tip of the runway, while group 3 respondents lived at a distance of more than 1,000 meters from the tip of the runway. the subdivision into groups according to the distance of their residence to the runway was based on the following considerations: 1. previous research by hartono(17) in the same geographical area showed that at a distance of less than 500 meters from the runway, the noise intensity was 96.57 db, as measured with the wecpnl scale. at a distance of more than 1000 meters from the runway, the noise intensity was 51.56 db on the wecpnl scale, while at a distance of 500 to 1000 meters from the runway, the noise intensity was 74. 42 db measured with the wecpnl scale.(18) 2. the area around the airport with a noise i n t e n s i t y l e v e l o f m o r e t h a n 8 0 d b w e c p n l i s n o t r e c o m m e n d e d f o r settlement and was therefore chosen as the exposure area, whereas the area with a noise intensity level of less than 56 db wecpnl was used as the control area, since the area is safe for settlement ( d e c r e e o f d i r e c t o r g e n e r a l o f a i r transportation no. skep/109/vi, for the year 2000). measurement of noise exposure the noise exposure was measured using a sound level meter (extech model 407735, japan) and rated according to wecpnl. in each of the two study areas measurement of noise was conducted at three different points with a portable sound level meter (slm), and the acoustic physical parameter was measured in db with a load. the slm was placed with its filter parallel to the subject’s ears. the slm was set up at its maximum function of value to measure the peak noise level of aircraft passing over the areas so that the background noise level could be blocked. the acoustic physical parameter was recorded based on the peak noise level occurring at aircraft take-off and landing, and the time of occurrence of noise level was also recorded. the noise level was rated by using the wecpnl (weighted equivalent continuous perceived noise level) scale, according to the following equation: wecpnl = db (a) + 10 log n-27 n = n1 + 3n2 + 10n3 db (a): average decibel score of each peak level of aircraft activity in a day. n : number of aircraft arrivals and departures in 24 hours. n1 : number of aircraft arrivals and departures b e t w e e n 0 7 . 0 0 a n d 1 9 . 0 0 we s t e r n indonesia time n2: number of aircraft arrivals and departures b e t w e e n 1 9 . 0 0 a n d 2 2 . 0 0 we s t e r n indonesia time n3: number of aircraft arrivals and departures b e t w e e n 2 2 . 0 0 a n d 0 7 . 0 0 we s t e r n indonesia time the measurements in db (a) were then converted into wecpnl in accordance with the number of aircraft passing over the area in 24 hours. biochemical measurements after the subjects had been selected, 4 ml of venous whole blood was taken from the respondents, between 7.00 to 08.00 western indonesian time. subsequently the plasma cortisol level was measured by means of enzyme-linked immunosorbent assay (elisa). the number of nk cells was determined as follows: peripheral blood mononuclear cells (pbmc) were separated from 10 ml of the whole blood using ficoll-hypaque densitygradient centrifugation (30 minutes, 20oc, 400 x g). the pbmc were washed twice with pbs and suspended in rpmi 1640 medium (gibco, univ med vol.29 no.3 156 hartono cortisol decreases natural killer invitrogen) containing 10% (vol/vol) fetal bovine serum, penicillin (100 iu/ml) and streptomycin (100 µg/ml), stored at 4oc until required for analysis. nk cells were enumerated by three-color immunophenotyping using appropriate combinations of monoclonal antibodies (pharmingen, san diego, ca), these being conjugated fluorescein isothiocyanate (fitc), phycoerythrin (pe) and perpridininchlorophyll protein (percp). briefly, a sample of 1 x 106 pbmc was mixed with saturating amounts of monoclonal antibody conjugated with fitc (anti-cd16), pe (anti-cd56) and percp (anti-cd3). after being washed twice with pbs, the stained cells were passed through a facscan flow-cytometer (becton dickinson). t h e n u m b e r o f n k c e l l s i n p b m c w a s calculated as a percentage of cd16+cd56+cd3cells.(13,14,16) nk cell activity was measured by a nonradioactive method, which was a modification of the procedure conducted by andalib et al.(19) the lymphocytes were separated by means of t h e f i c o l l h y p a q u e g r a d i e n t t e c h n i q u e (lymphoprep, norway). lymphocytes were isolated, washed, and brought to a concentration of 5 x 105 cells/ml in rpmi 1640 + 10% fcs (gibco, germany). atcc-k562 erythromyelocytic leukemia cells as the target cells were maintained in continuous suspension culture in rpmi 1640 + 10% fcs, supplemented with l-glutamine, 100 µg/ml streptomycin and 100 u/ml penicillin. a working solution was prepared by adding 0.5 µg/ml propidium iodide (pi, sigma) in rpmi 1640 + 10% fcs. the lymphocytes (effectors) and k562 leukemia cells (target cells) were mixed and cultured in the same tube, with a target to effector ratio of 50:1. briefly, the tubes containing the mixed cells were centrifuged for 7 minutes at 250 x g at room temperature, then kept at 37oc for 10 minutes in a water bath, after which the mixed cells were resuspended. in the working solution, a concentration of 1 x 105 cells/ml was prepared to avoid recycling of nk cells. the samples were then incubated for 1.5 hours at 37oc, under 5% co2, then the cell concentration brought to 1 x 106 cells/ml and the samples were ready for flow-cytometry. the resulting measurements were read twice by t w o d i ff e r e n t o b s e r v e r s . to m o n i t o r t h e spontaneous death rate of nk cells, target cells only (without effector cells) were incubated. the final concentration of 1 x 105 cells/ml has been running as control. the cells were analyzed with a facscalibur flow cytometer (becton dickinson, palo alto, ca, usa). nk cell activity was calculated based on the percentage of dead target cells (k562) in the tube containing effector cells, subtracted by the percentage of dead target cells in the control tube (without effector cells), divided by the total number of cells (100%), and subtracted by the dead target cells in the control tube (without effector cells), as shown in figures 1-3.   r2 figure 1. comparison between living k562 target cells, labeled in red color, and dead target cells, labeled in green color, in group 1 figure 2. comparison between living k562 target cells, labeled in red color, and dead target cells, labeled in green color, in group 2   r 157 t h e d o t p l o t h a s b e e n d e f i n e d a s fluorescence light-2 (ssc-height) versus fluorescence light-1 (pi) detectors that show the fluorescence intensity in distinctive stained cell populations (i.e. gated specific populations). dead k562 cells form a well-defined population with distinctive fluorescence staining with propidium iodide (green gated). the dot-plot is illustrated the representative for dead (green) and live target cell population (red) based on propidium iodide dye staining, in comparison with control cells (red population only) for k562. the calculation of cytotoxicity is based on the two different gated cells with the proportion of cell population based on the acquisition data on system. data analysis the study data were analyzed by analysis of variance (anova) to verify the differences in cortisol level, number of nk cells, and nk cell activity, among the three groups of r e s p o n d e n t s . p e a r s o n ’s p r o d u c t m o m e n t correlation test was used to investigate the correlation of cortisol level with number of nk cells and activity of nk cells. ethical clearance the study participants were subject to the e t h i c a l c l e a r a n c e r e l a t e d m e a s u r e s a n d procedures. ethical clearance was obtained from the ethical review committee, school of medicine, sebelas maret university. the r e s e a r c h s t u d y w a s a l s o s u b j e c t t o confidentiality and anonymity principles towards the data on the respondents. results t h e d a t a o n n o i s e i n t e n s i t y l e v e l (wecpnl), cortisol level, nk cell numbers and activity for each group are presented in table 1. the study showed that at a distance of less than 500 meters from the runway, the noise intensity was 92.29 db, while at a distance of 500 to 1,000 meters from the runway the noise intensity was 71.49 db, and at a distance of more than 1,000 meters from the runway the univ med vol.29 no.3 table 1. noise intensity level (wecpnl), circulating cortisol level, and number and activity of nk cells in each group a, b, c real difference in post hoc test using lsd test completed with homogenous subsets with á = 0.05. notes: group 1, respondents residing less than 500 meters from tip of runway at noise intensity of 92.29 db group 2, respondents residing between 500 and 1,000 meters from tip of runway at noise intensity of 71.79 db group 3, respondents residing more than 1,000 meters from tip of runway at noise intensity of 52.17 db figure 3. comparison between living k562 target cells, labeled in red color, and dead target cells, labeled in green color, in group 3. 158 hartono cortisol decreases natural killer n o i s e i n t e n s i t y w a s 5 2 . 1 7 d b , a l l d b measurements being on the wecpnl scale. the mean cortisol levels of the respondents in areas 1, 2, and 3, were 13.25 µg/dl, 12.19 µg/ dl, and 10.16 µg/dl respectively, which were significantly different (p=0.018). follow up with the post hoc test (á =0.05) showed that there was a significant difference between cortisol levels in the different groups, with the highest levels in area 1 and the lowest in area 3 (p<0.05). based on table 1, it can be seen that the mean numbers of nk cells (cd56+cd16+cd3) of the respondents in groups 1, 2, and 3 were 18.80%, 17.52%, and 12.88% respectively. these results were significantly different (p=0.038) and follow up with the post hoc test (á=0.05) showed that there was a significant d i f f e r e n c e b e t w e e n n k n u m b e r s i n t h e different groups of respondents (p<0.05). t h e a c t i v i t y o f n k c e l l s o f t h e respondents of groups 1, 2, and 3 were 12.50%, 17.20%, and 22.33% respectively. the results of the anova test showed that there was a significant difference in the mean activity of nk cells among the three groups (p=0.000). after the anova test was continued with the post hoc test, the results were similar to those on cortisol level. there was a significant d i ff e r e n c e i n m e a n a c t i v i t y o f n k c e l l s between group 1 and group 3, between group 1 and group 2, and between group 2 and group 3 (p<0.05). the results of pearson’s product moment correlation test showed that there was a s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n circulating cortisol levels with the number of n k c e l l s ( c d 5 6 +c d 1 6 +c d 3 -) [ r = 0 . 5 4 7 ; p<0.05] (table 2) and a negative (or inverse) correlation between circulating cortisol levels and nk cell activity (r=–0.578; p<0.05), signifying that cortisol level is inversely proportional to nk cell activity. the increase in nk cell numbers due to noise exposure had a negative correlation with nk cell activity, but the correlation was less strong (r=-0.343 and p<0.05) (table 2). discussion from the test results we can conclude that exposure to aircraft noise at an intensity level of 71.49 db (wecpnl) for more than one year acted as a stressor which increased the mean cortisol level of the treatment groups compared to that of the control group (intensity level of 52.17 db). in the group with aircraft noise at an intensity level of 92.29 db (wecpnl) the noise acted as a stressor, resulting in a higher increase in mean cortisol level than was the case with aircraft noise at an intensity level of 71.49 db (wecpnl). the aforementioned results are similar to those reported by cheng and ariizumi (2007) who did an experiment with balb/c mice at a noise intensity level of 90 db, with a length of exposure of five hours per day for four weeks. the mean cortisol level of the treatment group of mice exposed to noise for four weeks was 4.25 µg/dl, which was higher than that of the control group, with mean cortisol level of 1.60 µg/dl.(20) this is in line with the results of a research study conducted by dhanalakshmi et al., who reported that cold stress applied to *p< 0.05 table 2. pearson’s correlation coefficient between cortisol level and number and activity of nk cells 159 white rats resulted in increased plasma cortisol levels, which affected their immune system.(21) it is known that continuous recurrent aircraft noise will bring about stress. this chronic stress will increase the cortisol and catecholamine levels through hpa and sam pathways.(6,11,22) the increase in cortisol level via the glucocorticoid receptors will inhibit production of several cytokines generated by nk cells (ifn-á, ifn-â, ifn-ã, il-10, gmc s f, a n d t n f â ) . i n t e r f e r o n i s a m a j o r regulator of nk cells.(6,10) ifn-ã functions to inhibit the proliferation of nk cells through the inhibition of il-4, thus decreased inf-ã production will cause increased proliferation of nk cells. (10,23,24) t h e r o l e o f i l 2 i s t o i n c r e a s e t h e p r o l i f e r a t i o n o f n k c e l l s t h r o u g h t h e interleukin-2 receptors (il-2ráâã) which are expressed by nk cells of cd56bright and il2ráâã receptors which are expressed by nk cells of cd56dim. in relation to the proliferative response, the two receptors have a high proliferation response toward il-2 at a low dosage and do not have any responses if both receive a high dosage of il-2, particularly il2 r á â ã. the decreased level of il-2 (lowdosage) due to the inhibition of glucocorticoid activity will result in increased proliferation of nk cells.(10,24) several findings of animal model studies show that the increase in glucocorticoids due to stress in the long term resulted in a decreased activity of nk cells.(6,25,26) the activity of nk cells is influenced by tnf and the interferons (ifn á, â, and ã), which cause an increase in the cytolytic function of nk cells. ifn-á brings a b o u t a h i g h e r i n c r e a s e t o t h e c y t o l y t i c functions than ifn-ã. other lymphokines also have effects on nk cells. il-12 and il-2 synergistically increase the cytotoxicity of nk cells. nk cells are also able to lyse cells with the aid of il-2. on the one hand il-2 is potent induction factor for cytokine production by nk cells. on the other hand, il-2 is also a growth f a c t o r f o r n k c e l l s a n d p l a y s a r o l e i n increasing the cytotoxicity and migration of nk cells.(10,12,27) the increase in cortisol level due to stress in the long term will inhibit the activity of nfêb (nf-kappa beta). due to such inhibition, several cytokines generated by nk cells (ifná, ifn-â, ifn-ã, il-10, gm-csf, and tnf-â) and il-2 and il-4 generated by t cells will decrease in terms of their production. the decrease in the levels of cytokines tnf, ifn, il-2 and il-12, which is caused by inhibition of cortisol activity, will cause a decrease in nk cell activity.(10,12,23) these results are similar to those reported by andalib et al. in a study on 45 women suffering from chronic stress due to recurrent spontaneous abortion.(19) their study showed that there was a high level of stress in women with recurrent spontaneous abortion, and such a stress condition resulted in decreased activity of nk cells. similar results were also reported by morikawa et al., suzui et al. and nagao et al.(13,14,16) conclusion there was a relationship of circulating cortisol levels with natural killer cell activity and numbers among women with aircraft noise stress in the area of adi sumarmo airport, solo, boyolali. based on the results of the study, preventive measures are needed to deal with the aircraft noise to prevent further negative impacts on residents around adi sumarmo international airport, solo, boyolali. acknowledgment the author would like to thank prof. dr. adi heru husodo, dr. msc from the public health department, faculty of medicine, gadjah mada university, prof. marsetyawan, dr. hnes., msc., phd from the histology department, faculty of medicine, gadjah mada university and suharyana, phd from the d e p a r t e m e n t o f p h y s i c s , f a c u l t y o f univ med vol.29 no.3 160 hartono cortisol decreases natural killer mathematics and sciences, sebelas maret university, for fruitful discussions throughout the research study. references 1. passchier v w, passchier wf. noise exposure and public health. environ health perspect 2000;108:123-31. 2. haines mm, stansfeld sa, job soames rf, berglund b, head j. a follow-up study of effects of chronic aircraft noise exposure on child stress responses and cognition. inter j epidemiol 2001; 30:839-45. 3. spreng m. central nervous system activation by noise. noise health 2000;7:49-57. 4. ader r. on the development of psychoneuroimmunology. euro j pharmacol 2000;405:167-76. 5. glaser r, kiecolt gj. stress damage immune system and health. discov med 2005;5: 165-9. 6. padgett d, glaser r. how stress influences the immune response.trends immunol 2003;24:4448. 7. ronald de. noise, brain and stress. endocrine reg 2003;37:51-68. 8. stansfeld sa, matheson mp. noise pollution: non-auditory effects on health. british med bull 2003;68:243-57. 9. melamed s, luz j, green ms. noise exposure, noise annoyance and their relation to psychological distress, accident and sickness absence among blue-collar workers-the cordis study. lsr j med sci 1992;28:629-35. 10. cooper ma, fehniger ta, caligiuri ma. the biology of human natural killer-cell subsets. trends immunol 2001;22:633-40. 11. kiecolt gj, robles tf, heffner kl, loving tj, glaser r. psycho-oncology and cancer: psychoneuroimmunology and cancer. euro society med oncol 2002;31:165-9. 12. megan ac, todd af, sarah ct, kenneth sc, bobak ag, tariq g, et al. human natural killer cells: a unique innate immunoregulatory role for the cd56 bright subset. blood 2001;97:3146-51. 13. morikawa y, higashiguchi kk, tanimoto c, hayashi m, oketani r, miura k, et al. crosssectional study on the relationship of job stress with natural killer cell activity and natural killer cell subsets among healthy nurses. j occupl health 2005;47:378-83. 14. suzui m, kawai t, kimura h, takeda k, yagita a, okumora k, et al. natural killer cell lytic activity and cd56 dim and cd56 bright cell distribution during and after intensive training. j appl physiol 2004;96:2167-73. 15. kanemi o, zhang x, sakamoto y, nagatomo r. acute stress reduces intraparenchymal lung natural killer cells via beta-adrenergic stimulation. clin exp immunol 2005;139:25-34. 16. nagao fm, takeda k, yagita h, okumura k. mobilization of nk cells by exercise: downmodulation of adhesion molecules on nk cells by catecholamines. am j physiol regulatory integrative comp physiol 2000; 279:1251-6. 17. hartono. the effect of aircraft noise exposure to the number of lymphocytes among people in the area of adi sumarmo airport solo. environ 2006; 7:20-4. 18. hartono, isna q, margono. the effect of aircraft noise exposure on the histologic structure of duodenal layer in rat (rattus norvegicus). environ 2007;8:33-6. 19. andalib a, rezaie a, oreizy f, shafiei k, baluchi s. a study on stress, depression and nk cytotoxic potential in women with recurrent spontaneous abortion. iran j allergy asthma immunol 2006;5:9-16. 20. cheng z, ariizumi m. modulation of immune functions and oxidative status induced by noise stress. j occup health 2007;49:32-8. 21. dhanalakshmi s, srikumar r, manikandan s, parthasarathy nj, devi rs. antioxidant property of triphala on cold stress induced oxidative stress in experimental rats. j health science 2006;52: 843-7. 22. guyton ac, hall je. textbook of medical physiology. 11th ed. pennsylvania: elsevier inc.; 2006. 23. srikumar r, parthasarathy nj, manikandan s, muthuvel a, rajamani r, sheeladevi r. immunomodulatory effect of thripala during experimentally induced noise stress in albino rats. j health science 2007;53:142-5. 24. loza mj, perussia b. differential regulation of nk cells proliferation by type i dan type ii ifn. j immunol 2004;16:23-33. 25. gan x, zhang l, solomon gf, bonavida b. mechanism of norepinephrine-mediated inhibition of human nk cytotoxic functions: inhibition of cytokine secretion, target binding, and programming for cytotoxicity. brain behav immun 2002;16:227–46. 26. otagiri a, wakabayashi i, shibasaki t. selective corticotropin-releasing factor type 1 receptor antagonist blocks conditioned fear-induced release of norepinephrine in the hypothalamic 161 paraventricular nucleus of rats. j neuroendocrinol 2000;12:1022-6. 27. kehrl jh, ducovich m, whalen g, katz p, fauci as, green wc. novel interleukin-2 (il-2) univ med vol.29 no.3 receptor appears to mediate il-2 induced activation of natural killer cells. j clin inves 1998;81:200-5. oktavianus 19 *departement of histology faculty of medicine islamic university of indonesia yogyakarta **departement of anatomy faculty of medicine islamic university of indonesia yogyakarta correspondence dr. ika fidianingsih, m.sc *departement of histology faculty of medicine islamic university of indonesia jl. kaliurang km 14.5 sleman yogyakarta phone: +62274-898470 ext.2033 email: ikafidy@yahoo.com univ med 2014;33:19-26 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background cancer is a dreadful disease caused by abnormal and uncontrolled cell division. annona muricata l, also known as soursop, is useful as an anticancer herbal medication since its leaves, seeds and fruits contain active compounds called annonaceous acetogenins. the objective of this study was to scientifically justify the traditional application of soursop for anticancer treatment in the community, by comparing the antiproliferative effect of annona muricata l leaf, seed and fruit aqueous extracts on t47d breast cancer cells. methods this study used an experimental post test trial with control group design infusions of soursop leaves, seeds, and fruits collected from kaliurang, sleman district, yogyakarta were used for cytotoxicity tests on t47d cells, in comparison with tamoxifen as standard cancer therapy. proliferative inhibition was determined by 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide [mtt] assay. the parameter of proliferative inhibition was ic 50 which is defined as 50% proliferative inhibition ability of soursop and tamoxifen. significant differences between groups were determined at p<0.05 by kruskal-wallis test. results the leaves, fruits, and seeds annona muricata and tamoxifen were proven to be able to inhibit t47d cell proliferation. the ic 50 of annona muricata leaf, seed, fruit aqueous extracts and tamoxifen were 31,384.21 µg/ml; 1.528,800 µg/ml; 329,194.81 µg/ml and 114.52 µg/ml, respectively (p=0.016). the ic 50 of annona muricata aqueous extract was significantly different from that of tamoxifen. conclusions the proliferative inhibition of soursop leaves against t47d breast cancer cells is higher than that of soursop fruits and seeds. the leaves, fruits, and seeds of annona muricata aqueous extract was less toxic compared to tamoxifen. keywords: annona muricata l, cytotoxic, t47d breast cancer cells annona muricata aqueous extract suppresses t47d breast cancer cell proliferation ika fidianingsih* and ety sari handayani** 20 fidiyaningsih, handayani annona muricata suppresses t47d ekstrak air annona muricata l menghambat proliferasi sel kanker payudara t47d latar belakang kanker merupakan penyakit yang menakutkan karena adanya proliferasi sel yang abnormal dan tidak terkendali. annona muricata atau sirsak terutama daun dan bijinya telah diteliti bermanfaat sebagai antikanker karena mengandung senyawa aktif terutama annonaceous acetogenins. buah sirsak juga mengandung annonaceous acetogenins, namun buah sirsak yang mempunyai rasa lebih enak, belum banyak diteliti efeknya dalam membunuh sel kanker. penelitian ini bertujuan untuk membandingkan efek ekstrak air dari daun, biji dan buah sirsak terhadap penghambatan proliferasi sel kanker payudara t47d. metode sebuah rancangan eksperimental pasca perlakuan digunakan pada studi ini. infusa buah, biji dan daun sirsak dilakukan uji sitotoksik terhadap sel t47d dengan pembanding tamoksifen. penghambatan pertumbuhan dilihat dengan metode 3-(4,5-dimethyl thiazol-2-yl)-2,5-diphenyl tetrazolium bromide [mtt] assay. parameter penghambatan pertumbuhan digunakan ic 50 yaitu kemampuan bahan uji dalam menghambat proliferasi sebanyak 50%. uji kruskal-wallis digunakan untuk menguji perbedaan sitiosik antara kelompok perlakuan. tingkat kemaknaan yang digunakan besarnya p<0,05. hasil buah, daun, biji sirsak dan tamoksifen mampu menghambat proliferasi sel t47d. ic 50 dari buah, daun, biji sirsak sangat berbeda secara bermakna dengan tamoksifen, masing-masing besarnya 329.194,81 µg/ml; 1.528.800,00µg/ ml; 31.384,21µg/ml dan 114,52 µg/ml (p=0,016). kesimpulan penghambatan proliferasi ekstrak air terhadap sel kanker payudara t47d paling tinggi berturut-turut berasal dari daun, kemudian buah dan biji sirsak. ekstrak air dari buah, biji dan daun sirsak kurang toksik dibandingkan tamoksifen. kata kunci: annona muricata l, sitotoksik, sel kanker payudara t47d abstrak introduction cancer is one of the many diseases causing public health problems in the world as well as in indonesia. the number of people living with cancer in the world was 12.8 million in 2008, 7.6 million of which died.(1) if the trend continues, 21.3 million new cancer cases will be discovered in 2030 and almost half of them will die.(2) in indonesia, the proportion of cancer cases (10.2%) has a tendency to increase. cancer is claimed as the seventh leading cause of death (5.7%) in indonesia after stroke, tuberculosis, hypertension, injury, perinatal deaths, and diabetes mellitus. according to sistem informasi rumah sakit (sirs), in the year 2007 breast cancer ranked first among hospitalized patients in indonesia (16.85%), with a prevalence of 26 per 100,000 women.(3) cancer therapy applied mainly in the later stages of cancer is still a problem in the world as well as in indonesia. the signs and symptoms of 21 cancer are almost undetectable in the early stages, hence, patients with cancer are usually diagnosed suffering from cancer in the later stages. according to the study of ng et al.,(5) 63% of cancer patients at dharmais cancer center in jakarta presented with tnm stages iii or iv.(4) the cancer patients in the later stages who receive standard therapy still have a poor outcome. recently, there have been a large number of herbals showing chemopreventive potential, thus becoming attractive modalities to be examined and developed continuously. in the future, herbal medicines are expected to be the solution to the problem of poor treatment outcomes of cancer. therefore, the indonesian government has been supporting the development of herbal medicines for cancer therapy by ministerial decree (sk menkes no 381/menkes/ sk/iii/2007). in addition, complementary and alternative medicines (cam) such as herbals, are believed by the public to be safe and to have less side effects.(6) c am use is common among cancer patients in indonesia as well as in the united states,(7,8) but many of the herbal medicines are not based on clear evidence. one of the herbal medicines preferred by the community for cancer is soursop ( annona muricata l). soursop has been studied as an anticancer m e d i c a t i o n b e c a u s e i t c o n t a i n s a c t i v e c o m p o u n d s c a l l e d a c e t o g e n i n s . ( 9 ) t h e acetogenins in soursop leaves and seeds used as anticancer medication are selective, which means that normal cells are not killed. the cytotoxic effect of acetogenins from sour sop leaves and seeds has been studied in vitro on m a n y c a n c e r c e l l l i n e s , s u c h a s h u m a n hepatoma, lung carcinoma, human breast solid tumor, prostate adenocarcinoma, pancreatic carcinoma, colon adenocarcinoma, human lymphoma and multi-drug resistant human breast adenocarcinoma.(10,11) unfortunately, the aqueous extract of soursop leaves and seeds that people commonly consume have not been studied for its anticancer properties. this is in contrast with the proved anticancer effect of ethanolic(1214) and butanolic soursop extracts,(15) or of soursop essential oil.(16) in addition, soursop fruits, with their delicious taste, have also not been studied as anticancer medication. actually, the fruits of annona muricata had been reported to contain the acetogenin cis-annoreticuin,(17) while the cis-annoreticuin of annona montana was reported to exhibit cytotoxicity against a human hepatoma carcinoma cell line (hep g2). ( 1 8 ) t h e p r e s e n t s t u d y c o m p a r e s t h e antiproliferative effects of aqueous extracts of annona muricata fruits, leaves and seeds on t47d breast cancer cells, with the objective to provide scientific proof of the soursop anticancer effect. methods research design this research used an experimental post test trial with control group design and was conducted from march to october 2013 at integrated research testing laboratory gadjah mada university. plant material the leaves, seeds and fruits of soursop were obtained from kaliurang, sleman district, yogyakarta, indonesia. they were taxonomically identified by the laboratory of pharmacognosy, faculty of mathematics and natural sciences, islamic university of indonesia. extraction procedure t h e p l a n t m a t e r i a l w a s d r i e d f o r approximately 96 hours in a drying cabinet and powdered. five grams of the powdered material (fruits, seeds, or leaves) was added to 50 ml of distilled water, heated for 15 minutes to obtain an infusion, which was filtered and evaporated down to 1 ml (stock solution = 5 g/1 ml). intervention cytotoxic testing of soursop was carried out with initial concentrations of 1000 ìg/ml, 10,000 univ med vol. 33 no.1 22 fidiyaningsih, handayani annona muricata suppresses t47d ì g / m l, and 50,000 ì g / m l, but did not yield any ic 50 values. the last cytotoxic test on soursop was performed with an initial dose of 300,000 ì g / m l (figure 1), which was prepared from 60 ì l soursop stock solution. cytotoxic testing of tamoxifen was performed with an initial dose of 1000 ì g / m l (figure 2), taken from 100 ìl stock [tamoxifen stock = 10 mg/1 ml, dissolved in dimethyl sulfoxide (dmso)]. antiproliferative assay proliferative inhibition was determined by cytotoxic testing using the mtt assay on 96well microplates (figures 1 and 2). t47d breast cancer cells, which are estrogen receptorpositive, were obtained from the integrated research testing laboratory (laboratorium pengujian penelitian terpadu universitas gadjah mada). cytotoxic test results were read by elisa reader at 595 nm. percentage cytotoxicity of soursop and tamoxifen was calculated by the formula: % cytotoxicity = 100{[(a-b) – (c-b)]/(a-b)} x 100% [a= od of control cell; b = od of medium; c= od of treated cell] statistical analysis the experiments were performed in triplicate. cytotoxicity percentages are presented as mean ± standard deviation. the percentage of inhibition of each test material was converted into dose-responsiveness curves using probit analysis to obtain the ic 50 of each test material. significant differences between groups were determined at p<0.05 by kruskal-wallis test. figure 1. microplate scheme of soursop cytotoxic test u1-u10: medium + t47d cells + soursop fruit extract (at doses of 300,000; 150,000; 75,000; 37,500; 18,750; 9,375; 4,687.5; 2343.75; 1171.875; 585.9375 µg/ml). i1-i10: medium + t47d cells + soursop seed extract (at doses of 300,000; 150,000; 75,000; 37,500; 18,750; 9,375; 4,687.5; 2343.75; 1171.875; 585.9375 µg/ml). d1-d10: medium + t47d cells + soursop leaf extract (at doses of 300,000; 150,000; 75,000; 37,500; 18,750; 9,375; 4,687.5; 2343.75; 1171.875; 585.9375 µg/ml). ks = medium + t47d cells; km = medium only figure 2. microplate scheme of tamoxifen cytotoxic test t1-t7 = positive controls (medium + t47d cells + tamoxifen) (at doses of 1000; 500; 250; 125; 62.5; 31.25 µg/ml); ks = medium + t47d cells; km = medium only 23 ethical clearance the study protocol was approved by the medical and health research ethics committee (mhrec), faculty of medicine, gadjah mada university – dr. sardjito general hospital (reference number: ke/fk/259/ec). results cytotoxic testing was performed three times, starting at a dose of up to 1,000 µg/ml, up to 10,000 µg/ml, and up to 50,000 µg/ml. however, the soursop fruit, seed and leaf aqueous extracts at such doses could not inhibit t47d cell proliferation. the cytotoxic tests at an initial dose of 300,000 µg/ml showed the greatest proliferative inhibition with soursop leaf (88.45%) at a dose of 75,000 µg/ml. the lowest proliferative inhibition was found in soursop seed extract, with 36.28% inhibition at a dose of 300,000 µg/ml (table 1). the proliferative inhibition of tamoxifen was better than those of the aqueous extracts of soursop leaves, seeds, and fruits. tamoxifen at a dose of 500 µg/ml was able to inhibit proliferation of t47d cells by 99.91% (table 2). the mean ic 50 values show that the lowest dose was for tamoxifen (114.52 µg/ml), followed by soursop leaf, fruit and seed (table 1). the ic 50 value of tamoxifen was significantly different from the ic 50 values of the soursop aqueous extracts, showing that the latter are less cytotoxic (table 3). discussion we report the finding of an antiproliferative effect of aqueous extract of soursop leaves on t47d cells but only at high doses (mean ic 50 = 31,384 µg/ml). we also noted that there was a significant difference in the ic 50 values of soursop leaf aqueous extract and tamoxifen. a previous study with soursop leaf butanol extract obtained an ic 50 value of 29.2 µg for mda-mb-435s cells and 30.1 µg for hacat cells, while the ic 50 value of soursop leaf ethanol extract was 17,149 for t47d cells. the results of this study showed that soursop leaf aqueous extract is less cytotoxic, because its acetogenin content is less than that of the ethanol extract.(19) if people have to consume aqueous extracts of soursop leaves, they will require higher doses to get any cytotoxic effect. table 2. proliferative inhibition of tamoxifen on t47d cells table 1. proliferative inhibition of (%) soursop on t47d cells values represent mean ± s.d. values represent mean ± s.d. (%) univ med vol. 33 no.1 24 fidiyaningsih, handayani annona muricata suppresses t47d in this study, the aqueous extract of soursop seeds also showed less cytotoxic a c t i v i t y, h a v i n g a v e r y h i g h i c 5 0 v a l u e (1,528,800). to our knowledge there have been no studies demonstrating the ability of soursop seed aqueous extract to kill cancer cells, except a t h i g h d o s e s . ( 2 0 ) h i g h d o s e s o f a n n o n a muricata seeds (1000 mg/kg bw) decreased the viability of ovarium cancer cells in rats, but to a lesser extent than vinblastin.(20) in contrast, organic and aqueous extracts of defatted annona squamosa (custard apple) seeds tested o n d i ff e r e n t h u m a n t u m o r c e l l l i n e s f o r antitumoral activity, induced apoptosis in mcf7 and k-562 cells, but not in colo-205 cells.(21) the aqueous extract of soursop fruits was able to inhibit proliferation of t47d cells, in accordance with previous studies. day et al(22) reported selective inhibition of human breast cancer cell growth by an acetone extract of graviola (soursop) fruits in vitro and in vivo involving downregulation of egfr expression. the aqueous extract of cherimoya (annona chirimola) fruit pulp presented antitumoral activity in drosophila melanogaster.(23) in the present study, the ic 50 of soursop fruit was better than that of soursop seed because the fruit has a higher content of annonacin (the main acetogenin). a dichloromethane extract of a n n o n a m u r i c a t a s e e d s c o n t a i n e d o n l y annoreticuin-9-one, while the pulp (flesh) of the fruits yielded cis-annoreticuin and sabadelin.(19) according to a literature search, the cytotoxic effect of soursop is due to the acetogenin fraction and soursop ethanol extract possesses anti-inflammatory activity as shown table 3. mean ic 50 values of td47d cells by treatment groups soursop and tamoxifen i n e x p e r i m e n t a l a n i m a l s . ( 2 4 , 2 5 ) m o r e o v e r, acetogenins also have an inhibitory effect on nadh ubiquinone oxidoreductase, which is an important enzyme in oxidative phosphorylation reactions, resulting in lack of atp in the cells.(10) in fact, the aqueous extract of soursop leaves has been studied in vivo as an antioxidant to lower oxidative stress in the mice liver induced by streptozotocin.(27) our study has shown that the aqueous extract of soursop fruit has a better inhibitory effect on the proliferation of t47d cells than the aqueous extract of soursop seed, but both are less cytotoxic to t47d cells than tamoxifen. this means that for cancer self-treatment by the community, large amounts of soursop fruit have to be consumed. future research in experimental animals is needed to determine the exact dose of soursop aqueous extract that can kill cancer cells. conclusions proliferative inhibition of aqueous extract of soursop leaves against t47d cells is the highest compared to that of the fruits and seeds. the ic 50 value of soursop aqueous extract against t47d cells is less toxic than tamoxifen. acknowledgements the authors would like to thank the faculty of medicine, islamic university of indonesia for financial support of this research. we would also like to extend our gratitude to ms tri yuliati for helping us in the implementation of this study. values represent mean ± s.d. 25 references 1. jemal a, bray f, center mm, ferlay j, ward e, forman d. global cancer statistics. ca cancer j clin 2011;6:69–90. 2. world health organization international ageing for research on cancer. globocan 2008: cancer incidence, mortality, prevalence and disability-adjusted life years (dalys) worldwide. lyon: iarc press;2010. 3. departemen kesehatan. laporan nasional riset kesehatan dasar 2007. jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan ri; 2008. 4. ng ch, pathy bn, taib na, teh yc, mun ks, amiruddin a, et al. comparison of breast cancer in indonesia and malaysia – a clinicopathological study between dharmais cancer centre jakarta and universiti malaya medical centre, kuala lumpur. asian pac j cancer p 2011;12:2943-6. 5. keam b, im sa, kim hj, oh dy, kim jh, lee sh, et al. prognostic impact of clinicopathologic parameters in stage ii/iii breast cancer treated with neoadjuvant docetaxel and doxorubicin chemotherapy: paradoxical features of the triple negative breast cancer. bmc cancer 2007;7:111. 6. olaku o, white jd. herbal therapy use by cancer patients: a literature review on case reports. eur j cancer 2011;47:508–14. 7. anderson jg, taylor g. use of complementary therapies for cancer symptom management: results of the 2007 national health interview survey. j altern complement med 2012;18:235– 41. 8. wasito h. meningkatkan peran perguruan tinggi melalui pengembangan obat tradisional. mimbar 2008;24:117-27. 9. mishra s, ahmad s, kumar n. sharma bk. annona muricata (the cancer killer): a review. tgjpr 2012;2:1613-8. 10. gholse yn, yadav sr. developments in nutraceuticals for chemoprevention: a review. ijpt 2012;4:1950-73. 11. yukes je, balick mj. dominican medicinal plants: a guide for health care providers. 2nd ed. new york: the new york botanical garden; 2010. 12. ezirim au, okochi vi, james ab, adebeshi oa, ogunnowo s, odeghe o. induction of apoptosis in myelogenous leukemic k562 cells by ethanolic leaf extract of annona muricata l. global j res med plants indigen 2013;2:142–51. 13. rachmani epn, suhesti ts, widiastuti r. the breast of anticancer from leaf extract of annona muricata against cell line in t47d. int j appl sci technol 2012;2:157-64. 14. hamizah s, roslida ah, fezah o, tan kl, tor ys, tan ci. chemopreventive potential of annona muricata l leaves on chemicallyinduced skin papillomagenesis in mice. asian pac j cancer p 2012;13:2533-9. 15. george vc, kumar drn, rajkumar v, suresh pk, kumar ra. quantitative assessment of the relative antineoplastic potential of the nbutanolic leaf extract of annona muricata linn. in normal and immortalized human cell lines. asian pac j cancer p 2012;13:699-704. 16. owolabi ms, ogundajo al, dosoky ns, setze wn. the cytotoxic activity of annona muricata leaf oil from badagary, nigeria. ajeonp 2013; 1:1-3. 17. consolacion r, geneveve s, oscar t, ming-jaw d, chien-chang s. acetogenins from annona muricata. phcog j 2012;4:32-7. 18. liaw cc, chang fr, wu cc, chen sl, bastow kf, hayashi ki, et al. nine new cytotoxic monotetrahydrofuranic annonaceous acetogenins from annona montana. planta med 2004;70: 948–59. 19. champy p, melot a, guerineau v, gleye c, fall d, hoglinger gu, et al. quantification of acetogenins in annona muricata linked to atypical parkinsonism in guadeloupe. mov disord 2005;20:1629-33. 20. adewole so, ojewole jao. protective effects of annona muricata linn. (annonaceae) leaf aqueous extract on serum lipid profiles and oxidative stress in hepatocytes of streptozotocintreated diabetic rats. afr j trad cam 2009;6:3041. 21. pandey n, barve d. phytochemical and pharmacological review on annona squamosa linn. ijrpbs 2011;2:1404-12. 22. dai y, hogan s, schmelz em, ju yh, canning c, zhou k. selective growth inhibition of human breast cancer cells by graviola fruit extract in vitro and in vivo involving downregulation of egfr expression. nutr cancer 2012;63:795801. 23. nandhakumar e, indumathi p. in vitro antioxidant activities of methanol and aqueous extract of annona squamosa (l.) fruit pulp. j acupunct meridian stud 2013;6:142-8. 24. sousa ov, vieira gd, pinho jjrg, yamamoto ch, alves ms. antinociceptive and antiinflammatory activities of the ethanol extract of univ med vol. 33 no.1 26 fidiyaningsih, handayani annona muricata suppresses t47d annona muricata l. leaves in animal models. int j mol sci 2010;11:2067-78. 25. foong cp, hamid ra. evaluation of antiinflammatory activities of ethanolic extract of annona muricata leaves. braz j pharmacogn 2012;22:1301-7. 26. ukwubile ca. phytochemical screening and anti-ovarian cancer properties of annona muricata linn. (annonaceae) seed ethanol extract. ijpfr 2012;2:9-17. yenny 132 *department of pharmacology, medical faculty, trisakti university **department of pharmacology faculty of medicine, university of indonesia correspondence adr. yenny department of pharmacology, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2801 email : stasia_mk@yahoo.com univ med 2008; 27: 132-42. the benefits and risks of echinacea in treatment of common cold and influenza july-september, 2008july-september, 2008july-september, 2008july-september, 2008july-september, 2008 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 abstract universa medicina yenny* and zunilda djanun** the common cold is one of the world’s most prevalent illnesses in adults and children. this upper respiratory tract infection is caused by different kinds of agents. effects on health, well-being, and productivity are significant. preparations made from plants of the genus echinacea are widely used for the prevention and treatment of colds. three species of echinacea used for medicinal purposes are echinacea purpurea, echinacea angustifolia, and echinacea pallida. the beneficial effects of echinacea are thought to be due to its immunomodulatory activity, most notably activation of macrophages, polymorphonuclear leukocytes and natural killer cells. many clinical trials, including a number of blind randomized trials, have reported health benefits. although these studies show beneficial effects, recommendations on echinacea use cannot be made due to lack of product standardization and variability in dosage, sample size and methodological quality used in the trials. therefore, well-designed studies with consistent standardized measures are required for assessment of the efficacy of echinacea in treatment of common cold and influenza. keywords: echinacea, immunomodulation, common cold, influenza introduction the common cold is one of the most prevalent illnesses in the united states. the infection is self-limited, usually resolving within a week, but a number of viruses may persist more than three weeks, because of the large number of viral strains involved. the infection causes considerable economic loss, due to loss of 40% of work time and 30% of school time. adults experience an average of 2 4 i n f e c t i o n s a n n u a l l y, w h e r e a s c h i l d r e n experience 6-10 infections a year.(1) a study conducted in the united states in 2003 showed that americans spend 3 billion dollars annually on medical visits and over-the-counter (otc) d r u g s . a t l e a s t o n e t h i r d o f p a t i e n t s a r e prescribed antibiotics, although these drugs are not effective in viral infections. this not only adds to the costs, but also contributes to the emergence of bacterial antibiotic resistance.(2) 133 univ med vol.27 no.3 t h e u s e o f h e r b a l s u p p l e m e n t s h a s i n c r e a s e d b y m o r e t h a n 1 0 0 % i n t h e l a s t decade.(3) one of the factors accounting for this situation is the high cost of prescription drugs and the increased awareness of people of the side effects of these drugs. in addition, there is a widely held belief in the community that h e r b a l p r e p a r a t i o n s a r e n a t u r a l a n d consequently safer to consume. in reality there is evidence that the increased usage of herbal preparations are accompanied by an increase in side effects, drug interactions, and deaths. this is due to the fact that many of the commercially available herbal preparations have not been tested in respect with efficacy, safety, and purity o f t h e i r c o n t e n t s . t h e f o o d a n d d r u g administration (fda), which issues licenses for the use of otc drugs, does not evaluate and license preparations of plant origin. echinacea is one of the herbal preparations that is increasingly being used, and for years has been the second most sold medication in the united states. to the 300 million us dollars spent on herbal preparations in the united states, echinacea contributes 40 million us d o l l a r s . (4) o n e o f t h e r e a s o n s w h y p e o p l e consume echinacea preparations is for the prevention of common cold and influenza, and for enhancing the immune system.(5) the common cold and influenza t h e c o m m o n c o l d i s a n a c u t e nasopharyngitis caused by more than 100 kinds of viruses. more than 50% of colds is caused by rhinoviruses, 15% by coronaviruses and 5% by adenoviruses.(6) i n f e c t i o n w i t h r h i n o v i r u s e s i s characteristically limited to the nasopharynx, but may also affect the middle ear and the paranasal sinuses. the rhinovirus grows in the narrow temperature range (33-35°c) that is associated with the upper respiratory tract. the lower respiratory tract has a higher temperature and consequently is less conducive to viral growth. the virus is transmitted by direct contact from one individual to another, by contact with contaminated surfaces of objects ( e g . t e l e p h o n e r e c e i v e r h a n d l e s , s t a i r c a s e railings), and by inhalation of large-sized aerosol particles.(6) the rhinovirus binds to receptors for intracellular adhesion molecules 1 (icam-1) on the surface of nasopharyngeal epitelial cells. the infected cells release interleukin-8 (il-8), a strong chemoattractant that is capable of stimulating the release of proinflammatory mediators, such as kinins and prostaglandins. the presence of these mediators may lead to increased vasodilation, vascular permeability, a n d s e c r e t i o n o f e x o c r i n e g l a n d s , w h i c h ultimately give rise to the classic symptoms of the common cold, such as nasal congestion, rhinorrhea, and sneezing. the severity of cold symptoms is correlated with the concentration of il-8.(6) rhinoviral infections are generally selflimited. the symptoms commonly appear within 1-2 days after inoculation, and reach a peak within 2-4 days, although some reports state that symptoms may appear within less than 24 hours after exposure. the symptoms are frequently heralded by itching or pain in the throat, f o l l o w e d b y s n e e z i n g , n a s a l d r i p , n a s a l congestion, and general malaise. the body temperature is usually normal. initially, the nasal secretions are clear, thin and copious, then become more mucoid and purulent. if cough is present, it is usually mild and may persist for more than two weeks. uncomplicated colds usually resolve within 10 days.(1) complications may be secondary, as a result of other disorders accompanying upper respiratory infections, such as asthma, cystic fibrosis, chronic bronchitis, or infections of the lower respiratory tract in infants, the elderly, and immunocompromized patients. purulent 134 sputum or lower respiratory tract infections may indicate something more serious than rhinoviral infections alone. viral pneumonia is another potential complication; it is commonly mild and m a y r e s o l v e w i t h i n a f e w w e e k s w i t h o u t treatment, but in a number of cases it may worsen and require hospitalization. the rhinoviral infection may also spread to the sinuses or the ear, and cause excessive secretion of mucus. the ostium of the sinus or the auditory meatus may become blocked by accumulated mucus and provide a substrate for t h e m u l t i p l i c a t i o n o f b a c t e r i a a n d o t h e r microorganisms. it has been reported that 80% of children with otitis media recovers even without the use of antibiotics.(7) excessive use of systemic antibiotics (particularly penicillin d e r i v a t i v e s ) i n d u c e s i g n i f i c a n t a n t i b i o t i c resistance in the two pathogenic bacteria most commonly isolated from the nasopharynx of c h i l d r e n w i t h o t i t i s m e d i a , n a m e l y streptococcus pneumoniae and haemophilus influenzae.(1) i n f l u e n z a i s c a u s e d b y a v i r u s t h a t comprises three serotypes (a, b, and c), but only types a and b are associated with the human disease that is called flu. the virus is divided into a number of subgroups on the basis o f t h e i r c h a r a c t e r i s t i c a n t i g e n s . m u t a t i o n occurring in types a and b give rise to new viral strains, due to transfer of antigens from one virus to another (antigenic drift). as a result of mutation, antibody formed against previous strains have limited protective effect against newer strains of the virus.(6) i n f l u e n z a e p i d e m i c s a r e g e n e r a l l y associated with certain serotypes. however, it is still possible for multiple viral strains to simultaneously cause infection in the same l o c a t i o n . t h e u n i t e d s t a t e s e x p e r i e n c e s influenza epidemics every 2-3 years, mostly caused by influenza virus type a. influenza type b commonly causes a milder illness and does not experience antigenic drift as readily as does influenza virus type a.(6) the incubation period of influenza is 1-4 d a y s . i n m i l d c a s e s o f f l u , t h e s y m p t o m s resemble those of the common cold (eg. sore throat, runny nose); conjunctivitis may also occur, but in general the individual rapidly has a high fever and chills, cough, body aches, headache, and photophobia. the respiratory symptoms include sore throat, rhinitis, and productive or dry cough. children may also suffer from nausea, vomiting, or abdominal pain; in infants the symptoms of influenza may r e s e m b l e a s e p s i s l i k e s y n d r o m e . a c u t e symptoms of influenza commonly resolve within 2-3 days, but the fever may persist for more than five days. in uncomplicated cases, the patient commonly recovers after 3-7 days; however, cough and general malaise may persist for weeks.(1) all of the complications occurring in the common cold may also be found in influenza. although rarely encountered in influenza, encephalitis may also develop. the virus enters the blood stream, then relocates to the brain, causing inflammation of the brain tissues and meninges. in an attempt to combat the infection, the leukocytes invade the tissues of the brain, causing edema and tissue destruction. the s y m p t o m s t h a t d e v e l o p a r e f e v e r, s e v e r e headache, drowsiness, nuchal rigidity, muscle weakness, or convulsions. a comparison of the c h a r a c t e r i s t i c s o f t h e c o m m o n c o l d a n d influenza may be seen in table 1. ta b l e 2 l i s t s t h e p o p u l a t i o n g r o u p s identified as being at high risk for suffering from influenza. these groups should have first priority in receiving influenza prophylaxis or therapy. yenny, djanun echinacea in common cold 135 univ med vol.27 no.3 characteristics common cold influenza etiologic agent >100 viral strains; most commonly rhinovirus 3 strains of influenza virus: influenza virus a, b, and c site of infection upper respiratory tract upper and lower respiratory tract onset of symptoms insidious: 1-3 days acute: within hours fever and chills rare, temperature <38.3°c typical, temperature >38.3°c, lasting 2-4 days headache frequent, usually mild typical, more severe body aches mild, if any typical, frequently severe and affecting whole body cough, congestion of chest mild to severe common, may become severe sore throat common, usually mild occasionally found nasal drip & obstruction very frequent, with sneezing occasionally found tiredness, weakness mild, if any common, may be severe and last >2-3 weeks exhaustion never frequent, usually at onset of illness season throughout the year, peaks in winter most cases in november through february effect of antibiotics none, except in secondary bacterial infection none, except in secondary bacterial infection table 1. differences in characteristics of the common cold and influenza(1) echinacea (echinacea spp.) echinacea is a plant species that has since long been utilized as a medication by the indigenous peoples of north america and the settlers. echinacea was used for wounds, insect bites, infections, toothache, joint pains, and as an antidote against rattlesnake bites. in 1916adults > 65 years children < 2 years pregnant women individuals treated for extended periods in health care facilities inividuals with cardiovascular disease individuals requiring regular medical visits or extended periods of hospitalization due to chronic metabolic diseases (eg. diabetes mellitus), renal dysfunction, hemoglobinopathies or immunosuppression (eg. hiv) individuals suffering from various conditions capable of impairing respiratory function or secretion or increasing risk of aspiration children and adults receiving long-term aspirin therapy (due to risk of reye’s syndrome) table 2. populations at high risk for complications of influenza(1) 1950 echinacea was included in the national formulary of the united states. although the popularity of this medication has been waning since 1920, echinacea continues to be utilized and investigated in europe, especially germany. in 1980 echinacea was again widely used when consumers were beginning to be interested in 136 medications capable of enhancing the immune system in conditions of decreased immune function, such as in acquired immunodeficiency s y n d r o m e ( a i d s ) a n d c a n c e r. s i n c e t h e n echinacea was again being widely used for viral infections, because of its immunostimulant properties. (8) currently echinacea is being promoted for prevention and treatment of r e s p i r a t o r y t r a c t i n f e c t i o n s , i n c l u d i n g t h e common cold and influenza.(9) echinacea is also known under the name of purple coneflower. the plant is an herbaceous perennial (a plant capable of living more than two years) that grows in the western and central parts of the united states, southern canada, and europe, especially germany. echinacea is a member of the family compositae/asteraceae. its maximal height is 50-180 cm, depending on the species. up to date nine echinacea species h a v e b e e n f o u n d . t h e t h r e e s p e c i e s u s e d medicinally are e. angustifolia, the narrowleafed purple coneflower; e. purpurea, the eastern purple coneflower; and e. pallida, the p a l e p u r p l e c o n e f l o w e r. o f t h e t h r e e , e . purpurea is the one most extensively cultivated and utilized, because all parts of the plant (roots, leaves, flowers, seeds) may be utilized and also because this species is easy to cultivate. the parts of e. angustifolia and e. pallida that are u s e d a s m e d i c i n e a r e t h e r o o t s a n d t h e rhizomes.(10) echinacea preparations and their indications in germany, herbal medicines are widely u s e d , a n d a c o m m i s s i o n , t h e g e r m a n commission e, has been assigned the task of studying the data on herbals.(11,12) for internal use, the aerial parts of e. purpurea (the parts of the plant above the ground, thus excluding the roots) have been approved for treating the common cold, flu-like symptoms, fever, chronic respiratory tract infections (eg. bronchitis), urinary tract infections, inflammation of the mouth and pharynx, and recurrent infections. the plant is used externally for superficial wounds and burns. in addition, the root of e. pallida is used in supportive therapy of fever and the common cold. the german commission e has not yet approved e. angustifolia for medicinal use. currently there are more than 800 phytopharmacological products containing e c h i n a c e a . a l a rg e v a r i e t y o f e c h i n a c e a p r e p a r a t i o n s a r e a v a i l a b l e c o m m e r c i a l l y, involving different species of echinacea, various parts of the plant, with a number of added products, such as vitamin c and propolis.(13) echinacea is mostly sold in the form of powders, extracts, tinctures, and teas. the phenolic components contained in echinacea are active c o n s t i t u e n t s e x h i b i t i n g i m m u n o s t i m u l a n t , antihyaluronidase, antiviral, antioxidant, and antinflammatory effects. there is no agreement on the dosage of echinacea, and each company gives a different dosage on the label.(13) there i s a l s o n o a g r e e m e n t o n w h e n t o s t a r t consumption of echinacea, and the duration of the course. the duration of treatment of the common cold varies from 7 to 10 days. the physicians’ desk reference (pdr) states that a course for prevention of recurrent infections should not exceed eight weeks and should be about 1-2 weeks for acute infections. the oral dose for short-term treatment should be 3-4 times daily, whilst for long-term treatment it should be 2 times daily.(12) up to now echinacea was regarded as a fairly safe medication to consume. (13) the majority of reported side effects are allergic r e a c t i o n s , u s u a l l y s k i n r a s h e s . t h e s t u d y c o n d u c t e d b y p a r n h a m e t a l . ( 1 4 ) o n 1 , 2 3 1 children treated with echinacea showed that the most frequently reported side effect of the h e r b a l p r e p a r a t i o n s i s b a d t a s t e o f t h e m e d i c a t i o n , a n d l e s s f r e q u e n t l y n a u s e a , vomiting, sore throat, abdominal pain, diarrhea, and difficulty in swallowing. taylor et al.(15) yenny, djanun echinacea in common cold 137 univ med vol.27 no.3 performed a study on children between 2-11 years of age and found an increase in the occurrence of skin rashes in children receiving echinacea, compared with those not receiving the medication. another study reported on 51 c a s e s o f a l l e rg y p o s s i b l y a s s o c i a t e d w i t h echinacea, among which 26 were thought to be d u e t o i m m u n o g l o b u l i n e m e d i a t e d hypersensitivity reactions.(16) in view of the immunostimulant effects of echinacea, the german commission e states that echinacea should not be used in individuals w i t h i m m u n e r e l a t e d c h r o n i c p r o g r e s s i v e d i s e a s e s , s u c h a s a u t o i m m u n e d i s e a s e s , tuberculosis, multiple sclerosis, and aids. this warning was issued not on the basis of clinical evidence, but only on the theory that the mechanisms of inflammation in these diseases may be exacerbated by the immunostimulatory properties of echinacea. (12) more concrete evidence is needed on this issue. as is the case with nearly all medicines, the safety of echinacea usage in pregnancy has n o t b e e n d e t e r m i n e d . t h e r e a r e v e r y f e w relevant data available. a prospective controlled study was conducted on 206 pregnant women c o n s u m i n g e c h i n a c e a , c o m p a r e d t o 2 0 6 pregnant women used as controls. stillbirths, chromosmal abnormalities, and malformations were found in comparable numbers in both groups. the results of the study indicated that the use of echinacea during organogenesis was not associated with an increased risk of major malformations.(17) however, the study still has limitations in statistical power and methodology. the systemic review by perri et al.(18) showed that echinacea is not teratogenic when used in p r e g n a n c y. t h e u s e o f e c h i n a c e a d u r i n g lactation needs further investigation. mechanism of action t h e p r e c i s e m e c h a n i s m o f a c t i o n o f echinacea is not fully understood. the main constituents of echinacea are (i) alkylamides and polyacetylenes, (ii) derivatives of caffeic acid, (iii) polysaccharides, and (iv) glycoproteins. most of the alkylamides are isobutylamides that are capable of causing a burning sensation in the mouth. the derivatives of caffeic acid give rise to several components, such as caftaric acid, chlorogenic acid, cyanarin, echinacoside, and chicoric acid. chicoric acid is the main phenolic component found in e. purpurea, whereas echinacoside is the main phenolic component found in e. angustifolia and e. pallida. chicoric a c i d r e p o r t e d l y h a s i m m u n o s t i m u l a n t a n d phagocytic properties, is an antihyaluronidase, and has a protective effect against antioxidants that induce destruction of collagen, and exerts an anti-inflammatory action. echinacea also protects collagen against damage-inducing reactive oxygen species. chicoric acid and echinacoside are the constituents most frequently e n c o u n t e r e d i n v a r y i n g c o n c e n t r a t i o n s , depending on the species of echinacea.(19) the roots of e. purpurea contain chicoric acid in h i g h c o n c e n t r a t i o n s , b u t d o n o t c o n t a i n echinacoside. in contrast, the roots of e. pallida and e. angustifolia contain echinacoside in l a r g e a m o u n t s . t h e i m m u n o m o d u l a t o r y mechanisms of echinacea may assist in reducing t h e s y m p t o m s o f u p p e r r e s p i r a t o r y t r a c t infections through activation of macrophages, leukocytes, and granulocytes. immunomodulatory effects e. purpurea is well-known for its effects on the immune system. stimulation of various components of the immune system, such as macrophages, other monocytic cells, and natural killer (nk) cells, and modulation of cytokines have also been repeatedly demonstrated in v i t ro . ( 2 0 , 2 1 ) t h e m e c h a n i s m s o f i t s i n v i v o immunomodulatory effects are not yet fully u n d e r s t o o d . o n e t h e o r y s t a t e s t h a t immunosupressive effects occur on exposure to 138 allergens, diseases, malnutrition, drugs, toxins, o r p s y c h o l o g i c a l o r s o c i a l s t r e s s e s . administration of echinacea may bolster a weakened immune system and restore health. the term immunomodulation appears to be more appropriate for the effects of echinacea, because in the immune system echinacea reportedly stimulates a large number of complex c o m p o n e n t s w i t h o u t c l e a r l y e n h a n c i n g o r impairing the functions of the immune system. several of the immune activities are beneficial, w h i l s t o t h e r s a r e d e t r i m e n t a l . t h e t e r m immunomodulation conveys the meaning of a reduction in dangerous host reponses such as irritation or inflammation.(22) many studies have been performed to investigate the effects of echinacea on human leukocytes, either by adding echinacea to a l e u k o c y t e e x t r a c t , o r b y a d m i n i s t e r i n g echinacea to human subjects and subsequently preparing a leukocyte extract. these studies demonstrate increased neutrophil chemotaxis and enhanced bactericidal activities against staphylococcus; there were also raised levels of tumor necrosis factor (tnf) α, interleukin1 (il-1), and il-6. addition of a polysaccharide extract of an echinacea plant cell culture to i m m u n e c e l l s w i l l r e s u l t i n e n h a n c e d phagocytosis, chemotaxis, and oxidative bursts i n n e u t r o p h i l s a n d m a c r o p h a g e s . t h e polysaccharide constituents of echinacea do not activate b cells and do not stimulate antibody production. chicoric acid is a constituent with immunomodulatory effects that is capable of enhancing in vitro and in vivo phagocytosis. this substance also protects cells against free radicals capable of inducing degradation of c o l l a g e n . e c h i n a c o s i d e d o e s n o t h a v e i m m u n o m o d u l a t o r y p r o p e r t i e s , b u t h a s a protective effect against destruction of collagen by reactive oxygen. constituents of echinacea such as echinacoside and chicoric acid have the highest free radical scavenging activity. the c a f f e o y l d e r i v a t i v e s c o m m o n l y f o u n d i n echinacea protect cells against superoxide and hydroxyl radicals. polyunsaturated alkylamides found in e. angustifolia act as antiinflammatoty s u b s t a n c e s b y i n h i b i t i n g m i c r o s o m a l c y c l o o x y g e n a s e a n d 5 l i p o x y g e n a s e activities.(22) clinical trials of echinacea in respiratory infections many studies have been conducted with variable results on the effects of echinacea in reducing the symptoms and duration of illness of acute respiratory infections. the following studies revealed positive results of echinacea on upper respiratory tract infections. o n e s t u d y t h a t w a s u n d e r t a k e n f o r assessing the duration of the common cold, recruited 80 adults, who received e. purpurea extract or placebo from the onset of cold symptoms until their resolution. the mean duration of illness was 6 days in the group receiving echinacea and 9 days in the placebo group (p = 0.0112).(23) in a randomized double-blind controlled study involving 282 adults, the effects of an echinacea purpurea formulation (1 unit = 0.25 mg/ml alkamide, 2.5 mg/ml chicoric acid, and 25 mg/ml polysaccharide) were compared to those of a placebo. the proportions in the preparation had been extrapolated on body weight from studies in rats that demonstrated a n e n h a n c e m e n t i n a l v e o l a r m a c r o p h a g e functions. this study was conducted for a period of seven days after the onset of cold symptoms. on the day of onset 10 units were administered, which was followed by 4 units daily for the following seven days. the severity of symptoms was measured using a 10-point scale (0 = minimal; 9 = maximal). the echinacea group showed a 23.1% lower reduction in severity of s y m p t o m s t h a n t h a t i n t h e p l a c e b o g r o u p (p<0.01).(24) yenny, djanun echinacea in common cold 139 univ med vol.27 no.3 in a follow-up study conducted by the same investigators on 150 adults, using the same echinacea preparation, 8 units (5 ml/unit) were administered on the first day, followed by 3 u n i t s d a i l y f o r s e v e n d a y s . f a s t i n g b l o o d glucose samples were collected before and during the illness. the results of the study showed a reduction in total symptoms of illness and an increase in the numbers of red cells, monocytes, neutrophils, and nk cells in the group receiving echinacea compared with the placebo group.(25) a randomized double-blind controlled clinical trial on 430 children aged 1-5 years used a chizukit preparation containing 50 mg/ml echincea, 50 mg/ml vitamin c, or placebo, in a dosage of 5.0 ml in children aged 1-3 years and 7.5 ml in children aged 4-5 years, twice daily for 12 weeks. in the group receiving the herbal preparation there was a reduction of 36.2% in the number of episodes of respiratory infection, compared with the group receiving placebo. the weakness of this study is that the preparation used was unrefined and contained other substances that may have influenced the results of the study.(26) schoop et al.(27) performed a meta-analysis o f s t u d i e s i n v e s t i g a t i n g t h e e f f i c a c y o f e c h i n a c e a e x t r a c t s i n p r e v e n t i n g t h e development of cold symptoms, experimentally induced using rhinovirus. from a total of 234 papers that were identified, 231 were dropped from the analysis because in those studies the common cold occurred spontaneously. the results of the meta-analysis indicated that standardized echinacea extracts were effective in preventing cold symptoms when compared with placebo. conversely there were several studies reporting that echinacea had not been proven to be effective. a randomized double-blind controlled study, with alfalfa as placebo, was undertaken by barrett et al.(28) on 148 school children with recently acquired common cold. they were given capsules containing a mixture of unrefined e. purpura herbs (25%) and roots (25%) and the roots of e. angustifolia (50%) in a dosage of 1 gram 6 times daily on the first day of illness, and three times daily on the following days, for a maximal period of 10 days. there were no significant differences in severity a n d d u r a t i o n o f s y m p t o m s b e t w e e n t h e echinacea group and the placebo group. a limitation of this study was the use of unrefined echinacea and young healthy subjects. another limitation was that the placebo used was in r e a l i t y n o t a n i n e r t s u b s t a n c e , b e c a u s e i t contained the active substances of alfalfa. this bias may have reduced the difference in efficacy between the group on echinacea and the group o n p l a c e b o , s u c h t h a t t h e a d v a n t a g e s o f echinacea could not be made evident in this study. another placebo-controlled clinical trial used a total of 148 students, who were given 100 mg e. purpurea (as the freeze-dried pressed juice from the aerial parts of the plant) or a placebo, three times daily until the symptoms of illness resolved or until the fourteenth day. no statistical difference was apparent between both groups. the study performed by yale et al.,(29) using the pressed juice from the aerial parts of the plant, indicated that there were no beneficial effects in the use of e. purpurea within 24 hours of onset of the common cold for reducing the symptoms or severity of the illness. to compensate for the variability of the c o l d v i r u s , s p e r b e r e t a l . ( 3 0 ) a d m i n i s t e r e d rhinovirus type 39 in advance, before randomly assigning the subjects to the echinacea or placebo group. the pressed juice of the aerial parts of e. purpurea was given for seven days before and after the subjects had been infected w i t h r h i n o v i r u s . t h e r e s u l t s o f t h e s t u d y revealed that the use of echinacea before and 140 after exposure to rhinovirus type 39 did not reduce the rate of rhinovirus infection. there was a tendency for reduction of symptoms on days 2-7 in subjects receiving echinacea, when compared with those receiving placebo, but this was statistically not significant. the limitation of this study lies in its statistical power, which was too low to detect differences. the randomized study by turner et al.(31) on 437 volunteers, who received extracts of e. angustifolia roots or a placebo, showed no significant differences in rate of viral infection or severity of symptoms. upper respiratory infections also constitute a health problem in children. to reduce the symptoms of illness, t h e s e c h i l d r e n a r e g i v e n d e c o n g e s t a n t s , a n t i h i s t a m i n e s , a n d c o u g h m e d i c i n e s . unfortunately, however, there is scant evidence demonstrating the efficacy of the drugs in children under the age of 12 years. this fact prompted a study by taylor et al.(15) on the efficacy and safety of e. purpurea in combating infection in children. the results of the study showed that use of the pressed dried juice from the aerial parts of e. purpurea did not cure respiratory tract infections in children aged 211 years, and revealed an increased incidence of skin rashes in the group receiving echinacea. although this study failed to show a reduction in symptoms of respiratory tract infections, there was a statistically significant reduction in the frequency of attacks of infection. linda et al.(32) performed a systemic review o f t h e c o c h r a n e d a t a b a s e , u n d e r t h e t i t l e “echinacea for preventing and treating the common cold”. a total of 16 trials were selected according to existing criteria, from 40 previous studies. the sixteen studies were divided into t h r e e c a t e g o r i e s : f i v e p l a c e b o c o n t r o l l e d prevention trials, three controlled prevention trials without treatment, and eight placebocontrolled treatment trials. the total number of participants in the sixteen trials was 3,396. the large heterogeneity in the products used and the poor quality of reports caused difficulties in assessing the limitations in methodologies of the studies. however, there was some evidence that e. purpurea preparations derived from the aerial parts may be effective for initial treatment of the common cold in adults, but the results were not always consistent. there was no clear evidence that other echinacea preparations were effective, and the effects of echinacea on respiratory tract infections in children were not proven. the side effects of echinacea were infrequently found, but one study reported the occurrence of skin rashes in children. a s u m m a r y o f s e v e r a l s t u d i e s o n t h e benefits of echinacea in the common cold and influenza is listed in table 3. author year total subjects results schulten (23) 2001 80 adults effective goel (24) 2004 282 adults effective cohen (26) 2004 430 healthy children effective barret (28) 2002 148 children with cold ineffective yale (29) 2004 148 students with cold ineffective sperber (30) 2004 48 adults ineffective turner (31) 2005 437 adults ineffective taylor (15) 2003 524 healthy children ineffective table 3. summary of several studies pro and contra the efficacy of echinacea in the common cold and influenza yenny, djanun echinacea in common cold 141 univ med vol.27 no.3 conclusions extracts of echinacea plants are widely used in the united states for prevention and t r e a t m e n t o f r e s p i r a t o r y t r a c t i n f e c t i o n s . however, the available evidence from clinical trials on its efficacy is inconsistent. assessment of the efficacy of echinacea preparations is difficult to perform due to limited resources for comparing the available preparations. data on the use of echinacea in pregnancy and lactation are still limited, thus its use should be avoided. the use of echinacea for the prevention of the common cold is not yet supported by adequate data, so that it is not recommended. references 1. roxas m, jurenka j. cold and influenza: a review of diagnosis and conventional, botanical, and nutritional considerations. alter med rev 2007; 12: 25-48. 2. frederick am, monto as, nightingale b, sarnes m. the economic burden of non-influenza-related viral respiratory tract infections in the united states. arch intern med 2003; 163: 487-94. 3. kelly jp, kaufman dw, kelly k, rosenberg l, anderson te, mitchell aa. recent trends in use of herbal and other natural products. arch intern med 2005; 165: 281-6. 4. blumenthal. herb sales down in mainstream market, up in natual food stores. herbal gram 2002; 55: 60. 5. simasek m, blandino da. treatment of the common cold. am fam physician 2007; 75:51520. 6. rajnik m, murray c, hospenthal dr. rhinoviruses. available at: http:// www.emedicine.com/med/topic2030.htm. accessed october 15, 2007. 7. marcy sm. general information and practitioner guidelines for otitis media. apua newsletter 1999. available at: http://www.tufts.edu/med/apua/ practitionersaomguidelines.html accessed october 15, 2007. 8. klinger b. echinacea. am fam physician 2003; 67: 77-80. 9. national center for complementary and alternative medicineechinacea. herbs at a glance. nccam publication. 2005. available at: http:// www.nccam.nih.gov/health/echinacea. accessed october 15, 2007. 10. mills s, bone k. principle and practice of phytotherapy: modern herbal medicine. london: churchil livingstone; 2000. 11. blumenthal m, gruenwald j, hall t, riggins c, rister r, kle s. german commission e monograph: medicinal plants for human use. austin, texas: american botanical council; 1998. 12. gruenwald j, brendler t, jaenicke c. pdr for nonprescription drugs, dietary supplements, and herbs: the definitive guide to otc medications. 27th ed. montvale: thomson; 2006. 13. anonymous. echinacea: cold comfort. consumer rep 2004; 69: 30-3. 14. parnham m. benefits and risks of the squeezed sap of the purple coneflower (echinacea purpurea) for long-term oral immunostimulant therapy. immunomodulatory agents from plants. boston: birkhauser verlag; 1999. 15. taylor ja, weber w, standish l, quinn h, goesling j, mcgann m. efficacy and safety of echinacea in treating upper respiratory tract infections in children: a randomized controlled trial. jama 2003; 290: 2824-30. 16. mullins rj, heddle r. adverse reactions associated with echinacea: the australian experience ann allergy asthma immunol 2002; 88: 42-51. 17. gallo m, sarkar m, waisze a, peterzak k, comas b, smith m, et al. pregnancy outcome following gestational exposure to echinacea: a prospective controlled study. arch intern med 2000; 160: 31413. 18. perri d, dugoua jj, mills e, koren g. safety and efficacy of echinacea (echinacea angustifolia, e. purpurea, and e pallida) during pregnancy and lactation. can j clin pharmacol 2006; 13: e262-7. 19. pellati f, benvenuti s, margo l. analysis of phenolic compounds and radical scavenging activity of echinacea. j pharm biomed anal 2003; 35: 289-301. 20. rininger ja, kickner s, chigurupati p, mclean a, franck z. immunopharmacological activity of echinacea preparations following simulated digestion on murine macrophages and human peripheral blood mononuclear cells. j leukoc biol 2000; 68: 503-10. 142 yenny, djanun echinacea in common cold 21. mccann, solco a, liu y, macaluso f, murphy pa, kohut ml. cytokine and interferon-modulating properties of echinacea spp root tincture stored at -20°c for 2 years. j interferon cytokine res 2007; 27: 425-36. 22. barrett b. medicinal properties of echinacea: a critical review. phytomedicine 2003; 10: 66-86. 23. schulten b, bulitta m, ballering-bruhl b, kostler u, schafer m. efficacy of echinacea purpurea in patients with common cold: a placebo-controlled, randomized, double-blind clinical trial. arzneimittelforschung 2001; 51: 563-68. 24. goel v, lovlin r, barton r, lyon mr, bauer, lee td. efficacy of a standardized echinacea preparation (echinilin) for the treatment of the common cold: a randomized, double-blind, placebo-controlled trial. j clin pharm ther 2004; 29: 75-83. 25. goel v, lovlin r, chang c, slama jv, barton r, gahler r, et al. a proprietary extract from the echinacea plant (echinacea purpurea) enhances systemic immune respone during common cold. phytother res 2005; 19: 689-94. 26. cohen h, varsano i, kahan e. effectiveness of an herbal preparation containing echinacea, propolis, and vitamin c in preventing respiratory tract infections in children. arch pediatr adolesc med 2004; 158: 217-21. 27. schoop r, klein p, suter a, johnston sl. echinacea in the prevention of induced rhinovirus cold: a meta analysis. clin ther 2006; 28: 174-83. 28. barrett bp, brown rl, locken k, maberry r, bobula ja, d’alessio d, et al. treatment of the common cold with unrefined echinacea: a randomized, double-blind, placebo-controlled trial. ann intern med 2002; 137: 939-46. 29. yale sh, liu k. echinacea purpurea therapy for the treatment of the common cold: a randomized, double-blind, placebo-controlled clinical trial. arch intern med 2004; 164: 1237-41. 30. sperber s, shah l, gilbert r. echinacea purpurea for prevention of experimental rhinovirus colds. clin infect dis 2004; 38: 1367-71. 31. turner rb, bauer r, woelkart k, hulsey tc, gangemi d. an evaluation of echinacea angustifolia in experimental rhinovirus infection. n engl j med 2005; 354: 341-8. 32. linda k, barrett b, wolkart k, bauer r, melchart d. echinacea for preventing and treating the common cold (review). cochrane database of systematic review 2006, issue 1. art no.: cd000530. doi: 10.1002/14651858.cd000530.pub2. alvina 84 abstract *medical profession study program, medical faculty, trisakti university correspondence angela febriannie, s.ked jl. pramuka no. 1010, rubai pakanbaru, riau phone: 081159617 univ med 2010;29:84-9 prevalence of headache and impact on anxiety in adults angela febriannie*, renobulan sanusi*, reny fahdiyani* and riani dwianasari* may-august, 2010may-august, 2010may-august, 2010may-august, 2010may-august, 2010 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 vol.29 no.2 universa medicina headache is an extremely common symptom that may have profound impact on peoples’ functioning and quality of life. the two most common primary headache disorders, migraine and tension-type headache, have an annual prevalence of respectively 11% and 40% in adults. the objective of this study was to estimate the prevalences of headache and anxiety in primary care patients and the impact of headache on anxiety in these patients. a crosssectional study was conducted on 140 adult patients attending a primary health center. outcome measures included headache impact test scores and zungself rating anxiety scale. the mean age of the respondents was 34.6 ± 5.2 years (range 22 44 years). sixty one respondents (43.6%) in the preceding month had suffered from headaches that affected their lives, and 91 respondents (65.0%) had anxiety symptoms and a raw anxiety score of more than 40. there was a significant association between headaches affecting the lives of the respondents and the anxiety resulting from the headache (p=.0001). among 16 respondents with attacks of headaches that severely affected their lives, 87.5% were disturbed by anxiety. in conclusion, there was a relatively high proportion of patients in the primary care setting experiencing headaches that potentially affected their lives and had an impact on their anxiety. it may be appropriate to encourage patients to inform their health-care providers about their headaches and to encourage providers to identify patients with frequent headaches. keywords: anxiety, headache, headache impact test, migraine, adults introduction h e a d a c h e i s a n e x t r e m e l y c o m m o n symptom that may have a profound impact on peoples’ functioning and quality of life. the t w o m o s t c o m m o n p r i m a r y h e a d a c h e d i s o r d e r s , m i g r a i n e a n d t e n s i o n t y p e headache, have an annual prevalence of respectively 11% to 40% in adults.(1,2) chronic daily headache (cdh) is a frequent disorder, with a worldwide prevalence of 3.9–4.7%.(3) it produces severe disability in a quarter of patients suffering from the condition, leading to overuse of analgesic medications in 24– 85 univ med vol.29 no.2 34%, with only 25–50% seeking medical assistance.(4) cdh is a public health problem, but it is necessary to know its risk factors to clarify the pathophysiology of the disorder and to guide treatment, as well as to facilitate the development of prevention strategies. multiple risk factors have been described for c d h i n d i ff e r e n t c l i n i c a l o r p o p u l a t i o n settings.(5,6) previous prospective studies of specific headache types have identified a number of predictors of incidence. lyngberg et al.(7) in t h e i r 1 2 y e a r f o l l o w u p o f a g e n e r a l p o p u l a t i o n s a m p l e f o u n d t h e f o l l o w i n g predictors of migraine incidence: younger a g e , f e m a l e g e n d e r, f a m i l y h i s t o r y o f migraine, existing frequent tension-type headache (>14 headache days per year) and having a high work load. depression has also been identified as a predictor of migraine but n o t o f o t h e r s e v e r e h e a d a c h e s a n d t h i s relationship is bidirectional, with migraine also predicting depression.(8) headaches have been cross-sectionally found to be associated with anxiety, depression, and musculoskeletal pain.(9,10) h e a d a c h e i s a s y m p t o m o f v a r i o u s neurological and psychiatric disorders. the age range of 18-25 years is at 37.1% the most frequently subject to headaches, compared with the age range of 45-65 years at 27.8%.(11) only recently has attention been paid to the functional impact of migraine and headache, and it has been slow to be applied in clinical settings. the first questionnaire developed to document this impact was the headache impact questionnaire (himq).(12) migraine disability assessment (midas) was derived from himq, which was not very user friendly for routine application.(13) the headache impact test (hit)-6 was developed after midas. the main objective of both instruments is to identify and stratify patients requiring special attention in terms of treatment. the headache recall period is 4 weeks in hit-6 versus 3 months in midas and does not include days of absence or disability, but rather a semiquantitative assessment of disability. hit-6 is routinely used on websites.(14) a study on headache identification in patients at risk for anxiety disorders conducted in pittsburgh also used subjects in their productive years, with mean age of 40.4 ± 5.6 years and an age range of 27-53 years.(15) several studies have reported the prevalence of anxiety and depression in patients with chronic headache. the functional impact of chronic headache was estimated as severe by 74% of patients, and 75.7% suffered from anxiety.(16) the objective of the present study was to estimate the prevalence of headache and anxiety in a primary care setting. a secondary objective was to assess the impact of headache on anxiety in adults by means of the headache impact test (hit). methods research design an observational cross-sectional study was conducted at the public health center, tebet subdistrict, south jakarta, from 14 december 2009 to 5 february 2010. study subjects the subjects of the study were recruited among patients attending the tebet subdistrict public health center in south jakarta based on the following inclusion criteria: age 20 50 years, having suffered from headaches in the preceding month, and willing to participate. the age range 20 to 50 years was chosen because this period constitutes the productive years. the sample was chosen by means of nonprobability sampling, specifically consecutive sampling, of all patients at the public health center in the tebet subdistrict of south jakarta. data collecting data on patients’ age, gender, level of education and occupation were collected using questionnaires. 86 measurements the headaches attacking the patients during the preceding month were assessed by means of the hit-6 instrument, which is designed “to measure the impact of headaches on a person’s ability to function on the job, at school, at home, and in social situations.” in this test, numerical values for each response are added to produce summary scores ranging from 36 to 78, with scores of 49 points or fewer being described as “your headaches seem to be having little or no impact on your life”. scores of 50-55 were evaluated as “your headaches seem to be having some impact on your life”, suggesting that the headaches should not make the subjects “miss time from family, work, school, or social activities”. in contrast, scores of 56-59 mean that “your headaches are having a substantial impact on your life”, carrying the risk of the subject “experiencing severe pain and other symptoms, causing her/him to “miss some time from family, work, school, or social activities”. a score of 60 or more points is explained as “your headaches are having a very severe impact on your life”, with the additional advice to the patient not to allow the disabling pain and other symptoms interfere in the enjoyment o f l i f e ( f a m i l y, w o r k , s c h o o l o r s o c i a l activities).(17) the hit-6 was found to be reliable and valid among a heterogeneous population of those with headache, and had good internal consistency and reliability among patients from a headache-specialty clinic.(18-20) anxiety was assessed using the zung selfrating anxiety scale.(21) with each self-rating scale, there were 20 questions being completed by the researcher during the interview. each question was assigned a maximum score of 4, and the minimum and maximum responses for each patient were 20 and 80, respectively. the higher the score, the greater the symptoms associated with anxiety. the diagnosis of anxiety symptoms was made if the raw score was more than 40. data analysis the chi square test was used to analyze the difference in prevalence of headache and anxiety, where p < 0.05 was considered to be statistically significant. results mean age of the 140 subjects was 34.6 ± 5.2 years, with an age range of 22 to 44 years. there were 54 (38.6%) males and 86 (61.4%) females in the study sample. a total of 65 subjects (46.4%) had been educated at an smu (senior high school) or equivalent. ninety-one r e s p o n d e n t s ( 6 5 % ) w e r e e m p l o y e d . t h e average raw anxiety score of these patients was 32.1 ± 6.8. anxiety symptoms were present in 91 patients (65.0%) who had a raw anxiety score of more than 40. as shown in table 1, in 79 (56.4%) of the respondents, the headaches were in the category of “no or little impact on life” and 61 (43.6%) of respondents had headaches which had “impact on life”, from mild to severe. of these 61 patients, there were 28 ( 4 4 . 2 % ) w h o n e e d e d p h y s i c i a n b a s e d interventions. the results of the chi square test showed that there was a significant association between headaches that impacted on the lives of the patients and their anxiety. of the sixteen repondents with headaches that had a very severe impact on their lives, 87.5% suffered from anxiety. (table 2) table 1. distribution of headache categories of respondents headache category n (% ) no or little impact on life som e im pact on life substantial impact on life very severe impact on life 79 (56.4) 33 (23.6) 12 (8.6) 16 (11.4)   87 univ med vol.29 no.2 ta b l e 3 i n d i c a t e s t h a t t h e r e w a s n o relationship between the type of headache and the anxiety (p=0.9172) experienced by the respondents. the type of headache most frequently experienced by the respondents was migraine headache at 42.6% (26/61). among the respondents with anxiety, a total of 23.1% (21/91) had migraine headache. discussion our findings demonstrate that headache is a common symptom among patients seen at primary health centers, with 43.6% of the respondents in our study in the preceding m o n t h h a v i n g e x p e r i e n c e d h e a d a c h e s impacting on their lives. however, these headaches were not further analyzed for associations with chronic daily headache (chd). different results were found in the study at the university of north carolina (unc) family medicine center (fmc), where 58% of the respondents reported having had headaches in the preceding month(22) and 9% of these reported a frequency of headaches consistent with chronic daily headache (cdh). o u r s t u d y a l s o i n d i c a t e d t h a t 4 2 . 6 % respondents had headaches of the migraine type. our study results differed from those of a study on subjects with mean age of 40.4 ± 13.0 years, where 21% of the respondents had m i g r a i n e h e a d a c h e s . ( 1 5 ) r e g a r d i n g t h e association of headaches with anxiety, our study results demonstrated that 23.1% of respondents with anxiety also suffered from migraine headache. similar results were obtained in an italian study showing that anxiety was reported by 18.4% of patients with migraine, 19.3% of patients with tension-type h e a d a c h e , a n d 1 8 . 4 % o f p a t i e n t s w i t h c o m b i n e d m i g r a i n e a n d t e n s i o n t y p e h e a d a c h e s . ( 2 3 ) t h e e x a c t n a t u r e o f t h e relationship between migraine and anxiety remains unclear. although chronic headaches induce anxiety, it is likely that anxiety develops n o t o n l y a s a c o n s e q u e n c e o f r e c u r r e n t headache, but also of other disorders. the headache disorders are a group of heterogeneous conditions that result in a spectrum of disability within and among different individuals.(2) our findings suggest that headache disorders are relatively underdiagnosed in the primary care setting, but this is not entirely due to healthcare providers’ *chi-square test table 3. distribution of anxiety by type of headache in respondents type of headache* anxiety p=0.9172 yes (n= 49) no (n=12) tension headache cluster headache migraine headache 17 (80.9%) 11 (78.6%) 21 (80.7%) 4 (19.1%) 3 (21.4%) 5 (19.3%)   table 2. relationship between headaches and anxiety in respondents *chi-square test headache* anxiety p=0.008 yes (n= 91) no (n=49) no or little impact on life som e im pact on life substan tial impact on life very severe impact on life 42 (53.2%) 25 (75.8%) 10 (83.3%) 14 (87.5%) 37 (46.8%) 8 (24.2%) 2 (16.7%) 2 (12.5%)   88 possible inattention to headache conditions. rather, it appears that a substantial proportion o f p a t i e n t s b e l i e v e t h a t t h e i r h e a l t h c a r e providers do not know that they experience headaches. possible explanations for this i n c l u d e p a t i e n t s ’ p e r c e p t i o n t h a t t h e i r headaches or headache-related disabilities are not sufficiently important or debilitating to warrant discussion with their providers. a limitation of the present study is that our subjects were patients attending a public health center, who cannot be said to represent the whole population of the community. another limitation is the cross-sectional design used in our study, which cannot establish cause-effect relationships between headaches and anxiety, due to lack of temporal factors between both variables. it is therefore recommended to conduct further research studies using a prospective cohort study that is capable of determining cause-effect relationships between headaches and anxiety. conclusions a relatively high proportion of patients in the primary care setting experienced attacks of headaches that had an impact on anxiety. anxiety was also experienced by the majority of respondents, around one quarter of whom had migraine headaches. acknowledgements the investigators express their gratitude to the head of the tebet subdistrict public health center, dr. amnur r. kayo, for his guidance in the present study. we also thank the respondents who were willing to participate in this study. references 1. breslau n, rasmussen bk. the impact of migraine: epidemiology, risk factors, and comorbidities. neurology 2001;56: s4–12. 2. bjorner jb, kosinski m, ware je jr. calibration of an item pool for assessing the burden of headaches: an application of item response theory to the headache impact test (hit). qual life res 2003;12:913–33. 3. wang sj, fuh jl, lu sr, liu cy, jsu lc, wang pn, et al. chronic daily headache in chinese elderly. neurology 2000;54:314–9. 4. lu sr, fuh jl, chen wt, juang kd, wang sj. chronic daily headache in taipei, taiwan: prevalence, follow-up and outcome predictors. cephalalgia 2001;21:980–6. 5. stewart wf, scher ai, lipton rb. stressful life events and risk of chronic daily headache: results from the frequent headache epidemiology study. cephalalgia 2001;21:279-82. 6. boardman hf, thomas e, millson ds, croft pr. psychological, sleep, lifestyle, and comorbid associations with headache. headache 2005;45:657–69. 7. lyngberg ac, rasmussen bk, jørgensen t, jensen r. incidence of primary headache: a danish epidemiologic follow-up study. am j epidemiol 2005;161:1066–73. 8. breslau n, schultz lr, stewart wf, lipton rb, lucia vc, welch kma. headache and major depression: is the association specific to migraine? neurology 2000;54:308–13. 9. zwart ja, dyb g, hagen k, odegard kj, dahl aa, bovim g, et al. depression and anxiety disorders associated with headache frequency. the nord-trøndelag health study. eur j neurol 2003;10:147–52. 10. hagen k, einarsen c, zwart ja, svebak s, bovim g. the co-occurrence of headache and musculoskeletal symptoms amongst 51050 adults in norway. eur j neurol 2002;9:527–33. 11. rueda-sánchez m, díaz-martínez la. prevalence and associated factors for episodic and chronic daily headache in the colombian population. cephalalgia 2008;28:216–25. 12. stewart wf, lipton rb, simon d, von korff liberman j. reliability of an illness severity measure for headache in population sample of migraine sufferers. cephalalgia 1998;18:44–51. 13. stewart wf, lipton rb, kolodner k, liberman j, sawyer j. reliability of the migraine disability assessment score in population-based sample of headache sufferers. cephalalgia 1999;19:107–14. 14. magnoux e, freeman ma, zlotnik g. midas and hit-6 french translation: reliability and correlation between tests. cephalalgia 2007;28:26–34. 15. marcus da. identification of patients with headache at risk of psychological distress. headache 2000;40:373-6. 89 univ med vol.29 no.2 16. donnet a, lanteri-minet m, guegan-massardier e, mick g, fabre n, géraud g, et al. chronic cluster headache: a french clinical descriptive study. j neurol neurosurg psychiatry 2007;78: 1354-8. 17. bayliss ms, batenhorst as. the hit-6: a user’s guide. lincoln, ri: quality metric, inc; 2002. 18. kosinski m, bayliss ms, bjorner jb, ware je, garber wh, batenhorst a, et al. a six-item shortform survey for measuring headache impact: the hit-6. qual life res 2003;12:963-74. 19. ware je, kosinski m, bjorner jb, bayliss ms, batenhorst a, dahlof c, et al. applications of computerized adaptive testing (cat) to the assessment of headache impact. qual life res 2003;12:935-52. 20. kawata ak, coeytaux rr, devellis rf, finkel ag, mann jd, kahn k. psychometric properties of the hit-6 among patients in a headachespecialty practice. headache 2005;45:638-43. 21. zung wwk. a rating instrument for anxiety disorders. psychosomatics 1971;12:371-9. 22. coeytaux rr, linville jc. chronic daily headache in a primary care population: prevalence and headache impact test scores. headache 2007; 47:7-12. 23. beghi e, allais g, cortelli p, d’amico d, de simone r, d’onofrio f, et al. headache and anxiety-depressive disorder comorbidity: the hadas study. neurol sci 2007;28:s217–9. albendazole increases serum retinol concentration in primary schoolchildren with low serum retinol 121 *biomedical sciences master program, faculty of medicine, university of indonesia **department of parasitology, faculty of medicine, trisakti university ***department of parasitology, faculty of medicine, university of indonesia ****department of parasitology, faculty of medicine, tarumanagara university correspondence: dr. jane florida kalumpiu, m.biomed faculty of medicine, trisakti university jl. kyai tapa no. 260 grogol, jakarta 11440 phone:+6221-5672731 ext.2311 email: jane.mikejane@gmail.com univ med 2015;34:121-8 doi: 10.18051/univmed.2016.v35.121-128 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background soil-transmitted helminth (sth) infection is still a health problem in indonesia. vitamin a deficiency (vad) is often found together with sth. the effect of the deworming program on the vitamin a status of primary school children is unknown. the objective of this study was to evaluate the effect of albendazole on serum retinol concentrations in primary school children. methods an experimental study of pre-post test design without control was carried out on 118 children from grades 3-5 at one primary school in north jakarta, from july to september 2014. flotac examination was used to determine sth infections and high performance liquid chromatography was performed to measure serum retinol concentration. stool and blood samples were taken twice: before and three weeks after treatment with albendazole 400 mg single dose for three consecutive days given to all children either infected or uninfected. paired t-test was used to analyze the data. results of the 118 children, the prevalence of sth was 71.2%. the prevalence of low retinol serum (<1.05 µmol/l) was 17 (17.2%) and of mild vad was 2 (2.0%). albendazole given in 3 consecutive days could not increase serum retinol concentrations (p=0.055). serum retinol concentration significantly increased after albendazole treatment in children with low retinol serum at baseline (p=0.001). conclusions in children with low serum retinol concentration, treatment with albendazole increased retinol concentration. integrated deworming and vitamin a supplementation strategies should be evaluated in this population. keywords: albendazole, retinol, vitamin a status, primary schoolchildren albendazole increases serum retinol concentration in primary schoolchildren with low serum retinol jane florida kalumpiu,*,** yenny djuardi,*** taniawati supali,*** and ria buana*,**** doi: http://dx.doi.org/10.18051/univmed.2015.v34.121-128 122 kalumpiu, djuardi, supali, et al albendazole and retinol concentration pemberian albendazol meningkatkan kadar retinol dalam serum pada murid sekolah dasar dengan kadar retinol rendah latar belakang infeksi soil-transmitted helminthes (sth) masih merupakan masalah kesehatan di indonesia. prevalensi kurang vitamin a (kva) sering ditemukan bersamaan dengan sth. penelitian ini bertujuan untuk menilai pengaruh pemberian albendazol terhadap konsentrasi retinol dalam serum pada anak usia sekolah. metode penelitian eksperimental pra-pasca perlakuan tanpa kontrol dilakukan pada murid sekolah dasar kelas 3-5 di salah satu sdn di jakarta utara. sebanyak 118 subjek ikut serta pada penelitian, tetapi hanya 88 subjek yang dapat diikuti sampai selesai studi. pemeriksaan flotac dilakukan untuk menentukan infeksi dan intensitas sth. konsentrasi retinol dalam serum diukur menggunakan pemeriksaan kromatografi cair kinerja tinggi. sampel tinja dan darah diambil dua kali yaitu sebelum dan tiga minggu sesudah pengobatan dengan albendazol 400 mg dosis tunggal selama tiga hari berturut-turut yang diberikan pada semua anak. uji t-pasangan digunakan untuk analisis data. hasil dari 118 subjek didapatkan prevalence sth sebesar 71,2%. prevalence subjek dengan kadar retinol yang rendah (<1,05 µmol/l ) ditemukan sebesar 17 (17,2%) anak, dan kva ringan (<0,70 µmol/l) sebanyak 2 (2,0%). pemberian albendazol selama 3 hari berturut-turut tidak meningkatkan kadar retinol dalam serum (p=0,055). ternyata pada subjek yang kadar retinolnya rendah, pemberian albendazol mampu meningkatkan secara bermakna konsentrasi retinol dalam serum (p=0,001). kesimpulan pada subjek dengan kadar retinol yang rendah, pemberian albendazole mampu meningkatkan kadar retinol serum. pengobatan abendazol untuk sth harus dilakukan bersamaan dengan vitamin a untuk mengatasi kva pada murid sekolah dasar. kata kunci: albendazol, retinol, status vitamin a, murid sekolah dasar abstrak introduction helminthiasis is still one of the health problems in the world, particularly in developing tropical and subtropical countries, including indonesia.(1) it has been estimated that around two thousand million people are infected with at least one of these parasites, with the highest prevalence found in poor areas with poor sanitation and hygiene.(1,2) in indonesia helminthiasis is widespread in both rural and urban areas. a study covering several asian and african countries showed in 2010 that the prevalence of soil-transmitted helminths (sths) in indonesia was 24.6%.(3) young children are also disadvantaged by one or more of the soil-transmitted helminths (sths): ascaris lumbricoides (roundworm), trichuris trichiura (whipworm), and ancylostoma duodenale/necator americanus (hookworms).(4) although not fatal, helminthiasis has a wide impact, resulting among other things in malnutrition, anemia, abnormal cognitive functions, and decreased school performance so 123 univ med vol. 34 no.2 that it may decrease the quality of indonesian human resources.(1) the strategy recommended by the who to reduce the morbidity resulting from sths is the administration of anthelmintics of the benzimidazole group, i.e. albendazole 400 mg or mebendazole 500 mg single dose. these drugs should be given periodically once or twice a year, depending on the baseline sth prevalence found in primary school children.(1) the world health organization (who) has recommended that the delivery of vitamin a capsules and anthelmintic tablets should be simultaneous as worm infections and vitamin a deficiency (vad) have the same geographical distribution.(5) vitamin a, a fat soluble vitamin found in different forms such as retinol and provitamin a carotenoids, is essential for cell proliferation, immune functions, vision, gene expression, and excretion of growth hormone.(6) moreover, vitamin a derivatives play a major role in the development of the central nervous system. in addition, it helps maintain the integrity of the skin and mucous membranes that act as a barrier to pathogens.(7) previous in vivo studies have shown beneficial effects of vitamin a supplementation on tissue inflammation and innate and adaptive immunity in humans by downregulating inflammatory responses, enhancing immunoglobulin production, promoting the differentiation of t and b cells and modulating cytokine production.(7) an estimated 4.5 billion people of the world are at risk of intestinal parasites, and 1.2 billion might be infected with roundworms, such as ascaris lumbricoides.(8,9) vitamin a supplementation showed no effect on reinfection with a. lumbricoides (or: 1.11, 95% ci: 0.79-1.56).(10) with regards to a reduction of a. lumbricoides infection intensity, only one lowquality evidence with moderate-to-weak positive effects was found.(11) vitamin a increased the odds of having improved levels of il-4, which is part of the th2 response against helminths, in one study (strong positive effect, high quality evidence).(10) a study in bangladesh on children aged 2460 months showed that administration of albendazole only at a dose of 400g/day at baseline and 4 months later, was able to increase serum retinol concentrations.(12) studies of preschool-age children in bangladesh, india, and indonesia, however, showed that combined deworming and supplementation with beta-carotene, an inactive form of vitamin a, resulted in the greatest rise in serum retinol levels, as compared to children treated with deworming alone or beta-carotene supplementation exclusively.(12,13) while there is some evidence to suggest an added benefit to combined deworming and vitamin a supplementation, a number of studies in similar settings have either been unable to reproduce this positive outcome or have not specifically tested the combined intervention.(14) thus, while the who and the united nations children’s fund (unicef) recommend that benzimidazole anthelminthics can be safely coadministered with vitamin a, the potential synergies of deworming with vitamin a supplementation are neither well documented nor consistent. there is a need for additional studies, especially randomized clinical trials, given the frequent coexistence of dietary deficiency and infection in endemic areas.(15) the aim of the present study was to evaluate the effect of albendazole administration on serum retinol concentration in primary school children. methods design of the study an experimental study of pre-post test design without control was carried out from july to december 2014 in a state primary school in north jakarta. study subjects primary school children aged 9-11 years were recruited for this study. the inclusion criteria for the study subjects were: (a) apparently healthy without a history of chronic illness, and (b) willing to take daily one dose of albendazole during the study. in calculation of the sample size, the level of significance (α) was 0.05 two-sided, and the power (β) was 0.90. the selection of mean differences was taken 25% after albendazole 124 kalumpiu, djuardi, supali, et al albendazole and retinol concentration supplementation. taking into account a 20% dropout rate and the design effect, the sample size was increased to 71 children. the children participated in this study with the informed consent of their parents. intervention each of the children participating in this study were given a single oral dose of albendazole chewable tablets of 400 mg (indofarma) for three consecutive days. the anthelminthic tablets were chewed directly in front of the investigators. stool sample collection stool samples were collected in stool pots given on the previous day to the children through their respective home room teachers. the subjects had been given explanations on the stool collection method. for stool sample collection the children were given three days, after which the stool samples were collected directly by the team of investigators and transported to the laboratory. the stool sample collection was performed twice during the study, i.e. at baseline and three weeks after the last treatment. stool examination by the flotac technique one gram of stool sample was weighed, 10 ml of 5% formaline solution was added, and stored at room temperature waiting for the examination. all collected stool samples were examined by the flotac technique at the parasitology laboratory, faculty of medicine, university of indonesia. to one gram of stool sample was added 11 ml distilled water and the mixture homogenized and centrifuged at 1500 rpm for three minutes, and the supernatant discarded. a volume of 11 ml flotac solution (sucrose) was added and the mixture homogenized. five ml of the homogenate was placed into the flotac instrument, centrifuged at 1000 rpm for 5 minutes. the positive results of microscopic examination was presented as epg (eggs per gram). serum â-carotene determination blood samples were drawn from the antecubital vein by an experienced phlebotomist to the amount of 2-3 ml directly into a vacutainer tube containing a clot activator and separator gel and stored in a cool box containing icepacks and protected from light. the blood samples were then transported to the laboratory to be centrifuged and the serum was placed in a serum cup and stored in a freezer at minus 20°c for further examination. blood samples were drawn twice, at baseline and three weeks after administration of the anthelminthic. determination of serum retinol concentration was performed at the seameo laboratory, faculty of medicine, university of indonesia, by high performance liquid chromatography (hplc). to 40 µl of the serum sample to be assessed was added 100 µl of extraction solution (acetonitryl-methanol 1:1), manually shaken for one minute, and subsequently centrifuged at 12.000 rpm and 4°c for three minutes. approximately 20 µl of the supernatant was injected into the hplc apparatus (flow rate 1 ml/minute, column novapack c18 (3.9 x 150 mm), wavelength 325 nm). then pooled serum of known value was injected as control for every 10 or 20 samples. serum retinol concentration was presented in µmol/l. normal serum retinol concentration is >1.05 µmol/l.(16) data analysis to compare the baseline and follow-up values within the treatment groups, a paired ttest was used. significance was defined as p<0.05. ethical clearance all procedures to be performed in this study had been explained to the respective teachers, parents, and children, and had obtained ethical clearance from the health research ethics committee, faculty of medicine, university of indonesia. results of the 118 children recruited, complete data were available from 88 (88.9%) after three months. thirty children did not complete the trial for various reasons, including sickness, and refusal to take supplements (figure 1). 125 univ med vol. 34 no.2 there were about equal numbers of males and females (49.2% and 50.8%), and the percentage of children with sth infection was 71.2%. demographically, the school children of state primary school 07 (sdn 07) resided not far from school. the majority of the children’s parents were laborers (76.3%) while the remainder were business people, employees, fishers, and drivers. the highest educational level of the parents varied from primary school to senior high school, the majority (41.5%) being junior high school graduates (table 1). the blood samples collected at baseline (before treatment) was 99 and after treatment 88. at baseline mean serum retinol concentration was 1.38 ± 0.35 µmol/l. at baseline there were 1 7 ( 1 7 . 2 % ) c h i l d r e n w i t h s e r u m r e t i n o l concentrations of <1.05 µmol/l (mild vitamin a deficiency) and there were 2 (2.0%) children with serum retinol concentrations of <0.70 µmol/ l (severe vitamin a deficiency). after treatment mean serum retinol concentration was 1.45 ± 0.39 µmol/l, which was not significantly different from mean serum retinol concentration at baseline (p=0.055) (table 2). it was found that in subjects with baseline serum retinol concentrations below normal (<1.05 µmol/l), albendazole administration was able to significantly increase serum retinol concentrations (p=0.001) (table 2). based on infection status at baseline, mean serum retinol concentration before and after albendazole administration were not significantly different. this was shown by subjects with and without sth infection (table 2). discussion our study results showed that subjects with sth infection accounted for 71.2%, which is similar to the results of a study in pahang, malaysia, in which subjects who were infected with at least one sth species comprised 98.6%.(6) soil-transmitted helminthiases caused by ascaris lumbricoides, trichuris trichiura and hookworm are among the most common neglected tropical diseases (ntds) by infecting about 1,200 million people worldwide with the greatest morbidity being among children and mothers of childbearing age.(17,18) the main impact of sth infections is their association with malnutrition, vitamin a deficiency (vad) and iron deficiency anaemia (ida),which may figure 1. flow of subject participation during study 118 subject s screened by inclusion and exclusion criteria 88 subjects albendazole moni toring for 3 months 30 incomplete data due t o illness and refusing to take supplement table 1. distribution of sociodemographic characteristics, infection status, and retinol concentrations at baseline (n=118) 126 kalumpiu, djuardi, supali, et al albendazole and retinol concentration table 2. retinol concentration before (n=118) and after intervention (n=88) have effects at the community level with regard to work and productivity in adults and growth, learning and school performance in children.(19) in our study the prevalence of subjects with mild vitamin a deficiency (having a serum retinol concentration of <1.05 µmol/l) was 17.2% and the prevalence of subjects with severe vitamin a deficiency (with a serum retinol concentration of <0.70 µmol/l) was 2%. the prevalence in the population with low serum retinol (0.70 µmol/l or below) can be used to assess the severity of vitamin a deficiency in most age groups as a public health problem. there is said to exist a mild public health problem if the prevalence of subjects with low serium retinol (0.70 µmol/l or below) is 2.0-9.0%, moderate if 10.0-19.0% and severe if 20.0% or more.(16) the population in our study had a mild public health problem with regard to vitamin a deficiency. our study showed an increase in serum retinol concentration after administration of albendazole, although the increase was not significant from that before administration of albendazole. similar results were shown in a study on preschool children in bangladesh, in which oral albendazole administration at a dose of 400 mg did not significantly increase serum retinol concentration.(20) in communities in which vitamin a deficiency coexists with soil helminth infection, deworming alone and in combination with vitamin a supplementation has been explored as a means to correct or reduce deficiency in infected children. a population study in uttar pradesh, india, found that twice-yearly deworming alone of lightly infected children did not result in either significant weight gain or a reduction in mortality.(21) studies of preschool-age children in bangladesh, india, and indonesia, however, showed that combined deworming and supplementation with betacarotene, an inactive form of vitamin a, resulted in the greatest rise in serum retinol levels, as compared to children treated with deworming alone or beta-carotene supplementation exclusively.(4,20) while there is some evidence to suggest an added benefit to combined deworming and vitamin a supplementation, a number of studies in similar settings have either been unable to reproduce this positive outcome or have not specifically tested the combined intervention.(14) thus, while the who and the united nations children’s fund (unicef) recommend that benzimidazole anthelminthics can be safely coadministered with vitamin a the potential synergies of deworming with vitamin a supplementation are neither well documented nor consistent.(22) there is a need for additional studies, especially randomized clinical trials, given the frequent coexistence of dietary deficiency and infection in endemic areas. one of the limitations of our study was the lack of controls, so that the obtained results could not be compared with that of controls. the proportion of subjects that was successfully followed up to the completion of the study was still below 90.0%, which may have influenced the obtained study results. there is a need for additional studies, especially randomized clinical trials, given the frequent coexistence of dietary deficiency and infection in endemic areas. 127 univ med vol. 34 no.2 conclusions the sth prevalence in primary school children is still relatively high. albendazole administration is capable of increasing serum retinol concentrations in subjects with low serum retinol concentrations. multi-pronged approaches are probably necessary to prevent and control the negative effects of sth infections, including anthelminthic drugs, and, possibly improved nutrition. confict of interest the authors declare that they have no competing interests. acknowledgement the investigators thank prof dra taniawati supali and dr. yenny djuardi, phd. for guidance and consultations during the study. thanks are also due to prof dr dr adi hidayat, ms for his willingness to edit this article. daftar pustaka 1. world health organization. eliminating sth as a public health problem in children. geneva: world health organization;2012. 2. knoop s, steinman p, keiser j, et al. nematode infections soil-transmitted helminths and trichinella: infect dis clin n am 2012;26:34158. 3. pullan rl, smith jl, jasrasaria r, et al. global numbers of infection and disease burden of soil transmitted helminth infections in 2010. parasites vectors 2014;7:37. doi: 10.1186/17563305-7-37. 4. albonico m, allen h, chitsulo l, et al. controlling soil-transmitted helminthiasis in pre-school age children through preventive chemotherapy. plos negl trop dis 2008;2: e126. doi: 10.1371/journal.pntd.0000126. 5. world health organization. preventive chemotherapy in human helminthiasis. world health organization. 2006. 6. al-mekhlafi hm, anuar ts, al-zabedi em, et al. does vitamin a supplementation protect schoolchildren from acquiring soil-transmitted helminthiasis? a randomized controlled trial. parasites vectors 2014;7:367. doi: 10.1186/17563305-7-367. 7. hurst rj, else k. retinoic acid signalling in gastrointestinal parasite infections: lessons from mouse models. parasite immunol 2012;34:351– 9. 8. bethony j, brooker s, albonico m, et al. soiltransmitted helminth infections: ascariasis, trichuriasis, and hookworm. lancet 2006;367: 1521-32. 9. brooker s, clements ac, bundy da. global epidemiology, ecology and control of soiltransmitted helminth infections. adv parasitol 2006;62:221-61. 10. long kz, rosado jl, montoya y, et al. effect of vitamin a and zinc supplementation on gastrointestinal parasitic infections among mexican children. pediatrics 2007;120:e846– e855. 11. payne lg, koski kg, ortega-barria e, et al. benefit of vitamin a supplementation on ascaris reinfection is less evident in stunted children. j nutr 2007;137:1455–9 12. haque r, ahmed t, wahed ma, et al. low-dose ß-carotene supplementation and deworming improve serum vitamin a and ß-carotene concentration in preschool children of bangladesh. j health popul nutr 2010;28:23037. 13. albonico m, allen h, chitsulo l, et al. controlling soil-transmitted helminthiasis in pre-school-age children through preventive chemotherapy. plos negl trop dis 2008;2: e126. doi: 10.1371/journal.pntd.0000126. 14. tanumihardjo sa, permaesih d, muhilal. vitamin a status and hemoglobin concentrations are improved in indonesian children with vitamin a and deworming interventions. eur j clin nutr 2004;58:1223-30. doi: 10.1038/sj.ejcn. 1601953. 15. rajagopal s, hotez pj, bundy dap. micronutrient supplementation and deworming in children with geohelminth infections. plos negl trop dis 2014;8:e2920. doi:10.1371/ journal.pntd.0002920. 16. world health organization. serum retinol concentrations for determining the prevalence of vitamin a deficiency in populations. geneva: world health organization; 2011. 17. surin j. the burden of moderate-to-heavy soiltransmitted helminth infections among rural malaysian aborigines: an urgent need for an integrated control programme. parasites vectors 2011;4:242. 128 kalumpiu, djuardi, supali, et al albendazole and retinol concentration 18. world health organization. soil-transmitted helminthiases. eliminating soil-transmitted helminthiases as a public health problem in children: progress report 2001–2010 and strategic plan 2011–2020. geneva: world health organisation; 2012. 19. yap p, utzinger j, hattendorf j, et al. influence of nutrition on infection and re-infection with soil-transmitted helminths: a systematic review. parasites vectors 2014;7:229. doi:10.1186/17563305-7-229. 20. haque r, ahmed t, wahed ma, et al. low-dose â-carotene supplementation and deworming improve serum vitamin a and â-carotene concentrations in preschool children of bangladesh. j health popul nutr 2010;28:2307. 21. awasthi s, peto r, read s, et al. population deworming every 6 months with albendazole in 1 million pre-school children in north india: devta, a clusterrandomized trial. lancet 2013; 381:1476–86. 22. world health organization. how to add deworming to vitamin a distribution. geneva: world health organization;2004. senjaya 127 a 55 year old man with progressive neurologic deficits may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina sendjaja muljadi*a, and hardy senjaya** *department of neurology, medical faculty, trisakti university; head of neuroscience centre, medistra hospital, jakarta ** department of anatomy, mecical faculty, trisakti university, jakarta correspondence adr. sendjaja muljadi, sps department of neurology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11410 telp. 021-5672731 ex.2806 univ med 2009;28:127-32 abstract at least 50 percent of the decline in functional abilities associated with the elderly is caused by neurological conditions, particularly vascular dementia, such as occurs in binswanger disease. binswanger disease is a rare condition, characterized by acute strokes with symptoms and signs compatible with lacunar infarction. the disease onset is commonly between 55 and 75 years. the majority of patients with binswanger disease have chronic hypertension and other putative factors including diabetes mellitus, polycythemia, thrombocytosis, hyperlipidemia, hyperglobulinemia and pseudoxanthoma elasticum, increased fibrinogen levels and the antiphospholipid antibody syndrome. we report the case of a 55 year-old man, who suffered from involuntary movements of his right arm, slight hemiparesis on the right side, and also had dementia. he had a history of high blood pressure and laboratory tests showed that he had diabetes mellitus. the brain magnetic resonance imaging showed irregular white matter abnormalities with multiple lacunar infarcts in the basal ganglia and pons. the clinical picture is characterized by acute strokes, followed by involuntary movements and also dementia. therefore we decided to diagnose it as binswanger disease. after discharge from hospital, the patient has not returned for follow-up. keywords: binswanger disease, irregular white matter abnormalities, multiple lacunar infarcts introduction a g i n g i s a s s o c i a t e d w i t h a d e c l i n e i n functional abilities and neurologic conditions are responsible for at least 50 percent of this decline. neurologic conditions especially vascular dementia and stroke are the leading causes of disability and institutionalization in the elderly. a distinctive form of small vessel disease causing dementia is ischemic white matter disease or binswanger’s disease. the concept that dementia is a group of cognitive disorders with many underlying subtypes and pathophysiological causes was first defined by otto binswanger, alois alzheimer, and emil kraepelin at the close of the 19th century. however, at the end of the 20th century, t h e d e s c r i p t i v e c r i t e r i a , i n c i d e n c e , a n d 128 muljadi, senjaya progressive neurologic deficits prevalence for many of these dementias are still subject to controversy.(1) one subtype, labeled binswanger ’s disease by alzheimer in 1902, referred to arteriosclerotic subcortical white matter changes.(2) binswanger’s disease (bd), one of the most common forms of vascular dementia in the elderly, is also called subcortical vascular d e m e n t i a , o r s u b a c u t e a r t e r i o s c l e r o t i c encephalopathy. the clinical features of bd are h i g h l y v a r i a b l e a n d t h e p s y c h i a t r i c a n d behavioral manifestations have been less well described. patients with bd have dementia and various clinical and radiological abnormalities, such as focal cerebrovascular deficits, and subcortical cerebral dysfunction. they also have a variety of vascular risk factors.(3-5) p a t h o l o g i c a l l y t h e r e a r e w i d e s p r e a d , microscopic areas of damage to the deep layers of white matter in the brain. the disorder is characterized by prominent brain atrophy with ventricular dilation, diffuse demyelination of the white matter, and a scattering of lacunar infarcts in the basal ganglia and white matter. the brain damage is the result of thickening and narrowing ( a t h e r o s c l e r o s i s ) o f a r t e r i e s t h a t f e e d t h e subcortical areas of the brain.(6) a t h e r o s c l e r o s i s ( p o p u l a r l y k n o w n a s “hardening of the arteries”) is a systemic process that affects blood vessels throughout the body. it begins late in the fourth decade of life and increases in severity with age.(6) cerebral blood vessels undergo profound changes with aging. in the cerebral cortex and hippocampus cerebral capillaries are reduced in number and have thickened and fibrotic basement membranes.(7) these alterations reduce cerebral perfusion, deplete cerebrovascular reserves, and increase the susceptibility of the brain to vascular insufficiency and ischemic injury.(8) chronic hypertension is associated with profound changes in the structure and function o f c e r e b r a l b l o o d v e s s e l s . h y p e r t e n s i o n promotes atherosclerosis in cerebral arteries(9) and induces lipohyalinosis, a pathological process characterized by fibrinoid necrosis of the vascular wall.(9) functional changes include regionally reduced resting cbf and impaired cerebrovascular reactivity.(10) therefore functional hyperemia is attenuated in hypertensive patients. the following is a case report of binswanger’s disease as encountered by the authors. case report a 55-year old man who came from a remote area, was admitted to our clinic in july 2002. h e c o m p l a i n e d o f i n v o l u n t a r y s w i n g i n g movements of his right arm. before visiting us, this patient had been treated by the neurologist of the local hospital and was diagnosed as having cardiovascular disease (cvd)-ischemic stroke. two weeks later when this patient was treated in the same hospital, the patient’s family noticed that his memory became worse and also that he appeared to have difficulty in moving his limbs. he had a history of hypertension, and took anti-hypertensive drugs irregularly. he did not know whether he had diabetes mellitus or not. on physical examination in our clinic at the time of admission the patient had a good general performance and was fully alert with blood pressure 170/100 mmhg, and pulse 92x/min. there were no signs of meningeal iritation and no papilledema on bilateral funduscopy. the pupils were of equal size and of normal shape, l i g h t r e f l e x + / + . t h e r e w a s s l i g h t r i g h t h e m i p a r e s i s a n d o c c a s i o n a l l y i n v o l u n t a r y m o v e m e n t s o f t h e r i g h t a r m w e r e s e e n . physiologic reflexes were normal and there were n o p a t h o l o g i c a l r e f l e x e s . a t t h e t i m e o f admission our working diagnosis was cvdischemic stroke. we observed this patient as an inpatient and he underwent several diagnostic tests as shown in table 1. 129 echocardiography: valvular heart disease ( m i l d m o d e r a t e a o r t i c r e g u rg i t a t i o n ) . transcranial color doppler sonography (tccd): a l l t h e c e r e b r a l c i r c u l a t i o n ( a n t e r i o r a n d p o s t e r i o r ) s h o w e d l o w d o p p l e r v e l o c i t y. generalized cerebral atherosclerosis. chest xray: normal chest. brain magnetic resonance figure 2. t2 weighted mri showing irregular white matter abnormalities and multiple lacunar infarctions in basal ganglia. figure 1. t2 weighted mri showing infarction in pons i m a g i n g ( m r i ) : i r r e g u l a r w h i t e m a t t e r abnormalities and multiple lacunar infarcts in basal ganglia and pons (figure 1 and 2). no evidence of hydrocephalus (figure 3). after a careful study of his brain mri, we considered the patient to be a case of binswanger disease. this patient received pentoxyphylline univ med vol. 28 no.2 table 1. hematologic laboratory data 130 muljadi, senjaya progressive neurologic deficits 300 mg in 500 ml asering/12 hr, piracetam inj. 3 g four times daily, cilostazol (oral) 50 mg twice daily, donepezil 5 mg once daily, quinapril hcl (accupril) 10 mg once daily, glipizide 5 mg once daily. after discharge from hospital, the patient has not returned for follow-up. discussion binswanger’s disease (encephalopathy or leucoaraiosis) is a rare disorder of presumed vascular etiology, characterized by progressive d e m e n t i a a n d p s e u d o b u l b a r p a l s y. t h e h i s t o p a t h o l o g y s h o w s t h i n n i n g a n d hyalinization of the walls of small penetrating arteries and occasionally amyloid angiopathy in the basal ganglia and periventricular regions. associated with diffuse hemispheric demyelination resulting from chronic ischemic disease, there is axonal loss and gliosis, probably due to anoxia as a result of sclerotic changes.(11) the age of onset of senile dementia of the binswanger’s type is typically in the sixth and seventh decades of life, and men and women appear to be affected equally often. the incidence of the disease is difficult to cite given the controversies regarding its clinical features but has been reported to range from 3 to 12 percent in the elderly. (12) this patient came to our clinic from a remote area with swinging involuntary movements of his right arm. before he visited us, he had been treated at the local hospital as an ischemic stroke patient. when the patient was under treatment, his family noticed that his memory became worse and that he appeared to have difficulty in moving his limbs. the brain mri showed irregular white matter abnormalities and multiple lacunar infarcts in the basal ganglia and also in the pons (figure 1 and figure 2). there was no evidence of hydrocephalus (figure 3). this patient had hypertension and diabetes mellitus and tccd examination indicated t h e i m p r e s s i o n o f g e n e r a l i z e d c e r e b r a l atherosclerosis. the pathogenesis of bd and the relation between bd and the multiple lacunar infarctions that accompany the white matter lesions remain unclear. however, it is likely that chronic cerebral ischemia is common to these two conditions. in addition, it has been suggested that these white m a t t e r l e s i o n s m a y b e c a u s e d b y a r t e r i a l hypertension and a subsequent dysfunction of the b l o o d b r a i n b a r r i e r. l i p o h y a l i n o s i s ( a r t e r i o l o s c l e r o s i s ) , i n d u c e d b y c h r o n i c hypertension or diabetes or both, is the most common finding in the small vessels of deep brain from patients with white matter disease.(13) bd occurs equally in both sexes with onset between 55 and 75 years. the clinical picture is characterized by acute strokes with symptoms and signs compatible with lacunar infarction. subacute onset of focal neurological deficits may develop over a period of several days, and there is a s t e p w i s e p r o g r e s s i o n o f m o t o r, c o g n i t i v e , intellectual, and behavioral deficits during a 5–10 year period. corticospinal and extrapyramidal signs may also occur. the majority of patients with bd have chronic h y p e r t e n s i o n t h a t c o u l d c a u s e d e g e n e r a t i v e figure 3. t2-weighted mri showing no evidence of hydrocephalus 131 changes in the penetrating arteries of the white matter. other putative factors include diabetes m e l l i t u s , p o l y c y t h e m i a , t h r o m b o c y t o s i s , h y p e r l i p i d e m i a , h y p e rg l o b u l i n e m i a , a n d pseudoxanthoma elasticum, increased fibrinogen levels and increased serum viscosity, and the antiphospholipid antibody syndrome. this patient had the clinical picture of bd, such as acute stroke, extrapyramidal signs (involuntary movements), and dementia. he also had a history of hypertension and diabetes mellitus. after complete diagnostic examinations we d e c i d e d t o e s t a b l i s h t h e d i a g n o s i s o f binswanger’s disease in this patient. although we could not find the other putative factors such as polycythemia, thrombocytosis, increased fibrinogen levels and others, all his symptoms (high blood pressure, diabetes mellitus and more importantly, the brain mri) were typical of binswanger disease. bd is a progressive disease for which there is no cure. changes may be sudden or gradual and then progress in a stepwise manner. bd can often coexist with alzheimer ’s disease. if progression of bd can be halted or slowed with treatments then there is that much more time that the patient can properly function and retain his or her personality. until more studies can be done, however, all this is speculation and bd is still a sentence of life-imprisonment and eventual death to the body.(14) the victims of binswanger’s disease have gradually progressive aspontaneity, slowed cognition, frequent falls, upper motor neuron signs, and possibly seizures. in the future, newer laboratory findings may support the diagnosis of bd during life, such as magnetization transfer images, (15) ischemia on diffusion-weighted mri,(16) slowed arterial-venous transit times by transcranial sonography,(17) and decreased cerebral reactivity to apnea during cognitive tasks.(18) conclusion this patient was unique in having extreme bd without accompanying lacunae or cystic lesions, as are usually seen in the presence of hypertensive cerebrovascular arteriolosclerosis. references 1. roman gc. a historical review of the concept of vascular dementia: lessons from the past for the future. alzheimer dis assoc disord 1999;13(suppl 3):s4–s8. 2. hurley ra, tomimoto h, akiguchi i, fisher re, taber kh. binswanger’s disease: an ongoing controversy. j neuropsychiatry clin neurosci 2000;12:301-4. 3. tomimoto h, akiguchi i, wakita h, osaki a, hayashi m, yamamoto y. coagulation activation in patients with binswanger disease. arch neurol 1999;56:1104-8. 4. román gc. new insight into binswanger disease. arch neurol 1999;56:1061-2. 5. nagaratnam n, nagaratnam k. psychiatric and behavioral aspects of dementia of the binswanger type. am j alzheimer’s dis other demen 1998; 13:173-8. 6. national institute of neurological disorders and stroke. ninds binswanger’s disease information page. available at: http://www.ninds.nih.gov/ disorders/binswangers/binswangers.htm . accessed july 1, 2009. 7. farkas e, luiten pg. cerebral microvascular pathology in aging and alzheimer’s disease. prog neurobiol 2001;64:575–611. 8. fernando ms, simpson je, matthews f, brayne c, lewis ce, barber r, et al. white matter lesions in an unselected cohort of the elderly: molecular pathology suggests origin from chronic hypoperfusion injury. stroke 2006;37:1391–8. 9. droste dw, ritter ma, dittrich r, heidenreich s, wichter t, freund m, et al. arterial hypertension and ischaemic stroke. acta neurol scand 2003;107:241–51. 10. iadecola c, davisson rl. hypertension and cerebrovascular dysfunction. cell metab 2008;7:476–84. univ med vol. 28 no.2 132 muljadi, senjaya progressive neurologic deficits 11. gilroy j. binswanger disease. in: basic neurology. 3rd ed. u.s.a, new york: the mcgraw-hill companies, inc; 2000.p.261-2. 12. olsen cg, clasen me. senile dementia of the binswanger’s type. american academy of family physicians. available at: http://www.aafp.org/afp/ afpprinter/981200ap/olsen.html?print=yes. accessed july 1, 2009. 13. palumbo v, boulanger jm, hill md, inzitri d, buchan am. leukoaraiosis and intracerebral hemorrhage after thrombolysis in acute stroke. neurology 2007;68:1020-4. 14. kohshiro f, sadaharu t, hiroaki h, mitsuko s. binswanger’s disease: clinical and computed tomograph neuroradiological study of seven cases. psychogeriatrics 2005;5:127-46. 15. hanyu h, asano t, sakurai h, iwamoto t, takasaki m, shindo h, et al. magnetization transfer ratio in cerebral white matter lesions of binswanger’s disease. j neurol sci 1999;166:85– 90. 16. choi sh, na dl, chung cs. diffusion-weighted mri in vascular dementia. neurology 2000;54:83– 9. 17. puls i, hauck k, demuth k, horowski a, schlieber m, dörfler p, et al. diagnostic impact of cerebral transit time in the identification of microangiopathy in dementia: a transcranial ultrasound study. stroke 1999;30:2291-5. 18. lim gt, mendez mf, bronstein yl, jouben-steele l, vinters hv. clinicopathologic case report: akinetic mutism with findings of white matter hyperintensity. j neuropsychiatry clin neurosci 2002;14:214-21. alvina 102 abstract *department of pharmacology, medical faculty, trisakti university jakarta correspondence dr. elly herwana, mbiomed department of pharmacology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5672731 ext.2801 email : elly_herwana@yahoo.com univ med 2011;30:102-10. multimicronutrient supplementation in older persons decreased zinc deficiency but not serum tnf-ααααα elly herwana* and yenny* may-august, 2011 may-august, 2011 may-august, 2011 may-august, 2011 may-august, 2011 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 vol.30 no.2 universa medicina the increase in the population of older persons needs to be accompanied by increased quality of healthcare in older persons, particularly a decrease in the incidence of infections. impaired immune responses are common in older adults, and immune senescence likely contributes to the increased incidence of infectious diseases in the older persons. the aging process decreases the immune response and many studies have been conducted to explain the role of supplementation with various micronutrients, such as vitamin c, vitamin e, β-carotene and zinc, on the immune response. the aim of the present study was to assess the influence of 6 months of multi-micronutrient (mmn) supplementation on zinc and tumor necrosis factor (tnf-α) levels in older persons. a randomized controlled trial was conducted on 78 older persons, who were divided into two groups. the treatment group received mmn supplementation containing 40 mg elemental zinc, 120 mg ascorbic acid, 6 mg β-carotene, 15 mg α-tocopherol and 400 µg folic acid and the control group 400 mg calcium carbonate. the study did not demonstrate that 6 months of mmn supplementation resulted in significant differences between both groups, with respect to total protein, albumin, globulin, and tnf-α levels. in contrast, mmn supplementation significantly decreased the proportion of older persons with zinc deficiency in the treatment group, in comparison with the control group. the present data suggest that in older persons with relatively good immune and protein status, improvement of the immune status by mmn supplementation may be difficult and at best limited. key words: multimicronutrients, tnf-α, protein, zinc, older persons introduction technological advances and improved community healthcare services have led to an increased number of patients surviving fatal conditions, resulting in a steadily growing population of older persons. in the year 2025 the world population of older persons is predicted to exceed one billion, with the majority living in developing countries.(1) in indonesia, the number of older persons in the year 2000 was 14.6 million, increasing to 19.1 103 univ med vol.30 no.2 million in 2010, and estimated to reach 29.9 million in 2025.(2) the increase in the population of older persons has led to the need for measures to improve their quality of health, particularly by reducing their risk of infection. older persons generally have an increased risk of infection and a decreased functioning of the immune system. they tend to have increased morbidity of chronic diseases concurrently with the decline in organ functions due to the aging process. one study reported that 50-80% of older persons aged 65 years and over suffer from more than one chronic disease, thus affecting their quality of health.(3,4) nutritional factors also play a role in the health status of older persons and are particularly important for immune functions.(5) micronutrient supplementation has been mostly associated with its effect on immunity and infectious disease. in malnutrition, which is commonly accompanied by micronutrient deficiency, many abnormalities of immune function have been encountered. according to surveys performed in india, europe, the united states, and canada, around 35% of older persons have micronutrient deficiencies.(6) for an efficiently functioning immune system, adequate micronutrient intakes are required, as micronutrient deficiencies depress immunity. this condition causes the body to become more susceptible to infection, thus increasing morbidity and mortality in older persons. conversely, infection may also cause micronutrient deficiencies due to reduced n u t r i e n t i n t a k e s o r t h r o u g h c h a n g e s i n metabolic pathways.(7) oxidative stress has long been recognized as an important factor in several infectious diseases associated with degenerative processes, such as atherosclerosis, cardiac disorders, neurodegenerative disease, immunological disorders, and even the aging process itself.(8) various reactive oxygen species (ros) have been recognized, i.e. o2 .-, h2o2, and .oh, which are continuously generated in vivo under aerobic conditions. inflammatory cytokines, such as tumor necrosis factor-α (tnf-α) have long been known to generate ros in large numbers. (8) tnfα is a proinflammatory c y t o k i n e m a i n l y p r o d u c e d b y a c t i v a t e d m a c r o p h a g e s . s e v e r a l s t u d i e s h a v e demonstrated that zinc deficiency affects the production or the biological activity of tnfα. on the other hand, zinc supplementation given to normal healthy subjects and to cardiac patients resulted in reduced oxidative stress and serum tnf-α levels.(9,10) however, the effect of micronutrient supplementation on serum zinc, protein and tnf-α levels in healthy older persons has not been fully clarified. aging is associated with decreased immune responses and increased frequency of infections, and it may be assumed that older persons suffer from inapparent infections, such that they can be said to have received “natural vaccination”. infection causes t-cell activation, accompanied by the release of serum tnf-α, such that serum tnfα levels can be taken as a parameter of infection. advancing age is probably also associated with failure of control of tnf-α production, and plays a role in infectious events. (11) any beneficial effect of multimicronutrient (mmn) supplementation would be expected to increase serum zinc and protein l e v e l s , w h i l e d e c r e a s i n g t n f α l e v e l s . therefore the aim of the present study was to determine whether mmn supplementation in naturally immunized older persons increased serum zinc and protein levels, and decreased tnf-α levels. methods research design this study was an experimental study d e s i g n e d a s a r a n d o m i z e d d o u b l e b l i n d c o n t r o l l e d t r i a l , c o n d u c t e d i n m a m p a n g prapatan subdistrict, south jakarta, from 1 july 2006 up to 28 february 2007. 104 subjects the target population comprised older p e r s o n s r e s i d e n t i n m a m p a n g p r a p a t a n subdistrict, south jakarta. inclusion criteria were: male and female older persons aged 60 years and older, in good health, capable of communication, capable of walking without support, and willing to participate in the present study. exclusion criteria were individuals with severe or terminal disease, and patients with diabetes mellitus. the sample size was expected to detect a 15% difference in plasma tnf-α concentration, with a 2-sided p value of 0.05, and an approximate power of 80%. the sample size was computed statistically and was determined to comprise 80 subjects. sample selection was by multistage cluster and simple random sampling. randomization the subjects were randomly assigned into the mmn supplemented or the control group, using envelopes, each of which contained 2 smaller envelopes (1 assigning a subject to mmn treatment and 1 assigning a subject to p l a c e b o ) . t h e m m n g r o u p r e c e i v e d a supplement containing 40 mg elemental zinc (as zinc gluconate), 120 mg ascorbic acid, 6 mg βcarotene, 15 mg α-tocopherol (d-α-tocopheryl acid succinate) and 400 ì g folic acid. the control group received a preparation containing 400 mg calcium carbonate. random allocation was performed by means of a block-of-6 design, for which the sequence of random numbers was generated using microsoft excel. intervention supplementation was performed once daily up to 6 months and administered by field workers at home visits. participants in the t r e a t m e n t g r o u p r e c e i v e d s u p p l e m e n t s containing mmn, as explained above, and the control group received supplements containing calcium. both types of supplement were identical in form, color, and taste/flavor, so as to be superficially indistinguishable. subjects were considered to have dropped out of the study after failing to take 10% of supplements or by missing two weeks of supplementation. both mmn and control capsules were supplied by pt ikapharmindo laboratories (jakarta). data collection t h e c h a r a c t e r i s t i c s o f a g e , g e n d e r, educational status, marital status, and health status were collected by interviews using questionnaires that had been tested in a p r e l i m i n a r y t r i a l . c o l l e c t i o n o f s u b j e c t characteristics was done by field workers at b a s e l i n e ( s t a r t o f s t u d y ) a n d a f t e r supplementation for 6 months. measurements body mass index (bmi) was calculated by dividing body weight (kg) by height (m) squared. body weight was determined using sage scales accurate to 0.1 kg, while height was measured by means of microtoise accurate to 0.1 cm. for asian populations, bmi is classified into the following categories: underweight (<18.5 kg/m2), normal (18.5–22.9 kg/m2), overweight (23.0–27.5 kg/m2), and obese (> 27.6 kg/m2).(12) biochemical measurements from each subject 10 ml of venous blood was collected after an overnight fast of 10 hours, for laboratory determination of total protein, albumin and globulin, hemoglobin, zinc and tnf-α. determinations were performed twice, i . e . b e f o r e a n d a f t e r 6 m o n t h s o f supplementation. measurement of tnf-α concentration (pg/ml) was performed by means of an elisa kit (invitrogen). assessment of total protein (g/dl), albumin (g/dl) and globulin (g/dl) was also done using elisa. plasma zn levels were determined by flame atomic a b s o r p t i o n s p e c t r o m e t r y u s i n g s t a n d a r d methodology. a cut-off of 10.7µmol/l zinc was taken as an index of zinc deficiency.(13) herwana, yenny multimicronutrient in older persons 105 univ med vol.30 no.2 ethical clearance older persons willing to participate in the study were informed of the aims and protocol of the study. each of the subjects was informed that their participation in this study was voluntary and that they had the right to withdraw at will from the study at any time without giving any reasons. after the subjects understood the i n f o r m a t i o n g i v e n a n d w e r e w i l l i n g t o participate, they were asked to sign an informed consent form approved by the commission on research ethics of the medical faculty, trisakti university. statistical analysis demographic differences between mmn and control groups were examined by t-test (age) and chi-square test (gender, educational level, and marital status). for other variables, t-tests were used to compare group differences when variables were normally distributed. analyses were performed with spss for windows, version 15.0 (spss inc, chicago, il). the manufacturer of the supplements was not involved in the study design and data analysis. the authors had full and unrestricted rights to analyze, interpret, and publish the results. results from 1 july 2006 to 28 february 2008, a total of 100 older persons underwent screening. as shown in figure 1, 90 older persons as study subjects were randomly assigned to two treatment groups: 45 were assigned to the mmn group and 45 were assigned to the control group. after 6 months of intervention, 12 older persons dropped out of the study (3 in the mmn group and 9 in the control group). the most common reason for withdrawal was gastrointestinal upset (2 subjects in the mmn group and 6 subjects in the control group). a total of 42 subjects in the mmn group and 36 subjects in the control group participated in the study up to completion and all were included in the data analysis. figure 1. flowchart of the study design and subject participation throughout the study (mmn = multi-micronutrient) 6 months of follow‐up excluded ( n=10) not meeting inclusion criteria (n= 6) refused to participate (n= 4) enrollment (n= 90) allocation mmn group (n=45) control (n=45) assessed for eligibility (n=100) 6 months of follow‐up lost to follow‐up (n=3) ‐ discontinued intervention (n=2) ‐ moved (n=1) lost to follow‐up (n=9) ‐ discontinued intervention (n=6) ‐ moved (=3) analyzed (n=42) analyzed (n=36) 106 baseline characteristics of the study subjects are listed in table 1. age, gender, level of education, marriage status were similar at baseline among the treatment groups (p>0.05). baseline mean bmi was not significantly different in both study groups (p=0.874). compliance with the study protocol was confirmed by pill counts and about 95% of the subjects in the two groups had good compliance. serum total protein, albumin, globulin and tnf-α levels at baseline are presented in table 2. obtained values of total protein, albumin, globulin and tnf-α levels were not significantly different in both groups (p>0.05). the proportion of subjects with zinc deficiency was significantly higher in the mmn group (50%) in comparison with the control group (13.9%) (p=0.01). baseline zinc levels in the mmn group (11.24 ± 2.01 µmol/l) were lower than those in the control group (12.19 ± 1.59) (p=0.024). the number of mmn subjects with zinc deficiency was significantly higher than the control subjects (p=0.01). after administration of 6 months of mmn supplementation, values for total protein, albumin, globulin, and tnf-α levels were not variable supplement (n=4 6) control (n=36) p total protein (g/dl)§ 7.15 ± 1.07 7.11 ± 1.17 0.367† albu min (g/dl) § 3.97 ± 0.61 3.89 ± 0.60 0.952† globulin(g/d l) § 3.18 ± 0.63 3.24 ± 0.67 0.846† tnf-α (pg/ml) § 2.32 ± 2.11 1.89 ± 2.25 0.670 zinc (µmol/l) § 11.24 ± 2.0 12.19 ± 1.59 0.024†* zinc deficien cy (<10.7 µmol/l) 50.0% 13.9% 0.011‡* table 2. zinc, protein and tnf-á levels at baseline, by treatment groups † p values by independent t-test ; ‡ p value by chi-square test; * significance; § mean ± sd table 1. baseline descriptive statistics of the subjects by treatment groups ‡p values by chi-square test; †p value by independent t-test; §mean ± sd herwana, yenny multimicronutrient in older persons 107 univ med vol.30 no.2 significantly different between mmn and control groups (p>0.05) (table 3). in the group receiving mmn supplementation for 6 months the proportion of subjects with zinc deficiency was reduced to 33.3%, while that in the control group remained at baseline level. discussion o u r s t u d y s h o w e d t h a t m m n supplementation administered to older persons for six months resulted in a nonsignificant effect on plasma total protein, albumin, globulin, and tnf-α. differing results were found in the study by prasad et al. on oral supplementation of zinc gluconate (45 mg elemental zn/d) for 12 months to 49 subjects in the age range of 55-87 years. their study found that the ex vivo generation of tnf-α decreased significantly in the zinc s u p p l e m e n t e d s u b j e c t s a n d i n c r e a s e d significantly in the placebo group. it was also observed that tnf-α in the placebo group increased with time, suggesting that this increase may be an effect of increasing age.(8) these differences in outcomes presumably occurred because our study subjects were not vaccinated against influenza and pneumonia, unlike the subjects in prasad’s study,(8) in whom vaccination against influenza and pneumonia stimulated their immune system. a few years previously, prasad et al.(9) were the first to demonstrate that zinc supplementation to healthy volunteers significantly decreased oxidative stress. in their study, oxidative stress was assessed by lipid peroxidation and dna oxidation, and also by determining nf-κb activation induced by tnf-α in cultures of mononuclear cells isolated from the subjects. a more recent study in turkey evaluated the effect of zinc supplementation in children with heart disease at a dose of 30 mg elemental zinc given for 28 days after administration of influenza vaccine. among 49 recruited subjects, a total of 44 children completed the study. the results o f t h i s tu r k i s h s t u d y s h o w e d t h a t z i n c supplementation significantly decreased serum tnfα l e v e l s , c o m p a r e d w i t h t h e n o n supplemented group.(10) on the other hand, our finding that mmn supplementation did not affect plasma protein and tnf-α levels was similar to the results of a study by albers et al.(14) using a murine sensitization model. the investigators studied the effects of micronutrient supplementation on markers of adaptive and innate responses, including serum tnf-α, after sensitization and subsequent challenge of the experimental animals with di-nitro-chlorobenzene (dncb). the diets of the experimental animals were supplemented with a single antioxidant, comprising either one of the vitamins a, c, and e, or either one of the minerals zinc and s e l e n i u m . t h e r e w e r e n o s i g n i f i c a n t differences in tnf-α levels between all antioxidant groups and the control group. the tnf-α level for the zinc group was 164 ± 15 pg/ml, compared to 175 ± 13 pg/ml for the control group. table 3. zinc, protein and tnf-á levels after 6 months of supplementation, by treatment groups † p value by independent t-test; ‡ p value by chi-square test; * significance; § mean ± sd 108 the inability of mmn supplementation to improve the immune functions of our study subjects may have been due to the large number of zinc-deficient subjects. at baseline, 50% of the subjects in the mmn group had zinc d e f i c i e n c y, b u t a f t e r 6 m o n t h s o f m m n s u p p l e m e n t a t i o n t h e p r o p o r t i o n o f z i n c deficient subjects, although reduced, still amounted to 33.3%, in comparison with the control group, where it remained at its baseline level of 13.9%. many animal studies have shown that zinc deficiency decreases resistance to a wide range of pathogenic microorganisms, p r o b a b l y d u e t o i m p a i r m e n t o f i m m u n e r e s p o n s e s . ( 1 5 ) t h e z i n c d e f i c i e n c y i n o u r subjects was presumably due to changes in dietary preferences, income, and total energy intake, leading to decreased zinc intake, below the recommended dietary allowance (rda) of 12–15 mg/d in many older adults.(16) nutrition is an important determinant of the health of older persons, which is closely associated with increased morbidity of chronic a n d a c u t e d i s e a s e s in t h e s e p e r s o n s . malnutrition in older persons frequently r e m a i n s u n d e t e c t e d ; f o r d e t e c t i o n o f m a l n u t r i t i o n , s e v e r a l o f t h e f o l l o w i n g parameters need to be evaluated: loss of w e i g h t , a b n o r m a l b o d y m a s s i n d e x , m i c r o n u t r i e n t d e f i c i e n c i e s , a n d r e d u c e d nutrient intakes.(17) the types of undernutrition frequently found in older persons are protein and energy deficiencies. epidemiological data indicate that 5-10% of older persons have m a l n u t r i t i o n , a n d t h e p r e v a l e n c e o f m a l n u t r i t i o n i n c r e a s e s u p t o 2 6 % i n hospitalized patients with acute infections.(18) the changes in bioavailability also play a role in micronutrient deficiencies of older persons. around 24-40% of older persons suffer from a t r o p h i c g a s t r i t i s , l e a d i n g t o i m p a i r e d a b s o r p t i o n o f m i c r o n u t r i e n t s i n o l d e r persons.(19) micronutrients are required as cofactors and for modulating enzyme activity in metabolism.(20) micronutrient deficiency may affect metabolic pathways mediated by various enzymes and for enzyme synthesis adequate levels of protein are required. i n o u r s t u d y, 6 m o n t h s o f m m n supplementation had no effect on total protein, albumin, and globulin levels in the blood. this is likely due to the fact that baseline data did not reveal deficiency of total protein. the aging process results in a reduced immune response in older persons. however, there are other factors apart from the aging process itself that may influence the immune response in older persons. such factors as environmental factors, exposure to antigens, metabolic processes, and infections, may induce activation of immune c e l l s a n d r e l e a s e o f p r o i n f l a m m a t o r y cytokines.(15) the immune response is also affected by micronutrient deficiencies, which are frequently encountered in older persons. data on a population of home-dwelling older persons of age 75+ years indicate that around one-third to one-half of older persons have m i c r o n u t r i e n t d e f i c i e n c i e s . t h e s t u d y conducted by fabian et al.(21) reported low mean energy and micronutrient intakes in older persons, as compared with young adults. there w a s a l o w p r o p o r t i o n o f p r o t e i n i n t a k e compared with that of carbohydrates, while fat intake tended to be high. in addition, mean intakes of micronutrients, such as calcium, folic acid, vitamins a, b, c and d, and the minerals magnesium, iron, zinc, copper, and manganese, were also low.(22) it would be interesting to determine the potential of mmn s u p p l e m e n t a t i o n f o r i m p r o v i n g i m m u n e functions and reducing the incidence of i n f e c t i o n s i n o l d e r p e r s o n s . i n t h e u s , s u p p l e m e n t s a r e u s e d b y a b o u t 4 0 % o f individuals, including older persons. however, a l t h o u g h t h e p r e v a l e n c e o f s u p p l e m e n t consumption in older persons in the us is a p p a r e n t l y s u b s t a n t i a l , m i c r o n u t r i e n t deficiencies are still frequently found in older persons. the factors influencing nutritional disorders in older persons comprise economic, social, mental, environmental, physiological, and health-related factors.(23) herwana, yenny multimicronutrient in older persons 109 univ med vol.30 no.2 the present study did not succeed in demonstrating the expected reduction of tnfα l e v e l s a s a n o u t c o m e o f m m n s u p p l e m e n t a t i o n . t h e s e r e s u l t s m a y b e explained by the fact that tnf-α levels are influenced by many other factors in addition to micronutrients. other influencing factors of equal importance are socio-economic factors and p l a s m a z i n c l e v e l s . t h i s s t u d y w a s conducted on older persons with poor socioeconomic status, zinc deficiency, and low educational status, potentially decreasing the e x p e c t e d o u t c o m e s . f u r t h e r m o r e , o l d e r p e r s o n s w i t h o r w i t h o u t m i c r o n u t r i e n t deficiencies have reduced immune responses as a result of the aging process itself.(25,26) one of the limitations of our study was that it did not determine dietary intakes of the subjects, thus was unable to determine their nutritional status. mmn supplementation an sich was found to be inadequate for increasing immune function, and plasma protein and zinc levels. another limitation of this study was the duration of supplementation of less than 12 months, unlike that used by prasad et al.(8) conclusions mmn supplementation of zinc, ascorbic acid, β-carotene, α-tocopherol and folic acid for 6 months did not improve protein and tnf-α l e v e l s , b u t w a s c a p a b l e o f r e d u c i n g t h e proportion of older persons with baseline zinc deficiency. improvement of immune parameters in individuals with a generally good immune and protein status but poor in zinc levels may b e d i ff i c u l t t o o b t a i n w i t h n u t r i t i o n a l intervention. acknowledgement we express our gratitude to the medical faculty, trisakti university, for the funding of this study, and thank the respondents who were willing to participate in this study. references 1. mobbs c. the merck manual of geriatrics, section 2, chapter 2, biology of aging. available at: http://www.merck.com/pubs/mm_geriatrics/ sec2/ch 2.html. accessed july 23, 2007. 2. badan pengembangan pembangunan nasional, badan pusat statistik, united nations population fund. proyeksi penduduk indonesia 2000-2025. jakarta: pengembangan pembangunan nasional, badan pusat statistik, united nations population fund;2005. 3. hoffman c, rice d, sung hy. persons with chronic conditions: their prevalence and costs. jama 1996;276:1473-9. 4. yenny, herwana e. the prevalence of chronic disease and quality of life in elderly people. univ med 2006;25:164-71. 5. field cj, johnson ir, schley pd. nutrients and their role in host resistance to infection. j leukoc biol 2002;71:16-32. 6. berkow rl, wang d, larrick jw, dodson rw, howard th. enhancement of neutrophil superoxide production by preincubation with recombinant human tumor necrosis factor. j immunol 1987;139:3789-91. 7. maggini s, wintergerst es, beveridge s, hornig dh. selected vitamins and trace elements support immune function by strengthening epithelial barriers and cellular and humoral immune responses. br j nutr 2007;98 suppl 1:s29-35. 8. prasad as, beck fwj, bao b, fitzgerald jt, snell dc, steinberg jd, et al. zinc supplementation decreases incidence of infection in the elderly: effect of zinc on generation of cytokines and oxidative stress. am j clin nutr 2007;85:83744. 9. prasad as, bao b, beck fwj, kucuk o, sarkar fh. antioxidant effect of zinc in humans. free radic biol med 2004;37:1182–90. 10. yalcin ss, engur-karasimav d, alehan d, yurdakok k, ozkutlu s, coskun t. zinc supplementation and tnf-α levels in vaccinated cardiac patients. j trace elem med biol 2011; doi:10.1016/j.jtemb.2011.03.002. 11. lesourd b. nutritional factors and immunological ageing. proc nutr soc 2006;65:319-25. 12. world health organization. appropriate body mass index for asian populations and its implications for policy and intervention strategies. lancet 2004;363:157-63. 13. maret w, sandstead hh. zinc requirements and the risks and benefits of zinc supplementation. j trace elem med biol 2006;20:3–18. 110 14. albers r, antoine jm, bourdet-sicard r, calder pc, gleeson m, lesourd b, et al. markers to measure immunomodulation in human nutrition intervention studies. br j nutr 2005;94:452–81. 15. prasad as. effects of zinc deficiency on immune functions. j trace elem exp med 2000;13:1-20. 16. meydani sn, barnett jb, dallal gf, fine bc, jacques pf, leka ls, et al. serum zinc and pneumonia in nursing home elderly. am j clin nutr 2007;86:1167–73. 17. wells jl, dumbrel ac. nutrition and aging: assessment and treatment of compromised nutritional status in frail elderly patients. clin interv aging 2006;1:67-79. 18. mucci e, jackson shd. nutritional supplementation in community-dwelling elderly people. ann nutr metab 2008;52 suppl 1:s337. 19. russell rm. factors in aging that effect the bioavailability of nutrients. j nutr 2001;131suppl 4:s1359-61. 20. shenkin a. micronutrients in health and disease. postgrad med j 2006;82:559-67. 21. fabian e, elmadfa i. nutritional situation of elderly in the european union: data of the european nutrition and health report. ann nutr metab 2008;52 suppl 1:s57-61. 22. tucker kl, buranapin s. nutrition and aging in developing countries. j nutr 2001;131: s241723. 23. haase h, rink l. the immune system and the impact of zinc during aging. immunity ageing 2009;6:9-26. 24. tam m, gomez s, gonzalez-gross m, marcos a. possible roles of magnesium on the immune system. eur j clin nutr 2003;57:1193-7. 25. hamer dh, semportegui f, estrella b, tucker kl. micronutrient deficiencies are associated with impaired immune response and higher burden of respiratory infections in elderly equadorians. j nutr 2009;139:113-20. 26. marti l, cervera c, filella x, marin jl, almela m, moreno a. cytokine release patterns in elderly patients with systemic inflammatory response syndrome. gerontol 2007;53:239-44. herwana, yenny multimicronutrient in older persons alvina 8 abstract stenosis of the spinal canal can be caused by trauma, degenerative processes, and tumors, causing a neurological deficit. if the neurological deficit could be detected or diagnosed earlier, the late complications such as quadriparesis could be prevented. the torg ratio can be used to find evidence of cervical canal stenosis on lateral plain film, as it has the advantage of not being affected by magnification. the purpose of this study was to determine the torg ratio for normal subjects using lateral plain films of cervical vertebrae. this cross-sectional study was done at the department of radiology fkui/rsupn-cm jakarta, starting from september 16 – 20, 2008. the study included 98 subjects, aged 20 – 40 years, were the mean age of the subjects was 27.4 years (sd ± 5.4). all participants were subjected to measurement of the torg ratio by cervical lateral plain film. the mean torg ratio of normal subjects was 0.99 for males and 1.06 for females. the mean torg ratio of several ethnicities were 1.04 for javanese, 1.02 for sundanese, 1.01 for betawi, and 0.99 for other ethnicities. the mean torg ratio of our subjects is different from that of other people, such as pakistanis, singaporeans, and koreans. the mean torg ratios of ethnicities are not significantly different from one another. therefore, the torg ratio can be relied upon to predict narrowing of the cervical spinal canal in the sagittal plane. keywords: measurement, cervical lateral plain film, torg ratio *department of histology medical faculty trisakti university **department of radiology hosana medical hospital correspondence adr. david tjahjadi, msc. department of histology, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2701 email: davesaboch@yahoo.com univ med 2010;29:8-13 torg ratios based on cervical lateral plain films in normal subjects david tjahjadi*a and mz onibala** january-april, 2010january-april, 2010january-april, 2010january-april, 2010january-april, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina introduction the bodies of the cervical vertebrae are smaller in size compared with those of other vertebrae and function to protect the spinal cord, support the head, and allow movement o f t h e h e a d ( i n f l e x i o n , e x t e n s i o n , a n d rotation). the cervical vertebrae consist of 7 bones, two of which, i.e. cervical vertebrae 1 (c1) and 2 (c2), are different in form compared to the other five. the cervical canal, which is situated posteriorly to the vertebral body, has an almost cylindrical shape, and within it are the spinal cord running along c1-7, and the brachial plexus, which passes outwards at c56. stenosis, or narrowing, of the spinal canal has been previously associated with neurologic injury. the spinal canal-to-vertebral body ratio, 9 or torg ratio, is one method used to determine the presence of spinal stenosis.(1-3) cervical canal stenosis may cause a neurological deficit, with neck stiffness as the earliest symptom. if the deficit increases in severity, or treatment is delayed, there will be neck pain radiating to the shoulder and down the arm, corresponding to the distribution of the affected nerve root.(4) because of the possibility of a serious negative impact due to delayed treatment, this condition has to be detected as early as possible. if the potential for a neurological deficit can be detected in its early stages, risk management for quadriplegia may be avoided. torg et al.(5) have introduced a ratio called the torg ratio (spinal canal-vertebral body ratio), that is not affected by magnification, and is measured on lateral plain films of cervical vertebrae. this measure is a ratio determined by the distance from the center of the posterior aspect of the vertebral bodies c3c7 to the spinal laminar line (the diameter of the spinal canal) divided by the anteroposterior diameter of the vertebral body anterior to the canal. a ratio of > 1.0 signifies absence of stenosis of the spinal canal, but a ratio of < 0.8 indicates the presence of cervical spinal c a n a l s t e n o s i s . s u k e t a l . ( 6 ) f r o m k o r e a determined the torg ratio of the cervical vertebrae (c3-c7) of 90 normal korean adults (without any abnormality of the cervical vertebrae) and found mean ratios of 1.02 for females and 0.9 for males. a study of torg ratios conducted in pakistan in 100 males and females, yielded an almost identical result as in the korean study, with mean ratios of 1.08 for females and 0.95 for males.(7) both studies used a sample of dried human bone specimens f o r d i r e c t m e a s u r e m e n t o f s p i n a l c a n a l diameter and anteroposterior diameter of the vertebral body. lim et al.(8) state that the sagittal diameter of the cervical spinal canal and the size of v e r t e b r a l b o d y a r e d i ff e r e n t i n f e m a l e s compared with those in males. this study was conducted with a sample size of 80 males and females of various ethnic groups in singapore, using torg ratios measured on cervical lateral plain films, resulting in a mean ratio of 0.9 in males and 0.99 in females. the anthropometric structure of indonesians differs from that of americans, japanese, pakistanis, europeans, as well as koreans, necessitating determination of the normal torg ratios specifically for indonesians as indicator of the presence of spinal canal stenosis.(6-8) r a d i o l o g y h a s a s u b s t a n t i a l r o l e i n assessment of spinal canal stenosis, starting with plain films, and proceeding to the more sophisticated radiological modalities. the plain film has a strong potential for playing a role in assessment of spinal canal stenosis. a survey in the united states reports that the vertebral plain film has become a routine procedure (96%) in patients with severe blunt trauma and the standard for investigation of acute trauma of the vertebrae. the cross-table lateral plain film has a specificity of up to 97%, whilst its sensitivity is up to 96% in acute fracture due to cervical trauma, when compared with the ct scan.(9,10) the cross-table lateral plain film may be used for indicating spinal canal stenosis in traumatic as well as nontraumatic cases, but direct measurement of the spinal canal (based on the distance from the midpoint/center of the posterior aspect of the vertebral body to the spinal laminar line) is considerably affected by the magnification factor, due to differences in focus-to-film distance. it was reported that a difference in focus-to-film distance between 1 and 1.5 m will change the size of the spinal canal by 0.5 cm, thus in this case the torg ratio is a better measure.(11) the plain film is a standard radiological modality present in all hospitals in indonesia and the cost is relatively low. in trauma patients in whom assessment of the spinal canal is necessary for rapid detection of cervical canal stenosis, the torg ratio is used. in developing countries such as indonesia not all hospitals h a v e r a d i o l o g i c a l m o d a l i t i e s s u c h a s computerized tomography and mri. univ med vol.29 no.1 10 to date there are no definite torg ratios for indonesians for determining the presence o f s p i n a l c a n a l s t e n o s i s r e s u l t i n g i n neurological deficits. based on this fact, the investigators conducted the present study with the objective of ascertaining the torg ratio in normal subjects, by studying the cervical lateral plain film. methods research design t h e r e s e a r c h w a s d e s i g n e d a s a n observational cross-sectional study. subjects volunteer subjects were recruited from t h e d e p a r t m e n t o f r a d i o l o g y, c i p t o m a n g u n k u s u m o h o s p i t a l , j a k a r t a f r o m september 16 – 20, 2008. the inclusion criteria were: (i) individuals with inactive epiphyseal growth centers and without neurological manifestations, such as neck pain or paresthesia associated with movement of the neck; loss of sensibility corresponding to a dermatome; muscular weakness and atrophy; (ii) and on cervical lateral plain film: normal position of the cervical vertebrae, without evidence of listhesis; no evidence of osteophytes on the vertebral bodies (or only minimal osteophytic growth); no evidence of lytic or blastic lesions or fractures; no evidence of intervertebral disc compression; and intact pedicles. subjects who reported a history of cervical spine injury were excluded from the study. data collection d a t a c o l l e c t i o n i n t h i s s t u d y w a s performed at the department of radiology, cipto mangunkusumo hospital, jakarta. the vertebrae assessed consisted of the cervical vertebrae 3 to 7 (c3-c7). c1 and c2 were not included because of their different shape compared with the other cervical vertebrae, and also because cases of cervical stenosis are more common between c4 – c6.(7,8) the measurements of the cervical spinal canals and vertebral bodies were taken as described above and the torg ratio for each cervical vertebra was calculated by dividing t h e d i a m e t e r o f t h e s p i n a l c a n a l b y t h e anteroposterior diameter of the corresponding vertebral body.(6) for consistency of results, the measurements were performed by a single i n v e s t i g a t o r w i t h t h e s a m e m e a s u r i n g instrument. statistical analysis independent student’s t-test and analysis of variance (anova) were conducted to determine if significant differences existed among the selected variables. the statistical package for the social sciences (version 15.0, spss inc, chicago, il) was used for all statistical analyses. all statistical analyses were conducted in the null form, and the alpha level of 0.05 was determined a priori as statistically significant. results a total of 98 volunteers were willing to participate in this study and among these subjects there were more males than females, namely 58 males (59.2%) and 40 females (40.8%). the youngest subject was 20 years old and the oldest 39 years, while the mean age of the subjects was 27.4 ± 5.4 years. the torg ratio the results of the measurement of the midsagittal diameter of the spinal canal and the anteroposterior diameter of the cervical vertebral bodies c3 to c7 in males and females, and the calculated torg ratio may be seen in table 1. in males the mean torg ratio was 0.99 and in females 1.06. to find a difference in torg ratios between males and females, the investigators performed the t-test, and found that the mean torg ratio was not significantly different between males and females (p = 0.577). tjahjadi, onibala torg rations in normal subjects 11 gender canal body torg ratio p male (n=58) 1.88 1.89 0.99 0.577 female (n=40) 0.99 1.82 1.06 table 1. mean values of cervical variables according to gender c a l c u l a t i o n o f m e a n to r g r a t i o s associated with occupation was also performed for cervical vertebrae c3 to c7, the results of which can be seen in table 2. in physicians and students the mean torg ratio was 1.05, in workers and employees 1.04, in drivers / security / cleaning service personnel 1.01, and in housewifes 1.04. differences in torg ratios between occupations were tested by anova. from the anova results it was found that the m e a n torg r a t i o s w e r e n o t s i g n i f i c a n t l y different between occupations (p = 0.662). the difference of mean torg ratio by ethnicity may be seen in table 3. in javanese the mean torg rato was 1.04, in sundanese 1.02, in betawi 1.01, and in other ethnicities 0.99. to find a difference in mean torg ratios b e t w e e n e t h n i c g r o u p s , t h e i n v e s t i g a t o r performed anova and found that the mean torg ratio was not significantly different between ethnic groups (p=0.421). the differences of mean torg ratio by age group may be seen in table 4, where in the age group below 25 years the mean torg ratio was 1.03, in the age group 25-30 years 1.05, in the age group 31-34 years 1.03, and in the age group over 35 years 0.96. anova testing yielded a significant difference in torg ratios between age groups (p<0.05). the mean torg ratio was greatest in the age group over 35 years. occupation canal body torg ratio p physicians+ 1.89 1.80 1.05 0.662 employees+ 1.84 1.79 1.04 drivers+ 1.88 1.87 1.01 housewifes 1.77 1.73 1.04 table 2. mean values of all cervical variables according to occupation ethnicity canal body torg ratio p javanese 1.88 1.80 1.04 0.421 sundanese 1.81 1.77 1.02 betawi 1.87 1.86 1.01 other ethnicities 1.85 1.86 0.99 table 3. mean values of all cervical variables according to ethnicity age group canal body torg ratio p < 25 1.86 1.82 1.03 0.038 25 – 30 1.89 1.81 1.05 31 – 34 1.90 1.80 1.07 35+ 1.74 1.84 0.96 table 4. mean values of all cervical variables according to age group univ med vol.29 no.1 12 discussion cervical canal stenosis is a disorder that may cause neurological deficits, commonly r e s u l t i n g f r o m c e r v i c a l t r a u m a i n hyperextension or hyperflexion as well as in axial loading. the signs and symptoms of neurological deficits may be in the form of sensory or motor impairment, which if not detected promptly will cause more severe neurological deficits.(10-12) a number of investigators have conducted various studies to detect the presence of cervical stenosis by simple measurement of the cervical canal diameter using cervical lateral plain film.(7) however, the existence of these various reports giving different cervical canal diameters resulted in several different normal ranges. (7,9) in contrast, the spinal canal to vertebral body ratio introduced by torg et al.(5) is not affected by magnification, and is measured on lateral plain films of cervical vertebrae. a ratio of > 1.0 signifies absence of stenosis of the spinal canal, but a ratio of < 0.8 indicates the presence of cervical spinal canal stenosis. a study on 14 male subjects aged 24.4 ± 2.5 years found a torg ratio of less than 0.80 in at least one vertebral level in 93% of subjects. these differences can be attributed to the fact that the latter study used mri in determining measurements.(1) the torg ratio for diagnosis of stenosis of the cervical canal has been used by many researchers(12,13) with the objective of providing reference values for use by clinicians in diagnosis of cervical canal stenosis. t h e v a r i o u s m e t h o d s u s e d b y t h e s e investigators for assessing the torg ratio were lateral plain film,(4) ct scan and mri in living subjects as well as cadavers,(7,14) and direct measurement on dried bony specimens.(7) to date no studies had been conducted on the torg ratio in indonesia. in our study we measured the torg ratio in normal subjects with a total of 98 individuals, consisting of 58 males and 40 females, using cervical lateral plain film. in our study the mean torg ratios for m a l e s a n d f e m a l e s w e r e 0 . 9 9 a n d 1 . 0 6 , respectively. the results indicate the existence of differing values, the torg ratio in indonesian males being larger than that of pakistani males (0.95), whereas indonesian females have a l o w e r to rg r a t i o c o m p a r e d w i t h t h a t i n pakistani females (1.08).(8) the mean torg ratio of indonesians is also larger compared with that of korean males (0.93) and females (1.02)(4) and that of singaporean males and females (0.90 and 0.99 respectively).(8) because our subjects, particularly those living in jakarta, come from various ethnic groups, we also did a comparison of the mean torg ratio on the basis of ethnicity. the mean torg ratios of various ethnicities are listed in table 3. after performing anova to find a difference in mean torg ratio between ethnic g r o u p s , i t w a s f o u n d t h a t t h e r e w a s n o significant difference between these groups. t h i s i n d i c a t e s t h a t t h e e t h n i c g r o u p s p a r t i c i p a t i n g i n t h i s s t u d y r e p r e s e n t a homogeneous population. using anova, the mean torg ratios based on age group showed a significant difference. the variable apparently causing the difference was the mean diameter of the cervical canal in the age group of 35 years, which turned out to be narrower in comparison with the other age groups. this may be due to the smaller size of the sample in the 35 year age group and its poor homogeneity. in this study the investigators did not use the tukey nor shaeffer tests for finding the cause of the difference, because of the purely descriptive nature of this study. one limitation of our study is that it included a small number of normal subjects. we a n a l y z e d 9 8 n o r m a l s u b j e c t s , s o w e acknowledge that it is difficult to generalize the results to the entire indonesian population. another limitation of this study is its clinical r e l e v a n c e b e c a u s e r a d i o g r a p h s a r e l e s s sensitive as a screening mechanism than mri. tjahjadi, onibala torg rations in normal subjects 13 conclusions in normal subjects there was no difference in torg ratio between genders. however, the torg ratio was significantly different between younger and older age groups, where subjects in older age groups had a lower torg ratio. therefore, the torg ratio can be relied upon to predict narrowing of the cervical spinal canal in the sagittal plane. references 1. tierney rt, maldjian c, mattacola cg, starub ss, sitler mr. cervical spine stenosis measures in normal subjects. j athl train 2002;37:190-3. 2. ilyas m. dislokasi interfasetal bilateral. [bilateral interfacetal dislocation]. j med nus 2005;24: 127-34. 3. baehr m, frotscher m. topical diagnosis in neurology 4th ed. thieme: stuttgart; 2005. 4. pavlov h, torg js, robie b, jahre c. cervical spinal stenosis: determination with vertebral body ratio method. radiology 1987;164:771–5. cited by tierney rt, maldjian c, mattacola cg, straub sj, sitler mr. cervical spine stenosis measures in normal subjects. j athl train 2002;37:190–3. 5. torg js, corcoran ta, thibault le, pavlov h, sennett bj, naranja rt jr, et al. ccervical cord neurapraxia: classification, pathomechanics, morbidity, and management guidelines. j neurosurg 1997;87:843-50. 6. suk ks, kim kt, lee jh, lee sh, kim j, kim jy. reevaluation of the pavlov ratio in patients with cervical myelopathy. clin orthopaed surg 2009;1:6-10. 7. maqbool a, athar z, hussain l. midsagittal diameter of cervical spine and torg’s ratio of the cervical spine in pakistanis. pak j med sci 2003; 19:203-10. 8. lim jk, wong hk. variation of the cervical spinal torg ratio with gender and ethnicity. spine 2004;4:396–401. 9. nadalo la. spinal stenosis. available at: http:// www.emedecine.com. accessed september 2, 2009. 10. bailes ge, petschauer m, guskiewicz km, marano g. management of cervical spine injutries in athletes. j athl train 2007;42:126-34. 11. rao rd, currier bl, albert tj, bono cm, marawar sv, poelstra ka, et al. degenerative cervical spondylosis: clinical syndromes, pathogenesis, and management. j bone joint surg am 2007;89:1360-78. 12. prasad ss, o’malley m, caplan m, shackleford im, pydisetty rk. mri measurements of the cervical spine and their correlation to pavlov’s atio. spine 2003;28:1263-8. 13. kelly jd, aliquo d, sitler mr, odgers c, moyer ra. association of burners with cervical canal and foraminal stenosis. am j sports med 2000; 28:214-7. 14. koyanagi i, iwasaki y, hida k, akino m, imamura h, abe h. acute cervical cord injury without fracture or dislocation of the spinal column. j neurosurg 2000;93:15-20. univ med vol.29 no.1 harsono 18 seizure threshold, hormones and anti-epileptic drugs department of neurology medical faculty, gadjah mada university / dr. sardjito hospital yogyakarta correspondence prof. dr. harsono, sp.s(k) department of neurology medical faculty, gadjah mada university bulak sumur 55281 email : harsono_jombor@yahoo.com universa medicina 2008; 27: 18-28. january-march, 2008january-march, 2008january-march, 2008january-march, 2008january-march, 2008 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 abstract harsono the concept of seizure threshold holds that everyone has a certain balance between excitatory and inhibitory forces in the brain. a low seizure threshold makes it easier for epilepsy to develop and easier for someone to elicit single seizure. the opposing effects of estrogen (proconvulsant) and progesterone (anticonvulsant) on seizure threshold have been noted in animal and human studies. estrogen has been shown to lower the seizure threshold. in contrast to estrogen, several studies have confirmed the anticonvulsive effects of progesterone and its metabolite. antiepileptic drugs (aeds) are used to prevent or interrupt seizures. limitation of sustained repetitive neuronal firing via blockade of voltage-dependent sodium channels, enhancement of gaba-mediated inhibition, and blockade of glutamatergic excitatory neurotransmission are the mechanisms of anti-epileptic drugs in preventing seizures. aeds that induce hepatic cytochrome (cy) p450 enzymes can increase the metabolism of sex hormones and make hormonal contraception ineffective. in addition, aeds may even increase seizure frequency or severity or change the seizure type. keywords: seizure threshold, hormones, anti-epileptic drugs, proconvulsant, anticonvulsant universa medicina introduction the international league against epilepsy ( i l a e ) a n d t h e i n t e r n a t i o n a l b u r e a u f o r e p i l e p s y ( i b e ) h a v e c o m e t o c o n s e n s u s definitions for the terms epileptic seizure and epilepsy. an epileptic seizure is a transient o c c u r r e n c e o f s i g n s / o r s y m p t o m s d u e t o abnormal excessive or synchronous neuronal activity in the brain. epilepsy is a disorder of t h e b r a i n c h a r a c t e r i z e d b y a n e n d u r i n g predisposition to generate epileptic seizures and by the neurobiologic, cognitive, psychological, and social consequences of this condition.(1) physiologically, epilepsy is defined as a disorder in which the balance between cerebral excitability and inhibition is tipped toward uncontrolled excitability. there is now clear evidence that there are distinct differences between the immature and mature brain in the pathophysiology and consequences of seizures. both the enhanced excitability of the immature brain compared with mature brain and the unique pathologic consequences of seizures are related 19 universa medicina vol.27 no.1 to the sequential development and expression of essential signaling pathways. although the immature brain is less vulnerable than the mature brain to seizure-induced cell death, seizures in the developing brain can result in irreversible alterations in neuronal connectivity. developing n o v e l s t r a t e g i e s t o t r e a t a n d a v e r t t h e consequences of seizures in children will require further understanding of the unique mechanisms of seizure initiation and propagation in the immature brain.(2) epilepsy is no more prevalent in women than in men but there are experimental data from animals, and observational studies in humans, which show that seizures can be influenced by “female” hormones.(3) seizures patterns are often related to the reproductive cycles of a woman with epilepsy. seizures are influenced by changes in hormone levels, which occur during t h e m e n s t r u a l c y c l e a n d t h r o u g h o u t t h e reproductive life of women with epilepsy. establishing on early accurate diagnosis and initiating appropriate medical treatment may decrease seizure recurrence, reduce the number of anti-epileptic drug (aed) trial, and minimize the impact of seizures on the patient’s quality of life.(4) the importance of the following description is to disclose the relationship between seizure threshold, hormones and anti-epileptic drugs. clinical practice in epilepsy, especially the principles of treatment, should consider the r e l a t i o n s h i p . t h e u n d e r s t a n d i n g i n s u c h relationship will encourage physicians to be more rational in prescribing anti-epileptic drugs. seizure threshold the concept of seizure threshold holds that e v e r y o n e h a s a c e r t a i n b a l a n c e ( p r o b a b l y genetically determined) between excitatory and inhibitory forces in the brain. the relative proportions of each determine whether a person has a low threshold of seizures (because of the higher excitatory balance) or a high threshold (because of a greater inhibition). regarding this view, a low seizure threshold makes it easier for epilepsy to develop and easier for someone to elicit single seizure. threshold means “the place or point of beginning”, “the outset”, “the lowest point at which a stimulus begins to produce a sensation”, or “the minimal stimulus that produces excitation of any structure, eliciting a motor response”. meanwhile, as originally describe by j. hughlings jackson in 1870, a seizure is an “excessive discharge of nerve tissue on muscle”. jackson went on to say that “this discharge occurs in all degrees, it occurs with all sorts of conditions of ill-health, at all ages, and under innumerable circumstances”.(5) these comments by jackson are as true now as they were 130 years ago. epileptic seizures are one of the most common, and frightening, neurologic conditions that occur in children.(2) normally, an action potential occurs in neuron 1 when the membrane potential is depolarized to its threshold level. discharges in neuron 1 also may influence the activity of its neighbor, neuron 2. for example, a delay of several milliseconds from an action potential in n e u r o n 1 m a y g i v e r i s e t o a n e x c i t a t o r y postsynaptic potential (epsp) in neuron 2. if cell 3, an inhibitory interneuron, also is activated by a discharge from neuron 1, then the activity in neuron 2 will be modified by an inhibitory postsynaptic potential (ipsp) that overlaps in time with the epsp. the recorded event will be summed epsp-ipsp sequence. if the ipsp occurs earlier, perhaps coincident with the epsp, the depolarizing effect of the epsp will be d i m i n i s h e d . i n t h i s w a y, w e c a n e n v i s i o n inhibition as “sculpting” or modifying ongoing excitation. if this concept is extrapolated to thousands of interconnected neurons, each influencing the activity of many neighbors, it is easy to see how an increase in excitation or 20 decrease in inhibition in the system could lead to hyper synchronous epileptic firing in a large area of brain. normally, neurons fire single a c t i o n p o t e n t i a l s a l o n e o r i n r u n s , a n d excitability is kept in check by the presence of powerful inhibitory influences.(6) the term seizure refers to a transient a l t e r a t i o n o f b e h a v i o r d u e t o a b n o r m a l synchronized and repetitive bursts of firing of neurons in the central nervous system. different definitions emphasize different features of an e p i l e p t i c s e i z u r e : n a t u r e o f o n s e t a n d t e r m i n a t i o n , c l i n i c a l m a n i f e s t a t i o n s , a n d enhanced neuronal synchrony. some previous definitions also considered issues of etiology, classification, and diagnosis, although none of these strictly fits within the task of a definition.(1) seizure threshold is changed by thyroid hormones in experimental animal and human. seizures occur in patients with graves disease or with excess administration of thyroxin for h y p o t h y r o i d i s m . t h y r o i d h o r m o n e s h a v e profound effects on several aspects of early brain development and seizure threshold. increased level of myelin and thyroid hormones could increase the excitability of the central nervous system (cns) by lowering the threshold for various types of stimuli.(7) more than 50% of all partial epilepsy originates from foci in temporal lobe structures. the high incidence of temporal lobe foci may occur because of the low seizure threshold found in many temporal lobe structures, especially in the limbic structures of the mesial temporal lobe.(8) hormones general view male and female sexuality and reproductive functions are complex systems with cortical, limbic system, hypothalamic, pituitary, and end organ interactions. sexual steroids are produced in the sexual glands, the adrenals, and the brain. they undergo interconversion in the brain, bind to different brain areas, and have multiple effects behaviorally and neurologically. progesterone, estrogen and testosterone have neuroendocrine effects that alter epileptogenicity. seizure frequency may change throughout the life cycle as a result of hormonal status. changes in the central control, peripheral hormone levels, and or medication effects may all contribute to decreased libido, potency, and fertility. aeds interact with hormone-binding metabolism, resulting in altered human reproductive function. aeds alter contraceptive hormone treatment.(9) hormone such as estradiol, progestin, and androgen that are secreted by the ovaries can have a profound effect on seizure vulnerability. estradiol has long been known to decrease seizure threshold. although a preponderance of clinical and experimental data suggests that estradiol has proconvulsant effects, antiseizure effects of estrogen have also been reported. indeed, the precise impact of estradiol (and other steroid hormones) on seizure sensitivity may relate to both proximate levels and changes in those levels. the effects of progestin and androgens on seizure appear clear, with both g e n e t i c a l l y i n c r e a s i n g s e i z u r e t h r e s h o l d s . nonetheless, some reports suggest that progestin or androgen either lack antiseizure effects or that they can have proconvulsant effects.(10) wo m e n w i t h e p i l e p s y f a c e a d d i t i o n a l c h a l l e n g e s . s o m e a e d s r e d u c e l e v e l s o f physiologic ovarian sex steroid hormones and m a y r e d u c e t h e e ff i c a c y o f c o n t r a c e p t i v e steroids. women with epilepsy have a greater risk for syndromes associated with infertility, such as hypothalamic-pituitary axis disruption, polycystic ovary-like syndrome, and anovulatory cycles.(11) the menstrual cycle is fundamentally a n e u r o l o g i c a l e v e n t . n o r m a l r e p r o d u c t i v e f u n c t i o n r e q u i r e s a n i n t e r a c t i o n b e t w e e n harsono seizure threshold and anti-epileptic 21 universa medicina vol.27 no.1 hypothalamic, pituitary and ovarian hormones. gonadotropin releasing hormone (gnrh) is secreted in a pulsatile fashion by neurons in the a r c u a t e n u c l e u s o f t h e h y p o t h a l a m u s . i t s secretion is modified by catecholamines e.g., d o p a m i n e w h i c h a c t s a s a n i n h i b i t o r a n d norepinephrine which is a stimulator. the gnrh travels to the gonadotropic cells of the anterior pituitary through the portal system where it binds to receptors stimulating the production of f o l l i c l e s t i m u l a t i n g h o r m o n e ( f s h ) , a n d luteinizing hormone (lh). in children secretion of gnrh is extremely low, increasing slowly over the 3-4 years prior to the development of menses.(12) ovarian steroid hormones alter excitability of neurons of the central nervous system. estrogen reduces inhibition at the gamma aminobutyric acid (gaba a ) receptor, enhances e x c i t a t i o n a t t h e g l u t a m a t e r e c e p t o r, a n d increases the number of excitatory neuronal s y n a p s e s . p r o g e s t e r o n e n h a n c e g a b a mediated inhibition, increases gaba synthesis, and increases the number of gaba a receptors. in animal models of epilepsy, estrogen increases and progesterone decreases the likelihood that a seizure will occur.(13) e s t r o g e n a n d p r o g e s t e r o n e a ff e c t t h e c e n t r a l n e r v o u s s y s t e m – p r i m a r i l y, t h e regulation of endocrine functions and sexual behavior. experiments in animals have shown that the ovarian sex steroid hormones also modulate the seizure threshold.(14) the opposing e f f e c t s o f e s t r o g e n ( p r o c o n v u l s a n t ) a n d p r o g e s t e r o n e ( a n t i c o n v u l s a n t ) o n s e i z u r e threshold have been noted in animal and human studies. levels of these hormones fluctuate throughout the menstrual cycle, and in some women with epilepsy, these fluctuations may be related to the occurrence of seizures around the time of menses or an increase in seizures in relation to the menstrual cycle, also known as catamenial epilepsy.(15) across the menstrual cycle, estradiol is elevated in the second half of the follicular phase and increases to a peak at midcycle, while progesterone is primarily elevated during the luteal phase and declines before menstruation b e g i n s . t h e c o n t r a s t i n g e f f e c t s o f t h e s e hormones in activating or depressing central nervous system (cns) function, respectively, may have implications for behavior or perhaps even epilepsy across the cycle.(15,16) estrogen estrogen has been shown to lower the seizure threshold in laboratory animals by altering calcium influx at the cell membrane, reducing chloride influx at the gaba a receptor, and acting as an agonist at glutamate receptors in regions of the hippocampus.(12) there are three biologically active forms of estrogen i.e., (i) 17b-estradiol, dominant in pre-menopausal women, (ii) estriol, the major form of estrogen during pregnancy, and (iii) estrone, which is prevalent after menopause. estradiol has been shown in many studies to have significant proconvulsant effect. it facilitates various forms of induced seizures and has been shown to worsen seizures in women with epilepsy. on a cellular level, estradiol, aside from its normal reproductive effects, enhances neural excitation a n d s u p p r e s s e s i n h i b i t i o n . i t a l s o c r e a t e s changes in the physical properties of some neurons (increase in excitatory dendritic spine d e n s i t y i n t h e h i p p o c a m p u s ) , r e s u l t i n g i n increased potential for seizures. it has been observed that there is a relationship between the ratio of estrogen to progesterone and the level of seizure occurrence. an increase in this ratio during certain periods in the menstrual cycle could create the increase in seizure s u s c e p t i b i l i t y o b s e r v e d i n c a t a m e n i a l epilepsy.(17) epilepsy, especially temporal lobe epilepsy (tle), adversely affects testicular endocrine 22 function. the mechanism by which a brain d i s o r d e r c a u s e s a t e s t i c u l a r d i s o r d e r i s inexplicable. one mechanism by which this may o c c u r i s t h a t s u b t l e d e r a n g e m e n t s i n hypothalamic in pituitary function, caused by nearby epileptic discharges, produces elevation in circulating hormones, such as estrogen or prolactin. estrogen can induce sex-hormonebinding globulin production in the liver, and thus, reduce free fraction of testosterone. on the other hand, carbamazepine may increase the negative effects of epilepsy on serum level of reproductive hormones.(18) progesterone in contrast to estrogen, several studies have c o n f i r m e d t h e a n t i c o n v u l s i v e e f f e c t o f progesterone and its metabolite. progesterone enhances chloride influx at the gaba a receptor and reduces glutamate-mediated excitation, thereby increasing the seizure threshold. it also enhances the synthesis of gaba and of the gaba a receptor subunit. the clinical sequelae of these findings are seen most often during the menstrual cycle.(14) progesterone possesses anticonvulsive properties. the level of this hormone drops near the end of the menstrual cycle, leaving women more vulnerable to catamenial epilepsy. recent studies have shown how progesterone protects women against seizures. progesterone plays two roles in the brain. first, it binds to progesterone receptors in the brain, which help regulate the reproductive functions. second, progesterone gets metabolized to allopregnanolone in the brain called a neurosteroid. allopregnanolone plays a c r u c i a l r o l e i n s e i z u r e p r o t e c t i o n . t h e withdrawal from this neurosteroid, which occurs during the menstrual cycle, could provoke s e i z u r e s . c o n s e q u e n t l y, n e u r o s t e r o i d r e p l a c e m e n t c o u l d b e a n o v e l t h e r a p e u t i c approach for catamenial epilepsy.(17) the oral progestogen-only contraceptive pill should not be used in women taking enzymeinducing aeds because effective levels of progestogen cannot be guaranteed. however, the progestogen depot injection medroxyprogesterone acetate (depo-provera) does not undergo first p a s s m e t a b o l i s m a n d i s a v e r y u s e f u l contraceptive in these circumstances. there has been disagreement about the frequency of administration but most guidance now suggests shortening the usual 12 weeks to 10 weeks in women on enzyme-inducing anti-epileptic medication. long duration implants of the progestogen etonogestrel or levonorgestrel are not thought to be reliable in conjunction with enzymeinducing drugs on the basis of case reports of contraceptive failure. intrauterine devices with progestogen levonorgestrel are effective because their action is primarily via a direct effect on the endometrium.(3) water balance and hormones in 1931 mcquarrie & peeler reported their observations on water balance in epileptic patients.(19) early observations of an association between cerebral edema and convulsions led to a series of experiments in the early 20th century investigating the effects of water ingestion on seizures. excessive water ingestion and the antidiuretic hormone vasopressin provoked seizures in patients with epilepsy, while negative water balance produced by fluid restriction had the opposite effect. these findings suggested that neuronal cell membrane permeability was defective in epilepsy and that water imbalance may underlie catamenial epilepsy. however, the study by ansel & clarke revealed no significant difference in body weight, sodium metabolism, or total body water was found between women with perimenstrual seizures and healthy controls or between epileptic women with and without catamenial tendencies.(20) harsono seizure threshold and anti-epileptic 23 universa medicina vol.27 no.1 hormonal therapy oral contraceptives isolated cases of improved seizure control have been reported in women taking oral c o n t r a c e p t i v e s . i n t h e o n l y d o u b l e b l i n d , p l a c e b o c o n t r o l s t u d y, t h e o r a l s y n t h e t i c progestin norethisterone was ineffective in nine women with perimenstrual seizures.(15) medroxyprogesterone acetate (mpa) mpa has been shown to reduce seizures in small numbers of women with epilepsy. some women experience an increase in seizures during the interval between discontinuation of mpa and resumption of regular ovulatory cycles. this may be related to unopposed estrogen exposure during anovulatory cycles.(15) natural progesterone in contrast to oral synthetic progestin, which has been shown to be ineffective, herzog has found natural progesterone to be effective in women with focal epilepsy and catamenial tendencies. average monthly seizure frequency declined by 54% to 68% during the 3-month treatment periods and by 62% to 74% after 3 years. adverse effects, including transient fatigue and depression, resolved within 48 hours of dose reduction.(15,21) neurosteroids n e u r o s t e r o i d s a r e s t e r o i d s t h a t a r e synthesized locally in the brain and have a strong a n d r a p i d e f f e c t o n n e u r a l e x c i t a b i l i t y. ganaxolone, 3alpha-hydroxy-3beta-methyl5alpha-pregnan-20-one, is a neuroactive steroid, or neurosteroid, that modulates the gaba a receptor complex. it is a synthetic analogue of allopregnanolone, a progesterone metabolite, w h i c h h a s b e e n s h o w n t o p o s s e s s b r o a d spectrum anticonvulsant properties. recently it has been discovered that allopregnanolone is a positive modulator of gaba a receptors. gaba is the primary inhibitory neurotransmitter in the brain. allopregnanolone has specific, distinct binding sites on gaba a receptors that are separate from those for gaba, benzodiazepines, and barbiturates. at normal physiological levels, it is sufficient to activate these receptors. this s u g g e s t s t h a t a n a b r u p t “ w i t h d r a w a l ” o f allopregnanolone at the onset of menstruation could decrease the inhibitory effect and possibly exacerbate seizures.(22-24) thyrotropine-releasing hormone thyrotropine-releasing hormone (trh) has been successfully used for treating children with neurologic disorders including epilepsy. the effectiveness of trh and a trh analog has been reported in west syndrome, lennox-gastaut s y n d r o m e , a n d e a r l y i n f a n t i l e e p i l e p t i c e n c e p h a l o p a t h y t h a t w e r e i n t r a c t a b l e t o a n t i c o n v u l s a n t s a n d a d r e n o c o r t i c o t r o p i c hormone (acth). however, the peptide has not been widely studied as a treatment of intractable epilepsy outside japan. trh is safe for children and effective in some cases of west syndrome a n d l e n n o x g a s t a u t s y n d r o m e . t r h i s considered as a possible new strategy fro treating west syndrome and lennox-gastaut syndrome prior to acth therapy, especially for the patient with an infection, immunosuppression, or severe organic lesions in the brain. the mechanism of anti-epileptic action may differ from those of o t h e r a n t i e p i l e p t i c d r u g s ( a e d s ) . o n e possibility is that trh may act as an antiepileptic through a kynurenine mechanism, considering that kynurenic acid acts as an antagonist on the n-methyl-d-aspartate receptor complex. the adverse affects of trh therapy are transient urinary retention, irritability, sleepiness, worsening of involuntary movements, tremor, tachycardia or bradycardia, appetite loss, nausea, and vomiting. however, the adverse effects are infrequent and minimal.(25) 24 other hormonal agents the anti-estrogen clomiphene citrate, the synthetic androgen danazol, and the synthetic gonadotropin agonists triptorelin and goserelin have been effective in reducing seizure in small series. however, the use of these agents is limited because of the potential for significant adverse effects, and consultation with a reproductive endocrinologist or gynecologist is suggested before their use.(15) anti-epileptic drugs aeds are used to prevent or interrupt seizures. they act via three mechanisms i.e., (i) limitation of sustained repetitive neuronal firing via blockade of voltage-dependent sodium channels, (ii) enhancement of gaba-mediated inhibition, and (iii) blockade of glutamatergic excitatory neurotransmission.(26) several aeds t a r g e t a s p e c t s o f t h e i n h i b i t o r y s y s t e m . phenobarbital and benzodiazepines bind to different sites on the gaba a receptor. these drugs enhance inhibition by allowing increased chloride influx through the gaba receptor; phenobarbital by increasing the duration of chloride channel openings and benzodiazepine increasing the frequency of openings. vigabatrin is an example of a “designer drug” that was created to target a specific pathophysiologic mechanism. vigabatrin inhibits the gaba degradatory enzyme, gaba transaminase, t h e r e b y i n c r e a s i n g t h e a m o u n t o f g a b a a v a i l a b l e t o p a r t a k e i n i n h i b i t o r y neurotransmission.(6) other aeds affect aspects of neuronal excitation. phenytoin, carbamazepine, and lamotrigine block voltage-dependent sodium channels and reduce the ability of neurons to fire repetitively. ethosuximide, used primarily for absence seizures, blocks a unique calcium current that is present only in thalamic neurons, preventing them from firing in an oscillatory fashion and recruiting neocortical neurons into spike-wave patterns. several new aeds are said to alter the function of n-methyl-d-aspartate (nmda) receptors (lamotrgine and felbamate) or non nmda receptors (topiramate).(6) effects of aeds on male sexual hormones and function medications might have a direct effect on gonadal function. for example, spermatogenesis sensitive to a variety of factors (temperature, diet, alcohol, stress) including drugs. aeds such as carbamazepine and phenytoin have been shown to directly inhibit testosterone production by the leydig cells in vitro. these studies demonstrated a differential effect by each of the aeds on the metabolic pathway of sex steroid hormones.(27) herzog et al(28) reported their study on differential effects of aed on sexual function and hormones in men with epilepsy. they compared sexual function and reproductive hormone levels among 85 men with localizationrelated epilepsy who took carbamazepine (25 patients), phenytoin (25 patients), lamotrigine (25 patients), 10 untreated patients for at least 6 months (no aed) and 25 controls. sexual function scores (s-scores), hormone levels (bioactive testosterone, estradiol), hormone ratios (bioactive testosterone/bioactive estradiol), and gonadal efficiency (bioactive testosterone/ luteinizing hormone) were compared among the five groups. the conclusions were that sexual function, bioavailable testosterone levels, and gonadal efficiency in men with epilepsy who took lamotrigine were comparable to control and untreated values and significantly greater than with carbamazepine or phenytoin treatment.(28) c a r b a m a z e p i n e , o x c a r b a z e p i n e , a n d v a l p r o a t e a r e a s s o c i a t e d w i t h s p e r m abnormalities in men with epilepsy. in addition, valproate-treated men with generalized epilepsy who have abnormal sperm may have reduced harsono seizure threshold and anti-epileptic 25 universa medicina vol.27 no.1 testicular volume. however, no specific aed can be solely implicated as causing reproductive abnormalities; the common features of the subjects was that they had epilepsy, and the a e d s p r o d u c e d f u r t h e r v a r i a t i o n s ( o r correction) in parameter. in addition, there is no specific reason readily used to advise individual male patients about the risk of impotence or infertility.(29) herzog et al(28) found total and non-sex hormone-binding globulin (shbg)-bound serum estradiol levels to be significantly higher among phenytoin-treated men with epilepsy than among untreated epileptic men or normal control subjects. a significant linear correlation between serum concentrations of biologically active estradiol and phenytoin, but not albumin or hepatic enzymes, suggests a direct medication effects rather than an indirect cause mediated via drug-induced hepatic dysfunction. estradiol exerts a potent inhibitory influence on luteinizing hormone secretion and plays a major role in negative feedback in men as well as women. suppression of luteinizing hormone secretion results in hypogonadotropic hypogonadism. chronically low free testosterone leads to t e s t i c u l a r f a i l u r e a n d h y p e r g o n a d o t r o p i c hypogonadism. this may explain the frequent o c c u r r e n c e o f b o t h o f t h e s e r e p r o d u c t i v e endocrine disorders in men with epilepsy.(21,30) effects of aeds on female sexual hormones a n t i c o n v u l s a n t s t h a t i n d u c e h e p a t i c cytochrome (cy) p450 enzymes can increase the metabolism of sex hormones and make hormonal contraception ineffective. anticonvulsant drugs i n t h i s c a t e g o r y i n c l u d e c a r b a m a z e p i n e , phenobarbital, phenytoin, and primidone. two of the newer anticonvulsant also may interfere with contraceptive efficacy. oxcarbazepine at doses in excess of 1,200 mg/day and topiramate at doses above 200 mg/day can induce cyp450 enzymes. women receiving these anticonvulsants and oral contraceptives should consider taking higher dosage formulations of oral contraceptives. women receiving a cyp450 enzyme-inducing anticonvulsant have at least a 6% failure rate per year for oral hormonal contraceptive pills. gabapentin, lamotrigine, levetiracetam, and tiagabine do not reduce sex-steroid hormone levels and should not reduce contraceptive efficacy.(31) women wishing to take an enzyme-inducing aed with the combined oral contraceptive should be advised to take at least 50 ug of ethinylestradiol and to report any breakthrough bleeding. if it occurs the dose of estrogen should be increased to 75 or 100 ug. “tricycling” the oral contraceptive – that is, taking three months consecutively followed by a four day break, is also advised if the woman wishes to continue with the combined oral contraceptive, although there is no good evidence to back this up. absence of breakthrough bleeding does not necessarily mean that the contraception is e ff e c t i v e . i f m a x i m u m p r o t e c t i o n a g a i n s t pregnancy is desired barrier methods (for example, condom or diaphragm), should be used in addition to the oral contraceptive pill, or the type of contraception changed to one unaffected by enzyme induction.(3) previous observations have indicated that reproductive endocrine disorders are common a m o n g p a t i e n t s w i t h e p i l e p s y. va l p r o a t e treatment is associated with hyperandrogenism, polycystic ovaries, and obesity in women. carbamazepine may also induce endocrine d i s o r d e r s , w h i l e t h e h o r m o n a l e ff e c t s o f oxcarbazepine are poorly known.(32) drugs that stimulate hepatic metabolism may directly affect the serum concentration of e n d o g e n o u s s e x s t e r o i d s a n d v i c e v e r s a . fluctuations of aed concentrations across the menstrual cycle have been reported. women with c a t a m e n i a l s e i z u r e s t a k i n g p h e n y t o i n o r phenytoin and phenobarbital were found to have 26 table 1. aeds reported to increase seizures(34,35) lower aed concentrations despite taking higher doses of the drugs. the phenytoin concentration was significantly lower during menses in women with perimenstrual seizures compared with women who had seizures unrelated to menses, and levels were lower and clearance was greater during menses than during the periovulatory period in women with perimenstrual seizures.(16) paradox effects of aeds aeds may even increase seizure frequency or severity or change the seizure type (table 1). it may be difficult to be certain that an aed e x a c e r b a t e s o r w o r s e n s a s e i z u r e t y p e , particularly with partial seizures, but accurate counts of seizures preceding the introduction of a new aed may help the clinician identify this adverse effect. aeds that increase gaba levels may worsen or exacerbate some generalized seizures. toxic levels of some aeds, such as phenytoin, can increase seizures that were controlled initially. because aeds may worsen seizures, one of the first considerations in choosing an aed is the efficacy for patient’s type of seizure or epilepsy.(33) summary threshold can be defined as lowest point a t w h i c h a s t i m u l u s b e g i n s t o p r o d u c e a sensation, or a minimal stimulus that produces excitation of any structure, eliciting a motor response. a low seizure threshold makes it easier for epilepsy to develop and easier for someone to elicit single seizure. the ovarian sex steroid hormones such as estradiol, progestin, and androgen modulate the seizure threshold. estradiol decreases seizure threshold; progestin and androgen genetically increase seizure threshold. the e f f e c t o f e s t r o g e n ( p r o c o n v u l s a n t ) a n d p r o g e s t e r o n e ( a n t i c o n v u l s a n t ) o n s e i z u r e threshold have been noted in animal and human s t u d i e s . a d u l t w o m e n w i t h e p i l e p s y f a c e additional issues stemming from seizures and aeds that require neurologists to be well v e r s e d i n m e d i c a l s p e c i a l t i e s o u t s i d e o f epileptology, in particular gynecology. the opposing effects of estrogen (proconvulsant) and progesterone (anticonvulsant) on seizure t h r e s h o l d h a v e b e e n n o t e d i n a n i m a l a n d human. level of these hormones fluctuates t h r o u g h o u t t h e m e n s t r u a l c y c l e . i n s o m e women with epilepsy these fluctuations may be related to the occurrence of seizures around the time of menstruation or an increase in seizures in relation to the menstrual cycle. in prescribing aeds, it is important to be aware of potential drug-to-drug anddrug-tohormone interactions, especially in women taking hormonal contraceptives. hormone therapy may be associated with an increase in seizure frequency in menopausal women with epilepsy, and women who have had a catamenial seizure pattern may have increased seizure frequency during perimenopause. harsono seizure threshold and anti-epileptic 27 universa medicina vol.27 no.1 references 1. fisher r, van emde boass w, blume w, elger c, genton p, lee p, et al. epileptic seizures and epilepsy: definitions proposed by the international league against epilepsy and international bureau for epilepsy. epilepsia 2005; 46: 470-2. 2. holmes gl, ben-ari y. the neurobiology and consequences of epilepsy in the developing brain. ped res 2001; 49: 320-35. 3. jackson m. epilepsy in women. practical neurol 2006; 6: 166-79. 4. cohen j. new-onset epilepsy in women: an indication for a newer antiepileptic drug? adv stud nurs 2004; 2: 177-82. 5. jackson jha study of convulsions. transactions of the saint andrews graduate association 1870; 3: 162–204. cited by: holmes gl. ben-ari. the neurobiology and consequences of epilepsy in the developing brain. ped res 2001; 49: 320-35. 6. stafstrom ce. the pathophysiology of epileptic seizures: a primer for pediatricians. pediatr rev 1998; 19: 342-51. 7. su yh, izumi t, kitsu m, fukuyama y. seizure threshold in juvenile myoclonic epilepsy with graves disease. epilepsia 1993; 34: 488-92. 8. abdelmalik pa, burnham wm, carlen pl. increased seizure susceptibility of the hippocampus compared with the neocortex of the immature mouse brain in vitro. epilepsia 2005; 46: 356-66. 9. penovich pe. the effects of epilepsy and its treatment on sexual and reproductive function. epilepsia 2000; 41(suppl. 2): s53-s61. 10. harden cl, baker ga, frye ca, montouris gd, pennell pb, zupancm. epilepsy and ovarian hormones. emory university school of medicine. 2005. 11. morrell mj. reproductive and metabolic disorders in women with epilepsy. epilepsia 2003; 44 (suppl.4): 11-20. 12. yerby ms. neurological management of women with epilepsy. north pacific epilepsy research center, oregon 2002. 13. morrell mj. epilepsy in women. am fam physician 2002; 66: 1489-94. 14. hwang jy, morrell mj. coping with epilepsy in women: sexual function, fertility, pregnancy, and bone stability are affected. women’s health in primary care 1998; 1: 520-7. 15. foldvary-schaefer n, harden c, herzog a, falcone t. hormones and seizures. clev clin j med 2004; 71(suppl.2): s11-s8. 16. smith ss, woolley cs. cellular and molecular effects of steroid hormones on cns excitability. clev clin j med 2004; 71(suppl.2): s4-s10. 17. reddy ds. pharmacotherapy of catamenial epilepsy. indian j pharmacol 2005; 37: 288-93. 18. bauer j, blumenthal s, reuber m, stoffel-wagner b. epilepsy syndrome, focus location, and treatment choice affects testicular function in men with epilepsy. neurology 2004; 62: 243-6. 19. mcquarrie i, peeler db. the effects of sustained pituitary antidiuresis and forced water drinking in epileptic children. a diagnostic and etiologic study. j clin invest 1931: 915–40. cited by foldvary-schaefer n, harden c, herzog a, falcone t. hormones and seizures. clev clin j med 2004; 71(suppl.2): s11-s8. 20. ansell b, clarke e. epilepsy and menstruation. the role of water retention. lancet 1956; 2: 12325. cited by foldvary-schaefer n, harden c, herzog a, falcone t. hormones and seizures. clev clin j med 2004; 71(suppl.2): s11-s8. 21. herzog ag. psychoneuroendocrine aspects of temporolimbic epilepsy-part ii: epilepsy and reproductive steroids. psychosomatics 1999; 40: 102-8. 22. mcauley jw, moore jl, reeves al, flyak j, monaghan ep, data j. a pilot study of the neurosteroid ganaxolone in catamenial epilepsy: clinical experience in two patients. epilepsia 2001; 42 (suppl 7): 85. 23. reddy ds. pharmacology of endogenous neuroactive steroids. crit rev neurobiol 2003; 15: 197-234. 24. lambert jj, belelli d, peden dr, vardy aw, peters ja. neurosteroid modulation of gaba a receptors. progr neurobiol 2003; 71: 67-80. 25. takeuchi y, takano t, abe j, takikita s, ohno m. thyrotropin-releasing hormone: role in the treatment of west syndrome and related epileptic encephalopathies. brain & dev 2001; 23: 662-7. 26. bittigau p, sifringer m, genz k, reith e, pospischil d, govindarajalu s, et al. antiepileptic drugs and apoptotic neurodegeneration in the developing brain. proc natl acad sci u s a. 2002; 99: 15089-94. 27. edwards he, maclusky nj, burnham wm. epileptic seizures: do they cause reproductive 28 dysfunction? univ toronto med j 2000; 77: 10411. 28. herzog ag, drislane fw, schomer dl, pennell pb, bromfield eb, dworetzky ba, et al. differential effects of antiepileptic drugs on sexual function and hormones in men with epilepsy. neurology 2005; 65: 1016-20. 29. isojarvi jit, lofgren e, juntunen kst, pakarinen aj, päivänsalo m, rautakorpi i, et al. effect of epilepsy and antiepileptic drugs on male reproductive health. neurology 2004; 62: 24753. 30. herzog ag, levesque l, drislane f, ronthal m, neuroendocrine unit, charles a. dana research institute, beth israel hospital, and the department of neurology, harvard medical school, boston, massachusetts, u.s.a schomer dl. phenytoininduced elevation of serum estradiol and reproductive dysfunction in men with epilepsy. epilepsia 1991; 32: 550-3. 31. morrell mj. health concerns related to anticonvulsant drugs and reproductive and metabolic health. female patient suppl 2004: 23. 32. rattya j. reproductive endocrine effects of antiepileptic drugs with special reference to valproate. dissertation; oulu; oulu university press 2000. 33. greewood rs. adverse effects of antiepileptic drugs. epilepsia 2000; 41(suppl. 2): s42-s52. 34. berkovic sf. aggravation of generalized epilepsies. epilepsia 1998; 39(suppl. 3): s11-s4. 35. elger ce, bauer j, schermann j, widman g. aggravation of focal epileptic seizures y antiepileptic drugs. epilepsia 1998; 39(suppl.3): s15-s8. harsono seizure threshold and anti-epileptic diana1 100 gastrointestinal symptoms and sleep disturbance in female nurses may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina diana samara* *department of occupational medicine medical faculty, trisakti university correspondence dr. diana samara, mkk department of occupational medicine medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11410 telp. 021-5672731 ex.2101 email: davidwp@cbn.net.id univ med 2009;28:100-5 abstract sleep disturbance is a common symptom in the general population. an association between sleep disturbances and functional gastrointestinal (gi) disorders has been reported by several investigators. gi symptoms are more common among people with sleep disturbance in female nurses. to explore this issue further, a study using a cross sectional design was conducted to determine whether unexplained gi symptoms are more common in female nurses with self-reported sleep disturbance. this study was conducted from february through april 2007 in hospital x in central jakarta. a total of 152 female nurses participated in this study. a questionnaire consisting mainly of items concerning sleep disturbance was distributed to the subjects. significant associations were observed between gastrointestinal symptoms such as anorexia and constipation and sleep disturbance. constipation was significantly more common in female nurses with sleep disturbance (prevalence ratio=6.1;95% c.i. 1.76 – 20.56), but the association between shift work and sleep disturbance was not statistically significant (prevalence ratio=1.67;95% c.i. 0.53-5.24). both constipation and anorexia are more prevalent in female nurses with self-reported sleep disturbance. further research to understand the associations between gi symptoms and sleep disturbance is warranted. keywords: gastrointestinal symptoms, sleep disturbance, female nurses introduction sleep disturbance is a common symptom in the general population, with an estimated 50% or more of american adults experiencing one or more symptoms that indicate insomnia at least a few nights per week.(1) insomnia, in turn, causes significant morbidity, as evidenced by the increased need for general medical and mental health treatment for emotional problems. (2) conceivably, abdominal pain could be an etiology of sleep disturbance if it causes patients difficulty going to sleep or awakens them from sleep. conversely, a specific sleep disturbance might lead to a functional gastrointestinal d i s t u r b a n c e . ( 3 ) g a s t r o i n t e s t i n a l d i s e a s e i s 101 univ med vol.28 no.2 associated with shift work and insufficient sleep is often the reason for leaving shift work.(4) shift workers commonly go to sleep when everybody else has woken up and started working. to adjust to this change may be a sufficiently severe problem for the shift worker. this results in sleep disturbance, such that shift work is usually associated sleep disturbance, which leads to disorders of the immune system, causing the shift worker to become susceptible to flu, rhinorrhea, or other health problems. as many as 20 percent of workers in industrialized nations are shift workers in other words, people who work either at night or on rotating shifts.(5) shift-work sleep disturbance, defined as a primary complaint of insomnia or excessive sleepiness temporally associated with a work period that occurs during the habitual sleep phase, has been diagnosed in as many as 10 percent of shift workers.(6) shift workers overall appear to be at increased risk for peptic ulcer disease, coronary heart disease, insulin resistance, and the metabolic syndrome.(7-9) reports on the impact of shift work on health and well-being have shown a progressive increase and evolution over the years.(10) shift w o r k i s f r e q u e n t l y a s s o c i a t e d w i t h gastrointestinal and cardiovascular disorders. other complaints commonly occurring in shift workers with sleep disturbance are excessive sleep, insomnia, disturbed sleeping schedules, d e c r e a s e d p e r f o r m a n c e , d i ff i c u l t i e s i n interpersonal relations, and irritable moods or depression. a cross-sectional study conducted by ohida et al(11) in 11 japanese hospitals in young female nurses found no significant association between t h e f r e q u e n c y o f n i g h t s h i f t s a n d s l e e p disturbance. occupational sleep disturbance was defined as a complaint of insomnia or excessive sleep associated with work periods (usually at night), occurring during normal sleeping hours. the chief complaint in sleep disturbance is the feeling of unfreshness upon awakening and difficulty in staying awake in the post-shift period, particularly when the major sleep period starts in the morning between 06:00 and 8:00. the combination of decreased quality and quantity of sleep is associated with medical and social problems. shift workers commonly suffer f r o m g a s t r o i n t e s t i n a l a n d c a r d i o v a s c u l a r disturbances.(12) and often report sleep-related s y n d r o m e s s u c h a s c h r o n i c g a s t r i t i s , gastroduodenitis, peptic ulcer and colitis.(13) a high proportion of shift workers are employed in the health care and transportation industries. to explore this issue a study was conducted to determine whether gastrointestinal symptoms and sleep disturbance are more common among female nurses. methods research design the study used a cross-sectional design and was conducted in hospital x in central jakarta from february to april 2007. study subjects all female nurses working in hospital x who were willing to participate and agreed to give informed consent were included in the study. measurements a s s e s s m e n t o f s l e e p d i s t u r b a n c e w a s p e r f o r m e d w i t h a n i n s t r u m e n t f o r s l e e p disturbance, comprising duration of sleep, dreaming, depth of sleep, time needed for falling asleep, waking during the night, time needed for going back to sleep, pattern of awakening in the morning, and feeling of being refreshed on awakening. individuals categorized as having sleep disturbance were those with a score of over univ med vol.28 no.2 102 table 1. characteristics of subjects and risk of sleep disturbance *reference group 8 – 22, out of a range of 8 – 33. sleep disturbance was associated with shift work, age, occupation, and gastrointestinal disorders. data analysis data were processed by means of the spss for windows version 10 and stata version 6.0. logistic regression analysis was used for determining an association between various risk factors for sleep disturbance, with significance level set at p=0.05. results the participants in this study were 152 in number, with minimum age of 22 years (3.9%) and maximum age of 55 years (0.7%). as to marital status, 61 subjects were single (40.1%) whilst 91 subjects were married (59.9%). table 1 shows that the majority of nurses were under 35 years of age (79%) and that age group, marital status, work station, and work shift did not constitute risk factors for the occurrence of sleep disturbance. among the risk factors listed in table 2, the univariate risk factors causing sleep disturbance w e r e a n o r e x i a a n d c o n s t i p a t i o n , w h i c h significantly caused sleep disturbance. anorexia resulted in a five-fold increase in the risk of sleep disturbance, compared with nurses without anorexia (pr 5; 95% ci = 1.90 – 13.13). in nurses with constipation there was a seven-fold increase in the risk of sleep disturbance, compared with nurses without constipation (pr 7.12; 95% ci = 2.22 – 22.80). both factors were subsequently subjected to multivariate analysis using the enter method of logistic regression, of which the results are presented in table 3. from table 3 above it is evident that anorexia and constipation were risk factors for sleep disturbance. anorexia caused a four-fold i n c r e a s e i n t h e r i s k o f s l e e p d i s t u r b a n c e , compared with the nurses without anorexia (pr 4.37; 95% ci = 1.59 – 12.07). in nurses with constipation there was a six-fold increase in the risk of sleep disturbance, compared with the nurses without constipation (pr 6.01; 95% ci = 1.76 –20.56) samara gastrointestinal and sleep disturbance 103 univ med vol.28 no.2 *reference group table 2. gastrointestinal disorder and risk of sleep disturbance table 3. final model of risk factors of sleep disturbance *reference group discussion a total of 24 or 26.18% out of 152 nurses suffered from sleep disturbance. a similar prevalence was found by ohayon et al. in that more night-shift workers had difficulty in falling asleep, compared with non-shift workers (20.1% : 12.0%). in ohayon’s study it was found that workers on night-shift had a shorter duration of sleep than those not on shifts.(14) 104 the national sleep foundation has defined insomnia as any of the following: difficulty falling asleep, waking a lot during the night, waking too early with inability to get back to sleep, or waking up feeling unrefreshed.(1) in the present study shift work was not a r i s k f a c t o r f o r t h e o c c u r r e n c e o f s l e e p disturbance, although 26.18% of shift workers had sleep disturbance. the evidence points to anorexia and constipation as risk factors for sleep disturbance. anorexia caused an increase of 4.37 times in the risk of sleep disturbance, whereas constipation resulted in an increase of 6.01 times in the risk of sleep disturbance. uniquely, sleep disturbance in turn caused an increase of 7.12 times in the risk of anorexia and a five-fold increase in the risk of constipation, compared with those without sleep disturbance. thus it is apparent that sleep disturbance increases the risk of anorexia and constipation, and vice versa. this may be due to the fact that individuals suffering from daily sleep disturbance do not pay attention to their diet, whilst persons with anorexia and constipation become anxious at sleeping time, thus disturbing their sleep. in the present study age, marital status, o c c u p a t i o n , w o r k s h i f t , d i a r r h e a , n a u s e a / vomiting, abdominal distention, epigastric pain, and eructation were not risk factors for sleep disturbance. that work shift was not a risk factor for sleep disturbance may be due to the fact that the majority of nurses working on shifts were accustomed to shift work and thus did not suffer from sleeping problems. the study by chung et al. showed that disturbance in sleep quality was not caused by shift schedule or pattern, but by “morningness” or “eveningness” in relation to sleeping times.(15) the study conducted by chan(16) in 163 nurses demonstrated that more than 70% of the nurses had sleep disturbance, with older age and gastrointestinal disorders being risk factors for sleep disturbance. around 79.6% of the nurses were under 35 years of age and 19% of these had sleep disturbance, whereas among the 31 nurses in the age group over 35 years only one had sleep disturbance, because most of these older nurses were not on shift work; 63.2% of them worked at the outpatient clinic without night shifts. similar results were obtained by sivertsen et al using a historical cohort design, where the results showed that sleep disturbance was associated with somatic diagnoses and somatic symptoms.(17) there are several limitations in the present study, as assessment of sleep disturbance was obtained by self-report rather than clinical diagnosis, and as the instrument did not include an item on duration of sleep disturbance. the absence of the duration criterion may have reduced the specificity of the measurements and thus underestimated the true association. selfreport instruments are prone to error and residual confounding cannot be ruled out. conclusion the present study on the relationship between gastrointestinal symptoms and sleep disturbance provided evidence of an association i n f e m a l e n u r s e s b e t w e e n a n o r e x i a a n d c o n s t i p a t i o n o n t h e o n e h a n d a n d s l e e p disturbance on the other hand. to determine which of them is the cause and which the result, or whether they contribute to each other, future studies should be focussed on intervention by treatment of gastrointestinal symptoms. references 1. national sleep foundation. sleep in america poll. washington, dc. available at: http://www. sleepfoundation.org/2003poll.cfm. accessed august 1, 2009. 2. vege ss, richard locke iii g, weaver al, farmer sa, joseph melton iii l, talley nj. functional gastrointestinal disorders among people with sleep samara gastrointestinal and sleep disturbance 105 univ med vol.28 no.2 disturbances: a population-based study. mayo clin proc 2004;79:1501-6. 3. fass r, fullerton s, tung s, mayer ea. sleep disturbances in clinic patients with functional bowel disorders. am j gastroenterol 2000;95:1195-200. 4. akerstedt t. shift work and disturbed sleep/ wakefulness. occup med 2003;53:89–94. 5. basner rc. shift-work sleep disorder the glass is more than half empty. n engl j med 2005;353:3535. 6. drake cl, roehrs t, richardson g, walsh jk, roth t. shift work sleep disorder: prevalence and consequences beyond that of symptomatic day workers. sleep 2004;27:1453-62. 7. knutsson a. health disorders of shift workers. occup med 2003;53:103-8. 8. karlsson b, knutsson a, lindahl b. is there an association between shift work and having a metabolic syndrome? results from a population based study of 27,485 people. occup environ med 2001;58:747-52. 9. merijanti lt, samara d, tandean r, harrianto r. the role of night shift work on blood pressure among healthy female nurses. univ med 2008;27:65-71 10. giovanni costa shift work and occupational medicine: an overview. occup med 2003;53:83–8. 11. ohida t, kamal amm, sone t, ishii t, uchiyama m, minowa m, et al. night-shift work related problems in young female nurses in japan. j occup health 2001;43:150–6. 12. garbarino s, bellke m, costa g, violani c, lucidi f, ferrilo f, et al. brain function and effects of shift work: implication for clinical neuropharmacology. neuropsychobiology 2002;45:50-6. 13. scott aj. shift work and health. prim care 2000;27:1057-79. 14. ohayon mm, lemoine p, ab veronique, dreyfus m. prevalence and cosequences of sleep disorders in a shift worker population. j psychosom res 2002;53:577-83. 15. chung mh, chang fm, yang ch. sleep quality and morningness-eveningness of shift nurses. j clin nurs 2009;18:279-84. 16. chan mf. factors associated with perceived sleep quality of nurses working on rotating shifts. j clin nurs 2009;18:285-93. 17. sivertsen b, overland s, neckelmann d, glozier n, krokstad n, le pallesen s, et al. the long-term effect of insomnia on work disability. the hunt2 historical cohort study. am j epidemiol 2006;163:1018–24. alvina 177 abstract low back pain (lbp) is a common problem in adult life, since despite its benign nature it is commonly associated with incapacity, productivity loss due to sick leave, and correspondingly high costs to the individual worker. psychosocial and lifestyle factors and work-place exposures have been implicated in the onset of symptoms. heavy physical work, static work postures, frequent bending and twisting, lifting and postural movements, repetitive work, and whole body vibrations are occupational factors associated with lbp. the usual classification of lbp is related to the duration of the complaints (acute, subacute, and chronic). however, these terms fail to take into account several clinically important aspects of the course of lbp, which is frequently recurrent and thus neither acute nor chronic. more realistically, lbp should be classified as specific and nonspecific. approximately 90% of lbp cases have no identifiable cause and is designated nonspecific lbp. however, despite its high prevalence, the etiology and nature of nonspecific lbp are not yet well understood. its pathophysiology remains complex and multifaceted. multiple anatomic structures and elements of the lumbar spine (e.g. bones, ligaments, tendons, discs, and muscles) are all suspected of playing a role. many of these components of the lumbar spine have sensory innervations that can generate nociceptive signals in response to tissuedamaging stimuli. other causes could be neuropathic (e.g. sciatica). some cases of lbp most likely involve mixed nociceptive and neuropathic etiologies. keywords: nonspesific back pain, biomechanical, sciatica, nociceptive, neuropathic *department of anatomy, department of occupational medicine, medical faculty, trisakti university correspondence dr. ridwan harrianto, mh.sc.(om),spok department of anatomy, department of occupational medicine, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2101 email: riddharr@cbn.net.id univ med 2010;29:177-87. biomechanical aspects of nonspecific back pain ridwan harrianto* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction low back pain (lbp) is defined as a symptom complex consisting of pain and muscle tension or stiffness in the lumbar region localized below the costal margin and above the inferior gluteal folds, with or without pain radiating into the legs (sciatica) (figure 1).(1) lbp is a frequently occurring disorder, with more than 85% of individuals having ever experienced lbp in her/his lifetime.(1,2) about two thirds of adults suffer from low back pain at some time. low back pain is second to upper respiratory problems as a symptom-related reason for visits to a physician.(3) the highest prevalence of lbp occurs in the age range of 45-64 years among workers of both genders.(4) 178 harrianto biomechanical of nonspesific low back pain according to the national center for h e a l t h s t a t i s t i c s l b p p o s e s i m p o r t a n t socioeconomic problems to the workers’ community, the most frequent being disability of persons below 45 years of age.(5) adults with low back pain are often in worse physical and mental health than people who do not have low back pain: 28% of adults with low back pain r e p o r t l i m i t e d a c t i v i t y d u e t o a c h r o n i c condition, as compared to 10% of adults who do not have low back pain. also, adults reporting low back pain were three times as likely to be in fair or poor health and more than four times as likely to experience serious psychological distress as people without low back pain.(5) other studies state that in the united states lbp is responsible for the annual loss of 149 million working days, with 102 m i l l i o n w o r k i n g d a y s b e i n g l o s t d u e t o occupational lbp. it is estimated that the compensation costs for loss of working time amounts to $3000 per insurance claim.(4) figure 1. mannequin with shaded area indicating site of lbp symptoms.(1) patients with an attack of lbp commonly recover spontaneously after one month at the latest,(6) while several studies report that 90% of lbp cases recover within 6 weeks, 77% within 7 weeks, and the remaining cases become chronic lbp cases.(6) however, the study conducted by van den hoogen et al. suggests that 70% of patients still suffered from back pain after 4 weeks, 48% after 8 weeks, 35% after 12 weeks, and 10% after 1 year. (7) a population-based, prospective cohort study conducted by cassidy et al. showed that o n l y 1 . 0 % d e v e l o p e d i n t e n s e a n d 0 . 4 % developed disabling lbp after 12 months follow-up. resolution occurred in 26.8%, and 40.2% of episodes persisted. of those that recovered, 28.7% had a recurrence within 6 months, and 82.4% of it was mild lbp.(8) only around 5% of acute lbp becomes chronic lbp and results in disability. pengel et al. reported an improvement in disability of 58% in acute lbp, and found that 82% of patients returned to work within one month.(9) cases of lbp with recovery times of less than 6 weeks are designated acute, those with recovery times between 6 weeks and 3 months are called subacute, while chronic cases do not recover within 3 months and not infrequently lead to disability, such that the individual is unfit for work.(10) however, these terms fail t o t a k e i n t o a c c o u n t s e v e r a l c l i n i c a l l y important aspects of the course of lbp, w h i c h i s f r e q u e n t l y r e c u r r e n t a n d t h u s neither acute nor chronic. more realistically, lbp should be classified as specific and nonspecific.(7) lbp may occur as a result of excessive physical stress on normal spinal structures, or of normal physical stress on abnormal spinal structures. lbp cases with underlying organic disease, either spinal or nonspinal, generally identifiable by radiological abnormalities of the spine, are classified as specific lbp.(2) the study by koes et al. in the netherlands found that of all patients with lbp in primary care, 4% was identified with compression fractures, 179 3% with spondylolisthesis, 0.7% was caused by tumors or tumor metastases, 0.3% was due to ankylosing spondylitis, and 0.01% due to infection, while the remaining 90% had nonspecific lbp (nslbp).(11) epidemiology t h e l a rg e v a r i a t i o n i n m e t h o d s a n d diagnostic criteria for lbp, and the wide range of work types and physical activity performed, result in the wide range of figures obtained by epidemiological studies. the point prevalence of lbp in the general population has been reported by several studies as being in the range of 14-28,(12) while the reported 12-month prevalence is 35%.(7) the overall prevalence of lbp in workers, as reported by a number of studies, ranged between 15% and 30%,(12) while the overall 12-month prevalence in firsttime workers and first-time sufferers of lbp was 19%.(13) the study conducted by omokhodion and sanya on administrative workers with lbp in ibadan, nigeria, found a 12-month prevalence of 38% and a point prevalence of 20%.(14) the 1 2 m o n t h p r e v a l e n c e o f l b p i n i r a n i a n industrial workers is 21%,(13) in construction workers 30%(15) in long-distance taxi drivers 51%,(16) and in personal car drivers 53%.(17) several studies of the nursing profession yielded high prevalence rates, with 12-month prevalences of 30%-76%,(18) while the point prevalences were 15.5-54.7%.(15,17) risk factors the risk factors of influence are age, gender, education, body mass index, and length of employment.(3,8.10) daily habits may also c o n s t i t u t e r i s k f a c t o r s f o r l b p, s u c h a s smoking, alcohol consumption, sports and daily activities of living.(8.10) other factors, such as repetitive movements, vibration, parity and psychosocial stress, may also play a role in the development of lbp.(4,13) although a number of studies on various specific industrial groups suggested that the role of the above risk factors was less significant, most studies indicated a strong association between lbp on the one hand and mechanical exposure and poor working posture on the other.(11,19) approximately 80-90% of these disorders were caused by lumbar strain/sprain,(5) that could be triggered by acute or cumulative trunk injuries.(2) several highly important risk factors for lbp have been reported, such as manual material handling, which is work requiring s t r e n u o u s p h y s i c a l a c t i v i t y, e . g . l i f t i n g , l o w e r i n g , p u s h i n g , p u l l i n g , t h r o w i n g , s u p p o r t i n g , m o v i n g o f l o a d s ( 4 0 % ) , o r involving postures with frequent bending or stooping at work (20%), and prolonged static sitting or standing (20%).(13,20) the study by tousignanti et al. reported that the types of work necessitating frequent exposure to manual material handling activities have a 2.05 times higher risk for lbp than work with frequent exposure to manual material handling activities.(21) harkness et al. reported that workers lifting loads of more than >24 lb with two hands, those lifting loads of more than >23 lb at or above shoulder level, those pulling loads of >55 lb, and those kneeling or squatting at work for >15 minutes had twice the risk for lbp than workers who never performed these activities.(1) other investigators reported that work involving frequent flexing and/or rotation of the trunk had a 2.2x higher risk, while carrying loads of >25 kg had a 1.5x higher risk.(22) although various physical activities in t h e w o r k p l a c e h a v e b e e n i d e n t i f i e d a s significant risk factors for lbp prevalence, the quantitative relationship of both intensity and duration of exposure with lbp prevalence is still unclear. one of several studies in the netherlands reported that there is a doseresponse relationship between lifting and carrying loads of more than 10 kg and lbp risk. workers who performed lifting and carrying of loads for 7.5-15 minutes daily had a 2.13 times higher risk for lbp than those who performed lifting and carrying of loads for 0 – 7.5 minutes daily, while for workers univ med vol.29 no.3 180 harrianto biomechanical of nonspesific low back pain who performed lifting and carrying of loads for 15-30 minutes and for more than 30 minutes, the respective risks were only 1.38 and 1.33 times the risk for workers who p e r f o r m e d t h e s e a c t i v i t i e s f o r 0 – 7 . 5 minutes.(23) the study of chen et al. on longdistance taxi drivers in taiwan reported that static sitting (driving > 4 hours/day) had a 1.78 fold risk for lbp.(16) however, the same group of investigators in their 1998 – 2006 review reported that although 8 studies found evidence for an association between static sitting at work and development of lbp, one study failed to find such an association, thus chen et al. concluded that in the latter study static sitting was not the sole risk factor for lbp. anatomy of the locomotor apparatus of the trunk the spinal column is s-shaped, being concave in the thoracic region and convex in the lumbar region. this shape confers elastic p r o p e r t i e s o n t h e t r u n k f o r a b s o r b i n g downward compression forces when jumping and lifting loads. intervertebral disc (ivd)s are situated between two adjacent vertebrae, starting at ivd c2-c3 (between cervical vertebrae c2 and c3) down to ivd l5-s1 (between l5 and s1), giving a total of 23 ivds. an ivd consists of an outer annular rim, known as the annulus fibrosus, which is a plate of concentrically arranged fibrous connective tissue sheets, with the collagen fibers running obliquely in each sheet, but in different directions in each subsequent sheet, thus forming a strong fibrous ring. the central part of the disc is called n u c l e u s p u l p o s u s , c o n s i s t i n g o f s o f t fibrocartilage-like tissue with the consistency of foam rubber, thus exerting strong pressures to hold apart the vertebrae immediately above and below. this pressure develops in reaction to the weight of the body and the load being lifted. at the posterior part of each vertebra there is an opening in apposition with similar openings of the vertebrae above and below, forming the spinal canal for protection of the spinal cord. the spinal canal also contains openings for the passage of spinal nerve roots to all parts of the body. the risk factors for lbp are closely associated with the anatomical structure and function of the trunk and with the intensity of physical activity. the role of these risk factors c a n b e e x p l a i n e d b y b i o m e c h a n i c a l mechanisms of the locomotor apparatus of the human body. heavy physical work involving frequent truncal movements requires a larger a m o u n t o f e n e rg y o r p h y s i c a l s t r e n g t h , resulting in compressive stress loading on muscles, ligaments, nerves, blood vessels, bones and joints, particularly in the lumbar region. the compressive stress loading in turn gives rise to fatigue and constitutes repetitive microtraumas to these anatomical structures. muscles, nerve roots, dura mater, posterior longitudinal ligaments, facet joints, joint capsules, periosteum, vertebrae, and the fibers of the outer layers of the annulus fibrosus have a somato-sensory innervation and are therefore sensitive to pain stimuli. stimulation of the locally distributed pain fibers of the sensory nerves produces the sensation of pain in the lumbar region. several anatomical structures at these sites, such as the fibers of the inner l a y e r s o f t h e a n n u l u s f i b r o s u s , n u c l e u s pulposus and ligamentum flavum are resistant to pain stimuli. biomechanics of manual material handling and truncal motion in a person standing upright, the load of the body mass is carried by the five lumbar vertebrae and is particularly concentrated at ivd l5-s1. even without the person carrying a load, the compression forces exerted by the body mass results in a forward moment of forces, because the center of mass of the body is located slightly anterior to ivd l5-s1. therefore, maintaining the upright position of the body requires contraction of the erector 181 s p i n a e m u s c l e s ( s a c r o s p i n a l , q u a d r a t u s lumborum, longissimus dorsi, and multifidus muscles) and the flexors of the lower limb (gluteal and hamstring muscles), leading to traction and torsional forces on all spinous processes of the lumbar vertebrae, especially around ivd l5-s1 (figure 2).(24) when an individual is lifting a heavy load, the above mechanism causes ivd l5-s1 to r e c e i v e l a rg e r c o m p r e s s i o n f o r c e s t h a t ultimately result in tearing of the annulus fibrosus and allow prolapse of the nucleus pulposus, known as hernia nucleus pulposus (hnp), with pain radiating into the back due to compression of the spinal nerve roots in this location. although cases of hnp capable of inducing neurogenic pain are rare, mild cases are a relatively frequent occurrence. slight stretching or tearing of the outer layer of the annulus fibrosus, partial prolapse of the nucleus pulposus compressing the posterior longitudinal ligaments, or degeneration of the intervertebral articular surfaces, may lead to irritation of fine unmyelinated sensory nerve fibers in these locations, thus causing lumbar pain, termed mechanical or discogenic pain, which occasionally may be as severe as neurogenic pain. ong et al. in their australian study (25) reported that athletes participating in t h e o l y m p i c g a m e s w h o s u ff e r e d f r o m lumbago actually had degeneraon of the lumbar ivds, 36% of this abnormal group having severe degeneration. the degeneration became more severe in the caudal direction, and was most frequently found in the l5-s1 segment. ivd narrowing was also found mainly at ivd l5-s1, while hnp was most common in the lower lumbar ivds. in 58% of l5-s1 ivds there was displacement of the disc, in most cases causing disc bulging. flexion, extension and rotation of the trunk of an individual at work is a cumulative motion of the whole trunk, but actually the greater part of the motion is performed by the lumbar vertebrae. the lumbar ivds assume the role of synarthroses, with the nucleus pulposus functioning as rotational axis of the vertebrae when performing rotation, flexion, extension, lateral bending, and pulling and pushing movements, with 80-90% of these movements occurring at ivds l4-l5 and l5-s1. rotation of the vertebrae sets up shear stresses that rotate the external portion of the ivd, most of the shear occurring at the outer l a y e r s o f t h e a n n u l u s f i b r o s u s . f l e x i o n , e x t e n s i o n , a n d l a t e r a l b e n d i n g e x e r t compression stresses and tension in the annulus fibrosus at the sites of apposition. several force components parallel to the ivd, such as pushing and pulling of loads, produce shear stresses that give rise to slipping in the univ med vol.29 no.3 figure 2. compression forces of body mass load and the load supported by all spinous processes of the lumbar vertebrae, especially around intervertebral disc l5-s1.(24) b=load, s=sacrospinal muscles, g=gluteal muscles, h=hamstring muscles. 182 harrianto biomechanical of nonspesific low back pain direction of the forces. in load lifting the lumbar vertebrae act as levers, therefore contraction of the muscles of the back and buttock also sets up compressive and rotational stresses in the ivds. the combination of lifting and truncal motion causes larger intradiscal stresses at l5-s1 in comparison with those caused solely by lifting movements. axial loading of short duration is resisted by tensioning of the annular ligament fibers in the ivds, but axial loading of longer duration leads to stretching of the annulus fibrosus and induces larger compression stresses in the surface layers of the vertebral body, leading to pain due to injury to these anatomical structures. ligaments act as fixation points for joints, thus limiting their movements. in lumbar flexion the greatest tension is found within the interspinous and supraspinous ligaments, followed by the intrascapular ligaments and the ligamentum flavum. in lumbar extension the g r e a t e s t t e n s i o n o c c u r s i n t h e a n t e r i o r l o n g i t u d i n a l l i g a m e n t . l a t e r a l b e n d i n g produces the greatest tension in the ligaments contralateral to the direction of bending, while rotation results in the capsular ligaments undergoing the greatest tension.(13) the strains occurring in these ligaments increase the pain induced by the compressive and rotational stresses in the ivds. biomechanics of prolonged static standing and sitting the ivd has no blood supply and obtains its nutrients by diffusion from the adjacent tissues, for which process motion is a necessary condition, while static body posture, such as prolonged sitting or standing, decreases the nutrient flow. the lumbar vertebrae are positioned vertically on the sacrum, thus the magnitude of lumbar lordosis depends on the vertical position of the sacrum and is indicated by the magnitude of the lumbosacral angle. the vertical position of the sacrum depends on the rotation of the pelvis. in individuals standing upright, the thorax has a convex anterior curvature; when the lumbosacral angle is >40o, there is lumbar hyperlordosis, while with increasing relaxation of the upright position t h e p e l v i s r o t a t e s p o s t e r i o r l y a n d t h e lumbosacral angle diminishes. in the sitting position the pelvis rotates posteriorly and the magnitude of the lumbosacral angle decreases to – 5o, decreasing with increasing slumping. in the standing position the pelvic flexor m u s c l e s a n d t h e e r e c t o r s p i n a e m u s c l e s contract, while the pelvic extensors undergo relaxation (stretch) in order to stabilize the pelvis. the result is that the pelvis is tilted forward, thus increasing the lumbosacral angle. in this condition the center of equilibrium of the body tends to move forwards, and lumbar lordosis occurs, so as to maintain the upright position of the trunk and head. prolonged contraction of the erector s p i n a e m u s c l e s ( q u a d r a t u s l u m b o r u m , longissimus dorsi, and multifidus muscles), and the pelvic flexors (gluteal and hamstring muscles), results in weakness or stiffness and occasionally cramps in the pelvic muscles and s u s t a i n e d s t r e t c h i n g o f t h e s a c r o s p i n a l ligament, giving rise to pain.(7) the superior articular processes of a vertebra form facet joints with the inferior articular processes of the vertebra immediately above. the facet joints make an angle of 450 with the horizontal plane, which causes them to resist intervertebral shear forces, while the ivds resist compressive stresses. in a person lifting a load, the compression forces cause narrowing of the ivds and are transmitted to the facet joints, such that these sustain a heavy load, resulting in shear stresses. in the standing position, lumbar hyperlordosis of the vertebrae l e a d s t o s l a c k e n i n g o f t h e s u p r a s p i n o u s ligaments, such that the ligaments cannot prevent forward displacement of the lumbar vertebrae, causing the two adjacent vertebrae to slide against one another due to the greater compression forces on the superior endplates 183 of the ivd. narrowing of the facet joints leads to friction between their articular processes, facilitating the development of facet joint osteoarthritis, and giving rise to back pain. in the sitting position the pelvic extensors (iliopsoas) and the abdominal wall muscles contract, while the pelvic flexors (hamstring and gluteal muscles) and the erector spinae undergo relaxation (stretching), such that the pelvis is tilted backwards, resulting in a decreased lumbosacral angle and decreased lumbar lordosis, which may even turn into a kyphosis in order to maintain the upright position of the trunk and head. in this position the potential elasticity of the trunk in absorbing downward forces is lost, the compression forces are directly transmitted downwards, such that the intradiscal stresses at l5-s1 are greater in sitting than in standing. the large intradiscal stresses at l5-s1 in kyphosis compress the anterior portion of the ivd, while the posterior portion stretches, such that the annulus fibrosus (degenerated due to p o o r n u t r i t i o n ) p r o l a p s e s p o s t e r i o r l y, c o m p r e s s i n g t h e p o s t e r i o r l o n g i t u d i n a l ligaments and giving rise to pain. correlation of organic abnormalities with clinical manifestations of nonspecific back pain on the basis of the organic abnormalities underlying nslbp and for clinical purposes, several diagnostic entities are recognized, such as low back strain, piriformis syndrome, iliolumbar syndrome, discogenic pain, facet joint syndrome and sacroiliac syndrome. myofascial pain syndrome/low back strain muscles that are exposed to prolonged physical stress, because of considerably long periods of contraction and relaxation in a static p o s i t i o n , e x t r e m e l y r a p i d r e p e t i t i v e movements, or forced vigorous contractions, may undergo spasm and shortening, thus increasing their tone and tension, such that they may contract of their own accord without neural stimulation. these contractions may be prolonged and demonstrable on emg. in these conditions the muscle fibers undergo injury and inflammation, with a compromized blood supply. the injured muscles may regenerate if the damage is slight and the physical stress is discontinued, but with severe damage or p e r s i s t e n t p h y s i c a l s t r e s s , t h e m u s c l e s degenerate and are replaced by fibrotic tissue. these fibrotic sites may become trigger points (trps), palpable as tender nodules 3-6 mm in diameter. the pain arising from stimulation of these trps is called myofascial pain, and may be due to direct or indirect trauma, excessive or repetitive physical activity, or faulty posture.(1) the condition is called myofascial p a i n s y n d r o m e / c h r o n i c t h o r a c o l u m b a r syndrome (low back strain), which appears as sudden pain or as mild back pain that gradually worsens. the pain usually persists on one side of the back, and results in marked tenderness in the gluteal region and/or paralumbal regions, with a positive patrick test. piriformis syndrome the piriformis muscle runs from the facies pevina of the sacrum to the greater trochanter of the femur, dividing the greater sciatic foramen into the suprapiriformis and infrapiriformis foramina, through the latter of which the sciatic nerve passes from the pelvic cavity into the leg. in blunt trauma to the sacroiliac or gluteal region, e.g. in a person falling on the buttocks, there is hemorrhage and hematoma in or around the piriformis muscle, followed by spasm and stiffness of this muscle, thus irritating the sciatic nerve and resulting in the piriformis syndrome.(26) a c c o r d i n g t o s e v e r a l s t u d i e s , t h e incidence varies between 0.33% and 6%.(27) this syndrome may also result from arthritis and periarthritis of the sacroiliac joint, from excessive use of the pelvic rotators (e.g. in individuals with severe physical activity, soccer players, or athletes), and from repetitive univ med vol.29 no.3 184 harrianto biomechanical of nonspesific low back pain injury to the sciatic nerve, such as occurs in workers with prolonged sitting postures. the resulting pain, muscle spasm, or stiffness around the sacroiliac joint, greater trochanter, or iliopsoas muscle, is occasionally accompanied by pain radiating into the leg, leading to difficulty in walking. the pain may also appear on stooping or lifting. on physical examination a sausage-shaped mass and tenderness are found around the injured piriformis muscle, with positive lrs test and occasionally fibrosis of the gluteal muscle. the lesions are usually unilateral, but may be bilateral; paresthesia and numbness are rarely f o u n d . t h e s y n d r o m e i s d i ff i c u l t t o differentiate from ischialgia due to hnp. iliolumbar syndrome the iliolumbar ligament is a strong ligament connecting the transverse process of l5 with the internal lip of the iliac crest. injury or inflammation of this ligament results in acute lbp, subsequent to execution of an inapproriate movement or to blunt trauma. this syndrome is the most frequent cause of lbp, w i t h a p r e v a l e n c e o f u p t o 4 3 % . ( 2 8 ) t h e iliolumbar syndrome is also frequently called iliolumbar ligament syndrome, iliac crest pain syndrome, and multifidus triangle syndrome. the pain is felt on the medial side of the iliac crest, with the patient generally being able to indicate the precise location of the pain. the pain may be induced or increased by lateral flexion of the trunk or by the stair-step test. the syndrome commonly occurs in individuals whose occupation requires prolonged standing or sitting. there is marked tenderness at the site of pain, with positive lrs and patrick tests. discogenic pain repetitive compressive and rotational stresses on the ivds, particularly at l5-s1, lead to in degeneration of the annulus fibrosus, with solitary or multiple tears. the tears may b e m a r g i n a l , t a n g e n t i a l , o r r a d i a l , b u t fortunately may undergo autorepair. in the nucleus pulposus the degenerative process is manifested by dehydration and fragmentation of the nucleus into sequestra, with its normally firm consistency (resembling that of lobster meat) turning into a soft mass mixed with gas b u b b l e s . te a r s o f t h e a n n u l u s f i b r o s u s , particularly radial tears, facilitate prolapse of the sequestra of the nucleus pulposus. the posterolateral angle is the thinnest and weakest portion of the annulus fibrosus, such that ivd bulges due to prolapse of the nucleus pulposus compresses the posterior longitudinal ligament, stimulating the fine unmyelinated sensory nerve fibers in these locations, and inducing back pain known as mechanical or discogenic pain. kuslich et al. conducted a study on 193 candidates for hnp or spinal stenosis surgery. the investigators were able to elicit the pain by means of blunt surgical instruments or lowvoltage electric current in 30% of those stimulated at the paracentral area of the a n n u l u s f i b r o s u s a n d i n 1 5 % o f t h o s e stimulated at the central part.(28) mechanical or discogenic pain may ultimately cause tears in the annulus fibrosus, manifesting as hnp, due to compression of the spinal nerve roots. sacroiliac syndrome compression forces from the spinal c o l u m n a r e t r a n s m i t t e d t o t h e p e l v i c (innominate) bones by way of the sacroiliac joint. although apparently locking the pelvic girdle for transmission of downward loads to the femoral head, actually the sacrum tends to be displaced forwards and out of the pelvic girdle, due to its trapezoid shape that is narrower at the rear. in the sacroiliac syndrome the forward shift of the sacrum results in stretching of the ligaments inserting into the s a c r u m , n a m e l y t h e s a c r o s c i a t i c a n d sacrotuberous ligaments, giving rise to sudden pain radiating from the sacroiliac joint to the back and the posterior thigh(figure 3).(29) in contrast to discogenic pain, the pain is never centrally located, but commonly to one 185 figure 3. the sacrum tends to be pushed forwards when lifting loads.(29) a = sacrum, b = ilium, c = pubis, d = position of intervertebral disc between s1–l5 side. there is pain upon standing on one leg, limited movement at the sacroiliac joint, and tenderness over the joint, with positive patrick test. facet joint syndrome prolonged and continuously applied compression forces on the ivd result in ivd degeneration, described as narrowing of the ivd. the biomechanical axis of the ivd promotes the transmission of compression forces posteriorly to the facet joints. therefore the facet joints receive a larger distributed load from compression forces, in comparison to other parts of the vertebra. prolonged injury to the articular surface results in inflammation o f t h e j o i n t f o l l o w e d b y f o r m a t i o n o f osteophytes to compensate for the increased biomechanical stress in order to stabilize the j o i n t . o s t e o a r t h r i t i s o f t h e f a c e t j o i n t s manifests itself as lbp and rigidity of the joints, accompanied by secondary muscle spasm.(11) s i m i l a r t o t h e c a s e o f f a c e t j o i n t s , compressive loading on the sacroiliac joint also allows the formation of osteophytes and the development of osteoarthritis in the joint. parmar et al. found one case of an osteophytic bridge across the anterosuperior border of the left sacroiliac joint,(30) while kumar et al. found 4 cases of osteophytes at the inferior border of the sacroiliac joint, where all cases complained of back pain and recovered after surgery.(31) prevention health education and training for applying biomechanical principles of the body and adequate medical care of the back need to be instituted as primary and secondary preventive measures. analysis of daily physical activities needs to be carried out to study the relationships between the disease symptoms and influencing factors, such as occupation, hobbies, and sports, by evaluation of postures in standing, sitting, driving, load lifting, or performing other physical activities. work-related activities should be planned t o m i n i m i z e t h e a m o u n t o f w o r k t o b e univ med vol.29 no.3 186 harrianto biomechanical of nonspesific low back pain performed requiring prolonged sitting or standing, monotonous motions, and repeated stooping and truncal rotation. particularly for the working community, in the future jobs should be redesigned, by minimizing the frequency and duration of work postures capable of inducing physical stress, eliminating monotonous jobs, regulating rest periods to regain use of the muscles, joints and ligaments. there is also a need for training to improve work techniques. furthermore, there should be a stage by stage evaluation of performance. c l e a r l y w o r d e d i n s t r u c t i o n s f o r p r e v e n t i n g l b p s y m p t o m s s h o u l d b e mandatory reading material for all manual handling workers. strength testing should be carried out particularly when hiring new workers for manual handling jobs. every lbp event experienced by the workers should be reported, to allow early medical evaluation in order to reduce the occurrence of more severe and irreversible health problems. adequate evaluation and counseling performed on workers with previous lbp events is urgently required. conclusions in general nslbp is due to heavy work in connection with manual material handling, such as lifting, lowering, pushing, and pulling of heavy loads, and is also associated with frequent or prolonged bending of the body, stooping, sitting, and standing, or other unnatural postures. although the organic abnormalities of nslbp are difficult to clarify, it is firmly believed that biomechanical stress of the lumbar vertebrae due to changes in the center of gravity of the body, followed by compensatory changes in posture, will give rise to pain. tension and strains or injury to muscles, ligaments, vertebral and pelvic articular surfaces, spinal cord, and spinal nerve roots, are some of the causes of the symptoms. on the basis of the pathophysiology and the c l i n i c a l i m p l i c a t i o n s , n s l b p m a y b e categorized into a number of diagnostic entities. analysis of daily physical activities and training should be carried out, to institute positive behavioral changes in work-related postures, presumably leading to reduced risks of recurrent lbp. references 1. harkness ef, macfarlane gj, nahit es, risk factors for new-onset low back pain amongst cohorts of newly employed workers. rheumatology 2003;42:959-68. 2. zanni gr, wick jy. eliminating myths and elucidating realities. j am pharm assoc 2003;43:357-62. 3. deyo ra, weinsten jn. low back pain. n engl j med 2001;344:363-70. 4. manek nj, macgregor aj. epidemiology of back disorders: prevalence, risk factors, and prognosis. curr opin rheumatol 2005;17:134-140. 5. national centers for health statistics, chartbook on trends in the health of americans 2006, special feature: pain. available at: http://www. cdc.gov/nchs/data/hus/hus06.pdf. assessed august 15, 2010 6. coté pdc, baldwin ml, johnson wg. early pattern of care for occupational back pain. spine 2005;30:581-7. 7. van den hoogen hjm, koes bw, eijk jtm. on the course of low back pain in general practice: a one year follow up study. ann rheum dis 1998; 57:13-9. 8. cassidy jd, côté pdc, carroll, lj, kristman v. incidence and course of low back pain episodes in the general population. spine 2005;30:281723. 9. pengel lhm, herbert rd, maher cg, refshauge km. acute low back pain: systemic review of its prognosis. bmj 2003;327:323-7. 10. cunningham cc, flynn ta, toole cm, ryan rg, gueret pwj, bulfin s, et al. llow back pain and occupation among irish health service workers. occup med 2006;56:447-54. 11. koes bw, tulder mw, thomas s.clinical review diagnosis and treatment of low back pain. bmj; 2006:332:1430-4. 12. van tulder mw, tuut m, pennick v, bombardier c, assendelft wj. quality of primary care guidelines for acute low back pain. spine 2004; 29:e357-62. 187 13. ghaffari m, alipour a, jensen i, farshad aa, vingard e. low back pain among iranian industrial workers. occup med 2006;56:455-60. 14. omokhodion fo, sanya ao. risk factors for low back pain among office workers in ibadan, southwest nigeria. occup med 2003;53:287-9. 15. latza u, karmaus w, sturmer t, neth a, rehder u. cohort study of occupational risk factors of low back pain in construction workers. occup environ med 2000;57:28-34. 16. chen sm, liu mf, cook j, bass s, lo sk. sedentary lifestyle as a risk factor for low back pain: a systematic review. int arch occup environ health 2009;82:797-806. 17. toshihiko s, yuichi k, atsumasa u. effect of driving on low back pain. occup med 2006;56: 494-6. 18. omokhodion fo, umar u.s, ogunnowo b.e prevalence of low back pain among staff in a rural hospital in nigeria. occup med 2000;502:10710. 19. maul i, laubli t, kripstein a, kruger h. course of low back pain among nurses: a longitudinal study across eight years. occup environ med 2003;60:497-503. 20. liu wcv, monroy ds, prevalence of back discomfort and estimates of back load in two manufacturing facilities. ann occup hyg 1998; 42:549-56. 21. tousignanti m, tougas g, rossignol m, goulet l. development of a systemic observation protocol of physical exposure of the back: a preliminary study. ann occup hyg 2002;46:31727. 22. hoogendoorm we, bongers pm, de vet hcw, van mechelen v, bouter im.. comparison of two different approaches for the analysis of data from a prospective cohort study: an application to work related risk factors for low back pain. occup environ med 2002;59:459-65. 23. jansen jp, burdorf a. effect of measurement strategy and statistical analysis on dose-response relations between physical workload and low back pain. occup environ med 2003;60:942-7. 24. harrianto r, editor. bahaya kerja ergonomi. dalam: buku ajar kesehatan kerja. jakarta: penerbit buku kedokteran egc;2010.p.175-244. 25. ong a, anderson j, roche j. a pilot study of the prevalence of lumbar disc degeneration in elite athletes with lower back pain at sydney 2000 olympic games. br j sport med 2003;37:2636. 26. jawish rm, assoum ha, khamis cf. anatomical, clinical and electrical observations in piriformis syndrome. j orthop surg res 2010;5:3. 27. papadopoulus ec, hkan sn. piriformis syndrome and low back pain : a new classification and review of the literature. ortoped clin north am 2004;35:65-71. 28. kuslich sd, ulstrom cl, michael cj. the tissue origin of low back pain and sciatica: a report of pain response to tissue stimulation during operations on the lumbar spine using local anesthesia. orthop clin north am 1991;22:1817. 29. bridger rs, editor. introduction to ergonomics, singapore: mcgraw-hill book co; 1995. 30. parmar ka, solomon m, loefler a, dalton s. bridging osteophyte of the anterosuperior sacroiliac joint as a cause of lumbar back pain. br j sport med 2004;38:e33. 31. kumar b, sriram kg, george c. osteophyte at the sacroiliac joint as a cause of sciatica: a report of four cases. j orthop surg 2002;10:73–6. univ med vol.29 no.3 alvina 29 abstract osteoporosis is characterized by lower bone mineral density (bmd) and microarchitectural degeneration, which tends to increase bone fragility and fracture risk. bone microstructure depends on interactions between the mineral atoms, which may perform substitution or incorporation into bone crystals, and may dynamically take over the function of calcium or may become a complementary part. the mineral atoms may also become a composite in the hydroxyapatite crystals. the aim of this study was to find an association between the bone microstructure and hydoxyapatite crystal structure in osteoporosis, in comparison to normal bone. the subjects of this study were surgery patients at the department of orthopedics of ulin general hospital in banjarmasin and other centers. inclusion criteria consisted of the presence of fracture of trabecular bone, normal or osteoporotic bmd values, and no past history of chronic disease. bone was obtained from fracture patients during surgery. the characteristics of the hydroxyapatite crystals were analyzed by x-ray diffraction (xrd) and the microarchitecture by scanning electron microscopy (sem). sem showed degeneration of the microarchitecture of osteoporotic bone, in comparison with normal bone. on xrd there was a peak of hydoxyapatite crystals only and no other crystal phases. diffraction patterns showed a larger crystal size in osteoporotic bone as compared to normal bone, indicating higher porosity. it may be concluded that there is a difference in crystal size and morphologic distribution of hydoxyapatite in osteoporotic bone, determining bone microarchitecture. keywords: microarchitecture, hydroxyapatite, osteoporosis *department of orthopedics, ulin regional general hospital, faculty of medicine, lambung mangkurat university, banjarmasin **department of biology, faculty of mathematics and natural sciences, brawijaya university, malang ***department of orthopedics, syaiful anwar regional general hospital, faculty of medicine, brawijaya university, malang ****department of orthopedics, hasan sadikin hospital, faculty of medicine, padjadjaran university, bandung *****x-ray diffraction laboratory, central laboratory, faculty of mathematics and natural sciences, malang state university correspondence dr. zairin noor, spot(k) department of orthopedics, ulin regional general hospital jl. a. yani km 2 no.43 banjarmasin 70233 email: noorzairin@yahoo.com univ med 2011;30:29-35. assessment of microarchitecture and crystal structure of hydroxyapatite in osteoporosis zairin noor*, sutiman b sumitro**, mohammad hidayat***, agus hadian rahim**** and ahmad taufiq***** january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina 30 noor, sumitro, hidayat, et al microarchitecture and crystal structure introduction the characteristic features of osteoporosis are low bone mineral density (bmd) and m i c r o a r c h i t e c t u r a l d e g e n e r a t i o n , w h i c h increases the fragility of bone and the risk of fractures.(1-3) in developing or less developed countries the prevalence of osteoporosis is still not known with certainty, due to a lack of studies. in asian population segments of those over 50 years old, osteoporosis of the lumbar spine has a prevalence of 11%-37% in women and of 5.4%-37.4% in men; osteoporosis of the femoral neck accounts for 2% of women and 6.3%-11.4% of men; and osteoporosis of the hip affects 16% of women and 3.8%-24.3% of men.(4-9) according to the white paper issued by t h e i n d o n e s i a n o s t e o p o r o s i s s o c i e t y ( p e rh i m p u n a n o s t e o p o ro s i s i n d o n e s i a , perosi), the prevalence of osteoporosis in 2007 was 28.8% in men and 32.3% in women.(10) these percentages were consistent with the results of an analysis of osteoporosis risk by the nutritional research and development center (pusat penelitian dan pengembangan gizi, puslitbang gizi) of the department of health (depkes) in cooperation with fonterra brands indonesia, in 2005 2 in 5 indonesians were at risk for osteoporosis.(11) the paradigm assumed in taking measures for improving bmd is an adequate intake of calcium, although field data show inconsistent results. the study by prentice et al.(12) revealed that osteoporosis was rarely found in the gambian population, although having a low daily dietary calcium intake. a similarly low fracture rate is found in calcium-deficient asian populations. on the other hand, a high incidence of fractures was found in western populations, which have a high calcium consumption. therefore it must be concluded that bone strength and elasticity does not solely depend on bone density but also on bone quality.(13) mammalian bone is a mineralized tissue comprising solid phases of calcium phosphate mineral and organic matrix, with a crystal structure similar to that of the geological mineral hydroxyapatite, ca10(po4)6(oh)2. biological hydroxyapatites have multiple substitutions and deficiencies at all ionic sites.(14) the microstructure of a material strongly affects its physical characteristics, such as s t r e n g t h , d u c t i l i t y, h a r d n e s s , c o r r o s i o n r e s i s t a n c e , a n d b e h a v i o r a t l o w o r h i g h temperatures. the microstructural development of bone is determined by the role of the mineral atoms. each single atom may be substituted in the bone crystals or may be incorporated into them, and may dynamically replace the function of calcium or may complement calcium and simultaneously affect the elasticity and strength of bone. bone microstructure has frequently been studied in experimental animals as well as in humans,(15-17) but there have been few studies of the microstructure of osteoporotic bone.(18) substitution and incorporation determines the pattern and quality of growth of the bony matrix, which ultimately affects the atomic configuration of the hydroxyapatite complex and interaction of collagen with hydroxyapatite, thus affecting the characteristics of the bone. in the study of ren et al.(19) it was found that substitution of ca2+ ions by zn2+ ions in the hydroxyapatite structure significantly decreased the crystal size proportionally with increasing zinc concentrations. the implication was that zinc inhibited crystallization and crystal growth in hydroxyapatite, which was consistent with the findings of miyaji et al.(20) this phenomenon is believed to be due to the difference in size of ca2+ and zn2+ ions. zn2+ ions have an ionic radius of 0.74 å, which is significantly smaller than the ionic radius of ca2+ of 0.99 å, thus the substitution of ca2+ by zn2+ results in a decrease in the lattice parameters and reduction in crystal lattice volume, leading to impaired crystal growth.(21) the current study on the crystallization profile of hydroxyapatite in osteoporotic bone, in comparison with normal bone, is the first of its kind in indonesia. the aim of this study was 31 to evaluate the relationship between the crystallization profile of hydroxyapatite and the microstructural profile of osteoporotic bone, compared to normal bone, leading to an understanding of the role of mineral atoms as the basic components of bone composite in the course of osteoporosis. methods research design a laboratory observational study using cross-sectional design was conducted to assess the microstructure and crystal profile in osteoporotic bone. study subjects p a t i e n t s u n d e r g o i n g s u rg e r y a t t h e d e p a r t m e n t o f o r t h o p e d i c s o f t h e u l i n regional general hospital in banjarmasin and at other centers. the inclusion criteria for this study were: i) presence of fracture of trabecular bone (in patients undergoing surgery); ii) normal or osteoporotic bmd values; and iii) no past history of chronic disease. tools for bone measurements during the surgery bone samples were t a k e n f o r t h e f o l l o w i n g i n v e s t i g a t i o n s / assessments: i) bone microstructure by means of scanning electron microscopy (sem); ii) characteristics of hydroxyapatite crystals in bone by x-ray diffraction (xrd); and iii) bmd. on the basis of the bmd scores, the subjects were assigned to two groups, i.e. the normal group and the osteoporosis group. according to calculations performed with epi info version 6, the mininum sample size was 32 for á =95% and â =80%. assessment of bmd was performed at ulin regional general hospital in banjarmasin and syaiful anwar regional general hospital in malang. sem and xrd were conducted in the physics laboratory and the central laboratory of the faculty of mathematics and natural sciences (fmipa), malang state hospital. xrd was also performed on standard hydroxyapatite crystals. characterization of the x-ray diffraction r e s u l t s w a s p e r f o r m e d b y m e a n s o f pa n a n a l y t i c a l x ’ p e r t p r o m p d , f o r osteoporotic and normal bone. subsequent a n a l y s i s w a s b y m e a n s o f t h e s o f t w a r e programs high score plus, crystal maker and ddview, complemented with the latest version of pdf2. diffraction spectra were recorded at an angle of 2θ, from 200 to 60o, with a cu-kα radiation source (wave length = 1.54056 å, 40 ma, 40 kv) and step size of 0.05o. mean apatite crystal size in osteoporotic and normal bone was evaluated by means of the scherrer equation.(22) rietvield refinement analysis was obtained from spectrum details of osteoporotic and normal bone apatite samples. r i e t v e l d r e f i n e m e n t a n a l y s i s w a s performed by means of the high score plus program. space groups, cell parameters, atomic p o s i t i o n s , a n d h y d r o x y a p a t i t e t h e r m a l parameters were introduced in an initial structural model. rietveld refinement was performed in several stages. in the first stage, scale factors and background were refined, followed by refinement of other parameters, respectively comprising profile parameters, z e r o s h i f t , a s y m m e t r i c p a r a m e t e r s , c e l l parameters, preferential orientation, atomic c o o r d i n a t e s , t h e r m a l p a r a m e t e r s , a n d occupancy factors. ethical clearance the study was approved by the health r e s e a r c h e t h i c s c o m m i t t e e f a c u l t y o f medicine university of brawijaya. all the subjects were informed of the purpose of the study and were requested to sign an informed consent form. statistical analysis d a t a w e r e a n a l y z e d b y a n a l y s i s o f variance followed by tukey when appropriate. a p value < 0.05 was considered significantly. univ med vol. 30 no.1 32 noor, sumitro, hidayat, et al microarchitecture and crystal structure results the results of xrd characterization are presented in figure 1, indicating that only the hydroxyapatite crystal peak was present and that no other crystal phases were detected. from search and match tests it was found t h a t a l l b o n e s a m p l e s , b o t h n o r m a l a n d osteoporotic, had a crystal phase with a hexagonal structure and space groups p63/m and 176. application of the crystal maker program yielded the crystal structure depicted in figure 2. figure 1. xrd patterns of osteoporotic bone sample (red), normal bone (blue) and standard hydroxyapatite crystals (green) figure 2. crystal structure of bone legend: o-h = red; ca = gray (large); p = gray (small); o = blue 33 from figure 2 it is apparent that o-h groups are located at the corners of the crystal lattice, whereas ca, p, and o atoms are located within the crystal lattice space or volume, in a highly regular manner. the hexagonal crystal structure has the crystal lattice parameters a = b ≠ c, with angles á = â = 900 and ã = 1200. according to crystal geometry, this structure possesses a relatively high stability. crystal sizes are presented in detail in table 1. anova test concluded that there is significantly different of crystal size in all groups (p=0.000). post hoc test concluded there is significantly different of crystal size in all groups (p=0.000). the microarchitecture of osteoporotic bone is significantly different from that of normal bone. figure 3 depicts sem images at 200x magnification, and figure 4 depicts sem table 1. cystal sizes of standard hydroxyapatite, osteoporotic bone and normal bone images at 3000x showing trabeculae with large perforations surrounded by resorption cavities. in the trabecular structure there are stump s t r u c t u r e s a n d t h e i n n e r s u r f a c e o f t h e remaining cavities appear flat and thin (a). in normal bone the resorption cavities have not yet formed stump structures and the trabecular walls are still thick with a crumpled surface (b). discussion in connection with the abovementioned results, the mechanical properties of bone should not be viewed as a table of constants, but rather as a function of various factors.(23) osteoporosis is a process progressing to the amorphic that is difficult to characterize.(24) figure 3. trabeculae in osteoporotic (a) and normal (b) bone at 200x magnification univ med vol. 30 no.1 34 noor, sumitro, hidayat, et al microarchitecture and crystal structure figure 4. trabeculae in osteoporotic (a) and normal bone (b) at 3000x magnification at 3000x magnification, osteoporotic bone consists of parallel strands of collagen fibrils with intervening furrow-like resorption cavities, and also broken fibrils (a). normal bone lacks strands and presents predominantly closely packed hill-like structures (b) in summary, the atomic mineral make-up of the bone composite determines the crystal size, which is the basis for the development of bone microarchitecture. furthermore, the results of this study also indicate that although the crystal structure in osteoporotic bone and normal bone may be of similar hexagonal geometry, at the most basic level the crystal size and bone porosity are also important in determining bone quality, either physical, mechanical, or otherwise. a limitation of this study is that sem only reveals bone structure in two dimensions, necessitating further studies using micro-ct reconstruction as a three-dimensional analytical tool for osteoporotic bone as compared to normal bone. the crystal size also needs to be investigated three-dimensionally by means of atomic force microscopy. conclusion crystal size and morphologic distribution of hydroxyapatite in osteoporotic bone differs from those of normal bone and determine the microarchitecture of bone. the larger crystal size in osteoporotic bone is a result of the atomic make-up of the composite. the porosity of bone is proportional to the crystal size, where with a larger crystal size there is increased porosity. this is the reason why normal bone in this study exhibits a smaller crystal size (has a lower porosity) in c o m p a r i s o n w i t h o s t e o p r o t i c b o n e . t h i s phenomenon may be explained by the fact that within the bone structure the larger crystal size also leads to larger-sized interstitial cavities. the larger-sized cavities within the bone increase the total number of voids, such that ultimately the void ratio also increases. it is the void ratio that increases the porosity, as shown by the agreement between xrd and sem results. according to physical fact and logic, materials with a higher porosity level have poorer mechanical properties, possessing greater fragility, lower hardness, and lower flexibility, in consequence of the weaker bonds between the bone crystals. the present study found a reduction in bone mass and deterioration of its microarchitecture, which is comparable with the findings of shen.(18) 35 acknowledgement the author thank mr. abdulloh fuad and ms. zulaikha in malang state of university for sample preparation. references 1. 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samara*, purnamawati tjhin**, and magdalena wartono** *occupational health department ** anatomy department medical faculty, trisakti university, jakarta correspondence adr. ridwan harrianto, mh.sc.(o.m).sp.ok occupational health department medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2101 email: riddharr@cbn.net.id univ med 2009;28:170-8 abstract disorders of the musculoskeletal system constitute a considerable health problem in industrialized societies. low back pain (lbp) remains a common and costly problem among the workers. workplace injuries, primarily musculoskeletal disorders, are a persistent problem for nursing. a cross-sectional study was conducted to investigate prevalence of lbp as well as the potential risk factors associated with lbp. these potential risk factors include individual characteristics, job duration, type of work, and manual handling knowledge and practice. seventysix subjects consisting of 30 nurses and 46 administrative workers were recruited into the study. symptoms of lbp cases were assessed by means of a simple questionnaire, and the diagnosis of lbp was confirmed by clinical examination. the study showed that the overall point prevalence rate of lbp was 42.1%, while the overall 12-month prevalence rate was 69.7%. occupational group and manual handling practice were independent predictors for point prevalence of lbp. nursing occupation significantly increased the risk of point prevalence rate of lbp by 2.703 (95% c.i. or 1.046 – 6.984) compared to administrative workers. nurses with frequent manual handling practice had a 2.917-fold higher risk of developing a point prevalence of lbp, although this risk was statistically not significant (or=2.917;95% c.i. 0.094-3.003). the study indicates that an association exists between manual handling practice and lbp prevalence. keywords: low back pain, manual material handling, working pattern introduction l o w b a c k p a i n ( l b p ) i s a p r o b l e m frequently complained of by every individual, particularly workers. the frequency of lbp approaches that of flu,(1) as more than 85% of persons has at one time in his or her lifetime ever suffered from lbp.(1,2) in an american study 171 univ med vol.28 no.3 50% of workers suffered yearly from lbp.(2) another study stated that lbp in the united states was responsible for the loss of 149 million work-days per year, with 102 million work-days lost due to occupational lbp. it was estimated that the cost of lost work time reached an amount of $3000 for each insurance compensation claim.(3) lbp is a sharp or dull pain or muscular stiffness occurring in the back, i.e. the region between the lower costal margins and gluteal folds, the pain being either localized or radiating into the lower extremities (ischialgia).(4,5) koes et al.(5) in the netherlands stated that among all patients with lbp 4% was identified with compression fractures, 3% with spondylolisthesis, 0.7% due to tumors or tumor metastases, 0.3% due to ancylosing spondylitis, and 0.01% caused by infection, while among the remaining cases more than 90% had no identifiable cause and was classified as non specific lbp. (2) lbp is designated as acute lbp if it disappears in less than 6 weeks, as subacute lbp if it persists for 6 weeks to 3 months and as chronic lbp if it persists for more than 3 months. chronic lbp frequently leads to invalidity, such that the individual concerned is unfit for work.(4) the recurrence rate of lbp is also high (80%), where the 12-month recurrence rate is around 20-44%.(1) the point prevalence rate in the general population was reported by several studies to be around 14-30%,(6) whilst the point prevalence rate in workers was 15-30%.(7) the study by omokhodion and sanya(8) among office workers in nigeria revealed an annual/12-month lbp prevalence of 38%, while the point prevalence w a s 2 0 % . t h e a n n u a l p r e v a l e n c e r a t e s i n industrial workers(7) and construction workers(9) are 21% and 30.9%, respectively, whilst the point prevalence rate in workers packing detergent powder is 36.8%.(10) back pain is also a very prevalent condition among health care workers. for health care workers in different specialities the 12-month prevalence for pain in the lumbar spine has been reported as being as high as 76%. (11,12) a number of studies on nursing occupations yielded 12-month prevalences of 30%,(6) 69%(13) and 73-76%(14) while the point prevalence was 54.7%.(14) studies investigating occupational risk factors for lbp have tended to focus on workrelated mechanical risk factors with many using job title as a proxy measure of physical load. a study reported increased associations between mechanical factors, such as manual handling, handling heavy loads and frequent bending and twisting, and the risk of lbp.(15) this work pattern, termed manual material handling (mmh), is frequently encountered in n u r s i n g o c c u p a t i o n s , s u c h a s l i f t i n g a n d transferring of patients, walking or moving, abnormal postures of the trunk (bowing, lateral bending, torsion of the trunk) and pushing, p u l l i n g t r o l l e y s a n d m a n i p u l a t i n g h e a v y equipment.(16,17) more recently, work-related psychosocial factors have also been considered as risk factors for lbp.(18,19) there is evidence that individuals who physically transfer or lift objects (manual material handling) on a regular basis increase the strain on their backs and the r i s k o f d e v e l o p i n g b a c k p a i n . i n m a n y occupations, it is difficult to avoid this. the present study aims to measure the prevalence rate o f l b p a m o n g n u r s e s a s c o m p a r e d t o administrative workers and its association with work-related patient handling activities. methods design this was a cross-sectional study in which n u r s e s a n d a d m i n i s t r a t i v e w o r k e r s w e r e recruited. 172 research location and time frame the study was conducted at the medical faculty, trisakti university from september 2006 until february 2007. subjects subjects were nurses with a job duration of several years in a private hospital in jakarta, who w e r e a t t e n d i n g b i o m e d i c a l c o u r s e s a t t h e medical faculty, trisakti university, as part of the strata i program in nursing science, while administrative personnel of the medical faculty, trisakti university funtioned as the control group. all participants consented to become study subjects and signed an informed consent form for this study. exclusion criteria were nurses without nursing jobs or administrative workers without administrative work, persons with traumatic back pain, and persons with a past history of renal disease, tuberculosis of the spine or malignancies. the 12-month prevalence rate estimate of 70%(15) for low back pain was used to determine sample size. assuming the 12-months prevalence rate in administrative workers to be 35%, it was estimated that 29 subjects were required to have 80% power of detecting a risk associated with lbp (95% confidence interval).(20) data collection data collection was conducted by the investigators, using a questionnaire regarding the respondents’ demographic characteristics, type of work, and job duration. the diagnosis of lbp was established on the basis of past or current attacks of lbp at the present work location in the previous year, and/or tenderness of the paralumbal/gluteal/sacroiliac musculature, with or without a positive straight-leg raising test, and with or without radiation of the pain into the legs. assessment of knowledge of mmh techniques was performed by presenting the subjects with six illustrations of work posture in manual handling tasks, selected from “ergonomic checkpoints: practical and easy-to-implement solutions for improving safety, health and working conditions”.(21) this method has been u s e d b y p r e v i o u s i n v e s t i g a t o r s f o r t h e i r studies,(10) where the subjects were asked to choose between right or false and to give their r e a s o n s . s c o r i n g w a s a c c o m p l i s h e d b y evaluating aeach question according to the following criteria. a score of 0 was given if the answer was considered completely incorrect, a score of 1 for a nearly correct answer, and a score of 2 for correct answers. the scores for all six answers were then summed and the resulting total was categorized as follows: a total score of 7–12 was considered evidence of an understanding of the mmh techniques presented in the questionnaire, whereas a total score of 0–6 denoted ignorance of the mmh techniques in question. measurement of height and weight of the subjects was by means of smic instruments made in china. physical examination was performed by the investigators, while the diagnosis of lbp was established on the basis of the previously defined criteria. data analysis data were entered into the statistical package for the social sciences (spss v 15) software program. nominal, binary and interval data were analysed using descriptive statistics. bivariate analysis was used to examine for the associations between age, gender, job duration, manual handling, prevalence of low back pain, with occupational group. chi-square tests were used for categorical data and independent-t tests for continuous data. data were further analysed using multivariate logistic regression techniques to explore for independent predictors of point prevalence of lbp. harrianto, samara, tjin, et al low back pain among workers 173 univ med vol.28 no.3 results a total of 76 respondents were recruited into this study, comprising 30 nurses (3 male and 27 female nurses) and 46 administrative workers (26 male and 20 female workers). the youngest subject was 20 years of age and the eldest 60 years, with a mean age of 34.8 years and a standard deviation (sd) of 9.6. the majority of the subjects, viz. 44 (57,9%) had an academic educational background, 5 subjects had studied at primary school or junior high, comprising 2 with a primary school certificate and 3 with a junior high school certificate, whereas 27 subjects were educated at senior high school. the mean job duration was 10.6 years (sd = 8.6). m i n i m u m j o b d u r a t i o n w a s o n e y e a r a n d maximal job duration 37 years. approximately 67.1% of respondents had poor knowledge of manual handling techniques, and 71.1% rarely had manual handling practice. on t he d ay of the examination, the point prevalence of lbp was 42.1% and the 12-month prevalence of lbp 69.7% (table 1). characteristics n (%) gender male female occupational group nursing administration job duration group (yrs) < 10 > 10 job duration (yrs, mean ± sd) age groups (yrs) < 35 > 35 age (yrs, mean ± sd) manual handling knowledge yes no manual handling practice yes no point prevalence of lbp yes no annual prevalence of lbp yes no 29 (38.2) 47 (61.8) 30 (39.5) 46 (60.5) 46 (60.5) 30 (39.5) 10.6±8.6 44 (57.9) 32 (42.1) 34.8±9.6 25 (32.9) 51 (67.1) 22 (28.9) 54 (71.1) 32 (42.1) 44 (57.9) 53 (69.7) 22 (30.3) table 1. profile of respondents (n=76) occupational group characteristics nursing (n=30) administration (n=46) p gender male female 3 (10.3%) 27 (57.4%) 26 (89.7%) 20 (42.8%) 0.0000 age, yrs, mean (sd) 31.8 (8.2) 36.8 (10.1) 0.0281 job duration, yrs, mean (sd) manual handling knowledge yes no manual handling practice yes no point prevalence of lbp yes no annual prevalence of lbp yes no 7.9 (6.2) 16 (64.0%) 14 (27.5%) 22 (100.0%) 8 (14.8%) 17 (53.1%) 13 (29.5%) 24 (45.3%) 6 (21.6%) 12.4 (9.6) 9 (36.0%) 37 (72.5% 0 (0.0%) 46 (85.2%) 15 (46.9%) 31 (70.5%) 29 (55.7%) 17 (78.4%) 0.0271 0.002 0.0000 0.0380 0.1162 table 2. profile of respondents by occupational group (n=76) 174 males were significantly more frequently employed as administrative workers (89.7%) in comparison with nurses (42.8%). on average the administrative workers were older (36.8 ± 10.1) than the nurses (31.8 ± 8.2). regarding type of occupation, the point prevalence of lbp was significantly higher among nurses (53.1%) compared with that of administrative workers (46.9%). manual hanling knowledge and manual handling practice was significantly better in nurses than in administrative workers (table 2). the occurrence of a significant difference in point prevalence between nurses and administrative workers requires further analysis. a number of risk factors of point prevalence of lbp were examined, namely gender, age group (<40 and >40 years), job duration (<10 and >10 years), type of occupation, manual handling knowledge and manual handling practice. table 3 indicates that occupational group and manual handling practice were independent predictors for point prevalence of lbp. nursing occupation significantly increased the risk of point prevalence of lbp by 2.703 (95% c.i. or 1.046 – 6.984) compared to administrative workers. respondents who did frequent manual handling practice had a 3.501fold significantly higher risk of point prevalence of lbp compared with those who had no frequent manual handling practice (95% c.i.; or 1.241 – 9.849). based on the analytical results on table 3 a stratification of the respondents should be performed by occupation (table 4). nurses with frequent manual handling p r a c t i c e h a d a 2 . 9 1 7 f o l d h i g h e r r i s k f o r developing a point prevalence of lbp compared with nurses with infrequent manual handling practice, but the risk was statistically not significant (or=2.917;95% c.i. 0.094-3.003). risk factors odds ratio (or) 95% confidence interval or gender female male age group (yrs) < 40 > 40 job duration group (yrs) < 10 > 10 occupational group nursing administration manual handling knowledge poor good manual handling practice frequent rare 1.672 reference 0.900 reference 1.087 reference 2.703 reference 1.460 reference 3.501 reference 0.643 – 4.351 0.357 – 2.268 0.429 – 2.756 1.046 -6.984 0.545 – 3.912 1.241 – 9.849 table 3. risk factors of point prevalence of lbp in respondents (n=76) harrianto, samara, tjin, et al low back pain among workers 175 univ med vol.28 no.3 discussion the point prevalence of lbp was 42.1% in all respondents and the annual prevalence of lbp 69.7%. these study results were similar to those of a study in iran with an annual prevalence of lbp of 84% in industrial workers.(7) the present study showed a point prevalence rate of lbp and an annual prevalence rate of lbp in nurses of 53.1% and 46.9%, respectively. these results are consistent with a study in hongkong nurses where of the 377 nurses interviewed, 153 (40.6%) reported having lbp within the last 12 months.(22) in comparison, taiwanese nurses had a point prevalence rate of lbp of 66.0%.(23) however, the results obtained in our study showed a point prevalence of lbp and an annual prevalence rate of lbp that were greater than those found in irish nurses (respectively 15.5% and 30%).(6) the higher point prevalence of lpb found in the present study is presumably due to the fact that the diagnosis was established by a c o m b i n a t i o n o f i n t e r v i e w s a n d p h y s i c a l examination, as the symptoms and signs arising in mild cases that may be found on physical examination are frequently ignored by the patients. our study showed an annual prevalence rate of lbp in nurses which was not significantly different from the rate in administrative workers (respectively 45.3% and 55.7%). these lbp annual prevalence rates suggest that lbp prevalence among health service workers is no greater than that in the general population. this is consistent with the findings of maui et al.(14) who found no difference in lbp prevalence between nursing and non-nursing groups despite the fact that nursing is generally regarded as a high-risk occupation for lbp. however, contrary results were obtained in a study in hongkong indicating a high occurrence rate of lbp in nurses (40.6%).(22) such a high occurrence rate may be accounted for by the nature of nursing w o r k . t h e r e s u l t s o f o u r s t u d y c l e a r l y demonstrates that the annual prevalence rate of lbp is higher than the point prevalence rate of lbp. this fact suggests that lbp is chronic and recurrent in nature. the difference in lbp prevalences indicate a difference in assessment of lbp. two previous studies indicated that the risk factors for lbp d i ff e r w i t h h o w t h e l b p i s d e f i n e d a n d measured.(24,25) the day-to-day work routines proved to be a risk factor in the development of the point prevalence of lbp. nurses had a higher risk of s u ff e r i n g f r o m l b p i n c o m p a r i s o n w i t h administrative workers. in connection with mmh practice, the group of workers with table 4. manual handling practice as risk factor of point prevalence of lbp by occupational group (n=76) *all respondent in the administrative group rarely had manual handling practice, therefore the odds ratio could not be determined occupational group exp(β) 95% confidence interval exp(β) nursing (n=30) manual handling practice frequent rare 2.917 reference 0.094 – 3.003 administration manual handling practice* --- 176 f r e q u e n t m a n u a l h a n d l i n g p r a c t i c e h a d a significantly higher risk of lbp. stratification analysis showed that in nurses with frequent manual handling practice the risk of developing lbp was higher than in those with infrequent m a n u a l h a n d l i n g p r a c t i c e , a l t h o u g h t h e difference was not statistically significant. this may be because the assumption that formed the basis for the calculation of sample size was i n c o r r e c t . t h e a s s u m p t i o n w a s t h a t t h e prevalence of lbp in administrative workers would be 50% lower than in nurses, whereas actually in this study the lbp prevalence in administrative workers was 20% greater than in nurses. thus the sample size in this study was unable to demonstrate a significant difference between nurses with frequent manual handling practice and those with infrequent manual handling practice. the foregoing demonstrate that the technical knowledge of posture and work methods in occupations requiring heavy physical activity, such as lifting, lowering, pushing, pulling, throwing, supporting, transferring weights, or postures with frequent bending and/ or bowing, and sitting or standing still, may in the long term decrease the risk of lbp. h o w e v e r, a t t h e m o m e n t t h e r e i s controversy rather than agreement among the existing study results. one systemic review found no evidence that training with or without lifting equipment is effective in the prevention of back pain or consequent disability. either the advocated techniques did not reduce the risk of back injury or training did not lead to adequate change in lifting and handling techniques.(26) the study of hignett(27) reported that intervention strategies relying exclusively on training in correct manual handling techniques did not reduce the risk of lbp. the study performed by martimo et al.(28) revealed that training in correct manual handling techniques with or without mechanical aids was unable to prevent the occurrence of lbp or invalidity. other studies h a v e s t a t e d t h a t m u l t i p l e i n t e r v e n t i o n s comprising mmh training accompanied by administrative interventions, such as improving work policies and procedures, and by the use of m e c h a n i c a l w e i g h t l i f t i n g a i d s , a n d b y ergonomical redesign of behavior, equipment and work environment may reduce the risk of lbp.(29,30) conclusions a major finding in this study was the high p r e v a l e n c e o f l b p a m o n g n u r s e s a n d administrative workers. there was an association b e t w e e n ‘ m a n u a l h a n d l i n g p a r a c t i c e a n d occupation on the one hand, and point prevalence of low back pain on the other, especially in nurses. these findings have important implications for the prevention of occupational lbp, particularly for nurses. good posture and correct transferring techniques in ward situations should be reinforced with hands-on practice performed on nurses’ common types of clients. acknowledgements we would like to express our gratitude to the dean and vice-deans of the medical faculty, trisakti university, for the funding of the present s t u d y a n d t h e i r c o n t i n u i n g s u p p o r t u n t i l completion of this study. we also thank all respondents consenting to participate in this study. references 1. zanni gr, wick jy. eliminating myths and elucidating realities. j am pharm assoc 2003;43: 357-62. 2. patel at, ogle aa. diagnosis and management of acute low back pain. am fam physician 2000;61: 1779-86. harrianto, samara, tjin, et al low back pain among workers 177 univ med vol.28 no.3 3. cote pdc, baldwin ml, johnson wg. early pattern of care for occupational back pain. spine 2005;30: 581-7. 4. manek nj, macgregor aj. epidemiology of back disorders: prevalence, risk factors, and prognosis. curr opin rheumatol 2005;17:134-40. 5. koes bw, tulder mw, thomas s.clinical review diagnosis and treatment of low back pain. bmj 2006;332:1430-4. 6. cunningham c, flynn t, blake c. low back pain and occupation among irish health service workers. occup med 2006;56:447-54. 7. 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19. linton sj. occupational psychological factors increase the risk for back pain: a systematic review. j occup rehabil 2001;11:53–66. 20. browner ws, newman tb, cummings sr, huley sb. estimating sample size and power: the nittygritty. in: hulley sb, cummnings sr, browner ws, grady d, hearst n, newman tb, editors. designing clinical research: an epidemiological approach. 2nd ed. new york: lippincott willims & wilkins;2001. p.65-91. 21. international labor office. ergonomic checkpoints: practical and easy-to-implemen solutions for improving safety, health and working conditions. geneva: ilo publications;1999. 22. yip yb. a study of work stress, patient handling activities and the risk of low back pain among nurses in hong kong. j advanced nursing 2001;36:794804. 23. feng ck, chen ml, mao if. prevalence of and risk factors for different measures of low back pain among female nursing aides in taiwanese nursing homes. bmc musculoskeletal disorders 2007;8: 52-60. 24. violante fs, fiori m, fiorentini c, risi a, garagnani g, bonfiglioli r, et al. associations of psychosocial and individual factors with three different categories of back disorder. j occup health 2004;46:100-8. 25. ozguler a, leclerc a, landre mf, pietri-taleb f, niedhammer i. individual and occupational determinants of low back pain according to various definitions of low back pain. j epidemiol community health 2000;54:215-20. 26. martimo kp, verbeek j, karppinen j, furlan ad, kuijer ppfm, viikari-juntura e, et al. effect of training and lifting equipment for preventing back pain in lifting and handling: systemic review. bmj 2008;336:429-31. 27. hignett s. intervention strategies to reduce musculoskeletal injuries associated with handling patients: a systemic review. occup environ med 2003;60:e6. 178 28. martimo kp, verbeek jh, karppinen j, furlan ad, kuijer ppfm, viikari-juntura e, et al. manual material handling advice and assistive devices for preventing and treating back pain in workers. cochrane database syst rev 2007:cd005958. 29. carrivic pjw, lee ah, yau kkw. cosultative team to assess manual handling and reduce the risk of harrianto, samara, tjin, et al low back pain among workers occupational injury. occup environ med 2001;58: 339-4. 30. koopelaar e, knibbe jj, miedema hs. determinants of implementations on pateient handling in healthcare: a systematic review. occup environ med 2009;66:353-60. oktavianus 179 *faculty of medicine, yarsi university, jakarta **faculty of medicine and health sciences, muhammadiyah university of jakarta ***faculty of medicine, maranatha christian university, bandung correspondence susi endrini, ssi., msc., phd faculty of medicine yarsi university cempaka putih jakarta 10510 phone : +6221-4213065 email: susi.endrini@yarsi.ac.id/ sendrini@yahoo.com univ med 2014;33:179-84 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background plant-derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. they are freely available natural compounds that can be safely used to prevent various ailments. plants have been the basis of traditional medicine throughout the world for thousands of years and are providing mankind with new remedies. the objective of this study was to determine the cytotoxicity of soursop (anona muricata linn) leaves and pearl grass (hedyotis corymbosa (l.) lam.) on the hormone-dependent human breast carcinoma michigan cancer foundation-7 (mcf-7) cell line. methods this study used two types of solvents (water and ethanol) in the extraction process and two incubation times (24 hours and 48 hours) in the mtt assays to analyze the cytotoxic effects of both plants. results preliminary results showed that the ethanolic extract of soursop leaves (se) displayed cytotoxic effects against mcf-7 on 24and 48-hour incubation times with ic 50 values of 88.788 µg/ml and 14.678 µg/ml, respectively. ethanolic pearl grass extract (pe) showed similar results, with ic 50 values of 65.011 µg/ ml on 24-hour incubation time and 52.329 µg/ml on 48-hour incubation time against mcf-7 cell line. however, the water extract of both plants displayed lower cytotoxic effect against mcf-7 cell line. conclusion the ethanolic extract of both plants displayed cytotoxic effect against mcf-7. soursop (anona muricata linn) leaves have the strongest cytotoxic activity against mcf-7 breast cancer cell line. keywords: microculture tetrazolium salt assay, hedyotis corymbosa, annona muricata, mcf-7 annona muricata leaves have strongest cytotoxic activity against breast cancer cells susi endrini*, suherman**, and wahyu widowati*** 180 endrini, suherman, widowati soursop leaf cytotoxic activity against breast cancer daun sirsak mempunyai aktifitas sitotoksik paling kuat terhadap kultur sel kanker payudara latar belakang senyawa-senyawa yang diturunkan dari tumbuhan memiliki sejarah yang panjang dalam penggunaan klinis, toleransi pasien yang lebih baik dan juga penerimaannya. senyawa-senyawa tersebut adalah senyawa alami yang tersedia secara bebas yang dapat digunakan secara aman untuk mencegah berbagai penyakit. tanaman menjadi dasar sistem pengobatan tradisional di seluruh dunia selama ribuan tahun dan memberikan obat baru kepada manusia. penelitian ini dilakukan untuk menentukan efek sitotoksisitas daun sirsak (annona muricata linn ) dan rumput mutiara (hedyotis corymbosa (l.) lam.) terhadap kultur sel karsinoma payudara manusia tergantung hormon michigan cancer foundation-7 (mcf-7). metode dalam studi ini, kami telah menggunakan dua jenis pelarut (air dan etanol) dalam proses ekstraksi dan dua jenis waktu inkubasi (24 jam dan 48 jam) dalam tes mtt untuk menganalisis efek sitotoksik dari kedua tanaman . hasil hasil awal menunjukkan bahwa ekstrak etanol daun sirsak (se) yang ditampilkan efek sitotoksik terhadap mcf 7 pada waktu 24 jam dan 48 jam inkubasi dengan masing-masing nilai ic 50 adalah 88,788 µg/ml dan 14,678 µg/ ml. di sisi lain, ekstrak etanol rumput mutiara (pe) juga menunjukkan hasil yang serupa dengan nilai ic 50 65,011 µg/ml pada 24 jam waktu inkubasi dan 52,329 µg/ml pada waktu 48 jam inkubasi terhadap mcf-7. namun, ekstrak air dari kedua tanaman menampilkan efek sitotoksik yang lebih rendah terhadap mcf-7. kesimpulan daun sirsak (annona muricata linn) mempunyai aktifitas sitotoksik paling kuat terhadap kultur sel kanker payudara mcf – 7. kata kunci: uji microculture tetrazolium (mtt), hedyotis corymbosa, anona muricata, mcf-7 abstrak introduction breast cancer incidence in asia is escalating more rapidly than in the west. breast cancer is one of the most prevalent cancers in indonesian females.(1) in the other hand, the plant derived herbal compounds have a long history of clinical use, better patient tolerance and acceptance. they are freely available natural compounds that can be safely used to prevent various ailments. plants have been the basis of traditional medicine throughout the world for thousands of years and are providing mankind with new remedies.(2) in the case of human cancers, thus far, nine plantderived compounds have been approved for clinical use as anticancer drugs in the united states. in the cancer drug discovery program, a p a r a d i g m b a s e d o n e t h n o b o t a n i c a l a n d ethnopharmacological data would be more economical and beneficial for identifying potential anticancer molecules than mass screening of plant species.(3) indonesians have known a lot of traditional medicinal plants, among which are soursop and pearl grass. pearl grass (hedyotis corymbosa (l.) lam.) (synonym: oldenlandia corymbosa linn.) 181 of the rubiaceae family, is a weedy annual herb. the plant is known to clear heat and toxins, activate blood circulation, promote diuresis and relieve stranguria. it is also active against appendicitis, hepatitis, pneumonia, cholesystitis, urinary infection, cellulites and snake bite. chinese folk medicine prescribes the plant for treatment of skin sores, ulcers, sore throat, bronchitis, gynecologic infections and pelvic inflammatory diseases.(4) the anticarcinogenic properties of methanolic extracts of pearl grass have been published in our previous study.(5) in the present study we have continued our study using different solvents (water and ethanol) to achieve better results. annona muricata (common name: soursop) is a lowland tropical fruit-bearing tree in the annonaceae family. other common names are graviola and guanábana (sometimes shortened to guanába). related species include cherimoya (a. cherimola) and sugar-apple (a. squamosa), and paw paw (asimina triloba). the soursop is native to tropical central and south america and the caribbean, but is now widely cultivated in tropical areas worldwide, including southern florida and southeast asia, from sea level to altitudes of around 1150 meters. soursop has numerous traditional medicinal uses in south america and the caribbean, and has become a popular nutritional medicinal supplement. fruit, seeds, bark, leaves, and roots have all been used to treat intestinal parasites, coughs (including a s t h m a a n d b r o n c h i t i s ) , l i v e r a i l m e n t s , inflammation, diabetes, and hypertension. the seeds are insecticidal and a preparation from the leaves has been used to kill headlice and bedbugs.(6) numerous studies on plants from the annonaceae family have been carried out. a. montana has cytotoxic effect on the human liver c a n c e r c e l l l i n e h e p g 2 . (7) t h e s e e d s o f a.crassiflora have high antioxidant activity.(8) the purpose of this study was to determine the cytotoxic effects of pearl grass and soursop leaf extracts using different solvents by the microculture tetrazolium salt (mtt) assay on the hormone-dependent human breast carcinoma michigan cancer foundation-7 (mcf-7) cell line. methods t h i s s t u d y h a s b e e n d o n e u s i n g a n experimental research design and conducted in i n t e g r a t e d r e s e a r c h l a b o r a t o r y, ya r s i university jakarta from january to october 2013. plant materials and extractions for preparing the ethanolic extract, dried whole pearl grass (1 kg) and soursop leaves (1 kg) were extracted with 70% ethanol at room temperature. the extracts were then filtered through a whatman no. 1 filter paper. the collected filtrates were evaporated to dryness under vacuum at 40°c using a rotary evaporator (n-1000v-w, eyela, japan). the extraction methods were according to ali et al. (9) with slight modifications. after evaporation, the yield of dried ethanolic extracts (se and pe) was about 10% of the original plant samples. for preparing the water extract, dried whole pearl grass (1 kg) and soursop leaves (1 kg) were extracted with water at room temperature. the extracts were then filtered through a whatman no. 1 filter paper. the filtrates were freeze-dried and the dried water extracts (sw and pw) were collected. cell cultures the mcf-7 cell line was obtained from the american type culture collection (atcc, usa). the cells were grown in dulbecco’s m o d i f i e d e a g l e m e d i u m ( g i b c o , u s a ) supplemented with 10% of fetal calf serum, 100 iu/ml penicillin and 100 µg/ml of streptomycin (gibco, usa) using 25 cm2 flasks (nunc, denmark), in a co 2 incubator (sanyo, japan) at 37°c. mtt assay the viability of cells was determined with trypan blue. exponentially growing cells were univ med vol. 33 no.3 182 endrini, suherman, widowati soursop leaf cytotoxic activity against breast cancer harvested, counted by hemocytometer and diluted with medium, yielding a concentration of 1 x 105 cells/ml. from this cell suspension, 100 µl was pipetted into 96-well microtiter plates (nunc, denmark) and incubated for 24 h in a 5% co 2 incubator (sanyo, japan) at 37°c. the diluted range of test extracts were 0.468, 0.937, 1.875, 3.750, 7.5, 15 and 30 µg ml-1. after adding the extract samples, new medium was added to make up to a final volume of 200 µl per well. the plate was then incubated in the 5% co 2 incubator at 37°c for 24 and 48 h. then 20 µl of mtt reagent (roche, usa) was added into each well. the plate was incubated again for 4 h in the sanyo co 2 incubator at 37°c. after incubation, 200 µl solubilization solution (roche, usa) was added into each well. the cell was then left overnight at 37°c in the 5% co 2 incubator. finally, the absorbance was read with the elisa reader (lx-800). results as mentioned above, the ethanolic extracts and the water extracts of the studied plants were analyzed for their cytotoxic effects on the mcf7 cell line by the mtt assay using different incubation times. the mcf-7 standard curve used for calculation of ic 50 values is shown in figure 1. the ic 50 values against the mcf-7 cell line of the ethanolic extract of soursop leaves (se), the water extract of soursop leaves (sw), the ethanolic extract of pearl grass (pe) and the water extract of pearl grass (pw) for different incubation time (24 hours and 48 hours) are shown on table 1. doxorubicin has been used table 1. ic 50 values of extracts on 24 hours and 48 hours incubation time there were significant differences between groups a, b, c, and d at 24 and 48 hours. p-value was calculated using a one-way anova test with a value of less than 0.05 indicating significance figure 1. standard curve of mcf-7 183 as a positive standard, with ic 50 values of 0.147 µm and 0.000325 µm against mcf-7 on 24and 48-hour incubation times, respectively. there were significant differences between groups of ic 50 values (p<0.05). the strongest cytotoxic activity was shown by soursop leaves. discussion this study has shown that the ethanolic extract of soursop leaves (se) has potential cytotoxic effects on mcf-7 cells and that the effect increased with incubation time. the highest cytotoxic effect of this extract was displayed by the lowest ic 50 value that has been achieved. rachmani et al.(10) and fidianingsih et al.(11) have reported that annona muricata was cytotoxic against t47d cells. paul et al.(2) found that hela cells treated with 75 µg of a crude leaf extract of annona muricata showed 80% cell inhibition. they also detected in the crude leaf extract of annona muricata several bioactive compounds, such as anonaine, friedelin, isolaureline, a n n o n a m i n e , a n o m u r i n e , k a e m p f e r o l , asimilobine, quercetin, and xylopine. however, the water extract of soursop leaves (sw) did not display the potential cytotoxic activities. on the other hand, pearl grass has also displayed similar results. the ethanolic extract of pearl grass (pe) has potential cytotoxic effect on mcf-7 cell lines. however, the cytotoxic effect of its ethanolic extract was lower compared to the methanolic extract from our previous study.(5) lee et al.(12) reported that pearl grass extract had a significantly inhibited cell growth and induced apoptosis in colo 205 (colon cancer), hep3b (hepatocellular carcinoma) and h460 (lung cancer) cell lines. andriyani et al.(13) have reported that pearl grass extract displayed cytotoxicity against t47d cells. the ethanolic e x t r a c t o f p e a r l g r a s s l e a v e s h a s s h o w n significant anticancer activity on k562 human leukemia cell lines.(14) based on our previous study, the anticarcinogenic properties of pearl grass could be due to its high antioxidant activities. according to sasikumar et al.,(15) pearl grass extract exhibited high antiradical activity against abts, nitric oxide and hydroxyl radicals, with ec 50 values of 150, 130, and 170 ìg/ml, respectively. the pearl grass extract also enhanced the hepatoprotective in albino rats.(16) the alcoholic extract of pearl grass has also shown significant antiulcer activity against aspirin in rats.(17) pearl grass extract were reported to have a number of common bioactive constituents, including ferulic acid, flavonols, flavones, vanillic acid, syringic acid, and caffeic acid.(18) another study has also reported that the methanolic extract of pearl grass contains many bioactive chemical constituents including alkaloids, glycosides, terpenoids, steroids, flavonoids and tannins.(19) two groups of isolated c o m p o u n d s f r o m w h o l e p l a n t s a r e t h e genoposides and iridoid glycosides.(20) the iridoid g l y c o s i d e s h a v e s h o w n a v a r i e t y o f pharmacological activities, such as antioxidant, analgesic, antibacterial, antimalarial, anticancer and hepatoprotective effects.(21) the mechanisms of the cytotoxic effects of both plants are being studied. the different ic 50 values from different solvent extraction methods may have been due to the bioactive compounds of both plants. the anticarcinogenic properties of a combination of both plants are being studied to explore better treatments for breast carcinoma with inflammation. conclusions the strongest cytotoxic properties against the mcf-7 cell line were displayed by soursop leaves with ethanol as a solvent in the extraction process and 48-hour incubation time. the different ic 50 values from different solvent extraction methods may have been due to the bioactive compounds of both plants. acknowledgement we gratefully acknowledge the financial support of the directorate general for higher education, ministry of national education, univ med vol. 33 no.3 184 endrini, suherman, widowati soursop leaf cytotoxic activity against breast cancer republic indonesia, for decentralization research grant (hibah unggulan perguruan tinggi) to yarsi university for 2012-2013. references 1. ng ch, phaty nb, taib na, teh yc, mun ks, amiruddin a, et al. comparison of breast cancer in indonesia and malaysia: a clinico-pathological study between dharmais cancer centre, jakarta, and university malaya medical centre, kuala lumpur. asian pacific j cancer prev 2011;12: 2943-6. 2. paul j, ganan r, jayadeepa rm. anticancer activity of graviola, an exciting medicinal plant extract vs various cancer cell lines and a detailed computational study on its potent anti-cancerous leads. curr top med chem 2013;13:1666-73. 3. cochrane cb, nair pkr, melnick sj, resek ap, ramachandran c. anticancer effects of annona glabra plant extracts in human leukemia cell lines. anticancer res 2008;28:965-72. 4. patel td, jain v, dodia r. oldenlandia corymbosa l: a phytopharmacological review. int j phytophar 2014;4:79-82. 5. endrini s. antioxidant activity and anticarcinogenic properties of “rumput mutiara” {hedyotis corymbosa (l.) lam.} and “pohpohan” {pilea trinervia (roxb.) wight}. j medicinal plant res 2011;5:3715-8. 6. courteau j. annona muricata. encyclopedia of life. available at: http://www.eol.org/. accessed june 6, 2012. 7. liaw cc, chang fr, chen sl, wu cc, lee kh. novel cytotoxic monotetrahydrofuranic annonaceus acetogenins from annona montana. bioorg med chem 2005;13:4767-76. 8. roesler r, catharino rr. malta lg. antioxidant activity of annona crassiflora: characterization of major components by electrospray ionization mass spectrometry. food chem 2007;104:104854. 9. ali am, macjen m, hamid m, lajis nh, el ss, murakoshi m. antitumor promoting and antitumor activities of the crude extract from leaves of juniperus chinensis. j ethnopharmacol 1996;53:165-9. 10. rachmani epn, suhesti ts, widiastuti r, aditiyono a. the breast of anticancer from leaf extract of annona muricata against cell line in t47d. int j appl sci technol 2012;2:157-64. 11. fidianingsih i, handayani es. annona muricata aqueous extract suppresses t47d breast cancer cell proliferation. univ med 2014;33:19-26. 12. lee hz, bau d, kuo cl, tsai ry, hen yc, chan yh. clarification of the phenotypic characteristics and anti-tumor activity of hedyotis diffusa. am j chin med 2011;39:20113. 13. andriyani r, risdian c, udin z. cytotoxicity assay from fractions of hedyotis corymbosa extract against breast cancer cell line t47d. indones j cancer chemoprev 2011;2:182-6. 14. pandey k, sharma pk, dudhe r. anticancer activity of parthenium hysterophorus and oldenlandia corymbosa lam by srb method. scientific reports 2012;6:1-3. 15. sasikumar jm, maheshu v, aseervatham gs, darsini dt. in vitro antioxidant activity of hedyotis corymbosa (l.) lam. aerial parts. indian j biochem biophys 2010;47:49-52. 16. chimkode r, patil mb, jalalpure s, pasha ty, sarkar s. a study of hepatoprotective activity of hedyotis corymbosa linn, in albino rats. anc sci life 2009;28:32-5. 17. mammen d, daniel m, sane rt. identification of pharmacognostic and phytochemical biomarkers to distinguish between hedyotis corymbosa (l) lam and its adulterant, glinus oppositifolius (l) adc. res j pharm biol chem sci 2011;2:649-56. 18. hussain za. kumaresan s. phytochemical and antimicrobial evaluation of oldenlandia corymbosa. asian j plant sci res 2013;3:1558. 19. noiarsa p, ruhirawat s, otsuka h, kanchanapoom t. chemical constituents from oldenlandia corymbosa l of thai origin. j ant med 2008;62:249-50. 20. agrawal s. evaluation of antiulcer activity of oldenlandia corymbosa (l). int j res dev pharm sci 2013;2:363-7. 21. sivapraksam ssk, karunakaran k, kuppusamy s, subashini ts. a review on phytochemical and pharmacological profile of hedyotis corymbosa linn. int j pharm sci rev res 2014;26:320-4. murad1 51 * department of microbiology, ** department of community medicine, *** department of medical pharmacology, medical faculty trisakti university, jakarta correspondence aprof. dr. murad lesmana department of microbiology medical faculty trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2611 email: jmurad@cbn.net.id univ med 2008; 27: 51-6. transport media for shigella include buffered glycerol saline (bgs), and caryblair (cb). however being a liquid medium bgs may leak or spill during transport and thus may cause contamination. the other concern is the 30% concentration of glycerol in the bgs which may be inhibitory to some susceptible shigella species. this study was conducted to determine the best and safe transport media for shigella. rectal swab samples were obtained from 289 dysenteric patients and transported to the laboratory in cary-blair (cb) transport medium, standard buffered glycerol saline (bgs), bgs with the addition of 0.5% agar (bgs-a), and bgs with the addition of 0.5% agar and reduced glycerol to 15% (bgs-m). recovery rates between cb, bgs, bgs-a and bgs-m and their combinations were compared. the overall prevalence of shigella recovered from any of the four tubes was 24.9% (72/289). cb and bgs-m recovered shigella in 54 out of 289 patients (18.7%), cb and bgs-a in 50 (17.3%), and cb and bgs in 49 (17.0%), while cb, bgs, bgs-a, and bgs-m alone gave positive shigella in 30 (10,4%), 29 (10.0%), 34 (11.8%) and 46 (15.9%), respectively. this study suggests that a minor modification to the bgs raised the recovery rate of shigella. keywords: transport media, shigella, isolation comparative analysis of transport media for isolating shigella april-june 2008april-june 2008april-june 2008april-june 2008april-june 2008 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 abstract universa medicina murad lesmana*a, oktavianus ch. salim**, elly herwana***, paul bukitwetan*, and julius e. surjawidjaja* introduction shigellosis occurs both in epidemic and endemic forms in children and adults and remains a major public health problem in developing countries, causing high morbidity and mortality, particularly in children.(1,2) specific diagnosis of shigella infection depends on the isolation and identification of the organism from microbiological cultures of stool or rectal swab sample. it is important to laboratory examination that care should be taken to collect suitable fecal specimen, to use appropriate transport medium, and to ensure that specimen is transported to laboratory in a timely manner. 52 lesmana, salim, herwana, et al transport media for shigella shigellae are fastidious microorganisms and survive poorly in the environment. they remain viable for a limited time outside the human body, therefore, specimens for culture should b e p r o c e s s e d a s s o o n a s p o s s i b l e a f t e r collection.(3) delays of specimen process can result in substantial under-detection of the organism since it will reduce the number of viable shigella. (4) if a delay is anticipated b e t w e e n c o l l e c t i o n a n d p r o c e s s i n g o f specimens, transport medium should be used. transport medium is most commonly used for specimens collected for enteric pathogens analysis, and many media have been developed for this purpose.(5) for many years transport media commonly used for shigella-containing stool include buffered glycerol saline (bgs) and cary-blair (cb).(6-9) bgs has been widely used without undergone any major changes since it was developed and later perfected by sachs in 1939.(10) although cb is probably the best overall transport medium for diarrheal stool or rectal swab samples,(7) for the recovery of shigella, bgs was reported to be a better medium than cb.(3,11,12) one disadvantage of bgs, however, is that being a liquid medium it may leak or spill during transport and thus may c a u s e e n v i r o n m e n t a l c o n t a m i n a t i o n w i t h bacterial pathogens of the specimen being transported. the other concern is the 30% concentration of glycerol in the bgs which may be inhibitory to some susceptible shigella species. a literature search failed to find a transport medium for shigella which has a smaller concentration of glycerol other than the bgs. recent publications on bgs and other transport media were also scarce. to determine the best and safe transport media for shigella, we evaluated cb medium, standard traditional bgs as described and modified by sachs,(10) and two modified bgs media, which were (i) bgs with the addition of 0,5% agar without any other changes (bgsa), and (ii) bgs with the addition of 0,5% agar a n d r e d u c e d g l y c e r o l c o n t e n t t o a f i n a l concentration of 15% (bgs-m). methods preparation of transport media cb (becton dickinson, sparks, md) was prepared according to the direction provided by the manufacturer. standard bgs (bgs) was prepared from the formula according to the method described elsewhere.(13,14) for bgs-a, a 0.5% agar was added to the formula of standard bgs, and for bgs-m a 0.5% agar was added to the formula of standard bgs and the glycerol content was reduced to a final concentration of 15% (table 1). table 1. formula of buffered glycerol saline (bgs) and its modifications 53 collection and transportation of specimens from september 2005 through november 2007, a shigella surveillance was conducted in jakarta, involving community health center. consenting patients of all age groups presenting with dysenteric diarrhea (stool with blood and/ or mucus) were invited to participate. four rectal swabs were collected from each patient and each swab was placed in cb, bgs, bgsa and bgs-m, respectively. swab samples in transport media were held in the refrigerator (20 to 80c) until the end of the normal working day, at which they were transported in a coolbox to the microbiology laboratory of trisakti medical faculty. the swabs were processed immediately. bacteriological procedure swab specimen in transport medium was i n o c u l a t e d d i r e c t l y o n t o t h e s u r f a c e o f macconkey (mac) agar (difco, becton dickinson, sparks, md) and xylose lysine des oxycholate ( xl d) agar plate (difco, becton dickinson, sparks, md). plates were incubated at 370c, aerobically, for 20-24 hours. t h r e e s u s p i c i o u s c o l o n i e s ( n o n l a c t o s e fermenting) from each culture media were p i c k e d a n d i d e n t i f i e d b y c o n v e n t i o n a l biochemical test.(15) serological confirmation of isolates was performed by slide agglutination method employing group-specific shigella antisera (difco, becton dickinson, sparks, md). rectal swab specimen in a particular transport medium was considered negative for shigella when there were no growth on both mac and xld. statistical analysis chi-square test to compare the proportions of positive samples were used to determine the significant differences in isolation rates for individual and combined transport media. a p r o b a b i l i t y v a l u e p < 0 . 0 5 w a s c o n s i d e r e d statistically significant. results a total of 289 rectal swab samples from patients presenting with dysenteric diarrhea were obtained and processed. shigella was recovered from 72 (24.9%) of the rectal swab s a m p l e s . t h e p r o p o r t i o n a l d i s t r i b u t i o n o f shigella species was presented in table 2. s h i g e l l a f l e x n e r i w a s t h e m o s t p r e v a l e n t subgroup isolated with a rate of 17.3% (50/ 289), followed by s. sonnei with 7.3% (21/289) a n d s . b o y d i i , w i t h 0 . 3 % ( 1 / 2 8 9 ) . n o s . dysenteriae was isolated. both s. flexneri and s. sonnei comprised 98.6% (71/72) of all shigella isolated in this study. table 2. isolation of shigella spp. from rectal swab samples (n=289) in 4 transport media univ med vol.27 no.2 54 lesmana, salim, herwana, et al transport media for shigella i n r e l a t i o n t o t h e m e d i a u s e d f o r transportation of the specimen, shigella flexneri was recovered from 10.7% (31/289) rectal swab samples preserved and transported in bgs-m, followed by those in bgs and bgs-a each with 7.6% isolation rate, and in cb with 6.9%. shigella sonnei was isolated from 9 (3.1%) rectal swab samples preserved and transported in cb which was almost similar to that in bgs (2.4%), while bgs-a and bgs-m yielded 4.2% and 5.2% s. sonnei isolates, respectively. for both s. sonnei and s. flexneri, bgs-m with 15.9% (46/289) isolation rate provided the highest result compared to the other three transport media. for the isolation of s. flexneri the combination of cb+bgs-m (13.1%) was slightly better than those of cb+bgs (11.8%) and cb+bgs-a (11.4%), whereas for s. sonnei it was cb+bgs-a (5.5%) which gave higher isolation rate. overall isolation rate of shigella from specimens preserved and transported in cb was 10.4%, in bgs was 10.0%, in bgs-a was 11.8% and in bgs-m was 15.9%. compared to cb and bgs, bgs-m was significantly superior (p<0.05) in the total recovery of shigella species, but it was not significantly different than bgs-a (p>0.05). the combination of cb and b g s m p r o v i d e d 1 8 . 7 % i s o l a t i o n r a t e o f shigella, whereas the combination of cb and bgs, cb and bgs-a yielded 17.0% and 17.3%, respectively. the combination of cb and bgsm demonstrated a slightly better isolation rate f o r s h i g e l l a , h o w e v e r, i t d i d n o t d i f f e r s i g n i f i c a n t l y f r o m o t h e r c o m b i n a t i o n s o f transport media (p>0.05). discussion for over five decades since sachs(10) made a modification on the bgs from its original formulation this medium has been widely used without undergone any changes for preservation and transportation of shigella species in stool or rectal swab specimen. for recovery of shigella, the efficiency of bgs was reportedly superior than cb.(3,11,12) very few studies evaluated this medium were available since approximately over 20 y e a r s a g o . ( 3 , 1 2 ) m a y b e b e c a u s e b g s w a s c o n s i d e r e d t o b e t h e b e s t a n d c o n v e n i e n t transport medium for shigella in stool that further evaluations were not required. study conducted in 1997 by bonten et al(11) was the most recent on bgs which involved fecal flora of non-shigella. they compared the recovery of bacteria from stool specimens or rectal swabs that had been frozen using three preservatives including cb and bgs and concluded that bgs performed better than cb. being a liquid medium bgs is more likely t o s p i l l o r l e a k d u r i n g t r a n s p o r t a n d t h e specimen may contaminate the environment with enteric pathogens, however, there has been no attempt to minimize the risk. by the addition o f 0 . 5 % a g a r w h i c h m a d e t h e m e d i u m s e m i s o l i d , t h e p r o b l e m o f s p i l l a g e a n d contamination has been able to be avoided. thus, buffered glycerol saline with the addition of 0.5% agar (bgs-a) is a safer transport medium than the standard formula. our data shows that 0.5% agar in the standard bgs also increased slightly the yield of shigella species in the stool or rectal swab specimen from 10.0% (in bgs) to 11.8% (in bgs-a). it appears that agar not only served as a solidifying agent but increased the preserving capacity of the medium for shigella as well. the standard bgs contains glycerol at 30% concentration. bgs was a better medium to cb for preservation and transportation of shigella a s r e p o r t e d b y s e v e r a l i n v e s t i g a t o r s . ( 6 8 , 1 5 ) according to bonten et al(11) the stabilizing e ff e c t s o f g l y c e r o l i n b g s e x p l a i n e d t h e superiority of this medium to cb. our study shows that the total isolation rates of shigella 55 from the rectal swab specimens preserved in the standard bgs was no difference than that obtained from cb. the isolation rate, however, increased significantly from 10.0% to 15.9.0% (p<0.05) when the content of glycerol in the bgs was reduced to a final concentration of 15% in addition to the incorporation of agar (0.5%) such as that in the bgs-m (table 2). it appears that the relatively high concentration of glycerol (30%) in the standard bgs was inhibitory to s o m e s u s c e p t i b l e s h i g e l l a s p e c i e s . t h e inhibitory effect of 30% glycerol was reported on vibrio parahaemolyticus and to a lesser degree on salmonella as well.(16) in the long-term storage of bacterial isolates, glycerol was also used and added into the broth medium, namely trypticase soy broth, but in a lower concentration than that in the transport medium. in this maintenance medium the concentration of glycerol was between 15% to 20%.(9,17,18) the presence of glycerol as a preservant in a storage medium was associated with organism survival. glycerol protects the intracellular environment o f t h e o rg a n i s m . ( 1 7 ) c o m p a r e d t o c b , t h e i s o l a t i o n r a t e o f s h i g e l l a a l s o i n c r e a s e d significantly (p<0.05) when using bgs-m as transport medium for fecal or rectal swab specimen. in most studies of diarrheal diseases we usually employed two transport media, bgs specially for shigella and cb for the isolation of other enteric pathogens. specimen in bgs and cb were both processed for shigella. our data show that the combination of cb and bgs-m was better than other combinations although the differences were minimum. conclusion the use of cb and bgs may underestimate the true shigella prevalence, whereas a minor modification to the bgs, namely the bgs-m, raised the recovery rate of shigella. references 1. lee h, kotloff k, chukaserm p, samosornsuk s, chompook p, dee jl, et al. shigellosis remains an important problem in children less than 5 years of age in thailand. epidemiol infect 2005; 133: 46974. 2. ashkenazi s. shigella infections in children: new insights. semin pediatr infect dis 2004; 15: 24652. 3. niyogi sk. shigellosis. j microbiol 2005; 43: 13343. 4. surjawidjaja je, salim oc, bukitwetan p, lesmana m. perbandingan agar macconkey, salmonellashigella dan xylose lysine deoxycholate untuk isolasi shigella dari usap dubur penderita diare. univ med 2007; 26: 57-63. 5. lesmana m, surjawidjaja je, herwana e, salim oc, bukitwetan p. distribusi serotipe dan pola resistensi antibiotika dari isolat salmonella nontifoid di jakarta. univ med 2006; 25: 7-14. 6. clemens j, kotloff k, kay b. generic protocol to estimate the burden of shigella diarrhoea and dysenteric mortality. geneva. department of vaccine and biologicals, world health organization. 1999. 7. oyofo ba, lesmana m, subekti d, tjaniadi p, larasati w, putri m, et al. surveillance of bacterial pathogens of diarrhea disease in indonesia. diagn microbiol infect dis 2002; 44: 227-34. 8. agtini md, soeharno r, lesmana m, punjabi nh, simanjuntak c, wangsasaputra f, et al. the burden of diarrhea, shigellosis, and cholera in north jakarta, indonesia: findings from 24 months surveillance. bmc infect dis 2005; 17: 341-50. 9. fulla n, prado v, duran c, lagos r, levine mm. surveillance for antimicrobial resistance profiles among shigella species isolated from a semirural community in the northern administrative area of santiago, chile. am j trop med hyg 2005; 72: 851-4. 10. sachs a. difficulties associated with the bacteriological diagnosis of bacillary dysentery. j roy army med corps 1939; 73: 235-9. 11. bonten mjm, nathan c, weinstein ra. recovery of nosocomial fecal flora from frozen stool specimens and rectal swabs, comparison of preservatives for epidemiological studies. diagn microbiol infect dis 1997; 27: 103-6. 12. wells jg, gk morris. evaluation of transport methods for isolating shigella spp. j clin microbiol 1981; 13: 789-90. univ med vol.27 no.2 56 lesmana, salim, herwana, et al transport media for shigella 13. difco laboratories. the difco mannual, 11th ed. detroit, mich. difco laboratories; 1998. 14. chapin kc, lauderdale tl. reagents, stain, and media: bacteriology. in: murray pr, baron ej, jorgensen ph, pfaller ma, yolken rh, editors. manual of clinical microbiology. 8 th ed. washington, dc.: american society for microbiology; 2003. p. 354-83. 15. bopp ca, brenner fw, fields pi, wells jg, strockbine na. escherichia, shigella, and salmonella. in: murray pr, baron ej, jorgensen ph, pfaller ma, yolken rh, editors. manual of clinical microbiology. 8th ed. washington, dc.: american society for microbiology; 2003. p. 65471. 16. chun d, seol sy, tak r, park ck. inhibitory effect of glycerin on vibrio parahaemolyticus and salmonella. app microbiol 1972; 24: 675-8. 17. reimer lg, carroll kc. procedures for the storage of microorganisms. in: murray pr, baron ej, jorgensen ph, pfaller ma, yolken rh, editors. manual of clinical microbiology. 8 th ed. washington, dc.: american society for microbiology; 2003. p. 67-73 18. knapp js, koumans eh. neisseria and branhamella. in: murray pr, baron ej, pfaller ma, tenover fc, yolken rh, editors. manual of clinical microbiology. 7th ed. washington, dc.: american society for microbiology; 1999. p. 586603. martiem1 17 effect of aerobic exercise on blood lipid levels in postmenopausal women january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina martiem mawi* *physiology department, medical faculty, trisakti university correspondence dr. martiem mawi physiology department, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta barat phone: 021-5672731 ext.2803 email: martiemmawi@yahoo.com univ med 2009; 28: 17-24 abstract high blood total cholesterol (tc), low-density lipoprotein cholesterol (ldl-c) and triglycerides (tg), and low concentrations of high-density lipoprotein cholesterol (hdl-c) are related to risk for coronary heart disease (chd) development. growing evidence indicates that physical exercise can prevent at least some of the negative effects on health associated with post. the purpose of this study was to evaluate the effect of regular aerobic exercise for 12 weeks on the levels of total cholesterol (tc), low-density lipoprotein cholesterol (ldl-c), highdensity lipoprotein cholesterol (hdl-c), and triglycerides. an experimental study was conducted comprising 62 postmenopausal women, aged 50-70 years, not on hormonal therapy, consuming a regular diet, living in east and south jakarta, and willing to perform aerobic exercises regularly. the results of this study showed that all four lipid levels differed significantly between the control group and the intervention group, the respective mean levels ± sd for tc being 228.0 ± 39.7 mg/ dl vs. 171.6 ± 18.4 mg/dl, (p = 0.000); for ldl-c 149.0 ± 36.9 mg/dl vs. 97.7 ± 17.8 mg/dl, (p = 0.000); for hdl-c 50.9 ± 3.9 mg/dl vs. 71.5 ± 6.7 mg/dl, (p = 0.000); and for triglycerides 150.5 ± 67.5 mg/dl vs. 95.0 ± 37.8 mg/dl (p = 0.000). thus practitioners recommending exercise for coronary artery disease risk reduction in postmenopausal women. keywords: aerobic, exercise, blood lipid levels, postmenopausal women introduction c o r o n a r y h e a r t d i s e a s e ( c h d ) i s t h e leading cause of death and disability in both m e n a n d w o m e n . ( 1 ) t h e i n c i d e n c e o f cardiovascular disease is low in premenopausal w o m e n , b u t i n c r e a s e s i n p o s t m e n o p a u s a l women, to a level similar to that in men.(2) thirty percent of american women aged 65 years and above suffer from coronary arterial disease. after the onset of menopause, women have increased levels of total cholesterol, triglycerides, and low-density lipoprotein c h o l e s t e r o l ( l d l c ) c o m p a r e d w i t h t h e i r 18 mawi exercise on blood lipid levels premenopausal counterparts. epidemiological studies have shown a relationship between elevated cholesterol and coronary arterial disease.(3) e v e r y w o m a n w i l l e x p e r i e n c e menopause. (4) illnesses that often occur in postmenopausal women are cardiovascular disease, osteoporosis, cancer and stroke.(2,5,6) risk factors for cardiovascular disease include h y p e r c h o l e s t e r o l e m i a , h y p e r t e n s i o n , a n d smoking.(2,4) high levels of total cholesterol ( t c ) , l o w d e n s i t y l i p o p r o t e i n c h o l e s t e r o l (ldl-c), triglycerides, and a low level of highdensity lipoprotein-cholesterol (hdl-c) are risk factors for coronary arterial disease. hdlc is a strong predictor of coronary arterial d i s e a s e . r e s e a r c h i n t h e u s f o u n d t h a t a decrease of 1 mg/dl hdl-c would increase the risk of coronary arterial disease by 2-3 %.(7) coronary arterial disease increases in postmenopausal women because of a decrease in the levels of estrogen, and the occurrence of o v a r i a n d e g e n e r a t i o n i n p o s t m e n o p a u s a l women.(7-9) estrogen has protective properties on the cardiovascular system by increasing h d l c , a n d d e c r e a s i n g t c , l d l c a n d t r i g l y c e r i d e s , ( 1 0 1 2 ) w h e r e a s a d e c r e a s e o f estrogen decreases hdl-c, and increases tc, ldl-c and triglycerides. overall, the reduced estrogen level will accelerate the atherosclerotic process in the coronary arteries.(2) i n s o m e p o s t m e n o p a u s a l w o m e n , hypercholesterolemia is also present. typically, t c i n c r e a s e s b y 5 7 % , l d l c 1 0 1 2 % , triglyceride 8-10%, and hdl-c decreases by 4-6%. according to robergs, women who exercise regularly four times or more per week show a decrease in the risk of coronary arterial disease.(13) physical conditioning could have an effect on coronary arterial disease through aerobic exercise, such as static ergo cycle, gymnastics, and walking. regular exercise in postmenopausal women could increase hdlc and decrease tc, ldl-c, and triglycerides. besides those effects, exercise could also decrease hypertension, blood glucose, overweight, stress, and menopausal symptoms, and could increase physical fitness.(14) the results of one study showed that there was no increase of hdl-c in postmenopausal women after 40 weeks of aerobic walking exercise. these results may be due to the short duration and low intensity of exercise done in t h e s t u d y. t h e h d l c w i l l i n c r e a s e significantly if the duration of the aerobic exercise per week is long enough.(15) a different result was obtained in germany, where ldl-c in postmenopausal women after 4 years of intensive aerobic exercise did not show any significant difference compared to the control g r o u p . ( 1 4 ) i n p o s t m e n o p a u s a l w o m e n , t h e duration of exercise per week will slightly increase the hdl-c compared to the intensity of exercise; the duration of exercise should be around 12 weeks, and the intensity of exercise should be moderate.(16) the objective of this study was to determine the levels of blood lipids after regular aerobic exercise for 12 weeks in postmenopausal women. methods research design the study design was an experimental randomized non-blinded controlled trial. research subjects the subjects were women aged between 50-70 years, living in east and south jakarta, with a time window of 3 years after menopause, being in good health (not suffering from any a i l m e n t s , s u c h a s d i a b e t e s m e l l i t u s , l i v e r disease, heart disease, stroke, or lung and kidney diseases), not having received any hormonal therapy in the past three years, and consuming a balanced diet. 19 figure 1. screening of postmenopausal women intervention with aerobic exercise the subjects in the intervention group performed aerobic exercises for 12 weeks with a frequency of 4 times a week. before and after each round of exercises, blood pressure was m e a s u r e d . f o r t h e a e r o b i c e x e r c i s e s , a n e rg o c y c l e ( m o n a r k , f i n l a n d ) w a s u s e d t o measure the speed and loading, and the duration o f e x e r c i s e w a s t i m e b y a s t o p w a t c h . a n electrocardiograph (cardiopax, nihon kohden, japan) was used for monitoring of the heart rhythm and a polar (vantage nv tm, polar electro oy, finland) for checking of the heart rate before, during, and after the exercises. the exercises lasted for 25 minutes, of which the first 5 minutes were used for warming-up, the next 15 minutes for the main exercise, and the last 5 minutes for cooling down. the pulse rate was monitored until a value of 60% x (220 – age) had been achieved. for the first 2 weeks the exercises were conducted without addition of loads, and then loading was applied and gradually increased from 0.5 kp to 0.75 kp, and maximally to 1 kp. the weight was adjusted according to the health of the subject. if during the exercise, the pulse increased to 170 times per minute or more, or if the subject had headache, fatigue, or palpitations, then the exercise was terminated, and the subject was excluded from the study. a subject who had not completed the exercise for 12 weeks was also excluded from the study. the control group continued their habitual life style. measurement of variables r e c o r d e d s u b j e c t c h a r a c t e r i s t i c s comprised age, duration of menopause, and previous or current diseases, such as liver disease, diabetes mellitus, heart disease, stroke, or lung and kidney disease. the participants were also subjected to physical examination, resting 12-lead ecg recording, and fasting and 2-hour post-prandial blood glucose, blood pressure, and body mass index [weight (kg) d i v i d e d b y h e i g h t ( m 2) ] m e a s u r e m e n t s . a n t h r o p o m e t r i c m e a s u r e m e n t s i n c l u d e d standing height and weight. the subjects’ height (without shoes) was recorded to the nearest 0.5 cm, whilst recording of weight (with minimal clothing) was to the nearest 0.1 kg. height was univ med vol.28 no.1 20 mawi exercise on blood lipid levels taken using an anthropometer (microtoise ruban 2.20, france) and weight with a well-calibrated spring balance (secca 770 alpha, germany). those subjects who completed the above measurements were further examined at a certified laboratory in jakarta for their lipid p r o f i l e , n a m e l y t c , l d l c , h d l c a n d triglycerides. the subjects were required to have fasted 14 hours prior to the examination, w h e n 3 m l o f b l o o d w a s t a k e n f r o m t h e antecubital vein, and heparin or edta was added, or the blood was centrifuged to obtain serum. the tc was measured by chod pap enzymatic calorimetry using roche reagents. hdl-c as well as ldl-c concentrations were determined using a homogenous enzymatic method with daiichi reagents. triglyceride level was measured by the gpo-pap method u s i n g r o c h e r e a g e n t s . a l l o f t h e a b o v e assessments of lipid levels were performed using modular p 800, roche, germany. according to the national cholesterol education program, the upper normal limits of serum lipid concentrations are as follows: tc 2 0 0 – 2 3 9 m g / d l , l d l c 1 3 0 1 5 9 m g / d l , triglyceride 150-199 mg/dl, and hdl-c > 60 mg/dl. the present research had been approved b y t h e e t h i c a l c o m m i t t e e o f t h e m e d i c a l faculty, trisakti university. before starting the study, the subjects were given an informed consent form to fill out. after the subjects had expressed their consent by signing the form, they were given an interview and examination. statistical analysis the normality of the data on tc, ldl-c, hdl-c, triglyceride, age, bmi and duration of menopause at baseline was tested using the kolmogorov-smirnov test. if the result of the normality test showed that the data had a normal distribution, then the independent t-test was performed. independent t-test analysis was also used to test the differences in tc, ldl-c, hdlc and triglyceride concentrations between the intervention and control groups. a p value of < 0.05 (2-tailed) was considered to be significant. all analyses were performed using the spss/ p c s t a t i s t i c a l p r o g r a m ( v e r s i o n 1 2 . 0 ) f o r windows. results among the one hundred postmenopausal women who were available, ten refused to be included in the study and 28 did not fulfill the inclusion criteria, thus bringing the final sample size to 62. they were divided into two groups, thirty-one subjects were randomly assigned to t h e c o n t r o l g r o u p a n d a n o t h e r 3 1 t o t h e intervention group (figure 1). the normality tests on tc, ldl-c, hdl-c, triglyceride, age, bmi and duration of menopause at baseline were done using the kolmogorov-smirnov test. the results of the normality tests showed that tc (p = 0.607), ldl-c (p = 0.253), hdl-c (p = 0.959), triglyceride (p = 0.079), age (p = 0.959), bmi (p = 0.607), and duration of m e n o p a u s e ( p = 0 . 9 5 9 ) h a d a n o r m a l distribution. to compare baseline values of important characteristics, namely tc, ldl-c, hdl-c, t r i g l y c e r i d e , a g e , b m i a n d d u r a t i o n o f menopause, the independent t-test was used. the results showed no differences between the two groups with regard to tc, ldl-c, hdlc, triglyceride, age, bmi and duration of menopause at baseline. the mean age at baseline of the control group of 59.9 ± 5.4 years was not significantly different from that of the intervention (exercise) group of 59.9 ± 4.3 years (p = 0.959). similarly, mean bmi and mean duration of menopause in the control group of 26.2 ± 4.5 kg/m2 and 9.7 ± 5 . 6 y e a r s , r e s p e c t i v e l y, d i d n o t d i f f e r significantly from the corresponding variables 21 in the intervention group, being respectively 24.8 ± 4.4 kg/m2 (p = 0.198) and 10.5 ± 5.9 years (p = 0.425). mean tc, ldl-c and hdl-c concentrations of the control group (210.3 ± 34.7 mg/dl, 135.0 ± 28.1 mg/dl, and 54.8 ± 11.1 mg/dl, respectively) also showed no significant differences with their c o u n t e r p a r t s i n t h e i n t e r v e n t i o n g r o u p (respectively 202.3 ± 33.1 mg/dl, 128.0 ± 27.7 mg/dl, and 55.9 ± 13.2 mg/dl), the corresponding p values being p = 0.355, p = 0.345, and p = 0.733. mean triglyceride concentration of the control group was 137.0 ± 56.1 mg/dl, which was not significantly different from that of the intervention group of 110.0 ± 54.0 mg/dl (p = 0.063) (table 1). a f t e r e x p o s u r e t o 1 2 w e e k s o f a e r o b i c exercise, the tc concentration of the control group was 228.0 ± 39.7 mg/dl whereas that of the intervention group was 171.6 ± 18.4 mg/dl, indicating a significant difference in tc levels between the two groups (p = 0.000). the ldl-c concentration in the control group of 149.0 ± 36.9 mg/dl was also significantly different from that in the intervention group of 97.7 ± 17.8 mg/dl (p = 0.000). similarly, hdl-c concentrations were significantly different between the control group (50.9 ± 3.9 mg/dl) and the intervention group (71.5 ± 6.7 mg/dl) with p = 0.000, while the same was true for triglyceride concentrations, being 150.5 ± 67.5 mg/dl and 95.0 ± 37.8 mg/dl, respectively (p = 0.000) (table 2). note: bmi = body mass index; tc = total cholesterol; ldl-c = low density lipoprotein cholesterol; hdl-c = high density lipoprotein cholesterol table 1. age, bmi, tc, ldl-c, hdl-c, triglycerides and menopause duration at baseline by intervention groups table 2. mean of bmi, tc, ldl-c, hdl-c and triglycerides after intervention by intervention groups note: tc = total cholesterol; ldl-c = low density lipoprotein cholesterol; hdl-c = high density lipoprotein cholesterol univ med vol.28 no.1 22 mawi exercise on blood lipid levels discussion the results of this study showed that 12 weeks of aerobic exercise decreased tc, ldlc, triglyceride and increased hdl-c levels of postmenopausal women. these results are consistent with the those of one study on early postmenopausal women who exercised over 26 months and showed significant exercise effects on serum levels of tc and triglycerides.(17) similar investigations in exercise studies have been rather heterogeneous and depend on exercise intensity, duration, and frequency. increases in hdl-c levels were observed if regular exercise intensity levels exceeded 80% hr max, but not at lower intensity levels.(18) the hdl-c increases with moderate intensity exercise for 60 minutes with frequency of 2 to 4 times per week(19) in exercises of moderate to light intensity of more than 30 minute duration, the energy needed for the exercises is mainly generated from fat by lipolytic enzymes and mitochondrial activity. it can be concluded from several studies that in postmenopausal women moderate intensity exercise of long duration and sufficient weekly frequency is recommended for increasing hdl-c. fahlman et al.(15) reported increased hdlc l e v e l s , a n d d e c r e a s e d t c , l d l c , a n d triglyceride concentrations at week 10 of an aerobic training program at 70% heart rate submaximal in elderly women. regular exercise four times or more per week in postmenopausal women could increase hdl-c and decrease tc, ldl-c, and triglycerides. besides those effects, exercise could also reduce hypertension, blood glucose, stress and menopausal symptoms.(13,14) for postmenopausal women it is recommended to perform dynamic types of aerobic exercises, namely static bicycle, swimming, jogging and walking. these exercises are beneficial both physically and psychologically. physically, there will be increases in the number of red blood cells, capillary ratio and amount of muscle fiber as well as blood flow during e x e r c i s e , a n d a l s o i n c r e a s e d h d l c a n d fibrinolytic blood activity to decrease the risk of atherosclerosis. there is a positive relation between exercise and decreased incidence of chd.(13) a meta-analysis indicated that the effect of aerobic training resulted in a 2.53-mg/dl ( 0 . 0 6 5 m m o l / l ) e l e v a t i o n o f n e t h d l c change.(20) this is potentially of substantial importance in public health, although the effect of reducing cardiovascular risk by increasing h d l c l e v e l m i g h t b e s m a l l e r t h a n t h a t obtained through use of medications such as fibrates or niacin.(21) exercise increases the capacity of skeletal muscle to oxidize both glycogen and fatty acids to carbon dioxide and water. this responsive mechanism to increase fatty acid oxidative capacity is related to increased fatty acid tissue release and enzymes related to the transport and degradation of fatty tissue. fatty acids enter the krebs cycle and e l e c t r o n t r a n s p o r t s y s t e m , a n d t h e mitochondrial changes that occur are facilitated by the enzymes involved; all have a role in increasing skeletal muscle oxidative potential for fat metabolism. (22) during sub-maximal work more than 60% of the lipids utilized comes from intramuscular fat stores during muscle contraction due to low muscle glycogen. with submaximal work, an athlete oxidizes more fat than glycogen compared to a non-athlete. moreover, during exercise with submaximal effort, the amounts of lactic acid stored are less and fatigue is decreased.(13) a limitation of this study is its non-blinded randomized design. however, randomization of exercise trials is much more difficult compared with placebo-controlled pharmaceutical studies because exercise studies cannot be blinded. participants in exercise studies often refuse to be randomized to the study arm that they do 23 not prefer. people who do not want to exercise will drop out immediately. even worse, those subjects randomized to the control group who i n i t i a l l y w a n t e d t o e x e r c i s e m a y e x e r c i s e without reporting, causing a bias. conclusion a e r o b i c e x e r c i s e i n p o s t m e n o p a u s a l women of 50-70 years performed routinely for 12 weeks with a frequency of 4 times weekly and a duration of 25 minutes for each round of exercise, was able to reduce lipid levels, which are an important coronary heart disease risk factor. acknowledgments t h e a u t h o r s w o u l d l i k e t o t h a n k t h e trisakti university and the medical faculty that funded the study. we would also like to thank the women who participated in this study. references 1. welty fk. cardiovascular disease and dyslipidemia in women. arch intern med 2001; 161: 514-5. 2. rosano gmc, vitale c, voltererrani m. menopause and cardiovascular disease: the evidence. climacteric 2007; 10 (suppl 1): 19-24. 3. bittner v. estrogens, lipid and cardiovascular disease: no easy answers. j am coll cardiol 2000; 37: 431-3. 4. white wb. drospirenone with 17 ß estradiol in the postmenopausal woman with hypertension. climacteric 2007; 10 (suppl 1): 25-31. 5. ahlborg hg, johnell o, turner ch, rannevik g, karlsson mk. bone loss and bone size after menopause. n engl j med 2003; 349: 327-34. 6. lobo ra. menopause and stroke and the effects of hormonal therapy. climacteric 2007; 10 (suppl 2): 27-31. 7. assmann g, nofer jr. atheroprotective effects of high density lipoprotein. ann rev med 2003; 54: 321-41. 8. bulun se, nelson lr. estrogen production and action. semin reprod endocrinol 1999; 4: 34958. 9. expert panel on detection, evaluation, and treatment of high blood cholesterol in adults. executive summary of the third report of the national cholesterol education program (ncep) expert panel on detection, evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii). jama 2001; 285: 2486-97. 10. de aloyso d, gambacciani m, meschia m, pansini f, modena ab, bolis pf, et al. the effects of menopause on blood lipid and lipoprotein levels. the icarus study group. atherosclerosis 1999; 147: 147-53. 11. hinderliter l. cholesterol control. am j clin nutr 2001; 74: 730-6. 12. molina pe. endocrine physiology. new york: mcgraw hill; 2004: 207-43. 13. robergs ra, keteyian sj. fundamentals of exercise physiology: for fitness, performance, and health. 2nd ed. san francisco: mcgraw hill; 2003. p. 512 14. kemmler w, engelke k, von stengel s, wienneck j, laurer d, kalender wa. long-term four-year exercise has a positive effect on menopausal risk factors: the erlanger fitness osteoporosis prevention study. j strength cond res 2007; 21: 232-9. 15. fahlman mm, boardley d, lambert cp, flinn mg. effects of endurance training and resistance training on plasma lipoprotein profiles in elderly women. j gerontol a biol sci med sci 2002; 57: b54-60. 16. vella ca, kravitz l, janot jm. a review of the impact of exercise on cholesterol levels. am heart j 1999; 119: 277-83. 17. kemmler w, dirk lauber d, weineck j, hensen j, kalender w, engelke k. benefits of 2 years of intense exercise on bone density, physical fitness, and blood lipids in early postmenopausal osteopenic women. arch intern med 2004; 164: 1084-91. 18. haddock bl, hopp marshak hp, mason jl, blix g. the effect of hormone replacement therapy and exercise on cardiovascular disease risk factors in postmenopausal women. sports med 2000; 29: 3949. 19. sunami y. impact of exercise on cholesterol. am heart j 1999; 119: 277-83. univ med vol.28 no.1 24 mawi exercise on blood lipid levels 20. kodama s, tanaka s, saito k, shu m, sone y, onitake f, et al. effect of aerobic exercise training on serum levels of high-density lipoprotein cholesterol: a meta-analysis. arch intern med. 2007; 167: 999-1008. 21. birjmohun rs, hutten ba, kastelein jj, stroes es. efficacy and safety of high-density lipoprotein cholesterol increasing compounds: a meta-analysis of randomized controlled trials. j am coll cardiol 2005; 45: 185-97. 22. powers sk, howley et. exercise physiology 6th ed. new york: mcgraw hill; 2007. made 87 prof. sulianti saroso infectious disease hospital correspondence dr. dr. i made setiawan, sp.a prof. sulianti saroso infectious disease hospital jl. baru sunter permai raya jakarta utara 14340 phone : 021-6401412 univ med 2008; 27: 87-97. leptospirosis is a world-wide zoonotic disease, especially in tropical and subtropical regions. the disease is found in rural and urban areas with poor environmental conditions. the spectrum of human disease ranges from subclinical infection to severe clinical disease with multi-organ failure (weil’s disease) and high mortality rate, and depends both on the host and the infecting serovar. leptospirosis may occur either sporadically or in the context of an outbreak and is commonly related to occupational or recreational activities that involve direct or indirect contact with the urine of animal species that are reservoirs of the disease. the disease infects man through contact with contaminated environments or direct contact with carrier animals such as rats, dogs, cattle, pigs, etc. leptospirosis has clinical symptoms similar to such diseases as dengue, malaria, typhoid, influenza, thus laboratory methods are required for early detection to facilitate appropriate treatment of patients. the diagnosis of leptospirosis should be considered in any patient presenting with an abrupt onset of fever, chills, conjunctival suffusion, headache, myalgia, and jaundice. suspicion is further increased if there is a history of occupational or recreational exposure to infected animals or to an environment potentially contaminated with animal urine. treatment commonly consists of administration of antibiotics such as penicillin, ampicillin, amoxicillin, tetracycline and doxycycline. prevention by immunization is uncommon, as the available vaccines are ineffective in preventing the disease. in general, prevention is by avoiding environmental exposure. keywords: leptospirosis, clinical symptoms, laboratory, treatment clinical and laboratory aspect of leptospirosis in humans april-june 2008april-june 2008april-june 2008april-june 2008april-june 2008 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 abstract i made setiawan universa medicina introduction leptospirosis in humans is caused by leptospira bacteria transmitted through bites of infected animals, contaminated water and soil, or through occupations in the diary industry and agriculture. (1,2) there have been reports of leptospira infection in tourists after travelling and in sportsmen after participating in matches in areas endemic for leptospirosis.(3) the clinical manifestations of the disease are extremely variable. the majority of patients (90%) show mild symptoms similar to those of influenza, while the rest have very severe symptoms 88 setiawan clinical aspect of leptospirosis consisting of hepatic, renal, pulmonary and central nervous system abnormalities.(4,5) the clinical symptoms of leptospirosis are similar to those in other diseases, such as hepatitis, influenza, dengue hemorrhagic fever, and typhoid fever,(2,5,) so that it is extremely difficult to establish a definite diagnosis of l e p t o s p i r o s i s . to d a t e , t h e m o s t r e l i a b l e diagnostic test is the microagglutination test ( m at ) . h o w e v e r, t h i s t e s t i s e x t r e m e l y c o m p l i c a t e d , b e c a u s e i t r e q u i r e s e x p e r t personnel and a large number of live serovars o f t h e b a c t e r i u m . t h e r e f o r e a s e a r c h f o r alternative tests is mandatory. the treatment of this disease is actually extremely simple, with the administration of penicillin g, doxycycline, ampicillin, or amoxicillin. the prognosis is relatively good, but in severe cases the mortality rate is around 5-52%.(4,6) leptospira was first d i s c o v e r e d s i m u l t a n e o u s l y i n j a p a n a n d germany from autopsy specimens of patients diagnosed as having had yellow fever.(4) the problem of differentiating jaundice due to leptospirosis and yellow fever continued to exist until the deaths of stokes and noguchi while investigating the causative organism.(4) epidemiology leptospirosis is endemic throughout the world. the majority of infections in the tropics are mainly encountered in the rainy season,(5,7) while in the subtropics the disease is mostly found in summer. epidemics usually occur in seasons with a heavy rainfall.(8) in nature leptospira is found in carrier animals suffering from chronic renal disease, so that during the entire lifespan of the animals their urine continuously contains leptospira organisms. infection usually occurs through direct or indirect contact with infected animal urine or tissues.(4) the most important animals as a source of transmission are rats, followed by dogs, cattle and pigs.(9) the disease i s c o m m o n l y t r a n s m i t t e d i n d i r e c t l y a s a n occupational hazard, such as in abattoir workers, meat vendors, workers in the fruit and meat canning industries, and in farmers.(2) there have been reports of swimming pools contaminated with leptospira, so that a large number of swimmers were affected with leptospirosis.(1) in the tropics, individuals most frequently infected are farmers in contact with wet soils contaminated with leptospira-containing urine, e.g. rice and potato farmers. it has also been reported from isreal that troops stationed near the jordan river have been infected with leptospira. lastly the presence of leptospirosis has been reported in t o u r i s t s t r a v e l l i n g i n a r e a s e n d e m i c f o r leptospira.(3,4) microbiology leptospires are tightly coiled spirochetes, usually 0.1 mm by 6 to 0.1 by 20 mm, but occasional cultures may contain much longer cells. the helical amplitude is approximately 0.1 to 0.15 mm, and the wavelength is approximately 0.5 mm. the cells have pointed ends, either or both of which are usually bent into a distinctive hook (figure 1).(10) on the basis of genetic homology, there are currently more than one recognized species, figure 1. electron micrograph of leptospira interrogans fixed on a 0.2 µm filter(10) 89 which proves the existence of an extremely variable expression of the leptospira genome. based on a taxonomy currently being developed, l e p t o s p i r a i s d i v i d e d i n t o 1 2 s p e c i e s : l . alexanderi, l. biflexa, l. borgpetersenii, l. fainei, l. inadai, l. interrogans, l. kirschneri, l. noguchi, l. santarosai, l. weilii, l. meyeri, and l. wolbachii. all known species are divided into pathogens, intermediates, and saprophytes. the terms l. interrogans and l. biflexa have traditionally been used for distinguishing pathogenic and saprophytic strains. pathogenesis although the pathology due to leptospirosis has been known for a long time, not much is known about its pathogenesis. leptospira invades the body through abrasions in the skin, mucous membranes, and conjunctiva. the bacteria may also enter through inhalation of macroscopic aerosol droplets. transmission through food is unimportant, but may allow leptospira to enter by way of the mucous membranes. leptospira entering the blood may spread to all organs of the body.(4) the variety of clinical manifestations seen in patients is due to extensive vasculitis throughout the body. the symptoms occuring are extremely variable, with an onset from one day up to four weeks after exposure. recovery may take more than two months, or even more than six months.(11) there are two phases in the pathogenesis of leptospirosis: the leptospiremic phase and the immune phase. as mentioned previously, leptospira enters the human body through abrasions on the surface of the skin, mucous membranes, and conjunctiva. subsequently the o rg a n i s m s e n t e r t h e c i r c u l a t i o n , u n d e rg o replication and are carried to various organs in the body, thus causing extensive clinical symptoms. infection through the respiratory a n d g a s t r o i n t e s t i n a l t r a c t s i s c o n s i d e r e d insignificant.(12) h e p a t i c d a m a g e t a k e s t h e f o r m o f disorganization rather than injury to hepatocytes. leptospira invades and damages the hepatic sinusoids, spaces of disse, and parenchymal cells. the bacterium may also enter the spaces between the parenchymic cells and damage the canaliculi, thus causing bilirubin overflow into t h e s y s t e m i c c i r c u l a t i o n a n d s u b s e q u e n t h y p e r b i l i r u b i n e m i a , g e n e r a l l y w i t h o u t a n accompanying increase in hepatic enzymes. there is also disorganization or blockage of the sinusoids, while the kupffer cells are commonly swollen, containing leptospira organisms and red blood cells. in the liver there are also focal h e m o r r h a g e s , p e t e c h i a e a n d i n t e r s t i t i a l edema.(4,11) the pathologic findings encountered in the kidneys are due to hypoxia. in addition there are lesions in the kidneys appearing concurrently with impairment of renal function, thus being suggestive of intracellular damage due to toxins. t h e r e i s a l s o v a s c u l i t i s a n d h e m o r r h a g e accompanied by interstitial edema, necrosis of the tubular epithelium, and damage to the basal membrane.(11) in severe cases, the kidneys are swollen and yellowish, particularly in the [areas around the] cortical vessels. histologically, in the tubulointerstitial areas there is diffuse infiltration of lymphocytes, plasma cells and polymorphs, accompanied by localized areas of tubular necrosis. (4) the renal damage was subsequently demonstrated by the presence of abnormal urinalysis findings, i.e. proteinuria, sterile pyuria, hematuria, along with hyaline and granular casts. serum urea and creatinine levels also tended to be elevated.(12) leptospira may also attack the lungs, leading to hemorrhage, dyspnea, hemoptysis, and respiratory failure.(5) a great number of cases have died from respiratory failure, without renal and hepatic abnormalities; no inflammatory processes were found in the lungs of these patients.(8) hemorrhages were found throughout univ med vol.27 no.2 90 setiawan clinical aspect of leptospirosis the respiratory system, including the trachea, alveoli, and interstitium. the pulmonary vessels showed endothelial injury, and leucocytic and platelet thrombi, thus suggesting infarction. the occurrence of hypoxia will increase the vascular damage thus causing recurrent hemorrhages. in t h e a l v e o l i a n d b r o n c h i t h e r e a r e h y a l i n e membranes and occasionally fragments of leptospira or intact leptospiral organisms. in these cases the patient’s condition will rapidly deteriorate, resulting in death.(13) at postmortem, the heart frequently shows interstitial myocarditis with inflammation of the conducting system, coronary arteritis, and acute aortitis.(5,13,14) approximately 90% of known cases are mild in nature and self-limited. when not treated with antibiotics, the spirochetemic phase generally will be followed by the immune phase. clinical symptoms in general, the clinical symptoms are e x t r e m e l y v a r i a b l e , r a n g i n g f r o m m i l d subclinical symptoms with seroconversion, to very severe disease symptoms. clinically there are two recognized leptospirosis syndromes, i.e. self-limited systemic disease in about 90% of infections, and an extremely fatal leptospirosis accompanied by renal failure, hepatic failure, and hemorrhagic pneumonitis.(4) the course of illness is divided into two phases, i.e. the septichemic and immune phases. in the septichemic phase the bacteria are present in the circulation and subsequently spread to all organs; this is followed by the immune phase with grave symptoms (figure 2).(13) in severe illness, the line separating both phases is indistinct. occasionally the patients show symptoms only in the second phase.(4) t h e d i s e a s e c o m m o n l y h a s a m e a n incubation period of 10 days (5-14 days). the septichemic phase is of acute onset, characterized by an elevated temperature 38 to 40°c.(4) the clinical symptoms depend on the organs affected. the most prominent systemic findings are high fever with chills, sweating, nausea/vomiting, headache, joint pains, backache, wide-spread myalgia, particularly in the thigh and calf muscles, conjunctival suffusion, disturbances of vision, diarrhea, and skin rash.(2,3,7,15) occasional signs are lymphadenopathy, splenomegaly, and hepatomegaly. the acute phase usually terminates after 5 to 7 days. if the leptospirosis has affected the lungs, there is coughing that may lead to hemoptysis, and dyspnea leading to respiratory failure, such that the patient requires respiratoryassist devices.(8) the patient frequently also shows jaundice as a result of leptospira invasion of the liver. severe illness is commonly accompanied by manifestations of the central nervous system, such as severe headache, photophobia, visual disturbances, lowering of consciousness and delirium. aseptic meningitis occurs in 5-24% o f p a t i e n t s . o p h t h a l m i c f i n d i n g s i n c l u d e conjunctival suffusion, iritis, iridocyclitis, chorioretinitis, chorioditis, photophobia, blurred vision, and ocular pain. the ophthalmic findings appear a few days up to 18 months after acute i n f e c t i o n . ( 1 5 ) g a s t r o i n t e s t i n a l c o m p l a i n t s comprise abdominal pain, decreased appetite, vomiting, diarrhea, hematemesis, leading to fatal gastrointestinal hemorrhage. occasionally, there are symptoms of cholecystitis without biliary calculi, and pancreatitis.(16) there have also been reports of hemorrhages in cardiac muscle, causing disturbances of cardiac rhythm demonstrable on electrocardiographic examination, and resulting in sudden death.(11,14) nevertheless, reports on deaths in the acute phase are rare. the acute phase is followed by the immune phase, which commonly ends within 4-30 days. the disappearance of leptospira from the blood and cerebrospinal fluid takes place concurrently w i t h t h e a p p e a r a n c e o f i g m a n t i b o d i e s . leptospires may be found in nearly all tissues 91 and organs, and are detectable in urine up to a number of weeks, depending on the severity of the illness. the following findings may still be encountered in the immune phase: jaundice, renal f a i l u r e , c a r d i a c a r r h y t h m i a s , p u l m o n a r y disorders, aseptic meningitis, conjunctival s u f f u s i o n ( w i t h o r w i t h o u t h e m o r r h a g e ) , photophobia, ocular pain, adenopathy, and hepatosplenomegaly.(4,14) aseptic meningitis, either symptomatic or asymptomatic, is a characteristic sign of the immune phase, and is found in more than 80% of patients. in areas endemic for leptospira, the majority of aseptic meningitis cases are due to infection with leptospira. the patient complains o f e x t r e m e l y s e v e r e f r o n t a l a n d t e m p o r a l h e a d a c h e s , w i t h o r w i t h o u t l o w e r i n g o f consciousness. occasionally the presence of grave neurological complications have been figure 2. course of illness in leptospirosis(13) reported, such as coma, transverse myelitis, or the guillain-barre syndome.(4) the most severe form developing after the acute phase is weil’s disease, marked by hepatic and renal failure. in the acute phase occasionally a more severe illness develops, with fever of more than 40°c, followed by severe hepatic abnormalities, renal f a i l u r e , c a r d i a c a r r h y t h m i a s , a n d s h o c k appearing within an extremely short period of time, with no signs of improvement.(4,14) there is also acute renal failure marked by symptoms of uremia and oliguria appearing suddenly in the immune phase, and demonstrated by abnormal laboratory results. frequent pulmonary signs are hemoptysis and dyspnea leading to respiratory f a i l u r e . r a d i o l o g i c a l l y, s e v e r e p u l m o n a r y abnormalities are predominantly found in the lower lobes, developing from small snowflakelike solid nodules into patchy alveolar infiltrates. univ med vol.27 no.2 92 setiawan clinical aspect of leptospirosis extensive pulmonary consolidation is rarely encountered.(8,13,14) heart failure is seldom found; however, electrocardiographic abnormalities are frequent. more than 50% of patients on cardiac monitoring experience cardiac arrhythmias, i n c l u d i n g a t r i a l f i b r i l l a t i o n , f l u t t e r a n d tachycardia, premature ventricular contractions, and ventricular tachycardia.(4,14) prognosis t h e r e a r e m a n y f a c t o r s a ff e c t i n g t h e prognosis of patients with leptospirosis. in severe cases the mortality rate may reach 5-52%.(4,11) there are 5 factors determining the ocurrence of death, i.e. dyspnea, oliguria, a leucocyte count exceeding 12,900/ml, repolarization abnormalities on electrocardiograph (ecg), and the presence of alveolar infiltrates on chest radiographs. the mortality rate is higher in adults than in children,(17) and is not associated with the infecting serovar,(18) while in general those succumbing are the elderly. laboratory investigations the results of peripheral blood examination in the acute phase are not specific, but indicate t h e p r e s e n c e o f b a c t e r i a l i n f e c t i o n . t h e e r y t h r o c y t e s e d i m e n t a t i o n r a t e ( e s r ) i s generally raised, and there is thrombocytopenia and leucocytosis. leptospira organisms are detectable in the blood and cerebrospinal fluid in the acute phase. leptospira may also be found in the urine from the fifth to the seventh day after onset of symptoms. urinalysis results show mild proteinuria and pyuria, with or without hematuria, and hyaline and granular casts.(4,12) renal abnormalities are demonstrable by the hematological findings of elevated blood urea nitrogen levels, but not exceeding 100 mg/dl, and raised serum creatinine concentrations around 2 to 8 mg/dl. there is a reduced platelet count without signs of disseminated intravascular c o a g u l a t i o n ( d i c ) ; i n c o n t r a s t , s i g n s o f progressive renal disease are present.(4,14,18) cerebrospinal fluid examination in a severe immune phase will show increased cell counts, but not more than 500/mm 3, consisting of lymphocytes. protein is slightly increased, about 5-100 mg/ml, while glucose concentrations are within normal limits.(4) patients with severe jaundice will show hepatic pathological findings disproportionate to the visible jaundice. the s e r u m i n d i r e c t b i l i r u b i n c o n c e n t r a t i o n i s increased up to 80 mg/dl, with only a slight increase in the transaminases, i.e. alanine and aspartate aminotransferases. in contrast to viral hepatitis, the increase is rarely more than 200 µ/l. the jaundice will gradually disappear.(4,14) diagnosis a diagnosis of leptospirosis solely on the basis of clinical findings is extremely difficult to establish, as there are many diseases with similar findings. consequently, a definite diagnosis requires laboratory investigations. there are numerous laboratory methods suitable for diagnosis of human leptospirosis, such as (i) direct detection of leptospira in specimens using dark-field microscopy or polymerase chain reaction (pcr), (ii) detection of leptospira in specimens after prior culture, using dark-field microscopy or pcr, (iii) detection of antibodies u s i n g t h e m i c r o a g g l u t i n a t i o n t e s t ( m at ) , enzyme-linked immunosorbent assay (elisa) and various other methods.(4,11,13) direct method using dark-field microscopy t h i s r e q u i r e s e x p e r t a n d e x p e r i e n c e d laboratory personnel and yields low positive results (has low sensitivity). the specimens for examination are usually collected from tissues or fluids such as blood, urine, cerebrospinal fluid, or are cultured beforehand. specimens most frequently yielding positive results are 93 blood specimens, particularly those collected in the early phases. leptospira organisms are rarely found in the urine, as the leptospires are not resistant to acid environments. the collected s p e c i m e n s m a y a l s o b e c u l t u r e d p r i o r t o e x a m i n a t i o n . h o w e v e r, e x a m i n a t i o n o f microscopic specimens that must be cultured in advance is very time-consuming, because l e p t o s p i r a g r o w s v e r y s l o w l y i n c u l t u r e , requiring up to several weeks, such that this test cannot be used for rapid diagnosis.(11) detection of leptospira using pcr leptospira may also be detected by using the pcr. the results obtained are of high sensitivity and specificity, as pcr is capable of detecting leptospira organisms in numbers as low as 10.(19) another advantage of this test is its capacity for detecting the disease in the earlier phases. in addition, pcr is able to detect leptospiral dna in cerebrospinal and intraocular fluids, at a time when leptospiral antibodies have n o t y e t e n t e r e d t h e b l o o d s t r e a m . ( 2 0 ) t h e d i s a d v a n t a g e s o f t h i s t e c h n i q u e a r e i t s r e q u i r e m e n t f o r e x p e r t a n d e x p e r i e n c e d personnel, and the high price of the equipment, s o t h a t i t i s n o t a l w a y s a v a i l a b l e i n a l l l a b o r a t o r i e s , e s p e c i a l l y i n d e v e l o p i n g countries.(11) detection of leptospira antibodies using mat the diagnosis of leptospirosis is generally e s t a b l i s h e d b a s e d o n d e t e c t i o n o f s e r u m antibodies using the mat method. however, this technique is extremely complicated, as it requires expert and experienced personnel, a large number of live serovars, and a knowledge of the serovars prevalent within the particular area. the results obtained frequently arrive too late and are incorrect. this technique requires live leptospires, thus posing extreme dangers to the laboratory personnel involved. moreover, crossreactions are frequent and the test is difficult to s t a n d a r d i z e . ( 4 , 11 ) a l t h o u g h m at h a s m a n y weaknesses, it is still being used world-wide as a diagnostic standard for leptospirosis. samples should be collected serially in duplicate, one sample in the acute phase and one in the convalescent phase. if the titer of the second sample is four-fold that of the first, then the test is considered positive. occasionally, only one sample is collected.(11) evaluation of the result of one sample only is more difficult, because it r e q u i r e s k n o w l e d g e o f t h e m i n i m u m t i t e r considered positive. this minimum titer is different for each [geographic] area, depending on the seroepidemiological data for that area. generally in areas with frequent epidemics, the minimum level of antibodies is higher than in non-epidemic areas.(13) in the united states an antibody titer of over 1:200 is already considered p o s i t i v e , w h e r e a s f o r t r o p i c a l d e v e l o p i n g countries with a relatively high number of leptospirosis cases, a positive level of over 1:800 i s r e c o m m e n d e d . t h e a n t i b o d y t i t e r i n convalescent patients may reach 1:800 and this may commonly last up to 13 months, while a titer of 1:192 may last up to seven years after infection.(21) detection of leptospira using elisa elisa is a recent diagnostic technique c a p a b l e o f m o r e s p e c i f i c a l l y d e t e c t i n g leptospira antibodies, comprising both igm and igg. igm antibodies can be detected on day 2 to day 6 after onset of symptoms, with a sensitivity of 100% and a specificity of 94%, in comparison to mat. however, in about 29% of patients igm antibodies can be detected earlier. igg antibodies are detectable on day 5 to day 8. igm antibody responses occur in all patients, while igg antibody responses occur in less than 90% of patients.(22) based on above findings, the lepto dipstick was developed. the examination of igm using this technique does not require electricity for cooling equipment or refrigerators. this univ med vol.27 no.2 94 setiawan clinical aspect of leptospirosis technique is extremely easy to perform and the costs are low. the dipstick test uses a broadly reactive leptospira antigen, fixed on a solid support. when dipped into serum containing igm-antileptospiral antibodies, the anti-igm-dye conjugate forms an antigen-antibody complex, thus giving rise to a colored band, which may subjectively be assigned a score from 1 to 4. the ratio of acute to convalescent serum titers in each case may be used for establishing the diagnosis.(23) recently the lepto dipstick has been evaluated in 12 countries from 5 continents on 2,665 serum samples from 1,057 patients.(24) in the early phases of the disease, the lepto dipstick had a sensitivity of 86-35% and a specificity of 99-86%. the results of samples collected at a later time indicated that on average its sensitivity increased to 87%, from 60%. based on these facts, it is suggested that the test be used p a r t i c u l a r l y i n c o u n t r i e s w i t h c u r r e n t l y inadequate laboratory facilities. differential diagnosis leptospira exhibits extremely variable clinical findings, so that it is difficult to differentiate from other diseases. particularly in the tropics the incidence of diseases mimicking leptospirosis is very high, thus misdiagnoses are extremely frequent.(25) the climatic condition for the occurrence of epidemics of leptospirosis is identical to that for dengue fever epidemics, i.e. the rainy season. in the early stages both diseases are very difficult to differentiate, presenting with h e a d a c h e , e l e v a t e d b o d y t e m p e r a t u r e a n d myalgia. another disease to be differentiated from leptospirosis is hemorrhagic fever with renal syndrome (hfrs) caused by hantavirus, found in asia and europe accompanied by renal disorders. hfrs also exhibits acute fever, a f f e c t s t h e k i d n e y s a n d i s o c c a s i o n a l l y accompanied by pulmonary disorders. the source of transmission of this disease is also the r a t , t h u s h f r s e p i d e m i c s m a y o c c u r concurrently with those of leptospirosis, i.e. in the rainy season.(26) other hantaviruses, e.g. the south american andes virus, also present syndromes similar to leptospirosis, such as pulmonary vascular abnormalities, shock, and the occurrence of the acute pulmonary failure syndrome, resulting in death. this infectious disease may also be of endemic character.(27) f u r t h e r m o r e , r i c k e t t s i o s e s a l s o h a v e a n epidemiological profile similar to leptospirosis. treatment theoretically leptospira is sensitive to antibiotics, and the antibiotic considered having adequate potency as antileptospiral drug is tetracycline. tetracycline is capable of reducing the duration of fever and other symptoms, but it cannot prevent the occurrence of renal and hepatic abnormalities. penicillin is more effective in r e d u c i n g t h e d u r a t i o n o f f e v e r a n d o t h e r symptoms, especially if administered within the first four days after onset of symptoms. this antibiotic is capable of preventing hepatic and renal abnormalities, and also nuchal rigidity and hemorrhage, but it cannot reverse existing organ damage. in severe cases the administration of 1,500,000 – 2,000,000 units of intravenous penicillin every six hours is recommended. in children penicillin g may be given, 100,000 units/ kg body weight/day for 5-7 days, which may reduce the duration of illness. doxycycline given in oral doses of 100 mg twice daily reduces the duration and severity of illness, and can be used in prophylaxis of leptospira infection. however, at the time this study was undertaken, there were no severe cases of leptospirosis, thus using this antibiotic in severe cases is still of dubious merit.(24) a m p i c i l l i n m a y a l s o b e g i v e n a s a n antileptospiral drug. the results of a study in children given 100 mg/kg body weight/day for 95 5-7 days indicate that this drug may reduce the duration of illness. however, extreme care is called for when administering antibiotics, because these drugs may induce the jarischherxheimer reaction, comprising acute fever, h e a d a c h e s a c c o m p a n i e d b y m y a l g i a , a n d hypotension. the reaction commonly occurs 45 hours after administration of intravenous penicillin. the pathogenetic mechanism is still unclear, but the reaction is considered to be due t o l y s i s o f l e p t o s p i r a o r g a n i s m s b y t h e antibiotic, thus resulting in the release of toxins, which in turn cause induction of cytokines. h o w e v e r, o c c u r r e n c e o f t h i s r e a c t i o n i s extremely rare.(29) in severe cases, in addition to administration of antibiotics, supportive therapy is indicated for dehydration, hypotension, hemorrhage, renal failure, pulmonary disorders, hepatic disorders, and disorders of the central nervous system. administration of corticosteroids should be considered for prevention of chronic sequelae.(11) prevention p r e v e n t i o n o f l e p t o s p i r a i n f e c t i o n comprises avoidance of high risk exposures, use of protective clothing, immunization, and c h e m o p r o p h y l a x i s . ( 4 ) w h e n w o r k i n g i n a contaminated enviroment, e.g. farmers in wet soils at high risk of leptospira infection, it is advisable to don protective wear, such as boots and rubber gloves. for short-term prevention of leptospira infection, as when entering an area endemic for leptospirosis, use of chemoprophylactic drug is suggested. administration of doxycycline 200 mg weekly for 4 weeks may prevent leptospiral infection.(29) prophylaxis may be more beneficial if administered to communities in areas with epidemics (outbreaks).(30) in developed countries such as europe and the united states, immunization of humans for prevention of leptospirosis has not been widely p r a c t i s e d . i m m u n i z a t i o n w i t h p o l y v a l e n t vaccines has already been undertaken in far eastern countries, such as china and japan, where the number of leptospirosis cases are relatively high, the majority of the populations being farmers. in france, a monovalent vaccine containing the icterohemorrhagiae serovar has been used in humans, while in cuba a polyvalent vaccine has been developed for immunizing humans.(11) long-term prevention of human leptospirosis by vaccination is apparently not effective. this is due to the wide variation in lipopolysaccharides of the respective serovars, such that the antibodies produced are also extremely variable. each area has a specific range of prevalent serovars, differing from other areas. this represents a challenge to the development of human vaccines capable of preventing all serovars in a given area.(11) conclusions l e p t o s p i r o s i s h a s e x t r e m e l y v a r i a b l e symptoms such that it is difficult to differentiate from other diseases with similar symptoms. this disease is extremelty difficult to diagnose, as the techniques for examination are very complicated, require expert and experienced personnel, and are of rather low sensitivity, with the consequence that occurrences of this disease are notified only rarely. leptospirosis is treated by administration of antibiotics, such as penicillin, tetracycline, ampicillin, and amoxicilllin. prevention by immunization is as yet unsatisfactory, thus the currently best method is by avoiding exposure to contaminated environments. references 1. nardone a, capek i, baranton g, campese c, postic d, vaillant v, et al. risk factors for leptospirosis in metropolitan france: results of a univ med vol.27 no.2 96 setiawan clinical aspect of leptospirosis national case-control study, 1999-2000. clin infect dis 2004; 39: 751-3. 2. smythe l, dohnt m, symonds m, bernett l, moore m, brookes d, et al. review of leptospirosis notifications in queensland and australia: january 1998-june 1999. commun dis intell 2000; 24: 153-7. 3. morgan j, bornstein sl, karpati am, bruce m, bolin ca, austin cc, et al. outbreak of leptospirosis among triathlon participants and community residents in springfield, illinois, 1998. clin infect dis 2002; 34: 1593-9. 4. levett pn. leptospirosis. in: mandel gl, bennett je, dolin r, editors. principle and practice of infectious diseases. 6th ed. vol.2. philadelphia, pennsylvania, elsevier churchill livingstone; 2005. p. 2789-94. 5. karande s, kulkarni h, kulkarni m, de a, varaiya a. leptospirosis in children in mumbai slums. indian j pediatr 2002; 69: 855-8. 6. levett pn. usefulness of serologic analysis as a predictor of the infecting serovar in patients with severe leptospirosis. clin infect dis 2003; 36: 44752. 7. myint ksa, gibbons rv, murray ck, rungsimanphaiboon k, supornpun w, sithiprasasna r, et al. leptospirosis in kamphaeng phet, thailand. am j trop med hyg 2007; 76: 135-8. 8. trevejo rt, rigau perez jg, ashford da, mcclure m, gonzalez cjg, amador jj, et al. epidemic leptospirosis associated with pulmonary haemorrhage-nicaragua 1995. j infect dis 1998; 178: 1457-63. 9. ismail tf, wasfi m, abdul-rahman b, murray ck, hospenthal dr, abdel-fadeel m, et al. retrospective serosurvey of leptospirosis among patients with acute febrile illness and hepatitis in egypt. am j trop med hyg 2006; 75: 1085-9. 10. collins ra. leptospirosis. biomed scientist 2006; 116-21. 11. plank r, dean d. overview of the epidemiology, microbiology, and pathogenesis of leptospira spp. in humans. microb infect 2000; 2: 1265-76. 12. katz al, ansdell ve, effler pv, middleton cr, sasaki dm. assessment of the clinical presentation and treatment of 353 cases of laboratory-confirmed leptospirosis in hawaii, 1974-1998. clin infect dis 2001; 33: 1834-41. 13. levett pn. leptospirosis. clin microbiol rev 2001; 14: 296-326. 13. marotto pcf, nascimento cmr, neto je. acute lung injury in leptospirosis: clinical and laboratory feature, outcome, and factors associated with mortality. clin infect dis 1999; 29: 1561-3. 14. yersin c, bovet p, merien f, wong t, panowsky j, perolat p. human leptospirosis in the seychelles (indian ocean): a population-based study. am j trop hyg 1998; 59: 933-40. 15. chu km, rathinam r, namperumalsamy p, dean d. identification of leptospira species in the pathogenesis of uveitis and determination of clinical ocular characteristics in south india. j infect dis 1998; 177: 1314-21. 16. guarner j, shieh wj, morgan j, bragg sl, bajani md, tappero jw, et al. leptospirosis mimicking acute cholecystitis among athletes participating in a triathlon. human pathol 2001; 32: 750-2. 17. lopes aa, costa e, costa ya, sacramento e, deoliveira junior arr, lopes mb, et al. comparative study of the in-hospital case-fatality rate of leptospirosis between pediatric and adult patient age groups. rev inst med trop sao paulo 2004; 46: 19-24. 18. maciel eap, athanazio da, reis eag, cunha fq, queiros a, almeida d, et al. high serum nitric oxide levels in patients with severe leptospirosis. acta tropica 2006; 100: 256-60. 19. smythe ld, smith il, smith ga, dohnt mf, symond ml, barnett lj, et al. a quantitative pcr (taqman) assay for pathogenic leptospirosis spp. bmc infec dis 2002; 2: 13-9. 20. oliveira maa, caballero ol, vago ar, harskeerl ra, romanha aj, pena sdj, et al. diagnostics, typing and identification: lowstringency single specipic primer pcr for identification of leptospira. j med microbiol 2003; 52: 127-35. 21. romero ec, caly cr, yasuda ph. the persistence of leptospiral agglutinin titres in human sera diagnosed by the microscopic agglutination test. rev inst med trop sao paulo 1998; 40: 183-4. 22. croda j, ramos jg, matsunaga j, queiroz a, homma a, riley lw, et al. leptospira immunoglobulin like proteins as a serodiagnostic marker for acute leptospirosis. j clin microbiol 2007; 45: 1528-34. 23. tansuphasiri u, deepradit s, phulsuksombati d, tangkanakul w. a test strip igm dot-elisa assay using leptospiral antigen of endemic strain for serodiagnosis of acute leptospirosis. j med thai 2005; 88: 391-8. 97 24. smit hl, ananyina yv, cherhsky a, dancel l, lai-afat rfm, chee hd, et al. international multicenter evaluation of the clinical utility of a dipstick assay for detection of leptospira-specific immunoglobulin m antibodies in human serum specimens. j clin microbiol 1999; 37: 2904-9. 25. suriptiastuti. re-emergence of chikungunya: epidemiology and roles of vector in the transmission of the disease. univ med 2007; 26: 101-10. 26. evander m, eriksson i, pettersson l, juto p, ahlm c, olsson ge, et al. puumala hantavirus viremia diagnosed by real-time reverse transcriptase pcr using samples from patients with hemoohagic fever and renal syndrome. j clin microbiol 2007; 45: 2491-7. 27. chu y, owen rd, gonzalez lm, jonsson cb. the complex ecology of hantavirus in paraguay. am j trop med hyg 2003; 69: 263-8. 28. gonsales cr, casseb j, monteiro eg, paula-neto jb, fernandez rb, silva mv, et al. use of doxycycline for leptospirosis after hight-risk exposure in san paulo, brazil. rev inst med trop 1998; 40: 59-61. 29. levett pn. leptospirosis. clin microbiol rev 2001; 14: 296-326. 30. sehgal sc, suguman ap, murhekar mv, sharma s, vijayachari p. randomized controlled trial of doxycycline prophylaxis against leptospirosis in an endemic area. ints j antimicr agents 2000; 13: 249-55. univ med vol.27 no.2 may-august 2023 universa medicina vol.42no.2 pissn: 1907-3062 / eissn: 2407-2230 source of household water as main risk factor of soil transmitted helminth infections among elementary school pupils in wamena district, jayawijaya regency, papua semuel sandy1* and tri nury kridningsih2 abstract background soil-transmitted helminth (sth) infections are caused by three types of worms: ascaris lumbricoides, trichuris trichiura, and hookworms. soil transmitted helminth infections have significant health and socioeconomic implications for communities in developing countries, particularly in indonesia. this study aims to determine the prevalence and risk factors of sth infections among elementary school pupils in wamena district. methods the study design used was a cross-sectional design involving 317 elementary school pupils. the children were interviewed using a structured questionnaire that included demographic information, hygiene, and environmental sanitation data of the respondents. stool samples were collected from all consenting participants in sterile plastic containers and were analyzed within 24 hours of collection, using the kato-katz method. the data were analyzed using bivariate statistical analysis (chi-square) and multivariate analysis (logistic regression). results the prevalence of helminthiasis among elementary school pupils was 19.9% (63/317). sth prevalence of mild category was 17.4% (55/317). the risk factors for sth infections among school children were the habit of not washing hands with soap after defecation, with an odds ratio of 5.04 [95% ci (2.22-11.48)], and the source of water for household use, with an odds ratio of 7.22 [95% ci (3.66-14.22)]. conclusion the prevalence of helminthiasis was found to be 19.9% (63/317), with an sth prevalence of mild category. risk factors for sth infections included the habit of not washing hands with soap after defecation and the source of water for household use. keywords: risk factors, soil transmitted helminths, elementary school pupils, wamena 1research center for public health and nutrition, national research and innovation agency, jayapura selatan papua, indonesia 2balai penelitian dan pengembangan kesehatan papua, papua, indonesia *correspondence: research center for public health and nutrition, national research and innovation agency kawasan kerja bersama (kkb) brin jayapura, jl. isele kampung waena, distrik heram, jayapura selatan, papua, indonesia email: mercury.sandy56@gmail.com orcid id: 0000-0002-7300-4761 date of first submission, april 25, 2023 date of final revised submission, july 23, 2023 date of acceptance, july 27, 2023 this open access article is distributed under a creative commons attribution non commercial-share alike 4.0 international license original article 160 doi: http://dx.doi.org/10.18051/univmed.2023.v42:160-172 copyright@author(s) https://univmed.org/ejurnal/index.php/medicina/article/view/1466 cite this article as: sandy s, kridningsih tn. source of household water as main risk factor of soil-transmitted helminth infections among elementary school pupils in wamena district, jayawijaya regency, papua. univ med 2023;42: 16072. doi: 10.18051/univmed.2023. v42:160-172 mailto:mercury.sandy56@gmail.com https://orcid.org/0000-0002-7300-4761 https://orcid.org/0000-0002-0131-3987 https://univmed.org/ejurnal/index.php/medicina/article/view/1466 161 univ med vol. 42 no. 2 introduction soil-transmitted helminth infections, commonly known as worm infections, are among the most common neglected tropical diseases affecting children in lowand middle-income countries.(1–3) the most common type of worm infection is caused by soil-transmitted helminths. the largest burden of sth infections occurs in populations living in areas with limited access to sanitation and clean water, as well as poor standards of hygiene.(4) according to data from the world health organization (who), it is estimated that over 1.5 billion people, or 24% of the world’s population, are infected with sth.(5) additionally, over 260 million preschool-age children and more than 654 million school-age children live in areas wh e r e t h es e pa r a s it e s a r e tr a ns mi t te d intensively, requiring treatment and preventive interventions. the highest number of sth infections occurs in the asian region,(5,6) and south ea s t as ia ha s r ep or te d t he hi gh est p r e va l e nc e of st h i n f e c t i o ns i n r e c e nt decades.(4) the species of sth that infect h uma n s inc lu d e as c a r i s l um br i c o i de s , trichuris trichiura, necator americanus, and ancylostoma duodenale. these intestinal nematodes produce thousands of eggs per day, which are then excreted in the feces of infected individuals, contaminating the soil in areas with poor sanitation. sth infections are caused by intestinal nematodes that are transmitted to humans through soil contaminated with feces containing the eggs of ascaris lumbricoides, trichuris trichiura, and hookworms. these worm eggs enter the digestive tract along with contaminated food and drinking water due to poor environmental sanitation and hygiene practices.(7,8) soil-transmitted helminth infections can occur in all age groups, but school-age children are usually the most affected. infections of ascaris lumbricoides and trichuris trichiura are more common in children aged 5-14 years,(9) with a decrease in infection intensity and frequency during adulthood. this is due to changes in exposure, acquired immunity, or a combination of both. on the other hand, hookworm infections also occur in children, but the intensity and frequency generally remain high in adulthood, even in the elderly.(10) soil-transmitted helminth infections in school-age children have a detrimental impact on their growth, intellectual development, and cognitive abilities, which are undergoing rapid and ac tive growth.(11) the impact of sth infections also leads to reduced resistance in children to o ther dise ases and i ncrea sed vulnerability to nutritional deficiencies, due to low nutrient intake, malabsorption, and blood loss caused by the infection.(12) worm infections are also a contributing factor to stunting in children. stunting, or being short for age, is considered a good indicator of chronic malnutrition in children.(13) children who are stunted are more likely to become short-statured adults. sth i nf e c t i ons h a ve b e e n a s s oc ia t e d w i th malnutrition, including an increased risk of nutritional anemia, energy-protein deficiency, and growth impairments in children.(14,15) the results of the 2013 basic health research (riskesdas) showed that there are still households in indonesia where the final disposal of feces is not into a latrine or septic tank. the proportion of households practicing open defecation is still quite high. the five provinces with the highest proportion of feces not being disposed into septic tanks are papua (65.4%), east nusa tenggara ( 65.3%), we st nusa tenggara (49.7%), west sumatra (46.1%), central kalimantan (44.9%), and west sulawesi (44.1%).(16) data from the 2018 riskesdas stated that the proportion of unsafe household feces management for toddlers in papua is still r e l a ti ve l y hi gh a t 6 7. 8 % . unsa f e f e c e s management means that feces of toddlers is disposed of anywhere (including trash bins) or t ha t t od dle r s a r e c le a ne d i n a n y p l a c e . meanwhile, the practice of using a latrine for defecation and proper handwashing behavior in pa pua i s s til l low, a t 5 5. 8% and 2 6.7%, 162 r e s p e c t i ve ly. ( 1 7 ) t he h i gh pr op o r t i on o f households in papua practicing open defecation and the lack of access to sanitation facilities contribute to the development and transmission of sth infections in papua. the world health organization (who) targeted the control of sth infections in children to reduce morbidity by the year 2020 through a school-based deworming program. although the global target was to eliminate morbidity due to sth infections in children by 2020, these infections remain a significant health problem in indonesia, including papua province. the prevalence of worm infections in indonesia va r i e s bet we e n 2 .5 % a n d 6 2% , a n d h a s increased in children.(18) the control of worm infections is carried out through the mass drug administration (mda) p r o gr a m, w hi c h must be c o nt i n uo us l y implemented until the prevalence decreases below 10%. mass drug administration for worms is conducted twice a year in areas with high prevalence and once a year in areas with moderate prevalence. the drugs used for mda of worms are albendazole and mebendazole in the form of chewable tablets. the target age groups for deworming programs are toddlers, preschool-age children, and school-age children. deworming treatment needs to be accompanied by improved hygiene, sanitation, and healthy behaviors to prevent reinfection.(18) papua, as one of the indonesian provinces, has a tropical climate and humidity that supports t he d e ve lo p me n t o f sth l ar va e a n d t he maturation of sth eggs. socioeconomic factors such as lack of access to clean water sources, p oo r sa nit a t i on , l a c k o f per so n a l a n d environmental hygiene, education, poverty, barefoot walking habits, and nutritional status may increase the risk of sth infections in this region. epidemiological data related to sth infections among primary school-age children in papua, including in jayawijaya regency, are still very scarce. the reported prevalence of sth infections in papua includes 24.30% in jayapura regency and 32.90% in boven digoel regency (survey re p or t on wo r m in f e c ti o ns by th e environmental health control technical center ambon). furthermore, in keerom regency, the prevalence of sth infections among primary school children in arso district was 23.2% for ascariasis, 4.9% for trichuriasis, and 7.6% for hookworm infection.(19) the prevalence of helminthiasis in jayapura city is 50%.(20) research on risk factors for sth infections has been extensively conducted in indonesia, including demographic factors (gender, ethnicity, age, parental education, and occupation), hygiene practices (handwashing habits, footwear use, and playing with soil), and environmental sanitation (availability of toilets at home, flooring type, water sources). these studies have yielded significant results, showing variations in the impact of each risk factor variable.(1,19–21) in contrast, a study conducted by chadijah et l.(22) reported no significant association of knowledge level, environmental sanitation, and hygiene behavior with helminth infection among schoolaged children in palu. the study conducted by liena et al.(23) also mentioned that environmental sanitation factors did not have a significant association with sth infection among schoolaged children in moyudan health center, sleman regency. fu r t he r mor e , a s s e s si n g t he c u r r e nt prevalence and intensity of infection and identifying the associated risk factors of sths infection is vital to guide implementers, public health planners, stakeholders, and policymakers to plan and design precise intervention strategies to eliminate sths. therefore, this study aimed to assess the current prevalence, infection intensity, and identify the associated risk factors for sths among elementary school pupils of jayawijaya regency. methods study site the study was conducted in the jayawijaya regency, specifically in the wamena district. sandy, kridningsih soil-transmitted helminth infections 163 univ med vol. 42 no. 2 the selection of the wamena district as the research location was based on its significance in terms of economic and educational activities wi t h i n the j a ya wi j a ya re ge nc y. it w a s considered representative of the population of elementary school pupils in the jayawijaya regency. research design the study design was cross-sectional, and the research was conducted from january to december 2021. research subjects the required sample size in research is calculated based on the lemeshow equation.(24) the prevalence of worm infections in indonesia is 45-65%.(25) the sample size was calculated using the prevalence of 30.6% for sth infection among elementary school children, as found in the study conducted by yuwono et al.(26) in sorong district. the sample size was calculated based on a 30.6% proportion (p=0.306) with a 95% confidence level (1.96) and a margin of error of 5% (d=0.05). the sample size formula used was as follows: n=z²-α*p*(1–p)/d².(24) the calculation resulted in a minimum sample size of 326 samples, to account for non-response rate, an additional 5% of the sample size (16 samples) was added, resulting in a total of 342 samples.(27) the inclusion criteria for the study sample were elementary school pupils in grades 1-6, aged 612 years, of both genders. the exclusion criteria were pupils who were unwilling to participate in the interview and provide stool samples. the selection of school locations was done using simple random sampling, by creating a list of schools in the wamena district. the selection of child samples in schools was carried out using simple random sampling, by creating a list of school-aged children from teachers/school principals. data collection data collection was conducted using a structured questionnaire to obtain information related to demographic data (gender, age), socio-economic factors (mother’s education, mother’s employment status), hygiene practices, and environmental sanitation. face-to-face interviews with schoolchildren and their parents. the interviews were conducted with elementary school children in the presence of their parents or guardians, following prior permission and consent to participate in the research. stool sample examination the stool sample collection technique involved distributing stool containers to all selected school-aged children. the provided stool containers were labeled with the respective student’s name and date. stool samples were processed using a single kato-katz smear and examined under a microscope to detect the presence of intestinal helminth eggs. the intensity of infection was quantified as the number of eggs per gram (epg) of stool, following the guidelines established by the world health organization.(28) the intensity of sth infection is categorized according to who guidelines. the categories of intensity of ascaris lumbricoides infection are mild (14,999 epg), moderate (5000-49,999 epg), and severe (>50,000 epg). categories of degree of trichuris trichiura infection are mild (1-999 epg), moderate (1000-9.999 epg), and severe ( > 1 0 ,0 00 epg) . ca te go r i e s of d e gr e e of hookworm are mild (1–1.999 epg), moderate (2.000–3.999 epg), and severe (4.000 epg).(29) ethical consideration this research has obtained ethical approval for health research from the health research and development agency, ministry of health, indonesia, with the reference number: lb 02.01/2/ke.271/2021. data analysis descriptive analysis was conducted to depict the characteristics of the study population, including the prevalence of sth infections. the chi-square test was employed to examine the 164 relationships between variables. in the univariate analysis, the dependent variable was the prevale nce of sth inf ections, wh ile the independent variables included demographic and socioeconomic factors, hygiene practices, and environmental sanitation. independent variables with a significant value of p<0.25 related to the prevalence of sth infections were included in the multivariate logistic regression analysis to identify risk factors for sth infections. for each statistically significant factor, the odds ratio (or) and 95% confidence interval (ci) were c a l c u l a t e d t h r o ugh mu l ti va r i a te l o gi s t ic regression analysis. results the number of samples obtained at the research site was less than the expected minimum sample size of 326 samples, as only 317 samples met the criteria. this was due to many respondents being unwilling to provide stool samples due to feelings of disgust or discomfort, as well as a lack of participation f r om pa r e nts / gua r di a n s i n t he r e s e a r c h . additionally, some students were not present at the research site during the data collection period. an overview of the relationship between risk factors for sth infection can be seen in table.1. the chi-square test results indicated significant associations between sth infections among school-aged children and various risk factors, including ethnicity, the habit of washing hands with soap after defecation, main water source, and floor type. microscopic examination of the morphology and quantitative measurement of sth eggs was conducted using the kato-katz method on 317 stool samples collected from elementary school children in the wamena district of jayawijaya regency. the r esults of the stool sample examination can be seen in table 2. the stool examination results indicated that ascaris lumbricoides was the most prevalent type of worm found, with an infection rate of 10.4%. several other types of worm eggs were also detected in the stool samples, albeit at lower percentages, including trichuris trichiura ( 1 . 3% ) , ho okwo r ms ( 2 .2 % ) , en t e rob i us vermicularis (2.2%), and hymenolepis nana (0.3%). furthermore, the stool samples from the children exhibited cases of multiple infections, wi t h t h e pr e s e n ce of eggs f ro m as c a r i s lumbricoides, trichuris trichiura, hookworms, enterobius vermicularis, and hymenolepis nana. these findings demonstrate variations in the pattern of worm infections among primary school children in wamena district. the quantitative examination of sth egg counts using the kato-katz method in stool samples was further categorized based on infection intensity using criteria established by the world health organization, which includes mild, moderate, and severe infection categories. table 3 presents the results of the examination on the intensity of sth infections among primary school children in wamena district, where the infection intensity falls within the categories of mild and moderate intensity. fu r t he r logis t i c r e gr e s s i on a na lys i s revealed that the habit of washing hands with soap after defecation and main water source were significant risk factors for sth infection (refer to table 4). interaction tests conducted between each independent variable and the d e pe nd e n t va r i a b le d i d n ot de mo ns t r a te significant associations (p<0.05). discussion microscopic examination of stool samples from primary school-aged children in wamena district, jayawijaya regency, revealed the presence of several types of worms, including ascaris lumbricoides, trichuris trichiura, hookworms, enterobius vermicularis, and hymenolepis nana. the prevalence of worm infections among school-aged children in this location was 19.9% (63/317), with 17.4% (55/ 317) attributed to sth. the most commonly found infection in school-aged children in sandy, kridningsih soil-transmitted helminth infections 165 univ med vol. 42 no. 2 variable sth infections status p-value positive (n =55) negative (n=262) demographic characteristics sex male 32 (18.5) 141 (81.5) 0,544 female 23 (16.0) 121 (84.0) age group (years) 5 – 8 25 (14.5) 148 (85.5) 0.135 φ 9 – 12 30 (20.8) 114 (79.2) ethnicity papua 20 (30.8) 45 (69.2) 0,001 §φ nonpapua 35 (13.9) 217 (86.1) number of household members 2-4 25 (22.3) 87 (77.7) 0.841 φ >4 30 (14.6) 175 (85.4) mother’s education status low education 36(20.7) 13 8(79.3) 0.083 φ high education 19(13.3) 124 (86.7) mother’s employment status unemployed 29 (23.0) 97 (77.0) 0.030 §φ employed 26 (13.6.0) 165 (86.4) hygiene habit of washing hands before eating no 3 (20.0) 12 (80.0) 0.499 yes 52 (17.2) 250 (82.8) habit of washing hands with soap after defecation no 17 (48.6) 18 (51.4) 0.001 §φ yes 38 (13.5) 244 (86.5) nail clipping habit no 15 (21.4) 55 (78.6) 0.307 yes 40 (16.2) 207 (83.8) finger sucking habit no 49 (17.7) 228 (82.3) 0.722 yes 6 (15.4) 33 (84.6) habit of biting fingernails no 49 (18.4) 218 (81.6) 0.276 yes 6 (12.0) 44 (88.0) wearing shoes/slippers no 9 (22.0) 32 (78.0) 0.404 yes 46 (16.7) 230 (83.3) play with soil/dirt no 36 (16.6) 181 (83.4) 0.598 yes 19 (19.0) 81 (81.0) habit of frequent snacks outside home/school no 18 (15.4) 99 (84.6) 0.480 yes 37 (18.5) 163 (81.5) sanitation latrine type latrines without septic tanks 10 (23.3) 33 (76.7) 0.271 latrines with septic tanks 45 (16.4) 229 (83.6) main water source river water/rain/spring water/lake water/pond water 28 (48.3) 30 (51.7) 0.0001 § tap water/well water/bottled water 27 (10.4) 232 (89.6) floor type soil/wooden planks/bamboo 21 (45.7) 25 (54.3) 0.0001 § cement/ceramic floors 34 (12.5) 237 (87.5) family members who have experienced worms no 48 (16.4) 245 (83.6) 0.112 φ yes 7 (29.2) 17 (70.8) table 1. socio-demographic, hygiene, and sanitation factors associated with soil-transmitted helminth infection in elementary school pupils in wamena district, jayawijaya regency (n=317) data presented as n (%);sth : soil-transmitted helminth; § bivariate statistical analysis chi square test (significant, p value <0.05); φ risk factors followed by multivariate statistical analysis logistic regression test (p<0.25) 166 wa men a dist ric t was ca used by as caris lumbricoides. these findings are consistent with a study conducted by sandy et al.,(19) among school-aged children in arso district, keerom regency, where the prevalence of sth wa s r e po r t e d a s 29 . 9%, wi t h as c a r i s lumbricoides being the most prevalent at 23.3%. however, these findings differ from a study conducted by yuwono et al.,(26) in sorong re genc y, wes t pa p ua , wh i c h r e p o r te d a prevalence of sth infections among primary school children of 30.6%. the types of worms infecting primary school children in this area i nc lu d e as c a ri s l um br i co id e s , tri c h u ri s trichiura, hookworms, and strongyloides stercoralis. trichuris trichiura infection was most commonly observed in school-aged children at this location. a study conducted by salma et al.(30) revealed that 16.5% of schoolaged children in mayamuk subdistrict, sorong regency, were infected with one or more types of st h, including ascaris lumbricoides, tri c hu ri s tr i c hi ur a, h o okwo r ms, a nd strongyloides stercoralis. trichuris trichiura infection was the most prevalent both in single and multiple infections. ascaris lumbricoides infection was more common in children, while hookworm infection predominantly affected adults. trichuris trichiura infection was usually found alongside ascaris lumbricoides infection. however, strongyloides stercoralis infection was not detected, possibly due to the kato-katz table 2. prevalence of soil transmitted helminth species among elementary school pupils in wamena district, jayawijaya regency (n=317) note : sth : soil-transmitted helminth stool examination positive (n) prevalence (%) sth infections 55 17.3 non-sth infections 8 2.5 total worm infection (sth infections + non-sth infections) 63 19.9 infection types: mono-infection 52 16.4 dual-infections 9 2.8 triple infections 2 2.2 worm species ascaris lumbricoides 33 10.4 trichuris trichiura 4 1.3 hookworm 7 2.2 ascaris lumbricoides + trichuris trichiura 7 2.3 ascaris lumbricoides + hookworm 1 0.3 ascaris lumbricoides + enterobius vermicularis 1 0.3 ascaris lumbricoides + trichuris trichiura + hymenolepis nana 1 0.3 ascaris lumbricoides + hookworm + enterobius vermicularis 1 0.3 enterobius vermicularis 7 2.2 hymenolepis nana 1 0.3 ascaris lumbricoides trichuris trichiura hookworm mild intensity (1–4.999 epg) moderate intensity (5.000 – 49.999 epg) severe intensity (≥ 50.000 epg) mild intensity (1–999 epg) moderate intensity (1.000– 9.999 epg) severe intensity (≥ 10.000 epg ) mild intensity (1–1.999 epg) moderate intensity (2.000 – 3.999 epg) 42 (13.2%) 2(0.6%) 0(0%) 12(3.8%) 0(0%) 0(0%) 9(2.8%) 0(0%) table 3. intensity of sth infection among elementary school pupils in wamena district, jayawijaya regency (n=317) note : sth : soil-transmitted helminth sandy, kridningsih soil-transmitted helminth infections 167 univ med vol. 42 no. 2 method’s limited effectiveness in detecting the eggs of this worm.(1) the intensity of sth infections among school-aged children in wamena district was found to be predominantly mild for ascaris lumbricoides at 13.2% (42/317) and moderate at 0.6% (2/317). these findings are consistent with a study conducted by quiroz et al.,(31) where the majority of sth infections among school-aged children in colombia were classified as being of mild intensity for trichuris trichiura and hookworms and of mild to moderate intensity for ascaris lumbricoides. another study by ayu et al.(32) found a relatively high prevalence of sth infections among school-aged children in karangasem district, bali, categorized as mild to moderate intensity. another study by pasaribu et al.,(1) indicated mild intensity sth infections among school-aged children in north sumatra.. although the intensity of sth infections is low, variable n unadjusted models adjusted models odds ratio (or) 95% ci p-value odds ratio (or) 95% ci p-value age group (years) 5 – 8 173 0.64 0.32-1.29 0.212 9 – 12 144 1 ethnicity papua 65 1.17 0.48-2.81 0.733 nonpapua 252 1 number of household members 2-4 people 112 1.74 0.87-3.47 0.117 >4 people 205 1 mother education status low education 174 0.75 0.34-1.67 0.485 high education 143 1 mother’s employment status unemployed 126 2.02 0.93-4.38 0.076 employed 191 1 habit of washing hands with soap after defecation no 35 5.19 2.05-13.10 0.0001 5.04 2.22-11.48 0.0001* yes 282 1 1 family members who have experienced worms no 293 1.73 0.59-5.06 0.316 yes 24 1 main water source river water/rain/spring water/lake water/pond water 58 5.20 2.01-14.43 0.0001 7..22 3.66-14.22 0.0001* tap water/well water/ bottled water 259 1 1 floor type soil/wooden planks/bamboo 46 1.89 0.72-4.91 cement/ceramic floors 271 1 table 4. multivariate analysis of logistic regression test of risk factors associated with sths infection in school-age children in wamena district, jayawijaya regency *multiple logistic regression test (significant, p < 0.05); sth : soil-transmitted helminth 168 in the long term, sth infections can have detrimental effects on the health and quality of life of children.(33) the low intensity of sth infections among school-aged children in wamena district can be attributed to the integrated deworming program conducted by the district health office and community health centers, which includes the administration of anthelmintic drugs in conjunction with filariasis treatment once a year. the r isk factors showing significant associatio ns with sth infect ions among elementary school pupils include ethnicity, mother’s occupation status, handwashing habits with soap after defecation, main water source for household use, and floor type (table 1). ethnicity was found to have a significant relationship with sth infections in children, with a higher prevalence among the papua ethnic group compared to the non-papua ethnic group. furthermore, the mother’s occupation status was also found to be associated with sth infections, with a higher prevalence among unemployed mothers. the occupation of the parents, particularly those involved in farming, has close contact with the soil, which may be contaminated with sth eggs or larvae. parents with higher education generally have better knowledge of clean and healthy living behaviors compared to parents with lower education levels. additionally, educated parents are more likely to understand how to provide proper nutritional intake for their family members.(34) the risk factor of handwashing habits with soap after defecation showed a highly significant association with sth infections among children. children who did not practice handwashing with soap after defecation had a higher prevalence of sth infections (table 1). however, other variables such as handwashing before meals, nail cutting activities, thumb sucking, nail biting, wearing footwear outside the house/while playing, playing on the ground, and buying snacks outside the house/school did not show a significant relationship with sth infections. intestinal parasites can be transmitted through dirty hands, which serve as a medium for the entry of parasite eggs into the body.(35) the lack of availability of soap may be a reason why c h i l dr e n do no t wa s h t he ir ha nd s a f t e r defecation. these findings are supported by a study conducted by ayu et al.(32) among schoolaged children in ngis village, karangasem district, bali. a study on sth infections conducted by pasaribu et al.(1) in agricultural a r e a s i n nor t h suma t r a sho we d t h a t handwashing after defecation in toilets reduces the risk of sth infection. a different study by wiryadana et al. (36) on school-aged children in karangasem, bali, reported that the habit of handwashing with water and soap did not have a significant association with sth infection. data from riskesdas reported that proper handwashing behavior in papua is still very low, at only 26.7%.(17) in jayawijaya district itself, the proportion of correct handwashing behavior is only 18.78%. efforts to control helminthiasis can be made by improving health promotion through a family-based approach and through school health interventions, emphasizing the importance of handwashing with water and soap at five critical times: before meals, after using the toilet, before food preparation, after handling children’s waste, and before feeding children.(18) the risk factor of household environmental sanitation also has an influence on sth infections among children. it was found that the main water source used has a highly significant association with sth infections. households that use river/rain/spring water as their main water source have a higher prevale nce of sth infection (table 1). contamination of human and animal feces can pollute water sources through p oo r e n vi ro nme n t a l sa ni t a t i on , t h e r e b y transmitting sth eggs and larvae.(37) these findings are consistent with a study by konstantin et al.(38) in maluku, which found a bivariate association between water sources and sth infections among school-aged children in aru island, maluku. a study by sugianto et.al.(39) on school-aged children in antiga, bali also reported an association between water sources and sth sandy, kridningsih soil-transmitted helminth infections 169 univ med vol. 42 no. 2 infection. according to data from the central statistics agency, in urban areas, approximately 86.18% of the population has septic tank toilets, 5.38% have pit latrines, and 8.44% dispose of their feces into rivers/open fields/gardens/other areas. in contrast, in rural areas, 7.84% of the population has septic tank toilets, 16.5% have pit latrines, and 75.51% dispose of their feces into rivers/open fields/gardens/other areas. this indicates that sanitation in jayawijaya district is still very minimal because a significant percentage of the population does not have proper septic tank disposal for feces.(40) the risk factor of the type of house flooring is a ls o signi fi ca nt ly as so ci ate d wi th t he transmission of sth infections, with children living in houses with dirt/wood/bamboo floors having a higher prevalence of sth infections (table 1). data from the central statistics agency indicates that the indigenous population of jayawijaya district still maintains strong customs and culture. this can be seen from the significant number of indigenous papuan people who still live in traditional houses such as honai houses. generally, honai houses have thatched or grass roofs and are approximately 2.5 meters tall. the walls are made of wood and have a single small door but no windows, providing protection against the cold mountain air in papua.(40) the flooring of honai houses, which consists of wooden planks or soil with the addition of dry grass or straw, can increase the risk of sth transmission. this is because the presence of planks and dry grass/straw can create a damp environment underneath, which has the potential for the development of sth eggs and larvae. this study has several limitations, including the insufficient participation of children and parents in the research. data collection on household environmental sanitation observations could not be conducted due to the ongoing covid-19 pandemic at the time of data collection. the clinical implications of helminth infections have long-term effects, including decreased cognitive abilities, anemia, nutritional status, and stunting in children. this study was conducted to provide data on the prevalence of sth infections among school-aged children in wamena district, emphasizing the need for the health department to continue implementing d e wo r mi ng p r o gr a ms a n d e du ca t i n g t he community about the importance of healthy and hygienic behaviors, as well as environmental sanitation. evaluation of deworming programs can be carried out by conducting st h infection examinations among school-aged children to ensure the availability of prevalence data. in the future, studies on the impact of helminth infections on anemia, cognitive abilities, nutritional status, and stunting among schoolaged children, as well as examinations of environmental sanitation related to water sources and fecal contamination, should be conducted. in efforts to control helminth infections, a holistic and sustainable approach is essential. this includes education on hygiene practices, improving sanitation, regular health monitoring, a n d a w a r en e s s c a mp a i gn s i n vo l vi n g t he community and local authorities. with these measures, it is expected that the prevalence and negative impact of helminth infections on children in wamena district, jayawijaya regency can be reduced. in future research, there are several important directions to explore regarding soiltransmitted helminth (sth) infections among elementary school pupils in wamena district, jayawijaya regency, papua. firstly, the role of water sources as a main risk factor for sth infections should be investigated further. evaluating the effectiveness of interventions to improve water supply systems or implement water treatment methods in reducing sth infection risk would be essential. promoting hygienic practices among school pupils through behavior change interventions, such as proper handwashing, footwear usage, and minimizing contact with contaminated soil, should also be e xplo re d. a ss es sing the impa ct of th ese interventions on reducing sth infection rates would guide the development of effective health 170 education programs. lastly, investigating the role of environmental sanitation, such as improved sanitation facilities, in reducing sth transmission would be significant for prevention and control efforts. conclusion the prevalence of helminthiasis among school-aged children in wamena district is in the mild category. t he intensity of sth infections in school-aged children is also classified as being in the mild category. the risk factors associated with sth infections among school-aged children in jayawijaya district are the habit of not washing hands with soap after defecation and the source of water for household use. conflict of interest no conflict of interest funding this study was funded by the biomedical research and development institute of papua in the fiscal year 2021. acknowledgements we would like to express our gratitude to the head of the research and development center for health research in papua, the head of the provincial health office of papua, the head of the health office of jayawijaya district, and the head of the wamena city health center. we also extend our thanks to the staff of 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ajpcr.2019.v12i6.32941. 40. badan pusat statistik kabupaten jayawijaya. statistik daerah kabupaten jayawijaya 2020. ba d a n pus a t st at i s t ik k a bu p a t e n jayawijaya; 2020. sandy, kridningsih soil-transmitted helminth infections eka1 69 a retrospective study of spontaneous intracranial hemorrhage may-august, 2009may-august, 2009may-august, 2009may-august, 2009may-august, 2009 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 vol.28 no.2 universa medicina eka j. wahjoepramono* *department of neurology, faculty of medicine, pelita harapan university siloam hospital, karawaci tangerang correspondence dr. eka j. wahjoepramono, spbs department of neurology, faculty of medicine, pelita harapan university siloam hospital, karawaci tangerang univ med 2009;28:69-76 abstract spontaneous intracerebral hemorrhage (sich) is a serious disease despite progressing medical knowledge. sich appears suddenly without warning, unlike ischemic strokes that are often preceded by a transient ischemic attack. outcome is determined by the initial severity of the bleeding; mortality and morbidity of sich are high. the aim of this study was to describe the characteristics of type, location, and outcome of sich. a retrospective review was conducted on the records of 2042 cases admitted to a private hospital in karawaci, tangerang, between 1 january 1996 to 31 december 2008. analysis was done on type, location, and the final outcome measures by the glasgow outcome scale (gos). the results of the study showed that the most prevalent type of sich was hypertensive stroke, amounting to 1698 cases (83.1%), and the least commonly encountered type was dural fistula totaling 5 cases (0.3%). sich due to hypertensive stroke frequently occurred in the basal ganglia (50.8%) comprising the putamen, caudate nucleus and globus pallidus. on average, the outcome at the time of dismissal was good, where 105 cases (88.2%) were gos 4 and 5. sich requires prompt and appropriate management. therefore the signs and symptoms of intracranial hemorrhage should be promptly recognized and followed by appropriate ancillary examinations in order to promptly determine the management required, including possible surgical interventions. keywords : spontaneous intracranial hemorrhage, hypertensive stroke, arteriovenous malformations, prognosis introduction intracranial bleeding is a serious clinical event because of the high rates of fatality and disability that it causes. spontaneous intracranial hemorrhage (sich) is an acute hemorrhage occurring in the cerebral parenchyma, that may spread into the cerebral ventricles or even into the subarachnoid space on rare occasions. this type of hemorrhage accounts for 10-15% of all attacks of stroke and has a higher mortality than i s c h e m i c s t r o k e . i t o c c u r s m a i n l y d u r i n g 70 wahjoepramono spontaneous intracranial hemorrhage exertion, due to increased blood flow to the brains. spontaneous intracerebral hemorrhage (sich) must be considered separately from postt r a u m a t i c c e r e b r a l h e m o r r h a g e ( t c h ) , i n particular extraor epidural hematoma, subdural hematoma (sdh), subarachnoid hemorrhage (sah) and cerebral contusion. as the clinical presentation of ischemic atherosclerotic events is similar, the differential diagnosis relies on imaging techniques such as computerized t o m o g r a p h y ( c t ) a n d n u c l e a r m a g n e t i c resonance (nmr). the rate of sich is about 0.10.2 cases/1,000 population/year in the united states, with a trend for progressive increase correlated to the aging of the population.(1) sich represents about 15% of stroke cases. cerebral hemorrhages (ch) can be divided into four major c a t e g o r i e s : ( i ) c a p s u l a r o r t y p i c a l , ( i i ) intracerebral or atypical, localized in the frontal lobe or at the level of the parieto-temporal carrefour, (iii) cerebro-meningeal hemorrhage w i t h b l o o d l e a k a g e i n t h e v e n t r i c u l a r o r subarachnoid space, and (iv) small cerebellar, thalamic, pontine or bulbar bleeding. (2) sich can be divided into primary and secondary events. primary sich accounts for about 80% of cases and is mainly due to spontaneous rupture of a small vessel wall already damaged by arterial h y p e r t e n s i o n o r a m y l o i d a n g i o p a t h y. ( 2 ) s e c o n d a r y s i c h i s d u e t o d i s r u p t i o n o f congenital arteriovenous fistulas or aneurysms, neoplasia, or inherited or acquired (iatrogenic) coagulation disorders. risk factors for primary sich are arterial hypertension, alcohol abuse, heavy meals, emotional stress, physical exertion, prolonged exposure to sun, and pregnancy. genetic risk factors such as the mutation for the α s u b u n i t o f f a c t o r x i i i o r l i p o p r o t e i n e polymorphisms are under study. one of the main and most frequent causes of sich is hypertension, both acute and chronic, with degenerative changes in the cerebral vessel wall.(1,5) cerebral amyloid angiopathy (caa) is also one of the main causes of sich and the most frequent cause of lobar hemorrhage in the elderly.(4) the second most frequent cause of sich is blood vessel pathology,(4-5) such as cerebral aneurysms, avms, cavernomas, and angiographically occult vascular malformations (aovms), eg. dural arteriovenous fistulas and venous malformations.(2,3) coagulation disorders, including patients on previous anticoagulation t h e r a p y, t h r o m b o l y t i c t h e r a p y, l e u k e m i a , thrombocytopenia, also play a role in the development of sich. patients with abnormal liver functions are also at high risk of suffering from sich, because the liver plays a role in the p r o d u c t i o n o f c o a g u l a t i o n f a c t o r s a n d fibrinogen.(6) the incidence of intracranial hemorrhage increases significantly at ages over 55 years, and there will be a twofold rise in the risk for each additional decade up to the age of 80 years. above 80 years, the risk will increase 25-fold and more males than females are affected.(5) in the united states, afro-americans have a higher risk compared with caucasians. the prevalence of hemorrhagic stroke ranges from 17.2% in malaysia to 39% in surabaya, indonesia.(7) the a i m o f t h i s s t u d y w a s t o d e s c r i b e t h e characteristics of type, location, and outcome of the sich. methods subjects and design this study was designed as a retrospective review of case records. medical records of all patients with sich admitted to the neurology department of a private hospital in karawaci, ta n g e r a n g b e t w e e n 1 j a n u a r y 1 9 9 6 t o 3 1 december 2008 were screened. 71 univ med vol.28 no.2 data collection and grading system f o r e a c h r e c o r d , d a t a w e r e e x t r a c t e d concerning the patient’s age, chronic medical conditions and concurrent medications. relevant demographic, clinical, laboratory, and functional outcome data were collected during the study. p h y s i c a l a n d n e u r o l o g i c a l f i n d i n g s u p o n admission were noted. computed tomography (ct) of the brain was performed upon admission for all patients. cephalic ct scan is the initial examination of choice that may be performed rapidly and is capable of producing a hyperdense i m a g e o f b l o o d i n t h e b r a i n p a r e n c h y m a immediately after the occurrence of hemorrhage. the ct scan can accurately identify small hemorrhages, even those only a few millimeters i n d i a m e t e r. c t a n g i o g r a p h y ( c ta ) a n d magnetic resonance angiography are performed in patients with sich due to cerebral aneurysm. surgical criteria were established according to the algorithm of decision in force at the author’s institution, based on the recommendations of the st r o k e c o u n c i l o f t h e a m e r i c a n h e a r t a s s o c i a t i o n . ( 8 ) t h e m o s t c o m m o n l y u s e d classification of the severity of sich is that a c c o r d i n g t o h u n t a n d h e s s , w h e r e t h e differentiation is based on the clinical symptoms, and the classification of the international cooperative study, which is based on the glasgow outcome scale (gos). there is also a c l a s s i f i c a t i o n b a s e d o n t h e p r e s e n c e o f neurological deficits. the classification of hunt and hess is as follows: (i) grade i, patient is asymptomatic, with mild headache, and mild nuchal rigidity, (ii) grade ii, patient has paresis of the cranial nerves, moderate to severe headache, and nuchal rigidity, (iii) grade iii, patient has mild focal deficits, is lethargic or confused, (iv) grade iv, patient is stuporous, with moderate to severe hemiparesis, and early decerebration, and (v) grade v, patient is in coma, has decerebrate posture, and is moribund. the grade must incremented by one if there is serious systemic disease, such as hypertension and diabetes mellitus.(9) all cases p r e s e n t i n g w i t h i n t r a c e r e b r a l h e m o r r h a g e required prompt surgical intervention, whilst the remaining patients underwent elective surgery, among them 6 cases who were first given embolization, followed by excision. at the time of dismissal, the patients were assessed on functional outcome, using the glasgow outcome scale (gos). based on previous literature, gos = 1–3 was considered as unfavorable outcome and gos = 4–5 as favorable outcome, thus distinguishing whether or not patients were able to live independently.(10,11) all clinical ratings were performed by trained staff and neurologists, who were unaware of the sich results. statistical analysis a univariate analysis was used to describe the type, location and outcome of the sich. results a total of 2042 cases were collected at a private hospital in karawaci, tangerang, from 1996 to 2008, where the most prevalent type of hemorrhage was hypertensive stroke (1698 cases o r 8 3 . 1 % ) , a n d t h e l e a s t c o m m o n w a s hemorrhage due to dural fistula (5 cases or 0.3%). (table 1) *avms: arteriovenous malformations table 1. total number of cases of sich at a private hospital from 1996 to 2008 type of hemorrhage n (%) hypertensive stroke 1698 (83.1) cerebral aneurysm 220 (10.8) avms* 119 (5.8) dural fistula 5 (0.3) total 2042 72 wahjoepramono spontaneous intracranial hemorrhage s i c h d u e t o h y p e r t e n s i v e s t r o k e frequently occurs in the basal ganglia (50%) comprising the putamen, caudate nucleus and globus pallidus. next in order of frequency are hemorrhages in the internal capsule (19.4%), thalamus (8.6%), cerebellum (4.7%) and brain stem (4.5%) [particularly the pons] (table 2). a m o n g t h e p a t i e n t s w i t h c e r e b r a l aneurysm (76.8%) were in their productive years (below 50 years of age). the majority of the aneurysms were of moderate size (41.5%), followed by large (37.8%) and small (11%) aneurysms. the most common site of t h e a n e u r y s m s i n t h i s s e r i e s w a s i n t h e posterior communicating artery with a total of 82 cases (37.3%), followed by the anterior communicating artery with 77 cases (35%). t h e n u m b e r o f d e a t h s i n t h i s s e r i e s o f observations was as high as 5% (11 cases), among which 4.1% (9/220) had a hunt and hess grade iv and v on admission. average length of stay was 2-3 weeks. among 119 cases of sich due to avms, 62 (52.1%) were males and 57 (47,9%) were females, with a mean age of 28.9 years (range: 1 2 5 9 y e a r s ) . tw o o f t h e p a t i e n t s w e r e pregnant women, with gestational age of 6 and 9 weeks, respectively. most cases (50%) p r e s e n t e d w i t h h e m o r r h a g e r e q u i r i n g immediate surgery, 36 cases (30%) complained of seizures, and the remaining 24 cases (20%) had progressive headache. on average in the present study, the outcome at the time of dismissal was good, where 195 cases (88.2%) were glasgow outcome scale (gos) 4 and 5. all had previously been grade 1, 2 and 3, including the two pregnant women. seven patients (5.8%) with grade 4 suffered from hemiparesis and required supportive care. at follow-up in the o u t p a t i e n t c l i n i c , t h e s e p a t i e n t s s h o w e d improvement and were still under physiotherapy. three patients (2.5%) with grade 5 and a large hematoma in the basal ganglia remained in the vegetative state and on follow-up ct scan showed extensive infarction. three patients with gos grade 4 died of operative complications in the form of air embolism (2.5%). a ct scan of a patient with sich is shown in figure 1. in patients with cerebral aneurysm cta and mra was performed (figure 2). figure 3 shows the results of ct scan and cta in a patient with cerebral aneurysm. in patients with avms, cta was also performed (figure 4). figure 1. ct scan showing intracerebral hemorrhage extending intraventricularly location of sich n (%) basal ganglia 897 (52.8) internal capsule 330 (19.4) thalamus 315 (18.6) brain stem (pons) 77 (4.5) cerebellum 79 (4.7) table 2. location of sich due to hypertensive stroke in 1698 cases at a private hospital in karawaci, tangerang (1996-2008) 73 univ med vol.28 no.2 discussion sich is a common and serious clinical emergency. the retrospective design of this study must be taken carefully into account to give the right weight to the figures and rates of our study. i n p a r t i c u l a r, s i n c e w e m a y h a v e m i s s e d asymptomatic events, or even worse, fatal events, in outpatients, our figures could be an underestimate and should be considered as the lower limit of an undefined range. this study group is representative of patients with sich in g e n e r a l a s r e f l e c t e d b y t h e a n a t o m i c a l distribution of the ich, which is in accordance figure 2. cta and mra of a cerebral aneurysm figure 3. ct scan and cta of a patient with cerebral aneurysm figure 4. cta in a patient with avms 74 wahjoepramono spontaneous intracranial hemorrhage with a previous study in south israel.(12) the majority of sich locations are in the basal ganglia. consistent results were found in a number of other studies.(3-5) sich frequently occurs in the basal ganglia (50%) comprising the putamen, caudate nucleus and globus pallidus. next in order of frequency are hemorrhages in the internal capsule (19.4%), thalamus (8.6%), cerebellum (4.7%) and brain stem (4.5%) [particularly the pons]. sich is responsible for 10-15% of acute stroke.(13,14) the common causes of sich are h y p e r t e n s i o n , a n e u r y s m s , a r t e r i o v e n o u s malformations (avms), coagulopathies and vasculopathies.(15) in patients with sich due to avms, the majority of cases (50%) presented with hemorrhage, requiring immediate surgical i n t e r v e n t i o n . a r o u n d 3 0 % p r e s e n t e d w i t h seizures and the remaining cases (20%) with progressive headache. the results of the present study did not differ appreciably from those of the study by van beijnum et al.(16) who found that approximately 50% of avms presented with hemorrhage, 25% with seizures and another 25% with other complaints, particularly headaches and neurological deficits. aneurysms, avms, cavernomas, dural arteriovenous fistulas, and venous malformations all can result in secondary s i c h . t h e h e m o r r h a g e d u e t o a r u p t u r e d aneurysm almost always has a subarachnoid component and often extends into the ventricles. there should be a high index of suspicion in young patients with frontal or temporal lobe c l o t s . a r t e r i o v e n o u s m a l f o r m a t i o n s a r e associated with an estimated mean annual hemorrhage risk of 4%.(17) intracranial avms are lesions of the cerebral blood vessels comprising 3 components, namely a cluster of blood vessels with dysplasia (the nidus), a feeding artery, and a draining vein. approximately 30-55% of patients with avms present with intracerebral hemorrhage. a number of studies found that the risk of hemorrhage in intracranial avms is up to 2-4% annually. this information is important for the neurosurgeon to enable him or her to give advice on the risk of hemorrhage that may possibly be experienced by the patient. in contrast to aneurysms, the risk of recurrent hemorrhage in avms is much lower, so that elective surgery may be performed, days or w e e k s a f t e r t h e i n i t i a l h e m o r r h a g e . t h e advantage of this procedure is that the hematoma has time to undergo lysis, after which the condition of the patient may occasionally improve, so as to facilitate the operation and r e d u c e t h e r i s k o f t h e o p e r a t i o n . a n o t h e r a d v a n t a g e i s t h a t t h e r e i s m o r e t i m e f o r e v a l u a t i n g t h e r e s u l t s o f t h e r a d i o l o g i c a l examination, such that an accurate diagnosis may be established. the type of sich most frequently found in this study was hypertensive stroke. in several studies, sich has been strongly linked to age, race, and hypertensive status. (18-20) associations have been less consistent with many other risk factors, including greater alcohol consumption, male sex, smoking, low physical activity, body mass index (both high and low), total cholesterol (both low and high), and diabetes. age, male sex, high alcohol intake, and hypertension had a statistically significant association with ich in one recent metaanalysis.(19) another meta-analysis of studies from the asian-pacific region found high blood pressure, smoking, low cholesterol, and possibly diabetes associated with greater ich risk.(21) conventional angiography is still the gold standard for diagnosis of vascular lesions.(22) although the risk of angiographic procedures is small, there are a number of points to be taken into consideration before performing these procedures. the location of the hemorrhage, age of the patient, history of hypertension, and clinical condition are the points to be noted before selecting this procedure. however, with the development of the minimally invasive ct angiography (cta) and mra, conventional 75 univ med vol.28 no.2 angiography examinations are now rarely used for diagnostic purposes, but more frequently for interventions.(22) on average in the present study, the outcome at the time of dismissal was good, where 88.2% of cases had a good outcome (gos 4 and 5). this is in contrast to a study from argentina, where 38.6% had a good outcome ( g o s 4 t o 5 ) , b u t 2 2 . 8 % s u r v i v e d w i t h significant impairment (gos 2 to 3).(23) since some patients make a late recovery following rehabilitation, it remains to be determined whether the sich score can predict the long-term outcome. conclusions based on the results of our retrospective study, hypertensive stroke was identified as the most prevalent type of sich, while the most frequent location of sich was in the basal ganglia. the majority of patients with sich had a good gos score. a prospective confirmation of these results is needed and is possibly worth being performed. references 1. broderick jp, adams hp jr, barsan w, feinberg w, feldmann e, grotta j, et al. guidelines for the management of spontaneous intracerebral hemorrhage: a statement for healthcare professionals from a special writing group of the stroke council, american heart association. stroke 1999;30:905-15. 2. nicolini a, ghirarduzzi a, iorio a, silingardi m, malferrari g, baldi g. intracranial bleeding: epidemiology and relationships with antithrombotic treatment in 241 cerebral hemorrhages in reggio emilia. haematologica 2002;87:948-56. 3. qureshi ai, thurim s, broderick jp, batjer hh. spontaneous intracerebral hemorrhage. n engl j med 2001;344:1450-60. 4. fewel me, thompson bg, hoff jt. spontaneous intracerebral hemorrhage: a review. neurosurg focus 2003;15:1-16. 5. greenberg ms. intracerebral hemorrhage. in: handbook of neurosurgery 5th ed. new york: thieme;2001.p.815-24. 6. yukihiko y, shigekazu t, ryuichi t, tetsuo k, osamu s, takashi m. liver dysfunction in 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in a pooled prospective study. stroke 2007;38:2718-25. 19. ariesen mj, claus sp, rinkel gje, a. algra a. risk factors for intracerebral hemorrhage in the general population : a systematic review. stroke 2003;34: 2060-6. 20. suh i, jee sh, kim hc, nam cm, kim is, appel lj. low serum cholesterol and haemorrhagic stroke in men: korea medical insurance corporation study. lancet 2001;357:922–5. 21. woodward m, huxley h, lam th, barzi f, lawes cm, ueshima h. a comparison of the associations between risk factors and cardiovascular disease in asia and australasia. eur j cardiovasc prev rehabil 2005;12:484–91. 22. xavier ar, qureshi ai, kirmani jf, yahia am, bakshi r. neuroimaging of stroke: a review. south med j 2003;96:367-79. 23. godoy da, piòero g, di napoli m. predicting mortality in spontaneous intracerebral hemorrhage: can modification to original score improve the prediction? stroke 2006;37:1038-44. alvina 146 body mass index as predictor of carpal tunnel syndrome among garment workers september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina lie t merijanti s.* *department of occupational medicine, medical faculty, trisakti university correspondence dr. lie t merijanti s., mkk department of occupational medicine, medical faculty, trisakti university jl kyai tapa no. 260, grogol jakarta 11440 phone: 021-5672731 ext.2802 email: lie_mery@hotmail.com univ med 2009;28:146-51 abstract of the nerve entrapment syndromes the most well-known is the carpal tunnel syndrome (cts) involving the median nerve. in the field of occupational medicine this condition is categorized as an occupational disease, for which industrial workers may claim compensation if the cts occurs as a consequence of their job. however, although several occupational risk factors have been suggested as causing the development of cts, a definitive role of work activities as the central cause of cts is unclear. the aim of the present study was to evaluate both nonoccupational and occupational factors associated with cts in garment homeindustry workers. a cross-sectional study was performed from april to june 2008 in the garment home industry in north jakarta. a total of 99 workers, consisting of 45 males and 54 females were included in this study. historical data were obtained from a self-administered detailed questionnaire, while anthropometric and provocative median nerve test variables were measured directly. the results showed that body mass index (bmi) was a significant predictor of cts in male workers (p=0.031), while the risk of cts was three-fold higher in female workers using hormonal contraceptives (prevalence ratio/pr = 3.3 ; 95% confidence of interval/ci = 1.0 – 10.5). in conclusion it appears that bmi and hormonal contraceptive use were cts predictors. keywords: carpal tunnel syndrome, garment home industry, body mass index, hormonal contraceptive introduction carpal tunnel syndrome (cts) is the most w e l l k n o w n n e r v e e n t r a p m e n t s y n d r o m e involving the median nerve. however, its symptoms are such that they are often overlooked by patients or misdiagnosed by their primary care p h y s i c i a n s , l e a d i n g t o p r o g r e s s i o n o f t h e condition so severe that sometimes even surgical options will be of little benefit. the prevalence 147 univ med vol.28 no.3 of cts is approximately 3.8% of the general population, women are 3 to 4 times more likely to develop the condition, and it effects both wrists in 50% cases.(1) cts is often described as an occupational disease and claimed as a basis for worker’s compensation.(2) work related cts is a complex and costly condition.(3) in alberta, t h e r e w e r e 7 2 9 4 d i s a b l i n g i n j u r y c l a i m s ( r e q u i r i n g t i m e a w a y f r o m w o r k o r j o b modification) and 2849 compensated lost time c l a i m s f o r i n j u r i e s t o t h e h a n d a n d w r i s t excluding fingers, although the proportion of these that were due to cts was not reported.(4) t h e n u m b e r a n d f r e q u e n c y o f w o r k e r ’s compensation cts claims increased by more than 500%, despite increased regulations and m o n i t o r i n g , a n d p r e s u m a b l y i m p r o v e d workplace conditions. currently, cts leads to more lost workdays than any other workplace injury.(2) according to a review of occupational p o p u l a t i o n s a w i d e r a n g e i s f o u n d i n t h e prevalence of cts (0.6-61%), with the lowest prevalence occurring in industrial workers and the highest in grinders, butchers, grocery store workers, and frozen food factory workers, as in all of these occupations the workers use high force, high repetitive gripping.(5) however, the pathology of cts remained unclear until phalen reported on his experience in treating 439 patients at the cleveland clinic during the 1950s and 1960s. however, because the majority of the patients were middle aged women and thus not employed in industrial occupations, phalen concluded that cts was not an occupational disease but was “idiopathic”. one important observation made by phalen was that repeated, forceful grasping hand movements seemed to aggravate the symptoms.(6) it has been reported that cts is associated with certain diseases and conditions such as diabetes, hypothyroidism, pregnancy, rheumatoid arthritis, and work related factors. in some cases, two or more of these risk factors may coexist, placing the individual at a higher risk of developing cts. although less than half of all cases of cts are identified as work related in medical claims, a definitive role of work activities as the central cause of cts is unclear. in most cases, individual factors are not the potentially important independent causal factors that they appear to be, but frequently rather obscure an underlying relation between workplace factors and cts. hormone related disorders and/or hormone supplementation have been implicated as potential risk factors for cts, while obesity may also contribute to risk for cts.(7) the purpose of this study was to evaluate the predictors of cts among garment home industry workers. methods study design from april to june 2008 a cross-sectional study was carried out among garment home industry workers, north jakarta. study subjects the study sample consisted of garment home industry workers in north jakarta meeting the inclusion criteria, i.e. workers who had been employed for more than two years and were willing to participate in the study by signing an informed consent form. based on the cts prevalance of the 3.8%,(1) at a confidence level of 95% and accuracy of 4%, using the survey study formula a sample size of 92 subjects was obtained.(8) the exclusion criteria were workers with a history of wrist injuries, rheumatoid arthritis, diabetes mellitus and pregnancy of over three months, the history being obtained by interview. a l l o f t h e w o r k e r s h a v e b e e n i n v i t e d t o participate. 148 merijanti carpal tunnel syndrome data collection all participants in the study filled out a questionnaire relating to gender, age, level of education, use of hormonal contraceptives, job characteristics including job duration, work hours and type of work, also the presence of r e l e v a n t s y m p t o m s o f c t s l i k e t i n g l i n g , numbness, or pain affecting areas innervated by the median nerve. assessments body weight was recorded to the nearest kilogram on barefoot subjects using health scale instruments, while body height was measured with a microtoise to the nearest centimeter without shoes. body mass index (bmi) was calculated as the weight (kg) divided by the height squared (m2), and defined by the following thresholds: underweight (<18.5 kg/m2), normal (18.5 22.9 kg/m2), overweight (23.0 27.5 kg/ m2), and obese (>27.6 kg/m2).(9) provocative tests used to isolate the median nerve included the phalen test and the tinel test. phalen’s sign is of use because it is considered a very good indicator for cts with high sensitivity and specificity, as it localizes the median nerve at the carpal tunnel.(10-12) the phalen test is performed by asking the respondent to hold his or her wrist in a flexed position at approximately 90 degrees. the test is considered a positive phalen’s sign if numbness or pain is felt by the patient within 30-60 seconds. tinel’s sign isolates the median nerve at the carpal tunnel by percussing it at the distal palmar crease. this also should elicit n u m b n e s s o r p a i n . ( 1 0 ) c t s w a s d i a g n o s e d a c c o r d i n g t o t h e n a t i o n a l i n s t i t u t e o f occupational safety and health (niosh) c r i t e r i a , c o m p r i s i n g o n e o f t h e f o l l o w i n g symptoms, i.e. numbness, pain, tingling or paresthesia in the afected area, accompanied by positive tinel’s or phalen’s test and history of occupational work. phalen’s and tinel’s signs, although they may assist in the diagnosis of cts,(11) are not pathomnemonic for the disorder. statistical analysis data were analyzed using spss version 15.0. comparison of the means of continuous variables was performed using the independent t-test, while the chi-square test was done to a n a l y z e t h e r e l a t i o n s h i p b e t w e e n s e v e r a l categorical variables. prevalence ratio was used to evaluate the strength of association between dependent and independent variables. results from a total of 105 garment home-industry workers, 99 participated in this study. six subjects were excluded because they had been employed for less than two years. the subjects consisted of 99 garment workers with a mean characteristics n = 99 age (yrs) 29.8 + 8.0 job duration (yrs) 7.8 + 5.9 work hours (hrs) 10.4 + 1.5 education primary education 91 (91.9%) secondary education 8 (8.1%) bmi overweight and obese 27 (27.3%) normal and underweight 72 (72.7%) hormonal contraceptive use yes 19 (19.2%) no 80 (80.8%) gender male 45 (45.6%) female 54 (54.4%) work type sewing 81 (81.8%) pressing and cutting 10 (10.1%) finishing 5 (5.1%) packing 3 (3.1%) table 1. summary of the characteristics of the participating subjects 149 univ med vol.28 no.3 age of 29.8 + 8.0 years and a mean job duration of 7.8 + 5.9 years. the 99 workers comprised 45 males and 54 females, among whom eigthy one (81.8%) worked as seamster/seamstress and ninety one (91.9%) had only primary education. of the 99 workers included in the study, 43 w o r k e r s w e r e d i a g n o s e d o f c t s . s o , t h e prevalence of cts in our sample was 43.4% (table 1). t h e r e l a t i o n s h i p b e t w e e n w o r k p l a c e factors, personal characteristics and cts in male workers are shown in table 2. there was a significant difference in body mass index (bmi) in male workers with cts, compared with those without cts (p=0.031). both in female workers with cts and those without cts, analysis revealed no significant difference in age, job duration, work hours, or bmi (table 3). among female workers it was found that the risk of cts was three-fold higher in workers using hormonal contraceptives (pr=3.3; 95% ci= 1.0 – 10.5) (table 4). discussion this study did not find any significant relationship between occupational risk factors and cts. however, among male workers bmi was a significant predictor of cts and use of variables cts present n=24 cts absent n=30 p age (years) 28.3 + 6.3 27.2 + 8.8 0.447 job duration (years) 7.1 + 5.4 5.8 + 6.1 0.768 work hours 10.1 + 1.5 10.2 + 1.6 0.220 body mass index 21.6 + 3.2 21.1 + 2.8 0.190 table 3. comparison of several continuous variables and cts in female garment home industry workers variables cts present n=24 cts absent n=30 prevalence ratio (95% ci) non contraceptive 12 23 3.3 (1.0 – 10.5) hormonal contraceptive 12 7 table 4. hormonal contraceptive use and cts in female garment home industry workers variables cts present n=19 cts absent n=26 p age (years) 35.1 + 6.7 30.2 + 7.6 0.124 job duration (years) 10.0 + 5.6 9.0 + 5.6 0.259 work hours 10.5 + 1.7 10.7 + 1.3 0.173 body mass index 20.5 + 3.3 21.3 + 2.7 0.031* table 2. comparison of several continuous variables and cts in male garment home industry workers * significant (p<0.05) 150 loss in obese patients, however, does not bring relief of the syndrome. women who are pregnant, taking oral contraceptives or going through menopause also are at increased risk, most likely due to hormonal changes. hormonal fluctuations in women play a role in cts. such fluctuations may cause fluid retention and other changes that cause swelling in the body. fluid retention is one reason why cts may develop during pregnancy. fortunately, carpal tunnel syndrome related to p r e g n a n c y a l m o s t a l w a y s i m p r o v e s a f t e r c h i l d b i r t h . ( 2 2 , 2 3 ) e s t r o g e n r e c e p t o r s a n d progesteron receptors are present in transverse carpal ligament and flexor tendon synovitis, suggesting a role for sex steroid hormones in the pathogenesis of cts disease.(24) however, the study by maghsoudipour et al.(7) could not find any association between hormonal contraceptive use and cts incidence. one limitation of the present study is that working conditions did not include ergonomic factors but only type of work, because garment home-industry workers are informal workers with irregular work, making it difficult to perform a job analysis. another limitation is that the diagnosis of cts was established without being confirmed by a nerve conduction test, which is considered the most accurate single test for cts. therefore the cts prevalence in this study has presumably been overestimated. conclusions in this study bmi was found to be a significant predictor of cts among males workers, whereas hormonal contraceptives increased the risk of cts in females workers. references 1. burke fd, ellis j, mckenna h, bradley mj. primary care management of carpal tunnel syndrome. j postgrad med 2003;79:433-7. merijanti carpal tunnel syndrome hormonal contraceptives was a predictor in females. the degree of work relatedness of cts reported in the literature ranged from very high to very low.(7) cts is accepted as a work related disorder by some authors.(11) occupational hand uses that are considered ergonomic risk factors for developing cts include those involving highly repetitive awkward wrist movements, high handgrip and pinch forces and those associated with high vibration.(2,14) however, this notion is controversial and many authors do not agree with it. (15) in the longest prospective longitudinal study by nathan, occupation was not associated with cts although being female and overweight was.(13) gedizlioglu et al.(18) found that in manual steel industry workers, m a n u a l w o r k w a s n o t a r i s k f a c t o r f o r development of clinical cts. in the present study, working conditions did not include ergonomic factors but only type of work, because garment home industry workers are classified as informal sector workers with uncertain job continuity. they mainly work when there is an order from clients and the types of garment they produce vary with each order. in addition, when there is no commission, the workers frequently return to their villages, with the result that they are not daily exposed to the same risk factors. the protective nature of informal employment is most likely attributable to decreased exposures to such risk factors and greater time for rest of the affected body areas.(19) bmi and hormonal contraceptive use were found to be significant predictors of cts in this study. the risk of cts was three-fold higher in female workers on hormonal contraceptives (pr= 3.3; 95% ci= 1.0 – 10.5). obesity has been associated with cts in previous studies. (20) a study conducted by geoghegan et al.(21) in the community found that obesity was a significant risk factor to cts. obesity and wrist shape correlate with a higher prevalence of carpal tunnel syndrome. weight 151 univ med vol.28 no.3 2. kao sy. carpal tunnel syndrome as an occupational disease. j am board fam pract 2003;16:533-42. 3. sperka p, cherry n, burnham r, beach j. impact of compensation on work outcome of carpal tunnel syndrome. occup med 2008;58:490-95. 4. alberta employment, immigration and industry. occupational injuries and diseases in alberta. lost time claims, disabling injury claims and claim rates. edmonton, alberta: government of alberta;2007. 5. van rijn rm, huisstede bma, koes bw, burdof a. association between work related factors and the carpal tunnel syndrome a systematic review. scand j work environ health 2009;35:19-36. 6. phalen gs. the carpal tunnel syndrome: 17 years experience in diagnosis and treatment of 654 hands. j bone joint surg am 1966;48:211-28. cited by kao sy. carpal tunnel syndrome as an occupational disease. j am board fam pract 2003;16:533-42. 7. maghsoudipour m, moghimi s, dehghaan f, rahimpanah a. association of occupational and non occupational risk factors with the prevalence of work related carpal tunnel syndrome. j occup rehabil 2008;18:152-6. 8. sastroasmoro s. dasar-dasar metodologi penelitian klinis. edisi 2. jakarta: cv agung seto;2002. 9. world health organization. appropriate body mass index for asian populations and its implications for policy and intervention strategis. lancet 2004;363; 157-63. 10. katz jn, simmons bp. carpal tunnel syndrome. n engl j med 2002;346:1807-12. 11. lajoie as, mccabe sj, thomas b, edgell se. determining the sensitivity and specificity of common diagnostic testa for carpal tunnel syndrome using latent class analysis. plast reconstr surg 2005; 116:502-7. 12. scanlon a, maffei j. carpal tunnel syndrome. j neuroscience nursing 2009;41:140-7. 13. nathan pa, isthvan ja, meadows kd. a longitudinal study of predictors of research defined carpal tunnel syndrome in industrial workers: findings at 17 years. j hand surg 2005;30:593-8. 14. wellman h, davis l, punnet l. work related carpal tunnel syndrome (wr-cts) in massachusetts 1992-1997: source of wr-cts, outcomes, and employer intervention practices. am j ind med 2004;45:139-52. 15. palmer kt, harris c, coggnon d. carpal tunnel syndrome and its relation to occupation: a systematic literature review. occup med 2007;57: 57-66. 16. stapleton mj. occupation and carpal tunnel syndrome. anz j surg 2006;76:494-6. 17. katz jn, simmons bp. carpal tunnel syndrome. n engl j med 2002;346:1807-11. 18. gedizlioglu m, arpaci e, cevher d, ce p, kulan ca, colak i, et al. carpal tunnel syndrome in turkish steel industry. occup med 2008;58:212-4. 19. cole dc, ibrahim s, shannon hs. predictors of work-related repetitive strain injuries in a population cohort. am j pub health 2005;95;1233-7. 20. werner ra, franzblau a, gell n, ulin ss, armstrong tj. a longitudinal study of industrial and clerical workers: predictors of upper extremity tendonitis. j occup rehabil 2005;15:37-46. 21. geoghegan jm, clark di, bainbridge lc, smith c, hubbard r. risk factors in carpal tunnel syndrome. j hand surg eur vol 2004;29:315-20. 22. solomon dh, katz jn, bahn r, mogun h, avorn j. nonoccupational risk factors for carpal tunnel syndrome. j gen intern med 1999;14:310-4. 23. ferry s, hannafoed p, warskyj m, lewis m, croft p. carpal tunnel syndrome: a nested case-control study of risk factors in women. am j epidemiol 2000;151:566-74. 24. toesca a, pagnotta a, zumbo a, sadun r. estrogen and progesterone receptors in carpal tunnel syndrome. cell biol int 2008;32:75-9. dr.sylvia 1 effect of dillenia suffruticosa extract on dengue virus type 2 replication *department of microbiology medical faculty trisakti university correspondence dr. sylvia y. muliawan, phd department of microbiology medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 ext. 2402 email: sylviaym2003@yahoo.com universa medicina 2008; 27: 1-5. january-march, 2008january-march, 2008january-march, 2008january-march, 2008january-march, 2008 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 vol.27 no.1 drugs of plant origin can have easy acceptability as they are non-toxic and inexpensive. dillenia suffruticosa (d.suffruticosa), also known as simpoh air in malaysia, has antibacterial and antifungal activity, but no reported antiviral activity. this led the investigation of this plant against dengue virus type 2 (dv2) replication. two type of samples: dv2-infected c6/36 cells and uninfected c6/36 cells were prepared. d.suffruticosa extract was prepared in water. the plant extract, d.suffruticosa, was subjected to c6/36 cells with following concentrations, 0.025, 0.05, 0.1, 0.2, 0.4 mg/ml and incubated for 2 hours before infected by dv2. immobilized non-linear ph gradient strips, ph 3-10 were used in isoelectric focusing, and 10% homogeneous gels were used in sodium dodecyl sulphatepolyacrylamide gel electrophoresis. the results were stained by silver staining for visualization. it was found that ns1 protein expression on infected c6/36 cells was down regulated in intensity of concentrations 0.025, 0.05 and 0.1 mg/ml. however, when higher concentration of d.suffruticosa extract were used (0.2 and 0.4 mg/ml), the cells exhibited slightly reduced ns1 protein spots, when compared to the above concentrations (0.025, 0.05, 0.1 mg/ml). d.suffruticosa extract has an inhibitory effect on the replication of dv2. non-structural ns1 could be used as a diagnostic marker and/or as a parameter to evaluate the effect of antiviral agents for dengue type 2 virus infection/replication. keywords: dillenia suffruticosa, dengue virus type 2, proteomics abstract sylvia y. muliawan introduction many types of information cannot be obtained from the study of genes alone. for example, it is proteins and not genes, that are responsible for the phenotypes of cells. it is impossible to elucidate mechanisms of disease, aging, and effects of the environment solely by study the genome. only through the study of p r o t e i n s c a n p r o t e i n m o d i f i c a t i o n s b e c h a r a c t e r i z e d a n d t h e t a r g e t s o f d r u g s identified.(1) there is an increasing interest in proteomics technologies now. from a simplistic pharmacological point of view, functional universa medicina 2 muliawan d.suffruticosa on dengue virus proteomics via the analysis of protein complexes would contribute to the identification of novel drug targets, the reconstruction of pathways and to understanding the mechanism of action and s i d e e ff e c t s o f t h e r a p e u t i c c o m p o u n d s . ( 2 ) information at the level of the proteome is critical for understanding the function of specific cell types and their role in health and disease.(3) the application of proteomics provides major opportunities to elucidate disease mechanisms and to identify new diagnostic markers and therapeutic targets.(4) by the very definition of proteomics, it is inevitable that complex protein m i x t u r e s w i l l b e e n c o u n t e r e d . t h e r e f o r e methods must exist to resolve these protein mixtures into their individual components so that the proteins can be visualized, identified, a n d c h a r a c t e r i z e d . ( 1 ) t h e p r e d o m i n a n t technology for protein separation and isolation by use of polyacrylamide gel electrophoresis. d i f f e r e n t i a l d i s p l a y p r o t e o m i c s f o r comparison of protein levels has potential application in a wide range of diseases. graves et al(1) believed that if the appropriate controls were performed, proteomics was an extremely powerful approach for addressing important p h y s i o l o g i c a l q u e s t i o n s . i n t h i s a r e a , t h e m i c r o b i o l o g i s t u s i n g p r o t e o m i c s h a s a significant advantage in cases when the genome of the microbe and even the specific isolate being investigated, is fully sequenced.(5) this clearly improves the success rate of protein identification. the quantitative study of protein expression between samples that differ by some variable is known as expression proteomics.(1) proteomics attempts to catalog and characterize these proteins, compare variations in their expression levels in health and disease, study their interactions, and identify their functional roles. the search for sources of new biologically active compounds is important for the discovery o f n e w d r u g s r e l a t i v e t o t h e t r e a t m e n t o f diseases. in many gardens, there may be plants that will provide suitable breeding sites for m o s q u i t o e s . t h e s e c o m e i n t h e f o r m o f phytotelmata (singular phytotelma), that is, non-aquatic plants that impound water. they include leaf axils, inflorescence, modified leaves, stem holes or depressions, fallen leaves and open fruits. some examples are bromeliads, bananas, taro, gingers, dillenia suffruticosa ( s i m p o h ) , a n d f i c u s a u r i c u l a t a ( f i c u s roxburghiana; ara). the leaf axils and inflorescences are often overlooked as potential breeding sites for mosquitoes. examples of mosquitoes breeding in leaf axils include members of the genera a e d e s , c u l e x , to p o m y i a , to x o r h y n c h i t e s , tripteroides and uranotaenia. drugs of plant origin can have easy acceptability as they are n o n t o x i c a n d i n e x p e n s i v e . t h e c u r r e n t l y a v a i l a b l e a n t i v i r a l c o m p o u n d s a n d t h e antibiotics all have major drawbacks such as a narrow spectrum of activity, limited therapeutic usefulness, and variable degrees of toxicity. any new agent, which could overcome some or all of these disadvantages, would be important. neem leaves (azadirachta indica juss) are traditionally being used as curative against certain fungal, bacterial diseases,(6) and as an antiviral against small pox, fowl pox, polio and herpes simplex.(7) mangrove plants are being used in folklore medicine for treatment of several diseases, such as anti-hiv activity.(8) a review of the literature concerning the s c r e e n i n g o f p l a n t e x t r a c t s f o r v a r i o u s biological activities revealed that there have b e e n o n l y a l i m i t e d n u m b e r o f s t u d i e s t o evaluate the screening of plants for antiviral activity; therefore, extensive studies in this area are justified. in this study, dillenia suffruticosa (d.suffruticosa) extract was evaluated against dengue virus type 2 replication. 3 materials and methods preparation of uninfected sample i n t h i s s t u d y , c 6 / 3 6 c e l l s f r o m t h e department of medical microbiology, faculty of medicine, university of malaya, malaysia were prepared as per the method described by muliawan. (9) briefly, the c6/36 cells were disrupted by repeated fast freezing, three times for 15 minutes, and subsequently pelleted by spinning at 4°c, 2000 rpm for 10 minutes. the pellet was resuspended with 1 ml lysis buffer (8m urea, 4% triton-x 100, 2% ipg-phosphate buffer 3-10), incubated for 1 hour at 4°c and again spun as above. the supernatant was stored at -20°c. dv2-infected c6/36 cells dengue virus type 2 (dv2) cells (newguinea c strain), obtained from the department of medical microbiology, faculty of medicine, university of malaya, malaysia were prepared as per the method described by muliawan.(9) briefly, virus stocks were used to infect 80% confluent cell monolayers and incubated at 28°c until cytopathic effect (cpe) was observed (up to day-4), at which stage the supernatant and cell monolayers were harvested. preparation of d. suffuticosa extract in water briefly, one hundred grams of each sample was soaked in 800 ml distilled water and heated at 60°c in a water bath for 4-5 hours, and then filtered it. the residue was washed with additional distilled water. the combined filtrate was concentrated using a freeze drying (heto.fd4.lab equipment). the concentrated water extract was used for the screening bioassay experiments. d.suffruticosa extract subjected to proteomics technique two-dimensional gel electrophoresis (2dge) was performed using the multiphore ii e l e c t r o p h o r e s i s s y s t e m b a s e d o n t h e recommended method by berkelman et al(10) and m u l i a w a n . ( 9 ) to d e t e r m i n e t h e e ff e c t o f d.suffruticosa extract on c6/36 cells infected with dv2 treated with low concentrations (serial dilutions of 0.025 to 0.1 mg/ml), and high concentrations (serial dilutions of 0.2 to 0.4 mg/ ml) of d.suffruticosa extract were loaded on the drystrip (ph 3-10, 11cm). the proteins separated on isoelectric focusing unit for the first dimension and were separated further according to their m o l e c u l a r w e i g h t o n 1 0 % h o m o g e n e o u s polyacrylamide gel on the second dimension and then visualized by silver staining. results the ns1 protein spots on dv2-infected c6/ 36 cells were reduced after treatment with d.suffruticosa extract, i.e. in concentration of 0.025 mg/ml (fig.1a); 0.05 mg/ml (fig. 1b); 0.1 mg/ml (fig. 1c) and in concentration 0.02 mg/ ml (fig. 2a); 0.4 mg/ml (fig. 2b) compared to infection c6/36 cells without treatment (fig. 3). and the result of ns1 protein expression of dv2infected c6/36 cells after treatment with high concentration of this extract (fig. 2a, 2b) revealed the ns1 protein spots were slightly reduced when compared to the ns1 protein expression when treated with low concentrations of this extract (fig. 1a, 1b and 1c). discussion d.suffruticosa, is known in malaysia as “simpoh air”.(11) in the state of perak, peninsular malaysia, d.suffruticosa is used as traditional medicine for its antibacterial and antifungal activities.(12) in this study, ns1 was used as a m a r k e r a n d p a r a m e t e r t o i n d i c a t e d e n g u e infection(9) and to evaluate the protective efficacy of d.suffruticosa extract against dv2 replication through expression proteomics. this s tudy universa medicina vol.27 no.1 4 muliawan d.suffruticosa on dengue virus showed that d.suffruticosa extract has the ability to down regulated ns1 protein expression in various low concentrations of 0.025-0.1 mg/ ml and in various high concentrations of 0.20.4 mg/ml. comparing the results of ns1 protein expression of dv2-infected c6/36 cells after treatment with different concentrations of this extract revealed the ns1 protein spots after treatment with various higher concentration (0.20.4 mg/ml) of this extract were slightly reduced when compared to the protein expression when treated with various lower concentration (0.0250.1 mg/ml) of this extract. since there were no previous reports of studies employing this technique and/or material, it could not obtain comparative data to support the results. a b figure 2. proteins profile of infected c6/36 cells treated by d. suffruticosa extract in concentrations 0.2 mg/ml (fig. 2a), and 0.4mg/ml (fig. 2b) figure 3. protein profile of infected c6/36 cells without treatment cb a figure 1. proteins profile of infected c6/36 cells treated by d.suffruticosa extract: in various concentrations of 0.025mg/ml (a), 0.05mg/ml (b), and 0.1mg/ml (c) of this extract 5 conclusion d.suffruticosa extract has the inhibitory potential against dv2 replication and the ns1 protein expression could be used as a diagnostic marker and/or as a parameter to evaluate the effect of the antiviral agents for dengue type 2 virus infection/replication. acknowledgements the author expresses her gratitude to prof. lam sk, prof. devi s, prof. hashim oh, and p r o f . yu s o f r f r o m f a c u l t y o f m e d i c i n e , university of malaya, kuala lumpur, malaysia, and also to prof. dr. chambers tj, saint louis university school of medicine, st. louis, usa for their support in this study. references 1. graves pr, haystead taj. molecular biologist’s guide to ptroteomics. microbiol mol biol rev 2002; 66: 39-63. 2. bauer a, kuster b. affinity purification-mass spectrometry powerful tools for the characterization of protein complexes. eur j biochem 2003; 270: 570-8. 3. srinivas pr, srivastava s, hanash s, wright gl. proteomics in early detection of cancer. clin chem 2001; 47: 1901-1. 4. chambers g, lawrie l, cash p, murray gi. proteomics: a new approach to the study of disease. j pathol 2000; 192: 280-8. 5. cash p. proteomica in medical microbiology. electrophoresis 2000; 21: 1187-201. 6. parida mm, upadhyay c, pandya g, jana am. inhibitory potential of neem (azadirachta indica juss) leaves on dengue virus type-2 replication. j ethnopharmacol 2002; 79: 273-8. 7. reddy ab, sethi ms. antiviral effects of some indigeneous plant extracts on vaccinia and fowl pox viruses on chick embryo fibroblasts. indian j exp biol 1974; 12: 572-9. 8. premnathan m, nakashima h, kathiresan k, rajendran n, yamonto n. in vitro anti-human immunodeficiency syndrome virus activity of mangrove plants. indian j med res 1996; 103: 278-81. 9. muliawan sy. profile of nonstructural glycoprotein ns1 as a diagnostic marker in dengue type 2 virus infection. univ med 2007; 26: 73-9. 10. berkelman t, stenstedt t. 2-d electrophoresis using immobilzed gradients: principles and methods. uppzala: ammersham pharmacia biotech inc; 1998. 11. low ks, lee ck, tan bf. quaternized wood as sorbent for reactive dyes. appl biochem biotechnol 2000; 87: 233-45. 12. wiart c, mogana s, khalifah s, mahan m, ismail s, buckle m, et al. antimicrobial screening of plants used for traditional medicine in the state of perak, peninsular malaysia. fitoterapia 2004; 75: 68-73. universa medicina vol.27 no.1 alvina 189 abstract *health research unit, faculty of medicine, university of padjadjaran, bandung **department of community medicine, united arab emirates university, al ain, united arab emirates ***former institution: leiden university medical center correspondence dr. edhyana sahiratmadja, phd health research unit, faculty of medicine, university of padjadjaran jl. eijkman no.38 bandung phone: 022-2033915 email: e.sahiratmadja@gmail.com univ med 2011;30:189-96. smoking habit as a risk factor in tuberculosis: a case-control study edhyana sahiratmadja* and nico nagelkerke**,*** september-december, 2011september-december, 2011september-december, 2011september-december, 2011september-december, 2011 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 vol.30 no.3 universa medicina indonesia is fifth in the tuberculosis (tb) prevalence globally and this country is one of the largest tobacco producers. smoking has been reported to be an important risk factor for tb and a reduction in smoking could be expected to have a significant impact on tb incidence and prevalence. however, studies from various countries yielded conflicting results. our study aims to explore the association between smoking and tb in indonesia as tb-endemic country. in two major cities of indonesia, jakarta and bandung, a case-control study had been conducted. tb was diagnosed based on who criteria including clinical presentation, and chest x-ray (cxr) examination, and confirmed by microscopic detection of acidfast bacilli in ziehl-nielsen stained sputum smears or by culture of m. tuberculosis. newly diagnosed smear-positive pulmonary tb patients (n=802) and their spouses (n=253) or sex-matched neighborhood controls (n=534) were interviewed about their smoking habits. an extensive questionnaire was used to collect data about smoking habits of both patients and controls. smoking categories were grouped into ever (for current/past smokers) and never. our study result showed that smoking appears not to be strongly associated with tb (or=0.99, 95% ci 0.761.31). the reasons for the effect heterogeneity remain to be elucidated as smoking is a lethal habit and should be well controlled. the need to incorporate tobacco cessation programs into tb treatment is strongly recommended to improve tb control. keywords: smoking, tuberculosis, aged >15 years introduction with approximately 7-8 million new cases and 2-3 million deaths annually, tuberculosis (tb) is one of the leading infectious causes of death worldwide.(1) although sufficiently high case detection and cure rates may ultimately eliminate transmission of the causative organism m. tuberculosis, this objective is currently out of reach due to difficulties of diagnosis, multidrug resistance, treatment adherence, and the hiv pandemic.(2,3) new control tools, including better diagnostics, new drugs, and a more effective vaccine are therefore badly needed. 190 sahiratmadja, nagelkerke smoking habit and tuberculosis another potential avenue for tb control is control of risk factors. several risk factors for tb are known, either affecting the risk of infection, or the risk of developing disease after infection; including socio-economic factors (e.g. crowding), concomitant diseases and infections (e.g. diabetes mellitus, and hiv), and behavioral factors (e.g. smoking).(4) a strong association between tobacco smoking and tb has been found in several studies carried out in various countries. meta-analysis of those studies concluded that smoking could be considered as an important risk factor for t h e d e v e l o p m e n t o f t b . (5 ) t h e r e i s a l s o evidence that some of the sex differences in w o r l d w i d e t b n o t i f i c a t i o n r a t e s c a n b e explained by sex differences in smoking.(6) interestingly, a systematic review of studies in four countries: china, india, uk and usa, found associations between both the risk of tb infection and smoking, and between tb disease a n d s m o k i n g , s u g g e s t i n g t h a t t h e l a t t e r association may be due to the increased risk of tb infection among smokers rather than an increased risk of tb disease.(7) the review included several high quality studies, carried out, inter alia in india, where the association appears to be particularly strong. for example, in a nested case-control study carried out in tamil nadu, india, a positive and dosedependent relationship was identified with odds ratios (or) being as high as 3.23 for the highest smoking category.(8) possibly the largest ever study on this association was carried out by gajalakshmi,(9) which positively identified smoking as a major risk factor for t b m o r t a l i t y i n m e n i n i n d i a . wi t h a n estimated relative risk of over 4, smoking would rank only second in strength among known risk factors for tb mortality after hiv i n f e c t i o n , w h i l e i n t e r m s o f p o p u l a t i o n attributable fraction, it may even rank first. in the same study, from a separate population survey, slightly more moderate relative risk estimates were obtained for the effect of smoking on past or present prevalent tb. for cigarettes the prevalence ratio was 1.7 for <10 cigarettes/day and 2.6 for heavier smokers. for bidis (a cigarette rolled in the leaf of another plant, temburni) these prevalence ratios were 2.9 (< 15/day) and 4.5 (15/day or more) respectively. other studies have also found a positive association between smoking and tb. for example, in a cohort study carried out in h o n g k o n g a h i g h l y s i g n i f i c a n t e ff e c t (adjusted hr 2.87) of current smoking on pulmonary tb was found.(10) in a hospitalb a s e d c a s e c o n t r o l s t u d y c a r r i e d o u t i n thailand again a strong positive relationship was found.(11) smoking was similarly found to be a risk factor for tb in a study from south africa,(12) although the strength of the effect (or=1.61) was smaller than that found in either india or hong kong. this might, in part, be attributable to the design used in the southafrican study, in which cases were deaths from smoking related conditions and controls deaths f r o m p re s u m a b l y n o n s m o k i n g r e l a t e d disorders. however, not all studies yielded positive associations between smoking and tb. for example, in chengdu, szetchuan province china, a positive association was identified, b u t o n l y i n a s s o c i a t i o n w i t h a l c o h o l consumption,(13) while, in a case-control study carried out in malawi, no association at all was identified.(14) in view of the heterogeneity of results and the non-experimental nature of all s t u d i e s , w i t h t h e r i s k o f c o n f o u n d i n g , combined with a lack of insight into the extent of publication bias, additional information on the association between smoking and tb would be valuable. as passive smoking was also found to constitute a risk factor, tobacco control could become one of the key pillars of tb control.(15) with an estimated incidence rate of 245/100.000 and a prevalence rate of 275/ 100.000, indonesia harbors over 10% of tb cases worldwide, which makes this country rank third in terms of tb prevalence.(3) in order 191 to further explore the effect of tobacco smoking on tb in indonesia as a tb-endemic country, we analyzed smoking data from a case-control s t u d y a s p a r t o f a l a rg e r s t u d y o n t h e immunogenetic basis of susceptibility to and d i s e a s e m a n i f e s t a t i o n o f m y c o b a c t e r i a l infections in indonesia. methods research design a case-control study was carried out from march 2001 to december 2005 in two major cities of indonesia, jakarta and bandung, as part of a larger study on the immunogenetic b a s i s o f s u s c e p t i b i l i t y t o a n d d i s e a s e manifestation of mycobacterial infections in indonesia, which is a collaborative study between indonesian institutes in jakarta and bandung and the netherlands. research subjects a total of 1020 newly diagnosed smear positive active pulmonary tb patients aged >15 years from tb outpatient clinics were enrolled. tb patients were recruited from perkumpulan pemberantasan tuberkulosis indonesia (ppti) in jakarta and from a tb clinic of rumah sakit hasan sadikin. briefly, tb was diagnosed based on who criteria including clinical presentation and chest x-ray (cxr) examination, and confirmed by microscopic detection of acid-fast bacilli in ziehl-nielsen stained sputum smears or by culture of m. tuberculosis. free anti-tb drug treatment was provided to all patients, consisting of a standard regimen of isoniazide, rifampicin, pyrazinamide and ethambutol (2hrze/4h3r3) according to guidelines of the national tb program. treatment was supervised once weekly by directly observed treatment program. active pulmonary tb p a t i e n t s w i t h h i vs e r o p o s i t i v e ( n = 1 0 ) , concomitant diabetes mellitus (dm, n=153), other co-morbidity such as heart diseases (n=7), and those with missing smoking data (n=48), were excluded in this current study. dm in this study was excluded because it is strongly associated with tb as described previously elsewhere.(16) in the same period, a total of 895 controls, i n c l u d i n g s p o u s e c o n t r o l s a n d n e i g h b o r controls were recruited. spouse controls were enrolled in view of the interest in passive smokers. control neighbors were selected randomly from individuals in the same rukun tetangga, the smallest residential unit or compound system in indonesia consisting of 15-30 households, and matched with patients of the same sex, age (+/10%), and socioe c o n o m i c b a c k g r o u n d . c o n t r o l s w e r e interviewed using the same standardized questionnaire, and subjected to physical and blood examination, and cxr scheme as tb cases. controls were excluded when they showed signs and symptoms suggestive of active tb as seen in the cxr or had a history of anti-tb therapy (n=46), concomitant dm or other co-morbidities (n=30), were hivseropositive (n=3 of 238 tested controls), or had missing smoking data (n=29). data collection in a part of an extensive questionnaire smoking habits were asked in both patients and controls. further we grouped the smoking category into ever (i.e. current and past) vs. never, and the outcome variable into tb or control. ever smoking was used, instead of current smoking, as tb patients may have quitted smoking because of their disease. unfortunately, no information was available on the time when quitters stopped smoking. ethical clearance written consent was obtained from all subjects, and the study protocol was approved by the ethical committees of the medical faculty, university of indonesia, jakarta and the medical faculty, university of padjadjaran, bandung. univ med vol.30 no.3 192 sahiratmadja, nagelkerke smoking habit and tuberculosis statistical analyses as spouse controls were counter-matched for gender, and because in bandung the information linking individual cases to controls was not adequately collected, we decided for our main data analysisto ignore the matching structure and to analyze the data using standard binary logistic regression, and to include the matching structure (jakarta only) only as an auxiliary analysis using conditional logistic regression. the level of statistical significance was set at p<0.05. results demographic data in total, newly diagnosed smear-positive pulmonary tb patients (n=802) and their spouse (n=253) or sex-matched neighborhood controls (n=534) were analyzed, distributed in two cities (jakarta and bandung) as shown in table 1. the mean age of all cases was 31.6 years ± s.d. 10.9 and 29.8 ± s.d. 10.6 for men and women, respectively. the mean age of all neighbor controls was 33.1 ± 12.0 years and 3 2 . 3 ± 11 . 4 y e a r s f o r m e n a n d w o m e n , respectively, while for all control spouses these numbers were 33.8 ± 11.7 years and 32.0 ± 10.2 years, respectively. data from participants in jakarta showed that bcg scar status in tb patients differed significantly from the control group, consisting of both neighbor and spouse controls (p=0.03; or 0.75; ci 0.59-0.97, table 1). the status of ‘no bcg scar’ does not mean that the participants never received bcg vaccination, as no medical record of bcg vaccination in the past was available. table 2 shows the bcg scar and no bcg scar group i n d i ff e r e n t a g e g r o u p s . b e t t e r m e d i c a l recording for nation-wide vaccination is recommended to study the extent of protection afforded by bcg vaccination. table 1. demographic data of the participants from two cities in indonesia, jakarta and bandung note: *not all controls in jakarta or bandung have data on bcg scar. the number is only from the participants with bcg data; **neighbor controls were not equally recruited. †† statistically significant (p<0.05) in jakarta participants and in total number of analyses 193 smoking data smoking categories were grouped into ever (for current/past smokers) and never. notably, most of the males were smokers and most of the females were non smokers (table 1). surprisingly, no significant difference between pulmonary tb and control groups was found. this is different from global findings showing that smoking is a significant risk factor. logistic regression of cases on age, age-squared, site (jakarta or bandung), sex, and ever smoking yielded no statistically significant association between any of the variables and tb, with an adjusted odds ratio (or) for the association between tb and smoking of 0.99 (95% ci 0.761.31). stepwise logistic regression with forward selection only yielded a (negative) statistically significant association between age and tb (data not shown). repetition of the analysis with exclusion of spousal controls, to reduce the risk of over-matching (exposure to passive smoking, highly concordant tb infection) gave similar results with an or of 0.93 (95% ci: 0.68-1.28). re-analysis using conditional logistic regression to take the matching into account using only data from jakarta again yielded no significant association between s m o k i n g a n d t b ( d a t a n o t s h o w n ) . t h e distribution of smoking habits in tb patients and controls in jakarta and bandung, two major cities in indonesia, is presented in table 3. table 3. the distribution of smoking habits in male and female pulmonary tb patients, compared to neighbour and spouse controls in two major cities in indonesia note. smoking categories were grouped into ever for current/past smokers and never; * logistic regression analysis of cases on site (jakarta or bandung), sex, and ever smoking, yielded no statistically significant association d tb; ** repetition of the analysis with spousal controls excluded, to reduce the risk of over-matching or (odds ratio). table 2. the distribution of bcg scar for age group among pulmonary tb patients and controls univ med vol.30 no.3 194 sahiratmadja, nagelkerke smoking habit and tuberculosis discussion indonesia is one of the biggest tobacco producers in the world and smoking-at least among men is very common.(17) indonesia has the third highest tb-prevalence worldwide after india and china, countries where smoking appears to be a strong risk factor.(18) if smoking would be a causal risk factor for tb in this high burden country as it appears to be in many other countries an enormous number of cases could be prevented through tobacco control.(19) in our present study in indonesia, however, no significant positive association between smoking and tb was found. thus, our data would seem to suggest that smoking is less of a risk factor for tb in indonesia than in many other places in the world. although the sample size of our study was only small compared to the studies in india and china, resulting in somewhat wide confidence intervals for effect parameters, our data seem to contradict a major effect of smoking on tb in this population. the question is how to explain this a p p a r e n t h e t e r o g e n e i t y i n t h e e f f e c t o f smoking. first, our study was not specifically designed to study this association, and smoking habits were not elicited in great detail, also no attempt was made to independently verify information. second, misclassification of smoking status may have biased the odds ratios towards unity. another explanation may be overmatching in our study. some of the controls in this study were spouses, and s m o k i n g i n s p o u s e s m a y b e c o r r e l a t e d , especially if passive smoking would have a similar effect as smoking itself. a study in china showed that passive smoking accounted for 13.7% of active tb and for 18.5% of culture-positive tb;(15) however, in our study only a minority of the controls were spouses and exclusion of spouse controls did not affect our conclusions. a fourth possibility may be that the heterogeneity in the effect of smoking i s r e a l , d u e t o d i ff e r e n c e s i n e x p o s u r e s resulting from smoking. for example, the type of tobacco smoked may differ, which could lead to different exposure to, and uptake of, various chemical substances and elements such as iron.(20) smoking habits differ markedly between countries. in india, for example, many people smoke (unfiltered) bidis, whereas in indonesia kretek (unfiltered, cloves containing) cigarettes are common.(17) cloves are known to have disinfecting properties, and may have immuno-modulatory effects as well.(21) also the average amount of tobacco smoked may differ among countries. fifth, either the associations between smoking and tb in other places may have been due to confounding, or conversely our failure to find an association may be due to confounding by an unmeasured variable. in indonesia most men smoke, but most women do not. this makes men who do not s m o k e , o r w o m e n w h o d o , s o m e w h a t exceptional, and the reason for not smoking by men, for instance, may be health related. smoking may be associated with other risk factors for tb, and these associations may d i ff e r a m o n g c o u n t r i e s , w h i c h m a k e s i t notoriously difficult to rule out or adjust for confounding. for example, it is well known that in many populations smoking and alcohol consumption are positively correlated. (22) unfortunately, we have no data on alcohol consumption to explore whether there are any interactions with alcohol consumption, but in view of the fact that the majority of indonesian population are muslims, this is probably low. other associations may exist, for example, it is still disputed whether the association between smoking and cervical cancer which has been observed in different populations is c a u s a l o r d u e t o c o n f o u n d i n g b y s e x u a l behavior.(23) similarly disputed is the protective effect of smoking on alzheimer’s disease.(23,24) however, confounding can also mask true associations, which might explain our failure to find an association. one may use hill’s (minimal) criteria to explore the plausibility of causality.(25) the association between tobacco and tb clearly 195 meets several of these criteria. for example, an association is highly plausible given that tobacco smoking affects the lungs, and is also known to have many effects on the human immune system and increases the risk of several other infections as well.(26,27) several studies, such as the two studies cited above carried out in india, also clearly identify a dose-response relationship, again strongly supporting a causal relationship. another important criterion is consistency, i.e. whether the result can be replicated in different settings or using different methodologies. here the support for a causal relationship between tobacco and tb appears somewhat less positive in view of the negative or mixed findings from malawi, chengdu and indonesia. case-control studies and other nonexperimental studies have many potential b i a s e s . (2 8 ) i n a d d i t i o n , t h e r e e x i s t s t h e possibility of publication bias, as studies identifying smoking as a risk factor would have greater potential public health impact than those that did not.(19) whether this indeed occurs is uncertain, since no registers of epidemiological studies exist as they do for clinical trials. nevertheless, large-scale studies such as the one carried out in india are unlikely to go unreported even when the results would show no association. obviously, no clinical trial to establish experimentally the effect of tobacco smoking on tb is possible, though experiments in vivo and in vitro with primary human alveolar macrophages exposed to cigarette smoke extract, nicotine, or acrolein showed an increased burden of intracellular m. tuberculosis.(29) conclusion tb is a multifactorial disorder. our finding showed that smoking appears not to be strongly associated with tb in indonesia. this inconsistency in smoking and tb findings needs to be further explored as it may provide insight into both the noxious effects of tobacco smoking and the pathogenesis of tb. it has little bearing on public health policy, however, since smoking is a lethal habit and should be controlled by all reasonable means. the need to actively incorporate tobacco cessation p r o g r a m s i n t o t b s e r v i c e s i s s t r o n g l y recommended to improve tb control. acknowledgement this study is an indirect result of the project “imunogenetic basis of susceptibility to and disease manifestation of mycobacterial infections”, financially supported by the royal academy of arts and sciences (knaw), the netherlands, project no 99med01. we would like to thank paul eijler for his support in finding archival data in the netherlands. we are grateful to the members of tb working group under scientific program of indonesian and nederland (spin); prof rhh nelwan, prof sangkot marzuki and bachti alisjahbana from indonesia, and prof thm ottenhoff, esther van de vosse, prof jwm van der meer and reinout van crevel from the netherlands for their continuous support. competing interests the authors declare that they have no competing interests. references 1. frieden tr, sterling tr, munsiff ss, watt cj, dye c. tuberculosis. lancet 2003;362:887-99. 2. lönnroth k, raviglione m. global epidemiology of tuberculosis: prospects for control. semin respir crit care med 2008;29:481-91. 3. world health organization. global tuberculosis control: surveillance, planning, financing. who report 2005. who/htm/tb/2005.349. world health organization, geneva 2005. 4. lienhardt c, fielding k, sillah js, bah b, gustafson p, warndorff d, et al. investigation of the risk factors for tuberculosis: a case-control study in three countries in west africa. int j epidemiol 2005;34:914-23. univ med vol.30 no.3 196 sahiratmadja, nagelkerke smoking habit and tuberculosis 5. maurya v, vijayan vk, shah a. smoking and tuberculosis: an association overlooked. int j tuberc lung dis 2002;6:942-51. 6. watkins re, plant aj. does smoking explain sex differences in the global tuberculosis epidemic? epidemiol infect 2006;134:333-9. 7. davies pd, yew ww, ganguly d, davidow al, reichman lb, dheda k, et al. smoking and tuberculosis: the epidemiological association and immunopathogenesis. trans r soc trop med hyg 2006;100:291-8. 8. kolappan c, gopi pg. tobacco smoking and pulmonary tuberculosis. thorax 2002;57:964-6. 9. gajalakshmi v, peto r, kanaka ts, jha p. smoking and mortality from tuberculosis and other diseases in india: retrospective study of 43000 adult male deaths and 35000 controls. lancet 2003;362:507-15. 10. leung cc, li t, lam th, yew ww, law ws, tam cm, et al. smoking and tuberculosis among the elderly in hong kong. am j respir crit care med 2004;170:1027-33. 11. ariyothai n, podhipak a, akarasewi p, tornee s, smithtikarn s, thongprathum p. cigarette smoking and its relation to pulmonary tuberculosis in adults. southeast asian j trop med public health 2004;35:219-27. 12. sitas f, urban m, bradshaw d, kielkowski d, bah s, peto r. tobacco attributable deaths in south africa. tob control 2004;13:396-9. 13. anderson, johnson c, palmer ph, chou cp, pang z, zhou d, et al. tobacco use among youth and adults in mainland china: the china seven cities study. public health 2006;120:1156-69. 14. crampin ac, glynn jr, floyd s, malema ss, mwinuka vk, ngwira bm, et al. tuberculosis and gender: exploring the patterns in a case control study in malawi. int j tuberc lung dis 2004;8:194-203. 15. leung cc, lam th, ho ks, yew ww, tam cm, chan wm, et al. passive smoking and tuberculosis. arch intern med 2010;170:28792. 16. alisjahbana b, van crevel r, sahiratmadja e, den heijer m, maya a, istriana e, et al. diabetes mellitus is strongly associated with tuberculosis in indonesia. int j tuberc lung dis 2006;10:606700. 17. aditama ty. kretek. the 13th world conference on tobacco or health building capacity for a tobacco-free world. july 12-15, 2006, washington, dc, usa available at: http:// 2006.confex.com/uicc/wctoh/techprogram/ meeting.htm. accessed july 1, 2011. 18. siddiqi k, lee ac. an integrated approach to treat tobacco addiction in countries with high tuberculosis incidence.trop med int health 2009;14:420-8. 19. ng n, padmawati rs, prabandari ys, nichter m. smoking behavior among former tuberculosis patients in indonesia: intervention is needed. int j tuberc lung dis 2008;2:567-72. 20. boelaert jr, gomes ms, gordeuk vr. smoking, iron, and tuberculosis. lancet 2003;362:1243-4. 21. raghavenra h, diwakr bt, lokesh br, naidu ka. eugenol the active principle from cloves inhibits 5-lipoxygenase activity and leukotrienec4 in human pmnl cells. prostaglandins leukot essent fatty acids 2006;74:23-7. 22. gajalakshmi v, peto r. smoking, drinking and incident tuberculosis in rural india: populationbased case-control study. int j epidemiol 2009; 38:1018-25. 23. wolf r, freedman d. cigarette smoking, sexually transmitted diseases, and hiv/aids. int j dermatol 2000;39:1-9. 24. almeida op, hulse gk, lawrence d, flicker l. smoking as a risk factor for alzheimer’s disease: contrasting evidence from a systematic review of case-control and cohort studies. addiction 2002; 97:15-28. 25. hill ab. the environment and disease: association or causation? proc r soc med 1965; 58:295-300. 26. arcavi l, benowitz nl. cigarette smoking and infection. arch intern med 2004;164:2206-16. 27. sopori m. effects of cigarette smoke on the immune system. nat rev immunol 2002;2:3727. 28. jiang j, liu b, nasca pc, zeng x, chen j, zou x, et al. smoking and risk of death due to pulmonary tuberculosis: a case-control comparison in 103 population centers in china. int j tuberc lung dis 2009;13:1530-5. 29. shang s, ordway d, henao-tamayo m, bai x, oberley-deegan r, shanley c, et al. cigarette smoke increases susceptibility to tuberculosisevidence from in vivo and in vitro models. j infect dis 2011;203:1240-8. alvina 34 abstract *department of obstetrics and gynaecology, medical faculty, trisakti university correspondence dr. raditya wratsangka, spog department of obstetrics and gynaecology medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2706 email: raditya@trisakti.ac.id; radityaw@indo.net.id univ med 2010;29:34-45 psychosocial aspect determines quality of life in postmenopausal women raditya wratsangka* january-aprl, 2010january-aprl, 2010january-aprl, 2010january-aprl, 2010january-aprl, 2010 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 vol.29 no.1 universa medicina introduction according to the population census of the year 2000, the number of indonesian women more than 50 years of age and presumably having entered menopausal age was 15.5 million and in the year 2020 it is estimated that the number of women at menopausal age will be 30.3 million or around 11.5% of the total population of indonesia.(1) apart from the increasing number of menopausal women as part of the group of elderly people among the indonesian as well as the world population, there are several other factors that cause the the postmenopausal period plays an important role in women’s life and gives rise to many physical and mental problems. a cross-sectional was conducted to determine the quality of life and its determinants, namely pyschosocial and lifestyle factors in postmenopausal women. one hundred and seventy-six postmenopausal women meeting the inclusion criteria were recruited. the menopause rating scale (mrs) questionnaire was used for the assessment of quality of life. one hundred and thirty-one (74.4%) subjects had complaints related to menopausal symptoms in 3 degrees of severity: mild (38.6%), moderate (30.7%) and severe (5.1%). urogenital symptoms were the most frequent complaints reported by the subjects (71.6%). the correlation between quality of life of postmenopausal women and several psychosocial and lifestyle factors, namely marital status, education, occupation, knowledge and attitude towards menopause, exercise, smoking and consumption of caffeine and alcohol was statistically not significant (p>0.05). by multiple logistic regression analysis, some of the determinants, such as the subjects’ attitude towards menopause (pr = 2.863; 95% ci: 0.578 – 14.185) and support from the husband and/or family (pr = 2.124; 95% ci: 0.979 – 4.610) did not reach statistical significance, but were still the most influencing factors on their quality of life. quality of life is worst in postmenopausal women and therefore counseling and support from husband and/or family are needed by the postmenopausal women in order to improve their quality of life. keywords: psychosocial, quality of life, postmenopause 35 univ med vol.29 no.1 menopausal problem to be in need of special concern. the declining steroidal hormone levels, especially estrogen, in the female body in the perimenopausal period, not only alters the menstrual pattern but also affects general health, such as is apparent in wrinkled skin, dry vagina (leading to dyspareunia), dysuria, p a l p i t a t i o n s , m i g r a i n e , h o t f l u s h e s a n d i n s o m n i a , c o l l e c t i v e l y k n o w n a s t h e climacteric syndrome. the prevalence of the c l i m a c t e r i c s y n d r o m e i n e u r o p e a n a n d american women ranges from 74 to 87.2%,(2) while in china and other asian nations the incidence is 10%.(3) the prevalence of hot flushes varies widely across populations and is strongly influenced by culture and ethnicity. in the united states, the study of women’s health across the nation (swan) surveyed more than 16,000 women and found that the prevalence of hot flushes was highest among a f r i c a n a m e r i c a n s ( 4 6 % ) , f o l l o w e d b y hispanics (34%), whites (31%), chinese (21%), and japanese (18%).(4) several factors strongly suspected of playing a role in the menopausal experience and quality of life in this period are culture-bound behaviors, such as diet, smoking, and exercise.(5-7) preventive measures for proactive maintenance of health and vitality that may be taken by women in facing the menopause, as recommended by the international menopause society (ims), relate to healthy behavior and lifestyle, such as exercise, decreased consumption of caffeine, salt, and sugar, reduction or cessation of smoking and alcohol consumption, taking certain vitamins and/or minerals, adequate rest and sleep, consumption of a balanced diet (low i n f a t ) a n d u n d e rg o i n g r e g u l a r h e a l t h checkups. (8) several longitudinal studies conducted with sufficiently large samples in populations of menopausal women revealed a s u b s t a n t i a l l y s t r o n g r e l e v a n c e b e t w e e n psychosocial factors and biomedical aspects. with the support of healthy behavior and lifestyle, social support of friends and family, the symptoms of menopause may be adequately managed such that medical intervention is unnecessary.(6,7) quality of life (qol) does not only relate to problems of physical health or absence of disease, and currently there is no universally accepted definition of “quality of life”. the definition of “quality of life” according to the who is the perception of an individual ‘of his/ her position in life in the context of the culture and value systems in which he/she lives, and in relation to his/her goals, expectations, standards and concerns’. (9) in the health context, qol that is related to the impact of illness or health disturbance has a number of situational and conditional aspects in the life of an individual. the menopause rating scale (mrs) is a health-related scale that was d e v e l o p e d i n r e s p o n s e t o t h e l a c k o f standardized scales for measuring the severity of aging symptoms and their impact on the hrqol in the early 1990s. actually, the first version of the mrs was to be filled out by the treating physician but methodological critics l e d t o a n e w s c a l e w h i c h c a n e a s i l y b e completed by the women themselves, not by their physician.(10) the aims of the mrs were to enable comparisons of the symptoms of a g i n g b e t w e e n g r o u p s o f w o m e n u n d e r different conditions, to compare severity of symptoms over time, and to measure changes preand post-treatment.(11-13) i n m a n y d e v e l o p e d c o u n t r i e s t h e menopausal problem and its symptoms or the climacteric syndrome have become a national health problem. currently in indonesia many studies and investigations have started to be conducted on the menopausal problem, but are still limited to the biomedical aspects, such the climacteric syndrome, and lipid profile and osteoporosis in menopausal women.(14-15) the present study was done in order to study the degree of qol of postmenopausal women and to determine the presence of an association between psycho-social aspects and lifestyle on the one hand and qol on the other. 36 methods research design t h i s w a s a c r o s s s e c t i o n a l s t u d y t o determine the presence of an association between psycho-social aspects and lifestyle of indonesian postmenopausal women and their qol. the study was performed at the cilandak district health center – south jakarta from 23 march to 25 april 2009. subjects of study a total of 176 subjects were recruited by non-probability (or consecutive) sampling in all service units of the cilandak district health center – south jakarta. all women in the age r a n g e o f 4 5 5 9 y e a r s h a v i n g e n t e r e d menopause (with cessation of menstruation of minimally one year) and meeting the inclusion criteria were involved in this study. the inclusion criteria were: literate and healthy women (capable of independently performing daily activities of life and engaging in verbal communication), not on menopause-related hormonal drugs within the previous three months, willing to participate in the study until completion after receiving the necessary information, and willing to sign an informed consent. the exclusion criteria were prior bilateral oophorectomy and/or hysterectomy and non-compliance with the study protocol. data collection the data collectors conducted interviews by means of a questionnaire comprising questions on sociodemography and economy, knowledge about and attitude towards the menopause, use of hormone replacement therapy and family support, and a number of habits or lifestyles such as exercise, dietary c o m p o s i t i o n , h a b i t u a l c o n s u m p t i o n o f phytoestrogen-containing foods, smoking, consumption of caffeine and alcohol. the hrqol of the menopausal women were assessed by means of the indonesian version of the mrs, that had been tested on its validity and reliability in indonesia, comprising three d i m e n s i o n s o r s u b s c a l e s o f m e n o p a u s a l s y m p t o m s , v i z . p s y c h o l o g i c a l , s o m a t o vegetative and urogenital.(16,17) this instrument consists of questions on 11 menopausal symptoms, the degree of which is expressed as a number from 0 (no symptoms) up to 4 (extremely severe symptoms), as stated by the subject in question based on her own perception.(14,15) the subject was considered to have no qol abnormalities if the abnormality was “absent/slight” or if the subject had a total score of 0–4 by mrs assessment criteria. the s u b j e c t w a s c o n s i d e r e d t o h a v e q o l a b n o r m a l i t i e s i f t h e a b n o r m a l i t y w a s “moderate/severe” or if the subject had a total score of >5 by mrs assessment criteria. data analysis the study data were analyzed with spss (statistical package for social sciences) version 15.0. bivariate chi-square tests were used for comparative analysis of each of the independent variables (psychosocial and lifestyle) and the dependent variable (quality of life of menopausal women), both of which were categorical data. the relationships between several independent variables and the dependent variable were analyzed in a multiple logistic regression model. p-value < 0.05 was taken as a cut-off for statistical significance and all tests were 2-sided. results the subjects recruited into this study totaled 176 women in the age range of 45-59 years, where the youngest subject was 45 years of age and the oldest 59 years, and the mean s u b j e c t a g e w a s 5 3 ± 3 . 4 6 y e a r s . t h e assessment results on the degree of qol of the subjects conducted by means of the mrs questionnaire showed that 131 (74.4%) of the subjects had qol abnormalities, comprising mild (38.6%), moderate (30.7%) and severe degrees (5.1%). wratsangka qol in postmenopausal women 37 univ med vol.29 no.1 vi e w e d f r o m t h e a s p e c t s o f t h e abnormalities experienced by the subjects, the u r o g e n i t a l a b n o r m a l i t i e s w e r e t h e m o s t frequently suffered by the subjects (126 women or 71.6%), i.e. abnormalities connected with sexual and urinary (voiding) activities, and m o r e t h a n 5 0 % a m o n g t h e m h a d t h e abnormalities in moderate and severe degrees. another aspect of the abnormalities that was also experienced by more than half of the subjects related to psychological abnormalities (114 women or 64.7%) and somatovegetative abnormalities (106 women or 60.2%) (table 2). in this investigation, a study was performed on the relationship of marital status, level of education, occupational status, activities of the subjects outside their occupation, and sources of household finance and health funds on the one hand and qol on the other. there was no significant difference in the prevalence of qol abnormalities (p>0.05) between the groups of subjects in marital status, occupational status, activities outside occupation, and household finance and health funds (table 3). therefore it may be stated that i n t h i s s t u d y t h e s e s o c i o e c o n o m i c a n d demographic factors had no relationship with qol abnormalities in postmenopausal women. in this study there was also no significant d i ff e r e n c e i n t h e p r e v a l e n c e o f q o l abnormalities (p>0.05) in respect to: (i) knowledge of the subjects about menopause: between the group of subjects who considered menopause to be an illness, those who viewed menopause as a normal/natural process not needing special attention, and those for whom menopause was a natural process with a risk of negative impacts; (ii) attitude of the subjects towards menopause: between the group of subjects with a positive and those with a negative attitude towards menopause (table 4). as may be seen from table 5, in this study there was also no significant difference in the prevalence of qol abnormalities between the various groups of subjects with respect to adequacy of information on the menopause, knowledge of the husband/partner about the menopause and family support in facing the menopause (p>0.05). degree of quality of life abnormality absent mild moderate severe aspect of abnormality n % n % n % n % psychological 62 35.2 70 39.8 31 17.6 13 7.4 somato-vegetative 70 39.8 74 42.0 26 14.8 6 3.4 urogenital 50 28.4 35 19.9 69 39.2 22 12.5 table 2. aspect and degree of quality of life abnormality of postmenopausal women table 1. assessment criteria of the menopause rating scale (16) assessment score quality of life abnormality psychological subscale somato-vegetative subscale urogenital subscale total absent/slight 0 – 1 0 – 2 0 0 – 4 mild 2 – 3 3 – 4 1 5 – 8 moderate 4 – 6 5 – 8 2 – 3 9 – 16 severe > 7 > 9 > 4 > 17 38 there was no significant difference in the prevalence of qol abnormalities between g r o u p s o f s u b j e c t s w i t h v a r i o u s h a b i t s / lifestyles, such as exercise, consumption of phytoestrogen-containing foods, smoking, and consumption of caffeine and alcohol (p > 0.05). in this study there was a significant difference (p<0.05) in the prevalence of qol abnormalities between groups of subjects who were always, occasionally, and not at all concerned about a balanced composition (carbohydrate, fat and protein) of their daily diet. in the group of subjects who never were concerned about balanced dietary patterns there were more qol abnormalities compared with the other groups. wratsangka qol in postmenopausal women table 4. relationship between knowledge and attitude of subjects on menopause and quality of life abnormalities in postmenopausal women quality of life abnormality yes no psychosocial aspect n % n % p* prevalence ratio knowledge about menopause abnormality/illness normal/natural process natural process with risk of negative impact 3 38 15 75.0 25.2 71.4 1 113 6 25.0 74.8 28.6 0.945 attitude towards menopause positive negative 117 14 73.1 87.5 43 2 26.9 12.5 0.339 2.573 (95% ci: 0.561 – 11.789) * chi-square test * chi-square test table 3. relationship between several sociodemographic and economic factors and quality of life abnormalities in postmenopausal women degree of quality of life abnormality yes no psychosocial aspect n % n % p* prevalence ratio marital status single married 26 105 76.5 73.1 8 37 23.5 26.9 0.933 0.873 (95% ci: 0.363 –2.098) level of education low high 42 89 79.2 72.4 11 34 20.8 27.6 0.440 0.686 (95% ci: 0.317 –1.484) occupational status unemployed employed 62 69 73.8 75.0 22 23 26.2 25.0 0.994 1.065 (95% ci: 0.541 –2.096) activities outside occupation absent present 57 64 77.0 71.9 20 25 23.0 28.1 0.547 0.764 (95% ci: 0.387 –1.509) source of household finance own/husband’s salary retirement funds/other 92 39 76.7 69.6 28 92 23.2 30.4 0.418 0.698 (95% ci: 0.343 –1.419) source of health funds insurance/employer support own funds 55 76 71.4 76.8 22 23 28.6 23.2 0.528 1.322 (95% ci: 0.670 –2.608) 39 univ med vol.29 no.1 quality of life abnormality yes no family factors n % n % p* adequacy of information on menopause inadequate adequate more than adequate 46 77 8 80.7 74.0 53.3 11 27 7 19.3 26.0 46.7 0.096 knowledge of husband/partner about menopause abnormality/illness normal/natural process natural process with risk of negative impact 4 92 9 80.0 72.4 90.0 1 35 1 20.0 27.6 10.0 0.453 family support none poor adequate 99 2 30 79.2 66.7 62.5 28 1 18 20.8 33.3 37.5 0,075 table 5. relationship between family factors and quality of life abnormalities in postmenopausal women * chi-square test * chi-square test table 6. relationship between lifestyle and quality of life abnormalities in postmenopausal women quality of life abnormality yes no lifestyle n % n % p* exercise yes, almost daily yes, occasionally none at all 23 79 29 74.2 71.8 82.9 8 31 6 25.8 28.2 17.1 0.43 balanced dietary composition yes, always yes, occasionally none at all 16 23 38 62.8 25.8 86.4 27 66 6 37.2 74.2 13.6 0.04 consumption of phytoestrogen/isoflavone yes, daily yes, occasionally none at all 95 35 1 72.5 79.5 100.0 36 9 0 27.5 20.5 0 0.54 smoking never in the past, but not now yes 121 7 3 73.8 87.5 75.0 43 1 1 26.2 12.5 25.0 0.69 caffeine yes, daily yes, occasionally none at all 50 64 17 83.3 72.7 60.7 10 24 11 16.7 27.3 39.3 0.07 alcohol yes, daily yes, occasionally none at all 1 4 126 100.0 57.1 75.0 0 3 42 0 42.9 25.0 0.48 40 among the 17 variables included in the bivariate selection, there were five with p < 0.25 and meeting the criteria for inclusion in the multivariate analysis, viz. attitude of subjects towards the menopause, adequacy of i n f o r m a t i o n o n t h e m e n o p a u s e , f a m l i t y support, balanced dietary composition and consumption of caffeinated beverages. the v a r i a b l e “ k n o w l e d g e o f s u b j e c t a b o u t menopause” was also included in the bivariate modeling in spite of p>0.25, because it was considered to be very important. multivariate analysis was performed on several independent variables (psychosocial factors and habit/lifestyle) against the dependent variable (qol). prior to this, bivariate selection was done and it was determined that the independent variables with p < 0.25 were to be included in the multivariate logistic regression analysis. in the multivariate analysis, as shown on table 7, the subjects’ attitude towards the menopause and family support for them in facing the menopausal changes played an important role in their qol. in the relationship between the subjects’ attitude towards the menopause and qol abnormalities, a prevalence ratio of 2.863 was found (95% ci: 0.578 – 14.185) between the group of subjects with a negative and those with a positive attitude towards menopause. this means that the subjects with a positive attitude towards menopause had a 2.8-fold greater probability of being free from qol abnormalities, in comparison with the subjects with a negative attitude towards menopause. in the relationship between family support for the subjects in facing the menopausal changes and the prevalence of qol abnormalities, prevalence ratios of respectively 2.124 (95% wratsangka qol in postmenopausal women quality of life abnormalities yes no independent variable n % n % p* prevalence ratio knowledge about menopause abnormality/illness normal/natural process natural process with risk of negative impact 3 38 15 75.0 25.2 71.4 1 113 6 25.0 74.8 28.6 0.767 0.879 0.536 1.087 (95% ci: 0.372-3.177) 0.433 (95% ci: 0.031-6.140) attitude towards menopause positive negative 43 2 26.9 12.5 117 14 73.1 87.5 0.198 2.863 (95% ci: 0.578-14.185) environmental support none poor adequate 99 2 30 79.2 66.7 62.5 28 1 18 20.8 33.3 37.5 0.159 0.057 0.854 2.124 (95% ci: 0.979-4.610) 1.291 (95% ci: 0.085-19.728) balanced dietary composition yes, always yes, occasionally none at all 27 66 38 62.8 74.2 86.4 16 23 6 37.2 25.8 13.6 0.301 0.124 0.328 0.407 (95% ci: 0.130-1.278) 0.595 (95% ci: 0.210-1.684) caffeinated beverages yes, daily yes, occasionally none at all 50 64 17 83.3 72.7 60.7 10 24 11 16.7 27.3 17.0 0.254 0.313 0.099 0.635 (95% ci: 0.263-1.535) 0.390 (95% ci: 0.127-1.193) table 7. results of multivariate logistic regression of quality of life by psychosocial aspects and lifestyle in postmenopausal women * chi-square test 41 univ med vol.29 no.1 ci: 0.979 – 4.610) and 1.291 (95% ci: 0.085 – 19.728) were found between the group of subjects who felt unsupported by their family a n d t h o s e w h o f o u n d t o b e p o o r l y a n d adequately supported. in spite of that, in this study it could not be proven that the factors of no support and poor support constituted risk f a c t o r s f o r q o l a b n o r m a l i t i e s i n postmenopausal women. discussion based on the qol assessments conducted in the study subjects by means of the mrs questionnaire, a total of 131 (74.4%) subjects were found to have qol abnormalities in mild, moderate and severe degrees. the majority of the subjects had qol abnormalities of mild ( 3 8 . 6 % ) a n d m o d e r a t e d e g r e e s ( 3 0 . 7 % ) . although the prevalence of qol abnormalities in postmenopausal women as found in this study cannot be generalized and cannot be considered to reflect the overall picture of i n d o n e s i a n p o s t m e n o p a u s a l w o m e n , t h e prevalence rates obtained may presumably increase our alertness to the problems of menopause and may provide the basis for further studies on a larger population. the investigators were unable to find reports of other studies on qol in postmenopausal indonesian women, particularly those using the mrs as the measuring instrument of qol of postmenopausal women. viewed from the aspect of abnormalities e x p e r i e n c e d b y t h e s u b j e c t s , u r o g e n i t a l abnormalities were the most frequent (71.6%), namely abnormalities related to sexual and urinary (voiding) activities. more than 50% of the subjects had urogenital abnormalities in moderate and severe degrees. the majority of subjects (80.7%) were married and some of them were still engaged in sexual activities w i t h t h e i r h u s b a n d s / p a r t n e r s , s u c h t h a t abnormalities of sexual activities at menopause may affect their qol. a turkish study yielded lower fugures, i.e. 10.8% of postmenopausal women had sexual problems, 15.0% urinary p r o b l e m s , a n d 7 . 8 % v a g i n a l d r y n e s s . ( 1 8 ) results similar to the turkish study were obtained by boekitwetan et al. in the elderly, where 19.5% har urinary tract infections.(19) from the psychological aspect most of the subjects (64.8%) also had psychological abnormalities. more than 60% of the subjects involved in the present study had somatovegetative abnormalities. hot flushes and night sweats were the most frequently experienced symptoms in postmenopausal women, in association with sleep disturbances, sensory a b n o r m a l i t i e s a n d d e c r e a s e d c o g n i t i v e functions. the turkish study yielded similar figures for somato-vegetative abnormalities, where hot flushes and sweating amounted to 50.7%, heart problems 22.9%, sleep problems 24.3%, feeling unhappy 22.6% and anxiety 30.7%.(18) these abnormalities may lead to s o c i a l a n d w o r k r e l a t e d p r o b l e m s , w i t h significantly lowered qol. the somatovegetative symptoms frequently cause the menopausal women suffering from them to visit their doctor and be prescribed drugs for treating the complaints, so that ultimately h e a l t h e x p e n d i t u r e s m a y c o n s t i t u t e a socioeconomic burden. in the present study the variables marital status, level of education, occupational status, accessory activities outside work, source of family finance and source of funds for health expenditures were not associated with qol abnormalities in postmenopausal women. the study results did not reveal the presence of a significant association between marital status and qol in postmenopausal women. however, a number of studies showed different results, where sociocultural features influence the presence and severity of menopausal symptoms and are not influenced by sociodemographic factors.(20,21) married subjects still needed sexual functions, such that abnormalities in sexual functions may be uncomfortable to them and disturb their relationship with their husbands. several prior studies indicated that 42 v a s o m o t o r s y m p t o m s a n d u r o g e n i t a l abnormalities were less frequently found in single (unmarried or widowed) women in comparison with married women.(17) in this study no association was also found between level of education and qol of postmenopausal women. most of the subjects (69.9%) had a high level of education (high school and university), and several studies have s h o w n t h a t v a s o m o t o r s y m p t o m s a n d palpitations were found more commonly in less-educated women.(4) the outcomes being evaluated in this study comprised not only v a s o m o t o r a b n o r m a l i t i e s , b u t a l s o q o l involving other abnormalities, so that they cannot indeed be compared with the results of other studies. in this study no significant difference in qol was found with regard to: (i) knowledge of the subjects about the menopause, i.e. between the group of subjects who regarded menopause as an illness, menopause as a normal/natural process not needing special attention, and menopause as a natural process with a risk of negative impacts, and (ii) attitude of the subjects towards menopause: between the group of subjects with a positive and those with a negative attitude towards menopause. the menopause is an event in the life of a woman when the menstrual cycle comes to an end, marked by physiological and psychosocial changes in her life. although the menopause is a normal event in a woman’s life, the individual perceptions are variable and some women need medical assistance to manage the symptoms of menopause. the transition into the menopause is a complex physiological process that is frequently accompanied by accessory effects of the aging process and social adaptation. (22) a number of women experience the climacteric syndrome, in the form of vasomotor, somatic and psychic complaints in the transition period towards menopause or the perimenopausal period. subsequently there will be an increased risk of degenerative diseases or chronic disorders such as osteoporosis, cardiovascular disease, colorectal cancer and lowered cognitive functions. the menopausal experience is strongly influenced by the perception of the women about the bodily changes and about how the immediate community around her perceives the menopause. the biomedical model of menopause is focused more on the biological aspects of decreased hormonal levels and on identification of climacteric symptoms. the menopause is considered to be characteristically a deficiency disorder that requires treatment in the form of replacement hormonal therapy, thus transforming the w o m a n i n t o a s i c k p a t i e n t , b e c a u s e t h e climacteric symptoms are believed to be associated with estrogen deficiency. this points out the need for dissemination of more accurate and comprehensive information to premenopausal women regarding all aspects of the menopause. the menopausal woman needs support from her neighbourhood in facing the changes taking place in the periand post-menopausal periods. in this study it is apparent that the majority (72.2%) of the subjects’ husbands/ partners also consider the menopause to be a n o r m a l a n d n a t u r a l p r o c e s s t h a t w i l l b e experienced by every woman, without realizing the risk of negative impacts due to the lowered estrogen levels, arising in the short term as well as in the long term. only 5.7% of the subjects’ husbands realized the risk of negative impacts of the menopause that may be experienced by their wife. a l t h o u g h a p p r o x i m a t e l y h a l f o f t h e subjects (59.1%) mentioned the adequacy of sources of information about the menopause, the abovementioned data indicate that most women or their partners did not know about the true nature of the menopause as a natural process with a risk of negative impacts. the majority of the subjects (59.1%) stated that they had adequate information about the menopause, which they obtained from their friends (35.8%) and health personnel (30.7%), wratsangka qol in postmenopausal women 43 univ med vol.29 no.1 and for the remainder from popular science books, the printed/electronic media and others. l a c k o f i n i t i a l i n f o r m a t i o n a n d c o r r e c t knowledge of the subjects, their partners or their neighborhood (including health personnel a n d m a s s m e d i a ) a b o u t t h e p r o b l e m s o f menopause was one of the causes of the s u b j e c t s ’ l o w n e e d f o r m o r e a d e q u a t e information regarding the menopause and its future impacts. the majority of the subjects (71%) also did not feel that they received special support from the neigborhood when facing physical and non-physical changes due to the menopause. the community, particularly the women, need to be better informed about the all of the problems of menopause. doctors and health personnel in general should be able to provide good support and counseling to women regarding the menopause, starting from menopausal symptoms and complaints and their short term as well as long term impacts, the steps that may be taken for managing those complaints and for preventing problems that might occur in the long term as a result of the menopause. most menopausal women need education, information about the menopause and support from an individual who is willing to listen to them about their experiences.(23) regular exercise, nutritionally balanced daily diets, and lesser or no consumption of caffeinare important for maintaining a high qol in menopausal women. women who exercise regularly and are non-smokers have a better score for positive mood in facing the changes that they experience. studies have found that physically less active women complain more of postmenopausal neurovegetative symptoms than do women who exercise regularly.(7) women who exercise regularly have a better mood profile, fewer somatic symptoms and fewer difficulties in concentration in comparison with l e s s p h y s i c a l l y a c t i v e w o m e n . p h y s i c a l excercise is known to be capable of increasing t h e l e v e l s o f â e n d o r p h i n s , w h i c h a r e neurotransmitters capable of influencing the regulation of body temperature, and it is the levels of â-endorphins that are lowered as a result of reduced estrogen concentrations in menopausal women. in this study no significant difference was found in qol abnormalities between the groups of subjects who exercise almost daily, exercise occasionally or never exercise (table 6). in this study habitual exercise was only quantitatively assessed, based on interviews or self-report by the subjects, making it difficult to measure the differences in biological effect of excercise done almost daily, occasionally, or not at all. phytoestrogens, which are commonly found in the daily food of the indonesian community, are substances whose structure mimick that of estrogens and are thus capable of binding estrogen estrogen receptors and functioning as selective estrogen receptor modulators (serm). several randomized double-blind studies that have been conducted o n t h e i n f l u e n c e o f p h y t o e s t r o g e n s u p p l e m e n t a t i o n r e p o r t e d a s t a t i s t i c a l l y significant reduction in the prevalence of hot flushes in the group of subjects receiving supplemental phytoestrogens.(24,25) with regard to the dietary composition of the subjects, as viewed from the balance between carbohydrate, fat and protein, the important role of dietary fat in the development of menopausal symptoms was indicated by the association between low fat intake and low prevalence of menopausal symptoms in asian populations, such as japanese and chinese women. a high intake of fat is associated with h i g h e r s e r u m l e v e l s o f e s t r o n e a n d dehydroepiandrosterone sulfate (dheas) in postmenopausal japanese women.(26) differing results were obtained in an interventional trial indicating that a low-fat diet had no effect on s e r u m d h e a s a m o n g p o s t m e n o p a u s a l australian women.(27) the statistically non-significant difference obtained in the present study may indeed indicate no association between various 44 abovementioned habits/lifestyles and qol a b n o r m a l i t i e s , o r m a y a l s o b e d u e t o shortcomings of the instrument used in this study, e.g. the qualitative assessment on the independent and dependent variables. the p r e s e n c e o r a b s e n c e o f a s t a t i s t i c a l l y significant difference is not always identical to a clinically significant difference. it is wellknown that the probability of obtaining a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e i s proportional to the sample size, such that with a larger sample size even extremely small or clinically irrelevant differences may become statistically significant. the results of multivariate analysis showed t h a t t h e s u b j e c t s ’ a t t i t u d e t o w a r d s t h e menopause and family/neighborhood support for them in facing menopausal changes play an important role in their qol. qol abnormalities were found with a 2.8-fold greater frequency in subjects with a negative attitude towards menopause, compared with those with a positive attitude. qol abnormalities in postmenopausal women were also more frequent in the group of subjects who felt totally unsupported by their neighborhood/family in facing menopausal changes, in comparison with those subjects who felt themselves to be poorly supported. in this study both abovementioned factors (negative attitude towards the menopause and lack of neighborhood support) cannot yet be considered r i s k f a c t o r s f o r q o l a b n o r m a l i t i e s i n postmenopausal women. conclusions i n t h i s s t u d y t h e m a j o r i t y o f postmenopausal women had qol abnormalities and the most commonly experienced symptoms were hot flushes, sweating, heart problems, sleep problems and anxiety. the high percentage of qol abnormalities among postmenopausal women necessitates adequacy of information and counseling by health personnel on the problems of menopause using a biopsychosocial approach. acknowledgements the investigators gratefully acknowledge the help provided by the head and staff of the cilandak health center and the health office of south jakarta in acquiring the necessary licenses and their full support during the conduct of this study. the investigators also thank the postmenopausal women who were willing to participate in this study. references 1. departemen kesehatan republik indonesia. survei kesehatan nasional 2001, laporan skrt 2001: studi morbiditas dan disabilitas. jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan ri; 2002. 2. hvas l, thorsen h, sondergaard k. discussing menopause in general practice. maturitas 2003; 46:139–46. 3. ho sc, ho sc, chan sg, yip yb, cheng a, yi q, chan c. menopausal symptoms and symptom clustering in chinese women. maturitas 1999;33: 219–27. 4. gold eb, sternfeld b, kelsey jl, brown c, mouton c, reame n, salamone l, stellato r. relation of demographic and lifestyle factors to symptoms in a multi-racial/ethnic population of women 40-55 years of age. am j epidemiol 2000; 152:463-73. 5. hvas l. positive aspects of menopause: a qualitative study. maturitas 2001;39:11-7. 6. hvas l, thorsen h, sondergaard k. discussing menopause in general practice. maturitas 2003; 46:139-46. 7. gold eb, block g, crawford, s, lachance l, gerald gf, miracle h, et al. lifestyle and demographic factors in relation to vasomotor symptoms: baseline results form the study of women’s health across the nation. am j epidemiol 2004;159:1189-99. 8. genazzani ar, gambacciani m, simoncini t. international menopause society expert workshop. menopause and aging, quality of life and sexuality. climacteric 2007;10:88–96. 9. alder em. how to assess quality of life: problems and methodology. in: schneider hpg, editor. hormone replacement therapy and quality of life. london: parthenon publishing;2002.p.11-21. 10. schneider hpg, strelow f, heinemann laj, thai dm. the menopause rating scale (mrs) scale: wratsangka qol in postmenopausal women 45 univ med vol.29 no.1 a methodological review. health qual life outcomes 2004;2:45-52. 11. schneider hpg, heinemann laj, rosemeier hp, potthoff p, behre hm. the menopause rating scale (mrs): reliability of scores of menopausal complaints. climacteric 2000;3:59-64. 12. schneider hpg, heinemann laj, rosemeier hp, potthoff p, behre hm. the menopause rating scale (mrs): comparison with kupperman index and quality of life scale sf-36. climacteric 2000; 3:50-8. 13. schneider hpg, rosemeier hp, schnitker j, gerbsch s, turck r. application and factor analysis of the menopause rating scale [mrs] in a post-marketing surveillance study of climen. maturitas 2000;37:113-24. 14. patria f. gambaran densitas tulang pada wanita menopause yang menderita osteopenia osteoporosis dan mendapat terapi fitoestrogen di rsupn cipto mangunkusumo (tesis). program pendidikan dokter spesialis obstetri dan ginekologi. jakarta: fakultas kedokteran universitas indonesia;2006. 15. lasmini ps. pengaruh isoflavon terhadap profil lipid pada perempuan menopause dan pasca menopause di jakarta (tesis). program pendidikan konsultan fertilitas endokrinologi reproduksi bagian obstetri dan ginekologi. jakarta: fakultas kedokteran universitas indonesia;2005. 16. heinemann, laj, potthoff, p, schneider hpg. international version of the menopause rating scale (mrs). health qual life outcomes 2003; 1:28. 17. heinemann laj, dominh t, strelow f. the menopause rating scale (mrs) as outcome measure for hormone treatment? a validation study. health qual life outcomes 2004;2:67. 18. budikoðlu r, õscan c, eri”lu, yanik f. quality of life and postmenopausal symptoms among women in a rural district of the capital city of turkey. gynecol endocrinol 2007;23:404-9. 19. boekitwetan p, surjawidjaja je, aidilfit m, lesmana m. multimicronutrient suplementation and asymptomatic urinary tract infections in the elderly. univ med 2009;28:25-33. 20. kowalski i, rote d, bans c, dietrich k. women’s attitude and perception towards menopause in different cultures. cross-cultural and intra-cultural comparison of pre-menopausal and postmenopausal women in germany and in papua new guinea. maturitas 2005;51:227-35. 21. dennerstein l, lehert p, guthrie jr, burger hg. modeling women’s health during menopausal transition: a longitudinal analysis. menopause 2007;14:53-62. 22. nelson hd. menopause. the lancet 2008;371: 760-70. 23. armitage gd, suter e, verhoef mj. women’s needs for cam information to manage menopausal symptoms. climacteric 2007;10:21524. 24. upmatis dh, lobo r, bradley l. vasomotor symptom relief by soy isoflavone extract tablets in postmenopausal women: a multicenter, doubleblind, randomized placebo controlled study. menopause 2000;7:236-42. 25. faure ed, chantre p, mares p. effects of standardized soy extract on hot flushes: a multicenter, double-blind, randomized placebocontrolled study. menopause 2002;9:329-34. 26. nagata c, nagao y, shibuya c, kashiki y, shimizu h. fat intake is associated with serum estrogen and androgen concentrations in postmenopausal japanese women. j nutr 2005; 135:2862–5. 27. dowsett m, donaldson k, tsuboi m, wong j, yates r. effects of the aromatase inhibitor anastrozole on serum oestrogens in japanese and caucasian women. cancer chemother pharmacol 2000;45:35–9. editorial i vaccines for prostate cancer : a new era? julius i mulia department of pharmacy, medical faculty, trisakti university univ med vol. 28 no.3 prostate adenocarcinoma is the most prevalent type of noncutaneous cancer in the western world, with an estimated 218,890 new cases and 27,050 deaths in the united states in 2007. currently prostate cancer is detected by measurement of prostate-specific antigen (psa), a serine protease synthesized by the prostatic epithelium. psa is an organ-specific and tumor-associated antigen (taa) but it is not tumor-specific.(1) partly because of increased cancer screening with psa, prostatic cancer may now be diagnosed when it is still localized. localized tumors of the prostate are generally treated with radical prostatectomy, external-beam radiation therapy (ebrt), brachytherapy, or watchful waiting. unfortunately, up to 30%-40% of patients fail local therapy. the standard treatment of recurrent or metastatic disease is androgen-deprivation therapy (adt), but this is only a temporary measure as in the majority of cases the cancer ultimately becomes hormone refractory, the condition being termed androgen-independent prostate cancer (aipc) or hormone refractory prostate cancer (hrpc), which then progresses rapidly. the only available nonpalliative therapy for androgenindependent prostate cancer is docetaxel in combination with prednisone. however, adt given prior to the onset of clinical symptoms results in rising psa levels with castrate levels of testosterone, often with a relatively low tumor burden. this systemic treatment earlier in the disease course combined with effective palliative chemotherapy is implicated in the improvement in median survival time of patients with aipc from an average of about 12 months to about 17-18 months.(2) clearly there is a need for the development of novel, molecularly targeted therapies of aipc. it is therefore not surprising that the last decades have seen the development and testing of several types of prostate cancer vaccines as alternative therapeutic modalities. metastatic prostate cancer is characterized by relatively small lesions within lymph nodes, bone marrow, and other sites within the immune system. therefore prostate cancer may be more susceptible to vaccine immunotherapy than tumors that are characterized by bulkier or less accessible lesions.(3) prostate cancer is well suited for an analysis of the efficacy of cancer vaccines because prostate tumors are relatively slow-growing, and early diagnosis of recurrence is frequent. the doubling time of serum psa acts as a surrogate marker for disease prognosis and outcome. a range of prostate cancer-associated antigens have been identified and characterized, such as secreted proteins psa and prostatic acid phosphatase (pap), cell surface proteins prostate-specific membrane antigens (psma) and intracellular proteins. early ii univ med clinical studies have shown that patients will mount immune responses to the prostate cancer-associated antigens post vaccination.(1,4) a number of approaches to prostate cancer immunotherapy have been investigated, such as whole tumor cell, dendritic cell (dc), and poxvirus vaccines.(2) tumor cell vaccines can be prepared from autologous cells from the patient’s own tumor specimen or from cultured allogeneic tumor cells. autologous cell vaccines contain tumor-specific antigens and taas, but their preparation is time-consuming and at times difficult due to tumor necrosis and other conditions decreasing the percentage of tumor cells. on the other hand, allogeneic cell vaccines are relatively easy to prepare and usually consist of one or more tumor cell lines and may also contain several tumor-specific antigens and/or taas. the cell lines can also be infected with vectors that express cytokine genes such as granulocyte-macrophage colony-stimulating factor (gm-csf), or co-stimulatory molecule genes such as b7-1 or b7-2, to enhance immunogenicity of the tumor cells. however, they may not contain essential antigens for a particular patient and there is the potential induction of alloimmunity.(1) a tumor cell vaccine in advanced clinical trial testing is gvax which consists of two irradiated allogeneic prostate cancer cell lines engineered to secrete gm-csf. its mode of action is proposed as the uptake of the ex vivo x-irradiated tumor cells by antigen-presenting cells (apc) and crosspresentation of tumor-associated antigens to t cells in draining lymph nodes. two phase ii clinical studies have been completed in patients with asymptomatic metastatic hrpc, resulting in median survival times of 20 to 35 months.(4) therapeutic vaccines based on dendritic cells (dcs) carrying tumor antigens have emerged as a promising strategy to initiate an immune response against tumor cells. dc vaccines contain dendritic cells, which are potent apc that have the ability to stimulate t-lymphocyte immune responses in animals and in humans. dcs are capable of activating cd4and cd8-positive t lymphocytes by antigen presentation on their surface major histocompatibility complex (mhc) class i and class ii molecules. dc vaccines are obtained by leukapheresis from peripheral blood mononuclear cells and then cultured in the presence of cytokines before being reinfused into the patient. this process must be conducted for each patient. apart from the difficult preparation process, dc vaccines also have the disadvantage of high cost. one of the most advanced vaccine approaches is sipuleucel-t, which is not a pure autologous dc vaccine but rather a mixture of white cells.(1) sipuleucel-t consists of autologous antigen-presenting cells and a fusion protein composed of prostatic acid phosphatase and gm-csf. in 2006, the results were published on a placebo-controlled, randomized phase iii trial comparing sipuleucel-t and placebo in patients with metastatic, asymptomatic, hrpc. progressionfree survival (the primary endpoint) was not statistically significant, but overall survival between the vaccine and the placebo groups reached statistical significance.(2,4) the vaccine was denied food and drug administration (fda) approval due to its failure to achieve the primary endpoint, necessitating another study with revised endpoint. for this trial the fda decision may be published in the coming months of this year. to stimulate a t-lymphocyte response it is also possible to transfect dcs with tumor messenger rna (mrna). this method could be even more potent than loading dcs or apcs with peptides or proteins (as in sipuleucel-t) or transfection with plasmids or transduction with viral vectors.(1,2,5) poxviruses and fowlpox (modified to be replication-incompetent) have the ability to accept and express multiple transgenes and can thus be engineered to express not only tumor-associated antigens, but also various immunostimulatory molecules. one approach uses recombinant vaccinia and fowlpox iiivol. 28 no.3 vectors containing the transgenes for psa and three human co-stimulatory molecules (cd80, intracellular adhesion molecule 1, and lymphocyte function–associated antigen 3, designated “tricom”). recent phase i and ii trials involving patients with metastatic and locally advanced prostate cancer have shown clinical responses and drops in serum psa.(2) references 1 doehn c, böhmer t, kausch i, sommerauer m, jocham d. prostate cancer vaccines current status and future potential. biodrugs 2008;22:71-84. 2 tarassoff cp, arlen pm, gulley jl. therapeutic vaccines for prostate cancer. oncologist 2006;11:451-62. 3 durso rj, andjelic s, gardner jp, margitich dj, donovan gp, arrigale rr, et al. a novel alphavirus vaccine encoding prostate-specific membrane antigen elicits potent cellular and humoral immune responses. clin cancer res 2007;13:3999-4008. 4 schlom j, gulley jl, arlen pm. role of vaccine therapy in cancer: biology and practice. curr oncol 2007;14:238-45. 5 sousa-canavez jm, canavez fc, leite krm, camara-lopes lh. therapeutic dendritic cell vaccine preparation using tumor rna transfection: a promising approach for the treatment of prostate cancer. genet vacc ther 2008;6:1-7. asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts 96 *department of dermatovenereology, faculty of medicine, tarumanegara university **biomedical sciences doctoral program, faculty of medicine, university of indonesia ***centre for pharmaceutical and medical technology, agency for the assessment and application of technology, ministry of research, technology, and higher education ****department of dermatovenereology, faculty of medicine, university of indonesia correspondence: dr. linda yulianti wijayadi, spkk department of dermatovenereology faculty of medicine, tarumanegara university jalan letjen. s.parman no.1 grogol jakarta barat 11440 email : lindajuliantiwijayadi@gmail.com univ med 2015;34:96-103 doi: 10.18051/univmed.2016.v35.96-103 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background asiatiocoside, a saponin component isolated from centella asiatica can improve wound healing by promoting the proliferation of human dermal fibroblasts (hdf) and synthesis of collagen. the skin-renewing cells and type i and iii collagen synthesis decrease with aging, resulting in the reduction of skin elasticity and delayed wound healing. usage of natural active compounds from plants in wound healing should be evaluated and compared to retinoic acid as an active agent that regulates wound healing. the aim of this study was to compare and evaluate the effect of asiaticoside and retinoic acid to induce greater cell proliferation and type i and iii collagen synthesis in human dermal fibroblast. methods laboratory experiments were conducted using human dermal fibroblasts (hdf) isolated from human foreskin explants. seven passages of hdf were treated with asiaticoside and retinoic acid at several doses and incubated for 24 and 48 hours. cell viability in all groups was tested with the mtt assay to assess hdf proliferation. type i and iii collagen synthesis was examined using the respective elisa kits. analysis of variance was performed to compare the treatment groups. results asiaticoside had significantly stronger effects on hdf proliferation than retinoic acid (p<0.05). the type iii collagen production was significantly greater induction with asiaticoside compared to retinoic acid (p<0.05). conclusion asiaticoside induces hdf proliferation and type i and iii collagen synthesis in a timeand dose-dependent pattern. asiaticoside has a similar effect as retinoic acid on type i and type iii collagen synthesis. keywords: asiaticoside, type i collagen, type iii collagen, fibroblast, proliferation, retinoic acid asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts linda yulianti*,**, etik mardliyati***, kusmarinah bramono****, and hans joachim freisleben** doi: http://dx.doi.org/10.18051/univmed.2015.v34.96-103 97 univ med vol. 34 no.2 asiaticoside menginduksi proliferasi sel dan sintesis kolagen pada fibroblas dermis kulit manusia latar belakang asiatiocosida merupakan komponen saponin yang diisolasi dari centella asiatica yang bermanfaat untuk mempercepat penyembuhan luka dengan meningkatkan proliferasi dan sintesis kolagen dalam fibroblas dermis kulit manusia. proses pembaharuan sel kulit dan sintesis kolagen tipe i dan iii menurun dengan proses penuaan, elastisitas kulit berkurang dan penyembuhan luka melambat. penggunaan senyawa aktif alami dari herbal dalam penyembuhan luka dievaluasi dan dibandingkan dengan asam retinoat sebagai bahan aktif yang mengatur penyembuhan luka. tujuan penelitian ini adalah untuk membandingkan dan mengevaluasi pengaruh asiaticosida dan asam retinoat terhadap proliferasi fibroblas dan sintesis kolagen tipe i dan iii pada dermis kulit manusia. metode sebuah penelitian eksperimental laboratorik menggunakan sel dermis fibroblas kulit manusia yang diisolasi dari eksplan kulit. generasi ke tujuh fibroblas dermis kulit manusia dipajankan dengan beberapa dosis asiaticosida dan asam retinoat, diinkubasi selama 24 dan 48 jam. viabilitas sel diuji dengan uji mtt untuk menilai proliferasi fibroblas kulit manusia. sintesis kolagen tipe i dan iii diperiksa menggunakan elisa kit, masing-masing dievaluasi setelah 24 dan 48 jam. analisis uji anova digunakan untuk menguji perbedaan antara kelompok perlakuan. hasil penelitian ini menunjukkan, asiaticosida menginduksi proliferasi fibroblas dermis kulit manusia lebih tinggi secara bermakna dibandingkan asam retinoat (p<0,05). asiaticosida menginduksi sintesis kolagen tipe iii lebih tinggi secara bermakna dibandingkan asam retinoat (p<0,05). kesimpulan asiaticosida mampu menginduksi proliferasi fibroblas dermis kulit manusia dan sintesis kolagen tipe i dan iii yang dipengaruhi oleh dosis dan waktu inkubasi. asiaticosida mempunyai efek yang sama seperti asam retinoat pada sintesis kolagen tipe i dan iii kata kunci: asiaticosida, kolagen i, kolagen iii, fibroblas, proliferasi, asam retinoat abstrak introduction skin wound healing is a complex process carried out by cells that are involved in inflammation, re-epithelialization, angiogenesis, granulation, tissue formation and differentiation, and deposition of interstitial matrix. in general, wound healing can be divided into three stages: inflammation, proliferation and remodeling.(1) centella asiatica (l.)urb. (ca) has been widely used as traditional herbal medicine in malaysia, india and nepal as part of the traditional ayurvedic medicine for hundreds of years.(2,3) it is commonly known as pegaga in malaysia, pegagan in indonesia and as penny wort or gotu kola in america.(2-4) this tropical plant can be used as “longevity herb” or herbal anti-aging cosmetic (5,6) and traditionally serves various medicinal purposes such as wound healing, treatment of asthma, ulcers, leprosy, lupus erythematosus, psoriasis, venous disorders, for memory improvement, and as antidepressant, antibacterial, antifungal, and anti-cancer agent.(79) 98 c e n t e l l a a s i a t i c a h a s b e e n u s e d traditionally to improve wound healing.(3-5) centella asiatica ethanolic extract treated wounds can epithelialize faster and have greater collagen content.(9) the biologically active ingredients in ca are triterpenes, namely asiatic a c i d , m a d e c a s s i c a c i d , a s i a t i c o s i d e a n d madecassoside.(8-10) asiaticoside isolated from ca promotes fibroblast proliferation and extracellular matrix synthesis in wound healing (11,12) by enhancing collagen formation and angiogenesis.(12,13) in particular, asiaticoside and madecassoside stimulate type i and type iii collagen, respectively.(12,14) a study by hadi et al.(13) indicated that allogenic human dermal fibroblasts (hdf) are viable in peripheral blood. monolayer co-culture in vitro can induce synthesis of type i and iii collagen, the major structural and functional components of the skin. asiaticoside and retinoic acid can regulate (i.e. stimulate or inhibit) the production of these collagens.(11,14) i n s k i n , r e t i n o i d s a r e i n v o l v e d i n keratinocyte cell differentiation, epidermal cell adhesion and corneocyte exfoliation from the surface of epidermis. more specifically to the d e r m i s , r e t i n o i d s r e g u l a t e f i b r o b l a s t proliferation, induce angiogenesis and play an essential role in the synthesis of collagen and elastin fibrils.(14) natural products contain a wealth of interesting pharmaceutically active compounds and various active agents are expected to be beneficial in wound healing and as preventive anti-aging cosmetics. (1) the stimulation of collagen synthesis is an important criterion for plant extracts to be considered as a potential ingredient in skin care anti-aging products.(11,12) due to its ability to stimulate collagen synthesis, ca has been used in skin care products for anti-aging, restoring skin firmness a n d e l a s t i c i t y a n d i m p r o v i n g s k i n appearance.(11,12) lu et al. (10) reported that asiaticoside isolated from centella asiatica promotes fibroblast proliferation and extracellular matrix s y n t h e s i s i n w o u n d h e a l i n g , t h e p r e c i s e mechanism, however, being still only partially understood. the aim of this study was to evaluate fibroblast proliferation and type i and iii collagen synthesis by asiaticoside as an active compound from ca in comparison to retinoic acid, a wellknown active ingredient in the prevention of skin aging. methods research design in the present study, experiments on fibroblast proliferation were conducted at the laboratory of the centre for pharmaceutical and medical technology, agency for the assessment and application of technology, from january 2013 to april 2013, and on collagen synthesis at the integrated laboratory of medicine, yarsi university, from april 2014 to june 2014. preparation of centella asiatica extract centella asiatica (ca) aqueous and ethanolic extracts (tawangmangu strain) and centella asiatica chitosan nanoparticles were prepared in the laboratory of the agency for the assessment and application of technology. centella asiatica was encapsulated in the chitosan nanoparticles by ionotropic gelation. then, proliferative and cytotoxic effects of nanoencapsulated centella asiatica extracts was evaluated in vitro on human dermal fibroblasts (hdf). the mtt proliferation assay was performed with the encapsulated ca extracts in comparison with the unencapsulated ca extracts and with empty chitosan nanoparticles (cnp) as controls. cell culture and treatment human dermal fibroblast cultures were established individually from preputial skin of an 8 year-old child and prepared at yarsi laboratorium by dr. indra kusuma. the seventh passages of cultured cells were used for all experiments. normal human dermal fibroblasts (nhdf) were grown in roswell park memorial institute (rpmi) medium containing 10% fetal yulianti, mardliyati, bramono, et al asiaticoside and cell proliferation 99 univ med vol. 34 no.2 b o v i n e s e r u m ( f b s ) a n d 1 % p e n i c i l l i n streptomycin. cells were reseeded at a ratio of 1:3 and density of 1 x 106 cell/ml and subjected to treatment with 0.3125 mg/ml, 0.625 mg/ml, 1.25 mg/ml, 2.5 mg/ml, 5 mg/ml and 10 mg/ ml of asiaticoside or retinoic acid. the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (mtt) assay was repeated three times using mtt as substrate, which is actively absorbed by fibroblasts and then reduced by mitochondrial succinate dehydrogenase under the addition of nicotinamide adenine dinucleotideh (nadh) from water-soluble mtt to insoluble purple formazan. the number of viable cells was then evaluated at 24 and 48 hours after treatment, as an indicator of fibroblast proliferation.(17) obtained values were then normalized against the control group, which was taken as 100%. type i and iii collagen synthetic activity by human dermal fibroblasts collagen synthesis was analyzed using elisa kit for human collagen type i and iii, alpha 1 from cusabio. fibroblast collagen synthetic activity was evaluated using elisa, 24 and 48 hours after treatment and compared with untreated hdf as negative control. results for collagen synthesis were reported in µg/ml. statistical analysis f o r s t a t i s t i c a l e v a l u a t i o n o f h d f proliferation and collagen type i and iii synthesis, one-way anova as well as tukey’s post hoc analysis was performed when one-way anova results were significant. significance was set to p<0.05, and for highly significant differences to p<0.001. results ta b l e 1 s h o w s t h a t a f t e r 2 4 h o u r s , asiaticoside at all concentrations had stronger effects on hdf proliferation than retinoic acid. the optimal stimulatory effect was observed at 0.625 mg/ml. in statistical analysis with t ab le 1 . m ea n d is tr ib u ti o n o f h d f p ro li fe ra ti o n a n d c o ll ag en s y n th es is b y a si at ic o si d e an d r et in o ic a ci d t es t co m po un d co nc en tr at io n fo r h d f p ro li fe ra ti on ( m g/ l ) : i : 0. 31 25 ;i i : 0. 62 5; ii i : 1. 25 ;i v : 2 .5 0; v : 5 .0 ;v i : 10 .0 t es t co m po un d co nc en tr at io n fo r co ll ag en s yn th es is ( µg /m l ): i : 0 .0 25 ;i i : 0. 5; ii i : 0. 1 100 anova test the difference between asiaticoside and retinoic acid was significant (p<0.05). after 48 hours, asiaticoside has its strongest effect at 0.625 mg/ml vs. retinoic acid (p<0.025). the one-way anova test results indicated that between the asiaticoside dose groups there were significant differences in fibroblast proliferation after 24 and 48 hours of treatment (p<0.001). the post hoc tukey test found significant differences in the asiaticoside groups at 24 hours, between dose i and ii, ii and iii, ii and iv, ii and v, ii and vi, iii and iv, iii and vi (table 2). according to the one-way anova test, between the retinoic acid dose groups there were significant differences in fibroblast proliferation after 24 hours and 48 hours of treatment (p <0.001). the post hoc tukey test found significant differences in the retinoic acid groups at 24 hours, between dose i and ii, i and iii, i and v, i and vi, ii and iii, ii and iv, ii and v, ii and vi, iii and v, iii and vi (table 3). type i collagen synthesis by hdf after 24 and 48 hours of treatment at all three doses of asiaticoside and retinoic acid, was observed to actually be lower than controls. however, the differences were statistically not significant. for type iii collagen production, we observed the greatest induction at 24 hours with asiaticoside treatment at 2.50 ì g / m l. on the other hand, at 48 hours the maximum effect was achieved by retinoic acid at 0.50 ì g / m l. table 2. post hoc tukey multiple comparisons on hdf proliferationby asiaticoside asiaticoside concentration (mg/l) : i: 0.3125; ii: 0.625; iii: 1.25; 28.80 iv: 2.50; v: 5.0; vi: 10.0 yulianti, mardliyati, bramono, et al asiaticoside and cell proliferation 101 univ med vol. 34 no.2 table 3. post hoc tukey multiple comparisons on hdf proliferation by retinoic acid retinoic acid concentration (mg/l) : i : 0.3125; ii : 0.625; iii : 1.25; iv : 2.50; v : 5.0; vi : 10.0 discussion our results indicate that 24 hours after treatment, asiaticoside has a stronger effect on fibroblast proliferation at all concentrations compared to retinoic acid. this is in accordance to the previous study by lu et al.,(10) in which they reported alterations in col18a1, col1a2, and col3a1 gene profile, as well as in the fibroblast cell division cycle, in response to asiaticoside stimulation. gimemo et al.(15) investigated the timedependency effect of four different retinoids in the µm dose range on human dermal fibroblasts cultivated in vitro. this study showed that retinol and retinal at concentrations greater than 20 µm, can damage cells as evaluated by lactate dehydrogenase activity released into the culture medium, and induce oxidative stress and apoptosis in human dermal fibroblasts. type i and iii collagens are major structural and functional components of skin connective t i s s u e . t h e r e f o r e , w e i n v e s t i g a t e d t h e intracellular and extracellular modifications of type i and iii collagen, as in the cross-linked step by asiaticoside stimulation.(11) hadi et al.(13) investigated allogeneic hdf viability in peripheral blood mononuclear co-culture and reported that allogeneic hdf were able to secrete type i and iii collagen. our study showed that 102 asiaticoside and retinoic acid induced the synthesis of type i collagen in hdf after 24 and 48 hours of incubation, but after 24 hours 0.05 µ g / m l o f a s i a t i c o s i d e i n d u c e d i n h d f significantly higher type iii collagen synthesis compared to retinoic acid. our experiment indicated that after 24 hours, type iii collagen synthesis in hdf was significantly higher after treatment with asiaticoside compared to retinoic acid and controls (without treatment). maximal effect was achieved at 2.50 µg/ml of asiaticoside, but after 48 hours retinoic acid had a greater effect than asiaticoside at the same dose.(7) a study in malaysia showed that ca extract has a dose-dependent stimulatory effect on collagen synthesis, which was enhanced threefold over controls at a dose of 50 mg/ml.(5) compared to our experiment, another study in malaysia by hashim et al.(4) evaluated the effect of centella asiatica on collagen synthesis in hdf, compared to vitamin c. the effect of ca extract was stronger than that of vitamin c, which was used as a positive control. the ca extract at 50 mg/ml showed three-fold collagen enhancement, whereas vitamin c had a two-fold collagen enhancing response.(4) lu et al.(10) showed that asiaticoside isolated from ca induced collagen iii synthesis with maximum effect at 48 hours. in our study, both asiaticoside and ra showed greatest effects at 48 hours. this is supported by the previous study of wu et al.,(16) which reported significant elevation of procollagen iii level following s t i m u l a t i o n w i t h l o w c o n c e n t r a t i o n s o f asiaticoside and madecassoside. moreover, research by pareda et al.(17) indicated a dosedependent in vitro stimulation on collagen synthesis by green coffea arabica (gco). it was found that collagen synthesis was enhanced 1.8 fold in human dermal fibroblasts than in negative controls after 48 hours of treatment with 3.125 mg/ml and 6.25 mg/ml gco. these results demonstrated a more pronounced stimulatory effect of biosynthetic compounds on collagen p r o d u c t i o n . n e v e r t h e l e s s , w i t h f u r t h e r improvement, we expect a similar stimulatory performance of asiaticoside. song et al. ( 1 8 ) c o n d u c t e d a s t u d y o f madecasoside (one of the active compounds of centella asiatica) and found that this compound induces apoptosis of keloid fibroblasts in a mitochondrial-dependent pathway, in addition to inhibiting type i collagen synthesis. our study with asiaticoside matches this observation, with greater type iii collagen and lower type 1 collagen synthesis in human dermal fibroblast cultures of preputium explants of young children (4-8 years). ju-lin et al.(19) had previously reported that asiaticoside induces the expression and activation of the tgf-â signaling pathway in hyperthropic scars, while inhibiting smad7. this is the underlying mechanism behind the decreased type i collagen production. in the present study, however, we isolated human dermal fibroblast from one subject only. therefore, this may not fully represent some genetic variations related with collagen synthesis. a clinical study by kaji et al.(20) concluded that topical retinol treatment for 24 weeks improved fine wrinkles associated with natural aging, and that increased collagen production by retinol is the most likely reason behind it. clinical application of the present study will be done to i n v e s t i g a t e t h e e ff e c t o f a s i a t i c o s i d e i n accelerating wound healing. another possible clinical study is to investigate the anti-aging effect of asiaticoside after topical treatment, in comparison with retinoic acid. conclusions asiaticoside induces hdf cell proliferation and collagen synthesis in a timeand dosedependent manner. asiaticoside as an active agent of centella asiatica has a similar effect as retinoic acid on type i and iii collagen synthesis in human dermal fibroblasts. conflict of interest the authors have no conflict of interest. yulianti, mardliyati, bramono, et al asiaticoside and cell proliferation 103 univ med vol. 34 no.2 acknowledgements the authors wish to thank intan razari and dani for laboratory assistance, dr. indra kusuma for hdf cell culture and dr. pras for providing pure asiaticoside from the agency for the assessment and application of technology (badan pengkajian dan penerapan teknologi bppt). references 1. davis sc, perez r. cosmeceutical and natural products: wound healing. clin dermatol 2009; 27:502-6. 2. chanchal d, swarniata s. novel approaches in herbal cosmetics. j cosmet dermatol 2008;7:8995. 3. tiwari s, geniot s, gambhir is. centella asiatica: a concise drug review with probable clinical uses. j stress physiol biochem 2011;7: 39-42. 4. hashim p, sidek h, helan mhm, et al. triterpene composition and bioactivities of centella asiatica. molecules 2011;16:1310-22. 5. pitella f, dutra rc, junior dd. antioxidant and cytotoxic activities of centella asiatica (l) urb. int j mol sci 2009;10:3713–21. 6. paocharoen v. the efficacy and side effects of oral centellla asiatica extract for wound healing promotion in diabetic wound patients. j med assoc thai 2010;93:s166–9. 7. zainol na, voo sc, sarmidi mr, et al. profiling of centella asiatica (l.) urban extract. malaysian j anal sci 2008;12;322-7. 8. gohil kj, patel ja, gajjar ak. pharmacological review on centella asiatica: a potential herbal cure-all. indian j pharm sci 2010;72:546-56. 9. bhavna d, jyoti k. centella asiatica: the elixir of life. ijrap 2011;2:431-8. 10. lu l, ying k, wei s, et al. asiaticoside induction for cell-cycle progression, proliferation and collagen synthesis in human dermal fibroblasts. int j dermatol 2004;43:801-7. 11. loc nh, tam an nt. asiaticoside production from centella (centella asiatica l. urban) cell culture. biotechnol bioprocess engineering 2010;15:1065–70. 12. lintner k, chamberlin cm, modun p, et al. cosmeceuticals and active ingredients. clin dermatol 2009;27:461-8. 13. hadi rs, kusuma i, sandra y. allogenic human dermal fibroblast are viable in peripheral blood mononuclear co-culture. univ med 2014;33:3442. 14. jurzak m, latocha m, goiniczek k. influence of retinoids on skin fibroblasts metabolism in vitro. acta pol pharm drug res 2008;1:85-95. 15. gimeno a, zarogoza r, vivosese i. retinol at concentration greater than the physiological limit, induces oxidative stress and apoptosis in human dermal fibroblast. exp dermatol 2004; 13:45-54. 16. wu f, bian d, xia y, et al. identification of major active ingredients responsible for burn wound healing of centella asiatica herbs. j evid based complement altern med 2012. article id 848093, 13 pages. doi:10.1155/2012/848093. 17. pareda mdcv, dieamant gdc, eberlin s, et al. effect of green coffea arabica l. seed oil on extracellular matrix components and waterchannel expression in in vitro and ex vivo human skin models. j cosmetic dermatol 2009;8:5862. 18. song j, day y, bian d, et al. madecassoside induce apoptosis of keloid fibroblast via a mitochondria–dependent pathway. drug dev res 2011;72:315-22. 19. ju-lin x, shao-hai q, tian-zeng l, et al. effect of asiaticoside on hypertrophic scar in the rabbit ear model. j cutan pathol 2009;36:234-9. 20. kaji r, kwak hsr, schumacker we, et al. improvement of naturally aged skin with vitamin a (retinol). arch dermatol 2007;143: 606-12. editorial i the benefit of physical activity for old and young richard tjan editor univ med vol. 29 no.2 there is now ample scientific evidence that elderly persons, will benefit from physical activity in a similar manner as the younger generations. regular physical activity of at least moderate intensity reduces the risk of cardiovascular major events, leading to the conclusion that physical inactivity is a major cardiovascular risk factor. the underlying molecular mechanism may be that moderate physical activity leads to a reduction of oxidative stress.(1) in a prospective cohort of 21,094 men (mean age, 53 years) without known coronary heart disease at baseline in the physicians’ health study, it was shown that vigorous physical activity (defined as ‘exercise to the point of breaking a sweat’) was associated with a decreased risk of heart failure, whereas elevated bmi (even in the pre-obese range) was associated with an increased risk.(2) numerous studies have also demonstrated the benefits of daily physical activity for the elderly in maintaining functional independence, defined as ‘the capacity to carry out activities of daily living’. paterson and warburton in 2010 conducted a systematic review of the relationship between physical activity of healthy community-dwelling elderly (age range 66 84 years) and functional limitations, disability, or loss of independence by analysis of prospective cohort studies reported in the literature. among 2,309 citations from electronic bibliographical databases (medline, embase, cinahl, psycinfo, cochrane library, sportdiscus) the investigators analyzed 66 prospective studies and concluded that “regular aerobic activity and short-term exercise programmes confer a reduced risk of functional limitations and disability in older age”.(3) not only relatively healthy elderly, but also those who are frail or severely deconditioned may benefit from exercise. in a 12-month exercise program, 65 old japanese elderly (median age 85 years) with minor disabilities, including those who had to be assisted in their activities of daily living, showed significant improvements in lower limb strength.(4) similarly, a study of frail institutionalized nonagenarians up to 96 years of age demonstrated increased muscle strength, size and functional mobility after 8 weeks of training.(5) on the other hand, physical activity in the elderly may occasionally result in arrhythmias. the elderly therefore should use equipment that promotes safety, such as treadmills with handrails and cycle ergometers. although the risk of fatalities is indeed minimal (2 fatalities per 1.5 million patient-hours of exercise in cardiac rehabilitation programs), it should still be borne in mind by the elderly when conducting physical activity.(1) the general rule is to conduct exercise according to ones capability and to increase it gradually. if a person is not yet ready for a given activity, she or he should not perform it. conducting daily physical activity should not overload the organ systems of the body. the elderly should also refrain from climbing activities without adequate precautions, as advanced age is associated with deterioration of balance and increased risk of ii univ med vol. 29 no.2 falls. elderly with conditions that can be exacerbated by exercise, such as active fever, unstable angina, uncontrolled diabetes, and congestive heart failure, should undergo appropriate tests before participating in an exercise program.(6) the current physical activity recommendation for healthy elderly as well as for younger age groups is at least 30 minutes of moderate physical activities, such as brisk walking, which may be augmented by vigorous physical activity, such as jogging. the recommendation should be interpreted as being a minimum prescription for the general population.(1) in the year 2007, the american college of sports medicine and the american heart association recommended that “the promotion of physical activity in older adults should emphasize moderate-intensity aerobic activity, muscle-strengthening activity, reducing sedentary behavior, and risk management”.(7) in the same year the national heart foundation of australia advocated 30 minutes or more of moderate-intensity daily physical activity for persons with cardiovascular disease.(8) in summary, the elderly of all ages should engage in at least moderate daily physical activity in order to maintain functional independence and quality of life as long as possible, within the lifespan determined by their genetic makeup. in addition, they should consume nutritionally adequate diets and have adequate sleep, as nutritional status and circadian rhythms are known to affect physical fitness. if daily physical activity can prolong the useful life of an elderly, the young should also take note and conduct as a minimum the recommended levels of daily physical activity and hopefully may all of us become healthy nonagenarians and centenarians. references 1. ignarro lj, balestrieri ml, napoli c. nutrition, physical activity, and cardiovascular disease: an update. cardiovasc res 2007;73:326–40. 2. kenchaiah s, sesso hd, gaziano jm. body-mass index and vigorous physical activity and the risk of heart failure among men. circulation 2009;119:44–52. 3. paterson dh, warburton der. physical activity and functional limitations in older adults: a systematic review related to canada’s physical activity guidelines. int j behav nutr phys act 2010;7:38. 4. taguchi n, higaki y, inoue s, kimura h, tanaka k. effects of a 12-month multicomponent exercise program on physical performance, daily physical activity, and quality of life in very elderly people with minor disabilities: an intervention study. j epidemiol 2010;20:21-9. 5. fahlman mm, topp r. increasing physical activity in the deconditioned person: the role of resistance training. ethn dis 2004;14:s25-7. 6. nelson me, rejeski wj, blair sn, duncan pw, judge jo, king ac, et al. physical activity and public health in older adults: recommendation from the american college of sports medicine and the american heart association. med sci sports exerc 2007;39:1435–45. 7. briffa t, maiorana a, allan r. on behalf of the executive working group and national forum participants. national heart foundation of australia physical activity recommendations for people with cardiovascular disease. sydney: national heart foundation of australia; 2007. meiyanti1 49 the role of triptans in the management of migraine january-april, 2009january-april, 2009january-april, 2009january-april, 2009january-april, 2009 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 vol.28 no.1 universa medicina meiyanti* *department of pharmacology, faculty of medicine, trisakti university correspondence dr. meiyanti, sp.fk department of pharmacology, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: 021-5672731 ext.2805 univ med 2009; 28:49-58 abstract migraine is one of the most prevalent disorders seen in clinical practice today and also a major cause of disability in the workplace. the prevalence of migraine is highest during the years of peak productivity, ie, between the ages of 25 and 55 years. the triptans are a group of selective 5-hydroxtriptamine (ht) 1 serotonin receptor agonists that activate the 5-ht 1b/1d receptor and possibly also the 5-ht 1a dan 5-ht 1f receptors. to date 7 subclasses of serotonin receptors have been identified, namely subclasses 5-ht 1 to 5-ht 7 . triptan causes cranial vasoconstriction, inhibits peripheral trigeminal activity and the trigeminal afferents. with its triple action, triptans can control acute attacks of migraine. triptan is contraindicated in patients with previous ischemic or coronary artery disease, cerebral or peripheral vascular disease and other cardiovascular disorders. triptans should be given immediately after an acute attack of migraine. the triptans are useful in the management of an acute migraine, but are not indicated for preventive therapy of migraine. several new advances in migraine management have been made in regard to the recognition of the disease, the pathogenesis of migraine, and the phenomenon of central sensitization. more treatment options become available to patients and prescribers, the impact of such therapy on worker productivity will become more important in determining the value of such interventions. keywords: tiptans, migraine, serotonin reseptor introduction m i g r a i n e i s a n e u r o l o g i c a l s y n d r o m e characterized by changes in body perception, headaches and nausea. the most commonly e n c o u n t e r e d c l i n i c a l m a n i f e s t a t i o n s a r e periodically recurrent headaches, the attacks of which initially are unilateral, but at some point in time may become bilateral or total. migraine o c c u r s a s a r e s u l t o f t r i g e m i n o v a s c u l a r abnormalities. (1) for clinical purposes, the international headache society (ihs) has divided headaches into 2 categories, i.e. primary and secondary headaches. migraine is classified as a primary headache, i.e. not due to organic or structural abnormalities.(2) 50 meiyanti triptans in migraine migraine affects 17% of women and 6% of men in the united states. before puberty, the prevalence and incidence is higher in boys than in girls. in those aged over 12 years, there is an increased prevalence of migraine in females and males. at ages over 40 the incidence declines, except in perimenopausal women. overall, the prevalence in females is higher than in males. the ratio of females to males increases from 2.5:1 at puberty to 3.5:1 at the age of 40 years, then decreases. the incidence of migraine with aura reaches a peak in boys at around 5 years of age and in girls at around 12-13 years. in the united states, the incidence of migraine in white females is higher than in asian females.(2) poor socio-economic conditions are said to be associated with migraine. studies have shown that migraineurs experience lower quality of life than the general population,(3,4) and that attack frequency is inversely related to quality-of-life scores.(5) studies have also shown that effective treatment of migraine has a positive impact on health-related quality of life.(6) on the basis of the clinical manifestations, there are 3 distinctive phases in migraine, namely triggering of acute attacks, aura and headaches.(7) migraine with aura is a hereditary disorder, in which several genes are involved. in familial hemiplegic migraine, a dominant h e r e d i t a r y d i s o r d e r, t h e l o c u s h a s b e e n identified in the 19q gene. the 19q gene codes for p/q calcium channels cacna1a. a second gene, atp1a2, located on chromosome 1q23, was discovered several years ago. the atp1a2 gene encodes an á-subunit of the sodium/ p o t a s s i u m p u m p l o c a t e d i n t h e n e u r o n a l membrane.(8) one complication associated with migraine is stroke, because migraine is associated with an increased risk of ischemic stroke. other complications of migraine comprise mental health problems, such as depression, manic depression, anxiety and panic disorders. for aborting acute attacks of migraine, anti-migraine drugs may be used, such as nonsteroidal anti-inflammatory drugs, triptans, or ergotamine. on the other hand, for the prevention migraine attacks, the drugs used are beta blockers, calcium channel inhibitors, antidepressants, and anticonvulsants.(9) through continuous research in migraine, scientists have been able to identify the serotonin receptors. figure 1. chemical structure of triptans(12) 51 currently there are 7 recognized subclasses of s e r o t o n i n r e c e p t o r s , i . e . s u b c l a s s e s 5hydroxtriptamine (ht) 1 to 5-ht 7 . the 5-ht 1 receptor consists of 5 subtypes, namely a, b, d, e and f.(10) the accumulated knowledge about serotonin receptors ultimately led to the discovery of triptans. chemical structure of triptans triptans are serotonin (5-ht) receptor agonists, that are selective for 5-ht 1 . triptans activate the 5-ht 1b/1d receptors and possibly also the receptors for 5-ht 1a dan 5-ht 1f .(10,11) these drugs are derivatives of indol, with substitutions at positions 3 and 5. the chemical structure of triptans is shown in figure 1.(12) a number of studies have concluded that the trigger for the initial attacks of migraine involves the brain stem as migraine generator, which is associated with genetic abnormalities. in the phase following the initial attack, auras and headaches develop. when the migraine generator is triggered, the cerebral blood flow will be reduced, followed by supression of cortical wave propagation. in patients with a reduction in cerebral blood flow below a critical level, the symptoms of aura will appear. in addition to noradrenaline and acetylcholine, immunohistochemical studies have uncovered several vasodilation transmitters in perivascular n e r v e s t h a t s u p p l y t h e i n t r a c r a n i a l b l o o d vessels, including serotonin (5-ht), vasoactive intestinal peptide (vip), nitric oxide (no), substance p, neurokinin a dan cgrp. no may be involced in the pathophysiology of migraine, and inhibition of no synthesis may be used in the treatment of migraine. in several cases, vasodilation causes changes in blood volume, followed by changes in cardiac stroke volume, with consequent enhanced pulsation of the blood vessels. enhanced pulsation will be detected by receptors in the vessel wall and figure 2. pathophysiology of migraine(7) univ med vol.28 no.1 52 meiyanti triptans in migraine results in increased activity of the perivascular s e n s o r y t r i g e m i n a l n e r v e s , w h i c h c a u s e s headaches and other symptoms (figure 2).(7) a c c o r d i n g t o a d d i t i o n a l e v i d e n c e , serotonin and dopamine stimulate the stage of t h e i n f l a m m a t o r y p r o c e s s t h a t i n v o l v e s endothelial cells, mast cells and platelets. this inflammatory reaction causes vasodilation and perivascular reaction. receptors for 5-ht are r e c o g n i z e d a s b e i n g t h e m o s t i m p o r t a n t r e c e p t o r s i n t h e m e c h a n i s m o f h e a d a c h e . several symptoms associated with migrainous headaches, such as nausea (80%), yawning, irritability, hypotension and hyperactivity may be linked to dopamine receptor activation.(1,13) there are 2 hypotheses that may explain the mechanisms of action of triptan in the pathogenesis of migraine. the first hypothesis states that the 5-ht 1b receptor has the ability to induce vasoconstriction of intracranial blood vessels, including arteriovenous anastomoses. i n m i g r a i n e t h e r e i s d i l a t a t i o n o f c a r o t i d arteriovenous anastomoses in the head, the causes of which are currently still not known with certainty. a total of 80% of the carotid arterial flow is reportedly shunted through anastomoses located in the scalp and the ears. there will be extravasation of blood from the capillaries, which subsequently causes cerebral ischemia and hypoxia. according to this version of the pathophysiology of migraine, an effective antimigraine drug must be able to close the shunt and reverse the cerebral blood flow.(10,11,13) triptan interacts with 5-ht 1d and 5-ht 1b receptors and has no or only low affinity for other 5-ht receptors. the drug is not active against α1-, α2-, and β-adrenergic, dopamine, cholinergic muscarinic and benzodiazepine receptors. the effective dose of triptan is determined by its affinity for the 5-ht 1b and 5ht 1d receptors, whilst the affinity of triptan for 5-ht 1a or 5-ht 1e receptors has no influence on its effective dose.(7,10) the second hypothesis states that 5-ht 1d agonists inhibit release of proinflammatory neuropeptides on perivascular nerve terminals. in the pathophysiology of migraine, headaches are not solely caused by cranial vasodilation, but also involve an inflammatory mechanism known as neurogenic inflammation. arterial dilatation causes traction on the perivascular nerve fibers, resulting in depolarization of the fibers, which induces an action potential that is conducted to the central nervous system. in addition, depolarization also results in the release of neuropeptides from the nerve fibers around the artery. the released neuropeptides are substance p from the c fibers and calcitonin gene-related peptide (cgrp) from the aδ fibers, ultimately causing increasing dilatation of the artery and producing pain.(10,11,14,15) from both of the above hypotheses and several studies that have been conducted, it may be concluded that triptan has 3 mechanisms of a c t i o n , n a m e l y i n d u c t i o n o f c r a n i a l v a s o c o n s t r i c t i o n , i n h i b i t i o n o f p e r i p h e r a l trigeminal activity, and inhibition of trigeminal afferents.(11,14,16) with these three actions, triptan can control acute attacks of migraine. the following illustration shows the site of action of triptan in the trigeminovascular system (figure 3).(11) another benefit of triptan is its ability to r e l i e v e t h e n a u s e a a n d v o m i t i n g w h i c h frequently accompany migraine. this is because triptan acts on the 5-ht 1d receptor that is l o c a t e d i n t h e s o l i t a r y t r a c t n u c l e i , t h u s i n h i b i t i n g t h e c e n t e r s f o r n a u s e a a n d vomiting.(10,14) there are seven kinds of triptans that have been studied and synthesized for use as acute a n t i m i g r a i n e d r u g s , i . e . s u m a t r i p t a n , z o l m i t r i p t a n , n a r a t r i p t a n , r i z a t r i p t a n , almotriptan, eletriptan and frovatriptan.(7,10) with the increasing numbers of available drugs, some guidance is necessary in the selection of 53 those triptans that have the greatest chances o f s u c c e s s i n t h e m a n a g e m e n t o f a c u t e migraine. sumatriptan reaches a peak plasma c o n c e n t r a t i o n w i t h i n 1 2 m i n u t e s a f t e r subcutaneous administration and 1-2 hours after oral administration. the bioavailability of subcutaneous preparations approach 97%, whilst for oral or intranasal preparations it is only 14-17%. the elimination half-time of the drug is 1-2 hours. sumatriptan is metabolized mainly by mao-a, and its metabolites are excreted in the urine. the plasma protein binding of sumatriptan is around 14%.(7,10,17,18) f o r z o l m i t r i p t a n , t h e p e a k p l a s m a concentration occurs about 1.5-2 hours after administration of the oral preparation, which h a s a b i o a v a i l a b i l i t y o f a r o u n d 4 0 % . zolmitriptan is converted into the n-desmethyl form, an active metabolite that has an affinity for 5-ht 1b and 5-ht 1d receptors several times higher than that of zolmitriptan. the half-life of zolmitriptan and is metabolite is 2-3 hours.(7,10,17,18) rizatriptan has its peak plasma concentration i n 1 1 . 5 h o u r a f t e r o r a l a d m i n i s t r a t i o n . i t s bioavailability is around 45%, and the main m e t a b o l i c p a t h w a y i s t h r o u g h o x i d a t i v e deamination by mao-a. plasma protein binding of rizatriptan is about 14 %.(7,10) a l m o t r i p t a n a n d e l e t r i p t a n r e a c h t h e i r respective peak plasma concentrations in 1.4-3.8 and 1-2 hours after administration of the oral p r e p a r a t i o n . t h e b i o a v a i l a b i l i t y o f t h e o r a l preparation of almotriptan is 80%, whilst that of eletriptan is 50%. the elimination half-times of almotriptan and eletriptan are 3.3-3.7 and 3.6-5.5 hours, respectively. almotriptan is metabolized by cytochrome p-450 and mao-a, and eletriptan by cyp3a4.(11,14) figure 3. site of action of triptan11 univ med vol.28 no.1 54 meiyanti triptans in migraine for frovatriptan the characteristics are as follows: peak plasma concentration is reached i n 2 4 h o u r s a f t e r o r a l a d m i n i s t r a t i o n ; bioavailability of the oral preparation is 2430%; elimination is through the kidneys and liver, with a half-time of 25 hours and over; the drug is not metabolized by mao-a.(7,11,18) the side effects of the administration of 5-ht1 agonists may be ser ious or minor. serious side effects rarely occur, and may take t h e f o r m o f c o r o n a r y a r t e r i a l v a s o s p a s m , myocardial ischemia, atrial and ventricular arrhythmias, or myocardial infarction. these side effects usually occur in patients with underlying risk factors for coronary heart disease. however, generally the side effects of triptan in the treatment of acute migraine are minor. oral administration of triptan may cause paresthesia; asthenia and weakness; flushing of the face; sensations of oppression, fullness or p a i n i n t h e c h e s t , n e c k a n d l o w e r j a w ; drowsiness; nausea; vertigo and diaphoresis. on subcutaneous administration, the majority of patients has reported minor pain and burning sensation at the injection site.(10,11,14,16) triptan is contraindicated in patients with a history of ischemia or vasospasm of the c o r o n a r y a r t e r i e s , p e r i p h e r a l o r c e r e b r a l v a s c u l a r d i s e a s e , o r o t h e r c a r d i o v a s c u l a r disorders. however, triptan is still safer than other drugs commonly used for managing migraine, e.g. ergot preparations, which are n o n s p e c i f i c s e r o t o n i n a g o n i s t s . t h e vasoconstrictive effect of the coronary arteries are mediated by 5-ht2a receptors, whilst t r i p t a n h a s o n l y w e a k a c t i v i t y f o r t h e s e receptors.(7,9-11) triptan is also contraindicated in patients with uncontrolled hypertension, as it causes an a c u t e , a l b e i t u s u a l l y s m a l l , r i s e i n b l o o d pressure. on the other hand, triptan is not contraindicated in patients with controlled hypertension. naratriptan is contraindicated in patients with severe renal or hepatic disorders, for which rizatriptan may be given, but under special monitoring.(7,9) use of triptans in migraine triptan is the drug of choice for aborting attacks of migraine. it is reportedly effective, safe and well-tolerated in migraine. treatment with triptan should be given immediately after the onset of a migrainous attack. triptan is effective in the treatment of acute migraine, with or without aura, but it is not indicated for use in the prevention of migraine. the oral preparations are the safest, but difficult to use in patients suffering from nausea or vomiting d u r i n g t h e a t t a c k s o f m i g r a i n e . ( 1 9 2 1 ) t h e i n d i v i d u a l r e s p o n s e s t o t r i p t a n a r e unpredictable, therefore selection of a suitable drug for a patient is usually on a trial-and-error basis. if the selected triptan does not succeed in effecting a cure or if there are side effects limiting its use, the patient could possibly have a better response to another triptan.(9) the injectable preparation sumatriptan 6 mg is the most effective treatment of acute migraine, but also has more frequent side effects, and requires skill in injecting the drug. the injection may be repeated once in one 24hour period if the initial dose fails to abort the attack.(7,10,22) the onset of action of sumatriptan nasal s p r a y s i s a p p r o x i m a t e l y 1 5 m i n u t e s . t h e recommended dosage is between 5-20 mg, which may be repeated after 2 hours, until the maximal dose of 40 mg/24 h is reached.(10) the recommended oral dose of sumatriptan is 25-100 mg, which may be repeated after 2 hours until a total dose of 200 mg in one 24hour period is reached. zolmitriptan is given orally with a dose of 1.25-2.5 mg, repeatable after 2 hours, up to the maximal dose of 10 mg/ 24 h. on the other hand, the oral dose of naratriptan is 1-2.5 mg, which must not be 55 repeated until 4 hours have passed since the previous dose was given. the maximal dose in o n e 2 4 h o u r p e r i o d m u s t n o t e x c e e d 5 mg.(7,9,17,18) the recommended oral doses of rizatriptan and almotriptan are 5-10 mg and 6.25-12.5 mg, respectively, to be repeated if necessary after 2 hours, up to the maximal dose of 30 mg/24 h for rizatriptan and 25 mg/24 h for almotriptan. the triptans are not recommended for c o n c o m i t a n t a d m i n i s t r a t i o n w i t h e r g o t preparations or other triptans within one 24hour period, because all of them stimulate the serotonergic receptors of the cerebral and c o r o n a r y b l o o d v e s s e l s . ( 7 ) s u m a t r i p t a n , rizatriptan and zolmitriptan are contraindicated in patients on monoamine oxidase inhibitors s u c h a s m o c l o b e m i d e . ( 2 3 ) t h e g r o u p o f m o n o a m i n e o x i d a s e i n h i b i t o r s r e t a r d t h e metabolism of sumatriptan, rizatriptan and z o l m i t r i p t a n , a n d r a i s e t h e p e a k b l o o d concentration of these triptans. naratriptan, eletriptan, and frovatriptan are not metabolized by monoamine oxidase and as such are not contraindicated for concomitant administration with monoamine oxidase inhibitors. almotriptan is mainly eliminated through the kidneys and metabolized by cytochrome cyp 450, and only a small percentage is metabolized by monoamine oxidase, and thus is also not contraindicated for use with monoamine oxidase inhibitors.(7,9,18) p r o p a n o l o l i n c r e a s e s t h e b l o o d concentration of rizatriptan through its effect on the monoamine oxidase-a system, such that the dose of rizatriptan needs to be reduced if adiministered with propanolol. a dose of 5 mg of rizatriptan on concomitant administration with propanolol results in an equal blood concentration as a dose of 10 mg rizatriptan given without propanolol. the maximal dose of rizatriptan is also modified to 15 mg/24 h on concomitant administration with propanolol, w h i l s t i n p a t i e n t s w i t h o u t p r o p a n o l o l t h e m a x i m a l d o s e i s 3 0 m g / 2 4 h o u r s . t h i s interaction does not occur between rizatriptan and other b-blockers or between other triptans and propanolol.(14,16) e l e t r i p t a n i s m e t a b o l i z e d m a i n l y b y cytochrome p-450 cyp3a4 and eliminated from the brain by the p-glycoprotein pump. drugs that induce or inhibit this system may modify the pharmacokinetics of eletriptan, a l t h o u g h t h e c l i n i c a l i m p o r t a n c e o f t h i s interaction is not fully understood.(16) t h e u s f d a h a s n o w r e p o r t e d t h e serotonin syndrome in a number of people t a k i n g t r i p t a n s c o n c u r r e n t l y w i t h s o m e antidepressants, particularly selective serotonin a n d s e r o t o n i n – n o r e p i n e p h r i n e r e u p t a k e inhibitors (ssris and snris, respectively).(24) the serotonin syndrome is an idiosyncratic r e a c t i o n t h a t m a y o c c u r w h e n t r i p t a n i s combined with serotonin-specific reuptake inhibitors (ssris). the serotonin syndrome consists of changes in the triad of mental status (e.g. confusion), dyautonomia (e.g. diaphoresis, d i a r r h e a , h y p e r t e n s i o n , a n d f e v e r, a n d neuromuscular symptoms (e.g. myclonus and t r e m o r ) , b u t t h i s s y n d r o m e o c c u r s o n l y rarely.(14,16,25) the symptoms of the serotonin syndrome (box 1)(25) often have a rapid onset, usually within hours of the initiation or dose change of a drug, but occasionally up to several weeks after.(26,27) the majority of triptan clinical trials has a similar research design, i.e. randomized, double blind, and controlled, and thus metaanalyses may be conducted using data from these trials. in 2001 a meta-analysis was done on 53 triptan clinical trials involving 24,089 patients in the age group of 18-65 years with m o d e r a t e a n d s e v e r e a t t a c k s o f m i g r a i n e according to the international headache society criteria. the patients had been treated with one oral triptan preparation in a predertermined therapeutical dose. in the meta-analysis, all univ med vol.28 no.1 56 meiyanti triptans in migraine table 1. comparison of oral sumariptan 100 mg with other oral triptans.(28) legend: the equal sign (=) signifies a value equal to that of sumatriptan 100 mg; the plus sign (+) means superior or higher than sumatriptan 100 mg; two plusses (++) signify increased superiority; the minus sign (-) means inferior or less than sumatriptan 100 mg recovery in 2 hours: indicates that the patient responded to treatment by recovering from moderate or severe pain into having mild pain or none at all. pain-free in 2 hours: the patient became pain-free within 2 hours without additional drugs and there was no recurrence of the headache within 24 hours. consistency: the patient responded in minimally 2 out of 3 treated attacks of migraine. tolerability: in how far the patient could tolerate the side effects of the drug which medically were not important but clinically disturbing, e.g. flushing of the face and sensations of oppression in the chest. box 1. signs and symptoms of serotonin syndrome(25) autonomic hyperactivity • abnormal blood pressure: in moderate cases, severe hypertension in severe cases, hypotension • dilated pupils • diarrhea • fever, diaphoresis, shivering • tachycardia, tachypnea, dyspnea mental status changes • agitation, nervousness, hypervigilance, insomnia • confusion, agitated incoherent speech, delirium • semicoma or coma neuromuscular abnormalities • akathisia, mydriasis, impaired coordination • myoclonic twitching, tremors, ataxia, rigidity, hyperreflexia, clonus (including ocular clonus) • seizure 57 triptan preparations with differing dosages were compared as to efficacy, consistency, and tolerability, with sumatriptan 100 mg as the standard. frovatriptan was excluded from the m e t a a n a l y s i s , b e c a u s e t h e d a t a w e r e n o t available and its efficacy was considered to be less than that of other triptans.(28,29) the safety of a drug is not identical to its tolerability. the occurrence of side effects of the drug in patients on triptan therapy is related to its tolerability and does not affect its safety. a b s e n c e o f s i d e e f f e c t s d o e s n o t i m p l y increased safety. the safety of triptan should be placed in the context of endangering patients at risk for underlying cardiovascular disease. from various studies the fact emerged that longterm use of triptans is safe in patients without contraindications.(16) conclusions migraine is a disorder with a sufficiently high prevalence which affects both women and men. the management of migraine is divided into management for aborting the acute attack and management for prevention of attacks of migraine. the triptans are one group of the drugs of choice in acute attacks of migraine. t h e t r i p t a n s a r e a l l e ff e c t i v e i n a b o r t i n g m i g r a i n e h e a d a c h e s , a s t h e y h a v e s i m i l a r p h a r m a c o d y n a m i c e f f e c t s . t h e o r a l preparations rizatriptan 10 mg, eletriptan 80 mg, and almotriptan 12.5 mg have a better efficacy than do other oral triptan preparations in the management of acute migraine. references 1. blanda m. headache, migraine. available at : http:/ /www.emedicine.medscape.com. accessed august 20, 2008. 2. srivastava ss. pathophysiology and treatment of migraine and related headache. available at: http:/ /www.emedicine.medscape.com. accessed august 20, 2008. 3. lipton rb, liberman jn, kolodner kb, bigal me, dowson a, stewart wf. migraine headache disability and health-related quality-of-life: a population-based case-control study from england. cephalalgia 2003; 23: 441-50. 4. magnusson je, becker wj. migraine frequency and intensity: relationship with disability and psychological factors. headache 2003; 43: 104959. 5. terwindt gm, ferrari md, tijhuis m, groenen sm, picavet hs, launer lj. the impact of migraine on quality of life in the general population: the gem study. neurology 2000; 55: 624-9. 6. lofland jh, johnson ne, batenhorst as, nash db. changes in resource use and outcomes for patients with migraine treated with sumatriptan: a managed care perspective [published correction appears in arch intern med. 1999; 159:2228]. arch intern med 1999; 159: 857-63. 7. villalon cm, centurion d, valdivia lf, vries p, saxena pr. migraine: pathophysiology, pharmacology, treatment and future trends. curr vasc pharmacol 2003; 1: 71-84. 8. li th, wong lk. advances in headache diagnosis and treatment. medical progress 2009; 161-5. 9. srivastava ss. medication for migraine headache: a review of adverse effect profile and safety. headache 2004; 44: s 31-9. 10. sanders-bush e, mayer se. 5-hydroxytryptamine (serotonin): receptor agonists and antagonits. in: hardman jg, limbird le, gilman ag, editors. goodman & gilman’s the pharmacologic basis of therapeutics. 10th ed. new york:mc graw-hill; 2001. p. 269-90. 11. goadsby pj, lipton rb, ferrari md. migraine current understanding and treatment. n engl j med 2002; 346: 257-70. 12. spierings elh. mechanism of migraine and action of antimigraine medications. med clin north am 2001; 85: 943-58. 13. aurora sk. pathophysiology of migraine. available at: http://www.touchneurology.com. accessed april 28, 2009. 14. tepper sj. safety and rational use of the triptans. med clin north am 2001; 85: 959-70. 15. reuter u, moskowitz ma. new insights into migraine pathophysiology. curr opin neurol 2006; 19: 294-8. univ med vol.28 no.1 58 meiyanti triptans in migraine 16. jamieson dg. the safety of triptan in the treatment of patients with migraine. am j med 2002; 112: 135-40. 17. tfelt-hansen p, de vries p, saxena pr. triptans in migraine: a comparative review of pharmacology, pharmacokinetics and efficacy. drugs 2000; 60: 1259-87. 18. jhee ss, shiovitz t, crawford aw, cutler nr. pharmacokinetics and pharmacodynamics of the triptan antimigraine agent: a comparative review. clin pharmacokinet 2001; 40: 189-205. 19. vincenza s, kweiss k, wall am, pilson cm. pharmacologic management of acute attacks of migraine and prevention of migraine headache. ann intern med 2002; 137: 840-9. 20. oldman ad, smith la, mcquay hj, moore ra. pharmacological treatments for acute migraine: quantitative systematic review. pain 2002; 97: 24757. 21. waeber c. emerging drugs in migraine treatment. expert opin emerg drugs 2003; 8: 437-56. 22. kaniecki r. headache assessment and management. jama 2003; 289: 1430-3. 23. armstrong sc, cozza kl. triptans: med-psych drug-drug interactions update. psychosomatics 2002; 43: 502–4. 24. us food and drug administration. information for healthcare professionals: selective serotonin reuptake inhibitors (ssris), selective serotonin– norepinephrine reuptake inhibitors (snris), 5hydroxytryptamine receptor agonists (triptans). available at: www.fda.gov/cder/drug/info sheets/hcp/triptanshcp.htm. accessed sept 14, 2008. 25. wooltorton e. triptan migraine treatments and antidepressants: risk of serotonin syndrome. cmaj 2006; 175: 8745. 26. boyer ew, shannon m. the serotonin syndrome. n engl j med 2005; 352: 1112-20. 27. birmes p, coppin d, schmitt l. serotonin syndrome: a brief review. cmaj 2003; 168: 143942. 28. ferrari md, roon kl, lipton rb, goadsby pj. oral triptans (serotonin 5-ht 1b/1d agonist) in acute migraine treatment: a meta-analysis of 53 trials. lancet 2001; 358: 1668-75. 29. ferrari md, goadsby pj, roon ki, lipton rb. triptans (serotonin, 5-ht 1b/1d agonists) in migraine: detailed results and methods of a metaanalysis of 53 trials. cephalalgia 2002; 22: 633-58. oktavianus 185 *department of biochemistry, faculty of medicine, universitas islam indonesia **anatomic pathology laboratory, faculty of medicine, universitas gadjah mada correspondence dr. asri hendrawati, msc department of biochemistry, faculty of medicine, universitas islam indonesia kaliurang road km 14,5 yogyakarta email: asri_xabi@yahoo.com univ med 2014;33:185-91 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background diabetes mellitus (dm) is a group of metabolic diseases which are characterized by hyperglycemia, resulting in various complications. a major macrovascular complication of dm is cardiac failure due to cardiomyopathy. hyperglycemia increases oxidative stress, so an oxidative-stress reducing therapeutic agent is required, e.g. the antioxidant quercetin. the aim of this study was to assess the effects of quercetin in reducing damage to cardiomyocytes of type 2 diabetic rats. methods this research is an experimental study using 40 rats. with simple random allocation, rats were divided into eight groups, then type 2 diabetes mellitus was induced using streptozotocin (5 rats per group). the test material was quercetin given at doses of 5, 20 and 80 mg/kgbw/day orally for 4 weeks. each single dose of quercetin was given in combination with glibenclamide 5 mg/ kgbw/day. after 4 weeks the rats were decapitated and the cardiac tissues taken to quantify the percentage of cell damage after hematoxylin-eosin staining (he). results quercetin at a dose of 80 mg/kgbw/day can lower cardiomyocyte damage better than quercetin at doses of 5 or 20 mg/kgbw/day. a combination of quercetin and glibenclamide can significantly lower levels of cardiomyocyte damage better than quercetin without glibenclamide (p<0.05). conclusion quercetin at a dose of 80 mg/kgbw/day with or without glibenclamide can lower damage to cardiomyocytes of type 2 diabetic rats. thus quercetin might serve as a valuable protective agent in cardiovascular inflammatory diseases in diabetic rats. keywords: quercetin, glibenclamide, cardiomyocytes, type 2 diabetes mellitus, rats quercetin reduce cardiomyocytes damage in type 2 diabetic rats asri hendrawati* and nadhir** 186 hendrawati, nadhir quercetin reduce cardiomyocytes damage kuersetin menurunkan kerusakan sel otot jantung pada tikus diabetes melitus tipe 2 latar belakang diabetes melitus (dm) merupakan kelompok penyakit metabolik yang ditandai hiperglikemia. berbagai komplikasi karena dm disebabkan oleh hiperglikemia. komplikasi makrovaskuler utama dm adalah gagal jantung karena kardiomiopati. hiperglikemia menyebabkan peningkatan stress oksidatif sehingga diperlukan terapi untuk menurunkan stress oksidatif seperti antioksidan kuersetin. penelitian ini bertujuan untuk menilai efek kuersetin dalam menurunkan kerusakan sel otot jantung tikus dm tipe 2. metode penelitian eksperimental menggunakan kontrol dilakukan pada 40 ekor tikus. secara alokasi random sederhana, tikus dibagi menjadi 8 kelompok yang diinduksi menggunakan streptozotocin menjadi dm tipe 2 (5 ekor per tikus kelompok). bahan uji adalah kuersetin dosis 5, 20 dan 80 mg/kgbb/hari peroral selama 4 minggu. masing-masing dosis kuersetin diberikan tunggal dan kombinasi dengan glibenklamid 5 mg/kgbb/hari. perlakuan diberikan selama 4 minggu. kemudian tikus didekapitasi dan diambil jantungnya untuk dihitung persentase kerusakan selnya setelah pewarnaan hematoksilin-eosin (he). hasil kuersetin dosis 80 mg/kgbb/hari menurunkan kerusakan sel otot jantung lebih baik dari kuersetin dosis 5 atau 20 mg/kgbb/hari. pemberian kombinasi kuersetin bersama glibenklamid menurunkan tingkat kerusakan sel otot jantung lebih baik secara bermakna dibandingkan kelompok tanpa kombinasi dengan glibenklamid (p<0,05). kesimpulan kuersetin dosis 80 mg/kgbb/hari dengan atau tanpa kombinasi glibenklamid mampu menurunkan kerusakan sel otot jantung tikus dm tipe 2. penelitian ini membuktikan kuersetin merupakan agen yang bermanfaat untuk mencegah kerusakan sel otot jantung pada tikus dm tipe 2. kata kunci: kuersetin, glibenklamid, sel otot jantung, diabetes melitus tipe 2, tikus abstrak introduction diabetes mellitus (dm) is a metabolic disease characterized by hyperglycemia, causing damage, dysfunction, and failure of organs, such as the eyes, kidneys, heart, and blood vessels.(1) in 2005 dm had reportedly affected 135 million world inhabitants (around 3% of the world population), including the urban and rural poor.(2) the number of dm patients in the world is projected to reach 522 million in the year 2030.(3) in indonesia there are around 8.4 million people with dm or around 5% of its population and this number is estimated to reach around 21.3 million in 2030.(4) chronic hyperglycemia in dm causes glucose autooxidation into free radicals, such as superoxides that can easily react with cells, so causing cell damage and even cell death.(5) diabetes mellitus is a high risk factor for heart failure, similar to coronary heart disease and hypertension. heart failure in dm is mainly caused by death of cardiomyocytes (diabetic cardiomyopathy). the major cause of cardiac 187 muscle death is principally attributed to increased production of reactive oxygen species (ros) in dm patients.(6) c u r r e n t l y, a d m i n i s t r a t i o n o f o r a l hypoglycemic agents is less effective in reducing oxidative stress in dm patients.(5) therefore, administration of antioxidants such as quercetin plays an important role in the treatment of dm to protect the organs, including cardiac muscle, against free radical-induced damage. quercetin, a flavonoid compound that has been intensively studied in vivo and in vitro, exerts a hypoglycemic effect and reduces the risk of obesity.(7) quercetin and other flavanoids have been shown to increase the synthesis and activity of antioxidant enzymes, such as catalase, in reducing oxidative stress.(7) quercetin has a protective effect on pancreatic â -cells and reduces oxidative stress. quercetin increases insulin secretion and protects pancreatic â -cells against oxidative damage via the extracellular signal-related kinase (erk 1/2) pathway.(8) the aim of the present study was to evaluate the ability of quercetin to prevent cardiomyocyte damage in type 2 diabetic rats. methods design of the study this was an experimental study of post test control group design. the study period was between february and june 2014. experimental animals the experimental animals used were 40 three-month old male wistar rats weighing between 150 and 250 grams each. the sample size was calculated with federer’s formula: (n1) (t-1) >15, giving for 8 groups the minimal number of 4 animals per group.(9) this study used 5 animals per group. the animals were kept and tested in the central inter-university laboratory of gadjah mada university. experimental protocol the rats were assigned by simple random allocation into 8 groups of 5 rats, in whom type 2 dm was induced by a single intraperitoneal injection of streptozotocin at a dose of 60 mg/ kgbw, dissolved in 100 mm cold citrate buffer at ph 4.5. fifteen minutes after streptozotocin administration, the rats were given a single intraperitoneal injection of nicotinamide at a dose of 120 mg/kgb. one week after streptozotocin i n d u c t i o n , t h e f a s t i n g b l o o d g l u c o s e concentration of the rats was determined. rats with a fasting blood glucose of more than 126 mg/dl were considered to be diabetic.(10) the diabetic rats were then subjected to the interventions, where group 1 (k1) received daily oral sodium carboxymethyl cellulose (cmc) as placebo, group 2 (k2) diabetic rats received glibenclamide 5 mg/kgbw/day once daily,(11) group 3 (k3) were given quercetin 5 mg/kgbw/ day, group 4 (k4) diabetic rats quercetin 20 mg/ kgbw/day, (12) group 5 (k5) diabetic rats quercetin 80 mg/kgbw/day, group 6 (k6) quercetin 5 mg/kgbw/day plus glibenclamide 5 mg/kgbw/day, group 7 (k7) diabetic rats quercetin 20 mg/kgbw/day plus glibenclamide 5 mg/kgbw/day, and group 8 (k8) received quercetin 80 mg/kgbw/day plus glibenclamide 5 mg/kgbw/day. the interventions were administered daily to the diabetic rats by a single gavage. the interventions were administered for 4 weeks.(12) histopathologic examination after 4 weeks of treatment, all rats were decapitated and their hearts taken for preparation of hematoxylin-eosin (he) stained sections. normal cardiomyocytes showed centrally located oval nuclei, surrounded by regular miofibrils. damaged cells were characterized by alterations in the size and shape of the nuclei, w h i c h b e c a m e m o r e r o u n d e d o r s q u a r e , surrounded by fibrotic tissue.(13) cardiac muscle damage was expressed as the percentage (%) of square/rounded nuclei in five high power (400x) fields under the light microscope.(14) determination of fasting blood glucose the rats were fasted for 12 hours before their blood was collected for determination of univ med vol. 33 no.3 188 hendrawati, nadhir quercetin reduce cardiomyocytes damage blood glucose. the blood was drawn from the orbital sinus. the blood glucose concentration o f t h e r a t s w a s m e a s u r e d u s i n g a spectrophotometer. this instrument is based on the principle of oxidation of glucose by glucose oxidase, resulting in release of hydrogen p e r o x i d e , w h i c h t h e n r e a c t s w i t h 4 aminoantipyrine and phenol. this reaction is catalyzed by peroxidases and produces the brownish-yellow quinoneimin. color intensity is measured at 500 nm wavelength. the intensity of the color produced is proportional to the glucose concentration. data analysis the between-group differences in cardiac cell damage was tested by one-way anova, followed by post hoc lsd. ethical clearance this study received ethical clearance from the medical and health research ethics committee (mhrec), faculty of medicine, gadjah mada university, yogyakarta, with reference number ke/fk/90/ec. results a l t h o u g h t h e r e w e r e n o s i g n i f i c a n t differences in the weight of the rats (p=0.528) between the intervention groups at baseline, there were significant differences in fasting glucose concentrations (p=0.005) (table 1). the results of multiple comparison lsd showed differences in fasting blood glucose concentrations between k1 and k5, between k1 and k7, and between k6 and k8 (data not shown). the degree of cardiomyocyte damage was d e t e r m i n e d b y m i c r o s c o p i c s t u d y o f cardiomyocyte histology after hematoxyllineosin (he) staining. normal cardiomyocytes were recognized by their centrally located oval nuclei, with regular miofibrils surrounding them. damaged cells were characterized by alterations in the size and shape of the cell nuclei, which became more rounded or square, surrounded by an accumulation of fibrotic tissue. the histologic picture of he-stained normal and damaged cardiomyocytes are shown in figures 1 and 2, respectively. table 1. means of body weights and fasting blood glucose concentrations by intervention group at baseline notes: *significant one-way anova if p<0.05. k1: diabetic rats on placebo/day, k2: diabetic rats on glibenclamide 5mg/ kgbw/day, k3: diabetic rats on quercetin 5 mg/kgbw/day, k4: diabetic rats on quercetin 20 mg/kgbw/day, k5: diabetic rats on quercetin 80 mg/kgbw/day, k6: diabetic rats on quercetin 5 mg/kgbw/day and glibenclamide 5mg/kgbw/day, k7: diabetic rats on quercetin 20 mg/kgbw/day and glibenclamide 5mg/kgbw/day, k8: diabetic rats on quercetin 80 mg/ kgbw/day and glibenclamide 5mg/kgbw/day. figure 1. healthy rat cardiomyocytes stained with he, showing oval cell nuclei surrounded by regular miofibrils oval nucleus regular miofibril 189 figure 2. damaged cardiomyocytes of diabetic rats receiving placebo (he stain), showing many cell nuclei with more rounded shapes and fibrosis of miofibrils around the nuclei without normal miofibril morphology after 4 weeks of intervention, there were no significant between-group differences in mean body weight of the rats (p=0.116). however, there were significant differences in fasting blood glucose concentrations between intervention groups after 4 weeks of intervention (p=0.003) (table 2). multiple comparison lds analysis showed significant differences in fasting blood glucose concentration between k1 and k4 and between k1 and k8. there were also significant d i ff e r e n c e s i n f a s t i n g b l o o d g l u c o s e concentrations between k3 versus k4 and k8, as well as between k4 and k6, and between k6 and k8 (data not shown). in the determination of cardiomyocyte damage, there were significant differences in the mean percentages between the intervention groups (p=0.000). the lowest mean percentage of cardiomyocyte damage was found in k8 and the highest in k1 (table 2). discussion the present study found a significant weight loss in the group of rats with induced dm, one of the clinical signs of which is unexplained weight loss. in type 2 dm there is insulin resistance resulting in decreased cellular glucose uptake. the cells cannot utilize sufficient amounts of glucose as a source of energy, inducing a compensatory utilization of noncarbohydrates as energy source, such as fat and protein reserves. this leads to a compensatory increase in lipolysis and a compensatory decrease in lipogenesis, thereby resulting in weight loss.(15) the present study found that quercetin alone at 80 mg/kgbw/day was effective in reducing the blood glucose concentration of diabetic rats, as compared with 5 mg/kgbw/day or 20 mg/kgbw/ day. quercetin has previously been shown to significantly reduce blood glucose concentration in patients with dm.(16) in another study, oral quercetin at a dose of 100 mg/kgbw/day given for 7 weeks significantly reduced fasting and 2hour postprandial blood glucose concentration table 2. means of weight, fasting blood glucose, and cardiac cell damage after 4 weeks of intervention by intervention group notes: *significant one-way anova if p<0.05. k1: diabetic rats on placebo/day, k2: diabetic rats on glibenclamide 5mg/ kgbw/day, k3: diabetic rats on quercetin 5 mg/kgbw/day, k4: diabetic rats on quercetin 20 mg/kgbw/day, k5: diabetic rats on quercetin 80 mg/kgbw/day, k6: diabetic rats on quercetin 5 mg/kgbw/day and glibenclamide 5mg/kgbw/day, k7: diabetic rats on quercetin 20 mg/kgbw/day and glibenclamide 5mg/kgbw/day, k8: diabetic rats on quercetin 80 mg/ kgbw/day and glibenclamide 5mg/kgbw/day. univ med vol. 33 no.3 190 hendrawati, nadhir quercetin reduce cardiomyocytes damage in diabetic rats. the ability of quercetin in reducing blood glucose concentration is thought to be due to its ability to inhibit the enzyme αglucosidase that plays a role in carbohydrate digestion in the small intestine.(17) t h e g r o u p o f d i a b e t i c r a t s g i v e n a combination of quercetin 20 mg/kgbw/day and glibenclamide 5 mg/kgbw/day and that given quercetin 80 mg/kgbw/day plus glibenclamide 5 mg/kgbw/day had significant reductions in mean fasting blood glucose concentration. this indicates that the combination of quercetin 20 mg/kgbw/day and glibenclamide is more e ff e c t i v e i n d e c r e a s i n g b l o o d g l u c o s e concentration than glibenclamide 5 mg/kgbw/ day or quercetin alone at 20 mg/kgbw/day. a previous study showed that a combination of honey (as antioxidant) and glibenclamide more e ff e c t i v e l y d e c r e a s e d b l o o d g l u c o s e concentration and oxidative stress, as compared with glibenclamide only. the study showed that glibenclamide alone was not significant in preventing pancreatic damage from oxidative stress.(18) in our study, oral quercetin at 80 mg/ kgbw/day for 4 weeks and the combination of quercetin 20 mg/kgbw/day or 80 mg/kgbw/day with glibenclamide 5 mg/kgbw/day were effective in decreasing fasting blood glucose concentration of the rats. in the present study it was found that quercetin 5 mg/kgbw/day was capable of a b e t t e r r e d u c t i o n o f t h e p e r c e n t a g e o f cardiomyocyte damage than placebo, being equivalent to glibenclamide 5 mg/kgbw/day. quercetin at a dose of 20 mg/kgbw/day decreased cardiomyocyte damage better than quercetin 5 mg/kgbw/day, glibenclamide 5 mg/ kgbw/day or placebo, but was equivalent to the combination of quercetin 5 mg/kgbw/day plus glibenclamide 5 mg/kgbw/day. quercetin may protect cardiomyocytes against damage, because of its ability to decrease oxidative stress in diabetic rats. in a previous study it was found that quercetin at doses of 50 mg/kgbw/day and 80 mg/kgbw/day for 45 days was capable of significantly decreasing oxidative stress in diabetic rats.(19) quercetin is able to promote the activity of the transcription factor nuclear factor erythroid 2-related factor 2 (nrf2) t o e n h a n c e p r o d u c t i o n o f e n d o g e n o u s a n t i o x i d a n t s c a p a b l e o f d e c r e a s i n g f r e e radicals.(20) in the present study, quercetin at a dose of 80 mg/kgbw/day was the best in decreasing cardiomyocyte damage than quercetin at lower doses, with or without glibenclamide. the administration of quercetin 5 mg/ kgbw/day, 20 mg/kgbw/day, or 80 mg/kgbw/ day in combination with glibenclamide 5 mg/ kgbw/day was shown to significantly decrease cardiomyocyte damage to a higher degree than did the quercetin doses without glibenclamide. this is due to the ability of quercetin in decreasing the numbers of free radicals that damage the cardiomyocytes, assisted by the action of glibenclamide in reducing blood glucose concentration, so decreasing the n u m b e r s o f f r e e r a d i c a l s p r o d u c e d b y hyperglycemia. a previous study stated that combining standard hypoglycemic drugs with antioxidants was more effective in decreasing free radicals and oxidative stress.(18) from our study results, it is hoped that quercetin may be used as adjunctive treatment for type 2 dm, so decreasing its complications and improving the quality of life of the patients. before this is possible, however, attention should be given to other variables capable of affecting quercetin bioavailability, e.g. its absorption and distribution, which are different in experimental animals and humans. a limitation of the present study was the lack of attention to the amount of feed consumed by the experimental animals, which may affect the bioavailability of quercetin as well as the clinical condition of the diabetic animals. during the intervention, the type and amount of feed given to each of the rats was identical, but some rats ate all their daily feed, whereas others left some of their feed untouched. 191 conclusions quercetin at a dose of 80 mg/kgbw/day protected cardiomyocytes better against damage in type 2 diabetic rats than did quercetin at doses of 5 and 20 mg/kgbw/day, glibenclamide 5 mg/ kgbw/day, or placebo. the combination of q u e r c e t i n a n d g l i b e n c l a m i d e p r o t e c t e d cardiomyocytes better against damage in type 2 diabetic rats than quercetin alone. acknowledgments the researchers wish to express their gratitude to the research and community service unit, faculty of medicine, universitas islam indonesia, yogyakarta for the funding of this study. references 1. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2012;35:64-71. 2. murugesh kv, yeligar dk, dash p, sengupta bc, maityt tk. antidiabetic, antioxidant and antihyperlipidemic status of heliotropium zeylanicum extract on streptozotocin-induced diabetic rats. biol pharm bull 2006;29:2202-5. 3. perkumpulan endokrinologi indonesia (perkeni). konsensus pengelolaan dan pencegahan diabetes mellitus tipe 2 di indonesia 2006. pb. perkeni: jakarta. 4. whiting dr, guariguata l, weil c, shaw j. idf diabetes atlas: global estimates of the prevalence of diabetes for 2011 and 2030. diabetes res clin pract 2011;94:311-21. 5. karasu c. glycoxidative stress and cardiovascular complications in experimentallyinduced diabetes: effects of antioxidant treatment. open cardiovasc med j 2010;4:24056. 6. murarka s, movahed mr. diabetic cardiomyopathy. j card fail 2010;16:971-9. 7. aguirre l, arias n, maraculla mt, gracia a, portillo mp. beneficial effects of quercetin on obesity and diabetes. open nutraceuticals j 2011;4:189-98. 8. youl e, bardy g, magous r. quercetin potentiates insulin secretion and protects ins-1 pancreatic â-cells against oxidative damage via the erk1/2 pathway. br j pharmacol 2010;161: 799-814. 9. david ah. evaluation of the oral toxicity of formaldehyde in rats. univ med 2008;27:10612. 10. barik r, jain s, qwatra d, joshi a, tripathi gs, goyal r. antidiabetic activity of aqueous root extract of ichnocarpus frutescens in streptozotocin-nicotinamide induced type-ii diabetes in rats. indian j pharmacol 2008;40:1922. 11. rabbani si, devi k, khanam s. protective role of glibenclamide against nicotinamidestreptozotocin induced nuclear damage in diabetic wistar rats. j pharmacol pharmacother 2010;1:18–23. 12. parabathina rk, raja gv, rao mn, rao gs, rao ks. cardioprotective effects of vitamin e, morin, rutin and quercetin against doxorubicininduced oxidative stress of rabbits: a biochemical study. j chem pharm res 2010;2:754-65. 13. thent zc, lin ts, das s, zakaria z. histological changes in the cardiac and the proximal aorta in experimental diabetic rats fed with piper sarmentsoum. afr j tradit complement altern med 2012;9:396-404. 14. fineschi v. cardiac pathology in death from electrocution. int j legal med 2006;120:79-82. 15. lai mh. antioxidant effects and insulin resistance improvement of chromium combined with vitamin c and e supplementation for type 2 diabetes mellitus. j clin biochem nutr 2008; 43:191-8. 16. kelly gs. quercetin. altern med rev 2011;16: 172-94. 17. kim sh, jo sh, kwon yi, hwang jk. effects of onion (allium cepa l.) extract administration on intestinal á-glucosidase activities and spikes in postprandial blood glucose levels in sd rats model. int j mol sci 2011;12:3757-69. 18. erejuwa oo, sulaiman sa, wahab ms, sirajudeen kns, salleh ms, gurtu s. effect of glibenclamide alone versus glibenclamide and honey on oxidative stress in pancreas of streptozotocin-induced diabetic rats. int j appl res nat prod 2011;4:1-10. 19. mahesh t, menon vp. quercetin alleviates oxidative stress in streptozotocin-induced diabetic rats. phytother res 2004;18:123–7. 20. moskaug jo, carlsen h, myhrstad m, blomhoff r. molecular imaging of the biological effects of quercetin and quercetin-rich foods. mech ageing dev 2004;125:315-24. univ med vol. 33 no.3 alvina 54 abstract asymptomatic bacteriuria (abu) is one of the bacterial infections most frequently found in women in all age groups, particularly in postmenopausal women. around 10-25% of women suffer from symptomatic urinary tract infection (uti) all their life. several risk factors are associated with the occurrence of abu in females, such as a history of uti, sexual activity, diabetes mellitus, personal hygiene, sexual hygiene, and menopausal status. the aim of this study was to determine the most important risk factor of influence on abu in premenopausal and postmenopausal women at the pasar minggu primary health center. this was an analytical cross-sectional observational study. the study sample consisted of premenopausal and postmenopausal women aged 40-65 years visiting a primary health center of the pasar minggu subdistrict, south jakarta, who were selected by consecutive sampling. the data analysis used prevalence ratios and logistic regression. the results of the study showed that the prevalence of positive bacteriuria in females was 45%. sexual activity, sexual hygiene, menopausal status of the females, and personal hygiene were risk factors of abu. personal hygiene was the most influential risk factor on the occurrence of abu in both premenopausal and postmenopausal women. since in this study abu was present in 45% of women, thus making it a common disorder in middleaged and older women, it is recommended that the primary care physician directly administer therapy to women with symptoms of uti without performing laboratory analysis. keywords: asymptomatic bacteriuria, personal hygiene, sexual activity, women *professional study program, medical faculty, trisakti university correspondence farasita ambarwati, sked. professional study program, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 univ med 2011;30:54-62. personal hygiene and asymptomatic bacteriuria in women aged 40 to 65 years farasita ambarwati*, maysarah elfani*, apriani margastutie*, and nurul mahirah binti mohd. dahuri* january-april, 2011january-april, 2011january-april, 2011january-april, 2011january-april, 2011 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 vol.30 no.1 universa medicina introduction urinary tract infection (uti) is a bacterial infection most frequently found in women and with greater frequency in older than in younger women,(1) and particularly in postmenopausal women. in young and middle-aged women the prevalence of uti is around 5%, rising rapidly with advancing age.(2) in spite of the high prevalence of bacteriuria in postmenopausal women, the predisposing factors of uti in this group has not been extensively studied in comparison with premenopausal women. however, reports from epidemiological and 55 univ med vol.30 no.1 case-control studies indicate that nonsecretory status and urological factors such as residual v o l u m e , r e d u c e d u r i n a r y f l o w, a n d incontinence, are closely associated with recurrent uti, particularly in postmenopausal women.(3) in the us the annually recorded numbers of patients with uti attending clinics is around 8 million, with a recurrence rate of 25% 50%.(3,4) the underlying mechanism of recurrence has been only partially elucidated, but there is a close association between impairment or cessation of estrogen production and increased risk of uti.(4) postmenopausal vaginal changes lead to clinical manifestations that play a role in uti. more than 50% of women older than 60 years of age experience a variety of urogenital symptoms.(2) utis may be divided clinically into symptomatic and asymptomatic infections. the infection is called symptomatic if there is significant bacteriuria (>100.000 cfu/ml) accompanied by clinical symptoms, such as dysuria, frequent micturition, and strangury, with or without fever and lower back pain. the infection is called asymptomatic if significant bacteriuria is found in apparently healthy children or adults without symptoms pointing to uti.(1) the climacteric, more commonly known as menopause, is the transition period between the reproductive phase and the senile phase (senium), occurring as a result of declining ovarial functionality. the premenopausal period is the period between the age of 40 years and the onset of the climacteric phase. the postmenopausal phase is the period from 12 months after menopause up to the senium.(5) in t h e p o s t m e n o p a u s a l p e r i o d t h e v a g i n a l epithelium is atrophic, due to lack of estrogen. epithelial atrophy is also found in the urethra, frequently leading to the urethral syndrome, which includes bacterial urethritis.(2,5,6) in the majority of utis the etiology is commonly found to be bacterial. several studies have reported a higher prevalence of bacteriuria in postmenopausal w o m e n c o m p a r e d w i t h t h a t i n y o u n g o r premenopausal women, (2,4) but data from developing countries are scarce. it is unclear whether women in developing countries have a higher frequency of uti and more severe symptoms, when compared to women in developed countries.(6) there are also reports stating that the prevalence of bacteriuria in premenopausal women is higher than in postmenopausal women, as it tends to be c o r r e l a t e d w i t h t h e f r e q u e n c y o f s e x u a l r e l a t i o n s , u s e o f s p e r m i c i d a l a g e n t s , contraception by means of diaphragms, and history of past utis, but data on the prevalence rate of bacteriuria are still lacking.(7) it has also been reported that diabetic women have a higher risk of asymptomatic bacteriuria (abu) and uti,(8,9) although it is unclear whether postmenopausal diabetic women have a higher r i s k o f b a c t e r i u r i a i n c o m p a r i s o n w i t h nondiabetic women. some investigators view a b u a s a c o m p l i c a t i o n o f d i a b e t e s i n women.(10,11) our study is based on the contradictory results of previous studies and the lack of data on the prevalence of bacteruria in preand p o s t m e n o p a u s a l w o m e n , e s p e c i a l l y i n indonesia. additionally this study also aims to find a relationship between risk factors of abu in premenopausal and postmenopausal women. methods research design the present study was an analytical observational study with cross-sectional approach, conducted from 3 january until 21 january 2011 at pasar minggu subdistrict primary health center, south jakarta. research subjects and sampling t h e s t u d y s u b j e c t s w e r e p r e a n d postmenopausal women on their first visit to the pasar minggu subdistrict primary health center, south jakarta, recruited by nonp r o b a b i l i t y c o n s e c u t i v e s a m p l i n g . t h e 56 inclusion criteria were: i) women in the age range of 40-65 years, with monthly periods (premenopausal women) or without monthly periods in the last year (postmenopausal women), and volunteering to participate. e x c l u d e d f r o m t h e s t u d y w e r e menstruating or pregnant women, and those on antibiotic therapy for infections. sample size the optimal sample size needed in this study was calculated according to the following formula:(12) ( ) ( ) ( ) ( ) 2 21 221111 1112n ⎥ ⎥ ⎦ ⎤ ⎢ ⎢ ⎣ ⎡ − −+−−− = ππ ππππππ βα zz for á = 0.05, zá = 1.96 and for â = 0.20, zâ = 0.845. the prevalence of bacteriuria in p o s t m e n o p a u s a l w o m e n i s 0 . 0 9 a n d i n premenopausal women 0.03,(13) giving a sample size of 50 subjects in each group. data collection t h i s s t u d y u s e d a q u e s t i o n n a i r e a s research instrument for collecting personal data (name, age, marital status, education, occupation, income), sexual activity, history of uti, incontinence, and diabetes mellitus. personal hygiene was assessed by means of a questionnaire consisting of 4 items on hygienic m e a s u r e s t a k e n b y t h e r e s p o n d e n t s i n connection with abu. sexual hygiene was assessed using a questionnaire of 4 items, comprising measures taken by the respondents in connection with sexual relations with their partner. sexual activity was measured by asking the respondents about the frequency of sexual relations with their partner within the last month. personal hygiene, sexual hygiene, and sexual activity was divided into two categories, i.e. excellent and poor. laboratory methods collection of urine specimens before collecting the urine specimens, the external genitalia were washed with soap and water, then rinsed with clean water. the initial urinary stream was discarded and only 20 ml of the midstream urine was collected in a sterile container.(14) transport of urine specimens the containers holding the urine specimens were labeled with the identity of the subject, and were subsequently placed in a thermos bottle filled with ice for transport to the microbiology laboratory of the medical faculty, trisakti university within 4 hours after collection. the urine specimens that could not be transported in time were stored in a cooling box at the study location and were sent to the laboratory within 24 hours. micobiological culture upon arrival at the laboratory the urine specimens were directly processed according to standard conventional methods.(1) volumes of 0.01 and 0.001 ml noncentrifuged urine were plated on blood agar and mcconkey agar. the agar plates were then incubated under aerobic conditions at 350 c for 20-24 hours, after which a count was made of the growing colonies, and the mean number of colonies per ml urine was calculated. identification of the isolates were done according to standard methods. colony counts of <10.000 cfu/ml were considered not indicative of bacteriuria; colony counts in the range of 10.000 – 100.000 cfu/ml were probably due to contamination; while colony counts of >100.000 cfu/ml definitely indicated bacteriuria.(14) abu is a microbiologic diagnosis on urine specimens collected with minimal contamination and rapidly transported to the laboratory to limit bacterial growth. ambarwati, elfani, margastutie, et al personal hygiene of asymptomatic bacteriuria 57 univ med vol.30 no.1 data analysis u n i v a r i a t e a n a l y s i s w a s u s e d f o r explaining the frequency distribution curve against the variables under study, while for determining the presence of an association of s e v e r a l k e y v a r i a b l e s w i t h a s y m t o m a t i c bacteriuria the prevalence ratio (pr) was used. logistic regression is a statistical method which can be used to estimate the probability of disease for a given covariate pattern. the results of logistic regression lead naturally to an estimate of the odds ratio but may also be used to estimate the pr.(15) the statistical tests were considered significant for p <0.05. the statiscal package for the social sciences version 15.0 (spss inc., chicago, il) was used for the statistical analysis. ethical clearance a f t e r w e r e c e i v e d p e r m i s s i o n a n d administrative clearance from the head of the pasar minggu subdistrict primary health center, the subjects meeting the inclusion criteria were entered into the study after giving written informed consent. initially the subjects were informed of the purpose and the general outlines of the study. after the subjects understood and agreed they were given an informed consent form for signing. the present study received ethical clearance from the ethics commission of the medical faculty, trisakti university. results this study recruited 100 women who were willing to participate in the study. fifty women in the age range of 40-50 years and 50 women a g e d 5 1 6 5 y e a r s w e r e a s s i g n e d t o t h e premenopausal and postmenopausal groups, respectively. mean age of the subjects was 50.5 ± 6.55 years, 54% of subjects had higher education (sma + university) and 80% still had a partner. the prevalence of diabetes was 26% (table 1). subjects with uti were 46 in number (46%) and those with excellent sexual hygiene totaled 64%. in this study the overall number o f s u b j e c t s w i t h a b u , a m o n g b o t h premenopausal and postmenopausal women, was 45 (45%). the results of bacteriological culture showed that escherichia coli was the principal causative organism of bacteriuria in women, being present in 26 subjects (57.8%). the variable n = 100 age (years) marital status single (unmarried + widowed) married educational level low (uneducated + primary school) intermediate (junior high) high (senior high + university) employment history employed unemployed menopausal status premenopause postmenopause history of diabetes mellitus present absent sexual hygiene excellent poor personal hygiene excellent poor sexual activity frequent rare/none history of uti present absent history of incontinence present absent asymptomatic bacteriuria present absent 50.5 ± 6.55 20 (20) 80 (80) 8 (8) 38 (38) 54 (54) 48 (48) 52 (52) 50 (50) 50 (50) 26 (26) 74 (74) 64 (64) 36 (36 88 (88) 12 (12) 46 (46) 54 (54) 46 (46) 54 (54) 51 (51) 49 (49) 45 (45) 55 (55) table 1. description of demographic characteristics, history of diabetes mellitus and uti, and sexual activity of the respondents 58 remaining infections were caused by klebsiella spp. (15.6%), staphylococcus epidermidis (20.0%), enterococcus spp. (2.2%), and bacillus spp. (4.4%) (table 2). ta b l e 3 p r e s e n t s a s u m m a r y o f t h e relationship between several key variables and the prevalence of abu in female subjects. in subjects with past uti the risk of bacteriuria was 3.05 times that of subjects without past uti (pr=3.05;95% c.i. 1.84-5.05). similarly, subjects with frequent sexual activity with their partners had a risk of abu of 1.6 times that of subjects who rarely or never had seksual relations with their partners (pr=1.60;95% c.i. 1.03-2.49). differring results were found in subjects with excellent sexual and personal hygiene, where the risk of abu was lower than that of subjects with poor sexual and personal hygiene. ambarwati, elfani, margastutie, et al personal hygiene of asymptomatic bacteriuria table 2. distribution of causative bacteria of abu in the respondents (n=45) table 3. relationship between several key variables and abu in female subjects abbreviations: pr: prevalence ratio; ci: confidence interval; uti: urinary tract infection 59 univ med vol.30 no.1 we constructed a series of multivariable logistic regression models based on a priori hypotheses about potential risk factors. in the final model, 5 variables continued to be associated with abu: sexual activity, sexual hygiene, personal hygiene, history of uti, and menopausal status (table 4). a history of uti (or, 0.131; 95% ci, 3.25-5.42) was strongly associated with abu. premenopausal women h a d a l o w e r r i s k o f a b u t h a n d i d postmenopausal women. discussion this study investigated potential risk factors for abu among women aged between 40 and 65 years. although abu is common, its prevalence in populations varies widely w i t h a g e , g e n d e r, a n d t h e p r e s e n c e o f g e n i t o u r i n a r y a b n o r m a l i t i e s . i n h e a l t h y women, the prevalence of bacteriuria increases w i t h a d v a n c i n g a g e , f r o m 1 % a m o n g s c h o o l g i r l s t o > 2 0 % a m o n g h e a l t h y community-dwelling women older than 80 years.13). the findings in our study were similar; there was a prevalence of abu of 45% in women in the 45-65 year age group, while postmenopausal women had a higher risk of a b u t h a n p r e m e n o p a u s a l w o m e n . a f t e r menopause, the vagina undergoes various changes that lead to vaginal colonization by uropathogenic e. coli and to the development of bacteriuria.(16) vaginal symptoms of frequent occurrence in the early postmenopausal period reportedly affect around 30% of women, increasing up to around 47% in the late postmenopausal period.(17) before menopause the vaginal fluid is acid in reaction, but subsequently the ph becomes neutral, thus enabling the proliferation of enteric bacteria a s s o c i a t e d w i t h u t i . ( 5 ) f u r t h e r m o r e , postmenopausal women reportedly also suffer from symptoms of incontinence without prior urinary bladder dysfunction or neurological abnormalities.(18) the declining estrogen level that is part of the menopausal process and is considered to be a risk factor for bacteriuria, results in the loss of the normal vaginal flora, which consists predominantly of lactobacilli. this triggers a rise in vaginal ph and increases the colonization of the introitus by e. coli.(19) the vaginal and urethral epithelia are estrogen r e s p o n s i v e , a s t h e y h a v e a c o m m o n embryological origin, and function to protect these organs against infection. however, with a d v a n c i n g a g e t h e s e e p i t h e l i a u n d e rg o degenerative changes,(4,6) causing a decline in their regulatory capacity in postmenopause and facilitating infection. however, it has been s u g g e s t e d b y h o o t o n ( 2 0 ) t h a t i n h e a l t h y postmenopausal women the incidence of uti is probably lower than in premenopausal women. in agreement with the generally accepted view that risk factors for uti in women include sexual activity, our study also found that women with frequent sexual activity had a higher risk of abu. sexual activity of the women with their partners constituted a general risk factor for the occurrence of abu. in addition, the results of our study indicate that history of past uti is one of the important risk factors for abu in female subjects aged 40 to 65 years. several studies also found that a history of previous uti is an important risk factor for asymptomatic uti in young and postmenopausal women.(7,21) t h e m o s t f r e q u e n t l y e n c o u n t e r e d microorganism causing uti in this study was escherichia coli with a prevalence of 57.8% table 4. multivariable logistic regression model with selected variables for abu abbreviations: ci: confidence interval; or: odds ratio; uti: urinary tract infection 60 in female subjects, followed by klebsiella pneumoniae. studies on the etiology of bacteriuria have found that escherichia coli is the causative agent in more than 80% of all utis and causes both abu and symptomatic uti. ( 2 2 2 4 ) uropathogenic e. coli possess various virulence traits, enabling these bacteria to attack and infect the normal urinary tract.(25) the reported virulence traits include the presence of fimbriae or pili, which function as l i g a n d s f o r g l y c o l i p i d a n d g l y c o p r o t e i n receptors in uroepithelial cells.(26-28) another risk factor for abu in the present study was a past history of urinary incontinence in the female subjects. studies on behavior in connection with uti or bacteriuria are very few in number.(22) the behavior in question include personal hygiene, sexual hygiene and sexual activity, all of which are risk factors for abu, both in p r e m e n o p a u s a l a n d p o s t m e n o p a u s a l women.(22,29) our study indicates that female subjects with excellent sexual hygiene had a far lower risk than those with poor sexual hygiene. perineal hygiene is important in women, who should be encouraged to shower after sexual intercourse to reduce colonization by fecal and perineal organisms.(30) although there are many similarities among the various uti risk factors in preand postmenopausal women, the single basic factor distinguishing both groups is their sexual activity. as stated in several reports, one of t h e d o m i n a n t u t i r i s k f a c t o r s i n premenopausal women is their sexual activity and use of spermicides.(19,22,29) diabetes melitus results in disturbances of the immune system and other organ functions, thus facilitating the occurrence of infection, among which the most frequent is uti.(10,31) it has previously been reported by several studies that advancing age with added burden of diabetes may increase the risk of uti in an individual.(29) however, our study results differred in this respect, as a history of diabetes mellitus in women was not a risk factor for abu, although we did not enquire in more detail about the treatment given to the study subjects. indeed, in subjects who were not treated regularly for their diabetes mellitus, the risk of abu was higher than in subjects under regular treatment, although there was no increase in the odds of uti in women with diabetes who did not receive treatment.(32) the fact that the rate of abu increases with advancing age may be explained by the low estrogen levels, atrophy of the vaginal epithelium, impaired voiding, and changes in personal as well as sexual hygiene.(33,34) a b u m a y a l s o r e s u l t i n l o n g t e r m cardiovascular-renal damage, the possibility of which should always be kept in mind, and a complete search for all possible causes of abu and treatment modalities should be done to reduce this important cause of chronic disease. to minimize potential bias in this study, particularly to prevent misclassification of abu, we used a case definition of bacteriuria, namely a bacterial count of >100.000 cfu/ml, t h a t i s m o r e t h a n 9 5 % s p e c i f i c f o r t h i s diagnosis.(14) since the population consisted of female patients attending the health center, the r e s u l t s m a y n o t b e g e n e r a l i z e d t o o t h e r populations. conclusions in the population of the present study, the risk factors for women in the age range of 40 65 years reflect the health status of their transitional period towards old age. history of uti and incontinence, personal and sexual hygiene, sexual activity, and menopausal status were all associated with a higher risk of abu. p r o s p e c t i v e s t u d i e s i n d i ff e r e n t p a t i e n t populations are needed to better understand the risk factors of uti. acknowledgements we wish to express our gratitude to the staff of the department of community health, ambarwati, elfani, margastutie, et al personal hygiene of asymptomatic bacteriuria 61 univ med vol.30 no.1 medical faculty, trisakti university for their guidance and support in conducting this study and writing this report. to dr. wida wildani and staff of the pasar minggu primary health 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risiko kesakitan dan kematian. lanjut usia (lansia) yang menderita dm seringkali juga mengalami penyakit lainnya, ketidakmampuan fisik, gangguan psikososial dan fungsi kognisi, serta meningkatnya pelayanan kedokteran. pada akhirnya, komplikasi yang terjadi akan mempengaruhi kualitas hidup lansia. metode penelitian ini bertujuan untuk memperoleh data tentang besarnya prevalensi dm dan hubungannya dengan kualitas hidup lansia di masyarakat. penelitian potong lintang dilakukan terhadap 101 lansia melalui wawancara terstruktur, pemeriksaan fisik, antropometri, pemeriksaan gula darah dan penilaian kualitas hidup lansia berdasarkan whoqol-bref. hasil hasil penelitian menunjukkan domain 4 (kondisi lingkungan) mempunyai rata-rata skor tertinggi (14,1 ± 1,8) dan domain 2 (kondisi psikologi) mempunyai rata-rata skor terendah (12,9 ± 1,9). lansia pria mempunyai rata-rata skor lebih tinggi pada domain kesehatan fisik dan hubungan sosial dibandingkan dengan lansia wanita. prevalensi dm sebesar 15,8% didapatkan pada kelompok usia 60-70 tahun dan lansia wanita memiliki prevalensi lebih tinggi dari lansia pria. rata-rata skor domain kondisi lingkungan lebih tinggi bermakna pada lansia yang tidak menderita dm dan rata-rata skor kesehatan fisik lebih tinggi bermakna pada lansia yang menderita obesitas. semakin besar indeks massa tubuh maka skor domain kesehatan fisik akan semakin meningkat secara bermakna. semakin tinggi kadar gula darah puasa maka skor domain kesehatan lingkungan akan semakin menurun secara bermakna. kesimpulan kadar gula darah yang tinggi menurunkan kualitas hidup lansia. intervensi pada lansia yang mengalami dm harus direncanakan untuk memperbaiki kualitas hidup lansia. kata kunci : diabetes melitus, lanjut usia, whoqol-bref *bagian ilmu penyakit dalam fakultas kedokteran universitas trisakti korespondensi dr. rita khairani, msc bagian ilmu penyakit dalam fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2707 email: rita_khairani@yahoo.co.id universa medicina 2007; 26: 18-26 universa medicina januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 19 universa medicina vol. 26 no.1 prevalence of diabetes mellitus and the relationship with quality of life of older people in the community rita khairani background diabetes mellitus (dm) is a common condition associated with greater risks of illness and death. elderly who suffered from dm will also has other disease such as physical disability, psycho-social disorder and cognitive function disorder, it is also increase the admission to hospital. finally, the complication of dm will influence the quality of life in elderly. the study objective was to obtain data of dm prevalence and the association with quality of life in older people. methods a cross-sectional study was done in 101 older people by a structured interview, physical examination, anthropometry, blood glucose measurement, and quality of life based on whoqol-bref. results this study showed that domain 4 (environment) has the highest score (14.1 ± 1.8) and domain 2 (psychological) has the lowest score (12.9 ± 1.9). men has the higher score than women in domain 1 (physical health) and domain 3 (social relationship). the prevalence of dm was 15.8% and has found in age group of 60-70 year old and women has higher prevalence than men. mean score of domain 4 (environment) was significantly higher in older people thas has no dm compare with older people with dm. mean score of domain 1 (physical health) was significantly higher in older people with obesity. the higher score of body mas index will increase domain 1 (physical health) significantly and the higher score of fasting blood glucose levels will decrease domain 4 (environment) significantly. conclusions high blood glucose levels decreased quality of life of the elderly. interventions for older adults with dm could be designed to improve the quality of life of the elderly. keywords : diabetes mellitus, prevalence, older people, whoqol-bref *department of internal medicine medical faculty trisakti university correspondence dr. rita khairani, msc department of internal medicine medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2707 email: rita_khairani@yahoo.co.id universa medicina 2007; 26: 18-26. abstract pendahuluan pertumbuhan penduduk lanjut usia (lansia) di indonesia tercatat sebagai paling pesat di dunia dalam kurun waktu tahun 1990-2025. pada tahun 2005 terdapat 17.767.709 jiwa atau 7,97% dari total populasi, akan menjadi sekitar 25,5 juta orang pada tahun 2020 atau sebesar 11,37 persen dari jumlah penduduk, itu berarti jumlah lansia di indonesia akan berada di peringkat empat dunia sesudah cina, india dan amerika serikat.(1) sedangkan di dki jakarta, berdasarkan data survei kesehatan nasional 2001 lansianya berjumlah 641.124 jiwa atau 8,64% 20 dari keseluruhan penduduk dki jakarta yang berjumlah 7.423.379 jiwa.(2) diabetes melitus (dm) merupakan keadaan yang seringkali dikaitkan dengan meningkatnya risiko kesakitan dan kematian. insidens dan prevalensi dari dm semakin meningkat dan pada tahun 2030 diperkirakan prevalensi dm di seluruh dunia akan meningkat. menjadi dua kali lipat.(3) dm tipe 2 didapatkan pada 85-90% dari total penderita dm dan seringkali ditemukan pada lansia. prevalensi dm tertinggi didapatkan pada penduduk berusia ≥60 tahun dengan i n s i d e n s t e r t i n g g i j u g a d i d a p a t k a n p a d a kelompok usia tersebut.(4) hasil penelitian the canadian study of health and aging (chsa) m e n u n j u k k a n p r e v a l e n s i d m b e s a r n y a 12,1%.(5,6) menurut survei yang dilakukan world h e a l t h o rg a n i z a t i o n ( w h o ) , i n d o n e s i a menempati urutan ke-4 dengan jumlah penderita dm terbesar di dunia setelah india, cina dan amerika serikat.(3) pada tahun 2000 terdapat 8,4 j u t a p e n d e r i t a d m d a n p a d a t a h u n 2 0 3 0 diperkirakan meningkat menjadi 21,3 juta penderita. dm telah menyebabkan sekitar 60% kematian dan 43% kesakitan di seluruh dunia.(3) l a n s i a y a n g m e n d e r i t a d m t i p e 2 membutuhkan perawatan yang berbeda dengan penderita dm tipe 2 usia yang lebih muda. penelitian eksperimental pada lansia yang menderita dm tipe 2 lebih sedikit dibandingkan kelompok usia lebih muda. dengan demikian hasil penelitian pada subjek yang lebih muda tidak dapat diekstrapolasikan pada lansia.(7) lansia yang menderita dm seringkali juga mengalami penyakit lainnya, ketidakmampuan f i s i k ( p h y s y i c a l d i s a b i l i t y ) , g a n g g u a n p s i k o s o s i a l d a n f u n g s i k o g n i s i , s e r t a meningkatnya pelayanan kedokteran.(8) pada a k h i r n y a , k o m p l i k a s i y a n g t e r j a d i a k a n mengganggu kualitas hidup lansia. sehat menurut definisi who adalah suatu keadaan sehat sejahtera yang menyeluruh secara fisik, mental dan sosial, tidak terbatas hanya terbebas dari penyakit. hal ini berarti bahwa m e n e n t u k a n s e s e o r a n g s e h a t , t i d a k h a n y a terbebas dari suatu indikasi penyakit tertentu melainkan juga sehat sejahtera yang dapat dinilai d a r i p e n g u k u r a n k u a l i t a s h i d u p y a n g d i h u b u n g k a n d e n g a n k e s e h a t a n . ( 3 ) w h o mendefinisikan kualitas hidup sebagai persepsi individu di kehidupan mereka dalam konteks k e b u d a y a a n d a n n o r m a k e h i d u p a n d a n hubungannya dengan tujuan, harapan, standar dan perhatian mereka. hal ini dipengaruhi oleh kesehatan fisik, mental, psikologi, kepercayaan pribadi dan hubungan sosial mereka dengan lingkungan sekitar.(9) pengukuran kualitas hidup menurut who (the world health organization quality of life-bref/whoqol-bref) terdiri dari dua b a g i a n , y a i t u i ) k u a l i t a s h i d u p s e c a r a keseluruhan dan ii) kualitas kesehatan secara umum. pada kualitas kesehatan secara umum terdapat 24 item yang dibagi menjadi 4 area/ domain yaitu kesehatan fisik meliputi 7 item, kondisi psikologis meliputi 6 item, hubungan sosial meliputi 3 item dan kondisi lingkungan meliputi 8 item. kuesioner whoqol telah diterima secara luas dan dapat dijadikan alat yang akurat dan sahih untuk menilai kualitas hidup lansia.(10-11) penelitian tentang hubungan antara dm dan kualitas hidup pada lansia sudah dilakukan di berbagai negara. instrumen yang digunakan untuk mengukur kualitas hidup tersebut adalah health related quality of life (hrqol). namun h a s i l p e n e l i t i a n m e n u n j u k k a n a d a n y a kontradiksi, beberapa penelitian menunjukkan a d a n y a p e r b a i k a n k u a l i t a s h i d u p s e t e l a h diberikan pengobatan dm, (12,13) sedangkan p e n e l i t i a n l a i n n y a m e n u n j u k k a n a d a n y a perbaikan fungsional.(14,15) a d a n y a h a s i l p e n e l i t i a n y a n g t i d a k konsisten ini memerlukan penelitian lebih lanjut tentang hubungan antara dm dan kualitas hidup p a d a l a n s i a d i m a s y a r a k a t . p e n e l i t i a n i n i 21 universa medicina vol. 26 no.1 bertujuan untuk memperoleh informasi tentang besarnya prevalensi dm dan hubungannya dengan kualitas hidup pada lansia di masyarakat. metode desain penelitian penelitian ini menggunakan rancangan potong lintang (cross sectional) untuk mencapai tujuan penelitian. populasi penelitian populasi penelitian adalah para lansia (usia ≥60 tahun) yang ada di kecamatan mampang kotamadya jakarta selatan sampel sampel penelitian ini merupakan sebagian dari penelitian utama yang dilakukan oleh fakultas kedokteran universitas trisakti. p e m i l i h a n s u b s a m p e l d i l a k u k a n d e n g a n menggunakan tabel random dari seluruh sampel. kriteria inklusi adalah lansia pria dan wanita y a n g b e r u s i a ≥6 0 t a h u n d a n m a s i h d a p a t beraktivitas. kriteria eksklusi adalah lansia yang m e n d e r i t a p e n y a k i t b e r a t s e h i n g g a h a r u s berbaring di tempat tidur. pengumpulan data lansia terpilih diberikan penjelasan tentang tujuan penelitian dan pemeriksaan yang akan d i l a k u k a n d a n d i m i n t a k e s e d i a a n d e n g a n menandatangani informed consent. mula-mula d i l a k u k a n w a w a n c a r a k e p a d a l a n s i a y a n g bersedia berpartisipasi dalam penelitian ini oleh petugas pewawancara. wawancara dilakukan dengan menggunakan kuesioner terstruktur yang dilakukan oleh peneliti dan paramedis dari p u s k e s m a s k e c a m a t a n m a m p a n g y a n g sebelumnya mengikuti pelatihan, dan uji coba kuesioner dilakukan untuk validasi instrumen yang digunakan. pengisian kuesioner dilakukan setiap hari senin sampai hari jumat setiap minggunya. pasien yang berhasil diwawancara diminta untuk datang ke puskesmas kecamatan m a m p a n g p a d a h a r i s a b t u . s u b y e k y a n g termasuk dalam kelompok yang diambil sampel darahnya diminta puasa 10-12 jam pada malam hari sebelum pengambilan darah. keesokan harinya pasien datang ke puskesmas untuk diperiksa tekanan darah, nadi dan pemeriksaan antropometri yang meliputi berat badan, tinggi badan, lingkar perut dan tinggi lutut. pasien yang telah puasa diambil sampel darah vena untuk pemeriksaan laboratorium yang meliputi kadar gula darah puasa dan 2 jam sesudah makan. pemeriksaan laboratorium dilakukan di laboratorium klinik x. pengumpulan data dilakukan mulai bulan november 2005 sampai dengan januari 2006. pengukuran antropometri indeks massa tubuh (imt) dan rasio lingkar p i n g g a n g / p a n g g u l ( r l p p ) a d a l a h d u a pengukuran antropometrik yang paling sering digunakan untuk menilai obesitas dan obesitas sentral.(16,17) imt diperoleh dari berat badan (kg) dibagi tinggi badan (m2). berdasarkan konsensus ahli endokrinologi indonesia (perkeni), dikatakan berat badan lebih (imt 23,0-24,9 kg/ m2), obesitas i (imt 25,0 29,9 kg/m2) dan obesitas ii (imt ≥30 kg/m 2). (18-20) obesitas abdominal bila rlpp untuk pria 0,9 dan wanita 0,8.(16) atau bila lingkar panggul pria >90 cm dan wanita >80 cm.(21) kriteria dm k r i t e r i a d i a g n o s i s d m b e r d a s a r k a n american diabetes association (ada-2004) yaitu bila gula darah puasa (gdp) ≥126 mg/dl. gdp terganggu bila gdp 100-125 mg/dl, dan normal bila gdp <100 mg/dl, atau sedang menjalani pengobatan dm.(22) whoqol-bref intsrumen yang digunakan untuk mengukur kualitas hidup lansia adalah whoqol-bref. 22 whoqol-bref terdiri dari 24 item yang dibagi dalam 4 domain yaitu kesehatan fisik (domain 1), kondisi psikologi (domain 2), h u b u n g a n s o s i a l ( d o m a i n 3 ) d a n k o n d i s i lingkungan (domain 4). item yang dinilai dari kondisi fisik meliputi: i) rasa nyeri, ii) perasaan tidak nyaman, iii) energi untuk kehidupan sehari-hari, iv) kelelahan, v) mobilitas, vi) aktivitas sehari-hari dan v) kondisi kerja. halhal yang dinilai dari kondisi psikologi meliputi: i) perasaan positif, ii) perasaan negatif, iii) kepuasan diri, iv) kemampuan berpikir dan konsentrasi, v) penampilan diri, dan vi) merasa diri berarti. item yang dinilai dari hubungan sosial meliputi: i) hubungan dengan orang lain, ii) kehidupan seksual dan iii) dukungan sosial. item yang dinilai dari kondisi lingkungan meliputi: i) sumber keuangan, ii) ketersediaan i n f o r m a s i , i i i ) r e k r e a s i d a n a k t i v i t a s menyenangkan, iv) lingkungan sekitar rumah, v) akses pelayanan kesehatan dan sosial, vi) perasaan aman, vii) lingkungan fisik dan viii) transportasi. selain keempat domain tersebut di atas terdapat dua hal yang dinilai tersendiri yaitu kualitas hidup secara umum dan kualitas kesehatan secara umum. domain tidak dihitung bila ≥20% pertanyaan tidak terjawab.(9,10) pengolahan dan analisis data pengolahan data menggunakan program k o m p u t e r st a t i s t i c a l p ro g r a m f o r s o c i a l science (spss) versi 11,0. untuk mengetahui adanya korelasi antara kadar gula darah dan kualitas hidup dipakai uji pearson’s product moment correlation. tingkat kemaknaan yang digunakan besarnya 0,05. hasil s e b a n y a k 1 0 1 l a n s i a ( 3 5 p r i a d a n 6 6 w a n i t a ) b e r h a s i l d i k u m p u l k a n s e l a m a pelaksanaan penelitian ini. rata-rata usia lansia pria besarnya 66,8 ± 5, 9 tahun dan lansia wanita 65,6 ± 6,2 tahun, rata-rata imt pada lansia pria 23,8 ± 4,6 kg/m2 dan lansia wanita 24,5 ± 3,9 kg/m2, rata-rata lingkar perut pada lansia pria 82,9 ± 12,3 cm dan lansia wanita 84,9 ± 10,7 cm, rata-rata gula darah puasa pada lansia pria 105,1 ± 27,5 mg/dl dan lansia wanita 114,76 ± 39,4 mg/dl prevalensi obesitas besarnya 41,6%, obesitas lebih banyak d i t e m u k a n p a d a l a n s i a w a n i t a ( 4 7 % ) dibandingkan lansia pria (37,1%).. prevalensi dm tipe 2 pada lansia besarnya 15,8%. dm lebih banyak ditemukan pada lansia wanita (18,2%) dibandingkan lansia pria (11,4%) (tabel 1). tabel 1. karakteristik demografik dan laboratorik dari lansia (n=111) 23 universa medicina vol. 26 no.1 dari ke empat domain whoqol-bref terlihat domain kondisi lingkungan mempunyai rata-rata skor tertinggi (14,1 ± 1,8) dan domain kondisi psikologi mempunyai rata-rata skor terendah (12,9 ± 1,9). lansia pria mempunyai rata-rata skor lebih tinggi pada semua domain dibandingkan dengan lansia wanita (tabel 2). dm tipe 2 semuanya dialami oleh lansia berusia 60-70 tahun, dan tidak didapatkan pada lansia berusia >70 tahun. prevalensi dm tipe 2 p a d a l a n s i a w a n i t a ( 7 5 , 0 % ) l e b i h b e s a r dibandingkan lansia laki-laki (25,0%). (tabel 3) r a t a r a t a s k o r k e e m p a t d o m a i n d a r i kualitas hidup lansia penderita dm tipe 2 lebih r e n d a h d i b a n d i n g k a n d e n g a n l a n s i a t i d a k menderita dm tipe 2. perbedaan ini bermakna pada domain kondisi lingkungan (p=0,044) (tabel 4). rata-rata skor kualitas hidup lansia yang mengalami obesitas tidak berbeda dengan lansia yang tidak mengalami obesitas, tetapi terdapat perbedaan bermakna pada domain kesehatan f i s i k ( p = 0 , 0 2 6 ) . r a t a r a t a s k o r d o m a i n kesehatan fisik lansia yang obese lebih besar dibandingkan lansia yang tidak obese. (tabel 5) analisis regresi ganda menunjukkan imt sebagai prediktor dari kualitas hidup lansia pada domain kesehatan fisik. semakin besar imt maka skor domain kesehatan fisik akan semakin meningkat secara bermakna. sedangkan kadar gula darah puasa merupakan prediktor dari kualitas hidup pada domain kondisi lingkungan. semakin tinggi kadar gula darah puasa maka skor domain kesehatan lingkungan akan semakin menurun secara bermakna (tabel 6). tabel 3. distribusi penderita dm tipe 2 berdasarkan usia dan jenis kelamin tabel 4. kualitas hidup pada lansia menderita dan tidak menderita dm tipe 2 * bermakna tabel 2. nilai rata-rata skor whoqol-bref berdasarkan domain pada lansia 24 pembahasan hasil penelitian menunjukkan prevalensi obesitas dan dm tipe 2 lebih banyak didapatkan pada lansia wanita dibandingkan lansia pria (47% vs 37,1%) dan (18,2% vs 11,45). weta dkk menemukan lansia overweight (imt 25,0 – 29,9 kg/m2) di jakarta utara pada tahun 19961997 sebesar 35% pada lansia pria dan 31,7 % pada lansia wanita, obesitas (imt ≥30 kg/m2) sebesar 3,8% pada lansia pria dan 7,7% pada lansia wanita.(23) di korea, kim menemukan overweight (imt 25,0 – 29,9kg/m 2) lansia sebesar 23,4% pada dewasa pria dan 24,9% pada dewasa wanita, sedangkan yang obesitas (imt ≥30 kg/m2) sebesar 1,7% pada dewasa pria dan 3,2% pada dewasa wanita.(24) dilihat dari semua domain, domain kondisi lingkungan mempunyai rata-rata skor tertinggi (14,1 ± 1,7) dan domain kondisi psikologi mempunyai rata-rata skor terendah (12,9 ± 1,9). lansia pria mempunyai rata-rata skor lebih tinggi pada domain kesehatan fisik dan hubungan sosial dibandingkan dengan lansia wanita. sedangkan domain kondisi psikologis dan kondisi lingkungan mempunyai rata-rata skor yang sama pada lansia pria dan wanita. p e n e l i t i a n n a u m a n n p a d a l a n s i a menunjukkan hasil serupa, domain kondisi lingkungan memiliki rata-rata skor tertinggi (14,5 ± 2,3) dan domain kondisi psikologi memiliki rata-rata skor terendah (11,7 ± 3,5), lansia pria mempunyai rata-rata skor lebih tinggi pada 3 domain, kecuali domain hubungan sosial.(25) prevalensi dm tipe 2 sebesar 15,8% dan semuanya didapatkan pada kelompok usia 6070 tahun dan tidak didapatkan pada lansia berusia >70 tahun. prevalensi dm tiga kali lebih besar pada lansia wanita. hasil penelitian the canadian study of health and aging (chsa) menunjukkan prevalensi dm pada lansia sebesar 12,1% dan terdapat kecenderungan menurunnya i n s i d e n s d m p a d a u s i a s a n g a t t u a ( > 8 0 tahun).(5,6) tabel 5. kualitas hidup pada lansia berdasarkan obesitas * bermakna tabel 6. imt dan gula darah puasa sebagai prediktor dari kualitas hidup pada lansia usia 60-70 tahun * bermakna 25 universa medicina vol. 26 no.1 dari uji korelasi whoqol-bref dengan l a n s i a y a n g m e n d e r i t a d m a t a u t i d a k menderita, didapatkan rata-rata skor domain kondisi lingkungan lebih tinggi bermakna pada lansia yang tidak menderita dm (p=0,044), rata-rata skor domain kesehatan fisik dan kondisi psikologik lebih tinggi pada lansia yang tidak menderita dm, sedangkan rata-rata skor domain hubungan sosial sedikit lebih tinggi pada lansia yang menderit a dm. edelman m e l a p o r k a n h a s i l p e n e l i t i a n n y a y a n g m e n g g u n a k a n i n s t r u m e n h r q o l , p a d a kelompok usia 45-64 tahun, tidak ada perbedaan bermakna antara kelompok yang menderita dm dengan yang tidak menderita dm baik pada komponen fisik atau mental.(26) kesimpulan prevalensi dm tipe 2 besarnya 15,8% dan didapatkan pada kelompok usia 60-70 tahun. semakin besar imt maka skor domain kesehatan fisik akan semakin meningkat secara bermakna. semakin tinggi kadar gula darah puasa maka skor domain kesehatan lingkungan akan semakin menurun secara bermakna. daftar pustaka 1. perkumpulan keluarga berencana indonesia. prosiding seminar dan lokakarya pengembangan pusat pelayanan lanjut usia. oktober 2001. 2. badan penelitian dan pengembangan kesehatan departemen kesehatan. survei kesehatan nasional (surkesnas) 2001. jakarta; 2002. 3. wild s, sicree r, roglic g, king h, green a. global prevalence of diabetes: estimates for the year 200 and projections for 2030. diabetes care 2004; 27: 1047-53. 4. ubink-veltmaat lj, bilo jhg, groenier kh, houweling st, rischen ro, meyboom-de jong b. prevalence, incidence and mortality of type 2 diabetes mellitus revisited: a prospective population-based study in the netherlands (zodiac-1). eur j epidemiol 2003; 18: 793-800. 5. rockwood k, philips s, tan mh, mcdowell i. prevalence of diabetes mellitus in elderly people in canada. age aging 1998; 27: 573-7. 6. rockwood k, awalt e, macknight c, mcdowell i. incidence and outcomes of diabetes mellitus in elderly people: report from the canadian study of health and aging. cmaj 2000; 162: 769-72. 7. rosenstock j. management of type 2 diabetes mellitus in the elderly. drugs aging 2001; 18: 3144. 8. gregg ew, beckles gl, williamson df, leivelle sg, langlois ja, engelgau mm, et al. diabetes and physical disablity among older u.s. adults. diabetes care 2000; 23: 1272-7. 9. world health organization. whoqol, measuring quality of life. programme on mental health. division on mental health and prevention of substance abuse. geneva: world health organization; 1997. 10. world health organization. whoqol-bref: introduction, administration, scoring and generic version of the assessment. field trial version. programme on mental health. geneva: world health organization; 1996. 11. ohaeri ju, olusina ak, al-abassi ahm. factor analytical study of the short version of the world health organization quality of life instrument. psychopathology 2004; 37: 242-8. 12. tovi j, engfeldt p. well being and symptoms in elderly type 2 with poor metabolic control: effect of insulin treatment. pract dia intl 1998; 15: 737. 13. mehta z. quality of life in type 2 diabetic patients is affected by complication but not by intensive policies to improve blood glucose or blood pressure control (upkds 37). diabetes care 1999; 22: 1125-36. 14. bourdel-marchasson i, dubroca b, manciet g, decamps a, emeriau jp, durtiques jf. prevalence of diabetes and effect on quality of life in older frence living in the community: the paquid epidemiological survey. j am geriatr soc 1997; 45: 295-301. 15. wandelll pe, tovi j. the quality of life of elderly diabetic patients. j diabetes complications 2000;14:25-30. 16. rosenthal ad, jin f, shu xo, elasy ta, chow wii, ji bt, et al. body fat distribution and risk of diabetes among chinese women. int j obes. 2004; 28: 594-9. 17. deshmukh pr, maliye c, bharambe ms, dongre ar, kaur s, garg bs. does waist-hip ratio 26 matter?a study in rural india. regional health forum 2005; 9: 28-35. 18. waspadji s. diabetes melitus: mekanisme dasar dan pengelolaannya yang rasional. in: soegondo s, soewondo p, subekti i, editors. penatalaksanaan diabetes melitus terpadu. jakarta: balai penerbit fkui; 2005. p. 29-42. 19. who expert consultation. appropriate body-mass index for asian populations and its implications for policy and intervention strategies. the lancet 2004; 363: 157-63. 20. pua yh, ong ph. anthropometric indices as screening tools for cardiovascular risk factors in singaporean women. asia pac j clin nutr 2005; 14: 74-9. 21. misra a, vikram nk, gupta r, pandey rm, wasir js, gupta vp. waist circumference cut off points and action levels for asian indians for identification of abdominal obesity. int j obes 2006; 30: 106-11. 22. american diabetes association. diagnosis and classification of diabetes mellitus. position statement. diabetes care 2006; 29: s43-8. 23. weta wi, savitri s, lukito w, lestiani l, kamso s. body fat distribution and lipids profile of elderly in southern jakarta. asia pacific j clin nutr 2000; 9: 256-63. 24. kim y, suh yk, choi h. bmi and metabolic disorders in south korean adults: 1998 korea national health and nutrition survey. obes res 2004; 12: 445-53. 25. naumann vi, byrne gj. whoqol-bref as a measure of quality of life in older patients with depression. int psychogeriatr 2004; 16: 159-73. 26. edelman d, olsen mk, dudley tk, harris ac, oddone ez. impact of diabetes screening on quality of life. diabetes care 2002; 25: 1022-6. prothrombine and activated partial thromboplastin time are prolonged in hepatic cirrhosis 26 *department of clinical pathology, faculty of medicine, diponegoro university, semarang correspondence edward kurnia setiawan limijadi department of clinical pathology, faculty of medicine, diponegoro university jl. dr sutomo no. 16-18 semarang 50244 mobile: +62838 6230 5904 email: liemsianhok@yahoo.com univ med 2016;35:26-32 doi: 10.18051/univmed.2016.v35.26-74 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 prothrombine and activated partial thromboplastin time are prolonged in hepatic cirrhosis edward kurnia setiawan limijadi*, lisyani budipradigdo suromo*, and imam budiwiyono* background chronic hepatitis and hepatic cirrhosis are chronic liver diseases that cause disorders of liver function, such as the formation of platelets and coagulation factors (prothrombin time/pt and activated partial thromboplastin time/aptt). chronic hepatitis in the long term can develop into hepatic cirrhosis. the aim of this study was to determine platelet count, pt, and aptt as indicators in the progression of chronic hepatitis towards hepatic cirrhosis. methods a cross-sectional study was conducted on 50 patients with chronic hepatitis and hepatic cirrhosis in semarang city regional general hospital, telogorejo hospital and kariadi general hospital. the platelet count was measured with a sysmex xp-100, while pt and aptt was measured with a sysmex ca-1500 coagulometer. the mann whitney test was applied to analyze the difference in platelet count, pt, and aptt between chronic hepatitis and hepatic cirrhosis. results median, minimum, and maximum values of platelet count, pt and aptt in chronic hepatitis were 284.000/µl, 210.000/µl, 390.000/µl; 10.6 sec, 9.5 sec, 13.6 sec; and 30.5 sec, 24.2 sec, 46.4 sec, respectively, and in hepatic cirrhosis they were 96.300/µl, 48.200/µl, 133.800/µl; 27.5 sec, 11.9 sec, 44.7 sec; and 55.6 sec, 31.3 sec, 72.0 sec, respectively. there was a significant difference the reduction of platelet count, and the prolongation of pt and aptt in chronic hepatitis compared to hepatic cirrhosis (p=0.000). conclusions prothrombine time and aptt were prolonged and platelet count was decreased in hepatic cirrhosis subjects. the three parameters may be used to evaluate the progression of chronic hepatitis towards hepatic cirrhosis. keywords: platelet count, pt, aptt, chronic hepatitis, hepatic cirrhosis doi: http://dx.doi.org/10.18051/univmed.2016.v35.26-32 27 univ med vol. 35 no.1 introduction chronic liver disease comprises chronic hepatitis kronis and hepatic cirrhosis.(1) with regard to hepatic cirrhosis, the morbidity and mortality rates have significantly increased in developing countries, particularly as a result of hepatitis viruses b and c.(2,3) anatomically, hepatic cirrhosis consists of extensive fibrosis with nodule formation in all parts of the liver, and not limited to one lobule.(4) chronic liver disease causes decreased liver f u n c t i o n , a n d f u r t h e r m o r e d e c r e a s e d t h r o m b o p o i e t i n s y n t h e s i s , l e a d i n g t o thrombocytopenia. thrombocytopenia in chronic l i v e r d i s e a s e i s a l s o c a u s e d b y p l a t e l e t sequestration from portal hypertension and the suppression of platelet production in the bone marrow.(5) mihai et al.(6) assert that chronic hepatitis c is associated with thrombocytopenia; the more severe the chronic hepatitis, the more severe the resulting thrombocytopenia. sylvester et al.(7) state that abnormalities of platelet count occur in liver disease associated with hepatitis virus b infection. in hepatic cirrhosis, the platelet count is lower than in chronic hepatitis. the synthesis of coagulation factors that play a role in hemostasis will be reduced in chronic liver disease. routine coagulation tests, i.e. simultaneous determination of protrombine time (pt) and activated partial thromboplastine time (aptt) are required to evaluate both procoagulant pathways. pt and aptt are prolonged in chronic liver disease.(8) tagwa et al.(9) state that pt and aptt are prolonged in patients with hepatic cirrhosis, as compared with healthy persons, indicating the occurrence of abnormalities of hemostasis in these patients.(9) ahmadhameed et al.(10) state that prolonged pt and aptt indicate lack of coagulation factors. abnormalities of platelet count, pt, and aptt in chronic hepatitis have not been much studied previously. the purpose of present study was to determine the three above-mentioned parameters as indicators of the progression of chronic hepatitis towards hepatic cirrhosis. methods study design a cross-sectional study was conducted from march to may 2014 at the ambulatory clinic and hospital wards of semarang city regional general hospital, telogorejo hospital and kariadi general hospital. study subjects the sample size for each group was calculated from the formula of the difference of two means as follows (11): at an effect size of 0.8, zα of 1.96 and zâ of 0.84, the sample size in each group was 25. s e l e c t i o n o f t h e s t u d y s u b j e c t s w a s performed by non-random consecutive sampling. chronic hepatitis was diagnosed on the basis of hbsag positive tests for more than 6 months in the case of chronic hepatitis b infection and on the basis of anti-hcv positive tests for more than 6 months in the case of chronic hepatitis c infection. hepatic cirrhosis was diagnosed from the clinical picture, comprising the signs of liver cell failure, i.e. edema, jaundice, spider nevi, pectoral alopecia, gynecomastia, liver damage, ascites, loss of pubic hair, palmar erythema, and testicular atrophy in males, and the signs of portal hypertension, i.e splenomegaly, esophageal v a r i c e s , c a p u t m e d u s a e , a s c i t e s , a n d hemorrhoids. on usg examination of the abdomen, there are nodules, irregularities, increased echogenicity, and liver atrophy, with positive hbsag and/or anti-hcv test results. the respondents in this study were 21-60 years old; male and female; body temperature within normal limits (36.8-37.2uc); normal sleeping and resting pattern, normal eating and drinking pattern (not alcoholic); regular menstruation in females, and agreeing to participate in the study. excluded from this study were patients on anticoagulants, dextran, isoniazide, quinidine, phenylbutazone; having a 2 21 βα 21 )µ(µ z(z 2nn       − + == 28 history of malignancies, joint disease, skin rash, hemophilia, anemia; menstruating females, and patients with lipemic and hemolyzed blood samples. questionnaire one study questionnaire was handed out to each of the respondents, who were requested to fill it in completely. the questionnaire contained the respondent’s identity, history of illness associated with chronic hepatitis and hepatic cirrhosis, and history of other illnesses. in case a questionnaire was not completely filled-in, the investigators completed it with data from the medical record and from an interview with the respondent. laboratory examination venous blood samples were collected from the respondents with chronic hepatitis and hepatic cirrhosis to determine the platelet count, pt, and aptt. the blood sample was collected from the median cubital vein to a volume of 5 ml, drawn into a vacutainer tube containing 3.2% edta and sodium citrate, and then homogenized. subsequently the blood sample was transported to the laboratory of dr. kariadi general hospital, semarang, for examination. the blood in the citrate tube was centrifuged at 3000 rpm for 5 minutes. the platelet count, pt, and aptt were determined without delay, using a sysmex xp-100 instrument and a sysmex ca1500 coagulometer and corresponding reagents. the normal platelet count was taken as 150.000450.000/µl, while the reference value of pt was 9.8-12.1 seconds and that of aptt was 26.437.5 seconds.(12) statistical analysis the collected data were edited, coded, and entered into the required computer program. the difference in platelet count, pt, and aptt values between chronic hepatitis and hepatic cirrhosis was tested with the mann-whitney test, since the data were not normally distributed. the degree of significance used was 0.05. ethical clearance written informed consent was obtained from all respondents in this study and the identity of the patients was kept secret. permission to conduct the study was obtained from the commission for medical research ethics, faculty of medicine, diponegoro university/dr. kariadi general hospital, semarang, under no. 609/ec/fk-rsdk/2014. results the median age of the chronic hepatitis patients was 47 years, with minimum and m a x i m u m v a l u e s o f 2 2 a n d 6 0 y e a r s , respectively, whereas the median age of the hepatic cirrhosis patients was 55 years, with minimum and maximum values of 35 and 60 years, respectively. not all study subjects knew if they had been diagnosed as having chronic hepatitis and hepatic cirrhosis. the minimum and maximum values of the duration of hepatitis in the group with chronic hepatitis were 7 months and 252 months, respectively. the minimum and maximum values of the duration of hepatitis in the group with hepatic cirrhosis were 108 months and 168 months, respectively. the minimum and maximum values of the duration of hepatic cirrhosis were 3 months and 72 months, respectively (table 1). there were more males than females among the subjects with chronic hepatitis and hepatic cirrhosis. the number of males with chronic hepatitis was 76%, which was higher than the number of females with chronic hepatitis, this being 24%. the number of males with hepatic cirrhosis was 72%, which was higher than the number of females with hepatic cirrhosis, namely 28%. the study results regarding the type of hepatitis are presented in table 1, where it is apparent that the percentage of hepatitis b was 88% and that of hepatitis c was 12%. the percentages of each type of hepatitis were identical between hepatic cirrhosis and chronic hepatitis groups. limijadi, suromo, budiwiyono prothrombine time prolonged in cirrhosis 29 univ med vol. 35 no.1 table 1. distribution of respondent characteristics by hepatitis status the platelet count in hepatic cirrhosis subjects was significantly reduced in comparison w i t h t h e c h r o n i c h e p a t i t i s s u b j e c t s . t h e prothrombine time and aptt were significantly prolonged in hepatic cirrhosis subjects in comparison with the chronic hepatitis subjects (p=0.000) (table 2). discussion the results of our study showed the median age of patients with chronic hepatitis and hepatic cirrhosis to be 47 and 55 years, respectively. essentially similar results were obtained in the study by sajjadieh et al,(13) who found that the mean age of patients with chronic hepatitis was 44.8 years, and that of patients with hepatic cirrhosis was 43.2 years. differing results were obtained in the studies by elzouki et al.(14) and fawad et al.(15) the present study is in agreement with the study of the ministry of health, republic of indonesia, showing that hepatitis has the highest prevalence of 1.4% in the age group of 45-54 years.(16) the median age of the hepatic cirrhosis subjects disagrees with that of daniel, who states that hepatic cirrhosis is more prevalent in the age group of 45-54 years.(17) the indonesian liver researchers association (perhimpunan peneliti hati indonesia, pphi) writes that the mean age of patients with hepatic cirrhosis is 44 years, which lies within the interval between the minimum and maximum.(18) this study is similar to the study by rahmat et al.(19) who found that hepatic cirrhosis is more prevalent between the ages of 36 and 60 years. table 2. differences in platelet count, pt and aptt between chronic hepatitis and hepatic cirrhosis *: statistical testing using the mann-whitney test 30 in our study there were more males with chronic hepatitis than females. this situation agrees with the study by elzouki et al.(14) who state that the prevalence of hepatitis was higher in males than in females. the ministry of health of the republic of indonesia states that males have a higher hepatitis prevalence than females.(16) in the present study, the number of male patients with hepatic cirrhosis was higher than that of female patients. this is in agreement with ratib et al.(20) who state that the prevalence of hepatic cirrhosis was higher in males than in females. infection with hepatitis b virus was more prevalent than that with hepatitis c virus in the chronic hepatitis and hepatic cirrhosis groups. this is similar to that obtained by kiah et al.(21) and marc et al.,(22) in that hepatitis b infection was more frequent than hepatitis c infection. this agrees with the report written by the national institute for health and care excellence (nice),(23) that chronic hepatitis b in its course will develop into hepatic cirrhosis and the report by kenneth (24) that patients with chronic hepatitis c will develop cirrhosis. the platelet count is lower in hepatic cirrhosis than in chronic hepatitis. the difference in platelet count between chronic hepatitis and hepatic cirrhosis in our study is similar to that found in the study by sylvester et al.,(7) namely that the platelet count in hepatic cirrhosis is significantly lower than in chronic hepatitis. tagwa et al.(9) writes that the platelet count in hepatic cirrhosis is significantly reduced, in c o m p a r i s o n w i t h t h e c o n t r o l g r o u p . thrombocytopenia may occur in hepatic cirrhosis through several mechanisms, i.e. splanchnic sequestration, reduced activity of thrombopoetin (tpo), depression of bone marrow by chronic infection with hepatitis virus, interferon antiviral therapy, and autoantibodies against platelet surface antigens.(5,25) rafique et al.(26) state that pt and aptt are significantly prolonged in hepatic cirrhosis, in comparison with chronic hepatitis, which is similar to our study results. tagwa et al.(9) also state that pt and aptt values in hepatic cirrhosis are significantly different from those in the chronic hepatitis group. bikha et al.(27) write that pt and aptt are significantly prolonged in patients with hepatic cirrhosis, in accordance with the severity of the disease. the prothrombine time and aptt in hepatic cirrhosis may be prolonged as a result of several factors. firstly, decreased factor vii followed by decreases in factors v and x and prothrombin.(28) vitamin k deficiency may occur, thus inhibiting the synthesis of factors vii, ix, and x.(29) the differences in platelet count, pt, and aptt between chronic hepatitis and hepatic cirrhosis are caused by the severity of hepatic injury, which is different in both situations. although there is abnormal liver function in chronic hepatitis, this function can still be compensated, since not all hepatic tissues are replaced by fibrous tissue. hepatic cirrhosis differs from chronic hepatitis, since in hepatic c i r r h o s i s t h e l i v e r f u n c t i o n c a n n o t b e compensated anymore, because there is a diminishing amount of hepatic tissue, while the majority has been replaced by fibrosis. the significant differences in the three parameters between chronic hepatitis and hepatic cirrhosis can be used as the basis for evaluating the condition of the patients / for follow-up. we recommend that patients with chronic hepatitis, according to the instructions of the physician, undergo periodic determination of the three parameters for evaluation of the disease, so that the development of hepatitis into hepatic cirrhosis may be known at an earlier point in time. on the basis of our study results, if the platelet count starts to decrease, the patient has entered the hepatic cirrhosis state. the prolonged prothrombine time and aptt indicate that the patient is starting to enter the hepatic cirrhosis state. one limitation of this study was that we did not perform a further evaluation of the exact timing of the decrease in platelet count, and the prolongation of pt and aptt, before the chronic limijadi, suromo, budiwiyono prothrombine time prolonged in cirrhosis 31 univ med vol. 35 no.1 hepatitis progresses to hepatic cirrhosis. further studies on the three parameters using a cohort design may be conducted to evaluate the degree of severity of chronic hepatitis before progressing to hepatic cirrhosis, so that the exact timing of the change may be known. conclusion platelet count, pt and aptt may be used to evaluate the progress of chronic hepatitis to hepatic cirrhosis. conflict of interest the investigators confirm that there was no conflict of interest. acknowledgement the investigators wish to thank the laboratory analysts and nurses at dr. kariadi general hospital, semarang, telogorejo hospital, semarang, and semarang city regional general hospital, who assisted in this study in the collection and examination of the samples. references 1. ivanova l, russev v. chronic liver disease and parenterally transmitted hepatitis viruses. eur j inflamm 2007;5:1-6. 2. ricardo r, rui tm, miguel s. classification and staging of chronic liver disease from multimodal data. ieee trans biomed eng 2012;60:133644. 3. guntur h. perspektif masa depan imunologi infeksi. surakarta: sebelas maret university press; 2006. 4. sherlock s, dooley j, editors. disease of the liver and biliary system. 11th ed. usa: blackwell science; 2002. 5. hiromitsu h, toru b, ken s, et al. management of throombocytopenia due to liver cirhosis: a riview. world j gastroenterol 2014;20:2595605. 6. olariu m, olariu c, olteanu d. thrombocytopenia in chronic hepatitis c. j gastrointestin liver dis 2010;19:381-5. 7. sylvester cn, obike i. quantitative platelet abnormalities in patients with hepatitis b virusrelated liver disease. gastroenterol res 2009;2: 344-9. 8. reverter jc. abnormal hemostasis tests and bleeding in chronic liver disease: are they related? j thromb haemos 2006;4:717-20. 9. tagwa ye, fathelrahman mh. assessment of platelets count and coagulation parameters among sudanese patients with liver cirrhosis. rmj 2013;38:215-8. 10. ahmadhamed a, samina n, saeed s, et al. an assessment of coagulation parameters in liver cirrhosis. biomedica 2006;22:74-7. 11. dawson b, trapp rg. basic & clinical biostatistics. 4th ed. new york: mc graw-hill; 2014. 12. siemens. thromborel s. germany: siemens 2008:1-2. 13. sajjadieh m, viunytska l. prothrombin time in patients with and without fibrotic chronic liver disease. int j pathol 2008;8:1-5 14. elzouki an, smeo mn, sammud m, et al. prevalence of hepatitis b and c virus infections and their related risk factors in libya: a national seroepidemiological survey. east mediterr health j 2013;19:589-99. 15. fawad k, sulaiman s, ihteshamud dq, et al. hepatitis b virus infection among different sex and age groups in pakistani punjab. virology j 2011;8:1-5. 16. badan penelitian dan pengembangan kesehatan kementrian kesehatan ri. riset kesehatan dasar (riskesdas) kementrian kesehatan ri. jakarta: badan penelitian dan pengembangan kesehatan; 2013. 17. daniel hc. chronic liver disease and cirrhosis deaths in california. california: california department of health services; 2006. 18. perhimpunan peneliti hati indonesia (pphi). sirosis hepatis; 2013. 19. rahmat h, ramli ha, elvie l. hubungan gambaran usg pada penderita sirosis hati dengan fibrosis skor di bagian radiologi rsup prof. dr. r. d. kandou manado periode januari 2013 – desember 2013. e-clinic; 2014;2:1-9. 20. ratib s, west j, crooks cj, et al. diagnosis of liver cirrhosis in england, a cohort study, 1998– 2009: a comparison with cancer. am j gastroenterol 2014;109:190-8. 21. kiah h, syarif hd, edhiwan p, et al. penatalaksanaan hepatitis b kronik. majour 2010;1:1-7. 22. marc gg, doris bs, david lt, et al. hepatology: diagnosis, management, and treatment of hepatitis c. an update 2009;49:1335-74. 32 23. national institute for health and care excellence (nice). hepatitis b chronic. united kingdom: health and social care directorate; 2013. 24. kenneth es. perspective advanced liver disease: what every hepatitis c virus treater should know hepatitis. ias-usa 2011;19:121-5. 25. nezam a, john mh, robert b, et al. thrombocytopenia associated with chronic liver disease. j hepatol 2008;48:1000-7. 26. rafique am, badar a, riaz aq. the importance of abnormal coagulation tests and management of bleeding in chronic liver disease. mc 2011;17:65-8. 27. bikha rd, mir aat, aneela ar, et al. coagulopathies in patients with liver cirrhosis. world appl sci j 2012;17:1-4. 28. jody lk. hemostatic defects in end stage liver disease. crit care clin 2005;21:563-87. 29. senzolo m, burra p, cholongitas e, et al. new insights into the coagulopathy of liver disease and liver transplantation. world j gastroenterol 2006;12:7725-36. limijadi, suromo, budiwiyono prothrombine time prolonged in cirrhosis julius 57 perbandingan agar macconkey, salmonella-shigella, dan xylose lysine deoxycholate untuk isolasi shigella dari usap dubur penderita diare julius e. surjawidjaja*, oktavianus ch. salim**, paul bukitwetan* dan murad lesmana* *bagian mikrobiologi dan **bagian ilmu kedokteran komunitas fakultas kedokteran universitas trisakti korespondensi aprof. dr. julius e. surdjawidjaja, sp.mk bagian mikrobiologi fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2601 email: fkusakti@indosat.net.id universa medicina 2007; 26: 57-63. universa medicina april-juni 2007april-juni 2007april-juni 2007april-juni 2007april-juni 2007 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 latar belakang umumnya media untuk isolasi shigella dari tinja terdiri dari media diferensial seperti macconkey (mac) dan media selektif seperti agar salmonella-shigella (ss), xylose-lysine-deoxycholate (xld), dan hektoen enteric (hea). untuk isolasi kuman enterik digunakan kombinasi media dengan selektivitas sedang dan sangat selektif, tetapi belum ada keseragaman mengenai media atau kombinasi media yang baik. penelitian ini bertujuan untuk membandingkan media mac, ss, dan xld serta mengetahui media mana yang paling sensitif untuk isolasi shigella. metode usap dubur dari penderita diare ditanamkan pada agar mac, ss dan xld. lempengagar diinkubasi pada suhu 370c selama 24 jam. koloni tersangka (nonlactose fermenting) diambil dan ditanamkan ke media biokimia untuk identifikasi shigella. uji serologi dilakukan untuk konfirmasi dengan menggunakan serum anti spesifik (difco laboratories, detroit, mi). program epi info versi 6 (center for disease control and prevention) digunakan untuk analisis statistik. hasil sebanyak 1027 usap dubur dari penderita diare dibiakkan pada agar mac, ss, dan xld. hasil isolasi untuk shigella secara keseluruhan adalah 8,4%, terdiri dari s. flexneri 6,2%, s. sonnei 1,9%, s. boydii 0,2% dan s. dysenteriae 0,2%. derajat isolasi shigella pada agar mac adalah sebesar 5,1%, pada ss 4,8%, dan pada xld 7,1%. kombinasi dari media biakan menunjukkan bahwa 6,5% dari isolat shigella diperoleh dari mac+ss, 8,1% dari mac+xld, dan 7,9% dari ss+xld. dari 86 usap dubur yang positif untuk shigella, 20 (22,7%) isolat berasal ari lempeng agar xld saja, 5 (5,8%) dari ss saja, dan 6 (7,0%) dari mac saja. kesimpulan untuk isolasi s. flexneri dan s. sonnei, xld adalah media yang paling sensitif. mac+xld merupakan kombinasi media diferensial dan selektif yang paling sensitif untuk isolasi kuman shigella. kata kunci: media biakan, shigella, diare abstrak 58 comparison of macconkey, salmonella-shigella, and xylose lysine deoxycholate agar for isolation of shigella from rectal swab of diarrheal patients julius e. surjawidjaja*a, oktavianus ch. salim**, paul bukitwetan* and murad lesmana* background generall, isolation of shigella from stool specimen required differential and selective media such as macconkey (mac), salmonella-shigella (ss), xylose-lysinedeoxycholate (xld), and hektoen enteric (hea). to obtain high recovery rate of enteric pathogens, a combination of moderately inhibitive and highly selective media is used. unfortunately, none of these media were choose as the best media by clinical laboratories. the objective of this study was to compare mac, ss, and xld media to determine its sensitivity for isolating shigella. metode rectal swab from diarrheal patients was cultured on mac, ss and xld agar and the plates were incubated at 370c, for 24 hr. suspected shigella colonies appeared as non-lactose fermenting were selected and subcultured in biochemical media for the identification. serologic test for confirmation of shigella identification was performed by using specific anti-sera from difco (difco laboratories, detroit, mi). a software package, epi info version 6, (center for disease control and prevention) was used for statistical analysis. results a total of 1027 rectal swabs from diarrheal patients were obtained and cultured on mac, ss, and xld agar. overall, shigella was isolated from 8.4% specimens, comprised of s. flexneri, 6.2%, s. sonnei, 1.9%, s. boydii, 0.2% and s. dysenteriae, 0.2%. the isolation rate for shigella on mac was 5.1%, on ss was 4.8%, and on xld was 7.1%. combination of media showed that mac+ss yield 6.5% of shigella isolates, mac+xld 8.1%, and ss+xld 7.9%. out of 86 positive rectal swab samples for shigella, 20 (22.7%) isolates were recovered from xld only, 5 (5.8%) from ss only and 6 (7.0%) from mac only. conclusion for isolation of s. flexneri and s. sonnei, xld was the most sensitive media. results of the mac+xld was the best differential combination and selective media for maximum isolation of shigella. keywords: culture media, shigella, diarrhea *department of microbiology and ** department of community medicine, medical faculty, trisakti university correspondence aprof. julius e.surjawidjaja, dr, sp.mk department of microbiology, medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2402 email: fkusakti@indosat.net.id universa medicina 2007; 26: 57-63. pendahuluan spesies shigella merupakan kuman yang telah dikenal menyebabkan diare yang serius dan setiap tahunnya menyebabkan sekitar 164,7 juta abstract kasus shigellosis di seluruh dunia.(1) di antara kuman-kuman patogen enterik penyebab diare, shigella dikenal berperan dalam kejadian diare inflamatorik dan disenteri serta menyebabkan problem kesehatan global dengan morbiditas dan surjawidjaja, salim, bukitwetan, dkk isolasi shigella dari usap dubur 59 m o r t a l i t a s y a n g t i n g g i d i n e g a r a n e g a r a berkembang.(1) masalah shigellosis di indonesia menjadi menarik oleh karena pada lima tahun terakhir shigella menduduki urutan pertama sebagai penyebab diare bakterial diikuti salmonella di tempat kedua dan vibrio cholerae di tempat ketiga, padahal sebelumnya v. cholerae adalah patogen enterik predominan yang menduduki tempat teratas sebagai etiologi diare.(2) angka isolasi shigella di indonesia berkisar antara 46%,(2,3) angka ini lebih kecil bila dibandingkan dengan yang dilaporkan dari negara berkembang lain seperti bangladesh, afrika dan malaysia.(46) mungkin angka isolasi ini harusnya lebih tinggi dari yang dilaporkan mengingat bahwa upaya biakan bakteriologi tidak dilakukan secara optimal di indonesia karena biayanya yang mahal. di samping itu, ada banyak faktor lain yang berperan di dalam sistem biakan seperti m i s a l n y a p r o s e d u r p e n g a m b i l a n s a m p e l , p e n a n g a n a n d a n t r a n s p o r t a s i s a m p e l k e laboratorium. semuanya itu menentukan hasil isolasi bakteri. pada dasarnya, konfirmasi diagnosis klinis shigellosis memerlukan isolasi dan identifikasi kuman patogen enterik tersebut. sistem ini melibatkan penggunaan media biakan, baik media diferensial maupun media selektif.(7) selama bertahun-tahun ada banyak variasi media biakan yang dirancang untuk isolasi s h i g e l l a d a n b e r b a g a i k o m b i n a s i t e l a h dilaporkan dari berbagai laboratorium. (8-10) namun demikian, beberapa di antara media tersebut ternyata justru menyebabkan hambatan terhadap pertumbuhan dari shigella sedangkan media non-selektif acapkali memungkinkan t u m b u h n y a m i k r o o r g a n i s m e n o n p a t o g e n s e h i n g g a m e n e k a n k u m a n k u m a n p a t o g e n enterik.(11) secara tradisional media untuk isolasi shigella dari tinja atau usap dubur terdiri dari m e d i a y a n g b e r s i f a t d i f e r e n s i a l s e p e r t i macconkey (mac) dan media selektif seperti agar salmonella-shigella (ss), agar xyloselysine-deoxycholate (xld), dan agar hektoen enteric (hea). umumnya digunakan kombinasi media biakan dengan selektivitas sedang dan sangat selektif untuk isolasi shigella dari p e n d e r i t a d i a r e . m e s k i p u n t e l a h b a n y a k dilaporkan penggunaan media biakan tersebut, tetapi masih belum ada kesepakatan mengenai media atau kombinasi media mana yang paling sensitif dan memberikan hasil paling tinggi serta relatif murah untuk isolasi shigella.(7-10) untuk s u a t u d a e r a h a t a u l a b o r a t o r i u m , p i l i h a n penggunaan media isolasi primer shigella dapat berbeda dari daerah atau laboratorium lainnya dan sangat tergantung pada tipe spesies yang prevalen di tempat tersebut. (11) seharusnya variasi penggunaan media ini tidak terjadi, dan ada lempeng agar media biakan yang optimal untuk seluruh tipe shigella secara universal. ini akan dapat mengurangi penggunaan media secara berlebihan yang menyebabkan mahalnya biaya untuk memproses tinja atau usap dubur dalam upaya mendeteksi adanya kuman-kuman e n t e r i k s e p e r t i s h i g e l l a . b e b e r a p a s t u d i melaporkan bahwa performa agar ss untuk i s o l a s i s p e s i e s s h i g e l l a l e b i h i n f e r i o r dibandingkan dengan mac dan xld, (11,12) namun demikian ss agar digunakan di banyak tempat dan merupakan media yang telah mapan di dalam sistem biakan untuk isolasi shigella.(7) agar ss ini untuk waktu lebih dari 10 tahun tidak pernah dievaluasi efektifitasnya terhadap shigella meskipun dari hasil-hasil laboratorium frekuensi isolasi shigella tetap rendah saja.(11,12) bahan dan cara media media yang digunakan dalam penelitian ini adalah xylose-lysine-deoxycholate (xld) agar (difco, becton dickinson, sparks, md), macconkey (mac) agar (difco, becton universa medicina vol.26 no.2 60 dickinson, sparks, md), dan salmonella shigella (ss) agar (difco, becton dickinson, s p a r k s , m d ) . m a c , s s , d a n x l d d i b u a t berdasarkan petunjuk pabrik, yang dicantumkan di masing-masing botol media tersebut. bahan pemeriksaan dan proses biakan sampel berupa usap dubur diperoleh dari penderita diare yang datang ke puskesmas mampang dan tebet. sampel diambil tanpa melihat beratnya penyakit (ringan, sedang atau berat). usap dubur diambil pada saat penderita datang berobat dan sebelum diberi pengobatan antibiotika. usap dubur dimasukkan ke dalam media transpor cary blair dan disimpan di lemari es sampai dikirimkan ke laboratorium mikrobiologi fakultas kedokteran universitas trisakti. transportasi bahan pemeriksaan (cary blair yang mengandung usap dubur) dilakukan dengan menggunakan kotak pendingin (termos). segera setelah sampai di laboratorium, usap dubur ditanamkan pada lempeng agar mac, ss and xld. lempeng-lempeng agar diinkubasi pada suhu 370c selama 24 jam. koloni-koloni tersangka (non-lactose fermenting) dipilih untuk diambil dan ditanamkan ke media biokimia untuk identifikasi dan karakterisasi.(7) uji serologi dilakukan untuk konfirmasi spesies shigella dengan menggunakan serum anti spesifik (difco laboratories, detroit, mi). biakan persemaian tidak dilakukan terhadap shigella oleh karena tidak banyak memberikan isolat tambahan d i b a n d i n g k a n d e n g a n m e t o d e b i a k a n langsung.(3,7) analisis data perbedaan derajat isolasi shigella pada masing-masing lempeng agar secara sendiris e n d i r i a t a u d a l a m g a b u n g a n ( k o m b i n a s i ) ditentukan kemaknaannya dengan menggunakan chi-square untuk membandingkan proporsi sampel positif. program epi info versi 6 (center for disease control and prevention) digunakan di dalam perhitungan statistik. hasil s e b a n y a k 1 . 0 2 7 u s a p d u b u r t e l a h dikumpulkan dari penderita diare yang datang berobat pada dua puskesmas di wilayah jakarta selatan (mampang dan tebet) antara bulan o k t o b e r 2 0 0 4 s a m p a i d e n g a n j u n i 2 0 0 6 . shigella spp. berhasil diisolasi sebesar 86 (8,4%) dari media mac+ss+xld, sedangkan media ss menghasilkan isolat shigella paling rendah (4,8%) (tabel 1). mayoritas isolat yang paling banyak dan menonjol adalah s. flexneri (6,2%), diikuti oleh s. sonnei (1,9%), dan s. dysenteriae (0,2%) serta s. boydii (0,15%) dalam jumlah yang sama besarnya (tabel 1). surjawidjaja, salim, bukitwetan, dkk isolasi shigella dari usap dubur 61 jumlah terbesar dari bahan pemeriksaan yang positif untuk s. flexneri didapatkan dari lempeng agar xld dengan angka sebesar 5,2% (53/1027) yang secara bermakna lebih besar dari pada hasil isolasi dari lempeng agar lainnya (p<0,0001) yaitu agar mac (3,7%) dan ss (3,8%). derajat isolasi untuk s. flexneri pada lempeng agar mac dan ss tidak berbeda bermakna, namun, s e c a r a n o m i n a l , j u m l a h s . f l e x n e r i y a n g didapatkan dari lempeng agar ss (39 isolat) sedikit lebih besar dari pada di mac (38 isolat). dari 86 usap dubur yang positif untuk shigella, 20 (22,7%) isolat diperoleh dari lempeng agar xld, 5 (5,8%) dari ss dan 6 (7,0%) dari mac. derajat isolasi dari s. flexneri pada kombinasi agar mac+xld (6.0%) and ss+xld (5,8%) secara bermakna lebih besar (p<0,01) dari pada derajat isolasinya pada kombinasi mac+ss ( 5 , 0 % ) , t e t a p i p a d a m a c + x l d d a n p a d a ss+xld tidak berbeda bermakna (p>0,05). derajat isolasi dari s. sonnei pada agar xld adalah 1,8%. ini secara bermakna lebih besar (p<0,0001) dari pada yang ditunjukkan oleh mac (1,0%) dan ss (0,8%) dan sama baiknya dengan kombinasi mac+ss, untuk isolasi s. sonnei. untuk agar mac dan ss, tidak ada perbedaan bermakna dalam isolasi s. sonnei (p>0,05). kombinasi lempeng agar manapun y a n g m e n g a n d u n g x l d ( m a c + x l d o r ss+xld) memperlihatkan derajat isolasi yang lebih tinggi dari s. sonnei (1,8%) dari pada kombinasi lainnya seperti mac+ss (1,2%) (p<0,001). d e r a j a t i s o l a s i d a r i s . b o y d i i d a n s . dysenteriae pada agar mac (masing-masing 0,2%) lebih besar dari pada di agar xld dan ss (masing-masing 0,1%). kecuali perbedaan bermakna (p<0,05) derajat isolasi dari s. boydi dan s. dysenteriae pada ss dan xld dengan mac, perbedaan antara lempeng agar lain tidak bermakna (p>0,05) (tabel 1). untuk isolasi s. boydii dan s. dysenteriae. kombinasi dari mac+ss dan mac+xld hanya sedikit lebih baik dari pada kombinasi ss+xld. tabel 1 juga menunjukkan bahwa di antara ketiga media b i a k a n t e r s e b u t , x l d a d a l a h y a n g p a l i n g s u p e r i o r d a n s e c a r a b e r m a k n a l e b i h b a i k (p<0,0001) dari pada mac dan ss dalam total isolasi semua jenis spesies shigella. derajat isolasi keseluruhan dari shigella pada xld adalah 7.1% (73/1027), sedangkan pada mac dan ss, masing-masing secara berurutan adalah 5 , 1 % d a n 4 , 8 % . k e p e k a a n k o m b i n a s i mac+xld dalam isolasi shigella (8,1%) hampir tidak berbeda dari kombinasi lempeng biakan mac+ss+xld yang memberikan hasil isolasi sebesar 8,4%, sedangkan mac+ss d e n g a n 6 , 5 % d e r a j a t i s o l a s i u n t u k s e m u a s h i g e l l a a d a l a h y a n g t e r b u r u k ( p < 0 , 0 0 1 ) kepekaannya dibandingkan dengan kepekaan kombinasi lempeng agar lainnya. pembahasan altwegg et al(12) melaporkan bahwa derajat isolasi untuk shigella secara keseluruhan pada lempeng agar mac dan xld, masing-masing adalah sebesar 86% dan 91% sedangkan pada ss sebesar 77%. ini sesuai dengan yang kami dapatkan pada penelitian ini di mana xld menempati urutan efisiensi biakan paling tinggi sebesar 85% (73/86) dari seluruh isolat shigella yang didapatkan, disusul dengan mac (60%) dan terakhir ss (57%). pilihan media biakan untuk isolasi shigella mungkin tidak sama untuk suatu tempat atau daerah, tergantung dari jenis shigella yang prevalen untuk daerah tersebut. suatu media yang baik dan mendukung pertumbuhan suatu galur shigella tertentu, tidak selalu efektif untuk j e n i s g a l u r s h i g e l l a l a i n n y a . ( 11 1 3 ) ketidakserasian ini sering kali menimbulkan keraguan apabila sebuah laboratorium harus memilih dan menentukan jenis media yang akan d i g u n a k a n d i d a l a m m e n g e r j a k a n b a h a n pemeriksaan untuk isolasi shigella. akan sangat universa medicina vol.26 no.2 62 ideal bilamana perbedaan di dalam pemilihan dan penggunaan media untuk shigella dapat dihilangkan dan suatu media atau kombinasi media yang umum, dapat digunakan secara s e r a g a m . h a l i n i a k a n m e n y e d e r h a n a k a n kebutuhan bahan biakan dan waktu pemeriksaan l a b o r a t o r i u m y a n g p a d a a k h i r n y a j u g a m e n u r u n k a n b i a y a . m e s k i p u n d e m i k i a n , d i a n j u r k a n a g a r u n t u k m e l a k u k a n b i a k a n shigella dari tinja atau usap dubur, selalu disertakan agar mac dalam sistem isolasi t e r s e b u t k a r e n a b e b e r a p a g a l u r s h i g e l l a mungkin mengalami hambatan pada media yang lebih selektif.(12) echeverria et al (11) melaporkan bahwa derajat isolasi dari s. flexneri secara bermakna lebih tinggi pada agar mac dari pada agar ss, sedangkan untuk s. sonnei, agar mac dan ss memberi hasil yang tidak berbeda. berbeda dengan yang mereka(11) laporkan, hasil yang kami peroleh adalah agar mac dan agar ss hampir sama dalam mendeteksi s. flexneri, akan tetapi untuk s. sonnei, mac memberikan d e r a j a t i s o l a s i y a n g s e d i k i t l e b i h t i n g g i d i b a n d i n g k a n d e n g a n a g a r s s , m e s k i p u n p e r b e d a a n i n i t i d a k b e r m a k n a ( p > 0 , 0 5 ) . s a y a n g n y a , e c h e v e r r i a e t a l ( 11 ) t i d a k memberikan data mengenai performa xld meskipun dikatakannya bahwa xld telah digunakan secara memuaskan untuk isolasi shigella. bopp, et al(7) juga mengingatkan supaya berhati-hati menggunakan media ss terutama untuk isolasi s. dysenteriae tipe i, karena agar s s t e r b u k t i m e n g h a m b a t d a n m e n e k a n pertumbuhan galur shigella ini. kecuali itu, juga dilaporkan bahwa galur shigella pembawa r-factor mengalami hambatan pada lempeng agar ss. efesiensi lempeng media biakan yang r e n d a h j u g a d i p e r l i h a t k a n o l e h a g a r s s terhadap isolasi s. sonnei. keadaan ini sesuai dengan yang kami temukan pada penelitian ini yaitu bahwa dari seluruh media biakan yang digunakan, ss hanya memberikan hasil isolasi untuk s. sonnei sebesar 0,8% saja (tabel 1). semua laporan mengenai efisiensi lempeng a g a r b i a k a n u n t u k s h i g e l l a m e n u n j u k k a n bahwa xld adalah yang paling atas urutannya sedangkan ss adalah yang paling rendah. di dalam prosedur pengerjaan bahan pemeriksaan tinja atau usap dubur untuk deteksi kumankuman enteroabacteriaceae, agar ss tidak lagi d i c a n t u m k a n s e b a g a i m e d i a y a n g direkomendasikan untuk digunakan karena nilainya tidak memadai seperti yang pernah dilaporkan selama ini.(7,14) media ss dianggap s a n g a t m e n g h a m b a t p e r t u m b u h a n d a n dilaporkan bahwa tiga perempat dari galur shigella yang diuji pada lempeng agar ss mengalami tekanan atau hambatan(11) sehingga banyak yang tidak dapat tumbuh pada lempeng agar biakan ini. penelitian kami juga menunjukkan bahwa untuk isolasi s. flexneri dan s. sonnei, agar xld adalah yang paling sensitif dibandingkan agar mac dan ss, sedangkan untuk s. boydii and s. dysenteriae, mac adalah yang terbaik. akan tetapi kesimpulan ini tidaklah pasti mengingat bahwa jumlah kedua tipe shigella yang didapatkan tersebut (s. dysenteriae dan s. boydii) hanya sedikit, masing-masing dua untuk s. dysenteriae dan s. boydii. kebanyakan laboratorium menggunakan baik media biakan diferensial maupun media selektif dalam upaya isolasi kuman shigella atau kuman dari famili enterobacteriaceae lainnya. media biakan yang paling umum digunakan adalah mac, ss dan xld. dengan penggunaan kombinasi dua media biakan diharapkan akan dapat diperoleh derajat isolasi yang cukup tinggi. pada penelitian ini, kombinasi mac+xld merupakan sistem kombinasi media biakan yang paling sensitif untuk isolasi shigella, di mana derajat isolasi yang didapatkan untuk shigella adalah 96,5% (63/86) (tabel 1). mac+xld adalah kombinasi media biakan yang dianjurkan surjawidjaja, salim, bukitwetan, dkk isolasi shigella dari usap dubur 63 digunakan untuk mendapatkan hasil isolasi shigella secara optimal dan menjadi kombinasi media biakan baku untuk isolasi shigella dari bahan pemeriksaan tinja atau usap dubur. kesimpulan biakan xld merupakan media yang paling sensitif untuk isolasi s. flexneri dan s. sonnei. mac+xld merupakan kombinasi media biakan y a n g p a l i n g s e n s i t i f u n t u k i s o l a s i k u m a n shigella. ucapan terima kasih kami mengucapkan terima kasih kepada pimpinan fakultas kedokteran universitas trisakti yang telah memberikan dana sehingga penelitian ini dapat dilaksanakan. terima kasih juga kami sampaikan kepada staf puskesmas tebet dan mampang, jakarta selatan, atas bantuan dalam pengumpulan bahan pemeriksaan dari penderita diare. daftar pustaka 1. kotloff kl, winickoff jp, ivanoff b, clemens jd, swerdlow dl, sansonetti pj, et al. global burden of shigella infections: implications for vaccine development and implementation of control strategies. bull who 1999; 77: 651-66. 2 oyofo ba., lesmana m, subekti d, tjaniadi p, larasati w, putri m, et al. surveillance of bacterial pathogens of diarrhea disease in indonesia. diagn microbiol infect dis 2002; 44: 227-34. 3. subekti d, oyofo ba, tjaniadi p, corwin al, larasati w, putri m, et al. shigella spp. surveillance in indonesia: the emergence or reemergence of s. dysenteriae. emerg infect dis 2001; 7: 1-4. 4. khan ai, huq s, malek ma, hossein mi, talukder ka, faruque as, et al. shigella serotypes among hospitalized patients in urban bangladesh and their antimicrobial resistance. epidemiol infect 2004; 132: 773-7. 5. shapiro lr, kumar l, phillips-howard p, wells jg, adcock p, brooks j, et al. antimicrobial resistant bacterial diarrhea in rural western kenya. j infect dis 2001; 183: 1701-4. 6. lee ws, puthucheary sd. bacterial pathogens isolated in childhood diarrhea in kuala lumpur the changing trend. med j malaysia 2002; 57: 12. 7. bopp ca, brenner fw, fields pj, wells jg, strockbine na. in: murray pr, baron ej, jorgensen jh, pfaller ma, yolken rh, editors. escherichia, shigella, and salmonella. manual of clinical microbiology 8th ed. washington dc: american society for microbiolog; 2003, p. 65471. 8. el-gendy a, el-ghorab n, lane em, elyazeed ra, carlin nia, mitry mm, et al. identification of shigella flexneri subserotype 1c in rural egypt. j clin microbiol 1999; 37: 873-4. 9. bogaerts j, verhaegen j, munyabikali jp, mukantabana b, lemmens p, vandeven j, et al. antimicrobial resistance and serotypes of shigella isolates in kigali, rwanda (1983-1993): increasing frequency of multiple resistance. diagn microbiol infect dis 1997; 28:165-71. 10. khalil k, khan sr, mazhar k, kauser b, lindblom g-b. occurrence and susceptibility to antibiotics of shigella species in stool of hospitalized children with bloody diarrhea in pakistan. am j trop med hyg 1998; 58: 800-3. 11. echeverria p, sethabur o, pitarangsi c. microbiology and diagnosis of infections with shigella and enteroinvasive escherichia coli. rev infect dis 1991; 13 (suppl 4): s220-5. 12. altwegg m, buser j, von graevenitz a. stool cultures for shigella spp.: improved specificity by using macconkey agar with xylose. diagn microbiol infect dis 1996; 24: 121-4. 13. maddocks s, olma t, chen s. comparison of chrom agar salmonella media and xylose-lysine desoxycholate and salmonella-shigella agars for isolation of salmonella strains from stool samples. j clin microbiol 2002; 40: 2999-3003. 14. farmer jj. enterobacteriaceae: introduction and identification. in: murray pr, baron ej, jorgensen jh, pfaller ma, yolken rh, editors. escherichia, shigella, and salmonella. manual of clinical microbiology. 8th ed. washington dc: american society for microbiolog 2003. p. 654-71. universa medicina vol.26 no.2 yefta 157 effect of range of motion and isometric strengthening exercises on grip strength and hand function in rheumatoid arthritis patients october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 universa medicina yefta daniel bastiana,*,**,a, angela bm tulaar**, surjanto hartono*** and zuljasri albar**** *department of anatomy, medical faculty, trisakti university **department of rehabilitation medicine, ***medical research unit, ****rheumatology division, department of internal medicine, faculty of medicine, university of indonesia correspondence adr yefta daniel bastian, sprm department of anatomy, medical faculty, trisakti university, jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2101 email: yeftabastian@yahoo.com univ med 2008; 27: 157-64 abstract in previous studies, duration of hand exercises in patients with rheumatoid arthritis (ra) had widely varying ranges, from 3 weeks to 4 months. an experimental study was conducted to evaluate the effect of range of motion (rom) and muscle strengthening exercises for 6 weeks on grip strength and hand function in ra patients. seventeen patients with chronic ra were randomly assigned to a treatment group and a control group. the treatment group (n=8) was given muscle strengthening exercises and heat therapy using paraffin baths 3 times a week at the hospital and rom exercises once a day at home for 6 weeks. the control group (n=9) was given only paraffin baths 3 times a week. after 6 weeks, there were significant differences in hand function (p=0.003), right and left grip strength (p=0.000 and p=0.001) and rom in the interventional group only. rom and isometric strengthening exercises significantly improved grip strength and hand function in patients with ra, while no impact was found when the patients were given paraffin baths only. in view of the small size of the study population, there is a need for further studies with larger populations. keywords: hand exercise, grip strength, rheumatoid arthritis introduction r h e u m a t o i d a r t h r i t i s ( r a ) i s a n inflammatory chronic systemic disease, which i s p a r t i c u l a r l y m a n i f e s t e d a t t h e s y n o v i a l membrane of diarthrodial joints and can result in destruction of the involved joints.(1) it is present in 0.5% to 1% of the general population, twice as often in women, and the age at disease onset is mainly between 45 and 65 years.(2) the clinical picture of ra is characterized by pain, fatigue, disability, and reduced quality of life. 158 bastian, tulaar, hartono, et al grip strength and hand function the course of the disease is often unpredictable, and the symptoms may vary from day to day. the main goals of treatment for ra are to prevent or control joint damage, prevent loss o f f u n c t i o n , a n d d e c r e a s e p a i n . ( 3 ) d e s p i t e substantial progress in the pharmacological and surgical interventions over the last decade, many patients with ra will still experience disability, pain, psychological distress, fatigue, and poor quality of life.(4) reduced levels of physical performance has been found to be associated with ra. patients with ra have been shown to have reduced muscle strength and aerobic capacity. impairments, disabilities, and h a n d i c a p s a s s o c i a t e d w i t h r a c a n b e devastating, leading to pain, activity restriction, and diminished quality of life, while placing a strain on the health care system and society.(5) besides pharmacological and surgical interventions, conventional therapies such as physical therapy, occupational therapy, and c o m p r e h e n s i v e r e h a b i l i t a t i o n a n d s e l f m a n a g e m e n t p r o g r a m s a r e c o m m o n l y a n d f r e q u e n t l y u s e d i n t e r v e n t i o n s . d e s p i t e d i f f e r e n t p a t h o p h y s i o l o g i c a l p r o c e s s e s , patients suffering from ra experience pain and a gradual decline in muscle strength, eventually resulting in loss of function and quality of life. increasing evidence shows that p h y s i c a l e x e r c i s e i m p r o v e s f u n c t i o n a n d prevents loss of function in ra.(6) owing to a fear of enh ancing joint inf lammation and accelerating cartilage destruction, it has been advocated that exercise in active ra should be restricted to gentle assisted range of motion (rom) exercises. on the contrary, exercise w a s f o u n d t o h a v e b e n e f i c i a l e f f e c t s o n f u n c t i o n , p a i n a n d m u s c l e s t r e n g t h . a n intensive exercise program consisting of rom strengthening and aerobic exercises is more effective than a conservative exercise program, a n d d o e s n o t h a v e d e l e t e r i o u s e ff e c t s o n disease activity.(7) in ra patients with active disease, an inpatient program with frequent exercise therapy was found to be superior to usual care regarding disease activity as well as muscle strength. long term high impact exercise has been proven to be beneficial regarding function and muscle strength in p a t i e n t s w i t h l o w d i s e a s e a c t i v i t y i n a n outpatient setting. in this study, exercise also did not increase disease activity.(8) hand function is recognized as being important to those diagnosed with ra, because reduction in muscle power and grip can lead t o i n c r e a s i n g d i f f i c u l t i e s i n p e r f o r m i n g activities of daily living. o’brien et al gave hand strengthening and stretching exercises as a home program for 6 months and showed significant results compared with stretching and joint protection.(9) theoretically, the effect of strengthening exercises can be expected after 2-3 weeks because of neural adaptation, but muscle adaptation itself can be seen after minimally 4 weeks of training.(10) the aim of this study was to examine the e f f e c t o f r o m i s o m e t r i c s t r e n g t h e n i n g exercises, combined with therapeutic heating using paraffin baths on grip strength and hand function in ra patients. methods research design this study was a single-blind, randomized controlled trial and conducted between january and may 2006. subjects subjects were ra patients in the subacute phase who had already been treated, aged 2070 years, male or female, meeting the criteria of the american college of rheumatology.(11) additional inclusion criteria were : (i) having joint involvement in the hand, which may be recognized from intrinsic muscle atrophy and/ 159 univ med vol.27 no.4 or a hand function index (hfi) score of 535.(12) (ii) never done structured rom and hand strengthening exercise before; and (iii) willing to be involved in the study. exclusion criteria were: (i) presence of peripheral nerve problems or muscle disease accompanied with atrophy; (ii) hand muscle weakness; (iii) sensory problems in the hand; (iv) finger amputation, open wound, fracture and contracture; (v) memory and cognitive problems. additional exclusion criteria were: (i) severe hypertension with systolic blood pressure of more than 150 mmhg and diastolic blood pressure of more than 100 mmhg; (iii) h a n d d e f o r m i t y r e l a t e d t o r a , i n c l u d i n g radioulnar or metacarpophalangeal (mcp) subluxation, boutonniere and swan neck; and (iii) doing strenuous grip activities in their activity of daily living, such as washing clothes. the study protocol was approved by the committee of medical research ethics of the faculty of medicine, university of indonesia. interventions all subjects meeting the inclusion and exclusion criteria, after having been given an explanation about the study program and the mechanisms of joint protection, were randomly assigned to the intervention group or the control group, using optimal allocation with a simple randomization. subjects in the intervention group were given heat therapy using paraffin baths 3 times a week at cipto mangunkusumo hospital, followed by isometric strengthening exercises. strengthening exercises were done b y o p p o s i n g t h e r e s i s t a n c e g i v e n b y t h e r e s e a r c h e r ’s h a n d o r b y p u t t y, w i t h o u t performing any movement of the joints. muscle contraction was sustained for 6 seconds and repeated up to 6 times for each joint, alternately for the right and left hands. every subject in this group also did rom exercises once a day at home. these were performed over the 6-week study period. subjects in the control group only had therapeutic heating using paraffin baths 3 times a week at the hospital and did not do any home exercises. all outcome assessments were undertaken at baseline and 6 weeks following randomization. outcome measures hand function was assessed using the hfi by giving a score according to the ability of the subject to do some finger activity then totally summed up. the minimum score is 4 and the maximum 42. the lower the score, the better the hand function. rom was measured using a goniometer, and grip strength (gs) of the right and left hands was measured using a modified sphygmomanometer. the sphygmomanometer was modified by rolling up the cuff and securing it within a bag made of nonstretch material so that when inflated to a specific point, the cuff attains a constant circumference of 6 inches. for each hand, gs was measured 3 times alternately. the best values were taken for each gs. gs measurements were done using a standardized protocol in which the subject had to sit with the shoulder in neutral rotation, the e l b o w f l e x e d 9 0 0, t h e f o r e a r m i n n e u t r a l position, the wrist extended 300, and the ulnar d e v i a t e d 1 5 0. t h e p r e s s u r e o f t h e sphygmomanometer should be adjusted to 20 mmhg before taking any measurements. statistical analysis descriptive statistics was done to know the distribution of the variables age, gender, education level, job and medication. changes i n o u t c o m e m e a s u r e s w e r e e x a m i n e d b y calculating 95% confidence intervals of the d i f f e r e n c e b e t w e e n b a s e l i n e a n d e n d l i n e scores. between-group differences in score changes were determined by student’s t test for unpaired samples. the significance level was set at 0.05. 160 bastian, tulaar, hartono, et al grip strength and hand function results subjects seventeen subjects were involved in this study, with eight subjects in the intervention group and nine in the control group. all subjects successfully followed the study to completion. subjects’ compliance in getting treatment in the hospital in the intervention group was 93%, while that in the control group was 79%. in the interventional group, subjects’ compliance to do rom exercises at home was 96.4%. all subjects were right handed. the age of the subjects ranged from 20 to 70 years (table 1). the majority of the subjects, totalling 14 p a t i e n t s ( 8 2 . 3 % ) w e r e f e m a l e a n d o n l y 3 patients were male. from the educational perspective, 8 subjects had an educational level of high school (47%) and 7 subjects had a master degree (41.2%). hand function h a n d f u n c t i o n b e f o r e a n d a f t e r intervention can be seen in table 2. hfi before and after intervention between the two groups showed non-significant results, in which the p value was more than 0.05. grip strength grip strength before and after intervention can be seen in table 2. gs before and after intervention between interventional and control group showed non-significant results in which the p value for right and left gs was more than 0.05. figure 1. flowchart of the number of subjects who completed the study ra patients attending rheumatology outpatient departmen (n=54) declined (n=33) : died (n=1) fail to be contacted (n=3) not interested (n=14) mobility difficulty (n=12) moved to another city (n=3) assessed for eligibility (n=21) participants randomized (n=17) not randomized (n=4) not meet inclusion criteria intervention group (n=8) control group (n=9) intervention group analysed after 6 weeks (n=8) control group analysed after 6 weeks (n=9) 161 univ med vol.27 no.4 range of motion r a n g e o f m o t i o n b e f o r e a n d a f t e r intervention can be seen in table 3. rom before and after intervention between the intervention and control group showed nonsignificant results for all joints. using the paired t-test, it was found that there was a significant improvement in rom for 12 areas of joints in the intervention group and 2 areas of joints in the control group. discussion the greatest prevalence of ra is in the 5059 year subgroup and 82.3% are female. this reconfirms the literature stating that ra is frequently found in the fourth and fifth decades and the incidence of ra is higher in females.(2) hfi was used in this study to measure hand function before and after intervention, because it has been proved to be a simple and fast tool for evaluating function and disease activity in ra.(12) between the two groups, there was no s i g n i f i c a n t d i f f e r e n c e b e f o r e a n d a f t e r intervention. in the interventional group, there was a significant difference in hand function before and after intervention (table 2), but there was none in the control group. this indicated that there were significant differences of hand function after intervention in both groups, but the difference was higher in the interventional g r o u p . h f i s h o w e d b e t t e r i m p r o v e m e n t characteristic intervention (%) n=8 control (%) n=9 age (year) 20-29 1 (12.5) 1 (11.1) 30-39 2 (25) 2 (22.2) 40-49 2 (22.2) 50-59 4 (50) 1 (11.1) 60-70 1 (12.5) 3 (33.3) sex female 7 (87.5) 7 (77.8) male 1 (12.5) 2 (22.2) education junior high school 1 (12.5) 1 (11.1) high school 4 (50) 4 (44.4) master degree 3 (37.5) 4 (44.4) occupation house wife 2 (25) 2 (22.2) retired 2 (25) 2 (22.2) civil servant 1 (11.1) teacher 3 (37.5) 2 (22.2) student 1 (11.1) unemployed 1 (12.5) 1 (11.1) medication methothrexate 8 (100) 6 (66.7) steroids 5 (62.5) 3 (33.3) nsaids* 5 (62.5) 5 (55.6) others** 2 (25) 4 (44.4) table 1. demographic and clinical data of 17 patients participating in the study at baseline *nsaids = non steroid anti inflammation drugs; **others (sulcolon and chloroquine) 162 bastian, tulaar, hartono, et al grip strength and hand function h fi = h an d fu nc tio n in de x; r g s= ri gh t g ri p st re ng th ; l g s= le ft g ri p st re ng th ; n s= n ot s ig ni fi ca nt (p >0 .0 5) in te rv en ti on g ro up c on tr ol g ro up p ar am et er b as e lin e e nd li ne m ea n di ff er en ce w it hi n gr ou p p b as e lin e e nd li ne m ea n di ff er en ce w it hi n gr ou p p m ea n di ff er en ce be tw ee n gr ou p p h fi 16 .8 8 ± 9. 46 14 .6 3 ± 10 .1 6 2. 25 ± 0 .7 0. 00 3 17 .1 1 ± 5. 75 15 .4 4 ± 4. 82 1. 67 ± 0 .9 3 0. 15 3 0. 58 ± 0 .2 3 ns r g s 89 .5 0 ± 18 .4 5 98 .0 0 ± 18 .7 3 8. 5 ± 0. 28 0. 00 0 89 .1 1 ± 15 .3 3 87 .5 6 ± 12 .7 2 1. 55 ± 2 .6 1 0. 72 5 6. 95 ± 2 .3 3 ns lg s 82 .7 5 ± 30 .6 3 91 .2 5 ± 29 .9 5 8. 5 ± 0. 68 0. 00 1 85 .3 3 ± 18 .1 9 88 .0 0 ± 13 .2 7 2. 67 ± 4 .9 2 0. 31 6 5. 83 ± 4 .2 4 ns ta bl e 2. h an d fu nc ti on a nd g ri p st re ng th b ef or e an d af te r in te rv en ti on ta bl e 3. j oi nt r an ge o f m ot io n be fo re a nd a ft er i nt er ve nt io n w =w ri st ; r =r ig ht ; l =l ef t; f= fl ex io n; e =e xt en si on ; m c p= m et ac ar po ph al an ge al ; p ip = pr ox im al in te rp ha la ng ea l; ns = no t s ig ni fi ca nt ( p > 0. 05 ) in te rv en ti on al g ro up c on tr ol g ro up p ar am et er b as e lin e e nd li ne m ea n di ff er en ce w it hi n gr ou p p b as e lin e e nd lin e m ea n di ff er en ce w it hi n gr ou p p m ea n di ff er en ce be tw ee n gr ou p p r w e 53 .7 5 ± 13 .8 2 60 .6 3 ± 15 .6 8 6. 88 ± 1 .8 6 0. 00 1 55 .0 0 ± 13 .9 2 52 .5 0 ± 10 .0 0 2. 5 ± 3. 92 0. 34 7 4. 38 ± 2 .0 6 ns r m c p1 f 61 .5 0 ± 14 .9 2 67 .5 0 ± 14 .5 3 6. 00 ± 0 .3 9 0. 01 3 58 .6 7 ± 15 .1 3 60 .3 3 ± 13 .9 6 1. 66 ± 1 .1 7 0. 60 5 4. 34 ± 0 .7 8 ns lm c p2 f 85 .0 0 ± 8. 42 90 .2 5 ± 5. 39 5. 25 ± 3 .0 3 0. 02 7 86 .0 0 ± 5. 57 88 .1 1 ± 4. 14 2. 11 ± 1 .4 3 0. 10 0 3. 14 ± 1 .6 ns lm c p2 e 20 .0 0 ± 9. 26 24 .2 5 ± 6. 45 4. 25 ± 2 .8 1 0. 08 5 20 .6 7 ± 8. 89 25 .3 3 ± 10 .0 5 4. 66 ± 1 .1 6 0. 01 9 0. 41 ± 1 .6 5 ns lm c p3 e 20 .0 0 ± 9. 26 24 .2 5 ± 6. 45 4. 25 ± 2 .8 1 0. 08 5 20 .8 9 ± 9. 06 24 .6 7 ± 9. 90 3. 78 ± 0 .8 4 0. 03 3 0. 47 ± 1 .9 7 ns lm c p4 f 84 .5 0 ± 8. 80 88 .2 5 ± 5. 90 3. 75 ± 2 .9 0. 03 5 83 .7 8 ± 9. 46 87 .2 2 ± 7. 14 3. 44 ± 2 .3 2 0. 06 0 0. 31 ± 0 .5 8 ns lm c p5 f 82 .5 0 ± 10 .9 9 88 .2 5 ± 4. 95 5. 75 ± 6 .0 4 0. 04 0 86 .4 4 ± 7. 67 86 .6 7 ± 8. 00 0. 23 ± 0 .3 3 0. 89 2 5. 52 ± 5 .7 1 ns r m c p4 e 84 .5 0 ± 8. 99 84 .7 5 ± 9. 07 0. 25 ± 0 .0 8 0. 04 4 20 .0 0 ± 8. 66 21 .7 8 ± 9. 08 1. 78 ± 0 .4 2 0. 08 6 1. 53 ± 0 .3 4 ns lm c p4 e 18 .7 5 ± 9. 91 24 .2 5 ± 6. 45 5. 5 ± 3. 46 0. 03 6 22 .0 0 ± 9. 54 24 .7 8 ± 10 .0 2 2. 78 ± 0 .4 8 0. 08 5 2. 72 ± 2 .9 8 ns r pi p3 f 99 .2 5 ± 10 .7 9 10 4. 00 ± 9 .7 2 4. 75 ± 1 .0 7 0. 03 2 10 2. 67 ± 8 .3 1 10 4. 56 ± 6 .9 1 1. 89 ± 1 .4 0. 17 6 2. 86 ± 0 .3 3 ns r pi p4 f 10 1. 00 ± 1 1. 95 10 6. 63 ± 1 1. 55 5. 63 ± 0 .4 0. 02 1 10 2. 67 ± 1 0. 10 10 7. 33 ± 5 .4 1 4. 66 ± 4 .6 9 0. 07 1 0. 97 ± 4 .2 9 ns r pi p5 f 99 .0 0 ± 8. 35 10 5. 00 ± 9 .4 4 6. 00 ± 1 .0 9 0. 01 2 10 2. 33 ± 1 0. 37 10 2. 44 ± 7 .4 5 0. 11 ± 2 .9 2 0. 96 9 5. 89 ± 1 .8 3 ns lp ip 3f 10 0. 25 ± 1 5. 51 10 4. 88 ± 1 4. 52 4. 63 ± 0 .9 9 0. 02 6 10 5. 67 ± 7 .2 8 10 8. 33 ± 4 .7 4 2. 66 ± 2 .5 4 0. 05 5 1. 97 ± 1 .5 5 ns lp ip 4f 10 0. 75 ± 1 3. 39 10 6. 75 ± 1 0. 31 6. 75 ± 3 .0 8 0. 00 2 10 5. 33 ± 7 .3 5 10 7. 44 ± 5 .5 0 2. 11 ± 1 .8 5 0. 13 0 4. 64 ± 1 .2 3 ns 163 univ med vol.27 no.4 especially by a combination of therapeutic exercises and heating. it is important to note that hfi measures the ability of the wrist and f i n g e r s t o m o v e i n t h e i r r o m , t h u s improvement in rom is indicated by a higher hfi. there was no significant improvement in right and left grip strengths between the two groups. there was a significant difference in right and left grip strengths in the interventional group (table 2), but not in the control group. it is known from the literature that strengthening exercises can have effect after a minimum of 4 weeks.(10) this was proved in the isometric strengthening of quadriceps muscle in patients with osteoarthritis of the knee.(13) other studies showed that exercise in ra patients needed a longer time to take effect, such as in the study by hakkinen for 2 years.(14,15) myositis in ra patients can result in muscle weakness, and can be confirmed by muscle biopsy, in which there is type ii muscle atrophy, acute myositis and focal necrosis. medications such as steroid agents also can result in myopathy.(16) table 1 indicates that 62.5% of subjects in the interventional group and 33.3% of subjects in the control group took steroid agents. in the control group, right gs decreased after intervention, while left gs increased. this may be due to the great variability in clinical manifestations, joint involvement, disease course a n d r e s p o n s e t o t r e a t m e n t . t h e r e w a s n o significant difference between the two groups. rom before and after intervention in the i n t e r v e n t i o n a l g r o u p s h o w e d s i g n i f i c a n t differences in 12 joints (table 3), while in the control group there were differences in only 2 joints.this may be due to the variability in baseline characteristics between the two groups. in the interventional group, rom improvement may have been the result of the heating and rom exercises that were done at home. in the control group, rom improvement could have been the result of therapeutic heating. as has been commonly recognized, the effect of heating is to improve tissue extensibility, decrease joint stiffness and pain, and help reduce infiltrate resolution in the inflammation.(17) there were no significant differences between the 2 groups in hfi, gs and rom, possibly as a result of the limited number of subjects in this study. the duration of this study which was only 6 weeks may have been one of t h e f a c t o r s r e s u l t i n g i n a n o n s i g n i f i c a n t difference in gs and rom between the two groups. moreover, rom exercises in this study were done only once a day. in this study, joint pain was not assessed. it is well-known that pain can stimulate reflex inhibition of muscular contraction, but the investigators attempted to reduce pain by giving the patient education about joint protection and b y m e a s u r i n g t h e g s u s i n g a m o d i f i e d sphygmomanometer.(18,19) conclusion r o m a n d i s o m e t r i c s t r e n g t h e n i n g exercises combined with therapeutic heating using paraffin baths for 6 weeks in ra patients can increase hand function. gs and rom better than therapeutic heating using paraffin baths alone. references 1. goronzy jj, weyand cm. arthritis rheumatoid: epidemiology, pathology, and pathogenesis. in: klippel jh, editor. primer on the rheumatic diseases. 12th ed. atlanta: arthritis foundation; 2001. p. 632-3. 2. stenstrom ch, minor ma. evidence for the benefit of aerobic and strengthening exercise in rheumatoid arthritis. arthritis rheum 2003; 49: 428–34. 3. american college of rheumatology subcommittee on rheumatoid arthritis. guidelines for the 164 bastian, tulaar, hartono, et al grip strength and hand function management of rheumatoid arthritis: 2002 update. arthritis rheum 2002; 46: 328–46. 4. astin ja, beckner w, soeken k. psychological interventions for rheumatoid arthritis: a metaanalysis of randomized controlled trials. arthritis rheum.2002; 47: 291–302. 5. kobelt g, eberhardt k, johansson b. economic consequences of the progression of rheumatoid arthritis in sweden. arthritis rheum 1999; 42: 347– 56. 6. de jong z, vliet vlieland tpm. safety of exercise in patients with rheumatoid arthritis. curr opin rheumatol 2005; 17: 177–82. 7. van den ende chm, breedveld fc, le cessie s, dijkmans bac, de mug aw, hazes jmw. effect of intensive exercise on patients with active rheumatoid arthritis: a randomised clinical trial. ann rheum dis 2000; 59: 615–21. 8. bulthuis y, drossaers-bakker kw, taal e, raskan j, oostveen j, van’t pad bp, et al. arthritis patients show long term benefits from 3 weeks intensive training directly following hospital discharge. rheumatology 2007; 46: 1712-7. 9. o’brien, av, jones p, mullis r, mulherin d, dziedzic k. conservative hand therapy treatments in rheumatoid arthritis – a randomized controlled trial. rheumatology 2006; 45: 577-83. 10. kisner c, colby la. resistance exercise. in: therapeutic exercise foundations and techniques. 4th ed. philadelphia; f.a. davis; 2002. p. 68-9. 11. arnett fc, edworthy sm, bloch da, mcshane dj, fries jf. the american rheumatism association 1987 revised criteria for the classification of rheumatoid arthritis. arthritis rheum 1988; 31: 315–24. 12. kalla aa, smith pr, brown gmm, meyers ol, chalton d. responsiveness of keitel functional index compared with laboratory measures of diseases activity in eheumatoid arthritis. br j rheumatol 1995; 34: 141-9 13. widjanantie sc. pengukuran fungsi lutut dengan time up and go test dan stair climbing test pada latihan isometrik otot kuadrisep pasien osteoartritis lutut (thesis). jakarta: program studi ilmu rehabilitasi medik, fakultas kedokteran universitas indonesia; 2006. 14. hakkinen a, sokka t, hannonen p. a home-based two year strength training period in early rheumatoid arthritis led to good long-term compliance: a fiveyear follow up. arthritis rheum 2004; 51: 56-62. 15. hakkinen a, sokka t, kautiainen h, kotaniemi a, hannonen p. sustained maintenance of exercise induced muscle strength gains and normal bone mineral density in patients with early rheumatoid arthritis: a five year follow up. ann rheum dis 2004; 63: 910-6. 16. hicks je, joe go, gerber lh. rehabilitation of the patient with inflammatory arthritis and connective tissue disease. in: delisa ja, gans bm, walsh ne, bockenek wl, frontera wr, et al, editors. 4th ed. philadelphia: lippincot williams & wilkins; 2005. p. 737. 17. basford jr. therapeutic physical agents. in: delisa ja, gans bm, walsh ne, bockenek wl, frontera wr, et al, editors. 4th ed. philadelphia: lippincot williams & wilkins; 2005. p. 255. 18. harris ed. clinical features of rheumatoid arthritis. in: ruddy s, harris ed, sledge cb, editors. kelley’s textbook of rheumatology. 6th ed. philadelphia: w.b. saunders; 2001. p. 969, 984. 19. harris ed. treatment of rheumatoid arthritis. in: ruddy s, harris ed, sledge cb, editors. kelley’s textbook of rheumatology. 6th ed. philadelphia: w.b. saunders; 2001. p. 1004-18. alvina 162 abstract menopause is a natural event to be experienced by every woman and appears to be associated with increased risk for developing type 2 diabetes mellitus. it has been postulated that a diet high in legumes may be beneficial for the prevention of glycosuria. these unique components of soy have been shown to have beneficial effects on glucose and insulin homeostasis. the objective of the present study was to examine the possibility of an association between soy food consumption and risk of glycosuria in postmenopausal women. a cross-sectional study was conducted among 94 postmenopausal women attending mampang prapatan health center, mampang prapatan subdistrict, south jakarta. anthropometric measurements were taken using standard instruments and dietary intake was assessed with a validated food-frequency questionnaire. a dipstick semiquantitative assay for urinary glucose was used to define glycosuria (positive). the results showed that 12.8% of postmenopausal women had glycosuria and mean isoflavone intake was 69.5 ± 61.4 g/d. intake of isoflavone was inversely associated with risk of glycosuria. compared to women in the lowest quintile of isoflavone intake (q1), postmenopausal women in the highest quintile (q5) had a lower risk for glycosuria of 0.30 times that of q1, which risk was however statistically not significant (or = 0.30; 95% ci = 0.02 – 3.14). in conclusion, isoflavone may play a role in the prevention of glycosuria (an important indicator of diabetes) among postmenopausal women. keywords : isoflavone, intake, glycosuria, postmenopausal women *professional study program, medical faculty, trisakti university correspondence nurmillah, sked. professional study program, medical faculty, trisakti university jl. kyai tapa no.260 grogol jakarta 11440 univ med 2010;29:114-24. soyfood consumption and risk of glycosuria in post-menopausal women nurmillah*, irena stefani yolantha*, and victor livinus* september-december, 2010september-december, 2010september-december, 2010september-december, 2010september-december, 2010 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 vol.29 no.3 universa medicina introduction s o m e f a c t s a b o u t m e n o p a u s e a r e applicable to all women, since menopause is a natural event to be experienced by every woman. the common denominator is cessation of menstrual cycles. on the other hand, in r e l a t i o n t o t h e s y m p t o m s a n d r i s k s f o r osteoporosis, cardiovascular diseases, cancer and alzheimer disease, every woman is unique. menopausal women suffer from deleterious effects of lowered estrogen levels, including r e d u c t i o n o f b o n e m a s s , m e n o p a u s a l symptoms, and hypercholesterolemia. these effects are pronounced during early post m e n o p a u s e b e c a u s e o f d r a s t i c e s t r o g e n reduction, and tend to become attenuated during late post menopause. menopause 163 univ med vol.29 no.3 appears to be associated with a decrease in p a n c r e a t i c i n s u l i n s e c r e t i o n a s w e l l a s increased insulin resistance. these changes are thought to contribute to the increased risk for developing type 2 diabetes mellitus (dm) after menopause. the prevalence of type 2 dm has been increasing rapidly worldwide;(1) thus knowledge of risk factors and protective factors associated with type 2 dm is essential for the development of prevention strategies. phytoestrogens can potentially alleviate hypoestrogen related deleterious effects. soy and isoflavone dietary supplements are regularly used by millions of north americans for s y m p t o m s a s s o c i a t e d w i t h m e n o p a u s a l t r a n s i t i o n o r f o r o t h e r p u r p o r t e d h e a l t h b e n e f i t s . ( 2 ) p h y t o e s t r o g e n s a r e n a t u r a l l y occurring plant compounds that are structurally and functionally similar to estradiol. there are four major classes of phytoestrogens: i) isoflavones, found in soy beans and soy products; ii) lignans, found in whole grains, cereals and oilseeds; iii) flavonoids, found in some fruits and legumes; and iv) coumestans found in bean and alfafa sprouts.(3) isoflavones are phytoestrogens with the most potent estrogenic activity, comprising genistein, daidzein and glycitein found in soybeans, the l a t t e r b e i n g a m a j o r d i e t a r y s o u r c e o f isoflavones. asians consume 10–100 times more isoflavones than do westerners, and osteoporosis-related fractures are less frequent in asian than western communities, possibly b e c a u s e o f t h e l a rg e q u a n t i t i e s o f phytoestrogen-rich soybeans and vegetables in the asian diet.(4) soybean, a member of the legume family, is a rich source of plant protein, fiber, vitamins, minerals, and phytoestrogens (isoflavones).(5) these unique components of soy have been shown to have beneficial effects on glucose and insulin homeostasis. animal studies have d e m o n s t r a t e d t h a t s o y p r o t e i n a n d s o y isoflavones improve glycemic control, lower insulin requirement, and increase insulin sensitivity.(6) similar benefits have also been documented in several intervention studies with soy supplements (soy protein, soy fiber, and soy isoflavones), but results have been inconsistent.(7,8) one cross-sectional study examined the effect of usual dietary intake of isoflavones on glucose metabolism and observed an inverse association with insulin level.(9) an animal study revealed that soy protein and isoflavone content could improve control of glycemic levels, and increase insulin sensitivity. similar benefits were documented in several studies with soy-derived supplements, but the results were inconsistent. the main point is that soy food has a low glycemic level, which is linked to blood glucose levels.(10,11) the present study a i m e d t o d e t e r m i n e t h e e x i s t e n c e o f a n association between isoflavone intake and the risk of glycosuria in postmenopausal women in mampang prapatan subdistrict, south jakarta. methods design of the study a cross-sectional study was conducted from 16 january 2010 to 2 february 2010 to determine the possiblity of an association between soyfood intake and glycosuria in postmenopausal women at a primary health center in mampang prapatan sub-district, south jakarta. study subjects the subjects who participated in this study were postmenopausal women attending the primary health center at mampang prapatan subdistrict, south jakarta. the inclusion criteria used in this study were: female with cessation of menstruation for one year or longer, able to communicate, and willing to participate in this study. data collection personal interviews were conducted to o b t a i n i n f o r m a t i o n o n d e m o g r a p h i c a n d 164 socioeconomic features, tobacco and alcohol u s e , p h y s i c a l a c t i v i t y, m e n s t r u a l a n d reproductive history, lifetime occupational history, as well as medical history, including diabetes mellitus, coronary heart disease, s t r o k e , h y p e r t e n s i o n , a n d o t h e r c h r o n i c diseases ever diagnosed by a physician, along with use of selected medicines, including postmenopausal hormones. anthropometric measurements, including weight and height, were performed on all study participants according to a standard protocol. body mass index (bmi) was calculated as weight in kilograms divided by the square of height in meters. bmi levels were categorized as normal weight (< 23.0 kg/m2), overweight or obese (> 2 3 . 0 k g / m 2) a c c o r d i n g t o t h e c r i t e r i a established for asian populations.(11) dietary assessment data on nutrient intake were obtained by p e r s o n a l i n t e r v i e w u s i n g a s o y f o o d q u e s t i o n n a i r e l i s t i n g 1 4 s o y f o o d i t e m s r e s p o n s i b l e f o r t h e m a j o r i t y o f s o y consumption, comprising steamed tofu, dried tofu, bandung tofu, soft tofu, steamed tempeh, tempe bacem, regular soy milk, tahu kuning, soy bean sangria, fried soy beans, steamed soy b e a n s , s o y s a u c e , t a h u b u n g k u s , f r i e d tempeh.(12,13) study participants were asked to r e c a l l t h e f r e q u e n c y o f c o n s u m p t i o n o f individual food items (number of times per day, week, month, and year) and estimated portion size, using local weight units (50 g) or natural units (cup, bowl) per unit time. the usa fda f o o d c o m p o s i t i o n ta b l e ( 1 3 ) w a s u s e d t o estimate the intake levels of isoflavone for study participants. urinary glucose test a dipstick semi-quantitative assay for random urinary glucose was performed on study participants who gave a spot urine sample. a cutoff point of greater than trace for urinary glucose was used to define glycosuria (positive) in this study. sample size the number of subjects required for this study was calculated on the basis of the formula for sample size for surveys with a prevalence of glycosuria of 0.1, alpha level of 0.05 and acceptable margin of error for proportion being estimated of 0.05.(14) the sample was taken by consecutive non-random sampling, where persons attending the health center and meeting the inclusion criteria were designated as respondents until the required sample size was obtained. statistical analysis study participants were divided into five categories according to quintile distribution of total soy protein intake (an equivalence of total soy food intake) among the study participants. the lowest quintile was treated as the reference group. logistic regression analysis was used to obtain maximum likelihood estimates of the odds ratios (ors) and their 95% confidence intervals (cis) to measure the association between soy food consumption and glycosuria. variables adjusted for multiple regression models included age, education, regular physical activity during the past 5 years, and bmi. all statistical tests were based on twosided probability using spss, version 15.0. results t h e s t u d y p a r t i c i p a n t s w e r e 9 4 postmenopausal women with mean age of 58.7 ± 7.7 years (range 43-89 years). around half of the women (59.6%) was married, 85.1% was u n e m p l o y e d , 6 8 . 1 % h a d a l o w l e v e l o f education and 97.9% did not smoke (table 1). for most respondents (40.4%) the duration of menopause was more than 10 years, normal and underweight bmi (<22.9) accounted for 50% of respondents, and 56.4% of respondents performed moderate activity (table 2). glycosuria was present in 12.8% of respondents, while mean isoflavone intake was 69.5 ± 61.4 g/d (table 3). nurmillah, yolantha, livinus soyfood consumption and glycosuria 165 univ med vol.29 no.3 table 4 shows that compared to women in the lowest quintile of isoflavone intake (q1), postmenopausal women in the highest quintile of isoflavone (q5) had a 0.30 times lower risk for glycosuria, but the difference was not statistically significant (or = 0.30;95% ci = 0.02 – 3.14). discussion in the present study, the prevalence of glycosuria in postmenopausal women with mean age of 58.7 ± 7.7 years was 12.8%, differing from that of a large cross-sectional study of chinese women, where the prevalence was 8.2%.(11) in our study, postmenopausal women with higher isoflavone intakes had a l o w e r r i s k o f g l y c o s u r i a , a l t h o u g h t h e association was not statistically significant. however, in the abovementioned chinese cross-sectional study there was a significant inverse association between soy intake and glycosuria among postmenopausal women.(16) our study found a mean isoflavone intake of 69.5 ± 61.4 g/d. in traditional asian diets, soy intake is usually high, with a mean level of 100 g/d, which corresponds to an approximate soy protein intake of 10 g/d.(15) there are few studies on the relationship between isoflavone intake and glycosuria in postmenopausal women in indonesia. glycosuria is a strong predictor of diabetes, and our results suggest a potential role for soy foods in the prevention of diabetes. although no previous study has evaluated regular consumption of soy foods in relation to the risk of diabetes, our findings are supported by the existing evidence from animal and human studies showing that several major components of soy foods have beneficial effects on glucose and insulin homeostasis. animal studies have revealed that feeding soy protein results in elevated glucose disposal rates, decreased pancreatic insulin release, and increased hepatic insulin removal.(16) although glycosuria is a powerful predictor of diabetes, its value in diagnosis of diabetes has been factors n (%) glycosu ria posi tive negative isofl avone intake (g/d) * quintile 1 quintile 2 quintile 3 quintile 4 quintile 5 12 (12.8) 82 (87.2) 69.5 ± 61.4 16.9 ± 5.8 31.2 ± 4.9 50.2 ± 8.6 87.1 ± 13.1 159.2 ± 75.0 table 3. glycosuria prevalence and isoflavone intake in postmenopausal women (n=94) * mean ± s.d. table 2. distributions of duration of menopause, bmi dan activity of respondents (n=94) factors n (%) duration of menopause (years) 1 – 5 5 – 10 > 10 body mass index ≤ 22.9 = 23.0 activity inacti ve moderate high 29 (30.9) 27 (28.7) 38 (40.4) 47 (50) 47 (50) 34 (36.2) 53 (56.4) 7 (7.4) demographic n (%) age (years)* level of education ** low medium high employmen t unemployed employed mari tal status married widowed single 58.7 ± 7.7 64 (68.1) 19 (20.2) 11 (11.7) 85.1 14.9 59.1 39.4 1.1 table 1. distribution of demographic characteristics of postmenopausal women (n=94) *mean ± sd; **low: no education, primary school; medium: junior high school; high: senior high school and academy/university 166 questioned. in a large prospective, populationbased study of middle-aged chinese women, a h i g h e r i n t a k e o f l e g u m e s , s o y b e a n s i n particular, was associated with a reduced risk of type 2 dm.(17) soy intake is generally low in western populations, which limits the ability o f e p i d e m i o l o g i c s t u d i e s t o d e t e r m i n e associations between soy intake and type 2 dm in these populations. phytoestrogens have a molecular structure similar to that of estrogens and are weakly e s t r o g e n i c i n c o n d i t i o n s o f e n d o g e n o u s estrogen depletion.(18) among postmenopausal w o m e n , p r e v i o u s s t u d i e s h a v e f o u n d a n a s s o c i a t i o n b e t w e e n i n t a k e s o f d i e t a r y isoflavone and isoflavone supplements on the one hand and improved insulin resistance on the other.(8,19) in addition, there is evidence that soy consumption in postmenopausal women also increases sex hormone-binding globulin level,(20) the latter being inversely associated with impaired glucose tolerance. in a doubleblind, randomized, placebo-controlled trial involving 203 postmenopausal chinese women aged 48 to 62 years, the investigators found that soy isoflavone supplementation for a period of one year presumably could decrease fasting glucose levels.(21) in contrast, a 6-mo r a n d o m i z e d c o n t r o l l e d t r i a l c o u l d n o t convincingly demonstrate that soy protein with or without isoflavone supplementation had favorable effects on glycemic control and insulin sensitivity among postmenopausal hong kong chinese women with prediabetes or early untreated diabetes.(22) soy isoflavones, as a major category of p h y t o e s t r o g e n , c a n b i n d t o t h e e s t r o g e n receptors á and â, although more strongly to the latter.(23) existing evidence has shown that isoflavones could act as estrogen agonists or antagonists, depending on the target tissues,(24) d o s e s a n d t y p e s o f i s o f l a v o n e s , ( 2 5 ) a n d endogenous circulating sex hormone profile.(20) a previous cross-sectional study in shanghai r e p o r t e d t h a t s o y i n t a k e w a s a s s o c i a t e d d i ff e r e n t l y w i t h g l y c o s u r i a b e t w e e n premenopausal and postmenopausal women, i n d i c a t i n g t h a t m e n o p a u s a l s t a t u s a n d endogenous estrogen levels may modify the phytoestrogen activity of soy.(11) legumes have been indirectly linked to a protective role in the development of type 2 dm as components of a prudent diet, which is associated with a lower risk of type 2 dm.(27,28) conversely, in another study, high consumption of legumes was linked to a dietary pattern associated with an increased risk of type 2 dm.(29) n e v e r t h e l e s s , o u r s t u d y h a s s e v e r a l limitations. the primary concern of this study is the accuracy of the method used to detect urinary glucose. a published study, however, has demonstrated that the dipstick assay for urinary glucose is quite accurate, showing a very good agreement with the quantitative e s t i m a t i o n ( k c o e ff i c i e n t = 0 . 8 ) . ( 3 0 ) t h e p o s s i b i l i t y o f o t h e r s o u r c e s o f b i a s i s minimized because of the use of validated ffq. owing to the inherent limitations of a crosssectional study design, the temporal sequence o f s o y f o o d i n t a k e a n d d e v e l o p m e n t o f glycosuria cannot be firmly established in this study. isoflavone intake (g/d) n or ( 95% ci) quintile 1 quintile 2 quintile 3 quintile 4 quintile 5 18 19 19 19 19 1.0 0 (r eference gr oup) 1.0 1 (0.24 – 6.13) 1.0 0 (0.23 5.72) 0.6 3 (0.16 – 4.38) 0.3 0 (0.02 – 3.14 table 4. odds ratio (or) and 95% confidence interval (ci) for glycosuria associated with isoflavone intake among postmenopausal women nurmillah, yolantha, livinus soyfood consumption and glycosuria 167 univ med vol.29 no.3 conclusions regular isoflavone intake was inversely associated with the risk of glycosuria among postmenopausal women, suggesting a potential protective effect of soy foods in the prevention of diabetes. acknowledgements we thank the health workers and the postmenopausal women of mampang prapatan subdistrict for their important contributions to the study. we also thank prof. dr. dr. adi hidayat, ms for his helpful comments and suggestions on the manuscript. references 1. zimmet p. globalization, coca-colonization and the chronic disease epidemic: can the doomsday scenario be averted? j intern med 2000;247:301– 10. 2. henderson v. isoflavones: food for thoughtful consideration. menopause 2003;10: 189-90. 3. branca f, lorezentti s. health effects of phytoestrogens. forum nutr 2005;57:100-11. 4. somekawa y, chiguchi m, ishibashi t, aso t. soy intake related to menopausal symptoms, serum lipids, and bone mineral density in postmenopausal japanese women. obstet gynecol 2001;97:109-15. 5. chester ea. soy for cardiovascular indications. am j health syst pharm 2001;58: 663–6. 6. lavigne c, marette a, jacques h. cod and soy proteins compared with casein improve glucose tolerance and insulin sensitivity in rats. am j physiol endocrinol metab 2000;278:491–500. 7. hermansen k, sondergaard m, hoie l, carstensen m, brock b. beneficial effects of a soy-based dietary supplement on lipid levels and cardiovascular risk markers in type 2 diabetic subjects. diabetes care 2001;24:228–33. 8. jayagopal v, albertazzi p, kilpatrick es, howarth em, jennings pe, hepburn da, et al. beneficial effects of soy phytoestrogen intake in postmenopausal women with type 2 diabetes. diabetes care 2002;25:1709–14. 9. goodman-gruen d, kritz-silverstein d. usual dietary isoflavone intake is associated with cardiovascular disease risk factors in postmenopausal women. j nutr 2001;131: 12026. 10. foster-powell k, holt sh, brand-miller jc. international table of glycemic index and glycemic load values. am j clin nutr 2002;76:556. 11. zhou bf. cooperative meta-analysis group of the working group on obesity in china. predictive values of body mass index and waist circumference for risk factors of certain related diseases in chinese adults: study on optimal cutoff points of body mass index and waist circumference in chinese adults. biomed environ sci 2002;15:83-96. 12. fred hutchinson cancer research center. soy food questionnaire;2002. available at: http:// www.fhcrc.org/science/shared_resources/ nutrition/other_questionnaires/soy.pdf. accessed december 12, 2009. 13. nutrient data laboratory beltsville human nutrition research center agricultural research service u.s. department of agriculture. usda. database for the isoflavone content of selected foods. release 2.0. baltimore: u.s. department of agriculture agricultural research service; 2008. 14. bartlett je ii, kotrlik jw, higgins cc. organizational research: determining appropriate sample size in survey research. info technol learn perform j 2001;19:43-50. 15. nagata c, takatsuka n, shimizu h. soy and fish oil intake and mortality in a japanese community. am j epidemiol 2002;156:824-31. 16. lavigne c, marette a, jacques h. cod and soy proteins compared with casein improve glucose tolerance and insulin sensitivity in rats. am j physiol endocrinol metab 2000;278:491-500. 17. villegas r, gao yt, yang g, li hl, elasy ta, zheng w, et al. legume and soy food intake and the incidence of type 2 diabetes in the shanghai women’s health study. am j clin nutr 2008;87: 162-7. 18. bhathena sj, velasquez mt. beneficial role of dietary phytoestrogens in obesity and diabetes. am j clin nutr 2002;76;1191-201. 19. goodman-gruen d, kritz-silverstein d. usual dietary isoflavone intake is associated with cardiovascular disease risk factors in postmenopausal women. j nutr 2001; 131:12026. 20. pino am, valladares le, palma ma, mancilla am, yanez m, albala c. dietary isoflavones affect sex hormone-binding globulin levels in postmenopausal women. j clin endocrinol metab 2000;85:2797-800. 168 21. ho sc, chen ym, ho sss, woo jlf. soy isoflavone supplementation and fasting serum glucose and lipid profile among postmenopausal chinese women: a double-blind, randomized, placebo-controlled trial. menopause 2007;14: 905-12. 22. liu zm, chen ym, ho sc, ho yp, woo j. effects of soy protein and isoflavones on glycemic control and insulin sensitivity: a 6-mo doubleblind, randomized, placebo-controlled trial in postmenopausal chinese women with prediabetes or untreated early diabetes. am j clin nutr 2010;91:1394-401. 23. morito k, hirose t, kinjo j, hirakawa t, okawa m, nohara t ,et al. interaction of phytoestrogens with estrogen receptors alpha and beta. biol pharm bull 2001; 24:351-6. 24. safe sh, pallaroni l, yoon k, gaido k, ross s, saville b, et al. toxicology of environmental estrogens. reprod fertil dev 2001;13:307-15. 25. mueller so, simon s, chae k, metzler m, korach ks. phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor alpha (eralpha) and beta (erbeta) in human cells. toxicol sci 2004;80:14–25. 26. glazier mg, bowman ma. a review of the evidence for the use of phytoestrogens as a replacement for traditional estrogen replacement therapy. arch intern med 2001;161:1161-72. 27. fung tt, willett wc, stampfer mj, manson je, hu fb. dietary patterns and the risk of coronary heart disease in women. arch intern med 2001; 161:1857-62. 28. van dam rm, rimm eb, willett wc, stampfer mj, hu fb. dietary patterns and risk for type 2 diabetes mellitus in u.s. men. ann intern med 2002;136:201-9. 29. heidemann c, hoffmann k, spranger j, klipstein-grobusch k, möhlig m, pfeiffer af, et al. a dietary pattern protective against type 2 diabetes in the european prospective investigation into cancer and nutrition (epic)–potsdam study cohort. diabetologia 2005;48:1126-34. 30. chaturvedi s, jain n, bhatia a. evaluation of semi-quantitative methods for protein and sugar estimation in urine. indian j pathol microbiol 2001;44:399-401. nurmillah, yolantha, livinus soyfood consumption and glycosuria riany 143 *department of neurology, medical faculty, trisakti university correspondence dr. riani indiyarti, sp.s department of neurology, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2806 univ med 2008; 27: 143-9 occipito-cervical meningioma in pregnancy july-september, 2008july-september, 2008july-september, 2008july-september, 2008july-september, 2008 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 abstract universa medicina riani indiyarti* meningiomas are tumors that are believed to be derived from the cells and vascular elements of the meninges, and grow intracranially or in the vertebral canal. they are most common in women. the growth of meningiomas is stimulated by female sex hormones and thus may progress more rapidly in pregnant women and in women with breast cancer. the patient was a pregnant 39-year old woman (g4p3a0) of 8 months gestation. the clinical symptoms and signs were progressive upper motor neuron quadriparesis, diminished sensory functions from the level of c2 downwards, and loss of bladder and rectal control. brain and cervical computed tomography (ct) scans done 2 months before admission showed no abnormalities. induced delivery was terminated by forceps extraction, resulting in a baby of 2,100 g with apgar score 7/9. after delivery, postcontrast magnetic resonance imaging (mri) showed a large contrast-enhancing tumor mass of intradural-extramedullary location in the right occipito-cervical region. the tumor had a meningeal tail, extended into the right posterior fossa and caudally to the level of c3, with compression of the spinal cord. the patient underwent a nontotal resection to remove a tumor that microscopically had the features of a meningioma. keywords : meningioma, pregnancy introduction a meningioma is a tumor that is derived from cellular or vascular elements of the meningeal membranes covering the brain and spinal cord. this type of tumor is the second most common of primary intracranial tumors. a r o u n d 1 5 – 3 0 % o f a l l b r a i n t u m o r s a r e meningiomas, which increase in frequency above the age of 40 years. the tumor is rarely found in children (in only 15% of all cases), and is more predominant in females than in males, in the ratio of 3 : 1. meningiomas may be located intracranially or within the vertebral canal, in a ratio of 6 : 1. around 90% of meningiomas occur in the head, on the cerebral hemispheres, at the base of the skull or the inferior aspect of the brain, such as the brain stem proximal to the spinal cord. meningiomas may also occur on the meningeal coverings of the optic nerve or i n t h e s p i n a l c o r d . ( 1 5 ) t h e m a j o r i t y o f meningiomas have an intradural-extramedullary 144 location.(6) high cervical, spinal, or intradural meningiomas are rare lesions, generating a gradual neur ological decline, with severe deficits or acute loss of spinal function.(7) the incidence of meningioma in pregnant women is comparable with that in non-pregnant women of the same age group. the clinical presentation of headache, vomiting, or seizures could be m i s d i a g n o s e d a s h y p e r e m e s i s g r a v i d a r u m during early pregnancy or as eclampsia during late pregnancy. an abnormal fundoscopic examination, visual impairment, focal seizures, and lateralizing neurological deficits support the diagnosis of an intracranial mass lesion and s h o u l d p r o m p t f u r t h e r i n v e s t i g a t i o n w i t h magnetic resonance imaging (mri) to confirm t h e d i a g n o s i s . ( 8 ) i n t h i s p a p e r, a c a s e o f meningioma in pregnancy and its diagnostic and therapeutic management is presented. case description a 39-year-old women, g4p3a0, of 8 months gestation, presented with paresis of all four e x t r e m i t i e s b e g i n n i n g t w o m o n t h s b e f o r e a d m i s s i o n . t h e p a t i e n t h a d a l r e a d y b e e n admitted to another hospital, where brain and cervical computed tomography (ct) scans were performed with normal results. subsequently the patient was treated by alternative medicine, but the symptoms became worse. there were no history of trauma, radiation therapy or a family history of a similar illness. on physical examination there were upper motor neuron quadriparesis with power of 2, physiological reflexes -/-, pathological reflexes + / + , c l o n u s + / + , h y p e s t h e s i a f r o m t h e c 2 dermatome downwards, and urinary and fecal incontinence. neurofibromatosis was not found. laboratory examination yielded a hemoglobin concentration of 10.7 g/dl, white cell count 8 x 109/l, red cell count 3.8 x 1012/l, hematocrit 0.32 l/l, platelet count 256 x 109/l, blood group o rh+, total protein 61 g/l, albumin 38 g/l, globulin 28 g/l, sgot 27 iu/l, sgpt 30 iu/l, ureum 1.1 mmol/l, creatinine 90 mmol/ l, sodium 133 mmol/l, potassium 34 mmol/l, chloride 109 mmol/l, random serum glucose 9 . 7 m m o l / l . d e l i v e r y w a s i n i t i a t e d b y i n d u c t i o n ; t h e r e w a s c o m p l e t e c e r v i c a l dilatation, but the patient could not bear down adequately and the delivery was terminated by forceps extraction; the infant weighed 2100 g and had an apgar score of 7/9. postcontrast magnetic resonance imaging (mri) performed after delivery showed an extensive contrast-enhancing tumor mass, of intradural-extramedullary location in the right occipito-cervical region, having a meningeal tail, extending into the right posterior fossa and caudally to the level of c3, accompanied by compression of the spinal cord (figure 1, figure 2). the patient underwent a nontotal resection with pathological diagnosis of meningioma. discussion m e n i n g i o m a s a r e t h e m o s t c o m m o n primary intracranial neoplasms in the adult population, representing about 24% of all brain tumors.(9) they originate from the meningeal membranes covering the brain and spinal cord. ninety percent of meningiomas are benign, whereas the remaining 10% are atypical or malignant. although designated as ‘benign’, brain tumors may cause disability or even be life-threatening. in a number of cases, benign meningiomas grow slowly and may attain a large size (depending on their location) before causing symptoms or signs. other meningiomas grow more rapidly. in most patients with meningioma, there is only one tumor, but occasionally several tumors are found at different locations in the brain and spinal cord.(1-5) 145 figure 2. sagittal mri section showing tumor with meningeal tail, extending into posterior fossa and caudally to level of c3 figure 1. axial mri section showing extensive tumor mass, with intradural extramedullary location in right ocipito-cervical region, and compression of spinal cord univ med vol.27 no.3 146 meningiomas may be graded according to the world health organization classification of tumors of the central nervous system, which uses histological grades to communicate a “stage of malignancy” that will predict biologic behavior. grade i tumors are generally well circumscribed, slowly progressing, and can often be cured by resection; grade ii lesions are typically infiltrative with low proliferation, but have a higher likelihood of recurrence; grade iii tumors are histologically malignant and generally require more aggressive adjuvant t h e r a p y ; a n d g r a d e i v t u m o r s a r e h i g h l y malignant and can be rapidly fatal.(16) the current who grading of meningioma into three categories is as follows:(15,16) i) grade i: benign meningioma does not fulfill criteria of grade ii and iii; comprises histologic variants other than clear cell, papillary and rhabdoid tumors. the most c o m m o n h i s t o l o g i c a l s u b t y p e s a r e meningothelial, fibrous (fibroplastic), and transitional (mixed) meningiomas. less common subtypes are psammomatous, a n g i o m a t o u s , m i c r o c y s t i c , s e c r e t o r y, lymphoplasmacytic-rich, and metaplastic meningiomas. grade i tumors account for the majority (90%) of all meningiomas. ii) grade ii: atypical meningioma mitotic index of 4 or more per 10 high-power f i e l d s ; o r a t l e a s t 3 o f t h e f o l l o w i n g parameters in an otherwise grade i tumor: sheeting architecture (loss of whorling and/or fascicles), small cells with high nuclear/cytoplasmic index, macronucleoli, hypercellularity, spontaneous necrosis; or brain invasion; or clear cell meningioma; or chordoid meningioma. grade ii tumors account for 5-7% of all meningiomas. iii) g r a d e i i i : a n a p l a s t i c ( o r m a l i g n a n t ) meningioma mitotic index of 20 or more p e r 1 0 h i g h p o w e r f i e l d s ; o r f r a n k anaplasia (obviously malignant cytology, eg. resemblance to sarcoma, carcinoma, or melanoma); or papillary meningioma; or r h a b d o i d m e n i n g i o m a . m a l i g n a n t meningiomas are rare, accounting for 3-5% of all meningiomas. a high grade is associated with a poor prognosis. patients with grade i meningioma usually have a good prognosis, with 80% or more patients experiencing no progressivity or recurrence of tumors after initial therapy. patients with grade ii or grade iii tumors have a higher risk of recurrence; patients with grade ii tumors will experience a recurrence within 5 y e a r s , a n d g r a d e i i i p a t i e n t s w i l l h a v e a recurrence within 2 years.(1,3) the etiology of meningioma is as yet not known with certainty, but possibly both genetic and environmental factors are of influence. conditions associated with a higher risk of m e n i n g i o m a a r e t h e f o l l o w i n g : i ) neurofibromatosis. in about 40–80% of these tumors there is a deletion in the long arm of c h r o m o s o m e 2 2 , a t t h e l o c u s o f t h e neurofibromatosis 2 (nf2) gene. nf2 is a tumor suppressor gene at 22q12 that is inactive in 40% of sporadic meningiomas. patients with nf2 and familial non-nf2 syndromes may develop multiple meningiomas, commonly at a young age; ii) radiation therapy. patients receiving radiation therapy of the head have higher risk of meningiomas, particularly 10-20 years after t h e r a p y ; i i i ) h i s t o r y o f h e a d t r a u m a . meningioma is found at the site of previous trauma; iv) female hormones and breast cancer. meningiomas are more common in women, especially postmenopausal women on hormonal r e p l a c e m e n t t h e r a p y. m e n i n g i o m a s m a y increase in size during pregnancy, and there is also an association between meningiomas and breast cancer. the occurrence of a meningioma during pregnancy is extremely rare. the signs and symptoms of a meningioma may initially appear 147 d u r i n g t h e s e c o n d o r t h i r d t r i m e s t e r o f pregnancy and then subside in the postpartum period. the most frequent symptoms of a brain tumor such as headache, visual disturbance, nausea and vomiting are often attributed to pregnancy itself and hence the diagnosis may be delayed. it is believed that the presence of h o r m o n e r e c e p t o r s , m a i n l y p r o g e s t e r o n e receptors, are responsible for the worsening symptoms of meningioma.(10,11) the fluctuations in the hormonal milieu of pregnancy as a result of increase in the blood volume, redistribution o f b o d y w a t e r b e t w e e n i n t r a c e l l u l a r a n d e x t r a c e l l u l a r f l u i d c o m p a r t m e n t s a n d t h e influence of steroid hormones may increase the tumor size and peritumor edema. m e n i n g i o m a s h a v e r e c e p t o r s f o r s e x h o r m o n e s a n d o t h e r l i g a n d s , i n c l u d i n g progesterones, estrogens, androgens, dopamines dan a receptors for platelet derived growth factor. using specific immunohistochemical and molecular biological techniques it was found that estrogen receptors are only expressed in approximately 10% of meningiomas and only at very low levels. in contrast, progesterone and androgen receptors are present in approximately t w o t h i r d s o f m e n i n g i o m a s . i n m u l t i p l e meningiomas, the number of progesterone r e c e p t o r s i s h i g h e r t h a n t h a t i n s o l i t a r y m e n i n g i o m a s . p r o g e s t e r o n e r e c e p t o r s i n meningiomas are identical to those found in mammary carcinomas.(1-4,12) the present case concerns a meningioma that occurred during p r e g n a n c y, w i t h o u t a h i s t o r y o f p r e v i o u s t r a u m a , r a d i a t i o n o r t h e p r e s e n c e o f neurofibromatosis, leading to the supposition that female hormonal factors in pregnancy had affected the progression of the tumor. meningiomas cause symptoms and signs by a number of mechanisms, namely irritation of the underlying cortex, pressure on the brain and cranial nerves, hyperostosis and/or invasion of the surrounding soft tissues, or induction of vascular injury in the brain. in general, the symptoms of meningioma consist of headache, vomiting, seizures and focal neurological deficits. the site of the lesion affects the clinical symptoms. depending on the location of the l e s i o n , a m e n i n g i o m a m a y c a u s e v i s u a l disturbances, hearing loss, changes in mental state, paralysis of the limbs, or even cause hydrocephalus, when the tumor is blocking the flow of cerebrospinal fluid.(1-5,13) in the case described here, the meningioma was located in the occipito-cervical region, with pressure on the motor pathways (corticospinal tracts) of the spinal cord, resulting in upper motor neuron quadriparesis. the tumor also exerted pressure on the sensory pathways ( s p i n o t h a l a m i c t r a c t a n d d o r s a l c o l u m n ) , causing hypesthesia from the c2 dermatome downwards and pressing on the autonomic pathways, giving rise to fecal and urinary incontinence. extramedullary tumors of the occipito-cervical region may also include neoplasms of the occipital bone, which are in general rather infrequent when compared to other bones of the skull. these occipital bone t u m o r s c o m p r i s e s u c h d i v e r s e e n t i t i e s a s chondroblastoma, ossifying fibroma, atypical juvenile epidermoid tumor, giant cell tumor in the background of von recklinghausen disease, and primary lymphoma.(13) t h e d i a g n o s i s o f m e n i n g i o m a i s e s t a b l i s h e d b y n e u r o l o g i c a l e x a m i n a t i o n , f o l l o w e d b y a n c i l l a r y p r o c e d u r e s s u c h a s c o m p u t e r i s e d t o m o g r a p h y ( c t ) , m a g n e t i c resonance imaging (mri), angiography or biopsy. in the ct scan, the lesion may appear isodense or hyperdense, and hyperostosis or erosion of bones may be found and occasionally e v e n i n t r a t u m o r a l c a l c i f i c a t i o n . a f t e r administration of contrast, the tumor will be of higher intensity than the surrounding tissues. cystic cavities may also be found, which may represent tumor necrosis, previous hemorrhage, 148 cystic degeneration, or entrapped cerebrospinal fluid. mri of meningioma yields an isointense or hypointense image on t1-weighted sequence, and will appear isointense or hyperintense on t2. enhancement occurs after administration of contrast, and occasionally a ‘dural tail’ appears. the presence of a heterogenous enhancement, irregular tumor boundaries, and edema are indicative of an anaplastic tumor. on magnetic resonance spectroscopy a choline/creatine ratio may be found illustrating the proliferative potential of the lesion; if there is a peak at 1.5 ppm, a meningioma should be suspected. on digital substraction angiography (dsa), pial vessels will be seen supplying the peripheral areas and dural vessels supplying the center of the lesion. in addition, a ‘sunburst’ appearance of enlarged ‘dural feeders’ and ‘prolonged vascular stain’ may be found. it is essential to p e r f o r m e n d o v a s c u l a r a n g i o g r a p h y f o r visualizing the tumor vasculature and impairment o f v i t a l v a s c u l a r s t r u c t u r e s . p r e o p e r a t i v e endovascular embolization in ‘vascular feeders’ of the external circulation may be beneficial, because it reduces the risk of hemorrhage. resection should be performed as soon as p o s s i b l e a f t e r e m b o l i z a t i o n t o r e d u c e t h e likelihood of revascularization of the tumor.(1-5,12) the management of meningioma includes surgical therapy, radiation therapy (stereotactic radiosurgery or stereotactic radiotherapy), or c o n s e r v a t i v e m a n a g e m e n t f o r s m a l l asymptomatic tumors, consisting of close observation without therapy. the choice of therapy is based on location of the tumor, tumor size, symptoms and signs, and age and medical condition of the patient. in a tumor indicated for operation, the therapeutic goal is total resection. however, for tumors in a virtually inaccessible location, the resection frequently cannot be performed maximally, and the patient w i l l c o n t i n u e t o h a v e c l i n i c a l s y m p t o m s . recurrence and survival rate depend on extent of tumor resection and histological grade. postoperative recurrence within 10 years may be estimated from the extent of resection according to the simpson scale: i) grade 1: total resection, including dura and skull, recurrence 9%; ii) grade 2: total resection with coagulation of dural adhesions, recurrence 19%; iii) grade 3: total resection without coagulation of dural adhesions, recurrence 29%; iv) grade 4: subtotal resection, recurrence 29%; and v) grade 5: decompression, recurrence 40%. administration of corticosteroids before a n d a f t e r o p e r a t i o n s i g n i f i c a n t l y r e d u c e s m o r t a l i t y a n d m o r b i d i t y r a t e s o f s u rg i c a l r e s e c t i o n . p a t i e n t s w i t h s u p r a t e n t o r i a l meningioma should receive anticonvulsant t h e r a p y. f o r m e d i c a l m a n a g e m e n t o f meningioma, mifepristone and hydroxyurea m a y b e g i v e n a s a n t i p r o g e s t e r o n e s . administration of cox-2 and 5-lo inhibitors is still under investigation, as is administration of temozolomide in patients with recurrent meningioma and incomplete resection. the use of interferon a as an angiostatic may also be considered. indications for radiotherapy of meningiomas are tumors unsuitable for total r e s e c t i o n , a n d r e c u r r e n t , i n o p e r a b l e , o r histologically malignant meningiomas.(1-5,14) at 32 weeks of pregnancy a consensus was reached to terminate the pregnancy, considering that the fetus would be capable of survival o u t s i d e t h e m a t e r n a l e n v i r o n m e n t . i t w a s decided to perform induction of labor to initiate uterine contractions and opening of the uterine cervix. the delivery was terminated by forceps extraction, because of inability of the patient to bear down, due to impairment of voluntary nerves from the brain to the corresponding muscles. cesarean section was not performed, due to financial considerations and the presence of quadriparesis and immobilization, leading to fears that postoperative recovery would be suboptimal. 149 the prognosis of patients undergoing total resection of meningioma is commonly quite good. however, incompletely excised malignant and multiple meningiomas tend to recur.(3) in the present case the tumor could not be totally resected due to the difficult location of the tumor, but as much as possible of the tumor was removed to relieve pressure on the brain stem a n d s p i n a l c o r d , w h i c h c o u l d h a v e f a t a l consequences. conclusions the management of a pregnant patient with a b r a i n t u m o r s h o u l d b e t a i l o r e d t o t h e individual according to the circumstances. in this patient with meningioma the pregnancy could continue to term and delivery without endangering the mother or the fetus. references 1. uptodate patient information: meningioma. 2007. available at: http//patients.uptodate.com/ topic.asp?file=cancer/10844. accessed october 4, 2007. 2. neurosurgery facts about meningiomas. available at: http://www.brighamandwomens.org/ neurosurgery/meningioma/meningiomafacts.aspx. accessed october 4, 2007. 3. wahjoepramono ej. tumor otak. cetakan pertama. tangerang: penerbit fakultas kedokteran universitas pelita harapan; 2006. 4. gilroy j. basic neurology. 3rd ed. new york: mcgraw-hill; 2000. 5. adams rd, victor m, ropper ah. principles of neurology. 7th ed. new york: mc graw-hill; 2001. 6. clarke mj, krauss we. cervical meningioma masquerading as schwannoma: two cases and review literature. j neurosurg sci 2006; 50: 41-4. 7. iacob g, dabu a, iana g, chirita ã, julea i. spinal cervical, c2-c3, intradural, meningothelial meningioma – a case report. rom j neurosurg 2004: 1: 79-80. 8. kanaan i, jallu a, kanaan h. management strategy for meningioma in pregnancy: a clinical study. skull base 2003;13: 197-203. 9. balki m, manninen ph. craniotomy for suprasellar meningioma in a 28 week pregnant woman without fetal heart rate monitoring. can j anesth 2004; 51: 573–6. 10. swensen r, kirsch w. brain neoplasms in women: a review. clin obstet gynecol 2002; 45: 904-27. 11. smith js, quiãjones-hinojosa a, harmon-smith m, bollen aw, mcdermott mw. sex steroid and growth factor profile of a meningioma associated with pregnancy. can j neurol sci 2005; 32: 122-7. 12. mayo clinic. meningioma 2007. available at: http:/ /www.mayoclinic.com/health/meningioma/ ds00901/dsection=4. acccessed october 4, 2007 13. castellano-sanchez aa, alexis jb, poppiti rj, nanes m, villalobos ee. pathologic quiz case: an unexpected occipital tumor. arch pathol lab med 2001; 125: 301-3. 14. cancer backup. meningioma. 2007. available at: http://www.cancerbackup.org.uk/cancertype/ brain/typesofbraintumor/meningioma. accessed october 4, 2007. 15. lamszus, k. meningioma pathology, genetics and biology. j neuropathol experim neurol 2004; 63: 275-86. 16. brat dj, parisi je, kleinschmidt-demasters bk, yachnis at, montine tj, boyer pj, et al. surgical neuropathology update: a review of changes introduced by the who classification of tumours of the central nervous system. 4th ed. arch pathol lab med 2008; 132: 993-1007. harsono 194 *department of neurology faculty of medicine gadjah mada university / dr. sardjito hospital yogyakarta correspondence prof. dr. harsono, sp.s(k) department of neurology, faculty of medicine gadjah mada university bulak sumur 55281 email : harsono_jombor@yahoo.com univ med 2008; 27: 194-203 the quality of life of epileptic patients october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 abstract universa medicina harsono* epilepsy has historically been surrounded by prejudice and myth, and associated with many misconceptions. even today, people with epilepsy experience psychosocial problems, especially in their relationships and employment. age, seizure severity and frequency, stigma of epilepsy, social deprivation, fear and anxiety, factors responsible for inadequate treatment, treatment gap, and healthcare needs may affect the quality of life of epileptic patients. improving the diagnosis, treatment, prevention, and social acceptability are important factors in achieving the objective of the management of epilepsy. treatment of epilepsy refers to any intervention that is intended to restore health status including quality of life. quality of life is recognized as an important outcome in epilepsy treatment. quality of life has been reported to vary across epileptic patients with different clinical, demographic, and socio-economic variables. seizure type and frequency have been found to be significant predictors of quality of life scores. for measuring quality of life in clinical practice, such as in epileptic patients, a wider range of properties is required to ensure that a measure can be used routinely. these include validity, appropriateness and acceptability, reliability, responsiveness to change, and interpretability. keywords: quality of life, epilepsy, stigma, anxiety, treatment gap introduction epilepsy is the most common serious neurological disorder affecting people with intellectual disabilities (mental retardation), with prevalence ranging from 20-40%, 30 times higher than the general population rate. threequarters of affected individuals in the general population become seizure free on anti-epileptic drug (aed) therapy. on the other hand, epilepsy in people with intellectual disabilities is more difficult to manage, although clinical guidelines have recently been developed by a working group of the international association for the scientific study of intellectual disability. chronic epilepsy may also be associated with psychiatric, behavioral and socioeconomic s e q u e l a e , a n d w i t h i n c r e a s e d r i s k o f hospitalization and failures of community care. i t i s p a r t i c u l a r l y i m p o r t a n t , t h e r e f o r e , t o 195 univ med vol.27 no.4 u n d e r s t a n d t h e f u n c t i o n a l a n d b e h a v i o r a l consequences of epilepsy in this population.(1,2) quality of life (qol) is recognized as an important outcome in epilepsy treatment, and various instruments have been developed to assess qol in epilepsy. the last 20 years has seen an increased interest in identifying the factors that affect qol for individuals with epilepsy. the great majority of this research has focused on the impact of medical interventions o n q o l . a t t h e s a m e t i m e , i n c r e a s e d understanding has developed regarding the significant impact that physical, social, and psychological factors make on the qol and life satisfaction of people with epilepsy. in an effort to better understand the relationships of the physical and psychosocial factors to each other and to qol among people with epilepsy, the authors developed and evaluated an exploratory m o d e l . t h e r e s u l t s h a v e i m p l i c a t i o n s f o r rehabilitation professionals who work with people with epilepsy.(3) typically, studies exploring the impact of epilepsy treatments on qol compare the mean score of instruments among various treatment groups and assess whether the differences are statistically significant. however, it is difficult to interpret the importance of mean changes in qol, regardless of their statistical significance. this is because aggregate data group convey no information about the number of individuals in a group who experience clinically important change. for example, when the mean change for the group is not statistically significant or when it is lower than a prespecified minimum threshold, clinicians may erroneously conclude that the treatment has no important effects.(4,5) qol has been reported to vary across e p i l e p t i c p a t i e n t s w i t h d i ff e r e n t c l i n i c a l , demographic, and socioeconomic variables. with regard to clinical variables, seizure type and frequency have been found to be significant p r e d i c t o r s o f q o l s c o r e s . t h e r o l e o f demographic variables such as age, sex, and education, as well as socioeconomic status in determining qol is less clear. in some studies a g e , s e x , a n d e d u c a t i o n d i d n o t c o r r e l a t e significantly with the qol. several studies have highlighted that patients with epilepsy are m o r e l i k e l y t o b e u n d e r e m p l o y e d o r u n e m p l o y e d , a n d h a v e l o w e r r a t e s o f marriage.(5,6) many factors influence the qol of people with epilepsy, including impact of age and seizures, stigma, social deprivation, fear a n d a n x i e t y, t h e p r e s e n c e o f c o g n i t i v e o r psychiatric problems, and others.(7) bishop and his colleagues also concluded from their study that epilepsy has a broad impact on a patient’s life, cutting across interpersonal, intrapersonal, and extrapersonal domains.(8) referring to the above description, the f o l l o w i n g d i s c u s s i o n w i l l f o c u s o n t h e tendencies of decreased qol of epileptic patients. understanding such phenomena will h e l p p h y s i c i a n s t o d o a p p r o p r i a t e a n d c o m p r e h e n s i v e m a n a g e m e n t o f e p i l e p t i c patients. the purpose of epilepsy management is not merely to maintain the patient free of seizures but also to keep the qol of the epileptic patients as defined by the world health organization (who). general considerations according to the definition of the who, “health is a state of complete physical, mental, a n d s o c i a l w e l l b e i n g a n d n o t m e r e l y t h e absence of disease or infirmity.” a disturbance of health can affect all or some aspects or subsets covered by this definition. treatment refers to any intervention that is intended to restore health.(9) the international league against epilepsy (ilae), the international bureau for epilepsy (ibe), and the who in june 1997 launched the global campaign against epilepsy. the major objective of this campaign is to bring epilepsy 196 “ o u t o f t h e s h a d o w s ” b y i m p r o v i n g t h e diagnosis, treatment, prevention, and social a c c e p t a b i l i t y o f t h i s w o r l d w i d e d i s o r d e r, especially in developing countries. it also looks for “a removal of barriers to a better qol for those with the disease”.(9,10) people with epilepsy living in europe or in north america have significant impairment in their qol. seizure severity and frequency a n d a e d s a p p e a r t o b e p a r t i c u l a r l y problematic.(10,11) qol is impaired in tunisian people with epilepsy. a prospective study was conducted of patients with refractory epilepsy being admitted to an inpatient video-eeg m o n i t o r i n g u n i t . t h e i m p a c t o f c l i n i c a l variables (age, sex, marital status, seizure f r e q u e n c y, d u r a t i o n a n d t y p e o f s e i z u r e d i s o r d e r, s e i z u r e l o c a l i z a t i o n , n u m b e r o f aeds, depression) on qol was analyzed. this study showed that depression was a powerful predictor of qol.(12) the influencing factors differ from the previously published data. s e v e r a l p o s s i b l e r e a s o n s s u c h a s f a m i l y support and cultural and religious beliefs are p r o p o s e d t o e x p l a i n t h e c r o s s c u l t u r a l differences.(13) meanwhile, epidemiologic data on qol of epileptic patients in indonesia are not yet available. measurement of qol the underlying reason for using qol measures in clinical practice is to ensure that treatment plans and evaluations focus on the patient rather than the disease. qol is not the only way to measure patient-centered outcome; measures of disability, social interaction and support, and psychological wellbeing may be a l s o a p p r o p r i a t e . q o l m e a s u r e s a r e n o substitute for measuring outcomes associated with disease but are an adjunct to them.(14) qol measures have eight potential uses in aiding routine clinical practice. they can be u s e d t o p r i o r i t i z e p r o b l e m s , f a c i l i t a t e communication, screen for potential problems, i d e n t i f y p r e f e r e n c e s , m o n i t o r c h a n g e s o r response to treatment, and train new staff. they can also be used in clinical audit and in clinical governance. the first five of these items are of immediate value in clinical encounter, while the last three contribute to training, reviewing care, and improving care in the future.(14) the danish qol survey is based on the philosophy of life known as the integrative quality-of-life (iqol) theory. it comprises eight different quality-of-life concepts, ranging from the superficially subjective via the deeply e x i s t e n t i a l t o t h e s u p e r f i c i a l l y o b j e c t i v e . definitions of what makes up a good life are bound to be subject to numerous and diverging interpretations. the iqol theory states that the qol emanates from (1) well-being “how are you feeling now?”, (2) satisfaction with life “how satisfied are you with life now?”, (3) happiness “how happy are you now?”, (4) meaning in life “how meaningful is your life now?”, (5) inner balance and biological order “how balanced (your inner equilibrium and state of health) are you now?”, (6) realizing life potential “how well are you realizing your d e e p e s t d re a m s a n d d e s i re s n o w ? ” , ( 7 ) fulfillment of needs “how well are your needs being fulfilled now?”, and (8) objective factors (ability, societal norms) “how many of the following societal norms do you fulfill now?”.(15) t h e o b j e c t i v e q o l c o m p r i s e s a l l nonsubjective aspects of life related to external status and achievement, measured in terms of the norms that are dominant in the culture of the respondent. as theories of objective qol typically tend to be theories of lists, in which many things and qualities a person ought to possess are enumerated, such a list was also constructed. it is possible to make many other lists. however, as stated in the methodological criteria, theses lists must be based on the theory or overall philosophy of life.(15) harsono epileptic patients 197 univ med vol.27 no.4 in line with the measurement of qol, epilepsy-related quality of life (erqol) is assessed with administration of the quality of life in epilepsy (qolie)-31. this instrument was developed by cramer et al, derived from the longer qolie-89 developed by devinsky et al. t h e q o l i e 3 1 i s a s e l f a d m i n i s t e r e d questionnaire designed for completion by the patients themselves. this instrument includes 7 of the 17 qolie-89 subscales, i.e. seizure worry, overall qol, emotional well-being, energyfatigue, cognitive functioning, medication effects, social function, and health status. responses can be scored to provide subscale scores and a total score. higher scores represent better function.(4,16) for measuring qol in clinical practice, such as in epileptic patients, a wider range of properties is required to ensure that a measure can be used routinely. these include validity (does the instrument measure what it is intended to measure, such as qol?), appropriateness and acceptability (is the measure suitable for its i n t e n d e d u s e ? ; t h i s i s c r u c i a l i n c l i n i c a l practice), reliability (does the measure produce the same results when repeated in the same population?) responsiveness to change (does the measure detect clinically meaningful changes?; t h i s i s s o m e t i m e s c a l l e d s e n s i t i v i t y ) a n d i n t e r p r e t a b i l i t y ( c a n r e s u l t s f r o m t h e measurement be interpreted clinically and are they relevant?).(14) impact of age, seizure severity and frequency patient age was inversely related to qol in the areas of physical functioning and physical r o l e l i m i t a t i o n . ( 1 7 ) t h e l o c a t i o n , t y p e , a n d f r e q u e n c y o f s e i z u r e s m a y c o n t r i b u t e t o psychosocial problems. seizures may affect the temporal lobes or limbic structures, which are crucial areas for emotions and coping, as well as cortical areas, which are necessary for cognitive and physical functioning. studies have demonstrated that frequency and type of seizure injuries correlate with seizure severity and frequency. patients often report that complex partial seizures in which the patient is partially aware of altered emotions, hallucinations, e x p e r i e n t i a l p h e n o m e n a , o r b e h a v i o r a r e especially frightening. patients with more f r e q u e n t s e i z u r e s e x p e r i e n c e t h e g r e a t e s t burdens; their healthcare costs are five times g r e a t e r t h a n t h o s e o f p a t i e n t s w i t h w e l l controlled seizures. greater seizure severity was also correlated with poorer qol in many studies of adolescents and adults. however, even patients with well-controlled seizures report that their condition affects their life in many ways.(16) as seizure frequency increases, patients showed more impaired qol in the areas of physical functioning, vitality, general health, mental health, and social functioning.(17,18) the above description indicates that older age and frequency as well as severity of seizures have a significant impact on qol and healthcare costs of the epileptic patients. those variables should be considered properly in the management of epileptic patient. stigma of epilepsy epilepsy has historically been surrounded by prejudice and myth, and associated with many misconceptions. even today, people with epilepsy experience psychosocial problems, e s p e c i a l l y i n t h e i r r e l a t i o n s h i p s a n d employment. these problems are usually not directly related to the severity of the seizures, but are based on misconceptions about the condition. there is a vicious circle concerning attitudes towards people with epilepsy. the negative attitudes and fear displayed by the public and employers towards people with epilepsy lead them to continue to conceal their diagnosis. this in turn makes changes to the social attitudes and increased acceptance of people with epilepsy a slow process. these 198 problems are universal but are greatest in the developing world where a majority of the patients with epilepsy receive no diagnosis or t r e a t m e n t . e d u c a t i o n p l a y s a k e y r o l e i n i n c r e a s i n g p r o f e s s i o n a l k n o w l e d g e a b o u t epilepsy as a treatable brain disorder and in reducing, and ultimately preventing, social exclusion of patients with epilepsy.(19) stigma and factors that contribute to it should be addressed as the top priority in epilepsy self-management and advocacy. these factors include (a) lack of awareness, (b) lack of timely, complete, and accurate information, ( c ) m i s p e r c e p t i o n s , ( d ) t h e b r o a d , v a r i e d spectrum of disability among people with epilepsy, (e) over-concern about safety and over-protection limiting choices and options, (f) social tolerance for stigma and discrimination, ( g ) i n s u f f i c i e n t r e s e a r c h o n s t i g m a a n d psychosocial aspects of epilepsy, (h) liability concerns, and (i) fear.(19) mielke et al. in their study showed that people with epilepsy face various psychosocial challenges, including such simple functions as getting from one location to another and maintaining employment even when seizures are well controlled. (20) st i g m a o f e p i l e p s y i s c o n s t r u c t e d b y misconception about the condition amongst the community, not limited to lay people, but also in the well-educated groups. this misconception can be waived by continuous counseling either by direct activities or dialogue, such as seminars, symposia, and small-group discussions, or by indirect action, i.e. leaflets, flyers, daily new papers. the effect of stigma, which is somewhat cross-cultural, has also been reported in studies among epileptics in nigeria. (21) in nigeria, epilepsy is associated with shame and social isolation, and patients often present late for hospital care. most patients would have visited traditional and religious healers before finally presenting for orthodox medical care, and indeed, many combine the various treatment modalities. this practice often affects patients psychosocial functioning, resulting in low qol.(22) social deprivation the relation between illness, health and social deprivation has been well documented. previous studies dating from nearly 60 years ago have shown that this finding remains valid for psychiatric illness, although there is debate as to the direction of causality and whether the relation is specific to diagnosis.(23)patients with epilepsy tolerate higher levels of unemployment than the general population and it would be expected that this will impact on deprivation. unemployment is regarded as a key variable in all deprivation indices and has been identified independently as a strong indicator of health need. patients with epilepsy are also more likely to have unskilled jobs and consequently to be of lower social class. it is likely therefore that a positive association between epilepsy and social deprivation will exist, which may be i n c r e a s e d b y t h e c o n f o u n d i n g e f f e c t o f psychiatric illness and learning disability. epilepsy is well documented as a comorbidity of both these conditions. social relationship and community living epilepsy is associated with a wide range of markers of social and economic disadvantage, including poor academic achievement and low income. because of this association, it is often assumed that people who are socially and economically deprived are more likely to develop epilepsy. this hypothesis is supported to some extent by the observation that the incidence of epilepsy is higher in developing countries than in developed countries.(24) epilepsy can affect the way a person develops healthy relationships and independent living skills (box 1). harsono epileptic patients 199 univ med vol.27 no.4 these social issues, or indirect costs, can have greater impact than the direct costs of m e d i c a l c a r e . m a n y p a t i e n t s a n d f a m i l y members say “it is more than just the seizures” when explaining the challenges of living with epilepsy. community resources can address the burden of living with epilepsy from broader perspectives, helping people identify their strengths and available sources of support. state vocational rehabilitation departments, local epilepsy centers, and epilepsy foundation affiliates can provide a range of employment assistance, support services, information, case management, and educational programs.(7) the background of social deprivation related to epilepsy comprises misconception about the condition as well as the consequences o f t h e i l l n e s s . s o c i a l d e p r i v a t i o n c a n b e minimized by proper management of the patients and providing sheltered workshops or vocational rehabilitation for the patients according to their capacities and disabilities. local epilepsy centers, organizations for epileptic patients, and epilepsy foundations will be useful to assist the p a t i e n t s i n c l o s i n g t h e i r g a p s i n s o c i a l relationships and community living. fear and anxiety for people with epilepsy, fear can be enormous and often is overlooked. in the survey of fisher and associates, 45% of respondents listed fear as the worst thing about living with epilepsy.(25) in addition, austin identified fear and anxiety as common in children with newonset seizures. patients may fear dying during a seizure, suffering brain damage, having a seizure in public, losing control, or losing their friends, their driver ’s license, or their job. women with epilepsy may fear for their safety during or after a seizure. in addition, they may wonder about their ability to successfully bear or rear children; unfortunately, many women with epilepsy are still erroneously advised not to have children.(26) the psychological impacts of seizures are fear and anxiety which disturb the patients’ activities of daily living. in the long run, fear and anxiety may worsen the qol of epileptic patients. factors responsible for inadequate treatment various studies in developing countries h a v e s h o w n t h a t m a n y c o n s t r a i n t s a n d difficulties hinder the adequate treatment of epilepsy. these factors are not restricted to one p a r t i c u l a r s o c i a l s e c t o r b u t e x i s t i n t h e economic, political, and cultural frameworks of s o c i e t i e s . d i f f e r e n t p e r c e p t i o n s a n d understanding, lack of prioritization, lack of i n f r a s t r u c t u r e a n d s t r u c t u r a l a d j u s t m e n t programs, supply of anti-epileptic drugs, and c h o i c e o f d r u g a r e t h e p r o m i n e n t f a c t o r s r e s p o n s i b l e f o r i n a d e q u a t e t r e a t m e n t i n developing countries.(27) cultural beliefs vary from country to country and may influence individuals’ healthseeking strategies. for example, people may not seek treatment with aed if epilepsy is not seen as a condition that can be treated by western medicine. this is equally true of people in both box 1. issues in social relationships and community living for patients with epilepsy (7) 1. personal adjustment to epilepsy 2. sexual issues 3. education and employment 4. recreational opportunities 5. disclosure of epilepsy to employers 6. stigma and discrimination 7. independent living 8. transportation 9. respite care 200 industrialized and developing countries. despite its importance and the existence of an ofteneffective remedy, epilepsy is not generally recognized as a public health priority. where there are low budgets for health, resources are inevitably prioritized for conditions perceived to be more important than epilepsy, such as infectious diseases. lack of infrastructure and structural adjustment programs are linked to and may further accentuate the lack of priority. supply of aed is an important factor. ideally, the choice of aed for each patient should be based on seizure type and/or syndrome as well as the individual person’s needs. unfortunately, in most developing countries both the choice and supply of drugs are limited. phenobarbitone h a s b e c o m e w h o ’s f r o n t l i n e a e d i n developing countries, where it is the most c o m m o n l y p r e s c r i b e d a e d . p h e n y t o i n , carbamazepine, and valproate are up to 5, 15, a n d 2 0 t i m e s a s e x p e n s i v e , r e s p e c t i v e l y. questions have, however, been raised about its suitability with respect to its efficacy and the p r o f i l e o f a d v e r s e e ff e c t s . i n d e e d , i l a e commissions have argued that the who list of essential drugs needs to be discussed further, as the status of phenobarbitone seems to be based on economic factors rather than on efficacy and suitability.(27) there are various factors responsible for inadequate treatment, ranging from a simple reason such as individual noncompliance in taking aed to a complicated situation involving socio-cultural, political and economical aspects. these factors should be considered in the management of epileptic patients. p o t e n t i a l r e a s o n s f o r t h e treatment gap level of health care development the level of health care development is important because it influences knowledge about the true nature of the problem and its potential solutions. it also influences the means to reduce the treatment gap. whether the means available will be sufficient depends on the demand. thus it might be argued that health care should be primarily directed toward prevention. however, in the first place, the prevalence of symptomatic epilepsies is less than one third of a l l c a s e s . n o t a l l s y m p t o m a t i c c a s e s a r e attributable to preventable causes, such as deficient perinatal care or cysticercosis. it should also be stated that before the effects of prevention are felt, incidence cases, which occurred before prevention became effective, will have on average a duration of 13 years (the r a t i o o f p r e v a l e n c e o v e r i n c i d e n c e d a t a ) . therefore closure of the seizure treatment gap remains a high priority.(9) culture beliefs cultural values affect people’s healthseeking strategies. if people see epilepsy as caused by something that is not natural or biomedical, then treatment through western medicine may not be sought.(28) such beliefs may mean that people with epilepsy seek treatment by traditional or faith healers or that continued compliance with aeds is difficult to obtain. these decisions may be rational within these people’s cultures, but they may also lead to epilepsy being untreated or not treated successfully. it should also be mentioned that levels of literacy and knowledge will impinge on cultural beliefs insofar as choices arising from alternative beliefs, or evidence to back up c h o i c e s , a r e l i m i t e d i f t h e m e a n s o f communication and acquiring background knowledge are restricted.(29) economy as some of the sections concerning health care and financial coverage for each region i n d i c a t e , t h e p r o v i s i o n o f a e d s t h r o u g h harsono epileptic patients 201 univ med vol.27 no.4 government-funded schemes may in some cases be impossible. it should also be stated that many developing countries’ health sectors are hindered by world bank structural adjustment programs, so that what little income they have is spent on debt repayment rather than on health expenditure. although this is not the only sector to be adversely affected.(9) distance from health facilities the problem of distance from health care facilities is in fact a problem of the know-how available at the community health care level. it has been shown that a reasonable level of seizure treatment can be achieved by primary health care workers. however, notwithstanding the objections to vertical programs, sufficient back-up for the primary health care workers should be available to give epilepsy care its proper place among their many other duties. some participants argued that epilepsy care w o u l d b e b e t t e r o f f , i f u n d e r t a k e n b y community-based rehabilitation programs.(2) supply of aeds distance may also play a role in the lack of a sustained supply of prescribed aeds. aeds are used to prevent or interrupt seizures.(30) the majority of developing countries also have an extremely limited choice of drugs, with newer aeds being mostly unavailable. however, the reasons for this are more based on income (both personal and government) and inequalities than on geography and distance. the problem is compounded by the evil of counterfeit drugs.(9) lack of prioritization although epilepsy is an important factor in the burden of neurologic and noncommunicable disease and effective treatment often exists, it is not generally recognized as a public health priority. epilepsy has to compete with many other conditions and illnesses. such competition is even harsher where there are low health budgets, so that resources are prioritized for conditions perceived to be more important, such a s m a l a r i a , h i v / a i d s , t u b e r c u l o s i s e t c . although not wanting to detract from the scale of such communicable diseases, it may be more cost-effective to treat epilepsy than to treat these other conditions.(9) political will of the national as well as local government is needed to solve this problem. such political will can be initiated by the ministry of health (as a national policy) and then implemented by the primary, secondary and tertiary health care. the political will should have comprehensive matters such as diagnostic, therapeutic and restorative services. identification of healthcare needs s u c c e s s f u l m a n a g e m e n t o f e p i l e p s y involves many factors (box 2). patients and families should be taught basic skills, such as observing and recording seizures, managing adverse drug effects, identifying and managing stress and other triggers, and maintaining personal safety. the ability to record seizures and identify triggers may lead to behavioral o r l i f e s t y l e c h a n g e s t h a t i m p r o v e s e i z u r e control. it is vital that both physician and patient understand medication management issues, such as identifying optimal times to m a k e m e d i c a t i o n o r d o s a g e c h a n g e s a n d knowing how to respond to adverse effects and h o w t o e n h a n c e c o m p l i a n c e . p h y s i c i a n s handling epileptic patients with uncontrolled seizures may have to consider many factors when choosing or changing treatment. women have unique issues because their epilepsy and general health needs vary across their life span; they depend on primary care physicians to a s s i s t i n c o o r d i n a t i n g c a r e a c r o s s m a n y specialties.(7) 202 conclusions the qol of epileptic patients tend to be poorer compared with healthy people. the background of such tendencies consists of a g e , m e d i c a l , s o c i o c u l t u r a l , e c o n o m i c a l , p s y c h o l o g i c a l , p o l i t i c a l a n d g e o g r a p h i c a l a s p e c t s . h e n c e , m a n a g e m e n t o f e p i l e p t i c patients should be based on a comprehensive and integrated intervention in line with the achievement of health status as defined by the who. successful management of epilepsy involves many important factors such as early diagnosis, choosing the appropriate aed, c o m p l i a n c e i n t a k i n g a e d , a n d s o c i a l a c c e p t a b i l i t y. h o w e v e r, q o l i s a h i g h l y individual concept rather than that of a group or community. in addition to the successful management of epileptic patients, the following issues are important and should be kept in mind: age, sex, stigma of epilepsy, frequency and severity of seizures, fear and anxiety, social deprivation, factors responsible for inadequate treatment, potential reasons for the treatment gap, and healthcare needs. references 1. espie ca, watkins j, curtice l, espie a, duncan r, ryan j, et al. psychopathology in people with epilepsy and intellectual disability; an investigation of potential explanatory variables. j neurol neurosurg psychiatry 2003; 74: 1485-92. 2. cyrlac n, sureskumar pn, kunhikoyamu am, girija as. social factors and psychopathology in epilepsy. neurol india 2002; 50: 153-57. 3. bishop m, berven nl, hermann bp, chan f. quality of life among adults with epilepsy: an exploratory model. rehabil couns bull 2002; 45: 87-95. 4. wiebe s, matijevic s, eliasziw m, derry pa. clinically important change in quality of life in epilepsy. j neurol neurosurg psychiatry 2002; 73: 116-20. 5. djibuti m, shakarishvili r. influence of clinical, demographic, and socioeconomic variables on quality of life in patients with epilepsy: findings from a georgian study. j neurol neurosurg psychiatry 2003; 74: 570-73. 6. herodes m, oun a, haldre s, kaasik ae. epilepsy in estonia: a quality of life study. epilepsia 2001; box-2. healthcare issues for patients with epilepsy (7) 1. coping with the diagnosis 2. observing and recording seizures 3. identifying potential triggers and high-risk times 4. maintaining personal safety 5. handling seizures emergencies 6. managing adverse drug effects 7. understanding the nonmedical options for seizure management 8. managing concomitant illness 9. understanding the relationship between seizures and hormonal states 10. practicing family planning 11. managing pregnancy and menopause 12. maintaining bone health harsono epileptic patients 203 univ med vol.27 no.4 42: 1061-73. 7. shafer po. improving the quality of life in epilepsy. postgrad med 2002; 111: 1. available at: http:// www.postgradmed.com/issues/2002/01_02/ shafer.htm 10/18/2004. accessed september 2, 2008. 8. bishop m, allen ca. the impact of epilepsy on quality of life: a qualitative analysis. epilepsy behav 2003; 4: 226-33. 9. meinardi h, scott ra, reis r, sander jwas. ilae commission report: the treatment gap in epilepsy: the current situation and ways forward. epilepsia 2001; 42:136-49. 10. reynolds eh. the ilae/ibe/who epilepsy global campaign history: international league against epilepsy – international bureau for epilepsy. epilepsia 2002; 43:9-11. 11. stavem k, loge jh, kaasa s. health status of people with epilepsy compared with the general reference population. epilepsia 2000; 41: 85-90. 12. boylan ls, flint la, labovitz dl, jackson sc, starner k, devinsky o. depression but not seizure frequency predicts quality of life in treatmentresistant epilepsy. neurology 2004; 62: 258-61. 13. leidy nk, elixhauser a, vickrey b, means e, willian mk. seizure frequency and the healthrelated quality of life of adults with epilepsy. neurology 1999; 53: 162-66. 14. higginson ij, carr aj. measuring quality of life: using quality of life measures in the clinical setting. bmj 2001; 322: 1297-300. 15. ventegodt s, merrick j, andersen nj. measurement of quality of life iii. from the iqol theory to the global, generic seqol questionnaire. sci world j 2003; 3: 972-91. 16. cramer ja, arrigo c, van hammee, gauer lj, cereghino jj. effect of levetiracetam on epilepsyrelated quality of life. epilepsia 2000; 41: 868-74. 17. mrabet h, mrabet a, zouari b, ghachem r. healthrelated quality of life of people with epilepsy compared with a general reference population: a tunisian study. epilepsia 2004; 45: 838-43. 18. malgren k. epilepsy care across europe and key concerns. neurological disorders: long-term healthcare strategies 2003; 44: 54-6. 19. american epilepsy society. living well with epilepsy. report of the national conference on public health and epilepsy; 1997. 20. mielke j, sebit m, adamolekun b. the impact of epilepsy on quality of life of people with epilepsy in zimbabwe: a pilot study. seizure 2000; 9: 25964. 21. olley bo. psychological and seizure factors related to depression and neurotic disorders among patients with chronic epilepsy in nigeria. afr j med sci 2004; 33: 39-44. 22. mosaku ks, fatoye fo, komolafe m, lawal m, ola ba. quality of life and associated factors among adults with epilepsy in nigeria. int psychiatry med 2006; 36: 469-91. 23. morgan cli, ahmed z, kerr mp. social deprivation and prevalence of epilepsy and associated health usage. j neurol neurosurg psychiatry 2000; 69: 13-7. 24. jacoby a, buck d, baker g, mcnamee p, grahamjones s, chadwick d. uptake and costs of care for epilepsy: findings from a uk regional study. epilepsia 1998; 39: 776-86. 25. fisher rs, vickrey bg, gibson p, hermann b, penovich p, scherer a, et al. the impact of epilepsy from the patient’s perspective i: descriptions and subjective perceptions. epil res 2000; 41: 39-51. 26. austin jk. concerns and fears of children with seizures. clin nurs pract epil 1993; 1: 4-10. 27. scott ra, lhatoo sd, sander jwas. the treatment of epilepsy in developing countries: where do we go from here? bull who 2001; 79: 344-51. 28. shorvon sd, farmer pj. epilepsy in developing countries: a review of epidemiological, sociocultural and treatment aspects. epilepsia 1988; 29: s36-45. 29. watts ae. a model of managing epilepsy in a rural community in africa. bmj 1989; 298: 805-7. 30. harsono. seizure threshold, hormones and antiepileptic drugs. univ med 2008; 27: 18-28. oktavianus 138 *department of public health and community medicine, faculty of medicine jenderal soedirman university **staff at magetan district health office, magetan, east java correspondence: dr. yudhi wibowo, m.ph department of public health and community medicine, faculty of medicine jenderal soedirman university jl. dr. gumbreg no. 1, mersi east purwokerto 53 112 phone: +62281-622022 email: fk@unsoed.ac.id or dryudhiwibowo2005@gmail.com univ med 2015;34:138-48 doi: 10.18051/univmed.2016.v35.138-148 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background malaria is re-emerging because of imported cases and the presence of potential vectors that can transmit and spread malaria. malaria is a health problem in banyumas district. mapping the spread of infectious diseases is epidemiologically important. the purpose of this study was to determine the relationship between the variables and the epidemiology of malaria that were spatially modeled using the geographic information system (gis). methods this was a case-control study with ratio of 1:1. cases were malaria-positive patients and controls were people without malaria, as diagnosed by microscopic examination. minimum sample size was 139 per group and total sample size was 282 people. chi-square was used to test the relationship between the variables, and gis modeling to determine the spatial distribution of malaria cases. results there were significant relationships between level of income below minimum wage, not using mosquito nets, not using wire netting, not using insect repellents, habit of going out at night, history of malaria, cattle sheds not located between woods and residential area, history of going to endemic areas, residence at distances <1000 m from plantations, bushes, swamps and puddles, with incidence of confirmed malaria (p<0.001). the group of cases living <1000 meters from plantations numbered 141 (100%). conclusions malaria incidence is clustered and buffers around plantations at <1000 m. malaria hot spots are displayed as risk maps that are useful for monitoring and spatial targeting of prevention and control measures against the disease. keywords: risk factors, spatial analysis, malaria incidence incidence of malaria is clustered and buffers around plantations: a spatial analysis yudhi wibowo*, agung saprasetya dwi laksana*, joko mulyanto*, madya ardi wicaksono*, and agus y purnomo** doi: http://dx.doi.org/10.18051/univmed.2015.v34.138-148 139 univ med vol. 34 no.2 kejadian malaria kluster dan bufer di sekitar perkebunan : model analisis spasial latar belakang malaria adalah penyakit yang muncul dan muncul kembali karena kasus impor dan potensi vektor yang dapat menularkan dan menyebarkan malaria. malaria adalah masalah kesehatan di kabupaten banyumas. pemetaan penyebaran penyakit menular secara epidemiologi adalah penting. tujuan penelitian ini adalah untuk menentukan hubungan antara variabel dan epidemiologi malaria yang dimodelkan secara spasial menggunakan sistem informasi geografis (sig). metode penelitian ini merupakan studi kasus-kontrol dengan perbandingan 1: 1. kasus adalah semua penderita malaria (+) dan kontrol adalah orang-orang dengan tidak didiagnosis malaria dengan pemeriksaan mikroskopis. besar sampel minimum 139 per kelompok dan besar sampel total penelitian adalah 282 orang. chi-square digunakan untuk menguji hubungan antara variabel dan pemodelan menggunakan sig untuk menentukan distribusi spasial kasus malaria. hasil ada hubungan yang bermakna antara tingkat pendapatan kurang upah minimum, tidak menggunakan kelambu, tidak menggunakan kain kawat kasa, tidak menggunakan obat nyamuk, kebiasaan keluar pada malam hari, riwayat penyakit malaria, tata letak kandang ternak tidak di antara hutan dengan perumahan, riwayat bepergian ke daerah endemis, jarak rumah dengan perkebunan, semak-semak, rawa dan genangan air <1000 m dengan kejadian konfirmasi malaria (p<0,001). kelompok kasus yang tinggal <1000 meter dari perkebunan adalah 141 (100%) dari kasus. kesimpulan kejadian malaria adalah kluster dan bufer di sekitar perkebunan yang <1000 m. pemetaan malaria ditampilkan sebagai peta risiko yang bermanfaat untuk pemantauan dan penargetan spasial tindakan pencegahan dan pengendalian penyakit. kata kunci: faktor risiko, analisis spasial, kejadian malaria abstrak introduction each year indonesia’s 230 million people collectively suffer at least several million cases of malaria caused by all four known species of human plasmodium.(1) eastern indonesia faces the challenges of providing adequate and equitable health services to a largely remote, r u r a l p o p u l a t i o n . h e a l t h i n t h e e a s t e r n indonesian province of nusa tenggara timur (ntt) is generally poor, with high incidence of malaria, high infant mortality rate (54/1000, compared with 44/1000 nationally), and child malnutrition averaging 39% and reaching 50% in some areas.(2) one of the malaria-endemic areas in indonesia is central java province. the m a l a r i a m o r b i d i t y r a t e ( a n u a l p a r a s i t e index=api) was 0.1 ‰ in 2011. the number of malaria positive patients in 2010 was 3,300, increasing to 3,467 people in 2011. in the year 2011, the case fatality rate for malaria was 0.12%.(3) 140 banyumas is one of the malaria endemic districts in central java province. according to data from the malaria control program at banyumas district health office, an increase in malaria positive patients occurred from 2007 till 2010. in 2010, the highest number of people with malaria was 623 cases. in 2011, 423 of the 1,278 clinical malaria cases were found to be malaria positive.(3) the number of malaria positive patients is the highest in the region, especially at the watuagungvillage health center tambak i with 146 cases. the second largest number of patients was in the bogangin and banjar penepen village health center sumpiuh ii comprising 117 people. in 2010 an outbreak of malaria occurred in banyumas, in ketanda village (14 malaria positive patients), sumpyuh sub-district, and in watuagung village (30 malaria positive p a t i e n t s ) , ta m b a k s u b d i s t r i c t . b a s e d o n laboratory tests it is known that the most abundant species is plasmodium falcifarum. several studies have shown that malaria infection is influenced by environmental factors s u c h a s t e m p e r a t u r e , r a i n f a l l , h u m i d i t y, elevation, and factors related to demography. specifically, temperature and rainfall act as limiting factors on the development of anopheles mosquitoes which are the intermediate hosts in the transmission of malaria parasites.(4) transmission dynamics of malaria does not recognize administrative boundaries. the increasing mobility of people from one area to another increases due to advancement and ease of transportation. this increases the risk of malaria transmission due to displacement of the population employed in agriculture, plantations and mining. individuals working in the forest are at risk of malaria, especially if staying in the forest at night, as found in studies conducted in central vietnam by thanh et al.(5) this is supported by the results of research conducted in central vietnam by thang et al.(6) stating that work in the forest and the home environment are risk factors for the incidence of malaria. risk factors for malaria in indonesia, among others, are the geography, gender, education and employment.(7) mapping the spread of infectious disease is epidemiologically important. spatial analysis is useful to determine the pattern of distribution of the disease, the high risk areas, the source of disease transmission and disease risk factors in terms of areal, making efforts to c o n t r o l t h e d i s e a s e a n d i n t e r r u p t i n g t h e transmission chain, so that the later can be done properly. the geographic information system (gis) is a technology to store, manipulate, analyze, and display geographically referenced information. outside of healthcare, it has been shown to be valuable in a wide range of situations, such as u r b a n p l a n n i n g , e n v i r o n m e n t a l r e s o u r c e m a n a g e m e n t , e m e rg e n c y p l a n n i n g , a n d t r a n s p o r t a t i o n f o r e c a s t i n g . (8) t h e u s e o f geographic information systems with spatial statistical techniques is useful for measuring the magnitude of the risk of malaria incidence and identify the environmental factors that influence the re-emergence of malaria disease risk factors, so that public health planning should be focused on the maximum spatial cluster region.(9) control management based on regional spatial data is one of the essential ways to control the disease, e s p e c i a l l y i f b a c k e d u p b y d a t a a n d epidemiological investigation of outbreaks. the purpose of this study was to determine the relationship between risk variables and the epidemiology of malaria in banyumas that is spatially modeled using the gis. methods research design this study was an observational analytic study using case-control design with a ratio of 1: 1. research was carried out within 6 months (may to october 2014) and located at the banyumas, kemranjen, sumpyuh and tambak health centers, banyumas district. research subjects the population in this study comprised all banyumas residents who had positive malaria wibowo, laksana, mulyanto, et al incidence of malaria is clustered 141 univ med vol. 34 no.2 by microscopic examination, with the results recorded in the register of banyumas district health office in 2012. the control group were people living in the same district who did not have malaria, as diagnosed by microscopic examination (and recorded in the register of banyumas district health office). exclusion criteria were positive malaria cases who at the time of the study had died or moved outside the study area. the sample size was determined based on a 0.05 significance level and power of 80%, with the proportion in the control group being 0.5 and an odds ratio (or) of 2 obtained, giving a minimum sample size of 139 per group, so that in this study the total sample size was 282 people. (10) the study was conducted in all regions of the sub-districts of banyumas with cases of malaria, especially tambak, sumpyuh, kemranjen and banyumas health center, because in 2012 the regions had the highest malaria positive case rates in banyumas. outbreaks of malaria in 2010 also occurred in this region. all of the malaria positive cases in 2012 were included in the group of cases and controls matched by age and sex. data collection data were collected by interview, using a questionnaire comprising items on level of income, use of mosquito nets, wire netting, mosquito repellents, habit of going out at night, history of malarial illness, the location of cattle sheds, a history of travel to endemic areas, d i s t a n c e o f r e s i d e n c e s ( s e t t l e m e n t s ) t o plantations, bushes, swamps and puddles. geographic information system the geographic information system is a kind of software that can be utilized for the i m p o r t a t i o n , s t o r a g e , m a n i p u l a t i o n , a n d presentation of geographic information products and their attributes. geographic information system is software that serves to collect, store, display and connect spatial data about a geographical phenomenon to be analyzed and the results are communicated to the user as a basis for decision making. the coordinates of the respondents’ homes were determined using gps garmin etrex 10. spatial analysis spatial modeling was performed using computer assistance and mapping software. buffer analysis of the incidence of malaria in banyumas in 2012 was used to determine the environmental buffer zone, the suspected breeding sites of the malaria vectors, and the mosquito flying distance to a live subject with p o s i t i v e m a l a r i a . t h e a n a l y s i s o f t h e environmental buffer zone included rivers, f o r e s t s , r a i n f e d f i e l d s , i r r i g a t e d f i e l d s , plantations, pastures, fields and bushes. through a spatial query mechanism, a spatial element was taken in the form of a line 1000 m from the environmental buffer, which then generated new spatial elements such as polygons that cover the environmental buffer. based on the scope of the polygon, the number of subjects with positive malaria within the scope of the polygon buffer is counted. mean center analysis was used to determine the geographic center (central case) of malaria in banyumas in 2012. from an analysis using gis software the mean center was obtained. the center was obtained from the mean values of x and y that are calculated to obtain the centroid point of malaria cases in banyumas. the projection of the point data is used to measure distances accurately. the mean center value of the x and y point was derived from the calculation of the mean values of x and y and then the x and y coordinates were stored. central points used are the mean centers, and dispersion refers to the spread of the mean centers bounded by polygons. fields surrounding the polygon centroid were determined based on the distance calculated by using the weighted average of the center of all malaria positive subjects. nearest neighbor analysis is an approach to look at the pattern of spread of malaria cases in banyumas 2012. the calculated average nearest neighbor (ann) score is used to 142 determine the pattern of malaria incidence in banyumas 2012. ann can be calculated using the formula: ann= ï o / ï e. ïo : observed mean distance between each feature and their nearest neighbor, ïe : expected mean distance for the features given a random pattern. however, in practice ann values are calculated using gis software. ann values are inferred based on the value of the expected mean distance, and is represented by: (1). ann = 1 indicating the occurrence of a random pattern, (2). ann <1 indicating the incidence of crowding (clustered), (3). ann >1 indicating the incident spread (dispersed). regarding the incidence of malaria in banyumas in 2012, there were 140 subjects with positive malaria, therefore m = 140. because the area of banyumas is 1327.60 km2, the value of a = 1327.60. the distance between each occurrence is calculated using the euclidean distance. ann values are calculated using gis software. to determine the presence of a spatial pattern, tests of significance were used: (i). h0: there is no spatial pattern of malaria cases in banyumas in 2012, há: there is a spatial pattern of malaria cases in banyumas in 2012, (ii). significance level : á, (iii). critical area h0 is rejected if z score > z á/2 = 2.58 z score < z á/2 = -2.58. the standard deviational ellips (sde) is used to analyze trends and patterns of dispersion of malaria cases. standard deviation ellips is a summary of central tendency and dispersion in two dimensions, as well as a directional trend. there are two points that are used as the basis for the distribution of point locations on sde. they are central tendency and dispersion. central tendency uses the center of the mean and dispersion refers to the spread of the mean center bounded by the ellipse. standard deviational ellipse models can be used to gain a better understanding of the geographical aspects of the phenomenon and identify the cause of an event, based on specific geographic patterns.(11) data analysis an odds ratio was used to examine the relationship berween variables, with significant variables being included into a model for multiple logistic regression. ethical clearance t h i s s t u d y w a s a p p r o v e d b y t h e commission on health research ethics, medical school, jenderal soedirman university (unsoed) with ref no: 100/iec/vii/2014. results the analysis using gis software found a z score of -11.17. because z score = -11 17 <2.58 we rejected h0. it can be concluded that there is a spatial pattern of malaria cases in banyumas in 2012. because the expected mean distance = 0.44 < 1 it can be concluded that the pattern of malaria incidence in banyumas in 2012 is clustered. the confirmed participation rate of 141 malaria cases from 214 (65.9%), is sufficient to represent the study and 34.1% of the confirmed cases was not able to participate in this research because the case had died or moved away with no known address. most species of mosquitoes captured in the four endemic villages are of the genus culex spp totalling 106 individuals (44.7%) in both indoor and outdoor captures. species of the genus anopheles were quite diverse, from the most common type, namely an. vagus (36.3%), an. barbirostris (7.2%), an. kochi (4.6%), an. maculatus (4.2%) and an. aconitus (1.3%). an. aconitus, an. maculatus and an. balabacensis are most commonly found in watuagung village. regarding the characteristics of the respondents, in the group of cases of confirmed malaria, 53.2% were male, 73.8% were aged 1560 years, 54.6% less educated, 84.4% income < regional minimum wage (rgw) and 47.5% were farmers. results of bivariate analysis showed statistically significant differences (p<0.001) wibowo, laksana, mulyanto, et al incidence of malaria is clustered 143 univ med vol. 34 no.2 between the independent variables and the dependent variable (incidence of confirmed malaria) in banyumas as presented in table 1. the independent variables were: income < minimum wage, not using mosquito nets, not using wire netting, not using mosquito repellents, habit of going out at night, history of malaria, cattle sheds not located between woods and residential area, history of going to endemic areas, residence at distances < 1000 m from plantations, bushes, swamps and puddles. results of multivariable analysis showed three independent variables that have the highest precision, i.e. location <1000 m from puddles, not using mosquito nets and not using insect repellent, and are associated with a statistically significant incidence of confirmed malaria cases as presented in table 2. table 1. relationship between independent variables and incidence of confirmed malaria or : odds ratio; ** cannot be calculated because the cell value = 0; rmw : regional minimum wage table 2. multivariate logistic regression of selected independent variables with incidence of confirmed malaria rmw : regional minimum wage 144 figure 1. distribution map of malaria cases and controls in banyumas district 2012 from the results of the spatial analysis based on the analysis of average nearest neighbor distance, it is known that the distribution of malaria incidence in banyumas in 2012 was clustered, with z score = -17.74 sd and p-value <0.001 as presented in figure 1. from the results of the analysis of cases the buffer was found for 141 (100%) of cases to be in the area <1000 meters from plantations as presented in figure 2. results of analysis of the sde in banyumas showed the axis to be oriented in the northwestsoutheast direction. from the corresponding elliptical area on the map can be predicted the possibility of a subsequent spread of malaria upon the presence of risk factors and the absence of supportive intervention services/health workers as shown in figure 3. discussion according to erhart et al. (12) the risk of malaria infection was higher in men than in women because of the influence of gender and wibowo, laksana, mulyanto, et al incidence of malaria is clustered 145 univ med vol. 34 no.2 figure 2. malaria map of banyumas district 2012, subjects with positive malaria residing in plantation buffer figure 3. standard deviational ellipse of malaria map of banyumas district 2012 146 their behavior. in developing countries, an activity that involves the movement of people, including workers in the plantation colonies, increases the risk of malaria transmission. regarding characteristics of respondents in the cases of confirmed malaria, 47.5% were clove farmers, rubber farmers, and coconut tree farmers (locally called penderes,i.e. tappers of the sap of coconut inflorescences to be made into brown sugar). the species an. aconitus, an. maculatus and an. balabacensis were most commonly found in watuagung village. the habitats of an. maculatus are in open hilly areas and mountains where forests have been cleared and is located in the hilly areas of forest.(13) the breeding sites of an. maculatus are puddles that get sunlight, various types of ponds, lakes, marshes, along the banks of slow-flowing streams, ditches and rice fields near the hills.(14) in the confirmed malaria case group, 84.4% had an income <rmw. according to dale et al. (15) low income affects fulfillment of living needs, including adequate housing for the residents, health care, and nutritious foods. the malaria risk is increased 2.5 times in people living in houses with shoddy construction (cannot protect occupants from mosquito bites). not using mosquito nets at night increases the risk of malaria cases and is in accordance with lozano et al.(16) the use of mosquito nets at night can prevent mosquito bites and to enhance the protection, the recommended type of mosquito nets are nets that have been impregnated with the insecticide permethrin.(17) the use of insecticide-treated nets proved to be able to protect humans from the bite of mosquitoes / malaria vectors.(18,19) not using wire netting will increase the risk of suffering from malaria 10.55 times and these results are consistent with the research of kuswanto,(20) who conducted research on malaria risk factors in banyumas in 2005. those not using insect repellent are at 6.46-fold increased risk to suffer from malaria. our study results were in accordance with the results of the study of kuswanto.(20) our results shows that the habit of going out at night increases the risk of suffering from malaria 4-5 times. activities outside the home until late at night in an area with exophilic and exophagic malaria vectors increases the risk of malaria infection. malaria transmission is highly dependent on the habits of anopheles mosquitoes and the intensity of malaria transmission is associated with the frequency of a person’s activity in areas that allow exposure to the bite of the anopheles mosquito containing sporozoites. history of travel to endemic areas increases the risk of malaria 8.5 times. mobility or migration plays a role in moving all ecotypes of malaria, and the spread of new strains with anti-malarial drug resistance, increasing the transmission of malaria from one place to another. mobility to endemic areas results in an increase in the cases of imported malaria. the presence of plantations is significantly associated with malaria incidence. changes in the balance of the forest ecosystem as a result of the clearing of forests for residential land, plantations, and agriculture contributes greatly to increased malaria transmission and epidemics.(21) the presence of shrubs <1000 m is statistically significantly associated with the incidence of malaria. during the rainy season the presence of anopheles habitats not associated with shrubs but positively correlated with the presence of agricultural land, grassland, and shrubs in agricultural areas is a risk factor for malaria incidence. poor irrigation systems allow the growth of shrubs that could potentially be a breeding ground for anopheles. approximately 75.2% of the cases are in the area <1000 m of water pools and statistically significantly associated with the incidence of malaria. the results of the study show that residents who live near stagnant water (<1000 m) had 2.1 times the risk of malaria infection because stagnant water is where anopheles sp. multiply. the closer the vector breeding sites are to the residential population, the greater the risk of malaria incidence.(22) the analysis of sde in banyumas showed the axis to be oriented in the northwest-southeast wibowo, laksana, mulyanto, et al incidence of malaria is clustered 147 univ med vol. 34 no.2 direction. from the corresponding elliptical area on the map, it can be predicted that the possibility of the spread of malaria is in that direction if it is supported by the presence of risk factors and the absence of intervention from the health department. after the implementation of the planned malaria control programs the malaria cases in banyumas continued to decline from 2012 to 2013, the number of cases decreasing from 214 to 40 cases. the results showed that the variables: level of income <rmw, not using mosquito nets, not using wire gauze, not using insect repellent, habit of going out at night, history of malaria illness, cattle sheds not located between plantations and settlements, a history of travel to endemic areas, distance to the plantations, shrubs, swamps and stagnant water <1000 m, are associated with the incidence of malaria. results of multivariable analysis showed that level of income <rmw, not using mosquito nets, habit of going out at night, not using insect repellent, history of travel to endemic areas, location of plantations and water pools <1000m from residential buildings, are associated with the incidence of malaria in the malaria districts.the incidence of malaria in banyumas in 2012 was clustered, with mean and median centers located in the sumpyuh district. malaria in the buffer area in banyumas in 2012 occurred near the plantations with <1000 m radius. limitations of this study are that biological factors (e.g. existence, type, density and mapping of mosquito vectors) and environmental factors (e.g. temperature, humidity, and weather) that influence the incidence of malaria were not examined in this study. a shortcoming of the analytic-observational study with case-control approach is recall bias, since exposure risk factors have already occurred and to obtain information about these risk factors the investigators must rely solely on the memory of respondents. selection bias occurs because of the selection of cases based solely on traffic data of patients who went to health services (health centers and hospitals). controls were selected from surrounding communities among those who did not suffer malaria cases and came from one village, making them less representative of the actual population of controls. regarding measurement of the coordinates of the respondents using gps who live around the forest, it is quite difficult to get an accuracy of under 10 meters, so that the investigators had to use a measurement accuracy of under 20 m. for further research, it is necessary to conduct research related to the causes of malaria (plasmodium sp.), especially on behavioral aspects, vector bionomics and migration in banyumas. conclusions the incidence of malaria in banyumas is clustered and buffers around plantations that are located <1000 m from residential buildings. malaria hot spots are displayed as risk maps that are useful for monitoring and spatial targeting of prevention and control measures against the disease. conflict of interest the authors declare that they have no competing interests. acknowledgement acknowledgements are due to jenderal soedirman university, institute for research and community service, faculty of medicine & health sciences, banyumas district health office, and health centers for their cooperation and assistance. references 1. elyazar irf, hay si, baird jk. malaria distribution, prevalence, drug resistance and control in indonesia. adv parasitol 2011;74: 41175. doi: 10.1016/b978-0-12-385897-9.000021. 2. fisher rp, myers ba. free and simple gis as appropriate for health mapping in a low resource 148 setting: a case study in eastern indonesia. int j health geogr 2011;10:15. doi: 10.1186/1476072x-10-15. 3. dinas kesehatan kota banyumas. laporan hasil kegiatan program pengendalian penyakit malaria kabupaten banyumas tahun 2013. purwokerto: dinas kesehatan kota banyumas; 2013. 4. kazembe ln, kleinschmidt i, holtz th, et al. spatial analysis and mapping of malaria risk in malawi using point-referenced prevalence of infection data. int j health geogr 2006;5:41. doi: 10.1186/1476-072x-5-41. 5. thanh pv, hong nv, van nv, et al. epidemiology of forest malaria in central vietnam: the hidden parasite reservoir. malar j 2015;14:86. doi: 10.1186/s12936-015-0601-y. 6. thang nd, erhart a, speybroeck n, et al. malaria in central vietnam: analysis of risk factors by multivariate analysis and classification tree models. malar j 2008;7:28. doi: 10.1186/ 1475-2875-7-28. 7. ompusunggu s, tati s, dewi rm. faktor risiko malaria di indonesia (analisis data riskesdas 2007). bul penelit kesehat 2009; suppl 11-22. 8. parmanto b, paramita mv, sugiantara w, et al. spatial and multidimensional visualization of indonesia’s village health statistics. int j health geogr 2008;7:30. doi: 10.1186/1476-072x-7-30. 9. zhang w, wang l, fang l, et al. spatial analysis of malaria in anhui province, china. malar j 2008;7:206. doi: 10.1186/1475-2875-7-206. 10. tilaki-haijan m. sample size estimation in epidemiologic studies. caspian j intern med 2011;2:289-98 11. eryando t, susanna d, pratiwi d, et al. standard deviational ellipse (sde) models for malaria surveillance, case study: sukabumi districtindonesia, in 2012. malar j 2012; 11 suppl 1: 130. doi: 10.1186/1475-2875-11-s1-p130. 12. erhart a, ngo dt, phan vk, et al. epidemiology of forest malaria in central vietnam: a large scale cross-sectional survey. malar j 2005;4:58. 13. ndoen e, wild c, dale p, et al. relationships between anopheline mosquitoes and topography in west timor and java, indonesia. malar j 2010; 9:242. doi: 10.1186/1475-2875-9-242. 14. sinka me, bangs mj, manguin s, et al. the dominant anopheles vectors of human malaria in the asia-pacific region: occurrence data, distribution maps and bionomic précis. parasit vectors 2011;4:89. 15. dale p, sipe n, anto s, et al. malaria in indonesia: a summary of recent into its environmental relationship. southest asian j trop med public health 2005;36:1-13. 16. lozano dr, rodriguez mh, betnzos-reyes af, et al. individual risk factors for plasmodium vivax infection in the residual malaria transmission focus of oaxaca, mexico. salud publica de mexico 2007;49:199-209. 17. badan penelitian dan pengembangan departemen kesehatan ri. laporan nasional riset kesehatan dasar tahun 2010. jakarta: badan penelitian dan pengembangan departemen kesehatan ri;2010. 18. winskill p, rowland m, mtove g, et al. malaria risk factors in north-east tanzania. malar j 2011; 10:98. doi: 10.1186/1475-2875-10-98. 19. alemu a, tsegaye w, golassa l, et al. urban malaria and associated risk factors in jimma town, south-west ethiopia. malar j 2011;10:173. 20. kuswanto. analisis faktor-faktor risiko kejadian malaria di kecamatan kemranjen kabupaten banyumas [tesis]. semarang: program pascasarjana universitas diponegoro;2005. 21. mushinzimana e, munga s, minakawa n, et al. landscape determinants and remote sensing of anopheline mosquito larval habitats in the western kenya highlands. malar j 2006;5:13. 22. kazwaini m, martini s. tempat perindukan vektor, spesies nyamuk anopheles, dan pengaruh jarak tempat perindukan vektor nyamuk anopheles terhadap kejadian malaria pada balita. j kesehatan lingkungan 2006;2:173-82. wibowo, laksana, mulyanto, et al incidence of malaria is clustered inverse correlation between serum albumin 75 department of internal medicine, faculty of medicine, university of indonesiarsupn cipto mangunkusumo, jakarta, indonesia correspondence : dr. ifael yerosias mauleti, sppd department of internal medicine, faculty of medicine, university of indonesiarsupn cipto mangunkusumo, jakarta, indonesia email: ifaelbsdpapdi@gmail.com univ med 2016;35:75-80 doi: 10.18051/univmed.2016.v35.75-80 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 inverse correlation between serum albumin and serum lactate in adults with dengue ifael yerosias mauleti*, suhendro*, leonard nainggolan*, and martin rumende* background dengue infection is an acute viral infection, in the natural history of which plasma leakage may occur, resulting in shock followed by tissue hypoxia, with death as the final outcome if not treated properly. the purpose of this study was to determine the correlation of the hematocrit, serum albumin concentration, and the presence of pleural effusion or ascites, with hyperlactatemia in adult dengue patients. methods a cross-sectional study was conducted on 62 subjects. the inclusion criteria were: diagnosed dengue viral infection, age >14 years, fever during three consecutive days, and hyperlactatemia. serum albumin was measured on an advia 1800 analyzer using the bromocresol green method. the lactate oxidase method was used to determine serum lactate levels. pleural effusion and/or ascites was determined using an ultrasound scanner (xario ssa-660 a, toshiba, japan). the pearson correlation test was used to analyze the data. results there was no significant correlation between the hematocrit (r=0.11; p=0.301), serum albumin (r=0.003;p=0.981), and pleural effusion or ascites (r=0.75; p=0.692) with serum lactate levels. however, in patients aged >30 years there was a significant inverse correlation between serum albumin and lactate levels (r =0.663;p=0.026). conclusion this study demonstrated a significant inverse correlation between albumin and serum lactate levels in dengue patients aged > 30 years. this can aid in the early recognition and prompt management of at-risk patients to reduce morbidity and mortality. keywords: dengue infection, serum lactate level, hematocrit, albumin, adult dengue patients doi: http://dx.doi.org/10.18051/univmed.2016.v35.75-80 76 mauleti, suhendro, nainggolan, et al serum albumin and lactate in dengue introduction according to data for the year 2013, the number of dengue hemorrhagic fever (dhf) cases in indonesia was 101,218, with an incidence rate of 41.25/100,000 inhabitants, and a case fatality rate of 0.7%, while the numbers of provinces and districts or cities with dengue infections were 33 (100%) and 436 (87.73%), respectively.(1,2) infection with dengue virus may present with a wide clinical spectrum of symptoms, from asymptomatic to symptomatic (dengue fever /dd, dhf, dengue shock syndrome/dss).(3,4) plasma leakage occurs in the critical phase, i.e. on days 3 to 5 of fever, thus lasting 2 times 24 hours.(5,6) if the occurrence of plasma leakage is not recognized earlier and appropriate fluid therapy is not immediately instituted, the complication of dengue hemorrhagic fever, i.e. dengue shock syndrome, will follow, thus increasing the mortality rate of patients with dengue hemorrhagic fever.(7,8) plasma leakage resulting in tissue hypoxia is determined from the hematocrit (ht) and plasma protein concentration (especially albumin), and from the presence of pleural effusion and/or ascites upon imaging, using both plain x-rays of the thorax and ultrasonography of the abdomen.(912) the study of santosa et al.(11) on children with hemorrhagic fever showed an increase in the hematocrit of 20% or higher, which value is currently considered to indicate the occurrence of significant plasma leakage, and was found to correspond to a serum lactate level of >2.4 mmol/ l (sensitivity 79.31%, specificity 77.42%). the conclusion of abovementioned study was that serum lactate level may be used to determine the occurrence of significant plasma leakage in children with dengue hemorrhagic fever. plasma leakage is defined as an increase in hematocrit of >20% above the reference range or the presence of clinical evidence of fluid accumulation.(13) the advantage of determining serum lactate level is that a single assessment can be used for early recognition of plasma leakage, which usually occurs on days 3 to 5 of fever, and for evaluation of the adequacy of fluid therapy in patients with dengue hemorrhagic fever.(11,14) studies such as that of santosa et al.(11) have never been performed on adult patients with dengue infection. no information has been found about clinical studies on the hematocrit, serum albumin concentration, pleural effusion and/or ascites for determination of tissue hypoxia as a result of early plasma leakage in adult patients with dengue infection. therefore the purpose of the present study was to determine the occurrence of tissue hypoxia as a result of early plasma leakage in adult patients with dengue infection by assessment of the hematocrit, serum albumin concentration, pleural effusion and/or ascites, and by testing their correlation with the serum lactate level. methods research design this study was of cross-sectional design and was carried out by collecting data from the medical records of patients with dengue infection who had attended cipto mangunkusumo national central general hospital, jakarta and persahabatan central general hospital, jakarta, from 1 april 2014 until 15 march 2015. subjects of the study data collection was performed by consecutive non-random sampling. the inclusion criteria were patients with a diagnosis of dengue virus infection, aged >14 years, with 3 days fever, and hyperlactatemia. the exclusion criteria were patients with cancer, congestive heart failure, chronic obstructive pulmonary disease, anemia, pneumonia, pericarditis, pleuritis, chronic renal disease, sepsis, hepatic cirrhosis, and undernutrition. the sample size was determined with the correlation coefficient formula,(15,16) i.e. ( ) ( )[ ] 31/r1n 0.5 zz n 2 β-1/2-1 +      −+ + = rι α where n=sample size, z 1-α/2=1.96, z1-β=0.1, and r=0.4. the minimum total sample size required was 62 patients. 77 l a b o r a t o r y a n d u l t r a s o n o g r a p h i c investigations dengue ns-1 antigen was detected using standard diagnostics dipsticks, yielding either positive or negative results. the hematocrit was determined on a sysmex xe – 2100 hematology analyzer by the impedance method, with the flowcytometric results expressed as percentages (%). serum albumin was determined on an advia 1800 analyzer by the bromocresol green method and the results expressed in g/dl. serum lactate concentration was measured on the advia 1800 analyzer by the lactate oxidase method and the results expressed in mmol/l. serum glutamic oxaloacetic transaminase (sgot) and serum glutamic pyruvic transaminase (sgpt) concentrations were determined on a cobas 501 analyzer using an enzymatic method and the results expressed in u/l. pleural effusion and ascites were determined using an ultrasound scanner (xario ssa-660 a, toshiba, japan) with the results given as present or absent. ethical clearance this study obtained ethical clearance from the health research ethics committee, faculty of medicine, university of indonesia, under no. 549/un2.f1/etik/2015. data analysis the kolmogorov-smirnov test was used to determine the normality of the hematocrit and serum albumin data. to determine a correlation of the hematocrit, serum albumin concentration, pleural effusion and/or as cites with serum lactate concentration, the pearson test was used. data analysis was performed with a computer program. the statistical tests were said to be significant if p<0.05. results from a search of the medical records, 70 patients were found, but those meeting the inclusion criteria were only 62 in number. among these, 20 patients came from cipto mangunkusumo national central general hospital, jakarta and 42 were from rsup persahabatan central general hospital, jakarta. among the selected subjects, there were 34 (55%) males and 28 (45%) females, with a mean age of 23.7 ± 8.1 years. after the subjects had been grouped according to age, the age group of <20 years had 25 patients (40%), the age group of 20-29 years had 26 patients (42%), the age group of 30-39 years 7 patients (11%), the age group of 40-49 years 3 patients (5%) and the age group of 50-59 years 1 patient (2%). mean sgot and sgpt concentrations were 80.6 ± 77.7 u/l and 44.0 ± 34.9 u/l, respectively, while mean serum albumin and serum lactate concentrations were 3.9 ± 0.3 g/dl and 2.6 ± 0.8 mmol/l, respectively (table 1). in males, the hematocrit (r=0.235; p=0.181) and serum albumin concentration (r=-0.116; p=0.515) were not significantly correlated with serum lactate level. a similar nonsignificant correlation was shown by the females. on subgroup analysis, the serum albumin concentration of patients aged >30 years had a significant inverse correlation with serum lactate level (r=-0.663; p=0.026) (table 2). this inverse table 1. distribution of general characteristics of subjects (n=62) legend: %: percentage, sd: standard deviation univ med vol. 35 no.2 78 mauleti, suhendro, nainggolan, et al serum albumin and lactate in dengue correlation indicates that the decrease in serum albumin concentration was correlated with increased serum lactate level in subjects with dengue aged 30 years and over. discussion tissue hypoxia may occur in patients with dengue infection who have plasma leakage, which frequently occurs in the critical phase, i.e. on days 3 to 5 of fever.(5,6) if this tissue hypoxia is not recognized earlier, to be managed by appropriate and aggressive fluid therapy, it will increase the number of complications and the mortality rate of these patients.(7,8) currently serum lactate is used as a biomarker to evaluate tissue hypoxia in patients with sepsis.(16) the serum lactate level of a patient is a biomarker for the severity of systemic hypoperfusion, and does not depend on organ failure and shock.(17) increases in arterial or central venous lactate level can also predict in-hospital mortality in a number of disorders, such as infection, sepsis, liver disease, trauma, and cardiac arrest.(18) our study results showed no relationship between the hematocrit and serum lactate level. to ascertain the presence of tissue hypoxia as a result of early plasma leakage in patients with dengue infection, the hematocrit has to be determined.(19,20) in addition, to ascertain the presence of tissue hypoxia as a result of early plasma leakage, the serum albumin concentration has also to be assessed. on subgroup analysis, a statistically significant inverse correlation was found between serum albumin and serum lactate concentrations in patients >30 years of age. this signifies that a decrease in serum albumin concentration is accompanied by an increase in serum lactate level in patients with dengue aged 30 years and over. before the present study was conducted, there was no information on studies of the relationship between serum albumin and lactate level in patients with dengue. patients with severe dengue frequently present with severe plasma leakage and severe organ involvement. the age of >40 years was independently associated with severe dengue in thailand.(21) this is consistent with a previous retrospective analysis from france, where plasma leakage was the most common presentation for adults with severe dengue and that the age of >37 years was a predictor of plasma leakage.(22) one limitation of this study was its crosssectional design, which precludes any assumption of a cause-and-effect relationship. to clarify the role of serum lactate level as indicator of tissue hypoxia in patients with dengue infection, a cohort study should be conducted, so as to minimize the risk of missing data and reduce bias. the clinical implication of our study results is that in adult patients with dengue aged >30 years, serum lactate level should be determined as biomarker for detection of tissue hypoxia resulting from early plasma leakage. use of this biomarker may facilitate early recognition and timely treatment of patients with severe dengue, thereby reducing mortality, hospital burden, and unnecessary hospitalizations as a result of dengue. conclusions the present study identified a significant inverse correlation between serum albumin and lactate concentrations in adult patients with table 2. results of correlation analysis between subgroups and serum lactate level 79 dengue infection aged >30 years. this can aid in the early recognition and prompt management of at-risk patients to reduce morbidity and mortality. conflict of interest the authors declare that they have no competing interests. acknowledgment the investigators thank the managing directors (chief executive officers) of cipto mangunkusumo national central general hospital (rsupn ciptomangunkusumo), jakarta and persahabatan central general hospital (rsup persahabatan), jakarta, for permission to collect patient data in their respective hospitals. references 1. kementrian kesehatan republik indonesia. profil kesehatan indonesia 2013. jakarta: kementrian kesehatan republik indonesia 2013. 2. karyanti mr, uiterwaal cs, kusriastuti r, et al. the changing incidence of dengue haemorrhagic fever in indonesia: a 45-year registry-based analysis. bmc infect dis 2014;14: 412. doi: 10.1186/1471-2334-14-412. 3. malavige gm, fernando s, fernando dj, et al. dengue viral infections. postgrad med j 2004;80:588-601. 4. murray nea, quam mb, wilder-smith a. epidemiology of dengue: past, present and future prospects. clin epidemiol 2013;5:299-309. 5. martina be, koraka p, osterhaus ad. dengue virus pathogenesis: an integrated view. clin microbiol rev 2009;22:564-81. 6. world health organization. dengue guidelines for diagnosis, treatment, prevention and control. geneva: world health organization;2013. 7. da silva mmc, gil lhvg, de azevedo marques et jr, et al. potential biomarkers for the clinical prognosis of severe dengue. mem inst oswaldo cruz 2013;108:755-62. 8. conroy al, gelvez m, hawkes m, et al. host biomarkers are associated with progression to dengue haemorrhagic fever: a nested casecontrol study. int j infect dis 2015;40:45-53. 9. world health organization. handbook for clinical management of dengue. geneva: world health organization; 2012. 10. yacoub s, wills b. predicting outcome from dengue. bmc med 2014;12:147. doi: 10.1186/ s12916-014-0147-9. 11. santosa d, alwi eh, idjradinata ps. validitas kadar laktat darah dalam mendeteksi kebocoran plasma pada infeksi virus dengue anak. majalah kedokteran indonesia 2011;61:58-62. 12. michels m, sumardi u, de mast q, et al. the predictive diagnostic value of serial daily bedside ultrasonography for severe dengue in indonesian adults. plos negl trop dis 2013;7:e2277. 13. thanachartwet v, wattanathum a, oer-areemitr n, et al. diagnostic accuracy of peripheral venous lactate and the 2009 who warning signs for identifying severe dengue in thai adults: a prospective observational study. bmc infect dis 2016;16:46. doi 10.1186/s12879-016-1386-5. 14. puspanjono mt, latie at, sastroasmoro s, et al. comparison of serial blood lactate level between dengue shock syndrome and dengue haemorrhagic fever (evaluation of prognostic value). paediatr indones 2007;47:150-55. 15. madiyono b, moeslichan smz, sastroasmoro s, et al. perkiraan besar sampel. dalam sastroasmoro s, ismael s, editors. dasar-dasar metodologi penelitian klinis. 4th ed. jakarta: sagung seto 2011:p. 348-82. 16. shapiro ni, howell md, talmor d, et al. serum lactate as a predictor of mortality in emergency department patients with infection. ann emerg med 2005;45:524-8. 17. mikkelsen me, miltiades an, gaieski df, et al. serum lactate is associated with mortality in severe sepsis independent of organ failure and shock. crit care med 2009;37:1670–7. 18. okorie on, dellinger p. lactate: biomarker and potential therapeutic target. crit care clin 2011; 27:299–326. 19. de azeredo el, monteiro rq, de-oliveira pinto lm. thrombocytopenia in dengue: interrelationship between virus and the imbalance between coagulation and fibrinolysis and inflammatory mediators. mediators inflamm 2015; article id 313842, 16 pages http://dx.doi.org/10.1155/2015/313842. 20. balasubramanian s, janakiraman l, kumar ss, et al. a reappraisal of the criteria to diagnose plasma leakage in dengue hemorrhagic fever. indian pediatr 2006;43:334-9. univ med vol. 35 no.2 80 mauleti, suhendro, nainggolan, et al serum albumin and lactate in dengue 21. thanachartwet v, oer-areemitr n, chamnanchanunt s, et al. identification of clinical factors associated with severe dengue among thai adults: a prospective study. bmc infect dis 2015;15:420. doi: 10.1186/s12879015-1150-2. 22. thomas l, brouste y, najioullah f, et al. predictors of severe manifestations in a cohort of adult dengue patients. j clin virol 2010;48: 96–9. oktavianus 112 *biomedical science department, faculty of medicine, muhammadiyah university of semarang, indonesia **maternal fetal medicine department, kk women’s and children hospital, singapore ***antenatal medical clinic department kk women’s and children hospital, singapore ****maternal fetal medicine department, kk women’s and children hospital, singapore correspondence: dr. yanuarita tursinawati, msi, med biomedical science department, faculty of medicine, muhammadiyah university of semarang, jl. wonodri sendangraya 2a, semarang, central java, indonesia phone: +62243551894 email : yanuarita.tursinawati11@gmail.com univ med 2015;34:112-20 doi: 10.18051/univmed.2015.v34.112-120 abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 backgound invasive prenatal diagnosis (pnd) through amniocentesis and chorionic villus sampling (cvs) can detect down syndrome. pregnant women usually experience a variety of psychological responses associated with invasive pnd. this study is intended to assess depression, anxiety and stress levels and the factors related to their psychological responses in pregnant women with invasive prenatal diagnosis of down syndrome. methods a cross sectional study was conducted at kandang kerbau women’s and children’s hospital, singapore. the psychological responses of 70 women undergoing pnd were assessed by depression anxiety stress scale 21 (dass 21) questionnaire. a multiple linear regression analysis was used to analyze association between knowledge and perceived risk with psychological responses (ci 95% and significance value p<0.05). results more than half of the participants had normal anxiety (55.7%), stress (72.8%), depression levels (65.8%). the results revealed significantly higher level of anxiety in women with gestational age >13 weeks who had pursued amniocentesis. women with no previous children had higher levels of depression and stress. women who pursued amniocentesis had significantly higher anxiety scores compared to women undergoing cvs (p=0.015). conclusions women’s psychological responses are associated with gestational age, type of procedure and parity. the level of anxiety increased in women who underwent amniocentesis for diagnosis of down syndrome. knowledge and perceived risk of having a baby with down syndrome do not seem to have psychological effects to women. keywords: prenatal diagnosis, psychological responses, down syndrome, pregnant women amniocentesis increases level of anxiety in women with invasive prenatal diagnosis of down syndrome yanuarita tursinawati*, serene thain**, christina choi***, and george seow heong yeo**** 113 univ med vol. 34 no.2 amniocentesis meningkatkan derajat ansietas pada ibu hamil yang menjalani diagnosis prenatal invasif sindrom down latar belakang ibu hamil biasanya akan menunjukkan respon psikologis yang bervariasi berkaitan dengan prosedur diagnosis prenatal invasif yang berupa amniocentesis dan chorionic villus sampling (cvs) pada sindrom down. penelitian ini bertujuan menilai level depresi, kecemasan dan stress serta faktor faktor yang mempengaruhi respon psikologis ibu hamil yang menjalani diagnosis prenatal invasif pada ibu hamil yang menjalani diagnosis prenatal invasif sindrom down. metode studi cross sectional dilakukan di rumah sakit ibu dan anak kandang kerbau, singapura. respon psikologis 70 ibu hamil dinilai dengan kuesioner depression anxiety stress scale 21 (dass 21). analisis regresi linier digunakan untuk menganalisis hubungan antara pengetahuan dan perkiraan resiko (perceived risk) dengan respon psikologis (dengan interval kepercayaan 95% dan nilai signifikan p<0,05). hasil lebih dari setengah partisipan memiliki level normal dari depresi (65,8%), kecemasan (55,7%) dan stress (72,8%). hasil menunjukkan adanya tingkat kecemasan lebih tinggi secara signifikan pada ibu hamil yang menjalani amniocentesis dibandingkan ibu hamil yang menjalani cvs (p=0,015). ibu hamil yang yang belum pernah memiliki anak memiliki tingkat depresi dan stress lebih tinggi secara signifikan. ibu hamil yang menjalani prosedur amniocentesis memiliki nilai kecemasan secara bermakna dibandingkan ibu hamil yang menjalani cvs (p<0,015). kesimpulan tingkat respon psikologis ibu hamil yang lebih tinggi dapat dihubungkan dengan usia kehamilan, jenis prosedur dan paritas. tingkat kecemasan yang lebih tinggi ditemukan pada ibu hamil yang menjalani amniocentesis. pengetahuan dan perceived risk tampaknya tidak berpengaruh terhadap respon psikologis ibu hamil. kata kunci : diagnosis prenatal invasif, respon psikologis, sindrom down, ibu hamil abstrak introduction prenatal diagnosis (pnd) through amniocentesis and chorionic villus sampling (cvs) has been performed in singapore for several years. before pregnant women undergo invasive prenatal diagnosis, they should ideally be counseled by a trained nurse counselor or their obstetrician to obtain sufficient information about the indications for pursuing invasive testing, details of the procedural technique, risks of the invasive procedure, as well as the accuracy and limitations of the results. despite receiving counseling, women usually experience a variety of psychological responses such as depression, anxiety and stress, deriving from their fears of procedural complications, pain, or worries over possible adverse outcomes from the final test results.(1,2) the study by allison et al.(3) revealed that women undergoing pnd procedures experienced higher levels of stress and anxiety. a study by el hage et al.(4) also proved that women in the amniocentesis group expressed significantly higher level of anxiety and depression than those in the control group. women’s psychological responses towards invasive prenatal diagnosis can 114 be influenced by factors such as their knowledge and perceived risk of having an abnormal baby. there are conflicting reports related to the a s s o c i a t i o n b e t w e e n k n o w l e d g e a n d psychological responses. several studies claim t h a t p r e g n a n t w o m e n w i t h i n a d e q u a t e information and less knowledge about prenatal testing had higher anxiety scores than women with more knowledge of prenatal diagnosis.(5) on the contrary, a study by rowe et al.(6) claims that anxiety levels of pregnant women who were well informed were not significantly different from those who were poorly informed.(6) in addition, psychological responses are also influenced by women’s perceived risk of having an abnormal baby. women who perceive themselves to be at greatest risk of having an abnormal baby tend to have higher anxiety levels.(7) women’s knowledge, perceived risk and psychological responses of invasive testing influence their decisions on whether or not to pursue the test. there is a need to understand women’s psychological responses in order to provide psychological support, sufficient information and clear communication of risk to help them make informed decisions about the procedure. all women are at risk of having a baby with a chromosomal abnormality, the most common being down syndrome. screening tests determine if the baby has an increased risk of having a particular problem such as down syndrome or a neural tube defect. they are not diagnostic and an increased risk result does not mean the baby will definitely be affected. none are perfect, sometimes screening tests miss the conditions they are meant to detect. if a screening test picks up an increased risk, then there are diagnostic tests such as cvs, amniocentesis or ultrasound, that can sort out whether or not the baby has the disorder. prenatal diagnostic tests should not be considered routine, but rather offered as a choice to women.(8) therefore, this study is conducted to assess depression, anxiety and stress status and their relationship with invasive pnd of down syndrome. methods research design and setting an analytic study with cross sectional design was performed to assess women’s psychological responses towards invasive prenatal diagnosis of down syndrome and the factors related to their psychological responses s u c h a s s o c i o d e m o g r a p h i c a n d o b s t e t r i c characteristics, knowledge and perceived risk. this study was conducted in kandang kerbau women’s and children’s hospital (kkh), singapore between may 2012 and july 2012. research subjects all patients who had been counseled by a trained nurse counselor or their obstetrician and were proved to be at high risk for the first trimester screening test or the second trimester maternal serum screening test and opted for invasive pnd were recruited for this study during the period of research. a total of 70 pregnant women who consisted of a convenience series of women referred for amniocentesis and cvs to the kkh, singapore, constituted the sample. we included all women without considering whether or not it was their first pnd procedure and included only singleton pregnancies. we excluded women with incapacity to consent or to understand the study. only the participants who were sufficiently fluent in english were studied to complete the questionnaires. measurements each participant was administered a questionnaire that included sociodemographic characteristics such as age, marital status, career and education levels, and obstetric characteristics such as parity, gravidity and history of previous cvs procedures. the next questionnaire consisted of three parts. the first part of the questionnaire contained several questions about women’s knowledge of the invasive procedure, risk of procedure, waiting time for the results and possible findings from the invasive test. knowledge was assessed by a tursinawati, thain, choi, et al amniocentesis and invasive prenatal diagnosis 115 univ med vol. 34 no.2 modified version of a questionnaire by schoonen et al.,(9) which consisted of 15 questions that participants had to answer with either “true”, “false” and “do not know”. the “correct” answer was given 1 point, while “not correct” and “do not know” was given 0 points. scores between 12 and 15 were categorized as high level of knowledge, scores between 9 and 11 as moderate level of knowledge and scores between 0 and 8 as low level of knowledge. the second part of the questionnaire consisted of a question that asked patients to indicate their perceived risk of having a baby with down syndrome on a likert scale. the question was written as follows: “based on your own views and opinions, how would you rate your chances of having a baby with down syndrome?” (1= no chance to 5 = very high chance). the measurement results were divided into three groups, i.e. high risk, low risk and no chance. the last part was performed to assess women’s psychological responses which were measured by short term depression anxiety stress scale 21 (dass 21). the dass 21 consisted of three subscales: depression, anxiety and stress. each subscale contained seven items which related to a symptom of depression, anxiety and stress. the participants were asked to consider how much each statement applied to them in the past week. each item was scored from 0 (did not apply to me at all) to 3 (applied to me very much). the total answer points for each category (depression, anxiety and stress) were summed up, and multiplied by 2. based on recommended cutoff scores, the interpretation of dass 21 was as follows: depression: 0-9 (normal), 10-13 (mild), 14-20 (moderate), 21-27 (severe) and > 28 (extremely severe). anxiety: 0-7 (normal), 8-9 (mild), 10-14 (moderate), 15-19 (severe) and > 20 (extremely severe). stress: 0-14 (normal), 1518 (mild), 19-25 (moderate), 26-33 (severe) and >37 (extremely severe). data analysis independent two sample t-test was used to compare the mean values of knowledge, perceived risk and psychological responses between different groups (age, gestational age, occupation, educational level, parity, type of procedure, history of previous procedure), p<0.05 was considered as statistically significant. ethical approval written ethical approval was obtained from the ethical review board of the kandang kerbau women’s and children’s hospital (kkh), singapore. informed consent was obtained before the participants filled in the questionnaires. results the sociodemographic and obstetric characteristics such as parity, gravidity and history of previous procedure of the participants are presented in table 1. among these women, 41.4% who were > 35 years old pursued amniocentesis/cvs. most of participants who accepted this procedure were chinese (71.4%), married (94.3%), career women (71.4%) and graduated from college (74.3%). the majority of these never had experience with amniocentesis/ cvs before (85.7%). table 2 shows that 52.9% of participants had a high level of knowledge and low perceived risk of having a baby with down syndrome. the psychological responses of all participants are shown in table 3. two thirds of the participants scored in the normal range of the dass 21 depression subscale (65.8%). more than half of the participants also had normal ranges of the anxiety (55.7% of participants) and stress (72.8% of participants) either in the cvs or the amniocentesis group. we also examined the association between sociodemographic and obstetric characteristics with psychological responses. women with no previous children had significantly higher levels of depression (p=0.015) and stress (p=0.002) compared to women who had previous children. our results also showed that women with gestational age >13 weeks had higher anxiety levels than those at <13 weeks (p=0.043). women who pursued amniocentesis also had significantly higher anxiety scores than those pursuing cvs (p=0.015) (table 4). 116 discussion invasive prenatal diagnosis may result in a range of psychological responses such as depression, anxiety and stress which can be influenced by many factors. several studies proved that a higher level of knowledge was associated with a lower level of emotional distress and anxiety.(5) on the contrary, our study revealed that the association between women’s table 1. distribution of sociodemographic and obstetric characteristics by invasive prenatal diagnosis of down syndrome table 2.distribution of knowledge, perceived risk for invasive procedure of down syndrome in pregnant women tursinawati, thain, choi, et al amniocentesis and invasive prenatal diagnosis 117 univ med vol. 34 no.2 table 3. distriburtion of psychological responses in pregnant women knowledge and their psychological responses was statistically insignificant. in our study, 52.9% o f p a r t i c i p a n t s p o s s e s s e d h i g h l e v e l s o f knowledge about the invasive procedure. this proper knowledge is believed to be obtained from adequate counseling from the nurse counselor or kkh obstetrician. pregnant women should be provided with sufficient information in the counseling session, so they are able to make informed decisions to undergo invasive pnd. in the counseling session, the counselor also explains to the patients the actual risk of the prenatal screening test. however, women may have their own perception about the risk which may be influenced by many factors such as age, education, socioeconomic, and psychological table 4. association of sociodemographic and obstetric characteristics and psychological responses in pregnant women 118 factors.(10,11) women’s perceived risk of having a baby with down syndrome were significant determinants of their anxiety level as reported by berg et al.(7) participants who perceive themselves as having a lower or no chance for having a baby with down syndrome might be influenced by their own optimism or avoidance. optimism is often associated with the reduction, elimination or management of the stressors d u r i n g p r e g n a n c y. ( 1 2 , 1 3 ) c o n t r a r y t o t h a t hypothesis, our study revealed that women’s perceived risk for having a baby with down syndrome did not affect their level of depression, anxiety or stress. this might be due to a lack of u n d e r s t a n d i n g o f t h e r i s k o r p o o r r i s k information. most of the participants did not demonstrate depression, anxiety and stress. these participants may have used adaptive rather than maladaptive coping strategies to cope with the stressful situation.(14) women who are optimistic tend to use problem focused coping strategies more frequently which are characterized by acceptance of the situation, humor or showing evident positive results on their life.(12,15,16) a previous study by podolska et al.(17) revealed that pregnant women who used emotion-concentrated styles of coping in stressful situations increased the risk of perinatal depression. thus, further study is needed to know the kind of coping strategy that participants use to cope with their stress. more than half of the participants endorsed the normal state of depression/anxiety/stress in the dass21 subscale, which might also indicate that the invasive procedure had less influence on their psychological status. apart from the knowledge and perceived risk, the results of this study revealed that psychological responses were significantly associated with other factors, such as gestational age and type of procedure. women who pursued amniocentesis demonstrated higher levels of anxiety than women who had cvs. our results are similar to those of other studies, confirming that women pursuing invasive pnd experienced higher levels of anxiety and stress.(18,19) a disadvantage of the amniocentesis procedure is that it is performed after 17 weeks of gestational age, and the final results are available relatively late in pregnancy. the waiting time for the test results coupled with the late gestational age of the pregnancy may make patients more distressed, particularly since termination of pregnancies in singapore is only feasible before 24 weeks gestational age. the waiting time for having the pnd test results is approximately one month, which can be a time when women do not feel happy about their pregnancy, repress the pregnancy, and avoid to think about the pregnancy until the good results of the pnd are confirmed.(20) the patients’ raised anxiety level may be caused by the possible increased risk of maternal morbidity associated with late termination or due to the pregnant woman’s attachment to the fetus.(21,22) maternal fetal attachment starts in early pregnancy, increases during the pregnancy and seems to be most intensive in the last trimester.(23) this theory is in line with our results revealing that the more advanced the gestational age, the higher the anxiety level. the strength of the emotional bond between mother and baby by the second trimester of pregnancy when amniocentesis was performed may be considered to be the cause of the women’s anxiety. another factor that may influence psychological responses is parity. women with no previous child exhibited significantly more psychological distress than women who had previous children. our results confirm previous studies by csaba et al.(2) and sockol et al.(24) which found that higher degrees of anxiety are associated with nullipara (first time mothers). physiological, social, financial problems and emotional changes may be potential sources of stress and worry in pregnant women and may be heightened if the pregnancy is at risk for an unhealthy baby. based on the results of our study, women with no previous children are more susceptible to psychological distress because it is their first time to become parents. some limitations of this study should be acknowledged. firstly, the sample size was tursinawati, thain, choi, et al amniocentesis and invasive prenatal diagnosis 119 univ med vol. 34 no.2 limited and used convenience sampling strategy. this study was of cross sectional design, in which psychological responses and risk factors were assessed simultaneously, and it is probable that women simply reflect the presence of depression, anxiety and stress at that time. another limitation of this study is the lack of sociodemographic diversity because this study was located in one hospital. conclusions this study demonstrates that anxiety levels i n c r e a s e d i n w o m e n w h o u n d e r w e n t amniocentesis for diagnosis of down syndrome. women who demonstrate severe and extremely severe degrees of depression, anxiety and stress s h o u l d b e a t t e n d e d t o a n d s u p p o r t e d b y professionals to help them to cope their stress. further research needs to be conducted in order to observe the development of psychological responses of women over the course of their pregnancy. acknowledgements this research is a part of the attachment program under the collaboration between kk women’s and children’s hospital, singapore and the medical faculty of diponegoro university, semarang sponsored by beasiswa unggulan bpkln, ministry of education, republicof indonesia. we are grateful to dr.abe susanto, prof. dr. sultana mh faradz, phd and dr. tri indah winarni for their support and constant encouragement extended throughout this program. we would like to extend our thanks to wei xing for providing comments on this manuscript. we are thankful to the genetic counselors (ms.wendy goh, ms.tanjong, ms. kelly) and all staff in the antenatal diagnosis center at kk hospital for their valued support and assistance. last, we would like to express our gratitude to the women who participated in this research. conflict of interest the authors declare that they had no support from any organization for the submitted work, no financial support, and no other relationship or activities that could appear to have influenced the submitted work. references 1. kowalcek i. stress and anxiety associated with prenatal diagnosis. best pract res 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its impact on mental and physical well being. clin pract epidemiol ment health 2012;6:25-9. doi: 10.2174/174501790100601 0025. 13. nes ls, segerstrom sc. dispositional optimism and coping: a meta-analytic review. pers soc psychol rev 2006;10:235–51. 14. milic bb, tamara md, karin ko, et al. stress and anxiety in relation to amniocentesis: do women who perceive their partners to be more involved in pregnancy feel less stressed and anxious? croat med j 2010;51:137-43. 15. rasmussen hn, wrosch c, scheier mf, et al. self-regulation processes and health: the importance of optimism and goal adjustment. j personal disord 2006;74:1721-48. 16. schou i, ekeberg o, ruland cm. the mediating role of appraisal and coping in the relationship between optimism-pessimism and quality of life. psychooncology 2005;14:718-27. 17. zm podolska, majkowicz m, sipak so, et al. ways of coping in stressful situation and anxiety state or anxiety trait among women with symptoms of perinatal depression. ginekol pol 2009;80:201-6. 18. v beck, enzlin p, done e, et al. psychosocial aspects of invasive fetal therapy as compared to prenatal diagnosis and risk assessment. prenat diagn 2013;33:334-40. 19. sarkar p, bergman k, o’connor tg, et al. maternal antenatal anxiety and amniotic fluid cortisol and testosterone: possible implication for foetal programming. j neuroendocrinol 2008;20: 489-96. 20. durand ma, stiel m, boivin j, et al. information and decision support needs of parents considering amniocentesis: interviews with pregnant women and health professionals. health expect 2010;13:125-38. doi: 10.1111/j. 1369-7625.2009.00544.x. 21. coleman pk, coyle ct, rue vm. late term elective abortion and susceptibility to posttraumatic stress symptoms. j pregnancy 2010. article id 130519, 10 pages, 2010. doi: 10.1155/2010/130519. 22. kersting a, kroker k, steinhard j, et al. psychological impact on women after second and third trimester termination of pregnancy due to fetal anomalies versus women after preterm birtha 14 month follow up study. arch womens ment health 2009;12:193-201. 23. alhausen jl. a literature update on maternalfetal attachment. j obstet gynecol neonatal nurs 2008;37:315-28. doi: 10.1111/j.1552-6909. 2008.00241.x. 24. sockol le, c neil e, jacques pb. the relationship between maternal attitudes and symptoms of depression and anxiety among pregnant and postpartum first-time mothers. arch womens ment health 2014;17:199-212. tursinawati, thain, choi, et al amniocentesis and invasive prenatal diagnosis maria lina 111 * pusat aplikasi teknologi isotop dan radiasi (patir), badan tenaga nuklir national (batan) ** bagian mikrobiologi, fakultas kedokteran, universitas indonesia korespondensi adra. maria lina rosilawati, m.biomed pusat aplikasi teknologi isotop dan radiasi (patir), badan tenaga nuklir nasional (batan) jl. cinere pasar jumat po box 7002 jkskl jakarta 12070 email: marialinarosilawati@yahoo.com universa medicina 2007; 26: 111-9. universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 abstrak maria lina rosilawati*a dan budiman bela** latar belakang teknik biologi molekuler seperti teknik reverse transcription – polymerase chain reaction (rt-pcr) dot blot hybridization dengan pelacak dna berlabel biotin dapat mendeteksi human immunodeficiency virus (hiv) dalam serum darah. teknik ini selanjutnya dapat diterapkan untuk skrining hiv donor jaringan biologi terutama dari bank jaringan riset badan tenaga nuklir nasional (batan), seperti amnion, allograft steril radiasi, melalui darahnya. penelitian ini bertujuan untuk membandingkan sensitivitas metode rt-pcr hibridisasi dot blot dengan pelacak dna berlabel biotin dan rt-pcr elektroforesis gel agarosa untuk deteksi hiv. metode penelitian ini menggunakan serum darah dari rumah sakit ketergantungan obat (rsko) fatmawati. jumlah serum yang dipakai sebanyak 55 sampel terdiri dari 5 sampel negatif hiv hasil uji serologi dengan rapid test dan 50 sampel dengan enzyme linked immunoassay (elisa). ekstraksi rna hiv sampel darah dilaksanakan menggunakan kit rna viral extraction sedangkan teknik one step rt-pcr digunakan untuk amplifikasi dna. hasil hasil penelitian menunjukkan pada 55 sampel yang diuji baik dengan teknik rtpcr elektroforesis gel agarosa maupun rt-pcr hibridisasi dot blot, 43 sampel positif mengandung hiv. hasil rt-pcr hibridisasi dot blot jauh lebih jelas dibanding dengan rt-pcr-elektroforesis gel agarosa. hal ini terlihat munculnya dot hitam tebal pada film sedangkan pada gel agarosa pita dna tampak tipis untuk beberapa sampel positif hiv yang sama kesimpulan teknik rt-pcr hibridsasi dot blot dengan pelacak dna berlabel biotin lebih sensitif dibanding dengan rt-pcr elektroforesis gel agarosa untuk mendeteksi hiv. kata kunci : hiv, rt-pcr, hibridisasi dot blot, serum darah teknik reverse transcription – polymerase chain reaction (rt-pcr) dan hibridisasi dot blot dengan pelacak dna untuk deteksi human immunodeficiency virus (hiv) dalam serum darah 112 rosilawati deteksi hiv * center for the application of isotopes and radiation technology, national nuclear energy agency. ** department of microbiology, faculty of medicine, university of indonesia correspondence adra. maria lina rosilawati, m.biomed center for the application of isotopes and radiation technology, national nuclear energy agency jl. cinere pasar jumat po. box 7002 jkskl jakarta 12070. phone: 7690709 ext. 214 email: marialinarosilawati@yahoo.com universa medicina 2007; 26: 111-9. use of reverse transcription – polymerase chain reaction (rt-pcr) and dot blot hybridization with biotinylated labeled dna probe techniques to detect human immunodeficiency virus (hiv) in blood serum abstract maria lina rosilawati*a and budiman bela** background human immunodeficiency virus (hiv) in blood serum can be detected by means of molecular biology techniques such as reverse transcription – polymerase chain reaction (rt-pcr) and dot blot hibridization with a biotinylated labeled dna probe. the techniques subsequently can be applied to screen hiv from blood of biological tissues such as radiation sterilized amnions and. allografts especially from national nuklir energy agency research tissue bank. purpose of this research is, to detect hiv by comparing the sensitivity of the rt-pcrdot blot hybridization with a biotinylated labeled dna probe and rt-pcr-agarose gel electrophoresis methods. methods samples used in this research were blood sera from fatmawati drug dependent hospital. the amount of the samples were 55 blood sera consisted of 5 negative and 50 positive hiv samples which were analysed with the rapid test and enzyme linked immunoassay (elisa) serological assays, respectively. hiv rna in blood serum was extracted by means of rna viral extraction kit while one step rt-pcr technique was performed for dna amplification process. results results of this research revealed that the hiv positive could be detected on 43 of 55 samples either by rt-pcr agarose gel electrophoresis or rt-pcr dot blot hybridization techniques. the detection result of rt-pcr dot blot hybridization appeared much more clear than rt-pcr agarose gel electrophoresis techniques. several samples with positive hiv appeared as thick black dots on the film whereas on agarose gel the same samples performed as thin dna bands. conclusion based on the results obtained, the rt-pcr dot blot using biotinylated labeled probe techniques are more sensitive than rt-pcr agarose gel electrophoresis techniques to detect hiv in blood serum. keywords : hiv, rt-pcr, dot blot hybridization, blood serum pendahuluan human immunodeficiency virus (hiv) adalah penyebab acquired immunodeficiency s y n d ro m e ( a i d s ) , y a n g m e r u s a k s i s t e m kekebebalan tubuh sehingga tubuh mudah diserang penyakit lain. ada 2 tipe hiv yaitu hiv-1 dan hiv-2, akan tetapi sebagian besar 113 infeksi disebabkan hiv-1 karena tipe tersebut lebih virulen.(1) sejak ditemukannya, virus tersebut menjadi perhatian dunia karena sindrom yang ditimbulkan dan meningkatnya jumlah pengidap. berdasarkan data who/unaids tahun 2006, dinyatakan 39,5 juta orang hidup dengan hiv dan diperkirakan 8,6 juta orang terdapat di asia dengan sekitar 960.000 orang t e r i n f e k s i b a r u h i v p a d a t a h u n 2 0 0 6 , d i antaranya 630.000 orang meninggal akibat aids.(2) di indonesia infeksi hiv cenderung meningkat dari tahun ke tahun. sampai akhir september 2006 dilaporkan jumlah penderita aids ada 6987 orang dan 1651 (23,63%) di antaranya meninggal dunia. jumlah pengidap hiv dan aids yang dilaporkan lebih sedikit dibanding kondisi sebenarnya. departemen kesehatan mengestimasikan tahun 2006 jumlah orang tertular hiv di indonesia antara 169.000 – 216.000 di mana 46% adalah injection drug u s e r ( i d u ) . ( 3 ) b a r u b a r u i n i p b b m e m p e r k i r a k a n p e n g i d a p h i va i d s d i indonesia telah mencapai 180.000 orang.(4) u n t u k m e n g e t a h u i a p a k a h s e s e o r a n g m e n g i d a p h i v d i p e r l u k a n p e m e r i k s a a n penunjang di laboratorium. metode deteksi y a n g d a p a t d i g u n a k a n u n t u k p e m e r i k s a a n t e r s e b u t a n t a r a l a i n t e k n i k e n z y m e l i n k e d immunoassay (elisa), western blot , dan immunofluorescence assay.(5,6,7) teknik tersebut mendeteksi antibodi terhadap hiv. kelemahan metode tersebut tidak dapat mendeteksi virus pada window period, suatu periode di mana seseorang telah terinfeksi hiv tetapi tubuh belum membentuk antibodi yaitu sekitar 2–4 minggu, bahkan kadang-kadang sampai 3 bulan sehingga berdasarkan uji untuk mendeteksi antibodi, hasilnya negatif.(8-11) pada periode tersebut, pengidap dapat merupakan sumber penularan hiv. virus dapat dideteksi dengan teknik polymerase chain reaction (pcr) dalam p e r i o d e t e r s e b u t , m e s k i p u n d a l a m j u m l a h kecil.(8,12,13) sehubungan dengan penyediaan jaringan biologi steril radiasi seperti allograft maupun amnion oleh bank jaringan riset badan tenaga nuklir nasional (batan), metode d e t e k s i i n i j u g a s a n g a t d i p e r l u k a n u n t u k p e n y e d i a a n j a r i n g a n y a n g a m a n a p a b i l a ditransplantasikan ke resipien.(12-14) penelitian simonds et al,(14) menyatakan organ maupun jaringan dari donor seorang yang meninggal d e n g a n h i v n e g a t i f s e c a r a s e r o l o g i y a n g kemudian ditransplantasikan, 7 orang resipien terinfeksi hiv-1 setelah transplantasi. hasil autopsi dari penderita aids menunjukkan adanya infeksi hiv dalam plasma dan sumsum tulang belakang tetapi dalam fragmen tulang yang sudah diproses virus tidak terdeteksi. hasil proses pcr memperlihatkan adanya dna hiv dalam allograft tersebut yang telah diproses.(13) metode reverse transcription – polymerase chain reaction (rt-pcr) dengan hibridisasi menggunakan pelacak dna spesifik adalah metode deteksi alternatif berdasarkan adanya rna hiv, dapat mendeteksi adanya virus dalam darah secara langsung baik secara kualitatif (ada a t a u t i d a k v i r u s d a l a m d a r a h ) ( 1 2 ) m a u p u n kuantitatif (jumlah virus/jumlah copy rna d a l a m d a r a h ) . ( 1 5 , 1 6 ) b e b e r a p a p e n e l i t i a n menyatakan metode deteksi hiv yang sensitif dan spesifik adalah pcr yang dilanjutkan dengan teknik hibridisasi menggunakan pelacak dna spesifik.(12,17) tujuan penelitian adalah menguasai metode d e t e k s i h i v d a l a m s e r u m d a r a h h a s i l u j i serologi, dengan teknik rt-pcr & hibridisasi dot blot berlabel biotin. teknik ini selanjutnya akan diterapkan untuk deteksi hiv dalam sampel darah jaringan biologi. bahan dan metode sampel penelitian sampel berupa serum darah yang diperoleh dari rumah sakit ketergantungan obat (rsko) fatmawati, jakarta. jumlah sampel adalah 5 universa medicina vol.26 no.3 114 rosilawati deteksi hiv serum darah negatif hiv dengan pemeriksaan serologi secara rapid test dan 50 serum positif hiv secara elisa. serum disimpan pada suhu 4 oc d i l a b o r a t o r i u m r u m a h s a k i t t e m p a t pengambilan sampel lebih dari 30 jam. hal ini dikarenakan pengambilan sampel darah secara bertahap disesuaikan dengan jumlah pasien y a n g a d a d a n j u g a k e t e r b a t a s a n f a s i l i t a s laboratorium rumah sakit. sampel yang sudah t e r k u m p u l ( 1 0 1 5 s a m p e l p e r m i n g g u ) dikirimkan ke laboratorium patir-batan kemudian disimpan pada suhu -20oc (sekitar 3-4 bulan) sebelum dilakukan proses ekstraksi rna. ekstraksi rna e k s t r a k s i r n a d i l a k s a n a k a n d e n g a n menggunakan mini kit dari rna qiaamp viral extraction (qiagen, jerman). sel virus dalam serum darah dilisis dengan larutan bufer lisis y a n g a n t a r a l a i n m e n g a n d u n g g u a n i d i n e tiosianat dengan ph 6,4. pengikatan rna virus dilakukan dengan memasukkan sel virus yang telah dilisis dalam serum ke dalam qiaamp spin column yang dilapisi dengan membran s i l i c a g e l , d i t a m b a h k a n e t a n o l a b s o l u t selanjutnya disentrifugasi. rna pada membran dicuci dengan bufer pencuci yang antara lain mengandung guanidine hidroklorida, sodium azida dan kemudian dielusi dengan bufer elusi yaitu air bebas rnase ya mengandung sodium azida. rna yang diperoleh dapat disimpan pada freezer suhu -20oc. proses rt-pcr te k n i k rtp c r d i l a k u k a n d e n g a n menggunakan kit one step rt-pcr (qiagen). proses reverse transcription rna menjadi cdna dan amplifikasi cdna dilaksanakan dalam 1 tabung. campuran pereaksi rt-pcr (premix) terdiri dari 1x larutan bufer, 0.4 mm masing-masing dntp, 1x larutan q, 0,6 µm tiap primer, 20 unit rnase inhibitor, 2,0 µl one step rt-pcr enzyme mix dan air bebas rnase. rna hasil ekstraksi kemudian ditambahkan ke dalam campuran tersebut. kontrol positif yang digunakan adalah plasmid pnl43 (klon hiv subtype b) sedangkan premix dan bufer elusi dipakai sebagai kontrol negatif. volume master m i x t e r s e b u t a d a l a h 5 0 µ l . p r i m e r y a n g digunakan adalah hi-976c (3’tct gca gct tcc tca ttg atg g5’) dan hi-853f (3’cag cat tat cag aag gag cca c5’) y a n g d i r a n c a n g o l e h b , p e n e l i t i b a g i a n mikrobiologi, fakultas kedokteran universitas indonesia. primer tersebut dirancang dari bagian yang conserved pada daerah gag genom h i v1 ( s u a t u g e n y a n g m e n y a n d i p r o t e i n nukleocapsid hiv) berasal dari 8 strain hiv yang dianggap mewakili setiap tipe. tahap a m p l i f i k a s i m e l i p u t i p r o s e s re v e r s e transcription selama 30 menit pada suhu 37oc, initial pcr activation step pada suhu 95oc selama 15 menit. tahapan selanjutnya untuk setiap siklus terdiri dari denaturasi selama 30 detik 94oc, annealing pada suhu 56oc, 30 detik dan extension pada suhu 72oc, 45 detik. jumlah s i k l u s y a n g d i p e r l u k a n a d a l a h 4 0 s i k l u s . extended extension selama 7 menit pada suhu 72oc merupakan tahap akhir dari proses pcr. proses elektroforesis hasil rt-pcr dideteksi dengan teknik elektroforesisi gel agarosa 1,5% dalam larutan buffer tris-borat-edta (tbe). visualisasi h a s i l e l e k t r o f o r e s i s d i l a k u k a n d e n g a n uv transilluminator setelah gel diwarnai dengan larutan etidium bromida. hibridisasi dot blot, konjugasi dan visualisasi f r a g m e n d n a h a s i l p c r s e t e l a h didenaturasi dengan pemanasan, dispotkan pada membran nilon hybond n+ menggunakan dot blotter . dna pada membran difiksasi dengan pemanasan pada suhu 80oc selama 2 j a m d a n k e m u d i a n d i l a k u k a n p r o s e s 115 prehibridisasi dalam larutan 5x sspe, 5x l a r u t a n d e n h a r d t d a n 0 , 5 % s d s . p r o s e s hibridisasi dilakukan pada suhu 50oc selama 1-2 jam dengan menggunakan larutan yang s a m a y a n g d i t a m b a h d e n g a n p e l a c a k oligonukleotida berlabel biotin [biotin teg tdg grg gac ayc arg sag cha trc a – 3 ’ ; d ( a g t ) , r ( a g ) , y ( c t ) , s ( g c ) , h(act)] sehingga konsentrasi pelacak tersebut menjadi 10 pmol/ml. pelacak tersebut juga d i r a n c a n g o l e h p e n e l i t i y a n g s a m a y a n g m e r a n c a n g p r i m e r d a l a m p e n e l i t i a n i n i . membran selanjutnya dicuci 2 kali dengan larutan 2 x sspe, 0,1% sds dan 1 kali dengan larutan 1 x sspe, 0,1% sds. proses konjugasi dilaksanakan dengan menambahkan streptavidin p e ro k x i d a s e pada membr an dalam larutan pencuci sehingga konsentrasi enzim tersebut menjadi 10µg/ml selama 1 jam pada suhu ruang. pencucian membran dilakukan 4 kali dalam larutan dan suhu yang sama. hasil hibridisasi d i d e t e k s i d e n g a n m e n a m b a h k a n l a r u t a n e n h a n c e c h e m i l u m i n e s c e n s ( e c l ) p a d a membran selama 1 menit. visualisasi dilakukan dengan memaparkan membran pada hiperfilm dalam kaset selama 1-2 jam dalam ruang gelap. f i l m k e m u d i a n d i p r o s e s d e n g a n l a r u t a n developer selama 1-5 menit dan larutan fixer s e l a m a 2 5 m e n i t , d i c u c i d e n g a n a i r d a n dikeringkan. hasil hasil rt-pcr dna sampel serum darah dan kontrol positif (pnl43) dengan primer oligonukleotida hi-976c dan hi-853f dapat dilihat pada gambar 1. dari 50 sampel serum darah yang positif secara elisa, 43 sampel dan kontrol positif (pnl43) menunjukkan hasil rt-pcr positif yaitu adanya fragmen/pita d n a b e r u k u r a n 11 8 b p p a d a h a s i l elektroforesis gel agarosa. (gambar 1: gel bagian atas (a), lajur 2-6, 8; gel bagian bawah (b) lajur 1, 3, 4, 6-8). fragmen dna berukuran tersebut tidak tampak pada hasil negatif rtpcr (gambar 1: gel a, lajur 7; gel b, lajur 2 & 5). sampel negatif secara rapid test juga menunjukkan hasil negatif dengan rt-pcr. pelacak oligonukleotida dalam penelitian ini dirancang dari sekuen dna spesifik yang terletak di dalam fragmen dna hasil rt-pcr dengan primer hi976c dan hi853f. hasil rt-pcr yang dilanjutkan dengan hibridisasi dot blot dengan pelacak tersebut dapat dilihat pada gambar 2. universa medicina vol.26 no.3 gambar 1. hasil rt-pcr dan elektroforesis gel agarosa dna pnl43 dan sampel serum darah dengan primer hi 976 dan hi 853 gel a : lajur 1 : marker hae iii�x174 lajur 2 : pnl43 (kontrol positif) lajur 3-6 : sampel serum darah (hasil elisa +) lajur 7 : sampel serum darah (hasil rapid test -) lajur 8 : sampel serum darah (hasil elisa +) gel b : lajur 1 : dna pnl43 (kontrol positif) lajur 2 & 5 : sampel serum darah (hasil rapid test -) lajur 3, 4, 6-8 : sampel serum darah (hasil elisa +) 1 2 3 4 5 6 7 8 116 rosilawati deteksi hiv dot berwarna hitam pada film menunjukkan hasil positif hiv, sedangkan tidak adanya dot berwarna hitam menunjukkan tidak adanya hiv pada sampel darah (gambar 2: dot a6, a10, a11, b2, b4, b6, b7, b10, b11, c8, c9). dari 50 sampel darah positif dengan pemeriksaan elisa, 43 sampel (86%) menunjukkan hasil positif dengan rt-pcr akan tetapi fragmen/pita dna hasil rt-pcr pada beberapa sampel positif terlihat tipis pada gel agarosa (gambar 1: gel a, lajur 5, 6 : gel b, lajur 3, 6). jumlah sampel yang sama, 43 sampel menunjukkan juga hasil positif dengan metode rt-pcr yang dilanjutkan dengan hibridisasi dot blot dengan pelacak berlabel biotin, akan tetapi terlihat semua hasil positif menunjukkan dot hitam tebal termasuk pita dna yang terlihat tipis pada gel agarosa tersebut di atas (gambar 2: dot a9, b3, b8, c2). ketebalan dot dna pada film dan pita dna pada gel agarosa menunjukkan jumlah 1 2 3 4 5 6 7 8 9 10 11 12 a2 : pnl43 (kontrol positif) a3 –a5 , a7-a9 b1, b3, b5, b8, b9, b12 sampel serum darah c1 – c7, c10 (hasil rt-pcr +, elisa +) a6, a10, a11, b6 : sampel serum darah (hasil pcr , rapid test -) b2, b4, b7, b10, b11, c8 : sampel serum darah (hasil pcr -, elisa +) c9 : kontrol negatif keterangan: gambar 2. hasil rt-pcr dan hibridisasi dot blot dengan pelacak oligonukleotida berlabel biotin virus. makin tebal dot atau pita dna makin banyak jumlah virus yang terdeteksi. pembahasan dari hasil penelitian ini terlihat 7 dari 50 sampel serum darah positif hiv dengan teknik e l i s a , m e n u n j u k k a n h a s i l n e g a t i f h i v menggunakan baik dengan teknik rt-pcr elektroforesis gel agarosa maupun dengan rtpcr hibridisasi dot blot dengan pelacak berlabel biotin. beberapa faktor yang mempengaruhi k e t i d a k s e s u a i a n h a s i l d e t e k s i h i v s e c a r a serologi dan molekuler antara lain kemungkinan k a r e n a p e n y i m p a n a n s p e s i m e n s e t e l a h pengambilan darah, jenis spesimen, primer yang digunakan pada rt-pcr. adanya antibodi yang bereaksi silang dengan antigen host atau antigen dari virus-virus yang terkait, juga menyebabkan hasil positif palsu secara serologi.(17) penurunan 117 rna hiv terjadi 30 jam setelah pengambilan darah pada suhu ruang (20 -23 oc) dan stabil dalam plasma pada suhu tersebut selama 24 jam.(18,19) untuk menghindari degradasi rna hiv penyimpanan plasma darah dilakukan pada 4oc selama 30 jam atau -70 atau -80oc untuk jangka lama sampai 1 tahun.(16,20) rna hiv paling stabil dalam plasma dibanding dalam darah (whole blood) dan serum.(20) penurunan rna hiv dalam plasma darah yang disimpan p a d a 2 0 oc d a n 8 0 oc d a l a m 1 2 m i n g g u penyimpanan, tidak menunjukkan perbedaan nyata.(20) jenis spesimen yang digunakan dalam penelitian ini adalah serum darah. spesimen yang paling baik untuk pemeriksaan rt-pcr adalah plasma darah karena mengandung rna hiv 30–80% lebih banyak dari pada serum.(20) menurut sebire et al(16) jumlah rna hiv dalam serum 50% lebih kecil dari plasma karena banyak virion terjebak dalam jalinan fibrin dari bekuan darah saat proses pemisahan d a r a h d e n g a n s e r u m . s e n s i t i v i t a s d a n spesifisitas teknik deteksi hiv secara molekuler seperti rt-pcr sangat ditentukan oleh primer yang digunakan. primer yang dipakai dalam penelitian ini adalah hi-976c & hi-853f dari daerah yang conserved pada gen gag hiv. dalam penelitian ini terlihat sensitivitas baik metode rt-pcr & elektroforesis gel agarosa maupun rt-pcr & hibridisasi dot blot dengan pelacak berlabel biotin adalah 86% apabila dibandingkan dengan metode elisa. meskipun demikian dilihat dari ketebalan pita dna hasil elektroforesis dibandingkan dengan dot hitam hasil hibridisasi dot blot pada beberapa sampel yang sama, menunjukkan teknik hibridisasi dot blot lebih sensitif. sensitivitas rt-pcr dengan menggunakan primer daerah yang conserved pada gen gag tersebut lebih tinggi dibandingkan dengan menggunakan primer dari gen pol ( p r o t e a s e ) y a i t u m a s i n g m a s i n g 8 0 , 8 d a n 65,4%. tingkat sensitivitas tersebut diperoleh dengan membandingkan metode deteksi hiv tersebut dengan pemeriksaan serologi secara rapid test.(21) pemeriksaan hiv berdasarkan deteksi antibodi seperti elisa mempunyai kelemahan karena dapat memberikasn hasil p o s i t i f a t a u n e g a t i f p a l s u . r e a k s i s i l a n g antibodi dari infeksi virus dna, hepatitis karena alkohol, vaksinasi influenza, vaksinasi hepatitis b dapat memberikan hasil positif palsu.(22,23,24) penelitian gasasira et al(25) menunjukkan p e r k e m b a n g a n r e s p o n i m u n y a n g k u r a n g terhadap malaria pada orang-orang dengan usia muda, sangat memungkinkan adanya stimulasi sel b nonspesifik yang memproduksi antibodi yang bereaksi silang dengan antigen hiv-1 pada keadaan tidak ada infeksi hiv. metode rt-pcr elektroforesis dan rtpcr hibridisasi dot blot merupakan metode deteksi hiv yang sangat diperlukan untuk mengatasi keterbatasan metode elisa terutama p a d a w i n d o w p e r i o d . s e h u b u n g a n d e n g a n penyediaan jaringan biologi seperti amnion, allograft steril radiasi, diperlukan jaringan yang berkualitas tinggi, aman jika ditransplantasikan antara lain bebas hiv. menurut fideler et al,(12) hilmy dan lina,(26) allograft dari donor yang terinfeksi hiv yang diiradiasi sinar gamma sampai dengan 25 kgy, dosis yang direkomendasikan international atomic energy agency (iaea) untuk produk kesehatan tidak merusak gen hiv. dna hiv masih terdeteksi dengan menggunakan teknik yang sensitif dan spesifik seperti pcr, tetapi tidak terdeteksi pada dosis 30 dan 40 kgy. jumlah sampel negatif secara serologi dalam penelitian ini sangat terbatas, sehingga belum ditemukan pengidap hiv dalam keadaan window period, yang akan membuktikan teknik rt-pcr & elektroforesis gel agarosa dan rt-pcr & hibridisasi dot blot dengan pelacak berlabel biotin lebih sensitif dari metode elisa. dari penelitian ini terlihat deteksi rt-pcr d e n g a n h i b r i d i s a s i d o t b l o t m e n g g u n a k a n universa medicina vol.26 no.3 118 rosilawati deteksi hiv pelacak berlabel biotin lebih sensitif dibanding d e n g a n e l e k t r o f o r e s i s g e l a g a r o s a d e n g a n pewarnaan larutan etidium bromida. teknik tersebut juga dapat digunakan untuk mendeteksi hiv dalam sampel dengan jumlah banyak sehingga sangat efisien untuk studi surveillance. beberapa penelitian membuktikan penggunaan pelacak dna berlabel radioisotop pada proses hibridisasi ternyata lebih sensitif dan prosedur yang digunakan lebih sederhana dibandingkan d e n g a n p e l a c a k d n a n o n i s o t o p . ( 2 7 ) o l e h karenanya, penggunaan pelacak dna berlabel radioisotop (32p) pada proses hibridisasi dot blot u n t u k d e t e k s i h i v a k a n d i g u n a k a n p a d a penelitian selanjutnya. kesimpulan d e t e k s i h i v d e n g a n t e k n i k rtp c r hibridisasi dot blot menggunakan pelacak dna b e r l a b e l b i o t i n p a d a s a m p e l s e r u m d a r a h ternyata lebih sensitif dibanding dengan rt-pcr e l e k t r o f o r e s i s g e l a g a r o s a m e n g g u n a k a n pewarnaan larutan etidium bromida pemilihan sampel berupa plasma, penyimpanan pada suhu y a n g t e p a t , k e m u n g k i n a n a k a n l e b i h meningkatkan sensitivitas deteksi hiv secara molekuler. ucapan terima kasih terima kasih kepada dr. elly watty, sp.pk, r s k o f a t m a w a t i a t a s b a n t u a n n y a d a l a m penyediaan sampel darah. kepada sdr. rika heryani dan almaida, patir-batan atas bantuan teknis dalam pelaksanaan penelitian ini. daftar pustaka 1. o’brien wa, pomerantz rj. hiv 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hiv-1 test result in a low risk screening setting of voluntary blood donation. jama 1998; 280: 1080-5. 7. lane hc. human immunodeficiency virus. in : rose nr, macario ec, folds jd, lane hc, nakamura rm, editors. manual of clinical laboratory immunology. 5th ed. washington d.c.: asm press; 1997. p. 763-809. 8. abravaya k, esping c, hoenle r, gorzowski j, perry r, kroeger p, et al. performance of a multiplex qualitative pcr lcx assay for detection of human immunodeficiency virus type 1 (hiv1) group m subtypes, group o, and hiv-2. j clin microbiol 2000; 38: 716-23. 9. goldsby ra, kindt tj, osborne ba. aids and other immunodeficiencies. in: kuby immunology. 4th ed. new york: w.h freeman and company. 2000. p. 485. 10. legge a. direct hiv testing of donated blood is inevitable. bmj 1997; 314: 1433. 11. lackritz em, satten ga, aberle-grasse j. estimated risk of transmission of human immunodeficiency virus by screened blood in the united states. n engl j med 1995; 333: 1721-5. 12. fideler bm, 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clin microbiol 1998; 36: 14719. 21. fatmawati dn. reverse transcription polymerase chain reaction (rt-pcr) sebagai uji alternatif untuk deteksi infeksi human immunodeficiency virus type 1 (hiv-1) (tesis). jakarta: program pendidikan dokter spesialis i, fakultas kedokteran, universitas indonesia; 2006. 22. anonymous. human immunodeficiency virus (aids). bremancos diagnostic. inc. available at: http://www.bremancos.com. accessed june 19, 2004. 23. mylonakis e, paliou m, lally m, flanigan tp, rich jd. laboratory testing for infection with the human immunodeficiency virus: established and novel approaches. am j med 2000; 109: 568-76. 24. pasquier c, sandres-saune k, mansury jm, puissant b, viraben r, spenato n, et al. virological exploration of individuals with discordant hiv screening tests. j clin virol 2004; 30: 218-23. 25. gasasira af, dorsey g, kamya mr, havlir d, kiggundu m, rosenthal pj, et al. false-positive results of enzyme immunoassay for human immunodeficiency virus in patient with uncomplicated malaria. j clin microbiol 2006; 44: 3021-4. 26. hilmy n, lina m. effect of ionizing radiation on viruses, proteins and prions. in: phillips go, von versen r, strong dm, nather a, editors. advances in tissue banking. singapore: world scientific publishing co. pte. ltd. 2001. p. 358-75. 27. international atomic energy agency (iaea). organization of radioisotopes based molecular biology laboratory (iaea-tecdoc-1528). vienna, austria: iaea: december 2006. universa medicina vol.26 no.3 frequent coconut milk intake increases the risk of vascular disease in adults 149 *center for community health intervention technology, health research and development board, ministry of health, republic of indonesia **center for clinical epidemiology and applied health technology, health research and development board, ministry of health, republic of indonesia correspondence: sulistyowati tuminah, s.si center for community health intervention technology, health research and development board, ministry of health, republic of indonesia jl. percetakan negara no. 29, jakarta pusat e-mail : sulistyowati@litbang.depkes.go.id/ sdarjoko@yahoo.com univ med 2015;34:149-58 doi: 10.18051/univmed.2016.v35.149-158 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background coconut milk is an essential ingredient of the indonesian cuisine. in view of its saturated fatty acid content, coconut milk has frequently been blamed as a cause of cardiovascular disease. the objective of this study was to determine any association between coconut milk intake and vascular disease among adults. methods a cross-sectional study was conducted in 2011-2012 on adults aged 2565 years. self-reported health and nutritional data were collected by means of questionnaire-based interviews, with the aid of 3-d food models. medical data comprised blood pressure and anthropometry (waist circumference and body mass index), neurological status, ecg, blood glucose and lipid profile. data analysis was done using multiple logistic regression. results a total of 4,187 respondents fullfilled the inclusion criteria. frequent coconut milk intake (>3 times/week) significantly increased the risk of vascular disease by 1.3-fold compared with infrequent coconut milk intake (<2 times/week) (or=1.33; 95% ci: 1.11-1.60; p=0.002) after controlling for age, gender, hypertension, diabetes mellitus and stress. the influence of coconut milk intake on vascular disease risk was lower than that of age, gender, hypertension and diabetes mellitus. conclusion frequent coconut milk intake increased the risk of vascular disease in adults. further investigations are needed about the effect of coconut milk intake on noncommunicable diseases. keywords: coconut milk intake, vascular disease, adult frequent coconut milk intake increases the risk of vascular disease in adults sulistyowati tuminah*, and marice sihombing** doi: http://dx.doi.org/10.18051/univmed.2015.v34.149-158 150 tuminah, sihombing coconut milk risk vascular disease asupan santan yang sering meningkatkan risiko terjadinya penyakit vaskuler pada orang dewasa latar belakang santan merupakan salah satu bahan dalam masakan indonesia. di sisi lain, santan yang merupakan produk kelapa yang sering dianggap sebagai penyebab penyakit pembuluh darah vaskuler karena banyak mengandung asam lemak jenuh. tujuan penelitian ini adalah untuk menentukan hubungan antara asupan santan dengan penyakit vaskuler (pv) [stroke atau penyakit jantung koroner/pjk] pada orang dewasa. metode rancangan penelitian yang digunakan adalah rancangan potong lintang (cross sectional) yang dilaksanakan antara bulan januari 2011 sampai desember 2012 pada responden yang berusia 25-65 tahun. pengumpulan data dilakukan dengan cara: 1). wawancara tentang kesehatan masyarakat menggunakan kuesioner dan wawancara gizi menggunakan kuesioner serta food model, 2). pemeriksaan kesehatan yang meliputi: a). pengukuran tekanan darah dan antropometri (lingkar perut, berat dan tinggi badan), b). pemeriksaan nerologi dan rekam jantung (ekg), c). pemeriksaan darah laboratorium (kadar glukosa darah puasa dan 2 jam post prandial, kolesterol total, hdl, ldl dan trigliserida). regresi logistik ganda digunakan untuk analisis data. hasil data yang memenuhi kriteria inklusi sebanyak 4.187 responden. asupan santan yang sering (≥3 kali/minggu) meningkatkan risiko untuk mengalami penyakit vaskuler secara bermakna sebesar 1,3 kali (or=1,33; 95% ci: 1,11-1,60; p=0,002) dibandingkan dengan asupan yang jarang (<2 kali/minggu) setelah dikontrol oleh umur, jenis kelamin, hipertensi, diabetes dan stres. namun besaran risikonya terhadap penyakit vaskuler tersebut masih lebih kecil dibandingkan dengan faktor umur, jenis kelamin, hipertensi dan diabetes. kesimpulan asupan santan yang sering meningkatkan risiko mengalami penyakit vaskuler pada orang dewasa. penelitian lebih lanjut mengenai asupan santan terhadap penyakit tidak menular masih sangat diperlukan. kata kunci: asupan santan, penyakit vaskuler, dewasa abstrak introduction coconuts have a high content of saturated fatty acids, which is the reason why coconuts and coconut products are frequently categorized as “bad fat” and a cause of heart disease. this is in spite of the fact that the majority of the saturated fatty acids are medium chain fatty acids (c8-c12) with features and metabolism that are different from those of saturated fatty acids of animal origin. medium chain saturated fatty acids in coconuts are not enzymatically hydrolyzed, but can be directly absorbed by the intestinal wall and carried in the circulation to be metabolized in the liver. in the liver, these medium chain fatty acids are converted into energy only and not into cholesterol or adipose tissue.(1) the fatty acid composition of coconut oil has been determined, but only little is known about other coconut products, such as coconut milk, coconut water, coconut cream, and grated coconut meat. coconut water has a low fat content, coconut milk contains 24% fat, whereas coconut cream and grated coconut meat have a fat content of approximately 34%.(2) coconut milk is a product made from coconut meat (endosperm) by 151 univ med vol. 34 no.2 pressing grated coconut meat with water. it has a milky color, being an emulsion of water, protein and oil. the fat content of coconut milk is around 17%, approximately 90-92% of which is saturated. although coconut milk fat is more saturated than most oils and fats, around 2/3 of the saturated fatty acids are medium chain fatty acids. an experimental study by ejike et al.(3) showed that coconut milk did not increase the risk of cardiovascular disease in consumers of coconut milk as food or medication. a case-control study on coronary heart disease (chd) was conducted in the minangkabau community, known for its high coconut consumption. in this study, the controls and cases received a total saturated fatty acid intake of 27 grams/day, equivalent to 129 grams of coconut milk or 31.5 gram of coconut oil. the study results showed that the cases had a high meat intake, such as fish, eggs, beef, chicken, and cow’s milk. the cases with a meat intake of more than 210 grams (upper quartile) had a higher risk of chd in comparison with those with a meat intake below 108 grams (lower quartile). meat intake, total protein, total cholesterol and total carbohydrate are predictors of chd.(4) the indonesian basic health research (riskesdas) data indicate that the highest proportion of deaths from noncommunicable disease is caused by vascular disease, with a mortality rate of 20.5%, comprising stroke (15.4%) and ischemic heart disease (5.1%).(5) the riskesdas results for the province of west java also show that bogor city has a prevalence of 12.1% for heart disease and 1.1% for stroke, which are both higher than the national prevalence of 8.2% fort heart disease and 0.9% for stroke.(6) the purpose of the present study was to find any association between coconut milk intake and vascular disease in adults. in this study, vascular disease was limited to stroke and chd. methods research design this study forms part of an umbrella cohort study on risk factors of noncommunicable diseases (frptm). the study was of crosssectional design and was conducted between january 2011 and december 2012. study subjects the respondents were 25-65 year old permanent residents of five villages (kelurahan) in central bogor subdistrict, bogor city, consisting of kelurahan kebon kalapa, kelurahan babakan pasar, kelurahan babakan, kelurahan ciwaringin and kelurahan panaragan. the inclusion criteria were: 1). males and females, 2). respondents aged 40 years and older who had undergone electrocardiography, 3). respondents aged under 40 years with a history of heart disease or hypertension, or were active smokers, who had undergone electrocardiography, 4). respondents aged 25-65 years who had experienced specific stroke symptoms or sequelae and had undergone neurological examinations. the number of respondents who fulfilled the inclusion criteria was 4.187 persons. data collection data were collected by the who steps (7) method, comprising 1). interviews using a community health questionnaire (on sociodemographic characteristics, medical history of the respondents and their families, and risk factors) and nutritional questionnaire (on frequency and number of foods consumed), 2). measurement of blood pressure and physical or anthropometric assessment (weight, height, and abdominal circumference), 3). laboratory tests (fasting and 2-hour postprandial blood glucose levels, total cholesterol, hdl cholesterol, ldl cholesterol, and triglycerides), 4). electrocardiography and neurological examination. at the time of interview, the respondents were asked if they had ever experienced several symptoms of stroke. those who admitted having had specific stroke symptoms or those who had stroke sequelae were asked to undergo neurologic examination. respondents were diagnosed as having had a stroke if they had a positive history of stroke and had visible stroke sequelae. the diagnoses were 152 tuminah, sihombing coconut milk risk vascular disease confirmed by a neurologist. respondents aged 40 years and over and respondents under 40 years of age who were active smokers or had hypertension or were known to have a history of heart disease, were asked to undergo electrocardiography. the recording was performed by means of an elitech 300g electrocardiograph using the minnesota code and the results were read by a specialist in cardiology and vascular diseases.(8) respondents were diagnosed as having chd if the ekg was abnormal, after confirmation of the diagnosis by the cardiologist. in the present study patients with vascular disease were defined as those who on medical examination were diagnosed as having stroke or chd. vascular disease was categorized as “yes” and “no” (not a vascular disease). respondents were categorized as having a family history of vascular disease if one or more family members had been diagnosed as having vascular disease or had died from vascular disease. family history of vascular disease was categorized as present and absent. emotional stress was assessed using a self reporting questionnaire with 20 items (srq-20).(9) respondents were said to experience stress if they answered “yes” to minimally 6 out of 20 questions in the instrument. the age of the respondents was calculated from the difference in years between their last birthday and date of birth. age was categorized into 4 groups, namely 25-34 years, 35-44 years, 45-54 years and 55-65 years. gender of the respondents was divided into 2 categories, male and female. smoking behavior was divided into 2, i.e. smoker status and smoking frequency. smoking status was divided into 3 categories, i.e. non-smoker, ex-smoker (daily and occasionally) and active smoker (daily and occasionally). smoking frequency was also divided into 3 categories, i.e. never, occasional smoker (past and current) and daily smoker (past and current). intake of high risk foods coconut milk intake was determined by an enumerator from an academy of nutrition using dietary recall and food frequency questionnaire (ffq). coconut milk intake was divided into 4 categories, i.e. never, 1-2 times/week, 3-6 times/ week and >7 times/week. in this analysis, coconut milk intake was divided into 2 categories, i.e. <2 times/week (never and 1-2 times/week) and >3 times/week (3-6 times/week and >7 times/week). high risk food in this study comprised processed meat, eggs, milk, fried snacks, and frying oil. data on consumption of high risk food was obtained by dietary recall and ffq, and were then divided into into 2 categories, i.e. <2 times/week (never and 1-2 times/week) and >3 times/week (3-6 times/week and >7 times/week), except for intake of frying oil. intake of frying oil was categorized into 3, i.e. <2 times/day, 2-3 times/day and >3 times/day. measurements weight (w) was determined using and digital scales. respondents in light clothing were weighed barefoot in the standing position. height (h) was measured in the standing position using a microtoise. body mass index (bmi) was calculated from the weight (w) and height (h) using the formula: bmi = w (kg) / h (m)2 based on the bmi classification for asians according to the international obesity task force (iotf) (10) was divided into 5 categories. these were: underweight (bmi <18.5), normal (18.522.9), overweight (23-24.9), obese i (>25) and obese ii (>30). in this advanced analysis, bmi was divided into 2 categories only, obese (>25) and non-obese (<25). abdominal circumference was measured using non-elastic measuring tape by first determining the midpoint between the lower margin of the last palpable rib and the top of the iliac crest. from this midpoint the measuring tape was then placed horizontally around the abdomen and the result was recorded in cm. the measurement was made at the end of a normal expiration. if the respondents had a prominent abdomen, the measurement was made over the most prominent portion. central obesity was divided based on the cutoff point of abdominal circumference, differentiated by gender. 153 univ med vol. 34 no.2 respondents were categorized as having central obesity if their abdominal circumference was >90 cm for males and >80 cm for females.(10) in the present study, blood pressure was measured twice at 3-minute intervals on the right arm by means of a digital sphygmomanometer. if the two systolic or diastolic blood pressure measurements differed by >10 mm/hg, then a third measurement was made after a break of 10 minutes. hypertension was categorized according to the 8th joint national committee (jnc) guideline for the year 2014. for systolic blood pressure, the categories were normal (<120 mmhg), prehypertension (120-139 mmhg), grade i hypertension (140-159 mmhg), grade 2 hypertension (>160 mmhg). the diastolic blood pressure categories were normal (<80 mmhg), prehypertension (80-89 mmhg), grade i hypertension (90-99 mmhg), and grade ii hypertension (>100 mmhg).(11) blood pressure measurements were performed by paramedics. respondents were said to have hypertension if they had a previous diagnosis of hypertension or were currently taking antihypertensive drugs, or the current blood pressure measurements were above normal. in the present study blood pressure was categorized into 2, i.e. “yes” (hypertension) and “no”. laboratory investigations for determination of blood glucose and lipids, the respondents were asked to fast for 1214 hours from the previous night up to the time of taking blood samples in the morning. the respondents were only allowed to drink water. from the fasting respondents blood samples were drawn from the cubital vein, then the respondents were given a solution of 75 grams of glucose in 250 ml water. to respondents with known diabetes, a 300 calorie food supplement was given instead of the glucose solution. subsequently, 2 hours after glucose loading, a second blood sample was drawn from the cubital vein. evaluation of blood glucose status was carried out according to the guidelines of the national institute of diabetes and digestive and kidney diseases. fasting blood glucose (fbg) was divided into 3 categories, i.e. normal <99 mg/dl, abnormal fasting blood glucose tolerance 100-125 mg/dl, and high fasting blood glucose >126 mg/dl. two-hour postprandial blood glucose was divided into 3 categories, i.e. normal <139 mg/dl, abnormal 2hour postprandial blood glucose tolerance 140199 mg/dl, and high 2-hour postprandial blood glucose >200 mg/dl. respondents were diagnosed as having diabetes mellitus if they had been diagnosed as having diabetes mellitus by health personnel or had fasting blood glucose levels of >126 mg/dl or 2-hour postprandial blood glucose levels of >200 mg/dl.(12) determination of blood lipid levels comprised determination of total cholesterol, ldl cholesterol, hdl cholesterol, and triglycerides. evaluation of blood lipid status was performed according to the guidelines of the national cholesterol education program (ncep 2004) as follows: total cholesterol level was divided into 2 categories, i.e. normal (<200 mg/dl) and high (>200 mg/dl). triglyceride level was divided into 2 categories, i.e. normal (<150 mg/dl) and high (>150 mg/dl). ldl cholesterol level was divided into 2 categories, i.e. normal <100 mg/dl) and high (>100 mg/dl). hdl cholesterol level was divided into 2 categories by gender, i.e. normal for females >50 mg/dl, and for males >40 mg/ dl. respondents were considered to have dyslipidemia if total cholesterol >200 mg/dl, triglycerides >150 mg/dl, ldl cholesterol >100 mg/dl, and hdl cholesterol <50 mg/dl (females) or <40 mg/dl (males).(13) blood chemistry investigation was performed by prodia laboratory in bogor city. data analysis the data were analyzed by a bivariate test (chi square) to determine any association between coconut milk intake and a number of risk factors. significantly associated variables were then analyzed by multiple logistic regression. the level of significance used was 0.05. ethical clearance the protocol of this study was accorded ethical clearance from the ethics commission of 154 tuminah, sihombing coconut milk risk vascular disease the health research and development board, ministry of health, republic of indonesia. results the proportion of vascular disease increased with advancing age, and was twice as high in female respondents than in males (table 1). with regard to biological factors, the proportion of patients with vascular disease was higher in respondents who had central obesity, belonged to the obese category, or had hypertension, dm and dyslipidemia, as compared with respondents with normal biological factors (p=0.000). similarly, the proportion of patients with vascular disease was higher in respondents with a family history of vascular disease in comparison with those without a family history (p=0.048). therefore all these risk factors were included in the multivariate analysis (table 1). the results of bivariate analysis showed that with respect to smoker status, respondents who had more vascular diseases were smokers, either current smokers or ex-smokers (30.5%), in comparison with those who were non-smokers. regarding smoking frequency, respondents who had more vascular diseases were those who smoked, either daily or occasionally (29.1%), in table 1. patients with vascular disease by modifiable and nonmodifiable factors 155 univ med vol. 34 no.2 table 2. distribution of vascular disease patients by behavioral factor comparison with those who never smoked. the proportion of respondents with emotional disturbances had more vascular diseases in comparison with respondents without emotional disturbances (p=0.001) (table 2). according to frequency of consumption of high risk foods, all foods were included in the multivariate analysis, with the exception of eggs (p=0.371) and milk (p=0.715), since these foods did not show a significant association. the highest proportion of patients with vascular disease was found among respondents who consumed coconut milkcontaining foods >3 times/week (19.1%). however, the highest proportion of patients with vascular disease was also found among respondents who consumed prepared meat (18.8%) and fried snacks (18.6%) <2 times/week. the highest proportion of frying oil intake was 23 times/day (19.4%). according to the results of multivariate analysis (table 3), coconut milk intake of >3 times/week significantly increased the risk of vascular disease by 1.3 fold in comparison with coconut milk intake with a frequency of <2 times/ week, after controlling for other risk factors i.e. age, gender, hypertension, diabetes and stress (or=1.33; 95% ci: 1.11-1.60; p=0.002). however, the risk of coconut milk intake for vascular disease was lower than that of age, gender, hypertension, and diabetes mellitus. 156 tuminah, sihombing coconut milk risk vascular disease discussion in this study it was found that frequent coconut milk intake (≥3 times/week) increases the risk of vascular disease as compared with infrequent intake (<2 times/week). this signifies that coconut milk intake ≥3 times/week is already predictive, i.e. increases the risk of suffering from vascular disease, after controlling for other risk factors, i.e. age, gender, hypertension, diabetes mellitus, and stress. coconut milk intake <2 times/ week may be said to be within safe consumption limits. however, the effect of coconut milk containing much saturated fatty acid on the risk of vascular disease is still low in comparison with the four other risk factors, i.e. age, gender, hypertension, and diabetes mellitus. one study showed similar results, where consumption of moderate quantities of coconut oil, as a part of routine diet, may not contribute to the risk for coronary artery disease, either directly by affecting the lipid profile or indirectly by aggravating oxidative stress.(14) saturated fatty acids are fatty acids without double bonds in their hydrocarbon chain, are stable, do not readily oxidize, and are not converted into trans fatty acids or other deleterious compounds. coconuts and their products, such as coconut oil and coconut milk, contain much medium chain saturated fatty acids that are readily absorbable, digestible, and can be rapidly metabolized, therefore they are not found in the circulation, and cannot form vascular plaques (are not atherogenic).(15) the effect of certain foods on chd cannot be predicted merely on the basis of their saturated fatty acid content, since the saturated fatty acids themselves may have an effect on the heart and blood vessels. in addition, most sources of saturated fatty acids have contents that affect the risk of chd.(16) the study conducted by yamagishi et al.(17) found a protective effect of saturated fatty acid intake on total stroke [hr=0.77; 95% ci: 0.650.93; trend p=0.002] and ischemic stroke [hr=0.84; 95% ci: 0.67-1.06; trend p=0,08], but was predictive of myocardial infarction [hr=1,39; 95% ci: 0,93-2,08; trend p=0,046] although the latter effect was not significant. similar findings results were revealed in a metatable 3. association of several risk factors and vascular disease *adjusted odds ratio controlled for variables 157 analysis of prospective epidemiological studies conducted by siri-tarino et al.(18) the investigators found no significant evidence to conclude that saturated fat intake was associated with increased risk of vascular disease, such as chd, stroke and cardiovascular disease (cvd). the study conducted by ekanayaka et al.(19) found that supplementation of coconut milk significantly reduced ldl cholesterol levels and increased hdl cholesterol levels. according to silalahi and nurbaya,(20) coconut oil and coconut milk do not increase ldl cholesterol and are protective for chd. this due to the role of hdl cholesterol in reversing cholesterol transport from the cell membrane to the liver, allowing the liver to remove excess cholesterol from peripheral tissues, thus protecting the organs from atherosclerosis. the results of the study of griel and etherton (21) showed that increases in hdl cholesterol levels also significantly reduced the total cholesterol to hdl cholesterol ratio. a reduction of one unit in the total cholesterol to hdl cholesterol ratio signifies a reduction in the risk of myocardial infarction by 53%. therefore it may be stated that intake of saturated fatty acidcontaining coconut milk is not a risk factor of vascular disease. in the advanced analysis of the baseline data of the cohort study on risk factors of noncommunicable diseases (frptm) there were several limitations. one of these is that in the data collection on smoking, ex-smokers were not asked the number of cigarettes that they had consumed as active smokers. therefore the data on smoking were assigned to 2 variables i.e. smoking status and frequency. smoking status was divided into 3 categories, i.e. non-smokers, ex-smokers (daily and occasional) and active smokers (daily and occasional). smoking frequency was also divided into 3 categories, i.e. never smokers, occasional smokers (past and current) and daily smokers (past and current). furthermore, among the participants in the frptm cohort study there were more females than males, thus influencing the results of the analysis. from the results of this study, the community is expected to take measures for preventing vascular disease as early as possible, particularly with respect to healthy life style, i.e. by performing physical activity (sports), refraining from smoking, and avoiding alcohol consumption. in addition, dietary intake should be of nutritional value and healthy, to avoid suffering from vascular disease. measures for control of vascular disease should be focused on individuals (particularly female pre-elderly and elderly persons) with hypertension, diabetes mellitus, and stress. more studies should be conducted on consumption of coconut products, especially coconut milk, that are related to noncommunicable diseases such as stroke, chd, or diabetes mellitus, due to the high coconut milk consumption by the communities of various regions of indonesia. conclusions frequent coconut milk intake (≥3 times/ week) increases the risk of vascular disease, but the magnitude of the risk is still lower than that for other risk factors such as age, gender, hypertension, and diabetes mellitus. conflict of interest the authors declare that there is no conflict of interest in this study and in the advanced analysis. acknowledgements the research team hereby wish to express their utmost gratitude to dr. eddy darma and dr. siti robiah of the bogor health service, dr. lily, dr. irma, dr. atik, ms tri yuliani, ms anisa, and ms yuli of all cooperating puskesmas in the villages (kelurahan) of kebon kalapa, ciwaringin, panaragan, babakan and babakan pasar. thanks are also due to ms ida paulina of prodia laboratory, prof.dr.dr. budi setianto, sp.jp and prof.dr.dr. bambang bs, sp.jp of harapan kita hospital, and dr. m. kurniawan, sp.s of cipto mangunkusumo national general univ med vol. 34 no.2 158 tuminah, sihombing coconut milk risk vascular disease hospital for guidance and directions in this study. we also thank all health cadres of kelurahan kebon kalapa, ciwaringin, panaragan, babakan and babakan pasar, for their excellent cooperation in data collection, dr. iwan ariawan of fkm-ui for his guidance and directions in data analysis, so that the results can benefit all parties, particularly decision and policy makers, and all st-frptm colleagues for their cooperation in conducting this study. references 1. sulistyowati t. efek perbedaan sumber dan struktur kimia asam lemak jenuh terhadap kesehatan. buletin penelitian kesehatan 2010;38:43-51. 2. pehowich dj, gomez av, barnes ja. fatty acid composition and possible health effect of coconut constituents. west indian med j 2000;49:12833. 3. ejike cecc, nwankwo oo, ijeh ii. consumption of coconut milk did not increase cardiovascular disease risk in mice. int j current res 2010;6:63-4. 4. lipoeto ni, agus z, oenzil f, et al. dietary intake and the risk of coronary heart disease among the coconut-consuming minangkabau in west sumatra, indonesia. asia pac j clin nutr 2004;13:337-84. 5. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar indonesia 2007 : laporan nasional. jakarta: badan penelitian dan pengembangan kesehatan;2008. 6. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar indonesia 2007: laporan provinsi jawa barat. jakarta: badan penelitian dan pengembangan kesehatan;2008. 7. world health organization. the who stepwise approach to noncommunicable disease risk factor surveillance (steps). geneva: world health organization;2014.. 8. prineas rj, crow rs, zhu-ming z, editors. the minnesota code manual of electrocardiographic findings. 2nd ed. london: springer;2010. 9. scholte wf, verduin f, van lammeren a, et al. psychometric properties and longitudinal validation of the self-reporting questionnaire (srq-20) in a rwandan community setting: a validation study. bmc medical research methodology 2011;11:116. 10. razak f, anand ss, shannon h, et al. defining obesity cutoff points in a multiethnic population. circulation 2007;115:2111-8. 11. american academy of family physicians. jnc 8 guidelines for the management of hypertension in adults. am fam physician 2014;90:503-4. 12. the national diabetes information clearinghouse. diabetes overview. bethesda, md: the national diabetes information clearinghouse;2008. 13. lorenzo c, williams k, hunt kj, et al. the national cholesterol education program–adult treatment panel iii, international diabetes federation, and world health organization. definitions of the metabolic syndrome as predictors of incident cardiovascular disease and diabetes. diabetes care 2007;30:8-13. 14. sabitha p, vaidyanathan k, vasudevan dm, et al. comparison of lipid profile and antioxidant enzymes among south indian men consuming coconut oil and sunflower oil. indian j clin biochem 2009;24:76-81. 15. sulistyowati t. efek asam lemak jenuh dan asam lemak tak jenuh “trans” terhadap kesehatan. media penelitian pengembangan 2009;19 suppl 2:s13-20. 16. astrup a, dyerberg j, elwood p, et al. the role of reducing intakes of saturated fat in the prevention of cardiovascular disease: where does the evidence stand in 2010? am j clin nutr 2011;93:684-8. 17. yamagishi k, iso h, kokubo y, et al. dietary intake of saturated fatty acids and incident stroke and coronary heart disease in japanese communities: the jphc study. eur heart j 2013; 34:1225-32. 18. siri-tarino pw, sun q, hu fb, et al. metaanalysis of prospective cohort studies evaluating the association of saturated fat with cardiovascular disease. am j clin nutr 2010;91: 535-46. 19. ekanayaka rai, ekanayaka nk, perera b, et al. impact of a traditional dietary supplement with coconut milk and soya milk on the lipid profile in normal free living subjects. j nutr metab 2013; article id 481068, 11 pages http:/ /dx.doi.org/10.1155/2013/481068 2013:1-11. 20. silalahi j, nurbaya s. komposisi, distribusi, dan sifat aterogenik asam lemak dalam minyak kelapa dan kelapa sawit. j indon med assoc 2011;61:453-7. 21. griel ae, kris-etherton pm. tree nuts and the lipid profile: a review of clinical studies. br j nutr 2006;96 suppl 2:s68–78. diana1 72 department of occupational medicine, medical faculty, trisakti university correspondence adr. diana samara, mkk department of occupational medicine, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2802 email : davidwp@cbn.net.id univ med 2008; 27: 72-7. the association between shift work and body mass index among female nurses april-june 2008april-june 2008april-june 2008april-june 2008april-june 2008 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 abstract universa medicina diana samaraa, lie t. merijanti, reza tandean, and ridwan harrianto possibly due to a disturbance of biological rhythms, shift work is associated with several health problems, particularly an increased risk of coronary heart disease (chd). obesity is a well known independent cardiovascular risk factor and has been shown to be more prevalent among shift workers. the objective of this study was to examine the association between shift work and body mass index in nurses working in one hospital in central jakarta. a cross-sectional study was conducted among 152 female nurses, ranging from 22 to 55 years of age, with determination of body mass index (bmi), and systolic and diastolic blood pressure (sbp and dbp). the results show a mean age of 30.9 ± 7.2 years and mean job experience of 8.9 ± 7.6 years. there was no significant difference in the prevalence of gastrointestinal disorders between shift and day workers. mean bmi (23.1 ± 3.6) and mean job experience (6.9 ± 5.9 years) in shift nurses were significantly different from those in day workers (bmi = 24.8 ± 3.8; job experience 15.4 ± 8.4 years). in shift working nurses the bmi increased significantly in proportion to job experience (p=0.014). in conclusion, the present study has proven that job experience significantly increased bmi in shift working nurses. keywords : shif work, body mass index, job experience, female nurses introduction s h i f t w o r k c a n b e d e f i n e d a s w o r k performed outside the normal daylight hours. shiftwork has been empirically linked to a variety of diseases, although evidence does not suggest an effect on all-cause mortality.(1) three p a t h w a y s h a v e b e e n i m p l i c a t e d i n t h e relationship between shiftwork and disease: (i) disruption of circadian rhythms (leading to sleep/ wake disturbances, desynchronization of internal processes, and increased susceptibility to disease); (ii) disturbed socio-temporal patterns (resulting from atypical work hours leading to 73 univ med vol.27 no.2 family problems, reduced social support, and stress); and (iii) unfavorable changes in health behaviors (increased smoking, poor diet, and irregular meals).(2) many workers complain of g a s t r o i n t e s t i n a l p r o b l e m s r e s u l t i n g f r o m irregularly timed meals and poor quality of food, as well as increased consumption of soft drinks and caffeine-containing beverages.(3,4) circadian disturbances affecting the intake, digestion, and absorption of food are thought to play a major etiological role, but loss of sleep, fatigue, and the social stress of shiftwork may also be implicated. typically, shift workers have h i g h e r l e v e l s o f g a s t r i c s y m p t o m s ( e . g . indigestion, heartburn, constipation, loss of appetite, and nausea) than day workers, even with control for demographic, job, and lifestyle variables.(5) gastrointestinal disorders are more common in shift workers, who complain of abdominal pain and alteration in bowel habit. there is strong evidence linking shift work to peptic ulcer disease and in lesser degree to c o r o n a r y h e a r t d i s e a s e . ( 6 ) s h i f t w o r k i s associated with an increased risk of coronary heart disease (chd), as reported in several studies performed on shift workers.(7) obesity is a well known independent cardiovascular risk factor, and interestingly, it has been shown to be more prevalent among shift workers.(8) similarly, a more marked weight gain has been reported in shift-working subjects.(9-11) obesity suffer from a high risk of insulin resistance and its metabolic complications, such as type 2 diabetes mellitus, hypertriglyceridemia, low levels of high density lipoprotein cholesterol, and hypertension.(12) shift work is the most widely used tool of working time organization, as it enables round-the-clock activities not only in relation to rigid technological conditioning (e.g. chemical and steel industries, power plants) and necessary social services (e.g. h e a l t h s e r v i c e s , t r a n s p o r t , e l e c t r i c i t y, telecommunications), but also to support of productive and economic choices (e.g. textile, paper, food, mechanical industry, banking). in the medical field, physicians, nurses and other ancillary staff are expected to be performing their jobs or to be on-call on a 24-hour basis. there have been few studies undertaken in indonesia on the extent to which shift work is related to health problems among health care workers, and this has been a neglected area in o c c u p a t i o n a l h e a l t h a n d s a f e t y i s s u e s i n hospitals. the objective of the present study was to investigate the association between shift work schedules and health problems, particularly body mass index (bmi). methods research design a cross-sectional study was conducted during februari – april 2007. study subjects t h e p a r t i c i p a n t s w e r e f e m a l e n u r s e s working in one hospital in central jakarta. from a total of 187 female nurses employed by the h o s p i t a l , 1 5 2 h e a l t h y n u r s e s ( f r e e o f gastrointestinal disorders) participated in this study. thirty five nurses were excluded because of gastrointestinal disorders. data collection the investigators explained the purpose of the research and requested cooperation from e a c h p a r t i c i p a n t . a n a n o n y m o u s s e l f administrated questionnaire was used to collect the required data from each participant. the distribution and collection of questionnaires was performed through the head nurse. the items included in the questionnaire related to subject characteristics, e.g. age, gender, education, marital status, working unit, job experience, and working system (day worker/shift worker). in this study, shiftwork was defined as any regular 74 employment outside the usual working hours, defined arbitrarily to be between 08.00 hours and 16.00 hours. for gastrointestinal problems, several questions were combined into one, e.g. “which one of the following problems have you experienced continuously?”, viz. decreased appetite, constipation, diarrhoea, nausea, peptic ulcer, indigestion and eructation. measurements systolic and diastolic blood pressure (sbp and dbp) readings were recorded to the nearest 5 m m o f m e r c u r y a s t h e m e a n o f t w o measurements with the subjects seated, using a m e r c u r y s p h y g m o m a n o m e t e r w i t h a n appropriate cuff size. hypertension was defined according to who criteria.(13) body weight was measured using the sega portable scale and recorded to the nearest 0.1 kg. height was measured on barefooted subjects to the nearest c e n t i m e t e r u s i n g t h e m i c r o t o i s . b m i w a s calculated as weight in kilograms divided by the s q u a r e o f h e i g h t i n m e t e r s . f o r a s i a n populations, bmi is classified into the following categories: underweight (<18.5 kg/m2), normal (18.5–22.9 kg/m2), overweight (23.0–27.5 kg/ m2), and obese (≥ 27.6 kg/m2).(14) statistical analysis statistical analyses were performed using spss version 10.0 and epi info 2000. the chisquared test was used to assess univariate associations between shift variables and reported problems, and student’s t test for comparing continuous variables. analysis of variance was applied for comparing bmi and duration of job experience. the level of statistical significance was set at 0.05. results table 1 summarizes the characteristics of the participating nurses. the mean age was 30.9 ± 72.2 years, 91 nurses (59.9%) were married and mean job experience was 8.9 ± 7.6 years. since only 2.6% of the nurses had a degree or higher qualification, the study population could not be regarded as being highly educated. approximately 76.3% (116/152) of the nurses were engaged in a rotating shift schedule. table 2 presents the reported prevalence of shiftwork-related gastrointestinal problems, in which the difference in prevalence between shift a n d d a y w o r k e r s d i d n o t r e a c h s t a t i s t i c a l significance (p > 0.05). mean sbp and dbp were not significantly different between shift workers and day workers. the mean bmi in the group of shift workers was significantly lower (23.1 ± 3.6) compared with the group of day workers (24.8 ± 3.8) (p=0.012). mean job experience among shift workers was significantly shorter duration (6.9 ± 5.9 years) in comparison with day workers (15.4 ± 8.4 years). (table 3) table 1. characteristics and work pattern of nurses in the hospital under study (n = 152) samara, merijanti, tandean, et al shift work and body mass index 75 univ med vol.27 no.2 table 2. prevalence of shiftwork-related gastrointestinal problems reported among the nurses t h e s e d a t a w e r e s u b j e c t e d t o f u r t h e r analysis to determine the influence of job experience on bmi, by using one-way analysis o f v a r i a n c e a n d tu c k e y h s d m u l t i p l e comparison. one-way analysis of variance determined the values of bmi distributed for classes of job experience in day workers and shift w o r k e r s . o n a v e r a g e t h e b m i i n c r e a s e d significantly with job experience in the shift workers (p=0.014), but not in the day workers (p=0.644). (table 4) the tuckey hsd multiple comparison showed that the mean difference (-3.2613) was statistically significant between nurses with job experience of less than 5 years and 16-30 years. (table 5) table 3. comparison of the continuous variables age, job experience, bmi, sbp, and dbp between day and shift workers table 4. means and sd of bmi distributed for classes of job experience in day workers and shift workers (152 subjects) 76 discussion this study has demonstrated that mean bmi was lower in shift workers than in day workers. however, after adjustment for job experience, the bmi was significantly higher in shift workers compared with day workers. the results are in agreement with the study done by karlssona et al,(8) indicating a significantly higher bmi in shift workers in comparison with non-shift workers. obesity was also more common in shift workers than in day workers. a longitudinal observational study among 469 nurses aged 2158 years showed that exposure to night work can l e a d t o w e i g h t g a i n a n d o v e r w e i g h t . ( 11 ) essentially identical results were obtained in offshore workers, viz. that continued exposure to day-night shift work gives rise to increased bmi. another study on shift workers revealed the ocurrence of weight gain, where body weight in shift workers increased 4.3 kg as compared with 0.9 kg in day workers (p=0.02).(15) there was a tendency for shift workers to report eating more and taking more naps than did the day workers. a cohort study on 377 shiftworkers and nons h i f t w o r k i n g c o n t r o l s s h o w e d a p o s i t i v e relationship between bmi and waist-to-hip ratio (whr) in comparison to duration of shiftwork experience, with adjustment for age.(16) the study undertaken by di lorenzo et al. on 319 workers in southern italy indicated that shift work was responsible for excessive body mass index.(17) however, nakamura et al(18) did not find any difference in body weight when comparing day workers and shift workers, although workers i n a t h r e e s h i f t r o t a t i o n h a d a n i n c r e a s e d abdominal to hip ratio, indicating a higher proportion of central obesity among shift workers. a possible explanation for these findings could be insufficient adjustment for p o s s i b l e c o n f o u n d i n g v a r i a b l e s , s u c h a s socioeconomic factors. another possibility is that the duration of follow-up could influence the change in body weight as suggested by karlsson.(8) shift workers commonly have a disturbed appetite dan tend to disregard food quality and quantity; they tend to consume “junk” food instead of complete nutritionally balanced meals.(19) the present study did not find any differences in prevalence of hypertension b e t w e e n s h i f t a n d d a y w o r k e r s , w h i c h i s consistent with previous studies.(20) the shift system in hospital x, where the nurses were employed, had work shifts of 6 hours duration rotated as follows: the first shift lasted from 08.00 to 14.00 local time, the second from 14.00 until 20.00, and the third w a s f r o m 2 0 . 0 0 t o 0 8 . 0 0 . e a c h s h i f t w a s maintained for 2 days, with one day off after 6 working days. there is still a difference of opinion as to which system is the better one: weekly or twice weekly shifts, as each has its strengths and weaknesses. conclusions shift nurses tend to have a gain in bmi proportional to job experience. however, no significant differences in sbp and dbp were found in shift workers as compared to day workers. samara, merijanti, tandean, et al shift work and body mass index table 5. multiple comparisons of bmi between the class job of experience in the shift workers 77 univ med vol.27 no.2 acknowledgements this study was supported by the medical faculty of trisakti university. we extend our gratitude to all nurses who participated in this s t u d y, a n d t o d r. m . f a d h i l r a s a d , e n t specialist, as head of the center of education and training at tarakan hospital, jakarta. references 1. knutsson a. health disorders of shift workers. occup med 2003; 53: 103-8. 2. knutsson a, boggild h. shiftwork and cardiovascular disease: review of disease mechanisms. rev environ health 2000; 15: 35972. 3. caruso cc, lusk sl, gillespie bw. relationship of work schedules to gastrointestinal diagnoses, symptoms, and medication use in auto factory workers. am j ind med 2004; 46: 586-8. 4. morshead dm. stress and shift work. occup health saf 2002; 71: 36-8 5. costa g. the 24-hour society: between myth and reality. j hum ergol 2001; 30: 15-20. 6. harrington jm. health effects of shift work and extended hours of work. occup environ med 2001; 58: 68-72. 7. knutsson a, hallquist j, reuterwall c, theorell t, akerstedt t. shift work and myocardial infarction: a case–control study. occup environ med 1999; 56: 46-50. 8. karlsson b, knuttsson a, lindahl b. is there an association between shift work and having a metabolic syndrome? results from a population based study of 27485 people. occup environ med 2001; 58: 747-52. 9. geliebter a, gluck me, tanowitz m, aronoff nj, zammit gk. work-shift period and weight change. nutrition 2000; 16: 27-9. 10. yamada y, kameda m, noborisaka y, suzuki h, honda m, yamada s. excessive fatigue and weight gain among cleanroom workers after changing from an 8-hour to a 12-hour shift. scand j work environ health 2001; 27: 318–26. 11. parkes kr. shift work and age as interactive predictors of body mass index among offshore workers. scand j work environ health 2002; 28: 64–71. 12. pusparini. central obesity, metabolic syndrome and type 2 diabetes mellitus. univ med 2007; 26: 195204. 13. chalmers j, macmahon s, mancia g, whitworth j, beilin l, hansson l, et al. world health organization international society of hypertension guidelines for the management of hypertension. guidelines subcommittee of the world health organization. clin exp hypertens 1999; 21: 100960. 14. world health organization. appropriate bodymass index for asian populations and its implications for policy and intervention strategies. lancet 2004; 363: 157-63. 15. niedhammer i, lert f, marne mj. prevalence of overweight and weight gain in relation to night work in a nurses’ cohort. int j obes relat metab disord 1996; 20: 625-33. 16. van amelsvoort lg, schouten eg, kok fj. duration of shiftwork related to body mass index and waist to hip ratio. int j obes relat metab disord 1999; 23: 973-8. 17. di lorenzo l, de pergola g, zocchetti c, abbate nl, a basso1, pannacciulli n, et al. effect of shift work on body mass index: results of a study performed in 319 glucose-tolerant men working in a sourthern italian industry. int j obes 2003; 27: 1353-8. 18. nakamura k, shimai s, kikuchi s. shiftwork and risk factors for coronary heart disease in japanese blue-collar workers: serum lipids and anthropometric characteristics. occup med 1997; 47: 142-6. 19. costa g. shift work and occupational medicine: an overview. occup med 2003; 53: 83-8. 20. bøggild h, knutsson a. shiftwork, risk factors and cardiovascular disease. scand j work environ health 1999; 25: 85-99. rina1 57 department of community medicine, medical faculty, trisakti university correspondence dr. rina k.kusumaratna, m.kes department of community medicine, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2504 email : rkusumaratna@yahoo.com univ med 2008; 27: 57-64. impact of physical activity on quality of life in the elderly april-june 2008april-june 2008april-june 2008april-june 2008april-june 2008 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 vol.27 no.2 abstract universa medicina rina k. kusumaratna the rapid growth of the elderly population is having an impact on the maintenance of a healthy mental and physical state. the aim of this study was to assess the correlation of physical activity and quality of life in the elderly. the participants in this study were 21 male and 80 female elderly persons (66.4 ± 6.3 years, mean ± sd) recruited from the community in 2 subdistricts in south jakarta. data were collected on the quality of life (qol) using the whoqol-bref questionnaire that contains 26 items, 24 of them making up the 4 domains of physical health, psychological health, social relationships, and environment. the short form (sf) international physical activity questionnaire (ipaq) was used to measure the habitual practice of physical activities of the elderly. subjects were separated into groups with low (n = 25), moderate (n= 37) and high activity level (n = 39), according to their position on the physical activity scale. the qol scores in all four domains were significantly higher (p < 0.05) in the group reporting a higher level of physical activity. after adjustment for age, the more active group had higher values in all four qol domains as follows: physical health (p = 0.027), psychological health (p = 0.000), social relationships (p = 0.005), and environment (p = 0.000). healthy older adults who had higher physical activity had higher qol scores than those who were physically less active. therefore, incorporating more physical activity into the lifestyles of sedentary or minimally active elderly persons may improve their qol. keywords : physical activity, quality of life, elderly introduction age-associated changes in humans are having an impact on physiological ageing and pathophysiological processes. increased life expectancy is associated with an increase in multiple chronic conditions that could translate into functional disability and need for assistance in daily life activities. the longer life span could lead to declining health conditions, reduced mobility, depression, isolation and loneliness. the determinants important to the health and functioning of the elderly are demographic, social and environmental factors, including 58 kusumaratna physical activity and quality of life physical activity and dietary habits. over the past 15 years there has been growing evidence for the positive effect of exercise interventions on motor impairments(1) and selected mobility functions(2,3) in older home-dwelling people. thus, exercise interventions should also improve perceived functioning and well-being, while regular physical activity could support and increase one’s capability to ward off potential age-related diseases. for example, regular exercise could reduce the risk of bone fractures a n d i n f l u e n c e t h e f u n c t i o n i n g o f t h e cardiovascular system. physical activity was also shown to be effective in the prevention of type-2 diabetes.(4) at present the sedentary lifestyle has become a common feature at any age, while ageing leads to reduced activity levels and to further decline in the physical activity option. regular exercise training in elders can have a positive effect on muscle function and also contribute to the activities of daily living. it is a well known fact that strength training in older people improves balance and gait speed, enhances bone health and decreases many of the risk factors of an osteoporotic fracture. quality of life (qol) is a measure of how well people live.(5) in gerontology, research on qol has been used as an umbrella term to describe a number of outcomes believed to be of importance in the lives of elderly people.(6) the world health organization quality of life group (whoqol group) has developed an i n s t r u m e n t t o e v a l u a t e q u a l i t y o f l i f e (whoqol-bref) with 24 questions that evaluate quality of life in different domains: physical health, psychological health, social r e l a t i o n s h i p s , a n d e n v i r o n m e n t . ( 7 ) t h e whoqol-bref assessment was developed as a short version of the whoqol-100 for use in situations in which time is restricted or the burden on the respondent must be minimized, and in large epidemiologic studies and clinical trials.(8) a positive correlation has been reported between the degree of physical activity and health related quality of life (hrqol) in old age.(9) results are inconclusive as to whether h r q o l v a r i e s w i t h t h e t y p e o f e x e r c i s e undertaken.(10) the aim of this study was to assess the correlation between physical activity and quality of life in the elderly. methods research design a cross-sectional study was performed from january to may 2006. subjects participants in this study comprised a total of 101 subjects (eighty females and twenty-one males) ≥ 60 years of age, who were active members of senior citizens associations in 7 subdistricts of the tebet area in south jakarta. subjects had to meet the following criteria : i) be in apparent health, ii) be well nourished, iii) be able to communicate, and iv) be able to give a written informed consent. excluded from the study were persons who had been using a walking aid (crutches, cane, or wheelchair) or who were unable to perform physical activity for medical reasons. the collected demographic data included gender, age, marital status, educational level, and household income level. in addition, physical activity, quality of life, and body mass index variables were collected. measurements physical activity the short form (sf) international physical activity questionnaire (ipaq) was used to measure the habitual practice of physical activities of the elderly.(11,12) the sf ipaq was designed for use in surveillance studies, in which 59 time is limited, and consists of 7 items for estimating the time spent in performing physical a c t i v i t i e s ( m o d e r a t e t o v i g o r o u s ) a n d i n inactivity (sitting). the sf ipaq form assesses p h y s i c a l a c t i v i t y u n d e r t a k e n a c r o s s a comprehensive set of domains, including leisure time physical activity, domestic activities, work-related and transport-related physical activity, and sedentary behavior (sitting). data obtained by either short or long ipaq forms can be reported as categorical or as continuous scores. categorical scores are expressed as low, moderate or high levels of physical activity, while continuous scores are given as metabolic equivalent-minutes (met-minutes) per week. participants indicated the number of times in the past seven days which they spent in walking, and in performing moderate and vigorous activities, and also the average amount of time spent per day in aforementioned activities. the questionnaire took approximately 10 minutes to complete and a trained research assistant was present to help the participants. computation of the total score required summation of the duration in minutes and frequency in days per w e e k o f w a l k i n g , m o d e r a t e i n t e n s i t y a n d vigorous-intensity activities. physical activity (pa) data obtained from sf ipaq were used f o r c o m p u t i n g m e tm i n u t e s p e r w e e k , calculated as the met intensity multiplied by the duration (in minutes) of each activity over the seven day period. the following met values and formulae for computation of metminutes were taken from the ipaq validity and reliability study:(12) (i) walking met-minutes/ week = 3.3 * walking minutes * walking days; ( i i ) m o d e r a t e m e tm i n u t e s / w e e k = 4 . 0 * moderate-intensity activity minutes * moderateintensity days; (iii) vigorous met-minutes/ week = 8.0 * vigorous-intensity activity minutes * vigorous-intensity days. individuals are considered to be highly a c t i v e i f t h e y p e r f o r m v i g o r o u s pa o n a minimum of three days accumulating to at least 1,500 met-min/week, or seven days of any combination of walking, moderate or vigorous pa accumulating to a total of at least 1,500 met-min/week. the pa-status (low, moderate, and high activity level) was defined according to the ipaq scoring protocol.(13) the categories were adjusted to the recommendations of the centers for disease control (cdc).(14) quality of life quality of life was measured using the whoqol-bref assessment, which is a selfreported questionnaire that contains 26 items, where each item represents 1 facet. the facets are defined as those aspects of life that are considered to have contributed to a person’s qol. among those 26 items, 24 of them make up the 4 domains of physical health (7 items), p s y c h o l o g i c a l h e a l t h ( 6 i t e m s ) , s o c i a l relationships (3 items), and environment (8 items), whereas the other 2 items measure overall qol and general health.(8) the version of the whoqol-bref assessment has been v a l i d a t e d b y s a l i m e t a l . ( 1 5 ) w a s f i r s t summarized to a 4-domain construct (physical h e a l t h , p s y c h o l o g i c a l h e a l t h , s o c i a l relationships, environment) according to the guidelines for the whoqol-bref.(16) scores were calculated by taking the mean score for a l l i t e m s i n c l u d e d i n e a c h d o m a i n a n d multiplying by a factor of 4. the score for each domain therefore ranged from 4 to 20, with a higher score indicating a better qol. body mass index body weight was measured to the nearest 0 . 1 k g u s i n g a p l a t f o r m m o d e l e l e c t r o n i c weighing scale (seca 770 alpha, hamburg, germany), with the subject wearing a minimum of clothing. height was measured to the nearest 0.1 cm with a microtoise, with the subject standing as straight as possible with the back univ med vol.27 no.2 60 kusumaratna physical activity and quality of life against the wall. body mass index (bmi) was calculated as the weight (kg) divided by the square of the height (m). for asian populations, bmi is classified into the following categories: underweight (<18.5 kg/m2), normal (18.5–22.9 kg/m2), overweight (23.0–27.5 kg/m2), and obese (≥ 27.6 kg/m2).(17) statistical analysis data were entered to spss format using version 15.0. chi-square tests were performed o n c a t e g o r i c a l v a r i a b l e s t o d e t e r m i n e differences between males and females in the p h y s i c a l a c t i v i t y a n d b m i c a t e g o r y. independent t-test was used to compare the age difference between males and females, while a n a l y s i s o f c o v a r i a n c e w a s p e r f o r m e d t o d e t e r m i n e w h e t h e r d i f f e r e n c e s i n q o l measurements between the low, moderate and high level groups persisted after adjusting for age. statistical significance was set at p< 0.05. measurements are reported as mean ± standard deviation (sd). ethical clearance the study protocol and related documents were approved by the ethical committee of the medical faculty of trisakti university. results the participants were 101 elderly persons (80 female and 21 male) aged 60 to 85 years. (table 1). the subjects were on average 66.4 ± 6.3 years old, 61 (60.4%) were married, 73 (82%) had at least a 6-year education, 35 were of normal weight (34.7 %), while 44 (43.6 %) were overweight. the mean values for physical health, psychological health, social relationships and environment were 13.7 ± 2.4, 14.6 ± 2.6, 13.6 ± 2 . 7 , a n d 1 3 . 3 ± 2 . 4 , r e s p e c t i v e l y. t h e calculated physical activity values based on met categories are shown in table 2. each participant could select more than one exercise which they had been doing in the last 7 days. in the category of light physical activity, most of the elderly reported to have been walking in the morning for 30 minutes, for a total of 5 times weekly. in the moderate activity category, most had a preference for cycling, conducted once weekly for 15 to 30 minutes. in the category of vigorous physical activity, the majority conducted fitness exercises (senam jantung sehat) at the health center two times a w e e k , w i t h a 3 0 m i n u t e d u r a t i o n , f o r t h e p u r p o s e o f p r o m o t i n g c a r d i o r e s p i r a t o r y fitness. analysis yielded a statistically significant difference between males and females in age (p=0.000), but not in physical activity category (p=0.108). (table 3) table 1. characteristics of participants 61 after adjusting for age (table 4), the following four domains of qol remained higher in the vigorous activity group: physical domain (p = 0.027), psychological domain (p = 0.000), social relationship domain (p = 0.005) and environment domain (p = 0.000). on average the qol as measured in the physical health, psychological health, social relationships, and environmental domains increased significantly with the level of physical activity. table 2. physical activity categories, frequency and duration table 4. adjusted quality of life values in subjects with low, moderate and high levels of physical activity. values are expressed as mean and se table 3. physical activity category and age by gender univ med vol.27 no.2 62 kusumaratna physical activity and quality of life the tukey hsd multiple comparisons s h o w e d t h a t t h e s c o r e s f o r t h e p h y s i c a l , p s y c h o l o g i c a l , s o c i a l r e l a t i o n s h i p , a n d environment domains were significantly higher in the high activity group, when compared to the low activity group (p= 0.039; p=0.000; p=0.015; p=0.000) and the moderate activity group (p=0.029;p=0.000;p=0.005;p=0.000) (table 5). discussion the findings of our study suggest an increased quality of life in those elderly who had reported higher physical activity levels. the q o l s c o r e s i n a l l f o u r d o m a i n s w e r e significantly higher (p < 0.05) in the high physical activity group. several studies have shown that organized vigorous physical activity can benefit qol in healthy populations(18-20) an active lifestyle preserves physical function in older adults,(20) which may possibly contribute to higher levels of qol scores in domains related to physical health. the present study supports this notion, as the group having higher physical activity levels had greater values in all qol domains (i.e., physical health, psychological health, social relationship and environment) c o m p a r e d w i t h t h e i r m o r e s e d e n t a r y counterparts. after adjusting for age, the more active group had higher values in the following four qol domains: physical health (p = 0.029), p s y c h o l o g i c a l h e a l t h ( p = 0 . 0 0 0 ) , s o c i a l relationship (p = 0.005), and environment (p = 0.000). this suggests that the qol domains are positively associated with physical activity, independent of the age of the elderly. increased longevity is always associated with an increase in multiple chronic conditions a n d s u b s e q u e n t l y c o u l d d e t e r i o r a t e i n t o functional disability and need for assistance. h e a l t h a n d f u n c t i o n i n g o f t h e e l d e r l y i s i n f l u e n c e d b y m a n y f a c t o r s , c o m p r i s i n g demographic, social and environmental factors, including physical activity and dietary intake. f o r m a i n t e n a n c e o f t h e i r h e a l t h a n d independence, the elderly commonly attempt to explore health information and behavioral changes, thus an important aspect of self-care behavior involves physical activity and diet, which contributes to the health and quality of l i f e o f t h e e l d e r l y. t h i s i s t h e r e a s o n f o r conducting programs for the elderly at the health center, where most of the activities consist in supplying health information related to chronic diseases, performing physical examinations, cardiac fitness training (senam jantung sehat) and also exercises for osteoporosis prevention. t h e p r e s e n t s t u d y s h o w s s i g n i f i c a n t differences in frequency and duration of each table 5. multiple comparisons of physical, psychological, social relationship, and environment domains between physical activity groups in the elderly 63 exercise category of physical activity. the greatest frequency in the vigorous activity category was 1-2 times per week of 30 minutes duration, in the moderate category once a week for 15-30 minutes, and in the light category 5 times per week for 15 minutes. exercise has a positive impact on muscle function and could support the activities of daily living, including daily self-care. most of the participants were women, thus they generally have to perform some household work and daily self-care, especially if they do not have a caregiver or are not living with their children. p e r i o d i c a l p a r t i c i p a t i o n i n m o d e r a t e physical activity could delay functional decline and the onset of chronic diseases in the healthy elderly and limit their effects in those who are already chronically ill. active living also enhances mental health and promotes social contacts, while among the economic benefits of being active could be a reduction in medical costs and demands for health services. our findings h a v e i m p l i c a t i o n s f r o m a p u b l i c h e a l t h perspective, especially for health programs for the elderly, which are conducted at the health center as the vanguard of the health referral s y s t e m a t c o m m u n i t y l e v e l . p o l i c i e s a n d programs on ageing should be addressed to improving quality of life and health status through conduction of regular physical activity p r o g r a m s t h a t h a v e b e n e f i c i a l e ff e c t s i n preventing chronic and age-associated diseases that might be a burden to the elderly. the main limitation of this study is the utilization of a cross-sectional design, which does not allow for a true causal relationship to be established. although physical activity may affect qol, alternatively it is also possible that qol has an impact on physical activity. selfreported physical activity is subject to reporting bias and physical activity questionnaires do not accurately quantify activity-related energy expenditure. the development of an appropriate correction factor, which could be applied to q u e s t i o n n a i r e s s u c h a s t h e i pa q , w o u l d overcome systematic bias and provide a more reliable and valid measure of energy expenditure estimation. at the population level the ipaq has the potential to standardize physical activity measurement. used in conjunction with abovementioned correction factor the ipaq would be of great value for international comparison and estimating the burden of disease associated with physical inactivity.(22) conclusion higher physical activity associated with i n c r e a s e d o f q o l s c o r e s i n a l l d o m a i n s independent of the age of the elderly. acknowledgements the author extends her gratitude to all persons who participated in this research, in particular the medical students who assisted in the study, and her colleagues who provided continuous support during the realization of this research project. references 1. brown m, sinacore dr, ehsani aa, binder ef, holloszy jo, kohrt wm. low-intensity exercise as a modifier of physical frailty in older adults. arch phys med rehabil 2000; 81: 960-5. 2. alexander nb, galecki at, grenier ml. taskspecific resistance training to improve the ability of activities of daily living-impaired older adults to rise from a bed and from a chair. j am geriatr soc 2001; 49: 1418-27. 3. binder ef, schechtman kb, ehsani aa. effects of exercise training on frailty in community-dwelling older adults: results of a randomized, controlled trial. j am geriatr soc 2002; 50: 1921-8. 4. drewnowski a, evans wj. nutrition, physical activity, and quality of life in older adults. j gerontol 2001; 56a: 89-94. univ med vol.27 no.2 64 kusumaratna physical activity and quality of life 5. cella d, nowinski cj. measuring quality of life in chronic illness: the functional assessment of chronic illness therapy measurement system. arch phys med rehabil 2002; 83: s10-s17. 6. rejeski wj, mihalko sl. physical activity and quality of life in older adults. j gerontol a biol sci med sci 2001; 56: 23-35. 7. the whoqol group. development of the world health organization whoqol-bref quality of life assessment. psychol med 1998; 28: 551-8. 8. skevington sm, lotfy m, o’connell ka. the world health organization’s whoqol-bref quality of life assessment: psychometric properties and results of the international field trial. a report from the whoqol group. qual life res 2004; 13: 299-310. 9. stewart kj, turner kl, bacher ac. are fitness, activity, and fatness associated with health-related quality of life and mood in older persons? j cardiopulm rehabil 2003; 23: 115-21. 10. king ac, pruitt la, phillips w, oka r, rodenburg a, haskell wl. comparative effects of two physical activity programs on measured and perceived physical functioning and other health-related quality of life outcomes in older adults. j gerontol a biol sci med sci 2000; 55: m74-m83. 11. booth m. assessment of physical activity: an international perspective. res q exerc sport 2000; 71: 114-20. 12. oja p, craig cl, marshal a, sjorstrom m, bauman ae, booth ml, ainsworth be, et al. international physical activity questionnaire: 12 country reliability and validity. med sci sports exerc 2003; 35: 1381-95. 13. ipaq research committee. guidelines for data processing and analysis of the international physical activity questionnaire (ipaq): short and long forms. 2005. available at: http://www.ipaq.ki. se. accessed october 10, 2007. 14. pate rr, pratt m, blair sn, haskell wl, macera ca, bouchard c, et al. physical activity and public health. a recommendation from the centers for disease control and prevention and the american college of sports medicine. jama 1995, 273: 4027. 15. salim oc, sudharma n, kusumaratna rk, hidayat a. validity and reliability of world health organization quality of life-bref to assess the quality of life in the elderly. univ med 2007; 26: 27-38. 16. the whoqol group. development of the world health organization whoqol-bref quality of life assessment. psychol med 1998; 28: 551-8. 17. world health organization. appropriate bodymass index for asian populations and its implications for policy and intervention strategies. lancet 2004; 363: 157-63. 18. stewart al, mills km, sepsis pg, king ac, mclellan by, roitz k, et al. evaluation of champs, a physical activity promotion program for older adults. ann behav med 1997; 19: 35361. 19. acree ls, longfors j, fjeldstad as, fjeldstad c, schank b, nickell kj, et al. physical activity is related to quality of life in older adults. health qual life outcomes 2006; 4: 37-42. 20. rejeski wj, brawley lr, schumaker sa. physical activity and health-related quality of life. exerc sport sci rev 1996; 24: 71-108. 21. king mb, whipple rh, gruman ca, judge jo, schmidt ja, wolfson li. the performance enhancement project: improving physical performance in older adults. arch phys med rehabil 2002; 83: 1060-9. 22. maddison r, mhurchu cn, jiang y, vander hoorn s, rodgers a, lawes cmm, et al. international physical activity questionnaire (ipaq) and new zealand physical activity questionnaire (nzpaq): a doubly labelled water validation. int j behav nutr phys act 2007; 4: 62-70. directly observed treatment increases drug compliance in lymphatic filariasis mass drug administration 119 *postgraduate program, faculty of medicine, gadjah mada university, yogyakarta **department of parasitology faculty of medicine jenderal soedirman university ***department of parasitology, faculty of medicine, gadjah mada university, yogyakarta ****faculty of veterinary medicine, gadjah mada university, yogyakarta correspondence: dr. tutik ida rosanti, m.kes. department of parasitology faculty of medicine jenderal soedirman university jl. dr. gumbreg no.1 purwokerto jawa tengah mobile: +62811 2600 255 email: tutikidarosanti@yahoo.com univ med 2016;35:119-27 doi: 10.18051/univmed.2016.v35.89-95 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 directly observed treatment increases drug compliance in lymphatic filariasis mass drug administration tutik ida rosanti*,**, sugeng juwono mardihusodo***, and wayan t. artama**** backgroud. mass drug administration (mda) has been one of the strategies for lymphatic filariasis elimination. since the start of implementation in 2011 in pekalongan, no study on mda acceptability has yet been done. the objectives of the study were to determine the microfilaria rate (mf rate) and the risk factors of drug compliance in the mda program. methods a longitudinal study was conducted at pabean region, pekalongan city. there were 90 household heads as subjects, who were selected by proportional cluster random sampling. microfilaria rate (mf rate) was determined by finger blood examination. drug compliance was measured using questionnaires and observation sheets. drug compliance observers, filariasis counseling participation, and presence of filariasis patients were factors influencing drug compliance. results mf rate in 2015 was 1.35% and drug compliance rate was 86.80%. reasons for failing to take drugs were fear of side effects (50%), refusals (25%), laziness (16.7%), and perceiving the drug to be useless (8.3%). the chisquare test shows a significant difference between the presence of drug compliance observer and compliance (p=0.006). filariasis counseling participation and presence of filariasis patients did not show a significant difference with drug compliance (p= 0.986). conclusions families as the source of observers was associated with increased filariasis drug compliance. it is therefore essential to address the issues linked to low compliance to make the program more efficient and achieve the goal of filariasis elimination. keywords: filariasis, medical compliance, mass drug administration doi: http://dx.doi.org/10.18051/univmed.2016.v35.119-127 120 rosanti, mardihusodo, artama directly observed treatment in filariasis introduction lymphatic filariasis or elephanthiasis is a chronic communicable disease that is caused by filarial worms infecting the lymphatics and lymph nodes. the disease is marked by the clinical symptoms of swelling of the hands, feet, and scrotum, and causes life-long disability and disfigurement to the patients. although it is not fatal, this disease causes economic loss and social stigmatization for the patients, family and country.(1) to date, lymphatic filariasis still constitutes a public health problem in indonesia and the world. indonesia is one of the 11 countries endemic for lymphatic filariasis in south east asia with an average prevalence rate of 19%, distributed over 337 districts /cities.(2) pekalongan is one of the cities with endemic lymphatic filariasis in central java with wuchereria bancrofti as causative organism and culex quinquefasciatus mosquitoes as vectors.(3) fingerprick blood surveys (fbs) in 2010 showed that several kelurahan (villages) had high microfilaria (mf) rates of 5.54%, 2.39%, 3.39%, and 2.39% in the kelurahan of bumirejo, tegalrejo, pabean, and bandengan, respectively.(3) as an endemic area of lymphatic filariasis, pekalongan has carried out mass drug administration (mda) for filariasis since 2011. the purpose of mass drug administration for filariasis is to eliminate filariasis by treating the source of infection, namely persons who have microfilaria in their blood. the presence of microfilaria in the blood of patients is a source of infection for other individual in the neighborhood and for the mosquito vectors of filariasis. the transmission of filariasis may be reduced or inhibited through elimination of microfilarial reservoirs by means of mass drug administration and reducing contact between humans and the vectors.(4) mass drug administration in the filariasis elimination program in indonesia aims to interrupt filariasis transmission by reducing the mf rate to less than 1% and reducing mean microfilarial density.(5) mass drug administration for the elimination of lymphatic filariasis is the strategy of interrupting the filariasis transmission chain by a single annual administration of dietylcarbamazine citrate (dec) tablets to all individuals above 2 years of age. the coverage and compliance levels are important to inhibit transmission for a period of 4-6 years, which is the mean reproduction time of adult filarial worms.(6) a study by karoma et al.(7) reported that upon instituting the third mda round, the program succeeded in reducing the mf rate by 88.5%, from 2.6% to 0.3% and in reducing mean microfilarial density by 17.59 mf/ml, whereas the study by eigege et al.(8) showed that a 90% reduction in mf rate from 3.17% to 0.32% only occurred after implementation of the 8th mda round. a systematic review in india identified a coverage-compliance gap in many mda programs, along with potentially correctable causes. overall mda coverage rates ranged from 48.8% to 98.8% and compliance rates from 20.8% to 93.7%. among 31 mdas, there were only 10 with a compliance level of e”65% (five of 20 mdas in rural and two of 12 mdas in urban areas).(9) another study showed that 99% of the study participants received dec and albendazole tablets during mda, of which only just above a quarter actually consumed the drugs.(10) the gap between mda coverage rates and compliance rates, as reported in previous studies, needs to be subjected to further reasearch to find the causative factors. the purpose of the present study was to assess the impact of the mda program and determine the factors associated with noncompliance in taking antifilariasis drugs. methods study design this was a longitudinal study that was carried out from september 2014 until may 2015. study subjects the study subjects were residents of an area with chronic filariasis patients and were recorded 121 as recipients of antifilariasis drugs in the filariasis mass drug administration program for the years 2014 and 2015. mass drug administration for filariasis was performed under the coordination of the pekalongan city health service, in accordance with the who recommendations, namely using a single dose of diethyl carbamazine citrate (dec) (three 100 mg tablets for persons weighing 50 kg) with the addition of a single 400 mg dose of albendazole.the population for the assessment of filariasis drug compliance included all household heads (if there was no household head, he or she was represented by a household member) who were residents of the pabean region, north pekalongan district, pekalongan city, and comprised 1378 households. sample size calculation was by means of the minimal sample size formula:(11) p)(1 p z1)(n d n p)p(1 z n α/z1 22 α/z1 2 −+− − = − − according to the formula, if p (estimated prevalence of filariasis drug taking practice) = 0.5 and d (precision) = 0.1, then the minimal sample size was 89.90, which with the addition of possible drop outs became 91. the sample was recruited by proportional cluster random sampling. the sample size for each rt was determined using the formula according to sugiyono,(12) i.e. based on this formula, the obtained sample size for rt 2 rw i was 10 household heads, for rt 4 rw i 12 household heads, rt 3 rw ii 7 household heads, rt 1 rw iii 29 household heads, rt 2 rw iii 16 household heads, and rt 1 rw iv 17 household heads. the samples were determined based on an interval with the formula: based on this formula, the samples were determined with an interval between 5 to 6. data collection procedures the questionnaire comprised personal data of the study subjects (name, age, gender, address, 1nx n x n = n n i = presence/absence of chronic disease, pregnant/ non-pregnant, lactating/non-lactating), reasons for drug noncompliance, variables associated with drug compliance in the mda program (the presence of drug compliance observers, type of the drug compliance observers, history of participation in filariasis counseling, and the presence of chronic filariasis patients in the residential area of the study subjects). drug compliance observers were persons who reminded the study subjects to take the antifilariasis drugs distributed by puskesmas (community health center) personnel, observed the subjects swallowing the drugs, and reported eventual occurrence of side effects. drug compliance observers comprised health cadres and family members. observation sheets were used to record the results of observation of the study subjects regarding their compliance in taking antifilariasis drugs. the compliance in taking antifilariasis drugs in the filariasis mass drug administration program was determined by interviews and filling-in of observation sheets. observation of drug compliance was performed by the investigators assisted by 5 trained health cadres and 5 personnel of dukuh puskesmas, pekalongan city. before performing the interviews and filling-in of observation sheets, comprehension and equality of perception was checked for each question and observation item. laboratory examination equipment and materials used in the determination of mf rate consisted of microscope glass slides, blood lancets, tissue paper rolls, alcohol swabs, gloves, slide boxes, giemsa stock solution, measuring glasses, survey forms, dropper pipettes, pencils, aquar bottled water for preparing giemsa solution. t h e s a m p l e s i z e f o r t h e m f r a t e determination was 300 persons. the sample was recruited by gathering the residents who were t h e s u r v e y t a rg e t s a n d r e s i d e d i n t h e neighborhood of clinical cases of bancroftian filariasis. the identification of microfilariae in the blood used the fbs technique. the sample univ med vol. 35 no.2 122 rosanti, mardihusodo, artama directly observed treatment in filariasis collection was performed in the pabean region in the neighborhood of clinical cases of bancroftian filariasis at night, from 21.00 up to 01.00 western indonesian time. the blood samples were collected from the tips of the second or third fingers, which were pricked with sterile lancets until bleeding. the first drop of blood was swabbed away and the next three drops of blood (60 µl) were placed on a clean glass slide. subsequently the blood was spread out with the corner of another slide. the blood slide was left to air-dry overnight at room temperature and subsequently stained with giemsa. the mf rate was calculated by dividing the number of residents whose blood were positive for microfilariae by the number of examined blood slides multiplied by one hundred percent. the fbs population comprised adults who had resided in the area for 2 years or more. statistical analysis univariate analysis was performed on variables of respondents’ characteristics, consisting of age, gender, compliance in taking antifilariasis drugs in the years 2014 and 2015, accompanied with reasons for drug noncompliance, and type of drug compliance observer. the mf rates for the years 2014 and 2015 were expressed as percentages of reductions or increases. bivariate analysis on the variables presence of drug compliance observer, history of subject participation in filariasis counseling, and the presence of patients with chronic filariasis in the neighborhood of the homes of the study subjects, in relation to drug compliance in the filariasis mda program was performed using the chisquare test. ethical clearance written informed was obtained from all respondents, while keeping their identities confidential. the study was conducted after obtaining ethical clearance from the ethics commission, faculty of medicine, gadjah mada university, yogyakarta, with reference no. ke/ fk/869/ec. results after filariasis mass drug administration, i.e.the 4th year of mda program, there occured a reduction in mf of 3,57% from 1.4% to .35%, while in 2015, a reduction in mf rate of 76.29% occurred, changing form 1.35% to 0.32%. according to table 1, the majority (18 or 19.8%) of the respondents belonged to the age group of 31-36 years, while 72 respondents (79.1%) were females. the majority of the respondents were employed as batik workers who worked at home and in the batik factories in pekalongan city. they were 60 (65.9%) in number and 38 of the respondents (41.7%) had finished primary school or equivalent. table 2 shows that overall mda drug compliance in 2014 was 86.8%, whereas in 2015 it was 74.7%. mda respondents who had noncadres as drug compliance observers were more compliant in taking the mda antifilarial drugs in 2014 and 2015, in comparison with respondents table 1. distribution of sociodemographic characteristics of respondents (n=91) 123 table 2. distribution of drug compliance in mda program for 2014 and 2015, by type of drug compliance observer dco = drug compliance observer; nc = noncompliant; c = compliant whose drug compliance observers were cadres, with compliance rates of 96.7% in 2014 and 93.3% in 2015. the majority of the 91 respondents of the filariasis mda program in 2014 and 2015, namely 61 respondents (67%) had cadres as drug compliance observers, while the remainder (30 respondents or 33%) had non-cadres originating from families as drug compliance observers. according to table 3, the most frequent reason for mda antifilariasis drug noncompliance in 2014 and 2015 was the fear of drug side effects, the percentage of responses being 50.0% and 39.1%, respectively. table 4 shows that all variables, namely type of drug compliance observer, participation in counseling and presence of patients with filariasis in residential area were not significantly correlated with mda drug compliance in 2014, with p values of 0.095, 0.154 and 0.757, respectively. similarly, for the mda program in 2015, participation in counseling and the presence patients with filariasis in residential area were also not significantly correlated with mda antifilariasis drug table 3. reasons for drug noncompliance in the mda program for 2014 and 2015 (n=91) compliance, with p values of 0.325 and 1.000, respectively. however, among the respondents with non-cadre observers, it was found that 93.3% of subjects complied in drug taking in 2015, which was significantly higher than the percentage of subjects with cadres as observers, their drug compliance being 65.6% (p=0.009). discussion filariasis mass drug administration is implemented in lymphatic filariasis endemic a r e a s , w h i c h a r e a r e a s w i t h m f r a t e s o f approximately 1%. the goal of mass drug administration is to simultaneously kill all microfilariae in the blood of every resident, so as to interrupt the transmission chain. mass drug administration in the framework of lymphatic filariasis elimination aims to reduce the mf rate to less than 1% and to reduce the mean microfilarial density. our study showed that following filariasis mda in 2014, i.e. the 4th annual round, there was a reduction in mf rate of 0.05%, from 1.4% to 1.35%, while in 2015 univ med vol. 35 no.2 124 rosanti, mardihusodo, artama directly observed treatment in filariasis table 4. relationship of type of drug compliance observers, counseling participation, and presence of filariasis patients in residential area with mda drug compliance in 2014 and 2015 dco = drug compliance observer there was a reduction in mf rate of 23.70%, from 1.35% to 0.32%. these results signify that upon implementation of the mda program in the 5th year, the area in question had ceased to be a filariasis endemic area. the success of this filariasis elimination program was supported among other factors by the relatively high overall drug compliance level, i.e. 86.8% in 2014 and 74.7% in 2015. kumar and sachan state that filariasis mass drug administration using a combination of dec and albendazole has the potential to attain the elimination target, if the drug coverage is more than 75%.(13) the results of the present study are in line with the study by kalimuthu et al.,(14) who state that following implementation of the fifth annual mda round in south india, there was a reduction in mf rate of 72.5%, supported by a drug compliance rate of 86.32%. vaishnav et al.(15) reported on the success of a filariasis elimination program after implementation of 4 annual mda rounds, with support from attainment of drug compliance of greater than 85%. the success of this mda program was supported by excellent preparation of activities, full government support, involvement of institutions of medical education, increased capacity of the executive team and evaluation of the impact through a survey. in contrast, jones et al.(16) state that implementation of an mda program of up to 9 rounds, did not succeed in liberating tanzania from its filariasis endemic status. although there was a reduction in filarial antigen prevalence of 34.6%, from 49% to 14.4%, the presence of filarial antigen upon examination of the blood of the population showed that there still was active filariasis transmission in these areas. the continual transmission of filariasis was due to low drug compliance in its mda program. the residents who were actively involved and complied in taking antifilariasis drugs accounted for less than 50% of the total number of residents. 125 drug noncompliance in the mda program in the pabean area was mostly caused by fear of drug side effects. the respondents were afraid of suffering from dizziness, nausea, and muscle pain after taking the drugs, causing them to be incapacited as batik factory workers, since nearly all were batik factory workers. the respondents were more concerned with the economic loss that they had to bear if they suffered from drug reactions after taking the antifilariasis drugs. kumar and sachan stated that one of the reasons for failure to attain 100% compliance in consuming antifilariasis drugs in the mda program is that the group of asymptomatic patients harboring microfilariae refuse to take antifilariasis drugs, because of the development of fever after ingesting dec, which signifies inappropriate dissemination of information.(13) hussain et al.(10) state that failure to attain drug compliance in the mda program is due to absence of health personnel during distribution of antifilariasis drugs. among 99% of subjects who are recipients of antifilariasis drugs in india, only around 25% take the drugs, which agrees with the mda program in the pabean area of pekalongan. the health personnel from the puskesmas were not all present during the distribution of antifilariasis drugs to the residents and did not observe the taking of the antifilariasis drugs by the residents. actually the antifilariasis drugs were handed over to the health cadres, to be subsequently distributed to the residents.(17-19) the mda program in the pabean region used a combination of dec, albendazole and paracetamol, administered once annually for a minimum of 5 years. the dose of dec is 6 mg/ kgbw, albendazole 400 mg for all age groups, and paracetamol 10 mg/kgbw. dec kills microfilariae within several hours after being ingested. this is accomplished by paralyzing the microfilarial musculature, thus dislocating them from their normal habitat, and by altering the composition of the microfilarial surface membranes, so that they are readily destroyed by the host defense mechanisms. dec is rapidly absorbed by the alimentary tract, reaches peak plasma concentrations after 4 hours, and is completely excreted in urine within 48 hours. albendazole acts to kill adult filarial worms and enhance the effect of dec in killing adult worms and microfilariae without additional adverse reactions.(20) the drugs dec and albendazole are safe drugs that are well-tolerated by most people. however, occasionally drug reactions may occur, particularly in infections with brugia malayi and brugia timori. the reactions may take the form of systemic and local reactions. systemic reactions occur only within the first 3 days after taking of the drugs and are self-limited. they occur due to an individual’s immune response against disintegrating microfilariae. the greater the number of disintegrating microfilariae, the more severe the reaction that may develop. systemic reactions comprise headaches, fever, nausea, vomiting, muscle pains, joint pains, and itching. local reactions against bancroftian filariasis are in the form of scrotal cutaneous nodules caused by dead adult worms, which is the most frequently occurring local reaction. in addition, lymphadenitis, lymphangitis, orchitis, abscess, ulceration, and lymphedema may occur. local reactions are caused by the death of adult worms and may develop up to 3 weeks after mass drug administration.(5,16) the presence of drug compliance observers, participation in counseling, and the presence patients with chronic filariasis in the residential area of the respondents are factors for which an association with antifilariasis drug compliance was investigated. in this study, drug observers are persons who remind the study subjects to take the antifilariasis drugs, who observe the taking of the drugs by the study subjects, and who report the development of side effects. drug compliance observers in this study consisted of health cadres and non-cadres, the latter being family members. all study subjects who complied by ingesting the antifilariasis drugs in this study were accompanied by drug compliance observers (pmo). our study shows that among the subjects with non-cadre drug compliance observers, 93.3% univ med vol. 35 no.2 126 rosanti, mardihusodo, artama directly observed treatment in filariasis were compliant in taking antifilariasis drugs in 2015, which was significantly higher than the subjects who had cadres as drug compliance observers, among whom only 65.6% were drugcompliant. to eliminate lymphatic filariasis from an endemic area within four to six annual rounds of mda, at least 65% of the at-risk population should receive and take the recommended drugs (albendazole/ivermectin or albendazole/diethyl carbamazine) under directly observed therapy.(21) the filariasis elimination effort through the mda program requires the involvement and compliance of all community members in endemic areas, especially among the households. the presence of residents who are not taking drugs in the filariasis mda program may constitute a possible source of microfilariae for other individuals and for the mosquito vectors. therefore it essential that all household and community members have an attitude of mutual interest and accountability in drug compliance. this is particularly true since the mda program is carried out for 4-6 years, corresponding to the reproductive period of the adult worms. this long period will inevitably lead to feelings of being fed up and bored, particularly those who do not feel the direct benefits of the treatment. thus there is a need for reciprocal support and reminding between household and community members in endemic areas that the filariasis elimination effort is a common responsibility. the effort to delve into the reasons for noncompliance in taking antifilariasis drugs using the interview technique constitutes a limitation of this study, particularly for study subjects who refuse to take the antifilariasis drugs. the presence of noncompliant residents in taking antifilariasis drugs during the filariasis mda program in pekalongan city, will particularly result in the possibility of a continued presence of sources of filarial infection, which may inhibit the filariasis elimination effort in this area. further studies on the reasons for drug noncompliance in the filariasis mda program should use a qualitative study design involving antifilariasis drug noncompliant subjects by means of in-depth interviews. conclusions in 2014 there was a reduction in mf rate of 0.05%, from 1.4% to 1.35%, and in 2015 a reduction in mf rate of 23.70%, from 1.35% to 0.32%. the presence of drug compliance observers from the family circle is the most important variable associated with antifilariasis drug compliance. conflict of interests the investigators state that they had no conflict of interest in this study. acknowledgement the investigators express their gratitude to the health service of pekalongan city, the head of dukuh puskesmas and staff, to th health cadres of the pabean area who assisted in this study, especially in mobilizing the residents for the filariasis fingerprick blood survey. references 1. world health organization. progress report 2000-2009 and strategic plan 2010-2020 of the global program to eliminate lymphatic filariasis: halfway towards eliminating lymphatic filariasis. geneva: world health organization;2010. 2. kementrian kesehatan ri. epidemiologi penyakit kaki gajah (filariasis) di indonesia. jakarta: ditjen pp & pl kemenkes ri;2009. 3. ramdhani t, soeyoko, sumarni s. culex quinquefasciatus sebagai vektor utama filariasis limfatik yang disebabkan wuchereria bancrofti di kelurahan pabean kota pekalongan. j ekol kes 2010;9:1303-10. 4. de souza dk, koudou b, kelly-hope la, et al. diversity and transmission competence in lymphatic filariasis vectors in west africa, and the implications for accelerated elimination of anopheles-transmitted filariasis. parasit vectors 2012;5:259. 127 univ med vol. 35 no.2 5. kementerian kesehatan ri. pengobatan masal filariasis. jakarta: ditjen pp & pl kemenkes ri; 2009. 6. nujum zt. coverage and compliance to mass drug administration for lymphatic filariasis elimination in a district of kerala, india. int health 2011;3:22-6. 7. koroma jb, sesay s, sonnie m, et al. impact of three rounds of mass drug administration on lymphatic filariasis in areas previously treated for onchocerciasis in sierra leone. plos negl trop dis 2013;7:e2273. 8. eigege a, kal a, miri e, et al. long-lasting insecticidal nets are synergistic with mass drug administration for interruption of lymphatic filariasis transmission in nigeria. plos negl trop dis 2013;7:e2508. 9. babu bv, babu gr. coverage of and compliance with mass drug administration under the programme to eliminate lymphatic filariasis in india: a systematic review. trans r soc trop med hyg 2014;108:538-49. 10. hussain ma, sitha ak, swain s, et al. mass drug administration for lymphatic filariasis elimination in a coastal state of india: a study on barriers to coverage and compliance. infect dis poverty 2014;3:31. 11. lwanga sk, lemeshow s. sample size determination in health studies: a practical manual. geneva: world health organization; 1991. 12. sugiyono. metode penelitian kuantitatif, kualitatif dan r & d. bandung: cv.alfabeta; 2011. 13. kumar a, sachan p. measuring impact on filarial infection status in a community study : role of coverage of mass drug administration. tropical biomedicine 2014;31:225-9. 14. kalimuthu m, sunish ip, nagaraj j, et al. residual microfilaremia in rural pockets of south india after five rounds of dec plus albendazole administration as part o the lf elimination campaign. j vector borne dis 2015;52;182-4. 15. vaishnav kg, desai hs, srivastava pk, et al. impact of mass drug administration on elimination of lymphatic filariasis in surat city india. j comm dis 2012;44:251-9. 16. jones c, tarimo ds, malecela mn. evidence of contiuned transmission of wuchereria bancrofti and associated factors despite nine rounds of ivermectin and albendazole mass drug administration in rufiji district tanzania. tanzania j health res 2015;17:1-9. 17. world health organization. global programme to eliminate lymphatic filariasis: progres report 2014. weekly epidemiol rec 2015;90:489-504. 18. krentel a, fischer pu, weil gj. a review of factors that influence individual compliance with mass drug administration for elimination of lymphatic filariasis. plos negl trop dis 2013;7: e2447. 19. ghosh s, samantha a, kole s. mass drug administration for elimination of lymphatic filariasis: recent experiences from a district of west bengal, india. trop parasitol 2013;3:6771. 20. fernando sd, rodrigo c, rajapakse s. current evidence on the use of antifilarial agents in the management of bancroftian filariasis. j trop med 2011, article id 175941, 12 pages. doi: 10.1155/ 2011/175941. 21. offei m, anto f. compliance to mass drug administration programme for lymphatic filariasis elimination by community members and volunteers in the ahanta west district of ghana. j bacteriol parasitol 2014;5:180. doi: 10.4172/2155-9597.1000180. nurbuanto 179 pterygium: degeneration, exuberant wound healing or benign neoplasm? september-december, 2009september-december, 2009september-december, 2009september-december, 2009september-december, 2009 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 vol.28 no.3 universa medicina nurbuanto tradjutrisno* *department of ophthalmology, gatot subroto central military hospital, jakarta correspondence dr. nurbuanto tradjutrisno,spm department of ophthalmology, gatot subroto central military hospital jl. abdul rahman saleh no.24, jakarta pusat phone: 021-3441008 email: ntradjutrisno@gmail.com univ med 2009;28:179-87 abstract pterygium is a condition characterized by the encroachment of a fleshy triangle of conjunctival tissue into the cornea. despite various studies, the pathogenesis of pterygium remains unclear. chronic exposure to sun is the only factor of which the role has been clearly documented by epidemiologic and in vitro studies. recent studies have provided data such as loss of heterozygosity and microsatellite instability, decreased apoptosis, increased growth factors, increased p53 expression, telomerase activity, and presence of oncogenic viruses, which support the concept that pterygia can be considered a neoplastic condition. some of results are contradictory and some features were only found in a proportion of pterygia; this may be due to differences in methodology and study population, but may also be indicative of multiple causative factors. this implies that the pterygium should be viewed as a final common outcome of various etiological factors, such as geography and ethnicity, with ultraviolet radiation-induced damage as the main factor. thus the pterygium remains an enigma, having the features of an exuberant growth but also those of a benign neoplasm. in this connection there is a need for a unifying theory capable of explaining all of the characteristics of the pterygium. keywords: pterygium, apoptosis, growth, neoplasm introduction pterygium is a chronic disease that is characterized by an aggressive invasion of fibrovascular tissue into the cornea, and is one of the most common ocular surface diseases in tropical and subtropical zones.(1) many suffer from this aggressive disease and its prevalence in the general population has been estimated to be 7– 13%.(2,3) two frequently encountered lesions of the conjunctiva are pterygia and pingueculae(4) pterygia have been known since antiquity and theories about their pathogenesis have been proposed for a comparable length of time. one of the theories that should be mentioned is that of fuchs, described in 1892.(5) fuchs postulated 180 tradjutrisno pterygium that the pingueculum is the forerunner of the pterygium. modern ophthalmology usually d e s c r i b e p t e r y g i a a s d e g e n e r a t i o n s o f t h e conjunctiva originating from pingueculae, which have been considered as precursors of pterygia. however, kanski(4) is of the opinion that pterygia may represent a response to chronic dryness and ultraviolet exposure, and does not mention any relationship with pingueculae.(4) the association of pterygia with chronic exposure to sunlight and dryness had also been noted.(6) the term “pterygium” (pteron = a wing) usually refers to a triangular lesion on the conjunctiva, but may also designate winglike or weblike structures in other locations, such as the n e c k o r e l b o w s . t h u s t h e p t e r y g i u m o f ophthalmology should properly be called “ocular pterygium” or “ophthalmic pterygium”. omission of the label “ocular” may result in the inclusion of non-ocular pterygia in internet searches based on the key word “pterygium”, eg. multiple pterygium syndrome.(7) in connection with recent advances in research on pterygia, this mini-review aims to summarize some of the current views on the pathogenesis of pterygia. pterygium morphology a pterygium is usually described as a fleshy triangular growth located in the interpalpebral fissure, most frequently on the nasal side. the lesion starts growing from limbal (or corneal) stem cells in the limbal epithelium and invades the cornea centripetally, eventually covering the pupil. a typical pterygium consists of the apex or head, the collarette or collar, the body and the edges. the apex is the part of the pterygium that invades the cornea as a raised whitish growth firmly adherent to the cornea. behind the apex is the collar, which represents the limbal portion of the pterygium and is continuous with the apex. the apex is preceded by a hood or cap, which is a crescent-shaped grayish avascular patch on the corneal epithelium around the apex. in front of the hood under the corneal epithelium there are small irregular areas of grayish opacity called fuchs’ patches, and a fine yellow-green crescentshaped line (stocker ’s line) resulting from hemosiderin (iron) deposits in bowman’s layer. stocker’s line is a morphological marker of a chronic inactive pterygium; in rapidly advancing lesions there is no time for the accumulation of the iron. the body is the main fleshy part of the pterygium, trapezoidal in shape and highly vascularized. it has winglike lateral extensions (the edges) consisting of conjunctival folds that demarcate the pterygium from the normal conjunctiva.(6,8) the iron deposition in stocker’s line has also been said to originate from tear film lactoferrin.(9) t h e m a i n h i s t o l o g i c a l f e a t u r e s o f a pinguecula are elastotic degeneration of the collagen fibers of the conjunctival stroma, thinning of the overlying epithelium, and occasionally calcification. a pterygium has essentially the same features as a pinguecula, but with more neovascularization and thickening of the conjunctival epithelium. in addition, there is also destruction of bowman’s layer and the superficial corneal lamellae. an in vivo study of 20 primary pterygia from 15 patients by confocal microscopy using t h e n e w l y d e v e l o p e d h e i d e l b e rg re t i n a l tomograph ii (hrt-ii) with cornea module. their observations showed that the vascularization of the pterygium formed loops at the leading edge of the head of the pterygium and did not extend into the healthy cornea.(6) in front of the pterygial h e a d g r o u p s o f c e l l s c o u l d c o m m o n l y b e observed that were more refringent than the corneal epithelial cells. these cell clusters had indistinct outlines and corresponded to fuchs’ patches (ilôts de fuchs). occasionally the region in front of the head took the form of an irregular refringent band, which was more marked in active pterygia. 181 i n t e r m s o f g r o w t h c h a r a c t e r i s t i c s , a pinguecula may occasionally enlarge very slowly, but seldom requires surgical excision. in contrast, a pterygium ultimately progresses towards the visual axis, when surgical excision is indicated. recurrence of pterygia after surgical removal is a particularly difficult problem, for which may solutions have been proposed, such as postoperative administration of beta-radiation, thiotepa, or mitomycin, none of them being completely satisfactory.(9) ocular surface biology the normal ocular surface is composed of two types of epithelia, conjunctival and corneal, each with a distinct cellular phenotype. the nonkeratinized conjunctival epithelium contains mucin-expressing goblet cells, whereas the corneal epithelium is a non-keratinized stratified epithelium expressing the cornea-specific keratins k3 and k12 and has mucin-expressing non-goblet superficial epithelial cells. the l i m b u s o r c o r n e o s c l e r a l j u n c t i o n i s t h e transitional zone between the conjunctiva and cornea, and contains corneal stem cells, also known as limbal stem cells. this finding was a major advance in ocular surface epithelial biology, as this discovery led to many other research studies.(10) in 1997 it was proven conclusively that corneal progenitor cells are localized in the limbus, by culturing limbal cells from patients w i t h c o m p l e t e l o s s o f t h e c o r n e o l i m b a l epithelium into sheets of corneal cells and subsequently restoring the patients damaged cornea by grafting of autologous corneal sheets.(11) characteristics of pterygia t h e c o m m o n l y a c c e p t e d f e a t u r e s o f pterygia may be summarized as follows: pterygia univ med vol.28 no.3 figure 1. pterygium morphology(6) legend: t = head of pterygium; c = collar of pterygium; c = body of pterygium; arrow = progressive zone of fuchs; arrowhead = fuchs’s patch 182 tradjutrisno pterygium originate in the limbal epithelium from altered limbal basal epithelial cells as a result of ultraviolet (uv) radiation induced damage. the limbal cells invade the cornea centripetally followed by conjunctival epithelium. bowman’s layer is dissolved under the leading edge of the pterygia, while vascularization occurs in the conjunctiva adjacent to pterygia. it should also be noted that pterygia have a high recurrence rate.(12) pathogenesis of pterygium currently, theories on the pathogenesis of pterygium cannot give a completely satisfactory explanation of this enigmatic lesion. theories that have been proposed include inflammatory influence, degeneration of connective tissue, genetic instability, angiogenesis, redox related toxicity, cellular proliferation, aberration of apoptosis, exuberant wound healing, altered lipid metabolism, mast cell infiltration, and stem cell dysfunction.(13) dushku and reid(14) found nuclear p53 expression without apoptosis in the limbal epithelia of pterygia, limbal tumors, and most pingueculae. a study also found overexpression of p53 in 7 of 14 pterygia.(15) subsequently d u s h k u e t a l . ( 1 2 ) d e m o n s t r a t e d e l e v a t e d expression of matrix metalloproteinases (mmps) i n p t e r y g i u m c e l l s . m m p s a r e u b i q u i t o u s proteolytic enzymes that are controlled by natural antagonists, the tissue inhibitors of metalloproteinases (timps). the mmps modify or degrade extracellular matrix components in normal and remodeling tissues, and are found in many diseases. these enzymes are secreted by a variety of cell types including fibroblasts and are categorized into five groups: collagenases ( m m p 1 , m m p 8 , m m p 1 3 ) , g e l a t i n a s e s (mmp-2, mmp-9), stromelysins (mmp-3, mmp-10, mmp-21, mmp-22), membrane type mmps.(16) proposed that pterygia actually consist of two tumors, where the main tumor is the pterygium itself, while the second tumor is formed by a group of fibroblasts (the pinguecula) within the pterygium.(12) this condition is also seen in other ocular and skin tumors with elastotic stromal degeneration. the pterygium arises from uv-induced alterations in the p53 gene of limbal (corneal) basal epithelial cells, whereas the pinguecula originate from mutations in the elastin gene of limbal fibroblasts. the figure 2. pterygium (arrow) 183 fibroblasts make abnormal elastotic material and express multiple types of mmp, which are also produced by the limbal epithelial cells. t h e p 5 3 g e n e m u t a t i o n s a r e a c t i v e mutations resulting in a damaged programmed cell death mechanism (apoptosis), thus leading to acquisition of mutations in other genes. this allows for the multistep development of pterygia from p53-expressing limbal basal epithelial cells. t h e p 5 3 m u t a t i o n s m a y a l s o l e a d t o overproduction of tgf-b via the p53-rb-tgfb pathway; thus pterygia may be considered to be tgf-b secreting tumors. excess secretion of tgf-b results in elevated mmp expression by pterygium cells (altered limbal basal epithelial cells) as well as by activated normal fibroblasts under the invading limbus epithelium, both leading to dissolution of bowman’s layer. some of these cytokine-activated fibroblasts migrate anterior to the leading edges of pterygia between bowman’s layer and the basement membrane of the corneal basal cells to form little islands of fibroblasts (ilôts de fuchs) that make mmps, again helping to dissolve bowman’s layer.(12) however, in a more recent study, the investigator found no p53 gene mutations in caucasian pterygia. in their revised theory, the accumulation of p53 protein without causing apoptosis may be the result of inactivation of the p53 protein.(17) in contrast, their molecular analysis of p53 gene mutations in eight chinese pterygia detected mutations in all samples, with six substitutions and two deletions.(18) additionally, a study showed statistically significant overexpression of p53 in around 35% of 31 ecuadorian pterygia, which were also 8-hydroxy deoxyguanosine (8-ohdg) immunoreactive.(19) moreover, a study in brazil found abnormal p53 expression in 14 of 36 pterygial specimens, and hpv dna in 21 of them.(20) support for dushku’s statements on the role of mmps in pterygia may be found in the results of a chinese study, in which the investigators reported overexpression of mmp-1 and mmp-9 i n p r i m a r y p t e r y g i u m . t h e y a l s o f o u n d overexpression of tgf-b1, which may lead to hyperplasia of collagen fibers and accumulation o f m e t a m o r p h i c f i b e r s . ( 2 1 ) i n c o n t r a s t , t h e brazilian study on mmp-9 expression in 42 primary and 19 recurrent pterygia found no significant difference in mmp-9 expression between the pterygia and normal conjunctiva.(16) pterygia do not erode through the full thickness cornea and thus are distinct from malignant tumors. in their view, the pterygium is a mixed soft tissue tumor that is strongly associated with non-ionizing uv radiation.(13) as the investigators note, in this respect the pterygium is highly unusual, because uvinduced tumors in humans are generally of epithelial origin, while uv radiation effects on keloids, if any, are commonly inhibitory. the pterygium is also unusual because researchers have been unable to discover an analogous tumor in animals or reconstitute the disease in organ cultures. the implication is that pterygium involves species-specific mechanisms mediated by in vivo cell-cell or cell-matrix interactions. the molecules involved in this disease may have therefore arisen from divergent evolution in the h u m a n o c u l a r s u r f a c e . t h e o r i g i n o f t h e epithelium overlying pterygial lesions to be still unclear, although suspected to be conjunctival. this view is at variance.(12) who maintain that in the pterygium the migrating limbus is followed by conjunctival epithelium, implying that the epithelium covering the body of the pterygium is of conjunctival origin.(12,13) the global gene expression study used the u133a microarray, and the microarray data were subsequently validated by relative quantitative real time polymerase chain reaction (qpcr) and immunohistochemistry. the study revealed that several genes were upregulated, while apostosis genes were downregulated in pterygium. the upregulated genes included fibronectin (fn1), univ med vol.28 no.3 184 tradjutrisno pterygium carcinoembryonic antigen (ceacam5 or cea), cd24, sparc, microseminoprotein (msmb) and tff1. the downregulated genes coding for apoptosis were tgm2, igfbp3 and dusp1. this down-regulation of apoptosis in pterygium reinforced the over-proliferative tendency of the lesion.(13) a s i m i l a r s t u d y h a d b e e n c o n d u c t e d previously by john-aryankayalil et al.(22) in this study, over expression was found of novel and previously identified extracellular-matrix-related, proinflammatory, angiogenic, fibrogenic, and oncogenic genes in human pterygium. there was one notable difference, in that the clic2 gene was down regulated in a study,(22) but significantly upregulated in other study.(13) the winglike shape of a pterygium and the mechanisms supporting its centripetal growth have also been investigated. the concomitant breakdown of the limbal barrier and subsequent conjunctivalization of the cornea may explain the shape and formation of a primary pterygium. because the growth pattern of pterygia mirrors the radial arrangement of corneal nerves, advanced the hypothesis that directional growth of pterygia may be facilitated by neuropeptides.(23) in support of their hypothesis, the researchers f o u n d t h a t s u b s t a n c e p a t n a n o m o l a r c o n c e n t r a t i o n s i n d u c e d c e l l m i g r a t i o n i n pterygium fibroblasts and vascular endothelium in a dose-dependent fashion, which was inhibited by an nk1 receptor antagonist. on the other hand, pterygium epithelial cells were not migratory in these experiments. they therefore concluded that nk1 receptors are present in pterygia and that substance p is a potent chemoattractant for pterygium fibroblasts and vascular endothelial cells. thus substance p may contribute to the shape of pterygia through its profibrogenic and angiogenic action.(23) uv irradiation of biological tissues may induce a direct phototoxic effect or an indirect effect through formation of radical oxygen species (ros). ros damage cellular proteins, lipids, and dna in a process known as oxidative stress. in addition, ros can induce cyclooxygenase 2 (cox 2) formation via activation of the nf-kb signaling pathway. both ros and cox 2 were found to play the most important role in uvrelated cutaneous carcinogenesis. because pterygium is currently considered to be a uvrelated tumor, a study in taiwan demonstrated cyclooxygenase-2 expression in the epithelium of 90 pterygia.(24) uv radiation-induced dna lesions caused by ros have been associated with initiation and progression in a multistage model of carcinogenesis. a most typical oxidative lesion product is 8-ohdg, since guanine is the most easily oxidized naturally occurring base. 8-ohdg frequently mispairs with adenine during dna replication, leading to g-c to t-a transversion, and thus gives rise to mutations. it has also been established that it is a sensitive marker of oxidative dna damage. g-c to t-a transversions, induced by the presence of 8-ohdg during dna replication, have also been observed in the ras oncogene and p53 in human skin cancers of sunexposed areas and in uv-induced mouse skin cancers. in their study of 31 ecuadorian pterygia, perra et al. (19) found 8-ohdg in 21 (68%) specimens, and p53 expression in 11 (35%). a study had previously reported the ocurrence of a g-c to t-a transversion in exon 6, codon 213 of p53, which is typical of oxidative lesions, and also the presence of 8-ohdg in 23% of 52 pterygia samples.(25) the in vivo confocal microscopy study of gheck et al.(6) substantially confirmed previous observations on the features of pterygia. in their view, ultraviolet radiation causes micro traumas that are responsible for the inflammatory lesions. certain uv-sensitive tumor supressor genes (p53) may play a role in the pathogenesis of pterygia, whilst other factors, such as dust, wind, heat and dryness, are of secondary importance. their theory is based on localized areas of 185 dryness of the conjunctiva (dellen) due to destruction of the lipid layer of the tear film, leading to an increased evaporation. these dellen i n d u c e a c i c a t r i z i n g r e s p o n s e w i t h t h e proliferation and growth of inflammatory conjunctival tissues. it is notable that the mucus produced by the pterygium differs from normal conjunctival mucus. the cellular proliferation, inflammation, modification of conjunctival tissue and angiogenesis are dependent on the presence of growth factors such as fibroblast growth factor (fgf), platelet-derived growth factor (pdgf), transforming growth factor (tgf) and tumor necrosis factor-α (tnf-α), some of which are produced by the fibroblasts.(6) a study found increased telomerase activity in pterygia compared with normal conjunctival tissues.(26) they studied 27 tissue samples from pterygia, in which 14 epithelial tissues from pterygia (51.9%) were positive for telomerase activity, whereas telomerase activity was positive in only three of 9 normal conjunctival tissues (33.3%), although there was no significant statistical difference between pterygium-covered conjunctival epithelia and normal conjunctival epithelial tissues. telomerase functions by maintaining a constant telomere length, thus ensuring the retention of chromosomal stability attributed to functional telomeres and alters the cell’s biological clock, effectively preventing a cell from aging. telomerase activity is usually found in immortal cells such as germline cells, stem cells, malignant cells, cells from some types of benign and premalignant skin tumors, and also in cutaneous tissues chronically exposed to uv radiation. because pterygia are believed to be also caused by chronic uv radiation, park et al.(26) searched for telomerase activity in pterygia, w i t h t h e a b o v e m e n t i o n e d r e s u l t s . t h e researchers suggest that the difference in telomerase activity between normal conjunctival epithelium and that of pterygium may be caused by an activation of telomerase or a clonal expansion of telomerase positive cells due to chronic sun exposure. to elucidate the role of angiogenesis in the pathogenesis of pterygia, aspiotis et al. (27) investigated the microvessel density and the expression of vascular endothelial growth factor (vegf) and thrombospondin-1 (tsp-1) in tissue sections of 52 pterygia and 7 normal conjunctiva. the researchers found a higher microvessel density and an increased level of vegf in pterygia as compared to normal conjunctiva, and a statistically significant positive correlation b e t w e e n m i c r o v e s s e l d e n s i t y v a l u e s a n d pterygial vegf expression. on the other hand, there was a low level of tsp-1 expression in approximately half of the pterygia. the contribution of cellular immunity to pterygium pathogenesis was studied in turkey by tekelioglu et al.(28) who found higher rates of intercellular adhesion molecule-1 (icam-1)positive cells, vascular cell adhesion molecule1 (vcam-1)-positive cells, human leucocyte antigen (hla)-dr-positive cells and cd4 and cd8 lymphocytes in pterygium tissue samples. f i n a l l y, i n a m o r e r e c e n t r e v i e w o f p t e r y g i u m p a t h o g e n e s i s , a p a r t f r o m t h e previously mentioned factors, such as uv radiation, limbal stem cells, oxidative stress, growth factors and cytokines, the possible pathogenetic roles of altered phospholipase d, human papilloma virus (hpv) involvement and ki-ras proto-oncogene mutations are discussed.(9) pterygium: benign neoplasm? p t e r y g i u m a n d p a p i l l o m a a r e b e n i g n conjunctival tumors that can be treated by surgical excision. however, recurrences are frequently observed, and recurrent lesions tend to grow more rapidly than primary lesions. a l t h o u g h t h e e t i o l o g y o f p t e r y g i u m a n d papilloma has not been established, human papillomavirus (hpv) is thought to be one of the causative factors.(29) another study found tradjutrisno pterygium 186 tradjutrisno pterygium unifying theory capable of explaining all of the c h a r a c t e r i s t i c s o f t h e p t e r y g i u m . a s b o t h exuberant growth and benign neoplasm are essentially cellular proliferations, whether normal or aberrant, we have to a wait further studies for a solution to this interesting scientific puzzle. references 1. coroneo mt, di girolamo n, wakefield d. the pathogenesis of pterygia. curr opin ophthalmol 1999;10:282–8. 2. gazzard g, saw sm, farook m, koh d, widjaja d, chia se, et al. pterygium in indonesia: prevalence, severity and risk factors. br j ophthalmol 2002;86:1341–6. 3. wong ty, foster pj, johnson gj, seah sk, tan dt. the prevalence and risk factors for pterygium in an adult chinese population in singapore: the tanjong pagar survey. am j ophthalmol 2001;131: 76–83. 4. kanski jj. clinical ophthalmology: a systematic approach. 5th ed. butterworth-heinemann: edinburgh;2003. 5. fuchs e. uber das pterygium. albert von graefes arch klin exp ophthalmol 1892;38:1. cited by gheck l, dupas b, denion e, amar n, baudouin c. apport de la microscopie confocale in vivo à l’étude des ptérygions. [advantages of the in vivo confocal microscope for investigation of the pterygium]. j fr ophtalmol 2007;30:3-10. 6. gheck l, dupas b, denion e, amar n, baudouin c. apport de la microscopie confocale in vivo à l’étude des ptérygions. [advantages of the in vivo confocal microscope for investigation of the pterygium]. j fr ophtalmol 2007;30:3-10. 7. vogt j, harrison bj, spearman h, cossins j, vermeer s, cate ln, et al. mutation analysis of chrna1, chrnb1, chrnd, and rapsn genes in multiple pterygium syndrome/fetal akinesia atients. am j hum genet 2008;82:222–7. 8. buratto l, phillips rl, carito g. clinical anatomy. in: pterygium surgery. buratto l, phillips rl, carito gc, editors. thorofare:slack;2000.p.17-20. 9. detorakis et, spandidos da. pathogenetic mechanisms and treatment options for ophthalmic pterygium: trends and perspectives. intl j mol med 2009;23:439-47. hpv in 50% of 10 pterygia with the same types as in conjunctival papillomas. they suggested that persistent conjunctival hpv may possibly play a part in the recurrence of pterygia post excision.(30) in addition, rodrigues et al.(20) hpv dna in 21 of 36 pterygia in brazil, along with a b n o r m a l 5 3 e x p r e s s i o n i n 1 4 o f t h e 3 6 s p e c i m e n s , a n d c o n c l u d e d t h a t a b n o r m a l expression of p53 and hpv dna are required co-factors for the development of pterygium. the different and sometimes contradictory results of various studies and the fact that some features, eg. p53 mutations, were only found in a p r o p o r t i o n o f p t e r y g i a , m a y b e d u e t o d i ff e r e n c e s i n m e t h o d o l o g y a n d s t u d y populations. however, it may also be indicative of multiple causative factors, implying that the pterygium may not be a distinct disease entity but should rather be viewed as a final common outcome of various etiological factors, such as geography and ethnicity, with uv radiationinduced damage as the main factor. in connection with ethnicity, the variable findings of p53 protein expression in pterygia by several study groups might be explained by the observation that the frequency of p53 mutations in cancer can vary among different races. ueda et al.(31) d e m o n s t r a t e d t h a t t h e i n c i d e n c e o f i m m u n o r e a c t i v i t y f o r p 5 3 i s r e l a t e d t o differences in development of pterygium with race as well as environmental factors. conclusions from the abovementioned observations and theories it may be concluded that the pterygium remains an enigma, having the features of an exuberant growth but also those of a benign neoplasm. the previously held opinion that the pterygium is a degenerative condition should in all probability be revised, but not (yet) discarded. at present there is a plethora of data, but no 187 10. di girolamo n, tedla n, kumar rk, mccluskey p, lloyd a, coroneo m, et al. culture and chracterisation of epithelial cells from human pterygia. br j ophthalmol 1999;83:1077-82. 11. pellegrini g, traverso ce, franzi at, zingirian m, cancedda r, de luca m. long-term restoration of damaged corneal surfaces with autologous cultivated corneal epithelium. lancet 1997;349: 990-3. 12. dushku n, john m, schultz gs, reid tw. pterygia pathogenesis: corneal invasion by matrix metalloproteinase expressing altered limbal epithelial basal cells. arch ophthalmol 2001;119: 695-6. 13. tong l, chew j, yang h, ang lpk, tan dth, beuerman rw. distinct gene subsets in pterygia formation and recurrence: dissecting complex biological phenomenon using genome wide expression data. bmc med genomics 2009;2:14. 14. dushku n, reid tw. p53 expression in altered limbal basal cells of pingueculae, pterygia, and limbal tumors. curr eye res 1997;16:1179-92. 15. weinstein o, rosenthal g, zirkin h, monos t, lifshitz t, argov s. overexpression of p53 tumor suppressor gene in pterygia. eye 2002;16:619–21. 16. schellini sa, hoyama e, oliveira de, bacchi ce, padovani cr. expressão da metaloprotease de matriz-9 no pterígio [matrix metalloproteinase-9 expression in pterygium]. arq bras oftalmol 2006; 69:61-4. 17. schneider bg, john-aryankalayil m, rowsey jj, dushku n, reid tw. accumulation of p53 protein in pterygia is not accompanied by tp53 gene mutations. exp eye res 2006;82:91-8. 18. tsai yy, cheng yw, lee h, tsai fj, tseng sh, chang kc. p53 gene mutation spectrum and the relationship between gene mutation and protein levels in pterygium. mol vis 2005;11:50-5. 19. perra mt, maxia c, corbu a, minerba l, demurtas p, colombari r, et al. oxidative stress in pterygium: relationship between p53 and 8-hydroxydeoxyguanosine. mol vis 2006;12:136-42. 20. rodrigues fw, arruda jt, silva re, moura kkvo. tp53 gene expression, codon 72 polymorphism and human papillomavirus dna associated with pterygium. gen mol res 2008;7:1251-8. 21. ke m, sun z, wang l. expression of mmp-1, mmp-9 and tgf-ß1 in human pterygium and its clinical significance. faseb j 2007;21:711-18. 22. john-aryankalayil m, dushku n, jaworski cj, cox ca, schultz g, smith ja, et al. microarray and protein analysis of human pterygium. mol vis 2006; 12:55-64. 23. chui j, di girolamo n, coroneo mt, wakefield d. the role of substance p in the pathogenesis of pterygia. invest ophthalmol vis sci 2007;48:4482– 9. 24. chiang cc, cheng yw, lin cl, lee h, tsai fj, tseng sh, et al. cyclooxygenase 2 expression in pterygium. mol vis 2007;13:635-8. 25. tsai yy, cheng yw, lee h, tsai fj, tseng sh, chang kc. p53 gene mutation spectrum and the relationship between gene mutation and protein levels in pterygium. mol vis 2005;11:50-5. 26. park tk, jin kh. telomerase activity in pterygial and normal conjunctival epithel-ium. korean j ophthalmol 2000;14:85-9. 27. aspiotis m, tsanou e, gorezis s, ioachim e, skyrlas a, stefaniotou m, et al. angiogenesis in pterygium: study of microvessel density, vascular endothelial growth factor, and thrombospondin-1. eye 2007;21:1095–101. 28. tekelioglu y, turk a, avunduk am, yulug e. flow cytometrical analysis of adhesion molecules, tlymphocyte subpopulations and inflammatory markers in pterygium. ophthalmologica 2006;220: 372–8. 29. piras f, moore ps, ugalde j, perra mt, scarpa a, sirigu p. detection of human papillomavirus dna in pterygia from different geographical regions. br j ophthalmol 2003;87:864-6. 30. gallagher mj, giannoudis a, herrington cs, hiscott p. human papillomavirus in pterygium. br j ophthalmol 2001;85:782–4. 31. ueda y, kanazawa s, kitaoka t, dake y, ohira a, ouertani am, et al. immunohistochemical study of p53, p21 and pcna in pterygium. acta histochem 2001;103:159-65. univ med vol.28 no.3 combination of five clinical data as prognostic factors of mortality after ischemic stroke 68 *faculty of medicine, duta wacana christian university/ bethesda hospital, yogyakarta correspondence: dr. rizaldy taslim pinzon, sps, m.kes faculty of medicine, duta wacana christian university jl. dr. wahidin sudiro husodo no.. 5–25 yogyakarta 55224 email: drpinzon17@gmail.com univ med 2017;36:68-76 doi: 10.18051/univmed.2017.v36.68-76 pissn: 1907-3062 / eissn: 2407-2230 received august 8, 2016 accepted for publication april 25, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract combination of five clinical data as prognostic factors of mortality after ischemic stroke background the mortality rate after ischemic stoke is influenced by various factors. prognosis after ischemic stroke can be predicted using a scoring system to help the doctor to evaluate patient’s condition, neurologic deficits, and possible prognosis as well as make appropriate management decisions. the objective of this study was to identify the factors which determine mortality rates in patients after ischemic stroke and to determine the prognosis of ischemic stroke patients using the predictive mortality score. methods this was a nested case control study using data from the stroke registry and medical records of patients at the neurology clinic of bethesda hospital yogyakarta between 2011-2015. data was analysed using simple and multiple logistic regression analysis. the scoring was analyzed using receiver-operating characteristic (roc) curve and the cut-off point using area under the curve (auc). results multiple logistic regression analysis showed a significant association between mortality of ischemic stroke patients and age (or: 4.539, 95% ci: 1.974-10.439, p<0.001), random blood glucose (or: 2.692, 95% ci: 1.580-4.588, p<0.001), non-dyslipidemia (or: 2.313, 95% ci: 1.3953.833, p=0.001), complications (or: 1.609, 95% ci: 1.019-2.540, p=0.041), risk of metabolic encephalopathy (or: 2.499, 95% ci: 1.2445.021, p=0.010) and use of ventilators (or: 17.278, 95% ci: 2.015148.195, p=0.009). conclusions age, high random blood glucose level, complications, metabolic encephalopathy risk and the use of ventilators are associated with mortality after ischemic stroke. the predictive mortality score can be used to assess the prognosis of patients with ischemic stroke. keywords: ischemic stroke, causes of mortality, predictive mortality score doi: http://dx.doi.org/10.18051/univmed.2017.v36.68-76 rizaldy taslim pinzon*, fransiska theresia meivy babang*, and esdras ardi pramudita* universa medicinajanuary-april, 2017 vol.36 no.1 original article 69 univ med vol. 36 no.1 introduction ischemic stroke is an episode of neurologic dysfunction that is caused by focal infarction of the brain, spinal cord and retina.(1) each year the number of stroke patients is increasing. the prevalence of stroke (with the criterion of having been diagnosed by health personnel) of 8.3 per 1000 in the indonesian basic health research survey for 2007 (riskesdas 2007) increased to 12.1 per 1000 in 2013 (for stroke respondents aged 15 years and above).(2) from data at the stroke registry of bethesda hospital for 2014 it is known that each trimester the number of patients with stroke is increasing. of 2460 stroke patients at bethesda hospital between 2011 and 2013, a total of 9.47% had died.(3) there are many factors that influence the cause of death in patients with ischemic stroke. age, stroke subtype, atrial fibrillation, history of previous strokes/transient ischemic attack, albumin, creatinine, von willebrand factor and ß-thromboglobulin are the factors that can predict death in patients with ischemic stroke.(4) the study of mogensen et al.(5) on cause-specific mortality after stroke found that hypertension, smoking and alcohol consumption were not associated with cause-specific death. previous studies reported that antihypertensive treatment directly after acute ischemic stroke may increase the mortality rate.(6) other studies state that hypertension is the factor that is most frequently found in stroke patients. the factors that influence the prognosis of stroke patients are age, mean corpuscular volume (mcv), folic acid, and erythrocyte sedimentation rate (esr). advanced age, high mcv and low folic acid concentration in stroke patients are associated with a poor prognosis.(7) çomoðlu et al.(8) in their study state that esr level of patients at admission do not reflect stroke severity and cannot be used as a useful predictor for evaluating the short term prognosis of patients with acute ischemic stroke. the predictive factors of mortality in stroke patients are still varied and subject to debate. the prognosis of ischemic stroke can be predicted by scoring. the use of a scoring system can help doctors in evaluating the initial condition of the patients, giving a rapid and accurate evaluation about deficits in stroke, thus facilitating communication between doctors and saving time for triage and treatment of the patients.(9) the objective of this study was to identify the factors that influence mortality in patients with ischemic stroke and determine the prognosis of these patients using the predictive mortality score. methods design of study this independent nested case control study was conducted at bethesda hospital, yogyakarta, from february until march 2016 using secondary data from the stroke registry and medical records at bethesda hospital, yogyakarta, from 2011 up to and including 2015. study subjects the study subjects were patients with ischemic stroke over 40 years of age who had been diagnosed by head ct scan from 2011 to 2015 at bethesda hospital, yogyakarta, whose data in the stroke registry and the medical records were complete, and had not been discharged against medical advice. sample size was c a l c u l a t e d a c c o r d i n g t o t h e f o r m u l a f o r differences in proportions of two populations. based on the formula for sample size calculation, the sample comprised 179 deceased patients and 179 live patients so that the total sample size required was 358 patients. measurements the age of the patients was calculated from the number of years from birth up to the last data collection at the hospital. the level of consciousness was assessed with the glasgow coma scale (gcs) or noted in the medical record as compos mentis or somnolence or stupor or 70 coma. muscle strength was assessed on the medical research council (mrc) scale. systolic blood pressure, diastolic blood pressure, random blood glucose and leukocyte count were obtained from the results of examination at admission. patients were declared to be at risk for metabolic encephalopathy due to intoxication, renal disorders (uremic encephalopathy), pulmonary or hepatic disorders (hepatic encephalopathy) from the changes in electrolyte or enzyme c o n c e n t r a t i o n s o r u r e u m / c r e a t i n i n e concentrations (sodium concentration <125 mmol/l or sgot or sgpt or ureum or creatinine concentration >3 times the normal concentrations). patients were declared have a history of hypertensive disease if they had ever been diagnosed with hypertension or consumed antihypertensive medications, or had blood pressures of >140/90 mmhg at 2 readings. patients were declared to have atrial fibrillation by a cardiologist on the basis of their ekg. patients were declared to have a history of dyslipidemia if they had ever been diagnosed with dyslipidemia or consumed statins/fibrate, or their cholesterol concentration was >200 mg/dl or triglycerides >150 mg/dl (fasting) or hdl <40 mg/dl or ldl >100mg/dl. patients were declared to have a history of diabetes mellitus if they had ever been diagnosed with diabetes mellitus or consumed antidiabetics, or from the results of laboratory examination of random blood glucose of ≥200 g/dl or fasting blood glucose of ≥126 g/dl. the complications that were investigated in this study were the other disorders that emerged during hospitalization of the stroke patients and were noted in the medical records, i.e. gastrointestinal bleeding, dysphagia and urinary tract infections. data analysis data were analyzed by descriptive analysis and statistical analysis. descriptive analysis comprised characteristic data of all patients, w h e r e a s s t a t i s t i c a l a n a l y s i s c o m p r i s e d univariate, bivariate and multivariate analysis and validation tests using receiver-operating characteristic (roc) curves, whereas for cutoff point the area under the curve (auc) was used. the discrimination model was determined from the area under the roc curve that was calculated by nonparametric methods. the area under the roc curve is a measure of total predictive discrimination to separate patients with good outcomes from patients with poor outcomes. a roc curve area of 0.5 indicates no discrimination, and an area of 1.0 indicates perfect discrimination. determination of the cutoff point in this study was evaluated by logistic regression to obtain the largest area under the curve.(10) ethical clearance this study was observational on secondary data from the registry. for this study ethical clearance was obtained from the research ethics committee, faculty of medicine, duta wacana christian university, under no. 152/ c/16/ fk/ 2016. results table 1 above shows the baseline characteristics of all study samples that were analyzed descriptively. a total of 358 patients meeting the inclusion and exclusion criteria were recruited into this study, consisting of 203 male patients (56.7%) and 155 female patients (43.3%). the most numorous subjects were in the age range of >70 years, comprising 121 patients (33.8%). the most frequent onset to admission time was >24 hours (44.1%). the relationship of predictive factors of mortality with the dependent variables listed in table 2 above shows that the variables that have a significant relationship with mortality in ischemic stroke patients (p<0.05) are age, level of consciousness, muscle strength, side of weakness, systolic blood pressure, diastolic blood pressure, leukocyte count, random blood glucose, history of dyslipidemia, complications, risk of metabolic encephalopathy and use of ventilators. pinzon, babang, pramudita prognostic factors of mortality and stroke 71 univ med vol. 36 no.1 table 1. baseline characteristics of all patients 72 pinzon, babang, pramudita prognostic factors of mortality and stroke table 1. cont. table 2. relationship between predictive factors of mortality and death of ischemic stroke patients 73 univ med vol. 36 no.1 table 2. cont. table 3. multivariate analysis with logistic regression of mortality risk factors 74 the data in table 3 on multivariate analysis with logistic regression list the variables that have a significant relationship with mortality in ischemic stroke patients, i.e. age, random blood glucose concentration, non-dyslipidemia, complications, risk of metabolic encephalopathy and use of ventilators (p<0.05). the results of the analysis show that there are variables that are associated with mortality in ischemic stroke patients, i.e. age (or: 4.539, 95% ci: 1.974-10.439, p<0.001), random blood glucose (or: 2.692, 95% ci: 1.580-4.588, p<0.001), non-dyslipidemia (or: 2.313, 95% ci: 1.395-3.833, p<0.001), complications (or: 1.609, 95% ci: 1.019-2.540, p=0.041), risk of metabolic encephalopathy (or: 2.499, 95% ci: 1.244-5.021, p=0.010) and use of ventilators (or: 17.278, 95% ci: 2.015-148.195, p=0.009). the discrimination value of the scoring model in this study was 0.692; (95%ci 0.638-0.746; p<0.001). the larger the auc the better the overall accuracy of the test. in this study the auc value lies between 0.50 and 0.75, thus it can be said that the overall accuracy of the test is fair. the value of p<0.001 shows that the test is proven to be able to predict the mortality. discussion based on the results of the study it is known that with advancing age, the mortality risk in stroke patients is also increased. patients who are over 50 years old have a higher mortality risk as compared with patients who are less than 50 years old. according to the study by fonarow et al.(11) elderly patients have a higher prevalence o f d i s o r d e r s s u c h a s a t r i a l f i b r i l l a t i o n , hypertension, coronary heart disease, a history of stroke and a smoking habit. elderly patients commonly die in hospital instead of at home, for each 10-year increase in age. patients with high random blood glucose have a 2.6 times higher mortality risk compared with patients with normal random blood glucose. from a study on focal experimental ischemia by piironen et al.(12) it is known that hyperglycemia can increase the size of the infarct and decrease the therapeutic window of recanalization. patients without dyslipidemia have a 2.3 times higher mortality risk than do patients with dyslipidemia. that dyslipidemia is a factor that reduces the mortality risk in patients with ischemic stroke is still a controversial issue. according to sohail et al.(13) high concentrations of triglycerides and low-density lipoprotein (ldl) and low concentration of high-density lipoprotein (hdl) are associated with high scores on the modified rankin scale (mrs), with severe stroke and poor clinical outcomes. the results of the aforementioned study contradict those of the study by muhammad et al.(14) who showed that patients with high total cholesterol concentrations have good clinical outcomes, since the cholesterol functions as a buffer for neutralizing free radicals and preventing injury to neural tissues. patients with complications such as gastrointestinal bleeding, urinary tract infections, and dysphagia have a 1.6 times higher mortality r i s k c o m p a r e d w i t h p a t i e n t s w i t h o u t complications. according to a study conducted by ogata et al.(15) gastrointestinal bleeding after the onset of stroke can cause a decrease in hemoglobin concentration, neurological damage, poor functional results and death. another complication, i.e. urinary tract infection occurring during hospitalization of the stroke patients, such as put forward by poisson et al.(16) have a characteristic impact, i.e. increased duration of hospital stay, increased health care costs, exposure to intravenous antibiotics, the risk of bacteriemia, decreased neurological status, poor clinical outcomes, and increased mortality risk. p a t i e n t s w i t h a r i s k o f m e t a b o l i c encephalopathy have a 2.4 times higher mortality risk compared with patients without a risk of metabolic encephalopathy. the study conducted by rodrigues et al.(17) about ‘hyponatremia in the prognosis of acute ischemic stroke’ showed that hyponatremia is associated with mortality in patients with acute ischemic stroke. according pinzon, babang, pramudita prognostic factors of mortality and stroke 75 univ med vol. 36 no.1 to zhang et al.(18) the serum urea concentration of patients with acute ischemic stroke who are treated with intravenous tissue plasminogen activator (r-tpa) is independently associated with poor clinical results and death of the patients. p a t i e n t s w h o u s e v e n t i l a t o r s d u r i n g hospitalization have a 17.2 times higher mortality risk compared with patients who do not use ventilators. the study by mayer et al.(19) states that two-thirds of stroke patients who use mechanical ventilators die during hospitalization and the majority of patients who survive experience severe disabilities. patients who experience coma or worsening of their clinical condition after intubation have an extremely low chance of survival. the discrimination value of the scoring model in this study is 0.692 (95% ci 0.638-0.746; p<0.001). the larger the auc the better the overall accuracy of the test. in this study the auc value lies between 0.50 and 0.75, thus it may be said that the overall test accuracy is fair. this value shows that this model is proven to be able to predict mortality. one study demonstrates the importance of staffing levels and having a reasonable catchment population/stroke service as key factors associated with a better mortality outcome in stroke, which is one of the biggest killers globally.(20) variable staff composition in hospitals was not investigated in our study and is one of the limitations of the study. another limitation of the present study is the nested case control design using secondary data, i.e. from the stroke registry and medical records of ischemic stroke patients at bethesda hospital, yogyakarta. a limitation that is difficult to avoid is that the investigators were unable to control the condition and quality of the measurements performed by other persons in the past, because of the use of secondary data. it is hoped that clinicians may be able to perform a good evaluation of the condition of the patients and appropriately determine the prognosis of these patients so that the patients’ condition can be reported more accurately to their families. if in the future similar studies will be conducted, it is suggested that they use a different study method, e.g. a prospective cohort study using primary data so that the patients may be directly followed up. conclusions age, high random blood glucose concentration, complications, risk of metabolic encephalopathy, and use of ventilators are associated with mortality in ischemic stroke patients. the management of ischemic stroke should focus on the prevention of complications, thus decreasing mortality risk. conflict of interest the authors declare that there is no conflict of interest with the parties associated in this study. acknowledgements the authors thank the duta wacana christian university for the funding of this study and the staff of bethesda hospital, yogyakarta, for the use of their facilities during the study and for their kind assistance. contribution rtp, tb and ea contributed to prepare the manuscript. rtp contributed to design the research, and responsible for the final content rtp, tb and ea contributed to collected and analyzed the data; rizaldy pinzon contributed to wrote the manuscript, and all authors read and approved the final manuscript. references 1. sacco rl, kasner se, broderick jp, et al. on behalf of the american heart association stroke council, council on cardiovascular surgery and anesthesia, council on cardiovascular radiology and intervention, council on cardiovascular and stroke nursing, council on 76 epidemiology and prevention, council on peripheral vascular disease, and council on nutrition, physical activity and metabolism . an updated definition of stroke for the 21st century: a statement for healthcare professionals from the american heart association/american stroke association. stroke 2013;44:2064-89. doi: 10.1161/str.0b013e318296aeca. 2. kementerian kesehatan ri. riset kesehatan dasar (riskesdas 2013). jakarta; badan penelitian dan pengembangan kesehatan, kementerian kesehatan ri; 2013. 3. pinzon r. profil stroke: gambaran tentang pola demografi, faktor resiko, gejala klinik, dan luaran klinis pasien stroke. yogyakarta: betha grafika; 2014. 4. carter am, catto aj, mansfield mw, et al. predictive variables for mortality after acute ischemic stroke. stroke 2007;38:1873-80. doi: 10.1161/strokeaha.106.474569. 5. mogensen ub, olsen ts, andersen kk, et al. cause-specific mortality after stroke: relation to age, sex, stroke severity, and risk factors in a 10-year follow-up study. j stroke cerebrovasc dis 2013;22:e59–65. 6. ivanov a, ambreen m, korniyenko a. permissive hypertension in acute ischemic stroke: is it a myth or reality? j am coll cardiol 2015;suppl.10s:a1344. 7. aksoy d, inanir a, ayan m, cevik b, et al. predictor of mortality and morbidity in acute ischemic stroke. arch neuropsychiatry 2013;50: 40-4. 8. çomoðlu ss, çilliler ae, güven h. erythrocyte sedimentation rate: can be a prognostic marker in acute ischemic stroke. turkish j cerebrovasc dis 2013;19:18-22. doi: 10.5505/tbdhd.2013. 32042. 9. kasner se. clinical interpretation and use of stroke scales.lancet neurol 2006;5:603–12. 10. fan j, upadhye s, worster a. understanding receiver operating characteristic (roc) curves. can j emerg med 2006;8:19-20. pinzon, babang, pramudita prognostic factors of mortality and stroke 11. fonarow gc, reeves mj, zhao x, et al. agerelated differences in characteristics, performance measures, treatment trends, and outcomes in patients with ischemic stroke. circulation 2010;121:879-91. 12. piironen k, putaala j, rosso c, et al. glucose and acute stroke.stroke 2012;43:898-902. 13. sohail, a, khatri ia, mehboob n. effect of dyslipidemia on severity and outcome of stroke using mrs scores in northern pakistani population. rmj 2013:384;345-50; 14. muhammad d, javed m, sheikh ga. acute ischemic stroke; correlation between higher total cholesterol level and high barthel index score in patients. professional med j 2015;22:276-80. 15. ogata t, kamouchi m, matsuo r, et al. gastrointestinal bleeding in acute ischemic stroke: recent trends from the fukuoka stroke registry. cerebrovasc dis extra 2014;4:156–64. 16. poisson sn, johnston sc, josephson sa. urinary tract infections complicating stroke: mechanisms, consequences, and possible solutions. stroke 2010;414:e180-4. 17. rodrigues b, staff i, fortunato g, et al. hyponatremia in the prognosis of acute ischemic stroke. stroke cerebrovasc dis 2014;23:850-4. doi: 10.1016/j.jstrokecerebrovasdis.2013.07.011. 18. zhang y, churilov l, meretoja a, et al. elevated urea level is associated with poor clinical outcome and increased mortality post intravenous tissue plasminogen activator in stroke patients. j neurol sci 2013;332:110-5. doi: 10.1016/j.jns.2013.06.030. 19. mayer sa, copeland d, bernardini gl, et al. cost and outcome of mechanical ventilation for life-threatening stroke. stroke 2000;31:2346-53. 20. myint pk, bachmann mo, loke yk, et al. important factors in predicting mortality outcome from stroke: findings from the anglia stroke clinical network evaluation study. age ageing 2017;46:83–90. doi: 10.1093/ageing/ afw175. low vitamin c intake increases risk of pre-eclampsia 128 *department of nutrition, faculty of medicine, diponegoro university, semarang **department of environmental health, faculty of public health diponegoro university, semarang ***department of public health nutrition, faculty of public health diponegoro university, semarang correspondence: rifatul masrikhiyah department of nutrition, faculty of medicine, diponegoro university, semarang email: rifatul.masrikhiyah@yahoo.com univ med 2016;35:128-34 doi: 10.18051/univmed.2016.v35.128-134 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 low vitamin c intake increases risk of pre-eclampsia in high pesticide exposure area rifatul masrikhiyah*, suhartono**, and martha irene kartasurya*** background pre-eclampsia is the largest direct cause (42%) of maternal mortality death in brebes district in 2013. the cause of pre-eclampsia is not known with certainty, oxidative stress being one of its causes. the aim of this study was to determine macroand micronutrient intakes that do not correspond to the needs, as pre-eclampsia risk factors in post-partum women. methods the research was an observational study of case control design. study subjects were 20-35 year-old post partum women consisting of 55 cases (pre-eclampsia) and 55 controls (non-pre-eclampsia). nutrient intakes were measured using semi-quantitative food frequency questionnaires and data on levels of pesticide exposure, body mass index (bmi), midupper arm circumference (muac) and education were collected from structured interviews. data was analyzed by chi-square tests and multiple logistic regression. results there was no difference in age and muac between the cases and controls. low vitamin c intake (or: 20.93; 95% ci: 2.72 – 161.36), high polyunsaturated fatty acid intake (or: 10.50; 95% ci: 2.47 – 44.57), and low vitamin e intake (or: 8.57; 95% ci: 2.07 – 35.46) were pre-eclampsia risk factors after controled for pesticide exposure, bmi and education. low vitamin c intake played the most important role in the incidence of preeclampsia. conclusion low vitamin c intake played the most important role toward preeclampsia. greater caution is needed when issuing recommendations regarding the consumption of vitamin supplements in pregnancy, as high doses of some vitamins may be deleterious. keywords: pre-eclampsia, vitamin c, vitamin e, polyunsturated fatty acids, pesticide exposure doi: http://dx.doi.org/10.18051/univmed.2016.v35.128-134 129 univ med vol. 35 no.2 introduction pre-eclampsia still constitutes a pubic health problem in developed and developing countries, contributing toward global maternal and perinatal morbidity and mortality.(1) pre-eclampsia affects 5-10% of all global pregnancies. the prevalence of pre-eclampsia in developing countries reaches 16.7% and around 40-60% is estimated to contribute toward maternal mortality in developing countries.(2) the world health organization estimates that the incidence of preeclampsia is seven-fold higher in developing countries (2.8% of live births) than in developed countries (0.4%).(3) according to the health service of brebes district, in 2012 the direct causes of the maternal mortality rate (mmr) in this district were hemorrhage 22%, pre-eclampsia 42%, infection 8%, heart failure 14%, dyspnea 4%, ruptured ectopic pregnancy 2%, ectopic pregnancy 2%, post curettage 2% and others 4%.(4) oxidative stress results from an imbalance between free radical production and the antioxidant defense system (5) and is one of the causes of preeclampsia. nutrients may influence oxidative stress by increasing free radicals/decreasing antioxidants/providing substrates for the formation of free radicals. a case-control study on 453 pregnant women (151 cases and 302 controls) showed that women with mid-upper arm circumference (muac) of >25.6 cm had two and a half times greater risk for pre-eclampsia than pregnant women with muac of <25.6 cm.(6) according to a study conducted by kartasurya (7) on 200 pregnant/post partum women (50 pre-eclampsia cases and 150 controls), in women with pre-pregnancy body mass index (bmi) scores of >23 kg/m2, the risk of pre-eclampsia was four and a half times greater than in women with pre-pregnancy bmi scores of <23 kg/m2. lipids are involved in the generation of free radicals and polyunsaturated fatty acids (pufa) tend to be oxidized by free radicals and reactive oxygen species (ros). a high pufa intake may render the organism more susceptible to lipid peroxidation, which is strongly suspected to play a important role in the causation of maternal endothelial dysfunction and the appearance of clinical symptoms of pre-eclampsia. (8-10) according to the study by clausen et al.(11) an increased intake of pufa in pregnant women increases the risk of pre-eclampsia. antioxidants may decrease the level of lipid peroxides by limiting or inactivating their formation. free radicals may be treated by administration of antioxidants such as vitamins a, c and e. low intakes of vitamins a, c and e may cause an imbalance between free radicals and antioxidants in the body. a previous study found that low intakes of vitamins a, c and e may increase the risk of pre-eclampsia.(12) since protein status affects glutathione concentrations, a low protein intake contributes toward oxidative stress, as a result of low glutathione concentrations. low glutathione concentrations affect the antioxidant effects of glutathione itself, causing free radicals to become uncontrolled, thus resulting in oxidative stress. the results of a study in pregnant women with pre-eclampsia showed that the glutathione concentrations in their blood were lower than in normal pregnant women.(13) a total of 104 pregnant women with preeclampsia and of 206 pregnant women at high risk of, but not suffering from, pre-eclampsia, were studied in second trimester pregnancies. this study showed that there was no relationship between nutritional intake and the development of pre-eclampsia (or other effects) when evaluated for 7 days.(14) a review and bivariate meta-analysis of 36 studies involving 1,699,073 pregnant women showed bmi to be a weak predictor of preeclampsia.(15) brebes district in central java is one of the largest producers of shallots (bawang merah) in indonesia, and has a high pesticide utilization. pesticide exposure may cause health disorders in pregnant women. the study conducted by saldana 130 et al.(16) found that pesticide exposure is a risk factor of pre-eclampsia. based on these facts the present study aimed to determine the macroand micronutrient intakes that do not correspond to needs, as risk factors of pre-eclampsia in postpartum women residing in areas of high pesticide exposure. the present study differs from previous studies in the location/site of the study, since it was conducted in brebes district in an area of high pesticide exposure, where no previous studies had been conducted on the intakes of lipids, proteins and antioxidants (vitamins a, c and e) in relation to pre-eclampsia. methods research design this study was of observational case control design and was conducted from february – march 2016 at 3 puskesmas (public health centers) in brebes district, central java, i.e. puskesmas brebes, wanasari and bulakamba. research subjects the subjects in this study were women of productive age (20-35 years) who were up to 3 months post partum. the number of subjects in this study was 110, consisting of 55 cases and 55 controls. this number was obtained based on total sampling in the three puskesmas (brebes, wanasari and bulakamba), where 55 subjects were found for the group of cases and 55 subjects for the group of controls. the inclusion criteria were women of productive age (20-35 years), agreeing to participate in the study, being 3 months post partum and diagnosed as having had preeclampsia in pregnancy (as cases), normal pregnant women (as controls), and residing in the catchment area of bulakamba, wanasari and brebes subdistricts, brebes district. the exclusion criteria for the subjects in this study were women with twin pregnancies and with diabetes mellitus. pre-eclampsia was diagnosed by health personnel by the criteria of a blood pressure of >140/90 mmhg after 20 weeks of pregnancy and of proteinuria. anthropometric measurements measurement of bmi and muac of the women before pregnancy was performed by health personnel. the bmi of women before pregnancy was obtained from pre-pregnancy body weight divided by height, while muac was measured at the start of pregnancy. nutrient intake measurements nutrient intakes were obtained from interviews conducted by the investigators using the semi-quantitative food frequency questionnaire. the assessed nutrient intakes comprised total lipids, pufa, and vitamins a, c and e. the nutrient adequacy level was calculated using the nutrisurvey 2007 program and based on the indonesian recommended dietary allowances (irda) of 2013. the cut-off point for low protein intake was categorized at <80% of irda and for adequate protein intake at >80% of irda.(17) low intake of vitamins a, c and e was categorized at <77% of irda and adequate intake at >77% of irda.(18) the intakes of pufa and total lipids were categorized as high at >110% of irda and adequate at <110% of irda.(17) exposure to pesticides the level of pesticide exposure was obtained through structured interviews, using a list of questions for the interviews and forms for willingness to participate in the study. the level of pesticide exposure was divided into two categories, i.e. mild and severe, with total scores of 1-11 and 12-22, respectively. statistical analysis chi square and independent t tests were used to determine differences between cases and controls. simple logistic regression and multiple logistic regression were used to examine the risk factors of pre-eclampsia. the level of significance used was 0.05. ethical clearance this study was conducted after obtaining approval of the ethics commission under no. 051/ ec/fk-rsdk/2016. masrikhiyah, suhartono, kartasurya vitamin c intake and pre-eclampsia 131 univ med vol. 35 no.2 results table 1 shows that with regard to age and level of education of the study subjects, there were no significant differences between cases and controls (p=0.245 and p=0.096). table 2 shows the results of the analysis of pre-eclampsia risk factors. the results of simple logistic regression showed that there was a relationship between intakes of nutrients (protein, pufa, and vitamins a, c and e), bmi, and level of pesticide exposure with the incidence of preeclampsia. low intake of protein (or: 5.37; 95% ci: 1.43 – 20.08), high intake of pufa (>110% of irda) (or: 6.55; 95% ci: 2.62 – 16.41), low intake of vitamin a (or: 4.84; 95% ci: 1.28 – 18.26), low intake of vitamin c (or: 4.35; 95% ci: 1.33 – 14.24), low intake of vitamin e (or: 12; 95% ci: 4.15 – 34.69), bmi >23 kg/m2 (or: 2.50; 95% ci: 1.10 – 5.67), severe pesticide exposure (or: 3.34; 95% ci: 1.26 – 8.82) are risk factors of the incidence of pre-eclampsia. the variables that were used as candidates in the multiple logistic regression analysis were those that were significant in the simple logistic regression analysis. these comprised the intakes of protein, pufa, vitamin a, vitamin c, vitamin e, pesticide exposure, and bmi. table 1. characteristics of study subjects, by cases (n=55) and controls (n=55) *independent t-test; ** chi-square test table 2. simple logistic regression analysis of risk factors of pre-eclampsia irda: indonesian recommended dietary allowances 132 a low vitamin c intake was the risk factor with the most important role on the incidence of pre-eclampsia (or: 20.93; 95% ci: 2.72161.36). other risk factors that also played a role on the incidence of pre-eclampsia were high intake of pufa(or: 10.50; 95% ci: 2.4744.57), low vitamin e intake (or: 8.57; 95% ci: 2.07 –35.46), and severe pesticide exposure (or: 5.83; 95% ci: 1.31 – 25.94) (table 3). discussion low protein intake was a risk factor for the incidence of pre-eclampsia, but was statistically not significant. the results of this study are in agreement with a case control study conducted on 113 women with gestational hypertension and 150 healthy pregnant women in tehran, that did not show any significant differences in protein intake between the two groups.(19) however, the results of our study differed from those of one study on pregnant or post partum women in semarang and another study on pregnant women in makassar, with and without pre-eclampsia, where it was found that low protein intake was a risk factor of preeclampsia in pregnancy.(7,20) similarly, a study that was conducted in one indian hospital found that low protein intake was a risk factor of preeclampsia.(21) low protein intake contributes toward oxidative stress that subseaquently causes preeclampsia. protein status affects glutathione concentration, where low protein intake leads to low glutathione concentrations. glutathione is the substrate for the enzyme catalyzing the reduction of ros and free radicals. glutathione has an important antioxidant capacity and plays an essential role in the detoxification of endogeneous metabolic products, including lipid peroxides, and of xenobiotic compounds.(22) low glutathione concentrations increase oxidative stress that causes pre-eclampsia. high intake of pufa (>110% of irda) is a risk factor of the incidence of pre-eclampsia. this is in line with the study by clausen et al.(11) who found that high intake of pufa in pregnant women increased the risk of pre-eclampsia. lipids are involved in the generation of free radicals. pufa in lipid membranes are the main targets of lipid peroxidation by ros. pufa are degraded by free radicals, thus producing end products termed malondialdehydes (mda), which may be used for determining the level of oxidative damage caused by lipid peroxidation. therefore, high pufa intake may render the organism more susceptible to lipid peroxidation. lipid peroxidation is strongly suspected to play an important role in causing endothelial dysfunction and the development of clinical symptoms of pre-eclampsia.(8-10) the results of our study showed that low intakes of vitamin a, vitamin e and vitamin c are risk factors for the incidence of preeclampsia. the results of this study agree with those of a study conducted in yogyakarta, where it was found that pregnant women with a low consumption of vitamins a, c, and e, and beta carotene were at risk for pre-eclampsia.(12) vitamins a, c and e act as antioxidants by donating electrons to free radicals for binding to single (unpaired) electrons of free radicals without becoming new free radicals. free radicals may be treated with the administration table 3. multiple logistic regression for risk factors of pre-eclampsia masrikhiyah, suhartono, kartasurya vitamin c intake and pre-eclampsia 133 univ med vol. 35 no.2 of antioxidants. an imbalance between free radicals and antioxidants causes oxidative stress.(5) oxidative stress is considered an important element in the pathogenesis of preeclampsia resulting in endothelial dysfunction and ultimately inducing the syndrome of preeclampsia.(23) the results of our study show that low vitamin c intake increases the risk of preclampsia. these results are consistent with those of a cohort study conducted on danish pregnant women, showing that the risk of severe pree c l a m p s i a / e c l a m p s i a m a y d e c r e a s e w i t h increasing vitamin c intake.(25) however, a review of twenty-nine trials involving 24,300 women who took vitamin c supplements during pregnancy concluded that vitamin c supplements do not prevent pregnancy problems, including stillbirth, neonatal mortality, preterm birth, preeclampsia or low birthweight babies.(26) there is no agreement on the optimal gestational time for measurement of vitamin c intake. however, the second half of pregnancy may reflect vitamin c i n t a k e i n p l a c e n t a l d e v e l o p m e n t , s i n c e trophoblast invasion is completed by this time. the time of complete placentation is not generally considered to be the most probable gestational time point associated with oxidative stress.(25) in the present study, severe level of pesticide exposure is a risk factor for the incidence of preeclampsia, which agrees with the results of a study in the usa where it was found that women who were exposed to pesticides during pregnancy had a greater risk of pre-eclampsia than did pregnant women who were not exposed to pesticides.(16) pesticides are one of the sources of free radicals. when free radical production increases and exceeds the capacity of the antioxidant defense system in the body, a condition occurs called oxidative stress.(5) oxidative stress is c o n s i d e r e d a n i m p o r t a n t e l e m e n t i n t h e pathogenesis of pre-eclampsia that results in endothelial dysfunction and ultimately causes the syndrome of pre-eclampsia, although its role has not yet been completely analyzed.(23) a limitation of this study was that only nutrient intakes, pesticide exposure and bmi were measured, whereas there are still many other uncontrollable or unavoidable factors that affect pre-eclampsia in pregnant women of productive age, such as immunity, cardiovascular adaptation, and genetic factors. for assessment of nutrient intakes, this study used the semiquantitaive ffq, which may still contain information bias or recall bias. in this study we d i d n o t m e a s u r e s e r u m n o o r m d a concentrations to determine the level of oxidative s t r e s s i n t h e w o m e n . t h e p u b l i c h e a l t h implications of our findings are that they confirm the current dietary recommendations for intakes of vitamin a, c, e in pregnancy. conclusions l o w v i t a m i n c i n t a k e p l a y s a m o s t important role toward pre-eclampsia. however, greater caution is also needed in recommending the consumption of vitamin supplements in pregnancy, as high doses of some vitamins may be deleterious. conflict of interests there are no conflicts of interest (financial, personal, political, intellectual or religious). acknowledgement we thank kabupaten brebes health office staff and heads of puskesmas wanasari, brebes and bulakamba for facilitating and the research process providing secondary data and the study subjects for their participation. references 1. national high blood pressure education program working group on high blood pressure in pregnancy. report of the national high blood pressure education program working group on high blood pressure in pregnancy. am j obstet gynecol 2000;183:s1-22. 134 2. osungbade ko, ige ok. public health perspectives of pre-eclampsia in developing countries: implication for health system strengthening. j pregnancy 2011. article id481095, 6 pages. http://dx.doi.org/10.1155/ 2011/481095. 3. trogstad l, magnus p, skjærven r, et al. previous abortions and risk of pre-eclampsia. int j epidemiol 2008;37:1333-40. 4. world health organization. make every mother and child count, in the world health report 2005. geneva: world health organization;2005. 5. dinas kesehatan kabupaten brebes. angka kematian ibu (aki). laporan dinas kesehatan kabupaten brebes. brebes;2013. 6. powers sk, jackson mj. exercise-induced oxidative stress: cellular mechanisms and impact on muscle force production. j physiol rev 2008; 88:1243-12. 7. endeshaw m, ambaw f, aragaw a, et al. effect of maternal nutrition and dietary habits on preeclampsia: a case-control study. int j clin med 2014;5:1405-16. 8. kartasurya mi. pre-eclampsia risk factors of pregnant women in semarang, indonesia. ijsbar 2015;22:31-7. 9. hung th, bruton gj. hypoxia and reoxygenation: a possible mechanism for placental oxidative stress in pre-eclampsia. taiwanese j obstet gynecol 2006;43:189-200. 10. borekci b, aksoy h, ozturk n, et al. correlation between calprotectin and oxidized ldl in preeclampsia. turkey j med sci 2009;39:191-5. 11. gupta s, aziz n, sekhon l, et al. lipid peroxidation and antioxidant status in preeclampsia : a systematic review. obstet gynecol survey 2009;64:750-9. 12. clausen t, slott m, solvoll k, et al. high intake of energy, sucrose, and polyunsaturated fatty acids is associated with increased risk of preeclampsia. am j obstet gynecol 2001;185:451– 8. 13. nofrisa mp, hartini tns, hakimi m. kurang asupan vitamin a, c, e dan beta karoten meningkatkan kejadian preeklampsia di rsup dr. sardjito, yogyakarta. gizi indon 2010;33: 136-42. 14. rani na, naidu jn, krishnamma. evaluation of nitric oxide and reduced glutathione levels in pre-eclampsia. ijbritish 2014;1:13-7. 15. al-ahdab ma. the relationship between dietary intake and pre-eclampsia. j am sci 2013;9:185203. 16. cnossen js, leeflang mmg, de haan ee, et al. accuracy of body mass index in predicting preeclampsia: bivariate meta-analysis. bjog 2007;114:1477-85. 17. saldana tm, basso o, baird dd, et al. pesticide exposure and hypertensive disorders during pregnancy. environ health perspect 2009;117: 1393-6. 18. hardinsyah. angka kecukupan energi, protein, lemak dan serat makanan. prosiding widyakarya pangan dan gizi viii; 2004 mei 17-19; jakarta; 2004.p.323-27. 19. gibson rs. principle of nutritional and assessment. 2nd ed. new york: oxford university press;2005. 20. kazemian e, dorosty-motlagh ar, sotoudeh g, et al. nutritional status of women with gestational hypertension compared with normal pregnant women. hypertens pregnancy 2013;32:146–156. doi: 10.3109/10641955. 2013.784782. 21. nuryani, ade am, citrakesumasari, et al. hubungan pola makan, sosial ekonomi, antenatal care dan karakteristik ibu hamil dengan preeklampsia di kota makassar. media gizi masyarakat indonesia 2013;2:104-12 22. bej p, chhabra p, sharma ak, et al. determination of risk factors for pre-eclampsia and eclampsia in a tertiary hospital of india: a case control study. j family med prim care 2013;2:371–5. 23. lee dh, steffes mw, jacobs dr jr. can persistent organic pollutants explain the association between serum gamma glutamyl transferase and type 2 diabetes? diabetologia 2008;51:402–7. 24. cindrova-davies t. gabor than award lecture 2008: pre-eclampsia – from placental oxidative stress to maternal endothelial dysfunction. placenta 2009;23:55-65. 25. klemmensen ak, tabor a, østerdal ml, et al. intake of vitamin c and e in pregnancy and risk of pre-eclampsia: prospective study among 57,346 women. bjog 2009;116:964–74. 26. rumbold a, ota e, nagata c, et al. vitamin c supplementation in pregnancy. cochrane database of systematic reviews 2015, issue 9. art. no.: cd004072. doi: 10.1002/14651858. cd004072.pub3. masrikhiyah, suhartono, kartasurya vitamin c intake and pre-eclampsia recombinant vascular endothelial growth factor 121 192 *department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret/ rsud dr. moewardi, surakarta correspondence: dr. dr. sri sulistyowati, sp.og(k) department of obstetrics and gynecology rsud dr. moewardi jl. kol. sutarto 132 surakarta phone: +62271 665145 email: elis_spog@yahoo.co.id univ med 2016;35:192-8 doi: 10.18051/univmed.2016.v35.192-198 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 recombinant vascular endothelial growth factor 121 decreases vascular cell adhesion molecule-1 in murine pre-eclampsia model placenta sri sulistyowati*, john arianto sondakh*, eric edwin yuliantara*, supriyadi hari respati*, and soetrisno* background preeclampsia is one of the major contributors to maternal and fetal morbidity and mortality. imbalance of soluble fms-like tyrosine kinase (sflt-1) as anti-angiogenic factor and vascular endothelial growth factor (vegf) as pro-angiogenic factor plays a role in the pathogenesis of preeclampsia. endothelial dysfunction in preeclampsia causes vascular cell adhesion molecule-1 (vcam-1) to be expressed on its surface. this study aims to evaluate the effect of recombinant vegf-121 on vcam-1 expression in the placenta of a murine preeclampsia model. methods an experimental analytical study conducted from february until march 2016 in the biomedical laboratory, faculty of veterinary medicine, airlangga university. the study sample consisted of 30 pregnant mice, divided into three groups, i.e. 10 normal pregnant mice, 10 mice with preeclampsia model and 10 mice with preeclampsia model and recombinant vegf-121 therapy. all animals were subjected to immunohistochemical examination of vcam-1 expression in their placentas. the results were assessed semiquantitatively according to a modified remmele method. data analysis was done using one-way anova and tukey’s multiple comparisons method. results mean vcam-1 expression in normal (0.97 ± 0.54%) murine placentas, compared with placentas (2.94 ± 0.96%) of murine preeclampsia models (p=0.000), while mean vcam-1 expression in placentas of murine preeclampsia models with vegf intervention was 2.14 ± 0.68% (p=0.030). conclusion recombinant vegf-121 can reduce vcam-1 expression in placentas of murine preeclampsia models. the present study has shown the potential benefits of vegf therapy, justifying serious consideration of this therapeutic approach for use in women with preeclampsia. keywords: recombinant vegf-121, vcam-1, preeclampsia, pregnant murine doi: http://dx.doi.org/10.18051/univmed.2016.v35.192-198 193 univ med vol. 35 no.3 introduction preeclampsia is pregnancy that is associated with hypertension occurring after 20 weeks of gestation. preeclampsia is still the main contributor to maternal and fetal morbidity and mortality.(1) the incidence of preeclampsia is 210% of all pregnancies in the world and according to the who is 7-fold greater in developing than in developed countries.(2) preeclampsia is also associated with 10% of perinatal and neonatal causes of death. (3) between 30–40% of preeclampsia cases in indonesia result in the death of the pregnant mothers and 30–50% cause perinatal deaths. in rsud dr. moewardi, surakarta, the mortality rate among pregnant mothers in 2012 caused by preeclampsia was 19 out of 30 pregnant mothers and in 2013 12 out of 21 pregnant mothers.(4) preeclampsia can cause complications in the mother, i.e. acute kidney disease, liver failure, antepartum hemorrhage, postpartum hemorrhage, eclampsia and maternal death.(5) complications in the fetus that may be caused by preeclampsia are among others: intrauterine fetal death (iufd), intrauterine growth restriction (iugr), fetal distress, and also increased risk of respiratory distress and low birth weight associated with prematurity.(6) preeclampsia is described as a syndrome specific to pregnancy that can influence all organ systems. to date no effective preventive treatment of preeclampsia is available. termination of pregnancy or delivery is considered the best measure, particularly in cases of preeclampsia with complications.(7) preeclampsia presumably occurs in two stages. the first stage is asymptomatic and is characterized by abnormal placental development in the first trimester, particularly in angiogenesis, causing placental insufficiency and release of placental material into the maternal circulation. in the endothelialization process there occur cytotrophoblast abnormalities and inadequate invasion of the spiral arteries in the myometrium. poor placentation results in placental ischemia and hypoxia. release of placental material triggers the clinical picture of the second stage, i.e. the symptomatic stage. in this stage there occurs the development of signs of hypertension, renal abnormalities and proteinuria, and damage in other end organs, leading to histological, functional, and metabolic placental abnormalities, that are thought to play a major role in the pathophysiology of preeclampsia.(8) in the pathogenesis of preeclampsia there is presumably an imbalance between the proangiogenic placental growth factor (plgf) and vascular endothelial growth factor (vegf) on the one hand and the angiogenesis inhibitors (antiangiogenic factors) sflt-1 and soluble endoglin (seng). the balance between plgf and vegf as pro-angiogenic factors and sflt-1 and seng as anti-angiogenic factors is important in influencing the processes of angiogenesis, vasculogenesis and placental development during pregnancy.(9,10) one of the facts that constitute strong evidence for the role of an imbalance between proand anti-angiogenic factors in preeclampsia is the increase in soluble fms-like tyrosine kinase-1 (sflt1). the aforementioned imbalance can be detected before the clinical diagnosis of preeclampsia. currently measurement of serum sflt-1 levels and the ratio of sflt-1 to plgf may be used as predictors of preeclampsia. an sflt-1/plgf ratio of <38 can be used as a strong predictor of preeclampsia in women who are clinically at risk.(11) placental ischemia in experimental animals causes reduced uterine perfusion pressure (rupp) that produces increased levels of sflt-1 and decreased levels of free vegf in the circulation.(12) in endothelial dysfunction, on the endothelial surface adhesion molecules are expressed, such as vascular cell adhesion molecule-1 (vcam-1) and intercellular cell adhesion molecule-1 (icam1). in the supernatants of endothelial cell cultures that have been incubated with the serum of patients with preeclampsia, increases are found in soluble vcam-1, but not in other adhesion molecules such as icam-1 and e-selectin. therefore vcam-1 presumably plays a role in preeclampsia.(13,14) 194 recombinant vegf-121 is an exogenous vegf that plays a role in vasculogenesis and angiogenesis, since there are vegf receptors (vegfr-1) in the endothelial wall. administration of recombinant vegf for the treatment of preeclampsia may result in reduced sflt-1 levels in the blood of experimental animals, clinically reduced blood pressure, histopathological improvement of endothelial function, and reduction of placental hypoxia.(15,16) recombinant vegf therapy can neutralize the increased sflt-1 in preeclampsia, thus reducing sflt-1 concentration and resulting in a return to normal angiogenesis.(3) the vegf-121 therapy in a murine model of preeclampsia has the effects of reducing blood pressure, improving albumin and creatinine, placental histology, and glomerular endotheliosis.(15) thus far there have not been many studies on recombinant vegf-121 therapy in relation to vcam-1 expression as marker of endothelial dysfunction in preeclampsia. the present study aimed to evaluate the influence of recombinant vegf-121 administration on vcam-1 expression in the placenta of a murine model of preeclampsia. methods design of the study this was an experimental analytical study, conducted from february until march 2016 in the experimental animal house, biomedical laboratory, faculty of veterinary medicine, airlangga university. animal model the experimental animal study used murine placentas that meeting the inclusion criteria, i.e. originating from female swiss mice (mus musculus) obtained from center veterinaria farma center, surabaya. the mice were 3 months old, healthy, and weighing 20 to 25 grams. the sample size based on the replication formula of steel et al.(17) was 30 mice that were divided into 3 groups, i.e. group 1 (k1) comprising normal pregnant mice, group 2 (k2) comprising mouse preeclampsia models and group 3 (k3) comprising mouse preeclampsia models on recombinant vegf-121 therapy. on day 16 of murine gestation, all animals from the three groups underwent an operation in which their placentas were removed. the reason for collecting the placentas on day 16 was the assumption that this period was comparable to the second trimester of pregnant humans, in which the manifestations of preeclampsia are already present. impregnation of the female mice was performed by estrus synchronization, in which female 3-month old adult mice weighing 20 to 25 grams were injected with 5 iu of pregnant mare serum gonadotropin (pmsg), and 48 hours later injected with 5 iu human chorionic gonadotropin (hcg). the female mice that were already in synchronized estrus were mated with 7-months old male mice weighing ± 60 grams. seventeen hours after mating the female mice can be diagnosed as pregnant if there is a copulatory plug filling their vagina from cervix to vulva. on day 1 of pregnancy, the whole sample was randomly divided into three groups, i.e. a group of 10 normal pregnant mice (k1) kept without intervention and 2 groups, each comprising 10 pregnant mouse as models of preeclampsia (k2 and k3). the mice were converted into a preeclampsia model by the intravenous administration on days 1 to 4 of anti qa-2 antibody at a dose of 10 ng. on days 12 to 15 of pregnancy, the mice in group k3 received a single dose of recombinant vegf-121 (125 mg/ kg body weight). on day 16 of pregnancy, the mice from all three groups were terminated by euthanasia using ketamine and subsequently underwent necropsy. after the abdominal cavity of each animal was opened, the placenta was removed and placed into a pot containing 10% neutral buffered formalin. the placenta was then subjected to immunohistochemical examination. the reagent kits used were anti-qa2 antibody (5k44), xeno-freetm recombinant mouse vegf121 125 mg/ kg body weight, pregnant mare serum gonadotropin (pmsg) 600 pg, chorulon human chorionic gonadotropin (hcg) 1500 iu and bioss vcam. sulistyowati, sondakh, yuliantara, et al vascular endothelial growth factor 195 univ med vol. 35 no.3 immunohistochemistry preparations were obtained from each of the surgically removed murine placentas, made into paraffin blocks and stained with hematoxylineosin (he). histological sections were prepared by fixing the placentas in 10% neutral buffered formalin, then cutting them up and placing them in plastic containers. the specimens were further dehydrated in graded alcohols of 70%, 80%, 90%, absolute alcohol i, absolute ii, each immersion lasting 2 hours. the specimens were then cleared in xylol, molded into paraffin blocks and stored in the refrigerator. the aforementioned blocks were then cut into 5–6 ì m sections in a microtome. the sections were floated in warm water at a temperature of 60°c to stretch the sections to prevent folds in the tissues. the preparation were then lifted out and placed on a glass slide for staining with hematoxylin and eosin (he). subsequently the sections were examined under a nikon eclipse cy1 light microscope at 1000x magnification. immunohistochemical examination was performed to evaluate vcam-1 expression, calculated as the percentage of positive cells per field of view. data analysis data were analyzed semiquantitatively according to a modification of the remmele method, where the remmele scale index (immunoreactive score, irs),(18) is the product of the percentage of immunoreactive cells and the color intensity of these cells. the analytical technique used in this study was the one-way anova statistical test. in case of a significant difference, the analysis was continued with the post hoc tukey test, at a significance level of 5% (p<0.05). ethical clearance ethical clearance was obtained from the research ethics commission, faculty of veterinary medicine, airlangga university, under no. 419-to, dated 18 march 2015. results in table 1 k1 has a mean vcam-1 score of 0.97 ± 0.54% per field of view, group k2 a mean score of 2.94 ± 0.96% per field of view, while group k3 has a mean score of 2.14 ± 0.68% per field of view (p=0.000). the mean vcam-1 score between the three groups differed significantly. mean vcam differed significantly between group k1 and group k2 (p=0.00), group k1 differed significantly from group k3 (p=0.001) and group k2 differed significantly from group k3 (p=0.035) (table 2). from these study results it may be concluded that recombinant vegf-121 administration reduced vcam-1 expression in the preeclampsia model placentas. in figure 1 it is apparent that there was no dominant chromogen pattern as a marker of vcam-1 expression in the endothelium of normal pregnant murine placentas (k1), whereas in murine preeclampsia model placentas (k2) and murine preeclampsia model placentas treated with table 1. mean vcam-1 in normal pregnant murine placentas (k1), murine preeclampsia model placentas (k2) and murine preeclampsia model placentas treated with recombinant vegf-121 (k3) table 2. results of post-hoc analysis using tukey’s procedure k1=vcam-1 in normal pregnant murine placentas; k2=murine preeclampsia model placentas; k3=murine preeclampsia model placentas treated with recombinant vegf-121 196 figure 1. vcam-1 expression in normal pregnant murine placentas (k1), murine preeclampsia model placentas (k2) and murine preeclampsia model placentas treated with recombinant vegf-121 (k3) recombinant vegf-121 (k3) may been seen chromogen patterns (indicated by white arrows) showing vcam-1 expression in the endothelium of murine placentas. the chromogen pattern in k-2 (endothelium of pregnant murine preeclampsia model placentas) was more dominant than in k-3 (endothelium of pregnant murine preeclampsia model placentas treated with recombinant vegf-121), the latter showing lower vcam-1 expression. discussion in this study mean vcam-1 expression in the group of normal pregnant mice was significantly lower than in the preeclampsia model mice. this demonstrates that in preeclampsia there occurs endothelial dysfunction because of significantly increased vcam-1 expression. endothelial dysfunction in preeclampsia is characterized by expression of adhesion molecules on the endothelial, i.e. vcam-1 and icam-1. increases in the concentrations of soluble vcam1 are found in the supernatant of endothelial cell cultures that were incubated with the serum of patients with preeclampsia, but no increases in other adhesion molecules such as icam-1 and e-selectin. therefore it is presumed that vcam1 plays a role in preeclampsia.(13) this study agrees with other studies that increased vcam-1 indicates the possibility of altered endothelial activity and therefore of endothelial damage in preeclampsia. increased vcam-1 indicates activation of endothelial cells.(19) recombinant vegf-121 has receptors called vegfr-1 in the endothelial wall, which may also bind sflt-1 more strongly. recombinant vegf-121 binds to vegfr-1, thus triggering angiogenesis. recombinant vegf-121 therapy may also neutralize the effect of increased sflt-1 in preeclampsia, in that binding of sflt-1 to proangiogenic factors reduces circulating sflt-1 and effects a return to normal angiogenesis.(3) administration of recombinant vegf-121 for treatment of preeclampsia in experimental animals can lead to reduction in sflt-1 concentrations in the circulation, clinical reduction in blood pressure, histopathological improvement in endothelial function and decreased placental hypoxia.(15,16) the studies of li et al,(15) bergmann et al,(20) gilbert et al,(21) mateus et al,(16) and woods et al,(7) on administration of vegf-121 in experimental animals show similar significant results, such as reduced systolic blood pressure and reduced renal damage. in the present study mean vcam-1 expression in the group of murine preeclampsia models given recombinant vegf121 was significantly lower than in the group of murine preeclampsia models alone. this shows that administration of or therapy with recombinant vegf-121 significantly reduces vcam-1 expression, signifying improvement of endothelial function in preeclampsia. chronic infusion of vegf-121 in rats with experimentally induced rupp improves glomerular filtration rate and endothelial function, reduces high blood pressure and prevents the onset of hypertension in late gestation.(21) in the study of agarwal and karumanchi (22) on rats as preeclampsia models, administration of vegf-121 shows improvement in glomerular filtration rate and endothelial function, and also reduces the blood pressure sulistyowati, sondakh, yuliantara, et al vascular endothelial growth factor 197 univ med vol. 35 no.3 associated with placental ischemia and high sflt1 expression. in our study we did not investigate sflt-1 expression, therefore treatment with recombinant vegf-121 could not show improvement in the balance between the pro-angiogenic vegf and the anti-angiogenic sflt-1 in the form of a reduction of the endothelial dysfunction occurring in preeclampsia. conclusions recombinant vegf-121 may reduce vcam-1 expression in murine preeclampsia model placentas. administration of recombinant vegf-121 may be one alternative for developing preeclampsia therapies to reduce maternal and fetal morbidity and mortality. conflict of interest the authors state that there is no conflict of interest in connection with the study, the authors, and/or the publication of this paper. acknowledgments we express our gratitude to all staff members of the experimental animal house, biomedical laboratory, faculty of veterinary medicine, airlangga university, who assisted in the preparation of the murine preeclampsia models. references 1. osungbade ko, ige ok. public health perspectives of preeclampsia in developing countries: implication for health system strengthening. j pregnancy 2011; article id 481095, 6 pages. http://dx.doi.org/10.1155/ 2011/481095. 2. kooffreh me, ekottm, ekpoudom do. the prevalence of pre-eclampsia among pregnant women in the university of calabar teaching hospital, calabar. saudi j health sci 2014;3: 133-6. 3. maynard se, karumanchi sa. angiogenic factors and preeclampsia. semin nephrol 2011; 31:33-46. 4. sulistyowati s, soetrisno, kartika nh. ekspresi human leukocyte antigen–c di trofoblas dan natural killer cell di desidua pada preeklampsia berat. jurnal kedokteran brawijaya 2016;29:5963. 5. roberts cl, ford jb, algert cs, et al. population-based trends in pregnancy hypertension and pre-eclampsia: an international comparative study. bmj open 2011;24:e000101. doi: 10.1136/bmjopen-2011-000101. 6. aabidha pm, cherian ag, paul e, et al. maternal and fetal outcome in pre-eclampsia in a secondary care hospital in south india. j family med prim care 2015;4:257–60. 7. woods ak, hoffmann ds, weydert cj, et al. adenoviral delivery of vegf 121 early in pregnancy prevents spontaneous development of pre-eclampsia in bph/5 mice. hypertension 2011;57:94-102. 8. roberts jm, hubel ca. the two stage model of preeclampsia: variation on the theme. placenta 2009;30 suppl a:s32-7. 9. levine rj, lam c, qian c, et al. soluble endoglin and other circulating antiangiogenic factors in preeclampsia. n engl j med 2006;355: 992-1005. 10. gu y, lewis df, wang y. placental productions and expressions of soluble fms-like tyrosine, soluble endoglin, and placental growth factor in normal and preeclamptic pregnancies. j clin endocrinol metab 2008;93:260-6. 11. zeisler h, llurba e, chantraine f, et al. predictive value of the sflt-1:plgf ratio in women with suspected preeclampsia. n engl j med 2016;374:13-22. doi: 10.1056/nejmoa 1414838. 12. warrington jp, george em, palei ac, et al. recent advances in the understanding of the pathophysiology of preeclampsia. hypertension 2013;62:666-73. 13. sulistyowati s, abadi a, hood j, et al. the influence of low hla-g protein expression on hsp-70 and vcam-1 profile in preeclampsia. indonesian j obstet gynecol 2010;34:185–90. 14. sulistyowati s, abadi a, wijiati m. low class ib (hla-g/qa2) mhc protein expression against hsp-70 and vcam-1 profile on preeclampsia: an observation on experimental animal mus musculus with endothelial dysfunction model. indonesian j obstet gynecol 2010;34:103–6. 15. li z, zhang y, ying ma j, et al. recombinant vascular endothelial growth factor 121 attenuates hypertension and improves kidney damage in a rat model of preeclampsia. hypertension 2007;50:686-92. 198 16. mateus j, bytautiene e, lu f, et al. endothelial growth factor therapy improves preeclampsialike manifestasions in a murine model induced by overexpression of svegfr-1. am j physiol heart circ physiol 2011;301:h1781-7. 17. steel rg, torrie jh, dickey da. principles and procedures of statistics: a biometrical approach. 3rd ed. new york: mcgraw-hill companies; 1997. 18. halon a, donizy p, biecek p, et al. her-2 expression in immunohistochemistry has no prognostic significance in gastric cancer patients. sci world j 2012; article id 941259, 6 pages. doi: 10.1100/2012/941259. 19. anwar ad, achmad th, krisnadi sr, et al. polimorfisme c1167t gen reseptor tipe ii transforming growth factor-â , kadar soluble endoglin dan vascular adhesion molecule-1 pada preeklamsia. mkb 2010;42:115-22. 20. bergmann a, ahmad s, cudmore m, et al. reduction of circulating soluble flt-1 alleviates preeclampsia-like symptoms in a mouse model. j cell mol med 2001;14:1857-67. 21. gilbert js, verzwyvelt j, colson d, et al. recombinant vegf 121 infusion lowers blood pressure and improves renal function in rats with placental ischemia-induced hypertension. hypertension 2010;55:380-5. 22. agarwal i, karumanchi sa. preeclampsia and the anti-angiogenic state. pregnancy hypertens 2011;1:17-21. sulistyowati, sondakh, yuliantara, et al vascular endothelial growth factor mayanti1 143 universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 penatalaksanaan tuberkulosis pada kehamilan meiyanti* *bagian farmasi fakultas kedokteran universitas trisakti korespondensi dr. meiyanti, sp.fk bagian farmasi fakultas kedokteran universitas trisakti jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5672731 eks.2805 universa medicina 2007; 26: 14351. tuberkulosis masih menjadi masalah kesehatan di dunia demikian juga tuberkulosis pada kehamilan. insidens tuberkulosis pada kehamilan makin meningkat. tuberkulosis pada kehamilan mempunyai gejala klinis yang serupa dengan tuberkulosis pada wanita tidak hamil. diagnosis mungkin ditegakkan terlambat karena gejala awal yang tidak khas. tuberkulosis tidak mempengaruhi kehamilan dan kehamilan tidak mempengaruhi manifestasi klinis dan progesivitas penyakit bila diterapi dengan regimen kemoterapi yang tepat dan adekuat. pemberian regimen yang tepat dan adekuat ini akan memperbaiki kualitas hidup ibu, mengurangi efek samping obat-obat tuberkulosis terhadap janin dan mencegah infeksi yang terjadi pada bayi yang baru lahir. obat anti tuberkulosis yang diberikan dibagi dalam 2 golongan yaitu obat lini pertama dan lini kedua. obat lini pertama, kecuali streptomisin dapat digunakan pada tuberkulosis pada kehamilan. penggunaan streptomisin dan obat lini kedua (kanamisin, etionamid, kapreomisin) sebaiknya dihindari pada wanita hamil karena efek samping yang akan terjadi pada janin, kecuali dalam keadaan resistensi beberapa obat. kata kunci : tuberkulosis, kehamilan, obat antituberkulosis abstrak meiyanti tuberkulosis pada kehamilan 144 management of tuberculosis in pregnancy meiyanti* abstract *department of pharmacy medical faculty trisakti university correspondence dr. meiyanti, sp.fk department of pharmacy medical faculty trisakti university jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5672731 ex.2805 universa medicina 2007; 26: 143-51. tuberculosis as well as tuberculosis in pregnancy continues to be a major health problem in the world. it has been reported that incidence of tuberculosis in pregnancy is increasing. the symptoms of tuberculosis in pregnancy are similar to those of general women. thus, the diagnosis may be delayed due to the unspecified symptoms.tuberculosis does not affect the pregnancy so as pregnancy does not interfere the clinical manifestation and progressiveness of disease if accompanied by adequate chemotherapy. the administration of appropriate regimen could improve the quality of mothers’ life, reduce the side effects on foetus and prevent the infection of a newborn baby. antituberculosis drugs are divided into 2 groups: the first line and the second line. the first line drugs with the exception of streptomycin can be used to treat tuberculosis in pregnancy. the usage of streptomycin and second line drugs (kanamycin, etionamid, kapreomycin) should be avoided because their side effects on foetus except in the case of multidrug resistance (mdr). keywords : tuberculosis, pregnancy, antituberculosis drugs pendahuluan tu b e r k u l o s i s ( t b c ) m a s i h m e n j a d i masalah kesehatan di seluruh dunia, demikian juga tuberkulosis pada kehamilan. insidens tbc pada kehamilan adalah 1/10.000 kehamilan. penelitian pada tahun 1985-1990 di new york, memperlihatkan insidens tbc pada kehamilan adalah 12 kasus per 100.000 kelahiran dan pada tahun 1991-1992 insidens meningkat menjadi 95 kasus per 100.000 kelahiran.(1) penelitian di london tahun 1997-2001, menunjukkan 32 wanita hamil menderita tbc, dengan insidens 252/100.000 kelahiran. lima puluh tiga persen didiagnosis sebagai tbc ekstrapulmonal, 38% tbc pulmonal dan 9% tbc ekstra dan intra pulmonal.(2) p a d a m a s a s e b e l u m d i t e m u k a n n y a kemoterapi, didapatkan kematian sampai 70% d i s e b a b k a n o l e h t b c p a d a w a n i t a u s i a reproduksi. setelah kemoterapi ditemukan i n s i d e n s t b c m e n i n g k a t k e m b a l i , h a l i n i d i k a r e n a k a n t i m b u l n y a b e r m a c a m m a c a m f a k t o r, s a l a h s a t u n y a i n f e k s i h u m a n immunodeficiency viral (hiv).(3) tbc pada kehamilan mempunyai gejala klinis yang serupa dengan tbc perempuan tidak hamil. diagnosis mungkin ditegakkan terlambat karena gejala awal yang tidak khas. keluhan yang sering ditemukan batuk, demam, malaise, penurunan 145 berat badan dan hemoptisis.(3,4) pemeriksaan p e n u n j a n g d a l a m h a l i n i p e m e r i k s a a n u j i tuberkulin diikuti oleh foto toraks merupakan pemeriksaan yang dianjurkan pada kelompok tbc risiko tinggi. f a k t o r l a i n y a n g b e r p e r a n a d a l a h pemberian regimen terapi yang tepat. risiko yang dihadapi oleh ibu dan janin lebih besar bila t i d a k m e n d a p a t k a n p e n g o b a t a n t b c dibandingkan risiko pengobatan itu sendiri. pemberian regimen kemoterapi yang tepat dan adekuat akan memperbaiki kualitas hidup ibu, mengurangi efek samping obat anti tuberkulosis (oat) terhadap janin dan mencegah infeksi yang terjadi pada bayi yang baru lahir.(4,5) hubungan antara tuberkulosis dan kehamilan pengaruh tbc pada kehamilan tergantung dari beberapa faktor antara lain: lokasi penyakit (intra atau ekstrapulmonal), usia kehamilan, status gizi ibu dan ada tidaknya penyakit penyerta.(6) beberapa studi menyatakan terdapat hubungan antara tbc dan meningkatnya risiko berat badan lahir rendah, kelahiran preterm, kehidupan perinatal sampai pada kematian bayi.(3) jika pemberian oat dimulai pada awal kehamilan akan memberikan hasil yang sama seperti pasien yang tidak hamil, tetapi bila diagnosis dan penanganan terlambat terjadi peningkatan angka morbiditas bayi 4 kali lipat dan peningkatan kelahiran preterm sebesar 9 kali lipat.(7) selama kehamilan dapat terjadi transmisi basil tbc ke janin. transmisi biasanya terjadi s e c a r a l i m f a t i k , h e m a t o g e n a t a u s e c a r a langsung. janin dapat terinfeksi melalui darah yang berasal dari infeksi plasenta melalui vena u m b i l i k a l i s a t a u a s p i r a s i c a i r a n a m n i o n , komplikasi seperti ini jarang terjadi. tbc yang terjadi disebut sebagai tbc kongenital.(8) tbc k o n g e n i t a l h a r u s d i b e d a k a n d e n g a n t b c postnatal. cantwell et al (9) mengemukakan tentang kriteria diagnosis tbc pada bayi dengan salah satu kriteria berikut yaitu adanya lesi, kompleks primer di hati, infeksi tbc pada plasenta atau endometrium pada minggu pertama kehidupan serta dapat disingkirkannya transmisi postnatal. gejala mungkin terlihat saat lahir tetapi biasanya pada minggu kedua dan ketiga. pada pemeriksaan fisis didapatkan hepatomegali (76%), gangguan pernafasan (72%), demam (48%) dan limfadenopati (38%). gambaran foto toraks mungkin normal segera setelah lahir tetapi b e r j a l a n p r o g r e s i f d e n g a n c e p a t d i s e r t a i pembentukan kavitas.(8) apabila memungkinkan dilakukan biakan tuberkel basil pada plasenta. uji tuberkulin tidak banyak membantu karena hasil negatif pada awalnya dan menjadi positif dalam waktu 1-2 bulan. pemeriksaan lain seperti b a s i l t a h a n a s a m ( b ta ) d a n b i a k a n p a d a jaringan atau cairan lambung.(3-5) deteksi tbc p a d a i b u m e r u p a k a n h a l p e n t i n g u n t u k pemberian pengobatan adekuat sehingga risiko serius yang terjadi pada janin dan bayi baru lahir dapat dikurangi. d i a g n o s i s t u b e r k u l o s i s pa d a kehamilan diagnosis tbc pada kehamilan sama d e n g a n t b c t a n p a k e h a m i l a n . d i a g n o s i s m u n g k i n t e r l a m b a t d i t e g a k k a n k a r e n a manifestasi klinis yang tidak khas, tertutup oleh gejala-gejala pada kehamilan.(5,8) good et al(10) melaporkan bahwa dari 27 wanita hamil dengan p e m e r i k s a a n b i a k a n s p u t u m y a n g p o s i t i f , didapatkan 74% gejala batuk, 41% penurunan berat badan, 30% demam, malaise dan lelah, 19% batuk darah dan 20% tanpa gejala. oleh karena itu perlu dilakukan penapisan pada perempuan hamil dengan risiko tinggi terkena tbc melalui pemeriksaan antenatal. pemeriksaan yang dianjurkan adalah uji tuberkulin, sputum bta dan pemeriksaan biakan.(8) universa medicina vol. 26 no.3 meiyanti tuberkulosis pada kehamilan 146 p e n ata l a k s a a n t b c pa d a kehamilan p e n a t a l a k s a n a a n p a s i e n t b c p a d a kehamilan tidak berbeda dengan tbc tanpa kehamilan. hal-hal yang harus diperhatikan adalah pemberian oat yang bisa menimbulkan efek teratogenik terhadap janin. penatalaksanaan secara umum terbagi atas penderita dengan tbc aktif dan tbc laten.(8,11,12) wanita hamil dengan tbc aktif biasanya diterapi dengan tidak mempertimbangkan trisemester kehamilan. oat yang digunakan tidak berbeda dengan wanita yang tidak hamil.golongan utama oat seperti isoniazid, rifampisin, etambutol digunakan secara luas pada wanita hamil. obat-obat tersebut dapat melalui plasenta dalam dosis rendah dan tidak menimbulkan efek teratogenik pada janin. (8,11-13) pada pemberian isoniazid sebaiknya diberikan piridoksin 50 mg/hari untuk m e n c e g a h t e r j a d i n y a n e u r o p a t i p e r i f e r. pemeriksaan fungsi hati sebaiknya dilakukan s a a t p e m b e r i a n i s o n i z i d d a n r i f a m p i s i n . pemberian vitamin k dilakukan pada akhir trismester ketiga kehamilan dan bayi yang baru lahir.(12) pada kasus multidrug resistant (mdr) digunakan pirazinamid, akan tetapi pirazinamid tidak digunakan secara rutin pada wanita hamil karena terdapat efek teratogenik. paraamino salisilat (pas) telah digunakan secara aman pada wanita hamil akan tetapi obat tersebut ditoleransi tubuh secara buruk.(13) tuberkulosis laten adalah pasien dengan uji tuberkulin positif dan secara klinis tidak ada tanda-tanda terjadi tuberkulosis aktif.(11) terapi pada tbc laten tergantung faktor risiko dan hasil konversi uji tuberkulin. pemberian terapi pada tbc laten biasanya ditunda sampai 2-3 bulan setelah kelahiran.(11,12) pada pasien yang mempunyai risiko kontak dengan individu bta positif dan infeksi hiv, terapi diberikan setelah trisemester pertama pada kehamilan dengan konversi uji tuberkulin positif dalam 2 tahun terakhir. sedangkan pada wanita hamil dengan tbc laten yang sebelumnya telah diterapi s e c a r a a d e k u a t t i d a k m e m e r l u k a n t e r a p i profilaksis isoniazid (300 mg selama 6-12 bulan).(1) penatalaksanaan tbc pada wanita hamil harus diberikan secara tepat dan adekuat, serta mencegah timbulnya efek samping teratogenik pada janin. pasien tbc aktif dengan sputum bta positif diberikan isoniazid, rifampisin, etambutol dan piridoksin selama 9 bulan pada populasi risiko tbc rendah. pada populasi dengan risikotbc tinggi dan adanya resisten o b a t a n t i t b c t i n g g i p e r l u p e n a m b a h a n pirazinamid.(11-14) p a s i e n d e n g a n u j i t u b e r k u l i n p o s i t i f , sputum bta negatif, biakan negatif dan foto t o r a k s m e n u n j u k k a n i n f i l t r a t a t a u a d a n y a k a v i t a s , d i b e r i k a n i s o n i a z i d , r i f a m p i s i n , etambutol dan piridoksin selama 9 bulan. sedangkan bila pada foto toraks terlihat proses penyakit yang telah menyembuh (terdapat kalsifikasi pada kelenjar getah bening dan lesi parenkim), dilakukan observasi pada pasien. pengobatan diberikan secara tepat setelah melahirkan atau diberi pengobatan profilaksis dengan isoniazid dan piridoksin selama 9 bulan y a n g d i m u l a i p a d a t r i s e m e s t e r k e d u a kehamilan.(11-14) pasien dengan konversi uji tuberkulin t e r b a r u p o s i t i f , f o t o t o r a k s n o r m a l s e r t a p e m e r i k s a a n b a k t e r i o l o g i s n e g a t i f , m a k a d i l a k u k a n o b s e r v a s i s e l a m a k e h a m i l a n , pengobatan diberikan setelah melahirkan atau dengan pemberian profilaksis isoniazid dan p i r i d o k s i n s e l a m a 9 b u l a n d i m u l a i p a d a trisemester kedua kehamilan.(12,13) pasien dengan resistensi organisme maka diberikan isoniazid, rifampisin, etambutol, pirazinamid sesuai dengan uji sensitivitas. pada pasien dengan ketidakmampuan mentoleransi i s o n i a z i d d a n r i f a m p i s i n , m a k a d i b e r i k a n etambutol atau obat lain yang tersedia.(15) 147 obat antituberkulosis selama kehamilan oat yang diberikan dibagi atas 2 golongan yaitu obat lini pertama (first line) dan obat lini kedua (second line). rifampisin merupakan obat lini pertama yang terutama bekerja pada sel yang sedang tumbuh, tetapi juga memperlihatkan efek pada sel yang sedang tidak aktif (resting cell). bekerja dengan menghambat sintesa rna m. tuberculosis sehingga menekan proses awal pembentukan rantai dalam sintesa rna. bekerja di intra dan ekstra sel. pada konsentrasi 0,005 0,2 mg/l akan menghambat pertumbuhan m. tuberculosis secara in vitro. obat ini juga menghambat beberapa mycobacterium atipikal, bakteri gram negatif dan gram positif. secara in vitro, rifampisin dapat meningkatkan aktivitas s t r e p t o m i s i n d a n i s o n i a z i d t e r h a d a p m . tuberculosis dan juga mempunyai mekanisme post antibiotic effect terhadap bakteri gram negatif.(16) diabsorpsi dengan baik melalui saluran cerna, absorpsi rifampisin dapat berkurang bila d i b e r i k a n b e r s a m a m a k a n a n . a b s o r p s i rifampisin akan berkurang 30% jika diberikan bersama dengan antasida. pemberian antasida akan meningkatkan ph lambung dan akan m e n g u r a n g i p r o s e s d i s s o l u t i o n r i f a m p i s i n sehingga akan menghambat absorpsi. rifampisin dengan mudah didistribusikan ke sebagian besar organ, jaringan, tulang, cairan serebrospinal dan cairan tubuh lainnya termasuk eksudat serta kavitas tuberkulosis paru. obat ini menimbulkan warna orange sampai merah bata pada urin, saliva, feses, sputum, air mata dan keringat. volume distribusi 1 l/kg bb, ikatan protein plasma 60-80%, waktu paruh 1-6 jam dan akan memanjang bila terdapat gangguan fungsi hepar. metabolisme terjadi melalui deasetilasi dan hidrolisis, sedangkan ekskresinya terutama melalui empedu. dapat melewati barier plasenta dan dapat dijumpai konsentrasi rendah di asi. rifampisin melewati plasenta dengan kadar yang sama dengan ibu. pada akhir trismester ke3 rasio konsentrasi pada tali pusat dan ibu besarnya 0,12 0,33.(17) studi yang dilakukan pada tikus, hewan pengerat dan kelinci dengan pemberian dosis 2,5 10 kali dosis yang masuk ke uterus tidak menunjukkan peningkatan kelainan kongenital. pada 442 perempuan hamil y a n g m i n u m r i f a m p i s i n , t e r m a s u k 11 9 perempuan yang terpajan selama trismester pertama tidak terdapat peningkatan kelainan janin secara bermakna. beberapa studi yang menunjukkan insidens malformasi rata-rata 1,8 4,4% pada 204 kehamilan.(10) pada kelinci telah d i l a p o r k a n t e r j a d i s p i n a b i f i d a d a n c l e f t palates.(17) efek samping ringan dapat timbul pada pemberian rifampisin antara lain: sindrom kulit seperti gatal-gatal kemerahan, sindrom flu berupa demam, menggigil, nyeri tulang dan sindrom perut berupa nyeri perut, mual, muntah dan kadang-kadang diare. efek samping yang berat tetapi jarang terjadi adalah sindrom respirasi, purpura, anemia hemolitik yang akut, syok dan gagal ginjal. efek samping ringan sering terjadi pada saat pemberian berkala dan dapat sembuh sendiri atau hanya memerlukan pengobatan simtomatik. efek samping pada bayi b a r u l a h i r j u g a d i d a p a t k a n h e m m o r rh a g i c disease of the newborn sehingga dianjurkan pemberian profilaksis vitamin k.(12,18) isoniazid (inh) menghambat biosintesis asam mikolat yang merupakan unsur penting dinding sel mycobacterium. menghilangkan sifat tahan asam dan menurunkan jumlah lemak yang terekstraksi oleh metanol dari mycobacterium. hanya kuman yang peka yang menyerap obat ke dalam selnya dan proses ini merupakan proses aktif. bersifat bakterisid, dapat membunuh 90% populasi kuman dalam beberapa hari pertama pengobatan.(16) inh mudah diabsorpsi pada pemberian oral maupun parenteral. kelarutan inh dalam lemak tinggi, berat molekul rendah universa medicina vol. 26 no.3 meiyanti tuberkulosis pada kehamilan 148 dan melalui plasenta serta mudah mencapai janin dengan kadar hampir sama dengan ibu. pada penelitian, setelah pemberian inh dosis 100 mg jangka pendek sebelum kelahiran didapatkan rasio konsentrasi tali pusat dan ibu sebesar 0,73. kadar puncak dicapai dalam waktu 1-2 jam setelah pemberian oral. di hati, inh terutama m e n g a l a m i a s e t i l a s i , d a n p a d a m a n u s i a kecepatan metabolisme ini dipengaruhi oleh faktor genetik (asetilator cepat/lambat) yang secara bermakna mempengaruhi kadar obat dalam plasma dan masa paruhnya. waktu paruh berkisar 1-3 jam. mudah berdifusi ke dalam sel d a n s e m u a c a i r a n t u b u h . a n t a r a 7 5 9 5 % diekskresikan melalui urin dalam waktu 24 jam dan seluruhnya dalam bentuk metabolit.(16) isoniazid tidak bersifat teratogenik janin, meskipun konsentrasi yang melewati plasenta cukup besar. pada studi yang dilakukan pada h e w a n t i d a k m e n u n j u k k a n r e t a r d a s i pertumbuhan serta peningkatan malformasi pada tikus dan kelinci dengan dosis 60 kali dosis manusia.(11) efek samping berat berupa hepatitis dapat timbul pada kurang lebih 0,5 % penderita. bila terjadi ikterus, hentikan pengobatan sampai ikterus hilang. efek samping yang ringan dapat b e r u p a : t a n d a k e r a c u n a n p a d a s a r a f t e p i , kesemutan, nyeri otot atau gangguan kesadaran. efek ini dapat dikurangi dengan pemberian piridoksin (dengan dosis 5-10 mg per hari atau dengan vitamin b kompleks). efek samping pada bayi baru lahir dilaporkan adanya perdarahan ( h e m m o r r h a g i c d i s e a s e o f t h e n e w b o r n ) sehingga dianjurkan pemberian profilaksis vitamin k sebelum kelahiran.(12,14,16) etambutol (emb) merupakan inhibitor arabinosyl transferases (i,ii,iii). arabinosyl transferase terlibat dalam reaksi polimerisasi arabinoglycan, yang merupakan unsur esensial d a r i d i n d i n g s e l m y c o b a c t e r i u m . a f i n i t a s terhadap arabinosyl transferase iii lebih kuat dibandingkan lainnya. arabinosyl transferase digunakan untuk menjadikan emb-cab operon. hal ini menyebabkan metabolisme sel terhambat dan sel mati. gangguan sintesis arabinoglycan mengubah barier sel, lipofilik meningkatkan aktivitas obat yang bersifat seperti rifampisin dan ofloksasin.(19) dinding sel mycobacterium spp sangat dibutuhkan untuk pertumbuhan dan kelangsungan hidup organisme di penjamu. dinding sel mycobacterium terdiri dari mycolic acid, arabinoglycan dan peptidoglycan. dinding s e l m e r u p a k a n l a p i s a n l i p i d b i l a y e r d a n asimetris.(19) hampir semua galur m. tuberculosis dan m . k a n s a s i i s e n s i t i f t e r h a d a p e t a m b u t o l . etambutol tidak efektif untuk kuman lain. etambutol pada konsentrasi 1-5 ìg/ml akan m e n g h a m b a t p e r t u m b u h a n m . t u b e rc u l o s i s secara in vitro. etambutol ini tetap menekan pertumbuhan m.tuberculosis yang telah resisten terhadap isoniazid dan streptomisin. etambutol dosis 15 mg/kg bb ini hanya aktif terhadap sel yang bertumbuh dengan khasiat tuberkulostatik, sedangkan pada dosis 25 mg/kg bb bersifat bakterisidal. penggunaan etambutol tunggal, ditemukan sputum basil tahan asam (bta) n e g a t i f d a l a m 3 b u l a n , t e t a p i d i t e m u k a n resistensi 35% dari kasus dan frekuensi relaps lebih tinggi.(16) efektivitas pada hewan coba sama dengan isoniazid. invivo, sukar menciptakan resistensi terhadap etambutol dan timbulnya lambat. resistensi bakteri terhadap etambutol terjadi akibat mutasi embb, emba dan embc, kode untuk arabinosyl transferase. resistensi ini timbul bila etambutol diberikan tunggal. pada p e m b e r i a n o r a l s e k i t a r 7 5 8 0 % e t a m b u t o l d i s e r a p d i s a l u r a n c e r n a . m a k a n a n t i d a k mempengaruhi absorpsi obat. kadar puncak plasma dicapai dalam waktu 2-4 jam setelah p e m b e r i a n . d o s i s t u n g g a l 2 5 m g / k g b b menghasilkan kadar plasma sekitar 2-5 ìg/ml dalam 2-4 jam, kurang dari 1 ìg dalam 24 jam. masa paruh eliminasinya 3-4 jam dan dapat 149 memanjang sampai 8 jam pada pasien dengan gangguan fungsi ginjal. etambutol secara bebas melewati plasenta dengan cord to maternal serum ratio adalah 0,75. penelitian pada kelinci terdapat efek monoftalmia sedangkan pada tikus terjadi penurunan kesuburan.(17) rata-rata malformasi yang dilaporkan pada 638 bayi yang dilahirkan oleh ibu yang mendapat etambutol selama kehamilan adalah 2,2%. s e c a r a t e o r i e t a m b u t o l m e n y e b a b k a n kemungkinan toksisitas pada mata. hal ini diyakinkan kembali dengan penilaian pada 6 janin yang mengalami abortus pada minggu 512 kehamilan, tidak didapatkan gangguan pada sistem optik embrional.(20) pirazinamid (pza) adalah suatu prodruk, y a n g m e m e r l u k a n k o n v e r s i e n z i m pirazinamidase (dihasilkan oleh mikobakterial tertentu) menjadi bentuk aktif asam pirazinoat, masuk ke dalam sitoplasma m. tuberculosis secara difusi pasif, mengalami konversi oleh enzim nikotinamidase/pirazinamidase menjadi bentuk aktif asam pirazinoat (poa).(16) pza lebih aktif terhadap basil tuberkel semidorman k a r e n a s i s t e m p o m p a e f l u k s y a n g l e m a h dibandingkan dengan basil sedang bertumbuh c e p a t , d i m a n a p o m p a e f l u k s l e b i h a k t i f . peradangan akut akan menurunkan ph akibat produksi asam laktat oleh sel-sel inflamasi, hal ini menguntungkan aktivitas pza. berkurangnya peradangan akan meningkatkan ph lingkungan basil tuberkel yang berakibat pada peningkatan konsentrasi hambat minimal pza. kuman dalam keadaan dorman tidak dapat dipengaruhi karena pada saat itu ambilan pza tidak terjadi. b a n y a k p e n e l i t i a n m e n y a t a k a n d a y a sterilisasi obat ini dalam makrofag, dengan k o n s e n t r a s i ≥ 2 0 µ g / m l m e n g h a m b a t b a s i l tuberculosis intraseluler. efek bakteriostatik atau bakterisidal terhadap m. tuberculosis tergantung dosis (konsentrasi pza), serta lamanya paparan terhadap makrofag yang terinfeksi m. tuberculosis. pada berbagai studi dan laporan tidak ditemukan efek teratogenik yang bermakna pada hewan dan malformasi janin pada pasien yang telah diterapi.(8,10,17,18) penggunaan pza pada wanita hamil telah direkomendasikan oleh international union against tuberculosis and lung disease secara rutin, namun di amerika dilarang karena tidak a d a n y a d a t a y a n g a d e k u a t m e n g e n a i e f e k teratogeniknya.(20) efek samping utama dari penggunaan obat ini adalah hepatitis, juga dapat terjadi nyeri sendi dan kadang-kadang dapat menyebabkan serangan arthritis gout yang kemungkinan d i s e b a b k a n b e r k u r a n g n y a e k s k r e s i d a n penimbunan asam urat. pemberian intermiten dapat mengurangi kejadian tersebut. efek samping lain adalah anoreksia, mual, muntah, disuri, demam dan reaksi hipersensitivitas. streptomisin melewati plasenta dengan cepat sampai ke sirkulasi janin dan cairan amnion s e r t a m e n c a p a i k a d a r k u r a n g d a r i 5 0 % dibandingkan kadar ibu.(8) efek samping yang dilaporkan dari berbagai studi pada hewan y a i t u o t o t o k s i s i t i . tu l i k o n g e n i t a l t e l a h dilaporkan terjadi pada bayi yang terpajan selama dalam kandungan, walaupun tidak ada h u b u n g a n y a n g p a s t i t e n t a n g m e k a n i s m e o t o t o k s i s i t i d e n g a n p a j a n a n s e l a m a kehamilan.(5,8) hasil penelitian menggunakan a u d i o g r a m m e n u n j u k k a n 5 0 a n a k t i d a k mengalami gangguan, 2 dari 33 anak dengan kehilangan pendengaran, sampai 4 dari 13 anak dengan tes kalorifik tidak normal. hal ini merupakan kejadian ototoksisiti yang berasal dari pajanan selama dalam kandungan.(8) penelitian lain menyimpulkan streptomisin dapat menyebabkan kerusakan sistem vestibular dan kerusakan nervus kranialis ke 8. pada n e g a r a b e r k e m b a n g d i a n j u r k a n t i d a k menggunakan streptomisin selama kehamilan.(12) dosis streptomisin 0,75 1 g/hari selama 14-21 hari selanjutnya 1g 3 kali seminggu secara intramuskular. universa medicina vol. 26 no.3 meiyanti tuberkulosis pada kehamilan 150 kanamisin merupakan obat lini kedua dan m e r u p a k a n v a r i a s i d a r i a m i n o g l i k o s i d a , mempunyai efek samping yang sama dengan streptomisin dan sebaiknya tidak digunakan pada kehamilan kecuali pada mdr. dosis yang diberikan 15 mg/kg, bb diberikan 3-5 kali s e m i n g g u i n t r a m u s c u l a r. ( 8 , 1 2 , 2 1 ) e t i o n a m i d mempunyai penetrasi yang baik ke semua jaringan termasuk cairan serebrospinal. pada penelitian yang dilakukan pada tikus dan kelinci tidak ditemukan peningkatan kerusakan sistem saraf pusat. pada tikus putih didapatkan efek pada tulang rangka (dosis 5-10 kali normal) sedangkan terjadi retardasi pertumbuhan pada h e w a n p e n g e r a t . ( 8 ) e t i o n a m i d d i n y a t a k a n potensial bersifat teratogenik dan sebaiknya dihindari penggunaan pada kehamilan kecuali jika dibutuhkan pada kasus mdr-tb. efek samping lainnya seperti hepatitis, neuritis optik dan neuritis perifer. dosis 0,5 1 gram/hari d a l a m d o s i s t e r b a g i . ( 8 , 1 6 , 2 1 ) k a p r e o m i s i n merupakan obat lini kedua yang diberikan secara i n t r a m u s k u l a r. k a p r e o m i s i n s e b a i k n y a digunakan jika diperlukan, biasanya untuk mdr-tb.(3,21) dosis yang diberikan 0,75 1 gram/hari selama 2-6 bulan, selanjutnya 1 gram 3 kali seminggu. kesimpulan tu b e r k u l o s i s t i d a k m e m p e n g a r u h i kehamilan dan kehamilan tidak mempengaruhi m a n i f e s t a s i k l i n i s d a n p r o g r e s i t i v i t a s tuberkulosis bila diterapi dengan tepat dan adekuat. penggunaan regimen pengobatan yang tepat dan adekuat dapat memperbaiki kualitas hidup ibu hamil dan menghindari efek samping ke janin dan bayi yang baru lahir. penggunaan obat streptomisin dan obat lini kedua dihindari pada wanita hamil karena efek samping terhadap janin, kecuali dalam keadaan mdr. daftar pustaka 1. tripahty sn. tuberculosis and pregnancy. int j gynaecol obstet 2003; 80: 247-53. 2. kothari a, girling j. tuberculosis and pregnancy: result of a study in a high prevalence area in london. eur j obstet gynecol 2006; 126: 48-55. 3. small pm, fujiwara pi. management of tuberculosis in the united states. n engl j med 2001; 345: 189-99. 4. khilnani gc. tuberculosis and pregnancy. indian j chest dis allied sci 2004; 46: 105-11. 5. frieden tr, sterling tr, munsiff ss, watt cj. tuberculosis. lancet 2003; 362: 887-96. 6. arora vk, gupta r. tuberculosis and pregnancy. ind j tub 2003; 50: 13-6. 7. figueroa dr, arredondo jl. pregnancy and tuberculosis: influence of treatment on perinatal outcome. am j perinatol 1998; 15: 303. 8. ormerod lp. tuberculosis in pregnancy and the puerperium. thorax 2001; 56: 494-9. 9. cantwell, shehab zm, cosello am, et al. brief report: congenital tuberculosis. n engl j med 1994; 330: 1051-4. 10. efferen ls. tuberculosis and pregnancy. curr opin pulm med 2007; 13: 205-11. 11. adis internasional editors. managing pregnant women with tuberculosis. j paed obstet gynaecol 2002: 25-8. 12. cdc. treatment of tuberculosis. mmwr 2003; 52. 13. bothamley g. drug treatment for tuberculosis during pregnancy: safely considerations. drug safety 2001; 24: 553-65. 14. world health organization. global tuberculosis programme: global tuberculosis control. who report 2000. 15. miller ks, miller jm. tuberculosis in pregnancy: interactions, diagnosis and management. clin obstet gynecol 1996; 39: 120-42. 16. petri jr wa. drugs used in the chemotherapy of tuberculosis. in: hardman jg, limbird le, gilman ag, editors. goodman & gilman’s the pharmacology basis of therapeutics. 10th ed. new york: mc graw-hill; 2001. p.1273-94. 17. holdiness mr. transplacental pharmacokinetics of the antituberculosis drugs. j clin pharmacikinetic 1987; 13: 125-9. 151 18. api tb consensus guidelines 2006. management of pulmonary tuberculosis, extra pulmonary tuberculosis and tuberculosis in special situations. j assoc physician india 2006; 54: 219-34. 19. mikusova k, slayden ra, besra gs, brennan pj. biogenesis of the mycobacterium cell wall and the site of action of ethambutol. j antimicrob chemother 1995; 39: 2484-9. universa medicina vol. 26 no.3 20. haas dw, prez rm. current treatment and management. in: rossman md, mac gregor rr, editors. tuberculosis clinical management and new challenges. new york: mc graw-hill; 2000. p. 187-219. 21. queesland tuberculosis control centre. guidelines for treatment of tuberculosis in pregnancy. 2006. laboratory data predictor of severe dengue 19 *tropical and infectious diseases consultant, pondok indah hospital, jakarta **division of tropical and infectious diseases, department of internal medicine, faculty of medicine, indonesia university, cipto mangunkusumo national hospital, jakarta, indonesia ***general practitioner, pondok indah hospital, jakarta ****department of clinical pathology, pondok indah hospital, jakarta correspondence: dr. dr. suhendro, sppd-kpti division of tropical and infectious diseases, department of internal medicine, faculty of medicine, indonesia university, cipto mangunkusumo national hospital, jakarta email: suhendro@ui.ac.id univ med 2017;36:19-24 doi: 10.18051/univmed.2017.v36.19-24 pissn: 1907-3062 / eissn: 2407-2230 received february 21, 2016 accepted for publication april 7, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract combination of three laboratory data as predictor of severe dengue in adults : a retrospective cohort study suhendro suwarto*,**, surya ulhaq***, and bing widjaja**** background dengue infection is a worldwide health problem. treatment is symptomatic, since no specific treatments are available which are effective in early detection. the roles of various clinical and laboratory findings were studied to predict severity of dengue infection. however, risk factors for severe dengue have not been fully investigated. thus, the aim of the study was to develop a combination of risk factors based on patient routine laboratory investigations to predict dengue infection severity in adults. methods this retrospective cohort study was conducted at a private hospital in jakarta, indonesia, from september 2010 to april 2016. complete blood count, ast ratio, and albumin were recorded. logistic regression was used to analyze the data. the receiver operating characteristic (roc) curve analysis was used to determine the optimal cut-off values of each parameter. results a total of 191 patients with non-severe dengue and 20 patients with severe dengue were included in this study. a multivariate analysis showed that hemoconcentration ≥15.625% (adjusted or, 6.28; 95% ci, 2.01-19.57), platelet count ≤15,500/ml (adjusted or, 3.18; 95% ci, 1.08-9.38), and ast ratio ≥5.01 (adjusted or, 3.93; 95 % ci, 1.36-11.35) were associated with severe dengue. the roc of the logistic regression model was 85.9% (95% ci 77.8-94.0). conclusion the combination of hemoconcentration, thrombocytopenia, and an elevated ast ratio is a risk factor for severe dengue in adults. the application of these findings may help to optimize the allocation of resources, which leads to a more appropriate and effective for the management of patients with severe dengue. keywords: hemoconcentration, thrombocytopenia, ast ratio, severe dengue doi: http://dx.doi.org/10.18051/univmed.2017.v36.19-24 universa medicinajanuary-april, 2017 vol.36 no.1 original article 20 suwarto, ulhaq, widjaja laboratory data predictor of severe dengue introduction severe dengue is a serious complication in dengue-infected patients and is associated with increased mortality. a previous study observed patients for 5 years and showed that of 12,321 patients with severe dengue, 1,062 patients had died. (1) according to the world health organization (who), severe dengue is defined by severe bleeding, dengue shock syndrome, acute respiratory distress syndrome, aspartate aminotransferase (ast), alanine aminotransferase (alt) ratio ≥1000 u/l, or organ dysfunction.(2) this dengue guideline proposed criteria for diagnostic warning signs that are risk factors for severe dengue. the warning sign criteria include clinical syndrome (pain in the abdomen, unrelenting vomiting, fluid accumulation, bleeding in the mucosa, lethargy, restlessness or hepatomegaly more than 2 cm) and laboratory parameters (increment of hemoconcentration simultaneous with a rapid decrease of thrombocytopenia).(2) one of these criteria, fluid accumulation, can be detected in clinical practice using abdominal usg.(3) however, this risk factor was determined in a group of pediatric patients.(4,5) in addition, an increment of hemoconcentration simultaneous with a rapid decrease of thrombocytopenia was not defined.(6) a recent study in adult patients with dengue showed that four laboratory parameters such as hematocrit, platelets, albumin, and increased ast ratio at a critical phase could be used to identify which patients are at risk for developing plasma leakage. an increase in ast provides better association with plasma leakage compared with alt.(7,8) these four variables are included in routine laboratory tests used in clinical practice, and are diagnostic predictors of plasma leakage.(7) however, the association between these four laboratory parameters and severe dengue has not been investigated. knowledge of severe dengue risk factors is crucial in identifying patients who are at risk for developing severe dengue. therefore, we conducted a study to investigate the association of hematocrit, platelets, albumin, and ast ratio with severe dengue in adult patients. methods research design a retrospective cohort study was conducted at pondok indah hospital, jakarta from september 2010 to april 2016. research subjects adult dengue patients 14 years of age or older who had a positive non-structural protein 1 (ns-1) antigen test result were included in this study. patients’ medical records from september 2010 to april 2016 were reviewed in this study. the exclusion criterion was incomplete data in medical records. the sample size was calculated using the formula for the difference between two population means by estimating the hematocrit population variance of 1.64,(9,10) assuming a difference in the population mean of 0.5. with 80% power to detect a 5% difference, the minimum total sample size required was 206 patients. laboratory analysis parameters such as age, sex, and laboratory parameters: complete blood count, ast, and albumin were recorded. ast and albumin tests were performed during the critical phase. the ast values were then converted to the ast ratio. this was calculated by dividing the ast value by the upper reference limit (here, the ast upper reference limit was 37 u/l).(7) hemoconcentration was calculated based on a previously published formula.(7) the criteria for severe dengue were based on dengue guidelines proposed by the who in 2009, and included severe bleeding, dengue shock syndrome, acute respiratory distress syndrome, ast ≥1000 u/l, or organ dysfunction.(2) ethical approval t h i s s t u d y w a s a p p r o v e d b y t h e management of pondok indah hospital and the 21 health research ethics committee faculty of m e d i c i n e u n i v e r s i t a s i n d o n e s i a c i p t o mangunkusumo hospital. statistical analysis association between the laboratory parameter variables (i.e., hematocrit, platelets, albumin, and ast ratio) and severe dengue was analyzed using a non-parametric mann–whitney u test. all laboratory parameters that had a significant association with severe dengue based on a bivariate analysis were then subjected to the receiver operating characteristic (roc) curve analysis to determine the optimal cut-off values of each variable. these cut-off values were used to classify the subjects using nominal variables. the nominal variables were then analyzed by logistic regression using a backward selection to obtain an adjusted odds ratio (or) for the predictors of severe dengue. spss version 20.0 (ibm corp., armonk, ny, usa) and stata version 14.0 (stata corp station, tx, usa) were used to perform the statistical analysis. results there were 211 infected dengue patients and 20 of these patients had severe dengue. clinical characteristics are presented in table 1. we found that hematocrit, platelets, albumin, and ast ratio have a significant association with severe dengue based on a bivariate analysis (table 1). the best cut-off value for each variable based on an roc analysis is presented in table 2. this cut-off value was used to classify subjects using nominal variables. all nominal variables were then subjected to logistic regression analysis. we found that hemoconcentration ≥15.625% (adjusted or=6.28; 95% ci 2.01-19.57), platelet count ≤15,500/µl (adjusted or= 3.18; 95% ci 1.089.38), and ast ratio ≥5.01 (adjusted or, 3.93; 95% ci, 1.36-11.35) were associated with severe dengue (table 3). table 1. distribution of clinical characteristics of patients with non-severe dengue and severe dengue adata presented as median (interquartile range); * p<0.05 by mann whitney test table 2. receiver operating characteristic curve of the laboratory parameters as predictors of severe dengue and cut off values for optimal sensitivity and specificity table 3. multiple logistic regression of the laboratory parameters to predict severe dengue univ med vol. 36 no.1 22 suwarto, ulhaq, widjaja laboratory data predictor of severe dengue the roc of the logistic regression model was 85.9% (95% ci 77.8-94.0%; figure 1). discussion in 2009, the who recommended criteria for warning signs in dengue-infected patients to identify patients with severe dengue.(2) the use of abdominal usg to detect pleural effusion or ascites as a marker for plasma leakage is not a routine procedure in the management of dengue infection.(11) hematocrit, platelet count, albumin concentration, and ast ratio are laboratory parameters that are suggested to be checked as part of dengue patient management. these laboratory parameters were associated with plasma leakage.(7,12) previous studies reported that the plasma leakage was most commonly detected in the critical phase, thus, in the present study, we collected data of the laboratory parameters measured in the critical phase, i.e., one day after defervescence.(13,14) this study showed that a combination of hemoconcentration, platelet count, and ast ratio was associated with severe dengue. however, a previous study reported that an increase in hematocrit of ≥20% is not sensitive for detecting plasma leakage and that it occurred infrequently in severe dengue.(6,9,15) suwarto et al.(7) showed that the cut-off point for hemoconcentration of ≥15.1% is a better predictor of plasma leakage and minimizes the risk of under-diagnosing patients who are at risk for developing severe dengue. in the present study, we suggest a similar cut-off point as the previous study,(7) which is ≥15.625%, as a risk factor for severe dengue. we found that a platelet count ≤15,500/µl was associated with severe dengue. this result is compatible with a previous study that reported that platelet counts <20,000/ml were strongly associated with severe dengue.(15) the patients who have severe thrombocytopenia, as indicated by a platelet count <20,000/ml, have a high risk of bleeding.(16) bivariate analysis showed that albumin levels are significantly lower in severe dengue patients compared to patients without severe dengue. however, based on multivariate analysis, this association did not persist. several studies reported low levels of albumin in dengueinfected patients without warning signs.(17-19) this figure 1. receiver operating characteristic (roc) curve of the logistic regression model 23 previous finding may explain a less significant role for albumin in severe dengue. the present study showed that an elevated ast ratio ≥5.01 was a risk factor of severe dengue. hepatic dysfunction is known to be associated with severe dengue. the cause of elevated ast levels in severe dengue is not fully understood, and possible mechanisms include a direct effect of dengue virus, dysregulation of immune response, necrosis of the liver cell, and ischemia or hypoxia result in liver injury.(20,21) however, further study is needed to confirm the mechanism of hepatic dysfunction in dengue. our study suggested that hemoconcentration, thrombocytopenia, and an elevated ast ratio is a risk factor for developing severe plasma leakage. this result is consistent with that of a previous study reporting that the combination of fluid accumulation that can be detected by a b d o m i n a l u s g, e l e v a t e d a s t a n d thrombocytopenia was associated with severe dengue.(22) limitations of this study include its retrospective nature, and lack of clinical data such as abdominal pain, vomiting, mucosal bleeding, lethargy, and hepatomegaly that may potentially influence the predictors of severe dengue. conclusion this study presents the novel finding that a lower cut-off point for hemoconcentration than that suggested by who, the presence of severe thrombocytopenia, and an increased ast ratio ≥5.01 are good criteria to identify patients at risk of severe dengue. competing interests no reported conflicts of interest. acknowledgments t h e a u t h o r s w o u l d l i k e t o t h a n k suhariyanto for assistance with data collection. contribution ss, su and bw contributed to drafting the manuscript. ss contributed to design the research, and responsible for the final content. ss, su and bw contributed to collected, analyzed and interpretatethe data. ss contributed to wrote the manuscript, and all authors read and approved the final manuscript. references 1. moraes gh, de fatima duarte e, duarte ec. determinants of mortality from severe dengue in brazil: a population-based case-control study. am j trop med hyg 2013;88:670-6. 2. tdr and world health organization. dengue: guidelines for diagnosis, treatment, prevention and control. new edition. geneva: world health organization;2009. 3. bandyopadhyay d, chattaraj s, hajra a, et al. a study on spectrum of hepatobiliary dysfunctions and pattern of liver involvement in dengue infection. j clin diagn res 2016;10: oc21-6. 4. narvaez f, gutierrez g, perez ma, et al. evaluation of the traditional and revised who classifications of dengue disease severity. plos negl trop dis 2011;5:e1397. doi: 10.1371/ journal.pntd.0001397. 5. alexander n, balmaseda a, coelho ic, et al. multicentre prospective study on dengue classification in four south-east asian and three latin american countries. trop med int health 2011;16:936-48. 6. thein tl, gan vc, lye dc, et al. utilities and limitations of the world health organization 2009 warning signs for adult dengue severity. plos negl trop dis 2013;7:e2023. doi: 10.1371/ journal.pntd.0002023. 7. suwarto s, nainggolan l, sinto r, et al. dengue score: a proposed diagnostic predictor for pleural effusion and/or ascites in adults with dengue infection. bmc infect dis 2016;16:322. doi: 10.1186/s12879-016-1671-3. 8. mahmuduzzaman m, chowdhury as, ghosh dk, et al. serum transaminase level changes in dengue fever and its correlation with disease severity. mymensingh med j 2011;20:349-55. 9. michels m, sumardi u, de mast q, et al. the predictive diagnostic value of serial daily bedside univ med vol. 36 no.1 24 suwarto, ulhaq, widjaja laboratory data predictor of severe dengue ultrasonography for severe dengue in indonesian adults. plos negl trop dis 2013;7:e2277. doi: 10.1371/journal.pntd.0002277. 10. wan x, wang w, liu j, et al. estimating the sample mean and standard deviation from the sample size, median, range and/or interquartile range. bmc med res methodol 2014;14:135. 11. potts ja, thomas sj, srikiatkhachorn a, et al. classification of dengue illness based on readily available laboratory data. am j trop med hyg 2010;83:781-8. 12. fernando s, wijewickrama a, gomes l, et al. patterns and causes of liver involvement in acute dengue infection. bmc infect dis 2016;16:319. doi: 10.1186/s12879-016-1656-2. 13. srikiatkhachorn a. plasma leakage in dengue haemorrhagic fever. thromb haemost 2009;102: 1042-9. 14. srikiatkhachorn a, krautrachue a, ratanaprakarn w, et al. natural history of plasma leakage in dengue hemorrhagic fever: a serial ultrasonographic study. pediatr infect dis j 2007;26:283-90. 15. jayaratne sd, atukorale v, gomes l, et al. evaluation of the who revised criteria for classification of clinical disease severity in acute adult dengue infection. bmc res notes. 2012;5: 645. doi: 10.1186/1756-0500-5-645. 16. makroo rn, raina v, kumar p, et al. role of platelet transfusion in the management of dengue patients in a tertiary care hospital. asian j transfus sci 2007;1:4-7. 17. itha s, kashyap r, krishnani n, et al. profile of liver involvement in dengue virus infection. natl med j india 2005;18:127-30. 18. roy a, sarkar d, chakraborty s, et al. profile of hepatic involvement by dengue virus in dengue infected children. n am j med sci 2013;5:4805. 19. jagadishkumar k, jain p, manjunath vg, et al. hepatic involvement in dengue fever in children. iran j pediatr 2012;22:231-6. 20. tan ss, bujang ma. the clinical features and outcomes of acute liver failure associated with dengue infection in adults: a case series. braz j infect dis 2013;17:164-9. 21. samanta j, sharma v. dengue and its effects on liver. world j clin cases 2015;3:125-31. 22. flamand c, fritzell c, prince c, et al. epidemiological assessment of the severity of dengue epidemics in french guiana. plos one 2017;12:e0172267. doi: 10.1371/journal.pone. 0172267. underweight increases tuberculosis 4 *department of nutrition, faculty of medicine, diponegoro university, semarang **science program study of nutrition, muhammadiyah university semarang correspondence: galuh chandra irawan department of nutrition, faculty of medicine, diponegoro university gedung g, jln. prof. soedarto, sh, tembalang semarang 1269 email: chandragaluh50@gmail.com univ med 2017;36:4-10 doi: 10.18051/univmed.2017.v36.4-10 pissn: 1907-3062 / eissn: 2407-2230 received october 22, 2016 accepted for publication april 7, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract underweight increases the risk of pulmonary tuberculosis in adult galuh chandra irawan*, ani margawati*, and ali rosidi** background tuberculosis (tb) is a leading cause of morbidity and mortality, especially in middleand low-income countries. the risk of developing tb may be related to nutritional status. socioeconomic and behavioral factors are also shown to increase the susceptibility to tb infection. the objective of this study was to determine nutritional factors as risk factors of pulmonary tb in adult. methods this was an observational study of case control design. the study subjects were community members consisting of 19 adult cases of pulmonary tuberculosis and 38 controls. data on nutritional intakes were obtained by semiquantitative food frequency questionnaire (ffq), while smoking behavior, history of dm, body mass index, education, and income were obtained by structured interviews. the data were analyzed by independent t-test and logistic regression for calculation of the odds ratio (or). results the bivariate test showed that the adequacy levels for energy (or=6.8; 95% ci: 1.51-30.54), protein (or=5.1; 95% ci: 1.52-17.14), vitamin a (or=4.2; 5% ci: 1.31-13.54), vitamin c (or=3.8; 95% ci: 1.21-12.36), selenium (or=4.2; 95% ci: 1.34-13.58), body mass index (or=4.4; 95% ci: 1.32-14.35) and smoking behavior (or=3.7; 95% ci: 1.15-11.9), were significant risk factors for pulmonary tuberculosis. multiple logistic regression test showed that low body mass index (<18.5 kg/m2) (or=6.0; 95% ci: 1.32-27.18) was a the most influential risk factor of pulmonary tuberculosis. conclusion low body mass index is the most influential risk factor for pulmonary tuberculosis incidence in adult. nutrition profile in adult is an important determinant of tb incidence. keywords: pulmonary tuberculosis, body mass index, energy, smoking behavior, adult doi: http://dx.doi.org/10.18051/univmed.2017.v36.4-10 universa medicinajanuary-april, 2017 vol.36 no.1 original article 5 univ med vol. 36 no.1 introduction pulmonary tuberculosis is a directly transmissible disease caused by mycobacterium tuberculosis. mycobacterium tuberculosis mostly attacks the lungs, but may also affect other organs. (1) pulmonary tuberculosis is a communicable disease that is still attracting global attention, since to date there is no single country that is free of pulmonary tuberculosis.(1) the number of patients with pulmonary tuberculosis in the world is increasing annually. the results of a who survey in 2013 showed that there were approximately 9 million patients with pulmonary tuberculosis, with more than half (56%) living in southeast asia and the western pacific region.(1) indonesia currently ranks third as one of the countries with the largest numbers of pulmonary tuberculosis cases in the world after china and india, with the number of patients reaching 325,582 in 2013.(1) the prevalence of tuberculosis in central java ranks fifth at 0.4%. (2) banjarnegara district in 2013 had a prevalence of 0.3% per 100.000 inhabitants.(3) the number of pulmonary tuberculosis patients in 2014-2016 in karangkobar subdistrict showed a fluctuating trend, with 14 cases in 2014, 123 suspected and 30 confirmed cases in 2015 and 19 cases in may 2016.(3) apart from home environmental health factors, nutritional status is also associated with the incidence of pulmonary tuberculosis.(4) inadequate intakes of protein and energy result in excessive utilization of the energy reserves of the body, thus causing weight loss and biochemical abnormalities.(5) one study states that energy adequacy level affects nutritional status.(6) undernutrition may increase the risk of pulmonary tuberculosis, since poor nutritional status may undermine the immune system and facilitate aggravation of the tuberculosis.(7) pulmonary tuberculosis may cause nutritional deficiency from decreased appetite, thus influencing nutrient intakes, with consequent changes in metabolic processes. the a s s o c i a t i o n b e t w e e n p o o r n u t r i t i o n a n d pulmonary tuberculosis has been demonstrated by the bcg vaccine trial conducted in the us in 1 9 6 0 a n d i t h a s b e e n e s t i m a t e d t h a t undernourished children have a two-fold risk of pulmonary tuberculosis as compared with wellnourished children.(8) nutrients such as vitamin a and zinc are micronutrients that play an important role in immunity. in addition, other antioxidant compounds such as selenium, vitamin c, vitamin d, vitamin e and iron may assist in maintaining the host defences in a healthy condition, by preventing oxidative processes and inflammatory responses.(8) smoking behavior is also a factor that is associated with pulmonary tuberculosis incidence, in addition to nutrient intakes.(9) one study showed that there is an association between smoking and pulmonary tuberculosis incidence and that smokers have a 3.1-fold higher risk for pulmonary tuberculosis than nonsmokers.(9) up to the present, pulmonary tuberculosis has been associated solely with poor nutrition caused by environmental factors and the epidemiological spread of the disease, so that there is a need for a more specific study on nutrient intakes, in order to know the nutrient adequacy levels at risk for pulmonary tuberculosis.(10) previous studies on nutritional status as risk factor have not shown consistent results. a study conducted in rural india stated that there was no association between body mass index and pulmonary tuberculosis incidence.(11) therefore a study is needed that aims to determine nutritional status as risk factor for pulmonary tuberculosis incidence in adults. methods research design the study was of case-control observational design and conducted at puskesmas karangkobar in banjarnegara district, central java, between july and august 2016. research subjects the subjects of this study were adult males and females aged 19-63 years, consisting of 19 cases of pulmonary tuberculosis and 38 controls 6 (non-tuberculosis cases), yielding a total of 57 subjects. the total number of study subjects was arrived at on the basis of a total sampling of cases that were registered during the previous 6 months at puskesmas karangkobar as patients with pulmonary tuberculosis. the inclusion criteria in this study were subjects diagnosed with pulmonary tuberculosis who were positive for acid-fast bacilli and had positive radiographs, community members with suspect pulmonary tuberculosis, and healthy community members living with pulmonary tuberculosis patients, who resided in the catchment area of puskesmas karangkobar, banjarnegara district, and were willing to participate in the study. the exclusion criteria were pulmonary tuberculosis patients with complications. assessment of nutrient intakes nutrient intakes were obtained from the r e s u l t s o f i n t e r v i e w s p e r f o r m e d b y t h e investigators using semi-quantitative food frequency questionnaires. the assessed nutrient intakes comprised energy, protein, vitamin a, vitamin c, and selenium. nutrient adequacy levels were calculated using the nutrisurvey 2007 program and were based on the indonesian recommended daily allowances (irda) for 2013. energy and protein adequacy levels were categorized as poor if <90% of irda and optimal if ≥90% of irda.(12) vitamin a, vitamin c and selenium adequacy levels were categorized as poor if <77% of irda and optimal if ≥77% of irda.(13) anthropometric measurements assessment of body mass index (bmi) of the study subjects was performed by the investigators. the bmi values of the study subjects were obtained from measurements of body weight before receiving treatment for 6 months and divided by height. bmi was categorized as underweight if <18.5, normal if ≥18.5 – 24.9, and overweight if >25 – 29.9.(14) smoking habits smoking behavior was obtained through structured interviews. the instruments used were the list of interview questions and the informed consent form. the associated data were presence of active smoking behavior at a frequency of more than once on the day previous to the establishment of the diagnosis of pulmonary tuberculosis.(15) statistical analysis data analysis was performed by means of the independent t-test and logistic regression. multiple logistic regression was performed to control for possible confounding factors and to identify factors that are independently associated with undernutrition. a p value of less than 0.05 was used to declare the presence of a statistically significant association between variables. ethical clearance this study was conducted after obtaining approval from the ethical commission under no.774/ec/fk-rsdk/2016. results table 1 shows that for the characteristics of age and gender there were no differences (p>0.05) between the group of cases and the group of controls. table 1. demographics distribution of cases (n=19) and controls (n=38) irawan, margawati, rosidi underweight increases tuberculosis 7 univ med vol. 36 no.1 table 2 lists the risk factors for pulmonary tuberculosis. poor adequacy levels for energy and protein were risk factors for pulmonary tuberculosis incidence, with respective or values of 6.8 (95% ci=1.51 – 30.54) and 5.1 (95% ci=1.52 – 17.14). poor adequacy levels for vitamin a, vitamin c and selenium were also r i s k f a c t o r s f o r p u l m o n a r y t u b e r c u l o s i s incidence, with respective or values of 4.2 (95% ic=1.31 – 13.54), 3.8 (95% ic=1.21 – 12.36), and 4.2 (95% ic=1.34 – 13.58). in addition, body mass index of <18.5 kg/m2 (or=4.4; 95% ic=1.32 – 14.35) and smoking behavior (or=3.7; 95% ic= 1.15 – 11.91) were risk factors for pulmonary tuberculosis incidence. body mass index of <18.5 kg/m2 was the major risk factor for pulmonary tuberculosis incidence (or=6.0; 95% ci=1.32 – 27.4) (table 3). discussion poor adequacy levels for energy were more frequently found in the group of cases than in the group of controls, whereas optimal adequacy levels for energy were more frequently found in the group of controls than in the group of cases. a similar study in the province of nusa tenggara timur found that respondents with poor energy intakes were closely associated with pulmonary tuberculosis incidence.(16) the body uses 60-70% of the total energy requirement for maintaining its basic functions, called the basal metabolism. the energy is required for the basal metabolism and such body functions as digestion, processing, and absorption of food in the digestive tract, moving, walking, working, and other activities. inadequate energy consumption leads to table 2. bivariate logistic regression of several nutritional risk factors for pulmonary tuberculosis table 3. multiple logistic regression of significant variables of pulmonary tuberculosis 8 excessive utilization of the energy reserves of the body, thus resulting in weight loss and biochemical abnormalities.(5) in the group of cases only 8 subjects had an optimal adequacy level for protein, whereas 11 of the 19 subjects had poor adequacy levels for protein. low protein intake is a risk factor for pulmonary tuberculosis incidence, eg. energyprotein deficiency is associated with abnormal cell-mediated immunity, phagocytic function, complement system, immunoglobulin a secretion, and cytokine production.(7) this is in agreement with a study conducted in africa, showing that the majority of pulmonary tuberculosis patients have chronic energy-protein deficiency. (17) vitamin a deficiency as seen from its adequacy levels was also found in the group of cases in comparison with the group of controls. this was because there were more subjects (79.4%) with optimal adequacy levels for vitamin a in the group of controls than in the group of cases. inadequate vitamin a is a risk factor for pulmonary tuberculosis incidence. the present study is in line with another study conducted in the community of nusa tenggara timur province, stating that low vitamin a intake carries a 3.2fold risk of pulmonary tuberculosis.(16) on the other hand, a study conducted in delft, western cape, south africa, found no relationship between vitamin a intake and pulmonary tuberculosis incidence.(18) in the group of cases, 57.9% of the community members had poor adequacy levels for vitamin c, whereas in the group of controls, 26.3% had poor adequacy levels for vitamin c. this shows that in the group of controls the adequacy level for vitamin c was higher than that in the group of cases. the study by johnkennedy et al.(19) reported that the vitamin c level in patients with tuberculosis tends to be low, which results in decreased total serum antioxidant levels in patients with tuberculosis. the low antioxidant level is caused by inadequate intake and increased free radicals in the phagocytosis of mycobacterium. these results agree with the study by madhavi et al.,(20) who found that tuberculosis patients receiving high doses of vitamin c for 2 weeks showed increased total antioxidants. these results are supported b y a s t u d y i n b a n d u n g , r e p o r t i n g t h a t administration of vitamin c is of benefit for increasing the antioxidant level in patients with pulmonary tuberculosis.(21) poor adequacy level for selenium is a risk factor for pulmonary tuberculosis incidence. this may be seen from the number of subjects with pulmonary tuberculosis who had poor adequacy levels for selenium, namely 52.2%, when compared with the number of healthy subjects who had poor adequacy levels for selenium, i.e. only 28.9%. our study results are supported by a study in india, stating that selenium levels in the blood of patients with pulmonary tuberculosis are lower than in healthy persons.(21) selenium supplementation may improve the antioxidant status in patients with pulmonary tuberculosis on standard treatment, thus assisting the healing process.(22) low body mass index was more frequently found in the group of cases, while in the group of controls only a few had low body mass index. the majority of the controls had normal body mass index, while some had a higher than normal body mass index. body mass index may be said to be a risk factor for pulmonary tuberculosis. the results of a study in aceh showed a significant association between nutritional status (body mass index) and pulmonary tuberculosis incidence.(4) a similar study conducted in indian communities also found an association between body mass index and pulmonary tuberculosis.(24) however, a study in connection with body mass index among adult females in rural india showed no differences in body mass index before and after 6 months of treatment.(11) the study also showed no differences in body mass index among male patients who dropped out of the study.(11) this was caused by a low motivation for taking food among patients with pulmonary tuberculosis. smoking behavior was frequently found both in the group of cases and in the group of controls, since mountain communities are avid smokers, irawan, margawati, rosidi underweight increases tuberculosis 9 univ med vol. 36 no.1 for warming the body, in spite of its impact on their health. smoking increases the risk of infection with mycobacterium tuberculosis and the risk of the disease developing and leading to death among patients with pulmonary tuberculosis.(25) this signifies that smoking behavior is a risk factor for pulmonary tuberculosis incidence. our study results showed an association between smoking and pulmonary tuberculosis incidence, with smokers being 3.1 times at higher risk of pulmonary tuberculosis in comparison with non-smokers.(8) a cohort study in rural china found no significant association between smoking b e h a v i o r a n d p u l m o n a r y t u b e r c u l o s i s incidence.(15) another study also reported no significant association between smoking behavior and pulmonary tuberculosis incidence, because smoking was counterbalanced by high nutritional intakes, thus decreasing the risk of infection with pulmonary tuberculosis.(26) a limitation of the present study lies in the use of the semiquantitative ffq to determine the intakes of the nutrients under study, thus requiring a strong recall capacity. another limitation is the lack of assessment of serum albumin, vitamin a, vitamin c and selenium. the study was of case-control design, therefore the occurrence of recall bias may have influenced the collected data. this study proves that low nutrient intake is a risk factor for contracting tuberculosis. nutritional supplementation should be aimed at those individuals with poor nutritional status, in order to improve it, which will reduce the risk of contracting tuberculosis. conclusions low body mass index is a risk factor for pulmonary tuberculosis, after controlling for energy adequacy level. further studies are required to evaluate the effect of nutritional supplementation on tuberculosis incidence in adults. conflict of interest there was no conflict of interest between the authors and the study subjects. acknowledgments we thank the head and staff of puskesmas karangkobar, banjarnegara district, central java and the pulmonary tuberculosis patients and their families who allowed the principal investigator to conduct the study and obtain the data at the aforementioned puskesmas. contributors gci contributed to drafting the manuscript and the study design. gci, am, and ar contributed to data collection, analysis and interpretation. gci and am contributed revising manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. world health organization. global tuberculosis report. geneva: world health organization. 2014. 2. kementerian kesehatan republik indonesia. riset kesehatan dasar (riskesdas) tahun 2013. jakarta: kementerian kesehatan republik indonesia; 2013. 3. dinas kesehatan provinsi jawa tengah. riset kesehatan dasar (riskesdas) tahun 2013. semarang: dinas kesehatan provinsi jawa tengah; 2013. 4. mulyadi, suangkupon r, dermawan i. profil penderita tuberkulosis paru di pesisir pantai aceh barat daya (kajian di puskesmas biangpidie). j respir indones 2011;31:105-8. 5. hatta m, supriatmo, ali m, et al. comparison of zinc-probiotic combination therapy to zinc therapy alone in reducing the severity of acute diarrhea. paediatrica indonesiana 2011;51:1-6. 6. manalu hs. faktor-faktor yang mempengaruhi kejadian tb paru dan upaya penanggulangannya. j ekologi kesehatan 2010; 9:1340-4. 10 7. world health organization. global tuberculosis control: surveillance, planning, financing. report of who expert committee. who technical report series. geneva: who;2007. 8. narasimhan p, wood j, macintyre cr, et al. risk factors for tuberculosis. pulm med 2013. article id 828939, 11 pages. http://dx.doi.org/10.1155/ 2013/828939. 9. alavi-naini r, sharifi-mood b, maliheh m. association between tuberculosis and smoking. int j high risk behav addict 2012;1:71-4. 10. cegielski jp, mcmurray dn. the relationship between malnutrition and tuberculosis: evidence from studies in humans and experimental animals. int j tuberc lung dis 2004;8:286-98. 11. bhargava a, chatterjee m, jain y, et al. nutritional status of adult patients with pulmonary tuberculosis in rural central india and its association with mortality. plos one 2013;8:e77979. doi: 10.1371/journal.pone. 0077979. 12. hardinsyah. angka kecukupan energi, protein, lemak dan serat makanan. prosiding widyakarya pangan dan gizi viii; 2004 mei 17-19; jakarta; 2004.p.323-27. 13. gibson rs. principles of nutritional assessment. 2nd ed. new york: oxford university press; 2005. 14. maro i, lahey t, mackenzie t, et al. low bmi and falling bmi predict hivassociated with tuberculosis: a prospective study in tanzania. int j tuberc lung dis 2010;14:1447–53. 15. chen w, shu w, wang m, et al. pulmonary tuberculosis incidence and risk factors in rural areas of china: a cohort study. plos one 2013;8:e58171. doi: 10.1371/journal.pone. 0058171. 16. pakasi ta, kariadi e, wibowo y, et al. vitamin a deficiency and other factors associated with severe tuberculosis in timor and rote islands, east nusa tenggara province, indonesia. eur j clin nutr 2009:63:1130–5. 17. dheda k, schwander sk, zhu b, et al. the immunology of tuberculosis: from bench to bedside. respirology 2010;15:433-50. 18. lombardo cc, rina s, visser me. the nutritional status of patients with tuberculosis in comparison with tuberculosis-free contacts in delft, western cape. s afr j clin nutr 2012; 25:180-5. 19. johnkennedy n, onyinyechi as, chukwunyere nne. the antioxidant status and lipid peroxidation product of newly diagnosed and 6 weeks follow up patients with pulmonary tuberculosis in owerri, imo state, nigeria. asian pacific j tropical dis 2011;2:292-4. 20. madhavi m, samudram p, kumar h, et al. effect of antioxidant vitamins c dan e supplementation on its plasma levels and on lipid profile in pulmonary tuberculosis patients. am j infect dis 2009;5:263-72. 21. zakiyyah er, gurnida da, kartasasmita cb. pengaruh pemberian vitamin c terhadap perubahan kadar total antioksidan serum pada penderita tuberkulosis paru. sari pediatri 2014; 16:2-7. 22. ramakrishnan k, sharma sp, shenbagarathai r, et al. serum selenium levels in pulmonary tuberculosis levels with and without hiv/aids. retrovirology 2009;6:p76. doi: 10.1186/17424690-6-s2-p76. 23. seyedrezazadeh e, ostadrahimi a, mahboob s, et al. effect of vitamin e and selenium supplementation on oxidative stress status in pulmonary tuberculosis patients. respirology 2008;13:294-98. doi: 10.1111/j.1440-1843. 2007.01200.x. 24. gupta kb, gupta r, atreja a, et al. tuberculosis and nutrition. lung india 2009;26: 9-16. 25. wijaya aa. merokok dan tuberkulosis. j tuberculosis indones 2012;8:18-23. 26. leung cc, lam th, ho ks, et al. passive smoking and tuberculosis. arch intern med 2010;170:287-92. irawan, margawati, rosidi underweight increases tuberculosis rina 113 *department of community medicine, medical faculty, trisakti university correspondence dr. rina k.kusumaratna, m.kes. department of community medicine, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2504 email : rkusumaratna@yahoo.com univ med 2008; 27: 113-24 gender differences in nutritional intake and status in healthy free-living elderly july-september, 2008july-september, 2008july-september, 2008july-september, 2008july-september, 2008 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 vol.27 no.3 abstract universa medicina rina k. kusumaratna* malnourishment in the older population is reported to be a consequence of inadequate food intake, underlying diseases, with females being at higher risk and economically more vulnerable. the objective of this study were to determine the patterns of gender differences in nutritional intake and status among free-living elderly in the jakarta urban area. a cross sectional design was performed on freeliving elderly people aged 60 years and older in south jakarta. a total of 298 freeliving elderly were interviewed on demography and social economic status, and assessed on anthropometric measures, two-day nonconsecutive dietary recall and biochemical indices using fasting blood samples. this study showed that most significant gender differences were in daily energy and nutrient intakes. males had more energy intake 1246.6 kcal compared to female 1043.1 kcal and consumed more carbohydrate 168.1 g, protein 39.7 g, total vitamin a 12229.7 µg, vitamin c 22.3 mg, iron 6.87 mg, and zinc 4.9 mg food sources. older males had better status indices for anemia than did older females (p=0.009). older persons who coresided with family had better dietary nutrient intakes than those living in nuclear families, except for protein, total vitamin a and zinc food sources. there are gender differences in energy, dietary nutrient intake, nutritional status and health status in free-living elderly. both genders were deficient in all macronutrients and selected micronutrients, especially iron and zinc. keywords : gender, nutritional intake, healthy elderly introduction the rapid increase in the number of the elderly population due to longevity has been dramatic throughout the world. the percentage of the elderly population over 60 years old will be doubled in 2025, and around 80% of the elderly will be in developing countries.(1,2) indonesia is one of the nations with a large population of aging people, thus maintaining a healthy physical and mental status in the elderly population and will be one of the crucial aspects 114 kusumaratna nutritional intake in elderly activity as dominant determinants of quality of life (qol) and well-being.(13) a balanced diet and variety in nutritional intake will ensure that the body has adequate nutrients. because nutrition is a modifiable risk factor, attempts should be made to devise p r e v e n t i v e n u t r i t i o n a l s t r a t e g i e s a i m e d a t improving the quality of life and consequently reducing the utilization of unnecessary health care resources. the purpose of the present study w a s t o d e t e r m i n e t h e p a t t e r n s o f g e n d e r differences in nutrient intake and status among healthy free-living elderly. methods study design t h e s t u d y w a s c o n d u c t e d a s a c r o s s sectional study at one public health center area in south jakarta, and the data were collected from july to september 2007. subjects the subjects were aged 60 years and over and living at mampang prapatan district south jakarta. the study area was chosen because of p r e v i o u s a n d c u r r e n t s t u d i e s o n t h e h i g h numbers of inactive members of posyandu lansia (integrated health care for the elderly) in district health centers of south jakarta. the total population of mampang prapatan district was approximately 103,136 persons with an estimated 15,9% (16,415) of residents aged 60 years and above. most of elderly had a moderate to low social economic background. st u d y p a r t i c i p a n t s w e r e r e c r u i t e d b y announcements made in the district health center and in sub-district public health centers that were under the jurisdiction of the district health center, through cadres in the community where the study was conducted. to identify eligible elderly subjects, we performed a survey in eight hamlets to collect a list of names of the of public health challenges. the elderly have a d e c r e a s e d r e s i s t a n c e a g a i n s t d i s e a s e , a n d g e n e r a l l y h a v e i n a d e q u a t e n u t r i t i o n a n d inadequate financial and social support. most of them face risks of disease and unhealthy environment. the incidence of infections and chronic disease, such as atherosclerosis and diabetes mellitus, have been shown to rise with increasing age. older persons also have the risk of low nutrient intake, both for macronutrients or micronutrients. a proportion of the elderly have deficiencies of several micronutrients, such as vitamin e, vitamin c, folic acid, iron and zinc, which have an important role in maintaining the immune function. (2,3) most studies on health and nutritional status among older persons have been conducted in developed countries and very few in developing countries. in indonesia there are limited data on the elderly population as one of the high risk targets that have been reported. several studies among the u r b a n e l d e r l y i n j a k a r t a h a v e r e p o r t e d deficiencies of iron, thiamine and folic acid intake(4) and a high prevalence of low body mass index.(5-7) intake of high quality nutrients will ensure that the body has sufficient nutrients to maintain its function of preventing deficiency and delaying the onset of chronic diseases. poor nutritional status in older people is multifaceted and includes the physiological, psychological and social changes associated with aging that a f f e c t f o o d i n t a k e , b o d y w e i g h t a n d a r e exacerbated by the occurrence of illness. g e n d e ra s s o c i a t e d d i f f e r e n c e s i n nutritional status and health complaints among o l d e r p e r s o n s h a v e b e e n n o t e d i n s e v e r a l studies.(8-12) thus gender should be considered to be a determinant factor. recently, there has been a shift in the concept of healthy and successful aging, from a focus on number of years to the level of quality of life. this shift has introduced to lead the healthy lifestyle, especially food intake and habitual physical 115 elderly who were eligible according to the following inclusion criteria: participants had to be 60 years or older (verified by their national identification card (ktp) or other papers), h e a l t h y a n d f r e e l i v i n g , a b l e t o v e r b a l l y communicate and willing to participate. the subjects were a part of the “daily multi m i c r o n u t r i e n t s s u p p l e m e n t a t i o n i n u p p e r respiratory infection” (supel) study. a total of 298 elderly subjects was enrolled on this study. during home visits we provided the subjects with detailed information on the study, and if they agreed to participate in the study, they were invited to join and sign a written informed consent form. a face-to-face interview for each participant was conducted by the cadres a s t r a i n e d i n t e r v i e w e r s . e a c h c a d r e w a s responsible for one hamlet area (around 35 elderly subjects) to obtain data on the field study and assist in filling out the questionnaire. data collection and measurement d e m o g r a p h i c a n d s o c i a l e c o n o m i c characteristics, health status, and dietary intake were collected from each participant, using a pre-tested questionnaire. the demographic indicators included age and gender of the participant. social economic status (ses) of the participant was indicated by years of schooling, history of past occupation and information on financial support. health status the self-reported history of health status w a s d i v i d e d i n t o t w o c a t e g o r i e s : ( i ) degenerative diseases and (ii) acute infection. for the category of degenerative diseases, the r e s p o n d e n t s w e r e a s k e d i f t h e y h a d b e e n suffering from hypertension, diabetes mellitus o r r h e u m a t i s m o r a c o m b i n a t i o n o f t h e s e diseases. for acute infections, the participants were asked if they had suffered from upper respiratory tract infection, diarrhea or fever or a combination of the two, in the last month as reference period. dietary assessment dietary intake data were collected by a trained nutritionist through a home visit to each elderly participant in the initial two weeks of the study. the dietary intake was assessed using a prospective 2-day non-consecutive and semiquantitative food frequency questionnaire.(14) each participant was asked to recall all the foods and drinks consumed the day before (for 24-h recall) and a frequency checklist of foods was used to estimate the regular daily intake of foods, with the last month as reference period (for sqffq). both assessments included dietary intake o f f o o d s o u r c e s o f m a c r o n u t r i e n t s a n d micronutrients by describing the foods and portion sizes in as much detail as possible. threedimensional food models were used as an aid in recognizing several foods and portion sizes, on the assumption of the presence of memory impairment in older persons. portion sizes were c o r r e l a t e d t o s t a n d a r d p o r t i o n s u s i n g t h e reference standard of indonesian foods. dietary adequacy was calculated based on indonesian recommended daily allowance (rda), and the indonesian food composition tables were used to convert foods into nutrients. nutrient analysis was performed by the nutrisurvey for windows program according to erhardt et al. (2005).(15) anthropometric assessment anthropometric assessment methods were used to measure weight, height (derived kneeheight by standard equation) and waist-hip ratio. at the time of measurement, the elderly wore minimal clothing. a portable scale was used to measure weight to the nearest 0.1 kg (platform model electronic weighing scale seca 770 a l p h a , h a m b u rg , g e r m a n y ) . st a t u r e w a s measured to the nearest 0.1 cm with a caliper f o r k n e e h e i g h t ( r o s s c a l i p e r, u s a ) a n d univ med vol.27 no.3 116 kusumaratna nutritional intake in elderly c a l c u l a t e d w i t h c h u m l e a ’s e q u a t i o n . t h e subjects were seated with the knee bent at 900 and the foot at 900 relative to the leg.(16) body mass index (bmi) was calculated as the weight (kg) divided by the square of the height (m2). for asian populations, bmi is c l a s s i f i e d i n t o t h e f o l l o w i n g c a t e g o r i e s : underweight (<18.5 kg/m2), normal (18.5 – 22.9 kg/m2), overweight (23.0 – 27.5 kg/m2), and obese (>27.6 kg/m2).(17) biochemical assessment from each participant, 8 ml of fasting venous blood was drawn from the antecubital vein between 8 to 10 am for assessment of hemoglobin (hb), serum zinc and total serum protein concentrations. the blood sample was divided into two vacutainer tubes (becton dickinson), one tube receiving 3 ml of venous blood with added edta anticoagulant for determination of hemoglobin level, and the other tube 5 ml of blood without edta for zinc and total protein determination. the hemoglobin c o n c e n t r a t i o n b y t h e c y a n m e t h e m o g l o b i n method was used in screening for anemia. for serum zinc and protein determination, serum was separated out from the 5 ml of venous blood by centrifugation at 3500 rpm for 1 0 m i n u t e s . s u b s e q u e n t l y t h e s e r u m w a s divided into 2 tubes, (i) for serum zinc, 1 ml of serum was taken with a special pipette and tube to avoid contamination (all tubes used had to be free of trace elements) and stored at or below -200c; and (ii) for total serum protein, 500 ì m l of serum was used. f o r d e t e r m i n a t i o n o f s e r u m z i n c concentration, the serum sample was centrifuged at the maximum speed of 15,000 rpm for 15 minutes and analyzed by atomic absorption s p e c t r o f o t o m e t r y ( a a s ) a c c o r d i n g t o t h e manufacturer’s instructions (perkin elmer). the aas instrument used was gbc 933 aa, with 1000 mg timisol zinc standard reagent (merck 1.09953). the resulting absorbance was read using a zn lamp with ë = 213.9 nm. total serum protein measurement was conducted on the 500 ìml serum sample using a t r x 7 0 1 0 a u t o m a t i c a n a l y z e r. n o r m a l threshold values used in this study were as follows: hemoglobin >12 g/l for the female elderly and >13 g/l for the male elderly;(18) for both genders: zinc status >10.7 µmol/l;(13,19) total protein range 6.4-8.3 g/dl and albumin range 3.4-4.8 g/dl (prodia clinical pathological laboratory cut-off values). ethical considerations before the study, the selected elderly subjects and their family were informed by the cadres about the purpose and protocol of the study. assurance was given to the subjects that their participation was voluntary, and that they could at any time withdraw from the study w i t h o u t a n y s a n c t i o n . i f t h e y a g r e e d t o participate in the study, they were invited to join and return the written informed consent form (legalized by signature for literate subjects or by thumb-print for illiterate subjects). the protocol and informed consent form had been approved by the ethical committee of the medical faculty, trisakti university. statistical analysis descriptive statistics were calculated and expressed as mean and standard deviation (for a normal distribution), median and range (for other k i n d s o f d i s t r i b u t i o n ) , a n d p r o p o r t i o n s . significant differences in general characteristics, a n t h r o p o m e t r i c , d i e t a r y a n d b i o c h e m i c a l measures between females and males were analyzed using independent t, chi square and m a n n w h i t n e y t e s t s , d e p e n d i n g o n t h e normality of the distribution. a p value of less than 0.05 (p < 0.05) was considered to be statistically significant. data analysis was performed using spss for windows version 15. 117 variables female (n=189) male (n=109) p-value mean age (sd) * range 64.8 (4.65) 60 83 65.1 (4.53) 60 85 0.351 educational background (%) ** none 69 (36.5) 11 (10.1) 6 yr education 87 (46.0) 55 (50.5) > 9 yr education 33 (17.5) 43 (39.4) 0.000 history of occupation (%) ** entrepreneur 10 (5.3) 22 (20.2) laborer 3 (1.6) 25 (22.9) government employee merchant unemployed 4 (2.1) 26 (13.8) 146 (77.2) 16 (14.7) 12 (11.0) 34 (31.2) 0.000 financial support (%) ** regular 132 (69.8) 68 (62.4) non-regular 57 (30.2) 41 (37.6) 0.187 health-fund sources (%) ** insurance 17 (9.0) 17 (15.6) non-insurance 172 (91.0) 92 (84.4) 0.084 note: * mann whitney test; **chi-square test table 1. baseline characteristic of the 60 to 85 year old female and male free-living elderly results a total of 109 male and 189 female elderly subjects were enrolled in the study. selected characteristics of the free-living elderly are presented in table 1 according to gender. the subjects in the study area were mostly f e m a l e s ( a p p r o x i m a t e l y 6 3 % ) . m e a n a g e between genders was slightly different, with a maximum age of 83 years for females and 85 years for males, and males tending to be older than females. the proportion of literate persons and the history of occupation as young adults were significantly different (p<0.05) between female and male elderly. almost 36% of female s u b j e c t s w e r e i l l i t e r a t e a n d 7 7 % w e r e unemployed as young adults, and carried out only household work as their daily activity. there were no differences between genders for sources of financial support, the majority obtaining financial support from their children for their monthly expenditures on food, health services and household needs. mean body mass index (bmi) values were identical in both genders, and the majority of subjects was categorized as having a normal bmi (table 2). table 2. anthropometric variables of the 60 to 85 year old female and male free-living elderly note: *independent-t test variables female (n= 189) mean (sd) male (n =109) mean (sd) p-value weight (kg) * 49.1 (10.5) 56.8 ( 9.8) 0.000 height (cm) * 151.1 (4.2) 161.3 (4.4) 0.000 bmi (kg/m2) * 21.6 (4.3) 21.9 (3.6) 0.529 waist circumference (cm) * 78.1 (11.7) 81.2 (10.7) 0.022 univ med vol.27 no.3 118 kusumaratna nutritional intake in elderly variables female (n=189) male (n=109) p-value degenerative diseases (%) * hypertension 40 (21.2) 15 (13.8) 0.113 diabetes mellitus 12 (6.3) 7 (6.4) 0.980 rheumatism 36 (19.0) 23 (21.1) 0.668 morbidity in past of 1 month (%) * upper respiratory infection 62 (32.8) 36 (33.0) 0.968 diarrhea 30 (15.9) 5 (4.6) 0.004 fever 41 (21.7) 23 (21.1) 0.905 table 4 . health status of the 60 to 85 year old female and male free-living elderly note : *chi–square test variables female n (%) male n (%) p-value size of house (m2) * < 80 m2 111 (58.7) 71 (65.1) > 80 m2 78 (41.3) 38 (34.9) 0.275 status of house * own house 150 (79.4) 90 (82.6) does not own house 39 (20.6) 19 (17.4) 0.501 co-residence * nuclear family 16 (8.5) 17 (15.6) extended family 173 (91.5) 92 (84.4) 0.059 table 3. pattern of living of the 60 to 85 year old female and male free-living elderly note: * chi-square test overall, the proportion of underweight (bmi <18.5 kg/m2) among female and male subjects was 27% and 20%, respectively. the proportions of overweight (bmi 23.0–27.5 kg/ m2) were different between females (22%) and males (28%). overweight was more prevalent in males than in females, but obesity occurred more in females (13%) than in males (8%). moreover, there were significant differences in weight, height and waist-hip ratio among f e m a l e s a n d m a l e s ( p < 0 . 0 5 ) . m a l e s w e r e significantly heavier, taller and had a higher waist-hip ratio than females older. both genders have a high risk of cardiovascular disease due to hip-waist circumference ratio. the pattern of living of the subjects is shown in table 3. no differences were found b e t w e e n g e n d e r s o n s i z e o f h o u s e , l i v i n g arrangement and co-residence. the majority of older persons lived in a house of less than < 80 square-meters and preferred to co-reside with their extended family (siblings or relatives). of those who lived in a nuclear family, 4% lived alone; males tended to live in a nuclear family in comparison to females. the health status of the elderly is shown in table 4. the majority of self-reported health problems among the participants were not significantly different between females and males. most of them had disorders that were diagnosed as degenerative diseases, namely hypertension, diabetes mellitus and rheumatism. however, a higher proportion of the females reported suffering from degenerative diseases than did the males, i.e. 46.5% and 41.3% respectively. 119 variables female (n=189) male (n=109) p-value energy (kcal) %< rda 1043.16 (345.88)* 65 1246.65 (437.71)* 61 0.000 carbohydrate (g) %< rda 142.62 59.4 168.09 55 0.000 protein (g) %< rda 33.98 68 39.72 66 0.000 vitamin a (µg re) %< rda 1122.95 0 1229.70 0 0.626 vitamin e activity (mg) % < rda 0.00 100 0.00 100 0.428 vitamin c (mg) % < rda 14.40 19 22.30 25 0.048 fe (mg) % < rda 5.81 48 6.87 53 0.134 zn (mg) %<rda 3.89 41 4.93 37 0.001 table 5. daily energy and nutrient intake by the 60 to 85 year old female and male free-living elderly note : * mean (sd) no gender difference was found in complaints of upper respiratory infection, diarrhea, and fever in the past month. the prevalence of diarrhea appeared mostly to be experienced significantly by female elders (p<0.05). in general, the differences in proportion of morbidity of acute illness between females and males were quite high. table 5 lists the energy intake among the subjects. almost 93% of older persons of both genders were found to have a lower energy intake compared to the indonesian rda (angka kecukupan gizi). females had a lower reported intake of energy and nutrients than males. the energy intake of the females was 65% of rda and that of the males was 61%, which affected their carbohydrate and protein intake. females had significantly lower dietary intakes of energy (p=0.000), carbohydrate (p=0.000), protein ( p = 0 . 0 0 0 ) , v i t a m i n c ( p = 0 . 0 4 8 ) a n d z i n c ( p = 0 . 0 0 1 ) , i n c o m p a r i s o n t o m a l e s . b o t h g e n d e r s t e n d e d t o h a v e a n i n a d e q u a t e micronutrient intake, except for vitamin a. variables female (n=188) male (n=108) p-value zinc (µmol/l) zinc deficiency (%) 11.05 ± 2.1 59 (31.2) 12.36 ± 5.3 23 (21.1) 0.06 hemoglobin (g/dl) anemia status (%) 12.67 ± 1.4 52 (27.5) 14.17 ± 1.5 46 (42.2) 0.009 protein status (g/dl) (%) <6.4 (low) 55 (29.3) 24 (22.2) 6.4 8.30 (normal) 94 (50.0) 70 (64.8) >8.30 (high) 39 (20.7) 14 (13.0) 0.043 albumin status (g/dl) (%) <3.4 (low) 43 (22.9) 14 (13.0) 3.4 4.8 (normal) 142 (75.5) 84 (77.8) >4.8 (high) 3 (1.6) 10 (9.3) 0.037 table 6. biochemical laboratory measurements of the 60 to 85 year old female and male free-living elderly univ med vol.27 no.3 120 kusumaratna nutritional intake in elderly variables* 60-70 years (n=242) > 70 years (n=56) p value energy (kkal) 1069.95 (843.15; 1342.90) 1022.30 (792.07; 1257.92) 0.356 carbohydrate 153.71 (123.52; 194.91) 141.50 (122.04; 181.56) 0.257 protein 35.20 (26.20; 48.40) 35.58 (27.89; 44.94) 0.817 total vitamin a (µg re) 1155.0 (804.31; 1863.28) 1364.75 (757.46; 1840.30) 0.784 total vitamin e activity (mg) 0.00 (0.00; 0.09) 0.00 (0.00; 0.09) 0.020 vitamin c 17.21 (6.45; 65.33) 15.55 (5.64; 57.88) 0.797 fe (mg) 6.28 (3.71; 10.59) 6.68 (4.06; 14.35) 0.310 zn (mg) 4.23 (3.06; 5.65) 4.14 (3.25; 5.36) 0.975 variables* nuclear family/alone (n=33) extended family (n=265) p value energy (kkal) 1098.26 (919.47; 1298.38) 1056.11 (827.93; 1322.37) 0.743 carbohydrate 154.58 (135.65; 193.26) 149.88 (121.3; 190.58) 0.462 protein 33.75 (26.83; 45.10) 35.46 (26.37; 48.14) 0.782 total vitamin a (µg re) 1087.97 (792.08; 1865.35) 1214.90 (804.08; 1851.00) 0.813 total vitamin e activity (mg) 0.00 (0.00; 0.09) 0.00 (0.00; 0.09) 0.946 vitamin c 25.05 (6.40; 43.35) 16.90 (6.25; 65.60) 0.702 fe (mg) 6.89 (3.50; 10.71) 6.22 (3.81; 11.44) 0.807 zn (mg) 3.96 (2.95; 5.47) 4.18 (3.12; 5.61) 0.623 note :* mann whitney test, median (25th percentile; 75th percentile) table 8. nutrient intake by age group and co-residence variables 60-70 years (n=242) > 70 years (n=56) p value bmi underweight 57 (23.6) 15 (26.8) normal 96 (39.7) 24 (42.9) overweight 60 (24.8) 12 (21.4) obese 29 (12.0) 5 (8.9) 0.826 table 7. body mass index by age group and gender the majority of biochemical indices for serum zinc, hemoglobin level and total serum protein were categorized as normal. however, z i n c d e f i c i e n c y i s m o r e p r e v a l e n t a m o n g females than among males (31% vs. 21%), while anemia was more prevalent among males (42% vs. 27.5%). low protein and albumin status was more prevalent among females than males (p < 0.043 and < 0.037 respectively). thus there w e r e g e n d e r d i ff e r e n c e s i n m i c r o n u t r i e n t deficiency and protein status (table 6). there were no significant differences between nutritional status in each age group. however, 23.6% of the younger elderly (< 70 yr) and 26.8% of the more advanced age group (>70 yr) were in the bmi category of less than 18.5 kg/m2 (underweight). overweight and obese were more prevalent among the younger subjects (table 7). there was no significant difference in nutrient intake between age groups, except for food sources of vitamin e (p<0.05); both age groups have lower dietary intakes. however, the younger subjects had higher intakes of energy, carbohydrate, total vitamin a, vitamin c and food sources of zinc than the older ones (table 8). 121 no significant difference was found in nutrient intakes between the two categories of co-residence. older persons who lived in a nuclear family tended to have lower nutrient intakes of protein, total vitamin a and food sources of zinc, compared with those who lived in an extended family. discussion this study presents information on gender differences in patterns of nutrition, health condition and living condition among healthy free-living elderly residents in an urban area of jakarta. sixty-three percent of the population a r e f e m a l e s a s t h e m a j o r i t y o f t h e o l d e r p o p u l a t i o n , a n d p r e s u m a b l y f e m a l e s w i l l c o n t i n u e t o b e t h e m a j o r i t y. i n f o r m a t i o n regarding years of schooling and history of past occupations indicates that most of the elderly f e m a l e s a r e i l l i t e r a t e ( 3 7 % ) , w i t h l e s s opportunity or participation in economically remunerative work in the past. this is the same phenomenon that has been predicted in the un r e p o r t ( 2 0 0 2 ) ( 1 9 ) a n d t h e r e p o r t o n “ o l d e r population in indonesia” by unfpa (2006),(1) in that elderly women constitute the majority of the older population in most countries. this fact could be interpreted to mean that the elderly f e m a l e s a r e d e p e n d e n t a n d v u l n e r a b l e t o financial and economic security. most of the older persons receive regular financial support from their pension and from their children to pay for their monthly expenditure. the findings of this study on the pattern of living are that most of the elderly parents live in their own household in co-residence with their family. as in most asian cultures, the norm is that the older persons co-reside with their family members and are cared for by them. coresidence is the main component of family s u p p o r t a n d t h e b e s t a s p r o x y i n d i c a t o r. depending on the situation, most older persons were co-residing with daughters, younger siblings or other close relatives.(20,21) however, the percentage of older persons living without children, whether alone or with their spouse, is very small. in terms of gender differences, coresidence with children had similar patterns in females and in males. from their self-reported health status, older females were at higher risk of illness for both degenerative diseases and acute infections, as compared to older males. older persons are known to be more vulnerable to a higher incidence of morbidity; however, the higher incidence of chronic diseases or acute infections will alter the need for health services to manage the diseases of old age. although this will have an impact on nutritional status of the elderly, in this study there were no differences between younger and older subjects in suffering from illness, but the younger elderly were more vulnerable than the older ones. regarding anthropometric measures, the present study demonstrates that although most members of the elderly population are of normal weight, 29% of them has under-nutrition. this prevalence shows that under-nutrition is quite high among older persons, and this is identical to the results of other studies that had been c o n d u c t e d a m o n g t h e o l d e r p o p u l a t i o n worldwide.(12,13,22) no difference was found in the proportions of bmi categories between younger and older subjects. in this study it a p p e a r s t h a t t h e e l d e r l y p o p u l a t i o n h a s a predisposition for overweight and obesity. obesity and high waist circumference values in t h e o l d e r p e r s o n c o n s t i t u t e r i s k f a c t o r s predisposing to degenerative disease, such as heart disease and diabetes. t h e p r e s e n t s t u d y d e m o n s t r a t e s a n inadequacy of dietary intake for energy and selected nutrients, resulting in a high prevalence of risk of malnutrition based on gender. the r e c o m m e n d e d d a i l y e n e r g y i n t a k e i s univ med vol.27 no.3 122 kusumaratna nutritional intake in elderly approximately 2050 kcal for older males and 1600 kcal for older females (based on the i n d o n e s i a n r d a ) . t h e l o w e r t h a n recommended levels of energy and selected nutrient intakes proves that there is a tendency for low consumption in this study. the older person has a risk of inadequate micronutrient intake, except for vitamin e. the main factors known to lead to decreased nutritional intake are chronic diseases, declines in absorption and m e t a b o l i s m i n t h e g a s t r o i n t e s t i n a l t r a c t , d e p r e s s i o n a n d l o w i n c o m e , ( 2 , 2 3 ) w h i c h subsequently could lead to decreased resistance to infection in the elderly. most of the mean values of nutrient intake were below the indonesian rda for the older person. this low energy intake did not adversely affect the protein intake. the data on regular daily consumption in this study (not shown) indicate that the majority of the protein intake is obtained mainly from a plant based diet, such as soybean products (tempeh and tofu). similar findings in kusumaratna et al. (2005)(24) showed that tempeh and tofu were the major sources of protein intake for daily consumption (68%) in most of elderly persons. our results regarding the low dietary intake of macronutrients and selected micronutrients in both genders are confirmed by the biochemical indices of lower serum protein and albumin levels, and higher anemia and zinc deficiency status. in both g e n d e r s , t h e p r e v a l e n c e o f a n e m i a w a s significantly higher in subjects with lower biochemical indices. anemia was found in 42% of males and 28% of females. hemoglobin l e v e l s w e r e b e l o w t h e c u t o f f p o i n t recommended by who in 5.4% and 4.4% of aged males and females, respectively.(11) anemia is due mostly to low intake of food sources of iron and/or low bioavailability of dietary iron, based on the patterns of regular consumption of staple food. the decrease in hemoglobin levels that occurs with aging is generally not due to the commonly recognized causes of anemia, such as iron deficiency or chronic disease, but probably to a decrease in the production of blood cells that might related to the process of aging.(25,26) similar predisposing factors due to zinc deficiency have been found in this study. from the proportion of daily nutrient intakes (table 4) it is apparent that elderly males tend to have lower dietary intakes of vitamin c a n d f o o d s o u r c e s o f i r o n a s c o m p a r e d t o females. in both genders the low intake of animal products, known to be the best food sources for iron and zinc, might have resulted in low dietary intake analysis and biochemical indices. this finding is supported by previous studies, namely the european zenith study(27), the study on free-living elderly in japan(28) and the study on free-living elderly in southjakarta.(7) among the micronutrients, zinc is known to be essential in the elderly, for its impact on immune functions. in the present study, among the older population we have observed gender differences in zinc status, total protein and albumin concentrations, with a higher number of deficiencies in females. conclusions among healthy free-living elderly there was a high prevalence of undernourished and lower dietary intakes of macronutrients and micronutrients. responsiveness to the risks a s s o c i a t e d w i t h t h e s e d e f i c i e n c i e s a m o n g healthy free-living elderly should be increased for a healthy aging of the elderly. references 1. unfpa. population ageing in east and south-east asia: current situation and emerging challenges. papers in population ageing no.1. unfpa, thailand, 2006. 123 2. meydani a, ahmed t, meydani sn. aging, nutritional status and infection in the developing world. nutr rev 2005; 63: 233-46. 3. lesourd bm. nutrition: a major factor influencing immunity in the elderly. j nutr health aging 2004; 8: 28-35. 4. juguan ja, lukito w, schultink w. thiamine deficiency is prevalent in selected group of urban indonesian elderly people. j nutr 1999; 129: 36671. 5. rabe b, thamrin mh, gross r, solomons nw, schultink w. body mass index of the elderly derived from height and from armspan. asia pac j clin nutr 1996; 5: 79-83. 6. budiman, djaya n. status gizi dan pola makan kelompok lanjut usia di dki jakarta in: gambaran kesehatan pada masyarakat lanjut usia di dki jakarta dan hubungan dengan determinannya. jakarta: monograph of fakultas kedokteran unika atmajaya; 2007. p. 74-88. 7. kusumaratna rk, salim oc, sudharma is. dietary intake and zinc status differences between male and female elderly of south jakarta community. universa medica 2007; 26: 179-85. 8. bates cj, prentice a, finch s. gender differences in food and nutrient intakes and status indices from the natonal diet and nutrition survey of people aged 65 years and over. eur j clin nutr 1999; 53: 694-9. 9. ledikwe jh, wright hs, mitchell dc, jensen gl, friedmann jm, still cd. nutritional risk assessment and obesity in rural older adults: a sex difference. am j clin nutr 2003; 77: 5518. 10. foote ja, giuliano ar, harris rb. older adults need guidance to meet nutritional recommendations. j am coll nutr 2000; 19: 62830. 11. olivares m, hertrampf e, capurro mt, wegner d. prevalence of anemia in elderly subjects living at home: role of micronutrient deficiency and inflamation. eur j clin nutr 2000; 54: 834-9. 12. kabir zn, ferdous t, cederholm t, khanam ma, streatfied k, wahlin k. mini nutritional assessment of rural elderly people in bangladesh: the impact of demographic, socio-economic and health factors. public health nutr 2006; 9: 968-74. 13. sempertegui f, estrella b, elmieh n, jordan m, ahmed t, rodriquez a, et al. nutritional, immunological and health status of the elderly population living in poor neighborhoods of quito, ecuador. brith j nutr 2006; 96: 845-53. 14. gibson rs. principles of nutritional assessment. 2nd ed. new york : oxford university; 2005. 15. erhardt jg, mack h, sobeck u, biesalski hk. βcarotene and α-tocopherol concentration and antioxidant status in buccal mucosal cells and plasma after oral supplementation. bmj 2002; 87: 471-75. 16. lohman tg, roche af, martorell r. anthropometric standardization reference manual. champaign, illinios: h. human kinetics books; 1988. 17. world health organization. appropriate body mass index for asian populations and its implications for policy and intervention strategies. lancet 2004; 363: 157-63. 18. world health organization. nutritional anaemias. who tech rep ser no. 405. geneva: world health organization; 1988. 19. united nation. the ageing of the world’s population. geneva: united nation; 2002. 20. arifin en. living arrangements of older persons in east java, indonesia. asia pac pop j 2006; 21: 93-112. 21. mujahid g. population ageing in east and southeast asia, 1950-2050: implication for elderly care. asia pac pop j 2006; 19: 25-43. 22. nyaruhuda cn, msuya jm, matrida e. nutritional status, functional ability and food habits of institutionalized and non-institutionalized elderly people in morogoro region, tanzania. east afr med j 2004; 81: 248-53. 23. wahlqvist ml, kouris-blazos a. requirements in maturity and ageing. in: wahlqvist ml, editor. food and nutrition australasia, asia and the pacific. 2nd ed. melbourne: allen & unwin pty ltd; 1997. p. 344-66. 24. kusumaratna rk, salim o, hidayat a. status zinc dan selenium pada masyarakat lanjut usia di dki jakarta. in: gambaran kesehatan pada masyarakat lanjut usia di dki jakarta dan hubungan dengan determinannya. jakarta: monograf fakultas kedokteran unika atmajaya; 2005. p. 89-97. 25. fleming dj, jacques pf, tucker ki, massaro jm, d’agostino rb, wilson pwf, et al. iron status of the free-living elderly framingham heart study cohort: an iron-replete population with a high prevalence of elevated iron stores. am j clin nutr 2001; 73: 638-46. 26. sibai am, zard c, adra n, baydoun m, hwalla n. variations in nutritional status of elderly men and women according to place of residence. gerontol 2003; 49: 215-4. univ med vol.27 no.3 124 kusumaratna nutritional intake in elderly 27. sanchez ma, favier ih, meunier n, toti e, zaccaria m, brandolini-bunlon m, et al. zinc intake and status in middle-aged and older european subjects: the zenith study. eur j clin nutr 2005; 59: s37-s41. 28. kogirima m, kurasawa r, kubori s, sarukura n, nakamori m, okada s, et al. ratio of low serum zinc levels in elderly japanese people living in the central part of japan. eur j clin nutr 2007; 61: 375-81. rita k 205 *bagian ilmu penyakit dalam fakultas kedokteran universitas trisakti **alumni fakultas kedokteran universitas trisakti korespondensi dr.nurman phd, sp.pd, kgeh bagian ilmu penyakit dalam fakultas kedokteran universitas trisakti jl.kyai tapa no.260 grogol jakarta barat 11440 telp.5677231 eks.2707 email: nurman_achmad@yahoo.com universa medicina 2007; 26: 205-15 universa medicina oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 perlemakan hati non alkoholik a. nurman* dan margareta a. huang** abstrak spektrum patologi klinik dari perlemakan hati non alkoholik (phna) terentang dari simple steatosis sampai non alkoholic steatohepatitis (nash). simple steatosis memiliki perjalanan klinik yang relatif jinak, namun nash dapat berkembang ke sirosis dan karsinoma hepatoseluler. phna umumnya asimtomatik dan harus dicurigai pada pasien-pasien dengan obesitas abdominal dan diabetes melitus tipe 2. peningkatan alanine aminotransferase (alt) serum dan/atau gambaran perlemakan hati pada usg sering kali merupakan temuan pertama yang mengarahkan diagnosis ke phna. diagnosis phna ditegakkan setelah menyingkirkan penyakit hati kronis yang lain; diagnosis definitif hanya dibuat dengan biopsi. peningkatan alt umumnya tidak melebihi 5 kali batas atas nilai normal. adanya ikterus, bilirubinuria dan demam juga menyingkirkan diagnosis phna. sebagian besar steatosis hepatik tidak berkembang menjadi fibrosis atau steato hepatitis, 30-40% akan menjadi fibrosis dalam periode 4 tahun dan +15% akan berkembang menjadi sirosis. penatalaksanaan phna tidak ditargetkan pada hepar saja, melainkan harus holistik, berpusat pada perbaikan resistensi insulin dan gangguan metabolisme terkait, yang dimulai dengan modifikasi gaya hidup. berat badan harus diturunkan secara bertahap.vitamin e dan asam ursodeoksikolat diberikan karena dapat menurunkan alt. metformin dan/atau rosiglitazone dapat diberikan pada pasien tanpa diabetes yang nyata, karena obat-obat ini tidak menyebabkan hipoglikemia, dan dapat meningkatkan sensitivitas insulin. perburukan fungsi hati juga harus dicegah dengan menghindari zat-zat hepatotoksik. kata kunci : perlemakan hati, obesitas abdominal, diabetes tipe 2 206 nurman, huang perlemakan hati non alkoholik *department of internal medicine medical faculty trisakti university **alumnus of medical faculty trisakti university correspondence: adr.a.nurman phd, sp.pd, kgeh department of internal medicine medical faculty trisakti university jl.kyai tapa no.260 grogol, jakarta barat phone. 5677231 ext.2707 email: nurman_achmad @yahoo.com universa medicina 2007; 26: 205-15 the cilinicopathologic spectrum of non alcoholic fatty liver disease (nafld) ranges from simple steatosis to non alcoholoic steatohepatitis (nash) .simple steatosis has a relatively benign cilinical course,but nash can progress to cirrhosis and hepatocellular carcinoma. nafld is often asymptomatic and its presence must always be considered in patients with abdominal obesity and type 2 diabetes mellitus. elevation of serum alanin aminotransferase (alt) level and/or fatty liver appearance on ultrasound are often the 1st clues toward the diagnostic of nafld. the diagnostic of nafld is established after excluding other causes of chronic liver diseases, such as viral hepatitis, alcoholic liver disease, drug-induced liver disease, etc. however, only liver biopsy establishes the definitive diagnosis. alt elevation usually does not exceed five times upper normal limit. should alt elevation exceed five times upper normal limit, other diagnosis must be considered. management of nafld is not solely directed at nafld itself, but should focus on improving insulin resistance and related metabolic dearangements as well, which start with life-style modification. body weight should be reduced gradually by diet and exercise, as exercise increase glucose uptake in skeletal muscle cells. vitamine e and ursodeoxylate acid are often administrated due to their economical cost and capable of reducing alt level, eventhough histological improvement may not always take place. metformin and/or rosiglitazone can be given to patients without overt diabetes as these drugs do not cause hypoglycemia but improve insulin sensitivity. avoiding hepatotoxic agents is necessary to prevent further deterioration of liver function. keywords : fatty liver, abdominal obesity, diabetes type 2 non alcoholic fatty liver disease a. nurman*a and margareta a. huang** abstract pendahuluan b e b e r a p a t a h u n b e l a k a n g a n i n i perlemakan hati nonalkoholik (phna) mulai mendapatkan banyak perhatian sebagai suatu kesatuan klinis. resistensi insulin adalah faktor penting yang mendasari phna. (1) seiring dengan meningkatnya prevalensi dan insidens obesitas dan sindrom metabolik, prevalensi dan i n s i d e n s p h n a p u n s e m a k i n m e n i n g k a t . obesitas dan sindroma metabolik sudah menjadi suatu pandemi, tidaklah mengherankan (2,3) bahwa phna menjadi penyebab tertinggi penyakit hati kronis di negara-negara maju m a u p u n b e r k e m b a n g . ( 4 ) b a h k a n p h n a i n i dianggap sebagai komponen hati dari sindrom metabolik.(5) jaringan adiposa sendiri mampu memproduksi berbagai sitokin dan hormon yang disebut adipokin atau adipositokin. deposisi lemak, selain pada phna, juga didapatkan p a d a b e n t u k p e r l e m a k a n h a t i y a n g l a i n , m i s a l n y a p e r l e m a k a n h a t i a l k o h o l i k , perlemakan hati yang disebabkan malnutrisi kalori-protein, dan lain-lain. 207 epidemiologi prevalensi dan insidens pasti phna sejauh ini belum dapat ditentukan karena biopsi hati sebagai modalitas diagnosis definitif tidak m u d a h d i k e r j a k a n d a l a m b e r b a g a i s t u d i epidemiologis. pada sebagian besar penelitian, kadar alanin aminotransferase (alt) dan ultrasonografi digunakan sebagai modalitas utama untuk menegakkan diagnosis kerja phna, n a m u n m o d a l i t a s t e r s e b u t t i d a k m a m p u membedakan dua spektra histologis phna, y a i t u p e r l e m a k a n h a t i s e d e r h a n a ( s i m p l e steatosis) dan steatohepatitis nonalkoholik (nonalcoholic steatohepatitis/nash). diperkirakan bahwa 36,9% dari penduduk amerika serikat menderita pnha dan 5,7% menderita nash. prevalensi bervariasi menurut etnis di mana lebih tinggi pada orang kulit putih dan hispanik dan lebih rendah pada afroamerika. sebuah survei di amerika serikat(4) menunjukkan bahwa pada 5,4% dari 15.676 sampel didapatkan peningkatan alt yang tidak berhubungan dengan infeksi virus hepatitis, a l k o h o l i s m e d a n i ro n o v e r l o a d , d a n berhubungan dengan indeks massa tubuh (imt), lingkar pinggang, kadar trigliserida dan insulin p u a s a y a n g l e b i h t i n g g i , d i a b e t e s t i p e 2 , hipertensi, dan high density lipoprotein (hdl) yang lebih rendah. studi dionysos di italia utara menemukan bahwa prevalens abnormalitas tes fungsi hati yang diduga disebabkan oleh phna adalah lebih dari 5%,(3) sedangkan di cittanova, sebuah kota kecil di italia selatan, prevalens a b n o r m a l i t a s t e s f u n g s i h a t i y a n g d i d u g a disebabkan oleh phna adalah sebesar 3,1% dari seluruh populasi.(6) tampaknya perbedaan ini disebabkan oleh pola diet yang berbeda antara i t a l i a u t a r a d a n d a e r a h m e d i t e r e a n . d i indonesia, suatu penelitian di pinggiran kota jakarta(7) mendapatkan prevalens phna 30,6%, terbanyak pada usia pertengahan (37,2%). hal ini tidak berbeda dengan prevalens phna di negara maju. patogenesis phna p a t o g e n e s i s p h n a b e l u m t e r u n g k a p sepenuhnya. resistensi insulin adalah faktor y a n g m e n d a s a r i p h n a . p e n e l i t i a n y a n g dilakukan oleh marceau et al(8) menunjukkan bahwa semakin banyak komponen sindrom metabolik pada seorang pasien, semakin tinggi pula risiko steatosis pada pasien tersebut. di lain pihak, hanley et al (9) dalam penelitiannya mendapatkan bahwa kadar alt dan aspartat aminotransferase (ast) berkorelasi erat dengan insidens diabetes melitus tipe 2. meskipun demikian, resistensi insulin bukanlah satusatunya faktor penting pada phna. perlemakan hati timbul akibat kombinasi berbagai faktor, a n t a r a l a i n d i e t t i n g g i k a r b o h i d r a t y a n g mencetuskan sintesis de-novo asam lemak bebas di hati, stres oksidatif, fungsi mitokondria yang cacat dan sitokin-sitokin proinflamasi seperti adipokin yang dihasilkan oleh jaringan adiposa viseral.(10) ini meliputi (sitokin pro inflamasi seperti) interleukin-6 (il-6) dan tumor necrosis factor (tnf)-α sama seperti leptin, resistin dan juga adiponektin. adiponektin merupakan polipeptid yang bekerja sebagai antidiabetik dan antiaterogenik yang berkorelasi kuat dengan sensitivitas insulin sistemik. penurunan kadar adiponektin dalam plasma berkaitan dengan peningkatan imt, penurunan sensitivitas insulin, profil lemak dalam plasma yang aterogenik, p e n i n g k a t a n k a d a r p e t a n d a i n f l a m a s i d a n p e n i n g k a t a n r e s i k o u n t u k p e n y a k i t k a r d i o v a s k u l e r. o l e h k a r e n a i t u k a d a r adiponektin dapat digunakan sebagai suatu i n d i k a t o r u n t u k s i n d r o m a m e t a b o l i k . ( 11 ) timbunan lemak di sitoplasma menjadikan hepatosit lebih rentan terhadap agresi-agresi e k s t e r n a l , y a n g k e m u d i a n m e n c e t u s k a n apoptosis.(12) belakangan ini telah diteliti pula p e r a n f l o r a u s u s d a l a m p a t o g e n e s i s steatohepatitis. pertumbuhan bakteri usus yang berlebihan dapat memperberat stres oksidatif pada hepar,(13) karena proses fermentasi yang universa medicina vol. 26 no.4 208 nurman, huang perlemakan hati non alkoholik dilakukan bakteri menghasilkan banyak etanol. selain itu, lipopolisakarida yang dihasilkan b a k t e r i m e r a n g s a n g p e m b e n t u k a n s i t o k i n proinflamasi. p h n a t e r d i r i d a r i b e b e r a p a s p e k t r a histologis. pada awal penyakit ini didapatkan steatosis makrovesikular dengan pendorongan nukleus ke pinggir sel hepatosit. steatosis atau perlemakan hati ini terjadi akibat akumulasi trigliserida di hepar. trigliserida tersebut dibentuk oleh asam lemak bebas dari makanan maupun lipolisis perifer, dan juga secara de novo. adanya resistensi insulin meningkatkan lipolisis, sehingga lebih banyak asam lemak bebas yang ditranspor ke hepar. bila terjadi kerusakan sel-sel hati, terjadilah peradangan (steatohepatitis) yang diperantarai berbagai sitokin. stres oksidatif dipercayai sebagai pencetus steatohepatitis. secara histologis terlihat pembengkakan hepatosit (ballooning), sebukan sel neutrofil dan limfosit, dan kadangkadang tampak badan inklusi dalam sitoplasma, y a i t u b a d a n m a l l o r y. s e l a n j u t n y a , steatohepatitis dapat diikuti dengan fibrosis perisinusoidal, yang kemudian dapat meluas dan menjadi sirosis. perubahan histologis yang s e r u p a d i d a p a t k a n p a d a p e r l e m a k a n h a t i alkoholik. bila phna berlanjut ke sirosis, maka s t e a t o s i s b e r k u r a n g , b a h k a n m e n g h i l a n g , sehingga pada biopsi hati yang mengalami sirosis ini tidak tampak lagi perlemakan hati. sebagian besar sirosis kriptogenik diduga berasal dari phna ini. beberapa penelitian akhir-akhir ini mengindikasikan bahwa phna dan resistensi insulin dapat meningkatkan risiko k a r s i n o m a h e p a t o s e l u l a r. s t u d i s t u d i l o n g i t u d i n a l m a s i h d i b u t u h k a n u n t u k mengkonfirmasi hal ini. perjalanan penyakit steatosis hepatik merupakan penampilan yang paling sering dan pada sebagian besar pasien tidak berkembang menjadi fibrosis atau steatohepatitis.(14) insidens fibrosis pada pasien dengan phna adalah sama seperti yang tampak pada penyakit hati menahun dengan 30-40% menunjukkan fibrosis yang lanjut dan sekitar 15% menunjukkan sirosis.(8,13,14) studi tentang progres dari phna dari steatohepatitis ke steatohepatitis dengan fibrosis masih terbatas dan sebagian besar merupakan data retrospektif, dilaporkan pada 30-40% timbul fibrosis selama periode ±4 tahun.(15) bila nash berkembang menjadi sirosis, derajat steatosis berkurang dan bahkan dapat menghilang.demikian pula tanda tanda yang lain dari nash bisa berkurang dan p e m e r i k s a a n h i s t o l o g i h a t i d a p a t h a n y a menunjukkan bland inactive cirrhosis keadaan ini mengarah ke sirosis kriptogenik. banyak pasien dengan sirosis kriptogenik memiliki tanda t a n d a k i l i n i s d a r i s i n d r o m m e t a b o l i k . pengamatan akhir-akhir ini menunjukkan bahwa risiko dekompensasi hati pada pasien sirosis yang berhubungan dengan phna lebih rendah daripada sirosis dengan hepatitis c. sayang data yang menyangkut mortalitas pasien dengan phna sangat terbatas dan bersifat retrospektif. data yang terkini memberikan kesan bahwa s e s e o r a n g d e n g a n s i r o s i s k r i p t o g e n i k d a n karsinoma hepatoseluler memiliki resistensi insulin yang lebih besar ,yang memberikan kesan b a h w a p h n a d a n r e s i s t e n s i i n s u l i n b i s a menambah risiko untuk karsinoma hepatoseluler. dapat disimpulkan dari data mortalitas yang ada bahwa risiko kematian dalam 5-10 tahun dari steatosis atau nash kemungkinan adalah sangat rendah tetapi akan meningkat s e c a r a b e r m a k n a d e n g a n b e r k e m b a n g n y a fibrosis yang lanjut dan sirosis. gambaran klinis phna biasanya ditemukan secara tidak sengaja pada waktu pemeriksaan kesehatan berkala, baik dari gambaran usg maupun tes fungsi hati yang abnormal. mayoritas pasien tidak mempunyai keluhan. rasa tidak nyaman 209 di abdomen kanan atas dan kelelahan dapat saja dikeluhkan, namun keluhan-keluhan tersebut tidak spesifik untuk phna. phna berhubungan erat dengan resistensi insulin, maka obesitas abdominal sering ditemukan, demikian pula hipertensi, yang juga merupakan komponen sindrom metabolik. pada palpasi abdomen dapat ditemukan hepatomegali. ikterus dan stigmata sirosis tidak ditemukan, kecuali bila phna sudah sampai pada tahap sirosis. gambaran laboratorium p e n g u k u r a n k a d a r alt s e r u m a d a l a h m o d a l i t a s s k r i n i n g y a n g d i g u n a k a n u n t u k diagnosis phna secara presumtif pada banyak studi populasi. meskipun cukup berguna untuk skrining, pengukuran kadar alt serum ini tidak sempurna dalam hal sensitivitas dan spesifisitas. kadar alt yang normal dapat ditemukan pada pasien dengan steatohepatitis lanjut atau sirosis. sudah diketahui pula bahwa kadar alt tidak berkorelasi dengan derajat kerusakan hepar. selain itu, masih ada perbedaan mengenai batas atas kadar alt yang normal. pada umumnya, peningkatan alt pada phna tidak melebihi 5 kali batas atas nilai normal.(16) kadar ast juga meningkat, namun ast kurang spesifik untuk h e p a r d i b a n d i n g k a n d e n g a n a lt, d a n peningkatannya pun tidak setinggi alt, sehingga rasio ast: alt kurang dari 1. rasio as: alt yang lebih dari 2 dapat memperkuat dugaan terhadap pha. dengan adanya resistensi insulin, gambaran laboratorium sindroma metabolik sering ditemukan pada pasien phna. diagnosis gambaran histologis sering kali tidak dapat m e m b e d a k a n p e n y e b a b p e r l e m a k a n h a t i , sehingga diagnosis harus ditegakkan dengan a n a m n e s i s y a n g t e l i t i . d i a g n o s i s p h n a ditegakkan dengan dua komponen: i) adanya bukti perlemakan hati dan/atau steatohepatitis, dan ii) eksklusi penyebab lain perlemakan hati, s e p e r t i a l k o h o l i s m e . ( 1 5 ) d i a g n o s i s p a s t i ditegakkan dengan biopsi hati, tetapi hal ini tidak mudah dilakukan. risiko biopsi tidak sebanding dengan manfaat yang didapatkan; selain itu b i o p s i k u r a n g d a p a t d i a n d a l k a n k a r e n a pengambilan sampel jaringan yang tidak tepat dan teknis lainnya. namun biopsi tetap menjadi satu-satunya alat untuk membedakan tiap-tiap spektrum histologis phna. modalitas diagnostik yang paling banyak digunakan adalah usg. usg bersifat non invasif, berguna untuk penilaian kwalitatif, mempunyai sensitivitas dan spesifisitas masingmasing sebesar 82-94% dan 82%.(15) gambaran tekstur hepar yang terang (bright liver) dan deep attenuation pada usg menunjukkan perlemakan hati. salah satu keterbatasan usg adalah bahwa hati tidak dapat divisualisasikan dengan baik pada pasien-pasien dengan obesitas,dan tidak cukup sensitif untuk mendeteksi steatosis ringan misalnya yang mengenai <33% dari hepatosit dan tidak mampu untuk membedakan subtipe d a r i p n h a . s e l a i n i t u p a d a u s g d a p a t ditemukan hepatomegali dan visualisasi yang berkurang dari vena-vena porta dan vena-vena hepatika. kadang-kadang sulit membedakan steatosis dan fibrosis. computed tomography (ct), magnetic re s o n a n c e i m a g i n g ( m r i ) d a n m a g n e t i c resonance spectroscope (mrs) telah digunakan untuk menilai pasien phna. walau semuanya dapat memberikan pengukuran yang akurat dari h a t i , k e m a m p u a n n y a u n t u k m e m b e d a k a n berbagai subtipe phna masih terbatas.(16) ct berguna untuk mendeteksi steatosis. densitas hati berkurang dengan bertambah beratnya s t e a t o s i s . c t j u g a d a p a t m e n d e t e k s i splenomegali dengan adanya hipertensi portal yang mencurigakan adanya fibrosis yang lanjut pada phna. terdapat bukti yang menyatakan fast gradient echo sequence mri merupakan penilaian steatosis hati yang akurat dan cepat.(17) universa medicina vol. 26 no.4 210 nurman, huang perlemakan hati non alkoholik di masa yang akan datang, mri memberikan kepada kita suatu modalitas yang akurat dan berisiko rendah yang berguna untuk memantau jangka panjang pasien phna. (18) bilamana b i o p s i t i d a k d a p a t d i l a k u k a n , g a m b a r a n radiografik dari steatosis hepatik mendukung diagnosis phna, namun modalitas radiologik yang ada saat ini belum mampu secara akurat mendiagnosis tipe progresif dari phna seperti nash atau stadium dari fibrosis hati. pada waktu yang akan datang, teknik-teknik modern seperti ultrasonografi kontras dengan levovist (schering ag, berlin, jerman) dan transient elastography (fibroscan, echoscans, paris, perancis) dapat menyumbangkan modalitas diagnostik yang akurat.(19) dari modalitas-modalitas yang baru ini, transient elastography mengukur kekakuan hati dengan cara yang tanpa menimbulkan rasa nyeri, reproducible dan dapat memperkirakan fibrosis pada pasien dengan infeksi hcv. namun pada populasi phna yang disertai obesitas, cara ini sukar digunakan dan kurang akurat.(20) biopsi hati dan penilaian histologik m o d a l i t a s k l i n i k , l a b o r a t o r i u m d a n radiologik belum mampu mendiagnosis nash dengan akurat, maka penilaian histologik dan biopsi hati pada phna masih penting. dengan biopsi hati selain untuk menegakkan diagnosis pasti dari nash dan menentukan stadium fibrosis dari hati, dapat juga memberikan informasi mengenai prognosis. namun terdapat beberapa masalah yang berkaitan dengan biopsi hati dan peranan biopsi hati ini dalam praktek klinik masih kontroversi. satu problem dengan penggunaan biopsi hati untuk mendiagnosis n a s h a d a l a h b e l u m a d a n y a k e s e p a k a t a n mengenai kriteria minimal untuk diagnosis histologik dari phna. akhir-akhir ini the national institute of diabetes and digestive and kidney disease (niddk), nash clinical research network mengembangkan pendekatan standar untuk penilaian histologik nash21, dengan menggunakan sistem skor nonalcoholic fatty liver disease (nafld) yang didasarkan atas 3 gambaran patologik : steatosis, inflamasi lobuler dan degenerasi gelembung (degeneration ballooning). skor terentang dari 0–8 di mana skor 5 ekivalen dengan nash, skor 3 atau 4 merupakan batas nash dan skor 2 atau kurang setara dengan non-nash nafld. walaupun skor phna belum divalidasi s e c a r a p e n u h , c a r a i n i d a p a t m e m b e r i k a n pendekatan yang standar pada uji klinik pasien nash. keterbatasan lain dari biopsi hati adalah p o t e n t i a l s a m p l i n g e r ro r, y a n g d a p a t menyebabkan interpretasi dari ahli patologi yang bervariasi.(22) akhirnya tindakan biopsi adalah mahal dan invasif dengan mortalitas 0,01%, dan komplikasi 0,3%. (23) selain itu kebanyakan pasien phna adalah asimtomatik dan enggan u n t u k d i l a k u k a n b i o p s i . o l e h k a r e n a i t u k e p u t u s a n u n t u k m e l a k u k a n b i o p s i h a r u s dilakukan secara individual setelah penyakit lain disingkirkan. sampai biomarker noninvasif untuk diagnosis nash telah ditemukan, adalah bijaksana untuk mencadangkan biopsi hati pada pasien dimana terdapat bukti-bukti sindrom metabolik dan peningkatan yang menahun dari enzim-enzim hati meskipun telah diberikan pengobatan-pengobatan untuk diabetes tipe 2, hiperlipidemia dan obesitas. algoritma untuk diagnosis perlemakan hati alkoholik (pha) dapat dilihat pada gambar 1. pendekatan noninvasif b e r b a g a i p e t a n d a s e r u m t e l a h dikombinasikan dengan parameter klinik untuk mengembangkan model-model fibrosis pada nash yang prediktif. namun pada banyak pasien tes-tes ini tidak konklusif dan belum divalidasi penuh pada pasien-pasien phna.(24) petanda-petanda patogenik dari fibrosis hati (termasuk thioredoksin, faktor nekrosis tumor, leptin dan adiponektin) telah dipelajari juga, 211 namun belum ditunjukkan adanya hubungan yang jelas dengan stadia fibrosis.(25-27) di masa yang akan datang, biomarker diagnostik dapat merupakan bagian dari serum-based assay dan digunakan dengan dikombinasikan dengan modalitas pemindaian yang lebih baru. diagnosis banding sebelum menegakkan diagnosis phna, penyebab peningkatan alt/ast lainnya harus d i s i n g k i r k a n . p h n a d a n p e r l e m a k a n h a t i alkoholik (pha) tidak dapat dibedakan dengan pemeriksaan histologis maupun pencitraan, anamnesis yang teliti mengenai konsumsi alkohol harus dilaksanakan. konsumsi alkohol yang lebih dari 20 g/hari (untuk perempuan) atau 30g/ hari (untuk laki-laki) mengarahkan diagnosis ke pha. (14) namun anamnesis bersifat sangat subjektif, sehingga kurang dapat diandalkan. selain itu, konsumsi alkohol dapat memperburuk p h n a , d a n s e b a l i k n y a p h n a j u g a d a p a t memperburuk pha dan penyakit hati lainnya. h e p a t i t i s v i r u s , h e p a t i t i s i m b a s o b a t d a n hepatitis autoimun juga harus disingkirkan s e b e l u m m e n e g a k k a n d i a g n o s i s p h n a , meskipun steatosis tidak jarang menyertai keadaan-keadaan tersebut. hepatitis a akut yang tipikal ditandai dengan ikterus/peningkatan bilirubin, bilirubinuria dan demam. bila gejalagejala tersebut tidak ada, peningkatan alt/ast yang lebih tinggi daripada pha (lebih dari 5 kali batas atas normal) dan petanda virus yang positif universa medicina vol. 26 no.4 gambar 1. algoritma diagnostik untuk penilaian phna(15) 212 nurman, huang perlemakan hati non alkoholik dapat menjadi petunjuk adanya hepatitis virus akut. umumnya, bila kadar alt di atas 300 iu/ l , h a r u s d i c a r i p e n y e b a b p e n y e b a b l a i n peningkatan alt.(14) penatalaksanaan walaupun sudah banyak obat yang diteliti untuk mengurangi derajat perlemakan hati atau menurunkan alt, sampai sekarang ini tidak ada penatalaksanaan yang spesifik untuk phna. mengingat resistensi insulin berperan besar u n t u k t i m b u l n y a p h n a , p e n a t a l a k s a n a a n idealnya tidak berfokus pada perbaikan fungsi hati saja, namun juga pada resistensi insulin itu sendiri. penurunan berat badan dengan diet dan o l a h r a g a t e r a t u r m e m b a n t u p e r b a i k a n sensitivitas insulin. beberapa studi klinik membuktikan bahwa penurunan berat badan berhasil menurunkan kadar alt serum, bahkan memperbaiki histologi hepar.(28) penurunan berat badan sebesar 10% dari berat badan semula boleh menjadi target awal penatalaksanaan phna,(29) walaupun hal ini belum menjadi suatu konsensus resmi.penurunan berat badan secara bertahap dapat memperbaiki histologi hepar. pada pasien-pasien dengan perlemakan hati berat, penurunan berat badan secara cepat dan drastis malah memperburuk inflamasi portal dan fibrosis. fenomena paradoks ini mungkin d i s e b a b k a n o l e h m o b i l i s a s i l e m a k y a n g meningkatkan asam lemak bebas yang beredar dalam darah. asam lemak bebas yang banyak ini menyebabkan ‘lipotoksisitas’ pada hepar. karena stres oksidatif juga mendasari phna, berbagai macam antioksidan telah diuji coba dalam berbagai penelitian, salah satunya adalah vitamin e, yang banyak digunakan dalam penatalaksanaan phna. vitamin e cukup murah dan relatif tidak berefek samping. sebuah studi di jepang membuktikan bahwa á-tocopherol ( 3 0 0 m g / h a r i ) m e m p e r b a i k i h i s t o l o g i steatohepatitis, menurunkan kadar plasma transforming-growth factor â1 (suatu marker yang berkorelasi dengan inflamasi dan fibrosis hepar), dan menurunkan alt serum.(30) pada pasien pediatri, penurunan alt secara bermakna juga terlihat setelah pemberian vitamin e sebesar 400-1200 iu per hari selama 4-10 bulan.(31) asam ursodeoksikolat adalah suatu bahan sitoprotektif agent yang mencegah kerusakan membran hepatosit. sebuah studi yang dilakukan l i n d o r d k k m e m b a n d i n g k a n e f e k a s a m ursodeoksikolat dengan plasebo, (32) namun ternyata keduanya tidak menunjukkan perbedaan bermakna pada biopsi. pada studi sebelumnya, penurunan alt, ã-gt, fosfatase alkali dan derajat steatosis terlihat secara bermakna setelah penggunaan asam ursodeoksikolat dibandingkan dengan klofibrat.(33) efek asam ursodeoksikolat terhadap perbaikan histologi hati memang tampaknya masih belum konsisten, meskipun demikian tetaplah rasional memberikan obat ini bila efek yang diharapkan adalah menurunkan enzim hati. penelitian-penelitian dalam beberapa tahun belakangan ini mengindikasikan bahwa insulin sensitizer adalah agen terapeutik yang cukup menarik. sebuah studi klinik di italia pada pasienpasien phna nondiabetik(34) membandingkan efek metformin (2 gram/hari) dengan vitamin e atau diit. dibandingkan dengan vitamin e, metformin lebih unggul dalam hal penurunan alt, sedangkan bila dibandingkan dengan diet saja, metformin tidak lebih unggul secara bermakna dalam hal penurunan alt dan imt. d a r i h a s i l b i o p s i d i d a p a t k a n p u l a b a h w a metformin secara bermakna menurunkan lemak hepar, nekroinflamasi dan fibrosis. rosiglitazone adalah insulin sensitizer b a r u , y a n g m e r u p a k a n a g o n i s p e ro x i s o m e proliferators-activated receptor-ã (ppar-ã). obat ini meningkatkan kerja insulin di jaringan perifer maupun hepar. pada sebuah uji coba di finlandia(35) pada subjek-subjek diabetik dengan phna, rosiglitazone (8 mg/hari) menurunkan 213 lemak hepar dan meningkatkan ambilan glukosa ( t e r u t a m a d i j a r i n g a n p e r i f e r ) , s e d a n g k a n m e t f o r m i n t i d a k . m e t f o r m i n h a n y a meningkatkan ambilan glukosa di hepar tanpa m e m p e n g a r u h i k a n d u n g a n l e m a k h e p a r. sibutramine cukup efektif untuk menurunkan berat badan dengan memperpanjang masa satiety. selain itu, sibutramine mempunyai efekefek metabolik yang menguntungkan, seperti p e n u r u n a n h b a 1 c , p e r b a i k a n s e n s i t i v i t a s insulin, penurunan leptin dan peningkatan adiponectin, sehingga tampaknya obat ini juga berguna untuk pasien-pasien phna.(36) telah diteliti pula peran losartan pada phna di jepang.(37) ternyata losartan secara s i g n i f i k a n m e n u r u n k a n k a d a r p l a s m a transforming growth factor â1, salah satu p e t a n d a f i b r o s i s h a t i . s e l a i n i t u , l o s a r t a n memperbaiki histologi hepar dan menurunkan k a d a r a lt. n a m u n h a l i n i m a s i h h a r u s dikonfirmasikan dengan studi-studi selanjutnya. selain dengan obat-obatan yang telah diuraikan di atas, penatalaksanaan phna juga mencakup penatalaksanaan penyakit hati pada umumnya. pasien harus menghindari alkohol dan substansisubstansi lain yang dapat memperburuk fungsi h e p a r. b i l a s u d a h t i m b u l s i r o s i s , m a k a penatalaksanaan difokuskan pada pencegahan progresivitas dan komplikasi penyakit. pada pasien-pasien dengan penyakit hati kronis dan obesitas sekaligus, penurunan berat badan dan olah raga menurunkan kadar enzim hepar dan memperbaiki kualitas hidup.(38) penurunan berat badan pada pasien-pasien dengan hepatitis v i r u s ( 3 9 ) b u k a n h a n y a m e n g u r a n g i d e r a j a t s t e a t o s i s d a n k a d a r a lt, t e t a p i j u g a memperbaiki fibrosis walaupun virus tetap ada. tantangan dan kendala masih banyak tantangan dan kendala yang dihadapi dalam penanganan phna. perlemakan hati sederhana dan steatohepatitis yang lebih p r o g r e s i f h a n y a d a p a t d i b e d a k a n s e c a r a histologis dengan biopsi, yang sulit dikerjakan dan menjadi kendala dalam banyak penelitian. s e j a u h i n i b e l u m a d a p e t a n d a ( s u r ro g a t e marker) fibrosis yang cukup sensitif maupun spesifik. selain itu kadar alt serum pun dapat n o r m a l p a d a s i r o s i s . d e n g a n a d a n y a keterbatasan-keterbatasan tersebut, sulit untuk mengidentifikasi pasien-pasien dengan risiko sirosis yang tinggi dan yang perlu mendapatkan terapi intensif. peran biopsi hati untuk diagnosis dan mengarahkan terapi masih menjadi pro dan kontra. di satu sisi, bila terlihat fibrosis dan sirosis pada sampel jaringan, pasien dan dokter dapat lebih termotivasi untuk mengatasi phna. di sisi lain, modifikasi faktor risiko pun sudah harus dimulai tanpa mempedulikan temuan histologis, (40) dan pada umumnya kelainankelainan metabolik pada pasien phna (misalnya dislipidemia, toleransi glukosa terganggu) adalah target terapi yang lebih penting daripada phna itu sendiri. kesimpulan pnha umumnya asimtomatik dan harus dicurigai pada pasien dengan obesitas dan d i a b e t e s t i p e 2 . a lt m e n i n g k a t d a n p a d a ultrasonografi abdomen didapatkan gambaran perlemakan.hati. diagnosis ditegakkan setelah menyingkirkan kausa penyakit hati kronis yang lain seperti alkohol, virus, obat-obatan. namun diagnosis definitif adalah dengan biopsi hati yang sejauh ini masih menjadi kendala. sejauh ini belum ada petanda fibrosis yang cukup sensitif dan spesifik sebagai pengganti biopsi hati. penatalaksanaan harus holistik berpusat p a d a p e r b a i k a n r e s i s t e n s i i n s u l i n d a n metabolisme. berat badan harus diturunkan bertahap dengan diet dan olah raga. vitamin e dan asam ursodeoksikolat dapat diberikan karena menurunkan alt; metformin dan atau rosiglitazon dapat meningkatkan sensitifitas insulin. universa medicina vol. 26 no.4 214 nurman, huang perlemakan hati non alkoholik daftar pustaka 1. marchesini g, brizi m, morselli-labate am, bianchi g, bugianesi e, mccullough, et al. association of nonalcoholic fatty liver disease with insulin resistance. am j med 1999;107:450-5. 2. nomura h, kashiwagi s, hayashi j, kajiyama w, tani s, goto m, et al. prevalence of fatty liver in a 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tiikainen m, hakkinen am, korheninnikova e, nyman t, makimattila s, yki-jarvinen h. effects of rosiglitazone and metformin on liver fat content, hepatic insulin resistance, insulin clearance, and gene expression in adipose tissue in patients with type 2 diabetes. diabetes 2004; 53: 2169-76. 36. filippatos td, kiortsis dn, liberopoulos en, mikhailidis dp, elisaf ms. a review of the metabolic effects of sibutramine. curr med res opin 2005; 21: 457-68. 37. yokohama s, yoneda m, haneda m, okamoto s, okada m, aso k, et al. therapeutic efficacy of an angiotensin ii receptor antagonist in patients with nonalcoholic steatohepatitis. hepatol 2004; 40: 1222-5. 38. hickman ij, jonsson jr, prins jb, ash s, purdie dm, clouston ad, et al. modest weight loss and physical activity in overweight patients with chronic liver disease results in sustained improvements in alt, fasting insulin and quality of life. gut 2004; 53: 413-9. 39. hickman ij, clouston ad, macdonald ga, purdie dm, prins jb, ash s, et al. effect of weight reduction on liver histology and biochemistry in patients with chronic hepatitis c. gut 2002; 51: 89-94. 40. campbell ms, reddy kr. the evolving role of liver biopsy. aliment pharmacol ther 2004; 20: 249-59. universa medicina vol. 26 no.4 oktavianus 77 universa medicina may-august, 2015 vol.34 no.2 2015: the beginning of the end of the war against malaria? richard tjan editor i n m a y 2 0 1 5 t h e 6 2 th wo r l d h e a l t h assembly formulated a global malaria strategy for 2016-2030 aiming to “reduce the global disease burden by 40% by 2020, and by at least 90% by 2030. it also aims to eliminate malaria in at least 35 new countries by 2030”.(1) as a reminder, it was 60 years ago that the eighth world health assembly decided in 1955 to shift from malaria control to malaria eradication, with the aim to make many areas of free of malaria “within 10 to 15 years”.(2) this has yet to be accomplished in many malaria endemic countries such as indonesia, where the earliest program was the malaria eradication program of 1959, evolving into the malaria control program, the roll-back malaria program, and finally in 2012 into the malaria elimination program.(3) in view of the ever-present insecticideresistance in the mosquito vector and antimalarial drug resistance in the malaria parasite, a longterm goal has been the development of malaria vaccines. there is currently no commercially available malaria vaccine, but the best candidate seems to be the rts,s/as01 vaccine.(4) the rts,s vaccine was developed by glaxosmithkline biologicals (gsk), with a d d i t i o n a l f u n d i n g b y t h e p r o g r a m f o r appropriate technology in health malaria vaccine initiative (path-mvi), which received a grant from the bill and melinda gates foundation. the vaccine is now manufactured by gsk under the trade name mosquirix.(4) the rts,s/as01 vaccine has been shown to protect children and infants older than 6 weeks against plasmodium falciparum malaria and has an acceptable side-effect profile. furthermore, the vaccine can be administered safely with other childhood vaccines, and provides protection against severe malaria. rts,s/as01 vaccine efficacy against severe falciparum malaria in young children (5 to 17 months of age at first vaccination) was around 50% in multicenter phase 3 trials.(4,5) according to the final study results, vaccination with the 3-dose primary series reduced clinical malaria cases by 28% in young children and 18% in infants to the end of the study. administration of a booster dose 18 months after the primary series reduced the number of of clinical malaria cases in young children by 36% and in infants (aged 6-12 weeks at first vaccination) by 26% to the end of the study. however, the vaccine efficacy decreased over time after the booster dose.(4,6) around 80% of study subjects used insecticide-treated bed nets, therefore these study results were achieved i n t h e p r e s e n c e o f e x i s t i n g a n t i m a l a r i a measures.(4) the goal of the rts,s vaccine is to prevent the sporozoites that are introduced by a mosquito bite into the blood from entering liver cells for maturation and multiplication.(4,5) the active substance in the current malaria vaccine is rts,s, a recombinant antigen expressed in saccharomyces cerevisiae. the 78 univ med 2015;34:77-8. doi: http://dx.doi.org/10.18051/univmed.2015.v34.77-78 rts,s antigen consists of the rts and s proteins, that spontaneously assemble into mixed polymeric particles intracellularly. the rts p o l y p e p t i d e c o n t a i n s t h e p. f a l c i p a ru m circumsporozoite protein (cs), including the putative universal t cell epitope, fused to the hepatitis b virus surface antigen (s).(7,8) the final vaccine for administration is obtained by reconstituting a lyophilized preparation of rts,s antigen with the adjuvant as01 and has to be injected intra-muscularly, within 6 hours of reconstitution.(7) the rts,s vaccine has now received a positive opinion from the committee for medicinal products for human use (chmp) of the european medicines agency (ema), which was announced on 24 july 2015. accordingly, the who may reach a policy recommendation for rts,s by the end of 2015.(4) since it has only limited efficacy, the rts,s vaccine is to be regarded as an additional tool for malaria control, to be used with established control measures, s u c h a s i n s e c t i c i d e s , b e d n e t s , a n d antimalarials.(6) in view of emerging parasite resistance against artemisine antimalarials, any addition to the armamentarium against malaria should be welcomed, however slight its efficacy, as long as standard antimalaria measures are not neglected and their funds are not diverted. let us give the rts,s/as01 vaccine the opportunity to prove itself in real-life situations and hope that the who malaria control goals for 2030 may be achieved. references 1. world health organization. world health assembly agrees global malaria strategy and programme budget 2016-17. geneva: world health organization;2015. 2. mayo cw, brady fj. the eighth world health assembly. public health rep 1955;70:1057-60. 3. kusriastuti r, surya a. new treatment policy of malaria as a part of malaria control program in indonesia. acta med indones 2012;44:265-9. 4. the path malaria vaccine initiative (mvi). fact sheet: the rts,s malaria vaccine candidate (mosquirixtm);2015. 5. the rts,s clinical trials partnership. a phase 3 trial of rts,s/as01 malaria vaccine in african infants. n engl j med 2012;367:2284-95. doi: 10.1056/nejmoa1208394. 6. rts,s clinical trials partnership. efficacy and safety of rts,s/as01 malaria vaccine with or without a booster dose in infants and children in africa: final results of a phase 3, individually randomised, controlled trial. lancet 2015;386: 31-45. 7. glaxo smith kline biologicals and path malaria vaccine initiative. rts,s/as01 candidate malaria vaccine-summary for the sage meeting;2009. 8. stoute ja, kester ke, krzych u, et al. longterm efficacy and immune responses following immunization with the rts,s malaria vaccine. j infect dis 1998;178:1139-44. universa medicina may-august, 2015 vol.34 no.2 aloe vera improves total antioxidants 34 *department of biology, faculty of mathematics and natural sciences, semarang state university correspondence: drh. wulan christijanti, msi department of biology, faculty of mathematics and natural sciences, semarang state university kampus fmipa jl. raya sekaran gunungpati semarang phone: 024 86458295 mobile: +62815 6536 227 email: wchristijanti@gmail.com univ med 2017;36:34-41 doi: 10.18051/univmed.2017.v36.34-41 pissn: 1907-3062 / eissn: 2407-2230 received december 9, 2016 accepted for publication april 7, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicinajanuary-april, 2017 vol.36 no.1 aloe vera extract reduces 8-oxo-2'-deoxyguanosine levels and improves total antioxidants in streptozotocin-induced diabetic rats wulan christijanti*, aditya marianti*, and wiwi isnaeni* background diabetes mellitus is a chronic disease caused by lack of insulin production in the pancreas or by insulin resistance, the disease being characterized by elevated blood glucose levels. hyperglycemia in diabetes could lead to oxidative stress due to the rise in 8-oxo-2'-deoxyguanosine (8-oxo-dg) levels and the decrease in levels of total antioxidant status (tas). the purpose of this study was to assess the effect aloe vera extract on 8-oxodg level and total antioxidant status in diabetic rat testis. methods this was an experimental laboratory study with 20 rat samples which were divided into 4 groups (1 control group and 3 treatment groups). diabetes was induced in the rats by streptozotocin (stz) at 65 mg/kgbw. the diabetic rats were then treated for 28 days with aloe vera extract at 0 mg (p0), 200 mg rind (p1), 200 mg pulp (p2), respectively. the level of 8 -oxo-dg was measured by elisa and total antioxidant status with 2,2' -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid (abts). data were analyzed with anova and least significant difference advanced test at p<0.05. results 8-oxo-dg levels were significantly different between the control group and both p0 and p2, but not between the control group and p1. among the treatment groups the 8-oxo-dg levels were significantly different. mean total antioxidant status was significantly different between control and treatment groups, and also between treatment groups (p<0.05). conclusions aloe vera extract reduced free radicals (level of 8-oxo-dg) and increased the total antioxidant status in diabetic rat testis. keywords: aloe vera, 8-oxo-dg, total antioxidants, diabetes mellitus doi: http://dx.doi.org/10.18051/univmed.2017.v36.34-41 original article 35 univ med vol. 36 no.1 introduction h y p e rg l y c e m i a i n d i a b e t e s l e a d s t o oxidative stress resulting from stimulation of the polyol pathway, the formation of advanced glycation end products (ages), protein kinase c (pkc) and the further formation of reactive oxygen species (ros). (1) increased ros production in diabetics could potentially a c c e l e r a t e t h e o x i d a t i v e d a m a g e t o macromolecules such as lipids, proteins and dna. mitochondria and the nucleus are the main targets of oxidative stress, the former having a variety of enzymes to fix dna modifications due to induction of oxidants.(2) 8-oxo-7,8-dihydro2'-deoxyguanosine (8-oxo-2'-deoxyguanosine, 8oxo-dg) modified by ros is a sensitive index of oxidative dna damage.(3) a c c o r d i n g t o s e v e r a l s t u d i e s , t h e concentrations of 8-oxo-dg of serum (4) and urine of patients (5) as well as from tissues of diabetic rats were significantly higher than normal.(6) another study states that there is a positive correlation of insulin resistance with oxidative stress parameters (malondialdehyde/mda and 8-oxo-dg), and a negative correlation with the antioxidant enzyme (superoxide dismutase/ sod).(7) levels of 8-oxo-dg, which is more stable than dna reflect the generation rate and repairs.(8) o x i d a t i v e s t r e s s o c c u r s w h e n t h e production of free radicals is higher than the capacity of cellular antioxidants. hyperglycemia raises oxidative stress, therefore, theoretically, diabetes patients can benefit from antioxidant supplementation. the beneficial effects of antioxidants have been reported in animal models o f d i a b e t e s a n d i n d i a b e t e s p a t i e n t s . ( 9 ) antioxidants are divided into endogenous antioxidants, such as sod, glutathione (gsh), vitamins c and e, and exogenous antioxidants ( v i t a m i n e , v i t a m i n c , c a r o t e n o i d s a n d flavonoids).(10) administration of vitamin e has been shown to be beneficial in preventing cell damage by inhibiting lipid peroxidation, protein oxidation, protein glycation, and platelet a g g r e g a t i o n . a d e c r e a s e i n p l a s m a f 2 isoprostanes was seen in type 2 diabetic patients after six weeks of supplementation with vitamin e 600 mg/day.(11) constituent antioxidants from plant material act as free radical scavengers, and can be found in various food sources such as fruits, vegetables and tea.(12) s o u r c e s o f e x o g e n o u s a n t i o x i d a n t supplements from plants such as aloe (aloe vera), contain natural components such as phenols, flavonoids, vitamin c and vitamin e, and have been proven to reduce the levels of mda and increase the antioxidant capacity.(13) a l o e v e r a e x t r a c t h a s a n a s s o r t m e n t o f pharmacological properties which encompasses protection against radiation and antiviral, antibacterial, laxative, antioxidant, antii n f l a m m a t i o n , a n t i c a n c e r, a n t i d i a b e t i c , antiallergic, and immunostimulant properties amongst others.(14) besides, the provision of aloe vera in diabetic rats showed improvement by increasing the survival of cells of the islets of langerhans.(15) research by mohapatra et al.(16) showed that aloe vera extract at 150 and 300 mg/kgbw can increase the levels of sod and gsh and c a n d e c r e a s e l i p i d p e r o x i d e s ( m d a ) i n comparison with diabetic controls. flavonoids are presumably able to bind transition metal ions, which play a role in glycation, to prevent the formation of a metal catalyst to form the hydroxyl radical, or a related species of h 2 o 2 . a study showed that a l o e v e r a g e l e x t r a c t significantly increased plasma total antioxidants in healthy volunteers.(17) there is insufficient scientific evidence on the efficacy of indonesian aloe vera extract prepared from local cultivars and on its mechanism of action. additional work is required to probe into the antidiabetic, antimicrobial, and antioxidant properties of indonesian aloe vera extract. the purpose of this study was to assess the effect of aloe vera extract on the levels of 8oxo-dg and total antioxidant status in the testis of diabetic rats. 36 methods research design this study used an experimental post-test only control group design and was conducted in the animal breeding house, while laboratory measurements of the variables were performed in the food and nutrition center at the interuniversity center (pusat antar universitas, pau), gajah mada university, yogyakarta, from june until september 2016. plant material fresh aloe vera leaves were collected from bandungan district, semarang, in june 2016 and identified at the plant morphology laboratory o f t h e b i o l o g y d e p a r t m e n t , f a c u l t y o f mathematics and natural sciences, semarang state university. preparation of aloe vera gel extract aloe vera gel was prepared from aloe vera leaf according to a modification of a previously published procedure.(13) mature, healthy and fresh leaves of aloe vera having a length of approximately 60 cm were washed with fresh water. the leaves were cut lengthwise into two pieces, then the thick epidermis and the solid gel in the center of the leaf were homogenized in an electric blender. the homogenate sample was extracted using 96% ethanol. the filtrate was collected and evaporated to form a gel under reduced pressure in a rotary evaporator. the gel was stored in small containers in the refrigerator until further use. an aqueous suspension was prepared by dissolving suitable amounts of the ethanolic aloe vera leaf extract to obtain the desired concentration. the herbal solutions were prepared freshly each time and administered intragastrically. the dosing schedule used was once per day. experimental animals the number of experimental animals per group was the minimum sample of five.(18) a total of 20 male wistar rats weighing 150-200 g were randomly divided into four groups (one control group and three diabetic groups). the rats were obtained from the food and nutrition center at pusat antar universitas (pau), gajah mada university. the animals were acclimatized for 7 days, maintained on 12 hour light-dark cycles with standard rat feed and water ad libitum. induction of experimental diabetes after fasting, the rats in the diabetic group were induced into the diabetic state by intra peritoneal streptozotocin (stz) at 65 mg/kgbw on day 8. (19) seventy-two hours after the induction, blood glucose measurements were performed, to determine the presence of a hyperglycemic condition with blood glucose levels of ≥200 mg/dl. experimental procedure the rats were randomized into four groups; group 1 (k, controls) received distilled water; group 2 (p0) received stz 65 mg/kgbw and distilled water; to group 3 (p1) was administered stz 65 mg/kgbw and 200 mg/kgbw of rind extract, while group 4 (p2) received stz 65 mg/ kgbw and 200 mg/kgbw of pulp extract. treatment with aloe vera extract was performed orally for 28 days, during which period the rats received feed and water ad libitum. blood was collected in microhematocrit tubes and was used for the estimation of glucose levels on the day before and three days after streptozotocin induction and after four weeks of treatment with aloe vera extract. the blood glucose level of the serum samples was measured by the enzymatic glucose oxidase-peroxidase (godpap) colorimetric method. the animals were sacrificed by cervical decapitation. a transverse abdominal incision was made and the testes were removed from the rats and cleansed of blood. the testes were homogenized with phosphate buffer and centrifuged at 3600 rpm to collect the supernatant. christijanti, marianti, isnaeni aloe vera improves total antioxidants 37 univ med vol. 36 no.1 measurements of 8-oxo-dg an total antioxidants t h e s u p e r n a t a n t s w e r e u s e d f o r t h e e s t i m a t i o n o f 8 o x o d g l e v e l s a n d t o t a l antioxidant status. 8-oxo-dg levels (ng/ml) were estimated using ht 8-oxo-dg elisa kit ii (cat # 4380-096-k). total antioxidant level was measured with abts. the tas was estimated using a kit (cat# cs0790). to the wells of a 96-well plate was added 125 ml of reagent 1 (assay buffer) and 7.5 ml of either the assay standard (standard 1, water; standard 2, 0.01 mmol trolox equivalent/l solution) or the sample, and the ûrst absorbance was read at 450 nm. [troloxtm is a water-soluble vitamin e analog and serves as a standard or control antioxidant.] then, 18.75 ml of reagent 2 (2,2'azino-bis (3-ethylbenzothiazoline-6-sulfonic acid/abts) was added to all of the wells and the second absorbance was read exactly after 10 min. the ∆a was calculated as the difference between the first and second absorbance readings of either the sample or the standard or the blank, as the case may be. the difference (a1) between the ∆a of the standard 1 and the ∆a of the sample was calculated. similarly, the difference (a2) between the ∆a of the standard 1 and the ∆a of the standard 2 was calculated. the tas in the tissue was equal to the a1/a2 ratio and expressed in mmol/l. statistical analysis data were processed and analyzed using spss version 16 software. the statistical analysis used was one way anova and lsd at a level of significance of 0.05. ethical clearance the study was conducted after obtaining ethical clearance from dr. kariadi central general hospital and the faculty of medicine, diponegoro university in semarang, under no. 699/ec/fk-rsdk/2016. results blood glucose levels before and three days after stz induction and after four weeks of treatment with aloe vera extract are presented in table 1. the blood glucose levels before induction showed no significant differences between all groups (p=0.172). three days after stz induction, the glucose level in p0, p1, and p2 reached >200 mg/dl, indicating that the induction had successfully created diabetic rats. moreover, treatment with aloe vera extract for four weeks significantly decreased blood glucose levels from >200 mg/dl to 103.39 ± 5.80 mg/ dl (p1) and 125.89 ± 5.01 mg/dl (p2) (p<0.05). on determination of serum 8-oxo-dg concentration, it was found that mean 8-oxo-dg concentrations of the control and treatment groups were 3.34 ± 0.26 ng/ml, 18.55 ± 3.18 ng/ml, 4.80 ± 0.25 ng/ml and 9.06 ± 0.15 ng/ml, respectively (table 2). thus serum 8-oxo-dg concentration in the control group (k) was significantly lower than in the diabetic groups (p=0.000). the free radical level in the form of 3.34 ng/ml 8-oxo-dg in the control group was significantly different from that in the treatment groups (p <0.05), except group p1. among the treatment groups (p0, p1 and p2) the free radical level also showed significant differences. table 1. blood glucose level in non diabetic and diabetic rats after four weeks of treatment with aloe vera extract k: distilled water; p0: stz 65 mg/kgbw and distilled water; p1: stz 65 mg/kgbw and 200 mg/kg of rind extract; p2: stz 65 mg/kgbw and 200 mg/kgbw of pulp extract; stz = streptozotocin; significantly different (p<0.05) 38 on determination of serum total antioxidants it was found that the differences were statistically significant (p=0.000). thus serum total antioxidants of the control and treatment groups were 1.79 ± 0.12 mmol/l, 0.62 ± 0.13 mmol/l, 0.88 ± 0.10 mmol/l and 1.29 ± 0.17 mmol/l, respectively. total antioxidant status at 1.79 mmol/l in the control group was significantly different from that of the treatment groups, and was also significantly different between the treatment groups (p<0.05) (table 2). the positive effects of aloe vera extract in reducing the levels of 8-oxo-dg and increasing the total antioxidant status of diabetic rats are presented in figure 1. from the graph it can be seen that the maximum level of 8-oxo-dg in diabetic rats was 19.75 ng/ml. treatment with aloe vera extract decreased the level to 4.80 ng/ ml and 9.06 ng/ml, respectively. the aloe vera extract exerted a positive effect in increasing tas in diabetic rats from 0.62 to 0.88 and 1.29, respectively. discussion the present study showed that the blood glucose levels in diabetic rats were significantly higher than those in non diabetic rats. aloe vera rind and pulp extract at a dose of 200 mg/kgbw/ day for four weeks given to diabetic rats could significantly decrease the blood glucose level towards normality (<200 g/dl). the effect of aloe vera extract on glycemic control in animal models has been proven by a number of authors.(20-22) it has also been reported that aloe vera extract can stimulate the regeneration of christijanti, marianti, isnaeni aloe vera improves total antioxidants table 2. differences in 8-oxo-dg level and total antioxidant concentrations between the control and treatment groups k: distilled water; p0: stz 65 mg/kgbw and distilled water; p1: stz 65 mg/kgbw and 200 mg/kgbw of rind extract; p2: stz 65 mg/kgbw and 200 mg/kgbw of pulp extract. tas = total antioxidant status; significantly different (p<0.05) figure 1.graph of 8-oxo-dg levels and total antioxidant status (tas) note: sat = indonesian term for tas 39 univ med vol. 36 no.1 pancreatic β cells and improve serum antioxidant enzyme levels, thus preventing pancreatic cell damage in diabetics.(16) the 8-oxo-dg levels in diabetic rats in the present study were significantly higher than in normal rats or treatment groups receiving aloe vera extract. the 8-oxo-dg levels increased to maintain the homeostasis mechanism in response to excessive dna damage. analysis of the levels of urinary 8-oxo-dg may reflect on dna repair mechanisms.(23) however, hyperglycemia in dm stimulates ros, which leads to the activation of ros generation and oxidative damage. these r e s u l t s a r e s i m i l a r t o t h o s e o f p r e v i o u s reports.(24,25) the free radicals produced by the normal metabolic processes are maintained in balance by endogenous and exogenous antioxidants that act as free radical scavengers. oxidative stress occurs when the concentration of free radicals exceeds the detoxifying capacity of cellular antioxidants.(26) increased oxidative stress as a hyperglycemic condition may be one factor in the emergence of complications of diabetes. oxidative damage is generally associated with the formation of oh that can cause oxidative damage to cellular components such as lipids, proteins and dna.(27) the reduced levels of 8-oxo-dg in the groups of diabetic rats treated with aloe vera extract show that aloe vera extract contains antioxidants that can protect cells from free radical influence during hyperglycemia. the hypoglycemic effect of aloe vera which is obtained by stimulating pancreatic β cell regeneration, increases the resistance of the cells, mitochondrial activity, and insulin production.(2,15) in addition, inhibition of pancreatic amylase activity in the gut reduces the breakdown of carbohydrates into glucose.(21) oxidative stress that occurs because of a hyperglycemic state causes lipid peroxidation of the cell membrane, thus damaging the dna. active oxygen and other agents that generate free oxygen radicals are able to induce a variety of lesions in the dna that cause deletions, mutations and other genetic effects. defects that are specific for the dna have shown that the sugar and basic groups making up the dna are susceptible to oxidation.(28) in the present study, mean tas in the diabetic groups was lower than that in the control group. the diabetic groups p1 and p2 receiving aloe vera extract had a higher total antioxidant status than the diabetic group p0 not receiving the extract. aloe vera extract has a tendency to increase the total antioxidant levels in diabetic rats along with a significant increase in the levels of glutathione, superoxide dismutase (sod), catalase, glutathione peroxidase and glutathiones-transferase in the liver and kidneys of diabetic rats.(13) these results are similar to those of previous reports, where enzyme activity of sod, catalase, and glutathione increased significantly after administration of ethanolic aloe vera extract to prevent glycation.(29) the hyperglycemic condition in diabetes increases the concentration of free radicals that reduce endogenous antioxidants. humans and animals have an antioxidant pool consisting of enzymatic antioxidants such as sod, glutathione peroxidase (gpx), glutathione reductase (gr), a n d c a t a l a s e ( c at ) , a n d n o n e n z y m a t i c antioxidants including glutathione (gsh), v i t a m i n c a n d v i t a m i n e . ( 2 7 ) e x o g e n o u s antioxidants obtained from outside the body include micronutrients, vitamin e, vitamin c, minerals, carotenoids and flavonoids. ( 1 0 ) superoxide dismutase, catalase and glutathione peroxidase reduce ros by converting ros into stable compounds. the role of sod is to catalyze the transformation of superoxide radicals (o 2 ’”-) to hydrogen peroxide (h 2 o 2 ), which is then converted by the enzyme catalase into water and oxygen molecules.(30) the presence of free radicals (ros) can be overcome by involving both exogenous antioxidants and endogenous origin. antioxidants are a group of substances that even at low concentrations would significantly impede or delay the oxidative process.(28) one treatment that was developed in the management of diabetes used antioxidant therapy either from foods or 40 supplements. foods such as vegetables and fruits are known to have many components of active substances that are useful for the treatment. aloe vera, which is known as one of the plants that have antioxidant components such as phenolics, coumarins, flavonoids, polyfunctional organic acids, and tocopherol, can reduce lipid p e r o x i d a t i o n i n c e l l m e m b r a n e s , ( 2 6 ) thus i n c r e a s i n g t h e c e l l u l a r a n t i o x i d a n t s . ( 1 3 ) consumption of vitamin e has been shown to p r e v e n t c e l l d a m a g e b y i n h i b i t i n g l i p i d peroxidation, protein oxidation, protein glycation and platelet aggregation.(31) a limitation of this study was that it did not analyze other factors that influence oxidative stress, such as hormones and inflammatory status, and did not examine seminogram parameters in hyperglycemic conditions. the clinical implication of this study is that oxidative stress may presumably stimulate apoptosis in testicular germ cells, thus affecting the quality of the produced sperm. further research needs to be done to determine the factors affecting the quality of sperm (that are highly susceptible to oxidative stress) in order to reveal the fertility status of the male diabetic. conclusion the study demonstrated that aloe vera rind and pulp extracts can prevent the effect of oxidative stress in diabetic rat testis by lowering the concentration of free radicals (8-oxo-dg) and improving the tas. conflict of interest none declared. acknowledgement the author wish to thank dipa pnbp unnes (daftar isian pelaksanaan anggaran pendapatan negara bukan pajak universitas negeri semarang) for the year 2016, which has provided the research funds. contributors wc contributed to drafting the manuscript and design of the study. wc and am contributed to data collecting, analysis and interpretation. ad and wi contributed revising the manuscript. wc wrote the manuscript and all authors read and approved the final version of the manuscript. references 1. giacco f, brownlee m. oxidative stress and diabetic complications. circ res 2010;107: 1058-70. 2. turrens jf. mitochondrial formation of reactive oxygen species. j physiol 2003;552:335–44. 3. sun j, lou x, wang h, et al. serum 8-hydroxy22 -deoxyguanosine (8-oxo-dg) levels are elevated in diabetes patients. int j diabetes dev ctries 2011;35:368-71. 4. chan ss, byung sm, kyong sp, et al. serum 8-hydroxy-guanine levels are 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medicine. 4th ed. oxford: clarendon press;2007. 28. kumar s. free radicals and antioxidants: human and food system. adv appl sci res 2011;2:12935. 29. ramachandraiahgari rmy, somesula sr, adi pj, et al. protective role of ethanolic extract of aloe vera antioxidant properties on liver and kidney of streptozotocin-induced diabetic rats. digest j nanomaterials biostructures 2012;7: 175–84. 30. darmawan h. production of ros and its effects on mitochondrial and nuclear dna, human spermatozoa, and sperm function. med j indones 2007;16:127-33. 31. minamiyama y, takemura s, bito y, et al. supplementation of α-tocopherol improves cardiovascular risk factors via the insulin signalling pathway and reduction of mitochondrial reactive oxygen species in type ii diabetic rats. free radic res 2008;42:261-71. oktavianus 213 *department of anatomy, medical school, medical and health sciences, general soedirman university **genetics laboratory, biological faculty, general soedirman university ***department of biochemistry, medical school, medical and health sciences, general soedirman university correspondence dr. fitranto arjadi, mkes. department of anatomy, medical school, medical and health sciences, general soedirman university jl. dr gumbreg no 1 mersi purwokerto 53122 phone: +62281 622022 email: f.arjadi@yahoo.com univ med 2014;33:213-20 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background hypertension is associated with endothelial-dependent vasodilation disorders, due to reduced nitric oxide (no) availability and excessive angiotensin ii (angii) activation. the objective of this study was to determine the association between matrix metallopeptidase 9 (mmp-9) gene polymorphism and hypertension in ethnic javanese in the 40-80 year age group. methods this was a case-control study on 50 prolanis patients of family doctors meeting the inclusion criteria and 50 controls without hypertension. subjects were hypertensive patients with constant systolic arterial pressure of >140 mmhg and diastolic arterial pressure of >90 mmhg, confirmed in three successive measurements the observed parameters were degree of mmp-9 polymorphism, and no and ang-ii levels. matrix metallopeptidase 9 polymorphism was determined by polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) using the smai restriction enzyme. mmp-9 polymorphisms were indicated by variation in band patterns. degree of polymorphism in cases and controls were compared with no and ang-ii levels in both groups. data analysis was done using independent t-test. results the heterozygous (3 band) to normal (2 band) mmp-9 genotype ratio was 3:1 in hypertensives, but balanced in controls. in hypertensives, heterozygous ga and homozygous aa genotype frequencies were respectively 3.198 and 1.548 times higher than that of the gg genotype (p=0.008 and p=0.726). there was a statistically significant differences of no and ang-ii levels between cases and controls (p=0.000 and p=0.000; respectively). conclusion matrix metallopeptidase 9 gene polymorphisms in hypertensive ethnic javanese are associated with no and angiotensin ii levels. keywords: hypertension, ethnic javanese, mmp-9 gene polymorphism, adults metalloproteinase-9 gene variants and risk for hypertension among ethnic javanese fitranto arjadi*, saefuddin ‘aziz**, and alfi muntafiah*** 214 arjadi, ‘aziz, muntafiah metalloproteinase-9 gene and hypertension variasi genetik gen penyandi metalloproteinase-9 dan risiko hipertensi pada etnis jawa latar belakang. hipertensi berhubungan dengan gangguan vasodilatasi yang tergantung endotel, akibat penurunan kesediaan nitrit oksida (no) dan terjadinya aktivasi angiotensin ii (ang ii), yang berlebihan.tujuan penelitian adalah untuk menentukan adanya hubungan antara polimorfisme gena matrix metallopeptidase 9 (mmp-9) dan hipertensi pada pasien etnik jawa usia 40 higga 80 tahun. metode digunakan rancangan kasus control yang mengikut sertakan 50 kasus hipertensi prolanis dokter keluarga yang memenuhi kriteria inklusi. kontrol merupakan pasien prolanis sebanyak 50 orang dengan usia 40 hingga 80 tahun yang tidak menderita hipertensi. kasus adalah pasien hipertensi yang dicirikan dengan tekanan sistolik dan diastolik melebihi 140/90 mmhg, yang dikonfirmasi dengan tiga kali pengukuran. parameter yang diamati adalah no dan angi ii. polimorfisme gena mmp-9 ditentukan dengan metode polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) menggunakan enzim restriksi smai. tingkat polimorfisme antara kasus dan kontrol kemudian dibandingkan dengan kadar no dan ang ii. data dianalisis menggunakan uji t independen. hasil kasus hipertensi memiliki rasio heterozigot (3 pita) mmp-9 3 kali lebih besar dibandingkan genotipe normal (2 pita). pada polimorfisme gena mmp-9, penderita hipertensi kemungkinan memiliki genotipe heterozigot ga, 3,198 kali lebih besar daripada genotip gg (p=0,008) dan memiliki genotip aa, 1,548 kali lebih besar daripada memiliki genotip gg walaupun secara statistik tidak bermakna (p=0,726). terdapat perbedaan kadar no dan ang ii yang bermakna antara kasus dan kontrol (masing-masing dengan p=0,000 dan p=0,000). kesimpulan terdapat polimorfisme gena mmp-9 pada pasien etnik jawa dengan hipertensi. polimorfisme gena mmp9 berhubungan secara bermakna dengan kadar no dan ang ii. kata kunci : etnis jawa, hipertensi, polimorfisme gena mmp-9, dewasa abstrak introduction the results of basic health research (riskesdas) of the ministry of health of indonesia in 2007 showed that hypertension has the highest mortality rate (31.9%) and that the ethnic group with the highest numbers are the javanese. (1) in central java, hypertension patients rank fourth among indonesians (37%).(2) in banyumas, the number of hypertensive patients always higher in comparison with other d i s e a s e s . (3) m a n y g e n e s a r e i n v o l v e d i n h y p e r t e n s i o n , s u c h a s t h e m a t r i x metalloproteinase (mmp) gene that has a higher risk in connection with peripheral resistance. peripheral resistance leads to changes in cardiovascular structures, including myocardial hypertrophy and fibrosis resulting from changes i n e x t r a c e l l u l a r c o l l a g e n . m a t r i x metalloproteinase is responsible for the balance between tissue matrix and proteolysis of extracellular matrix components such as elastin, proteoglycan and collagen, that have an important role in blood vessel remodeling.(4) a 215 high level of metalloproteinase-9 (mmp-9) affects the thickness of the tunica media of blood vessels and decreases the possibility of arterial stiffness.(5) a meta-analysis by niu et al.(6) s h o w e d t h e c o n n e c t i o n o f m m p 9 g e n e polymorphism with coronary heart disease. matrix metalloproteinase 9 is responsible for a balanced tissue matrix and contributes to vascular stiffness. matrix metalloproteinase 9 gene polymorphisms which lead to an increase in mmp-9 can cause differences in individual susceptibility to hypertension.(7) a decrease in nitric oxide (no) production in endothelial cells activates the extracellular matrix of the renal tubuli, namely the metalloproteinase-2 (mmp2) matrix and mmp-9 matrix, its induction of microvascular dilation in renal glomeruli and renal tubuli in order to increase filtration and decrease blood pressure. the interstitial connective of blood vessel and capillary endothelial basement membrane contains ultrastructural elastin and collagen, but the imbalance between mmp and its inhibitor, tissue inhibitor of metalloproteinase (timp), will lead to increased expression of mmp-2 and mmp9. the next process of mmp-2 and mmp-9 is degrading elastin and collagen and replacing it with more rigid oxidized collagen. this process is exacerbated in polymorphisms of the mmp-9 gene at a distance of 277 nucleotides, where guanine is substituted by adenine, so causing the mmp-9 activation to occur continuously. polymorphisms of the mmp-9 gene, located in the 20q12.2-13.1 chromosome, are found at promoter, coding, and untranslated regions.(8) on the other hand, angiotensin ii (ang ii) and no have an important role in the blood pressure feedback regulation mechanism. ang ii controls the expression of endothelial cell nitric oxide synthase (enos), which produces no. no s y n t h e s i s r e s u l t s i n s m o o t h m u s c l e c e l l v a s o c o n s t r i c t i o n , w h i l e n o r e d u c e s t h e expression of ang ii receptors in endothelium causing vasodilation of blood vessels.(9) the study of mmp-9 gene polymorphisms in ethnic caucasians showed that the base change c to t in the mmp-9 gene promoter at -1562 increases the risk of heart disease due to an i n c r e a s e i n s y s t o l i c a n d d i a s t o l i c b l o o d pressures.(10) another study on ethnic indians showed a correlation between mmp-9 gene polymorphism and hypertension and also the occurrence of left ventricular damage in the heart. ( 11 ) i n t h e b r a z i l i a n p o p u l a t i o n , a polymorphism of the mmp-9 gene is associated with high cholesterol levels that indicate a relation with abnormalities of fat metabolism and heart disease.(12) however, the presence of polymorphisms of mmp-9 genes in ethnic han chinese was not associated with the incidence of stroke or intracerebral hemorrhage.(13) t h e d i ff e r e n c e s i n f r e q u e n c y o f polymorphisms of mmp-9 genes with the incidence of hypertension in several human ethnic groups are interesting for further study. in indonesia there are many ethnic groups and one of the largest are the javanese. besides, basic molecular analysis of polymorphisms of mmp9 genes in hypertension patients of javanese ethnicity has not been carried out. therefore it is important to determine the effects of mmp-9 gene polymorphism in hypertension patients of javanese ethnicity. the objective of this study was to determine the association between mmp9 gene polymorphism and hypertension in ethnic javanese in the age group of 40-80 years. methods research design an observational study with case-control design was conducted from july to december 2012. the research was carried out in the research laboratory of general soedirman university in purwokerto, central java. research subjects the case group consisted of 50 patients of family doctors associated with the prolanis p r o g r a m ( c h r o n i c d i s e a s e m a n a g e m e n t program, program pengelolaan penyakit kronis) in banyumas in collaboration with pt univ med vol. 33 no.3 216 arjadi, ‘aziz, muntafiah metalloproteinase-9 gene and hypertension askes (the national health insurance agency). the inclusion criteria for the case group were: male or female, ethnic javanese (3 generations), aged >40 years, with blood pressure of >140/90 and not taking antihypertensive medications. the control group consisted of 50 persons meeting the following inclusion criteria: male or female, ethnic javanese (3 generations), aged > 40 years, with normal cholesterol, triglyceride, blood glucose levels, and normal body mass index ( b m i ) . a l l p a r t i c i p a n t s w e r e d i a g n o s e d according to their medical history and clinical symptoms by clinicians under supervision of a consultant before being assigned to the case group or control group. blood sample collection a volume of 6 ml of venous blood from each participant was collected in a tube and used for dna isolation and analysis of serum no and ang ii levels. blood for determination of no and angii was collected in an eppendorf tube with anticoagulant and centrifuged at 4.000 rpm for 10 minutes, so that the yellowish serum forming the supernatant was separated from the erythrocytes. the determination of no levels by the griess’ method by calculating nitrate and nitrite concentrations uses the formula: [nitrate + nitrite] [ì m] = [a 540-y] [200 µl] x dilution slope sample volume dna extraction extraction of dna used the method of miller et al.(14) packed cells were lyzed with red cell lysis buffer and then centrifuged at 4000 rpm for 15 min at 4°c to pellet out the nucleated cells (wbcs). nucleated cells were subjected to detergent (10% sds) and protease (proteinase k) treatment in sodium chloride-edta buffer and left at 37°c overnight on a shaker. proteins were subsequently salted out with 5m nacl, then pelleted out by centrifugation at 4000 rpm at room temperature for 15 min. to the supernatant, which was transferred to another tube, ethanol was added to precipitate the dna. the isolated dna was stored in te (tris-hcl edta) buffer in 4°c for further use. the concentration and purity of the isolated dna was measured by spectrophotometry. genotype determination the polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) procedure for polymorphism of mmp-9 genes started with the amplification of mmp-9 genes by using the forward primer r279q f: 5'atg ggt caa aga aca gga-3’ and reverse primer r279q r: 5'ggt aga cag ggt gga gg-3’, producing 277-bp long products.(15) t h e p c r c o n d i t i o n s i n c l u d e a n i n i t i a l denaturation of 950c 5 minutes, 35 cycles consisting of denaturation 940c 30 seconds, annealing 580c 30 seconds, elongation 72 0c 45 seconds and final elongation 720c 10 minutes. the pcr product was cut with the smai restriction enzyme. digestion results were analyzed by electrophoresis using agarose gel 5% and visualized with ethidium bromide under uv light. aa electrophoresis results were not cut, gg was cut into 2 fragments of 181 bp and 96 bp, while ca was cut into 3 fragments of 277 bp, 181 bp and 96 bp. statistical analysis data were expressed as means of absolute number (percentage), and independent-t test was used to determine the differences between individuals carrying polymorphisms of nos3 genes and mmp-9 genes in hypertension patients and if the distribution was abnormal we continued with the mann whitney test. ethical clearance all procedures have been approved by the medical and health research ethics committee (mhrec), faculty of medicine, gadjah mada university (approval letter reference number: ke/fk/734/ec). 217 results table 1 shows the association between p o l y m o r p h i s m o f m m p 9 g e n e s w i t h hypertension of the 100 subjects who participated in the study. the genotypes gg and ga were separated with smai enzyme at its restriction sites and yielded 2 and 3 bands, respectively. on the other hand, there was no smai enzyme restriction site in the aa genotype, so aa appeared as 1 band. in subjects of the case group, the highest frequencies was for the ga genotype (62%), followed by the gg genotype (28%), while the aa genotype had the lowest frequencies (10%). in contrast, among the control subjects, the greatest number was in the gg genotype (52%), followed by the ga genotype (36%) and aa genotype (12%). in hypertensive, the ratio of heterozygous (3 band) to normal (2 band) mmp-9 genotype was 3:1. the control group had a balanced ratio of heterozygous and normal genotype. in hypertensive patients the frequencies of heterozygous ga and homozygous aa genotypes were respectively 3.198 and 1.548 times higher than the frequencies of the gg genotype (at p=0.008 and 0.726, although the latter was not statistically significant). statistical analysis showed that there was a significant of angiotensin ii and no levels between cases and controls (p=0.000 and p=0.000; respectively) (table 2). figure 1 shows the electrophoresis results of pcr-rflp products to detect genotype variation in mmp-9 genes. the m marker ref20, aa with the band size of 277 bp is not cut by the restriction enzyme, gg is cut into two bands 181 bp and 96 bp, while ga is a combination of one uncut band and two cut bands, producing three bands of sizes 277 bp, 181 bp, and 96 bp. after completion of the pcr-rflp, some samples were sequenced at the eijkman laboratory, jakarta. the mmp-9 pcr fragment products show that sample m1 is an uncut band (figure 2). sample k11 forms two bands because it is cut by the smai enzyme, while sequence gi7427228 is a human mmp-9 gene sequence from the gene bank. the nucleotide sequences at site 96 in bold print show the variation between sequences and the nucleotide cccggg sequence (underlined) is that recognized by smai. discussion regarding mmp-9 gene polymorphism, h y p e r t e n s i o n p a t i e n t s m a y h a v e h i g h e r frequencies of the heterozygous genotype ga ( 6 2 % o r 3 . 1 9 8 t i m e s ) t h a n h o m o z y g o u s genotypes (gg genotype and aa genotype). on the other hand, the group without hypertension (control) have smaller genotype frequencies of h e t e r o z y g o u s g e n o t y p e g a ( 1 8 % ) t h a n table 1. association of mmp-9 gene polymorphisms between case and control groups table 2. comparison of no and angiotensin ii levels between case and control groups univ med vol. 33 no.3 218 arjadi, ‘aziz, muntafiah metalloproteinase-9 gene and hypertension homozygous frequencies of gg (52%). this result is the same as the report on brazilians with hypertension by mazzoti et al.,(12) among whom the frequency of the mmp-9 heterozygous genotype reached 70.3%. however, fan et al.(16) reported that heterozygous allele frequencies in mmp-9 polymorphisms both in the control group and case group of chinese stroke patients showed approximately equal percentages (< 25%). our results showed, similarly to all other studies that have been done before, that the aa genotype frequency is always the lowest, both in case and control groups. this indicates that the aa genotype is a genetic factor causing hypertension due to g->a (guanine to adenine) base changes. in the present research, the aa genotype is not visible as a clinical sign of hypertension because the mmp-9 enzyme may have vasodilator and vasoconstrictor effects. the mmp-9 enzyme is able to degrade the extracellular matrix which can lead to vasodilation in the early phase but not to vascular stiffness. the excessive action of the mmp-9 enzyme may eventually lead to continuous blood vessel stiffness, causing hypertension. therefore the aa genotype is not a risk factor in the early stages of hypertension, but continued activity can cause mmp-9 to b e c o m e a r i s k f a c t o r f o r c a r d i o v a s c u l a r disease.(17) the polymorphism of mmp-9 genes and the lack of tissue inhibitor of metalloproteinase (timp) can cause the excessive mmp-9 enzyme activity that will convert elastin and collagen into more rigid forms.(18) polymorphism of mmp-9 in the promoter and coding sequences affects the mmp-9 levels and activity, which will strongly increase and cause arterial stiffness, both in men and women,(17) therefore the mmp-9 gene affects cardiovascular mortality, especially in patients with the 279 gln allele.(19) the differences in age and gender also influence the response of mmp9.(17) m a t r i x m e t a l l o p r o t e i n a s e 9 w a s significantly associated with no, which means that there are significant differences in no levels between mmp-9 gene polymorphism groups. on the other hand, alp et al.(20) reported that hypertension in turkish people is not related to polymorphisms occurring both in mmp-9 and nos3. our study also showed that mmp-9 polymorphism was not significantly associated with angiotensin ii. the result of no significant differences between polymorphism mmp-9 and ang ii in hypertension cases in our study is unclear. the tendency to increased blood pressure in the elderly subjects is a normal phenomenon unrelated to hypertension. with respect to the emergence of coronary heart disease, no is able to regulate mmp-9 activity through guanylyl-cyclase dependent and independent pathways, chemically through protein modification by reactive nitrogen species, biologically through modulation of mmp-9/ timp-1 balance via soluble guanylyl-cyclasedependent pathways, and proteolytically through the regulation of mmp-1 and mmp-13 which can cleave the prodomain of mmp-9.(21) figure 1. electropherogram of mmp-9 gene fragment pcr products. note: pb = base pair(s) figure 2. sequencing products of genotypic variation in mmp-9 genes 219 inhibition of nitric oxide synthase (nos) can increase the expression of mmp-9, so decreasing no levels will increase mmp-9 activity.(22) the increased activity of mmp-9 enzyme causes vasodilation and extremely excessive activity will lead to changes in elastic t i s s u e i n t h e b l o o d v e s s e l s , c a u s i n g vasoconstriction.(18) the results of previous studies on mmp-9 promoter polymorphism showed that the t allele results in a higher promoter activity compared with the c allele, because of the preferential binding of a transcription repressor protein to the c allele.(23) the t allele has also been associated with higher plasma mmp-9 levels in patients with coronary artery disease (cad).(19) angiotensin ii increases the expression of mmp-9 genes by inducing monocytes, through the g protein/non-g pathway, mobilization of c a 2+, m i t o g e n a c t i v a t e d p r o t e i n k i n a s e s (mapk), receptor and non-receptor tyrosine kinase, janus family kinases-signal transducers and activators of transcription-suppressors of cytokine signaling (jak-stat-s), small g proteins (ras, rho, rac, and others), and activation of nadph oxidase, which further increases mmp-9.(24,25) the increased levels of mmp-9 cause matrix degradation in the tunica intima of blood vessels that are involved in vasodilation. the condition of excessive mmp9 enzymes alter the elastic tissue of blood vessels to become stiff and cause vasoconstriction.(26) in the present study, for some effects the explanation of mmp-9 polymorphism acting synergistically in the vasoconstriction process is not sufficient, so these effects might be influenced by other factors. this study only examined the polymorphism in the promoter region of mmp-9 without measuring the mmp-9 enzyme levels. the age range of the subjects and the number of subjects have no significant influence on the results of our study. however, the knowledge of the relationship of mmp-9 polymorphisms, no and angii levels in the javanese population in connection with essential hypertension opens opportunities for early detection of hypertension by determination of biomarkers. conclusion in ethnic javanese hypertension patients, there occurs polymorphism of mmp-9 genes, an association between polymorphism of mmp-9 genes, angiotensin and no levels. acknowledgements we would like to thank the head of health research and development ministry of health of indonesia, through the risbin iptekdok program for 2012 as funder, along with eijkman m o l e c u l a r b i o l o g y i n s t i t u t e a s r e s e a r c h supervisor. we also like to thank the prolanis patients in several family clinics in banyumas who willingly became our research subjects. references 1. sugiharto a. faktor-faktor risiko 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22. knipp bs, ailawadi g, ford jw, peterson da, eagleton mj, roelofs kj, et al. increased mmp9 expression and activity by aortic smooth muscle cells after nitric oxide synthase inhibition is associated with increased nuclear factor-êb and activator protein-1 activity. j surg res 2004;116: 70-80. 23. alp e, menevse s, tulmac m, kan d, yalcin r, erkan af, et al. lack of association between matrix metalloproteinase-9 and endothelial nitric oxide synthase gene polymorphisms and coronary artery disease in turkish population. dna cell biol 2009;28:343-50. 24. yaghooti h, firoozrai m, fallah s, khorramizadeh m. angiotensin ii induces nfêb, jnk and p38 mapk activation in monocytic cells and increases matrix metalloproteinase-9 expression in a pkc-and rho kinase-dependent manner. braz j med biol res 2011;44:193-9. 25. knipp bs, ailawadi g, ford jw, peterson da, eagleton mj, roelofs kj, et al. increased mmp9 expression and activity by aortic smooth muscle cells after nitric oxide synthase inhibition is associated with increased nuclear factor-êb and activator protein-1 activity. j surg res 2004;116: 70-80. 26. medley tl, kingwell ba, gatzka cd, pillay p, coleet tj. matrix metalloproteinase-3 genotype contributes to age-related aortic stiffening through modulation of gene and protein expression. circ res 2003;92:1254-1261. women and stroke patients are more at risk for fall10 *research and development center of community health efforts **health resource and services center, health research and development agency, ministry of health, repubic of indonesia correspondence address sulistyowati tuminah jl. percetakan negara 29, jakarta pusat email: sdarjoko@yahoo.com / watidarjoko@gmail.com univ med 2016;35:10-8 doi: 10.18051/univmed.2016.v35.10-18 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 women and stroke patients are more at risk for fallrelated injury among older persons sulistyowati tuminah*, woro riyadina*, and aprildah nur sapardin** background in indonesia, the prevalence of injury has increased from 7.9% in 2007 to 8.2% in 2013. among older persons, falls were the main cause of injury. the objective of this study was to determine the major risk factors and the magnitude of the risk for fall-related injury in older persons. methods a study of cross-sectional design was conducted on 4,222 respondents aged 60 years and over. injury was defined as fall-related injury occurring in the previous 12 months that disturbed activity daily living (adl). data collection was done by: 1) interview about sociodemographics and health (including hearing impairment, non-communicable diseases and injury) using a questionnaire, 2) measurement of blood pressure and anthropometry (body mass index), 3) examination of vision in the right and left eyes using tumbling e cards and pinhole eye covers. data were analyzed using the chi square test and multiple logistic regression with 0.05 significance level. results after controlling for age, occupation, vision disorder, educational level and economic status, older women had a 2.0-fold increased risk for fallrelated injury compared to men (aor=2.3; 95% ci: 1.93-2.73; p=0.000); subjects with a history of stroke had a 2.1-fold increased risk for fallrelated injury compared to those without a history of stroke (aor=2.07; 95% ci: 1.38-3.09; p=0.000). educational level was a confounding factor. conclusion women and stroke sufferers were at higher risk of fall-related injury among older persons. prevention of fall-related injury should be done by older persons through periodic control of their health condition. keywords: injury, stroke, fall, older persons doi: http://dx.doi.org/10.18051/univmed.2016.v35.10-18 11 univ med vol. 35 no.1 introduction the prevalence falls dan fall-related injuryin populasi lanjut usia (lansia) semakin meningkat. falls are one of the causes of death from injury among older persons. around 2030% of older persons with moderate and severe injury may suffer from loss of independence, decrease in quality of life and eventually death.(1) in indonesia, the prevalence of injury showed a slightly increasing trend from 7.9 percent in riskesdas 2007 to 8.2 percent in riskesdas 2 0 1 3 . ( 2 , 3 ) o f t h e p e r c e n t a g e o f i n j u r e d respondents, the proportion of falls (40.9%) was the second greatest cause of injury after land transport accidents (47.7%).(3) the risk factors of falls are categorized as internal and external. internal factors comprise age, gender, problems of balance associated with w a l k i n g s t y l e , f u n c t i o n a l a n d c o g n i t i v e impairment, visual problems, and comorbidities such as depression, stroke, parkinsonism, and arthritis. external factors are closely associated with home hazards, such as slipping, stumbling, and falling from loss of balance, e.g. wet floors, slippery floors, uneven floor surfaces, inadequate lighting, absence of handholds, absence of walking aids, and others.(4-7) the study results of rodriguez et al.(8) showed that falls were more frequently experienced by female older persons and older persons aged 80 years and above. according to jalayondeja,(9) falls are common occurrences in healthy older persons and are more frequently experienced by stroke patients. in individuals with stroke, the incidence of firsttime falls ranged from 3.8 to 22%, whereas the incidence of falls was around 1.3-6.5 times in the year following a stroke event. data on fall-related injury in riskesdas 2007 and 2013 were respectively representative of data from the national level down to the district level in indonesia. however in riskesdas 2007, data on fall-related injury comprised all types of fall-related injury, including slight injury, so that its prevalence was greater.(10) this is in contrast with the data obtained in riskesdas 2013, which were focussed exclusively on fallrelated injury that impaired the activities of daily living (adl), and which constitute the novelty of the present study. the objective of the present study was to determine the most dominant risk factors of fall-related injury among older persons in indonesia and the magnitude of the risk. methods design of the study this advanced analysis of a subset of the riskesdas 2013 data was of cross sectional design. the complete advanced analysis consisting of the writing of the proposal, protocol, and a request for data to the data management laboratory (lab mandat), data analysis, and writing of the report, was conducted from january to december 2014. study subjects the respondents who were selected for the riskesdas 2013 sample came from 33 provinces. the inclusion criteria were: 1) male and female respondents, 2) aged 60 years and above (older persons), 3) respondents who in the previous 12 months experienced fall-related injury that impaired their adl. the number of respondents meeting the inclusion criteria was 4,222 persons. interviews the interviews were conducted on elderly respondents aged 60 years and above. the age of the respondents was the difference in years between the last birthday and the birth date. these data were of ordinal scale and were divided into 3 categories, i.e. 60-69 years, 70-79 years, 80 years, and above. the dependent (outcome) variable was fall-related injury and was obtained through 2 questions: “within the last 12 months, did the respondent experience events that resulted in injuries?” and “what were the causes of these injuries?”.(11) the injuries referred to were injuries that impaired adl. the responses to the experiencing of injuries were divided into 2 categories, i.e. “yes” (experienced injuries) and 12 “no”, while the cause of injury that was the focus of the present study was falls. in this case falls were causes of unintentional injury and resulted in the person falling to the ground, e.g. gliding, stumbling, falling from heights.(12) the causes of injuries were grouped into 2 categories, i.e. “due to falls” and “not due to falls” (land transport accidents, injuries from sharp/blunt objects, burns/scalds, animal attacks/bites, etc.). the study sample consisted of the respondents who answered “yes” (had experienced injuries) and “falls” as the cause. other independent variables that were collected comprised socio-economic factors (education, occupation, and economic status), biological factors (gender, history of illness, health status, and physical condition). the education of the respondents was the final completed educational stage of the respondents who received diplomas. the education of the respondents were grouped into 3 categories, i.e. low (no schooling/never went to school, did not finish primary school and finished primary school), middle (finished junior high school and finished senior high school), and high (finished d1/d2/d3 and finished tertiary education). the occupation of the respondents was a composite of their occupational status and the type of their main occupation. the main occupation of the respondents was grouped into 5 categories, i.e. non-employed, employee, entrepreneur, farmer/ fisher/laborer and others. the economic status was determined according to the quintile of ownership index, which was calculated from several variables of ownership of durable goods. the economic status was grouped into 5 categories, i.e. lowest, lower middle, middle, upper middle, and upper. what was meant by “history of illness of the respondents” in this case were the diseases suffered by the respondents based on the diagnosis established by health personnel (physicians/nurses/midwifes). for example, diagnosis of hypertension, or consumption of anti-hypertensive medications, coronary heart disease (chd) or heart failure, renal disease, diseases of the joints/rheumatism or stroke. respondents were said to have a history of hypertension if they were diagnosed as having hypertension by health personnel or had never been diagnosed as having hypertension by health personnel but consumed anti-hypertensive medications.(3) the responses for history of illness was divided into 2 categories, i.e “yes” and “no”. what was meant by “health status of respondents” in this analysis was the health condition of the respondents at the time of data collection, e.g. presence of high blood pressure (hypertension), visual or hearing impairment. the information on blood pressure and visual impairment was obtained by measurement or examination, whereas the information on hearing impairment was obtained from the interviews based on the report of the respondents. the response options were 1) yes, in one ear; 2) yes, in both ears; 3) yes, fluctuating hearing impairment; 4) no hearing impairment; 8) do not know. in this analysis, hearing impairment was grouped into 2 categories, i.e. “yes” (response code 1, 2, or 3) or “no” (response code 4 or 8).(13) measurements blood pressure was determined using an omron ia1 digital tensimeter on the left arm, and performed twice with a 2-3 minute interval. if the results of the two determinations differed >10 mm/hg for systolic or diastolic blood pressure, a third measurement was done after a rest of 2-3 minutes. high blood pressure (hypertension) was grouped according to the guidelines of the 8th joint national committee (jnc) for 2014.(14) however, in this study blood pressure was categorized into 2, i.e. high (hypertension stage 1 and 2) and normal (normotension and prehypertension). for hypertension associated with health status, the respondents were said to have hypertension if they had a history of hypertension or had hypertension at the time of examination or if the blood pressure at the time of measurement was tuminah, riyadina, sapardin women risk for fall related injury 13 univ med vol. 35 no.1 not high because they had previously taken antihypertensive medications. if at the time of m e a s u r e m e n t t h e b l o o d p r e s s u r e o f t h e respondents was not high but the respondents had previously consumed anti-hypertensive m e d i c a t i o n s , t h i s w a s c a l l e d c o n t r o l l e d hypertension. the physical condition in this case was seen from the body mass index (imt) status that was obtained from the anthropometric measurements (weight and height). weight was measured using camry digital scales with a capacity of 150 kg and precision of 100 g. the respondents were recommended to wear thin or the lightest clothing, and were weighed in the standing position without footwear. height was measured in the upright position using a multifunctional height measuring instrument with a capacity of 2 meters and precision of 0.1 cm. the body mass index was obtained by first measuring weight and height, then calculating with the formula: bmi = weight (kg) / height (m)2 in general, obesity was divided into 5 categories obtained from the bmi classification for asians according to the international obesity task force (iotf).(15) these obesity categories were underweight (bmi <18.5), normal (bmi 18.5-22.9), overweight (bmi 23.0-24.9), obese 1 (bmi 25.0-29.9) and obese 2 (bmi 30 or higher). in this study the bmi was grouped into 2 categories only, i.e. obese (bmi 25 or higher) and non-obese (bmi less than 25). visual examination visual impairment may be determined by v i s u a l a c u i t y t e s t i n g o n b o t h e y e s . t h e instruments used were a 6-m measuring tape, one set of tumbling e cards (of great, medium, and small sizes), and eye-shields with a pinhole. the eye examination was started on the right eye of the respondent, without a pinhole. the response options for the visual examination results were as follows: 1) capable of seeing small e cards (at a distance of 6m); 2) not capable of seeing small e cards, but capable of seeing medium e cards (at a distance of 6m); 3) not capable of seeing medium e cards, but capable of seeing large e cards (at a distance of 6m); 4) not capable of seeing large e cards (at a distance of 6m), but capable of seeing large e cards (at a distance of 3m); 5) not capable of seeing large e cards at a distance of 3m; 6) cannot be examined. if there is slight visual impairment (code 2), low vision (code 3 and 4) or blindness (code 5), then it is recommended to perform a pinhole examination. after the columns for examination without and with pinhole of the right eye have been completely filled in, this is followed by examination of the left eye. visual impairment was grouped into 2 categories, i.e. yes and no. respondents were said to have visual impairment (yes) if both eyes had codes 2, 3, 4, or 5. respondents were said not to have visual impairment if either one of the eyes had code 1.(13) data analysis the data were analyzed using: 1) bivariate analysis (chi square) to determine differences between the proportions of the dependent and independent variables; 2) multiple logistic regression analysis to determine the relationship between females and stroke patients with regard to fall-related injury in older persons, after controlling for confounding variables. ethical clearance this protocol of the basic health research (riset kesehatan dasar, riskesdas) for the year 2013 has been accorded ethical clearance from the ethics commission of the health research and development agency, ministry of health, repubic of indonesia. results the data on fall-related injury in older persons that could be analyzed comprised 4,222 cases (65.4%). based on biological factors (table 1), the proportion of older persons with fall-related injury was increased with increasing age. there were more female elderly with fall14 related injury (75.8%) than male elderly. with regard to physical condition, there were no significant differences between the proportion of obese older persons with fall-related injury and that of the non-obese. on the other hand, with regard to history of illness, the proportion of older persons with fall-related injury was larger in respondents who had been diagnosed by health personnel as having hypertension/consumed anti-hypertensive medications, heart failure, renal disease, diseases of the joints/rheumatism or stroke. similarly with respondents who had coronary heart disease, although the differences in proportion were not significant. while according to the health status of the respondents at the time of data collection, there were more older persons with fall-related injury among the respondents with hypertension, and with visual or hearing impairment. from the results of the bivariate analysis, the variables that could be included in the multivariate analysis were age, gender, history table 1. frequency distribution of fall-related injury in older persons by biological factors tuminah, riyadina, sapardin women risk for fall related injury 15 univ med vol. 35 no.1 of illness (hypertension/medications, heart failure, renal disease, diseases of the joints/ r h e u m a t i s m a n d s t r o k e , h e a l t h s t a t u s (hypertension, visual impairment and hearing i m p a i r m e n t ) , e d u c a t i o n , o c c u p a t i o n a n d economic status. the highest proportion of older persons with fall-related injury was found among the respondents with low educational level, unemployed, with lowest econmoic status (table 2). the results of multivariate analysis showed that the risk of older persons for being injured by falls increased with increasing age, with the magnitude of the risk (aor=1.94;95% ci: 1.462.58; p=0.000) for the age group of 80 years and older. female older persons had a 2.3-fold i n c r e a s e d r i s k f o r f a l l r e l a t e d i n j u r y (aor=2.30;95% ci: 1.93-2.73; p=0.000) in comparison with male older persons. older persons with a history of stroke had a 2.1-fold i n c r e a s e d c h a n c e o f f a l l r e l a t e d i n j u r y (aor=2.07;95% ci: 1.38-3.09; p=0.000) in comparison with older persons without stroke. older persons with visual impairment had a 1.4fold increased chance of suffering from fallrelated injury (aor=1.36; 95% ci: 1.14-1.64; p=0.001) in comparison with older persons without impaired vision (table 3). discussion falls in older persons are the main cause of morbidity and mortality from unintentional injury in the united states in 2006, accounting for 45.4%.(16) this figure is lower than the prevalence of fall-related injury in older persons (aged >60 years) in the year 2013 in indonesia (65.4%). this may be caused by the relative low socio-economic conditions of the indonesian community so that an advanced age older persons must still be more actively working to meet their necessities of life. from the aspect of age, the risk of older persons to experience fall-related injury increases with advancing age. this agrees with the study by pi et al.(17) in china, who found that in the majority of cases falls occurred in individuals who were over 80 years of age. however, in the study conducted by orces (18) the investigators found different results. this may be because in i n d o n e s i a m a n y o l d e r p e r s o n s h a v e l o w educational level and lower-middle economic status, so that in their old age they have to continue working hard. in general, persons with low educational level perform more physical work. with increasing age, the body becomes physically weaker through exhaustion. therefore table 2. associations between socio-economic factors and fall-related injury in older persons 16 their risk of experiencing fall-related injury also increases. according to gender, female older persons have a 2.3-fold greater risk of fall-related injury than do males. this is in agreement with the study results of kuang et al.(19) showing that females have a 2.3 fold greater risk of falls. this is because female older persons are at higher risk for osteoporosis than are males. the presence of osteoporosis increases the risk for fractures after falls.(10,20) on the other hand, our results differ from the study results of grundstrom et al.(16) who found a higher risk of fall-related injury in males than in females. with regard to history of illness, older persons who ever had stroke had a 2.1-fold increased risk of experiencing injury from falls. similar results were obtained by thurman et al.(21) and orces (18) who found that subjects with table 3. multiple logistic regression analysis of risk factors for fall-related injury in older persons tuminah, riyadina, sapardin women risk for fall related injury stroke had a greater risk of falls in comparison with subjects without stroke. this is because in general stroke attacks decrease the functions of the patient’s extremities and disturb the body balance so increasing the risk of falls. older persons with visual impairment have a 1.4-fold higher risk of fall-related injury in comparison with older persons without impaired vision. this is in accordance with the study results of kuang et al.(19) who found that visual impairment increases the risk of falls almost 2-fold in comparison with normal subjects. this is because at their age (60 years and above), the older respondents usually have visual impairment and move less actively, thus decreasing their strength and balance, which increases their risk of falls. our study results showed that educational level was not a risk factor for falls, which differs 17 univ med vol. 35 no.1 control their health status by routine blood pressure measurements, periodic ophthalmologic examinations, positive socializing such as participating in health gymnastics or religious studies (pengajian) to prevent boredom, and using walking aids if necessary. based on the results of this study, the ministry of health and the regional government should cooperate in increasing community health services by maximizing the functioning of community health centers (puskesmas) in monitoring and guiding the community, particularly those families at high risk of poor health (both healthy older persons and those with chronic diseases). conclusion older women and those with a history of stroke are at greater risk for fall-related injury. conflict of interest there was no conflict of interest in this study. acknowledgment the authors express their deep-felt gratitude to: prof. dr. bastaman basuki and dr. iwan ariawan of fkm-ui who provided guidance and directions in the analysis of the riskesdas data, so that the results may be of benefit to all parties, particularly to decision and policy makers. our colleages of the advanced analysis team for their cooperation so that this study could proceed smoothly and succesfully. references 1. jamebozorgi aa, kavoosi a, shafiee z, et al. investigation of the prevalent fall-related risk factors of fractures in elderly referred to tehran hospitals. med j islamic republic iran 2013;27:23-30. 2. riyadina w. profil cedera akibat jatuh, kecelakaan lalu lintas dan terluka benda tajam/ tumpul pada masyarakat indonesia. jurnal penyakit tidak menular. 2009;1:1-11. from the study results of hayashi et al.(22) in japan and qader et al.(23) in iraq. these investigators found that the risk of injury from falls was significantly higher in older persons with low educational level in comparison with those of older persons with high educational level. in contrast, with regard to the lowest ownership index (socioeconomic status), our study showed similar results with those of the studies by hayashi et al(22) and qader et al,(23) in that the risk of falls in older persons with low socio-economic status was significantly greater than in older persons with high socio-economic status. this is because the respondents with high socio-economic status have better welfare, nutrition, and health services. nonworking older persons have a 1.6-fold greater risk of fall-related injury in comparison with working older persons. these results agree with those of the iraqi study, in that the risk of fall-related injury in non-working older persons is significantly greater in comparison with older persons of working status. psychological factors, especially depression, as a result of having no occupation, which is caused by age and health conditions, are important risk factors for the occurrence of falls.(23) one limitation of this study is the lack of data on impairment of balance so that it cannot explain the role of balance in the risk of falls in older persons. in addition, the lack of information on the conditions of the site of injury, e.g. multistory buildings, slippery floors, roads with potholes or uneven surfaces and others, so that these cannot be used as further information on the role of environmental factors on the risk of falls. there is a need for further studies to determine the role of environmental conditions on fall-related injury. to reduce the resulting morbidity and mortality, internal preventive measures need to be taken, i.e. behavioral changes for a healthy life style, such as consumption of healthy foods to avoid diseases such as stroke, limiting salty foods to avoid hypertension, managing emotions to avoid stress, sufficient rest and routine physical exercise.(23) it is important for older persons to 18 3. kementerian kesehatan. pokok-pokok hasil riskesdas 2013. jakarata: badan penelitian dan pengembangan kesehatan kementerian kesehatan;2014. 4. lok n, akin b. domestic environmental risk factors associated with falling in elderly. iranian j publ health 2013;42:120-8. 5. damian j, pastor-barriuso r, valderrama-gama e, et al. factors associated with falls among older adults living in institutions. bmc geriatrics 2013;13:6. 6. sabatini sn, kusuma he, tambunan l. faktor eksternal risiko jatuh lansia: studi empiris. prosiding temu ilmiah iplbi; 2015 okt 30-31; manado, sulawesi utara; 2015.p.1-6. 7. reed-jones rj, solis gr, lawson ka, et al. vision and falls: multidisciplinary review of the contribution of visual impairment to falls among older adults. maturitas 2013;75:22-8. 8. rodriguez ig, fraga gp, de azevedo barros mb. falls among the elderly: risk factors in a population-based study. rev bras epidemiol 2014;705-18. doi: 10.1590/1809-45032014000 30011. 9. jalayondeja c, sullivan pe, pichaiyongwongdee s. six-month prospective study of fall risk factors identification in patients post-stroke. geriatr gerontol int 2014;14:778-85. 10. riyadina w. cedera akibat jatuh pada penduduk usia lanjut yang mengalami obesitas di indonesia. jurnal penyakit tidak menular 2009; 1:73-83. 11. williams sj, kowal p, hestekin h, et al. prevalence, risk factors and disability associated with fall-related injury in older adults in lowand middle-income countries: results from the who study on global ageing and adult health (sage). bmc medicine 2015;13:1-12. doi: 10.1186/s12916-015-0390-8. 12. kameel mh, abdulmajeed aa, ismail se. risk factors of falls among elderly living in urban suez – egypt. pan african med j 2013;14:26. doi:10.11604/pamj.2013.14.26.1609. 13. kementerian kesehatan ri. pedoman pemeriksaan riset kesehatan dasar. jakarta: badan penelitian dan pengembangan kesehatan; 2013. 14. american academy of family physicians. joint national committee (jnc) 8 guidelines for the management of hypertension in adults. am fam physicians 2014;90:503-4. 15. gray lj, yates t, davies mj, et al. defining obesity cut-off points for migrant south asians. plos one 2011;6: e26464. doi:10.1371/ journal.pone.0026464. 16. grundstrom ac, guse ce, layde pm. risk factors for falls and falls-related injuries in adults 85 years of age and older. arch gerontol geriatr 2012;54: 421-8. 17. pi hy, hu mm, zhang j, et al. circumstances of falls and fall-related injuries among frail elderly under home care in china. int j nursing sciences 2015;2:237-42. available at: http:// dx.doi.org/10.1016/j.ijnss.2015.07.002. 18. orces ch. prevalence and determinants of falls among older adults in ecuador: an analysis of the sabe i survey. current gerontol geriatr res 2013;1-7. article id 495468, 7 pages. http:/ /dx.doi.org/10.1155/2013/495468. 19. kuang tm, tsai sy, hsu wm, et al. visual impairment and falls in the elderly: the shihpai eye study. j chin med assoc 2008;71:467-72. 20. prihatini s, vita km, abas bj, et al. faktor determinan risiko osteoporosis di tiga provinsi di indonesia. media litbang kesehatan 2010;20: 91-9. 21. thurman dj, stevens ja, rao jk. practice parameter: assessing patients in a neurology practice for risk of falls (an evidence-based review): report of the quality standard subcommittee of the american academy of neurology. neurology 2008;70:473-9. 22. hayashi t, kondo k, suzuki k, et al. factors associated with falls in community-dwelling older people with focus on participation in sport organization: the japan gerontological evaluation study project. bio med research int 2014;1-10. 23. qader maa, amin rm, shah sa, et al. psychological risk factors associated with falls among elderly people in bagdad city. iraq open j prev med 2013;3:441-5. tuminah, riyadina, sapardin women risk for fall related injury cadmium increase prostate specific antigen 42 *department of public health and community medicine faculty of medicine jenderal soedirman university purwokerto correspondence: dr.dr.nendyah roestijawati, mkk department of public health and community medicine faculty of medicine jenderal soedirman university purwokerto jl. dr. gumbreg no. 1 mersi purwokerto phone: +62281 622022 fax: +62281 624990 email: nendyahr@yahoo.com univ med 2017;36:42-8 doi: 10.18051/univmed.2017.v36.42-48 pissn: 1907-3062 / eissn: 2407-2230 received february 2, 2017 accepted for publication april 20, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract blood cadmium levels increase prostate specific antigen and insulin-like growth factor-1 among cadmium exposed workers nendyah roestijawati*, lintje setyawati maurits*, and sugiyanto* background cadmium (cd) is a heavy metal that is classified as a human carcinogen (group ia), one of the cancers that it can cause being prostate cancer. the development of prostate cancer on a molecular basis involves oncogenes such as insuline-like growth factor-1 (igf-1). prostate cancer can be detected in the laboratory through the examination of prostate specific antigen (psa). the present study aimed to determine the relationship of cd levels with levels of psa and igf-1 in exposed and unexposed workers. methods the study design was cross sectional. the subjects of the studycame from two groups of workers, ie. the group of cd exposed workers who were welding shop workers and the group of unexposed workers who were office workers. the minimum samplesize was 85 people. the independent variable was blood cd level. the dependent variables were psa and igf1 levels. blood cd levels were measured by atomic absorption spectrometry (aas), while psa and igf-1 were measured using elisa. data analysis was performed using the mann-whitney test and the spearman correlation test. results mean blood cd level in the exposed workers was 6.5 µg/l and in the unexposed workers 2.15 µg/l. there was a relationship between blood cd and psa levels (p<0.05) and between blood cd levels and igf-1 (p <0.05). conclusions there was a relationship of blood cd with psa and igf-1 levels.among workers. psa and igf-1 could be a biochemical markers of disease control in cadmium exposed workers. keywords: cadmium, prostate specific antigen, insuline-like growth factor-1, workers doi: http://dx.doi.org/10.18051/univmed.2017.v36.42-48 universa medicinajanuary-april, 2017 vol.36 no.1 original article 43 univ med vol. 36 no.1 introduction cadmium (cd) is a heavy metal that has carcinogenic effectson humans (group ia) according to the international agency of research on cancer (iarc). cadmiumis carcinogenic in humans, as has been determined based on studies in humans and experimental animals. several studiesofcd exposed workers claim significant increases in lung cancer, although there is still controversy about disturbing exposures to other metals, such as arsenic or nickel.(1) in addition to lung cancer, cd is also suspected to cause prostate cancer in humans, because cd accumulates in the prostate and testes, as well as in the kidneys and liver. the study showed increased cd in the blood and prostate of mice injected with cd. the amount of cd accumulated in the prostate is approximately 50% of the total cd in the body and is linearly associated with the amount of cd accumulated in the liver and testis.(2) research by guzel et al.(3) proved the effect of cd exposure on prostate cancer, with their results showing elevated cd levels in patients with prostate cancer. the development of prostate cancer on a molecular basis involves many gene expressions, suchas 1q24-1q25 (hpc1),1q42-q43(pcap), xq27-q28 (hpcx), 20q13 (hpc20), and mutations in tumor suppressor genes such as p53, pten, cdkn1b, mx11, nkx3.1, glutathione s-transferase gene (gstp-1) and several oncogenes such as insulin-like growth factor-1 (igf-1).(4) igf-1 can cause cancer through inhibition of apoptosis and stimulation of cell proliferation. epidemiological studies reported a positive correlation of igf-1 with prostate cancer.(5) the laboratory indicator for prostate cancer is prostate specific antigen (psa). cd is currently one of the most extensive occupational and environmental pollutants. some studies suggest a relationship between cd and psa. the results of a study by de coster et al.(6) showed a significant correlation between urinary cd levels of ≥p90 (1.24 µg/l) and psa levels in the population group aged 50-65 years living in nine areas with different levels of air pollution.on the contrary, the results of a study by pizent et al.(7) found no significant association between blood cd and psa in men with a history of work unexposed to cd. this indicates that a relationship will be found between cd and psa at high cd levels but not at low cd levels. natural conditions of cd release into the environment are due to the weathering of sediments from the effects of weather, erosion and volcanic activity. on the other hand, under anthropogenic conditions cd is released into the environment due to human activity. relations between cd and igf-1 have been investigated by chen et al.(8) the results showed a proportional relationship between cd and igf-1 levels, with higher cd levels at high igf-1 levels. workers in a large variety of occupations, especially those who are involved in the manufacture of alloys and batteries, and nonferrous metal smelting and refining, are exposed to high levels of cadmium through inhalation of dust and fumes, and incidental ingestion of dust from contaminated hands, cigarettes or foods.(9) the high levels of blood cd are influenced by workplace environmental factors, foods/beverages and tobacco. the cd exposed workers comprise such groups as police officers, smelter workers, and workers in metal casting and welding.in nature, cd, such as electroplating, mining, smelting, use of pesticides, fertilizers, etc.(10) welding shop workers are at risk of exposure to cd. cd sources of exposure among welding shop workers are from cd fumes generated in the welding process. cd levels in the air depend on the workplace environment for welding filler metals, the welding techniques used and the condition of the workplace. high levels of environmental cd lead to high levels of cd in the blood that can cause prostate cancer. several e p i d e m i o l o g i c s t u d i e s i n v e s t i g a t i n g t h e a s s o c i a t i o n b e t w e e n c d e x p o s u r e a n d susceptibility to prostate cancer have yielded inconsistent findings. high levels of cd in exposed workers was proven by the research of 44 moitra et al.,(11) which showed a significant difference in blood and urine levels of cd in jewelry factory workers (5.8 µg/dl) as compared with those in unexposed workers, such as jewelry sales employees (0.41 µg/dl). the indonesian national standard (sni) for cd exposure in the workplace is 10 µg/m3 for metallic cd and 2 µg/ m3 for cd in the form of compounds.in men, cd was associated with cancers of the lung and pancreas and with non-hodgkin lymphoma, but not with prostate cancer.(12) the present study aimed to determine the relationship of cd levels with levels of psa and igf-1 in cd exposed and unexposedworkers. methods research design this study was an observational analytic study of cross sectional design. the research was carried out in 8 months (january to august 2015) and was located in purwokerto and banyumas city, banyumas district. research subject the subjects of the research came from two groups of workers, one group consisting of cd exposed workers and the other group consisting of unexposed workers. the cd exposure group were welding workers and the group of workers unexposed to cd were office workers. thesample size was determined based on a 0.05 significance level and power of 80%, with correlation of 0.3, giving a minimum sample size of 85 people. samples were selected by consecutive sampling in accordance with the inclusion criteria, which weremales over 30 years of age and having worked in the workplace for a minimum of 6 months. the exclusion criteria wasacquiring an acute illness (elevated body temperature) during the last 4 months. laboratory analysis blood samples were collected from the median cubital vein.prior to blood sampling, the subjects were asked to fast for 10 hoursand not to have sexual intercourse during 2x24 hours. blood sampling was performed at 7:00 a.m. to 9:00. approximately 10 ml of blood was drawn into small purple-top vacutainer tubes. following clotting, the blood tube was centrifuged at 2500– 3000 x g for 5 min. the plasma supernatant was placed in a sterile acid-washed, metal ion–free microfuge tube, and stored frozen at -20°c until needed for the assays.(13) cadmium levels were determined using s t a n d a r d l a b o r a t o r y p r o t o c o l s . i n b r i e f , specimens were acid digested under high pressure in a microwave oven and then assayed for cadmium in a perkin elmer 3110 (waltham, ma, usa). we used a calibration curve prepared from a serially diluted cadmium standard (merck, germany).(13) psa and igf-1 levels were determined using human elisa kit (sunred biotechnology company, ltd, shanghai), based on the principle of the double-antibody sandwich technique, and assayed on an elisa reader 270 (biomeureux, france). laboratory analysis took place at the laboratory of the institute for research, faculty of medicine, jenderal soedirman university, purwokerto. data analysis data analysis to determine differences between groups was by means of the mannwhitney test, because the data were not normally distributed. data analysis to determine whether there was a correlation, was done using the spearman correlation test. ethical clearance t h i s s t u d y w a s a p p r o v e d b y t h e commission on health research ethics, faculty of medicine, jenderal soedirman university (unsoed) with ref no: 062/kepk/iii/2014. results the study was conducted on 85 subjects, consisting of 40 cd exposed workers and 45 unexposed workers. the average age of the cd roestijawati, maurits, sugiyanto cadmium increase prostate specific antigen 45 univ med vol. 36 no.1 exposed workers was higher than that of the unexposed workers. there was no significant d i ff e r e n c e i n a g e b e t w e e n e x p o s e d a n d unexposedworkers (p>0.05).the averageof duration of work of thecd exposed workers was longer than that of the unexposed workers. there was no significant difference in duration of work between the exposed and unexposedworkers ( p > 0 . 0 5 ) . t h e r e w a s n o d i ff e r e n c e i n characteristics between exposed and unexposed workers, as presented in table 1. statistical analysis of blood cd, psa and igf-1 ofthe groups showed differencesin levels of blood cd and psa between cd exposed workers and unexposed workers (p<0.05) but no difference in level of igf-1 between cd exposed and unexposed workers (p>0.05), as presented in table 1. the relationship between blood cd and psa was statistically significant (r=0.445; p<0.05), being weakly positive. similarly, the relatioship between blood cd and igf-1 was statistically significant (r=0.218; p<0.05), being also weakly positive, as presented in table 2. further analysis found a statistically significant relationship between igf-1 and psa (r=0.502; p<0.05), the relationship being moderately positive, as presented in table 2. discussion the present study showed statistically significant differences in the levels of cd between exposed and unexposed workers. cd levels in exposed workerswere higher than those in unexposedworkers. the results of this study was similar with those of golbabaei et al.(14) in iran, who found differences in levels of urinary cd in welders and controls. the results of our study on exposed workers showed that blood cd concentration was 6.5mg/ l. these resultsare different with those of the study by idham (15) on welders in an automotive plant in jakarta, with the blood cd levels ranging from 1.28 µg/l up to 43.33 µg/l, and mean of 14.29 µg/l. the differences in these results are due to the different characteristics of the workshops. the weldingprocess in idham’s research was conducted in a closed room with a ventilation system, whereas the welding process in the present study was conducted in an open l o c a t i o n s o t h a t t h e m e t a l f u m e s immediatelyevaporate into the surrounding air. according golbabaei et al.(14) welders working in an enclosed space with poor ventilation are at a 150 times higher risk than are welders in open space. table 1.subject characteristics and difference of blood cd, psa and igf-1 between groups *significant 0.05, tested by mann-whitney test table 2. correlation between blood cd, psa and igf-1 (n=85) *significant p<0.05 (2-tailed), tested by spearman test 46 the mean blood cd of unexposed workers was 2.34 µg/l. sources of cd exposure in this group are the environment, cigarettes, foods and beverages, which contain cd. a total of 53% of the subjects were smokers. according to the national research council (us) subcommittee on zinc cadmium sulfide, the average cadmium intake through food varies between countries and individuals. intake of nonindustrial cd in rural areas is estimated to be 10-60 µg/day, and in a polluted area in japan it was reported to be higher (500 µg/day). cd intake from cigarettesis estimated at 0.1-0.2 ìg per cigarette, so it can be shown that in individuals who smoke 20-40 cigarettes/day, the intake of cd is 2-8 ìg/day. the results of the study by he et al.(16) in china found that mean exposure to cd was 16.7 µg/ day or 33.8% of the daily tolerable intake. foods and cigarettes are a major source. cd exposure from food was 12.8 µg/day, while that from cigarettes was 3.9 mg/day. most of the unexposed workers reside and work in urban areas. the results of research by mohmandet al.(17) was that one of the heavy metals contained in the air is cd. cd levels in urban areas are higher than in rural areas. according to the national research council (us) subcommittee on zinc cadmium sulfide, in america, people who breathe 20 m3 of air per day and use 10% of their time outside their homesare estimated to absorb 0.1-0.8 µgcd/day in urban areas and less than 0.02 µg/day in rural areas. the results of ourstudy showed differences in psa levels between exposed and unexposed workers. the results of the analysis of the correlation between blood cd and psa were found to be statistically significant. the results of our study are similar to those of zeng et al.(18) in china, who found that subjects with a positive digital rectal examination and psa blood levels above normal limits, have blood cd levels above normal. the mechanism of the effect of cd on psa levels is to date still unclear. there is some conjecture about the mechanism of the effect of cd on plasma psa. several studies have strongly suggested that the effect of cd on the occurrence of prostate cancer is through changes in the expression of apoptosis regulator cells and inhibition of cell apoptosis. barriers to apoptosis of cells are characterized by decreased expression of the caspase 3 gene, p53 oncogenes, decreased rad51 and increased gadd45 oncogenes and the growth factors c-jun and igf1rb.(18,19) prostate cell proliferation caused by exposure to cd increases the psa synthesis in prostate cells. the prostate is the main source of psa production. an increase in serum psa indicates prostate pathology. results of research by mok et al.(20) in korean males found a relationship of increased risk of prostate cancer with psa. the risk of prostate cancer increased by 7% for each increase of 1 ng/ml psa. the results of our study differ from those of wu et al.(21) who found that psa levels are inversely associated with blood cadmium (bcd) and urinary cadmium (ucd) levels.multivariate logistic regression analysis showed that men with psa ≥4.0 ng/ml had an odds ratio (or=0.4; 95% ci=0.1-0.9) to have bcd of >0.49 µg/ l. the present study showed a weakly significant association between blood cd levels and igf-1. these results support the study of chen et al.(8) who found increased levels of igf1 in the liver cells of zebra fish that had been exposed to cd and under observation for 12 and 24 hours. the present study showed a moderately significant association between igf-1 and psa. according to research by saikali et al.(22) igf-1 significantly increased the invasive capacity of du145 prostate cancer cells in vitro. the data indicates a regulatory role of igf-1 signalling via both the phosphatidylinositol-3 kinase (pi3k) and the mitogen activated protein kinase ( m a p k ) p a t h w a y s i n d u 1 4 5 p r o s t a t e carcinoma cells. phosphorylation of the key elements of these pathways, akt and the mapk, following igf-1 treatment, confirmed the role of the pi3-k and mapk pathways in igf-1 signalling and correlated with invasive capacity. roestijawati, maurits, sugiyanto cadmium increase prostate specific antigen 47 univ med vol. 36 no.1 a meta-analysis by rowlands et al.(23) confirms that raised circulating lgf-i is positively associated with prostate cancer risk. these resultswere different from those of borugian et al.(24) who observed no association of prostate cancer risk with prediagnostic igfiand a positive association with prediagnostic igfbp-3 and prostate cancer, but only for the subgroup of black males and only in the third quartile. the interaction between igf-i and igfir is regulated by the igf-binding proteins (igfbps), igfbp-3 being the most abundant. at tissue level, igfbp-3 regulates the mitogenic activity and inhibits the anti-apoptotic effect of igf-i and has been linked to induction of apoptosis. in prostate cancer patients with undetectable psa levels, their igfbp-3 levels are expected to be higher, since igfbp-3 is a substrate of psa and is proteolytically cleaved by it. the inverse correlation between psa and igfbp-3 could make igfbp-3 a possible biochemical marker of disease control in prostate cancer patients.(25) the present study has limitations such as not performing measurements on igfbp-3 and not doing more clinical examinations, such as a digital rectalexaminations for confirmation of any abnormality in the prostate of subjects with psa levels above 4 ng/l. the clinical implication of this research is that igf-1 may be used as biomarker of prostate cancer. to achieve these objectives more research is needed that examines the sensitivity and specificity of igf-1 for the early detection of prostate cancer. research is also needed to d e t e r m i n e o t h e r b i o m a r k e r s o f p r o s t a t e carcinogenesis caused by cd exposure by a less expensive and equally valid method for prevention of occupational diseases. prevention can be done through controls to ensure that welding done in a confined space is provided good ventilation, through measurement of cd in the working environment, through education of workers in healthy practices in hygiene, through periodical medical checks and the use of personal protective equipment. conclusions the present study showed an association of blood cd with psa and igf-1. psa and igf1 can be used for screening of occupational diseases in workers exposed to cd. conflict of interest the authors declare that they have no competing interest. acknowledgement acknowledgements are due to the ministry of research, technology, and higher education, republic of indonesia (kemenristek dikti) for r e s e a r c h g r a n t s , t o j e n d e r a l s o e d i r m a n university and the institute for research and community service, faculty of medicine, for their cooperation and assistance. contribution nr contributed to the design the research, drafting the manuscript and responsible for the final content. nr, s, and lsm contributed to collected, analyzed and interpretate the data. nr contributed to wrote the manuscript, and all authors read and approved the final manuscript. references 1. sorahan t. lung cancer 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2015;41:110-5. doi: 10.1590/s1677-5538.ibju.2015.01.15. roestijawati, maurits, sugiyanto cadmium increase prostate specific antigen restu 133 *papua health and biomedical research and development institute, ministry of health **department of parasitology, faculty of medicine, gadjah mada university correspondence semuel sandy, m.sc, apt papua health and biomedical research and development institute, ministry of health (balai penelitian dan pengembangan biomedis papua) jl. kesehatan no.10 dok ii. jayapura phone: +62967-534539 fax: +62967-534697 e-mail: mercury.sandy56@gmail.com univ med 2014;33:133-40 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background in indonesia, there is still a high prevalence of hookworm infection, especially in poor areas with poor sanitation. the number of helminthic diseases in keerom regency was about 599 cases in 2010. this number is bound to increase due to the low sanitation, hygiene and socio-economic status of the people in the regency. the children are a group at risk for contracting infections, especially intestinal worms, which affect the child’s physical growth and intelligence. the objective of this study was to determine the risk factors of hookworm diseases in elementary school students. methods a cross-sectional study was conducted on 224 elementary school students. demographic data were obtained by questionnaire, comprising gender, parental socio-economic status, household sanitation, and personal hygiene. body mass index was calculated by measurement of body weight and height. and hemoglobin concentration was measured using a quick check hb-meter. stool samples were microscopically examined using the kato-katz method. we used chi-square and logistic regression to find predictors of hookworm infections, with level of significance at p<0.05. results the number of hookworm infection was 6.7% and the risk factor of hookworm infection among elementary school students was the habit of using footwear outdoors [or 5.3; 95% ci 1.7-17.7; p=0.004]. conclusion the use of footwear outdoors was a predictor of hookworm infections in elementary school children. an effective and efficient intervention program is needed to prevent and eradicate hookworm infection among primary school children. key words: hookworm, sanitation, hygiene, anemia, primary school children footwear as a risk factor of hookworm infection in elementary school students semuel sandy*, sri sumarni**, and soeyoko** 134 sandy, sumarni, soeyoko footwear a risk hookworm infection penggunaan alas kaki sebagai faktor risiko infeksi cacing tambang pada murid sekolah dasar latar belakang prevalensi infeksi cacing tambang di indonesia masih cukup tinggi terutama di daerah miskin yang memiliki sanitasi lingkungan yang buruk. jumlah kasus kecacingan di kabupaten keerom yaitu 599 kasus pada tahun 2010, dan tentunya kasus ini akan bertambah mengingat sanitasi, higiene dan status sosial-ekonomi masyarakat rendah. anakanak merupakan kelompok berisiko untuk tertular infeksi cacing yang berdampak pada pertumbuhan fisik dan intelegensia anak. penelitian ini bertujuan untuk menentukan prevalensi dan faktor risiko infeksi cacing tambang pada murid sekolah dasar (sd) metode desain penelitian potong lintang (cross sectional) mengikut sertakan 224 murid sd. pengambilan data demografi menggunakan kuesioner meliputi jenis kelamin, status sosial ekonomi orang tua murid, sanitasi lingkungan rumah, higiene murid pengukuran antropometri untuk mengukur status gizi dan kadar hemoglobin (hb) menggunakan quick chek hb-meter. spesimen tinja diperiksa menggunakan metode kato-katz. analisis data menggunakan statistik bivariat (chi square) dan regresi logistik untuk menentukan variabel prediktor. hasil jumlah infeksi cacing tambang sebesar 6,7% (n=224). hasil analisis regresi logistik menunjukkan faktor prediktor terjadinya infeksi cacing tambang adalah kebiasaan menggunakan alas kaki saat bermain di luar rumah dan sekolah [or 5,3; (95% ki 1,7-17,7); p=0,004]. kesimpulan kebiasaan menggunakan alas kaki saat diluar rumah merupakan faktor risiko infeksi cacing tambang pada murid sd di kabupaten keerom. diperlukan suatu intervensi yang efektif dan efisien untuk mencegah dan memberantas infeksi cacing tambang kata kunci: cacing tambang, sanitasi, higiene, anemia, murid sekolah dasar abstrak introduction infection by soil transmitted helminths (sth) is a neglected tropical disease caused by worms of the species ascaris lumbricoides, a n c y l o s t o m a d u o d e n a l e , a n d tr i c h u r i s trichiura. soil transmitted helminth infection depends on socio-economic conditions and is frequently found among children in developing countries. a high prevalence of sth infection is found in poor village areas, with poor sanitation and hygiene, where there are no toilets, allowing pollution of the soil with infective worms.(1,2) hookworm infection is a soil transmitted helminthiasis because it needs soil in the growth process of the worm larva to become infective (filariform larva).(3) man is the sole host for hookworm (a. duodenale and n. americanus), since these worms do not have an intermediate host. the hookworm infection is known as a n c y l o s t o m i a s i s ( f o r i n f e c t i o n w i t h a . duodenale) and as necatoriasis (for infection with n. americanus). hookworm infection is a symptomless chronic infection that can cause microcytic hypochromic anemia.(4) hookworms infect humans by way of infective (filariform) larvae which penetrate the skin (through the sides of toes and the skin pores 135 of legs and hands).(3) children and gardeners are at risk of hookworm infection, because children like to play with soil and frequently do not use footwear when outdoors, and gardeners are exposed to filariform larvae if they do not wear boots or gloves when working in the garden.(2) soil transmitted helminth infection occurs in almost all parts of the world. it is estimated that about 2 billion people are infected by these parasitic worms. generally, this disease is found among poor people with poor environmental sanitation. the world health organization (who) estimates that 250 million people are infected by ascariasis, 151 million by hookworm, 100 million by strongyloidasis and 45.4 million by trichuriasis.(5) school-age children are at high risk of becoming infected by this contagious disease.(6) the factors of poverty, environmental sanitation, and immunity also contribute to infection by these worms through soil. the detrimental effects of these parasitic and contagious worms can be various and harmful, such as impeding physical growth of the children, disturbing their activities, and decreasing their ability to receive lessons at school. the chronicity of these infections can decrease the quality of human resources.(7) in indonesia, soil transmitted helminthic infections pose health problems in urban and semi-urban areas with low socio-economic level, poor environmental sanitation, and poor hygiene. generally, the data on the prevalence of worm infections in indonesia depend on geography, climate, human behavior, level of education, and personal hygiene. the tropical climate of indonesia substantially supports the growth of the hookworm, as the high humidity, light intensity, and rainfall influence the process of the eggs maturing to become infective larvae.(8) keerom regency is a region in the papua province of indonesia with a surface area of 9.365 km2. its geographic location is between longitude 1400 15’ 00’’ 1400 0’ 0’’ e and latitude 20 37’ 00’’ 40 00’ 00’’ s.(9) it also has a tropical climate with high rainfall, annual average temperatures of 30.50c 35.10c, 80-89% humidity, consisting for 99.9% of soft soils and 0.42% peaty soils, with soil ph values between 5.0 – 8.3.(10) the geographic condition and physical environment substantially supports the spread of hookworm infection through soil.(3,11) the number of helminths found in the regency in 2011 was about 1.2%.(9) the prevalence of hookworm infection has a high probability of increasing, based on the traditional lifestyle of the local farmer community, poor environmental sanitation, and the existence of people who still defecate in gardens or near their homes. the aim of this study was to estimate the prevalence of hookworm infection among elementary school students and identify the risk factors affecting this infection. methods research design the present study used a cross-sectional analytic observational design and was conducted in the arso district, keerom regency, papua province, from september until december 2012. research subjects the research subjects were thirdand sixth-grade elementary school students meeting the inclusion and exclusion criteria, and having signed a statement agreeing to join the research ( i n f o r m e d c o n s e n t ) . t h e t h r e e l a r g e s t elementary schools in the area were selected for the study, comprising inpres elementary school 01 arso i, inpres elementary school 02 arso i and inpres elementary school pir i. the sample size for this study was calculated by using the single proportion formula at 95% confidence interval (ci) level (z (1-α/2) = 1.96), an expected prevalence of 19.4 % and 5% marginal error.(12) the calculated total sample size was 241 students. however, the total number of students joining the research, filling the questionnaire and delivering their stool samples for examination, was 224. univ med vol. 33 no.2 136 sandy, sumarni, soeyoko footwear a risk hookworm infection data collection a structured questionnaire was distributed among the participating pupils for the collection of demographic information, such as age, gender, socio-economic status of their parents, household sanitation, and personal hygiene. the items on parental socio-economic status were educational level, occupation, and income of the parents. on the subject of sanitation, the questions asked were type of toilets, location of toilets, and main water source of household. personal hygiene questions comprised habitual use of footwear when playing, using soil as medium for playing, the habit of washing hands before eating, and the habit of washing hands a f t e r d e f e c a t i n g . b o d y w e i g h t ( k g ) w a s measured to the nearest 0.1 kg using omron digital scale and height body (cm) was measured to the nearest 0.1 cm using a microtoise. the children were measured wearing light uniforms, without shoes, belts, caps or any other material that could interfere with their actual height and weight. subjects were categorized as having wasting and normal anthropometric nutritional indices. wasting was defined as < -2 weight for age z-score [waz score].(3) collection of stool samples and laboratory testing the pupils were educated on the causes of intestinal helminthic infections among schoolage children and they were convinced that every child should be free from such infections, thus the necessity of participating in the research work was appreciated by them. thereafter, wide mouthed corked sterile containers were given to the pupils for the collection of their stool samples at home. the pupils were taught how to collect the stool samples, with the aid of their teachers. the stool samples received from the children were properly labelled and transported in a cold box to the laboratory of the papua h e a l t h a n d b i o m e d i c a l r e s e a r c h a n d development institute, ministry of health. the samples that could not be analyzed immediately were preserved using 10% formalin until they could be examined.(14) stool analysis was performed using the kato-katz technique by malachite green staining to detect hookworm e g g s . we u s e d m i c r o s c o p i c a n a l y s i s f o r identification of hookworm eggs. the results were categorized as positive if the kato-katz slide contained hookworm eggs. for the determination of the hemoglobin concentration of the pupils, finger-prick blood samples of the pupils were collected and the hemoglobin concentration measured with a rapid test quick check hb meter, using capillary tubes containing 20 µl of finger prick blood. subjects were categorized as having anemia if the hemoglobin concentration was <11.5 g/dl. ethical clearance ethical clearance for this research was issued by the medical and health research ethics committee, faculty of medicine, gadjah mada university. statistical analysis the data analysis was by descriptive statistics, and differences between two variables were tested by bivariate statistics (chi square and fisher’s test). statistical significance was considered to be achieved at p<0.05. logistic regression was performed to identify the predictor factors of hookworm infections. results the distribution of hookworm infection in elementary school students of arso district on the basis of gender can be seen in table 1. the number of hookworm cases was found to be greater in male students (7.8%) than in female students (5.8%). there was a significant relationship between hookworm infection and the habit of using footwear while playing or being outside the house (p<0.05). there was a risk of hookworm infection in elementary school students who did not use footwear when playing outdoors. the result of the logistic regression 137 table 1. distribution of risk factors of hookworm infection in elementary school students *chi square test (significant if p<0.05); pam = water purification plant; d3, s1, s2,s3 = tertiary degrees, equivalent to undegraduate diploma, bachelor, master, phd univ med vol. 33 no.2 risk fac tor hookworm infecti on odds rati o (or) confidenc e i nterval (ci) p value positive n (% ) negative n (% ) gender male 8 (7.8) 95 (92.2) 0.729 0.255-2.084 0.554 female 7 (5.8) 114 (94.2) anemia anemia (>11.5 g/ dl) 3 (10.7) 25 (89.3) 0.543 0.143-2.060 0.410 normal (<11.5 g/ dl) 12 (6.1) 184 (93.9) nutritiona l status wasting ( <2 s d ) 11 (6.2) 167 (93.8) 1.446 0.438-4.769 0.517 normal ( > -2 sd) 4 (8.7) 42 (91.3) type of toilet toil et without septic tank 4 (5.7) 66 (94.3) 1.269 0.390-4.134 0.781 toil et with sept ic tank 11 (7.1) 143 (92.9) loca tion of toilet out side the house 17 (17.7) 79 (82.3) 1.749 0.911-3.358 0.091 insi de the house 35 (27.3) 93 (72.7) main water source untreat ed water (wel l water, river wa ter, rain water) 12 (8.4) 131 (91.6) 0.420 0.155-1.534 0.177 treat ed water (pi ped wa ter from pam / bottled water) 3 (3.7) 78 (96.3) type of house floor noncemented floor (soil, board, bamboo) 5 (5.0) 95 (95.0) 1.667 0.511-5.045 0.362 cemented/ ceramic til e floor 10 (8.1) 114 (91.9) parents’ educati onal level low (element ary school, junior hi gh school, senior high school) 11 (6.4) 160 (93.6) 1.187 0.362-3.896 0.777 hi gh (d3, s1, s2, s3) 4 (7.5) 49 (92.5) parents’ job non-empl oye e (farmer, trader) 7 (5.4) 122 (94.6) 1.603 0.560-4.584 0.376 employee (empl oyee of pri vate firm, ci vil servant) 8 (8.4) 87 (91.6) parents’ income low (<rp 3,000,000) 13 (6.7) 182 (93.3) 1.037 0.222-4.850 1.000 hi gh (> rp 3,000,000) 2 (6.9) 27 (93.1) habi t of using foot wear when pla ying yes 9 (4.6) 185 (95.4) 0.195 0.064-0.595 0.007* no 6 (20.0) 24 (80.0) using soil as medium for playing yes 5 (9.8) 46 (90.2) 0.564 0.184-1.734 0.341 no 10 (5.8) 163 (94.2) habi t of washing hands before eat ing using water 2 (4.2) 46 (95.8) 1.834 0.399-8.423 0.744 using water and soap 13 (7.4) 163 (92.6) habi t of washing hands aft er defecat ing using only wat er 4 (8.9) 41 (91.1) 0.671 0.203-2.215 0.509 using water and soap 11 (6.1) 168 (93.9) 138 sandy, sumarni, soeyoko footwear a risk hookworm infection was that the predictor of hookworm infection in this study was the habit of using footwear when playing outdoors [or 5.3; 95% ci 1.7-17.7; p=0.004 ] (data not shown). discussion examination for hookworm eggs was done by means of the kato-katz method, which is a widely used diagnostic stool providing estimates of population prevalence and infection intensity. the accuracy of the kato-katz method to detect hookworm eggs was reported by knopp et al.(15) t o b e h i g h e r t h a n t h a t o f t h e f l o ta c technique. another study on the accuracy of the kato-katz technigue for hookworm eggs was done by funk et al.,(16) who showed that the prevalence of hookworm infection in elementary school students was 6.7%. similar results were obtained in an indian study, which found the prevalence hookworm infections to be about 6.3%.(17) in the present study, male children were more infected than females. this is presumably caused by the different playing habits of male and female children, such as walking on the ground without footwear, using soil as medium for playing, playing with balls, jump ropes, and marbles. the children also have the habit of taking off the footwear (shoes) when going home.(1,2,7) this is in agreement with the results of a study conducted by unike et al.(13) in nigeria, where male children were more infected than females, although the difference in infection rate was not significant. however, contrasting results were obtained in a malaysian study showing that hookworms were more frequent in female children.(18) there was no significant relationship of hookworm infection effects with nutritional status and anemia. the finding for anemia is similar to that obtained in the satak malaysian study, showing no significant association between anemia and hookworm infection.(2) in addition, the finding for nutritional status in our study is similar to the results obtained in pahang, malaysia, showing no association of nutritional status with soil-transmitted helminth infection.(19) there is the possibility of finding symptoms of malnutrition in round worm infection of children, while anemia is probably found in children infected by malaria and hookworm.(11,20) that there is no significant relationship between hookworm infection and anemia may be because generally the number of infected children is smaller, and there are other causative factors of anemia in children, such as nutritional intake. the finding on anemia in our study is also in agreement with that obtained in turkey of no association between anemia and parasitic worm infections.(21) environmental sanitation has an important role in worm infections that are transmitted t h r o u g h t h e s o i l , e s p e c i a l l y h o o k w o r m infection,(22) where poor sanitation increases the risk of this infection. the bivariate statistical test results of risk factors in environmental sanitation found no significant relationship with hookworm infection. this is presumably because of the small number of hookworm infection cases in this study, whereas a large number of cases are needed to find relationships at population level. another study showed that water source and the habit of handwashing with soap were s i g n i f i c a n t l y a s s o c i a t e d w i t h h o o k w o r m infection.(7) the present study found no significant relationship between socio-economic status of the parents and hookworm infection. high educational level of parents does not guarantee that their children are free from hookworm infection. although parents with high educational level, income, and job status are different from the lower classes of the community, generally all community members have gardening as a side job. therefore members of the higher class also have direct contact with the soil and their children can be exposed to hookworm infection, because after school they help their parents in the garden.(1,13,14) the risk factor of personal hygiene plays an important role in hookworm infection transmission. the bivariate analysis results show 139 a significant relationship for the risk factor of hygiene, i.e. the habit of using footwear outdoors. the latter habit is very important to avoid exposure to soil polluted by infective (filariform) larvae. the behavior of children of using sandy soil as playing medium has a high risk of this worm infection.(7,23) this is in contrast with the results of a study conducted by nasr et al.(24) and kattula et al.,(17) where use of footwear and s h o e s o u t d o o r s w a s n o t a s s o c i a t e d w i t h hookworm infection. there are several limitations of this study as potential sources of bias. one source may have been the administration of the socioeconomic survey forms. the children answered questions about their personal, household, and health behavior characteristics on their own, while the same information was collected about children by asking their parents. since only a single stool sample per child was collected in this study, using the kato-katz preparation to determine infection status, there is presumably a significant underestimation of the infection. this is because a single sample may miss infection in an individual as a result of the temporal variation in egg excretion over hours and days.(25) the cross-sectional study did not describe the effects of socio-economic factors on the children’s nutritional and parasitic status. a longitudinal cohort study would be necessary to provide information about a community’s social, educational, economic and behavioral characteristics.(21) our study shows that hookworm infections still pose a significant health challenge to children in the poor rural areas of papua. intestinal parasitic infections have detrimental effects on survival, appetite, growth and physical fitness of children. we hope that this study may motivate the government to the deworming of soil transmitted helminths in school-age children and provide educational information for preventing soil transmitted helminth and other intestinal parasite infections. when supporting school health programs, hopefully parents may become involved in deworming and dietary activities which may increase their care about the importance of health information and education.(21) conclusion the prevalence of hookworm infection among elementary school students was 6.7%. the habit of using footwear was a risk factor in transmission of hookworm infection to these elementary school students. acknowledgement the researchers thank the head of the department of parasitology, medical faculty, gadjah mada university, prof. dr. supargiyono, dtm&h, su, sp.par.k, ph.d, the head of the institute of health biomedical research and development papua dr. lidwina salim, m.si, the headmasters of inpres elementary school arso 01, arso 02, and inpres elementary school pir i, and the chief of national unity, politics and public protection board papua, keerom regency (limnas kabupaten keerom). references 1. ahmed a, al-mekhlafi hm, choy sh, ithoi i, aladhroey a, abdulsalam am, et al. the burden of moderate-to-heavy soil transmitted helminth infections among rural malaysian aborigines: an urgent need for an integrated control programme. parasites vectors 2011;4:242-9. doi:10.1186/17563305-4-242. 2. ahmed a, al-mekhlafi hm, choy sh, ithoi i, aladhroey a, abdulsalam am, et al. the nutritional impacts of soil-transmitted helminth infections among orang asli schoolchildren in rural malaysia. parasites vectors 2012;5:119-28. doi:10.1186/1756-3305-5-119. 3. loukas a, constan sl, bethony jm. immunology of hookworm infection. fems immunol med microbiol 2005;43:115-24. 4. soedarto. the textbooks of medical parasitology. sagung seto, jakarta, 2011. 5. world health organization. helminth control in school-age children: a guide for managers of control programmes. 2nd ed. geneva: world health organization; 2011 univ med vol. 33 no.2 140 sandy, sumarni, soeyoko footwear a risk hookworm infection 6. avad sb, hiware cj. soil transmitted helminthiasis among school age children in aurangabad district, maharashtra state, india. dama internasional 2012;1:31-3. 7. alemu a, atnafu a, addis z, shiferaw y, teklu t, 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2004;118:151-8. 22. ziegelbauer k, speich b, mäusezahl d, bos r, keiser j, utzinger j. effect of sanitation on soiltransmitted helminth infection: systematic review and meta-analysis. plos med 2012;9:e1001162. doi:10.1371/journal.pmed.1001162. 23. ratnayaka rmkt, wang zj. prevalence and effect of personal hygiene on transmission of helminth infection among primary schoolchildren living in slums. int j multidisciplinary res 2012;2: 1-12. 24. nasr na, al-mekhlafi hm, ahmed a, roslan ma, bulgiba a. towards an effective control programme of soil-transmitted helminth infections among orang asli in rural malaysia. part 2: knowledge, attitude, and practices. parasites vectors 2013; 6:28. doi: 10:1186/17563305-5-119. 25. wang x, zhang l, luo r, wang g, chen y, medina a, et al. soil transmitted helminth infections and correlated risk factors in preschool and school aged children in rural southwest china. plos one 2012;7: e45939. doi:10.1371/ journal.pone.0045939. decreased magnesium level and membrane potential of glaucoma patients 105 *department of medical laboratory science, faculty of health science, imo state university owerri, imo state, nigeria **department of optometry, faculty of health science, imo state university, owerri, imo state, nigeria ***department of microbiolgy, faculty of science, imo state university owerri, imo state, nigeria †department of medical laboratory services, federal medical centre, umuahia abiastate, nigeria correspondence: dr. johnkennedy nnodim department of medical laboratory science, faculty of health science, imo state university owerri, imo state, nigeria, phone: +238034237000 email : johnkennedy23@yahoo.com univ med 2016;35:105-9 doi: 10.18051/univmed.2016.v35.105-109 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 decreased magnesium level and membrane potential of glaucoma patients nnodim johnkennedy*, nsonwu magnus**, obioma elemba je**, nwadike constance*, edward ukamaka*, njoku-obi treasure***, and jimoh raifu folorunso† background one of the most important sight-threatening eye disorders in ophthalmology is glaucoma. together with age related macular degeneration it is the most common cause of irreversible blindness. magnesium (mg) is considered to improve the ocular blood flow in glaucoma patients and could protect the retinal ganglion cell against oxidative stress. membrane potential refers to the difference in electrical potential between the intracellular and the extracellular of a biological cell. the objective of this study was to determine the mg serum level and membrane potential in glucoma subjects. methods a case-control study was conducted in eighty confirmed glaucoma patients and eighty normal subjects between 50 to75 years were selected. the serum sodium, potassium and calcium were estimated using randox kit. while membrane potential was determined by calculation using nerst equation. magnesium levels were determined by atomic absorption spectrophotometer technique. an independent t-test was used to analyze the data. results the results obtained showed that the level of magnesium serum (1.11 ± 0.14 mg/dl) and membrane potential (176.40 ± 42.00 j) was significantly decreased in glaucoma as compared to the controls (2.4 ± 0.68 mg/dl) and (251.76 ± 191.99) (p<0.05). the serum sodium, potassium and calcium were also significantly decreased among glaucoma subjects compared to control (p<0.005). conclusion the result probably suggests, that decrease in magnesium aggravates glaucoma while decrease in membrane potential could cause poor energy transmission and hence affect ocular blood flow. hence, decreased magnesium and membrane potential levels contributes greatly to glaucoma. keywords: magnesium, membrane potential, glaucoma doi: http://dx.doi.org/10.18051/univmed.2016.v35.105-109 106 johnkennedy, magnus, elemba, et al membrane potential in glaucoma introduction m a g n e s i u m ( m g ) i s a n a b u n d a n t intracellular cation. it is considered as a cofactor in most enzymatic reactions in the body. about 50% of mg is present in bones, tissues and organs while 1% in the blood stream. it is associated with many metabolic processes such as maintenance of normal cell membrane function, energy metabolism, as well as production of nucleic acids. also, mg tends to act as a natural physiologic calcium channel blocker.(1) it is part of many enzymes which play essential roles in carbohydrate, protein, and fat metabolism. mg is considered to improve the ocular blood flow in glaucoma patients and could protect the retinal ganglion cell against oxidative stress.(2) it posses advantageous effects via neuronal and vascular mechanisms, which could serve as an attractive therapeutic agent in glaucoma. additionally, mg plays a critical role in maintaining normal nerve and muscle function, normal heart rhythm, neuromuscular conduction, muscular contraction, vasomotor tone, normal blood pressure, bone integrity, as well as glucose and insulin metabolism.(3) mg is associated with many metabolic processes such as maintenance of normal cell membrane function, energy metabolism, and production of nucleic acids. on the other hand, patients with glaucoma could affect membrane potential.(4) membrane potential refers to the difference in electrical potential between the intracellular and the extracellular of a biological cell. the membrane potential result from the interaction between the membrane and the actions of two types of trans membrane proteins embedded in the plasma membrane.(5) there is a reduction of sodium ion and potassium ion concentration gradients in glaucoma. this could be associated with increased permeability of these ions. glaucoma is a group of eye disorders in which the optic nerve at the back of the eye is gradually damaged. in some people this disorder could be as a result of increased pressure inside the eye or as a result of poor blood supply to the vital optic nerve fibres. it is characterized by chronic optic neuropathy leading to gradual loss of vision. (6) glaucoma is considered as a condition of elevated intraocular pressure (iop), however, other risk factors may play a role in the pathogenesis of glaucomatous optic nerve damage. vascular dysregulation in ocular blood flow and oxidative stress are important risk factors for glaucomatous retinal ganglion cell loss. in spite of the fact that anyone can get glaucoma, some people may have an increased risk, especially those with a family history of glaucoma, diabetes, migraine, short sightedness (myopia), long sightedness (hyperopia), eye injuries, blood pressure, past or present use of cortisone drugs (steroids).(7) also, disturbed ocular blood flow and oxidative stress could contribute to glaucoma. it is also the leading cause of blindness in nigeria particularly in imo state. glaucoma is the leading cause of irreversible blindness globally and a growing public health concern because of an aging global population. if glaucoma is detected early enough, it could be possible to arrest the gradual progression with medical and surgical means.(8) glaucoma tends to induce oxidative stress which affects the magnesium status.(9) owing to scarcity of information on magnesium and membrane potential, this study tends to determine the magnesium and membrane potential among glaucoma patients aged 50 to 75 years. methods research design a case-control study was carried out in the genera hospital owerri, imo state nigeria research subjects eighty patients attending general hospital owerri who were 50 to75 years of age and were confirmed of glaucoma participated. exclusions were defined as having had ocular surgery in the month prior to their appointment and/or having secondary glaucoma associated with ocular or 107 systemic disorders. subjects provided informed consent prior to enrollment in the study. measurements after obtaining informed consent, subjects were provided a routine ophthalmic examination of both eyes including visual acuity assessment, intraocular pressure measurement, visual field examination, external slit lamp examination, and dilated retinal exam to confirm glaucoma. biochemical assay in all subjects, 5ml of venous blood was collected into a non-anticoagulated tubes. the sample were spun in a wisterfuge (model 684), centrifuge at 1000g for 10 minutes and the serum c o l l e c t e d i n t o a c l e a n d r y b i j o u b o t t l e . magnesium and membrane potential were estimated. the serum sodium, potassium and calcium were estimated using randox kit. while m e m b r a n e p o t e n t i a l w a s d e t e r m i n e d b y calculation using nerst equation. magnesium levels were determined by atomic absorption spectrophotometer technique as described by addison.(10) statistical analysis the results were expressed as mean ± standard deviation. the statistical evaluation of data was performed by using students ttest. the level of significance was calculated at p<0.05. ethical clearance informed consent of the participants was obtained and was conducted in line with the ethical approval of the hospital. table 1. magnesium and membrane potential levels in glaucoma and control *significantly different from control at p<0.05 results the serum sodium level in glaucoma subjects (134.00 ± 2.88 nmol/l) was significantly lower compared to the control healthy subjects (139.75 ± 4.00 nmol/l) (p=0.003). the potassium serum level in glaucoma subjects (3.10 ± 2.88 nmol/l) was also significantly lower than the control healthy subjects (3.58 ± 0.41) (p=0.042). the level of mg and membrane potential, were significantly affected in glaucoma when compared with the control (p=0.047) (table 1). discussion i n t h i s s t u d y, i t w a s o b s e r v e d t h a t m a g n e s i u m i n g l a u c o m a s u b j e c t s w e r e significantly decreased when compared with control. the is in line with the work of de valk ( 11 ) w h o f o u n d t h a t p a t i e n t s w i t h s e v e r e retinopathy have a lower plasma magnesium level compared to patients without retinopathy. mg tends to play an important function in insulin homeostasis. dietary mg intake and serum mg levels were found to be inversely correlated with fasting serum insulin levels.(6,12) also, serum mg concentration is known to be decreased in diabetic than in non diabetic patients.(13) the reduction in serum magnesium was correlating with retinopathy and hypertension.(14) it may be an added advantage in clinical practice to periodically monitor serum magnesium levels in glaucoma patients. if serum magnesium is reduced, an intervention to increase dietary intake of magnesium may be beneficial. univ med vol. 35 no.2 108 johnkennedy, magnus, elemba, et al membrane potential in glaucoma glaucoma is characterized by chronic optic neuropathy leadingtogradual vision loss. magnesium is the major intracellular divalent cation. it is usually characterized by the presence of at least two of the three hallmarks including elevated intraocular pressure, optic disc cupping, and visual field loss.(6) the normal concentrations of extracellular magnesium and calcium are essential for normal neuromuscular activity. although, the intracellular magnesium forms a key complex with atp and it is a crucial cofactor for a wide range of enzymes, transporters and nucleic acids required for normal cellular function, replication and energy metabolism.(11) the serum mg play an important function in the development and normal functioning of the eye. magnesium decrease has been linked to multifocal necrosis in the retinal pigment epithelium. the low level of magnesium has been found to be correlated with pigmentaryretinal degenerations such as kearns-sayre syndrome and retinitis pigmentosa.(15,16) magnesium could be essential for maintenance of healthy ocular surface in the prevention of infections as well as d r y n e s s a n d i n i n f l a m m a t o r y s t a t u s o f conjunctiva.(2,17) the decrease in serum and erythrocyte magnesium level is associated with advanced retinopathy.(18) hence, preventing low serum magnesium in glaucoma by supplementing magnesium may be beneficial. in the same vein, it was observed that the levels of serum levels sodium and potassium were significantly decreased in glaucoma when compared with healthy controls. this results were in line with a prospective case-control study in a subpopulation of 18 patients with glaucoma, and 24 control subjects age range 30–62 years were selected from the hospital staff. this study found the serum sodium level in glaucoma subjects (138.0 ± 2.1 nmol/l) was significantly lower compared to the control healthy subjects (140.0 ± 2.4 nmol/l) (p=0.006). and potassium serum level in glaucoma subjects (3.7 ± 0.3 nmol/l) was also significantly lower than the control healthy subjects (4.0 ± 0.4) (p=0.002).(5) the level of potassium ions is low in the extracellular environment while that of sodium is high. it is relevant to note that the sodium potassium atpase and sodium potassium ion pump maintains the concentration as well as inherent electrochemical equilibrium between the extracellular and intracellular environment. this pump takes out three sodium ions for every two potassium ions taken into the cell. therefore, this increased potassium concentration helps to draw water molecules into the cell.(19) our study showed that the membrane p o t e n t i a l w a s s i g n i f i c a n t l y d e c r e a s e d i n glaucoma patients when compared with the control. this is an agreement with the work of he et al.(20) that the mitochondrial membrane potential level was decreased in glaucomatous trabecular meshwork cells. one limitation of this study was that we did not perform a further evaluation of the exact age of glaucoma among the patients. the decrease in mg and membrane potential was not correlated to period of glaucoma. further studies on the parameters using a cohort design may be conducted to evaluate the degree of severity of glaucoma, so that the exact period of the change may be known. conclusion it is quite obvious that the level of serum magnesium and membrane potential decreased in glaucoma .therefore, supplementation with diets or foods rich in magnesium could be beneficial to glaucoma patients. conflict of interest the investigators confirm that there was no conflict of interest. acknowledgement the investigators wish to thank the laboratory scientists, medical doctors and nurses at general hospital, owerri nigeria who 109 assisted in this study in the collection and examination of the samples. references 1. naik ns, lamani s, devarmani ss. the role of serum magnesium level in type 2 diabetes mellitus. int j res med sci 2015;3:556-9. 2. wishal d, ramdas wd, wolfs rcw, et al. nutrient intake and risk of open-angle glaucoma: the rotterdam study. eur j epidemiol 2012; 27:385–93. doi: 10.1007/s10654-012-9672-z. 3. matthias b, schulze m, heidemann c, et al fiber and magnesium intake and incidence of type 2 diabetes. arch intern med 2007;167:956-65. 4. mittag tw, danias j, pohorenec g, et al. retinal damage after 3 to 4 months of elevated intraocular pressure in a rat glaucoma model. invest ophthalmol vis sci 2000;41:3451–9. 5. pechere-bertsch a, sunaric-megevand, haefliger i, et al. renal sodium handling in patients with normal pressure glaucoma. clin sci 2007;112:337–44. doi: 10.1042/cs200600 82. 6. ekici f, korkmaz f, karaca ee, et al. the role of magnesium in the pathogenesis and treatment of glaucoma. isrn 2014;10:1155-9. 7. edward u, nnodim jk, akabogu ui, et al. immunoglobulin expressions in glaucoma patients attending general hospital owerr. res j med med sci 2015;10:33-5. 8. leffler ct, schwartz sg, hadi tm, et al. the early history of glaucoma: the glaucous eye (800 bc to 1050 ad). clin ophthalmol 2015;9:20715. 9. chrysostomou v, rezania f,trounce ia, crowston jg. oxidative stress and mitochondrial dysfunction in glaucoma. curr op pharmacol 2013;13:12–5. 10. addison c. clinical biochemistry: magnesium (serum, plasma). london: the association for clinical biochemistry and laboratory medicine; 2012. 11. de valk hw. magnesium in diabetes mellitus. neth j med 1999;54:139-46. 12. mozaffarieh m, flammer j. new insights in the pathogenesis and treatment of normal tension glaucoma. current opin pharmacol 2013;13: 43–9. 13. mozaffarieh m, flammer j. is there more to glaucoma treatment than lowering surv ophthalmol 2007;52:174–9. 14. smith jm, lowe rf, fullerton j, et al. an integrative review of the side effects related to the use of magnesium sulfate for pre-eclampsia and eclampsia management bmc pregnancy childbirth 2013;13:43–9. 15. katsanos kh, elisaf m, bairaktari e, et al. severe hypomagnesemia and hypoparathyroidism in kearns-sayre syndrome. am j nephrol 2001;21:150–3. 16. mayama c. calcium channels and their blockers in intraocular pressure and glaucoma. eur j pharmacol 2013;739:96–105. 17. musso cg. magnesium metabolism in health and disease. int urol nephrol 2009;41:357–62 18. liang sy, lee lr. retinitis pigmentosa associated with hypomagnesaemia. clin exp ophthalmol 2010;38:645–7. 19. bookchin rm, etzion z, sorette m, et al. identification and characterizationof newly recognized population of high na+, low k+ lowdensity sickle cell and normal cells. proc natl acad sci usa 2000;97:8045–50. 20. he y, leung kw, zhang yh, et al. mitochondrial complex i defect induces ros release and degeneration in trabecular meshwork cells of poag patients: protection by antioxidants. invest ophthalmol vis sci 2008; 49:1447–58. univ med vol. 35 no.2 campylobacter species in adult patients of ardabil, campylobacter species in adult patients of ardabil, iran 59 *molecular biology research center, baqiyatallah university of medical sciences, tehran, iran **young researchers and elite club, tabriz branch, islamic azad university, tabriz, iran correspondence: daryoush babazadeh, dvm, phd candidate, school of veterinary medicine, shiraz university, shiraz, ir iran email: daryoush.babazadeh@shirazu.ac.ir univ med 2017;36:59-67 doi: 10.18051/univmed.2017.v36.59-67 pissn: 1907-3062 / eissn: 2407-2230 received march 29, 2017 accepted for publication april 25, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract contact with poultry and animals increases risk of campylobacter infections in adults of ardabil province, iran background the acute gastroenteritis caused by campylobacteriosis is known as one of the common infectious diseases with worldwide distribution. the aim of this study was to detect campylobacter species in stool samples by routine culturing and polymerase chain reaction (pcr) and explore the risk factors in adult subjects in east azerbaijan province of iran. methods a cross sectional study involving 1010 adult subjects, from whom stool samples were collected. samples with inflammatory criteria like fecal leukocytes (wbc ≥5) were selected and isolated through fecal lactoferrin detection test. the wbc ≥5 and lactoferrin positive samples were selected for campylobacter detection by culture and pcr methods. the required information consisting of gender, age, place of habitation, and contact with poultry and animals were asked and recorded. chi-square test and prevalence ratio (pr) was used to analyze the data. results of 1010 stool samples, 231 (22.9%) had wbc ≥5, and from these samples 58 (25.1%) were positive by culturing and 61 (26.4%) by pcr. subjects having habitual contact with animals and poultry had increased risk of campylobacter infections by 1.65 times compared with subjects without contact with animals and poultry (pr=1.65; 95% ci: 1.07-2.68). conclusions detection of campylobacter infections by pcr was more sensitive in adults. investigation of campylobacter prevalence in ardabil showed this bacterium should be viewed as one of the possible pathogens in inflammatory diarrheal cases. people having habitual contact with animals should check the health of the animals regularly and not consume food from suspected sources. keywords: campylobacter, culture, lactoferrin, patient, pcr, prevalence doi: http://dx.doi.org/10.18051/univmed.2017.v36.59-67 reza ranjbar* and daryoush babazadeh** universa medicinajanuary-april, 2017 vol.36 no.1 original article 60 ranjbar, babazadeh campylobacter species in adult patients of ardabil, iran introduction infections of the gastrointestinal tract are strongly associated with morbidity and even mortality in children and elderly people.(1,2) recently, food and water-borne outbreaks of gastrointestinal and diarrheal illness due to various microbial pathogens in iran have been reported in many studies. (3-6) one of the most common and important causes of infectious diarrheal illness and acute gastroenteritis in adults and children with worldwide distribution is campylobacteriosis.(7) campylobacter is well recognized as the leading cause of bacterial foodborne diarrheal disease worldwide.(8) c a m p y l o b a c t e r i o s i s i s e n d e m i c i n developing countries and the major sources of human infections are food and environmental contamination.(9) campylobacter contamination can be the result of consumption of suspected animal sources of food and dairy products.(10) environmental contamination can be caused by domestic animals and poultry (as natural reservoirs of campylobacter species). (11) poultry carcasses in stores that are not under sanitary monitoring and consumption of undercooked p o u l t r y m e a t a r e i m p o r t a n t c a u s e s o f outbreaks.(12) the occurrence of this disease is usually higher in older persons over 75 years of age, young children under 4 years of age and young adults who are 20-40 years old.(13,14) person-toperson transmission of this disease is extremely rare.(15) developing countries often do not have national surveillance programs for controlling this infection. according to various reports, the incidence of campylobacter in developing countries is between 5 and 20 percent.(16) a study showed that in rural households, the presence of poultry manure, uncovered litter in house yards and lack of barriers to keep animals out of houses have been identified as risk factors for campylobacter infection in children.(17) another study identified exposure to domestic animals as a sufficient risk factor for infection.(18) the clinical symptoms of enteritis caused by campylobacter species range from watery, non-bloody and non-inflammatory diarrhea to severe inflammatory diarrhea with abdominal pain and fever; however c. jejuni typically shows acute and self-limited signs.(19) diagnosing campylobacter infection will not be possible from routine clinical signs and usually there is co-infection with multiple pathogens such as escherichia (e. coli), listeria monocytogenes, staphylococcus aureus, cronobacter sakazakii, salmonella enterica, vibrio cholera, and shigella and yersinia species.(20-23) red blood cells and fecal leukocytes have been found in the majority of the stool samples of infected patients and the peripheral white blood cell (wbc) count is slightly elevated.(24) l a b o r a t o r y m e t h o d s f o r d i a g n o s i n g campylobacter enteritis have been successfully developed, such as polymerase chain reaction (pcr), genotyping methods like ribotyping, pulsed-field gel electrophoresis and elisa for detecting dna and antigens in stool samples and also specific culturing of the organism from fecal specimens.(25-28) the ability of pcr to amplify minute amounts of specific microbial dna sequences has made it a powerful molecular tool.(5,29) it is reported that multiplex pcr diagnostic tools are fast, inexpensive and sensitive for campylobacter species.(30) the present study aimed to determine the prevalence of gastroenteritis related to campylobacter species and explore the risk factors in adult patients of ardabil province, iran. methods research design a cross sectional study was conducted in ardabil province from june to december 2016. ardabil province is in northwest iran, having ardabil city as its capital and largest city, and common borders with the provinces of zanjan, east azerbaijan, and gilan, and the republic of azerbaijan. 61 univ med vol. 36 no.1 sample collection a total of 1010 stool samples were collected from adult patients (517 men and 493 women aged 18-70 years) who were referred with acute diarrhea to the central laboratory of ardabil, bu-ali central laboratory, ardabil university of medical sciences, ardabil, iran. patients who had consumed antibiotics before sampling, who were not satisfied with the investigation, were less than 18 years and more than 70 years old, and had a history of noninfectious diarrhea, were excluded from the study. the fresh samples with inflammatory criteria like fecal leukocytes (wbc ≥5 under a field of light microscope x 400) were selected and isolated through the fecal lactoferrin detection test as described in previous studies.(31) lactoferrin detection in fecal samples has been confirmed for diagnosis of inflammatory bowel disease (ibd). intestinal inflammation may be caused by bacterial infection or ibd and is directly related to the disease activity and severity.(31) the wbc ≥5 and lactoferrin positive samples were selected for campylobacter detection by culture and pcr methods. the necessary information like age, gender, daily contact with animals (pets, exotic animals, poultry, small and large animals) and place of living was asked and recorded. culture of sample the fresh samples were enriched on campythio medium (that is suggested as a holding medium for samples suspected to contain campylobacter spp. when immediate inoculation cannot be performed) for 1-2 hours and then cultured on a campylobacter selective agar (merck, germany), containing 5% defibrinated sheep blood, trimethoprim, polymyxin, and vancomycin for inhibiting growth of e. coli, pseudomonas, pasteurella, klebsiella, shigella, salmonella, enterobacter, streptococcus, clostridium, proteus and corynebacterium. the samples were incubated at 42°c for 48 hours in a microaerophilic atmosphere (85% n 2 , 10% co 2 and 5% o 2 ). only the suspected colonies were examined by phase-contrast microscope and gram staining technique for motility and morphology. for further investigation, the suspected colonies also were cultured on blood agar plates, and incubated under microaerophilic conditions at 42°c for 48 h. hippurate hydrolysis, catalase activities, oxidase test, and also susceptibility to 30 µg discs of nalidixic acid and cephalothin (mast co., england) were used for confirming the results. dna extraction dng plus kit (cinnagen, iran) was used according to the commercial recommendations for extracting the dna of samples.(32) the extracted dna was stored at -20°c. multiplex pcr assay strains of c. jejuni atcc 29428 and c. coli atcc 33559 were prepared at tabriz university of medical sciences, tabriz, iran, as positive controls. the target genes hipo and asp were targeted for c. jejuni and c. coli respectively. the oligonucleotide primers of the study are presented in table 1. the reaction mixture consisted of 25 µl multiplex master mix (qiagen, iran), 0.5 µl asp primer (50 pmol µl -1), 1.0 µl hip primer (100 pmol µl -1), 0.5 µl bovine serum albumin [bsa] (10 mg/ml) (promega, iran), 4.5 µl eluted dna (0.1 µg) and sterile water to a final volume of 50 µl. the pcr amplification cycle used was heat denaturation at 95°c for 10 min, 35 cycles with denaturation at 94°c for 50 seconds, annealing at 55°c for 40 seconds, extension at 72°c for 50 seconds, and a final extension at 72°c for 4 min. all pcr products were analyzed by gel electrophoresis on 1.8% agarose gel and stained with 0.1 µg/ml ethidium bromide for visualization. the predicted product size for hipo primer was 735 bp and for asp primer 500 bp.(33,34) ethical clearance the present research was carried out after ethical approval of the research ethics committee of tabriz university of medical sciences, tabriz, iran under the ethics code no. 5/4/7518 according 62 ranjbar, babazadeh campylobacter species in adult patients of ardabil, iran t o t h e d e c l a r a t i o n o f h e l s i n k i ( h t t p : / / www.ufrgs.br/hcpa/gppg/helsin5.htm). statistical analysis data analysis was carried out using statistical package for social sciences (spss) software (version 16, chicago, usa). the relationship of the prevalence of campylobacter with other quality variables was computed by chisquared test and prevalence ratio. p values less than 0.05 were reported as statistically significant. results a total of 711 out of 1010 samples were noninflammatory diarrheal cases (wbc less than 5) and 299 samples were inflammatory cases (wbc more than 5). all inflammatory samples were tested for lactoferrin and 231 cases were positive. the prevalence of campylobacter species obtained by pcr and culture methods was 6% and 5.7%, respectively. sixty one (26.4%) out of 231 lactoferrin positive samples were positive for campylobacter infection by multiplex pcr and 58 (25.1%) samples were positive by specific culture. there was no significant correlation between different ages and prevalence of campylobacter (p=0.977). the highest prevalence of campylobacter species was seen in 18-30 years old patients (culture results: 6.3% and pcr results: 6.6%) and the lowest number of positive samples was found in 60-70 years old patients (culture results: 5.7% and pcr results: 5.7%). the average contamination rates per case were 6% and 5.7% among all collected samples which were indicated by pcr and specific culture methods respectively. the highest contamination rate was recorded in the age range of 18-30 years (6.6%) and the lowest contamination rate was recorded in 40-70 year old patients (5.7%) (table 2). there was no significant correlation between gender and prevalence of campylobacter (p≥0.05). the contamination of females (culture results: 5.5% and pcr results: 5.7%) was insignificantly lower than that of males (culture results: 6.0% and pcr results: 6.4%) (p=0.638). the contamination rate in male patients (6.3%) was higher than in females (5.7%) (table 2). table 2. relationship of age and gender with campylobacter species detected by culture and pcr (n=1010) pcr = polymerase chain reaction; c/p = contaminated based on pcr results/all subjects with certain age table 1. primers used for amplification of target genes of campylobacter species isolated from adult subjects 63 univ med vol. 36 no.1 the results of campylobacter prevalence in patients with daily contact with animals and poultry (343 patients), in patients with daily contact with animals but not poultry (249 patients) and in patients without any contact with animals (667 patients) are shown in table 3. in subjects who had habitual contact with animals and poultry the risk of campylobacter infections increased by 1.65 times compared to subjects who had no contact with animals and poultry (pr=1.65;95% ci:1.07-2.68).there was no significant correlation between contact with animals and prevalence of campylobacter in the collected samples (pr=1.28; 95% ci:0.75-2.18) (table 3). the lowest contamination rate among all patients was seen in patients who had no contact with animals (5%) and the higher contamination rates were seen respectively in patients who had daily contact with domestic animals and poultry (8.2%) and in patients who had daily contact with animals but not poultry (7.2%). the rate of campylobacter infection in patients who were referred from villages and cities was indicated by fecal culture and pcr-positive infections as shown in table 3. there was no significant correlation between living in city or village and prevalence of campylobacter (pr=0.69;95% ci 0.43-1.13). the contamination rates in rural and urban patients were 7.3% and 5.1% respectively. discussion investigation of campylobacter prevalence in ardabil showed that this bacterium should be viewed as one of the possible pathogens in inflammatory diarrheal cases. the 6% and 5.7% prevalence of campylobacter species in ardabil province was obtained in the present research by pcr and culture methods, respectively. previous findings in some cities of iran indicated 12.4% in semnan,(3) 8% in tehran,(35) 10% in hamedan (36) and 6.4% in zanjan.(37) differences in prevalence rates probably are related to pathogenic agents or living conditions in different regions. the prevalence of c. jejuni and c. coli and mixed infections among all patients with gastroenteritis were in agreement with previous f i n d i n g s o f r e s e a r c h e r s w h o d e t e c t e d campylobacter by multiplex pcr method and reported a higher prevalence of c. jejuni than c. coli.(38,39) rapid detection of campylobacter species is extremely important to ensure food and water safety. multiplex pcr is one of the possible and trustworthy molecular methods for indicating the prevalence of bacterial diseases and especially campylobacter infection in a single sample.(29, 30) there are other possible methods for detection of campylobacter infection like specific culture under specific conditions.(16) the hipo and asp table 3. relationship of subjects’ contact with animals and poultry and place of habitation with campylobacter species detected by culture and pcr (n=1010) pcr = polymerase chain reaction; c/p = contaminated based on pcr results/all subjects with certain age; pr= prevalence ratio 64 ranjbar, babazadeh campylobacter species in adult patients of ardabil, iran genes were targeted in the present investigation, since the hipo gene has been represented as the most widely validated gene for the identification of c. jejuni and is highly conserved in c. jejuni strains,(26) while the asp gene is highly specific for c. coli and encodes aspartokinase.(34) although several researches have targeted the rrna genes for genus and species identification of campylobacter they might have found a lower specificity due to the high level of conservation among closely related species.(26) currently fecal lactoferrin has majored as one of the important factors for investigating fecal samples suspected of bacterial infections.(37) acute bacterial infections in the gastrointestinal system or severe bowel diseases will increase fecal lactoferrin. it is reported that the fecal lactoferrin value was is higher in patients with campylobacter (10.32 µg/g) and salmonella (11.17 µg/g).(40) the results of the present study revealed the presence of campylobacter species in 26.4% and 25.1% of lactoferrin positive samples as determined by pcr and specific culture methods respectively. the present results revealed that the age and gender of adult patients are not effective factors in the prevalence of campylobacteriosis. however a higher contamination rate was seen in younger patients than in older patients. these findings are in accordance with the previous report of samia et al. from egypt who indicated that gender is not an effective factor in the prevalence of this infection and that younger patients had more infections with campylobacter species.(39) however some researchers believed that the occurrence of this disease was higher in young children, young adults and oldest elderly patients.(2,13,14) as gender and age did not have a major role in morbidity in the present study, the living conditions and location of habitations were not significantly associated with the causes of contamination. however, patients who lived in villages and had daily contact with animals or poultry showed a higher prevalence ratio of disease than patients who lived in the cities. regardless of the present findings, it is reported t h a t t h e g a s t r o e n t e r i t i s p r e v a l e n c e a n d contamination rate of campylobacter species isolated from patients who lived in urban areas were higher than in patients from rural areas, which is confirmed by pcr and lactoferrin assay.(37) there are various reports about the prevalence of gastroenteritis in relation to campylobacteriosis in rural and urban patients t h a t i n d i c a t e d d i ff e r e n t p r e v a l e n c e s o f infections.(13) the presence of campylobacter species in stool samples and egg shells of poultry as the normal flora has been demonstrated.(41,42) in a 10-year study in switzerland (from 2002 to 2012) the prevalence of campylobacter in animals was indicated and it has been found that chicken are the most common reservoir/source of campylobacter infection, with average contamination of 70.9%, whereas cattle have been named as the second most common source, with a rate of 19.3%, while dogs and pigs were other sources with rates of 8.6% and 1.2% respectively.(11) the presence of campylobacter species as normal flora in stool and eggs of poultry is one of the major causes that might affect the prevalence of campylobacteriosis in patients.(38) also there are some reports that found this pathogen in the normal flora of other animals.(43) regardless of present findings, in two studies the possible effect of poultry on incidence of campylobacter disease was denied; but consumption of unpasteurized milk and ingestion of under cooked chicken were significantly associated with infection.(12,39) reducing the number of campylobacter in poultry carcasses will greatly decrease the risk of infection in consumers.(26,44) one of suggested methods for controlling the campylobacter species as normal flora in poultry is using feed additives like probiotics and synbiotics. it is reported that a mixture of fructooligosaccharides and a galactooligosaccharide plus one bifidobacterium strain (b. longum subsp. longum pcb133) can significantly reduce the c. jejuni concentration in poultry feces.(45) 65 univ med vol. 36 no.1 there were several limitations in our study. some limitations are inherent in the structure of a cross sectional study, in which the association between risk factors and campylobacter infection cannot be explained as a cause and effect. the risk associations determined in this study are most appropriately understood as relative indicators of risk. a larger prospective epidemiological study with active case finding might circumvent the limitations of the present study design. conclusions i n t h i s s t u d y, i n v e s t i g a t i o n o f c a m p y l o b a c t e r p r e v a l e n c e s h o w e d t h i s bacterium should be viewed as one of the possible and important pathogens in inflammatory diarrheal cases. persons who have habitual contact with animals and poultry are at increased risk of campylobacter infections and should check the health of the animals regularly and not consume food from suspected sources. conflict of interest all authors declare to have no conflict of interest. acknowledgments the authors thankful to the dey laboratory, laboratory of bu-ali hospital, ardabil university of medical sciences for their support and supplying the instruments and samples. we also give thanks to dr. veghar hejazi for her wonderful scientific insights. present study is sponsored by research deputy of baghiatollah university of medical sciences. contribution rr contributed to the design of the project, prepared the laboratory equipment for pcr and performed the practical procedure of this method. db contributed to the design of the 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microbial pathogens: a how-to guide. new microbiol 2014;37:1-15. 26. guyard-nicodème m, rivoal k, houard e, et al. prevalence and characterization of campylobacter jejuni from chicken meat sold in french retail outlets. int j food microbiol 2015;203:8-14. doi: 10.1016/j.ijfoodmicro. 2015.02.013 27. rokosz n, rastawicki w, wo³kowicz t. microbiological diagnosis of infections caused by campylobacter jejuni and campylobacter coli in humans. postepy hig med dosw 2014; 68:48-56. 28. karami a, ranjbar r, ahmadi z, et al. rapid detection of different serovares of salmonella entrica by multiplex pcr. iran j pub health 2007;36:38-42. 29. ranjbar r, afshar d, mehrabi ta. development of multiplex pcr for simultaneous detection of three pathogenic shigella species. iran j public health 2014;43: 1657–63. 30. el-adawy h, hotzel h, tomaso h, et al. elucidation of colonization time and prevalence of thermophilic campylobacter species during turkey rearing using multiplex polymerase chain reaction. poult sci 2012;91:454-9. 31. sipponen t. diagnostics and prognostics of inflammatory bowel disease with fecal neutrophil-derived biomarkers calprotectin and lactoferrin. dig dis 2013;31:336-44. 32. harzandi n, jamshidi s, dezfulian m, et al. molecular detection and speciation of campylobacter species in children with gastroenteritis using polymerase chain reaction in bahonar hospital of karaj city. int j enteric pathog 2015;3:e21796. doi: 10.17795/ijep 21796. 33. persson s, olsen ke. multiplex pcr for identification of campylobacter coli and campylobacter jejuni from pure cultures and directly on stool samples. j med microbiol 2005;54:1043-7. 34. debruyne l, samyn e, de brandt e, et al. comparative performance of different pcr assays for the identification of campylobacter 67 univ med vol. 36 no.1 jejuni and campylobacter coli. res microbiol 2008;159:88–93. doi: 10.1016/j.resmic.2007. 11.020. 35. feizabadi mm, dolatabadi s, zali mr. isolation and drug resistant patterns of campylobacter strains cultured from diarrheic children in tehran. jpn j infect dis 2007;60:217-9. 36. rastyani s, alikhani my, sedighi i, et al. campylobacter jejuni and campylobacter coli in children with acute diarrhea in health centers of hamadan, iran. avicenna j clin microbiol infect 2015;2:e29791. doi: 10.17795/ajcmi29791. 37. mobaien a, moghaddam f, talebi s, et al. studying the prevalence of campylobacter jejuni in adults with gastroenteritis from northwest of iran. asian pac j trop dis 2016;6:957-60. doi: 10.1016/s2222-1808(16)61164-7. 38. al amri a, senok ac, ismaeel ay, et al. multiplex pcr for direct identification of campylobacter spp. in human and chicken stools. j med microbiol 2007;56:1350-5. doi: 10.1099/jmm.0.47220-0. 39. girgis sa, rashad ss, othman hb, et al. multiplex pcr for identification and differentiation of campylobacter species and their antimicrobial susceptibility pattern in egyptian patients. int j curr microbiol app sci 2014;3:861-75. 40. chen cc, chang cj, lin ty et al. usefulness of fecal lactoferrin in predicting and monitoring the clinical severity of infectious diarrhea. world j gastroenterol 2011;17:4218-24. 41. sasaki y, maruyama n, zou b, et al. campylobacter cross-contamination of chicken products at an abattoir. zoonoses public health 2012;60:134–40. doi: 10.1111/j.1863-2378. 2012.01509.x. 42. jonaidi-jafari, n, khamesipour f, ranjbar r, et al. prevalence and antimicrobial resistance of campylobacter species isolated from the avian eggs. food control 2016;70:35-40. 43. shahrokhabadi r, rahimi e, mommtaz h, et al. prevalence of campylobacter jejuni and coli in sheep carcasses by using cultural and pcr methods. zahedan j res med sci 2013;15:2833. 44. westrell t, ciampa n, boelaert f, et al. zoonotic infections in europe in 2007: a summary of the efsa-ecdc annual report. eurosurveill 2009; 14:785–94. 45. baffoni l, gaggìa f, di gioia d, et al. a bifidobacterium-based synbiotic product to reduce the transmission of c. jejuni along the poultry food chain. int j food microbiol 2012; 157:156–61. doi: 10.1016/j.ijfoodmicro.2012. 04.024. irwan juss 165 *emergency department, pluit hospital north jakarta correspondence dr. erwan jus, mars emergency department, pluit hospital north jakarta telp. 021-6685070 email : erwanjus@yahoo.co.id univ med 2008; 27: 165-73 factors influencing length of stay in the emergency department in a private hospital in north jakarta october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 abstract universa medicina erwan jus* length of stay (los) is a key measure of emergency department (ed) throughput and a marker of quality objectives. time studies that assess ed services may help to clarify the causes of prolonged ed service to patients. the objective of this study was to analyse factors influencing los in the ed of private hospital x. this study was conducted at the ed of private hospital x in north jakarta. one hundred and seventy one adult patients were admitted via the over a period of three weeks in march 2008. data recorded included characteristics of patients, service time of nurses, service time of physicians, and overall ed los. anova and independent t-test were performed to determine factors associated with ed los. elderly patients, patients in emergency status, patients with letters of referral, electrocardiogram (ecg) training for physicians and advance cardiac life support (acls) training for nurses were significantly associated with duration of service in the ed. mean ed los was 50 ± 20.7 minutes. patient category and characteristics of physicians and nurses are important independent variables that influence the ed’s duration of service. future research is necessary to determine how these and other factors can be incorporated into a model for predicting ed los. keywords : length of stay, emergency department, quality objective introduction patient visits to the emergency department (ed) have recently increased rapidly. in 1990 ed services in america increased 106% from those in 1980, while in the year 2002 ed visits reached 110.2 million and increased by 23% from the 90 million visits occurring in 1992.(1) in new south wales in 1994 1995 1.5 million patients came to the ed for treatment. in 1999 2000 their numbers increased to 1.7 million, signifying an increase of 10.7% from 1994 1995.(2) in indonesia in 2005 visits to eds of all hospitals was recorded as being 3.6 million, out of 30 million admissions in all hospitals. among 166 jus length of stay these cases presenting to eds, 1.7 million (46.5%) were treated, 51,000 (1.4%) referred, a n d 1 . 9 m i l l i o n ( 5 1 . 5 % ) d i s c h a rg e d . t h e mortality in indonesian eds was up to 0.6%.(3) in the whole jakarta area the number of ed visits was 154, 810 or 5.9% of all hospital admissions (2.6 million). the number of ed patient deaths in jakarta was 1% in 2005.(3) in the united states 34% of visits to eds was classified as requiring treatment within 15 minutes, and only 10% was classified as nonurgent cases.(4) the demand of patients for quality health service has steadily increased, particularly in e d s , w h i c h a r e t h e s e r v i c e g a t e w a y o f a h o s p i t a l . t h e p r o b l e m s i n e d s a r e v e r y complex. the large number of concurrent visits to eds has resulted in overcrowding of eds. overcrowding in eds occur when the demand for services exceeds ed capacity to provide quality care in the appropriate time frame. this situation results in patients not being correctly treated, causing them ultimately to voice their complaints. in canada overcrowded eds have led to errors and delays in therapy, patient dissatisfaction, loss of control on the staff and prolonged waiting time.(5) the ed of private hospital x has adopted t h e q u a l i t y m a n a g e m e n t s y s t e m o f t h e international organization for standardization (iso). it is essential to ascertain whether the iso quality management system has indeed improved the quality of ed service by analyzing the factors affecting ed los. increased number of visits to eds are accompanied by demands on the ed as the health service gateway, to provide immediate service of professional quality. the ed of private hospital x has used triage time and service time of patients as quality objectives of ed service. the objective of this study was to determine the factors influencing los in the ed of private hospital x. methods design of study t h i s s t u d y w a s p e r f o r m e d u s i n g a c r o s s sectional design to determine factors affecting time for achievement of quality objectives in the ed of private hospital x. location and time of study t h i s s t u d y w a s c o n d u c t e d i n t h r e e successive weeks between 1 march 2008 and 21 march 2008 at the ed of private hospital x, north jakarta. population and sample the population in this study comprised all patients presenting to the ed of private hospital x in north jakarta. sampling was done by means of sample blocks within three successive weeks in march 2008. the number of samples taken was minimally consistent with the size of the sample or the number of samples obtained during the study period. inclusion criteria were patients presenting for treatment to the ed of private hospital x who required hospitalization and had to receive an intravenous fluid drip (ivfd). data collection the collected data comprised times for achievement of quality objectives, including service times of nurses and physicians, and patient data in the category referrals and triage. c h a r a c t e r i s t i c s o f a g e , g e n d e r, l e n g t h o f e m p l o y m e n t , a n d t r a i n i n g o f n u r s e s a n d physicians were also collected. data were collected on morning, evening and night shifts by observers who had been given a short training course on data collection methods. measurements the total duration of patient service or los is the time needed by the patient from arrival 167 univ med vol.27 no.4 until discharge from ed. service time was one of the indicators of the quality of ed health s e r v i c e . tr i a g e d i v i d e d p a t i e n t s i n t o t w o categories on the basis of their emergency status. statistical analysis the collected data were processed by the data-processing software program statistical package for social science (spss). univariate and bi var iate analyses wer e conducted to a c h i e v e t h e o b j e c t i v e s o f t h e s t u d y. t h e statistical tests used were anova (analysis of variance) and independent t-test, with a level of significance of 0.05. results one hundred and seventy one patients were recruited for the study, ranging in age from 15 to 95 years. the mean total duration of service (mean ed los) in the ed of private hospital w a s 5 0 ± 2 0 . 7 m i n u t e s , w i t h a m i n i m u m duration of 15 minutes and a maximum of 134 minutes. if the standard of total duration of ed service taken to be 60 minutes, then the mean total duration of patient service in the ed is already substandard. around 73.1% of patients had a total duration of service of less than 60 minutes, while the remaining 26.9% had a total duration of more than 60 minutes. younger patient tended to obtain quicker service compared with older patients. mean total duration of service for patients aged below 21 years was 38 minutes, while that for patients aged 65 years and over was 60 minutes. these values show that total duration of patient service tended to lengthen with increasing age of the patient. the difference in total duration of ed service based on patients’ age was statistically significant (p=0.001) (table 1). out of 171 patients, 41.5% (71 patients) came to the ed with a letter of referral, while 58.5% (100 patients) did not. among the patients bringing a letter of referral, the mean ed service time was 43.6 minutes, whereas in those without a letter of referral the mean ed service time was 55.1 minutes (table 1). therefore it is apparent that patients without letter of referral had a significantly longer total duration of service in the ed of private hospital x, compared to patients with a letter of referral (p=0.001). table 1. mean duration of patient service based on age, referral and triage in the ed of private hospital x patient’s characteristic n (171 ) mean duration of service (min) sd p 37 38.0 15.3 32 46.8 18.6 39 53.0 21.6 26 53.7 20.4 age (yr) < 21 21 – 30 31 – 50 51 – 60 > 60 37 60.4 20.6 0.001 71 43.6 20.2 letter of referral yes no 100 55.1 19.8 0.001 33 67.2 23.7 triase emergency no 138 46.3 17.8 0.001 168 jus length of stay a m o n g t h e 1 7 1 p a t i e n t s , 1 9 . 3 % ( 3 3 patients) were emergency cases and 80.7% (138 patients) were not. for patients with emergency status, mean total duration of service was 67.2 minutes and for those without emergency status, mean total duration of service was 46.3 minutes (table 1). the patients with emergency status had a significantly longer total duration of service total compared with those without (p=0.001). duration of patient service by physicians’ characteristics the duration of service by a physician is the total response time of the physician and the time of examination of the patient by the physician. mean duration of patient service by physicians was 12.9 minutes, with a minimum duration of 2 minutes and a maximum of 65 minutes. in 57.3% of cases, the duration of service by physicians was < 10 minutes and in 42.7% it was > 10 minutes. mean duration of service by physicians in the age group of < 35 years was 14.0 minutes, which was longer compared with 11.9 minutes by physicians in the age group of > 36 years (table 2). however, the difference in mean total d u r a t i o n o f s e r v i c e b y d o c t r o s w a s n o t statistically significant (p=0.221). the same result was obtained for mean duration of service by physicians in the ed of private hospital x, based on the gender of the physicians. based on table 2, male physicians have a shorter mean duration of service in the ed of private hospital x, compared with female p h y s i c i a n s , b u t t h e d i f f e r e n c e w a s n o t statistically significant (p=0.827). ppgd* : penanggulangan penderita gawat darurat (ppgd)/management of emergency patients atls** : advance life support training ecg*** : electro cardio gram table 2. mean duration of patient service by physicians’ characteristic in the ed of private hospital x physician’s characteristics n (171 ) mean duaration of service (min) sd p 82 14.0 12.5 age (yr) ≤ 35 > 36 89 11.9 9.1 0.221 gender male female 119 52 12.8 13.2 9.5 13.7 0.827 85 13.9 12.7 53 11.9 8.6 length of employment (yr) < 4 4-6 > 6 33 11.9 9.1 0.490 ppgd* yes no 142 29 13.3 11.1 7.0 11.5 0.312 atls** yes no 123 48 13.0 12.9 10.8 11.0 0.959 104 14.8 13.2 ecg*** yes no 67 11.7 8.9 0.067 169 univ med vol.27 no.4 from table 2 it is apparent that there is no difference in mean total duration of patient service based on length of employment as a physician (p=0.490). this indicates that the duration of service by physicians in the ed of private hospital x has been standardized in spite of differences in length of employment. among the physicians who had followed training for management of emergency patients penanggulangan penderita gawat darurat (ppgd), the mean duration of patient service was 13.3 minutes, which was longer compared with physicians without ppgd training, who had a mean duration of 11.1 minutes. however, the difference was statistically not significant (p=0.312). similarly, for physicians who had training for advance trauma life support (atls), the mean duration of patient service was 12.9 m i n u t e s , p r a c t i c a l l y i d e n t i c a l t o t h a t o f physicians without atls training, whose mean duration was 13.0 minutes. additionally, the physicians who had ecg training, has a mean duration of patient service of 11.7 minutes, less than the mean duration of 14.8 minutes for physicians without ecg training. duration of patient service by characteristics of nurses the duration of patient service by nurses in t h e e d i s t h e n u r s e ’s r e s p o n s e t i m e f o r examination of vital signs of patients by the nurse and length of time needed by the nurse in setting up an ivfd. mean duration of patient service was 14.7 minutes, with a minimum of 8 minutes and a maximum of 30 minutes. in around 66.1% of cases, the duration of patient service was < 10 minutes and in 33.9% it was > 10 minutes. nurse’s characteristics n (171) mean duration of service (min) sd p 45 14.1 3.1 58 14.9 5.0 age (yr) ≤ 25 26 – 30 31 – 35 ≤ 36 44 24 15.0 15,0 5.2 4.2 0.785 gender male female 68 103 14.6 14.8 4.8 4.3 0.713 length of employment (yr) ≤ 7 > 7 135 36 15.0 13.7 4.6 4.1 0.117 ppgd* yes no 19 152 14.7 14.7 3.3 4.6 0.976 117 13.8 3.0 acls** yes no 54 15.2 5.0 0.067 table 3. mean duration of patient service by nurse’s characteristic in the ed of private hospital x ppgd* : penanggulangan penderita gawat darurat (ppgd)/management of emergency patients acls** : advance cardiac life support 170 jus length of stay the mean duration of patient service by nurses aged 25 years or below was 14.1 minutes, which was shorter compared with nurses in the 26-30 year age group, whose mean duration of service was 14.9 minutes, and with nurses older than 30 years, with a mean duration of 15 minutes (table 3). however, the difference was statistically not significant (p=0.785). the mean duration of patient service by female nurses was 14.8 minutes, equal to that of male nurses, with a mean of 14.6 minutes, but the difference was statistically not significant (p=0.713). mean duration of patient service by nurses with length of service of 7 years or less was 15.0 minutes, while for nurses with length of service of more than 7 years it was 13.7 minutes (table 3). this indicates that nurses with a long period of employment had a shorter mean duration of service, but the difference was statistically not significant (p=0.117). among nurses who had ppgd training, the mean duration of patient service was 14.7 minutes, equal to that of nurses without ppgd training, with mean duration of 14.7 minutes. among nurses who had advances cardiac life support (acls) training the mean duration of patient service was 15.2 minutes, which was longer than the mean duration of 13.8 minutes among nurses without acls training, but the difference was statistically not significant (p=0.067). discussion this study demonstrates that the utility of time studies in identifying and quantifying factors that prolong ed los. the study obtained a mean duration of patient service in the ed of 50 minutes, while the minimum was 15 minutes and the maximum 134 minutes. this means that since the arrival of the patient until his/her discharge from the ed, a time period of 50 minutes was required. thus it is apparent that in 73.1% of cases the patient service time in the ed was less than 60 minutes, but around 26.9% of cases still required more than 60 minutes. data of activities in the ed of rsud bantul (bantul general hospital) indicate that management of medical emergencies in the ed requires a mean time period of 30 minutes, or 15 minutes above standard quality of service based on emergency response time rate. (6) however, the results of the present study differs from studies conducted in three metropolitan hospitals in melbourne, which showed that the overall mean of ed los was 7.96 hours.(7) the melbourne studies had more prolonged ed los, because the patients arriving at the eds were patients requiring hospitalization. t h e n a t i o n a l h o s p i t a l a m b u l a t o r y medical care survey (nhamcs) obtained a mean los of 167.4 minutes for outpatients, while for hospitalized patients the mean time period required was 364.4 minutes.(8) a study by gardner showed that the median los for hospitalized patients was 255 minutes and for outpatients 210 minutes.(9) the results of the present study showed that mean duration of ed patient care in private hospital x was already below the quality objective of the ed of private hosital x of 60 minutes. however, closer examination revealed a difference in mean patient care based on patient’s age, letter of referral, and level of e m e rg e n c y / t r i a g e o f t h e p a t i e n t . yo u n g e r patients tended to have a shorter duration of ed service compared with older patients. the mean duration of service was 38 minutes for patients below 21 years, and tended to increase with patient’s age, reaching 60 minutes in patients over 65 years old. the difference in duration of e d s e r v i c e b a s e d o n p a t i e n t ’s a g e w a s statistically significant. these results are similar to those of studies in canada, where annually more than one million canadians are hospitalized through the 171 univ med vol.27 no.4 ed. the canadian patients tended be elderly p e r s o n s s u f f e r i n g f r o m v a r i o u s s e v e r e conditions and on average had longer hospital stays than other inpatients.(10) the centers for disease control and prevention (cdc) reported an increase in ed visits by adults, especially those aged 65 years and over.(11) a study in u.k.showed that the proportion of patients requiring ed los of more than 4 hours was 39.0% and belonged to the age group of >85 years.(12) from the results of this study it may be concluded that patients without a letter of referral have longer service times compared with patients having a letter of referral. this was probably due to the physician on duty being able to focus on administration of therapy, because in the letter of referral the patient’s identity, diagnosis and therapy had already been specified. patients with emergency status had a longer duration of service compared with patients without emergency status. these results are similar to those of canadian studies stating that ed los depends on the severity of the patient’s illness. data from 2003-2004 in canada showed that patients with more severe disease stayed longer in the ed compared with patients with milder disease. ed los resulting from triage indicates that patients with canadian triage and aculty scale (ctas) i (requiring resuscitation/emergency care) had a median los of 161 minutes compared with the median for ctas v (non-emergency) of 67 minutes. this difference shows that the more complex a patient’s health problem, the larger the number of diagnostic tests required and the longer the observation time, compared with conditions that can be managed by the ed physicians themselves. it was also proved that the median los increases with advanced age. the increased los in ed patients with more severe illness has also been reported in australia in 2003-2004. patient who underwent emergency triage had a los of more than 3 hours (195 minutes) compared with those undergoing nonemergency triage, with a los of 75 minutes.(13) this study showed that the duration of patient service by ed physicians is 12.9 minutes. research in canada has shown that the service time of their physicians is 3-4 patients per hour.(14) the study by millar indicates that mean service time of ed physicians is 17.9 minutes.(15) studies in the ed of rsud bantul show that mean medical emergency handling time is 12.51 minutes, with most frequent mean handling time of 10 minutes. these results indicate that handling of emergencies was not delayed and could prevent deaths in up to 30% of emergency cases.(6) canadian studies have associated total service time of ed physicians per patient visit with gender, age, vital signs and glasgow coma scale (gcs), mode of arrival at ed, triage level, accompanying diseases of patient and hospital working procedures, demonstrating that the most important predictors influencing service time of physicians are working procedure, triage level, arrival by ambulance, gcs, patient’s age, accompanying disease and number of visit priorities.(16) service time of patients by ed nurses in private hospital x is 14.7 minutes. studies in new zealand yielded mean patient service time by nurses of 49 minutes per visit. on average, patients with an emergency triage code had longer ed los, meaning that non-ambulatory patients take up more of the nurse’s time.(17) research in america has demonstrated that the most critically ill or injured patients get a quicker response from nurses and physicians, although with varying lengths of time, but the overall of service rate by nurses was up to 80%.(18) the results of studies on canadian nurses indicate that nurses take care of between 1-2 patients per hour.(5) the results of this study show that the significant factors influencing service time of 172 jus length of stay ed patients are patient category, physicians’ charracteristics and nurses characteristics. patient category (as independent variable) includes age, presence or absence of referral letter, and level of emergency or triage of patient. nurses’ characteristics (as independent variable) include age, length of service and acls training, while physicians’ category (as independent variable) includes ekg training. the current study results are consistent with studies in america stating that the factors influencing ed los (emergency departement length of stay) from the study sample included patient’s age, patient’s diagnosis, consultation, multiple radiological examinations, delays in a d m i s s i o n ( h o s p i t a l i z a t i o n ) , n u r s e s b e i n g occupied, family members staying at bedside, o b s e r v a t i o n o f p a t i e n t , p a t i e n t r e q u i r i n g stabilization, beds not ready (not made), and p a t i e n t r e c e i v i n g i v f d . t h e r e w a s n o significant ed los factor noted as being the result of initial examination by the physician.(19) limitations of the study a major limitation of this study was that t h e s t u d y w a s c r o s s s e c t i o n a l a n d n o t p r o s p e c t i v e . t h i s i n v e s t i g a t i o n f a i l e d t o delineate causal relationships between predictor v a r i a b l e s a n d l o s . e d p r o c e s s e s a r e i n t e r d e p e n d e n t a n d s u b j e c t t o e x t e r n a l influences; therefore, improvement in one area may not shorten los, and meaningful change may involve adjusting a combination of many factors, the identification of which may be difficult. a final concern is that the generalizability o f o u r f i n d i n g s m a y b e l i m i t e d b e c a u s e sociodemographic factors, ed work processes, ed management structures, availability of specialty consultation and inpatient services all vary across sites and would be expected to influence ed los. conclusions e m e rg e n c y d e p a r t m e n t l o s w a s associated with elderly status, letter of referral, p a t i e n t t r i a g e , p h y s i c i a n ’s a n d n u r s e ’s characteristics. strategies for reducing ed los, which presumably will lead to ed cost-savings, should target elderly patients. importantly, they m a y a l s o p r e v e n t p a t i e n t m o r b i d i t y a n d mortality. references 1. freeman j. emergency department overcrowding: peering through the holes in the safety net. can j emerg med. 2007; 9: 378-9. 2. new south wales. emergency department services plan. nsw public health bulletin; 2001. 3. department of health (depkes), republic of indonesia. hospital statistics in indonesia. series 1: service activity. jakarta: department of health ri; 2006. 4. centers for disease control and prevention. latest data on emergency department visits. centers for disease control and prevention; 2004. 5. canadian association emergency physicians (caep). position statement on emergency department overcrowding. caep; 2007. 6. pranowo kth . pengaruh waktu penatalaksanaan kegawatdaruratan medis terhadap mutu pelayanan di instalasi gawat darurat rsud bantul. cermin dunia kedokteran 2006; 152: 15-9. 7. liew d, liew d, kennedy mk. emergency department length of stay independently predicts excess inpatient length of stay. med j aust 2003; 179: 5246. 8. daniel ah, j mc connell k. stay and predictive demographic characteristics. west j emerg 2007; 8, article 21. available at : http://repositories cdlib.org/wetjem/vol8/iss 3/art 21. accessed june 2, 2008. 9. gardner rl. factors associated with longer ed length of stay. am j emerg med 2007; 25: 643-50. 10. dawson h, weerasooriya j, webster g. hospital admissions via the emergency department: implications for planning and patient flow. health care quart 2008; 11: 20-2. 11. centers for disease control and prevention. visits 173 to u.s emergency departments at all-time high. centers for disease control and prevention; 2005. 12. downing a, wilson rc, cooke mw. which patients spend more than 4 hours in the accident and emergency department. j publ health 2004; 26: 172-6. 13. canadian institution for health information. understanding emergency department wait times: who is using emergency departments and how long are they waiting? canadian institution for health information; 2005. 14. carte aj, chochinov ah. a systematic review of the impact of nurse practitioners on cost, quality of care, satisfaction and wait times in the emergency department. can j emerg med 2007; 9: 286-95. 15. millar rk. estimating physician workload on the pediatric emergency department (thesis). calgary: university of calgary; 2007. 16. innes gd. perspective time study derivation of emergency physician workload predictor. can j emerg med 2005; 7: 299-308. 17. gabolinscy b. triage code: a predictor of nursing care in the emergency department (thesis). auckland: university of technology; 2005. 18. yoon p, steiner i, reinhardt g. 2003. ‘analysis of factors influencing length of stay in the emergency department’. can j emerg med 2003; 5: 155-62. 19. weinsier ts. factors affecting emergency department length of stay of patients admitted to telemetry and critical care units (dissertation). miami: florida international university; 1999. univ med vol.27 no.4 woro1 64 profil keparahan cedera pada korban kecelakaan sepeda motor di instalasi gawat darurat rsup fatmawati woro riyadina*a dan ita puspitasari subik** *puslitbang biomedis dan farmasi balitbangkes dep.kes. ri. **igd rsup fatmawati korespondensi a woro riyadina, m.kes. puslitbang biomedis dan farmasi balitbangkes depkes. r.i. jl. percetakan negara no.23a jakarta pusat telp. 021-4244693 email: w_riyadina@litbang.depkes.go.id universa medicina 2007; 26: 64-72. universa medicina april-juni 2007april-juni 2007april-juni 2007april-juni 2007april-juni 2007 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 latar belakang proporsi disabilitas dan case fatality rate cedera akibat kecelakaan lalu lintas masih tinggi (25%). cedera akibat kecelakaan lalu lintas merupakan penyebab utama disabilitas dan mortalitas di negara berkembang. tujuan penelitian ini adalah untuk menggambarkan profil keparahan cedera yang dialami oleh korban kecelakaan lalu lintas sepeda motor yang masuk ke bagian instalasi gawat darurat rumah sakit umum pusat (igd rsup) fatmawati. metode penelitian ini merupakan bagian dari penelitian “pengembangan surveilans cedera akibat kecelakaan lalu lintas pada pengendara sepeda motor.” pengumpulan data dilakukan selama 1 bulan yaitu selama bulan oktober 2005. responden adalah pengendara sepeda motor baik sebagai pengemudi maupun penumpang yang mengalami kecelakaan lalu lintas dan masuk ke igd rumah sakit. pengumpulan data dilakukan dengan metode wawancara terhadap korban kecelakaan maupun pengantar dengan menggunakan formulir register cedera. hasil korban kecelakaan sepeda motor yang masuk ke igd rsup fatmawati selama kurun waktu 1 bulan sebanyak 138 orang. karakteristik korban adalah mayoritas laki-laki 73,9%, berumur sekitar 21-30 tahun 43,5%, pendidikan setingkat smu 59,4% dan bekerja sebagai pegawai swasta 55,8%. kondisi korban yang mengalami cedera ringan sekitar 52,2%, cedera parah 47,8%. daerah cedera meliputi kepala 55,1% dengan commotio cerebri 6,5%, kaki 12,3% dan lutut/tungkai bawah 9,4%. jenis luka meliputi luka terbuka 42,0%, patah tulang 18,0% dan luka lecet 14,5%. faktor-faktor yang berbeda bermakna dengan keparahan cedera adalah waktu terjadinya kecelakaan (malam hari) dan kecepatan kendaraan ≥ 60 km/jam (p<0,05). kesimpulan korban kecelakaan sepeda motor yang masuk ke igd rsup fatmawati kebanyakan mengalami cedera di bagian kepala dengan luka terbuka, dan kondisi korban yang parah cukup besar yaitu 47,8%. kata kunci: keparahan, cedera, kecelakaan, sepeda motor, rsup fatmawati abstrak 65 universa medicina vol.26 no.2 pendahuluan kecelakaan lalu lintas merupakan masalah kesehatan masyarakat di seluruh negara di dunia, khususnya di negara berkembang.(1) kecelakaan l a l u l i n t a s m e n e m p a t i u r u t a n k e 9 p a d a di s a b i l i t y ad j u s t e d li f e ye a r ( d a ly ) d a n diperkirakan akan meningkat menjadi peringkat ke 3 di tahun 2020 (1) sedangkan di negara profile of the severity of motorcycle injury that admitted to emergency department of fatmawati hospital woro riyadina*a and ita puspitasari subik** bankground proportion of disabilities and case fatality rates related road traffic accident injury are still high in the development countries. the objective of the study was to describe profile of fatality of motorcycle injury that entered to emergency department of fatmawati hospital. methods the study was part of the research “development of road traffic accident surveillance on motorcyclist“. data were analyzed from victims of motorcycle accident who entered to emergency department of fatmawati hospital in south of jakarta during the month of oktober 2005. data collected by interview using injury registry form with victims, or witnesses. results victims of motorcycle accident who admitted to emergency department of fatmawati hospital during one month were 138 cases. the majority of victims were males (73.9%), aged between 21-30 years (43.5%), high school graduates (59.4%) and workers (55.8%). victims who suffered mild injuries were 52.2%, severe 47.8%. parts of the body injured were head (55.1%) with commotio cerebri 6.5%, legs (12.3%) and knee-lower leg 9.4%. type of injuries were excoriasi (42.0%), fracture (18.0%) and superficial (14.5%). accident in the night and speed ≥ 60 km/hour had significant differenced with the severity of motorcycle injury (p<0.05). conclusion the majority victims of motorcycle accident who admitted to emergency department of fatmawati hospital have head injuries with excoriasi and severe condition was 47.8%. keywords: severity, injury, accident, motorcycle, fatmawati hospital * center of biomedical research & development and pharmacy, indonesia remising of health **admitted to emergency department of fatmawati hospital correspondence a woro riyadina, m.kes. center of biomedical research & development and pharmacy, indonesia remising of health jl. percetakan negara no.23a jakarta pusat telp. 021-4244693 email: w_riyadina@litbang.depkes.go.id universa medicina 2007; 26: 64-72. abstract berkembang menempati urutan ke-2.(2) pada tahun 2002, kecelakaan lalulintas merupakan penyebab kematian urutan kesebelas di seluruh dunia. rate dari road traffic fatality penduduk paling rendah di negara maju di eropa (11,0 per 1 0 0 . 0 0 0 ) , d a n p a l i n g t i n g g i d i n e g a r a berpenghasilan rendah dan menengah di eastern mediterrean (26,4 per 100.000) dan afrika (28,3 per 100.000).(3) cedera akibat kecelakaan 66 lalu lintas adalah penyebab utama kematian dan disabilitas (ketidakmampuan) secara umum terutama di negara berkembang.(4) proporsi disabilitas dan case fatality rate (cfr) cedera akibat kecelakaan lalu lintas masih tinggi. cfr tertinggi dijumpai di beberapa negara amerika latin (41,7 per 100.000 penduduk), asia (21,9 d a n 2 1 , 0 p e r 1 0 0 . 0 0 0 p e n d u d u k d i k o r e a s e l a t a n s e r t a t h a i l a n d ) . ( 3 ) d i i n d o n e s i a sebagian besar (70%) korban kecelakaan lalu l i n t a s a d a l a h p e n g e n d a r a s e p e d a m o t o r. ( 5 ) kontribusi sepeda motor terhadap kecelakaan di indonesia adalah 80,3% (14.223 kasus dari 17.732) dan di jakarta ialah 59,2% (2403 kasus dari 4065).(6) kecelakaan lalu lintas disebabkan oleh faktor manusia (host), faktor kendaraan (agent) dan faktor lingkungan (enviroment) yang saling berkaitan antara satu faktor dengan faktor yang l a i n . f a k t o r f a k t o r t e r s e b u t b e r p e n g a r u h terhadap tingkat keparahan cedera yang dialami oleh korban kecelakaan disamping beberapa faktor yang lain seperti faktor penanganan cedera baik di pra rumah sakit dan di rumah s a k i t . p e n e l i t i a n i n i b e r t u j u a n u n t u k m e n g g a m b a r k a n p r o f i l k e p a r a h a n c e d e r a korban kecelakaan sepeda motor dan faktor risikonya. metode rancangan penelitian rancangan penelitian yang digunakan adalah potong lintang (cross sectional). lokasi penelitian penelitian dilakukan di bagian instalasi gawat darurat (igd) rumah sakit umum pusat ( rsup) fatmawati yang merupakan salah satu rumah sakit besar di wilayah dki jakarta yang berlokasi di jakarta selatan. rsup fatmawati termasuk ke dalam katagori rumah sakit tipe a. subyek penelitian sampel atau subyek penelitian adalah semua pengendara sepeda motor (pengemudi atau penumpang/pembonceng) baik yang masih hidup maupun yang sudah meninggal akibat korban kecelakaan lalu lintas yang datang atau dibawa ke igd rsup fatmawati. pengumpulan data pengumpulan data dilakukan dengan cara wawancara terhadap subyek atau pengantar menggunakan formulir registri cedera oleh perawat igd dan dilengkapi oleh petugas rekam medis yang sudah dilatih. kegiatan pengumpulan data dilakukan selama kurun waktu 1 bulan yaitu dari tanggal 1 sampai d e n g a n 3 1 o k t o b e r 2 0 0 5 . va r i a b e l y a n g d i k u m p u l k a n t e r d i r i a t a s f a k t o r m a n u s i a (karakteristik responden, pemakaian helm, k o n s u m s i a l k o h o l ) , f a k t o r k e n d a r a a n ( k e c e p a t a n k e n d a r a a n ) , f a k t o r l i n g k u n g a n (kondisi jalan dan cuaca) dan faktor pelayanan kesehatan (penanganan pra dan di rumah sakit). klasifikasi cedera b e r d a s a r k a n i n t e r n a t i o n a l c o d e o f diseases (icd)-10(7) klasifikasi cedera daerah atau bagian tubuh yang mengalami cedera terbagi dalam 10 katagori, yaitu bagian (1) kepala, (2) leher, (3) dada, (4) perut, pungung, pinggang dan panggul, (5) bahu dan lengan a t a s , ( 6 ) s i k u d a n l e n g a n b a w a h , ( 7 ) pergelangan tangan, (8) sendi,pinggul, tungkai atas, (9) lutut dan tungkai bawah dan (10) pergelangan kaki. sedangkan katagori jenis cedera menurut klasifikasi icd-10(6) terbagi dalam 17 tipe yaitu (1) superfisial (luka lecet), (2) luka terbuka, (3) patah tulang (termasuk gigi), (4) dislokasi, sprain dan strain, (5) cedera syaraf (sumsum tulang belakang), (6) cedera pembuluh darah, (7) cedera otot dan tendo, (8) cedera mata, (9) cedera jantung (organ intra abdomen), (10) cedera organ thoraks lainnya riyadina, subik cedera kecelakaan sepeda motor 67 universa medicina vol.26 no.2 (pelvis), (11) komosio cerebri, (12) kontusio, laserasi dan perdarahan otak, (13) perdarahan e p i d u r a l , ( 1 4 ) p e r d a r a h a n s u b d u r a l , ( 1 5 ) r e m u k , ( 1 6 ) a m p u t a s i , d a n ( 1 7 ) l a i n n y a . keparahan cedera akibat kecelakaan sepeda m o t o r d i s i n i d i k a t a g o r i k a n m e n j a d i d u a kelompok yaitu cedera parah dan tidak parah. pengelompokan tersebut dilakukan atas dasar pertimbangan beberapa hal yaitu skor glasgow coma scale (gcs), skor trauma, sifat dan daerah cedera menurut icd-10 serta tindakan medis utama yang dilakukan terhadap korban kecelakaan sepeda motor analisis statistik a n a l i s i s p e r s e n d i g u n a k a n u n t u k menggambarkan distribusi dari masing-masing v a r i a b e l d a n u j i c h i s q u a re u n t u k membandingkan proporsi masing-masing faktor terhadap keparahan cedera. hasil selama kurun waktu 1 bulan di igd rsup f a t m a w a t i d i d a p a t k a n k o r b a n k e c e l a k a a n sepeda motor sebanyak 138 orang. dari jumlah t e r s e b u t y a n g m e n e m p a t i p o s i s i s e b a g a i pengemudi sebanyak 97 orang (70,3%) dan sebagai penumpang/pembonceng sebanyak 41 orang (29,7%). adapun korban kecelakaan yang mengalami cedera ringan (tidak parah) 72 orang (52,2%), dan cedera parah sebanyak 66 orang (47,8%). di antara 138 kasus sebanyak 2 (1,4%) orang meninggal. karakteristik korban kecelakaan sepeda motor karakteristik korban kecelakaan sepeda motor yang masuk ke igd rsup fatmawati digambarkan pada tabel 1. korban kecelakaan sepeda motor sebagian besar umurnya berkisar antara 21-30 tahun (43,5%), diikuti kelompok umur 31-40 tahun (25,4%) dan umur antara 1120 tahun sebanyak 16,7%. korban kecelakaan sepeda motor yang masuk ke igd rsup fatmawati proporsinya seimbang antara korban yang berstatus belum tabel 1. karakteristik korban kecelakaan sepeda motor 68 kawin dan yang sudah kawin yaitu masingmasing 69 orang (50%). hal tersebut sesuai jika dibandingkan dengan kelompok umurnya karena kebanyakan korban berada di rentang umur 2130 tahun. ti n g k a t p e n d i d i k a n s e b a g i a n b e s a r setingkat smu atau tingkat pendidikan sedang yaitu 59,4%, dan selanjutnya diikuti oleh tingkat pendidikan rendah (setingkat sd dan smp) yaitu s e k i t a r 2 5 , 4 % . s t a t u s p e k e r j a a n k o r b a n kecelakaan sepeda motor lebih dari separuhnya adalah pegawai swasta yaitu sebanyak 77 orang ( 5 5 , 8 % ) . a d a p u n s u m b e r b i a y a u n t u k pengobatan dan tindakan medis selama di rumah sakit bak yang langsung bisa pulang atau dirawat inap hampir 90% ditanggung sendiri oleh korban kecelakaan. pemanfaatan biaya dari asuransi jasa raharja masih sangat rendah yaitu hanya sekitar 0,7%. korban kecelakaan sepeda motor yang masuk di igd fatmawati paling banyak mendapatkan tindakan medis diobati dan langsung bisa pulang sebanyak 52,5%, diikuti oleh diobati dan dirawat inap sekitar 27,5% dan yang pulang paksa sebanyak 14%. adapun korban yang mengalami tindakan intervensi bedah sebanyak 4 orang (2,9%). tindakan bedah terdiri dari bedah syaraf (otak) sebanyak 75% dan bedah tulang sekitar 25%. daerah dan jenis cedera daerah dan jenis cedera yang dialami oleh k o r b a n k e c e l a k a a n s e p e d a m o t o r a k a n menentukan tingkat keparahan cederanya. distribusi prosentase daerah cedera tercantum dalam tabel 2. daerah atau bagian tubuh yang dominan mengalami cedera pada korban kecelakaan sepeda motor adalah kepala yaitu sebanyak 55,1%, selanjutnya adalah bagian pergelangan kaki yaitu sekitar 12,3% dan bagian lutut dan tungkai bawah yaitu sekitar 9,4%. cedera kepala selalu menempati urutan teratas dari cedera yang dialami oleh pengendara sepeda motor. hal tersebut berhubungan dengan pemakaian helm sebagai alat pelindung kepala, baik dari jenis h e l m m a u p u n d a r i c a r a p e m a k a i a n n y a . sedangkan untuk cedera di kaki dan lutut menunjukkan perlunya dirancang pelindung bagi bagian tubuh tersebut untuk bisa mengurangi tabel 2. distribusi daerah cedera korban kecelakaan sepeda motor berdasarkan klasifikasi icd-10 tabel 3. distribusi jenis cedera akibat kecelakaan sepeda motor menurut klasifikasi icd-10 korban minimal mempunyai satu macam atau jenis cedera dan kebanyakan merupakan multiple injury (cedera lebih dari satu jenis dan satu daerah) riyadina, subik cedera kecelakaan sepeda motor 69 universa medicina vol.26 no.2 risiko cedera yang lebih parah. adapun jenis cedera akibat kecelakaan sepeda motor disajikan dalam tabel 3. urutan jenis cedera yang paling banyak dialami oleh korban kecelakaan sepeda motor adalah luka terbuka (42,0%), patah tulang (18,1) dan superfisial/lecet (14,5%). adapun untuk cedera kepala yang dominan adalah k o m o s i o c e r e b r i y a i t u s e k i t a r 6 , 5 % d a n kontusio, laserasi dan perdarahan dalam otak sebanyak 3,6%. korban kecelakaan sepeda motor minimal mempunyai satu jenis luka dan p a d a u m u m n y a m e n g a l a m i c e d e r a y a n g k o m p l e k ( m u l t i p l e i n j u r y ) b a i k d a r i j e n i s maupun daerah cedera. keparahan cedera keparahan cedera dapat dipengaruhi oleh beberapa faktor diantaranya meliputi faktor e k s t e r n a l ( f a k t o r l i n g k u n g a n ) d a n f a k t o r internal seperti manusia. faktor lingkungan fisik meliputi jenis dan kondisi jalan, cuaca dan waktu kejadian. adapun faktor internal antara l a i n y a k n i p e r i l a k u p e n g e n d a r a s e p e r t i pemakaian helm, kecepatan mengendarai dan k o n s u m s i a l k o h o l . p e r b e d a a n p r o p o r s i keparahan cedera terhadap faktor lingkungan fisik disajikan pada tabel 4. lingkungan fisik disini adalah sarana fisik yang meliputi kondisi jalan tempat lalu lintas kendaraan, cuaca dan waktu pada saat terjadi kecelakaan. tabel 4 menunjukkan bahwa perbedaan p r o p o r s i k e p a r a h a n c e d e r a y a n g b e r b e d a bermakna (p<0,05) hanya terhadap faktor waktu kejadian kecelakaan. kecelakaan yang terjadi di malam hari mempunyai proporsi yang l e b i h t i n g g i k e p a r a h a n c e d e r a n y a ( 5 9 % ) dibandingkan kejadian kecelakaan di siang hari (41,6%). sedangkan korban kecelakaan sepeda motor yang mengalami cedera tidak parah proporsinya lebih tinggi apabila waktu kejadian kecelakaan pada siang hari. proporsi korban yang mengalami cedera parah di jalan dua arah tampak lebih tinggi (49,4%) dibandingkan d e n g a n d i j a l a n s a t u a r a h ( 3 9 , 6 % ) t e t a p i perbedaannya tidak bermakna (p>0,05). arah arus kendaraan dua arah berisiko lebih tinggi mengakibatkan kecelakaan yang lebih parah dibandingkan dengan jalan dengan arus satu arah. cuaca hujan saat terjadi kecelakaan mempunyai proporsi cedera parah yang lebih tinggi (64,7%) dibandingkan dengan cuaca c e r a h a t a u t i d a k h u j a n ( 4 6 , 9 % ) m e s k i p u n perbedaannya tidak bermakna (p>0,05). tabel 4. perbedaan proporsi keparahan cedera berdasarkan faktor lingkungan fisik 70 p e n g e n d a r a s e p e d a m o t o r a d a l a h pengemudi dan penumpang (pembonceng). perbedaaan proporsi keparahan cedera dengan faktor perilaku manusia dapat dilihat pada tabel 5. tabel 5 menunjukkan bahwa dari beberapa faktor perilaku pengendara yang mempunyai perbedaan proporsi keparahan cedera berbeda bermakna (p<0,05) adalah kecepatan kendaraan ≥ 60 km/jam. sepeda motor yang melaju dengan kecepatan tinggi (≥ 60 km/jam) mempunyai proporsi mengalami cedera parah lebih tinggi (77,8%) dibandingkan dengan kecepatan rendah/lambat (< 60 km/jam) yaitu 40,5%. pembahasan mayoritas korban kecelakaan sepeda motor menimpa kelompok umur usia muda (21-30 tahun) yang merupakan umur produktif. hasil ini sesuai dengan hasil penelitian terdahulu yaitu kelompok yang berisiko terjadi kecelakaan adalah usia 15-25 tahun(8) dan kematian akibat kecelakaan lalu lintas lebih dari separohnya terjadi pada kelompok umur dewasa muda umur antara 15-44 tahun.(9) dari sudut pandang beban ekonomis hal tersebut merupakan faktor yang berpengaruh terhadap produktifitas dan kualitas hidup korban kecelakaan pasca kejadian. korban kecelakaan yang mengalami cedera parah dan mengakibatkan cacat yang permanen akan menurunkan kualitas hidup untuk menyongsong masa depan. hal tersebut menyebabkan beban fisik, psikologis dan ekonomi yang tidak sedikit. beberapa hasil penelitian menunjukkan bahwa korban kecelakaan sepeda motor mayoritas berjenis kelamin laki-laki dibandingkan dengan p e r e m p u a n , d e m i k i a n j u g a d e n g a n h a s i l penelitian ini. jumlah korban laki-laki (73,9%) kira-kira 3 kali lebih banyak dari jumlah korban perempuan (26,1%). hasil ini sesuai dengan studi who yang menunjukkan bahwa 73% dari korban kecelakaan lalu lintas yang fatal adalah laki-laki.(9) hal tersebut dapat dijelaskan karena adanya faktor mobilitas yang lebih tinggi serta s e p e d a m o t o r m e r u p a k a n k e n d a r a a n y a n g p a l i n g b a n y a k d i p i l i h s e b a g a i s a r a n a transportasi . riyadina, subik cedera kecelakaan sepeda motor tabel 5. perbedaan proporsi keparahan cedera berdasarkan perilaku manusia 71 universa medicina vol.26 no.2 tingkat pendidikan yang pernah diraih oleh korban kecelakaan sepeda motor kebanyakan setingkat smu atau tingkat pendidikan sedang yaitu 59,4%, dan selanjutnya diikuti oleh tingkat pendidikan rendah (setingkat sd dan smp) yaitu sekitar 25,4%. hasil ini sesuai dengan hasil penelitian dari suwandono pada tahun 2001 bahwa mayoritas korban kecelakaan sepeda motor adalah berpendidikan setingkat smu.(5) tingginya proporsi korban yang mengalami pulang paksa karena kemauan sendiri beralasan k e b e r a t a n a t a s b i a y a p e r a w a t a n . k e a d a a n tersebut menggambarkan bahwa korban cedera parah yang tidak mendapatkan penanganan m e d i s y a n g t e p a t d a m p a k n y a a k a n d a p a t menyebabkan kecacatan pada anggota tubuh s e c a r a p e r m a n e n . h a l t e r s e b u t d a p a t menurunkan kualitas hidupnya. sedangkan tingginya proporsi korban kecelakaan sepeda motor yang mengalami jenis luka terbuka dan patah tulang menunjukkan tingkat keparahan cedera yang membutuhkan tindakan medis, perawatan yang lama di rumah sakit dan waktu rehabilitasi pasca pengobatan. hal tersebut akan menyebabkan beban bagi korban kecelakaan baik finansial dan waktu yang tidak sedikit dan dampak dari kejadian biasanya berakibat pada kecacatan yang permanen (menetap). sedangkan d a m p a k d a r i c e d e r a k e p a l a ( k o m o s i o d a n kontusio) kebanyakan menimbulkan kelainan di bagian syaraf yang mengakibatkan adanya gangguan mental. keadaan tersebut secara otomatis akan menurunkan tingkat kualitas hidup untuk masa depannya. penelitian pada 1534 penderita berusia 17-69 yang masuk di rs d i o x f o r d m e n u n j u k k a n s e b a n y a k 1 , 5 % menderita cedera berat dan 21% cedera ringan. ternyata setelah 3 bulan ditemukan posttraumatic stress disorder (pstd) dan ansietas serta depresi.(10) keparahan korban kebanyakan akibat kecelakaan yang terjadi pada malam hari.hal ini senada dengan hasil penelitian di new z e a l a n d b a h w a a d a t i g a f a k t o r y a n g meningkatkan risiko cedera lebih parah yaitu kondisi mengantuk, tidur kurang dari 5 jam dalam sehari dan mengendara antara jam 02.00 – 05.00.(11) waktu malam hari suasananya lebih gelap dan lalu lintas biasanya sudah mulai sepi. kondisi tersebut menyebabkan pengendara sepeda motor mengemudikan kendaraannya dengan kecepatan tinggi (≥ 60 km/jam), kurang waspada dan kurang hati-hati. faktor tersebut b e r i s i k o t e r j a d i n y a k e c e l a k a a n y a n g menyebabkan cedera yang lebih parah. risiko u n t u k t e r j a d i n y a k e m a t i a n d a n c e d e r a meningkat seiring dengan kenaikan kecepatan mengemudi.(12) kecepatan kendaraan 20 meter per jam mempunyai risiko 5% menyebabkan kematian sedangkan untuk kecepatan 85 meter per jam meningkatkan risiko kematian menjadi 8 5 % . ( 1 3 ) k e c e l a k a a n s e p e d a m o t o r a k i b a t k e c e p a t a n t i n g g i s a a t m e n g e m u d i k a n k e n d a r a a n n y a p a d a u m u m n y a m e n g a l a m i cedera parah dikarenakan mengalami benturan hebat dan kuat yang memungkinkan korban terlempar jauh dari titik kejadian. hasil yang tidak berbeda didapatkan di kathmandu, nepal, m e n u n j u k k a n s e l a m a t a h u n 1 9 8 1 2 0 0 3 kematian akibat kecelakaan lalulintas setiap tahunnya meningkat sebesar 3,88.(14) kondisi fisik jalan yang rusak maupun yang mulus tidak berbeda bermakna terhadap proporsi keparahan cedera. kondisi jalan yang mulus mendorong pengendara sepeda motor untuk memacu kecepatan sedangkan untuk kondisi jalan yang rusak pengendara sepeda motor biasanya kurang berhati-hati dalam mengemudikan kendaraannya meskipun dengan k e c e p a t a n y a n g r e n d a h . f a k t o r p e r i l a k u b e r k e n d a r a b e r p e r a n p e n t i n g t e r h a d a p keparahan cedera. faktor manusia mempunyai p r o p o r s i t e r t i n g g i ( 9 0 % ) s e b a g a i f a k t o r penyebab terjadinya kecelakaan lalu lintas.(7) h a l i n i t e r k a i t d e n g a n p e r i l a k u m a n u s i a (pengendara) saat mengemudikan kendaraan. 72 kesimpulan korban kecelakaan sepeda motor yang masuk ke igd rsup fatmawati kebanyakan mengalami cedera di bagian kepala dengan luka terbuka, dan kondisi korban yang parah cukup besar yaitu 47,8%. untuk itu perlu ditingkatkan manajemen kegawatdaruratan dan sarana di igd untuk penanganan cedera tersebut. daftar pustaka 1. nantulya vm, reich mr. the neglected epidemic: road traffic injuries in developing countries. bmj 2002; 324: 1139-41. 2. coats tj, davies g. prehospital care for road traffic casualties. bmj 2002; 324: 1135-8. 3. peden m, scurfiled r. sleet d. worlds report on road traffic injury prevention. geneva: world health organization; 2004. 4. world health organization. statistics of road traffic accident. geneva: un publications; 2000. 5. suwandono a. road traffic collision in urban indonesia, epidemiology and policy opportunities. jakarta badan penelitian dan pengembangan kesehatan, departemen kesehatan ri; 2002. 6. polri. prevensi dan reduksi kecelakaan sepeda motor di jalan raya. seminar diskusi penyusunan sistem surveilans cedera akibat kecelakaan lalu lintas pada pengendara sepeda motor, cisarua, 15 agustus 2005. 7. world health organization. international classification of diseases 10th revision (icd-10). geneva: world health organization; 2006. 8. ayuthya rsn. bohning d. risk factors for traffic accident in bangkok metropolis: a case-reference study. southeast asian j trop med public health 1997; 28: 881-5. 9. world health organization. world report on road traffic injury prevention. who; 2004. available at http://www.who.int/violence-injury-prevention. accessed december 2, 2006. 10. mayou ra, black j, bryant b. unconsciousness, amnesia and psychiatric symptoms following road traffic accident injury. br j psy 2000; 177: 540-5. 11. connor j, norton r, ameratunga s, robinson e, civil i, dunn r, et al. driver sleepiness and risk of serious injury to car occupant: population-based control study. bmj 2002; 324: 1125. 12. baker sp, o’neill b, ginsburg mj, li g. the injury fact book. 2nd ed. new york: oxford university press; 1992. 13. charlton r, smith g. how to reduce the toll of road traffic accidents. j r soc med 2003; 96: 4756. 14. poudel kt, nakahar s, poudel ks, ichikawa m, wakai s. traffic fatalities in nepal. jama 2004; 291: 2542. riyadina, subik cedera kecelakaan sepeda motor c:\users\jurnal fkusakti\docume 31 *department of physiology, faculty of medicine, islamic university of indonesia, yogyakarta **faculty of medicine, islamic university of indonesia, yogyakarta ***department of histology, faculty of medicine, islamic university of indonesia, yogyakarta correspondence: dr. titis nurmasitoh, msc department of physiology, faculty of medicine, islamic university of indonesia, yogyakarta email: titisnurmasitoh@gmail.com date of first submission, june 10, 2017 date of final revised submission, april 9, 2018 date of acceptance, april 10, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina intermittent fasting decreases oxidative stress parameters in wistar rats (rattus norvegicus) titis nurmasitoh*, shindy yudha utami**, endah kusumawardani**, abdulhalim ahmad najmuddin**, and ika fidianingsih*** background chronic and degenerative diseases are closely related to modern lifestyles that tend to be deficient in physical activity but excessive in food intake. one method used to overcome this problem is dietary restriction through intermittent fasting. intermittent fasting decreases the risk of chronic and degenerative diseases, e.g. by lowering oxidative stress. oxidative stress can be determined from the malondialdehyde (mda) levels and lipid profile in the blood. the present study aimed to determine the effect of intermittent fasting on plasma mda levels and lipid profile of wistar rats (rattus norvegicus). methods this research was a laboratory experimental research using a post-test control group design. twenty four male wistar rats (rattus norvegicus) were randomly assigned to 4 groups, ie control group (k), fasting group (p), non-fasting obese group (otp), and fasting obese group (op). fasting treatment given in this research was intermittent fasting, comprising one day of fasting for 12 hours alternating with one day of normal feed ad libitum in the span of 8 weeks. after completion of treatment, blood was taken transcardially for examination of mda levels and lipid profiles using spectrophotometry. results there were significant between-group differences in mda levels and lipid profiles (p <0.05 for all parameters). groups treated with intermittent fasting had lower levels of mda, total cholesterol, triglycerides, and ldl than those without intermittent fasting. the hdl levels were higher in the intermittent fasting group than the other groups. conclusion intermittent fasting has an effect on the oxidative stress parameters of wistar rats (rattus norvegicus). keywords: intermittent fasting, diet restriction, mda, lipid profile, oxidative stress, rats doi: http://dx.doi.org/10.18051/univmed.2018.v37.31-38 original article pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 cite this article as: nurmasitoh t, utami sy, kusumawardani e, najmuddin aa, fidianingsih i. intermittent fasting decreases oxidative stress parameters in wistar rats (rattus norvegicus). univ med 20 18;3 7:3 1-8. d oi : 10 .18 05 1/ univmed.2018.v37.31-38 32 nurmasitoh, utami, kusumawardani, et al intermittent fasting and oxidative stress introduction the lifestyle changes occurring in various community groups also influence their health status. in post-industrial and industrializing countries, including indonesia, the number of cases with obesity is continually increasing.(1) the prevalence of obesity in indonesian females increased by 32.9% from 2007 up to and including 2013. the prevalence of adult male inhabitants with obesity was 19.7% in 2013, which was higher than the 13.9% in 2007.(2) globally, the proportion of adults with body mass index (bmi) of 25kg/m2 increased from 1980 to 2013, i.e. from 28.8% to 36.9% in males and from 29.8% to 38% in females.(3) obesity is closely associated with modern lifestyles that tend to be deficient in physical activity and excessive in food consumption. changes in dietary pattern also contribute to the cases of obesity. currently the public tends to consume foods excessive in calories but very deficient in the essential nutrients actually required by the body.(2-4) the cases of obesity are caused by errors in nutritional management and are strongly suspected to increase the risk for degenerative disease s, such as dia betes, arteriosclerosis, hypertension, cardiovascular disease, stroke, heart failure, cancer, etc.(4,5) a number of studies demonstrated that a variety of diseases caused by obesity are associated with oxidative stress and involve inflammatory mechanisms.(6,7) a number of studies have been conducted to prevent obesity. one of the frequently used methods is dietary restriction.(7,8) dietary restriction is applied by various methods and in various diseases with sufficiently diverse parameters. dietary restriction is thought to be associated with a decre ased risk for cardiovascular disease, cancer, and a number of other chronic disea ses through var ious mechanisms, one of them being a decrease in oxidative stress. oxidative stress is determined by mea suring malo ndia ldehyde ( mda) concentrations and the lipid profile in the blood.(7,9,10) the methods of dietary restriction of the various studies are sufficiently diverse. for instance, there are studies that restrict the feeding frequency or daily amount of calories in healthy rats,(11,12) and the percentage of intake in rats with induced diabetes mellitus.(10) there are also various models of dietary restriction that are used in given regions or community groups, such as the okinawan diet,(1) the ramadan fast,(9) the greek orthodox fast,(9) and the daniel fast.(9) previous studies on intermittent fasting also used different techniques and caloric restrictions from those of the present study. intermittent fasting as used in the present study, comprising one day of fasting for 12 hours alternating with one day of the normal diet, is still infrequently used. the parameters under study are also diverse, such as superoxide dismutase (sod), (7 ) ros production,(11,13) redox status,(11,13) mda,(7,11) insulin sensitivity,(14) diabetes mellitus,(7) cancer,(15) nutritional status,(16) lipid profile,(10) lifespan and anti-aging,(9,17) memory,(12) and others. the majority of these studies state that dietary restriction has a positive effect on the body. among these studies, those using dietary restriction by the method of intermittent fasting such as applied in the present study (one day fasting for 12 hours and one day normal diet) with parameters of oxidative stress, i.e. increase in mda concentrations and lipid profile in rats, have not been carried out. one study using the method of intermittent fasting that is similar to this study was conducted by fidianingsih et al.,(18) but these investigators looked for the effect of intermittent fasting on the histologic structure of various organs associated with the immune system. the purpose of the present study was to evaluate the effect of intermittent fasting on oxidative stress marker s in rats, i.e. the concentrations of mda, total cholesterol, triglycerides, low-density lipoprotein (ldl), and high-density lipoprotein (hdl). 33 univ med vol.37no.1 methods research design this was an experimental laboratory study with post-test control group design. the study was conducted at the integrated research laboratory, faculty of medicine, universitas islam indonesia (uii) for 6 months, from june to december 2015. subjects of the study this study used 24 male wistar rats (rattus norvegicus) aged around 8 weeks and weighing 160-260 grams, which were obtained from the test animal laboratory, integrated research and testing laboratory (lppt), gadjah mada university (ugm), yogyakarta. the number of test animals was adjusted using the 3r principle (replacement, refinement, and reduction), to minimize the use of experimental animals, and was calculated with the following resource equation: (19) e=n-t, where e = constant between 10-20, n = intended number of animals per group multiplied by the number of intervention groups, while t = number of intervention groups (4 in this study). if the number of animals per group = 6 then e=(6x4)-4=20, which is still in the range of 10-20. therefore, the sample size or total number of test animals is 24.(19) intervention previous to the intervention, the rats were adapted for 7 days. the test animals were placed in cages in a room with controlled light intensity, using a 12hour light-dark cycle. during the adaptation period, feed and drinking water were given ad libitum. in the second week (after completion of the adaptation), the rats were grouped randomly into 4 groups, i.e. control group (k), fasting group (p), non-fasting obese group (otp), and fasting obese group (op). each of the groups consisted of 6 rats that were assigned randomly by lot to place them in the designated groups. group k was given standard rat feed and plain drinking water from the onset to the end of the study (12 weeks). group p was subjected to intermittent fasting, i.e. one day of fasting for 12 hours and one day of normal diet, from the onset to the end of the study according to the protocol of fidianingsih et al.(18) group otp was the group on high fat feed for 4 weeks followed by 8 weeks of normal feed and plain drinking water until the end of the study. group op was the group on high-fat feed for 4 weeks followed by 8 weeks of intermittent fasting until the end of the study. before undergoing the intervention, all rats were weighed to determine their weight at baseline. the rats were weighed every day to determine changes in body weight from day to day. the weighing was performed every morning using rat scales and the weights recorded. in addition to weighing, the body length of the rats was measured according to the procedure of novelli et al.(20) the rats were measured in the supine position from the nose to the anus using a ruler. the body length was determined twice, i.e. at the start and end of induction of obesity to determine the body mass index (bmi) of the rats, using the formula: body weight (gram)/length2 (cm2). the fasting of the groups p and op was intermittent fasting, i.e. the rats were fasted for 12 hours from 18.00 to 06.00, then were not fasted on the second day, again fasted on the third day, and not fasted on the fourth day, etc. on nonfasting days, the rats were given feed and drinking water ad libitum. in group p, the fasting was performed from the start of the intervention, whereas in group op, the fasting was performed for 8 weeks after the rats received the high-fat feed for 4 weeks. the feeds for the control group and non-fasting groups were given on average at 10% of body weight per day or around 20 grams/ day (10 grams was given in the morning and 10 grams in the evening). induction of obesity was performed using high-fat feed, in the form of a feed mixture consisting of rat feed with the addition of shortening (1/10 of the feed by weight), ½ egg yolk, and 7 grams of white rice until the rats attained obesity, i.e. when the bmi >0.68 g/cm2.(20) 34 nurmasitoh, utami, kusumawardani, et al intermittent fasting and oxidative stress a b c group c (n=6) f (n=6) nfo (n=6) fo (n=6) p* mda (nmol/ml) 2.11 ± 0.18 1.67 ± 0.19 03.15 ± 0.14 2.52 ± 0.14 0.000 cholesterol (mg/dl) 128.83 ± 1.26 119.71 ± 1.48 152.43 ± 2.50 138.69 ± 2.69 0.000 triglycerides (mg/dl) 100.58 ± 2.96 89.93 ± 3.22 117.94 ± 2.54 109.61 ± 2.61 0.000 ldl (mg/dl) 50.28 ± 2.22 38.57 ± 2.22 58.31 ± 1.91 55.63 ± 1.66 0.000 hdl (mg/dl) 48.35 ± 2.18 56.09 ± 2.09 36.18 ± 1.62 42.21 ± 1.58 0.000 table 1. mean mda, total cholesterol, triglycerides, ldl, and hdl group c: control group, group f: fasting group, group nfo: non-fasting obese group, group fo: fasting obese group * results of one-way anova laboratory analysis at the end of the study, blood was drawn transcardially from each rat using a 3-ml syringe and around 2 ml was placed in an edta tube. su bs e q ue nt l y, t h e b l oo d sa mp l e s we r e transported to the nutritional laboratory of the in t e r -u ni ve r si ty ce n t e r ( pu s at an t ar universitas, pau), ugm, yogyakar ta to determine the concentrations of mda, total cholesterol, triglycerides, and ldl by enzymatic s pe c t r op hot o me tr y. t he b l ood w a s t h e n centrifuged to obtain the supernatant. to every 100 microliters of serum, 2.45 ml of tca (trichloracetic acid) and 2.45 ml of tba (thiobarbituric acid) were added, then the mixture was heated at 100ºc for 20 minutes. subsequently, the mixture was centrifuged for 10 minutes at 8000 rpm. the absorbance of the obtained supernatant was determined with a blank of 2.45 ml tca and 2.45 ml tba based on the included standard curves. statistical analysis the distribution of the obtained numerical data was determined with the saphiro-wilk test, f o l l ow e d by le ve ne ’s t e st t o a s s e ss t he homogeneity of the data. one-way anova was used to determine between-group differences in the mean concentrations of mda, total cholesterol, triglycerides, ldl, and hdl. if there was a significant difference, a multiple comparison post-hoc test was done. t he difference was considered significant if p<0.05. ethical clearance the present study was approved by the ethics committee, faculty of medicine, uii, as stated in letter of ethical clearance no. 18/ ka.kom.et/70/ke/iii/2015. results before the start of the intervention, the randomly grouped rats were weighed to make sure that they were in the same condition at the start of the study. mean weights of the rats were analyzed by anova to determine significant d if f e r e nce s i n bo dy we i ght b e f o r e t he intervention. analysis using one-way anova showed no significant difference in the weight of the rats between groups at baseline, indicating that the randomization successfully equalized the weights among the four groups. after the induction of obesity for 4 weeks, the mean bmi of the rats in the induction groups (otp and op) was more than 0.68, whereas the control group showed a mean bmi of less than 0.68.(20) after completion of the induction, the intervention was continued for each group according to schedule until 8 weeks afterward. on the last day, blood was drawn from the heart of each rat to a volume of 2 ml to determine the mean concentrations of mda, total cholesterol, triglycerides, ldl, and hdl, as shown in table 1. t he c o n ce nt r a t i on s o f m da, t ot a l cholesterol, triglycerides, and ldl at the end of the study were highest in group otp (the group with induction of obesity without fasting) and lowest in group p (fasting without induction of obesity). on the other hand, the highest hdl concentration was shown by the rats in the fasting group and the lowest concentration by 35 mean difference p mda c f 0.6433 0.000 nfo -0.84 0.000 fo -0.21 0.000 f nfo -1.285 0.000 fo -0.655 0.000 nfo fo 0.8283 0.000 cholesterol c f 12.7511 0.000 nfo -19.9689 0.000 fo -6.2289 0.000 f nfo -29.0939 0.000 fo -15.3539 0.000 nfo fo 17.3661 0.000 triglycerides c f 14.076 0.000 nfo -13.934 0.000 fo -5.599 0.000 f nfo -24.5823 0.000 fo -16.2473 0.000 nfo fo 11.7627 0.000 low-density lipoprotein c f 14.1375 0.000 nfo -5.5958 0.000 fo -2.9225 0.000 f nfo -17.3042 0.000 fo -14.6308 0.000 nfo fo 5.1025 0.000 high-density lipoprotein c f -5.4663 0.000 nfo 14.4437 0.000 fo 8.412 0.000 f nfo 22.1804 0.000 fo 16.1487 0.000 nfo fo -3.7613 0.000 table 2. post hoc lsd analysis group c: control group, group f: fasting group, group nfo: non-fasting obese group, group fo: fasting obese group the rats in group otp (induction of obesity without fasting). the results of one-way anova showed that there was a significant dif ference in the concentrations of mda, total cholesterol, triglycerides, ldl, and hdl between the groups (p<0.05). the post hoc test using lsd showed a significant difference (p<0.05) between the groups for all parameters (table 2). the concentrations of mda, cholesterol, triglycerides, ldl, and hdl in the fasting groups differed significantly with those in the non-fasting groups. discussion intermittent fasting is one of the dietary restriction methods. several studies state that dietary restriction may influence health in general, prevent pr emature aging with all of its consequences, prevent degenerative disease, cognitive deficit, etc.(5,9) this is based on the fact that various diseases, particularly degenerative diseases, are frequently caused by lifestyle, in this case including uncontrolled dietary patterns.(1) in addition, dietary restriction, such as alternateday fasting and calorie restriction may decrease the risk of various chronic diseases, such as diabetes mellitus, heart disease, and cancer, and at the same time prevent their occurrence.(10) therefore dietary restriction is expected to be able to improve diseases that are the result of uncontrolled lifestyle. there are various dietary restr iction methods, one of them being intermittent fasting, which itself has been developed into various models.(1,9) the method of intermittent fasting was first introduced by carlson and hoelzel, who found that rats subjected to intermittent fasting had increased life spans.(11) intermittent fasting may significantly improve oxidative stress parameters as seen in the lower concentr a tions of mda, total cholesterol, triglycerides, and ldl, and the higher concentrations of hdl. in the fasting groups, the oxidative stress parameters showed the most excellent results compared with the other groups. administration of intermittent fasting to groups with induced obesity also showed significant improvement in oxidative stress parameters, as shown in the decreased concentrations of mda, total cholesterol, triglycerides, and ldl, and the increased concentrations of hdl compared with the obese groups that were not subjected to fasting. the results of the present study showed that plasma mda concentrations in rats subjected to intermittent fasting were lower compared to the groups not subjected to intermittent fasting. this univ med vol.37no.1 36 nurmasitoh, utami, kusumawardani, et al intermittent fasting and oxidative stress study is in line with a previous study carried out by chausse et al.,(11) who found that intermittent fasting decreased the redox status of specific organs, particularly the liver and brain, so preventing oxidative damage in these organs. more specifically, intermittent fasting decreases the mda concentrations in the hearts of the rats. this shows that oxidative stress in rat hearts may be minimized through intermittent fasting. malondialdehyde (mda) is the main product of lipid peroxidation and is used to determine the amount of reactive oxygen species (ros) produced by oxidative stress.(6) this agrees with the theory on the effect of dietary restriction, including intermittent fasting. intermittent fasting is frequently associated with the prevention of oxidative stress in the body. it is well known that excessive and unlimited dietary calories promote lipid peroxidation. polyunsaturated fatty acids are easily oxidized into free radicals and other reactive compounds such as h 2 o 2 . lipid peroxidation is a mechanism of cellular injury and is used as an indica tor of oxidative stress in cells a nd tissues.(7,11) lipid endoperoxides originating from polyunsaturated fatty acids are unstable and are decomposed into a number of complex compounds, including reactive carbonyls, particularly mda.(6) restriction of calories and control of the amount of calories entering the body are expected to prevent and control oxidative stress in the body. therefore, the concentrations of mda formed in the body also become lower.(6,7) the results of the present study also showed lower total cholesterol, triglyceride, and ldl concentrations in rats subjected to intermittent fasting compared with groups not subjected to intermittent fasting. in agreement with the present study, klempel also state that in humans, intermittent fasting combined with calorie restriction showed effective results in weight reduction in female subjects with obesity. in addition, this method is also effective in decreasing the risk for cardiovascular disease such as coronary heart disease. the decreased risk for cardiovascular disease is indicated by the decreased concentrations of plasma lipids and ldl.(8) in addition, chen et al.(14) also state that a restriction to 68% of carbohydrates may decrease the concentrations of ldl and total cholesterol. similarly with the present study, park et al. also conducted a study by dividing rats into 3 groups, i.e. the control group, the group on a high fat diet, and the group on a high fat diet followed with caloric restriction. the calorie restriction that was applied was the administration of 60% of the number of calories in the normal daily feeds. in the group subjected to caloric restriction, there was a decrease in triglyceride and total cholesterol concentrations, improvement of the leptinadiponectin ratio, a decrease in the expression of no-synthetase, cyclooxygenase-2, nf-e2related factor, and heme oxygenase-1.(21) increased dietary intake of energy or fat will increase lipogenesis and subsequently increases the formation of free fatty acids. the free fatty acids are mobilized from adipose tissue into the liver, where they bind to glycerol to form triglycerides. the higher the fat consumption, the higher the triglyceride concentration. induction with high fat feeds causes increased cholesterol synthesis and ultimately cholesterol accumulation in the blood (hypercholesterolemia). if the blood cholesterol concentrations increase, the ldl as cholesterol transporter to the cells throughout the body also increases. subsequently, the accumulation of cholesterol is followed by free radical activity causing oxidative damage in a number of tissues. in addition, ldl is also a readily oxidized substance. oxidized ldl may bind to macrophages and subsequently form foam cells, so causing a therosclerotic lesions. hypercholesterolemia may cause atherosclerosis, two of the complications being cardiac infarction and stroke. restriction of dietary intake is expected to decrease the concentrations of triglycerides, ldl, as well as total cholesterol.(222 4 ) in this study we also found higher hdl concentrations in the rats subjected to intermittent fasting compared with the groups not subjected to intermittent fasting. diets high in carbohydrates 37 and polyunsaturated fatty acids decr ease apolipoprotein a-1, which is the main component of hdl and functions to decrease oxidized ldl concentrations. intermittent fasting causes decreased intake of calories so that the production of apolipoprotein a-1 may be optimal and subsequently may increase hdl concentrations in the blood.(25,26) the results of the present study are in line with those of chen et al. (14) who state that dietary restriction for carbohydrates and calories in wistar rats was able to increase hdl concentrations and improve other lipid profile parameters. the decrease in triglycerides in the groups on intermittent fasting is the result of the decreased regulation of the hepatic lipogenetic system. restriction of dietary intake may also increase the activity of lipoprotein lipase (lpl) so increasing triglyceride clearance in the blood vessels. increases in lpl also increase the catabolism of lipoproteins rich in triglycerides, so resulting in the transfer of esters, apoproteins, and phospholipids to form hdl.(14) the study of ma et al.(27) also found that mice subjected to dietary interventions alternating between high fat diets and normal showed good results in the prevention and therapy of the metabolic syndrome. the longer the periods of the normal diets replacing the high fat diets, the better the results. ma et al.(27) divided the groups into 5+1 (five days of high fat diet alternated by one day of normal diet), 5+2 (five days of high fat diet alternated by two days of normal diet), and 5+3 (five days of high fat diet alternated by three days of normal diet). in addition to affecting lipid metabolism, calorie restriction also has an influence on the brain, memory, and other systems. kuhla et al. also state that caloric restriction (by giving only 60% of the normal daily requirement) in the long term of up to 74 weeks may prevent a decrease in working memory, cognitive function, and spatial memory in mice. caloric restriction also causes a decrease in locomo tor activity, motor coordination, and increased anxiety in rats.(12) based on the results of this study and of previous studies, there is a need to conduct univ med vol.37no.1 further studies to determine the formulation of dietary and caloric restriction as well as the most effective method of intermittent fasting for the reduction of oxidative stress. in addition, further explorations should be carried out with regard to the parameters that may be affected by this restriction of intake. the negative effects that may occur in this intervention should also be investigated. the present study still has its limitations, such as the absence of baseline data, although there are control groups for comparison. the clinical implication of the present study is that dietary restriction may be beneficial in cases of excessive obesity. conclusions intermittent fasting may decrease oxidative stress as shown by the decrease in mda, total cholesterol, triglyceride, and ldl concentrations. acknowledgments the authors express their gratitude to the directorate of research and public service, universitas islam indonesia, for the funding and to ernadita budiastuti for technical assistance in this study . conflict of interest there was no conflict of interest in the present study . contributors tn, syu, ek, aan, and if contributed equally to the design of the study, drafting of the manuscript, data collection and analysis, and revising the manuscript. all authors read and approved the final manuscript. references 1. willcox dc, willcox bj, todoriki h, et al. the okinawan diet: health implications of a lowcalorie, nutrient-dense, antioxidant-rich dietary 38 nurmasitoh, utami, kusumawardani, et al intermittent fasting and oxidative stress pattern low in glycemic load. j am coll nutr. 2009; 28(suppl4):s500–16. 2. balitbangkes. riset kesehatan dasar (riskesdas 2013). jakarta: kementrian kesehatan ri; 2013. 3. ng m, fleming t, robinson m, et al. global, the regional and national prevalence of overweight and obesity in children and adults 1980-2013: a systematic analysis. lancet 2014;384:766–81. doi: 10.1016/s0140-6736(14)60460-8. 4. jain s, singh sn. calorie restriction: an approach towards obesity management. j nutr disord ther 2015;1(s1). doi: 10.4172/2161-0509.1000s1-006. 5. klenk j, rapp k, ulmer h, et al. changes of body mass index in relation to mortality: results of a 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alternate-day fasting and chronic disease prevention: a review of human and animal trials. am j clin nutr 2007;86:7– 13. 11. chausse b, vieira-lara ma, sanchez ab, et al. intermittent fasting results in tissue-specific changes in bioenergetics and redox state. plos one 2015;10:1–13. doi: https://doi.org/10.1371/ journal.pone.0120413. 12. kuhla a, lange s, holzmann c, et al. lifelong caloric restriction increases working memory in mice. plos one 2013;8:7. doi: https://doi.org/ 10.1371/journal.pone.0068778. 13. walsh me, shil y, van remmen h. the effects of dietary restriction on oxidative stress in rodents. free radic biol med 2014;66:88–99. 14. chen jh, ouyang c, ding q, et al. a moderate low-carbohydrate low-calorie diet improves lipid profile, insulin sensitivity and adiponectin expression in rats. nutrients 2015;7:4724–38. 15. lv m, zhu x, wang h, et al. roles of caloric restriction, ketogenic diet and intermittent fasting during initiation, progression and metastasis of cancer in 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the high diet fed rats. med inflamm 2012;2012:1-11. doi: 10.1155/2012/984643. 22. alagwu ea, okwara je, nneli ro, et al. effect of honey intake on serum cholesterol, triglycerides and lipoprotein levels in albino rats and potential benefits on risks of coronary heart disease. niger j physiol sci 2011;26:161–5. 23. nurmasitoh t, pramaningtyas md. honey improves lipid profile of diet-induced hypercholesterolemic rats. univ med 2015;34: 177–86. doi: 10.18051/univmed.2016.v35. 177-186. 24. tomkin gh, owens d. ldl as a cause of atherosclerosis. open atheroscler thromb j 2012; 5:13–21. 25. annema w, von eckardstein a. high-density lipoproteins. circ j 2013;77:2432–48. doi: https:/ /doi.org/10.1253/circj.cj-13-1025. 26. das b, mishra t. role of hdl-c in health and disease. jiacm 2012;13:218-22. 27. ma y, gao m, liu d. alternating diet as a preventive and therapeutic intervention for high fat diet-induced metabolic disorder. sci rep 2016; 6:26325. doi: 10.1038/srep26325. c:\users\jurnal fkusakti\docume 3 *pharmacology department faculty of medicine tarumanagara university **dermatovenereology department faculty of medicine tarumanagara university ***public health department faculty of medicine tarumanagara university correspondence: dr. oentarini tjandra, m.biomed, mpd. ked department of pharmacology faculty of medicine tarumanagara university jl. letjen. s. parman no. 1 grogol jakarta barat 11440 email: uunfang@gmail.com fax: +6221-5663126 date of first submission, july 14, 2017 date of final revised submission, february 6, 2018 date of acceptance, february 11, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina green tea moisturizer improves skin hydration in elderly oentarini tjandra*, linda j wijayadi**, and marcella e rumawas*** background dry skin is a major skin health problem in elderly. green tea, which has an antioxidant effect, has recently been used as an active ingredient in moisturizing creams; yet the effect has not been well studied. this study compares the skin hydration effect of green tea and vitamin e moisturizer among elderly. methods this quasi-experimental study involved 60 elderly living in tresna werda budi mulia 4 social institution, jakarta. using the runve hl 611 skin analyzer, skin capacitance was measured prior to experiment and every following week during the 5-week application of green tea and vitamin e skin moisturizer on both forearms. the consecutive measurement data were analyzed using generalized estimating equation to compare the relative changes in skin hydration between the two moisturizer groups over 5 weeks of intervention. results the green tea moisturizer showed more significant increases in skin hydration level than the vitamin e moisturizer at all measurement sites on right arm and proximal left arm (p= 0.021), and medial and distal left arm (p= 0.034). skin hydration levels significantly changed over time at proximal (p=0.021), medial (p=0.006) and distal (p=0.006) right arm, and medial left arm (p=0.021). a parallel trend of skin hydration improvements for both moisturizer groups indicated no correlation between the moisturizer type and the duration of usage (p >0.05) in all measurement locations. no side effects were observed during application period in both groups. conclusion routine use of a moisturizer containing green tea may improve skin hydration in elderly. keywords: green tea moisturizer, vitamin e, skin hydration, elderly original article cite this article as: tjandra o, wijayadi lj, rumawas me. green tea moisturizer improves skin hydration in elderly. univ med 2018;37:3-12. doi: 10.18051 /univmed.2018.v37.3-12 pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2018.v37.3-12 january-april, 2018 vol.37no.1 4 tjandra, wijayadi, rumawas green tea moisturizer and skin hydration introduction the skin is the outermost and largest organ of the human body, while one of its major roles is as a protective barrier against microorganisms and as the body’s temperature regulator. skin dryness increases the risk of developing several skin diseases, such as atopic dermatitis, irritant contact dermatitis, allergic contact dermatitis, ichthyosis, and psoriasis.(1) the protective function of the skin is primarily performed by the epidermis, of which the stratum corneum is the outermost part.(2) the stratum corneum is a dynamic structure that c o nt i n uo u sl y r e s p on ds t o va ri ou s d a i l y exogenous factors. the epidermal layer of the skin protects the body against many external stressors including physical (mechanical, thermal, ultraviolet rays) and chemical (solvents, surfactants, xenobiotics).(3) it also protects the s ki n f r o m be i n g d e h yd r a t ed t hr ou gh transepidermal water loss (tewl).(4) every year, the older population in indonesia increases in number. statistics for 1980 recorded seven million older individuals in indonesia. in 2006, this number tripled to 19 million people, and in 2010 increased to approximately 23 million people.(5) it has been predicted that in 2025 and 2050, the number of elderly in indonesia will rise to 34,592,000 and 67,353,000 people respectively.(5) this will cause a parallel increase in the number of skin-related diseases, especially because older individuals have a thinner stratum corneum, fewer hair follicles, and lower skin hydration level as compared to the younger.(3) skin dryness may increase tewl, which in turn will increase skin dehydration.(6) moisturizers are the most common skincare products that are often used to reduce dryness of the skin by minimizing skin friction, softening the skin, and filling skin cracks.(7) the usage of moisturizers containing vitamin e for maintaining skin hydration has been well studied. it acts as an antioxidant and inhibits the formation of lipid peroxides, and thus prevents skin aging.(8) recently, green tea has been introduced as an active component in moisturizers. both green tea and vitamin e have antioxidant effects that h ave be e n r e la t ed to del a yi ng t h e a ging process.(9-11) a study showed that green teac o nt a i n i ng c o sme t i c f or mu l at io n s ha ve pronounced moisturizing effects and skin microrelief improvement.(12) catechins are the major antioxidants in gre en tea (camellia sinensis or camellia assamica), but since they do not penetrate the skin well, the application of green tea in cosmetic products has so far been limited.(13) a review of numerous studies with green tea (camellia s i ne n s i s) h a s c o n c l ud e d t h a t b o th or a l consumption and topical application of green tea protect against inflammation, and chemical and uv-induced carcinogenesis.(14) a st udy t o e va lua te the h uman skin penetration of epigallocatechin-3-gallate (egcg) and quercetin from green tea and gingko biloba extracts vehiculated in cosmetic formulations showed that both compounds exhibited good skin penetration and retention, which can favor their skin effects.(15) meanwhile in india, vegetable oils have been used as skin moisturizers, especially in neonates and children due to their proven efficacy, low cost, easy availability and good safety profile.(16) our study was to evaluate the effectiveness of green tea moisturizer cream in elderly persons, whose skins are dehydrated, making them more sensitive and fragile than young people’s. there are promising results with the use of green tea for several dermatologic conditions.(17) however, the efficacy of topical green tea h a s no t al wa ys b e e n c o nf ir me d. on e randomized, double-blind, controlled, clinical trial involving 40 women with moderate photoaging, who were treated with topical green tea extract, showed no statistically significant clinical improvements in these women.(18) this study evaluated the effect of a green tea containing moisturizer on skin hydration in older people. 5 methods research design a quasi-experimental controlled study was conducted amongst elderly living in tresna werda budi mulia 4 social institution, jakarta, from february 2015 until january 2016. research subjects all subjects were aged 60 years or above, and included both me n and w omen. t he eligibility criteria were absence of dermatitis, erythema, psoriasis, and other skin diseases or tattoos on the arms, and currently not under any local or systemic therapy. individuals were excluded if they developed any skin allergy or skin disease during the intervention period or if they did not comply with the study protocol. the sample size was obtained using the formula : (19) where α = 5 %, so the z α = 1.96; β = 20%, so that z β = 0.84; the mean differenced was 5. the optimal sample size for each group was 30. intervention the participants were randomly allocated to the green tea group and the vitamin e control group. moisturizers containing green tea or vitamin e were distributed inside two identical standard containers, weighing 12.5 grams each. trained caregivers applied the moisturizer on the participant’s right and left forearms, twice a day after showering for 5 weeks. each of the subjects’ volar surface of the forearms was divided into three sites, i.e. proximal, medial, and distal, with application area of 2x2 cm each. each measurement area was approximately four square centimeters. measurements the skin hydration level was measured with the runve hl 611 skin analyzer at baseline and every week during the 5-week intervention. the skin analyzer had to be placed on the skin with constant pressure until both contacting poles depressed the skin. after three to five seconds, the measurement result would appear on the lcd s c r e en. tr e a t me n t a pp l ic a t io n w a s monitored daily by evaluating the used container and by cross-checking information between the respondents and the field workers. dropouts were those allergic to any ingredient in the moisturizer, those with skin diseases, severe medical condition, reluctant or incompliant subjects, and participants who passed away during experimental period or transferred to another social institution. relative changes in skin hydration were evaluated from the baseline value, and were expre ssed as pe rcent dif f erences in skin hydration between each week of follow-up and baseline. statistical analysis to evaluate the repeated measurements, we used the generalized estimating equation (gee) statistical procedure. data distribution was found to be normal. potential confounding factors related to skin hydration were evaluated, such as age; sex; frequency of showering (minimally twice a day); whether subjects had been using other moisturizers, eucalyptus oil, balm or powder; whether any skin disorder was present; and liquid intake (in cups/day). the statistically significant was used at p < 0.05. ethical clearance the study was approved by the human subjects committee of trisakti school of medicine 8/ker/fk/v/2015. each participant had given an informed consent. at the start of the study 62 subjects were recruited, among whom 60 were found to meet the inclusion and exclusion criteria. during the five-week study period, there were no dropouts. at the end of the study there were 60 remaining subjects, comprising 26 subjects in the green tea group and 36 subjects in the vitamin e group (figure 1).   2 xx sd zz 2nn 21 βα 21          univ med vol. 37 no.1 6 tjandra, wijayadi, rumawas green tea moisturizer and skin hydration the overall rate of compliance over 5 weeks of intervention was 69.8% in both groups (60.5% in the green tea group, and 79.1% in the vitamin e group; p=0.061). baseline characteristics of the respondents are shown in table 1. the mean age of the respondents was 75.4 ± 8.5 years, and most participants (73%) were women. there were no significant skin hydration differences between the groups at baseline, and in some factors that are of potential influence on skin hydration (p>0.05). the green tea group had a significantly higher relative change of skin hydration than the vitamin a b c figure 1. flowchart of the research subjects green tea moisturizer (n = 26) vitamin e moisturizer (n = 34) p value age (years) 71.5 ± 7.6 70.3 ± 9.4 0.611 sex women 20 (76.9) 24 (70.6) 0.582 men 6 (23.1) 10 (29.4) showering min. twice/d ‡ 25 (96.2) 32 (94.1) 0.731 routine application moisturizer ‡ 0 3 (8.8) 0.251 eucalyptus oil‡ 4 (15.4) 5 (14.7) 0.944 balm‡ 1 (3.8) 2 (5.9) 0.732 powder‡ 1 (3.8) 3 (8.8) 0.625 skin disorders ‡ 4 (15.4) 10 (29.4) 0.204 liquid intakes (cup /d) 4.7 ± 2.1 4.2 ± 2.3 0.381 % right arm skin hydration proximal 39.0 ± 3.87 39.9 ± 4.56 0.452 medial 37.8 ± 4.39 38.3 ± 4.59 0.703 distal 37.5 ± 4.29 37.9 ± 4.24 0.692 % left arm skin hydration proximal 39.4 ± 4.51 39.6 ± 4.07 0.811 medial 38.2 ± 4.19 38.1 ± 4.51 0.971 distal 38.0 ± 4.90 38.4 ± 4.48 0.692 table 1. baseline characteristic of the study population data presented as mean ± sd, except for sex, showering, routine application, skin disorders and liquid intakes (n,%) † differences between the moisturizer groups were calculated by two groups independent –t-test (numerical data) or pearson chi-square / fisher exact-test (categorical data) 7 green tea moisturizer (n = 26) vitamin e moisturizer (n = 34) p value right arm proximal week 1 10.40 ± 3.16 1.88 ± 2.84 0.502 week 2 6.22 ± 2.29 3.85 ± 1.95 0.431 week 3 4.43 ± 2.64 0.18 ± 2.37 0.193 week 4 3.92 ± 2.89 4.47 ± 2.53 0.881 week 5 7.08 ± 2.97 6.90 ± 2.36 0.964 mean (wk 1 to 5) 5.25 ± 2.34 2.82 ± 2.86 0.021 medial week 1 9.16 ± 2.85 2.50 ± 2.56 0.082 week 2 7.84 ± 2.56 4.58 ± 2.18 0.341 week 3 4.42 ± 3.09 0.16 ± 2.79 2.282 week 4 3.55 ± 2.81 3.61 ± 2.46 0.981 week 5 5.28 ± 2.64 6.35 ± 2.31 0.763 mean (wk 1 to 5) 4.88 ± 3.37 2.54 ± 2.40 0.021 distal week 1 9.93 ± 2.58 0.93 ± 2.31 0.014 week 2 7.52 ± 2.63 4.20 ± 2.25 0.341 week 3 5.05 ± 3.15 1.68 ± 2.84 0.124 week 4 0.23 ± 3.58 3.22 ± 3.22 0.472 week 5 5.04 ± 2.19 6.49 ± 1.92 0.621 mean (wk 1 to 5) 4.32 ± 3.47 2.27 ± 2.11 0.021 left arm proximal week 1 10.24 ± 3.07 3.17 ± 2.75 0.092 week 2 8.14 ± 2.63 2.99 ± 2.24 0.143 week 3 3.97 ± 2.81 0.03 ± 2.53 0.302 week 4 2.96 ± 2.81 3.71 ± 2.46 0.841 week 5 4.89 ± 2.74 5.77 ± 2.39 0.812 mean (wk 1 to 5) 4.90 ± 2.79 2.57 ± 2.67 0.021 medial week 1 9.04 ± 2.82 2.83 ± 2.54 0.111 week 2 7.66 ± 2.46 5.01 ± 2.09 0.421 week 3 4.37 ± 3.03 0.26 ± 2.73 0.324 week 4 3.36 ± 2.72 4.09 ± 2.38 0.842 week 5 5.37 ± 2.62 6.82 ± 2.29 0.681 mean (wk 1 to 5) 4.84 ± 2.90 3.14 ± 2.59 0.034 distal week 1 7.64 ± 2.55 0.27 ± 2.29 0.021 week 2 4.58 ± 2.62 0.89 ± 2.24 0.292 week 3 0.57 ± 2.75 3.82 ± 2.47 0.241 week 4 1.65 ± 3.03 1.89 ± 2.65 0.953 week 5 2.55 ± 2.42 2.87 ± 2.11 0.922 mean (wk 1 to 5) 2.71 ± 2.86 0.32 ± 2.46 0.021 table 2. relative changes from baseline in skin hydration during 5 weeks of intervention by treatment groups data presented as mean ± sd e group at all measurement locations (p=0.021 for proximal, medial and distal right arm, and proximal left arm; p = 0.034 for medial and distal left arm) (table 2, figures 1 and 2). within each of the two groups, skin hydration improved during the 5 weeks of intervention, indicating the presence of a time effect (duration of moisturizer application) in skin hydration changes, especially at the proximal (p=0.021), medial (p=0.006) and distal right arm (p=0.006), as well as at the medial left arm (p=0.021). at all measurement sites, we found no correlation between the effects of the moisturizer and the duration of intervention on the relative changes in skin hydration. both moisturizers showed a parallel trend in the relative change of skin hydration during the univ med vol. 37 no.1 8 tjandra, wijayadi, rumawas green tea moisturizer and skin hydration figure 2. relative changes of skin hydration of the right arm during 5 weeks of intervention according to moisturizer group vertical bars indicate 95% ci. mean relative changes in skin hydration are expressed as percentage of the baseline value of the difference between follow-up and baseline values. changes in skin hydration over time were analyzed using generalized estimating equations. p group<0.05 indicates the group effect (difference between the two groups); p time <0.05 indicates the time effect (change within-subject over time) and p group*time indicates group by time interaction (different trend of change over time between the two groups). results are presented for 60 participants at alltime points (26 in the green tea moisturizer group; and 34 in the vitamin e moisturizer group). overall 5-week intervention (p>0.05 for the moisturizer and time). additional adjustments for some factors related to skin hydration did not change overall results. furthermore, we observed that over the 5-week study period, both groups did not have any objective signs or subjective complaints that might be interpreted as being the side effects of the moisturizer. discussion t hi s st udy s ho ws t ha t mois t u r i ze r application is crucial for the elderly skin. it prevents pruritus, thus maintaining the highest degree of skin integrity. the elderly tend to acquire a drier skin, therefore proper moisturizer usage is necessary to avoid dry skin-related 9 figure 3. relative changes of skin hydration of the left arm during 5 weeks of intervention according to moisturizer group vertical bars indicate 95% ci. mean relative changes in skin hydration are expressed as percentage of the baseline value of the difference between follow-up and baseline values. changes in skin hydration over time were analyzed using generalized estimating equations. p group <0.05 indicates the group effect (difference between the two groups); p time <0.05 indicates the time effect (change within-subject over time) and p group*time indicates group by time interaction (different trend of change over time between the two groups). results are presented for 60 participants at alltime points (26 in the green tea moisturizer group; and 34 in the vitamin e moisturizer group). problems, such as pruritus. other studies have also emphasized on improving and maintaining elderly skin hydration with topical or oral supplementations. in their study, mahmood et al.(16) found that application of the combination of green tea plus lotus extract as moisturizer lead to a greater improvement in skin hydration than did each ingredient alone. in another study, chiu et al. (18) found that consuming green tea polyphenol with milk is good for the elderly, because catechin from green tea will bind to the milk protein casein and form a catechin-casein complex. the bioavailability of the catechincasein complex in the digestive tract is better than catechin alone, therefore greater absorption levels of the antioxidant will be achieved. this antioxidant is crucial for skin integrity. a study by lee at al.(19) on asian female subjects aged univ med vol. 37 no.1 10 tjandra, wijayadi, rumawas green tea moisturizer and skin hydration 32-56 years discovered that fermented green tea consumption has a cooling effect on women’s peripheral skin temperature, which is useful for people with cold hypersensitivity. these studies were similar to our study in their objective, which was to maintain skin hydration in order to keep the skin barrier intact. however, they slightly differ from our study in the methods used, the longer-term study design, and the use of more advanced measuring equipment. with increasing age and presumably also aging process, the need for moisturizer application increases. our study showed that among 60 participants, approximately only 2.3 – 7.0% had been using moisturizers before this study was conducted. skin disorders were found in 20.9% of participants in both groups such as atopic dermatitis, chronic lichen simplex, scabies, ichthyosis, and tinea corporis. in subjects with skin disorders, the measurement was still done at the three locations (proximal, medial, distal), because the results showed that the skin hydration level between both arms at the three locations were not significantly different at baseline. as an anti-aging substance, vitamin e has proved to be a stable, easy to formulate, and reasonably priced emollient. vitamin e acts as an antioxidant substance that prevents lipid peroxidase activities in cells and soft tissues, skin softening, and that reduces inflammation caused by reactive oxygen species, as well as being a topical skin photoprotective. (20) vitamin e as alpha tocopherol or tocopherol acetate is also used in over-the-counter drugs in 1-5% concentrations to hydrate the stratum corneum and increase the water binding capacity of the skin.(20,21) green tea leaves contain around 30–35% polyphenols, which comprise a number of catechins (flavan-3-ols). catechins consist of 4 main components, i.e . epicatechin ( ec), epigallocatechin (egc), epicatechin gallate (ecg), and epigallocatechin gallate (egcg). epigallocatechin gallate is the most effective antioxidant component among the four types mentioned and prot ects the skin from photocarcinogenes is and uv-induced phototoxic ity, (2 2-24 ) a nd also r educes skin hyperpigmentation.(25) polyphenols in green tea may inhibit the activity of collagenase and increase the biosynthesis of collagen in fibroblasts to further protect the skin from free radicals.(9,26,27) moisturizers protect the skin through occluding the tewl process by creating a protective layer above the stratum corneum. another mechanism is by increasing water a b s or p t i on i nt o th e s t r a tu m c or ne u m (humectant), and filling the gap between desquamating keratinocytes to create a softer skin (emollient). moisturizers also act as protein rejuvenator to improve the water storage of the epidermis. (2,7) our clinical trial showed that both vitamin e and green tea extract may be used as an active component of moisturizers, as both substances may act as an antioxidant in moisturizers and in anti-photoaging. the latter further reduces uv exposure that may lead to dry skin. be t we e n th e t wo s t ud y gr ou ps , n o statistically significant difference was observed. the two groups applied the moisturizer after showers, twice daily. strict surveillance was done by the field officer because most of the subjects were elderly, who presumably have some degree of memory impairment. the application of the moisturizers by the field workers was monitored by measuring the remaining amount of each moisturizer by the end of the week. daily application by the field workers and subject compliance were important aspects in this study, since the moisturizers require regular usage in order to achieve a good skin hydration status. under-treatment of dry skin may give rise to multiple skin disorders, and the resulting itch may cause the patient to scratch, causing secondary skin infections. the management principle of dry skin is to restore the integrity of the stratum corneum, and application of moisturizers may help improving skin hydration status, especially in the elderly. moisturizers act as occlusive, humectant, and emollient substances that improve the water content of the stratum corneum.(2,4) 11 in our study, no side effects were observed following application of either moisturizer to the study participants. there were no objective side effects such as mild erythema and allergic reactions by the second and fifth week after the initiation of intervention. subjective side effects such as itch, pain, or stinging sensation were also not reported. this result showed that it is safe to apply moisturizer containing either vitamin e or green tea extract. since in practice the subjects had to apply t he moi s tur ize r by t h e ms e lve s , per so na l compliance is very important. significant moisturization effects can only be achieved with regular usage. one experimental study showed that low compliance reduces the effectivity of moisturizer s in improving skin hydration status.(20) in our study, the compliance status of the study participants was associated with improvement of skin hydration status in both groups. on the other hand, incompliance of several subjects and the significant number of drop-outs were identified as the limitation of this study. the study aimed to educate and persuade the elderly to apply moisturizers to keep their skin hydrated. further studies in the future should utilize a better skin analyzer, in order to obtain more specific and standardized data. conclusion this research suggests that routine use of moisturizers improves skin hydration in the elderly. this effect may be more prominent with the usage of moisturizers containing green tea as compared to those containing vitamin e. conflict of interest the authors have no conflict of interest. aknowledgements the authors are thankful to irene dorthy, md, sheilla khonada, s.ked, jonathan, s.ked, and andy, s.ked. contributors ljw conception and design of study, ot acquisition of data and drafting the manuscript, mer analysis and interpretation of data. all authors read and approved the final manuscript. references 1. baroni a. epidermal barrier function: clinical implications and therapeutic. clin dermatol 2012; 30:255-366. 2. del rosso jq. moisturizer and barrier repair formulations. in: draelos zd, dover js, alam m, editors. cosmeceuticals. 3rd ed. philadelphia: elsevier;2016.p.81-9. 3. chu dh. development and structure of skin. in: wolff k, katz s, paller a, et al, editors. fitzpatrick’s dermatology in general medicine. 8th ed. new york: mcgraw-hill; 2012.p.58-75. 4. draelos zd. new treatments for restoring impaired epidermal barrier permeability: skin barrier repair creams. clin dermatol 2012;30:345-8. 5. badan pusat statistik. proyeksi penduduk indonesia 2010-2035. jakarta: badan pusat statistik indonesia;2013. 6. verdier-sevrain s, bonte f. skin hydration: a review on its molecular mechanisms. j cosmet dermatol 2007;6:75-82. 7. loden m. effect of moisturizers on epidermal barrier function. clin dermatol 2012;30:286-96. 8. datta hs, mitra sk, paramesh r. theories and management of aging: modern and ayurveda perspectives. evid based complement alternat med 2011;528527:1-6. 9. sandeep k, nisha s, schweta, et al. green tea polyphenols: versatile cosmetic ingredients. ijarpb 2012;1:348-62. 10. lintner k, mas-chamberlin c, mondon p, et al. cosmeceuticals and active ingredients. clin dermatol 2009;27:461-8. 11. chanchal d, swarnlata s. novel approaches in herbal cosmetics. j cosmet dermatol 2008;7:8995. 12. gianeti md, mercurio dg, campos pm. the use of green tea extract in cosmetic formulations: not only an antioxidant active ingredient. dermatol ther 2013;26:267-71. 13. wisuitiprot w, somsiri a, ingkaninan k, et al. in vitro human skin permeation and cutaneous metabolism of catechins from green tea extract and green tea extract-loaded chitosan microparticles. int j cosmet sci 2011;33:572-9. univ med vol. 37 no.1 12 tjandra, wijayadi, rumawas green tea moisturizer and skin hydration 14. binic i, lazarevic v, ljubenovic m, et al. skin aging: natural weapons and strategies. evid based complement alternat med 2013; 827248:110. 15. belo se, gaspar lr, maia campos pm, marty jp. skin penetration of epigallocatechin-3-gallate and quercetin from green tea and ginkgo biloba extracts vehiculated in cosmetic formulations. skin pharmacol physiol 2009;22:299-304. 16. sarkar r, podder i, gokhale n, et al. use of vegetable oils in dermatology: an overview. int j dermatol 2017;56:1080-6. 17. white po, tribout h, baron e. protective mechanisms of green tea polyphenols in skin. oxidative med cell longevity 2012, article id 560682, 8 pages. doi: 10.1155/2012/560682. 18. chiu ae, chan jl, kern dg, et al. double blinded,placebo-controlled trial of green tea extracts in the clinical and histologic appearance of photoaging skin. dermatol surg 2005;31:855– 60. 19. sudigdo s, sofyan i. dasar-dasar metodologi penelitian klinis 4th ed. jakarta: sagung seto;2011. 20. mahmood t, akhtar n. combined topical application of lotus and green tea improves facial skin surface parameters. rejuvenation res 2013; 16:91-7. 21. chiu hf, lin ty, shen yc, et al. improvement of green tea polyphenol with milk on skin with respect in healthy adults: a double-blind placebocontrolled randomized crossover clinical trial. food funct 2016;7:893-901. 22. lee e, lee bj, ha j, et al. efficacy of fermented green tea on peripheral skin temperature: a randomized and placebo-controlled clinical study. j cosmet dermatol 2016;15:226-30. 23. thiele jj, ekanayake-mudiyanselage s. vitamin e in human skin: organ-specific physiology and considerations for its use in dermatology. mol aspects med 2007;28:646-67. 24. baumann l. tocopherol (vitamin e). in: weisburg e. cosmeceuticals and cosmetic ingredients. new york: mcgraw-hill;2014.p.182-7. 25. baumann l. green tea. in: weisburg e. cosmeceuticals and cosmetic ingredients. new york: mcgraw-hill;2014.p.137-43. 26. zink a, traidl-hoffmann c. green tea in dermatology – myths and facts. j dtsch dermatol ges 2015;13:768-75. 27. yusuf n, irby c, katiyar sk, et al. photoprotective effects of green tea polyphenols. photodermatol photoimmunol photomed 2007;23:48-56. c:\users\jurnal fkusakti\docume 105 *national institute of health research and development, ministry of health, republic of indonesia correspondence: woro riyadina national institute of health research and development, ministry of health, republic of indonesia jalan percetakan negara no.29 jakarta 10560 kotak pos 1226 email: w.riyadina02@gmail.com mobile : +62812 9767 3074 date of first submission, march 24, 2017 date of final revised submission, june 21, 2018 date of acceptance, june 25, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina motorcycle accident injuries are more severe than other land transportation injuries woro riyadina djoeworo*, dian perwitasari tasono hadi*, and sulistyowati tuminah darjoko* background injuries due to traffic accidents are the leading cause of death and disability, especially in developing countries. the proportion of injuries in indonesia tends to increase. the cause of the injury is dominated by motorcycle accidents (70-85%). the aim of this study was to compare the severity of motorcycle injuries with that of other land transportation injuries in indonesia. methods a study of a cross-sectional design was performed involving 15,849 subjects from 33 provinces in indonesia. the dependent variable was the severity of injury and the main independent variable was transportation mode (motorcycle and other vehicles). other independent variables included were respondent characteristics (sex, age, education, occupation, economic status) and health status (stroke, hypertension, heart disease, impaired hearing and vision). chi-square test and multiple logistic regression were used to analyze the data. results the contribution of motorcycle transportation mode to the severity of the injury was 85.5%. the proportion of severity of injury due to motorcycle transportation was higher (9.0%) than to other land transportation modes (7.5%). the severity of injuries related to motorcycle transportation was 1.1 times greater (aor=1.1 95% ci 1.0-1.3) than that other related to vehicles. the determinants of severity of injury due to motorcycle transportation were age and sex. conclusion motorcyclists are at higher risk of having injuries than persons using other modes of transportation. motorcyclists need to increase the safety of riding and to be more discriminating in the selection of the safest mode of transportation, by taking age and sex into consideration. keywords: injury, severity, motorcycle, land transportation doi: http://dx.doi.org/10.18051/univmed.2018.v37.105-114 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cite this article as: djoeworo wr, hadi dpt, darjoko st. motorcycle accident injuries are more severe than other land transportation injuries. univ med 2018; 37:105-14. doi: 10.18051/univmed. 2018.v37.10 5-1 14 106 djoeworo, hadi, darjoko motorcycle accident injuries introduction injuries due to land transportation are a challenging public health problem and are estimated to become the fifth main health problem globally in the year 2013.(1) more than two-thirds of cases of injury occur in developing countries.(1) mortality due to injury is projected to increase from 5.1 million to 8.4 million (9.2% of global mortality) and is estimated to rank third in disability -adjusted life years (dalys) in the year 2020.(2) injury ranks second as a cause of death in indonesia. the problem of injury in developing countries causes an economic loss of 2% of the gross development product (gdp),(3) and the severity of injury contributes to 15-55% of deaths.(4) studies found that families of low economic status are at high risk for becoming poor after a family member experiences a transportation accident as compared with families of high economic status.(5) injury due to traffic or land transportation accidents is the main cause of death and general disability particularly in developing countries. the prevalence of injury shows an increasing trend from 25.9% in 2007 (6) to 47.7% in 2013.(7) in indonesia the number of motorcyclists with unsafe riding behavior increases by 11% each year.(8) the vehicles that contribute to around 7085% of the causes of land transportation injury are motorcycles.(9) in malaysia, 70% of accidents involve motorcycles, and accounts for 60% of all road fatalities.(10) the results of the basic health research survey (riskesdas) for 2013 report the propor tion of inj ur y due to motor cycle transportation to be 40.6%.(7) disability and death due to injury are closely associated with the degree of severity experienced. death due to injury from motorcycle transportation involving drivers and passengers account for 58.7%.(10) the results of a study in kenya show that motorcycles as a mode of public transport are the main cause of severity and death due to injury.(11) there are three main principles in the prevention and control of injury, i.e. 1) reduce the probability of occurrence of injury (primary prevention), 2) reduce the severity of the injurious event (secondary prevention ) and 3) reduce the effects that occur after the injurious event (tertiary prevention). primary prevention means the total elimination of the incidence of trauma so that it does not occur at all. secondary prevention is the reduction of the severity of t r a u ma t hat o c c u r s . te r t i a r y pr e ve n t i on comprises the efforts carried out to reduce the consequences of trauma after it occurs, e.g. a trauma management system (coordination of emergencies, identification of the center of emergency and integration of rehabilitation services).(12) these efforts at trauma prevention need information on the degree of severity of the injury experienced, especially that due to land transportation. studies in nigeria report that motorcycles account for 67% of all traffic accident victims who are referred to hospitals (13) while in tanzania the total is 37.2%.(14) in thailand, a total of 80% of victims of motorcycle transport accidents who are referred to hospitals have head injuries.(15) the results of one study state that motorcycle drivers are at 26-fold greater risk of dying from accidents as compared with car drivers, and at 5-fold greater risk of injury. the degree of severity of injury of drivers due to motorcycle accidents is 6 times more severe than that of car passengers.(16) several studies have shown consistent results, in that motorcycle accidents are the largest contributors to the incidence of land transportation injury and cause more severe inj u ry co mp ar ed to o the r mo de s o f la nd transportation. however, there are not many analytical studies on the degree of severity of injury by combining the parts of the body affected by injury and the type of wounds based on the criteria of the 10th e dition of the international classification of diseases (icd).(17) the increasing trend in the incidence of land transportation accidents and injuries, particularly those caused by motorcycles, results in the need for comparative information on the degree of 107 severity of injury due to motorcycles and other modes of land transportation. the aim of the present study was to compare the degree of severity of injury due to motorcycles and other land transportation modes. methods research design the present study was an observational study of cross-sectional design that used secondary data from riskesdas 2013 for 33 provinces and was conducted from january to december 2014. study subjects the subjects of the present study were residents who had ever experienced la nd transportation injury. the inclusion criteria were the completeness of data for the dependent and independent variables, while the exclusion criterion was the presence of extreme data (outliers), e.g. when there were wounds on all parts of the body (recall data). the sample size was based on public health data from 33 provinces, which were the data of the riskesdas 2013 survey that fulfilled the inclusion and exclusion criteria and were ready for analysis. the sample of 15,894 persons was the sample size calculated with lemeshow’s formula for estimation of proportions,(18) with a proportion of land transportation injury of 7.7%.(6) measurements all variables were collected from interviews using structured questionnaires in accordance wi th t he riskesdas 2 013 da ta c oll ection guidelines.(19) injury was defined as the physical damage to the human body caused by forces that could not be tolerated and could not be foreseen.(20) the cases of injury were obtained from interviews. the injuries asked were events that the respondents of all ages experienced during the last 12 months. injury in riskesdas was defined as the experiencing of injury causing disturbance of daily activities. for cases of injury in which the event occurs more than once in 12 months, the case of injury asked was the most severe one accor ding to the r espondent’s statement. especially for the variables of vision and hearing impairments, physical examination was performed according to the guidelines for biomedical measurement and examination (pe doman penguk uran dan pe meriks aan biomedis) of riskesdas 2013.(21) in the present study, the severity of the injury was a combination of two variables, i.e. the body part involved in the injury and the type of injury, that are further categorized into severe and non-severe injury according to the criteria of diagnostic classification of injury of icd10.(15) the injury was categorized as severe if the number of injured body parts was 4 out of 6 (head-neck, breast-back, abdomen-waist-hip, upper extremity/hand, lower extremity/leg), or if the injury was in the form of fractures or concussion or amputation of an extremity. with regard to the characteristics of the respondents (age, sex, education, employment and economic status), age was categorized into 8 age groups (0-4; 5-14; 15-24; 25-34; 35-44; 45-54; 55-64; >65 years), while sex was categorized into male and female. educational level was categorized into 3 groups, i.e. low (no schooling – elementary school – junior high school) middle (senior high school); high (diploma – tertiary education). employment was categorized into 6 groups, i.e. not yet employed (<10 yr), unemployed (10 yr), employed ( gove r n me nt , p r i va t e b u si n e s s, p o li c e ) , entrepreneur, farmer/laborer/fishermen, and other. economic status was categorized into quintiles 1–5. quintiles were calculated based on the conversion of assets according to the categories of the central statistics agency (bps). there were 2 types of disease that were analyzed, i.e. stroke and coronary heart disease, each with the categories yes and no. there were 2 types of impairment of the special senses that we r e a na lyze d , i .e . vi s i on a nd h e a r in g impairments, each with the categories yes and no. univ med vol. 37 no.2 108 djoeworo, hadi, darjoko motorcycle accident injuries a b c data analysis differences in severity of injury due to motorcycles in comparison with other land transportation were analyzed with the chi-square test. the risk of severity of injury in relation to other factors was determined by multivariate analysis using the multiple logistic regression test. data were analyzed by means of the spss program, at level of significance p<0.05 and 95% confidence interval. ethical clearance this study used secondary data from riskesdas 2013, which received ethical clearance from the commission for health research ethics (komisi etik penelitian kesehatan, kepk) of the agency for he alth research and development (badan pene litian dan pengembangan kesehatan, balitbangkes), ministry of health, republic of indonesia (kemenkes ri) under no. lb.02.01/5.2/ke.006/ 2013. results the different proportions of the severity of injury according to the mode of transportation are presented in table 1. the number of data analyzed was 15,894, divided into injury due to motorcycles with 13,524 data (85.09%) and injury due to other land transportation with 2,370 (14.91%). subjects with severe injury due to motorcycles were 1,214 in number (9.0%) and that due to other land transportation 178 (7.5%). of all 1,392 cases with severe injury (8.8%), injury caused by motorcycles accounted for 1214 (9.0%), which was significantly higher than that caused by other land transportation, with 178 cases (7.5%) (p=0.021). a comparison of the proportions of the severity of injury between motorcycles and other land transportation, by subject characteristic, may be seen in table 2. there were significant differences in the proportions of the severity of injury due to motorcycles in comparison with other land transportation for the following respondent characteristics, i.e. age group, educational level, type of employment, and economic status. the proportion of severity of injury by age group showed significant differences (p<0.05) in the age groups of 5–14, 25–34, and 35–44 years, respectively. the proportion of severity of injury due to motorcycles appeared to be higher in the age group of 5–14 years as compared with injury due to other land transportation. on the other hand, in the age groups of 25–34 and 35–44 years, the proportion of severity of injury was greater in other land transportation as compared with motorcycles. the proportion of severe injury due to motorcycles in both sexes was higher than the severity due to other land transportation. in comparing the degree of severity of motorcycle accidents in both sexes, it appears that the severity of the injury was higher in males than in females, although the differ ence was not significant. based on educational level, a significantly different proportion of severity of injury due to motorcycles as compared with other land transportation was seen with low education (no schooling–elementary school), whereas with higher educational level there was no difference. the employment status of “not yet employed (children)” showed a significant difference (p<0.021) for the severity of injury due to motorcycles as compared with other land transportation. mode of transportation severity of injury p value severe (n=1,392) not severe (n=14,502) motorcycles (n=13,524) 1214 (9.0%) 12310 (91.0%) 0.021 other land transportation (n=2,370) 178 (7.5%) 2192 (92.5%) table 1. distribution of severity of injury due to motorcycles and other land transportation modes (n=15,894) 109 univ med vol. 37 no.2 table 2. distribution of severity of injury due to motorcycles and other land transportation, by subject characteristic (n=15,894) *legend: low educational level: no schooling, elementary school, and junior high school; middle educational level: senior high school; high educational level: academy/tertiary education the health status in the advanced analysis was the health condition of the respondents of having a disease or ill-health. the diseases or ill-health in question were those suspected of being associated with the injury, i.e. stroke, coronary heart disease, and vision and hearing impairments. the differences in the proportions of severity of injury due to motorcycles and other l a n d t r a n spo r t a t i on by h e a l t h s t a t u s a r e presented in table 3. the health status that was suspected to be associated with severity of injury comprised history of stroke, coronary heart disease, and impairment of the special senses (hearing and vision). the health status that showed a significant difference (p<0.05) in the severity of the injury was impairment of vision only. however, for impairment of vision the proportion of severity of the injury was even higher in other land transportation modes as compared with motorcycles. t he se ve ri t y of i nj u r y d ue t o l a n d transportation was differentiated into 2 groups based on transportation mode, i.e. severity of characteristic severity of injury motorcycles (n=13,524) other land transportation (n=2,370) p value severe n (%) not severe n (%) severe n (%) not severe n (%) age (years) 0 – 4 11(6.7) 152 (93.3) 5 (3.8) 128 (96.2) 0.309 5 – 14 120 (7.8) 1425 (92.2) 41 (3.4) 1154 (96.6) 0.000* 15 – 24 413 (8.0) 4741 (92.0) 25 (8.1) 285 (91.9) 0.915 25 – 34 239 (8.4) 2603(91.6) 28 (12.7) 192 (87.3) 0.035* 35 – 44 195 (9.9) 1775 (90.1) 28 (15.6) 151 (84.4) 0.021* 45 – 54 134 (11.5) 1032 (88.5) 19 (11.7) 143 (88.3) 0.896 55 – 64 35 (20.5) 136 (79.5) 14 (18.7) 61 (81.3) 0.080 65 + 1214 (9.0) 12309 (91.0) 179 (7.6) 2190 (92.4) 0.863 sex male 887 (9.7) 8234 (90.3) 122 (8.2) 1370 (91.8) 0.065 female 327(7.4) 4076 (92.6) 57 (6.5) 822 (93.5) 0.355 educational level low 513 (9.3) 4998 (90.7) 110 (6.1) 1700 (93.9) 0.000* middle 638 (8.8) 6653 (91.2) 58 (11.4) 452 (88.6) 0.053 high 63 (8.7) 659 (91.3) 9 (18.4) 40 (81.6) 0.081 employment unemployed 499 (8.7) 5207 (91.3) 68 (6.9) 923 (93.1) 0.055 employed (government, private business, police) 186 (9.0) 1874 (91.0) 18 (13.1) 119 (86.9) 0.126 entrepreneur 165 (9.4) 1592 (90.6) 25 (15.8) 133 (84.2) 0.017* farmer, laborer, fishermen 272 (9.2) 2680 (90.8) 41 (13.6) 261 (86.4) 0.018* other 45 (9.5) 427 (90.5) 5 (8.9) 51 (91.1) 1.000 not yet employed (children) 47 (8.2) 529 (91.8) 21 (2.9) 706 (97.1) 0.000* economic status quintile 1 123 (8.8) 1274 (91.2) 22 (8.1) 250 (91.9) 0.814 quintile 2 205 (8.8) 2132 (91.2) 34 (7.5) 420 (92.5) 0.410 quintile 3 284 (9.3) 2779 (90.7) 39 (7.3) 494 (92.7) 0.163 quintile 4 347 (9.1) 3466 (90.9) 34 (5.4) 592 (94.6) 0.002* quintile 5 255 (8.7) 2660 (91.3) 48 (9.9) 435 (90.1) 0.389 110 djoeworo, hadi, darjoko motorcycle accident injuries injury due to motorcycles and that due to other land transportation. the determinants of severity of the injury that were entered as variables in the multivariate analysis were the variables with p<0.250. determinants contributing to the severity of injury due to motorcycle s as compared with other land transportation are presented in table 4. the severity of injury due to motorcycles as compared with other land transportation was influenced by age and sex. the motorcycle transportation mode had a 1.1-fold higher risk (aor) (95% ci: 1.0–1.3) for severe injury as compared with other land transportation modes, after adjustment for age and sex. these results may be interpreted to mean that the probability of an individual to experience severe motorcycle injury was 52.4%. the factor of age had a relatively large contribution to the severity of injury due to motorcycles. the results of the multivariate analysis showed that the risk (odds ratio) of having severe injury increased with age. health status severity of injury p value motorcycles other land transportation severe n (%) not severe n (%) severe n (%) not severe n (%) stroke yes 10 (23.3) 33 (76.7) 1 (14.3) 6 (85.7) 1.000 no 1,204 (8.9) 12,277 (91.1) 177 (7.5) 2,186 (92.5) 0.023 coronary heart disease yes 8 (11.9) 59 (88.1) 1 (11.1) 8 (88.9) 1.000 no 1,206 (9.0) 12,252 (91.0) 177 (7.5) 2,184 (92.5) 0.021 impairment of hearing yes 56 (11.6) 427 (88.4) 9 (8.4) 98 (91.6) 0.397 no 1,158 (8.9) 11,883 (91.1) 170 (7.5) 2,094 (92.5) 0.033 impairment of vision yes 311 (10.2) 2,734 (89.8) 58 (13.7) 366 (86.3) 0.035* no 903 (8.6) 9576 (91.4) 121 (6.2) 1,826 (93.8) 0.000 *legend: variables stroke and coronary heart disease from 15 yr upwards, variables impairment of hearing and impairment of vision from 6 yr upwards table 3. differences in severity of injury due to motorcycles and other land transportation, by health status (n=15,894) determinant severity of injury crude odds ratio 95% confidence interval adjusted odds ratio 95% confidence interval mode of transportation motorcycles other land transportation 1.3 1 1.1 – 1.6 reference 1.1 1 1.0 – 1.3 reference age (years) 0 – 4 5 – 14 15 – 24 25 – 34 35 – 44 45 – 54 55 – 64 65 1 1.1 1.6 1.7 2.1 2.3 2.9 4.7 reference 0.7 – 1.9 0.9 – 2.6 1.0 – 2.9 1.2 – 3.5 1.4 – 3.9 1.7 – 5.1 2.6 – 8.7 1 1.1 1.5 1.6 1.9 2.2 2.8 4.3 reference 0.6 – 1.9 0.9 – 2.5 0.9 – 2.7 1.1 – 3.3 1.3 – 3.7 1.6 – 4.8 2.4 – 7.9 sex male female 1.3 1 1.2 – 1.5 reference 1.3 1 1.2 – 1.5 reference table 4. determinants of the severity of injury due to motorcycles as compared with other land transportation 111 univ med vol. 37 no.2 in the age group of 65 years, the risk for severe injury due to motorcycles was 4.3 times greater than in the age group of 0-4 years (or= 4.3;95% ci: 2.4–7.9). sex was also a determinant of severity of injury due to motorcycles . males had a 1.3-fold higher risk of having a severe injury (95% ci: 1.2–1.5) as compared with females, or the probability for severity of the injury was 56.5%. discussion the present study demonstrated that injury due to motorcycles is more severe than that due to o th er l and tr anspo rt at io n mo de s. t he motorcycle transportation mode accounts for the largest proportion of land transportation injury. t he 20 1 3 da t a o f t h e na t i ona l tr a ff ic m a n a ge me n t cent e r ( nt m c) ( 2 2 ) o f t he indonesian police (polri) comprise information that motorcycles are still the transportation mode most frequently involved in accidents, i.e. 119,568 out of 168,000 cases (71.17%) as compared with other transportation modes. the n umbe r of se ver e ly wo unde d vic t ims of accidents is reportedly 5000 cases (2.9%).(22) this situation is similar to that in malaysia, where 70% of ac cid en t vic ti ms ar e mot orc yc le drivers.(10) the results of our study showed that in general the proportion of severity of injury due to land transportation was 8.8%. based on transportation mode, it is apparent that the p r o po r t i on of s e ve r i ty o f i nj u r y du e t o moto r c yc l e s wa s s igni f i ca nt ly h igh e r a s compared with other land transportation modes. these results showed that the proportion of severity of injury was far higher as compared with the data on severity (severe wounds) of the police, because the assessment of severity was based only on observation of the condition of the victims by police officers at the scene of an accident (tempat kejadian perkara, tkp). the difference lies in the evaluation of the severity of an injury, in that the assessment performed by police officers at the tkp was by physical examination only, whereas in our analysis the severity of the injury was determined ba sed on medical diagnostic criteria, i.e. according to the icd-10 criteria. the proportion of severity of injury due to motorc ycles was higher in the groups of respondents of age 5-14 years, male, of low education, and with economic status at quintile 4. the proportion of severity of injury in the age gr ou p o f 5-14 ye a r s w a s h i ghe r f o r t he motorcycle transportation mode as compared with other land transportation modes. the results of our analysis cannot be compared with the police data, where the majority of the victims of land transportation accidents are dominated by t he a ge gr o up of 16 – 30 ye a rs a nd l a c k information on the severity of injury.(22) the severity of injury due to traffic accidents is significantly associated with the age of the drivers.(23) the results of a study on the registry of injuries due to motorcycle accidents in fatmawati central general hospital showed the age range of the majority of victims to be 21-30 years,(24) but data on severity were not reported. meanwhile, the results of advanced analysis of riskesdas 2007 data on injury due to traffic accidents also showed a wider age range for the majority of victims, i.e. 15-59 years and there were no reports yet on categories of severity of injury. this was due to the fact that in riskesdas 2007 there was no differentiation between motorcycle accidents and accidents due to other land transportation modes.(25) the proportion of severity of injury was more frequent in males and in children (not yet employed). the results of a study in developing countries show that a ccident victims are predominantly males.(26) these results are consistent with those of a study in kenya, where the ratio of males to females with injury due to motorcycles was 3.4 : 1. males are at higher risk for severe injury because the majority are motorcycle riders.(15) originating from a younger age group, males are more frequently involved in traffic accidents than do females, i.e. around 77%. around 89% of deaths and severe injuries 112 djoeworo, hadi, darjoko motorcycle accident injuries experienced by males are caused by motorcycle accidents.(26) according to economic level by quintile, apparently the proportion of severity of inj ury due to motorcycles was higher as compared with other land transportation modes in the quintile 4 groups (middle economic status). these results are similar to those from india, where motorcycle injury was more frequent in the groups of middle and high economic status.(27) the present study also showed that severity of injury is associated with health status. the results of our advanced analysis showed that impairment of vision contributed to a higher proportion of severity even in accidents due to other land transportation as compared with mot o r c yc l es ( p < 0. 0 5) . t h e r es u l ts o f a ta iwan ese s tud y re por t th at poor he al th conditions had a 1.68-fold higher risk for severe injury. bad or less favorable health conditions potentially increase the risk of more severe injury.(28) the results of our multivariate analysis showed that severity of injury due to motorcycles was higher as compared with severity due to other land transportation after adjustment for sex and age. injury due to motorcycles had a 1.3-fold more severe (or=1.3;95% ci: 1.1–1.6) and a 1.1-fold higher adjusted odds ratio (aor=1.1;95% ci 1.0–1.3) as compared with with the odds ratios for other land transportation after adjustment for sex and age. these results may be interpreted as showing that the probability of an individual having severe injury due to motorcycles was 52.5%. these results are in line with an australian study, where injury due to motorcycles have a 29-fold higher degree of severity and a 37-fold fatality rate as compared with injury due to other types of vehicles.(29,30) the severity of injury due to motorcycles has the highest rank of hospital visits of 79.9%, followed by injury due to car accidents of 10.7% and other land transportation modes of 9.4%.(31) the victims of motorcycle accidents also experience a trauma rate of 64.9%.(32) motorcycle riders have a 26-fold higher risk of death and a 5-fold higher risk of injury as compared with car drivers and passengers. in the us in 2013 it was found that the fatality rate of injury due to motorcycles was 6 times greater than that due to cars.(16) the results of a study in finland in 2000 and 2012 showed that the number of motorcycles increased up to 170% and 186%, respectively.(33) motorcycle crashes are 37 times more lethal than automobile crashes.(34) the determinants of severity of injury due to motorcycles are age and sex. the determinant of age showed that aging increases the risk of severe motorcycle injury, with the highest risk experienced by those aged 65 years. this may be due to physiological changes in aging that result in longer reaction or perception times, resulting in delayed driving behavior of older persons, so that they become at risk for more severe injury.(35) the results of a wisconsin study report that injury due to motorcycles was more severe for older riders.(23) our study has several limitations such as the unequal completeness of the data for all variables. there are several variables such as employment that were asked only to respondents who were 15 years old and above. the criteria of severity were limited to the number of injured body parts without information on length of hospitalization due to the injury. this was due to the fact that the available data on length of hospitalization consisted of the total length of hospitalization of all injuries experienced during one year, whereas the collected data on injury were limited to the most severe injury according to the statements of the respondents. the concept of causality (cause-and-effect) between risk factors/determinants that antedate the outcome (severity) cannot be explained in this analysis due to the cross-sectional design of the riskesdas (risk factors and outcomes viewed in the same time period). the factors of vehicles, environment, and behavior of drivers were not collected in the riskesdas so that the present study was unable to consider these contributing factors. the clinical implications of the severity of injury due to motorcycles and other land transportation modes are that it may result in disability and premature or early death that 113 should be preventable. disability may decrease productivity. early death may reduce the life expectancy of an individual who may actually h a ve a l o ng l i f e sp a n . t he r e su l t s o f a n epidemiological study state that 13% of subjects who survive a land transportation accident experiences post-traumatic stress.(12) however, the results of our study suggest that further studies should be done that are focused on persons of age 65 years and over. the factors associated with elderly traffic injuries in indonesia should also be explored further. conclusions injury due to motorcycles is more severe as compared with other land transportation. for motorcycle riders, there is a need to increase the safety of driving and to be more discriminating in the selection of safer land transportation modes, by taking age and sex into consideration. conflict of interest there was no conflict of interest for this article. acknowledgment the authors extend their utmost gratitude to prof. dr. bastaman basuki, mph, spkp and dr. iwan ariawan, ms for their cooperation in the analysis team by providing input, guidance, and counseling so that this article could be wellorganized. contributors wrd contributed to the idea for this article, the technical points of data analysis, and writing of the manuscript draft. dpth contributed to data collection and analysis. std contributed to revising the manuscript. all authors read and approved the final draft of the manuscript. references 1. mathers cd, loncar d. projections of global mortality and burden of disease from 2002 to 2030, plos med 2006;3:e442. doi: 10.1371/journal.pmed. 0030442. 2. peden m, scurfield r, sleet d, et al. world 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27: 150-6 magnetic resonance cholangiopancreatography: practical experience in 30 subjects october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 abstract universa medicina a. nurman* magnetic resonance cholangiopancreatography (mrcp) is a noninvasive method of imaging the biliary and pancreatic ducts. no special patient preparation is required but the usual contraindications to mr scanning apply. the diagnostic performance of mrcp in most biliary tract diseases is similar to that of more invasive techniques of direct cholangiography such as endoscopic retrograde cholangiopancreatography (ercp). the objectives of this study were to investigate the diagnostic efficacy of mrcp in patients with abdominal pain with lesser likelihood of having choledochal stone and to determine whether use of mrcp could eliminate the need for purely diagnostic endoscopic retrograde cholangio-pancreatography (ercp). a total of 30 patients with suspected biliopancreatic pathology from several hospital was studied retrospectiely between january 2007 and december 2008 in jakarta. the sensitivity and specificity of mrcp was 92.59% (95% confidence interval, 74.25 98.71%) and 66.67% (95% confidence interval, 12.53 – 98.23%), respectively. the positive predictive value of mrcp for all biliary pathology was 96.15% (95% confidence interval, 78.42 – 99.79%) the negative predictive value of mrcp was 50.00% (95% confidence interval, 9.19 – 90.81%). mrcp seems to be effective in diagnosing patients with abdominal pain with lesser likelihood of having choledochal stone. keywords : magnetic resonance cholangiopancreatography, biliary, pancreatic introduction cholangiography is often used in patients suspected of having biliary tract and pancreatic disorders to uncover the exact diagnoses of the disease. endoscopic retrograde cholangiopancreatography (ercp) was considered the golden standard method for the diagnosis of these diseases, but carries a potential risk of c o m p l i c a t i o n s , i n c l u d i n g p a n c r e a t i t i s , haemorrhage, particularly from sphyncterotomy s i t e s , a n d d u o d e n a l p e r f o r a t i o n . ( 1 3 ) t h e procedure may not always be successful. ercp is highly sensitive and specific, but is invasive 151 nurman cholangiopancreatography and inconvenient for the patient, requiring sedation and contrast (with minimal risk of a l l e r g i c r e a c t i o n ) , a n d a s s o c i a t e d w i t h significant morbidity (5–10%) and mortality ( < 1 % ) . ( 4 ) a t p r e s e n t m a g n e t i c r e s o n a n c e cholangiopancreatography (mrcp) begins to r e p l a c e e r c p a s a s c r e e n i n g m e t h o d f o r p a t i e n t s s u s p e c t e d o f h a v i n g b i l i a r y obstruction due to biliary calculus. mrcp was first described by wallner et al. in 1991.(5) the use of single-shot fast sequences in a breathhold period provides heavily t2-weighted sequences allowing thick slices and avoiding secondary reconstructions and artifacts. this t e c h n o l o g i c d e v e l o p m e n t h a s l e d t o i t s widespread use.(6,7) as a result, mrcp is an easy, quick, noninvasive test accessible to all patients who do not have contraindications. mrcp is also useful in patients with failed or incomplete ercp. there is also a role for mrcp in the e v a l u a t i o n o f p a t i e n t s p r i o r t o s u r g i c a l procedures, to plan what kind of surgical p r o c e d u r e w i l l b e u n d e r t a k e n , a n d t o demonstrate the alteration the bile ducts after biliodigestive surgery. mrcp is non-invasive for studying the biliary tree and requires no ionizing radiation or iodinated contrast. it gives high-resolution projectional images of the common bile duct (cbd) with no known hazards in the absence of incompatible foreign bodies. the aim of this study was to investigate the diagnostic validity of mrcp in assessing symptomatic patients suspected of having hepatopancreaticobiliary diseases. methods study design this was a retrospective cohort study of a consecutive group of patients who underwent mrcp during a period of 2 years (2007 – 2008). patients patients were identified from several hospitals in jakarta over a total period of 24m o n t h s . c o l l e c t e d d a t a i n c l u d e d c l i n i c a l presentation, liver function tests, abdominal ultrasound, and mrcp findings, and procedurerelated complications. mrcp technique all mrcp images were obtained by using a 1.5-t superconducting magnet with a gradient s t r e n g t h o f 5 0 m t / m ( g e n e r a l e l e c t r i c , milwaukee, wis, usa), with a body-phased array coil through the liver and pancreas. the mean time of the mrcp examination with e v a l u a t i o n w a s 1 5 m i n u t e s . m r c p w a s performed by using thick slab single shot fast spin echo (ssfse), with selective fat saturation. the first sequence afforded a single image with a dimension of 40 mm, and exhibited the following parameters: tr/te/fa (2800/1100/ 180), matrix 240 x 256, field of view of 300 mm, and acquisition time of 7 seconds. the second sequence yielded 13 contiguous 5-mm slices and presented the following parameters: tr/te/fa (10.92/87/180), matrix 256 x 224, field of view of 280 mm, and acquisition time of 19 seconds. statistical analysis the data were entered in a microsoft excel database. the results of mrcp were analysed a g a i n s t l a b o r a t o r y f i n d i n g s , u l t r a s o u n d examinations and the final diagnoses for the entire study population. sensitivity, specificity, positive and negative predictive values were individually calculated in the usual manner. results thirty patients underwent mrcp and were eligible for further evaluation during the 24month period studied, of whom 15 (50%) were 152 univ med vol.27 no.4 female and 15 (50%) were male with median a g e o f 5 6 y e a r s ( r a n g e 2 6 – 7 5 y e a r s ) . abdominal pain was the predominant symptom at presentation (90%) with 5 subjects having associated jaundice. pancreatitis (n=6) and c h o l a n g i t i s ( n = 2 2 ) w e r e t h e o t h e r m o s t common features. ultrasound u l t r a s o u n d s c a n o f t h e h e p a t o b i l i a r y system was the primary investigation in 30 patients, of whom 10 (33.3%) showed evidence of cholelithiasis and 2 (6.7%) of biliary sludge. other abnormalities seen were dilated cbd, liver cirrhosis (lc), dilated gall bladder (gb), dilated extrahepatic bile duct (ehbd), dilated intrahepatic bile duct (ihbd), and choledochal cyst in fifteen patients. no abnormality was observed in 3 patients. mrcp o f 3 0 p a t i e n t s r e f e r r e d , m r c p w a s s u c c e s s f u l l y p e r f o r m e d i n a l l ( 1 0 0 . 0 % ) patients. final diagnosis the final diagnoses of the patients were: i) choledochal cysts in 3 patients (10%); ii) c h o l e d o c h a l s t o n e / s l u d g e i n 5 p a t i e n t s (16.7%); iii) gall bladder stone in 8 patients (26.7%) (figure 1); iv) intrahepatic stone 1 patient (3.3%);v) distal obstruction of the common bile duct due to mass in 7 cases (23.3%), and follow up ct scan/mri revealed c a r c i n o m a o f t h e p a n c r e a s i n 5 p a t i e n t s (16,7%) and carcinoma of the papilla in 2 patients (6.7%); vi) stricture of the common bile duct post laparoscopic cholecystectomy in 1 patient (3.3%); viii) klatskin tumor in 1 patient (figure 2); (3,3%); viii) post hepaticojejunostomy operation in choledochal cyst in 1 patient (3%); and ix) no abnormality in 3 (10%) patients (figure 3). figure 3. normal mrcp figure 2. klatskin tumor figure 1. mrcp in gall bladder stone 153 nurman cholangiopancreatography accuracy of mrcp a t o t a l o f 3 0 p a t i e n t s w i t h c o m p l e t e comparative data were considered for detailed analysis. the sensitivity and specificity of mrcp was 92.59% (95% confidence interval, 74.25 – 98.71%) and 66.67% (95% confidence interval, 12.53 – 98.23%), respectively. the positive predictive value (having the disease) of mrcp for all biliary pathology was 96.15% (95% confidence interval, 78.42 – 99.79%) the negative predictive value of mrcp (truly disease free) was 50.00% (95% confidence interval, 9.19 – 90.81%) (table 1). t h e r e w e r e 1 8 g a l l b l a d d e r s t o n e s , choledochal cyst, and common bile duct sludge among various positive diagnoses, based on all investigations (table 2). discussion mrcp is a noninvasive method of imaging the biliary and pancreatic ducts and until now this technique continues to be improved. the basic principle underlying mrcp is that body fluids, such as bile and pancreatic secretions, h a v e h i g h s i g n a l i n t e n s i t y i n h e a v i l y t 2 weighted magnetic resonance (i.e. they appear white), whereas background tissues such as the surrounding liver and flowing blood generate little signal (i.e. they appear dark). as a result of this combination of imaging characteristics, mrcp provides optimal contrast between the hyperintense signal of the bile and pancreatic secretions and the hypointense signal of the table 1. sensitivity and specificity of mrcp as evidenced against gold standard procedures final diagnosis mrcp positive negative total positive negative 25 2 1 2 26 4 total 27 3 30 background tissue (solid organs), while blood vessels have no measurable signal. on these images the intrinsic fluids of the the biliary and pancreatic ducts make up the cholangiogram and pancreatogram. at present, ercp is considered the gold standard method for the diagnosis of ductal c a l c u l u s , b u t c a r r i e s a p o t e n t i a l r i s k o f complications, including pancreatitis, and bleeding, particularly from sphincterotomy sites and duodenal perforation.(8) most of the patients presented with the chief complaint of right upper quadrant or epigastric pain, with or without jaundice, or were jaundiced patients w i t h o r w i t h o u t u p p e r a b d o m i n a l p a i n . abdominal ultrasound (us) and ct scan were mostly used as an initial evaluation in these patients. in these cases the roadmap of the biliary tract and the pancreatic duct is needed. the next examination will be the choice of to decide on ercp or mrcp.(9) diagnosis number true positive gall bladder stone choledochal cyst carcinoma pancreas obstructed hepatojejunistomy false negative common bile duct sludge gall bladder stone 13 3 7 2 1 1 total 27 table 2. different diagnoses among true positive and false negative of results of mrcp 154 univ med vol.27 no.4 if the compaint was due to biliary disease, i.e. choledochal stone or stricture/dilatation obstruction of the bile or pancreatic disease, it was easy to find the dilatation of the bile duct on abdominal us/ct. in these cases ercp will be the choice because it can be followed by therapeutic endoscopy i.e. sphincterotomy and s t o n e e x t r a c t i o n , s t e n t / n a s o b i l i a r y t u b e installment etc.the choice of imaging will be e r c p w h e n i t i s a s s u m e d t h a t i t w i l l b e followed by therapeutic endoscopy.(10) if the complaints were due parenchymal liver disease, there would be no dilatation nor abnormality of the biliary tract or pancreatic duct, such that it will not be followed by therapeutic endoscopy, thus mrcp would be the choice. mrcp would be chosen when the likelihood for therapeutic intervention were l e s s , h e n c e u n n e c e s s a r y e r c p i s h e r e b y avoided. this study showed that the sensitivity and positive predictive value of mrcp for the diagnosis is extremely high, being 92.59% (95% confidence interval, 74.25 98.71%) and 96.15% (95% confidence interval, 78.42 – 99.79%) respectively. a prospective study in spain found that mrcp had a sensitivity of 91% and positive predictive value of 89%, similar to our study.(11) but the specificity of 66.67% (95% confidence interval, 12.53 – 98.23%) was lower than that obtained in the study by calco et al (84%). the negative predictive value in the present study was also low (50.0%; 95% confidence interval, 9.19 – 90.81%), compared to the spanish study (88%). no special patient preparation is required, but the usual contraindications to mr scanning apply.(1,12) patients with cardiac pacemakers, neurostimulators, or ferromagnetic aneurysm clips are excluded. the examination is usually performed after the patient has fasted for several hours to allow filling of the gallbladder and e m p t y i n g o f t h e s t o m a c h . n o s e d a t i o n i s required, and no hospitalization is needed. in s o m e p a t i e n t s c l a u s t r o p h o b i a m i g h t b e a p r o b l e m . ( 1 3 ) o r a l c o n t r a s t a g e n t s a r e n o t administered, no intravenous contrast agents are needed, and there is no radiation exposure.(10) mrcp is an ideal imaging method for patients with allergies to iodide/iodine-based contrast, or those with a general history of atopy, and f o r p r e v e n t i n g t h e o c c u r e n c e o f c o n t r a s t nephropathy.(10) in this study as in that from a different center,(10) the procedures were performed with the indication as screening in patients with right u p p e r q u a d r a n t / e p i g a s t r i c p a i n w i t h n o dilatation/equivocal dilatation of the common bile duct with/without gall bladder stone, or with normal/disturbed liver function test, with the goal to avoid unnecessary ercp. in acute pancreatitis ercp is avoided, because ercp carries potential risk of complication including p a n c r e a t i t i s , b l e e d i n g p a r t i c u l a r l y f r o m s p h i n c t e r o t o m y s i t e s , a n d d u o d e n a l perforation.(14) if dilatation of bile duct was found then ercp would be the choice and subsequent sphincterotomy and extraction of choledochal stone will be retrogradely performed. mrcp is an alternative to diagnostic ercp for the imaging of the bile tree and the pancreatic ducts. a major feature of mrcp is that is not a therapeutic procedure, while in contrast ercp is used for both diagnosis and treatment.(1) mrcp is the only modality that allows imaging of these ducts in the basal state, as extrinsic contrast agents are not used,(10) hence more accurately displays the native calibre of the duct than ercp. because of its noninvasive nature, it does not carry the risks and complications a s s o c i a t e d w i t h e r c p a n d p e r c u t a e o u s transhepatic cholangiography (ptc).(1) in mrcp there is no morbidity nor mortality like in ercp/ptc. mrcp is particularly useful w h e r e e r c p i s d i ff i c u l t , h a z a r d o u s o r impossible(12) to perform, ie in patients with 155 nurman cholangiopancreatography anatomical/structural abnormalities, such as gastroenteric anastomosis or gastrojejunostomy. in this series mrcp was performed in a patient a f t e r h e p a t i c o j e j u n o s t o m y b e c a u s e i t w a s impossible to perform ercp in this setting. in some situations mrcp maybe preferable to ercp, such as situations where mrcp may give more informations than ercp (eg hilar b i l i a r y s t r i c t u r e s , l e s i o n s a s s o c i a t e d w i t h complete pancreatic or biliary duct cut-off). mrcp is a non invasive tool that is suitable in p a t i e n t s s u s p e c t e d o f h a v i n g t o h a v e pancreaticobiliary disease, where there is no l i k e l i h o o d o r l i t t l e p o s s i b i l i t y t o p e r f o r m t h e r a p e u t i c i n t e r v e n t i o n , e g p a t i e n t s w i t h asymptomatic cholelithiasis without clinical evidence of clinical bile duct disease, like the presence of jaundice and abnormalities of liver fuction tests. mrcp is a non invasive imaging technique, seems to be highly accurate for the presense of o b s t r u c t i o n , ( 1 5 , 1 6 ) b u t i s l e s s a c c u r a t e a t differentiating malignant from benign causes of obstruction.(10) it is almost as good as ercp in the diagnoses of common bile duct stones (cbds), although the ability of mr to detect s m a l l s t o n e s i n n o n d i l a t e d d u c t s m a y b e limited.(10,17) mrcp may be considered as a new gold standard for the investigation of cbds and permits reservation of ercp to patients with a high probability of therapeutic intervention.(10) the eropean association of laparoscopic surgeons consider mrcp to be the standard diagnostic test for patients with an intermediate p r o b a b i l i t y o f c b d s . ( 1 7 ) t h e p r e s e n c e o f intracranial metallic clips, claustrophobia, or morbid obesity might preclude mrcp.(4,15) conclusions magnetic resonance cholangiopancreatography seems to be effective in the diagnosis of patients with abdominal pain with lesser likelihood of having choledochal stone. it plays a fundamental role in patients with a low or i n t e r m e d i a t e r i s k o f c h o l e d o c h o l i t h i a s i s , c o n t r i b u t i n g t o t h e a v o i d a n c e o f p u r e l y diagnostic ercp. references 1. mac eneaney p, mitchell mt, mc dermoth r. update on magnetic resonance cholagio pancreatography. gastroenterol clin n am 2002; 31: 731-46. 2. ballinger pw, frank ed. biliary tract and gallbladder. in: merrill’s atlas at radiographic position and radiologic procedures. vol. ii. missouri: mosby; 2003. p. 69-116. 3. karani j. the biliary tract. in: sutton d, editor. textbook of radiology and imaging. vol.1. london: churchill livingstone; 2003. p. 711-36. 4. shanmugam av, beattie gc, yule sr, reid w, london ma. is magnetic resonance cholangiopancreatography the new goldstandard in biliary imaging ? br j radiol 2005; 78: 888-93. 5. wallner bk, schumacher ka, weidenmaier w, friedrich jm. dilated biliary tract: evaluation with magnetic resonance cholangiopancreatography with a t2-weighted contrast-enhanced fast sequence. radiology 1991; 18: 805-8. 6. mougenel jl, hudziak h, ernst o. evaluation of a new sequence of magnetic resonance cholangiopancreatography in thick cut and one shot acquisition. gastroenterol clin biol 2000; 24: 888– 95. 7. gallix bp, régent d, bruel jm. use of magnetic resonance cholangiography in the diagnosis of choledocholithiasis. abdom imaging 2001; 26: 21– 7. 8. topal b, van de moortel m, fieuws s, vanbeckevoort d. the value of magnetic resonance cholangio pancratography in predicting common bile duct stones in patients with gallstone disease. br j surg 2003; 90: 42–7. 9. wijaya pm. magnetic resonance cholangiopancreatography for biliary obstruction. 7th hepatobiliary surgical update and workshop on therapeutic endoscopy. jakarta; 2006. 10. barrish ma, yuccel ek, ferrucci jt. magnetic resonance cholangio pancreatography. n engl j med 1999; 341: 258-64. 156 univ med vol.27 no.4 11. calco mm, bufanda l, calderon a, heras i, cabriada jl, bernal a, et al. role of magnetic resonance cholangiopancreatography in patients with suspected choledocholithiasis. mayo clin proc 2002; 77: 422-8. 12. romagnuolo j, bardon m, rahme e, joseph l, reinhold c, barkun an. magnetic resonance cholangiopancreatography: a meta analysis of test performance in suspected biliary disease. ann inter med 2003; 139-7: 547-57. 13. kaltenhaler e, vergel yb, chilcot j, thomas s, blakeborough t, walters sj, et al. a systemic review and economic evaluation of magnetic resonance cholangiopancreatography compare with diagnostic endoscopic retrograde cholangiopan-creatography. health technology assessment; 2004. availabe at: http://www.hta.ac.uk/exec.summ /summ810.htm. accessed august 1, 2008. 14. leung j. fundamentals of ercp, in advanced digestive endoscopy: endoscopic retrograde cholangiopancreatography. in: cotton pb, leung j, editors. 1 st ed. massachusets: blackwell publishing; 2006. p. 17-80. 15. georgopulos sk, schwartz lh, jarnagin wr, gerdes h, breite i, fong y, et al. comparison of magnetic resonance and endoscopic retrograde cholangiography in malignant pancreaticobiliary obstruction. arch surg 1999; 134: 1002-7. 16. fulcher as,turner ma, capps gw, zfass am, baker k. half-fourier rare magnetic resonance cholangiopancreatography: experience in 300 subjects. radiology 1998; 207: 21-32. 17. williams ej, green j, beckingham i, park r, martin d, lombard m. guidelines of the management of common bile duct stones. gut 2008; 57: 1004-21. c:\users\jurnal fkusakti\docume 205 *department of clinical pathology, faculty of medicine, trisakti university, jakarta,indonesia **department of occupational medicine, faculty of medicine, trisakti university, jakarta, indonesia ***department of clinical pathology, faculty of medicine, trisakti university, jakarta,indonesia †department of pharmacology, faculty of medicine, trisakti university, jakarta, indonesia correspondence : prof. dr. dr. pusparini, sp.pk department of clinical pathology, faculty of medicine, trisakti university jl. kyai tapa no 260, grogol, jakarta 11440, indonesia phone: +62-21-5655786, fax: +62-21-5660706 email: pusparini_sppk@yahoo.com date of first submission, may 29, 2017 date of final revised submission, november 15, 2017 date of acceptance, november 16, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 decreased adiponectin level in adults with central obesity and low 25-hydroxy vitamin d level pusparini*, lie tanu merijanti**, alvina lesmana***, and meiyanti† background vitamin d insufficiency is associated with obesity and central obesity, which is in turn associated with the accumulation of visceral fat. visceral fat secretes adiponectin which has a role in the pathogenesis of the metabolic syndrome, diabetes mellitus and cerebrovascular disease. the aim of this study was to determine the relationship between serum 25-hydroxy vitamin d [25(oh)d] level and adiponectin level in adults with central obesity. methods this study was a cross sectional study with 80 subjects with central obesity and 80 healthy subjects as controls. the age of the subjects ranged from 55 to 65 years. the data collection included anthropometry and blood biochemistry (lipid profile, fasting blood glucose, 25(oh)d, and adiponectin). the data were analyzed using independent t test with p <0.05. results mean waist circumference and 25(oh)d level in the central obesity group were 94.9 ± 7.8 cm and 14.3 ± 6.6 ng/ml, respectively, and in the control group 72.4 ± 5.0 cm and 19.2 ± 8.6 ng/ml respectively. there were significant differences in adiponectin level between the central obesity group and the control group at the 25(oh)d level of <20 ng/ml (p=0.001), but no significant differences between the two groups at the 25(oh)d level of 20 ng/ml (p=0.538). conclusion the central obesity subgroup with 25(oh)d level of <20 ng/ml have lower adiponectin levels compared with the control group. this study demonstrates the role of adiponectin as a link between insulin resistance and vitamin d status in adults. keywords: vitamin d deficiency, central obesity, adiponectin, adult doi: http://dx.doi.org/10.18051/univmed.2017.v36.205-213 original article cite this article as: pusparini, merijanti lt, lesmana a, et al. decreas ed adiponectin level in adults with central obesity and low 25-hydroxy vitamin d level. univ med 2017;36:205-13. doi: 1 0 . 1 8 0 5 1 / un i vm e d . 2 0 1 7 .v 3 6 . 2 0 5 2 1 3 pissn: 1907-3062 / eissn: 2407-2230 206 pusparini, merijanti, lesmana, et al adiponectin 25-hydroxy vitamin d introduction obesity is a global problem in developed as well as developing countries. there was a two-fold increased prevalence of obesity from 1980 up to 2014.(1) the rapid increase in prevalence of obesity is associated with changes in life style and dietary pattern. obesity is associated with various disease risk factors such a s s uc h as di a b e t e s , h yp e r te n s i on a n d cardiovascular disease.(2) the initial stage of type 2 diabetes mellitus and the metabolic syndrome is central obesity. central obesity is the condition of an individual’s waist circumference of 80 cm for females for females and 90 cm for males according to asia-pacific criteria.(3) adipose tissue is an endocrine organ that se cre tes a number of pro tei ns kno wn as adipokines. one of the adipokines is adiponectin that is known to play a role in the pathogenesis of the metabolic syndrome. the physiological role of adiponectin is not yet clear.(4) one of several hypotheses that is put forward to clarify t he p r o t ec t i ve r ol e o f a d i po ne c ti n i n cardiometabolic disorders is among others that adiponectin has anti-inflammatory effects, increases insulin sensitivity, increases glucose u pt a ke a t t h e c e l l ul a r l e ve l , d e c r e a se s gluconeogenesis in the liver, and increases the formation of adipocytes.(5-7) a low adiponectin level is frequently associated with obesity, insulin resistance, hypertension and cardiovascular disease (cvd).(4) a low adiponectin level in the blood also is a strong marker of the probability of the metabolic syndrome and type 2 diabetes mellitus.(7) in obesity there is a change in protein secretion in adipose tissue, such as a decrease in the anti-inflammatory adiponectin, while other adipokines produced by adipose tissue such as t umo r n e c r os i s f a c t o r  ( t n f-) a nd interleukin-6 (il-6) are increased.(5,6) obesity is associated with low vitamin d and adiponectin levels and an increase in cvd and mortality. the vitamin d level is associated with the body mass index (bmi) and adiponectin. the higher the bmi of an individual, the lower the vitamin d and adiponectin levels.(8) vitamin d deficiency is a common health problem found throughout the world and is found in around one third to one half of the young adult up to the elderly population.(9) the vitamin d level is influenced by exposure to sunlight, seasonal changes, dietary intake, clothing habits, body composition, ethnicity and genetic factors.(6) vitamin d deficiency is caused by inadequate exposure to sunlight and by a dietary intake that is low in vitamin d. vitamin d deficiency is most frequently associated with musculoskeletal disorders. in addition, vitamin d deficiency is also one of the risk factors for hypertension, various types of cancer, autoimmune disease, type 2 diabetes mellitus and cvd.(9) the results of the study by setiati (10) with 74 females as subjects showed a prevalence of vitamin d deficiency of 31.5%, whereas the results of the study by green et al.(11) on females aged 18-40 years showed a prevalence of vitamin d deficiency of 63%. the results of these two studies showed that although indonesia is located in a region with high exposure to sunlight, this does not guarantee the absence of vitamin d de fi cie nc y i n th is coun tr y. vit amin d deficiency contributes to the development of chronic diseases, such as cvd, hypertension and diabetes mellitus.(10) there are many reports of studies on the role of vitamin d and its relationship with obesity that triggers chronic disease, but the underlying mechanism of this relationship is still a matter o f d e b a t e pa r t ic ul a r ly on th e r ol e o f adiponectin.(6) the study of bidulescu et al.(8) showed that the relationship between vitamin d and adiponectin levels is influenced by ethnicity, gender, and body mass index (bmi). the study of foong et al.(12 ) showed that vitamin d insufficiency is associated with a younger age, gender, abdominal obesity and the metabolic syndrome.(12) the relationship of adipokines with vitamin d is studied from the aspect of their role in obesity. a meta-analysis on the effect of vitamin d supplementation on adiponectin and leptin levels also could not observe an association among 9 randomized controlled trials.(6) 207 for women aged 55-65 years, weight gain and obesity are their major health risks.(13) in a prospective, observational study designed to analyze risk factors for fracture in an ambulatory population aged >55 years, 73 (88%) had evidence of osteopenia or osteoporosis (t-score <-1.5) and/or low 25(oh)d.(14) the present study aimed to determine the relationship between 25(oh)d and adiponectin levels in the serum of adults with central obesity aged 55-65 years. methods research design this was a cross-sectional study conducted at puskesmas kecamatan mampang, south jakarta, from march to june 2014. research subjects the study subjects consisted of two groups, i.e. 80 subjects with central obesity and 80 healthy subjects without central obesity as controls. the sample size was obtained using the formula: where α = 5 %, for one-sided hypothesis so that zα = 1.64; β = 10%, so that zβ = 1.28; r 1 = r 2 = 0.36.(8) the minimal sample size was 63, rounded upwards to 80. for the control group the number of subjects was also taken as 80. subjects were selected by simple random sampling. the inclusion criteria were: males and f e ma l e s age d 55 -6 5 ye a r s , agr e e i n g t o participate in this study by signing informed consent, not suffering from stroke and other terminal diseases such as malignancy, renal failure, and myocardial infarction. the exclusion c r i t e r i a we r e c u r r e nt c on s umpt i o n of corticosteroids, hormonal replacement therapy, and vitamin d supplements. data collection based on the demographic data the elderly study subjects aged 55 – 65 years were selected from five kelurahan in kecamatan mampang, so ut h j a ka r t a . su bj e c t s wh o a gr e e d t o participate in this study were asked to give a statement of their willingness to participate by signing informed consent. the questionnaires were filled in by interviewers who had previously been trained with regard to the objectives of the study and the filling in of the questionnaires. the i nt e r vie w e r s we r e c a dr e s a t pu s k e s ma s kecamatan mampang with an educational level of at least senior high school or equivalent. measurements the study subjects who met the inclusion and exclusion criteria were asked to come to the study location after an overnight fast of 12 hours before blood collection for laboratory examination. measurement of blood pressure (with a mercury sphygmomanometer), pulse rate (by stopwatch), temperature, weight, height, and waist circumference, and also blood sample collection were performed by 2 previously trained nurses. laboratory analysis at baseline, laboratory examinations were performed using venous blood samples of 10 ml each for determination of fasting glucose and lipid profile, while the remaining sera of the blood samples were stored for determination of adiponectin and 25(oh)d. the 25(oh)d level wa s de te r min e d b y i n di r e c t co mpe t i t i ve chemiluminescence immunoassay (clia),(15) while adiponectin level was determined by sandwich immunoassay.(16) vitamin d deficiency was defined as a serum 25(oh)d level below 20 ng/ml or 50 nmol/l. a 25(oh)d level in the range of 21-29 ng/ml (52-72 nmol/l) was designated vitamin d insufficiency while a level of 30 ng/ ml w as de e me d su ff ic ie nt o r normal.(9) the laboratory examinations were performed at prodia laboratories, jakarta. univ med vol. 36 no.3             3 2 r1/r11nr1/r11n 0.5 zβzα n2n1 2211          208 pusparini, merijanti, lesmana, et al adiponectin 25-hydroxy vitamin d a b c ethical clearance the study was performed after approval of the procedure by the ethical committee, faculty of medicine, trisakti university, jakarta, under registration number 51/ker/fk/05/2013. at recruitment specially trained field workers explained the study protocol to the study subjects before requesting their written informed consent. statistical analysis tests on the normality of the variable data using the kolmogorov-smirnov test showed that the data were normal ly distribute d. t he characteristics of the subjects were presented as mean and standard deviation. to evaluate differentiation between 25(oh)d and adiponectin concentrations in central obesity and control subjects, the independent t-test was used. results a total of 189 subjects aged 55-65 years agreed to participate in this study, among whom 29 subjects did not meet the inclusion and exclusion criteria, among others 10 subjects who were older than 65 years, 5 subjects who were younger than 55 years, 2 subjects who were consuming blood viscosity reducing agents such as aspirin, 2 subjects who had once suffered from stroke, 1 subject who had liver disorder and 9 who did not appear on the first day of the study. the study respondents who came for physical and laboratory examinations, and met the inclusion and exclusion criteria, were in total 160 subjects, consisting of two groups, i.e. 80 subjects with central obesity and 80 subjects without central obesity (waist circumference less than 80 cm for females and less than 90 cm for males) as the control group. characteristics of study subjects mean age of the study subjects in both groups were not significantly different, i.e. 59.1 ± 2.9 years in the central obesity group and 58.8 ± 2.5 years in the control group. in the central obesity group the majority of subjects were male (62.0%) whereas in the control group the majority of subjects were female (52.9%). the most frequent educational level in the central obesity group was senior high school (34.2%) whereas in the control group the levels were elementary school and senior high school (29.4%). the majority of subjects were still employed, i.e. 53.2 % in the central obesity group and 64.7% in the control group. in the central obesity group the mean waist circumference of the males and females were 95.1 ± 7.2 cm and 9 4. 9 ± 8. 3 c m, r e s pe c ti ve l y, wh i c h w a s significantly different with the control group, where the mean waist circumference of the males was 71.5 ± 4.8 cm and that of the females 73.5 ± 5.4 cm (p=0.000). mean bmi in the central obesi ty group was 27.2 ± 3.4 kg/m2, being significantly different with the mean bmi of the control group of 20.9 ± 2.2 kg/m2 (p=0.000). mean systolic blood pressure in the central obesity group was 136.5 ± 21.3 mmhg, being significantly different with that of the control group of 114.1 ± 13.4 mmhg (p=0.000). daily dietary intake of vitamin d was not significantly different between the central obesity group and the control group. the demographic data of the study subjects can be seen in table 1. biochemical parameters lipid prof ile, i.e. the levels of total c h ol e s t e ro l , hd l c h ol e s t e r ol a n d l dl cholesterol was not significantly different between the central obesity group and the control group, except for the triglyceride level (p=0.027). mean triglyceride level in the control group was lower than in the central obesity group (95.6 ± 45.8 mg/dl vs 152.5 ± 77.7 mg/dl). the vitamin d level in the central obesity group was significantly lower compared with the control group (p=0.013). the most frequent vitamin d level in both groups was <20 ng/ml, the p e r c e n t a ge s be i n g 86 .2 % a nd 5 2. 9 % , respectively, in the central obesity group and the control group (p=0.000). the adiponectin level in the central obesity group was 4.7 ± 0.26 ng/ 209 ml, which was significantly lower compared with that in the control group of 6.8 ± 0.72 ng/ ml (p=0.003) (table 2). the present study found vitamin d deficiency in 111 subjects (69.4%) in both groups combined. adiponectin levels in vitamin d central obesity subgroups table 3 shows adiponectin levels in the vitamin d level subgroups of <20 ng/ml and 20 ng/ml. the higher the vitamin d level the univ med vol. 36 no.3 characteristic central obesity controls p value (n=80) (n=80) age (years) 59.1 ± 2.9 58.8 ± 2.5 0.684 gender [n (%)] female 31 (38.0) 42 (52.9) 0.259 male 49 (62.0) 38 (47.1) education [n (%)] did not attend school 6 (6.3) 0 (0) did not finish elementary school 11 (13.9) 14 (17.6) elementary school 22 (27.6) 24 (29.4) 0.708 junior high school 12 (15.2) 19 (23.5) senior high school 27 (34.2) 23(29.4) academy/tertiary education 1 (1.3) 0 (0) employment status [n (%)] employed 38 (46.8) 28 (35.3) non-employed 42 (53.2) 52 (64.7) 0.763 waist circumference (cm) 94.9 ± 7.8 72.4 ± 5.0 0.000* males 95.1 ± 7.2 71.5 ± 4.8 0.000* females 94.9 ± 8.2 73.5 ± 5.4 0.000* hip circumference (cm) 98.3 ± 7.5 88.0 ± 5.9 0.000* weight (kg) 65.3 ± 10.2 49.8 ± 6.2 0.000* height (cm) 154.8 ± 6.9 154.1± 8.6 0.731 body mass index (kg/m2) 27.2 ± 3.4 20.9 ± 2.2 0.000* systolic blood pressure (mmhg) 136.5 ± 21.3 114.1 ± 13.4 0.000* diastolic blood pressure (mmhg) 84.2 ± 10.7 70.0 ± 6.4 0.000* pulse rate (per minute) 77.0 ± 10.0 77.9 ±11.3 0.749 nutrient intake/day energy (kcal) 1866 ± 566.2 1675 ± 553.1 0.000* carbohydrate (g) 326.5 ± 101.7 275.6 ± 97.5 0.000* protein (g) 38.7 ± 18.5 38.3 ± 17.9 0.873 fat (g) 58.7 ± 33.4 54.6 ± 31.2 0.789 vitamin d (mg) 6.5 ± 2.4 6.6 ± 2.3 0.832 table 1. distribution of sociodemographic characteristics and clinical features in the two groups *p< 0.05 significance (independent t-test). data presented as mean ± sd, except for gender, education and employment table 2. biochemical parameters in the central obesity and control groups *p<0.05 significance (independent t-test). data presented as mean ± sd except for vitamin d status biochemical parameter central obesity control p value (n=80) (n=80) lipid profile total cholesterol (mg/dl) 202.3 ± 34.3 205.9 ± 41.6 0.776 triglycerides (mg/dl) 152.5 ± 77.7 95.6 ± 45.8 0.027* hdl cholesterol (mg/dl) 47.7 ± 8.9 53.3 ± 7.7 0.074 ldl cholesterol (mg/dl) 133.9 ± 31.6 142.8 ± 34.1 0.431 fasting glucose (mg/dl) 108.1 ± 55.3 95.5 ± 28.8 0.409 vitamin d (ng/ml) status [n,%)] 14.3 ± 6.6 19.2 ± 8.6 0.013* <20 ng/ml 69 (86.2) 42 (52.9) 0.000* 20 ng/ml 11 (13.8) 38 (47.1) adiponectin (ng/ml) 4.7 ± 0.26 6.8 ± 0.72 0.003* 210 pusparini, merijanti, lesmana, et al adiponectin 25-hydroxy vitamin d higher also the adiponectin level. this was seen in both the central obesity group and the control group. in the central obesity subgroup with vitamin d level of <20 ng/ml, the adiponectin levels were significantly lower than in the control group (p=0.001), whereas in subjects with vitamin d levels of 20 ng/ml there was no significant difference in adiponectin levels between the central obesity and control groups (p=0.538). discussion our study results showed a relatively high prevalence of vitamin d deficiency, i.e. 69.4% in all subjects involved in this study. vitamin d deficiency was 86.2% in the central obesity group and 52.9% in the control group. our study results showed significantly lower levels of vitamin d in subjects of the central obesity group compared with those in the controls. both groups showed a mean vitamin d level below 20 ng/ ml whic h den otes vita min d def ic ie ncy according to the criteria used by holick.(9) the prevalence of vitamin d deficiency of 69.4 % in the present study was similar to the study results of al-dabhani et al.(17) i.e. 64%. aldabhani et al.(17) conducted a study on 1205 subjects consisting of 702 females and 503 males, with mean age of 39.9 ± 10.9 years. the study of al-dabhani et al.(17) also found in subjects with the metabolic syndrome lower serum vitamin d levels compared with the nonmetabolic syndrome group.(17) the same was also reported by shafinaz et al.(18) in kuala lumpur, malaysia, who found that 67.4% of adult females with mean age of 48.5 ± 5.2 years had vitamin d levels below 20 ng/ml.(18) the study by zhen et al.(19) in china on subjects aged 40-75 years showed that 75.2% subjects had vitamin d levels of <20 ng/ml.(19) indonesia is country that is located at the equator where sunlight is nearly always available throughout the year, so that no vitamin d deficiency is expected to be found or at most at a very low prevalence compared with western countries where continuous sunlight is not always. regarding the habits associated with exposure to sunlight, the majority of subjects answered that they are exposed to sunlight in the morning (data not shown). the daily duration of exposure to sunlight was mostly 10-15 minutes for the central obesity group (33.7%) and >15 minutes for the control group (31.25%). in addition, it was also seen that the time of exposure to sunlight was mostly from 8 to 8.30 am for the central obesity group and from 7 to 7.30 am for the control group. this shows that the majority of subjects are still of the opinion that the best sunlight is in the morning, although the study of holick et al.(9) showed that exposure to sunlight from 10 am to 13 pm twice weekly for 5-30 minutes on the arms and legs was adequate for the daily requirement of vitamin d. the use of tightly covered clothing was found in only 22.5% of the central obesity group and in 25% of the control group. sunblocking agents were also not much used by the subjects of our study, so that pre su ma bly the vitamin d de ficiency was mostly due to inadequate exposure to sunlight. in the present study there was no significant difference in dietary vitamin d intake between the central obesity group and the control group and both groups tended to have a dietary intake of low vitamin d content, so that the most of the vitamin d was obtained from parameter adiponectin p value central obesity control n=80 n=80 25 (oh) d (ng/ml) <20 4.4 ± 0.23 7.1 ± 1.2 0.001* 20 5.8 ± 0.9 6.5 ± 0.9 0.538 table 3. adiponectin by 25(oh)d level and central obesity *p<0.05 significance (independent t-test); data presented as mean ± sd 211 univ med vol. 36 no.3 exposure to sunlight. food sources rich in vitamin d, such as salmon, tuna, and mackerel are not frequently consumed by indonesians. mean vitamin d levels of the subjects with central obesity was lower than those in the control group. this is in line with the results of the study by johnson et al.(20) who reported that subjects with obesity had low levels of vitamin d, particularly in the males compared with the females, with an odds ratio of 1.41 (95% ci: 1.171.70). other studies that showed low levels of vitamin d in subjects with obesity were among others the studies conducted by shafinaz et al.,(18) oommen et al.,(21) and tosunbayraktar et al.(22) in our study we found a mean adiponectin level of 4.7 ± 0.26 ng/ml in subjects with central obesity, which was significantly lower than the mean adiponectin level of 6.8 ± 0.72 ng/ml in the control group. this showed that subjects with central obesity had lower adiponectin levels compared with the controls. adiponectin is also known by the name of adipocyte complement related protein of 30 kda, which is secreted by adipose tissue. adiponectin may activate peroxisome proliferator-activated receptor α (ppar-α), stimulate oxidation of free lipids and decrease the triglyceride content of muscular tissue. in the liver, it is only the intact adiponectin molecule that activates adenosine mono phosphate kinase ( ampk ) that further reduc es the molecules involved in gluconeogenesis and increases the phosphorylation of acetyl coenzyme carboxylase (acc) and oxidation of fatty acids. adiponectin activates ppar-α then stimulates the oxidation of fatty acids and decreases the triglyceride content of the liver. all these changes increase the in vivo insulin sensitivity.(23) the triglyceride levels in the controls was significantly lower than those in the central obesity group. this shows that in the control group a low adiponectin level was associated with a high triglyceride level, and vice versa, a high level of adiponectin was associated with a low triglyceride level. adiponectin is one of several adipokines that are produced by mature adipocytes. adiponectin aids the uptake of glucose and oxidation of free fatty acids in muscle and inhibits gluconeogenesis in the liver, so increasing insulin sensitivity. the level of adiponectin is low in individuals with obesity.(24) the study of a relationship between vitamin d and adiponectin levels is due to several causes; first, vitamin d receptors are present on preadipocytes so that there is the possibility that 1,25 (oh) 2 d regulates the expression of the adiponectin gene.(25) second, 25 (oh) 2 d is a hormone that is thought to play a role in the regulation of tumor necrosis factor α (tnf-α) production. tnf-α is a factor that plays a important role in the synthesis of adiponectin.(6,11,26) our study results showed that in the control group the adiponectin levels were significantly higher compared with the central obesity group. in the vitamin d subgroups of <20 ng/ml and 20 ng/ml, the adiponectin levels in the control group were higher compared with the central obesity group. these results agree with those of a study by si et al.(27) on elderly aged above 65 years, showing that adiponectin levels were higher in the group of patients without the metabolic syndrome compared with patients with the metabolic syndrome. in the aforementioned study it was concluded that the most influential factors on adiponectin leve ls wer e bmi, waist circumference, waist to hip ratio, visceral fat, visceral fat ratio and 2 hour insulin levels.(28) the study results of wang et al.(28) who carried out a study on 3,193 subjects aged 50-70 years showed that adiponectin levels were negatively correlated in the subjects with the metabolic syndrome. a limitation of this study is the crosssectional study design, so that this study cannot clarify any cause-and-effect relationship. there is a need for an experimental study with subjects who receive adequate exposure to sunlight and do not use vitamin d supplementation, to determine the role of adiponectin in connection with the low levels of vitamin d in subjects with central obesity. the present study showed a prevalence of vitamin d def iciency and insufficiency in the study subjects that was higher than 61.6%. this indicates the need for education 212 pusparini, merijanti, lesmana, et al adiponectin 25-hydroxy vitamin d on the importance of exposure to sunlight, particularly in subjects with central obesity and on the increased awareness of the community about low levels of vitamin d in the majority of the present study subjects, although indonesia is a country with high exposure to sunlight. further investigations are needed to better understand the role of vitamin d in the occurrence of central obesity. conclusion the level of adiponectin in central obesity with 25(oh)d level of <20 ng/ml was lower compared with the control group in adults. early detection of 25(oh)d and adiponectin levels can prevent the increasing risk of cardiovascular disease. conflict of interest all authors have disclosed any actual or potential competing interests regarding the submitted article and the nature of those interests. acknowledgements we thank the participants in this study for their cooperation. this study was supported by the faculty of medicine, trisakti university, jakarta, indonesia contributors p and ltm contributed to the design of the study. p, ltm, m, and a contributed to collection of samples and data. p and ltm analyzed and interpreted the data and performed statistical analysis. p, ltm, m, and a contributed to drafting and revising the manuscript. all authors read and approved the final manuscript. references 1. james wp. the epidemiology of obesity: the size of the problem. j intern med 2008;263:336-52. 2. zhang p, wang r. gao c, et al. prevalence of central obesity among adults with normal bmi and its association with metabolic diseases in northeast china. plos one 2016;11:e0160402. doi: 10.1371/jounal.pone.0160402. 3. singla p, bardolol a, parkash aa. metabolic effects of obesity: a review. world j diab 2010;1: 76-88. 4. zhang j, kho p, kuliesh ov, et al. adiponectin, resistin and leptin : possible markers of metabolic syndrome. endocrinol metab syndr 2015;4:212. doi: 10.4172/2161-1017.1000212. 5. huang fy, del rio navarro be, perez-ontiveros ja, et al. effect of six month lifestyle intervention on adiponectin, resistin and soluble tumor necrosis factor–α receptors in obese adolescents. endocr j adv pub 2014. doi: 10.1507/endocrj. ej14-0157. 6. hamid mehmood ztn, papandreaou d. an updated mini review of vitamin d and obesity: adipogenesis and inflammation state. j med sci 2016;15:526-32. 7. jaramillo pl. the role of adiponectin in cardiometabolic diseases: effects of nutritional interventions. j nutr 2016;146:422s-6s. 8. bidulescu a, morris aa, stoyanova n, et al. association between vitamin d and adiponectin and its relationship with body mass index: the meta health study. front pub health 2014;2:193. doi: 10.3389/fpubh.2014.00193. 9. holick m. vitamin d deficiency. n engl j med 2007;357:266-81. 10. setiati s. pengaruh sinar ultraviolet b matahari terhadap konsentrasi vitamin d dan hormone parathyroid pada perempuan usia lanjut indonesia. j kes masy nas 2008;2:147-53. 11. green tj, skeaff cm, rockell jep, et al. vitamin d status and its association with parathyroid hormone concentration in women of child bearing age living in jakarta and kuala lumpur. ejcn 2008;62:373-8. 12. foong mm, bulgiba a. high prevalence of vitamin d insufficiency and its association with obesity and metabolic syndrome among malay adults in kuala lumpur, malaysia. bmc public health 2011;11:735. http://www.biomedcentral. com/1471-2458/11/735. 13. friedenreich cm, biel rk, lau dcw, et al. case control study of the metabolic syndrome and metabolic risk factors for endometrial cancer. epidemiol biomarkers prev 2011;20:2384-95. 14. seton m, jackson v, lesser ke, et al. low 25hydroxyvitamin d and osteopenia are prevalent in persons > or = 55 yr with fracture at any site: a prospective, obeservational study of persons 213 fracturing in the community. j clin densitom 2005; 8:454-60. 15. enko d, kriegshauser, stolba r, et al. method evaluation study of a new generation of vitamin d assays. biochem med 2015;25:203-12. 16. liu d, schuster t, baumann m, et al. comparison of immunoassays for the selective measurement of human high molecular weight adiponectin. clin chem 2009;55:568-72. 17. al-dabhani k, tsilidis kk, murphy n, et al. prevalence of vitamin d deficiency and association with metabolic syndrome in a qatari population. nutrition & diabetes 2017;7:e263. doi: 10.1038/nutd.2017.14. 18. shafinaz id, moy fm. vitamin d level and its association with adiposity among multi ethnic adults in kuala lumpur, malaysia: a cross sectional study. bmc public health 2016;16:232. doi: 10.1186/s12889-016-2924-1. 19. zhen dh, liu lj, guan ch, et al. high prevalence of vitamin d deficiency among middle-age and elderly individuals in northwestern china: its relationship to osteoporosis and lifestyle factors. j bone 2015;71. doi: 10.1016/j.bone.2014.09.024. 20. johnson lk, hofso d, aasheim et, et al. impact of gender on vitamin d deficiency in morbidly obese patients: a cross sectional study. eur j clin nutr 2012;66:83-90. 21. oommen a, al zahrani ih. association of obesity with vitamin d deficiency and the clinical implications. int j res med sci 2015;3:3262-5. 22. tosunbayraktar g, bas m, kut a, et al. low serum 25(oh)d levels are associated to higher bmi and metabolic syndrome parameter in adult subjects in turkey. afri health sci 2015;15:1161-9. 23. norman aw. mini review: vitamin d receptor new assignments for an already busy receptor. endocrinology 2006;147:5542-8. doi: 10.1210/ en.2006-0946. 24. liu e, meigs jb, pittas ag, et al. plasma 25hydroxyvitamin d is associated with markers of the insulin resistant phenotype in nondiabetic adults. j nutr 2009;139:329-34. 25. lee s, lee dk, choi e, et al. identification of functional vitamin d response element in the murrine insig-2 promoter and its potential role in the differentiation of 3t3-l1 preadipocytes. mol endocrinol 2005;19:399-408. 26. wimalawansa sj. vitamin d deficiency is a surrogate marker for visceral fat content, metabolic syndrome, type 2 diabetes, and future complications. j diabetes metab disord control 2016;3:00059. doi: 10.15406/jdmdc.2016.03.00059. 27. si ek, yea ek, kyong hy, et al. plasma adiponectin levels in elderly patients with prediabetes. endocrinol metab 2015;30:326-33. doi: 10.3803/ enm.2015.30.3.326. 28. wang j, li hx, franco oh, et al. adiponenctin and metabolic syndrome in the middle-aged and elderly chinese. obesity 2008;16:172-8. doi: 10.1038/oby2007.42. univ med vol. 36 no.3 knowledge of hiv-aids a dominant factor of antiretroviral therapeutic adherence in women with hiv-aids 129 *psychiatric department, faculty of medicine, atma jaya catholic university of indonesia **medical profession program, faculty of medicine, atma jaya catholic university of indonesia correspondence: dr. dr. surilena, spkj(k) psychiatric department, faculty of medicine, atma jaya catholic university of indonesia jl. pluit raya no. 2, jakarta 14440. email: surilenahasan@yahoo.co.id / surilena@atmajaya.ac.id phone: +62812-9457690 univ med 2015;34:129-37 doi: 10.18051/univmed.2016.v35.129-137 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2015may-august, 2015may-august, 2015may-august, 2015may-august, 2015 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 vol.34 no.2 background antiretroviral therapy adherence (art adherence) is a factor significantly extending life expectancy of people living with hiv/aids. the objective of this study was determine several factors on art adherence in women infected with hiv/aids. methods a cross-sectional study involving 99 women with hiv/aids who were infected through their sexual partner or spouse was conducted in dharmais hospital between march and august 2014. the instruments used were demographic and self-esteem questionnaires, hamilton rating scale for depression, hamilton rating scale for anxiety, knowledge, perception of art benefits and limitations, family support, peer support as well as assessment of art adherence. the knowledge questionnaire has been validated with cronbach’s alpha = 0.823. data were analyzed using chisquare test and multivariate logistic regression. results a total of 99 women with hiv/aids participated in the study, with an age range of 3060 years and mean age of 36 ± 3.72 years. a total of 57.58% of participants showed poor art adherence. multivariate logistic regression analysis showed that knowledge, art side effects, depression, peer support and arv availability significantly affected art adherence (p<0.05). the most dominant factor affecting art adherence was knowledge, with or = 64.02 (95% ci 4.99-670.12). conclusion with good knowledge about hiv/aids infection, art benefits, and possible arv side effects, women living with hiv/aids are expected to carry out art adherence according to the recommended rules. keywords: antiretroviral therapy, adherence, knowledge of hiv-aids, hiv/aids, women knowledge of hiv-aids a dominant factor of antiretroviral therapeutic adherence in women with hiv-aids surilena* and jean valeri** doi: http://dx.doi.org/10.18051/univmed.2015.v34.129-137 130 surilena, valeri hiv-aids antiretroviral therapeutic adherence pengetahuan tentang hiv/aids merupakan faktor dominan terhadap kepatuhan terapi antiretroviral pada perempuan terinfeksi hiv/aids latar belakang kepatuhan terapi antiretroviral (art) merupakan salah satu faktor yang dapat memperpanjang umur harapan hidup orang dengan hiv/aids secara bermakna. penelitian ini bertujuan untuk menentukan beberapa faktor yang berperan terhadap kepatuhan art pada perempuan yang terinfeksi hiv/aids. metode studi cross-sectional pada 99 perempuan yang terinfeksi hiv/aids dari pasangan seksual atau pasangan tetapnya, dan dilaksanakan di unit terpadu aids rs dharmais, antara bulan maret-agustus 2014. instrumen yang digunakan pada penelitian ini adalah kuesioner demografik, self-esteem questionnaire, hamilton rating scale for depression, hamilton rating scale for anxiety, pengetahuan, persepsi manfaat art, persepsi hambatan art, dukungan keluarga, dukungan teman sebaya dan kepatuhan art. kuesioner pengetahuan telah di validasi dengan nilai cronbach’s alpha sebesar 0,823. data di analisis dengan chi-square test dan regersi logistik ganda. hasil sebanyak 99 perempuan dengan hiv/aids berpartisipasi dalam studi dengan rentang usia 30-60 tahun dan rerata usia adalah 36 ± 3,72 tahun. sebanyak 57,58% responden menunjukkan kepatuhan art buruk. analisis regresi logistik ganda menunjukkan bahwa pengetahuan, efek samping art, depresi, dukungan sebaya dan ketersediaan arv memiliki hubungan yang signifikan terhadap kepatuhan art (p<0,05). faktor yang paling dominan yang mempengaruhi kepatuhan art adalah pengetahuan dengan nilai or = 64,02 (95% ci 4,99-670,12). kesimpulan pengetahuan yang baik tentang infeksi hiv/aids, manfaat art dan efek samping obat arv yang dapat terjadi, diharapkan perempuan yang mengalami infeksi hiv/aids mampu menjalankan kepatuhan art sesuai aturan yang dianjurkan. kata kunci: terapi antiretroviral, kepatuhan, pengetahuan tentang hiv/aids, hiv/aids, perempuan abstrak introduction the united nation global report on the aids epidemic showed that there are 1.7 million women in asia infected with hiv and 35% of them are infected from their sexual partner or their spouse. the number of women infected with hiv/aids is increasing each year along with the increase in the number of men doing unsafe sex, who eventually will transmit hiv/aids to their sexual partner or spouse.(1) the indonesian ministry of health reported that in 2010 there were 404.600 people with hiv/aids (among whom 42% women).(2) in 2014, the indonesian ministry of health reported that there were 64% men and 36% women (6,539 housewives) infected with hiv/aids.(3) increased hiv transmission to housewives can increase the risk o f t r a n s m i s s i o n t o t h e i r i n f a n t s , w h i c h consequently results in increased perinatal aids patients.(2,3) the psychosocial burden of housewives infected with hiv/aids is larger than their physical burden, ranging from changes in selfimage, transmission of hiv/aids to their husband and children and the guilt felt after the 131 univ med vol. 34 no.2 death of the husband or the children, becoming the bread winner after the death of the husband. the psychosocial burden of the women will extremely affect their survival and may cause emotional and mental health problems, which may lead to mental emotional disorder and poor medication adherence.(4) a study demonstrated that 45% of people living with hiv/aids who experienced depression had poor medication adherence compared to 25% of those who did not. depressive disorders can increase risky behaviors that facilitate hiv transmission to others, because dormant hiv can become active again and depression accelerates the rate of hiv disease.(5) the indonesian national aids commission in 2011 reported that the program of hiv/aids care and treatment in indonesia is still not effective. the commission estimated that the number of people living with hiv in indonesia was around 300,000, yet those who received art were only 30,000 people and about 200,000 people were thought to be infected with hiv/ aids but had not been diagnosed, since they had not undergone hiv tests.(6) in 2012, the indonesian ministry of health reported that a decrease in art adherence was associated with increased morbidity and hospital admissions as well as increased costs of care. poor adherence to antiretroviral therapy is one of the most important factors that prevent successful hiv/ aids treatment.(3,6) antiretroviral therapy must be continued for life and adherence is necessary. a good level of adherence of > 95% is required to obtain successful antiretrovital therapy. a high adherence level is quite difficult to maintain after long term treatment.(7) there are some factors affecting art adherence, including internal and external factors. internal factors include age, educational level, survival motivation, perception about hiv/aids and art, depression, anxiety, beliefs, self-image, self-esteem, self-efficacy, and presenting opportunistic infections, while external factors are level of knowledge, family economic status, support from the case manager, medical team, society, and family, arv medication availability, access to art health c a r e , a rt s i d e e ff e c t s , s t i g m a a n d discrimination.(6,7) our study aimed to determine factors affecting art adherence such as knowledge, depression, anxiety, perception about art benefits and limitations, self-esteem, family support, peer support, art side effects, and arv availability, in women infected from their sexual partner or spouse. methods research design a cross-sectional study was conducted on women infected with hiv/aids who visited the outpatient clinic of aids comprehensive diagnostic unit at dharmais hospital between march and august 2014. research subjects the inclusion criteria were women infected with hiv/aids through their partner/spouse, aged 30-60 years, literate in the indonesia langguage or able to read and write, and without history of substance abuse/alcohol dependency or abuse, and had ongoing arv therapy. the exclusion criteria were women with psychosis, and those who refused to sign informed consent. the sampling technique was by consecutive nonrandom sampling; with an anticipated 10% drop out, the number of respondents was 99 women infected with hiv/aids.(8) measurements the respondent filled in demographic and self-esteem questionnaires,(9) the latter consisting of 10 questions with four possible answers, with a score of >15 for high self-esteem and a score of <15 for low self-esteem. the hamilton rating scale for depression/hdrs (10) consists of 17 questions, with a score of < 7 indicating negative depression, and a score of > 7 indicating positive depression. the hamilton rating scale for anxiety/hars (11) consists of 14 questions, a score of 0-13 indicating negative anxiety and a 132 surilena, valeri hiv-aids antiretroviral therapeutic adherence score of >14 positive anxiety. the peer support test (12) consists of 10 questions, with a score of >12 for positive peer support, and a score of <12 for negative peer support. all instruments in this study have been previously validated, including the knowledge questionnaire with cronbach’s alpha of 0.823 and the art adherence questionnaires (self-report), with a sensitivity of 94.4%, specificity 88.9%, and a cutoff value of 95 (>95% good art adherence and <95% low adherence).(13) the knowledge questionnaire consists of 23 questions with three possible answers, a total score of <29 indicating poor knowledge, a score of >30-45 indicating moderate knowledge, and a score of >46 indicating good knowledge.(13) statistical analysis in this study, bivariate analysis was performed using chi square test and multivariate analysis using stepwise logistic regression. statistical analysis was done using spss software program version 17.0. ethical clearance this research fulfilled the international ethical guidelines for ethical review in the faculty of medicine, atma jaya catholic university. results in this study, there were 99 women who were infected with hiv/aids through their partner/spouse, with age range of 30-60 years and mean age of 36.03 ± 3.72 years. in this study there were no respondents who dropped out. the m a j o r i t y o f s u b j e c t s h a d t h e f o l l o w i n g characteristics: high school graduates (44.4%), married (98.7%), housewives (54.5%), medium income (57.6%), and more than one child (82.8%). in the study, art adherence was measured by the self-report. there were 57 (57.6%) subjects with poor art adherence (<95%) (table 1). table 1. socio-demographic characteristics and art adherence art : anti retroviral therapy c h i s q u a r e a n a l y s i s s h o w e d t h a t knowledge, perception of art benefits and side effects, self-esteem, depression, peer support, art availability, have a significant effect on art adherence (p<0.05). chi-square analysis also showed that perception of art limitations, anxiety, and access to art did not have a significant effect on art adherence (p >0.05) (table 2). this study showed that 60.6% of the subjects had moderate knowledge about the category of hiv/aids infection, art benefits and side effects, with 56.7% of subjects having 133 univ med vol. 34 no.2 low art adherence. a total of 32.4% of the subjects claimed not to know the timeline of progression of hiv infection to aids if not given treatment, 54% lacked understanding of antiretroviral drug resistance, and 38% did not know the side effects of antiretroviral drugs and the duration of antiretroviral therapy that has to be followed (table 2). this study showed that subjects with poor perception of the benefits of art had poor art adherence (57.6%) . hence, 19% of the subjects stated to lack confidence that art therapy is beneficial for the disease and would not reduce the risk of opportunistic infections (tuberculosis, herpes, and others). most subject perceptions regarding art limitations (55.6%) were in the moderate category and 58.7% of subjects had poor art adherence. there were 51.2% of subjects who reported being bored by routine therapy, 41% table 2. association between factors affecting art adherence (n=99) *p < 0.05; art : anti retroviral therapy found it difficult to stop art implementation and 39% of subjects were afraid of becoming unemployed because they were often absent from work to undergo art (table 2). this study showed that of the subjects who experienced a rt s i d e e ff e c t s , 8 2 . 8 % h a d p o o r a rt adherence. in this study, 64.7% of subjects had a high score for the category of availability of antiretroviral drugs and 43.7% had low art adherence. regarding access to art, the majority of subjects (46.5%) had a poor access to art and 86.9% had low art adherence. the subjects claimed that arv drugs are given free by the indonesian government, but that hospital administration, other drugs, and laboratory tests are quite expensive, the cost of which they have to pay themselves. the subjects also had to spend quite expensive transportation costs of around rp 35,000-40,000 per hospital visit (table 2). 134 surilena, valeri hiv-aids antiretroviral therapeutic adherence the subjects with low self esteem and poor art adherence accounted for 71.2%. subjects with anxiety disorders (62.6%) tended to have poor art adherence, and 77.1% of subjects had depression and poor art adherence. (table 2). in this study, the majority of subjects (71.7%) did not have peer support. poor adherence is more prevalent in subjects without peer support (66.2%) than in subjects with peer support (35.7%). the subjects claimed that they were not sure where to go to get the support of their peers and that they needed the support of other friends who are infected with hiv/aids or who are not infected, of people in the neighborhood, to share information about hiv/aids and its treatment and support each other, to remind them to take antiretroviral drugs regularly and not do it furtively, to provide loans in case of a shortage of antiretroviral drugs, so that they may optimistically face their hiv/aids and be motivated on a regular basis, especially with regard to antiretroviral treatment (table 2). multiple logistic regression analysis showed that there were five significant variables (p<0.05). these variables are good knowledge (or=64.02; 95% c.i. 4.998-670.120), side effects to antiretroviral therapy (or=22.06; 95% c.i. 1.456-83.275), depression (or= 57.07 ( 9 5 % c . i . 1 . 0 7 7 1 2 . 3 4 5 ) , p e e r s u p p o r t (or=46.01; 95% c.i. 1.098-13.768), and availability of antiretroviral drugs (or= 12.18; 95% ci, 1.890-68.210) (table 3). table 3. multivariate analysis assessing the association of several variables and art adherence discussion there were 99 subjects analyzed in this study, i.e. all women who were infected with hiv/aids from their regular sexual partner/ spouse and had no history of drug/substance abuse. in indonesia, it is predicted that the number of women infected with hiv/aids will be increasing. the indonesian ministry of health reported in 2012, that the number of housewives infected with hiv will increase each year, while the number of commercial sex workers infected with hiv is declining. it is assumed that this results from transmitted hiv infection from their partner/spouse who have high-risk behavior.(3) antiretroviral therapy must be continued life long and adherence is necessary.(7) this study showed that 57.6% of subjects had poor art adherence (<95%) and approximately 56% of subjects had a history of often receiving antiretroviral drugs not on schedule.(12) research in 2013 on 55 outpatients with hiv/aids at dr m djamil hospital in padang showed that 25.5% subjects had low art adherence (<95%).(14) the indonesian ministry of health reported that in 2014, art adherence in women who are infected with hiv/aids in indonesia is still low. they often receive the antiretroviral drugs late or forget to take them on a regular basis, because they have to care for their children or the sick husband, to become the backbone of the family, to hide their hiv/ aids infection status from 135 univ med vol. 34 no.2 their children or other family members so that they have to take the medications furtively, and there are some hospital staff who behave less friendly to them when they come for health services.(15) this study tested the most powerful factor in influencing adherence to art. the findings in this study proved that the variable knowledge about hiv/aids and art is the most powerful f a c t o r i n i n f l u e n c i n g a rt a d h e r e n c e . multivariate statistical analysis with backward stepwise method showed a value of or=64.02 (ci 95%: 4.998-670.120) on knowledge in the good category. subjects who had knowledge about hiv/aids and whose art adherence was good, tended to be 64 times more likely to have high art adherence, compared with subjects with poor knowledge. the results of this study are in accordance with those the study of martoni et al.(14) which showed that the most dominant factor was knowledge associated with art adherence in people infected with hiv/aids. people infected with hiv/aids with good knowledge about the infection rate of hiv/aids and its treatment have a 9 times greater probability to reach good art adherence. risha’s (16) study showed that a good knowledge about the disease and its treatment can provide support and motivation to carry out the treatment and taking medication. women who are infected with hiv/aids and who have a good knowledge about hiv/aids and antiretroviral therapy will tend to have a good degree of art adherence. they can also change their behavior to control disease conditions and adhere to treatment. multivariate statistical analysis with backward stepwise method showed subjects without depression have a 57 times higher tendency to have high art adherence compared with subjects with depression. these results were similar to those of jenna (17) who reported a significant correlation between depression and arv non-adherence. depression affected patient’s self-care and contributed to degradation of physical and mental health, causing someone to feel helpless, unworthy, hopeless, and to have a lesser degree of motivation to survive and to achieve therapy adherence, which may lead to decline of the immune system and exaggeration of hiv/aids.(18) mazzafero and murray states that depression may be a predictor of poor art adherence, increased sexual risk behavior, failure of antiretroviral treatment, hiv progression, and high mortality. people infected with hiv/aids who are depressed are almost twice as likely to have problems in adherence to antiretroviral therapy (art) than those who do not experience depression.(19) research by mary et al.(20) showed that women infected with hiv/aids with chronic depressive symptoms have twice the risk of death than women without depression. health professionals should maintain early detection of emotional mental disorders in women infected with hiv/aids in order for the disease to receive serious treatment and not to have a wide impact on the patients’ health and life. on multivariate statistical analysis with backward stepwise method it was found that subjects with peer support have a 46 times higher tendency to achieve high adherence compared with subjects without peer support. the role of peer support is very essential, i.e. helping patients living with hiv/aids to ease the loneliness and to deal with the problems, helping to build selfesteem and confidence, sharing resources, ideas and information (about benefits of arv therapy, updated treatment, local health support, etc.) as well as affecting adherence to art treatment.(21) another study reported that a peer support group, which is a group of two or more people infected with hiv/aids or those who have been directly affected, is significantly correlated with higher art adherence.(22) multivariate statistical analysis with backward stepwise method showed that in subjects without experienced art side effects, the probability of implementing art adherence is 22.06 times higher than in subjects with experienced art side effects. when people with hiv/aids at the start of therapy experience side effects and do not have education or good knowledge about the benefits and side effects of 136 surilena, valeri hiv-aids antiretroviral therapeutic adherence art, they do not know how to overcome the side effects and do not have the skills to cope with the side effects, and may possibly feel reluctant to implement therapy in accordance with the rules or may even decide to quit the treatment, which will increase the numbers of those lost to follow-up or stopping therapy.(23) multivariate statistical analysis with backward stepwise method showed that subjects who were in the high category of arv drugs availability were 12 times more likely to implement high art adherence. the indonesian ministry of health reported that the provision of antiretroviral drugs cannot be done at every desired location, since because antiretroviral drugs are expensive, their distribution should be regulated through locations that have been determined to be easily controlled. drug policies to provide free antiretroviral drugs proved to have a substantial impact in reducing morbidity, mortality and increasing the quality of life of people living with hiv.(3,7) health care systems that are difficult to access, expensive health financing, muddled bureaucracy, and the availability of antiretroviral drugs in only a few hospitals as determined by the government could lead to patients having difficulty accessing antiretroviral therapy programs and may have an impact on poor art adherence.(24) in women infected with hiv/aids who have a good knowledge about hiv/aids, the benefits of art adherence, art side effects, and duration of antiretroviral treatment, it will be easier to change their perception and behavior towards hiv/aids and its treatment so as to prevent the occurrence of mental disorders and to achieve good art adherence. therefore, they need psycho-education and counseling on a regular basis to improve their knowledge and help overcome the psychological problems that occur in women infected with hiv/aids. thus the global vision agreed upon by various countries in the world in the management and treatment of hiv / aids, which was to “achieve zero” (getting to zero) in 2015, comprising zero new hiv/aids infections, zero discrimination, a n d z e r o a i d s r e l a t e d d e a t h s , c a n b e achieved.(6,16) this study has limitations in that some respondents were not confident and were ashamed to be invited to communicate or when asked to fill out questionnaires. to address this, the researchers accompanied the respondents to fill out questionnaires and to be interviewed by nurses from the local hospital. another limitation was the time required (around 35 minutes) for filling out the questionnaires and interviewing each respondent. conflict of interest the authors report no conflict of interest. conclusions the main factors affecting antiretroviral therapeutic adherence in women infected with hiv/aids in dharmais hospital was the patient’s knowledge about therapy. strict adherence to antiretroviral therapy is key to sustained hiv suppression, reduced risk of drug resistance, improved overall health, quality of life, and survival, as well as decreased risk of hiv transmission. acknowledgement we thank all women who were infected with hiv/aids through their partner/husband and received antiretroviral therapy in dharmais hospital, who were willing to participate in this research and all health care staff of dharmais hospital who have helped in this research. references 1. joint united nations programme on hiv and aids (unaids). global aids response country progress report. reporting period january 2010 to december 2011. geneva: unaids;2012. 2. departemen kesehatan r.i. direktorat jenderal pemberantasan penyakit menular dan penyehatan lingkungan langsung. pedoman nasional tata laksana klinis infeksi hiv dan 137 terapi antiretroviral pada orang dewasa. jakarta: departemen kesehatan r.i. direktorat jenderal pemberantasan penyakit menular dan penyehatan lingkungan langsung;2010. 3. departemen kesehatan r.i. direktorat jenderal pemberantasan penyakit menular dan penyehatan lingkungan langsung. statistik kasus hiv/aids di indonesia dilaporkan sampai dengan desember 2014. jakarta: departemen kesehatan r.i. direktorat jenderal pemberantasan penyakit menular dan penyehatan lingkungan langsung;2015. 4. bor r, du plessis p. the impact of hiv/aids on families: an overview of recent research. am j psychology 2011;15:413-27. 5. penzack sr, redy ys, grimsely sr. depression in patients with hiv/aids infection. am j health-syst pharm 2010;57:376-401. 6. world health organization, unaids & komisi penanggulangan aids nasional. odha dan akses pelayanan kesehatan dasar di indonesia. 2010-2014. available at: http://www.spiritia.or.id /dokumen/odha-akses.pdf. accessed december 9, 2014. 7. filho lf, nogueira sa, machado es, et al. factors associated with lack of antiretroviral adherence among adolescents in a reference centre in rio de janeiro, brazil. jias 2009;19: 685-8. 8. valeri j, surilena, yanto b, et al. family support is not a risk factor of self-esteem in hiv/aids women. univ med 2015;34:61-7. 9. della. cognitive behavior therapy untuk meningkatkan self-esteem pada mahasiswa universitas indonesia yang mengalami distres psikologis. [tesis]. jakarta: fakultas psikologi, universitas indonesia;2012. 10. hamilton m. development of a rating scale for primary depressive illness. am j psychiatry 2009;8:233-9. 11. sumanto r, marsito. hubungan tingkat nyeri dan tingkat kecemasan pada pasien post operasi section caesarea di rsu gombong. j ilmiah kesehatan keperawatan 2011;7:83-90. 12. leserman j, barroso j, pence bw, et al. trauma, stressful life events and depression predict hivrelated fatigue. aids care 2008;20:1258-65. doi: 10.1080/09540120801919410. 13. surilena, irawati r, irwanto, et al. the effect of rational emotive behaviour therapy (rebt) on antiretroviral therapeutic adherence and mental health in women infected with hiv/aids. acta med indones 2014;4:283-91. 14. martoni w, helmi a, raveinal. faktor-faktor yang mempengaruhi kepatuhan pasien hiv/ aids di poliklinik khusus rawat jalan bagian penyakit dalam rsup dr. m. djamil padang periode desember 2011maret 2012. j farmasi andalas 2013;1:48-52. 15. djauzi s. arv for adult; implementation in primary health care. aids 2014;29:111-6. 16. risha. faktor-faktor yang mempengaruhi tingkat kepatuhan pengobatan arv pada odha di rumah sakit umum daerah tugu rejo dan rumah sakit umum panti wilasa citarum semarang. j penelitian 2011;4:122-7. 17. jenna m. depression greatly impairs treatment adherence. jias 2010;7:321-42. 18. folkman. stress and coping caregiving partners of women with aids. psychiatr clin north america 2006;17:35-52. 19. mazzafero ke, murray pz. depression, stress, and social support as predictors of hiv/aids in young women. j adolesc health 2009;39:337-44. 20. rotheram-borus mj, stein ja, jiraphongsa c, et al. benefits of family and social relationships for thai parents living with hiv. prev sci 2010; 11:298-307. 21. kremer h, ironson g, porr m. factors contributing to arv non adherence among plwhaa attending ctc at ligula hospital in mtwara region tanzania. aids 2009;24:103135. 22. filho lf, nogueira sa, machado es, et al. factors associated with lack of antiretroviral adherence among adolescents in a reference centre in rio de janeiro, brazil. int j std aids 2008;19:685-8. doi: 10.1258/ijsa.2008.008017. 23. tuldra a, fumaz cr, ferret m1j. prospective randomized two-arm controlled study to determine the efficacy of a specific intervention to improve long-term adherence to highly active antiretroviral therapy. jias 2011;25:221-8. 24. gifford al, bormann je, shively mj, et al. predictors of self-reported adherence and plasma hiv concentrations in patients on multidrug antiretroviral regimens. jias 2012;23:386-95. univ med vol. 34 no.2 rita k 179 *department of community medicine, medical faculty trisakti university correspondence adr. rina k. kusumaratna, m.kes. department of community medicine, medical faculty trisakti university jl. kyai tapa no.260 grogol jakarta barat phone: 021-5672731 ex. 2504 email : rkusumaratna@yahoo.com universa medicina 2007; 26: 179-85 universa medicina oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 dietary zinc intake and zinc status differences between male and female elderly of south jakarta community rina k. kusumaratna*a, oktavianus ch.salim*, and novia i. sudharma* abstrak introduction the elderly have a greater risk of zinc deficiency compared to younger adults. this condition may be reflected by a lowered zinc intake and reduced zinc absorption in the elderly. the aim of the study was to explore the dietary zinc intake and zinc status differences between male and female elderly. methods a cross-sectional study was conducted included eighty-nine free-living subjects, aged above 60 years, apparently healthy and ambulatory. a two day and non consecutive diet record was used to assess energy and nutrient intake of the elderly. it was combined with a semi-quantitative food frequency questionnaire (sq-ffq), with food model that was also used to quantify the food pattern on each food frequency item. serum zinc concentrations were measured by using atomic absorption spectrum photometry. results the total energy intake and normal serum zinc concentration in both genders of free-living elderly were mostly below the recommended dietary allowance. the mean serum zinc concentration did not differ significantly between female (13.7 µmo/l) and male elderly (13.9 µmo/l). mostly the intake of zinc was below two thirds of the rda it presented on intake of zinc in males was much less compared to female. overall, the prevalence of zinc deficiency appeared low among the free-living elderly in south jakarta. conclusions the prevalence of zinc deficiency was relatively low in healthy elderly. prevalence of zinc deficiency and zinc intake were lower in female compared to male elderly. keywords: zinc, intakes, free-living, elderly 180 kusumaratna , salim, sudharma zinc intake and zinc status in elderly *bagian ilmu kedokteran komunitas fakultas kedokteran universitas trisakti korespondensi adr. rina k.kusumaratna, m.kes. bagian ilmu kedokteran komunitas, fakultas kedokteran universitas trisakti jl. kyai tapa no.260 grogol jakarta barat 11410 telp. 021-5672731 eks.2504 email: rkusumaratna@yahoo.com universa medicina 2007; 26: 179-85. latar belakang risiko terjadinya defisiensi seng pada lanjut usia (lansia) lebih besar dibandingkan usia dewasa muda. keadaan ini direfleksikan oleh rendahnya asupan dan absorpsi seng pada lansia. penelitian ini bertujuan untuk membedakan asupan dan status seng antara lansia laki-laki dan perempuan. metode sebanyak 89 lansia berusia 60 tahun ke atas berbadan sehat dan mampu melakukan aktifitas secara mandiri diikut sertakan pada studi yang menggunakan rancangan potong silang (cross-sectional). asupan energi total dan kadar seng diukur menggunakan catatan diet selama dua hari berturut-turut. pengukuran ini dikombinasi dengan semi-quantitative food frequency questionnaire (sq-ffq) dan model makanan. kadar seng dalam serum diukur menggunakan atomic absorption spectrum photometry. hasil rata-rata kadar seng dalam serum pada lansia perempuan (13,7 µmo/l) tidak berbeda bermakna dengan lansia laki-laki (13,9 µmo/l). asupan energi total dan kadar seng dalam serum lebih rendah dibandingkan dari jumlah yang dianjurkan baik pada lansia laki-laki maupun perempuan. sebagian besar asupan seng besarnya duapertiga lebih rendah dari angka kecukupan gizi (akg), asupan seng pada lansia laki-laki lebih rendah dibandingkan lansia wanita. secara keseluruhan prevalensi defisiensi seng di antara komunitas lansia di jakarta selatan adalah rendah. kesimpulan studi ini menunjukkkan prevalensi defisiensi seng yang relatif rendah. asupan dan status seng lebih rendah pada lansia perempuan dibandingkan lansia laki-laki. kata kunci : seng, asupan, komunitas, lanjut usia perbedaan asupan dan status seng antara laki-laki dan perempuan lanjut usia di komunitas jakarta selatan rina k. kusumaratna*a, oktavianus ch.salim*, dan novia i. sudharma* abstract introduction in general, the elderly due to inadequate m i c r o n u t r i e n t i n t a k e a r e s u s c e p t i b l e t o physical inactivity, infection, and chronic diseases. inadequate nutrition intake will have an impact on an individual’s health status. nutritional disorder might also develop not just because of inadequate intake, but also due to the body’s inability to utilize nutrients; even though, the individual consumes appropriate a m o u n t s a n d a v a r i e t y o f f o o d s . g e n d e r differences of choice in nutrient intake among the elderly could indicate differences in values o f h e m a t o l o g i c a l a n d c l i n i c a l a n a l y s i s . micronutrient deficiency such as iron, vitamin 181 a and iodine are wellestablished in developing countries. presently, zinc deficiency has also been recognized by a number of experts as an important public health issue. clinical zinc deficiency was first described in early 1960s, and since then it has been recognized as a c o m m o n d e f i c i e n c y i n h u m a n s . ( 1 , 2 ) z i n c deficiency has been observed in animal and human studies that could decrease resistance to infectious diseases. zinc as one of the essential trace element has recently been of i n t e r e s t b e c a u s e o f i t s f u n c t i o n i n t h e maintenance of human health and nutrition statuses. elderly individuals are at greater risk of zinc depletion than younger adults that might be reflected in lower zinc intake and reduced zinc absorption due to aging. zinc deficiency has also been reported in the european zenith study that showed zinc deficiencies in healthy, free-living, late middle-age and older persons, even with a low prevalence.(3) study in central japan also found zinc deficiency in free-living of elderly.(4) major sources of zinc are animal based products that generally are consumed in small amounts; moreover, the asian diet which is based on whole-grain cereals and other plant foods is less bio-available due to its relatively high content of phytate, a compound that inhibits zinc absorption. the elderly who avoid flesh foods due to some reason, might be at risk of poor zinc status because of its reduced bioavailability in the daily diet. vitamin and mineral deficiency (vmds) as stated in a united nation report, includes deficiency of vitamin a, iron, iodine, zinc, folic acid that have affected over 2 billion people in the world. this condition is mostly caused by diets deficient in vitamins and minerals that the body needs, and become worse by losses or poor absorption related to illness. these situations are found in every part of the world, and the global databases report has estimated that 20% of the world’s population is at risk of zinc deficiency.(5) zinc supplementation could be a low cost strategy in improving physiological and cognitive functions in the elderly. the aim of this study was to compare the intake and status of zinc between male and female elderly, and whether there is a difference in dietary intake based on gender. method research design a cross-sectional study was conducted in a sub-district health center in south jakarta. subjects using demographic information system t h a t c o n t a i n e d t h e h e a l t h c e n t e r p a t i e n t database, potentially eligible elderly were identified and invited to participate in the study. the participants were residents living in the d i s t r i c t a n d t h e i r m a i n o c c u p a t i o n w a s pensioner or retired government employee or house wife. the number and selection of the elderly was determined by using a simple random sampling on a finite population. three hundred persons aged 60 years and older was gathered by cluster random sampling from 7 villages health center which was under the jurisdiction of mampang prapatan district health center. these were later screened, and finally only one-hundred and fifty elderly were assessed on their dietary intake and fasting blood sample. the eligibility of subjects in the study was gender both male and female, aged 60 years above, ambulatory and apparently h e a l t h y d u r i n g e x a m i n a t i o n . e a c h o f t h e p a r t i c i p a n t s w a s r e q u e s t e d t o f i l l o u t a n informed consent form to ensure that they agreed to fully participate in the study. data collection was conducted from november 2005 to february 2006. however, from the 150 elderly who were eligible for the study only eighty-nine subjects had complete records that universa medicina vol. 26 no.4 182 kusumaratna , salim, sudharma zinc intake and zinc status in elderly could be matched between their biological analysis sample and the dietary assessment. biological measurements all blood samples were collected after overnight fasting. for zinc analysis, samples were collected using serum of blood in a tube of zinc-free nitric acid, and diluted with zinc-free water. samples were centrifuged at a maximum speed of 15,000 rpm for 5 minutes. serum zinc w e r e d e t e r m i n e d b y a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( a a s ) w i t h z e e m a n background corrected (gbc 933 aa, with ë 213, 9 nm). dietary intake a two day and non consecutive diet record was used to assess energy and nutrient intake of the elderly. it was combined with a semiquantitative food frequency questionnaire (sqffq), with food model that was also used to quantify the food pattern on each food frequency item. the calculated nutrient intake of the sample was compared with the “indonesian recommended dietary allowance” (indonesian rda) for the particular age and gender group, (female 1,850 kcal and male 2,200 kcal). the amount of various food consumption reported was recorded then converted into energy and nutrient intakes per day. values less than two thirds of the recommended intake are considered as a risk of deficiency of the respective nutrients. the rda of zinc intake was based on recommended rdas for zinc for southeast asia.(6) statistical analysis data were reported as mean, standard deviation and frequency distribution. food c o n s u m p t i o n a n a l y s i s u s e d n u t r i s u r v e y calculation. all statistical analysis was done with spss windows version 11.5. normal distribution of data was checked using the kolmogorovsmirnov test. independent-t test was used to compare the zinc intake and concentration between male and female elderly. ethical clearance study protocol was reviewed and sanctioned by the medical faculty ethics commission. results the result of one-sample kolmogorov smirnov test presented that the distribution of the d a t a w a s n o r m a l . t h e s a m p l e s w e r e predominately females than males (69.7% vs 30.3%) and 85.4% were mostly 60–70 years in both gender. the total energy intake was mostly below of the recommended dietary allowance based on the indonesian rda in both gender, 57.7% females and 48.7% males respectively. the intakes of zinc between females and males were inadequate compared to the rda. male was twice more likely than female categorized in the less than two thirds of the rda. there were similar findings on zinc sources intake between dietary recall and semi-quantitative food frequency (sq ffq) (table 1). no significantly difference between energy and zinc intake and gender. note : zinc rda/akg (mg/day) a=4.4 ; b=6.5 females (n = 62) males (n energy (kcal/d) 1068.98 ± 313 1072.08 zinc (mg/d) 4.11 ± 1.8 3.9 ± <2/3 kg (%) 25.8%a 70% table 1. mean daily energy and zinc intakes of free-living elderly by gender (mean ± s.d.) 183 the mean serum zinc concentration did not differ significantly between female (13.7 µmo/ l) and male elderly (13.9 µmo/l). the percentage of subjects based on gender that showed a serum zn concentration below 10.7 µmol/l, which is considered as the cut-off level for zinc deficiency was 3.2% in females and 7.4% in males. there were more male subjects that had lower serum zinc concentration compare to female, almost double, with an average of 4.5% for all subjects. (table 2) discussion zinc is an essential trace element for human and all forms of life, thus in the elderly zinc has an essential function in immunity, age related changes of bone mass, cognitive functions and oxidative stress, and a leading cause of blindness in people over the age of 65 years. one function of zinc as a micronutrient is as antioxidant that has the capability of protecting cells from the damaging effects of free radicals released in the body. other studies of human subjects have reported that zinc deficiency in individuals has declined the sensitivity of taste bud and could be restored by zinc supplementation.(4,7-9) this study of free living elderly showed that most of the subjects had normal serum zinc concentration. however, the mean daily intake of zinc sources was mostly below two thirds of the rda for older people above 60 years, namely 37% females and 74% males. based on the result as shown in table 1, it could be reported that free-living elderly dominantly had less zinc intake. this was due to the source of zinc intake mainly from the daily food diet, there was no i n d i c a t i o n o f a n y o t h e r a d d i t i o n a l z i n c supplements being used. the same results were shown in a study conducted by sibai(11) and ervin rb. (12) however, the present study showed different results compared to the study in japan b y k o g i r i m a e t a l , ( 4 ) t h e z e n i t h s t u d y b y andriollo sanchez m et al,(3) galan p, et al(13) and paik hy et al.(14) in the last study, the intake of zinc of the subjects was adequate. zinc intake of females was higher compared to males (4.11 ± 1.8 mg/d vs 3.9 ± 1.7 mg/d ). this data could reflect the specific differences in dietary habits. from dietary assessment, female elderly consumed more animal protein as a source of zinc than males (33.38 ± 15.3 mg/day vs 28.86 ± 12.8 mg/day). i n t h e p r e s e n t s t u d y, s e r u m z i n c concentration in the female and male elderly were 13.67 ± 2.3 µmol/l vs 13.98 ± 2.6 µmol/l, respectively. this result was almost similar to o t h e r s t u d i e s t h a t h a v e b e e n c o n d u c t e d earlier.(3,14,16,17) those results did not reflect the w i d e r a n g e o f d i f f e r e n c e s i n s e r u m z i n c concentration values between the genders. percentage of elderly that were below the cut-off level for zinc deficiency showed that males were twice more lower than females. is the risk of zinc deficiency in males much higher than females? to explore this finding further, a study should be done to prove this tendency based on the baseline data of the present study on assessing zinc status of the healthy free-living elderly. the result of this study also showed that there was a low prevalence of zinc deficiency universa medicina vol. 26 no.4 table 2. zinc status in free-living elderly by gender (mean ± s.d.) females (n = 62) serum zn (µ mol/l) 13.7 ± 2.3 <10.7 µ mol/l (%) in serum 3.2% 184 kusumaratna , salim, sudharma zinc intake and zinc status in elderly in the free-living elderly. the same result was shown in the zenith study, that showed a low prevalence of zinc deficiency but differed in the japanese study,(2) that reported a high p r e v a l e n c e o f z i n c d e f i c i e n c y a m o n g t h e japanese aged, and the de jong et al(17) study in new zealand also reported that twelve percent among of new zealand elderly women had zinc deficiency. the present data showed contradictive findings with the first study that was conducted in five municipalities in jakarta that assessed the zinc concentration of freeliving elderly. the study reported that 67% of f r e e l i v i n g e l d e r l y i n j a k a r t a h a d z i n c deficiency. i n o u r s a m p l e s , t h e r e w e r e a l s o contradictive findings between the results of zinc intake and corresponding zinc concentration. majority of the elderly were below 2/3 of rdas zinc intake, but had above the cutoff level of zinc concentration. there could be a bias in assessing the food intake of the elderly, such as memory bias. because all the subjects were above 60 years aged, then they had to memorize what they had been eating in a day and the next day. for the elderly to memorize two days and non consecutively on what they had eaten could have created difficulties for the elderly. thus, the differences of the findings could be due to lack of memory on the part of the subjects. conclusion the findings of the study in free-living e l d e r l y i n s o u t h j a k a r t a s h o w e d a l o w prevalence of zinc deficiency but had similar results with their corresponding zinc intake daily. for further study, assessing of dietary intake should use food weighing namely what the subject actually ate daily to reduce errors caused by memory bias. dietary assessment instruments used must also accurately capture and reflect elderly dietary intakes. acknowledgement this study was conducted with the support of funding from the medical faculty of trisakti university. the dietary survey was supported b y s e a m e o t r o p m e d n u t r i t i o n a l f i e l d surveyor. the author would like to thank all of those who participated and assisted in the study. references 1. prasad as. zinc: mechanism of host defence 1-3. j nutr 2007; 137: 1345-50. 2. hambidge km, krebs nf. zinc deficiency: a special challenge 1-2. j nutr 2007; 137: 1101-6. 3. andriollo-sanchez m, hininger-favier i, meunier n, toti e, zaccaria m, brandolini-bunlon m, et al. zinc intake and status in middle-aged and older european subjects: the zenith study. eur j clin nutr 2005; 59 (suppl 2): s37-41. 4. kogirima m, kurasawa r, kubori s, sarukura n, nakamori m, okada s, et al. ratio of low serum zinc levels in elderly japanese people living in the central part of japan. eur j clin nutr 2007; 61: 375-81. 5. international zinc nutrition consultative group. vitamin and mineral deficiencies technical situation analysis : a report for the ten year strategy for the reduction of vitamin and mineral deficiencies (editorial). food nutr bull 2007; 28 (suppl): s160-s4. 6. de jong n. sensible aging: impaired sensory functioning in elders (dissertation). wageningen : wageningen university; 1999. 7. meunier n, m o’connor j, maiani g, cashman kd, secker dl, ferry m, et al. importance of zinc in the elderly: the zenith study. eur j clin nutr 2005; 59 (suppl 2): s1-4. 8. hidayat a. manfaat supplementasi seng (zinc) pada kelompok lanjut usia. universa med 2002; 21: 146. 9. stewart-knox bj, simpson eea, parr h, rae g, polito a, intorre f, et al. zinc status and taste acuity in older europeans: the zenith study. eur j clin nutr 2005; 59 (suppl 2): s31-6. 10. institute life science international recommended dietary allowances (rda): harmonization in southeast asia. ilsi 2005. 185 11. sibai am, zard c, adra n, baydoun m, hwalla n. variations in nutritional status of elderly men and women according to place of residence. gerontol 2003; 49: 215-4. 12. erven rb, kennedy-stephenson j. mineral intakes of elderly adult supplement and nonsupplement users in the third national health and nutrition examination survey. j nutr 2002; 132: 3422-7. 13. galan p, viteri fe, bertrais s, czernichow s, faure h, arnaud j, et al. serum concentration of â-carotene, vitamins c and e, zinc and selenium are influenced by sex, age, diet, smoking status, alcohol consumption and corpulence in a general french adult population. eur j clin nutr 2005; 59: 1181-90. 14. paik hy, joung h, lee jy, lee hk, king jc, keen cl: serum extracellular superoxide dismutase activity as an indicator of zinc status in humans. biol trace elem res 1999; 69: 45-57. 15. hotz c, peerson jm, brown kh. suggested lower cut-offs of serum zinc concentrations for assessing zinc status: reanalysis of the second national health and nutrition examination survey data (1976-1980). am j clin nutr 2003; 78: 756-64. 16. age-related eye disease study research group. the effect of five-years zinc supplementation on serum zinc, serum cholesterol and hematocrit in persons randomly assigned to treatment group in the age-related eye disease study: areds report no.7. j nutr 2002; 132: 697-702. 17. de jong n, gibson rs, thomson cd, ferguson el, mckenzie je, green tj, et al. selenium and zinc status are suboptimal in a sample of older new zealand women in a community-based study. j nutr 2001; 131: 2677-84. universa medicina vol. 26 no.4 oktavianus 27 *bagian ilmu kedokteran komunitas fakultas kedokteran universitas trisakti korespondensi adr. novia i. sudharma bagian ilmu kedokteran komunitas fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2504 email: sudharmanovi77@yahoo.com universa medicina 2007; 26: 27-38. latar belakang jumlah populasi dan umur harapan hidup lanjut usia (lansia) semakin meningkat akibat perbaikan dari pelayanan kesehatan masyarakat dan intervensi kedokteran. dengan semakin meningkatnya umur harapan hidup, maka sangat penting untuk memperbaiki kualitas hidup lansia. world health organization (who) telah mengembangkan sebuah instrumen untuk mengukur kualitas hidup seseorang yaitu who quality of life -bref (whoqol-bref). whoqol-bref terdiri dari empat domain yaitu fisik, psikologik, sosial dan lingkungan. penelitian ini bertujuan untuk menilai validitas dan reliabilitas dari whoqol-bref sebagai instrumen untuk mengukur kualitas hidup lansia. metode sebuah rancangan potong silang dilakukan di kecamatan mampang, jakarta selatan. selama dua bulan (oktober dan november 2005) sebanyak 306 lansia (usia ≥60 tahun, mampu berjalan, mampu berkomunikasi, dan tidak dalam kedaan terminal) bersedia ikut serta dalam penelitian. sepuluh pewancara yang sudah dilatih mengumpulkan informasi tentang umur, jenis kelamin, pendidikan, status pernikahan, dan kualitas hidup. hasil hasil penelitian menunjukkan distribusi skor setiap domain dari whoqol-bref adalah simetris dan tidak didapatkan efek floor atau ceiling. validitas diskriminan, validitas konstruk, dan konsistensi internal menunjukkan hasil yang baik dari skor keempat domain. namun terdapat dua item pertanyaan yaitu tentang perasaan negatif dan kepuasan hidup seksual yang menunjukkan korelasi yang lemah dengan domain psikologik dan sosial. kedua pertanyaan ini harus dimodifikasi supaya lebih mudah dipahami oleh lansia. kesimpulan whoqol-bref merupakan instrumen yang valid dan reliable untuk mengukur kualitas hidup lansia, tetapi masih diperlukan sedikit perbaikan. kata kunci : validitas, reliablitas, kualitas hidup, lanjut usia abstrak universa medicina januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 validitas dan reliabilitas world health organization quality of life-bref untuk mengukur kualitas hidup lanjut usia oktavianus ch. salim*, novia i. sudharma*a, rina k. kusumaratna* dan adi hidayat* 28 salim, sudharma, kusumaratna, dkk validitas dan reliabilitas who qol-bref validity and reliability of world health organization quality of life – bref to assess the quality of life in the elderly oktavianus ch. salim*, novia i. sudharma*a, rina k. kusumaratna* and adi hidayat* abstract background the population of elder people are increasing and life expectancy has been extended due to improved public health services and medical interventions. because increased life expectancy in the elderly has been assured, it is important to improve their quality of life (qol). in measuring qol, the world health organization (who) has developed the who quality of life–bref (whoqol-bref). the whoqol-bref was developed in the context of four domains of qol: physical, psychological, social and environmental. the objective of this study was to evaluate the validity and reliability of the whoqolbref to assess the qol in the elderly. methods a cross-sectional design was conducted in mampang sub-district, south jakarta. during 2 months (october and november 2005) assessment, 306 elderly (age ≥60 years, mobile, well communication and not in terminal condition) agreed to participate in the study. information on age, gender, education, marriage status, and quality of life were collected by ten trained interviewers. results this study showed that the score distribution in each domain of the whoqol-bref was symmetrical with no floor or ceiling effect. furthermore, all domain scores indicated excellent discriminant validity, construct validity, and good internal consistency. nevertheless, two items related to negative feelings and sexual activity had a weak correlation with psychological and social domain. the two items may need to modify to avoid misunderstanding for older people. conclusion with few modification the whoqol-bref assessment was valid and reliable for measuring qol in the elderly. keywords : validity, reliability, quality of life, elderly *department of community medicine, medical faculty trisakti university correspondence adr. novia i. sudharma department of community medicine, medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2504 email: sudharmanovi77@yahoo.com universa medicina 2007; 26: 27-38. pendahuluan survei kesehatan nasional tahun 2001 menunjukkan jumlah penduduk lanjut usia (lansia) yang berumur 60 tahun ke atas besarnya 9 , 6 % d a r i s e l u r u h j u m l a h p e n d u d u k d i indonesia.(1) angka harapan hidup penduduk indonesia (laki-laki dan perempuan) semakin meningkat dari 67,8 tahun pada periode 20002005 menjadi 69,8 tahun pada periode 20052010.(2) seiring dengan meningkatnya umur harapan hidup penduduk indonesia, maka populasi penduduk lansia juga akan meningkat. populasi lansia di indonesia pada tahun 2005 29 diperkirakan mencapai angka kurang lebih 18,7 juta orang (8,5% dari seluruh jumlah penduduk). indonesia termasuk negara kelima yang akan memiliki populasi lansia yang tinggi setelah cina, india, amerika serikat, dan meksiko.(3) s u d a h d i k e t a h u i b a h w a d e n g a n bertambahnya usia, sistem kekebalan akan semakin berkurang. hal ini mengakibatkan meningkatnya penyakit pada lansia, baik akut m a u p u n k r o n i k . m e n i n g k a t n y a g a n g g u a n / p e n y a k i t p a d a l a n s i a d a p a t m e n y e b a b k a n perubahan pada kualitas hidup mereka. yang dimaksud dengan kualitas hidup menurut world health organization (who) adalah persepsi seseorang dalam konteks budaya dan norma yang sesuai dengan tempat hidup orang tersebut serta berkaitan dengan tujuan, harapan, standar dan kepedulian selama hidupnya.(4) kualitas hidup seseorang merupakan fenomena yang multidimensional. (5) betapa pentingnya berbagai dimensi tersebut tanpa melakukan evaluasi sulit menentukan dimensi mana yang penting dari kualitas hidup seseorang. seseorang seringkali berpendapat semua aspek dari kualitas hidup sama pentingnya. untuk mengukur kualitas hidup seseorang who telah membentuk who quality of life (qol) group. kelompok ini telah melakukan penelitian di 15 negara yang berbeda budaya, norma dan adat istiadatnya. dengan demikian who telah berhasil mengatasi hal yang paling kontroversial tentang emics dan etics dengan mengaplikasikan sebuah kuesioner yang sama p a d a b e r b a g a i b u d a y a y a n g b e r b e d a . ( 6 ) p e n g u k u r a n k u a l i t a s h i d u p d i l a k u k a n m e n g g u n a k a n i n s t r u m e n wo r l d h e a l t h organization quality of life-100 (whoqol1 0 0 ) . ( 7 ) w h o q o l 1 0 0 t e r d i r i d a r i 1 0 0 pertanyaan yang mencakup 25 segi (facets) dan sudah diterjemahkan dalam berbagai bahasa di 15 negara tersebut. kemudian who menyusun whoqol-bref yang merupakan versi singkat dari whoqol-100. whoqol-bref dapat digunakan bila waktu yang diperlukan untuk menyelesaikan 100 pertanyaan terlalu lama dan tingkat dari segi (facets) secara rinci tidak diperlukan, misalkan pada survei epidemiologi d a n p e r c o b a a n k l i n i k . h a s i l p e n e l i t i a n menggunakan whoqol-100 di 15 negara menunjukkan beberapa pertanyaan valid untuk menyusun whoqol-bref. (8) whoqolbref terdiri dari 24 facets yang mencakup 4 domain dan terbukti dapat digunakan untuk mengukur kualitas hidup seseorang. keempat domain tersebut adalah: i) kesehatan fisik (physical health) terdiri dari 7 pertanyaan; ii) psikologik (psychological) 6 pertanyaan; iii) h u b u n g a n s o s i a l ( s o c i a l re l a t i o n s h i p ) 3 pertanyaan; dan iv) lingkungan (environment) 8 pertanyaan. whoqol-bref juga mengukur 2 facets dari kualitas hidup secara umum yaitu: i) kualitas hidup secara keseluruhan (overall quality of life); dan ii) kesehatan secara umum (general health). walaupun whoqol-bref ini sudah digunakan di 23 negara pada usia dewasa namun penggunaan whoqol-bref untuk lansia masih belum banyak dilakukan, khususnya di indonesia. penelitian ini bertujuan untuk menilai validitas dan reliabilitas dari whoqol-bref untuk mengukur kualitas hidup pada lansia. metode rancangan penelitian penelitian whoqol-bref ini dilakukan dengan menggunakan rancangan potong silang di kecamatan mampang jakarta selatan. subyek penelitian kriteria inklusi yang digunakan adalah lansia berusia 60 tahun ke atas, mampu berjalan t a n p a b a n t u a n o r a n g l a i n , m a m p u berkomunikasi/berbicara, tidak dalam keadaan terminal (menderita keganasan), tidak menderita demam saat dilakukan wawancara maupun 30 salim, sudharma, kusumaratna, dkk validitas dan reliabilitas who qol-bref pemeriksaan dan bersedia ikut serta dalam penelitian dengan menandatangani lembar persetujuan (inform consent). besar sampel sampel optimal yang diperlukan pada penelitian ini dihitung menggunakan rumus besar sampel untuk studi potong silang,(9) yaitu: kualitas hidup yang lebih baik. skor dari domain dihitung dengan mengalikan rata-rata setiap facet dengan 4. domain tidak diberikan skor, bila ≥ 20% pertanyaan tidak dijawab oleh responden. analisis statistik frekuensi, reliabilitas dan korelasi a n a l i s i s f r e k u e n s i d i l a k u k a n u n t u k m e n g g a m b a r k a n d i s t r i b u s i d a r i s e t i a p pertanyaan. efek floor dan ceiling dihitung untuk menyajikan persentase dari subyek yang memberikan respons nilai terendah dan tertinggi dari kemungkinan skor setiap item instrumen whoqol-bref. efek floor dan ceiling > 20% dari besar sampel adalah bermakna, yang mengindikasikan pengukuran hanya terbatas pada beberapa item pertanyaan dari whoqolbref.(10) konsistensi internal setiap domain dinilai mengunakan cronbach’s α dan kontribusi dari setiap pertanyaan terhadap α total. interitem correlations untuk setiap domain dan korelasi dari setiap item dengan domainnya juga dihitung. nilai cornbach’s α paling sedikit 0,7 u n t u k m e n u n j u k k a n r e l i a b i l i t a s s e b u a h instrumen, namun pada studi eksploratori dapat diturunkan menjadi 0,6.(11) validitas diskriminan (discriminant validity) kemampuan dari skor setiap domain untuk membedakan responden yang sehat dan sakit diuji dengan membandingkan nilai rata-rata (mean) kedua kelompok menggunakan uji-t.(5) validitas konstruk keempat domain dari whoqol-bref diuji validitasnya menggunakan analisis faktor (principal component analysis) untuk menilai instrumen ini sahih sebagai instrumen untuk mengukur kualitas hidup pada lansia. setiap p e r t a n y a a n h a r u s m e m p u n y a i n i l a i f a k t o r loading minimal ± 0,3 untuk dapat diikut sertakan ke dalam struktur sebuah domain. 2 2 d qxpxzα n = zα pada tingkat kepercayaan sebesar 95% besarnya 1,96 p = prevalensi lansia yang kualitas hidupnya kurang sebesar 23,6% (data dari penelitian pendahulan) q = prevalensi lansia yang kualitas hidupnya baik sebesar 76,4% d = kesalahan yang dapat diterima sebesar 0,05 berdasarkan rumus di atas besar sampel optimal yang adalah 288. pengumpulan data s e p u l u h p e t u g a s y a n g s u d a h d i l a t i h melakukan wawancara menggunakan kuesioner yang sudah diuji coba terlebih dahulu. data yang dikumpulkan meliputi usia, jenis kelamin, pendidikan, status pernikahan, dan kualitas hidup. instrumen whoqol-bref diberikan kepada responden untuk diisi dan bila ada halh a l y a n g k u r a n g j e l a s d a p a t d i b a n t u o l e h pewawancara. pengumpulan data dilakukan antara bulan oktober sampai november 2005. skoring dari whoqol-bref whoqol-bref yang sudah diterjemahan ke dalam bahasa indonesia diberikan skor yang mencakup empat domain: i) fisik terdiri dari 7 pertanyaan, ii) psikologik 6 pertanyaan, iii) hubungan sosial 3 pertanyaan dan iv) lingkungan 8 pertanyaan. setiap pertanyaan diberikan nilai 1 sampai 5, dan nilai lebih tinggi merupakan 31 faktor loading ± 0,4 termasuk kategori lebih penting (more important), loading > ± 0,5 praktis bermakna (practically significant).(12) sedangkan nilai eigenvalue digunakan untuk analisis peran setiap domain terhadap struktur whoqol-bref. nilai eigenvalue harus >1,0, adalah bermakna, untuk menyatakan sebuah domain mampu menjelaskan variasi dari itemitem pertanyaan domain tersebut.(12) hasil responden yang dianalisis terdiri dari 87 ( 2 8 , 4 % ) l a n s i a l a k i l a k i d a n 2 1 9 ( 7 1 , 6 % ) perempuan dan usia berkisar antara 60-85 tahun dengan rata-rata besarnya 66,4 ± 6,1 tahun. sebanyak 167 (54,6%) masih tinggal bersama pasangannya dan 137 (44,8%) sudah sendiri (duda/janda). pendidikan formal sebagian besar 1 7 6 ( 5 8 , 5 % ) b e r p e n d i d i k a n r e n d a h ( t i d a k sekolah, tidak tamat sd dan tamat sd) dan 71 (23,2%) berpendidikan tinggi (smu, akademi, dan perguruan tinggi). sebanyak 82 lansia menyatakan dalam keadaan sehat dan tidak m e n g a l a m i g a n g g u a n p e n y a k i t , b a i k a k u t maupun kronik dan 224 lansia menyatakan mengalami berbagai jenis penyakit. rata-rata skor dari domain fisik besarnya 12,8 ± 1,6; 12,7 ± 1,7 untuk domain psikologik; 13,1 ± 2,4 untuk domain hubungan sosial dan 13,7 ± 1,9 untuk domain lingkungan. untuk setiap domain nilai median mendekati nilai mean, hal ini berarti distribusi dari nilai setiap domain hampir simetris mendekati distribusi normal (meskipun uji shapro-wilk untuk normalitas menunjukkan nilai p < 0,05 untuk setiap domain, data tidak disajikan). persentase dari nilai ceiling dan floor untuk setiap domain cukup rendah (< 20%) berkisar antara 0,3 18,3% (tabel 1). n a m u n p e r s e n t a s e n i l a i c e i l i n g d a r i pertanyaan whoqol-bref tentang rasa sakit menghalangi aktivitas (nomor 3) dan perasaan negatif (nomor 26) masing-masing besarnya 37,6% dan 45,1% (data tidak disajikan). kedua pertanyaan tersebut memerlukan jawaban yang terbalik dibandingkan 24 pertanyaan lainnya. konsistensi internal cornbach’s ααααα untuk mengukur konsistensi internal dari instrumen digunakan cornbach’s α, inter-item correlation dan korelasi antar domain (tabel 2). s e p e r t i t e r l i h a t p a d a ta b e l 2 , k o e f i s i e n cornbach’s α dari domain besarnya berkisar antara 0,41 sampai 0,77; domain fisik dan lingkungan menunjukkan konsistensi internal yang kuat (> 0,70) antara setiap item di kedua domain tersebut. konsistensi internal domain p s i k o l o g i k b a i k , s e d a n g k a n p a d a d o m a i n hubungan sosial lemah (< 0,5). setiap item dari d o m a i n f i s i k , p s i k o l o g i k d a n l i n g k u n g a n berkontribusi terhadap domainnya masingmasing seperti terlihat dari besarnya koefisien cornbach’s α bila item tersebut dihilangkan. tabel 1. distribusi skor keempat domain dan faset kualitas hidup serta kesehatan secara umum dari whoqol-bref pada lansia 32 salim, sudharma, kusumaratna, dkk validitas dan reliabilitas who qol-bref nilai koefisien cornbach’s α setiap item berkisar antara 0,69 0,75 untuk domain fisik dan 0,73 0,77 untuk domain lingkungan. pada domain psikologik nilai koefisien cornbach’s α b e r k i s a r a n t a r a 0 , 5 3 0 , 7 2 . d o m a i n hubungan sosial dihitung berdasarkan 3 item sedangkan domain lainnya terdiri dari 6-8 item. n i l a i k o e f i s i e n c o r n b a c h ’s α k e t i g a i t e m domain hubungan sosial berkisar antara 0,10 0,69. item kehidupan seksual bila dihilangkan m e n u n j u k k a n c o r n b a c h ’s α s e b e s a r 0 , 6 9 , berarti item tersebut merupakan item yang k u r a n g m e n d u k u n g i n s t r u m e n w h o q o l bref. sedangkan cornbach’s α item perasaan n e g a t i f b e s a r n y a 0 , 7 2 b i l a i t e m t e r s e b u t dihilangkan. analisis diskriminan ta b e l 3 m e n y a j i k a n h a s i l a n a l i s i s diskriminan yang membandingkan rata-rata nilai setiap domain antara lansia yang sehat dan s a k i t . a n a l i s i s d i s k r i m i n a n m e n u n j u k k a n adanya perbedaan rata-rata skor domain fisik, psikologik dan lingkungan yang bermakna antara responden sehat dan sakit. rata-rata skor domain hubungan sosial pada lansia yang sakit lebih rendah dibandingkan yang sehat, tetapi secara statistik tidak bermakna. validitas konstruk k u a l i t a s h i d u p s e s e o r a n g m e r u p a k a n ukuran yang kompleks dan multidimensional, u n t u k i t u s k o r d a r i s e t i a p d o m a i n d a r i tabel 2. konsistensi internal setiap domain diukur menggunakan cornbach’s α 33 tabel 3. diskriminan analis uji-t setiap domain berdasarkan status kesehatan lansia * mean ± sd ** bermakna * bermakna tabel 4. validitas hubungan antara domain dan pertanyaan yang umum (standardized â ) (n=306) whoqol-bref dapat dibandingkan dengan pertanyaan umum tentang kualitas hidup. hasil analisis regresi ganda dengan skor kualitas hidup secara umum dan kesehatan umum sebagai variabel tergantung dan keempat domain sebagai variabel bebas menunjukkan domain psikologik merupakan prediktor terbaik untuk kualitas hidup secara umum (beta = 0,444 dan p=0,000). sedangkan domain fisik (beta = 0,241, p=0,002) m e r u p a k a n p r e d i k t o r p a l i n g b e r p e n g a r u h terhadap kesehatan umum diikuti dengan domain psikologik (beta 0,188, p=0,023) (tabel 4). secara keseluruhan keempat domain secara bermakna mampu menjelaskan variasi dari pertanyaan umum tentang kualitas hidup secara umum dan kesehatan umum (p=0,000). seperti yang diharapkan, analisis faktor menunjukkan korelasi yang sedang (moderate) dengan nilai koefisien korelasi sebesar 0,5 -0,7 a n t a r a s e t i a p f a s e t / i t e m p e r t a n y a a n d a n domainnya masing-masing. namun terdapat 7 item yang berkorelasi dengan domain yang bukan merupakan domainnya. item energik, mobilitas, aktifitas sehari-hari, dan kemampuan bekerja yang merupakan item pertanyaan dari domain fisik berkorelasi sedang (moderate) d e n g a n d o m a i n p s i k o l o g i k d a n d o m a i n lingkungan. sedangkan item kepuasan diri dari domain hubungan psikologik berkorelasi sedang (moderate) dengan domain lingkungan. item hubungan personal dan dukungan sosial dari domain hubungan sosial berkorelasi sedang (moderate) dengan domain lingkungan. namun terdapat dua item pertanyaan yaitu tentang perasaan negatif (r = 0,212) dan kehidupan seksual (r = 0,121) yang mempunya korelasi yang sangat lemah dengan domainnya masingmasing (tabel 5). u n t u k m e n g u j i s t r u k u r f a k t o r d a r i whoqol-bref digunakan analisis principal component analysis dengan rotasi varimax. hasil analisis disajikan pada tabel 6. tabel 6 menunjukkan sebagian besar pertanyaan dari w h o q o l b r e f m e m p u n y a i n i l a i f a k t o r loading yang > 0,5, berarti pertanyaan tersebut bermakna untuk dimasukkan ke dalam domain yang bersangkutan. secara umum cutt-off point dari faktor loading setiap pertanyaan besarnya m i n i m a l 0 , 3 u n t u k d i m a s u k k a n k e d a l a m domainnya. ternyata item nomor 26 tentang s e r i n g m e m i l i k i p e r a s a a n n e g a t i f s e p e r t i kesepian, putusa asa, cemas dan depresi nilai faktor loading besarnya 0,28, sedikit di bawah cutt-off point. 34 salim, sudharma, kusumaratna, dkk validitas dan reliabilitas who qol-bref tabel 5. struktur domain dari whoqol-bref pada lanjut usia sedangkan item pertanyaan nomor 21 tentang kepuasan terhadap kehidupan seksual n i l a i f a k t o r l o a d i n g b e s a r n y a 0 , 3 1 , y a n g termasuk kategori lemah (weak). untuk menilai kuatnya setiap domain untuk menjelaskan k e p e n t i n g a n n y a d a l a m s t r u k t u r i n s t r u m e n whoqol-bref perlu dihitung eigenvalue dan v a r i a n s s e t i a p d o m a i n ( ta b e l 7 ) . ta b e l 7 menunjukkan nilai eigenvalue keempat domain >1, yang berarti item pertanyaan dari keempat domain dapat menjelaskan domain tersebut. domain sosial mempunyai nilai eigenvalue yang paling kecil (1,588) dibandingkan domain lainnya. secara keseluruhan keempat domain ini b e r m a k n a u n t u k m e n j e l a s k a n v a r i a s i d a r i pertanyaan-pertanyaan instrumen whoqolbref sebesar 52,9 – 61,4%. pembahasan jumlah sampel pada penelitian ini besarnya 306 lansia. jumlah ini memenuhi kritera dari who yang menyatakan, untuk mengaplikasikan instrumen whoqol-bref diperlukan besar sampel minimal 300.(13) namun ratio antara lansia laki-laki dan perempuan besarnya 1: 2,5; ini tidak sesuai dengan anjuran who yaitu 1:1, mengingat penelitian dilakukan pada lansia yang 35 tabel 6. faktor loading setiap item dari whoqol-bref p = nomor pertanyaan populasi lansia laki-laki lebih kecil dibandingkan perempuan (ratio 0,9/1).(2) efek floor dan ceiling untuk setiap domain cukup rendah (< 20%) berkisar antara 0,3 – 18,3%. gambaran ini menunjukkan bahwa respons lansia terhadap ke26 pertanjaan dari whoqol-bref cukup baik. sebuah instrumen harus menghasilkan efek tabel 7. nilai eigenvalue dan persentase varians setiap domain dari whoqol-bref floor dan ceiling yang minimal supaya dapat direkomendasikan untuk mengukur sebuah fenomena.(14) namun terdapat 2 pertanyaan yaitu pertanyaan nomor 3 tentang “seberapa jauh rasa sakit mencegah aktivitas anda” dan pertanyaan nomor 26 tentang “perasaan negatif yang anda miliki” yang mempunyai nilai ceiling masing36 salim, sudharma, kusumaratna, dkk validitas dan reliabilitas who qol-bref m a s i n g s e b e s a r 3 7 , 6 % d a n 4 5 , 1 % . k e d u a pertanyaan ini sebenarnya memerlukan jawaban yang terbalik. hasil studi ini tidak berbeda dengan penelitian yang dilakukan di 23 negara yang menunjukkan pertanyaan nomor 3 tentang “seberapa jauh rasa sakit mencegah aktivitas a n d a ” m e m p u n y a i n i l a i c e i l i n g s e b e s a r 3 5 , 2 % . ( 1 5 ) s t u d i p a d a l a n s i a d i ta i w a n menunjukkan pertanyaan nomor 3 mempunyai nilai ceiling yang tidak berbeda sebesar 31%.(16) mengingat respons lansia terhadap pertanyaanpertanyaan lain tidak menunjukan efek floor dan ceiling yang >20%, maka perlu dipertimbangkan untuk mengubah kedua pertanyaan (nomor 3 dan 26) ini menjadi pertanyaan yang positif supaya dapat lebih dipahami oleh lansia. rata-rata keempat domain whoqolbref pada penelitian ini tidak berbeda dengan rata-rata domain lansia di taiwan(16) tetapi lebih baik dibandingkan lansia yang mengalami depresi.(17) setelah diberikan pengobatan antidepresant selama delapan minggu, terdapat perubahan dari kualitas hidup penderita. u n t u k m e n g u j i a p a k a h i t e m i t e m pertanyaan dari instrumen whoqol-bref mampu mengukur kualitas hidup seseorang d i g u n a k a n a n a l i s i s k o n s i s t e n s i i n t e r n a l . instrumen whoqol-bref menunjukkan nilai konsistensi internal yang baik (cornbach’s α > 0,7) kecuali pada domain hubungan sosial besarnya cornbach’s α = 0,41. nilai cornbach’s α umumnya rendah (< 0,7) bila sebuah domain hanya terdiri dari beberapa item pertanyaan. domain hubungan sosial hanya terdiri dari 3 pertanyaan sedangkan domain fisik, psikologik dan lingkungan terdiri dari 6-8 pertanyaan. hasil yang tidak berbeda ditunjukkan pada penelitian yang dilakukan oleh yang et al(18) pada penderita d e n g a n t r a u m a p a d a t u l a n g b e l a k a n g (cornbach’s α= 0,54). namun penelitian di 23 negara menunjukkan nilai cornbach’s α yang lebih besar 0,68 (range antara 0,51 – 0,77), nilai cornbach’s α terendah didapatkan pada subyek penelitian di norwegia.(14) walaupun subyek penelitian di 23 negara berusia antara 12–97 tahun (rata-rata 45 ± 16 tahun), namun hasil analisis konsistensi internal tidak menunjukkan adanya perbedaan dengan penelitian yang dilakukan pada lansia di jakarta. sedangkan penelitian yang dilakukan di poliklinik jiwa dewasa, poliklinik neurologi dan poliklinik rehabilitasi medik rscm jakarta menunjukan nilai cornbach’s α domain hubungan sosial sebesar 0,65 yang lebih besar dari hasil studi pada lansia ini.(19) subyek penelitian pada studi di rscm berusia antara 18-79 tahun yang berbeda dengan usia subyek penelitian ini (lansia). hasil-hasil penelitian ini menunjukkan secara keseluruhan whoqol-bref reliable (konsisten) untuk mengukur kualitas hidup pada lansia. analisis diskriminan menunjukkan ratarata skor keempat domain pada lansia yang sehat lebih besar dibandingkan lansia yang sakit. validitas diskriminan menunjukan adanya perbedaan skor yang bermakna pada domain fisik, psikologik dan lingkungan, tetapi tidak b e r m a k n a s e c a r a s t a t i s t i k p a d a d o m a i n hubungan sosial. studi yang dilakukan di taiwan(15) dan 23 negera lainnya(14) menunjukkan h a s i l y a n g k o n s i s t e n , w h o q o l b r e f m e r u p a k a n i n s t r u m e n y a n g v a l i d u n t u k mengukur kualitas hidup seseorang. analisis regresi ganda dari keempat domain sebagai variabel bebas dengan dua pertanyaan umum sebagai variabel tergantung menunjukkan keempat domain berkontribusi secara bermakna untuk menjelaskan variasi dari kualitas hidup s e c a r a k e s e l u r u h a n . d o m a i n p s i k o l o g i k merupakan prediktor terbaik untuk kualitas hidup secara umum (overall quality of life) dan d o m a i n f i s i k m e r u p a k a n p r e d i k t o r p a l i n g berpengaruh terhadap kesehatan umum (overall health satisfaction) (tabel 4). penelitian yang dilakukan di polandia menggunakan instrumen whoqol-bref menunjukkan hasil yang k o n s i s t e n , d o m a i n p s i k o l o g i k m e r u p a k a n 37 kontributor utama terhadap kualitas hidup s e c a r a u m u m d a n d o m a i n f i s i k t e r h a d a p kesehatan umum.(20) whoqol-bref secara teoritis mencakup empat domain yaitu fisik, psikologik, hubungan sosial dan lingkungan. pada studi ini, analisis faktor menunjukkan korelasi yang sedang (moderate) antar setiap faset/item pertanyaan dengan domainnya masing-masing (r= 0,5-0,7 tabel 5). namun terdapat 7 item pertanyaan yang berkorelasi dengan domain yang bukan merupakan domainnya. hal ini dapat terjadi mengingat tingkat pendidikan lansia sebagian b e s a r 1 7 6 ( 5 8 , 5 % ) b e r p e n d i d i k a n r e n d a h , sehingga dapat menimbulkan persepsi yang b e r b e d a t e n t a n g a r t i s e b u a h p e r t a n y a a n mengenai kondisi fisik, psikologik, hubungan sosial dan lingkungannya. pertanyaan mengenai “ s e b e r a p a j a u h a n d a m e n i k m a t i h i d u p ” (pertanyaan nomor 5) dan “bagaimana kepuasan anda terhadap diri” (pertanyaan nomor 19) dapat diartikan sebagai kondisi kesehatan fisiknya. walaupun demikian tidak satupun pertanyaan yang menunjukkan korelasi yang kuat dengan domain yang bukan merupakan d o m a i n n y a . a n a l i s i s f a k t o r ( p r i n c i p a l component analysis) digunakan untuk menilai validitas konstruk dari instrumen whoqolbref untuk mengukur kualitas hidup pada l a n s i a . s e b a g i a n b e s a r p e r t a n y a a n d a r i w h o q o l b r e f m e m p u n y a i n i l a i f a k t o r l o a d i n g > 0 , 5 , b e r a r t i p e r t a n y a a n t e r s e b u t bermakna untuk dimasukkan ke dalam domain yang bersangkutan. hanya item pertanyaan nomor 26 tentang sering memiliki perasaan negatif seperti kesepian, putus asa, cemas dan depresi nilai faktor loading besarnya 0,28 berarti item ini perlu dimodifikasi supaya lebih mudah dipahami oleh lansia. hal yang sama p a d a i t e m p e r t a n y a a n n o m o r 2 1 t e n t a n g kepuasan terhadap kehidupan seksual nilai faktor loading besarnya 0,31, yang termasuk kategori lemah (weak). kedua item pertanyaan ini harus dikaji lebih lanjut dan disesuaikan dengan norma masyarakat di indonesia untuk meningkatkan penggunaan instrumen whoqolbref pada lansia. misalkan untuk pertanyaan t e n t a n g k e p u a s a n k e h i d u p a n s o s i a l d a p a t diperbaiki menjadi: “apakah anda merasa puas dengan kehidupan seksual anda, termasuk hubungan baik dengan teman-teman dan bukan saja hubungan seksual (sexual intercourse)”. keempat domain dari whoqol-bref mampu menjelaskan variasi dari data yang dikumpulkan sebesar 52,9 61,42%. keempat domain ini m a m p u m e m p r e s e n t a s i k a n k u a l i t a s h i d u p seseorang secara keseluruhan.(21) d i s t r i b u s i k e 2 6 p e r t a n y a a n d a r i whoqol-bref adalah simetris dan hasil penelitian menunjukkan instrumen whoqolbref valid dan reliable untuk mengukur k u a l i t a s h i d u p p a d a l a n s i a . s a x e n a d a n o’connell(22) menyatakan bahwa kemampuan cross-cultural dari instrumen whoqol-bref merupakan suatu keunggulan dan mendukung premis yang menyatakan instrumen ini dapat digunakan sebagai alat screening. whoqolbref merupakan suatu instrumen yang valid dan reliable untuk digunakan baik pada populasi l a n s i a m a u p u n p o p u l a s i d e n g a n p e n y a k i t tertentu. instrumen ini telah banyak digunakan di berbagai negara industri maupun berkembang pada populasi penderita hati dan paru-paru yang kronik sebagai alat screening. kesimpulan s e c a r a k e s e l u r u h a n w h o q o l b r e f merupakan instrumen yang valid dan reliable untuk mengukur kualitas hidup pada lansia. pada setiap domain dari whoqol-bref d i s t r i b u s i s k o r h a m p i r s i m e t r i s d a n t i d a k didapatkan efek floor atau ceiling. namun perlu dilakukan modifikasi pada pertanyaan tentang seringnya lansia memiliki perasaan negatif seperti kesepian, putus asa, cemas dan kepuasan terhadap kehidupan seksual supaya lebih mudah dimengerti. 38 salim, sudharma, kusumaratna, dkk validitas dan reliabilitas who qol-bref daftar pustaka 1. departemen kesehatan r.i. survei kesehatan nasional 2001. laporan skrt 2001 : studi morbiditas dan disabilitas. jakarta : badan penelitian dan pengembangan kesehatan departemen kesehatan r.i. ; 2002. 2. badan perencanaan pembangunan nasional, badan pusat statistik, united nations population fund. proyeksi penduduk indonesia 2000 – 2025. jakarta: bappenas, bps, unfpa; 2005. 3. united nations population division. world population prospects: the 2002 revision. united nations: new york; 2003. 4. the whoqol group. development of the whoqol: rationale and current status. int j mental health 1994; 23: 24-56. 5. skevington sm, mc arthur p, somerset m. developing items for the whoqol: an investigation of contemporary beliefs about quality of life related to health in britain. br j health psychol 1997; 2: 55-72. 6. warner r. the emics and etics of quality of life assessment. soc psychaitry psychiatr epidemiol 1999; 34: 117-21. 7. the whoqol group. the world health organization quality of life assessment (whoqol): development and general psychometric properties. sos sci med 1998; 41: 1403-9. 8. the whoqol group. development of the world health organization whoqol-bref quality of life assessment. psychol med 1998; 28: 551-58. 9. bordens ks, abbott bb. using survey research. in: research design and methods: a process approach. 3rd ed. london: mayfield publishing company; 1996. p. 181-215. 10. hsueh ip, wang ch, shue cf, hsieh cl. comparison of psychometric properties of three mobility measures for patients with stroke. stroke 2003; 34: 1741-5. 11. streiner dl, norman gr. reliability. in: health measurement scales: a practical guide to their development and use. 2nd ed. qxford: oxford university pres; 2000. p. 104-27. 12. hair jf, anderson re, tatham rl, black wc. factor analysis. in: multivariate data analysis. 5th ed. london: prentice-hall international inc.; 1998. p. 87-138. 13. world health organization. introduction, administration, scoring and generic version of the assessment: field trial version. geneva: world health organization; 1996. 14. alexander m, berger w, buchholz p, walt j, burk c, lee j, et al. the reliability, validity, and preliminary responsiveness of the eye allergy patient impact questionnaire (eapiq). health qual life outcomes 2005; 3: 67-73. 15. skevington sm, lofty m, o’connel ka. the world health organization’s whoqol-bref quality of life assessment: psychometric properties and results of the international field trial, a report from the whoqol group. qual life res 2004; 13: 299-310. 16. hwang hf, liang wm, chiu yn, lin mr. suitable of the whoqol-bref for community-dwelling older people. age aging 2003; 32: 593-600. 17. naumann vj, byrne gja. whoqol-bref as a measure of quality of life in older patients with depression. int psychogeriatr 2004; 16: 159-73. 18. jang y, hsieh cl, wang yh, wu yh. a validity study of the whoqol-bref assessment in persons with traumatic spinal cord injury. arch phys med rehabil 2004; 85: 1890-5. 19. wukandari wd, wibisono s, kusumawardhani a, irmansyah. penentuan validitas whoqol-100 dalam menilai kualitas hidup pada pasien rawat jalan di rscm (versi indonesia). jiwa 2005: xxxviii: 17-48. 20. jaracz k, kalfoss m, gorna k, baczyk g. quality of life in polish respondents: psychometric properties of the polish whoqol-bref. scand j caring sci 2006; 20: 251-60. 21. skevington sm, sartorius n, amir m. developing methods for assessing quality of life in different cultural settings: the history of the whoqol instruments. soc psychiatry psychiatr epidemiol 2004; 39: 1-8. 22. saxena s, o’connell k. a commentary: crosscultural quality of life assessment at the end of life. gerontologist 2002; 42: 81-5. oktavianus 1 universa medicina january-april, 2016 vol.35 no.1 global emergence of zika virus richard tjan editor z i k a v i r u s ( z i k v ) b e l o n g s t o t h e flaviviruses (family flaviviridae), which includes dengue, yellow fever, west nile, and japanese encephalitis viruses. zika virus was isolated in 1947, in the zika forest near kampala, uganda, from one of the rhesus monkeys used as sentinel animals in a yellow fever research program.(1) previous to 2007, zikv was present in a f r i c a a n d a s i a (i n d i a , m a l a y s i a , t h e philippines, thailand, vietnam, and indonesia) without causing outbreaks. since then zikv has caused a number of outbreaks, the first occurring in 2007 on yap island in the federated states of micronesia, followed in 2013-2014 by outbreaks in french polynesia, new caledonia, the cook islands, and easter island, and finally spreading in 2015-2016 to central and south america. a number of the patients in french polynesia had severe disease with neurological symptoms, such as the guillain-barré syndrome.(1-3) in brazil, there were an estimated 440,000 to 1.3 million cases of autochthonous zikv infection up to december 2015.(4) up to this time zikv disease appeared to be a mild illness,(2) but the brazilian zikv cases were reportedly associated with microcephaly in fetuses born to zikv-infected mothers.(4) t h e a b o v e m e n t i o n e d c l u s t e r s o f microcephaly, guillain-barré syndrome, and other neurological disorders associated in time and place with zikv have been declared by the who to constitute a public health emergency of international concern (pheic), based on the advice of an emergency committee of the international health regulations and other experts. in this connection, the emergency committee has explained that the advice was made “on the basis of what is not known” about the neurological findings associated with zikv, since zikv disease itself is relatively mild and 80% of infected persons are asymptomatic.(5) three recommendations were made by the committee, as follows: to conduct standardized and enhanced surveillance of microcephaly in areas of known zika virus transmission; to investigate the etiology of confirmed clusters of microcephaly and neurological disorders and to determine any causative association with zikv, o t h e r f a c t o r s , a n d c o f a c t o r s ; t o t a k e precautionary measures for the prevention of arboviral infection, which should also result in the prevention of chikungunya and dengue outbreaks.(5) c o n c e r n i n g z i k v s u s c e p t i b i l i t y t o desinfectants, the virus cannot be neutralized by 10% ethanol, but is killed by potassium permanganate, ether, and temperatures above 60°c.(1) as to the clinical picture, the majority of persons infected with zikv are asymptomatic. if symptoms develop, the most common are fever, h e a d a c h e , m y a l g i a / a r t h r a l g i a , e d e m a o f extremities, maculopapular rash, retro-orbital pain, conjunctivitis, lymphadenopathy. some p a t i e n t s m a y h a v e v e r t i g o o r d i g e s t i v e disorders.(1,3) there is no specific treatment or vaccine.(1,3) the only treatment is supportive (rest, fluids) and symptomatic (analgesics, antipyretics).(1,3) it should be noted that the fever may be subjective, the patient having a normal temperature as measured with a thermometer.(2) 2 univ med 2016;35:1-2. doi: http://dx.doi.org/10.18051/univmed.2016.v35.1-2 prevention of zikv infection is by personal protection against mosquito bites and eradication of mosquitoes (anti-vectorial prevention). at the community level, prevention consists in decreasing the number of egg-laying sites by drying, isolating, or treating with insecticides.(3) .diagnostic tests for zikv include pcr for viral rna, immunoassays such as elisa to detect specific antibody, and the plaque reduction neutralization assay. in general, in diagnostic testing for flavivirus infections, two serum samples should be collected, one sample collected in the acute phase and one sample collected 2 to 3 weeks afterwards. for pcr, the samples should be collected within 10 days of onset. an elisa developed by the centers for disease control and used on yap island sera from patients with previous flavivirus infections, yielded more frequent cross-reactivity with dengue virus than with yellow fever, japanese encephalitis, murray valley encephalitis, or west nile viruses. the plaque reduction neutralization assay is more specific than immunoassays, but may still show cross-reactive results in secondary flavivirus infections.(1) as observed by hayes(1) in 2009, travel and commerce may spread the virus across large distances, as witnessed by the 2007 outbreak on yap island, an isolated spot in the pacific, far to the north of indonesia. the medical community, especially public health officers, should never relax their efforts at surveillance. already in 2009, in connection with the yap island outbreak, duffy(2) stated that “clinicians and public health officials should be aware of the risk of further expansion of zika virus transmission.” hayes in 2009 also warned against complacency, citing the case of the west nile virus, previously regarded as relatively benign, before causing large outbreaks of neuroinvasive disease in romania and north america.(1) the brain lesions in microencephalic infants a r e p r o b a b l y a s s o c i a t e d w i t h t h e h i g h l y neurotropic character of zikv in young mice, in whom there is neuronal degeneration, cellular infiltration, and softening of the brain.(6) from the medical practitioners point of view, zikv disease should now be included in the differential diagnosis of dengue. zika virus disease is also epidemiologically similar to dengue, therefore control and prevention of the disease should be performed by avoiding mosquito bites and reducing the numbers of mosquito vectors, as recommended by the emergency committee.(5) in conclusion, the zika pandemic has emphasized the fact that the impact of human behaviors such as air travel on ecologic balance can result in the unexpected emergence of many infectious agents that are now lying dormant, necessitating integrated research into their ecosystems.(7) references 1. hayes eb. zika virus outside africa. emerg infect dis 2009;15:1347-50. 2. duffy mr, chen t-h, hancock wt, et al. zika virus outbreak on yap island, federated states of micronesia. n engl j med 2009;360:253643. 3. ioos s, mallet hp, leparc goffart i, et al. current zika virus epidemiology and recent epidemics. med mal infect 2014;44:302-7. 4. mlakar j, korva m, tul n, et al. zika virus associated with microcephaly. n engl j med 2016; doi: 10.1056/nejmoa1600651. 5. heymann dl, hodgson a, sall a, et al. zika virus and microcephaly: why is this situation a pheic?. the lancet 2016;387:719-20. available at: http://dx.doi.org/10.1016/s0140-6736(16) 00320-2. accessed 20 februray, 2016. 6. marcondes cb, ximenes mffm. zika virus in brazil and the danger of infestation by aedes (stegomyia) mosquitoes. rev soc bras med trop 2015. available at: http://dx.doi.org/10.1590/ 0037-8682-0220-2015. accessed 20 february, 2016. 7. fauci as, morens dm. zika virus in the americas — yet another arbovirus threat. n engl j med 2016;374;7. doi: 10.1056/nejmp1600 297. universa medicina january-april, 2016 vol.35 no.1 oktavianus 199 *division of endocrinology & metabolic disease, department of internal medicine, faculty of medicine, university of sumatera utara, medan correspondence dr. muhammad fahmi hidayat division of endocrinology & metabolic disease, department of internal medicine, faculty of medicine, university of sumatera utara, jl. dr. mansyur no. 5 medan, sumatera utara 20155 email: amiek1313@yahoo.com univ med 2014;33:199-204 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background type 2 diabetes mellitus is a heterogeneous disease which is characterized by variable degrees of insulin resistance and impaired insulin secretion. uric acid has been identified as a marker for a number of metabolic and hemodynamic abnormalities. in diabetic patients, there is biochemical interaction between serum glucose and purine metabolism, with increased excretion of uric acid during hyperglycemia and glycosuria. the objective of this study was to evaluate the correlation of serum uric acid levels with glycated hemoglobin (hba1c) and blood glucose levels in type 2 diabetes patients. methods a cross sectional study from may until july 2014 had been done in 82 type 2 diabetes patients. serum was analyzed for uric acid, fasting and 2-hour post prandial blood glucose and lipid profile. spearman correlation test was used to assess associations of hba1c, fasting and post prandial blood glucose with serum uric acid levels. results there was a negative correlation of hba1c with serum uric acid levels (r=0.407; p=0.000) and of fasting and post prandial blood glucose with serum uric acid levels (r=-0.338; p=0.002 and r=-0.318; p=0.004, respectively). conclusions serum uric acid level was associated with hba1c, fasting and post prandial blood glucose suggesting a significant role of serum uric acid in the deterioration of glucose toleration. keywords: uric acid, hba1c, type-2 dm elevated uric acid level decreases glycated hemoglobin in type 2 diabetes mellitus muhammad fahmi hidayat*, santi syafril*, and dharma lindarto* 200 hidayat, syafril, lindarto uric acid level and glycated hemoglobin peningkatan kadar asam urat menurunkan hemoglobin terglikasi pada diabetes melitus tipe 2 latar belakang diabetes melitus tipe 2 adalah penyakit heterogen dengan karakteristik derajat resistensi insulin dan gangguan sekresi insulin yang bervariasi. asam urat telah diidentifikasi sebagai penanda pada sejumlah abnormalitas metabolik dan hemodinamik. resistensi insulin dapat menyebabkan hiperinsulinemia yang meningkatkan reabsorpsi sodium dan air, termasuk asam urat dari ginjal. pada pasien diabetes melitus, dijumpai interaksi biokimiawi antara glukosa serum dan metabolisme purin, dengan peningkatan ekskresi asam urat selama hiperglikemia dan glikosuria. penelitian ini bertujuan untuk menetukan adanya hubungan antara kadar asam urat serum dengan hba1c, glukosa puasa dan 2-jam post prandial pada pasien diabetes melitus tipe 2. metode penelitian potong lintang dilakukan pada 82 pasien diabetes tipe 2 antara bulan mei dan juli 2014. serum diambil dan dilakukan pemeriksaan kadar asam urat, kadar glukosa darah puasa dan 2-jam post prandial dan profil lipid. uji korelasi spearman digunakan untuk menilai korelasi antara hba1c, kadar glukosa darah puasa dan post prandial dengan kadar asam urat serum. hasil dijumpai korelasi negatif antara hba1c dengan kadar asam urat (r=-0,407; p=0,000), kadar glukosa darah puasa dan post prandial dengan kadar asam urat serum (masing-masing, r=-0,338; p=0,002 dan -0,318; p=0,004). kesimpulan kdar asam urat berhubungan dengan kadar hba1c, glukosa darah puasa dan post prandial. hal ini menunjukkan peran asam urat pada keadaan memburuknya toleransi glukosa. kata kunci: asam urat, hba1c, dm tipe-2 abstrak introduction according to the american diabetes association (ada) diabetes mellitus is a group o f m e t a b o l i c d i s e a s e s c h a r a c t e r i z e d b y hyperglycemia resulting from defects in insulin secretion, insulin action, or both.(1) uric acid has been identified as a marker for a number of metabolic and hemodynamic abnormalities. insulin resistance can lead to hyperinsulinemia which increases reabsorption of sodium and water, including uric acid from the kidneys.(2,3) a meta-analysis showed that the risk of type 2 diabetes was increased by 6% per 1 mg/dl increment in serum uric acid level.(4) choi et al.(5) and kaur et al.(6) reported that the serum uric acid levels increased with moderately increasing levels of glycated hemoglobin (hba1c) <7% and then decreased with further increasing levels of hba1c >7% (a bell-shaped relation). meanwhile meera et al.(7) reported a negative correlation of serum uric acid with blood glucose in type 2 diabetes. previous studies reported that serum uric acid in type 2 diabetes was lower when compared with pre-diabetes and healthy population, especially in diabetes mellitus patients with blood glucose levels of >10 mmol/l (>180 mg/dl). 201 t h i s p r o b a b l y r e f l e c t s t h e b i o c h e m i c a l interaction between serum glucose and purine metabolism, with increased excretion of uric acid during hyperglycemia and glycosuria.(8) rodriguez et al .(9) also reported that the incidence of gout among diabetes patients was lower, as compared with individuals with no diabetes and the inverse association was stronger with type 1 diabetes than with type 2 diabetes and was stronger among men than women. nan et al.(10) found a linear correlation between serum uric acid with fasting and 2-hour post prandial b l o o d g l u c o s e w h e r e s e r u m u r i c a c i d concentration increased with increasing fasting blood glucose levels up to 7.0 mmol/l, but notably decreased when fasting blood glucose over 7.0 mmol/l, with an increasing trend in the uric acid concentration at 2-hour post prandial glucose <10.0 mmol/l and a decreasing trend at 2-hour post prandial glucose >10.0 mmol/l. on the basis of the abovementioned information, considering that there are contrary results and only few studies that have analyzed the relationship between serum uric acid and hba1c in type 2 diabetes, the objective of the present study was to determine the correlation of serum uric acid levels with hba1c, fasting and 2-hour post prandial blood glucose in type 2 diabetes patients. methods research design a cross-sectional study was conducted at the department of internal medicine of h. adam malik hospital, medan, from may 2014 to july 2014. research subjects the study population comprised type 2 diabetes patients who were treated at the outpatient and inpatient clinics of the department of internal medicine. the study sample consisted of individuals from the study population who met the inclusion criteria, which were: males or females >17 years of age, having type 2 diabetes, and willing to participate in the research. the exclusion criteria were: type 1 diabetes, anemia (males with hb <12 g/dl or females with hb <11 g/dl), gouty arthritis, and serum creatinine >1.5 mg/dl. the study subjects were selected by consecutive non-random sampling. we used the sample size calculation method to determine the minimum single sample using the coefficient of correlation.(11) to calculate the sample size, we used a previous study by gill et al.(12) who r e p o r t e d a c o e ff i c i e n t o f c o r r e l a t i o n o f approximately -0.532. the calculated sample size was 82, which was estimated to be adequate to detect a 30% difference between the two groups using a two-tailed test, an alpha of 0.05 and power of 80%. data collection all study subjects were interviewed using a questionnaire for age, gender, history and duration of diabetes. subjects were measured for height, weight and body mass index (bmi). the blood pressure was measured in a quiet room with the participant seated. laboratory analysis laboratory studies comprising routine blood parameters, serum uric acid, fasting and 2-hour post prandial blood glucose, hba1c and lipid profile [total cholesterol, low density lipoprotein (ldl) cholesterol, and high density liprotein (hdl) cholesterol], were measured with the cobas automated analyzer. statistical analysis the kolmogorov-smirnov normality test was used to assess the normality of distribution of the data. the pearson correlation test was used to assess correlations of hba1c, fasting and post prandial blood glucose with serum uric acid for normally distributed data, and the spearman correlation test for non-normally distributed data. statistical analysis was performed with the spss statistical package for windows, version 17.0, a n d a p v a l u e o f < 0 . 0 5 w a s c o n s i d e r e d statistically significant. univ med vol. 33 no.3 202 hidayat, syafril, lindarto uric acid level and glycated hemoglobin ethical clearance the study protocol was approved by the health research ethical committee, faculty of medicine, university of sumatera utara. all study subjects signed written informed consent after having been informed about the aims and benefits of the study. results during the study period 82 study subjects with diabetes mellitus type 2 were obtained consisting of 40 males (48.8%) and 42 females (51.2%), with mean age of 59.12 ± 9.25 years old and mean body mass index of 27.33 ± 3.69 kg/m2. from measurement of serum uric acid we found mean uric acid levels of 6.69 ± 2.12 mg/ dl. mean hba1c was approximately 8.24 ± 1.83%, while mean fasting blood glucose and 2h o u r p o s t p r a n d i a l b l o o d g l u c o s e w e r e approximately 168.6 ± 74.3 mg/dl and 290.91 ± 103.89 mg/dl, respectively (table 1). using the spearman correlation test, we found a fairly significant negative correlation of hba1c, fasting and post prandial blood glucose level with serum uric acid levels (r=-0.407; p=0.000, r=-0.338; p=0.002, and r=-0.318; p=0.004, respectively) (table 2). discussion in this cross sectional study involving 82 type 2 diabetes mellitus subjects we found a fairly significant negative correlation between hba1c and serum uric acid levels in type 2 diabetes patients, where with higher uric acid values the hba1c levels actually decreased. gill et al.(12) showed a contrary result, where hba1c, serum insulin and serum uric acid parameters were found to be increased in patients with type table 1. baseline characteristics of diabetic type 2 subjects (n=82) table 2. correlation of serum uric acid with glycated hemoglobin, fasting blood glucose, and 2-hour post prandial blood glucose in diabetic type 2 subjects 203 2 diabetes mellitus as compared to their levels in controls. in newly diagnosed diabetic patients serum uric acid levels linearly increased with an increase in hba1c levels. previous studies showed that uric acid was found to be of prognostic value in patients with type 2 diabetes mellitus and confirmed coronary artery disease, where uric acid increased all-cause mortality and cardiac mortality risk by approximately 29% and 15%, respectively, for each standard deviation increase in the logarithmic scale of uric acid level.(13) choi et al.(5) and kaur et al.(6) reported that serum uric acid levels increased with moderately increasing levels of glycated hemoglobin (hba1c) <7% and then decreased with further increasing levels of hba1c >7% (a bell-shaped relation). the study by cervantes et al.(14) showed that the serum concentration of uric acid had a positive relationship with the total phase of insulin secretion, even in the state prior to hyperuricemia and thus uric acid can play an important role in the function of the beta cell in patients with type 2 diabetes. a study in diabetic patients found a negative and inverse relationship between serum uric acid and hba1c. hence, in that study uric acid was found to be a potential biomarker of the glucose metabolism. (15) another study in sarajevo showed there were no significant correlations between uric acid and both hba1c levels and bmi in type 2 and type 1 dm.(16) our study also found a fairly significant negative correlation of serum uric acid with fasting blood glucose and 2-hour post prandial blood glucose, which agrees with the study by meera et al.(7) who reported a negative correlation of serum uric acid with blood glucose in type 2 diabetes patients. the study by nan et al.(10) showed a linear correlation between serum uric acid with fasting and 2-hour post prandial blood glucose but only with increasing fasting blood glucose levels up to 7.0 mmol/l and post prandial blood glucose levels up to 10 mmol/l, and then serum uric acid notably decreased with fasting blood glucose over 7.0 mmol/l and post prandial blood glucose over 10 mmol/l. fan et al.(17) reported that serum uric acid in individuals with impaired fasting glucose and/or impaired hba1c was strongly associated with 2-hour post prandial blood glucose, independent of fasting plasma glucose and hba1c and other established risk factors, where a 1.53 mg/dl increment of serum uric acid was significantly associated with a 36% higher risk for 2-hour newly diagnosed diabetes in multivariate logistic regression. there are a number of hypotheses to explain the above negative correlation between uric acid and blood glucose. first, it probably reflects the biochemical interaction between serum glucose and purine metabolism, with increased excretion of uric acid (uricosuria) during hyperglycaemia and glycosuria.(8) another hypothesis is that an increase in the inflammatory response in dm may have a direct protective effect on the incidence of gout and hyperuricemia that directly produces an intense inflammatory response against urate crystals which have anti-oxidant and free radical effects.(9) moreover, there are conditions that interfere with purine catabolism or synthesis of uric acid due to a chronic inflammatory process.(18,19) uric acid has been also known as an antioxidant. this antioxidant property might preserve or delay apoptosis and improve â-cell function in diabetics and augment insulin secretion.(20) further studies are required on the role of uric acid as antioxidant in the diabetic state. a limitation of this study was the absence of direct assessment of risk factors that may affect the outcomes of uric acid levels both in purine catabolism and chronic inflammatory systemic response and the presence of poor metabolic control, renal hyperfiltration and overt nephropathy necessitating further research of these factors in the future. conclusion serum uric acid level was associated with hba1c, fasting and post prandial blood glucose, univ med vol. 33 no.3 204 hidayat, syafril, lindarto uric acid level and glycated hemoglobin suggesting a significant role of serum uric acid in the deterioration of glucose toleration. acknowledgement we thank all study subjects who agreed to participate in the present study and all colleagues who gave their advice to improve the manuscript. references 1. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2013;36:67-74. 2. krishnan e, pandya bj, chung l, hariri a, dabbous o. hyperuricemia in young adults and risk of insulin resistance, prediabetes and diabetes: a 15-years follow up study. am j epidemiol 2012;176:108-16. 3. kramer ck, muhlen dv, jassal sk, connor eb. serum uric acid levels improve prediction of incident type 2 diabetes in individuals with impaired fasting glucose: the rancho bernardo study. diabetes care 2009;32:1272-3. 4. qin lv, meng xf, he ff, chen s, su h, xiong j, et al. high serum acid and increased risk of type 2 diabetes: a systemic review and metaanalysis of prospective cohort studies. plosone 2013;8:e56864. 5. choi hk, de vera ma, krisnan e. gout and the risk of type 2 diabetes among men with a high cardiovascular risk profile. rheumatology 2008;47:1567-70. 6. kaur a, kukreja s, malhotra n, neha. serum adenosine deaminase activity and its correlation with glycated haemoglobin levels in patients of type 2 diabetes mellitus. j clin diagnos res 2012;6:252-6. 7. meera ks, vasudha kc, sushmitha j. the study of serum uric acid levels in non dependent diabetes mellitus (niddm). asian j pharm biol res 2011;1:260-6. 8. choi hk, ford es. hemoglobin a1c, fasting glucose, serum c-peptide and insulin resistance in relation to serum uric acid levels the third national health and nutrition examination survey. rheumatology 2008;47:713-7. 9. rodriguez lag, soriano lc, choi hk. impact of diabetes against the future risk of developing gout. ann rheum dis 2010;69:2090-4. 10. nan h, pang z, wang s, gao w, zhang l, ren j, et al. serum uric acid, plasma glucose and diabetes. diabetes vasc dis res 2010;7:40-6. 11. madiyono b, moeslichan s, sastroasmoro s, budiman i, purwanto sh. perkiraan besar sampel. in: dasar-dasar metodologi penelitian klinis. 4th ed. editors: sastroasmoro s, ismael s. jakarta: sagung seto; 2011. p.348-81. 12. gill a, kukreja s, malhotra n, chhabra n. correlation of the serum insulin and the serum uric acid levels with the glycated haemoglobin levels in the patients of type 2 diabetes mellitus. j clin diagnos res 2013;7:1295-7. 13. ndrepepa g, braun s, king l, cassese s, tada t, fusaro m, et al. prognostic value of uric acid in patients with type 2 diabetes mellitus and coronary artery disease. clin sci 2013;124:25968. 14. cervantes jar, zavala mgr, ortiz mg, abundis em, sandoval cv, chavez at, et al. relationship between serum concentration of uric acid and insulin secretion among adults with type 2 diabetes mellitus. int j endocrinol 2011. article id 107904. doi: 10.1155/2011/107904. 15. aziz kma. effect of serum uric acid in augmentation of insulin secretion and improvement of hba1c in diabetic patients: proposed statistical regression models for uric acid, hba1c and insulin. j res diabetes 2014. article id 237887. doi:10.5171/2014.237887. 16. babic n, avdagic n, music m, huskic j, dizdarevic-bostandzic a, dervisevic a, et al. serum uric acid levels in type 1 and type 2 diabetic patients. folia medica 2013;48:43-9. 17. fan hq, tang w, wang zx, wang sj, qin yh, fu q, et al. association of serum uric acid with 2-hour postload glucose in chinese with impaired fasting plasma glucose and/or hba1c. plosone 2013;8:e67759. 18. li c, hsieh mc, chang sj. metabolic syndrome, diabetes and hyperuricemia. curr opin rheumatol 2013;25:210-6. 19. puddu p, puddu gm, cravero e, vizioli l, muscari a. the relationships among hyperuricemia, endothelial dysfunction and cardiovascular diseases: molecular mechanisms and clinical implications. j cardiol 2012;59:23542. 20. bowman gl, shannon j, frei b, kaye ja, quinn jf. uric acid as a cns antioxidant. j alzheimer’s dis 2010;19:1331-6. c:\users\jurnal fkusakti\docume 166 *department of medical laboratory science, faculty of health sciences and technology, nnamdi azikiwe university, nnewi campus, anambra state nigeria **department of chemical pathology, faculty of medicine, nnamdi azikiwe university, nnewi campus, anambra state nigeria ***education department, medical laboratory science council of nigeria †department of biochemistry, faculty of biological sciences, anambra state university, owerri, nigeria ††department of human biochemistry, faculty of basic medical sciences, nnamdi azikiwe university, nnewi campus, nigeria cor r espo nde nc e augustine chinedu ihim department of medical laboratory science, faculty of health sciences and technology, college of health sciences, nnamdi azikiwe university, nnewi campus, p.m.b. 5001, anambra state, nigeria email: ac.ihim@unizik.edu.ng date of first submission, september 18, 2 01 7 date of final revised submission, october 31, 2017 date of acceptance, november 1, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 short-term aerobic exercise does not change kidney function in students of nnamdi azikiwe university, nigeria ihim augustine chinedu*, patrick chinedu obi**, egbe josephat udoka*, nosakhare osakue*, donatus f. ozuruoke***, emmanuel i. nwobodo†, samuel c. meludu††, and okeke chizoba okechukwu* background exercise has been known to have beneficial effects on human health. the kidneys play an important role in regulating acid-base and water-electrolyte balance disturbances induced by exercise. the objective of this study was to investigate the effect of short-term aerobic exercise (volleyball training) on the kidney function of apparently healthy individuals. methods an experimental study of pre-post test design was conducted involving 41 amateur volleyball players, comprising 22 males and 19 females. they were randomly divided into seven different teams. each team trained for at least 45 minutes for four consecutive days for two weeks. both pre-and postexercise blood pressure (bp) was measured using an automatic blood pressure measuring device omron 907 (omron, hoofddorp, netherlands). likewise, both preand post-exercise blood samples were collected into lithium heparin tubes and centrifuged at 3000 rpm for 10 minutes and the plasma separated into plain tubes. electrolytes were analysed using ion selective electrode machine (sfri 4000, germany), urea using modified berthelot method, creatinine using jaffe-slot method and uric acid using the uricase method and estimated glomerular filtration state (egfr) was calculated using the modification of diet in renal disease (mdrd) formula. results the mean levels of preand post-exercise systolic blood pressure, creatinine, urea, sodium, potassium, chloride, bicarbonate and egfr did not differ significantly (p>0.05). however, serum uric acid was significantly increased (p<0.05), while diastolic bp significantly decreased after exercise (p<0.05). conclusion the study showed that short-term moderate intensity aerobic exercise does not have any significant effect on the renal functions. keywords: short-term, aerobic exercise, kidney function, students doi: http://dx.doi.org/10.18051/univmed.2017.v36.166-172 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: chinedu ia, obi pc, udoka ej, et al. short term aerobic exerci se d oes not cha nge kidn ey function in students of nnamdi azikiwe university, nigeria. univ med 2017;36: 166-72. doi: 10.18051/univmed.2017. v36.166-172 167 univ med vol. 36 no.3 introduction several studies have reported that exercise is associated with both long-term and short-term metabolic, physiological and hemodynamic changes which can impact either positively or negatively on the health of the exercising subjects. physical activity has been shown to h a ve a p osi t ive e f f e c t o n man y c h r on i c conditions such as cardiovascular disease, chronic kidney disease and diabetes.(1) the benefits of physical activity to renal health have been demonstrated in a few studies but there are still questions which remain unanswered. one of the unanswered questions is related to the longitudinal effects of physical activity on renal function. the few studies which have sought to address this question in humans have revealed conflicting results.(2,3) a study investigated the association between physical a c t i vit y, me a su r e d ob j e c t i vel y wi t h a n accelerometer, and kidney function, and showed that total and light physical activities were found t o b e po s it i ve l y a s so c i a t e d wi t h ki d ne y function.(4) however another study in middle-aged women showed that six months of aerobic training does not induce significant change in estimated glomerular filtration rate and blood urea nitrogen.(5) students in the environment of anambra state, nigeria, are daily involved in physical activities, some of which have become part of their lifestyle (e.g long distance walking, climbing staircases, climbing down and up the hilly environment of the school etc) and some others as recreational activities (volleyball, badminton, soccer and jogging). specifically, volleyball playing is becoming an interesting sport of choice for students in this community. however, no study has been done with regard to its effect on the kidney function in the e nvi ro n me n t o f a na mb ra st a te , n i ge r ia . therefore, this study is designed to assess the a c u t e o r sh or t-te r m e f f e c t s of e x e r c i s e (volleyball practice) on the kidney function of apparently healthy students of nnamdi azikiwe university, nigeria. methods research design this was an experimental pre-post test designed to assess the effect of short term exercise on the kidney function and was conducted from july to september 2016. study area the study was carried out in the college of health sciences, nnamdi azikiwe university, nnewi, anambra state, nigeria. this is a multifaculty college comprising the faculties of health sciences and technology, medicine and basic medical sciences. each of these faculties is made up of numerous departments such as medical laboratory science, anatomy, nursing, medical rehabilitation, medical radiography, physiology, medical biochemistry, pharmacology, community medicine etc. it has sporting facilities including a football field, volleyball court, tennis court etc. study subjects the study participants were students of the college of health sciences, nnamdi azikiwe university nnewi, anambra state. a total of forty-one (41) student amateur volleyball players comprising 22 males and 19 females who volunteered to participate in the study, were recruited for this study. the inclusion criteria were apparently healthy students within the age range of 17–28 years, who play volleyball and were willing to participate in the study. the following subjects were excluded; those suffering from any renal disease or hypertension, those who sustained any form of musculoskeletal injury, alcoholics, smokers and those on any form of medication including herbal remedies. sampling was done by stratified sampling technique, where students were grouped into strata according to their departments. students 168 were selected from each department of the faculty using simple random sampling. exercise potocol t he s ub j e c ts wer e i nstr uc t ed n ot t o participate in any form of sports for one-week. after the one week of rest, the subjects were randomly divided into six (6) teams comprising 6 participants each, while the seventh team was comprised of 5 participants. each team was subjected to volleyball training for a minimum time of 45 mins for four consecutive days in oneweek for two-week periods. measurements anthropometric parameters such as the h e igh t an d we ight of e a ch s ub j e c t we r e measured using a standard stadiometer and a weighing scale before the exercise session and their body mass index (bmi) were calculated from it. subjects’ dietary pattern and lifestyle were also obtained using a well-structured questionnaire. a pre-exercise blood pressure was measured by taking two blood pressure (bp) readings on the right arm placed at the heart level, using an automatic blood pressure measuring device omron 907 (omron, hoofddorp, netherlands) after the subjects had rested for at least 5 minutes in a sitting position upon arrival to the volleyball court. t he measurements were taken 60 seconds apart and the average systolic and diastolic bp were recorded and used for data analyses. postexercise bp were also taken after exercise on the last day. laboratory analysis five ml each of pre-exercise and postexercise blood samples was drawn from the ante-cubital vein of each of the participants before the commencement of the exercises. the blood samples were collected into lithium heparin anticoagulant containers before commencement of the exercise session and on the last day of the session after exercise. the blood sample was then centrifuged at 3000 rpm for 5 minutes and then the plasma was extracted and kept frozen until biochemical analyses were carried out. urea estimation was carried out using the modified urease-berthelot methods.(6) the jaffeslot method was us ed to estimate the concentration of creatinine.(7) estimation of uric acid was determined by the uricase based methods.(8) the electrolytes assayed include sodium (na +), potassium (k +), chloride (cl-) and bicarbonate (hco3) and this was done using ion selective electrode (ise). the estimated glomerular filtration rate (egfr) was calculated using an online calculator for the modification of diet in renal disease (mdrd) formula for adults.(9) the formula is given as: gfr (ml/min /1.73m2) = 186 x [plasma creatinine (µmol/l)]-1.154 x [age]-0.203 x [1.210 (if black)] x [0.742 (if female)]. statistical analysis statistical package for social science students (spss) version 16.0 was used for the analysis of the results. data were presented as mean ± standard deviation (sd) and student’s paired t-test and independent t-test were used to determine level of significance set at p<0.05. ethical consideration ethical approval was sought and obtained from the ethics committee of the faculty of health sciences, nnamdi azikiwe university (no 2011614024p). an informed consent of all participants was obtained before they were recruited for the study. results t he r e su l t s o f a n t hr op ome t r i c measurements of the participants showed that there was no statistically significant difference in the mean values of the male and female participants’ age (23.23 ± 2.30 years; 21.74 ± 1.33 years), height (1.76 ± 0.05 m; 1.67 ± 0.06 m) and bmi (23.05 ± 1.68 kg/m2; 22.39 ± 3.22 chinedu, obi, udoka, et al short term aerobic exercise and kidney function 169 univ med vol. 36 no.3 a b c kg/m2), respectively (p>0.05) except in their weight (71.23 ± 7.19 kg; 62.37 ± 10.04 kg) (p<0.05) (table 1). as shown in table 2, their diastolic bp was significantly lower after exercise (p=0.021). however, the pre and post exercise systolic bp showed no significant difference (p=0.087). t he p o st e xe r c i se ur i c ac i d me a n concentration was higher than the pre-exercise va lue (p=0.011 ). however, there was no significant difference in the mean values of electrolytes, urea and creatinine (p>0.05). there was also no significant difference in the mean values of the participants’ gfr before and after exercise (p=0.548) (table 2). discussion several reports have shown that duration and/or intensity of exercise elicit different effects on minerals metabolism and that inadequate status of the body mineral composition can lead to a diminution of performance and endurance both in sportsmen and rats.(10,11) longer duration of exercise is needed to elicit a significant decrease in some biochemical parameters. (12) during aerobic exercise, systolic bp increases, since as the exercise intensity increases the heart works harder to pump more oxygenated blood to the muscles. at the same time, diastolic bp remains relatively stable and may even decrease slightly. on average, men have higher bp than women during aerobic exercise.(13) such changes could have transient effects on the gfr as well as the renal function. the findings from this work revealed that during short-term volleyball exercise, diastolic pressure decreased significantly. this drop in the diastolic pressure can be attributed primarily to the vasodilation of the arteries from the exercise bout which causes a reduction in peripheral resistance. it could also be due to a decrease i n the blood volume caused by dehydration from water loss after exercise. this finding is in consonance with the findings of syme et al.(14) who conducted a study and showed a decrease in diastolic blood pressure post exercise. however, there was also a small parameter male (n=22) female (n=19) p value age (years) 23.23 ± 2.30 21.74 ± 1.33 0.087 weight (kg) 71.23 ± 7.19 62.37 ± 10.04 0.021* height (m) 1.76 ± 0.05 1.67 ± 0.06 0.611 bmi (kg/m2) 23.05 ±1.68 22.39 ± 3.22 0.624 table 1. the distribution of mean values of anthropometric measurements in the subjects bmi : body mass index; significant values set at p<0.05; *significant parameters pre-exercise (n=41) post-exercise (n=41) p value sbp (mmhg) 112.71 ± 9.00 116.10 ± 12.74 0.087 dbp(mmhg) 79.98 ± 11.61 76.39 ± 8.20 0.021* na+ (mmol/l) 139.14 ± 2.36 138.41 ± 2.56 0.143 k+ (mmol/l) 3.61 ± 0.30 3.61 ± 0.46 0.991 cl(mmol/l) 102.89 ± 6.59 101.44 ± 2.01 0.154 hco3 (mmol/l) 21.81 ± 2.58 22.70 ± 2.55 0.078 urea (mmol/l) 3.60 ± 0.88 3.79 ± 0.83 0.133 uric acid (mmol/l) 326.47 ± 109.20 380.04 ± 54.58 0.011* creatinine (µmol/l) 72.36 ± 14.24 73.22 ±18.72 0.717 gfr (ml/min/1.73m2) 135.51 ± 54.96 129.27 ± 27.50 0.548 sbp= systolic blood pressure; dbp= diastolic blood pressure; gfr= glomerular filtration rate; significant values set at p<0.05. *significant table 2. the blood pressure and renal function test of the subjects before and after exercise 170 but insignificant increase in their systolic blood pressure. the mean concentrations of electrolytes after the exercise session as compared to the pre-exercise values, showed no significant change. it was observed that although there was a slight decrease in the mean concentrations of sodium and chloride ions after the volleyball exercise, this decrease was not significant. this slight but insignificant decrease in sodium can be attributed to minute loss of sodium in sweat d ur ing t he e x e rc is e . al s o t h e s l i ght b u t insignificant reduction in the chloride ion can be explained by the fact that during exercise, reduction in sodium is accompanied by reduction in chloride as both sodium and chloride ions are the main constituents of sweat. it was also found that there was a positive correlation between the preand post-exercise concentrations of sodium and chloride. these findings were in agreement with those of baydil,(15) who reported that the change in the mean concentration of sodium and chloride ions in individuals who exercised to the point of exhaustion were not significant. however, another study showed that e x e r c i s e r e du c e d s o di u m a n d c hl o r i de concentration significantly.(16) during exercise, muscle k+ efflux increases with increasing exercise intensity, and therefore maximal exercise induces a marked elevation in arterial plasma potassium ion.(17) however, the present study revealed that there was no change between the pre-exercise mean concentration of potassium ion and the post-exercise concentration. this can be explained by the fact that the changes in extracellular potassium ion concentrations are initially buffered by movement of potassium into and out of the skeletal muscle, a phenomenon known as internal potassium balance. it may be also have been due to the fact that the exercise was moderate in intensity and lasted for forty-five minutes. this does not support the observations made by several studies (15,18) that reported a significant increase in potassium concentration following exercise. similarly, the post-exercise bicarbonate concentration changes in this work showed a small but insignificant increase when compared to the pre-exercise value. t his increase can be attributed to the physiological re-absorptive adjustment made by the kidneys in order to buffer the blood ph which tends to acidity as a result of increased co 2 production from respiration during the exercise session. this observation is in conformity with the findings made by ugwuja et al.,(19) who reported that exercise caused no significant change in the serum concentrations of bicarbonate. glomerular filtration is one of the key functions of the kidney and assessing the glomerular filtration rate has important clinical implications. previous work reported a decrease in gfr after exercise.(20) according to the report, this decrease is dependent on the exercise intensity. this finding can be explained by the fact that during exercise, the renal blood flow tends to reduce as a result of the effect of the sympathetic nervous system and the release of catecholamines which induces the reduction of blood flow to the kidneys, which in turn de cr ea se s th e gl omeru la r fi ltr a tion r at e. however, the work by ayca et al.(21) revealed that the glomerular filtration rate of individuals performing volleyball exercise decreased slightly but not significantly. this insignificant decrease can be attributed to the moderate intensity and short duration of the exercise and possibly to the use of serum creatinine and mdrs formula in calculating gfr. furthermore, the present study showed that the mean concentrations of creatinine and u re a be f or e an d af te r e xe rc is e wer e not significantly increased. this can be attributed to the moderate intensity and short duration of the exercise and also to the reduced renal blood perfusion and slight decrease observed in the gfr of the participants after the volleyball exercise. a previous study conducted on halfmarathon athletes reported an increase in gfr and creatinine clearance which contradicts the observation made in our study.(22) chinedu, obi, udoka, et al short term aerobic exercise and kidney function 171 univ med vol. 36 no.3 in the present study a significant increase in the mean concentration of uric acid of the participants was observed. this is in line with the findings of dunlap et al.(23) the increase can be explained by the fact that during short and long term exercise, the increase in uric acid levels serves as a protective mechanism against exercise induced oxidative stress. it was also observed in the present study that the pre-creatinine concentration correlated positively and significantly with age, height, weight and bmi, whereas no correlation was found between these parameters and the preexercise blood urea concentration. this is in accordance with previous studies.(24,25) meyer e t a l . ( 2 6 ) r e po r t e d t h a t s e r u m c r e a ti n i ne concentration varies closely with the skeletal muscle mass. also the study showed that the post-exercise concentration of creatinine was correlated negatively and significantly with the post-exercise gfr value, whereas there was no significant correlation between the preexercise concentrations. the limitation of this study was that the exercise was a short term plan and could have affected the study results that there was no significant change in the kidney function of the participants. moreover the intensity of the volley ball training was not considered. a prospective study in this line should endeavour to consider using a larger sample size, engage participants on long term exercise (volley ball) for a month and ensure that training intensity is consistent. conclusion this study found that short-term aerobic exercise (volleyball training) caused slight but insignificant changes in the renal function tests, showing that invariably it caused no renal function impairment. conflict of interest the authors declare that no conflict of interest exists. acknowledgement we appreciate greatly the management of reene medical diagnostic centre, awada obosi, idemili north lga, anambra state for granting us permission to use their laboratory for the biochemical analysis of the samples. contributors aci, smc and no contributed to draft the ma nu s c r ip t . aci, s mc, no a n d do f contributed to conception and design of the study. ef, eni and oco contributed to acquisition of the data. smc, aci and pco contributed to data analysis and interpretation. aci, ej, rni, oco and smc contributed to revising the manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. cigolle ct, blaum cs, halter jb. diabetes and cardiovascular disease prevention in older adults. clin ger med 2009;25:607-41. 2. white sl. physical inactivity and chronic kidney disease in australian adults: the aus diab study. nutr metab cardiovascular dis 2009;12:125-30. 3. robinson-cohen c. physical activity and rapid decline in kidney function among older adults. arch int med 2009;169:2116-23. 4. hawkins ms, sevick ma, richardson cr, et al. association between physical activity and kidney function: national health and nutrition examination survey. med sci sports exerc 2011;43:1457-64. 5. bijeh n, farahati s. the effect of six months of aerobic training on renal function markers in untrained middle-aged women. intl j sport stud 2013;3:218-24. 6. pandya d, nagrajappa ak, ravi ks. assessment and correlation of urea and creatinine levels in saliva and serum of patients with chronic kidney disease, diabetes and hypertension: a research study. j clin diagn res 2016;10:zc58-6. 7. kakkar m, kakkar r. a comparative study on the estimation of serum creatinine levels by jaffe’s and enzymatic methods at different levels of serum bilirubin. int j clin biochem res 2017;4:305-30. doi: 10.18231/2394-6377.2017.0071. 172 8. zhao y, yang x, lu w, et al. uricase based methods for determination of uric acid in serum. microchim acta 2009;164:1–6. doi: 10.1007/s00604 -008-0044-z. 9. levey as, bosch jp, lewis jb, et al. modification of diet in renal disease study group: a more accurate method to estimate glomerular filtration rate from serum creatinine: a new prediction equation. ann intern med 1999;130:461-70. 10. nielsen fh, lukaski hc. update on the relationship between magnesium and exercise. magnes res 2006;19:180-9. 11. rakhra g, masih d, vats a, et al. copper, iron, zinc and magnesium status of physically active young indian males. j sports phys educ 2015;2: 25-32. 12. ihim ac, meludu sc, obiodinukwe cm, et al. effect of short-term exercise on serum cortisol and lipid profile in young male of college of health sciences, nnamdi azikiwe university, nnewi campus. indian j basic ap med res 2015;4:2138. 13. brooks ga, fahey td, baldwin km, editors. human bioenergetics and its applications. in: exercise physiology. 4th ed. mc graw hill; new york;2005.p.122-5. 14. syme an, blanchard be, guidry ma, et al. peak systolic blood pressure on a graded maximal exercise test and the blood pressure response to an acute bout of submaximal exercise. am j cardiol 2006;98:938-43. 15. baydil b. serum macro-micro element response to acute maximal physical exercise. world appl sci j 2013;2:945-9. 16. anastasiou ca, kavouras sa, arnaoutis g, et al. sodium replacement and plasma sodium drop during exercise in the heat when fluid intake matches fluid loss. j athletic train 2009;44:11723. 17. harmer ar, patricia ar, michael j, et al. effects of sprint training on extrarenal potassium regulation with intense exercise in type 1 diabetes. j appl physiol 2006;100:26-34. 18. cinar v, baltaci ak, mogulkoc r. effect of exhausting exercise and calcium supplementation on potassium, magnesium, copper, zinc and calcium levels in athletes. pak j med sci 2009;25: 238-42. 19. ugwuja se, obeagu ei, ochei kc, et al. effect of physical exercises on serum electrolyte. j dent med sci 2014;13:118-21. 20. morales ap, sampaio-jorge f, da cruz rangel lf, et al. cardiopulmonary performance during maximal exercise in soccer players with alterations in renal function. j hum kinet 2017;57:107-15. doi: 10.1515/hukin-2017-0052. 21. ayca b, sener a, apikogl-rabus s, et al. the effect of exercise on urinary gamma-glutamyl transferase and protein levels of volley ball players. j sports med phys fitness 2006;46:623-7. 22. lippi g, schena f, salvagno gl. acute variation of estimated glomerular filtration rate following a half-marathon run. int j sports med 2008;29:94851. doi: 10.1055/s-2008-1038745. 23. dunlap ke, reynolds aj, duffy lk. total antioxidant power in sled dogs supplemented with blueberries and the comparison of blood parameters associated with exercise. comp biochem physiol a mol integr physiol 2006;143: 429-34. 24. traynor j, mactier r, geddes cc, et al. how to measure renal function in clinical practice. bmj 2006;333: 733-737. 25. lamb ej, tomson cr, roderick pj. estimating kidney function in adults using formulae. ann clin biochem 2005;42:321-45. 26. myers gl, miller wg, coresh j. recommendations for improving serum creatinine measurement: a report from the laboratory working group of the national kidney disease education program. clin chem 2006;52:5-18. chinedu, obi, udoka, et al short term aerobic exercise and kidney function oktavianus 43 *division of endocrinology and metabolic, department of internal medicine, faculty of medicine, university of sumatera utara medan **division of hematology oncology medic, department of internal medicine, faculty of medicine, university of sumatera utara medan correspondence dr. mardiya sari division of endocrinology and metabolic, department of internal medicine, faculty of medicine, university of sumatera utara jl. bunga lau no.17 medan sumatera utara 20136 fax: 6261-8363009 email: dr.mardiya@gmail.com univ med 2014;33:43-8 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background mean platelet volume (mpv) shows the average size of platelets in the circulation and can be used to assess the activity of platelets. increased of mpv can be sign of atherotrombosis and can be used to assess the risk of vascular complications such as retinopathy diabetic (rd). the aim of this study was to determine correlation between glycated haemoglobin (hba1c), mpv with degree of retinopathy in diabetes mellitus (dm) patients. methods a cross sectional study from june until july 2013 was conducted involving 77 dm subjects. anamnesis, laboratory examination and funduscopy had been done in all subjects. one way anova was used to assess the differences between mpv with other parameters to the degree of retinopathy and continued with bonferroni test to assess the differences between the degree of retinopathy with the parameters that significantly different in one way anova. results one way anova showed significance difference of mean mpv between normal funduscopy group compared than proliferative diabetic retinopathy (pdr) group (9.57 ± 0.63 fl vs 10.45 ± 0.51 fl, p=0.044). pdr group were older (p=0.001), longer suffered from dm (p=0.001) and hypertension (p=0.011). bonferroni test showed no significance difference of mean mpv between normal funduscopy versus non-proliferative diabetic retinopathy (npdr) group (p=0.290) and npdr versus pdr (p=0.409). conclusion there was a significance differences between mpv with the degree of retinopathy. platelets may play a role in the pathogenesis of vascular complications and that mpv can be used as simple parameter to assess the vascular events in dm. keywords: retinopathy, mean platelet volume, diabetes mellitus, adults mean platelet volume increases in proliferative retinopathy among diabetes mellitus subjects mardiya sari*, dharma lindarto*, and dairion gatot** 44 sari, lindarto, gatot mean platelet volume in retinopathy mean platelet volume meningkat pada retinopati proliferatif di antara subyek diabetes melitus latar belakang mean platelet volume (mpv) merupakan gambaran ukuran rata-rata trombosit di sirkulasi dan dapat digunakan untuk menilai aktivitas trombosit. peningkatan nilai mpv dapat menggambarkan aterotrombosis dan dapat digunakan untuk menilai risiko komplikasi vaskular seperti retinopati pada penderita diabetes mellitus (dm). tujuan penelitian ini adalah untuk menentukan adanya hubungan antara glycated haemoglobin hba1c, mpv dan derajat retinopati pada penderita dm. metode penelitian potong lintang dilakukan dengan mengikut sertakan 77 penderita dm yang dilaksanakan pada bulan juni juli 2013. anamnesis, pemeriksaan laboratorium dan funduskopi dilakukan pada semua subjek. one way anova digunakan untuk menguji perbedaan antara mpv dan parameter lainnya dengan derajat retinopati dan dilanjutkan dengan uji bonferroni untuk menguji perbedaan antara derajat retinopati dengan parameter yang berbeda signifikan pada analisis one way anova. hasil one way anova menunjukkan perbedaan signifikan nilai mean mpv pada grup funduskopi normal dibandingkan grup proliferative diabetic retinopathy (pdr) (9,57± 0,63 fl vs 10,45 ± 0,51 fl, p=0,044). grup pdr berusia lebih tua (p=0,001), lebih lama menderita dm (p=0,001) dan disertai dengan hipertensi (p=0,011). uji bonferroni menunjukkan tidak dijumpai perbedaan signifikan mean mpv antara grup funduskopi normal versus non-proliferative diabetic retinopathy (npdr) (p=0,290) dan grup npdr versus pdr (p=0,409). kesimpulan ada perbedaan yang signifikan antara mpv dengan derajat retinopati. trombosit berperan dalam patogenesis terjadinya komplikasi vaskular sehingga mpv dapat digunakan sebagai parameter yang sederhana untuk menilai komplikasi vaskuler pada dm. kata kunci: retinopati, mean platelet volume, diabetes melitus, dewasa abstrak introduction diabetes mellitus (dm) according to the american diabetes association (ada) is a group of metabolic diseases with characteristic of hyperglycemia that occurs due to abnormal insulin secretion, insulin action or both. dm is a major global health problem.(1) one of the most common microvascular complications of dm is diabetic retinopathy (dr). diabetic retinopathy is a chronic progressive, potentially sight-threatening disease of the retinal microvasculature associated with the prolonged hyperglycaemia and other conditions linked to diabetes mellitus such as hypertension.(2) it is the most common cause of new blindness in adults with range age of 20–74 years. almost all of patients with type 1diabetes and over than 60% of patients with type 2 diabetes will develop to retinopathy after two decades of suffering from dm, even 25% of patients with type 2 diabetes had been suffered from retinopathy at the time of diagnosis dm. dr associated with poor blood sugar control, duration of dm and hypertension.(3) 45 numerous studies have been done on platelet parameters and function in diabetes and most had shown increased platelet aggregation and activated platelet state. several things may explain this occurrence are due to the formation of immature platelets with larger size and more r e a c t i v e i n t h e b o n e m a r r o w, m e t a b o l i c environment of dm and blood vessel damage.(4,5) mean platelet volume (mpv) is a picture of the average size of platelets in the circulation and can be used to assess the activity of platelets.(6,7) shimoidara et al.(7) reported higher mpv in prediabetes subjects compared to normal subjects and the increase of mpv was strongly correlated with fasting blood sugar in prediabetes subjects. some studies carried out by papanas et al.,(8) zuberi et al.(9) and jindal et al.(10) reported that mpv was higher in dm rather than nondm subjects. demirtunc et al.(11) and kodiatte et al.(12) reported that mpv had significant correlation with glycated haemoglobin (hbaic). ates et al.(13) reported that the increase of mpv had significant correlation with the degree of retinopathy and play a role in the pathogenesis of vascular complications so that mpv could be useful in monitoring the progression of dm. however, kodiatte et al.(12) and hekimsoy et al.(14) found no significant correlation between the increase of mpv with the degree of retinopathy. this study was conducted because so far researchers have not found any study in indonesia that report the correlation between hba1c, mpv and degree of retinopathy in dm patients. methods design of the study this study was an analytical of crosssectional design and was carried out at h adam malik hospitals, medan between june until july 2013. study subjects the target population comprised patients with dm. diagnosis of dm was established using the ada criteria. inclusion criteria were subjects with aged >17 years in both male and female and agreed to follow the study. exclusion criteria were diabetes patients who were taking anti-platelet and anticoagulant, malignancies, anemia (hb <12 g/dl for males and <11 g/dl for females) and pulmonary tuberculosis. the study s u b j e c t s w e r e s e l e c t e d b y n o n r a n d o m consecutive sampling. we used the sample size calculation method with the estimation of coefficient correlation from previously study r=0.39. we calculated that at least 68 subjects to enter the study with á =0.05 (z á =1.96) and power of study 90% (z â =1.282). data collection all subjects were interviewed by means of a questionnaire whose items included the subject’s name, age, gender, duration of diabetes, history of hypertension, medication that were taking, smoker or non smoker. the subject’s weight was measured using omron scales and height was determined by a kenko height measuring instrument. direct fundoscopy was carried out and classified as normofunduscopy, non-proliferative diabetic retinopathy (npdr) and proliferative diabetic retinopathy (pdr). biochemical measurements all subjects had been fasting for 10 hours and blood samples were taken from vena mediana cubiti for biochemical measurements. full blood count, fasting plasma glucose (fpg), t w o h o u r s p o s t p r a n d i a l b l o o d g l u c o s e , glycated haemoglobin (hba1c) and lipid profile [total cholesterol, triglycerides, low density lipoprotein (ldl) cholesterol, high density lipoprotein (hdl) cholesterol] of all subjects were measured using automatic cobass 6000 c501 system. mpv (fl) involved in the direct measurement of platelet size from the whole blood. data analysis one way anova was used to assess the differences between the degree of retinopathy and univ med vol. 33 no.1 46 sari, lindarto, gatot mean platelet volume in retinopathy v a r i o u s p a r a m e t e r s t h a t w e r e n o r m a l l y distributed and the kruskal-wallis test was used for parameters that are not normally distributed. bonferroni test was used to assess the parameters that significantly different by one way anova or. statistical analysis was performed using spss version 15.0, with the level of significance set at p<0.05. ethical clearance the study protocol was approved by the health research ethical committee of north sumatra university, faculty of medicine. all study subjects signed written informed consent after having been informed about the aims and benefits of the study. results there were 77 subjects met the inclusion criteria and willing to participate in this study. among the 77 subjects, 33 (42.86%) were males and 44 (57.14%) were females with range of age 42-75 years. seventeen (22.0%) subjects of normal funduscopy, 50 (65.0%) subject of npdr and 10 (13.0%) subjects of pdr. pdr group were significantly older (64.90 ± 6.89 years; p=0.001), longer suffered from dm (18.6 ± 6.7 years; p=0.0001). in the pdr group, the mean of mpv was higher (10.45 ± 0 . 5 1 f l ; p = 0 . 0 4 6 ) c o m p a r e d t h a n n o r m a l funduscopy group and npdr group. mean fpg, 2-hour post-prandial and hba1c were higher in group npdr and pdr compared normofundus but not statistically significant (table 1). bonferroni test showed significance different of mean mpv between normal funduscopy group versus pdr (p=0.044), but there were no significance different of mean mpv between normal funduscopy group versus npdr (p=0.290) and npdr versus pdr group (p=0.409) (table 2). tabel 1. distribution of demographic and clinical characteristics subjects by funduscopic groups values are numbers (%); mean ± sd fpg=fasting plasma glucose; pppg=post prandial plasma glucose; bmi= body mass index; hdl= high density lipoprotein; ldl= low high density lipoprotein; npdr=non-proliferative diabetic retinopathy; pdr=proliferative diabetic retinopathy; * significant 47 discussion this study found that pdr group had the highest mpv value compared than npdr and normofunduscopy group. this is consistent with the study conducted by ates et al.(13) that found a positive correlation between mpv values with degrees of retinopathy in diabetic patients. sustained hyperglycemia leads to a series of interrelated alterations that can cause endothelial dysfunction and vascular lesion in diabetic c o m p l i c a t i o n s . f o r m a t i o n o f a d v a n c e d glycalation end products, activation of protein kinase c and disturbance in polyol pathways are mechanisms by which increased glucose induces vascular abnormalities.(15-18) it can be explained because dm is a prothrombotic that chronically activate platelets, activate the coagulation system and decrease the ability of fibrinolysis. these phenomena, together w i t h i m p a i r e d p r o s t a n o i d m e t a b o l i s m , phosphoinositide turnover and enhanced calcium mobilization contribute to enhanced risk of small vessel occlusions.(17,19) mpv is an indicator of the average size and activity of platelets. mpv value above the upper limit normal suggests that platelets in the circulation were younger, bigger and more reactive to aggregate because it will secrete more serotonin, â-thromboglobulin and produce more thromboxane a2 than normal platelets. this will produce a procoagulant effect and lead to vascular complications. thus, platelets may assume an important role in signaling of the d e v e l o p m e n t m i c r o a n d m a c r o v a s c u l a r complications in dm such as retinopathy.(16-18) as it suggests a relationship between the increase of mpv with retinopathy. so mpv can be used as a simple economical test in the monitoring of dm and thereby help curb the morbidity and mortality.(9) occlusions and micro aneurysms result in hypoxia in diabetic retinopathy which is a strong stimulus for new vessel formation. vascular endothelial growth factor, which is released in r e s p o n s e t o h y p o x i a , s t r o n g l y i n d u c e s neovascularization. platelet-derived growth f a c t o r, t r a n s f o r m i n g g r o w t h f a c t o rb e t a , epidermal growth factor, insulin-like growth factor-1, growth hormone and basic fibroblast growth factor induce collagen synthesis and c a u s e p r o l i f e r a t i v e r e t i n o p a t h y v i a neovascularization. diabetic retinopathy begins with mild non-proliferative changes characterized by increased vascular permeability and advances t o m o d e r a t e t o s e v e r e n o n p r o l i f e r a t i v e retinopathy characterized by vascular occlusions, microaneurysms, punctual hemorrhages, cotton wool spots and to a more severe form, the proliferative retinopathy, which is characterized by neovascularization.(2,3,13) several studies s h o w e d p l a t e l e t s a c c u m u l a t e i n r e t i n a l vasculature and induce the release of local growth factors by causing inflammation.(5) however, studies conducted by kodiatte et al.(12) and hekimsoy et al.(14) had not found significance correlation between the increase of mpv with the duration of dm and had not found significance different of mpv in diabetic retinopathy. in contrast, studies conducted by demirtunc et al.(11) and kodiatte ta et al.(12) had found significance correlation between mpv and hba1c. limitation of this study are the small size of the study population, being restricted to one central hospitals and did not consider the administration of insulin or anti-diabetic drug that were taken by the subjects. as some previous studies reported that there are a number of antidiabetic drugs can inhibit hyperagregation of platelet. thus, further studies are needed to assess this. table 2. multivariat comparisons with bonferroni test variable funduscop ic p val ue mpv (fl) n ormal vs npdr 0.290 n ormal vs pdr 0.044* npdr vs pdr 0.409 npdr=non-proliferative diabetic retinopathy; pdr= proliferative diabetic retinopathy univ med vol. 33 no.1 48 sari, lindarto, gatot mean platelet volume in retinopathy conclusion t h i s s t u d y d e m o n s t r a t e d t h a t m p v increases in proliferative retinopathy among subjects with diabetes melittlus. mpv can be used as a simple economical test in the monitoring of dm and thereby help curb the morbidity and mortality. acknowledgments we thank to all study subjects who agreed to participate in the present study and all colleagues who assisted in the recruitment of study subjects. references 1. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2013;36:67-74. 2. the royal collage of ophthalmologists. diabetic retinopathy guidelines. london: scientific department the royal college of ophthalmologists;2012. 3. fong ds, aiello l, gardner fw, king gl, blankenship gl, cavallerano jd, et al. retinopathy in diabetes. diabetes care 2004;27 suppl 1:s 84-7. 4. yngen m. platelet function in diabetes mellitus. intern j endocrinol 2005;12:109-16. 5. yamashiro k, tsujikawa a, ishida s, usui t, kaji y, honda y, et al. platelets accumulate in the diabetic retinal vasculature following endothelial death and suppress bloood retinal barrier breakdown. am j pathol 2003;163:25359. 6. kosus a, kosus n, duran m, turhan no. assessment of mean platelet volume of pregnant woman with gestasional diabetes mellitus and impaired glucose tolerance as a marker of future cardiovaskular disease risk. br j diabetes vasc dis 2010;10:233-7. 7. shimodaira m, niwa t, nakajima k, kobayashi m, hanyu n, nakayama t. correlation between mean platelet volume and fasting plasma glucose levels in prediabetic and normoglycemic individuals. cardiovasc diabetol 2013;12:14. doi: 10.1186/1475-2840-12-14. 8. papanas n, symeonidis g, maltezos e, mavridis g, karavageli e, vosnadikis t, et al. mean platelet volume in patients with type 2 diabetes mellitus. platelets 2004;15:475-8. 9. zuberi bf, akhtar n, afsar s. comparison of mean platelet volume in patients with diabetes mellitus, impaired fasting glucose and non diabetic subjects. singapore med j 2008;49:1146. 10. jindal s, gupta s, gupta r. platelet indices in diabetes mellitus: indicators of diabetic microvascular complications. hematology 2011; 16:86-9. 11. demirtunc r, duman d, basar m, bilgi m, teomete m, garip t. the relationship between glycemic control and platelet activity in type 2 diabetes mellitus. j diabetes complications 2009;23:89-94. 12. kodiatte ta, manikyam uk, rao sb, jagadish tm, redy m, lingaiah hkm, et al. mean platelet volume in type 2 diabetes mellitus. j lab physicians 2012;4:5-9. 13. ates o, kiki i, bilen h, keles m, kocer i, kulacoglu dn, et al. association of mean platelet volume with the degree of retinopathy in patients with diabetes mellitus. eur j gen med 2009;6: 99-102. 14. hekimsoy z, payzin b, ornek t, kandogan g. mean platelet volume in type 2 diabetic patients. j diabetes complications 2004;18:173-6. 15. ferroni p, basili s, falco a, davi g. platelet activation in type 2 diabetes mellitus. j thromb haemost 2004;2:1282-91. 16. ferreiro jl, gomez ja, angiolillo dj. platelet abnormalities in diabetes mellitus. diabetes vasc dis res 2010;7:251-9. 17. kakouros n, rade jj, kourliouros a, resar jr. platelet function in patients with diabetes mellitus: from a theoritical to a practical perspective. intern j endocrinol 2011. article id 742719. doi:10.1155/2011/742719. 18. schneider dj. factors contributing to increased platelet reactivity in people with diabetes. diabetes care 2009;32:525-7. 19. wilkinson cp, ferris fl, klein re, lee pp, agarhd cd, davis m, et al. global diabetic retinopathy project group: proposed international clinical diabetic retinopathy and diabetic macular edema disease severity scales. ophtalmol 2003; 110:1677-82. rita k 120 *pusat penelitian dan pengembangan biomedis dan farmasi, badan penelitian dan pengembangan kesehatan departemen kesehatan r.i. korespondensi adr. lusianawaty tana, ms, sp.ok pusat penelitian dan pengembangan biomedis dan farmasi, badan penelitian dan pengembangan kesehatan departemen kesehatan r.i. jl. percatakan negara no.23a jakarta telp. 021-6500266 email: lusianawaty@litbang.depkes.go.id universa medicina 2007; 26: 120-8 universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 merokok dan usia sebagai faktor risiko katarak pada pekerja berusia ≥≥≥≥≥ 30 tahun di bidang pertanian lusianawaty tana*a, laurentia mihardja*, dan lutfah rif’ati* abstrak latar belakang ruang lingkup penelitian ini adalah peranan faktor merokok dan katarak pada pekerja di bidang pertanian di kabupaten karawang. katarak adalah kelainan mata berupa kekeruhan lensa, yang dapat mengganggu penglihatan bahkan sampai buta. 16% dari jumlah buta katarak di indonesia terjadi di usia produktif. salah satu tujuan penelitian adalah mendapatkan hubungan antara faktor merokok dengan katarak dalam rangka memperlambat katarak. metode rancangan adalah belah lintang. sampel penelitian adalah petani dan keluarganya di kecamatan teluk jambe barat kabupaten karawang, dengan usia 30 tahun ke atas, yang terpilih secara purposive random sampling. data diperoleh dengan wawancara, pemeriksaan dan pengukuran. diagnosis katarak ditentukan oleh dokter spesialis mata dengan ophthalmoscope tanpa midriatika. hasil di samping faktor usia, faktor merokok mempunyai hubungan positif dengan katarak. katarak berhubungan positip dengan merokok. semakin berat derajat merokok maka semakin tinggi katarak. kesimpulan usia dan merokok merupakan faktor risiko yang berhubungan positif dengan katarak pada pekerja di bidang pertanian. kata kunci: katarak, petani, merokok, karawang 121 universa medicina vol. 26 no.3 *pusat penelitian dan pengembangan biomedis dan farmasi, badan penelitian dan pengembangan kesehatan departemen kesehatan r.i. korespondensi adr. lusianawaty tana, ms, sp.ok centre biomedic and farmacy research and development national institute of health research and development ministry of health r.i. jl. percatakan negara no.23a jakarta phone. 021-6500266 email: lusianawaty@litbang.depkes.go.id universa medicina 2007; 26: 120-8 background the purpose of this study assesed a relation of smoking factor to the cataract in agriculture farmers and their families, in teluk jambe barat in karawang subdistrict. methods a cross sectional designed study was done, on 2005, with 1223 samples aged 30 years and up among agriculture farmers and their families in 4 villages, selected with purposive random sampling methods. data collection was done through interview, measurement, and examination. cataract was diagnosed through ophthalmoscopic examination without midriatic by ophthalmologists. results the study found that besides age factor, the smoking factor had a positive relation to the cataract. the cataract in the heavier smoker’s group was higher than the cataract in the light smoker’s group. conclusions age and smoking were the risk factors of cataract in agriculture farmers. keywords: smoking, cataract, farmers, karawang smoking and age as risk factors of cataract in agriculture farmers aged 30 years and over lusianawaty tana*a, laurentia mihardja*, and lutfah rif’ati* abstract pendahuluan katarak adalah kekeruhan pada lensa mata y a n g d i s e b a b k a n o l e h a d a n y a p e m e c a h a n p r o t e i n a t a u b a h a n l a i n n y a a k i b a t p r o s e s o k s i d a s i d a n f o t o o k s i d a s i . k a t a r a k t i d a k menimbulkan gejala rasa sakit tetapi dapat mengganggu penglihatan, dari penglihatan kabur sampai menjadi buta.(1-3) jumlah buta katarak di indonesia, terdapat 16% buta katarak pada usia produktif (40-54 tahun), pada hal sebagai penyakit degeneratif buta katarak u m u m n y a t e r j a d i p a d a u s i a l a n j u t . ( 2 ) h a s i l s u r v e i k e s e h a t a n r u m a h ta n g g a s u r v e i kesehatan nasional (skrt-surkesnas) tahun 2001 menunjukkan prevalensi katarak di indonesia adalah sebesar 4,9%. prevalensi katarak di jawa bali sebesar 5,5% lebih tinggi dibandingkan dengan daerah indonesia lainnya. prevalensi katarak di daerah pedesaan 6,29% lebih tinggi jika dibandingkan daerah perkotaan 4,5%.(4) prevalensi katarak di jawa barat tahun 1993-1996 sebesar 6,2%-9,7%.(3) etiologi katarak masih tidak jelas dan dihubungkan dengan banyak faktor.(5) penyebab katarak yang utama adalah proses alamiah 122 metode rancangan penelitian rancangan penelitian yang digunakan adalah belah lintang (cross sectional). subyek penelitian populasi adalah petani dan keluarganya di teluk jambe barat kabupaten karawang. petani d a n k e l u a rg a n y a d i p i l i h s e b a g a i p o p u l a s i p e n e l i t i a n d e n g a n p e r t i m b a n g a n b a h w a p e n d u d u k d i d a e r a h i n i s e b a g i a n b e s a r mempunyai pekerjaan sebagai petani, yang terpajan sinar matahari pada saat melakukan pekerjaannya. responden dipilih berdasarkan purposive random sampling, dengan kriteria inklusi yaitu berumur minimal 30 tahun ke atas, berdomisili dan beraktifitas di daerah karawang, dan bersedia ikut serta dalam penelitian dengan menandatangani informed consent. kriteria eksklusi adalah menderita penyakit berat, demensia, buta karena cacat tidak mempunyai kedua bola mata dan sudah operasi katarak/lensa mata pada kedua mata. besar sampel ditentukan dengan rumus : dengan bertambah lanjutnya usia menimbulkan perubahan pada mata. banyak faktor lain yang b e r p e n g a r u h t e r h a d a p t e r j a d i n y a k a t a r a k , a n t a r a l a i n p e n y a k i t d i a b e t e s m e l i t u s , pemakaian steroid yang lama, kelainan bawaan metabolisme, pajanan kronis terhadap sinar ultra violet (sinar matahari), riwayat katarak p a d a k e l u a r g a , m y o p i a , a l k o h o l , n u t r i s i , m e r o k o k , d e r a j a t s o s i a l e k o n o m i , s t a t u s pendidikan, dan multivitamin.(5-8) sebagai salah satu faktor risiko katarak, m e r o k o k b e r h u b u n g a n d e n g a n t e r j a d i n y a peningkatan katarak, yaitu antara 1,5 sampai 2,9 kali dibandingkan yang tidak merokok.(9-12) pada tahun 1997, indonesia termasuk dalam lima negara pengkomsumsi rokok terbesar di dunia, dengan urutan china, amerika, jepang, rusia dan indonesia. menurut world health organization south east asia regional office ( w h o s e a r o ) t a h u n 2 0 0 0 , i n d o n e s i a menduduki peringkat ke 4 jumlah perokok terbanyak di dunia.(13) hasil survei sosial ekonomi nasional ( s u s e n a s ) t a h u n 2 0 0 4 m e n u n j u k k a n 3 5 % penduduk berusia 15 tahun ke atas adalah perokok, 28% adalah perokok tiap hari dan 6% perokok kadang-kadang. penduduk yang mantan p e r o k o k a d a l a h 4 % . p e n d u d u k p e d e s a a n sebanyak 37% adalah perokok, lebih tinggi dibandingkan penduduk perkotaan yang sebesar 32%.(14) s e b a g a i u p a y a u n t u k m e n u r u n k a n prevalensi katarak perlu tindakan pencegahan y a n g s e s u a i d e n g a n f a k t o r r i s i k o y a n g berhubungan dengan katarak pada penduduk indonesia. tindak pencegahan terjadinya katarak antara lain adalah dengan mengurangi pajanan terhadap faktor perusak, salah satunya adalah rokok. untuk mencegah terjadinya katarak perlu d i l a k u k a n p e n e l i t i a n y a n g b e r t u j u a n mengidentifikasi merokok dan usia sebagai faktor risiko katarak pada pekerja dibidang pertanian. 2 2 d pqzα n = keterangan : n = besar sampel minimal α= 0,05 p = perkiraan proporsi dari kepustakaan = 7,5% 3 q= 1-p d= toleransi error x p toleransi error = 20% n=1223 orang pengumpulan data p e n g u m p u l a n d a t a d i l a k u k a n d e n g a n w a w a n c a r a d a n p e m e r i k s a a n . wa w a n c a r a tana, mihardja, rif’ati risiko katarak petani 123 universa medicina vol. 26 no.3 menggunakan kuesioner yang menanyakan usia, jenis kelamin, pendidikan, pekerjaan, merokok, jumlah batang rokok yang dihisap, dan lama merokok. untuk merokok dibedakan derajat b e r a t r i n g a n m e r o k o k b e r d a s a r k a n i n d e k s brinkman,(15) yang merupakan perkalian antara batang rokok yang dihisap perhari dan lama merokok. wawancara dilakukan oleh 4 orang pewawancara. p e m e r i k s a a n y a n g d i l a k u k a n a d a l a h pemeriksaan visus dan pemeriksaan mata. p e m e r i k s a a n v i s u s a d a l a h m e r u p a k a n p e m e r i k s a a n u n t u k m e n g e t a h u i t a j a m p e n g l i h a t a n , d e n g a n m e n g g u n a k a n k a r t u snellen dan pinhole, dan diperiksa oleh petugas medis dari balai kesehatan mata masyarakat cikampek. pemeriksaan mata untuk diagnosis adanya katarak atau tidak, dilakukan oleh 2 o r a n g d o k t e r s p e s i a l i s m a t a d e n g a n ophthalmoscope (tanpa midriatika). analisis data analisis data menggunakan program epi info. tingkat kemaknaan sebesar 0,05. persetujuan etik persetujuan etik penelitian diperoleh dari k o m i s i e t i k p e n e l i t i a n k e s e h a t a n b a d a n p e n e l i t i a n d a n p e n g e m b a n g a n k e s e h a t a n departemen kesehatan ri. hasil responden yang memenuhi kriteria inklusi dan eksklusi sebanyak 1223 orang, dengan karakteristik seperti terlihat pada tabel-tabel berikut ini : gambaran deskriptif responden penelitian sebaran karakteristik responden penelitian terlihat pada tabel 1. responden berusia ratarata 48,9 tahun, laki-laki sebanyak 52,2% dan perempuan sebanyak 47,8%, dengan tingkat tabel 1. sebaran karakteristik responden berdasarkan usia, jenis kelamin, pendidikan, dan merokok (n = 1223) karakteristik responden n % umur 30-44 494 40,4 45-54 320 26,2 55-64 251 20,5 65+ 158 12,9 jenis kelamin laki-laki perempuan 639 584 52,2 47,8 tingkat pendidikan tidak sekolah/tamat sd tamat sd tamat sltp tamat smu-pt 879 292 33 19 71,9 23,9 2,7 1,6 perokok merokok sampai kini eks perokok tidak merokok 566 76 581 46,3 6,2 47,5 derajat merokok pada kelompok yang masih merokok (indeks brinkman) ringan sedang berat 335 203 28 27,4 16,6 2,3 pendidikan tidak pernah sekolah atau tidak tamat sd mencapai hampir 72%. persentase responden yang merokok sampai kini sebesar 46,3% sedangkan mantan perokok sebesar 6 , 2 % . r e s p o n d e n y a n g m a s i h m e r o k o k besarnya 566 orang, 27,4% adalah perokok r i n g a n , 1 6 , 6 % p e r o k o k s e d a n g , d a n 2 , 3 % perokok berat. responden dengan lama merokok lebih dari 20 tahun sebanyak 45,6%, dan responden yang merokok rata-rata lebih 40 batang perhari sebanyak 3,1%, yang merokok rata-rata 20-40 batang perhari sebanyak 28%. (tabel 2) 124 k a s u s k a t a r a k y a n g d i p e r o l e h p a d a pemeriksaan dokter mata mencapai 464 orang (37,9%), sedangkan yang tidak katarak sebesar 759 orang (62,1%). pada kelompok usia 30-44 tahun yang menderita katarak sebesar 4,7%, pada kelompok usia 45-54 tahun yang menderita katarak sebesar 30,3%, pada kelompok 55-64 tahun yang menderita katarak sebesar 76,9% dan pada 65 tahun ke atas yang menderita katarak s e b e s a r 9 5 , 6 % . h a s i l p e m e r i k s a a n v i s u s responden dengan katarak yang termasuk dalam p e n g l i h a t a n n o r m a l 1 3 7 o r a n g ( 2 9 , 5 % ) , g a n g g u a n p e n g l i h a t a n r i n g a n 2 2 5 o r a n g (48,5%), penglihatan buruk 77 orang (16,6%), dan buta 25 orang (5,4%). hubungan bivariat antara beberapa variabel dan katarak hubungan antara faktor usia dan merokok dengan katarak terlihat pada tabel 3. risiko terjadinya katarak pada responden berusia 55 tahun ke atas 30,6 kali lebih tinggi dibandingkan responden berusia 30-54 tahun. pada responden yang merokok risiko terjadinya katarak 2,17 kali lebih tinggi dibandingkan responden yang bukan perokok. tabel 3. hubungan antara faktor usia dan merokok dengan katarak (n = 1223) tabel 4. hubungan antara derajat perokok dengan katarak (n = 566) * dibandingkan dengan perokok ringan tabel 2. sebaran karakteristik responden yang pernah merokok (n=642) berdasarkan lama dan jumlah rokok yang dihisap perhari tana, mihardja, rif’ati risiko katarak petani 125 universa medicina vol. 26 no.3 hubungan antara derajat perokok dengan katarak terlihat pada tabel 4. risiko terjadinya katarak pada responden perokok derajat sedang 1,57 kali lebih tinggi dibandingkan responden perokok ringan. pada responden perokok berat risiko terjadinya katarak 4,85 kali lebih tinggi dibandingkan responden perokok ringan. analisis regresi logistik ganda faktor usia dan merokok dengan katarak. analisis regresi logistik ganda faktor usia dan merokok terhadap katarak terlihat pada tabel 5. usia dan merokok berhubungan secara b e r m a k n a d e n g a n p e n i n g k a t a n t e r j a d i n y a katarak. apabila dibandingkan antara faktor u s i a d a n m e r o k o k , m a k a f a k t o r u s i a berhubungan lebih kuat dalam meningkatkan katarak dibandingkan faktor merokok. pembahasan prevalensi katarak pada salah satu atau kedua mata besarnya 37,9 dan yang tidak katarak sebanyak 62,1%. pada kelompok usia 30-44 tahun, persentase katarak paling rendah s e b e s a r 4 , 7 % , d e n g a n m e n i n g k a t n y a u s i a persentase katarak semakin meningkat dan pada usia diatas 65 tahun didapatkan persentase katarak sebesar 95,6%. prevalensi katarak di indonesia menurut skrt-surkesnas 2001 sebesar 4,99% dan di jawa bali sebesar 5,48%. prevalensi katarak di daerah pedesaan sebesar 6,29%, lebih tinggi dibandingkan daerah perkotaan sebesar 4,5%.(2) pada penelitian ini prevalensi yang diperoleh lebih tinggi dibandingkan hasil penelitian lain. hal ini dapat dijelaskan pada penelitian ini usia responden adalah 30 tahun ke atas. rata-rata usia adalah 48,9 tahun, dan jumlah responden dengan usia 50 tahun ke atas sebanyak 45,95%, sedangkan responden pada penelitian skrtsurkesnas 2001 adalah semua umur. pada penelitian ini apabila ditinjau dari hubungan bivariat antara usia dan katarak, maka terlihat adanya peningkatan persentase katarak dari kelompok usia 30-54 tahun ke kelompok usia 50 tahun ke atas. apabila ditinjau dari hubungan antara usia dan merokok, maka terlihat makin tua responden makin banyak yang merokok. apabila dibandingkan dengan penelitian lain, h a s i l p a d a p e n e l i t i a n i n i s e s u a i d e n g a n kepustakaan yaitu persentase katarak meningkat secara bermakna sesuai dengan peningkatan usia. penyebab katarak yang utama adalah proses alamiah dengan bertambah lanjutnya usia menimbulkan perubahan pada mata.(16-19) w h o m e l a p o r k a n b a h w a h u b u n g a n katarak dengan proses ketuaan telah diketahui sejak dulu. usia dikatakan merupakan faktor risiko utama terjadinya katarak. katarak senilis dikatakan sebagai suatu penyakit idiopatik, yang umum terjadi pada usia di atas 50 tahun, prevalensinya cenderung meningkat sesuai dengan bertambahnya usia.(16) tabel 5. analisis regresi logistik ganda faktor usia dan merokok dengan katarak (n = 1223) 126 hasil penelitian ini diperoleh lebih tinggi dibandingkan hasil penelitian survei kesehatan i n d e r a p e n g l i h a t a n d a n p e n d e n g a r a n o l e h depkes, yaitu prevalensi katarak pada kelompok usia 19-54 tahun 3,5%, pada kelompok 55-64 tahun sebesar 33,4% dan pada kelompok 65 tahun ke atas sebesar 62,2%. hasil penelitian ini juga lebih tinggi bila dibandingkan dengan p e n e l i t i a n l a i n d i a m e r i k a s e r i k a t , y a n g menunjukkan katarak pada usia 55-64 tahun sebesar hampir 40%, pada usia 65-74 tahun sebesar 70%, dan pada usia 79-84 tahun lebih dari 90%.(20) perbedaan ini mungkin disebabkan o l e h a d a n y a f a k t o r f a k t o r l a i n y a n g m e m p e n g a r u h i k a t a r a k . k e p u s t a k a a n menyebutkan adanya faktor-faktor penyebab katarak, yang dapat berasal dari dalam dan dari luar tubuh, termasuk faktor demografik dan lingkungan. faktor dalam tubuh sendiri antara lain adalah faktor usia, jenis kelamin, etnis, dan genetik. faktor dari luar tubuh antara lain adalah faktor pajanan kronis terhadap ultra violet, infra merah, atau sinar matahari, merokok, nutrisi, myopia, alkohol derajat sosial ekonomi, status pendidikan dan multivitamin.(19-23) pada penelitian ini ditinjau dari hubungan bivariat antara faktor merokok dengan katarak maka terlihat bahwa katarak pada responden perokok 2,17 kali lebih tinggi secara bermakna dibandingkan dengan katarak responden bukan p e r o k o k . a p a b i l a d i t i n j a u d a r i i n d e k brinkman (15) yang merupakan derajat berat ringan perokok, maka terlihat katarak pada perokok sedang lebih tinggi secara bermakna dibandingkan katarak pada perokok ringan, dari or terlihat 1,57 kali lebih tinggi dibandingkan perokok ringan. katarak pada perokok berat l e b i h t i n g g i s e c a r a b e r m a k n a ( 4 , 8 5 k a l i ) dibandingkan katarak pada perokok ringan, sedangkan katarak pada perokok sedang lebih tinggi secara bermakna (1,6 kali) dibandingkan katarak pada perokok ringan. penelitian ini sesuai dengan kepustakaan yang melaporkan bahwa salah satu faktor yang mempengaruhi terjadinya katarak adalah faktor merokok.(5,6,16) penelitian lain menyebutkan bahwa jumlah rokok juga mempengaruhi peningkatan risiko terjadinya katarak, dilaporkan perokok dengan j u m l a h l e b i h 2 0 b a t a n g s e h a r i a k a n meningkatkan risiko menjadi katarak hampir 2 kali lipat lebih tinggi.(16) hubungan dosis respon terlihat sesuai dengan penelitian yang telah dilakukan oleh beberapa peneliti.(10-12,24) pada penelitian ini, makin berat derajat merokok maka katarak y a n g t e r j a d i m a k i n t i n g g i , s e s u a i d e n g a n penelitian lain yang melaporkan bahwa makin banyak jumlah rokok yang dihisap, maka risiko terjadi katarak makin tinggi. apakah hasil ini dipengaruhi oleh usia, karena ditinjau dari hubungan antara merokok dengan usia adalah b e r b e d a b e r m a k n a y a i t u r e s p o n d e n y a n g merokok lebih banyak yang berusia 55 tahun ke atas dibandingkan yang tidak merokok. namun dari hasil analisis multivariat yang t e r l i h a t b a h w a s e l a i n u s i a , m a k a f a k t o r merokok katarak mempunyai hubungan kuat dengan katarak. apabila ditinjau dari indeks b r i n k m a n , y a n g m e r u p a k a n d e r a j a t b e r a t r i n g a n n y a m e r o k o k d a n m e r u p a k a n h a s i l perkalian antara lama merokok dan jumlah batang rokok yang dihisap, maka terlihat bahwa makin berat derajat merokok responden makin tinggi katarak yang terjadi. s u a t u p e n y u l u h a n d i p e r l u k a n u n t u k meningkatkan pengetahuan, sikap dan praktek y a n g b a i k t e r h a d a p f a k t o r f a k t o r y a n g berhubungan dengan peningkatan katarak salah satunya adalah faktor merokok, dengan harapan dapat memperlambat/mencegah terjadinya katarak. (12) dengan penyuluhan diharapkan dapat merubah pengetahuan, sikap dan praktek responden terhadap rokok, yaitu berubah dari yang merokok menjadi tidak merokok. tana, mihardja, rif’ati risiko katarak petani 127 universa medicina vol. 26 no.3 kesimpulan usia dan merokok merupakan faktor risiko yang berhubungan positif dengan katarak. p e r s e n t a s e k a t a r a k m e n i n g k a t d e n g a n m e n i n g k a t n y a u s i a . p e r s e n t a s e k a t a r a k meningkat pada perokok dibandingkan yang bukan perokok. makin berat derajat merokok, makin tinggi persentasi katarak. ucapan terima kasih a t a s b a n t u a n b e r b a g a i p i h a k s e l a m a penelitian, kami mengucapkan terima kasih khususnya kepada dr. tjahjono gondhowiardjo phd,spm dan dr. agus suwandono, mph, dr.ph sebagai konsultan pada penelitian ini dan kepada kepala dinas kesehatan kabupaten karawang, kepada para kepala desa wanasari, wanajaya, wanakerta dan karang ligar atas kerjasamanya selama pelaksanaan penelitian ini. daftar pustaka 1. vaughan d, asbury t. general ophthalmology. alih bahasa waliban, hariono b. jakarta; widya medika; 1990. 2. departemen kesehatan ri. rencana strategis nasional penanggulangan gangguan penglihatan dan kebutaan (pgpk) untuk mencapai vision 2020. jakarta. 2003. 3. sub ditbina kesehatan mata, ditjen binkesmas depkes ri. laporan operasi katarak massal dengan bantuan cbm di 8 propinsi (sumatera utara, sumatera barat, jawa barat, sulawesi utara, sulawesi selatan, kalimantan timur, nusa tenggara barat dan nusa tenggara timur), 1999. 4. departemen kesehatan republik indonesia. gangguan kesehatan indera penglihatan dan pendengaran. analisis data morbiditasdisabilitas, skrt-surkesnas 2001. direktorat jenderal bina kesehatan masyarakat direktorat kesehatan khusus dan badan penelitian dan pengembangan kesehatan, sekretariat surkesnas. jakarta. 2004. 5. taylor a, nowell t. oxidative stress and antioxidant function in relation to risk for cataract. adv pharmacol 1997; 38: 515-36. 6. indonesian second country in south-east asia region to launch national vision 2020 programe. who experts plan regional strategy for preventable blindness for next twenty years. available at http://www.home.earthlink.net/blindworld/research/4-04-12-04.htm. accesed august 20, 2004. 7. xu l, cui t, zhang s, sun b, zheng y, hu a et al. prevalence and risk factors of lens opacities in urban an rural chinese in beijing. ophthalmology 2006; 113: 747-55. 8. hennis a, wu sy, nemesure b, leske mc; barbados eye studies group. risk factors for incident cortical and posterior subcapsular lens opacities in barbados eye studies. arch ophthalmol 2004; 122: 525-30. 9. rowe ng, mitchell pg, cumming rg. diabetes, fasting blood glucose and age-related cataract: the blue mountains eye study. ophthalmic epidemiol 2000; 7: 103-14. 10. krishnaiah s, vilask, shamanna br, rao gn, thomas r, balasubramaniah d. smoking and its association with cataract: results of the andhra pradesh eye disease study from india. inves ophthalmol vis sci 2004; 122: 564-72. 11. hiller r, sperduto rd, podgor mj, wilson pw, ferris fl, colton t, et al. cigarette smoking and the risk of development of lens opacities. the framingham studies. arch ophthalmol 1997; 115: 1113-8. 12. christen wg, glynn rj, ajani ua, schaumberg da, buring je, hennekens ch, et al. smoking cessation and risk of age related cataracts in men. jama 2000; 284: 713-6. 13. aditama ty. masalah merokok dan penanggulangannya. yayasan penerbitan ikatan dokter indonesia (yp-idi) bekerjasama dengan perhimpunan dokter paru indonesia (pdpi) lembaga menanggulangi masalah merokok (lm3). jakarta. 2001. 14. badan penelitian dan pengembangan kesehatan departemen kesehatan. survei sosial ekonomi nasional (susenas) 2004-substansi kesehatan. status kesehatan, pelayanan kesehatan, perilaku hidup sehat dan kesehatan lingkungan. jakarta. 2004. 128 15. brinkman gl, voates jr eo. the prevalence of chronic bronchitis in an industrial population. am rev respir div 1962; 47-54. 16. world health organization. management of cataract in primary health care services. 2nd edition. geneva, 1996. 17. manson je, christen wg, seddon jm, glynn rj, hennekens ch. a prospective study of alcohol consumption and risk of cataract. am j prev med 1994; 10: 156-61. 18. christen wg, liu s, schaumberg da, buring je. fruit and vegetable intake and the risk of cataract in women. am j clin nutr 2005; 81: 1417-22. 19. hennis a, wu sy, nemesure b, leske mc. barbados eye studies group. arch ophthalmol 2004; 122: 525-30. 20. weintraub jm, willett wc, rosner b, colditz ga, seddon jm, hankinson se. smoking cessation and risk of cataract extraction among us women and men. am j epidemiol 2002; 155: 72-9. 21. leske mc, wu sy, hennis a, connell am, hyman l. diabetes, hypertension, and central obesity as cataract risk factors in a black population. the barbados eye study. ophthalmol 1999; 106: 35-41. 22. kuang tm, tsai sy, hsu wm, cheng cy, liu jh. epidemiologic study of age-related cataracts among an elderly chinese population in shihpai, taiwan. ophthalmology 2003; 110: 108995. 23. jacques pf, moeller sm, hankinson se, chylack lt jr, rogers g, tung w, et al. weight status, abdominal adiposity, dabetes, and early agerelated lens opacities. am j clin nutr 2003; 78: 400-5. 24. lindblad be, hakansson n, svensson h, philipsoon b, wolk a. intensity of smoking and smoking cessation in relation to risk of cataract extraction: a prospective study of women. am j epidemiol 2005; 162: 73-9. tana, mihardja, rif’ati risiko katarak petani oktavianus 36 *division of nephrology and hypertension, department of internal medicine, faculty of medicine, university of sumatera utara medan correspondence dr. andy luman division of nephrology and hypertension, department of internal medicine, faculty of medicine university of sumatera utara jl. dr. mansyur no. 5 medan sumatera utara 20155 email: andyluman@yahoo.com univ med 2014;33:36-42 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background a substantial proportion of uncomplicated, treated primary hypertensive patients have failure of hypertension treatment. the enzyme creatine kinase (ck) regenerates adenosine triphosphate in striated muscle, myocardium and blood vessels. several studies showed that serum ck was found to be a predictor of blood pressure in the general population. high tissue ck precedes hypertension in populations with high hypertension risk. the hypothesis of this study was to examine that ck increases in uncontrolled hypertension subjects. methods a cross-sectional study from november until december 2013 was conducted in 82 adult hypertensive patients with mean age of 61.96 ± 10.76 years. patients’ histories were taken and their blood pressure and body mass index were measured. serum was analyzed for serum ck, random blood glucose and lipid profile. independent t test and mann whitney was used to assess differences in mean ck and other variables between hypertension categories. chi-square test was also used to assess differences between ck tertiles and status of hypertension. results there was a significant difference in mean ck between controlled hypertension (81.83 ± 29.70 u/l) and uncontrolled hypertension groups (132.17 ± 55.91 u/ l). the number of subjects in the high ck tertile (>109.33 u/l) was significantly higher in the uncontrolled hypertension group (81.5%), compared with the controlled hypertension group (18.5%) (p=0.0001). conclusion creatine kinase level increases in adults with uncontrolled hypertension. measuring serum levels of creatine kinase may help doctors to spot patients whose hypertension will be difficult to treat. keywords: uncontrolled hypertension, creatinine kinase, adult creatine kinase increases in adults with uncontrolled hypertension andy luman* and abdurrahim rasyid lubis* 37 univ med vol. 33 no.1 kreatin kinase meningkat pada subjek dewasa dengan hipertensi tidak terkontrol latar belakang dijumpai banyak pasien hipertensi primer yang berobat dan tanpa komplikasi mengalami kegagalan pengobatan hipertensi. enzim kreatin kinase (ck) membentuk adenosine triphosphate pada otot rangka, jantung dan pembuluh darah. beberapa penelitian menunjukkan ck serum merupakan prediktor tekanan darah pada populasi umum. kreatin kinase jaringan yang tinggi mencetuskan hipertensi dan dijumpai pada populasi resiko tinggi hipertensi. hipotesis penelitian ini adalah untuk menguji bahwa ck meningkat pada subjek dengan hipertensi yang tidak terkontrol. metode penelitian potong lintang dilakukan antara bulan november hingga desember 2013 pada 82 pasien hipertensi dewasa usia 61,96 ± 10,76 tahun. anamnesis, pengukuran tekanan darah dan indeks massa tubuh dikumpulkan dari semua subjek. serum diambil dan dilakukan pemeriksaan ck, kadar gula darah sewaktu dan profil lipid. uji t-independen dan mann-whitney menilai perbedaan rerata kadar ck dan variabel lainnya terhadap kategori hipertensi. uji chi-square juga digunakan untuk menilai perbedaan tertile ck dengan status hipertensi. hasil jumlah subjek dengan tertil tinggi lebih besar secara bermakna pada kelompok hipertensi tidak terkontrol (81,5%) dibandingkn kelompok hipertensi terkontrol (18,5%) (p=0,0001). terdapat perbedaan rata-rata ck yang bermakna antara kelompok hipertensi terkontrol (81,83 ± 29,70 u/l) dan hipertensi tidak terkontrol (132,7 ± 55,91 u/l) (p=0,000). kesimpulan kadar ck meningkat pada dewasa dengan hipertensi tidak terkontrol. pemeriksaan kadar ck sangat diperlukan pada subjek dengan hipertensi yang sulit dikendalikan. kata kunci: tekanan darah, hipertensi, kreatin kinase, dewasa abstrak introduction hypertension is a leading risk factor for heart disease, stroke, and kidney failure. because hypertension is the most common primary diagnosis in the united states, it is not suprising that blood pressure measurement is one of the most common reasons for a visit to the doctor‘s office, and antihypertensive medications are among the most commonly written prescriptions.(1) blood pressure was controlled only in an estimated 50.1% of all patients with hypertension in the national health and nutrition examination survey (nhanes) 2007-2008, with most of the improvement since 1988 occurring after 19992000. while control of hypertension is on the rise, prevalence of total hypertension continues to increase.(2,3) a substantial proportion of treated hypertensive patients do not achieve blood 38 pressure control. in general, these patients tend to be obese, older, or have diabetes and endorgan damage. however, many patients have uncomplicated, primary hypertension, and it is not well explained why these patients respond poorly to drug therapy, even when patient’s adherence and physician’s therapeutic inertia are taken into account.(4-6) the enzyme creatine kinase (ck) is thought to enhance pressor responses through rapid regeneration of adenosine triphosphate (atp), as the enzyme catalyzes the reversible transfer of the high-energy phosphate moiety (p) between creatine and adenosine diphosphate (adp). the rate of transfer of the phosphoryl group by ck is greater than the maximum rate of atp generation by oxidative phosphorylation a n d g l y c o l y s i s t o g e t h e r, e n s u r i n g r a p i d resynthesis of atp. adenosine triphosphate synthesized by ck is preferentially used to fuel highly energy-demanding processes such as sodium retention, cardiovascular contractility, as well as remodeling of arteries.(7) a study by brewster et al.(8) showed that serum ck was a main predictor of blood pressure in the general population, independent of age, sex, bmi, or ethnicity, with a crude systolic blood pressure (bp) increase of 14 mmhg per log ck increase, without evidence of muscle damage. a study by oudman et al.(9) showed that high tissue ck precedes hypertension and anti-hypertensive therapy lowers high tissue ck in animal models, whereas incubation of human resistance arteries w i t h a c k i n h i b i t o r r e d u c e s v a s c u l a r contractility. another study by brewster et al.(10) showed that tissue ck is high in population s u b g r o u p s w i t h h i g h h y p e r t e n s i o n r i s k , including in skeletal, cardiac, and vascular muscle. on the basis of the abovementioned information, considering the high proportion of uncontrolled hypertensive patients and the role of ck in hypertension, we tested the hypothesis that creatinine kinase increases in uncontrolled hypertension among subjects taking antihypertensive medications. methods research design a cross-sectional study was conducted at the department of internal medicine of h. adam malik hospital and dr. pirngadi hospital, medan, from november 2013 to december 2013. research subjects t h e s t u d y p o p u l a t i o n c o m p r i s e d hypertensive patients using anti-hypertensive medications. the inclusion criteria of the subjects were males or females >18 years of age and taking anti-hypertension drugs for at least 1 m o n t h . t h e e x c l u s i o n c r i t e r i a w e r e neuromuscular disease, renal impairment with glomerular filtration rate (gfr) <60 ml/ minute/ 1.73 m2, and use of statins. the subjects were divided into the controlled and uncontrolled hypertension groups. the controlled hypertension group comprised subjects on anti-hypertensive drugs for more than 1 month with systolic blood pressure (bp) of <140 mmhg (<130 mmhg for subjects with diabetes) and diastolic bp of <90 m m h g ( < 8 0 m m h g w i t h d i a b e t e s ) . t h e uncontrolled hypertension group consisted of subjects on anti-hypertensive drugs for more than 1 month with systolic bp of >140 mmhg (>130 mmhg with diabetes) and/or diastolic bp of >90 mmhg (>80 mmhg with diabetes).(11) the study subjects were selected by consecutive nonrandom sampling. we used the sample size c a l c u l a t i o n m e t h o d f o r c o m p a r i n g t w o independent proportions.(12) to calculate the sample size, we used a previous study by oudman et al.(9) who reported a proportion of hypertension of approximately 0.268 in the low ck population group. the calculated sample size per group was 41, which was estimated to be adequate to detect a 30% difference between the two groups using a two-tailed test, an alpha of 0.05 and power of 80%. data collection all study subjects were interviewed using a questionnaire for age, gender, history of luman, lubis creatine kinase in uncontrolled hypertension 39 univ med vol. 33 no.1 diabetes, duration of hypertension, use of antihypertensive drugs, and levels of medication adherence. the latter was assessed with the morisky medication adherence scale-8 of 8 questions, in which three levels of adherence were considered (low: scores of 0 to <6; medium: 6 to <8; high: 8).(13) subjects were measured for height, weight, and body mass index (bmi). the blood pressure was measured in a quiet room with the participant seated. an appropriately adjusted cuff size was used on the non-dominant arm, which was supported at h e a r t l e v e l a n d t h e b l o o d p r e s s u r e w a s c a l c u l a t e d a s t h e m e a n o f t h e f i r s t t w o c o n s e c u t i v e r e a d i n g s , w i t h a 1 5 m i n u t e difference in measurement. laboratory analysis laboratory studies included serum ck, random blood glucose, lipid profile [total cholesterol, low density lipoprotein (ldl) cholesterol, and high density lipoprotein (hdl) c h o l e s t e r o l ] m e a s u r e d w i t h t h e c o b a s automated analyzer. table 1. comparison of demographic, clinical and laboratory characteristics of study population achi square; b mann whitney; c independent t test; *values represent mean ± sd, and numbers (%) ace inhibitor= angiotensin converting enzyme inhibitor; arb= angiotensin receptor blocker; bmi= body mass index; ccb= calcium channel blocker; ck= creatine kinase; hdl= high density lipoprotein; ldl= low density lipoprotein 40 statistical analysis kolmogorov-smirnov normality test was used to assess normal distribution of the data. the independent t-test was used to establish differences in mean ck and other variables between hypertension categories for normally distributed data, and the mann-whitney test for non-normally distributed data. the chi-square test was used to assess differences between ck tertiles and status of hypertension. statistical analyses were performed with the spss statistical package for windows, version 17.0, a n d a p v a l u e o f < 0 . 0 5 w a s c o n s i d e r e d statistically significant. ethical clearance the study protocol was approved by the health research ethical committee, faculty of medicine, university of sumatera utara. all study subjects signed written informed consent after having been informed about the aims and benefits of the study. results during the study period 41 study subjects for each group were obtained. by using the kolmogorov smirnov test, we found that the data for age, duration of hypertension, ck level, random blood glucose, total cholesterol, and ldl cholesterol were non-normally distributed, while bmi and hdl cholesterol were normally distributed. using the chi-square test, based on the status of hypertension categories, we found a significant difference for gender (p=0.002) and number of anti-hypertensives used (p=0.015) versus status of hypertension. the mann whitney test showed a significant difference in mean ck between controlled and uncontrolled hypertension groups (p=0.0001) (table 1). by using the chi square test, we found significant differences between ck tertiles with status of hypertension. the number of subjects in the high ck tertile (>109.33 u/l) was significantly higher (81.5%) in the uncontrolled hypertension group, compared with the controlled hypertension group (18.5%) (p=0.0001) (table 2). discussion in this study involving 82 subjects we found that ck level increases significantly in subjects with uncontrolled hypertension. a similar result was found by oudman et al.(9) in that elevated levels of ck increase the incidence risk of hypertension and that subjects with uncontrolled hypertension have higher ck levels compared with normotensive subjects and those with controlled hypertension. in one of their studies, brewster et al.(14) found that subjects with idiopathic hyperckemia showed greater hypertension risk than controls. a contrary result from the study by johnsen et al.(15) did not find differences in ck levels between those with controlled and uncontrolled hypertension; however, the study objective of these investigators was to analyze associations between ck levels and blood pressure in a normal population, not in a hypertensive population. in the study by brewster et al.(8) it was found that serum ck activity was independently table 2. creatine kinase levels in controlled and uncontrolled hypertension groups luman, lubis creatine kinase in uncontrolled hypertension 41 univ med vol. 33 no.1 associated with blood pressure in a multiethnic population. a linear regression analysis found an increase in systolic bp and diastolic bp, respectively, of 8.0 mmhg (95% ci 3.3-12.7) and 4.7 mmhg (95% ci 1.9-7.5) per log ck increase after adjustment for age, sex, bmi, and ethnicity. the study by johnsen et al.(15) showed similar results, i.e. that serum ck activity in a caucasian normal population was independently associated with blood pressure, where a one unit increase in log ck was associated with a 3.3 mmhg (95% ci 1.4-5.2) increase in systolic bp and a 1.3 mmhg (95% ci 0.3-2.3) increase in diastolic bp. in an another study, brewster et al.(16) showed that low ck was associated with a 73% higher incidence of fainting in a random population sample. our study showed similar results to another study, where serum ck was s i g n i f i c a n t l y h i g h e r i n t h e u n c o n t r o l l e d hypertension group and systolic bp and diastolic bp were significantly correlated with ck levels. a number of physiological mechanisms are involved in the maintenance of normal blood pressure, and their derangement may play a part in the development of essential hypertension. it is probable that a great many interrelated factors contribute to the raised blood pressure in hypertensive patients, and their relative roles may differ between individuals. among the factors that have been intensively studied are salt intake, obesity, insulin resistance, the renin-angiotensin system, the sympathetic nervous system, genetics, endothelial dysfunction, low birth w e i g h t a n d i n t r a u t e r i n e n u t r i t i o n , a n d neurovascular anomalies.(17) the ck system is of particular importance in tissues that display high and variable rates of atp turnover, including skeletal muscle, the cardiovascular system, brain, and the kidney. in these tissues the enzyme provides atp for muscle contraction and ion transport.(10) high serum ck activity was previously postulated as a genetic factor that could explain the higher blood pressures found in black people, a population s u b g r o u p w i t h a g r e a t e r p r e v a l e n c e o f hypertension and its complications. pressor responses were proposed to be enhanced via increased atp availability for cardiovascular contractility, renal sodium retention, and capillary rarefaction of skeletal muscle.(18) the enzyme ck is abundantly expressed in both the mitochondrion and the cytosol. at the mitochondrial site, it facilitates the formation of creatine phosphate, which is transported by ck to subcellular locations of high-energy demands. here, ck is tightly bound in the immediate proximity of atp-utilizing enzymes, such as na+/k+-atpase and ca2+-atpase at membranes, and myosin light-chain kinase and myosin atpase at the contractile proteins, where it rapidly provides atp to these enzymes. creatine kinase thus fuels highly energydemanding processes such as sodium retention, cardiovascular contractility, and remodeling of arteries. relatively high activity of the enzyme, particularly in resistance arteries, is thought to enhance pressure responses and lead to higher blood pressure levels.(8) a limitation of this study was the small size of the study population, being restricted to two central hospitals. as a follow-up to this study, a cohort study should be performed to determine causal relationships of ck level increments with control of blood pressure in hypertension. measuring serum levels of ck may help doctors to spot patients whose hypertension will be difficult to treat. conclusion creatine kinase level increases in adults with uncontrolled hypertension. it is hoped that scientific interest will explain the possible involvement of the ck in the pathophysiology of the hypertension. acknowledgement we thank all study subjects who agreed to participate in the present study and all colleagues who gave their advice to improve the manuscript. 42 references 1. blumenfeld jd, liu f, laragh jh. primary and secondary hypertension. in: brenner and the rector‘s the kidney. 9th ed. philadelphia: elseviersaunders;2012.p.1670-751. 2. egan bm, zhao y, axon rn. us trends in prevalence, awareness, treatment, and control of hypertension, 1998-2008. jama 2010;303: 2043-50. 3. olives c, myerson r, mokdad ah, murray cjl, lim ss. prevalence, awareness, treatment, and control of hypertension in united states counties, 2001-2009. plos one 2013;8:e60308. 4. egan bm, zhao y, axon rn, brzezinski wa, ferdinand kc. uncontrolled and apparent treatment-resistant hypertension in the united states, 1998 to 2008. circulation 2011;124:104658. 5. brewster lm, van montfrans ga, kleijnen j. systematic review: anti-hypertensive drug therapy in black patients. ann intern med 2004; 141:614-27. 6. gu q, dillon cf, burt vl, gillu rf. association of hypertension treatment and control with allcause and 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2009;19:231-6. 17. beevers g, lip gyh, brien eo. abc of hypertension: the pathophysiology of hypertension. bmj 2001;322:912-6. 18. brewster lm, clark jf, van montfrans ga. is greater tissue activity of creatine kinase the genetic factor increasing hypertension risk in black people of sub-saharan african descent?. j hypertens 2000;18:1537-44. luman, lubis creatine kinase in uncontrolled hypertension c:\users\jurnal fkusakti\docume 50 abstract universa medicina flatfoot decreases school functioning among children <11 years of age yuke damayanti*, panji fortuna hadisoemarto**, and irma ruslina defi*** background flatfoot is a musculoskeletal abnormality commonly found in children that could result in a reduction of quality of life because of its related symptoms and functional abnormalities. this study aimed to determine the association between flatfoot and the quality of life among children 5-18 years of age. methods a cross-sectional design was used in this study of children aged 5-18 years. the diagnosis of flatfoot was made in accordance with the chippauxsmirak index classification, whereas general and domain-specific quality of life were measured using the indonesian version of pedsql™ that has been previously validated. mann whitney test was used to compare quality of life between children with flatfoot and normal feet, along with other variables that potentially may influence a child’s quality of life. results a total of 79 out of 120 subjects was used to analyze the data. there was a statistically significant difference in the quality of life score in the school functioning domain between children with flatfoot (64.46 ± 18.22) and those with normal feet (75.90 ± 17.29) in children <11 years of age (p=0.029). apart from this, the study did not find any statistically significant differences in the total quality of life score or in other quality of life domains between the two groups. conclusions flatfoot significantly decreases children’s quality of life in the school functioning domain. early detection and correction of flatfoot may be necessary to improve the educational attainment of children suffering from this condition. keywords: musculoskeletal deformity, flatfoot, quality of life, children doi: http://dx.doi.org/10.18051/univmed.2018.v37.50-56 original article pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 *undergraduate medical education program, faculty of medicine, padjadjaran university, bandung, indonesia **department of public health, faculty of medicine, padjadjaran university, bandung, indonesia ***department of physical and rehabilitative medicine, faculty of medicine, padjadjaran university/ dr. hasan sadikin general hospital, bandung, indonesia correspondence: yuke damayanti faculty of medicine, padjadjaran university jalan raya bandung-sumedang km. 21, sumedang, indonesia phone: +62 857 2166 5551 email: yuke.damayanti27@gmail.com date of first submission, november 2, 2017 date of final revised submission, april 12, 2018 date of acceptance, april 18, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cit e th is a rtic le a s: d amayanti y, hadisoemarto pf, defi ir. flatfoot decreases school functioning among children <11 years of age. univ med 20 18;3 7:5 0-6. d oi : 10 .18 05 1/ univmed.2018.v37.50-56 51 january-april, 2018 vol.37no.1 introduction flatfoot or pes planus is the relatively common condition in which the whole or almost the whole foot contacts the ground as a result of flatness of the medial longitudinal arch.(1,2) for example, a study in surakarta found that the prevalence of flatfoot in elementary school s t ud e n ts wa s 2 7. 4% o f 1 08 9 s tu d e nt s examined.(3) similarly, the study conducted by ezema et al.(4) in nigeria found a prevalence of 22.4% among 474 children aged 6-10 years. a ta i wa ne s e st u dy e ve n s ho we d th a t t h e prevalence of flatfoot attained 59% among 2083 children aged 7-12 years.(1) around 65% of children with flatfoot experience symptoms, such as pain, with some 68% having functional limitation.(5) continuous pain accompanied by functional limitations may be the cause of disturbed learning activities, d if f i c u l ty i n so c i a l c o n ta c t s, d e c r e a se d participation in recreational activities, increased needs for health services, and ultimately may decrease quality of life.(6) assessment of quality of life may be performed to determine the magnitude of the impact of symptoms and also the functional limitations in a person with flatfoot. quality of life itself is a multidimensional comprehensive construct that may further be subdivided into domains, such as quality of physical, emotional, and social functioning.(7) assessment of quality of life in children may also evaluate the impact of functional limitations in more specific aspects, including the ability of the children to go to school or to play.(8) a study on the quality of life of children with flatfoot was conducted in the uk by kothari et al.(9) who showed a significant decrease in the quality of life in the physical, emotional and school domains in children with flatfoot compared with children with normal feet. on the other hand, a study in colombia did not find a significant difference in the quality of life of children aged 6-12 years with different types of foot arch.(10) these inconsistent study results call for further studies. therefore, there is a need to conduct a study to compare the quality of life of children with flatfoot aged 5-18 years. methods design of the study the present study used secondary data taken from a cross-sectional study on children’s health that was conducted in soreang subdistrict, bandung district, from september october 2016. subjects of the study children aged 5-18 years with neutral and flatfoot posture were recruited for the study. the exclusion criteria were presence of neurological abnormalities, lower limb surgery, and current use of orthoses. the prevalence of children with poor quality of life was 0.25 (11) and for µ=0.05 and error level=0.05, the required minimal sample was 120. two villages in soreang subdistrict, i.e. pamekaran and sadu, were selected on the basis of the number of children with recorded data at the posyandu and the excellent activity of the health cadres. subsequently 20 children in each of the age groups of the pedsql™ (12) quality of life inventory (5-8, 9-12, and 13-18 years) were selected from each village to ensure a uniform subject distribution. data collection the cadres in each village invited the parents of the candidate subjects who resided in the area to their homes to complete the quality of life survey at the appointed days. for children aged 5-7 years and children who were unable to read, the questions were read out and the questionnaire filled in by the investigators in accordance with the guidelines. on the other hand, for children aged 8-18 years, the filling in of the questionnaire was performed by the parents and the children themselves, so that the responses of the children could be confirmed by the responses of the parents. 52 variable flatfoot (n=32) normal (n=47) p value mean age (±sd) 8.7 (2.5) 11.8 (3.4) <0.001* gender male female 17 (53.1%) 15 (46.9%) 19 (40%) 28 (60%) 0.266 bmi (±sd) 18.21 (4.57) 17.50 (2.82) 0.395 educational level  elementary school ˃ elementary school 30 (93.8%) 2 (6.2%) 28 (59.6%) 19 (40.4%) 0.001* education of father  elementary school ˃ elementary school 10 (31.2%) 22 (68.8%) 17 (36.2%) 30 (63.8%) 0.651 education of mother  elementary school ˃ elementary school 12 (37.5%) 20 (62.5%) 19 (40.4%) 28 (59.6%) 0.796 village pamekaran sadu 15 (46.9%) 17 (53.1%) 16 (34%) 31 (66%) 0.252 table 1 demographic characteristics of study subjects damayanti, hadisoemarto, defi flatfoot and school functioning children children who completed the questionnaire on the quality of life inventory were then invited again for taking their footprints to measure the medial arch. the study subjects included in the analysis were those who had data on quality of life and footprint results. assessment of foot shape assessment of foot shape was performed by f inal stage reside nt physicians at the department of physical health science and rehabilitation, dr. hasan sadikin hospital, ba n du n g. br i e f l y, t h e a ss e ss me n t w a s performed by taking the children’s footprint on a sheet of paper. the foot shapes of the children were then grouped into flatfoot or normal feet, based on the chippaux-smirak index (csi), which is the ratio of the minimum width of the f o o t a r c h t o t h e ma x i mu m w idt h o f t he me t a t ar s a l s . ( 1 3 ) a va l ue o f csi 4 5 wa s categorized as flatfoot.(13,14) assessment of quality of life the assessment of quality of life was per formed using the questionnaire of the pediatric quality of life inventory (pedsql™) 4.0 generic core scales.(13–15) pedsql™ is an instrument of quality of life measures in children, that may be used in healthy children as well as children with given medical conditions such as a s thma , c a nc er, or ga str oi n te st i na l t ra c t disorders.(16,1) there are two forms of the instrument, i.e. reports of parents on the children (parent proxy-report) or reports of the children themselves (child self-report).(13) in addition to the total quality of life score, pedsql™ also assesses the children’s quality of life in five different functioning domains, i.e. physical, psychosocial, emotional, social, and school functioning. pedsql™ has been translated into 60 languages, including the indonesian language, and all language versions have been validated. statistical analysis to determine if the condition of flatfoot influences the quality of life score, a multiple linear regression test was performed. the total quality of life score and the quality of life score in each pedsql™ domain was used as dependent variable and foot shape (flatfoot or normal) as an independent variable. other variables that potentially influence quality of life in children, i.e. age, gender, parental occupation, educational level of the children, and body mass index, was used in the model as control variable. the level of statistical significance was set at p-value = 0.05. 53 january-april, 2018 vol.37no.1 mean p value flatfoot normal physical functioning 81.84 ± 14.7 79.80 ± 14.09 0.488* social functioning 80.80 ± 19.23 83.69 ± 17.46 0.642* emotional functioning 64.53 ± 19.97 66.38 ± 14.51 0.634 school functioning 66.09 ± 18.03 72.60 ± 16.08 0.161* psychosocial functioning 70.16 ± 16.21 73.98 ± 12.50 0.247 total 74.99 ± 13.38 76.11 ± 11.29 0.975* table 2 comparison of mean quality of life between children with flatfoot and normal foot *analysis: mann whitney u-test a b c flatfoot normal p value age <11 years physical functioning 80.80 ± 15.24 78.17 ± 16.68 0.563 social functioning 79.07 ± 20.00 77.50 ± 21.58 0.799* emotional functioning 63.03 ± 20.51 65.90 ± 16.37 0.594 school functioning 64.46 ± 18.22 75.90 ± 17.29 0.029* psychosocial functioning 68.45 ± 16.67 72.50 ± 15.53 0.388 quality of life 73.63 ± 13.82 74.45 ± 14.14 0.838 age 11 years physical functioning 89.08 ± 7.85 81.25 ± 11.51 0.179 social functioning 92.50 ± 5.00 89.37 ± 10.03 0.612 emotional functioning 75.00 ± 12.90 66.80 ± 12.98 0.228 school functioning 77.50 ± 13.22 69.58 ± 14.58 0.319 psychosocial functioning 81.66 ± 4.08 75.34 ± 9.02 0.185 quality of life 84.23 ± 1.88 77.62 ± 7.87 0.111 table 3. comparison of quality of life between children with flatfoot and normal foot by age ethical clearance the primary and secondary studies passed ethical clearance and were approved by the et hic s commis sion o n hea lth re se arc h, padjadjaran university, bandung, under nos. 790/un6.c1.3.2/kepk/pn/2015 and 334/ un6.c.10/kepk/pn/2017. results of 120 children who became the subjects of this study, 79 (65.8%) had complete quality of life data and footprints, with statistically equal gender distribution. a total of 32 (40.5%) subjects had flatfoot. the mean age of the children with flatfoot (8.7 ± 2.5 years) was significantly different from that of the children with normal feet (11.8 ± 3.4 years) (p=0.001). most of the children with flatfoot were still in elementary school or kindergarten. there was no significant difference between the two groups i n me a n bo dy ma ss i n de x a nd pa r e n t a l educational level (table 1). there were no significant differences in quality of life or in functionings (table 2). therefore a stratified analysis should be conducted based on age of the children. the results of a stratified analysis based on the children’s age showed that in children aged <11 years, the quality of life in the school functioning domain was significantly lower in the flatfoot group compared with the normal group. in the other functioning domains no significant differences were found, showing that school functioning decreased significantly in the flatfoot group (table 3). on the other hand, in children aged 11 years no significant decrease in school functioning was found in the flatfoot group. 54 damayanti, hadisoemarto, defi flatfoot and school functioning children discussion the results of this study showed that flatfoot was significantly correlated with decreased quality of life in the school functioning domain in children aged <11 years. on the other hand, flatfoot was not correlated with quality of life in the other domains and the total quality of life score. flatfoot in children is known to cause symptoms such as pain in the sole and also to cause functional limitations such as limitations in standing, walking and running.(5,18) pain that is continuously present and the functional limitations may result in abnormal school functioning in the children.(6) the fact that flatfoot was not correlated with total quality of life and domains other than school functioning may be explained by the pain resulting from flatfoot, that may more frequently appear in school as a result of the children having to wear shoes. in addition, the pain may also more frequently appear in school because of the limited opportunities for the children to rest their feet at the onset of pain. a literature search found at least one study showing an association between the appearance of pain in flatfooted children and the use of shoes, and a number of studies showing that flatfoot causes increased internal activity and fatigue of the leg muscles and ultimately pain.(19) similar results were found by kothari et al.(20) who showed that flatfoot may affect quality of life. however, their study found that children with flatfoot also had a lower quality of life in physical and emotional functioning domains as compared with children with normal feet. these differences may be explained as a result of the selection of study subjects in the uk who mostly came from orthopedic clinics, whereas in the present study the subjects came from the community. in addition, the paired t-test used in previous studies also did not control for other possible confounding variables.(20) the influence of location on the quality of life of the children also cannot be eliminated. most locations in developed countries differ in many respects from such locations as the villages of sadu and pamekaran in bandung district, that may influence the quality of life of children with flatfoot. this includes, but is not limited to, differences in the quality of health services and in the activity of the children in school. these results from bandung district also show a significant mean difference in mean total quality of life and several specific domains among the children residing in the villages of sadu and pamekaran. although the variables that potentially may confound the association between quality of life and foot shape were entered into a regression equation, only 16-27% of the variability of the quality of life scores could be explained. this shows that there were other variables affecting the quality of life domains that were present in the regression model. these other factors were among others pathological condition, physical activity, sedentary lifestyle, sleep duration, and the food consumed by the children, that were not assessed in this study.(21–23) although the present study did not find implications of flatfoot on the children’s quality of life in general, the influence flatfoot on school functioning may have practical significance and may form the basis for early interventions in p a t i e nt s wi th f l a t f o ot . in oth e r wo r d s, interventions in children with flatfoot may confer benefits in the form of improved academic performance and, furthermore, improved quality of human resources. the possible interventions are among others the fitting of foot orthoses, selection of appropriate footwear, and, if necessary, surgery. however, the public health significance of flatfoot and early interventions for this condition need to be researched further. generalization of the study results to the population of children outside the study sample should also be done carefully, because of the possibility that selection bias from the selection of subjects residing nearby the homes of the health cadres may not be fully eliminated. 55 january-april, 2018 vol.37no.1 conclusions in th is stu dy s ampl e, f la tf oo t ha d a sta tistically significant with a decreased children’s quality of life score in the school functioning domain. early detection and management of this condition in school children may be necessary and may contribute to educational attainment in children with flatfoot. conflict of interest the authors declare that there is no conflict of interest with the parties concerned in this study. acknowledgments this study formed part of the final tasks in completion of the graduate study of the first author with support from an academic leadership grant under leadership of professor dany hilmanto. the authors wish to thank nadiah ikhsani for her input on the methods section. contributors yd and ird contributed to drafting of the manuscript and composing the study proposal. pfh contributed to data collection and revising the manuscript. yd, pfh and ird read and approved the final manusrcipt. references 1. chang jh, wang sh, kuo cl, et al. prevalence of flexible flatfoot in taiwanese school-aged children in relation to obesity, gender, and age. eur j pediatr 2010;169:447–52. doi: 10.1007/s00431-009-1050-9. 2. nurzynska d, di meglio f, castaldo c, et al. flatfoot in children: anatomy of decision making. ital j anat embryol 2012;117:98–106. 3. wardanie s, herawati i, wahyuni. prevalensi kelainan bentuk kaki (flatfoot) pada anak usia 612 tahun di kota surakarta. universitas muhammadiyah surakarta; 2013. 4. ezema ci, abaraogu uo, okafor go. flatfoot and associated factors among primary school children: a cross-sectional study. hong kong physiother j 2013;5:1–8. 5. benedetti mg, ceccarelli f, berti l, et al. diagnosis of flexible flatfoot in children: a systematic clinical approach. orthopedics 2011;34:94. doi: https:// doi.org/10.3928/01477447-20101221-04. 6. odell s, logan de. pediatric pain management: the multidisciplinary approach. j pain res open 2013;6:785–90. 7. riazi a, shakoor s, dundas i, et al. health-related quality of life in a clinical sample of obese children and adolescents. heal qual life outcomes 2010;8: 134. 8. svedberg p, eriksson m, boman e. associations between scores of psychosomatic health symptoms and health-related quality of life in children and adolescents: a cross-sectional study. health qual life outcomes 2013;11:176. 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j pediatr gastroenterol nutr 2012;54:664-71. doi: 10.1097/ mpg.0b013e31823c9b88.2012;54:664–71. 19. wilson jmm. synopsis of causation: pes planus. uk: ministry of defence; 2008. 20. mosca vs. flexible flatfoot in children and adolescents. j child orthop 2010;4;107–21. 21. kothari a, stebbins j, zavatsky ab, et al. healthrelated quality of life in children with flexible flatfeet: a cross-sectional study. j child orthop 2014;8:489–96. 22. petersen as, hagglof bl, bergstrom ei. impaired health-related quality of life in children with recurrent pain. pediatr 2009;124:e759-67. doi: 10.1542/peds.2008-1546. 23. chen g, ratcliffe j, olds t, et al. bmi, health behaviors, and quality of life in children and adolescents: a school-based study. pediatr 2018;133 868–74. doi: 10.1542/peds.2013-0622. oktavianus 52 *radiology study program, faculty of medicine, diponegoro university/ dr. kariadi hospital, semarang **department of radiology, faculty of medicine, diponegoro university/ dr. kariadi hospital, semarang correspondence dr. margaretha vianny, sp.rad. radiology study program, faculty of medicine, diponegoro university/ dr. kariadi hospital, semarang email: margarethavianny@gmail.com univ med 2015;34:52-60 doi: 10.18051/univmed.2016.v35.52-60 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background hepatocellular carcinoma (hcc) incidence is increasing in asia and africa. locoregional minimally invasive transarterial chemoembolization (tace) is the palliative therapy of choice, improving survival rate. adequate lipiodol dose calculation in tace is necessary to produce good therapeutic effect. lipiodol retention pattern can predict tace therapeutic effect. this study aimed to determine correlation of lipiodol volume/tumor volume (l/v) ratio and lipiodol volume/tumor diameter (l/d) ratio with lipiodol retention pattern in post-tace ct-scans of hcc patients. methods this cohort prospective study was done from november 2013 to march 2014 on eighteen hcc patients with post-tace therapy in dr.kariadi hospital, semarang, fullfilling inclusion and exclusion criteria. lipiodol retention pattern was observed on 28 days post-tace and classified as type i (lipiodol accumulation in tumor and surrounding area), type ii (homogenous accumulation in tumor only), and type iii (partial accumulation). the spearman correlation test was used to determine any relationships between the various variables studied. results spearman correlation test showed that lipiodol volume had significant moderate correlation with lipiodol retention pattern (r=-0.684; p=0.002). both l/v and l/d ratios had moderately significant correlation with lipiodol retention pattern (r=0.511; p=0.030; and r=0.518; p=0.028, respectively). conclusion correlations of l/v ratio l/d ratio with lipiodol retention pattern were both moderately significant. lipiodol dose calculation based on l/v ratio is suggested considering the irregular three-dimensional form of the tumor, making volumetric measurement more appropriate. keywords: tace, lipiodol, lipiodol retention, hepatocellular carcinoma lipiodol retention pattern predicts transarterial chemoembolization therapeutic effect in hepatocellular carcinoma margaretha vianny*, gunawan santoso**, and eddy soedijanto** doi: http://dx.doi.org/10.18051/univmed.2015.v34.52-60 53 univ med vol. 34 no.1 pola retensi lipiodol meramalkan efek terapi lokoregional pada keganasan hepar latar belakang karsinoma hepatoseluler (khs) merupakan keganasan hepar dengan insidensi meningkat di asia dan afrika. terapi lokoregional (transarterial chemoembolization) (tace) dengan lipiodol dan kemoterapi menjadi terapi paliatif pilihan untuk meningkatkan angka kelangsungan hidup. dosis lipiodol pada tace harus adekuat agar efek terapi baik. penelitian ini bertujuan menentukan adanya korelasi dan membandingkan rasio volume lipiodol/ volume tumor (l/v) dan volume lipiodol / diameter tumor (l/d) dengan pola retensi lipiodol pada ct-scan khs dengan terapi tace pada penderita khs. metode desain penelitian adalah kohort prospektif menggunakan 18 penderita khs dengan terapi tace yang memenuhi kriteria inklusi dan eksklusi di rs dr.kariadi semarang selama november 2013 hingga maret 2014. pola retensi lipiodol pada ct scan 28 hari pasca tace diamati dan diklasifikasikan menjadi tipe i (akumulasi area tumor dan sekeliling tumor), ii (akumulasi homogen pada tumor), dan iii (akumulasi parsial). uji korelasi spearman digunakan untuk menguji hubungan berbagai variabel yang diteliti. hasil uji korelasi rank spearman menunjukkan volume lipiodol berkorelasi sedang dengan pola retensi lipiodol (r=0,684; p=0,002). korelasi signifikan dengan kekuatan korelasi sedang juga ditemukan antara rasio l/v dengan pola retensi lipiodol (r=0,511; p=0,030); dan antara rasio l/d dengan pola retensi lipiodol (r=0,518; p=0,028). kesimpulan korelasi antara rasio l/v dengan pola retensi lipiodol dan rasio l/d dengan pola retensi lipiodol sama-sama bermakna dan kekuatan korelasinya hampir sama. penghitungan dosis lipiodol berdasarkan rasio l/v lebih dianjurkan mengingat bentuk tumor irreguler dan memiliki dimensi ruang, sehingga lebih tepat pengukuran berdasarkan volume. kata kunci : tace, lipiodol, pola retensi lipiodol, karsinoma hepatoseluler abstrak introduction hepatocellular carcinoma (hcc) is the most frequent primary malignancy of the liver, being number six among the most frequent neoplasms found in the whole world. it is more prevalent in males, with a male-to-female ratio of 4:2.(1-3) hcc incidence is highest in east asia and sub-saharan africa.(3) in indonesia, hcc has a substantial incidence of 4.5%, with a 6% mortality rate and fourth rank among cancers in males. this pattern of findings is associated with an approximately 80% incidence of viral hepatitis infection, and with liver cirrhosis and other hcc risk factors.(3,4) hcc ranks third among fatal cancers in the world, with a mortality rate of 600.000 per year and survival times of less than 8 months after diagnosis.(5-7) among the major therapeutic modalities of hcc are surgery, percutaneous radiofrequency ablation (prfa), percutaneous ethanol injection ( p e i ) , a n d t r a n s c a t h e t e r a r t e r i a l c h e m o 54 embolization (tace), radiation therapy, and chemotherapy.(8-10) in the last two decades, nonsurgical therapeutic interventions such as tace have been inceasingly used for unresectable hcc.(9,10) lo et al.(11) give the survival rate of hcc patients undergoing tace as 57%, which is higher than their 32% survival rate without tace. the cumulative survival rate of hcc patients receiving tace therapy is 90.6% with a median survival time of 46.6 months.(12) radiological investigations play an important role in establishing the diagnosis and treatment of hcc. multiphasic ct scans and mri are important imaging modalities in hcc diagnosis and follow-up, particularly to visualize contrast enhancement patterns.(13,14) other modalities that can be used are ultrasonography and laboratory investigation of the alpha-fetoprotein serum marker.(15) transcatheter arterial chemoembolization is a minimally invasive chemotherapeutic method applied to the target tumor via the hepatic artery, followed by embolization of its branches supplying blood to the tumor.(16) the combination of chemotherapy with an embolant places a higher concentration of the chemotherapeutic agent in longer contact with the target organ and reduces systemic effects.(17) transcatheter arterial chemoembolization combined with lipiodol as embolant has been reported to be one of the most effective palliative treatment for hcc.(11,12) the dose of lipiodol should be adequate to yield good therapeutic effect, but it must not be excessive, since the lipiodol may undergo reverse flow to the normal liver parenchyma, decreasing liver function and resulting in liver cirrhosis. there is no agreement yet in the literature on the volume of lipiodol to be used in tace. to date the dose has been based on tumor diameter or maximally 20-30 ml for a tumor diameter larger than 10 cm. apart from the diameter, a tumor has shape and volume, therefore the lipiodol dose in the tumor does not depend exclusively on its diameter.(18) the lipiodol accumulation or retention pattern in the tumor area on post-tace ct scans are reported to play a role in predicting the efficacy of tace for hcc.(19) the disease free survival rate is higher and the recurrence rate lower in patients with a lipiodol accumulation pattern in the area surrounding the tumor on post-tace ct scans.(10) there have been several previous studies on the relationship between tace-treated hcc and lipiodol retention pattern on ct scan. kawaguchi et al.(10) investigated the lipiodol accumulation pattern in post tace hcc patients in connection with therapeutic efficacy. they found that the lipiodol accumulation pattern evaluated by ct scans immediately after tace was a strong indicator of tace therapeutic efficacy. matsuo et al.(20) conducted a study to find the optimal lipiodol dose based on lipiodol accumulation pattern and histopathologic picture. they found a correlation of lipiodol accumulation on ct scan with degree of necrosis on histologic examination and suggested an optimal lipiodol dose d > d (if tumor diameter d<5 cm) with a maximal dose of 10 ml. cheng et al.(18) conducted an investigation on lipiodol dose adjustment in tace on the basis of the hcc blood supply on ct scan, and found that the lipiodol dose may be determined from the blood supply pattern and tumor diameter. the lipiodol dose was adjusted to tumor diameter or 1.5 times the tumor diameter and maximally 20 ml (tumor diameter <10 cm) up to 30 ml (tumor diameter >10 cm). the present study is the first to be conducted on the relationship of lipiodol volume used in tace, tumor volume, tumor diameter, and lipiodol retention pattern. the purpose of this study was to determine any correlation of, on the one hand, the tace lipiodol volume to tumor volume ratio and the lipiodol volume to tumor diameter ratio as measured on baseline ct scan, with on the other hand, the lipiodol retention pattern on ct scan evaluated 28 days post-tace, in accordance with the hcc evaluation protocol. evaluation of the lipiodol retention pattern may be predictive of antitumor effect or tace success rate. this study did not involve patient survival rate or histopathologic picture of the lesion. vianny, santoso, soedijanto lipidiol retention in hepatocellular carcinoma 55 univ med vol. 34 no.1 methods design of study this study was observational analytic, of prospective cohort design without controls. the study was carried out in dr. kariadi central general hospital, semarang, starting from october 2013 until march 2014. study subjects the study subjects were patients with a diagnosis of hcc based on ct scan results, in accordance with the criteria of the european association for the study of the liver (easl) and european organisation for research and treatment of cancer (eortc). the diagnosis of hcc was based on the size of the primary lesion, i.e. the presence of a hepatic mass of more t h a n 2 c m w i t h c h a r a c t e r i s t i c a r t e r i a l v a s c u l a r i z a t i o n v i s i b l e o n a t l e a s t t w o radiological modalities (usg, ct, mri) or the presence of a hepatic mass identified by one radiological modality and increased serum alphafetoprotein (afp) of >400 ng/ml. ( 21-23) the diagnosis of hcc may be established by a specialist in radiology or internal medicine. the study subjects were hcc patients meeting the following inclusion criteria: i) no tumor invasion to the portal vein and inferior vena cava, or other metastases; ii) hb > 9 g/dl, leukocyte count < 11.000, serum creatinine < 1.5 mg/dl, total bilirubin < 3 mg/dl; iii) child-pugh criteria a or b, i.e. hcc with or without decreased liver function that can be compensated by the patient,(24) and iv) a ct scan picture showing rapid enhancement (increase in hounsfield unit score) in the arterial phase when the contrast agent starts to fill the hepatic artery followed by wash out (rapid decrease in hounsfield unit score) in the portovenous and equilibrium phase when the contrast has entered the portal vein and inferior vena cava. the exclusion criteria for the study subjects were: i) hcc subjects not having the ct scan exactly 28 days post-tace; ii) presence of arteriovenous fistulae in hcc vascularization. the sample size was determined using the f o r m u l a f o r s a m p l e s i z e c a l c u l a t i o n f o r proportions with proportion of tace efficacy of 0.84, at α=0.05, yielding a minimal sample size of 18. the study sample was selected by consecutive non-random sampling, on the basis of patient admittance at dr. kariadi hospital, semarang. determination of hcc diameter and volume on ct scan tumor diameter and volume before tace therapy were measured by multiphase ct scan in the cranio-caudal direction using nonionic contrast medium at a concentration of 300 mg/ ml, a dose of 1.5-2 ml/kgbw, with maximal dose of 150 ml. the contrast medium was administered by intravenous injection in the extremities or by a 20g cannula, using the mallinckrodt optivantage power injector at flow rates of 4-5 ml/s. the scans were performed for the whole hepatic region in the early arterial phase (25 s), late arterial phase (35 s), portal phase (60-70 s), and equilibrium phase (120-180 s), starting at the time of contrast injection. evaluation of the scan results was by multiplanar reconstruction using a siemens syngo dynact work station. ct parameters: collimation 64 x 0 . 6 m m , s l i c e t h i c k n e s s 3 m m , i m a g e reconstruction increment 0.625 mm, 120 kv, 200-250 mas. tumor diameter (d) was measured by drawing a center line through the widest area of the hcc tumor image as seen on ct scan and was expressed in centimeters (cm). tumor volume (v) was measured by manual drawing using a computer mouse following the tumor contour lines in all sections or slices, then the area of the lesion was calculated by computer software to yield the tumor volume in cubic centimeters (cm3). lipiodol volume in tace tace was performed according to standard procedures using a dsa artisz (siemens) fluoroscopy instrument with arterial access via 56 the common fermoral vein using a 6f sheath. access to the celiac trunk was achieved using a simmons 5f catheter followed by a 2.7f microcatheter to access the lobar or segmental artery that constitutes the feeding artery to the t u m o r. a f t e r c a t h e t e r p l a c e m e n t , t h e chemotherapeutic drug was injected and embolization was done using lipiodol until a substatic flow was obtained, with minimal tumor blush, when the lipiodol had occupied the whole tumor area and the injected lipiodol flow slowed down. the injection was performed manually to minimize retrograde flow to the arterial vascularization of other segments. the volume of lipiodol (l) was obtained from the amount of lipiodol administered in tace therapy until circulatory stasis was seen in the hcc (in ml) or maximally 30 ml. the standard optimal lipiodol dose according to matsuo et al.(20) is d>d (for tumors < 5 cm); and maximally 10 ml. on the other hand, the lipiodol dose according to cheng et al.(18) is adjusted to tumor diameter or 1.5 times tumor diameter, and maximally 20 ml (for tumors <10 cm) up to 30 ml (for tumors >10 cm). the ratio of lipiodol volume to tumor volume (l/v ratio) was calculated by comparing the lipiodol volume administered during tace to the tumor volume on diagnostic ct scan, expressed in ml/cm3. the ratio of lipiodol volume to hcc tumor diameter (l/d ratio) was determined by comparison of lipiodol volume administered during tace to hcc tumor diameter on diagnostic ct scan, expressed in ml/cm. lipiodol retention pattern the lipiodol retention pattern was evaluated o n c t s c a n s 2 8 d a y s p o s t ta c e u s i n g procedures, and contrast medium amounts and concentrations similar to those used for ct scans before tace therapy. the observed lipiodol retention pattern was then classified into 3 types (figure 1).(10) type i, lipiodol accumulation in the whole tumor and surrounding area. lipiodol and chemotherapeutic agent fill the whole tumor and surrounding area. according to studies, patients with this type of retention have higher survival rates. type ii, homogenous accumulation in whole hcc tumor area. in this type, the lipiodol and chemotherapeutic agent can fully enter the tumor, with an area of tumor necrosis of up to 98%, and is considered to represent an adequate antitumor effect. type iii, accumulation in the hcc tumor area only. this type is correlated with a less than optimal distribution of the chemotherapeutic agent. data analysis the spearman correlation test was used to determine any relationships between the various variables studied (lipiodol volume, tumor volume, tumor diameter and their ratios versus lipiodol retention pattern). the correlation was considered statistically significant if p<0.05. a b c figure 1. classification of lipiodol retention in hcc patients on tace.(10) a. type i : accumulation in whole hcc tumor and surrounding area b. type ii : homogenous accumulation throughout the hcc tumor area c. type iii : accumulation in part of hcc tumor area only vianny, santoso, soedijanto lipidiol retention in hepatocellular carcinoma 57 univ med vol. 34 no.1 results during the study period 18 hcc patients were encountered who met the research criteria. the median age of the study sample was 59 years, with minimum of 46 years and maximum of 77 years. the age distribution of the study sample was as follows: < 50 years 2 subjects (11%), 51-60 years 10 subjects (55%), 61-70 years 3 subjects (17%), and >70 years 3 subjects (17%). most of the study subjects were male, 16 in total (89%), while females totaled only 2 (11%). the 28-day post-tace ct scans showed variable lipiodol retention patterns, comprising type i (lipiodol accumulation in whole tumor and s u r r o u n d i n g a r e a ) , t y p e i i ( h o m o g e n o u s accumulation in tumor), and type iii (partial accumulation in tumor). the most frequent lipiodol retention pattern was of type iii, accounting for 13 subjects (72.2%), followed by type i with 3 subjects (16.7%), and type ii with 2 subjects (11.1%). in this study the median tumor volume was 231.38 cm3, the largest tumor volume was 668.2 cm3 and the smallest 3.7 cm3, while the median tumor diameter was 8.18 cm3, the largest diameter 14.24 cm3 and the smallest 2.5 cm3. the maximal lipiodol volume in this study was 25 ml, administered to one tumor with a volume of 668.2 cm3 and a diameter of 13.8 cm, and one with a volume of 522.25 cm3 and diameter of 10.3 cm. the minimum lipiodol volume in this study was 5 ml, administered to one tumor with a volume of 3.7 cm3 and a diameter of 2.5 cm. the lipiodol retention types differed in their median l/v and l/d ratios. the highest l/v ratio of 1.28 ml/cm3 was found in lipiodol retention pattern type i, followed by type ii with a ratio of 0.11 ml/cm3, while the lowest ratio was for type iii, namely 0.04 ml/cm3. the l/d ratio had a similar data distribution, with the highest l/d ratio in lipiodol retention pattern type i, namely 2.64 ml/cm, followed by type ii with 2.07 ml/ cm, and the lowest in type iii with 1.83 ml/cm (table 1). there was a significant moderately moderate negative correlation of lipiodol volume, tumor volume, and tumor diameter with lipiodol retention pattern (r=-0.68, p=0.002; r=-0.63, p=0.005;and r=-0.67, p=0.002, respectively). however, the correlation of lipiodol volume/hcc tumor volume and lipiodol volume/tumor diameter ratios with lipiodol retention pattern was positive significant of moderate correlation(r=0.51; p=0.030) and negative moderate correlation (r=-0.53; p=0.028) (table 2). table 1. lipiodol volume, tumor volume, tumor diameter, lipiodol volume/hcc tumor volume ratio, and lipiodol volume/hcc tumor diameter ratio, by lipiodol retention pattern *values are median tabel 2. correlation of lipiodol volume/tumor volume (l/v) and lipiodol volume/tumor diameter (l/d) with lipiodol retention pattern 58 discussion this study involved 18 subjects consisting of 16 males (89%) and 2 females (11%), with the youngest 46 years of age and the oldest 77 years, and the majority between 51-60 years. this agrees with the literature, in that hcc incidence is higher in males than in females, with a ratio of 5:1 and mean age of 50-70 years.(3,7) altekruse et al.(1) and the american cancer society revealed that the higher hcc incidence in males is associated with behavior or life style as risk factors for hcc, such as alcohol consumption, smoking, and drug abuse by injection.(1) the tumor with the largest volume in this study did not always have the largest tumor diameter or received the largest lipiodol volume, whereas the largest tumor diameter also did not always have the largest tumor volume and the largest lipiodol volume. this was caused by the irregular tumor shape, such that increases in tumor volume and tumor diameter were not always proportional, but tended to be linear in the diagram, and this was also the case with the lipiodol volume administered to the tumor. the lipiodol retention pattern in the tumors varied from type i to type iii. of the 18 study subjects, most had type iii retention patterns, totalling 13 subjects (72.22%), followed by type i with 3 subjects (16.67%), and type ii with 2 subjects (11.11%). these findings differed from the results of kawaguchi et al.(10) who found that in 117 hcc tumors the most frequent retention pattern was of type ii (42%), followed by type i (35%), the least frequent being type iii (22%). in our study the finding of type iii as the most frequent retention type may have been caused by the large tumor size in the majority of study subjects and the negative correlation of lipiodol volume, tumor volume, and tumor diameter with lipiodol retention pattern. therefore the larger lipiodol volume, tumor volume, and tumor diameter, tended to result in low lipiodol retention (type iii). the larger tumors will form arteriovenous microfistulae, such that the lipiodol entering the hepatic artery cannot accumulate but on the contrary will flow into the venous system. o u r s t u d y r e s u l t s s h o w e d a s i g n i f i c a n t relationship of lipiodol volume/tumor volume ratio and lipiodol volume/tumor diameter ratio with lipiodol retention pattern. the positive correlations showed that higher l/v and l/d ratios tend to yield type i retention, where lipiodol fills the whole tumor and surrounding area, while lower l/v and l/d ratios tend to result in type iii retention. therefore, with increasingly higher l/v and l/d ratios there will be an optimal lipiodol accumulation in the tumor and adequate therapeutic results. however, it should be borne in mind that according to the literature the maximal dose of lipiodol is 30 ml. the lipiodol dose should be adequate to yield good therapeutic effects, but should not be excessive, since the lipiodol may then undergo a reverse flow to the normal liver parenchyma, thus decreasing liver functions, resulting in liver cirrhosis.(18) lipiodol in tace functions as anticancer drug transporter, and acts to enhance the antitumor effect of anticancer drugs. it may also a c t a s e m b o l a n t , c a u s i n g i s c h e m i a a n d lymphatic drainage problems, finally resulting in tumor necrosis. decreasing retention of lipiodol by the tumor (approaching type iii r e t e n t i o n ) i n d i c a t e s l e s s t h a n o p t i m a l distribution of the chemotherapeutic agent, with a small area of necrosis, indicative of a poor therapeutic response. increasing lipiodol r e t e n t i o n ( a p p r o a c h i n g t y p e i r e t e n t i o n ) i n d i c a t e s o p t i m a l d i s t r i b u t i o n o f t h e chemotherapeutic agent, with a large area of necrosis and excellent therapeutic response.(9,10) some of the factors that may influence the lipiodol retention pattern are among others: tumor size, volume of lipiodol administered, vascular permeability, lymphatic flow, and tumor necrosis.(9) the lipiodol retention pattern has been reported to play a role in predicting the effect of tace on hcc.(19) the diseasefree survival rate is higher and the recurrence rate lower in patients with type i lipiodol accumulation pattern.(10) vianny, santoso, soedijanto lipidiol retention in hepatocellular carcinoma 59 univ med vol. 34 no.1 the type i lipiodol retention pattern is obtained from the higher median l/v and l/d ratios in comparison with the other types of retention (l/v ratio = 1.28 ml/cm3; l/d ratio = 2.64 ml/cm). the type ii lipiodol retention pattern has lower median l/v and l/d ratios of 0.11 ml/ cm3 and 2.09 ml/cm, respectively; while the type iii retention pattern has the lowest median l/v and l/d ratios of 0.04 ml/cm3 and 1.83 ml/cm, respectively. this picture also supports the positive correlation results, where higher l/v and l/d ratios tend to approach type i retention patterns. therefore high l/d and l/ v ratios are expected to increase the effectivity of curative treatment. the retention patterns that are considered good are types i and ii, with l/v ratios in the range of 0.11 – 1.28 ml/cm3 and l/ d ratios from 2.07 to 2.64 ml/cm. the l/d ratio in the present study was higher than that of cheng et al.(18) who stated that lipiodol volumes equal to 1.5 times the tumor diameter yield curative effects. however, in their study the lipiodol dose was not determined from tumor volume or l/v ratio. the correlation values of l/v and l/d with lipiodol retention are almost identical. in spite of this, dose calculation on the basis of volume is superior to tumor size measurement, because it can compensate for actual tumor shape, particularly in irregularly-shaped tumors that cannot be represented by their largest diameter only.(25,26) initially, the who issued guidelines for tumor size determination using the largest diameter of the tumor. in line with technological advances, prasad et al.(26) reported on hcc volume measurement using three-dimensional ct that can give a true picture of post-tace tumor response, particularly for tumors of irregular shape. the european organization for research & treatment in oncology and the national cancer institute of the united states also recommend the technique of tumor volume determination by ct scan for the evaluation of tace.(24,25,27) therefore it can be stated that lipiodol dose calculation based on l/v ratio is better for succesful tace. however, in hospitals where tumor volume determination by ct scan reconstruction software cannot be done, the l/ d ratio may still be used, as it is correlated with l/v. the significant correlations of l/v and l/ d ratios with lipiodol retention pattern indicate the importance of lipiodol dose calculation based on volume as well as tumor diameter, with the expected formation of good lipiodol retention patterns on 28-day post-tace ct scans. the ct scan is an important imaging modality for tumor diagnosis, determination of tumor diameter and volume, and evaluation of hcc response on tace therapy. o u r s t u d y d i d n o t p e r f o r m s a m p l e stratification according to tumor size, which might have resulted in different and varying l/ v and l/d ratios, depending on the size of the tumors. in the future we expect to conduct studies on hcc tumors with tumor size stratification. larger tumor size tends to lead to arterio-venous microfistula formation, such that the lipiodol entering the hepatic artery cannot accumulate but is drained by the venous system, resulting in type iii lipiodol retention.(9) to obtain good retention patterns, a larger lipiodol dose should be used, but limited by the maximal dose. in large tumors the maximal dose is usually insufficient to fill the whole tumor area, causing circulatory stasis. this causes optimal differences in l/v and l/d r a t i o s b e t w e e n l a rg e a n d s m a l l t u m o r s . furthermore, we expect future studies to allow us to deduce the therapeutic efficacy from the patient’s clinical improvement and liver function test results. conclusion the correlations of both l/v ratio and l/d ratio with lipiodol retention pattern were significant and of similar strengths. 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of unresectable hepatocellular carcinoma after tace. world j gastroenterol 2010;16:2038-45. 26. prasad sr, jhaveri ks, saini s, et al. ct tumor measurement for therapeutic response assessment: comparison of unidimensional, bidimensional, and volumetric techniques initial observations. radiol 2002;225:416-9. 27. guan ys, hu y, liu y. multidetector-row computed tomography in the management of hepatocellular carcinoma with transcatheter arterial chemoembolization. j gastroenterol hepatol 2006;21:941-6. vianny, santoso, soedijanto lipidiol retention in hepatocellular carcinoma adi priyana 152 universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 * bagian patologi klinik fakultas kedokteran universitas trisakti korespondensi dr. dr. adi priyana, sp.pk bagian patologi klinik fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 021-5672731, eks. 2404 email: adipr@indo.net.id universa medicina 2007; 26:152-9. abstrak osteoporosis adalah kelainan tulang yang kronik progresif akibat berkurangnya massa dan kekuatan tulang sehingga menjadi predisposisi meningkatnya risiko fraktur. pertanda tulang dapat memberikan gambaran proses remodelling yang sedang terjadi. pemeriksaan ini meliputi pertanda resorpsi tulang yang dilakukan oleh osteoklas dan pertanda formasi tulang yang dilakukan oleh osteoblas. diagnosis untuk osteoporosis yang dianggap baku adalah pemeriksaan bone mass density (bmd) tetapi bmd tidak dipakai untuk monitoring pengobatan karena membutuhkan waktu lama (2 tahun) untuk melihat perbedaan gambaran massa tulang. analisis pertanda tulang dapat memberikan gambaran proses remodeling yang sedang terjadi dalam waktu yang lebih singkat (3 – 6 bulan). artinya analisis pertanda tulang dapat memantau dan menilai respons pengobatan, prognosis penderita dengan risiko osteoporosis, mencari penyebab berkurangnya tulang secara cepat, memilih pengobatan yang sesuai, memantau pasien dengan pengobatan kortikosteroid dan mempelajari patogenesis osteoporosis. dalam tulisan ini akan diuraikan beberapa analisis pertanda tulang seperti alkaline phosphatase (alp), osteocalcin, p1-np dan ß-crosslaps dan kegunaannya pada tatalaksana osteoporosis. kata kunci: osteoporosis, pertanda tulang, osteocalcin, p1-np dan ß-crosslaps peran pertanda tulang dalam serum pada tatalaksana osteoporosis adi priyana* 153 universa medicina vol. 26 no.3 *department of clinical pathology faculty of medicine university of trisakti correspondence dr. dr. adi priyana, sp.pk department of clinical pathology medical faculty trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone : 021-5672731 ex.2404 email: adipr@indo.net.id universa medicina 2007; 26: 152-9. osteoporosis is a skeletal disorder with reduced bone density and strength. this lead increased risk of fracture. osteoporosis can be classified as primary and secondary osteoporosis. primary osteoporosis is found in menopaused women due to reduced estrogen hormone, and in men due to reduced calcium. many primary diseases can cause secondary osteoporosis. bone mass density can be considered as a standard examination for osteoporosis but it is still controversial because it need more than 2 years to differentiate bone mass. but bone marker examination can give description of remodelling process in bones only for 3–6 months, such as bone resorption by osteoclast and bone formation by osteoblasts. bone marker analysis can support the diagnosis of osteoporosis, therapeutic response, identify the causal factors, select the mode of therapy, and study the pathogenesis of osteoporosis. in this review some bone markers such as alkaline phosphatase (alp), osteocalcin, p1-np, and β-crosslaps and their use in osteoporosis will be described. keywords: osteoporosis, bone marker, osteocalcin, p1-np dan ß-crosslaps role of serologic bone marker in osteoporosis management adi priyana* abstract pendahuluan jumlah penduduk indonesia akan meningkat lebih dari empat kali sejak tahun 1990–2025. m e n i n g k a t n y a d e r a j a t k e s e h a t a n a k a n mengakibatkan terjadinya ledakan penduduk u s i a l a n j u t t e r u t a m a w a n i t a p a d a u s i a menopause. tulang adalah organ tubuh yang selalu mengalami proses pergantian (turnover), tulang lama diganti dengan tulang baru yaitu terjadi proses resorpsi dan proses formasi. bila terjadi ketidakseimbangan antara proses resorpsi tulang oleh osteoklas dan proses formasi oleh osteoblas maka akan terjadi osteopenia.atau osteoporosis. osteoporosis adalah kelainan tulang yang bersifat kronik progresif akibat berkurangnya massa tulang sehingga terjadi p e n u r u n a n k e k u a t a n t u l a n g y a n g m e n j a d i p r e d i s p o s i s i m e n i n g k a t n y a r i s i k o f r a k t u r. struktur tulang bagian dalam tampak perbedaan k e p a d a t a n t u l a n g a n t a r a n o r m a l d e n g a n osteoporosis (gambar 1). gambar 1. gambaran tulang pada orang normal dan osteoporosis (5) 154 kualitas tulang ditentukan oleh empat faktor penting yaitu densitas tulang (bone density), ukuran tulang, pergantian tulang (bone turnover) dan geometri tulang (mikroarsitektur). banyak faktor yang mempengaruhi pembentukan tulang seperti kalsium dan vitamin d, hormon paratiroid, kalsitonin yang diproduksi kelenjar tiroid, estrogen dan testosteron. faktor yang meningkatkan kerja osteoklas sehingga terjadi bone resorption adalah hormon glukokortikoid. osteoporosis diklasifikasikan dalam 2 kelompok y a i t u o s t e o p o r o s i s p r i m e r d a n s e k u n d e r. osteoporosis primer terdapat pada wanita menopause (post menopause osteoporosis) karena berkurangnya hormon estrogen. pada pria usia lanjut (senile osteoporosis) akibat berkurangnya kalsium. osteoporosis sekunder disebabkan oleh berbagai penyakit primer s e p e r t i p e n y a k i t c u s h i n g , h i p e r t i r o i d , hiperparatiroid, hipogonadisme, gagal ginjal k r o n i s , k u r a n g a k t i v i t a s , a l k o h o l i s m e , p e m a k a i a n o b a t g o l o n g a n k o r t i k o s t e r o i d , perokok dan kelebihan kafein.(1-4) osteoporosis sendiri umumnya tidak menimbulkan keluhan tetapi manifestasi klinisnya yang berat adalah terjadinya fraktur akibat berkurangnya kekuatan tulang yang menjadi predisposisi meningkatnya risiko fraktur. pemeriksaan bone mass density (bmd) merupakan pemeriksaan baku emas (gold standard) oleh world health organization (who) untuk diagnosis osteoporosis, tetapi kurang bermanfaat untuk memantau pengobatan osteoporosis.(1-4) analisis pertanda tulang te r d a p a t e m p a t f a k t o r p e n t i n g u n t u k menilai kualitas tulang yaitu densitas tulang ( b o n e d e n s i t y ) , p e r g a n t i a n t u l a n g ( b o n e turnover), ukuran tulang dan geometri tulang ( m i k r o a r s i t e k t u r ) . p e rg a n t i a n t u l a n g a t a u remodelling terdiri dari resorpsi tulang oleh o s t e o k l a s d a n p e m b e n t u k a n t u l a n g o l e h osteoblas. pergantian tulang yang meningkat akan mempercepat porositas tulang. kombinasi p e m b e n t u k a n d a n r e s o r p s i t u l a n g a k a n menentukan massa tulang dan osteoporosis. jeannettee(6) menyatakan bahwa terdapat lebih dari 75 juta penderita osteoporosis di amerika, eropah dan jepang dengan kejadian fraktur 1,3 juta setiap tahunnya. teknik yang baik untuk menilai densitas tulang adalah dengan m e t o d e p e m e r i k s a a n d u a l e n e rg y x r a y a b s o r p t i o m e t r y ( d x a ) d a n q u a n t i t a t i v e computerized tomography (ct).(7-9) kedua metode tersebut memiliki tingkat kesalahan hanya 0,5 – 2 persen dan dianjurkan untuk d i p a k a i s e b a g a i a l a t d i a g n o s i s . wa l a u p u n pemeriksaan ct lebih sensitif tetapi lebih banyak terjadi radiasi sehingga para ahli lebih memilih pemeriksaan dxa. pemeriksaan densitas t u l a n g d i a n j u r k a n d i l a k u k a n p a d a w a n i t a perimenopause yang akan memulai pengobatan dengan hormon, pasien yang lama memakai glukokortikoid, hiperparatiroid dan sebagai alat u n t u k m e m a n t a u m e r e k a y a n g d i b e r i k a n pengobatan untuk osteoporosis. meskipun pemeriksaan dxa memiliki presisi tinggi untuk menentukan bagian tulang yang mengalami osteoporosis, tetapi pemeriksaan dxa hanya dapat melihat proses penurunan massa tulang pada tahap lanjut. banyak peneliti lebih memilih a n a l i s i s p e r t a n d a t u l a n g u n t u k m e n g e l o l a osteoporosis.(9-11) kini dikenal pemeriksaan pertanda tulang (bone marker) seperti ostocalcin, β-crosslaps, p1np dan alkali fosfatase. berbeda dengan pemeriksaan bmd, tujuan analisis pertanda tulang adalah untuk memantau dan menilai respons pengobatan, diagnosis penderita dengan r i s i k o o s t e o p o r o s i s , m e n c a r i p e n y e b a b berkurangnya tulang secara cepat, memilih pengobatan yang sesuai, memantau pasien d e n g a n p e n g o b a t a n k o r t i k o s t e r o i d d a n mempelajari patogenesis osteoporosis.(1-4) p e r t a n d a t u l a n g d a p a t m e m b e r i k a n gambaran proses remodelling yang sedang priyana pertanda tulang pada osteoporosis 155 universa medicina vol. 26 no.3 terjadi. pemeriksaan ini meliputi pertanda resorpsi tulang (resorption bone marker) yang dilakukan oleh osteoklas dan pertanda formasi tulang (formation bone marker) yang dilakukan oleh osteoblas. pemeriksaan pertanda tulang dapat dilakukan terhadap bahan serum atau urin.(4) dilakukan pada usia >40 tahun, sebelum d i b e r i k a n t e r a p i a n t i r e s o r p s i o r a l ( o b a t penghambat penyerapan tulang) dan 3 bulan s e t e l a h t e r a p i a n t i r e s o r p s i o r a l , u n t u k m e n g e t a h u i k e b e r h a s i l a n p e n g o b a t a n . ( 6 8 ) a p u r v a e t a l ( 1 2 ) m e n g a n j u r k a n m e m a k a i pertanda tulang sebagai cara untuk mengelola osteoporosis dengan alasan: (i) dapat menilai p r o s e s r e m o d e l i n g d a n t i n g k a t k e c e p a t a n h i l a n g n y a t u l a n g ; ( i i ) b m d t i d a k m a m p u memprediksi risiko fraktur pada pengobatan o s t e o p o s o s i s ; ( i i i ) d a p a t m e n i l a i r e s p o n s pengobatan dalam 3–6 bulan. pemeriksaan pertanda tulang sebaiknya dilakukan dalam keadaan puasa walaupun hanya terdapat sedikit perbedaan kadarnya dengan setelah makan.(13-15) pemeriksaan pertanda tulang dapat dikerjakan dengan bahan urin atau serum. bahan tersebut dapat disimpan pada suhu kamar selama 48 jam, 2–8oc selama seminggu dan bila dibekukan masih dapat diperiksa setelah 6 bulan.(16-18) jung(19) membuktikan bahwa analisis pertanda tulang dapat dipakai sebagai alat untuk memantau metastase ke tulang dari karsinoma prostat. termasuk pertanda pembentukan tulang adalah alkali fosfatase, osteocalcin, p1nterminal propeptide (p1np). pertanda resorpsi tulang adalah pyridium cross-links, collagen i c-terminal telopeptide ß (ß-crosslaps). akan diuraikan disini alkaline phosphatase (alp), osteocalcin, p1-np dan ß-crosslaps karena ketiganya dapat diperiksa di laboratorium dari bahan darah dan urin.(20-23) alkaline phosphatase serum alkaline phosphatase (alp) terdiri dari beberapa isoensim yang terdapat pada banyak organ seperti hati, tulang, ginjal, usus dan placenta. alp hati dan tulang kadarnya tinggi dalam serum sehingga banyak dipakai u n t u k m e n i l a i p r o s e s m e t a b o l i s m e t u l a n g khususnya menilai dan memantau aktivitas osteoblas dan untuk menilai kelainan pada hepatobilier. nilai normal: pria 90–239 µ/l dan wanita di bawah 45 tahun 76–196 µ/l dan wanita >45 tahun 87–250 µ/l.(8) l e b i h s p e s i f i k l a g i b i l a m e l a k u k a n pemeriksaan isoensim dari alp karena akan m e n g g a m b a r k a n k e l a i n a n m a s i n g m a s i n g isoensim yang diproduksi oleh organ tertentu. reid ir et al(16) memakai pemeriksaan pertanda t u l a n g i n i s e b a g a i m a r k e r o s t e o b l a s p a d a p e n g o b a t a n p e n y a k i t p a g e t d e n g a n b i s p h o s p h o n a t . p e m e r i k s a a n i s o e n s i m dilakukan dengan cara khusus yaitu teknik elektroforesis dan kadar isoensim alp tulang adalah 20–120 µ/l. procollagen type 1 amino-terminal propeptide (p1np) lebih dari 90% matriks organik tulang berisi type 1 collagen yang akan dibentuk menjadi tulang. type 1 collagen berasal dari type 1 procollagen yang dihasilkan fibroblas dan osteoblas. type 1 procollagen mengandung n-amino dan c carboxy terminal propeptida yang akan diuraikan oleh ensim protease selama terjadi perubahan procollagen menjadi collagen dan kemudian dibentuk matriks tulang (gambar 2). pertanda tulang p1np merupakan indikator s p e s i f i k d a n a l a t p r e d i k t o r u n t u k m e n i l a i pembentukan tulang. p1np dilepas selama pembentukan type 1 collagen dan akan masuk ke dalam aliran darah.(20,21) pasien yang diobati dengan pengobatan anabolik akan meningkat k a d a r n y a ( g a m b a r 3 ) . b r a s o ( 2 3 ) m e m a k a i p e r t a n d a t u l a n g i n i d a l a m p e n g o b a t a n karsinoma prostat. nilai normal p1np: 5–1200 µg/l atau ng/ml.(24,25) 156 gambar 2. p1np dilepas ke dalam darah dan urine pada saat pembentukan tulang(26) gambar 3. pemantauan pengobatan anabolik meningkatkan kadar p1np(26) gambar 4. ß-crosslaps dilepas dari kolagen tipe 1 pada saat resorpsi tulang(27) priyana pertanda tulang pada osteoporosis 157 universa medicina vol. 26 no.3 ß-crosslaps saat ini telah dikembangkan pemeriksan ßc r o s s l a p s y a n g d a p a t d i g u n a k a n s e b a g a i marker resorpsi tulang yang sensitif dan spesifik. sensitivitas mencapai >70% dan spesifisitas 80%. ß-crosslaps adalah hasil pemecahan protein kolagen tipe 1 yang spesifik tulang dan m e r u p a k a n p r o d u k m e t a b o l i s m e a t a u p e m b o n g k a r a n t u l a n g s e c a r a l a n g s u n g . p e r o m b a k a n t u l a n g y a n g d i l a k u k a n o l e h osteoklas akan menghancurkan kolagen tipe 1 dan terbentuk bentuk α dan ß. bentuk ß ini disebut ß-crosslaps dan kadarnya dapat diukur dari serum, plasma atau urin (gambar 4). kadar ß-crosslaps dipengaruhi usia, jenis kelamin dan siklus sirkadian dengan puncak tengah malam dan kadar terendah sore hari. diduga pada saat puncak kadarnya 66% lebih tinggi dibandingkan dengan kadar rata-rata. pemeriksaan ß-crosslaps dapat dipakai sebagai alat pemantau terapi terutama pada pengobatan dengan anti-resorptif seperti bisphosphonate. dalam waktu 3 minggu seharusnya terjadi penurunan kadar ß-crosslaps dalam darah atau urin sehingga dokter yang memberi pengobatan dapat memprediksi hasil pengobatan (gambar 5). pemeriksaan kadar ß-crosslaps lebih sensitif dalam menilai perbaikan metabolisme tulang dibandingkan dengan pemeriksaan bmd.(8) gambar 5. ß-crosslaps sebagai pertanda sensitif pada pengobatan antiserorptif(27) teknik pemeriksaan ß-crosslaps dengan c a r a e l e c t r o c h e m i l u m i n e s c e n t s a n d w i c h immunoassay. nilai normalnya: pria usia 30– 50 tahun: 0,016 – 0,584 ng/ml. usia 50–70 tahun: 0,104 – 0,704 ng/ml sedang usia >70 t a h u n : 0 , 1 0 4 – 0 , 8 5 4 n g / m l . wa n i t a premenopause: 0,025 – 0,573 ng/ml dan pasca menopause: 0,104 – 1,008 ng/ml.(26,27,28) n-mid osteocalcin o s t e o c a l c i n m e r u p a k a n p r o t e i n n o n kolagen yang terdapat paling banyak dalam t u l a n g d a n d i p r o d u k s i s e l o s t e o b l a s . osteocalcin berperan penting dalam proses m i n e r a l i s a s i d a n p r o s e s h o m e o s t a s i s i o n k a l s i u m . m a k a p e m e r i k s a a n o s t e o c a l c i n m e r u p a k a n p a r a m e t e r y a n g b a i k u n t u k menentukan gangguan metabolisme tulang pada saat pembentukan tulang dan penggantian t u l a n g ( b o n e t u r n o v e r ) . p e m e r i k s a a n osteocalcin sering dipakai sebagai biomarker awal pada pengobatan obat pembentuk tulang dan untuk menilai efektivitas hasil pengobatan. hasil pemeriksaan osteocalcin cukup akurat dan stabil dalam menilai proses pembentukan tulang. metode pemeriksaan osteocalcin adalah enzyme-linked immunosorbent assay (elisa). n i l a i n o r m a l n y a a d a l a h : 1 0 , 1 ± 9 , 4 n g / ml.(11,17,19) 158 pengobatan banyak obat yang bisa dipakai baik untuk pengobatan osteoporosisnya maupun untuk mengurangi dampak negatif akibat osteoporosis seperti biphosphonate, kalsium dan vitamin d, estrogen, selective estrogen receptor modulator, kalsitonin, strontium ranelate, raloxifene, alendronate dan hormon paratiroid.(1,28-30) terapi sulih hormon masih merupakan pilihan antara pemakaian estrogen kimiawi atau estrogen alami (fitoestrogen) tetapi beberapa peneliti menganjurkan pemakaian terapi sulih hormon untuk penderita osteoporosis.(1,2, 30-33) perubahan gaya hidup seperti menu diet banyak mengandung kalsium dan vitamin d, sinar matahari dan banyak latihan fisik yang memakai beban ringan.(24,28,34,35) kesimpulan osteoporosis adalah kelainan tulang akibat berkurangnya kekuatan tulang yang menjadi p r e d i s p o s i s i m e n i n g k a t n y a r i s i k o t e t a p i osteoporosis memberikan manifestasi klinisnya yang berat yaitu terjadinya fraktur. pemeriksaan bone mass density (bmd) dianggap sebagai pemeriksaan baku emas (gold standard) oleh who untuk diagnosis osteoporosis tetapi k u r a n g b e r m a k n a d a l a m m e m a n t a u h a s i l p e n g o b a t a n . p e t a n d a t u l a n g m e r u p a k a n pemeriksaan pelengkap dan pengukuran bmd pada penatalaksanaan osteoporosis. manfaat potensial dari pertanda tulang adalah untuk m e n e n t u k a n p e m b e r i a n p e n g o b a t a n d a n memantau efektifitas pengobatan. daftar pustaka 1. mauck k, clarke bl. diagnosis, screening, prevention and treatment of osteoporosis. mayo clin proc 2006; 81: 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a, wells g, wilan. metaanalysis of therapies for postmenopausal osteoporosis. endocr rev 2002; 23: 508–16. oktavianus 35 *master in biomedical sciences program, faculty of medicine, university of indonesia **department of histology, faculty of medicine, trisakti university ***department of histology, faculty of medicine, university of indonesia ****department of biochemistry and molecular biology, faculty of medicine, university of indonesia *****doctoral program in biomedical sciences, faculty of medicine, university of indonesia correspondence dr. kartini, m.biomed. department of histology, faculty of medicine, trisakti university jl. kyai tapa no 260 grogol jakarta 11440 phone:+6221-5672731 email: kartiniedwin@yahoo.co.id univ med 2015;34:35-42 doi: 10.18051/univmed.2016.v35.35-42 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background prematurity refers to live births before 37 weeks of gestation, wherein the baby is born before the body and its organ systems achieve perfect maturity, and this disorder is still a global problem. the high incidence of prematurity is a problem in developing and also in developed countries. certain conditions accompanying pregnancies like preeclampsia, infection, and placental insufficiency, may trigger uterine hypoxia, causing premature birth. the placental condition is related to the intra-uterine fetal condition. in prolonged placental hypoxia, there occurs a compensatory mechanism, i.e. an increase in placental angiogenesis. this study aimed to evaluate the effect of hypoxia on fetal blood vessel count as compensatory mechanism for tissue hypoxia. methods an observational-analytical cross-sectional design using paraffin blocks of conserved premature placentas, comprising 31 samples of hypoxic premature placentas and 28 samples of non-hypoxic premature placentas, selected using non-random consecutive sampling. the samples were made into slides and stained with hematoxylin-eosin for assessment of histological structure, including fetal blood vessel count and integrity, villus conditions, syncytiotrophoblastic nuclear changes, and syncytiotrophoblastic nuclear aggregation. mann-whitney test was used to compare the difference of blood vessel count between groups. results assessment of histological structure showed a significant increase in fetal blood vessel count in the hypoxic group [8.00 (5-15)] as compared with the non-hypoxic group [7.50 (3-15)]. conclusion the hypoxia in premature placentas caused an increase in the number of fetal blood vessels as a form of compensation for disturbed oxygen homeostasis. keywords: premature, placenta, hypoxia, hif-1α fetal blood vessel count increases in compensation of hypoxia in premature placentas k. kartini,*,** ahmad a. jusuf,*** sri widia a. jusman,**** m. ekawati,***** and ani r. prijanti,**** doi: http://dx.doi.org/10.18051/univmed.2015.v34.35-42 36 kartini, jusuf, jusman, et al blood vessel count increases in hypoxia jumlah pembuluh darah fetus meningkat sebagai kompensasi pada plasenta bayi prematur yang hipoksia latar belakang prematuritas merupakan kelahiran bayi dalam kondisi hidup pada usia kehamilan kurang dari 37 minggu dengan berat badan lahir kurang dari 2500 gram, sehingga bayi lahir sebelum tubuh dan sistem organnya mencapai maturitas yang sempurna, dan kelainan ini masih menjadi masalah global. kejadian prematuritas tidak hanya terjadi di negara berkembang tetapi juga di negara maju. beberapa kondisi ibu hamil seperti preeklampsia, infeksi, insufisiensi plasenta, dll, dapat memicu keadaan hipoksia dalam rahim sehingga menyebabkan kelahiran prematur. keadaan plasenta menggambarkan kesejahteraan janin intra uteri. pada plasenta yang mengalami hipoksia lanjut terjadi mekanisme kompensasi yang salah satunya adalah peningkatan angiogenesis plasenta. penelitian ini bertujuan menilai pengaruh hipoksia terhadap jumlah pembuluh darah fetus sebagai kompensasi terhadap keadaan hipoksia jaringan. metode sebuah studi observasional analitik potong silang menggunakan bahan biologis tersimpan (bbt) plasenta prematur dalam bentuk blok parafin. sebanyak 31 bbt plasenta prematur yang hipoksia (h) sebagai kasus dan 28 bbt plasenta prematur non-hipoksia (n) sebagai kontrol, dipilih secara non random-consecutive sampling, kemudian dilakukan prosesing pewarnaan hematoksilin-eosin untuk dinilai struktur histologis plasenta prematur yang meliputi penilaian jumlah pembuluh darah fetus, integritas pembuluh darahnya, keadaan vilus, perubahan pada inti sinsisiotrofoblas, dan adanya agregasi inti sinsisiotrofoblas. uji mann-whitney digunakan untuk membandingkan jumlah pembuluh darah antara kedua kelompok. hasil penilaian struktur histologis menunjukkan adanya perbedaan bermakna (p<0,05) pada jumlah pembuluh darah fetus antara kelompok hipoksia [8,00 (5-15)] dibandingkan kelompok non-hipoksia [7,50 (3-15)]. kesimpulan pengaruh hipoksia terhadap plasenta prematur menyebabkan peningkatan jumlah pembuluh darah fetus sebagai bentuk kompensasi dalam upaya memelihara homeostasis oksigen. kata kunci: prematur, plasenta, hipoksia, hif-1α abstrak introduction the millennium development goals (mdgs) is a declaration of the who signed by 191 member countries in 2000, containing 8 goals to be achieved in the year 2015. the fourth and fifth goals are to reduce the mortality rate of the underfives and increase the quality of health of their mothers during delivery. (1) it is expected that in the year 2015 a reduction of up to two-thirds of the mortality rate of underfives will be achieved. one of the current global problems that has not been well-managed is prematurity. prematurity refers to live births before 37 weeks of gestation, with birth weights of less than 2500 grams, such that the baby is born before its body and organ systems have achieved perfect maturity. the complications of premature live births are the cause of nearly half of neonatal 37 deaths through the world, and currently ranks second after pneumonia as the second cause of mortality among children under the age of five years.(2,3) the estimated premature birth rate in 184 countries in 2010 ranged from 12.3 to 18.1 million live births.(2) a report from the world health organization mentions that each year around 15 million babies in the world (more than 1 in 10 births) are born premature. (3) more than 1 million of these babies die not long after birth. this makes prematurity an urgent priority in the effort to reduce the pediatric mortality rate in 2015 and succeeding years. the processes of embryogenesis, fetal development and perinatal infant survival are much dependent on maternal health and the development of a normal placenta.(4) several disorders of pregnancy, such as pre-eclampsia, infections, and placental insufficiency may induce hypoxic conditions in the uterus such that the baby is born premature. in a placenta with prolonged hypoxia, compensatory mechanisms take place, one of which is increased placental angiogenesis.(5) angiogenesis is regulated by the transcription factor hypoxia inducible factor (hif) (5,6) in an attempt to maintain oxygen homeostasis. one of the target genes of hif-1α is vascular endothelial growth factor (vegf), which induces the formation of new blood vessels (angiogenesis) in compensation for reduced oxygen levels. . (6) t h e s t u d y b y h e c h t e t a l . (7) found histological abnormalities of the placenta in premature babies in the form of inflammation or infarction. the team of mongia revealed histomorphological changes in the placenta of mothers with anemia, i.e. reduced placental weight, decreased number of cotyledons, i n c r e a s e d s y n c y t i a l k n o t s , i n c r e a s e d cytotrophoblast proliferation, thickening of the b a s e m e n t m e m b r a n e , a n d f o r m a t i o n o f vasculosyncytial membranes.(8) the study of mongia and co-workers found that in a group of anemic mothers a total of 23% experienced premature labor.(8) there have to date been no histological studies on hypoxic placentas in premature infants. the objective of the present study was to evaluate the effect of hypoxia on the histological structure of the placenta in premature infants. methods study design this study was of observational-analytical cross-sectional design. the study was conducted in the histological laboratory, department of histology, faculty of medicine, university of indonesia, from july to december 2014. study samples the study samples consisted of paraffin blocks of conserved premature placentas and included 31 hypoxic and 28 non-hypoxic specimens (hypoxia was diagnosed by blood gas analysis and/or pulse oxymetry of the premature infants, if po 2 <30 mmhg and/or o 2 saturation <60%). the sampling technique used was consecutive sampling. placental examination the paraffin blocks containing the placental specimens were serially cut at 5 µm thicknesses. the coded samples were subjected to routine histological processing (deparaffinizationrehydration-hematoxylin-eosin-dehydrationxylol) to obtain histological preparations of quality. the stained samples were micrographed (optilab professional edition, department of histology, faculty of medicine, university of indonesia) under 4x, 10x, and 40x objective l e n s e s . t h e e v a l u a t e d h i s t o m o r p h o l o g y comprised several parameters, i.e. fetal blood vessel count, fetal blood vessel integrity (good or poor), condition of villi (intact or disrupted), changes in syncytiotrophoblastic nuclei, and aggregation of syncytiotrophoblastic nuclei in adaptation to reduced oxygen levels. evaluation of the fetal blood vessels was by means of the image raster program (measurement of villus diameter and blood vessel count). fetal blood univ med vol. 34 no.1 38 kartini, jusuf, jusman, et al blood vessel count increases in hypoxia vessel count was determined from three free villi measuring around 150-300 µm and the blood vessels counted were those in cross section with v i s i b l e e n d o t h e l i a l c e l l s . h i s t o l o g i c a l examination was performed by the double blind method. statistical analysis comparison of fetal blood vessel counts in the two experimental groups (hypoxic and nonhypoxic) was done with the mann-whitney test, while the other histomorphological features were analyzed by chi-square and fisher’s exact tests. the level of significance was set at p<0.05. ethical clearance the study protocol was approved by the committee of health research ethics, faculty of medicine, university of indonesia/cipto mangunkusumo hospital, under no. 506/h2.f1/ etik/2014. results the mann-whitney test results showed significant differences in fetal blood vessel count in the two groups (p<0.05) (table 1). the chi-square and fisher’s exact tests showed no correlation between hypoxic status and other described histomorphologic features (p>0.05) (table 2 and figure 1). discussion the placenta is a vital organ for normal fetal growth and development.(9-11) the placental condition reflects the welfare of the fetus while in the uterus. (12) the purpose of the present study was to evaluate the effect of hypoxia on the histological structure of the placenta in premature infants. the fetal blood vessel count was determined to find any effects of hypoxia on angiogenesis in t h e p r e m a t u r e h y p o x i c p l a c e n t a a s a compensatory response to reduced oxygen levels. from our study results we conclude that there is a significant difference in fetal blood vessel count between the two groups. one study concluded that the vascular morphology of the fetal placenta in humans adapts in the same way to various forms of hypoxic stress. (9) increased numbers of capillaries in the terminal villi are found in hypoxic placentas of mothers residing in highlands as well as in those with iron deficiency anemia. dilatation of capillary sinusoids accompanied by thinning of the villus membrane is also a form of adaptation to hypoxia. the study of soni on term placentas of mothers with anemia also yielded similar findings, in that there was an increase in the total number of fetal capillaries per villus accompanied by capillary dilatation, in proportion with the severity of anemia.(9) table 1. comparison of blood vessel count in hypoxic and non-hypoxic groups table 2. correlation of hypoxic status with histomorphologic description 39 figure 1: histologic structure of premature placenta, he 400x;[a,b] integrity of fetal blood vessels; [c,d] condition of villi; [e,f] nuclear changes; [g,h] syncytiotrophoblastic aggregations. left panels: hypoxic placenta; right panels:non-hypoxic placenta univ med vol. 34 no.1 40 kartini, jusuf, jusman, et al blood vessel count increases in hypoxia infection plays an important role in premature birth.(2) intra-amnionic infection has been found as the main predisposing factor in around 50% of cases of prematurity and only around 8-25% of pregnant mothers show symptoms and signs of infection.(13) from several studies it was evident that the most frequent cause of premature birth is chorioamnionitis. (7) the lesions of an acute inflammatory reaction show edema of the villi that may affect trophoblastic permeability/integrity, intravascular pressure, as well as capillary integrity. (13) in our study we found poor vascular integrity and disrupted villi in both hypoxic and non-hypoxic groups. from the results of statistical analysis we concluded that there was no correlation between hypoxic status and vascular integrity or villus conditions. in advanced pregnancy there are variations in the thickness of the syncytiotrophoblast, which in several places may even appear thin or be i n v i s i b l e b e c a u s e o f t h e f o r m a t i o n o f a vasculosyncytial membrane. the thickness of the villus membrane is correlated with the efficiency of placental functioning in supporting fetal growth. a thin villus membrane is apparently one of the features associated with decreased placental functioning.(14) the disrupted villi found in both groups may have been caused by the thinness and fragility of the premature placenta, probably as a result of inflammation. nutrient intake and exchange of gases and metabolic waste products occur through the syncytiotrophoblast lining the intervillous space of the placenta and being in direct contact with t h e m a t e r n a l c i r c u l a t i o n . ( 11 , 1 5 , 1 6 ) t h e syncytiotrophoblast is a layer of multinuclear trophoblastic cells that plays an important role in supporting placental functioning, because it is involved in active maternofetal transfer, catabolism and resynthesis of proteins and lipids, synthesis of various hormones, transfer of gases and water by diffusion, facilitation of glucose transfer, and active transfer of amino acids and electrolytes. (16,17) the syncytiotrophoblast has a relatively high natural rate of cellular turnover, in which the syncytiotrophoblastic cells continously undergo apoptosis and are replaced by cytotrophoblasts.(15) the apoptotic nuclei accumulate at the villus surface, forming a syncytial knot which in turn is released into the maternal circulation.(15) i n t h e p r e s e n t s t u d y, t h e t e r m syncytiotrophoblastic aggregation refers to both syncytial knots and syncytial sprouts, which are aggregations of syncytiotrophoblastic nuclei protruding on the villus surface, since both structures are difficult to distinguish from one another under the light microscope. according to baergen, the term syncytial knot is more frequently used to represent the other features. it is used to evaluate placental age and uteroplacental perfusion.(17) the number of syncytial knots is significantly higher in term placentas of women residing in highlands in comparison with those residing in lowlands. the syncytial knot is an aggregation of nonproliferating syncytiotrophoblasts that forms an anuclear vasculosyncytial membrane. (10) a study suggested that the increase in syncytial knots is an attempt to reduce the diffusion distance for oxygen from maternal blood to fetal plasma. (10) several studies also proved that the increase in syncytial knots is a compensatory mechanism to hypoxic stress induced by altitude or disease. (10) in the present study both the hypoxic and nonhypoxic groups showed syncytiotrophoblastic aggregations. from statistical analysis we concluded that there was no correlation between hypoxic status and syncytiotrophoblastic aggregations (figure 1). however, our study was u n a b l e t o d e t e r m i n e w i t h c e r t a i n t y a n y differences in the number of syncytial knots. wi t h a d v a n c e d p r e g n a n c y t h e syncytiotrophoblastic nuclei may show signs of degenerative abnormalities as early signs of infarction.(17) in the present study, we found only 2 hypoxic (6%) and 3 non-hypoxic samples ( 11 % ) s h o w i n g c h a n g e s o f v i l l u s syncytiotrophoblastic nuclei. the nuclei appear denser and smaller (condensation) and in several villi the nuclei are fragmented. these nuclear changes may be the result of apoptosis. statistical analysis revealed that there was no c o r r e l a t i o n b e t w e e n h y p o x i c s t a t u s a n d 41 syncytiotrophoblastic nuclear changes. from various studies it is known that oxidative stress induces placental lesions resulting in apoptosis and necrosis of the syncytiotrophoblast.(16,18) h o w e v e r, d e t e r m i n a t i o n o f a p o p t o s i s i n syncytiotrophoblastic cells requires special staining. overall, the histological structure of the premature placentas in the two groups was apparently similar. there was only a difference in fetal blood vessel count, which was higher in the hypoxic group compared with the nonhypoxic group. this probably indicates the occurrence of compensatory changes in the form of slightly increased fetal blood vessel count in the hypoxic group. the results of the present study differred from those of other studies with respect to the description of the hypoxic premature placenta. this may be due to the l i m i t a t i o n s o f t h e s a m p l e d a t a , s u c h a s uncertainty regarding the actual occurrence of hypoxia in the placentas, since the criteria of hypoxia depended solely on oxymetry and gas analysis of fetal blood; whether the hypoxia was acute or chronic; conditions of the samples, e.g. the presence of other factors affecting the determination of infant hypoxia levels; the probability of cellular compensatory mechanisms to the hypoxia, such that the hypoxia did not yet produce significant changes in placental structure. these compensatory mechanisms may have occurred at the molecular level, thereby affecting placental functioning only. b u r t o n a n d j a u n i a u x r e v e a l e d t h e occurrence of stress in the endoplasmic reticulum of placentas from cases of intrauterine growth restriction (iugr) as a result of abnormal p l a c e n t a l p e r f u s i o n ( m o s t l y d u e t o p r e e c l a m p s i a ) . ( 1 9 ) st r e s s i n t h e e n d o p l a s m i c reticulum will activate proteins to improve homeostasis in the organelles through a number of integrated mechanisms. one of the first pathways to be activated is the phosphorylation of eukaryotic initiation factor-α (eif2α) that subsequently blocks the initiation of protein translation in ribosomes. the inhibition of protein translation leads to reduced levels of several kinases and proteins involved in the regulation of the cell cycle. other evidence states that stress in the endoplasmic reticulum is followed by decreased cyclin d1 levels, which results in reduced levels of protein kinase b. protein kinase b (akt) / mammalian target of rapamycin (mtor) is a central regulator of cell proliferation that also regulates amino acid transporters in the human placenta. therefore the decrease in cyclin d1 is presumably associated with reduced cell proliferation rate.(19) burton and jauniaux found multiple blocks in the protein translation process that are consistent with akt-mtor signaling in pathological placentas. this blockade causes a reduction in cell proliferation that results in the phenotype of the small placenta.(19) this is in agreement with the suggestions of baergen (17) and nagi,(20) that the small placenta is associated w i t h p r e m a t u r e b i r t h . to d e t e r m i n e t h e occurrence of stress in the endoplasmic reticulum of premature placentas requires further study. the present study has many limitations because the only structure that was evaluated was the placental parenchyma. comprehensive evaluation should be performed of the basal region, chorionic plate, placental parenchyma, and umbilical region. determination of the blood vessel count will be more accurate if special capillary endothelial markers are used, e.g. cd 34 . we hope that the results of the present study may be used as a starting point for further studies to find preventive factors of premature births. conclusions hypoxia induces histological structural changes in the form of significantly increased numbers of fetal blood vessels. acknowledgement we thank the board of research, faculty of medicine, trisakti university, and dr. dr. ani retno p., ms, and also all who contributed to the completion of this study. univ med vol. 34 no.1 42 kartini, jusuf, jusman, et al blood vessel count increases in hypoxia references 1. world health organizatin. mdg 4: reduce child mortality. geneva: world health organization; 2013. 2. blencowe h, cousens s, chou d, et al. born too soon: the global epidemiology of 15 million preterm births. reprod health 2013;10(suppl 1):52:1-14. 3. world health organization. born too soon: the global action report on preterm birth. geneva: world health organization; 2012. 4. hutter d, kingdom j, jaeggi e. causes and mechanisms of intrauterine hypoxia and its impact on the fetal cardiovascular system: a review. int j pediatr 2010. doi: 10.1155/2010/ 401323. 5. zamudio s, wu y, letta f, et al. human placental hypoxia-inducible factor-1α expression correlates with clinical outcomes in chronic hypoxia in vivo. am j pathol 2007;170:2171-9. 6. oladipupo s, hu s, kovalski j, et al. vegf is essential for hypoxia-inducible factor-mediated neovascularization but dispensable for endothelial sprouting. pnas 2011;108:132649. 7. hecht j, allred e, kliman h, et al. histological characteristics of singleton placentas delivered before the 28th week of gestation. pathology 2008; 40:372-6. 8. mongia sm, jain sk, yadav m. placenta: the wonder organ. j indian acad forensic med 2011; 33:140-2. 9. soni r, nair s. study of histological changes in placenta of anaemic mothers. iosr-jdms 2013; 9:42-6. 10. saga z, minhas la, rana r. effects of altitude on morphology of human placenta with special reference to terminal vili and syncytial knots. int j pathol 2008;6:26-9. 11. biswas s. placental changes in idiopathic intrauterine growth restriction. oa anatomy 2013;1:1-7. 12. majumdar s, dasgupta h, bhattacharya a. a study of placenta in normal and hypertensive pregnancies. j anat soc india 2005;54:34-8. 13. salafia c, popek e. inflammatory and vascular placental pathology. glob libr women’s med 2008. doi: 10.3843/glown.10152. 14. kiran n, zubair a, khalid h, et al. morphometrical analysis of intervillous space and villus membrane thickness in maternal anaemia. j ayub med coll abbottabad 2014;26: 207-11. 15. carter am. maintaining the integrity of trophoblast during growth of the placenta. focus on “insulin-like growth factor i and ii regulate the life cycle of trophoblast in the developing human placenta”. am j physiol cell physiol 2008;294:1303-4. 16. bainbridge sa, belkacemi l, dickinson m, et al. carbon monoxide inhibits hypoxia/ reoxygenation-induced apoptosis and secondary necrosis in syncytiotrophoblast. am j pathol 2006;169:774-83. 17. baergen r. manual of pathology of the human placenta. 2nd ed. new york: springer; 2011. 18. belkacemi l, bainbridge sa, dickinson m, et al. glyceryl trinitrate inhibits hypoxia/ reoxygenation-induced apoptosis in the syncytiotrophoblast of the human placenta. am j pathol 2007;170:909-20. 19. burton g, jauniaux e. the maternal circulation and placental shape: villus remodeling induced through haemodynamics and oxidative and endoplasmic reticulum stress. in: burton g, barker d, maffett a, et al, editors. the placenta and human developmental programming. cambridge: cambridge university press;2011. p.161-74. 20. nagi a. a monograph placenta examination and pathology. biomedica 2011;27:81-99. serum lactate as predictor and diagnostic biomarker 213 *department of internal medicine, faculty of medicine, university of indonesia-rsupn cipto mangunkusumo, jakarta indonesia **division of tropical and infectious disease, department of internal medicine, faculty of medicine, university of indonesiarsupn cipto mangunkusumo, jakarta, indonesia ***clinical epidemiology and evidence-based medicine (ceebm) unit, faculty of medicine, university of indonesiarsupn cipto mangunkusumo, jakarta, indonesia correspondence: dr. rika bur, sp.pd-kpti department of internal medicine, faculty of medicine, university of indonesiarsupn cipto mangunkusumo, jakarta, indonesia jl. salemba raya no.6, jakarta phone: +62812 9927 394 email: krekot09@gmail.com univ med 2016;35:213-21 abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 serum lactate as predictor and diagnostic biomarker of plasma leakage in adult dengue patients rika bur*, suhendro suwarto**, widayat djoko santoso**, and kuntjoro harimurti*,*** background dengue fever (df) and dengue hemorrhagic fever (dhf) are differentiated by the occurrence in dhf of plasma leakage into the interstitial space as shown by pleural and peritoneal effusion, hemoconcentration, and intravascular hypovolemia. perfusion dysfunction causes anaerobic metabolism, which leads to increased serum lactate. this study was to determine serum lactate as prognostic predictor and diagnostic biomarker of plasma leakage in adult dengue patients. methods a cross-sectional retrospective cohort study was conducted on 57 adult dengue patients hospitalized in the internal medicine ward of cipto mangunkusumo hospital and persahabatan hospital in jakarta. serum lactate was examined to determine its mean difference between df and dhf. the data was analyzed by independent t-test and the cut-off points were identified for presence as well as absence of plasma leakage, then the receiver operating characteristics (roc) curve was used to determine sensitivity and specificity. results mean serum lactate was significantly higher in dhf than in df. from the roc curve, the cut-off point for serum lactate as prognostic predictor on day 3 of fever was ≥2.65 mmol/l with auc of 0.626 (95% ci 0.4800.772; p=0.108). the cut-off point for diagnostic biomarker of plasma leakage on day 5 of fever was ≥2.55 mmol/l with sensitivity 66.6%, specificity 54.2%, and auc 0.668 (95% ci 0.550-0.826; p=0.016). conclusion there was a significant difference in serum lactate between df and dhf. in the critical phase, serum lactate of ≥2.55 mmol/l could be used as plasma leakage diagnostic marker of low accuracy. keywords: dengue virus infection, plasma leakage, serum lactate, adult doi: 10.18051/univmed.2016.v35.213-221 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license doi: http://dx.doi.org/10.18051/univmed.2016.v35.213-221 214 bur, suwarto, santoso, et al serum lactate and dengue introduction dengue infection remains a global health problem. over half of the world population is thought to live in areas at risk of transmission, and recent estimates suggest that around 400 million infections occur annually, of which 100 million are clinically apparent.(1) in 2010, the indonesian ministry of health reported 150,000 cases of dengue hemorrhagic fever (dhf) with mortality of 1317 cases.(2) in 2013, more than 110,000 dhf cases with mortality of 871 cases were reported.(3) plasma leakage is the main process in dhf of which clinicians should be aware.(4) plasma leakage usually occurs when fever is decreased in the critical phase and reaches its peak on the fifth day after the onset of fever.(5-7) in the critical phase, plasma leakage can lead to worse conditions that can cause shock and death. since plasma leakage is the main characteristic of dhf or dengue shock syndrome (dss), it will be a great indicator for determining dhf severity.(8) according to the who a case of dhf is defined if there is plasma leakage which can be detected by hematocrit elevation of >20% from baseline and or hematocrit reduction from baseline of at least 20% after fluid resuscitation, accompanied by signs of fluid accumulation in the interstitial space, such as pleural effusion and ascites.(9) the latter can be detected through chest radiography and abdominal usg, which however requires expertise and is expensive. alternatively, plasma leakage can also be seen from laboratory tests for hypoalbuminemia.(10) regrettably, in daily practice hematocrit baseline values are not readily available and calculation of the degree hematocrit elevation is difficult. furthermore, baseline hematocrit level may also be affected by conditions such as anemia, which can lead to a misleading diagnosis and inadequate therapy.(11,12) serum lactate may reportedly be used as a marker of tissue hypoxia caused by systemic hypoperfusion. (13) serial serum lactate measurement can predict shock and multi-organ failure (mof), and high lactate levels can be used as predictors of survival or mortality in patients with trauma or sepsis.(14) however, studies regarding the use of serum lactate level as dengue severity predictor and diagnostic biomarker of plasma leakage are still scarce especially in adult patients, therefore further studies are needed. studies about the role of serum lactate in diagnosing dengue fever (df) and predicting plasma leakage have been conducted in children.(15,16) incidence rates of dengue shock syndrome (dss) are higher in children than in adults. the key factor that distinguishes this condition is associated with vascular fragility.(17) the role of serum lactate as diagnostic biomarker in adult dengue patients was reported previously by thanachartwet et al.(18), who found that a cut-off value at plasma venous lactate levels of ≥2.5 mmol/l, with sensitivity and specificity of 65.0 % and 96.2 %, respectively, could identify severe dengue in adult patients. as prognostic factor, they suggested the combination of procalcitonin level ≥0.7 ng/ml and serum lactate ≥2.5 mmol/l for predicting dss.(19) the present study was intended to re-evaluate the role of serum lactate as predictor of dengue severity and diagnostic biomarker of plasma leakage in adult indonesian dengue patients. methods research design this retrospective cohort study used secondary data (medical records) of adult dengue patients who were enrolled in ongoing research related to dengue fever in cipto mangunkusumo hospital and persahabatan hospital in jakarta. the study was conducted between april 2015 and march 2016. study subjects data collection was performed by consecutive non-random sampling. the inclusion criteria were patients with a diagnosis of dengue virus infection through positive ns1 antigen (a non structural protein of the dengue virus) tests on the first and second day of fever, above 14 215 univ med vol. 35 no.3 years of age, and hospitalized in the internal medicine ward of cipto mangunkusumo hospital and persahabatan hospital between 2014 and 2015. the exclusion criteria were pregnant patients and those with incomplete medical records. dengue hemorrhagic fever patients were defined by hematocrit elevation of >20% on the fifth day as compared to baseline and or the presence of pleural effusion or ascites by abdominal usg. we used the sample size calculation method for the comparison of two independent means and the diagnostic test formula.(20,21) for sample size calculation by the two independent means formula, we used the study by santosa et al.,(8) who reported a mean and standard deviation of 2.81 ± 0.62 in the plasma leakage group (n=19) and of 2.36 ± 0.46 in the non-plasma leakage group (n=30). in our study, the calculated sample size per group was 17, which was estimated to be adequate to detect a difference in means of 0.44 between the two groups using a two-tailed test, an alpha of 0.05 and power of 90%. therefore the total minimum sample was 35 patients. using the formula for diagnostic test,(21) we found that the total minimum sample size was 57 patients, which was estimated to be adequate for a sensitivity of 90%, precision of 10% and dengue prevalence of 0.61.(22) from the results of the two formulas, we used the largest calculated minimum sample size, which was 57 patients. measurements data taken at the time of admission were the patients’ identity such as name, age, gender, date of birth and address. from the patients’ history we retrieved data about onset of fever and other clinical symptoms such as nausea, vomiting, heartburn, headache and myalgia. vital signs such as blood pressure, heart rate, respiratory rate, and temperature were obtained through physical examination. data taken during hospitalization were bleeding manifestations, such as positive tourniquet tests, petechiae, gingival bleeding, epistaxis, menometrorrhgia, hematemesis, and melena. from the physical examination we obtained the presence or absence of hepatomegaly. the presence or absence of as cites and pleural effusion was determined by usg. laboratory analysis this study measured serial complete blood counts and serum lactate, aspartate transaminase (ast) and alanine transaminase (alt) on the third and fifth days after the onset of fever. statistical analysis all data was inserted into the patient forms and processed according to the aim of this study. subject characteristics were descriptively displayed in table form with spss (statistical package for the social sciences) version 20.0. independent t-test was used to analyze the differences in mean serum lactate on the third and fifth days and in mean basic laboratory test results between df and dhf patients. an analysis to determine the diagnostic and prognostic role of serum lactate was done by using the receiver operating characteristics (roc) curve and calculating the area under the roc (auc) with 95% confidence interval. the serum lactate cutoff points for diagnosis and prognosis of plasma leakage were obtained by determining the highest sensitivity and specificity values from the roc. to determine the accuracy of the diagnostic test, we also calculated the sensitivity, specificity, positive and negative predictive value (ppv and npv), as well likelihood ratios. prognostic ability was calculated by absolute risk (ar) and relative risk (rr) at 95 % ci for plasma leakage in dengue patients with serum lactate above the cut-off point. ethical clearance this study used secondary data from medical records therefore informed consent was not necessary. this study has been approved by the health research ethics committee, faculty of medicine, university of indonesia, and the cipto mangunkusumo national central general hospital (rsupn cipto mangunkusumo) under no. 412/un2.f1/etik/2015. all data were kept confidential by the researchers. 216 bur, suwarto, santoso, et al serum lactate and dengue results a total of 57 adult dengue patients were enrolled in the study. the median age of the patients was 23 years (interquartile range [iqr]: 11) and 35 (61.4%) were male. the most common complaint besides fever was nausea (78.9%), while the most common bleeding manifestation was a positive tourniquet test (59.7%). the characteristics of the subjects are summarized in table 1. during monitoring, we found that laboratory findings such as hemoglobin, hematocrit, leukocytes, and platelets on days 3 and 5 corresponded to the course of dengue. however, we found no significant differences in basic laboratory workup results between df and dhf patients, except the leukocyte count on day 5 and ast level on day 3 (table 2). on day 3, we found that mean serum lactate level was statistically higher in dhf patients than in df patients (2.88 table 1. distribution of characteristics and clinical features of subjects (n=57) * patients may have reported more than one symptom ** iqr : interquartile range table 2. distribution of mean of laboratory blood parameters between dengue fever and dengue hemorrhagic fever adata presented as mean±standard deviation 217 univ med vol. 35 no.3 ± 0.59 mmol/l vs. 2.55 ± 0.43 mmol/l; p=0.028). serum lactate in dhf patients remained higher than in df patients on day 5 (2.84 ± 0.55 mmol/ l vs. 2.40 ± 0.55 mmol/l; p=0.005) (table 2). we used roc analysis as a test for the association between serum lactate on the third day and the presence of plasma leakage on the fifth day to determine the role of serum lactate as a predictor of plasma leakage. the results of the analysis showed that serum lactate cannot be used as predictor of plasma leakage (auc 0.626; 95% ci 0.480-0.772; p=0.108) (figure 1). the cutoff point of serum lactate on the third day of ≥2.65 mmol/l had sensitivity of 42.5%, specificity 50.0%, ppv 62.5%, npv 54.2%, which was also associated with a 1.20-fold increased risk (95% ci 0.76-1.91) to develop plasma leakage in the table 3. diagnostic value of serum lactate levels for dengue severity critical phase, but this finding was not statistically significant (table 3). however, the roc on serum lactate and the presence of plasma leakage on the fifth day showed that serum lactate on the fifth day can be used as diagnostic tool for plasma leakage in dhf patients (auc 0.688; 95% ci 0.5500.826; p=0.016) (figure 2). the cut-off point of serum lactate on the fifth day of ≥2.55 mmol/ l had the highest sensitivity (66.6%; 95% ci 0.50-0.80) and specificity (54.2%; 95% ci 0.350.72). this cut-off value had ppv 67% (95% ci 0.50-0.80), npv 54% (95% ci 0.35-0.72), positive likelihood ratio lr (+) 1.45 and negative likelihood ratio lr (-) 0.62 (table 3). therefore it can be concluded that serum lactate had a low diagnostic accuracy. figure 1. area under the receiver operating characteristic curve (auc) for serum lactate level ≥2.65 mmol/l on day 3 as predictor of plasma leakage was 0.626 (95% ci: 0.480-0.772; p=0.108) 218 bur, suwarto, santoso, et al serum lactate and dengue discussion lactate is a marker for tissue hypoxia that may be the result of anaerobic metabolism mainly due to hypoperfusion.(13) serum lactate laboratory tests had been incorporated in surviving sepsis campaign (ssc) guideline 2012 as important workup for all suspected patients with sepsis.(23) the ssc used as cut-off value a serum lactate level of 4 mmol/l as marker for severe sepsis. hyperlactatemia is associated with increased mortality in septic patients, therefore it can be used as prognostic factor. normalization of lactate level also has been suggested as one of the therapeutic goals of sepsis management.(23) while the role of serum lactate in septic patients had been well established, the role of serum lactate as biomarker in dengue patients was understudied, especially in adult patients. in dhf patients the main characteristic finding is plasma leakage from increased vascular permeability, resulting in interstitial edema, pleural effusion, ascites, hemoconcentration, and intravascular hypovolemia. these conditions cause tissue hypoperfusion, which lead to impaired oxygen supply and activation of anaerobic metabolism.(18) based on dengue pathogenesis, we assessed the role of serum lactate in predicting and diagnosing plasma leakage in adult dengue patients. in this study, we included 57 adult dengue patients consisting of 22 df cases and 35 dhf cases. most of our patients were young male adults, who reported nausea as the most common chief complaint and positive tourniquet test as the most common bleeding manifestation. there are many studies assessing the factors associated with severe dengue or dss.(24,25) the who proposed a list of warning signs, such as abdominal pain, persistent vomiting, mucosal bleed, and lethargy, for predicting severe dengue and the need for strict observation. however, a study by thanachartwet et al found that the sensitivity and specificity of the 2009 who warning signs (except the presence of clinical fluid accumulation) were too low for identifying severe dengue at admission.(18) in our study, among the laboratory parameters we found no significant differences between df and dhf patients, except leukocyte count on day 5 and ast level on day 3. this finding may have been caused by the adequate fluid resuscitation received by the patients. serum lactate values on day 3 and day 5 were found to be significantly higher in dhf patients than in df patients. a higher lactate level figure 2. auc for serum lactate level ≥2.55 mmol/l as diagnostic biomarker of plasma leakage on day 5 was 0.668 [ci 95% 0.550-0.826; p=0.016] 219 univ med vol. 35 no.3 may reflect the degree of severity of plasma leakage in dhf patients compared to df. sirikutt et al.(16) also reported that serum lactate was found to be elevated in children with dhf and/or dss. hyperlactatemia, in addition to being caused by anaerobic metabolism due to hypoperfusion from plasma leakage, may also be caused by impaired lactate clearance from hepatic impairment.(16,26) therefore, serum lactate may be considered as a marker for the severity of adult dengue patients. we analyzed the sensitivity and specificity of various cut-off values using roc to determine the best cut-off point for serum lactate on the third day as a plasma leakage predictor. serum lactate on the third day of ≥2.65 mmol/l was shown to be nonsignificant in predicting plasma leakage on the fifth day, having a sensitivity of 42.5%, specificity 50.0%, ppv 62.5%, and npv 54.2%. it can be concluded that serum lactate is nonsignificant as a predictor of plasma leakage in the critical phase. a prospective observational study from thailand found that the combination of procalcitonin level of ≥0.7 ng/ml and serum lactate level of ≥2.5 mmol/l resulted in a sensitivity of 81.2%, specificity of 84.4%, ppv 56.5%, npv 94.7% to predict dss and/or organ failure.(19) the diagnostic accuracy can be improved if combined with the finding of clinical fluid accumulation, resulting in a sensitivity of 90% and specificity of 87.6%. these differing findings were presumably the result of differences in the degree of plasma leakage caused by differences in the timing of blood sample collection for serum lactate measurement. the abovementioned study in thailand used the lactate level at admission for diagnostic accuracy assessment,(19) but we uniformly take blood samples on day 3 which is generally thought as the beginning of plasma leakage. this is presumably done because the degree of plasma leakage is still minimal on day 3, since plasma leakage in the form of third space fluid accumulation on ultrasound is mostly detected on the fourth and fifth days.(27) another possible explanation is that because our study was of retrospective design, the patients may have received fluid therapy so that the intravascular volume had been replaced and met the needs for microcirculation. when perfusion is maintained the lactate level may decrease.(28) according to the 1997 who guideline, plasma leakage is determined by the presence of hemoconcentration as reflected by increased hematocrit level of ≥20% and/or thrombocytopenia of <100,000/mm3. a limitation to these criteria is that baseline hematocrit values are commonly not available, and may be misleading in the presence of anemia or hemorrhage and during the early phase of resuscitation.(11,12) based on roc, the cut-off point of serum lactate ≥2.55 mmol/l was determined as plasma leakage diagnostic, with sensitivity of 66.6%, specificity 54.2%, ppv 67% and npv 54%. our cut-off value was slightly different from that reported in the prospective observational study in thailand which found that the cut-off value of serum lactate level of ≥2.5 mmol/l was optimal to identify severe dengue, having a sensitivity of 65%, specificity of 96.2%, ppv of 76.5%, and npv of 93.5%.(18) the difference in cut-off values can be explained by the different timing of serum lactate sampling, since we used the serum lactate value on day 3 while the study from thailand used the lactate value on day 5. one limitation of our study was its retrospective cohort design which may be associated with a risk of bias and confounding factors. we suggest that a prospective study be conducted, with blinding of the treating physicians and investigators to the results, to minimize the risk of missing data and bias. for further research, we recommend serial daily serum lactate measurement starting before the third day of the onset of fever, and six-hourly serum lactate measurement during the critical phase until the recovery phase. the clinical implication of our study is that according to the supporting evidence, serum lactate level on the fifth day can be used as diagnostic biomarker of plasma leakage, apart from radiological workup. therefore early identification and treatment of severe dengue can made, thus resulting in decreased mortality rates. 220 bur, suwarto, santoso, et al serum lactate and dengue conclusions we found that serum lactate level on the third and fifth days of fever was higher in dhf patients compared to df patients. serum lactate level on the third day cannot be used for predicting plasma leakage in the critical phase. serum lactate on the fifth day can be used for diagnosis of plasma leakage in the critical phase with low accuracy. conflict of interest the authors declare no competing interest. acknowledgement the authors thank the director of cipto mangunkusomo national central general hospital and persahabatan general hospital jakarta for permission to collect patient data in their respective hospitals. references 1. bhatt s, gething pw, brady oj, et al. the global distribution and burden of dengue. nature 2013; 496:504-7. 2. kementerian kesehatan republik indonesia. demam berdarah dengue. jakarta: pusat data dan surveilans epidemiologi kementerian kesehatan republik indonesia;2010. 3. kementerian kesehatan republik indonesia. profil kesehatan indonesia 2013. jakarta: kementerian kesehatan republik indonesia; 2014. 4. srikiatkhachorn a. plasma leakage in dengue haemorrhagic fever. thromb haemost 2009;102: 1042-9. 5. simmons cp, farrar jj, chau nv, et al. dengue. n engl j med 2012;366:1423-32. 6. lee yr, liu mt, lei hy, et al. mcp-1, a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells. j gen virol 2006;87:3623-30. 7. yacoub s, mongkolsapaya j, screaton g. the pathogenesis of dengue. curr opin infect dis 2013;26:284-9. 8. santosa d, alwi eh, idjradinata ps. validitas kadar laktat darah dalam mendeteksi kebocoran plasma pada infeksi virus dengue anak. majalah kedokteran indonesia 2011;61:58-62. 9. world health organization. dengue: guidelines for diagnosis, treatment, prevention and control. geneva: world health organization;2009. 10. villar-centeno la, diaz-quijano fa, martinezvega ra. biochemical alterations as markers of dengue hemorrhagic fever. am j trop med hyg 2008;78:370-4. 11. potts ja, thomas sj, srikiatkhachorn a, et al. classification of dengue illness based on readily available laboratory data. am j trop med hyg 2010;83:781-8. 12. kuo mc, lu pl, chang jm, et al. impact of renal failure on the outcome of dengue viral infection. clin j am soc nephrol 2008;3:13506. 13. fuller bm, dellinger rp. lactate as a hemodynamic marker in the critically ill. curr opin crit care 2012;18:267-72. 14. krishna u, joshi sp, modh m. an evaluation of serial blood lactate measurement as an early predictor of shock and its outcome in patients of trauma or sepsis. indian j crit care med 2009; 13:66-73. 15. puspanjono mt, latief a, tumbelaka ar, et al. comparison of serial blood lactate level between dengue shock syndrome and dengue hemorrhagic fever (evaluation of prognostic value). paediatrica indonesiana 2007;47:151. 16. sirikutt p, kalayanarooj s. serum lactate and lactate dehydrogenase as parameters for the prediction of dengue severity. j med assoc thai 2014;97 suppl 6:s220-31. 17. halstead sb. the world health organization definition of dengue hemorrhagic fever is inadequate for clinical and epidemiological purposes. in: pascol g, hoffman sl, editors. dengue tropical medicine: science and practice. 5th vol. london: imperial college press; 2008.p. 427-31. 18. thanachartwet v, wattanathum a, oer-areemitr n, et al. diagnostic accuracy of peripheral venous lactate and the 2009 who warning signs for identifying severe dengue in thai adults: a prospective observational study. bmc infect dis 2016;16:46. doi: 10.1186/s12879-016-1386-5. 19. thanachartwet v, desakorn v, sahassananda d, et al. serum procalcitonin and peripheral venous lactate for predicting dengue shock and/or organ failure: a prospective observational study. plos negl trop dis 2016;10: e0004961. doi:10.1371/ journal.pntd.0004961. 20. madiyono b, moeslichan smz, sastroasmoro s, et al. perkiraan besar sampel. in: sastroasmoro s, ismael s, editors. dasar-dasar metodologi 221 penelitian klinis. 5th ed. jakarta: sagung seto; 2014.p.352-87. 21. hajian-tilaki k. sample size estimation in diagnostic test studies of biomedical informatics. j biomed informatics 2014;48:193-204. 22. nainggolan l. pengembangan sistem skor sebagai prediktor kebocoran plasma pada demam berdarah dengue: peran stnfr-1, vegf, svecadherin dalam patofisiologi kebocoran plasma [disertasi]. jakarta: universitas indonesia;2012. 23. dellinger rp, levy mm, rhodes a, et al. surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock: 2012. crit care med 2013;41:580637. 24. huy nt, van giang t, thuy dhd, et al. factors associated with dengue shock syndrome: a systematic review and meta-analysis. plos negl trop dis 2013;7:e2412. doi: 10.1371/journal. pntd. 25. thanachartwet v, oer-areemitr n, chamnanchanunt s, et al. identification of clinical factors associated with severe dengue among thai adults: a prospective study. bmc infect dis 2015;15:420. 26. sterling sa, puskarich ma, jones ae. the effect of liver disease on lactate normalization in severe sepsis and septic shock: a cohort study. clin exp emerg med 2015;2:197-202. 27. premaratna r, ragupathy a, miththinda jk, et al. timing, predictors, and progress of third space fluid accumulation during preliminary phase fluid resuscitation in adult patients with dengue. int j infect dis 2013;17:e505-9. 28. bakker j, nijsten mwn, jansen tc. clinical use of lactate monitoring in critically ill patients. ann intensive care 2013;3:12. forns index as a useful noninvasive predictor of 199 *division of gastroenterohepatology, department of internal medicine, faculty of medicine, university of sumatera utara, medan correspondence: dr. rizqi arini siregar division of gastroenterohepatology, department of internal medicine, faculty of medicine, university of sumatera utara jl. bunga lau no.17 medan sumatera utara 20136 fax: +6261 8363009 mobile: +62813 7043 3555 email: arini_siregar@yahoo.com univ med 2016;35:199-205 doi: 10.18051/univmed.2016.v35.199-205 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 forns index as a useful noninvasive predictor of esophageal varices in liver cirrhosis rizqi arini siregar*, leonardo b. dairi*, and gontar alamsyah siregar* background rupture and bleeding from esophageal varices are major complications of portal hypertension and associated with a high mortality rate. noninvasive serum markers of liver fibrosis could be used as predictors of esophageal varices in cirrhotic patients. the objective of this study was to assess the performance of forns index as a noninvasive predictor in diagnosing esophageal varices. methods a cross-sectional study was done in 51 cirrhotic patients who were admitted to adam malik hospital, medan. demographic and clinical data were recorded and laboratory tests were performed, so that forns index could be calculated. the difference between forns index and size of esophageal varices as determined by endoscopy was tested by independentt and mann-whitney analysis. the diagnostic performance was assessed using sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), accuracy, likelihood ratios and areas under the receiver operating characteristic curves (auroc). results of the 51 patients with esophageal varices included in this study, the size of esophageal varices comprised f2 (37.3%), f3 (33.3), and f1 (29.4%). most patients were of child-plug c type (52.6%). there was a significant difference between forns index and grade of esophageal varices. the auroc for forns index was 0.717 (95% ci: 0.561 0.872) and the cutoff >7.92 was highly predictive to diagnose large esophageal varices with a sensitivity of 63.9%, specificity of 73.3%, ppv of 85.2%, npv of 45.8% and accuracy of 71.7%. conclusion forns index was significantly increased in large esophageal varices. forns index is a good noninvasive predictor of esophageal varices in cirrhotic patients. keywords: forns index, non-invasive parameter,esophageal varices, cirrhotic patients doi: http://dx.doi.org/10.18051/univmed.2016.v35.199-205 200 siregar, dairi, siregar predictor of esophageal varices introduction cirrhosis, the end stage of chronic liver disease, is the most common cause of portal hypertension.(1) esophageal varices is a major complication that frequently appears in more than 90% of liver cirrhosis patients. among diagnosed cases of liver cirrhosis, about 60% have some degree of esophageal varices.(2) the mortality at each bleeding episode is between 10-20%.(3) one-year survival is only about 63%.(4) i n c i r r h o t i c p a t i e n t s , s c r e e n i n g f o r esophageal varices is highly recommended and extremely important because it is closely linked to the scheme of nonselective betablocker therapy or endoscopic prophylaxis to prevent variceal bleeding.(5) the current screening method is endoscopy, which is performed every 2 to 3 years in patients without esophageal varices, every 1 to 2 years in those with mild varices, and annually in those with decompensated cirrhosis. however, periodical endoscopic checkup is extremely expensive and frequently associated with complications such as bleeding and perforation. in addition, not every kind of health service has endoscopic facilities, along with the limitation of competition to do the endoscopic checkup. therefore there is a need in relation to portal hypertension for a noninvasive checkup procedure that can identify the presence of esophageal varices in patient with liver cirrhosis.(6) some noninvasive methods have been proposed to serve as markers for evaluating the degree of liver fibrosis and esophageal varices, including serum markers, such as aspartate aminotransferase (ast) to platelet ratio index (apri), fib-4, forns index and lok score,(7-9) transient elastography,(10) magnetic resonance elastography (11) and acoustic radiation force impulse imaging.(12,13) common tests previously validated as predictors of liver fibrosis, such as aspartate aminotransferase-to-platelet ratio (apri), a s p a r t a t e a m i n o t r a n s f e r a s e t o a l a n i n e aminotransferase ratio (aar), fib-4, fi, king, lok, forns, and fibro index scores, are primarily based on regular laboratory tests and readily available demographic data, and do not need any special experience in imaging techniques.(14,15) they are more convenient and economical in clinical practices. forns index is a serum biomarker that is used to predict liver fibrosis and is predicted to be able to replace liver biopsy or endoscopy in detecting esophageal varices. forns index is based on the variables of platelet count, age, gamma gt, and total cholesterol. some studies suggest that there is a relationship between liver fibrosis, portal hypertension, and esophageal varices, whereas other studies are trying to carry out research about forns index in predicting esophageal varices.(7) based on the above, the objective of this study was to determine the association of esophageal varices and forns index and the diagnostic accuracy of forns index in liver cirrhosis patients. methods research design a cross-sectional study was conducted on patients with liver cirrhosis at adam malik hospital, medan, from september to december 2014. research subjects males or females aged >18 years, having liver cirrhosis, and agreeing to sign informed consent, were included in this study. patients who previously had variceal bleeding, endoscopic therapy (ligation or sclerotherapy), surgical treatment for portal hypertension (transjugular intrahepatic portosystemic shunt, tips), or were on beta blocker treatment, were excluded from the study. the formula used to calculate the sample size for hypothesis testing was: 2 ao 2 aaβ)(1oo 2 α(1 )p(p )p(1pz)p(1pz n −      −+− = −− 201 univ med vol. 35 no.3 where: z(1-α/2) = raw deviate of alpha, for α = 0.05 � z(1-α/2) = 1.96 z(1-β) = raw deviate of beta, for β = 0.10 � z(1-β) = 1.282 p 0 – pa = significant difference in proportions = 0.15 p 0 = proportion of liver cirrhosis cases at a number of hospitals in indonesia � 3.5% = 0.035 pa = estimated proportion of liver cirrhosis cases under study = 0.185 minimal sample size = 33 persons per group the subjects’ personal history was assessed through anamnesis and physical checkup, radiological checkup, laboratory checkup consisting of determination of platelet count, gamma-glutamyl transferase (gamma gt) c o n c e n t r a t i o n , a n d t o t a l c h o l e s t e r o l concentration, and endoscopic checkup. esophageal varices were classified into three types: f1 (small straight esophageal v a r i c e s ) , f 2 ( s l i g h t l y e n l a rg e d t o r t u o u s esophageal varices occupying less than one-third of the esophageal lumen), and f3 (large coilshaped esophageal varices occupying more than one-third of the esophageal lumen).(16) serum biochemical markers venous blood samples were taken from all participants after an overnight fast (8–12 hours). the samples were tested in the laboratory to determine aspartate aminotransferase (ast) and alanine aminotransferase (alt) levels, gammaglutamyl transferase (gamma gt), total cholesterol and platelet (plt) count. to assess the gamma gt level we used a kinetic assay with (gamma-l-glutamyl)-p-nitroanilide and glycylglycine as substrates. to assess total cholesterol we used r1 reagents and the cobas mira® automatic analyzer. forns index the formula used to calculate the forns index was 7.811 3.131 x ln [platelet count (109/ l)] + 0.781 x ln [gamma gt(iu/l)] + 3.467 x ln [age (years)] – 0.014 x [cholesterol (mg/dl).(17) data analysis to display a descriptive overview of the patients’ basic data a tabulation system was used. the independent t-test was used for normally distributed data, and mann whitney test for nonnormally distributed data. statistical analysis was performed using spss with significance limit of p<0.05. to obtain the cut-off points for forns index, the area under the receiver operating characteristic curve (auroc) was used. in this study, we also performed diagnostic tests to obtain the sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv), likelihood ratio for positive tests (lr +), likelihood ratio for negative tests (lr -), and accuracy (acc). to evaluate the severity of liver cirrhosis we used the child pugh score: 202 siregar, dairi, siregar predictor of esophageal varices ethical clearance ethical clearance (permission to carry out research) was obtained from the health sector research committee of the faculty of medicine, university of north sumatra. results in this study, the total number of participants consisted of 51 patients with liver cirrhosis who had met the inclusion criteria. most patients were male (34 persons or 66.7%). the mean age of the patients involved in this study was 52.04 ± 12.33 years. respondents with hepatitis b comprised 30 persons (58.8%), hepatitis c 2 persons (3.9%), while those with a negative result for hepatitis b or c comprised 19 persons (37.3%). respondents with child pugh a consisted of 10 persons (13.2%), with child pugh b 26 persons (34.2%), and child pugh c 40 persons (52.6%). the medians of platelet count and gamma gt of the patients in this study were respectively 104 (31144)x109/l and 66 (6-530) u/l, while mean total cholesterol was 149.12 ± 67.55 mg/dl. regarding the size of the esophageal varices, which were graded based on endoscopic checkup results, there were esophageal varices of f1 size in 15 people (29.4%), f2 size in 19 people (37.3%), and f3 size in 17 people (33.3%) (table 1). there was a significant difference in mean forns index between large esophageal varices and small esophageal varices, with the forns index of large esophageal varices (8.47 ± 2.05) being significantly higher than that of the small esophageal varices (6.37 ± 2.68) (p=0.016) (table 2). from the receiver operating characteristics (roc) curve, the best forns index cut off value was determined to be 7.92. the forns index value of >7.92 in the diagnosis of large sized esophageal varices had the following characteristics: sensitivity 63.9%, specificity 73.3%, ppv 85.2%, npv 45.8%, lr (+) 2.4, lr (-) 0.49, and diagnostic accuracy 71.7% (figure 1). discussion among the 51 study subjects, the most common cause of liver cirrhosis (58.8%) was hepatitis b infection. this agrees with the study conducted by limquiaco et al,(18) where the most common cause of liver cirrhosis was chronic hepatitis b (40%) infection. however, different results were found in a us study showing hepatitis c virus (hcv) infection and alcohol abuse to be the two leading causes of liver cirrhosis.(19) this was also the case with the report of stefanescu et al.,(7) where the most common cause of liver cirrhosis (49.78%) was chronic hepatitis c infection. these differences may caused by different research locations, since table 1. distribution of demographic characteristics of respondents (n=51) 203 univ med vol. 35 no.3 the hepatitis c prevalence is higher in europe and the us. for the child pugh classification, limquiaco et al.(18) mention that child a accounted for 3%, child b for 73%, and child c for 23%. stefanescu et al.(7) found values of 75.9%, 18.4%, and 5.7% for child a, child b and child c, respectively. in our study we found that child a comprised 13.2%, child b 34.2%, and child c 52.6%. the high prevalence of child c in this study was due to its being conducted at a referral hospital, since most of the patients referred to this institution had advanced disease. our study showed that the proportion of esophageal varices for f1, f2, and f3 was 29.4%, 37.3%, and 33.3%, respectively. a previous study on 40 subjects showed that the proportion of esophageal varices for f1, f2, and f3 was 32.5%, 42.5% and 25.0%, respectively.(20) table 2. difference in biochemical characteristics between small and large esophageal varices (a)independent t-test (b)mann whitney test; *significant; *ast: aspartate aminotransferase; alt: alanine aminotransferase; pt: protrombin time ;inr: international normalized ratio figure 1. forns index roc in predicting the grade of varices 204 siregar, dairi, siregar predictor of esophageal varices mild to moderate thrombocytopenia occurs in 49-64% of patients with liver cirrhosis. the etiology of thrombocytopenia is multifactorial, including increased platelet sequestration in the spleen in congestive splenomegaly, which is caused by portal hypertention, decreased production of thrombopoietin, as well as platelet destruction as a result of immune processes or antibodies.(21) there was a significant difference in mean platelet count between the group of large esophageal varices and the group of small esophageal varices, with a significantly lower mean platelet count among large esophageal varices as compared with small esophageal varices. this is in line with a previous study reporting that low platelet count was an independent risk factor or predictor for the presence of esophageal varices and their size.(22) the study conducted by stefanescu et al.(7) evaluated four non-invasive methods that had previously been known as predictors of liver fibrosis grade, for identifying esophageal varices and their size, in comparison with endoscopic checkup as a standard procedure. one of the evaluated methods was forns index. these investigators found that the latter method can be a predictor of the presence of esophageal varices, particularly those of the larger sizes. in their study the investigators found that for detecting esophageal varices of any size, forns index (cutoff >7.297) had an auroc of 0.648, whereas in detecting large sized esophageal varices, forns index (cut-off >8.538) had an auroc of 0.645. however, this is the disadvantage of using serum markers of liver fibrosis, in which the variables used in calculating a non-specific score describing liver abnormalities, can be affected by other disease conditions of the patients. the present study had different cut-off values for diagnosing large esophageal varices as compared to the study results of stefanescu et al.(7) we found that the lower cut-off value of >7.92 in the present study may be affected by different mean variable values included in the forns index valuation, such as the younger age of the study sample, lower mean gamma gt, and higher mean platelet count than that reported in the study by stefanescu et al. (7) moreover, there is a difference in sample size which may be a limitation of our study, since our sample was much smaller than that of the study of stefanescu et al.,(7) which involved 231 persons. the predictive value of the forns index cufoff value of >7.92 in diagnosing large esophageal varices, having 63.9% sensitivity, 73.3% specificity, 85.2% ppv, 45.8% npv, and an accuracy of up to 71.7%, can be used as one of the noninvasive methods of choice for detection of esophageal varices. in addition, forns index is expected to be used as a basis in giving â-blocker treatment as a primary prevention against the limitation of a physician’s competence to perform endoscopic checkup.(1) conclusion this study found a correlation between the grade of esophageal varices and forns index in liver cirrhosis patients. forns index is a noninvasive parameter that can be used to predict the grade of esophageal varices in cirrhotic patients. conflict of interest the authors state that there are no conflicts of interest with regard to this manuscript. acknowledgement we thank all study subjects who agreed to participate in the present study and all colleagues who gave their advice to improve the manuscript. references 1. biecker e. portal hypertension and gastrointestinal bleeding: diagnosis, prevention and management. world j gastroenterol 2013; 19:5035-50. 2. ashkenazi e, kovalev y, zuckerman e. evaluation and treatment of esophageal varices in the cirrhotic patient. imaj 2013;15:109-15. 3. turon f, casu s, hernandez-gea v, et al. variceal and other portal hypertension related 205 bleeding. best pract res clin gastroenterol 2013;27:649-64. 4. stokkeland k, brandt l, ekbom a, et al. improved prognosis for patients hospitalized with esophageal varices in sweden 1969–2002. hepatology 2006;43:500-5. 5. de franchis r. evolving consensus in portal hypertension. report of the baveno iv consensus workshop on methodology of diagnosis and therapy in portal hypertension. j hepatol 2005; 43:167-76. 6. de franchis r. revising consensus in portal hypertension: report of the baveno v consensus workshop on methodology of diagnosis and therapy in portal hypertension. j hepatol 2010;53:762-8. 7. stefanescu h, grigorescu m, lupsor m, et al. a new and simple algorithm for the noninvasive assessment of esophageal varices in cirrhotic patients using serum fibrosis markers and transient elastography. j gastrointestin liver dis 2011;20:57-64. 8. friedrich-rust m, wunder k, kriener s, et al. liver fibrosis in viral hepatitis: noninvasive assessment with acoustic radiation force impulse imaging versus transient elastography. radiology 2009;252:595-604. 9. garcia-tsao g, sanyal aj, grace nd, carey w and the practice guidelines committee of the american association for the study of liver diseases, the practice parameters committee of the american college of gastroenterology. prevention and management of gastroesophageal varices and variceal hemorrhage in cirrhosis. hepatology 2007;46:922-38. 10. friedrich-rust m, ong mf, martens s, et al. performance of transient elastography for the staging of liver fibrosis: a meta-analysis. gastroenterology 2008;134:960-974. 11. talwalkar ja, yin m, venkatesh s, et al. feasibility of in vivo mr elastographic splenic stiffness measurements in the assessment of portal hypertension. am j roentgenol 2009;193: 122-7. 12. grgurevic i, cikara i, horvat j, et al. noninvasive assessment of liver fibrosis with acoustic radiation force impulse imaging: increased liver and splenic stiffness in patients with liver fibrosis and cirrhosis. ultraschall med 2011;32:160-6. 13. bota s, sporea i, sirli r, et al. spleen assessment by acoustic radiation force impulse elastography (afri) for prediction of liver cirrhosis and portal hypertension. med ultrason 2010;12:213-7. 14. vallet-pichard a, mallet v, nalpas b, et al. fib4: an inexpensive and accurate marker of fibrosis in hcv infection: comparison with liver biopsy and fibrotest. hepatology 2007;46:32-6. 15. cross tj, calvaruso v, maimone s, et al. prospective comparison of fibroscan, king’s score and liver biopsy for the assessment of cirrhosis in chronic hepatitis c infection. j viral hepat 2010;17:546-54. 16. farber e, fischer d, eliakim r, et al. esophageal varices: evaluation with esophagography with barium versus endoscopic gastroduodenoscopy in patients with compensated cirrhosis—blinded prospective study. radiology 2005;237:535-40. 17. forns x, ampurdanes s, llovet jm, et al. identification of chronic hepatitis c patients without hepatic fibrosis by a simple predictive model. hepatology 2002;36:986-92. 18. limquiaco j, daez r, gloria rr, et al. clinical predictors of bleeding from esophageal varices: a retrospective study. phil j of gastroenterology 2006;2:103-11. 19. shoreibah m, anand bs, singal ak. alcoholic hepatitis and concomitant hepatitis c virus infection. world j gastroenterol 2014;20:1192934. 20. mifune h, akaki s, ida k, et al. evaluation of esophageal varices by multidetector-row ct: correlation with endoscopic red color sign. acta med okayama 2007;61:247-54. 21. sebastiani g, tempesta d, fattovich g, et al. prediction of oesophageal varices in hepatic cirrhosis by simple serum noninvasive markers: results of a multicenter, large-scale study. j hepatol 2010;53:630-8. 22. suk tk. revision and update on clinical practice guideline for liver cirrhosis. the korean j hepatol 2012;18:1-21. univ med vol. 35 no.3 editorial 1 glycated albumin for glycemic control of diabetic patients on hemodialysis alvina department of pathology clinic, medical faculty, trisakti university regular assessment of glycemic control in diabetic patients is very important, because it reduces the incidence of complications and determines prognosis and quality of life of the patients.(1) uncontrolled and advanced diabetes may lead to complications, such as kidney disease, heart attacks, and stroke, which are on the increase worldwide. patients with diabetic nephropathy account for almost 50% of dialysis patients in the us, while cardiovascular disease is the main cause of death of patients with complicated diabetes.(2) assessment of glycemic control status in diabetic patients may be performed by determining the glycation of several proteins, which is increased in diabetic patients as compared with nondiabetic patients. among these glycated proteins, glycated hemoglobin (hba1c) is the gold standard index for glycemic control in the treatment of patients with diabetes.(3) hba1c is the condensation product of hemoglobin a and glucose, and its level is defined as the percentage of hemoglobin in the circulation that is bound to glucose.(1,2) measurement of hba1c concentration in diabetic patients with nephropathy may be inaccurate (yield lower values) due to shortened erythrocyte life span, which causes decreased chemical binding of glucose to erythrocytes, resulting in low hba1c concentrations.(2) glycated albumin (ga) may be an alternative marker for glycemic control in diabetic patients undergoing hemodialysis that is more accurate than hba1c.(1,2,4) since human serum albumin turnover is faster (with a half life of 15-20 days) than that of hemoglobin, determination of glycated albumin yields a glycemic control index over a shorter period of time (2 weeks) in comparison with hba1c (4 months). hba1c is not suitable for evaluation of glycemic control in the short term, due to the long erythrocyte life span of 120 days.(5) hba1c is also inaccurate for determination of glycemic control status in patients with anemia and variant hemoglobins.(3) in hemodialysis patients with low hemoglobin concentrations, administration of high doses of erythropoietin may shorten the erythrocyte life span, thus also lowering hba1c levels. this is in contrast with glycated albumin, which is not associated with hemoglobin turnover or erythropoietin dose.(2) a variety of data indicate that hba1c is not an ideal index for glycemic control in diabetic patients undergoing hemodialysis and receiving erythropoietin, as approximately 90% of dialysis patients receive erythropoietin therapy.(1) fukuoka et al. reported that glycated albumin (ga) was correlated with life span in japanese dialysis patients with diabetes and end stage renal disease (esrd).(6) glycated albumin is a ketoamine formed by the binding of albumin and glucose in a nonenzymatic oxidation reaction. measurement of glycated albumin is not influenced by hemoglobin metabolic disorders. the percentage (%) of glycated albumin is calculated from (glycated albumin/total serum univ med vol. 33 no.1 2 univ med vol. 33 no.1 albumin) x 100. determination of this parameter is a more accurate test for glycemic control in diabetic patients on hemodialysis and the percentage of glycated albumin is an accurate predictor of morbidity and mortality in diabetic patients undergoing hemodialysis.(2) in the study by inaba et al. on hemodialysis patients with diabetes, the degrees of glycemic control based on hba1c values were categorized as follows: excellent (hba1c <6%), good (6.0 <hba1c <7%), fair (7.0 < hba1c <8.0%), and poor (hba1c > 8%). since ga values were three times greater than hba1c, glycemic control based on ga was categorized as: excellent (ga <18%), good (18.0 < ga <21.0%), fair (21.0 < ga <24.0%) and poor (ga >24.0%).(1) glycated albumin is used for glycemic control in diabetic patients on hemodialysis, because ga is not affected by renal anemia.(3) ga also contributes directly to vascular damage and is therefore better than hba1c in predicting the development of vascular complications in diabetic patients on hemodialysis, although determination of ga also has its limitations, e.g. in liver disorders and metabolic disorders of albumin.(1) references 1. inaba m, okuno s, kumeda y, yamada s, imanishi y, tabata t, et al. glycated albumin is a better glycemic indicator than glycated hemoglobin values in hemodialysis patients with diabetes:effect of anemia and erythropoietin injection. j am soc nephrol 2007;18: 896-903. 2. peacock tp, shihabi zk, bleyer aj, dolbare el, byers jr, knovich ma, et al. comparison of glycated albumin and hemoglobin a1c levels in diabetic subjects on hemodialysis. kidney int 2008;73:1062-8. 3. koga m, kasayama s. clinical impact of glycated albumin as another glycemic control marker. endocr j 2010;57:751-62. 4. freedman bi, shihabi zk, andries l, cardona cy, peacock tp, byers jr, et al. relationship between assays of glycemia in diabetic subjects with advanced chronic kidney disease. am j nephrol 2010;31:375-9. 5. takahashi s, uchino h, shimizu t, kanazawa a, tamura y, sakai k, et al. comparison of glycated albumin (ga) and glycated hemoglobin (hba1c) in type 2 diabetic patients: usefulness of ga for evaluation of short-term changes in glycemic control. endocr j 2007;54:139-44. 6. fukuoka k, nakao k, morimoto h, nakao a, takatori y, arimoto k, et al. glycated albumin level predict long term survival in diabetic patients undergoing haemodialysis. nephrology 2008;13:278-83. oktavianus 118 *department of veterinary anatomy, veterinary faculty, airlangga university (universitas airlangga) **department of phytochemistry and pharmacognosy, pharmacy faculty airlangga university ***department of parasitology, veterinary faculty, airlangga university (universitas airlangga) correspondence dr. eka pramyrtha hestianah, drh, m.kes department of veterinary anatomy, veterinary faculty, airlangga university (universitas airlangga) kampus c unair, jl. mulyorejo, surabaya 60115 phone: +6231-5992785 email : ekamirta@gmail.com univ med 2014;33:118-25 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background people have been using curcuma aeruginosa rhizome as a traditional herbal medicine as appetite stimulant, without realizing its side effects. herbal plants contain tens to hundreds of compounds, some of which are toxic. the aim of this research was to determine which toxic compound of curcuma aeruginosa rhizome has an impact on apoptosis and parp-1 expression of hepatocytes in male mice. methods eighty eight male balb c mice were divided into 10 groups treated respectively with curcuma aeruginosa rhizome cloroform extract, methanol extract, essential oil, infusion, and press juice, at dosages of 0.004g/kgbw and 0.06g/kgbw, and 1 control group. the treatment was given orally once a day for 10 days and on the 11th day, the research animals were sacrificed, and their liver taken for histopathologic slide preparation with apopteq detection kit, and immunofluorescence. compounds in curcuma aeruginosa rhizome were analyzed with gas chromatography/mass spectrometry. the data obtained were analyzed by one-way anova, and partial least squares to determine which compounds had an impact on murine hepatocytes. results the result of one way anova showed that the chloroform groups at dosages of 0.004g/kgbw and 0.06g/kgbw showed the highest apoptosis of mice’s hepatocytes (p<0.05). there were significant differences in parp-1 expression between control and treatment groups. the highest parp-1 expression was in the essential oil group at a dosage of 0.06g/kg bw (p<0.05). conclusion curcuma aeruginosa rhizome given to mice orally causes necrosis of mice’s hepatocytes. key words : apoptosis, parp-1 expression, hepatocyte, mice toxic compounds of curcuma aeruginosa causes necrosis of mice hepatocytes eka pramyrtha hestianah*, idha kusumawati**, lucia tri suwanti***, and sri subekti**** 119 univ med vol. 33 no.2 senyawa toksik ekstrak temu ireng menyebabkan nekrosis dari hepatosit mencit latar belakang temu ireng merupakan tumbuhan obat yang banyak digunakan sebagai obat tradisional, seperti penambah nafsu makan, tanpa menyadari efek samping yang ditimbulkannya. tumbuhan obat mengandung banyak bahan-bahan yang dapat bersifat toksik. penelitian ini bertujuan untuk menentukan senyawa toksik dari rimpang temu ireng yang berpengaruh terhadap terjadinya apoptosis, dan ekspresi parp-1 hepatosit pada mencit jantan. metode mencit jantan balb c sebanyak 88 ekor dibagi menjadi 10 kelompok perlakuan terpapar ekstrak kloroform, ekstrak metanol, minyak atsiri, infusa, perasan rimpang temu ireng dengan dosis 0,004g dan 0,06 g/kgbb pada masingmasing kelompok dan 1 kelompok kontrol. perlakuan diberikan sekali sehari selama 10 hari dan hari ke 11 mencit dikorbankan, diambil organ hepar, dibuat sediaan histopatologi dengan apopteq detection kit, dan imunofloresen. senyawa dalam rimpang temu ireng dianalisis dengan gc/ms. data yang diperoleh dianalisis dengan anova oneway, dan partial least squares (pls) untuk menentukan senyawa yang berpengaruh. hasil hasil analisis anova one way menunjukkan rata-rata apoptosis hepatosit tertinggi pada kelompok yang diberikan chloroform dengan dosis 0,004 g and 0,06 g/kgbb (p<0,05). terdapat perbedaan bermakna ekspresi parp-1 hepatosit antara kelompok kontrol dan kelompok perlakuan. ekspressi parp-1 tertinggi pada kelompok minyak atsiri dosis 0,06 g/kgbb (p<0,05) kesimpulan rimpang temu ireng yang diberikan per oral pada mencit menyebabkan terjadinya nekrosis hepatosit mencit. kata kunci : apoptosis, ekspresi parp-1, hepatosit,mencit abstrak introduction herbal plants are widely used to promote and rehabilitate health, and to prevent and cure disease.(1) one of the herbal plants that has often been used is temu ireng (curcuma aeruginosa roxb). it has been reported that the compounds contained in chloroform and methanol extracts of curcuma aeruginosa rhizome can cause uterine relaxation, while in case of alopecia, curcuma aeruginosa rhizome hexane extract of can stimulate hair growth.(2,3) people think that traditional medicines/ herbal plants such as curcuma aeruginosa rhizome may be safely consumed, as they do not have negative effects on the liver or kidneys. people believe and see curcuma aeruginosa rhizome as worm killer, appetite stimulant, phlegm cleaner, reliever of sore throat, and others. these uses of curcuma aeruginosa rhizome are supported by research conducted in indonesia or overseas, but most studies are s t i l l l i m i t e d t o t h e p h a r m a c o l o g y a n d phytopharmacy of herbal plants based on their use by some people with functions that are empirically tested.(4-6) the opinion that herbal plants are totally safe and free from side effects is wrong. herbal 120 plants contain tens to hundreds of compounds, some of which are toxic. two types of toxic effect have been reported, firstly, that caused b y i n t r i n s i c f a c t o r s o f t h e h e r b a l p l a n t s themselves such as excessive dosage, allergic reaction e.g. vomiting, inflamation, diarrhea. secondly, that caused by external factors such as mistakes or failure of medicine preparation, mistakes in identifying the herbal plants, and contamination.(7) the world health organization (who) states that inconsistent compound content of herbals and toxic compounds contained in herbal p l a n t s c a n c a u s e t o x i c e ff e c t s . (8) to x i c compounds in traditionally consumed medicines can cause nephrotoxicity, damage to epithelial cells of the small intestine, and also damage to hepatocytes.(9-11) the present research study was conducted because there has not been any clinical research on the toxic compounds in curcuma aeruginosa. compounds in a herbal plant can be separated in a common way, by bioassay-guided fractionation, which needs modern separation techniques such as chromatography combined with mass spectrography, followed by nuclear m a g n e t i c r e s o n a n c e ( n m r ) t o e l u c i d a t e structure. the technique requires special skills, is very difficult and time-consuming, and needs abundant materials.(12-14) for that purpose, this research uses chemometrics partial least squares (pls) by correlating chromatogram profiles of compounds contained in curcuma aeruginosa rhizome with toxic effects observed from apoptosis and expression of poly (adpribose) polymerases-1 (parp-1). the research aimed to find toxic marker compounds in curcuma aeruginosa rhizome which impact on apoptosis and parp-1 expression of male murine hepatocytes. methods research design this experimental study was carried out at the laboratory of pathology, veterinary medical faculty, the laboratory of pharmacognosy and phytochemistry, pharmacy faculty, airlangga university (universitas airlangga), and the laboratory of biological central science, brawijaya university. the study was conducted from march to august 2010. animals experimental animals were two-month old male balb c mice, weighing from 25g to 30g, obtained from pusat veterinaria farma, surabaya. the animals were given food and water ad libitum. intervention eighty eight male mice were divided into 10 groups, respectively treated with curcuma a e r u g i n o s a r h i z o m e c h l o r o f o r m e x t r a c t , methanol extract, curcuma aeruginosa rhizome essential oil, infusion, and press juice; and one control group. the treated groups were given 2 types of oral dosage of curcuma aeruginosa rhizome extract at 0.004g/kgbw and 0.06g/ kgbw, based on ethnomedicine. the control group was given sodium carboxyl methyl cellulose (cmcna) + distilled water. the treatment was given orally once a day for 10 days. on the 11th day, the experimental animals were sacrificed. plant sample curcuma aeruginosa rhizomes were collected from jombang, and identified at balai k o n s e r v a s i tu m b u h a n k e b o n r a y a purwodadi, pasuruan. preparation of extract five kilograms of curcuma aeruginosa rhizome was sliced into pieces of 0.3-0.5 cm and distilled for the essential oil. in addition, 500 g of dry powdered curcuma aeruginosa rhizome was extracted four times by maceration at room temperature with 5 liters of chloroform, followed by four times maceration with methanol. for preparation of the crude drug infusion, t h e r h i z o m e s w e r e b o i l e d i n a p a n a t a hestianah, kusumawati, suwanti, et al toxic compounds of curcuma aeruginosa 121 univ med vol. 33 no.2 temperature of 90o c for 15 minutes, then cooled and strained. curcuma aeruginosa press juice w a s o b t a i n e d b y g r a t i n g f r e s h c u rc u m a aeruginosa rhizomes and pressing with clean flannel cloth. the curcuma aeruginosa rhizome infusion and squeezed juice were then freezedried. measurement of apoptosis and parp-1 expression data on the percentage of apoptotic hepatocytes in the histopathologic slides processed with the apoptosis detection kit, were collected from 1500 hepatocytes, examined in several high-power fields.(15,16) parp-1 data were collected by checking preparations with immunofluorescence analysis using the olympus fv1000/fv10-asw model confocal laser scanning microscope, at ex (peak excitation wave length) of 495 nm, and em (peak emission wave length) of 519 nm, based on the intensity of the light emitted by the hepatocytes. the light intensity was displayed in graph form, then 3 highest peaks were taken from the graphs to get the average score and use it as data. metabolite profiling data were obtained by identifying compounds from the curcuma aeruginosa extract, based on fragmentation and matching of mass spectra obtained from every form of extract and compared to the us national institute of standards and technology (nist) mass spectral database. data analysis the data obtained were analyzed with oneway anova at a significance level of p<0.05 and chemometrics partial least squares (pls) to determine which compounds exert influence on hepatocytes. ethical clearance ethical approval for this study was obtained from the research ethics committee, veterinary medicine faculty, airlangga university, surabaya. results the data on histopathological apoptosis and parp-1 expression in hepatocytes of the male mice receiving curcuma aeruginosa rhizome extract, can be seen in table 1. the results of one-way anova analysis on hepatocyte apoptosis showed no significant difference between the control group and the methanol group at a dosage of 0.004 g/kgbw; and between the essential oil groups at dosages of 0.004 g/kgbw and 0.06 g/kgbw (p>0.05). duncan post hoc analysis showed that the highest proportion of apoptosis of mice’s hepatocytes was in the chloroform groups at dosages of 0.004 g/kgbw and 0.06 g/kgbw (p<0.05). while the results of one-way anova on parp-1 expression showed significant differences in all treatment groups, the essential oil group at a dosage of 0.06 g/kgbw showed the highest mean parp-1 expression of 3369.77 ± 194.16% (p<0.05). compounds contained in the five types of c u rc u m a a e r u g i n o s a r h i z o m e e x t r a c t (cloroform extract, methanol extract, essential oil, infusion, pressed curcuma aeruginosa rhizome) juice, obtained by means of gas chromatography–mass spectrometry (gc/ms) and tested by means of partial least squares (pls), can be seen in figure 1, where the names of the compounds are not shown. from the results of pls analysis, three compounds were obtained that were correlated with murine hepatocyte apoptosis and parp-1 expression. these were 9-methyltetracyclo [7.3.1.0(2.7). 1(7.11)] tetradecane; epicurzerenone; and cis1,3-dimethyl-2-methylene cyclohexane. discussion table 1 shows that the five types of curcuma aeruginosa rhizome extract given to the experimental animals did not show significant differences in apoptosis between the control group and the essential oil groups at dosages of 0.004 g/kgbw and 0.06 g/kgbw. this indicates 122 that curcuma aeruginosa rhizome causes necrosis of the hepatocytes, as can be seen from the mean parp-1 expression score of 3187.24 and 3369.77%. the highest hepatocyte apoptotic activity occurred at a mean of 1.33% with the choloroform extract of curcuma aeruginosa rhizome. apoptosis is the physiologic process of programmed cell death occurring in all organs, but apoptotic overactivity indicates cell death or necrosis.(17) partial least square analysis (figure 1) test results showed the presence of apoptosis ( a ) , w h e r e c o m p o u n d n u m b e r 6 1 , 9 methyltetracyclo[7.3.1.0(2.7).1(7.11)]tetradecane, had the strongest correlation, as seen from the short distance between the point representing the compound and the point representing hepatocyte apoptotic activity. the correlation of compound 61 resulting from gc/ms analysis with that resulting from pls showed that there figure 1. pls test result of compounds contained in curcuma aeruginosa rhizome note: a = apoptosis; parp = parp-1 expression extract type control 0 chloroform methanol essential oil infusion squeezed juice a b a b a b a b a b apoptosis (%) 0.30 ± 0.13 a 1.49 ± 0.22 d 1.33 ± 0.24 d 0.29 ± 0.04 a 1.13 ± 0.13 c 0.31 ± 0.14 a 0.32 ± 0.22 a 0.77 ± 0.13 b 0.96 ± 0.12 c 0.72 ± 0.15 b 1.05 ± 1.14 c parp-1 (%) 1280.14 ± 232.20 a 2324.78 ± 198.19 b 3166.47 ± 146.17 f,g 2588.38 ± 181.55 c 2840.42 ± 170.20 d 3187.24 ± 169.84 f,g 3369.77 ± 194.16 g 3028.98 ± 216.50 d,e,f 2854.60 ± 229.62 d,e 3076.89 ± 306.29 e,f 3141.48 ± 279.76 f,g table 1. mean percentages of hepatocyte apoptosis and parp-1 expression by five types of curcuma aeruginosa rhizome extract all dosages given as g/kg body weight. means with differing superscript within rows were significantly different at the p<0.05 based on one way anova analysis and duncan post hoc multiple comparisons note: a=0.004 g/kgbb; b=0.06 g/kgbb hestianah, kusumawati, suwanti, et al toxic compounds of curcuma aeruginosa 123 univ med vol. 33 no.2 because the bioactive compounds of curcuma aeruginosa rhizome are unknown, it is difficult to determine the quality of the product. however, the compounds contained in curcuma aeruginosa rhizome can be known due to developments in chemical analysis. a clear profile chromatogram which is the profile of the chemical composition of curcuma aeruginosa rhizome extract can be known by means of gas c h r o m a t o g r a p h y c o m b i n e d w i t h m a s s spectroscopy (gc-ms) using a chemometric approach. the structure of a chemical compound can be known from the nist database of mass spectra of chemical compounds.(24) the use of 5 different types of curcuma aeruginosa rhizome extracts was proved to be able to induce parp-1 expression of murine hepatocytes. post hoc duncan test results showed that the group of mice exposed to curcuma aeruginosa rhizome essential oil at a d o s a g e o f 0 . 0 6 g / k g b w h a d t h e h i g h e s t hepatocyte parp-1 expression, with a mean of 3369.771. overexpression of parp-1 in this research indicated that the cells suffered from necrosis.(18) from pls analysis, there were several compounds which correlated with parp-1 expression activity, but those with the strongest correlation were compounds numbers 26 and 38, i.e. cis-1,3-dimethyl-2-methylene cyclohexane and epicurzerenone. the strength of correlation with parp-1 expression activity can be seen from the short distance between the point representing the compound and the point representing hepatocyte parp-1 expression activity. shorter distances indicate stronger correlation between both activity points. correlation between the compounds in curcuma aeruginosa rhizome as a result of gc/ ms analysis with those from pls showed that there was a positive role of compounds number 26 (cis-1,3-dimethyl-2-methylene cyclohexane) and number 38 (epicurzerenone) in hepatocyte parp-1 expression activity. the term “positive” means that the compound point and parp-1 expression activity point (parp) are located in was a positive role of 9-methyl tetracyclo [7.3.1.0(2.7).1(7.11)] tetradecane on hepatocyte apoptotic activity. this means that large amounts o f t h e c o m p o u n d c o n t a i n e d i n c u rc u m a aeruginosa rhizome will be associated with high apoptotic activity. apoptotic overactivity indicates abnormal cellular death, as shown by the significant differences in parp-1 expression between control and treated groups (table 1). poly (adp-ribose) polymerase (parp-1) is an enzyme group that can repair dna damage, but if parp-1 is too active, it can lead to cell death.(18,19) therefore it is necessary to carry out research on compounds which play a role in h e p a t o c y t e a p o p t o s i s b a s e d o n pa r p 1 expression. traditional medicines have toxic effects on specific organs. moreover, there is little information about the chronic toxic effects due to prolonged use and the dosis consumed, which can cause acute or chronic intoxication.(20,21) curcuma aeruginosa rhizome is often used as an appetite stimulant for children without considering the dosage. the results of a previous study by hestianah et al.(22) showed that longer administration of curcuma aeruginosa rhizome will worsen hepatocyte necrosis, while the compounds responsible for cell death are unknown. an in vitro study showed that curcuma aeruginosa rhizome increases the percentage of cell death in baby hamster kidney (bhk-21) fibroblast cell cultures.(23) one influencing factor in obtaining the chromatogram profile that can show the chemical content of an extract is the method and solvent used in extraction, so different solvents were used in this research. the solvents used were chloroform, methanol and water for extraction by maceration, while steam destillation was used to obtain the essential oil. in addition, manual pressing was used to obtain the extract without added solvent. these five types of extracts were meant to provide totally different chemical compositions, to obtain different toxic effects so that the correlation between chemical content and toxic effect could be obtained. 124 the same quadrant. it also means that a larger amount of cis-1,3-dimethyl-2-methylene cyclohexane and epicurzerenone contained in curcuma aeruginosa rhizome extract would be also followed by higher parp-1 expression activity. according to gc/ms analysis, cis-1,3dimethyl-2-methylene cyclohexane was obtained from the squeezed juice of curcuma aeruginosa rhizome, with a relative percentage area of parp1 expression of 29.61%. on the other hand, epicurzerenone was obtained from the methanol extract, essential oil, and press juice, with relative percentage areas of respectively 6.59%, 27.78%, and 8.48%. oneway anova analysis showed that mice exposed to essential oil at 0.06g/kgbw had the highest parp-1 expression, due to the relatively high epicurzerenone percentage area in curcuma aeruginosa rhizome essential oil (27.78%). the relative percentage area of parp1 expression of 29.61% for cis-1,3-dimethyl-2methylene cyclohexane was larger than that for epicurzerenone, but the former compound did not not show as much parp-1 expression activity as epicurzerenone. this might have been caused by the interaction of some compounds in curcuma aeruginosa rhizome which had a positive correlation with parp-1 expression. the interaction could presumably decrease the effect of cis-1,3-dimethyl-2-methylene cyclohexane on hepatocyte parp-1 expression activity. the univariate analysis results showed that there were differences in hepatocyte parp-1 expression due to the different extract dosages (0.004g/kgbw and 0.06g/kgbw). this was related to the epicurzerenone compound contained in curcuma aeruginosa rhizome which had a positive role and strongly correlated with hepatocyte parp-1 expression activity. therefore increased dosages of essential oil of c u rc u m a a e ru g i n o s a r h i z o m e w o u l d b e followed by increased parp-1 expression activity. the same would also apply to cis-1,3dimethyl-2-methylene cyclohexane compounds contained in the press juice of curcuma aeruginosa rhizome. the curcuma aeruginosa rhizome extract contains toxic compounds, which when given to mice are absorbed in the small intestine and from there to the liver. the compounds penetrate the hepatocyte cell membrane and reduce the activities of the cells, resulting in structural changes and rupture of organelles. parp-1 has a role in the life and death of cells under various conditions of stress. parp-1 activity requires that nicotinamide adenine dinucleotide (nad+) be hydrolyzed to adp-ribose, leading to reduction of nad+ and depletion of atp. mitochondria are organelles for the production o f atp f o r t h e c e l l s , s o t h a t d a m a g e t o mitochondria will influence cell survival.(18,19) lack of atp causes damage to the hepatocyte that is called necrosis. the finding of toxic compounds in curcuma aeruginosa rhizome in this study will give the society information about the dangers of using this rhizome and can be used as a guide in consuming curcuma aeruginosa rhizome. finally, this study was carried out to inform that curcuma aeruginosa rhizome is safe when used as a traditional herb. a limitation of this study was in the collection of c u rc u m a aeruginosa rhizomes that grow well in the same area and can be harvested at the same age in sufficient quantities to conduct the research. conclusion curcuma aeruginosa rhizomes containing t h e t o x i c m a r k e r c o m p o u n d s o f 9 m e t h y l t e t r a c y c l o [ 7 . 3 . 1 . 0 ( 2 . 7 ) . 1 ( 7 . 1 1 ) tetradecane; epicurzerenone; and cis-1,3d i m e t h y l 2 m e t h y l e n e c y c l o h e x a n e a r e correlated with mice hepatocyte parp-1 expression. acknowledgement we thank the staff of the laboratory of histology, faculty of veterinary medicine, airlangga university, for their support towards the completion and publication of this study. hestianah, kusumawati, suwanti, et al toxic compounds of curcuma aeruginosa 125 univ med vol. 33 no.2 references 1. badan pom ri. standardisasi ekstrak tumbuhan obat indonesia, salah satu tahapan penting dalam pengembangan obat asli indonesia. info pom 2005;6:1-4. 2. thaina p, tungcharoen p, wongnawa m, reanmongkol, subhadhirasakul s. uterine relaxant effect of curcuma aeruginosa roxb. rhizome extracts. j ethnopharmacol 2009;121:43343. 3. pumthong g, asawanonda p, varothai s, jariyasethong v, triwongwaranat d, 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isolation and characterization of bioactive compounds from plants extracts. afr j tradit complement altern med 2011;8:1-10. 14. tringali c, editor. isolation, characterisation and biological properties. in: bioactive compounds from natural sources. 1st ed. new york: taylor & francis;2004.p.1-30. 15. bursch w, wastl u, hufnagl k, schulte-hermann r. no increase of apoptosis in regressing mouse liver after withdrawal of growth stimuli or food restriction. toxicol sci 2005;85:507-14. 16. xia w, wang z, wang q, han j, zhao, hong z, et al. roles of nad+ / nadh and nadp+/nadph in cell death. curr pharm des 2009;15:12-9. 17. mcgavin md, zacharyjf, editors. pathology basic of veterinary disease. 4th ed. st.louis, missouri: mosby elsevier;2007. 18. kehe k, raithel k, kreppel h, jochum m, worek f, thiermann h. inhibition of poly (adp-ribose) polymerase (parp) influences the mode of sulfur mustard (sm)-induced cell death in haca t cells. arch toxicol 2008;82:461-70. 19. vodenicharov md, 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zinc deficiency as risk factor for stunting 11 *department of nutrition, faculty of medicine, diponegoro university semarang **science program study of nutrition, muhammadiyah university, semarang correspondence : salsa bening department of nutrition, faculty of medicine, diponegoro university semarang gedung g, jln. prof soedarto, sh, tembalang, semarang 1269 email: b3n.salsa@gmail.com univ med 2017;36:11-8 doi: 10.18051/univmed.2017.v36.11-18 pissn: 1907-3062 / eissn: 2407-2230 received october 27, 2016 accepted for publication april 20, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract zinc deficiency as risk factor for stunting among children aged 2-5 years salsa bening*, ani margawati*, and ali rosidi** background stunting is a nutritional problem in the form of linear growth disturbance caused by low intake of macroand micronutrients. the prevalence of stunting in children aged 2-5 years in semarang is higher in well-to-do families (67.2%) than in poor families (32.8%). the purpose of this study was to analyze the role of low adequacy levels of vitamin c, iron, zinc and low food expenditure as risk factors of stunting among children aged 2-5 years. methods this was a case-control study with a sample of 71 cases (stunting) and 71 controls (no stunting). the collected variables were adequacy levels of vitamin c, iron, zinc and level of food expenditure. nutrient intake data were obtained with the semiquantitative food frequency questionnaire (ffq), while data on food expenditure were obtained through a food expenditure questionnaire. data analysis was performed using simple and multivariate logistic regression method. results the results of the simple logistic regression showed that low levels of vitamin c ( or=2.97; 95% ci=1.41 – 6.31), iron (or=2.87; 95% ci=1.44 – 5.71) and zinc (or=9.24; 95% ci=2.02 – 42.12) were risk factors of stunting. multivariate analysis showed that the risk factor that most affected stunting was a low level of zinc (or=6.39; 95% ci=1.34 – 30.33). a low level of food expenditure was not proven to influence the incidence of stunting. conclusions a low level of zinc was the risk factor that most affects stunting in children aged 2-5 years. preventive strategies to prevent stunting and promote healthy eating is recommended. keywords: stunting, risk factors, zinc, iron, children 2-5 years doi: http://dx.doi.org/10.18051/univmed.2017.v36.11-18 universa medicinajanuary-april, 2017 vol.36 no.1 original article 12 bening, margawati, rosidi zinc deficiency as risk factor for stunting introduction stunting is one of the nutritional problems in the form of linear growth disturbance due to low nutrient intakes and chronic infectious diseases.(1) stunting in the underfives is usually less realized since the difference in height of children with stunting and normal children at such an age is not too apparent. based on the criteria of the world health organization (who), stunting is indicated by a height-for-age z-score of less than -2 standard deviations (sd).(2) in indonesia, according to data from the basic health research (riset kesehatan dasar, riskesdas), the prevalence of stunting increased from 35.6% in 2010 to 37.2% in 2013, comprising 28% ultrashort and 19.2% short underfives.(3) the prevalence of stunting in the underfives in central java in 2014 reached 33.9%. on the other hand, data from the semarang city health service show that according to the results of the monitoring of nutrional status survey (pemantauan status gizi) based on the heightfor-age index in semarang city the prevalence of stunting in the underfives is 4.03% and in children aged 2-5 years 20.37%.(4) an important component that plays a role in growth is the intake of the micronutrients vitamin c, iron and zinc. a study in egypt on vitamin c intake in pregnant women showed that the vitamin c concentration in the blood of pregnant women has a significant positive impact on neonatal anthropometrics and placental weight.(5) vitamin c is required in the growth process through its role in collagen synthesis. in a person with vitamin c deficiency, the latter will inhibit the formation of structural proteins and collagens, thus inhibiting the growth process.(6) in this connection, few studies have been conducted on vitamin c as a risk factor of stunting in the underfives. a study on the relationship between iron intake and stunting incidence carried out by hidayati (7) found a difference in iron intake of children with stunting and of those without stunting in urban slums. iron deficiency is associated with decreased immune function, which is measured by changes in several components of the immune system. however, a study in vietnam on the effect of iron and zinc supplementation in infants showed no significant effect on stature.(8) a study conducted at puskesmas klungkung found that low serum zinc concentration was a risk factor for short stature.(9) on the other hand, the study of taufiqurrahman (10) showed that zinc was not a risk factor for stunting incidence in the underfives in the province of west nusa tenggara (nusa tenggara barat, ntb). the study by gibson (11) on children 6-12 years of age reported a significant difference between serum zinc concentration of male children of short stature and that of male children of normal stature, but no difference in female children. the study by mozaffari-koshravi (12) showed that zinc supplementation only affected the height of male children aged 2-5 years . i n a d d i t i o n , t h e e c o n o m i c f a c t o r o f household poverty results in an inability to meet food requirements.(13,14) candra (15) states that a budget of less than rp 25,000.00 per day results in the inability of the household to have access to a sufficient quantity and quality of food for meeting daily requirements and is a risk factor for stunting in the underfives, with an or of 2.43. however, a different study in semarang found that per capita income was not correlated with the level of consumption of energy and protein in children aged 2-5 years.(16) semarang is the capital of central java and has a community of diverse economic status. according to data obtained from the health service of semarang city, there are 32.8% underfives with stunting from low households and 67.19% underfives with stunting from well-to-do households.(4) this shows that stunting is higher in children of wellto-do households at a ratio of 2:1 as compared with children of low households, pointing to a disparity, since in well-to-do households there should be fewer children with stunting. the present study was conducted in children aged 2-5 years since this age group has passed 13 the first thousand days of life and has been weaned of exclusive breastfeeding, thus being at highest risk of low parenting. the majority of previous studies on stunting used the variables of energy and protein macronutrient intakes, with significant results. in contrast, the present study used the variables of vitamin c, iron and zinc micronutrient intakes and the level of food expenditure. in view of the inconsistent results of previous studies and the fact that several variables had not been investigated, it was considered necessary to conduct a study with the objective of analyzing low intakes of vitamin c, i r o n , a n d z i n c , a n d a l o w l e v e l o f f o o d expenditure as risk factors for stunting in children aged 2-5 years. methods research design the study was of observational case-control design and was conducted in the catchment area of puskesmas bangetayu, genuk district, semarang city, from august until september 2016. research subjects the subjects of this study were children aged 2-5 years who met the inclusion criteria and were domiciled in genuk district, semarang city, at a ratio of cases to controls of 1:1. the cases were children aged 2-5 years who had stunting and a height-for-age z-score (haz) of <-2sd, while the controls were children aged 25 years with a height-for-age z-score of ≥-2 sd. the respondents in this study were the mothers of the children who had been selected as study subjects. the inclusion criteria in this study were children aged 2-5 years who resided in genuk district, had a growth curve card (kartu menuju sehat, kms), were living with their own parents and agreed to be included in the study sample. the exclusion criteria in this study were children of premature birth, those with physical defects, were out of town at the time the study was conducted, and withdrew from the study sample. the study sample was calculated using or values of several previous study variables(10,15) thus obtaining a minimal sample size of 71 subjects for each group after correction for possible dropouts (10%). recruitment of the subjects was by consecutive non-random sampling. anthropometric measurements height was measured with a stadiometer to an accuracy of 0.1 cm. stunting was categorized as a height-for-age z-score of <-2 sd and normal stature as a height-for-age z-score of ≥2.(2) nutrient intake measurements the level of adequacy for vitamin c, iron and zinc were determined using the semiquantitative ffq. nutrient adequacy level was determined by the nutrisurvey 2007 program, then calculated on the basis on the indonesian recommended daily allowances (irda) for 2013. the level of adequacy for vitamin c, iron and zinc were categorized as adequate if the intake was ≥77% irda and low if the intake was <77% irda.(17) level food expenditure the household food expenditure is the amount used for the food consumption of all household members for one month divided by the number of household members. the level of food expenditure was categorized as low if the food expenditure was <rp437,520.00 per capita, a n d h i g h i f t h e f o o d e x p e n d i t u r e w a s ≥rp437,520.00 per capita.(18) statistical analysis simple and multivariate logistic regression was used to analyze the data at a significance level á of <0.05. ethical clearance this study was carried out after obtaining approval from the ethics commission, faculty of medicine, diponegoro university, semarang no. 852/ec/fk-rsdk/viii/2016. univ med vol. 36 no.1 14 bening, margawati, rosidi zinc deficiency as risk factor for stunting results the majority of study subjects were female (55.63%). table 1 lists the characteristics of the study subjects, i.e. age, weight, height, z-score and monthly income. there were no differences in age and monthly income of the study subjects (p>0.05) in the group of cases and the group of controls. with regard to weight, height and zscore of the study subjects, there was a difference (p<0.05) between the group of stunted and the group of non-stunted. table 2 shows that the proportion of children with a low level of adequacy for vitamin c was greater in the group of stunted (42.3%) t h a n i n t h e n o n s t u n t e d g r o u p ( 1 9 . 7 % ) (or=2.97; 95% ci=1.41 – 6.31). likewise, the proportion of children with a low level of adequacy for iron was greater in the group of stunted (69%) than in the non-stunted group (43.7%) (or=2.87; 95% ci=1.44 – 5.71). in addition, the proportion of children with a low level of adequacy for zinc was greater in the group of stunted (21.1%) than in the non-stunted group (2.8%) (or=9.24; 95% ci=2.02 – 42.12). finally, the proportion of children with a low level of food expenditure was greater in the group of stunted non-stunted (81.7%) than in the group (74.6%) (or=1.51; 95% ci=0.67 – 3.38). multivariate analysis performed on 3 variables, i.e. levels of adequacy for vitamin c, iron and zinc. the results of the multivariate logistic regression analysis presented in table 3 showed that the variable with the most important role on stunting incidence was a low adequacy level for zinc. a low adequacy level for iron had a 2.13fold significantly higher risk for stunting incidence than a adequate adequacy level for iron (or=1.20; 95% ci=1.03-4.37). a low adequacy level for zinc had a 6.39-fold significantly higher risk for stunting incidence than a adequate adequacy level for zinc (or=6.39; 95% ci=1.34-30.33). table 1. characteristics of the study subjects by stunted and non-stunted children * independent t-test; ** mann-whitney test table 2. simple logistic regression analysis of several variables for the stunted and non-stunted children 15 discussion a low adequacy level for vitamin c was a risk factor for stunting in children aged 2-5 years. the results of this study was in agreement with the study in cairo on children aged 2-5 years, which showed that mean vitamin c intake was significantly lower in children with stunting than in controls.(19) differing results were found in a study on primary school students in tehran, iran, s h o w i n g n o s i g n i f i c a n t d i ff e r e n c e s f o r micronutrient intakes (vitamin c, iron and zinc) between children with stunting and those without stunting.(20) these differing results was caused by a difference in age, in which the subjects of the tehran study were primary school students with a mean age of 82 ± 4 months. differing nutrient intakes and environments may also lead to differing study results. vitamin c is important for the synthesis of collagen, fibers and structural proteins. collagen is required for the formation of bones and teeth and wound repair. vitamin c is required in the growth process through its role in collagen synthesis, through hydroxylation of proline and lysine into hydroxyproline, an important material for collagen synthesis, and a protein affecting the structural integrity of the cells in all connective tissues, such as cartilage. normal collagen cannot be formed without the presence of vitamin c. (6) children with vitamin c deficiency will experience inhibition the formation of structural proteins and collagens thus inhibiting the growth process. a low adequacy level for iron was a risk factor for stunting in children aged 2-5 years. these study results are in line with the results of a study conducted in kepulauan nusa tenggara reporting that iron and zinc were the strongest variables on stunting incidence in the underfives (24-59 months).(10) iron deficiency is associated with decreased immune functions, as measured with changes in several components of the immune system that occur in iron deficiency.(21) the consequence of the changes in immune functions is lowered r e s i s t e n c e t o w a r d i n f e c t i o u s d i s e a s e . ( 2 2 ) underfives with iron deficiency are more suceptible to microorganisms, because iron deficiency is closely associated with damaged functional capacity of the immune defense mechanism that is important for the entry of infectious disease.(23) iron plays a role in the immune system. the immune responses by the t lymphocytes will be disturbed upon decreased production of cells, which is caused by decreased dna synthesis. dna synthesis is caused by disturbances of ribonucleotide reductase which requires iron for its ordered functioning, so that iron deficiency may cause disorders of the immune system. children with immune system disorders are more susceptible to infectious diseases, thus affecting nutritional status.(24) a low adequacy level for zinc is a risk factor for stunting in comparison with respondents with a adequate adequacy level for zinc. our study results are in agreement with the study conducted by hidayati,(7) stating that low zinc intake carries a 2.67-fold higher risk for stunting incidence in children. kenneth (25) revealed in a study on children in peru that children who received zinc supplementation had weights which were 0.41 kg higher in comparison with children who did not receive zinc and proved that zinc deficiency may inhibit growth. zinc plays an important role in the structure and function of biomembranes, and zinc is an important component of several enzymes that regulate cell growth, protein and dna synthesis, e n e rg y m e t a b o l i s m , r e g u l a t i o n o f g e n e transcription, hormone concentrations and table 3. multivariate-adjusted odds ratios for stunting across iron and zinc intakes univ med vol. 36 no.1 16 bening, margawati, rosidi zinc deficiency as risk factor for stunting metabolism of growth factors.(26) zinc commonly plays a role in the synthesis of growth hormones. a growth hormone that plays a role in growth is insulin-like growth factor-1 (ig-f) which functions to increase cellular growth. zinc also plays a role in human immune functions.(27) m a r a s m i c i n f a n t s w h o r e c e i v e z i n c supplementation show increases in defensive responses. children in developing countries who received zinc supplementation showed decreases in the incidence and duration of acute and chronic diarrhea.(28,29) a low level of food expenditure was not a risk factor for stunting. in the present study the mean income in the group of cases was rp 2,023,267.00 and in the group of controls rp 1,938,014.00. on average the group of controls allocated a greater amount of funds for food than did the group of cases, so that the types and amounts of nutrients consumed by the group of controls was more diverse and considerable in comparison with the group of cases. thus the proportion of total nutrients seen in this study in the group of controls was higher than in the group of cases. the results of this study are in line with the study in semarang, where the per capita income was not associated with level of energy and protein consumption in children aged 2-5 years.(16) a low food expenditure in households results in a diet with low diversity, nutritiousness, and balance, which affects dietary consumption patterns.(30,31) low availability of foods may result in low realization of nutrient intakes in households.(32) a decrease in the quality of household food consumption that is characterized by limited purchases of foods that are sources of proteins, vitamins and minerals will lead to undernutrition.(33) the low adequacy level for zinc is the most important variable affecting stunting in children aged 2-5 years. iron and zinc play a role in the immune system.(23) the immune responses by t lymphocytes will be disturbed upon a decrease in the production of cells, which is caused by decreased dna synthesis, so that iron and zinc deficiencies may cause disorders of the immune system.(26) children with disorders of the immune system are more susceptible to infectious disease, thus affecting nutritional status.(29) a limitation of this study was the use of a retrospective case control design, with the possibility of recall bias in data collection, particularly on the variables of food intakes. in this case the recall capacity of the respondents will considerably affect the study results. in future, studies may be conducted using other less biased instruments, such as biochemical assessment, so that the obtained results may be more valid. the public health implication of this study is to recommend vitamin c, iron and zinc intakes in children. finally, stunting is a heterogeneous multi-factorial disorder and besides dietary factors, other variables, such as hereditary factors and metabolic conditions must be considered. conclusions a low adequacy level for zinc is the most influential factor for stunting incidence in children aged 2-5 years. this study demonstrates the necessity of continued coordination of interventions to reduce multiple micronutrient deficiencies for optimal growth of children aged 2-5 years. conflict of interest there was no conflict of interest in this study (financial, personal, political, intellectual or religious). acknowledgments the authors wish to express their gratitude to the head of kantor kesatuan bangsa, politik, dan perlindungan masyarakat (kesbangpol) of semarang city, the head of the health service (dinas kesehatan) of semarang city and the head of the health center (puskesmas) of bangetayu, who permitted the authors to collect 17 data for our study. we also thank the cadres of the integrated service post (posyandu) of bangetayu, the respondents and all who provided assistance. contributors sb contributed to drafting the manuscript and the study design. sb, am, ar contributed to data collection, analysis and interpretation. sb and am contributed revising manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. chirande l, charwe d, mbwana h, et al. 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university press; 2005. 18. badan pusat statistik kota semarang. rata-rata pengeluaran per kapita sebulan dan komposisi konsumsi tahun 2008-2015. semarang: badan pusat statistik;2016. 19. mikhail wza, sobhy hm, el-sayed hh, et al. effect of nutritional status on growth pattern of stunted preschool children in egypt. acad j nutr 2013;2:1-9. doi: 10.5829/idosi.ajn.2013. 2.1.7466. 20. esfarjani f, roustaee r, mohammadi-nasrabadi f, et al. major dietary patterns in relation to stunting among children in tehran, iran. j health popul nutr 2013;31:202-10. 21. thompson j, biggs ba, pasricha sr. effects of daily iron supplementation in 2 to 5 year old children: systematic review and meta-analysis. univ med vol. 36 no.1 18 bening, margawati, rosidi zinc deficiency as risk factor for stunting pediatrics 2013;131:739-53. doi: 10.1542/peds. 2012-2256. 22. lozoff b. iron deficiency and child development. food nutr bull 2007;28:s560-71. 23. mayo-wilson e, imdad a, junior j, et al. preventive zinc supplementation for children, and the effect of additional iron: a systematic review and meta-analysis. bmj open 2014;4: e004647. doi: 10.1136/ bmjopen-2013-004647. 24. pasricha sr, hayes e, kalumba k, et al. effect of daily iron supplementation on health in children aged 4-23 monts: a systematic review and meta-analysis of randomised controlled trials. lancet global health 2013;1:e77-86. doi: 10.1016/s2214-109x(13)70046-9. 25. kenneth hb, daniel lr, joanne ea, et al. comparison of the effects of zinc delivered in a fortified food or a liquid supplement on the growth, morbidity, and plasma zinc concentrarion of young peruvian children. am j clin nutr 2007;85:538-47. 26. lee yl, lee cy, grzechnik a, et al. rna polymerase instability couples cellular growth to metal availability. mol cell 2013;51:105-15. doi:10.1016/j.molcel.2013.05.005. 27. maggio m, de vita f, lauretani f, et al. igf-1 the cross road of the nutritional, inflammatory and hormonal pathways to frailty. nutrients 2013;5:4184-205. 28. salam ra, das jk, bhutta za. current issues and priorities in chilhood nutrition, growth and infections. j nutr 2015;145:s1116s-22. 29. perignon m, fiorentino m, kuong k, et al. stunting, low iron status and parasite infection are significant risk factors for lower cognitive performance in cambodian school-aged children. plos one 2014;9e112605. doi: 10.1371/journal.pone.0112605. 30. ramli, kingsley ea, kerry j, et al. prevalence and risk factors for stunting and severe stunting among under-fives in north maluku province of indonesia. bmc pediatrics 2009;9:64. doi: 10.1186/1471-2431-9-64. 31. amsalu s, tigabu z. risk factors for severe acute malnutrition in children under the age of five: a case-control study. ethiop j health dev 2008;22: 21-5. 32. zhou h, wang xl, ye f, et al. relationship between child feeding practices and malnutrition in 7 remote and poor counties, p r china. asia pac j clin nutr 2012;21:234-40. 33. paudel r, pradhan b, wagle rr, et al. risk factors for stunting among children: a community based case control study in nepal. kathmandu university med j 2012;10:18-24. f:\36 no 2\yolanda.pmd 150 *department of child health, faculty of medicine, atma jaya university correspondence: natharina yolanda department of child health, faculty of medicine, atma jaya university phone:+62821 2200 9982 email: natharinayolanda@hotmail.com date of first submission, february 13, 2 01 7 date of final revised submission, march 31, 2017 date of acceptance, august 1, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 pediatric idiopathic hypercalciuria with bilateral nephrolithiasis and hypertensive urgency natharina yolanda* and irene* background pediatric nephrolithiasis is an important cause of morbidity worldwide. even with low prevalence, nephrolithiasis can cause detrimental longterm effects on kidney function. idiopathic hypercalciuria is the most common cause of renal stones in children. this case report presents a pediatric patient with bilateral nephrolithiasis and hydronephrosis associated with underlying idiopathic hypercalciuria. case description we report on a 17-month-old boy presenting with two days of high fever, irritability, nausea, and poor feeding. the patient had been diagnosed with bilateral nephrolithiasis at the age of 6 months. on examination, the patient was febrile and hypertensive. laboratory evaluation revealed anemia, leukocytosis, normokalemia, normocalcemia, and normal glomerular filtration rate. blood gases were within normal limits. urinalysis revealed ph 5.0, hematuria, pyuria, and bacteriuria. urine culture was positive for proteus mirabilis and enterobacter gergoviae. electrolyte analysis from urine samples revealed hypercalciuria. ultrasonography found stones at the left ureteropelvic junction with corresponding grade i hydronephrosis and stones in the right lower calyx without hydronephrosis. the patient was treated with antibiotics, an antihypertensive agent, thiazide diuretics, and was referred to a pediatric urologist. conclusion this case can be considered as idiopathic. most children with renal stones have metabolic risk factors including enteric, endocrinologic, or renal sources. metabolic evaluation is mandatory in pediatric renal stone patients and hypercalciuria is an important etiology to be evaluated. ignorance of the underlying metabolic abnormality or failure in its identification in pediatric urolithiasis could lead to catastrophic long-term effects. keywords: idiopathic hypercalciuria, pediatric nephrolithiasis, renal stones doi: http://dx.doi.org/10.18051/univmed.2017.v36.150-156 case report cite this article as: yolanda n, irene. pediatric idiopathic hypercalciuria with bilateral nephrolithiasis and hypertensive urgency. univ med 2017;36:1506. doi: 10.18051/univmed.2017.v36. 1 5 0 -1 5 6 pissn: 1907-3062 / eissn: 2407-2230 151 univ med vol. 36 no.2 introduction pediatric nephrolithiasis is an important cause of morbidity worldwide, with detrimental long-term effects on kidney function. recent data provide evidence th at the incidence of nephrolithiasis in children is rising. in the united states, pediatric nephrolithiasis accounts for 1 per 1,000 to 7,600 pediatric hospital admissions.(1-3) data from indonesia a re not available. approximately 50% of these patients have an onset around 5 years of age. one characteristic feature of pediatric stone disease is the frequency of an underlying metabolic etiology. it has been reported that 50% of children aged <10 years with stone disease have an underlying metabolic disorder.(4) underlying metabolic risk factors include enteric, endocrinologic, and renal sources. hypercalciuria is the most common cause of stones in children, representing up to 50% of metabolic risk factors identified, followed by hypocitraturia. in children with nephrolithiasis or nephrocalcinosis, hypercalciuria is found in 28– 79% of cases. most causes of hypercalciuria are idiopathic, both sporadic and familial.(2) hypercalciuria with nephrolithiasis is associated with a wide variety of symptoms. the most common symptoms are abdominal pain (53– 75%), gross hematuria (14–33%), and dysuria (15%), although 15% of cases present as an asymptomatic, incidental radiologic finding.(3,4) urinary tract infection (uti) is also a common associated symptom, affecting 8–45% children with nephrolithiasis.(1) in infants, symptoms of nephrolithiasis may not be specific (irritability, nausea, fever, hematuria) and uti may be the presenting sign up to 75% of the time. it is particularly importa nt to rec ognize the combination of obstructing stone and uti as this clinical scenario can quickly lead to sepsis.(1) although urolithiasis may affect children of all ages, little information is available regarding preschool children.(5) the aim of this case report was to present a pediatric patient with bilateral nephrolithiasis and hydronephrosis associated with underlying idiopathic hypercalciuria and to emphasize the importance of looking deeper than the stone to find the underlying abnormalities. case report a 17-month-old boy presented with two days of fever, with a maximum of 40.4oc. additional symptoms were irritability, nausea, and poor feeding. previously, the patient had been diagnosed with bilateral nephrolithiasis at the age of 6 months, the initial presentation being hematuria and high fever. at that time, urinalysis revealed 12–15 white blood cells (wbc) per high power field, 8–10 red blood cells (rbc) per high power field, and crystals. leukocyte esterase and nitrite tests were negative. abdominal ultrasonography (usg) revealed multiple renal stones in the left lower calyx (0.3, 0.6 and 0.7 cm in diameter) without hydronephrosis and multiple renal stones in the right lower calyx (0.4 and 0.5 cm in diameter) without hydronephrosis. two months after the initial symptoms, the patient spontaneously passed two white stones, each 0.3 cm in diameter. stone analysis was positive for calcium, oxalate, phosphate, and cystine. there had been two episodes of urinary tract infection since the diagnosis of nephrolithiasis was made. each episode was treated with outpatient antibiotics. family history of stone disease, renal dysfunction, gout, or arthritis were denied. on examination, the patient was irritable, with blood pressure of 160/90 mmhg (>p 99 + 5 mmhg), heart rate of 128 beats per minute, respiratory rate of 36 times per minute, and temperature of 38.4oc. conjunctivae, skin, and the oral mucous membrane were pale. there was no tenderness at the costovertebral angle. the patient has no phimosis and was circumcised. laboratory evaluation revealed microcytic hypochromic anemia consistent with chronic disease (hemoglobin level 8.5 g/dl, hematocrit level 28%, mcv 64 fl, mch 19.2 pg, mchc 25 g/dl, reticulocyte 1.4%, ferritin 26.4 ng/ml, and serum iron 49 mg/dl), leukocytosis (wbc 24,700/ml with ba nd predominance), 152 normokalemia (serum potassium 3.4 mmol/l), normocalcemia (serum ionized calcium 1.3 mmol/ l), and normal glomerular filtration rate (gfr 83 ml/min/1.73m2). urinalysis revealed ph 5.0, pyuria (60–70 wbc per high power field), and gram negative rods. leukocyte esterase and nitrite were negative. urine culture from bladder catheterization was positive for proteus mirabilis (>100,000 colony-forming units (cfu)/ml) and enterobacter gergoviae (1,000 – 10,000 cfu/ ml). electrolyte analysis from 24-hour urine samples revealed hypercalciuria (urinary calcium 400 mg/kg/day, ten times the normal value). repeat us found stones of 7 mm x 3 mm in the left uretheropelvic j unction (upj) with corresponding grade i hydronephrosis and stones of 2 mm in diameter in the right lower calyx without hydronephrosis. the patient was diagnosed as having urinary tract infection with predisposition of bilateral nephrolithiasis ca used by idiopathic hypercalciuria. complications were hypertensive urgency, hydronephrosis, and anemia of chronic disease. the hypertensive crisis was treated initially with oral nifedipine and controlled with oral captopril. uti was treated with intravenous cefotaxime, with the fever subsiding after 72 hours, when the intravenous antibiotic was shifted to oral cefixime. the antibiotics were given for a total of 10 days. t he patient was given prophylactic antibiotics and then referred to a pediatric urologist for further management. discussion epidemiologic studies have shown a progressive increase in the incidence of pediatric urolithiasis (pu) worldwide over the few decades.(6) pu affects children of all ages and seems to primarily appear in the first years of life, which might be due to the fact that it is frequently based on tubulopathies or inborn errors of metabolism.(7) the true burden in indonesia is not known due to limited data; to date , here have been few case reports on this topic. the present case is important because there have been not many similar cases reported in indonesia; this report is intended to be a trigger for doctors to increase their awareness and knowledge regarding the need of an appropriate search for the underlying metabolic abnormalities and their treatment in pediatric patients with urolithiasis. pediatric urinary stone disease is a relatively rare pathology, with overall incidence of 1–2%.(8) predisposing causes can be recognized in >75% of children and adolescents with urolithiasis and even the first stone episode deserves a comprehensive examination. approximately 40% of children with urolithiasis have a positive family history of renal stones and most of them have a metabolic background.(9) this disease is associated with considerable morbidity with reported recurrence rates ranging from 6.5–44% and appears to be highest in children with metabolic abnormalities.(8) a history of high fever and non-specific symptoms, pyuria, hematuria, and positive urine culture in this young child previously diagnosed with nephrolithiasis is consistent with uti, specifically pyelonephritis. proteus mirabilis is one of the most common urinary pathogen. the patient’s history of recurrent urinary infections indicates the need for an attempt to identify predisposing factors. some risk factors can be eliminated for this patient, which are female gender, uncircumcised male, toilet-training, and constipation. the mechanism of uti in patients with hypercalciuria is due to the damage of the epithelium caused by microcrystal aggregates formed secondary to hypercalciuria, which provides a nidus for bacterial sequestration.(5) in this patient, nephrolithiasis was identified by us to be a risk factor. based on a previous study, nephrolithiasis is more likely to be found in young children whereas ureteral stones are more likely to be found in older children. most pediatric renal stones are found in the upper urinary tract.(10) several other studies noted a slight predominance of boys in pediatric urolithiasis, with a boy:girl ratio of 1.3:1.0. if the stone is identified in the urinary tract but not passed in the urine, an evaluation of anat omic and metabolic yolanda, irene pediatric idiopathic hypercalciuria 153 univ med vol. 36 no.2 abnormalities is recommended. younger children are more likely to have an underlying metabolic risk factor. based on several studies, the most common metabolic disorders are hypercalciuria and hyperuricosuria. however, there is no significant difference in frequency of metabolic abnormalities between patients aged <3 years and 3 years. the risk factors for urolithiasis depend on the geographic a re a, genetic s, and environmental factors (climate and dietary habits).(5) spontaneous elimination of stones was found to occur in 46% of patients, with an average size of 5.3 mm. the probability of spontaneous elimination is reduced if the stone is larger than 3–4 mm in diameter.(12) it was noted that children older than 5 years were more prone to spontaneous evacuation. (1 3) if the stone has passe d spontaneously, stone chemical analysis will identify specific stone composition and lead to further workup. struvite stone is associated with infection; a urine sample for culture must be taken. urinary cystine should be measured if a cystine stone is found. calcium oxalate or calcium phosphate stones must be followed by complete metabolic evaluation, which for pediatric nephrolithiasis comprises urinalysis, urine culture, spot urine to measure calcium-to-creatinine ratio, serum chemistries (electrolytes, uric acid, creatinine, calcium, phosphorus, bicarbonate, and magnesium), and 24-hour urine (ph, volume, calcium, oxalate, uric acid, cystine, citrate, magnesium, sodium, phosphorus, and potassium adjusted for urinary creatinine). analysis of our patient’s stone revealed calcium, oxalate, phosphate, and cystine. the most probable main component of this patient’s stone is calcium oxalate and/or calcium phosphate, which is consistent with data from most of the literature. appr oximately 60–90 % of patients with urolithiasis form calcium stones, most of which are composed primarily of calcium oxalate or, less often, calcium phosphate. the majority of calcium stones form in otherwise healthy individuals without systemic disease.(6,14) struvite constitutes 1–18% of the stones in developed countries; uric acid constitutes 5–10%, cysteine 1–5%, and mixed stones 4%.(6) based on metabolic evaluation, this patient had hypercalciuria with normal serum calcium and normal renal function (normocalcemic hypercalciuria).(15) hypercalciuria is considered idiopathic if the serum calcium level is normal and known possible causes of normocalemic h yp e r c a lc iu r i a c a n be e xc l ude d. so me differentials of normocalcemic hypercalciuria are idiopathic hypercalciuria, hyperparathyroidism, f u r o s e mi de or c or t i c os t e r o id t h e r a p y, immobilization, bartter syndrome, syberth syndrome, early vita min d toxicity, limb fracture, thyrotoxicosis, and distal renal tubular acidosis (rta).(16) idiopathic hypercalciuria (ih) is a common metabolic abnormality in children. the gene responsible for familial ih has not been identified, but appears to be transmitted in an autosomal dominant manner.(12) the normal upper limit for calcium excretion in children is generally considered to be 4 mg/kg/day and is best measured from 24-hour urine collection,(15) which is recommended to exclude diurnal fluctuations related to intake of food and beverages.(9) the urinary calcium-to-creatinine ratio (uca/ucr ratio) can be used as a screening test if 24-hour urine collection cannot be done. a ratio exceeding 0.21 in random voided urine can be defined as hypercalciuria.(15) in this patient, some etiology differentials for hypercalciuria can be excluded through the history and physical examination. the patient did not have a history of furosemide or corticosteroid therapy or immobilization, nor a family history of hereditary hypercalciuria, or limb fracture. the history and physical examination did not reveal signs of thyrotoxicosis or sarcoidosis. rta that is associated with renal stones is distal rta (type i), having the clinical findings of hyperchloremic acidosis, minimum urine ph of >5.5, and low to normal plasma potassium, which were absent in this patient. normal serum electrolytes ruled out renal tubular acidosis and bartter syndrome.(15) theref ore, the pr oba bility of idiopathic hypercalciuria can be declared. 154 idiopathic hypercalciuria can have a familial predisposition or can occur in sporadic form. in the familial form, the inheritance is compatible with autosomal dominant transmission. (6 ) idioptahic hypercalciuria results from either a combination of increased gastrointestinal calcium absorption from a direct increase in calcium absorption or thr ough excess 1,25dihydroxyvita min d-mediated calc ium absorption; decreased renal absorption of either calcium or phosphorus; or enhanced bone resorption. formal assessment for subtypes of hypercalciuria in children on a regular basis is not mandatory, but it can be utilized as a reasonable framework to methodically approach an evaluation of a medically complex child with hypercalciuria.(17) in children, calcium intake should not be limited. in fact, low calcium diets have been associated with increased risk of nephrolithiasis while high dietary calcium intake has been associated with decreased risk of stones in adults. this is likely due to the ability of dietary calcium to decrease the intestinal absorption of oxalate.(18) the r isk of nephrolithiasis increases progressively with the greater levels of ih. in 85% of children with ih, renal microlithiasis has been reported in follow-up us. most cases of ih have non-specific symptoms and are commonly associated with symptoms of urolithiasis. ih can manifest as lower urinary tract signs and symptoms probably due to microcrystallization causing injury to the urinary tract epithelia. the typical presentations of urolithiasis in children are (1) intense colicky abdominal pain, (2) gross hematuria with or without pain, (3) urinary infection, (4) asymptomatic stones, or (5) persistent microscopic hematuria, which consists of 5 or more rbcs per hpf in 3 of 3 consecutive urine specimens obtained at least 1 week apart.(16) gross hematuria in ih is often transient, or persists for several days.(15) this patient’s symptoms– transient macroscopic hematuria and recurrent uti—were associated with the urolithiasis. considering the history of recurrent uti in this patient, a prophylactic antibiotic was indicated to prevent reinfection. prophylactic antibiotic using either the sulfamethoxazoletrimethoprim or nitrofurantoin at one third of the normal therapeutic dose once a day is often effective. prophylaxis with amoxicillin or cephalexin also may be effective, but the risk of breakthrough uti is higher because bacterial resistance may be induced.(3) this patient’s condition warrants urgent urologic surgery consultation since the stone was associated with urinary obstruction and recurrent pyelonephritis. hypercalciuria in this patient is probably idiopathic, but the treatment for the hypercalciuric condition has to be done to prevent recurrent stone formation. for long-term management or prevention of recurrence, clinical treatment is needed. treatment aims to reduce or prevent the emergence of new stones or prevent existing ones to increase in size, thus reducing morbidity and the need for surgical intervention. the clinical treatment includes nonpharmacological and pharmacological treatment. the non-pharmacological approach, i.e. increasing the fluid intake and dietary adequacy, is the most important approach.(14) an increased fluid intake under close monitoring is critical in preventing super saturation of the urine. dietary sodium restriction along with a high-potassium, l ow -o xa la t e d i e t i s r e c omme n de d . ( 1 9 ) pharmacotherapy is recommended if the fluid and dietary approach has failed. the most common drug used is a thiazide diur etic, such as hydrochlorothiazide (1–2 mg/kg/day).(12) in addition to fluid intake and diet modification, thiazide diuretics remain the major therapy for calcium stones associated with ih. thiazide drugs reduce calcium excretion by increasing calcium absorption in the proximal tubule and stimulating calcium reabsorption in the distal tubule.(14) between 25–50% of childr en with nephrolithiasis are estimated to undergo surgical management. the most common options are extracorporeal shockwave lithotripsy (eswl), retrograde intrarenal surgery (rirs), and percutaneous nephrolithotomy (pcnl). open surgery is rarely needed. (1 9) esw l is yolanda, irene pediatric idiopathic hypercalciuria 155 univ med vol. 36 no.2 recommended for small upper tract stones, in children without cystinuria or stones secondary to obstructive uropathy. current guidelines recommend eswl and pcnl as first line treatment for renal stones <2 and >2 cm in diameter respectively. however, rirs is gaining more acceptance as a first line alternative option for renal stones up to 2 cm or more. rirs can reduce complications related to pcnl, namely urinary sepsis and bleeding.(20) long-term complications of pediatric nephrolithiasis are hydronephrosis, recurrent urinary infection associated with renal scarring, renal failure, anemi a, and hypertension. previously, renal scarring has been reported to occur in 8% of children with urolithiasis.(5) the prognosis for nephrolithiasis depends on the primary diagnosis and adherence to therapy, but recurrence rates generally are high when the condition is left untreated. recurrence of pediatric urolithiasis ranges from 6.5 – 44% with a mean interval of 3–6 years. without follow-up and medical intervention, recurrence rates have been reported as high as 50% within 5–6 years.(8) a limitation of this report was the incomplete illustration of the clinical course of the patient, i.e. the long-term follow-up to identify recurrences and complications. we also did not do further investigations in the family to identify a possible familial cause. conclusions hypercalciuria is the most common cause of stones in children, in which most of them are idiopathic. treatment of the stones and the underlying hypercalciuria is important to prevent recurrent stone for mation and long-term complications. it should always be kept in mind that the renal stone is the sign of the disease, but not the disease it self. conflict of interests competing interests: no relevant disclosures. acknowledgment the authors thank the postgraduate students on pediatric internship, visakha irawan and maria patricia, for helping with the data collection. contributors ny contr ibuted to data collection, manuscript drafting, and analysis of the case. i contributed to analysis of the case and revision of manuscript. both authors read and approved the final manuscript. references 1. kokorowski pj, hubert k, nelson cp. evaluation of pediatric nephrolithiasis. indian j urol 2010; 26:531-5. 2. sas dj. an update on the changing epidemiology and metabolic risk factors in pediatric kidney stone disease. clin j am soc nephrol 2011;6: 2062-8. 3. elder js. urinary tract infections. in: kliegman r, stanton b, js geme, schor n, editors. nelson textbook of pediatrics, 19th ed. philadelphia: elsevier;2011.p.1785-9. 4. valentini rp, lakshmanan y. nephrolithiasis in children. adv chronic kidney dis 2011;18:3705. doi: 10.1053/j.ackd.2011.07.002. 5. elmaci am, ece a, akin f. clinical characteristics and metabolic abnormalities in preschool-age children with urolithiasis in southeast anatolia. j ped urol 2013;xx:1-5. 6. sharma ap, giller g. epidemiology of pediatric urolithiasis. indian j urol 2010;26:51-22. 7. habbig s, beck bb, hoppe b. nephrocalcinosis and urolithiasis in children. kidney int 2011; 80:1278–91. doi: 10.1038/ki.2011.336. 8. koyuncu h, yencilek f, ertuhan s, et al. clincal course of pediatric urolithiasis: follow-up data in a long-term basis. int urol nephrol 2011; 43:7-13. 9. hoppe b, kemper mj. diagnostic examination of the child with urolithiasis or nephrocalcinosis. pediatr nephrol 2010;25:403-413. 10. asl as, maleknejad s. pediatric urolithiasis: an experience of a single center. ikjd 2011;5:30913. 11. alpay h, ozen a, gokce i, et al. clinical and metabolic features of urolithiasis and 156 yolanda, irene pediatric idiopathic hypercalciuria microlithiasis in children. pediatr nephrol 2009; 24:2203e9. 12. copelovitch l. urolithiasis in children. pediatr clin n am 2012;59:881-96. 13. milošević d, batinić d, turudić d, et al. demographic characteristics and metabolic risk factors in croatian children with urolithiasis. eur j pediatr 2014;173:353-9. 14. amancio l, fedrizzi m, bresolin nl, et al. pediatric urolithiasis: experience at a tertiary care pediatric hospital. j bras nefrol 2016;38:90-8. 15. gillespie rs. stapelton fb. nephrolithiasis in children. pediatr rev 2010;25:131-40. 16. esfhani st, madani a, siadati aa, et al. prevalence and symptoms of idiopathic hypercalciuria in primary school children of tehran. iran j pediatr 2007;17:353-8. 17. srivastava t, schwaderer a. diagnosis and management of hypercalciuria in children. curr opin pediatr 2009;21:214-9. 18. jackson ec, reeber ma. urolithiasis in children: treatment and prevention. curr treat options peds 2016;2:10–22. doi: 10.1007/ s40746-016-0041-0. 19. chu di, tasian ge, copelovitch l. pediatric kidney stones—avoidance and treatment. curr treat options peds 2016;2:104-11. 20. salerno a, nappo sg, matarazzo e, et al. treatment of pediatric renal stones in a western country: a changing pattern. j pediatr surg 2013; 48:835-9. c:\users\jurnal fkusakti\docume 88 *papua biomedical research and development station (balai litbang biomedis papua) correspondence : semuel sandy balai litbang biomedis papua jl. kesehatan no.10 dok ii. jayapura email: mercury.sandy56@gmail.com) mobile: +62815 2764 4234 date of first submission, december 28, 2017 date of final revised submission, august 2, 2018 date of acceptance, august 6, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina habit of cooking pork on hot stones as main risk of cysticercosis semuel sandy*, antonius oktavian*, hanna s kawulur*, mirna widiyanti*, iman hs sasto*, and yustinus maladan* background cysticercosis is an infectious disease caused by the larval form of taenia solium (cysticercus cellulosae) and has been ranked as the most important food-borne parasite of humans in terms of public health, socioeconomic and trade impact. cysticercosis is still a health problem in papua and is inseparable from socio-cultural factors, hygiene and environmental sanitation. the aim of this study was to investigate the seroprevalence of cysticercosis and the risk factors that contribute to cysticercosis. methods a cross-sectional study was conducted in march-november 2016 involving 800 subjects. demographic data and risk factors were collected using questionnaires. cysticercosis serological examination was performed by means of the magnetic microsphere bead immunoassay technique coupled with rt24h recombinant protein to detect serum rt24h cysticercosis specific antibodies. the data obtained were analyzed by bivariate test (chi-square) and logistic regression. results cysticercosis seroprevalence in papua was 3.6% (284/7 874). the logistic regression analysis found that the risk factors playing the role of predictor were cooking pork with hot stones [or=3.06; 95%ci: 2.19-4.28; p=0.000], nail hygiene [or=2.05; 95%ci: 1.57-2.67; p=0.000], consumption of raw vegetables or salads [or=0.52; 95%ci: 0.30-0.91; p=0.022], use of river water for washing foods [or= 1.92; 95%ci: 1.39-2.64; p=0.000]. conclusions cooking pork with hot stones was the main risk factor of cysticercosis. suspected cases of t. solium in pigs should be confirmed by molecular methods. both taeniasis and human cysticercosis should be notifiable and surveillance in animals should be improved. keywords: seroepidemiology, cysticercosis, taenia solium, pork, papua doi: http://dx.doi.org/10.18051/univmed.2018.v37.88-96 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cite this article as: sandy s, oktavian a, kawulur hs, et al. habit of cooking pork on hot stones as main risk of cysticercosis. univ med 2018;37:88-96. doi: 10.18051/univmed.2018.v37.889 6 89 univ med vol. 37 no.2 introduction cysticercosis is a parasitic infectious disease caused by the larval stage of taenia solium that can attack human tissues or organs such as the muscles, eyes, heart, lungs, and brain.(1) the sources of cysticercosis transmission are patients with t. solium taeniasis, where the stools of the patients that contai n the ta peworm eggs contaminate the environment, and if the eggs are ingested they can cause cysticercosis in pigs as well as in humans. cystic ercosis in the subcutaneous tissues can be observed by the formation of subcutaneous nodules, while in the brain the presence of t. solium cysts cause neurocysticercosis with epileptic seizures as the most frequent sign.(2,3) neurocysticercosis is a public health problem in developing countries. the presence of t. solium cysts in the brain results in a disorder of the central nervous system, causing secondary epilepsy in the maj ority of patients. neurocysticercosis has been reported as the cause of around 20-50 % of secondary epilepsy in several countries in the world. (4) the incidence of taeniasis/cysticercosis is highest in africa, southeast asia, central and south america, and europe.(3,5) in the southeast asian region research has been conducted on the prevalence of taeniasis and cysticercosis, e.g. in indonesia, the philippines, singapore, vietnam and burma. in indonesia, endemic areas of t. solium are west irian, north sumatra, bali, flores and timor.(6) however, taeniasis/cysticercosis has also been reported in lampung, j aka rta, east j ava, east nusa tenggara, west kalimantan, east kalimantan, north sulawesi, south sulawesi and southeast sulawesi.(7) taeniasis/cysticercosis in papua was initially encountered in the paniai district in 1970-1972, then spread to the regions east of the jayawijaya mountain range into baliem valley and other districts, including the districts of jayawijaya, tolikara, puncak jaya, pegunungan bintang, and the areas borderin g papua new guinea (png).(7,8) the prevalence of cysticercosis in the papua mountains is around 20.8%, while the prevalence of taeniasis is 7%.(9) humans are infected with taeniasis because of consumption of undercooked or raw t. solium larvae-containing pork, while cysticercosis occurs due to human consumption of food or water contaminated with t. solium eggs. cysticercosis may also occur from autoinfection in patients with taeniasis.(5) a study on the risk factors of taeniasis/ cysticercosis in vietnam reported that consuming undercooked or raw pork, raw vegetables or salads, and unboiled water are important positive factors for the spread of the disease, whereas hand washing with soap before meals and after defecation is important in its prevention. (10) the risk factors of taeniasis/cysticercosis transmission in africa, asia, latin america, and southeast asia are generally similar, i.e. community culture, poor environmental sanitation, low level of personal hygiene, low educational level, free-roaming pigs, consumption of undercooked or raw pork, and consumption of raw vegetables or salads.(5,10) similarly, the risk factors of taeniasis/cysticercosis transmission in papua are associated with the consumption of undercooked pork and vegetables, low educational level on hygiene, and free-roaming pigs.(11) however, there is a need for further study on the management of pork consumption. the aim of this research was to determine the risk factors that ar e important predictors of cysticercosis in humans. methods research design this was a cross-sectional research project o f t h e pap ua bi ome d i c a l re se a r c h a nd development station (balai litbang biomedis papua) that was conducted in march 2016 in 10 districts of papua province, i.e. the districts of biak numfor, paniai, nabire, deiyai, nduga, intan jaya, jayawijaya, yalimo, mamberamo pusat, and lannya jaya. the reason why these 1 0 d i s t r i ct s we r e s e l e c t e d wa s b e c a u s e historically the initial cases of taeniasis and cysticercosis were found in the paniai district, 90 then spread to the east to pegunungan bintang district, while to the west they spread to the districts of deiyai, nabire, teluk wondama and manokwari (figure 1). research subjects the research subjects were respondents above the age of 5 years from among the residents who were domiciled around pig farms. sample collection was performed by purposive sampling by considering community spots of pig farmers. the size of the rese arch sample was calculated by the central agency for statistics of papua province (badan pusat statistik provinsi papua), using the cross-sectional study equation with consideration of the design effect o f t he st u dy. ( 1 2 ) t he p r e va le n c e ( p ) of cysticercosis in papua was 20-25.6% (based on considerations of the papua province animal    (x)k x ep 1.05 x deffp1 p z n 2 2   figure 1. cysticercosis sero-epidemiologic locations and sample size by district, papua 2017. legend: 1. biak numfor (n=800), 2. nabire (n=798), 3. deiyai (n=798), 4. paniai (n=800), 5. nduga (n=797), 6. jayawijaya (n=799), 7. lannya jaya (n=800) 8. mamberamo tengah (n=800). 9. intan jaya (n=800), 10. yalimo (n=682) br e e di n g a n d he a l t h ser vi c e ( di n a s peternakan and kesehatan hewan provinsi papua), with a margin of error (e) of 20%, confidence interval of 95% (95%ci), mean h ou s e ho l d p o pu l a t i on ( x ) of 4 p e r s o ns , anticipated drop-outs of 5%, and research design effect (deff) of 2. therefore the study sample size was 800 individuals. data collection collection of demographic data and risk f a c t or s wa s p e r f o r me d by me a n s of questionnaires, comprising the respondents’ gender, education, habit of washing hands with water and soap before meals, habit of washing 91 univ med vol. 37 no.2 a b c hands with water and soap after defecation, nail hygiene, source of water for washing vegetables, consumption of raw vegetables, and habit of using hot stones for cooking pork. interviews with 5-12 year-old respondents were performed in the presence of their parents or of adults residing in the same house. laboratory assay t he d i a gno si s o f c ys ti c e rc o si s wa s established by drawing a blood sample of 4 drops (an estimated 400 µl of blood sample) from the fingertips of the respondents using whatman grade no.3 filter paper that was then dried and placed in silica gel-containing plastic bags. serologic assays of serum specimens were performed by means of magnetic microsphere bead immunoassay for detection of specific circulating antibody against the recombinant cysticercosis protein rt24h using magpix lu mi ne x equ i pme n t . ( 1 3) t he a s s a y wa s a multiplex assay using the cysticercosis protein antigen rt24h (genscript lot.# u7761bi2801) on microsphere beads according to the luminex protocol. the samples were prepared using the luminex immunoassay technique (14,15) i.e. the blood sample on filter paper was dissolved in 1 x pbs solution (sigma, cat.# p4417-100 tab); 0.3% tween-20 (sigma, cat.# p1379 100 ml) and 0.1% nano 3 (himedia, cat.# grm1038), so that the sample concentration was 1:10. a volume of 50 µl rt24h magnetic microsphere mixture (50 µl microparticles/µl in 1x pbs; 0.3% tween-20; 5% skim milk) was pipetted and placed in round-bottomed microplate wells (costar, cat.# 3792). subsequently 50 µl of the s a mpl e was a d de d t o t he w e ll s ( s a mp le concentration 1:100 in 1 x pbs; 0.3% tween20; 5% skim milk) and incubated at room temperature for 30 minutes using the microplate shaker (800 rpm). washing with pbs 1 x; 0.3% tween-20 was performed twice manually using luminex magnetic microplate washer. binding of the recombinant rt24h protein complex with specific circulating antibody was detected by adding 50 µl biotinylated mouse anti-igg4 (clone hp6025, affinity purified, southern biotech, birmingham, al, cat.number 920008) dissolved in 1 x pbs, 1% bsa, 0.05% nano 3 (concentration 1:200) then incubated for 30 minutes on the microplate shaker (800 rpm). subsequently washing was done 3 times then detection of the fluorescent color was performed by adding 50 µl r-phycoerythrin-labeled streptavidin conjugate [(invitrogen, cat.# s866) dissolved in 1x pbs-1% bsa, 0.05% (sigma ca t . # a 96 4 7-1 0 g) na n 3 s o t h a t t h e concentration was 1:250] then incubated for 30 minutes. after incubation washing was done 3 times then the mixture was suspended in 125 µl 1x pbs, 1 % bsa, 0.05% nano 3 , and the median fluorescence intensity (mfi) was read on the magpix luminex using the luminex software 4.0.(16) data validity control was performed with recombinant rt24h antigen as cysticercosis antigen standard. the data were interpreted by observing the data output of the luminex magpix in the form of mfi values and quantitative data of magnetic beads that had reacted. the sera of patients with positive cysticercosis were used as cysticercosis posit ive contro ls. t he cu t-off valu e was determined using cysticercosis-negative serum, by measuring the mean mfi and its standard deviation (sd), where eventually the cut-off value = mean mfi + 2*sd of the mfi negative standard. the serologic assay results were interpreted as positive if the mfi > cut-off value, then the cysticercosis serologic score was determined (0= negative cysticercosis, 1= positive cysticercosis) for logistic regression analysis. statistical analysis the collected questionnaire data were processed using epi data ver. 3.1, then bivariate screening analysis was done with chi-square test at significance of p<0.05, followed by logistic regression analysis to determmine the causal predictors of cysticercosis in the community at 95% confidence level and significance of p<0.05. 92 ethical clearance this research obtained ethical clearance from the health research ethics commission of badan litbangkes under no. lb.02.01/5.2/ ke.086/2016. results the laboratory test results for 7,874 serum s a mpl e s sho we d t h a t t he c yst i c e r c os i s seroprevalence in papua was 3.6%. the highest seroprevalence was found in memberamo tengah district (16.0%), followed by nduga (6.9%; 55/797), yalimo (4.0%; 27/682), nabire (2.6%; 21/798), paniai (2.4%;19/800), lannya jaya (1.6%;13/800), jayawijaya (0.9%;7/799), biak numfor (0.8%;6/800), intan jaya (0.8%; 6/800), and deiyai (0.3%; 2/798) (table 1). the relationship between risk factors and cysticercosis in the 10 districts of papua may be seen in table 2. table 2 shows that there was a significant relationship of educational level, nail hygiene, vegetable salads, use of river water for washing vegetables, and cooking pork on hot stones with the incidence of cysticercosis (p<0.05). the significant risk factors were education (p=0.041), nail hygiene (p=0.021), consumption of raw vegetables or salads (p=0.049), river as water source (p=0.000), and cooking pork on hot stones (p=0.000), which were further analyzed by logistic regression, for determining which risk factors constituted causal predictors of cysticercosis in the community. the results of the logistic regression analysis may be seen in table 3. table 3 shows that cooking pork on hot stones constituted the greatest risk factor for the occurrence of cysticercosis (or= 3.06;95% c.i. 2.19-4.28). discussion the results of this study shows that the cysticercosis seroprevalence in papua was 3.6%. this does not differ much from the results of previous studies in several districts in the central mountain range of the papua province, where the seroprevalence in puncak jaya and pegunungan bintang was 2-2.6 % and in the paniai and jayawijaya districts 20.8-29.2%.(17) on-location observation of the respondents’ hygiene still found dirty and long fingernails. the results of logistic regression analysis shows a significant relationship between nail hygiene and incidence of cysticercosis [or=2.05; 95% c.i. 1.57-2.67). these results show that dirty f i n ge r na il s c a r r y a 2. 0 5-f ol d r i s k of cysticercosis. this is due to the fact that long fingernails will readily accumulate dirt, such as soil, if children play with soil or adults work in their gardens or after defecation if they do not thoroughly wash their hands. dirt under the fingernails is difficult to remove by hand washing only without picking and removing it. this may be a risk factor for the adherence of t. solium eggs from contaminated soil, so that they can cause cysticercosis.(18) this is also supported by mwanjali et al.(19) who reported that poor nail hygiene constitutes a risk factor for cysticercosis infection. district n cysticercosis seroprevalence % biak numfor 800 6 0.8 nabire 798 21 2.6 deiyai 798 2 0.3 paniai 800 19 2.4 nduga 797 55 6.9 jayawijaya 799 7 0.9 lannya jaya 800 13 1.6 mamberamo tengah 800 128 16 intan jaya 800 6 0.8 yalimo 682 27 4.0 total 7,874 284 3.6 table 1. cysticercosis seroprevalence by districts in papua 93 univ med vol. 37 no.2 risk factors cysticercosis serology total p valueseropositive seronegative n % n % n % gender male female 130 154 3.6 3.6 3 493 4 097 96.4 96.4 3 623 4 251 100 100 0.935 education low (no schooling, elementary school) middle to high (junior high school, senior high school, d1, d2, d3, s1) 200 84 3.9 3.0 4 898 2 692 96.1 97.0 5 098 2 776 100 100 0.041* hand washing with water and soap before meals no yes 156 128 3.6 3.7 4 230 3 359 96.4 96.3 4 386 3 487 100 100 0.788 hand washing after defecation no yes 162 122 3.6 3.6 4 342 3 247 96.4 96.4 4 504 3 369 100 100 0.954 nail hygiene not clean clean 184 100 3.3 4.4 2 192 5 398 96.7 95.6 5 582 2 292 100 100 0.021* vegetables made into salads or consumed raw no yes 270 14 3.7 2.2 6 964 620 96.3 97.8 7 234 634 100 100 0.049* river as source of water no yes 232 52 3.2 7.6 6 960 630 96.8 92.4 7 192 682 100 100 0.000* cooking pork on hot stones no yes 49 235 1.8 4.6 2 677 4 913 98.2 95.4 2 726 5 148 100 100 0.000* table 2. relationship between several risk factors and cysticercosis *p with chi-square test risk factors β p odds ratio 95% confidence interval cooking pork on hot stones (1) 1.12 0.000 3.06 2.19 4.28 vegetables made into salads or consumed raw (1) -0.64 0.022 0.53 0.30 0.91 river as water source (1) 0.65 0.000 1.92 1.39 2.64 nail hygiene (1) 0.72 0.000 2.05 1.57 2.67 table 3. results of multiple logistic regression analysis of risk factors correlated with incidence of cysticercosis the r esults of our study show that consumption of raw vegetables or salads was a risk factor for cysticercosis. similar results were obtained in a laotian study where consumption of uncooked vegetables was a risk factor for cysticercosis.(20) a study by sah et al.(21) found that the use of river water for consumption was significantly associated with incidence of cysticercosis. a study by van de et al.(22) in vietnam reported that the sources of cysticercosis transmission are consumption of raw vegetables and fresh fruit, half-cooked pork, and unboiled drinking water. unprotected river streams or water wells may become contaminated with t.solium eggs,(23) which can survive in water for long periods of time so becoming a source of 94 cysticercosis transmission in pigs and humans.(1,5) different results were obtained by nkouawa et al.(24) who reported that there was no significant relationship of the use of river water and vegetable consumption with cysticercosis. the cooking of pork on hot stones still allows the tapeworm larvae to survive, so that they can cause taeniasis and indirectly constitute a factor in the transmission of cysticercosis if humans consume the half-cooked and taeniasis-infected pork. patients with taeniasis may become sources of cysticercosis tra nsmission and inf ect themselves (autoinfection) as well as pigs and humans in their neighborhood.(25) the results of our study show that the risk factor of cooking pork on hot stones has a significant relationship with the prevalence of cysticercosis. this may be because the vegetables and roots that are cooked together with the pork are contaminated with tapeworm eggs from the soil around the cooking site.(28) consumption of raw or halfcooked t. solium larvae-containing pork is an indirect factor, since it causes taeniasis, which results in contamination of the environment with t.solium eggs, but causes cysticercosis upon autoinfection. this is supported by the study of khaing et al.(23) who reported that the risk factors of cysticercosis transmission are among others community cooking resulting in consumption of half-cooked or raw meat. a limitation of this study is its crosssectional design so that it cannot evaluate any cause-and-effect relationships. the collection of the samples was performed on respondents residing near pig farming communities, so that there is a high probability that local respondents residing far from district cities were not included. inter view bias ma y occur fr om the large community gatherings at the time of visit to the respondents. the implication of this study is that there are still cases of taeniasis and cysticercosis to be found in papua, indicating that there are still neglected diseases in indonesia, so requiring cross-sectoral management for the prevention and control of disease transmission. the use of the magnetic microbead immunological assay is of great help in doing surveys with large samples, so saving time and expenditure. cysticercosis studies or surveys should not only be conducted in humans but also in pigs for early identification of porcine cysticercosis to obtain more epidemiologic data for this disease. the magnetic microbead immunoassay with recombinant rt24h protein for the detection of serum cysticercosis antibodies in pigs should also be performed. this method requires only small a mou n t s o f se r um so si mp l if yi n g t he management and transportation of the samples from distant locations. conclusion this study demonstrated that cooking pork on hot stones was the main risk factor for cysticercosis. the main problem for control of cysticercosis lies in reducing the risk factors. acknowledgments the authors extend their heartfelt gratitude to the heads of badan litbangkes ri, dr. sukwan hamdali, phd, dra. rita m dewi, mkes, and dr. masri s maha, the heads of balai litbang biomedis papua, limnas papua, provincial health ser vice (dinas kese hatan prov in si) and distr ict he alth services in papua, the heads of all puskesmas, community leaders/village heads (kepala kampung) and all community members of papua province who participated in the study. conflict of interest there is no conflict of interest in this study contributors ss and ao 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research and development for biomedicine papua, ministry of health **microbiology division, faculty of medicine, gadjah mada university ***biomedical and health basic technology, jakarta, ministry of health, republic of indonesia correspondence mirna widiyanti institute of health research and development for biomedicine papua, ministry of health jln. kesehatan no.10 dok ii jayapura papua phone: +62967-534389 fax : +62967-534697 email: ninawidhy@gmail.com univ med 2014;33:49-57 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background human immunodeficiency virus (hiv) is a retrovirus that infects human immune system. hiv is genetically differentiated into hiv-1 and hiv-2. hiv1 is classified into 3 groups : m,n and o. m group is divided into 9 subtypes, namely : a, b, c, d, f, g, h, j and k. hiv subtype mapping would become a good reference to study the epidemiology of hiv-1. hiv phylogenetic analysis required to understand virus origin, geographical distribution and tracking parameter of hiv transmission process in sample area. the objective of this study was to determine hiv-1 subtype distribution and the relationship between hiv-1 subtypes from with genbank hiv isolate based on envelope gene fragment. methods a cross setional design was conducted involving 36 subjects in jayapura between january to march 2013. blood samples were taken and analyzed using reverse transcription polymerase chain reaction (rt-pcr), nested pcr and confirmed with sequencing. phylogenetic analysis performed along 400 bp of envelope gene fragment. the data were analyzed with bioedit ver.7 and mega 5.1. software. result two types of hiv-1 subtype were found in jayapura regency. crf01_ae was confirmed in 31 patients (86.1%) and subtype b in 5 patients (13.9%). phylogenetic analysis showed phylogenetic relationship between crf01_ae from jayapura with subtype from singapore and thailand, while subtype b from jayapura were closely related with subtype from united states and united kingdom conclusion this study reflect the independent introduction of multiple hiv-1 strains into papua, with the rapid spread in the majority of infected patients tested of single strain of hiv-1 (crf01_ae). keywords: hiv-1 subtype, phylogenetic analysis, crf01_ae, subtype b, papua subtypes and phylogenetic analysis of human immunodeficiency virus-1 in jayapura mirna widiyanti*, tri wibawa**, and holy arif wibowo*** 50 widiyanti, wibawa, wibowo phylogenetic of human immunodeficiency virus-1 analisis subtipe dan filogenetik human immunodeficiency virus-1 di jayapura latar belakang human immunodeficiency virus (hiv) adalah retrovirus yang menginfeksi sistem kekebalan tubuh manusia. secara genetik hiv dibedakan menjadi hiv-1 dan hiv-2. hiv-1 diklasifikasikan kedalam 3 grup yaitu : m, n, dan o. grup m dibagi menjadi 9 subtipe yaitu: a, b, c, d, f, g, h, j dan k. pemetaan subtipe dapat menjadi rujukan dalam perkembangan epidemi hiv-1. analisis filogenetik juga diperlukan untuk mengetahui asal-usul virus, distribusi geografis dan parameter pelacakan proses transmisi hiv pada area sampel. penelitian ini bertujuan untuk menentukan distribusi subtipe hiv-1 dan hubungan kekerabatan isolat hiv-1 jayapura dengan isolat hiv genbank berdasarkan fragmen gen envelope metode sebuah rancangan potong lintang (cross sectional) dilakukan pada 36 subjek di jayapura antara bulan januarimaret 2013. sampel darah dianalisis menggunakan teknik reverse transcription polymerase chain reaction (rtpcr), nested pcr dan dikonfirmasi dengan sekuensing. analisis flogenetik dilakukan berdasarkan fragmen gen sampul (envelope) sepanjang 400 pasangan basa (bp). hasil dianalisis dengan program bioedit dan mega 5.1. hasil dua subtipe ditemukan di kabupaten jayapura. 31 pasien (86.1%) crf01_ae dan 5 pasien (13.9%) subtipe b. analisis filogenetik menunjukkan hubungan antara crf01_ae jayapura dengan singapura dan thailand, sedangkan subtipe b dari jayapura memiliki hubungan dengan isolat dari amerika dan inggris. kesimpulan penelitian ini merefleksikan introduksi independen beberapa strain hiv-1 ke papua, melalui penyebaran cepat dengan mayoritas pasien yang terinfeksi strain hiv-1 adalah crf01_ae. kata kunci : subtipe hiv-1, filogenetik, crf01_ae, subtipe b, papua abstrak introduction acquired immunodeficiency syndrome (aids) is a collection of symptoms caused by infection with the human immunodeficiency virus (hiv). this retrovirus attacks and destroys thelper (cd4+) lymphocytes, causing the immune system of patient to weaken and become more s u s c e p t i b l e t o v a r i o u s i n f e c t i o n s a n d malignancies.(1) there are two types of hiv that are genetically differentiated, hiv-1 and hiv2.(2) hiv-1 is a common virus found in all parts of the world (africa, america, europe and asia).(3) hiv-1 is classified into three major phylogenetic groups: m, n and o.(4-6) group m is subdivided phylogenetically into nine subtypes: a, b, c, d, f, g, h, j and k.(7-8) in addition, 48 new subtypes of hybrid virus called circulating recombinant forms (crf) have been found in the world, including crf01_ae, crf02_ag and crf03_ab.(9) the crf01_ae subtype is distributed widely in southeast asia.(10) hiv-1 subtype variation could be a potential problem for public health in terms of the 51 transmission and prevention strategies of hiv/ aids. hiv subtype is an important molecular marker to monitor the changes in the hiv epidemic in the world.(11) hiv-1 subtypes have different distributions between one region to another, where geographical factors can affect the subtypes differences. in indonesia, the number of cases of hiv/ aids continually increases every year. a total of 103,759 of hiv and 43,347 of aids cases were recorded at the end of march 2013.(12) papua is the third highest-ranking province for hiv/aids, with 10,881 hiv and 7795 aids cases being found.(13) research in jakarta and east java showed that crf01_ae subtype transmission is limited to syringe sharing among drug users and promiscuous sexual intercouse of the heterosexual population.(14) papua is one of the provinces with the highest number of hiv/aids cases compared with other regions in indonesia. the cases may be increasing, since the regional expansion in papua allows mobilization of the people in great numbers. this study was conducted in papua because there were no research data related to hiv subtype distribution and only limited information regarding genetic characteritics of the virus. jayapura is one of the regencies in papua with hiv/aids cases. in jayapura regency there were reportedly 936 people consisting of 537 women and 399 men who were hiv/aids positive, and among these there were 150 deaths. there have been no reports on hiv-1 subtype d i s t r i b u t i o n a n d i t s r e l a t i o n s h i p w i t h demographic characteristics and risk factors in jayapura. therefore, it was considered important to determine the subtypes of hiv-1 and their relationship with demographic characteristics and risk factors in jayapura regency in order to obtain basic data in hiv-1 subtype mapping, as a reference standard in hiv epidemiology and clinical applications. hiv-1 subtype is associated with clinical manifestations and antiretroviral resistance. identifying the association between hiv-1 subtype and clinical therapy can assist clinicans in improving treatment strategies for hiv infection.(15) furthermore, hiv phylogenetic analysis is also needed for determining the origin of the virus and its geographical distribution, and f o r t r a c k i n g p r o c e s s p a r a m e t e r s o f h i v transmission in the sample area. the objective of the present study was to determine hiv-1 subtype distribution and the relationship between hiv-1 subtypes and genbank hiv isolates based on envelope gene fragment, to obtain basic data in hiv-1 subtype mapping as a reference standard in hiv epidemiology and clinical application. methods research design an analytic cross-sectional study was conducted on hiv/aids patient undergoing routine care at yowari hospital vct unit, from january to march 2013. research subjects a total of 36 hiv/aids patients were selected based on consecutive sampling among those patients meeting the inclusion criteria. included in the study were men and women with hiv/aids diagnosed by 2 laboratory tests (rapid test and elisa), after the subjects agreed to participate in the study with signed informed consent. phylogenetic analysis blood serum samples of the 36 patients were collected after written informed consent. laboratory tests included rna extraction, reverse transcription polymerase chain reaction (rt-pcr), nested pcr, electrophoresis, and the results were confirmed by sequencing. hiv rna was extracted from serum and amplified using two pairs of primer. the primers used in rtpcr were: tcttaggagcagcaggaagca ctatggg as gp 40 forward primer, aacga caaaggtgagtatccctgcctaa as gp41 reverse primer: acaattattgttgg tatagt gcaacca as gp46 forward primer, and univ med vol. 33 no.1 52 widiyanti, wibawa, wibowo phylogenetic of human immunodeficiency virus-1 variable subtype total a e b age <35 year 24 5 29 >35 year 7 0 7 sex male 17 1 18 female 14 4 18 tribe papua 29 5 34 non-papua 2 0 2 occupat ion unemployed 12 0 12 goverment officer 5 0 5 farmer 9 2 11 student 2 2 4 la bour 3 1 4 table 1. subtype and demographic variable at yowari’s hospital ttaaacctatcaagctcctactatcata as gp47 reverse primer.(14) to determine hiv-1 subtype, the hiv-1 gene region encoding glycoprotein 41 (gp41) on the viral envelope was amplified. region 41 is a reliable marker for h i v1 s u b t y p e g r o u p i n g . ( 1 6 ) n e s t e d p c r products were confirmed by electrophoresis and sequencing. sequencing results were compared with the hiv-1 sequences available in genbank and analyzed with blast. phylogenetic relationships between hiv-1 isolates were analyzed with molecular evolutionary genetic analysis (mega) version 5.1. software and bioedit ver.7. the results were then presented in a phylogenetic tree. rna extraction, rt-pcr and sequencing were performed at the national institute of health research and development, ministry of health. sequencing results were analyzed using bioedit software. the initial stages performed during the analysis process were trimming (cutting) unreadable sequencing results, and combining readable forward and reverse primer sequences for confirmation. the next stage was the basic local alignment search tool (blast) process, of which the goal is to obtain the hiv virus genotypes and subtypes. blast was performed by using the internet at two genbank sites for confirmation, i.e. national center for biotechnology information (ncbi) blast at www.ncbi.nlm.nih.gov and hiv sequence database at www.hiv.lanl.gov. data analysis the results were analyzed descriptively. nucleotide sequences of hiv sequencing results were then compared with the hiv virus isolates from the united states, canada, thailand, china, vietnam, singapore, accessible via genebank. phylogenetic analysis was performed to determine the phylogenetic relationship between hiv isolates from jayapura district and isolates from genbank by the joining neighboor analysis method using the mega version 5.1. program. the phylogenetic relationship was then visualized with the njplot program. ethical clearance ethical approval was obtained from medical and health research ethics commitee (mhrec), faculty of medicine, gadjah mada university. results hiv-1 subtype distribution at yowari hospital is shown in table 1. among the 36 subjects in this study, two subtypes were found, namely subtype ae in 31 patients (86.1%) and s u b t y p e b i n 5 p a t i e n t s ( 1 3 . 9 % ) . t h e phylogenetic relationship diagram between subtype b isolates from jayapura and references isolates from genbank is shown in figure 1. the p h y l o g e n e t i c t r e e o f j a y a p u r a s u b t y p e crf01_ae isolates and genbank isolates can be seen in figure 2. discussion the preponderance of subtype crf01_ae in jayapura in comparison with subtype b is similar to findings of previous studies in jakarta and east java.(14) in addition, the study conducted by foley et al.(17) in timika (papua) on 16 hiv 53 figure 1. phylogenetic relationship between indonesian origin hiv subtype b and hiv subtype b reference isolate from other countries with neigbour joining analysis from mega 5.1 software patients also found a higher prevalence of subtype crf01_ae (87.5%) in comparison with s u b t y p e b ( 1 2 . 5 % ) . c r f 0 1 _ a e i s predominantly found in southeast asia and is widespread in thailand and cambodia, where the number of persons with hiv is as high as one million.(11) crf01_ae has also been reported in china, vietnam, malaysia, myanmar, laos, taiwan, philippines and singapore.(19) historically, subtype b was generally found in western europe, north america, latin america, japan, australia, and new zealand. the molecular subtype b in this study was included in the american cluster sequences, because of its close genetical relationship to the latter. this had also been found in previous studies on subtype b sequences from indonesia. the global distribution of hiv-1 subtype is more related to social change, the economy, immigration and international travel compared with differences in the nature or the power transmission of the v i r u s . i n a d d i t i o n t o t h e a b o v e f a c t o r s , geographical factors and population mobility also play an important role in the spread and genetic diversity of hiv-1. (18) the geographically strategic position of jayapura has led to a high vulnerability of its residents to hiv infection. the mobility of local residents, non-indigenous persons, and sex workers from timika to jayapura is one factor in the transmission chain, including the possibility of transmission of subtypes crf01_ae and b. results from blast data on subtype b isolates in jayapura showed a similarity with isolates from the usa and the united kingdom. subtype b isolated from jayapura shares the same roots with both isolates from usa and united kingdom, as shown in figure 1. subtype b isolates from jayapura and the reference isolates were divided into two clusters. cluster 1 was occupied by the usa isolates and isolates from jayapura. isolate number 18 had the same root as the us isolates, whereas isolates 15 and 5 were in one cluster with us isolates but separate from the reference isolates and formed their own branch of the cluster. cluster 2 was occupied by the uk isolates and isolates from jayapura, where isolates 28 and 10 had the same roots as the uk isolates. the phylogenetic relationship between subtype b isolates from jayapura and the genbank reference isolates is shown in the phylogenetic tree analyzed with neighbour joining.(19,20) based on data from blast, sequenced crf01_ae isolates from jayapura has a similarity with sequenced isolates from univ med vol. 33 no.1 54 widiyanti, wibawa, wibowo phylogenetic of human immunodeficiency virus-1 figure 2. phylogenetic relationship between indonesian origin hiv subtype crf01_ae and hiv subtype crf01_ae reference isolate from other countries with neigbour joining analysis from mega 5.1 software thailand (genbank access code: jn388200, a f 0 0 7 0 7 0 3 , ay 9 6 1 5 9 0 , g q 9 1 6 5 7 0 a n d jn388190), china (genbank access code: g u 4 7 5 0 3 6 , h m 2 1 5 4 1 9 , h q 6 9 9 9 3 , gu475020), vietnam (genbank access code: fj185244, fj185248, fj185252, hq245652), singapore (genbank access code: hq539264, h q 5 3 9 3 2 7 , h q 5 3 9 3 3 2 , h q 5 3 9 3 11 ) a n d cambodia (genbank access code: dq518410). sequenced isolates from these five countries were subsequently used as reference isolate. from the phylogenetic tree (figure 2) it is apparent that the hiv-1 subtypes ae are divided into several clusters, with the indonesian subtype ae originating from jayapura occupying the same roots as isolates from singapore, thailand, vietnam and china. indonesian isolates and genbank original isolates were divided into several clusters. cluster 1 was occupied by isolates from indonesia, singapore and thailand. the first branch was occupied by indonesian and 55 singaporean isolates, three indonesian isolates (number 9, 33 and 31) were grouped into one single root, while number 7 isolate was in the same root as reference isolates from singapore. the second branch was occupied by singaporean and indonesian isolates, with isolates number 3 and 25 being in the same roots as isolates from singapore. the third, fourth, fifth and sixth branches were occupied by isolates from indonesia (number 4, 27, 36, 19, 21, 13, and 34), which were separated from the reference isolates and formed new branches. the seventh branch was occupied by thai and indonesian isolates, with isolates 8, 1, and 11 occupying the same roots as the thai isolates. the eighth branch was occupied by isolates from indonesia (number 22, 23, 32) which were separate from the reference isolates and formed their own branch in the cluster. cluster 2 was occupied by isolates from indonesia, vietnam and china. the first branch was occupied by indonesian isolate number 16 and 35, with both isolates were separate from the reference isolates. the second branch was occupied by isolates from china and vietnam, while the third branch was occupied by vietnamese and indonesian isolates, where isolate number 12 was in one root with the isolates from vietnam. cluster 3 was occupied by isolates from thailand, singapore, vietnam, china and indonesia. indonesian isolate number 24 was in one root with the isolates from china. cluster 4 was occupied by isolates from indonesia, where isolates number 6, 29, 2, 14, 26 and 30 were separate from the reference isolates and formed a new cluster. the next clusters were occupied by isolates from china, cambodia and indonesia, which were individually separate in their own cluster. furthermore, all isolates (originating from indonesia, thailand, china, singapore, vietnam and cambodia) showed significant genetic distances against hiv-1 nc_001802 complete genome isolate. thus, these results might explain the possibility that hiv-1 nc_001802 isolate is not the ancestor of the isolates from indonesia and other southeast asian countries. ncbi blast molecular analysis showed a close relationship between the sequences of crf01_ae from jayapura regency with sequences from southeast asian countries (thailand, vietnam, cambodia and singapore). from this point of view it is suggested that subtype crf01_ae in jayapura came from southeast asia countries (singapore and or other surrounding countries). the transmission may have been the result of intensive interactions between peoples in southeast asia.(14) the introduction of the virus into high-risk groups c o u l d q u i c k l y s p r e a d t h e d i s e a s e t o t h e surrounding areas. phylogenetic analysis studies i n ti m i k a ( p a p u a ) s h o w e d t h a t s u b t y p e crf01_ae from timika has a close genetic relationship with viruses from thailand, and that subtype b from timika has a close relationship with american and european isolates.(17) phylogenetic analysis showed that hiv-1 isolates in jayapura regency were both locally transmitted viruses and viruses imported from other regions. hiv-1 viral isolates from jayapura were not spread uniformly, which means that the viral isolates derived from imported as well as local viruses. imported viruses would be randomly distributed among the reference isolates. however, in the phylogenetic tree a larger monophyletic group of local transmission was seen to appear, suggesting a more local transmission from a single source of infection, so that the subtypes obtained from the samples were almost entirely crf01_ae. due to the dynamic nature of hiv epidemics, it is suggested to conduct periodic hiv subtype studies, so as to update the information for program planning in education, prevention, medication, and vaccine manufacture. conclusions this study found that there were two hiv1 subtypes in jayapura, subtype crf01_ae in the majority of samples and subtype b in a univ med vol. 33 no.1 56 widiyanti, wibawa, wibowo phylogenetic of human immunodeficiency virus-1 minority. phylogenetic analysis showed a phylogenetic relationship between subtype crf01_ae from jayapura and subtypes from singapore and thailand, while subtype b from jayapura was closely related to subtypes from the united states and the united kingdom. cultural norms for sexual behavior within papuan society also need to be explored in order to understand which educational programs promoting behavior change are most warranted. acknowledgements the authors gratefully acknowledge their indebtedness to nicodemus barends, jetty kalembang and yowari vct clinic staff for helping and supporting this study, and to lidwina salim, vivi setyawati and colleagues at the prof. oemijati national laboratory, national institute of health research and development, ministry of health, for their help and support of this project, without which this work would not have been possible. references 1. grant i, sacktor n, mcarthur j. hiv neurocognitive disorders. in: gendelman he, grant i, everall i, lipton sa, swindells s, editors. the neurology of aids. 2nd ed. london: oxford university press;2005.p.357-70. 2. buonaguro l, tornesello ml, buonaguro fm. human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications. j virol 2007;81:10209-19. 3. world health organization united nations aids. hiv prevalence map: a global view of hiv infection;2010. available at: http://www. unaids.org. accessed february 25, 2014. 4. abecasis ab, annemarie mw, dimitri p, jurgen v. hiv-1 subtype distribution and its demographic determinants in newly diagnosed patients in europe suggest highly compartementalized epidemic. retrovirology 2013;10:1-13. 5. yebra g, mulder, holguin a. description of hiv1 group m molecular epidemiology and drug resistance prevalence in equatorial guinea from migrants in spain. plos one 2013;8:e64293. 6. djoko df, rimoin aw, vidal n, tamoufe u, wolfe nd, butel c, et al. high hiv-1 type 1 group m pol diversity and low rate of antiretroviral resistance mutations among the uniformed services in kinshasa democratic republic of the congo. aids res hum retroviruses 2011;27:323-9. 7. chaplin b, elisen g, idoko j, onwujekwa d. impact of hiv type-1 subtype on drug resistance mutation in nigeria patients failing first-line theraphy. aids res hum retroviruses 2011;27: 71-80. 8. buonaguro l, tagliamonte m, tornesello ml, buonaguro fm. genetic and phylogenetic evolution of hiv-1 in a low subtype heterogenecity epidemic: the italian example. retrovirology 2007;4:341-9. 9. los alamos national laboratory. hiv circulating recombinant forms (crfs); 2012. available at: http://www.hiv.lanl.gov/content/ sequence/hiv/crfs/crfs.html. accessed july 2, 2012. 10. boonchawalit b, jullaksorn d, uttiyong j, yowang a. molecular evolution of hiv-1 crf01_ae env in thai patients. plos one 2011; 6:1-11. 11. srisuphanut m, sukeepaisarnchaeron w, kucherer c, pauli g. epidemiology of hiv-1 subtypes in infected patients from northeastern thailand. southeast asian j tropmed public health 2004;35:641-6. 12. direktorat jenderal pemberantasan penyakit menular dan penyehatan lingkungan. statistik kasus hiv/aids di indonesia. jakarta: kementerian kesehatan ri.;2013. 13. dinas kesehatan provinsi papua. statistik kasus hiv/aids di papua. jayapura: bidang pemberantasan penyakit menular; 2012. 14. wibowo ha, setyawaty v, salwati e. epidemiologi molekuler genotipe hiv-1 pada odha di jawa timur dan dki jakarta. buletin penelitian kesehatan 2011;39:1-9. 15. santoro mm, perno cf. hiv-1 genetic variability and clinical implication. isrn microbiology 2013. article id 481314, 20 pages. doi.org/10. 1155/2013/481314. 16. zhang h, hoffman f, he j, he x, kankasa c, west jt, et al. characterization of hiv-1 subtype c envelope glycoproteins from perinatally infected childrens with different courses of disease. retrovirology 2006;3:1-15. 17. foley b. donegan e, silitonga n, wignall f, busch m, delwart e. importation of multiple hiv type 1 strains into west papua indonesia (irian jaya). aids res hum retroviruses 2001; 17:1655-9. 57 18. gifford rj. oliveira t, rambat a, pybus og, dunn d, vandamme am, et al. phylogenetic surveillance of viral genetic diversity and the envolving molecular epidemiology of hiv type 1. j virol 2007;81:13050-6. 19. michu e. a short to phylogeny reconstruction. plant soil environ 2007;53:442-6. 20. harrison cj, langdale ja. techniques for molecular analysis: a step by step guide to phylogeny reconstruction. plant j 2006;45:56172. univ med vol. 33 no.1 formula jamu antihipertensi and captopril are 81 *department of pharmacology, faculty of medicine, sultan agung islamic university **department of physiology, faculty of medicine, sultan agung islamic university ***department of histology, faculty of medicine, sultan agung islamic university †department of anatomy, faculty of medicine, sultan agung islamic university ‡department of clinical pathology, faculty of medicine, sultan agung islamic university correspondence : dr. atina hussaana, msi. apt. department of pharmacology, faculty of medicine, sultan agung islamic university jl.raya kaligawe km.4, semarang, jawa tengah email: atinahussaana@unissula.ac.id univ med 2016;35:81-8 doi: 10.18051/univmed.2016.v35.81-88 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 formula jamu antihipertensi and captopril are equally effective in patients with hypertension atina hussaana*, hadi sarosa**, ulfah dian indrayani***, chodidjah†, bagas widiyanto*, and danis pertiwi‡ background hypertension is the main cause of morbidity and mortality associated with cardiovascular diseases. many herbs/spices appear to have significant effects in favorably modulating high blood pressure. a jamu formulation containing 6 plant extracts, formula jamu antihipertensi (fja), has been described previously. this research study aimed to evaluate the effect of fja as antihypertensive agent in mild to moderate hypertensive patients. methods this double-blind experimental study was conducted in 40 hypertensive patients, who were randomized into two groups. the first group was treated with oral administration of 2 g fja and the second group 25 mg captopril daily for 4 weeks. systolic and diastolic blood pressure as well as liver and kidney function were followed up every week. independentt test and two-way anova were used to analyze the data with a level of significance of 0.05. results the results showed that after the treatment, fja and captopril were capable of significantly decreasing systolic and diastolic blood pressure (p <0.05). the decrease in systolic blood pressure between the two groups was identical (p>0.05), while the decrease in diastolic blood pressure was greater in the fja group than in the captopril group (p <0.05). conclusion from this study it may be concluded that administration of fja for 4 weeks is equally effective as captopril in reducing systolic and diastolic blood pressure in patients with mild and moderate hypertension. thus, natural plants and herbs can be our source of drugs, with fewer side effects and better bioavailability for treatment of hypertension in the future. keywords: jamu, antihypertensives, captopril, hypertension patients doi: http://dx.doi.org/10.18051/univmed.2016.v35.81-88 82 hussaana, sarosa, indrayani, et al jamu antihipertensi in hypertension introduction efforts for the development and utilization of medicinal plants or traditional medications to treat various disorders, particularly degenerative diseases, is continuously attracting attention from the government, educational institutions, investigators, businessmen, and the community. the government, in this case the ministry of health, has issued guidelines for the utilization of herbal medications (jamu) at community healthcare facilities, namely that they shall meet the requirements of being safe, beneficial, and standardized. in addition, by minister of health regulation (permenkes) no. 003/menkes/per/ i/2010,(1) the government provided a solution for the acceleration of jamu utilization in formal healthcare services through saintifikasi jamu (jamu “scientification”) programs, which are healthcare-based clinical observational studies to provide scientific evidence for the properties of jamu. in spite of this, the results from those varied programs have not yet been capable of reaching the expected goals, namely the acceptance and use of traditional medications in formal healthcare services. several factors are acting as constraints, such as the lack of scientific evidence in support of the efficacy and safety of the use of traditional medications. the use of herbal medicine as a treatment modality has significantly increased over the last decade.(2) this is due to several factors, the principal of which is that herbal medicine is a cheaper alternative with fewer undesired side effects.(3) however, the increased desire to use herbal treatment is not a reflection of the economic status of an individual from a certain region or a country. indeed, 70% of the population in developed nations have resorted to complementary and alternative medicine (cam) for treatment purposes, and herbal medicine forms a large proportion of its application.(4) as one of the efforts to overcome abovementioned constraints, a preliminary study was conducted to develop the formula jamu antihipertensi (fja) [antihypertension herbal formulation] consisting of 6 medicinal plant extracts. the fja comprises three blood pressure reducing components, i.e. the leaves of apium graveolens l leaves (celery),(5,6) orthosiphon aristatus (java tea, cat’s whiskers, kumis kucing) (7) and centella asiatica (asiatic pennywort), (8,9) and three components for improving physical fitness, i.e. the leaves of phyllanthus niruri l. (meniran) (10) and the rhizomes of temu lawak (curcuma xanthorrhiza roxb.) and turmeric (curcuma domestica val.). these six herbal components have been recommended by the national commission for scientification of jamu (komisi nasional saintifikasi jamu).(11) the herbal formulation using these six simplicia have been scientifically tested as to their clinical antihypertensive benefits.(12) one study on hypertensive patients receiving antihypertension jamu, was capable of reducing systolic and diastolic blood pressure by 12.67% and 2.33%, respectively, after 7 days of use.(13) from the results indicating the potential of fja composed of the above six plant extracts, it was deemed necessary to conduct a further clinical study to determine the efficacy of fja against patients with mild to moderate hypertension. methods design of the study this experimental study was designed as a double blind, randomized controlled trial. oral fja treatment was administered to patients with mild to moderate hypertension for one month, and compared with captopril. this study was carried out at the faculty of medicine, sultan agung islamic university, semarang, and the sultan agung hospital, semarang, from 2014 to 2015. study sample patients with mild to moderate hypertension were recruited from the environs of east semarang. the minmal sample size was calculated with the following formula: ( ) ( ) 7.8 10 1.1196.196.1 2 xx sdzβzα 2 21 =      +=      − + 83 in this study, the sample size was taken as 40 persons meeting these inclusion criteria: diagnosed with mild to moderate hypertension (systolic blood pressure = 140-180 mmhg, diastolic blood pressure = 90-109 mmhg), not consuming other drugs or herbals for study and clinical observation, and not suffering from other diseases. exclusion criteria: having dyslipidemia and /or metabolic disorders. interventions the formula jamu antihipertensi (fja) used in this study was made in capsule form. the fja capsules consisted of extracts of apium graveolens l, orthosiphon aristatus, centella asiatica, phyllanthus niruri l., curcuma xanthorrhiza roxb. (temu lawak) and curcuma domestica val.(kunyit) rhizomes. the extracts used were standardized extracts of each of the plants, obtained from pt industri jamu borobudur, semarang. the standard hypertensive drug used as control was captopril 25 mg in tablet form, which were then made into capsules. administration of treatments patients were block-randomized into two groups, i.e. one group receiving a dose of 2 g fja and the second group receiving 25 mg captopril daily. the treatment was administered daily in divided doses for 1 month. to ensure blinding of the treatment, the shape, color, size, and odor of the fja and captopril capsules were made identical and placed in identical packaging containers with identical labeling. the treatment codes were applied to the packaging containers at a location not easily seen by the patients. the coding of the packaging containers, safekeeping of the code, and the random allocations were carried out by an investigator who did not examine the patients. blood pressure measurements blood pressure measurements in this study were performed before treatment and at the end of each week, using a mercury sphygmomanometer. the blood pressure of each patient was measured by three physicians, from whose results the mean blood pressure was calculated. measurement of serum sgpt, sgot, urea and creatinine concentrations to ensure blinding, the hepatic and renal function tests were done at the sarana medika clinical laboratory, semarang. the urea concentration was determined using 2 µl serum kwith the addition of 200 µl bun reagent (ureakit s 180) and incubated for 15 minutes. absorbance was read at a wave length of 340 nm. the normal urea concentration was taken as 7-21 mg/dl. t h e c r e a t i n i n e c o n c e n t r a t i o n w a s determined using 17 µl serum with the addition of 150 µl creatinine reagent (creatinine liquicolor, human) and incubated for 3 minutes. absorbance was read at a wave length of 500 nm. the normal creatinine concentration was taken as 0.5-1.3 mg/dl. the serum glutamic pyruvic transaminase (sgpt) concentration was determined using 20 µl serum with the addition of 200 µl sgpt reagent (alat (gpt) fs, diasys diagnostic systems, germany) and incubated for 10 minutes. absorbance was read at a wave length of 340 nm. the normal sgpt concentration was taken as <31 u/l. the serum glutamic oxaloacetic transaminase (sgot) concentration was determined using 20 µl serum with the addition of 200 µl sgot reagent (asat ( g o t ) f s , d i a s y s d i a g n o s t i c s y s t e m s , germany) and incubated for 10 minutes. absorbance was read at a wave length of 340 nm. the normal sgpt concentration was taken as <32 u/l. ethical clearance this study was conducted after obtaining ethical clearance from the ethics committee, faculty of medicine, sultan agung islamic university. statistical analysis the independent t-test was used to draw conclusions as to mean differences in blood pressure between both treatment groups at univ med vol. 35 no.2 84 hussaana, sarosa, indrayani, et al jamu antihipertensi in hypertension baseline and at the end of the treatment. a twoway anova was used to compare the mean differences of blood pressure between the treatment groups and duration of intervention. the significance level was fixed at 5% level. results forty patients with mild to moderate hypertension were involved in this study. during a follow-up period of 4 weeks, there were 6 dropouts for various reasons, such as typhoid fever necessitating ingestion of 5 medications, psychological reasons, and non-compliance in taking the treatment capsules. the flow of participants from recruitment until analysis is shown in figure 1. of the 34 patients with mild to moderate hypertension participating in this study up to its completion, their characteristics in the two treatment groups are summarized in table 1. table 1 shows that among the patients who participated in this study, the groups receiving the fja and captopril interventions were dominated by female patients aged 50 years or above. there were no significant differences between the patients in the fja group and the captopril group. this indicates that the randomization was successful in distributing the other variables between the two treatment groups, apart from the treatment variables. the results of systolic and diastolic blood pressure measurements of the participating patients with mild baseline hypertension during the first three weeks of follow-up and at the completion of the treatment in the fourth week, are illustrated in figure 2. mean systolic and diastolic blood pressure of the participants at the start of the study (baseline) did not differ between the fja and captopril treatment groups. both interventions equally reduced the systolic and diastolic blood pressure. after receiving the interventions for 4 weeks, both treatment groups showed significant differences in systolic and diastolic blood pressure (p<0.05) (figure 2). the two-way anova results indicated that there was no significant interaction between treatment groups and duration of treatment for systolic blood pressure (p=0.066). however, there was a significant interaction between figure 1. flow of participants in formula jamu antihipertensi clinical trial 85 treatment groups and duration of treatment for d i a s t o l i c b l o o d p r e s s u r e ( p = 0 . 0 1 5 ) . t h e significant difference was between 1 week versus 2 weeks of treatment (p=0.006) and between 1 week versus 3 weeks of treatment (p=0.000) (table 2). there was no difference in mean serum sgot, sgpt, urea and creatinine concentrations at baseline. the measurements were repeated at figure 2. mean systolic and diastolic blood pressure of patients with mild baseline hypertension, during follow-up and at the completion of the formula jamu antihipertensi (fja) treatment for 4 weeks table 1. distribution of the characteristics of several variables, by treatment group at base-line fja = formula jamu antihipertensi; normal urea = 7-21 mg/dl; normal creatinine= 0.5-1.3 mg/dl; normal serum glutamic pyruvic transaminase (sgpt) <41 u/l; normal serum glutamic oxaloacetic transaminase (sgot) <32 u/l the end of treatment in the fourth week. these are presented in table 3, showing that at the end of treatment, the urea, creatinine and sgpt levels did not differ significantly between the fja and captopril groups. on the other hand, the final sgot concentration in the fja group was significantly higher than in the captopril group, although the increased sgot concentration was still within the normal range (table 3). univ med vol. 35 no.2 86 hussaana, sarosa, indrayani, et al jamu antihipertensi in hypertension discussion the results of this study constitute evidence t h a t f j a h a s a n a n t i h y p e r t e n s i v e e ff e c t comparable with that of captopril. fja could reduce the systolic blood pressure to an equal extent as did captopril and could even more effectively reduce the diastolic blood pressure than captopril. these results are in accord with those of the saintifikasi jamu antihipertensi (sja) study using the same 6 plant simplicia as fja. these “scientification” results also proved that sja can more effectively reduce the blood pressure as compared with captopril, although the difference is not significant.(13) the combination used in fja comprises herbal extracts acting on the target organs and herbals capable of improving fitness. the herbals affecting the cardiovascular system in this f o r m u l a t i o n a r e a p i u m g r a v e o l e n s l , orthosiphon aristatus, and centella asiatica. other herbals in the formulation are phyllanthus niruri l, curcuma xanthorrhiza roxb and curcuma domestica val, which are expected to improve fitness, thus indirectly affecting the blood pressure. the effects of fja are the synergistic effects of apium graveolens l, orthosiphon aristatus, and centella asiatica. apium graveolens l has been demonstrated to have hypotensive, negative inotropic and chronotropic effects through stimulation of muscarinic receptors.(14) the apigenin contained in apium graveolens l has vasodilator and diuretic effects by decreasing the sodium stores of the body, thus reducing blood volume and cardiac output.(15) orthosiphon aristatus is capable of inhibiting the activity of angiotensin converting enzyme (ace).(16) o. aristatus also contains potassium which can increase renal flow and sodium excretion, so that the blood pressure decreases.(17) the hypotensive effect of centella asiatica is the result of its triterpenoid content, which affects the reninangiotensin-aldosterone system (raas) and inhibits ace and vasoconstriction.(18) the active principles of the herbals apium graveolens l, normal urea = 7-21 mg/dl; normal creatinine= 0.5-1.3 mg/dl; normal serum glutamic pyruvic transaminase (sgpt) < 41 u/l; normal serum glutamic oxaloacetic transaminase sgot) < 32 u/l table 3 . mean urea, creatinine, sgpt and sgot concentrations after 4 weeks of treatment, by treatment group table 2. comparison of mean systolic and diastolic blood pressures after 1, 2, 3 and 4 weeks of treatment, by treatment group sbp= systolic blood pressure; dbp= diastolic blood pressure; fja1= formula jamu antihipertensi after 1 week of treatment; c1= captopril after 1 week of treatment; fja2= formula jamu antihipertensi after 2 weeks of treatment; c2= captopril after 2 weeks of treatment; fja3= formula jamu antihipertensi after 3 weeks of treatment; c3= captopril after 3 weeks of treatment; fja4=formula jamu antihipertensi after 4 weeks of treatment; c4= captopril after 4 weeks of treatment 87 orthosiphon aristatus, and centella asiatica that are responsible for inhibiting ace activity are the flavonoids.(19) as a whole, the plant extracts in fja act synergistically in reducing blood pressure through a combination of vascular smooth muscle relaxation and muscarinic receptor stimulation, which mediate the endothelial release of relaxing factors, inhibition of influx and utilization of extracellular calcium ions by vascular smooth muscle, and attenuation of the force and degree of myocardial contractions, and through a diuretic effect. the three other extracts contained in fja, i.e. phyllanthus niruri l, curcuma xanthorrhiza roxb and curcuma domestica val. are potential analgetic anti-inflammatory immunomodulators (aai). the effect of curcuma xanthorrhiza roxb is to confer physical fitness, accelerate metabolism and improve liver function, curcuma d o m e s t i c a s t i m u l a t e s d i g e s t i o n , w h i l e phyllanthus niruri l improves the body defenses.(20) the results of this study also showed that fja did not disturb hepatic and renal functions. this may be because the active principles of c u rc u m a d o m e s t i c a va l . a n d c u rc u m a xanthorrhiza roxb are curcuminoids that are active antioxidant, anti-inflammatory and hepatoprotective compounds.(21) similarly the active quercetin flavonoid content of phyllanthus niruri l has antioxidant and hepato-protective effects.(22,23) a large percentage of hypertensive patients are certainly treatable by appropriate adaptation of life-style changes, such as a shift from a sedentary (couch potato) setting to a more active, exercise-orientated healthy lifestyle. in addition t o t h e a b o v e , i n g e s t i o n / c o n s u m p t i o n o f functionally bioactive nutrients, such as the a f o r e m e n t i o n e d p h e n o l i c r i c h a n d o t h e r botanical-derived pharmacological agents, would be beneficial to normal health.(24) therefore, such judicious modifications will not only ameliorate hypertension, but simultaneously diminish the onset of atherothrombogenic diseases (coronary a r t e r i a l d i s e a s e , p e r i p h e r a l a r t e r i a l , a n d cerebrovascular diseases). moreover, the burden on scarce national health budgets will decrease. this study found no increase in body fitness or health status of the patients treated with fja. on average, during the course of this study, the patients registered no complaints in relation to adverse or other uncomfortable effects of the administered treatment. the results of this study may add to the body of evidence regarding the actions and safety of fja, so that this preparation may be administered to patients with mild to moderate hypertension. however, this study suffered from many limitations including the short duration of treatment and the sample participants were relatively small. conclusions from this study it may be concluded that administration of fja for 4 weeks is equally effective as captopril in reducing systolic and diastolic blood pressure in patients with mild and moderate hypertension. thus, natural plants and herbs can be our source of drugs, with fewer side effects and better bioavailability for treatment of hypertension in the future. conflict of interest the investigators had no conflicting interests in this study. acknowledgements thanks are due to the directorate for guidance of research and community service (direktorat pembinaan penelitian dan pengabdian kepada masyarakat, dirbinlitabmas) of the directorate general of higher education (ditjen dikti) for the funding of this study through a grant from the higher education quality research (penelitian unggulan perguruan tinggi, pupt) fund for the year 2015. univ med vol. 35 no.2 88 hussaana, sarosa, indrayani, et al jamu antihipertensi in hypertension references 1. kementerian kesehatan ri. peraturan menteri kesehatan republik indonesia nomor 003/ menkes/per/2010 tentang saintifikasi jamu dalam penelitian berbasis pelayanan kesehatan. jakarta; 2010. 2. frishman wh, beravol p, carosella c. alternative and complementary medicine for preventing and treating cardiovascular disease. dis mon 2009;55:121–92. doi: 10.1016/ j.disamonth.2008.12.002. 3. susalit e, agus n, effendi i, et al. olive (olea europaea) leaf extract effective in patients with stage-1 hypertension: comparison with captopril. phytomedicine 2001;18:251–8. doi: 10.1016/ j.phymed.2010.08.016.4. 4. world health organization. traditional medicine. geneva: world health organization. fact sheet no 134;2008. 5. vergara-galicia j, jimenez-ramirez lá, tunsuarez a, et al. vasorelaxant activity of extracts obtained from apium graveolens: possible source for vasorelaxant molecules isolation with potential antihypertensive effect. asian pac j trop biomed 2013;3:776–9. 6. moghadam am, imenshahidi m, mohajeri sa. antihypertensive effect of celery seed on rat blood pressure in chronic administration. j med food 2013;16:558–63. 7. manshor nm, dewa a, asmawi mz, et al. vascular reactivity concerning orthosiphon stamineus benth-mediated antihypertensive in aortic rings of spontaneously hypertensive rats. int j vasc med 2013, article id 456852, 8 pages http://dx.doi.org/10.1155/2013/456852. 8. gohil kj, patel ja, gajjar ak. pharmacological review on centella asiatica: a potential herbal cure-all. indian j pharm sci 2010;72:546-56. doi:10.4103/0250-474x.78519. 9. tabassum n, ahmad f. role of natural herbs in the treatment of hypertension. pharmacogn rev 2011;5:30-40. doi:10.4103/0973-7847.79097. 10. bharati d, rawat s, sharma p, et al. evaluation of in vivo efficacy of aqueous leaf extract of phyllanthus niruri in diabetic hypertensive rats. ann clin exp hypertension 2015;3:1031-7. 11. supriyatna, moelyono mw, iskandar y, et al. mengenal obat herbal pemahaman obat herbal untuk fitoterapi. bandung: unpad pres;2013. 12. ahmad af. analisis penggunaan jamu untuk pengobatan pada pasien di klinik jamu hortus medicus tawangmangu.[tesis]. jakarta: universitas indonesia; 2012. 13. febriyanti rm, maesaroh i, supriyatna, et al. analisis farmakoekonomi saintifikasi jamu antihipertensi, antihiperglikemia, antihiperkolesterolemia, dan antihiperurisemia. ijpst 2014;1:39-46. 14. brankovic s, kitic d, radenkovic m, et al. hypotensive and cardioinhibotory effects of the aqueous and ethanol extracts of celery (apium graveolens, apiaceae). acta medica medianae 2010;49:13-6. 15. zhang yh, park ys, kim tj. endotheliumdependent vasorelaxant and antiproliperative effects of apigenin. gen pharmacol 2002;35:3417. 16. shafaei, mun’im a, lukmanto d, et al. inhibition of angiotensin converting enzyme (ace) activity by some indonesia edible plants. ijpsr 2013;6:1054-9. 17. adam y, somchit mn, sulaiman mr, et al. diuretic properties of orthosiphon stamineus benth. j ethnopharmacol 2009;124:154-8. 18. larson aj, adaramoye oa, anjos rm, et al. hypotensive and endothelium-independent vasorelaxant effects of methanolic extract from centella asiatica in rats. j ethnopharmacol 2012;124:457-62. 19. iswantini d, trisilawati o, rahminiwati m, et al. formula antihipertensi (>60%) captoril dari bahan aktif flavonoid pegagan, tempuyung, kumis kucing dan sambiloto dan budidaya untuk meningkatkan kandungan flavonoid (>1,5%). lppm-ipb darmaga;2010. 20. siswoyo h. formularium jamu untuk anti hipertensi, hiperglikemia, hiperurisemia, dan hiperkolestrolemia. jakarta: badan penelitian dan pengembangan kesehatan kementrian kesehatan ri;2011. 21. akram m, shahab-uddin, ahmed a, et al. curcuma longa and curcumin: a review article. rom j biol plant biol 2010;55:65–70. 22. bhattacharjee r, parames cs. protein isolate from the herb, phyllanthus niruri l. (euphorbiaceae), plays hepatoprotective role against carbon tetrachloride induced liver damage via its antioxidant properties. food chem toxicol 2007;45:817–26. 23. harish r, shivanandappa t. antioxidant activity and hepatoprotective potential of phyllanthus niruri. food chem 2006;95:180–5. 24. anwar ma, al disi ss, eid ah. antihypertensive herbs and their mechanisms of action: part ii. front pharmacol 2016;7:50. doi: 10.3389/fphar.2016.00050. oktavianus 126 *department of radiology, faculty of medicine, trisakti university, jakarta **department of radiology, diponegoro university / dr. kariadi central general hospital, semarang ***department of internal medicine, diponegoro university / dr. kariadi central general hospital, semarang correspondence dr. caecilia marliana, sp.rad department of radiology, faculty of medicine, trisakti university jl. kyai tapa no. 260, grogoljakarta 11440 phone: 6221-5672731 ext 2501 e-mail: caecilia.marliana@yahoo.com univ med 2014;33:126-32 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background accurate identification of patients with deep vein thrombosis (dvt) is critical, as untreated cases can be fatal. it is well established that the specificity of the clinical signs and symptoms of dvt is low. therefore, clinicians rely on additional tests to make this diagnosis. there are three modalities for dvt diagnosis; clinical scoring, laboratory investigations, and radiology. the objective of this study was to determine the correlation of plasma d-dimer concentration with the ultrasonographic non-compressibility ratio in patients with dvt in the lower extremities. methods this research was a cross-sectional observational study. the sample comprised 25 subjects over 30 years of age with clinically diagnosed dvt in the lower extremities. in all subjects, d-dimer determination using latex enhanced turbidimetric test was performed, as well as ultrasonographic non-compressibility ratio assessment of the lower extremities. data were analyzed using pearson’s correlation at significance level of 0.05. results mean plasma d-dimer concentration was 2953.00 ± 2054.44 µg/l. the highest mean non-compressibility ratio (59.96 ± 35.98%) was found in the superficial femoral vein and the lowest mean non-compressibility ratio (42.68 ± 33.71%) in the common femoral vein. there was a moderately significant correlation between plasma d-dimer level and non-compressibility ratio in the popliteal vein (r=0.582; p=0.037). in the other veins of the lower extremities, no significant correlation was found. conclusion the sonographic non-compressibility ratio is an objective test for quick and accurate diagnosis of lower extremity dvt and for evaluation of dvt severity. key words: deep vein thrombosis, d-dimer, non-compressibility ratio, age >30 years the non-compressibility ratio for accurate diagnosis of lower extremity deep vein thrombosis caecilia marliana*, a. gunawan santoso**, and santosa*** 127 univ med vol. 33 no.2 non-compressibility ratio untuk keakuratan diagnosis trombosis vena dalam pada tungkai bawah latar belakang identifikasi yang akurat pada pasien dengan trombosis vena dalam (tvd) sangat penting, karena bila tidak ditangani dengan baik bisa berakibat fatal. diagnosis tvd secara klinis sangat sulit, sebab itu klinisi membutuhkan pemeriksaan tambahan untuk menegakkan diagnosis. terdapat tiga modalitas yang digunakan untuk diagnosis adanya tvd, skor prognosis klinis, pemeriksaan laboratorium dan pemeriksaan radiologi. penelitian ini bertujuan untuk menentukan adanya korelasi kadar d-dimer dengan non-compressibility ratio pada sonografi tvd tungkai bawah. metode penelitian observasional dengan desain belah lintang mengikutsertakan 25 orang penderita tvd secara klinis, berusia 30 tahun keatas. pemeriksaan kadar d-dimer dalam plasma menggunakan metode latex enhanced turbidimetric test. ultrasonografi pada tungkai bawah dilakukan untuk mengukur non-compressibility ratio vena tungkai bawah . analisis data dengan uji korelasi pearson pada tingkat kemaknaan 0.05. hasil rata-rata kadar d-dimer plasma besarnya 2953,00 ± 2054,44 µg/l. nilai rata-rata non-compressibility ratio tertinggi ditunjukkan pada vena femoralis superfisial sebesar 59,96 ± 35,98% dan terendah pada vena femoralis komunis sebesar 42,68 ± 33,71%. didapatkan korelasi moderat yang bermakna antara kadar d-dimer plasma dengan non-compressibility ratio pada vena poplitea (r=0,582; p=0,037). pada vena-vena tungkai bawah lainnya tidak terdapat hubungan yang bermakna. kesimpulan non-compressibility ratio secara sonografi merupakan pemeriksaan obyektif yang dapat digunakan untuk menghasilkan diagnosis secara cepat dan akurat adanya tvd tungkai bawah dan untuk evaluasi berat-ringan tvd. kata kunci: trombosis vena dalam, d-dimer, non-compressibility ratio, usia >30 tahun abstrak introduction for the last 25 years the annual incidence of venous thromboembolism (vte) in the usa has remained stable at approximately 900,000 cases, with 300,000 deaths from pulmonary embolism.(1,2) first-time vte incidence is around 1 per 1000 per year in the adult population, and after 45 years of age vte increases sharply to 5-6 cases per 1000 persons 80 years of age.(3) there is no significant difference in its incidence in males and females.(4) according to more recent prospective studies, sex has no significant impact on vte prevalence.(5,6) studies on deep vein thrombosis (dvt), one of the manifestations of vte, have been focused only on one to a few variables in their cohort such as the distribution and extent of thrombosis, and their association with risk factors. in dvt of the lower extremities, thrombi are formed in veins of the femoral and/or crural regions. accurate identification of patients with dvt is essential, since if dvt is not well managed, it may have fatal results. the condition is very serious because it can result in permanent damage and can cause pulmonary embolism.(3) there are three modalities used in the diagnosis of dvt, i.e. the clinical prognostic 128 score, laboratory investigations, and radiological examination.(7) in the clinical prognostic score, the following features are taken into account: p r e s e n c e o r a b s e n c e o f l o w e r e x t r e m i t y paralysis, orthopedic surgical history, swelling of the lower extremity, pitting edema, superficial venous collaterals, and presence of cancer.(8-10) the d-dimer test for the presence of cross-linked d fragments of fibrin, indicative of ongoing thrombus formation, has been applied in an algorithm for evaluation of suspected vte.(11) according to several case reports, negative results for d-dimer eliminate the need for further e x a m i n a t i o n s , s u c h a s u l t r a s o n o g r a p h y (usg).(12,13) kurklinsky et al.(13) sought to find an association between the d-dimer value and sonographic thrombus volume, and concluded that in spite of the many clinical prognostic scores in existence, determination of d-dimer fibrin is a sensitive test for venous thrombosis and is correlated with thrombus volume. the most common radiological examination performed for the diagnosis of dvt is b-mode usg, which can rapidly give information on the presence or absence of a thrombus.(14) tsao et al.(15) studied the non-compressibility ratio in dvt in relation to the clinical prognostic score, and found that usg with compression is a rapid and objective examination to evaluate the severity of the formed thrombus. the noncompressibility ratio is found from the diameter of the lower extremity veins before and after compression. (14-15) there are 3 types of dvt results on sonographic compression, i.e. patent, partial occlusion, and total occlusion. the patent type is easily diagnosed if there is total collapse of the vascular lumen. total occlusion is also easily seen from the persistent dilation of the venous lumen upon compression. the only type of venous thrombosis, the severity of which is difficult to determine, is the partially occlusive type. (14) in measuring the venous diameter, the transducer should be gently pressed on the region to be examined, making sure to avoid accidental compression of the adjacent arterial structure.(16-17) in view of the above background, the purpose of the present study was to determine the relationships between d-dimer concentration and usg non-compressibility ratio in thrombi of the lower extremities. methods design of the study t h i s s t u d y u s e d a c r o s s s e c t i o n a l observational design and was carried out at dr. kariadi central general hospital, semarang, from january to may 2014. study subjects the subjects of this study were patients with dvt of the lower extremities, undergoing d-dimer determination at the clinical pathology d e p a r t m e n t a n d u s g a t t h e r a d i o l o g y department of dr. kariadi central general hospital, semarang, and meeting the following inclusion criteria: being >30 years of age, having a high clinical prognostic score, and being a new case. the exclusion criteria were: being on a n t i c o a g u l a n t t h e r a p y, h a v i n g s e v e r e hemorrhage, infection, or edema of upper extremities, and history of previous dvt. the sample size was calculated from the correlation test formula with a minimum sample size of 25. the subjects were selected by consecutive sampling, which is based on the patients’ presentation at dr. kariadi hospital, semarang. data collection patients referred from the internal medicine clinic of dr. kariadi central general hospital, semarang, with signs and symptoms of dvt of the lower extremities, were interviewed using a questionnaire containing items on age, sex, and history of the present disease. these patients were subjected to determination of d-dimer concentration and usg of the lower extremity veins. marliana, santoso, santosa, et al non-compressibility ratio for deep vein thrombosis 129 univ med vol. 33 no.2 determination of d-dimer concentration plasma d-dimer concentration in the venous blood samples was determined in the clinical pathology department of dr kariadi hospital, by means of the latex enhanced turbidimetric test, using the sysmex ca-1500 coagulometer, with a cut-off value of 500 ng/ ml. measurement of non-compressibility ratio the veins of the lower extremities of the subjects were examined by b-mode usg with a transducer linear frequency of 7.5 mhz. the lower extremity veins examined were the common femoral vein, superficial femoral vein, popliteal vein, and anterior and posterior tibial veins. in measuring the venous diameter, the transducer was pressed gently on the region to be examined, taking care to avoid compression of the adjacent arterial structure. the diameter of the vein was recorded before and after compression. the non-compressibility ratio was obtained from the formula (figure 1): (2) (diameter of vein after compression/diameter of vein before compression) x 100. data analysis data analysis was descriptive and bivariate analytic using the pearson correlation test at a significance level of 0.05. figure 1. non-compressibility ratio of venous thrombosis using sonographic transverse sectioning. a. diameter of vein before compression. b. diameter of vein after compression.(2) ethical clearance this study obtained ethical clearance from the commission on health research ethics, faculty of medicine, diponegoro university/dr. kariadi central general hospital, semarang. results total sample size was 25, with the youngest subject 38 years of age and the oldest 86 years. most of the subjects (11 or 44.0%) were found in the age group of >61 years, and 20 (80.0%) variable n (% ) demogra phic characteristic age (years) < 50 5160 > 61 s ex male female acc ompanying cl inical features hypertensi on diabetes mellit us malignancy obesit y hormona l contra cepti on history of sur gery 6 (24.0) 8 (32.0) 11 (44.0) 5 (20.0) 20 (80.0) 14 (46.6) 2 (6.7) 10 (33.2) 2 (6.7) 1 (3.4) 1 (3.4) table 1. distribution of demographic characteristics and accompanying clinical features in dvt subjects (n=25) 130 table 2. mean d-dimer concentration and non-compressibility ratio in dvt subjects (n=25) values are mean ± sd that the mean age of patients with dvt was 62 years.(18,19) the majority of dvt patients in the present study (80%) were female, which differs from studies reporting no difference in dvt prevalence in males and females up to 45 years of age.(19) this topic is however still subject to c o n t r o v e r s y. r i s k f a c t o r s p r e s u m a b l y a c c o u n t i n g f o r s e x d i f f e r e n c e s a r e o r a l contraceptive use, pregnancy, and history of abortion or premature births. more recent prospective studies did not find differences in prevalence of venous thromboembolism (vte) between sexes, although vte may be more prevalent in premenopausal women.(6,20) the higher prevalence of vte in premenopausal women may be caused by the fact that women are particularly affected by hormones that may increase the levels of blood clotting factors and facilitate venous thrombus formation. our study results showed that the noncompressibility ratio was significantly but moderately correlated with plasma d-dimer levels in the popliteal veins of dvt patients. plasma d-dimer determination appears to be a simple method with high clinical utility for excluding suspect acute recurrent dvt.(21) a negative d-dimer test result in a patient who is clinically not suspected of having dvt, can be used to exclude the diagnosis of dvt, without having to resort to usg testing. in an emergency situation requiring a quick and available diagnostic test for dvt, the noncompressibility ratio may be used, as it is significantly correlated with plasma d-dimer concentration. on account of its high high sensitivity (95%), specificity (99%), and table 3. relationship between non-compressiblity ratio and d-dimer concentration in dvt subjects values are pearson correlation coefficients (r); *significant at p<0.05 marliana, santoso, santosa, et al non-compressibility ratio for deep vein thrombosis of the subjects were female. the majority of the subjects (14 or 46.6%) had hypertension (table 1). mean d-dimer concentration was 2953.00 ± 2054.44 µg/l. the highest mean noncompressibility ratio of 59.96 ± 35.98% was found for the superficial femoral vein and the lowest ratio of 42.68 ± 33.71% was for the common femoral vein (table 2). t h e r e w a s a m o d e r a t e l y s i g n i f i c a n t association between d-dimer concentration and non-compressiblity ratio in the popliteal vein (r=0.582; p=0.037) (table 3). the other veins of the lower extremities did not show a significant association. discussion in this study, the highest proportion of patients with lower extremity dvt (44.0%) was in the ≥61 year age group. essentially similar results were obtained in several studies showing 131 univ med vol. 33 no.2 accuracy (98%), venous doppler sonography is now the initial test used to determine the possibility of dvt.(15) determination of the non-compressibility ratio of thrombosed veins constitutes an objective test for assessing dvt severity and admitting patients for evaluation of potential adverse outcomes. although d-dimer testing and several algorithms based on clinical criteria can be used for the diagnosis of dvt, there are increasing numbers of duplex ultrasonography (dus) tests being performed annually. this may be a reflection of the importance of avoiding medicolegal issues and of time constraints for conducting physical examinations. however, dus has a reduced accuracy in diagnosing distal thrombosis and is mainly available during regular clinic or hospital radiology department hours. a limitation of the present study was the r e s t r i c t e d a r e a o f t h e u s g s c a n s f o r determination of the non-compressibility ratio, with an exclusive focus on thrombi in the superficial femoral, common femoral, popliteal, anterior tibial, and posterior tibial veins, thus being incapable of excluding the presence of thrombi in veins outside the scan area. conclusion plasma d-dimer level is correlated with the sonographic non-compressibility ratio of the popliteal vein in dvt of the lower extremities. references 1. heit j, petterson t, farmer s, bailey k, melton l. trends in incidence of deep vein thrombosis and pulmonary embolism: a 35-year population-based study. blood 2006;108:430a. 2. heit j, cohen a, anderson f. estimated annual number of incident and recurrent, non-fatal and fatal venous thromboembolism (vte) events in the us. blood 2005;106:267a. 3. cushman m. epidemiology and risk factors for venous thrombosis. semin hematol 2007;44:629.doi: 10.1053/j.seminhematol.2007.02.004. 4. riddle dl, wells ps. diagnosis of lower-extremity deep vein thrombosis in outpatients. phys ther 2004;84:729-35. 5. kahn s, shrier i, julian j, ducruet t, arsenault l, miron mj, et al. determinants and time course of the post thrombotic syndrome after acute deep venous thrombosis. ann intern med 2008;149: 698-707. 6. prandoni p, noventa f, ghirarduzzi a, pengo v, bernardi e, pesavento r, et al. the risk of recurrent venous thromboembolism after discontinuing anticoagulation in patients with acute proximal deep vein thrombosis or pulmonary embolism : a prospective cohort study in 1626 patients. haematologica 2007;92:199-205. 7. geno m. diagnostic assessment of deep vein thrombosis and pulmonary embolism. am j med 2005;118:s3-12. 8. goodacre s, stevenson m, wailoo a, sampson f, sutton aj, thomas s. how should we diagnose suspected deep-vein thrombosis? qjm 2006;99: 377-88. 9. falanga a, zacharski l. deep vein thrombosis in cancer: the scale of the problem and approaches to management. ann oncol 2005;16:696-701. 10. subramaniam mr, chou t, heath r, allen r. importance of pretest probability score and ddimer assay before sonography for lower limb deep venous thrombosis. am j roentgenol 2006; 186:206-12. 11. wada h, kobayashi t, abe y, hatada t, yamada n, sudo a, et al. elevated levels of soluble fibrin or d-dimer indicate high risk of thrombosis. j thromb haemost 2006;4:1253-8. 12. sahakian gd, claessens ye, allo jc, kansao j, kierzek g, pourriat jl. accuracy of d-dimers to rule out venous thromboembolism events across age categories. emergency med int 2010, article id 185453,4 pages.doi:10.1155/2010/185453. 13. kurklinsky ak, kalsi h, wysokinski we, mauck kf, bhagra a, havyer rd, et al. fibrin d-dimer concentration, deep vein thrombosis symptom duration, and venous thrombus volume. angiology 2006;62:253-6. 14. chen jd. ultrasonography in the evaluation of low-extremity deep vein thrombosis. j chin med assoc 2011;74:1-2. 15. tsao jh, tseng cy, chuang jl, chen yc, huang hh, yi-hong chou yh, et al. non-compressibility ratio of sonography in deep venous thrombosis. j chin med assoc 2010;73:563-7. 16. masuda em, kistner rl. the case for managing calf vein thrombi with duplex surveillance and selective anticoagulation. dis mon 2010;56:60113. 17. righini m, paris s, le gal g, laroche jp, perrier a, bounameaux h . clinical relevance of distal deep vein thrombosis. review of literature data. thromb haemost 2006;95:56-64 . 132 marliana, santoso, santosa, et al non-compressibility ratio for deep vein thrombosis 18. ouriel k, green r, greenberg r, clair d. the anatomy of deep venous thrombosis of the lower extremity. j vasc surg 2000;31:895-900 . 19. poulikidis kp, gasparis ap, labropoulos n. prospective analysis of incidence, extent and chronicity of lower extremity venous thrombosis. phlebology 2014;29:37-42. 20. kahn s, shrier i, julian j, ducruet t, arsenault l, miron mj, et al. determinants and time course of the post thrombotic syndrome after acute deep venous thrombosis. ann intern med 2008;149: 698–707. 21. rathbun sw, whitsett tl, raskob ge. negative d-dimer result to exclude recurrent deep venous thrombosis: a management trial. ann intern med 2004;141:839-45. c:\users\jurnal fkusakti\docume 13 *department of physiology faculty of medicine university of islam indonesia correspondence: rokhima lusiantari department of physiology faculty of medicine university of islam indonesia jl. kaliurang km. 14.5 ngemplaques, sleman, yogyakarta 55584 mobile: +62813 9161 2612 rokhimalusi@uii.ac.id date of first submission, june 12, 2017 date of final revised submission, february 12, 2018 date of acceptance, february 15, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina shortening tends to increase aortic foam cell count and wall thickness in male wistar rats rokhima lusiantari*, miranti dewi pramaningtyas*, titis nurmasitoh*, rachmi hidayati pattimura*, and anggita dewanti* background shortening is widely used as raw material for bread and other foods. hypercholesterolemia increases aortic arch foam cell formation and abdominal aortic wall thickness. this study aimed to determine the effect of shortening on the number of aortic arch foam cells and abdominal aortic wall thickness in rats. methods this study was of experimental posttest control group design. twenty four male wistar rats were randomized into 4 groups. the negative control group (c-) received standard feed, the positive control group (c + ) standard high-fat feed, group t 1 shortening and standard feed at a ratio of 1:5 and group t 2 shortening and standard feed at a ratio of 1:10. the interventions were given for 6 weeks through gavage. the foam cell count in the aortic arch and the thickness of the abdominal aortic wall were measured. oneway anova test was used to analyze the data. results there was no significant difference in the mean foam cell count of the aortic arch between the four groups c(7.17 ± 4.17), c + (9.33 ± 7.01), t 1 (11.83 ± 4.88) and t 2 (9.33 ± 6.80) (p=0.598). the mean thickness of the abdominal aortic wall between the four groups c(741.98 ± 60.67m), c + (714.29 ± 90.59m), t 1 (838.90 ± 75.86 m), and t 2 (749.88 ± 99.37m) also was not significantly different (p=0.110). conclusion shortening tends to increase the foam cell count of the aortic arch and the thickness of the abdominal aortic wall of rats. keywords: shortening, histopathology, aortic arch, abdominal aorta, rats doi: http://dx.doi.org/10.18051/univmed.2018.v37.13-18 original article ci te t his arti cle as: lus iant ari r, praman ingt yas md, nur masi toh t, pattimura rh, dewanti a. shortening tends to increase aortic foam cell count and wall thickness in male wistar rats. univ med 2018;37:13-8. doi: 10.18051 /univmed.2018.v37.13-18 pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 14 lusiantari, pramaningtyas, nurmasitoh, et al shortening and wall thickness in rats introduction cardiovascular disease is one of the causes of mortality and disability in cases of noncommunicable dise ase (ncd) in various countries.(1,2) non-communicable disease, such as cardiovascular disease, is a main problem not o nl y in d eve l ope d c o un t r ie s but al so in developing countries. in 2014, russia and the us had the highest mortality rates from ischemic heart disease.(3) according to the global status report on non-communicable disease, in 2008 more than 36 million persons died (63% of total mortality) as a result of non-communicable disease in countries with lower to middle per capita incomes.(4) cardiovascular disease may be caused by a number of factors, one of them being hyperlipidemia that may occur as a result of consumption of a high fat diet.(5) hyperc holesterolemia is a condition characterized by a high cholesterol concentration in the blood. hypercholesterolemia may cause the accumulation of fat and oxidized low density lipoprotein (ldl) cholesterol in blood vessel walls,(6) leading to the formation of plaques in the blood vessels and the occurrence of ischemic heart disease. hypercholesterolemia may also increase oxidative stress in various organs, such as the liver, heart, and kidneys.(6-8) several studies on animal models of hypercholesterolemia have been conducted, in which the condition was induced by means of intravenous adrenalin, duck egg yolk, and lard. however, these studies yielded less significant and consistent re sults in causing hypercholesterolemia in rats. in addition, the use of lard is constrained by ethical considerations, while cost is also a problem with standard high fat feeds.(9,10) in this connection, shortening may also be used as inducer of hypercholesterolemia, since it is of relatively low cost and easily available.(11) fat is widely used as raw material of bread and other foods.(12,13) according to nurmasitoh and pramaningtyas, shortening may increase total cholesterol concentration, triglycerides and ldl in the blood of male wistar rats.(11) shortening, which is made by hydrogenation, has a high trans fat content.(12,13) trans fat increases the ldl concentration and decreases the high density lipoprotein (hdl) concentration in the blood, so increasing the risk for atherosclerosis. oxidized ldl may induce the formation of atherosclerotic plaques that ultimately lead to cardiovascular disorders, one of them being acute coronary syndrome. hypercholesterolemia may promote the formation of atherosclerotic lesions, the onset of which is marked by an increase in foam cell count. hypercholesterolemia may also cause an increase in the thickness of blood vessel walls.(6,14,15) to date there are still few studies on the effect of shortening on foam cells and aortic wall thickness. therefore the purpose of the present study was to evaluate the effects of stepped doses of shortening on foam cell count and abdominal aortic wall thickness in male wistar rats. methods study design this was an experimental post-test only controlled study, that was conducted in the physiology laboratory and the integrated research laboratory, faculty of medicine, universitas islam indonesia, from may to december 2016. animals and experimental procedure the study involved 24 male wistar rats aged 2-3 months and weighing 200-250 grams that were obtained from the integrated research and testing la bor atory (lppt ) gadj ah mada university. the sample size was calculated using the federer formula (n-1) (t-1)15, where n=sample size for ea ch interve ntion and p=number of interventions. previous to the intervention, the rats were adapted for 7 days. the rats were put in cages of 40 cm x 20 cm x 20 cm and subjected to 12-hour light and dark cycles. the rats were randomly divided into 4 groups, i.e. a negative control group (c), a 15 positive control group (c + ) and two intervention groups (t 1 and t 2 ). group creceived ad ii standard rat feed containing 51% carbohydrates, 15% crude protein, 3-7% crude fat, 6% crude fiber, 7% ash, 0.9-11% calcium, 0.6-0.9% p ho sp hor, 12% wa te r, anti biot i cs, and a coccidiostat. group c + received standard high fat feed, while groups t 1 and t 2 received shortening and standard feed at a ratio of 1:5 and 1:10, respectively. the intervention was administered via the oral route by gavage for 6 weeks in the physiology laboratory. upon completion of the intervention, the animals were terminated. prepration of histological slides after the rats were terminated, their aortic arch and abdominal aorta were removed, fixed in formalin for 2 days, dehydrated in alcohol 70%, and transported to the integrated research laboratory, faculty of medicine, universitas islam indonesia, where they were made into histological slides. the aortic arch and abdominal aorta of each rat were block paraffinized, microtomed, and stained with hematoxylin-eosin (he). the histological slides were evaluated with respect to foam cell count, using an olympus cx41 binocular microscope connected to an optilab camera, with which images were taken of 10 fields of view at 40x magnification. the foam cell count was counted using a cell count application. the foam cells that were counted were those located in the tunica intima and tunica media. the thickness of the abdominal aortic wall was measured from the lamina intima up to the lamina media in 8 fields of view in a clockwise direction (at 12.00, 13.30, 15.00, 16.30, 18.00, 19.30, 21.00, 22.30) using optilab and micrometer scale.(10) data analysis the test used was one-way anova, with the level of significance set at p<0.05, followed by a post-hoc tukey test to determine betweengroup differences.(16) ethical clearance t he pr e se nt s t ud y o bt a i n ed e t hi c a l clearance from the ethics committee faculty of medicine universitas islam indonesia under no. 03/ka.kom.et/70/ke/ii/2016. results the results of the present study shows that the greatest increase in foam cell count was in t he i n t e r ve nt i o n gr o up t 1 r e c e i vi n g t he combination of shortening and standard feed at the ratio of 1:5, followed by groups c + , t 2 and c (figure 1). although t 1 had the greatest increase in foam cell count as compared to group c-, the results of the statistical analysis were still not significant (p=0.598). in agreement with the increase in foam cell count, the greatest abdominal aortic wall thickness was in intervention group t 1 , followed by groups t 2 , c , and c + (figure 2). although t 1 had increased abdominal aortic wall thickness as compared to group c-, the results of the statistical analysis were not yet significant (p=0.110). the complete set of data on mean aortic ar ch foam cell count and abdominal aortic wall thickness is shown in table 1. univ med vol. 37 no.1 variable intervention group p value c– ( n=6) c+ (n=6) t1 (n=6) t2 (n=6) foam cell count 7.17 ± 4.17 9.33 ± 7.01 11.83 ± 4.88 9.33 ± 6.80 0.598 aortic wall thickness (µm) 741.98 ± 60.67 714.29 ± 90.59 838.90 ± 75.86 749.88 ± 99.37 0.110 table 1. mean aortic arch foam cell count and abdominal aortic wall thickness by intervention group c-: negative control (ad ii standard feed), c + : positive control (standard high fat feed), t 1 : shortening + ad ii standard feed (1:5 ratio), t 2 : shortening +ad ii standard feed (1:10 ratio) 16 lusiantari, pramaningtyas, nurmasitoh, et al shortening and wall thickness in rats discussion the present study shows that the use of high fat feeds comprising a combination of shortening and standard feed at a ratio of either 1:5 or 1:10 did not yet show a significant increase in foam cell count. these results are in line with previous studies reporting that administration of high fat feeds in consisting of 0.2% egg yolk, 2% cholic acid, fat 5% goat and 92.8% standard feed in amounts of 20 gram/day was proved to be able to form foam cells but did not yet show a significant increase in foam cell count in the control group and the groups receiving the aforesaid high fat feeds.(17) these results differ from those of a study on the administration of lard, which showed an increase in foam cell count.(10) the differing results may very well be the result of a difference in the induction period of the test animals. in the a b c figure 1. photomicrographs of aortic arch cross sections of wistar rats, he staining, 40 x magnification. a: negative control group on standard feed ad ii. b: positive control group on standard high fat diet. c: group t1 receiving shortening and standard feed ad ii at the ratio of 1:5. d : group t2 receiving shortening and standard feed ad ii at the ratio of 1:10.red arrows: foam cells latter study the induction period was 8 weeks, whereas in our study the induction period was 6 weeks. an induction period of less than 8 weeks may cause less than optimal foam cell formation. this is also referred to in the study of nurmasitoh and pr aman ingtyas (1 1) who found that administration of shortening for 14 days to wistar rats already showed a significant increase in lipid profile.(11) however, as a result of the shorter induction period, the increase in lipid profile could not yet lead to significant structural vascular changes, i.e. an increase in wall thickness and foam cell count. atherosclerosis occurs as a result of the conversion of low density lipoprotein (ldl) to oxidized ldl, causing endothelial lesions. oxidized ldl and plasma molecules also undergo extravasation into the endothelial space where they are trapped so that they may become 17 cytotoxic, proinflammatory, chemotactic, and proatherogenic. endothelial injury facilitates the entr y of monocytes that adhere in the subendothelial space and subsequently become macrophages. active macrophages will secrete an oxygen species toxin that increases ldl oxidation, leading to the development of foam cells. in addition, the endothelium becomes more active, with a decrease in no synthesis and development of endothelial stiffness.(18,19) our study also showed that the use of a combination of shortening in standard feed at a ratio of 1:5 or 1: 10 also does not yet show an increase in vessel wall thickness. this supports the results of a pr e vious study where administration of daun katuk (sauropus androgynus or sweetleaf), cholic acid and cholesterol for 8 weeks, also did not have significant effects on vessel wall thickness.(20) univ med vol. 37 no.1 figure 2. photomicrographs of abdominal aorta cross sections of wistar rats, he staining, z x magnification a: negative control group on standard feed ad ii. b: positive control group on standard high fat diet. c: group t1 receiving shortening and standard feed ad ii at the ratio of 1:5. d : group t2 receiving shortening and standard feed ad ii at the ratio of 1:10 d another study also states that fat induction in wistar rats significantly increases the lipid concentration in the 22nd week.(21) a limitation of the present study is the relatively short intervention period so that the results were less than maximal. therefore there is a need for further studies using higher induction doses and longer intervention periods, so that the results will be according to theory. conclusion shortening tend to increase the aortic arch foam cell count and abdominal aortic wall thickness in male wistar rats. conflict of interest there was no conflict of interest. 18 lusiantari, pramaningtyas, nurmasitoh, et al shortening and wall thickness in rats acknowledgement we thank the uppm, faculty of medicine universitas islam indonesia for the funding of this study. we also express our utmost gratitude to ernadita budi astuti, fauzi ahmad, angkit daru prihatmoko for their technical assistance during the course of this study. thanks are also due to all parties who contributed to the completion of this study. contributors rl and mdp contributed to the design of the study and drafting of the manuscript. tn and rhd contributed to data collecting and analysis. ad contributed to revising the manuscript. all authors read and approved the final manuscript. references 1. carmen g, iliescu c. modern risk stratification in coronary heart disease. j med life 2011;4:377-86. 2. rohilla a, dagar n, rohilla s, et al. hyperlipidemiaa deadly pathological condition. int j curr pharm res 2012;4: 15-8. 3. finegold ja, asaria p, francis dp. mortality from ischaemic heart disease by country, region, and age: statistics from world health organisation and united nations. int j cardiol 2013;168: 93445. 4. world health organization. global status report on noncomunicable disease 2010. geneva: world health organization; 2011. 5. harikumar k, althaf sa, kishore kb, et al. a review on hyperlipidemic. int j nov trends pharm sci 2013;3: 69–80. 6. bonomini f, rodella lf, rezzani r. metabolic syndrome, aging and involvement of oxidative stress. aging dis 2015;6:109–20. doi: 10.14336/ ad.2014.0305. 7. csonka c, sarkozy m, pipicz m, et al. modulation of hypercholesterolemia-induced oxidative/ nitrative stress in the heart. oxid med cell longev 2016;2016:1-23. doi: 10.1155/2016/3863726. 8. alkhamees oa. protective effects of vitamin-p and vitamin-c on hypercholesterolemia-induced oxidative hepatic damage and lipid profile changes in female rats: a comparative study. j appl pharm sci 2013;3:99–105. 9. dewi ncp, probosari e. pengaruh pemberian ekstrak kacang hijau (phaseolus radiatus) terhadap kadar kolesterol ldl serum tikus hiperkolesterolemia. j nutr coll 2013;2:585-92. 10. maramis r, kaseke mk, tanudjadja gn. gambaran histologi aorta tikus wistar dengan diet lemak babi setelah pemberian ekstrak daun sirsak (annona muricata). j e-biomedik 2014;2:431-35. 11. nurmasitoh t, pramaningtyas md. honey improves lipid profile of diet-induced hypercholesterolemic rats. univ med 2015;34:17786. doi: 10.18051/univmed.2016.v35.177-186. 12. mamat h, hill se. effect of fat types on the structural and textural properties of dough and semi-sweet biscuit. j food sci technol 2014;51:1998-2005. 13. jissy jacob, leelavathi k. effect of fat-type on cookie dough and cookie quality. j food eng 2007;79:299–305. 14. tomkin gh, owens d. ldl as a cause of atherosclerosis. open atherosclerosis thromb j 2012;5:13-21. 15. eckel rh, borra s, lichtenstein ah, et al. understanding the complexity of trans fatty acid reduction in the american diet. circulation 2007; 115:2231-46. 16. dahlan s. statistik untuk kedokteran dan kesehatan edisi 6. epidemiologi indonesia. jakarta; 2014. 17. welinsa f, asni e, malik z, et al. histopatologi arteri torasika rattus novergicus strain wistar jantan setelah 8 minggu pemberian diet aterogenik. jom fk 2014;2:1-11. 18. bentzon jf, otsuka f, virmani r, et al. mechanisms of plaque formation and rupture. circ res 2014; 114;1852-1866. doi: 10.1161/circresaha.114. 302721. 19. porth cm, grossman s, editors. porth’s pathophysiology concepts of altered health states. 9th ed. philadelphia: wolters kluwer health: lippincott williams & wilkins; 2014. 20. callisa o, handayani d, tritisari kp. pengaruh pemberian tepung daun katuk terhadap penebalan dinding aorta tikus wistar. indones j human nutr 2015;2:85-90. 21. heriansyah t. pengaruh berbagai durasi pemberian diet tinggi lemak terhadap profil lipid tikus putih (rattus norvegicus strain wistar) jantan. j kedokt syah kuala 2013;13:114-50. silvy 73 profile of nonstructural glycoprotein ns1 as a diagnostic marker in dengue type 2 virus infection sylvia y. muliawan* abstract background several studies in dengue on cloned or cell culture passaged in wild and attenuated virus have been performed, but each report described different sites of nucleotide and amino acid changes possibly associated with virulence. the aim of this study was to investigate protein expression of dengue type 2 virus infected c6/36 cells through two-dimensional gel electrophoresis followed by n-terminal edman degradation sequencing to identify target proteins. methods two type of samples; dengue type 2 virus-infected c6/36 cells and uninfected c6/36 cells were prepared. immobilized non-linear ph gradient strips, ph 3-10 were used in isoelectric focusing, and 10% homogeneous gels were used in sodium dodecyl sulphate polyacylamide gel electrophoresis. the target protein spots were subsequently transferred onto polyvinyldifluoride membrane by western blotting and visualized by coomassie brilliant blue for n-terminal sequencing purpose. results this study revealed four target protein spots in dengue type 2 virus-infected c6/36 cells that were not present in uninfected c6/36 cells. the n-terminal sequencing result was d-s-g-c-v-v-s-w-k-n-k which was identical to nonstructural glycoprotein ns1 (swissprot-database) associated with the replication process of flaviviruses. conclusion nonstructural ns1 might be used as a diagnostic marker and/or as a parameter to evaluate the effect of antiviral agents for dengue type 2 virus infection/replication. keywords: dengue type 2 virus, non structural ns1 protein, proteomics *department of microbiology medical faculty trisakti university correspondence dr. sylvia y. muliawan, phd department of microbilogy medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2402 email: sylviaym2003@yahoo.com universa medicina 2007; 26: 73-9. universa medicina april-juni 2007april-juni 2007april-juni 2007april-juni 2007april-juni 2007 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 74 muliawan nonstructural glycoprotein ns1 profil glikoprotein “non structural” ns1 sebagai marker pada diagnosis infeksi virus dengue tipe 2 sylvia y. muliawan latar belakang pada dengue telah dilakukan beberapa penelitian yang menggunakan cara cloning atau subkultur sel baik yang berasal dari virus yang delemahkan maupun yang tidak dilemahkan, tetapi masing-masing hasil memperlihatkan perubahan asam amino dan nukleotida pada tempat yang berbeda yang berhubungan dengan virulensi. tujuan penelitian adalah untuk menyelidiki ekspresi protein virus dengue tipe 2 yang di kultur pada sel c6/36 melalui two-dimensional gel electrophoresis. cara sampel yang digunakan terdiri dari dua tipe, yaitu kultur sel c6/36 yang diinfeksi dengan virus dengue tipe 2 dan kultur sel c6/36 yang tidak diinfeksi. pada teknik isoelectric focusing digunakan immobilized non-linear ph gradient strips dengan ph 3-10 yang diikuti dengan sodium dodecylsulphate-polycrylamide gel electrophoresis. selanjutnya protein target yang diperoleh dipindahkan ke polyvinyldifluoride membrane dengan metode western blot dan dilakukan pewarnaan dengan coomassie brilliant blue untuk keperluan n-terminal sequencing. hasil hasil yang diperoleh pada ekspresi protein adalah 4 titik ekstra yang ditemukan pada sel c6/36 yang diinfeksi virus dengue tipe 2, tetapi tidak ditemukan pada sel c6/36 yang tidak diinfeksi. hasil n-terminal sequencing menunjukkan urutan sekuens yang identik dengan nonstructural glycoprotein sesuai dengan swissprot-database yaitu d-s-g-c-v-v-s-w-k-n-k. sekuens ini berkaitan dengan proses replikasi flaviviruses. kesimpulan nonstructural ns1 dapat digunakan sebagai alat bantu diagnosis dan atau parameter untuk mengevaluasi efek antivirus terhadap infeksi atau proses replikasi virus dengue tipe 2. kata kunci: virus dengue tipe 2, protein non structural ns1, proteomik *bagian mikrobiologi fakultas kedokteran universitas trisakti korespondensi dr. sylvia y. muliawan, phd bagian mikrobiologi fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2402 email: rsylviaym2003@yahoo.com universa medicina 2007; 26: 73-9. abstrak introduction dengue fever (df), dengue hemorrhagic fever (dhf) and shock syndrome (dss) are mosquito-borne infectious diseases that have b e c o m e m a j o r i n t e r n a t i o n a l p u b l i c h e a l t h concerns. df and dhf/dss occur in tropical and subtropical regions around the world, predominantly in urban and semi-urban areas.(1) a c c o r d i n g t o wo r l d h e a l t h o rg a n i z a t i o n (who) estimates, its incidence has increased by a factor of 30 over the last 50 years.(2) it occurs in tropical areas and affect up to 100 million people each year,(3,4) including 500,000 75 cases of dhf and around 30,000 deaths, mostly among children.(5) it is now endemic in more than 100 countries (the americas, the eastern mediterranean, southeast asia, and the western pacific) and poses a threat to more than 2.5 billion people.(6) the african continent seems less affected by the dengue virus.(5) an epidemic o f d h f i n t h e n e w wo r l d t o o k p l a c e i n venezuela in 1989; isolates of den-1, den-2 and den-4 were obtained during this epidemic, but the most severe cases were associated with den-2 infections.(10) dengue viruses belong to the flavivirus genus of the flaviviridae family. flaviviruses are enveloped, single stranded r n a v i r u s e s . t h e d e n g u e g e n o m e i s approximately 11 kb in size and contains a single open reading frame, which encodes a precursor polyprotein and is flanked by two nontranslated regions (5’ and 3’ ntr). coand posttranslational proteolytic cleavage of the precursor results in the formation of three structural proteins, capsid (c), membrane (m), and envelope (e), and seven nonstructural (ns) proteins, ns1, ns2a, ns2b, ns3, ns4a, n s 4 b , a n d n s 5 . a m o n g t h e m , t h e nonstructural protein ns1 related to replication process of flaviviruses. unusually for a viral glycoprotein, ns1 is produced both membraneassociated and secreted forms(5) both of which are highly immunogenic and can elicit high-titre antibody.(11) ns1 has a predicted molecular mass of 46 to 50 kilodalton(12) due to n-linked carbohydrate chains at position 130 and 207 and 12 invariant cysteine residues.(13) ns1 is not incorporated into the virion and therefore does not elicit virus-enhancing antibodies.(14) the precise function of dengue ns1 protein remains unclear.(15) p r o t e o m i c s i s w i d e l y a c c e p t e d a s a technology in the post genomic era to identify proteins that are either involved in a specific cellular process (cell map proteomics) or exhibit an altered expression profile as the r e s u l t o f s o m e c h a n g e s i n p h y s i o l o g i c a l c o n d i t i o n ( e x p r e s s i o n p r o t e o m i c s ) . s i n c e proteins play a central role in the life of an o r g a n i s m , p r o t e o m i c s i s i n s t r u m e n t a l i n discovery of biomarkers, such as markers that indicate a particular disease. t h e d e v e l o p m e n t o f t w o d i m e n s i o n a l e l e c t r o p h o r e s i s ( 2 d e ) w a s a m a j o r s t e p forward of the science in proteomics to identify the proteins of interest. the 2-de is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells, tissue, or other biological samples. (16) one problem that had to be overcome was the lack of sensitive protein sequencing technology. the f i r s t m a j o r t e c h n o l o g y t o e m e rg e f o r t h e identification of proteins was the sequencing of proteins by edman degradation.(17) followed by image analysis and mass spectrometry to quantitify and to characterize proteins.(18) the aim of this study was to investigate protein expression of dengue type 2 virus-infected c6/ 3 6 c e l l s t h r o u g h t w o d i m e n s i o n a l g e l electrophoresis followed by n-terminal edman d e g r a d a t i o n s e q u e n c i n g t o i d e n t i f y t a rg e t proteins. material and methods preparation of uninfected sample c 6 / 3 6 c e l l s f r o m t h e d e p a r t m e n t o f medical microbiology, faculty of medicine, u n i v e r s i t y o f m a l a y a , m a l a y s i a w e r e m a i n t a i n e d i n r p m i 1 6 4 0 m e d i u m ( f l o w laboratories, u.k.) supplemented with 2% fetal calf serum, and incubated at 28°c. the c6/36 cells were disrupted by repeated fast freezing, three times for 15 minutes, and subsequently pelleted by spinning at 4°c, 2000 rpm for 10 minutes. the pellet was twice washed with 1 ml cold-pbs (phosphate saline buffer), spun as above, then resuspended with 1 ml lysis buffer (8m urea, 4% triton-x 100, universa medicina vol.26 no.2 76 muliawan nonstructural glycoprotein ns1 2% ipg-phor buffer 3-10), incubated for 1 hour at 4°c and again spun as above. the supernatant was stored at -20°c. dv2-infected c6/36 cells dengue virus-2 (new-guinea c strain) was obtained from the department of medical microbiology, faculty of medicine, university of malaya, malaysia. virus stocks were used to infect 80% confluent cell monolayers in rpmi1640 supplemented with 2% fetal calf serum and incubated at 28°c until cytopathic effect (cpe) was observed (up to day-4), at which stage the supernatant and cell monolayers were harvested. cell processing used the same method as described for uninfected cells. two-dimensional gel electrophoresis two-dimensional gel electrophoresis (2dge) was performed using the multiphore ii e l e c t r o p h o r e s i s s y s t e m b a s e d o n t h e r e c o m m e n d e d m e t h o d b y a m e r s h a m biosciences.(19) sample was diluted in buffer containing 8m urea, 0.5% (v/v), triton x-100, 0.5% (v/v), ipg-phor buffer 3-10 and 12 mm dithiothreitol (dtt). pre-cast immobilized dry strips ph 3-10 (amersham biosciences) were rehydrated overnight in the same solution. the strips were aligned in a tray that was filled with mineral oil (amersham biosciences). isoelectric focusing was performed in three steps by varying the voltage as suggested by the manufacturer. for the second dimension, the strips were incubated for 15 minutes in a solution consisting of 6m urea, 1% (w/v) sds, 30% (v/v) glycerol and 0.3m dtt dissolved in 0.05m tris-hcl (ph 6.8). this was followed by incubation for another 15 minutes in a similar solution but containing 50mm iodoacetamide instead of dtt. st r i p s w e r e p l a c e d o n a s o d i u m d o d e c y l sulphate-polyacylamide gel electrophoresis (sds-page) 10% homogeneous gel (amersham biosciences) and electrophoresed for 100 minutes. visualization step was done by silver staining. for determination of molecular weight of the target proteins, marker with low range w a s r u n t o g e t h e r o n s d s pa g e w i t h t h e molecular weight kit in range of 45 kda and 66 kda (bio-rad manufacturer). immundblotting and immund detection western blotting was performed according to the standard method.(20) nova blot kit of m u l t i p h o r e i i e l e c t r o p h o r e s i s s y s t e m (pharmacia, uppasala, sweden) was used to transfer the protein to nitrocellulose membrane (protran-schlicher and schuell-0.45 µm). the unit was run at 0.8ma/cm2 for 2 hours. after transfer was completed, immunodetection procedure was performed using hyperimmune anti rabbit sera against dv2 and amplified a l k a l i n e p h o s p h a t a s e g o a t a n t i r a b b i t immunoblot assay kit (bio-rad). the reaction was stopped by washing the membrane with distilled water. preparation of sample for edman sequencing western blotting was performed as per standard method.(20) nova blot kit of multiphore i i e l e c t r o p h o r e s i s s y s t e m ( p h a r m a c i a , uppasala, sweden) was used to transfer the p r o t e i n t o p o l y v i n y l d i f l u o r i d e ( p v d f ) membrane (0.45 µm). to vasilitate the nterminal sequencing process, the infected c6/36 cells were run together on sds-page with the molecular weight kit. after wards, the protein spots from sds-page were transferred to pvdf membrane and visualized with coomassie brilliant blue staining (figure 2), then sent for n-terminal edman degradation sequencing (midwild analytical, usa). results t h i s i n v e s t i g a t i o n o f d e n g u e p r o t e i n expression revealed four target protein spots on 77 dv2-infected c6/36 (figure 1a), but not on uninfected c6/36 (figure 1b). the result of immunoblotting and immunodetection of infected c6/36 cells showed the target protein spots (figure 2a) were picked by antibody against dv2 and were visualized as a purple color (figure 2b). the molecular weight of the protein spots in infected c6/36 cells was 50 kda in (figure 3a) and immunoblotting of infected c6/36 cells onto pvdf membrane with coomassie blue staining for n-terminal sequencing purpose was s h o w e d f i g u r e 3 b . n-terminal sequencing determined the protein spots to be d-s-g-c-vv-s-w-k-n-k (midwild-analytical,usa), which was identical to nonstructural glycoprotein ns1 ( s w i s s p r o t d a t a b a s e ) a s s o c i a t e d w i t h t h e replication process of flaviviruses. discussion in this study, the aim of this study was to observe the protein expression of uninfected and infected c6/36 cells by dv2 through 2-dge. this study found protein spots in dv2-infected c6/36 cells which were absent in uninfected c6/ 36 cells (figure 1). immunoblotting was done to prove the target protein spots originally came from dv2 infection. based on swiss-prot database, the 50 kda protein spots turned out to have the following sequence: d-s-g-c-v-vs-w-k-n-k, which was determined to be homologous to non structural ns1. glycoprotein ns1, present in all flaviviruses, appears to be essential for virus viability. ns1 is produced both membrane-associated an secreted forms(5) both of which are highly immunogenic and can elicit high-titre antibody.(11) this is presumably because ns1 is present on the surface of infected cells and is efficiently secreted.(21) in the post genomic era, proteins are coming back into focus because it has been realized again than whole genome sequence information alone is not s u ff i c i e n t t o e x p l a i n a n d p r e d i c t c e l l u l a r phenomena, as it is largely the proteins that execute and control the majority of cellular activities. there are several reasons for focusing on the analysis of proteins, such as the level of mrna expression frequently does not represent the amount of active protein in a cell(22) or the g e n o m e s e q u e n c e d o e s n o t d e s c r i b e p o s t t r a n s l a t i o n a l m o d i f i c a t i o n , w h i c h m a y b e essential for protein function and activity. in this study, the protein spots did not migrate to the s a m e l e v e l , p r o b a b l y d u e t o t h e d i ff e r e n t g l y c o s y l a t i o n p a t t e r n s . ( 1 3 ) n s 1 p r o t e i n expression through proteomics technique might be of use as a diagnostic marker and/or as a parameter for evaluation of the efficacy of antiviral agents in dv2 infection. the value of using ns1 as a surrogate marker of infection is supported by young et al who assessed the potential of capture elisa as a diagnostic assay(23) and huang et al who showed high level expression of recombinant dengue viral ns1 protein used as a diagnostic antigen.(24) there are no known reports from other researchers on this protein using identical or similar techniques. conclusion the application of ns1 separated by 2dge may provide an alternative approach for monitoring or as a parameter for evaluation the e ff i c a c y o f a n t i v i r a l a g e n t s a g a i n s t d v 2 infection/replication. acknowledgements the author expresses her gratitude to prof. lam sai kit, prof. shamala devi, prof onn hashim, prof. rohana yusof from faculty of medicine, university of malaya, kuala lumpur, malaysia and also to associate prof. dr. thomas j. chambers, saint louis university school of medicine, st.louis, usa for their keen interest and support in this study. universa medicina vol.26 no.2 78 muliawan nonstructural glycoprotein ns1 figure 1. protein expression of (a) infected c6/36 cells with dv2 and (b) uninfected c6/36 cells (a) (b) figure 2. protein expression of (a) infected c6/36 cells with dv2 and (b) immunoblotting and immunodetection of infected c6/36 cells with specific antibody against dv2 onto nitrocellulose membrane (a) (b) 97kda 66kda 45kda 30kda figure 3. protein expression of (a) infected c6/36 cells with the molecular weight of target protein spots and (b) immunoblotting of infected c6/36 cells onto pvdf membrane with coomassie blue staining (a) (b) 79 references 1. cisneros a, diaz-bdillo a, cruz-martinez g, tovar r, amirez-palacios lr, jimenez-rojaz f, et al. dengue 2 genotypes in the state of oaxaca, mexico. arch virol 2006; 151:113-25. 2. world health organization. epidemic and pandemic alert and response impact of dengue. world health organization, geneva, switzerland 2005. available at: http:/www.who.int/csr/disease/ dengue/impact/en/index.html. accessed 18 april, 2006. 3. gubler d. dengue virus and dengue hemorrhagic fever. j clin microbiol rev 1998; 11: 480-96. 4. mcbride wjh, ohmann hb. dengue viral infections; pathogenesis and epidemiology. microbes infect 2000; 2:1041-50. 5. dussart p, labeau b, lagathu g, louis p, nunes mrt, rodrigues sg, et al. evaluation of an enzyme immunoassay for detection of dengue virus ns1 antigen in human serum. clin and vac immunol 2006; pp.1185-9. 6. guzman mg, kourl g. dengue: an update. lancet infect dis 2002; 2:33-42. 7. kalayanarooj s, vaughn dw, nimmannitya s, green s, suntayakorn s, kunentrasai n, et al. early clinical and laboratory indicators of acute dengue illness. j infect dis 1997; 176:313-21. 8. hommel d, talarmin a, deubel v, reynes jm, drouet mt, sarthou jl, et al. dengue 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adv virus res 2003; 59:141-75. 15. huang jl, huang jh, shyu rh, teng cw, lin yl, kuo md. high level expression of recombinant dengue viral ns1 protein and its potential use as a diagnostic antigen. j med virol 2001; 65:553-60. 16. anderson ng, anderson nl. twenty years of twodimensional electrophoresis: past, present and future. electrophoresis 1996; 17: 443-53. 17. edman p. a method for the determination of the amino acid sequence of peptides. arch biochem biophys 1949; 22: 475-83. 18. corthals gl, wasinger vc, hochstrasser df, sanchez jc. the dynamic range of protein expression: a challenge for proteomic research. electrophoresis 2000; 21: 1104-15. 19. berkelman t, stenstedt t. 2-d electrophoresis principles and methods, 1998. 20. shuib as, chua ct, hashim oh. sera of iga nephropathy patients contain a heterogeneous population of relatively cationic alpha-heavy chains. nephron 1998; 78:290-5. 21. wengler g. the carboxy-terminal part of ns3 protein of the west nile flavivirus can be isolated as a soluble protein after proteolytic cleavage and represents an rna-stimulated ntpase. virology 1991; 184: 707-15. 22. anderson l, seilhamer j. a comparison of selected mrna and protein abundances in human liver. electrophoresis 1997; 18: 533-7. 23. young pr, hilditch pa, bletchly c, halloran w. an antigen capture enzyme-linked immunosorbent assay reveals high levels of the dengue virus protein ns1 in the sera of infected patients. j clin microbiol 2000; 38:1053-7. 24. huang jl, huang jh, shyu rh, teng cw, lin yl, kuo md. high level expression of recombinant dengue viral ns1 protein and its potential use as a diagnostic antogen. j med virol 2001; 65: 553-60. universa medicina vol.26 no.2 c:\users\jurnal fkusakti\docume 160 *departmen t of oph thalmology, faculty of medicine, christian university of indonesia, jakarta/ cikini cci hospital jakarta correspondence: dr. gilbert ws simanjuntak, dr, spm(k) department of ophthalmology, faculty of medicine christian university of indonesia jalan ps sutoyo cawang jakarta indonesia email: retinaid@yahoo.com telephone : +8211168237 date of first submission, july 19, 2017 date of final revised submission, september 26, 2017 date of acceptance, september 29, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 macular hole surgery under retrobulbar local anesthesia increases visual acuity gilbert ws simanjuntak*, golda a m simanjuntak*, and reinne natali christine* background macular hole surgery can be done under general or local anesthesia, and general anesthesia is still the standard procedure. the objective of this study was to investigate the results of macular hole surgery under local anesthesia. methods a retrospective study on medical records of patients who had undergone macular hole surgery under local anesthesia. funduscopic examination of macular hole had been performed and all other diseases with deterioration of visual acuity eliminated. surgery was performed under retrobulbar local anesthesia, using 2 ml of 2% lidocaine mixed with 3 ml bupivacaine in one syringe. vitreous tamponade used 20% sulfur hexafluoride (sf 6 ). demographic and clinical characteristics, visual acuity before and after surgery, and improvements that occurred were assessed. results no significant interruption occurred during surgery. mean age of the patients was 53.89 ± 13.3 years (22-74 years), with mean duration of symptoms of 48.83 ± 100.51 weeks (1-560 weeks). the patients comprised 20 males (55.6%) and 16 females (44.4%). there were improvements in visual acuity after surgery in 31 patients (86.1%), the rest (13.9%) had identical or lower visual acuity after surgery. a total of 20 patients (55.6%) had postoperative visual acuity of 6/18 or better. initial mean preoperative visual acuity was 0.08 ± 0.08 (0.01 0.29) and improved after the surgery to 0.38 ± 0.26 (0.00 1.00), the improvement being statistically significant (p=0.000). conclusion retrobulbar anesthesia is an effective and safe method in macular hole surgery. macular hole surgery can be done well under local anesthesia, and visual acuity improved significantly. keywords: macular hole, anesthesia, blindness, visual acuity, vitrectomy doi: http://dx.doi.org/10.18051/univmed.2017.v36.160-165 original article cite this article as: simanjuntak gws, simanjuntak gam, christine rn. macular hole surgery under retrobulbar local anesthesia increases visual acuity. univ med 2017;36:160-5. doi: 10.18051 /univmed.2017.v36.160-165 pissn: 1907-3062 / eissn: 2407-2230 161 univ med vol. 36 no.3 introduction macular holes may occur spontaneously, presumably due to a dehiscence on the macular surface.(1) other possible causes are trauma, cystoid macular edema, and abnormal macular traction such as in vitreomacular traction syndrome, epiretinal membrane or tractional retinal detachment.(2) usually a macular hole is accompanied by localized retinal detachment around the hole, and a tiny retinal operculum attached to the vitreous that exerts traction upon it. a macular hole can be directly seen and diagnosed at fundoscopy.(1,2) macular hole management is by surgery. before 1991, the disease could not be resolved and ended with visual impairment. after kelly and wendel (2) reported the results of vitrectomy surgery with a gas tampon, which succeeded in closing the macular hole, the experts competed t o r e po r t go od r e s u l ts a f t e r vi t r e c t o my. vitrectomy is generally done under general anesthesia. lo c a l a ne st h e s ia t e c h ni q ue s f o r vitreoretinal surger y include retrobulbar, subtenon, peribulbar, and topical anesthesia.(3) although the advantages of local anesthesia are well known and include more rapid return to ambulation for the patient, ability to perform the surgery as an outpatient procedure, avoidance of complications of general anesthesia, and quicker surgery, it has been the subject for some controversy.(4)  in indonesia, vitrectomy under local anesthesia was reported to be a standard in one of the hospitals.(5 ) operation under local anesthesia will make for faster and safer patient mobilization.(6) patients’ difficulty to sleep on their stomach for at least a week is very burdensome, especially for those who have problems with the spine. as an alternative to gas, some surgeons have recommended silicone oil. each technique has its advantages and disadvantages. the visual improvement after surgery is the more important issue and also the goal.(3,6) however, the visual experience of the patients while under local anesthesia was not well recognized or understood until a few years ago. most patients undergoing vitreous surgery under local anesthesia experience a variety of intraoperative visual sensations.(7) the purpose of this study was to investigate the results of macular hole surgery under local anesthesia. methods research design this was a retrospective analytic study by collecting medical records of patients who underwent macular hole surgery under local anesthesia at cikini cci hospital over a period of 12 years (2004 – 2015). ophthalmologic examination regular ophthalmologic examinations were performed which included examination of corneal clarity, especially of the central area, lens clarity; iris examination and to find anterior and posterior synechiae if any. visual acuity (va) before surgery was checked and pre operative best-corrected va of all patients was 0.08 ± 0.08. the posterior segment examination was performed thoroughly and carefully, looking at the conditions of the peripheral retina and the macula specifically using a 90d lens with magnification and estimating the size of the hole by comparing with the caliber of the veins coming out of the optic nerve (n. ii); looking at the condition of the optic nerve, and examining the intraocular pressure (iop). as far as possible, the macular hole size was predicted clinically by using optical coherence tomography (oct). if the hole was calculated to be more than 600 microns in diameter, then the operation was canceled or if the patient still requested surgery then the less promising postoperative vision prognosis was explained. surgery for macular hole t he ope ra t io n wa s p er f or me d un de r retrobulbar local anesthesia, using 2 ml of 2% 162 lidocaine mixed with 3 ml of bupivacaine in one syringe. when necessary, an additional 2 ml of lidocaine was used for subtenon injection during surgery towards the optic nerve. the pupils were dilated maximally by dripping 1% tropicamide (mydriatil) and 10% efrisel at intervals of 10 minutes for a minimum of 3 times, before surgery. vitrectomy was done as cleanly as possible. if there was a retinal tear or peripheral lesion, a laser was performed. internal limiting membrane (ilm) staining was performed with trypan blue in an air tampon, and was left for one minute then the ilm peeled with forceps. 20% sulfur hexafluoride (sf 6 ) gas was given after surgery as vitreous tamponade. after surgery, the p a t i e nt ’s sl e e pi n g p o si t i on wa s t i l t e d contralaterally toward the unoperated eye for 2 days, and from the third day until the seventh was made completely downward except for eating and toilet activities. measurements any important clinical findings obtained during and after surgery were recorded. patients were instructed to make follow up visits at 1, 7 and 14 days after surgery in the first month. afterwards every 1-2 months for the first 6 months. postoperative treatment consisted of topical steroids and antibiotics that were gradually reduced for 1 month, and oral antiglaucoma medications for 2 weeks. tamponade used was sf 6 20% gas. a va and intraocular pressure examination before surgery and at every postoperative visit was made by opticians who were unaware of the type of surgery or p at ie nt i ll n ess . pat ie n ts wh o e x pe r i e nc e cataracts, will be operated two months after macular surgery. only patients with a full threemonth follow-up were included in this study. subjects were not included if they had obscured media or optic nerve damage. statistical analysis independent t-test was used to analyze the data. normality of the data was tested with the kolmogorov-smirnov test, at 95% confidence interval. ethical clearance informed consent was obtained after the patients clearly understood the risks involved, including the anatomical and functional success rates and the actions required if a second operation was required. the christian university of indonesia institutional ethics committee granted approval for this study following the tenets of the declaration of helsinki (no. 17/ pen/km/2017). results there were 48 medical records of patients who had undergone macular hole surgery at cikini cci hospital from 2004-2015. a total of 12 medical records were not evaluated due to incomplete records, or incomplete follow up visit for three months. a total of 36 medical records were included, and all had undergone macular hole surgery under local anesthesia. during surgery, there was no additional anesthesia, either subtenon, subconjunctival or retrobulbar injection. based on the surgical reports, there were no significant complaints such as patient discomfort during surgery, or other complaints that interfered with surgery, such as inability of patients to calm down, shortness of breath, frequent movements, or fear. the operation was performed with 20g instrument and the sclerotomy incision was closed with one stitch of 8.0 vycril. all patients were filled with sf 6 20% gas tamponade, no patients had spinal disorders, so that they could sleep on their stomach for a minimum of 1 week, and all patients made complete follow up visits. the subjects’ mean age was 53.89 ± 13.30 years (22-74 years), with a mean macular hole duration of 48.83 ± 100.51 weeks (1-560 weeks). the subjects consisted of 20 males (55.6%) and 16 females (44.4%). there was va improvement after surgery in 31 patients (86.1%), in the remaining (13.9%) the va was the same or simanjuntak, simanjuntak, christine macular hole surgery under local anesthesia 163 univ med vol. 36 no.3 worse after surgery. a total of 20 patients (55.6%) had a va of 6/18 or better after surgery. the pre-operative va was 0.08 ± 0.08 (0.01 0.29), and improved after surgery to 0.38 ± 0.26 (0.00 to 1.00), statistically significant (p=0.00) (table 1). as a detailed illustration, a funduscopic view and an optical coherence tomography (oct) examination of one patient before and after the surgery, is presented. it appeared that the hole and localized retinal detachment becomes closed and attached, respectively, after surgery. there was improvement in vision after surgery (figure 1). discussion retinal surgery, including macular hole surgery, is generally done unde r gener al anesthesia. only a small portion of the center serves the surgery under local anesthesia as the primary choice. there are several commonly used local anesthetic agents. bupivacaine has the advantage of a block effect of long duration, but its onset is slow when used singly under anesthesia.(8) although lidocaine has a rapid onset of analgesia and akinesia, the effect is shorter.(9) therefore, a mixture of balanced volumes of bupivacaine and lidocaine is often used to achieve rapid onset of analgesia with prolonged effect. this effect may be extended during surgery under local anesthesia by subtenon injection, or during general anesthesia in pediatric patients.(10,11) the study of zhou et al.(9) found that the combination of lidocaine and bupivacaine has a time of onset of analgesia of 101.51  56.94 minutes and onset of akinesia of 141.00  62.69 minutes. local anesthesia is also an alternative to general anesthesia in other medical procedures. this has been reported in cardiac procedures,(12) cerebral biopsy,(13) and surgery for umbilical hernias.(6) related to the cooperativeness of the patients in ocular surgery, pain perception is positively influenced by female gender, rural place of residence, younger age of patient, and lower preoperative visual acuity.(14) recovery time under local anesthesia was almost insignificant compared to general anesthesia, and this recovery time may differ between drugs used,(15) or procedures.(16) one of the disadvantages of retrobulbar injections under local anesthesia is the possibility of accidentally penetrating the eyeball.(17) characteristic sex (n,%) male female age (years) duration of symptoms (weeks) visual acuity (va) initial va final va p value 20 (55.6) 16 (44.4) 53.89 ± 13.30 48.83 ± 100.51 0.08 ± 0.08 0.38 ± 0.26 0.001 table 1. characteristics of patients undergoing macular hole surgery under local anesthesia (n=36) figure 1. (a). funduscopic view with macular hole, accompanied by a localized retinal detachment around the macula; (b). oct results clearly show the full thickness macular hole, va of 0.1; (c). the operating result shows the macular hole closed, with improved va, 0.5 a b c 164 fear of the patient moving spontaneously during surgery, uneasiness and pain was not found in the present surgical report records. the possibility of such disturbance maybe existed but was not to be regarded either as complicating the operation or as significant, and therefore not recorded. another possibility is that the patients had received complete information, so that they were quieter on the operating table. this may be seen from the 86.1% improvement of vision after surgery. these improvements were significant when compared with the results of existing operations in other countries, ranging from 42 to 100%, with a mean of 65.2%.(18) in the present study it can be seen that 55.6% of subjects obtained a visual acuity 6/18 or better after surgery. in comparison, in the study by chu et al.,(8) the surgery was a combination of pars plana vitrectomy, inverted internal limiting membrane flap technique and intraocular gas tamponade for macular hole–induced retinal detachment, the resulting retinal reattachment rate was 100% and the rate of macular hole closure was 73.3%. the mean of best-corrected visual acuity (bcva), converted to the logarithm of the minimal angle of resolution (logmar), improved from 1.73 ± 0.69 to 1.16 ± 0.51 logmar (p<0.05). in the present study the number of male and female subjects was comparable, whereas in other study settings, the females were almost twice the number of males.(18) possible demographic factors in indonesia, where patients in cikini cci hospital generally come from areas outside java are also worth c onsi dering. t he current prevalence of blindness in indonesia of 3.2% is in line with rapid assessments of avoidable blindness (raab) research results in several provinces.(19) among these cases of blindness, there was blindness due to retinal disease, such as macular diseases. in comparison, it has been estimated that there are new cases of retinal disorders of 17,500 25,000 per year.(20,21) this was evidenced by the results above, that macular surgery under local anesthetic can be performed successfully, resulting in va improvement of 86.1%. our study was limited by the small sample size and retrospective nature. a multivariate study with a larger number of patients is needed to confirm our findings. conclusion this study demonstrated that macular hole surgery can be done under local anesthesia with a significant visual acuity improvement. conflict of interest none. no authors have any financial/ conflicting interests to disclose. acknowledgement the authors wish to express their gratitude to the faculty of medicine, christian university of indonesia, for supporting this study. contributors gwss contributed to the concept and de sign of the study, data analysis and/or interpretation. gams contributed to data acquisition and drafting. rnc revised the manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. johnson wm. perifoveal vitreous detachment and its macular complication. trans am ophthalmol soc 2005;103:537-67. 2. liu w, grzybowski a. current management of traumatic macular hole. j ophthtalmol 2017;17:18. 3. seidenari p, santin g, milani p, et al. peribulbar and retrobulbar combined anesthesia for vitreoretinal surgery using ropivacaine. eur j ophthalmol 2006;16:295–9. 4. chan vm, loo avp, tam bsm, et al. topical anesthesia in posterior vitrectomy. retina 2000; 20:563–4. 5. simanjuntak gws. surgical result of pseudophakic retinal detachment in cikini cci simanjuntak, simanjuntak, christine macular hole surgery under local anesthesia 165 univ med vol. 36 no.3 hospital-school of medicine christian university of indonesia. j ophthalmol indones 2009;7:52-5. 6. jairam ap, kaufmann r, muysoms f, et al. the feasibility of local anesthesia for the surgical treatment of umbilical hernia: a systematic review of the literature. hernia 2017;21:223-31.doi: 10.1007/s10029-017-1577-z. 7. prasad n, kumar cm, patil bb, et al. subjective visual experience during phacoemulsification cataract surgery under sub-tenon’s block. eye 2003;17:407-9. 8. chu b, tsang s, yuen c. a combination of pars plana vitrectomy, inverted internal limiting membrane flap technique and intraocular gas tamponade for macular hole-induced retinal detachment. hkj ophthalmol 2016;20:109-13. 9. zhou y, tong y, wang y, et al. a prospective, randomised, double-masked comparison of local anaesthetic agents for vitrectomy. br j ophthalmol 2017;0:1-6. doi:10.1136/bjophthalmol-2016309780. 10. chhabra a, sinha r. subramaniam r, et al. comparison of sub-tenon’s block with i.v. fentanyl for paediatric vitreoretinal surgery. br j anaesth 2009;103:739-43. 11. ghai b, ram j, makkar jk, et al. subtenon block compared to intravenous fentanyl for perioperative analgesia in pediatric cataract surgery. anesth analg 2009;108:1132-8. 12. jabbar a, khurana a, mohammed a, et al. local versus general anesthesia in transcatheter aortic valve replacement. am j cardiol 2016;1;118:171216. 13. quick-weller j, konczalla j, duetzmann s, et al. general anesthesia versus local anesthesia in stereotactic biopsies of brain lesions: a prospective randomized study.world neurosurg 2017;105:16-20. 14. omulecki w, laudanska-olszewska i, synder a. factors affecting patient cooperation and level of pain perception during phacoemulsification in topical and intracameral anesthesia. eur j ophthalmol 2009;19: 977–83. 15. mir ah, shah nf, din mu, et al. effectiveness of sodium thiopentone, propofol, and etomidate as an ideal intravenous anesthetic agent for modified electroconvulsive therapy. saudi j anaesth 2017;11:26-31. doi: 10.4103/1658-354x. 197339. 16. jain ra, shetty an, oak sp, et al. effect of avoiding neuromuscular blocking agents during maintenance of anaesthesia on recovery characteristics in patients undergoing craniotomy for supratentorial lesions: a randomised controlled study. indian j anaesth 2017;61:42-7. doi: 10.4103/0019-5049.198408. 17. wadood ac, dhillon b, singh j. inadvertent ocular perforation and intravitreal injection of an anesthetic agent during retrobulbar injection. j cataract refract surg 2002;28:562-5. 18. simanjuntak gws. surgical approach for macular diseases. makassar: pertemuan ilmiah tahunan dokter spesialis mata;2008. 19. das t, ackland p, correia m, et al. is the 2015 eye care service delivery profile in southeast asia closer to universal eye health need! int ophthalmol 2017;2: 1-10. doi: 10.1007/s10792-0170481-y. 20. gupta op, benson we. the risk of fellow eyes in patients with rhegmatogenous retinal detachment. curr opin ophthalmol 2005;16:1758. 21. mitry d, charteris dg, fleck bw, et al. the epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations. br j ophthalmol 2010;94:678-84. c:\users\jurnal fkusakti\docume 75 *department of microbiology, faculty of medicine, trisakti university **department of microbiology, faculty of medicine, university of indonesia ***department of dermatovenereology, cipto mangunkusumo hospital correspondence: dr. ida effendi, spmk department of microbiology, faculty of medicine, trisakti university, jl. kyai tapa 260, jakarta 11450, indonesia phone: +6221 5672731 fax: +6221 5660706 email: idaeffendi@trisakti.ac.id date of first submission, february 6, 2018 date of final revised submission, april 19, 2018 date of acceptance, april 19, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina multiplex nested polymerase chain reaction for treponema pallidum using blood is more sensitive than using serum ida effendi*,**, yeva rosana**, andi yasmon**, and wresti indriatmi*** background syphilis is a multistage disease transmitted primarily through sexual intercourse. nowadays, the polymerase chain reaction (pcr) test for treponema pallidum has been widely used and is expected to overcome problems in diagnostic tests for syphilis. the treponema pallidum pcr is influenced by type of specimens, pcr methods and target genes. this study aimed to assess the use of blood and serum in multiplex nested pcr for treponema pallidum, targeting the 23s rrna. methods a cross-sectional study was conducted from april 2015 april 2016. sampling was carried out consecutively among patients with clinical features of secondary syphilis who came to sexually transmitted disease (std) clinics in jakarta. all sera were also tested with rapid plasma reagin (rpr) and treponema pallidum hemagglutination assay (tpha) assay, which was considered as the gold standard for this study. we determined the sensitivity and specificity of the multiplex nested pcr for treponema pallidum using blood and serum. results pcr test was performed on 122 clinical specimens (61 blood and 61 serum). the positive results of pcr test on blood was 22.95% and serum was 6.56%, while the positive results of serology was 68.85%. the sensitivity of treponema pallidum multiplex nested pcr on blood was 30.95% compared to serum 9.52% (p=0.006). pcr test on blood is able to detect 3.25 times higher than serum. conclusion the use of blood has a higher proportion of positives compared to serum in treponema pallidum multiplex nested pcr using 23s rrna target gene. keywords: secondary syphilis, treponema pallidum, multiplex nested pcr test, blood, serum doi: http://dx.doi.org/10.18051/univmed.2018.v37.75-84 original article pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 cite this article as: effendi i, rosana y, yasmon a, et al. multiplex nested polymerase chain reaction for treponema pallidum using blood is more sensitive than using serum. univ med 2018;37:7584. doi: 10.18051/univmed.2018.v37. 7584 76 effendi, rosana, yasmon, et al multiplex nested pcr for treponema pallidum introduction syphilis is one of the multistage diseases that are transmitted mainly through sexual intercourse. this dise ase shows diverse clinical manifestations, a protracted course, and serious complications. syphilis constitutes a global health problem particularly in developing countries, including indonesia.(1) since this disease was first known in the fifteenth to twentieth centuries, the prevalence of syphilis had already decreased, but in the last few decades, the incidence of early syphilis in various countries in america, europe, and asia was reported to be again on the increase.(1, 2) in indonesia, based on a survey conducted by the integrated surveillance of behavioral biology program (surveilans terpadu biologi perilaku, stbp) in the years 2007 and 2011 on the population most at risk, there was an increase in the prevalence of syphilis.(3) this occurred particularly in the population most at risk, i.e. males who had sexual intercourse with males (msm), from a prevalence of 4% in 2007 to 13% in 2011. the highest prevalence of syphilis was found in the transvestite population, i.e. a total of 28%.(3) according to data from the who in 2008, the prevalence of syphilis in southeast asian countries was 12.3 million, i.e. 6.2 millions in women and 6.1 millions in males. (4) in 2012, who estimated that there were 6 million new cases of syphilis each year in the whole world and most case s occu rre d in developing countries.(5) the diagnosis of syphilis is established based on anamnesis, physical examination of the patient and accessory examination. the difficulty in diagnostic testing of syphilis is due to the fact that treponema pallidum cannot be cultured in vitro.(6,7) microscopic examination may directly evaluate the presence of treponema pallidum, but requires a special microscope or special staining, and cannot differentiate between treponema species. at present, laboratory facilities for microscopic examination of syphilis are very limited. in addition, experienced laboratory technicians are required to evaluate treponema pallidum by microscopy. serologic tests are diagnostic tests for syphilis that are commonly used, including in indonesia. the extremely sensitive nontreponemal tests are followed by the more specific treponemal confirmatory tests. however, there are several limitations in nontreponemal or treponemal tests, as they may show false positive, false negative, or dubious results. at present, the polymerase chain reaction (pcr) for treponema pallidum is widely used and is expected to be able to decrease the problems in diagnostic testing of syphilis. the published studies on the treponema pallidum pcr test comprise conventional pcr, real-time pcr, nested pcr and multiplex pcr, with various target genes specific for treponema pallidum, such as the target genes pola, tpp47, and bmp. at present, the pcr methods have become the diagnostic tests recommended by the cdc and the united kingdom for detecting treponema pallidum, in addition to microscopy.(8,9) the treponema pallidum pcr test may use various types of clinical specimen depending on the course of the disease. the sensitivity and specificity of the molecular pcr test results are affected by the type of specimen, the pcr method, and the target gene used.(10,11) spe cimens that ma y be used for the treponema pallidum pcr test in secondary syphilis are exudates from syphilitic lesions and blood. the sensitivity of pcr with smears from lesions of secondary syphilis reaches 80% and the specificity 98.6%.(12) the skin lesions in secondary syphilis are extremely infectious. however, not all patients with secondary syphilis show skin lesions; in addition, sample collection from skin lesions of patients with secondary syphilis is more difficult, invasive, and causes discomfort in the patients. blood is the specimen that may used for examination in all stages of syphilis.the number of treponema pallidum in the blood is highest in 77 early syphilis.(13, 14) in addition to the complete set of blood components (whole blood), other blood components may also be used as specimen for the treponema pallidum pcr, such as serum, plasma, and peripheral blood mononuclear cells (pbmc). cruz et al.(13) found that blood is the best type of specimen for detecting treponema pallidum. grange et al.(10) reported on the higher proportion of positives for blood as compared with serum, plasma, and pbmc for nested pcr testing in secondary syphilis. in contrast to that reported by cruz et al.(13) and by gayet-ageron et al. (15) reported that serum was better than blood for detecting treponema pallidum using pcr in secondary syphilis (47% and 36%). the difference between the results of cruz et al.(13) and grange et al.(10) on the one hand and gayet-ageron et al.(15) on the other, is thought to be due to factors of the pcr test system used, such as difference in target gene and primer design. the purpose of 23s rrna with the multiplex nested pcr method is to detect two-point mutations causing treponema pal lidum resistance to azithromycin, one of the alternative drugs of choice for syphilis.(16) therefore this study uses multiplex nested pcr to determine the better specimen between blood and serum for the application of this test. methods research design a cross-sectional study was performed from april 2015 to april 2016. study subjects the study subjects were screened with consecutive methods, i.e. patients aged >18 years with the clinical picture of secondary syphilis who attended the dermatoveneorology polyclinic in cipto mangunkusumo hospital, the sexually transmitted disease (std) clinic of the pasar rebo subdistrict puskesmas, the std clinic of the tambora subdistrict puskesmas and the indonesian familty planning association (perkumpulan keluarga b erencana indonesia, pkbi) procare clinic. patients who had received treatment were excluded from the study. sample size determination the sample size was obtained by considering the proportion of positives of the pcr test with blood samples of secondary syphilis to be 38%.(10) the sample size determination used the following formula: univ med vol. 37 no.1       2 2p2p1 p2q2p1q1zβ2pqzα n2n1            where n1=n2= sample size zα= 1.96 zβ= 0.842 p1 = proportion of standard effect 1 p2 = proportion of investigated effect (clinical judgment) [proportion of positives ] p = ½ (p1+p2) p2 = 38% =0.38 q2 = 1-p2 = 0.62 p1-p2 = 25% = 0.25 p1 = 0.63 q1 = 0.37 p = 0.50 q = 0.5 so that the minimal sample size was 61 per group. intervention from all patients, blood was drawn from the vein in the cubital fossa into two vacutainer tubes using sterile 23g ¼ inch needles. the blood was drawn to a volume of 3-4 ml into the red-capped tube without anticoagulant and to a volume of 12 ml into the violet-capped tube containing edta anticoagulant. the blood in the tube without anticoagulant was processed into serum, which was used for the rpr and tpha serological tests. whole blood and serum were used for treponema pallidum multiplex nested pcr testing. 78 effendi, rosana, yasmon, et al multiplex nested pcr for treponema pallidum a b c measurements the rpr and tpha tests were performed quantitatively to det er mine the titration concentrations. the serological test results in patients with the picture of clinical secondary syphilis were considered to be positive if an rpr titer of >1:8 and a tpha titer of >1:80 were obtained. a positive treponema pallidum multiplex nested pcr test was shown by a 187bp band on visualization in 3% agarose gel after electrophoresis at 100 volts for 30 minutes, as internal control indicating the presence of treponema pallidum. if bands of 130 bp and 100 bp were also found, these show the presence of mutations at 2 points in the dna sequence of azithromycin-resistant treponema pallidum. laboratory analysis qualitative rpr testing used a commercial kit (rpr aim cat. no. e rpr 2) on the sera of all patients according to the protocol of the kit. the results that were reactive on qualitative rpr testing were to be followed with the quantitative rpr test. all samples subjected to the rpr test were confirmed with the quantitative tpha (tpha aim cat. no.tpha e 100) according to the protocol of the kit. the pcr test used the dna resulting from the extraction of blood and serum that were stored at a temperature of -35oc. blood was extracted according to the protocol of the roche kit (high pure pcr dna template preparation kit). serum was extracted according to the protocol of the qiagen kit (qiagen qiamp dna mini kit). the multiplex nested pcr test used treponema pallidum 23s rrna target genes and was already optimized using 3 (three) pairs of primers.(16) the pcr 1 reaction used one pair of primers (forwardreverse) for detecting the 23s rrna component of treponema pallidum. the reaction mixture with total volume of 20 µl consisted of distilled water (dnase-free water) 7.5 µl, 10x hotstar pcr buffer 2.0 µl, 25 µm mgcl 2 0.2 µl, 10 µm dntp mix 0.4 µl, 5x q solution 4.0 µl, 10 µm pcr1 f primer 0.4 µl, 10 µm pcr1 r primer 0.4 µl, and hotstar dna polymerase enzyme 0.1 µl. a volume of 5 µl dna was introduced to the reaction mixture as template. the pcr 1 reaction consisted of a tota l of 25 cycles at a predenaturation temperature of 95ºc for 15’, denaturation at 95ºc for 30", annealing at 58ºc for 30", elongation at 72ºc for 30", and final extension at 72ºc for 10’. the pcr 2 reaction used two pairs of primer (forward-reverse) with the purpose of amplifying the dna sequence of treponema pallidum where there are mutation points to identify azithromycin-resistant treponema pallidum based on the mutation patterns of adenine bases into guanine in the dna sequence positions 2058 and 2059 of the 23s rrna gene. the reaction mixture at a total volume of 20 µl consisted of distilled water (dnase-free water) 9.2 µl, 10x hotstar pcr buffer 3.0 µl, 25 µm mgcl 2 0.2 µl, 10 µm dntp mix 0.4 µl, 5x q solution 4.0 µl, 10 µm pcr 2058f primer 0.9 µl, 10 µm pcr 2058r primer 0.5 µl, 10 µm pcr 2059f primer 0.3 µl, 10 µm pcr 2059r primer 0.8 µl, and hotstar dna polymerase 0.3 µl. a volume of 1 µl pcr 1 products were introduced into the mixture as a template. the pcr 2 reaction comprised 35 cycles at predenaturation temperature of 95ºc for 15’, denaturation at 95ºc for 30", annealing at 61ºc for 30", elongation at 72ºc for 1’ and final extension at 72ºc for 5’. the positive controls used were 194 bp-long positive synthetic dna controls, which were used in the pcr 2 reaction. the negative controls in the pcr 1 reaction used dnase free water. the pcr 2 products were subjected to electrophoresis using 3% agarose. the marker used was the 50 bp marker. the agarose gel was subjected to electrophoresis at 100 volts for 30 minutes, then stained with gel red staining and visualized using uv radiation with the biorad® geldoc™ ez imager. the pcr 2 products were shown as bands of 187 bp, 130 bp and 100 bp, respectively. statistical analysis the collected data were processed with the spss software version 20 and microsoft excel 79 2007. to obtain the significance of the treponema pallidum multiplex nested pcr test results, sensitivity and specificity were used to evaluate the diagnostic test. ethical clearance this study obtained ethical clearance from the health research ethics committee, faculty of medicine,universitas indonesia, under no. 158/ un2.f1/etik/2015. this study also obtained approval from cipto mangunkusumo hospital (rscm), the jakarta one-door integrated service board (badan pelayanan terpadu satu pintu jakarta), recommendation and research permit from the jakarta health service head office (dinas kesehatan jakarta), a nd recommendation and research permit from the west jakarta health service sub-office (suku dinas kesehatan jakarta barat). results the collected study sample numbered 122 clinical specimens, consisting of 61 blood specimens and 61 sera from 61 patients with the clinical picture of secondary syphilis. the study subjects were predominantly males with median age of 26 years and overall age range of 18-40 years. a total of 88.5% (54 study subjects) had a homosexual orientation, i.e. males who had sexual intercourse with males. the majority of study subjects, i.e. 54.10% (33 of 61) had a reactive hiv status (table 1). all sera (61 specimens) were serologically tested for syphilis. a total of 42 of the 61 showed positive results for the rpr and tpha tests, signifying that the proportion of positives of the serological tests for secondary syphilis in the present study was 68.85%. the treponema pallidum multiplex nested pcr test was performed for all blood and serum specimens. in blood, the test showed positive results in 14 of 61 specimens (22.95%), which were marked by the presence of a 187 bp band on visualization with agarose gel. on the other hand, only 4 of the 61 serum specimens (6.56%) were positive. figure 1 shows visualization of the pcr test results on agarose as seen under ultraviolet light. univ med vol. 37 no.1 table l. demographic and clinical characteristics of the study subjects (n=61) characteristic n (%) sex male female 58 (95.08) 3 (4.92) sexual orientation homosexual (msm) heterosexual transvestite 54 (88.52) 6 (9.84) 1 (1.64) status hiv reactive hiv nonreactive hiv no data clinical manifestation maculopapular erythema genital ulcer moth-eaten alopecia uveitis condyloma lata 33 (54.10) 24 (39.34) 4 (6.56) 61 (100) 11 (18.03) 4 (6.56) 3 (4.92) 2 (3.28) figure 1. visualization of positive electrophoresis results band 187 bp specimen no. 15 treponema pallidum multiplex nested pcr test in blood; k= negative control, k+ = positive control, m =50 bp marker; numbers indicate the numbers of specimen teste 80 effendi, rosana, yasmon, et al multiplex nested pcr for treponema pallidum the results of the treponema pallidum multiplex nested pcr test using blood and serum were compared with the results of the serological tests. the proportion of positives of the treponema pallidum multiplex nested pcr test using blood was 22.95%, compared with the proportion of positives of the serological tests of 68.85%. nearly all patients with positive blood results on the treponema pallidum multiplex nested pcr test had positive serological test results. there was one patient (5.26%) whose blood test result on multiplex nested pcr was positive, but whose serological test results did not support secondary syphilis (rpr titer >1:8 and tpha titer 1:80). the proportion of positives in the treponema pallidum multiplex nested pcr test for serum was 6.56% compared with the positive serological test results of 68.85%. all patients’ sera with positive treponema pallidum multiplex nested pcr test results also had serological test results that supported secondary syphilis. treponema pallidum multiplex nested pcr for blood showed a sensitivity of 30.95% and a specificity of 94.7% (table 2), while for serum the test showed a sensitivity of 9.52% and a specificity of 100% (table 3). under the conditions of the treponema pallidum multiplex nested pcr test in this study, it was found that the test for blood was able to detect treponema pallidum 3.25 times higher positive serological test results, compared with serum, and statistically showed a significant difference. discussion the study subjects were predominantly males, particularly the homosexuals, i.e. male who had sex with males (msm). according to the results of a survey by the integrated surveillance of behavioral biology program (surveilans terpadu biologi perilaku, stbp) in 2011 in 23 large indonesian cities, the highest prevalence of syphilis was in the transvestite group, but the msm were the most at risk population group for infection with syphilis.(3) if compared with the stbp surveys of 2007 and 2011, the prevalence of syphilis increased 3-fold in the msm population.(3) the increased prevalence of syphilis in the msm group had also been reported by several studies in developed countries.(17) the treponema pallidum multiplex nested pcr test using blood was able to detect treponema pallidum at 22.95%. the proportion of positives of the test using blood was lower than in other studies. the treponema pallidum pcr test using blood has been evaluated in several studies.(8,18) the proportion of positives of the pcr tests vary among a number of table 2. treponema pallidum multiplex nested pcr test in blood and serological tests of the patients (n=61) table 3. treponema pallidum multiplex nested pcr test in serum and serological tests of the patients (n=61) serological test total positive negative t.pallidum multiplex nested pcr blood positive 13 1 14 negative 29 18 47 total 42 19 61 serological test total positive negative t.pallidum multiplex nested pcr blood positive 4 0 4 negative 38 19 57 total 42 19 61 81 studies on patients with secondary syphilis, which comprise the studies conducted by martin et al.,(18) tipple et al.,(8) and grange et al.(10) with a proportion of positives of 44%, 58% and 38%, respectively. in these studies the pcr test with the target gene pola resulted in the detection of treponema pallidum using blood in patients with reinfection of syphilis, pr imary syphilis, secondary syphilis, and latent syphilis. in metaanalytic studies, the sensitivity of pcr tests using blood in patients with secondary syphilis reached 52.2%.(19,20) the treponema pallidum multiplex nested pcr test performed on serum could detect treponema pallidum at 6.56%. the proportion o f p o si ti ve s of t he tre p on e m a pa ll id u m multiplex nested pcr test using serum was lower than in other published studies. the study by gayet–ageron et al.(15) and grange et al.(10) found that the sensitivity of serum examination in secondary syphilis was 47% and 15%, respectively. both studies used the same target gene tpp47. the difference in the proportion of positives in the treponema pallidum multiplex nested pcr test using blood and serum with the other studies may presumably have been caused by the different target genes used, the pcr method, the sample preparation, the active phase of the disease, and the presence of immunodeficiency. the use of the treponema pallidum target gene and the differing clinical specimens affected the results of the multiplex nested pcr.(13, 14, 21) the target gene and pcr method used in this study refers to the target gene and pcr method used by gultom et al.(16) the study by gayet–ageron et al.(20) used the 47kda treponema pallidum target gene with the real-time pcr method. the stud y by gra nge et al. ( 10 ) use d th e tp p47 treponema pallidum target gene with the nested pcr method. in the meta-analytic study by gayet-ageron et al.(9) that compared the use o f t he ta r get ge n e s tpp 47 a n d p ola, t he investigators stated that the most used target ge ne in molecul ar stu dies of tre pone ma pallidum is the tpp47 gene (78%). from the results of the comparison it was apparent that both target genes, the tpp47 as well as the pola gene showed the same accuracy. there have been no previous publications on the use of the 23s rrna target gene to detect treponema pallidum in molecular pcr tests. spirochetemia may be detected in the blood particularly in the active phase. in the course of syphilis the highest concentration of bacteria are found in the secondary stage.(22) in the present study, the low number of treponema pallidum detected with multiplex nested pcr, was pr esumably because the concentration of treponema pallidum in the blood of the patients were below the detection threshold of the pcr test or there was dna damage in the specimens that were not immediately processed. the presence of immunodeficiency in the patients will affect the proportion of positives of the treponema pallidum multiplex nested pcr test.(23) in this study the majority of the patients showed reactive hiv results (54.09%). the pcr test successfully detected treponema pallidum in one patient with reactive hiv, the rpr test results showed a high titer (1:16 to 1:64), but the tpha test yielded a low titer (1:20 to 1:80). this patient was diagnosed as having syphilis with the serological tests, which needs to be confirmed. in patients with reactive hiv, the immune response may be suppressed so that the formation of antitreponemal antibodies is inhibited. as a result, the tpha test shows a low or negative titer. (2 3,24 ) in cont rast, the cond ition of immunodeficiency causes a reduction in the numbers and ability of the macrophages to eliminate pathogenic bacteria, so that the number of treponema pallidum may be higher in the blood circulation, particularly in the active phase of syphilis.(22) high spirochetemia will increase the sensitivity of the pcr. in the present study, the majority of patients who showed positive results for the treponema pallidum multiplex nested pcr test both in blood and serum had a reactive hiv status. the purpose of the treponema pallidum pcr test is to detect treponema pallidum dna. univ med vol. 37 no.1 82 effendi, rosana, yasmon, et al multiplex nested pcr for treponema pallidum various specimens may be used for the pcr test, but not all specimens have the same diagnostic value. the differing processes in the b lo od and ser um ma y p re su mab ly ca use differences in the numbers of bacteria that are present. in the blood, all blood components are preserved and treponema pallidum exists in greatest numbers. in serum, there is separation of the cellular components from the liquid as a result of the centrifugation process. treponema pallidum may most probably be precipitated together with the cellular components, so that the number of treponema pallidum in serum is not as great as in the blood. this is thought to be the cause of the higher proportion of positives of the treponema pallidum multiplex nested pcr test in the blood as compared with serum. based on the published results of a metaanalysis, the highest sensitivity of the treponema pallidum pcr of 78.40% was obtained from the examination of smears of lesions in primary syphilis.(23) in examinations using blood samples the sensitivity of pcr in primary and secondary syphilis was moderate.(15,20) the low proportion of positives in the treponema pallidum pcr test shows that the pcr test should not be used to rule out the diagnosis of syphilis in patients with the clinical picture of secondary syphilis. the pcr test is more useful as a confirmatory test, particularly in serological testing with dubious results.(20) in specimens with positive serological test results, the sensitivity of the trepone ma pallidum multiplex nested pcr test using blood was 30.95%, whereas when using serum, the sensitivity of treponema pallidum multiplex nested pcr test was only 9.52%. the results of this study are supported by the study conducted by grange et al.(10) who found proportions of positives of serum and blood of 15% and 38%, respectively, in the pcr test for treponema pallidum in patients with secondary syphilis. one of the limitations in this study was the fact that no microscopic examination was performed as gold standard for the diagnosis of syphilis for comparison with the multiplex nested pcr test, due to the difficulties in collecting exudates from lesions of syphilis in all patients. the study was able to show the best specimens between blood and serum of patients with the clinical picture of secondary syphilis for the treponema pallidum multiplex nested pcr test with the 23s rrna target gene to assist clinicians in determining the diagnosis and instituting rapid and appropriate management. further studies are required using molecular tests with different target genes and pcr methods for blood with positive serological test results, to explain the low proportion of positives in the treponema pallidum multiplex nested pcr test. conclusions the sensitivity of the treponema pallidum multiplex nested pcr test using blood specimens is 3.25 times higher than using serum, i.e. 30.95% compared with 9.52%. in patients with the clinical picture of secondary syphilis, using blood is better than using serum for the treponema pallidum multiplex nested pcr test. conflict of interest co mp e t i ng i nt e r e s t s: n o r e l e va nt disclosures. acknowledgement this study was funded by hibah pupt of the directorate of higher education (dikti) 20152016. we thank the department of microbiology, faculty of medicine university of indonesia (fkui) and department of dermatoveneorology, faculty of medicine university of indonesia cipto mangunkusumo hospital (fkui-rscm), std clinic cipto mangunkusumo hospital, std clinic puskesmas pasar rebo, std clinic puskesmas tambora and pkbi pro care clinic jatinegara for all assistance and cooperation in this study. 83 contributors ie, yr, and ay designed the study. ay and wi supervised fieldwork and gathered data and samples. ie and wi did statistical analyses. ie, yr and ay wrote the first draft of the report, with revisions and input from wi. all authors contributed to revisions and approved the final version. references 1. wijesooriya ns, rochat rw, kamb ml, et al. global burden of maternal and congenital syphilis in 2008 and 2012: a health systems modelling study. lancet glob health 2016;4:e525–33. 2. braxton j, carey d, davis d, et al. sexually transmitted disease surveillance 2014. atlanta, georgia: u.s. department of health and human services , centers for disease control and prevention national center for hiv/aids, viral hepatitis, std, and tb prevention division of std prevention; 2015. 3. kementerian kesehatan republik indonesia. surveilans terpadu biologi perilaku. jakarta: kementerian kesehatan republik indonesia dirjen pengendalian penyakit dan penyehatan lingkungan; 2011. 4. world health organization. global incidence and prevalence of selected curable sexually transmitted infections-2008. geneva: world health organization;2012. 5. world health organization. report on global sexually transmitted infection surveillance 2013. geneva: world health organization;2014. 6. tramont ec. treponema pallidum (syphilis). in: bennett je, dolin r, blaser mj, editors. mandell, douglas, and bennett’s principles and practice of infectious diseases. philadelphia: elsevier saunders;2015.p.3035-55. 7. zobaníkova m, mikolka p, čejková d, et al. complete genome sequence of treponema pallidum strain dal-1. standards genomic sci 2012;7:12-21. doi:10.4056/sigs.2615838. 8. tipple c, hanna mof, hill s, et al. getting the measure of syphilis: qpcr to better understand early infection. sex transm infect 2011;87:e485. doi: 10.1136/sti.2011.049494. .9 gayet-ageron a, combescure c, lautenschlager s, et al. comparison of the diagnostic accuracy of polymerase chain reaction targeting the 47kda protein membrane gene of treponema pallidum or the dna polymerase i gene: a systematic review and meta-analysis. j clin microbiol 2015; 53:3522-9. doi: 10.1128/jcm.01619-15. 10 grange pa, gressier l, dion pl, et al. evaluation of a pcr test for detection of treponema pallidum in swabs and blood. j clin microbiol 2012;50:54652. doi: 10.1128/jcm.00702-11. 11 casal cad, silva mod, costa ib, et al. molecular detection of treponema pallidum sp. pallidum in blood samples of vdrl-seroreactive women with lethal pregnancy outcomes: a retrospective observational study in northern brazil. revista da sociedade brasileira de medicina tropical 2011;44:451-6. 12. buffet m, grange pa, gerhardt p, et al. diagnosing treponema pallidum in secondary syphilis by pcr and immunohistochemistry. j invest dermatol 2007;127:2345–50. doi: 10.1038/ sj.jid.5700888. 13 cruz ar, pillay a, zuluaga av, et al. secondary syphilis in cali, colombia: new concepts in disease pathogenesis. plos negl trop dis 2010;4:e690. doi: 10.1371/journal.pntd.0000690. 14 ho el, lukehart sa. syphilis: using modern approaches to understand an old disease. j clin invest 2011;121:4584-92. 15 gayet-ageron a, toutus-trelllu nbl, lautenschlager s, et al. assessment of a real-time pcr test to diagnose syphilis from diverse biological samples. sex transm infect 2009;85:2649. doi: 10.1136/sti.2008.034314. 16. gultom da, rosana y, efendi i, et al. detection and identification of azithromycin resistance mutations on treponema pallidum 23s rrna gene by nested multiplex polymerase chain reaction. med j indones 2017;26:90-6. https://doi.org/ 10.13181/mji.v26i2.1543. 17. sharma s, chaudhary j, hans c. vdrl v/s tpha for diagnosis of syphilis among hiv sero-reactive patients in a tertiary care hospital. int j curr microbiol app sci 2014;3:726-30. 18. martin ie, tsang rsw, sutherland k, et al. molecular characterization of syphilis in patients in canada: azithromycin resistance and detection of treponema pallidum dna in whole-blood samples versus ulcerative swabs. j clin microbiol 2009;47:1668-73. 19. tipple c, jones r, mcclure m, et al. rapid treponema pallidum clearance from blood and ulcer samples following single dose benzathine penicillin treatment of early syphilis. plos negl trop dis 2015;10:e0003492. doi: 10.1371/ journal. pntd.0003492. 20. gayet-ageron a, lautenschlager s, ninet b, et al. sensitivity, specificity and likelihood ratios of pcr in the diagnosis of syphilis: a systematic univ med vol. 37 no.1 84 effendi, rosana, yasmon, et al multiplex nested pcr for treponema pallidum review and meta-analysis. sex transm infect 2013;89:251-6. 21. sato ns. laboratorial diagnosis of syphilis. syphilis recognition, description and diagnosis. croatia: intech;2011.p.87-108. 22. wu br, tsai ms, yang cj, et al. spirochetemia due to treponema pallidum using polymerasechain-reaction assays in patients with early syphilis: prevalence, associated factors and treatment response. clin microbiol infect 2014;20:o524-7. doi: 10.1111/1469-0691.12504. 23. leslie de, azzato f, karapanagiotidis t, et al. development of a real-time pcr assay to detect treponema pallidum in clinical sspecimens and assessment of the assay’s performance by comparison with serological testing. j clin microbiol 200745:93-6. doi:10.1128/jcm.01578-06. additional probiotic therapy lowers scorad index 68 division of allergy immunology, department of pediatrics, faculty of medicine, university of sumatera utara, medan correspondence : dr. jessica ekaputri division of allergy immunology, department of paediatrics, faculty of medicine, university of sumatera utara, jl. dr. mansyur no. 5 medan, sumatera utara 20155. email: jessicaekaputri58@gmail.com univ med 2016;35:68-74 doi: 10.18051/univmed.2016.v35.68-74 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 additional probiotic therapy lowers scorad index in children with atopic dermatitis jessica ekaputri*, rita evalina*, and melda deliana* background atopic dermatitis (ad) is a common skin disease that is usually chronic, relapsing, causing pruritus and frequent in children. the pathogenesis of ad involves genetic, immunological and environmental factors causing skin barrier dysfunction and dysregulation of the immune system. probiotic treatment has been claimed to offer several functional properties including stimulation of the immune system and plays an important role in ad. the objective of this study was to evaluate the effect of probiotic therapy on atopic dermatitis in children. methods a randomized controlled trial was conducted on 62 children suffering from ad from december 2015 to january 2016. ad severity was assessed based on the scoring of atopic dermatitis (scorad) index. subjects were divided into two groups consisting of 32 and 30 children, the probiotic (probiotic + emollient) and control (emollient) groups, respectively. scorad index was re-evaluated after 2 weeks of therapy. the data was analyzed using mann whitney test. results after the intervention, the mean scorad index in the probiotic group was significantly much lower than the control group (18.09 ± 8.59 vs 23.21 ± 8.71; p=0.001). the mean decrease in scorad index in the probiotic group was 40.4 %, much higher than the control group 25.2%. the number needed to treat (nnt) score of probiotic treatment was 5.3. conclusion the addition of probiotics to conventional therapy effectively lowers scorad index by 40.4% in atopic dermatitis. the impact of probiotics on scorad indices is thought to be attained by modification of the immunogenicity of potential allergens. keywords: atopic dermatitis, scorad, probiotic, children doi: http://dx.doi.org/10.18051/univmed.2016.v35.68-74 69 univ med vol. 35 no.2 introduction atopic dermatitis (ad), or eczema, is a common skin disease that is usually associated with other atopic diseases such as allergic rhinitis and asthma. ad is usually chronic, relapsing, causing pruritus and common in children, usually starting in early childhood.(1-3) ad comprises a broad clinical spectrum of mild to severe agerelated disorders.(4,5) ad pathogenesis involves genetic, immunological and environmental factors causing skin barrier dysfunction and dysregulation of the immune system.(6) scoring of ad (scorad) is a scoring system that was developed to assess the severity of atopic dermatitis by measuring the extent of the area involved, and the intensity of the subjective symptoms of pruritus and sleep disturbance.(7) ad requires a holistic assessment and its treatment remains a challenge, because it requires an efficient short-term acute symptom control, without affecting the overall treatment plan that is aimed at long-term stabilization, preventing flare-ups and avoiding the side effects of the treatment.(8.9) a probiotic is an oral supplement or food product that contains viable microorganisms in amounts sufficient to change the microflora of the host and have a potential beneficial effect on health.(10) supplying probiotics plays a role in the prevention and treatment of allergic disease, but the mechanism underlying the immunomodulatory effects is still uncertain.(11) the meta-analysis by elazab et al.(12) shows that probiotic therapy in the prenatal period and early life reduces the risk of atopic sensitization and total ige level in children but does not reduce the risk of asthma. the meta-analysis by boyle et al.(13) shows the opposite where probiotics have the effect of significantly decreasing the symptoms and severity of ad. another study showed different results, in that there was no benefit from supplementation with b. lactis or l. paracasei in the treatment of eczema, when given as an adjunct to basic topical treatment, and no effect on the progression of allergic disease from age 1 to 3 years.(14) the decrease in scorad indices caused by probiotics is thought to occur through modification of immunogenicity of potential allergens. the variable effects of probiotics on allergic diseases is likely influenced by the type of probiotic given, and by environmental and host factors.(15) on the basis of the abovementioned information, the purpose of the study was to evaluate the effect of the probiotic supplements to conventional therapy towards the scorad index of children with atopic dermatitis. methods research design a r a n d o m i s e d c o n t r o l l e d t r i a l w a s conducted on selected pediatric patients with atopic dermatitis at two posyandu (integrated health care stations) in medan from december 2015 to january 2016. research subjects the study population consisted of pediatric patients with atopic dermatitis. the research sample comprised the population that met the inclusion criteria, i.e. children aged 1 month to 5 years who satisfied the hanifin and rajka criteria for atopic dermatitis. the exclusion criteria were refusal of the children’s parents to provide medication, children with malnutrition and other skin diseases. we used the sample size c a l c u l a t i o n m e t h o d f o r c o m p a r i n g t w o independent proportions.(16) to calculate the sample size, we used a study by yesilova et al.(17) who reported a standard deviation of the scorad index of 13.4 and 6.1, respectively, in the probiotic therapy group and conventional therapy-only group. the calculated sample size per group was 28, which was estimated to be adequate to detect a 30% difference between the two groups using a two-tailed test, an alpha of 0.05 and power of 80%. measurements anamnesis, physical examination, and anthropometric measurements were performed, 70 including the measurement of height and weight of all research subjects. height was measured in an upright position without footwear, from the feet to the top of the head using a gea statue meter and expressed in meters (m). weight was also measured in an upright position using camry digital scales, and expressed in kilograms (kg). an assessment of body mass index (bmi) was also carried out, the bmi being expressed in units of kg/m2. all measurements were carried out at the time of the examination of the subject. the severity of atopic dermatitis of each group was rated based on scorad index. scorad is a clinical tool used to assess the extent and severity of eczema. dermatologists may use this tool before and after treatment to determine whether the treatment has been effective. intervention patients who met the inclusion criteria were included in the study and were divided into two groups by simple randomization i.e. a group that received a probiotic along with conventional therapy (probiotic group) and a group that received conventional therapy only (control group). the probiotic group was given a probiotic orally, administered twice daily for two weeks along with the conventional therapy such as topical emollients, applied three times daily after bathing. the probiotic was given twice a day in the form of a powder containing a mixture of l. acidophilus, b. longum, and s. thermophilus, each in amounts of 1x107 cfu/g. the control group was given conventional therapy such as topical emollients applied three times a day after bathing, for two weeks. the research samples were monitored for two weeks to assess the severity of atopic dermatitis based on scorad index after the intervention. statistical analysis the kolmogorov-smirnov normality test was used to assess the normal distribution of the data. the independent t-test was to be used on normally distributed data to establish the differences in scorad index between case and control groups before and after intervention, and the differences in the decline of scorad indices between the two groups, while for non-normally distributed data the mann-whitney test was to be used. the number needed to treat test was used to assess the effectiveness of probiotic therapy. statistical analysis was performed with the spss statistical package for windows, version 17.0, and a p-value of <0.05 was considered statistically significant. ethical clearance the study protocol was approved by the health research ethical committee, faculty of medicine, university of sumatera utara (695/ komet/fkusu/2015). all study subjects signed written informed consent after having been informed about the aims and benefits of the study. results during the study period, 62 subjects suffering from atopic dermatitis were obtained and divided into two groups. the probiotic group of 32 subjects were given a probiotic along with conventional therapy, while the remaining 30 subjects were only given conventional therapy (control group) (figure1). there were no significant differences in gender, mean age, and family history of atopy between the two study groups (p=0.422, 0.269, and 1.000; respectively) at base-line (table 1). before the intervention, the mean scorad index was 30.33 ± 12.96 in the probiotic group and 31.05 ± 10.54 in the control group, there being no significant differences between the scorad index of the two groups (p=0.522) (table 1). after the intervention, it appears that the mean scorad index in the probiotic group was significantly much lower than the control group (18.09 ± 8.59 vs 23.21 ±8.71, p=0.010). the mean decrease in scorad index in the probiotic group was much more than in the control group (12.23 ± 6.46 vs 7.83 ± 5.49, p=0.001) (table 2). ekaputri, evalina, deliana probiotic in atopic dermatitis 71 univ med vol. 35 no.2 figure 1. flow diagram table 1. demographics and family history of atopy at base-line by treatment group a chi square test, b mann whitney test, c fisher’s exact test the number needed to treat was 5.3, meaning that it takes five people to achieve an effect of improvement in the scorad values by giving probiotics (table 3). discussion the mean age of children with atopic dermatitis was 2.51 ± 1.59 years and 2.80 ± 1.37 years in the probiotic group and the control group, respectively. atopic dermatitis is known to be often found during early infancy with an incidence rate of 85% starting before the age of 5 years.(1.2) in the study no significant differences were found in mean age between the two groups. after the intervention, there was a significant difference in the mean scorad index between the two groups. the above results are similar to those found in the study conducted by yesilova et al.(17) regarding the administration of a probiotic containing b. bifidum, l. acidophilus, l. casei and l. salivarius which showed reductions in scorad score and in serum il-5, il-6, and ige levels of atopic dermatitis patients. likewise, the meta-analytical study by yao et al.(18) showed significantly lowered scorad scores between probiotic and placebo groups. the opposite was found in the meta-analytical study by boyle et al.(13) in which probiotics did not significantly reduce ad symptoms compared with placebo. other meta-analytical studies also found variable probiotic effects in ad.(14,20) highly heterogenous conditions, including differences in probiotic preparations, study populations and the final results of measurement can directly affect the comparative results of these studies. 72 previous studies only evaluated the decrease in scorad index and other parameters in ad. in the present study, an analysis of the number needed to treat (nnt) was conducted between the two groups and resulted in an nnt of 5.3, which means that it takes five children to get the effect of improving the value of scorad. this value indicates that the administration of the probiotic is quite effective as an adjunctive therapy to reduce the severity of atopic dermatitis. ad is found among 15-30% of children and 2-10% of adults, having increased up to 2 or 3 times in industrialized countries over the past three decades. atopic dermatitis often begins in early infancy (known as early onset ad). fortyfive percent of all cases of ad starts in the first 6 months of life, 60% in the first year and 85% before the age of 5 years.(1.2) the treatment of ad is intended to overcome itchiness, to repair the skin barrier and to reduce inflammation. the successful treatment of ad relies on a multi-disciplinary approach to education of patients and their families, skin care optimalization, anti-inflammatory medication and treatment of skin infections.(2) the relationship between atopy and the intestinal environment comes from the hypothesis that a deficiency of intestinal microflora in children with atopy will interfere with the required development of the immune response and the normal intestinal mucosal barrier.(21) the microbial intestinal flora or microbiome table 2. mean differences in scorad between groups after two weeks of intervention * mann whitney test table 3. number needed to treat of probiotic treatment contributes to the pathogenesis of allergic disease because of its effect on mucosal immunity. exposure to the normal microbial flora in early life results in the th-1 / th-2 lymphocyte balance changing in favor of th-1 cells.(22) according to the food and agriculture organization (fao)/world health organization (who), probiotics are defined as as live microorganisms which when administered in adequate amounts, confer a health benefit on the host. health benefits have mainly been demonstrated for specific probiotic strains of the following genera: lactobacillus, bifidobacterium, saccharomyces, enterococcus, streptococcus, pediococcus, leuconostoc, bacillus, escherichia coli.(23,24) these microorganisms are fermentative, obligate or facultative anaerobic, usually nonmotile, having different shapes and the majority typically producing lactic acid.(10) the levels of lactobacillus and bifidobacterium are significantly decreased in the fecal samples of infants and children with ad, demonstrating the therapeutic potential of probiotic supplementation in ad.(25) there are no specific directions for probiotic dose in atopic dermatitis. probiotic treatment is more often based on the directions of the probiotic manufacturers. in the present study, the probiotic given twice a day was a mixture of l.acidophilus, b.longum, and s.thermophilus, each to the amount of 1x107cfu/g. the probiotic was administered for 2 weeks to assess the severity of atopic dermatitis based on scorad index. ekaputri, evalina, deliana probiotic in atopic dermatitis 73 univ med vol. 35 no.2 this study has limitations which include lack of control and monitoring of patient compliance during the intervention period. monitoring was only done on the amount of drugs given to the patients, as shown by the fact that no medication was returned by patients during the study. the results of this study provide new insights into the role of immunologic factors in atopic dermatitis and administration of probiotics can modulate the immune system, thereby improving the symptoms of atopic dermatitis. a broader study is needed to determine the exact mechanisms and the benefits of probiotics in the treatment of atopic dermatitis. conclusions the addition of probiotics to conventional therapy effectively lowered the scorad index in atopic dermatitis compared to conventional therapy. this result could be a consideration to using probiotics for the treatment of atopic dermatitis. the impact of probiotics on scorad indices is thought to be attained by modification of the immunogenicity of potential allergens. conflict of interests no conflict of interest to be declared. acknowledgement we would like to thank all study subjects who participated in the study, and all colleagues who advised us in writing this report. references 1. bieber t. atopic dermatitis. n engl j med 2008;358:1483-94. 2. watson w, kapur s. atopic dermatitis. allergy asthma clin immunol 2011;7(suppl 1):s4. 3. beck la. atopic dermatitis. in: lichtenstein lm, busse ww, geha rs, editors. current therapy in allergy, immunology, and rheumatology. 6th edition. philadelphia: elsevier;2004. p.88-92. 4. daveiga sp. epidemiology of atopic dermatitis: a review. allergy asthma proc 2012;33:227-34. 5. akdis ca, akdis m, bieber t, et al. diagnosis and treatment of atopic dermatitis in children and adults: european academy of allergology and clinical immunology/ american academy of allergy, asthma and immunology/ practall consensus report. allergy 2006; 61:969-87. 6. eichenfield lf, tom wl, chamlin sl, et al. guidelines of care for the management of atopic dermatitis: section 1 diagnosis and assessment of atopic dermatitis. j am acad dermatol 2014; 70:338-51. 7. charman c, chambers c, williams h. measuring atopic dermatitis severity in randomized controlled clinical trials: what exactly are we measuring? j invest dermatol 2003;120:932–41. 8. darsow u, wollenberg a, simon d, et al. difficult to control atopic dermatitis. wao j 2013;6:6. 9. rubel d, thirumoorthy t, soebaryo rw, et al. consensus guidelines for the management of atopic dermatitis: an asia-pacific perspective. j dermatol 2013; 40:160-71. 10. thomas dw, greer fr. committee on nutrition: section on gastroenterology, hepatology and nutrition. clinical report: probiotics and prebiotics in pediatrics. pediatrics 2010;126: 1217-31. 11. fiocchi a, burks w, bahna sl, et al. clinical use of probiotics in pediatric allergy (cuppa): a world allergy organization position paper. waoj 2012;5:148-67. 12. elazab n, mendy a, gasana j, et al. probiotic administration in early life, atopy, and asthma: a meta-analysis of clinical trials. pediatrics 2013; 132:e666-e76. 13. boyle rj, bath-hextall fj, leonardi-bee j, et al. probiotics for the treatment of eczema: a systematic review. clin exp allergy 2009;39: 1117-27. 14. gore c, custovic a, tannock gw, et al. treatment and secondary prevention effects of the probiotics lactobacillus paracasei or bifidobacterium lactis on early infant eczema: randomized controlled trial with follow-up until age 3 years. clin exp allergy 2012;42:112-22. 15. prescott sl, bjorksten b. probiotics for the prevention or treatment of allergic diseases. j allergy clin immunol 2007;120:255-62. 16. madiyono b, moechlisan s, sastroasmoro s, et al. perkiraan besar sampel. in: sastroasmoro s, ismael s, editors. dasar-dasar metodologi penelitian klinis. 4th edition. jakarta: sagung seto;2011.p.348-381. 74 17. yesilova y, calka o, akdeniz n, et al. effect of probiotic on the treatment of children with atopic dermatitis. ann dermatol 2012;24:189-93. 18. yao tc, chang cj, hsu yh, et al. probiotics for allergic disease: realities and myths. pediatr allergy immunol 2010;21:900-19. 19. lee j, seto d, bielory l. meta-analysis of clinical trials of probiotics for prevention and treatment of pediatric atopic dermatitis. j allergy clin immunol 2008;121:116-21. 20. michail sk, stolfi a, johnson t, et al. efficacy of probiotics in the treatment of pediatric atopic dermatitis: a meta-analysis of randomized controlled trials. am allergy asthma immunol 2008;101:508-16. 21. ogden ns, bielory l. probiotics: a complementary approach in the treatment and prevention of pediatric atopic disease. curr opin allergy clin immunol 2005;5:179-84. 22. winkler p, ghadimi d, scherezenmeir j, et al. molecular and cellular basis of microflora-host interactions. j nutr 2007;137:766s-72s. 23. sekhon bs, jairath s. prebiotics, probiotics and synbiotics: an overview. j pharm educ res 2010; 1:13-30. 24. kaur ip, kuhad a, garg a, et al. probiotics: potential pharmaceutical applications. in: cho ss, finocchiaro et, editors. handbook of prebiotics and probiotics ingredients. florida: crc press;2010.p.381-408. 25. pan sj, kuo ch, lam kp, et al. probiotics and allergy in children: an update review. pediatr allergy immunol 2010;21:e659-e66. ekaputri, evalina, deliana probiotic in atopic dermatitis oktavianus 65 *department of ophthalmology faculty of medicine trisakti university jakarta correspondence dr. rose, spm, msc department of ophthalmology faculty of medicine trisakti university jl. kyai tapa no. 260 grogol jakarta 11440 phone : +6221-5672731 email: rose_149@yahoo.com univ med 2014;33:65-72 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background imbalance between oxidative processes and antioxidant defenses has been considered to play a role in cataractogenesis, particularly in diabetes patients. superoxide dismutase (sod) is an important precursor for oxidative stress in the human lens, and its activity is mainly dependent on the copper and zinc levels in the body. the aim of this study was to compare erythrocyte sod, erythrocyte zinc and total serum testosterone levels in male patients with early senile nuclear cataract and evaluate the correlations between the parameters in all subjects. methods a community-based study of cross-sectional design was conducted at cilandak district primary health center where 52 adult and 17 elderly men with early senile nuclear cataract were chosen as the study subjects. erythrocyte sod, erythrocyte zinc, serum testosterone, and fasting blood glucose (fbg) levels were measured in all subjects. nuclear cataract stage was assessed with the pentacam® instrument (oculus, germany). independent student t test and pearson’s correlation were used to analyze the results. results erythrocyte sod level was significantly decreased in elderly men compared to adult men (p=0.014). erythrocyte zinc, serum testosterone and fbg did not differ significantly in adult and elderly males (at p=0.304; p=0.145;and p=0.376, respectively). erythrocyte sod activity was significantly associated with erythrocyte zinc level (r=0.486; p=0.048). conclusions lower erythrocyte sod activity was found in elderly males than in adult males with early nuclear cataract. there was a relationship between erythrocyte sod and erythrocyte zinc level in elderly males with early nuclear cataract. keywords: superoxide dismutase, zinc, testosterone, fasting blood glucose, early nuclear cataract, elderly decreased erythrocyte superoxide dismutase in elderly men with early nuclear cataract rose* and erlani kartadinata* 66 rose, kartadinata superoxide dismutase in nuclear cataract superoksida dismutase eritrosit menurun pada laki-laki lanjut usia dengan katarak nuklear awal latar belakang ketidakseimbangan proses oksidasi dan pertahanan dari antioksidan, khususnya pada pasien diabetes melitus diduga mempengaruhi proses terbentuknya katarak. superoxide dismutase (sod) merupakan petanda stres oksidasi yang penting pada lensa manusia, dan fungsinya sebagian besar tergantung pada kofaktor cuprum dan seng (zinc). penelitian ini bertujuan untuk membandingkan kadar sod, seng di eritrosit dan testosteron total pada pria dengan katarak nuklearis awal dan mengevaluasi korelasi dari parameter tersebut. metode studi ini merupakan suatu studi berbasis komunitas dengan disain potong lintang yang dilakukan di puskesmas kecamatan cilandak dengan memilih 69 orang pria yang menderita katarak nuklearis awal. lima puluh orang terdapat dalam kelompok dewasa di bawah 60 tahun dan 17orang dalam kelompok lanjut usia. kadar sod, seng eritrosit, testosteron total dan gula darah puasa (gdp) dibandingkan pada kedua kelompok tersebut. derajat kekeruhan katarak nuklearis diperiksa menggunakan alat pentacam® (oculus, germany). uji t independent dan korelasi pearson digunakan untuk menganalisis data. hasil kadar eritrosit sod menurun secara signifikan pada usia lanjut dibandingkan pada kelompok pria dewasa (p=0,014). kadar eritrosit seng, testosteron, dan gdp tidak berbeda secara signifikan pada kedua kelompok (p=0,304; p=0,145; p=0,376 masing-masing). aktivitas sod eritrosit berhubungan secara signifikan dengan kadar eritrosit zinc (r=0,486; p=0,048) kesimpulan aktivitas eritrosit sod lebih rendah pada kelompok pria usia lanjut dengan katarak nuklearis awal dibandingkan dengan kelompok orang dewasa. pada lanjut usia dengan katarak nuklearis awal terdapat hubungan antara aktivitas sod eritrosit dan kadar seng eritrosit. kata kunci : superoxide dismutase, zinc, testosteron total, gula darah puasa, katarak nuklearis dini, lanjut usia abstrak introduction senile cataract is an age-related disorder, characterized by progressive lens opacification and globally is the leading cause of chronic visual impairment in the elderly. in 2010, the who reported that cataract accounts for 51% of all blindness in the world.(1) as the mean age of the global population is rising, cataract will not only pose a major economic burden for the patients but also increase mortality in elderly.(2) although cataract surgery is a successful way to cure the cataract, it is still unaffordable or inaccessible for many patients in developing countries. alternative methods to prevent or delay cataract development still remain a challenge for researchers. m u l t i p l e f a c t o r s i n v o l v i n g c o m p l e x i n t e r a c t i o n b e t w e e n v a r i o u s p h y s i o l o g i c processes contribute to the development of senile cataract.(3,4) senile cataract starts in the fourth decade as a consequence of aging, with the lens 67 losing its transparency, its nucleus becoming more rigid, and thicker, and its shape more resistant to change during accommodation, which is the basis of presbyopia. these may be the earliest observable symptoms of age-related cataract.(5,6) the biochemical cause of senile cataract is not fully known. numerous studies show that there are other possible risk factors involving oxidative mechanisms associated with agerelated cataract, such as gender, nutritional status, smoking, pollution, drugs, ultraviolet light exposure, occupational status, aging and diabetes.(7-9) so far, aging and diabetes remain the most significant contributors to cataract progression.(3,10) oxidative stress continues to be the leading mechanism of cataractogenesis.(11,12) oxidative stress releases free oxygen radicals which are neutralized in the lens by antioxidant enzymes, such as superoxide dismutase (sod), glutathione peroxides, catalase and by non-enzymatic antioxidants (ascorbate, carotenoids, vitamin e, the glutathione system).(13,14) a study showed that cataractous patients had significantly decreased erythrocyte sod activity.(15) during aging, antioxidant system activities continue to decrease. sod acts as a first order antioxidant enzyme and its function mainly depends on copper and zinc ions. zinc is one of the important cofactors in humans, and zinc deficiency is related to the sensitivity of oxidative stress. (16-18) as males age, there is also a decline in serum testosterone concentration, an increase in sex hormone binding globulin (shbg) concentration, and a decrease in free testosterone, but the hormonal decline in aging males is modest c o m p a r e d w i t h e s t r o g e n d e f i c i e n c y i n menopause.(19) researchers have shown that oxidative stress, which is caused by the accumulation of free radicals, plays a role in the pathogenesis of cataracts and that this process can be prevented by antioxidants. we undertook the present study to determine erythrocyte sod, erythrocyte zinc, and serum testosterone levels in adult and elderly males, and to evaluate any correlation between erythrocyte sod, erythrocyte zinc, and serum testosterone levels in males with early nuclear cataract. methods research design this was a community-based crosssectional study which was carried out from may to october 2011 at the cilandak district primary health center, south jakarta, indonesia. study subjects the study subjects were males aged >40 years, residing in cilandak district, jakarta, indonesia, and agreeing to participate voluntarily in this study. sixty nine male subjects who had early nuclear or incipient cataract in both eyes with best-corrected of visual acuity (bcva) of 1.0 were included in this study. we excluded subjects with mature cataract, glaucoma, diabetic retinopathy, and those unable to undergo complete ophthalmologic and laboratory examinations. data collection all participants were interviewed to collect their demographic data. based on age, the subjects were categorized into two groups, i.e. adult males (<60 years) and elderly males (≥60 years). the subjects underwent physical examination and routine eye examination, i n c l u d i n g r e f r a c t i v e c o r r e c t i o n , f u n d u s photography, intraocular pressure examination, and lens imaging for assessment of nuclear cataract using the pentacam® instrument (oculus, germany). this instrument uses a blue light-emitting diode to image the anterior segment of the eye. nuclear opacity was graded by quantifying the density of the lens opacification in three dimensions with the built-in rotating scheimpflug camera and analyzing with specific software. the total area of opacity was graded for both eyes. only early nuclear cataract (grade 1 and 2) was included in this study. univ med vol. 33 no.1 68 rose, kartadinata superoxide dismutase in nuclear cataract table 1. distribution of characteristics of subjects with early nuclear cataract (n=69) *values are mean ± sd; sod = superoxide dismutase laboratory measurements five-milliliter blood samples were drawn from a peripheral vein in all subjects and collected into 10 ml vacutainer tubes, containing ethylene diamine tetraacetic acid (edta), after 10 hours of fasting. fasting blood glucose (fbg) was assessed by spectrophotometry using the advia 1800 and bayer advia 180074024 glucose reagent (r1: atp, nad, sodium azide; r2: hexokinase, g6pd). detection limit: 0.6mg/dl, linearity: 0-700 mg/dl. conversion factor: mg/ dl x 0.05555 mmol/l. the glucose in the sample is phosphorylated by hexokinase to yield glucose6-phosphate (g6p), which is then oxidized by glucose-6-phosphodehydrogenase (g6pd). the oxidative reaction releases nicotinamide adenine dinucleotide (nad)h, and this is taken as a measure of the glucose concentration in the s a m p l e . e r y t h r o c y t e s o d a c t i v i t y w a s determined by a colorimetric enzymatic method, in which xanthine is oxidized to uric acid by xanthine oxidase (xod), releasing reactive oxygen species (ros), which is neutralized by sod. ros reduces 2-(4-iodophenyl)-3-(4nitrophenol)-5-phenyltetrazolium chloride (int) into a red formazan dye. the sod activity is measured by the degree of inhibition of this reaction, therefore formazan intensity is inversely related to sod activity. erythrocyte zinc was measured using flame atomic absorption spectrophotometry. the erythrocyte mass obtained from total blood was centrifuged three times at 4ºc in a centrifuge at 3000 x g for 15 minutes, after washing with 5 ml of 0.9% saline a n d h o m o g e n i z a t i o n . s u b s e q u e n t l y t h e e r y t h r o c y t e m a s s w a s e x t r a c t e d w i t h a micropipette, placed in demineralized eppendorf tubes, and stored at -20°c until required for zinc analysis. to determine the serum testosterone level we employed an electrochemiluminescent immunoassay (eclia) using roche elecsys r e a g e n t k i t c a t 11 7 7 6 0 6 1 ( r 1 : a n t i testosterone-ad-biotin, r2: testosteroneruthenium) on a 2010/cobas e601 analyzer. the s a m p l e t e s t o s t e r o n e i s a t t a c h e d t o a n t i t e s t o s t e r o n e a d b i o t i n . t h i s c o m p l e x i s immobilized in the solid phase and bombarded by electrons to induce chemiluminescence, which consitutes a measure of the testosterone concentration in the sample. ethical clearance written informed consent was obtained from all participants, and the study protocol was approved by the research ethics committee, faculty of medicine, trisakti university, jakarta. statistical analysis d a t a w e r e a n a l y z e d w i t h s p s s f o r windows version 17.00. descriptive data were expressed as mean ± sd; difference between the levels of sod, zinc and testosterone in both groups were tested using the independent t-test. pearson’s correlation was used to check the correlation between variables. the statistical level of significance was p<0.05. results a total of sixty nine participants were enrolled in this study. baseline characteristics of the study population are given in table 1. mean age, height, weight, and body mass index (bmi) were 53.69 ± 7.77 years (range 42–70 years), 162.25 ± 5.62 cm (range 150–176), 64.11 ± 11.44 kg (range 40–87), and 24.31 ± 3.95 kg/ m2, respectively. fifty two (75.36%) were adult 69 table 2. comparison of fbg, testosterone, sod and zinc levels in adult and elderly males with early nuclear cataract fbg = fasting blood glucose; sod = super oxide dismutase; values are mean ± sd; *significant males and 17 (24.64%) were elderly males. means of fbg, testosterone, sod and zinc were 111.73 ± 43.78 mg/dl, 511.26 ± 174.78 ng/dl, 196.66 ± 26.88 u/ml, and 81.02 ± 12.63 µg/dl, respectively. as shown in table 2, the erythrocyte sod level was significantly lower in elderly males (181.65 ± 26.05 u/ml) than in adult males (200.13 ± 26.31 u/ml) with early nuclear c a t a r a c t ( p = 0 . 0 1 4 ) . h o w e v e r, t h e f b g, testosterone and erythrocyte zinc levels were not significantly different between adult males and elderly males. the results presented in table 3 show that there was a significant correlation between erythrocyte sod and erythrocyte zinc levels in elderly males with early nuclear cataract (r=0.486; p=0.048). relationships between fbg and early nuclear cataract (r=-0.164; p=0.530) and between serum testosterone and early nuclear cataract (r=-0.476; p=0.085) were not significant. discussion our study showed that erythrocyte sod was decreased in elderly males with early nuclear cataract as compared to adult males. the human lens grows during life and undergoes dynamic changes in optical, physical and biochemical properties with age. aging is the most important risk factor for age-related nuclear cataract. (9,11) the aging process is accompanied by extensive oxidative damage to the crystalline lens. disturbed balance between oxidative processes and antioxidative defenses results in oxidative stress that can damage proteins, lipids, polysaccharides and nucleic acids of the lens.(4,18,20) some external factors which have been identified as risk factors for the development of nuclear cataract are smoking, excessive ultraviolet-b exposure and diabetes.(5,8,15) we found that the bmi in both groups with cataract was higher than that in the normal population, indicating that obesity is associated with cataract. however, it is not clear whether obesity simply causes cataract or r e l a t e s t o o t h e r a b n o r m a l i t i e s t h a t m a y contribute to cataract formation. (5,21,22) in this study, we found that mean fbg level was greater than 100 mg/dl. this represents an association between fbg and early nuclear cataract in males. this finding is in contrast to the results of the study by park et al.(23) who found that the elevated fbg appears to be associated with nuclear cataract only in women and not in males. (23) univ med vol. 33 no.1 table 3. correlation between erythrocyte sod, fbg, erythrocyte zinc and serum testosterone in elderly subjects with early nuclear cataract sod = super oxide dismutase; fbg = fasting blood glucose; values are r (pearson correlation); *significant 70 rose, kartadinata superoxide dismutase in nuclear cataract since the fbg level in the present study was inversely correlated with erythrocyte sod activity, we suggest that hyperglycemia may contribute to the development of early nuclear cataract, the glucose in the blood entering the aqueous humor and the lens, while erythrocyte sod activity decreases slightly with age.(4,24) the present study was an evaluation of erythrocyte sod, erythrocyte zinc and serum testosterone levels with respect to early nuclear cataract in adult and elderly males, based on laboratory examination. oxidative stress is difficult to monitor by direct examination of the blood, but can be estimated by measuring the antioxidant levels in the blood or erythrocytes. sod is an essential component of the second antioxidant system that can be evaluated from the red blood cell.(12,18,25) in the present study, the erythrocyte sod level declined in the elderly male group, showing that erythrocyte sod correlates with senile cataract. the increased sod in early nuclear in the adult male group may be a defensive response to the increased oxidation levels in the body in the early stages of nuclear cataract. that the sod level decreases with the aging process in elderly males may be a reflection of an adaptive protective response in the body system.(12) chakraborty et al.(16) found a decrease in plasma sod activity in senile cataract and a significant correlation of sod activity with serum zinc. in the study of virgolici et al.,(12) the investigators demonstrated that red blood cell sod activity was significantly increased in cataract patients,(12) although there was no description of the severity of the senile cataracts included in their study. in contrast, our study compared erythrocyte sod levels only in male subjects with early nuclear cataract. in erythrocytes, the level of zinc, an important co-factor of sod, was found to be significantly higher than the normal reference value of our laboratory. dawczynski et al.(26) reported that zinc concentration in mature cataracts was significantly higher than in c o r t i c o n u c l e a r c a t a r a c t s . j a m i l e t a l . ( 2 7 ) demonstrated that higher zinc levels were found in mature and hypermature cataracts as a consequence of increased protease activity in the lens. proteases in the human eye may be considered as metallo-enzymes which may lead to cataract formation.(27,28) one limitation of this study was the small number of subjects with the parameters of interest in the blood. a more complete approach is necessary to evaluate other antioxidant and external factors in cataract formation, involving a larger population with different types of agerelated cataracts, and also to assess the protective role of a healthy lifestyle and nutritional status in delaying cataractogenesis in elderly males. conclusions we found that in elderly males with early nuclear cataract, erythrocyte sod level was significantly decreased compared to adult males, whereas serum testosterone and erythrocyte zinc l e v e l s w e r e n o t s i g n i f i c a n t l y i n c r e a s e d . erythrocyte sod activity was correlated with erythrocyte zinc level and may be a good predictor of early nuclear cataract in elderly males. acknowledgements we would like to express our gratitude to all of the cilandak primary health center staff and all participants in this research. thanks are also due to pt. optik tunggal and staff for their contribution in providing the ophthalmic equipment and to prodia laboratory for the laboratory analyses. last but not least, we also thank the dean of the faculty of medicine, trisakti university for the funding of this study. references 1. world health organization. global data on visual impairments 2010. available at: http:// www.who.int/blindness/globaldatafinal forweb.pdf. accessed december 1, 2013. 2. cugati s, cunming rg, smith w, burlutsky g, mitchell p, wang jj. visual impairment, age71 related macular degeneration, cataract, and longterm mortality: the blue mountains eye study. arch ophthalmol 2007;125:917-24. 3. ocampo vvd, roy hr. senile cataract. available at: 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neurosci behav physiol 2008;38:887-90. 21. villareal dt, apovian cm, kushner rf, klein s. obesity in older adults: technical review and position statement of the american society for nutrition and naaso, the obesity society. obes res 2005;13:1849-63. 22. ye j, lou lx, he jj, xu yf. body mass index and risk of age-related cataract: a meta-analysis of prospective cohort studies. plos one 2014:9:e89923. doi:10.1371/journal.pone.00899 23. 23. park yh, shin ja, han k, yim hw, lee wc, park ym. gender difference in the association of metabolic syndrome and its components with age-related cataract: the korea national health and nutrition examination survey 2008-2010. plos one 2014;9:e85068. doi:10.1371/journal. pone.0085068. 24. kinoyama m, nitta h, hara s, watanabe a, shirao k. blood superoxide dismutase (sod) decrease following oral administration of plant sod to healthy subjects. j health sci 2007;53: 608-14. 25. kawai k, hayashi t, matsuyama y, minami t, era s. difference in redox status of serum and aqueous humor in senile cataract patients as monitored via the albumin thiol-redox state. jpn j ophthalmol 2010;54:584-8. 26. dawczynski j, blum m, winnefeld k, strobel j. increased content of zinc and iron in human cataractous lenses. biol trace element res 2002; 90:15-23. univ med vol. 33 no.1 72 rose, kartadinata superoxide dismutase in nuclear cataract 27. jamil sa, naseer sa, noreen k. comparison in effect of different metal ions, ph and reducing agent on the protease activity in human hypermature and mature cataract. j zhejiang univ sci b 2007;8:599-603. 28. gunduz g, gunduz f, yucel i, senturk uk. levels of zinc and magnesium in senile and diabetic senile cataractous lenses. biol trace element res 2003;95:107-12. rizaldy1 39 universa medicina januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 telaah kritis terapi risperidone untuk perbaikan perilaku pada gangguan spektrum autistik rizaldy pinzon*a, lucas meliala** dan sri sutarni** *smf saraf rsud dr. m.haulussy ambon maluku **bagian saraf fakultas kedokteran universitas gadjah mada korespondensi adr. rizaldy pinzon, m.kes.,sp.s staf medis fungsional saraf rsud dr. haulussy ambon, maluku telp. 0274-881008 email: medidoc2002@yahoo.com universa medicina 2007; 26: 39-45. abstrak autisme adalah sindroma klinik yang ditandai oleh gangguan interaksi sosial, hambatan komunikasi verbal, dan keterbatasan aktivitas serta minat. sampai saat ini, tidak ada terapi untuk autisme. pengobatan farmakologis terutama ditujukan untuk mengurangi agresivitas, perilaku obsesif kompulsif, dan perilaku melukai diri sendiri. telaah pustaka ini menunjukkan bahwa risperidone efektif dan aman untuk terapi tantrum, agresivitas, dan perilaku melukai pada anakanak autistik. namun risperidon tidak dapat memperbaiki kekurang anak dalam hal interaksi sosial dan komunikasi. diperlukan penelitian lebih lanjut untuk mengembangkan pengobatan yang efektif untuk memperbaiki gangguan intarksi sosial dan komunikasi pada penderita autistik. efektifitas yang menjanjikan dari resperidone ini memberikan harapan untuk mengobati anak-anak yang mengalami gangguan perilaku pada autisme. kata kunci : autistik, risperidone, anti psikotik, perilaku pinzon, meliala, sutarni risperidone dan gangguan spektrum austik 40 resperidone for the treatment for disruptive behaviors in autistic disorder: a critical appraisal rizaldy pinzon*a, lucas meliala** and sri sutarni** abstract autism is a clinical syndrome characterized by qualitative impairment of social interaction, verbal and nonverbal communication, imaginative activity, and a markedly restricted repertoire of activities and interests. no specific treatment is available that alters the course of the illness. the pharmacological treatments are used for decreasing aggressiveness, obsessive-compulsive behaviors and self-stimulating behaviors. this review showed that risperidone seems to be effective and well tolerated for the treatment of tantrums, aggression, or self-injurious behavior in children with autistic disorder. risperidone did not significantly change their deficit in social interaction and communication. further research is necessary to develop effective treatments for the core social and communicative impairments of autism. the encouraging efficacy outcomes achieved with this agent offer new hope for the management of behavioral symptoms exhibited by children with autism. keywords: autism, risperidone, anti psychotic, behaviour *neurology functional medical staf dr. m. haulussy hospital ambon, maluku **department of neurology medical faculty gadjah mada university correspondence adr. rizaldy pinzon, m.kes., sp.s neurology functional medical staf dr. m. haulussy hospital ambon, maluku telp. 0274-881008 email: medidoc2002@yahoo.com universa medicina 2007; 26: 39-45. pendahuluan autisme merupakan gangguan perkembangan yang terutama ditandai oleh ketidakmampuan dalam komunikasi, sosialisasi, dan imajinasi.(1) tatalaksana farmakologis tidak akan mengubah riwayat keadaan atau perjalanan gangguan autistik.(2) terapi farmakologi bukan merupakan pendekatan terapi yang utama, namun penggunaan terapi farmaka untuk gejala-gejala tertentu dapat membantu secara signifikan program terapi dan edukasi.(2) tu j u a n u t a m a p e n g g u n a a n t e r a p i f a r m a k o l o g i s p a d a a u t i s m e a d a l a h u n t u k mengendalikan gejala gangguan perilaku dan p s i k i a t r i k y a n g m u n c u l , s e h i n g g a a k a n meningkatkan kemampuan individu untuk berpartisipasi dalam sistem pendidikan, sosial, p e k e r j a a n , d a n k e l u a rg a , s e r t a m e m b a n t u keberhasilan program terapi lainnya.(2) obat-obat neuroleptik merupakan golongan obat yang secara luas digunakan pada autisme. penelitian morgan, et al(3) menunjukkan bahwa 86 dari 164 (52,4%) penderita autisme mendapat pengobatan neuroleptik. neuroleptik yang paling banyak digunakan adalah thioridizine (24,4%), haloperidol (22%), dan risperidone (22%). tinjauan pustaka ini akan membahas secara mendalam dasar rasional penggunaan anti psikotik atipikal untuk gangguan spektrum autistik, dan berbagai bukti ilmiah pendukungnya. efektivitas terapi digambarkan dengan nilai number needed to treat (nnt), yang menggambarkan jumlah pasien yang harus diterapi untuk mendapatkan satu pasien dengan efek yang diharapkan. nilai nnt semakin kecil menggambarkan semakin efektif suatu terapi.(4) 41 metode pelacakan kepustakaan pelacakan kepustakaan dilakukan dengan menggunakan internet, proquest database, dan pelacakan manual pada berbagai penelitian dan k a j i a n t e n t a n g t a t a l a k s a n a f a r m a k o l o g i s autisme dengan tahun publikasi 1995-2006. kata kunci yang dipergunakan adalah: autism, treatment, behaviour, randomized controlled trial (rct), dan risperidone. kriteria terpakai untuk dilakukan analisa kritis analisa kritis akan dilakukan pada artikel penelitian dengan karakteristik sebagai berikut: ( i ) s e m u a u j i k l i n i k a c a k , b u t a g a n d a , terkendali, yang meneliti tentang penggunaan terapi farmakologi risperidone pada penderita autisme dengan kontrol plasebo; (ii) naskah dalam bahasa inggris dan (iii) dipublikasikan a n t a r a t a h u n 1 9 9 5 2 0 0 6 . te l a a h k r i t i s dilakukan berdasar pada kajian dixon, et al(5) pada telaah kritis ini bukti-bukti ilmiah dari s u a t u p e n e l i t i a n d i k e l o m p o k k a n s e c a r a kuantitatif ke dalam tiga kelompok, yaitu: (i) pemaparan hasil, (ii) validitas seperti seleksi, pengukuran dan analisis statistik dan (iii) utilisasi. pengukuran efek terapi penetapan tingkat bukti ilmiah terhadap berbagai penelitian terapi yang ada didasarkan sesuai dengan panduan scotish intercollegiate guidelines network(6) (tabel 1). pengukuran efek terapi akan dilakukan sesuai dengan panduan guyatt et al(4) sebagai berikut: (tabel 2). tabel 1. derajat bukti ilmiah artikel terapi(6) tabel 2. pengukuran besar efek terapi dan efek samping akibat terapi(4) pinzon, meliala, sutarni risperidone dan gangguan spektrum austik 42 peningkatan keuntungan absolut adalah perbedaan rate absolut favorable outcome antara kelompok terapi dan kelompok kontrol. n n t a d a l a h j u m l a h p e n d e r i t a y a n g h a r u s diterapi untuk memperoleh satu pasien dengan r e s p o n y a n g m e n g u n t u n g k a n . n i l a i n n t d i d a p a t k a n d e n g a n m e n g h i t u n g 1 d i b a g i peningkatan keuntungan absolut, semakin kecil nnt adalah semakin baik. number needed to be harmed (nnh) digunakan untuk menilai efek samping terapi. nilai nnh didapatkan dengan menghitung 1 dibagi peningkatan risiko efek samping absolut, semakin besar nilai nnh adalah semakin baik.(5) hasil p a d a p e l a c a k a n p u s t a k a d i p e r o l e h 5 penelitian uji klinik yang membandingkan risperidone dan plasebo untuk terapi autisme pada anak-anak yang dapat diakses full text. tabel 3 dan 4 menunjukkan karakteristik 5 penelitian tersebut di atas. penelitian uji klinik dengan randomisasi dilakukan oleh mcdougle, et al (7) pada 31 penderita gangguan autisme dewasa. respon terapi diukur dengan global improvement scale dengan skala likert. perbaikan gejala didapatkan secara bermakna pada kelompok terapi risperidone dibanding plasebo (57% vs 0%, p <0,002). perbaikan gejala dijumpai pada p e n u r u n a n p e r i l a k u r e p e t i t i f , a g r e s i , kecemasan, depresi, dan iritabilitas. efek samping yang terjadi adalah sedasi ringan. pada seluruh pasien tidak didapatkan efek samping g a n g g u a n e k s t r a p i r a m i d a l d a n p e r u b a h a n gambaran ekg. tabel 3. karakteristik uji klinik dan tingkat bukti ilmiah penggunaan terapi risperidone pada autisme tabel 4. nilai telaah kritis penelitian terapi riperidone untuk autisme 43 tabel 6. telaah hasil penelitian risperidone untuk autisme p e n e l i t i a n u j i k l i n i k a c a k b u t a g a n d a (randomized clinical trial) risperidone lebih baru dilakukan oleh mccracken, et al(9) dengan subyek 101 anak autisme yang berusia antara 2-8 tahun. risperidone diberikan selama 8 minggu dengan dosis 0,5 mg sampai 3,5 mg per hari, dengan plasebo yang identik bagi kelompok kontrol. respon positif didefinisikan dengan pengurangan skor iritabilitas minimal 25%. hasil penelitian menunjukkan bahwa respon p o s i t i f s e c a r a b e r m a k n a d i d a p a t k a n p a d a kelompok terapi risperidone dibanding kelompok plasebo (69% vs 12%, p<0,01). penelitian aman, et al(10) pada 63 anak dengan autisme menunjukkan bahwa perbaikan p a d a s k a l a g l o b a l p a d a a k h i r p e n g a m a t a n tercapai pada 82,5% pasien. penghentian obat terjadi pada 6 pasien, 5 pasien akibat tidak ada perbaikan yang memuaskan, dan efek samping obat pada 1 orang pasien. h a s i l p e n e l i t i a n m c d o u g l e , e t a l ( 11 ) memperlihatkan terapi risperidone memberikan perbaikan pada perilaku sensori-motor, respon sensorik, dan afektif. terapi risperidone tidak bermakna untuk meningkatkan kemampuan berbahasa, dan kedekatan sosial. tabel 5 dan 6 menunjukkan karakteristik dan hasil penelitian uji klinik penggunaan risperidone untuk terapi autisme. pembahasan hasil kajian kritis pada dua penelitian uji klinik buta ganda penggunaan risperidone untuk autisme memperlihatkan nilai nnt sebesar 2. hal ini berarti setiap 2 pasien yang diterapi, a k a n d i d a p a t k a n e f e k r e s p o n t e r a p i y a n g menguntungkan pada 1 orang pasien. terapi risperidone untuk gangguan perilaku pada anak-anak dan remaja didukung pula oleh 3 uji klinik dengan jumlah sampel yang cukup besar. kedua hasil tersebut memperlihatkan bahwa terapi risperidone memberikan perbaikan gejala gangguan perilaku dan disruptif. tabel 5. hasil penelitian terapi risperidone pada autisme keterangan : rct = randomized controlled trial pinzon, meliala, sutarni risperidone dan gangguan spektrum austik 44 penelitian uji acak terkendali oleh reyes, et al(12) membandingkan penggunaan risperidone dan placebo pada 335 anak dan remaja dengan perilaku disruptif (bentuk gangguan perilaku yang sering teramati pada anak-anak dengan autisme). hasil penelitian menunjukkan bahwa gejala rekurensi pada kelompok risperidone s e c a r a b e r m a k n a l e b i h r e n d a h d a r i p a d a kelompok kontrol (27% vs 42,3%, p=0,002). penelitian findling, et al(13) pada 107 anak dengan gangguan perilaku disruptif dan tingkat intelegensi di bawah rata-rata memperlihatkan bahwa pemberian risperidone efektif untuk mengurangi gejala disruptif. efek samping utama yang muncul adalah mengantuk (33%), nyeri kepala (33%), dan pertumbuhan berat badan (21%). hasil serupa ditunjukkan pada penelitian uji klinik buta ganda oleh aman, et al(14) pada 118 anak dengan tingkat intelegensi di bawah rata-rata. pemberian risperidone efektif dalam memperbaiki gangguan perilaku. penelitian mccracken, et al(9) mendapatkan efek samping akibat terapi yang ringan, dan akan menghilang dengan sendirinya setelah beberapa m i n g g u . e f e k s a m p i n g y a n g u t a m a p a d a penggunaan risperidone adalah penambahan n a f s u m a k a n , m e n g a n t u k , k e l e l a h a n , d a n meneteskan air liur. efek samping mengantuk memiliki nilai nnh 3, yang berarti akan ada 1 orang yang mengalami efek samping mengantuk di antara 3 orang yang diterapi risperidone (tabel 7). efek samping konstipasi, pandangan kabur, mulut kering, dan mengantuk disebabkan oleh p e r a n g s a n g a n s i s t e m a n t i k o l i n e r g i k p a d a pemberian anti psikotik. sifat penghambatan histamin akan menyebabkan penambahan berat badan dan mengantuk. sifat antagonistik pada reseptor alfa satu akan menyebabkan penurunan tekanan darah, dizziness, dan mengantuk.(15,16) hasil yang konsisten ditunjukkan pada penelitian yang dilakukan pada anak-anak, pemberian resperidone mampu menaikkan berat badan sebanyak 2,7 kg dibandingkan kelompok kontrol 1,0 kg.(17) untuk mencegah terjadinya kenaikan berat badan anak-anak dianjurkan untuk diet dan melakukan aktifitas olahraga.(18) efek samping yang terjadi dapat sembuh sendiri (self-limiting) a t a u d a p a t d i t a n g g u l a n g i d e n g a n c a r a m e n g u r a n g i d o s i s p e m b e r i a n r e s p e r i d o n e . efektifitas yang menjanjikan dari resperidone ini memberikan harapan untuk mengobati anakanak yang mengalami gangguan perilaku pada autisme. kesimpulan a u t i s m e m e r u p a k a n k e l a i n a n y a n g kompleks, terutama ditandai oleh gangguan fungsi berbahasa, interkais sosial, dan gangguan tabel 7. telaah terhadap kejadian efek samping akibat penggunaan risperidone untuk terapi autisme(9) bermakna bila p < 0,05 45 perilaku. penatalaksanaan farmakologis dengan prinsip menyeimbangkan fungsi neurotransmiter m e r u p a k a n d a s a r p e n d e k a t a n t e r a p i y a n g rasional. beberapa penelitian menunjukkan efektivitas terapi antagonis sistem dopaminergik pada autisme. tiga buah uji klinik randomisasi membuktikan bahwa terapi risperidone efektif dan relatif aman untuk terapi autisme. daftar pustaka 1. herman a. neurobiological insights into infantile autism. in: the harvard brain. spring; 1996. p. 1925. available at: http://www.hcs.harvard.edu/~husn/ brain/vol3/contents.html. accessed april 1, 2006. 2. rapin i. autism: current concept. n engl j med 1997; 337: 97-104. 3. perry p, kuperman s. pediatric psychopharmacology: autism, clinical psychopharmacology seminar. university of iowa ; 2003. 4. guyatt gh, sackett dl, cook dj. how to use an article about therapy and prevention. jama 1995; 27: 59-63. 5. dixon ra, munro jf, silcocks pb. the evidence based medicine: critical appraisal for clinical problem solving. oxford: read educational and professional publishing ltd. oxford; 1997. 6. sign. level of evidence and recommendation. scotish intercollegiate guideline network, 2001. available at: http://www.sign.ac.uk. accessed april 7, 2006. 7. mcdougle cj, holmes jp, carlson dc, pelton gh, cohen dj, price lh, et al. a double-blind, placebocontrolled study of risperidone in adults with autistic disorder and other pervasive developmental disorders. ach gen psychiatry 1998; 53: 633-41. 8. masi a, cosenza a, mucci m, de vito g. risperidone monotherapy in preschool children with pervasive developmental disorders. j child neurol 2001; 16: 395-400. 9. mccracken jt, mcgough j, shah b, cronin p, hong d, aman mg, et al. risperidone in children with autism and serious behavioral problems. n eng j med 2002; 347: 314-1. 10. research units on pediatric psychopharmacology autism network. risperidone treatment of autistic disorder: longer term benefits and blinded discontinuation after 6 months. am j psychiatry 2005; 162:1361-9. 11. mcdougle mj, scahill l, aman mg, mccracken jt, tierney e, davies m, et al. risperidone for the core symptom domains of autism: results from the study by the autism network of the research units on pediatric psychopharmacology. am j psychiatry 2005; 162: 1142-8. 12. reyes m, buitelaar j, toren p, augustyns i. a randomized, double blind, placebo controlled study of risperidone maintenance treatment in children and adolescents with disruptive behavior disorder. am j psychiatry 2006; 163:402-10. 13. findling rl, aman mg, eerdekens m. long term, open label study of risperidone in children with severe disruptive behaviors and below average iq. am j psychiatry 2004; 161: 1677-84. 14. aman mg, smedt gd, derivan a, lyons b. double-blind, placebo-controlled study of risperidone for the treatment of disruptive behaviors in children with subaverage intelligence. am j psychiatry 2002; 159: 1337-46. 15. nestler ej, hyman se, malenka rc. molecular neuropharmacology: a foundation for clinical neuroscience. new york: mcgraw-hill companies; 2001. 16. jones hm, pilowsky ls. dopamine and antipsychotic drug action revisited. br j psychiatry 2002; 181: 271-5. 17. shea s, turgay a, carroll a, schulz m, orlik h, smith i, et al. risperidone in the treatment of disruptive behavioral symptoms in children with autistic and other pervasive developmental disorders. pediatrics 2004; 114: 634-41. 18. turgay a, binder c, snyder r, fisman s. longterm safety and efficacy of risperidone for the treatment of disruptive behavior disorders in children with subaverage iqs. pediatrics 2002; 110. judhy1 90 fathers help to improve breastfeeding practice: can indonesian fathers provide the same help? judhiastuty februhartanty,*a siti muslimatun*, and andi mariyasari septiari* * southeast asian ministers of education organization (seameo), tropical medicine and public health (tropmed), regional center for community nutrition (rccn), university of indonesia, jakarta korespondence ajudhiastuty februhartanty, msc seameo-tropmed rccn, university of indonesia, campus of ui salemba salemba raya no.6, jakarta 10430, indonesia po box 3852, jakarta indonesia 10038 phone:+62-21-31930205 fax:+62-21-3913933 e-mail:jfebruhartanty@seameorccn.org universa medicina 2007; 26: 90100. universa medicina april-juni 2007april-juni 2007april-juni 2007april-juni 2007april-juni 2007 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 the objectives of this paper are i) to explore factors influencing practices of timely breastfeeding initiation and exclusive breastfeeding with special attention on factors related to the roles of father; and ii) to elaborate possible factors influencing father’s roles in breastfeeding based on literatures from the developed world. studies conclude that father’s roles in supporting breastfeeding include participation in decision on infant feeding mode, involvement in child care taking and household tasks, as well as being the protector and the family provider. they also explore that the quality of couple relationship, parental knowledge and attitudes, characteristics of parental function, and the burden on the family economy are barriers which discourage fathers from being supportive towards breastfeeding. indonesian fathers may influence breastfeeding in the same way and also experience similar barriers. however, due to different exposure to norms, beliefs, and cultures, a specific study on this matter is very relevant to be carried out in indonesia prior to any breastfeeding promotion targeted to fathers. keywords: breastfeeding initiation, exclusive breastfeeding, father’s roles abstract 91 universa medicina vol.26 no.2 peran ayah untuk meningkatkan praktek pemberian asi: dapatkah ayah di indonesia melakukannya? judhiastuty februhartanty,*a siti muslimatun*, dan andi mariyasari septiari* makalah ini bertujuan untuk menggali literatur tentang i) faktor-faktor yang mempengaruhi inisiasi dini menyusui dan pemberian asi eksklusif, terutama faktor yang berkaitan dengan peran ayah, dan ii) faktor-faktor yang mempengaruhi peran ayah tersebut. studi-studi di negara maju menyimpulkan bahwa peran ayah dalam mendukung pemberian asi mencakup keikutsertaannya dalam pengambilan keputusan untuk pola pemberian makan bayi, perawatan/pengasuhan bayi, pekerjaan rumah tangga, dan kegiatan sebagai pencari nafkah keluarga. studi-studi lain juga dari negara maju menunjukkan bahwa kualitas kehidupan pernikahan, pengetahuan dan sikap, karakteristik parenting, dan beban ekonomi keluarga dapat menjadi kendala sehingga ayah tidak dapat memberikan dukungan yang semestinya terhadap pemberian asi. ayah di indonesia mungkin dapat memberikan dukungan serupa terhadap pemberian asi dan mengalami kendala yang mirip seperti yang dialami ayah di negara maju. namun karena mereka dilingkupi oleh norma, kepercayaan, dan budaya yang berbeda, maka sebuah studi tentang peran ayah di indonesia terhadap pemberian asi sangat diperlukan. kata kunci: inisiasi menyusui, pemberian asi eksklusif, peran ayah abstrak introduction b a s e d o n wo r l d h e a l t h o rg a n i z a t i o n ( w h o ) / u n i t e d n a t i o n c h i l d r e n ’s f u n d (unicef),(1) the current recommendations on b r e a s t f e e d i n g i n c l u d e t h e f o l l o w i n g k e y behaviors: i) early initiation of breastfeeding; ii) feeding of colostrum to the newborn; iii) exclusive breastfeeding for the first 0-6 months; and iv) continued breastfeeding through the second year. in response to the above global recommendation, this paper focuses on the elaboration of breastfeeding practices related to timely breastfeeding initiation and exclusive breastfeeding. with regards to breastfeeding initiation, who (2) stated that mothers and infants should not be separated after birth unless there is an unavoidable medical reason. optimally the infant should be left with the mother continuously from birth, and allowed to attach spontaneously to the breast whenever he shows signs of readiness to do so. an a r b i t r a r y b u t p r a c t i c a l m i n i m u m recommendation is for skin-to-skin contact to start within at most half-an-hour of birth and t o c o n t i n u e f o r a t l e a s t 3 0 m i n u t e s . f u r t h e r m o r e , w h o ( 3 ) d e f i n e s e x c l u s i v e l y breastfed infants as those who have received only breast milk from the mother or a wet nurse, or expressed breast milk, and no other liquids or solids with the exception of drops or syrups consisting of vitamins, mineral supplements, or medicines. in indonesia, early breastfeeding initiation was practiced only among 8.3% of infants within 30 minutes after birth,(4) 3.7-36% within * seameo tropmed rccn universitas indonesia korespondensi ajudhiastuty februhartanty, msc seameo-tropmed rccn, universitas indonesia, kampus ui salemba jl. salemba raya no.6, jakarta 10430, indonesia po box 3852, jakarta indonesia 10038 telp.021-31930205 fax. 021-3913933 e-mail:jfebruhartanty@seameorccn.org universa medicina 2007; 26: 90-100. 92 one hour,(5-6) and 27.1% within one day after d e l i v e r y. ( 5 ) t h e d a t a s u g g e s t t h a t e a r l y b r e a s t f e e d i n g i n i t i a t i o n i s n o t c o m m o n l y practiced. the delay in starting breastfeeding i m m e d i a t e l y i s a n i n d i c a t i o n t h a t s o m e prelacteal feed is given during the period between birth and initiation of breastfeeding. indonesian demographic and health survey in 2002-2003 found that the percentage of infants who received a prelacteal liquid was very high. as expected, infants were more likely to receive liquid than semi solid food before they are r e g u l a r l y b r e a s t f e d 4 5 % a n d 1 8 % respectively.(5) furthermore, results of national nutrition and health surveillance conducted by helen keller international/indonesia found that in 2002 25% of the infants in jakarta were exclusively breastfed for 1-3 months, 4% for 4-6 months and 1% for 5-6 months.(7) a more recent data found that exclusive breastfeeding practiced until 4-6 months in jakarta was 8.5%.(6) this means that a higher proportion of i n f a n t s a g e d b e l o w 6 m o n t h s h a v e b e e n introduced to liquid, semi solid and/or solid foods other than breast milk. furthermore, data from indonesian ministry of health shows that the average age of indonesian children given breast milk was 23.9 months.(4) this is similar t o t h e r e c o m m e n d a t i o n o f t h e i n n o c e n t i declaration(8) to give breast milk to the children up to the age of 24 months. other data reveals that although breastfeeding duration in general among indonesian children was 22.3 months but it was only 14.4 months in dki jakarta.(5) the d a t a s h o w t h a t a l t h o u g h b r e a s t f e e d i n g i s universally acknowledged as the most beneficial m e t h o d o f i n f a n t f e e d i n g a n d c o m m o n l y p r a c t i c e d , b r e a s t f e e d i n g p r a c t i c e s a s recommended (i.e. early breastfeeding initiation after birth, exclusive breastfeeding from birth until 6 months of age) are generally low. these sub-optimal practices on the two key b r e a s t f e e d i n g b e h a v i o r s m a y r e d u c e t h e prominent benefits of breastfeeding for child optimal growth and survival. such a benefit is reported that timely initiation of breastfeeding i s c o n s i d e r e d c r u c i a l s t a r t i n g p o i n t f o r s u c c e s s f u l b r e a s t f e e d i n g . ( 2 ) d e l a y e d breastfeeding initiation increased the risk of n e o n a t a l m o r t a l i t y d u e t o h y p o t h e r m i a attributed from delayed skin-to-skin contact.(9) delayed skin contact may also increase the risk of experiencing nosocomial infections among i n f a n t s . ( 1 0 ) e a r l y i n i t i a t i o n i s l i k e l y t o b e associated with a longer duration of exclusive breastfeeding and also longer duration of breastfeeding in general.(11) introduction of liquid, semi solid and/or solid foods before the infant reaches the age of 6 months has been proven to have effect on lower growth, (12) increase incidence and/or severity of a wide range of infectious diseases such as diarrhea(13) and respiratory tract infection.(14) breastfeeding also reduces risk of childhood obesity.(15) in short, benefits of breastfeeding are o u t s t a n d i n g . w h i l e t h i s u n d e r s t a n d i n g i s fundamental to address the high public health significance resulted from a simple and natural practice such as breastfeeding, many studies found that simply understanding the benefits of breastfeeding is not convincing enough to e n c o u r a g e n u r s i n g m o t h e r s t o p e r f o r m breastfeeding practices as recommended. this is due to the fact that factors influencing breastfeeding practices are multifaceted. and therefore, this paper ’s objective is to explore f a c t o r s i n f l u e n c i n g p r a c t i c e s o f t i m e l y b r e a s t f e e d i n g i n i t i a t i o n a n d e x c l u s i v e breastfeeding with special attention on factors related to the roles of father. this paper also elaborates possible factors influencing father ’s roles in breastfeeding based on literatures from the developed world. the information presented in this paper may be useful to portray the p o s s i b l e r o l e s o f i n d o n e s i a n f a t h e r s i n supporting breastfeeding. februhartanty, muslimatun, septiari indonesian fathers improve breastfeeding 93 universa medicina vol.26 no.2 factors influencing timely breastfeeding i n i t i a t i o n a n d e x c l u s i v e b r e a s t f e e d i n g practices: do fathers have influence on these practices? delayed initiation of breastfeeding is directly related to lack of antenatal exposure,(2) inappropriate attitudes and practice of health personnel,(2,16-17) unfriendly policy at hospital/ c l i n i c s e t t i n g t o b r e a s t f e e d i n g p r a c t i c e ( 2 ) including marketing of breast milk substitutes at maternity hospital/clinic.(5) improper social influences from the family, peer, and community shape the innate intention of the mothers for not breastfeeding,(10) thus hindering timely initiation of breastfeeding. f u r t h e r m o r e , f a c t o r s a s s o c i a t e d w i t h exclusivity and duration of breastfeeding include p o o r k n o w l e d g e a n d l a c k o f m o t h e r ’s confidence,(17-18) inadequate skills of appropriate breastfeeding techniques (i.e. positioning, latching on) and management of common p r o b l e m s d u r i n g l a c t a t i o n ( i . e . b r e a s t engorgement, perception of insufficient breast milk, sore nipple, etc),(19-20) lack of physical and psychological supports received during lactation period (i.e. from family members, peer, health p r o f e s s i o n a l s ) , ( 1 0 , 2 1 2 2 ) u s e o f t e a t s o r p a c i f i e r s , ( 2 , 2 3 ) m a t e r n a l e m p l o y m e n t , ( 1 7 , 2 4 ) exposure from various sources of information,(25) inappropriate culture and norms regarding exclusive breastfeeding among the family members, peer, and society.(17,26) data from the developed world state that most women who decide to breastfeed make this d e c i s i o n b e f o r e o r e a r l y i n p r e g n a n c y. ( 2 7 ) moreover, breastfeeding plans described by the mother at the time of delivery were significantly associated with the duration of breastfeeding.(23) time of planning to breastfeed does not seem to be an issue in indonesia since breastfeeding has always been regarded as a normative act. however, other factors may have played greater roles in influencing indonesian nursing mothers to practice breastfeeding as recommended. nurses/midwives,(17,22) family members,(17,28) and p a r t n e r ’s v i e w s ( 1 7 1 9 , 2 7 , 2 9 ) w e r e i m p o r t a n t influences for both breastfeeding initiation and continued breastfeeding. the latter influence is less elaborated in the indonesian context even though lawrence and lawrence(10) states that breastfeeding as any other types of child caring has been acknowledged as a triadic relationship among mother, father and the infant. some observational studies confirm this fact that father is one of the key influences in breastfeeding.(3031) furthermore, studies also conclude the influence of father particularly on breastfeeding decision,(18) breastfeeding initiation,(29,32) also duration and exclusivity of breastfeeding(24,33) and a risk factor for bottle feeding.(17,30) based on father ’s participation in the decision to breastfeed and their involvement in caretaking, that there are four styles of fathering i.e. involved, assistor, supervisor, and detached. involved fathers participate actively in the decision to breastfeed and in caring for their children. assistor fathers follow their wives’ lead regarding breastfeeding and they are actively involved in care taking. supervisor fathers are involved with the breastfeeding decision and they provide little child care or household help. detached fathers are not involved in child care or the breastfeeding decision.(34) exclusive breastfeeding in which its success i s i n f l u e n c e d b y t i m e l y i n i t i a t i o n o f breastfeeding(11) is considered time demanding by the nursing mothers,(21) thus women need supporters during postpartum period. right after delivery, support from health provider for early breastfeeding initiation is significant.(2) at this point, fathers or other family members who accompany the mothers during delivery inside or outside the delivery room may provide a source of emotional support. although such a practice is getting common recently among 94 indonesian fathers,(35) information on its effect to the mother psychological state, thus to timely breastfeeding initiation is lacking. furthermore, a hypothesis on this regard may be addressed in s u c h a w a y t h a t f a t h e r s w i t h a p p r o p r i a t e knowledge and attitude on the importance of timely breastfeeding initiation would be able to influence the hospital setting by directly asking f o r t h e r i g h t s o f t h e n e w b o r n b a b y t o b e immediately fed with the mother’s breast milk. furthermore, many studies have reported type of roles the nursing fathers can provide for supporting exclusive breastfeeding immediately after the birth of the infant up to the age of 6 m o n t h s a n d s u p p o r t i n g a n y b r e a s t f e e d i n g practices. these include paternal participation i n d e c i s i o n o n i n f a n t f e e d i n g m o d e 3 4 a n d involvement in child care taking.(24,29,34,36) child care includes developing comforting skills for the infant such as stroking, holding, carrying, cuddling,(10) changing baby’s diaper and less night sleep(37) caring for the infant when the mother works outside home(24) and involvement in infant feeding i.e. help mother express the breast milk, store it and feed it to the infant.(29,38) additionally, paternal roles are also identified within the family economy as they are the protector and the bread winner,(36) also provider o f g o o d n u t r i t i o n f o r t h e b r e a s t f e e d i n g mothers,(24) and assistance for some forms of household tasks.(6,39) other roles of fathers are related to couple (i.e. husband-wife) functions which include nurturance of marital relationship based on the satisfaction of a couple’s objective and subjective needs with the provision of mutual s u p p o r t a i m e d a t p e r s o n a l d e v e l o p m e n t (friendship), partnership in daily responsibilities and tasks (fellowship), and an affective and sexual relationship (love relationship). (33,39) conflicting couple’s relationship and parental function as well as lack of paternal knowledge may predispose fathers to suggest bottle feeding to the nursing mothers.(17,30) a study among low income fathers and nursing mothers in usa found that father’s roles in infant feeding include father involvement in feeding of expressed breast milk.29 this may be caused by the fact that fathers want to build closeness with the infant, and furthermore it is felt hurtful by the fathers to know that they can not duplicate the comfort the mother can provide to the infant while breastfeeding. this issue may not yet be the case felt by the indonesian fathers. however, it is very relevant to explore father ’s feeling of jealousy towards the mother and/or the infant. furthermore, such issues about expressed breast milk for infants whose mothers return to work or are away also need further investigation among indonesian fathers. norms and beliefs related to feeding the infant not directly from the mother’s breasts may or may not be favorable in this setting. the magnitude of male’s involvement specifically in pregnancy care is reported in the 2002-2003 indonesian demography and health survey (idhs) in which currently married men who have had at least one child were asked about pregnancy care of the mother of the last-born child and the health care of the child. findings in dki jakarta province show that during their wife’s last pregnancy, 48% of fathers talked to a health care provider about the pregnancy care and health of their wife. of these men, 45% talked with a health care provider about the types of foods his wife should eat during pregnancy, 46% talked about how much rest she should have during pregnancy, and 44% talked about types of health problems for which she should get immediate medical attention. fathers in their thirties live in urban areas, and those who are better educated are more likely than other fathers to talk with a health care provider about their wife’s health and care during pregnancy.(5) while breastfeeding should be prepared long enough during pregnancy, the idhs data show that breastfeeding topics were less discussed with the februhartanty, muslimatun, septiari indonesian fathers improve breastfeeding 95 universa medicina vol.26 no.2 fathers during antenatal visits. although many studies conclude the need to include fathers during antenatal counseling for influencing breastfeeding initiation and duration, it is substantial to investigate further the influence of paternal involvement during antenatal care to breastfeeding practices in indonesia. f a c t o r s i n f l u e n c i n g f a t h e r ’s r o l e s i n breastfeeding a growing concern on father ’s roles in breastfeeding is closely related to a deeper understanding on the challenges in fathering. over the last 30 years, fathers’ roles in caring for their children have been expanded by rapid and profound socioeconomic changes and by society’s evolving perceptions and expectations of fathers’ roles.(40) the dramatic movements of childbearing-aged women into workforce, the s o f t e n i n g o f s e x u a l s t e r e o t y p e s a f t e r t h e achievements of the women’s movement, and e x p r e s s e d l o n g i n g a m o n g m e n f o r d e e p e r meaning in their lives have brought men into close contact with their infants. fathering, as with mothering, exists simultaneously for the infant and the family. (41) this implies that parenting is equal for women and men. there are complementary activities for fathers and mothers. parents are not equally able to do all t h i n g s . t h e s t r e s s p l a c e d o n s h a r i n g responsibilities of parenthood implies an acrossthe-board division of labor. there is more to nurturing the infant than to feeding. the father therefore should play a very significant role with the infant. for instance, when the infant is fussy and does not need to be fed, comforting is often best done by the father. nonnutritive cuddling is best done by the father.(10) in the united states, father means more than “wage earner” or “provider” and now can include stay-at-home dad, caregiver of child and sharer of child care responsibilities.(40) in indonesian context, these phenomena seem to be emerging. a limited study among patrilineal and matrilineal families of rural households in indonesia found that 21% and 30% of fathers respectively were identified t o t a k e i m p o r t a n t r o l e s i n c h i l d c a r i n g responsibilities especially among those who live as nuclear families.(42) due to the economic burden, more women now are working. this leaves the consequence that father’s roles in child caring are expected to be more substantial. a review on the parenthood experiences during the child’s first year concluded that mothers felt satisfied and confident as a mother, are overwhelmed and drained for being primarily responsible for the child, struggled with the limited time available for herself and that fathers felt confident as a father and as a partner, drained with the new demands in life, are hurtful for being prevented from achieving closeness to the child, and being the protector and the primary provider of the family.(36) as such, pruett(41) states that parenthood is influenced first by past experience, spousal expectations, economics, personal and marital values and behaviors. a review study on supporting the father whose infant is being breastfed concluded that the quality of adult couple’s relationship is an important predictor of breastfeeding success because the foundational relationship in the family is the adult relationship between the mother and the father.(39,43) a study by falceto et al(33) found that a good couple relationship was associated with more paternal breastfeeding support and involvement in infant’s care but not in the breastfeeding practice. this finding is inconsistent with sullivan et al. (39) this is probably because falceto (33) included both couple’s function and parental function into couple’s relationship and sullivan(39) categorized h u s b a n d w i f e i n t e r a c t i o n f o r c o u p l e ’s relationship. an earlier study used a qualitative study to explore the experiences of 10 canadian fathers in dual-earner families after birth of their first infants in terms of redefining their roles as 96 spouses, workers, and fathers. the study found that the fathers’ experiences consisted of coping with many demands from children, partners, and jobs. these men redefined their roles as fathers a f t e r t h e i r p a r t n e r s r e t u r n e d t o f u l l t i m e employment.(44) in addition, a study in australia found that although rewarding, fathers found new or expanding fatherhood to be a significant challenge and time of change. major themes i n c l u d e d m a k i n g a c o m m i t m e n t , t a k i n g responsibility, negotiating responsibilities, developing and maintaining relationships, m a i n t a i n i n g f a m i l y i n t e g r i t y, b a l a n c i n g activities, and perceiving the self as father. work h a d a m a j o r i m p a c t o n f a t h e r s ’ a b i l i t y t o participate with their family and newborn. to manage, fathers sought to balance the demands of work and home, deal with stressors, manage their time, develop routines, and reprioritize. fathers developed a sense of themselves as fathers over time, building confidence and deriving satisfaction from their fathering role. a range of competing factors affected fathers’ ability to participate in the home with their newborn in the early weeks after birth.(45) furthermore, a qualitative study among midwives and nursing mothers in north jakarta reveals that fathers had strong influence in early introduction of formula milk as a result of father’s being unconfident to the sufficiency of the mother ’s breast milk and having improper perception towards baby’s cry. mothers who felt afraid of his command finally complied by supplementing breast milk with formula milk.(17) these suggest that fathers should also be the target for exposure to appropriate knowledge about breastfeeding and practical skill on how to overcome its difficulties when occur. in response to the above rationale, an observational study targeted to fathers and nursing mothers in south jakarta is currently in progress with the objectives of i) investigating father ’s roles which are most influential to support timely breastfeeding initiation and e x c l u s i v e b r e a s t f e e d i n g p r a c t i c e s ; a n d i i ) exploring its determinant factors that include q u a l i t y o f c o u p l e r e l a t i o n s h i p , p a r e n t a l knowledge and attitudes, characteristics of parental function, and socio-economic factors. lessons learned from the interventions involving fathers in the developed world interventions directly targeted to fathers h a v e b e e n i n i t i a t e d i n s o m e d e v e l o p e d countries(32,38,46-48) as presented in table 1. in s u m m a r y, t h e e x p e r i e n c e s r e f l e c t s o m e interesting strategies on how to approach the western fathers. each has its own challenges. however, these interventions were mainly addressed to raise awareness and improve knowledge related to breastfeeding techniques and benefits to male target audiences. except a study in italy which involved fathers to help manage common lactation-related problems e n c o u n t e r e d b y t h e i r f e m a l e p a r t n e r s , ( 3 8 ) exploration on what specific support a father should provide to the nursing mother and the infant during lactation period is not yet well addressed. likewise, a proper understanding on factors influencing father’s roles in supporting or not supporting breastfeeding practices must be addressed proportionally in order to match with the actual context of the father’s capacity since at the same time they are expected to providing various roles as parent, husband, and bread winner. if we are to copy the lessons learned from the existing interventions, a careful assessment and judgment on the used approaches should be in place for ensuring the acceptability and applicability of the approach in indonesia setting. approaching indonesian fathers may be even more challenging especially when issue to be discussed is related to child caring an issue which is probably still considered as outside of their “domain”. februhartanty, muslimatun, septiari indonesian fathers improve breastfeeding 97 universa medicina vol.26 no.2 t ab le 1 . t h e su m m ar y o f so m e in te rv en ti o n s in v o lv in g f at h er s 98 i n t h i s r e g a r d , r e l a t i o n s h i p b e t w e e n indonesian fathers and mothers may be worth i n v e s t i g a t i n g s i n c e t h i s i s r e l e v a n t f o r understanding flow of communication and path of decision making among indonesian couples. then, this information can be used to decide whether fathers may be well approached through the mothers or it is the other way around. conclusions interestingly, studies in the developed countries confirm that there are barriers which hinder fathers from being supportive towards breastfeeding practice. these barriers include the quality of couple relationship, parental knowledge and attitudes, characteristics of parental function, and the burden on the family economy. indonesian fathers may be able to provide similar supports as provided by their western counterparts. however, type of father ’s support expected by the indonesian mothers for influencing the breastfeeding practices should be explored. as such, a careful analysis on the barriers encountered by the indonesian fathers should be done as they may be different from those experienced by the westerners. this is due to the fact that norms and beliefs as well as prevailing cultures in indonesian society are of course different from those of the western s o c i e t y a n d k n o w n t o s t r o n g l y i n f l u e n c e someone’s actual actions. therefore, it is crucial to investigate types of father ’s roles that are strategically influencing breastfeeding practice and at the same time to explore types of father’s roles that match with the actual context of the father’s capacity as a father, a husband, and the bread winner of the family. acknowledgements the authors wish to thank the nestle foundation, switzerland for the financial contribution in the stage of literature review during the research proposal development. references 1. who/unicef. global strategy for infant and young child feeding. geneva, switzerland: who; 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(in indonesian). 26. abada tsj, trovato f, lalu n. determinants of breastfeeding in the philippines: a survival analysis. soc sci med 2001; 52: 71-81. 27. earle s. factors affecting the initiation of breastfeeding: implication for breastfeeding promotion. health prom intl 2002; 17: 205-14. 28. libbus k, bush ta, hockman nm. breastfeeding beliefs of low-income primigravidae. int j nurs stud 1997; 34: 144-50. 29. schmidt mm, sigman-grant m. perspectives of low-income fathers’ support of breastfeeding: an exploratory study. jne 1999; 31: 31-7. 30. bar-yam nb, darby l. fathers and breastfeeding: a review of literature. j hum lact 1997; 13: 45-50. 31. bentley me, caulfield le, gross sm, bronner y, jensen j, kessler la, et al. source of influence on intention to breastfeed among african-american women at entry to wic. j hum lact 1999; 15: 2734. 32. wolfberg aj, michels kb, shields w, o’campo p, bronner y, bienstock j. dads as breastfeeding advocates: results of a randomized controlled trial of an educational intervention. am j obstet gynecol 2004; 191: 708-12. 33. falceto og, giugliani erj, fernandes clc. couples relationships and breastfeeding: is there an association? j hum lact 2004; 20: 46-55. 100 34. gamble d, morse j. fathers of breastfed infants. jognn 1993; 22: 358-365. cited by: bar-yam nb, darby l. fathers and breastfeeding: a review of literature. j hum lact 1995; 13: 45-50. 35. sood s, chandra u, palmer a, molyneux i. measuring the effects of the siaga behavior change campaign in indonesia with populationbased survey results. maryland, usa: jhpiego; 2004. 36. nystrom k, ohrling k. parenthood experiences during the child’s first year: literature review. j adv nurs 2004; 46: 319-30. 37. gay cl, lee ka, and lee sy. sleep patterns and fatigue in new mothers and fathers. biol res nurs 2004; 5: 311-8. 38. pisacane a, continisio gi, aldinucci m, d’amora s, continisio p. a controlled trial of father’s role in breastfeeding promotion. electronic article. pediatrics 2005; 116: 494-8. available at http:// www.pediatrics.aappublications.org/cgi/content/ full/116/4/e494. accessed oktober 31, 2005. 39. sullivan ml, leathers sj, kelley ma. family characteristics associated with duration of breastfeeding during early infancy among primiparas. j hum lact 2004; 20: 196-205. 40. coleman wl, garfield c. committee on psychosocial aspects of child and family health. fathers and pediatricians: enhancing men’s roles in the care and development of their children. pediatrics 2004; 113: 1406-11. available at http:// www.pediatrics.org/cgi/content/full/113/5/1406. accessed august 7, 2006. 41. pruett kd. role of the father. pediatrics 1998; 102: 1253-61. available at http://www.pediatrics.org/cgi/ content/full/102/5/se1/1253. accessed october 18, 2006. 42. februhartanty j, fransisca do, roshita a, fahmida u, dianawati e. childcare of children under three years old in patrilineal and matrilineal families: a study among karo and minangkabau households. research report. jakarta, indonesia: seameotropmed rccn-ui; 2005. 43. jordan pl, wall vr. supporting the father when an infant is breastfed. j hum lact 1993; 9: 31-4. 44. hall wa. new fatherhood: myths and realities. public health nurs 1994; 11: 219-28. 45. st john w, cameron c, mcveigh c. meeting the challenges of new fatherhood during the early weeks. j obstet gynecol neonatal nurs 2005; 34: 180-9. 46. cohen r, lange l, slusser w. a description of a male-focused breastfeeding promotion corporate lactation program. j hum lact 2002; 18: 61-5. 47. ingram j, johnson d. a feasibility study of an intervention to enhance family support for breastfeeding in a deprived area in bristol, uk. midwifery 2004; 20: 367-79. 48. stremler j, lovera d. insight from a breastfeeding peer support pilot program for husbands and fathers of texas wic participants. j hum lact 2004; 20: 417-22. februhartanty, muslimatun, septiari indonesian fathers improve breastfeeding editorial 151 ebola virus – new threat to global health rina k. kusumaratna department of public health, faculty of medicine, trisakti university univ med vol. 33 no.3 ebola hemorrhagic fever is a fatal infectious disease of humans and primates. the disease is caused by single-stranded rna viruses belonging to the family filoviridae. the ebola virus started to emerge in 1976, in an outbreak that almost simultaneously attacked two countries, namely zaire and sudan. (1) around 500 cases were reported, with a case fatality rate of 88% in zaire and 53% in sudan. although occurring at the same time, the ebola viruses in the two countries were of different species, by serologic criteria as well as by sequence analysis.(2) the following ebola virus species have been found: zaire ebolavirus, sudan ebolavirus, cote d’ivoire ebolavirus, reston ebolavirus, and bundibugyo ebolavirus.(1) since september 2014, more than 4000 confirmed cases of ebola virus disease, with more than 2000 deaths, have been reported in west african countries, such as guinea, liberia, nigeria, senegal, and sierra leone.(3) the ebola outbreak started in guinea in desember 2013.(4) nine months after the occurrence of the first cases, there was a weekly increase in the number of reported cases and deaths. there is now a global warning for nations to be on the alert against the spread of the ebola virus. therefore the world health organization has declared the situation to be a public health emergency of international concern.(3) at the present time the ebola outbreak is developing and spreading, so there are substantial challenges in controlling the outbreak, halting its transmission, and providing clinical services to ebola virus patients. however, the detection of patients with ebola infection requires adequate diagnostic facilities, accompanied by treatment clinics and medicines, so as to help in containing the disease. the ebola virus infection is mainly spread by contact with body fluids of symptomatic patients, and its transmission can be prevented by early diagnosis, contact tracing, isolation of patients, patient care, and infection control.(4,5) since it became known that two american healthcare workers in liberia had become infected with the ebola virus, the ebola outbreak in west africa has attracted much attention and caused anxiety in many countries.(7) this is not the first viral outbreak in west africa that has caused concern in advanced countries. between 1928-1929, a yellow fever outbreak in west africa resulted in the deaths of prominent research workers from america, england, and japan.(7) although several ecological aspects of the virus are known, there is still much to be investigated in relation to its ecology. fruit bats are thought to be one of the reservoirs of the ebola virus, although other animals may also be susceptible to the ebola virus and be able to infect humans.(6) it is thought that infection in humans first occur at the time of exposure to body fluids from animal reservoirs of the virus, or intermediate animal hosts.(1) a study conducted by pourrut et al.(8) reported that 4% of bats in gabon were positive for immunoglobulins to the zaire ebolavirus. this seems to provide evidence of bats as carriers of ebola viruses and as a source of ebola infection. furthermore, 152 univ med vol. 33 no.3 person-to-person transmission may also occur through direct contact with body fluids from the patients. the incubation period of ebola virus disease is between 1 and 21 days. the signs and symptoms are acute fever, shivering, headache, and myalgia, followed by rashes, sore throat, nausea, vomiting, diarrhea, and abdominal pain.(1) around half of the patients show signs of hemorrhage, such as hemorrhage of the nasal cavity, hematuria, gastrointestinal hemorrhage, and vaginal hemorrhage in women. wamala et al.(1) report that around 59% of patients who die from ebola virus have hemorrhages. the case fatality rate of zaire ebolavirus and sudan ebolavirus varies from 53%-90%.(1) the outbreak occurring in the bundibugyo district, uganda, in august – december 2007 showed three successive transmission cycles. each transmission cycle lasted 6 weeks, with an interval of 3-11 days, and was marked by a typical peaking of the outbreak, followed by a gradual decline.(1) frieden et al.(9) list three types of preventive interventions. the first is strict control of the infection in healthcare settings, because the greatest risk of transmission is not the obvious one from the patients, but from delayed detection and isolation. the second is education of local communities regarding habits that may spread the disease, and the third is to avoid consumption of raw meat of wild animals. the ebola virus outbreak constitutes a serious warning that epidemics may occur anywhere and places every afflicted nation at risk. therefore it is essential to institute measures to stop its spread and its future threat, which is a moral obligation of members of the health profession, whether academicians, researchers, or health ministry officials. references 1. wamala jf, lukwago l, malimbo m, nguku p, yoti z, musenero m, et al. ebola hemorrhagic fever associated with novel virus strain, uganda 2007-2008. emerg infect dis 2010;16:1087-92. 2. jeffers sa, sanders da, sanchez a. covalent modification of the ebola virus glycoprotein. j virol 2002; 76:12463-72. 3. who ebola response team. ebola virus disease in west africa – the first nine months of the outbreak and forward projections. n engl j med 2014;37:148-95. 4. briand s, bertherat e, cox p, formenty p, kieny mp, myhre jk, et al. the international ebola emergency. n engl j med 2014:371:180-3. 5. pigott dm, golding n, mylne a, huang z, henry aj, weiss dj, et al. mapping the zoonotic niche of ebola virus in africa. elife 2014;3:e04395. doi: 10.7554/elife.04395. 6. world health organization. ebola virus disease: cuban medical team heading for sierra leone available at: http://www.who.int/csr/disease/ebola/en/. accessed october 1, 2014. 7. mitman g. ebola in a stew of fear. n engl med 2014. n engl j med 2014;371:1763-5. doi:10.1056/ nejmp1411244. 8. pourrut x, souris m, towner js, rollin pe, nichol st, gonzales jp. large serological survey showing co-circulation of ebola and marburg viruses in rousettus aegyptiacus. bmc infect dis. 2009;159. doi. 10.1186/1471-2334-9-159. 9. frieden tr, damon i, bell bp, kenyon t, nichol s. ebola 2014 new challenges, new global response and responsibility. n engl j med. 2014. doi: 10.1056/nejme141147. nuryani 174 validity and reliability of preschool language scale 4 for measuring language development in children 48-59 months of age october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 universa medicina nuryani sidarta*a, angela bm tulaar**, amendi nasution** and suryanto** *department of anatomy, medical faculty, trisakti university **department of physical medicine and rehabilitation cipto mangunkusumo hospital correspondence adr. nuryani sidarta, sp.rm department of anatomy, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2101 email: nuri_sid@yahoo.com univ med 2008; 27: 174-82 abstract prevalence rates for speech and language delay have been reported across wide ranges. speech and language delay affects 5% to 8% of preschool children, often persisting into the school years. a cross-sectional study was conducted in 208 children aged 48-59 months to determine the validity and reliability of the indonesian edition of the preschool language scale version 4 (pls4) as a screening tool for the identification of language development disorders. construct validity was examined by using pearson correlation coefficient. internal consistency was tested and repeated measurements were taken to establish the stability coefficient and intraclass correlation coefficients (icc) for test-retest reliability. for construct validity, the pearson correlation coefficient ranged from 0.151-0.526, indicating that all questions in this instrument were valid for measuring auditory comprehension (ac) and expressive communication skills (ec). cronbach’s alpha level ranged from 0.81-0.95 with standard error of measurement (sem) ranging from 3.1-3.3. stability coefficients ranged from 0.98-.0.99 with icc coefficient ranging from 0.97-0.99 both of which showed an excellent reliability. this study found that pls4 is a valid and reliable instrument. it is easy to handle and can be recommended for assessing language development in children aged 48-59 months. keywords : language development, pls4, validity, reliability introduction language is a conventional code that has been socialized as a tool for expressing ideas or concepts, while speech is verbal communication for expressing language.(1) language and speech development has been named by the experts as a n e x c e l l e n t i n d i c a t o r f o r a s s e s s i n g c h i l d development as a whole and cognitive ability and is associated with scholastic achievement.(2) rescola reported that the prevalence of language and speech disorders in children aged 2 4.5 175 univ med vol.27 no.4 y e a r s w a s a r o u n d 5 8 % a n d f o r l a n g u a g e development disorders only it was around 2.319%.(3) a number of studies even indicated that children below 5 years of age with language disorders that have not been treated correctly will have low verbal abilities, reading disorders and disorders of spelling and behavior.(4) roulstone (2003) found that children referred for speech therapy but not given direct intervention, within the next 12 months, two-thirds were still in the category of speech therapy.(5) it is however a pity that to date there has been no clarification about the consistency of clinicians in screening for language and speech disorders in children. one study stated that 43% of parents reported that their children (of age 10-35 months) never had been given assessment of development at the time their children visited the doctor for treatment and 30% of parents also reported that the pediatricians whom they visited had never discussed communication problems in their children. (6) what formed an impediment in screening language and speech abilities of children usually was a time problem, absence of a d e f i n i t i v e p r o t o c o l a n d t h e f a c t t h a t development problems are assumed to be less important than other complaints. in indonesia there are no complete data about the number of children experiencing language and speech disabilities. rahmanoe (1999) reported a prevalence of 52.8% of children having delayed language abilities at the growth and development clinic in palembang.(7) in semarang, a study by jaenudin found that the prevalence of children experiencing speech development problems was 4.7%.(8) data from t h e d e p a r t m e n t o f p e d i a t r i c s , c i p t o mangunkusumo hospital showed a value of 11.47%-12.9%.(9) there are many assessments of linguistic and speech disorders in children, but there has never been uniformity in screening techniques. the instruments that are often used in evaluating the communication skills in children are the ages and stages questionnaire, (10) clinical adaptative test/clinical linguistic and auditory milestone scale, (11) denver developmental screening test,(11) mcarthur communicative d e v e l o p m e n t a l i n v e n t o r y, ( 1 2 ) wa r d i n f a n t l a n g u a g e s c r e e n i n g te s t , a s s e s s m e n t , acceleration, and remediation (wilstaar),(13) f l u h a r t y p r e s c h o o l s p e e c h a n d l a n g u a g e screening test(11) and early language milestone scale.(11) the preschool language scale (pls) is a psychometric instrument that was designed to evaluate language ability in children from birth up to 6 years and 11 months(14,15) this instrument is a diagnostic tool which may be applied for identification of comprehensive and expressive ability in children and may also be utilized to assess changes over time in the language abilities of the child. the preschool language scale version 4 (pls4) can be used individually and includes assessment of ability in preverbal behavior and at the same time assesses the l a n g u a g e s k i l l s i n t h e f i e l d o f s e m a n t i c s , m o r p h o l o g y, s y n t a x , a n d i n t e g r a t i v e a n d preliterative abilities. the reliability and validity of pls4 has been the subject of a number of studies. considering the increasing number of children with language and speech disorders and the absence of a gold standard for evaluation of these disorders, the present study aims to determine the validity and reliability of the indonesian language edition of the pls4 as an instrument for evaluating language development in indonesian children. methods research design in this study an observational research design with cross-sectional approach was used for attaining the aims of this research. 176 study subjects participants in this study were children 4 8 5 9 m o n t h s o f a g e f r o m k i n d e rg a r t e n s ( n u r s e r y s c h o o l s ) i n s o u t h j a k a r t a , a n d participation had been approved by the parents of the subjects. non-cooperative children or those with abnormalities in vision and hearing and a history of neurological trauma in the central nervous system (cns) were to be d r o p p e d o u t f r o m t h e g r o u p o f s t u d y participants. respondents were recruited from four districts in south jakarta, namely tebet, pasar minggu, cilandak and mampang. from each district one kindergarten was selected by cluster random sampling. sample size the optimal sample size required for conducting this study was calculated on the basis of the prevalence of 16% of children with language disabilities, with an acceptable error of 0.05. based on the results of the calculation, the minimum sample size was 208 children, with equal proportions of boys and girls. instrument the pls4 is an individually administered test designed to identify young children from birth to 6 years 11 months old who have a language disorder or delay. it consists of two subscales for the assessments of auditory comprehension and expressive comprehension respectively.(15) the materials provided for the a d m i n i s t r a t i o n o f t h e t e s t i n c l u d e a n e x a m i n e r ’s m a n u a l t h a t s p e l l s o u t t h e administration, scoring and interpretation procedures; a record form that provides all the items of the test; a picture manual that contains color pictorial stimuli needed for many of the test items; and a box of manipulatives (e.g., ball, bowls, rattles, windup toys, cloth) needed for interactions with the child during test a d m i n i s t r a t i o n . i t c o n s i s t s o f 6 2 a u d i t o r y c o m p r e h e n s i o n t a s k s a n d 6 8 e x p r e s s i v e comprehension tasks to identify comprehension a n d e x p r e s s i v e l a n g u a g e s k i l l s . h o w e v e r, a l m o s t a l l o f t h e s e i t e m s / t a s k s a r e b e t t e r described as testlets than items as each of them contains anywhere between 2 to 8 related subitems/tasks. data analysis in this study calculations were performed on internal and external reliability of pls4. external reliability was assessed with the testretest method, either by the same examiner ( i n t r a c l a s s r e l i a b i l i t y ) o r b y d i f f e r e n t examiners (interclass reliability). internal r e l i a b i l i t y w a s o b t a i n e d b y c a l c u l a t i n g cronbach’s alpha where a minimum value of 0.7 was required to show reliability of an instrument.(15) the validity measured in this study was construct validity, where for each question the validity coefficient was to be measured by finding a correlation between the score for each question and the total score obtained. the validity coefficient required had to be larger than 0.138 to demonstrate validity o f a q u e s t i o n . ( 1 5 ) c o n s t r u c t v a l i d i t y w a s a s s e s s e d b y u s i n g p e a r s o n ’s c o r r e l a t i o n c o e ff i c i e n t f o r a c a n d e c . i n t e r n a l a n d e x t e r n a l v a l i d i t y w a s r e p r e s e n t e d b y cronbach’s alpha, the stability coefficient, and the intraclass correlation coefficient (icc) with a time interval between examinations of 7 days. results a total of 208 participants was recruited during conduction of the study, consisting of 106 (51%) boys and 102 (49%) girls aged between 48-59 months, with a mean of 55.54 ± 2.86 months. sidarta, tulaar, nasution, et al preschool language scale 4 177 univ med vol.27 no.4 reliability testing the reliability of pls4 in this study was to be assessed from the results of calculation of internal consistency (cronbach’s alpha), stability coefficient, and interclass correlation. with a total of respondents of 208 children, for the auditory comprehension domain an internal consistency was obtained of 0.95, with standard error of measurement (sem) of 3.3. for the e x p r e s s i v e c o m m u n i c a t i o n d o m a i n a cronbach’s apha of 0.92 with sem 3.0 was obtained, while for total language domain a smaller internal consistency value of .081 with sem 3.1 was obtained. ta b l e 1 b e l o w s h o w s t h e i n t r a c l a s s reliability scores. test and retest mean scores ± sd for auditory comprehension were almost identical, being 93.91 ± 10.08 and 93.55 ± 10.37, with a stability coefficient of 0.98. test and retest mean scores for expressive communication were 87.27 ± 10.96 and 87.73 ± 10.96, respectively, with a stability coefficient of 0.99, while mean total language scores were 90.64 ± 10.19 vs. 89.95 ± 10.41, with a stability coefficient identical to that obtained for the auditory comprehension domain. table 3. construct validity of auditory comprehension questions in pls4 question correlation ( r ) 36 0.196 37 0.188 38 0.298 39 0.291 40 0.264 41 0.187 42 0.361 43 0.396 44 0.359 45 0.347 46 0.306 47 0.234 48 0.449 49 0.349 50 0.366 51 0.403 52 0.360 53 0.388 54 0.407 55 0.518 56 0.382 57 0.151 58 0.196 59 0.188 60 0.298 61 0.291 62 0.264 test retest mean sd mean sd interclass correlation aditory comprehension 93.55 10.37 93 10.37 0.97 epressive communication 87.73 10.96 87.27 11.63 0.99 total languange 89.95 10.41 89.23 10.66 0.99 table 2. interclass reliability value of pls4 test retest mean sd mean sd stability coefficient aditory comprehension 93.91 10.08 93.55 10.37 0.98 epressive communication 87.82 10.96 87.73 10.96 0.99 total languange 90.64 10.19 89.95 10.41 0.98 table 1. intraclass reliability of pls4 178 table 4. construct validity of expressive communication questions of pls4 question correlation ( r ) 34 0.289 35 0.201 36 0.185 37 0.308 38 0.295 39 0.271 40 0.191 41 0.370 42 0.403 43 0.364 44 0.351 45 0.307 46 0.234 47 0.457 48 0.356 49 0.363 50 0.403 51 0.356 52 0.387 53 0.406 54 0.516 55 0.378 56 0.151 57 0.368 58 0.198 60 0.426 61 0.300 62 0.170 to assess interclass reliability, test results were obtained by two different examiners with an interval of 7 days. table 2 indicates that test and retest mean scores had an interclass correlation of 97% for the auditory comprehension domain and 99% for expressive communication and total language. results of analyses performed for each pls4 question (tables 3 and 4) indicated that the mean correlation for each question had an r value > 0.138. sidarta, tulaar, nasution, et al preschool language scale 4 table 6. distribution of values for auditory comprehension, expressive communication and total language in children aged 48-59 months with pls4 legend: acss (auditory comprehension standard score); ac-ae (auditory comprehension age equivalent); ecss (expressive communication standard score); ec-ae (expressive communication age equivalent); tlss (total language standard score); tlae (total language age equivalent); sd (standard deviation); sem (standard error of measurement) value minimum maximum median mean sd sem acss 65 125.0 99.0 97.8 9.6 3.3 ac-ae 32 75.0 55.0 55.7 6.3 3.5 ecss 50 119.0 95.0 94.4 10.3 3.0 ec-ae 1 69.0 53.0 53.0 7.1 3.6 tlss 50 115.0 96.0 95.8 10.5 3.1 tl-ae 11 69.0 53.0 54.1 7.0 3.7 acs ecs tls acs 0.68 0.86 ecs 0.68 0.90 tls 0.86 0.90 table 5. correlation between auditory comprehension, expressive communication and total language legend: acs (auditory comprehension standard score), ecs (expressive communication standard score), tls (total language standard score) 179 univ med vol.27 no.4 correlation between domains t h e c o r r e l a t i o n b e t w e e n t h e t h r e e language domains was also assessed in this study. the correlation between acs and ecs was 0.68, while the correlation between acs a n d t l s w a s 0 . 8 6 ( p < 0 . 0 5 ) . a l a r g e r c o r r e l a t i o n v a l u e w a s o b t a i n e d i n t h e association between ecs and tls of 0.90 (p<0.05) (table 5). distribution of scores in this study, the examiners also attempted to find the scores for auditory comprehension, expressive communication and total language. table 6 shows a mean score of 97.8 ± 9.6 for auditory comprehension, with a median of 99, a n d a m e a n s c o r e o f 9 4 . 4 ± 1 0 . 3 f o r t h e expressive communication domain, with a median of 95.0. for total language the mean score was 95.8 ± 10.5, with a median of 96.0. for each domain the median approximated the mean, indicating that the distribution of scores i n e a c h d o m a i n w a s a l m o s t s y m m e t r i c a l , approximating the normal distribution. discussion the time required for completing the pls4 test varied for each child, depending on whether or not the child was cooperative. in the present study of children aged 48-59 months, the time required ranged from 15-20 minutes. the results of this study differs from the assessment conducted by zimmerman, where the time r e q u i r e d r a n g e d f r o m 2 0 4 5 m i n u t e s . ( 1 4 ) screening instruments, which take about 10 minutes to administer, could offer a reasonable and standardized approach to screening for speech and language delay in primary care settings. however, screening with such a tool m u s t b e f o l l o w e d w i t h a m o r e t h o r o u g h diagnostic evaluation before implementing an appropriate intervention.(16) if the children were accompanied by a by nursemaid or a parent, they usually would appear to be calm and could concentrate more on answering the questions. in addition, the number of children examined in a given room was also of influence on the time required for completion of the test. if the number of children was too large, the concentration of the children could be disturbed by voices of other children. on the other hand, if only one child was examined in a room, the child would be visibly fearful so that it had difficulties in answering t h e q u e s t i o n s . a c c o r d i n g t o t h e s u p p l i e d reference book there is no standard size for examination rooms. the book only suggests that examination rooms are well-ventilated and have a background setting that is pleasing to the children, so that they feel comfortable when examined. t h e r e s u l t s o f t h i s s t u d y s h o w a cronbach’s alpha for components ac, ec, and tl respectively of 0.95, 0.92 and 0.81. a cronbach’s alpha larger than 0.7 demonstrates the high reliability of this instrument. in other words, this instrument has high degree of confidence, with a small random error. the results obtained in this study did not differ substantially from those of zimmerman in his study on validation and reliability of pls4 in 213 children, internal consistency values of 0.90, 0.93 and 0.95, respectively were obtained for ac, ec and tl.(14) on average, the results of both studies were comparable, as both yielded high values for cronbach’s alpha, proving that the psl4 instrument has a high reliability. cronbach’s alpha indicates the level of c o n s i s t e n c y o f t h e p l s 4 i n s t r u m e n t i n measuring language ability, but cannot be directly used to interpret individual language skills. to this end the examiner also determined the sem. this value will give information about the estimated deviations in language skills of a 180 given child from the test results obtained by that child. with high consistency values, the sem may be expected to become smaller. in this study the sem for ac was 3.3, while the sems for ec and tl were respectively 3.0 and 3.1. these values are slightly below the values obtained by zimmerman(14) in the validation study of pls4, where the investigators obtained sem values of 4.74, 3.97 and 3.21 for ac, ec and tl in the age group of 48-59 months. these sem values will give estimates of the possible range of scores obtainable by a child. for example if a child gets a score of 9.0 with sd of 10 and sem of 3, then the scores of the child range from 8.7 ± 10 up to 9.3 ± 10, signifying that the scores of the child may vary from 7.7 to 10.3. to test the external reliability of the pls4 the examiner also calculated the stability coefficient for intraclass reliability and the inter-class correlation (icc) for interclass reliability. the time interval used between the first and the second test was seven days. the reason for selecting this interval was because according to the manual the ideal time interval for the test and retest method is 2 to 14 days. the selected time interval was expected to m i n i m i z e t h e e ff e c t o f l e a r n i n g f r o m t h e administered questions, but the interval was not too long to allow changes in language skills of the child. in the present study mean test and retest scores for auditory comprehension were similar, namely 93.91 ± 10.08 and 93.55 ± 10.37. the difference in mean scores between the first and second test was 0.36 with a difference in sd of 0.25 and stability coefficient of 0.98. this value indicates that if the assessment had been done twice by the same examiner, there would have been a probability of 98% of the results being identical. the values obtained here are lower t h a n t h o s e i n t h e r e l i a b i l i t y s t u d y b y zimmerman(14) in 218 children (117 girls and 101 boys) aged 2 to 5 year and 11 months, where the mean ac was 96.0 ± 15.1 and 98.1 ± 17.9.(14) the differences in mean values in the study by zimmerman was 2.1, with difference in sd of 2.8 and stability coefficient of 0.85. similar results were obtained for the two other domains. mean test and retest scores for ec were 87.73 ± 1 0 . 9 6 a n d 8 7 . 2 7 ± 1 0 . 9 6 w i t h s t a b i l i t y coefficient of 0.99, while for tl they were 90.64 ± 10.19 and 89.95 ± 10.41, with the same stability value as for auditory comprehension. in comparison, in the study by zimmerman, obtained values for ec and tl for test and retest were 98.8±15.0 and 101±17.1, respectively 9 7 . 2 ± 1 6 . 2 a n d 9 9 . 7 ± 1 8 . 5 w i t h s t a b i l i t y coefficient for ac, ec and tl of 0.95 and 0.96, respectively. f o r a s s e s s m e n t o f i n t e rr e l i a b i l i t y, calculations of the icc values were done by two different examiners on one subject. the time interval between examinations was seven d a y s . to g u a r a n t e e t h e o b j e c t i v i t y o f t h e assessments, the examiners did not know the scores given by one another. after completion of the scoring process the inter-reliability values were calculated. the obtained icc for the ac domain was 0.974 and for ec and total language an icc of 0.99 was found. this means that a l t h o u g h t e s t e d b y d i ff e r e n t p e r s o n s , t h e probability of obtaining similar examination results was 97.4% with an error level of 2.6%. the same results were obtained in the study of pls4 validation when testing the scoring system for this instrument in 100 samples. every sample was assessed twice by different examiners and a correlation of 0.99 was found, indicating a high level of interclass reliability. this means that the scoring system in pls4 was w e l l c o n s t r u c t e d a n d s t i l l h a d a g o o d consistency even though examined by different persons. sidarta, tulaar, nasution, et al preschool language scale 4 181 univ med vol.27 no.4 after making statistical calculations for each of the questions found on the pls4 (tables 3 and 4) a range of correlation values was found for the auditory comprehension domain, from 0.17 to 0.52. for the expressive communication domain a correlation value of 0.151 to 0.516 was obtained, signifying that both the questions on auditory comprehension as well as those on expressive communication had a correlation value of more than 0.138. this indicates that a b o v e m e n t i o n e d q u e s t i o n s h a d c o n s t r u c t validity, from which it may be concluded that the questions found in this instrument had formed a good construct or framework in a s s e s s i n g t h e l a n g u a g e s k i l l s o f c h i l d r e n between 48-59 months of age. to f i n d a n a s s o c i a t i o n b e t w e e n t h e respective domains the pearson correlation test was performed. the correlation of acs with ecs was 0.676, while the correlation of acs with tls was 0.856. a higher correlation was obtained for the association of ecs with tls, being 0.903 with significant p value of < 0.05. this indicates the existence of a substantially high correlation of acs with ecs, because both d o m a i n s a r e p a r t o f l a n g u a g e a n d a r e interrelated. however, the correlation was not too strong because acs and ecs are also two different aspects of language. on the other hand, the correlation of acs-tls and ecs-tls had a higher correlation level, indicating that acs and ecs scores would subsequently determine the total language score. from the results in table 6 it can be seen that the scores for all three domains has a n o r m a l d i s t r i b u t i o n w h e r e t h e m e a n a n d medians are close together. in the auditory comprehension domain a range of 65 -125 was obtained, with mean score of 97.8 ± 9.6. this value is slightly lower than that obtained in a study on 2000 children in the united states, w h e r e a m e a n s c o r e o f 1 0 . 1 2 ± 1 3 . 8 w a s obtained.(17) however, if the normal range of scores is – 15 sd to + 1.5 sd, then the scores in indonesian children are still within the normal range, namely between 77 and 122. based on the above guidelines there were 12 children (58%) with scores below –1.5 sd. a study in spain revealed that the children under study performed approximately 1.5 sd below the mean.(18) f o r t h e d o m a i n o f e x p r e s s i v e communication the mean score obtained here was lower compared to the mean of african american children. in the present study the mean score was 94.4 ± 10.3, while in african american children the obtained score was 101.7 ± 11.8.(19) however, similarly as with the ac scores, the ec scores in indonesian children were still within the normal range. results of statistical calculations indicated that there were 8 people (38%) with standard values below – 1.5 sd. mean total language score obtained in this study was 95.8 ± 10.5 and the number of children having a score below – 1.5 sd was 25 (12%). however, this prevalence were not appreciably different from those obtained in the study by elmina harahap in children 3 to 24 months of a g e , w h e r e t h e p r e v a l e n c e o f c h i l d r e n experiencing language delay was 11.47%.(9) pls4 is frequently used for screening symptoms of neurodevelopmental delay (nd) and pervasive developmental disorder (pdd).(20) conclusions pls4 is a valid instrument with a high level of reliability. this instrument is easy to administer and is recommended as one of the i n s t r u m e n t s f o r e v a l u a t i o n o f l a n g u a g e development in children aged 48-59 months. overall, pls4 is a well-designed and carefully developed instrument, meeting standards of quality. 182 references 1. owen r.e. the territory. in: owen re. language development – nn introduction. boston: ally & bacon; 2001. p. 12-29. 2. catt hw, fey me, tomblin jb. zhang x. a longitudinal investigastion of reading outcomes in children with language impairments. j speech lang hear res 2002; 45: 1142-7. 3. rescorla l, alley a. validation of the language development survey (lds): a parent report tool for identifying language delay in toddlers. j speech lang hear res 2001; 434-45. 4. us preventive services task force. screening for speech and language delay in preschool children : recommendation statement. pediatrics 2006;117 : 497-501. 5. roulstone s, peters tj, glogowska m, enderby p. a 12-month follow up of preschool children investigating the natural history of speech and language delay. child care health dev 2003; 29: 245-55. 6. halfon n, olson i, inkelas m, lange i. summary statistics from the national survey of early childhood health, 2000. national center for health statistics.vital health stat 2002; 15: 1-34. 7. rahmanoe m. spektrum faktor lingkungan yang berpengaruh terhadap terjadinya gangguan berbicara pada anak. ika-rscm; 1999. 8. jaenudin e. stimulasi keluarga pada perkembangan bicara anak usia 6 sampai 36 bulan di kelurahan kuningan, semarang utara (thesis). semarang: ika-/rsup dr kariadi; 2000. 9. harahap r.e. perbandingan uji tapis bayley infant neurodevelopmental screener dengan uji tapis denver ii untuk deteksi dini keterlambatan perkembangan bayi usia 3-24 bulan dengan menggunakan baku emas griffiths mental development scales (thesis). jakarta: ika-rscm; 2006. 10. bricker d, squires j, mounts l. ages and stages questionnaires: a parent-completed, child-monitoring system. 2nd ed. baltimore: paul h: brookes; 1999. 11. nelson hd, nygren p, walker m, panoscha r. screening for speech and language delay in preschool children: systematic evidence review for the us preventive services task force. pediatrics 2006;117: e298-e319. 12. fenson l, pethick sj, renda c, cox jl, dale ps, reznick js. short-form versions of the macarthur communicative development inventories. appl psycholinguist 2000; 21: 95–6. 13. coulter l, gallagher c. piloting new ways of working: evaluation of the wilstaar programme. int j lang commun disord 2001; 36: 270–5. 14. zimmerman il, castilleja nf. the role of a language scale for infant and preschool assessment. mental retardation and developmental disabilities. res rev 2005; 11: 238-46. 15. hoi ks. review of the preschool language scale – fourth edition, pennsylvania state university, university park, pa. available at: http://suen.ed. psu.edu/~hsuen/pubs/buros11%20preschool% 20language%20scale.pdf ). accessed september 3, 2008. 16. nelson hd, nygren p, walker m, panoscha r. screening for speech and language delay in preschool children. rockville, md: agency for healthcare research and quality. 2006. evidence synthesis no. 41. available at: www.ahrq.gov/ clinic/uspstfix. htm. accessed september 3, 2008. 17. zimmerman il, steiner vg, pond re. preschool language scale, fourth edition (pls-4). technical report. available at: http://pearsonassess.com/nr/ r d o n l y r e s / 6 2 6 9 b 9 fa 1 c c f 4 d f 8 9 f 8 b 282863589c25/0/pls4_techreport.pdf. accessed august 30, 2008. 18. restrepo ma, silverman sw. validity of the spanish preschool language scale-3 for use with bilingual children. am j speech lang pathol 2001;10: 382-93. 19. huaqing qi, kaiser ap. the performance of lowincome african american children on the preschool language scale-3. j speech lang hear res 2003;46: 576-90. 20. rescorla l. language delay and behavioral/ emotional problems in toddlers: findings from two developmental clinics. j speech lang hear res 2007; 50: 1063-78. sidarta, tulaar, nasution, et al preschool language scale 4 c:\users\jurnal fkusakti\docume 173 *tuberculosis and lung disease research center, tabriz university of medical sciences, imam reza hospital, division of respiratory and critical care medicine, department of internal medicine, tabriz, iran correspondence : katrin buyukaghazadeh tuberculosis and lung disease research center, tabriz university of medical sciences, imam reza hospital, division of respiratory and critical care medicine, department of internal medicine, tabriz, iran email: katrinaghazadeh@gmail.com date of first submission, august 14, 2 01 7 date of final revised submission, november 2, 2017 date of acceptance, november 2, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 safety of bronchoscopic guided percutaneous dilatational tracheostomy in icu patients in tabriz city mohammad esmaeil hejazi*, mohammadamin rezazadehsaatlou*, leila namvar*, armin sadeghi*, veghar hejazi*, yasin hejazi*, and katrin buyuk aghazadeh* background tracheostomy is one of the most frequently performed procedures in intensive care units (icu). the widespread attraction of percutaneous dilatational tracheostomy (pdt ) is increasing in modern icu. bronchoscopic guidance seems to secure the safety of the technique. multiple studies done to explain characterize differences in complications and cost-effectiveness of open and percutaneous tracheotomy. the objective of this study was to evaluate the benefits of pdt using the ciaglia technique with bronchoscopic guidance. methods a total of 100 elective pdt using the ciaglia technique with a little modification were performed under flexible fiber optic bronchoscopic guide. the demographic variables were recorded, the underlying cause for patient’s referred to the center for pdt, and intraoperative as well as early postoperative complications. patients followed for several days after tracheostomy for early complications. results no severe complications related to pdt were noticed during and after the procedure. three patients had bleeding during incision and two led to subcutaneus hematoma. there were no other complications such as infection, emphysema and puncture of posterior wall. conclusions we recommended the use of endoscopic guidance bedside percutaneous tracheostomy using the ciaglia technique with a little modification because it is safe and simple to do without significant complications. pdt with bronchoscopic guidance is a safe and easy procedure that can be done at the bedside setting. keywords: percutaneous dilatational tracheostomy, bronchoscopy, complication doi: http://dx.doi.org/10.18051/univmed.2017.v36.173-178 original article cite this article as: hejazi me, rezazadehsaatlou m, namvar l, et al. safety of bronchoscopic guided percutaneous dilatational tracheostomy in icu patients in tabriz city. univ med 201 7;36:173-8. d oi : 10. 1805 1/ univmed.2017.v36.173-178 pissn: 1907-3062 / eissn: 2407-2230 174 hejazi, rezazadehsaatlou, namvar, et al guided percutaneous dilatational tracheostomy introduction tracheostomy is one of the oldest surgical procedures, described in hindu and egyptian texts dating back to 1000 bc.(1) tracheostomy usually performed in patients with difficult weaning from mechanical ventilation or somecatastrophic neurologic disorders, also many laryngeal infectious and neoplastic processes may require a surgical airway.(2,3) to avoid the complications of chronic endotracheal intubation, tracheostomy performed in patients requiring long-term intubation to facilitate pulmonary hygiene and weaning from the ventilator.(4) other indications include management of upper airway obstruction and copious secretionsand to minimize laryngeal injury.(5) in recent studies the benefits of early tracheostomy in patients who require extended period of mechanical ventilation compared with prolonged trans la r yngeal intubation. percutaneous dilatational tracheostomy (pdt) techniques have been described recently, these techniques providing the clinicians a chance to perform rapidly and easily tracheostomy atthebedside, the complications associated with this new procedurearen’t definedyet.(4) percutaneous dilatational tracheostomy is the standard airway access in critically ill patients who require prolonged mechanical ventilation.(6) percutaneous dilatational tracheostomy is a rapid, simple, easy to lear n and cost-ef fective procedure. it has many advantages over surgical tracheostomy. over the years, pdt has evolved as a safe bedside procedure that has enabled a temporary tracheostomy to be inserted by the intensive sits in icu.(7) ciaglia et al.(8) in 1985 introduced principle technique of contemporary percutaneous tracheostomy and refined in 1999 as ciaglia blue rhino (9) and 2005 as ciaglia blue dolphin.(10) the ciaglia blue dolphin or its modifications have been used increasingly in different patient populations.(11) both the surgical tracheostomy (st) and the pdt strategies provide advantages and disadvantages. sur gical tracheostomy immediately secures the newly established access to the airway. in contrast, the pdt is performed via a small skin incision followed by different dilatational mechanisms and insertion of the tracheal cannula. co nsequently, there is disagreement regarding which strategy offers the superior benefit/risk ratio for critically ill patients. a systematic review and meta-analysis to compare percutaneous with surgical strategy for tracheostomy showed that the choice of strategy is primarily made by the preference of the surgeon and not by evidence, as evidence for the strategy with the best parameters of safety is poor and conflicting.(12) over the past decade, the application of various imaging guidance modalities (flexible bronchoscopy, rigid bronchoscopy, and ultrasound) has further enhanced pdt and extended its use in a variety of patient populations.(13) the use of guidance bronchoscopy in pdt has not been adequately described. this study aimed to evaluate pdt with bronchoscopy guidance similar to cialgia with a little change in method to shorten the time and reduce the complications. methods study design and subjects in this clinical trial we prospectively analyzed the data from 100 patients who underwent tracheostomy during about 1.5 years period (from january 2016 to june 2017). all patients were from pulmonary icu of imam reza hospital and shahid madani hospital of tabriz university, iran. all patients need prolonged mechanical ventilation (more than 7 days) due to medical problems and require an artificial airway to prolonged ventilation, meanage was 59 years old, and a total of 100 patient were included.inclusion criteria were traditional indication to tracheostomy, along with having none of absolute or relative contraindications to pdt (coagulopathy, high positive end-expiratory pressure (peep) or inspired oxygen concentration (fio2) requirement, difficult anatomy, and hemodynamic instability). the exclusion criteria were obesity and patients with short neck or scar 175 in neck and distortion of trachea and large thyroid gland. performance of pdt all patients sedated with fentanyl (100-200 mcg/iv) and midazolam (3 to 5mg/iv) then neck is hyperextended by using a shoulder pad. bronchoscope is inserted true endotracheal tube (ett) and adjusted so that the lower edge of ett was visible, throughout the procedure the bronchoscope is in this position to see the all steps of procedure. local anesthesia is done by lidocaine 2% (3-5 cc). transverse incision (1.5 cm) is made 2 cm below cricoid cartilage and curved hemostat is used for separation of subcutaneous tissue. retraction done until fibromuscular tissue over laying the tracheal rings detected. needle inserted between the second and third tracheal rings, through the needle j shape guide wire directed toward the carina. the needle removed and a short 14-f dilator inserted over the guidewire then the dilator is removed with j guidwire in place. ciaglia blue rhino loaded on an 8f guiding catheter are inserted over the guidewire. dilator removed leaving the guiding catheter in place then the tracheostomy tube loaded over a dilator inserted in to the trachea and then guiding wire and catheter are removed. the br onchoscope in ser te d through the tracheostomy tube to ensure the placement of tube then fixation done by band (figure 1; a: incision; b: insertion of guidwire; c: insertion of small dilator; d: preparation of dilator; e: tracheostomy replacement; f: tracheostomy fixation). mean time for the procedure from skin incision to tracheostomy tube placement was 3 minutes. assessment of complications complications related to pdt, such as serious complication (cardiopulmonary arrest, trachoesophageal f istula, inter mediate complications and posterior tracheal wall perforation) and minor complication (bleeding, tube misplacement and emphysema) were documented. figure 1. stages of percutaneous dilatational tracheostomy a:incision; b: insertion of guidwire; c: insertion of small dilator; d: preparation of dilator; e: tracheostomy replacement; f: tracheostomy fixation univ med vol. 36 no.3 176 hejazi, rezazadehsaatlou, namvar, et al guided percutaneous dilatational tracheostomy a b c ethical clearance this study received ethical clearance from the health research ethics committee, faculty of medicine, tabriz university of medical sciences.all patients had given written consent for surgery (this study is retrospective study). results from 100 patients (34 female and 66 male with mean age 59.5 years old) who underwent percutaneous tracheostomy, morbidity rate directly attributed to the tracheostomy was 0%. no major early complications were found in all patients. in this technique there was small amount of bleeding (about 3-4 cc) and only one of cases had massive bleeding that caused stop of procedure and patient referred to surgical tracheostomy and three patient had minor bleeding from dilatation site that controlled without any complication; meanwhile of these 3 cases two patients had local hematoma that didn’t need any intervention and only conservative treatment had done. we didn’t found any other complications such as emphysema, infection, posterior wall of trachea perforation (table 1). the main underlying causes for the patients were: neurologic 58%, pulmonary disease 22%, metabolic and hypoxic encephalopathy 15%, rheumatologic problems 3%, other problems 2% (table 2). % totally (%) neurologic cva (swallowing impairment, ineffective cough, and prolonged intubation traumatic injury to cervical cord) multitrauma and head trauma with long time disturbance of consciousness and need to prolonged intubation neuromuscular guillain-barré syndrome myasthenia gravis multiple sclerosis 41 4 5 4 3 1 58 8 pulmonary respiratory failure and long-term ventilation due to copd status asmaticus lung parenchymal disease sepsis or multiple organ failure 15 1 3 3 22 hypoxic and metabolic encephalopathy mi with cpr cardiopulmonary arrest electric shock severe drug poisoning 10 1 1 3 15 rheumatologic behcet disease pulmonary renal disorders 1 2 3 others ketoacidosis lymphoma 1 1 2 table 2. underlying cause for patient’s referred to the center for percutaneous dilatational tracheostomies (pdt), tabriz, iran (n=100) mi= myocard infarct; cpr= cardiopulmonary resuscitation complications n (%) serious complications cardiopulmonary arrest trachoesophageal fistula intermediate complications posterior tracheal wall perforation 0 (0.0) 0 (0.0) 0 (0.0) 0 (0.0) minor complications bleeding tube misplacement emphysema 4 (4.0) 0 (0.0) 0 (0.0) table1 1. complications among 100 patients underwent percutaneous dilatational tracheostomies (pdt) in tabriz city 177 discussion during the last decades, it has been proven that the tracheostomy has popularities like reducing pulmonary dead space, easier weaning of pa tients from the ventilator, make the opportunity to clearing the pulmonary secretions caused various (pathological or non-pathological) conditions, and also improves the patient’s comfort.(4,12,14) one patient had massive bleeding that need to surgical intervention to control the bleeding, and 3 cases had mild bleeding without any need for further intervention; meanwhile, of these 3 cases, two patients had hematoma (total complication rate: 4%). these complications occurred at the beginning of this study and with progression didn’t shown these complication, so this method needs to expert team, and we suggest after doing at least about 5 to 8 tracheostomy under observation of skilled person he or she can do this procedure a lone without maj or complications. the related complication rates of this procedure in several studies were significantly larger than those observed in this study, ranging from 8.9 to >30%, (3 ,4,14 ) while the total complication in this study was found in 0.06% cases (6 patients). there was another important aspect in this study that skin incision before insertion of the needle help to reducing the time of dilatation and probability of infection. performing tracheostomy under bronchoscopy was safer than other methods and without major complications. the results of this study suggest that the present method perform bedside tracheostomy in patients who haven’t any contra indication for pdt. as previously reported the complications of pdt, such as bleeding, infection and hypoxia, are less common, but in this study all complications of pdt were among the minor complications. most are complications are minor. in a study by klotz et al.(12) the perioperative complication rate was 6%, with premature extubation as the most common problem. ea r ly postoperative complications were found in 5% of patients, with bleeding as the most common complication. tracheal stenosis was seen in only 1.6% of patients who survived their icu stay and were subsequently decannulated. however, the rate of these complications is low and death rarely occurs in this regard. it is reported three cases of extensive posterolateral longitudinal tracheal laceration that all were managed successfully with surgical repair.(14) pdt is becoming the preferred method for tracheostomy in many centres. however, the currently available evidence suggests that apart from the risk related to intrahospital patient transport, pdt is comparable to surgical tracheostomy (st). with respect to other complications, pdt is associated with lower infection and cost but higher chances of tube block and difficulty in decannulation compared to st.(15,16) bleeding is the most frequent complication in this method. as with all patients undergoing a surgery, it is paramount that the patient did not have a coagulopathy. we recommend that the platelet count, prothromb in time and partial thromboplastic time be measured before pdt in all patients. our study has some limitations. we collected data retrospectively, and our findings should be confirmed in prospective trials. and complications related to anticoagulants suspension were not present in our study. future investigations might also include the detailed analysis of the operating room utilization resources benefit from saving charges and using free operating room time space for additional elective procedures, examination of delayed complications, length of ventilation, and length of hospital stay. conclusion pdt reduced the frequency of incidence of infection and indicated lesser complication rate which was found in 4 patients (4%). performing pdt in icu should be considered as the main procedur e to be per forming in elective tracheotomy in critically adult patients. but further studies are recommended in larger population and univ med vol. 36 no.3 178 hejazi, rezazadehsaatlou, namvar, et al guided percutaneous dilatational tracheostomy with evaluation of more related parameters in this regard. conflict of interest there is no conflict of interest contributors meh conducted the research.kba and yh wrote the manuscript. ln, as and mr supervised the r esear ch. vh an d mr corr ected the manuscript. all authors read and approved the manuscript. references 1. frost ea. tracing the tracheostomy. ann otol rhinol laryngol 1976;85(5 pt.1):618-24. 2. nowak a, kern p, koscielny s, et al. feasibility and safety of dilatational tracheotomy using the rigid endoscope: amulticenter study. bmc anesthesiol 2017;7:17. doi: 10.1186/s12871-0170301-y. 3. putensen c, theuerkauf n, guenther u, et al. percutaneous and surgical tracheostomy in critically ill adult patients: a meta-analysis. critical care 2014;18:544. doi:10.1186/s13054-014-0544-7. 4. brass p1, hellmich m, ladra a, et al. percutaneous techniques versus surgical techniques for tracheostomy. cochrane database syst rev 2016; 7:cd008045. doi: 10.1002/14651858. cd008045. pub2. 5. kiran s1, eapen s1, chopra v1. a comparative study of complications and long term outcomes of surgical tracheostomy and two techniques of percutaneous tracheostomy. indian j crit care med 2015;19:82-6. doi: 10.4103/0972-5229.151015. 6. pilarczyk k, carstens h, heckmann j, et al. safety and efficiency of percutaneous dilatational tracheostomy with direct bronchoscopic guidance for thoracic transplant recipients. respir care 2016;61:235–42. doi: 10.4187/respcare.04128. 7. acharya d, nanjangud p. percutaneous tracheostomy. ind j resp care 2014; 3:488-96. 8. ciaglia p, firsching r, syniec c. elective percutaneous dilatational tracheostomy: a new simple bedside procedure; preliminary report. chest 1985;87:715-9, 9. ciaglia p. technique, complications, and improvements in percutaneous dilatational tracheostomy. chest 1999;115:1229-30. 10. gromann tw, birkelbach o, hetzer r. balloon dilatational tracheostomy: initial experience with the ciaglia blue dolphin method. anesth analg 2009;108:1862-6. 11. susarla sm, peacock zs, alam hb. percutaneous dilatational tracheostomy: review of technique and evidence for its use. j oral maxillofac surg 2012;70:74-82. 12. klotz r, klaiber u, grummich k, et al. percutaneous versus surgical strategy for tracheostomy: protocol for a systematic review and meta-analysis of perioperative and postoperative complications. syst rev 2015;4:105. doi: 10.1186/s13643-015-0092-5. 13. rajajee v, fletcher jj, rochlen lr, et al. real-time ultrasound-guided percutaneous dilatational tracheostomy: a feasibility study. crit care 2011; 15:r67. doi: 10.1186/cc10047. 14. byhahn c. percutaneous tracheostomy: the ciaglia techniques. in: servillo g, pelosi p,editors. percutaneous tracheostomy in critically illpatients. new york: springer international publishing switzerland;2016.p.37-41. 15. higgins km, punthakee x. meta-analysis comparison of open versus percutaneous tracheostomy. laryngoscope 2007;117:447-54. 16. mirski ma, pandian v, bhatti n, et al. safety, efficiency, and cost-effectiveness of a multidisciplinary percutaneous tracheostomy program. crit care med 2012;40:1827-34. , artesunate-tinospora combination treatment decreases nuclear factor kappa-b and intercellular adhesion 222 *master of biomedical science program, study program for specialist in neurology, faculty of medicine, brawijaya university/ rsud dr.saiful anwar malang **department of parasitology, faculty of medicine, brawijaya university, malang ***department of neurology, faculty of medicine, brawijaya university/ rsud dr.saiful anwar, malang correspondence: dr. nur izzati faculty of medicine, brawijaya university/rsud dr.saiful anwar malang jl. jaksa agung suprapto no.2, malang phone: +62341321297 mobile: +62811 3093 891 email: dr.nizzah@gmail.com univ med 2016;35:222-8 doi: 10.18051/univmed.2016.v35.222-228 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 artesunate-tinospora combination treatment decreases nuclear factor kappa-b and intercellular adhesion molecule-1 expression in mouse malarial models nur izzati*, loeki enggar fitri**, and mochammad dalhar*** background cerebral malaria is a severe form of malaria caused by brain ischemia. artesunate, an artemisinin derivative, is the standard who therapy for severe malaria. tinospora crispa (brotowali) is a traditional plant with antiinflammatory, antioxidant and antiparasitic properties. the aim of this study was to determine the effect of combinations of artesunate and t. crispa extract on nuclear factor kappa-b (nfκb) and intercellular adhesion molecule-1 (icam-1) expression in the brain of mouse malaria models. methods this was an experimental post-test only control group study using c57bl/ 6j mice infected with plasmodium berghei, divided into 7 groups: negative control, positive control, group receiving artesunate 32 mg/kgbw, group receiving tinospora extract 3.5 mg/kgbw, and three groups receiving combinations of artesunate 32 mg/kgbw and tinospora extract 2.5 mg/ kgbw, 3 mg/kgbw and 3.5 mg/bw, respectively. the expression of nfκb and icam-1 was measured by immunohistochemistry. one-way anova was used to analyze the data. results nfκb and icam-1 expression increased significantly in the positive controls compared to all other groups (p=0.000). nfκb expression was significantly lower in the groups receiving artesunate and tinospora at 3 mg/kgbw and 3.5 mg/kgbw, as compared with the artesunate only group (p=0.003; p=0.005) and the tinospora extract only group (p=0.001; p=0.003). nfκb expression in all combination treatment groups was similar to that in the negative controls (p>0.05), whereas icam-1 expression did not differ between single and combination treatment groups (p>0.05). conclusion the combination of artesunate and t. crispa extract is better in decreasing nfκb and icam-1 expression in the brain of mouse malaria models. keywords: artesunate, tinospora , icam-1, malaria, nfκb, mouse doi: http://dx.doi.org/10.18051/univmed.2016.v35.222-228 223 univ med vol. 35 no.3 introduction malaria is an important cause of death, especially in tropical countries. in 2013, there were 198 million cases of malaria in the world, resulting in 584,000 deaths. (1) the pathophysiology of cerebral malaria is not clearly understood. erythrocytes infected with p. falciparum and undergoing rupture release glycosyl phosphatidyl inositol (gpi) molecules from the parasite, thereby inducing tumor necrosis factor α (tnf-α). the latter activates the transcription of nuclear factor kappa-b (nfκb) which triggers the expression of various mediators of inflammation (including intercellular adhesion molecule-1, icam-1) and the innate immune response.(2) the world health organization malaria guidelines since 2006 have recommend the use of artemisinin-based combination theraphy (act) to prevent antimalarial drug resistance.(1,3) artesunate is an artemisinin derivative with endoperoxide structure that rapidly eradicates the asexual erythrocytic stages of p. vivax and p. falciparum. the drug is 10-100 times more potent in eradicating malarial parasites than other antimalarial drugs.(4) brotowali (tinospora crispa (l) miers) of the family menispermaceae is a traditional medicinal plant that is widely used as antimalarial, antipyretic, antidiabetic, antiinflammatory, and analgesic agent. t. crispa contains a bitter substance, i.e. tinocrisposide, and several alkaloids such as aporphine, berberine and palmatine.(5) in a study of 10 malaysian medicinal plants, using methanol extracts at concentratiosn of 0.03 µg/ml, t. crispa stem extract showed the highest in vitro antiplasmodial activity to p. falciparum at 90% growth inhibition.(6) berberine and palmatine significantly inhibit the activation of nfκb and decrease the expression of icam1 and transforming growth factor-beta 1 (tgfβ1) in mesangial cells of rats with diabetic nephropathy, while berberine also inhibits inducible nitric oxide synthase (inos) expression.(7,8) one study showed the protective effects of tinospora crispa stem extract on renal damage and hemolysis in plasmodium berghei infection.(9) there is no information on studies about the effect of t. crispa and artesunate administration on the expression of nfκb and icam-1 in the brain of mouse malaria models. the purpose of this study was to evaluate the effect of a combination of artesunate and tinospora stem extract on the expression of nfκb and icam-1 in the brain of mouse malaria models. methods design of the study this study used an experimental post-test only control group design and was conducted in the parasitology and biomedical laboratory, faculty of medicine, brawijaya university, malang, from january 2015 until february 2016. experimental animals the test animals used were mice of the c57bl/6j strain, which were obtained from the eijkman institute, jakarta, where randomization was performed. the inclusion criteria were: females, weighing 20-25 grams, aged 12-16 weeks. the sample size was determined using the formula p(n-1) ≥15, where p=number of intervention groups and n=number of replications.(10) all subjects were allocated by simple randomization into 7 treatment groups: group i (negative controls), group ii (positive controls), group iii (artesunate 32 mg/kgbw), group iv (tinospora extract 3.5 mg/kgbw), group v (combination treatment a=artesunate 32 mg/ kgbw and tinospora extract 2.5 mg/kgbw), group vi (combination treatment b=artesunate 32 mg/kg and tinospora extract 3 mg/gbw), and group vii (combination treatment c=artesunate 32 mg/kgbw and tinospora extract 3.5 mg/ kgbw). administration of artesunate 32 mg/ kgbw was by single daily intraperitoneal injection on days 4, 5, and 6 post infection,(11) while 224 izzati, fitri, dalhar artesunate-tinospora decreases nfκb and icam-1 tinospora extract was administered once daily by gavage on days 4, 5, and 6 post infection. dissection was performed on day 7 after inoculation. parasites and infection the mice were inoculated intraperitoneally with 1x107 p. berghei-infected erythrocytes. preparation and administration of t. crispa stem extract extraction was performed at the chemistry research center of the indonesian scientific institute (lipi), bandung. the extract was then subjected to phytochemical analysis and thin-layer chromatography (tlc). administration of tinospora extract at 2.5 mg/kgbw, 3 mg/kgbw, and 3.5 mg/kgbw was performed by dissolving the extract in 0.2 ml of distilled water and administering the solution orally by gavage, starting on day 4 and continuing for 3 days. immunohistochemistry of nfκκκκκb and icam-1 as an initial step, antigen retrieval with citrate buffer was performed, then immunohistochemical staining as follows: the slides were dripped with 3% h 2 o 2 in methanol, incubated for 15 minutes, and washed 3 times with pbs for 2 minutes each. then blocking of nonspecific protein was done. onto the slides were dripped nfκb primary antibody (nfκb in a 1:300 dilution in phosphate buffered solution (pbs) and 2% bovine serum albumin (bsa), and icam-1 primary antibody (icam-1 in a 1:50 diution in pbs-bsa). the slides were then incubated with secondary antibody and afterwards with streptavidin-horseradish peroxidase (sahrp) for 20 minutes. then diaminobenzidine (dab) and dab buffer were dripped in a ratio of 1:50. subsequently mayer’s hematoxylin and tap water were dripped in a ratio of 1:10, then the slides were rinsed with tap water and dried. evaluation expression of nfκb and icam-1 was observed under the microscope at 400x magnification in 10 fields of view. all nucleated cells expressing nfκb showed brown-colored nuclei and cytoplasms,(12) whereas cells expressing icam-1, particularly vascular endothelial cells, showed brown-colored cytoplasms.(13) statistical analysis the statistical analysis was performed using oneway anova followed by the tukey test to find the differing parameters, with level of significance set at p<0.05. ethical clearance the present study was approved by the ethics commission, faculty of medicine, brawijaya university, malang. results from the phytochemical and tlc tests it the respective total phenolic and flavonoid contents were found to be 43.34 ± 1.92% and 74.26 ± 1.32% of dry weight. the obtained values for nfκb and icam1 expression are shown in table 1. mean expression of nfκb and icam-1 was significantly different between the seven treatment groups (p=0.000; p=0.000). the lowest mean expression of nfκb and icam-1 was found in the artesunate-tinospora combination groups. the immunohistochemical test results on nfκb and icam-1 expression are shown in figures 1 and 2, respectively. the tukey test showed a significant difference in nfκb expression between the negative control group versus the positive control group (p=0.000), the artesunate group (p=0.001), and the tinosporaextract group (p=0.001), but no significant difference between the negative control group and each of the combination treatment groups (p=0.142; p=1,000; p=0.986). positive control group was significantly different from all other groups (p<0.05). the artesunate group was not significantly different from the tinosporaextract group (p=1.000) and the combination treatment a groups (p=0.192). there was 225 univ med vol. 35 no.3 significant difference in nfκb expression between the artesunate grup and the combination treatment b grup (p=0.003), the combination treatment c groups (p=0.005). in addition, there was significant difference between tinospora extract group and the combination treatment b (p=0.001), the combination treatment c groups (p=0.003). (data not shown). the tukey test showed a significant difference between the negative control group versus the positive control group (p=0.000), the artesunate group (p=0.005), and the tinospora extract group (p=0.045), but no significant difference between the negative control group and each of the combination treatment groups (p=0.057; p=0.176; p=0.453). group ii or positive control group was significantly different from all other groups (p<0.05). icam-1 expression in the artesunate group was not significantly different from that in each of the combination treatment groups (p=0.824; p=0.453; p=0.762). in addition, there was no significant difference in icam-1 expression between the tinospora extract group and each of the combination treatment groups (p=1.00; p=0.981; p=0.757) (data not shown). table 1. mean nfκb and icam-1 expression by treatment group i: negative control; ii: positive control; iii: artesunate 32 mg/kg bw; iv: tinospora extract 3.5 mg/kg; v: artesunate 32 mg/ kg bw + tinospora extract 2.5 mg/kg bw; vi: artesunate 32 mg/kg bw + tinospora extract 3 mg/kg bw; vii: artesunate 32 mg/kg bw + tinospora extract 3.5 mg/kg bw figure 1. nfκb expression by immunohistochemistry (400x) (a) negative control, (b) positive control, (c) artesunate, (d) tinospora extract, (e) combination treatment a, (f) combination treatment b, (g) combination treatment c. arrows indicate nfκb expressing cells f g d ec ba 226 discussion cerebral malaria is a form of severe malaria that is caused by brain ischemia and may result in death. the severity of cerebral malaria is assumed to be caused by dysregulation of homeostasis between proand anti-inflammatory conditions.(14-16) infection with plasmodium affects the erythrocytes, thus leading to parasitemia. the surface of the membrane of parasitized erytrocytes is covered with knobs called pfemp-1, which are surface antigens that bind to icam-1 in the vascular endothelium. the parasitized erytrocytes rupture and release toxic molecules from the parasite, i.e. glycosyl phosphatidyl inositol (gpi) molecules, which induce mononuclear cells to produce pro-inflammatory cytokines, such as tumor necrosis factor alpha (tnf-α), interleukin1 alpha (il-1α), il-6, il-10, il-1β, ifn-γ and others through activation of the transcription factor nfκb. these pro-inflammatory cytokines then induce the activation of nfκb,(2) thereby increasing nfκb proteins, which in the brain are predominantly expressed by microglia and astrocytes.(12) in the present study it was found that nfκb expression in the positive control group was exceedingly high and differed significantly from that in the treatment groups. in this study, upon administration of tinospora extract decreases in both nfκb and icam-1 expression were found. this is due to the fact that tinospora extract has antiinflammatory, antioxidant, and antiparasitic activities, which are mainly caused by phenolics, flavonoids, and alkaloids in all parts of the plant, such as leaves, fruits, seeds, roots, and bark.(5,17) in this study the sample of tinospora stem extract tested on tlc showed a content of total phenolics of 43.34% and total flavonoids of 74.26%, which are high for tinospora and represent its antiinflammatory and antioxidant properties. the antioxidant test conducted by ibrahim et al.(13) showed that a methanolic extract of t. crispa had figure 2. icam-1 expression by immunohistochemistry (400x) (a) negative control, (b) positive control, (c) artesunate, (d) tinospora extract, (e) combination treatment a, (f) combination treatment b, (g) combination treatment c. arrows indicate icam-1-expressing cells a b d e f g c izzati, fitri, dalhar artesunate-tinospora decreases nfκb and icam-1 227 univ med vol. 35 no.3 the highest antioxidant activity dependent on total flavonoid and phenolic content, and an activity against free radicals of 2,2 diphenyl 2 picryl hydrazyl hydrate (dpph). the excellent anti-inflammatory activity of berberine that has been observed in vitro and in vivo showed decreases in pro-inflammatory cytokines in the acute phase. the nfκb pathway plays an important role in the control of inflammation and is one of the main targets of the anti-inflammatory activity of berberine. in the nfκb signaling pathway, iκb kinase-β (ikk-β) may be activated by stimulation by inflammatory cytokines such as tnf-α. activation of ikk-β requires phosphorylation of serine (ser-181). administration of berberine substantially decreases ser-181 phosphorylation and ikk-β activation.(18,19) the anti-inflammatory and antioxidative activity of berberine is also mediated through the ampk and nrf2/ho pathways.(20) in vitro, berberine and palmatine may inhibit no production and nfκb activation in macrophages, that is stimulated by lps or tnf-α.(21) artesunate is a first-line antimalarial drug which has been demonstrated to be effective as compared with other artemisinin derivatives. however, its use in monotherapy may cause recrudescence, therefore it should be used in combination with other drugs.(1) tukey’s post hoc test showed that combination therapy caused a greater and significantly different decrease in nfκb expression as compared with the groups receiving monotherapy with artesunate or tinospora extract. according to previous studies, the pharmacokinetic properties of berberine are as follows: half-life (t 1/2 ) 945.8 minutes, time to reach maximum drug concentration (t max ) 15.0 minutes, peak plasma concentration (c max ) 20.0 ng/ml, area under plasma concentration from time point 0 until a given time point t (auc 0�t ) 4954.3 minutes-ng/ml, distribution volume 5124.3 l/kg and total body clearance (cl) 198.6 l/hour/kg.(22) in a previous study, the in vivo antimalarial effect of t. crispa extract at a dose of 80 mg/kg/ day showed inhibitory activity on the growth of p.yoelii.(23) in another study, a methanolic extract of t. crispa stems had in vitro anti-plasmodial activity against p. falciparum strain 3d7 with ic 50 values between 0.27 mg/ml and 0.29 mg/ml, while the effective inhibitory dose was 2.0 mg/ml, at which dose the parasitemia decreased to 7.62% and 5.85% after 48 and 72 hours, respectively, resulting in a mean reduction of parasitemia degree by 47.12% and 56.83%, respectively. (24) a limitation of the present study was the fact that the mice were sacrificed on day 7 only, therefore precluding comparison with a longer treatment period to find the effect of length of drug and herbal administration. further studies should be conducted to determine whether or not combination therapy of artesunate and tinospora extract also has inhibitory effects on other pro-inflammatory cytokines, apart from icam-1, in which the production depends on nfκb activation. in addition, there is a need to conduct studies on the benefits and safety of tinospora extract for human malaria through clinical trials and their stages or phases. conclusion combination therapy with artesunate and tinospora extract results in a higher reduction in nfκb and icam-1 expression in the brain of mouse malaria models as compared with artesunate therapy. conflict of interest none declared. acknowledgement the authors wish to express their gratitude to the head of the parasitology and biomedical laboratory, brawijaya university, malang, for the use of facilities in the completion of this study. references 1. world health organization. world malaria report. geneva: world health organization; 2015. 228 2. punsawad c, yaowapa m, urai c,et al. nuclear factor kappa b modulates apoptosis in the brain endothelial cells and intravascular leukocytes of fatal cerebral malaria. malaria j 2013;12:1-12. 3. patel jp, gami b, patel k. evaluation of in vitro schizontocidal properties of acetone extract of some indian medicinal plants. adv biol res 2010;4:253-8. 4. brown gd. the biosynthesis of artemisinin (qinghaosu) and the phytochemistry of artemisia annua l. 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rodseth rn. the endothelial glycocalyx: a review of the vascular barrier. anaesth 2014;69:777–84. 16. cabrales p, yuri cm, peng ko, et al. cerebral tissue oxygenation impairment during experimental cerebral malaria. virulence 2013;15;4:686-97. doi: 10.4161/viru.26348. 17. zhang l, ravipati as, koyyalamudi sr, et al. antioxidant and antiinflammatory activities of selected medicinal plants containing phenolic and flavonoid compounds. j agr food chem 2011;59:12361-7. doi: 10.1021/jf203146e. 18. moghaddam hk, baluchnejadmojarad t, roghani m, et al. berberine ameliorate oxidative stress and astrogliosis in the hippocampus of stz-induced diabetic rats. mol neurobiol 2013; 49:820-6. 19. wang y. attenuation of berberine on lipopolysaccharide-induced inflammatory and apoptosis responses in β-cells via tlr4independent jnk/nf-κb pathway. pharm biol 2013;52:532-8. 20. mo c, wang l, zhang j, et al. the crosstalk betweennrf2 and ampk signal pathways is important for the anti-inflammatory effect of 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batang tinospora crispa terhadap plasmodium falciparum galur 3d7 secara in vitro]. jurnal kedokteran brawijaya 2014;28:91-6. izzati, fitri, dalhar artesunate-tinospora decreases nfκb and icam-1 135 *department of neurology, faculty of medicine, trisakti university, jakarta **department of neurology, cipto mangunkusumo general hospital, faculty of medicine, university of indonesia, jakarta ***department of physiology, faculty of medicine, trisakti university, jakarta correspondence : dr. yudhisman imran, sp.s department of neurology faculty of medicine trisakti university jl. kyai tapa no. 260 – grogol jakarta 11440 phone : +62813 1945 9868 email: yudhisman.imran@trisakti.ac.id univ med 2016;35:135-42 doi: http://dx.doi.org/10.1805/ univmed.2016.v35.135-142 abstract universa medicina may-august, 2016 vol.35 no.2 high blood pressure tends to increase carotid intima-media thickness in adult females yudhisman imran*, pukovisa prawiroharjo**, and martiem mawi*** background atherosclerosis is initiated by endothelial dysfunction, as a result of increasing degradation of nitrit oxide by reactive oxygen species, thereby increasing oxydative stress. dyslipidemia is one of the risk factors of endothelial dysfunction. the aim of this study was to evaluate the relationship of blood pressure and serum lipid level with carotid intimamedia thickness (cimt) in subjects aged between 55-65 years. methods a cross sectional study was carried out in 52 male and female subjects aged between 55-65 years. age, gender, body mass index (bmi), blood pressure, lipid profile, and cimt were assessed in all subjects. the independent t-test was used to to analyze the relationship between all variables and cimt. the level of statistical significance was set at p<0.05 results mean age was 59.19 ± 3.68 years, prevalence of thickened cimt was 66.5%, and plaques were found in 9 subjects. there were no significant differences in age, bmi, systolic and diastolic blood pressure, and lipid profile between normal and thickened cimt (p>0.05). however, there was a significant difference in gender between the two groups (p=0.011). in females, mean systolic and diastolic blood pressure were higher in the thickened cimt group than in the normal cimt group, but the difference was not significant (p>0.05). conclusions our findings suggest that high blood pressure tends to increase cimt in female adults. an increasing value of cimt should be considered as a sign of cerebrovascular disease. keywords: atherosclerosis, blood pressure, lipid, carotid intima-media thickness, adult 136 imran, prawiroharjo, mawi blood pressure and carotid intima-media thickness introduction the increased longivity of the population is reflected in the high numbers of the elderly in the population. in indonesia in the year 2010, there were 18,037,009 elderly or around 7.59% of the population, and their number is projected to increase to 414% between the years 1990 and 2025.(1) the age of an individual advances with increasing oxidative stress of protein and lipid biomolecules. this increased oxidative stress will raise the risk of vascular diseases such heart disease and stroke, thus causing a decreased quality of life of the elderly.(2) the data in indonesia show a rising trend of stroke cases, with regard to mortality, morbidity, or disability. the age-related mortality rates are 15.9% (age group of 45-55 years), 26.8% (age group of 5564 years) and 23.5% (age group of >65 years).(3) atherosclerosis, one of the causative factors of ischemic stroke, is a chronic inflammatory process, which leads to the formation of the initial lesion. this lesion develops into a more complex one and may even cause an acute lesion such as a ruptured plaque. atherosclerosis commences with endothelial dysfunction in the form of thickening of the intima media complex (imc). the endothelial dysfunction is the result of decreased bioavailability of nitric oxide (no), which is known as a vasodilator resulting from increased no degradation by reactive oxygen species (ros). free radicals are produced continuously in the body for physiological purposes. if they are present in excessive amounts, they will cause a pathological condition known as oxidative stress.(4,5) in dyslipidemia there is an increase in the blood cholesterol concentration. total cholesterol may be categorized into several diagnostic lipoprotein profiles, comprising high density lipoprotein (hdl) cholesterol, low density lipoprotein (ldl) cholesterol, intermediate density lipoprotein (idl) cholesterol and very low density lipoprotein (vldl) cholesterol, chylomicrons remnants, and triglycerides. the optimal total cholesterol concentration is <200 mg/dl, that of ldl cholesterol <100 mg/dl, hdl cholesterol >40 mg/dl, and triglycerides <150 mg/dl.(6,7) the high ldl cholesterol concentration in t h e c i r c u l a t i o n a n d t h e d e c r e a s e i n t h e concentration of hdl cholesterol, which serves to transport cholesterol from the tissues to the liver, play a role in atherosclerosis. subsequently ldl cholesterol adheres to the damaged e n d o t h e l i u m a n d u n d e rg o e s o x i d a t i v e modification in the subendothelial space into oxidized ldl cholesterol (ox ldl), which has proinflammatory activity but may still be recognized by ldl receptors. initially, the endothelium attempts to repair itself by attracting t lymphocytes, monocytes and platelets to the site of injury. when the repair process fails, the endothelium becomes more permeable and the lymphocytes and monocytes migrate to the subendothelial space by a process of adhesion. the monocytes develop into macrophages and express scavenger receptors, thus forming foam c e l l s . t h e a b o v e m e n t i o n e d o x i d a t i v e modification comprises reactive oxygen species (ros) produced by endothelial cells and macrophages. the increase in ros leads to decreased nitric oxide (no) production and bioavailability, resulting in vasoconstriction and platelet aggregation.(5,8) an increase in carotid intima-media thickness (cimt) can be detected by noninvasive u l t r a s o u n d ( u s g ) t e c h n i q u e s . t h e u s g examination can evaluate the vessel wall structure, which has a characteristic double reflection with an intermediate hyperechoic zone, designated the intima media complex (imc), and used as a parameter of atherosclerosis. the imc value increases with advancing age. the normal imc value is <0.8mm for asian populations, and a thickening of 1mm is considered abnormal and is suspected as a lesion of lipid disorders.(9,10) one study investigating the association between cholesterol concentration and imc thickness was conducted by enomoto et al.(11) showing a significant association between hdl cholesterol and imc. differing results were 137 univ med vol. 35 no.2 shown by yang et al.(12) who found no significant association between mean imc and mean hdl cholesterol. these inconsistent study results call for futher research. therefore, the purpose of the present study was to differentiate serum lipid concentrations and carotid intima-media thickness (cimt) in males and females aged between 55-65 years. methods research design this was an analytical study of crosssectional design, conducted at the puskesmas of mampang prapatan district, south jakarta, from august 2013 until february 2014. study subjects the subjects of this study were elderly residents of mampang prapatan district, south jakarta, aged between 55-65 years, who were selected randomly. the inclusion criteria in this study were age between 55-65 years, agreeing to participate in this study, capable of active locomotion without walking aids, capable of answering the questions of the investigator on her/his own or with the aid of other persons. the exclusion criteria were a history of hypertension, diabetes mellitus, heart disease, stroke, smoking, consumption of cholesterol-lowering drugs and incomplete participation in the study. a total of 1 7 7 r e s p o n d e n t s w e r e i n t e r v i e w e d b y 8 interviewers (puskesmas cadres) using a questionnaire. then physical examinations and measurements of serum lipid concentration were performed. of the 177 respondents who had a l a b o r a t o r y e x a m i n a t i o n t h e r e w e r e 7 0 respondents who fulfilled the inclusion criteria. however, only 52 subjects agreed to undergo doppler ultrasound (usg) examination. meas u rem ent of carot id int im a-media thickness the protocol for cimt measurement has been published elsewhere.(13) carotid duplex usg was performed, in which the subjects were lying supine with the neck slighly extended and rotated away from the side to be examined. the 10 mhz linear transducer was placed on the neck and moved upwards from the supraclavicular region to the right and left mandibular angles. the measurement was performed on the distal part of the common carotid artery, i.e. on a 1cm long segment of the carotid bulb. on the screen a double-line would be visible, indicating the simultaneous capture of the near-wall and the far-wall. the cimt measurement results were reported in millimeters (mm). the instrument used was the mindray z6 digital u l t r a s o n i c i m a g i n g s y s t e m . t h e c i m t examination was performed on 52 subjects at the mampang prapatan district puskesmas. t h e l a b o r a t o r y e x a m i n a t i o n s w e r e performed in the prodia clinical laboratory, while the carotid doppler examination was carried out by the investigators. the cimt results were divided into two categories: normal if cimt was <0.8 mm and thickened if cimt was ≥0.8 mm.(14) measurement of body mass index height was measured by means of a portable microtoise and expressed in cm with a precision of 0.1 cm, weight was measured using sage portable scales and expressed in kg with a precision of 0.1 kg. body mass index (bmi) was calculated from the weight in kg divided by height in meters squared and divided into the following categories: underweight (<18.5 kg/m2), normal (18.5-22.9 kg/m2), overweight (23.0-27.5 kg/m2) and obesity (>27 kg/m 2). blood pressure measurement results were expressed in mmhg. measurement serum lipid concentrations a 10-ml blood sample was collected by venipuncture from the cubital vein of each subject, after an overnight fast of 12-14 hours, then the blood sample was centrifuged at 3000 rpm for 15 minutes. the serum was separated and placed in an automatic analyzer. serum lipids were determined directly in the prodia laboratory at each laboratory examination. the 138 imran, prawiroharjo, mawi blood pressure and carotid intima-media thickness normal total cholesterol concentration was defined at <200mg/dl, ldl cholesterol <100mg/dl, hdl >40mg/dl, and triglycerides <150mg/dl. determination of cholesterol concentration was performed by the chod-pap method, with a coefficient of variation of 1.19 and april 2016 as expiration date. for ldl and hdl cholesterol determinations, the coefficient of variations were 1.43 and 2, respectively, with expiration date 2 february 2016. triglycerides were measured by the gdhnad method, with a coefficient of variation of 1.27. glucose was determined by the godpap method, with coefficient of variation 1.05 and expiration date 1 january 2017. statistical analysis the comparison of lipid variables and cimt was done with the independentt test. the level of statistical significance was set at p<0.05. ethical clearance this study was given ethical clearance by the ethics commision, faculty of medicine, trisakti university (no. 55/ker/fk/05/2013). results the 52 subjects had a mean age of 59.19 ± 3.68 years and 28 (53.8%) were females, while 40.4% had finished primary school and 80.8% was unemployed. based on the results of body mass index measurements the mean bmi was 24.55 ± 3.42 kg/m2. mean systolic blood pressure was 138.17 ± 21.78 mmhg and mean diastolic blood pressure 83.07 ± 9.90 mmhg. a total of 45 (66.5%) subjects had thickened cimt (table 1). on lipid profile examination the mean total cholesterol (tc) concentration was 202.19 ± 35.66 mg/dl, mean ldl cholesterol 138.34 ± 32.36 mg/dl, mean hdl cholesterol 47.34 ± 8.53 mg/dl, and mean triglyceride (tg) concentration was 135.09 ± 66.03 mg/dl (table 1). there were plaques in 9 subjects with mean age of 60.05 ± 3.97 years. in comparison with the normal cimt group, the parameters of age, body mass index, systolic table 1. distribution of subjects by characteristics and biochemical indicators (n=52) cimt: carotid intima media thickness; bp: blood pressure; ldl: low density lipoprotein; hdl: high density lipoprotein blood pressure and levels of tc, tg and ldl in the group with thickened cimt did not show statistically significant differences (p>0.05). however, of the male subjects 24 (100.0%) had thickened cimt, thus differing significantly from the females, of whom 21 (75.0%) had thickened cimt (p=0.011) (table 2). the results of the analysis show a difference in gender between both cimt groups. stratification analysis of the females showed thickened cimt, while the systolic (143.33 ± 22.66 mmhg) and diastolic blood pressures (86.43 ± 8.68) tended to be higher in comparison with the normal cimt group, although the difference was statistically not significant (p>0.05) (table 3.) discussion our study results show a significantly greater cimt prevalence in males than in females. 139 univ med vol. 35 no.2 table 2. comparison between normal cimt group and thickened cimt group in terms of general information and biochemical indicators cimt: carotid intima media thickness; bp: blood pressure; ldl: low density lipoprotein; hdl: high density lipoprotein table 3. comparison between females of the normal and thickened cimt groups in terms of general information and biochemical indicators (n=28) cimt: carotid intima media thickness; bp: blood pressure; ldl: low density lipoprotein; hdl: high density lipoprotein differing results were shown in the study of guan et al.(15) where the prevalence of thickened cimt was significantly higher than in females. in our study, mean systolic blood pressure (sbp) and mean diastolic blood pressure (dbp) were higher in the thickened cimt group compared with the normal cimt group, but the difference was statistically not significant. different results were obtained in the study by guan et al.(15) showing that sbp was significantly higher in the thickened cimt group compared with the normal cimt group, but that dbp was not significantly different. another study showed different results, in that both systolic and diastolic bp were found to be positively associated with cimt.(16) high bp has been recognized as an important risk factor for cardiovascular and cerebrovascular diseases. although high bp is a major determinant of cimt, the associations between cimt and different types of abnormal bp in different populations remain unknown. in an investigation enrolling 129 subjects aged from 29 to 94 years, a positive association was shown between aging and cimt. in relation to systolic hypertension, a significant association was observed with cimt.(18) as a result of the systemic diffuse lesions of atherosclerosis, vital organs such as the heart, brain and kidney may undergo structural and functional alterations.(19) the diagnosis of subclinical atherosclerosis can be based on cimt measurement by ultrasonography, which may facilitate its assessment and early intervention. atherosclerosis is currently viewed as a multifactorial disease, where the most important pathogenic factors are lipid metabolic disorders, which play an important role in its development.(20) in atherosclerosis, endothelial dysfunction occurs 140 imran, prawiroharjo, mawi blood pressure and carotid intima-media thickness as a result of chronic inflammation, which includes decreased nitrite oxide (no) production, increased expression of prothrombotic factors, proinflammatory adhesion molecules, cytokines, and chemotactic factors, in addition to increased reactive oxygen species (ros). low no bioavailability increases the expression of several adhesion molecules, such as vascular cell adhesion molecule-1 (vcam-1), intracellular adhesion molecule-1 (icam-1), platelet endothelial adhesion molecule-1 (pecam-1), and integrins and selectins. furthermore, these adhesion molecules induce recruitment and adhesion of monocytes, t lymphocytes, and thrombocytes on the endothelium.(21,22) thickening of imc is also increased by various proinflammatory mediators and other types of oxidative stress. based on the obtained results, the limitations in this study were that no assessment was done on physical activity, dietary pattern, sociodemographic characteristics of the subjects, and on other oxidative biomarkers. the study method used was cross-sectional, so that no control was possible for confounding factors, other than those capable of causing bias. although the association of cimt and increased risk of cardiovascular events is an established fact, there is still insufficient evidence to indicate that decreased cimt will lead to a reduction in cvd. in addition, subclinical atherosclerosis is mostly considered a non-causal and nonspecific marker of atherosclerotic complications.(23) the use of standardized methods in cimt measurement is required for further investigations, since cimt reflects atherosclerosis in its initial stages. carotid plaque area may be a better measure of atherosclerosis than cimt or plaque thickness, since on average plaque area possibly increases at twice its rate of thickening.(24) it is recommended to conduct further studies of cohort design, with larger sample sizes and broader age ranges. the benefits will better felt if other biomarkers of oxidative stress are also evaluated. conclusions our study found that high blood pressure tends to increase cimt thickening in adult females. on persons with high blood pressure and increased total and ldl cholesterol, regular health checks should be performed, especially carotid ultrasound examination, for early identification and prevention, and delayed development of atherosclerosis. legend: a: plaque; b: imc figure 1. intima-media complex (imc)(9) 141 univ med vol. 35 no.2 conflict of interests none declared. acknowledgements the investigators thank the dean and vicedeans of the faculty of medicine, trisakti university, for moral and material support in the conduct of this study. thanks are also due to the head of mampang health center (puskesmas m a m p a n g ) a n d s t a ff , a n d t o t h e s t u d y participants. references 1. badan perencanaan pembangunan nasional, badan pusat statistik, united nations population funds. proyeksi penduduk indonesia 2010-2035. jakarta: badan pusat statistik;2013. 2. dawis ne. atherosclerosis an inflammatory process. j insur med 2005;37:72-5. 3. pokdi stroke perhimpunan dokter spesialis saraf indonesia (perdossi) guideline stroke 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population. j transl intern med 2013;1:36-9. 16. ayer jg, harmer ja, nakhla s, et al. hdl cholesterol, blood pressure, and asymmetric dimethylarginine are significantly associated with arterial wall thickness in children. arterioscler thromb vasc biol 2009;29:943-9. 17. acevedo m, kramer v, tagle r, et al. cardiovascular risk factors among young subjects with high carotid intima media thickness. rev med chil 2011;139:1322-9. 18. viana de freitas e, brandão aa, pozzan a, et al. study of the intima-media thickening in carotid arteries of healthy elderly with high blood pressure and elderly with high blood pressure and dyslipidemia. clin intervent aging 2008;3: 525–34. 19. uno k, nicholls sj. biomarkers of inflammation and oxidative stress in atherosclerosis. biomark med 2010;4:361-73. 20. morrison km, dyal l, conner w, et al. cardiovascular risk factors and non-invasive assessment of subclinical atherosclerosis in youth. atherosclerosis 2010;208:501-5. 21. sengupta d, bardhan j, mahapatra ab, et al. corelation between lipid profile and carotid intima media thickness in cerebral ischemia. indian j physiol pharmacol 2014;58:354-64. 22. lorenz mw, markus hs, bots ml, et al. prediction of clinical cardiovascular evants with carotid intima-media thickness. a systematic review and meta-analysis. circulation 2007;115: 459-67. 142 imran, prawiroharjo, mawi blood pressure and carotid intima-media thickness 23. touboul pj, hernandez rh, kucukoglu s, et al. carotid artery intima thickness, plaque and framinham cardiovasua score in asia, africa/ middle east latin america: the parc-aala study. int j cardiovasc imaging 2007;235:55767. 24. bartels s, franco ar, rundek t. carotid intimamedia thickness (cimt) and plaque from risk assessment and clinical use to genetic discoveries. perspectives med 2012;1:139-45. c:\users\jurnal fkusakti\docume 25 *department of public health and community medicine faculty of medicine jenderal soedirman university purwokerto correspondence: dr.dr.nendyah roestijawati, m.k.k department of public health and community medicine faculty of medicine jenderal soedirman university purwokerto jl. dr. gumbreg no. 1 mersi purwokerto phone: +62281 622022 fax. +62281 624990 email: nendyahr@yahoo.com date of first submission, november 6, 2 01 7 date of final revised submission, february 21, 2018 date of acceptance, february 22, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina high serum iron and zinc decrease glutathione s-transferase among women with breast cancer nendyah roestijawati*, dwi arini ernawati*, and diah krisnansari* background breast cancer in indonesia ranks second as the most common cancer found in women after cervical cancer. tumor cells express increased levels of antioxidant proteins to detoxify reactive oxygen species (ros). glutathione s-transferase (gst) is known to play a key role in the detoxification and reduction of ros. trace elements have nutritional benefits as essential cofactors for physiologic processes, but some can be toxic to humans. accumulated evidence suggests that deficiency or excess of certain trace elements may be associated with risk of chronic diseases including cardiovascular diseases, diabetes and cancer. the aim of this research was to determine the correlation of the trace elements iron and zinc with the oxidative stress level of gst activity in women with breast cancer. methods an observational analytic study with cross sectional design was conducted involving 35 breast cancer women. research subjects were women with breast cancer diagnosed by biopsy. measurements of iron and zinc levels were performed using atomic absorption spectrometry, gst activity was measured using spectrophotometry. data analysis was done with the pearson correlation test. results the results showed that there was a significant relationship of serum iron with gst (r=-0.487; p<0.05) and serum zinc with gst (r=-0.409; p<0.01). conclusion our study demonstrated that the higher zinc and iron level, the lower gst level among breast cancer women. it is recommended to use zinc, iron and gst levels as biomarkers for breast cancer and its progression. keywords: iron, zinc, glutathione s-transferase, breast cancer, women doi: http://dx.doi.org/10.18051/univmed.2018.v37.25-30 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: roestijawati n, ernawati da, krisnansari d. high serum iron and zinc decrease glutathione s-transferase among women with breast cancer. univ med 2018;37:25-30. doi: 10.18051/univmed.2018.v37.25-30 january-april, 2018 vol.37no.1 26 roestijawati, ernawati, krisnansari iron, zinc and glutathione s-transferase in breast cancer introduction breast cancer in indonesia ranks second as the most common cancer found in women after cervical cancer. the number of cases of breast cancer has steadily increased, from 5,297 cases in 2004, 7,850 cases in 2005, 8,238 cases in 2006, to 8,277 cases in 2007.(1) elevated rates of reactive oxygen species (ros) have been detected in almost all cancers, where they promote many aspects of tumor development and progression. however, tumor cells also express increased levels of antioxidant proteins to detoxify ros, suggesting that a delicate balance of intracellular ros levels is required for cancer cell function.(2) glutathione s-transferase (gst) is known to play a key role in the detoxification and reduction of reactive oxygen species (ros). according to research by huang et al. (3 ) expression of gst-pi, a phase ii detoxification enzyme, was up-regulated in breast cancers that exhibited higher oxidative stress and were associated with higher gst activity. trace elements have nutritional benefits as essential cofactors for physiologic processes, but some can be toxic to humans. accumulated evidence suggests that deficiency or excess of certain trace elements may be associated with risk of chronic diseases including cardiovascular diseases, diabetes and cancer.(4) since the recognition of the possibility of the direct or indirect influence of trace elements in the development and prevention of malignant diseases, the contribution of the trace elements has been under scrutiny.(5) trace elements have been implicated in the pathogenesis of breast cancer.(6) according to holanda et al.(7) increases in zinc concentration in the cellular compartment and reduction of this trace element in the blood of patients with breast cancer appear to alter the activity of metalloproteinases 2 and 9 that contribute to the occurrence of malignancy. however, new studies that determine the effectiveness of these inhibitors in breast carcinoma are still needed. research by jian et al.(8) showed that mice fed an iron-deficient diet had significantly higher tumor volumes and lung metastasis compared to those fed normal iron diets. iron deficiency mainly altered notch but not tgf-β and wnt signaling in the primary tumor, leading to the activation of epithelial mesenchymal transition ( e m t ) . t hi s wa s r e ve a l e d by i n c r e a s e d expression of snai1 and decreased expression of e-cadherin. importantly, correcting iron deficiency by iron therapy reduced primary tumor volume, lung metastasis, and reversed emt markers in mice. furthermore, the research found that mild iron deficiency was significantly associated with lymph node invasion in young breast cancer patients. on the other hand, research by silva et al.(9) showed that concentrations of trace elements are higher in neoplastic tissues (malignant and benign) when compared with normal tissues. thus, there is still inconsistency of research results on the effect of trace element level on breast cancer. the aim of this research was to determine the correlation of the trace elements iron and zinc with the oxidative stress level of gst activity in women with breast cancer. methods research design this study was an observational analytic study using crosssectional design. the research was carried out within 6 months (july to december 2016) and was located at a hospital in purwokerto, banyumas district. research subjects the subjects of the research were patients with breast cancer. samples were selected by consecutive sampling in accordance with the inclusion criteria of breast cancer diagnosis based on biopsy, never received chemotherapy and 27 willing to be a research subject. the sample size was 35, based on a 0.05 significance level and power of 10%, with a correlation coefficient of 0.5. laboratory analysis blood samples were collected from the median cubital vein. prior to blood sampling, subjects were asked to fast for 10 hours. blood sampling was performed from 7:00 to 9:00 a.m. approximately 10 ml of blood was drawn into small purple-top vacutainer tubes. following clotting, the blood tube was centrifuged at 2500– 3000g for 5 min. the plasma supernatant was placed into a sterile acid-washed, metal ion–free microfuge tube, and stored frozen at “20°c until required for the assay.(10) iron and zinc levels were determined using s t a nd a r d la b or a t or y p r o to c o l s. in br ie f , specimens were acid digested under high pressure in a microwave oven and then assayed for iron and zinc on a perkin elmer 3110 atomic absorption spectrometer (waltham, ma, usa). we used calibration curves prepared from serially diluted iron and zinc standards (merck, germany).(10) measurement of gst activity was done by the method of habig et al.(11) in this method the gst activity is measured spectrophotometrically by following the yellow coloration as a result of 1-chloro-2,4-dinitrobenzene conjugation with gsh by gst. laboratory analysis took place at the research laboratory, faculty of medicine, jenderal soedirman university, purwokerto. data analysis data analysis was to determine whether there was a correlation using the pearson correlation test. a p value of <0.05 was considered significant. ethical clearance t hi s s t ud y w a s a pp r o ve d b y t he commission on health research ethics, medical sc h oo l , j e n de r a l so e d i r ma n un i ve r si t y (unsoed) with ref no: 152/kepk/vii/2016. characteristics mean ± sd age (yr) 47.89 ± 12.49 serum iron (µg/dl) 131.14 ± 8.48 serum zinc (mg/l) 2.12 ± 0.72 gst activity (µmol/dl) 55.86 ± 16.84 table 1.distribution of subject characteristics variables gst activity (µmol /dl) p value serum iron (µg/dl) serum zinc (mg/l) r = 0.487 r = 0.409 0.003 0.015 table 2. correlation of serum iron and zinc with gst activity (n=35) *significant at 0.05, tested by pearson correlation test results the mean age of the subjects was 47.89 ± 12.49 years, with the youngest subject being 25 years old and the oldest 76 years. mean serum iron was 131.14 µg/dl, mean serum zinc 2.12 mg/l and mean gst content was 55.86 μmol/l as shown in table 1. the result of the correlation analysis of serum iron with gst showed a weak negative correlation ( p<0.05, r=-0.487) , while the correlation of serum zinc with gst also showed a weak negative correlation (p<0.01, r=-0.409) as shown in table 2. discussion mean serum zinc concentration in this study was 2.12 mg/l, which was higher than the standard laboratory reference of 0.75–1.3 mg/ l).(12) zinc is required for both normal cell survival and for cell death via its role in apoptosis. zinc may also regulate autophagy and other forms of survival due to its early sensitivity to cell stress. thus, zinc could play a central role, regulating apoptosis and autophagy as well as immune cell function. cancer cells are continuously stressed (genomic stress, endoplasmic-reticulum (er) stress, nutrient stress, oxidant stress, etc) and selected for survival (likely by autophagy). zinc has a spectrum of effects on cell death and january-april, 2018 vol.37no.1 28 roestijawati, ernawati, krisnansari iron, zinc and glutathione s-transferase in breast cancer a b c survival, where zinc depletion induces cell death via apoptosis (or necrosis if apoptotic pathways are blocked) while sufficient zinc levels allow maintenance of cell survival pathways such as autophagy and regulation of reactive oxygen species. cancer cells have up-regulated zinc importers, and most frequently have increased zinc levels, which allow them to survive. based on these notions, means to locally regulate zinc levels to promote immune cell survival and tumor apoptosis are in order.(13,14) the mean serum iron concentration in this study was 131.4 µg/dl, which was at the high end of the standard laboratory reference range of 40 – 155 µg/dl.(15) according to the study by wen et al.(16) in taiwan, elevated serum iron concentrations were associated with higher risks of cancer and cancer mortality. a swedish cohort study by gaur et al.(17) found that increased serum iron concentrations were associated with slightly higher risks of postmenopausal breast cancer. the mean gst activity in this study was 55.86 µmol/dl. glutathione-s-transferase (gst) is a cytopla smi c protei n re sponsible f or detoxification. the study by he et al.(18) showed that cell proliferation and migration could be enhanced in greater concentrations of gst, where gst directly bound to the surface of the cancer cells. glutathione-s-transferase activities were higher in the breast tumor than in the normal cytosolic fractions.(19) enginler et al.(20) reported significantly higher gst enzyme in healthy tissues compared to tumor samples from the human mammary gland in a comparison of t he ma li gna n t t umo r s a nd t h eir he a l th y surrounding tissues. our study found negative correlations of serum iron and zinc with gst. according to kasperczyk et al.,(21) in the group with high levels of calcium, magnesium, zinc, copper, iron, and selenium, the activity levels of glutathione peroxidase (gpx) and glutathione-s-transferase (gst) were lower than in the low level group. higher levels of the examined metals are associated with elevated oxidative stress accompanied by decreased activities of some of the antioxidant enzymes. iron is a central element of the heme molecule, which is a critical part of hemoglobin and essential for oxygen transport. heme is also a part of many essential enzymes such as catalase and the cytochrome series.(22) however, when in abnormal expression, they seem to contribute to several pathological processes, including tumor growth, invasion and metastasis. free iron is a pro-oxidant and can induce oxidative stress and dna damage. the carcinogenicity of iron has been demonstrated in animal models, and epidemiologic studies have shown associations with several human cancers. however, a possible role of excess body iron stores or of elevated iron intake in breast carcinogenesis has received little attention epidemiologically. over time a high intake of iron can lead to iron overload, which may disrupt iron homeostasis with a resulting increase in free iron, so contributing to the development of breast cancer. furthermore, body iron stores increase in women following menopause. reactive oxygen species produced by normal aerobic cellular metabolism can lead to the release of free iron from ferritin. in the presence of superoxide radicals and hydrogen peroxide, stored ferric iron (fe3+) is reduced to ferrous iron (fe2+), which catalyzes the formation of the hydroxyl radical (*oh). this in turn can promote lipid peroxidation, mutagenesis, dna strand breaks, oncogene activation, and tumor suppressor inhibition, increasing the risk of breast cancer. in addition to its independent role as a prooxidant, high levels of free iron may potentiate the effects of estradiol, ethanol, and ionizing radiation—three established risk factors for breast cancer.(23) zinc occurs in all living cells as a constituent of metalloenzymes involved in major metabolic pathways. zinc controls several enzymes of intermediary metabolism, dna and rna synthesis, gene expression, immunocompetence, and plays a significant role in homeostasis of hormone s.(24 ) high levels of zn increased oxidative stress in breast cancer. the study by 29 seraj et al. (25) showed that plasma zinc and malondialdehyde conce ntrations were significantly higher in patients as compared to the control group. it may also be suggested that increased oxidative stress in breast cancer patients may be attributed to the altered trace element level in such patients. one limitation of this study was the absence of normal subjects (not having breast cancer) as controls. thus there was no control data on the zinc, iron and gst concentrations for comparing with those of the study subj ects and for demonstrating that the relationships between zinc, iron and gst occur only in women with breast cancer. further studies need to be carried out using a controlled study design, e.g. a case control study design. conclusions in the present study, we showed a negative correlation between serum iron and zinc with gst. this finding result support previous research that high accumulation of iron and zinc increased oxidative stress and decreased oxidant enzyme.. conflict of interest the authors declare to have no conflict of interest. acknowledgement acknowledgements are due to jenderal soedirman university for funding and faculty of medicine for their cooperation and assistance. contributors nr, da, and dk contributed to drafting of the manuscript and acquisition of the data. da and dk contributed to conception and design of the study. da and dk contributed to revising the manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. departemen kesehatan republik indonesia. sistem kesehatan nasional. jakarta: departemen kesehatan republik indonesia;2008. 2. liou gy, storz p. reactive oxygen species in cancer. free radic res 2010;44:479-96. 3. huang j, tan ph, tan bk, et al. gst-pi expression correlates with oxidative stress and apoptosis in breast cancer. oncol rep 2004;12:921-5. 4. he k. trace elements in nails as biomarkers in clinical research. eur j clin invest 2011;41:98– 102. doi: 10.1111/j.1365-2362.2010.02373.x. 5. adaramoye oa, akinloye o, olatunji ik. trace elements and vitamin e status in nigerian patients with prostate cancer. afr health sci 2010;10:2-8. 6. suzana s, cham bg, ahmad rohi g, et al. relationship between selenium and breast cancer: a case-control study in the klang valley. singapore med j 2009;50:265-9. 7. holanda aon, de oliveira ars, cruz kjc, et al. zinc and metalloproteinases 2 and 9: what is their relation with breast cancer? rev assoc med bras 2017;63:78-84. doi: 10.1590/1806-9282.63.01.78. 8. jian j, yang q, shao y, et al. a link between premenopausal iron deficiency and breast cancer malignancy. bmc cancer 2013;13:307. https:// doi.org/10.1186/1471-2407-13-307. 9. silva mp, soave df, ribeiro-silva a, et al. trace elements as tumor biomarkers and prognostic factors in breast cancer: a study through energy dispersive x-ray fluorescence. bmc research notes 2012;5:194. https://doi.org/10.1186/17560500-5-194. 10. benoff s, hauser r, marmar jl, et al. cadmium concentrations in blood and seminal plasma: correlations with sperm number and motility in three male populations (infertility patients, artificial insemination donors, and unselected volunteers). mol med 2009;15:248–62. 11. habig wh, pabst mj, jakoby wb. glutathione s-transferases: the first enzymatic step in mercapturic acid formation. j biol chem 1974;249: 7130-9. 12. szkup m, jurczak a, brodowska a, et al. analysis of relations between the level of mg, zn, ca, cu, and fe and depressiveness in postmenopausal women. biol trace elem res 2017;176:56–63. 13. john e, laskow tc, buchser wj, et al. zinc in innate and adaptive tumor immunity. j transl med 2010;8:118. doi: 10.1186/1479-5876-8-118. 14. maret w. molecular aspects of human cellular zinc homeostasis: redox control of zinc potentials and zinc signals. biometals 2009;22:149-57. 15. gomella lg, haist hs, editors. laboratory january-april, 2018 vol.37no.1 30 roestijawati, ernawati, krisnansari iron, zinc and glutathione s-transferase in breast cancer diagnosis: chemistry, immunology, serology. in: clinician’s pocket reference: the scut monkey. 11th ed. new york: mcgraw-hill;2007.p.53-94. 16. wen cp, lee jh,tai yp, et al. high serum iron is associated with increased cancer risk. cancer res 2014;74:6589–97. 17. gaur a, collins h, wulaningsih w, et al. iron metabolism and risk of cancer in the swedish amoris study. cancer causes control 2013;24: 1393–402. 18. he s, liao tt, chen yt, et al. glutathione-stransferase enhances proliferation-migration and protects against shikonin-induced cell death in breast cancer cells. kaohsiung j med sci 2011;27: 477-84. 19. oguztuzun s, abu-hijleh a, coban t, et al. gst isoenzymes in matched normal and neoplastic breast tissue. neoplasma 2011;58:304-10. 20. enginler so, toydemir tsf, ates a, et al. examination of oxidative/antioxidative status and trace element level in dogs with mammary tumors. bulg j agric sci 2015;21:1086-91. 21. kasperczyk a, dobrakowski m, horak s, et al. the influence of macro and trace elements on sperm quality. j trace elem med biol 2015;30:1539. doi: 10.1016/j.jtemb.2014.12.007. 22. brewer jg. iron and copper toxicity in diseases of ageing, particularly atherosclerosis and alzheimer’s disease. exp biol med 2007;232:323– 35. 23. kabat gc, rohan te. does excess iron play a role in breast carcinogenesis? an unresolved hypothesis. cancer causes control 2007;18:1047– 53. 24. jamakala o, rani ua. amelioration effect of zinc and iron supplementation on selected oxidative stress enzymes in liver and kidney of cadmiumtreated male albino rat. toxicol int 2015;22:1–9. doi: 10.4103/0971-6580.172289. 25. seraj ak, shankhar m, raju kd, et al. antioxidants and lipid peroxidation status in women with breast cancer. int med j malaysia 2015;14:71-5. rita k 186 *bagian ilmu penyakit gigi dan mulut fakultas kedokteran universitas trisakti korespondensi adr.drg kartika wangsarahardja, mpd bagian ilmu penyakit gigi dan mulut fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks. 2501 universa medicina 2007; 26: 186-94. universa medicina oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 hubungan antara status kesehatan mulut dan kualitas hidup pada lanjut usia kartika wangsarahardja*a, olly v. dharmawan*, dan eddy kasim* abstrak latar belakang proporsi lanjut usia bertambah lebih cepat dibandingkan dengan kelompok usia lain terutama di negara yang sedang berkembang, dan usia harapan hidup juga meningkat. kesehatan mulut yang buruk berdampak negatif terhadap kualitas hidup pada usia lanjut, dan membutuhkan program kesehatan mulut secara intensif. metode penelitian ini menggunakan rancangan potong silang dan dilaksanakan di kelurahan cideng, tomang, dan jati pulo. tigaratus enampuluh delapan lanjut usia (umur > 60), mampu berjalan, mampu berkomunikasi, bersedia berpartisipasi dalam penelitian ini dambil sebagai sampel. informasi tentang umur, jenis kelamin, status pernikahan, pendidikan, pekerjaan, status sosio-ekonomi (penghasilan), dan kualitas hidup telah dikumpulkan oleh 6 orang pewawancara yang sudah dilatih. status kesehatan mulut dikumpulkan melalui pemeriksaan gigi-mulut oleh 3 orang dokter gigi. hasil indeks kesehatan gigi (dmft) rata-rata pada lanjut usia > 70 adalah 15,57 + 10,36, lebih tinggi dibandingkan dengan usia < 70 (12,31 + 9,72). dmft berhubungan lemah secara bermakna dengan domain persahabatan dan cinta kasih (intimacy) (domain 6) (r=0,151; p<0,05) dan kualitas hidup secara keseluruhan (r=0,135; p<0,05). tidak terdapat hubungan yang bermakna antara kesehatan gigi dan mulut dengan setiap domain kualitas hidup pada lansia. kesimpulan berdasarkan tingginya indeks dmft pada lanjut usia yang berdampak negatif terhadap kualitas hidup, maka diperlukan intervensi yang bertujuan memperbaiki kesehatan mulut. kata kunci : kesehatan mulut, kualitas hidup, lanjut usia 187 universa medicina vol. 26 no.4 *department of stomatology medical faculty trisakti university correspondence adr. drg. kartika wangsarahardja, mpd department of stomatology medical faculty trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks. 2501 universa medicina 2007; 26: 186-94. background the proportion of older people is growing rapidly, faster than other age groups especially in developing countries, and life expectancy has increased. poor oral health has a negative impact on quality of life of older people, and needs intensive oral health programme implementation. methods a cross-sectional design was conducted in cideng, tomang, and jati pulo villages. three hundred and sixty eight elderly (age > 60), mobile, with good communication and agreed to participate, included in this study. information on age, gender, marriage status, education, occupation, and socio-economic status (income), and quality of life were collected by six trained interviewers. oral health status information were collected by three dentists. result the average dmft of the older age >70 was 15.57 + 10.36, higher than those of <70 of age (12.31 + 9.72). there were a weak significant correlation between dmft with the intimacy domain (r=0.15; p<0.05) and quality of life (r=0.135; p<0.05). conclution due to the high index of the dmft in the elderly which effected negatively the quality of life, an intervention aimed at improving the oral health is needed. keywords : oral health, quality of life, elderly the corelation between oral health status and the quality of life in the elderly kartika wangsarahardja*a, olly v. dharmawan*, and eddy kasim* abstract pendahuluan proporsi penduduk lanjut usia (lansia) bertambah lebih cepat dibandingkan kelompok usia lain.(1) pada tahun 2002 terdapat sekitar 600 juta orang berusia 60 tahun ke atas dan akan menjadi dua kali lipat pada tahun 2025 bahkan pada tahun 2050 akan menjadi 2 milyar, 80 % d i a n t a r a n y a b e r m u k i m d i n e g a r a berkembang.(1,2) seiring dengan meningkatnya usia harapan hidup penduduk indonesia, maka populasi penduduk lansia (60 tahun ke atas) juga akan meningkat. populasi lansia di indonesia tahun 2005 diperkirakan akan mencapai angka kurang lebih 18,7 juta orang (8,5% jumlah penduduk). angka ini akan menjadikan indonesia m e n e m p a t i u r u t a n k e -4 t e r b a n y a k n e g a r a berpopulasi lansia setelah cina, india dan amerika.(3) hal ini merupakan tantangan kepada para perencana/perancang kebijakan kesehatan dan sosial, khususnya karena penyakit-penyakit k r o n i s s e p e r t i p e n y a k i t k a r d i o v a s k u l a r, 188 hipertensi, kanker dan diabetes banyak dijumpai pada lansia.(4) di negara berkembang, penyakit kronik dan ketidakmampuan (disability) pada lansia banyak terjadi yang dapat dikurangi d e n g a n u p a y a h e a l t h p ro m o t i o n u n t u k meningkatkan kualitas hidup.(5) bersamaan dengan bertambahnya usia, terjadi pula penurunan fungsi organ tubuh dan berbagai perubahan fisik. penurunan ini terjadi pada semua tingkat seluler, organ, dan sistem. hal ini mengakibatkan terjadinya peningkatan kejadian penyakit pada lansia, baik akut maupun kronik. meningkatnya gangguan penyakit pada lanjut usia dapat menyebabkan perubahan pada kualitas hidup lanjut usia. yang dimaksud dengan kualitas hidup menurut world health organization (who) adalah persepsi seseorang dalam konteks budaya dan norma yang sesuai dengan tempat hidup orang tersebut serta berkaitan dengan tujuan, harapan, standar dan kepedulian selama hidupnya.(6) k u a l i t a s h i d u p p a d a l a n s i a d a p a t dipengaruhi oleh berbagai faktor antara lain status kesehatan mulut. studi di negara maju m e n u n j u k k a n b a h w a g a n g g u a n m u l u t merupakan kelainan bersifat kronik yang sering d i j u m p a i p a d a l a n s i a s e p e r t i k a r i e s g i g i , kehilangan gigi dan penyakit periodontal.(7) gejala dari penyakit mulut dapat berupa rasa s a k i t , i n f e k s i d a n t e r g a n g g u n y a f u n g s i mengunyah, yang dapat menurunkan kualitas hidup lansia. kelainan kronik pada mulut lansia dapat terjadi akibat rendahnya kunjungan pemeriksaan ke pusat kesehatan gigi atau t e n a g a p r o f e s i k e d o k t e r a n g i g i l a i n n y a . ( 8 ) dampak negatif dari kesehatan mulut yang buruk terhadap kualitas hidup para lanjut usia merupakan masalah kesehatan masyarakat yang penting. di seluruh dunia (globally) kesehatan mulut yang buruk pada lansia terutama tampak dengan banyaknya gigi yang hilang (tooth loss), k a r i e s g i g i d a n p e n y a k i t p e r i o d o n t a l . ( 9 ) penelitian in bertujuan untuk menyelidiki hubungan antara status kesehatan mulut dan kualitas hidup pada lansia. metode rancangan penelitian penelitian potong silang (cross sectional) d i g u n a k a n u n t u k m e n j a w a b m a s a l a h d a n mencapai tujuan penelitian. lokasi penelitian ini dilakukan di 3 kelurahan : cideng, tomang, dan jatipulo kecamatan gambir, jakarta pusat antara bulan oktober 2006 sampai dengan juni 2007. subjek penelitian kriteria inklusi dari penelitian ini adalah : lanjut usia (usia 60 tahun ke atas), dalam keadaan mobil (mampu bergerak tanpa bantuan orang lain), tidak mengalami infeksi akut (panas tinggi). subjek penelitian yang bersedia menandatangani surat perjanjian (informed concent) diikutsertakan pada studi ini. kriteria eksklusi penelitian ini adalah tidak dapat melihat, tidak dapat berbicara, dan hanya mampu diam di tempat tidur. pemilihan subjek penelitian dilakukan secara convenient nonrandom sampling.(16,17) besar sampel b e r d a s a r k a n p e n e l i t i a n p e n d a h u l u a n prevalensi lansia yang mengalami kualitas hidup kurang besarnya 0,27. 2 2 e qxpxz n α = p = 0,27; q = 0.73, zα pada tingkat kepercayaan sebesar 95% besarnya 1,96; e = 0,05 n = (1,96)2 x 0,27 x 0,73/ (0,05)2 = 302 wangsarahardja, dharmawan, kasim kesehatan mulut da n kualitas hidup lansia 189 universa medicina vol. 26 no.4 pada tingkat kepercayaan sebesar 95 % dan memperhitungkan drop-out sebesar 15%, maka besar sampel optimal yang diperlukan studi ini adalah 350. pengumpulan data sebanyak 6 orang petugas pengumpul data yang sudah dilatih melakukan wawancara menggunakan kuesioner yang sudah di uji coba. data yang dikumpulkan meliput: tempat dan t a n g g a l l a h i r / u s i a , j e n i s k e l a m i n , s t a t u s pernikahan, pendidikan, pekerjaan, tempat dan t a n g g a l l a h i r / u s i a p a s a n g a n , p e n d i d i k a n pasangan, pekerjaan pasangan, penghasilan d a l a m k e l u a rg a / b u l a n , p e n g e l u a r a n d a l a m keluarga/bulan. status kesehatan mulut sebanyak 3 orang dokter gigi melakukan pemeriksaan status kesehatan mulut meliputi keluhan sakit gigi, perawatan gigi, pengetahuan tentang kesehatan gigi, perilaku menyikat gigi, kendala pelayanan kesehatan gigi meliputi akses, b i a y a , j a r a k d a n w a k t u y a n g d i p e r l u k a n / tersedia. pemeriksaan status kesehatan gigi mulut meliputi pemeriksaan gigi yang berlubang atau decay (dt), gigi yang hilang atau missing (mt), gigi yang telah ditumpat atau filling (ft) dan diperiksa ada atau tidak adanya karang gigi a t a u c a l c u l u s . ( 1 8 ) s t a t u s k e s e h a t a n m u l u t dikelompokkan sebagai berikut: sangat baik, baik, sedang dan buruk (indeks dmft very low < 5,0, low 5,0 – 8,9, moderate 9,0 – 13,9 dan high > 13,9).(19) kualitas hidup kualitas hidup lansia diukur menggunakan instrumen dari whoqol-old terdiri dari 6 d o m a i n : i ) k e m a m p u a n s e n s o r i ( s e n s o r y abilities), meliputi: kemunduran panca indera, penilaian terhadap fungsi sensoris, kemampuan m e l a k u k a n a k t i v i t a s , d a n k e m a m p u a n berinteraksi, ii) otonomi (autonomy), meliputi: kebebasan mengambil keputusan, menentukan m a s a d e p a n , m e l a k u k a n h a l h a l y a n g d i k e h e n d a k i , d i h a r g a i k e b e b a s a n n y a , i i i ) aktivitas pada masa lampau, kini dan yang akan datang (past, present and future activities), meliputi: hal-hal yang diharapkan, pencapaian keberhasilan, penghargaan yang diterima, pencapaian dalam kehidupan, iv) partisipasi s o s i a l ( s o c i a l p a r t i c i p a t i o n ) , m e l i p u t i : penggunaan waktu, tingkat aktivitas, kegiatan s e t i a p h a r i , p a r t i s i p a s i p a d a k e g i a t a n masyarakat, v) kematian dan keadaan terminal (death and dying), meliputi: jalannya/caranya meninggal, mengontrol akhir hidup, takut akan akhir hidup, merasakan sakit pada akhir hidup, dan vi) persahabatan dan cinta kasih (intimacy), meliputi : persahabatan dalam kehidupan, cinta dalam kehidupan, kesempatan untuk mencintai, dan kesempatan untuk dicintai. masing-masing pertanyaan mempunyai lima peringkat dari peringkat 1 sampai dengan peringkat 5, dengan kriteria berturut-turut: sama sekali tidak (1), sedikit (2), cukup (3), banyak (4), dan sangat banyak (5).(20) pengolahan dan analisis data setelah dilakukan pengumpulan data, setiap kuesioner dievaluasi oleh supervisor lapangan; b i l a d i t e m u k a n a d a n y a k e k u r a n g a n d a n kesalahan akan dikembalikan kepada petugas pengumpul data untuk diperbaiki/dilengkapi. k e m u d i a n d i l a k u k a n p e n g o l a h a n d a t a menggunakan komputer dengan program spss versi 13.0. penyajian data dilakukan secara tekstular dan tabular. untuk menguji adanya hubungan antara tingkat kesehatan mulut dan kualitas hidup lansia digunakan uji korelasi pearson bila data menyebar secara normal. bila data tidak tersebar secara normal digunakan uji korelasi spearman. semua uji statistik dilakukan pada tingkat kemaknaan sebesar 5%. 190 sedikit lebih kecil dibandingkan lansia berusia > 70 tahun (15,24 ± 9,60) (p=0,806). semakin tinggi pendapatan lansia ternyata indeks dt paling kecil (0,08 ± 0,11) dibandingkan dengan lansia yang berpenghasilan rendah (0,09 ± 0,12) dan sedang (0,08 ± 0,12), tetapi perbedaan ini tidak berbeda secara bermakna (p=0,840). sedangkan pada indeks dmft, pada lansia yang berpenghasilan sedang (16,46 ± 9,93) lebih tinggi dibandingkan rata-rata indeks dmft lansia berpenghasilan rendah (14,34 ± 9,25) dan yang berpenghasilan tinggi (15,41 ± 9,86) (p=0,167) (tabel 2). kemampuan otonomi (autonomy) (domain 2) kualitas hidup lansia berusia 60–69 tahun berhubungan lemah secara bermakna dengan i n d e k s d e c a y ( r = 0 , 1 5 0 ; p < 0 , 0 5 ) . i n d e k s kesehatan gigi (dmft) berhubungan lemah s e c a r a b e r m a k n a b a i k d e n g a n d o m a i n persahabatan dan cinta kasih (intimacy) (domain 6) (r=0,151; p<0,05) dan kualitas hidup secara keseluruhan (r=0,135; p<0,05). tidak terdapat hubungan yang bermakna antara kesehatan gigi dan mulut dengan setiap domain kualitas hidup pada lansia berusia > 70 tahun. pembahasan penyakit mulut merupakan salah satu kondisi kronik yang paling banyak dijumpai pada lansia. stacher menyatakan kesehatan mulut yang buruk sebagai ‘silent epidemic’.(10) penekanan bahwa kesehatan mulut tidak hanya berupa gigi yang sehat tetapi integral pada kesehatan umum dan pentingnya pencegahan p e n y a k i t k a r i e s g i g i s e r t a p e n y a k i t periodontal.(10) akibat dari penyakit ini yang meliputi rasa sakit, infeksi dan hilangnya fungsi, dapat menyebabkan menurunnya kualitas hidup l a n s i a . k e s e h a t a n m u l u t p e n t i n g u n t u k melengkapi kesehatan umum dan kesejahteraan seorang lanjut usia.(11) hasil sebanyak 368 lansia berhasil dikumpulkan, usia berkisar antara 60– 90 tahun. mereka yang rata-rata usianya 60–69 tahun banyaknya 6 1 , 1 % d a n > 7 0 t a h u n 3 8 , 9 % , l a k i l a k i b a n y a k n y a 2 9 , 6 % d a n p e r e m p u a n 7 0 , 4 % . sebagian besar menikah sebanyak 51,1%, berpendidikan tidak tamat sd 30,4%. sosial ekonomi rendah 58,7% dan tidak bekerja 69,0% (tabel 1). indeks dmft yang sangat rendah besarnya 15,5% (57/368) dan rendah 16,8 (62/368). rata-rata indeks mt pada lansia berusia 6069 tahun (0,39 ± 0,31) tidak jauh berbeda dibandingkan lansia berusia > 70 tahun (0,35 ± 0,30) (p=0,222). hal yang sama terlihat pada indeks dmft, rata-rata indeks dmft pada lansia berusia 60 69 tahun (14,99 ± 9,53) variabel n = 368 (%) usia (tahun) 60-69 > 70 225 (61,1) 143(38,9) jenis kelamin laki-laki perempuan 109 (29,6) 259 (70,4) status menikah tidak menikah duda/janda 188 (51,1) 10 (2,7) 170 (46,2) pendidikan tidak tamat sd sd smp sma >sma 112 (30,4) 93 (25,3) 68 (18,5) 80 (21,7) 15 (4,1) pekerjaan bekerja tidak bekerja 114 (31,0) 254 (69,0) sosial ekonomi rendah (< rp. 500.000) sedang (rp.500.000 – 1 juta) tinggi (> 1 juta) 216 (58,7) 106 (28,8) 46 (12,5) tabel 1. karakteristik demografi lanjut usia wangsarahardja, dharmawan, kasim kesehatan mulut da n kualitas hidup lansia 191 universa medicina vol. 26 no.4 tabel 2. rata-rata indeks decaying, missing dan filling gigi berdasarkan karakteristik demografi lanjut usia (n=368) a k s e s p a d a p e r a w a t a n k e s e h a t a n d a n kesehatan gigi yang layak dapat mengurangi angka kesakitan ( morbidity) dan kematian (mortality) yang dini, mempertahankan fungsi – fungsi dan meningkatkan kualitas hidup secara keseluruhan.(12) meskipun jelas keadaan ini mempunyai dampak luas termasuk gangguan kesehatan umum, kesejahteraan sosial dan mental lansia, usaha-usaha pelayanan kesehatan gigi dan mulut pada orang tua masih jauh dari m e m a d a i . s a l a h s a t u a l a s a n y a n g p a l i n g menonjol adalah bahwa orang tua menganggap disfungsi oral merupakan bagian dari proses alamiah dan konsekuensi usia lanjut sehingga para lansia menerima saja kondisi menurunnya kualitas hidup tersebut tanpa berupaya untuk mendapatkan pertolongan.(8) jumlah sampel pada penelitian ini besarnya 368 lansia dengan rasio antara laki-laki dan perempuan 109 berbanding 259. pada lanjut usia (>70 tahun) besarnya subyek 143 orang, indeks rata-rata dmft adalah 15,24 + 9,60, bila mengacu pada kriteria status kesehatan mulut berdasarkan gigi permanen pada lansia >70 tahun ini indeks rata-rata dmft tinggi (high >13,9).(19) sedangkan untuk usia 60-69 tahun adalah 12,31 + 9,72, termasuk sedang (moderate 9,0-13,9). 192 yang menjadi masalah adalah kurangnya perhatian dan ketiadaan biaya untuk mencari dan mendapatkan pelayanan kesehatan gigi dan mulut karena lebih dari setengah jumlah subyek berpendidikan rendah. hal ini mengakibatkan k u r a n g n y a p e r h a t i a n (a w a re n e s s ) t e n t a n g kesehatan gigi-mulut sehingga terabaikan. pendidikan yang rendah pada umumnya juga mengakibatkan status sosio-ekonomi khususnya penghasilan yang rendah pula. dari 368 subyek, 216 orang (58,7%) berpenghasilan kurang dari r p . 5 0 0 . 0 0 0 , h a n y a 1 0 6 o r a n g y a n g berpenghasilan sedang (rp. 500.000 1 juta). hal ini mengakibatkan subyek tidak mencari p e n g o b a t a n k a r e n a k e t i a d a a n b i a y a . d a r i p e n e l i t i a n va r g a s ( 1 8 ) d i d a p a t k a n k a r e n a kurangnya fasilitas pelayanan kesehatan gigi dan mulut di pedesaan amerika terutama bagi golongan sosio-ekonomi rendah tidak mendapat pelayanan/pengobatan gigi dan mulut. lansia dengan status sosio-ekonomi rendah yaitu yang tergolong miskin dan mendekati miskin (poor wangsarahardja, dharmawan, kasim kesehatan mulut dan kualitas hidup lansia tabel 4. korelasi status kesehatan gigi dan mulut dengan kualitas hidup lansia > 70 catatan : nilai dalam sel merupakan nilai koefisien korelasi pearson (r) tabel 3. hubungan antara status kesehatan gigi dan mulut dengan kualitas hidup pada lansia usia 60 – 69 tahun (n=368) catatan : nilai dalam sel merupakan nilai koefisien korelasi pearson (r) * bermakna (p<0,05) and near poor) mencapai 64%. hal tersebut m e n y e b a b k a n k e t i a d a a n b i a y a u n t u k mendapatkan perawatan kesehatan gigi-mulut yang memadai. mengingat kronisitas dari penyakit gigi-mulut dan bertambahnya jumlah lansia maka dampak kesehatan mulut terhadap k u a l i t a s h i d u p l a n s i a a k a n m e n j a d i l e b i h signifikan di tahun-tahun mendatang. meskipun para dokter telah melakukan estimasi mengenai efek kondisi kronik dan pengobatannya terhadap kualitas hidup lansia, pendekatan serupa belum pernah dilakukan di dunia kedokteran gigi. penelitian-penelitian yang a d a p a d a u m u m n y a h a n y a b e r k i s a r p a d a masalah-masalah obyektif tetapi sama sekali tidak menyinggung persepsi subyektif dari lansia mengenai kualitas hidupnya.(13) dalam penelitian i n i k u a l i t a s h i d u p l a n s i a d i t e l i t i d e n g a n menggunakan instrument kuesioner whoqolold. terlihat adanya hubungan antara status kesehatan gigi-mulut dengan kualitas hidup l a n s i a t e r u t a m a b i l a d i t i n j a u d a r i d o m a i n 193 universa medicina vol. 26 no.4 kemampuan otonomi. demikian pula terdapat hubungan antara gabungan keseluruhan domain dari domain pertama sampai dengan keenam yang disebut sebagai raw whoqol 24 items dengan status kesehatan mulut. locker(14) menjelaskan bahwa indikator kualitas hidup dalam kaitannya dengan kesehatan mulut menggunakan pengukuran seberapa besar masalah gigi dan mulut mempengaruhi fungsi normal kehidupan seseorang. penelitian oleh biazevic et al di brazil, menggunakan instrumen oral health impact profile (ohip) untuk meneliti kualitas hidup dalam kaitannya dengan kesehatan mulut. ( 1 5 ) akibat dari e d e n t u l i s m e , karies, penyakit-penyakit periodontal, ditambah dengan a k i b a t k o m o r b i d i t a s s e p e r t i d i a b e t e s d a n xerostomia, sangat dirasakan oleh para lansia dan memberikan akibat yang bermakna terhadap fisiknya, serta ekonomis dan psikologis. akibat dari penyakit oral yang memberikan dampak kepada kualitas hidup lansia meliputi berbagai keadaan termasuk mengunyah, makan dan bicara. selanjutnya dapat memberikan dampak berupa menurunnya interaksi sosial, rasa sejahtera, harga diri dan perasaan berguna. kesimpulan dmft rata-rata pada usia yang lebih lanjut (> 70) lebih tinggi dibandingkan dengan pada usia <70. kemampuan otonomy (autonomy) (domain 2) kualitas hidup lansia berusia 60 – 69 tahun berhubungan lemah secara bermakna dengan indeks decay. indeks kesehatan gigi (dmft) berhubungan lemah secara bermakna baik dengan domain persahabatan dan cinta kasih (intimacy) (domain 6) dan kualitas hidup secara keseluruhan. ucapan terima kasih terima kasih kepada professor graeme hawthorne and the australian wohqol field c e n t r e , d e p a r t m e n t o f p s y c h i a t r y, t h e university of melbourne atas izin penggunaan kuesioner whoqol-old. kepada pimpinan fakultas kedokteran universitas trisakti, t e r i m a k a s i h a t a s d u k u n g a n d a n a y a n g memungkinkan terlaksananya penelitian ini. daftar pustaka 1. united nations population division. world population prospects: the 2002 revision, new york, ny, usa: united nations, 2003. 2. world health organization. active ageing: a policy framework. geneva: who, 2002. 3. departemen kesehatan r.i. mengenai masalah mental lanjut usia. available from: http:// www.depkes.go.id/index.php. accessed februari 10, 2006. 4. world health organization. the world health report shaping the future. geneva: who, 2003 5. puska p, pietenen p, uusitalo u. influencing public nutrition for non communicable disease prevention from community intervention to national programme experiences from findland. public health nutrition 2002; 5: 245–51. 6. petersen pe, yamamoto t. improving the oral health of older people: the approach of the who global oral health programme. comm dent oral epidemiol 2005; 33: 81–92. 7. skevington sm, lotfy m, o’connell ka. the world health organization’s whoqolbref quality of life assessment: psychometric properties and results of the international field trial. a report from the whoqol group. qual life res 2004; 13: 299-310. 8. ahluwalia kp, sadowsky d. oral disease, burden, and dental services utilization by latino and african american seniors in northern manhattan. j comm health 2003; 28: 267-80. 9. mcgrath c, bedi r. can dental attendance improve quality of life. br dent j 2001; 190: 262-5. 10. u.s. department of health and human services. oral health in america: a report of the surgeon general. rockville, md: u.s. department of health and human services, national institute of dental and craniofacial research, national institute of health, 2000. 11. oral health in america: a report of the surgeon general. rockville, md: u.s. department of 194 health and human services, national institute of dental and craniofacial research, national institute of health, 2000. 12. healthy people 2000: national health promotion and disease prevention objectives – full report, with commentary. dhhs publication no. (phs) 91–50 212. washington, dc: u.s. department of health and human services, public health services, 1991. 13. ekanayake l, perera i. perceived need for dental care among dentate older individuals in sri lanka. spec care dentist 2005; 25: 199-205. 14. locker d, issues and measuring change in self – perceived oral health status. comm dent oral epidemiol 1998; 26: 41–7. 15. biazevicn mgh, michel–crosato e, iagher f. impact of oral health on quality of life among the elderly population on joacaba, santa catarena, brazil. braz oral res 2004; 16: 85-91. wangsarahardja, dharmawan, kasim kesehatan mulut da n kualitas hidup lansia 16. hulley sb, gove s, browner ws, cummings sr. choosing the study subjects: specification and sampling. in: hulley sb, cummings sr, editors. designing clinical research. baltimore: williams & wilkins; 1992. p. 18-30. 17. riegelman rk. studying a study and testing a test: how to read the medical evidence. 4th ed. philadelphia: lippincott williams & wilkins; 2000. 18. vargas om, yellowitz ja, hayes kl. oral health status of older rural adults in the united states. jada 2003; 134: 479-86. 19. petersen pe, bourgeois d, ogawa h, estupinanday s, ndiaye g. the global burden of oral diseases and risks to oral health. bull world health organ 2005; 83: 661-9. 20. power m, quinn k, schmidt s, and the whoqolold group. development of the whoqol-old module. qual life res 2005; 14: 2197-214. f:\36 no 2\sulistyowati.pmd 138 *center for public health research and development **center for health resource and services research and development correspondence: sulistyowati tuminah darjoko center for public health research and development jl. percetakan negara 29, jakarta pusat email: sdarjoko@yahoo.com / watidarjoko@gmail.com date of first submission, may 26, 2017 date of final revised submission, august 14, 2017 date of acceptance, august 15, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 hormonal contraception increases risk of breast tumor based on clinical breast examination among adult women sulistyowati tuminah darjoko* and aprildah nur sapardin** background in indonesia, cancer prevalence according to the basic health research 2013 was 1.4 per 1000 inhabitants and the most common cancer in hospitalized patients in 2010 was breast cancer (28.7%). hormonal contraception (hc) use increases the breast cancer risk, even though hc has been used by 210 million women in the world. we aimed to define the association of hc with breast tumors based on clinical breast examination (cbe). methods a case-control design using secondary data from the baseline of the cohort study on the risk factors of non-communicable disease (rfncd) in 2011-2012 in 5 villages in central bogor district, bogor city. samples consisted of 152 cases and 152 controls. cases comprised palpable tumors in one or both breasts cbe (+). controls had no tumors in both breasts / cbe(-). data were analyzed by logistic regression. results odds ratio (or) of cbe + was 1.83 (95% ci: 1.11-3.04; p=0.019) for hc user and 1.62 (95% ci: 1.01-2.60; p=0.044) for blood total cholesterol level <200 mg/dl. or of group cbe(+) was 1.01 (current smokers) and 0.49 (former smokers) compared with nonsmokers (p=0.082); or was also 1.21 for subjects with one child and 1.77 for those without children, compared with those who had 2 children (p=0.454). conclusion hormonal contraception use increases breast tumor risk 1.8-fold after controlling for total cholesterol, smoking status and parity. with the several limitations of this advanced analysis, investigations focused on types and duration of hc use are still necessary. keywords: hormonal contraception, breast tumor, adult women doi: http://dx.doi.org/10.18051/univmed.2017.v36.138-149 original article cite this article as: darjoko st, sapardin an. hormonal contraception increases risk of breast tumor based on clinical breast examination among adult women. uni v med 20 17;3 6:1 3 8 -4 9 . doi: 1 0 . 1 8 0 5 1 / un i vm e d . 2 0 1 7 . v3 6 . 1 3 8 1 4 9 pissn: 1907-3062 / eissn: 2407-2230 139 univ med vol. 36 no.2 introduction cancer is the principal cause of death in advanced countries and the second main cause of death in developing countries.(1) breast cancer is the most common cause of death from cancer in women (with 522,000 deaths in 2012) and is also the type of cancer occurring most frequently in women in 140 out of 184 countries.(2) the prevalence of cancer in indonesia based on interview results in the basic health research (riskesdas) for 2013 was 1.4 per 1000 population and cancer was the seventh leading cause of death (5.7%) from all-cause mortality. the most frequent type of cancer in hospitalized patients throughout indonesia in 2010 was breast cancer (28.7%).(3) the risk factors for breast cancer are among other things age, family history of breast cancer and reproductive factors characterized by exposure to sexual hormones (i.e. estrogen and progesterone in women).(4) both hormones are contained in hormonal contraceptives (hc). oral contraceptives contain estrogen and progesterone, whereas the mini pills, contraceptive injections, and implants contain progesterone.(5,6) the results of epidemiological and clinical research showed str ong evidence on the role of estrogen/ progesterone in the formation of breast cancer, but the exact mechanism of tumor formation is not yet completely understood. (7,8) according to urban et al., (9) there was an increased risk of breast cancer associated with the use of hc, i.e. pills and/or injections (or=1.66; 95% ci: 1.28 2.16; p<0.001), pills only (or=1.57; 95% ci: 1.03-2.40; p=0.04), injections only (or=1.83; 95% ci: 1.31-2.55; p<0.001). the results of an analysis by sihombing and sapardin (10) from data of the cohort study on risk factors of noncommunicable disease (rfncd) [studi kohor faktor risiko peny akit tidak menular (frptm)] showed that the use of contraceptive pills has a risk of 3.63-fold for causing tumors of the breast based on ultrasonography [usg] (95% ci: 1.63-8.10; p=0.002). this finding differs from that of an analysis by sirait et al.(11) from riskesdas 2007 data, who did not find a significant relationship between the use of contraceptive pills and tumor/cancer breast based on interviews (aor=0.74; 95% ci: 0.50-1.08; p=0.117). in indonesia, the percentages of hc users were: for injections 38.5%, pills 31%, and implants 12.3%.(10) the novelty of this study lies in the diagnosis of breast tumors by means of clinical breast examination (cbe), because in the rfncd cohort study the cbe results were a determinant for performing or not performing usg. the reason for selecting cbe as a method of early detection is because the study by zafar ( 1 2 ) s ho we d t ha t s t a nd a r di zed cbe c a n differentiate between benign and malignant tumors. structured cbe in patients with breast tumors has a sensitivity of 100% (95% ci: 0.81) and a specificity of 94.6% (95% ci: 0.860.97) and the likeli hood rat io fo r bre ast carcinoma is 17.8 (95 % ci: 7.6-41.7). the study by ravi and rodriguez (13) found that among 15 cases of malignancy in patients who underwent mammography followed by histopathological confirmation, cbe detected one c ase of malignancy that had been overlooked in the mammogram. the research carried out by khoda and kapa (14) in 50 female patients with clinically palpable breast lump(s) found on cbe, the lumps in 40 (80%) patients had benign and 8 (16%) had malignant features. however, 2 (4%) patients were found to be in the “suspicious” category. on histopathological examination, 36 benign tumors were confirmed as such, but 4 were found to be malignant. all of 8 malignant tumors found by cbe were confirmed by histopathological examination. in the analysis of data from the previous rfncd cohort study on tumors/cancers of the breast,(10) there was no definition of the contraceptive pill variable, because in the questionnaire there was no specific question to differ between respondents who used one type of hc and those who used more than one type of hc (combined hc). because of this limitation, hc as the main independent variable in the present paper was defined as contraception using pills or 140 injections or implants. results of several investigations showed that a higher total cholesterol concentration (tcc) has a decrease risk for tumors/cancers of the breast than a lower tcc. to determine the odds ratio of lower tcc (<200 mg/dl) against breast tumors, in this analysis high tcc (200 mg/dl) as another independent variable was positioned as a reference. in indonesia, data about breast tumors/ cancers are generally data from patients attending hospitals, who are usually already in an advanced stage. in 2016 the indonesia agency for research and development, ministry of health (badan penelitian dan pengembangan kesehatan) conducted a nationa l research of noncommunicable disease that focused on breast tumors in the community, but the report is still in the process of finalization. according to poosari et al.(15), epidemiological research and risk factors for breast cancer are very important in its prevention. the use of hc is a risk factor for breast cancer, but the magnitude of the risk is not yet clear. therefore, the aim of this further analysis was to determine the magnitude of the risk of hc use for the development of breast tumors based on cbe. methods design of the study a case-control study was conducted using secondary data from the baseline of the rfncd cohort study that had been performed in the years 2011-2012. subjects the respondents were permanent residents aged 25-65 years in 5 kelurahan (villages) of central bogor district – bogor city, i.e. kebon kalapa, babakan pasar, babakan, ciwaringin and panaragan.the inclusion criterion was: female respondents who had already undergone cbe during the execution of the rfncd cohort study and whose data were complete. the exclusion criterion was respondents who were pregnant or had been breastfeeding for equal or less than 6 months. the 152 cases with positive cbe results (cbe(+)) i.e. the presence of a tumor in one breast or both breasts, which were found in the rfncd cohort study, were all included in the study sample. this number has met the minimum sample requirements which calculated with the formula of hypothesis testing for the odds ratio for case control studies, with a level of significance of 95% (=5%) and power of 95% (=5%). the or that was considered to be significant was 3.6 and the estimated proportion effect in the controls was 0.657, which was taken from the study results of sihombing and sapardin.(10) the controls were respondents with negative cbe results i.e. no presence of a tumor in both breasts. the selection of the controls was performed in a ratio of 1:1 by simple random sampling, and they were not paired (unmatching), so that the analyzed data totalled 304 respondents, consisting of 152 cases and 152 controls.(16) questionnaire the sociodemographic data that were collected as a result of the questionnaire-based interviews consisted of: 1) age (<40 years and 40 years);(10) 2) education i.e. low (no formal education, not having finished elementary school, and finished elementary school), middle (junior high school and senior high school) and high (d3/ d4 and university);(17) 3) marital status i.e. have a partner (married) and have no partner (single or divorced/widowed).(15) this grouping was used because there were very few respondents with single status, comprising only 10 subjects (3.3%). data about risk factors consisted of: 1) smoking status, i.e. nonsmoking (never smoked), former smoking (occasionally or daily) and current smoking (occasionally or daily);(18) 2) mental/emotional disorder was measured using the self-reporting questionnaire instrument with 20 items of question (srq-20). the respondents were considered having a mental/emotional disorder if they answered “yes” to minimally 6 out of 20 questions in the instrument;(19) 3) parity/ darjoko, sapardin hormonal contraception and breast tumor 141 univ med vol. 36 no.2 number of deliveries was calculated from the total number of children that were born (own children), divided into 3 groups, i.e. “no children”, “1 child” and “2 children or more”;(20) 4) breastfeeding experience i.e. “breastfeeding for <6 months or never did breastfeeding”, and “breastfeeding for 6 months”;(21) 5) hc users (respondents who ever had sexual intercourse) were respondents who had ever used or were currently using contraceptive pills, injections or implants. the responses were divided into 2 categories, i.e. “yes” and “no”.(15) the use of hormone-replacement therapy (hrt ) and consumption of hormonal medications (treatment for infertility) was not incorporated in this analysis since the data were very few in number. regarding the foods/beverages that were habitually consumed, the respondents were asked according to the food frequency questionnaire (ffq); for example, consumption of milk, coconut milk, fried snacks and packaged beverages. each type was divided into two categories, i.e. “3 times per week (frequent)” and “<3 times per week (seldom)”.(22) measurements anthropome tr ic mea surement was performed according to the guide to examination and measurement of the rfncd cohort study (unpublished). the respondents were advised to use loose and thin clothing. body weight (bw) was measured using and type uc-322 digital scales with a capacity of 150 kg and precision of 50 g. the respondents were asked to stand without footwear. height was measured in the upright position using a “multifunction” measuring tool. the body mass index (bmi) was obtained by the following formula: bmi = bw (kg) / height (m)2 based on the classification of the south asian health foundation, the bmi is divided into 4 categories, i.e. underweight (bmi <18,5), normal (18.5-22.9), overweight (23-24.9), obesity (25).(23,24) in the present analysis the bmi was only assigned into 2 categories, i.e. 25 and <25.(10) clinical breast examination in the cbe procedure, visual inspection and palpation was performed according to the guide to examination and measurement of the rfncd cohort study (unpublished). the cbe positive respondents were those who had a palpable tumor in one breast or both breasts (right/left). the cbe was performed by the midwife of the primary health care (puskesmas) who had been trained by a specialist in oncologic surgery from dharmais hospital – jakarta. laboratory blood analysis for the determination of blood lipid concentration, the respondents were asked to fast for 12-14 hours, from the night before the examina tion until the ne xt mor ning. the respondents were only permitted to drink water. with the respondents in the fasting condition, venous blood samples were drawn from them. the lipid profile comprised tcc (<200 mg/dl and 200 mg/dl); triglycerides (<150 mg/dl and 150 mg/dl); low density lipoprotein cholesterol (ldl) (<100 mg/dl and 100 mg/dl); high density lipoprotein cholesterol (hdl) (50 mg/ dl and < 50 mg/dl). ( 25 ) blood c hemistry investigation was performed by prodia laboratory in bogor city. data analysis bivariate analysis (chi-square test) was performed to determine the presence or absence of a difference in proportions between the dependent and independent variables, followed by multiple logistics regression analysis to determine the level of hc user risk for the presence of breast tumors based on cbe, by controlling for other variables.(26) ethical clearance the rfncd cohort study had already received ethical clearance from the commission on health research ethics of the indonesia agency for research and development, ministry of health (badan penelitian dan pengembangan kesehatan) under no. ke.01.08/ec/485/2011 142 dated 10 august 2011 and no. ke.01.05/ec/394/ 2012 dated 11 may 2012. results among the 2955 female respondents who underwent cbe in the rfncd cohort study were found 152 cbe(+) cases (5.14%). the marital status of the majority of the respondents was “married/divorced/widowed” and only 10 (3.3%) of the 304 persons were single. among 231 contraceptive users, 184 persons (79.7%) were current users or had ever used hormonal family planning, comprising 165 persons (89.7%) with married marital status and 19 persons (10.3%) with divorced marital status. table 1 shows that more than half of the respondents, in both the cbe(+) and cbe(-) groups, were more than 40 years old, half of the cbe(+) group being of low educational level, with bmi of <25 and total cholesterol concentration (tcc) of <200 mg/dl. a large proportion of the cbe(+) group comprised respondent s with triglycer ide concentration of <150 mg/dl, ldl 100 mg/dl, and hdl 50 mg/dl, who were not yet in menopause. while in the cbe(-) group, more than table 1. characteristics of respondents based on positive and negative cbe *tumor diagnosis based on cbe : clinical breast examination darjoko, sapardin hormonal contraception and breast tumor characteristic cbe p value positive negative n % n % age (years) 0.340 <40 59 38.8 51 33.6 40 93 61.2 101 66.4 education 0.492 high 11 7.2 11 7.2 middle 78 51.3 68 44.7 low 63 41.4 73 48.0 marital status 0.882 married 124 81.6 125 82.2 single/divorced 28 18.4 27 17.8 bmi (kg/m2) 0.038 <25 79 52.0 61 40.1 25 73 48.0 91 59.9 total cholesterol (mg/dl) 0.021 200 71 46.7 91 59.9 ? 200 81 53.3 61 40.1 triglycerides (mg/dl) 0.156 150 14 9.2 22 14.5 <150 138 90.8 130 85.5 ldl (mg/dl) 0.216 100 123 80.9 131 86.2 <100 29 19.1 21 13.8 hdl (mg/dl) 0.720 <50 53 34.9 56 36.8 50 99 65.1 96 63.2 menopause 0.180 yes 45 29.6 56 36.8 no 107 70.4 96 63.2 143 univ med vol. 36 no.2 * tumor diagnosis based on cbe: clinical breast examination table 2. risk factors for breast tumors based on positive and negative cbe half were respondents with low educational level, with bmi 25 and tcc 200 mg/dl. a large proportion in the cbe(-) group were respondents with triglyceride concentration of <150 mg/dl, ldl 100 mg/dl, hdl 50 mg/dl and who were not yet in menopause. table 2 shows that both in cbe(+) and cbe(-) gr oups, a larger proportion were nonsmoking, had no stress, had 2 children, with a duration of breastfeeding of 6 months, without family history of breast cancer, with consumption of fried foods, milk, coconut milk and packaged beverages of <3 times per week. in the cbe(+) group, it was apparent that the percentage of hc users was double that of the non-users, while in the cbe(-) group, there wer e almost no differences between hc users and non-users. from the results of multivariate analysis (table 3), the odds ratio of hc users was 1.8fold greater (aor=1.83; 95% ci: 1.11-3.04) than those of non-users, after controlling for tcc, smoking status, and parity. the odds ratio in the cbe(+) group in respondents with tcc <200 mg/ dl was 1.6-fold greater (aor=1.62; 95% ci: 1.01-2.60) those that of the respondents with tcc 200 mg/dl. the or of current smoking was 1.01 (95% ci: 0.52-1.96) and those of former smoking 0.49 (aor=0.49; 95% ci: 0.26-1.93) , as cbe risk factor positive negative p value n % n % smoking status 0.188 nonsmoking 109 71.7 99 65.1 former smoking 20 13.2 32 21.1 current smoking 23 15.1 21 13.8 stress 0.803 no 107 70.4 105 69.1 yes 45 29.6 47 30.9 parity (no. of children) 0.728 2 children 111 73.0 117 77.0 1 child 29 19.1 25 16.4 without or as yet without a child 12 7.9 10 6.6 duration of breastfeeding 0.428 6 months 126 82.9 131 86.2 < 6 months/never 26 17.1 21 13.8 hormonal contraception 0.035 no 51 33.6 69 45.4 yes 101 66.4 83 54.6 family history of breast cancer 1.000 absent 146 96.1 146 96.1 present 6 3.9 6 3.9 consumption of fried foods 0.398 <3 times/week 103 67.8 96 63.2 3 times/week 49 32.2 56 36.8 milk consumption 0.435 <3 times/week 109 71.7 115 75.7 3 times/week 43 28.3 37 24.3 coconut milk intake 0.523 <3 times/week 112 73.7 107 70.4 3 times/week 40 26.3 45 29.6 consumption of packaged beverages 0.368 <3 times/week 122 80.3 128 84.2 3 times/week 30 19.7 24 15.8 144 table 3. multiple logistic regression analysis of risk factors for braest tumors in adult women * adjusted odds ratio controlled for variables in this table; ** tumor diagnosis based on cbe compared with res pondents who were nonsmoking, but this difference was statistically not significant. similarly with parity, although the adjusted odds ratio (aor) of cbe(+) respondents was 1.21 (95% ci: 0.65-2.25) for those who have only one child and 1.77 (95% ci: 0.69-4.53), for those who have no children, was higher than in the respondents with 2 children, this result was also not statistically significant. in this connection, smoking status and parity are confounding variables, that if excluded from the multivariate analysis causes changes in or of hc (as the principal independent variable) >10%. discussion the respondents who used hc had a 1.8fold greater risk for developing breast tumors as compared with those who were non-users. this is according to the study results of urban et al.(9) who showed an increase in the or of breast cancer in users of contraceptive pills and/or injections. poosari et al.(15) found an increased risk of breast cancer of 1.31 times in hc users, which was however statistically non-significant (95% ci: 0.65-2.65). in general, with regard to hc composition, the pills contain estrogen and progesterone, whereas the mini pills, injections and implants contain progesterone.(5,6) artoum et al.(27) state that estrogen contribute to the development of tumors by promoting cell proliferation and mutation or by increasing the probability of mutations that regulate growth and differentiation of mammary cells that may play an important role in the growth of breast cancers. according to sirait et al.,(11) the growth of mammary tissues is very sensitive to estrogen, therefore females with long-term exposure to estrogen will carry a high risk for the occurrence of breast cancer. breast cancer is characterized by the loss of estrogen receptors (ers) that is associated with aggressive pathology and a low level of estimated r ecovery (prognosis).(28) before the publication of the study results of women’s health initiative (whi) in the us in 2002, many experts were of the opinion that the increased risk of breast cancer observed in hrt research was due to the effect of estrogen. after the whi results had found, the focus changed to progesterone, which was considered to increase cell division and accumulation of damaged dna. the highest proliferative activity occurs in the luteal phase of the menstrual cycle (when the endogenous progesterone concentration is high).(29) daniel et al.(8) state that progestin added to hrt significantly increases the incidence of breast tumors and the breast tumor stage in females who are in menopause. therefore progesterone is no longer considered a completely darjoko, sapardin hormonal contraception and breast tumor risk factor crude or 95% ci p adj or 95% ci p value hormonal contraception no 1.00 reference 1.00 reference yes 1.65 1.04 – 2.62 0.035 1.83 1.11 – 3.04 0.019 total cholesterol 200 mg/dl 1.00 reference 1.00 reference <200 mg/dl 1.70 1.08 –2.68 0.022 1.62 1.01 – 2.60 0.044 smoking status non-smoking 1.00 reference 1.00 reference former smoking 0.57 0.31 – 1.06 0.074 0.49 0.26 – 1.93 0.028 current smoking 0.99 0.52 – 1.91 0.987 1.01 0.52 – 1.96 0.987 parity (no. of children) 2 children 1.00 reference 1.00 reference 1 child 1.22 0.68 – 2.22 0.507 1.21 0.65 – 2.25 0.555 none 1.27 0.53 – 3.05 0.600 1.77 0.69 – 4.53 0.234 145 univ med vol. 36 no.2 safe alternative. according to lanari,(30) more than 70% of breast cancers express the estrogen receptor alpha (erα) and respond to antiestrogen therapy. these cancers also express progesterone receptors that are reliable markers for estrogen receptors. the results of research by llanos et al.,(31) shah et al.,(32) melvin et al.(33) and ni et al.(34) showed that higher tcc has a less risk for breast tumors/cancers than lower tcc, therefore in the present further analysis tcc of 200 mg/dl (high) is positioned as reference for determining the odds ratio of tcc of <200 mg/dl (low). the multivariate results show that tcc <200 mg/dl actually increases the risk of breast tumors by 1.6-fold as compared with tcc of 200 mg/dl. this is in line with the study results of llanos et al.(31) who state that there is an inverse relation between the risk of breast cancer and tcc (or=0.46; 95% ci: 0.25-0.85), in other words, tcc in the cases was significantly lower (189.3 mg/dl) as compared with the controls (206.8 mg/ dl). similarly, the study results of shah et al.(32) found that higher tcc was significantly associated with a decreased risk of breast cancer (or=0.30; 95% ci: 0.12-0.76). while melvin et al.(33) and ni et al.(34) found that higher tcc has a lower risk for breast cancer, although results were not statistically significant, i.e. hr=0.97 (95% ci: 0.89-1.05) and rr=0.96 (95% ci: 0.86-1.07), respectively. these results differed with those of a study by kitahara et al.(35) who state that higher tcc (240 mg/dl) is positively associated with breast cancer (hr=1.17; 95% ci: 1.03-1.33). there are also the results of the study by hu et al.(36) who found that higher tcc increases the risk of breast cancer (or=1.45; 95% ci: 1.141.85). likewise, the study by peela et al.(37) found significantly increased tcc in patients with breast cancer. llaverias et al.(38) state that in general the role of cholesterol i n the initiation and development of tumors is very controversial. low cholesterol concentrations are known to be used as a cancer marker, and several types of cancer appear to decrease plasma cholesterol level. this is the result of increased utilization of cholesterol by the tumors for their development. thus the increased plasma cholesterol level accelerates the development and increases the aggressivity of the tumors. from the results of the study by mcdonell et al.(39) it is known that cholesterol does not play a direct role in tumor pathogenesis, but cholesterol or its derivatives function as marker molecule in cancer cells. cholesterol is the raw material for the biosynthesis of steroid hormones, one of them being estradiol (estrogen).(40,41) according to llaverias et al.(38) an important aspect that has to be considered when testing the correlation between plasma cholesterol and breast cancer is that the estrogen concentration is also associated with plasma hdl cholesterol. in the present analysis it was found that the relationship between smoking status and the risk of breast tumors was statistically not significant (aor=0.49) for former smoking and aor=1.01 for curr ent smoking, as compared with nonsmoking. comparatively identical results were found by xue et al.(18) in their research the hr=1.06 (95% ci: 1.01-1.11) for former smoking and hr=1.09 (95% ci: 1.02-1.17) for current smoking, after controlling for age at menopause, menopause status and use of hormones postmenopause as compared with nonsmoking. pasarelli et al.(42) even differentiate the risk of smoking between 1 year before and 1 year after diagnosis of breast cancer. individuals who for 1 year before diagnosis of breast cancer were active smoking, had a 1.3-fold higher risk of dying from breast cancer (hr=1.25; 95% ci: 1.13-1.37) as compared with nonsmoking. while 10% of females who continued to smoke after diagnosis, had a 1.7-fold higher risk of dying from breast cancer (hr=1.72; 95% ci: 1.13-2.60), as compared with nonsmoking. according to bjerkaas et al.,(43) several large prospective cohort studies have found that smoking can cause breast cancer, especially in females who smoke for an extended period of time, those who smoke a great number of cigarettes per day and those who smoke before delivering their first child. their research results showed that the mortality from breast cancer may indeed be low, but increases 146 significantly for active smoking (current) and former smoking (hr=1.15; 95% ci: 1.01-1.32 and hr=1.15; 95% ci: 1.02-1.30, respectively), as compared with nonsmoking. based on the study by bishop et al.(44) it is known that the relative mortality risk from breast cancer is 1.44 for current smoking (95% ci: 1.01-2.06) and 1.13 for former smoking (95% ci: 0.66-1.94), as compared with nonsmoking. in the present study it was found that respondents with 1 child had an aor=1.21 and that respondents who have no children, had an aor=1.77, which is greater than in respondents who have 2 children. however, these results were statistically not significant, sirait et al.(11) found that persons who have no children have a 1.97fold greater risk (95% ci: 1.24-3.14) and that those who have 1 child have a 1.64-fold greater risk (95% ci: 1.16-2.33) that is statiscally significant as compared with persons who have 4 children. with reference to the statement of shen et al.,(45) in that increased parity (the number of deliveries) is a protective factor against breast cancer, because parity decreases estrogen/ progesterone receptor-positive breast cancers and breastfeeding (lactation) decreases the risk of receptor positive or negative breast cancers or both. heys et al.(46) state that estrogen increases during pregnancy then decreases post-delivery until around 1 year. futhermore, breastfeeding temporarily decreases estrogen post-delivery. the concentration of estrogen during the ovulatory cycle is lower after the first pregnancy when compared with females who have never borne any children. this differs from the results of the study by sun et al.(20) who showed that subjects who had 1-2 children had a 1.32-fold greater risk of suffering from breast cancer (95% ci: 0.89-1.95), while subjects who had 3 children had a 1.77fold greater risk of suffering from breast cancer (95% ci: 1.18-2.66) as compared with subjects who had never borne any children. thus it can be explained that the exogenous steroid hormones (estrogen/progesterone) contained in hc promote tumor cell proliferation and accumulate dna damage.(29) low cholesterol concentrations are known to be able to become cancer markers. this is caused by the increased utilization of cholesterol by the tumors for their development. the increased concentrations of plasma cholesterol, which is the raw material for the biosynthesis of steroid hormones, accelerates the development and increases the aggressivity of tumors.(38) in addition, the substances contained in cigarettes, such as nitrosamine and nicotine, can also be carcinogenic. nitrosamine induces cancers by causing gene and/or dna mutations, while nicotine promotes cancer cell development.(47) meanwhile, parity (the total number of children) is a measure of a life-long exposure to endogenous steroid hormones (from pregnancy and delivery up to the lactation period). exposure to high concentrations of exogenous or endogenous steroid hormones is associated with the risk of breast cancer.(29) the limitations of the present study are as follows. first, there are no data on the total number of cigarettes smoked per week by the occasional smoking; second, there is no separate question for respondents who use one type of hc and for those who use more than one type of hc (combined hc) and for duration of use; third, there is no question about whether or not hc was used continuously; and fourth for respondents who had ever used hc, there is no question about the duration of cessation of hc use, because according to the study results of cibula et al.,(48) the effect of hc (pills) on the risk of breast cancer will disappear after cessation of the use of the contraceptive pills f or 5-10 yea rs. it is recommended that future studies, especially about questions on the type and duration of hc use may be more focused, so that the results will be increasingly improved. conclusion hormonal contraceptive (hc) users had a 1.8-fold increased risk of breast tumors after controlling for tcc (200 mg/dl=reference), smoking status and parity (2 chil dren= reference). darjoko, sapardin hormonal contraception and breast tumor 147 univ med vol. 36 no.2 conflict of interest the author declare that there is no conflict interest in this article. acknowledgement the authors wish to extend their utmost gratitude to dr. iwan ariawan (fkm-ui), dr. ekowati rahajeng, skm, m.kes and dr. delima, m.kes (badan penelitian dan pengembangan kesehatan) who provided guidance and direction, from the data analysis up to the preparation of the manuscript. contributors st contributed to the literature search, data analysis and writing of the draft. ans contributed to “data cleaning” b efore data analysis (particularly that associated with cbe) and literature search. all authors read and approved the final manuscript. references 1. jemal a, bray f, center mm, et al. global cancer statistics. ca cancer j clin 2011;61:69-90. doi: 10.3322/caac.20107. 2. international agency for research on cancer world 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36 no.2 46. heys m, jiang c, cheng kk, et al. life long endogenous estrogen exposure and later adulthood cognitive function in a population of naturally postmenopausal women from southern china: the guangzhou biobank cohort study. psychoneuroendocrinology 2011;36:864-73. doi: 10.1016/j.psyneuen.2010.11.009. 47. xue j, yang s, seng s. mechanisms of cancer induction by tobacco-specific nnk and nnn. cancers 2014;6:1138-56. doi: 10.3390/cancers 6021138. 48. cibula d, gompel a, mueck ao, et al. hormonal contraception and risk of cancer. hum reprod update 2010;16:631-51. rita k 160 *brain and spine centre, rumah sakit pantai indah kapuk, jakarta **departemen kardiologi, rumah sakit pantai indah kapuk, jakarta koresponden adr. sendjaja muljadi brain and spine centre, r.s. pantai indah kapuk, jakarta email : nsm@cbn.net.id universa medicina 2007; 26: 160-7. universa medicina oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 studi kasus: stroke iskemik dengan infark luas pada pasien laki-laki muda dengan stenosis mitral berat sendjaja muljadi*a, alfred sutrisno*, dan linda lison** abstrak latar belakang meskipun terdapat kecenderungan peningkatan insidens stroke pada usia dewasa muda, namun tetap lebih jarang dijumpai dibandingkan dengan usia pertengahan dan usia yang lebih tua. penyebab stroke iskemik pada pasien-pasien usia muda lebih heterogen dibandingkan dengan pasien-pasien yang lebih tua. deskripsi kasus kami melaporkan sebuah kasus, seorang laki-laki berusia 39 tahun yang mengalami kejang-kejang selama kurang lebih 30 menit dan terjadi kira-kira 1 jam sebelum pasien tersebut dibawa ke rumah sakit. dan pada saat itu pasien dalam keadaan somnolen, hasil pemeriksaan dengan pencitraan tomografi komputer kepala (brain ct scan) saat itu tidak menunjukkan adanya infark. akan tetapi 12 jam kemudian, dilakukan pemeriksaan pencitraan tomografi komputer kepala yang kedua, ternyata menunjukkan adanya infark yang luas di hemisfer kanan. pada pemeriksaan ekokardiografi menunjukkan adanya pelebaran atrium kiri, stenosis mitral pada katup-katup mitral, dengan luas area 0,94 cm2 dan skor mitral 14. elektro kardiografi (ekg) menunjukkan sinus ritmik. kesimpulan pasien tersebut di atas memiliki masalah besar dengan jantungnya dan menyebabkan terjadinya oklusi pada pembuluh darah di otak (pemeriksaan dengan pencitraan tomografi komputer kepala yang kedua menunjukkan adanya infark yang luas). pasien tersebut akhirnya meninggal setelah menjalani perawatan selama 6 hari di rumah sakit. prognosis seorang berusia muda dengan kelainan mitral bila menderita stroke iskemik tidak begitu baik. kata kunci : usia muda, infark luas, stenosis mitral, emboli 161 universa medicina vol. 26 no.4 *brain and spine centre, pantai indah kapuk hospital, jakarta **department of cardiology, pantai indah kapuk hospital, jakarta korespondensi adr. sandjaja muljadi brain and spine centre, pantai indah kapuk hospital, jakarta email: nsm@cbn.net.id universa medicina 2007; 26: 160-7 background although there is a steady rise in the incidence of stroke in young adults with increasing age, stroke are encountered less frequently than in the middle aged and elderly. the cause of ischemic stroke is more heterogenous among young adults than among older patients. case description we report a case of 39 years old man, who suffered from seizures about 30 minutes, one hour before admitted to the hospital and at that time he was somnolent, the brain ct scan didn’t show any infarction. but 12 hours later, the second brain ct scan showed the extensive infarction on the right hemi-cerebral. also the echocardiography showed a large left atrium, mitral stenosis in mitral valves, with area use 0.94 cm2 and mitral score 14. electro cardiogram (ekg) showed sinus rhythm. conclusion heart disease is a potent cause of stroke in all ages. this patient had a big problem with his heart and caused the occlusion (the second brain ct scan showed the extensive infarction). this patient passed away after 6 days hospitalization. the prognosis of ischaemic stroke in young adult with mitral stenosis not so good. keywords : young adult, extensive infarction, mitral stenosis, embolism case study : ischaemic stroke with diffuse infarct in young male patient with mitral stenosis sendjaja muljadi*a, alfred sutrisno*, and linda lison** abstract pendahuluan stroke dapat menyebabkan kelumpuhan atau bahkan mematikan dan dalam hitungan detik dapat mengubah nasib dan jalan hidup seseorang. dewasa ini stroke cenderung menjadi masalah kesehatan dikalangan masyarakat indonesia, terutama di kota-kota besar.(1) stroke merupakan istilah klinik penyakit peredaran darah otak ppdo) atau cerebrovascular disease (cvd) yang ditandai dengan timbulnya kelainan fungsi otak secara mendadak, menetap serta mempunyai kecenderungan memburuk, bahkan kematian dalam kurun waktu 24 jam pertama. penyebab kematian dalam minggu pertama disebabkan oleh pembengkakan jaringan otak akibat stroke yang luas di supratentorial atau di batang otak. stroke merupakan suatu manifestasi akhir kelainan-kelainan patologik pada pembuluh darah yang prosesnya secara bertahap dimulai j a u h s e b e l u m t e r j a d i n y a s e r a n g a n s t r o k e . pembuluh darah diotak mengalami degenerasi secara bertahap dari berbagai penyakit iskemik yang disebut “faktor risiko”.(2) i n s i d e n s i p e n y a k i t s e r e b r o v a s k u l a r meningkat menjadi 34/100.000 per tahun pada 162 muljadi, sutrisno, lison stroke iskemik dengan infark luas orang-orang yang berusia kurang dari 55 tahun. meskipun lebih dari 70% didapatkan perbaikan, emboli serebral dan perdarahan intrakranial akan membawa prognosis yang buruk pada pasien-pasien usia muda.(3) sekitar 4% dari stroke iskemik masih belum jelas etiologinya dan disebut stroke cryptogenic.(4) penyebab stroke iskemik lebih heterogen p a d a p a s i e n p a s i e n y a n g b e r u s i a m u d a d i b a n d i n g k a n d e n g a n p a s i e n p a s i e n y a n g b e r u s i a l e b i h t u a . ( 5 ) g a n g g u a n j a n t u n g m e r u p a k a n p e n y e b a b s t r o k e y a n g s a n g a t potensial pada segala usia terutama bila terjadi emboli yang berasal dari material-material yang bersumber dari jantung yang menuju ke otak maupun organ-organ lainnya. kurang lebih 20% stroke iskemik dan serangan otak sepintas (transient ischemic attack) disebabkan oleh emboli yang berasal dari jantung. substansi emboli yang paling mengancam adalah yang berasal dari fibrilasi atrial baik yang nonrematik maupun yang rematik, endokarditis infektif, katup jantung prothese, infark miokard yang baru, kardiomiopati dengan penebalan, t u m o r i n t r a k a r d i a l d a n s t e n o s i s m i t r a l rematik.(4-6) diskripsi kasus seorang laki-laki, 39 tahun dibawa ke rumah dengan keluhan demam dan flu, sedikit merasakan nyeri kepala, pusing seperti berputar dirasakan satu hari sebelum dibawa ke rumah sakit dan pasien tetap sadar pada saat itu. satu jam sebelum dibawa ke rumah sakit, pasien mengalami kejang-kejang selama kurang lebih 3 0 m e n i t d a n s e t e l a h i t u i a t i d a k d a p a t berkomunikasi dan tak sadarkan diri. pasien ini a d a l a h p e r o k o k b e r a t , d a n t i d a k p e r n a h menyadari bahwa ia mempunyai gangguan jantung, dan riwayat penyakit demam rematik semasa remaja. pada pemeriksaan fisik di ruangan gawat darurat yang dilakukan pada t a n g g a l 1 8 n o p e m b e r 2 0 0 4 , p u k u l 2 0 . 3 0 didapatkan kesadaran pasien somnolen, tekanan darah 90/40 mmhg, nadi 108x/menit, suhu 37,30 c, pernapasan 20x/menit, pupil bulat, isokor, refleks cahaya +/+, kesan gerakan kedua sisi anggota gerak sama aktif, refleks fisiologis (biceps/triceps/kpr/apr) +/+, refleks patologis -/-. pemeriksaan dengan pencitraan tomografi k o m p u t e r k e p a l a ( b r a i n c t s c a n ) t i d a k menunjukkan adanya infark (gambar 1), foto thoraks memperlihatkan rasio kardio-toraks lebih dari 60% dan kontur ganda (d o u b l e contour), serta pneumonia (gambar 2). pada pasien ini diberikan cairan infus (saline) 500 cc/8 jam, lalu dipasang selang nasogastric (nasogastric tube), diberikan fenitoin 100 mg 3x sehari, dan pasien dirawat diruang icu. pada pukul 24.00, kesadaran pasien menjadi soporous dan terjadi udem paru akut yang timbul akibat stenosis mitral berat yang ada. pemeriksaan ekokardiografi menunjukkan pelebaran atrium kiri, dan stenosis mitral pada katup-katup mitral. area 0,94 cm2, dan skor m i t r a l s e b e s a r 1 4 ( g a m b a r 3 d a n 4 ) . elektrokardiogram menunjukkan sinus ritmik. udem paru saat itu dapat dikontrol dengan ventilator, diuretik, morfin dan kepada pasien diberikan antibiotika untuk infeksi sekunder. meskipun demikian gejala-gejala neurologis semakin memburuk. pada pemeriksaan fisik yang dilakukan pada keesokan harinya (19 nopember 2004), pukul 09.00 didapatkan: keadaan umum tampak sakit berat, ventilator terpasang, kesadaran soporokomatous, pupil anisokor (kanan>kiri), dan refleks cahaya +“↓/ + “↓ ( m e n u r u n ) . p e m e r i k s a a n m o t o r i k memberikan kesan adanya hemiplegi sinistra, refleks fisiologis menurun pada kedua sisi dan refleks patologis -/(negatif/negatif). pemeriksaan dengan pencitraan tomografi komputer kepala ulang yang kedua menunjukkan adanya infark yang luas di sisi hemisfer kanan (gambar 5). 163 universa medicina vol. 26 no.4 gambar 1. hasil pencitraan tomografi komputer kepala pertama pada saat pasien pertama tiba di rumah sakit, tidak menunjukkan adanya tanda-tanda infark gambar 2. foto thoraks ap, menunjukkan rasio kardio-thoraks lebih dari 60%, pneumonia serta tanda-tanda bendungan paru 164 gambar 3. pemeriksaan ekokardiografi, pada m-mode setinggi katup mitral dengan pandangan aksis memanjang, terlihat penurunan ef mendatar dengan kalsifikasi daun katup gambar 4. pemeriksaan doppler ekokardiografi dengan area katup mitral 0.94 cm2 muljadi, sutrisno, lison stroke iskemik dengan infark luas 165 universa medicina vol. 26 no.4 gambar 5. hasil pencitraan tomografi komputer kepala yang kedua yang dilakukan 12 jam setelah pasien berada di rs, menunjukkan gambaran infark yang sangat luas disisi kanan pasien mendapatkan pengobatan : ♣ manitol 250 cc/30 menit — 3x sehari ♣ metil prednisolon injeksi 125 mg (iv), 4x sehari ♣ ranitidine injeksi 1 ampul (iv), 2x sehari ♣ phenitoin 100 mg (per oral), 3x sehari ♣ pentoxyphiline 600 mg/24 jam (iv) h a s i l p e m e r i k s a a n l a b o r a t o r i u m menunjukkan hasil normal, (meliputi: eritrosit/ lekosit/hb/ht/trombosit/kolesterol total/hdl/ ldl/trigliserida, juga diperiksa fibrinogen/aca igg dan igm dan kadar-kadar mineral: natrium/ kalium/klorida/kalsium. juga pemeriksaan analisa gas darah/ureum/kreatinin/protein total/ albumin/sgot/sgpt/∂ gt). pada siang hingga sore harinya, pukul 15.00 – 17.00, keadaan pasien semakin memburuk terus, dan pada hari berikutnya kesadaran pasien menjadi koma dalam, pupil mulai midriasis, dan refleks cahaya -/(negatif/negatif). ventilator tetap dalam keadaan terpasang. pergerakan motorik tidak dapat dibangkitkan, bahkan dengan rangsang nyeri sekalipun dan pada akhirnya pasien meninggal setelah menjalani perawatan selama 6 hari di rumah sakit. diskusi pasien adalah seorang laki-laki muda yang berusia 39 tahun, mengalami kejang-kejang kurang lebih 1 jam sebelum dibawa ke rumah sakit. pada saat itu kesadarannya pada tingkat somnolen. pencitraan tomografi komputer kepala pada saat itu tidak menunjukkan adanya 166 infark (gambar 1). foto toraks menunjukkan rasio kardio-toraks yang lebih dari 60% dan b e r k o n t u r g a n d a ( d o u b l e c o n t o u r ) s e r t a pneumonia (gambar 2). namun kira-kira 3 jam kemudian, tingkat kesadaran pasien menurun menjadi soporous. pemeriksaan ekokardiografi menunjukkan adanya pelebaran atrium kiri, stenosis mitral pada katup-katup mitral. area 0,94 cm2, skor mitral 14 (gambar 3 dan 4). pemeriksaan dengan pencitraan tomografi komputer kepala yang kedua (dikerjakan kurang lebih 12 jam kemudian dari pemeriksaan yang pertama) menunjukkan adanya infark luas di sisi h e m i s f e r k a n a n ( g a m b a r 5 ) . p e m e r i k s a a n pencitraan tomografi komputer kepala yang kedua ini menunjukkan adanya infark didaerah sirkulasi anterior total (biasanya disebabkan oleh oklusi dari cabang utama atau cabang dari arteri cerebri media), umumnya disebabkan oleh emboli yang berasal dari jantung.(5,6) stenosis p a d a a r t e r i s e r e b r i m e d i a m e r u p a k a n l e s i vaskuler yang paling sering dijumpai pada orang-orang asia yang menderita stroke. (7) pasien ini mempunyai masalah yang serius dengan jantungnya (stenosis mitral berat), oleh karena itu emboli dapat ditimbulkan dari jantung dan menyebabkan oklusi (pada cabang utama atau cabang dari arteri serebri media). adanya kejang-kejang pada masa dini, mengindikasikan buruknya prognosis.(3) penyebab terjadinya iskemik stroke pada pasien-pasien usia muda yang dirujuk ke rumah sakit (oleh karena emboli yang berasal dari jantung) ditunjukkan secara konsisten pada penelitian di italia yang meneliti 55 subjek berusia 17-45 tahun.(7) walaupun insidens stroke pada usia muda <35 tahun sangat kecil berkisar 1 0 , 1 / 1 0 0 . 0 0 0 , n a m u n a p a b i l a p e n d e r i t a mengalami kelainan jantung, serangan stroke dapat menyebabkan terjadinya kematian. (9) haruslah diakui bahwa banyak orang berusia muda tidak menyadari bahaya terjadinya stroke yang mungkin terjadi apabila ada keluarganya yang menderita kelainan jantung. faktor risiko tambahan pada pasien ini, yaitu ia adalah s e o r a n g p e r o k o k b e r a t . wo r l d h e a l t h organization juga menyatakan bahwa merokok merupakan faktor risiko terjadinya stroke.(10) sebuah penelitian pada kelompok berusia 18 sampai 30 tahun menunjukkan bahwa kelompok b e r u s i a m u d a t i d a k m e l a k u k a n p e r u b a h a n perilaku walaupun mereka mengetahui bahaya terjadinya stroke. mereka tetap merokok, tidak mengobati hipertensinya dan mempunyai berat badan berlebihan.(11) dari hasil penelitian naess dan kawan-kawan menunjukkan banyaknya faktor risiko melukiskan semakin beratnya proses patologi yang mendasari timbulnya stroke iskemik pada usia muda.(5) k a s u s i n i m e n u n j u k k a n k e l e m a h a n pemeriksaan dengan pencitraan tomografi komputer kepala, yaitu kegagalannya untuk m e l i h a t a d a n y a i n f a r k y a n g l u a s p a d a pemeriksaan yang pertama dan juga gejala yang dialami pasien pada saat itu tidak memberikan kesan adanya hemiparesis ataupun gejala lain selain kesadaran yang menurun (somnolen). p a s i e n i n i d i r u j u k k e b e d a h s a r a f ( u n t u k kemungkinan tindakan dekompresi), namun tidak a d a t i n d a k a n y a n g d i a m b i l , o l e h k a r e n a kesadaran pasien yang menurun sangat cepat (menjadi koma dalam). kami telah mencoba untuk mengurangi udema perifokal dengan manitol dan juga steroid (metilprednisolon), akan tetapi tidak menolong. setelah 6 hari masa perawatan di rumah sakit, akhirnya pasien tersebut meninggal dunia. kesimpulan kelainan jantung khususnya stenosis mitral yang berat sudah dihipotesakan merupakan faktor risiko untuk terjadinya emboli pada penderita usia muda. studi kasus menunjukkan tingginya prevalensi penderita stroke iskemik akibat kelainan katup mitral pada usia muda. muljadi, sutrisno, lison stroke iskemik dengan infark luas 167 universa medicina vol. 26 no.4 prognosis seorang berusia muda dengan kelainan mitral bila menderita stroke iskemik tidak begitu baik. daftar pustaka 1. ranakusuma tas. peran masyarakat pada penanggulangan stroke dalam upaya peningkatan dan pelestarian sumber daya manusia di indonesia. diajukan pada dies natalis ui ke xliv tingkat fkui jakarta, pebruari 1993. 2. misbach j, suryamiharja a, malik ms, sandia in. laporan penelitian survey stroke di jakarta suatu awal penelitian masalah stroke di indonesia menjelang tahun 2000. pusat penelitian penyakit tidak menular, badan penelitian dan pengembangan kesehatan, departemen kesehatan ri. 1995. 3. gilroy j. strokes in children and young adults. in: basic neurology. 3rd ed. usa. the mcgraw-hill companies, inc, 2000, 307-9. 4. da silva ceap, vale tc, leite fjb, casali tg, ramalho gdv, cruizeiro mm. ischemic stroke and patient foramen ovale: case report. arq neuropsiquiatr 2006; 64: 858-61. 5. naess h, andreassen w, thomassen l, nyland h, myhr km. do all young ischemic stroke patients need long term secondary preventive medication. neurology 2005; 65: 609-11. 6. warlow cp, dennis ms, gijn jv, hankey gj, sandercock pag, bamford jm, et al.. stroke: a practical guide to management. 1st ed. great britain: bath press; 1996. 7. wong ks, ng pw, tang a, liu r, yeung v, tomlinson b. prevalence of asymptomatic intracranial atherosclerosis in high-risk patients. neurology 2007; 68: 20358. 8. musolina m, la spina p, granata a, gallito g, leggiadro n, carerj s, et al. ischaemic stroke in young people: a prospective and long-term followup study. cerebrovasc dis 2003; 15: 121-8. 9. marini c, totaro r, de santis f, ciancarelli i, baldassarre m, carolei a. stroke in young adults in the community-based l’aquila registry. stroke 2001; 32: 52-6. 10. world health organization. the who stepwise approach to stroke surveillance geneva: world health organization; 2006. 11. larkin m. young people ignore warning of increased heart disease and stroke risk. the lancet 2002; 359: 1584. c:\users\jurnal fkusakti\docume 187 *department of psychiatry, faculty of medicine, atma jaya catholic university of indonesia **medical profession program, faculty of medicine, atma jaya catholic university of indonesia correspondence: dr. dr. surilena hasan, sp.kj(k) department of psychiatry, faculty of medicine, atma jaya catholic university of indonesia jl. pluit raya no. 2, north jakarta indonesia phone: +6221-6693168 fax: +6221-6606123 email: surilenahasan@yahoo.co.id or surilena@atmajaya.ac.id date of first submission, june 2, 2017 date of final revised submission, november 10, 2017 date of acceptance, november 13, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 high perceived discrimination and no family support increase risk of poor quality of life in gender dysphoria surilena hasan*, yovita alviany**, cerellia clarissa**, and sonia sudana kusuma** background family support and low discrimination perceptions are a factor in protecting against mental disorders and poor quality of life in male to female (mtf) gender dysphoric individuals. the aim of this study was to determine the role of family factors, peer support, self-esteem, perceived discrimination, depression, anxiety, and stress on the quality of life among mtf gender dysphoric individuals. methods a cross-sectional study was conducted involving 106 mtf gender dysphoric individuals of sanggar swara jakarta. respondents filled out questionnaires on demographics, whoql-bref, perceived discrimination, rosenberg self-esteem, family support, social support (ssq6), family relations (faces iii), and depression anxiety stress scale (dass). simple and multivariate logistic regression tests were used to analyze the data. results among the 106 mtf gender dysphoric individuals of sanggar swara jakarta with an age range of 18-45 years, 78.3% had no family support, 64.1% no peer support, 62.3% high perception discrimination, 64.1% low self-esteem, 36% extreme family relations, 44.3% depression, 59.4% anxiety, 35.8% stress and 62.3% poor quality of life. employment, perception of discrimination, self-esteem, family support, and anxiety were significantly associated with quality of life (p<0.05). multiple logistic regression analysis showed that perceived discrimination (odds ratio=13.89; 95% ci: 5.89-11.99), and family support (odds ratio=29.11; 95% ci: 2.45-8.21) were significantly associated with quality of life. conclusion high perceived discrimination and no family support increase the risk of poor quality of life in mtf gender dysphoric individuals. these findings suggest the need for prevention and intervention of stigmatization and discrimination that should have a special focus on families with mtf gender dysphoric individuals. keywords: quality of life, family support, perceived discrimination, gender dysphoria doi: http://dx.doi.org/10.18051/univmed.2017.v36.187-196 original article cite this article as: hasan s, alviany y, clarissa c, et al. high perceived discrimination and no family support increase risk of poor quality of life in gender dysphoria. univ med 2017;36: 187-96. doi : 10.18051 /univmed.2 017 . v36 .1 87 -19 6 pissn: 1907-3062 / eissn: 2407-2230 188 hasan, alviany, clarissa, et al perceived discrimination in gender dysphoria introduction lesbian, gay, bisexual, and transgender (lgbt) has been the hot topic of conversation in indonesian society for decades. the lgbt community has long existed in indonesia but only in recent decades has it become phenomenal. the lgbt community began to present itself in various mass media and some cases of samesex marriage emerged openly, along with the formation of communities with a clearly visible structure.(1) a transgender or gender dysphoria individual is an individual who expresses his gender identity as being opposed to his biological gender. there are 2 types of gender dysphoria communities, namely male to female (mtf) and female to male (ftm). the mtf gender dysphoria community is considerably more numerous than the ftm gender dysphoria community, with a 1:1 to 6.1:1 ratio. the number of gender dysphoric individuals continues to increase worldwide. it is estimated that by 2030, the number of gender dysphoric individuals will increase by two-fold.(2) survey in the united states by bockting, et al.(3) in 1093 gender dysphoria mtf (57.5%) and ftm gender dysphoria (42.5%) showed that there were 464 (56.5%) gender dysphoria mtf with high economic status. gender dysphoria mtf depression is more likely to be discriminated (verbal 37%, physical 20%, work 39%) compared to non-depressed mtf gender dysphoria. data from the kebaya ngo waria yogyakarta extended family showed that the number of mtf gender dysphoric individuals is 234 people spread over several areas in yogyakarta.(4) gender dysphoric individuals experience more psychosocial problems such as stigma, discrimination, social support and low family support compared to lesbian, gay, bisexual (lgb) individuals. the majority of indonesians (93%) still reject gender dysphoria and only 3% accept it. discrimination and shame fearing family reactions are two main reasons for gender d ysph o ri c pe o ple n ot t o r e p or t ca s e s o f discrimination they experience to relevant agencies, to be unwilling to document their case, and to be reluctant to seek mental health services. gender dysphoric individuals may experience dropping out of school because of discrimination and rejection causing gender dysphoric individuals to risk social isolation, problematic academic performance, bullying behavior, feelings of inadequacy to go to school with the biological gender status, experience ostracism, harassment (sexual, physical, verbal, and emotional), persecution, humiliation, difficulty in getting employment, rejection by health care providers, and a disrespectful public attitude both in the workplace and in public places.(5,6) the psychological aspect is strongly correlated to social stigma that ends up to the psychological pressure experienced by the gender dysphoria community. gender dysphor ia , discriminated, victimized, and stigmatized persons may be at risk of high perceived discrimination, negative self-image (feeling inferior, embarrassed, isolated and rejected by the environment, lonely, helpless, anxious, depressed, attempting suicide), emotional and mental health problems of behavior and deterioration of the quality of life. depression and suicide attempts are two of the most common transgender mental health problems.(5) the national center for transgender equality and national gay and lesbian task force survey shows that 41% of gender dysphoric individuals attempts suicide compared with 1.6% of the general population, 32% are depressed, and 29% have somatization disorders.(7) acceptance as well as family and community support provide a protective effect against many of the “threats” to the well-being of gender dysphoric individuals, including the risk of hiv-aids infection, depression and suicide. inadequate social and family support, especially from biological families, is often reported in the case of the individuals’ sense of security when in a public setting.(6.7) the phenomenon o f various gender dysphoria problems is increasing in indonesia. gender dysphoric individuals experience stigma and discrimination from various parties such as family, community, religion, culture and country. the role of the family is a protective factor for 189 ge nder dysphoria in the face of stigma, discrimination, sexual identity, and in lowering the risk of mental disorders and poor quality of life.(7) in the present study, the researchers wanted to determine the perceived discrimination in gender dysphoric individuals, which is the perception of gender dysphoric individuals of their sexual identity, unfair treatment, and stigma and discrimination from family, environment, and society. high perceived discrimination has an effect on poor adaptability and low self-esteem in the face of stigma and discrimination that can have further impacts on emotional mental disorders and poor quality of life.(8) early detection of perceived discrimination, self-esteem and family factors (family relationships, family support, parenting patterns) and social support for ge nder dysphoria in the face of stigma, discrimination and sexual identity, can be made for the prevention and early intervention of the impact inflicted. the aim of this study was to determine the role of family factors, peer support, self-esteem, perceived discrimination, depression, anxiety, and stress on the quality of life of mtf gender dysphoric individuals. methods research design the study design was cross sectional and the study took place in march november 2016 at the sanggar swara jakarta community. study subjects sanggar swara jakarta is one of the gender dysphoria community centers in jakarta with about 300 male to female (mtf) and 50 female to male (ftm) gender dysphoric individuals with an age range of 14-45 years, spread over the five districts of dki jakarta (north jakarta, south jakarta, west jakarta, east jakarta, central jakarta). the sample size in this study was 106 mtf gender dysphoric individuals obtained for unpaired categorical analysis and 10% drop-outs. the sample was obtained from all religions using cluster random sampling.(9) the inclusion criteria of this research were mtf gender dysphoric individuals targeted at the five jakarta swara areas (south, west, east, central, north) areas, aged 18-45 years, with no history of drug abuse, able to read and write, and agreeing to fill out questionnaires completely. excluded from this study were those who were unwilling to sign informed consent, or were suffering from severe mental disorders (psychotic disorder and mental retardation). measurements the instruments used in this study are the following eight questionnaires. the first is a demographic questionnaire consisting of age, educational level, occupation, and economic status. the second is the rosenberg self-esteem questionnaire consisting of 10 questions with 4 categories, where self-esteem is high if the total score is 15 and low if the total score is <15.(10) this study also used a perceived discrimination questionnaire consisting of 15 questions, where perceived discrimination is high if total score >13 and low if total score 13 with cronbach’s alpha of 0.890.(11) the questionnaire on family relations (faces iii) consists of 16 questions with 4 types of family cohesion and 4 types of family adaptability, which are further summarized in 3 types of family relationships, i.e. balanced, extreme, and mid-range family relations.(12) the family support questionnaire consists of 20 questions with supportive results if the total score 60 and non supportive when the total score <60.(13) the social support questionnaire (ssq6) consists of 6 questions, where the results are peer support if the total score 3 and no peer support when the total score <3.(14) the depression anxiety stress scales (dass) consists of 42 questions and is divided into 3 sub-scales, namely depression, anxiety and stress, each containing 14 questions. each sub-scale has a range of values 0-3. the total score is calculated by summing the scores of each sub-scale giving a score range of 0-42 for each sub-scale. total scores below 10,18,15 on anxiety depression and stress sequentially univ med vol. 36 no.3 190 hasan, alviany, clarissa, et al perceived discrimination in gender dysphoria a b c indicate normal levels, while total scores above 20, 14 and 25 in depression, anxiety and stress sequentially indicate very severe levels.(15) the whoql-bref questionnaire consists of 24 questions where the results are good quality of life if the total score 60 and poor quality of life when the total score <60.(16) statistical analysis the data of this study were analyzed using binary simple logistic regression analysis for the relationship of gender dysphoria quality of life with employment, perceived discrimination, selfesteem, family support, and anxiety. multiple logistic regression analysis was used for the relationship of quality of life with perceived d is c r i mi n at i on a nd f a mi l y s upp or t . t he relationships were considered to be of statistical significance at an error probability of less than 0.05 (p<0.05). ethical clearance this research has been approved by the ethical clearance committee, faculty of medicine, atma jaya catholic university of indonesia, under number 05/04/kep-fkuaj/ 2016april 18, 2016. results characteristics of participants in this study, there were 106 mtf gender dysphoric individuals of sanggar swara jakarta, with the age range of 18-40 years. in this study there were no respondents who dropped out. the maj ority of subj ect s had the following characteristics: 57.5% senior high school education, 69.8% low economic status, 64.2% employed, 35.8% residing in north jakarta. most of the respondents had the following pecentages of determinant factors: 62.3% high perceived discrimination, 64.1% low self-esteem, 44.3% depression, 59.4% anxiety and 35.8% stress. a total of 78.3% had no family support, 64.1% no peer support, 36.0% had extreme family relations and 62.3% poor quality of life (table 1). variables n (%) age (years) 18-24 34 (32.1) 25-31 36 (34.0)* 32-38 26 (24.5) 39-45 10 (9.40) education senior high school 55 (57.5)* junior high school 45 (42.5) occupation employed 62 (64.2)* non-employed 38 (35.8) family socio-economic status middle income (rp 3.100.000/month or above) 26 (30.2) low income (below rp 3.100.000/month) 74 (69.8)* region north jakarta 38 (35.8)* central jakarta 6 (5.70) west jakarta 13 (12.2 ) east jakarta 22 (20.7) south jakarta 27 (25.6) perceived discrimination high 66 (62.3)* low 40 (37.7) self-esteem high 32 (35.9) low 68 (64.1)* family support supportive 17 (21.7) non-supportive 83 (78.3)* peer support supportive 32 (35.9) non-supportive 68 (64.1)* family relations type balanced 18 (16.9) mid range 50 (47.1)* extreme 38 (36.0) depression negative 53 (55.7)* positive 47 (44.3) anxiety negative 37 (40.6) positive 63 (59.4)* stress negative 62 (64.2)* positive 38 (35.8) quality of life good 40 (37.7) poor 66 (62.3)* table 1. key characteristics of study respondents and other explanatory variables (n=106) note* = high percentage 191 table 2 shows that there were 5 variables (employment, perceived discrimination, selfesteem, family support, and anxiety) significantly associated with life quality (p<0.05). but there was no significant relationship of educational level, socioeconomic status, type of family relation, peer support, stress, and depression with quality of life (p>0.05). the study showed that 83.3% of employed respondents had poor quality of life and 16.7% of non employed respondents had poor quality of life. there were 31.8% of respondents with low perceived discrimination who had poor quality of life and 68.2% of respondents with high perceived discrimination who had poor quality of life. there were 40.9% of respondents with high self-esteem who had poor quality of life and 59.1% of respondents with low self-esteem who had poor quality of life. there were 72.7% of respondents without family support who had poor quality of life and 37.3% of respondents with family support who had poor quality of life. this study also showed that 45.5% respondents with anxiety had poor quality of life and 54.5% respondents without anxiety had poor quality of life. univ med vol. 36 no.3 table 2. correlation between factors affecting quality of life in mtf gender dysphoric individuals (n=106) or= odds ratio variable p value adjusted or@ 95% ci for exp(b) lower upper occupation family support perceived discrimination self-esteem anxiety socioeconomic status depression 0.201 0.000* 0.004* 0.069 0.169 0.109 0.089 1.79 29.11 23.89 11.61 6.78 1.89 3.78 1.925 5.899 2.455 5.201 12.221 9.760 10.675 3.422 11.990 8.210 23.901 26.701 14.109 21.670 table 3. factors associated with quality of life in mtf gender dysphoric individuals (n=106) note: ci= 95% confidence interval for the mean; p*= significant association; @adjusted odds ratio controlled for variables variables quality of life p value or 95% ci good n (%) poor n (%) education senior high school 23 (57.5) 38 (57.5) 0.301 2.4 11.9-45.9 junior high school 17 (42.5) 28 (42.5) occupation employed 13 (32.5) 55 (83.3) 0.000* 0.46 39.0-78.8 non-employed 27 (67.5) 11 (16.7) socioeconomic status middle 14 (35.0) 18 (27.3) 0.101 4.9 0.78-98.5 low 26 (65.0) 48 (72.7) perceived discrimination high 21 (52.5) 45 (68.2) 0.004* 12.70 29.8-76.55 low 19 (47.5 ) 21 (31.8) self-esteem high 11 (27.5) 27 (40.9) 0.001* 2.89 34.1-56.2 low 29 (72.5) 39 (59.1) depression negative 26 (65.0) 33 (50.0) 0.209 6.7 0.21-33.56 positive 14 (35.0) 33 (50.0) anxiety negative 13 (32.5) 30 (45.5) 0.002* 2.15 22.9-42.8 positive 17 (67.5) 36 (54.5) stress negative 21 (52.5) 47 (71.2) 0.602 1.8 0.81-92.7 positive 19 (47.5) 19 (28.8) family support support 14 (35.0) 18 (37.3) 0.000* 23.19 37.1-59.1 no support 26 (65.0) 48 (72.7) peer support support 14 (35.0) 14 (21.2) 0.407 3.5 0.42-67.5 no support 26 (65.0) 52 (78.8) family relations type balanced 6 (15.0) 13 (19.7) 0.605 0.306 2.9 1 (reference) 1.9 0.52-23.8 0.34-19.6 extreme 13 (32.5) 40 (60.6) mid range 21 (52.5) 13 (19.7) 192 hasan, alviany, clarissa, et al perceived discrimination in gender dysphoria the binary logistic regression test showed different proportions of quality of life between subjects who were employed and those who were non-employed (or=0.46, p=0.000), between subj ects with low and high per ceived discrimination (or=12.70, p=0.004), and between low and high self-esteem (or=2.89, p=0.001), without family support and with family support (or=23.19, p=0.000), and with and without anxiety (or=2.15, p=0.002), respectively (table 2). there were no significant relationships of educational levels, socio-economic status, type of family relations, peer support, stress, and depression with quality of life (p>0.05). a total of 57.5% of respondents who were senior high school graduates and 42.5% of respondents who were junior high school graduates had poor quality of life. the study also showed that 69.8% of respondents with low economic status and 30.2% respondents with middle economic status had poor quality of life. the study also showed that 60.6% of respondents with extreme family relationships and 19.7% respondents with balanced family relationships had poor quality of life. a total of 78.8% respondents without peer support had poor quality of life and 21.2% respondents with peer support had poor quality of life. the results of this study showed that 28.8% of respondents with stress had poor quality of life and 71.2% without stress had poor quality of life. there were 50% of respondents with depression who had poor quality of life and 50% of respondents without depression who had poor quality of life. the proportion of quality of life was not significantly different between subjects with senior and junior high school level (p=0301); low and middle economic status (p=0.101); extreme and balanced family type (p=0.605); peer support and no peer support (p=0.407); stress and no stress (p=0.602); depression and no depression (p=0.209) (table 2). multivariate analysis the results of multivariate logistic regression analysis showed that there were two influencing variables, i.e. perceived discrimination and family support. the variable perceived discrimination had an odds ratio of 13.89 (95%ci: 5.89-11.99), which means that respondents with high perceived discrimination had a 13.89-fold greater risk for poor quality of life. the variable family support had an odds ratio of 29.11 (95%ci: 2.45-8.21), which means that respondents without family support had a 29.11 times greater risk for poor quality of life (table 3). discussion the result of this study indicate that among the 106 mtf gender dysphoric individuals in the age range of 18-45 years, the majority of respondents have high school education, most are employed as sex workers, the rest work as salon employees, salon owners, and buskers. they have a low economic status (earning less than rp 3.100.000 /month) and most live in villages, occupying a narrow boarding-house room. research by prabawanti et al.(17) in mtf gender dysphoric individuals in dki jakarta showed that 42% had junior high school education, 60% were commercial sex workers (34.9% as sex worker variable p adjusted or@ 95% ci for exp (b) lower upper occupation family support perceived discrimination self-esteem anxiety socioeconomic status depression 0.201 0.000* 0.004* 0.069 0.169 0.109 0.089 1.79 29.11 23.89 11.61 6.78 1.89 3.78 1.925 5.899 2.455 5.201 12.221 9.760 10.675 3.422 11.990 8.210 23.901 26.701 14.109 21.670 table 3. factors associated with quality of life in mtf gender dysphoric individuals (n=106) note: ci= 95% confidence interval for the mean; p*= significant association; @adjusted odds ratio controlled for variables 193 for their main income and 24.4% for additional income). nuttbrock et al.(18) showed that most of the mtf gender dysphoria individuals experiences discrimination and tend to have no choice in running their lives in the community. the large number of constraints from the environment, access to education, access to health and the right to decent work leads to low-income, low-paid work, and sex work. the results of our study showed that 62.3% of respondents had high perceived discrimination, and 64.1% had low self-esteem. bazargan and galvan state (19) that discrimination experienced by transgender individuals may be physical or sexual violence, social exclusion, humiliation, being viewed as a sex worker and as hiv positive, discrimination at work and health services. gender dysphoric individuals suffering from discrimination and stigmatization have a low selfesteem that can have further impacts with emotional mental disorders, suicide and poor quality of life. the results of the present study indicate that 59.4% of respondents had anxiety, 44.3% depression and 35.8% stress. bockting et al.(3) showed 52% respondents with anxiety, 48.7% depression and 25.1% stress. depression and s ui c i d e a tt e mp t s a r e t he mos t c o mmo n c o nd i t i ons i n t r a n sge n de r me n ta l h e al t h problems. the survey of the national center for transgender equality and the national gay a n d l e s bi an ta s k fo r c e s t a t ed th a t t he prevalence of depression in transgender was 44.1%, among them 41% suicide attempts, while depression in the general population was 6.6% and suicide attempts 1.6%. there were more suicide attempts by transgender individuals who have lost their jobs due to a negative view of their transgender status, are targets of bullying or bullying at school, or victims of physical or sexual abuse.(20) the results of our study also showed that 78.3% of respondents did not receive family support, 64.1% did not receive social support, and 36% had extreme family relations. this study is in accordance with the study by simon et al.(21) which showed that 74% of gender dysphoric individuals did not have family support, 59.2% were not socially supported, and 27% had extreme family relations. gender dysphoric individuals who do not get family or social support have a greater risk of depression and suicidal ideation than those who get family or social support. two studies in india found that 20.1% of gender dysphoric individuals who did not get fa mi ly supp or t co uld e xp er ie nce gre at er discrimination and violence at home than the 0.4% who received family support. family rejection of gender dysphoric individuals took the form of judging them to be a “disgrace” to the family. relationship factors between family members and the environment can affect a f a mi l y’s re f u s a l o f ge nd e r d ys ph o r i a a s individuals “neighbors” that can trigger gender dysphoria behavior by the family and most gender dysphoric individuals leave the house. low family support is due to the inability of families to adapt and accept their gender dysphoria family members.(20,21) the results of the present study showed that 62.3% of respondents had poor quality of life. george (22) showed that gender dysphoric individuals without family support led them to become homeless, employed as sex worker, and make suicide attempt. the national transgender discrimination survey (ntds) showed that 19% gender dysphoric individuals were expelled from residence, more than 2.5 times more likely to be jailed, 4 times more likely to work as sex workers, 3 times more likely to be hiv positive and 4 times more likely to make suicide attempts. the national transgender discrimination su rve y als o s howed th at 28% of gender dysphoric individuals delayed seeking health services due to discrimination, 48% did not have sufficient funds, and 19% experienced denial of he alth services. limited access to health services occurs because rejection or prejudice and lack of knowledge of health care workers on gender dysphoric individuals, making it difficult for these to obtain appropriate and competent health services.(23) univ med vol. 36 no.3 194 hasan, alviany, clarissa, et al perceived discrimination in gender dysphoria our study showed that occupation, perceived discrimination, self-esteem, family support, and anxiety were significantly associated with quality of life. bar et al.(24) showed that most of the mtf gender dysphoric individuals experiences discrimination that tends to leave them no choice in running their lives in society. the many barriers of access to education and the right to decent work cause many of the gender dysphoric individuals to be of low income and low education, and employed in sex work. prabawanti et al.(17) argues that the frequently stigmatized and discriminated mtf gender dysphoric individuals are forced to become sex workers because it is difficult to compete for decent jobs in jakarta, with a degree of education that is limited to junior and senior high school. despite employment opportunities, transgender individuals are often denied work because of their identity as transgender individuals and are judged as a violation of community values and norms. gender dysphoria people who are employed as sex workers have poorer mental, physical, psychological and social health than gender dysphoria people who are not employed as sex worke rs, experience highe r stigma and discrimination and have a higher risk of drug abuse, physical and sexual abuse, as well as hiv disease or other sexually transmitted diseases that end in emotional and behavioral disorders and poor quality of life.(20.24) the result of our study showed there were two variables, i.e. perceived discrimination and family support that were associated with quality of life. the most dominant variable was family support, which affects the quality of life of transgender mtf individuals. the study by burgess et al.(25) suggested that unfavorable family support poses a 5.72 times greater risk for anxiety, while the risk of depression is 4.06 times, and suicide risk is 3.09 times compared with proper family support. family support has the strongest positive effect on mental health and the survival of transgender life. the majority of families will reject family members who take the liberty to express themselves as gender dysphoric individuals, who are judged as a disgrace to the family, as sinners, as exposing the shame of the family to the neighbors, and various other negative stigmata. family rejection of gender dysphoric individuals always occur, being only different in the type of rejection, from refusal to acknowledge them to refusal that leads to physical violence even to their being expelled from home.(26) nemoto et al.(27) stated that mtf gender dysphoric individuals who do not have family or social support have a greater suicide risk than those receiving social and family support. weissman’s (28) study reported that 54.8% of gender dysphoric individuals had high perceived discrimination. basar et al.(29) stated that high perceived discrimination, poor social support, and poor family support can affect self-esteem and gender dysphoria mental health by degrading the quality of life. prabawanti et al.(17) stated that mtf gender dysphoric individuals are often stigmatized and discriminated, forcing them to become sex workers because it is difficult to compete for employment in jakarta, when only having a high school education. although discriminatory acts are legally prohibited, mtf ge nder dysphoric individuals experience discrimination that causes difficulties in completing education, job search, housing ownership and access to general health services. wilson et al.(0) stated that 61% of mtf gender dysphoric individuals have difficulties in obtaining jobs due to stigma and discrimination, so they look to sex work for alternatives. the limitation of this study is the crosssectional design used, that cannot explain any causal relationship of perceived discrimination and family support with male to female (mtf) gender dysphoric individuals’ quality of life. further research is needed with longitudinal research design to be able to explain causal relationships. the p resent r esear ch was conducted on mtf gender dysphoric individuals in the community of sanggar swara jakarta spread over the five municipal areas of the dki jakarta special capital city area with unequal 195 numbers of respondents in the five regions. it is hoped that through the results of this research there may be early detection of psychological problems, stigma and discrimination, emotional mental disorders in transgender individuals and attempts at comprehensive intervention by involving the family or parents so as to achieve good quality of life for gender dysphoric individuals and their families. further research can be done on gender dysphoria of both male to female (mtf) or female to male (ftm) types on the role of family factors, especially family support for family members with gender dysphoria. family support and good family communication can help the gender dysphoria individual to face acceptance of their sexual identity, self-esteem, decrease per ceived discrimination, prevent emotional and behavioral mental disorders and obtain a good quality of life conclusion high perceived discrimination and no family support are high risk factors for emotional and behavioral mental disorders and poor quality of life for gender dysphoria people especially male to female (mtf) gender dysphoric individuals. conflict of interest there are no conflicts of interest acknowledgment we thank all male to female (mtf) gender dysphoric individuals in sanggar swara jakarta community who have been willing to participate in this research and all the health care staff of sanggar swara jakarta who have helped in this research. contributors s contributed to drafting and designing the study. s, ya, ccl and ssk contributed to conducting the study. s and ya supervised and/ or performed the data management and data entry. s, ya, cc and ssk contributed to analysis and interpretation of the data. all authors read and approved the final manuscript. references 1. bradford j, reisner sl, honnold ja, et al. experiences of transgender-related discrimination and implications for health: results from the virginia transgender health initiative study. am j public health 2013;103:1820–9. 2. abigail, s. regional feature: the gwl-ina: the formation of a national network of gay men, transgender and msm in 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doi:10.1371/journal.pone. 29. basar k, gokhan o, jale k. perceived discrimination, social support, and quality of life in gender dysphoria. j sex med 2016;7:1133-41. doi: 10.1016/j.jsxm.2016.04.071. 30. wilson ec, garofalo r, harris rd, et al. transgender female youth and sex work: hiv risk and a comparison of life factors related to engagement in sex work. aids behav 2009;13: 902–13. maria lina 1 deteksi mycobacterium tuberculosis dan resistensinya terhadap rifampisin dengan metode nested polymerase chain reaction (pcr) dan sequencing maria lina rosilawati* abstrak *pusat aplikasi teknologi isotop dan radiasi, badan tenaga nuklir nasional (batan), jakarta korespondensi dra. maria lina rosilawati, m.biomed pusat aplikasi teknologi isotop dan radiasi, badan tenaga nuklir nasional (batan) jl. cinere pasar jumat po box 7002 jkskl jakarta 12070 email: marialinarosilawati@yahoo.com universa medicina 2007; 26: 1-10. latar belakang dna rpoβ (rna polymerase sub unit β) mycobacterium tuberculosis dapat diamplifikasi secara spesifik dengan metode nested polymerase chain reaction (pcr). nested pcr yang dilanjutkan dengan sequencing dapat secara langsung diaplikasikan untuk mendeteksi m. tuberculosis dan menentukan mutasi pada gen rpoβ yang berkaitan dengan resistensinya terhadap rifampisin, pada isolat klinis maupun sampel sputum. metode dalam penelitian ini digunakan 20 isolat klinis dan 30 sampel sputum yang diamplifikasi dengan primer yang dirancang dari bagian gen rpoβ m. tuberculosis. metode fenol-kloroform dan metode boom masing-masing digunakan untuk ekstraksi dna isolat klinis dan sampel sputum. sequencing hanya dilakukan untuk hasil pcr dari sampel sputum. hasil dari 20 isolat klinis, 15 isolat positif terdeteksi sebagai m. tuberculosis dengan nested pcr, 4 isolat tergolong mycobacteria other than tuberculosis (mott) dan 1 isolat non-mycobacteria. hasil nested pcr pada 30 sampel sputum dengan 25 sampel basil tahan asam (bta) positif dan 5 sampel bta negatif, menunjukkan hasil positif pada 21 sampel. besarnya produk first-round dan second-round pcr masing-masing adalah 205 bp dan 157 bp. kesimpulan nested pcr dengan sequencing lebih sensitif dan spesifik dalam mendeteksi m. tuberculosis dan resistensinya terhadap rifampisin. kata kunci : mycobacterium tuberculosis, dna rpoβ, nested pcr, sequencing universa medicina januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 2 rosilawati resistensi mycobacterium tuberculosis pendahuluan tu b e r k u l o s i s ( t b c ) , p e n y a k i t i n f e k s i disebabkan mycobacterium tuberculosis sampai saat ini masih menjadi masalah serius di seluruh dunia, karena merupakan penyebab kematian tertinggi. setiap tahun diperkirakan 8 juta infeksi baru terjadi dan 2,5 sampai dengan 3 juta menimbulkan kematian.(1) dari seluruh kasus tbc di dunia, 38% terdapat di asia tenggara dan lebih dari 95% kasus tersebut terdapat di negara berkembang seperti india, indonesia, bangladesh, thailand, dan myanmar.(2) dalam annual report on global tb control 2003, who m e n y a t a k a n a d a 2 2 n e g a r a d i k a t e g o r i k a n sebagai high-burden countries terhadap tbc. indonesia termasuk peringkat ketiga tertinggi jumlah kasus tbc di dunia setelah india dan china.(3) b e r d a s a r k a n h a s i l s u r v e i k e s e h a t a n rumah tangga (skrt) tahun 2001,(4) tbc menduduki peringkat ketiga sebagai penyebab kematian (9,4% dari total kematian), setelah penyakit kardiovaskular dan sistem pernafasan. who memperkirakan 583.000 kasus baru tuberkulosis terjadi di indonesia setiap tahun detection and resistance of mycobacterium tuberculosis on rifampicin using nested polymerase chain reaction and sequencing method maria lina rosilawati* abstract background the rpoβ (rna polymerase sub unit β) dna of mycobacterim tuberculosis can be specifically amplified by using a nested pcr the nested pcr linked to dna sequencing was applied directly to detect m. tuberculosis and determine the rpoβ gene mutation related with the rifampicin resistance either in clinical isolates or sputa. methods samples used in this research were 20 clinical isolates and 30 sputa which were amplified with the region of rpoβ dna of m. tuberculosis. dna of clinical isolates and sputum samples were extracted by means of fenol-kloroform and boom’s methods, respectively. sequencing method was only applied for sputum samples. results of 20 clinical isolates, 15 isolates were positive for m. tuberculosis with nested pcr, 4 isolates were mycobacteria other than tuberculosis (mott) and 1 isolate was nonmycobacteria. the nested pcr could detect 21 sputum samples of 30 samples consisted of 25 samples with positif acid fast bacilli (afb) and 5 samples with negative afb. first-round and second-round pcr products were 205 bp and 157 bp, respectively. conclusion nested pcr in the sequencing was more sensitive and specific to detect m. tuberculosis and it resistance to rifampicin. keywords : mycobacterium tuberculosis, rpoβ dna, nested pcr, sequencing *center for the application of isotopes and radiances technology, jakarta correspondence dra. maria lina rosilawati, m.biomed *center for the application of isotopes and radiances technology, jakarta jl. cinere pasar jumat po box 7002 jkskl jakarta 12070 email: marialinarosilawati@yahoo.com universa medicina 2007; 26: 1-10. 3 d a n 1 4 0 . 0 0 0 m e n g a k i b a t k a n k e m a t i a n . ( 5 ) e s t i m a s i p r e v a l e n s i t b c d i i n d o n e s i a berdasarkan pemeriksaan mikroskopik bta positif sebesar 148,5 per 100.000 orang. (6) program pemberantasan tuberkulosis menjadi l e b i h r u m i t a k i b a t m u n c u l n y a k u m a n penyebabnya yang resisten terhadap obat anti tuberkulosis (oat) disebabkan penggunaan obat yang tidak tepat baik dosis maupun lamanya. d i a g n o s i s t u b e r k u l o s i s k h u s u s n y a tuberkulosis paru, dapat ditegakkan dengan pemeriksaan klinik (anamnesis terhadap keluhan p e n d e r i t a d a n h a s i l p e m e r i k s a a n f i s i k ) , pemeriksaan laboratorium, dan pemeriksaan radiologik. ketiga hasil pemeriksaan tersebut disatukan untuk diagnosis tuberkulosis. salah s a t u p e m e r i k s a a n l a b o r a t o r i u m a d a l a h mendeteksi kuman m. tuberculosis sebagai penyebabnya. pada umumnya metode yang digunakan adalah metode konvensional seperti pemeriksaan mikroskopik basil tahan asam (bta) dan pemeriksaan kultur. pemeriksaan mikroskopik cukup cepat dan ekonomis akan tetapi sensitivitas dan spesifitasnya masih k u r a n g s e d a n g k a n p e m e r i k s a a n k u l t u r memerlukan waktu yang cukup lama, sekitar 312 minggu.(7) oleh karenanya, untuk mengatasi keterbatasan tersebut, diperlukan metode deteksi m. tuberculosis yang cepat, sensitif dan spesifik seperti misalnya metode polymerase chain re a c t i o n ( p c r ) . a m p l i f i k a s i d n a m . tuberculosis dengan target sekuens yang berbeda telah banyak diteliti menggunakan pasangan primer dari bagian gen yang conserved seperti gen yang menyandi protein 38 kda, 65 kda, m t p 4 0 ( 8 1 0 ) a t a u p u n d a r i s e q u e n c e s i s i p a n / insertion sequence seperti is6110.(11) pengembangan metode pcr untuk deteksi m. tuberculosis telah mulai dilakukan dengan tujuan untuk meningkatkan sensitivitas yaitu d e n g a n n e s t e d p c r . m e t o d e t e r s e b u t menggunakan dua pasang primer dari bagian yang conserved dari genom bakteri tersebut.(12,13) nested pcr dapat dikaitkan dengan metode lain s e p e r t i n e s t e d p c r s s c p ( 1 4 ) n e s t e d p c r re v e r s e h y b r i d i z a t i o n ( 1 5 ) d a n n e s t e d p c r s e q u e n c i n g . ( 1 6 ) m e t o d e t e r s e b u t d a p a t m e n d e t e k s i t i d a k h a n y a k e b e r a d a a n m . tuberculosis dalam spesimen klinis akan tetapi juga dapat menentukan resistensinya tehadap oat, berdasarkan adanya mutasi gen penyandi sasaran oat pada m. tuberculosis seperti gen rpoβ untuk rifampisin. dalam penelitian ini digunakan metode nested pcr dan sequencing untuk mendeteksi m. tuberculosis dari isolat klinis dan dalam sampel sputum dan mengetahui adanya mutasi gen rpoβ yang berkaitan dengan resistensinya terhadap rifampisin. bahan dan cara sampel penelitian dalam penelitian ini digunakan 20 isolat klinis m. tuberculosis hasil isolasi dari pasien. dan 30 spesimen klinis berupa sputum. isolat k l i n i s d i d a p a t d a r i b a g i a n m i k r o b i o l o g i , fakultas kedokteran, universitas indonesia dan telah diuji secara konvensional dengan metode k u l t u r. s a m p e l s p u t u m d i p e r o l e h d a r i perkumpulan pemberantasan tuberkulosis indonesia (ppti), kebayoran baru, jakarta selatan dengan hasil mikroskopik bta positif (25 sampel) dan negatif (5 sampel). data dukung penderita meliputi jenis kelamin dan umur. p e n d e r i t a t e r d i r i d a r i 1 6 l a k i l a k i d a n 1 4 p e r e m p u a n d e n g a n u m u r m a s i n g m a s i n g berkisar 18-55 tahun dan 17-60 tahun. homogenisasi dan ekstraksi dna sputum isolat klinis yang tumbuh dalam media lowenstein jensen diekstraksi dnanya dengan memanen isolat bakteri tersebut dengan cara menambahkan larutan nacl 0,9%. ekstraksi dna dilakukan dengan cara seperti telah dilaporkan sebelumnya,(17) yaitu dengan melisis 4 rosilawati resistensi mycobacterium tuberculosis sel bakteri yang telah dipanen, dengan larutan te (tris-edta) 1 x, sds (sodium dodecyl s u l f a t e ) d a n p r o t e i n a s e k , k e m u d i a n ditambahkan larutan fenol-kloroform-isoamil alkohol (24:1). presipitasi dna dilaksanakan dengan menambahkan etanol dan sentrifugasi dengan kecepatan tinggi. untuk proses pcr, pelet dna yang diperoleh kemudian dilarutkan dengan larutan buffer te satu kali. proses homogenisasi dan dekontaminasi dilakukan untuk spesimen klinis (sputum) d e n g a n t u j u a n u n t u k m e m e k a t k a n s a m p e l s e h i n g g a m e m p e r b a n y a k j u m l a h b a k t e r i khususnya m. tuberculosis yang terkandung dalam sampel sputum dan untuk mengeliminasi mikroba lain selain mycobacteria. sputum dihomogenisasi dan didekontaminasi dengan larutan asetil-l-sistein, naoh dan na-sitrat, kemudian disenrtifugasi. metode untuk ekstraksi dna sputum adalah metode boom.(18) sel dilisis dengan larutan tris-hcl, guanidin tiosianat sebagai chaotropic agent, edta, dan triton x100. larutan diatom (pengikat dna), aseton, etanol 70% serta sentrifugasi dengan kecepatan tinggi digunakan untuk ekstraksi dan presipitasi dna. untuk proses pcr, dna hasil ekstraksi dielusi dengan buffer te satu kali. proses pcr dan elektroforesis amplifikasi dna hasil ekstraksi dilakukan d e n g a n n e s t e d p c r d a n d i l a k s a n a k a n d i departement of microbiology, seoul national university college of medicine, seoul korea. p r i m e r y a n g d i g u n a k a n a d a l a h t b 1 ( 5 ’ acgtggaggcgatcacaccgcagacgt3’) dan tb2 (5’tgcacgtcgcggacctcc a g c c c g g c a 3 ’ ) s e b a g a i o u t e r p r i m e r sedangkan sebagai inner primer tb3 (5’tcgccgcgatcaaggagttcttc-3’) dan tr8 (5’-tgcacg tcg cggacc tcca-3’). pada proses nested pcr digunakan campuran pereaksi yang sudah dikemas dalam tabung pcr (accu power pcr premix; bioneer, daejon, korea) yang terdiri dari 50 mm tris-hcl (ph 8,3), 40 mm kcl, 1,5 mm mgcl 2 , 250 µm dntp, 1u taq dna polymerase dan gel loading dye. konsentrasi akhir outer primer dan inner primer masing-masing 20 pmol dan 0,5 pmol. dna target, ditambahkan dalam campuran tersebut (1 tabung untuk 1 reaksi) sehingga volume menjadi 20 µl. program pada 1st round p c r d a r i n e s t e d p c r m e l i p u t i 1 s i k l u s denaturasi awal, yaitu pada 94oc, 5 menit; 15 siklus dengan tiap siklus terdiri dari denaturasi pada suhu 94oc, 30 detik, annealing 82oc, 30 detik, extension, 82oc, 30 detik. program ini langsung dilanjutkan dengan 2nd round pcr dengan 30 siklus dari denaturasi pada 94oc, 30 detik; annealing, 72oc, 30 detik, extension 72oc, 30 detik, dan tahap akhir adalah extended extension pada 72oc, 5 menit. hasil nested pcr dideteksi dengan teknik elektroforesis gel agarosa (1,5%). visualisasi dna dilakukan dengan uv transilluminator setelah gel diwarnai dalam larutan etidium bromida. sequencing produk pcr sequencing dilakukan hanya untuk 20 sampel sputum serta m. tuberculosis h 37 rv hasil n e s t e d p c r . p e r s i a p a n u n t u k s e q u e n c i n g dilakukan dengan memotong masing-masing dna sampel pada gel agarosa, yang dipurifikasi dengan qiaex ii agarose gel extraction. hasil p u r i f i k a s i k e m u d i a n s e q u e n c i n g d e n g a n m e n g g u n a k a n i n n e r p r i m e r t r 8 . d a n d i l a k s a n a k a n o l e h d e p a r t m e n t o f d n a sequencing, macrogen company, seoul, korea. hasil nested pcr dalam penelitian ini telah mengamplifikasi dna rpoβ m. tuberculosis. p r o d u k a m p l i f i k a s i p a d a f i r s t ro u n d p c r m e n g g u n a k a n p r i m e r t b 1 d a n t b 2 menghasilkan fragmen dna dengan ukuran 205 b p , s e d a n g k a n f r a g m e n u k u r a n 1 5 7 b p 5 merupakan produk pcr dengan primer tb3 dan tr8 pada second-round pcr (gambar 1 dan 2 ) . h a s i l p o s i t i f d a r i n e s t e d p c r y a i t u terdapatnya fragmen dna 205 bp atau 157 bp menunjukkan adanya m. tuberculosis. hasil nested pcr isolat klinis dan sampel sputum beserta hasil bta-nya dapat dilihat pada ta b e l 1 . d a r i 2 0 i s o l a t k l i n i s , 5 i s o l a t menunjukkan hasil negatif yaitu tidak terlihat fragmen dna baik fragmen 205 bp maupun 157 bp pada gel agarosa. isolat tersebut adalah r10 (gambar 1, lajur 10) dan z4, z9, z10, z11 (gambar 2 , lajur 4, 8, 9, 10). jadi isolat tersebut bukan m. tuberculosis. gambar 2. hasil amplifikasi bagian gen rpoβ isolat klinis m. tuberculosis dengan nested pcr dalam gel agarosa 1,5%. lajur m: marker dna 100 bp ladder; lajur 1-5 : isolat klinis z1-z5; lajur 6: isolat klinis z7; lajur 710: isolat klinis z8-z11 gambar 1. hasil amplifikasi bagian gen rpoβ isolat klinis m. tuberculosis dengan nested pcr dalam gel agarosa 1,5%. lajur m: marker dna 100 bp ladder; lajur 1-10: isolat klinis r1-r10 6 rosilawati resistensi mycobacterium tuberculosis pada tabel 1 terlihat hasil second round p c r d a r i 3 0 s a m p e l s p u t u m , 2 1 s a m p e l menunjukkan hasil positif mengandung m. tuberculosis yaitu terdapatnya fragmen 157 bp dan sebaliknya 9 sampel negatif (gambar 3). dari hasil pemeriksaan secara mikroskopik pada 9 sampel tersebut, 8 sampel menunjukkan hasil b ta + 1 s a m p a i d e n g a n + 2 . ta b e l 1 j u g a memperlihatkan hasil positif nested pcr pada 4 sampel dari 5 sampel sputum dengan bta negatif. hasil nested pcr yang dilanjutkan dengan sequencing fragmen 157 bp dari gen rpoβ dalam penelitian ini dilakukan hanya pada 20 strain m. tuberculosis dari sampel sputum dan m. tuberculosis h 37 rv sebagai strain standar. dari 20 strain m. tuberculosis tersebut, hanya 8 sampel dan m. tuberculosis h 37 rv yang dapat diinterpretasikan hasil sequencing sedangkan strain m. tuberculosis yang lain tidak dapat diinterpretasikan. b e r d a s a r k a n h a s i l s e q u e n c i n g , s t r a i n standar dan 8 strain m. tuberculosis dari sampel sputum ternyata tidak terjadi mutasi. strain2 tersebut kemungkinan besar masih sensitif terhadap rifampisin. pembahasan nested pcr isolat klinis menunjukkan hasil negatif pada 5 isolat dan diperkirakan kelima i s o l a t a d a l a h m y c o b a c t e r i a b u k a n m . t u b e rc u l o s i s ( m y c o b a c t e r i a o t h e r t h a n tuberculosis / mott) atau bukan mycobacteria (non-mycobacteria). dna rpoβ mott atau non-mycobacteria tidak teramplifikasi pada first round nested pcr dengan primer yang sama pada suhu annealing yang tinggi. ( 1 5 ) fragmen dna rpoβ berukuran 342 bp dapat diamplifikasi dengan menggunakan primer mf & mr dari 44 strain acuan (reference strain) mycobacteria, akan tetapi tidak ada amplifikasi pada dna non-mycobacteria.(19) tabel 1. hasil nested pcr isolat klinik dan sputum bta positif *) keterangan : + pita dna terlihat jelas; + tipis : pita dna terlihat tipis; sampel no. 1-20 : isolat klinik; sampel no. 21 – 40, no. 4547 dan 49 – 50 : sputum bta positif; sampel no. 41 – 44 dan 48 : sputum bta negatif 7 gambar 3. hasil amplifikasi bagian gen rpoβ m. tuberculosis dari sputum dengan nested pcr dalam gel agarosa 1,5%. lajur m: marker dna 100 bp ladder ; lajur 1: sampel sputum (s61); lajur 2: sampel sputum (s81); lajur 3: sampel sputum (s97) ; lajur 4: sampel sputum (s99); lajur 5: sampel sputum (s101); lajur 6: sample sputum (s102); lajur 7: sampel sputum (s90); lajur 8: sampel sputum (s91); lajur 9: sampel sputum (s94); lajur10: sampel sputum (s100); lajur 11: m. tuberculosis h37 rv (kontrol positif) oleh karenanya, untuk mengetahui apakah kelima isolat tersebut di atas tergolong dalam m y c o b a c t e r i a a t a u n o n m y c o b a c t e r i a , dilakukan juga amplifikasi dengan primer mf & mr. isolat 10r ternyata non-mycobacteria karena tidak ada fragmen dna 342 bp sebagai hasil amplifikasi, sedangkan 4 isolat lainnya ( z 4 , z 9 , z 1 0 , z 11 ) m e m p u n y a i f r a g m e n tersebut, menunjukkan isolat tersebut tergolong dalam genus mycobacteria. untuk konfirmasi 4 isolat tersebut adalah mycobacteria, hasil amplifikasinya direstriksi dengan enzim hindii. fragmen dna 342 bp tidak terestriksi (data tidak diperlihatkan) yang menunjukkan 4 isolat tersebut adalah mott. restriksi produk pcr dengan primer mf-mr dengan enzim hindii, dapat membedakan antara m. tuberculosis kompleks dan mott yaitu 2 fragmen (232 bp, 11 0 b p ) m e r u p a k a n h a s i l r e s t r i k s i m . tuberculosis kompleks, sedangkan untuk mott tidak direstriksi (342 bp).(20) dari hasil penelitian kim et al,(15) menunjukkan amplifikasi dengan first round-nested pcr menggunakan primer tb1 dan tb2 pada 48 isolat klinis memberikan hasil positif hanya pada 20 isolat sedangkan 28 isolat yang lain hasilnya negatif. hasil ini sesuai dengan hasil identifikasi dengan tes biokimia dan s e q u e n c i n g y a i t u 2 0 i s o l a t a d a l a h m . t u b e rc u l o s i s d a n 2 8 i s o l a t a d a l a h m o t t. amplifikasi juga tidak terjadi pada 19 strain a c u a n m y c o b a c t e r i a d a n 9 s t r a i n n o n mycobacteria. produk pcr 322 bp pada nested pcr dengan primer yang mengamplifikasi daerah gen yang menyandi protein 38-kda (protein antigen b) m. tuberculosis, hanya dapat mendeteksi m. tuberculosis kompleks dan tidak pada 10 strain acuan mott.(13) hasil deteksi 5 i s o l a t k l i n i s d a l a m p e n e l i t i a n i n i s e c a r a konvensional dengan kultur memberikan hasil positif, sedangkan dengan nested pcr negatif. hal ini menunjukkan nested pcr lebih spesifik dibanding dengan kultur. pada sampel sputum dengan bta + tetapi hasil nested pcr-nya negatif kemungkinan ini 8 rosilawati resistensi mycobacterium tuberculosis bukan terinfeksi m. tuberculosis akan tetapi infeksi oleh mott. identifikasi mycobacteria secara mikroskopik langsung dari sputum kurang sensitif dan apabila hasilnya positif, pemeriksaan tersebut tidak dapat mengidentifikasi species mycobacteria yang menunjukkan metode tersebut juga kurang spesifik.(7,8,21) hal ini juga terlihat pada hasil nested pcr yang positif pada sampel sputum tetapi hasil bta negatif. penelitian cheng e t a l ( 2 2 ) m e n u n j u k k a n h a s i l p e m e r i k s a a n mikroskopik dari 144 spesimen pulmonary dan extrapulmonary dari pasien dengan diagnosis klinis infeksi m. tuberculosis, hanya 25% positif pada pemeriksaan bta-nya sedangkan 80% hasil positif diperoleh dengan metode pcr. pendapat umum menyatakan pasien dengan bta negatif tidak berperan secara nyata menyebarkan infeksi akan tetapi dari hasil penelitian behr et al yang d i k u t i p o l e h g a r c i a q u i n t a n i l l a e t a l , ( 2 3 ) diperoleh 27% kasus tb di san francisco, california ditransmisi dari kasus dengan bta negatif. hasil sequencing produk nested pcr yaitu untuk mengetahui adanya mutasi pada gen rpoβ dari 20 strain m. tuberculosis dari sampel sputum hanya 8 strain yang dapat diinterpretasikan. k e m u n g k i n a n y a n g m e n y e b a b k a n t i d a k diperolehnya dna yang murni untuk proses secuencing saat dipurifikasi pada 12 strain dari sampel sputum tersebut, disebabkan terdapatnya dna hasil pcr yang multiband pada gel agarosa. mutasi tidak terjadi pada 8 strain, kemungkinan besar strain tersebut masih sensitif terhadap rifampisin atau mutasi terjadi di luar daerah 81 bp gen rpoβ. menurut heep et al yang dikutip oleh sajduda et al. (24) mutasi yang berkaitan dengan resistensi terhadap rifampisin dapat terjadi di luar daerah 81 bp gen rpoβ, meskipun hal ini jarang sekali terjadi dasar dari resistensi strain m. tuberculosis terhadap rifampisin adalah adanya mutasi pada gen rpoβ. beberapa peneliti menyatakan lebih dari 95% strain m. tuberculosis resisten rifampisin disebabkan adanya mutasi pada bagian dna 81 b p d a r i g e n r p oβ y a n g m e n y a n d i r n a polymerase sub unit β.(1,25,26) pada umumnya deteksi m. tuberculosis d e n g a n n e s t e d p c r d i g u n a k a n u n t u k meningkatkan sensitivitas dengan menggunakan beberapa macam primer yang dirancang dari b a g i a n g e n a t a u s e k u e n s s i s i p a n ( i s ) m . tuberculosis yang conserved. beberapa primer digunakan untuk nested pcr seperti bagian gen yang menyandi antigen protein 65 kda,(7) antigen protein b 38 kda,(13) mpb64 (major secreted protein specific to m. tuberculosis complex)(14) dan is6110.(27) metode nested pcr sequencing dalam penelitian ini dapat diaplikasikan langsung untuk mendeteksi selain adanya m. tuberculosis dalam spesimen klinis seperti sputum juga dapat mengetahui resistensinya terhadap rifampisin. kesimpulan nested pcr lebih spesifik dari kultur dalam mendeteksi m. tuberculosis. nested pcr lebih s e n s i t i f d a n s p e s i f i k d i b a n d i n g d e n g a n pemeriksaan mikroskopik untuk mendeteksi m. tuberculosis. nested pcr dengan mengunakan primer yang dirancang dari bagian gen rpoβ m. tuberculosis merupakan metode yang cepat, sensitif dan spesifik untuk mendeteksi m. t u b e rc u l o s i s d a n r e s i s t e n s i n y a t e r h a d a p rifampisin pada isolat klinis maupun langsung spesimen klinis seperti sputum. ucapan terima kasih penulis mengucapkan terima kasih kepada international atomic energy agency (iaea) atas bantuan dana melalui program technical cooperation (tc), kepada prof. yoon-hoh kook, md. ph.d., department of microbiology, seoul national university college of medicine, seoul, korea, atas ijin dan fasilitas laboratorium 9 untuk pelaksanaan penelitian ini, dan kepada sdr. rika heryani dan almaida atas bantuannya dalam penelitian ini. daftar pustaka 1. zhang m, yue j, yang yp, zhang hm, lei jq, jin rl, et al. detection of mutations associated with isoniazid resistance in mycobacterium tuberculosis isolates from china. j clin microbiol 2005; 43: 5477-82. 2. alamsyah b. epidemiologi genetic serta faktor resiko mycobacterium tuberculosis yang resisten inh dan atau rifampisin [disertasi]. jakarta: program doktor ilmu kesehatan masyarakat, program pascasarjana, fakultas kesehatan masyarakat, universitas indonesia; 2003. 3. world health organization. global tuberculosis control. who report: surveillance, planning, financing. geneva: world health organization; 2004. 4. departemen kesehatan ri. survei kesehatan rumah tangga (skrt). jakarta: departemen kesehatan ri; 2001. 5. direktorat jendral pemberantasan penyakit menular dan penyehatan lingkungan pemukiman 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rhienthong d, medo miranda a, udomsantisuk n, ellis k, van leeuwen j, et al. a more reliable pcr for detection of mycobacterium tuberculosis in clinical samples. j clin microbiol 1994; 32: 672-8. 12. wang jy, lee ln, chou cs, huang cy, wang sk, lai hc, et al. performance assessment of a nested pcr assay (the rapid bap-mtb) and the bd probetec et system for detection of mycobacterium tuberculosis in clinical specimens. j clin microbiol 2004; 42: 4599-603. 13. martins lc, paschoal ia, nowakonski av, silva sab, costa ff, ward ls. nested pcr using mpb64 fragment improves the diagnosis of pleural and meningeal tuberculosis. rev soc bras med trop 2000; 33: 1-7. 14. kim bj, lee kh, park bn, kim sj, park em, parkyg, et al. detection of rifampin resistant mycobacterium tuberculosis in sputa by nested pcr linked single strand conformation polymorphism and dna sequencing. j clin microbiol 2001; 39: 2610-7. 15. paluch-oles j. application of nested and reverse hybridization for diagnosis of central 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zwolska z, niemann s, et al. molecular characterization of rifampin and isoniazid resistant mycobacterium tuberculosis strains isolated in poland. j clin microbiol 2004; 42: 2425-31. 25. aragon lm, navarro f, heiser v, garrigo m, espanol m, coll p. rapid detection of specific gene mutations associated with isoniazid or rifampicin resistance in mycobacterium tuberculosis clinical isolates using non-fluorescent low-density dna microarrays. j antimicrob chemother 2006; 57: 825-31. 26. yuen lkw, leslie d, coloe pj. bacteriological and molecular analysis of rifampin resistant mycobacterium tuberculosis strains isolated in australia. j clin microbiol 1999; 37: 3844-50. 27. whelen ac, felmlee ta, hunt jm, williams dl, roberts cd, stockman l, et al. direct genotyping detection of mycobacterium tuberculosis rifampicin resistance in clinical specimens by using singletube heminested pcr. j clin microbiol 1995; 33: 556-61. maternal perception of sickness as a risk factor 156 *division of nutrition, faculty of medicine, diponegoro university, semarang **department of pediatrics, faculty of medicine, diponegoro university/ dr.kariadi central general hospital, semarang ***program in nutrition, muhamadiyah university, semarang correspondence: maria fatima dete dellu division of nutrition, faculty of medicine, diponegoro university, semarang. jl. dr. soetomo no. 18 (komplek zona pendidikan rsup dr. kariadi, gd. dekanat fk undip lt.2) email: mariafatimadellu@gmail.com univ med 2016;35:156-64 doi: 10.18051/univmed.2016.v35.156-164 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 maternal perception of sickness as a risk factor of stunting in children aged 2-5 years maria fatima dete dellu*, maria mexitalia**, and ali rosidi*** background stunting in children is caused by past nutritional problems, adversely affects the physical and mental characteristics of children and is a wellestablished child-health indicator of chronic malnutrition. socio-cultural factors can affect parenting, thereby indirectly affecting child growth. the objective of this study was to determine socio-cultural factors and parental short stature as risk factors of stunting in children aged 2 to 5 years. methods the study used a quantitative approach with case control design and a qualitative approach with in-depth interview. the study subjects were children aged 2-5 years, consisting of 45 cases of stunting and 45 controls (normal subjects). socio-cultural data were obtained through interviews with the mothers, while height was measured with a stadiometer. data were analyzed using chi square and logistic regression tests to calculate the odds ratio (or). results the logistic regression test showed that maternal perception of sickness as a curse (or=7.43; 95% ci: 2.37-23.21), stopping breastfeeding at <24 months (or=6.01;95% ci: 1.83-19.69) and low household expenditure for food (or=5.78;95% ci: 1.28-26.01) were risk factors of stunting incidence with a probability of 73.8%. the most dominant risk factor of stunting was maternal perception of sickness (or=7.43 95% ci: 2.37-23.21). conclusion maternal perception of sickness was the most dominant risk factor of stunting in children 2-5 years of age. a multidisciplinary approach is needed to address the range of raised issues and so combat stunting in children. keywords: stunting, socio-cultural, parental short stature, children aged 2-5 years doi: http://dx.doi.org/10.18051/univmed.2016.v35.156-164 157 introduction stunting or short stature is the condition in which the height-for-age index is less than -2 standard deviations (sd). a child with stunting will be shorter than a normal child of the same age.(1) the condition of stunting in children shows the presence of nutritional deficiencies and disease that occurred in their growth and development early in life.(2) the global prevalence of stunting to date is still relatively high, since in several countries more than half of children under 5 years of age are recorded as having retarded growth.(2) data from the indonesian basic health research (riskesdas) for the year 2013 showed that up to 37.2% of indonesian underfives suffered from stunting. the highest prevalence of stunting of 58% was found in the province of east nusa tenggara (nusa tenggara timur, ntt).(3) in this province, south central timor (timor tengah selatan, tts) district has in recent years had the highest prevalence of stunting among the underfives. the riskesdas for 2007 showed that the prevalence of stunting in the underfives in tts district was 57%, increasing up to 70.5% in 2013, with amanuban subdistrict as one of the subdistricts with the highest prevalence of stunting, i.e. 66.25%.(4) previous studies have concluded that the prevalence of stunting in children is caused by various factors. the study of nabuasa et al.(5) in children aged 24-59 months found a significant association between culture and the prevalence of stunting. this differs from a study in the medan area district, which found no relationship between family tradition/beliefs and nutritional status in children.(6) parental genetics, in this case parental height, is also of influence on the problem of stunting in children. parental height and growth pattern are the key to the growth pattern of their children. mothers with a height of <145 cm are at higher risk of having children with stunting compared with mothers with a height of >145 cm.(7) the study by solihin et al.(8) stated that maternal height is significantly associated with nutritional status (height-for-age index) of the underfives. however, this differs from the study by hanum (9) and kusuma (10) who found that maternal height is not associated with the nutritional status of the children. the present study focuses on the sociocultural variables of the local community and parental short stature, in this case the height of both parents. previous studies investigating the influence of socio-economic and socio-cultural variables on the nutritional status of the children, focused only on maternal height in relation to the prevalence of stunting in children. on the other hand, the present study used the height of both parents in relation to the prevalence of stunting, with the aim to determine socio-cultural factors and parental short stature as risk factors for the prevalence of stunting in children aged 2-5 years. methods design of the study the study design used a quantitative approach with an analytic-observational unmatched case-control design and a qualitative approach with in-depth interview and focus group disscusion (fgd). the study location was amanuban selatan subdistrict, timor tengah selatan district, nusa tenggara timur province. the study was conducted from february to april 2016. study subjects the subjects of this study were 90 children aged 2-5 years, consisting of 45 cases of stunting and 45 normal controls. the required sample size was calculated using the formula for analytic unmatched case-control studies with categorical variables. recruitment of the study subjects was by the simple random type of probability sampling. the subjects were selected from the intended study population of 415 children, who were then stratified into cases and controls, in accordance with the exclusion and inclusion criteria that had been decided upon. the inclusion univ med vol. 35 no.3 158 criteria in this study were children with stunting and normal children, living with their own parents, having a record of their birth weight, born at term with a weight of >2500 grams, and their parents agreeing for their children to be included in the study sample. categorization into children with stunting and normal children was based on the children’s height-for-age index expressed as zscores. the children were considered to have stunting if the height-for-age z-score was ≤-2 standard deviations (sd) and not to have stunting (or to be of normal height) if the height-for-age zscore was >-2 sd.(4) anthropometric measurements measurement of height was performed in the children who were the subjects of this study and in both of their parents, using a stadiometer with an accuracy of 0.1 cm. the measurement procedure was as follows: the subjects without footwear and headgear stood upright in contact with the measuring device and looked straight ahead, with the eyes parallel to the vertex. the operator moved the paddle of the measuring device downwards so that it rested lightly on the subject’s head, then read the result off the device. the evaluation of parental short stature was performed by measuring the height of both parents, then using the formula of midparental height (mph) to project the development of the height of their children based on the genetics of both parents. the calculated result was then plotted on a curve generated by the who anthro program.(11) data collection data on community socio-cultural characteristics were obtained by in-depth interviews with the respondents, i.e. the mothers of the subjects. data on individual perception about sickness and the customs for pregnant mothers were obtained by interviews using questionnaires. the investigators asked whether the parents, in this case the mothers of the subjects, believed that the sickness suffered by their children was or was not a curse, through a number of questions. maternal perception about sickness was categorized as perception of sickness as a curse and perception of sickness not as a curse. on the custom of food taboos for pregnant mothers, the investigators asked the mothers about their dietary habits during pregnancy, such as whether they did or did not observe taboos on certain foods or dishes that are forbidden by culture. the dietary habit of the pregnant mothers was categorized as observing food taboos and not observing food taboos. with regard to the custom of stopping breastfeeding, the investigators asked the mothers whether they believed or not that breastfeeding the children until the age of 24 months will interfere with the growth of the children. the custom of stopping breastfeeding was categorized as stopping breastfeeding at <24 months and stopping breastfeeding at ≥24 months. the question posed to the mothers about the custom of administering food to the newborn was whether or not there were certain foods that according to culture must be given to newborn infants. administration of foods to newborn infants was categorized as administering the foods and not administering the foods. data on the maternal educational level were obtained by asking the mothers about their level of formal education up to the time of interview. the educational level was categorized as low if the respondents did not have any education or received education up to primary school or junior high school, and as high if the respondents went from senior high school to tertiary education, either at diploma level or higher. as to household expenditure, the parents were asked about their weekly expenditure for food for consumption by all family members. the weekly expenditure was then multiplied by four to obtain the total monthly expenditure. the cutoff point was determined on the basis of the mean total expenditure. the level of household expenditure was categorized as low (<rp.921.250) and high (≥rp.921.250). data on maternal outdoor activity were also obtained by interviews using questionnaires. the dellu, mexitalia, rosidi maternal perception of sickness and stunting 159 investigators asked the mothers whether they were involved in certain activities so that they had less time to care for their children. maternal outdoor activity was categorized as present and absent. statistical analysis bivariate analysis was performed using the chi square test with 95% confidence interval (95% ci) (p<0.05) to find the p value and the odds ratio (or). multivariate analysis was by means of the logistic regression test. ethical clearance this study obtained ethical clearance no.008/ec/fk-rsdk/2016 from the ethics commission. results the proportions of males in the group of cases and controls were 68.9% and 51.1%), respectively. there were more farmers among the subjects’ fathers in the group of cases (91.1%) than in the group of controls (86.7%). the number of mothers who were housewifes or unemployed was greater in the group of cases (73.3%) than in the group of controls (51.1%). there was no difference in paternal height between the group of cases and the group of controls, with mean heights of 160.1 cm and 160.8 cm, respectively. on the other hand, there was a difference in maternal height between the group of cases and controls, with mean heights of 149.2 cm and 152.4 cm, respectively. the results of the analysis showed that the risk factors for the prevalence of stunting in children were maternal perception about sickness, with sickness being considered a curse (or=8.94;95% ci 3.26-24.46), the custom of stopping breastfeeding at <24 months (or=6.20; 95% ci 2.29-16.80), and low level of expenditure (or=6.22;95% ci 1.89-20.46) (table 1). univ med vol. 35 no.3 *significant (p<0.05) table 1. relationship between several risk variables and incidence of stunting 160 table 2. results of logistic regression of factors influencing incidence of stunting in children aged 2-5 years *significant (p<0.05), r2=0.49=49% on the basis of the results of bivariate analysis, the variables included in the multivariate analysis were individual perception, i.e. maternal perception about sickness, custom of stopping breastfeeding at <24 months, low level of household expenditure, and maternal outdoor activity (p<0.25). the results of the multivariate analysis showed that maternal perception about sickness, custom of stopping breastfeeding and level of household expenditure were risk factors for the prevalence of stunting. the variable of maternal outdoor activity was not a risk factor for the prevalence of stunting. the variable with the greatest influence or the dominant variable for the prevalence of stunting with highest or value w a s m a t e r n a l p e r c e p t i o n a b o u t s i c k n e s s (or=7.43;95% ci 2.37-23.21). this signifies that children whose mothers perceive sickness as a curse have a 7.43-fold greater probability to experience stunting than do children whose mothers do not perceive sickness as a curse (table 2). discussion maternal perception about sickness is the most dominant risk factor for the prevalence of stunting in children. the results of this study are in line with a study on underfives among the dawan clan in tts district, in which it was found that family beliefs in traditional ceremonies are a risk factor for the prevalence of short stature in children.(3) this study is also in line with the study of nabuasa who demonstrated a significant relationship between culture and the prevalence of stunting.(5) however, a study conducted in medan area district found no relationship between family traditions/beliefs and nutritional status of the children.(6) the perception of the community about the causes of a disease differred between regions, depending on the culture prevailing and developing in the community in question. the community of the tts district in general still believes in various traditional ceremonies, such as the ceremony of confession of sins, called naketi in the local language, which is a ceremony for the healing of sick persons. it is usually performed by the parents of sick children in the hope that after performing the ceremony, the children will recover. the results of the present study are supported by the results of in-depth interviews with principal informants and triangulation informants. these informants said that the care of sick children consists in bringing them to a doctor or healthcare facility, but if they do not recover, the cause of the sickness is sought on the basis of local beliefs, i.e. through the naketi ceremony. however, the children are frequently not brought to a hospital or medical facility, since the community is of the opinion that bringing the children to a medical facility is useless, because this will not heal the children, but on the other hand will worsen their sickness. sickness is caused not only by a disease but is according to them is also attributable to bad winds or parental obstruction. the custom of food taboos for pregnant mothers is not a risk factor for the prevalence of stunting. food taboo is the prohibition of consuming certain types of food, since their consumption will endanger the consumer. initially food taboos were meant to protect the health of dellu, mexitalia, rosidi maternal perception of sickness and stunting 161 the children and their mothers, but may actually become detrimental to the children’s health and nutrition, since the food taboos usually concern foods that are sources of essential nutrients for both the mothers and the children. the absence of a relationship between the custom of food taboos for pregnant mothers and the prevalence of stunting is caused by the higher total proportion of mothers in the group of cases and controls who did observe the food taboos when pregnant, in comparison with those who did not. these results show the occurrence of behavioral changes associated with food taboos. the custom of administering food to newborn infants is not a risk factor for the prevalence of stunting. this study agrees with that of aguayo et al.(12) who state that administering food to newborn infants had a significant but negative association with the prevalence of stunting. the custom of administering food to newborn infants is in principle associated with exclusive breastfeeding. the period of exclusive breastfeeding recommended by the who is the first 6 months. this recommendation was issued based on the results of a study revealing that exclusive breastfeeding up to 6 months has several benefits, such as a decrease in gastrointestinal and respiratory infections, that does not occur in exclusive breastfeeding up to 3 or 4 months.(13) the present study did not agree with the studies conducted by nadiyah and by muchina, who showed that children with prelacteal feeding were more at risk of stunting.(14,15) the absence of such a relationship in our study was caused by the greater total proportion (60.0%) of respondents who administered food to their newborn infants, both in the group of cases and the group of controls, in comparison with those who did not (40.0%). this signifies that among the subjects in the groups of cases and controls, there were still many who had received food as newborns or under the age of 6 months. the custom of stopping breastfeeding before the children are 2 years old (<24 months) is a risk factor for the prevalence of stunting in children. the present study is in agreement with previous studies stating that children who were breastfed for less than 24 months were more at risk for stunting, as compared to children who were breastfed for at least 24 months.(16,17) however, the present study is not in line with the study by kamal which showed that breastfeeding for less than 24 months is not a risk factor for the prevalence of stunting.(18) weaning is a term used to refer to the transition period in which infants still receive liquid food, either breast milk or formula milk, but are also gradually introduced to solid food. in england, the term weaning signifies the introduction of solid food along with milk (breast or formula milk), and not the process of separating the infants from their mother’s breasts.(19) the weaning process in children may start at different times. the who and unicef recommend that infants be breastfed up to the age of two years. in indonesia, there are certain cultural communities that wean the children before the age of 6 months or even after the age of 2 years. between the experts’ concept of weaning and weaning as understood by the community in tts district, there is a difference in definition. the community of tts district understands by weaning the definitive cessation of breastfeeding. when the children are able to consume the family foods or foods other than breast milk, they are not breastfed any longer and this weaning is called sole in the local language. in this process the infants are clearly separated from their mothers’ breasts. this is supported by in-depth interviews with the informants, who stated that if the children are able to consume family foods, they are not breastfed anymore, since this will impair their appetite. in addition, according to the informants, the longer the children are breastfed, the more dumb they become and the larger their buttocks. the custom of sole is one of the cultural traditions of the local community that up to the present time are still adhered to. the lack of breastfeeding or even the absence of breastfeeding up to 24 months has a profound impact, such as increased prevalence of diarrhea from unhygienic complementary foods or formula milk, poor nutrient adequacy for univ med vol. 35 no.3 162 children under the age of two years (baduta), allergic reactions in some of the children as a result of administration of formula milk or complementary feeding that is not suited to the condition of the children, and the increased household expenditure resulting from the purchase of formula milk. all these factors increase the probability of stunting in the children. maternal educational level is not a risk factor for the prevalence of stunting in children. this study is in line with the study of nasikah and margawati (20) who found that maternal educational level was not significantly associated with the prevalence of stunting. however, our study is not in line with previous studies showing that mothers with a low educational level are at risk for stunting in their children.(7,20) in fact, a higher maternal educational level will contribute to total family income and better social status, and will ultimately influence the nutritional status.(1) the level of household expenditure for food may be used to determine economic status. the results of this study showed that a low level of household expenditure for food is a risk factor for stunting in children. this study is in line with the study by yasmin, who state that lower household expenditure increases the risk of stunting.(7) households with low socio-economic status are more susceptible to food shortages because of their low food consumption and their limited access to food.(22) materrnal outdoor activity is associated with the length of time the mothers spend with their children, thereby providing good parenting for their children. our study showed that maternal outdoor activity is not a risk factor for the prevalence of stunting in children. this is in line with the study by lestari who found that maternal employment status had no significant relationship with the prevalence of stunting in children.(23) maternal outdoor activity may influence parenting pattern, either because the mothers are busy earning a living or are involved in time-consuming social activities. among mothers with an occupation, the probability of stunting in children is greater than among those without occupation.(20) parental short stature is not a risk factor for the prevalence of stunting in children. the results of this study is in line with the study of hanum(9) who found no relationship between parental height and prevalence of stunting. parental short stature, in this case parental height and growth pattern, was not a determinant of stunting in their children. parents of short stature do not automatically have children of short stature and vice versa. according to the hardy-weinberg law of equilibrium,(24) height is hereditary and is transmitted continously from one generation to the next. however, this herediatry trait may also be influenced by changes in diverse environmental factors such as socioeconomic status, dietary pattern and physical activity of the children. the absence in this analysis of a relationship between the stature of parents and stunting in their children was caused by the almost identical parental growth patterns in the group of cases as well as the controls, so that in the analysis no association was found. according to the who, the prevalence of stunting in children is the result of a complex interaction of various factors, among others household condition, environmental influence, socio-economics and culture.(25) the risk factors that were investigated in this study were only socio-cultural factors, namely factors that only indirectly cause stunting. on the other hand, there are still many other factors with a direct influence on stunting, such as dietary intake and infectious disease. in this study no in-depth investigation was carried out on parental short stature in relation to genetic factors. determination of parental stature was based only on measurement of parental height during the study, while no research was done on the height of the preceding generations. the occurrence of a relationship between maternal perception about sickness and the prevalence of stunting in children aged 2–5 years, show the need for intensive education to improve culture-related community concepts that tend to be detrimental to the community, particularly the underfives. further in-depth studies should be dellu, mexitalia, rosidi maternal perception of sickness and stunting 163 conducted on the relation of parental genetic factors with the prevalence of stunting in children, since height is the product of interaction between genetic and environmental factors. conclusion maternal perception of sickness was the most dominant risk factor of stunting in children 2-5 years of age. a multidisciplinary approach is needed to address the range of raised issues and so combat stunting in children. conflict of interest there were no conflicts of interest in this study , which was entirly self-financed. acknowledgments the authors wish to express their utmost gratitude to the study subjects and respondents who agreed to participate in this study. the authors also thank the supervisors and examinators who provided helpful advice for writing the study report. references 1. senbanjo io, oshikoya ka, odusanya oo, et al. prevalence and risk factors for stunting among school children and adolescents in abeokuta, southwest nigeria. j health pop nutr 2011;2: 364-70. 2. united nations children’s fund (unicef). tracking progress on child and maternal nutrition: a survival and development priority. new york: united nations children’s fund (unicef);2009. 3. kusumaningrum r. studi epidemiologi sosial kejadian stunting anak balita suku dawan di kabupaten timor tengah selatan (tts) (tesis). kupang: universitas nusa cendana;2014. 4. kementerian kesehatan republik indonesia (kemenkes ri). riset kesehatan dasar (riskesdas) tahun 2013. jakarta: kementerian kesehatan republik indonesia (kemenkes ri); 2013. 5. nabuasa cd, juffrie m, huriyati e. riwayat pola asuh, pola makan, asupan zat gizi berhubungan dengan kejadian stunting pada anak usia 24-59 bulan di kecamatan biboki utara, kabupaten timor tengah utara provinsi nusa tenggara timur. jurnal gizi dietetik indonesia 2013;1:3143. 6. yudi h. hubungan faktor sosial budaya dengan status gizi anak usia 6-24 bulan di kecamatan medan area, kota medan (tesis). medan: universitas sumatera utara; 2008. 7. yasmin g, lilik k, cesilla md. risk factors of stunting among school-children from eight provinces in indonesia. pakistan j nutr 2014;13: 557-66. 8. solihin rdm, anwar f, sukandar d. kaitan antara status gizi, perkembangan kognitif, dan perkembangan motorik pada anak usia presekolah. jurnal penelitian gizi makanan 2013;36:62-72. 9. hanum f. hubungan asupan dan tinggi badan ibu dengan status gizi anak balita. jurnal gizi pangan 2014;9:78-1059. 10. kusuma ke, nuryanto. faktor risiko kejadian stunting pada anak usia 2-3 tahun (studi di kecamatan semarang timur). j nutr coll 2013; 2:523-30. 11. barstow c, rerucha c. evaluation of short and tall stature in children. am fam physician 2015; 92:43-50. 12. aguayo vm, nair r, badgaiyan n, et al. determinants of stunting and poor linear growth in children under 2 years of age in india: an indepth analysis of maharashtra’s comperhensive nutrition survey. matern child nutr 2016;12: 121-40. 13. cahyani nww, widarsa ikt. penerapan analisis jalur dalam analisis faktor determinan eksklusivitas pemberian asi di wilayah kerja puskesmas payangan, gianyar. j comm health 2014;2:96-106. 14. nadiyah, briawan d, martianto d. faktor risiko stunting pada anak usia 0-23 bulan di provinsi bali, jawa barat, dan nusa tenggara timur. jurnal gizi pangan 2014;9:125-32. 15. muchina en, waithaka pm. relationship between breastfeeding practices and nutritional status of children aged 0–24 months in nairobi, kenya. african j food agric nutr dev 2010;10: 1-14. 16. teshome b, kogi-makau w, getahun z, et al. magnitude and determinants of stunting in children under-five years of age in food surplus region of ethiopia: the case of west gojam zone. ethiop j health dev 2009;23:98-106 17. fikadu t, assegid s, dube l. factors associated with stunting among children af age 24 to 59 months in meskan district, gurage zone, south univ med vol. 35 no.3 164 dellu, mexitalia, rosidi maternal perception of sickness and stunting ethiophia: a case-control study. bmc public health 2014:14;1-7. 18. kamal m. socio-economic determinants of severe and moderate stunting among under five children of rural bangladesh. mal j nutr 2011; 17:105–18. 19. more j. gizi bayi, anak dan remaja. yogyakarta. pustaka belajar; 2014. 20. picauly i, sarci mt. analisis determinan dan pengaruh stuntingterhadap prestasi belajar anak sekolah di kupang dan sumba timur, ntt. jurnal pangan gizi 2012;8:55-62. 21. nasikhah r, margawati a. faktor resiko kejadian stunting pada balita usia 24-36 bulan di kecamatan semarang timur. j nutr coll 2012;1:176-84. 22. dekker lh, mora-plazas m, marin c, et al. stunting associated with poor socioeconomic and maternal nutrition status and respiratory morbidity in colombian school children. food nutr bull 2010;31:242-50. 23. lestari w, margawati a, rahfiludin zm. faktor risiko stunting pada anak umur 6-24 bulan di kecamatan penanggalan kota subulussalam provinsi aceh. jurnal gizi indonesia 2014;3: 126-34. 24. nature education. hardy-weinberg equilibrium; 2014. available at: http://www.nature.com/ scitable/definition/hardy-weinberg-equilibrium122. accessed september 2, 2016. 25. stewart cp, iannotti l, dewey kg, et al. contextualizing complementary feeding in a broader framework for stunting prevention. maternal child nutrition 2013;9:27-45. oktavianus 43 *department of pharmaceutical chemistry, faculty of pharmacy, airlangga university, surabaya **department of molecular pharmacology, faculty of veterinary medicine, airlangga university, surabaya correspondence dr. juni ekowati, apt., m.si department of pharmaceutical chemistry, faculty of pharmacy, airlangga university kampus b unair jl. dharmawangsa dalam surabaya 60286 email: j_ekowati@yahoo.com univ med 2015;34:43-51 doi: 10.18051/univmed.2016.v35.43-51 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background many tumors express on their receptor tyrosine kinases vascular endothelial growth factor activity associated with angiogenesis. inhibition of angiogenesis through reduction of tyrosine kinase activity is a promising strategy for cancer therapy. the present study aimed to determine the mechanism and potency of ethyl p-methoxycinnamate (epmc) isolated from kaempferia galanga as angiogenesis inhibitor. methods a laboratory experimental study was conducted using chorio-allantoic membranes (cams) of nine-day old chicken eggs induced by 60ng basic fibroblast growth factor (bfgf). ethyl p-methoxycinnamate (epmc) potency was determined at dosages of 30, 60, 90 and 120 µg and compared with celecoxib 60 µg as reference drug and one negative bfgf-induced control group. neovascularization and endothelial cell count in cam blood vessels were evaluated. to predict the antiangiogenic mechanism of epmc, a docking study was performed with the molegro virtual docker program on tyrosine kinase as receptor (pdb 1xkk). results angiogenesis stimulation by bfgf was prevented significantly (p<0.05) by epmc at dosages of 30, 60, 90 and 120 µg and this activity was dose dependent. molecular docking showed interaction between epmc functional groups and tyrosine kinase amino acids at met766, met793, thr854, thr790, gln791 and ala743. there was an association between epmc antiangiogenic activity and docking study results. conclusions ethyl p-methoxycinnamate is a potential new angiogenesis inhibitor through interaction with tyrosine kinase. epmc could be a promising therapeutic agent for treatment of angiogenesis-related diseases. keywords: ethyl p-methoxycinnamate, chorio-allantoic membrane, angiogenesis, tyrosine kinase ethyl p-methoxycinnamate from kaempferia galanga inhibits angiogenesis through tyrosine kinase juni ekowati*, suko hardjono*, and iwan sahrial hamid** doi: http://dx.doi.org/10.18051/univmed.2015.v34.43-51 44 ekowati, hardjono, hamid kaempferia galanga inhibits angiogenesis etil p-metoksisinamat dari kaempferia galanga menghambat angiogenesis melalui interaksi dengan tirosin kinase latar belakang banyak tumor menunjukkan pada reseptor tirosin kinasenya ekspresi vascular endothelial growth factor yang berhubungan dengan angiogenesis. hambatan angiogenesis melalui pengurangan aktivitas tirosin kinase adalah strategi yang menjanjikan untuk terapi kanker. penelitian ini bertujuan untuk menentukan mekanisme dan potensi etil p-metoksisinamat (epmc) sebagai penghambat angiogenesis. metode sebuah studi eksperimental laboratorium dilakukan menggunakan chorio-allantoic membrane (cam) telur ayam tertunas berumur sembilan hari, yang diinduksi dengan basic fibroblast growth factor (bfgf) 60ng. potensi epmc diteliti pada dosis 30, 60, 90 dan 120 µg; dibandingkan dengan celecoxib 60 µg sebagai obat referensi dan satu grup tanpa perlakuan. neovaskularisasi dan sel endotel pembuluh darah baru dari cam dihitung dan dievaluasi. untuk prediksi mekanisme antiangiogenesis epmc, dilakukan studi doking pada reseptor tirosin kinase (pdb 1xkk) menggunakan program molegro virtual docker v.5.5 hasil stimulasi angiogenesis oleh bfgf pada cam dihambat secara signifikan (p<0.05) oleh epmc pada dosis 30, 60, 90 and 120 µg dan bersifat dose dependent. celecoxib dan epmc tersebut menyebabkan terjadinya lisis sel endotel dari cam. studi doking menunjukkan adanya interaksi antara gugus fungsi pada epmc dengan residu asam amino tirosin kinase pada met766, met793, thr854, thr790, gln791 dan ala743. studi doking dan aktifitas antiangiogenesis epmc tersebut menunjukkan hasil yang berhubungan. kesimpulan etil p-metoksisinamat merupakan senyawa yang berpotensi sebagai angiogenesis inhibitor melalui hambatan pada tirosin kinase. kata kunci : etil p-metoksinamat, korio allantois membran, angiogenesis, tirosin kinase abstrak introduction tyrosine kinase receptors of many tumors express vascular endothelial growth factor (vegf) activity connected with angiogenesis.(1) in the last decade, angiogenesis has been explored in depth as an interesting cancer therapeutic target, since angiogenesis is an important step in tumor growth and cancer metastasis.(2,3) d r u g r e s i s t a n c e , i n c r e a s e d t u m o r progression, and signs of drug toxicity such as b l e e d i n g , f a t i g u e , h y p e r t e n s i o n a n d gastrointestinal perforation are the main clinical problems that occur in patients treated with angiogenesis inhibitors.(1) thus, the use of natural h e r b a l a l t e r n a t i v e a g e n t s t o c o n s t r a i n angiogenesis is quite crucial in cancer treatment. ethyl p-methoxycinnamate (epmc), a major constituent of kaempferia galanga linn. (local name: kencur, fam. zingiberaceae) has been used as sunscreen,(4) analgesic (5) and antiinflammatory.(6,7) agent, as cyclooxygenase-2 (cox-2) inhibitor, and to treat fibrosarcoma in mice.(8) it is has been established that pge 2 production in preclinical breast and colon cancer 45 models is strongly influenced by cox-2 expression. cox-2 inhibition augments vegf pathway blockade to destroy angiogenesis and tumor growth, avoid metastasis, and increase overall patient survival.(9-11) it has also been reported that epmc exerts anti-angiogenic activity in zebrafish (12) and can block endothelial function.(6) one method for the study of anti-angiogenic activity is by means of the chorio-allantoic membrane (cam) model. the cam is an extra-embryonic membrane formed on day 4 of incubation by fusion of the chorion and the allantois. the cam contains extracellular matrix proteins that mimic the p h y s i o l o g i c a l c e l l e n v i r o n m e n t a n d vascularization of cancers. use of cam has the advantages of easy accessibility and low cost, and is less time consuming.(13,14) the present study was done because there have been no reports of the potency of epmc to inhibit angiogenesis in the cam model. he et al.(12) reported that epmc influences multiple molecular targets of angiogenesis, therefore we evaluated the possibility of epmcm e d i a t e d t y r o s i n e k i n a s e i n h i b i t i o n b y performing a virtual molecular docking study using the molegro virtual docker program (pdb 1xkk). the objectives of our study were to determine the antiangiogenic potency of epmc and to predict whether the mechanism of action of this compound is related to its interaction with tyrosine kinase. methods research design this laboratory experimental study was conducted from may 2014 until september 2014. the research was performed in three steps, i.e. molecular docking study, isolation of epmc from kaempferia galanga and antiangiogenic assay using the cam model. docking study molecular docking studies of epmc, celecoxib and the reference ligand fmm_91[a] (lapatinib tosylate) into the three-dimensional tyrosine kinase structure were carried out using molegro virtual docker (mvd) software version 5.5. the receptor tyrosine kinase structure was obtained from the protein data bank (pdb 1xkk). ligand preparation was done using chembio ultra software version 10.0.; the geometry was optimized by the mmff 94 method and saved in sybyl mol2 format. all ligands were placed into the binding site of 1xkk (cavity-1) by alignment with the reference ligand fmm_91[a]) and 10 independent runs were performed. (15,16)the binding affinity between ligand and enzyme (docking score) was predicted using rerank scores. higher negative scores (kcal/mol) indicate stronger ligandenzyme binding, with the ligand filling the same pocket in the enzyme as in fmm_91[a] docking. validation of the docking study was carried out by redocking fmm_91[a] into cavity-1 of 1xkk. the best docking results can be observed visually by comparing the structure of the docked m o l e c u l e s w i t h t h e c r y s t a l s t r u c t u r e o f fmm_91[a] into the binding site. isolation of ethyl-p-methoxycinnamate from kaempferia galanga kaempferia galanga linn. was collected and determined by the botanist at materia medica botanical garden park at batu (near malang), indonesia, in august 2013. epmc was isolated from kaempferia galanga linn. by extraction of the dried powder of the rhizomes (1kg) with petroleum ether until the filtrate was nearly transparent.(6) the extract was filtered and concentrated in a rotavapor model laborota 4000 vacuum evaporator (heidolph instruments, germany) at 25°c, and then kept in the freezer. the extract was carefully washed with n-hexane, and the n-hexane supernatant was then decanted. this process was repeated until the solvent was almost transparent.(6) the residue that remained after washings consisted of pale yellow, needlelike crystals, which were recrystallized with ethanol 70%, yielding white pure epmc crystals (10.2 g), with a sharp melting point of 49°c, as univ med vol. 34 no.1 46 ekowati, hardjono, hamid kaempferia galanga inhibits angiogenesis observed using a fisher-john melting point a p p a r a t u s . t h e c h e m i c a l s t r u c t u r e w a s characterized spectroscopically using a jasco fourier transform infra red (ft-ir) 5300 spectrophotometer and a nuclear magnetic resonance (nmr) hitachi r-1900 spectrometer, confirming its identity as epmc. the ir and nmr spectroscopy results were as follows: ir (kbr) 3007, 2980, 2935, 1705, 1630, 1602, 1253, 1209, 829 cm-1 ; [1h nmr (400 mhz, cdcl 3 ) ä: 3.82 (3h, s, 1xoch 3 ); 6.96 (1h, d, j = 15.9hz , 1xch alkene); 6.88 (2h, d, j = 8.8 hz, 2xch benzylic); 6.96 (1h, d, j = 15.9 hz, 1xch alkene); 7.46 (2h, d, j = 8.8 hz, 2xch benzylic); 4.24 (2h, q, 1xch 2 ); 1.32 (3h, t, j = 7.2hz, 1xch 3 ).[13c (1h) nmr (400 mhz, cdcl 3 ) ä : 14.4 (ch 3 ), 55.4 (och 3 ), 60.4 (ch 2 ), 114.4 (benzylic ch2), 115.8 (ch alkene), 129.8(ar-c), 144.3 (ch alkene), 161.2 (ar-c), 167.4 (carbonyl c)]. antiangiogenic activity of epmc twenty-four nine-day old chicken eggs were obtained from pusvetma surabaya, and incubated at 37ºc and 60-70% humidity. after 1 d of incubation, a 1 cm2 window was opened at the top of the egg and the air was aspirated from the air chamber with a rubber ball until the cam came in contact with the eggshell membrane. on all cams of the experimental groups, induction of new blood vessels was performed with bfgf 60 ng dissolved in 60 ml of stock rh bfgf at a concentration of 1 ng/ml in a laminar air flow hood under aseptic conditions. the eggs were distributed in six groups; four groups were treated with epmc at dosages of 30, 60, 90 and 120 µg; one group was treated with celecoxib 60 µg as the positive control group, and one group was left without any treatment except bfgf blood vessel induction as the negative control group. to verify epmc antiangiogenic activity, a cam assay was accomplished with certain modifications as described before.(13,17) sterile 5-mm diameter w h a t m a n q u a l i t a t i v e f i l t e rp a p e r d i s k s containing bfgf alone, or supplemented with epmc (at dosages of 30, 60, 90 and 120 µg) or with celecoxib 60 µg, were implanted onto the cam of each embryo. the window was resealed with adhesive tape and the eggs were returned to the incubator until day 11 of chick embryo development. after that, the upper eggshell was removed and neovascularization as positive parameter was characterized by capillary branching of the main blood vessels onto the bfgf-containing paper disks. the capillaries attached at the edges and on the upper and lower s u r f a c e s o f t h e p a p e r d i s k s . t h e neovascularization parameter was considered to be negative if no capillary blood vessel formation occurred on the edges and upper and lower surfaces of the disk. those capillaries within 2,5 mm around the filter paper disk were observed and photographed. neovascularization around the disk was counted by determining the number of capillary branching of the main cam blood vessels onto the disk. in addition to macroscopic observation as mentioned above, we also carried out microscopic observations in the following way: the cam was removed from the shell, 10% formalin was added as preservative, and the cam was then prepared into histological slides and stained with hematoxylin and eosin (he).(13,17,18) endothelial cell formation in the neovascular capillaries was observed in cam cross-sections using an inverted nikon h600l phase contrast microscope. endothelial cell counts were determined in five visual fields of each slide at 400x magnification and compared w i t h p o s i t i v e a n d n e g a t i v e c o n t r o l s f o r subsequent analysis. statistical analysis s i g n i f i c a n t d i ff e r e n c e s b e t w e e n t h e treatment and control group were assessed by one-way analysis of variance (anova), supported by lsd test. differences were considered significant at p<0.05. ethical clearance this assay was approved by the ethical commision of airlangga university. 47 results the results of the molecular docking study (rerank scores) were -87.84 kcal/mol (epmc); -92.3184 kcal/mol (celecoxib); -143.23 kcal/mol (fmm_91[a]), in which the best dock of epmc, celecoxib and fmm_91[a] into tyrosine kinase was conducted in cavity-1 of the receptor. their interaction with tyrosine kinase (cavity-1 pdb 1xkk) is shown in figure 1. lapatinip tosylate displayed hydrogen bonding interaction between the following pairs: oxygen of ether moiety vs. leu792; nitrogen of amino moiety and oxygen of furan ring vs thr790; nitrogen of quinazoline ring vs. lys745; oxygen of sulphonyl moiety vs. gln791 and lys852. celecoxib presented h y d r o g e n b o n d i n g i n t e r a c t i o n o f t h e sulphonamide moiety with ala743, gln791 and l e u 7 9 2 . h o w e v e r, i t s h o w e d s t e r i c a n d hydrophobic interaction of the trifluoromethyl moiety with leu844, of the aromatic ring with lys745, ala743, and met793, and of the sulphonamide moiety with val742, gln791 and leu792 amino acid residues. epmc showed hydrogen bonding interaction of the oxygen moiety with met793, and steric and hydrophobic interaction of the met766, thr854, thr790, gln791 and ala743 of tyrosine kinase. it was concluded that functional groups of epmc can interact with some tyrosine kinase amino acids, similar to those of celecoxib and lapatinib tosylate. tr e a t m e n t o f c a m b f g f i n d u c e d neovascularization with celecoxib 60 µg caused endothelial cell lysis of the blood vessels and reduced cam neovascularization. celecoxib suppressed cam neovascularization by 83.4 ± 5.3% and growth of endothelial cells in capillary branches by 76.8 ± 2.2%. the potency of epmc as angiogenic inhibitor was dose-dependent. at doses of 30, 60, 90 and 120 µg, epmc inhibited neovascularization by 50.3 ± 4.5%, 67.6 ± 4.9%, 77.6 ± 6.3 %, and 81.9 ± 3.2 % (p<0.05) respectively. at the doses of 30, 60, 90 and 120 µg, epmc inhibited endothelial cell counts in capillary branches of cam by 50.9 ± 4.1%, 55.4 ± 2.9 %, 66.1 ± 4.7%, and 73.2 ± 4.5%, respectively (p<0.05) (table 1). figure 1. interaction of epmc (a), celecoxib (b), and fmm_91 [a] (c) with amino acids residue of tyrosine kinase (pdb 1xkk) a b c table 1. antiangiogenesis activity of epmc by treatment group using cam model all values are represented as the mean ± sd (n=4) and indicate significant differences compared with the control-untreated group at p<0.05 univ med vol. 34 no.1 48 ekowati, hardjono, hamid kaempferia galanga inhibits angiogenesis the endothelial cell counts of cam bfgfinduced capillaries in the negative control group were higher than in the epmc-treated groups (figure 2). treatment of cam with epmc caused lysis of the endothelial cells (figure 3). the higher epmc doses caused more endothelial cell lysis and decreased cam neovascularization more strongly. epmc as an antiangiogenic agent at doses of 30 and 60 µg was less active compared to celecoxib 60 µg. there were a s t a t i s t i c a l l y s i g n i f i c a n t d i ff e r e n c e s i n antiangiogenic potency between epmc doses of 90 and 120 µg. there were also a significant differences between celecoxib 60 µg and epmc at doses 90 and 120 µg (p<0.05). discussion angiogenesis plays an important role in delivering oxygen and nutrients essential for tumor progression and invasion of surrounding tissues and vessels. the development of a new vascular network consists of several steps which are controlled by various endogenous stimulators and inhibitors.(18) antiangiogenic therapy has great potential for developing new cancer treatment methods in the future.(19) figure 2. (a) capillary of neovascularization of cam induced by bfgf 60ng (white arrow), some of capillary blood vessels moving into the disk implanted on cam; (b) capillary of neovascularization of cam induced by bfgf 60ng and treated with celecoxib 60 µg (white arrow), none of capillary blood vessels moving into the disk implanted on cam (black arrow); (c) capillary of neovascularization of cam induced by bfgf 60 ng and treated with epmc 60 µg (white arrow), none of capillary blood vessels moving into the disk implanted on cam (black arrow) figure 3. photomicrograph images of endothelial cells of cam using an inverted phase contrast microscope (nikon h600l; camera ds fi2 300 megapixel) at 400 magnification. cam induced by b bfgf (black arrow), the morphology of endothelial cells is good. (b) cam induced by b fgf and treated by celecoxib 60 µg. some of the endothelial cells are lyzed (white arrow). (c) cam induced by b fgf and treated by epmc 60 µg. it also appears that some of the endothelial cells are lyzed (white arrow) a b c 49 he et al.(12) found by semiquantitative reverse transcription polymerase chain reaction assay that epmc influences multiple molecular targets connected to angiogenesis. a recent study, showed the chemopreventive effect of epmc against fibrosarcoma in mice and epmc’s capability to interact with cox-2.(8) the results of our molecular docking study on epmc’s interaction with the tyrosine kinase receptor (pdb 1xkk) predict the probable mechanism of epmc as a cancer chemopreventive agent. molecular docking is a virtual method, in which the ligand is placed on the binding site in such a manner as to obtain the lowest potential energy. the docking process consists of testing the coordinate area of the target binding mode and scoring each possible ligand pose into that site. the lowest value of rerank scores is expected as the best binding mode.(15) our results are in line with those of previous studies and tyrosine kinase is one of the molecular targets of epmc for inhibition of angiogenesis. altun et al.(20) reported that tyrosine kinase inhibitors are excellent agents for treating colorectal cancer and angiogenesis in a cam model. it is known that cox-2 may indirectly p r o v o k e a n g i o g e n e s i s b y i n c r e a s i n g t h e production of angiogenic factors, such as vascular endothelial growth factor (vegf).(18,19) our study also supports the antiangiogenic activity of the cox-2 inhibitor celecoxib in the cam assay. several reports revealed that there is a significant association between cox-2 expression and vegf-c expression. the combination of celecoxib with tamoxiven is a potential drug for treatment of breast cancers expressing high levels of vegf.(18) celecoxib as cox-2 inhibitor has been reported to suppress angiogenesis and growth of secondary bone tumors.(19) celecoxib overturns the expression of vegf by constraining the production of pge 2 and the stimulation of some signal transduction pathways by which the expression of vegf is upregulated.(21) basic fgf as a proangiogenic agent causes chronic inflammation that markedly increases the risk for the development of cancer, and therefore angiogenesis and vegf signaling might pose an important link between inflammation and tumor development.(19,20) histopathological analysis showed the presence of inflammatory cells on cam induced by bfgf. even though not directly proinflammatory, bfgf synergistically forces inflammatory mediator-induced leukocyte recruitment, thus indicating the relationship between inflammation and angiogenesis.(22,23) angiogenesis is triggered by increased arachidonic acid production as a result of prostaglandin pge 2 metabolism catalyzed by cox-2. prostaglandin affects vascularization a n d t h e c a n c e r m e t a s t a t i c p r o c e s s t h a t encourages the expression of proangiogenic factors, such as vegf and fibroblast growth factor (fgf).(24) a previous research study reported that epmc blocked cox-2 expession in the t47d cell line and caused lysis of that cell line.(25) this fact strengthens our prediction of the ability of antiinflammatory substances like epmc to block the starting step of angiogenesis. therefore, the present study was designed to investigate the potential angiogenesis inhibitory effect of epmc on cam. the outcomes showed that epmc is a potent antiangiogenic substance, t h e r e f o r e i t c a n b e u s e d a s a c a n c e r chemopreventive agent, in agreement with our earlier report. although the antiangiogenic activity of epmc is lower than that of celecoxib, epmc can be used as lead compound for developing agents with higher antiangiogenic activities. epmc can also be combined with other antiangiogenic drugs, e.g. bevacizumab, or with cox-2 inhibitors, such as celecoxib, so reducing the dose and decreasing the side effects of the drugs.(1,9) one limitation of this study is the virtual experimental study of interactions between epmc and tyrosine kinase, which eliminated the lipophilic effect of the compound. so, in our future research the experiment will be conducted by in vitro studies based on the present results. we will also study the inhibition of vegf expression on cam using epmc. univ med vol. 34 no.1 50 ekowati, hardjono, hamid kaempferia galanga inhibits angiogenesis the clinical implication of this study is the availability of an easily obtainable natural compound as a cancer chemopreventive agent. ethyl p-methoxycinnamate is easily obtained from kaempferia galanga, therefore its use may lower the expected cost of cancer treatment and increase life expectancy. conclusions ethyl p m e t h o x y c i n n a m a t e h a s antiangiogenic potency and its activity is dosedependent. the antiangiogenic potency of epmc is associated with its inhibition of tyrosine kinase. acknowledgment we t h a n k t h e m i n i s t r y o f n a t i o n a l education of the republic of indonesia, and the rector of airlangga university, indonesia, for financial support in 2014. thanks are also due to prof. dr. siswandono, ms., apt for mvd program support. references 1. gotink kj, verheul hmw. anti-angiogenic tyrosinee kinase inhibitors: what is their mechanism of action? angiogenesis 2010;13:114. 2. filho al, lopes jm, schmitt fc. angiogenesis and breast cancer. j oncol 2010. doi: 10.1155/ 2010/576384. 3. kyzas pa, stefanou d, agnantis nj. cox-2 expression correlates with vegf-c and lymph node metastases in patients with head and neck squamous cell carcinoma. mod pathol 2005;18: 153-60. 4. athikomkulchai s, vaymhasuwan p, tunvichien s, et al. the development of sunscreen product from kaempferia galanga. j health res 2007;21:253-6. 5. kusumawati i, yusuf h. phospholipid complex as a carrier of kaempferia galanga rhizoma extract to improve its analgesic activity. ijpps 2011;3 suppl 1:s44-6. 6. umar mi, asnawi mz, sadikun a, et al. ethyl p-methoxycinnamate isolated from kaempferia galanga inhibits inflammation by suppressing interleukin-1, tumor necrosis factor-?and angiogenesis by blocking endothelial functions. clinics 2014;69:134-44. 7. aroonrerk n, kamkaen n. anti-inflammatory activity of quercus infectoria, glycyrrhiza uralensis, kaempferia galanga and coptis chinensis: the main components of thai herbal remedies for aphthous ulcer. j health res 2009; 23:17-22. 8. ekowati j, bimo at, sukardiman, et al. structure modification of ethyl p-methoxycinnamate and their bioassay as chemopreventive agent against mice’s fibrosarcoma. ijpps 2012;4 suppl 3:s 528-32. 9. greehough a, smart hjm, moore ae, et al. the cox-2/pge 2 pathway: key roles in the hallmarks of cancer and adaptation to the tumour microenvironment. carcinog 2009;30:377-86. 10. yao l, liu f, hong l, et al. the function and mechanism of cox-2 in angiogenesis of gastric cancer cells. j exp clin cancer res 2011;30:13. doi:10.1186/1756-9966-30-13. 11. roskoski r. sunitinib: a vegf and pdgf receptor protein kinase and angiogenesis inhibitor. biochem biophys res commun 2007; 356:323-8. 12. he zh, yue gg, lau cb, et al. antiangiogenic effects and mechanisms of trans-ethyl pmethoxycinnamate from kaempferia galanga l. j agric food chem 2012;60:11309-17. 13. ribatti d. the chick embryo chorioallantoic membrane as in vivo assay to study antiangiogenesis. pharm 2010;3:482-513. 14. lokman na, elder asf, ricciardelli c, oehler mk. chick chorioallantoic membrane (cam) assay as an in vivo model to study the effect of newly identified molecules on ovarian cancer invasion and metastasis. intern j molec sci 2012;13:9959-70. doi: 10.3390/ijms13089959. 15. kusumaningrum s, budianto e, kosela s, et al. the molecular docking of 1,4-naphthoquinone derivatives as inhibitors of polo-like kinase 1 using molegro virtual docker. j app sci 2014;4: 47-53. 16. diyah nw, ekowati j, siswandono. synthesis and anti tumor activity evaluation of n, n’dibenzoyl-n,n’-diethylurea against human breast cancer cell line (mcf-7). ijpps 2014;6: 315-8. 17. salamah n, sugiyanto, hartati ms, et al. isolasi dan identifikasi eurycomanone akar pasak bumi (eurycoma longifolia, jack) serta uji anti angiogenik. majalah farmasi indonesia 2009; 20:118-26. 18. kumar bnp, rajput s, dey kk, et al. celecoxib alleviates tamoxifen-instigated angiogenic effect 51 by ros-dependent vegf/vegfr2 autocrine signaling. bmc cancer 2013;13:273. doi: 10. 1186/1471-2407-13-273. 19. klenke fm, gebhard m, ewerbeck v, et al. the selective cox-2 inhibitor celecoxib suppresses angiogenesis and growth of secondary bone tumors: an intravital microscopy study in mice. bmc cancer 2006;6:9. doi:10.1186/1471-24076-9. 20. altun a, temiz tk, balci e, et al. effects of tyrosine kinase inhibitor e7080 and enos inhibitor l-nio on colorectal cancer alone and in combination. chin j cancer res 2013;25:57284. 21. wang l, chen w, xie x, et al. celecoxib inhibits tumor growth and angiogenesis in an othotopic implantation tumor model of human cancer. exp oncol 2008;30:42–51. 22. fox sb, generally dg, harris al. review breast tumor angiogenesis. breast cancer res 2007;9: 1-11. 23. zittermann si, issekutz ac. basic fibroblast growth factor (bfgf, fgf-2) potentiates leukocyte recruitment to inflammation by enhancing endothelial adhesion molecule expression. am j patol 2006;168:835-46. 24. sahin e. cyclooxygenase-1 in cancer and angiogenesis. angiology 2009;60:242-53. 25. ekowati j, tejo ba, susilo i, et al. microwave assisted synthesis of ethyl p-methoxycinnamate derivative and its activity to blockade cox-2 expression on breast cancer cell line. proceeding of the 3rd icpaps; 2013 june 18-19. yogyakarta. p.32-8. univ med vol. 34 no.1 oktavianus 58 *deptement of biochemistry, faculty of medicine, padjadjaran university, bandung **deptement of obstetrics and gynaecology, hasan sadikin hospital/ faculty of medicine, padjadjaran university, bandung ***deptement of pathology anatomy, hasan sadikin hospital/ faculty of medicine, padjadjaran university, bandung correspondence edhyana sahiratmadja, m.d. phd deptement of biochemistry faculty of medicine padjadjaran university jl. eijkman no. 38 bandung email: e.sahiratmadja@gmail.com univ med 2014;33:58-64 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background persistent infection of high risk genotypes of human papilloma virus (hrhpv) has been established as the etiological cause for cervical cancer, and the most prevalent genotypes that infect the cervical tissue are hpv-16 and hpv-18. however, hpv genotype profile has been shown to differ according to geographical distribution across the globe. the present study aimed to determine the hpv genotype distribution in cervical cancer patients from bandung, indonesia. methods during the period of july – november 2010 viral dna was extracted from randomly chosen cervical cancer biopsies and subjected to genotype determination using the diagnostic linear array genotyping test (roche). the distribution of hpv genotypes was explored and the prevalence of hpv genotypes was mapped. results of 96 cervical cancer tissue samples, 76 (79.2%) were histopathologically classified as squamous cell cervical carcinoma. due to the high cost of hpv genotyping tests, only twenty-five samples were randomly genotyped. almost 90% of the cervical cancer patients were multiply infected with hpv-16 in combination with hpv-18, hpv-45, or hpv-52. the hpv-16 genotype had the highest prevalence, all samples being infected with hpv-16. conclusion the cervical cancer cases were predominantly infected by multiple hrhpvs with hpv-16 as the major genotype among other hrhpvs, supporting the carcinogenic role of this hrhpv. therefore, screening for hrhpvs in the general population is urgently needed as a means of early detection of cervical cancer. keywords: cervical cancer, hpv-16, multiple hpv infections, bandung multiple human papilloma virus infections predominant in squamous cell cervical carcinoma in bandung edhyana sahiratmadja*, maringan d.l. tobing**, birgitta m. dewayani***, bethy s. hernowo***, and herman susanto** 59 univ med vol. 33 no.1 infeksi multipel human papilloma virus mendominasi kanker serviks tipe sel skuamosa di bandung latar belakang infeksi virus human papilloma (hpv) tipe risiko tinggi yang kronik merupakan salah satu penyebab kanker serviks pada wanita. genotipe yang tersering menginfeksi jaringan serviks adalah hpv genotipe 16 dan 18. walaupun demikian, profil genotipe dapat berbeda-beda di seluruh dunia, tergantung dari geografinya. penelitian ini bertujuan untuk menentukan distribusi genotipe hpv yang menginfeksi jaringan kanker serviks di bandung, indonesia. metode selama periode juli – november 2010, dilakukan isolasi dna virus hpv dari biopsi jaringan kanker serviks yang diambil secara acak, kemudian genotipe hpv ditentukan dengan menggunakan linear array hpv genotyping test (roche). selanjutnya dilakukan penelusuran distribusi genotipe hpv dan dibuat peta prevalensi genotipe hpv. hasil dari sejumlah 96 jaringan kanker serviks, 76 (79.2%) terdiagnosis secara histopatologis sebagai tipe squamous cell cervical carcinoma (scc). karena tingginya biaya uji genotipe hpv, penentuan genotipe hpv hanya dapat dilakukan pada 25 sampel scc yang diambil secara acak hampir 90% scc terinfeksi secara multipel oleh hpv16 dengan kombinasi hpv-18, hpv-45, atau hpv-52, sedangkan prevalensi hpv-16 pada jaringan kanker serviks mencapai 100%. kesimpulan infeksi multipel hpv dengan genotipe risiko tinggi mendominasi jaringan kanker serviks, sesuai dengan peran genotipe hpv resiko tinggi sebagai agen karsinogenik, dengan prevalensi tertinggi untuk hpv-16. untuk itu skrining genotipe risiko tinggi hpv menjadi hal yang sangat dibutuhkan sebagai upaya deteksi dini kanker serviks. kata kunci: kanker serviks, hpv-16, infeksi multipel hpv, bandung abstrak introduction cervical cancer is the second most prevalent female cancer after breast cancer, affecting more than 150,000 women worldwide annually, of which 80% occur in developing countries.(1) this cancer continues to impose a significant health burden in lowand medium-resourced countries of sub-saharan africa, latin america, and south and south east asia.(1) in indonesia, cervical cancer ranks first among gynecological cancers and has become a major health problem in this country.(2) chronic human papilloma virus (hpv) infection is strongly associated with the development of cervical cancer. hpv infections in the cervix are frequently associated with intraepithelial neoplasia and invasive squamous cell carcinomas (scc) with all their different histological variants i.e. large-cell keratinizing, large-cell nonkeratinizing and small-cell carcinoma.(3) hpv types 16, 18, 31, 52, and 58 are considered carcinogenic or high-risk types, including other hpv types such as hpv type 33, 35, 39, 45, 51, 56, 59, 68, 73, and 82.(4) other hpv types 26, 53, and 66 are considered 60 as probably carcinogenic, and hpv types 6, 11, 40, 42, 43, 44, 54, 61, 70, 72, 81 are low-risk types.(4) persistent infection by high-risk hpv plays a pivotal role in the etiogenesis of cervical cancer, particularly hpv types 16 and 18. these two viruses were consistently identified i n c e r v i c a l c a n c e r c a s e s , r e g a r d l e s s o f geographical distribution.(5) interestingly, in a meta-analysis report, women with normal cytology in the general population have a d i f f e r e n t d i s t r i b u t i o n o f h p v i n f e c t i o n compared to what is usually found in cervical cancer cases.(6) there is also a shift in hpv distribution spanning from normal cervical cytology in the general population, to cervical intraepithelial neoplasia (cin)1, cin2, cin3 and finally invasive cancer.(7,8) furthermore, studies in the capital city of indonesia, jakarta, have shown different results from those in other parts of the world, showing that in indonesia hpv-18 is more predominant in adenocarcinoma, followed by hpv-16.(9) studies in three other regions of indonesia have reported that even though hpv-16 and hpv-18 were equally common in the general population, hpv-52 was the most prevalent type.(10) thus, it is recommended that hpv-52 should be included in prophylactic hpv vaccination.(10) studies on the distribution of hpv types in a particular area are useful to determine the type of hpv vaccine necessary for that area. however, the indonesian archipelago covers a large area, making such a study unrealistic. a smaller study, covering a smaller area was undertaken to determine the distribution of hpv in cervical cancer in bandung, indonesia. methods research design this study was part of the hpv prevalence s u r v e y u n d e r t a k e n i n b a n d u n g a n d t h e surrounding areas which was conducted by the department of obstetrics and gynecology, hasan sadikin hospital, bandung, indonesia, from july to november 2010. study subjects and sample collection all newly-diagnosed cervical cancer patients during this period, with international federation of gynecology and obstetrics (figo) classification stage iia/b, were counseled to take part in the study. biopsies from the cervical t i s s u e s w e r e d i a g n o s e d b y t h e h o s p i t a l pathologists and classified according to the world health organization (who) classification.(11) hpv genotyping test after diagnosis, the tissues were sent to the dharmais national cancer hospital for hpv genotyping using linear assay hpv genotyping according to the manufacturer’s protocol (roche molecular diagnostics, branchburgh, new jersey, usa). the assay detects 37 highand low-risk hpv genotypes. the hpv genotypes detected include 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73 (mm9), 81, 82 (mm4), 83 (mm7), 84 (mm8), is39, and cp6108. briefly, dna was extracted from the tissue specimen using the amplilute liquid medium extraction kit (roche molecular diagnostics, branchburgh, new jersey, usa). the pcr amplicons were denatured using the d e n a t u r a t i o n s o l u t i o n a n d s u b j e c t e d t o hybridization with a single hpv genotyping strip that was coated with hpv type-specific and human â-globin probes. the biotin-labeled amplicons were hybridized to the probes only if t h e t y p e s p e c i f i c s e q u e n c e m a t c h e d t h e amplicons. the biotin-labeled amplicons were detected by colorimetric development and the results were read visually by comparing the pattern of colored lines to the provided reference guide. the available number of assays for this study was very limited due to high cost of hpv g e n o t y p i n g t e s t s . to m a k e t h e s a m p l e homogenous, we decided to randomize only the non-keratinizing cases for this study. ethical clearance ethical clearance was granted by the faculty of medicine, padjadjaran university, bandung. sahiratmadja, tobing, dewayani, et al multiple human papilloma virus 61 univ med vol. 33 no.1 results during the period of the study between july and november 2010, a total of 100 women who came to the outpatient clinic of the department of obstetrics and gynecology, hasan sadikin general hospital, bandung, indonesia were newly diagnosed with cancer of the cervix with figo classification iia/iib. they were all counseled to take part in the study and 96 finally agreed to be involved. histological examination of the cervical tissues confirmed that 76 (79.2%) were squamous cell carcinoma (scc), which were mostly of the non-keratinizing type (n=70, 92.1%). the distribution of biopsy classification of these patients is as shown in table 1. b i o p s y s a m p l e s c l a s s i f i e d a s n o n keratinizing and moderately differentiated samples (n=59) were then randomly genotyped (n=25). the tests revealed that all the samples were infected by hpv-16. however, only three samples showed a single infection by hpv-16. the rest of the samples (n=22) had multiple infections; hpv-16 infected the tissue in various combinations with other high-risk hpv types, with the most prevalent types being hpv-18, hpv-45 and hpv-52 as shown in table 2. lowrisk hpv type hpv-61 was also detected in combination with high-risk type hpv-16 (n=1). thus, our random genotyping of squamous cell carcinoma of the cervix during the period of study was dominated by high-risk type hpv-16, followed by high-risk hpv types 18, 45, and 52, occurring in equal percentages (table 3). discussion studies have shown that even a cervix with normal cytology may harbor early infection of human papillomavirus (hpv).(12) it is estimated that around 291 million women worldwide with normal cervical cytology are actually carriers of the hpv dna, of whom 32% are infected with hpv types 16, 18 or even with both.(6) however, hpv infection is usually a self-limiting infection as cell-mediated response immunity is mounted t o c l e a r t h e i n f e c t i o n a n d i n d u c e l e s i o n regression.(13) there seems to be a shift in hpv distribution from normal cytology to cin that develops into invasive cancer and it has been table 2. distribution of hpv genotypes of non-keratinizing moderately differentiated squamous cell carcinoma (n=25) nk scc= non-keratinizing moderately differentiated squamous cell carcinoma; *hpv-61 belongs to the low-risk hpv genotypes hpv infecti ons nk scc single infection hpv-16 3 12% multiple infections 22 88% hpv-16, -18 5 hpv-16, -18, -45 1 hpv-16, ----, -45, -52/ 33/38/ 58 6 hpv-16, -18, -45, -52/33/38/ 58 5 hpv-16, -18, -----, -52/33/38/58 2 hpv-16, ----------, -51 1 hpv-16, -18.-----, -51 1 hpv-16, --------, --------61* 1 hpv genotype nkscc % hpv-16 25 (100.0) hpv-18 14 (56.0) hpv-45 12 (48.0) hpv-52 13 (52.0 hpv-51 2 (8.0) hpv-61* 1 (4.0) table 3. the prevalence of hpv genotypes in 25 patients with non-keratinizing moderately differentiated squamous cell carcinoma nk scc is non-keratinizing moderately differentiated squamous cell carcinoma; *hpv-61 belongs to the low-risk hpv genotypes hi stop athology classi fic ation* n % adenocarcinoma 11 11.5 adenosqua mous carcinoma 1 1.0 squamous cel l carcinoma: keratinizing 6 6.3 non keratinizing moderate differentiated 59 61.5 poor differentiated 11 11.5 others 8 8.3 table 1. histopathological distribution of cervical cancer patients with figo stage iia/b (n=96) *classification according to who classification 62 found that the high-risk hpv type 16 remains the major hpv infection in cervical cancer.(8) the current study showed that hpv type 16 was detected in all non-keratinizing squamous cell cervical carcinomas from bandung, either as a single infection or in combination with other high-risk hpvs. the distribution in our biopsy samples from women with cervical cancer was similar to the meta-analysis conducted by munoz et al.(14) where the most common hpv types in the patients, in descending order of frequency, were types 16, 18, and 45. however, our results also showed the presence of hpv type 52 infection, occurring at an equal percentage as did types 18 and 45. these results are contrary to the result of a similar study from jakarta, indonesia.(10) the difference in the results between these two studies may be due to the different types of carcinoma found in each study. the study from jakarta has reported multiple infections that were mostly found in adenosquamous carcinomas,(15) while in bandung the cervical samples recruited were histopathologically classified as nonkeratinizing squamous cell cancer, with only around 10% being adenocarcinoma. the high number of multiple hpv infections in this area need to be further explored to have a better insight on hpv genotype distribution in indonesia. more studies on hpv genotypes in cervical cancer might give accurate prevalence rates of hpv infection in cervical cancer in bandung. moreover, the distribution of single hpv infections in the bandung samples also differs significantly when compared with the global data. this could be explained by the different techniques used in the hpv genotyping in each study. the study in jakarta had used the innoline probe assay prototype from innogenetics (gent, belgium) for the genotyping assay (10) while the study in bandung had used the linear a s s a y f o r d n a g e n o t y p i n g f r o m r o c h e molecular diagnostics (branchburgh, new jersey, usa). different hpv type-specific profiles may be observed during genotyping using different methods on the same sample.(16,17) consistency in the dna purification protocol is r e q u i r e d t o g u a r a n t e e i n t r a a s s a y reproducibility.(18) the limitation of the linear assay probe used in our study is in the detection of the hpv genotype 52. the assay cannot differentiate type 52 from types 33, 35 and 58. since this test can separately detect hpv 33, 35 and 58, we have concluded that our samples were co-infected with hpv 52 since no other bands were shown at the hpv 33, 35 and 58 locations. based on this assumption, 52% of our samples were co-infected by hpv-52. another limitation of this study is the limited number of biopsies screened for hpv genotyping. linear assay for hpv genotyping test is currently too costly, making it not readily available to the general population in bandung. it is clear that the most prevalent hpv infection in cervical cancer is the high-risk type hpv-16. therefore, hpv genotype screening for the general population is necessary to detect those that are at high risk to develop cancer of the cervix,(19) even though it is worth noting that a cervix with normal cytology may harbor early high-risk type hpv infection that can be cleared by natural immunity of the host. it is even more important that the current introduction of various products of hpv genotype detection into the health system will yield different distributions with each product specifically suitable for certain hpv types only.(16) it should borne in mind that the types of hpv associated with the cervical cancer that is prevalent in the local scenario would determine which hpv vaccine product is suitable for that particular region. to give such a vaccine to a population without knowing the hpv types in the area would be a wasteful effort. conclusions t h e c e r v i c a l c a n c e r c a s e s w e r e predominantly infected by multiple hrhpvs with hpv-16 as the major genotype, which is in accord with the role of hrhpvs as carcinogenic agents. therefore, screening for hrhpvs in the general population is urgently needed as a means of early detection of cervical cancer. sahiratmadja, tobing, dewayani, et al multiple human papilloma virus 63 univ med vol. 33 no.1 acknowledgements we are grateful to dr. sri hartini and dra. runingsih from the dharmais national cancer hospital for their contribution in the hpv genotyping test. we thank ms. nurul setia rahayu and dr. ani melani maskoen, from the health research unit laboratory, faculty of medicine, padjajaran university, bandung, for their technical support. thanks are also due to prof. zainul rashid from universiti kebangsaan malaysia for carefully reading the manuscript. this work was partially supported by research grant under the strategi nasional dikti for the year 2010. references 1. castellsagué x. natural history and epidemiology of hpv infection and cervical cancer. gynecol oncol 2008;110 suppl 2:s4-7. 2. aziz mf. gynecological cancer in indonesia. gynecol oncol 2009;20:8-10. 3. khenchouche a, sadouki n, boudriche a, houali k, graba a, ooka t, et al. human papillomavirus and epstein-barr virus coinfection in cervical carcinoma in algerian women. virol j 2013;10:340. doi:10.1186/1743422x-10-340. 4. bruni l, diaz m, castellsague x, ferrer e, bosch fx, de sanjosé s. cervical human papillomavirus prevalence in 5 continents: metaanalysis of 1 million women with normal cytological findings. j infect dis 2010;202:178999. 5. de sanjose s, quint wg, alemany l, geraets dt, klaustermeier je, lloveras b for retrospective international survey and hpv time trends study group. human papillomavirus genotype attribution in invasive cervical cancer: a retrospective cross-sectional worldwide study. lancet oncol 2010;11:104856. 6. de sanjosé s, diaz m, castellsagué x, clifford g, bruni l, munoz n. worldwide prevalence and genotype distribution of cervical human papillomavirus dna in women with normal cytology: a meta-analysis. lancet infect dis 2007;7:453-9. 7. sjoeborg kd, tropé a, lie ak, jonassen cm, steinbakk m, hansen m. hpv genotype distribution according to severity of cervical neoplasia. gynecol oncol 2010;118:29-34. 8. coutlée f, ratnam s, ramanakumar av, insinga rr, bentley j, escott n, et al. distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in canada. j med virol 2011;83:103441. 9. de boer ma, vet jn, aziz mf, cornain s, purwoto g, van den akker be, et al. human papillomavirus type 18 and other risk factors for cervical cancer in jakarta, indonesia. int j gynecol cancer 2006;16:1809-14. 10. vet jn, de boer ma, van den akker be, siregar b, lisnawati, budiningsih s, et al. prevalence of human papillomavirus in indonesia: a population-based study in three regions. br j cancer 2008;99:214-8. 11. lax s. histophatology of cervical precursor lesions and cancer. acta dermatovenerol alp panonica adriat 2011;20:125-33. 12. schiller jt, day pm, kines rc. current understanding of the mechanism of hpv infection. gynecol oncol 2010;118 suppl: s127. 13. stanley m. hpv-immune response to infection and vaccination. infect agent cancer 2010;5:19. doi:10.1186/1750-9378-5-19. 14. munoz n, bosch fx, de sanjose s, herrero r, castellsague x, shah kv, et al. epidemiologic classification of human papillomavirus types associated with cervical cancer. n engl j med 2003;348:518-27. 15. schellekens mc, dijkman a, aziz mf, siregar b, cornain s, kolkman-uljee s, et al. prevalence of single and multiple hpv types in cervical carcinomas in jakarta, indonesia. gynecol oncol 2004;93:49-53. 16. wong fky, ching jcy, chow jkf. comparison of the diagcor genoflow human papillomavirus array test and roche linear array hpv genotyping test. open virol j 2010;4:169–74. 17. panigoro r, susanto h, novel ss, hartini s, sahiratmadja e. hpv genotyping linear assay test comparison in cervical cancer patients: implications for hpv prevalence and molecular epidemiology in a limited-resource area in bandung, indonesia. asian pac j cancer prev 2013;14:5843-7. 18. dona mg, benevolo m, pimpinelli f, battista m, rollo f, stivali f. comparative evaluation of different dna extraction methods for hpv genotyping by linear array and innolipa. j med virol 2011;83:1042-7. 64 sahiratmadja, tobing, dewayani, et al multiple human papilloma virus 19. domingo ej, noviani r, noor mr, ngelangel ca, limpaphayom kk, thuan tv, et al. epidemiology and prevention of cervical cancer in indonesia, malaysia, the philippines, thailand and vietnam. vaccine 2008;26 suppl 12:m719. editorial 1 does aspirin have a role in preventing unprovoked recurrent thromboembolism? yenny department of pharmacology, faculty of medicine, trisakti university univ med vol. 32 no.1 venous thromboembolism (vte), comprising deep vein thrombosis (dvt) and pulmonary embolism (pe), is a frequently recurring and serious complication after cessation of anticoagulant therapy. several studies indicate that 10 years after cessation of anticoagulant therapy the risk of recurrent vte is 39.9% and nearly 50% for unprovoked vte.(1) administration of vitamin k antagonists has been shown to be effective in reducing the risk of recurrent vte but does not increase survival rate. cessation of anticoagulants before the recommended time is frequently due to the increasing risk of hemorrhage upon long-term use. to attain the recommended international normalized ratio (inr), laboratory investigation and adjustment of dosage are required.(2) aspirin retards platelet aggregation by inhibiting cyclooxigenase-1. low-dose aspirin is inexpensive and readily available, and does not require laboratory monitoring to evaluate its effectiveness. this drug has been shown to be effective for prevention of arterial vascular events and for primary prevention of vte in high-risk patients.(3,4) the recently published results of the warfarin and aspirin (warfasa) study(5) show that aspirin presumably plays a role in reducing the recurrence risk of unprovoked vte following a first-ever unprovoked vte after cessation of oral anticoagulant therapy, without increasing the risk of major hemorrhage. this multicenter investigator-initiated double blind placebo-controlled trial was carried out on patients with first ever unprovoked vte who had completed oral anticoagulant therapy for 6 to 18 months. the patients were randomly assigned to either aspirin 100 mg/day (n= 205) or placebo (n= 197) with the option to extend the duration of the study (median study duration 24.6 months). the results of this study showed that the percentage of vte recurrences in the group receiving aspirin was lower than that in the placebo group (6.6% vs 11.2% per year; hazard ratio = 0.58; 95% confidence interval [ci]= 0.36 – 0.92). adverse events were similar in both groups. the investigators acknowledge that this study was underpowered to be able to demonstrate an effect of aspirin on the incidence of ischemic heart disease or cardiovascular disease. therefore the results of this study cannot be applied to patients who require aspirin for prevention of arterial events. a systematic review comprising 17 studies showed that the mean incidence of arterial cardiovascular events per patient-year was higher in patients with unprovoked vte (0.46%; 95% ci=0.34 – 0.59), in comparison with provoked vte (0.35%; 95% ci=0.24 – 0.49).(6) a meta-analysis conducted on 6 studies showed that the risk of arterial cardiovascular events was higher in patients with unprovoked vte than in controls (incidence rate ratio [irr]= 1.87; 2 univ med vol. 32 no.1 95% ci=1.32-2.65] or in patients with provoked vte (irr=1.86; 95% ci=1.19 – 2.89). these studies showed that patients with unprovoked vte were at higher risk for arterial cardiovascular events in comparison with controls or patients with provoked vte after long-term follow-up.(6) the aspirin to prevent recurrent venous thromboembolism (aspire)(7) trial aimed to look for recurrences after first-ever unprovoked vte. this study also included patients with major vascular events and had a median study duration of 37.2 months. the patients received either aspirin 100 mg/day (n=411) or placebo (n=411). the results of this study demonstrated a lower vte recurrence in the aspirin group as compared with the placebo group. however, the decrease was statistically not signuificant (4.8% vs 6.5% per year; hazard ratio of aspirin = 0.74; 95% ci=0.52 – 1.05; p=0.09). in comparison with placebo, aspirin significantly reduced the rate of the two prespecified secondary composite outcomes. venous thromboembolism, myocardial infarction, stroke, or cardiovascular mortality decreased up to 34% (aspirin 5.2% vs placebo 8.0% per year; hazard ratio of aspirin = 0.66; 95% ci=0.48 – 0.92; p=0.01) and vte, myocardial infarction, stroke, major bleeding, or all-cause mortality decreased up to 33% (hazard ratio = 0.67; 95% ci=0.49 – 0.91; p=0.01). no statistically significant difference was found between the two groups with regard to bleeding and other serious adverse effects. in this study aspirin did not significantly reduce vte recurrence in comparison with placebo, but significantly reduced major vascular events. this study supports the benefit of aspirin therapy in preventing first-ever unprovoked vte when administered to patients after initial anticoagulant therapy. the warfasa and aspire trials provide evidence of the benefit of aspirin in the prevention of unprovoked vte, with the added of advantage of preventing major vascular events. apart from these studies, the guidelines on antithrombotic therapy and prevention of thrombosis issued by the american college of chest physicians (2012) recommend that patients with firstever unprovoked vte should be given oral anticoagulants for at least 3 months, targetting an international normalized ratio (inr) of 2.0 3.0. the decision for continuation or cessation of anticoagulants after this period should be made for each patient individually and balanced againts his or her hemorrhagic risk.(2) references 1. prandoni p, noventa f, ghirarduzzi a, pengo v, bernardi e, pesavento r. the risk of recurrent venous thromboembolism after discontinuing anticoagulation in patients with acute proximal deep vein thrombosis or pulmonary embolism: a prospective cohort study in 1,626 patients. haematologica 2007;92:199-205. 2. kearon c, akl ea, comerota aj, prandoni p, bounameaux h, goldhaber sz, et al. antithrombotic therapy for vte disease: antithrombotic therapy and prevention of thrombosis, 9th ed: american college of chest physicians evidence-based clinical practice guidelines. chest 2012;141:e419s-94s. 3. antiplatelet trialists’ collaboration. collaborative overview of randomised trials of antiplatelet therapy – iii: reduction in venous thrombosis and pulmonary embolism by antiplatelet prophylaxis among surgical and medical patients. bmj 1994;308:235-46. 4. antithrombotic trialists’ collaboration. collaborative meta-analysis of randomised trials of antiplatelet therapy for prevention of death, myocardial infarction, and stroke in high-risk patients. bmj 2002;324:7186. 5. becattini c, agnelli g, schenone a, eichinger s, bucherini e, silingardi m, et al. aspirin for preventing the recurrence of venous thromboembolism. n engl j med 2012;366:1959-67. 6. becattini c, vedovati mc, ageno w, dentali f, agnelli g. incidence of arterial cardiovascular events after venous thromboembolism: a systematic review and a meta-analysis. j thromb haemost 2010;8:8917. 7. brighton ta, eikelboom j, mann k, mister r, gallus a, ockelford p, et al. low dose aspirin for preventing recurrent venous thromboembolism. n engl j med 2012;367:1979-87. c:\users\jurnal fkusakti\docume 127 *department of ophthalmology, christian university of indonesia, jakarta correspondence: dr. gilbert ws simanjuntak, dr, spm(k) department of ophthalmology faculty of medicine christian university of indonesia jalan ps sutoyo, cawang jakarta, indonesia email: retinaid@yahoo.com http://orcid.org/0000-0002-9680-0605 date of first submission, march 17, 2018 date of final revised submission, july 20, 2018 date of acceptance, july 23, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina males tend to come earlier for eye care with the national health coverage programme in private hospitals gilbert ws simanjuntak*, jannes f tan*, and reinne natali christine* background cataract is the leading cause of blindness in indonesia, and poverty is a major barrier to having cataract surgery. increasing the proportion of adults that have regular, comprehensive eye examinations for cataract and other common eye health problems is one of the national health insurance (jkn) objectives. the objective of this study was to determine the impact of vision insurance on eye care utilization of cataract patients in private hospitals with the jkn system as social intervention. methods a cross-sectional study was conducted involving 230 cataract patients in a private hospital. inclusion criteria were patients with cataract surgery, diagnosis and follow up visit minimally two months after surgery. a multiple logistic regression was used to analyse the data. results initial visual acuity (va) was 0.13 ± 0.15 and final va was 0.91 ± 0.15. two patients had final va of 0.2 and 0.15, respectively, in which the first was associated with persistent corneal edema and diabetes mellitus, and the other with corneal scar. age, gender, and level of education were not associated with va at the first visit (p>0.05). further analysis using a multiple logistic regression model was found to be significant (p=0.0104), with gender being associated with preoperative vision, where males tend to come earlier to undergo cataract surgery/treatment. conclusion males tend to come earlier than females for cataract surgery with the national health insurance (jkn) programme, despite the social intervention of the programme. keywords: cataract, private hospital, universal health coverage, gender doi: http://dx.doi.org/10.18051/univmed.2018.v37.127-134 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cite this article as: simanjuntak gws, tan jf, christine rn. males tend to come earlier for eye care with the national health coverage programme in private hospitals. univ med 2018;37: 127-34. doi: 10.18051/univmed.2018. v37.1 27 -13 4 128 simanjuntak, tan, christine males come earlier for service with universal health introduction the prevalence of blindness in indonesia according to the rapid assessment of avoidable blindness (raab) survey in 2014-2016 reached 3%, making indonesia the country with the highest blindness prevalence in south east asia.(1) differing from the national prevalence, the prevalence of blindness in jakarta was 1.9%. the most common cause of blindness was cataract (>80%), a degenerative disease which can be easily cured by surgery.(2) there are some conditions which cause the high level of blindness, such as the demographic change with the increasing trend in the total population of elderly indonesians, so that the prevalence of diseases related to degenerative processes also increases. in addition, the cataract surgical rate (csr) in indonesia is low because of the inadequate number and uneven distribution of ophthalmologists. most ophthalmologists reside in big cities, for reasons such as convenience, their children’s education, facilities, and other considerations. therefore, people who live in remote areas tend to receive less health service, including eye health service, and this contributes to the national prevalence of blindness.(2) other obstacles that prevent patients from getting surgery are issues of knowledge, cost, location, the family’s approval that cannot be obtained, fear due to the lack of knowledge, and comorbidities such as diabetes mellitus (dm) or hypertension, that must be managed first.(2) ideally, the adequate number of ophthalmologists to serve a population group is one per 28,000 people, by looking at the ratio in canada which has a good healthcare system.(3) therefore ac cord ing to this ra tio, for j a kart a wi th 10,177,924 residents (4) 363 ophthalmologists are required. the number of hospitals in jakarta is 154,(5) one of which is the christian university of indonesia hospital (uki hospital) with three ophthalmologists. some hospitals in jakarta do not have ophthalmologists, therefore they refer the patients to hospitals which have such facilities. ever since the enactment of national health insurance in 2014, which is managed by the national health insurance (jaminan kesehatan nasional (jkn)), the number of hospital visits has increased. before that period, patients often came late for treatment because of financial reasons. the assumption that female are late in getting treatment is because males have priority over females in indonesia, due to financial reasons (out-of-pocket payment). so far there have been no reports of jkn’s social intervention on health services being linked to gender, in all areas of health services in indonesia. in order to get an overview of health services in the jkn era, an overview from the christian university of indonesia hospital (uki hospital) as a b type private hospital in regional area 1 will be given. this profile is needed in order to get a more comprehensive overview of j kn’s social intervention impact on gender, along with an overview of demographic barriers, educational background and results of surgery. methods research design this retrospective study used data of patients who sought treatment due to cataract, which were collected from the medical records of uki hospital, january december 2016. research subjects consecutive case study analysis was applied to collect clinical data from the medical records of patients with age-related cataracts. from the number of available data, it was expected that sufficient patients would be obtained to describe the population of patients who sought cataract treatment. the total sample per year was based on the calculation of 230 patients, which was according to the total number of surgical operations of 776 patients and the raab 2016 figure of 3%. the inclusion criteria were patients with diagnosis of cataract who had undergone surgery and visited the polyclinic for a follow-up examination at least 2 129 months after surgery. incomplete examination caused patients to be dropped out of the assessment. data collection the collected data covered patients’ identity, gender, age, education, visual acuity, systemic diseases, and further treatments required if necessary. education was recorded and classified as low if the patients’ last education was elementary school or if they did not attend school, and classified as high if the patients’ last education was middle school or higher. best corrected visual acuity before and after the surgery was examined using a snellen optotype and expressed in decimal units. visual acuity was classified as poor if the patient could only see with finger counting from 6 meters (<0.1) and classified as good if visual acuity was more than 0.1 with the best correction. eye examination the eye examination procedure in uki hospital was as follows. the patient had to do the registration first and would be examined in the clinic, including visual acuity measurement. slit-lamp examination was conducted to find out the cataract thickness, its conformity with the ability to see, and pupillar isocoria, while funduscopy was performed on selected patients. after surgery, the patient would undergo followu p e x a min at i on s a t l e a s t 3 t ime s t o ge t eyeglasses prescription. follow-up examinations after surgery were conducted on day 1, day 7, and 3 weeks later. the medications given after surgery were steroid and antibiotic drops, as well as oral antiglaucoma drugs at a dose of 2 tablets per day for 3 – 5 days in order to resolve the possibility of intraocular pressure (iop) increase. this was to anticipate the case of the viscoelastic injection not being totally aspirated after surgery. topical steroid dosage was tapered-off, starting at 6 times 1 drop daily, and reduced by 1 drop every 5 days until it was stopped. during the follow-up visit, intraocular pressure (iop) examination was also conducted. if a c o mpli c a t i o n o c c u r r e d , ap p r o pr i a te treatment would be given to resolve it. measurements syst e mi c d i se a se e x a mi n at i on wa s conducted by an internist and the patient would receive appropriate treatment. considering the surgery was conducted with local anaesthesia, standard examinations were performed, such as routine blood test and random glucose test, and electrocardiography if there was a suspicion of heart problems. the highest blood sugar level allowed for undergoing surgery was a random blood sugar level of <200g/dl. the highest blood p r e s s ur e ( bp) a l l o we d wa s sys t o l ic bp <180mmhg and diastolic bp <95mmhg. statistical analysis clinical and demographic data were processed using spss 21.0 (spss, chicago, il, usa). a chi-square test and multiple logistic regression were used to analyse the data. the results were considered significant if p<0.05. ethical clearance the study was conducted in compliance with the tenets of the declaration of helsinki. the christian university of indonesia hospital institutional review board granted approval for this study (no. 21/ pen/km/2017). results there were 776 cataract patients who sought treatment and received surgery in uki hospital in 2016. samples were randomly taken and a total of 230 medical records were taken and divided into 15-20 medical records per month. five patients could not be included due to incomplete follow-up examinations. the study sample consisted of 106 (47.1%) male patients and 119 (52.9%) female patients, wi th n o si gnif ican t dif fer ence in gender (p=0.424). mean age was 62.39 ± 10.25 years (range 31–83 years). there were 126 patients (56%) with middle high school (or equivalent) univ med vol. 37 no.2 130 simanjuntak, tan, christine males come earlier for service with universal health a b c education. based on the c lassification of educational level, there were 170 patients (75.6%) with high level of education (middle s c h oo l a n d h i gh e r ) . in t h i s stu d y, me a n preoperative acuity was 0.13 ± 0.15, which was classified as low vision and due to cataract, but also as preventable blindness. details are to be seen in table 1. there were 39 male patients and 47 female patients with poor visual acuity at the first treatment however, there was no significant difference in visual acuity between genders at the first visit (p=0.671). there were 126 patients (56%) with middle high school (or equivalent) education. out of 55 patients with low level of education, 25 patients (45.4%) had poor visual acuity (finger counting or worse) at the first visit. out of 170 patients with high level of education, 61 patients (35.9%) had poor visual acuity at the first visit. however, educational level was not associated with visual acuity at the first visit (p=0.204) (table 2). further analysis using multiple logistic regression was found to be a significant model, with gender as not a significant factor associated with preoperative vision (p=0.069), where males tend to come earlier for cataract surgery/ treatment. details of the variables analysed in logistic regression are shown in table 3. type of surgery and related factors phacoemulsification surgery was performed on 163 patients (72.4%) and extracapsular cataract extraction on 62 patients (27.6%). mean early visual acuity during examination was 0.13 ± 0.15, and after the surgery reached 0.91 ± 0.15. there were 20 patients who suffered from mild to moderate corneal edema on the first day visit, and there were no signs of edema in 19 patients on the 7th day visit. there were 2 patients with final visual acuity of 0.2 and 0.15, respectively. in the first patient with final visual acuity of 0.2, severe corneal edema was found, which did not disappear until the follow-up examination two months after the surgery. this patient also had dm for more than 30 years and hypertension. the second patient with final visual acuity of 0.15 had a c entral corne al scar, so corneal transplantation was needed. figure 1 shows the clinical pictures before and after the cataract surgery of one of the patients. characteristic n % gender : male female 106 119 41.6 58.4 age (years) 62.39 ± 10.25 (31 – 83) education high university middle-high school 44 126 19.6 56.0 low elementary school no education 39 16 17.3 7.1 surgery technique phacoemulsification extracapsular 163 62 72.4 27.6 early visual acuity 0.13 ± 0.15 final visual acuity 0.91 ± 0.15 financing jkn personal 221 4 98 2 table 1. characteristics of patients with cataract surgery, uki hospital 2016 jkn : jaminan kesehatan nasional (national health insurance) characteristics visual acuity p value 1/300 (n=86) >1/300 (n=139) gender male female educational level* high low 39 47 61 25 67 72 109 30 0.671 0.242 *education level : high : middle-high school and university; low : no education and elementary school table 2. gender and level of education with visual acuity on first visit 131 univ med vol. 37 no.2 systemic disease can affe ct surgical preparation especially in relation to the tight budget of jkn in each service, and also affect visual outcome after surgery due to posterior segment disorders. hhypertension was found in 61 patients (27.1%), diabetes mellitus (dm) in 17 patients (7.6%) and hypertension along with dm in 20 patients (8.9%). there were a total of 221 patients on the jkn programme and 4 patients on personal expense. there was one jkn patient who later underwent trabeculectomy surgery due to glaucoma (data not shown). discussion in this study the data shows that the total number of female cataract patients in uki hospital was slightly higher compared to that of the males (58% vs 41%). this agrees with several studies which show that the total number of female cataract patients is much higher than that of male patients. zetterberg et al.(6) report that according to evidence from epidemiologic data, cataract is more common in females than in males. this is not solely due to a higher rate of cataract extraction in females, as is the case in the western world. but several populationbased studies show that female have a higher p r e va l e nc e o f l e n s o pa c i ti e s , e sp e c i a l l y cortical.there is no firm evidence that lifestylerelated factors are the cause of this gender discrepancy. the focus has, therefore, been directed towards the role of estrogen in cataract formation. in a remote area far from cities in sumatera, a study conducted by husain et al.(7) found that females have a higher prevalence of nuclear and subcapsular posterior types of cataract. on the other hand, gender does not have any significant connection with prior visual acuity. as is already known, preoperative visual acuity is an important indicator for the progress towards eliminating cataract blindness. one study reported from nepal showed the same results about the relation between gender and preoperative visual acuity.(8) preoperative visual acuity also illustrates the community awareness toward s eye h ealt h ca re w her e upon t he decrease in eyesight as initial symptom of cataract, the patient should immediately visit a primary health care facility for his/her problems. this can be related to access to medical facilities. this study states that preoperative visual acuity in male patients is better than that in female patients. this is because male patients visit the medical facilities immediately after they find that they have blurred vision. in developing countries such as pakistan and india, it has been found that males have easier access to eye health s e r vi c e s , e s pe c i a l l y c a t a r a c t t r e a t me n t , variable or (β) p value educational level 1.09 0.897 age 1.04 0.101 gender 0.28 0.069 table 3. multiple logistic regression of several variables with preoperative vision a b figure 1. a. cataract patient before surgery with visual acuity of 0.3 b. clinical condition 7 days after phacoemulsification surgery, visual acuity 1.0 with correction 132 simanjuntak, tan, christine males come earlier for service with universal health compared to females. this is influenced by three factors, i.e. social cultural differences, access to primary health care facilities, and life expectancy rate.(9-12) courtright and lewallen (13) reported that females may not have freedom of movement, their need for eye care may not be considered as urgent or important as that of male family members, or they may not have financial decisionmaking authority within the family to pay for eye care treatment services. in addition, the woman’s child care responsibilities may make it difficult for her to leave home. in connection with the level of education, this does not statistically affect visual acuity prior to surgery. more than 35% of cataract patients come with visual acuity which is classified as severe visual impairment or blindness. this may happen because the other eye might still have good vision or in the elderly and retired persons, his/her poor visual acuity is considered enough for them to do daily activities at home. the beijing eye study states that there is no relationship of level of education with cortical cataract, retinal vein occlusion, systemic blood pressure, and intraocular pressure. however, the level of education is associated with myopia, decrease in nuclear cataracts and closed angle glaucoma.(14) more than 70% of surgeries conducted in uki hospital used the phacoemulsification technique which is the latest technique that delivers excellent surgical results in a short period. recovery of vision is obtained within 1x24 hours after surgery. if there is a complication in the form of posterior capsular rent, reimplantation of the lens after the complication can be managed in this hospital.(15) other complications, in the form of rhegmatogenous retinal detachment or macular hole, c an also be overcome under local anaesthesia.(16) in several cases, cataract surgery can be carried out with the extracapsular technique. this is still commonly used in hypermature/hard cataracts and/or old patients with low corneal endothelial cell counts. in general, the results of cataract surgery in uki hospital were good because more than 80% patients who underwent surgery had visual acuity better than 0.3 with a postoperative complication rate of under 5%. this fact is in accordance with studies in some asian countr ie s, in that both phacoemulsification and extracapsular techniques do not have significant differences in terms of the visual acuity achieved. the extracapsular technique is considered to be more efficient in terms of financing and to be widely applicable in developing countries.(17) these good results will surely have a role in reducing the number of persons with cataract blindness in jakarta in general. for patients, these results could increase their quality of life as well as productivity, while for hospitals these will be a good marketing tool. the cost of surgery should no longer be an obstacle for patients to obtain treatment or undergo surgery, ever since the national health insurance was enacted in 2014 and widely applied in both public and private hospitals.(18,19) the number of persons with blindness in jakarta reached 1.9% and >80% of these cases were caused by unoperated cataract. increasing the cataract surgical rate (csr) is the only effective way to reduce the number of persons with cataract blindness. in developing countries such as iran, where the number of persons with cataract is still quite high, increases in the number of cataract surgeries were made in order to raise the csr and it has been found that during 2000-2005 the number of cataract surgeries and csr increased from 526 to 1331 in these 5 years.(20) similar conditions were reported from china, where eliminating the backlog of patients with cataract blindness remains a formidable challenge in the urban areas of china. the widespread health insurance having somewhat removed the financial barriers to cataract surgery, it should have better coverage than at present, so educational efforts to further stimulate demand for surgery may be needed.(21,22) the efforts to increase the number of cataract surgeries begins with the education about the symptoms of cataract, the best type of surgery with quick and painless results, and the cost of which is covered by the national health insurance. 133 univ med vol. 37 no.2 cataract patients’ access to surgical services can be increased with the increase in the number of public and private hospitals which provide such services. it has to be kept in mind that cataract patients have limited vision, are mostly elderly and more often have degenerative/metabolic disease so that they have limited mobility and depend upon a caretaker. this is the reason why the provision of services near their home becomes very important.(23) by providing cataract surgery services, a hospital must make a large investment in advance for equipment, expensive materials, trained human resources, and maintenance of tools and instruments for cataract surgery. the cost of cataract surgery using the phacoemulsification technique in public type b hospitals in indonesia is rp 7,560,000. in india, the cost is around $120 to $195 and the total financing for cataract and low vision management in india is around $2.6 billion. this amount is considered effective and efficient because it is lower than the amount that the government has to spend on social welfare for those who are unable to work due to blindness.(20) discrepancies in financing and hospital claim issues are still a problem in the newly implemented j kn system. however, the existence of this health insurance system gives more opportunities for middle to lower classes to seek hospital treatment. before the jkn era, there was the assumption that males come earlier for treatment for financial reasons, since payment is out of pocket; males will also be prioritised for cultural reasons. the present study found that even with jkn’s social intervention which is predicted to eliminate this service barrier towards gender, gender bias is still to be found. this might be related to multiple factors and needs to be explored by further studies. our study has limitations in the assessment of jkn’s social interventions, such as the situation before and after the launching of jkn, whether jkn can change the outcomes of health services related to gender, socio-economic status, and other factors. gender itself might be affected by multiple factors, and needs to be explored by multivariate analysis using more data, which is beyond the scope of this study and needs further studies of different design. conclusion the national health insurance (jkn) implemented by the government impacts on the distribution of eye health services. in spite of the jkn’s social intervention programme which eliminates out of pocket payment and now makes services free, males still come earlier than females for cataract surgery. acknowledgment we thank yeni dwi lestari, m.d. for her suggestions. conflict of interest no conflict of interest was declared by the authors financial disclosure no financial disclosure was made by the authors. contributors gws contributed to the concept and design of the study, data analysis and/or interpretation. jft contributed to data acquisition and drafting. rnc revised the manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. das t, ackland p, correia m, et al. is the 2015 eye care service delivery profile in southeast asia closer to universal eye health. int ophthalmol 2018;38:469-80. doi: 10.1007/s10792-017-0481-y. 2. ministry of health r.i. road map for visual impairment control 2017-2030. jakarta: minstry of health r.i.;2017. 134 simanjuntak, tan, christine males come earlier for service with universal health 3. bellan l, buske l. ophthalmology human resource projections: are we heading for a crisis in the next 15 years? can j ophthalmol 2007;42:34– 8. doi:10.3129/can.j.ophthalmol.06-115. 4. biro pusat statistik provinsi dki jakarta. data penduduk jakarta. jakarta pusat: biro pusat statistik provinsi dki jakarta;2017. 5. dinas komunikasi, informatika dan statistik provinsi dki jakarta. data jumlah rumah sakit dan tempat tidur menurut jenis rumah sakit dki jakarta. jakarta: dinas komunikasi, informatika dan statistik provinsi dki jakarta;2011. 6. zetterberg m, celojevic d. gender and cataract – the role of estrogen. curr eye res 2015;40:16974. doi: 10.3109/02713683.2014.898774. 7. bani a, wang d, congdon n. early assessment of visual acuity after cataract surgery in rural indonesia. clin exp ophthalmol 2012;40:155-61. doi: 10.1111/j.1442-9071.2011. 02667.x. 8. gurung r, shreshta mk, muller a, et al. preoperative visual acuity of people undergoing cataract surgery in rural and urban nepal. clin exp ophthalmol 2011;39:501–5. doi: 10.1111/j. 1442-9071.2011.02524.x. 9. rao gn, khanna r, payal a. the global burden of cataract. curr opin ophthalmol 2011;22:4–9. doi: 10.1097/icu.0b013e3283414fc8. 10. lewallen s, mousa a, bassett k, et al. cataract surgical coverage remains lower in women. br j ophthalmol 2009;93:295–8. doi: 10.1136/bjo.2008. 140301. 11. mundy km, nichols e, lindsey j. socioeconomic disparities in cataract prevalence, characteristics, and management. semin ophthalmol 2016;31:35863. doi: 10.3109/08820538.2016.1154178. 12. lipton bj, decker sl. the effect of health insurance coverage on medical care utilization and health outcomes: evidence from medicaid adult vision benefits. j health econ 2015;44:320– 32. doi: 10.1016/j.jhealeco.2015.10.006. 13. courtright p, lewallen s. gender and eye health; why are we adressing gender issues in vision loss? cehj 2009;22:17-19. 14. xu l, wang yx, jonas jb. level of education associated with ophthalmic diseases: the beijing eye study. graefes arch clin exp ophthalmol 2010;248:49–57. doi: 10.1007/s00417-009-1204-5. 15. simanjuntak gws. reimplantasi lensa setelah komplikasi operasi katarak. j kes mas nas 2012;6:168-72. doi: http://dx.doi.org/10.21109/ kesmas.v6i4.95. 16. simanjuntak gws, simanjuntak gam, christine rn. macular hole surgery under retrobulbar local anesthesia increases visual acuity. univ med 2017;363:160-165. doi: 10.18051/univmed.2017. v36.160-165. 17. venkatesh r, tan cs, sengupta s, et al. phacoemulsification versus small incision cataract surgery for white cataract. j cataract refract surg 2010;36:1849–54. doi: 10.1016/j.jcrs.2010.05.025. 18. battle jf, lansingh vc, silva jc, et al. the cataract situation in latin america: barriers to cataract surgery. am j ophthalmol 2014;158:242–50. doi: 10.1016/j.ajo.2014.04.019. 19. le h, ehrlich j, venkatesh r, et al. a sustainable model for delivering high-quality, efficient cataract surgery in southern india. health aff 2016;35:1783–90. doi: 10.1377/hlthaff.2016.0562. 20. hashemi h, alipour a, mehravaran s, et al. five year cataract surgical rate in iran. optom vis sci 2009;86:890–4. doi: 10.1097/opx.0b013e3181 ae1cc6. 21. huang w, zheng y, wang l, et al. cataract surgery in urban southern china: the liwan eye study. invest ophthalmol vis sci 2012;53:7936-2. doi: 10.1167/iovs.10-5382. 22. meng q, xu l, zhang y, et al. trends in access to health services and financial protection in china between 2003 and 2011: a cross-sectional study. lancet 2012;379:805–4. doi: 10.1016/s01406736(12)60278-5. 23. cabric e, zvornicanin j, jusufovic v. the safety and efficacy of day care cataract surgery. med arh 2014;68:117-20. doi: 10.5455/medarh.2014.68. 117-120. increase neutrophil after repeated to artemisinin 49 *veterinary pharmacy laboratory, department of basic veterinary medicine, faculty of veterinary medicine, airlanggga university correspondence: dr. lilik maslachah, drh., m.kes. department of basic veterinary medicine, faculty of veterinary medicine, airlanggga university c campus mulyorejo surabaya 60115 phone: +6231-5992785 fax : +6231-5993015 email: lilik.maslachah@yahoo.com univ med 2017;36:49-58 doi: 10.18051/univmed.2017.v36.49-58 pissn: 1907-3062 / eissn: 2407-2230 received october 17, 2016 accepted for publication april 20, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract increase in neutrophil count after repeated exposure of plasmodium berghei-infected mice to artemisinin lilik maslachah* and rahmi sugihartuti* background leukocytes play an important role in the elimination of malaria infection. the leukocyte profile upon elimination of the malaria parasites that have been exposed to antimalarials and are subsequently capable of faster growth has not been researched. the aim of this research was to evaluate the role of mouse leukocytes in the elimination of parasites as shown by the leukocyte profile. methods an experimental research with post test only control group design was conducted involving 24 male mice of the swiss albino strain weighing 20 g -30 g, and 2.5 months old. they were randomized into four groups: two control groups (k1, kp) and two treatment groups (p1, p4). artemisinin at a dose of 0.04 mg/g body weight was given to the mice for 3 days, starting 2 days after infection. the leukocyte profile was observed on the 2nd, 5th, 8th, and 10th day after infection. the results were analyzed by two-way anova. results as shown in treatment control group kp and treatment group p4, p. berghei that had been passaged in the mice and were still viable after repeated exposure to artemisinin, may cause changes in leukocyte profile. on the 10th day of infection, the neutrophil percentage in group p1 showed a significantly different decrease when compared with the other groups (k1, kp and p4) (p<0.05). conclusion repeated exposure to artemisinin of mice infected with p. berghei can cause changes in neutrophil profile in mice . keywords: artemisinin, leukocytes, repeated passage, p. berghei doi: http://dx.doi.org/10.18051/univmed.2017.v36.49-58 universa medicinajanuary-april, 2017 vol.36 no.1 original article 50 maslachah, sugihartuti increase neutrophil after repeated to artemisinin introduction malaria is still a public health problem in more than 95 countries around the world including indonesia. in 2015 there were approximately 214 million cases of malaria with 438.000 deaths, mostly occurring in african children under 5 years of age.(1) the increased incidence of morbidity and mortality of malaria is rapid and widespread due to increased parasite resistance to antimalarial medications. the new drugs for malaria treatment which are used to date are artemisinin and its derivatives. the effect of these drugs is faster than that of other anti-malarial drugs because they have more complex mechanisms of action. however, there have been indications that the plasmodium parasite has become resistant to these drugs.(2) this was shown clinically in two patients in cambodia who were infected with p. falciparum, in whom the parasites had become resistant to artesunate.(3) the research results showed that there has been decreasing efficacy of the combination of artesunate-mefloquine in falciparum malaria in cambodia.(4) currently, the resistance of p. falciparum to artemisinin has been detected to have occurred in five countries including cambodia, lao people’s democratic republic, myanmar, thailand and vietnam.(1) the results of an in vitro study of p. falciparum that were repeatedly exposed to artemisinin showed increased inhibitory concentration 50 % (ic 50 ), dormant phenotypic changes, and faster growth of viable plasmodium evolving from dormant forms. moreover, artemisinin exposure also can cause mutations in the pfatpase6 gene.(5) it is believed that the artemisinin resistance occurs because of mutations in pfcrt, pfmdr1, pfatpase6 and pfk13.(6,7) the presence of parasite pressure on the use of medicines at subcurative doses will lead to the development of new parasite strains that can survive the medicine. the results of this research are to be viewed as indicating an emergency, because there will be a risk of the development of in vivo resistance in humans, which will be one of the health problems in the world. moreover, there is no substitute for artemisinin. malaria treatment failure with artemisinin and its derivatives will open an era of untreatable malaria. experimental in vivo studies using rodent malaria are used to support the translation of laboratory studies into clinical studies. since the pathologic mechanism for the spectrum of malaria in humans is not known clearly, these experimental studies can be used to explain the mechanism of artemisinin resistance in vivo by using mice that are infected with p. berghei. because of the resilience and virulence of the malaria parasites in antimalarial resistance, there is a need to do research to develop effective strategies for malaria control. however, it is really difficult to do research in endemic areas because there are many confounding factors, such as the cloning of multiple infections from infective mosquito bites, which is impossible to do in humans because of ethical factors. so that this research is using rodent malaria as an in vivo drug resistance model of human malaria by exposing p. berghei to artemisinin. the effective dose of artemisinin that is used is 99% (ed 99 : 200 mg/kg body weight of the mice), and is given repeatedly to the mice upon parasite passage. p. berghei i s o n e o f t h e s p e c i e s o f plasmodium that infect rodents. many studies of malaria use p. berghei with mice as hosts, because p. berghei has the same life cycle and morphology as the plasmodium parasites that infect humans. molecular analysis show that there are similarities between rodent malaria and p. falciparum malaria. in order to support in vivo studies on p. falciparum drug resistance, p. berghei can be used as a model.(8) results of research by darlina et al.(9,10) showed that leukocytes play a role in the protective mechanism during malaria infection, as is indicated by the increasing numbers of leukocytes, lymphocytes, and monocytes and decreasing rates of parasitemia in mice that have been infected with irradiated p. berghei. 51 o n e s t u d y s h o w e d t h a t a r t e m e t h e r, artesunate and artemisinin markedly decreased the leukocyte count after 24 h of treatment. artemether and artesunate showed the highest efficacy in rescuing mice with late-stage cerebral malaria and rapidly decreased leukocyte accumulation in the brain.(11) the results of the present study can give an idea of the leukocyte profile in hosts infected with plasmodium that have been repeatedly exposed to artemisinin. this is not possible in humans because of ethical considerations, therefore the results of the animal studies can be used for improvements in the treatment of malaria infection. leukocytes are part of the blood that are vital to the body defenses against infection. the objective of the present study was to evaluate the effect of artemisinin on leukocyte profile in mice infected with p. berghei, so that this research was conducted to determine the changes in leukocyte profile during the development of artemisinin resistance in mice infected with p. berghei upon passage of p. berghei that had been exposed repeatedly to antimalarial drugs, methods research design this research was an experimental research with post test only control group design. the parasite that was used to infect the mice was p. berghei anka strain. the experimental animals were 2.5 months old male swiss albino mice weighing 20g -30 g. the artemisinin was of analytical grade from sigma chemical co. leukocyte examinations were conducted at the faculty of veterinary medicine, airlangga university, from june to october 2015. research sample this research used twenty four male mice of the swiss albino strain, weighing 20 g -30 g, and 2.5 months old. each group consisted of 6 treated mice, with one mouse as the donor. the sample size was determined by federer ’s formula. the animals were obtained from the a n i m a l f a c i l i t y a t t h e ve t e r i n a r y a n d pharmaceutical center (pusat veteriner dan farmasi, pusvetma), surabaya. research procedure mice were intraperitoneally infected with mouse red blood cells (rbc) containing 1x105 p.berghei per 0.2 ml. the infection was detected by daily microscopic examination of the erythrocytes in thin blood smears taken from the tail vein of the mice and stained with 20% giemsa. the mice were randomized into four groups, consisting of two control groups (k1, kp) and two treatment groups (p1, p4). in the treatment group p1, the mice were infected with p. berghei and 48 hours later treated with a single administration of artemisinin. subsequently the parasites in the blood of p1 animals were passaged into another group of animals (p2), who were then given 2 artemisinin treatments. the procedure was repeated for groups p3 and p4, which were infected with blood from p2 and p3 donors, respectively, and received 3 and 4 artemisinin treatments, respectively. the control group k1 consisted of mice who were infected with p.berghei and were untreated (did not receive artemisinin), while treatment control group kp consisted of untreated mice who were infected with p.berghei that had previously been treated four times with artemisinin. details of the treatment are as follows: treatment group p1 comprised six mice that had been inoculated with rbc containing 1x105 p. berghei in 0.2 ml (d 0 ) and were then treated with the artemisinin “4-day test” (4-dt) at the ed 99 dose of 200mg/kgbw for 3 days, starting 48 hours after infection (d 2 ). after that, the parasitemia rate was monitored and calculated at 120 hours after infection. after the parasitemia rate was higher than 2 % of rbc, one mouse was used as donor of parasites to be passaged to 5 new mice. then, 48 hours post infection, the 5 new mice were treated artemisinin at the same ed 99 dose for 3 days and so on up to 4 passages (p2, p3, p4). the control group k1 contained univ med vol. 36 no.1 52 maslachah, sugihartuti increase neutrophil after repeated to artemisinin five mice inoculated with 0.2 ml rbc containing 1x105 p. berghei that had never been treated with artemisinin (d 0 ) and were left untreated. treatment control group (kp) contained six mice inoculated with 0.2 ml rbc containing 1x105 p. berghei that had previously been treated 4 times with artemisinin (d 0 ) and were left untreated. parasite development was followed in all treatment groups until 10 days after infection.(12,13) leukocyte differential count the leukocyte differential count was done t o r e p r e s e n t o n e o f t h e i m m u n e s y s t e m parameters that plays an important role in malaria infection. the leukocyte differential count was done at 48 hours, and on the 5th, 8th and 10th day post-infection, by making thin blood smears taken from the mouse tail vein, fixed with methanol for 5 minutes, put in a giemsa staining solution for 20 minutes, washed with running water with the slide in a tilted position, and dried in the air. the leukocyte differential count was determined under the light microscope at 1000x magnification. every 100 leukocytes that were found were counted and grouped into neutrophils, eosinophils, basophils, lymphocytes, and monocytes. the leukocytes were counted in a number of visual fields throughout the smear, the examination starting from the center of the smear, proceeding parallel to the edge of the smear, then moving back and forth until a total leukocyte count of 100 was reached. the proportions of the types of leukocytes was expressed in percentages.(10) data analysis the data on the leukocyte profile was processed with anova. if there were any significant differences, the multiple comparisons test was done using spss 17.0, with the level of significance set at 5% to determine differences in treatment. ethical clearance this research has been approved by the ethics committee of the faculty of veterinary medicine, airlangga university, on june 3rd 2015 with certificate number no. 464 ke. results table 1 and fig. 1a show the resulting neutrophil percentage in p4, which was highest among the other treatment groups, starting from the 2nd day until the 10th day post infection. the graph also shows that the infection on the 2nd day in the treatment control group kp (which was infected with p.berghei that had been treated 4 times with artemisinin and were then left untreated) and the treatment group p4 (that was infected with p.berghei and had been treated four times with artemisinin) increased at significantly different rates when compared with the control group k1 (mice that had been infected with p.berghei that had never been treated with artemisinin) and group p1 (infected with p.berghei and treated once with artemisinin). on the 5th day of infection, group kp showed significantly different increases in neutrophils when compared with the other three groups, namely groups k1, p1, and p4. on the 8th day of infection, the neutrophil percentage did not show any significant differences between all the groups (k1,kp,p1,p4). on the 10th day of infection, the neutrophil percentage in group p1 showed significantly different decreases when compared with the other groups (k1, kp and p4). table 1 and figure 1b show that the highest monocyte percentage was in group p4 when compared with the other treatment groups. the monocytes started to increase on the 2nd 8th days post infection and then decreased on the 10th day post infection, but the percentage was still higher than in the other treatment groups. these results show that on the 2nd day of infection the kp control group showed significantly different increases when compared with groups k1, p1, and p4. on the 5th and 8th days of infection the monocytes percentage did not show any significant differences between all groups (k,kp,p1,p4). on the 10th day of infection, group 53 p1 showed significant differences when compared with the other three groups (k1, kp, and p4). table 1 and figure 1c show that the highest lymphocyte percentage was in group p4 when compared with the other groups, starting on the 2nd day until the 10th day post infection. the results show that on the 2nd day of infection groups kp an p4 showed significant differences when compared with groups k1 and p1. on the 5th, 8th, and 10th days of infection, the lymphocyte percentage did not show any significant differences between all groups (k1, kp, p1, p4) table 1 and figure 1d show that the highest basophil percentage was in group p4 when compared with the other groups, starting on the 2nd day until the 5th day post infection then on the 8th day post infection, the basophil percentage decreased. these results show that on the 2nd day of infection the kp control group showed significantly different increases when compared with groups k1 and p1. on the 5th day of infection, group p4 showed significantly different increases in percentages of basophils when compared with the other three groups (k1,kp,p1). on the 8th day of infection, group p4 showed significantly different increases in percentages of basophils when compared with groups k1 and p1, but did not show any significant differences with group kp. on the 10th day of infection, the basophil percentage did not show any significant differences between all control and treatment groups (k,kp,p1,p4). table 1 and figure 1e show that the eosinophil percentage in group p4 was higher the 2nd day post infection. after that, the eosinophil percentage decreased. the image shows that on the 2nd day of infection the kp table 1. distribution of mean leukocyte count by control and treatment groups values are means ± sem. values for each day along the same rows with different superscript letters are significantly different (p<0.05); note: k1 = control group infected with p.berghei and untreated with artemisinin; kp = treatment controls (infected with p.berghei, treated 4 times with artemisinin and then untreated; p1 = treatment group infected with plasmodium berghei and treated with a single dose of artemisinin; p4 = treatment group infected with plasmodium berghei and treated 4 times with artemisinin univ med vol. 36 no.1 54 maslachah, sugihartuti increase neutrophil after repeated to artemisinin figure 1. leukocyte type percentage in control and treatment groups repeatedly exposed to artemisinin in mice infected with p.berghei. note: k1 = control group infected with p.berghei and untreated with artemisinin; kp = treatment controls (infected with p.berghei, treated 4 times with artemisinin and left untreated); p1 = treatment group infected with plasmodium berghei and treated with a single dose of artemisinin; p4 = treatment group infected with plasmodium berghei and treated 4 times with artemisinin (a) (b) (c) (d) (e) 55 control group showed significantly different increases when compared with groups p1 and p4. on the 5th and 8th days of infection, group p4 showed significantly different increases in eosinophil percentages when compared with the k1 control group, but did not show any significant differences with groups kp and p1. on the 10th day of infection, the eosinophil percentage did not show any significant differences between all control and treatment groups (k,kp,p1,p4). discussion in the present study we showed that neutrophil percentage increased from the 2nd day until the 5th day post infection with p. berghei. repeat treatments with artemisinin showed that neutrophils are cells that function as the first line of defense of the body against infection or pathogens entering the body (to eliminate p.berghei). mice infected with p. berghei and treated with a one-time dose of an antimalarial drug yielded the same results as mice treated with a one-time dose of artemisinin (p1), as is shown by the increasing number of neutrophils, monocytes and lymphocytes after more than 5 days of infection.(14) research with repeated exposure of artemisinin in mice as a model of in vivo in infections in humans has not been done before. cytokines and interleukins that are released after lysis of neutrophils will stimulate the bone marrow to release neutrophils so that the production of neutrophils is increased. malaria infections can induce both humoral and cellular immune responses. in the humoral immune response, antibodies provide protection against m a l a r i a i n f e c t i o n t h r o u g h a v a r i e t y o f mechanisms. antibodies can block the invasion of merozoites into erythrocytes and inhibit the growth of parasites in the erythrocytes, while cellular immunity against plasmodium plays a role in the erythrocytic stage. cellular immunity is activated by t helper (th) lymphocytes, which are a subset of t cells needed in the induction of immune responses against an invasion of foreign antigens. according to the type of cytokine produced, th lymphocytes are divided into th1 cells, which will activate cellular immunity and t h 2 c e l l s , w h i c h w i l l a c t i v a t e h u m o r a l immunity.(15) the increase in neutrophil percentage on the 8th day post infection was not significantly different between groups. on the 10th day post infection, the group of mice infected with p.berghei and treated with a single dose of artemisinin (p1) showed a decrease in neutrophils when compared with groups k1, kp and p4. this shows that neutrophils function as the first line of defense to eliminate p.berghei by phagocytizing parasites and neutrophils on the first days of infection or when the p.berghei attacks. this suggests that infections of longer duration will increase the percentage of parasitemia in the mice so that the high numbers of parasites in their bodies will suppress the immune system (immunosupression), causing the immune system to become inadequate and unable to respond properly. high parasitemia rates will improve the mechanism of parasite invasion and suppress the host immune system.(16) neutrophils play an important role in the immune response by modulating both cellular and humoral immunity via the synthesis and release of immunoregulatory cytokines. all neutrophil profiles in the groups showed that p.berghei which had been treated with repeated artemisinin showed differences when compared with p.berghei that had never been treated with artemisinin. this shows the infection had become more powerful because of the parasites’ ability to develop faster. this is consistent with in vitro studies that were performed by maslachah (17) who found that plasmodium which remained viable after repeated artemisinin exposure will increase the parasitemia rate and take a shorter time to become viable again when compared with the parasites in the treatment group which only received a one-time artemisinin exposure. monocytes are phagocytic cells that can destroy antigens by ingestion. moreover, univ med vol. 36 no.1 56 maslachah, sugihartuti increase neutrophil after repeated to artemisinin monocytes may contribute to delivery of antigens to the lymphocytes (antigen precenting cells, apc). the relatively high increases in the number of monocytes in mice that had been infected with p. berghei and exposed repeatedly to artemisinin, resulted in increased phagocytosis of the parasites, this being the body’s defense mechanism against p.berghei. repeat artemisinin therapy in the treatment group causes faster growth of p.berghei that remained viable from medicine exposure, so increasing the parasitemia rate and also the number of cells contributing to the elimination of the parasites, which will affect the monocyte profile. the treatment control group of mice that were infected p.berghei and had been treated four times with artemisinin and left untreated (kp) showed an increasing average percentage of monocytes that is higher when compared with the control mice that were infected with p. berghei and were not exposed to the medicine (k0). this significant difference is visible on the 2nd day post infection. this can be explained from the research results of maslachah,(17) who found that plasmodium that have been exposed to artemisinin and are still viable and grow normally, have a faster rate of intra-erythrocytic development, so that the increase in the average percentage of monocytes is a mechanism for defense against p.berghei. the monocyte profile from the 2nd to the 8th day post infection showed an increasing trend in the treated group of mice exposed to artemisinin, but after the 10th day of infection, there was a decrease, whereas in the group of control mice, the trend was decreasing or stable. this shows that after chronic infection, monocyte numbers will be decreased so that the monocytes function maximally in the first week of infection. the average percentage of lymphocytes in the treated groups of mice showed that had been exposed to artemisinin, seemed to decrease when compared with the control groups. this is because artemisinin treatment for 3 days with a therapeutic dose in mice infected with p. berghei caused suppression of plasmodium growth so that the growth of malaria parasites in all treatment groups was inhibited, as compared with the untreated control group. research conducted by teuscher et al. (18) in p. falciparum strains w2 and w2al80 also showed a decrease in parasitemia rate to 1% up to 8 days after medicine exposure, when it began to show increases in parasitemia rate, reaching more than 10% after 12 days. wi t h r e g a r d t o t h e p e r c e n t a g e o f lymphocytes on the 2nd day of infection, both the treatment control group kp and the treatment group p4 infected with p.berghei that had been repeatedly exposed to artemisinin, showed significant differences when compared with the control group k1 and the treatment group p1 infected with p. berghei and received no treatment and a one-time exposure to artemisinin, respectively this can be explained by the fact that lymphocytes play a role in the body defense mechanism against p.berghei. at the beginning of infection, p.berghei will stimulate the formation of antigen presenting cells (apcs). apcs will stimulate the mice to produce t lymphocytes that play a role in the immune response to destroy the plasmodium. however, after the 5th, 8th and 10th day of infection, the lymphocyte percentage showed no significant differences between all groups of mice. this shows that antigen presenting cells (apc) can affect the response of t lymphocytes during acute infection in the erythrocytic stage. i n t h e p r e s e n t s t u d y, b a s o p h i l s a n d eosinophils did not play a major role in infection by the parasite p. berghei. basophils have an important role in the allergic process, while eosinophils play a major role in the process of infection by parasitic worms.(19) the average value of basophils from this study ranged from 0 t o 0 . 4 % . t h e s e r e s u l t s d o n o t s h o w significantly different increases from the physiological value of basophils of 0 to 0.3%. the percentage of eosinophils was 0 to 5.6%, which did not show a significant increase from the physiological value of eosinophils of 0-4%. on the 2nd day post infection, the basophil and 57 eosinophil profiles in the treatment control group kp showed that the groups of mice infected with p. berghei and exposed 4 times to artemisinin had been able to stimulate the production of cells that play a role the immune system (basophils and eosinophils). this suggests that infection with p.berghei that have been exposed repeatedly can stimulate the immune system to work optimally by expressing all types of leukocytes in order to neutralize the infection. derivatives of artemisisnin (artemether and artesunate) are effective in rescuing mice with late state cerebral m a l a r i a a n d i n d e c r e a s i n g l e u k o c y t e accumulation in the brain.(11) many research studies of medicinal plants have shown them to have antimalarial activity by inducing the synthesis of red blood cells, white blood cells and platelets.(20,21) a limitation of this study is that the leukocyte profile data were obtained by using b l o o d s m e a r p r e p a r a t i o n s o n l y, w h e r e a s specialized tools are required such as heme analyzers to obtain results with better validity. the clinical implication of this study is that this exposure model of artemisinin with in vivo repeated passages in mice infected with p.berghei can be used as a basis to describe the functioning of the immune system against infections with p. berghei parasites which have been exposed to repeated artemisinin. the results of this study can provide a baseline leukocyte profile as one of the immune system’s lines of defense against malaria infections caused by p. berghei that have repeatedly been exposed to antimalarial drugs, as an in vivo model of malaria in humans. f u r t h e r r e s e a r c h i s n e e d e d o n t h e immunologic profile of cytokine-mediated tissue or organ damage. the leukocyte response occurs under normal physiological and pathological conditions. the manifestations of the leukocyte response to malaria infection are in the form of decreases or increases in one or several types of leukocyte, which can provide information and clinical clues that may help in the diagnosis of infections caused by plasmodium. conclusion repeated exposure to artemisinin in mice infected with plamodium berghei will cause changes in leukocyte profile, comprising neutrophils, lymphocytes, monocytes, basophils and eosinophils. conflict of interest the authors state that there is no conflict of interest with the other authors, publisher or study. acknowledgements we thank the dean of the faculty of veterinary medicine, airlangga university, for the provision of funding from the annual workplan and budget (rencana kerja dan anggaran tahunan, rkat) for this research. contribution lm contributed to drafting the manuscript and the design of the study. lm and rs contributes to data collection, analysis and i n t e r p r e t a t i o n . l m c o n t r i b u t e d r e v i s i n g manuscript for critically important content. all authors read and approved the final manuscript. references 1. world health organization. who global malaria programme: world malaria report 2015. geneva: world health organization;2015. 2. afonso a, hunt p, cheesman s, et al. malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (encoding the sarcoplasmic and endoplasmic reticulum ca2+ atpase) tctp, mdr1 and cg10. antimicrob agents chemother 2006; 50:480-9. 3. noedl h. evidence of artemisinin resisntant malaria in western cambodia. n engl j med 2008;359:2619-620. 4. wongsrichanalai c, meshnick sr. declining artesunate-mefloquine efficacy against univ med vol. 36 no.1 58 maslachah, sugihartuti increase neutrophil after repeated to artemisinin falciparum malaria on the cambodia-thailand border. emerg infect dis 2008;4:716-8. 5. maslachah l, dachlan yp, nidom ca, et al. profil fenotipik plasmodium falciparum galur papua 2300 akibat paparan antimalaria artemisinin in vitro. mkb 2015;47:1-9. 6. paloque l, ramadani ap, mercereau-puijalon o, et al. plasmodium falciparum: multi faceted resistance to artemisinins. malar j 2016;15:149. doi: 10.1186/s12936-016-1206-9. 7. njokah mj, kang jn, kinyua j, et al. in vitro selection of plasmodium falciparum pfcrt and pfmdr1 variants by artemisinin. malar j 2016;15:381. doi: 10.1186/s12936-016-1443-y. 8. craig ag, grau ge, janse c, et al. the role of animal models for research on severe malaria. plos pathogens 2012;8:2 e1002401. 9. darlina, raharjo t, nurhayati s. changes of leukocytes type in mice immunized with irradiated plasmodium berghei. national seminar, 16 november,yogyakarta;2011. 10. darlina, krisnanto t, fauzan a. haematopoietic response of mice infected with erythrocytic stadium of gamma irradiated plasmodium berghei. indonesian j nuclear sci technol 2012; 13:85-94. 11. clemmer l, martin yc, zanini gm, et al. artemether and artesunate show the highest efficacies in rescuing mice with late stage cerebral malaria and rapidly decrease leukocyte accumulation in brain. antimicrob agents chemother 2011;55:1383-90. 12. kiboi dm, irungu bn, langat b, et al. plasmodium berghei anka: selection of resistance to piperaquine and lumefantrine in a mouse model. exp parasitol 2009;122:196-202. 13. henriques g, martinelli a, rodrigues l, et al. artemisinin resistance in rodent malaria– mutation in the ap2 adaptor µ-chain suggest involvement of endocytosis and membrane protein trafficking. mal j 2013;12:118. 14. odeghe ob, uwakwe aa, monago cc. some biochemical and haematological studies on the methanolic extract of anthocleista grandiflora stem bark. int j appl sci technol 2012;2:58-65. 15. baratawidjaja kg, rengganis i, editors. imunologi dasar. edisi ke 9. jakarta: balai penerbit fakultas kedokteran universitas indonesia;2010. 16. onwuamah ck, phillip ua, odeigah pg. mouse mortality from a high plasmodium berghei inoculum density may be due to immune suppression in the host. int j med med sci 2010; 2:162-6. 17. maslachah l. efek paparan artemisinin berulang terhadap perkembangan plasmodium falciparum resisten in vitro [disertasi]. surabaya; universitas airlangga;2013. 18. teuscher f, chen n, kyle de, et al. phenotypic changes in artemisinin resistant plasmodium falciparum line in vitro: evidence for decreased sensitivity to dormancy and growth inhibition. antimicrob agent chemother 2012;56:428-31. 19. fabre v, beiting dp, bliss sk, et al. eosinophil deficiency compromises parasite survival in chronic nematode infection. j immunol 2009; 182:1577-83. 20. balogun ea, adebayo jo, zailani ah, et al. activity of ethanolic extract of clerodendrum violaceum leaves against plasmodium berghei in mice. agric biol j n am 2009;1:307-12. 21. anggraini r, widodo gp, ningsih d. antiplasmodium effect of mundu (garcinia dulcis kurz) cortex n hexane extract with leukocyte and hemoglobin amount parameter on swiss webster mouse infected with plasmodium berghei. indonesian pharmacy j 2011;8:45-52. family history of hypertension increases risk of 181 *rift valley university lancha campus, addis ababa; ethiopia **pse of health professionals, jhpiego, bahir dar; ethiopia ***bahir dar university, bahir dar; ethiopia †school of public health, bahir dar university, bahir dar; ethiopia †† institute of public health, university of gondar, gondar; ethiopia ‡rift valley university, lancha campus dean, addis ababa, ethiopia correspondence: mulualem endeshaw lecturer, research officer and chairperson of research committee, rift valley university, lancha campus addis ababa ethiopia email: emulualem@gmail.com univ med 2016;35:181-91 doi: 10.18051/univmed.2016.v35.181-191 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 family history of hypertension increases risk of preeclampsia in pregnant women: a case-control study mulualem endeshaw*, fantu abebe**, melkamu bedimo***, anemaw asrat†, abebaw gebeyehu††, and alemayehu keno‡ background preeclampsia is one of the leading causes of maternal and fetal morbidity and mortalities worldwide. despite extensive research, the underlying cause of preeclampsia remains poorly understood. this study aimed to offer compelling evidence on the important risk factors of preeclampsia in amhara region, ethiopia. methods a case control study was conducted in public health facilities of bahir dar city from september 2014 to january 2015. a total of 453 (151 cases and 302 controls) pregnant women were enrolled in this study. hemoglobin level and urinary tract infection (uti) status were collected from clinical notes. oral examination was performed by a dentist for detection of periodontal diseases. univariate and multiple logistic regression analysis was conducted to determine the relationship of all the independent variables with the outcome variable. a p-value <0.05 was declared statistically significant. result advanced maternal age (aor=4.79;95% ci 1.031-22.18), family history of hypertension (aor=11.16;95% ci 5.41-41.43), history of diabetes mellitus (aor=6.17;95% ci 2.11-20.33), uti in the current pregnancy (aor=6.58;95% ci 2.93-14.73), failure to comply with iron and folic acid supplement during pregnancy (aor=8.32;95% ci 3.35-20.62), lack of exercise (aor=3.33;95% ci 1.35-8.17), multiple pregnancy (aor=4.05;95% ci 1.57-12.27), anemia (aor=4.19;95% ci 1.2713.92), and periodontal disease or gingivitis (aor =3.51;95% ci 1.1410.83) were associated with preeclampsia. conclusion family history of hypertension was the most dominant risk factor for preeclampsia in pregnant women. encouraging pregnant women to have health seeking behavior during pregnancy would provide a chance to diagnose preeclampsia as early as possible. keywords: preeclampsia, risk factors, pregnancy, pregnant women, bahir dar city doi: http://dx.doi.org/10.18051/univmed.2016.v35.181-191 182 endeshaw, abebe, bedimo, et al family history of hypertension risk of preeclampsia introduction preeclampsia is a pregnancy complication recognized by new-onset gestational hypertension and proteinuria first detected after 20 weeks gestation with or without generalized edema.(1,2) preeclampsia is a multisystem disorder of unknown etiology and unique to pregnancy.(3) in severe cases, it can present with hemolysis, elevated liver enzymes, and low platelet count (hellp) syndrome (4) or eclampsia, that is the occurrence of convulsions associated with a high rate of maternal mortality.(5) preeclampsia, complicating 5% to 8% of pregnancies, has been traditionally defined by an elevated blood pressure (over 140/90 mmhg on at least 2 occasions at least 6 hours apart) and proteinuria, at or beyond 20 weeks’ gestation.(6) the onset of preeclampsia is only 5% from 20 weeks to 34 weeks of gestation, 90% from 34 weeks to the time of labor and delivery and the remaining 5% occurs postpartum within 48 hours after delivery.(7) although the characteristic placental pathology of preeclampsia is established during early pregnancy,(2) there are no reliable tools for early clinical diagnosis and no effective therapies to treat the disease and improve maternal and fetal outcomes. in developed countries, surveillance for preeclampsia through prenatal care using risk factors allows for early identification and intervention via delivery. this management is very effective at reducing maternal mortality rate in developed countries. however in developing countries, where inadequate prenatal care limits preeclampsia surveillance, maternal mortality is common, accounting for 50,000 deaths yearly.(8) ninety-nine (99%) percent of these deaths occur in the developing world and more than 50% in sub saharan africa.(9) therefore it is necessary to recognize the epidemiological and clinical risk factors to predict the disease before it threatens the survival of both mother and fetus. preeclampsia has remained a significant public health threat in both developed and developing countries contributing to maternal and perinatal morbidity and mortality globally.(8) the incidence of pre-eclampsia ranges from 3% to 7% for nulliparas and 1% to 3% for multiparas.(10,11) the world health organization (who) estimates the incidence of preeclampsia to be seven times higher in developing countries (2.8% of live births) than in developed countries (0.4%).(3) preeclampsia affects 5% to 10% of all pregnancies globally. the rates are lower in the developed world (about 3% to 5% of women), but the prevalence of preeclampsia in developing countries reaches up to 16.7% and it is estimated to account for about 40% to 60% of maternal deaths in developing countries.(12) maternal morbidity remains great with preeclampsia, which continues to be one of the leading causes for the admission of pregnant women to intensive care units in the world.(13) fetal morbidity and mortality also increase substantially in women with preeclampsia and it is a major cause of stillbirths and early neonatal deaths in developing nations.(13,14) although the cause for pre-eclampsia is still unclear, there does appear to be certain risk factors which were identified by a number of studies such as advanced maternal age,(15) illiteracy,(16) nulliparity,(17) twin pregnancy,(18) high maternal body mass index (bmi),(19) family history of hypertension,(20) renal disease and diabetes mellitus.(21) other evidences also showed that preeclampsia is more common among women who have histories of certain health conditions, such as previous history of preeclampsia, (22) diabetes,(23) urinary tract infection (uti),(24) periodontal disease,(25) anemia,(26,27) migraine headaches and sickle cell disease.(23) while smoking,(26) alcohol use,(28) partner change and lack of physical exercise during pregnancy (29) have been explained as risk factors. fruit and vegetable intake,(26) adequate antenatal follow up (15) and periconceptual and ongoing regular use of folate containing multivitamins and induced abortion (30, 31) have been associated with primary prevention of preeclampsia. studies on the determinant factors of preeclampsia are non-existent in ethiopia. hence, 183 we aimed to identify risk factors for the occurrence of preeclampsia in the study area. methods study design a multi-center unmatched case-control study was conducted among pregnant women who attended antenatal care (anc) or skilled delivery service in public health facilities in bahir dar city from september 2014 to january 2015. study area the study was conducted in bahir dar city, one of the three administrative cities and the capital city of the amhara region. amhara region is the second largest and second most populous region in ethiopia, with a population of nearly 20 million. the city is located 565 km northwest of addis ababa. according to bureau of finance and economic development report, the city hosts a population of 277,566 with a male to female ratio of 0.92. the average fertility rate in the region is 4.8. currently, 70% and 26% of pregnant women in the region are attending anc and skilled delivery services respectively.(32,33) the city has one referral hospital and six public health centers serving approximately 5 million people within and outside the city. the study involved all of these public health facilities in bahir dar city. study population all pregnant women who stayed at least 6 months in the region and attended antenatal care follow up and/or delivery during the study period at any of the public health facilities of bahir dar city, and met the inclusion criteria comprised the study population.cases were pregnant women diagnosed to have any form of preeclampsia/ eclampsia during anc follow up or delivery care by an obstetrician. preeclampsia was diagnosed after 20 weeks of pregnancy if they had a blood pressure of 140/90 mmhg or higher measured at least twice with 6 hours separation and protein in their urine confirmed by a laboratory test. controls were normotensive women delivered in the same health facilities at the same time as cases were enrolled. hence, to avoid misclassification of potential cases as controls, we waited to see the outcome of all the controls until 48 hours post-partum. cases were enrolled consecutively as they were diagnosed to have preeclampsia/eclampsia until the required sample size was obtained. for each case, two controls were selected by using a simple random sampling technique from the same health facility on the same day. sample size determination the sample size was determined using epi info version 3.5.3 stat calc calculator for two populations based on all the necessary assumptions for case control study by taking power of 85% and significance level of 5% and control to case ratio of 2:1. in ethiopia there are only few descriptive studies conducted concerning the prevalence of hypertensive disorders of pregnancy,(32,33) but no previous study has been done on determinant factors of preeclampsia in the country. therefore the sample size was determined by using the results of a previous study to identify risk factors of preeclampsia and select a variable that would yield maximum sample size (34) which was advanced maternal age. based on the assumption, the total sample size required for cases and controls, including 10% for nonresponse, was 156 and 312 respectively. measurements gingivitis and periodontitis were diagnosed based on the american academy of periodontology (aap)/american dental association (ada) classification, using the measurement method of ramfjord by a calibrated periodontal probe.(35) clean catch urine specimens were collected from each of the study participants. from all subjects recruited into the study a specimen of clean catch midstream urine was collected during anc follow up that was subjected to a dipstick test and urine microscopy for uti. maternal anemia was defined as hemoglobin univ med vol. 35 no.3 184 endeshaw, abebe, bedimo, et al family history of hypertension risk of preeclampsia concentration less than 11 g/dl. if a woman had been taking iron and folic acid (ifa) supplement daily, she would be considered compliant to ifa supplement as per the who recommendations.(37) exercise was defined as at least a conscious effort to stroll around participant’s home for not less than 20-30 minutes daily.(29) data analysis ibm spss version 20 was used for data entry and cleaning. then, univariate logistic regression analysis was conducted using stata version 12.0. backward stepwise unconditional logistic regression analysis was employed to determine the strength of association between predictive variables and the outcome variable and to control for the effect of confounding variables. crude odds ratio (cor) was reported for bivariate logistic regression and adjusted odds ratio (aor) was reported for the multivariate logistic regression model. a p-value <0.05 was declared statistically significant. ethical clearance ethical clearance was obtained from the bahir dar university school of public health ethical review board. the person in charge of each facility and care providers were informed and their agreement received before the onset of data collection. moreover, the purposes and importance of the study was explained and informed consent was secured, confidentiality was maintained at all levels of the study, and participant’s involvement in the study was on a voluntary basis. results a total of 453 verbally consented pregnant women who came for their antenatal follow up and skilled delivery service were enrolled in this study. the mean age of the study participants was 27.14 ± 5.73 years; the mean age for cases was 29.7 ± 6.28 years and for controls 24.6 ± 5.36 years. the age group of 25–29 years was the most table 1. crude odds ratios for the associations between socio-demographic characteristics and preeclampsia among pregnant women *unmarried, divorced and widowed; **median monthly income: $1=21 ethiopian birr (etb) based on the current exchange rate 185 common age group for cases and controls. there was a difference in area of residence for cases and controls, in that the percentage from urban areas was larger in the control group (64.2%) than in the cases group (54.3%). other sociodemographic characteristics of the cases were similar to those of the controls (table 1). a higher proportion of cases than controls reported that they had taken alcohol during their pregnancy. the proportion of cases and controls who reported to have ifa regularly during pregnancy was 65.6% and 93.7% respectively. similarly, the majority of cases (78.1%) and controls (84.1%) reported that they had engaged in physical exercise during their pregnancy. comparable proportions of cases and controls had their first pregnancy while participating in this study. almost all participants in both groups (96.0% of cases, 95.4% of controls) had visited an antenatal clinic during their pregnancy. an appreciably greater number of cases had family history of hypertension and history of diabetes mellitus than did the controls. similarly, the proportion of women who had uti, periodontal disease and anemia was higher among cases than controls (table 2). table 2. crude odds ratios for the associations between maternal life style, reproductive and medical risk factors and preeclampsia among pregnant women univ med vol. 35 no.3 186 endeshaw, abebe, bedimo, et al family history of hypertension risk of preeclampsia the simple binary logistic regression analysis in this study revealed several factors to be predictors of preeclampsia. women who reported to have drunk alcohol at least weekly during the index pregnancy and those women who changed their partner had an increased risk of preeclampsia as compared with those women who did not (cor=1.66; 95% ci: 1.122.46; and cor=2.10; 95% ci: 1.23-3.60, respectively) in the bivariate analyses, but the values turned out to be insignificant when adjusted in the multiple binary logistic regression model. women whose age was 35 years and above were almost five times more likely to develop preeclampsia than women whose age was less than 20 years. inadequate use ifa was found to be a risk factor for the development of preeclampsia (aor=8.32; 95% ci: 3.35-20.62). in addition, lack of exercise was found to be a risk factor for the occurrence of preeclampsia (aor=3.33; 95% ci: 1.35-8.17). similarly, those women who had a family history of hypertension and history of diabetes mellitus were more likely than their counterparts to develop preeclampsia (aor=11.16; 95% ci: 5.41-41.43; aor=6.17; 95% ci: 2.11-20.33, respectively). likewise, presence of uti, anemia and periodontal disease during pregnancy were independent risk factors for the incidence of preeclampsia (aor=6.58; 95% ci: 2.9314.73; aor=4.19; 95% ci: 1.2713.92; aor=3.51; 95% ci: 1.14-10.83, respectively). a family history of hypertension was the most dominant risk factor of preeclampsia. however, factors such as residence, education, marital status, monthly income, occupation, alcohol intake, primiparity, sexual partner change and anc follow up did not show any significant association with the incidence of preeclampsia in our study (table 3). discussion the main finding of this study was that women who reported a family history of hypertension were significantly associated with preeclampsia. our observation of an association between family history of chronic hypertension and risk of preeclampsia is consistent with several previous reports.(20) our results showed that diabetes mellitus was associated with the subsequent development of preeclampsia. the result of our study showing a relationship between preeclampsia and diabetes was also consistent with previous findings, diabetes mellitus was independently and significantly associated with an increased risk of preeclampsia.(21) the result of this study is biologically plausible for several reasons. first, epidemiological and clinical data document a close association between insulin resistance, type 2 diabetes, and hypertension.(32) in addition hyperinsulinemia has been shown to stimulate the proliferation of vascular smooth muscle cells, enhance acute sympathetic nervous system activity and modify transmembrane cation transport, as well as renal sodium retention, release of the potent vasoconstrictor angiotensin ii, and associated endothelial dysfunction. all of these alterations may contribute to blood pressure elevation and thus preeclampsia.(37) second, evidence from diverse settings suggests that family history of hypertension and diabetes are strongly and consistently related to biophysiological markers of vascular disorders. in women with a family history of hypertension, endothelial changes also appear to involve a relative deficiency in the production of nitric oxide, a vasodilator and inhibitor of platelet aggregation, along with increased production of endothelin-i, which is an extremely potent vasoconstrictor and activator of platelets. this shift in the production of locally acting vasoactive substances could enhance vasoconstriction in response to circulating pressor hormones. the net effect would be to cause widespread arteriolar constriction leading to hypoxic/ischemic damage in different vascular beds, systemic hypertension, and worsening placental ischemia.(38) these reports, when taken together with results from our study, suggest that women’s family history of chronic hypertension and diabetes is an important risk factor for preeclampsia. 187 table 3. multiple logistic regression: only significant covariates included into the final model determinants of preeclampsia among pregnant women univ med vol. 35 no.3 188 endeshaw, abebe, bedimo, et al family history of hypertension risk of preeclampsia the finding that women who acquired uti in the current pregnancy were associated with significantly increased risk of preeclampsia is also consistent with a previous study.(23) the increased risk of preeclampsia suggests that acute maternal infection may play a role in the pathogenesis of preeclampsia.(39) various hypotheses have been proposed to explain the mechanism by which maternal infection may be associated with preeclampsia. a key feature of preeclampsia is the greater systemic inflammatory response of women who develop the syndrome compared to women who have normal pregnancies (40) which suggest that inflammation may play an important role in the pathogenesis. acute infections such as uti are an important source of inflammation. thus, the underlying mechanism of infection may be indirect, by enhancing the maternal systemic inflammatory response. it may also include direct effects of infectious agents increasing the risk of acute uteroplacental atherosis, resulting in increased systemic inflammation and vascular endothelial dysfunction preceding the clinical onset of preeclampsia.(41) although the exact mechanism of the association is uncertain, our study found an increased risk of preeclampsia associated with acute uti. in this study having periodontal disease or gingivitis is positively associated with preeclampsia. this report is also in line with previous findings.(25) there is a large body of evidence pointing to infection as a key factor in adverse pregnancy outcomes.(42) thus, it appears that periodontal disease may play a nonspecific role in various adverse pregnancy outcomes. this study revealed that periodontal disease is an independent risk factor for preeclampsia. this would be of great public health importance because periodontal disease is both preventable and curable. although the result of this study showed that alcohol intake during pregnancy was not significantly associated with development of preeclampsia, another study reported opposite results.(28) the possible explanation for this variation may be the difference in the type of alcohol being taken, the amount and the frequency of drinking, and possibly residual confounding, due to insufficient or unmeasured lifestyle habits. lack of physical exercise during pregnancy posed a statistically significant relation with preeclampsia in our study. this result differred from the study conducted in ghana.(29) however, exercise of low to moderate intensity is beneficial for general health reasons to maintain or improve circulation and physical fitness, and perhaps reducing the risk of preeclampsia. it is well-established that the risk of preeclampsia is greater in twin rather than in singleton pregnancies (18) and we found a similar result in our study this may be due to the presence in twin pregnancies of circulating soluble fmslike tyrosine kinase 1 (sflt1), which is a circulating antiangiogenic molecule of placental origin playing a central role in preeclampsia by antagonizing placental growth factor (plgf) and vascular endothelial growth factor signaling in the maternal vasculature defect. (43) in twin pregnancies, circulating sflt1 levels were twice as high as those in singleton pregnancies. the increased serum sflt1 levels in twin pregnancies were correlated with increased placental weight. these findings suggest that the increased risk of preeclampsia in twin pregnancies may be due to increased placental mass that leads to increased circulating levels of sflt1. alternatively, relative placental hypoxia due to the increased size of the placenta is thought to play an important role for an increased secretion of sflt1.(43) similar to previous studies (30,31,44) we found that folic acid supplementation during pregnancy was associated with a lower risk of preeclampsia. the observed beneficial effect of folic acid supplementation on preeclampsia may be related to several factors, of which the first is placental implantation and development. a well implanted and developed placenta is essential for the health and wellbeing of the mother and the fetus. placental growth/development is a period of increased cell proliferation and differentiation. therefore, higher folate intakes may be required 189 to support appropriate placental implantation and growth and development in early pregnancy. the second factor is the effect of folic acid in lowering blood homocysteine levels (45) as hyperhomocysteinemia is a risk factor for a number of pregnancy complications including preeclampsia.(46) the third factor is the effect of folate in improving systemic endothelial function and therefore reducing the risk of such complications as preeclampsia.(47) there was a statistically significant relationship between advanced maternal age and preeclampsia in this study, which is in agreement with the results of previous studies.(15) sexual partner change did not confer an increased risk of preeclampsia in this study. contrary to this, the study conducted in gahanna reported opposite results.(29) this may be due to the low frequency of mothers who reported sexual partner change prior to the fourth month of the current pregnancy. the present study reported a statistically significant relationship between anemia and preeclampsia; similarly other studies conducted in sudan and india reported that severe anemia is significantly associated with preeclampsia.(26,27) the prevalence of anemia (<10.5mg/dl) in our study was 8.6% among cases and 2.3% among controls. in our study, the hemoglobin value was measured during the third trimester antenatal care booking, while the same result was found in the two abovementioned studies using hemoglobin values measured during the first trimester antenatal booking. the findings of this study should be viewed in light of the following limitations. any random and systematic measurement error in self-reported data might attenuate the associations observed in this study. assessment of risk factors was made at diagnosis, hence recall bias is inevitable. finally, the temporal sequence between exposure and disease may be difficult to establish (reverse causality). further community-based matched case control studies are needed for further exploration of the potential determinants of preeclampsia. conclusions a family history of hypertension was the most dominant risk factor of preeclampsia. it is concluded that these factors can be used as a screening tool for preeclampsia prediction and early diagnosis, allowing timely interventions to minimize deaths associated with severe preeclampsia/eclampsia. conflict of interest the authors declare no competing interest. acknowledgment the authors would like to thank rift valley university lancha campus for sponsoring this study. we also extend our appreciation to amhara regional health bureau, bahir dar city administration health office, and health facilities for facilitating the data collection process. finally the authors wish to thank the nurses abebaw alem and yalew fentahun for their assistance in data collection and the study participants for their 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metaanalysis. bmj 2010;341:c7017. doi: 10.1136/ bmj.c7017. 38. gambone jc, marsden de. essentials of obstetrics and gynecology. 4th ed. philadelphia (pa): elsevier saunders;2007. 39. eiland e, nzerue c, faulkner m. preeclampsia 2012. j pregnancy 2012;2012:586578. doi: 10.1155/2012/586578. 40. sibai b, dekker g, kupferminc m. preeclampsia. lancet 2005;365:785–99. 41. minassian c, thomas sl, williams dj, et al. acute maternal infection and risk of preeclampsia: a population-based case-control study. plos one 2013;8:e73047. doi: 10.1371/journal. pone.0073047. 42. parihar as, katoch v, rajguru sa, et al. periodontal disease: a possible risk factor for adverse pregnancy outcome. j int oral health 2015;7:137. 43. bdolah y, lam c, rajakumar a, et al. twin pregnancy and the risk of preeclampsia: bigger placenta or relative ischemia? am j obstet gynecol 2008;198:428.e1-e6. 44. endeshaw m, abebe f, bedimo m, et al. diet and preeclampsia: a prospective multicentre case-control study in ethiopia. midwifery 2015; 31:617-24. doi: 10.1016/j.midw.2015.03.003. 45. chuang cz, boyles a, legardeur b, et al. effects of riboflavin and folic acid supplementation on plasma homocysteine levels in healthy subjects. am j med sci 2006;331:65-71. 46. lindblad b, zaman s, malik a, et al. folate, vitamin b12, and homocysteine levels in south asian women with growth retarded fetuses. acta obstet gynecol scand 2005;84:1055–61. 47. mackenzie ke, wiltshire ej, gent r, et al. folate and vitamin b6 rapidly normalize endothelial dysfunction in children with type 1 diabetes mellitus. pediatrics 2006;118:242–53. univ med vol. 35 no.3 f:\36 no 2\kennedi.pmd 88 *department of medical laboratory science, faculty of health science, imo state university owerri, imo state, nigeria **department of biochemistry, odimegwu ojukwu university uli, anambra state, nigeria ***department of chemical pathology, nnamdi azikiwe university teaching hospital, nnewi, anambra state, nigeria correspondence : johnkennedy nnodim department of medical laboratory science, faculty of health science, imo state university owerri, imo state, nigeria email: johnkennedy23@yahoo.com phone: +238034237000 date of first submission, april 20, 2017 date of final revised submission, june 6, 2017 date of acceptance, june 7, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 membrane potential, serum calcium and serum selenium decrease in preeclampsia subjects in owerri johnkennedy nnodim*, nwodo emmanuel**, okorie hope*, constance nwadike*, edward ukamaka*, and onah christian*** background pre-eclampsia is a serious hypertensive condition of pregnancy associated with high maternal and fetal morbidity and mortality. women who have had pre-eclampsia have a greater risk of developing hypertension, stroke and ischemic heart disease in later life. the etiology of pre-eclampsia remains unclear. placental insufficiency plays a key role in the progression of this disease. the aim of this study was to determine membrane potential, serum calcium and serum selenium levels in preeclampsia subjects in owerri. methods a case control study involving 200 primigravida (100 preeclamptic and 100 apparently healthy) between the ages of 20 and 32 years attending general hospital owerri. fasting venous blood was collected for the determination of serum selenium and serum calcium while membrane potential was calculated using the nernst equation. the serum calcium was estimated using randox kit and serum selenium by atomic absorption spectrophotometry. the independent student t test was used for statistical analysis. results the results revealed that membrane potential and serum selenium as well as serum calcium were significantly decreased in preeclampsia when compared with the controls, at p<0.05. conclusion our study demonstrated that the decrease in membrane potential, serum calcium and serum selenium levels may play a critical role in the pathogenesis of pre-eclampsia. there may be a need for increasing the dietary intake of these essential trace metals during pregnancy to prevent pre-eclampsia in owerri. keywords: membrane potential, selenium, calcium, preeclampsia doi: http://dx.doi.org/10.18051/univmed.2017.v36.88-93 original article cite this article as: nnodim j, emmanuel n, hope o, nwadike c, ukamaka e, christian o. cmembrane potential, serum calcium and serum selenium decrease in preeclampsia subjects in owerri. univ med 2017;36:88-93. doi: 10.18051/univmed.2017.v36.88-93 pissn: 1907-3062 / eissn: 2407-2230 89 univ med vol. 36 no.2 introduction preeclampsia is a major complication of pregnancy and remains an important cause of b ot h ma ter na l a nd f e t a l mor ta li t y a nd morbidity.(1) preeclampsia is a pregnancy-related disease and its physiopathology probably consists of multiple factors such as abnormal plac enta tion, i mbala nc e of a ngi ogen es is regulators, and maternal immune maladaptation. it could begin early in pregnancy with a poor trophoblastic invasion of maternal spiral arteries, leading to subsequent placental ischaemia and insufficiency.(2) the maternal response, which usually shows in the second half of pregnancy, is characterized by an increased systemic inflammatory response with high levels of proi nf l a mma to r y c yt oki ne s . t h e r e s ul t i n g wi d e s pr e ad e n d ot h e l i a l d ys fu n c t i on i s responsible for the hypertension and proteinuria which are the main features of preeclampsia. also, it has been reported that maternal hormonal disturbance arising from sleep deprivation or circadian rhythm disruption could retard fetal growth or result in complications of pregnancy.(3) preeclampsia mainly occurs during the second and third trimester of pregnancy and still remains a common condition in which hypertension o c c u r s in pr e gn a n c y. pr e e c la mp si a i s characterized by having systolic blood pressure equal to or greater than 140mmhg and diastolic blood pressure equal to or greater than 90mmhg on at least two occasions six or more hours apart. also, proteinuria results in preeclampsia.(4) it has been reported that endothelial cell dysfunction and inflammation are considered to have a crucial role in the pathophysiologic mechanism of preeclampsia. increased oxidative stress during preeclampsia results in increased generation of free radicals, lipid peroxides, reactive oxygen species and superoxide anion ra di ca ls to c au se en do the li al inj ur y a nd disorders. oxidative stress is an imbalance between the production of reactive oxygen and the biological system’s ability to detoxify the reactive intermediates or easily repair the resulting damage.(5) every type of organism maintains a reducing environment within its cells, which is produced by enzymes that preserve the reduced state through a constant input of metabolic energy. alterations in this normal redox state can cause toxic effects through the production of peroxides and free radicals that damage all components of the cell, including proteins, lipids and deoxyribonucleic acid (dna). during preeclampsia, as a consequence of endothelial cell damage, increased amounts of reactive oxygen species are generated, including hydroxyl radicals, lipid peroxyl radicals, singlet oxygen and peroxynitrite formed from nitrogen oxide.(6) all these chemical groups behave as a unit and constitute the free radicals. hence preeclampsia involves generalized damage to the maternal endothelium with the release of vasoconstrictive factors.(7) a growing body of evidence supports the understanding that preeclampsia begins in the utero-placental unit, is amplified by oxidative stress, and ends in the maternal endothelium. (8) the precise mechanisms that lead to preeclampsia, which often occurs without warning and may follow a precipitous course, are not known. sodium levels are increased in preeclamptic p a t i e nt s c o mp a r e d t o n or ma l su b j e c t s. hypernatraemia observed in this group could be caused by sodium retention. (9) as a result, hypokalaemia inhibits potassium channels in the cell membrane and depolarizes the membrane. because of its electrogenic nature, the inhibition of the sodi um p ump i tsel f de crea ses the membrane potential.(10) trace elements such as zinc (zn), selenium (se) and copper (cu) display antioxidant activity, wh i l e o t he r s s u c h a s c a l c i um ( ca ) a nd magnesium (mg) are essential micronutrients.(11) the disturbance in the metabolism of these elements may be a contributing factor in the development of certain diseases such as preeclampsia observed in pregnant women.(12) the essential trace miner al se acting through selenoproteins/selenoenzymes has the capacity to reduce the risk of pre-eclampsia.(13) 90 low serum calcium may cause high blood pressure by stimulating parathyroid hormone and r e n i n r e le a se a n d a l so b y i nd uc in g vasoconstriction by increasing its level in vascular smooth muscle.(14,15) calcium might also have an indirect effect on smooth muscle function by increasing magnesium levels.(16) however, the role of calcium and selenium in pregnant women is still being discussed. in recent times, there has been an increasing prevalence in the incidence of pre-eclampsia globally but there are conflicting reports on the relationship between trace elements and preeclampsia. some studies have shown that changes in the levels of blood trace elements in pre -ecla mptic pati ents may impli cate its pathogenesis (17) while others have failed to show an association of blood levels of trace elements and prevalence of pre-eclampsia.(18) in this study, the level of membrane potential, serum calcium and serum selenium in women with preeclampsia were evaluated to provide information on their status in owerri imo state, nigeria. methods research design this case control study was carried out from february 2014 to april 2015 at general hospital owerri. research subjects the subjects of the study were 100 pregnant women within the age range of 20-32 years, who had been clinically diagnosed with preeclampsia during the third trimester (30-40 weeks), and were visiting general hospital owerri. subjects were selected on the basis of high blood pressure ( sys t oli c a n d d ias t oli c ), p rot ei n ur ia an d pathological edema, which are the diagnostic criteria of preeclampsia. as normal controls were taken 100 healthy normotensive pregnant women, also within the age range of 20-32 years, who were in their third trimester (30-40 weeks). patients with a past history of hypertension, diabetes, and renal disease were excluded from the study. the following information was recorded by the trial midwife: age, blood pressure; date of the last menstrual period/ gestational age; weight and height. blood collection in all subjects 4 ml of fasting venous blood was collected into plain and edta bottles. the serum was separated by centrifuging the whole blood in westerfuge (model 684) centrifuge at 5,000g for 10 minutes. after centrifugation, red blood cells were separated from the plasma, washed thrice with physiological saline and lysed with 1.0 ml of distilled, deionized water. the red cell haemolysates were stored frozen until analysis. biochemical assay the extracellular cation concentrations were estimated from serum samples while intracellular calcium concentrations were determined from lysed red blood cells. the serum calcium was estimated using randox kit ca590. while membrane potential was determined by calculation using the nernst equation. selenium wa s d e t e r mi ne d by a t omi c a bs o r p ti o n spectrophotometry. statistical analysis the values were expressed as mean ± standard deviation. the independent student ttest was used to calculate the significant differences at p<0.05. ethical clearance consent as well as ethical approval was obtained from the ethical committee of the general hospital owerri (gho/14/011). results the present study enrolled 200 pregnant women, among whom the mean age of women with pre-eclampsia was lower than that of controls (22.51 ± 3.42 yrs vs 23.55 ± 3.11 yrs) nnodim, emmanuel, hope, et al membrane potential decrease in pre-eclampsia 91 univ med vol. 36 no.2 but the difference was statistically not significant (p>0.05). mean body mass index (bmi) was higher in pre-eclampsia subjects than in controls (24.99 ± 2.11 kg/m2 vs 23.84 ± 2.41 kg/m2, but again the difference was statistically not significant (p>0.05). the gestational age was significantly lower in pre-eclampsia subjects (p<0.05) (table 1). systolic and diastolic blood pressures were significantly higher in the preeclampsia subjects (p<0.05). the levels of membrane potential, serum selenium and calcium and red cell calcium were decreased in preeclampsia when compared with the controls (p<0.05) (table 2). discussion in our study, bmi was higher in the preeclampsia group compared to the control group, but the difference was not significant. this result differs from the study of akhtar et al.(19) and kanagal et al.(20) who found a significantly higher bmi in the preeclampsia group. women with high bmi tend to have hypertension, as compared with women with low bmi.(20) preeclampsia is linked with increased util izati on o f a nt iox ida nts. a nt iox idan ts neutralize toxins and volatile free radicals that are referred to as atoms or groups of atoms possessing an unpaired electron. among these are related reactive oxygen species (ros) that lead to free radical generation, causing cascading chain reactions in biological systems.(3) in this study, the membrane potential was significantly decreased in preeclampsia when compared with normal pregnancy. this is consistent with a study that found that plasma from pre-eclampsia women also had a significant decrease in mitochondrial membrane potential compared with normal pregnancy.(21) this means that the energy in preeclampsia patients is very low, which is linked to their high blood pressure. in our study the serum calcium was significantly lower in the preeclampsia group, which is consistent with the work of kanagal et al.(20) essentially similar results were obtained in a study in india, showing that plasma calcium l e ve l s w e re s i gn i f i c a n t l y l owe r i n t he preeclampsia group as compared with the control group.(22) similarly, in our study the intraerythrocytic calcium levels were shown to be significantly lower in preeclampsia as compared with controls. consistent results were also obtained in the study by igwe et al.(23) in contrast, several studies showed that serum calcium levels in the group with preeclampsia were not significantly different from those in the group with normal pregnancy.(24,25) table 1. clinical characteristics of pre-eclampsia subjects and controls table 2. membrane potential, calcium and selenium level in preeclampsia subjects and controls clinical characteristic pre-eclampsia (n=100) controls (n=100) p value age (years) bmi (kg/m2) gestational age (weeks) systolic bp (mmhg) diastolic bp (mmhg) 22.51 ± 3.42 24.99 ± 2.11 35.26 ± 3.29 142.29 ± 6.81 94.02 ± 8.33 23.55 ± 3.11 23.84 ± 2.41 38.84 ± 4.01 115.10 ± 5.11 76.63 ± 6.00 p>0.05 p>0.05 p<0.05 p<0.05 p<0.05 parameter control (n=100) pre-eclampsia (n=100) p value serum selenium(/l) 69.18 ± 2.44 63.21 ± 4.87 p<0.05 red cell calcium (mmol/l) 0.64 ± 0.07 0.56 ± 0.15 p<0.05 serum calcium (mmol/l) 2.58 ± 0.05 1.73 ± 0.13 p<0.05 membrane potential (j) 241.88 ± 42.85 185.36 ± 52.00 p<0.05 92 furthermore, it was observed from our study that serum selenium level was significantly decreased in preeclampsia when compared with the control group. selenium plays an important role as a cofactor for the reduction of antioxidant enzymes such as glutathione peroxidase, an enzyme reacting with potentially harmful oxidizing agents in substances like haemoglobin. high levels of glutathione function in the blood are linked with longevity. deficiency of selenium ma y b e a s soc i a te d w it h t he se ve r i t y of preeclampsia. this is consistent with a case-control study in bangladesh with 74 preeclampsia patients as cases and 118 normotensive pregnant women as controls from the same gestational period. this study showed that mean serum selenium concentration in preeclampsia patients was significantly lower than that of healthy pregnant women.(26) the observed reductions in the calcium and selenium levels in pregnancy may be attributed, not only to preeclampsia, but also to increase in b lo o d vol u me , a nd t o po or min e r a l supplementation in pregnancy as well as their utilization for fetal growth. a limitation of our study was that we did not determine in detail the dietary history of our subjects, so that we did not know the intakes of calcium and selenium in our study subjects. further studies should be done to investigate the relationship between i nta ke s o f c a l ci u m, s e l en i um, a nd o th e r electrolytes in preeclampsia and the importance of calcium and selenium supplementation for the prevention of preeclampsia conclusions this study showed a significant reduction of membrane potential, serum calcium and selenium level in preeclampsia compared to normal pregnancy. this demonstrates the importance of monitoring the serum calcium and serum selenium concentrations during antenatal examinations for reducing the incidence of preeclampsia. conflict of interest there is no conflict of interest. contributions jn contributed to the conception and writing of manuscript.ne contribute to data collection and critical revision of article. oh and oc c on t r i bu t e d to d a t a a n a l ys i s a nd interpretation. cn and eu contributed to data collection. all authors read and approved the final manuscript. references 1. nnodim jk, ihim a, uduji hi. alterations in antioxidants enzymes and malondialdehyde status in preeclampsia. asian pacific j trop biomed 2012;s673–8. 2. kashinakunti sv, sunitha hk, gurupadappa k, et al. lipid peroxidation and antioxidant 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https://doi.org/10.1016/j.jtemb.2015.08.002. oktavianus 143 universa medicina september-december, 2016 vol.35 no.3 global viral hepatitis elimination by the year 2030 richard tjan editor according to a report by stanaway et al.(1) in 2016, the absolute burden and relative rank of viral hepatitis increased between 1990 and 2013. for example, the number of global deaths due to viral hepatitis increased from 0.89 million to 1.45 million, indicating a need for its reduction. in this connection, on 28 may 2016 the 69th world health assembly adopted the global health sector strategy on viral hepatitis for the period 2016–2021,(2) as outlined in the report a69/32 of the secretariat,(3) with the goal of eliminating viral hepatitis b and c by the year 2030. the global health sector strategy (ghss) on viral hepatitis has constructed a roadmap toward the elimination of viral hepatitis b and c, targeting five priority prevention and treatment interventions. prevention involves universal hepatitis b immunization of infants, prevention of mother-to-child transmission, increased injection safety and blood safety, and increased harm reduction, the implementation of which will contribute toward universal health coverage, which is the target for goal 3 of the 2030 agenda for sustainable development. in combination with treatment of chronic hepatitis, the goal is to achieve by the year 2030 a reduction in the incidence of viral hepatitis by 90% and mortality by 65%.(3,4) it is common public health knowledge that elimination and/or eradication of a communicable disease is governed by a number of factors related to the agent, host and environment. when the agent attacks only humans and when vaccines and/or treatments are available, the limiting factors are usually economic and political. this should also apply to the proposed viral hepatitis elimination program. however, since this program is still in its early stages, this editorial will focus on a comparison with the historically successful smallpox eradication program. since the success of the smallpox eradication program, the who has gathered valuable data and experience about infectious disease elimination/eradication programs. smallpox eradication was facilitated by the absence of an intermediate host or animal reservoir and of a carrier state. on the negative side, there was no virocidal drug against smallpox. eradication of smallpox was mainly achieved by active surveillance and rapid containment, replacing mass vaccination as the priority approach, in combination with political support and dedicated manpower. smallpox was officially declared eradicated in 1980 and was the first viral infectious to have been combated successfully through international cooperation on a global scale, leading to its demise. in the 1960s, the disease was still endemic in africa and asia. the disease was conquered through vaccination campaigns, surveillance and prevention measures. before 1967, the smallpox eradication strategy relied on mass vaccination, which was however ineffective in densely populated regions where containment measures proved more effective, so as to relegate mass vaccination to a secondary role. the experience gained was helpful in formulating other control, elimination and/or eradication programs. to quote the 2010 the who in commemorating the 30th 144 universa medicina anniversary of smallpox eradication: “the eradication of smallpox shows that with strong mutual resolve, teamwork and an international spirit of solidarity, ambitious global public health goals can be attained.”(5) it may or may not be coincidence that the smallpox eradication program came in the wake of an unsuccessful malaria eradication program (although at that time no malaria vaccine was available), while the 2016 hepatitis elimination program comes after the 2015 malaria elimination program. hopefully both programs will achieve their goals within the targeted time. as was the case with smallpox, viral hepatitis b and c are limited to humans only, but a problem in their elimination is the presence of a carrier state. however, one favorable point is the availability of drugs for the treatment of chronic viral hepatitis, with the prospect of reduced prices in the near future.(6) globally, immunization for the prevention of viral hepatitis b has achieved considerable success. in addition to the well-established hepatitis a and b vaccines developed more than 20 years ago, only recently a vaccine for hepatitis e has become available, although not on a wide scale.(1) in 2010, indonesia, brazil, and columbia proposed a global comprehensive viral hepatitis prevention and management program, with the who providing assistance in the development of national strategies, effective surveillance, vaccine development, and treatment.(7) by the end of 2014, the number of children worldwide who received three doses of hepatitis b-containing vaccine was around 80%. the vaccine is available in combination with other vaccines in the expanded programme on immunization (epi), and has been reported to be highly effective, inexpensive, and highly costeffective.(5) the indonesian immunization program has been compulsory for each infant (aged 0-11 months) to receive complete basic immunization, including hepatitis b vaccination. during the last 9 years the hepatitis b immunization coverage had always remained above 90%, while according to three indonesian basic health surveys the coverage ranged from 62% to 76%.(7) since effective oral medicines are available for the treatment of chronic hepatitis b in persons born before hepatitis b vaccines became available, elimination of hepatitis b is now possible. since no hepatitis c vaccine is in existence, treatment with oral medicines will be used for its elimination.(5) in april 2016, the indonesian ministry of health has set out the outlines of a program to eliminate mother-to-child hepatitis b transmission by the year 2020, along with hiv and syphilis and of a hepatitis c elimination program by 2030.(9) after the implementation of mass vaccination and treatment, combined with surveillance and containment, the next step is monitoring, evaluating and reporting the results, thus providing the necessary feedback to the program directors. here the completeness and reliability of the reports are essential. in the case of hepatitis virus c infections, even in the us statistical data on hcv disease are incomplete. according to the chronic hepatitis cohort study (checs) conducted at four us healthcare systems, hcv infection is greatly underdocumented on death certificates, since only 19% of all checs decedents and only 30% of those with recorded liver disease had hcv listed on their death certificates.(10) overall, the prospect of a successful outcome of the proposed viral hepatitis elimination program seems to be relatively good, although the ghss is of the opinion that “elimination of viral hepatitis epidemics will require new technologies and approaches”, since the existing medicines, technologies and service delivery approaches are likely to be inadequate to achieve the 2030 targets.(4) the main obstacle will presumably be economic,(1) especially in developing countries with multiple concurrent elimination programs (eg. malaria and viral hepatitis) in progress. these may require considerable outside financial assistance, although the ghss stresses national funding.(4) 145 univ med 2016;35:143-5. doi: http://dx.doi.org/10.18051/univmed.2016.v35.143-145 september-december, 2016 vol.35 no.3 references 1. stanaway jd, flaxman ad, naghavi m, et al. the global burden of viral hepatitis from 1990 to 2013: findings from the global burden of disease study 2013. lancet 2016. doi: http:// dx.doi.org/10.1016/s0140-6736(16)30579-7. 2. sixty-ninth world health assembly. global health sector strategies on hiv, viral hepatitis and sexually transmitted infections, for the period 2016–2021. resolution wha69.22. geneva: world health organization;2016. 3. sixty-ninth world health assembly. draft global health sector strategies viral hepatitis, 2016– 2021. report by the secretariat.a69/32. geneva: world health organizationl;2016. 4. world health organization. global health sector strategy on viral hepatitis 2016-2021: towards ending viral hepatitis. who/hiv/2016.06. geneva: world health organization;2016. 5. world health organization advocacy brief. combating hepatitis b and c to reach elimination by 2030. geneva: world health organization;2016. 6. world health organization. the smallpox eradication programme sep (1966-1980). geneva: world health organization;2010. 7. direktorat jenderal pengendalian penyakit dan penyehatan lingkungan, kementerian kesehatan ri. pedoman pengendalian hepatitis virus. jakarta : direktorat jenderal pengendalian penyakit dan penyehatan lingkungan, kementerian kesehatan ri;2012. 8. info datin kementerian kesehatan ri;2016. situasi imunisasi di indonesia. jakarta: pusat data dan informasi, kementerian kesehatan ri;2016. 9. biro komunikasi dan pelayanan masyarakat, kementerian kesehatan ri. sebagian besar kematian akibat hepatitis virus berhubungan dengan hepatitis b dan c kronis. jakarta: biro komunikasi dan pelayanan masyarakat, kementerian kesehatan ri; 2016. 10. mahajan r, xing j, liu sj, et al. for the chronic hepatitis cohort study (checs) investigators. mortality among persons in care with hepatitis c virus infection: the chronic hepatitis cohort study (checs), 2006–2010. clin infect dis 2014;58:1055–61. oktavianus 109 *department of biology and histology, faculty of medicine, muhammadiyah university, semarang **department of pathologic anatomy, faculty of medicine, diponegoro university, semarang ***department of microbiology, faculty of medicine, diponegoro university, semarang ****biomedical master study program, faculty of medicine, diponegoro university, semarang correspondence dr. afiana rohmani, msimed department of biology and histology faculty of medicine muhammadiyah university semarang jl. wonodri sendang raya no 2a semarang 50242 email: afi.darwis@yahoo.com univ med 2014;33:109-17 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background formalin is a xenobiotic that is now commonly used as a preservative in the food industry. the liver is an organ that has the highest metabolic capacity as compared to other organs. mangosteen or garcinia mangostana linn (gml) peel contains xanthones, which are a source of natural antioxidants. the purpose of this study was to evaluate the effect of mangosteen peel extract on formalininduced liver cell mortality rate and p53 protein expression in wistar rats. methods eighteen rats received formalin orally for 2 weeks, and were subsequently divided into 3 groups, consisting of the formalin-control group receiving a placebo and treatment groups 1 and 2, which were treated with mangosteen peel extract at doses of 200 and 400 mg/kgbw/day, respectively. the treatment was carried out for 1 week, and finally the rats were terminated. the differences in liver cell mortality rate and p53 protein expression were analyzed. results one-way anova analysis showed significant differences in liver cell mortality rate among the three groups (p=0.004). the liver cell mortality rate in the treatment group receiving 400 mg/kgbw/day extract was lower than that in the formalincontrol group. there was no p53 expression in all groups. conclusions garcinia mangostana linn peel extract reduced the mortality rate of liver cells in rats receiving oral formalin. involvement of p53 expression in liver cell mortality in rats exposed to oral formalin is presumably negligible. key words : formalin, mangosteen peel, cell death, p53, rats mangosteen peel extract reduces formalin-induced liver cell death in rats afiana rohmani*, sarjadi**, winarto***, and kisdjamiatun**** 110 rohmani, sarjadi, winarto, et al mangosteen extract liver cell death ekstrak kulit manggis menurunkan kematian sel hepar tikus wistar yang diinduksi formalin latar belakang formalin merupakan salah satu xenobiotik yang sekarang ini banyak ditemukan dalam industri makanan sebagai pengawet. hepar merupakan organ yang memiliki kapasitas metabolik tertinggi dibanding organ lain. kulit manggis atau garcinia mangostana linn (gml) mengandung xanton yang merupakan sumber antioksidan. tujuan penelitian ini adalah untuk menilai pengaruh ekstrak kulit gml terhadap jumlah kematian dan ekspresi protein p53 sel hepar tikus wistar yang diinduksi larutan formalin. metode delapan belas ekor tikus diberikan formalin peroral selama 2 minggu. tikus secara acak dibagi menjadi 3 kelompok. kelompok pertama sebagai kelompok kontrol (k) diberi placebo, kelompok kedua (perlakuan 1) dan kelompok ketiga (perlakuan 2) masing-masing diberi ekstrak kulit manggis 200 dan 400 mg/kgbb/hari. perlakuan diberikan selama 1 minggu dan diakhiri dengan terminasi tikus. penilaian sel hepar dilakukan dengan melihat dan menghitung sel mati berdasarkan perubahan struktur histopatologi sel hepar, yaitu yang ditandai dengan adanya salah satu dari inti yang piknotik, karioreksis maupun kariolisis. pemeriksaan ekspresi p53 dilakukan dengan melihat dan menghitung sel hepar yang positif terekspresi p53, yaitu sel dengan inti dan sitoplasma yang tercat coklat. hasil analisis one way anova menunjukkan perbedaan jumlah kematian sel hepar pada ketiga kelompok (p=0,004). jumlah kematian sel hepar kelompok perlakuan dosis 400 mg/kgbb/hari ternyata lebih rendah dibanding kelompok kontrol. ekspresi p53 tidak didapatkan pada ketiga kelompok. kesimpulan ekstrak kulit manggis mampu menurunkan jumlah kematian sel hepar tikus yang dipaparkan formalin secara oral. kemungkinan adanya keterlibatan p53 pada kematian sel hepar tikus yang dipaparkan formalin secara oral sangat kecil. kata kunci : formalin, kulit manggis, kematian sel, p53, tikus abstrak introduction formalin is a xenobiotic that is now commonly found in the food industry as a preservative. the concentrations used are frequently unknown, because they are not registered with and not monitored by the department of health and the local drug and food supervisory board (bpom). a survey c a r r i e d o u t b y b p o m d e m o n s t r a t e d t h e existence of a number of formalin-containing foods, such as raw noodles, meat balls, dried salted fish, and tofu.(1) formalin is a colorless liquid with a pungent odor, being a solution of approximately 37 percent formaldehyde in water, to which up to 15 percent methanol is added as a preservative. exposure to formaldehyde takes place by 4 routes, i.e. by inhalation and ingestion, and through the skin and eyes. formaldehyde 111 entering the body by inhalation or ingestion is rapidly metabolized, but rather slowly when entering through cutaneous or ocular exposure.(1) formaldehyde entering the body in amounts above toxic threshold levels will react with nearly all substances in the cells, depressing cellular f u n c t i o n s , a n d u l t i m a t e l y c a u s i n g o r g a n damage.(2) unmetabolized formaldehyde may react with tetrahydrofolate, producing single carbon atoms that are electrophilic and react strongly with macromolecules, including dna and proteins. single carbon atoms also react strongly with nucleophilic cell membranes, causing increased production of reactive oxygen species (ros) in the body, thus resulting in oxidative stress. oxidative stress may cause peroxidation reactions of membrane lipids, oxidation of proteins, including enzymes, and oxidation of dna, leading to oxidative damage and carcinogenesis.(3-5) several studies have demonstrated the influence of formalin in cellular damage. formalin administered to rats at a dose of 100 ppm for 1 week will cause damage to cell m e m b r a n e s , m a r k e d b y i n c r e a s e d malondialdehyde (mda) and morphologic changes in hepatic tissues, as determined by histopathology. the free radicals produced by formaldehyde, particularly hydroxyl radicals (oh-), cause peroxidation of unsaturated fatty acids in cell membranes, resulting in oxidative damage of liver cells.(2) repeated exposure of mice to fish containing formalin at oral doses of 0.2 and 0.5 ppm for 3 months resulted in focal necrosis of the liver and renal tubuli.(6) hepatic cells undergoing cellular stress may b e d e t e c t e d b y i m m u n o h i s t o c h e m i c a l determination of p53 protein expression. the p53 proteins are produced in the cells as monitors of biochemical signals that may indicate dna damage and mutations.(7) as a result, p53 may induce cell cycle arrest at g0 (preventing the cells from continuing the cell cycle, to give time for the dna to perform repairs), or cause apoptotic cell death.(7-9) expression of p53 is frequently used in various studies as indicator of cellular damage and cell death. mangosteen or garcinia mangostana linn is a tropical plant native to indonesia (where it originated from kalimantan) and to other southeast asian countries. mangosteen peel extracts have been extensively utilized by communities, especially in thailand, for t r e a t m e n t o f w o u n d i n f e c t i o n s , a m e b i c dysentery, and as anti-inflammatory agents.(10) mangosteens are fruits that have been called super fruits, because they have a nice taste, are rich in nutrients, have antioxidant effects, and may reduce the risk of illness in humans.(11) mangosteen peel has been demonstrated to contain polyphenols, which are phenolic compounds with natural antioxidant properties. phenolic compounds are organic compounds possessing at least one aromatic ring with one or more hydroxyl groups. they may exert antioxidant effects because of their ability to stabilize free radicals, by rapidly delivering a hydrogen atom to the free radicals. in this way, phenolic compounds may protect cells from oxidative damage.(12,13) one study stated that the compound alphamangostin from mangosteen peel extract inhibited cell death from cisplatin, and reduced ros production and p53 expression.(14) other s t u d i e s m e n t i o n e d t h a t a l p h a m a n g o s t i n decreased tumor growth and metastases in mammary carcinoma.(15) many studies have explained the antioxidant effects of mangosteen peel on the basis of its hydroxyl and superoxide radical scavenging property.(10) however, there h a v e b e e n n o s t u d i e s o n t h e e f f e c t s o f mangosteen peel on cell death resulting from formalin-induced chemical lesions. the objective of this study was to evaluate the effect of m a n g o s t e e n p e e l e x t r a c t o n p 5 3 p r o t e i n expression in liver cells and on the mortality rate of wistar rat liver cells after induction by formalin. univ med vol. 33 no.2 112 rohmani, sarjadi, winarto, et al mangosteen extract liver cell death methods design of the study this was an experimental laboratory study of post test control group design using wistar rats as experimental subjects. the study was carried out in september 2013 for a period of 3 weeks. the study setting was at the inter university center (pusat antar universitas) of gadjah mada university (pau ugm), yogyakarta, and the pathologic anatomy laboratory of diponegoro university (undip), semarang. experimental animals the male wistar rats used in this study were o b t a i n e d f r o m g a j a h m a d a u n i v e r s i t y, yogyakarta, according to the following inclusion criteria: weight 150-200 grams, age 12 weeks, in healthy and active condition, and with normal external anatomic features. exclusion criteria: rats dying or becoming ill during the study. the minimum number of animals required for each group was 5, thus for 3 groups 15 animals were needed. in anticipation of dropouts one animal was added to each group. the sample size were according to the research guidelines for evaluation the safety efficiency of herbal medicines in world health organization (who).(16) so in this study there were 6 animals in each group. preparation of mangosteen peel extract mangosteen peel (pericarp) was cleansed, cut up into small parts, covered with black cloth and left to dry thoroughly in the sun. the dried peel was then minced in a blender, and 100 grams dry weight of the peel was placed in a 1-liter erlenmeyer flask. after addition of 96% ethanol to a volume of 900 ml, the mixture was thoroughly stirred, then left overnight until all particulate matter had settled to the bottom of the flask. the supernatant containing the solvent was then passed through filter paper. the filtrate was placed in a 1-liter evaporation flask connected to an evaporator, and the water bath was completely filled with water. the complete setup, including the rotary evaporator a n d w a t e r b a t h ( s e t a t 9 0 oc ) , w a s t h e n c o n n e c t e d t o t h e m a i n s e l e c t r i c i t y. t h e ethanolic solution was left in the evaporator to separate from the active substances already present in the flask, until the ethanol had stopped dripping into the receiving flask (1.5 to 2 hours for 1 flask). the extract was then put in plastic bottles and freeze-dried. intervention the rats were adapted to feed for 1 week. t h e a n i m a l s w e r e k e p t i n a r o o m a t a temperature of 25 ± 2 oc and 65-70% humidity, with a 12-hour light and dark cycle. formalin was then administered for 21 days, after which the rats were randomly assigned to 3 groups. the grouping was done by simple random allocation to prevent bias due to variation in age and weight. the formalin-control group received a placebo, while treatment groups 1 and 2 received mangosteen peel extract by gavage at a dose of 200 mg/kgbw/day and 400 mg/kgbw/day, respectively, both placebo and extract being given for 1 week. the rats were terminated by cervical dislocation under anesthesia after 1 week of treatment. histological analysis the liver of each rat was measured and weighed, macroscopically examined and fixed in a vessel containing 10% buffered formalin, at a ratio of 1 part liver tissue and 9 parts 10% buffered formalin. the liver tissue was then processed by standard histological technique and stained with hematoxylin-eosin. the liver cells were examined histologically for structural changes, followed by counting those cells showing either pycnotic nuclei, karyorhexis, or karyolysis. the evaluation and cell count results were compared with those of our pathologist, and in case of differing results, a consensus was reached. 113 a figure 1. microscopic views of p53 expression in the three groups, viz. formalin-control group (a), treatment group 1 ( b) and treatment group 2 (c). no brown-colored cells were found in the three groups (magnification 400x) b c immunohistochemistry of p53 i m m u n o h i s t o c h e m i c a l s t a i n i n g w a s performed by the indirect method, to visualize cells with p53 protein expression. unstained liver slide preparations were ligated with p53 monoclonal antibody and peroxidase-labeled secondary antibody, then characterized by double staining with diaminobenzidine (dab) and hematoxylin-eosin. the slides were then examined and counted for cells with positive p53 e x p r e s s i o n , s h o w i n g b r o w n n u c l e i a n d cytoplasms. the evaluation and cell count results were compared with those of our pathologist, and in case of differing results, a consensus was reached. statistical analysis the collected data were processed with the spss computer program. the data were evaluated for normality by means of the saphiro wilk test. since the data in this study were normally distributed, testing for between-group differences was done by one-way anova, followed by post-hoc tukey hsd. the level of significance was set at 0.05. ethical clearance this study was carried out after obtaining ethical clearance from the commision for medical research ethics, faculty of medicine, diponegoro university, semarang. results t h e c o n s e n s u s r e s u l t s o f t h e immunohistochemical assessment were negative for p53 expression, since no brown-colored cells were found, both in the formalin-control group and the treatment groups (figures 1a, b, and c). univ med vol. 33 no.2 114 rohmani, sarjadi, winarto, et al mangosteen extract liver cell death table 1. differences in liver cell mortality rate between groups treatment 1 : mangosteen peel extract at 200 mg/ kgbw/ day; treatment 2 : mangosteen peel extract at 400 mg/kgbw/day; *significant p value figure 2 . histopathological examination of the three treatment groups, viz. control group(a), treatment 1 ( b) and treatment 2 (c) (magnification 400x) a b c the differences in liver cell mortality after administration of mangosteen peel extract in rats after induction with formalin showed that there were significant differences in mean numbers of liver cell deaths between the three intervention groups. the group receiving mangosteen peel extract at a dose of 400 mg/ kgbw/day for one week, after a 3-week administration of formalin, showed the lowest mean liver cell mortality rate at 610.67 ± 59.742 cells (p=0.004). the differences in results after post-hoc tukey hsd anlysis are presented in table 1. in the control group given only formalin, the cells had a high mortality in comparison with t h e t r e a t m e n t g r o u p s 1 a n d 2 . t h e histopathological picture of the control group shows a number of liver cells with pycnotic nuclei, while some of the cells had karyorhexis and karyolysis (figure 2a). in the treatment groups, however, a number of liver cells still had intact nuclei (figures 2b and 2c). c ontrol treatment 1 treatment 2 control p=0.398 p =0.003* treatment 1 p=0.398 p =0.046* treatment 2 p=0.003* p=0.04 6* 115 between the formalin-control group and treatment group 1 (mangosteen peel extract 200 mg/kgbw) there were no significant differences in the number of dead liver cells (p=0.398), showing that mangosteen peel extract at a dose of 200 mg/kgbw had no appreciable effect on cellular recovery from formalin lesions. however, between the control group and treatment group 2 (mangosteen peel extract 400 mg/kgbw) a significant difference was found (p=0.004), indicating that mangosteen peel extract at a dose of 400 mg/kg bb induced cellular recovery from formalin lesions. discussion from the results of liver cell mortality rate assessment in our study, it is apparent that mangosteen peel extract was capable of effecting repair of histological liver cell structures that had been exposed to formalin. these observations are in agreement with those of previous studies. the study by pedraza-chaverri et al.(12) showed that xanthones isolated from mangosteen peel had pharmacologic effects, such as antioxidant and anti-inflammatory effects. the xanthones gamma-mangostin and alpha-mangostin contained in mangosteen peel showed radical scavenging activity.(17) gammamangostin also had anti-inflammatory effects through inhibition of cyclooxygenase (cox) activity. the study conducted by kosem et al.(10) showed that methanolic mangosteen peel extract had antioxidant and cytoprotective effects on endothelial cells. cell death from formalin is a chemical lesion capable of causing cellular damage and d e a t h . s w e l l i n g o f t h e c e l l i s t h e f i r s t morphological change to be seen, and is reversible in nearly all forms of cellular lesion. in advanced stages due to progressive lesions, the cell passes the point of no return, and enters the irreversible stage of cell death. the signs of cell death are to be seen in the cell nucleus. the earliest nuclear change is karyolysis, with the basophilic chromatin becoming pale, which is due to activation of dnase from decreased cellular ph. the second change is pycnosis, with shrinkage of the nucleus and increased basophilic staining, where the dna condenses into a solid, basophilic, shrunken mass. the third possible change is karyorhexis, with pycnotic nucleus, or fragmentation and disappearance of the pycnotic nucleus.(18-20) r e v e r s i b l e c e l l u l a r l e s i o n s m a y b e d e c r e a s e d b y t h e a n t o x i d a n t a n d a n t i inflammatory effects of mangosteen peel, and do not progress to the stage of irreversibility. liver cells that are still in the stage of reversible damage may recover, so as to minimize cell death or irreversible cellular damage.(20) the results of histopathological examination showed that p53 protein expression was negative in all groups (figures 1a, b, and c). there have been many studies conducted on the process of cell death or cellular damage using p53 expression as indicator, such as in mammary adenocarcinoma. the studies in question showed increased p53 expression after a 3week administration of ant-nest extract to mice with mammary adenocarcinoma. this indicates that p53 is a trigger protein for apoptosis of mammary cancer cells.(15) expression of p53 is also seen in cisplatin-induced cells.(15) in our study there were no cells expressing p53 protein. this may have been caused by the lack of opportunity of the cellular death pathway due to formalin-induced chemical lesions to relay instructions for p53 activation. formalin lesions are traumatic chemical lesions causing cell damage and necrotic death resulting from ros production and histological hypoxia. cell death from formalin lesions is necrotic death without p53 involvement. the cell death in the present study was acute, being a traumatic chemical lesion directly affecting morphological cell structures. the cells in our study were normal cells without carcinogenic initiation, mutation, or chronic processes. the observations in our study were short term, whereas cellular dynamics are ongoing processes. this constitutes a limitation of our univ med vol. 33 no.2 116 rohmani, sarjadi, winarto, et al mangosteen extract liver cell death study, since it is possible that during the short o b s e r v a t i o n p e r i o d , t h e r e h a d b e e n n o opportunity for expression of p53 proteins leading to cell death. further studies are needed to confirm the involvement of apoptosis in cell death due to oral formalin exposure. with regard to clinical application, the present study is expected to become the foundation study for discovery of antioxidant herbal drugs, to be utilized in humans for the cure or prevention of free radical-induced tissue damage. conclusions mangosteen peel extract effects celullar recovery from formalin-induced chemical lesions. involvement of p53 expression in liver cell mortality rate in rats exposed to oral formalin is presumably negligible. acknowledgments the investigators wish to express their utmost gratitude to the late prof sarjadi sppa for his patience in helping and guiding the conduct and preparation of this study. may he receive the best place at his side, and may his knowledge be of benefit to mankind and forever be a tribute to his constant flow of good deeds. references 1. agency for toxic substance and disease registry (atsdr) division of toxicology and environmental medicine. addendum to the toxicological profile of formaldehyde. atlanta, ga 30333;2010. 2. mahdi c, aulaniam, widodo, sumarno. yogurt sebagai detoksidan yang efektif terhadap toksisitas formalin yang terpapar dalam makanan. jurnal protein 2007;15:10-1. 3. u.s. department of health and human servicespublic health service national toxicology program (ntp). report on carcinogens: background document for formaldehyde; 2009. available at: http://www.ntp.niehs.nih.gov/ntp/ roc/twelfth/2009/formaldehyde_bd_final.pdf. assessed january 12, 2012. 4. juzral j, nasni y. pengaruh pemberian tahu berformalin terhadap gangguan fungsi hati dan terbentuknya radikal bebas dalam tubuh tikus putih. jurnal sains teknologi farmasi 2008;13:18. 5. price sa, wilson lm. gangguan asam-basa. dalam: hartanto h, susi n, wulansari p, mahanani, editors. patofisiologi, konsep klinis proses-proses penyakit. edisi 6. jakarta: egc; 2005.hal.384-9. 6. kartikaningsih h. pengaruh paparan berulang ikan berformalin terhadap kerusakan hati dan ginjal mencit (mus musculus) sebagai media pembelajaran keamanan pangan [disertasi]. malang: universitas negeri malang; 2008. 7. vaseva am. p53 opens the mitochondrial permeability transition pore to trigger necrosis. cell j 2012;149:1536-48. 8. dharmayanti i. kajian biologi molekuler gen supressor tumor (p53) sebagai target gen dalam pengobatan kanker. wartazoa 2005;l13:99-107. 9. spike bt, wahl gm. p53 stem cells and reprogramming tumor supression beyond guarding the genome. genes cancer 2011;2:40419. 10. kosem n, han yh, moongkarndia p. antioxidant and cytoprotective activities of methanolic extract from garcinia mangostana. science asia 2007;33:283-92. 11. primchanien m, wuttavut k, sincernart k, omboon l, naronguhai p, neelobol n. antiproliferation, antioxidation and induction of apoptosis by garcinia mangostana on skbr3 human breast cancer cell line. j ethno pharmacol 2004; 90:161-7. 12. pedraza-chaverri j, cardenas-rodriguez n, orozco-ibarra m, perez-rojas jm. medicinal properties of mangosteen (garcinia mangostana). food chem toxicol 2008;46:322739. 13. nugroho ae. manggis (garcinia mangostana l) : dari kulit buah yang terbuang hingga menjadi kandidat suatu obat. j traditional med 2012;12:42. 14. perez s. the alpha-mangostin prevention on cisplatin-induced apoptotic death in llc-pk1 cells is associated to an inhibition of ros production and p53 induction. chem biol interaction 2010;188:144-50. 15. kurose h, shibata ma, linuma m, otsuki y. alterations in cell cycle and induction of apoptotic cell death in breast cancer cells treated with á-mangostin extracted from mangosteen pericarp. j biomed biotechnol 2012:672428. doi: 10.1155/2012/672428. 117 16. budiarto e. biostatistik untuk kedokteran dan kesehatan masyarakat. jakarta: egc; 2002. 17. ibrahim my, mariod aa, mohan s. alpha mangostin from garcinia mangostana linn an updated review of its pharmacological properties. arabian j chem 2014:2.11. doi. 10.1016/j.arabjc. 2014.02.011 18. robins sl, kumar v, cotran rs. jejas sel dan adaptasi. dalam: asroruddin m, hartanto h, darmaniah n, editors. buku ajar patologi. edisi 7 jakarta: egc;2007. hal. 3-31. 19. hairrudin, helianti d, widiastuti y. aktifitas fisik berat menyebabkan degenerasi sel hepatosit melalui mekanisme stres oksidatif. jurnal kedokteran brawijaya 2012;27:61-5. 20. vanlangenakker n, van den berghe t, krysko dv. festiens n, vandenbeele p. molecular mechanisms and pathophysiology of necrotic cell death. curr mol med 2008;8:207-20. univ med vol. 33 no.2 untitled-1 80 *departement of physiology, faculty of medicine, trisakti university, jakarta **magister program in biomedical sciences faculty of medicine, university of indonesia ***departement of physiology, faculty of medicine, university of indonesia correspondence: dr. donna adriani, m.biomed departement of physiology, faculty of medicine trisakti university jl. kyai tapa no. 260 grogol jakarta 11440 phone: +628991777777 email: donna.adriani@trisakti.ac.id date of first submission, march 9, 2017 date of final revised submission, june 6, 2017 date of acceptance, june 7, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 combination of aerobic exercise and hibiscus sabdariffa linn. increased nitric oxide in rats donna adriani*,**, minarma siagian***, and dewi irawati*** background hypertension and myocardial infarction account for the high rate of mortality globally. hibiscus sabdariffa (hs) linn. is rich in antioxidants and previous studies have demonstrated its anti-hypertensive effects. several studies show that regular physical activity is an important component to reduce cardiovascular mortality. the objective of this study was to evaluate the effects of a combination of aerobic exercise and hs extract on nitric oxide (no) and endothelin-1 (et-1) in rats. methods an experimental study was conducted on 36 male wistar rats, aged 4 weeks and 60-70 g in weight. the interventions were aerobic exercises and hs at 400 mg/kg bw/day administered for 4, 8 and 12 weeks. the rats were randomized into 12 groups: 3 control groups (c4, c8, c12), 3 aerobic exercise groups (a4, a8, a12), 3 hs groups (h4, h8, h12), and 3 combination groups [aerobic exercise and hs] (ha4, ha8, ha12). after 4, 8, and 12 weeks, the rats were sacrificed and their abdominal aorta was collected for determination of nitric oxide and et-1 concentrations. one way anova was used to analyze the data. results there was a significant difference in no levels between all groups, with the 4-week aerobic exercise group (a4) showing the highest no levels compared to the other eleven groups (p<0.05). in contrast, the et-1 levels were not significantly different between all groups. conclusions this study demonstrated that the combination of hs supplementation and aerobic exercise increases no in rats, and provided further evidence to the traditional use of the plant as an antioxidants agent. keywords: aerobic exercise, hibiscus sabdariffa linn., nitric oxide, endothelin-1, rats doi: http://dx.doi.org/10.18051/univmed.2017.v36.80-87 original article cite this article as: adriani d, siagian m, irawati d. combination of aerobic exercise and hibiscus sabdariffa linn. increased nitric oxide in rats. univ med 2017 ;36 :8 0 -7 . d oi : 10.1 805 1/ univmed.2017.v36.80-87 pissn: 1907-3062 / eissn: 2407-2230 81 univ med vol. 36 no.2 introduction cardiovascular disease (cvd) and stroke produce immense health and economic burdens in the united states and globally. it has been projected that by the year 2030, 41.4% of us adults will have hypertension (an increase of 8.4% from 2012 estimates) and 43.9% of the us population will have some form of cvd. cardiovascular disease is one of the three principal causes of death in america.(1) one of the underlying pathological processes of cardiovascular disease is atherosclerosis, which is initiated by endothelial dysfunction occurring in childhood due to various risk factors. according to the study of hong,(2) 50% of american children aged 10–14 years show initial signs of atherosclerosis as a result of oxidative stress. in the pr esent study, to pr event endothelial dysfunction in 5-week old rats (which are the equivalent of 10-year old children),(3,4) the animals were subjected to aerobic physical exercises and h. sabdariffa l. administration, separately or in combination. regularly performed physical exercise may improve endothelial dysfunction through increased blood flow, which results in higher vascular shear stress. the latter increases the phosphorylation of endothelial no synthase (enos), leading to increased enos activity. therefore the bioavailability of nitric oxide (no) is improved, so increasing no production and inhibiting the production of endothelin-1 (et1).(5-7) oxidative stress may cause a decrease in the bioavailability of no, so causing hypertension and atherosclerosis. the decreased no production increases binding of et-1 to its receptors (et a / et b ), so increasing calcium levels in vascular smooth muscle cells, resulting in vasoconstriction and ultimately hypertension.(6,7) one herb that has a strong antioxidant effect and can prevent oxidative stress is hibiscus sabdariffa (hs) linn.(8,9) antioxidants act as electron donors, so preventing oxidation. hibiscus sabdariffa linn. contains anthocyanins that act as antioxidants.(8) hibiscus sabdariffa linn. (malvaceae) (common name: roselle) is used in traditional medicine as a diuret ic and for treating hypertension, fever, and gastrointestinal and hepatic disorders. the fresh or dried buds, calyces, flowers, leaves, and petals of hs are also used as refreshing beverages, food items, or lotions.(10,11) the antihypertensive activity of hs could be possibly via the inhibition of angiotensinconverting enzyme activity (ace),(11) or by incr ementing the pr oduction of no an endothelium-derived relaxation factor (edrf).(12) two systematic reviews have been published on the effectiveness of hs for the treatment of hypertension, but both reviews found that the evidence available at the time of review was inconclusive.(13,14) nitric oxide (no) is often recognized as an important indicator of vascular health. this compound plays an important role in blood pressure regulation due to its vasodilating potency,(15) as well as its ability to inhibit aggregation of platelets and proliferation of vascular smooth muscle cells (vsmcs).(16) vascular smooth muscle cells participate in the pathogene sis of hype rtension, and their proliferation contributes to increased peripheral resistance by decreasing arterial diameters.(17) amongst the several vsmc growth inhibitors are nitric oxide (no) , c yclic guanosine monophosphate (cgmp), transforming growth factor-beta ( tgfb ), and adenosine monophosphate activated protein kinase (ampk).(18) a study examining the effects of a crude methanolic hs calyx extract on vascular reactivity in isolated aortas from spontaneously hypertensive rats, showed that hs increased no in these animals.(12) however, another study showed that administration of hs did not clearly increase no levels.(19) one study on the administration of polyphenols from hibiscus sabdariffa calyces at 125 mg/kg bw/day for 4 weeks to patients with metabolic syndrome showed a non-significant reduction in endothelin-1 levels.(20) however, no 82 adriani, siagian, irawati aerobic exercise and hibiscus sabdariffa linn. information could be obtained regarding the effect of a combination of physical exercise and he on nitric oxide and endothelin-1 levels in wistar rats. the study by padilla et al.(21) found that the abdominal aorta in rats is more susceptible to atherosclerosis than the thoracal aorta. the purpose of the present study was to evaluate the effect of a combination of physical exercise and hs on nitric oxide and endothelin-1 levels in rats at stages equivalent to childhood, adolescence, and adulthood in humans. methods research design the study was c a rried out a t the experimental animal laboratory, center for biomedical and basic health technology, agency for health research and development, ministry of health, republic of indonesia, and at the integrated laboratory, faculty of medicine, university of indonesia from july to november 2015. animals the experimental animals were 36 healthy male wistar strain rats (rattus norvegicus), 4 weeks of age and weighing around 60–70 grams. before performing aerobic physical exercises (groups a and ha), the rats were acclimatized by running treadmills for 1 week. before and during treatment, the rats were prevented from falling ill. the rats were fed a standard diet at portions corresponding to their age, and water ad libitum, and were kept in cages, with each cage containing 3 rats. the cages were kept clean and subjected to 12 hour light-and-dark cycles. the environmental temperature was kept at 23±1 oc. all other conditions were maintained in accordance with the ethics code of the commission on management and utilization of experimental animals. the sample size was determined with the federer formula: (t-1) (n-1) 15, where t = number of experimental groups (12); n=number of animals per group. the calculated sample size was 3 animals per group, giving a total of 36 animals. the 5-week old male wistar strain rats were randomized into 12 groups, i.e. groups 1 to 3 (4, 8and 12-week controls), groups 4 to 6 (aerobic physical exercise for 4, 8 and 12 weeks, respectively), groups 7 to 9 (hs at 400 mg/kgbw/ day for 4, 8 and 12 weeks, respectively), groups 10 to 12 (combination of aerobic physical exercise and hs administration at 400 mg/kgbw/day for 4, 8, and 12 weeks, respectively). intervention the a erobic physical exercises were performed in treadmills at gradually increasing speeds and maintained at moderate intensities in accordance with the protocol of hsu et al.(22) as adapted by utami.(23) the initial speed of the aerobic physical exercises for the groups of rats aged 5, 7 and over 9 weeks was 12 m/min, 15 m/ min, and 20 m/min, respectively, with each speed being maintained for 2 weeks. the frequency of aerobic physical exercise was 5 times per week, with a duration of 20 minutes and 90 seconds rest for each 5-minute run, to prevent fatigue.(23) preparation of hibiscus sabdariffa linn. ethanol extract the plant material was obtained from the research station for spice and medicinal plants (balai penelitian tanaman rempah dan obat, balitro). the hs calyces were dried and then ground to a degree of fineness of 3-4 mm. one kilogram of the ground sample was mixed with 5 liters of 96% ethanol for 3 hours. the mixture was shaken for 2–3 hours, then kept for 24 hours at room temperature, filtered and the filtrate dried in a rotavapor at 50 o–60 oc, yielding a concentrated extract, which was stored in the refrigerator until further use. an aqueous suspension was prepared by dissolving suitable amounts of the ethanolic hs extract to obtain the desired concentration. the hs solutions were prepared freshly each time and administered by gavage using a cannulated syringe. the dosing schedule used was once per day. the hs extract was administered for 5 days per week according to group, at an oral dose of 400 mg/kgbw/day. 83 univ med vol. 36 no.2 measurements and laboratory analysis the no levels were determined by means of a nitric oxide colorimetric assay kit (biovision, catalog #k262-200). the kit measures total nitrites in the sample in 2 stages, i.e. conversion of nitrates into nitrites using nitrate reductase, then conversion of nitrites into dark-violet colored azo compounds using griess reagent. the materials and reagents used were homogenates of rat abdominal aortas, nitrate standard solution, assay buffer, nitrate reductase, enzyme cofactor, enhancer, griess reagent r1, and griess reagent r2. subsequently the standard or calibration curve was constructed as follows: the standard working solution was 5 ml (100mm) of standard solution mixed with 495 ml of assay buffer. for the concentration of 0 nmol/well, 85 ml of assay buffer was used. for the concentration of 2 nmol/ well, 2 ml standard solution mixed with 83 ml assay buffer. for the concentration of 4 nmol/well, 4 ml standard solution mixed with 81 ml assay of buffer. for the concentration of 6 nmol/well, 6 ml standard solution mixed with 79 ml assay buffer. for the concentration of 8 nmol/well, 8 ml standard solution mixed with 77 ml assay buffer. for the concentration of 10 nmol/well, 10 ml standard solution mixed with 75 ml assay buffer. the assay buffer to be used as blanks, at a volume of 200 ml was pipetted into the wells, then standards (0, 2, 4, 6, 8, 10) and samples at 85 ml each were pipetted into the wells, 115 ml of assay buffer was added to each well (standards and samples), then 5 ml each of nitrate reductase and enzyme cofactor were successively added to each well (standards and samples). the plate was covered and incubated at room temperature for 1 hour to convert nitrate into nitrite. then 5 ml of enhancer was added to each well and the plate was incubated for 10 minutes (standards and samples). in addition, 50 ml of griess reagent r1 50 ml of griess reagent r2 were successively added to each well (standards and samples), after which the plate was incubated at room temperature for 10 minutes, and the absorbance read at the wavelength of 540 nm. after construction of the standard curve, the values of a=0.1535, b=-0.0004, and r=0.9998 were obtained. the no concentrations were calculated from the standard curve. the no concentrations were divided by the protein concentr ations in mg to give the protein concentrations in nmol/mg. the et-1 concentrations were assayed with the uscnk elisa kit (catalog no. cea482ra). specific monoclonal antibody against et-1 was pre-coated on the microplate. a competitive inhibition reaction then occurs between biotinlabeled et-1 (standard) or non-labeled et-1 (sample) and the specific anti-et-1 antibody coating on the microplate. after an incubation period, the unbound conjugate was washed away. then hrp-conjugated avidin was added to each well and the plate again incubated. the amount of bound hrp conjugate is inversely proportional to the et-1 concentration in the sample. after the addition of the substrate, the color intensity is inversely proportional to the et-1 concentration in the sample. the materials and reagents used were homogenates of rat abdominal aortas, 500 pg/ml of standard stock solution (standard mixed with 0.5 ml of standard diluent), detection reagent a, detec tion reagent b, wash solution, and tetramethylbenzidine (tmb) substrate. construction of the standard curve: for the standard solution concentration of 0 pg/ml, 3.90625 pg/ml of standard solution was used. for the standard solution concentration of 15.625 pg/ml, 300 l of standard solution at a concentration of 31.25 pg/ml was mixed with 0.6 ml standard diluent. for the standard solution concentration of 31.25 pg/ml, 300 ml of standard solution at a concentration of 62.5 pg/ml was mixed with 0.6 ml standard diluent. similarly, for the standard solution concentrations of 62.5 pg/ml and 125 pg/ml, 300 ml standard solutions at concentrations of 125 pg/ml and 250 pg/ml, respectively, were each mixed with 0.6 ml standard diluent. finally, for the standard solution concentration of 250 pg/ml, 300 ml of standard stock solution was mixed with 0.6 ml standard diluent. 84 assay procedur e: 50 ml volumes of standard solution at concentrations of 0; 3.90625; 15.625; 31.25; 62.5; and 125 pg/ml, were each pipetted into their respective wells. then 50 ml of detection reagent a was rapidly added to each well, the plate covered with plate sealer and shaken using the microplate shaker and incubated for 1 hour at 37oc. then the plate was washed 3 times, after which 100 ml of detection reagent b was added to each well and the plate incubated for 30 minutes at 37oc. after incubation, the plate was washed 5 times, then 90 ml of substrate solution was added to the wells and the plate incubated for 25-25 minutes at 37oc. finally, 50 ml of stop solution was added to the wells. the absorbances were read as soon as possible at a wavelength of 450 nm. after construction of the standard curve, the values of a=-0.4334, b=-0.3568, and r=0.9871 were obtained. the et-1 concentrations were divided by the protein values in mg to obtain protein values in nmol/mg. statistical analysis data analysis was performed by one-way anova using spss version 21 and a=0.05. ethical clearance this study received ethical clearance from the health research ethics committee, faculty of medicine, university of indonesia, under no. 796/un2.f1/etik/2015. results no concentrations a comparison of no and et-1 levels in the control groups (c) and treatment groups (a, h, and ha) for 4, 8 and 12 weeks is presented in table 1. there were significant differences in no levels between the control and treatment groups after 4 weeks and 12 weeks of intervention, but not after 8 weeks of intervention. the et-1 concentrations in the control groups, in the physical exercise groups, and in the hs groups tended to increase progressively, but the increases were not significantly different. a comparison of no and et-1 levels in the 12 treatment groups is presented in table 2. there were significant differences in no but not in et1 concentrations between all groups. the no levels in the aerobic physical exercise groups (a4, a8, a12) tended to decrease with increasing duration of the physical exercises. the no levels in the aerobic physical exercise groups were higher than those in the controls. the highest no level was in the 4-week aerobic physical exercise group (a4), while the lowest no level was in the control groups after 12 weeks of treatment (c12). the results of the multiple comparison analysis showed that the groups with significant differences were c4 and a4, c4 and a8, c4 and ha8, c8 and a4, c8 and a8, c8 and ha8, c12 and a4, c12 and a8, c12 and ha8, a4 and a12, a4 and h4, a4 and h8, a4 and ha4, a4 and ha12, a8 and a12, a8 and h4, a8 and h8, a8 and ha4, a8 and ha12, a12 and ha8, h4 and ha8, ha4 and ha8 ( p<0.05) (data not presented). discussion the no levels in the control groups (c4, c8, and c12) tended to decrease with increasing age. this agrees with the results of a study carried out by puzserova et al.(24) who explained that the aging process is closely related to a decrease in no production and bioavailability. the no levels in the aerobic physical exercise groups (a4, a8, and a12) tended to decrease with increasing age and duration of the physical exercises. lantz et al.(25) and trinity et al.(26) explained that increasing age causes a reduction in shear stress, due to greater vessel diameter. the no levels in the aerobic physical exercise groups were higher than those in controls. this result is in agreement with the study by utami (23) who found higher no levels in the aerobic physical exercise groups in comparison with the controls in juvenile rats. this is presumably because of increased enos mrna adriani, siagian, irawati aerobic exercise and hibiscus sabdariffa linn. 85 univ med vol. 36 no.2 t ab le 1 . n o a n d e t -1 l ev el s af te r 4 , 8 a n d 1 2 w ee k s o f in te rv en ti o n , b y t re at m en t g ro u p s l eg en d : c = c o n tr o l gr o u p s, a = a er o b ic e xe rc is e gr o u p s, h = h ib is cu s sa b d a ri ff a l in n. g ro up s, h a = g ro u p s tr ea te d w it h a co m b in at io n o f ae ro b ic e xe rc is e an d h ib is cu s sa b d a ri ff a l in n. n o : n it yr ic o xi de ; e t -1 : en do th el in -1 ; d at a ar e p re se nt ed as m ea n ± s d t ab le 2 . n o a n d e t -1 l ev el s b et w ee n a ll c o n tr o l an d t re at m en t g ro u p s l eg en d : c = c o n tr o l gr o u p s, a = a er o b ic e xe rc is e gr o u p s, h = h ib is cu s sa b d a ri ff a l in n. g ro up s, h a = g ro u p s tr ea te d w it h a c o m b in at io n o f ae ro b ic e xe rc is e an d h ib is cu s sa b d a ri ff a l in n . ; n o : n it yr ic o x id e; e t -1 : e nd o th el in -1 ; d at a ar e p re se n te d a s m ea n ± s d . 86 expression in the aortas after long-term physical exercise. consistent results were obtained in the study by ajay et al.(12) who showed that hs administration at 0.3 mg/ml increased no levels in the aortic ring from spontaneously hypertensive rats. these data suggests that the relaxant effects of hs involved activation of endothelium-derived nitric oxide (edno) /c yclic guanosine monophosphate (cgmp)-relaxant pathway activity in addition to the increased synthesis/ release of edno. the no levels in the hs groups (h4, h8, and h12) tended to increase with increasing age. this was presumably due to the prevention of oxidative str ess, which improved no bioavailability, so as to increase no production.(6,7) the no levels in the hs groups were higher compared to those in the controls in all age groups. however, the no levels in groups h4 and h8, respectively, were lower as compared to those in groups a4 and a8, respectively, presumably reflecting the inadequacy of the administered hs dose.the no levels in the groups with a combination of hs administration and aerobic physical exercises (ha4, ha8, and ha12) were found to be higher in adolescents as compared with children and adults. this is presumably due to the higher et-1 levels in adolescents. the no level in group ha4 was lower than that in the controls, probably because of competitive inhibition. according to this study, the et-1 levels in the control group s tended to incr ease progressively, but non-significantly. this is due to the decreased no bioavailability so as to cause increa sed et-1 production from physical inactivity.(6,7) hs extracts are generally considered to have a low degree of toxicity. studies demonstrate that hs consumption does not adversely effect liver and kidney function at lower doses, but may be hepatotoxic at extremely high doses in addition, electrolyte levels generally are not effected by ingesting hs extracts despite its diuretic effects.(27) hs has great potential to reduce risk factors associated with cardiovascular disease and merits further study. the present findings explain the anti-oxidant effect of hibiscus sabdariffa linn. in vivo, and provide further evidence for the traditional use of the plant as an anti-oxidant agent. conclusions this study demon strated that hs supplementation at 400 mg/kgbw and aerobic exercise increases no concentration in rats. conflict of interest competing interests: no relevant disclosures. acknowledgement the authors wish to express their gratitude to the faculty of medicine, trisakti university, for the funding of this study. contributors dakm, ms and diss contributed to the concept and design of the study, data acquisition, data analysis and/or interpretation. dakm, ms and diss drafted and revised the manuscript critically for important intellectual content. all authors read and approved the final manuscript references 1. mozaffarian d, benjamin ej, go as, et al. heart disease and stroke statistics-2015 update: a report from the american heart association. circulation 2015;131:4-132. 2. hong ym. atherosclerotic cardiovascular disease beginning in childhood. korean circ j 2010;40:1-9. 3. andreollo na, dos santos 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2014, article id 658479, 12 pages. doi: http://dx.doi.org/10.1155/2014/658479. 25. lantz j, renner j, länne t, karlsson m. is aortic wall shear stress affected by aging? an imagebased numerical study with two age groups. med eng phys 2015;37:265-71. 26. trinity jd, groot hj, layec g, et al. impact of age and body position on the contribution of nitric oxide to femoral artery shear rate. hypertension 2014;63:1019-25. 27. hopkins al, lamm mg, funk j, et al. hibiscus sabdariffa l. in the treatment of hypertension and hyperlipidemia: a comprehensive review of animal and human studies. fitoterapia 2013;85: 84–94. doi: 10.1016/j.fitote.2013.01. 003. c:\users\jurnal fkusakti\docume 57 *department of medical laboratory technology, faculty of nursing and health science, muhammadiyah university, semarang **department of pharmacy, dr kariadi hospital, diponegoro university, semarang correspondence: dr. budi santosa,s.km,m.si.med medical laboratory technology, faculty of nursing and health science, muhammadiyah university jl kedungmundu raya 18 semarang mobile: +62818 0586 7211 email: budisantosa@unimus.ac.id date of first submission, june 4, 2017 date of final revised submission, april 16, 2018 date of acceptance, april 18, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina aqueous ir bagendit rice leaf extract decreases reticulocyte count in lead-exposed rats budi santosa*, henna ria sunoko**, and andri sukeksi* background lead acetate may inhibit the enzyme aminolevulinate dehydratase (alad) resulting in decreased heme synthesis (and consequently in anemia) but in increased number of reticulocyte cells. ir bagendit paddy leaf water extract has a high metallothionein protein content which acts to bind to lead. the study objective was to determine whether aqueous ir bagendit rice leaf extract dosage variations prior to lead exposure decreases reticulocyte count in lead-exposed rats. methods the study was of randomized post test only control-group design involving a sample of 28 rats, that were randomized into 4 groups consisting of 1 control group and 3 treatment groups, daily administered with aqueous ir bagendit rice leaf extract of respectively 0.2; 0.4; 0.8 mg using a feeding tube up to week 13. lead exposure was also given using a feeding tube to both control and treatment groups at a dose of 0.5 g/kg bw/day, up to week 13. the reticulocyte count was then examined using supravital brilliant cresyl blue staining. the reticulocyte count was determined per 1000 erythrocytes and then converted into a percentage. kruskal wallis test followed with bonferroni test was conducted to figure out the differences between groups. results mean reticulocyte count decreased significantly, starting from the control group up to the third treatment group (15.48 ± 3.41; 12.25 ± 03.28; 10.45 ±1.47; 9.10 ± 2.35 average per unit) (p=0.02). the bonferroni test showed that the reticulocyte count was significantly decreased in the third treatment group (p=0.004). conclusion aqueous rice leaf extract significantly decreases reticulocytes in rats exposed to lead. keywords: ir bagendit, reticulocytes, exposed, lead, metallothionein, rats doi: http://dx.doi.org/10.18051/univmed.2018.v37.57-64 original article pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 cite this article as: santosa b, sunoko hr, sukeksi a. aqueous ir bagendit rice leaf extract decreases reticulocyte count in lead-exposed rats. univ med 2018;37: 57-64. doi: 10.18051/univmed.2018. v37.57-64 58 santosa, sunoko, sukeksi rice leaf extract and reticulocyte in rats introduction in indonesia the use of lead is still relatively high, although it has largely been abandoned in the case of automotive fuels. the principal sources of lead intoxication are vegetables contaminated as a result of the use of phosphate fertilizers, pesticides, and herbicides. in addition, the use of batteries, paints, cosmetics, jewelry, toys, gasoline, etc., also constitutes a source of lead intoxication because they use lead as raw material. lead exposure in humans may occur through leadcontaminated air, foods, and drinks. those at highest risk of lead intoxication are children, pregnant women, and industrial workers.(1) one of the symptoms of lead intoxication is anemia(2) that is accompanied by increased reticulocyte count, decreased hematocrit concentration, and the presence of erythrocyte inclusions, i.e. basophilic stippling.(3,4) the study conducted by sunoko (5) showed that the blood lead content at a concentration of 7 µg/dl may cause inhibition of enzyme activity for hemoglobin synthesis, leading to a subclinical effect that is marked by the occurrence of elevated concentrations of δ-aminolevulinic acid (δ ala) and protoporphyrin in children, resulting in anemia. the occurrence of anemia depends on the lead concentration that may inhibit the activities of ferrochelatase, aminolevulinic acid synthetase (alas), and aminolevulinic acid dehydratase (alad), three important enzymes associated with heme synthesis. exposure to lead at 10 µg/dl may already inhibit alad, causing accumulation of aminolevulinic acid, so resulting in failure of heme synthesis that carries the risk of anemia.(6) cases of anemia are usually also accompanied by an increase in reticulocyte count. a low reticulocyte count indicates aplasia or disorders of the bone marrow. peripheral blood smears may supply additional information in patients with anemia in each of the morphological aspects, particularly reticulocytes.(7) the reticulocyte count constitutes an important parameter in the diagnosis of anemia from lead exposure. reticulocytes are nonnucleated erythroid cells that may visualized with supravital staining that colors ribonucleic acid (rna). supravital stains that may be used are solutions of brilliant cresyl blue, new methylene blue, azure b, and acridine orange for visual methods. the reticulocyte count is an important component of the complete blood examination that is relatively accurate, simple, and practical for describing the number of erythrocytes produced by the erythropoietic system.(8) the benefit of the reticulocyte count is for knowing the erythropoietic activity. an absolute increase in reticulocyte count marks the presence of increased erythropoietic activity. in anemia, there is also increased erythropoietic activity, accompanied by an increase in reticulocyte count, which is inversely correlated with hemoglobin concentration, with higher reticulocyte counts indicating lower hemoglobin concentrations. in lead intoxication there is anemia so that the reticulocyte count is increased.(9) the study conducted by hariono (10) showed an increase in reticulocyte count since the 10th week after oral administration of lead acetate at 0.5 g/kg/bw/ day in rats for 16 weeks. the study by hariono (10) also showed the occurrence of anemia accompanied by an increase in δ aminolevulinic acid dehydratase (alad) activity, and a decrease in body weight. in heme biosynthesis, lead intoxication depresses enzyme activities at the starting point (alad), at midpoint (coproporphyrinogen oxidase), and at the e ndpoint (fer rochelatase ) of heme biosynthesis, thus leading to anemia. approximately 90% of the lead introduced into the circulation will enter the erythrocytes, where it acts as a pro-oxidant, causing oxidative stress that r esults in damage to the er ythrocyte membranes and shortening of the erythrocyte lifespan.(3,4) membrane damage and shortened erythrocyte lif espan cause a decrease in erythrocyte numbers and volume in the blood. this increases the erythropoietic activity that is marked by an increase in reticulocyte count. lead may also cause glucose-6 phosphate dehydrogenase (g-6pd) deficiency and inhibit pyrimidine-5’59 nucleotidase that may cause the accumulation of ribonucleic acid (rna).(3,4) preventive mea su res against lead intoxication need to be studied and developed so that its effects will not become more severe. to date lead intoxication is managed with chelating agents, e.g. ethyelenediamine tetra-acetic acid (edta) and 2,3-dimercaprol, but the use of these substances is curative, not preventive. chelating agents are substances that act to bind toxic metal ions, so forming complex structures that are readily excreted by the body, either intracellularly or extracellularly. dimercaprol has long been used for the treatment of lead and arsenic intoxication, but has dangerous side effects so that less toxic analogs need to be developed.(11) these preventive measures to be developed may use vegetable substances that can bind to lead. one of these is the aqueous extract of the leaves of the ir bagendit variety of rice (oryza sativa l). the leaves of ir bagendit rice have the highest metallothionein protein content among vegetable substances such as corn, soybeans, and green beans.(12) the metallothionein proteins can bind to lead in the tissues via their sulfhydryl groups.(13) metallothioneins can be synthesized in the liver as well as in the gastrointestinal wall through the absorption of various vegetable substances as well as micronutrients, such as zinc, and has been demonstrated to improve heme biosynthesis, hematopoiesis, epithelial degeneration and necrosis of the renal tubule, and basophilic stippling.(14,15) the study conducted by santosa et al.(16) reported that zinc supplementation administered in stepwise increasing daily doses to rats (rattus norvegicus) was able t o increase metallothioneins and that the effect had a positive correlation, i.e. the higher the administered zinc dose, the higher the mean metallothionein concentration. in a subsequent study, metallothioneins were proven to be able to prevent the inhibition of heme biosynthesis, hematopoiesis, and epithelial degeneration and necrosis of the renal tubule in rats exposed to lead.(14,15) this showed that metallothioneins are consistent metalbinding proteins in the tissues of all organisms. regarding vegetable substances, it turns out that metallothioneins are abundantly present in the aqueous extract of ir bagendit rice leaves.(12) with reference to the supplementation dose and the metallothionein content of ir bagendit rice leaves, administration of stepwise increasing doses for determining the efficacy of the doses should be considered, e.g. 10 mg per day, 20 mg per day and 40 mg per day in humans. these doses can be converted to doses for the test animals, becoming 0.2 mg, 0.4 mg, and 0.8 mg, respectively.(16) in the present study the event to be observed was not anymore the increase in reticulocyte count as a result of lead exposure, as was proven in previous studies that yielded consistent results, i.e. the presence of substantial numbers of reticulocytes, but whether the aqueous extract of ir bagendit rice leaves administered to lead-exposed rats may reduce the reticulocyte count. the objective of the present study was to determine the effect of the aqueous extract of ir bagendit rice leaves on the reticulocyte count in rats exposed to lead. methods study design this study used a randomized post-test only control group design.(17) the test animals (rattus norvegicus) were maintained and subjected to the interventions in the integrated research and testing laboratory (lppt), gadjah mada university, yogyakarta. the study was carried out from may to august 2017. experimental animals determination of the sample size (ss) was performed with the formula: ss = (t – 1) (r – 1) 15 where t = number of groups and r = number of test animals per group. the study used 3 intervention groups and 1 control group, so that t=4, (4-1)(r-1) 15 ——r 6. the number of rats used was 6 for each group (3 intervention groups and 1 control group) so that the total sample size used in this study was 24. to the number of rats univ med vol. 37 no.1 60 santosa, sunoko, sukeksi rice leaf extract and reticulocyte in rats a b c in each group, one rat was added in reserve to anticipate the possibility of the rats dying, so that the total sample size was 28 rats (rattus norvegicus). the selected rats were all males, aged 15 weeks and weighing 180-220 grams. the maintenance and intervention of the test animals (rattus norvegicus) was performed in the integrated research and testing laboratory (lppt), gadjah mada university, yogyakarta. the maintenance from the selection period up to and including the intervention period lasted 100 days. in the intervention period the rats received standard pellet feed, aqueous extract of ir bagendit rice leaves, and lead via feeding tubes of appropriate size. aqueous extract of ir bagendit rice leaves by simple random sampling, the 28 rats were divided into 4 groups i.e. 1 control group and 3 intervention groups. from the first week up to the 13th week, the intervention groups received aqueous extract of ir bagendit rice leaves in stepwise increasing doses, at 0.2, 0.4, and 0.8 ml/day, respectively, via feeding tube, whereas the control group did not receive the aqueous extract. the aqueous extract of ir bagendit rice leaves in question was an extract prepared by infusion. the concentrations chosen were 0.2 ml, 0.4 ml, and 0.8 ml, referring to the metallothioneins contained in these volume doses.(12) lead exposure exposure to lead acetate was administered orally for 100 days, using a feeding tube, at a dose of 0.5 g/kg bw/day in all groups, both control and intervention groups.(10) determination of the reticulocyte count on the la st day of the 13th week the reticulocyte count was determined in the control and intervention groups. specimens for the reticulocyte count consisted of whole blood with edta anticoagulant, as part of the normal fluid composition in the vacutainer. reticulocyte counting used blood smears with supravital brilliant cresyl blue staining. the reticulocytes were counted per 1000 erythrocytes in the blood smears and the count multiplied by 100% to convert the result into a percentage (%). the normal value of the reticulocyte count is 0-1%.(18) determination of the blood parameters was per formed at the health analyst clinical pathology laboratory, department of medical laboratory technology, faculty of nursing and health science, muhammadiyah university, semarang (unimus). data analysis data analysis for determining the optimal concentration of aqueous extract of ir bagendit rice leaves in decreasing the reticulocyte count in rats exposed to lead was conducted with the kruskal wallis test, followed by bonferroni’s test. ethical clearance the present study obtained ethical clearance from the ethics commission, faculty of medicine, universitas islam sultan agung (unissula) semarang, under no. 209/vi/2017/komisi bioetik. the results of the ethical clearance were sent to the head of the integrated research and testing laboratory (lppt), gadjah mada university, yogyakarta. and constituted approval for starting the study. results the reticulocytes are formed because of the accumulation of ribonucleic acid (rna) and ribosomes in the erythrocytes as a result of inhibition of the enzyme pyr imidine-5’nucleotidase in lead exposure. in figure 1, the control group k that did not receive aqueous extract of ir bagendit rice leaves had a higher reticulocyte count compared with the intervention groups that did receive the extract, i.e. intervention groups p1 (0.2 mg), p2 (0.4 mg), and p3 (0.8 mg). at baseline, the weight of the test animals did not show significant differences (p=0.202). this indicates that the performed block 61 randomization procedure was able to distribute the weights evenly among the four groups. after administration of the intervention, there was an increase in the weight of the test animals, particularly in the intervention groups (p=0.000). the results of the kruskal-wallis test showed a significant decrease in reticulocyte count after administration of the intervention for 13 weeks, particularly in the intervention groups receiving ir bagendit rice leaf extract at 0.8 ml/day (p=0.021) (table 1). according to the results of bonferroni’s test (table 2), the difference in the reticulocyte count was between the control group and intervention group p2 that received ir bagendit rice leaf extract at 0.4 mg (p=0.013) and between the control group and intervention p3 that received ir bagendit rice leaf extract at 0.8 mg (p=0.004). univ med vol. 37 no.1 intervention 2 intervention 3 control intervention 1 figure 1. reticulocyte count in control group and intervention groups 1,2, and 3 (magnification 1000x). arrowheads point to reticulocytes variable intervention p value k (n=7) p1 (n=7) p2 (n=7) p3 (n=7) base line body weight (g) 185.71 ± 2.39 189.6 ± 1.36 188.39 ± 1.07 187.50± 5.42 0.202 after intervention body weight (g) reticulocyte count (%) 211.72 ± 4.08 15.48 ± 3.41 218.80 ± 3.56 12.25 ± 3.28 217.70 ± 2.01 10.45 ± 1.47 214.67 ±1.01 9.10 ± 2.35 0.000 0.021 table 1. mean reticulocyte count and difference in body weight after the 13th week, by intervention group 62 santosa, sunoko, sukeksi rice leaf extract and reticulocyte in rats discussion in the present study there was a decrease in the mean reticulocyte count. the highest mean reticulocyte count was in the control group that did not receive ir bagendit rice leaf extract. the lowest mean reticulocyte count was in intervention group p2 that received ir bagendit rice leaf extract at the highest dose of 0.8 mg. statistically there was a significant difference in reticulocyte count be twee n the control group and the intervention groups. the higher the dose of the ir bagendit rice leaf extract, the lower the reticulocyte count in the rats exposed to lead. the results this study were supported by the study of hariono (10), in that exposure to lead at 0.5 g/kg/bw wa s proven to inc rease the reticulocyte count in the rats in the 10th week. the reticulocytes are marked by punctate forms and bleaching of the blue color in the erythrocytes that frequently occur in cases of lead exposure. lead inhibits pyrimidine-5'-nucleotidase and alters the function of the other nucleotides. lead can disturb heme synthesis, so changing the concentrations of enzymes and intermediates in heme synthesis or its derivatives. lead poisoning may cause an increased reticulocyte count in the blood.(19) the novelty of the present study compared with previous studies lies in the fact that the reticulocyte count may have decreased because of the administration of ir bagendit rice leaf extract in the rats exposed to lead. lead exposure may cause membr ane damage in the erythrocytes, so shortening their lifespan and decreasing the erythrocyte count. the bone marrow as the site of erythropoiesis will compensate for the decreased numbers of erythrocytes in the circulation by increasing the erythropoietic activity, causing an increase in immature erythrocytes in the circulation.(4,20) in this study, lead exposure without aqueous ir bagendit rice leaf extract resulted in high mean reticulocyte count, whereas lead exposure with aqueous ir bagendit rice leaf extract resulted in decreased me an retic ulocyte count. t his demonstrates that aqueous ir bagendit rice leaf extract was able to decrease the reticulocyte count in rats exposed to lead. the reticulocyte count is the number of non-nucleated erythrocyte with remains of rna. the reticulocyte count is considerably affected by the erythrocyte count and hemoglobin concentration. the lower the erythrocyte count and hemoglobin concentration, the higher the erythropoie tic activity that increases the reticulocyte count. in this study, increased erythropoietic activity was marked by an increased reticulocyte count in lead-exposed rats receiving the highest dose of aqueous ir bagendit rice leaf extract (0.8). however, the reticulocyte count in lead-exposed rats that did not receive aqueous ir bagendit rice leaf extract was lower compared with that in the rats that did receive the extract. there was a significant difference in reticulocyte count between the control group that only received lead exposure and the intervention groups that in addition to lead exposure also received aqueous ir bagendit rice leaf extract in stepwise increasing doses, with a positive correlation of reticulocyte count with the doses. increases in the doses of aqueous ir bagendit rice leaf extract was followed by increased reticulocyte count in rats exposed to lead. the study conducted by santosa and sunoko (12) reported that the aqueous leaf extract from several rice varieties containing the highest concentration of metallothioneins was that of the leaves of ir bagendit rice. metallothioneins act mean difference p value k p1 p2 p3 3.23 5.03 6.36 0.182 0.013 0.004 p1 p2 p3 p2 p3 1.79 3.12 1.33 0.652 0.263 0.834 table 2. bonferroni’s multiple comparison test of reticulocyte count after thirteenth week, by treatment group k : control; p1: intervention group receiving ir bagendit rice leaf extract at 0.2 ml/day; p2 : intervention group receiving ir bagendit rice leaf extract at 0.4 ml/day; p3 : intervention group receiving ir bagendit rice leaf extract at 0.8 mg/ml/day 63 to bind lead and can improve the erythropoietic activity as a result of exposure to lead. most of the lead in the blood is present in the erythrocytes, where 90% is in the cytoplasm and 10% in the membrane, particularly in lipids and lipoproteins.(4) the distribution of lead in the plasma and erythrocyte membrane is due to the presence of lead bound to cytoplasmic elements, transportation via the erythrocyte membrane, and excretion through the calcium pump. the toxic effect of lead in erythrocytes is due to destruction of the erythrocytes, accelerating the sequestration of the erythrocytes in the spleen via exposure to phosphatidylserine (ps) and then increasing erythrophagocytosis.(21) many investigators have reported that the principal mechanism of lead toxicity is the production of free radicals. reactive oxygen species (ros) react with cellular macromolecules (dna, proteins, lipids).(22) erythrocytes are very susceptible to induction of oxidative stress due to high lead exposure.(22,23) according to murray et al.(3) and luis et al.(4), approximately 90% of the lead introduced into the circulation will enter the erythrocytes. in the erythrocyte membrane there occur chemical reactions producing potentially toxic oxygen species called pro-oxidants. increases in the amount of pro-oxidants may cause oxidative stress. lead may also cause deficiency of the enzyme g-6pd (glucose -6 phosphate dehydrogenase) that may inhibit erythrocyte maturation in the bone marrow.(3,4) gugliotta et al. (24) investigated the effect of lead on the proteins in the erythrocyte membrane. their results showed a decreased erythrocyte count, with membrane permeability playing an important role in the decrease. an in vitro study on lead exposure of erythrocytes that was conducted by mrugesh et al.(25) proved that lead nitrate is a cytotoxic agent that may cause hemolysis even at extremely low concentrations. in the experimental laborator y study conducted by gugliotta,(24) heparinized blood with hematocrit concentration of 3% was incubated for 1 hour at 25° c in a medium containing increasing concentrations of lead chloride (0, 0.3, 0.5 and 1 µm). the results of the study showed an increase in erythrocyte permeability with consequent cellular damage and de ath, particularly at high lead concentrations, changes in morphology and membrane structure of the red blood cell, and decreased erythrocyte atp concentrations. the clinical implication of this study is that aqueous ir bagendit rice leaf extract was proven to decrease the reticulocyte count in rats exposed to lead, indicating that aqueous ir bagendit rice leaf extract dec reases the impact of lead exposure, and therefore needs further study for its application in humans. one limitation of this study is that the highest administered dose is 0.8 ml, so leaving open the question whether doses higher than 0.8 ml can still decrease the reticulocyte count. future developments of this study should be in the form of clinical trials on the use of aqueous rice leaf extract for the prevention of anemia in humans exposed to lead. the gene coding for the metallothionein content of the rice leaves should also be studied. conclusion aqueous ir bagendit rice leaf extract at a concentration of 0.8 ml is able to optimally decrease the reticulocyte count in rats exposed to lead. acknowledgment the authors thank the ministry research and technology, directorate of higher education (ristek dikti) for the funding of this study through grants. contributors bs formulated the design of the study, conducted the experiments, performed data analysis, drafted, revised and critically reviewed the manuscript. hrs contributed to data collection and analysis. as contributed to preparation of univ med vol. 37 no.1 64 santosa, sunoko, sukeksi rice leaf extract and reticulocyte in rats reagents and material, and to data analysis. all authors read and approved the final manuscript. references 1. national health & medical research council. blood lead levels: lead exposure and health effects in australia. national health & medical research council 2009. melboune: national health & medical research council;2009. 2. hegazy aa, zaher mm, abd el-hafez ma, et al. relation between anemia and blood levels of lead, copper, zinc and iron among children. bmc 2010;3:133-8. doi: https://doi.org/10.1186/17560500-3-133. 3. murray rk. porphyrin and bile pigments. in: murray rk, granner dk, rodwell vw, editors. harper’s illlustrated biochemistry. 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toxicol sci 2011;122:177-84. doi: http://doi.org/10.1093/ toxsci/kfr079. 22. almeida-lopes acb, peixe ts, mesas ae, et al. lead exposure and oxidative stress: a systematic review. rev environ contam toxicol 2016;236:193238. doi: 10.1007/978-3-319-20013-2-3. 23. kasperczyk a, słowińska-łożyńska l, dobrakowski m,et al. the effect of lead-induced oxidative stress on blood viscosity and rheological properties of erythrocytes in lead exposed humans. clin hemorheol microcirc 2014; 56:187-95. 24. gugliotta t, luca gd, romano p, rigano c, scuteri a , romano l. effects of lead chloride on human erythrocyte membranes and on kinetic anion sulphate and glutathione concentrations. cel mol biol lett 2012;17:586-97. 25. mrugesh t, dipa l, manishika g. effect of lead on human erythrocytes: an in vitro study. acta poloniae pharmaceutica-drug research 2011;68: 653-6. adi priyana 129 exon prediction on dna-genes of plasmodium falciparum based on coding sequence structure using hidden markov model suhartati agoes*a, dadang gunawan**, sardy s**, and hoedojo*** universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 abstract * electrical engineering department, faculty of industrial technology, trisakti university ** electrical engineering department, faculty of engineering, indonesia university *** department of parasitology, medical faculty, trisakti university korespondensi air. suhartati agoes, mt electrical engineering department, faculty of industrial technology, trisakti university jl. kyai tapa no.1, grogol jakarta 11440 telp. 08164836613 email: suhartati_agoes@yahoo.com universa medicina 2007; 26: 12936. background a hidden markov model (hmm) is used for exon prediction on dna of genes plasmodium falciparum that has a model structure based on exon region structure in coding sequence (cds). the objective research was to develop a new structure model to predict exon on dna-genes of plasmodium falciparum based on cds structure using the hmm system. methods model design in cds, between two exon regions can be found one intron region and the model state number is used for its region. its state number is used by separating start codon from first exon region and stop codon from the last exon region up to 9. the viterbi algorithm and the backward-forward method for transition as well as emission states are used for training process. furthermore, viterbi and baum-welch algorithms are used for the testing process. the correlation coefficient (cc) was used as performance indicator, as the ratio of the estimated state in the output and the original state in the input of the model. results the simulation results has shown that the cc values depend on the given of the backward-forward transition state values randomly. the model with state number 9 showed the highest average of cc values of 0.7289 for viterbi algorithm, and is 0.7166 for baum-welch algorithm. however, the lowest average of cc values has been found for the model with state number five. its values are 0.6735 by using viterbi algorithm and 0.6661 by using baum-welch algorithm. conclusion the new structure model based on hmm system was valid to predict exon on dna-genes of plasmodium falciparum. keywords: exon prediction, dna-gene, coding sequence, hidden markov model 130 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm prediksi ekson dna-gen plasmodium falciparum berdasarkan struktur coding sequence dengan menggunakan model hidden markov suhartati agoes*a, dadang gunawan**, sardy s**, dan hoedojo*** latar belakang sebuah hidden markov model (hmm) yang digunakan untuk memprediksi ekson gen dna palsmodium falciparum mempunyai struktur model berdasarkan struktur gen dna pada coding sequence (cds). penelitian ini bertujuan untuk mengembangkan sebuah model stuktur baru untuk prediksi ekson gen dna plasmodium falciparum berdasarkan struktur cds dengan menggunakan sistem hmm. metode rancangan model pada cds, di antara dua lokasi ekson dapat diketahui sebuah lokasi intron dan jumlah state model dapat dilakukan pada lokasi tersebut. jumlah state dilakukan dengan memisahkan codon start dari ekson pertama dan codon stop dari ekson terakhir hingga mencapai 9. algoritma viterbi dan metode backward-forward transisi state serta emisi state digunakan untuk proses training. sedangkan untuk proses testing menggunakan algoritma viterbi dan baum-welch. sebagai kinerja model digunakan correlation coefficient (cc) yang didapat dari perbandingan state estimasi pada output dan state asli pada input model. hasil hasil simulasi menunjukkan bahwa nilai cc tergantung pada pemberian nilai acak state transisi backward-forward. model dengan jumlah state 9, menunjukkan nilai cc rata-rata tertinggi adalah 0,7289 untuk algoritma viterbi dan 0,7166 untuk algoritma baum-welch. kesimpulan struktur model berdasarkan sistem hmm sahih untuk memprediksi ekson gen dna plamodium falciparum kata kunci : prediksi ekson, gen-dna, coding sequence, model hidden markov * jurusan teknik elektro fakultas teknologi industri universitas trisakti ** jurusan teknik elektro, fakultas teknik universitas indonesia *** bagian parasitologi fakultas kedokteran universitas trisakti korespondensi air. suhartati agoes, mt jurusan teknik elektro fakultas teknologi industri universitas trisakti jl. kyai tapa no.1, grogol jakarta 11440 telp. 08164836613 email: suhartati_agoes@yahoo.com universa medicina 2007; 26: 129-36. abstrak introduction i n c o d i n g s e q u e n c e ( c d s ) , t h e e x o n prediction on dna-gene is very important to find the protein structure. genome p. falciparum belongs to genome eukaryotic and has a long dna genome, consisting of several exons and introns alternately located. after the splicing process in dna sequence, some regions of exon in cds will be found to produce the protein.(1) 131 t h e h i d d e n m a r k o v m o d e l ( h m m ) structure is one of the model used to predict the exon in desoxyribonucleai acid (dna) which is based on the exon region structure in cds. the hmm structure of the model: start codon (methyanine/atg), exon, intron and stop c o d o n ( ta a , o r t g a , o r ta g ) h a s b e e n d e m o n s t r a t e d b y r a b i n e r, h e n d e r s o n a n d krogh.(2-4) this research developed a new model of structures for exon prediction on dna of genes plasmodium falciparum was based on the work of nicorici and anantharaman.(5,6) the significant difference of this model is it at least has two exon regions used for exon prediction like in cds. usually the 5’ boundary of introns in most eukaryotes contains the dinucleotide guanine-thymine (gt), and the 3’ boundary contains the dinucleotide adenine-guanine (ag). furthermore, in intron regions of the dinucleotide gt were separated to be nucleotide guanine (g) and thymine (t), and dinucleotide ag to be nucleotide a and g. method research design the hmm framework in this experiment used viterbi algorithm for the training process and both viterbi and baum-welch algorithms for the testing process. the same sequence has been done for the training and testing process. the program is written in matlab 7.0, and bioi n f o r m a t i c s ’ t o o l b o x t o g e n e r a t e d n a sequences in genbank format and has the functions of hmm training and testing. the first experiment for the model based on cds has been done with separated start codon (codon: atg) from first exon and stop codon (codon: taa, or tag, or tga) from the last exon, its model has the state number of 5 (figure 1). furthermore, the second experiment u s e d s e p a r a t e d d i n u c l e o t i d e g t a n d dinucleotide ag from the intron region, its model has the state number of 7 (figure 2). the last experiment in the model also used separated d i n u c l e o t i d e g t i n t o g a n d t s t a t e s a n d dinucleotide ag into a and g states; the state number of the model is 9 (figure 3). the emission values of the models can be performed a s t h e m a t r i x , t h e c o l u m n s a r e t h e b a s e s numbered on dna sequences and the rows are the states number of the models designed. the state transition values of the models can be shown also in the figures. samples a genome sequence of p. falciparum using 3d7 clone has 23-megabase nuclear genomes consisting of 14 chromosomes, encodes about 5,300 genes, and it has the most (a+t)-rich genome sequenced to date.(8-10) the data set of experiments for this simulation has 152 dna sequences of genes from genome plasmodium f a l c i p a r u m a t : c h r o m o s o m e 1 ( l o c u s : n c _ 0 0 4 3 2 5 ) , c h r o m o s o m e 2 ( l o c u s : n c _ 0 0 0 9 1 0 ) , c h r o m o s o m e 3 ( l o c u s : n c _ 0 0 0 5 2 1 ) , c h r o m o s o m e 4 ( l o c u s : n c _ 0 0 4 3 1 8 ) , c h r o m o s o m e 5 ( l o c u s : n c _ 0 0 4 3 2 6 ) , a n d c h r o m o s o m e 9 ( l o c u s : nc_004330) in genbank format.(11,12) genbank format describes the cds and original dna sequences of genes p. falciparum. in cds of genes contains at least two exon regions and maximum 10 exon regions. the minimum length sequence of genes is 684 base pairs (bp) and maximum length is 10095 bp. hmm provides a good probability method for discrete sequences model of data like dna sequences (alphabet of four letters: a, c, g and t). prediction of exon in dna of genes p. falciparum is based on exon region in cds and it has at least two exon regions. the structure of the model based on cds structure as shown in figure 4, which separated is start codon from the first exon and separated stop codon from the last exon. universa medicina vol. 26 no.3 132 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm in the inputs are dna sequences and set the state number of the sequences depends on the structure model. based on hmm method, the viterbi algorithm used for the training and needs the transition state values and emission values for the process. the value of transition state is random and the value of emission can be found with the distribution number of dna nucleotide in each states. the result of hmm training is the estimated transition state and emission state values. the estimated transition and emission values were used for hmm testing p r o c e s s w i t h vi t e r b i a n d b a u m we l c h algorithms. the results of hmm testing process are the estimated states of the model.(2) the parameter of performance indicator used the figure 3. the hmm structure with 9 states exon intron exonstart stop figure 4. the hmm structure based on cds 9 start e g t i a g 1 2 3 4 5 6 7 e stop 8 1 1 0.9 0.1 0.9 0.9 0.1 0.1 0.9 0.9 0.1 0.1 0.9 0.1 0.8 0.1 0.1 5 0 .9 1 e is ta rt s to p 2 3 4 1 0 .5 0 .5 0 .1 0 .8 1 0 .1 9 1 1 0 -6e figure 1. the hmm structure with 5 states s tart e g t i a g e stop 1 2 3 4 5 6 7 1 10.85 0.1 0.05 0.7 0.3 0.9 0.1 0.7 0.9 0.1 0.3 figure 2. the hmm structure with 7 states 133 correlation coefficient (cc), as a ratio of the estimated states from the output and the original states from the input of the model(1,7) and the formula is the following: where: tp= true positive; fp= false positive; tn= true negative; fn= false negative statistical analysis the emission matrix of the model with state number 5 is: e = [ 0.3333 0 0.3333 0.3333 ; 0.4342 0.1094 0.1507 0.3056 ; 0.4210 0.0599 0.0703 0.4487 ; 0.4215 0.1173 0.1622 0.2990 ; 0.5592 0 0.1075 0.3333 ] the emission matrix of the model with state number 7 is: e = [ 0.3333 0 0.3333 0.3333 ; 0.4342 0.1094 0.1507 0.3056 ; 0 0 0.5000 0.5000 ; 0.4210 0.0599 0.0703 0.4487 ; 0.5000 0 0.5000 0 ; 0.4215 0.1173 0.1622 0.2990 ; 0.5592 0 0.1075 0.3333 ] the emission matrix of the model with state number 9 is: e = [ 0.3333 0 0.3333 0.3333 ; 0.4342 0.1094 0.1507 0.3056 ; 0 0 1.0000 0 ; 0 0 0 1.0000 ; 0.4210 0.0599 0.0703 0.4487 ; 1.0000 0 0 0 ; 0 0 1.0000 0 ; 0.4215 0.1173 0.1622 0.2990 ; 0.5592 0 0.1075 0.3333 ] fn)fp).(tnfp).(tpfn).(tn(tp (fp.fn)-(tp.tn) cc ++++ = the transition state values are randomly, but the first state on the model has the minimum transition state value of 0 and the last state has the maximum transition state value of 1. on the other hand, the emission state values were found from the distribution number of dna nucleotide i n e a c h s t a t e d e p e n d i n g o n t h e m o d e l s . calculation of the cc is by using the above equation with the assumptions that the exon is positive and intron is negative. results based on exon regions structure in cds, the simulation results for the model with state number 5 above and transition state values randomly has found the average of cc value in table 1. table 2 and table 3 shows the average of cc values of the model with state number 7 and state number 9. the transition state value in the first state of all structures on the models is 0 and the last state is 1. the other transition state values except in table 2 are 0.1 and the h i g h e s t a v e r a g e o f c c v a l u e s f o r t h i s experiments are shown in figure 5. the model based on exon regions structure in cds for state number nine resulted in the highest average of cc. its values are 0.7289 by using viterbi algorithm and 0.7166 by using baum-welch algorithm. however, the lowest average of cc values has been found for the model with state number five. its values are 0.6735 by using viterbi algorithm and 0.6661 by using baum-welch algorithm. discussion all these were based on the model of exon regions structure in cds, at the state number nine resulted in the highest average of cc values. it is also shown that the emission state value of t h e m o d e l s h a v e t h e ( a + t ) r i c h g e n o m e sequences of genes plasmodium falciparum. universa medicina vol. 26 no.3 134 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm transition state values corr.coef.(cc) exps tr11 tr22 tr33 tr44 tr55 viterbi b-welch 1 0 0.5 0.9 0.80 1 0.7054 0.6750 2 0 0.5 0.9 0.81 1 0.7061 0.6759 3 0 0.5 0.9 0.82 1 0.7054 0.6750 4 0 0.5 0.9 0.83 1 0.7054 0.6750 5 0 0.5 0.9 0.84 1 0.7045 0.6741 6 0 0.5 0.9 0.85 1 0.7054 0.6750 7 0 0.5 0.9 0.86 1 0.7062 0.6744 8 0 0.5 0.9 0.87 1 0.0012 0.0015 9 0 0.5 0.9 0.88 1 0.0011 0.0013 10 0 0.5 0.9 0.89 1 0.0011 0.0013 table 1. the average of cc values for the model with state number 5 transition state values corr.coef.(cc) exps tr11 tr22 tr33 tr44 tr55 tr66 tr77 viterbi b-welch 1 0 0.4 0.2 0.8 0.10 0.7 1 0.6713 0.6643 2 0 0.4 0.2 0.8 0.20 0.7 1 0.6713 0.6643 3 0 0.4 0.2 0.8 0.30 0.7 1 0.6733 0.6660 4 0 0.4 0.2 0.8 0.40 0.7 1 0.6731 0.6660 5 0 0.4 0.2 0.8 0.50 0.7 1 0.6735 0.6661 6 0 0.4 0.2 0.8 0.60 0.7 1 -0.0642 -0.0729 7 0 0.4 0.2 0.8 0.59 0.7 1 -0.0568 -0.0639 8 0 0.4 0.2 0.8 0.58 0.7 1 0.6735 0.6661 9 0 0.4 0.2 0.8 0.56 0.7 1 0.6735 0.6661 10 0 0.4 0.2 0.8 0.55 0.7 1 0.6735 0.6661 table 2. the average of cc values for the model with state number 7 transition state values corr.coef.(cc) exps tr11 tr22 tr33 tr44 tr55 tr66 tr77 tr88 tr99 viterbi b-welch 1 0 0.3 0.2 0.2 0.80 0.10 0.10 0.70 1 0.7242 0.7124 2 0 0.3 0.1 0.1 0.79 0.10 0.10 0.70 1 0.7242 0.7124 3 0 0.3 0.1 0.1 0.78 0.09 0.09 0.70 1 0.7242 0.7124 4 0 0.3 0.1 0.1 0.78 0.20 0.20 0.70 1 0.7258 0.7120 5 0 0.3 0.1 0.1 0.80 0.20 0.20 0.80 1 0.7258 0.7120 6 0 0.3 0.1 0.1 0.80 0.15 0.15 0.74 1 0.7258 0.7120 7 0 0.3 0.1 0.1 0.80 0.15 0.15 0.80 1 0.7258 0.7120 8 0 0.3 0.1 0.1 0.80 0.15 0.15 0.70 1 0.7258 0.7120 9 0 0.8 0.2 0.2 0.80 0.10 0.10 0.70 1 0.6787 0.6755 10 0 0.9 0.2 0.2 0.80 0.10 0.10 0.70 1 0.0451 0.0437 table 3. the average of cc values for the model with state number 9 135 this can also be showed in their matrix values described above. in order to match with markov’s chain in eukaryotic gene structure for c o n n e c t i n g t h e e x o n a n d i n t r o n , t h e n t h e backward-forward method are used in this hmm system. the experiments were done by viterbi algorithm for the training process and the both viterbi and baum-welch algorithms for the testing process. a set of 152 sequences is then used as data to train the model and a different hmm is produced for each set of sequences. hmm have been previously used in sequence analysis to produce an hmm that represents a sequence profile (a profile hmm), to analyze sequence composition and patterns, to locate genes by p r e d i c t i n g o p e n r e a d i n g f r a m e o r c o d i n g sequence, and to produce protein structure predictions. this hmm generates sequences 0.62 0.64 0.66 0.68 0.7 0.72 0.74 c orrelation c oefficient (c c ) t he highest average of cc values viterbi 0.7061 0.6735 0.7258 ba um-we lch 0.6759 0.6661 0.712 5 7 9 w i t h v a r i o u s c o m b i n a t i o n s o f m a t c h e d , mismatched, insertions and deletions, and gives these a probability, depending on the values of the various parameters in the model. a total of 424 predicted protein-coding g e n e s a n d 3 t r n a g e n e s o f p l a s m o d i u m falciparum have been identified on chromosomes 2 and 3, the two chromosomes for which s e q u e n c i n g h a v e b e e n c o m p l e t e d . ( 1 3 , 1 4 ) approximately 37% (158 genes) of three genes have a readily identifiable homologue in another species. such similarity allows a function to be implied, with many of those identified being involved in parasite metabolism. interestingly, a comparison of the predicted protein sequences with their homologues in other species showed that the majority of plasmodium falciparum proteins have insertion of low complexity sequences, often runs of a single amino acid figure 5. the highest average of cc values of the experiments universa medicina vol. 26 no.3 136 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm r e s i d u e ( t y p i c a l l y a s p a r a g i n e s , l y s i n e o r glutamine acid), or tandem arrays of a short peptide repeat sequence. examples of such regions have been identified previously, and are polymorphic between different parasite isolates. perhaps it will open up new avenues for drug and vaccine development. perhaps it will make a genetically modified mosquito that cannot act as vector a reality once processes are in place to deal with ethical, legal and social issues. but any new developments are unlikely to happen before the 2010 deadline set for roll back malaria. new drugs based on knowledge about the genome will probably not be affordable to those countries that need it the most.(15) conclusion this research with a new structure model was using for exon prediction on dna-genes of plasmodium falciparum based on cds structure by using the hmm system. acknowledgment t h e a u t h o r s w o u l d l i k e t o t h a n k d r. herawati sudoyo, phd, dr. helena ms and hidayat trimarsanto b.sc from the eijkman institute, for their valuable suggestions. references 1. samatova n.f. computational gene finding using hmms. ut-battelle information center, 1201 oak ridge turnpike, suite 100, oak ridge, tn 37830. institute oak ridge national laboratory; 2003. 2. lawrence r. a tutorial on hidden markov models and selected applications in speech recognition. proceeding of the ieee. 1989; 77: 257-68. 3. henderson j, salzberg s, fasman k.h. finding genes in dna with a hidden markov model. j comput biol 1997; 4: 127-42. 4. krogh a. an introduction to hidden markov models for biological sequences. in computational methods in molecular biology. salzberg sl, searls db, kasif s. editors. denmark. center for biological sequence analysis, technical university of denmark. 1998. p. 45-63. 5. nicorici d, astola j, tobus i. computational identification of exons in dna with a hidden markov model. tampere international center for signal processing, tampere university of technology, finland. available at: http:// www.gensips.gatech.edu/processings/contributed/ cp2-06.pdf. accessed may 12, 2005. 6. anantharaman t. finding genes in genomic dna the genescan system. available at : http:// www.biostat.wisc.edu/bmi776. accessed june 6, 2005. 7. vaisman i. bioinformatics and gene discovery. bioinformatics tutorial. north carolina, united state: university of north carolina at chapel hill. 1998. 8. hall n, gardner m.j, hyman rw, lasonder e, wilson rjm, scherf a, et al. sequence of plasmodium falciparum chromosomes 1, 3-9 and 13. nature 2002; 419: 527-31. 9. gardner m.j, hall n, hyman rw, hinterberg k, mattei d, wellem te, et al. sequence of plasmodium falciparum chromosomes 2, 10, 11 and 14. nature 2002; 419: 531-4. 10. hyman rw, gardner m.j, hall n, de bruin d, scherf a, day kp, et al. sequence of plasmodium falciparum chromosome 12. nature 2002; 419: 534-7. 11. anastassiou d. genomic signal processing. ieee signal processing magazine 2001; 18: 4. 12. alphey l. dna sequencing. manchester uk : bios scientific publishers limited; 1997. 13. gardner m.j, hall n, fung e, white o, berriman m, hyman rw, et al. chromosome 2 sequence of the human malaria parasite plasmodium falciparum. science 1998; 282: 1126-32. 14. bowman s, lawson d, basham d, brown d, chillingworth t, churcher c. m, et al. the complete nucleotide sequence of chromosome 3 of plasmodium falciparum. nature 1999; 400: 5328. 15. anonymous. malaria after the genomes. the lancet 2002; 360: 1107. 129 exon prediction on dna-genes of plasmodium falciparum based on coding sequence structure using hidden markov model suhartati agoes*a, dadang gunawan**, sardy s**, and hoedojo*** universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 abstract * electrical engineering department, faculty of industrial technology, trisakti university ** electrical engineering department, faculty of engineering, indonesia university *** department of parasitology, medical faculty, trisakti university korespondensi air. suhartati agoes, mt electrical engineering department, faculty of industrial technology, trisakti university jl. kyai tapa no.1, grogol jakarta 11440 telp. 08164836613 email: suhartati_agoes@yahoo.com universa medicina 2007; 26: 12936. background a hidden markov model (hmm) is used for exon prediction on dna of genes plasmodium falciparum that has a model structure based on exon region structure in coding sequence (cds). the objective research was to develop a new structure model to predict exon on dna-genes of plasmodium falciparum based on cds structure using the hmm system. methods model design in cds, between two exon regions can be found one intron region and the model state number is used for its region. its state number is used by separating start codon from first exon region and stop codon from the last exon region up to 9. the viterbi algorithm and the backward-forward method for transition as well as emission states are used for training process. furthermore, viterbi and baum-welch algorithms are used for the testing process. the correlation coefficient (cc) was used as performance indicator, as the ratio of the estimated state in the output and the original state in the input of the model. results the simulation results has shown that the cc values depend on the given of the backward-forward transition state values randomly. the model with state number 9 showed the highest average of cc values of 0.7289 for viterbi algorithm, and is 0.7166 for baum-welch algorithm. however, the lowest average of cc values has been found for the model with state number five. its values are 0.6735 by using viterbi algorithm and 0.6661 by using baum-welch algorithm. conclusion the new structure model based on hmm system was valid to predict exon on dna-genes of plasmodium falciparum. keywords: exon prediction, dna-gene, coding sequence, hidden markov model 130 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm prediksi ekson dna-gen plasmodium falciparum berdasarkan struktur coding sequence dengan menggunakan model hidden markov suhartati agoes*a, dadang gunawan**, sardy s**, dan hoedojo*** latar belakang sebuah hidden markov model (hmm) yang digunakan untuk memprediksi ekson gen dna palsmodium falciparum mempunyai struktur model berdasarkan struktur gen dna pada coding sequence (cds). penelitian ini bertujuan untuk mengembangkan sebuah model stuktur baru untuk prediksi ekson gen dna plasmodium falciparum berdasarkan struktur cds dengan menggunakan sistem hmm. metode rancangan model pada cds, di antara dua lokasi ekson dapat diketahui sebuah lokasi intron dan jumlah state model dapat dilakukan pada lokasi tersebut. jumlah state dilakukan dengan memisahkan codon start dari ekson pertama dan codon stop dari ekson terakhir hingga mencapai 9. algoritma viterbi dan metode backward-forward transisi state serta emisi state digunakan untuk proses training. sedangkan untuk proses testing menggunakan algoritma viterbi dan baum-welch. sebagai kinerja model digunakan correlation coefficient (cc) yang didapat dari perbandingan state estimasi pada output dan state asli pada input model. hasil hasil simulasi menunjukkan bahwa nilai cc tergantung pada pemberian nilai acak state transisi backward-forward. model dengan jumlah state 9, menunjukkan nilai cc rata-rata tertinggi adalah 0,7289 untuk algoritma viterbi dan 0,7166 untuk algoritma baum-welch. kesimpulan struktur model berdasarkan sistem hmm sahih untuk memprediksi ekson gen dna plamodium falciparum kata kunci : prediksi ekson, gen-dna, coding sequence, model hidden markov * jurusan teknik elektro fakultas teknologi industri universitas trisakti ** jurusan teknik elektro, fakultas teknik universitas indonesia *** bagian parasitologi fakultas kedokteran universitas trisakti korespondensi air. suhartati agoes, mt jurusan teknik elektro fakultas teknologi industri universitas trisakti jl. kyai tapa no.1, grogol jakarta 11440 telp. 08164836613 email: suhartati_agoes@yahoo.com universa medicina 2007; 26: 129-36. abstrak introduction i n c o d i n g s e q u e n c e ( c d s ) , t h e e x o n prediction on dna-gene is very important to find the protein structure. genome p. falciparum belongs to genome eukaryotic and has a long dna genome, consisting of several exons and introns alternately located. after the splicing process in dna sequence, some regions of exon in cds will be found to produce the protein.(1) 131 t h e h i d d e n m a r k o v m o d e l ( h m m ) structure is one of the model used to predict the exon in desoxyribonucleai acid (dna) which is based on the exon region structure in cds. the hmm structure of the model: start codon (methyanine/atg), exon, intron and stop c o d o n ( ta a , o r t g a , o r ta g ) h a s b e e n d e m o n s t r a t e d b y r a b i n e r, h e n d e r s o n a n d krogh.(2-4) this research developed a new model of structures for exon prediction on dna of genes plasmodium falciparum was based on the work of nicorici and anantharaman.(5,6) the significant difference of this model is it at least has two exon regions used for exon prediction like in cds. usually the 5’ boundary of introns in most eukaryotes contains the dinucleotide guanine-thymine (gt), and the 3’ boundary contains the dinucleotide adenine-guanine (ag). furthermore, in intron regions of the dinucleotide gt were separated to be nucleotide guanine (g) and thymine (t), and dinucleotide ag to be nucleotide a and g. method research design the hmm framework in this experiment used viterbi algorithm for the training process and both viterbi and baum-welch algorithms for the testing process. the same sequence has been done for the training and testing process. the program is written in matlab 7.0, and bioi n f o r m a t i c s ’ t o o l b o x t o g e n e r a t e d n a sequences in genbank format and has the functions of hmm training and testing. the first experiment for the model based on cds has been done with separated start codon (codon: atg) from first exon and stop codon (codon: taa, or tag, or tga) from the last exon, its model has the state number of 5 (figure 1). furthermore, the second experiment u s e d s e p a r a t e d d i n u c l e o t i d e g t a n d dinucleotide ag from the intron region, its model has the state number of 7 (figure 2). the last experiment in the model also used separated d i n u c l e o t i d e g t i n t o g a n d t s t a t e s a n d dinucleotide ag into a and g states; the state number of the model is 9 (figure 3). the emission values of the models can be performed a s t h e m a t r i x , t h e c o l u m n s a r e t h e b a s e s numbered on dna sequences and the rows are the states number of the models designed. the state transition values of the models can be shown also in the figures. samples a genome sequence of p. falciparum using 3d7 clone has 23-megabase nuclear genomes consisting of 14 chromosomes, encodes about 5,300 genes, and it has the most (a+t)-rich genome sequenced to date.(8-10) the data set of experiments for this simulation has 152 dna sequences of genes from genome plasmodium f a l c i p a r u m a t : c h r o m o s o m e 1 ( l o c u s : n c _ 0 0 4 3 2 5 ) , c h r o m o s o m e 2 ( l o c u s : n c _ 0 0 0 9 1 0 ) , c h r o m o s o m e 3 ( l o c u s : n c _ 0 0 0 5 2 1 ) , c h r o m o s o m e 4 ( l o c u s : n c _ 0 0 4 3 1 8 ) , c h r o m o s o m e 5 ( l o c u s : n c _ 0 0 4 3 2 6 ) , a n d c h r o m o s o m e 9 ( l o c u s : nc_004330) in genbank format.(11,12) genbank format describes the cds and original dna sequences of genes p. falciparum. in cds of genes contains at least two exon regions and maximum 10 exon regions. the minimum length sequence of genes is 684 base pairs (bp) and maximum length is 10095 bp. hmm provides a good probability method for discrete sequences model of data like dna sequences (alphabet of four letters: a, c, g and t). prediction of exon in dna of genes p. falciparum is based on exon region in cds and it has at least two exon regions. the structure of the model based on cds structure as shown in figure 4, which separated is start codon from the first exon and separated stop codon from the last exon. universa medicina vol. 26 no.3 132 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm in the inputs are dna sequences and set the state number of the sequences depends on the structure model. based on hmm method, the viterbi algorithm used for the training and needs the transition state values and emission values for the process. the value of transition state is random and the value of emission can be found with the distribution number of dna nucleotide in each states. the result of hmm training is the estimated transition state and emission state values. the estimated transition and emission values were used for hmm testing p r o c e s s w i t h vi t e r b i a n d b a u m we l c h algorithms. the results of hmm testing process are the estimated states of the model.(2) the parameter of performance indicator used the figure 3. the hmm structure with 9 states exon intron exonstart stop figure 4. the hmm structure based on cds 9 start e g t i a g 1 2 3 4 5 6 7 e stop 8 1 1 0.9 0.1 0.9 0.9 0.1 0.1 0.9 0.9 0.1 0.1 0.9 0.1 0.8 0.1 0.1 5 0 .9 1 e is ta rt s to p 2 3 4 1 0 .5 0 .5 0 .1 0 .8 1 0 .1 9 1 1 0 -6e figure 1. the hmm structure with 5 states s tart e g t i a g e stop 1 2 3 4 5 6 7 1 10.85 0.1 0.05 0.7 0.3 0.9 0.1 0.7 0.9 0.1 0.3 figure 2. the hmm structure with 7 states 133 correlation coefficient (cc), as a ratio of the estimated states from the output and the original states from the input of the model(1,7) and the formula is the following: where: tp= true positive; fp= false positive; tn= true negative; fn= false negative statistical analysis the emission matrix of the model with state number 5 is: e = [ 0.3333 0 0.3333 0.3333 ; 0.4342 0.1094 0.1507 0.3056 ; 0.4210 0.0599 0.0703 0.4487 ; 0.4215 0.1173 0.1622 0.2990 ; 0.5592 0 0.1075 0.3333 ] the emission matrix of the model with state number 7 is: e = [ 0.3333 0 0.3333 0.3333 ; 0.4342 0.1094 0.1507 0.3056 ; 0 0 0.5000 0.5000 ; 0.4210 0.0599 0.0703 0.4487 ; 0.5000 0 0.5000 0 ; 0.4215 0.1173 0.1622 0.2990 ; 0.5592 0 0.1075 0.3333 ] the emission matrix of the model with state number 9 is: e = [ 0.3333 0 0.3333 0.3333 ; 0.4342 0.1094 0.1507 0.3056 ; 0 0 1.0000 0 ; 0 0 0 1.0000 ; 0.4210 0.0599 0.0703 0.4487 ; 1.0000 0 0 0 ; 0 0 1.0000 0 ; 0.4215 0.1173 0.1622 0.2990 ; 0.5592 0 0.1075 0.3333 ] fn)fp).(tnfp).(tpfn).(tn(tp (fp.fn)-(tp.tn) cc ++++ = the transition state values are randomly, but the first state on the model has the minimum transition state value of 0 and the last state has the maximum transition state value of 1. on the other hand, the emission state values were found from the distribution number of dna nucleotide i n e a c h s t a t e d e p e n d i n g o n t h e m o d e l s . calculation of the cc is by using the above equation with the assumptions that the exon is positive and intron is negative. results based on exon regions structure in cds, the simulation results for the model with state number 5 above and transition state values randomly has found the average of cc value in table 1. table 2 and table 3 shows the average of cc values of the model with state number 7 and state number 9. the transition state value in the first state of all structures on the models is 0 and the last state is 1. the other transition state values except in table 2 are 0.1 and the h i g h e s t a v e r a g e o f c c v a l u e s f o r t h i s experiments are shown in figure 5. the model based on exon regions structure in cds for state number nine resulted in the highest average of cc. its values are 0.7289 by using viterbi algorithm and 0.7166 by using baum-welch algorithm. however, the lowest average of cc values has been found for the model with state number five. its values are 0.6735 by using viterbi algorithm and 0.6661 by using baum-welch algorithm. discussion all these were based on the model of exon regions structure in cds, at the state number nine resulted in the highest average of cc values. it is also shown that the emission state value of t h e m o d e l s h a v e t h e ( a + t ) r i c h g e n o m e sequences of genes plasmodium falciparum. universa medicina vol. 26 no.3 134 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm transition state values corr.coef.(cc) exps tr11 tr22 tr33 tr44 tr55 viterbi b-welch 1 0 0.5 0.9 0.80 1 0.7054 0.6750 2 0 0.5 0.9 0.81 1 0.7061 0.6759 3 0 0.5 0.9 0.82 1 0.7054 0.6750 4 0 0.5 0.9 0.83 1 0.7054 0.6750 5 0 0.5 0.9 0.84 1 0.7045 0.6741 6 0 0.5 0.9 0.85 1 0.7054 0.6750 7 0 0.5 0.9 0.86 1 0.7062 0.6744 8 0 0.5 0.9 0.87 1 0.0012 0.0015 9 0 0.5 0.9 0.88 1 0.0011 0.0013 10 0 0.5 0.9 0.89 1 0.0011 0.0013 table 1. the average of cc values for the model with state number 5 transition state values corr.coef.(cc) exps tr11 tr22 tr33 tr44 tr55 tr66 tr77 viterbi b-welch 1 0 0.4 0.2 0.8 0.10 0.7 1 0.6713 0.6643 2 0 0.4 0.2 0.8 0.20 0.7 1 0.6713 0.6643 3 0 0.4 0.2 0.8 0.30 0.7 1 0.6733 0.6660 4 0 0.4 0.2 0.8 0.40 0.7 1 0.6731 0.6660 5 0 0.4 0.2 0.8 0.50 0.7 1 0.6735 0.6661 6 0 0.4 0.2 0.8 0.60 0.7 1 -0.0642 -0.0729 7 0 0.4 0.2 0.8 0.59 0.7 1 -0.0568 -0.0639 8 0 0.4 0.2 0.8 0.58 0.7 1 0.6735 0.6661 9 0 0.4 0.2 0.8 0.56 0.7 1 0.6735 0.6661 10 0 0.4 0.2 0.8 0.55 0.7 1 0.6735 0.6661 table 2. the average of cc values for the model with state number 7 transition state values corr.coef.(cc) exps tr11 tr22 tr33 tr44 tr55 tr66 tr77 tr88 tr99 viterbi b-welch 1 0 0.3 0.2 0.2 0.80 0.10 0.10 0.70 1 0.7242 0.7124 2 0 0.3 0.1 0.1 0.79 0.10 0.10 0.70 1 0.7242 0.7124 3 0 0.3 0.1 0.1 0.78 0.09 0.09 0.70 1 0.7242 0.7124 4 0 0.3 0.1 0.1 0.78 0.20 0.20 0.70 1 0.7258 0.7120 5 0 0.3 0.1 0.1 0.80 0.20 0.20 0.80 1 0.7258 0.7120 6 0 0.3 0.1 0.1 0.80 0.15 0.15 0.74 1 0.7258 0.7120 7 0 0.3 0.1 0.1 0.80 0.15 0.15 0.80 1 0.7258 0.7120 8 0 0.3 0.1 0.1 0.80 0.15 0.15 0.70 1 0.7258 0.7120 9 0 0.8 0.2 0.2 0.80 0.10 0.10 0.70 1 0.6787 0.6755 10 0 0.9 0.2 0.2 0.80 0.10 0.10 0.70 1 0.0451 0.0437 table 3. the average of cc values for the model with state number 9 135 this can also be showed in their matrix values described above. in order to match with markov’s chain in eukaryotic gene structure for c o n n e c t i n g t h e e x o n a n d i n t r o n , t h e n t h e backward-forward method are used in this hmm system. the experiments were done by viterbi algorithm for the training process and the both viterbi and baum-welch algorithms for the testing process. a set of 152 sequences is then used as data to train the model and a different hmm is produced for each set of sequences. hmm have been previously used in sequence analysis to produce an hmm that represents a sequence profile (a profile hmm), to analyze sequence composition and patterns, to locate genes by p r e d i c t i n g o p e n r e a d i n g f r a m e o r c o d i n g sequence, and to produce protein structure predictions. this hmm generates sequences 0.62 0.64 0.66 0.68 0.7 0.72 0.74 c orrelation c oefficient (c c ) t he highest average of cc values viterbi 0.7061 0.6735 0.7258 ba um-we lch 0.6759 0.6661 0.712 5 7 9 w i t h v a r i o u s c o m b i n a t i o n s o f m a t c h e d , mismatched, insertions and deletions, and gives these a probability, depending on the values of the various parameters in the model. a total of 424 predicted protein-coding g e n e s a n d 3 t r n a g e n e s o f p l a s m o d i u m falciparum have been identified on chromosomes 2 and 3, the two chromosomes for which s e q u e n c i n g h a v e b e e n c o m p l e t e d . ( 1 3 , 1 4 ) approximately 37% (158 genes) of three genes have a readily identifiable homologue in another species. such similarity allows a function to be implied, with many of those identified being involved in parasite metabolism. interestingly, a comparison of the predicted protein sequences with their homologues in other species showed that the majority of plasmodium falciparum proteins have insertion of low complexity sequences, often runs of a single amino acid figure 5. the highest average of cc values of the experiments universa medicina vol. 26 no.3 136 agoes, gunawan, sardy, et al exon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmmexon prediction by using hmm r e s i d u e ( t y p i c a l l y a s p a r a g i n e s , l y s i n e o r glutamine acid), or tandem arrays of a short peptide repeat sequence. examples of such regions have been identified previously, and are polymorphic between different parasite isolates. perhaps it will open up new avenues for drug and vaccine development. perhaps it will make a genetically modified mosquito that cannot act as vector a reality once processes are in place to deal with ethical, legal and social issues. but any new developments are unlikely to happen before the 2010 deadline set for roll back malaria. new drugs based on knowledge about the genome will probably not be affordable to those countries that need it the most.(15) conclusion this research with a new structure model was using for exon prediction on dna-genes of plasmodium falciparum based on cds structure by using the hmm system. acknowledgment t h e a u t h o r s w o u l d l i k e t o t h a n k d r. herawati sudoyo, phd, dr. helena ms and hidayat trimarsanto b.sc from the eijkman institute, for their valuable suggestions. references 1. samatova n.f. computational gene finding using hmms. ut-battelle information center, 1201 oak ridge turnpike, suite 100, oak ridge, tn 37830. institute oak ridge national laboratory; 2003. 2. lawrence r. a tutorial on hidden markov models and selected applications in speech recognition. proceeding of the ieee. 1989; 77: 257-68. 3. henderson j, salzberg s, fasman k.h. finding genes in dna with a hidden markov model. j comput biol 1997; 4: 127-42. 4. krogh a. an introduction to hidden markov models for biological sequences. in computational methods in molecular biology. salzberg sl, searls db, kasif s. editors. denmark. center for biological sequence analysis, technical university of denmark. 1998. p. 45-63. 5. nicorici d, astola j, tobus i. computational identification of exons in dna with a hidden markov model. tampere international center for signal processing, tampere university of technology, finland. available at: http:// www.gensips.gatech.edu/processings/contributed/ cp2-06.pdf. accessed may 12, 2005. 6. anantharaman t. finding genes in genomic dna the genescan system. available at : http:// www.biostat.wisc.edu/bmi776. accessed june 6, 2005. 7. vaisman i. bioinformatics and gene discovery. bioinformatics tutorial. north carolina, united state: university of north carolina at chapel hill. 1998. 8. hall n, gardner m.j, hyman rw, lasonder e, wilson rjm, scherf a, et al. sequence of plasmodium falciparum chromosomes 1, 3-9 and 13. nature 2002; 419: 527-31. 9. gardner m.j, hall n, hyman rw, hinterberg k, mattei d, wellem te, et al. sequence of plasmodium falciparum chromosomes 2, 10, 11 and 14. nature 2002; 419: 531-4. 10. hyman rw, gardner m.j, hall n, de bruin d, scherf a, day kp, et al. sequence of plasmodium falciparum chromosome 12. nature 2002; 419: 534-7. 11. anastassiou d. genomic signal processing. ieee signal processing magazine 2001; 18: 4. 12. alphey l. dna sequencing. manchester uk : bios scientific publishers limited; 1997. 13. gardner m.j, hall n, fung e, white o, berriman m, hyman rw, et al. chromosome 2 sequence of the human malaria parasite plasmodium falciparum. science 1998; 282: 1126-32. 14. bowman s, lawson d, basham d, brown d, chillingworth t, churcher c. m, et al. the complete nucleotide sequence of chromosome 3 of plasmodium falciparum. nature 1999; 400: 5328. 15. anonymous. malaria after the genomes. the lancet 2002; 360: 1107. smoking tends to decrease glutathione and increase malondialdehyde levels in medical students 89 *department of biochemistry and medicinal chemistry, faculty of medicine, sriwijaya university correspondence : subandrate department of biochemistry and medicinal chemistry, faculty of medicine, sriwijaya university jalan dr. m. ali komplek rsmh km 3,5 palembang 30126 phone and fax: +62711-373438 email: saratebasyam@yahoo.com univ med 2016;35:89-95 doi: 10.18051/univmed.2016.v35.89-95 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina may-august, 2016may-august, 2016may-august, 2016may-august, 2016may-august, 2016 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 vol.35 no.2 smoking tends to decrease glutathione and increase malondialdehyde levels in medical students safyudin* and subandrate* background smoking is the act of introducing toxic substances into the body. cigarette smoke contains chemicals that may cause several disorders, including cardiovascular disease, cancer, and chronic obstructive pulmonary disease. toxic substances in cigarette smoke have the potential to increase free radicals, malondialdehyde (mda) levels and to decrease endogenous antioxidant (glutathione/gsh) levels. this study aims to determine the relationship of smoking with plasma gsh and mda levels in medical students. methods this study was analytical observational with cross-sectional approach. the study was conducted from april to december 2015. the subjects in this study were medical students, consisting of 20 smokers and 20 nonsmokers. plasma gsh and mda levels were determined biochemically using sigma gsh assay kit and sigma mda assay kit. data was analyzed using the independent t test. results the results showed that there was no significant difference between mean gsh in smokers (1.74 ± 0.91 µmol/l) and nonsmokers (2.42 ± 1.19 µmol/l) (p=0.441). mean smokers mda level of 2.06 ± 1.39 nmol/µl was not significantly different compared with mean nonsmokers mda level (1.32 ± 0.90 nmol/µl) (p=0.092). conclusions smoking tends to decrease plasma gsh levels and increase plasma mda levels in medical students. smoking history could be evidence of oxidative stress and an impaired oxidant defense system. in particular, young smokers should quit promptly before health problems arise, so as to have the optimal benefits of cessation. keyword: free radicals, gsh, mda, smoking, medical students doi: http://dx.doi.org/10.18051/univmed.2016.v35.89-95 90 safyudin, subandrate smoking glutathione and malondialdehyde introduction smoking is the act of introducing toxic substances into the body. cigarette smoke inhaled by a smoker contains hazardous compounds that are detrimental to health. smoking is at present still a threat to global public health, particularly in developing countries such as indonesia. cigarette consumption in indonesia is steadily on the increase.(1) in 2007, the number of smokers in indonesia was 34.2% and in 2013 it became 36.2%.(1) the who even predicts that the number of smokers in indonesia will increase up to 45% in the year 2025, if the indonesian government does not issue a strict cigarette policy.(2) which is also astounding is that indonesia ranks fifth in cigarette consumption in the world after china, the us, russia and japan.(2) the results of a study by the ministry of health of the republic of indonesia for the year 2011 showed that around 31% of smokers in indonesia are non-university students and almost 75% of private university students in sumatra are smokers.(1,2) in the us, the age range of 18-24 years has the highest prevalence of smokers.(1) it is known that cigarette smoke contains toxic compounds such as tar, nicotine, lead, and carbon monoxide that can adversely affect health.(3) the habit of smoking increases blood lead concentrations.(4) in addition, smoking in the long run can cause disturbed lung function and can increase chronic obstructive pulmonary disease and skeletal muscle dysfunction.(5,6) the results of a study on experimental animals s h o w e d t h a t c i g a r e t t e s m o k e i n c r e a s e d pulmonary alveolar cell death.(7) the gaseous and particulate components of cigarette smoke are potential inducers of free radicals.(3,8) the results of a study showed the presence of free radicals in periodontal tissues of smokers.(8) the results of another study in rats showed that exposure to cigarettes, whether electronic or conventional, can disturb rat spermatogenesis through an increase in free radicals.(9) free radicals are extremely reactive compounds and thus easily react with other compounds such as lipids, dna and proteins. free radical oxidation causes tissue or organ damage so as to cause illness.(10,11) cigarettes are suspected of being one of the sources of increases in free radicals in the body and are associated with various chronic pulmonary and cardiovascular diseases. the increase in free radicals in the body causes oxidative stress.(3,8,9) oxidative stress is an increase in oxidants (free radicals) and/or decrease in antioxidants in the body. one of the markers that is frequently used to evaluate oxidative stress is the malondialdehyde (mda) concentration. mda is the end product of lipid peroxidation by free radicals.(10,11) an increase in mda concentration indicates an increase in free radicals in the body. apart from mda, another marker that is frequently used to evaluate oxidative stress is glutathione (gsh). gsh is one of the endogenous antioxidants that play a role in cleaving free radicals. in conditions of oxidative stress, the gsh concentration decreases.(3,9) in addition to mda and gsh, other markers that can be used to evaluate oxidative stress are superoxide dismutase (sod), glutathione peroxidase (gpx), catalase and carbonyl compounds.(9,10) several studies have shown that smoking is associated with increased aging, cell damage, and disease through the mechanism of oxidative stress. in smokers, there is a tendency for an increase in free radicals and a decrease in antioxidants to occur.(7,10,11) however, these studies used a considerably broad age range, from young to old. the study by omotoso et al.(12) in rats showed that exposure to cigarette smoke decreased plasma malondialdehyde concentrations. similarly, the study of koubaa et al.(13) reported that mda concentrations were lower in smokers than in nonsmokers. in contrast, a study on thirty healthy adult subjects (15 smokers and 15 nonsmokers) showed no significant changes in gsh and mda concentrations of smokers.(14) in view of the large numbers of smokers in indonesia particularly among university students and the differing results of previous studies, it 91 was felt necessary to conduct another study on the association of smoking with mda and gsh concentrations in smokers. methods research design this was an analytical observational study w i t h c r o s s s e c t i o n a l a p p r o a c h t h a t w a s conducted from april until december 2015 in the medical study program of the faculty of medicine, sriwijaya university, palembang, south sumatra. study subjects the study sample comprised all smoking students aged 18-22 years in the medical study program of the faculty of medicine, sriwijaya university, who met the inclusion criterion and did not meet the exclusion criteria. the inclusion criterion was healthy males, while the exclusion criteria were students who were ill or had a history of a metabolic disorder, such as diabetes mellitus and cardiac and renal disorders. the subjects to be included in the sample were recruited by identifying all smokers and selecting the subjects according to the inclusion criterion. the sample size was determined using slovin’s formula,(15) namely n = n / (1 + n × e2), where n is the population size, and e is the margin of error. applying a 5% error margin in slovin’s formula, the recommended minimum sample size was 40. the sample size that was obtained in this study was 20 smoking students and 20 nonsmoking students. the study subjects were selected using simple random sampling by lot after those involved agreed to participate in the study and had been given an explanation of the benefits and discomforts of the participation in this study. their willingness to participate in the study was recorded by signing informed consent. data collection c o l l e c t i o n o f t h e s a m p l e d a t a w a s performed by interviews using a questionnaire, which included the subjects’ identity (using initials only), age, gender, duration of smoking, and number of cigarettes per day. laboratory analysis a blood sample of 2 ml was drawn at 07.30 am west indonesian time from the left median cubital vein of each (nonfasting) study subject using a 3-ml syringe. the blood samples were then centrifuged at 5000 rpm for 2 minutes to obtain plasma, which was then stored at -20°c, before being used for measurement of gsh and mda concentrations. the plasma gsh and mda concentrations were determined in the biochemistry laboratory and the molecular biology laboratory, faculty of medicine, sriwijaya university. the plasma gsh and mda concentrations were measured biochemically using the sigma gsh and mda assay kits, respectively. d e t e r m i n a t i o n o f p l a s m a g s h concentrations was done biochemically using the sigma gsh assay kit. a volume of 200 µl plasma was deproteinized with 200 µl of 5% 5sulfosalicylic acid (ssa). the mixture was then vortexed and left standing for 10 minutes at 2– 8°c. subsequently the mixture was centrifuged at 10,000 g for 10 minutes to remove the p r e c i p i t a t e d p r o t e i n . t h e n 1 0 µl o f t h e supernatant was taken and mixed with 150 µl working mixture in each well of a 96-well plate. the mixture was incubated for 5 minutes at room temperature, then 50 µl of nadph solution was added and the absorbance was measured using a spectrophotometer at a wavelength of 412 nm. the gsh concentration was then determined on the gsh standard curve in units of µmol/l. determination of plasma mda concentrations was done biochemically using the sigma mda assay kit. a volume of 10 µl plasma was mixed with 500 µl of 42 mm h 2 so 4 and 125 µl phosphotungstic acid in a microcentrifuge tube. the mixture was vortexed and incubated at room temperature for 5 minutes, then centrifuged at 3,000 g for 3 minutes. the precipitate was univ med vol. 35 no.2 92 safyudin, subandrate smoking glutathione and malondialdehyde collected and resuspended in a 3,5-di-tert-4butylhydroxytoluene (bht)-distilled water solution and adjusted to a volume of 200 µl with water. afterwards, 600 µl of thiobarbituric acid (tba) solution was added to the suspension and incubated at 95°c for 60 minutes. the mixture was then cooled in an ice bath for 10 minutes. the 200 µl mixture was placed into a 96-well plate and the absorbance read using a spectrophotometer at 532 nm wave length. mda concentrations were then determined using the mda standard curve in units of nmol/µl. statistical analysis the data were processed and analyzed using spss version 16. the statistical analysis used w a s t h e i n d e p e n d e n t t t e s t a t a l e v e l o f significance of 0.05. ethical clearance the study was conducted after obtaining ethical clearance from the unit for bioethics and humaniora, mohammad hoesin central general hospital and the faculty of medicine, sriwijaya u n i v e r s i t y, p a l e m b a n g , u n d e r n o . 1 5 2 / kepkrsmhfkunsri/2015. results smoking study subjects had a mean age of 21.0 ± 1.07 years, mean duration of smoking of 5.0 ± 2.0 years, and mean number of cigarettes smoked per day of 9.0 ± 4.36 cigarettes. the subject characteristics can be seen in table 1. o n d e t e r m i n a t i o n o f p l a s m a g s h concentration it was found that mean gsh concentrations of the smoking and nonsmoking study subjects were 1.74 ± 0.91 µmol/l and 2.42 ± 1.19 µmol/l, respectively (table 2). thus plasma gsh concentration in smokers was lower than in nonsmokers, but the difference was statistically not significant (p=0.441). this means that smoking did not significantly decrease gsh concentration. o n d e t e r m i n a t i o n o f p l a s m a m d a concentration it was found that mean mda concentration of the smoking study subjects was 2.06 ± 1.39 nmol/µl and that of the nonsmoking study subjects 1.3 ± 0.90 nmol/µl. therefore, the plasma mda concentration in smokers was higher than in nonsmokers, but the difference was statistically not significant (p=0.092) (table 2). this indicates that smoking did not significantly increase plasma mda concentration. the correlation of duration of smoking and n u m b e r o f c i g a r e t t e s p e r d a y w i t h g s h concentration resulted in r=0.108 (p=0.650) and r=0.316 (p=0.175), respectively, which were statistically not significant. the correlation of duration of smoking and number of cigarettes per day with mda concentrations also resulted in r=-0.132 (p=0.579) and r=-0.117 (p=0.624), which again were statistically not significant (table 3). table 1. distribution of characteristics of smokers and nonsmokers table 2. differences in gsh and mda concentrations between smokers and nonsmokers gsh= glutathione; mda= malondialdehyde; *independent t-test; significant at p<0.05 93 discussion the smokers in this study were 18-22 years of age, with mean duration of smoking of 5 years. with a view to the mean duration of smoking, it may be estimated that the age of the study subjects when they started smoking was 13-17 years, i.e. when they were adolescents (in junior or senior high school). at such an age, social influence, selfactualization, labile personality, and high level of curiosity, are the causes that these adolescents start smoking. the results of this study was comparable to those of the studies conducted by reimondos et al.(16) and subandrate et al.(17) who found that the age of smoking initiation was around 13-15 years. the smokers in our study were habitual or routine smokers, with mean daily consumption of around 9 cigarettes. this is in agreement with previous studies reporting that on average the smokers consumed 10-11 cigarettes or one pack per day.(1,16) smoking is the act of introducing toxic substances into the body. when smoking, more than 2000 compounds enter the lungs in gaseous or particulate form. these toxic substances in the cigarette smoke, such as tar, nicotine, lead, carbon monoxide, nitrogen monoxide, benzopyrene, and nitrosamines, will subsequently be metabolized in the liver to yield oxidants or free radicals.(14,18) long-term and continual smoking leads to accumulation of these toxic substances in the body, so that there is an increase in free radicals, such as hydroxyl and superoxide radicals. the increase in free radicals causes an increase in oxidant markers such as mda and a decrease in antioxidant markers such as gsh, glutathione peroxidase, catalase, and superoxide dismutase.(10,19) in the present study, mean gsh concentration in smokers was lower than that in nonsmokers, but the difference was statistically not significant. corresponding results were found in the study conducted by abdul-rasheed and al-rubayee, showing nonsignificant decreases in glutathione concentrations in smokers as compared with those of nonsmokers.(20) however, different results were f o u n d i n a n o t h e r s t u d y, w h e r e g s h concentrations were lower in smokers than in nonsmokers.(21) this may have been caused by an increase in free radicals such as hydroxyl radicals, hydrogen peroxide, and superoxide anions, resulting from the accumulation of cigarette smoke oxidants.(10,11,18) the increase i n f r e e r a d i c a l s t r i g g e r s t h e a c t i v i t y o f endogenous antioxidants such as gsh, so as to reduce their plasma concentrations. gsh is a tripeptide with sulfhydryl groups, thus being capable of cleaving free radicals. gsh can quench free radicals directly or cleave hydroxyl radicals and superoxide anions by acting as cofactor of glutathione peroxidase, glutathione transhydrogenase and superoxide dismutase.(10,11) the results of our study also showed that mean mda concentration in smokers was higher than in nonsmokers, but the difference was statistically not significant. similar nonsignificant results were shown in the study conducted by erguder et al.(22) who found that serum mda concentration was not significantly different between smokers and nonsmokers. in contrast, different results were shown in a study involving 22-25 year-old students in india, in that there was a significant increase in serum mda concentration in smokers as compared with that of nonsmokers.(21) this may have been possible because longterm and frequent table 3. correlation of duration of smoking and number of cigarettes per day with gsh and mda concentrations gsh= glutathione; mda= malondialdehyde; r= pearson correlation coefficient; significant at p<0.05 univ med vol. 35 no.2 94 safyudin, subandrate smoking glutathione and malondialdehyde cigarette exposure leads to accumulation of toxic substances and superoxide radicals, so as to induce oxidative stress. mda is the end result of a chain of lipid peroxidation reactions by free radicals. increases in mda concentrations indicate that the cells undergo oxidative stress.(10,11) a limitation of this study was that it did not perform an analysis on or exclusion of other factors that affect plasma antioxidant and oxidant concentration, such consumption of certain drugs, antioxidant supplements, a diet high in fruits and vegetables, and physical activity. the cross-sectional design that was used in this study does not allow it to explain any cause-and-effect relationship of smoking with gsh and mda concentrations. therefore, further studies need to be conducted of cohort or case-control design that can include an analysis of all these factors. the body has the capacity to overcome free radicals, originating from within or from outside the body. in this study exposure to exogenous oxidants in the form of cigarette smoke did not have a significant impact on the increase in free r a d i c a l s a n d r e d u c t i o n i n e n d o g e n o u s antioxidants. this signifies that in healthy young adults, the impact of exogenous oxidant exposure can still be overcome. conclusions smoking tends to decrease plasma gsh concentration and increase plasma mda concentration in medical students. smokers are m o r e s u s c e p t i b l e t o o x i d a n t s t r e s s a s a consequence of insufficient antioxidant potential and greater oxidative burden. in particular, young smokers should quit promptly before health problems arise, so as to have the optimal benefits of cessation. conflict of interest there was no conflict of interest in this study. acknowledgement thanks are due to ahmad wirawan and the chairman of the class of 2012-2015 of the medical study program of the faculty of medicine, sriwijaya university, who assisted with sample recruitment. references 1. world health organization. who global report on trends in prevalence of tobacco smoking, 2015. geneva: world health organisation; 2015. 2. departemen kesehatan ri. riset kesehatan dasar tahun 2013. jakarta: badan penelitian dan pengembangan departemen kesehatan republik indonesia, 2013. 3. tirtosastro s, murdiyati as. kandungan kimia tembakau dan rokok. buletin tanaman tembakau 2010;2:33-43. 4. nurbaya f, wijayanti y. faktor risiko yang berhubungan dengan kadar timah hitam dalam darah. kemas 2010:6:51-6. 5. nurjanah, kresnawati l, mufid a. gangguan fungsi paru dan kadar nikotin pada urin karyawan yang terpapar asap rokok orang lain. kemas 2014;10:43-52. 6. degens h, gayan-ramirez g, van hees hwh. smoking-induced skeletal muscle dysfunction from evidence to mechanisms. am j respir crit care med 2015;191:620–5. 7. atik n, avriyanti e, iwan arj, et al. pengaruh lidah buaya (aloe vera l.) pada paru-paru tikus yang diinduksi asap rokok. majalah kedokteran bandung 2012;44:159–64. 8. reejamol mk, swaminathan m. estimation of lipid peroxides and antioxidants in smokers and non-smokers with periodontitis. king saudi univ j dent sci 2013;4:53–6. 9. hybertson bm, gao b, bose sk, et al. oxidative stress in health and disease: the therapeutic potential of nrf2 activation. mol aspects med 2011;32:234–46. 10. pasupathi p, saravanan g, farook j. oxidative stress bio markers and antioxidant status in cigarette smokers compared to nonsmokers. j pharm sci res 2009;1:55-62. 11. donohue jf. ageing, smoking and oxidative stress. thorax 2006;61:461–2. 12. omotoso go, kadir re, alabii jt, et al. exposure to cigarette smoke altered the cytoarchitecture and anti-oxidant activity of the frontal cortex in wistar rats. int j biol chem sci 2013;7:1595-60. 95 13. koubaa a, triki m, trabelsi h, et al. the effect of a 12-week moderate intensity interval training program on the antioxidant defense capability and lipid profile in men smoking cigarettes or hookah: a cohort study. sci world j 2015; 2015: 1-9. 14. chávez j, cano c, souki a, et al. effect of cigarette smoking on the oxidation/antioxidation balance in healthy subjects. rev latinoam hipertens 2006;1:33-7. 15. tejada jj, punzalan jrb. on the misuse of slovin’s formula. philippine statistician 2012; 61:129-36. 16. subandrate, safyudin, arifin m, et al. kadar superoksida dismutase mahasiswa perokok di program studi pendidikan dokter universitas sriwijaya. jurnal kedokteran yarsi 2015;23:7682. 17. reimondos a, utomo id, mcdonald p, et al. smoking and young adults in indonesia. australian demographic and social research institute, the australian national university; 2012. 18. barreiro e, peinado vi, galdiz jb, et al. cigarette smoke–induced oxidative stress. am j resp crit care med 2010;182:477–88. 19. rajalakshmi g, sudha g. reduced plasma antioxidant level in smoking asthmatic. int res j pharm 2013;3:142-4. 20. abdul-rashees of, al-rubayee wt. effects of cigarette smoking on lipid peroxidation and antioxidant status in iraqi men at baghdad city. int j basic appl sci 2013;2:47-50. 21. mahapatra sk, das s, dey sk, et al. smoking induced oxidative stress in serum and neutrophil of the university students. al ameen j med sci 2008;1:20-31. 22. erguder ib, ucar a, ariturk i, et al. the effects of cigarette smoking on serum oxidant status, and cholesterol, homocysteine, folic acid, copper, and zinc levels in university students. turk j med sci 2009;39:513-7. univ med vol. 35 no.2 c:\users\jurnal fkusakti\docume 115 *department of histology, faculty of medicine, islamic university of indonesia, yogyakarta correspondence: dwi nur ahsani,md,m.sc department of histology, faculty of medicine, islamic university of indonesia, yogyakarta jl. kaliurang km14,5 sleman yogyakarta. phone: (+62) 852 2903 0982. fax :(+62) 27 4898444 ext.2007. email: dwinurahsani@gmail.com or dwi.nurahsani@uii.ac.id date of first submission, december 15, 2017 date of final revised submission, june 22, 2018 date of acceptance, june 25, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina age-related changes of malondialdehyde, body weight and organ weight in male mice dwi nur ahsani* and ika fidianingsih* introduction aging is characterized by gradual impairment in all physiological functions. increases in free radicals and changes in organ morphology occur with aging. the purpose of this study was to determine age-related changes in serum free radicals, body weight, organ weights, and relative organ weights in male mice. methods an experimental animal study was performed on 25 male mice (mus musculus), which were randomized into 5 groups according to age at termination, i.e. 12 (group k1), 24 (k2), 32 (k3), 40 (k4) and 48 weeks (k5), respectively. retro-orbital venous blood was taken for examination of malondialdehyde (mda) levels. after termination, liver, heart, kidneys, testes, brain, thymus and spleen were weighed using an analytical balance. anova and kruskal wallis tests were used to analyze the data, with p<0.05 being considered significant. results significant changes were found with age in serum mda level (p=0.000), body weight (p=0.000), and weights of all organs except thymus (p>0.05) (liver p=0.023, heart p=0.000, kidneys p=0.002, testes p=0.000, brain p=0.012 and spleen p=0.006). significant changes in relative weight of brain (p=0.001) and spleen (p=0.049) were also found with age. conclusion this study demonstrated increases in serum mda levels, body weight, and weights of the liver, heart, kidneys, testes, brain and spleen with age. peak increases in weights of kidneys and thymus were found earlier than those in mda levels and weights of other organs. keywords : body weight, organ weight, malondialdehyde, age, male mice doi: http://dx.doi.org/10.18051/univmed.2018.v37.115-126 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cit e th is ar ticle as : ah sa n i dn, fidianingsih i. age-related changes of malondialdehyde, body weight and organ weight in male mice univ med 201 8;37 :1 1 5-2 6 . doi: 10.1 805 1/ univmed. 2018.v37.115-126 116 ahsani, fidianingsih age and malondialdehyde male mice introduction aging is a naturally occurring process in living organisms that is marked by increases in body functions up to a given age, when it is followed by decreases in body function, resulting in a variety of medical conditions that ultimately increa se an individual’s susceptibility to disease.(1) several studies showed that persons older than 63 years have at least one chronic disease. degenerative diseases commonly found in older persons are hypertension, visual disorders, anemia, diabetes mellitus, disorders of mobility, and diseases of the central nervous system such as dementia and stroke.(2-4) cellular aging is marked by increases in free radical concentrations in the body, causing damage to the structure of cellular lipids, proteins, and also dna. in physiological conditions, increases in free radicals are inhibited by antioxidant enzymes.(5) one study showed that a gi ng c a us e s d e c r e a s e d e x p re s si o n o f antioxidant proteins. western blot assays showed increased oxidative stress levels in the l i ve r. a gin g d e c r e a s e s t h e a c ti vi ti e s o f superoxide dismutase, catalase and glutathione and increased lipid peroxidation and 70 kilodalton heat shock protein (hsp70) levels in the brain and also in peripheral organs such as the liver, kidneys and heart.(6) one of the markers of oxidative stress is malondialdehyde (mda), which is a product of lipid peroxidation with mutagenic, cytotoxic, and carcinogenic properties, due to its inhibitory effect on antioxidant enzymes. therefore, increased mda concentration is frequently used as one of markers of disordered homeostasis and aging.(7) other studies showed that oxidative stress is associated with weight increase (obesity), which increases the risk for various degenerative disorders such as cardiovascular disease and the metabolic syndrome.(8,9) increased body weight also induces chronic inflammatory conditions wi t h a ge , u l t i ma t e ly c a u si ng or ga n degeneration.(10) morphological changes of an organ are important for identifying abnormalities of the organ and evaluating the phase of a disease in a patient. the weight of an organ is important to be recognized as clinical parameter in several disciplines, e.g. for determining the toxic effects of new drugs in pharmacology and the limits of r a d i a t i on e x p os ur e i n r ad i ol o gy. ( 11 ,1 2 ) morphological changes in an organ cause functional disorders in older persons. studies associated with age-related organ weight have been frequently conducted for various purposes. studies on the weight of internal or gans in humans are commonly performed by forensic clinicians in post-mortem situations to obtain the normal weights of organs i n r e l a t io n t o r a c e o r ge n de r. ( 1 3 -1 5 ) in experimental animals, studies on changes in organ mass are aimed at determining the agerelated developmental pattern of an organ and also the underlying mechanism of the aging process.(16-19) therefore, studies on experimental animals are far more feasible to be performed by investigators as compared with studies on humans. the patterns of changes in organ size differ between organs. a study on wistar rats showed that the volume of the kidneys increases up to t he a ge of 1 2 mo nt h s a nd de c r e a s e s afterwards.(18) the brain experiences a rapid increase in volume at the age of less than 20 weeks and thereafter the development becomes s ta t ic . ( 19 ) in hu ma ns , t h er e i s a n e gat i ve correlation between aging and the weight of the brain, kidneys, liver, and spleen.(14) on the other hand, a positive correlation is found between aging and morphological changes of the heart.(15) in hu ma n s, a gin g no t on l y ch a n ge s t h e morphology of the organs, but also increases body weight.(20) an in-depth study should be carried out in connection with the accumulation of free radicals and the morphological organ changes occurring with age. there is still controversy on the relationship of aging with increased free radical levels and functional organ changes. the study 117 by luceri et al.(21) showed that aging increases plasma reactive oxygen species (ros) and protein carbonyl concentrations, which are clearly seen in 15-month old rats. systemic increases in ros concentrations also cause damage to the liver, as shown in the increased dna oxidative markers in the liver (8-hydroxy22 -deoxyguanosine/8-ohdg) and decreased expression of the dna repair gene apyrimidinic endonuclease-1 (ape-1). on the other hand, the study by gomes et al.(22) showed no relationship b e t we e n i nc r e a s e d o xi d a t i ve s t r e ss a n d functional changes in the kidneys of aging rats. this was shown by the increases in free radicals in the kidneys (mda in urine and h 2 o 2 in the renal cortex and medulla) and in plasma mda in 52-week old rats, but without increases in creatinine and sodium concentrations. in view of the inconclusive study results, further studies are still needed. the present study aimed to determine the changes in plasma mda and free radical concentrations, as well as the age-related changes in body weight and organ weights in male mice. methods study design this study was an experimental animal study conducted from september 2016 until march 2017 at the physiology laboratory, faculty of medicine, universitas islam indonesia (fk uii). study subjects t he mi c e w e r e obt a i n e d f r o m t he physiology laboratory, fk uii. the mice received care at the fk uii research laboratory according to the principles of experimental animal welfare. all animals received feed and drinking water ad libitum and were kept in group cages, each containing one group of 5 mice. the experimental animals had adequate ventilation and were kept on a 12-hour light-dark cycle. all mice were offspring of one dam. a total of 10 male mice (mus musculus) aged 8 weeks and 15 male mice aged 16 weeks were entered into this study. there were 5 groups in this study, i.e. the groups of mice terminated at ages of 12 (k1), 24 (k2), 32 (k3), 40 (k4) and 48 weeks (k5), respectively. groups k1 and k2 comprised 8-week old mice that were kept until the previously determined ages of termination (12 and 24 weeks, respectively), whereas groups k3, k4 and k5 comprised 16-week old mice that were kept until the previously determined ages o f t e r mi na t i o n ( 32 , 4 0 a nd 48 we e ks , respectively). sample size calculation the number of experimental animals used in this study was determined by means of the formula of charan and khantaria (23) i.e. e= total number of animals – total number of groups. e is a cons tant within the r ange of 10-20. according to the calculation, the total number of animals that should be used in this study is 15-25 rats, with each group consisting of 3-5 mice. in this study it was decided to use 5 animals in each group, so that the total number of experimental animals used in this study was 25. there was no special treatment for the experimental animals. the study subjects (mice) were kept until the previously determined ages (12, 24, 32, 40, and 48 weeks). as part of their care, the mice received feed and drinking water ad libitum, were kept in group cages and were subjected to light-dark cycles. the mice used in this study were healthy purebred males without disabilities and had never been used for research. mice with abnormal behavior (those that were apparently weak, inactive, ill, or aggressive) or those that died in the course of the study, were not included. measurements and laboratory analysis on the day of termination determined previously, the mice were weighed on an animal b a l a nc e to ob t a i n d a t a on body we i gh t . subsequently the mice were anesthetized by intramuscular injection of 0.2 ml ketamine. then univ med vol. 37 no.2 118 ahsani, fidianingsih age and malondialdehyde male mice a b c 3-ml venous blood samples were drawn from the right and left retro-orbital veins using capillary tubes after the mice were unconscious. each blood sample was placed in an edta-containing eppendorf tube. after a sufficient volume of ve no u s b lo o d h a d be e n o bt a i n e d , t he experimental animals were terminated by transcardial perfusion, using physiologic saline (nacl) followed by pbs 10% formol fixative. the following organs were taken and dried on filter paper, namely the brain (cerebrum and cerebellum), heart, thymus, spleen, liver, both testes and both kidneys. after drying, each organ was weighed on an analytical balance and the organ weight recorded, expressed in grams. the blood samples obtained from each of the experimental animals were centrifuged to obtain the respective sera, which were taken with micropipettes and placed in separate eppendorf tubes. the sera were subsequently sent to the laboratory of the gadjah mada university inter-university center (pusat antar universitas universitas gadjah mada, pau fk u gm ) f o r me as u r e me nt o f m da concentrations, using the thiobarbituric acid reactive substances (tbars) assay. to each 100 microliters of serum was added 2.45 ml tric hloro-ac etic aci d (t ca) and 2.4 5 ml thiobarbituric acid (tba). the mixture was heated to 100ºc for 20 minutes, then centrifuged for 10 minutes at 8000 rpm. absorbance of the compound formed was determined from the standard curves. the mda concentration was expressed in nanomol/gram (nmol/g). statistical analysis the analysis was performed using the spss® for windows software. the study data were displayed in a table as mean ± standard deviation. the body weights and organ weights of the mice were expressed in grams (g) wh e r e a s t he m da co nc e nt r a t i o ns we r e expressed in nanomol/gram (nmol/g). mean relative organ weights were obtained from the ratio of mean organ weights to mean body weight of the mice in the same group. the data analysis started with the shapirowilk test of normality of data and levene’s test of homogeneity of data. on normally distributed and homogeneous data, one-way anova was used as test of significance, whereas on nonnormally distributed and/or non-homogeneous data the kruskal wallis test was used. one -wa y anova wa s pe r f o r me d t o determine the significance of the differences between mean body weight, mean weights of liver, heart, kidneys, and testes, and mean mda c o nc e nt r ati o n s i n e a c h gr oup . o ne -wa y an ova was a ls o u se d to de te rmi ne t he significance of mean organ weights of brain, liver, heart, kidneys, testes, and spleen relative to mean body weights of the mice. the nonparametric kruskal-wallis test was performed to determine the significance of the differences between means of the weights of the brain, thymus, spleen and mean relative weight of the thymus. the post-hoc lsd test was performed on data that were found to be statistically significant by the parametric one-way anova test (body weight, organ weights of liver, heart, kidneys, and testes, mda concentration and brain relative weight), whereas the mannwhitney test was performed on data that showed statistically significant results (weights of brain and thymus relative to body weight). ethical clearance ethical clearance for this experimental a ni ma l st udy w as gra nt ed un de r no. 10 / ka.kom.et/ 70/ke/xi/2016 before this study was conducted. results a total of 24 mice successfully completed the study up to the time points determined previously in the protocol. one mouse was removed from group k3 since it died before the time point determined (before 32 weeks). the body weights of the 24 mice were determined prior to necropsy. twenty four organs, comprising the brain, liver, heart, thymus, spleen, both 119 univ med vol. 37 no.2 kidneys and both testes, were collected at termination. twenty-three blood samples for mda determination were successfully obtained from 23 mice. no blood sample was collected from one mouse in the 32-week group (k3). t he a nova t e s t r e s u l t s sh o we d a significant difference in mean serum mda concentration (p=0.000) and mean body weight (p=0.000). significant differences in mean organ weight were encountered for nearly all organs [liver p=0.023), hear t (p=0.000), kidneys (p=0.002), testes (p=0.000), brain (p=0.012) and spleen (p=0.06), except the thymus (p>0.05)]. significant differences in relative mean organ weight was found for the brain (p=0.001) and spleen (p=0.049). with regard to the parameter of organ weight relative to body weight of the experimental animals, there were no significant differences in the relative organ weights of the liver, heart, kidneys, testes, and thymus (p>0.05, table 1). the post-hoc test showed significant differences in mda concentration and body weight between group k5 and all other groups (k1, k2, k3, k4) and between k3 and k4. with regard to the parameter of body weight, a significant difference was seen between k1 and all other groups, and between group k2 and groups k3 and k4 (table 2). in the post-hoc test, the parameter of organ weight showed a significant difference between group k1 and groups k4 and k5 for the liver, also between groups k1 and k5 versus all other groups and between k2 and k4 for the heart. there was a significant difference between group k1 and groups k3, k4, and k5, and between k2 and groups k3 and k5 with regard to the parameter of the weight of the kidneys. the weight of the testes showed a significant difference between group k5 and groups k1 and k2, between k1 versus k3 and k4, and between k2 and k4 (table 3). the weight of the brain relative to body weight showed a significant difference between group k1 versus all other groups and between k2 and k3 (table 4). the relative weight of the spleen showed a significant difference between k2 versus k4 and k5, and between k3 versus k4 and k5 (table 4). discussion this study showed that in the experimental animals aging was marked by an increase in mda concentration in the blood. this study parameter group p value k1 (n=5) k2 (n=5) k3 (n=4) k4 (n=5) k5 (n=5) mean ± sd mean ± sd mean ± sd mean ± sd mean ± sd mda (mmol/ml)§ 3.79 ± 0.76 3.854 ± 0.46 4.405 ± 0.3 3.21 ± 0.52 6.705 ± 0.20 0.000* body weight : bw (g) § 26.9 ± 2.19 34.7 ± 3.36 40.8 ± 5.95 40.5 ± 3.62 47.52 ± 4.28 0.000* organ weight : ow (g) liver § 1.43± 0.16 1.89 ± 0.49 1.91 ± 0.48 2.19 ± 0.3 2.27± 0.41 0.023* heart § 0.12 ± 0.02 0.14 ± 0.01 0.18 ± 0.06 0.19 ± 0.03 0.23 ± 0.01 0.000* kidneys § 0.32 ± 0.03 0.44 ± 0.18 0.68 ± 0.06 0.54 ± 0.09 0.60 ± 0.15 0.002* testes § 0.17 ± 0.03 0.22 ± 0.05 0.25 ± 006 0.3 ± 0.02 0.29 ± 0.04 0.000* brain §§ 7.00 ± 0.18 6.60 ± 0.20 12.75 ± 0.15 17.4 ± 0.20 18.80 ± 0.02 0.012* thymus §§ 10.10 ± 0.01 12.50 ± 0.03 15.13 ± 0.12 17.50 ± 0.02 7.80 ± 0.01 0.211 spleen §§ 10.20 ± 0.08 5.80 ± 0.03 8.75 ± 0.04 16.00 ± 0.03 21.00 ± 0.09 0.006* relative organ weight (ow/bw) liver § 0.053 ± 0.006 0.054 ± 0.009 0.046 ± 0.005 0.055 ± 0.011 0.047 ± 0.007 0.405 heart § 0.004 ± 0.001 0.004 ± 0.001 0.004 ± 0.001 0.005 ± 0.001 0.005 ± 0.000 0.611 kidneys § 0.012 ± 0.001 0.012 ± 0.004 0.017 ± 0.001 0.013 ± 0.002 0.012 ± 0.002 0.125 testes § 0.006 ± 0.001 0.006 ± 0.001 0.006 ± 0.001 0.007 ± 0.001 0.006 ± 0.001 0.188 brain § 0.015 ± 0.001 0.012 ± 0.001 0.009 ± 0.003 0.011 ± 0.001 0.01 ± 0.000 0.001* thymus §§ 0.004 ± 0.004 0.002 ± 0.000 0.002 ± 0.001 0.004 ± 0.001 0.006 ± 0.002 0.09 spleen § 0.002 ± 0.001 0.001 ± 0.001 0.002 ± 0.003 0.001 ± 0.000 0.001 ± 0.000 0.049* k1 : 12 weeks, k2 : 14 weeks, k3 : 32 weeks, k4 : 40 weeks, k5 : 48 weeks § : anova, §§: kruskal-wallis test, *: p<0.05 table 1. mda concentration, body weight, organ weight, relative organ weight of mice (mus musculus) in various groups 120 ahsani, fidianingsih age and malondialdehyde male mice indirectly also showed that the changes in antioxidant enzyme concentrations with aging could not overcome the increased free radical concentrations in the body. at baseline relatively constant mda concentrations were found, f o l l ow e d by s l i gh t l y i nc re a s e d m da concentrations in the group of 32 weeks (k3) and decreased mda concentrations in the group of 40 weeks (k4). at the age of 48 weeks a two-fold increase in mda concentrations was found as compared with both the ages of 12 and 24 weeks. the results of this study showing increased mda concentrations with aging are apparently similar to the results of the study by hamezah e t al ., ( 1 9 ) wh ic h s ho we d in c r e a s e d m da concentrations and decreased glutathione peroxidase (gpx) concentrations in older rats (27 weeks). the linear increase in mda concentration started in 18to 27-week old rats. on the other hand, the gpx concentrations decreased in the same age range, showing that lipid peroxidation increases with aging. this indicates that the preventive measures of the body by means of antioxidants (in this case gpx) was inadequate, as shown by the mutually inverse mda and gpx concentration graphs. the study by kim et al.(24) showed that increases in the concentrations of endogenous metabolites (lysophosphatidylcholines/lysopcs, comprising l ys o pc16 : 0, l ys o pc1 8 :0 , lys opc1 8: 2 , lysopc20:4, and lysopc 20:5, and linoleyl carnitine) lead to increases in inflammation (creactive protein/crp and interleukin-6/il-6), oxi da ti ve marke rs (o xidi zed low-density l i po p r ot ei n / ox -l dl, m da an d 8 -e p iprostaglandin f2alpha/8-epi-pgf 2α ) and brachial arterial stiffness (brachial-ankle pulse wave velocity/ ba-pwv) in 57-year old men. there was a positive correlation between changes in lysopc16:0 concentration with changes in the concentrations of 8-epi-pgf 2α , mda, crp, il6 a n d b a -pw v. t h i s s ugge st s th a t l ip i d peroxidation, and increased inflammation and vascular stiffness in aging humans is caused by increases in lysopc16:0. aging is also marked by increased body weight, which in this study was observed from the ages of 24 to 48 weeks. this increase was 1-2 times the baseline body weight in group k1. the increases in body weight in this study are in line with those in the study by chinedu et al. (20) parameter group mean difference p value mda level k1 k2 -0.06400 0.845 k3 -0.61500 0.089 k4 0.58000 0.089 k5 -2.91500* 0.000* k2 k3 -0.55100 0.124 k4 0.64400 0.061 k5 -2.85100* 0.000* k3 k4 1.19500* 0.003* k5 -2.30000* 0.000* k4 k5 -3.49500* 0.000* body weight k1 k2 -7.80000* 0.006* k3 -13.90000* 0.000* k4 -13.60000* 0.000* k5 -20.62000* 0.000* k2 k3 -6.10000* 0.033* k4 -5.80000* 0.031* k5 -12.82000* 0.000* k3 k4 0.30000 0.911 k5 -6.72000* 0.020* k4 k5 -7.02000* 0.011* table 2. lsd post-hoc test on mda concentration and body weight of mice (mus musculus) in various groups 121 univ med vol. 37 no.2 who showed that increases in body weight in humans are clearly found from age 13-19 years up to 40-59 years. in older age groups the rate in weight increase was similar to that of the group of 40-59 years. this shows that after the age of 40-59 years the rate of weight increase showed a static trend. in contrast to the latter study, we did not determine the body weight of mice older than 48 weeks, so that our study was unable to describe the trend observed in mice older than 48 weeks. dutta and sengupta (25) state that based on their respective life spans, 9-day old mice are the homologs of 1year old humans. therefore the ages of 12, 24, 32, 40, and 48 weeks in mice of the present study are homologous to the ages of 9, 19, 25, 31, and 37 years in humans. accordingly, when converted to human ages, the increase in mda concentrations in this study will be found around the age of 40 years. this agrees with the free radical theory which states that the defense mechanism against increased table 3. lsd post-hoc test on the weight of liver, heart, kidneys and testes of mice (mus musculus) in various groups organ weight group mean difference p value liver k1 k2 -0.46060 0.075 k3 -0.48610 0.077 k4 -0.76500* 0.006* k5 -0.84280* 0.003* k2 k3 -0.02550 0.923 k4 -0.30440 0.229 k5 -0.38220 0.135 k3 k4 -0.27890 0.296 k5 -0.35670 0.185 k4 k5 -0.07780 0.754 heart k1 k2 -0.02500 0.226 k3 -0.06010* 0.011* k4 -0.06940* 0.003* k5 -0.11340* 0.000* k2 k3 -0.03510 0.114 k4 -0.04440* 0.039* k5 -0.08840* 0.000* k3 k4 -0.00930 0.666 k5 -0.05330* 0.021* k4 k5 -0.04400* 0.040* kidneys k1 k2 -0.11820 0.135 k3 -0.35520* 0.000* k4 -0.21500* 0.010* k5 -0.28180* 0.001* k2 k3 -0.23700* 0.008* k4 -0.09680 0.217 k5 -0.16360* 0.044* k3 k4 0.14020 0.097 k5 0.07340 0.372 k4 k5 -0.06680 0.389 testes k1 k2 -0.05340 0.060 k3 -0.08200* 0.009* k4 -0.13340* 0.000* k5 -0.12340* 0.000* k2 k3 -0.02860 0.325 k4 -0.08000* 0.007* k5 -0.07000* 0.017* k3 k4 -0.05140 0.085 k5 -0.04140 0.160 k4 k5 0.01000 0.712 122 ahsani, fidianingsih age and malondialdehyde male mice free radicals in the human body will become less effective at the age of 40 years.(7) apparently this was the cause of the increased mda concentrations in group k5 of the present study. the increases in body weight that were found from the age of 24 to 48 weeks in the mice of this study are homologous to the increases in body weight in the study of chinedu et al.,(20) i.e. the age group of 19 to 40 years. these increases in body weight show the increasing development of the body and the increased intake by the body. this study showed an increase in the weight of the brain with increasing age. the increased brain weight apparently reflects increases in brain ventricular volume in the study of chen et al.(16) however, in contrast to the study by chen et al. (16) that showed an increasing trend in ventricular volume particularly at the age of fewer than 20 weeks, in our study the brain weight showed a linear increase with age (increased up to the age of 48 weeks). this is understandable because of the differences in the observed parameters. the parameter of brain weight in the present study was measured by weighing the cerebrum and cerebellum of the mice. therefore, the increases in brain weight in our study not only reflect increases in brain ventricular volume but also increases in other structures in both cerebral hemispheres as well as in the cerebellum. similar to the increase in brain ventricular volume, the volume of the cerebellum apparently also increases with age.(19) apparently both of these caused the increases in brain weight in this study. the present study showed increases in heart weight with increasing age, with a peak at the age of 48 weeks. the weight increase of the heart apparently depicts hypertrophy of the heart with age, accompanied by increases in extracellular connective tissue formation. the aging process causes a reduction in cardiomyocytes, and the cardiac hypertrophy and increased production of the extracellular matr ix with age is a compensatory mechanism to overcome the reduction in cardiomyocytes.(26) relative organ weight group mean difference p value brain ‡ k1 k2 0.003469* 0.007* k3 0.006096* 0.000* k4 0.003901* 0.003* k5 0.005413* 0.000* k2 k3 0.002627* 0.045* k4 0.000433 0.711 k5 0.001944 0.108 k3 k4 -0.002194 0.088 k5 -0.000683 0.582 k4 k5 0.001511 0.205 spleen ‡‡ k1 k2 0.05 k3 0.05 k4 0.35 k5 0.12 k2 k3 0.46 k4 0.01* k5 0.01* k3 k4 0.01* k5 0.01* k4 k5 0.25 table 4. post-hoc test on weights of brain and spleen relative to body weight of mice (mus musculus) in various ‡ : lsd post hoc test, ‡‡: mann whitney post hoc test 123 the results of our study showed increases in organ weight of the kidneys, liver, spleen and testes with increasing age. the increases in the weight of the kidneys, liver, and spleen in our study are in line with those of caglar et al.,(13) kim et al.(14) and melchioretto et al. (18), which are the results of autopsies on humans. the increases in the weight of the kidneys, liver, and spleen occurred concomitantly with increases in individual body weight and body mass index (bmi). (13,14,18) the spleen is subject to growth up to the age of 25 years in humans.(13) in line with abovementioned studies, the increased weight of the spleen in our study could be observed starting from the age of 40 weeks. the increase in the volume of the kidneys in older rats was apparently also marked by a decrease in renal function.(18) in contrast to the study results of melchioretto et al.(18) who state that increases in the volume of the kidneys are observable in rats from the age of 12 months, our study showed a peak increase in the weight of the kidneys occurring at the ages of 8 months (32 weeks) and 12 months (48 weeks). this may be due to the differences in experimental animals used. however, our study results are in line with those of kim et al.(14) who state that increases in the weight of the kidneys and liver in humans can be found in middle age. in line with the latter study, the increase in weight of both organs was found at the age of 48 weeks (homologous with 37 years in humans). this is apparently associated with the function of both organs that play a role in metabolic degradation. the present study also showed that aging does not cause statistically significant changes in the weight of the thymus. the peak increase in the weight of the thymus occurs at the age of 40 weeks. the decreases in the weight of the thymus in mice at the age of 48 weeks may have possibly occurred as a result of involution. this is supported by the increased activity of βgalactosidase in mice at the ages of 12 up to 18 months, this enzyme being one of the markers of aging. at these ages also there are increased markers of dna damage and failure of cell replication.(17) the present study showed that the increases in organ weight occurred at different points in time. the peak increases in the weight of the kidneys occurred at age 32 weeks whereas increases in the weight of the liver, heart, testes, brain and spleen occurred at age 48 weeks. this showed that only macroscopic changes occur in the majority of murine organs at age 48 weeks (homologous to age 37 years in humans). additionally, the present study aimed to determine the relative organ weight changes with increasing age. the relative organ weight is the ratio of organ weight to body weight. this study showed that there was a relative decrease in brain weight with increasing age. this was apparently due to atrophy of the gyri and widening of the sulci and cerebral ventricles.(14) in contrast to the study results on the weight of individual organs that showed increased brain weight with increasing age, there was a decreasing trend of the brain-to-body weight ratio. this showed that aging causes a far greater increase in body weight in comparison with the weight increase of the brain itself. ros induce vascular injury and organ dysfunction.(28) increased ros concentration is frequently associated with decreased elasticity, constriction, and relaxation of the blood vessels. aging causes increases in ros concentration that induce lipid peroxidation, resulting in cell membrane damage and ultimately dysfunction of cell-surface receptors and enzymes. increases in mda concentrations from lipid peroxidation will inactivate cellular proteins.(7) one study showed weight increase and hypertrophy of white adipose tissue (wat) with age in the internal organs and subcutaneous tissues.(29) secretion of the pro-inflammatory cytokines tnf-α and il-6 by adipocytes and accumulation of macrophages in adipose tissue causes a chronic systemic inflammatory condition (inflammaging),(30) resulting in dna damage and degeneration in various body tissues. other studies showed that aging increases foci of dna damage in various organs, such as the lungs, liver, and spleen. ultimately failure of dna repair causes apoptosis of the cells.(10,31) univ med vol. 37 no.2 124 ahsani, fidianingsih age and malondialdehyde male mice aging is also marked by the occurrence of disorders of metabolism. decreases in growth hormone (gh), and insulin growth factor-1 (igf-1) and increases in insulin resistance are recognized as the main markers for age-related disorders of metabolism.(30) igf-1 induces increased production of collagens and various other components of the extracellular matrix (ecm).(32) in the kidneys, chronic disorders of cellular metabolism decrease the expression of sirtuin-1 (sirt-1) that plays an important role i n c e l l s ur vi va l . t h e d e c r e a se d sirt-1 expression results in decreases in the function of blood vessels and also in the production of matrix metalloproteinase-14 (mmp-14),(33) both of w hi ch c a use t is su e fi br os is . ( 10 ,3 3) it i s apparently these conditions that underlie the age-related changes in organ weight. t he in ve st i ga t or s a r e a war e of t he numerous limitations of this study. first, this study used small number of data on aging in the experimental animals and the small number of study subjects in each group. the investigators also selected the parameter of body weight without using the parameter of body mass index (bmi) as one of the variables under study. further studies should be conducted to know the patt er ns of morpholo gical c hanges, both anatomical and histological, which occur during the lifetime of an individual. it is hoped that with the knowledge of the pattern of changes that occur with age in experimental animals, will come the knowledge of the pattern of changes in free radicals and in the weight of internal organs in humans. by knowing these patterns of changes, age-related standard normal values will be obtained for each internal organ and free radical in experimental animals in general and in humans in particular. conclusion there were significant age-related changes in serum mda, body weight, and the weights of the liver, heart, kidneys, testes, brain and spleen. the peak increase in the weights of the kidneys and thymus occurred earlier than that of the changes in mda concentration, body weight and the weights of several organs (liver, heart, testes, brain and spleen). the rate of increase of the weights of the brain and spleen was greater than that of the body weight. acknowledgment the authors wish to thank the technicians of the physiology laboratory, faculty of medicine, uii, who assisted in the course of this study. conflict of interest the investigators declare that there is no conflict of interest from the start of this study up to and including the writing up of the manuscript. this study was self-funded by the investigators. contributors dna and if contributed fully to the study design, data collection, writing and revision 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fibrosis of vascular senescence. j am soc nephrol 2014;25:276–91. doi: 10.1681/ asn.2013010069. 33. horn ma, trafford aw. aging and the cardiac collagen matrix: novel mediators of fibrotic remodelling. j mol cell cardiol 2016;93:175–85. http://dx.doi.org/10.1016/j.yjmcc.2015.11.005. oktavianus 192 *faculty of medicine, padjadjaran university **department of biochemistry, padjadjaran university ***department of obstetrics and gynecology, dr. hasan sadikin general hospital correspondence kevin dominique tjandraprawira faculty of medicine, padjadjaran university jalan raya jatinangor km 21 jatinangor west java, indonesia email: kevintjandraprawira@gmail.com phone: +62813-20221221 univ med 2014;33:192-8 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background cervical cancer ranks second among female cancers worldwide and is widely associated with human papilloma virus (hpv) infection. however, hpv infection progression is influenced by various host factors. epidermal growth factor (egf) is a host factor important for proper epithelial proliferation and development, and may play a role in cervical cancer progression. a functional a61g polymorphism in the egf gene has been hypothesized to alter egf concentration in vivo with increasing guanine content associated with greater egf level. however, a map of a61g polymorphism distribution is not available for any population, including indonesia. this study aims to determine the distribution of egf a61g polymorphism among cervical cancer patients at dr. hasan sadikin general hospital. methods a retrospective cross-sectional study was conducted between july-november 2010. included were 61 cervical cancer patients of various stages at dr. hasan sadikin hospital, who had previously undergone blood sample collection, dna isolation and finally genotyping for egf gene using illumina beadxpress®. chi-square test was used to analyse the data. results the egf a61g polymorphism was exhibited by 88.5% of patients (as genotypes a/g and g/g). the majority of patients with this polymorphism were of moderate severity (figo stage ii and iii, 42.6% and 38.1% respectively). patients with the polymorphism but with the lightest severity (figo stage i) accounted for 22.2% of the population. conclusion egf a61g polymorphism affected the majority of cervical cancer patients and that once stratified, the patients showed intermediate severity in terms of their cancerous growth. keywords: cervical cancer, egf, a61g polymorphism epidermal growth factor polymorphism most prevalent in stage ii cervical carcinoma kevin dominique tjandraprawira*, ramdan panigoro**, yudi mulyana hidayat***, herman susanto***, and edhyana sahiratmadja** 193 univ med vol. 33 no.3 polimorfisme a61g pada gen egf paling banyak pada karsinoma serviks stadium ii latar belakang kanker serviks merupakan kanker kedua terbanyak pada perempuan di seluruh kanker serviks hampir selalu diasosiasikan dengan infeksi human papilloma virus (hpv). akan tetapi, berbagai faktor penjamu (host) telah dibuktikan dibutuhkan untuk memungkinkan progresi infeksi ini kedalam suatu massa neoplastic. epidermal growth factor (egf) merupakan sebuah faktor pertumbuhan dari penjamu yang diperlukan untuk proliferasi dan diferensiasi dari jaringan epitel. sebuah polimorfisme a61g fungsional pada gen egf diduga dapat mengubah konsentrasi egf in vivo di mana konsentrasi guanin yang lebih tinggi pada genotip seseorang dikaitkan dengan konsentrasi egf yang lebih besar pula. akan tetapi, hingga sekarang belum ada peta distribusi polimorfisme a61g untuk populasi indonesia. penelitian ini bertujuan untuk menentukan distribusi polimorfisme a61g pada pasien kanker serviks stadium i-iv di rumah sakit dr. hasan sadikin. uji chi-square digunakan untuk analisis data. metode sebuah rancangan potong silang dilakukan antara bulan juli-november 2010. pengambilan darah serta isolasi dna dilakukan pada 61 pasien kanker serviks dengan berbagai stadium di rs dr. hasan sadikin dan kemudian, uji genotyping untuk gen egf menggunakan beadxpress® illumina. hasil hasil analisis menunjukkan bahwa polimorfisme a61g dimiliki oleh 88,5% pasien (dengan genotip a/g dan g/ g). di antara pasien-pasien yang memiliki polimorfisme ini, mayoritas memiliki tingkat keparahan sedang (stadium ii dan iii figo, 42,6% dan 31,8%). kemudian, hanya 22,2% pasien dengan polimorfisme ini yang memiliki tingkat keparahan ringan. kesimpulan penelitian ini menunjukkan bahwa polimorfisme a61g pada gen egf meliputi mayoritas pasien kanker serviks dan setelah distratifikasi, mayoritas pasien menunjukkan tingkat keparahan sedang. kata kunci: kanker serviks, egf, polimorfisme a61g abstrak introduction cervical cancer forms a huge burden in the epidemiology of female cancer, as it currently ranks second worldwide. its prevalence is estimated at 500,000 cases, with 270,000 deaths annually.(1) southeast asia, sub-saharan africa and south america today form the hot zones for high hpv prevalence. in indonesia, cervical cancer reached a crude incidence rate of 12.1 per 100,000 women back in 2008 and this corresponds to more than 13,000 cases of new cervical cancer cases every year in indonesia alone.(2) cervical cancer, as any other type of cancer, requires both host and environmental factors. the human papilloma virus is the prerequisite environmental factor and both single and multiple hpv infections in a single patient is no longer an oddity.(3) in fact, from a local study done at dr. hasan sadikin general hospital, hpv 16 is the most prevalent virus among patients, followed by hpv 18, 45 and 52.(3) once the virus infects the cervical epithelial cells, it then has its genes expressed and mature viral particles synthesized via the expression of various early proteins, namely e5, e6 and e7 194 proteins. (4-6) the early proteins allow the inactivation of the pro-apoptotic genes inherent to the host, which then allows an uninhibited mitotic proliferation of cervical epithelium.(7) hpv is a necessary yet insufficient factor,(8) as only interaction with host factors will allow such infection to proceed to a neoplastic growth. one of the proposed factors is epidermal growth factor (egf), a protein encoded by a gene on chromosome 4q25-2. once it binds to its receptor, epidermal growth factor receptor (egfr), it activates several signaling pathways, namely ras/raf/ mitogen-activated protein kinase (mapk) or phospho-inositide 3-kinase (pi3k), which under normal conditions are required to ensure proper proliferation and differentiation of epithelial tissues.(8-10) however, excessive activation of egfr or heightened production of egf may also incite tumorigenesis in cells of epithelial origin.(8) as egf is a protein, the promoter region of the egf gene often has the final say in determining the circulating levels of egf. there are variations, known as single nucleotide polymorphisms (snps) (11) in the promoter region that may be responsible for the increased levels of egf and up to this day, only one functional snp has been described. shahbazi et al.(12) were the first to describe the rs4444903 or a61g polymorphism in the 5’-untranslated region of the gene’s promoter. their results revealed that this particular snp, associated with prompting increased guanine content in one’s genotype, had a high correlation with malignant melanoma development. since then, various authors have also accredited this snp as being involved in the development of esophageal adenocarcinoma, glioma, colorectal cancer and hepatocellular carcinoma.(13) however, protective effects of this particular snp have also been noted in ovarian cancer and gastric cancer.(13, 14) cervical cancer is derived from epithelial cells and therefore its development has also been suggested to be influenced by this snp. until recently, however, results have been conflicting. a study in korea performed by kang et al.(15) did not find any correlation between this snp and greater cervical cancer development while a study in the usa performed by araujo et al. (16) revealed the opposite results. furthermore, it has also been revealed that the prevalence of this polymorphism is somewhat governed by racial origins, with asian populations possessing a greater prevalence of the g allele in their genotypes.(17) however, until recently there has been no studies to confirm this finding on indonesian populations. thus, this study seeks to discover the distribution of the a61g polymorphism in cervical cancer patients presenting to dr. hasan sadikin general hospital, bandung. methods research design t h i s s t u d y e m p l o y s a r e t r o s p e c t i v e observational study design and was conducted between july – november 2010. research subjects included were patients who presented to dr. hasan sadikin general hospital with cervical carcinoma of various severity and had undergone histologic examinations at the department of anatomic pathology resulting in an established diagnosis of cervical cancer. subtypes of cervical cancer, namely keratinizing squamous cell carcinoma, nonkeratinizing squamous cell carcinoma, small cell squamous cell carcinoma, adenocarcinoma and adenosquamous carcinoma, were all included in the diagnosis of cervical cancer. patient details regarding age, age at first sexual intercourse, parity and abortion were collected. of the initial study population, 135 patients had undergone genotyping and were initially included in the study. however, lack of data on patients’ age, age at first sexual intercourse, parity and abortion, led to the inclusion of only 61 patients in the end. total inclusive sampling was then employed on the final study population. tjandraprawira, panigoro, hidayat, et al epidermal growth factor polymorphism 195 univ med vol. 33 no.3 since this study used secondary data obtained from an earlier study done under the supervision of the oncology working group of dr. hasan sadikin general hospital, the following steps were not carried out in the present study and are here included for completeness only. dna isolation dna is extracted from collected blood samples by means of salting out methodology. the extracted dna, adjusted to 50 ng/ìl and transferred onto 96-well plates, is then been analyzed with the illumina beadxpress system. snp array analysis three specific oligonucleotide primers (opa) are prepared for each snp, comprising two asps (allele specific primers) and one lsp (locus specific primer). these primers are then allowed to hybridize with the genomic dna and pcr is then carried out, generating multiple amplicons with labels attached for all the different snps. the pcr products are then combined with the veracode bar-coded beads. these beads contain sequences corresponding to the lsps and they emit fluorescence. the fluorescence on each bead is then quantified and the signal emitted by the fluorescence indicates a particular address sequence. each address is an indicator toward a specific locus and the signals emitted by the beads are then translated to aa, bb or ab genotypes. the wild-type genotype for the egf gene is a/a while the nonw i l d t y p e g e n o t y p e s a r e a / g a n d g / g respectively. statistical analysis for statistical analyses the spss 17.0. software package (spss inc., chicago, il) was used. patients were stratified according to their genotypic profile as well as their diagnostic s t a g i n g a s d e t e r m i n e d a c c o r d i n g t o t h e classification laid out by figo in 2008.(18) the influence of the egf genotype on the main outcome variable, severity of cervical carcinoma, was assessed with chi-square test. ethical clearance the ethical clearance required for this study was granted in july 2011 by the health research ethics committee, medical faculty, padjadjaran university – dr. hasan sadikin general hospital bandung. results this study involved 61 cervical carcinoma patients of varying severity (stages i-iv) with d i v e r s e s o c i o d e m o g r a p h i c b a c k g r o u n d s , concerning aspects of age, age at first sexual intercourse, parity and abortion. in this study, the majority of patients (88.5%) had the a61g polymorphism on the egf gene, resulting in a skewness toward the alleles with guanine nucleotides. table 1 presents a summary of the demographic and clinical features of the study population along with the population’s genotypic distribution. furthermore, among those patients who possessed a61g polymorphism in their genotype, most were accounted as either stage ii or stage iii in their carcinomic severity, as there were 12 and 9 patients for a/g genotype, and 11 and 8 charac teristic n (%) p atient’s age, mean (sd) 48.43 (8.74) age at first intercourse, mean (sd) 16.56 (2.22) p ari ty, mean (sd ) 3.62 (1.72) abortions, mean (s d) 0.56 (0.79) s tage at diagnosis (figo) i 13 (21.31) ii 24 (39.34) iii 22 (36.07) iv 2 (0.03) genotype aa 7 (11.5) ag 29 (47.5) gg 25 (41.0) table 1 ditribution of the demographic and clinical characteristics of the patients (n=61) figo: international federation of gynecology and obstetrics sd: standard deviation 196 patients for g/g genotype, classified under stage ii and iii respectively. the severest of all, stage iv, contributed the smallest number of patients, as out of 61 patients only 2 were reported to fall in this category. moreover, once the patients under a/g and g/g genotypes were merged together, it is evident that those diagnosed under stage ii and iii were still highest in number (table 2). discussion overall, the results of this study managed to demonstrate that there was no uniform distribution of egf gene polymorphisms among patients with cervical carcinoma at dr. hasan sadikin general hospital. the results were toward a majority of patients possessing a61g polymorphism, which seemed to confirm the notion that there is a greater proportion of this polymorphism in asian populations.(17) to date, there have only been two other studies that investigated the relationship between the a61g polymorphism of the egf gene with regard to cervical cancer. both of these studies differed much in their subjects as one was done in korea and the other one was done in the united states. studies investigating the role of egf gene polymorphism on cervical cancer development have been very few in number and efforts to search for greater support regarding egf gene polymorphism distribution in cervical cancer patients reaped few results. the first of the two studies was done back in 2007and this was the first ever study to i n v e s t i g a t e t h e p o s s i b l e r o l e o f a 6 1 g polymorphism in affecting the development of cervical cancer.(15) this study involved 337 patients, with 168 patients acting as cases and 169 as controls matched for age. this study discovered that there was no significant difference in terms of the polymorphism distribution between the two groups and eventually the study concluded that the a61g polymorphism exerted protective effects in reducing individual susceptibility toward cervical carcinoma. nevertheless, the study did point out that the presence of a61g polymorphism was associated with an increased risk toward metastasis in those patients with established carcinoma, albeit without statistical significance. another contribution made by the study performed by kang et al.(15) is that they managed to analyze the distribution of egf genotypes between caucasian and east asian populations from previous reports. based on their analysis, they confirmed that there was a significant difference between these two populations in terms of egf genotype distribution, with results in tune with our study, as in their study, kang et al.(15) also observed that the majority of their patients had a61g polymorphism. the second study was performed in 2012 in the united states of america.(16) similar to the first study, this study also involved cancer vs. noncancer patients. the study was replete with the indication that the a61g polymorphism had a role to play in increasing the likelihood of developing more advanced cervical carcinoma and that this likelihood was more evident in g carriers. our current study is the first of its kind to be done on indonesian population and it is also an addition to an already limited population of studies conducted on a61g polymorphism and its role in cervical cancer. thus, there is the table 2 distribution of the egf genotypes (wild-type vs. guanine-containing genotypes), stratified according to the diagnostic staging by figo figo: international federation of gynecology and obstetrics tjandraprawira, panigoro, hidayat, et al epidermal growth factor polymorphism 197 univ med vol. 33 no.3 urgency to conduct more studies on this particular issue as there has not been an unequivocal conclusion on the overall effect of having more guanine on one’s genotype toward one’s risk of developing a more severe neoplastic growth. h o w e v e r, o u r s t u d y i s n o t w i t h o u t limitations. the first limitation is the number of the samples, which was limited to 61 individuals. this number is significantly smaller than the calculated minimum requirement of 97 samples and this number is also considerably minute compared to the studies done by previous researchers.(15,16,19) furthermore, lack of similar publications concerning egf gene and cervical cancer development proved to be a hindrance in terms of coming up with greater support for the study. finally, the status of this study, despite being the first of its kind in indonesia, is a problem in itself, as the results of this study cannot be compared with, nor confirmed by, other similar studies. conclusion the a61g polymorphism is evident in the majority of cervical carcinoma patients and most of these patients exhibited moderate severity in their carcinoma staging. acknowledgement the authors wish to thank dr. ani melani maskoen, drg.,m.kes and ms. nurul melani rahayu of molecular genetics laboratory at dr. hasan sadikin general hospital for having performed the initial sequencing of genetic material using the illumina beadxpress®. references 1. parkin dm, bray f, ferlay j, pisani p. global cancer statistics, 2002. ca cancer j clin 2005; 55:74-108. 2. who/ico information center on hpv and cervical cancer (hpv information centre). human papillomavirus and related cancers in indonesia. barcelona;2010. 3. sahiratmadja e, tobing mdl, dewayani bm, hernowo bs, susanto h. multiple human papillomavirus infections predominant in squamous cell cervical carcinoma in bandung. univ med 2014;33:58-64. 4. chauhan s, jaggi m, bell m, verma m, kumar d. epidemiology of human papilloma virus (hpv) in cervical mucosa. methods mol biol 2009;471:439-56. doi: 10.1007/978-1-59745416-2_22. 5. zur hausen h. papillomaviruses causing cancer: evasion from host-cell control in early events in carcinogenesis. j nat cancer inst 2000;92:6908. 6. zur hausen h. papillomaviruses and cancer: from basic studies to clinical application. nat rev cancer 2002;2:342-50. 7. ibeanu oa. molecular pathogenesis of cervical cancer. cancer biol ther 2011;11:295-306. 8. narayanan r, kim hn, narayanan nk, nargi d, narayanan b. epidermal growth factorstimulated human cervical cancer cell growth is associated with egfr and cyclin d1 activation, independent of cox-2 expression levels. int j oncol 2012;40:13-20. 9. wells a. egf receptor. int j biochem cell biol 1999;31:637-43. 10. singh ab, harris rc. autocrine, paracrine and juxtacrine signaling by egfr ligands. cell signal 2005;17:1183-93. 11. bag a, jyala ns, bag n. indian studies on genetic polymorphisms and cancer risk. ind j cancer 2012;49:144-62. 12. shahbazi m, pravica v, nasreen n, fakhoury h, fryer aa, strange rc, et al. association between functional polymorphism in egf gene and malignant melanoma. lancet 2002;359:397401. 13. li tf, ren kw, liu pf. meta-analysis of epidermal growth factor polymorphisms and cancer risk: involving 9,779 cases and 15,932 controls. dna cell biol 2012;31:568-74. 14. araujo ap, ribeiro r, pereira d, pinto d, sousa b, catarino r, et al. ovarian cancer and genetic susceptibility: association of a61g polymorphism in the egf gene. exp biol med 2009;234:241-5. 15. kang s, kim jw, park nh, song ys, park sy, kang sb, et al. epidermal growth factor 61 a/ g polymorphism and uterine cervical cancer. int j gynecol cancer 2007;17:492-6. 16. araujo ap, catarino r, ribeiro r, pereira d, pinto d, medeiros r. epidermal growth factor genetic variation associated with advanced cervical cancer in younger women. am j clin oncol 2012;35:247-50. 198 17. lanuti m, liu g, goodwin jm, zhai r, fuchs bc, asomaning k, et al. a functional epidermal growth factor (egf) polymorphism, egf serum levels, and esophageal adenocarcinoma risk and outcome. clin cancer res 2008;14:3216-22. 18. pecorelli s, zigliani l, odicino f. revised figo staging for carcinoma of the cervix. int j gynecol obstet 2009;105:107-8. 19. dahlan ms. besar sampel dan cara pengambilan sampel. jakarta: salemba medika;2013. tjandraprawira, panigoro, hidayat, et al epidermal growth factor polymorphism c:\users\jurnal fkusakti\docume 214 *department of anatomy, faculty of medicine, islamic university of indonesia yogyakarta correspondence dr. kuswati, m.sc department of anatomy, faculty of medicine, islamic university of indonesia jl. kaliurang km 14,5 yogyakarta phone: +62274 898444 mobile: +62 852 284 784 87 email : kuswatinugroho@gmail.com date of first submission, july 26, 2017 date of final revised submission, november 22, 2017 date of acceptance, november 22, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract propolis increases neuronal count in hippocampal area ca1 and prefrontal cortex in stressed rats kuswati*, ety sari handayani*, and zainuri sabta nugraha* background stress induces neuronal cell damage in the hippocampus and prefrontal cortex. propolis has a neuroprotective effect that can inhibit apoptosis and decrease neuronal cell count. this study aimed to determine the effect of propolis on neuronal cell count in hippocampal area ca1 and prefrontal cortex in sprague dawley rats with induced stress. methods a study of laboratory experimental design was conducted involving 24 male sprague-dawley rattus norvegicus. the animals were randomly divided into 4 groups, i.e. controls (k), and stress groups p1, p2 and p3. controls did not receive treatment, stress group (p1) received stress treatment, groups p2 and p3 received stress and propolis at 100 and 200 mg/kgbw, respectively. stress and propolis were given for 14 days, followed by termination. the number of neurons in the hippocampal area ca1 and prefrontal cortex were counted. one way anova was used to analyze the data. results the neuronal count in the hippocampal area ca1 and prefrontal cortex in the stress group (p1) was lower than in groups k, p2 and p3. there were significant differences in the neuronal count of the hippocampal area ca1 between p1 and p3 and p1 and k (p=0.019) and also in the neuronal count of the prefrontal cortex between p1 and p2, p3 and k (p=0.002). conclusions this study strongly suggest that propolis inhibits the decrease in neuronal count in in the hippocampal area ca1 and prefrontal cortex of sprague dawley rats with induced stress. the present study suggests a potential neuroprotective effect of propolis in the prevention of neurodegenerative disorders. keywords: propolis, hippocampus, prefrontal cortex, stress, rats doi: http://dx.doi.org/10.18051/univmed.2017.v36.214-220 original article universa medicinaseptember-december, 2017 vol.36 no.3 cite this article as: kuswati, handayani es, nugraha zs. propolis increases neuronal count in hippocampal area ca1 and prefrontal cortex in stressed rats. uni v med 20 17;3 6:21 4-20 . doi: 1 0 . 1 8 0 5 1 / un i vm ed . 2 0 1 7 . v 3 6 . 2 1 4 2 2 0 pissn: 1907-3062 / eissn: 2407-2230 215 univ med vol. 36 no.3 introduction the hippocampus plays an important role in learning and memory and is one of the brain regions that is affected by stress.(1) rats treated with various chronic stress models, by reducing sleep duration, immobilization stress and electrical stress, have a lower number of pyramidal cells in area ca3 of the hippocampus than do rats without stress treatment.(2) stress administered to rats 6 hours daily for 6 weeks results in changes in the hippocampal neurons. these changes comprise a reduction in the number and length of apical and basal dendrites, so causing atrophy of the neurons in area ca3 of the hippocampus.(3) chronic stress plays a role in the aging process in the hippocampus, marked by changes in the expression of several genes. exposure to stress for 10 days decreases the gene expression levels of sirtuin-1 and heat shock protein 70, but increases the gene expression levels of p-53, thioredoxin-interacting protein and nicotinamide adenine dinucleotide phosphate (nadph) oxidase.(4) in the prefrontal cortex, chronic stress increases the noradrenergic innervation and causes a decrease in working memory and interference with the connections between the prefrontal cortex and the hippocampus.(5) stress results in a decrease in the number of neurons (neuronal cell count) and b cell lymphoma 2 (bcl2) expression in the prefrontal cortex.(6,7) the combination of acute and chronic stress results in cytochrome c release from the mitochondria into the cytoplasm, activation of caspase-3 and increase in the number of cells undergoing apoptosis in the prefrontal cortex.(8) the results of the study by li et al.(9) demonstrates the occurrence of apoptosis and changes in the bcl2 associated x (bax) and the bcl-2/bax ratio in the prefrontal cortex of rat models of post traumatic stress disorder (ptsd). bachis et al.(10) report an increase in the number of cells expressing caspase 3 in the pyramidal frontal cortex layers of sprague dawley rats undergoing stress. rats that were given stress treatment by chronic social isolation for 6 weeks underwent an increase in apoptosis, a decrease in bcl-2 protein, an increase in bax, and an increase in the bax/bcl-2 ratio in the prefrontal cortex.(11,12) cao et al.(13) report that socially isolated rats have spatial and short term memory dysfunction, abnormal social functioning and anxiety. in addition, there was also increased active caspase 3 expression in the medial prefrontal cortex and hippocampus. propolis is one of the substances that are expected to be able to protect neurons from damage due to stress exposure.(14) propolis contains chrysin, caffeic acid phenethyl ester (cape) and caffeoylquinic acid (cqa) that have neuroprotective effects.(15,16) previous studies have proven that propolis may inhibit apoptosis in cell cultures of sh-sy5y neurons (a human neuroblastoma cell line) that were exposed to staurosporin, by inhibiting caspase-3 activity and apoptosis through the intrinsic pathway.(17) the aqueous extract of propolis decreases the production of lactate dehydrogenase (ldh) in cultures of the pc12 pheochromocytoma cell line as a result of exposure to h 2 o 2 . this shows that the aqueous extract of propolis has protective effects on the mitochondria and cell membranes of neurons. (18 ) pinocembr in is one of the flavanones found in propolis and honey with actions useful in preserving cognitive function.(19) in a recent animal study, pinocembrin ameliorated cerebral damage caused by global cerebral ischemia-reperfusion.(20) these neuroprotective eff ects were attrib uted to pinocembr in suppressing damaging biomarkers including oxidative stress and inflammation. one study found that chinese propolis inhibits neuronal ce ll death induced by endoplasmic reticulum stress that has been implic ate d in the pathogenesis of neurodegenerative and ischemic disorders.(17) long term administration of chrysin at 30 mg/kg showed reduced pathological changes and attenuated the chronic cerebral hypoperfusion induced cell loss in the cortex and hippocampal ca1 area of rats.(21) however, there is no 216 information available on the effect of propolis administration on the neuronal cell count in the hippocampal and prefrontal cortex areas. the present study aimed to evaluate the effect of propolis administration on the neuronal cell counts in the hippocampal area ca1 and the prefrontal cortex in sprague dawley rats receiving social isolation stress exposure. methods research design this was an experimental laboratory study with posttest only control group design. the study was conducted at the research laboratory, faculty of medicine, universitas islam indonesia (fk uii) from march to december 2016. research subjects the study subjects were 4 month-old male rattus norvegicus of the sprague -dawley strain, weighing 200-300 grams. the number of subjects required were calculated with the formula of charan and biswas (22) as follows: e = total number of animals total number of groups = 10 20 total number of animals = 20 total number of groups = 20 4 = 16 from the above calculation the number of subjects obtained was 16 rats. this method of sample size calculation is rather crude and should only be used if it cannot be done by power analysis. all experiments were performed in accordance with the standard guidelines for the care and use of laboratory animals. preparation of propolis extract the propolis preparation used in this study is a product available in the free market, with 1 ml containing 600 mg propolis. to 0.1 ml of the propolis preparation 0.9 ml distilled water was added thus giving a concentration of 60 mg/ml. this dilution was used to make up doses of 100 mg/kgbw and 200 mg/kgbw to be administered to the rats, in accordance with the study of lee et al.(23) stress procedure and propolis treatment in this study the social isolation stress model was applied by holding one rat per cage so that there was no interaction with the other rats.(24) this stress treatment was given for 14 d a ys . t h e r a t s we r e di vi de d by bl o c k randomization into 4 groups, each consisting of 4 rats (n=4). the groups comprised one control group (k), one stress group (p1) and two propolis groups (p2 and p3). the control group did not receive treatment, the stress group (p1) received stress treatment, while groups p2 and p3 received stress treatment and oral propolis administration by gastric tube at doses of 100 and 200 mg/kgbw, respectively. the control group and the stress group (p1) received distilled water. sample preparation on day 15 the rats were terminated and underwent transcardial perfusion. the rats were anesthesized by an intramuscular injection of ketamine at 100 mg/kgbw. the transcardial perfusion used normal saline at 100-200 ml until the returning perfusion fluid was clear. after perfusion, the brain was carefully dissected out and the portions containing the hippocampus and the prefrontal cortex were fixed in phosphatebuffered formalin for 24 hours. the portions of the brain containing the hippocampus and the prefrontal cortex were made into paraffin blocks and sectioned at 4 μm. the sectioning was done at the portion containing the hippocampus. from one rat brain 3 sample sections were made at distances of 240 m. hematoxyllin eosin staining was done according to the protocol at the research laboratory of fk uii, which briefly was as follows. the slide containing the tissue section was placed in a staining jar, deparaffinized, then washed twice in distilled water. subsequently the slide was placed in the hematoxyllin solution for 10 minutes, washed 3 times in distilled water, and checked under the microsc ope. if the hematoxyllin staining was adequate, the slide was washed in running water. subsequently the kuswati, handayani, nugraha propolis a neurodegenerative effect 217 univ med vol. 36 no.3 a b c preparation was placed in eosin and washed twice in distilled water. the slide was placed in the alcohol series of 70%, 80%, 90%, 95%, and 100% then dried with tissue paper. the dried slide was placed in xylol i, ii and iii for 5 minutes each, then mounted and observed under the microscope. neuronal cell counts in hippocampus and prefrontal cortex the preparation was observed under the light microscope at 400 x magnification. the cell count was performed in the whole hippocampal area ca1 and prefrontal cortex. subsequently the mean number of cells was calculated for one preparation per field of view with the formula: mean cell count = number of cells in the area under observation/number of fields of view. data analysis to compare the mean cell count per field of view between the groups, one-way anova was used, followed by a post hoc test to determine the differing groups. a p-value of less than 0.05 was considered statistically significant. ethical clearance this study was approved by the research ethic s committee, fac ulty of medic ine, universitas islam indonesia under no. 03/ ka.kom.et/70/ke/v/2016. results the resulting neuronal cell counts in hippocampal area ca1 and prefrontal cortex are presented in table 1. the results of one way anova showed a significant difference in neuronal cell count of hippocampal area ca1 between the four groups (p=0.019). the results of post hoc analysis showed significant differences between group p1 and the control group and group p3, respectively (table 2). significant differences in neuronal cell counts were also found in the prefrontal cortex (p=0.021). the results of post hoc analysis showed significant differences between group p1 and the groups k, p2 and p3, respectively (table 3). there were no significant differences between the control group k and groups p2 and p3. variable treatment groups p value k (n=4) p1 (n=4) p2 (n=4) p3 (n=4) neuronal cell count of hippocampal area ca1 neuronal cell count of prefrontal cortex 61.69 ± 9.89 86.80 ± 14.50 40.08 ± 2.59 50.03 ± 4.76 51.86 ± 11.11 88.11 ± 22.44 58.49 ± 14.61 89.63 ± 21.55 0.019 0.021 table 1. distribution of mean neuronal cell counts in hippocampal area ca1 and in the prefrontal cortex, by treatment groups k=control group (non-stress); p1=stress group; p2=stress group + propolis 100 mg/kgbw; p3=stress group + propolis 200 mg/kgbw group group p value k p1 p2 p3 0.003 0.139 0.620 p1 p2 p3 0.093 0.013 p2 p3 0.331 table 2. results of post hoc analysis of mean neuronal cell counts of hippocampal ca1 k=control group (non-stress); p1=stress group; p2=stress group + propolis 100 mg/kgbw; p3=stress group + propolis 200 mg/kgbw group group p value k p1 p2 p3 0.001 0.898 0.783 p1 p2 p3 0.001 0.001 p2 p3 0.898 k=control group (non-stress); p1=stress group; p2=stress group + propolis 100 mg/kgbw; p3=stress group + propolis 200 mg/kgbw table 3. results of post hoc analysis of mean neuronal cell counts of prefrontal cortex 218 discussion the results of this study showed that in the stress groups receiving propolis treatment (p2 and p3) the mean neuronal cell counts were higher than in the stress group receiving distilled water (p1). this showed that propolis treatment may inhibit the decrease in neuronal cell counts in hippocampal area ca1 and prefrontal cortex as a result of stress exposure. one study evaluated the effect of administration of propolis and caffeic acid phenylethyl ester (cape) on brain tissue of rats exposed to ionizing radiation and showed that propolis and cape would reduce the oxidative damage in the radiation-injured brain tissue.(25) the results of the study by reis et al.(26) showed that supplementation with oil extract of propolis (oep) at doses of 10, 30 and 50 mg/ kgbw administered intraperitoneally for 1 hour produced anxiolytic and antidepressant effects and improved locomotion in rats. another study showed that an aqueous extract of propolis decreased the production of ldh in cultures of neuronal-like pc12 cells as a result of exposure to h 2 o 2 .(18) this showed that aqueous extract of propolis has protective effects on mitochondria and neuronal cell membranes. in mice with scopolamine-induced neurotoxicity, administration of propolis at 100 mg/kgbw may improve memory, and inhibit acetylcholine esterase activity in the cerebral and hippocampal cortex.(27) this showed that propolis increased memory through inhibition of acetylcholine esterase activity in the hippocampus. therefore, propolis has the pharmacologic potential of a neuroprotective agent and may be used for treatment of amnesia in the elderly, alzheimer’s disease (ad) and other neurodegenerative diseases. propolis administration at a dose of 200 mg/ kgbw to wistar rats exposed to aluminum nitrate could inhibit lipid peroxidation in the liver, kidneys and neurons.(28) furthermore, propolis treatment could incr ease the c oncentrations of the antioxidant glutathione in brain tissues. propolis treatment could inhibit the decrease in atpase in the liver, kidneys and brain. propolis treatment could restore acetylcholine esterase activity in the brain after exposure to aluminum nitrate. addition of an ethanolic extract of propolis to neuronal cell cultures which had been exposed to the neurotoxic agents staurosporin and hydrogen peroxide, could inhibit the production of reactive oxygen species (ros) and caspase3 activity.(29) in rats with neurotoxicity induced by exposure to lead (pb) for 4 weeks and receiving oral propolis treatment, it was shown that propolis could inhibit the neurotoxic effects of pb. t his was appa rent in the normal acetylcholine esterase activity and the decrease in malondialdehyde (mda) concentrations in the brain. propolis increased the activities of reduced nicotinamide adenine dinucleotide (nadh), succinate dehydrogenase (sdh) and cytochrome c oxidase in mitochondria.(30) propolis contains cape that has been proven to have neuroprotective effects. cape inhibits neurodegeneration in dopaminergic neurons in the striatum and prevents the loss of dopamine in the 1-methyl-4-phenyl-1,2,3,6tetrahydropyridine (mptp) mouse model of parkinson’s disease. the neuroprotective effect of cape is associated with decreased inducible nitric oxide synthase (inos) and caspase 1 e x pr e s si o n. in vi t r o, ca pe i nh i bi t s t h e neurotoxicity induced by mpp+ (1-methyl-4phenylpyridinium), the neurotoxic oxidation product of mptp, and inhibits the release of cytochrome c and apoptosis inducing factor (aif) from the mitochondria.(15) cape reduces the neurotoxicity due to acrolein in ht22 murine hippocampal neuronal cells. cape inhibits ros production by neuronal cells of the hippocampus as a result of acrolein exposure. therefore, cape has antioxidant effects. administration of cape restores decreased glutathione levels from acrolein exposure in hippocampal neuronal cultures. cape inhibits the activation of p38 and jnk.(31) propolis contains caffeoylquinic acid ( cq a) , a n a nt i o xi d a n t c omp on e nt w i th neuroprotective effect in cultures of 1–42 kuswati, handayani, nugraha propolis a neurodegenerative effect 219 univ med vol. 36 no.3 treated sh-sy5y neuroblastoma cells. cqa increases the viability of 1–42 cells treated with sh-sy5y. cqa increases the expression of glycolytic enzyme mrna (phosphoglycerate kin a s e -1 ; pgk 1) a n d i nt r a c el l ul a r at p concentrations. cqa improves spatial learning and memory in mice.(16) ch r ys i n de c r e a s e s t h e a c t i vi ti e s o f superoxide dismutase and lipid peroxidase, and the levels of nitric oxide, tumor necrosis factor alpha, and interleukin-1β. administration of chrysin inhibits the expression of bax, bcl-2 and caspase-3.(32) the limitation of the present study is that only a histological examination was performed with counting of the number of neuronal cells in the brain and no biochemical analysis that may support the results of this study. the clinical implication of these study results is that propolis may be used to inhibit or prevent neuronal damage due to degeneration caused by stress exposure. further studies should be done to study the pharmacologic effects and the toxicity of propolis in order to support its benefits as a neuroprotective agent in brain tissue. conclusion administration of propolis may inhibit a decrease in neuronal cell count in hippocampal area ca1 and prefrontal cortex in rats with social isolation-induced stress. this study provides useful information for considering propolis as an alter native tre atment cho ice f or ne urode generative disorders. conflict of interest there were no conflicts of interest in the present study. acknowledgment the authors express their gratitude to the research and community service unit, faculty of medicine, islamic university of indonesia, who provided the funds for this study on hippocampal area ca1 through the lecturer grant scheme (skema hibah dosen). they also t ha nk t he di r e c t or a t e f o r res e a r c h a n d co mmu ni t y s e r vic e , un ive r s it a s is l a m indonesia, for the funds to conduct the study on the prefrontal cortex through the starting lecturer grant scheme (skema hibah dosen pemula). contributors k contributed to the design of the study, wrote the protocol and wrote the first draft of the manuscript. zsn contributed to revising the protocol. esh revised the manuscript. all authors read and approved the final manuscript. references 1. kim ej, pellman b, kim jj. stress effects on the hippocampus: a critical review. learn mem 2015; 22:411–6. 2. fitranto a, soejono sk, maurits ls, et al. jumlah sel piramidal ca3 hippocampus tikus putih jantan pada berbagai model stres kerja kronik. mkb 2014;46:197-202. 3. hemmalini, rao s. anti stress effect of centella asiatica leaf extract on hippocampal ca3 neurons a quantitative study. int j pharmacol clin sci 2013;2:25-32. 4. hidalgo acs, munoz mf, herrera aj, et al. chronic stress alters the expression levels of longevity-related genes in the rat hippocampus. neurochem int 2016;97:181-92. 5. arnsten aft. stress signalling pathways that impair prefrontal cortex structure and function. nat rev neurosci 2009;10:410–22. 6. kuswati, prakosa d, wasita b, et al. centella asiatica increases b-cell lymphoma 2 in rat prefrontal cortex. univ med 2015;34:10-6. 7. priyantiningrum ak, kuswati, handayani es. pengaruh ekstrak etanol centella asiatica terhadap jumlah sel neuron di cortex prefrontalis tikus yang diberi perlakuan stres. jkki 2015;6:198204. 8. filipovic d, zlatkovic j, inta d, et al. chronic isolation stress predisposes the frontal cortex but not the hippocampus to the potentially detrimental release of cytochrome c from mitochondria and the activation of caspase-3. j neurosci res 2011;89:1461-70. 220 9. li y, han f, shi y. increased neuronal apoptosis in medial prefrontal cortex is accompanied with changes of bcl-2 and bax in a rat model of posttraumatic stress disorder. j mol neurosci 2013;51: 127-37. 10. bachis a, cruz mi, nosheny, rl, et al. chronic unpredictable stress promotes neuronal apoptosis in the cerebral cortex. neurosci lett 2008;442:104-8. 11. djorjevic a, adzic m, djorjevic j, et al. chronic social isolation suppresses proplastic response and promotes proapoptotic signaling in prefrontal cortex of wistar rats. j. neurosci res 2010;88:252433. 12. djorjevic a, djorjevic j, elakovic i, et al. effect of fluoxetine on plasticity and apoptosis evoked by chronic stress in rat prefrontal cortex. eur j pharmacol 2012;693:37-44. 13. cao m, pu t, wang l, et al. early enriched physical environment reverses impairments of the hippocampus, but not medial prefrontal cortex of socially-isolated mice. brain behav immun 2017; 64:232-3. 14. de menezes da silveira ccs, fernandes lmp, silva ml, et al. neurobehavioral and antioxidant effects of ethanolic extract of yellow propolis. oxidative med cell longevity 2016, article id 2906953, 14 pages. 15. fontanilla cv, ma z, wei x, et al. caffeic acid phenethylester prevents 1-methyl-4-phenyl1 , 2 , 3 , 6 t e t r a h y d r o p y r i d i n e i n d u c e d neurodegeneration. neuroscience 2011;188:135– 41. 16. han j, miyamae y, shigemori h, et al. neuroprotective effect of 3,5-di-o-caffeoylquinic acid on sh-sy5y cells and senescenceaccelerated-prone mice 8 through the upregulation of phosphoglycerate kinase-1. neuroscience 2010;169:1039–45. 17. izuta h, shimazawa m, tazawa s, et al. protective effects of chinese propolis and its component, chrysin, against neuronal cell death via inhibition of mitochondrial apoptosis pathway in sh-sy5y cells. j agrict food chem 2008;56:8944-53. 18. jeong ch, jeong hr, kim do, et al. phenolics of propolis and in vitro protective effects against oxidative stress induced cytotoxicity. j agric life sci 2012;46:87-95. 19. kure c, t immer j, stough c. t he immunomodulatory effects of plant extracts and plant secondary metabolites on chronic neuroinflammation and cognitive aging: a mechanistic and empirical review. front pharmacol 2017;8:117. 20. saad ma, abdel salam rm, kenawy sa, et al. pinocembrin attenuates hippocampal inflammation, oxidative perturbations and apoptosis in a rat model of global cerebral ischemia reperfusion. pharmacol rep 2015;67: 115–22. 21. he xl, wang yh, bi mg, et al. chrysin improves cognitive deficits and brain damage induced by chronic cerebral hypoperfusion in rats. eur j pharmacol 2012;680:41–8. 22. charan j, biswas t. how to calculate sample size for different study designs in medical research? indian j psychol med 2013;35:121–6. 23. lee ms, kim yh, park ws. novel antidepressantlike activity of propolis extract mediated by enhanced glucocorticoid receptor function in the hippocampus. evidence-based complementary altern med 2013, article id 646039, 10 pages. 24. zlatkovic j, filipovic d. bax and b-cell-lymphoma 2 mediate proapoptotic signaling following chronic isolation stress in rat brain. neuroscience 2012;223:238-45. 25. alkis he, kuzhan a, dirier a, et al. neuroprotective effects of propolis and caffeic acid phenethyl ester on the radiation-injured brain tissue. intern j radiat res 2015;3:297-303. 26. reis jss, oliveira gb, monteiro mc, et al. antidepressantand anxiolytic-like activities of an oil extract of propolis in rats. phytomedicine 2014;21:466–72. 27. chen j, long y, han m, et al. water-soluble derivative of propolis mitigates scopolamineinduced learning and memory impairment in mice. pharmacol biochem behav 2008;90:441–6. 28. bhadauria m. combined treatment of hedta and propolis prevents aluminum induced toxicity in rats. food chem toxicol 2012;50:2487–95. 29. cardoso sm, ribeiro m, ferreira il, et al. northeast portuguese propolis protects against staurosporine and hydrogen peroxide-induced neurotoxicity in primary cortical neurons. food chem toxicol 2011;49:2862–68. 30. el-masry ta, emara am, shitany na. possible protective effect of propolis against lead induced neurotoxicity in animal model. j evol biol res 2011;3: 4-11. 31. huang y, jin m, pi r, et al. protective effects of caffeic acid and caffeic acid phenethyl ester against acrolein-induced neurotoxicity in ht22 mouse hippocampal cells. neurosci lett 2013;535: 146–51. 32. nabavi sf, braidy n, habtemariam s, et al. neuroprotective effects of chrysin: from chemistry to medicine. neurochem int 2015;90: 224-31. kuswati, handayani, nugraha propolis a neurodegenerative effect oktavianus 65 universa medicina may-august, 2016 vol.35 no.2 the burden of the complications of diabetes mellitus adi hidayat department of community medicine faculty of medicine, trisakti university diabetes mellitus (dm) is an important global cause of mortality, morbidity, and healthsystem costs,(1,2) with significant increases in its prevalence and number of cases in the last 30 years. it constitutes one of the four non communicable diseases (ncds) targeted for action by world leaders.according to who estimates, in 2014 there were 422 million adults with dm aged 18 years or older, with around half in the who south-east asia and western pacific regions.(3) the prevalence of dm and the number of adults with dm in lowand middle-income countries have over the last decade increased at a greater rate than in high-income countries. the global dm prevalence increased by more than 100% in men and by 60% in women, resulting in a predominance of males with dm in 2014 as compared with a higher dm female prevalence in 1980.(4) in 2012, there were a total of 3.7 million deaths, of which 34% were in age groups below 70 years. of these 3.7 million deaths, 1.5 million were caused by dm and 2.2 million by poorly controlled blood glucose, which increased the risks of cardiovascular and other diseases. the percentage of deaths under the age of 70 years caused by high blood glucose is higher in lowand middle-income countries than in high-income countries.(3) in indonesia, the 2007 and 2013 data reported by the basic health research showed a prevalence of dm of 6.9% for 2013, which is twice its prevalence in 2007.(5) according to the noncommunicable diseases risk factor collaboration (ncd-risc), the onset of type 2 dm should be prevented or delayed to reduce its global health and economic impact, since type 2 dm patients constitute the majority of cases worldwide. overweight, obesity, and lack of physical activity account for a large proportion of the global burden of dm.(6) the burden of dm increases in proportion to its complications and therefore control measures are an important goal for the prevention of the complications of dm. a number of factors may be noted that may be responsible for the higher increase in dm prevalence in low-income and middle-income countries as compared with high-income countries and the almost stationary dm prevalence in europe, particularly in the northwester parts. one factor is the substantial and relatively greater increase in adiposity as an important risk factor for dm in many low-income and middle-income countries than in europe and high-income asia pacific countries, especially in women.(7) another factor may be genetic susceptibility to dm or the occurrence of alterations in phenotype that are the result of inadequate fetal and childhood nutrition and growth.(4,8) a third factor may be that the better resourced health systems of europe and other high income countries are able to effect an earlier diagnosis of dm in high risk individuals, and use lifestyle and dietary modification or drugs to prevent or delay its onset.(9) the complications of poorly controlled dm frequently affect the patient’s health adversely and are commonly life-threatening, with acute complications significantly contributing to 66 universa medicina mortality, costs and poor quality of life. in the long term dm may result in cardiovascular, ocular, renal and neurologic disorders.(11) in regard to dm complications such as end-stage renal disease, cardiovascular events, lower-extremity amputations or pregnancy complications, there are no global estimates, although these conditions impact significantly at individual and population levels, making dm a serious threat to population health.(3) dm and its complications cause substantial economic loss to patients and their families, either directly through medical costs such as increasingly higher out-of pocket health-care payments, or indirectly through loss of earnings, associated with loss of productivity from disability and premature mortality. the high costs of care increase the risk of catastrophic medical expenditure.(10) this economic burden also affects the national and global health systems and economies, with a negative impact on the national gross domestic product (gdp)(3,10) the international diabetes federation (idf) estimates that in the period between 2003 and 2013 the total global healthcare expenditure on dm increased more than three times as a result of the increased number of individuals with dm and increased per capita expenditure on dm.(11) therefore population-based interventions are urgently needed, i.e. to prevent dm, increase the number of dm cases that are detected earlier, and prevent or delay the complications of dm through lifestyle and pharmacological interventions. ultimately, whether or not it is advisable to institute targeted interventions for high risk individuals, depends largely on the local context, such the availability of human and financial resources, as well as essential equipment and technology, to manage dm at the primary-care level. ideally, dm management should be part of ncd management and included in a universal health coverage package of essential services. dm management remains a top priority issue for the more than 420 million dm cases, although prevention is of importance. the who recommends a multidisciplinary approach, comprising patient education, medication, and consistent follow up. in addition, structured education programs, such as dose adjusted for normal eating (dafne) for individuals with type 1 dm, may improve life expectancy, dm related complications, and hypoglycemic awareness, and reduce the financial burden of disease management.(12) there is also an urgent need for structured self-management programs for delivery to large numbers of individuals, since 80–90% of cases are type 2 dm, which is linked to obesity and lifestyle. other measures include strengthening the primary healthcare system response to ncds, including diabetes and implementation of guidelines and protocols for improved diagnosis and management of dm in primary health care. furthermore, policies and programs should be established to ensure equal opportunties for accessing essential diagnostic and management technologies. finally, essential medicines such as human insulin should be made available and affordable to all who need them. the most important modifiable risk factors for type 2 dm are overweight, obesity, lack of physical activity and unhealthy dietary practices. smoking also increases the risk of both type 2 dm and its complications. control of these factors may decrease the occurrence of type 2 dm and reduce the complications of all types of dm. apart from promotion of a healthy diet and physical activity, reducing cigarette exposure will reduce dm complications and may result in reductions in type 2 dm. preventive measures for dm, such as life style changes, should be widely disseminated, particularly for highrisk individuals. these efforts may steadily reduce the complications of dm and the burden to be borne by the patient, the family, and the government. references 1. global burden of metabolic risk factors for chronic diseases collaboration. cardiovascular disease, chronic kidney disease, and diabetes mortality burden of cardiometabolic risk factors 67 from 1980 to 2010: a comparative risk assessment. lancet diabetes endocrinol 2014;2: 634–47. 2. seuring t, archangelidi o, suhrcke m. the economic costs of type 2 diabetes: a global systematic review. pharmacoeconomics 2015;33: 811–31. 3. world health organization. global report on diabetes. geneva: world health organization; 2016. 4. wandell pe, carlsson ac. gender differences and time trends in incidence and prevalence of type 2 diabetes in sweden—a model explaining the diabetes epidemic worldwide today? diabetes res clin pract 2014;106:e90–2. 5. pusat data dan informasi kementerian kesehatan r.i. situasi dan analisis diabetes. jakarta; pusat data dan informasi kementerian kesehatan r.i;2015. 6. gbd 2013 risk factors collaborators. global, regional, and national comparative risk assessment of 79 behavioural, environmental and occupational, and metabolic risks or clusters of risks in 188 countries, 1990–2013: a systematic analysis for the global burden of disease study 2013. lancet 2015;386:2287–323. univ med 2016;35:1-2. doi: http://dx.doi.org/10.18051/univmed.2016.v35.65-67 7. ncd risk factor collaboration (ncd-risc). trends in adult body-mass index in 200 countries from 1975 to 2014: a pooled analysis of 1698 population-based measurement studies with 19.2 million participants. lancet 2016;387:1377–96. 8. alharbi ns, almutari r, jones s, et al. trends in the prevalence of type 2 diabetes mellitus and obesity in the arabian gulf states: systematic review and meta-analysis. diabetes res clin pract 2014;106:e30–33. 9. farzadfar f, murray cj, gakidou e, et al. effectiveness of diabetes and hypertension management by rural primary health-care workers (behvarz workers) in iran: a nationally representative observational study. lancet 2012; 379:47–54. 10. smith-spangler cm1, bhattacharya j, goldhaber-fiebert jd. diabetes, its treatment, and catastrophic medical spending in 35 developing countries. diabetes care 2012;35: 319–26. 11. international diabetes federation. diabetes atlas, 6th ed. brussels, international diabetes federation;2014. 12. mcintyre hd. dose adjustment for normal eating: a role for the expert patient? diabetes metab j 2014;38:87-91. http://dx.doi.org/ 10.4093/dmj.2014.38.2.8. may-august, 2016 vol.35 no.2 oktavianus 1 universa medicina january-april, 2015 vol.34 no.1universa medicina january-april, 2015 vol.34 no.1universa medicina january-april, 2015 vol.34 no.1universa medicina january-april, 2015 vol.34 no.1universa medicina january-april, 2015 vol.34 no.1 benefits of smoking cessation for coronary heart disease patients adi hidayat department of community medicine, faculty of medicine, trisakti university cardiovascular disease (cvd) incidence increases with age and is frequently higher in the elderly.(1) therefore prevention of cvd in the elderly through management of risk factors is important in order to reduce the risk of coronary heart disease (chd). there are several risk factors of cvd that can be modified, such as smoking, physical activity, and unhealthy diet. cessation of smoking is the most potent measure to prevent thousands of cvd events and deaths.(2) smoking is a modifiable major risk factor that causes several types of disease resulting in death. various epidemiological studies in the last five decades have demonstrated a causal relationship between smoking on the one hand and chd and mortality on the other.(3) during these decades mortality from cvd had started to decrease in developed countries with the reduction in the number of smokers, but cvd is still the main cause of death. one study found that cessation of smoking in the elderly is still of benefit.(4) a metaanalytical study involving 503,905 subjects aged 60 years and older showed a significant association between smoking status and cvd mortality in smokers, with a hazard ratio of 2.07 (95%ci: 1.82 – 2.36) for current smokers and 1.37 (95%ci: 1.25-1.49) for former smokers in comparison with never-smokers.(5) the risk in smokers increased with increasing cigarette consumption in a dose-response manner, and decreased continously with time in former smokers after they ceased to smoke. an european study on 1,439 subjects aged ≥40 years who smoked >10 cigarettes per day showed a consistently high cv risk from smoking on the basis of the prediction models employed. according to the systematic coronary risk evaluation (score) model, the probability of death from cvd in the next 10 years was twice that of non-smokers.(6) consistent study results showing increased risks relative to cvd occurrence and mortality have become more important for eliminating smoking as the source of these studies and cvd m o r t a l i t y, t h r o u g h s m o k i n g c e s s a t i o n programs.(7) cessation of smoking as early as possible is associated with increased survival rate and quality of life, and reduces health care costs for smoking-related diseases. the principal benefit o f s m o k i n g c e s s a t i o n l i e s i n i m p r o v e d cardiovascular health.(8) communication on the risks of smoking and on the benefits of cessation of smoking is the most effective promotion effort. in order to eliminate the health and economic b u r d e n o f c v d , f u t u r e c o u n t r y s p e c i f i c interventions on smoking cessation should be based on improved community-based smoking cessation programs at the primary health level. s u c h m e a s u r e s a s c o u n s e l i n g a n d pharmacotherapy can be used by primary health practitioners to influence the results of smoking 2 cessation efforts. simple advice from gps has been shown to substantially increase smoking cessation rates.(9) references 1. riekard p, gharacholou sm, peterson ed. primary and secondary prevention of cardiovascular disease in older adults: a status report. clin geriatr med 2009;25:745–55. 2. aung mn, yuasa m, lorga t, et al. evidencebased new service package vs. routine service package for smoking cessation to prevent high risk patients from cardiovascular diseases (cvd): study protocol for randomized controlled. trials 2013;14:419-32. 3. us department of health and human services. the health consequences of smoking 50 years of progress: a report of the surgeon general. atlanta, ga: us department of health and human services, centers for disease control and prevention, national center for chronic disease prevention and health promotion, office on smoking and health, 2014. 4. gellert c, schottker b, muller h, et al. impact of smoking and quitting on cardiovascular outcomes and risk advancement periods among older adults. eur j epidemiol 2013;28:649–58. 5. mons u, muezzinler a, gellert c, et al. impact of smoking and smoking cessation on cardiovascular events and mortality among older adults: meta-analysis of individual participant data from prospective cohort studies of the chances consortium. bmj 2015;350:h1551. doi:10.1136/bmj.h1551. 6. mallaina p, lionis c, ro h, et al. smoking cessation and the risk of cardiovascular disease outcomes predicted from established risk scores: results of the cardiovascular risk assessment among smokers in primary care in europe (cvaspire) study. bmc public health 2013,13: 362. 7. gellert c, schottker b, muller h, et al. impact of smoking and quitting on cardiovascular outcomes and risk advancement periods among older adults. eur j epidemiol 2013;28:649–58. 8. mills ej, thorlund k, eapen s, et al. cardiovascular events associated with smoking cessation pharmacotherapies: a network metaanalysis. circulation 2014;129:28–41. doi:10. 1161/circulationaha.113.003961. 9. sadowski j, ruffieux c, cornuz j. self-reported smoking cessation activities among swiss primary care physicians. bmc fam pract 2009; 10:22. universa medicina january-april, 2015 vol.34 no.1 univ med 2015;34:1-2. doi: http://dx.doi.org/10.18051/univmed.2015.v34.1-2 c:\users\jurnal fkusakti\docume 179 *department of biochemistry and medical chemistry, faculty of medicine, sriwijaya university **medical study program, faculty of medicine, sriwijaya university ***medical study program, faculty of medicine, sriwijaya university †department of biochemistry and medical chemistry, faculty of medicine, sriwijaya university correspondence: subandrate department of biochemistry and medical chemistry, faculty of medicine, sriwijaya university jl. dr. m. ali komp. rsmh km 3,5 palembang sumsel 30126 phone and fax: +62711-373438 email: subandrate@unsri.ac.id date of first submission, june 20, 2017 date of final revised submission, november 8, 2017 date of acceptance, november 10, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 malondialdehyde levels are higher and glutathione levels are lower in preeclampsia than in normal pregnancies subandrate*, mia esta poetri afdal faisal**, nurul windi anggraini***, and sadakata sinulingga† background maternal mortality rate is still a health problem in indonesia. one major contributor to maternal mortality rate in indonesia is preeclampsia. one widely accepted theory is that preeclampsia is caused by oxidative stress. placental hypoxia or ischemia among preeclampsia patients is thought to be the cause of the formation of free radicals such as malondialdehyde (mda), which decreases endogenous antioxidants such as glutathione (gsh). t his stud y a ims to a sc er tain the diffe r enc e in plasma malo nd ia ld ehyd e and glutathione leve ls be twee n he a lth y a nd preeclamptic pregnant women. methods this was an observational analytic study of cross sectional design. research subjects were 30 normal (healthy) pregnant women (np), and 30 pre-eclamptic pregnant (pe) women. the measurement of plasma mda and gsh levels was done at the biochemistry and molecular biology laboratories, faculty of medicine, sriwijaya university using sigma-aldrich mda and gsh assay kits. mann whitney test was used to analyze the data. results subjects aged >35 years, with age of gestation >35 weeks and multipara was significantly higher in the pe group compared to the np group (p=0.016; p=0.01 and p=0.36; respectively). mda level was significantly higher in the pe group than in the np group (p=0.002). in contrast, gsh level was significantly lower in the pe group than in the np group (p=0.003). conclusion mda and gsh may reflect vascular complications of pe, and the ensuing increases in lipid peroxidation may play important pathogenic roles. keywords: free radical, gsh, normal pregnant, mda, preeclampsia doi: http://dx.doi.org/10.18051/univmed.2017.v36.179-186 original article cite this article as: subandrate, faisal meaf, anggraini nw, et al. malondialdehyde levels are higher and glutathione levels are lower in preeclampsia than in normal pregnancies. uni v med 20 17;3 6:17 9-86 . doi: 1 0 . 1 8 0 5 1 / un i vm ed . 2 0 1 7 . v 3 6 . 1 7 9 1 8 6 pissn: 1907-3062 / eissn: 2407-2230 180 subandrate, faisal, anggraini, et al malondialdehyde and glutathione in preeclampsia introduction the government and the obstetrics experts a n d pe r s on ne l i n ind o ne s i a a r e a lw a ys cooperating to improve the quality of public health care services. one of the parameters of success in obstetric service is the maternal mortality rate (mmr), as one of the sustainable development goals (sdg) of the who, i.e. a decrease in mmr below 70 per 100,000 live births, to be achieved up to the year 2030.(1) the results of the indonesian basic health survey (survei dasar kesehatan indonesia, sdki) in 2012 showed that the indonesian mmr actually increased to 359 per 100,000 live births.(2) one of the largest contributors to the mmr in developing countries such as indonesia is preeclampsia, in addition to eclampsia, bleeding and sepsis.(1,3) the mmr of south sumatra is still according to the results of the indonesian national socio-economic survey (survei sosial ekonomi nasional, susenas) for the year 2005, i.e. 262 per 1000 live births,(2) wh e r e a s t h e t a r ge t mm r of t h e w ho millennium development goals (mdgs) for 2015 is 102/100.000 live births.(1,3) preeclampsia is a syndrome encountered from the twentieth week of pregnancy up to 48 hours after delivery, and is characterized by hypertension and proteinuria.(4) the us national high blood pressure education progra m working group classifies preeclampsia as one of the categories of hypertension in pregnancy and uses the criterion of an increase in blood pressure of >140/90 mmhg with proteinuria of >300 mg in 24-hour urine.(4,5) preeclampsia is “the disease of theories”, because the initial cause of preeclampsia is not yet known with certainty. the ischemic placenta theory is one o f t h e h yp o th e s e s o n t he e ti ol o gy o f preeclampsia that is accepted by the experts.(4) the ischemic placenta theory explains that failure of trophoblastic invasion into the spiral a r t e r i e s le a d s t o a bn o r ma l iti e s o f th e uteroplacental blood supply so that there is diminished uteroplacental perf usion that ultimately causes the ischemic-hypoxic placenta to produce an excess of free radicals in the maternal circulation.(4,6,7) the increase in free radicals drastically shifts the normal condition to oxidative stress, in which the amount of free radicals in the body exceeds the neutralizing capacity of the body. oxidative stress is toxic and damages lipids, proteins, and dna.(7-9) oxidation of lipids by free radicals produces malondialdehyde (mda). to date mda is considered to be a good in-vivo oxidative stress marker.(8,10) oxidative stress actually does not occur if there is a balance between oxidants and antioxidants. one of the antioxidants that plays a role in quenching free radicals is reduced glutathione (gsh) that acts by preventing the formation of oxygen radicals.(11,12) oxidative stress in a pregnancy complicated by preeclampsia is thought to c ause an incre ase in mda concentration and a reduction in gsh concentration. this results in damage to the vascular endothelium and vital target organs.(12) several studies have demonstrated the occurrence of oxidative stress in placentas of patients with preeclampsia.(12-14) the study conducted by chandra et al.(11) shows that mean mda increases and mean gsh decreases significantly in preeclampsia, as compared with normal pregnancy. the study of suhail et al.(12) shows that increased mda concentration and decreased gsh concentration in preeclampsia causes fragility of the red blood cells. these studies differ from those carried out by bowen et al.(15) and dordevic et al.(16) bowen et al.(15) state that there is no reduction in antioxidants or an increase in lipid peroxidation (mda) in preeclampsia a s co mpared with normal pregnancy. the study carried out by dordević et al.(16) also shows that there is no difference in antioxidant (gsh) concentration between women with nor mal pr egna nc y and those with preeclampsia. since the results of these studies are still inconsistent concerning the differences in mda and gsh conce ntrations between nor mal pregnancies and preeclampsia, further studies 181 are necessary. the aim of the present study was to determine the differences in plasma gsh and mda concentrations between women with preeclamptic pregnancy (pe) and those with normal pregnancy (np). methods research design the present study was an observational analytic study of cross sectional design that was carried out in the department of obstetrics and gynecology of muhammadiyah palembang hospital, from september 2016 to february 2017. study subjects the study subjects were all pregnant women with preeclampsia and normal pregnant wo me n wh o ca me for tr eat me nt or we re hospitalized in the department of obstetrics and gyne col ogy, m uh a mma di yah pa l emban g hospital, who met the inclusion criteria and did not have the exclusion criteria of the study. the inclusion criteria were pregnant women aged 1840 years with a single live fetus, age of gestation of 20-40 weeks, and diagnosed as preeclampsia or normal pregnancy. the exclusion criteria were pregnant women with complications of other diseases (renal disorders, chronic hypertension, diseases of the liver, antepartum hemorrhage, heart disease and diabetes mellitus), smokers and alcohol consumers. sample size determination the sample size was determined with the f o r mu la f or t wo po p ul a t i o n me a n s , i . e . n 1 =n 2 =2[(z α +z β )s/(x 1 -x 2 )]2, where the value of z α is 5%, the value of z β is 10%, the value of s = 0.71 nmol/ml, and the value of x1-x2 = 0.54 nmol/ml.(11) the resulting sample size was 30 pregnant patients with preeclampsia and 30 normal pregnant patients. the selection of the sample for this study was by non-random consecutive sampling. in which all subjects who me t t h e st ud y c r i t e r i a we r e s e l e c t e d consecutively until the sample size was reached. the willingness of the patients to participate in the study was given by informed consent after the patients received clarification on the benefits and discomforts to the patients who participated in this study. data collection data collection was carried out by directly interviewing the patients and by looking at the data in the medical records using a check list with the patient’s name (initials), age, first day of the last menstrual period (lmp), age of pregnancy, parity, and diagnosis. the diagnosis of pregnancy with preeclampsia had been established by specialists in obstetrics and gynecology for those patients who met the conditions of age of pregnancy of more than 20 weeks, systolic blood pressure above 140 mmhg or diastolic blood pressure above 90 mmhg, and proteinuria.(4) laboratory analysis collection of laboratory samples was performed by drawing 3 ml of blood from the median cubital vein of the study subject and placing the blood sample in an ethylene-diamine tetra-acetic acid (edta)-containing blood collecting tube. the collected blood sample was centrifuged for 10 minutes at 600g, and part of the separated plasma was taken and placed in a l a be l e d t ube a nd st or e d a t -2 0 oc bef or e d e t e r mi na t i o n o f t h e m da a nd gsh concentrations. determination of the plasma mda and gsh concentrations was carried out in the biochemistry and molecular biology laboratories, faculty of medicine, sriwijaya university, using the procedures of the sigmaaldrich mda and gsh assay kits. the plasma mda concentration was determined biochemically using the sigmaaldrich mda assay kit. a volume of 10 µl plasma was mixed with 500 µl h 2 so 4 42 mm and 125 µl phosphotungstic acid. the mixture wa s vor t exe d a n d in c u ba te d a t r oo m temperature for 5 minutes, then centrifuged at univ med vol. 36 no.3 182 subandrate, faisal, anggraini, et al malondialdehyde and glutathione in preeclampsia a b c 13,000 g for 3 minutes. the precipitate was taken and resuspended in a solution of butylated hydroxytoluene (bht) in distilled water up to 200 µl. after that, 600 µl of thiobarbituric acid (tba) solution was added to the suspension and incubated at 95°c for 60 minutes. the mixture was cooled in ice water for 10 minutes. a volume of 200 µl of the mixture was placed on a 96well plate and its absorbance read with a spectrophotometer at a wavelength of 532 nm. the mda concentration in nmol/μl was then determined using the mda standard curve. t he pla sma gsh c onc en tr at io n wa s determined biochemically using the sigmaaldrich gsh assay kit. a volume of 200 µl plasma was mixed with 200 µl ssa 5%. the mixture was vortexed and left for 10 minutes at 2–8 °c. after that, the mixture was centrifuged at 10,000 g for 10 minutes. then, a volume of 10 µl of supernatant was taken and mixed with 150 µl working mixture in a 96-well plate. the mixture was incubated for 5 minutes at room temperature then 50 µl of nadph solution was added. then the absorbance was measured by spectrophotometer at a wavelength of 412 nm. the gsh concentration in μmol/ml was then determined using the gsh standard curve. statistical analysis the statistical analyses used were the mann-whitney test and the chi square test. significancy was considered for p<0.05. ethical clearance this study was carried out after obtaining ethical clearance from the unit on bioethics and humanities unit, faculty of medicine, sriwijaya university/rsmh palembang, under nos. 194 and 324/kepkrsmhfkunsri/2016. results the age of the pregnant women who were the study subjects varied from 18 to 40 years. in the pe group the number of subjects aged > 35 years was 7 (23.3%), which was significantly higher than the number of np subjects of 2 (6.7%) (p=0.016). mean age of gestation for all study subjects was 33.07 ± 5.89 weeks. the number of subjects with a pregnancy of >33 weeks was significantly greater in the pe group [26 (86.7%)] as compared with that in the np group [9 (30.0%)] (p=0.001). mean parity of all study subjects was 1.13 ± 1.00. the number of multipara were significantly higher in the pe group [10 (33.3%)] as compared with that in the np group [5 (16.7%)] (p=0.036). the demogr aphic characteristics and clinical features of the study subjects can be seen in table 1. in the present study the median, minimum, and maximum plasma mda concentration in the np group were 18.29, 8.80 and 21.93 nmol/μl, respectively. these values were significantly lower than those in the pe group, i.e. 20.66, 7.19 variable normal pregnancy (n=30) preeclampsia (n=30) p value age (years) <20 20-35 >35 4 (13.3) 24 (80.0) 2 (6.7) 0 (0.0) 23 (76.7) 7 (23.3) 0.016 age of gestation (weeks) 20-28 28-33 >33 12 (40.0) 9 (30.0) 9 (30.0) 0 (0.0) 4 (13.3) 26 (86.7) 0.001 parity nullipara primipara multipara 10 (33.3) 15 (50.0) 5 (16.7) 6 (20.0) 14 (46.7) 10 (33.3) 0.036 table 1. demographic and clinical features of the study groups *chi square test, significant if p<0.05; data are presented as n (%) 183 and 49.98 nmol/μl, respectively (p<0.002). the median, minimum, and maximum plasma gsh concentration in the np group were 11.46, 9.74 and 16.40 μmol/ml, respectively, which were significantly higher than those in the pe group, i.e. 10.15, 7.34, and 11.96 μmol/ml. respectively ( p = 0 .0 03) . t he p la sma m da a n d gsh concentrations can be seen in table 2. discussion the age of the subjects in the np and pe groups was still within the recommended reproductive age range i.e. 20 to 35 years. in the present study there was a tendency for preeclampsia to occur at a higher age than 35 years. at the age above 35 years, there is cellular degeneration causing structural and functional vascular changes so that the subjects are more susceptible to preeclampsia.(4,17) our study resul ts agree with those of the stu dy by lamminpaa et al.(17) in finland, who state that pregnant women of higher age (35 years) have a 1.5-fold greater tendency to suffer from preeclampsia as compared with pregnant women who are under 35 years of age. lisonkova and joseph state in their study that extreme age of women (<20 years or >35 years) is associated with the incidence of preeclampsia.(18) in our study, we found that preeclampsia occurred more frequently at a gestational age nearing full term. this finding is in line with the study of lisonkova and joseph, who concluded that >80% of preeclampsia cases were late onsetp r e e cl a mp si a ( lop) . ( 1 8 ) t h e ma j or i t y o f preeclampsia cases do occur near full term, but around 10% of cases occur before the 34th week of gestation. early-onset preeclampsia (eop) is pe o ccur r ing be fo re the 34 th wee k of gestation, whereas lop occurs at or after the 34th week of gestation.(4,19) increased age of gestation results in increased nutritional de mand s by th e f etus an d he modynamic changes in the mother. this may lead to vascular resistance so increasing the probability of placental hypoxia. advanced placental hypoxia causes the release of many toxic substances so that preeclampsia occurs.(18,19) our study shows that there were more multipara in the pe group than in the np group. preeclampsia tends to occur in nullipara and primipara rather than in multipara, because in the first pregnancy the formation of antibodies against placental antigens is incomplete so that the implantation of trophoblasts into the uterus is incomplete.(4,20) these results are in line with the study by paré et al.,(20) who state that multiparity is a risk factor for preeclampsia. in our study, the median plasma mda concentration in the preeclampsia group was significantly higher than in the normal pregnancy group. the study carried out by bakacak et al.(21) in turkey revealed a median plasma mda concentration that did not differ much from results of the present study, where the median plasma mda concentration in the preeclamptic pregnancy group was found to be higher than that of the group with normal pregnancies. consistent results were shown in the study of subakir et al.(22) who state that plasma mda concentration in patients with preeclampsia is significantly higher as compared with the control group. other studies carried out by chandra et al.(11)and rafeeinia et al.(23) also show similar univ med vol. 36 no.3 variable normal pregnancy (n=30) preeclampsia (n=30) p value mda (nmol/μl) median (min-max) 18.29 (8.80-21.93) 20.66 (7.19-49.98) 0.002 gsh (μmol/ml) median (min-max) 11.46 (9.74-16.40) 10.15 (7.34-11.96) 0.003 table 2. plasma mda and gsh concentrations in normal pregnancy and preeclampsia *mannwhitney test, significant if p<0.05, data presented as median range (minimum – maximum) 184 subandrate, faisal, anggraini, et al malondialdehyde and glutathione in preeclampsia results, i.e. mean plasma mda concentration in patients with severe preeclampsia turns out to be significantly different from the mean plasma mda concentration in normal pregnant women. however, different results were shown by bowen et al.(15) who state that there were no increases in lipid peroxidation products (mda) in women with preeclampsia and eclampsia as compared with normal pregnant women. t he ox i da ti ve st re ss un de r lyi n g t he occurrence of systemic inflammation in patients with preeclampsia indicates that there is an imbalance between free radic als and the antioxidant defense mechanism in the course of pregnancy.(6,19) the median plasma gsh concentration in the preeclampsia group was significantly lower than that in the group with normal pregnancy, indicating that there is more extensive oxidative stress in preeclamptic pregnancy. reactive oxygen species cause decreases in antioxidants in the cells.(12,17) similar results were shown in the study of atamer et al.(24) who state that lipid peroxides are significantly increased in pe while antioxidants are sign if ica ntly decr eased. however, different results were shown in the study by dordević et al.(16) showing that there is no difference in antioxidant concentration between normal pregnant women and women with preeclampsia. in pregnancies complicated with preeclampsia, the antioxidant glutathione (gsh) concentration decreases. oxidative stress may change the gsh to glutathione disulfide, oxidized glutathione (gssg) ratio, causing a shift of the gssg reduction-oxidation buffer towards gsh. in severe preeclampsia there is a decrease in the gsh/gssg ratio used to neutralize the increased mda concentration.(10-12) the placenta is known to be the main site of lipid peroxide production in patients with preeclampsia. the hydroxyl radical, the most prevalent radical found, is a very effective substance to effect peroxidation of the plasma membrane of various structures that are rich in the double-bonds of polyunsaturated fatty acids (pufas) .(6,7) pre eclampsia is a secondary systemic inflammation due to oxidative stress in the placenta, and its occurrence is associated with remodeling failure of the spiral arteries. oxidative stress occurring in patients with preeclampsia is probably due to ischemiar e p e r f us i on i n j ur y i n t h e p la ce n ta , t h a t subse quently leads t o r ele ase of var ious placental factors into the systemic circulation and ultimately induces the clinical manifestations o f p r e e c l amp s i a . imme d i a t e l y a f t e r t he occurrence of reperfusion injury in the placenta, the blood flow becomes adequate again and causes the release of various cytokines and other proinflammatory factors and reactive oxygen species (ros) particularly superoxides in very large amounts. ros, in this case superoxides, a r e e xt r e me l y r e a c t i ve a nd ha z a r d ou s substances, where ros may star t a lipid oxidation chain reaction that forms more free radicals, and causes the lipid peroxidation process to becomme more extensive.(6,7) another source of potential oxidative damage in patients wi t h pr e e c l a mps i a i s th e r e le a s e of autoantibodies against the angiotensin receptor (at1). these autoantibodies stimulate nadph oxida se, subseq uen tly ca usi ng i ncr eas ed production of ros. in addition, the placenta is also rich in macrophages, that may facilitate the overproduction of local free radicals in the placenta, such as reactive chlorine species (rcls).(6-8) in patients with preeclampsia placental angiogenc activity is significantly lower than in the placenta of normal pregnancy.(6,18) other studies are similar, as shown by the significant decreases in the mean value of endogenous a n t i ox i dan t s su c h a s gsh , su pe r ox i de dismutase, and glutathione peroxidase in p r e e c l a mp s ia c a se s a s c o mp a r e d wi t h normotensive pregnant women.(25) the significant difference between plasma mda and gsh concentration in preeclampsia and normal pregnant women shows the potential of using mda and gsh for early detection of 185 p r e e c l a mpt ic pr e gn a n c y so tha t e a r l y intervention may be performed to prevent p r e e c l a mpsi a or t he ma ni f e st a t i o n of complications. in addition, antioxidant therapy or a high-antioxidant diet may be given to pregnant women at high risk for preeclampsia. one of the limitations of this study is the cross sectional research design, so that the study c a n n ot d et e r mi ne a n y c a us e -a n d-e f f e c t association. further studies should be conducted of a prospective design, such as a cohort design. in addition, in this study there was no controlling for variables that possibly play a role in the pathophysiology of preeclampsia. there is a need to carry out studies involving diet, nutritional status, family history, history of pregnancy and other risk factors that may cause preeclampsia, to determine the possible mda and gsh concentrations that may be used as marker for the appropriate evaluation of the risk factors of preeclampsia. conclusions t he gr oup of pr e gn a n ci e s wi t h preeclampsia has a higher mda concentration and a lower gsh concentration as compared with the group with normal pregnancies. there is clear evidence for a key role of oxidative stress in preeclampsia. conflict of interest all authors have no conflict of interest in this study. acknowledgement we express our gratitude to the dean of the faculty of medicine, sriwijaya university (fk unsri) who provided the funds for the study in the form of a research grant for lecturers at fk unsri. thanks are also due to dr. dr. hj. aryani azis, sp.og(k) who facilitated the establishment of diagnoses and collection of samples at muhammadiyah palembang hospital. contributors s contributed to drafting of the manuscript. s, mepaf, nwa and ss contributed to data acquisition, analysis and/or interpretation. s contributed to revising the manuscript critically for important intellectual content. all authors read and approved the final manuscript. references 1. world health organization. maternal mortality. geneva: world health organization;2015. 2. statistics indonesia, national population and family planning board, kementerian kesehatan, and icf international. indonesia demographic and health survey 2012. jakarta, indonesia: bps, bkkbn, kemenkes, and icf international;2013. 3. say l, chou d, gemmill a, et al. global causes of maternal death: a who systematic analysis. the lancet global health 2014;2:323-33. doi: 10.1016/ s2214-109x(14)70227-x. 4. cunningham fg, gant nf, leveno kj, et al. obstetri williams. edisi ke-23. jakarta: egc;2010. 5. hutabarat ra, suparman e, fagey w. karakteristik pasien dengan preeklampsia di rsup prof. dr. r. d. kandou manado. ecl 2016;4: 31-5. 6. sánchez-aranguren lc, prada ce, riaño-medina ce, et al. endothelial dysfunction and preeclampsia: role of oxidative stress. front physiol 2014;5:1-11. doi: 10.3389/fphys.2014. 00372. 7. hung j. oxidative stress and antioxidants in preeclampsia. j chin med assoc 2007; 70:430-2. 8. niki e. lipid peroxidation: physiological levels and dual biological effects. free radic biol med 2009;47:469-84. 9. cencioni c, spallotta f, martelli f, et al. oxidative stress and epigenetic regulation in ageing and age-related diseases. int j mol sci 2013;14:1764363. 10. marbut mm, majeed mb, rahim sm, et al. estimation of malondialdehyde as oxidative factor and glutathione as early detectors of hypertensive pregnant women. tikrit med j 2009;15: 63-9. 11. chandra s, widodo ma, suwarto s, et al. kadar mda dan rasio gsh/gssh pada kehamilan normal, preeklampsia berat dan eklampsia di malang. jkb 2007;23:35-39. 12. suhail m, suhail mz, khan h. alterations in antioxidant and prooxidant balance in univ med vol. 36 no.3 186 subandrate, faisal, anggraini, et al malondialdehyde and glutathione in preeclampsia preeclampsia – impact on erythrocyte osmotic fragility. biochem med 2008;18:331-41. 13. sayyed a, sontakke a. study of lipid peroxidation and antioxidant status in preeclampsia. jkimsu 2013;2:69-76. 14. genc h, uzun h, benian a, et al. evaluation of oxidative stress markers infirst trimester for assessment of preeclampsia risk. arch gynecol obstet 2011;284:1367-73. 15. bowen rs, moodley j, dutton mf, et al. oxidative stress in pre-eclampsia. acta obstet gynecol scand 2001;80:719-25. 16. dordevic nz, babic gm, markovic sd, et al. oxidative stress and changes in antioxidative defense system in erythrocytes of preeclampsia in women. reprod toxicol 2008;25:213-8. 17. lamminpaa r, vehvilainen-julkunen k, gissler m, et al. s. preeclampsia complicated by advanced maternalage: a registry-based study on primiparous women in finland 1997–2008. bmc pregnancy childbirth 2012;12:1-5. doi: 10.3389/ fphys.2014.00372. 18. lisonkova s, joseph ks. incidence of preeclampsia: risk factors and outcomes associated with early-versus late-onset disease. am j obstet gynecol 2013;209:1-12. 19. valensise h, vasapollo b, gagliardi g, et al. early and late preeclampsia: two different maternal hemodynamic states in the latent phase of the disease. hypertension 2008;52:873-80. 20. pare es, parry tf, mcelrath d, et al. clinical risk factors for preeclampsia in the 21st century. obstet gynecol 2014;124:763-70. 21. bakacak m, kylync m, serin s, et al. changes in copper, zinc, and malondialdehyde levels and superoxide dismutase activities in pre-eclamptic pregnancies. med sci monit 2015;21:2414-20. 22. subakir sb, santosa dis, arleni. kadar mda dan hsp 70 pada plasenta penderita preeklampsia. makara kesehatan 2008;12:93-5. 23. rafeeinia a, tabandeh a, khajeniazi s, et al. serum copper, zinc and lipid peroxidation in pregnant women with preeclampsia in gorgan. open biochem j 2014;8:83-8. 24. atamer y, kocyigit y, yokus b, et al. lipid peroxidation, antioxidant defense, status of trace metals and leptin levels in preeclampsia. eur j obstet gynecol reprod biol 2005;119:60–6. 25. ozkaya o, mekin s, hakan k. serum malondialdehyde, erythrocyte glutation peroxidase, and erythrocyte superoxide dismutase levels in woman with early spontaneous abortion accompanied by vaginal bleeding. med sci monit 2008;14:47-51. c:\users\jurnal fkusakti\docume 1 universa medicina january-april 2018 vol.37 no.1 richard tjan editor the proportion of older persons worldwide is steadily increasing and by 2050 around 2 billion people will be 60 years of age and older.(1,2) as a consequence, in that year there will be 135 million people with dementia, particularly in lowand middle-income countries where 60% of the more than 47 million cases (2015 estimate) are living now.(2) therefore it is not surprising that on 29 may 2017 the seventieth world health assembly accepted the global action plan on the public health response to dementia 2017-2025. the aim of this plan is “to improve the lives of people with dementia, their families and the people who care for them, while decreasing the impact of dementia on communities and countries.”(3) in this connection, last december the who also initiated the global dementia observatory, an internet-focused monitoring service for dementia data, such as government policy, treatment and care infrastructure, and disease burden.(4) this initiative is part of the who plan to improve the health of the elderly, as discussed in the world report on ageing and health 2015,(5) where healthy aging is defined as “the process of developing and maintaining the functional ability that enables well-being in older age”. in this report, the following principle of attaining and maintaining elderly health is discussed, i.e. person centered and small-scale care, which emphasizes flexibility or doing away with the single rigid method of standardized care.(5) this approach is particularly needed in the care of persons with dementia, since there is currently no effective medical treatment available for this disorder.(6) previously, the first who ministerial conference on global action against dementia held in october 2015 outlined the principles and approaches to guide global efforts and the plan for a global dementia observatory,(5) the latter being realized in december 2017, as mentioned above. the most important types of dementia are alzheimer’s disease and vascular dementia. the first accounts for around 80% of cases, while the latter results from cardiovascular conditions, including hypertension, obesity, hyperlipidemia, diabetes, and also from smoking.(6) evidently the type of dementia that is preventable is vascular dementia, comprising around 20% of cases, by improved control of the risk factors. however, this is a long-term goal, since dementia requires one or more decades to become manifest (from around the age of 35-40 years up to old age at around 60 years and over). currently, prevention may be applied to older persons with normal cognitive function and to those with minimal cognitive impairment. for existing cases of dementia, the focus will be on personalized care.(2) for the general practitioner not directly connected with the care of older persons with dementia, the focus should be on giving practical advice both to the younger generation for the prevention of cognitive decline and to the older generation for retarding the onset of mild cognitive impairment and dementia. this should include reassurance that three-fifths of older persons do not develop dementia and that onefifth of dementia cases can be prevented by not smoking (or for smokers, by cessation of smoking), by weight control and control of blood pressure and blood glucose (for those with diabetes). last but not least, there should be an e mp ha sis o n a he al thy di et , s uc h a s th e mediterranean die t, and r egular physical activity.(6) references 1. united nations department of economic and social affairs. world population ageing 2017 highlights. new york: united nations; 2017. 2 univ med 2018;37:1-2. doi: http://dx.doi.org/10.18051/univmed.2018.v37.1-2 2. world health organization. the epidemiology and impact of dementia: current state and future trends. geneva: world health organization; 2015. 3. world health organization. seventieth world health assembly update; new realase. geneva: world health organization;2017. 4. editorial. dementia burden coming into focus. lancet 2017;390:2606. 5. world health organization. world report on ageing and health. geneva: world health organization; 2015. 6. ray s, davidson s. dementia and cognitive decline: a review of the evidence. age uk research; 2014. universa medicina january-april 2018 vol.37 no.1 basuki1 80 hubungan antara konsumsi iodium dan gondok pada siswi berusia 15-17 tahun basuki budiman*a dan iman sumarno* *pusat penelitian dan pengembangan gizi dan makanan, departemen kesehatan, bogor korespondensi a ir. basuki budiman, msa(ph) pusat penelitian dan pengembangan gizi dan makanan, departemen kesehatan r.i. jl. dr. semeru 63 bogor 16112 telp. 0251-321763 email: basukibudiman@yahoo.com universa medicina 2007; 26: 80-9. universa medicina april-juni 2007april-juni 2007april-juni 2007april-juni 2007april-juni 2007 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 latar belakang konsekuensi gangguan akibat kekurangan iodium (gaki) yang paling utama adalah kelainan neuropsikologi dalam masa pertumbuhan janin. remaja merupakan kelompok usia yang segera akan menghasilkan keturunan. penelitian ini bertujuan untuk menilai adanya hubungan antara konsumsi iodium dan prevalensi gondok pada remaja. metode penelitian ini dirancang menggunakan desain potong-lintang. sebanyak 300 siswa putri smu usia 15-18 tahun di kabupaten minahasa (mn, sulawesi), bukittinggi (bt, sumatera) dan gunungkidul (gk, jawa), berpartisipasi dalam penelitian ini. pembesaran tiroid (gondok) dipalpasi dengan mengikuti kriteria who/iccidd. data yang dikumpulkan termasuk sampel urin sesaat (casual urine), perihal kesukaan remaja mengudap, contoh garam dapur rumahtangga, dan penggunaan garam beriodium. hasil prevalensi gondok pada siswi sangat tinggi di tiga kabupaten, sebesar 51,0% (mn), 41,6% (bt) dan 31,1% (gk) searah dengan gradasi proporsi defisit iodium menurut konsentrasi iodium dalam urin (kiu) (<100 µg/l bertutur-turut: 38,1%; 24,3% dan 11,6%) yang berada dalam kisaran normal. kualitas garam beriodium di mn paling bagus (>20 ppm iodium = 56%) begitu pula jenis garam yang dikonsumsi (92,0% garam halus) dibandingkan dengan dua kabupaten lainnya (gk 90% garam bata; bt 61,5 % garam curai/krosok). kesimpulan konsumsi iodium berhubungan dengan prevalensi gondok pada siswi berusia 1517 tahun. kata kunci : goiter, siswi, konsumsi iodium abstrak 81 the relationship between iodine consumption and goiter in schoolgirls aged 15 to 17 years basuki budiman*a and iman sumarno* background iodine status of women in child bearing age before, during and after pregnancy is important due to the consequences of iodine deficiency on brain development. more attention should be given to group of girls 15 to 17 years of age because of their reproductive period. the objective of this study was to evaluate the relationship between iodine consumption and prevalence of goiter in schoolgirls aged 15 to 17 years. method a cross sectional design was conducted and a total of 300 high school students in 15 to 18 years old from three district minahasa (mn), bukittinggi (bt) and gunungkidul (gk)of different islands were participated in this study. thyroid volume was assessed by palpation according to who criteria. sub sample from those students were examined their urine iodine concentration (uic) and interviewed their food preferences. salt used in the home of corresponding students was also examined using wet digestion method. results the prevalence of goiter was similar with the gradation of iodine concentration in urine (uic) in these 3 districts (38.1µ.g/l; 24.3 µ.g/l and 11.6 µ.g/l). the quality of iodine salt comsumption in mn (>20 ppm iodium = 56%) was highest compared to gk and bt districts. conclusion iodine consumption was related with goiter prevalence in schoolgirls 15 to 17 years of age. keywords: goiter, iodine consumptions, school girl * the center for research and development in nutrition and food, the national institute of health research and development correspondence abasuki budiman, ir. msc(ph) the center for research and development in nutrition and food, the national institute of health research and development jl. dr. semeru 63 bogor 16112 phone. 0251-321763 email: basukibudiman@yahoo.com universa medicina 2007; 26: 80-9. abstract latar belakang penanggulangan masalah gangguan akibat kurang iodium (gaki) secara nasional sudah dilakukan sejak tahun 1975, melalui fortifikasi iodium dalam garam dan iodiol yang disuntikkan secara intramuskuler (iodiol im). pada awal tahun 1990-an diluncurkan kapsul minyak b e r i o d i u m d o s i s t i n g g i ( 2 0 0 m g ) d a n meninggalkan iodiol-intra-muskuler.(1) pemetaan m a s a l a h g a k i t a h u n 1 9 9 6 1 9 9 8 o l e h d e p a r t e m e n k e s e h a t a n ( 2 , 3 ) m e n g u a k f a k t a penurunan endemisitas gaki secara drastis. pada pemetaan ini diketahui penggunaan garam kio 3 di atas 30 ppm mencapai sekitar 77,2%(4) dan total goiter prevalence (tgr) turun dari 27 menjadi 9,8%. pada survei evaluasi gaki oleh intensified project-iodine deficiency disorder control (ip-iddc), departemen k e s e h a t a n ( 1 ) s e c a r a n a s i o n a l t a h u n 2 0 0 3 menunjukkan tgr 11,1% dan median iodium dalam urin dan proporsi ekskresi iodium dalam universa medicina vol. 26 no.2 82 urin di atas 300 µg/l sangat tinggi (35,4%).(5) bukti ini menunjukkan bahwa konsumsi iodium pada masyarakat sangat tinggi dan berkembang a n c a m a n h i p e r t i r o i d i ( i n d u c e d i o d i n e hyperthyroidi, iih), yang dapat bermanifestasi p e m b e s a r a n k e l e n j a r t i r o i d . ( 5 8 ) t e r u t a m a kelompok rentan, yaitu ibu usia reproduksi. beberapa penelitian terakhir di daerah endemis mengungkapkan kejadian hipertiroidi pada wanita usia subur. budiman, melaporkan kejadian hipertiroidi di kabupaten bekasi sebesar 10,0% pada wanita usia subur. daerah tersebut diidentifikasi sebagai daerah endemis defisiensi iodium (dedi) berat tahun 1996, dan sejak itu setiap tahun dilakukan profilaksi iodium dengan dosis masif (200 mg iodium) dan garam yang difortifikasi lebih dari 60 ppm. bahkan untuk wanita usia subur/produktif d i b e r i k a n i o d i u m 2 x 2 0 0 m g i o d i u m . pemberian garam iodium setara 50 ppm kio 3 selama tiga bulan ternyata dengan ’marker’ tiroksin serum ibu usia 17-35 tahun, telah menyebabkan kenaikan jumlah iodium lima kali lipat dengan kenaikan median tiroksin sebesar 36,5% dari 6,98 menjadi 9,30 µg/l. dengan marker ekskresi iodium dalam urin anak usia sekolah, intervensi iodium menaikkan proporsi b e r i s i k o h i p e r t i r o i d i s e b e s a r 8 , 1 % d a n menaikkan median konsentrasi iodium dalam u r i n ( k i u ) d a r i 2 0 7 m e n j a d i 2 4 5 µg / l ( 9 ) k o n s u m s i i o d i u m b e r l e b i h m e n u r u n k a n pelepasan hormon tiroid (t4 dan t3) sehingga konsentrasi hormon tirod dalam serum menurun dan menstimulasi tirotropin (tsh).(10) di zaire, penggunaan minyak beriodium sebanyak 480 m g / m l t i d a k m e n i m b u l k a n i i h , t e t a p i penggunaan garam beriodium (25% sampel menggunakan garam beriodium >50 ppm), mengakibatkan tirotoksikosis dan konsentrasi t s h t e r t e k a n ( s u p p r e s s e d ) s e m e n t a r a konsentrasi t4 dan t3 dalam serum mengalami penurunan atau tetap normal.(11) konsentrasi i o d i u m d a l a m u r i n d i t e m u k a n a n t a r a 2 0 0 sampai dengan 500 µg/l per hari. penderita tersebut menunjukkan simptom tirotoksikosis dan memerlukan pengobatan antitiroid. iih paling sering terjadi pada penduduk di dedi yang mengkonsumsi iodium secara berlebih.(12) remaja dalam keadaan tumbuh yang cepat dan membutuhkan energi dan zat gizi lebih banyak dari kelompok umur lain kecuali bayi. di indonesia, remaja mempunyai risiko tinggi terhadap defisiensi energi-protein, zat gizi besi, v i t a m i n a d a n z a t g i z i m i k r o l a i n n y a . metabolisme iodium ternyata diperngaruhi oleh status zat-zat gizi ini. defisiensi energi-protein m e n g h a m b a t m e t a b o l i s m e i o d i u m m e l a l u i pengaruh defisiensi energi-protein pada sistem endokrin yaitu terhadap berat kelenjar, struktur histologi, dan fungsi kelenjar tiroid walaupun d a l a m j a n g k a w a k t u y a n g c u k u p l a m a . mikronutrien lain seperti vitamin a, selenium (se) dan zinc (zn) berpengaruh terhadap sintesa hormon tiroid. di sudan, gaki dilaporkan berkaitan dengan defisiensi protein-energi, d e f i s i e n s i vi t a m i n a d a n a n e m i a . ( 1 3 ) s u p l e m e n t a s i v i t a m i n a d i l a p o r k a n meningkatkan efikasi iodium.(14-16) zat gizi besi ( f e ) b e r k a i t a n d e n g a n i o d i u m . wa l a u p u n mekanisme molekuler peran fe dengan iodium belum begitu jelas, namun beberapa penelitian menunjukkan korelasi antara kedua mineral tersebut.(17) defisiensi fe diduga berperan dalam m e t a b o l i s m e i o d i u m d a l a m s e l t i r o i d d a n defisiensi fe juga menurunkan efikasi profilaksis iodium. sintesa hormon tiroid membutuhkan kehadiran fe dan katalisa enzim tiroperoksiadse (tpo). anemia zat gizi besi (agb) menurunkan konsentrasi tiroksin dalam plasma. di samping i t u , p e n a n g g u l a n g a n d e f i s i e n s i g i z i b e s i dilaporkan meningkatkan efikasi iodium di cote d’lvoire.(18,19) defisiensi selenium merupakan bagian tak terpisahkan pada enzim glutation peroksidase (gsh-px) yang berpengaruh pada deiodinasi t4 menjadi t3.(20,21) konsentrasi zn, copper (cu), mangan (mn), dan se dalam budiman, sumarno konsumsi iodium dan gondok 83 plasma, eritrosit dan urin berubah-ubah sesuai dengan status tiroid. remaja terutama putri, rentan terhadap defisiensi gizi karena kebutuhan yang meningkat dan pandangan salah tentang postur tubuh menjadi pemicu kurang gizi pada remaja. iccidd(22) menyatakan bahwa kadar iodium dapat berkurang akibat proses pemasakan. oleh karena itu, masalah status iodium pada remaja putri menjadi penting untuk diketahui dan di indonesia masalah ini belum banyak diungkap. p e n e l i t i a n i n i b e r t u j u a n u n t u k mengidentifikasikan adanya hubungan antara konsumsi iodium dan goiter pada remaja putri berusia 15-17 tahun. metode rancangan penelitian penelitian ini dirancang secara kroseksional dan dilaksanakan di kabupaten bukittinggi (bt), s u m a t e r a b a r a t ; g u n u n g k i d u l ( g k ) , d i yogyakarta dan minahasa (mn), sulawesi utara. ketiga kabupaten ini dipilih karena diketahui perbedaan jumlah konsumsi cabe yang berbeda. masyarakat bt, sumatera barat pada umumnya dikenal sebagai pengonsumsi cabe merah (capsicum annuum); masyarakat mn, pengonsumsi cabe rawit (capsicum frutescens) dalam jumlah banyak; sedangkan masyarakat gk diketahui sedikit mengonsumsi cabe dan rasa m a n i s . k o n s u m s i g a r a m k i o 3 d e n g a n k o n s e n t r a s i 3 0 p p m a t a u l e b i h d i k e t i g a kabupaten ini berturut-turut 100%; 83,8% dan 100%. subjek penelitian remaja secara fisiologis berada dalam masa pertumbuhan cepat. untuk kemudahan operasional siswi smu kelas 1 dan 2 (usia antara 15-17 tahun) dipilih sebagai sampel. di setiap kabupaten dipilih satu smu yang jumlah siswinya terbanyak. sebanyak 100 siswi disetiap sekolah yang bersedia ikut serta dalam penelitian ini dipilih sebagai sampel. pemeriksaan kelenjar tiroid status tiroid siswi diperiksa pembesarannya secara perabaan (palpasi) dan palpasi dilakukan oleh pelatih palpasi nasional. pembesaran kelenjar tiroid ditentukan berdasarkan kriteria who/unicef/iccidd. (22) kelenjar tiroid dikatakan membesar jika pada waktu diraba (palpasi) terasa membesar dan pembesaran melebihi ujung ibu jari (terminal falang) subyek. pembesaran dikelompokkan sebagai berikut: sangat besar (grade iii) jika pembesaran kelenjar tiroid tampak menonjol pada leher bagian depan dan dapat dilihat pada jarak lebih dari lima meter. namun jika pembesaran tersebut tampak dalam jarak kurang dari 2 meter, posisi kepala dalam keadaan normal (tidak tengadah dan tidak tunduk) dan teraba maka masuk dalam grade ii. jika pembesaran tampak hanya pada posisi kepala tengadah dan teraba, maka masuk dalam g r a d e i . p a d a p o s i s i k e p a l a t e n g a d a h , pembesaran tidak tampak tetapi terasa kelenjar tiroid membesar melebihi ujung jari subyek maka masuk dalam grade ia. grade 0 untuk pembesaran tiroid yang tidak teraba atau jika teraba tidak lebih besar dari ujung ibu jari subyek. pemeriksaan iodium dalam urin konsumsi iodium konsentrasi iodium dalam urin (kiu) yang diambil secara sesaat, kiu tidak dapat menggambarkan status iodium secara individu karena keragaman antar waktu ( d a y t o d a y v a r i a t i o n d a n w i t h i n d a y variation).(22) urin diperiksa dari ekskresi urin sesaat (casual) yang dimintakan dari siswi sebanyak kurang lebih 10 ml. urin ditampung dalam botol kecil dengan tutup ganda dan diberi timol. menurut who/unicef/iccidd, (22) suatu wilayah dikatakan defisit iodium jika median kiu kurang dari 100 mg/l. dalam universa medicina vol. 26 no.2 84 sebaran kiu tersebut proporsi kiu <100 µg/l tidak lebih dari 50% atau proporsi kiu <50 µg/ l kurang dari 20%. di samping itu, endemisitas defisienssi iodium dapat pula ditentukan dengan proporsi goiter (total goiter rate, tgr) yaitu lebih dari 5%. daerah dengan tgr 5 19,9% termasuk daerah endemis defisiensi iodium ringan; 20,0 29,9% daerah endemis sedang; dan 30,0% ke atas daerah endemis berat pengukuran konsumsi iodium aktual secara kasar penggunaan garam di tingkat rumahtangga d i k u m p u l k a n m e l a l u i w a w a n c a r a d e n g a n kuesioner yang terstruktur dan observasi. rumahtangga yang diwawancara dipilih secara acak dari remaja puteri yang telah diwawancara dan dipalpasi. data yang dikumpulkan meliputi j e n i s g a r a m y a n g b i a s a d i p a k a i , t e m p a t penyimpanan garam, cara pemakaian dan jumlah g a r a m y a n g d i b e l i . c o n t o h g a r a m y a n g dikonsumsi rumahtangga dianalisis kandungan iodiumnya. analisis kandungan iodium dalam urin digunakan metode standar acid digestion metod. urin yang dibutuhkan sebanyak 250 µl dan dalam satu kali pemeriksaan dapat dilakukan 120 sampel sekaligus. jumlah garam yang dikonsumsi diperkirakan dari pertanyaan (i) kapan terakhir rumahtangga membeli garam (minggu), (ii) kapan garam tersebut diperkirakan habis dikonsumsi oleh rumahtangga (minggu) dan (iii) berapa banyak garam dibeli (gram). perkiraan konsumsi iodium (per orang per hari) dilakukan atas dasar kombinasi konsumsi garam rumahtangga dan kandungan iodium yaitu ratarata konsumsi garam dikalikan dengan rata-rata kandungan iodium, kemudian dibagi dengan tujuh. analisis statistik perbedaan rata-rata konsumsi cabe, garam d a n i o d i u m ( t e r c e r m i n d a r i k i u ) a n t a r kabupaten menggunakan analysis of variance ( a n o va ) s a t u a r a h d a n p e r b e d a a n a n t a r wilayah diuji dengan multiple comparison. analisis statistik dilakukan secara elektronik dengan menggunakan perangkat spss versi 6,0. hasil jumlah siswi yang bersedia menyerahkan contoh urin sebanyak 296 orang dari 306 siswi yang dipalpasi. siswi yang tidak menyerahkan urin karena sedang menstruasi. sebanyak 81 dari 306 siswi, secara random dikunjungi r u m a h n y a u n t u k m e w a w a n c a r a i i b u r u m a h t a n g g a a t a u o r a n g y a n g b i a s a menyiapkan makanan untuk keluarga perihal konsumsi garam, cabe dan masakan yang paling sering dimasak. pada tabel 1 disajikan status iodium siswi dan penggunaan garam beriodium dan tabel 2 menunjukkan hasil uji anova searah tentang konsentrasi iodium dalam urin, r a t a r a t a k o n s u m s i g a r a m d a n r a t a r a t a konsumsi cabe. tampak terdapat konsistensi antara total konsumsi iodium yang digambarkan melalui kiu, proporsi defisit konsumsi iodium (<100 µg / l ) d a n p r e v a l e n s i g o i t e r p a d a r e m a j a . p r o p o r s i d e f i s i t k o n s u m s i i o d i u m r e n d a h (11,6%), prevalensi goiter pada remaja juga r e n d a h ( 3 1 , 1 % ) . p r e v a l e n s i d e f i s i t t i n g g i (38,1%) proporsi remaja yang mengalami pembesaran kelenjar tiroid juga tinggi (51,0%) (tabel 1). perkiraan konsumsi iodium (per orang per hari) dilakukan atas dasar kombinasi konsumsi garam rumahtangga dan kandungan iodium (rata-rata konsumsi garam x rata-rata kandungan iodium 7). dengan cara ini remaja mn mengkonsumsi iodium (per orang per hari) paling tinggi (155,5 µg/l iodium), disusul remaja di gk (134,2 µg/l iodium) dan terendah bt (99,6 µg/l iodium). konsumsi iodium total kelompok remaja mn berbeda secara nyata dengan bt dan dengan gk (p=0,00) (tabel 2). budiman, sumarno konsumsi iodium dan gondok 85 namun keadaan ini tidak sesuai dengan asupan iodium hanya dari garam rumahtangga. jumlah garam rumahtangga yang dikonsumsi remaja di ketiga daerah secara statistik tidak berbeda bermakna (p=0,466). pada tabel 3 disajikan konsumsi cabe remaja di ketiga daerah penelitian. konsumsi cabe tertinggi di mn 178 gram per kapita per hari, menyusul remaja bk 101 gram per hari dan terendah di gk 92 gram per hari. urutan ini sesuai dengan prevalensi gondok di ketiga daerah. daerah dengan konsumsi cabe tertinggi (178 gram) mempunyai prevalensi gondok tertinggi (51,0%), dan daerah dengan konsumsi cabe terendah (92 gram) mempunyai prevalensi gondok terendah (31,1%). tabel 2. perbandingan kiu, konsumsi garam dan konsumsi cabe antara ketiga lokasi penelitian tabel 1. status iodium, penggunaan dan konsumsi garam berdasarkan lokasi penelitian universa medicina vol. 26 no.2 86 pembahasan g a k i d i k a t a k a n m e n j a d i m a s a l a h kesehatan masyarakat jika didapati pembesaran kelenjar tiroid (total goiter rate, tgr) lebih dari 5% dan dikatakan endemis berat jika tgr >30%. goiter remaja di tiga daerah >30%. hal i n i m e n u n j u k k a n b a h w a d e f i s i e n s i t e l a h berlangsung lama dan remaja adalah kelompok lapis kedua terpenting setelah wanita usia subur (wus) dalam siklus reproduksi. jika dalam beberapa tahun mendatang tidak dikoreksi, kemudian menikah, maka ia akan melahirkan bayi-bayi yang menderita gangguan akibat kekurangan iodium. konsekuensi terberat dari a d a l a h k e t e r b e l a k a n g a n p e r k e m b a n g a n neuropsikointelektual termasuk keterbelakangan mental dengan manifestasi yang berat adalah kretinism. tgr kurang menggambarkan status iodium m a s a k i n i , w h o / u n i c e f / i c c i d d ( 2 2 ) menganjurkan mengunakan marker biokimia kiu. masalah defisiensi iodium didefinisikan jika proporsi kiu <100 µg/l <50% dengan kiu < 50 µg/l <20%. median kiu antara 200-299 µg/l penduduk berisiko hiperttiroidi (iih) dalam kurun waktu 5-10 tahun kemudian dan median kiu>300 µg/l dinyatakan kelebihan konsumsi (ekses) dan penduduk berisiko menderita iih atau penyakit tiroid autoimun. status iodium dikatakan normal jika median kiu kurang dari 300 µg/l. konsumsi iodium yang diekspresikan dari kandungan iodium dalam urin (kiu) remaja di ketiga daerah penelitian termasuk pada kisaran normal. hal ini mengindikasikan bahwa p a d a s a a t p e n e l i t i a n d i l a k u k a n , s e c a r a biokimiawi konsumsi iodium remaja telah mencukupi walau ada sebagian remaja yang mengalami kekurangan. perlu diingat bahwa kiu sesaat adalah cerminan konsumsi wilayah, sedangkan cerminan konsumsi iodium individu perlu pengumpulan urin minimal 2 x 24 jam.(23) perkiraan asupan iodium yang dihitung dari rata-rata konsumsi garam dari rumahtangga dan kandungan iodium dalam garam (tabel 1). konsumsi garam per orang per hari di tiga kabupaten diperkirakan antara 5-8 g. jumlah ini mendekati hasil penelitian mustafa(24) dengan teknik marker-lithium di jawa timur (5,8 g/ o r a n g / h a r i ) , d a n l e b i h r e n d a h d a r i h a s i l penelitian abunain(25) yang hingga kini masih dijadikan acuan dep.kes (10 g). jumlah garam yang dikonsumsi di ketiga daerah secara statistik tidak berbeda, namun kualitas garam (jenis dan kandungan iodium >20 ppm) yang dikonsumsi di ketiga daerah berbeda. asupan iodium remaja mn paling banyak (155,5 µg/l), disusul remaja gk (134,2) dan bt (99,6). namun perkiraan asupan iodium ini tidak seiring dengan median kiu, yaitu yang paling tabel 3. distribusi makanan yang disukai remaja di daerah penelitian budiman, sumarno konsumsi iodium dan gondok 87 tinggi pada remaja gk (212) dan yang paling rendah mn (129). walaupun jumlah garam yang dikonsumsi secara statistik tidak berbeda di tiga daerah, namun dibandingkan dengan kedua daerah lainnya, kualitas garam di mn paling bagus dengan kandungan iodium >20 ppm p a l i n g t i n g g i ( w h o / i c c i d d merekomendasikan paling sedikit 15 ppm iodium; dep.kes menetapkan paling sedikit 30 ppm) dan jenis garam yang dikonsumsi pun p a l i n g b a i k ( g a r a m h a l u s ) s e h i n g g a p e n c a m p u r a n i o d i u m n y a l e b i h m e r a t a dibandingkan dengan iodium dalam garam bata a t a u g a r a m c u r a i / k r o s o k . j i k a k e b i a s a a n menambah garam pada saat mengudap (tabel 1) dipertimbangkan dalam memperkirakan asupan iodium, maka asupan remaja mn paling tinggi. gradasi proporsi defisit iodium (<100 mg/ l) dan gradasi median kiu di tiga daerah sesuai dengan gradasi proporsi pembesaran kelenjar tiroidnya, semakin tinggi konsumsi iodium dari garam semakin rendah proporsi pembesaran kelenjar tiroidnya.tampaknya ada hal yang menyebabkan gangguan metabolisme iodium sehingga remaja mn yang paling tinggi menderita gondok paling banyak dan walaupun perbedaan konsumsi iodium tidak ditemukan antara rata-rata konsumsi di bt dan gk, namun besar proporsi tampak berbeda. beberapa hal yang dapat menjelaskan f e n o m e n a i n i . p e r t a m a , r e m a j a m n b i a s a mengonsumsi garam, cuka dan cabe (rawit terutama) dalam jumlah banyak (konsumsi cabe remaja mn lebih banyak secara signifikan), bahkan makan pisang gorengpun dengan cabe dan garam. hal ini tidak dilakukan oleh remaja di bt dan gk. konsumsi iodium yang berlebih (ekses) pada remaja di mn karena kebiasaan menambah garam meja pada waktu mengudap, sehingga tiroid menghambat terjadi organifikasi iodium yang disebut efek wolff-chaikoff dan mempertahankan produksi hormon tiroid tetap n o r m a l . e f e k i n i s e b e n a r n y a b e r l a n g s u n g sekitar 48-50 jam, namun jika konsumsi iodium yang berlebih berlangsung terus, efek tersebut rusak dan terjadi hipotiroidi (iodine-induced hypothyrpoid) dan dapat menjadi permanen h i p o t i r o i d i . ( 8 ) k o n s u m s i i o d i u m b e r l e b i h menurunkan pelepasan t3 dan t3 dari kelenjar tiroid sehingga menurunkan konsentrasi t4 dan t3 dalam serum dan merangsang peningkatan h o r m o n t i r o t r o p i n ( t s h ) . ( 11 ) p e m b e s a r a n kelenjar tiroid dapat merupakan cerminan kelebihan konsumsi iodium (ekses) yang kronis. hasil penelitian di mn ini tampaknya mirip s e p e r t i y a n g t e r j a d i d i i n d i a d a n c i n a . p e n e l i t i a n d i i n d i a d i d a e r a h d e l t a mengindikasikan konsumsi berlebih (med kiu 225;115-525 µg/l) juga ditemukan goiter 38,2 (25-61) persen pada anak usia 6-12 tahun.(7) d i c i n a d i d a e r a h d e l t a j u g a y a n g 8 5 % penduduknya mengonsumsi iodium di atas 500 µg/l, goiter pada anak 6-15 tahun ditemukan sebesar 12-38%.(6) hal ini sesuai dengan hasil penelitian zimmermann.(26) kesimpulan dan saran kesimpulan prevalensi goiter pada remaja putri usia 15-17 tahun besarnya berkisar antara 31,1 % 51 % dan median konsentrasi iodium dalam urin antara 129,0 µg/l 212,5 µg/l . konsumsi cabe tertinggi di mn 177,9 gram/hari menyusul bt 102 gram/hari dan terendah di gk 92 gram/hari. terdapat konsistensi urutan prevalensi gondok dengan urutan konsentrasi iodium dalam urin d a n d e n g a n k o n s u m s i c a b e , t e t a p i t i d a k konsisten dengan estimasi konsumsi iodium saran perlu dipelajari lebih dalam hubungan antara konsumsi cabe, konsumsi iodium dan prevalensi gondok baik hipotiroidi maupun hipertiroidi universa medicina vol. 26 no.2 88 ucapan terima kasih penulis mengucapkan terima kasih kepada siswi dan guru sekolah daerah penelitian atas kerjasamanya dalam penelitian ini. terima kasih penulis sampaikan kepada tim pengumpul data dan tim peneliti atas kerja keras dan dedikasinya dalam penelitian ini. penulis tidak lupa menyampaikan terima kasih atas saran dan masukan prof. dr. adi hidayat, dr, ms untuk perbaikan artikel ini. daftar pustaka 1. djokomoelyanto r, setyawan h, dramaix m, hadisaputro s, soehartono t, delange f. the thyromobil model for standardized evaluation of iodine deficiency disorder control in indonesia. thyroid 2001; 11: 365-72. 2. muhilal, kartono d, permaesih d, latief d, tilden rl. national survey on iodine deficiency disorder (idd) 1996-1998. gizi indon 2000; 14: 1-8. 3. who, world bank, ministry of health-indonesia. iodine deficiency in indonesia-a detailed nationwide map of goiter prevalence. who 2001. malta. who/nhd/01.4. 4. permaesih d, latinulu s, kartono d, susanto d, the profile of iodized‘salt use in the district level in indonesia. gizi indon 2000; 14: 25-31. 5. departemen kesehatan. survai nasional pemetaan gangguan akibat kekurangan yodium (gaky). proyek intensifikasi penanggulangan gaky. evaluasi program gaki; 2003. 6. zhao j, wang p, shang l, sullivan km, van der haar f, maberly g. endemic goiter associated with high intake iodine. am j public health 2000; 90: 1633-5. 7. chandra ak, tripathy s, ghosh d, debnath a, mukhopadhyay s. iodine nutritional status & prevalence of goiter in sudarban delta of south24parganas, west bengal. indian j med res 2005; 122: 419-24. 8. markou k, geogorpoulos n, kyriazopoulou v, vagenakis ag. iodine-induced hypothyroidism. thyroid 2001; 11: 501-10. 9. budiman b, komari, saidin m. pengaruh pemberian garam beriodium terhadap kadar tiroksin pada ibu usia subur di daerah endemik defisiensi iodium. gizi indon 2005; 28: 32-9. 10. roti e and braverman le. iodine excess and thyroid function in nauman j, glinoer d, braverman le, hostalek u (eds). the thyroid and iodine. merck thyroid symposium, warsaw 1996. may 16-18; 7-17. 11. stanbury jb, ermans ae, bourdoux p, todd c, oken e, tonglet r el al. iodine-induced hyperthyroidism: occurence and epidemiology in iodine-induced hyperthyroidism-report of a workshop march 1996. thyroid 1998; 8: 83-100. 12. roti e, uberti e. iodine excess and hyperthyroidism. thyroid 2001; 11: 493-500. 13. elnour a, hambraeus l, eltom, m, dramaix m, bourdoux p. endemic goiter with iodine sufficiency: a possible role for the consumption of pearl millet in the etiology of endemic goiter. am j clin nutr 2000; 71: 59-66. 14. zimmermann mb, adou p, zeder c, torresani t, hurrel rf. persistence of goitre despite oral iodine supplementation in goitrous children with iron deficiency anemia in the cote d’ivoire. am j clin nutr 2000; 71: 88-93. 15. zimmermann mb, wegmuller r, zeder c, chaouki n, torresani t. the effect of vitamin a deficiency and vitamin a supplementation on thyroid function in goitrous children. j clin endocrinol metab 2004; 89: 5441-7. 16. zimmermann mb, wegmuller r, zeder c, chaouki n, beibinger r, hurrel rf et al triple fortification of salt with microcapsules of iodine, iron and vitamin a. am j clin nutr 2004; 80: 1283-90. 17. zimmermann mb, zeder c, chaouki n , torresani t, hurrel rf. dual fortification of salt with iodine and microcapsulated iron: a randomized, doubleblind, control trial in moroccan schoolchildren. am j clin nutr 2003; 77: 425-3212. 18. hess sj, zimmermann mb, adou p, torresani t, hurrel rf. treatment of iron deficiency in goitrous children improves the efficacy of iodized salt in cote d’lvoire. am j clin nutr 2002; 75: 743-8. 19. rajagopalan s, vinokumar m. effect of salt fortified with iron and iodine on the haemoglobin levels and productivity of tea pickers. fnb 2000; 323-9. 20. hawkes wc, keim nl. dietary selenium intakes modulates thyroid hormone and energy metabolism in men. j nutr 2003; 133: 3443-8. budiman, sumarno konsumsi iodium dan gondok 89 21. hotz cs,fitzpatrick dw, trick kd, l’abbe mr. dietary iodine and selenium interact to effect thyroid hormone metabolism of rats. j nutr 1997; 127: 1214-8. 22. iccidd/unicef/who.assessment of iodine deficiency disorders and monitoring their elimination. a guide for programme managers 2nd ed. who/nhd/01.1. 2001. 23. rasmussen lb, ovesen l, christiansen e. dayto-day and within day variation in urinary iodine excretion. euro j clin nutr 1999; 54: 401-7. 24. mustafa a, muslimatun s, untoro j, lan mcpj, kristianto y. determination of discretionary salt intake in an iodine deficient area of east javaindonesia using three different method. apjcn 2006; 15: 36-40. 25. abunain d. a study on profile iodized salt industry. an assessment of salt iodination activities in indonesia. ministry of industry-directorate of inorganic chemical industry in cooperation with unicef, jakarta. 1991. 26. zimmermann mb, ito y, hess sy, fujieda k, mulinari l. high thyroid volume in children with excess dietary iodine intakes. am j clin nutr 2005; 81: 840-4. universa medicina vol. 26 no.2 the changing paradigm of dyslipidaemia management 1 universa medicina january-april 2017 vol.36 no.1 the changing paradigm of dyslipidaemia management ruvan a i ekanayaka national hospital of sri lanka, institute of cardiology, colombo, sri lanka the 2013 american college of cardiology (acc)/american heart association (aha) guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults,(1) effected a change in the paradigm of dyslipidaemia management, in that it no longer advocated a target based therapy but instead posited a primary clinical basis for initiation of statin therapy. the intensity of statin therapy too was guided by clinical risk criteria rather than by absolute values of the lipid profile. many clinicians were uncomfortable with the new paradigm. the 2011 european society of cardiology (esc)/european atherosclerosis society (eas) guidelines for the management of dyslipidaemia (2) had given concrete lipid levels with reference to low density lipoprotein cholesterol (ldlc),high density lipoprotein cholesterol(hdl-c), triglycerides (tg) and nonhigh density lipoprotein cholesterol(hdl-c) etc. which provided goals towards which clinicians could aim with clarity of purpose. this type of target value based paradigm has been the norm for the past 15-20 years. hence the unhappy response to the new paradigm of the 2013 acc/aha guideline which eliminated target based management. clinicians as a group were familiar with managing metabolic diseases where laboratory testing gave an excellent guide regarding dosage, duration of therapyetc, which data would be accessible to both physician and patient. diabetes mellitus is a good example of the management scenario where concrete laboratory values override clinical risk stratification as guide to therapy. taking the 2011 esc/eas guidelines for t h e m a n a g e m e n t o f d y s l i p i d a e m i a a s a representative specimen of the clinical practice guidelines of the pre-2013 era, we find that the ldl-c is selected as the primary target of therapy. the justification is based on the dose dependant reduction in ischaemic events seen with lowering of ldl-c. it is statistically determined that 40% reduction of ldl-c is associated with a 22% reduction of morbidity and mortality due to ischemic heart disease (ihd).(3) absolute values are also given. a ldlc level of less than 70mg% or, a relative reduction of ldl-c 50% from baseline, seem to offer the best benefit in cardiovascular event reduction. the specific target for non hdl-c is given as a 30mg%. this parameter includes most if not all the atherogenic particles (chylomicron remnants, verylow-density lipoprotein (vldl), i n t e r m e d i a t e d e n s i t y l i p o p r o t e i n ( i d l ) , lipoprotein(a) (lp (a) ), and ldl. the 2011 esc/ eas guidelinedoes not set specific targets for hdl-c or triglycerides, as clinical trial evidence is lacking for determination of optimal values for these lipid constituents. the 2011 esc/eas guideline further emphasises that the specific statin is of little consequence and that the type of statin must be selected based on it’s ability to achieve the target ldl-c goal. up titration of the statin dose is recommended until the ldl-c goal is achieved and this is made mandatory. the 2013 acc/aha guideline did away with this mode of recommendations. the important determinant to effect the paradigm 2 universa medicina s h i f t m a n i f e s t e d i n t h e 2 0 1 3 a c c / a h a guideline, was the selection of evidence that was evaluated and utilized to prepare the guideline. whereas the previous guidelines had taken into consideration the randomized clinical trials(rct), epidemiological studies, basic science studies, clinical experience and genetics etc, the 2013 guideline utilized rcts published since 1960 (in addition to systematic reviews and meta analyses of rcts ). the expert panel studied 6 rcts regarding primary prevention and 19 rcts regarding secondary prevention. the expert panel found good evidence that statin benefited 4 clinical groups:1. individuals with clinical atherosclerotic cardiovascular disease (ascvd) 2. elevation of ldl-c ≥90mg% 3. 40-75 year old diabetics with ldl-c 70189mg% 4. no ascvd or diabetes mellitus (dm) but estimated 10year risk of ascvd ≥7.5%. however the expert panel could not find evidence from the rcts to recommended a specific ldl-c or non ldl-c target. most, if not all,the rcts had not titrated the statin therapy to achieve a target ldl-c level and the resultant effect was a dearth of rct evidence for a specific ldl-c target. in the absence of a goal for ldl-c, the clinician faces the problem of the intensity of statin therapy he needs to prescribe. the 2013 acc/ aha guideline classifies the intensity of statin therapy as high, moderate and lower. the expert panel used a calculated estimation of the percent ldl-c reduction for a specific statin – high intensity meant reducing ldl-l by 50%, moderate intensity by 30% 50% and lower intensity by <30%. thus the 2013 acc/aha guideline changed the paradigm of dyslipidaemia management from a target laboratory value based model to a cardiovascular risk based one. the baseline ldlc level and the achieved ldl-c level were not deemed important in the crucial decisions regarding the initiation and up titration of statin therapy. the latest updated guideline currently available is the 2016 esc/eas guideline for the management of dyslipidaemias.(4) the compilers of this guideline have carefully considered the basis on which the 2013 acc/aha guidelines were constructed but have opted to retain the older paradigm of reducing ldl-c towards a target goal. risk stratification takes an important place in the 2016 esc/eas guideline which has introduced a new risk category termed ‘very high risk’ group .hence in this guideline, risk categorization comprises of four levels. the very high risk group would need a ldl-c level of 70mg% or reduction of 50% from baseline ldlc; for the high risk group ldl-c of <100mg% and for the low to moderate risk group an ldl-c of <115mg%. the 2016 canadian cardiovascular society guideline for the management of dyslipidaemia for the prevention of cardiovascular disease in the adult (5) closely follows the paradigm set in the 2016 esc/eas guideline but introduces abdominal aortic aneurysm (>3cm in diameter) into the indicated conditions. the pleiotropic effects of statins are an important consideration in statin therapy irrespective of the baseline ldl-c levels, as the benefits of statin therapy exceed those predicted by cholesterol reduction alone. this well established fact is in favour of the 2013 acc/ aha paradigm for dyslipidaemia management. clinicians must therefore take the pleiotropic effect benefits into account when planning the treatment of aggressive atherosclerotic disease. although guidelines categorise risk on clinical grounds, atherosclerotic disease is dynamic and unstable by it’s very nature and this factor has not been adequately addressed in the dyslipidaemia guidelines. another problem area neglected in the guidelines is the relevance of dense ldl particles (6) and dysfunctional hdl.(7) these are considered highly atherogenic but rct evidence is lacking with regard to the targets and treatment modalities for either of these two lipid particles. even the normal values for lipid parameters which are 3 univ med 2017;36:1-3. doi: http://dx.doi.org/10.18051/univmed.2017.v36.1-3 january-april 2017 vol.36 no.1 routinely estimated in the lipid profile appear to be fluid in that different values are given for different clinical scenarios. it is therefore appropriate that the clinician use his clinical acumen to the maximum and not slavishly follow a given paradigm of dyslipidaemia management. he must be aware that both paradigms endorsed by the esc/eas and acc/aha have more similarities than differences and in fact could be complementary. it is the clinician who will know how the atherosclerotic disease has behaved in the past in a given patient and will be able to better predict how the disease will behave in the coming years (whatever the short comings of such a prediction might be). based on this data and following any guideline, the best therapeutic option for each individual patient must be selected. references 1. stone nj, robinson jg, lichtenstein ah, et al. acc/aha guideline on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk in adults. a report of the american college of cardiology/american heart association task force on practice guidelines. circulation 2014;129 (25 suppl. 2):s1-45. 2. reiner z, catapano al, graham i, et al. esc/ eas guidelines for the management of dyslipidaemias. eur heart j 2011;32:1769-818. 3. cholesterol treatment trialists’ (ctt) collaboration. efficacy and safety of more intensive lowering of ldl cholesterol: a metaanalysis of data from 170000 participants in 26 randomised trials. lancet 2010;376:1670– 81. 4. catapano al, graham i, de backer g, et al. esc/eas guidelines for the management of dyslipidaemias. eur heart j 2016; 37:2999-3058. 5. anderson tj, gregoire j, pearson gj, et al. canadian cardiovascular society guidelines for the management of dyslipidemia for the prevention of cardiovascular disease in the adult.canadian j cardiol 2016;32:1263-82. 6. wadhera rk, steen dl, khan i, et al. a review of low-density lipoprotein cholesterol, treatment strategies, and its impact on cardiovascular disease morbidity and mortality. j clin lipidol 2015;10:472-89. doi: 10.1016/j.jacl.2015.11. 010. 7. serban c, muntean d, mikhailids dp, et al. dysfunctional hdl: the journey from savior to slayer. clin lipid 2014;9:49-59. f:\36 no 2\sutrisno.pmd 131 *department of obstetrics and gynecology, faculty of medicine, sebelas maret university/ dr. moewardi general hospital surakarta correspondence: dr. soetrisno, spog(k) jl. slamet riyadi no. 381 surakarta phone : +62816672564 email: soetrisno_spogk@yahoo.com date of first submission, may 29, 2017 date of final revised submission, august 10, 2017 date of acceptance, august 11, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 l-arginine improves uterine spiral arterial wall thickness in mouse models of preeclampsia soetrisno*, sri sulistyowati*, and anwar sandi wibowo* background preeclampsia is a major cause of maternal and fetal morbidity and mortality. the imbalance of anti-angiogenic and angiogenic factors plays a role in endothelial dysfunction in preeclampsia. l-arginine is expected to improve the process of spiral artery remodeling. this study aims to examine the use of l-arginine to repair endothelial damage by measuring the thickness of uterine spiral arteries in mouse (mus musculus) models of preeclampsia. methods the researchers carried out an experimental study using 30 sixteen-day old pregnant swiss mice (in good health, weighing 20-25 grams), which were randomly divided into 3 groups (each consisting of 10 mice). the groups were as follows: 1) normal pregnancy k(-); 2) preeclampsia model k(+); and 3) preeclampsia model receiving l-arginine (p). the authors performed histopathological examination of the mouse placenta, which had been dissected, embedded in paraffin wax and subsequently stained with hematoxylin and eosin (he). the results were analyzed in spss v. 21 for windows using anova with tukey. results the mean thickness of spiral arteries in group k(-) was 53.95 + 26.96 m, in k(+) 96.50 + 16.66 μm, and in p 62.79 + 8.04 m. statistically, there were significant differences between groups k(-) and k(+) (p=0.001) and between k(+) and p (p=0.000), but non-significant differences between k(-) and p (p=1.000). conclusions the treatment with l-arginine proved to be effective in repairing endothelial damage by reducing intimal hyperplasia and atherosis and, in turn, the thickness of uterine spiral arteries in mouse models of preeclampsia. keywords: l-arginine, spiral artery thickness, preeclampsia, mouse doi: http://dx.doi.org/10.18051/univmed.2017.v36.131-138 original article ci te t his article as: soet risn o, sulistyowati s, wibowo as. l-arginine improves uterine spiral arterial wall thickness in mouse models of preeclampsia. univ med 2017;36: 131-7. doi: 1 0 . 1 8 0 5 1 / un i vm ed . 2 0 1 7 . v 3 6 . 1 3 1 1 3 7 pissn: 1907-3062 / eissn: 2407-2230 132 soetrisno, sulistyowati, wibowo l-arginine improves spiral arterial introduction given the fact that the exact cause of preeclampsia remains unclear, its prevention in high-risk women is still unsatisfactory. currently, preeclampsia is a major cause of high maternal and perinatal morbidity and mortality, especially in developing countries. preeclampsia affects approximately 2–8% of all pregnant women around the world.(1) hypertension, including preeclampsia, is being found in an increasing number of pregnant women and is becoming one of the most common medical complications in pregnancy. approximately 70% of women diagnosed with hypertension are affected by preeclampsia.(2,3) complications of the disease cause about 50,000 maternal deaths each year. in developing countries, maternal mortality rates reach 15% compared to 0–1.8% in developed countries, due to limited access to maternal health care in the f ormer. (4 ) in indonesia, the complications of preeclampsia account for approximately 30-40% of maternal deaths and about 30-50% of all perinatal deaths. in 2008, there were 37 cases of preeclampsia (out of 1956 deliveries) in dr. moewardi general hospital in surakarta with the maternal mortality rate at about 67.6%.(5) l-arginine is expected to play a role in preeclampsia prevention by relieving its systemic symptoms.(6,7) the exact mechanism of larginine in reducing and preventing the incidence of preeclampsia remains unknown. l-arginine allegedly improves the uterine spiral arteries’ remodeling processes, which are impaired in preeclampsia. according to ekambaram (8) and stuehr, (9) l-arginine has a role in the pathway o f l -a rgin i n e -ni t r i c o xi d e . impa i r e d endothelium-dependent responses have been reported in isolated blood vessels of women with preeclampsia.(10,11) this finding suggests that impaired production of endothelial nitric oxide (no) may play an important role in mediating the pathophysiological process of preeclampsia. given the ethical problems, it is impossible to conduct research using human uterus during the first trimester of pregnancy. in a rat model of preeclampsia, l-arginine, a substrate of no synthase enzymes, was shown to decrease blood pressure.(12) human data are controversial as far as the efficacy of l-arginine supplementation in ameliorating hypertension in pregnancy is concerned. a study of randomized placebocontrolled design has demonstrated that a short term iv infusion of l-arginine can significantly r ed u c e b l oo d p r e ss ur e. ( 1 3 ) ot he r p l ac e b o controlled trials have not shown an improvement i n b lo o d pr e s su r e wi t h o r a l l -a rgi ni n e supplementation,(14) but suggest an improvement in fetal outcomes.(15) in the present research, the authors decided to investigate the abovementioned problem in the laboratory mouse (mus musculus) because of its ability to adapt to the laboratory environment as well as its genetic resemblance to humans, which can be used as a comparison of preeclampsia in human pregnancies. this study aims to evaluate the effect of l-arginine to repair the damage of endothelial spiral arteries that undergo intima tunica hyperplasia and atherosis in a mouse model of preeclampsia. methods research design an experimental study, based on post-test control group design, conducted at the faculty of veterinary medicine, airlangga university, surabaya from november 2016 to january 2017. research subjects the researchers carried out the study using 30 sixteen-day old pregnant swiss mice, which were randomly divided into 3 groups (each consisting of 10 mice). the groups were as follows: normal pregnancy k(-); preeclampsia model k(+); and preeclampsia model receiving l-arginine (p). the laboratory technicians performed histopathological examination of the mouse placenta, which had been dissected, embedded in paraffin wax and subsequently stained with hematoxylin and eosin (he). the 133 univ med vol. 36 no.2 researchers’ goal was to examine differences in histopathological findings in uterine spiral arteries and subsequently compare the results in these groups. the inclusion criteria were as follows: animals in good health; 3 months old, and weighing approx. 20-25 grams. the exclusion criterion was death of an animal during the research. the mice (mus musculus, swiss strain) were obtained from pusat veterinaria farma in surabaya. experimental treatments at the beginning of the study, the researchers synchronized the estrus of all female mice by injecting them with 5 iu pregnant mare serum gonadotropin (pmsg). after 48 hours, the researchers injected them with 5 iu human chorionic gonadotropin (hcg), and then mated the mice with male mice (7 months old and weighing approx. 60 grams). the diagnosis of pregnancy was expected about 17 hours after mating with the presence of copulatory plugs. the researchers injected the k (+) and (p) groups with 10 ng of anti-qa2 intraperitoneally (ip) from the first day until the 4th day of pregnancy in order to establish preeclampsia models in accordance with the method designed by sulistyowati et al.(16) furthermore, the (p) group was given l-arginine 200 mg/kgbw/day orally using a gastric tube, on days 7–15 of pregnancy. preparation of the placental tissue on the 16th day of pregnancy, the researchers performed dissection in the three groups in order to examine placental tissue samples, which were embedded in paraffi n wax, stained with hematoxylin eosin (he), and viewed under the microscope at 400 x magnification. the reason for dissection and the preparation of slides on the 16th day of pregnancy is that this is assumed to correspond to the second trimester of pregnancy in humans, where the manifestations of preeclampsia arise. the assessed parameter was the thickness of the spiral artery walls (in micrometers, m). data analysis the results of the research have been analysed in spss v. 21 for windows using mannwhitney test with a significance level of 0.05 (confidence interval 95%). ethical clearance the study has been approved by the research ethics commission of the faculty of veterinary medicine, airlangga university, surabaya (ref. no. 648–ke). results the core tunica spiral artery thickness in normal pregnant mouse group k(-) was less than that in the preeclampsia model k(+). furthermore, the researchers found out that the spiral artery thickness was reduced whereas its diameter was increased in the preclampsia model (p), which received l-arginine treatment, in comparison to the preeclampsia model group k(+) (figures 13). the mean thickness of the spiral artery wall in group k(+) is (96.50 ± 16.66 m), higher than in the preeclampsia group p (62.79 ± 8.04 m) and group k(-) (53.95 ± 26.96 µm). the anova test result shows a significant difference in the mean thickness of the spiral artery wall in the three groups (p=0.001). based on the result of tukey’s multiple comparison test, it is shown that the mean thickness of the spiral artery wall in group k(+) is significantly different from group k(-) (p=0.024) and from the preeclampsia group p (p=0.004). the mean thickness of the spiral artery wall in group k(-) is not different from the preeclampsia group p (p=0.062) (table 1). discussion in preeclampsia, placental hypoperfusion results in severe stenosis and occlusion of the spiral arteries which affect the syncytial node of the placenta and lead to increased numbers of cytotrophoblasts; ch anges to the villous vascularization; calcification; obliterating 134 soetrisno, sulistyowati, wibowo l-arginine improves spiral arterial figure 1. spiral artery in normal pregnant mice k(-) figure 2. spiral artery in mice of control group k(+) 135 univ med vol. 36 no.2 endarteritis, atherosis, infarction, thrombosis and necrosis. blood vessel abnormalities affect blood flow in preeclampsia because spiral arteries are the arteries that supply the intervillous space. reduced blood flow in the intervillous space causes hypoxia, which can lead to obliterating endarteritis due to reduced supply of oxygen to the blood vessels, especially the arterioles. consequently, the smooth muscle cells of the tunica media migrate to the tunica intima and undergo a proliferat ion c harac ter ized by thickening of the tunica intima and, in turn, narrowing of the blood vessels.(17) in this research, the mean spiral artery thickness in the preeclampsia mouse model group was greater than normal and less than in the group treated with l-arginine, with the differences being statistically significant. however, there was no significant difference in the spiral artery thickness in normal pregnanci es compared to the preeclampsia group treated with l-arginine, which means that the l-arginine treatment was effective in reducing the spiral artery thickness and increasing its diameter. l-arginine acts as a precursor of nitric oxide and is converted to nitric oxide (no) and lcitrulline by nitric oxide synthase (nos) and can prevent the occurrence of preeclampsia. a study showed that dietary supplementation with a combination of l-arginine and antioxidants was associated with a significant reduction in the incidence of preeclampsia, compared with figure 3. spiral artery in mice of treated group (p) table 1. mean spiral artery thickness by treatment groups k(-)=normal; k (+)=preeclampsia; p=preeclampsia + l-arginine treatment groups p value k(-) (n=10) k(+) (n=10) p (n=10) spiral artery thickness (m) 53.95 ± 26.96 96.50 ± 16.66 62.79 ± 8.04 0.001 136 soetrisno, sulistyowati, wibowo l-arginine improves spiral arterial antioxidants alone and with placebo.(18,19) given that l-arginine is widely available as a dietary supplement, l-arginine can be viewed as potential therapy to prevent preeclampsia.(2,20,21) according to the studies conducted by han et al.(22) and li et al.,(23) the concentration of larginine is significantly reduced in women with pre-eclampsia compared to normal pregnancies. studies on animal models show that the larginine-no system undergoes dysregulation during pregnancy.(24,25) hypertension, proteinuria, intra-uterine growth restriction (iugr) and glomerular damage may occur due to inhibition of no synthesis. therefore, hypertension caused by inhibition of no synthesis may be treated by supplementation w ith l-arginine. (2 6,27 ) furthermore, given the fact that oxidative stress may play a key role in the development of endothelial dysfunction and preeclampsia in human pregnancies, the administration of larginine may improve uteroplacental circulation and reduce maternal blood pressure.(4,22,28) however, this study had limitations such as the blood pressure and urinary protein of the test animals could not be determined due to lack of equipment. therefore, the preeclamptic state of the mice was determined based on the instructions listed in the report of sulistyowati et al.(16) larginine has been found to be a potentially novel therapy for hypertension in pregnancy in order to prevent the incidence of preeclampsia in women at high risk for the disease. it is suggested that further research are conducted on the use of larginine in women at high risk for preeclampsia. conclusions the research has proved that l-arginine is effective in repairing endothelial damage by reducing the thickness of the uterine spiral arteries and, in turn, tunica intima hyperplasia and atherosis in a mouse model of preeclampsia. larginine may become a new treatment option for hypertension in pregnancy and the prevention of preeclampsia in high-risk women. conflict of interest the authors declare that there is no conflict of interest associated with this study, the authors, and/ or the publication of this article. acknowledgments the researchers would like to thank to the staff members of the faculty of veterinary medicine of airlangga university, surabaya, for the mating of the mice to get them pregnant, preparation of the mouse models of preeclampsia, and care of the pregnant mice until research sampling and preparation. thanks also to the anatomical pathology laboratory of the faculty of veterinary medicine of airlangga university, surabaya, who made the paraffin-embedded samples and pe rfor med histopathological observation on the research sample. contributors s participated in drafting the manuscript and write up. ss and sw par ticipated in the conception and design of the study, data collection, analysis and interpretations of the findings. all authors read and approved the final manuscript. references 1. world health organization. who recommendations for prevention and treatment of pre-eclampsia and eclampsia. geneva: world health organisation;2011. 2. burke sd, karumanchi sa. spiral artery remodelling in preeclampsia revisited. hypertension 2013;62:1013–4. 3. camacho e, silva ja, matos mg, et al. actividad de las enzimas anti oxidantes en el rinon de la rata con preeclampsia experimental. archivosven farmacol ter 2011;30:44–50. 4. creasy rk, resnik r, iams jd, et al. editors. maternal-fetal medicine: principles and practice. 7th ed. philadelphia, pa: saunders;2014. 5. cunningham fg, leveno kj, bloom sl, et al. editors. william obstetrics. 24th ed. new york: mcgraw hill education;2014. 137 univ med vol. 36 no.2 6. al-bayati ma, ahmad ma, khamas w. the potential effect of l-arginine 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roberts jm, hubel ca. the two stage model of preeclampsia: variation on the theme. placenta 2009;30 suppl a:s32–s7. doi: 10.1016/j.placenta. 2008.11.009. oktavianus 205 *faculty of mathematics and natural sciences, padang state university **faculty of medicine, andalas university ***faculty of agriculture, andalas university ****faculty of medicine, trisakti university *****faculty of science and technology, ar-raniry islamic state university, banda aceh correspondence drs. syamsurizal, m.biomed fakultas matematika dan ilmu pengetahuan alam universitas negeri padang komplek kampus unp air tawar padang, sumatera barat, indonesia phone:+628126709150 email: syam_unp@yahoo.co.id univ med 2014;33:205-12 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background the prevalence of type-2 diabetes mellitus (t2dm) has been increasing rapidly in developed and developing countries. many new loci associated with t2dm have been uncovered by genetic and genome-wide association studies, eg. the transcription factor 7-like 2 (tcf7l2) gene variant rs7895340 has been associated with type-2 diabetes mellitus. the aim of this research was to find an association between polymorphisms of tcf7l2 gene variant rs7895340 with t2dm in ethnic minangkabau. methods this was a case-control study using a consecutive sampling technique among ethnic minangkabau patients who came for treatment to the metabolic endocrinology polyclinic at dr. m. jamil hospital. analysis was performed on 132 subjects (66 t2dm patients and 66 controls). dna was isolated from venous blood samples and amplified using the amplification refractory mutation system polymerase chain reaction (arms-pcr) method to detect single nucleotide polymorphisms (snps) of tcf7l2 gene variant rs7895340. to determine the significance of association of snps of rs7895340 with t2dm in ethnic minangkabau, data analysis was performed using chi-square test. results the arms pcr for tcf7l2 gene variant rs7895340 found polymorphisms in 28 (42.42%) subjects with type 2 diabetes mellitus and in 15 (22.72%) nondiabetic subjects.there was a significant association between rs7895340 snps with the probability of t2dm in ethnic minangkabau (or=2.505, p=0.026). conclusions single nucleotide polymorphism rs7895340 of tcf7l2 gene can be used as genetic marker of type-2 diabetes mellitus in ethnic minangkabau. keywords: tcf7l2 gene, rs7895340 snp, type-2 diabetes mellitus, ethnic minangkabau transcription factor 7-like 2 as type-2 diabetes mellitus diagnostic marker in ethnic minangkabau syamsurizal*, yanwirasti**, asman manaf**, jamsari***, ml edy parwanto****, and arif sardi***** 206 transcription factor 7-like 2 sebagai petanda diagnostik untuk diabetes melitus tipe-2 pada etnik minangkabau latar belakang prevalensi diabetes melitus tipe 2 (dmt-2) semakin meningkat baik di negara berkembang maupun negara maju. penelitian mengenai hubungan genetik dengan berbagai kelainan masih perlu diteliti. varian gen transcription factor 7-like 2 (tcf7l2) yang diduga kuat berasosiasi dengan dmt-2 adalah snp rs7895340. kehadiran g alel dalam rs7895340 mengindikasikan meningkatnya kerentanan individu terhadap dmt-2. tujuan penelitian menentukan hubungan antara polimorfisme snp rs7895340 dari gen tcf7l2 dengan dmt-2 pada etnik minangkabau. metode penelitian ini merupakan penelitian kasus-kontrol. pemilihan sampel berdasarkan teknik consecutive sampling pada etnik minangkabau yang datang berobat ke poliklinik endokrinologi metabolik rsup dr. m. djamil. sebagai pembanding dipakai kelompok kontrol orang sehat tidak diabetes melitus tipe-2. kasus adalah subjek etnik minangkabau penderita dmt-2 sebanyak 66 orang dengan kontrol juga sebanyak 66 orang. dna diisolasi dari darah semua sampel, kemudian diamplifikasi menggunakan metoda amplification refractory mutation system polymerase chain reaction (arms-pcr) untuk mendeteksi single nucleotide polymorphisms (snps) rs7895340 pada gen tcf7l2. analisis hubungan gen tcf7l2 varian rs7895340 dengan diabetes melitus tipe-2 digunakan uji chi-square. hasil hasil arms-pcr pada gen tcf7l2 varian rs7895340 pada subyek diabetes mellitus tipe-2 ditemukan polimorfisme sebanyak 28 orang (42,42%), dan pada subyek tidak dmt2 (kontrol) hanya 15 orang (22,72%). hasil uji chisquare membuktikan adanya hubungan yang signifikan antara gen tcf7l2 varian rs7895340 dengan dmt-2 (or = 2,505, p=0,026). kesimpulan gen tcf7l2 varian rs7895340 dapat dijadikan petanda genetik untuk diabetes melitus tipe-2 pada etnik minangkabau. kata kunci: gen tcf7l2, rs7895340, diabetes melitus tipe-2, etnik minangkabau abstrak introduction diabetes mellitus (dm) is a complex metabolic disease caused by pancreatic cellular damage. it is clinically classified into four types: type 1 diabetes mellitus, type 2 diabetes mellitus (t2dm), gestational diabetes mellitus, and other t y p e s o f d i a b e t e s m e l l i t u s . t 2 d m i s characterized by the phenomenon of insulin resistence as a result of relative insulin deficiency.(1) the prevalence of t2dm has been increasing in developed and developing countries.(2-4) the prevalence of dm in indonesia is 4.1%, while in west sumatra for the year 2000 it was estimated to be 5.1%.(5) patients with diabetes commonly suffer from cardiovascular complications, which are the most common cause of death in patients with diabetes mellitus.(6-8) the diagnostic methods for dm are still being developed, e.g. using dna analysis. to perform dna analysis, data are required on genes associated with t2dm. several ethnic groups in the world have already gene banks for t2dm, e.g. in denmark, usa, uk, france, and india.(9) syamsurizal, yanwirasti, manaf, et al transcription factor 7-like 2 as diagnostic marker 207 through dna or gene analysis, patients with t2dm can be more rapidly and accurately diagnosed. although there are not many monogenic causes of diabetes, such as in maturity onset diabetes in the young (mody), several gene variants have been found through extensive research as causes of t2dm, among others pparg (pro12 ala), kcnj11, capn-10, and the genes for pc-1, pgc-1á , and irs-2.(9) recently the association of the transcription factor 7-like 2 (tcf7l2) gene with t2dm was mapped and consistent gene variants were found in several populations or ethnic groups. the tcf7l2 gene on chromosome 10q is strongly associated with t2dm (10) in ethnic danes, indians, and several asian ethnic groups.(9,11,12) the human tcf7l2 gene consists of 224429 bp and encodes a high mobility group (hmg) box that is a transcription factor for the wnt pathway. the protein transcription product is implicated in the homeostasis of glucose in the blood. its gene variants are associated with an increased risk for t2dm.(13) an appropriate marker or haplotype may furnish indications of increased susceptibility of an individual to t2dm, characterized by a minimum relative risk of 1.2-1.4. one variant of the tcf7l2 gene that is believed to be associated with t2dm is rs7895340, with acagttctagacacctagagagtaaa[a/ g]tgaagaagcctgttttcaggtttcc as sequence motif.(13) the presence of the g allele in rs7895340 is an indication of increased susceptibility to t2dm.(10,14,15) the aim of the present study was to evaluate tcf7l2 gene variant rs7895340 polymorphisms as diagnostic marker for t2dm in ethnic minangkabau. methods design of the study t h i s w a s a c a s e c o n t r o l s t u d y o n a population of ethnic minangkabau patients with type-2 diabetes mellitus who attended the metabolic endocrinology polyclinic at central general hospital dr. m. djamil. controls were taken from healthy ethnic minangkabau without t2dm. the study was carried out at the biomedical laboratory, faculty of medicine, andalas university, from february 2013 until april 2014. subjects of the study the subjects of the study were ethnic minangkabau who were healthy as well patients with t2dm. the diagnostic criteria for cases and controls were fasting blood glucose (fbg) a n d 2 h o u r p o s t p r a n d i a l b l o o d g l u c o s e (2hppbg). the diagnosis of t2dm was based on fbg >126 mg/dl, 2hppbg >200 mg/dl, that of nondiabetic controls fbg <100 mg/dl and 2hppbg <140 mg/dl. the inclusion criteria in this study were: ethnic minangkabau domiciled in west sumatra; b o t h p a r e n t s a n d a l l g r a n d p a r e n t s o f minangkabau ethnicity, and aged 40-60 years. the exclusion criteria were: type 2 prediabetes mellitus; malignancies; chronic disease such as h e p a t i c , r e n a l , p u l m o n a r y d i s e a s e s , a n d pregnancy. recruitment was by consecutive sampling among ethnic minangkabau patients with t2dm who came for treatment to the metabolic endocrinology polyclinic at central general h o s p i t a l d r. m . d j a m i l . h e a l t h y e t h n i c minangkabau without t2dm were used as controls. the sample size was determined from the formula;(16) where n = number of subjects per group; zá = 1.960 ; zâ = 0.842; p = expected proportion of cases p=1/2 (p1+p2); p1= estimated proportion of cases (42%) (3) ; p2 = proportion of controls (subjects without risk factors) (26%).(3) according to the formula, the sample size for each group was 62 (62 cases and 62 controls). in anticipation of drop-outs, each group was expanded by 5%, giving 66 subjects per group. univ med vol. 33 no.3 208 assay for single nucleotide polymorphisms of tcfl2 gene variant rs7895340 blood samples of 2 ml were collected from each subject in tubes containing edta (0.1 mmol/l) and stored at –20 oc before use. for dna extraction we used purelink genomic dna kits (invitrogen). testing for success and e ff e c t i v i t y o f g e n o m i c i s o l a t i o n w a s b y electrophoresis on agarose gel 1.5% at 100 volts for 30 minutes. the dna isolation products were then used as templates for in vitro reaction in the assay for single nucleotide polymorphisms (snps) of tcf7l2 gene variant rs7895340. primers for detection of tcf7l2 gene variant rs7895340 snps were constructed using a primer designer software. the tcf7l2 gene sequence used for primer contruction was obtained from the ncbi gene bank.(13) before application of the primers on the clinical samples, the primer constructs were subjected to computerized testing, to find any mispriming of primers against other regions of the homo sapiens genome. after no evidence of mispriming was found, the primer constructs were ready for synthesis into oligonucleotides. to evaluate the ability of the primers to detect tcf7l2 gene variant rs7895340 snps, amplification was done using the amplification refractory mutation system-polymerase chain reaction (arms-pcr) method. the pcr reaction was carried out with a pcr master mix using the invitrogen rtg kit. its principle consists of using three or more primers for simultaneous amplification of several dna regions. one of the three primers is the specific primer for identification of snp or mutating strains. the occurrence of an snp in a specific region can be detected by constructing a specific primer that can recognize the snp site. to d e t e c t t h e a r m s p c r d n a amplification products, electrophoresis was done on agarose 1.5%. a test sample without rs7895340 polymorphisms (wild type) produces two large fragments of ± 431 bp and ±209 bp, respectively. the presence of one fragment of ± 431 bp indicates a mutation or polymorphism in tcf7l2 gene variant rs7895340. all samples, consisting of 66 t2dm patients and 66 controls, were subjected to tests for tcf7l2 gene variant rs7895340 polymorphisms. selected samples were sequenced for checking and confirmation of the arms-pcr results. description of the primer and the assay for the ability to detect tcf7l2 gene rs7895340 snps were performed at the biomedical laboratory, faculty of medicine, andalas university. ethical clearance the present study obtained ethical clearance f r o m t h e f a c u l t y o f m e d i c i n e , a n d a l a s university. data analysis the significance of the association of tcf7l2 gene variant rs7895340 snps was analyzed by the chi-square test. results three primer constructs were made, i.e. rs78f, rs78r and rs78c. the primers rs78f and rs78c were forward primers, whereas rs78r was the reverse primer. primers rs78f and rs78r were used for dna amplification comprising a region of ± 431 bp (external primer). primers rs78c and rs78r were used for amplification of the region that will directly recognize the rs7895340 snp (internal primer, of ± 209 bp). the rs78f primer sequence was: 5'ccttgtgatgtcttctctcc-3', with gc= 50% and tm c=64oc. the rs78r primer sequence was: 5'-acatcttctctcctca ctc-3', with gc=50% and tm c=61 oc. the rs78c primer sequence was: 5'-gagacctag agagt gaag-3', with gc=50% and tm c=58 oc. visualization of the electrophoresis results (electrophoregram) of the pcr products for the primer pairs rs78c, rs78f and rs78r is presented in figure 1. syamsurizal, yanwirasti, manaf, et al transcription factor 7-like 2 as diagnostic marker 209 figure 1. visualization of arms-pcr assay using the primer pairs rs78c, rs78f and rs78r. d11 = sample code for t2dm patients; n11 = sample code for normal controls; l = 100 bp dna ladder in addition to the use of the arms-pcr method for recognizing rs7895340 snps, this study also used direct dna sequencing. after having been submitted to a blast search, the direct sequencing results yielded a query score of 200, indicating that the pcr results with the rs78 forward and reverse primers indeed originated from a fragment of the tcf7l2 gene. figure 2. alignment sequencing results with tcf7l2 variant rs7895340 snp motif; a. sample with polymorphism resulting in a�g base change; b. sample without polymorphism, with sequencing results identical to the rs7895340 snp motif. the sequencing results were then aligned with the rs7895340 snp nucleotide profile. the alignment results proved that the pcr products with rs12 forward and reverse primers indeed orignated from a fragment of the tcf7l2 variant rs7895340 snp, so that they could be used to detect the snp of interest. the sequencing results are shown in figure 2. the results of association analysis of tcf7l2 variant rs7895340 snp in 66 subjects with t2dm and 66 non-t2dm subjects of minangkabau ethnicity may be seen in table 1. discussion in this study we found a significant association of rs7895340 snp with the risk for t2dm in ethnic minangkabau. our results are consistent with those of several previous studies carried out on a number of ethnic groups in asia,(18) iceland,(15) and finland.(19) table 2 illustrates that rs7895340 is highly correlated with t2dm in ethnic minangkabau. this indicates that minangkabau individuals with an rs7895340 snp have a 2.5-fold probability to suffer from dm in comparison with individuals without the snp. univ med vol. 33 no.3 210 syamsurizal, yanwirasti, manaf, et al transcription factor 7-like 2 as diagnostic marker table 1. association of tcf7l2 variant rs7895340 snp with t2dm subjects the tcf7l2 gene, also known as tcf-4, belongs to the t-cell factor (tcf)/lymphoidenhancing factor family of the hmg box that comprise transcription factors for the wnt signaling pathway, a key component for the r e g u l a t i o n o f c e l l p r o l i f e r a t i o n a n d differentiation.(20) wnt activity is also important for lipid and glucose metabolism, proliferation and functioning of pancreatic beta cells, and production of the glucagon-like peptide-1 (glp1) incretin hormone. (21) together with the tcf7l2 nuclear receptor, wnt signaling is an important factor in glp-1 secretion by intestinal endocrine l-cells. a change in this pathway may trigger decreased glp-1 secretion, which then affects insulin secretion. glp-1, in close association with insulin, plays an important role in the homeostasis of glucose in the blood. it has been established that variation of the t c f 7 l 2 g e n e m a y i n d i r e c t l y t r i g g e r susceptibility to t2dm through alterations in glp-1 levels.(22) diabetes melittus type 2 is a degenerative disease that will appear in an individual carrying a genetic defect after long term genetic changes. with an appropriate diagnostic marker we may be able to predict the possibility of developing the disease at an early stage, which will aid in the prevention and treatment of the disease. on the basis of the obtained data, the tcf7l2 gene variant rs7895340 can be used as a diagnostic marker in ethnic minangkabau. the diagnosis of t2dm susceptibility can be established by detection of polymorphisms in rs7895340. the detection may be done by arms-pcr that can specifically recognize the snp-bearing region. the success of an arms-pcr assay in detecting mutations/snps is highly dependent on the specificity of the constructed primer. the choice of the correct primer will facilitate recognition and analysis of an snp in a sample. e r r o r s i n p r i m e r s e l e c t i o n r e s u l t i n t h e amplification of a different region, which will influence the quality of the assay results. nonspecificity of the selected primer for a given region or snp will preclude detection. therefore the primer construction process is an early and crucial step in snp detection. before being applied to a clinical sample, the primer should subjected to computerized testing, involving not only the site of primer adherence, but also other criteria that may influence the pcr assay. among the several criteria for primer construction to be considered are the tm and g+c content, which will affect the annealing temperature of the primer. other criteria are primer homology to template dna, the possibility of primer dimer and hairpin formation, and various intraand inter-molecular reactions capable of leading to erroneous pcr results.(17) from the enumeration of primer criteria, it may be seen that all three primers were in relatively ideal condition. visualization of the arms-pcr assay using the primer pairs rs78c, rs78f and r s 7 8 r c a n b e s e e n i n f i g u r e 1 . t h e electrophoresis results show the presence of 2 dna bands formed by amplification of the nont2dm (control) blood samples. both bands have estimated positions of ± 239 bp and ± 431 bp, respectively, while amplification of the t2dm templates yielded one band at a position of ± 431 bp. this indicates that in these conditions only the primers rs78f and rs78r were able to amplify the dna target, whereas amplification of primers rs78c and rs78r could not be 211 continued because of the prior occurrence of changes (snps) in the dna sequence of the tcf7l2 gene. the occurrence of an snp in a given region may be found with a primer construct that specifically recognizes the mutant site. this may be done by positioning the 3’ end of the primer exactly within the snp/point mutation sequence. the primer could not amplify the proposed target dna because of a nucleotide change in the template. as a result of the differing pcr target, the taq dna polymerase was unable to continue the amplification process in the region of interest.(17) on the basis of these data, it may be concluded that the arms-pcr assay may be used to detect snps in the tcf7l2 gene, particularly rs7895340 snps. however, this method has a limitation: the assay cannot possibly detect 100% of the snps in the tcf7l2 gene, which contains more than 25 types of snp.(15) in spite of this, its high specificity and sensitivity in detecting snps may be cited as one of the reasons why this method can be used. in comparison with other snp detection methods, arms-pcr has several advantages, being more economic and easily applied. it is faster in execution, since the whole process, from the preparation of reagents and equipment, addition of genomic dna (template), arms-pcr amplification, to agarose gel electrophoresis, can be completed in one day. its rapid application and ease of execution for detection of rs7895340 snps are essential for the prevention of t2dm. this method is relatively ideal to be applied in indonesia, since in contrast with other molecular m u t a n t d e t e c t i o n m e t h o d s ( s u c h a s t h e microarray technique), that use sophisticated equipment, are expensive, and need specialized expertise, the arms–pcr method is simpler. t h i s m e t h o d a p p l i e d t h e p r i n c i p l e s o f simultaneous pcr with a number of primers (multiplex pcr), which are generally available in almost every region in indonesia. although relatively simple, the arms–pcr technique still has a high sensitivity in detecting snps. conclusions the tcf7l2 gene variant rs7895340 may be used as a genetic marker for type 2 diabetes mellitus in ethnic minangkabau. in other words, the polymorphisms in the tcf7l2 gene variant rs7895340 can be used as an indicator of the r i s k f a c t o r c o n t r i b u t i n g t o i n d i v i d u a l susceptibility to type 2 diabetes mellitus in ethnic minangkabau. acknowledgements we hereby express our gratitude to the r e c t o r o f p a d a n g st a t e u n i v e r s i t y, w h o facilitated this study, so that it could be sponsored by a hibah bersaing grant for 2014 f r o m t h e d i r e c t o r a t e g e n e r a l o f h i g h e r education (dirjen dikti), ministry of education and culture, republic of indonesia. we also thank all staff at the biomedical laboratory, faculty of medicine, andalas university, who contributed towards the successful completion of this study. references 1. american diabetes association. diagnosis and classification of diabetes mellitus. diabetes care 2010;33 suppl 1:s62-9. 2. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes, estimate for the year 2000 and projection for 2030. diabetes care 2004;27:1047-53. 3. american diabetes association. standards of medical care in diabetes-2013. diabetes care 2013;36 suppl 1:s11-66. 4. negrato ca, dias jp, teixeira mf, dias a, salgado mh, lauris jr, et al. temporal trends in incidence of type 1 diabetes between 1986 and 2006 in brazil. j endocrinol investig 2010;33: 373-7. 5. perkumpulan endokrinologi indonesia. konsensus pengelolaan dan pencegahan diabetes melitus tipe 2 di indonesia. jakarta: pengurus besar perkumpulan endokrinologi indonesia; 2011. 6. orasanu g, plutzky j. the pathologic continuum of diabetic vascular disease. j am coll cardiol 2009;53:s35-42. univ med vol. 33 no.3 212 7. kemp tm, barr elm, zimmet pz, cameron aj, welborn ta, colagiuri s, et al. glucose, lipid, and blood pressure control in australian adults with type 2 diabetes. diabetes care 2005; 28:1490-2. 8. laing sp, swerdlow aj, slater sd, burden ac, morris a, waugh nr, et al. mortality from heart disease in a cohort of 23,000 patients with insulin-treated diabetes. diabetologia 2003;46: 760-5. 9. radha v, mohan v. genetic predisposition to type 2 diabetes mellitus among asian indians. indian j med res 2007;117:259-74. 10. stolerman es, manning ak, mcateer jb, fox cs, dupuis j, meigs jb, et al. tcf7l2 variants are associated with increased proinsulin/insulin ratios but not obesity traits in the framingham heart study. diabetologia 2009;52:614-20. 11. chang yc, chiu yf, ho llt, ting ct, shih kc, curb jd, et al. tcf7l2 genetic variants and progression to diabetes in the chinese population: pleiotropic effects on insulin secretion and insulin resistance. j mol med 2010; 33:183-02. 12. wu shl, hsieh ch, pei d, hung yj, kuo sw, lin e. association and interaction analyses of genetic variants in adipoq, enpp1, ghsr, pparγ and tcf7l2 genes for diabetic nephropathy in a taiwanese population with type 2 diabetes. nephrol dial transplant 2009;24: 3360–6. 13. national center for biotechnology information, u.s. national library of medicine. transcription factor 7 like 2 gen t-cell specific, hmg-box [homo sapiens (human)]; 2014. available at: http://www.ncbi.nlm.nih.gov/gene/6934. accessed june 6, 2014. 14. florez jc, jablonski ka, bayle n, pollin ti, bakker piw, shuldiner ar, et al. tcf7l2 polymorphisms and progression to diabetes in the diabetes prevention program. n engl j med 2006;355:241-50. 15. grant sf, thorleifsson g, reynisdottir i, benediktsson r, manolescu a, sainz j, et al. variant of transcription factor 7-like 2 (tcf7l2) gene confers risk of type 2 diabetes. nat genet 2006;38:320-3. 16. sastroasmoro s, ismael s. dasar-dasar metodologi penelitian klinis. jakarta: sagung seto;2010. 17. dieffenbach cw, dveksler gs, editors. pcr primer: a laboratory manual. 2nd edition. new york: cold spring harbor laboratory press; 2003. 18. peng s, zhu y, lu b, xu f, li x, lai m. tcf7l2 gene polymorphisms and type 2 diabetes risk: a comprehensive and updated meta-analysis involving 121174 subjects. mutagenesis 2013; 28:25-37. 19. scott lj, bonnycastle ll, willer cj, sprau ag, jackson au, narisu n, et al. association of transcription factor 7-like 2 (tcf7l2) variants with type 2 diabetes in a finnish sample. diabetes 2006;55:2649-53. 20. nelson wj and nusse r. convergence of wnt, β-catenin, and cadherin pathways. science 2004; 303:1483–7. 21. jin t. the wnt signalling pathway and diabetes mellitus. diabetologia 2008;51:1771-80. 22. smith u. tcf7l2 and type 2 diabetes-we wnt to know. diabetologia 2007;50:5-7. syamsurizal, yanwirasti, manaf, et al transcription factor 7-like 2 as diagnostic marker f:\36 no 2\aminah.pmd 94 *department of food and technology, faculty of nursing and health science, semarang muhammadiyah university semarang **department of food and agricultural product technology, faculty of agricultural technology, gadjah mada university, yogyakarta ***department of internal veterinary medicine, faculty of veterinary medicine, gadjah mada universityyogyakarta correspondence : siti aminah, s.tp., m.si faculty of nursing and health science, semarang muhammadiyah university semarang (unimus) jl. kedungmundu raya no. 18 semarang 50273 phone: +6224 76740295 fax : +6224 76740291 email: sitiaminah@unimus.ac.id date of first submission, march 29, 2 01 7 date of final revised submission, august 4, 2017 date of acceptance, august 7, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 consumption of elicited soybean sprout flour increases calcium level in serum of ovariectomized rats siti aminah*, suparmo**, sri naruki**, and hastari wuryastuty*** background the decline in estrogens could lead to an imbalance of bone formation and absorption as well as bone mineralization. phytoestrogens are known to have the potential to maintain bone health. calcium and phosphorus are essential components for bone mineralization. this study aimed to investigate the effect of soybean sprout flour consumption on serum calcium and phosphorus levels in ovariectomized rats. methods a randomized post-test-only control group design involving 25 female sprague-dawley rats aged 2 months, which were divided into five groups: (i) normal controls on standard feed diet ain-93 (c-n), (ii) ovariectomy controls on standard feed diet (ovx -n); (iii) ovariectomized rats on standard feed diet + ethinylestradiol (ovx -e), (iv) ovariectomized rats on standard feed diet + soybean flour (ovx -s); (v) ovariectomized rats on standard feed diet + soybean sprout flour (ovx -ss). the rats were treated for 6 weeks, then on the last day of treatment blood sampling was conducted. analysis of calcium and phosphorus in serum was performed using the arsenazo iii photometric method. data were analyzed using one-way anova. results mean serum calcium level was highest in the ovx-ss group (12.59 mg/ dl ± 1.32), while mean serum phosphorus level was highest in the c-n group (1.708 mg/dl ± 0.28). soybean sprout flour consumption had a significant effect on serum calcium levels (p=0.019), yet it had no effect on body weight and serum phosphorus levels of the rats. conclusion consumption of soybean sprout flour could positively affect serum calcium levels in rats. keywords : calcium, phosphorus, serum, rat ovariectomy, soybean sprout doi: http://dx.doi.org/10.18051/univmed.2017.v36.94-101 original article cite this article as: aminah s, suparmo, naruki s, wuryastuty h. consumption of elicited soybean sprout flour increases calcium level in serum of ovariectomized rats. univ med 2017;36:94-101. doi: 10.18051/univmed.2017.v36.941 0 1 pissn: 1907-3062 / eissn: 2407-2230 95 univ med vol. 36 no.2 introduction bone is a dynamic tissue constantly performing regenerating activity, i.e. absorption and replacement with new cells at a constant rate.(1) the main minerals of bone are calcium and phosphorus (50-70%) in the form of calcium hydroxyapatite [ca10 (po 4) 6 (oh) 2], while the rest are an organic matrix (20-40%); water (5-10%), and fat (3%).(2,3) both ca and p are impor ta nt miner als in the formation and metabolism of bone.(4) serum ca status is associated with ca absorption in the intestine, reabsorption in the kidneys, and resorption from bone.(5,6) ot he r c ompon ents in vo lve d in bone formation and metabolism include estrogen, parathyroid hormone, and vitamin d. estrogen plays an important role in bone metabolism, by promoting bone formation through increased activity of osteoblasts and by in habiting bone calcium resorption.(3) aloia and shieh(7) state that estrogen plays a role in calcium absorption through trans cellular pathways regulated by transient receptor potential vanilloid type 6 (trpv6) channel. trpv6 is expressed from calcitriol and vitamin d receptor (vdr). therefore, estrogen plays a very important role in bone metabolism. deficiency of estrogen in postmenopausal women could lead to increased resorption of ca and p in bone, decreased absorption of ca in the intestine, and increased excretion of ca and p. imbalances of absorption and excretion of ca and p could result in decreased ca and increased p in the blood. the imbalance in the absorption and excretion of ca and p in postmenopausal women can lead to decreased bone density so that the bone becomes brittle and easily fractured.(8) several studies have shown that serum calcium levels are significantly decreased in postmenopausal women. deficiency of estrogens causes calcium loss due to decreased absorption of calcium in the intestine and increased calcium resorption in the kidney.(9-11) ovariectomy causes increased excretion of calcium and phosphorus, resulting in decreased serum calcium and phosphorus levels in ovariectomized rats.(12) oneway to treat estrogen deficiency in postmenopausal women is by means of oral hormone replacement therapy (hrt). however, the use of hrt in the long term has aless favorable influence on women’s health, including triggering of cancer.(13) the alternative to synthetic hormone replacement is treatment with phytoestrogens.(14) soy is one of the food sources containing phytoestrogens, i.e. isoflavones, which have the potential of estrogen in bone me t a b ol i s m. ( 1 5 ) be s i de s a s a s ou r c e o f isoflavones, soy contains compounds, which could inhibit ca absorption, e.g.phytic acid.(16) the phytic acid component of soybeans could be reduced through germination. germinated soybeans have been proven to have better bioactive components than do soybeans. soybean sprouts are soy beans germinating under controlled conditions. compared to mature soybeans, soybean sprouts contain a substantial amount of protein and much higher amounts of various vitamins.(17) changes occurring in the germinated soybeans include increased levels of vitamin c, -tocopherol, crude fiber, and protein.(18) components of secondary metabolites such as polyphenols, isoflavones, phytosterols, flavonoids, polyphenols and the antioxidant capacity are also increased.(19) mugisha (20) reported that phytoestrogen components, especially soy and soybean sprout isoflavones provide are beneficial for bone health. phytoestrogens trigger protein synthesis, decrease the osteoprotegerin/receptor activator of nuclear factor-kappa  ligand (opg/rankl) rat io, and miner aliza tion by osteo blas ts. phytoestrogen administration could inhibit the differentiation and activation of osteoclasts.(21) phytoestrogens directly or indirectly affect the absorption of calcium. miao et al.(12) suggested t ha t a d min i st r a t i on o f i so fl a von e s to ovariectomized rats could prevent osteoporosis t hr ou gh i nhi b i t i on o f c a l c i um l o s s a nd improvement of parathyroid hormone (pth), which acts as a regulator of calcium absorption. 96 the process of elicitation is known to be able to increase secondary metabolite components of soybean sprouts.(22) according to saini et al.(23) elicited soybean sprouts have better nutraceutical potential than control sprouts. nacl is one of the abiotic elicitors, which could be used to improve the secondary metabolite components of soybean sprouts.(24,25) soybean sprouts are likely to have a better potential than ungerminated soybeans for bone health, especially in postmenopausal women. so far, there have been no reports about the effects of soybean sprout flour on ca and p levels in ovariectomized rats. this research aimed to study the potential effect of soybean sprouts after e li ci ta tio n on ca an d p i n the s er um of postmenopausal rats. the postmenopausal models were obtained through ovariectomy (ovary removal operation) of the rats, which were t he n e x p ec t e d t o e x pe r i e nc e e s tr oge n deficiency.(26) methods research design this research was of randomized post-testonly control gr oup design. research was conducted at the preclinical unit of the integrated research and testing laboratory (lppt), gadjah mada university [universitas gadjah mada (ugm)], yogyakarta. the study was conducted from february until may 2015. subjects of study the experimental animals used were female sprague dawley rats aged 2 months obtained from the preclinical unit of lppt, ugm, yogyakarta. sample size determination the sample size was determined based on the formula: s = (t-1) (r-1) 15, where t is the number of groups, and is the number of animals per group.(27) this study used three treatment groups in addition to two control groups, so that t = 5, giving (5-1) (r-1) 15, and resulting in (51) (5-1) = 16. therefore the number of rats (r) in each group was 5. the rats were randomly divided into the following 5 groups: (i) normal control group on standard feed diet ain-93m (c-n); (ii) ovariectomy control group (ovx c) on standa rd fee d diet ain-93m; ( iii) ovariectomy group on standard feed diet ain9 3m + e thi nyl e s t r a d i ol ( ov x -e ) , ( i v) ovariectomy group on standard feed diet ain93m + soybean flour (ovx -s); and (v) ovariectomy group on standard feed diet ain93m + soybean sprout flour (ovx -ss). preparation of the postmenopause rat model after the rats had been adapted for 1 week, in each of the four groups of rats (ii, iii, iv and v), their ovaries were surgically removed (ovariectomy). before the surgery, the rats were first anesthetized using a mixture of ketamine 10% at a dose of 5 mg/kgbw and xylazine 2% at a dose of 5 mg/kgbw, which was injected intramuscularly.(28) preparation of soybean sprout flour the soybeans used in this study were of the local yellow anjasmoro soybean variety obtained from the research institute for legumes and tubers, malang, east java. the soybean germination process began with the elicitation process (soaking) using a 2% nacl solution for 8 hours. germination was conducted for 36 hours in the dark (29) then the sprouts were dried and powdered. intervention evaluation of the potential influence of soybean sprouts on the serum levels of ca and p of the rats was done by comparing the groups of rats receiving either ethinylestradiol (lynoral 0.05 mg/tablet, sydna farma), soybean flour, or soybean sprout flour (groups iii; iv; and v respectively), as well as the two control groups (normal and ovariectomized controls). all rats received the standard feed ain-93m. all materials for intervention were prepared in soluble form. all treatments were given orally aminah, suparmo, naruki, et al soy sprouts increase serum calcium 97 univ med vol. 36 no.2 for 6 weeks, with the amount of soybean flour and soybean sprout flour given based on a dose of 10 g/g bw/day of isoflavones. the rats in group iii were treated with ethinylestradiolat 30 g/kg bw/day. during the experiment, the weight of each rat (expressed in grams) was checked every 2 days. the dose was adjusted to the weight gain of the rats. blood samples of ± 2 ml were taken o n th e l ast da y o f t r e a t me nt us i ng a microhematocrit tube through the orbital sinus. blood serum was separated using a centrifuge (thermo scientific, micro legend 12) at a speed of 3000 rpm for 15 min and then stored at -20° c until the laboratory analysis was performed. laboratory analysis analysis of serum ca and phosphorus was ca rr ie d out u sing a re age nt kit ( dia sys, germany) for the photometric arsenazo iii method. the analytical procedures were done according to the dyasis kit manual. ca levels were read using micro lab spectrophotometer 300 (elitech, france), at wave length () of 620 nm, while the p levels were read at  of 340 nm. both ca and p levels were expressed in mg/dl. statistical analysis the analysis of weight data as well as the serum levels of ca and p were performed using one-way anova with spss version 18. for the test results showing a significant effect, the test was continued with the lsd multiple comparison test. the significance level used was a p-value of <0.05. ethical clearance this study obtained ethical clearance from the commission on ethical clearance lppt ugm. no: 206/kec-lppt/xii/014. results body weight (bw) the mean bw of the rats may be seen in table 1. the results of statistical analysis showed that there was no difference in the bw of the rats in all experimental groups. table 1 shows that the post treatment bw of the rats in all groups was increased. when compared with the bw of the rats in the normal control group, all treatment groups had increased bw serum calcium and phosphorus levels serum calcium and phosphorus levels of the rats are shown in table 2. the treatment group receiving soybean sprouts showed the highest mean calcium level (12.59 ± 1.32 mg/dl), whereas the group treated with soybean flour showed the lowest mean calcium level (10.97 ± 0.68 mg/dl). the highest phosphorus levels were in the normal control group (1.708 ± 0.28 mg/ dl), while the lowest levels were in the group receiving soybeans (1.34 ± 0.29 mg/dl). the results of further statistic tests (table 2) showed that there was a significant effect of treatment on ca levels in serum (p=0.019), yet there was no significant effect on phosphorus levels in serum and the ca/p ratio (p=0.194). multiple comparisons analysis was done for calcium levels to determine which groups had significant differences. the ovx-ss group (treatment with soybean sprout flour) was significantly different from the other 4 groups discussion the study demonstrated that the highest increase in bw was in the treatment group receiving additional soy flour. although there was no difference in terms of bw increases based on statistical test results, in the group of ovariectomized rats there was a tendency to a higher percentage of bw than that in the normal control group. the results obtained in this study do not agree with those reported by park et al.(15) and shin et al.(30) that there was a significant increase of bw in the group of ovariectomized r a t s c o mpar e d t o t ha t i n c o nt r o l r a t s . ovariectomy is associated with increased abdominal fat in rats.the increase in bw in the bb-s ovx group was higher than that in the 98 other groups of ovariectomized rats. it was likely influenced by the greater amount of soybean flour than the amount of soybean sprout flour given. this was due to the treatment being based on the dose of isoflavones (daidzein), while the soy daidzein level (2.97 mg/g db) was lower than that in the soybean sprouts (6.33 mg/g db).the rise in body weight in the ovx-e group was l ow e s t , be c a us e on l y a sol ut i o n of ethynilestradiol was given as estrogen source, so there was no contribution of nutrients such as in soybeans and soybean spr outs. t he administered mean daily amounts of soybean flour and soybean sprout flour at the beginning of treatment were 0.44 g and 0.21 g, respectively, while at the end of treatment the amounts were 0.66 g and 0.32 g, respectively. the possibility exists that nutrients from both ingredients contributed to the body weight. table 2 shows that the levels of serum ca and p of the ovariectomized groups were lower than those of the normal control group, except in the ovx-ss group, where serum ca levels were higher than those of controls. several researchers have shown an association between ca and p levels in ovariectomized rats, as reported by mioa et al.(12) that ovariectomy increases urinary excretion of ca and p, and lowers the levels of ca and p in the serum of the rats. likewise, unis et al.(8) r eported that ovarie ctomized rats experience decreased serum ca, but no change in serum p levels. it is also reported by parket al.(15) and unis et al.(8) that mean serum levels of the minerals ca, p and mg in ovariectomized rats were significantly decreased compared to those of the non-ovariectomized control group. increased excretion of ca and p is likely associated with the role of estrogen on intestinal calcium absorption. the fall in estrogen may be the cause of the low absorption of ca and p in the intestine, so increasing the excretion of ca and p in the feces and urine. this explains why there is a relationship between the decrease of estrogen in the blood circulation and falling of ca and p absorption in the intestinal tract of ovariectomized rats. the decrease in serum calcium status is associated with decreased ovarian function in producing estrogen. estrogen could give either direct or indirect influence on the bone remodeling note: nc = normal controls; ovx-c = ovariectomized controls; ovx-e = ovariectomized rats on estradiol; ovx-s = ovariectomized rats on soybean flour; ovx-ss = ovariectomized rats on soybean sprout flour. data were analyzed with one-way anova.values presented are mean±sd. baseline =body weight of first day of adaptation; pretreatment = body weight after recovery from ovariectomy (2 weeks maintenance). postreatment =body weight at the end of treatment table 1. body weight of rats at baseline and end of treatment table 2. calcium and phosphorus levels in serum of rats after intervention for 6 weeks note: nc = normal controls; ovx-c = ovariectomized controls; ovx-e = ovariectomized rats on estradiol; ovx-s = ovariectomized rats on soybean flour; ovx-ss = ovariectomized rats on soybean sprout flour. data were analyzed with one-way anova. values presented are mean±sd. superscripted asterisk(*) shows significant differences in the row (p<0.05). multiple comparisons analysis for calcium levels aminah, suparmo, naruki, et al soy sprouts increase serum calcium variable groups p nc ovx-c ovx-e ovx-s ovx-ss baseline 109.70 ± 12.5 107.77 ± 14.33 107.98 ± 16.24 107.87 ± 14.10 107.98 ± 4.17 0.9 51 pretreatment 138.47 ± 7.06 138.21 ± 8.29 140.00 ± 13.40 134.67 ± 15.57 141.64 ± 16. 15 0.901 postr eatment 196.27 ± 18.66 200.81 ± 26.38 214.32 ± 21.13 186.62 ± 14.26 206.47 ± 16.17 0.1 28 variable groups p nc ovx-c ovx-e ovx-s ovx-ss serum calcium (mg/ dl) 11.36 ± 0.46 11.01 ± 0.65 11.03 ± 0.50 10.97 ± 0.68 12.59 ± 1.32 * 0.019 serum phosphor us (mg/ dl) 1.71 ± 0.28 1.53 ± 0.23 1.48 ± 0.22 1.34 ± 0.29 1.64 ± 0.1 7 0.194 ca/p rati o 6.83 ± 1.28 7.31 ± 0.95 7.57 ± 1.16 8.41 ± 1.52 7.79 ± 1.4 7 0.417 99 univ med vol. 36 no.2 arr angement. estrogens affe ct calc ium bioavailability and other endocrine activities involved in bone remodeling.(6) it has been proven that ovariectomized rats will experience the estrogen deficiency associated with menopause.(12) such conditions affect the absorption of calcium in the intestinal tract,(31) which is regulated by casrs (calcium sensing receptors) in the parathyroid glands. casr activities are modulated by pth, calcitonin, and 1,25-dihydroxyvitamin d.(32) ovariectomy could also trigger a decline in the number of vitamin d receptors in the jejunum, resulting in decreased cell response signaling of vitamin d in the intestine, leading to a decrease in intestinal calcium absorption.(33) women with osteoporosis experience decreased absorption of minerals caused by a decrease of 1,25-dihydroxy vitamin d (1,25(oh) 2d), which is known to be a regulator of bone mineral homeostasis. low levels of 1,25(oh) 2d will cause low serum calcium, yet increases pth resulting in increased bone resorption.(34) soybean flour and soybean sprout flour were given based on the dose of isoflavones, but the two materials had different effects on serum calcium in the rats. these effects were likely due to several possibilities: first, the action of isoflavones (which have estrogen-like activity) providing indir ect i nfluence on calc ium absorption. isoflavones are known to play a role in maintaining bone density through the inhibition of osteoclasts, and to stimulate bone formation, so that the process could inhibit bone resorption while the bone remodeling process could continue as normal.(35) miao et al.(12) reported that genistein could increase levels of serum minerals and reduce mineral excretion in the urine. the strength of the effect is dose dependent. second, soy contains anti-nutritional compounds including phytic acids that could inhibit the absorption of minerals such as phosphorus, calcium and iron.(36) phytic acid levels in soy will decrease in the germination process, so phytic acid level in soybean sprouts is lower than in soybean seeds.(37) thus, it is possible that the group of ovx-ss rats did not experience inhibition of calcium absorption by the phytic acid. then there is the role of bioactive components in soybeans such as vitamin c, vitamin e, and polyphenols that have antioxidant activity.(37) it is known that the content of bioactive components in soybean sprouts is higher than that of soybean seeds. ( 29 ) the antioxidative characteristics of soybean sprouts have a positive effect on rats experiencing oxidative stress due to ovariectomy. the condition affects calcium absorption as well as bone mineralization. some researchers suggest that the postmenopausal or ovariectomized rats will experience oxidative str ess indicated by the high levels of malonaldehyde (mda) and superoxide dismutase (sod) in serum.(38,39) a limitation of this study was that the soybean sprouts were given to the rats in the form of whole sprouts instead of as an extract. so, it is possible that not only the isoflavones may play a role, but also other bioactive components, thus affecting theresults of this study. the study also did not analyze phytic acid levels in the soybeans and soybean sprouts, so the phytic acid intake by the rats is unknown. the soybean sprouts in this study were prepared through the elicitation process to improve the bioactive components. the elicited soybean sprout products are potential for the maintenance of bone health, especially in postmenopausal women. the positive effects of soybean sprout consumption is strongly influenced by the amount and duration of consumption. further research is needed to assess the potential and safety of elicited soybean sprout extract to know and ensure the potential of the bioactive components of soybean sprouts in the maintenance of bone in particular and human health in general. conclusions post-menopausal rat models, which had been treated with soybean sprout flour for six weeks at a dose of 10 g/g bw/day, showed higher serum calcium levels. elicited soybean sprouts could potentially increase calcium levels in the serum of ovariectomized rats. 100 conflict of interest the authors declare that there is no conflict of interest acknowledgement the authors would like to thank the head and the technical implementation of laboratory, lppt ugm for their cooperation in the accomplishment of this study. contributors sa contributed to drafting the manuscript and the study design. sa and s contributed to acquisition of data and revising the manuscript critically for important intellectual content. sa, s, sn, and hw contr ibuted to data and interpretation. all authors read and approved the final manuscript. references 1. rauner m, stein n, hafbauer lc. basics of bone biology. in: pietschmann p, editor. principles of osteoimmunology – molecular mechanisms and clinical applications. new york: springer vienna; 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2013 dec 21; fatawa publishing;2013.p.162-72. 25. swigonska s, amarowicz r, król a, et al. influence of abiotic stress during soybean germination followed by recovery on the phenolic compounds of radicles and their antioxidant capacity. acta soc bot pol 2014;83:209-18. doi: 10.5586/ asbp.2014.026 26. komori t. animal models for osteoporosis. eur j pharmacol 2015;759:287-94. doi: 10.1016/j.ejphar. 2015.03.028. 27. supranto j. teknik sampling untuk survey dan eskperimen. jakarta: pt rineka cipta; cetakan keempat;2007.p.225-52. 28. hartiningsih, anggraini d, aji d. the response of metafisis distal femur ovariectomized rats consuming calcitiriol supplement. kedokteran hewan 2012;6:92-7. 29. rusydi mrm, azrina a. effect of germination on total phenolic, tannin, and phytic acid content in soy bean and peanut. int food res j 2012;19: 67-7. 30. shin jm, shin e, jo th, et al. effect of hwanggum (scutellaria radix) extract on ovariectomized osteoporotic rats. food sci biotechnol 2013;22: 493-9. 31. abu-taha m, rius c, hermenigildo c, et al. menopause and ovarictomy cause a low grade of systemic inflammation that may be prevented by chronic treatment with low doses of estrogen or losartan. immunology 2009;183:1393-402. 32. poulsen rc, kruger cm. soy phytoestrogens: impact on postmenopausal bone loss and mechanisms of action. nutr rev 2008;4:161021. 33. lips p. vitamin d and bone health. in: burckhardt p, dawson-hubhes b, weaver c, editors. nutritioal influences on bone health. lausanne, switzerland: springer 2010.p.115-25. 34. jumaa ss, ezzat-zadeh z, khalil da, et al. soy protein with or without isoflavones failed to preserve bone density in gonadal hormone– deficient male rat model of osteoporosis. nutr res 2012;32:604-700. 35. dardami ts, nurdiana, nurahmawati e. the effect of blackeyed peason osteoblas and osteoclast of rat with ovariectomy. j kedokteran brawijaya 2011;26:151-5. 36. al hasan sm , hassan m, saha s, et al. dietary phytate intake inhibits the bioavailability of iron and calcium in thediets of pregnant women in rural bangladesh: a cross-sectional study. bmc nutrition 2016;2:1-10. 37. xue z, wang c, zhai l, et al. bioactive compounds and antioxidant activity of mung bean (vigna radiata l.), soybean (glycine max l.) and black bean (phaseolus vulgaris l.) during the germination process. czech j food sci 2016; 34:68-78. 38. jiang jx. reactive oxygen species and oxidative stress in osteoclas to genesis, skeletal aging and bone diseases. j bone miner metab 2015;33: 359-70. 39. rodrigues mfc, stotzer us, domingos mm, et al. effects of ovariectomy and resistance training on oxidative stress markers in the rat liver. clinics 2013;68:1247-54. c:\users\jurnal fkusakti\docume 236 *department of nutrition, faculty of medicine, trisakti university correspondence: dr. verawati sudarma, spgk department of nutrition, faculty of medicine, trisakti university jl. kyai tapa no. 260, grogol jakarta barat 11440 mobile: +62811 9300 682 fax : +6221 5663232 email: y_sudarma@yahoo.com date of first submission, june 8, 2017 date of final revised submission, december 11, 2017 date of acceptance, december 14, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 high skeletal muscle mass is associated with increased serum 25(oh)d levels in elderly verawati sudarma* and lukman halim* background low vitamin d has been associated with various health problems. aging influences body composition, especially body fat and fat-free mass. anthropometric measurements, such as body weight (bw), body mass index (bmi), body fat (bf), skeletal muscle mass (smm), waist circumference (wc) and the waist-height ratio (whtr) represent body composition which many studies proposed will influence serum vitamin d [25(oh)d]. the objective of the present study was to determine which anthropometric measurements were determinants of 25(oh)d levels in elderly. methods a cross-sectional study was conducted involving 126 elderly (>60 years old) men and women at pusat santunan dalam keluarga (pusaka) central jakarta centers. anthropometric measurements [body mass index (bmi), skeletal muscle mass (smm), body fat (bf), and waist circumference (wc)] were determined by bioelectrical impedance analysis using the omron body composition monitor with scales (hbf-375, omron, japan). fasting blood samples were taken to measure 25(oh)d level by electrochemiluminescence immunoassay. multivariate linear regression was used to analyze the data. results the data showed that bmi, bf, and wc were higher than recommended, while smm and serum 25(oh)d were lower. when the analysis was done based on sex, there were significant differences in bf, smm, whtr, and serum 25(oh)d. in the linear regression multivariate analysis of log 25(oh)d with age and body anthropometric measurements, only smm reached significance level (β=0.019; p=0.025). conclusions this study demonstrated a positive association between skeletal muscle mass and serum levels of vitamin d in elderly. keywords : 25(oh)d, elderly, sex, anthropometric measurements doi: http://dx.doi.org/10.18051/univmed.2017.v36.236-242 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: sudarma v, halim l. high skeletal muscle mass is associated with increased serum 25(oh)d levels in elderly. univ med 2017;36:236-42. doi: 10.18051/univmed.2017.v36.236-242 237 univ med vol. 36 no.3 introduction vitamin d attracted much attention during the last years. the ideas about the importance of vitamin d and its connection with both communicable and noncommunicable diseases have made many researchers interested to do various studies. some of the diseases that have been linked to low vitamin d status are osteoporosis, cancer, the metabolic syndrome, a nd c ar di ovas cula r dise as e. ( 1) vit ami n d deficiency is a highly prevalent condition, even in indonesia, a tropical country with abundant sun exposure. vitamin d is a fat-soluble vitamin. there are two major forms of vitamin d, i.e. vitamin d 2 and vitamin d 3 . vitamin d 2 (ergocalciferol) is found in plants and can be consumed as a supplement or in fortified foods.(1) vitamin d 3 (cholecalciferol) is synthesized in the human epidermis or consumed in the form of natural or fortified food sources or as a supplement.(1) vitamin d in the form of 1,25(oh) 2 d is a hormone and the active form of vitamin d. directly or indirectly 1,25(oh) 2 d regulates over 200 genes, including those involved in renin production in the kidney, insulin production in the pancreas, release of cytokines from lymphocytes, production of cathelicidin in macrophages, induction of terminal differentiation, and growth and proliferation of both vascular smooth muscle cells and cardiomyocytes.(1,2) in di vi du al vi t a mi n d s t a tu s c a n b e estimated by measuring plasma 25-hydroxy vitamin d levels [abbreviated to 25(oh)d]. most experts define vitamin d deficiency as a 25(oh)d level of <20 ng/ml (50 nmol/l), vitamin d insufficiency as 21-29 ng/ml (525-725 nmol/ l) and optimal concentration of vitamin d as at least 30 ng/ml).(2) vitamin d deficiency may be due to inadequate dietary intake, inadequate exposure to sunlight, or malabsorption. during aging, body composition changes, leading to increased adiposity and decreased skeletal muscle mass (smm).(3) obesity, defined as excessive fat accumulation, is associated with low 25(oh)d. low levels of vitamin d in obesity has been suggested as due to sequestration in fat, increased clearance by a larger body-fat pool, and decreased sun exposure.(4) se vera l cr oss-sec tiona l st udie s ha ve investigated the relation between vitamin d to body mass index (bmi) dan body fat (bf), and those findings were inconsistent. han et al.(5) showed that there was a significant negative association between bf in adult men and women, but bmi and waist circumference (wc) were not significant in men. a meta-analysis showed a significant inverse weak correlation between serum vitamin d and bmi in the adult population, except for women living in developing countries.(6) a recent meta-analysis concluded that vitamin d deficiency is associated with obesity irrespective of age, latitude, cut-offs to define vitamin d deficiency and the human development index of the study location.(7) regarding the muscle mass, a study on subjects younger than 65 years showed that there was a statistically significant association between low 1,25(oh)d 2 levels and low muscle mass both in men and women.(8 ) a study on elderly concluded that lower 25(oh)d increased the risk of sarcopenia in older men and women.(9) the elderly have a strong need to maintain normal vitamin d status. it has been estimated that 20-100% of elderly men and women are vit a mi n d d e f ic ie nt . ( 2 ) co ns i de r i ng t h e importance of vitamin d and obesity in general health and the tendency to vitamin d deficiency in the elderly, this study’s objective was to determine the relationship between vitamin d and anthropometric measurements in the elderly population. methods research design this cross-sectional study was conducted i n th e pu s at san t un a n da l am ke lu a rg a (pusaka) non-institutional elderly care centers ii, v and x in central jakarta between januarymarch 2016. 238 research subjects subjects were men or women >60 years old and willing to participate in the study. exclusion criteria were: being bedridden, on dialysis and having consumed vitamin d supplements for the last 3 months. the number of subjects needed in the current study was 126, on the basis of r=0.455, with µ=0.05 and power = 90%.(10) the study subjects were selected by simple random sampling from a total of 245 elderly. written informed consent was obtained from each subject. anthropometric measurements the collected data included subj ect characteristics, such as age, sex, and history of family illness. height was measured using a stadiometer or as sitting knee height. body weight (bw), bf, and smm percentage was assessed by means of bioelectrical impedance analysis (bia) using the body composition monitor with scales (hbf-375, omron, japan). body mass index was calculated as weight (kg) divided by height (m) squared. waist circumference was measured with a non-stretch tape with a precision of one millimeter. the measurement was made at the level of the umbilicus, with the subject in the erect position, breathing silently. waist-toheight ratio (whtr) was calculated by dividing waist circumference by height in centimeters. serum 25(oh)d measurement the serum level of 25(oh)d is a good indicator of vitamin d, because it has a slower clearance than 1,25 (oh) 2 d.(11,12) venous blood samples of 5 ml were obtained in the morning after an overnight fast. blood was collected in evacuated tubes and stored at 2-8 oc and transported to the designated laboratory. serum 25(oh)d levels were determined using direct competitive chemoluminescent immunoassay (clia) (diasorin, stillwater, mn). this assay can detect levels ranging from 4.0 to 150 ng/ ml, b a s ed o n 3. 9 0% cv i nt e r -a s s a y pr ec ision . (1 3) t he e xa minat ion f or s er um 25(oh)d was done simultaneously at one point in time. statistical analysis we performed a sex-stratified analysis. the kolmogorov-smirnov normality test was used to assess the normality of distribution of all data. the differences between mean values for each tested variable between man and women were tested by independent student t test or mann-whitney test. multivariate linear regression analysis was u se d t o eva lu a t e a ss o c i a t i on s be t w e e n anthropometric measur ements and serum 25(oh)d. a p-value of <0.05 was considered statistically significant. ethical clearance ethical clearance no. 77/ker/fk/v/2015 was obtained from the ethics committee for medical research, faculty of medicine, trisakti university, prior to the study. results most of the subjects were women (86.5% vs. 13.5%). mean bmi was categorized as obese. mean wc was high for women but within a normal range for men. mean whtr was still in the normal category for people older than 60 years. the median of bf was high for men but in the normal range for women. median 25(oh)d concentration of all subjects indicated vitamin d deficiency (table 1). based on serum 25(oh)d, there were 80.2% subjects who had vitamin d sudarma, halim skeletal muscle mass and serum 25(oh)d variable sex men 17 (13.5%) women 109 (86.5%) age (years) 70.07 ± 6.39 body mass index (kg/m2) 23.41 ± 5.10 body fat (%) 33.83 ± 7.54 skeletal muscle mass (%) 21.72 ± 3.48 waist circumference (cm) 81.22 ± 1.75 waist-to-height ratio (whtr) 0.55 ± 0.08 25(oh)d (ng/dl) 13.9 (4.0 59.3) table 1. characteristics distribution of all subjects (n=126) *data presented as mean ± sd, except for 25(oh)d level as median (min.-max.) 239 univ med vol. 36 no.3 deficiency, 15.9% vitamin d insufficiency and only 4% reached a sufficient level of vitamin d. when an analysis was done by sex, there were significant differences in body fat, skeletal muscle mass, whtr, and serum 25(oh)d, while the other variables were not significantly different (table 2). in the multivariate linear regression analysis between log 25(oh)d and all variables, only smm correlated significantly with serum level of log 25(oh)d. (table 3). high skeletal muscle mass was associated with increased serum level of log 25(oh)d (β=0.019; p=0.025). discussion ou r s t udy s ho we d t ha t t he r e wa s a significant difference in bf percentage between men and women. this result agrees with one other study which showed that women have a higher fat tissue percentage (73.6%) compared to men (16.5%).(14) mean bmi in our study was categorized as obese grade 1 according to the classification for obesity in the asia-pacific population.(15) a study in mexico on 1,968 subjects older than 60 years showed that 62.3% of the subjects were obese.(14) in indonesia, the prevalence of overweight is higher in women (22.9%) compared with men (16.0%).(16)aging is related to changes in body composition, usually marked by a decrease in fat-free mass (ffm) and an increase in body fat mass (bfm). the wc in our study was 81.22 ± 11.75 cm. the simplest method to examine excess abdominal fat (central body fat) is wc, because it correlates with abdominal fat mass (subcutaneous and intra-abdominal). the whtr is a very simple, low-cost anthropometric measurement as an indicator of early health risk if the whtr is >0.5 for the age group between 40-50 years, while the critical value for people over 50 years is >0.6.(17-19) the whtr in our study was borderline, between 0.5 and 0.6, so that we can conclude that there is an increased health risk. the smm was significantly different between men and women. this was in line with the theory that there is sexual dimorphism of smm, with higher smm in men. t he recommended level of serum 25(oh)d is highera b variable men (n=17) women (n=109) p value age (years) 69.71 ± 7.50 70.15 ± 6.29 0.612 body mass index (kg/m2) 22.29 ± 4.88 23.57 ± 5.17 0.333 body fat (%) 24.17 ± 6.28 35.30 ± 6.60 0.0001* skeletal muscle mass (%) 27.39 ± 2.82 20.84 ± 2.66 0.0001* waist circumference (cm) 81.52 ± 13.48 81.18 ± 11.63 0.911 waist-to-height ratio (whtr) 0.51 ± 0.08 0.55 ± 0.07 0.043* 25(oh)d (ng/dl) 16.9 (12.4-59.3) 13.4 (4.7-30.1) 0.004** table 2. characteristics of subjects based on sex *p<0.05 t-test; ** p<0.05 mann-whitney; data presented as mean ± sd, except for 25(oh)d level as median (min.-max.) variable log 25(oh)d p value β beta age (years) 0.001 0.048 0.619 body mass index (kg/m2) 0.005 0.146 0.541 body fat (%) 0.001 0.047 0.745 skeletal muscle mass (%) 0.019 0.344 0.025* waist circumference (cm) 0.001 0.087 0.801 waist-to-height ratio (whtr) 0.232 0.099 0.785 table 3. multivariate linear regression analysis between the log-transformed 25(oh)d and other parameters *p<0.05; β: regression coefficient; beta: standardized regression coefficient 240 for people living in tropical countries. unfortunately, a high prevalence of vitamin d deficiency is also reported in tropical countries, including indonesia.(20) our study showed very low serum vitamin d levels in the elderly. a previous study in indonesia showed the same result, i.e. that there is a high prevalence of vitamin d deficiency in the elderly.(21) an increase in 24(oh)ase gene expression and in 1,25 (oh) 2 d 3 clearance with age has been reported. the study of angel et al.(22) showed an increase in the proportion of vitamin d deficiency in older age, especially in women. serum 25(oh)d was significantly different between men and women in our study, where women were found to be at higher risk of vitamin d deficiency. the study by huang et al. (23) showed that in the elderly, the female gender is a risk factor for low vitamin d status. the sex difference in correlation with vitamin d was also found in other studies.(24-26) the low serum level of 25(oh)d in our study might have been influenced by the great number of female participants. this may be explained by the observation that women generally prefer indoor activities and take more precautions against sunlight exposure. avoidance of sunlight exposure was found to be the reason for the low serum 25(oh)d, especially in women. moreover, most of the women in this study wear the hijab (headscarf) which will reduce sun exposure of the skin. to the best of our knowledge, no studies have examined the correlation between vitamin d level and whtr. although in our study the correlation between whtr was not statistically significant, it could be considered for further exploration. the multivariate linear analysis in our study showed that high skeletal muscle mass was significantly associated with increased serum 25(oh)d level. the study of ko et al.(25) showed similar results, in that there might be a positive relationship between 25(oh)d and smm. on the contrary, meng at al.(27) found no significant association between 25(oh)d and appendicular smm in elderly. other parameters in our study showed no significant association with log 25(oh)d. a linear regression analysis conducted by han et al.(5) also showed no association between bmi and wc, although bf was inversely associated with serum 25(oh)d. aging is related to changes in body composition, usually marked by a decrease in fat-free mass (ffm) and an increase in body fat mass (bfm). bmi may reasonably predict adverse outcomes in global population-based adult studies, but recent studies have demonstrated that bmi cutoffs ma y mi sre pr es ent t he d egr ee o f a dver se outcomes in the older population.(28,29) that is why, in older adults, bmi may be a suboptimal marker for adiposity. this study used bioimpedance analysis (bia) to assess body fat. bia is less accurate in older adults and can be influenced by food consumption, exercise, ethnicity, and certain medical conditions. a limitation of the present study is that food intake of the subjects was not assessed because nearly all of the subjects have the same meal pattern as part of pusaka policies. the use of whtr as a tool to assess visceral fat could be considered besides wc and bmi. most of the studies used bmi or wc to analyze obesity or fat content, a smaller number used bf analysis and only very few of the published studies used whtr. another limitation of the present study is the lack of data on physical activity. in the elderly, the tendency to vitamin d deficiency is relatively high, so that they should be advised to increase serum 25(oh)d by more direct sunlight exposure and higher intake of food sources of vitamin d. it is recommended that indonesians should have sunlight exposure for 25 minutes at 09.00, or 15 minutes at 09.0010.00 to have a vitamin d concentration of 2700 iu in the blood for each exposure, and minimally 3x/week to prevent the development of vitamin d deficiency.(26) the elderly should be advised to maintain their level of smm by consuming a moderate amount (25-30 g) of high-quality protein and do sudarma, halim skeletal muscle mass and serum 25(oh)d 241 univ med vol. 36 no.3 resistance exercises. (30) it is suggested that the elderly also maintain a normal body weight so that their bmi, wc, bf, whtr remain in the normal range. this can be done by showing healthy food models to the elderly and the pusaka policymakers. supplementation of either vitamin d2 or vitamin d3 can be used to treat and prevent vitamin d deficiency. conclusions our study demonstrated that high skeletal muscle mass is associated with increased serum levels of vitamin d in the elderly. further studies of prospective design are needed to examine whether vitamin d supplementation has beneficial effects on muscle mass among the elderly. conflict of interest co mp e t i ng i n t e r e s t s : n o r e l e va nt disclosures. the authors declare no conflict of interest in relation to this article. acknowledgement the authors wish to thank dr. dr. rina k k u s uma r a tna , m . k e s . f o r a dvi c e a n d suggestions in the successful completion of this study. contributors vs originated the idea for this study, did the resea rc h proposa l, dat a ana lysis and prepared the manuscript. lh contributed to the research proposal, reviewed the analysis and participated in the manuscript preparation. vs and lh conducted the data analysis and revised the manuscript. both authors read and approved the final version. references 1. holick mf. vitamin d deficiency. n engl j med 2007;357:266-81. 2. holick mf, binkley nc, bischoff-ferrari ha, et al. evaluation, treatment, and prevention of vitamin d deficiency: an endocrine society clinical practice guideline. j clin endocrinol metab 2011;96:1911-30. 3. batsis ja, mackenzie ta, bartels sj, et al. diagnostic accuracy of body mass index to identify obesity in older adults: nhanes 19992004. int j obes 2016;40:761-7. 4. kull m, kallikorm r, lember m. body mass index determines sunbathing habits: implications on vitamin d levels. intern med j 2009;39:256-8. 5. han ss, kim m, lee smm, et al. association between body fat and vitamin d status in korean adults. asia pac j clin nutr 2014;23:65-75. 6. saneei p, salehi-abargouei a, esmaillzadeh a. serum 25-hydroxyvitamin d levels in relation to body mass index: a systemic review and metaanalysis. obes rev 2013;14:393-404. 7. pereira-santos m, costa prf, assis amo, et al. obesity and vitamin d deficiency: a systemic review and meta-analysis. obes rev 2015;16:341-9. 8. marantes i, achenbach sj, atkinson ej, et al. is vitamin d a determinant of muscle mass and strength? jbmr 2011;26:2860-71. 9. visser m, deeg djh, lips p. low vitamin d and high parathyroid hormone levels as determinants of loss of muscle strength and muscle mass (sarcopenia): the longitudinal aging study amsterdam. j clin endocrinol metab 2008;88: 5766-72. 10. kuwabara a, tsugawa n, kondo h, et al. associations between serum 25-hydroxyvitamin d3 level and skeletal muscle mass and lower limb muscle strength in japanese middle-aged subjects. osteoporosis sarcopenia 2017;3:53-8. 11. palomer x, gonzález-clemente jm, blanco-vaca f, et al. role of vitamin d in the pathogenesis of type 2 diabetes mellitus. diab obes metabol 2008: 10:185–97. 12. wimalawansa sj. vitamin d in the new millennium. curr osteoporosis rep 2012;10:4–15. 13. sari dk, al rasyid h, lipoeto ni, et al. are avoiding sunlight exposure and low physical activity resulting micro evolution in tropical country women? scirj 2013;1:18-26. 14. sánchez-garcía s, garcía-peña c, duque-lópez mx, et al. anthropometric measures and nutritional status in a healthy elderly population. bmc public health 2007;7:2. doi: 10.1186/14712458-7-2. 15. who expert consultation. appropriate bodymass index for asian populations and its implications for policy and intervention strategies. lancet 2004;363:157-63. 242 16. sari k, mansyur m. female, live in urban, and the existence of a caregiver increased risk overnutrition in elderly: an indonesian national study 2010. health sci indones 2012;3:9e14. 17. schneider hj, friedrich n, klotsche j, et al. the predictive value of different measures of obesity for incident cardiovascular events and mortality. j clin endocrinol metab 2010;95:1777–85. 18. lee cm, huxley rr, wildman rp, et al. indices of abdominal obesity are better discriminators of cardiovascular risk factors than bmi: a metaanalysis. j clin epidemiol 2008;61:646–53. 19. browning lm, hsieh sd, ashwell a. systematic review of waist-to-height ratio as a screening tool for the prediction of cardiovascular disease and diabetes: 0.5 could be a suitable global boundary value. nutr res rev 2010;23:247–69. 20. handono k, ranuwijaya lk, fitri le, et al. the correlation between serum concentration of vitamin d with vitamin d receptor expression and disease activity in indonesian patients with systemic lupus erythematosus: a preliminary study. trop life sci res 2014;4:113-8. 21. hidayat r, setiati s, soewondo p. the association between vitamin d deficiency and type 2 diabetes mellitus in elderly patients. acta med indones 2010;42:123-9. 22. angel b, sanchez h, lera l, et al. vitamin d deficiency/insufficiency and obesity and metabolic disorders in community-living chilean elderly people. jarcp 2013;2:251-6. 23. huang c, huang ya, lai y, et al. prevalence and predictors of hypovitaminosis d among the elderly in subtropical region. plos one 12: e0181063. doi: https://doi.org/10.1371/journal. pone. 0181063. 24. lim js, kim km, rhee y, et al. gender-dependent skeletal muscle effects of vitamin d deficiency in a younger generation. j clin endocrinol metab 2012;97:1995-2004. doi: 10.1210/jc.2011-3098. 25. ko mj, yun s, oh k, et al. relation of serum 25hydroxyvitamin d status with skeletal muscle mass by sex and age group among korean adults. br j nutr 2015;114:1838-44. 26. setiati s, oemardi m, sutrisna b. the role of ultraviolet b from sun exposure on vitamin d and parathyroid hormone level in elderly women in indonesia. asian j gerontol geriatr 2007;2:12632. 27. meng l, man q, yuan l, et al. serum 25hydroxyvitamin d and elderly skeletal muscle mass and function in urban north china. asia pac j clin nutr 2017;26:849-55. 28. batsis ja, sahakyan kr, rodriguez-escudero jp, et al. normal weight obesity and mortality in united states subjects 60 years of age (from the third national health and nutrition examination survey). am j cardiol 2013;112:1592–8. 29. batsis ja, singh s, lopez-jimenez f. anthropometric measurements and survival in older americans: results from the third national health and nutrition examination survey. j nutr health aging 2014;18:123–30. 30. english kl, paddon-jones d. protecting muscle mass and function in older adults during bed rest. curr opin clin nutr metab care 2010;13:34-9. sudarma, halim skeletal muscle mass and serum 25(oh)d oktavianus 25 *department of basic veterinary medicine, faculty of veterinay medicine, airlangga university **department of parasitology, faculty of medicine, airlangga university ***department of parasitology, faculty of medicine, brawijaya university, malang correspondence drh. lilik maslachah, m.kes. department of basic veterinary medicine, faculty of veterinay medicine, airlanggga university kampus c mulyorejo surabaya 60115 phone: +6231-5992785 fax : +6231-5993015 email: lilik.maslachah@yahoo.com univ med 2015;34:25-34 doi: 10.18051/univmed.2016.v35.25-34 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background the presence of plasmodium falciparum resistance and decreased efficacy of artemisinin and its derivatives has resulted in the issue of malaria becoming increasingly complex, because there have been no new drugs as artemisinin replacements. the aims of this research were to evaluate in vitro changes in ultrastructural morphology of p. falciparum 2300 strain after exposure to artemisinin. methods the research used an experimental design with post test only control group. cultures of p. falciparum 2300 strain in one control and one mutant group were treated by exposure to artemisinin at ic 50 10-7 m for 48 hours. ultrastructural phenotypic examination of ring, trophozoite and schizont morphology and developmental stage in the control and mutant group were done at 0, 12, 24, 36, 48 hours by making thin blood smears stained with 20% giemsa for 20 minutes and examined using a microscope light at 1000x magnification. results dormant forms occurred after 48 hours of incubation with ic 50 10-7 m artemisinin in the control group. in the mutant group, dormant forms, trophozoites with blue cytoplasm and normal schizont developmental stages were seen. ultrastructural phenotypic morphology at 0, 12, 24, 36, 48 hours showed that in the control group dormant formation already occurred with exposure to ic 50 10-7 m, while in the mutant group dormant formation occurred only with exposure to ic 50 2.5x10-5 m. conclusion exposure to artemisinin antimalarials in vitro can cause phenotypic morphological changes of dormancy in p. falciparum papua 2300 strain. keywords : artemisinin, p. falciparum 2300, phenotype, resistance induction of plasmodium falciparum strain 2300 dormant forms by artemisinin lilik maslachah*, yoes prijatna dachlan**, chairul a.nidom*, and loeki enggar fitri*** doi: http://dx.doi.org/10.18051/univmed.2015.v34.25-34 26 maslachah, dachlan, nidom, et al plasmodium falciparum strain 2300 induksi bentuk dorman plasmodium falciparum galur 2300 oleh artemisinin latar belakang resistensi parasit p.falciparum dan penurunan efek artemisinin dan derivatnya menyebabkan masalah malaria menjadi semakin komplek, karena belum ada obat baru pengganti artemisinin. penelitian ini bertujuan untuk menilai efek paparan artemisinin terhadap morfologi ultrastruktur plasmodium falciparum galur 2300. metode penelitian menggunakan experimental design dengan post test only control group. kultur p. falciparum galur 2300 kelompok kontrol dan kelompok mutan diberikan perlakuan paparan artemisinin dengan dosis ic 50 10-7 m selama 48 jam, kemudian dilakukan pengamatan gambaran ultrastruktur p.falciparum galur 2300. pemeriksaan fenotipik ultrastruktur stadium perkembangan dan morfologi ring, trofosoit dan skizon pada kelompok kontrol dan kelompok mutan dilakukan setiap 12 jam setelah dipapar artemisinin. selanjutnya pada jam ke 0, 12, 24, 36, 48 dibuat hapusan darah tipis yang diwarnai dengan giemsa 20 % selama 20 menit dan dilakukan pemeriksaan menggunakan mikroskop cahaya perbesaran 1000x. hasil bentuk dorman terjadi setelah 48 jam diinkubasi artemisinin dosis ic 50 10-7 m pada p. falciparum 2300 kelompok kontrol sedangkan pada kelompok mutan p. falciparum 2300 gambaran fenotipik ultrastruktur terdapat stadium perkembangan bentuk dorman, trofozoit dengan sitoplama berwarna biru dan skizon normal. gambaran fenotipik ultrastruktur pada kelompok kontrol dan kelompok mutan pada jam ke 0, 12, 24, 36, 48 menunjukkan pada kelompok kontrol dengan paparan dosis ic 50 10-7 m sudah terjadi bentukan dorman sedangkan pada kelompok mutan dengan paparan dosis ic 50 2.5x10-5 m baru terjadi bentuk dorman. kesimpulan bentuk dorman terjadi di kelompok mutan hanya dengan peningkatan dosis paparan artemisinin. kata kunci : artemisinin, fenotip, p. falciparum galur 2300, resistensi abstrak introduction malaria is one of the infectious diseases with a global distribution, ranging from the tropics, subtropics to temperate climates. malaria is still a public health problem in more than 90 countries that are inhabited by 2.4 billion people or 40% of the world population. in 2008, the world health organization (who) estimated that there were approximately 243 million cases of malaria and 886.000 deaths because of malaria,(1) most of them in sub-saharan africa due to falciparum malaria in children under the age of five years. in addition to its impact on health, malaria imposes a heavy economic burden on individuals (2) and entire economies.(3) prevention efforts against malaria have been carried out, but the morbidity and mortality rates of malaria in some countries are still high. among the factors that cause difficulties in malaria p r e v e n t i o n , plasmodium r e s i s t a n c e t o antimalarial medications is the factor which is 27 univ med vol. 34 no.1 most difficult to overcome because of the occurrence of mutations in the genome of plasmodium that are difficult to control.(1) the most recent and currently used drugs for malaria therapy are artemisinin and its derivatives, but there have been indications that the plasmodium parasites are now resistant to these drugs.(4) a clinical study found two patients in cambodia who had been infected with plasmodium falciparum (p. falciparum) to be resistant to artesunate.(5) because of that, the eradication of malaria has become more complex and dangerous. it is one of the world health problems that have to date not yet been resolved because of the absence of artemisinin substitutes. the development of plasmodium resistance to antimalarial drugs which occurs faster than the development of new antimalarials becomes a consideration for seeking solutions to the accurate and efficient therapeutic management of malaria. resistance of p. falciparum to artemisin may be influenced by internal factors of p. falciparum, due to changes in the parasite itself to allow it to survive and adapt to environmental changes caused by drug exposure. the results of studies have pointed to the deceleration of the developmental life cycle and the induction of the expression of genes that code for proteins (protein overexpression) as one of the important mechanisms for the plasmodium parasite to free themselves from the effects of antimalarial medication and still be able to survive.(6) another research study was conducted by molecular monitoring of the genes that are involved in p. falciparum resistance to antimalarials.(7,8) the research results found that combined artemisinin resistance may be due to mutations in the p. falciparum adenine triphosphatase 6 (pfatpase6) gene.(7) although the mechanism of artemisinin resistance is unclear, it is suspected that there are changes in antimalarial resistance at phenotypic, proteomics, and genotypic levels in plasmodium. at the genotypic level, one of the changes are due to a mutation in the pfatpase6 gene and upregulation of expression of gene transcription.(9) the relationship of repeated a r t e m i s i n i n e x p o s u r e w i t h p h e n o t y p i c , proteomics and genotypic changes in chloroquine resistant p. falciparum has not been proved, necessitating the present study. previous research on the p. falciparum f32tanzania strain that was exposed for 3 years to artemisinin at low concentrations ranging from 0.01 µm up to 10 µm for 100 exposure times. drug-selected f32-art parasites were the result of treatment with high doses of artemisinin (art) for 48 h to 96 h, after which the parasites were returned to normal culture without the drug for 21 days until parasitemia reached 5%. the drug-selected parasite f32-art could recover from art treatment in a much shorter time than the f32-tanzania did and survive.(10) another research showed that p. falciparum strains gc06 and ch3-61 before and after selection with artemisinin at increasing concentrations from 0 to 20 nm and 0 to 100 nm, respectively, the ic 50 value of the selected and viable gc06 parasites increased. (11) this study was conducted to evaluate the effect of in vitro artemisinin exposure at ic 50 doses on the morphological changes in p. falciparum 2300 mutant strain. methods research design the research used an experimental design with post test only with control group. cultures of p. falciparum 2300 mutant strain were used in the control group and mutants. parasitemia and morphological examinations were conducted at the faculty of veterinary medicine, airlangga university, from march to october 2013. research sample the samples that were used as controls in this study were cultures of p. falciparum 2300 mutant strain that had become resistant to chloroquine, whereas for the mutant cultures of p. falciparum 2300 mutant strain were used that had become resistant to both chloroquine and artemisinin. 28 maslachah, dachlan, nidom, et al plasmodium falciparum strain 2300 experimental design the plasmodium cultures were divided into two treatment groups, i.e. the control group and the mutant. the control group was exposed to artemisinin in vitro at a concentration equivalent to ic 50 10-7 m, whereas the mutant was exposed to artemisinin at a dose of ic 50 2.5x10-5 m, both g r o u p s b e i n g e x p o s e d f o r 4 8 h o u r s . ultrastructural phenotypic examination of ring, trophozoite and schizont morphology and developmental stages were observed in both groups at 0, 12, 24, 36, 48 hours. culture and morphological examination cultures of p. falciparum 2300 mutant strain that had been stored in liquid nitrogen were thawed by the rowe method. one millimeter of the erythrocyte suspension was taken and mixed with 9 ml of complete medium plus 15% human serum type o, then put into a culture flask and incubated in a co 2 incubator at 37°c, under 5% co 2 , 5% o 2 and 90% n 2 . medium replacement was done carefully every 48 hours using a sterile pasteur pipette. a sample of the sediment was taken to make a smear to determine parasitemia, then 9 ml of medium was added to each culture flask and the culture was incubated again. phenotypic observations of the morphology a n d d e v e l o p m e n t a l s t a g e s o f t h e i n t r a erythrocytic cycle of p. falciparum 2300 strain were carried out on synchronized and nonsynchronized cultures that had been incubated for 48 hours. plasmodium cultures were divided into two treatment groups, i.e. the control group and mutant group with artemisinin exposure in vitro at ic 50 10-7 m for 48 hours. observations on developmental stages and morphology of ring, trophozoite and schizont stages in the control group exposed to artemisinin in vitro at ic 50 10-7 m a n d i n t h e m u t a n t g r o u p e x p o s e d t o artemisinin in vitro ic 50 at 2.5x10-5 m were done at 0, 12, 24, 36, 48 hours on thin blood smears stained with 20% giemsa for 20 minutes and examined using a light microscope at 1000x magnification.(6,12) data analysis morphological (ultrastructural) data of p. falciparum 2300 strain control and mutant g r o u p s w e r e c o m p a r e d a n d a n a l y z e d descriptively results figure 1 shows the differing percentages of growth inhibition of p. falciparum 2300 strain between the control and mutant groups, both of which had been exposed to artemisinin at 10-7 m concentration and incubated for 48 hours. the morphology of p. falciparum papua 2300 strains in the control and mutant groups before and after 48-hour exposure to artemisinin is presented in figure 2 below. the results of morphological description of artemisinin exposure every 12 hours in the control and mutant groups are presented in figures 3 and 4 below. as seen in figure 2, the morphology of p. falciparum 2300 strain were different in the control group as compared with the mutant g r o u p , b e f o r e a n d a f t e r t r e a t m e n t w i t h artemisinin at ic 50 10-7 m concentration and 48 hours of incubation. from figures 3 and 4 it is a l s o a p p a r e n t t h a t t h e d e v e l o p m e n t a l m o r p h o l o g i e s o f p. f a l c i p a r u m o f t h e synchronized control and mutant 2300 strains a f t e r e x p o s u r e t o a r t e m i s i n i n a t i c 50 concentrations and 12-hourly observation, were different from each other. discussion the results of the study showed that in the control group that was exposed to artemisinin at 10-7 m concentration and incubated for 48 hours, the growth percentage decreased to 35% and g r o w t h i n h i b i t i o n d e c r e a s e d t o 6 5 % . morphological description showed that there were dormant forms. the mutant group that was exposed to artemisinin at 10-7 m concentration and incubated for 48 hours had a growth 29 percentage of 92% and growth inhibition of 8%. morphological description also showed dormant forms, trophozoites with cytoplasm that still appear blue and the presence of normal schizonts containing merozoites with brownish black pigment (figures 1 and 2). the results of this study showed that the parent p. falciparum 2300 strain requires smaller artemisinin concentrations to show growth inhibition compared to the mutant p. falciparum 2300 strain that had become resistant to artemisinin, which required greater artemisinin concentrations to show growth inhibition of the parasites. it can already be seen from the morphological description that at the same artemisinin concentration (10-7 m), there are morphological changes in the mutant group when compared to the control group. in the mutant group, there are morphological changes in their ring, schizont and trophozoite developmental stages, which are normal. therefore, from the morphological description it can be concluded that to obtain the same dormant period a greater concentration of artemisinin is required. the results of this study also showed that the dormant period can occur in parasite strains that are resistant to artemisinin, but it needs a higher drug concentration for its induction. plasmodium parasites that are resistant to artemisinin cannot be induced into a dormant period if there had been tolerance to the drug concentration. it is possible that the dormant figure 2. parent and mutant p. falciparum morphology after being exposed for 48 hours to artemisinin at ic 50 dose (1000x magnification). giemsa staining. arrow color codes: black = dormant, red = ring, yellow = trophozoite, green = schizont, blue = merozoite. (a) parent p. falciparum 2300 strain, (b) parent p. falciparum 2300 strain after being exposed to artemisinin 10-7 m for 48 hours, (c) mutant p. falciparum 2300 strain, (d) mutant p. falciparum 2300 strain after being exposed to artemisin10-7 m for 48 hours figure 1. percentage growth inhibition of p. falciparum 2300 strains in control and mutants exposed to artemisinin c a b d univ med vol. 34 no.1 30 maslachah, dachlan, nidom, et al plasmodium falciparum strain 2300 0 hour 12 hours 24 hours 36 hours 48 hours control artemisinin at 10-7 m concentration figure 3. morphology of p. falciparum 2300 strain synchronized in the control group, treated with artemisinin at ic 50 10-7 m concentration, and monitored every 12 hours (1000x magnification). giemsa staining. black arrow = dormant, red = ring, yellow = trophozoite, green = schizont, blue = merozoite 31 univ med vol. 34 no.1 0 hour 12 hours 24 hours 36 hours 48 hours viable mutant artemisinin at 2.5x10-5 m concentration figure 4. morphology of p. falciparum 2300 strain synchronized in the viable mutant group, treated with artemisinin at ic 50 2.5x10-5 m concentration, and monitored every 12 hours (1000x magnification). giemsa staining. black arrow = dormant, red = ring, yellow = trophozoite, green = schizont, blue = merozoite 32 maslachah, dachlan, nidom, et al plasmodium falciparum strain 2300 parasites use innate mechanisms to survive the stressor, i.e. the drug concentration that can cause severe damage to the parasite, and that the dormant parasites can also be triggered when parasite growth is inhibited.(13,14) the results of this research are in agreement with those of research conducted on p.falciparum gc06 and ch3-61 strains before and after s e l e c t i o n w i t h a r t e m i s i n i n a t i n c r e a s i n g concentrations of 0 to 20 nm and 0 to 100 nm, respectively, where viable parasites showed an increase in ic 50 strain values after selection with artemisinin. ic 50 strain values increased in the first gc06 strain from 3.1 ± 0.1 nm to 12.5 ± 1.6 nm and in the first ch3-61 strains that from 28.8 ± 1.3 nm to 58.3 ± 4.5nm.(11) the results of research on p. falciparum tanzania f32 exposed to artemisinin for 3 years at low concentrations ranging from 0.01 µm up to 10 µm for 100 exposure times, resulting in the selection of f32-art strains, showed that at higher concentrations of artemisinin exposure (35 µm and 70 µm) for 96 hours, only the f32art strain was able to survive.(10) stage of development and morphology of the intra-erythrocytic cycle of p. falciparum 2300 strain that was synchronized in the control group, t r e a t e d b y a r t e m i s i n i n e x p o s u r e a t i c 5 0 concentration and observed every 12 hours, from 0 to 48 hours showed normal morphology development with faster growth compared with the group treated with artemisinin exposure at ic 50 concentration. the treatment group that was e x p o s e d t o a r t e m i s i n i n s h o w e d d o r m a n t morphology development with nuclear chromatin condensation and if able to survive exposure to artemisinin, it only survives up to 24 hours after exposure, with imperfect ring and trophozoite stages. the results of this study showed that cell cycle development in the control group proceeds normally while p. falcifarum in the mutant group that was not exposed to artemisin had a faster intra-erythrocytic life cycle. the increased growth of p. falciparum that had been exposed to artemisinin is caused by upregulation of gene transcription (multi-gene) that plays a role in cell cycle regulation, transport of substances from erythrocytes into the parasite, the enzymes involved in the biosynthesis of purines in dna synthesis and synthesis of proteins that play a role in the adaptation of parasites to the environment in the early stage of parasite growth (16-20 hours) during the development of the ring forms into trophozoites and at the end of development (36-40 hours) when trophozoites develop into schizonts.(10,15) i n t h e g r o u p t h a t w a s t r e a t e d w i t h artemisinin exposure at ic 50 concentration, there was decreased intra-erythrocytic development of p. falcifarum. the results of this study are comparable to those of research by veiga et al.(6) who performed mefloquine exposure on three strains of plasmodium (w2, 3d7 and fcb). compared with the control treatment, 40% of cell morphology showed retarded development. exposure to anti-malarial medications caused a 1.5-fold increase in pfmdr1, pfcrt, pfmrp1 and pfmrp2 gene expression. similarly, upon quinine exposure for 12 hours, there was a slowdown in the development of cell morphology. the results of this research demonstrate that a slowdown in the development of p. falciparum is a very important mechanism for the parasite to be able to escape from the influence of anti-malarial medications.(6,16) morphological dormancy in p. falciparum that have been exposed to artemisinin is a defense mechanism for the parasites to be able to survive from the exposure of artemisinin anti-malarial medication. the parasites will be able to grow normally after the pressure from the drug is removed. in this dormant period, parasites can survive in a few days by slowing down the process of metabolism to limit the effects of the medication, because in this dormant period there is no dna synthesis.(10,17-19) parasites that survive in the trophozoite stage from exposure to artemisinin have abnormal morphology, with formation of condensed cytoplasm. in p. falciparum that have been exposed to artemisinin for 48 hours, 33 m o r p h o l o g i c a l c h a n g e s o c c u r f r o m t h e trophozoite stage until the final phase of the life cycle. artemisinin exposure causes metabolic changes in the parasites that affect its growth and development, as can be observed from the morphological abnormalities. the changes start to occur at the trophozoite stage because at this stage, the parasites begin a process of active metabolism, growing larger in size from initial trophozoites into mature trophozoites. the nutrients extracted from the erythrocyte cytosol will be consumed quickly in the digestive vacuole that has started to form. the hemoglobin degradation process into oligopeptides and heme by proteolysis in the digestive vacuole is initiated to fullfil the nutritional needs of the parasite.(20,21) a b n o r m a l m o r p h o l o g y a n d g r o w t h inhibition after artemisinin exposure is also caused by the inhibition of parasite proteases (plasmepsin, falcipain and falcilysin) which are essential for parasite growth. research by bonilla et al.(22) showed that plasmodium k n o c k o u t m u t a n t s ( t r i p l e a n d q u a d r u p l e plasmepsins knockout mutants, pmko) in the protease enzyme have a deficiency in the endosomal vesicles that enter the digestive vacuole and produce multilamellar bodies, causing a deficiency in hemoglobin digestion and inhibition of hemozoin formation in the digestive vacuole, thus slowing growth. barriers artemisinin in endocytosis causes an inhibition of transport vesicle fusion into the digestive vacuole, so that the digestion of hemoglobin does not occur or is blocked. the relationship between endocytic transport and cellular signal transduction pathways that lead to inhibition of endocytosis will slow the growth of the parasites and result in reduction of parasitemia and parasite mortality.(23,24) one limitation of this research is that it was conducted only with the p. falciparum papua 2300 strain. a better way would have been to use more than one strain, so that the results could be compared. the implication of this research is that it may explain the mechanism of the development of resistance to artemisinin through phenotypic dormancy of p. falciparum which can cause a decrease in artemisinin efficacy and give rise to recrudescence and artemisinin treatment failure. further research needs to be done to visualize the ultrastructural changes of digestive vacuoles and mitochondria of p. falciparum by transmission electron microscopy (tem) and to determine changes at proteomic and genomic level, then performing an in vivo research in experimental animals as models of artemisinin resistance in humans. conclusion exposure to artemisinin antimalarials in vitro can cause phenotypic morphological changes of dormancy in p. falciparum papua 2300 strain. acknowledgments we would like to thank the directorate general of higher education (dirjen dikti), ministry of education and culture, republic of indonesia for bpps doctoral program 2009 in faculty of medicine airlangga university. references 1. world health organization. world health organization global report on antimalarial drug efficacy and drug resistance 2000-2010. geneva: world health organization; 2010. 2. chima ri, goodman ca, mills a. the economic impact of malaria in africa: a critical review of the evidence. health policy 2003;63:17-36. 3. sachs j, malaney p. the economic and social burden of malaria. nature 2002;415:680-5. 4. afonso a, hunt p, cheesman s, et al. malaria parasites can develop stable resistance to artemisinin but lack mutations in candidate genes atp6 (encoding the sarcoplasmic and endoplasmic reticulum ca2+ atpase) tctp, mdr1 and cg10. antimicrob agents chemother 2006;50:480-9. 5. noedl h. evidence of artemisinin resistant malaria in western cambodia. n engl j med 2008;359:2619-20. 6. veiga mi, ferreira pe, schmidt ba, et al. antimalarial exposure delays p. falciparum intra univ med vol. 34 no.1 34 maslachah, dachlan, nidom, et al plasmodium falciparum strain 2300 erytrocytic cycle and drives drug transporter genes expression. plos one 2010;5:e12408. 7. mugittu k, genton b, mshinda h, et al. molecular monitoring of plasmodium falciparum resistance to artemisinin in tanzania. malaria j 2006;5:126-128. 8. schonfeld m, miranda ib, schunk m, et al. molecular surveillance of drug resistance associated mutation of plasmodium falciparum in southwest tanzania. malaria j 2007;6:2. doi: 10.1186/1475-2875-6-2. 9. maslachah l. efek paparan artemisinin berulang terhadap perkembangan plasmodium falciparum resisten in vitro [disertasi]. program studi ilmu kedokteran program doktor. surabaya: fakultas kedokteran universitas airlangga; 2013. 10. witkowski b, lelievre j, barragan mjl, et al. increased tolerance to artemisinin in plasmodium falciparum is mediated by a quiescence mechanism. antimicrob agents chemother 2010;54:1872-7. doi: 10.1128/ aac.01636-09. 11. beez d, sanchez cp, stein wd, et al. genetic predisposition favors the acquisition of stable artemisinin resistance in malaria parasites. antimicrob agents chemother 2010;55:50-5. doi: 10.1128/acc.00916-10. 12. sanz lm, crespo b, de-cozar c, et al. p. falciparum in vitro killing rates allow to discriminate between different antimalarial mode of action. plos one 2012;7:e30949. 13. teuscher f, chen n, kyle de, et al. phenotypic changes in artemisinin resistant plasmodium falciparum line in vitro: evidence for decreased sensitivity to dormancy and growth inhibition. antimicrob agent chemother 2012;56:428-31. 14. cheng q, kyle de, gatton ml. artemisinin resistance in plasmodium falciparum: a process linked to dormancy. ijp 2012;2:249-55. 15. babbit se, altenhofen l, cobbold sa, et al. plasmodium falciparum responds to amino acid starvation by entering into a hibernatory state. pnas 2012;109:e3278-87. 16. tucker ms, mutka t, gil m, et al. in vitro recrudescence of plasmodium falciparum parasites suppressed todormant state by atovaquone aloneand in combination with proguanil. j trop med hygine 2005;99:62. 17. phadke ms, krynetskain nf, mishra ak, et al. glyceraldehyde 3-phosphate dehydrogenase depletion induces cell cycle arrest and resistance to antimetabolites in human carcinoma cell lines. j pharmacol exp ther 2009;331:77-86. 18. tucker ms, mutka t, sparks k, et al. phenotypic and genotypic analysis of in vitro selected artemisinin resistent progeny of plasmodium falciparum. antimicrob agent chemother 2012; 56:302-14. 19. lacrue an, scheel m, kennedy k, et al. effects of artesunate on parasite recrudescence and dormancy in the rodent malaria model plasmodium vinckei. plos one 2011;6:e26689. 20. rosenthal pj. antimalaria chemotherapy mechanism of action resistance and new direction in drug discovery. j antimicrob chemother 2003;51:1053. doi: 10.1093/jac/ dkg183 21. cowman af, berry d, baum j. the cellular and molecular basis for malaria parasite invasion of human red blood cell. jcb 2012;196:962-71. 22. bonilla aj, bonilla dt, yowell ac, et al. critical roles for digestive vacuole plasmepsins of plasmodium falciparum in vacuolar function. molecular microbiol 2007;65:64-75. 23. klonis n, ortiz mp, bottova i, et al. artemisinin activity against plasmodium falciparum requires hemoglobin uptake and digestion. pnas 2011;108:11405-10. 24. vieira a. endocytic transport and some of its implications for physiology, metabolism and disease. j physiobiochem metab 2012;1:2. editorial 73 statins and risk of diabetes mellitus richard tjan editor univ med vol. 33 no.2 statins are competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme a (hmg-coa) reductase, which reduces hmg-coa to mevalonate, the precursor of cholesterol via squalene. inhibition of hmg-coa reductase results in a decrease in cholesterol production. since 1987, when the united states federal drug administration (fda) approved lovastatin for clinical use,(1) statins have been widely used for secondary prevention of cardiovascular disease, particularly coronary heart disease (chd), which is associated with high levels of low-density lipoprotein (ldl) cholesterol. statins are also used in type 2 diabetes mellitus, since this carries a high risk of chd. statins have several adverse effects, to which must now be added new onset diabetes. in 2012 the fda issued a warning about the risk of newly developed diabetes mellitus in older persons, such that statin labels now include information on glycemic effects, including diabetes and increases in hemoglobin a1c or fasting plasma glucose.(2) according to the results of a recent meta-analysis involving 13,966 40+-year patients newly treated with statins between 1 january 1977 and 31 march 2011, a moderate but significant increase was found in the risk of new onset diabetes within the first two years of using regular higher potency statins (rosuvastatin >10 mg, atorvastatin >20 mg, and simvastatin >40 mg), compared with lower potency drugs. therefore these investigators caution clinicians regarding the use of higher potency statins in secondary prevention of cardiovascular disease.(2) the use of a new drug carries a “built-in time-bomb”, because nothing is known about its side effects, except for those revealed by animal tests and limited clinical trials. even a multicenter clinical trial cannot be expected to reveal all possible adverse reactions associated with a new drug. as an illustration, in patients without diabetes mellitus, more than 345 000 cases were needed to detect an increase in fasting blood glucose of 3 mg/dl as a result of statin use.(3) here is a verbatim quote from shah and goldfine: “for any prescription drug, the potential benefits to health must be balanced against potential risks. understanding these potential risks can help physicians and patients make informed decisions on whether to use a medication.” since the risk of statin-induced diabetes mellitus is important and unknown in the population of persons at lower risk of heart disease, it is considered prudent not to prescribe statins, except when diet and exercise cannot achieve ldl goals.(3) the mechanism by which statins induce diabetes in older persons has been recently uncovered. a canadian research team has shown that statins increase macrophage il-1β secretion, ndicating activation of the nucleotide-binding and oligomerization domain (nod)-like receptor pyrin domain 3 (nlrp3) inflammasome (caspase-1 inflammasome), which promotes insulin resistance, a precursor of type 2 diabetes. these investigators are of the opinion that the risk of statin-induced insulin 74 univ med vol. 33 no.2 resistance may be reduced by inhibiting the nlrp3/caspase-1 inflammasome, particularly in obese, hyperlipidemic patients who are often at risk for developing diabetes, but have to use statins.(4) in conclusion, although the risk of new diabetes mellitus with statin therapy may be considered to be minimal, the use of statins should only be prescribed by physicians for patients at risk for cardiovascular disease. however, when these patients are also at risk for diabetes mellitus, their blood glucose level should be monitored.(3) on the other hand, since statins may trigger new onset diabetes, presumably in predisposed persons, and since diabetes carries a risk of cardiovascular disease, even statins with the least side effects should not be used routinely for primary prevention, least of all as over-the-counter drugs. primum non nocere. references 1. endo a. a historical perspective on the discovery of statins. proc jpn acad 2010; 86:484-93. 2. dormuth cr, filion kb, paterson jm, james mt, teare gf, raymond cb, et al. higher potency statins and the risk of new diabetes: multicentre, observational study of administrative databases. bmj 2014;348:g3244. doi: 10.1136/bmj.g3244. 3. shah rv, goldfine ab. statins and risk of new-onset diabetes mellitus. circulation 2012;126:e282-e4. doi: 10.1161/circulationaha.112.122135. 4. henriksbo bd, lau tc, cavallari jf, denou e, chi w, lally js, et al. fluvastatin causes nlrp3 inflammasomemediated adipose insulin resistance. diabetes 2014. (published online ahead of print; june 11, 2014). adi priyana 11 perbandingan antara high density lipoprotein, lipoprotein(a) dan small dense low density lipoprotein sebagai parameter pertanda risiko penyakit jantung koroner adi priyana* universa medicina januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 abstrak latar belakang penyakit jantung koroner (pjk) merupakan penyebab kematian utama di indonesia seperti juga di berbagai negara industri di dunia. aterosklerosis merupakan penyebab utama dari pjk. faktor risiko utama sebagai penyebab aterosklerosis dan pjk adalah hipertensi, diabetes melitus, dislipidemia, kurang aktifitas dan merokok. beberapa faktor risiko lainnya adalah high density lipoprotein (hdl) kolesterol (hdl-k), low density lipoprotein (ldl) kolesterol (ldl-k), lipoprotein a [lp(a)], dan small dense ldl (sd-ldl). penelitian ini bertujuan untuk membandingkan hdl-k, lp(a) dan sd-ldl sebagai pertanda risiko terjadinya pjk. metode subyek penelitian dibagi dalam dua kelompok yaitu kelompok risiko mengikutsertakan 94 subyek yang memiliki minimal 2 faktor risiko pjk dan kelompok kontrol 49 subyek tanpa faktor risiko pjk. pemeriksaan sampel darah vena dilakukan pada subyek yang sebelumnya telah berpuasa 12–14 jam. pertanda faktor risiko yang diperiksa adalah hdl-k, lp(a) dan sd-ldl. tiga alternatif multivariat model dibandingkan sd-ldl, sd-ldl + hdl-k, dan sd-ldl, hdl-k + lp(a). hasil hasil penelitian menunjukkan sd-ldl merupakan pertanda risiko pjk yang akurat. ternyata lp(a) dan hdl-k kurang akurat bila digunakan sebagai pertanda risiko pjk dibandingkan dengan sd-ldl. kesimpulan small dense ldl merupakan pertanda risiko pjk yang akurat. perlu dilakukan penelitian lebih lanjut dengan mengikut sertakan lebih banyak penderita pjk dan kontrol dengan usia yang sebanding. kata kunci: penyakit jantung koroner, ldl kolesterol, lp(a) dan small dense ldl * bagian patologi klinik fakultas kedokteran universitas trisakti korespondensi dr. dr. adi priyana, sp.pk bagian patologi klinik fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 021-5672731, eks. 2404 email: adipr@indo.net.id universa medicina 2007; 26: 11-7. 12 priyana parameter pertanda risiko pjk pendahuluan p e n y a k i t j a n t u n g k o r o n e r ( p j k ) m e r u p a k a n p e n y e b a b k e m a t i a n u t a m a d i indonesia seperti juga di berbagai negara l a i n . ( 1 , 2 ) a t e r o s k l e r o s i s m e r u p a k a n d a s a r penyebab pjk. bila terdapat keadaan yang s a n g a t e r a t k a i t a n n y a s e b a g a i p e n y e b a b a t e r o s k l e r o s i s d i s e b u t f a k t o r r i s i k o aterosklerosis. faktor risiko tersebut dibagi dalam dua kelompok yaitu faktor risiko utama atau mayor (kebiasaan merokok, hipertensi >140/90 mmhg, kolesterol hdl <40 mg/dl, riwayat pjk dini, usia pria >45 tahun dan wanita >55 tahun. sedang faktor risiko minor adalah obesitas, stres dan hiperurisemia.(1-3) comparison between high density lipoprotein, lipoprotein (a) and small dense low density lipoprotein as a risk marker of coronary heart disease adi priyana* abstract background coronary heart disease (chd) is still the primary cause of death in indonesia and most industrialized countries. atherosclerosis is the most common cause of chd. the conventional risk factor for atherosclerosis and chd are diabetes mellitus, hypertension, dyslipidemia and smoking. on the other hand, lp(a) and small dense ldl (sd-ldl) have been considered as novel risk factors for chd. high density lipoprotein (hdl) cholesterol (hdl-c), as part of dyslipidemia, has been considered as independent risk factor for chd. the principal objective of the present study was to compare between hdl-c, lp(a) and sdl ldl as a risk marker of chd. methods the study subject were divided into two groups, risk group consist of 94 subjects who had ≥ 2 chd risk factors and control group recruited 49 subject who had no chd risk factors. both groups had fasted for 10-12 hours before their sera were collected. the sera were examined for hdl-c, lp(a) and sd-ldl. three alternative multivariate models were compared sd-ldl, sd-hdl + hdl-c, and sd-ldl, hdl-c + lp(a). results result of this study showed that the sd-ldl had the most significant and accurate as risk marker for chd. lp(a) and hdl-c were less accurate if used as risk marker for chd compare with sd-ldl. conclusions small dense ldl is an accurate risk marker for chd. further study must be done using larger sample size of chd subjects with similar age between risk and control groups. keywords : coronary heart disease, ldl cholesterol, lp(a) and small dense ldl *department of clinical pathology faculty of medicine university of trisakti correspondence dr. dr. adi priyana, sp.pk department of clinical pathology medical faculty trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone : 021-5672731 ex.2404 email: adipr@indo.net.id universa medicina 2007; 26: 11-7. 13 dari banyak penelitian eksperimental dan epidemiologis terbukti bahwa dislipidemia berperan penting pada kejadian aterosklerosis. berdasarkan pemeriksaan berbagai profil lipid, kini telah dikembangkan pemeriksaan pertanda faktor risiko pjk. pertanda faktor risiko pjk t e r s e b u t a n t a r a l a i n : k o l e s t e r o l t o t a l , l o w density lipoprotein (ldl) kolesterol (ldl-k), high density lipoprotein (hdl) kolesterol (hdl-k), rasio ldl/hdl-k, trigliserida, high s e n s i t i v e c re a c t i v e p ro t e i n ( h s c r p ) , lipoprotein (a) [lp(a)], homosisteinemia dan small dense (sd) ldl.(3-6) kadar kolesterol saja tidak akurat memprediksi risiko terjadinya pjk, karena merupakan jumlah dari berbagai jenis kolesterol yaitu ldl-k, hdl-k, v e r y l o w density lipoprotein (vldl), dan intermediate density lipoprotein (idl). dengan demikian u n t u k m e n e n t u k a n p e m b e r i a n p e n g o b a t a n didasarkan atas kadar hdl-k, ldl-k dan trigliserida.(6-8) dengan ditemukannya pemeriksaan pertanda risiko pjk, berarti telah dikembangkan usaha untuk diagnosis dini dan pengobatan dapat d i l a k u k a n l e b i h c e p a t d a n t e p a t . s e b u a h p e n e l i t i a n m e n u n j u k k a n k a d a r s d l d l berhubungan dengan sindroma metabolik dan m e n i n g k a t k a n r i s i k o t e r j a d i n y a p j k ( 6 ) . sedangkan penelitian lainnya menunjukkan k a d a r t r i g l i s e r i d a d a n l p ( a ) y a n g t i n g g i merupakan faktor risiko independen terhadap pjk baik pada laki-laki maupun perempuan.(6,7) studi prospektif di inggris pada laki-laki berusia 45 sampai 59 tahun menunjukkan kadar lipid (kolesterol total, hdl-k, dan trigliserida) berhubungan dengan risiko terjadinya pjk.(8) belum banyak diperoleh informasi tentang parameter yang akurat sebagai pertanda risiko p j k . p e n e l i t i a n i n i b e r t u j u a n u n t u k mendapatkan informasi pertanda risiko pjk yang paling tepat dengan membandingkan kadar hdl-k, sd-ldl dan lp(a) sebagai pertanda risiko pjk. metode rancangan penelitian penelitian dilakukan dengan menggunakan metode potong lintang (cross sectional). subyek penelitian s u b y e k p e n e l i t i a n d i b a g i d a l a m d u a kelompok, kelompok pertama (risiko) dengan minimal dua faktor risiko pjk terdiri dari 94 laki-laki dan wanita lansia (>65 tahun), masih aktif bergerak (mobile). yang dimaksud faktor risiko di sini adalah faktor risiko mayor seperti hipertensi, diabetes melitus, dislipidemia dan merokok. kelompok kedua adalah kontrol, berjumlah 49 pria dan wanita <45 tahun tanpa faktor risiko pjk sama sekali. pemeriksaan laboratorium semua subyek penelitian diharuskan tidak makan 12–14 jam sebelum pengambilan darah vena, hanya boleh minum air putih saja. diambil darah vena sebanyak 5 ml dalam keadaan puasa. darah vena didiamkan pada suhu kamar sampai m e m b e k u , k e m u d i a n d i s e n t r i f u s u n t u k mendapatkan serum. terhadap serum dilakukan pemeriksaan profil lemak seperti kolesterol, ldl-k dan hdl-k, trigliserida, lp(a) dan sdldl. pemeriksaan darah memakai alat architect ci 8200 buatan abbott. analisis statistik perbedaan rata-rata kadar profil lipid dan usia antara kelompok risiko dan kontrol diuji menggunakan uji-t independent. sedangkan perbedaan frekuensi laki-laki dan perempuan antara kedua kelompok diuji menggunakan uji chi-square. untuk menentukan profil lipid yang berkontribusi terhadap risiko terjadinya pjk digunakan analisis regresi logistik. nilai s e n s i t i v i t a s ( t r u e p o s i t i v e r a t e ) d a n 1 spesifisitas (false positive rate) dihitung untuk setiap pertanda risiko. 14 priyana parameter pertanda risiko pjk kemudian dibuat kurva reveiwer operating characteristic (roc) dengan menggunakan nilai sensitivitas sebagai sumbu y dan 1-spesifisitas s e b a g a i s u m b u x . r o c d i g u n a k a n u n t u k menguji nilai tambah dari ketiga jenis pertanda r i s i k o d a l a m m e m b e d a k a n s u b y e k y a n g mempunyai risiko dan tidak. area di bawah kurva roc menunjukkan ketelitian sebuah pertanda risiko untuk mempredikasi terjadinya pjk. semakin besar luas area di bawah kurva menunjukkan semakin baik kemampuan sebuah pertanda risiko memprediksi terjadinya pjk. sebuah area yang besarnya 0,5 menunjukkan p e r t a n d a r i s i k o t e r s e b u t t i d a k m a m p u m e m b e d a k a n a n t a r k e l o m p o k r i s i k o d a n kelompok kontrol. sedangkan area di bawah kurva roc sebesar 1,0 menunjukkan pertanda risiko secara sempurna mampu memprediksi terjadinya pjk.(9) analisis statistik dilakukan menggunakan program stata versi 8,0 dan tingkat kemaknaan yang digunakan besarnya 0,05. hasil pada tabel 1 terlihat nilai rata-rata hasil pemeriksaan lp(a), small dense ldl dan hdlk pada kelompok kontrol dan risiko. terlihat pada kelompok risiko rata-rata kadar lp(a) r e l a t i f l e b i h t i n g g i d i b a n d i n g k a n d e n g a n kelompok normal (18,2 dibanding 16,1), namun tidak berbeda bermakna (p = 0,4608). sementara kadar sd-ldl dan hdl-k terdapat perbedaan rata-rata yang bermakna pada kelompok kontrol lebih tinggi dibandingkan dengan kelompok risiko (masing-masing p = 0,0000 dan p = 0,0001). analisis regresi logistik ganda digunakan untuk menilai besarnya risiko terjadinya pjk dari setiap pertanda risiko (tabel 2). tabel 2 menunjukkan, or dari hdl-k sebelum dikoreksi merupakan pertanda rsiko terjadinya pjk, tetapi setelah dikoreksi pertanda risiko hdl-k tidak m a m p u m e m p r e d i k s i r i s i k o p j k s e c a r a bermakna (or = 0,9439 – 1,0132). tabel 2. odds rasio dari setiap pertanda risiko *or = 0dds rasio nilai adalah mean ± sd tabel 1. karakteristik dan pertanda risiko pjk berdasarkan kelompok kontrol dan risiko 15 o r d a r i l p ( a ) s e b e l u m d a n s e s u d a h dikoreksi bukan merupakan pertanda risiko yang mampu memprediksi risiko pjk secara b e r m a k n a . p e r t a n d a r i s i k o s d l d l b a i k s e b e l u m d a n s e s u d a h d i k o r e s i m a m p u memprediksi risiko pjk secara bermakna {or masing-masing besarnya 0,0011 (0,0004 – 0,0045) dan 0,0041 (0,0001 – 0,0164)}. untuk menetapkan pertanda risiko yang s e c a r a a k u r a t m a m p u m e m p r e d i k s i r i s i k o terjadinya pjk digunakan kurva roc yang diperoleh dari analisis regresi logistik ganda. ti g a m o d e l d i s a j i k a n u n t u k m e n e t a p k a n pertanda risko yang akurat yaitu i) model pertama hanya sd-ldl, ii) model kedua sdldl + hdl-k, dan iii) model ketiga sd-ldl + hdl-k + lp(a) (tabel 3). area di bawah kurva roc berdasarkan sdl d l ( m o d e l 1 ) b e s a r n y a 8 0 , 5 % , d a n penambahan pertanda risiko hdl-k (model 2) tidak menunjukkan peningkatan kemampuan untuk memprediksi risiko pjk (auroc = 80,9%). hal yang tidak berbeda ditunjukkan b i l a m e n a m b a h k a n p e r t a n d a r i s i k o l p ( a ) (model 3) besarnya auroc = 81,8%. hasil yang sama ditunjukkan bila pertanda risko d i g u n a k a n u n t u k m e m p r e d i k s i b a n y a k n y a subyek yang mempunyai risiko terjadinya pjk. pertanda risiko sd-ldl mampu memprediksi subyek yang mempunyai risiko pjk sebanyak 70, sedangkan penambahan pertanda risiko hdl-k dan lp(a) menunjukkan masing-masing sebanyak 73 dan 73 subyek. hal ini berarti satu p e r t a n d a r i s i k o s d l d l s u d a h m a m p u m e m p r e d i k s i s e c a r a a k u r a t s u b y e k y a n g mempunyai risiko pjk. semakin rendah nilai sd-ldl maka semakin tinggi risiko terkena pjk. diskusi hasil penelitian ini menunjukkan sd-ldl p a l i n g a k u r a t u n t u k m e n i l a i r i s i k o p j k dibandingkan lp(a) dan hdl-k. hasil yang konsisten ditunjukkan penelitian dari quebec cardiovascular study, sd-ldl meningkatkan risiko terjadinya pjk.(10) sd-ldl merupakan partikel yang highly atherogenic, semakin tinggi kadarnya semakin tinggi pula risiko terjadinya pjk. (11) st-pierre et al (11,12) pada penelitiannya menggunakan ldl partikel kecil (< 250ao) dalam kaitannya dengan penderita pjk membuktikan ada kaitan erat antara pjk dan ldl partikel kecil. analisis kurva roc pada studi ini mendukung konsep sd-ldl sebagai pertanda risiko yang akurat untuk terjadinya pjk. studi ini kurang mendukung lp(a) sebagai pertanda risiko pjk yang akurat. lp(a) sendiri lebih bersifat genetik karena perubahan gaya hidup, penurunan berat badan d a n o l a h r a g a t i d a k m e n u r u n k a n s e c a r a b e r m a k n a k a d a r l p ( a ) d a l a m s e r u m . ( 1 3 ) termasuk pengobatan dengan memakai statin dan bile acid sequestrans juga tidak berhasil menurunkan kadar lp(a). kadar lp(a) yang tinggi dalam waktu lama erat kaitannya dengan r i s i k o p j k , s t r o k e i s k e m i k d a n p e n y a k i t pembuluh darah perifer.(14) beberapa peneliti tabel 3. kurva area under roc (auroc) setiap model pertanda risiko model 1 : sd-ldl, model 2 : sd-ldl+hdl-k, model 3 : sd-ldl + hdl-k + lp(a) * kurva auroc menunjukkan area di bawah sensitivitas dan 1-spesifisitas, dalam persen ** jumlah subyek yang diprediksi risiko pjk 16 priyana parameter pertanda risiko pjk mengemukakan hal yang sama seperti hirano et al(15) dan beberapa peneliti dari amerika dan eropa. (16,17) sejumlah studi retrospektif kasus-kelola menujukkan adanya hubungan yang kuat antara meningkatnya kadar lp(a) dan terjadinya pjk.(4) n a m u n h a s i l s e m b i l a n s t u d i p r o s p e k t i f m e n u n j u k k a n h a s i l y a n g b e r b e d a , l p ( a ) merupakan faktor risiko independen terhadap pjk pada laki-laki. hasil yang tidak konsisten ini berkaitan dengan kurangnya standarisasi dan k e g a g a l a n p e n g u k u r a n i m m u n o a s s a y p a d a s e m u a i s o f o r m d a r i a p o ( a ) . ( 1 8 ) m e s k i p u n demikian, sebuah meta-analisis dari penelitian prospektif menunjukkan kadar lp(a) dalam plasma berhubungan dengan pjk baik pada lakilaki maupun wanita.(19) sebuah prospective e p i d e m i o l o g i c a l st u d y o f m y o c a rd i a l infarction (prime) yang mengikutsertakan 9 . 1 3 3 l a k i l a k i t a n p a r i w a y a t p j k a t a u menggunakan obat hipolidemik menunjukkan l p ( a ) s e b a g a i p r e d i k t o r r i s i k o t e r j a d i n y a pjk.(20) namun pada studi ini ternyata sd-ldl merupakan pertanda risiko pjk yang akurat, sedangkan lp(a) kurang akurat. walaupun harus diakui terdapat beberapa kelemahan dari studi ini. subyek yang diikutsertakan pada studi ini adalah mereka yang mempunyai risiko pjk d a n b u k a n p e n d e r i t a p j k , j u g a t e r d a p a t p e r b e d a a n u s i a y a n g b e r m a k n a a n t a r a kelompok risiko dan kontrol. kesimpulan kadar sd-ldl dalam serum merupakan pertanda risiko yang akurat untuk menentukan ada tidaknya risiko pjk. ternyata lp(a) dan hdl-k kurang akurat bila digunakan sebagai pertanda risko pjk dibandingkan dengan sdldl. perlu dilakukan penelitian lebih lanjut d e n g a n m e n g i k u t s e r t a k a n l e b i h b a n y a k penderita pjk dan kontrol dengan usia yang sebanding. ucapan terima kasih terima kasih kepada pimpinan fakultas kedokteran universitas trisakti atas dukungan dana yang diberikan untuk penelitian ini. kepada laboratorium klinik utama pramita, matraman, jakarta yang telah mengijinkan memakai alat architec ci 8200 abbott untuk pelaksanaan penelitian dan kepada p.t. abbott indonesia serta p.t. daichii indonesia yang telah membantu menyiapkan reagens penelitian. daftar pustaka 1. national cholesterol education program (ncep) expert panel on detection evaluation, and treatment of high blood cholesterol in adults (adult treatment panel iii). third report of the national cholesterol education program (ncep) expert panel on detection, evaluation and treatment of high blood cholesterol in adults (adult treatment panel iii) final report. circulation 2002; 106: 3143-421. 2. pb. perkeni. petunjuk praktis penatalaksanaan dislipidemia. jakarta: departemen ilmu penyakit dalam fkui; 2004. 3. kreisberg r, oberman a. medical management of hyperlipidemia/ dyslipidemia. j clin endocrin met 2003; 88: 2445-61. 4. carmena r, duriez p, fruchart jc. atherogenic lipoprotein particles in atherosclerosis. circulation 2004; 109 [suppl iii]: iii-2-7. 5. rizzol m, berneis k. low-density lipoprotein size and cardiovascular risk assessment. q j med 2006; 99:1-14. 6. tikac i, kimball bp, lewis g, uffelman k, steiner g. the severity of coronary atherosclerosis in type 2 diabetes mellitus is related to the number of circulating triglyceride-rich lipoprotein particles. arterioscler thromb vasc biol 1997; 17: 3633-8. 7. yarnell jwg, patterson cc, sweetnam pm, lowe gdo. haemostatic/inflammatory markers predict 10-year risk of ihd at least as well as lipids: the caerphilly collaborative studies. eur heart j 2004; 25: 1049-56. 8. greenberg rs, daniels sr, flanders wd, eley jw, boring jr iii, editors. diagnostic testing. in: medical epidemiology. 3rd ed. new york: lange medical books/mcgraw-hill; 2001. 17 9. lamarche b, lemieux i, despres jp. the small, dense ldl phenotype and the risk of coronary heart disease: epidemiology, pathophysiology, and therapeutic aspects. diabetes metab 1999; 25: 199211. 10. lamarche b, st pierre ac, ruel il, cantin b, dagenais gr, després jp, et al. a prospective, population-based study of low density lipoprotein particle size as a risk factor for ischemic heart disease in men. can j cardiol 2001; 17: 859–65. 11. st-pierre ac, ruel il, cantin b, dagenais gr, bernard pm, depres jp, et al. comparison of various electrophoretic characteristic of ldl particles and their relationship to the risk of ischemic heart disease. circulation 2001; 104: 2295-99. 12. st-pierre ac, cantin b, dagenais gr, mauriège p, bernard p-m, després j-p, lamarche b. lowdensity lipoprotein subfractions and the long-term risk of ischemic heart disease in men: 13-year follow-up data from the québec cardiovascular study. arterioscler thromb vasc biol 2005; 25: 553-9. 13. frolkis j. should one routinely screen for lipoprotein(a)? cleve clin j med 1999; 66: 465-8. 14. allan d. how, when and why to use apoplipoprotein b in clinical practice. am j cardiol 2002; 17: 48i-54i. 15. hirano t, yoshino g, adachi m. doxazosin reduces prevalence of small dense ldl and remnant-like particle cholesterol levels in nondiabetic and diabetic hypertensive patients. am j hypertens 2001; 14: 908-13. 16. pontrelli l, parris, adeli k. atorvastatin treatment beneficially alters the lipoprotein profile and incerase ldl lipoprotein particles diameter in patients with combined dyslipidemia and impaired fasting glucose type 2 diabetes. metabolism 2002; 51: 334-42. 17. yoshino g, hirano t. treatment of small dense ldl. j atheroscler thromb 2002; 9: 266-75. 18. seman lj, deluca c, jenner jl, cupples la, mcnamara jr, wilson pw, et al. lipoprotein(a) cholesterol and coronary heart disease in the framingham heart study. clin chem 1999; 45: 1039-46. 19. craig wy, neveux lm, palomaki ge, cleveland mm, haddow je. lipoprotein(a) as a risk factor for ischemic heart disease: meta analysis of prospective studies. clin chem 1998; 44: 23016. 20. luc g, bard jm, arveiler d, ferrieres j, evans a, amouyel p, et al. lipoprotein(a) as a predictor of coronary heart disease: the prime study. atherosclerosis 2002; 163: 377-84. inappropriate complementary feeding practice increases risk of stunting in children aged 12-24 months 146 *department of nutrition, faculty of medicine, diponegoro university semarang **department of public health, diponegoro university semarang correspondence: hijra department of nutrition, faculty of medicine, diponegoro university (komplek zona pendidikan rsup dr. kariadi, gd. dekanat fk undip lt.2) jl. dr. soetomo no. 18 semarang email: hijratulmarahsubanrio@gmail.com univ med 2016;35:146-55 doi: 10.18051/univmed.2016.v35.146-155 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 inappropriate complementary feeding practice increases risk of stunting in children aged 12-24 months hijra*, siti fatimah-muis*, and martha irene kartasurya** background in 2013, the prevalence of stunting in central sulawesi province was 41%, which was higher than the national prevalence of 37.2%. complementary feeding practice, infectious disease, history of exclusive breastfeeding, birth weight and birth length were assumed to contribute to the prevalence of stunting. the objective of the present study was to identify the risk factors for stunting among children aged 12 to 24 months. methods this study was conducted using a case control design. subjects were children aged 12-24 months, consisting of 58 cases and 58 controls. data were obtained with a body length infantometer. data on complementary feeding practices, history of exclusive breastfeeding, and history of infectious diseases were obtained using questionnaires. nutrient intakes were measured by semi quantitative food frequency questionnaires. data on birth weight and birth length were obtained from the buku kia (maternal and child health record). data were analyzed by chi-square and logistic regression tests. results there was no difference in sex, bmi, and parental education between cases and controls (p>0.05). after controlling for history of exclusive breastfeeding and birth weight, the risk factors for stunting were inappropriate complementary feeding practice in terms of quantity and quality (or=8.26; 95% ci: 2.69-25.44), history of diarrhea (or=4.73; 95% ci: 1.08-20.69), birth length (or=5.11; 95% ci: 1.69-15.46) and respiratory tract infection (or=5.30; 95% ci: 1.03-27.23). inappropriate complementary feeding practice was the most dominant factor for stunting. conclusion inappropriate complementary feeding practice increased the risk of stunting in 12-24 months old children by 8.26. this study confirms the need to scale up interventions during the first 2 years of life, including appropriate infant feeding practices. keywords: stunting, complementary feeding practices, diarrhea, children 12-24 months doi: http://dx.doi.org/10.18051/univmed.2016.v35.146-155 147 univ med vol. 35 no.3 introduction central sulawesi is one of the provinces with prevalence rates of stunting above the national prevalence and its stunting prevalence rate is categorized as serious.(1) the prevalence of stunting in the underfives in 2013 in central sulawesi was 41%, with a prevalence of stunting in the underfives in palu city of 35.5%. among the puskesmas (primary health centers) of palu city, puskesmas sangurara has the relatively high prevalence of stunting of 50%.(2) complementary feeding practice is important contributes to the prevalence ofstunting. complementary feeding that is of good quality and is quantitatively adequate is an important component in the diet of the underfives, since it contains contains macroand micronutrients that affect linear growth.(3) the age of 6 to 24 months is one of the most critical periods for linear growth, and is also the time of peak prevalence of stunting in developing countries, because of the high dietary requirements and the limited quality and quantity of complementary feeding.(4) complementary feeding refers to the introduction of safe and nutritious foods at the appropriate time.(5) in general, studies on complementary feeding in areas of food shortages show that fortification with micronutrients has only slight or no impact on growth. complementary feeding may increase linear growth and decrease stunting.(6) complementary feeding should preferably be adequate, safe, and appropriate to the age of the child. the period of introduction of complementary feeding that is coincident with the decrease in breastfeeding is the period of peak growth failure, nutrient deficiencies, and infectious diseases in children.(7-8) according to teshome et al.(9) too early introduction to complementary carries a 1.1-fold risk of stunting, whereas too late introduction to complementary carries a 2.2-fold risk of stunting. the present study on complementary feeding practice in differs from previous studies, in that it evaluated complementary feeding practice in terms of serving size (portion), provision of snacks, provision of foods of animal and vegetable origin, provision of formula milk, composition of the children’s meals, food preparation and storage by the mothers, maternal behavior when the children refuse to eat, provision of breastfeeding, adequacy level of energy, protein, zinc, iron, and vitamin a from the complementary feeding, all of which were lacking in the previous sudies. infectious diseases that are associated with linear growth are diarrhea and respiratory tract infection, and result in longterm effects in the form of growth deficit in height.(10) infectious disease may be the cause of decreased dietary intake. low dietary intake, decrease in nutrients as a result of vomiting, diarrhea, malabsorption and fever of long duration may cause nutrient deficiencies, and consequently infant and child growth and immune system abnormalities.(11) stunting is associated with respiratory disease and fever, because of increased metabolic requirements and decreased dietary intake during illness.(12) the purpose of the present study was to analyze inappropriate complementary feeding practice and infectious diseases as risk factors for stunting in children aged 12-24 months. methods study design the design of the study was observational with non-matched case controls. this study was conducted in the catchment area of puskesmas sangurara, palu city, central sulawesi, from february until april 2016. study subjects the subjects of this study were underfives aged 12-24 months, totaling 116 children, consisting of 58 stunted and 58 non-stunted children. the number of study subjects was calculated for an unpaired case control analytic study with categorical variables, using the following formula: [ ] 2 21 2 2211 21 )p(p qpqpzβ2pqzα nn − ++ == 148 based on the number of study subjects from previous studies using odds ratio,(13-14) the number of subjects for this study was 58 cases and 58 controls, so that the total number of subjects was 116. the diagnosis of stunting was established by health personnel at puskesmas sangurara. recruitment of the subjects was by consecutive non-random sampling. the inclusion criteria in this study were: children aged 12-24 months, having a height-to-age ratio of less than -2 sd for the cases and ≥-2 sd for the controls, with recorded birth weights and lenghts, living with both parents, and residing in the catchment area of puskesmas sangurara. the exclusion criteria were: children with physical or congenital disabilities, and children who were ill and had to be hospitalized during the study. measurements data on body length were obtained by means of an infantometer and were categorized into stunting and normal. data on complementary feeding practice in terms of quality, maternal knowledge and sanitation and hygiene, history of exclusive breastfeeding, and history of infectious diseases were obtained through interviews using structured questionnaires. data on complementary feeding practice in terms of quantity, i.e. nutrient intake from complementary feeding, were obtained using semiquantitative food frequency questionnaires. adequacy of nutrient intakes were determined from the daily dietary intake of the children as compared with indonesian recommended dietary allowances (irda). the nutrient intakes under study were adequacy level of energy, protein, zinc, iron, and vitamin a from the complementary feeding. the data on complementary feeding practice, which were obtained from 18 questions, were subsequently categorized into appropriate (total score = 1-14) and inappropriate (total score = 15-28). data on birth weight and birth length were obtained from the buku kia ( maternal and child health record). data on birth weight were categorized into low birth weight if the birth weight was <2500 grams and normal if the birth weight was ≥2500 grams. in addition, birth length was categorized as short if the birth length was <48 cm and normal if the birth length was ≥48 cm. data analysis the data analysis used the chi-square test and significance level á of ≤0.05 and the odds ratio (or). multivariate analysis by means of multiple logistic regression was used to determine the most dominant factors for stunting. ethical clearance this study obtained ethical clearance from the ethics commission, faculty of medicine, diponegoro university, semarang in february 2016. results complementary feeding practice was evaluated from several aspects, i.e. age of introduction of complementary feeding, frequency of provision, variety, servings (portions), provision of snacks, provision of animal and vegetable foods, provision of formula milk, composition of meals, food procurement and storage by the mothers, maternal behavior when the children refuse to eat, breastfeeding, and adequacy levels of energy, protein, zinc, iron, and vitamin a from the complementary feeding. the frequency distribution based on the 18 evaluation aspects of complementary feeding practice in terms of quality and quantity, may be seen in table 1, and the distribution of maternal knowledge and sanitation and hygiene in table 2. table 1 shows the evaluation aspects of complementary feeding in terms of quality and quantity. with regard to quality, between the cases and controls there was a difference in the frequency of complementary feeding, variety, portion size, provision of snacks, provision of animal and vegetable foods, provision of formula milk, and composition of meals (p<0.05). with regard to quantity, between the cases and controls there was a difference in the adequacy levels for hijra, muis, kartasurya inappropriate feeding practice and stunting 149 univ med vol. 35 no.3 table 1. distribution of complementary feeding practice in terms of quality and quantity energy, protein, iron, zinc, and vitamin a from the complementary feeding (p<0.05). table 2 shows the evaluation aspects of complementary feeding in terms of maternal konwledge and sanitation and hygiene. with regard to maternal knowledge, there was a difference in age of introduction of complementary feeding between cases and controls (p<0.05). however, between cases and controls there was no difference in maternal behavior when the child refuses to eat and in breastfeeding (p>0.05). with regard to sanitation and hygiene, between cases 150 table 2. distribution of complementary feeding practice in terms of maternal knowledge and sanitation and hygiene and controls there was no difference in preparation and storage of complementary food by mothers (p>0.05). table 3 shows that there were more children with inappropriate complementary feeding practice in the group of cases (79.3%) than in the control group (24.1%). mothers with inappropriate complementary feeding practice carried a risk of stunting that was 12.04-fold greater than those with appropriate complementary feeding practice (or=12.04; 95% ci: 5.02-28.89). there were more children suffering from uri in the last 2 months in the group of cases (70.7%) than in the control group (20.7%). children suffering from uri in the last 2 months had a 9.24 times greater risk of stunting when compared with those not suffering from uri in the last 2 months (or=9.24; 95% ci: 3.9521.64). there were more children suffering from diarrhea in the last 2 months in the group of cases (56.9%) than in the control group (10.3%). children suffering from diarrhea in the last 2 months had a 11.44 times greater risk of stunting when compared with those not suffering from diarrhea in the last 2 months (or=11.44; 95% ci: 4.24-30.85). there were more children without exclusive breastfeeding in the group of cases (81.0%) than in the control group (50.0%). children without exclusive breastfeeding had a 4.27 times greater risk of stunting when compared to children with exclusive breastfeeding (or=4.27; 95% ci: 1.85-9.84). children of low birth weight had a 5.16 times greater risk of stunting in comparison with those of normal birth weight (or=5.16; 95% ci: 1.7715.03). children with a birth length of <48 cm had a 6.91 times greater risk of stunting in comparison with those with a birth length of ≥48 cm (or=6.91; 95% ci: 3.05-15.62). the variables tested in the multiple logistic regression analyis were complementary feeding practice, history of uri, history of diarrhea, history of exclusive breastfeeding, birth weight, and birth length. the results of the multiple logistic regression analysis may been seen in table 4. the results show that the variables that constituted risk factors for stunting in children aged 12-24 months were complementary feeding practice, history of diarrhea, history of uri, and birth length. the most dominant risk factor was inappropriate complementary feeding practice (or=8.26; 95% ci: 2.69-25.44). hijra, muis, kartasurya inappropriate feeding practice and stunting 151 univ med vol. 35 no.3 discussion the frequency of complementary feeding in children with stunting tended to be lower than in normal children. the food frequency in children with stunting was very low and several subjects were found who had only one meal daily, in contrast to normal children who had 3 to 4 meals daily. this is in line with a study in nepal showing that a food frequency of less than 4 times daily was a risk factor of stunting, in which the children who had fewer than 4 meals daily carried a 3.60 times greater risk of stunting.(15) the majority of children with stunting did not consume a variety of foods. several subjects consumed only complementary foods of a certain brand and breast milk since the age of 6 months until 1 year. similar to the study in nepal, the present study shows that children with lower than the standard food variety had a 4.06 times greater risk of stunting.(15) the portion size in children with stunting tended to be smaller than in normal children. the results of our study shows that the majority of children with stunting consumed less than ½ portion at each meal, in contrast to normal children, most of whom consumed 1 full portion at each meal. these study results also show that normal children had more frequent snacks than children with stunting. most of the children with stunting were given a main dish of rice porridge or steamed rice, table 3. crude odds ratis with 95% confidence interval limits for stunting in children aged 12-24 months table 4. results of multiple logistic regression analysis 152 alternated only with breastfeeding, whereas normal children were given snacks such as biscuits of two popular brands. at the age of 12-24 months, the combination of breastfeeding and main dish only is inadequate to meet the energy requirements of the children for optimal growth, for which additional feeding is required such as snacks in the form of fruits or biscuits that contain adequate amounts of nutrients.(16) complementary feeding practice was also viewed from the aspect of composition of the meals. table 1 shows that the majority of subjects only consumed rice and fish without vegetables. this was due to the fact that these children did not like vegetables. several subjects were also found to consume only rice and vegetables without fish, because they did not like fish. however, there were also subjects who consumed rice only without fish or vegetables. the parents preferred to give sugar water, tea, and even coffee to their children. some children even received sugar water 3 times daily. the reason given by the parents was that their children did not like milk and that they could not afford to buy formula milk for their children, because of their low economic status. some subjects were also found who were not breastfed from the age of less than 1 year. the low adequacy level for energy was more frequent in children with stunting than in normal children. the present study is in line with a study on children aged 6-24 months in penanggalan district, subulussalam city, aceh province, which found that a low level of adequacy for energy was a risk factor of stunting. stunting occurs more frequently in children with a low adequacy level for energy.(13) the proportion of children with stunting was also higher in children with low adequacy level for protein. a study on children aged 2-5 years in kenya and nigeria found that inadequate protein intake was associated with stunting.(17) the low level of adequacy for protein in children with stunting in the our study was caused by inadequate consumption of fish, chicken, and beef, which are highly adequate sources of animal protein. some subjects only consumed fish once weekly, chicken once a month or at longer intervals, and beef once a year. in addition, vegetable protein was also occasionally consumed, because some children did not like to consume vegetables and fruits in adequate amounts (data not presented). the present study also shows that the proportion of children with stunting was higher in children with low adequacy levels for zinc. a study in klungkung district in children aged 2460 months showed that low zinc level was a risk factor of stunting. the risk of stunting in children with zinc deficiency was 16.1-fold.(18) the low adequacy level of zinc in children with stunting was caused by the low intake of foods with adequate zinc content, such as amaranth, fish and meat. the intake of iron was also exceedingly low in the group of children with stunting in comparison with normal children. this was caused by inadequate consumption of foods that are sources of iron, such as meat and fish. vitamin a is one of the important nutrients for growth. vitamin a deficiency causes children to be vulnerable to disease, so that their body defenses are reduced and their dietary intake becomes irregular. table 1 shows that the proportion of children with stunting was higher in children with low adequacy levels of vitamin a. a study in kenya in children aged 24-35 months found that the prevalence of stunting was 50% higher in children who did not consume vitamin a supplements, in comparison with children who did.(19) the present study found that too early or too late introduction of complementary feeding was more frequent in children with stunting than in normal children. a study in ethiopia in children aged 0-59 months showed that children with delayed complementary feeding had a 2.2 times greater risk of stunting, whereas too early introduction of complementary feeding had a 1.1 times greater risk of stunting.(20) the role of the mothers is very important in determining the nutritional status of their children, such as when the children have their meals, the mothers should pay attention to what is consumed by the children. some mothers only let their children cry or play hijra, muis, kartasurya inappropriate feeding practice and stunting 153 univ med vol. 35 no.3 when they refuse to eat, so that the portions consumed are very small, and some subjects were even found to consume 2-3 spoonful only. this is due to the fact that their mothers do not like their children to cry when given food and are afraid that their children will vomit when forced to eat. but some mothers persuade their children when they refuse to eat by giving them the food bit by bit so that their children will consume some. regarding the storage of the prepared food, in the group of children with stunting the food was more frequently left exposed in the covers, whereas in normal children, the mothers more frequently stored the food in a closed and clean place such as a pantry. storing food while letting them remain exposed may result in bacterial contamination of the food so that it may become a health hazard for the children. complementary feeding practice was more frequently inadequate in terms of quality and quantity, in comparison with maternal knowledge and sanitation and hygiene. the results of the present study are consistent with those of a study in banda aceh city on children aged 12-60 months, showing that children with inadequate complementary feeding had a 3.4 times greater risk of stunting.(14) nutrient intake plays an essential role in the growth process of children, so that to achieve optimal growth and development, nutrient intakes have to be met. by the age of 6 months, exclusive breastfeeding does not adequately meet the nutrient requirements of the children, so that additional sources of nutrients are needed from complementary feeding. provision of foods with inadequate nutrient intake, exceedingly low frequency of meals and portion size constitute the main causes of poor nutritional status in children. complementary feeding must provide sources of macroand micronutrients that play a role in linear growth in the underfives. food variety is also very important in maintaining the nutritional status of children. some subjects were found to consume only a single type of food even for a period of one week. in our study area, there is a special type of vegetable dish, namely moringa oleifera leaves (daun kelor) in coconut milk (santan), which is the type of vegetable dish most frequently consumed by the children. some children in one week consume only kelor leaves and rice without other foods. several evaluation aspects of complementary feeding practice that have been explained are essential in determining the quality and quantity of the provided complementary feeding. this is the reason why children with inappropriate complementary feeding practice have a 12.37 times greater risk of stunting. the risk of stunting in our study area was greater that that in previous studies. the prevalence of stunting may hopefully decrease to 20% (amounting to 127 million children) in 2025, which is the target of the world health assembly.(20) if the specific nutritional intervention package (provision to the mothers of folic acid, calcium, micronutrients, balanced protein and energy supplements, and promotion of breastfeeding and appropriate complementary feeding) will have been increased for a coverage of 90%, stunting will decrease and consequently also the mortality rate of the underfives.(21) children with uri in the last 2 months were more numerous in the group of cases than in the group of controls. the present study is in accord with a study in kupang, showing that children with a history of infectious disease had a greater probability of stunting than children without a history of infectious disease. stunting is associated with infectious disease, since during an illness the metabolic requirements increase, whereas the dietary intakes decrease, resulting in disturbed growth.(22) a history of diarrhea in the last 2 months was more frequent in the group of cases than in the group of controls. this study agrees with a study in nepal showing that diarrhea is a risk factor of stunting, where children with diarrhea have a 7.46 times greater risk of stunting.(15) diarrhea is associated with growth failure because of the occurrence of malabsorption of nutrients when the children have diarrhea. the age of 2 years is the age at which infectious diseases such as diarrhea carry an extremely high risk of stunting.(23) 154 children without exclusive breastfeeding were more numerous in the group of cases than in the group of controls. this study is similar to a study in ethiopia which showed that children with less than 6 months of breastfeeding had a greater risk of stunting than children with exclusive breastfeeding during the first 6 months.(24) a study in nepal showed that children who did not receive colostrum had a 3.40 times greater risk of stunting.(13) low birth weight infants tend to have intrauterine growth retardation because of poor maternal nutrition. low birth weight infants with subsequent inadequate nutrient intakes frequently have childhood infections in the growth period so that the growth of these children is retarded and ultimately results in stunting. these results are in agreement with those of a study in semarang city, in that low birth weight is a risk factor of stunting in children aged 1-2 years. children with a history of low birth weight have a 11.2 times greater risk of stunting than children with normal birth weight.(25) short birth length was more frequent in the group of cases than in the group of controls. this is in line with a study conducted in bogor district in 2013 on children aged 12 months, where it was found that birth length significantly affects stunting in children aged 12 months.(26) the most dominant risk factor of stunting in children aged 12-24 months in palu city, central sulawesi, was inappropriate complementary feeding practice in terms of quantity and quality. after controlling for the variables of history of exclusive breastfeeding and birth weight, the variables of birth length, complementary feeding practice, history of uri and history of diarrhea constituted the risk factors of stunting in children aged 12-24 months in palu city, central sulawesi. in the present study, the risk factors of stunting that were studied were only complementary feeding practice and adequacy levels of nutrients from complementary feeding, determined by means of semiquantitative ffq. this method carries informational bias (recall bias) since it is exclusively based on the capacity to remember. some mothers forgot what were the foods usually consumed by their children, so that they had to be questioned repeatedly. for health personnel, there is a need for improved dissemination of information in the form of health education to mothers of underfives on the quality and quantity of complementary feeding that is appropriate for consumption by children in their growth period so that it may prevent the risk of growth failure and deficiencies of certain nutrients that may be detrimental to the development of health and mental capacity of the children. in addition, health personnel need to improve dissemination of information to mothers of underfives so that they always practice behaviors of clean and healthy living (perilaku hidup bersih and sehat, phbs) to prevent the occurrence of infectious diseases, especially by maintaining food hygiene so that it may not be easily contaminated by bacteria. conclusion inappropriate complementary feeding practice was the must dominant risk factor of stunting in children aged 12-24 months old. this study confirms the need to scale up interventions during the first 2 years of life, including appropriate infant feeding practices. conflict of interest the authors declare that they have no conflict of interest with regard to the present study. acknowlegments we thank the head and staff of puskesmas sangurara, palu city, central sulawesi, who allowed us to conduct the study and obtain data at their puskesmas. thanks are also due to the parents of the study subjects for their cooperation in this study. references 1. kementerian kesehatan republik indonesia (kemenkes ri). riset kesehatan dasar hijra, muis, kartasurya inappropriate feeding practice and stunting 155 univ med vol. 35 no.3 (riskesdas) tahun 2010. jakarta: kementerian kesehatan republik indonesia;2010. 2. dinas kesehatan provinsi sulawesi tengah. laporan pemantauan status gizi (psg) tahun 2013. palu: dinas kesehatan provinsi sulawesi tengah;2013. 3. dewey kg, adu-afarwuah s. systematic review of the efficacy and effectiveness of compementary feeding interventions in developing countries. matern child nutr 2008;4:24-85. 4. shrimpton r, victora cg, de onis m, et al. worldwide timing of growth faltering: implications for nutritional interventions. pediatrics 2001;107:1-7. 5. world health organization (who). implementing the global strategy for infant and young child feeding. geneva: world health organization;2003. 6. lassi zs, das jk, zahid g, et al impact of education and provision of complementary feeding on growth and morbidity in children less than 2 years of age in developing countries: a systematic review. bmc public health 2013; 13:1-10. 7. victora cg, de onis m, hallal pc, et al. worldwide timing of growth faltering: revisiting implications for interventions. pediatrics 2010; 125: 473-480. 8. black re, allen lh, bhutta za, et al. maternal and child undernutrition: global and regional exposures and health consequences. lancet 2008;371:243-60. 9. teshome b, kogi-makau w, getahun z, et al. magnitude and determinants of stunting in children under-five years of age in food surplus region of ethiopia: the case of west gojam zone. ethiop j health dev 2009;23:98-106. 10. dewey kg, mayers dr. early child growth: how do nutrition and infection interact? maternal child nutr 2011;7:129–42. 11. adeladza at. the influence of socio-economic and nutritional characteristics on child growth in kwale district of kenya. afr j food agric nutr dev 2009;9:1570-90. 12. weisz a, meuli g, thakwalakwa c, et al. the duration of diarrhea and fever is associated with growth faltering in rural malawian children aged 6-18 months. nutr j 2011;10:1-4. 13. lestari w, margawati a, rahfiludin mz. faktor risiko stunting pada anak umur 6-24 bulan di kecamatan penanggalan kota subulussalam provinsi aceh. j gizi indonesia 2014;3:126-34. 14. al-rahmad ah, miko a, hadi a. kajian stunting pada anak balita ditinjau dari pemberian asi eksklusif, mp-asi, status imunisasi dan karakteristik keluarga di kota banda aceh. j kesehatan ilmiah nasuwakes 2013;6:169-84. 15. paudel r, pradhan b, wagle rr, et al. risk factors for stunting among children: a community based case control study in nepal. kathmandu university medical j 2012;10:1824. 16. imdad a, yakoob my, bhutta za. impact of maternal education about complementary feeding and provision of complementary foods on child growth in developing countries. bmc public health 2011;11:1-14. 17. stephenson k, amthor r, mallowa s, et al. consuming cassava as a staple food places children 2-5 years old at risk for inadequate protein intake, an observational study in kenya and nigeria. nutr j 2010;9:1-6. 18. mardewi kw. kadar seng serum rendah sebagai faktor risiko perawakan pendek pada anak (tesis). denpasar: universitas udayana;2014. 19. kimane-murage ew, ndedda c, raleigh k, et al. vitamin a supplementation and stunting levels among two year olds in kenya: evidence from the 2008-09 kenya demographic and health survey. int j child health nutr 2012;1: 135-47. 20. black re, victora cg, walker sp, et al. maternal and child undernutrition and overweight in lowincome and middle-income countries. lancet 2013;382:427-51. 21. bhutta za, das jk, rizvi a, et al. evidence based interventions for improvement of maternal and child nutrition: what can be done and at what cost? lancet 2013;382:452–77. 22. picauly i, toy sm. analisis determinan dan pengaruh stunting terhadap prestasi belajar anak sekolah di kupang dan sumba timur, ntt. j gizi pangan 2013;8:55-62. 23. dewey kg, mayers dr. early child growth: how do nutrition and infection interact?. matern child nutr 2011;7:129–42. 24. fikadu t, assegid s, dube l. factors associated with stunting among children of age 24 to 59 months in meskan district, gurage zone, south ethiopia: a case-control study. bmc public health 2014;14:1-7. 25. candra a, puruhita n, susanto jc. risk factors of stunting among 1-2 years old children in semarang city. media medika indonesiana 2011;45:206-12. 26. ernawati f, rosmalina y, permanasari y. pengaruh asupan protein ibu hamil dan panjang badan bayi lahir terhadap kejadian stunting pada anak usia 12 bulan di kabupaten bogor. penelitian gizi makanan 2013;36:1-11. suriptiastuti 101 re-emergensi chikungunya: epidemiologi dan peran vektor pada penyebaran penyakit suriptiastuti* *bagian parasitologi fakultas kedokteran universitas trisakti korespondensi adr. suriptiastuti, ms bagian parasitologi fakultas kedokteran universitas trisakti jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2305 universa medicina 2007; 26: 101-10. universa medicina april-juni 2007april-juni 2007april-juni 2007april-juni 2007april-juni 2007 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 vol.26 no.2 abstrak chikungunya (chik) adalah penyakit yang disebabkan oleh virus dari genus alphavirus, famili togaviridae, dan ditularkan melalui gigitan nyamuk aedes aegypti dan ae. albopictus yang terinfeksi oleh virus tersebut. penyakit ini pertama kalinya dilaporkan oleh robinson dan lumsden setelah terjadinya wabah di lembah makonde tahun 1952. seperti halnya dengan malaria dan dengue, penyakit ini telah menjadi endemis di negara-negara di afrika dan asia. dan telah menimbulkan wabah di tempat-tempat tersebut. chik ditandai oleh adanya tiga gejala khas (trias) yaitu demam, nyeri sendi (arthralgia) dan ruam kulit (rash). chik adalah penyakit yang bersifat dapat sembuh sendiri (self-limiting) dan tidak ada pengobatan yang spesifik untuk demam chik. upaya pengobatan hanya bersifat simtomatis, oleh karena itu pengendalian vektor merupakan usaha yang tepat pada pencegahan penyakit. kata kunci: chikungunya, vektor, epidemiologi 102 pendahuluan selama lebih dari satu dekade beberapa penyakit zoonotik dan virus yang bersifat vectorborne telah muncul menyerang berbagai daerah di asia tenggara dan pasifik barat. di antara p e n y a k i t v i r u s t e r s e b u t a d a l a h d e m a m c h i k u n g u n y a ( c h i k ) y a n g d i t u l a r k a n k e manusia melalui gigitan nyamuk aedes yang terinfeksi. chik bukan merupakan penyebab p e n y a k i t v e c t o r b o r n e u t a m a , n a m u n kemunculannya di daerah-daerah asia-pasifik telah menimbulkan berbagai masalah di bidang kesehatan masyarakat. demam chik yang mula-mula ditemukan di afrika, relatif umum dijumpai di bagian tenggara dan selatan benua asia sekitar tahun 1 9 6 0 a n . s e t e l a h m e n i m b u l k a n w a b a h d i berbagai negara di asia seperti india, sri langka, myanmar (burma, waktu itu) dan thailand, virus ini menghilang dan hanya re-emergence of chikungunya: epidemiology and roles of vector in the transmission of the disease suriptiastuti* *department of parasitology medical faculty, trisakti university correspondence adr. suriptiastuti department of parasitology medical faculty, trisakti university jl. kyai tapa no.260, grogol jakarta 11440 telp. 5672731 eks.2305 universa medicina 2007; 26: 101-10. abstract chikungunya (chik) fever is a disease caused by virus of the genus alphavirus in the family togaviridae which is transmitted to human by the bite of infected mosquito. the vector of this disease is the aedes aegypti and ae. albopictus. the disease was first described by robinson and lumsden in 1955 following an outbreak on the makonde plateau in 1952. like malaria and dengue, chik has been endemic in africa and asia and has caused epidemics in those places. the clinical features of chik fever include sudden onset of fever, arthralgia, and rash. chik is a selflimiting disease. there is no specific drug therapy available, treatment of chik fever is supportive. therefore, controlling the vector is an effective mean to prevent the disease. keywords: chikungunya, vector, epidemiology menyebabkan kasus-kasus sporadik saja yang berlanjut sampai tahun 1980an. (1) namun, wabah chik yang terbatas (localized) masih terjadi di negara-negara tersebut. virus juga menyebar ke indonesia dan untuk pertama kalinya ditemukan pada tahun 1982 -1985.(1) pada tahun 1995 wabah kembali menyerang thailand (2) dan tahun 1998-1999 mengenai malaysia.(1) ini adalah wabah yang pertama di malaysia meskipun sejak tahun 1960an antibodi terhadap virus chik telah banyak ditemukan di antara penduduk jasirah malaya dan serawak p e n y a k i t c h i k t e l a h m e n j a d i s e b u a h masalah global berhubung dengan eskalasi wabah di negara-negara seperti afrika, india dan asia tenggara. wabah ini merupakan akibat yang ditimbulkan oleh musim hujan yang memberikan suasana yang baik untuk nyamuk aedes berkembang biak di pemukiman urban di mana manusia merupakan hospes reservoir dari virus chik yang dibawa oleh nyamuk tersebut. suriptiastuti re-emergensi chikungunya 103 m e s k i p u n p e n y a k i t i n i t i d a k s a m p a i menyebabkan kematian, tetapi penderita dapat merasa sangat cemas oleh gejala-gejala yang terjadi. infeksi virus chik pada umumnya m e n i m b u l k a n s e r a n g a n m e n d a d a k d e n g a n demam dan nyeri sendi yang hebat pada daerah ekstremitas diikuti dengan kesulitan untuk menggerakkan sendi tersebut sehingga penderita seringkali menafsirkan kelainan sendi yang d i a l a m i i t u s e b a g a i k e l u m p u h a n . a d a n y a manifestasi klinis berupa perdarahan ringan khususnya pada kasus-kasus di daerah asia te n g g a r a d a n s u b k o n t i n e n i n d i a d i m a n a penyakit dengue endemis, infeksi chik tidak j a r a n g s a l a h d i d i a g n o s i s s e b a g a i d e m a m berdarah dengue. sayangnya, belum didapatkan vaksin untuk penyakit ini dan pengobatan spesifik juga belum ada sehingga sejauh ini pengobatan penyakit hanya ditujukan terhadap gejala-gejalanya saja. keprihatinan yang muncul berkaitan dengan penyakit ini adalah kecepatan dari infeksi chik ini menyebar dan mengenai banyak tempat di dunia. sejarah chikungunya c h i k a d a l a h p e n y a k i t i n f e k s i y a n g disebabkan oleh arbovirus dan disebarkan ke manusia melalui gigitan nyamuk aedes serta acapkali menimbulkan wabah. wabah yang terjadi di daerah urban bersifat sporadik tetapi karakteristiknya eksplosif. wabah tersebut kemudian menghilang dan timbul lagi berulang dalam interval yang tidak teratur.(3) penyakit chik untuk pertama kalinya dilaporkan oleh robinson(4) dan lumsden(5) dari kejadian sebuah w a b a h d i l e m b a h m a k o n d e , s e p a n j a n g perbatasan tanganyika dan mozambique, pada tahun 1952. nama chik sebenarnya berasal dari kata kerja bahasa makonde sendiri yaitu “ k u n g u n y a l a ” y a n g m e m p u n y a i a r t i “mengeringkan” (to dry up) atau “menjadi bengkok” (to become contorted).(4) selanjutnya robinson (4) menggunakan istilah tersebut secara lebih spesifik untuk menggambarkan orang yang menderita chik jalannya membungkuk sebagai akibat dari gejala artritis oleh penyakit tersebut. pada awalnya chik dijumpai dalam bentuk wabah di afrika, dan menyebabkan penyakit yang memberi gejalagejala seperti demam dengue. virus virus chik, atau dikenal juga sebagai “buggy creek virus” adalah virus yang termasuk dalam genus alphavirus dari famili togaviridae. alphavirus ini terdiri dari 30 spesies virus yang ditularkan oleh arthropoda (arthropod-borne v i r u s ) d i k e l o m p o k k a n m e n j a d i t u j u h serokompleks atas dasar data serologis.(6) secara serologis, virus chik termasuk subgrup semliki forest dari alphavirus. virus chik adalah s e b u a h v i r u s r n a b e r u l i r p o s i t i f d a n mempunyai selubung (enveloped) (gambar 1). suatu rna berulir-positif subgenomik yang dikenal sebagai 26s rna, identik dengan 3' dari rna genomik, ditranskip (transcribed) dari suatu perantara rna berulir-negatif. rna ini berfungsi sebagai mrna untuk sintesis dari p r o t e i n s t r u k t u r a l v i r u s . b i l a m e m a n d a n g susunan genomik dari alphavirus lainnya, genom chik dapat dinyatakan sebagai: 5’capnsp1-nsp2-nsp3-nsp4-(daerah ikatan)-c-e3e2-6k-e1-poly(a) 3' (gambar 2).(7) gambar 1. virus chikungunya universa medicina vol.26 no.2 104 suriptiastuti re-emergensi chikungunya pada saat ini ada 2 sekuens nukleotida lengkap dari virus chik yang sudah dikenali yaitu galur ross dan s27, keduanya diisolasi dari penderita wabah di tanzania tahun 1952.(8) sekuens nukleotida lengkap lainnya yang telah ditentukan adalah galur yang berasal dari wabah di senegal tahun 1983. analisis filogenetik berdasarkan sekuens parsial e1 dari isolat afrika dan asia menunjukkan akan adanya 3 filogrup virus chik, yaitu (i) semua isolat dari asia barat, (ii) isolat asia, dan (iii) isolat dari afrika tengah/timur.(8) siklus hidup alphavirus dimulai dengan perlekatannya pada reseptor pejamu lewat glikoprotein e2. virion kemudian menuju ke dalam vesikel sel pejamu melalui fusi membran antara virus dan vesikel pejamu, melepaskan nukleokapsid ke dalam sitoplasma. kemudian terjadi pelepasan genom rna untuk berlangsungnya sintesis genom rna yang baru. selanjutnya terjadi proses replikasi di dalam sel dan protein yang terbentuk kemudian bergerak ke permukaan sel pembentukan virion yang siap untuk dilepaskan dari dalam sel. di beberapa tempat di afrika, virus chik ditemukan dari nyamuk yang sifatnya zoofilik sehingga ada dugaan bahwa virus dapat berada di hewan seperti roden dan ternak. kecuali pada hewan-hewan tersebut, virus juga didapatkan pada tupai, chiroptera, dan tungau (ticks = alectorobius sonrai/). ditemukannya antibodi gambar 2. (a) strategi sekuensi untuk rna genomik chik. skala adalah dalam kilobasa. kotak menunjukkan daerah coding dari protein nonstruktural dan struktural. lokasi rna subgenomik ditunjukkan sebagai 26s rn. garis horisontal di kedua ujung menunjukkan daerah non-coding. seluruh genom diamplifikasi dengan cara rt-pcr untuk analisis sekuens bi-direksional; (b) struktur sekitar lintasan daerah i-poly (a) 105 spesifik pada roden dan burung mendukung dugaan bahwa suatu siklus sekunder terdapat pada hewan liar. di senegal, vektor sylvatic utama dari virus chik adalah sama dengan pada penyakit yellow fever, namun bentuk sylvatic ini belum pernah dijumpai sebagai bentuk yang dapat menularkan chik.(9,10) ada dugaan bahwa siklus liar tersebut mempunyai peranan dan dapat memberi kontribusi terhadap endemisitas chik di afrika. vektor vektor chik adalah nyamuk aedes yaitu ae. albopictus (gambar 3) untuk daerah rural atau pedesaan dan ae. aegypti untuk daerah urban atau perkotaan. aedes adalah genus nyamuk yang ditemukan di daerah tropik dan subtropik dan menjadi sangat berarti sebagai pembawa penyakit di masyarakat di banyak t e m p a t a t a u n e g a r a k a r e n a k e d e k a t a n n y a dengan manusia. secara morfologis, nyamuk ini menunjukkan pola yang khas yaitu adanya garis-garis putih yang di bagian samping dada membentuk gambaran seperti susunan dawai harpa (lyre-shaped), dengan palpi setengahnya putih dan probosis seluruhnya hitam.(9) pada umumnya nyamuk aedes beraktivitas pada pagi hari (mid-morning) antara jam 7.00 10.00 dan sore hari (late afternoon) jam 16.0019.00. nyamuk ini jarak terbangnya pendek, hanya sekitar kurang dari 200 m dan menyukai tempat-tempat dengan air bersih (fresh water) untuk berkembang biak. telur-telurnya tahan terhadap suasana kekeringan. nyamuk ae. a l b o p i c t u s m e r u p a k a n “ p e n g h u n i a s l i ” ( i n d i g e n o u s ) d a r i d a e r a h a s i a t e n g g a r a , kepulauan pasifik barat dan pulau-pulau di samudera hindia, yang pada beberapa puluh tahun belakangan telah menyebar ke afrika, daerah timur tengah, eropa dan amerika.(11) di beberapa tempat, nyamuk ae. albopictus ini terus berkembang dan telah mendesak ae. aegypti. variasi musiman (seasonal variation) dalam hal kepadatan populasi dan penyebaran (distribusi) spesies ditemukan pada ae. aegypti karena nyamuk ini peka terhadap perubahan suhu dan kelembaban yang ada di sekitar. umumnya populasi yang rendah dari nyamuk dijumpai pada musim kering dengan suhu rendah, sebaliknya mereka berkembang biak dengan cepat pada waktu suhu meningkat dengan kelembaban dan curah hujan yang tinggi.(9) musim hujan mendukung perkembangb i a k a n s t a d i u m a k u a t i k ( l a r v a d a n p u p a ) nyamuk dan melahirkan nyamuk-nyamuk muda. pepohonan dan bermacam tumbuh-tumbuhanan yang menjadi subur saat musim hujan juga memberi suasana teduh dan merupakan tempat y a n g b a i k b a g i n y a m u k d e w a s a u n t u k b e r i s t i r a h a t d a n m e l a k u k a n a k t i v i t a s perkembang-biakan. musim hujan dikaitkan dengan derajat prevalensi yang meningkat dari penyakit-penyakit yang ditularkan melalui gigitan nyamuk. di lingkungan perkotaan di mana banyak d i t e m u k a n a e . a e g y p t i , t e m p a t t e m p a t penampungan air menjadi faktor penting dalam perkembangan-biakan nyamuk tersebut. di universa medicina vol.26 no.2 gambar 3. nyamuk ae. albopictus 106 suriptiastuti re-emergensi chikungunya antara tempat-tempat penampungan, ada yang disukai nyamuk sebagai tempat perindukan (breeding place) seperti dilaporkan oleh banyak peneliti.(9-12) tempat plastik adalah yang paling cocok sebagi tempat perindukan di antara t e m p a t t e m p a t p e n a m p u n g a n a i r. te m p a t penampungan air berwarna hitam lebih disukai nyamuk baik jantan mmaupun betina, tetapi tempat dari logam tembaga (bronze) kurang cocok bagi perindukan nyamuk, mungkin karena unsur ion tembaga (copper) bersifat toksik. di samping itu, agaknya sifat logam yang mudah terpanasi menjadi salah satu faktor ketidakc o c o k a n t e m p a t t e m p a t d a r i l o g a m u n t u k perindukan nyamuk karena suhu di bagian dalamnya dapat mencapai suhu yang mematikan ( l e t h a l ) . ( 1 2 ) m e s k i p u n d e m i k i a n , t e m p a t p e n a m p u n g a n a i r d a r i l o g a m m a s i h d a p a t menjadi perindukan yang baik bagi ae. aegypti di bandingkan untuk ae. albopictus.(9) menurut shiram dan sehgal,(13) di daerah perkotaan ae. aegypti banyak dijumpai pada tempat-tempat penampungan air yang terbuat dari tanah liat, bata dan semen, disusul logam dan plastik. tetapi hendaknya perlu mendapat perhatian bahwa apapun bahan dari tempat penampungan air, tempat-tempat tersebut dapat menjadi p e r i n d u k a n n y a m u k s e h i n g g a d a p a t menyebarkan penyakit. epidemiologi virus chik telah banyak dilaporkan di hampir semua bagian benua afrika seperti yang diperlihatkan oleh derajat prevalensi antibodi igm terhadap virus ini dan isolasi virus dari nyamuk di negara-negara seperti pantai gading, republik afrika tengah, dan senegal.(10) di asia, wabah chik dilaporkan terjadi di daerah urban di mana ae. aegypti dan ae. albopictus menjadi vektornya.(1) daerah-daerah endemik chik di asia dan asia tenggara meliputi india, pakistan, myanmar, sri langka, philippines, indonesia dan malaysia.(1) bertambahnya rasa keprihatinan mengenai penyakit ini adalah karena kecepatan dan luas penyebarannya. sejumlah kejadian wabah banyak dilaporkan di b e b e r a p a n e g a r a . d i i n d i a , e p i d e m i y a n g pertama terjadi pada tahun 1963 di calcutta, kemudian menyusul wabah kedua tahun 1973. tetapi kemunculannya kembali (re-emergensi) terjadi di india selatan tahun 2005,(14) sejak itu w a b a h c h i k b e r k e m b a n g l u a s , m e n g e n a i banyak orang dan hingga saat ini jumlah kasus mencapai sekitar 180.000. penyebaran penyakit virus ini dilaporkan kebanyakan di bagian india selatan dekat samudera hindia dan teluk b e n g a l , s e d a n g k a n d i b a g i a n u t a r a t i d a k dijumpai adanya kasus chik. meskipun infeksi t i d a k s a m p a i m e n y e b a b k a n k e m a t i a n , bertambahnya jumlah kasus yang luar biasa b a n y a k n y a s e j a k t a h u n 2 0 0 5 i t u t e l a h menimbulkan kepanikan. pada 2006, diperkirakan sekitar 1,38 juta orang di sepanjang bagian selatan dan tengah i n d i a m e n u n j u k a n g e j a l a g e j a l a c h i k . ( 1 4 ) insidens penyakit mungkin lebih tinggi dari pada yang dilaporkan karena faktor-faktor seperti kesalahan dalam pengelompokan dan ketidaktepatan penggolongan penyakit. wabah yang terjadi di india pada 1963 dan 1973 disebabkan oleh virus jenis (genotipe) asia, tetapi epidemi chik tahun 2005 yang terjadi di pulau-pulau s a m u d e r a h i n d i a ( p u l a u p u l a u r e u n i o n , mayotte, mauritius, seychelles) dan yang terjadi di india 2006, disebabkan oleh virus genotipe afrika timur.(14) ada anggapan bahwa terjadinya mutasi virus, rendahnya derajat imunitas dari penduduk setempat, pengendalian vektor yang kurang memadai, dan globalisasi dalam masalah perdagangan dan perjalanan (travel), merupakan faktor yang menyebabkan terjadinya resurgensi infeksi. antara bulan maret dan april 2006, wabah chik dilaporkan terjadi di bagan pancor, perak, malaysia, mengenai lebih dari 200 orang 107 dan merupakan wabah kedua yang dilaporkan di malaysia.(15) terjadinya wabah di bagan pancor ini bertepatan dengan berjangkitnya wabah chik di samudera hindia. analisis filogenetik dari virus chik yang diisolasi dari penderita menunjukkan bahwa galur (strain) b a g a n p a n c o r m e m p u n y a i h u b u n g a n e r a t (closely related) dengan virus chik yang diisolasi pada wabah di klang tahun 1998. wabah ini merupakan peristiwa yang pertama kalinya virus chik diisolasi dan dilaporkan m e n i m b u l k a n g e j a l a g e j a l a p e n y a k i t d i malaysia.(16) penelitian-penelitian sebelumnya hanya dapat mendeteksi adanya antibodi virus chik pada populasi di daerah utara dan timur malaysia yang berbatasan dengan thailand di mana diketahui terdapat virus chik. sebuah sifat yang menonjol dari virus c h i k a d a l a h k e m a m p u a n n y a u n t u k menyebabkan wabah yang eksplosif di suatu daerah sebelum virus tersebut menghilang untuk periode beberapa tahun (sekitar 3-4 tahun). hal ini berbeda dengan keadaan virus dengue, meskipun memiliki vektor nyamuk yang sama dengan chik dalam penularannya, yaitu ae. a e g y p t i d a n a e . a l b o p i c t u s, v i r u s d e n g u e bersifat endemik. virus chik juga diketahui ditularkan melalui gigitan nyamuk golongan ae. furcifer-taylor yang mencari makanannya dari manusia dan primata.(10,17) adanya virus yang diisolasi dari primata dan hewan vertebrata lain seperti tupai dan kelelawar serta pada spesies nyamuk zoofilik mendukung eksitensi siklus transmisi silvatik (sylvatic transmission cycle) di benua afrika. keadaan inilah yang mungkin mempertahankan keberadaan virus di alam bebas pada tahun-tahun interepidemik. di asia, transmisi di daerah urban agaknya t e r j a d i t e r u t a m a d a r i a e . a e g y p t i d a n a e . albopictus ke manusia. tidak diketahui apakah di asia virus berada di alam bebas. juga belum ada hewan yang dapat ditentukan secara pasti menjadi reservoir virus meskipun ada dugaan m o n y e t m u n g k i n m e n j a d i p e j a m u c h i k . berbeda dengan dengue, transmisi transovarial dari virus chik di tubuh nyamuk belum dapat dibuktikan. perbedaan galur nyamuk aedes s e c a r a g e o g r a f i s b e r b e d a b e d a d a l a m kerentanannya terhadap infeksi dan dalam hal kemampuannya untuk menyebarkan virus. sifat ini mungkin menjadi salah satu faktor di dalam terjadinya endemisitas virus di suatu daerah. sampai saat ini sifat episodik dari wabah chik masih belum dapat dijelaskan, namun agaknya tergantung dari banyak faktor di antaranya adalah kerentanan pejamu (vektor dan manusia) terhadap infeksi, densitas nyamuk yang tinggi, dan masuknya virus dari daerah endemik lain. tetapi sebagaimana halnya dengan arbovirus lainnya, faktor-faktor seperti meningkatnya urbanisasi di banyak tempat, pemanasan global, dan intensitas transportasi, dapat mendorong m e n i n g k a t n y a p o p u l a s i n y a m u k a t a u menyebabkan terjadinya introduksi vektor ke suatu daerah geografis baru. keadaan ini di masa depan dapat mengubah epidemiologi chik seperti halnya dengan penyebarannya dari afrika ke asia. situasi wabah di indonesia peristiwa timbulnya wabah di indonesia d i p e r k i r a k a n d i m u l a i d e n g a n t e r j a d i n y a penyebaran virus chik sekitar tahun 1982-1985 dengan munculnya wabah chik di sumatera selatan (jambi) dan kalimantan barat (1982), jawa (yogyakarta, 1983), bagian selatan dan timur kalimantan (1983), nusa tenggara timur (1984), kepulauan maluku (1985), sulawesi selatan dan irian jaya (1985).(18) wabah pertama chik di indonesia dikenal sebagai “knuckle fever”. istilah ini berasal dari sebuah catatan di z a m a n b e l a n d a y a n g m e n u l i s k a n a d a n y a penyakit tersebut di batavia pada tahun 1779.(19) pada tahun 1973, suatu wabah penyakit demam yang tidak diketahui penyebabnya universa medicina vol.26 no.2 108 suriptiastuti re-emergensi chikungunya dilaporkan melanda samarinda dan balikpapan di sepanjang pantai timur pulau kalimantan. penyakit ini menunjukkan gejala-gejala yang cocok dengan infeksi chik.(20) sepuluh tahun kemudian, tepatnya tahun 1983, suatu rentetan epidemi chik terjadi di daerah istimewa yogyakarta, dengan attack rate sekitar 7090%.(3) delapan wabah lain yang mungkin sekali adalah chik dilaporkan terjadi di indonesia antara tahun 1982 dan 1985.(1) sebanyak 8.068 kasus dilaporkan selama t a h u n 1 9 8 3 d a r i 9 p r o p i n s i m e l i p u t i 2 9 kabupaten dan kota-kota di indonesia.(3) setelah masa tenang (hiatus) selama sekitar 20 tahun, antara 2001 sampai 2003 dilaporkan adanya 24 kejadian wabah chik di seluruh wilayah di indonesia.(20,21) re-emergensi epidemi chik yang terjadi di seluruh wilayah indonesia setelah masa tenang selama 20 tahun tidak berbeda dengan apa yang dialami sebelumnya oleh n e g a r a n e g a r a l a i n s e p e r t i i n d i a d a n thailand.(2,14,17) periode tenang tanpa aktivitas virus ini merupakan ciri epidemiologis yang membedakan chik dari kebanyakan penyakit virus vector-borne lain yang memiliki vektor dan dinamika transmisi yang serupa, terutama virus d e n g u e . p a d a u m u m n y a r e e m e rg e n s i d a n epidemi chik terjadi dalam siklus 7 atau 8 tahunan, meskipun demikian interval dua atau tiga dekade juga pernah dilaporkan dari negaran e g a r a s e p e r t i u g a n d a . ( 2 2 ) j u m l a h k a s u s meningkat sesuai dengan peningkatan umur dan perempuan lebih banyak dari pada laki-laki. ini kemungkinan besar menggambarkan keadaan di mana perempuan lebih banyak terpapar dengan aktivitas di lingkungan rumah pada siang hari sehingga risiko mendapat gigitan nyamuk aedes lebih besar.(20) meskipun kasus-kasus chik umumnya ditemukan di daerah urban dan suburban, kebanyakan episode wabah yang terjadi di indonesia pada waktu akhir-akhir ini mengenai daerah rural di jawa yang berpenduduk padat.(20) patogenesis dan gejala klinis infeksi chik merupakan penyakit infeksi yang melumpuhkan penderita ditandai dengan gejala demam, sakit kepala, mual, muntah dan nyeri otot dan sendi. dilaporkan bahwa attack rate pada populasi yang rentan berkisar antara 4 0 8 5 % d a n r a s i o s i m t o m a t i k t e r h a d a p asimtomatik adalah 1,2:1. (16) masa inkubasi penyakit antara 2-12 hari, tetapi umumnya 3-7 h a r i . s e t e l a h m a s a i n k u b a s i , s u h u b a d a n mendadak meningkat sampai 390-400c diikuti gejala menggigil yang intermiten. fase akut ini b e r l a n g s u n g 2 3 h a r i , s e l a n j u t n y a d e m a m menghilang untuk 1-2 hari dan kemudian timbul lagi sehingga memberi kurve demam dengan gambaran seperti “pelana” (saddle-back fever). demam pada chik secara tipikal berlangsung beberapa hari tetapi pada beberapa kasus rasa lemah (fatigue) yang menyertai penyakit ini dapat berlangsung lama seperti pada dengue, o’nyong-nyong, west nile atau infeksi arbovirus lain. selain demam, gejala klasik chik adalah myalgia, arthralgia, dan rash. arthralgia yang t e r j a d i s i f a t n y a p o l i a r t i k u l e r, b e r p i n d a h (migratory), dan terutama mengenai sendi-sendi k e c i l d a r i t a n g a n , p e r g e l a n g a n t a n g a n , pergelangan kaki dan kaki, sedangkan sendisendi besar sedikit saja dikenai. pembengkakan sendi terjadi sebagai akibat pengumpulan cairan. myalgia umum dan nyeri otot bagian belakang dan bahu adalah gejala yang biasa dijumpai. anak-anak jarang sekali mengalami nyeri sendi. penderita dengan manifestasi artikuler ringan biasanya bebas dari gejala nyeri sendi setelah beberapa minggu, tetapi mereka yang gejalagejala artikulernya berat memerlukan waktu b e b e r a p a b u l a n u n t u k s e m b u h s e c a r a menyeluruh. manifestasi kulit adalah tipikal untuk penyakit ini dan pada banyak penderita berupa kemerahan (flush) di daerah muka dan badan. suatu kelainan berupa ruam (rash ) m a k u l o p a p u l e r m e n g i k u t i g e j a l a f l u s h i n g 109 t e r s e b u t . r u a m m a k u l o p a p u l e r t e r u t a m a terdapat pada badan dan lengan tetapi telapak tangan dan kaki dapat juga dikenai. rasa gatal dan iritasi dapat menyertai erupsi ini. pada saat berlangsungnya keadaan akut dari penyakit, kebanyakan penderita mengeluh sakit kepala. fotofobia dan nyeri retro-orbital dapat dijumpai. pada bayi dan anak-anak, flushing yang jelas dan adanya ruam makulopapuler secara dini atau urtikaria adalah petunjuk yang bermanfaat. namun, gejala-gejala yang ditampilkan oleh chik sering kali secara klinis tidak dapat dibedakan dari demam dengue. isolasi yang simultan dari kedua virus tersebut dari sera penderita pernah dilaporkan sebelumnya dan merupakan petunjuk adanya infeksi gabungan (dual infection). (17) oleh karena itu sangat penting untuk membedakan infeksi virus chik dari demam dengue. secara umum, manifestasi perdarahan tidak dijumpai pada infeksi virus chik. pada infeksi yang tanpa komplikasi, gejala akut akan menghilang dalam waktu sekitar 10 hari dan mayoritas penderita akan sembuh secara total tetapi ada laporan mengenai beberapa kasus yang masih menderita nyeri sendi selama beberapa tahun setelah sembuh.(16) komplikasi yang serius sangat jarang dan kasuskasus yang fatal belum pernah secara konklusif ditemukan. uji diagnostik diagnosis pasti dapat dilakukan dengan uji laboratorium tetapi infeksi chik sudah harus dipikirkan bilamana terjadi wabah penyakit dengan tiga gejala (trias) utama yaitu demam, adanya ruam (rash) dan manifestasi reumatik. isolasi virus dapat dilakukan dengan inokulasi sel biakan nyamuk (mosquito cell culture), menyuntik nyamuk dengan sera penderita, inokulasi sel biakan mamalia atau mencit (suckling mice). viremia terjadi pada 48 jam pertama dari infeksi dan dapat dideteksi sampai hari keempat. antibodi igm dapat dideteksi dengan menggunakan metode elisa pada penderita yang telah sembuh dari infeksi chik dan akan menetap dalam kadar yang cukup tinggi selama 6 bulan. antibodi hambatan hemaglutinasi (haemagglutination inhibition antibodies) timbul sejalan dengan menurunnya v i r e m i a d a n s e m u a p e n d e r i t a a k a n memperlihatkan hasil positif pada hari ke-5 dan ke-7 masa sakitnya.(17) pengobatan sampai saat ini belum ada vaksin atau antiviral yang spesifik untuk chik. pengobatan masih bersifat simtomatik seperti istirahat, p e m b e r i a n c a i r a n u n t u k m e m p e r t a h a n k a n k e s e i m b a n g a n c a i r a n , o b a t o b a t u n t u k menurunkan panas badan (antipiretik). istirahat dianjurkan selama terdapat gejala sendi akut. setelah lewat masa akut, dapat diberikan aspirin untuk nyeri sendi. pada arthritis yang tidak dapat diobati dengan aspirin, klorokuin fosfat (250 mg/ hari) memberikan hasil yang cukup menjanjikan. beberapa studi menunjukkan bahwa klorokuin fosfat memiliki efek antiviral terhadap virus c h i k , n a m u n b e l u m k o n k l u s i f . ( 2 3 ) m a s i h dibutuhkan banyak penelitian sebelum dapat dipastikan mengenai aktivitas antiviral tersebut. kesimpulan perubahan global dalam aktivitas manusia serta faktor-faktor ekologis telah membawa dampak dengan terjadinya dan munculnya penyakit-penyakit infeksi dalam beberapa tahun terakhir ini, di antaranya adalah penyakit chik. tidak diketahui secara jelas mengapa terjadi penyebaran penyakit ini yang di beberapa tempat menimbulkan wabah. namun ada dugaan kuat bahwa berbagai perubahan yang terjadi secara global telah mendorong meningkatkan populasi nyamuk dan menyebabkan infeksi serta wabah. universa medicina vol.26 no.2 110 oleh karena itu, suatu pemahaman yang lebih baik mengenai vektor, virus dan penyakit yang ditimbulkannya untuk mengatasi dan melakukan upaya pencegahan terhadap penyakit tersebut. daftar pustaka 1. mackenzie js, chua kb, daniels pw, eaton bt, field he, hall ra, et al. emerging viral diseases of southeast asia and the western pacific. emerg infect dis 2001; 7(suppl): s497-s504. 2. thaikruea l, charearnsook o, reanphumkarnkit s, dissomboon p, phonjan r, ratchbud s, et al. chikungunya in thailand: a reemerging disease? southeast asian j trop med public health 1997; 28: 359-64. 3. berger sa. gideon: a comprehensive web-based resource for geographic medicine. int j health geogr 2005; 4: 10. 4. robinson m. an epidemic of virus disease in southern province, tanganyika territory, in 195253; i. clinical features. trans r soc trop med hyg 1955; 49: 28-32. 5. lumsden whr. an epidemic of virus disease in southern province, tanganyika territory, in 195253; ii. general description and epidemiology. trans r soc trop med hyg 1955; 49: 33-57. 6. van regenmortel mhv, fauquet cm , bishop dhl, carstens eb, estes mk, lemon sm, et al. virus taxonomy. seventh report of the international committee on taxonomy of viruses. san diego: academic press; 2000. 7. khan ah, morita k, parquet mc, hasebe f, mathenge eg, igarashi a. complete nucleotide sequence of chikungunya virus and evidence for an internal polyadenylation site. j gen virol 2002; 83: 3075-84. 8. hasebe f, parquet mc, pandey bd, mathenge eg, morita k, balasubramaniam v, et al. combined detection and genotyping of chikungunya virus by a specific reverse transcription-polymerase chain reaction. j med virol 2002; 67: 370-4. 9. okogun gra, nwoke beb, okere an, anosike jc, esekhegbe ac. epidemiological implications of preferences of breeding sites of mosquito species in midwestern nigeria. ann agric environ med 2003; 10: 217-22. 10. diallo m, thonnon j, traore-lamizana m, fontenille d. vectors of chikungunya virus in senegal: current data and transmission cycles. am j trop med hyg 1999; 60: 281-6. 11. gratz ng. critical review of the vector status of aedes albopictus. med vet entomol 2004; 18: 21527. 12. vezzani d, schweigmann n. suitablility of containers from sources as breeding sites of aedes aegypti (l.) in a cemetery of buenos aires city, argentina. mem inst oswald cruz 2002; 97: 78992. 13. shiram an, sehgal sc. aedes aegypti (l.) in port blair, andaman and nicholas islands distribution and larval ecology. j commun dis 1999; 31: 18592. 14. kalantri sp, joshi r, riley lw. chikungunya epidemic: an indian perspective. natl med j india 2006; 19: 315-22. 15. kumarasamy v, prathapa s, zuridah h, chem yk, norizah i, chua kb. re-emergence of chikungunya virus in malaysia. med j malaysia 2006; 61: 221-5. 16. sam i-c, abu bakar s. chikungunya virus infection. med j malaysia 2006; 61: 264-9. 17. kamath s, das ak, parikh fs. chikungunya. j assoc physicians india 2006; 54: 725-6. 18. carey de. chikungunya and dengue: a case of mistaken identity? j hist med allied sci 1971; 24362. 19. kanamitsu m, taniguchi k, urasawa s, ogata t, wada y, saroso sj. geographic distribution of arbovirus antibodies in indigenous human population in the indo-australian archipelago. am j trop med hyg 1979; 28: 351-63. 20. laras k, sukri nc, larasati rp, bangs mj, kosim r, djauzi, et al. tracking the re-emergence of epidemic chikungunya virus in indonesia. trans r soc trop med hyg 2005: 88:128-41. 21. edberg sc. global infectious diseases and epidemiology network (gideon): a web-based program for diagnosis and informatics in infectious diseases. clin infect dis 2005; 40: 123-6. 22. lanciotti rs, ludwigml, rwaguma eb, lutwama jj, kram tm, karabatson m, et al. emergence of o’nyong-nyong fever in uganda after a 35-year absence: genetic characterization of the virus. virology 1998; 252: 258-68. 23. center for disease control and prevention. chikungunya fever diagnosed among internatriomal travelers -united states 2005-2006. morb mortal wkly rep 2006; 55:1040-2. suriptiastuti re-emergensi chikungunya f:\36 no 2\editorial.pmd 77 universa medicina may-august 2017 vol.36 no.2 glycemic control for prevention of vascular complications in diabetic patients pusparini department of clinical pathology faculty of medicine trisakti university patients with type 1 diabetes (t1d) and type 2 diabetes (t2d) show an increased risk of vascular complications such as cardiovascular disease and an increased mortality rate resulting in a decrease in life expectancy of between 5-15 years, depending on age at diagnosis.there are several methods to establish the diagnosis of t2d. the american diabetes association (ada) recommends the following: i. fasting plasma glucose (fpg 126 mg/dl after no caloric intake for at least 8 hours), ii. two hour postprandial glucose (ppg 200 mg/dl after a 75 g oral glucose load), iii. glycated hemoglobin (hba1c) 6.5%, and iv. random plasma glucose 200 mg/ dl in patie nts with classic symptoms of hyperglycemia. fpg and ppg reflect the random glucose concentration, whereas hba1c reflects the mean blood glucose concentration of the previous 2 to 3 months. fpg and ppg contribute to hba1c, but the interrelationship of the three types of measurements varies between patients and is c omplex, while the risk of diabetic complications is still unclear.(1) there is strong epidemiological and pathophysiological evidence that hyperglycemia has a deleterious effect on cardiovascular profile. patients with t2d who have high glucose and hba1c levels are at high risk for cardiovascular complications. glycemic fluctuation in the blood is reportedly associated with an increased risk of t2d complications. glycemic fluctuation and chronic hyperglyce mia may trigger an inflammator y re sponse through increased endoplasmic reticulum stress and mitochondrial superoxide production. it has been known that the molecular pat hways under lying hyperglycemia, low-grade inflammation and oxidative stress play a role in the pathogenesis of endothelial dysfunction, which constitutes the initial step of atherogenesis. through this pathway, the initial atherogenesis induced by hyperglycemia may result in an increased probability for cardiovascular events in later life. the direct effects of glucose toxicity, oxidative stress and low-grade inflammation may be seenin decreased insulin sensitivity, reduction in the number of beta cells, and endothelial dysfunction, finally resulting in microand macrovascular complications.(2) there are several methods for measuring and recor ding glycemic fluc tuation. self monitoring of blood glucose (smbg) measures the daily glucose concentration at up to 8 time points. another method, the continuous monitoring of glucose (cmg) system measures glycemic fluctuation in greater detail by means of more frequent measurement of glucose concentration. measurement by smbg frequently has the limitation of nocturnal hypoglycemia so that the experts are increasingly recommending the use of cmg for recording the daily glucose profile in clinical practice, with a target glucose concentration range of 70-180 mg/dl, although this is not an ideal range of glucose concentration. this target is commonly used in daily practice and has been considered safe and realistic.(3) 78 universa medicina the target glucose concentrationrange is related to the risk of complications that may be experienced by the diabetic patient. a glucose concentration of above 180 to more than 400 mg/dl is categorized as high and hazardous, since it increases the risk of diabetic ketoacidosis, such as metabolic acidosis and ketonemia. on the other hand, low glucose concentrations below 70 mg/dl are also associated with various risks, such as convulsions, coma, and death.(4) the va r i ou s r is ks a s so c i a t e d w i th gl yc e mi c fluctuation consist of: 1. hypoglycemia, 2. macrovascular complications, and 3. oxidative stress as the mechanism of vascular damage. glycemic fluctuation includesh yperglycemic episodes and even hypoglycemia. a study on 29 patients with well-controlled t2d, who had hba1c levels of 7%, and were treated with me t f o r mi n a nd su l f o nyl ur e a , w it h cg m monitoring for 5 days, showed that 18 patients ( 6 2 % ) h a d a hi s t or y o f a sympt o ma ti c hypoglycemic episodes.(5) the evidence shows a relationship between severe hypoglycemia and increased morbidity and mortality rates, such as increased occurrence of microvascular and macrovascular complications, deaths from cardiovascular events and other causes of death.(5) glycemic fluctuation has been reported to cause cardiovascular complications in t2d. the study designated as diabetes epidemiology collaborative analysis of diagnostic criteria in europe (decode), which was conducted on more than 20,000 t2d patients, found that impaired glucose toleranceas determined by 2hour post-prandial glucose (the main contributor to glycemic fluctuation), increased the risk of cardiovascular and all-cause mortality.(6) diabetes is associated with a markedly increased risk of atherosclerotic vascular disorders, including coronary, cerebrovascular, and peripheral artery disease, which may account for the disabilities and high mortality rates in diabetics.(7) in addition, the diabete s interve ntion study f ound a relationship between poor control of postprandial glucose and increased risk of myocardial infarction and death in 994 patients with t2d.(3) a study in finland that had been conducted for 18 years showed a similar impact of t1d and t2d oncardiovascular mortality rate, with 5.2and 4.9-fold increased risks for type 1 and type 2 diabetes, respectively.glycemic fluctuation leads to excessive production of reactive oxygen species which has an impact on endothelial dysfunction.(4,8,9) rapid changes in both peak and diurnal glucose concentration is called glycemic fluctuation. currently glycemic fluctuation is a potential therapeutic target in t2d. in normal individuals, food intake causes a momentary increase in glucose concentration to a higher than basal level. but this does not apply to t2d, where the basal glucose concentration is already relatively high, as a re sult of fasting hyperglycemia, so that control of post prandial blood glucose is required to see an increase in post-prandial glucose concentration. in such patients the glucose concentration can vary widely throughout the day, although the target hba1c is well controlled. a study on t2d patients with wellcontrolled hba1c (hba1c <6.5%) who were using oral glucose lowering drugs, 24-hour monitoring of glucose concentration showed a unique pattern for each patient, with increased fluctuations in glucose concentration exceeding the target glucose range of 70-180 mg/dl.(3) there was no unanimous support among the avaiable studies for the suggestion that a reduction in glucose concentration may decrease morbidity and mortality rates from vascular complications. among the clinical trials investigating the relationship between glycemic control in t1d and t2d on the one hand and decreased risk of cardiovascular disease on the other hand, there were no convincing results in all of them. in the studies called action in diabetes and vascular disease: preterax and diamicron mr controlled evaluation (advance), the veterans affairs diabetes trial (vadt), and the action to control cardiovascular risk in diabetes (accord), there were no significant reductions in the risk of cardiovascular events in the treatment groups, 79 univ med 2017;36:77-9. doi: http://dx.doi.org/10.18051/univmed.2017.v36.77-79 may-august 2017 vol.36 no.2 these results being caused by increased mortality rates.(10) the negative results of these studies were caused by the fact that the accord study, which was specifically designed for cardiovascular outcomes, was also discontinued before the sample size had been reached, because of an unexpected increase in mortality rate in the intervention group. there were also several studies with such a low power that they failed to give clear results. such studies should be pooled in a meta-analytic studyso as to extract relevant information.(10,11) the assumption t hat trea tment of hype rglycemia can p revent all diabetic complications, inclu ding the macroand microvascular is still subject to debate. the results of several clinical trials show that glucose lowering therapies can be hazardous in certain conditions. some large scale clinical trials show that improvement in glycemic control is associated with decreased cardiovascular events, whereas hypoglycemia can increase death as a result of cardiovascular complications. accurate glycemic control for avoidin g hyperglyc emic and hypoglycemic events is recommended to prevent diabetic cardiovascular complications, whilethere is an individual target approach for hba1c level. the care of patients with diabetes requires comprehensive mana geme nt to pr event cardiovascular complications, including other cardiovascular risk factors besides glycemia. (2) references 1. american diabetes association. standard of medical care in diabetes 2016 abridged for primary care providers. clin diabetes 2016;34: 3–21. doi: 10.2337/diaclin.34.1.3. 2. mannucci e, dicembrini i, lauria a, et al. is glucose control important for prevention of cardiovascular disease in diabetes? diabetes care 2013;36:s259-63. 3. kohnert kd, heinke p, vogt l, et al. utility of different glycemic control metrics for optimizing management of diabetes. world j diabetes 2015; 6:17-29. 4. bergenstal rm, ahmann aj, bailey t, et al. recommendations for standardizing glucose reporting and analysis to optimize clinical decision making in diabetes: the ambulatory glucose profile. j diabetes sci technol 2013;7: 565-78. 5. simonyl g, csitari g, gasparics r, et al. silent hypoglycaemic episodes among well controlled type 2 diabetic patients treated with sulfonylureas. diabetologia 2015;58 suppl 1: s465-9. 6. decode study group, the european diabetes epidemiology group. glucose tolerance and cardiovascular mortality comparison of fasting and 2 hour diagnostic criteria. arch intern med 2001;161:397-405. 7. yamagishi s. cardiovascular disease in recent onset diabetes mellitus. j cardiol 2011;57:25762. 8. inzucchi se, bergenstal rm, buse jb, et al. management of hyperglycemia in type 2 diabetes, 2015: a patient centered approach. update to a position statement of the american diabetes association and the european association for the study of diabetes. diabetes care 2015;38: 140-9. doi: 10.2337/dc14-2441. 9. juutilainen a, lehto s, ronnemaa t, et al. similarity of the cardiovascular mortality in middle aged subjects. diabetes care 2008;31: 714-9. 10. the advanced collaborative group. intensive blood glucose control and vascular outcomes in patients with type 2 diabetes. n engl j med 2008; 358;2560-72. 11. hayward r, reaven pd, wiitala wl, et al. follow up of glycemic control and cardiovascular outcomes in type 2 diabetes. n engl j med 2015; 372:2197-205. restu 141 *center for public health technology and intervention, health research and development board, ministry of health, republic of indonesia **center for applied health technology and clinical epidemiology, health research and development board, ministry of health, republic of indonesia correspondence dra. woro riyadina, m.kes center for public health technology and intervention, health research and development board, ministry of health, republic of indonesia jl. percetakan negara no. 29, po box 1226 jakarta 10560 phone: hp: +62818787095, email:w_riyadina@yahoo.co.id univ med 2014;33:141-50 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background migraine is a primary headache causing substantial disability in patients. the prevalence of migraine in women is still high. menarche, menstruation, pregnancy, menopause, and the use of hormonal contraceptives and hormone replacement treatment may influence migraine occurrence. the aim of this study was to determine the major determinants of migraine in adult women aged 25-65 years. methods a cross-sectional study of 2,747 women from the baseline study “cohort study of risk factors for non-communicable diseases”. the dependent variable was migraine based on the diagnosis of health providers or symptoms. independent variables were demographic (age, marital status, education) and behavioral (smoking, diet, and stress) characteristics, metabolic disorders (obesity, hypertension, dyslipidemia), and hormonal factors (contraception and hormone therapy). data were collected through interviews (characteristics, health and hormonal status, diet), measurement (anthropometrics, blood pressure), and health examination (blood specimens, neurology). data were analyzed by chisquare test and multiple logistic regression. results migraine in adult women was found in 710 cases (25.8%) with symptoms of worsening with activity (15%), nausea and vomiting (13%), and photophobia/ phonophobia (4.1%). the main determinant of migraine in adult women was stress with a 2.47-fold risk [95% ci = 2.07 to 2.95] as compared with no stress, after controlling for smoking, menstruation and hormonal drug consumption. conclusion stress is a major determinant of migraine in adult women, therefore health programs should be instituted through health promotion, prevention and education to control stress. key words: migraine, stress, risk factors, adult women stress as a major determinant of migraine in women aged 25-65 years woro riyadina* and lelly andayasari** 142 riyadina, andayasari stress a determinant of migraine stres sebagai determinan utama terjadinya migren pada perempuan umur 25-65 tahun latar belakang migrain merupakan nyeri kepala primer yang menyebabkan disabilitas secara substansial bagi penderita. proporsi migrain pada perempuan masih tinggi. faktor menarke, haid, kehamilan, menopause, dan penggunaan obat kontrasepsi hormonal serta pengobatan sulih hormon dapat mempengaruhi kejadian migren. tujuan penelitian ini untuk menentukan determinan utama penyakit migren pada perempuan dewasa berusia 25-65 tahun. metode desain penelitian potong-lintang pada 2747 perempuan dari data baseline penelitian “studi kohor faktor risiko penyakit tidak menular”. variabel dependen adalah migren berdasarkan hasil diagnosis tenaga kesehatan atau gejala. variabel independen meliputi karakteristik demografi (umur, status kawin, pendidikan), perilaku (merokok, konsumsi makanan berisiko, dan stres), gangguan metabolik (kegemukan, hipertensi, dislipidemia) dan faktor hormonal (kontrasepsi dan terapi hormon). data dikumpulkan dengan metode wawancara (karakteristik, status kesehatan dan hormonal, diet), pengukuran (antropometri, tekananan darah) dan pemeriksaan (spesimen darah, neurologi). data dianalisis dengan uji chi-square dan uji regresi logistik ganda. hasil penyakit migren pada perempuan ditemukan pada 710 orang (25,8%), gejala penyerta nyeri kepala semakin hebat dengan aktivitas 15%, mual dan muntah 13% serta fotofobia/fonofobia 4,1%. determinan utama penyakit migren pada perempuan adalah stres dengan risiko 2,47 kali [95% ci = 2,07 – 2,95] dibandingkan tanpa stres, dengan mengontrol faktor merokok, mentruasi dan konsumsi obat hormon. kesimpulan determinan yang paling berperan terhadap migren adalah stres, sehingga program terkait perlu segera melakukan upaya promotif dan preventif melalui edukasi untuk mengendalikan faktor stres pada perempuan usia 25-65 tahun. kata kunci: migren, stress, faktor risiko, perempuan dewasa abstrak introduction migraine is a primary headache that is frequently misunderstood, poorly diagnosed, and seldom treated in clinical practice.(1) the s y m p t o m s o f m i g r a i n e a r e c o m m o n l y accompanied by nausea, vomiting, or excessive sensitiveness to light (photophobia). in many people, migraine causes pulsations occurring only on one side of the head.(2) migraine may cause substantial disability to the patients, their family, and the community, as a result of its high prevalence in the general population.(3) the respective prevalences of migraine in both males and females in spain,(3) taiwan,(4) and china (5) are 11.20%, 25.01%, and 9.3%.(4) its prevalences in taiwanese and spanish w o m e n a r e 1 5 . 0 7 % (3) a n d 1 5 . 9 4 % , respectively.(4) the spanish study showed that females have a 3.02-fold greater risk for migraine than males.(3) the prevalences of migraine by gender are said to be 43% for females and 18% for males, with a significant correlation being found between gender and migraine..(6) the main cause of migraine is still not known with certainty. it is believed that 143 migraine is caused by a combination of genetic and environmental factors. migraine is also influenced by several other factors, such as physiological (stress, fatigue, hunger, hormones) and dietary factors.(7) the main determinant of migraine in women has not been extensively studied. studies on migraine in women are few in number and do not completely reveal its determinants or factors. the existing studies state that the main factor influencing migraine is variation in female sex hormone levels.(8) genetic factors also influence the occurrence of migraine in women.(9) the aim of the present study was to find the main determinant of migraine in women aged 25–65 years. methods research design this was a study of cross-sectional design carried out in 5 kelurahan [villages] of central bogor district [kecamatan bogor tengah], bogor city, in 2011-2012. study subjects women aged 25–65 years were recruited into the study if they met the inclusion criteria (permanent resident, capable of communication, and independent) and the exclusion criteria (pregnant, refusing to participate). a subject was categorized as having migraine if she had been diagnosed by health providers or had unilateral headache accompanied by at least 1 of these 3 symptoms: aggravation by physical activity, nausea, and vomiting, and photophobia or phonofobia. the sample size was determined from the formula for estimation of proportions. after adjusting by a drop-out factor of 20%, the required sample size was 2747.(10) the sample w a s o b t a i n e d b y c o n s e c u t i v e s a m p l i n g (voluntary until the required size was reached) among households (permanent residents) selected from all rukun tetangga (rt) in all rukun warga (rw) of 5 kelurahan (kebon kalapa, babakan pasar, babakan, ciwaringin, and panaragan) in central bogor district [kecamatan bogor tengah], bogor city. data collection t h e s e l e c t e d s t u d y s u b j e c t s w e r e interviewed using questionnaires, consisting of public health questionnaires on demographic characteristics, health status (including mental or stress status), and nutritional questionnaires, comprising recall questionnaire and food frequency questionnaire (ffq). physical examination comprised determination of anthropometric measures (weight, height, and abdominal circumference) and blood pressure, in addition to laboratory investigations for fasting blood glucose and 2-hour blood glucose after a glucose load of 75 grams.(11) interviews the variables collected by interviews comprised the characteristics of the respondents ( a g e , g e n d e r, m a r i t a l s t a t u s , e d u c a t i o n , o c c u p a t i o n ) , b e h a v i o r ( s m o k i n g , a n d consumption of migraine trigger foods), mental health (stress), migraine status, and hormonal factors (consumption of hormonal drugs or hormone replacement therapy). age was categorized into 2 age groups, i.e. 25-44 years and 45–65 years. marital status was categorized into unmarried, married, and divorced/widowed. educational status of the respondents, based on length of education until completion, was categorized into 3 categories, viz. low (no education and primary school), intermediate (junior and senior high school) and high (d3 and sarjana). smoking habit was categorized into smoker (including ex-smokers) and non-smoker. mental health status was assessed by symptoms o f e m o t i o n a l d i s o r d e r ( s t r e s s ) , b y t h e respondents themselves answering questions on emotional disorder contained in the self reporting questionnaire (srq). respondents were considered to have symptoms of emotional disorder (stress) if they had at least 6 of the 20 symptoms. univ med vol. 33 no.2 144 riyadina, andayasari stress a determinant of migraine hormonal factors were defined as factors associated with a reproductive condition or status (menstruation) and use of hormonal contraceptives, hormonal drugs, and hormone replacement therapy. menstrual status was categorized as “yes” (if still menstruating) and “no” (if in menopause/ceased to menstruate). contraceptive use was categorized as “yes” (if using hormonal contraceptive pills, injections, or implants, and “no” (if not using them). hormonal therapy was defined as treatment or prevention by hormonal drugs or hormone replacement therapy. the categories were “yes” (if taking hormonal drugs or hormone replacement therapy) and “no” (if not taking hormonal drugs or hormone replacement therapy). measurements these comprised height and weight for calculation of body mass index (bmi) using the formula weight (weight in kg) /height2 (height in meters). bmi was categorized according to the classification of the world health organization into 4 categories, i.e. underweight (bmi <18.5), normal (bmi 18.5–22.9), overweight (bmi 23– 24.9) and obese (bmi >25). in the multivariate analysis obesity was categorized as obese (bmi >25) and non-obese (bmi <25). hypertension was defined as the condition of a systolic blood pressure of >140 mmhg and/or a diastolic blood pressure of >90 mmhg, based on the 2007 standards of the joint national committee (jnc) on prevention, detection, evaluation and treatment of high blood pressure.(12) metabolic abnormality was defined as the presence of metabolism-related disorders in an individual, comprising obesity, hypertension, dyslipidemia, and hyperglycemia. laboratory tests the laboratory tests were performed by regional private laboratories with iso 15189 and iso 9001 accreditation. all determinations were done on an automatic analyzer. total cholesterol concentration was determined by the colorimetric enzymatic hexokinase method. determination of high density lipoprotein (hdl) and low density lipoprotein (ldl) cholesterol in the blood was by direct homogeneous enzymatic method. triglyceride concentration was by the colorimetric enzymatic glycerol-3-phosphate method. determination of fasting blood glucose and 2-hour blood glucose after glucose loading was by the glucose hexokinase ii (gluh) method. dyslipidemia was defined as the condition in which blood total cholesterol, ldl cholesterol, and triglycerides were higher than normal, while hdl cholesterol was lower than normal. total cholesterol was categorized as “high” if >200 mg/dl and “normal” if <200 mg/dl. ldl cholesterol was categorized as “high” if >100 mg/dl and”normal” if <100 mg/dl. triglyceride level was categorized as “high” if >150 mg/dl and “normal” if <150 mg/dl. hdl cholesterol was categorized as “low” if <40 mg/dl in males and <50 mg/dl in females, while it was categorized as “normal” if >40 mg/dl (males) and >50 mg/dl (females). h y p e r g l y c e m i a w a s d e f i n e d a s t h e condition of the blood glucose level exceeding the standard. determination of hyperglycemia was based on blood glucose levels with a loading dose of 75 gram glucose. hyperglycemia was categorized as diabetes mellitus (dm) if the blood glucose was >200 mg/dl, as impaired glucose tolerance if the blood glucose was 140199 mg/dl, and as normal if the blood glucose was <140 mg/dl. dietary intake the habit of consumption of migraine trigger foods was determined on the basis of frequency of consumption of foods categorized as migraine triggers, such as milk, coconut milk (santan) and legumes. the dietary consumption was obtained by 24-hour recall and food frequency questionaire (ffq). ffq is a recall i n t e r v i e w m e t h o d a s k i n g a b o u t h a b i t u a l consumption on the basis of frequency of food consumption per week. the variable habitual consumption of trigger foods was categorized as “yes” (if consumed) or “no” (if not). 145 data analysis the data were analyzed using the chisquare test, for differences in proportions and and crude odds ratio. multiple logistic regression was used to find the main determinant associated with migraine in women after controlling for other determinants and calculating the adjusted odds ratio. ethical clearance this study was approved for ethical clearance by the commission on health research ethics of the health research and development board, ministry of health, republic of indonesia (litbangkes kemenkes ri). results a t o t a l o f 2 7 4 7 r e s p o n d e n t s w e r e successfully recruited into the study, among whom 710 respondents (25.8%) were diagnosed as having migraine. the accompanying symptom of headache aggravated by increasing activity was experienced by 15% respondents, nausea or vomiting by around 13.2%, and phonophobia or photophobia by 4.2%. table 1. distribution of migraine by respondent characteristics and risk behavior *significant at p<0.05 univ med vol. 33 no.2 146 riyadina, andayasari stress a determinant of migraine the distribution of women with migraine by respondent characteristics is presented in table 1. table 1 shows that characteristics and risk behavior have significant differences in the proportions (p<0.05) of the variables age, smoking, and emotional disorder (stress). the table 2. distribution of migraine by metabolic disorder and hormonal factors of respondents bmi = body mass index; hdl = high density lipoprotein; ldl = low density lipoprotein; dm = diabetes mellitus; igt = impaired glucose tolerance; *significant at p<0.05 metabolic disorder migraine p value yes (n=710) n o (n=2037) hyperten sion yes 241 25.3 710 74.7 0.680 no 469 26.1 1327 73.9 bmi obesity 382 24.6 1109 75.4 0.466 overweight 126 27.2 338 72.8 normal 189 27.4 500 72.6 un derweight 33 26.8 90 73.2 t otal ch olesterol high 361 24.7 1098 75.3 0.163 normal 349 27.1 939 72.9 ldl ch olesterol high 576 25.9 1648 74.1 0.912 normal 134 25.6 389 74.4 hd l cholesterol low 299 24.9 903 75.1 0.313 normal 411 26.6 1134 73.4 triglycerides high 101 24.3 314 75.7 0.466 normal 609 26.1 1723 73.9 blood glucose dm 49 23.0 164 77.0 0.078 igt 143 22.9 464 77.1 normal 523 27.1 1409 72.9 men strual status yes 503 28.1 1286 71.9 0.000* no 207 21.6 751 78.4 hormonal contraceptives yes 456 26.5 1264 73.5 0.322 no 254 24.7 773 75.3 hormonal replacemen t th erapy yes 14 27.5 37 72.5 0.749 no 696 25.8 2000 74.2 consumption of h ormonal drugs yes 27 37.5 683 25.5 0.028* no 683 25.5 1992 74.5 147 younger or young adult age group (25-44 years) had a higher proportion of migraine than did the 45+ age group (p=0.001). respondents with smoking habits had a higher proportion of m i g r a i n e t h a n n o n s m o k e r s ( p = 0 . 0 0 0 ) . respondents with migraine had more symptoms of emotional disorder (stress) than those without migraine (p=0.000). the distribution of migraine by metabolic disorder is presented in table 2. table 2 shows that all variables of metabolic disorders were not associated with migraine (p>0.05). this signifies that the factors of metabolic disorders (hypertension, obesity, total, ldl, and hdl cholesterol, triglycerides, and blood glucose) were not correlated with migraine prevalence. hormonal factors in women play an important role in body metabolism, such as the occurrence of a regular menstrual cycle at reproductive age. table 2 shows that among the hormonal factors (menstrual status, use of hormonal contraceptives, hormone replacement therapy, and consumption of hormonal drugs), the variables menstrual status and consumption of hormonal drugs had significantly different proportions (p<0.05). women who still had a menstrual cycle (were reproductive) had a higher proportion of migraine than those in menopause, while women taking hormonal drugs had a higher proportion of migraine than those not taking hormonal drugs. the results of multivariate analysis to find the main determinant of migraine in women are shown in table 3. table 3 shows from the results of bivariate (crude or) and multivariate a n a l y s e s ( a d j u s t e d o r ) t h a t t h e m a j o r determinants of migraine comprise stress (emotional disorders), smoking habit, menstrual status, and consumption of hormonal drugs. these determinants already showed a risk (crude or) in the bivariate analysis, and were again significantly correlated in the multivariate analysis. the results show that among the four determinants entering the multivariate analysis, the determinant smoking showed a decrease in risk from 1.87 (cor) to 1.53 (aor). the results may be summarized as indicating that the major determinants of migraine in women comprise stress, with a 2.47-fold risk (95% ci 2.07 – 2.95), smoking habit, with a 1.53-fold risk (95% ci 1.17 – 2.01), active menstrual cycle, with a 1.44-fold risk (95% ci 1.19 – 1.74), and consumption of hormonal drugs, with a 1.79-fold risk (95% ci 1.09 – 2.96). discussion in the present study, the proportion of migraine in women was around 25.8%, which agrees with the finding of a similar proportion table 3. determinants of migraine in women determinant migraine p value crude odds ratio (cor) 95% confidence interval adjusted odds ratio (a or) 95% confidence interval stress yes 2.50 2.00 – 2.98 2.47 2.07 – 2.95 0.000 no 1 referen ce 1 reference smoking yes 1.87 0.26 – 13.45 1.53 1.17 – 2.01 0.002 n o 1 referen ce 1 reference menstrual status yes 1.42 1.18 – 1.71 1.44 1.19 – 1.74 0.000 no 1 referen ce 1 reference con sumption of hormonal drugs yes 1.75 1.08 – 2.84 1.79 1.09 – 2.96 0.022 no 1 referen ce 1 reference univ med vol. 33 no.2 148 riyadina, andayasari stress a determinant of migraine (25.01%) in the taiwanese study.(4) this is in contrast with the studies conducted in spain and china, which found the lower prevalences of 11.02% and 9.3%, respectively.(3,5) t h e a c c o m p a n y i n g s y m p t o m m o s t frequently experienced by migraine patients was headache aggravated by activity (15%), followed by nausea and vomiting (13.2%), while the least frequent symptoms were phonophobia and photophobia (4.2%). these are all symptoms commonly developing in migraine without aura. in contrast, the headache in migraine with aura is usually accompanied by disturbances of vision and sensory equilibrium.(13) the number of accompanying symptoms apparently determine the severity of migraine without aura. studies have shown a significant correlation between s e v e r i t y o f m i g r a i n e a n d a c c o m p a n y i n g symptoms (photophobia, phonophobia, and nausea), which disturbs the functioning and quality of life of the patient.(14) characteristically, migraine of moderate degree is aggravated by increased activity and is associated with nausea, photophobia, and phonophobia.(15) according to subject characteristics and risk behavior, the determinants showing significant differences in proportions were age, smoking, and emotional disorder (stress). women in the age group of 25-44 years showed a higher proportion of migraine than those in the 45+-year age group. a number of previous studies state that younger age is a predictor of migraine onset.(16) with regard to smoking habit, women smokers in our study had a higher proportion of migraine than non-smokers. this is in accord with previous studies reporting the presence of an association between smoking habit and migraine, stating that the risk of migraine increases significantly with low educational level, unemployment, and smoking.(17) our migraineurs had more emotional symptoms than non-migraineurs. emotional disorders, such as stress or depression was associated with migraine onset, especially in women. similar findings were obtained by p e t e r l i n e t a l . i n s p a i n, ( 1 8 ) s h o w i n g t h a t depressive individuals with stress frequently suffered from migraine (25.71%), with a 3.64fold greater chance of experiencing migraine. stress activates the sympathetic nervous system and the hypothalamic pituitary adrenal axis, which are both associated with the feelings of an individual towards internal and external threats. a study carried out in sweden also states that stress is an important factor for the occurrence of migraine. anxiety and depression occurs twice as frequent in females as in males.(18) regarding metabolic disorders in women, such as hypertension, obesity, hyperglycemia, and dyslipidemia, in the present study no correlation was found between the metabolic syndrome and migraine upon further analysis. there is a study reporting significant differences between the metabolic syndrome and migraine for diabetes, abdominal circumference, and o b e s i t y, b u t n o t f o r h y p e r t e n s i o n a n d dyslipidemia.(19) these differing findings are p r e s u m a b l y d u e t o t h e f a c t s t h a t i n t h e abovementioned study the population consisted of males and females, that hyperglycemia was categorized on the basis of diabetic status, and that obesity was categorized on the basis of a bmi value of 30. migraine is strongly associated with hormonal factors, especially in women. in the case of hormonal factors, the present study shows that women with an active menstrual cycle (reproductive women) had more frequent migraine attacks than those in menopause. in women, migraine is associated with the menstrual period, resulting in the complication of headache associated with menstruation, usually at the time of ovulation, frequently during the menstrual period and seldom in the premenstrual period.(20) fluctuations in estrogen levels is said to be capable of triggering headache in women, especially migraine. migraine occurs (1) before or during the menstrual period (in which there is a large decrease in estrogen levels); (2) in pregnancy or menopause; (3) after consumption 149 of hormonal drugs, such as oral contraceptives and hormone replacement therapy.(21) women who use hormonal contraceptives (pills, injections, implants) and hormone replacement therapy, showed a significantly higher proportion of migraine than those who do not use these treatments. in the case of hormonal contraceptives, it has been shown that their use positively affects the occurrence of migraine, as a number of recent cross-sectional studies on large populations found that the use o f c o m b i n e d o r a l c o n t r a c e p t i v e s w a s significantly correlated with migraine.(22,23) hormonal drug and hormone replacement therapy showed a higher proportion of migraine in comparison with no hormonal therapy. our study also shows that the main determinants of migraine in women comprise stress (emotional disorders), smoking habit, menstrual status, and consumption of hormonal drugs. stress appears to have the highest risk than the other factors, as migraine in women is associated with stress with a 2.47-fold risk (95% ci 2.07 – 2.95). several previous studies reported that stress affects migraine and more frequently appears in women, since these are more susceptible to stressors.(24-26) the number of women smokers in our study was approximately 10%. our analyses showed that women smokers had a 1.53-fold increased risk (95% ci 1.17 – 2.01) of suffering from migraine than non-smokers. nicotine in cigarettes increases the risk of migraine with aura 1.8-fold (95% ci 1.0 – 3.2) and of migraine without aura 2.3-fold (95% ci 1.3 – 3.8).(27) smoking increases the risk of migraine 6.6-fold (95% ci 2.2 – 19.6) in comparison with no smoking, after c o n t r o l l i n g f o r t h e f a c t o r s o f a l c o h o l consumption, lack of sleep, stress, and number of household members. in the present study, migraine was also associated with menstrual status, women with active menstrual cycle having a 1.44-fold greater risk of migraine (95% ci 1.19 – 1.74) in comparison with women in menopause. in the case of hormonal drugs, women who consume hormonal drugs showed a 1.79-fold (95% ci 1.09 – 2.96) risk of suffering from migraine than women who were not on hormonal drugs. a previous study reported that the risk factors affecting migraine occurrence in women are associated with menarche, menstruation, pregnancy, use of hormonal contraceptives and hormone replacement therapy.(28) the present study has several limitations. the method of recall interview is constrained by the recall ability of the respondents and was not confirmed by special medical examination for the diagnosis of migraine. other limitations are the lack of diagnostic subcategories for migraine, such as episodic migraine, periodic migraine, migraine with aura and migraine without aura, the lack of a measure of dose response for the variables of concentration and duration, e.g. for smoking and hormonal drug consumption. conclusions the prevalence of migraine in women aged 25-65 years is 25.8% and a major determinant of migraine is stress. integrated programs should be instituted as promotive and preventive measures for migraine, through education of the community, with a focus on the control of stress (emotional disorder). acknowledgments the authors wish to express their gratitude to the department of neurology of dr. cipto mangunkusumo central general hospital (rscm), the atmajaya university, and the district health service and puskesmas in bogor city and their staff, for their cooperation in the research team of the cohort study of risk factors for non-communicable diseases. references 1. bigal me, lipton rb, stewart wf. the epidemiology and impact of migraine. curr neurol neurosci rep 2004;4:98-104. univ med vol. 33 no.2 150 riyadina, andayasari stress a determinant of migraine 2. jasmin l, zieve d and the adam editor team. migraine. pub med health;2012. 3. fernández-de-las-peñas c, hernández-barrera v, carrasco-garrido p, alonso-blanco c, palaciosceña d, jiménez-sánchez s, et al. populationbased study of migraine in spanish adults: relation to socio-demographic factors, lifestyle and co-morbidity with other conditions. j headache pain 2010;11:97-104. 4. chen yc, tang ch, ng k, wang sj. comorbidity profiles of chronic migraine sufferers in a national database in taiwan. j headache pain 2012;13:3119. 5. yu s, liu r, yang x, zhao g, qiao x, feng j. body mass index and migraine: a survey of the chinese adult population. j headache pain 2012;13:531-6. 6. ward tn. migraine diagnosis and pathophysiology. continuum (minneap minn). 2012;18:753-63. 7. piane m, lulli p, farinelli i, simeoni s, de filippis s, patacchioli fr, et al. genetics of migraine and pharmacogenomics: some considerations. j headache pain 2007;8:334-9. 8. karli n, baykan b, ertas m, zarifoglu m, siva m, saip s, et al. impact of sex hormonal changes on tension-type headache and migraine: a crosssectional population-based survey in 2, 600 women. j headache pain 2012 13:557-65. 9. chasman di, arttila v, buring je, ridker pm, schurks m, kurth t. selectivity in genetic association with sub-classified migraine in women. genet 2014;10:5-16; e1004366.doi:10.1371/ journal.open.1004366. 10. naing nn. determination of sample size. malaysian j medical sci 2003;10:84-6. 11. pusat tekonologi intervensi kesehatan masyarakat. studi kohor tumbuh kembang anak dan faktor risiko penyakit tidak menular tahun 2012: laporan akhir penelitian. jakarta: badan penelitian dan pengembangan kesehatan. kementrian kesehatan ri.;2013. 12. martin j. hypertension guidelines: revisiting the jnc 7 recommendations. j lancaster general hospital 2008;3:91-7. 13. leonardi m, mathers c. global burden of migraine in the year 2000: summary of methods and data sources. geneva: world health organization;2002. 14. freitag fg. migraine and associated symptoms: meeting the treatment challenge. cicago, illinois: nationale headache foundation;2008. 15. headache classification subcommittee of the international headache society. the international classification of headache disorders. 2nd. edn. cephalalgia 2004;24(suppl 1):1-151. 16. stewart wf, bigal me, kolodner k, dowson a, liberman jn. familial risk of migraine: variation by proband age at onset and headache severity. neurolory 2006; 66:344-8. 17. le h, tfelt-hansen p, skytthe a, kyvik ko, olesen j. association between migraine, lifestyle and socioeconomic factors: a population-based cross-sectional study. j headache pain 2011;12: 157-72. 18. peterlin bl, tietjen ge, brandes jl, rubin sm, drexler e, lidicker jr, et al. posttraumatic stress disorder in migraine. headache 2009;49:541–51. 19. hedborg k, anderberg um, muhr c. stress in migraine: personality-dependent vurnerability, life events, and gender are of significance. ups j med sci 2011;116:187-99. 20. guldiken b, guldiken s, taskiran b, koc g, turgut n, labayel l, et al. migraine in metabolic syndrome. neurologist 2009;15:55-8. 21. hutchinson s, silberstein sd. menstrual migraine: case studies of women with estrogen-related readaches. headache 2008;48:s131-41. 22. mayo clinic. disease and conditions migraine. available at: http://www.mayoclinic.org/diseases -conditions/migraine-headache/basics/causes/ con-20026358. accessed march 10, 2014. 23. nappi er, feld gsm, terreno e, pellegrineli a, viana m. hormonal contraception in woman with migraine, is progresteron-only better contraception choice?. j headache pain 2013;14: 66-71. 24. edovic k, wadiwalla m, engert v, pruessner jc. the role of sex and gender socialization in stress reactivity. dev psychol 2009;45:45-55. 25. bhurgri gr, buksh h, qureshi ma. frequency of depression in migraine patients. gomal j med sci 2009:7:106-8. 26. tietjen ge, brandes jl, digre kb, baggaley s, martin vt, recober a, et al. history of childhood maltreatment is associated with comorbid depression in women with migraine. neurology 2007;69:959-68. 27. sarker mab, rahman m, rashid ho, hossain s, kasuya h, sakamoto j, et al. association of smoked and smokeless tobacco use with migraine: a hospital-based case-control study in dhaka, bangladesh. tobbaco dis 2013;11:1521. 28. sacco s, ricci s, carolei a. migraine in women: the rule of hormones and their impact on vascular diseases. j headache pain 2012;13:177-89. yenny 183 *department of pharmacology, medical faculty, trisakti university correspondence dr. yenny department of pharmacology, medical faculty, trisakti university jl. kyai tapa 260 grogol jakarta 11440 telp 021-5672731 ext.2801 email: stasia_mk@yahoo.com univ med 2008; 27: 183-93 thiazolidinedione and cardiovascular risk in type 2 diabetes mellitus october-december, 2008october-december, 2008october-december, 2008october-december, 2008october-december, 2008 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 vol.27 no.4 abstract universa medicina yenny* cardiovascular disorders are the most common complications encountered in patients with type 2 diabetes mellitus (dm). the relationship is likely to be multifactorial and may also involve a specific, though ill-defined, diabetic cardiomyopathy. patients with heart failure accompanied by dm have a reduced cardiac output compared with patients without dm. thiazolidinediones (tzds) are agonists of peroxisome proliferator-activated receptor gamma (pparã) and have beneficial effects in the control of blood glucose and cardiovascular parameters, but the ability of these drugs to induce retention of plasma has to be taken into consideration in prescribing them for patients with dm at high risk of cardiovascular disease. the molecular mechanism of fluid retention by tzds has not been fully elucidated. available evidence indicate a possible role of epithelial sodium channels (enac) in causing the side effects of tzds. this paper will discuss the mechanism of enac in inducing fluid retention and the management to be applied for anticipating these side effects. keywords: thiazolidinedione, heart failure, type 2 diabetes, enac introduction epidemiologic studies have shown that around 10% of patients with type 2 diabetes mellitus (dm) experience heart failure. this prevalence rate is 2-4 times higher than that in p a t i e n t s w i t h o u t d m . ( 1 3 ) f u r t h e r m o r e , approximately 25% of patients in studies of heart failure suffered from dm,(2) while only around 0.5% of the general population suffered from both diseases.(3) patients with heart failure accompanied by dm have a reduced cardiac output compared with patients without dm. t h e o r a l a n t i d i a b e t i c a g e n t s c a l l e d t h i a z o l i d i n e d i o n e s ( t z d s , o r g l i t a z o n e s ) , c o n s i s t i n g o f r o s i g l i t a z o n e s ( r s g ) a n d pioglitazones, are agonists of peroxisome proliferator-activated receptor gamma (pparã) that act to increase insulin sensitivity.(4) in addition to lowering the blood glucose level, t z d s a l s o s h o w s b e n e f i c i a l e f f e c t s o n cardiovascular parameters, such as lipid levels, 184 yenny thiazolidinedione and cardiovascular blood pressure, biomarkers of inflammation, endothelial functions, and fibrinolytic status.(5) the beneficial effects of tzds on blood glucose and cardiovascular risk factors has resulted in the extensive use of these drugs in type 2 diabetic patients at high risk of cardiovascular disease. however, utilization of tzds is limited b y e x a c e r b a t i o n o f f l u i d r e t e n t i o n . t h e incidence of peripheral edema following use of tzds as monotherapy or in combination with other oral antidiabetic drugs is around 5% and may be up to 15% if tzds are used with insulin.(6) in extreme cases tzds can induce expansion of plasma volume such that it causes precipitation or exacerbation of pulmonary e d e m a a n d c o n g e s t i v e h e a r t f a i l u r e , complications commonly found in patients with type 2 diabetes.(1) the underlying mechanism for the effect of tzds in inducing plasma volume expansion and edema is still unclear. the molecular targets o f t z d s , n a m e l y p pa r γ , a r e d i f f u s e l y expressed in humans in all organs, including the kidneys.(7) tzds have a potential effect on the kidneys that is independent of their effects on glucose and lipid metabolism. in vitro and animal studies have demonstrated the agonist capabilities of pparγ in stimulating sodium r e a b s o r p t i o n i n t h e d i s t a l n e p h r o n s b y upregulating the expression and translocation of epithelial sodium channels (enac) in the collecting ducts.(8,9) f l u i d r e t e n t i o n i n d u c e d b y t z d s i s frequently resistant to loop diuretics, but may resolve upon discontinuation of the drug. it may also be possible to counteract expansion o f p l a s m a v o l u m e d u e t o t z d s b y u s i n g diuretics that act on the collecting ducts (e.g. spironolactone).(10) a meta-analysis conducted by nissen et al.(11) and sing et al.(12) showed t h a t t h e r s g t e n d t o i n c r e a s e t h e r i s k o f myocardial infarction by 42% in patients with type 2 diabetes. other meta-analyses have confirmed the risk of heart failure on rsg u s a g e . ( 1 3 ) i n c o n t r a s t t o r o s i g l i t a z o n e , pioglitazone in the ppospective pioglitazone clinical trial in macro vascular events study (proactive study) was capable of reducing m a c r o v a s c u l a r a t h e r o s c l e r o t i c r i s k , ( 1 4 ) r e c u r r e n t m y o c a r d i a l i n f a r c t i o n , ( 1 5 ) a n d r e c u r r e n t s t r o k e . ( 1 6 ) t h e c a r d i o v a s c u l a r protective effect of pioglitazone is supported by a meta-analysis conducted by lincoff et al.(17) the difference in ischemic risks of the two drugs is caused by the contrasting effects of these tzds on lipid profile; pioglitazone causes a reduction in low density lipoprotein (ldl) whereas rosiglitazone raises the ldl concentration.(18) in connection with the potential risk of rosiglitazone in causing myocardial infarction a n d t h e r i s k o f f l u i d r e t e n t i o n t h a t m a y precipitate heart failure when using tzds drugs, the food and drug administration (fda) h a s a d d e d a ‘ b l a c k b o x w a r n i n g ’ i n t h e information on prescribing of tzds.(19) this paper will discuss the mechanism of tzds in inducing fluid retention in connection with epithelial sodium channels (enac), and the management to be applied for anticipating these side effects. insulin resistance and role of pparγγγγγ insulin resistance develops long before the onset of clinical diabetes. the onset of insulin r e s i s t a n c e i s f r e q u e n t l y a c c o m p a n i e d b y obesity, particularly visceral obesity. adipose d y s f u n c t i o n l e a d s t o t h e d e v e l o p m e n t o f resistance to the anti-lipolytic effects of insulin, causing an increased level of plasma free fatty acids (ffa). the latter will induce insulin resistance in the liver and skeletal muscle, resulting in decreased glucose uptake by the tissues and increased gluconeogenesis. adipose 185 univ med vol.27 no.4 d y s f u n c t i o n a l s o l e a d s t o p r o d u c t i o n o f proinflammatory cytokines (e.g. tumor necrosis factorα (tnfα), interleukin (il)-6, and resistin in excessive amounts, which will increase the chances of insulin resistance, inflammation, atherosclerosis, and decrease secretion of insulin-sensitive cytokines, such as adiponectin, produced by adipose tissue.(20) p pa r γ r e c e p t o r s a r e m e m b e r s o f t h e nuclear receptor family that play a role in r e g u l a t i n g t r a n s c r i p t i o n f a c t o r s f o r g e n e s involved in uptake and storage of fatty acids, i n f l a m m a t i o n , a n d g l u c o s e h e m o s t a s i s . ( 2 0 ) pparγ has an important role in the normal differentiation and proliferation of adipocytes, as well as in ffa uptake and storage. various adipokines, such as adiponectin, tnfα and resistin, are egulated by ppa rγ a g o n i s t s . a d i p o n e c t i n i s a n a d i p o c y t o k i n e t h a t i s produced exclusively by adipose tissue and e n h a n c e s i n s u l i n s e n s i t i v i t y a n d a n t i atherogenic effects of insulin, whereas tnfα a n d r e s i s t i n i n d u c e i n s u l i n r e s i s t a n c e . figure 1. mechanisms of pparγ agonists in improving insulin resistance adiponectin levels are low in obese individuals and in those with type 2 dm.(21) there are several mechanisms of pparγ a g o n i s t s i n r e d u c i n g i n s u l i n r e s i s t a n c e . activation of pparγ receptors by their agonists can accelerate adipocyte differentiation and ffa uptake from visceral fat, followed by their storage in subcutaneous adipose tissue. this phenomenon results in reduced ffa levels r e s u l t i n g i n d e c r e a s e d i n s u l i n r e s i s t a n c e . furthermore, activation of pparγ by their agonists is thought to enhance expression and translocation of the glucose transporters (glu), glut1 dan glut4 to the cell surface, thus increasing glucose uptake by the liver and skeletal muscle and decreasing plasma glucose levels. another mechanism of pparγ agonists in reducing insulin resistance is by reducing p r o i n f l a m m a t o r y c y t o k i n e s ( t n f α , i l 1 , r e s i s t i n ) ( 2 2 ) a n d i n c r e a s i n g e x p r e s s i o n o f a d i p o n e c t i n b y a d i p o s e t i s s u e . ( 2 3 ) t h e mechanisms of pparγ agonists in improving insulin sensitivity are shown in figure 1. 186 yenny thiazolidinedione and cardiovascular pat h o g e n e s i s o f e d e m a o n t z d usage the mechanisms underlying the ability of tzds in inducing expansion of plasma volume and edema are still unclear. the role of renal mechanisms in causing induction of edema by tzds was first demonstrated by the study of s o n g e t a l . ( 2 4 ) t h e i r s t u d y r e p o r t e d t h a t administration of rosiglitazone for 3 days to sprangue dawley rats significantly reduced u r i n a r y v o l u m e ( u p t o 3 3 % ) a n d s o d i u m excretion (up to 44%). in addition the study also reported an increase in concentrations of na-k-atpase, nak 2 c l c o t r a n s p o r t e r s ( n k c c 2 ) , s o d i u m hydrogen exchangers (nhe3), aquaporin 2 ( a q p 2 ) , a n d a q u a p o r i n 3 ( a q p 3 ) . t h e s e findings suggested the occurrence of sodium transport in the proximal tubule and the thick ascending limb. o t h e r e v i d e n c e i n d i c a t i n g t h a t f l u i d retention induced by tzds is based on the activation of sodium transport in the distal n e p h r o n i s p r e s e n t e d h e r e . f i r s t l y, i n t h e kidneys pparγ is expressed at substantial concentrations in the collecting duct, and at l o w e r c o n c e n t r a t i o n s i n t h e g l o m e r u l u s , proximal tubule, and the microvasculature. secondly, in tissue culture of a human cortical c o l l e c t i n g d u c t c e l l l i n e , p pa rγ a g o n i s t s increased αenac concentrations on the cell surface. this increase in αenac occurs in parallel with increased mrna for serum and glucocorticoid regulated kinase 1 (sgk1), which could be abolished by administration of pparγ antagonists before the intervention. this effect is presumably due to binding of pparγ to specific response elements in the promoter region of sgk1. (8) thirdly, in vivo studies indicate that gi262570 (farglizar), a potent pparγ agonist, can stimulate water and sodium r e a b s o r p t i o n f r o m t h e d i s t a l n e p h r o n o f sprangue dawley rats by stimulating enac and na-k-atpase.(9) forthly, a study conducted on the collecting duct of pparγ knockout mice s h o w e d t h e d e v e l o p m e n t o f r e s i s t a n c e t o increases in body weight and plasma volume e x p a n s i o n i n d u c e d b y r o s i g l i t a z o n e , a s compared with rats expressing pparγ in their collecting ducts.(25) m e c h a n i s m o f s o d i u m r e a b s o r p t i o n i n t h e d i s ta l nephron the main site of sodium reabsorption in the renal tubule is the proximal tubule (>85%), b u t t h e d i s t a l n e p h r o n ( p a r t i c u l a r l y t h e aldosterone sensitive distal nephron) (asdn), where <10% of sodium reabsorption takes place, plays an important role in the regulation of plasma volume.(26) asdn is located at the e n d o f t h e d i s t a l t u b u l e , i n c l u d i n g t h e c o n n e c t i n g s e g m e n t , a n d t h e c o r t i c a l a n d medullary collecting duct (figure 2). sodium reabsorption in the asdn takes place through the epithelial sodium channel (enac). sodium reabsorption along the whole length of the nephron is controlled by basolateral na+-k+at p a s e a c t i v i t y, r e s u l t i n g i n d e c r e a s e d i n t r a c e l l u l a r s o d i u m c o n c e n t r a t i o n s a n d intracellular electronegativity. both conditions lead to an electrochemical difference for sodium influx through the apical membrane. sodium transport from the tubular lumen through the apical membrane of epithelial cells is mediated in the proximal tubule up to the thick ascending loop of henle by the sodium/ proton exchanger 3 (nhe3), in the distal tubule and connecting segment by the sodium-chloride cotransporter (ncct), and in the cortical and medullary collecting duct by the epithelial sodium channel (enac). 187 univ med vol.27 no.4 the na+-k+-atpase pump transports sodium through the basolateral membrane into the blood. the expression of the aldosterone sensitive distal nephron (asdn) occurs mainly at the site of enac expression. serum and glucocorticoid regulated kinase 1 (sgk1) is e x p r e s s e d a l o n g t h e w h o l e l e n g t h o f t h e nephron, and aldosteron potently induces sgk1 activation in the asdn. a sdn r egulates sodium and water hemostasis through its action on enac, a protein with an important role in regulating sodium reabsorption. enac consists of three subunits α, β, dan γ (figure 3), where enacα acts a a functional subunit whose activity is regulated by enacα and enacβ.(27) sodium reabsorption in the asdn occurs through expression of enac on the apical surface of the renal tubule and is associated with the role of the serum and glucocorticoid regulated kinases (sgk). sgk are a family of protein kinases b ( a l s o k n o w n a s p k b / a k t ) , t h a t p l a y a n important role in survival. currently there are 3 isoforms of sgk that have been identified as sgk1, sgk2, dan sgk3, whose functions are still not fully understood. these three kinases are said to be potent regulators of ion channel a c t i v i t y, t r a n s p o r t , a n d t h e t r a n s c r i p t i o n process.(27) sgk1 has been successfully identified as the key mediator in sodium reabsorption by the renal tubular epithelium. transcription of sgk1 is stimulated among others by stress or cell shrinkage (in renal epithelium by swelling of the cell), hormones (including mineralocorticoids, glucocorticoids), pparγ, high glucose levels, a n d o x i d a t i v e s t r e s s , a n d i s i n h i b i t e d b y heparin.(27) sgk1 is expressed at the site of expression of enac and mineralocorticoid r e c e p t o r s ( m r ) ( f i g u r e 4 ) . t h e s e l e c t i v e occupation of mr by aldosterone induces expression of sgk1 mrna. in addition to these mechanisms, activity of sgk1 as well as that of the other members of the pkb/akt family occurs t h r o u g h a c t i v a t i o n o f t h e p a t h w a y s f o r figure 2. expression of sodium transporter is specific for the renal segment and sgk1(26) figure 3. serum and glucocorticoid regulated kinase 1 (sgk1) as regulator of sodium reabsorption in the kidney(26) 188 yenny thiazolidinedione and cardiovascular phosphoinositol 3-kinase (pi3k) signaling and phosphoinositide-dependent protein kinase (pdk1). insulin itself is also an activator of the pi3k pathway. in synergy with aldosterone, both p a t h w a y s e n h a n c e s g k 1 p h o s p h o r y l a t i o n (figure 3). sgk1 subsequently integrates the pi3k signaling and mineralocorticoid pathways, leading to expression of enac on the asdn. in addition to aldosterone dan insulin, ppar-γ activation by its agonist may also stimulate transcription and activation of sgk1, which causes an increased expression of enac on the s u r f a c e o f t h e a p i c a l m e m b r a n e o f r e n a l tubules.(26,27) activation of enac may occur through aldosterone or insulin, and may also be induced by pparg agonists, such as rosiglitazone, through the role of serum and glucocorticoid regulated kinase 1 (sgk1) which induces enaca mrna expression, causing increased translocation of enac to the apical membrane. activation of sgk1 prevents degradation of enac by inactivating the ubiquitin ligase neural precursor cell expressed, developmentally d o w n r e g u l a t e d 4 2 ( n e d d 4 2 ) . n e d d 4 2 interacts with the py motif in enac, causing endocytosis and channel degradation (figure 4). t h e a b i l i t y o f s g k 1 i n i n d u c i n g e n a c expression is mediated by nedd4-2, which is c l o s e l y r e l a t e d t o e 3 u b i q u i t i n l i g a s e . interaction between enac and nedd4-2 causes inactivation of enac through ubiquitinylation and/ or endocytosis, followed by degradation b y l y s o z y m e . s g k 1 i t s e l f c a u s e s phosphorylation of nedd4-2, which impairs the ability of nedd4-2 to interact with enac. this causes an increased accumulation of enac on the plasma membrane leading to increased sodium transport.(26,27) figure 4. aldosterone and insulin both stimulate sodium transport in the collecting duct.(26) irs: insulin receptor substrate; pi3k: phosphoinositol 3 kinase; pip2: phosphoinositol bisphosphate; pip3: phosphoinositol triphosphate. 189 univ med vol.27 no.4 the permeability of the cell membrane to water is regulated by water channel proteins aquaporins (aqps). at present 10 species of a q p h a v e b e e n i d e n t i f i e d . ( 2 8 ) t h e m a i n aquaporin found in the kidney is aqp1-4. aqp1 and aqp2 function on the apical membrane, w h i l e a q p 3 d a n a q p 4 f u n c t i o n o n t h e basolateral membrane.(29) collectively these aqps provide a transcellular pathway for movement of water from the lumen of the collecting duct into the interstitium.(30) clinical applications an update of the consensus statement issued by the american heart association and european association for the study of diabetes (2008)(33) currently recommends tzds as a s e c o n d l i n e d r u g i n t h e a l g o r i t h m f o r m a n a g e m e n t o f d i a b e t e s t h a t c a n n o t b e successfully controlled by diet and life style modification or by metformin, as an alternative to insulin (the most effective) and sulfonylureas (the least expensive) for controlling blood glucose in patients with type 2 dm (figure 5). i n c o n n e c t i o n w i t h t h e p o t e n t i a l cardiovasculer risks (myocardiual infarction, heart failure) that may develop on tzds usage, there are three fundamental concepts that need to be kept in mind by the clinician in prescribing tzds for patients with type 2 dm.(34) first, the high risk of heart failure in patients with dm, because there are multiple cardiovascular risk f a c t o r s a c c o m p a n y i n g p a t i e n t s w i t h d m . second, the difference between true heart failure and heart failure induced by tzds; and third, the high long-term mortality in patients with dm. figure 5. algorithm for management of type 2 dm(33) 190 yenny thiazolidinedione and cardiovascular in connection with an association between tzds and risk of heart failure, it is essential to have a potential strategy for minimizing the risk of edema and/or heart failure in patients with type 2 dm on tzds. therefore, the concensus of the american heart association (aha) dan american diabetic association ( a d a ) ( 3 5 ) m a y b e u s e d a s a r e f e r e n c e i n administering tzds to patients with diabetes. t h e c o n c e n s u s r e c o m m e n d s t h a t b e f o r e starting tzds therapy, a complete evaluation should be performed of risk factors underlying e p i s o d e s o f h e a r t f a i l u r e , d r u g s c u r r e n t l y taken, and evidence of pre-existing edema or heart failure. the recommendations for tzds usage in connection with heart failure may be seen in figure 6. the recommendation also states that the presence of edema originating from noncardiac causes should not prevent use of tzds. in addition, there should be adequate monitoring for the presence of edema or heart failure, and the dosage used should be adjusted gradually to attain the hemoglobin a1c (hba1c) target. patients experiencing edema when using tzds should be screened for other causes of edema, i n c l u d i n g n e p h r o t i c s y n d r o m e , v e n o u s insufficiency, and use of other drugs, such as nonsteroidal anti-inflammatory drugs as well as calcium channel blockers (inhibitors). in diabetic patients without heart failure tzds should be prescribed according to the published guidelines. these guidelines do not prohibit use of tzds in patients with class i/ii figure 6. recommendations in connection with usage of thiazolidinedione and heart failure according to the aha and ada.(35) 191 functional heart failure according to the new york heart association (nyha), with a lower initial dose than usual for each drug (e.g. rosiglitazone 2 mg/day or pioglitazone 15 mg/ day), with the proviso that close monitoring should be performed for fluid retention and that use of tzds in patients with nyha class iii/ iv functional heart failure should be avoided.(35) potential therapy use of diuretics in the management of fluid retention induced by tzds has been evaluated by a number of investigators in case reports,(36) and by controlled clinical trials.(10) most of the case reports state that edema is commonly refractory to loop diuretics (furosemide) and that the symptoms generally resolve when use of tzds is discontinued. the clinical trial aiming to determine the efficacy of diuretics in the management of fluid retention induced by rsg was a multicenter parallel group open label randomized study of 381 patients with type 2 dm.(10) the trial used t h r e e k i n d s o f d i u r e t i c s w i t h d i f f e r e n t mechanisms of action, namely (i) furosemide, which inhibits the na-k-cl cotransporter in the thick segment of the ascending limb of henle, (ii) hydrochlorothiazide (hct), which inhibits the na-cl cotransporter in the distal tubule, and (iii) spironolactone, which is an enac inhibitor in the collecting duct. the results of the study showed that spironolactone is equally effective a s h y d r o c h l o r o t h i a z i d e i n r e d u c i n g f l u i d retention, while furosemide did not exhibit any s i g n i f i c a n t e f f e c t . t h e e f f e c t i v e n e s s o f spironolactone in reducing fluid retention may be related to the ability of this diuretic to impair the effects of fluid retention by pparγ in the collecting duct. hct also significantly reduced fluid retention due to rosiglitazone. the site of action of the thiazide diuretics is in the proximal part of the distal tubule, with inhibition of the na+/clcotransporter as mechanism of action, but this diuretic also inhibits the reabsorption of water and salt in the collecting tubule,(37) where pparγ is expressed in the nephron.(7) it is also conceivable that the thiazide diuretics may also antagonize the action of pparγ agonists at this site. this is in contrast to the loop diuretics, which do not show a significant effect in reducing fluid retention, presumably because their action is exclusivley in the thick segment segmen of the ascending limb of henle by inhibiting the na+-k+ clcotransporter, as this segment does not contain pparγ.(38) in addition to the ability of spironolactone in antagonizing p pa r γ i n t h e c o l l e c t i n g d u c t , a n o t h e r characteristic of spironolactone is the beneficial effect in ameliorating the left ventricular function and volume.(39) conclusions t z d s c o m p r i s i n g r o s i g l i t a z o n e a n d p i o g l i t a z o n e a r e a g o n i s t s o f p e r o x i s o m e proliferator-activated receptor gamma (pparγ) and have beneficial effects in controlling blood glucose and cardiovascular parameters. in addition to the beneficial effects, the ability of this drug to induce plasma volume expansion should be considered in prescribing tzds for patients with type 2 dm who also have a high cardiovascular risk. tzds should not be used in patients newly diagnosed as suffering from type 2 dm. in view of the risk of fluid retention when using tzds, it is imperative to consider the potential strategies for minimizing the risk of edema and/or heart failure in patients with type 2 dm who are receiving tzds therapy. the presence of edema due to other causes unrelated to heart failure should not prevent use of tzds. adequate monitoring is necessary for signs of edema or heart failure, and increased dosage should be adjusted gradually to attain the expected hba1c target. univ med vol.27 no.4 192 yenny thiazolidinedione and cardiovascular references 1. nichols ga, gullion cm, koro ce, ephross sa, brown jb. the incidence of congestive heart failure in type 2 diabetes: an update. diabetes care 2004; 27: 1897-84. 2. bell ds. heart failure: the frequent, forgotten, and often fatal complication of diabetes. diabetes care 2003; 26: 2433-41. 3. thrainsdottir is, aspelund t, thorgeirsson g, gudnason v, hardarson t, malmberg k. the association between glucose abnormalities and heart failure in the population based reykjavik study. diabetes care 2005; 28: 612-6. 4. yki-jarvinen h. thiazolidinediones. n engl j med 2004; 351: 1106-18. 5. parulkar aa, pendergrass ml, granda ayala r, lee tr, fonseca va. nonhypoglycemic effects of thiazolidinediones. ann intern med 2001; 134: 6171. [erratum in ann intern med 2001;135: 307]. 6. mudaliar s, chang ar, henry rr. thiazolidinediones, peripheral edema, and type 2 diabetes: incidence, pathophysiology, and clinical implications. endocr pract 2003; 9: 406-16. 7. guan y, zhang y, davis l, breyer md. expression of peroxisome proliferator-activated receptors in urinary tract of rabbits and humans. am j physiol 1997; 273: f1013-22. 8. hong g, lockhart a, davis b, rahmoune h, baker s, ye l, et al. ppargamma activation enhances cell surface enacalpha via up-regulation of sgk1 in human collecting duct cell. faseb j 2003; 17: 1966-8. 9. chen l, yang b, mcnulty ja, clifton lg, binz jg, grimes am. gi262570, a peroxisome proliferator-activated receptor γ agonist, changes electrolytes and water reabsorbtion from the distal nephron in rats. j pharmacol exp ther 2005; 312: 718-25. 10. karalliedde j, buckingham r, starkie m, lorand d, stewart m, viberti g, et al., for the rosiglitazone fluid retention study group. effect of various diuretic treatments on rosiglitazone induced fluid retention. j am soc nephrol 2006; 17: 3482-90. 11. nissen se, wolski k. effect of rosiglitazone on the risk of myocardial infarction and death from cardiovascular causes. n engl j med 2007; 56: 2457-71. 12. sing s, loke yk, furberg cd. long-term risk of cardiovascular events with rosiglitazone: a metaanalysis. jama 2007; 298: 1189-95. 13. home pd, pocock sj, beck-nielsen h, gomis r, hanefeld m, jones np, et al. record study group rosiglitazone evaluated for cardiovascular outcomes: an interim analysis. n engl j med 2007; 370: 1129-36. 14. dormandy ja, charbonnel b, eckland dj, erdmann e, massi-benedetti m, moules ik, et al. secondary prevention of macrovascular events in patients with type 2 diabetes in the prospective pioglitazone clinical trial in macrovascular events study (proactive study): a randomized controlled trial. lancet 2005; 366: 1279-89. 15. erdmann e, dormandy ja, charbonnel b, massibenedetti m, moules ik, skene am, et al. the effect of pioglitazone on recurrent myocardial infarction in 2.445 patients with type 2 diabetes and previous myocardial infarction. results from the proactive (proactive 05) study. j am coll cardiol 2007; 17: 1772-80. 16. wilcox r, bousser mg, betteridge dj, schernthander g, pirags v, kupfer s, et al. effects of pioglitazone inpatients with type 2 diabetes with or without previous stroke: results from proactive pioglitazone clinical trial in macro vascular events. stroke 2007; 38: 865-73. 17. lincoff am, wolski k, nicholls sj, nissen se. pioglitazone and risk of cardiovascular events in patients with type 2 diabetes mellitus: a metaanalysis of randomized trials. jama 2007; 298: 1180-8. 18. deeg ma, tan mh. pioglitazone versus rosiglitazone: effects on lipid, lipoproteins, and apolipoproteins in head to head randomized clinical studies. ppar res 2008; article id 520465, 6 pages. 19. u.s. food and drug administration: rosiglitazone maleate (marketed as avandia, avandamet, and avandary) information 2007. available at: http:// www.fda.gov/cder/drug /infopage/rosiglitazone/ default.html. accessed november 23, 2008. 20. steals b, fruchart jc. therapeutic roles of peroxisome proliferators activated receptor agonists. diabetes 2005; 54: 2460-70. 21. weyer c, funahashi t, tanaka s. hypoadipocectinemia in obesity and type 2 diabetes: close association with insulin resistance and hyperisulinemia. j clin endocrinol metab 2002; 86: 1930-5. 22. cabrero a, laguna jc, vazquez m. peroxisome proliferators activated receptors and the control of inflammation. curr drug targets inflamm allergy 2002; 1: 143-8. 193 23. maeda n, takahashi m, funahashi t, kihara s, nishizawa h, kishida k, et al. ppar-gamma ligands increase expression and plasma concentration of adiponection, an adipose-derived protein. diabetes 2001; 50: 2094-9. 24. song j, knepper ma, hu x, verbalis jg, ecelbarger ca. rosiglitazone activated renal sodium and water-reabsorptive pathways and lowers blood pressure in normal rats. jpet 2004; 308: 426-33. 25. zhang h, zhang a, kohan de, nelson rd, gonzalez fj, yang t. collecting duct-specific deletion of peroxisome proliferators activated receptor γ blocks thiazolidinedione-induced fluid retention. proc natl acad sci usa 2005; 102: 9406-11. 26. mccormick ja, bhalla v, pao ac, pearce d. sgk1: a rapid aldosterone-induced regulator of renal sodium reabsorption. physiology 2005; 20: 134-9. 27. lang f, bohmer c, palmada m, seebohm g, strutzseebohm n, vallon v. patho physiological significance of the serum and glucocorticoid inducible kinase isoforms. physiol rev 2006; 86: 1151-78. 28. firsov d, gautschi i, merillat am, rossier bc, schild l. the heterotetrameric architecture of the epithelial sodium channel (enac). embo j 1998; 17: 344-52. 29. agre p, king ls, yasui m, guggino wb, ottersen op, et al. aquaporin water channels-from atomic structure to clinical medicine. j physiol 2002; 542: 3-16. 30. knepper ma, wade jb, terris j, ecelbarger ca, marples d, mandon b. renal aquaporins. kidney int 1996; 49: 1712-7. 31. de lemos ja, mcguire dk, drazner mh. b-type natriuretic peptide in cardiovascular disease. lancet 2003; 362: 316-22. 32. ogawa s, takeuchi k, ito s. plasma bnp levels in the treatment of type 2 diabetes with pioglitazone. j clin endocrinol metab 2003; 88: 3993-6. 33. nathan dm, buse jb, davidson mb, ferrannini e, holman rr, sherwin r, et al. management of hyperglycemia in type 2 diabetes mellitus: a consensus update regarding the initiation and adjustment of therapy. update regarding thiazolidinediones: a consensus statement from the american diabetes association and the european association for the study of diabetes. diabetes care 2008; 31: 173-5. 34. erdmann e, wilcox rg. weighing up the cardiovascular benefits of thiazolidinedione therapy: the impact of increased risk of heart failure. eur heart j 2008; 29: 12-20. 35. nestro rw, bell d, bonow ro, fonseca v, grundy sm, horton es, et al. thiazolidinedione use, fluid retention, and congestive heart failure: a consensus statement from the american heart association and american diabetes association. circulation 2003; 108; 2941-8. 36. hirsch ib, kelly j, cooper s. pulmonary edema associated with troglitazone therapy. arch int med 1999; 159: 1811. 37. wilson dr, honrath u, sonnenberg h. thiazid diuretic effect on medullary collecting duct function in the rat. kidney int 1983; 23: 711-6. 38. shankar ss, brater c. loop diuretic: from the nak-2cl transporter to clinical use. am j physiol renal physiol 2003; 284: f11-21. 39. cicoira m, zanolla l, rossie a, golia g, franceschini l, brighetti g, et al. long-term, dosedependent effects of spironolacton on left ventrikel function and exercise tolerance in patients with chronic renal failure. j am coll cardiol 2002; 40: 304-10. univ med vol.27 no.4 editorial 69 hiv antiretroviral preexposure prophylaxis richard tjan editor univ med vol. 32 no.2 according to 3 field trials conducted in africa, one among african women and two among heterosexual couples, antiretroviral preexposure prophylaxis for prevention of hiv-1 has been shown to be effective.(1-3) in preexposure prophylaxis, persons without hiv infection are given an oral drug before they have sexual contact with hiv-infected partners.(4) the drug in question is tenofovir disoproxil fumarate (tdf), a prodrug of tenofovir.(1) on the basis of the 3 field trials, the antiviral drugs advisory committee of the food and drug administration has recommended a combination of antiretroviral drugs (tenofovir/emtricitabine) for preexposure prophylaxis of hiv.(5) tdf is currently indicated for the treatment of hiv in adults over 18 years of age or hepatitis b virus (hbv) infection in adults, or both. the drug is called a nucleotide reverse transcriptase inhibitor (nrti), preventing the synthesis of viral copies by hiv reverse transcriptase or hbv dna polymerase.(6) to retard the emergence of tdf resistance, the drug is usually given in combination with another antiviral, such as emtricitabine (ftc). nucleic acid testing for hiv virus when starting preexposure prophylaxis, may reduce the risk of resistance, but it is at present not an option in developing countries because of its high costs. rare but potentially serious adverse reactions to tdf are lactic acidosis and toxic effects on the liver and kidneys.(6) because administration of tdf to healthy noninfected persons implies using the drug for a prolonged period of many years, the long-term safety of tdf and the tdf-ftc combination has to be clearly established.(4) there is also a real possibility that preexposure prophylaxis may lead to relaxation of the customary precautions on the part of the sexual partners, such as engaging in increased risky sexual behavior or abandoning the use of conventional prophylactic measures (e.g. condoms). (4) this matter should be a problem for health educators. from a practical point of view, because of the potential of serious liver and kidney disease caused by tdf, the medical practitioner should prescribe preexposure prophylaxis only in high risk cases, and not for prevention of hiv in otherwise healthy individuals, e.g. blood bank personnel or dental practitioners with a low risk of exposure to hiv, which are currently not indicated. prescription should be done on an individual basis. indeed, the old hippocratic advice of not too readily prescribing any new modes of treatment, or in plain words wait and watchstill holds true. this is presumably one of the reasons for not blindly or overenthusiastically accepting hiv preexposure prophylaxis. 70 univ med vol. 32 no.2 references 1. van damme l, corneli a, ahmed k, agot k, lombaard j, kapiga s, et al. for the fem-prep study group. preexposure prophylaxis for hiv infection among african women. n engl j med 2012;367:411-22. 2. thigpen mc, kebaabetswe pm, paxton la, smith dk, rose ce, segolodi tm, et al. for the tdf2 study group. antiretroviral preexposure prophylaxis for heterosexual hiv transmission in botswana. n engl j med 2012;367:423-34. 3. baeten jm, donnell d, ndase p, mugo nr, campbell jd, wangisi j, et al. antiretroviral prophylaxis for hiv prevention in heterosexual men and women. n engl j med 2012;367:399-410. 4. cohen ms, baden lr. preexposure prophylaxis for hiv where do we go from here? n engl j med 2012;367:459-61. 5. mascolini m. fda panel endorses truvada for pre-exposure prophylaxis. international aids society, may 12, 2012. available at: http://www.iasociety.org/article.aspx? elementid=14505. accessed 13 august 2013. 6. world health organization. patient information leaflet. whopar part 3 11/2010, version 1.0. rita k 195 *bagian patologi klinik fakultas kedokteran universitas trisakti korespondensi dr. pusparini, sp.pk bagian patologi klinik fakultas kedokteran universitas trisakti jl.kyai tapa no.260 grogol jakarta 11440 telp.5677231 eks.2404 email: rini_puspa@gmail.com universa medicina 2007; 26: 195204 universa medicina oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007oktober-desember 2007 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 obesitas sentral, sindroma metabolik dan diabetes melitus tipe dua pusparini* abstrak sudah diketahui secara luas bahwa seseorang dengan obesitas mempunyai risiko tinggi untuk mengalami resistensi insulin dan komplikasi metaboliknya seperti diabetes melitus tipe 2 (t2dm), hipertrigliseridemia, penurunan kolesterol high density lipoprotein, hipertensi dan penyakit kardiovaskuler. akumulasi jaringan adiposa pada bagian tertentu di tubuh seperti di rongga perut menyebabkan peningkatan risiko terjadinya resistensi insulin sampai terjadinya sindroma metabolik. sindroma metabolik merupakan suatu abnormalitas metabolik yang melibatkan berbagai faktor yang saling berkaitan serta merupakan faktor risiko penyakit jantung koroner yang paling penting pada populasi modern. pengaturan produksi adipositokin berperan penting pada homeostasis metabolisme glukosa dan lipid. disregulasi produksi adipositokin pada obesitas sentral terlibat langsung pada patogenesis sindroma metabolik. penurunan berat badan atau pencegahan peningkatan berat badan merupakan cara terbaik mencegah terjadinya obesitas terutama obesitas sentral yang juga merupakan suatu cara mencegah terjadinya t2dm. edukasi mengenai komplikasi obesitas dan keterlibatan keluarga dalam pengobatan t2dm sangat penting. kata kunci : obesitas, sentral, sindroma metabolik 196 pusparini sindroma metabolik dan t2dm *department of clinical patology medical faculty, trisakti university correspondence: dr. pusparini, sp.pk department of clinical patology medical faculty trisakti university jl.kyai tapa no.260 grogol, jakarta phone. 5677231 ext.2404 email: rini_puspa@gmail.com universa medicina 2007; 26: 195-204 it is well recognized that persons with generalized obesity suffer from a high risk of insulin resistance and its metabolic complications, such as type 2 diabetes mellitus (t2dm), hypertriglyceridemia, low levels of high density lipoprotein cholesterol, hypertension and cardiovascular disease. accumulation of adipose tissue in a particular anatomical compartment as in abdominal cavity caused an excess risk of insulin resistance until caused metabolic syndrome. metabolic syndrome, which is defined by a metabolic abnormalities involve a cluster of risk factors of cardiovascular disease is an important risk factor among modern population. production control of adipocytokine is an important factor of homeostatis glucose and lipid metabolism. disregulation of adipocytokine production in central obesity has direct pathogenicity of metabolic syndrome. weight loss or prevention of increased body weight is a good intervention for preventing the obesity, especially central obesity and t2dm. education about complication of obesity and conscientious involvement by family members is necessary for t2dm treatment. keywords: obesity, central, metabolic syndrome central obesity, metabolic syndrome and type 2 diabetes mellitus pusparini* abstract pendahuluan di eropa prevalensi obesitas (indeks massa tubuh/imt >30 kg/m2) pada orang dewasa diperkirakan bervariasi antara 10-20% pada laki-laki dan 10-25% pada wanita. prevalensi tersebut meningkat sekitar 10-40% pada dekade terakhir di hampir sebagian besar negara eropa. berat badan lebih (overweight) dengan imt >25 k g / m 2 m e n c a p a i 5 0 7 0 % , t e r d a p a t p a d a s e b a g i a n b e s a r n e g a r a b e r k e m b a n g . ( 1 ) perkembangan tersebut membawa konsekuensi meningkatnya biaya pemeliharaan kesehatan m e n j a d i s e k i t a r 2 7 % y a n g d i s e b a b k a n peningkatan populasi obesitas. di inggris lebih dari 30.000 kematian terjadi akibat obesitas dan total biaya kesehatan diperkirakan mencapai 480 juta poundsterling. obesitas bukan hanya d i j u m p a i p a d a o r a n g d e w a s a , t e t a p i j u g a dijumpai pada anak-anak. di inggris, prevalensi berat badan lebih pada anak meningkat sejak 20 tahun yang lalu dan diperkirakan akan menjadi obesitas dan berat badan lebih pada dewasa muda.(1-2) wo r l d h e a l t h o rg a n i z a t i o n ( w h o ) menyatakan bahwa obesitas merupakan salah satu dari 10 kondisi yang berisiko di seluruh dunia dan salah satu dari 5 kondisi yang berisiko di negara berkembang.(3) obesitas disebabkan oleh dua faktor yaitu adanya peningkatan asupan makanan dan penurunan pengeluaran energi. u n t u k m e n j a g a b e r a t b a d a n y a n g s t a b i l diperlukan keseimbangan antara energi yang masuk dan energi yang keluar. hal yang menjadi 197 masalah adalah bahwa ternyata sangat sulit bagi s e s e o r a n g u n t u k m e n g a t u r a s u p a n d a n p e n g e l u a r a n e n e rg i n y a . a s u p a n m a k a n a n semakin meningkat karena ketersediaan beragam makanan siap saji yang makin bervariasi, mudah didapat, nikmat dan murah. di lain sisi aktivitas fisik masyarakat modern menjadi semakin berkurang dan kemajuan teknologi menyebabkan pada saat kerja maupun santai orang semakin mengurangi kegiatan fisik.(4) pernyataan obesitas s e b a g a i s u a t u p e n y a k i t m a s i h b a n y a k diperdebatkan, namun tidak diragukan lagi bahwa obesitas adalah stimulator utama untuk terjadinya berbagai penyakit terutama sindroma metabolik, diabetes melitus tipe 2 (t2dm) dan hipertensi. penyakit tersebut merupakan faktor r i s i k o p e n t i n g u n t u k t e r j a d i n y a p e n y a k i t kardiovaskuler. menurut who 40-60% pasien obesitas akan berkembang menjadi t2dm dan memiliki tekanan darah tinggi. diperkirakan peningkatan populasi obesitas di dunia akan meningkatkan penyakit kardiovaskuler dan merupakan penyebab kematian global terburuk pada abad ke-21.(4) the international diabetes federation (idf) menyatakan bahwa sindroma metabolik (sm) merupakan pemicu munculnya pandemik g l o b a l a n t a r a t 2 d m d a n p e n y a k i t kardiovaskuler. secara global insidens sm meningkat dengan cepat. prevalensi sm di dunia adalah 20-25% sedangkan di indonesia 13,3% dan tidak menutup kemungkinan prevalensi ini akan terus meningkat.(5) sejarah sindroma metabolik sm merupakan suatu kumpulan faktor risiko yang terdiri atas obesitas, hipertensi, hiperglikemia puasa dan dislipidemia yang dapat menyebabkan peningkatan risiko terjadinya t2dm dan penyakit kardiovaskuler.(6) reaven mengemukakan tentang the role of i n s u l i n re s i s t a n c e i n h u m a n d i s e a s e y a n g meliputi tiga topik utama yaitu (i) adanya sejumlah tanda-tanda dan gejala sehingga muncul sindroma yang disebut sindroma x. tanda dan gejala ini termasuk resistensi insulin, i n t o l e r a n s i g l u k o s a , h i p e r i n s u l i n e m i a , peningkatan trigliserida, very low density lipoprotein (vldl), penurunan kolesterol high density lipoprotein (hdl), dan hipertensi; (ii) sindroma ini umumnya meliputi resistensi insulin dan komponen lain yang berhubungan dengan resistensi insulin; (iii) dan dapat disimpulkan resistensi terhadap insulin yang menstimulasi asupan glukosa berperan penting pada prediksi kejadian penyakit jantung koroner (pjk).(7) kaplan mencetuskan the deadly quartet yang meliputi obesitas tubuh bagian atas, hipertensi, diabetes, dan hipertrigliseridemia sebagai penyebab sindroma yang berhubungan d e n g a n p e n i n g k a t a n r i s i k o p e n y a k i t k a r d i o v a s k u l e r. ( 8 ) ta h u n 1 9 9 1 , z i m m e t menyarankan obesitas sentral masuk dalam sindroma dan mengubah nama sindroma x m e n j a d i s i n d r o m a r e s i s t e n s i i n s u l i n a t a u m e t a b o l i c s y n d ro m e ( m e t s ) . ( 9 ) a l b e r t i d a n z i m m e t a t a s m e n y a t a k a n s i n d r o m a y a n g meliputi: (i) gangguan pengaturan glukosa atau diabetes; (ii) resistensi insulin; (iii) hipertensi; (iv) dislipidemia dengan trigliserida plasma >1,7 mmol/l dan atau kolesterol hdl ≤0,9 mmol/l pada laki-laki dan ≤1 mmol/l pada wanita; (v) obesitas sentral (laki-laki : waist to hip ratio > 0,9; wanita waist to hip ratio >0,85 dan atau body mass index (bmi) > 30 kg/m2 dan (vi) mikroalbuminuria.(9) nilai ambang batas yang digunakan untuk definisi obesitas sebaiknya dihubungkan dengan peningkatan risiko metabolik yang merugikan serta morbiditas dan mortalitas kardiovaskuler. himpunan studi obesitas indonesia (hisobi) telah melakukan studi untuk menentukan nilai ambang bmi dan waist circumference (wc) untuk populasi indonesia. studi dilakukan di bandung, karawang, semarang, solo, medan, universa medicina vol. 26 no.4 198 pusparini sindroma metabolik dan t2dm makasar, dan jakarta pada 5.978 orang (lakilaki 4.871, wanita 1.107) menunjukkan nilai ambang bmi dan wc untuk wanita 24,9 kg/m2 dan 82,5 cm, dan untuk laki-laki 24,9 kg/m2 dan 88,7 cm.(10) fisiologi obesitas penyimpanan lemak yang terdapat di tubuh ternyata bukan merupakan hasil kebiasaan buruk yang bersifat pasif. adiposa ternyata berperan pada pengaturan proses homeostasis energi, y a i t u s u a t u p r o s e s y a n g m e m b u t u h k a n keseimbangan antara asupan energi (asupan makanan) dan pengeluaran energi (metabolisme dan aktifitas fisik) serta jumlah cadangan energi dalam tubuh (massa lemak).(11) sistem biologi yang mengatur asupan makanan, mengontrol frekuensi dan jumlah makanan yang dimakan, s e r t a m e m p e r b a i k i k e s e i m b a n g a n y a n g terganggu, merupakan masalah yang kompleks dan belum dipahami dengan jelas.(11) te l a h d i l a p o r k a n a d a n y a d u a h o r m o n peptida yang diproduksi di saluran pencernaan yang diketahui mempengaruhi perilaku makan jangka pendek, sedangkan leptin dan insulin mengatur berat badan dalam jangka waktu hitungan bulan atau tahun (gambar 1). terdapat area di otak pada hypothalamus yaitu arcuate nucleus yang berperan menggabungkan aktivitas hormon-hormon tersebut di atas, memberikan s i n y a l k e p a d a t u b u h u n t u k m e n g a t u r kesimbangan asupan makanan dan penggunaan energi (gambar 2).(12) arcuate nucleus memiliki dua neuron utama dengan aksi yang berlawanan. neuron tipe pertama memproduksi neurotransmitter peptida yaitu neuropeptide y (npy) dan agouti related peptide (agrp), aktivasi neuron ini akan menstimulasi selera makan sambil mereduksi metabolisme. terdapat neuron lainnya yaitu neuron proopiomelanocortin (pomc) / cocaine and amphetamine regulated transcript (cart) yang akan melepaskan α melanocyte stimulating hormone (α msh) yang dapat menghambat keinginan untuk makan. ketika cadangan lemak gambar 1. beberapa hormon yang mengatur selera makan(12) gambar 2. pusat pengaturan selera makan(12) 199 dan konsentrasi leptin menurun, neuron npy dan agrp diaktivasi dan neuron pomc diinhibisi sehingga terjadi kenaikan berat badan.(12) hormon lain yang juga berperan dalam pengaturan berat badan adalah hormon insulin. reseptor insulin terdapat di seluruh bagian otak. penelitian lain mengatakan bahwa aksi hormon ini untuk menekan selera makan terjadi secara langsung pada arcuate nucleus. pemberian insulin ke dalam otak dekat arcuate nucleus dapat menghambat produksi npy, yang bekerja menstimulasi selera makan.(12) faktor penyebab terjadinya sm s i n d r o m a m e t a b o l i k m u n c u l s e b a g a i akibat dari interaksi antara kerentanan genetik dan pola hidup. definisi untuk sm berbedabeda dan masih diperdebatkan, tetapi semua s e t u j u b a h w a o b e s i t a s , r e s i s t e n s i i n s u l i n , d i s l i p i d e m i a d a n h i p e r t e n s i m e r u p a k a n komponen sm.(13) komponen utama sm adalah o b e s i t a s . o b e s i t a s m e r u p a k a n s u a t u peningkatan massa jaringan lemak tubuh yang terjadi akibat ketidakseimbangan antara asupan energi dengan keluaran energi. sel adiposit tidak hanya berperan pasif sebagai tempat metabolisme dan penyimpanan energi dalam bentuk trigliserida tetapi juga berperan sebagai kelenjar endokrin yang mensekresikan berbagai sitokin dan neuropeptida yang berperan dalam metabolisme.(14,15) pada keadaan obesitas terjadi gangguan keseimbangan adipositokin yang dilepaskan. s e l a d i p o s i t b e r u s a h a m e m p e r t a h a n k a n k e s e i m b a n g a n e n e rg i d e n g a n m e l e p a s k a n interleukin 6 (il-6), tumor necorsis factor -α (tnf-α) dan monocyte chemotatic protein-1 (mcp-1). pelepasan sitokin tersebut menandai a w a l i n f l a m a s i . o b e s i t a s d a p a t d i k a t a k a n merupakan bentuk inflamasi kronik. interleukin 6 dan tnf-α dapat memicu pembentukan creactive protein (crp) di hati. protein ini jika diproduksi terus menerus dapat memperburuk k o n d i s i i n f l a m a s i m e l a l u i a k t i v a s i k r o n i k terhadap sel endotel, akibatnya terjadi disfungsi endotel.(14,15) proses lipolisis yang tinggi menyebabkan jumlah stress oksidatif yang dihasilkan juga sangat tinggi. terjadi peningkatan jumlah re a c t i v e o x y g e n s p e c i e s ( r o s ) a k i b a t peningkatan aktivitas enzim oksidase dan disregulasi hormon adipositas. peningkatan s t re s s o k s i d a t i f m e n y e b a b k a n g a n g g u a n metabolisme, baik asupan glukosa pada otot maupun pada jaringan adipose, penurunan sekresi insulin dan kerusakan sel sehingga terjadi d i s f u n g s i e n d o t e l , a t e r o s k l e r o s i s s a m p a i akhirnya terjadi penyakit vaskuler. tubuh kita s e b e n a r n y a m e m i l i k i m e k a n i s m e d e f e n s i f terhadap stress oksidatif. superoksida dismutase (sod), glutathione peroxidase (gpx) dan k a t a l a s e m e r u p a k a n e n z i m y a n g d a p a t mendegradasi ros. superoksida dismutase m e n g u b a h s u p e r o k s i d a m e n j a d i h i d r o g e n peroksida (h 2 o 2 ) dan molekul oksigen (o 2 ). penurunan aktivitas sod merupakan penanda p e n t i n g s t r e s s o k s i d a t i f . p e n u r u n a n s o d meningkatkan risiko penyakit vaskuler.(16,17) perkembangan sm hingga menyebabkan penyakit vaskuler dan t2dm terjadi melalui b e b e r a p a t a h a p . b e b e r a p a p e n e l i t i a n menunjukkan bahwa disfungsi endotel dan kondisi inflamasi merupakan konektor utama terhadap kejadian penyakit vaskuler. lesi aterosklerotik dapat terjadi akibat induksi dari i n t e r a k s i a n t a r a k o n d i s i s t r e s s o k s i d a t i f , inflamasi, dan disfungsi endotel.(16,17) obesitas, resistensi insulin dan t2dm k i r a k i r a 8 0 % p a s i e n d e n g a n t 2 d m menderita obesitas, dan obesitas dihubungkan dengan resistensi insulin. jaringan adiposa menginduksi resistensi insulin melalui berbagai universa medicina vol. 26 no.4 200 pusparini sindroma metabolik dan t2dm mekanisme. adiposa dapat melepaskan asam lemak bebas yang dapat berpengaruh pada proses pembentukan sinyal insulin melalui mekanisme stimulasi terhadap isoform protein k i n a s e ( p k c ) . a s a m l e m a k b e b a s j u g a mempunyai kemampuan mengganggu pelepasan glukosa dari hepar.(18,19) obesitas viseral atau dikenal dengan obesitas sentral merupakan c o n t o h p e n i m b u n a n l e m a k t u b u h y a n g berbahaya karena lipolisis di daerah ini sangat efisien dan lebih resisten terhadap efek insulin d i b a n d i n g k a n a d i p o s i t d i d a e r a h l a i n . ( 1 8 2 0 ) j a r i n g a n a d i p o s a j u g a m e m b u a t d a n m e l e p a s k a n b e b e r a p a a d i p o s i t o k i n . adipositokin yang paling penting adalah tnfα, yang berperan menginduksi resistensi insulin melalui glucose transporter 4 (glut 4) dan meningkatkan pelepasan asam lemak bebas.(20) adiponektin yaitu adipositokin yang baru d i t e m u k a n d a n b e r p e r a n m e m p e n g a r u h i s e n s i t i v i t a s i n s u l i n . p e n u r u n a n k a d a r a d i p o n e k t i n p a d a t 2 d m d a n o b e s i t a s m e n u n j u k k a n a d a n y a k e t e r k a i t a n a n t a r a adiponektin, obesitas, dan t2dm. hal yang lebih mendukung hipotesis ini adalah bahwa tikus yang kekurangan adiponektin ditemukan menunjukkan resistensi terhadap insulin dan g e j a l a d i a b e t e s , s e d a n g k a n p e m b e r i a n adiponektin menyebabkan efek penurunan g l u k o s a d a n p e r b a i k a n k e a d a a n r e s i s t e n s i i n s u l i n . ( 1 9 ) s e l a i n i t u j u g a t e r d a p a t a n t i inflammatory cytokine interleukin-10 yang j u g a m e m p u n y a i e f e k a n t i d i a b e t o g e n i k . p e n u r u n a n k a d a r a d i p o s i t o k i n i n i j u g a menyebabkan terjadinya sm pada wanita.(19) o b e s i t a s s e n d i r i m e r u p a k a n k e l a i n a n poligenetik yang melibatkan interaksi gen dan lingkungan yang dapat menyebabkan t2dm. telah lama diketahui bahwa metabolic rate yang rendah dalam jangka waktu yang lama merupakan faktor risiko terjadinya obesitas. reseptor adrenergik yang terdapat di jaringan a d i p o s a t e r m a s u k l e m a k d i p e r u t d a p a t mempengaruhi pengaturan lipolisis dan suhu. studi pendahuluan pada orang pima indian memperlihatkan adanya mutasi w64r pada reseptor adrenergik yang berhubungan dengan terjadinya t2dm dini dan basal metabolic rate (bmr) yang rendah. beberapa studi lainnya juga memperlihatkan bahwa mutasi ini juga b e r h u b u n g a n d e n g a n p e n u r u n a n k a p a s i t a s s e k r e s i i n s u l i n d a r i s e l b e t a p a n k r e a s . ( 2 0 ) h o m e o s t a s i s g l u k o s a d i a t u r o l e h s e k r e s i insulin, efek insulin, produksi glukosa di hepar dan asupan glukosa seluler. reseptor insulin di hepar, otot dan jaringan adiposa dalam keadaan normal peka terhadap insulin. pada keadaan absorbtif, sekresi insulin sebagai respons terhadap peningkatan kadar glukosa darah menghambat produksi glukosa hepar dan mestimulasi penggunaan glukosa terutama di otot. selama proses pasca absorbtif (puasa), sekresi insulin menurun kembali ke keadaan b a s a l , d a n m e n g h a m b a t p r o d u k s i g l u k o s a (glukoneogenesis) di hepar ke keadaan yang lebih rendah untuk mempertahankan kadar glukosa darah puasa.(20) adanya peningkatan jaringan adipose biasanya diikuti keadaan resistensi insulin. resistensi insulin merupakan suatu fase awal abnormalitas metabolik sampai terjadinya intoleransi glukosa. resistensi insulin adalah suatu keadaan penurunan kemampuan jaringan y a n g s e n s i t i f t e r h a d a p i n s u l i n u n t u k memberikan respons yang normal terhadap i n s u l i n p a d a t i n g k a t s e l u l e r. p e n u r u n a n kemampuan tersebut dapat disebabkan karena faktor genetik, metabolik, dan nutrisi. adiposit viseral memberikan efek resistensi insulin ke k e a d a a n y a n g l e b i h b u r u k d i b a n d i n g k a n adiposit subkutan yang lain. pada kondisi awal intoleransi glukosa, insulin yang diproduksi sel β pankreas masih dapat melakukan kompensasi dengan meningkatkan sekresi insulin. keadaan hiperinsulinemia ini dapat mempertahankan kadar glukosa darah pada keadaan normal.(19,20) 201 sensitivitas terhadap insulin dan sekresi insulin merupakan dua kondisi yang berlawanan dan berkaitan secara proporsional. makin rendah sensitivitas insulin (makin tinggi resistensi insulin) makin banyak insulin yang disekresi. hasil dari keseimbangan antara sensitivitas insulin dan sekresi insulin adalah konstan dan d i g u n a k a n s e b a g a i s t a n d a r d a r i g l u c o s e disposition index. apabila sensitivitas insulin menurun, sekresi sel β pankreas harus meningkat untuk mempertahankan glucose disposition index. kegagalan sel β pankreas menyebabkan sekresi insulin tidak adekuat, sehingga terjadi transisi dari kondisi resistensi insulin ke diabetes yang manifes secara klinis. sebagai akibatnya proses awal penyakit ini berlangsung tanpa gejala, sampai terjadi kegagalan fungsi sel β pankreas dan pasien memerlukan terapi dengan obat atau insulin.(13) skrining individu dengan risiko t2dm diabetes melitus tipe 2 sering berlangsung tanpa gejala, seringkali terjadi komplikasi diabetes muncul sebelum gejala diabetes timbul. faktor risiko untuk t2dm meliputi berat badan berlebih dan obesitas, dan gejala resistensi insulin seperti pada keadaan akantosis nigrikans, pubertas prekoks, hipertensi, dislipidemia, dan sindroma ovarii polikistik. american diabetes a s s o c i a t i o n ( a d a ) m e m b e r i k a n p a n d u a n skrining diabetes untuk anak dengan bmi >85 persen untuk umur dan jenis kelamin, dengan 2 faktor tambahan (tabel 1).(13,20) ditemukannya kerusakan mikroangipopati pada pasien yang baru terdiagnosis t2dm menunjukkan bukti bahwa kerusakan sudah t e r j a d i s e j a k o n s e t d i a b e t e s . st u d i o t o p s i menunjukkan perubahan aterosklerotik vaskuler dapat dijumpai pada anak-anak dan luasnya aterosklerotik berkorelasi dengan faktor risiko seperti bmi dan kadar lemak. identifikasi dini pada anak dengan t2dm dapat meminimalkan komplikasi yang serius.(19,20) diagnosis t2dm te l a h d i k e t a h u i s e c a r a u m u m b a h w a standar diagnosis t2dm didasarkan pada nilai tabel 1. petunjuk tes untuk t2dm13 kriteria* berat badan lebih atau berisiko berat badan lebih • bmi >85% dari umur dan jenis kelamin atau • berat badan terhadap tinggi badan > 85%, atau • berat badan >120% dari tinggi badan ditambah dengan dua kriteria di bawah ini : faktor risiko : • riwayat keluarga t2dm pada keluarga ayah/ibu atau kakek/nenek • ras/etnis (american indian, black, hispanic asian/ pacific islander) • terdapat gejala resistensi insulin atau kondisi yang berhubungan dengan resistensi insulin (acantosis nigrikans, hipertensi, dislipidemia, sindroma polikistik ovarii) umur untuk memulai skrining : • 10 tahun atau onset pubertas bilamana pubertas dimulai pada usia yang sangat muda frekuensi tes : setiap 2 tahun tes : glukosa puasa *pertimbangan klinik harus dilakukan terhadap pasien dengan risiko tinggi yang tidak ditemukan kriteria di atas. glukosa puasa merupakan tes yang direkomendasikan oleh ada. tes alternatif meliputi glukosa sewaktu dan tes toleransi glukosa oral universa medicina vol. 26 no.4 202 pusparini sindroma metabolik dan t2dm glukosa puasa, glukosa sewaktu dan tes glukosa toleransi oral. kriteria diagnosis ini berlaku untuk usia anak maupun dewasa sama.(13,19) kadar glukosa puasa ditetapkan normal <100 mg/dl (6,11mm). pasien dengan kadar gl ukos a puasa antar a 100 dan 125 mg/dl menderita glukosa puasa terganggu. pasien d e n g a n k a d a r g l u k o s a p u a s a >1 2 6 m g / d l m e n d e r i t a d i a b e t e s . u n t u k m e n e g a k k a n diagnosis diperlukan dua kali peningkatan kadar glukosa darah pada hari yang berlainan. kadar g l u k o s a s e w a k t u > 2 0 0 m g / d l m e r u p a k a n keadaan diagnostik untuk diabetes bila pasien mempunyai gejala tambahan seperti poliuria, polidipsia, dan penurunan berat badan. selama tes toleransi glukosa oral, bila kadar glukosa 2 jam posprandial <140 mg/dl dikatakan pasien t e r s e b u t n o r m a l , > 1 4 0 d a n < 2 0 0 m g / d l dikatakan glukosa toleransi tes terganggu, dan >200 mg/dl dikatakan diabetes.(13) evaluasi awal pasien baru dengan diabetes melitus sangat penting untuk membedakan t1dm dan t2dm, dan untuk mengoptimalkan terapi. gejala klinik yang sangat membantu untuk membedakan t2dm dari t1dm adalah obesitas dan gejala resistensi insulin (akantosis nigrikans, hipertensi, sindroma ovarii polikistik). pasien dengan t2dm sering sekali mempunyai k a d a r c p e p t i d a y a n g t i n g g i . ti d a k ditemukannya autoantibodi terhadap insulin, sel islet dan asam glutamat dekarboksilase juga merupakan hal yang khas untuk sebagian besar kasus (tidak seluruh kasus) diabetes yang diklasifikasikan t2dm.(13,18) cara mengatasi t2dm perubahan gaya hidup b e b e r a p a p e n e l i t i a n m e m p e r l i h a t k a n keuntungan modifikasi gaya hidup terhadap progresivitas toleransi glukosa terganggu sampai menjadi t2dm. program pencegahan diabetes memperlihatkan di antara pasien dewasa dengan toleransi glukosa terganggu selama periode 3 tahun, diet rendah lemak yang dikombinasikan dengan latihan selama 15 menit per hari setiap minggu dapat mengurangi berat badan 5% sampai dengan 7% dan mengurangi risiko terjadinya t2dm 58% bila dibandingkan dengan yang tidak mendapat intervensi perubahan gaya hidup. penelitian lainnya memperlihatkan bahwa p e r u b a h a n g a y a h i d u p s e h i n g g a d a p a t mengurangi berat badan sebesar 10% dapat m e m p e r b a i k i p r o f i l l i p i d d a n s e n s i t i v i t a s insulin.(13,19,21) p e n u r u n a n b e r a t b a d a n d a n a t a u pencegahan peningkatan berat badan merupakan cara terbaik untuk mencegah terjadinya t2dm dengan segala komplikasinya. selain faktor di atas juga dianjurkan untuk melakukan skrining terhadap kesiapan keluarga untuk melakukan perubahan gaya hidup, pemberian edukasi m e n g e n a i k o m p l i k a s i m e d i s o b e s i t a s d a n k e t e r l i b a t a n k e l u a r g a d a l a m p e n g o b a t a n t2dm.(18-19, 21) terapi medikamentosa te r a p i a w a l t 2 d m t e r g a n t u n g d a r i beratnya manifestasi klinik. untuk pasien dengan hiperglikemia puasa sedang (126–200 mg/dl) dan hba1c <8,5% penatalaksanaan yang dianjurkan adalah perubahan gaya hidup d a n p e m b e r i a n a n t i d i a b e t i k o r a l s e p e r t i metformin. metformin merupakan satu-satunya obat yang lolos uji untuk pengobatan t2dm pada anak yang obesitas. metformin, obat oral antidiabetik golongan biguanid, memberikan efek menurunkan produksi glukosa hepar dan meningkatkan asupan glukosa pada jaringan p e r i f e r t e r u t a m a o t o t . p a s i e n d e n g a n h i p e rg i k e m i a b e r a t ( > 2 0 0 m g / d l ) , h b a 1 c >8,5% dan atau dengan ketosis, terapi harus diawali dengan pemberian insulin.(18-19) insulin harus diberikan bila kadar glukosa tidak dapat terkontrol setelah 3–6 bulan terapi metformin. beberapa bukti klinis menunjukkan 203 pasien dengan pemberian insulin secara dini memperbaiki kontrol glukosa dalam jangka waktu lama. hal itu mungkin menunjukkan adanya efek kerusakan yang ditimbulkan oleh kondisi hiperglikemia puasa pada sel β dan jaringan yang sensitive terhadap insulin.(13) hal-hal yang perlu diperhatikan pasien dengan t2dm dianjurkan untuk melakukan pemeriksaan kadar glukosa darahnya secara berkala, terutama pada tahap awal terapi, atau bila sedang menderita penyakit akut. kadar keton urin juga perlu dimonitor. kadar glukosa darah diharapkan dapat mencapai 70–100 mg/ dl. kadar hba1c diperiksa setiap 3 bulan dengan target terapi <7%.(13,18) diabetes juga berhubungan dengan keadaan hipertensi dan dislipidemia. tekanan darah sistolik dan diastolik dianggap normal bila < 90%. bila tekanan sistolik dan diastolik antara 90-95% dianggap prehipertensi. bila tekanan darah sistolik dan diastolik >95% disebut stadium 1 hipertensi, dan bila tekanan darah sistolik dan diastolik >99% ditambah 5 mmhg, pasien dimasukkan dalam stadium 2 hipertensi.(3,19) selain itu perlu juga diperhatikan kemungkinan terjadinya komplikasi mikrovaskuler dengan melakukan pemeriksaan kadar albumin urin dan fungsi ginjal setahun sekali.(19) kesimpulan asupan energi yang tidak seimbang dengan energi yang keluar menyebabkan terjadinya penimbunan lemak tubuh. distribusi lemak tubuh, terutama akumulasi di jaringan adiposa viseral merupakan faktor risiko kritis terhadap beberapa jenis penyakit seperti t2dm. obesitas viseral atau obesitas sentral lebih berbahaya karena lipolisis di daerah ini sangat efisien dan lebih resisten terhadap efek insulin dibandingkan dengan adiposit di daerah lain. obesitas sentral merupakan awal terjadinya intoleransi glukosa. keadaan ini biasanya ditandai dengan terjadinya resistensi insulin yaitu penurunan kemampuan jaringan yang sensitif terhadap insulin untuk memberikan respons yang normal terhadap insulin pada tingkat seluler sehingga akhirnya terjadi t2dm. untuk mengatasi t2dm hal yang paling penting diperhatikan adalah perubahan gaya hidup melalui pembatasan asupan energi yang masuk dan peningkatan pengeluaran energi misalnya dengan olahraga. daftar pustaka 1. seidel jc, rissanen am. time trends in world wide prevalence of obesity. in: bray ga, bouchard c, james wpt, eds. handbook of obesity. new york, ny: marcel dekker, 1998: 79-91. 2. world health organization. obesity: preventing and managing the global epidemic. report of a who consultation. who technical report series, 894. geneva, switzerland: world health organization, 2000. 3. hill jo, wyatt hr, reed gw, peter jc. obesity and the environment: where do we go from here? science 2003; 299: 853-5. 4. pi-sunyer x. a clinical view of the obesity problem. science 2003; 299: 859-60. 5. international diabetes federation. the idf consensus worldwide definition of the metabolic syndrome. available at: http://www.idf.org. accessed mei 2, 2007. 6. wannamethee sg, shaper ag, morris rw, whincup ph. measures of adiposity in the identification of metabolic abnormalities in elderly men. am j clin nutr 2005; 81: 1313-21. 7. reaven gm. role of insulin resistance in human disease. diabetes 1988; 37: 1595-607. 8. kaplan nm. the deadly quartet. upper body obesity, glucose intolerance, hypertrigliseridemia, and hypertension. arch intern med 1989; 149: 151420. 9. alberti kgmm, zimmet pz. for the who consultation, definition, diagnosis and classification of diabetes mellitus and its complications: part 1. diagnosis and classification of diabetes melitus. provision report of a who consultation. diabet med 1998; 15: 539-53. 10. sukmawati ir, harijanto t. optimal cut off value for obesity: using anthropometric indices to predict universa medicina vol. 26 no.4 204 pusparini sindroma metabolik dan t2dm atherogenic dyslipidemia in indonesia population. in 3rd national obesity symposium (nos iii) 2004. p. 87-8. 11. woods sc, seeley rj. understanding the physiolofy of obesity: review of recent development in obesitasity research. int j obesitas relat metab disord 2002; 26: suppl 4: s8-10. 12. kelner k, helmuth l. obesity-what is to be done. science 2003; 299: 845. 13. hannon ts, rao g, arslanian sa. childhood obesity and type 2 diabetes mellitus. pediatrics 2005; 116: 473-80. 14. hofbauer kg. molecular pathways to obesity. int j obesitas 2002; 26: 18-27. 15. fruhbeck g, ambrosi jg, muruzabal fj, burrell ma. the adipocyte: a model for intergration of encdocrine and metabolic signaling in energy metabolism regulation. am j physiol endocrinol metab 2001; 280: 827-47. 16. grey a. the reductive hotspot hypothesis: an update. arch biochem biophys 2000; 373: 295-301. 17. faraci fm, didion sp. vascular protection superoxide dismutase isoforms in the vessel wall. arterioscler thromb vasc biol 2004; 24: 136773. 18. watson ke, harmel alp, matson g. atherosclerosis in type 2 diabetes mellitus: the role of insulin resistance. j cardiovasc pharmacol therapeut 2003; 8: 253-60. 19. garg a. regional adiposity and insulin resistance. j clin endocrinol metab 2004; 89: 4206-10. 20. gastaldelli a, miyazaki y, pettiti m, buzzigoli e, mahankali s, ferrannini e, et al. separate contribution of diabetes, total fat mass, and fat topography to glucose production, gluconeogenesis, and glycogenolysis. j clin endocrinol metab 2004; 89: 3914-21. 21. zhu sk, wang zm, shen w, heymsfield sb, heshka s. percentage body fat ranges associated with metabolic syndrome risk: results based on the third national health and nutrition examination survey. am j clin nutr 2003; 78: 228-35. oktavianus 3 *physiology department, faculty of medicine yarsi university **anatomy department, faculty of medicine yarsi university correspondence dr. indra kusuma, m.biomed physiology department faculty of medicine yarsi university cempaka putih jakarta indonesia 10510 +6221-4206674 ext. 3091 email: indra.kusuma@yarsi.ac.id univ med 2013;32:3-10 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 background various products used in cellular therapy utilize tissue culture techniques requiring keratinocyte culture. an efficient and clinically acceptable keratinocyte culture system requires supplements with mitogenic activity. geraniin is a phytochemical with the potential as a supplement for expansion culture of keratinocytes. the objective of the present study was to verify the mitogenic activity of geraniin on human keratinocytes. methods this was an experimental study using two samples of human foreskin obtained by circumcision of a male child. epidermal keratinocytes were isolated from the foreskin samples and were divided into paired groups, comprising intervention and control groups. the intervention groups were cultured with geraniin supplementation, whereas the control groups with standard supplements, without the addition of geraniin. mitochondrial activity of the cells was evaluated by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide (mtt) proliferation assay. absorbance values in each of the groups was measured at 450 nm. data analysis was performed with the paired t-test. results geraniin supplementation significantly increased the keratinocyte proliferation rates at dosages of 0.8 to 3.1 µm. an increase of 57% in the proliferation rate was obtained at a dosage of 1.6 µm, while at a dosage of 12.5 µm toxic effects were starting to appear. geraniin presumably causes increased cellular energy status, resulting in increased proliferation rates. conclusion the findings in this study provide evidence in support of the utilization of geraniin as a supplement for expansion culture of keratinocytes. further studies may presumably identify the molecules acting as geraniin receptors and the intracellular mechanisms underlying the increase in proliferation rates. key words: keratinocyte, geraniin, foreskin, proliferation geraniin supplementation increases human keratinocyte proliferation in serum-free culture indra kusuma* and restu syamsul hadi** 4 kusuma, hadi geraniin and human keratinocytes suplementasi geraniin meningkatkan proliferasi kultur keratinosit manusia bebas serum latar belakang beberapa produk yang digunakan dalam terapi sel menggunakan tehnik rekayasa jaringan yang membutuhkan kultur keratinosit. sistem kultur keratinosit yang efisien dan dapat diterima oleh klinisi membutuhkan suplemen yang bersifat mitogenik. geraniin adalah suatu zat fitokimia yang memiliki potensi sebagai suplemen kultur ekspansi keratinosit. penelitian ini bertujuan untuk melakukan verifikasi aktivitas mitogenik geraniin terhadap keratinosit manusia. metode penelitian ini menggunakan desain eksperimental. dua sampel kulit preputium manusia diperoleh dari prosedur sirkumsisi pada anak laki-laki. keratinosit epidermal diisolasi dari kulit preputium dan dikultur dengan suplemen berupa epidermal growth factor dan bovine pitutary extract. keratinosit dibagi dalam kelompok berpasangan. kelompok perlakuan dikultur dengan tambahan suplemen geraniin, sedangkan kelompok kontrol dikultur dengan suplemen standar tanpa penambahan geraniin. aktivitas mitokondria sel dievaluasi dengan uji proliferasi 3-[4,5dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide (mtt). nilai absorbansi dari setiap kelompok diukur pada panjang gelombang 450 nm. analisis dilakukan menggunakan uji t pasangan. hasil suplementasi geraniin menunjukkan peningkatan proliferasi keratinosit secara signifikan pada dosis antara 0,8 sampai 3,1 µm. peningkatan proliferasi sebesar 57% dicapai pada dosis 1,6 µm sementara efek toksik mulai terlihat pada dosis 12,5 µm. geraniin menyebabkan peningkatan status energi yang mengakibatkan laju proliferasi sel yang lebih tinggi. kesimpulan temuan ini mendukung penggunaan geraniin sebagai suplemen kultur keratinosit untuk tujuan ekspansi. penelitian selanjutnya diharapkan dapat mengidentifikasi molekul yang berperan sebagai reseptor untuk geraniin dan mekanisme intraseluler yang mendasari peningkatan laju proliferasi sel. kata kunci: keratinosit, geraniin, preputium, proliferasi abstrak introduction human keratinocyte culture systems for the production of various epithelial cells initially required mouse fibroblast feeder layers and serum-containing media. serum-free culture without feeder layer was later described as using a c o l l a g e n c o a t i n g a n d m i t o g e n r i c h supplementation such as epidermal growth factor (egf) and bovine pituitary extract (bpe).(1,2) the use of basic fibroblast growth factor (bfgf), embryonic stem cell-conditioned medium, bone m o r p h o g e n e t i c f a c t o r 4 ( b m p 4 ) a n d transcription factor oct-4 was proved to be able to increase the total number of cells harvested and induce de-diferentiation, and was specifically directed to neuronal lineages.(3-5) recently, keratinocytes were succesfully reprogrammed to become induced pluripotent stem cells (ipscs).(6,7) 5 univ med vol. 32 no.1 keratinocytes were expanded to produce products such as cultured epithelial autografts (cea) and bilayered human skin equivalents (hse). bioreactors were developed to increase culture efficiency.(8) however, the use of serum and animal products in the culture system would cause the products to become unqualified for human therapy. therefore, the need to develop chemically-defined or plant-based mitogenic agents is increasing. geraniin is a phytochemical isolated from phyllanthus sp., also known as meniran in indonesia. phyllanthus urinaria l., locally known as rumput pacar by the dayak tunjung tribe of kalimantan, was traditionally used as medication for wounds and skin rashes.(9) studies on traditional herbs recognized that phyllanthus niruri was used to alleviate symptoms of ascites, urinary tract infection, enteritis, conjunctivitis, hepatitis, jaundice, and aphtae.(10,11) the isolated geraniin demonstrated inhibition of tumor necrosis factor (tnf)-á release and stimulation of celullar activities, such as proliferation, collagen biosynthesis and formation of extracelullar matrix proteins.(12,13) these properties of geraniin suggest that it may be a potential mitogenic agent for keratinocytes. a recent study by agyare et al.(12) shows that g e r a n i i n i s o l a t e d f r o m p h y l l a n t h u s muellerianus was able to increase proliferation of hacat cells, a human keratinocyte cell line. agyare et al.(12) did not use a normal human epidermal keratinocyte culture in their study. however, they revealed an increase in collagen production in dermal fibroblast culture. the present study was designed to evaluate the ability of geraniin to increase human keratinocyte proliferation rates in culture. this study focused on keratinocytes that are readily used for cellular therapy or as a component to construct the epidermal layer of hse. methods research design the present study using an experimental design was conducted from february to august 2012. cell culture cell culture and proliferation assays were conducted at the kimia farma cell culture laboratory, jakarta. two human foreskin samples were obtained after a circumcision procedure conducted at indonesia house of circumcision (ihc) in accordance with the manual provided by who.(14) keratinocytes, unless otherwise stated, were cultured in epilife medium supplemented with human keratinocyte growth supplement (hkgs, gibco; invitrogen) which contains bpe, human epidermal growth factor, hydrocortisone, insulin and transferrin. antibiotics and antimycotics used were a combination of penicillin-streptomycin (sigma) and fungizone (sigma). this supplementation will b e r e f e r r e d t o i n t h i s p a p e r a s s t a n d a r d supplementation. sample preparation the keratinocytes used in this study were d e r i v e d f r o m f o r e s k i n s c o l l e c t e d a f t e r circumcision procedures conducted on two children (6 and 8 years old). to obtain the proper number of cells, we performed keratinocyte expansion culture (figure 1). keratinocytes were subcultured 7 times, with 5 times population doubling at the third passage. collected samples were transferred to 10 ml epilife medium (gibco; invitrogen) to which 20 ug/ml gentamycin (sigma) and standard antibiotic-antimycotic combination1 were added, and then transported at 2-8oc to the laboratory. samples were refrigerated for 60 minutes before decontamination using povidone iodine and 70% ethyl alcohol (etoh).(12,15) samples were then rinsed using phosphate buffered saline (pbs, sigma). subcutaneous fat and blood clots were dissected away leaving the dermis and epidermis. the skin was then cut into strips and placed in a 60 mm dish containing 1 u/ml dispase ii (stem cell technologies) for overnight incubation at 2-8oc.(1,6) digested skin samples were placed in a dry 100 mm sterile glass petri dish cover. the epidermis was then peeled off and folded using a pair of fine 6 kusuma, hadi geraniin and human keratinocytes keratinocytes keratinocytes figure 1. primary keratinocytes for expansion culture (cobblestone morphology, 80-90% confluent, div 1, 20x axiocam; div: day in vitro) tweezers. the recovered epidermis was cut into smaller pieces and placed in a 15 ml tube containing 5 ml of 0.1% trypsin/edta (sigma) for a 30-minute incubation at 37o c.(15) trypsin/edta was deactivated using equal an amount of fetal bovine serum (fbs, sigma). the cell suspension was then triturated using a 10-ml serological pipette and passed through a 70–µm cell strainer (cascade biologix) into a 50-ml tube.(6,15) the collected cell suspension was then centrifuged at 1500 rpm for 7 minutes. the pellet was resuspended using pre-warmed epilife medium supplemented with hkgs (gibco; invitrogen). resuspended cells were then seeded (at 1x105 viable cells/sqcm) on 60mm culture dishes precoated with 10 ug/sqcm collagen type iv. (16) the primary culture (passage 0;p0) was incubated at 37o c with 5% co 2 and the medium was changed after 24 hours and then every other day. the culture reached 8 0 9 0 % c o n f l u e n c y i n 7 d a y s a n d w a s subcultured in a 1:3 split ratio. g e r a n i i n i s o l a t e d f r o m p h y l l a n t h u s urinaria to 98% purity by hplc was obtained from biopurify for use in this study. the agent was dissolved in 98% etoh prior to usage. the presence of etoh corresponding to the amount used in high dose geraniin experiments was e v a l u a t e d . t h e e x p e r i m e n t s h o w e d n o conclusive evidence of toxicity. therefore, the use of etoh presumably does not affect keratinocyte viability. other studies using agents dissolved in 98% etoh also showed no significant functional interference.(17) 20 mg of geraniin was dissolved in 98% etoh and kept at -20o c before use. the keratinocyte culture groups for experimentation were seeded on a 96-well plate overnight. geraniin solutions titrated for dose ranging were mixed with prewarmed epilife medium to replace the spent culture medium. keratinocytes were divided into 6 distinct groups with various dosages of geraniin supplementation. these groups were the positive control group, negative control group, and the four geraniin groups (at dosages of 0.8 ìm, 1.6 ì m, 3.12 ì m and 6.25 ì m, respectively). the negative control group was supplemented with 25 µm geraniin which is well above the geraniin toxic dose. the positive control group comprised keratinocytes cultured without geraniin. 7 univ med vol. 32 no.1 cell viability keratinocytes seeded on 60-mm culture dishes were subcultured by trypsinization and enumerated using a hemocytometer. subcultures were performed when the cultures reached 80% confluence. viable and non-viable cells from each subculture were identified using the trypan blue exclusion method. population doublings were calculated using: n=3.32 (log ucy-log i) + x (n= population doubling, ucy= cell yield, i= inoculum numbers, x=population doubling rate of inoculum) proliferation assay keratinocyte groups were incubated for 48 hours, then mitochondrial activity was assesed in triplicate by colorimetric methods using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium-bromide (mtt) assay (biovision) on a 96-well culture plate. a microplate reader with 450 nm filter was used to read the absorbance. cellular dehydrogenase activity (mtt assay) was used to quantify cell proliferation. figure 2. geraniin supplemented keratinocyte culture nc=negative controls; pc= positive controls; n=2 keratinocyte cultures for each data set. error bars represent sd. statistical significance *p<0.01, **p<0.05 data analysis data analysis was performed with excel (version 2007; microsoft). statistical significance was determined using the paired student t-test. ethical clearance ethical clearance was obtained from the ethical commission of yarsi university. informed consent was obtained from the patients’ parents results this study showed an increase in cell proliferation in a dose-dependent manner (figure 2). at a dosage of 1.6 µm there was a 57% increase in proliferation, compared to standard keratinocyte culture supplementation. optimal stimulation was achieved at dosages of 0.8 µm to 3.1 µm. geraniin supplementation clearly increased the keratinocyte proliferation rate. lower doses of geraniin did not increase the keratinocyte proliferation rate significantly. this study also revealed that geraniin supplementation started to reduce proliferation at 12.5 µm. to clarify the effect of high dose 8 kusuma, hadi geraniin and human keratinocytes geraniin supplementation, we evaluated cell v i a b i l i t y d a t a f r o m r o u t i n e k e r a t i n o c y t e subcultures. these data showed a quadruple number (32 ± 8.5; p<0.01) of non-viable cells recorded from subculture of flasks with 25 µm geraniin supplementation, compared to routine s u b c u l t u r e s ( 7 . 7 ± 4 . 6 ) o f n o n g e r a n i i n supplemented flasks. this finding confirms that 25 µm geraniin is toxic to keratinocyte cultures (figure 3). discussion our findings reveal that 1.6 µm of geraniin is able to increase the keratinocyte proliferation rate by 57%. a similar study showed that geraniin could increase the proliferation rate of hacat at a dose of 20 to 150 µm and induce celullar necrosis at >150 µm. increase in mitochondrial activity reflects increase of celullar energy status, which triggers the cell toward a higher proliferation rate.(12) these differences in proliferation rate and toxic dose could be attributed to differences in the purity of the geraniim used in these experiments. agyare et. al. used 5.5% geraniin derived from an aqueous extract of phyllanthus muellerianus leaves.(12) the present study uses geraniin derived from phyllanthus urinaria with a purity of >98% as determined by hplc. okabe et al. (13) showed that geraniin demonstrates inhibition of tnf-á release. tnfá is a proinflammatory cytokine that is produced by keratinocytes and is increased after uvb irradiation. recent findings show that tnf-á is able to diminish dna repair and cause cell cycle arrest.(18) these findings are in support of the ability of geraniin to increase cell viability by maintaining dna repair during periods of mitotic activity. the considerably lower population doubling in this study could be attributed to the age of the skin sources. most experiments use keratinoytes derived from neonatal foreskins, which are able to achieve 12-50 population doublings in 4 to 15 passages.(1,19,20) older sources of skin samples would be reflected by lower population doublings a n d e a r l i e r s e n e s c e n c e . t h i s s t u d y w a s figure 3. non-viable cells during routine subculture significant increase of non-viable cell number were observed at geraniin-supplemented flasks n=2 keratinocytes culture for each data set. statistical significance *p<0.01 9 univ med vol. 32 no.1 conducted using high passage keratinocytes (p56); therefore, the cells have undergone a certain extent of differentiation. in spite of this, geraniin supplementation was able to significantly increase the proliferation rate. this study was limited by the number of samples obtained. genetic differences in the population may cause different responses of keratinocytes toward geraniin supplementation. this study was also limited by the presence of s t a n d a r d k e r a t i n o c y t e s u p p l e m e n t a t i o n containing egf and bpe, both of which are potent mitogenic agents and are absolutely required to enable initial plating out of the keratinocytes. this study demonstrated the use of phytobased supplementation in cell culture systems, which are important in cell therapy and tissue engineering for the medical industry. these findings would prompt researchers in herbal study to look for other potential candidates that can be used not directly as therapeutical agent, but as a component in regenerative medicine industry. conclusion this study concludes that geraniin can be used as supplementation for keratinocyte expansion culture within optimal doses. further study should be directed to elucidating celullar mechanisms interacting with geraniin. research could also be directed to verify the effects of geraniin as a substitute for egf or bpe in keratinocyte supplementation. acknowledgements funding was provided by yarsi-hpeq grant program 006/hpeq-yarsi/ii/2012. we thank yurika sandra ulrike lichti, nimade swantari, and poengki dwi poerwantoro for valuable feedback, mahdian nur nasution for the circumcision procedures, ym lauda feroniasati, sakina l. gumilar, dwi octavia and ratih rinendyaputri for laboratory assistance. references 1. corte pd, verween g, verbeken g, rose t, jennes s, coninck ad, et al. feeder layerand animal product-free culture of neonatal foreskin keratinocytes: improved performance, usability, quality and safety. cell tissue bank 2012;13:17589. 2. coolen na, verkerk m, reijnen l, vlig m, bogaerdt aj, breetveld m, et al. culture of keratinocytes for transplantation without the need of feeder layer cells. cell transplant 2007;16:64961. 3. grinnell kl, bickenbach jr. skin keratinocytes pre-treated with embryonic stem cell-conditioned medium or bmp4 can be directed to an alternative cell lineage. cell prolif 2007;40:685-705. 4. grinnell kl, yang b, eckert rl, birkenbach jr. de-diferentiation of mouse interfollicular keratinocytes by the embryonic transcription factor oct-4. j investig dermatol 2006;127:37280. 5. sun x, fu x, han w, zhao y, liu h, sheng z. dediferentiation of human terminally differentiating keratinocytes into their precursor cells induced by basic fibroblast growth factor. bio pharm bull 2011;34:1037-45. 6. aasen t, belmonte j. isolation and cultivation of human keratinocytes from skin or plucked hair for the generation of induced pluripotent stem cells. nat prot 2010;5:371-82. 7. ohmine s, squillance k, hartjes k, deeds m, amstrong a, thatava t, et al. reprogrammed keratinocytes from elderly type 2 diabetes patients suppress senescence to acquire induced pluripotency. aging 2012;4:60-73. 8. kalyanaraman b, boyce st. wound healing in athymic mice with engineered skin subtitutes fabricated with harvested from automated bioreactor. j surg res 2009;152:296-302. 9. setyowati fm. etnofarmakologi dan pemakaian tanaman obat suku dayak tunjung di kalimantan timur. media litbang kesehatan 2010;20:104-12. 10. yuwono s. pengaruh pemberian meniran pada hati mencit yang diberi ccl4. cdk 2010;177:27881. 11. dalimartha s. atlas tumbuhan obat indonesia. jakarta: trubus agriwidaya; 2003. 12. agyare c, lectenberg m, deters a, petereit f, hensel a. ellagitannins from phyllantus muellerianus (kuntze) excell: geraniin and furosin stimulate cellular activity, differentiation, and collagen synthesis of human skin keratinocytes and dermal fibroblasts. phytomedicine 2011;18: 617-24. 10 kusuma, hadi geraniin and human keratinocytes 13. okabe s, suganuma m, imayoshi y, taniguchi s, yoshida t, fujiki h. new tnf-alpha releasing inhibitors, geraniin and corilagin in leaves acer nikoense, megusurino-ki. biol pharm bull 2001; 24:1145-48. 14. world health organization. manual for male circumcision under local anesthesia. geneva: world health organization; 2009. 15. lichti u, anders j, yuspa sh. isolation and shortterm culture of primary keratinocytes, hair follicle populations and dermal cells from newborn mice and keratinocytes from adult mice for in vitro analysis and for grafting to immunodeficient mice. nat prot 2008;3:799-810. 16. rantam fa, ferdiansyah, nasronudin, purwati, editors. stem cell exploration: methods of isolation andcculture. 1 st ed. surabaya: airlangga university press; 2009. 17. ma mt, zhang j, farooqui aa, chen p, ong wy. effects of cholesterol oxidation products on exocytosis. neurosci lett 2010;476:36-41. 18. faurschou a. role of tumor necrosis factor-a in the regulation of keratinocyte cell cycle and dna repair after ultraviolet-b radiation. dan med bull 2010;57:1-15. 19. li a, pouliot n, redvers r, kaur p. extensive tissue-regenerative capacity of neonatal human keratinocyte stem cells and their progeny. j clin invest 2004;113:390-400. 20. shariati srp, shokrgozar ma, vossoughi m, eslamifar a. in vitro co-culture of human skin keratinocytes and fibroblasts on a biocompatible and biodegradable scaffold. i b j 2009;13:169-77. rita k 168 *department of child health **department of microbiology, medical faculty trisakti university correspondence adr. h. widagdo, sp.a department of child health medical faculty trisakti university jl. kyai tapa no 260, grogol jakarta 11440 tel. 021-5672731 ext. 2705 fax 021-5660706 e-mail: widiant@centrin.net.id universa medicina 2007; 26:168-78 universa medicina oktober desember 2007oktober desember 2007oktober desember 2007oktober desember 2007oktober desember 2007 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 vol.26 no.4 clinical manifestations of upper respiratory tract infection in children at kalideres community health center, west jakarta widagdo*a, harmon mawardi*, ellen p. gandaputra*, firda fairuza*, rudy pou*, and paul bukitwetan** abstrak introduction the national household health survey showed that the incidence of upper respiratory tract infection (uri) in indonesia was high. the objectives of the study were to investigate the clinical manifestations of uri, its bacterial spectrum and sensitivity. methods a cross sectional study was carried out involving one hundred children with symptoms of uri i.e. fever, cough and or runny nose. the data of demography, physical sign, hematology, bacterial spectrum and sensitivity were collected. results the prevalence of uri was higher in male, younger age, smoker family, low educated, low income family, and polluted environment. the manifestations of uri were rhinopharyngitis (52%), pharyngitis (18%), rhinitis (12%), tonsilopharyngitis (10%), and tonsillitis (8%). the isolated bacteria were s. aureus, s. β hemolyticus, k. pneumoniae, c. diphtheriae, s. albus and s. anhemolyticus. s. aureus was higher in male than in female (p<0.01), while s. aureus, s. â hemolyticus, and c. bacterium diphtheriae were higher in preschool age children (p<0.01), and k. pneumoniae were higher in infants (p<0.01). s. aureus, and s. â hemolyticus were higher in children with under-nutrition, while in normal nutrition were of k. pneumonia and c diphtheriae (p<0.01). most bacteria were intermediate and resistant to fourteen tested antibiotics. conclusion the manifestations of uri were rhinopharyngitis (52%), pharyngitis (18%), rhinitis (12%), tonsilopharyngitis (10%), and tonsillitis (8%), each of which could be associated with the complication and accompanying disease. the pathogenic bacterial spectrum of the throat consisted of s. aureus, s. â hemolyticus, k. pneumonia, and c. diphtheriae. keywords: uri, children, clinical manifestations, bacterial sensitivity tests 169 universa medicina vol. 26 no.4 *bagian ilmu kesehatan anak, ** bagian mikrobiologi fakultas kedokteran universitas trisakti korespondensi adr. h. widagdo, spa bagian ilmu kesehatan anak fakultas kedokteran universitas trisakti jl. kyai tapa no.260 grogol jakarta barat 11410 telp. 021-5672731 eks.2705 email: widiant@centrin.net.id universa medicina 2007; 26: 168-78 latar belakang survei kesehatan rumah tangga (skrt) tahun 2001 menunjukkan angka kejadian infeksi saluran nafas atas (isna) di indonesia adalah tinggi. penelitian ini dilakukan untuk mengetahui manifestasi klinis isna serta bakteri usap tenggorok berikut uji sensitivitasnya. metode penelitian potong lintang dilakukan pada seratus pasien anak di puskesmas kalideres, jakarta barat, dengan keluhan isna berupa panas, batuk dan atau pilek. dilakukan pengumpulan data demografi, temuan fisik, laboratorium darah, biakan dan hasil uji sensitivitas. hasil kejadian isna pada anak terdapat lebih banyak pada anak laki, usia lebih muda, keluarga perokok, pendidikan rendah, kondisi ekonomi kurang, dan lingkungan berdebu. keluhan dan temuan fisik adalah demam, batuk, pilek, anoreksi, muntah, diare, sesak nafas, dan kejang. manifestasi isna meliputi rinofaringitis (52%), faringitis (18%), rinitis (12%), tonsilofaringitis (10%) dan tonsilitis (8%). penyakit penyulit dan penyerta antara lain otitis media, pneumonia, gizi-kurang, dan anemia. hasil biakan menunjukkan s. aureus terdapat lebih banyak pada anak laki dari perempuan (p<0,01), sedang s. aureus, s. â hemolitikus, dan k. difteri terdapat lebih banyak pada usia prasekolah (p<0.01). sebaliknya k. pneumonia ditemukan lebih banyak pada bayi (p<0,01). s. aureus dan s. â hemolitikus didapatkan lebih banyak pada anak gizi kurang, sedang k. pneumonia dan k difteri lebih banyak pada anak gizi baik (p<0.01). kuman memperlihatkan tingkat intermediate dan resisten terhadap 14 jenis antibiotik yang diuji. kesimpulan manifestasi klinis isna terbanyak adalah rinofaringitis (52%), faringitis (18%), rinitis (12%), tonsilofaringitis (10%) dan tonsilitis (8%). kata kunci: isna, anak, manifestatsi klinis, uji resistensi bakteri manifestasi klinis infeksi saluran nafas atas pada anak di pusat kesehatan masyarakat kalideres, jakarta barat widagdo*a, harmon mawardi*, ellen p. gandaputra*, firda fairuza*, rudy pou*, dan paul bukitwetan** abstract introduction the prevalence of upper respiratory tract infection (uri) in indonesia was 38.7% in infant, for under-five it was 42.2%, and 28.8% in children of 5 up to 14 years of age.(1) uri is an acute infections involving the nose, paranasal sinuses, pharynx, larynx, trachea, bronchi, acute 170 laryngotracheo-bronchitis (croup), epiglottitis, and otitis media.(2,3) these infections occur most common at about seven times per year among children and fall to two times per year in adult. assuming that each episode last about 4 days, then a 70 year old people may have spent about 1-2 years suffering from uri.(4) uri do not contribute significantly to deaths in children, but they cause considerable burden of disability.(2) the symptoms and signs of uri can be classified by the side from which symptoms originate and their functional effects. t h u s t h e c l i n i c a l pictures vary as does the site of the initial infection, but in some cases with uri may develop serious infections such as meningitis or pneumonia.(4) as widely reported, the most cause of uri is virus,(5,6) and the others are streptococci some of which are group a s. pyogenes, c. diphtheriae, n. gonorrhoeae, f u s o b a c t e r i a s p p . a n d s p i r o c h a e t e s , a n d chlamydia pneumoniae.(6) since the causes of uri are vary and the microbiological confirmation of diagnosis takes time, while the clinical course of the disease takes a relatively short period thence the problem arise is the establishment of diagnosis needed to treat the uri.(4) as the impact of such s i t u a t i o n i s t h e o c c u r r e n c e o f a n t i b i o t i c s overused with its bacterial resistant(2) and other u n t o w a r d e ff e c t s s u c h a s d e v e l o p m e n t o f eczema, wheeze, and allergic sensitization(7) that are difficult be hindered. the attempt of this study about uri was to investigate the clinical picture of uri including the bacterial spectrum of throat swab and its sensitivity to antibiotics. method research design a cross sectional design was carried out at kalideres community health center in west jakarta from 29 july 2003 to 9 october 2003. population and research subject the population was children who visit the c h i l d h e a l t h c l i n i c o f h e a l t h c e n t e r a t kalideres, west jakarta. three days in each week we came to the health center for recruiting maximally 4 children per day, from the visitors who had symptoms of uri. the children chosen were the first four patients who came for examinations of their uri, until a total of 100 samples were enrolled. data collections for the purpose of study the following data will be collected: the demographic data such as gender, age, parent education and monthly income, the clinical symptoms and signs included fever, cough, rhinorrhoea, sore throat, ear ache, tonsillar hypertrophy and hyperemia, pharyngeal h y p e r e m i a a n d e n g o r g e m e n t . l a b o r a t o r y measurement, included routine hematology ( h e m o g l o b i n , w h i t e b l o o d c e l l s , a n d i t s differential count), and microbiology was culture of throat swab and the sensitivity test of isolated bacteria. physical examinations the examinations of the patients were carried out on the day of visit i.e. the history t a k i n g , a n d p h y s i c a l e x a m i n a t i o n s w e r e performed by epg, ff, rp, under the supervision of hm. the clinical diagnosis of uri was made by the presence of general symptoms such as fever, cough, runny nose, headache, malaise and signs relating to the site of infection such as r h i n i t i s a r e n a s a l s e c r e t i o n , s n i f f l e s o r congestion. (8) pharyngitis was sore throat, pharyngeal hyperemia,(9) tonsillitis was swollen and hyperemic tonsil that may covered with exudates,(10) and infection of middle ear or otitis media was the presence of ear pain and bulging m e m b r a n e . ( 11 ) s i n u s i t i s w a s d i a g n o s e d a s persistent uri for 10-14 days associated with 3-4 consecutive of days purulent nasal discharge and cough.(12) widgdo, mawardi, gandaputra, et al upper respiratory tract infection in children 171 universa medicina vol. 26 no.4 laboratory examinations t h e h e m a t o l o g y e x a m i n a t i o n s w e r e p e r f o r m e d a t t h e l a b o r a t o r y d i v i s i o n o f kalideres health center, while microbiology examinations were carried out by the staffs of microbiology department supervised by pb. throat swabs were taken by scratching the tonsils and the posterior wall of the pharynx using sterile cotton buds, and avoided touching mucous membrane of the mouth. the specimens were put into the tube containing cary blair t r a n s p o r t m e d i a . t h e t u b e s c o n t a i n i n g specimens were collected in a bag containing d r y i c e , t h e n d i r e c t l y d i s p a t c h e d t o t h e department of microbiology for culture. upon t h e r e c e i p t , t h e s p e c i m e n w a s d i r e c t l y inoculated onto blood agar plate then was incubated in a candle-jar at 350 c for 24-48 hours. the colony grew on the blood agar plate was selected and identified according to the standard culture system. (13) the antibiotics sensitivity was determined by using of the disc diffusion method and the interpretation were made according to the criteria described by the national committee for clinical laboratory standards (nccls).(14) statistical analysis wi t h t h e u s e o f m i c r o s o f t e x c e l o f windows xp (2003) the relevant variables were totalized and converted to the percentage of the size of respective samples. the different of distribution of bacteria according each to gender, age, and nutrition were tested with the use of chi square test with p < 0.05 as the significant level. ethical clearance the participants were enrolled for the study with informed consent signed by their parents. the study will be initiated after the ethical clearance has been issued by the ethical committee of trisakti medical faculty. characteristics number of finding gender male (%) female (%) 58 42 child age (mo) infant (%) preschool age (%) school age (%) 39 ± 31 (4-120) 20 58 22 child number 2 ± 1 (1-6) family size 5 ± 2 (3-12) total children 2 ± 1 (1-7) education (y) father mother 9 ± 3 (0-12) 8 ± 3 (0-15) monthly income (idr 000) 544 ± 292 (100-1800) income per capita (idr 000) 135 ± 91 (11-600) table 1. demographic characteristics of the patients (n = 100) results during three months of the study a total sample of 100 children with uri visited the health center were enrolled. there were 19 patients refused the blood examinations. demographic characteristic was listed in table 1, mentioning such as gender of 58 boys and 42 girls, average age in month was 39 ± 31 (4-120 months) with the distribution as 21 infants, 57 preschool age, and 22 school age children, and the parents had low in education and social economy level. table 2 depicted the symptoms and signs such as fever, cough, runny nose, measurements, and upper respiratory findings. the study of hematologic value in 81 cases showed that the hemoglobin (g/dl) were 11.2 ± 1.0 (8.7-13.2) and prevalence of anemia was found 13.6% (11/81), mean white blood cells (103/dl) was 11.3 ± 4.9 (4.9-31.6) and the white blood cells of 10x103 or higher was found in 19% of cases, increased polymorphonuclear cells (pmnc) in 32%, and high mononuclear cells (mnc) in 54%. the erythrocyte sedimentation rate (esr) (cm/1 hr) was 20.1 ± 9.4 (1-50), and the esr higher than 10 was seen in 77% of cases. 172 table 3 showed the uri’s manifestation such as rhinopharyngitis 52%, the complication and accompanying diseases like pneumonia (3%), otitis media (2%), and under-nutrition was 49%. table 4 presented the bacterial spectrum of t h e t h r o a t c o n s i s t e d o f s . a u re u s , s . â hemolyticus, k. pneumonia, and c. diphtheriae. distribution of bacteria showed that s. aureus were higher in male than in female (p<0.01), while s. aureus, s. â hemolyticus, and c. bacterium diphtheriae were higher in preschool age children (p<0.01), on the other hand k. pneumoniae were higher in infants (p<0.01). (data not presented). table 5 showed the sensitivity of four pathogenic bacteria to 14 antibiotics being tested, and it showed that the bacteria were intermediate and resistant to all drugs, except c . d i p h t h e r i a e w h i c h w a s s e n s i t i v e t o a m o x i c i l l i n c l a v u l a n i c a c i d , c e f a d r o x i l , cefixime, and cotrimoxazol. widgdo, mawardi, gandaputra, et al upper respiratory tract infection in children findings number of cases (%) clinical manifestations rhinitis 12 tonsilitis 8 pharyngitis 18 rhinopharyngitis 52 tonsilopharyngitis 10 complications and accompanying diseases pneumonia, bronchitis 3 otitis media 2 enteritis, mild dehydration 1 febrile convulsion 1 anemia 12 under nutrition 49 tuberculosis 1 allergic diseases (rhinitis, urticaria, asthma) 4 cerebral palsy 1 table 3. clinical manifestations, complications, and accompanying diseases (n = 100) specifications findings (%) staphylococcus albus 32 staphylococcus aureus 34 streptococcus βhemolyticus 25 streptococcus γ-anhemolyticus 17 klebsiella pneumonia 20 corynebacterium diphtheriae 1 negative 7 table 4. result of the culture of the throat swab (n = 100) discussion t h i s s t u d y s h o w e d t h a t u r i m a i n l y occurred in boys than girls with the male-female ratio of 3:2, and the age of the children mainly were of preschool age, followed by school age and infant in the percentage of 57%, 22%, and 2 1 % , r e s p e c t i v e l y. s o m e s t u d i e s h a v e documented that boys had more uri than did girls and the highest age incidence was 6-23 mo.(15) kvaerner (2000) in oslo reported that symptoms and signs number of findings fever 100 runny nose 87 cough 97 anorexia 63 vomiting 34 diarrhea 10 headache 10 abdominal complaints 6 myalgia 4 convulsion 3 respiratory distress 2 respiration rate/ minute 24 ± 6 (13 40) heart rate / minute 99 ± 17 (58 146) body temperature (0c) 37.2 ± 0.8 (36 39) tonsil: enlargement & hyperemia 18 pharyngeal hyperemia 80 nasal secretion 64 submandibular swelling 2 abdominal distention/pain 8 table 2. the clinical characteristics of uri (n = 100) 173 universa medicina vol. 26 no.4 uri were common at 4 years old,(16) while other study also in oslo showed that uri was common in children of 10 years of age.(17) the factors that influenced the occurrence of uri in our studies were under-nutrition (49%), anemia (14%), large family size (65%), smoker family (74%), low level of education (73%) and low social economy (64%). studies in various country showed the predisposing factors of the e v i d e n c e o f u r i w e r e l a r g e f a m i l y s i z e , malnutrition, genetic such as down syndrome), and overcrowding(4) parental smoking,(4,15) young age,(4,15,19) pollution,(4,18) low birth weight(4,19) african/asian ethnicity and home dampness,(16) atopic disease,(16,19) and sharing bedroom with adult.(17) clinical presentation of uri depends on the site of the initial infection and the response of the mucosa such as simple necrosis that causes dryness, pain, and bleeding of the overlying vascular tissue, and the other possible response is over-secreting mucous afflicting sniffles, congestion, facial aching, and reduced hearing; the systemic symptoms may be related to direct pathogenic effects on skeletal muscles, or indirectly from metabolites of the immune response.(4) in accordance with the pathogenesis as stated by del mar (2000),(4) this study noted the clinical symptoms and signs were fever (100%), cough (97%), runny nose (87%), anorexia (63%), vomiting (34%), diarrhea (10%), dyspnea (2%), and convulsion (3%). the clinical signs were nasal secretion found in 52%, enlarged tonsil in 65% and hyperemic in 59%, and pharyngeal hyperemia in 50% of cases. then c l i n i c a l m a n i f e s t a t i o n s o f u r i w e r e a s rhinopharyngitis (52%), pharyngitis (18%), r h i n i t i s ( 1 2 % ) , t o n s i l o p h a r y n g i t i s ( 1 0 % ) , t o n s i l l i t i s ( 8 % ) , a n d t h e r e w e r e a l s o complications and accompanying diseases found in some cases (table 3). the studies abroad reported the difference in the clinical manifestations of uri such as common cold occurred in 58.3%, rhinitis 16.4%, t o n s i l o p h a r y n g i t i s 7 . 5 % a n d o t i t i s m e d i a 7.1%;(16) tonsilopharyngitis was found in 21.6%, and otitis media in 13.8%.(17) acute otitis media was the most common suppurative complications of uri, and the next were sinusitis, pneumonia, epiglottitis, peritonsilar abscess, periorbital cellulitis, and meningitis; while acute rheumatic fever and acute glomerulonephritis were another table 5. bacterial sensitivity of four microorganisms against 14 antibiotics tested 174 i m p o r t a n t c o m p l i c a t i o n s b e l o n g s t o n o n s u p p u r a t i v e t y p e o f p o s t s t r e p t o c o c c a l infection.(4) the most cause of uri was virus (40%), and the others were streptococci (25%) a third to half of which were group a s. pyogenes, less c o m m o n ( 1 2 % ) w e r e c . d i p h t h e r i a e , n . g o n o r r h o e a e , f u s o b a c t e r i a s p p . a n d s p i ro c h a e t e s , a n d c h l a m y d i a p n e u m o n i a e occupied 30% of patients with pharyngitis whenever the other cause was not found.(6) nokso-koivisto (2004) showed that the three most common viral causes of uri in children l e s s t h a n 2 y e a r s o l d w a s r h i n o v i r u s e s , e n t e ro v i r u s e s , a n d re s p i r a t o r y s y n c y t i a l viruses.(5) throat swab culture in our series showed positive in 93 cases (single bacteria in 57 and more than one in 36 cases), and negative in 7 cases. the bacteria were staphylococcus aureus, streptococcus β hemolyticus, klebsiella pneumoniae, and corynebacterium diphtheriae i n 3 4 % , 2 7 % , 2 0 % , a n d 1 % o f c a s e s , respectively, while staphylococcus albus and streptococcus non-hemolytic were found in 32% and 17% of cases. a m o n g t h e s o m a n y s p e c i e s o f staphylococcus, streptococcus, klebsiella, and corynebacterium most are normally found in the respiratory tract, but some others are pathogenic, s u c h a s s . a u re u s , s . e p i d e r m i d i s , a n d s . saprophyticus, s. pyogenes, s. dysgalactiae, and s. pneumoniae, k. pneumoniae, k. ozenae and k. rhinoscleromatis, and c. diphtheria.(20) the bacterial flora isolated from the culture of p h a r y n g e a l s w a b a n d m i d d l e e a r w e r e h . influenzae in 37-63%.(21-23) s. pneumoniae in 4058%,(21-25) non-typeable h. influenzae,(24) and m. c a t a r r h a l i s i n 2 6 8 0 % , ( 2 2 2 4 ) g ro u p a streptococci in 17% of cases.(26) s. aureus, s. pyogenes, k. pneumonia and c. diphtheria were tested for the sensitivity to 14 antibiotics, while such test was not done at s. albus and s. anhemolyticus, because the bacteria were considered as the saprophytes. the result of the sensitivity tests showed the evidence that the bacteria were of varying degree resistant to all antibiotics, except. c. diphtheria w h i c h w a s s t i l l s e n s i t i v e t o a m o x i c i l l i n clavulanic acid, cefadroxyl, cefixime, and cotrimoxazol. the study in thailand reported that 37% of streptococcus pneumoniae isolated from n a s o p h a r y n x o f c h i l d r e n w i t h u r i w e r e penicillin resistant(27) while in vietnam 39 to 87% of various pneumococcal strains were resistant to penicillin, cotrimoxazole, tetracycline, erythromycin, and cefotaxime,(28) and the report from korea mentioned that within 7 years period, there were increasing resistance rate of group a streptococci to erythromycin and clindamycin from 29 to 51% and 10 to 34%, respectively.(26) a collaborative study between eight latin american countries showed that s. pneumonia were penicillin-resistance in 30%, while to cefuroxime, cefachlor, and azithromycin, the bacteria were resistant in 9.2%, 20.8%, and 15.1% cases, respectively.(29) based on several studies it was concluded that the bacterial resistant related to the overused o f a n t i b i o t i c s i n t h e t r e a t m e n t o f u p p e r respiratory infections.(2,30-32) as much as sixteen to eighty nine percent of children with uri of whatever the causes who have visited family physician or pediatrician in various countries h a v e r e c e i v e d a n t i b i o t i c s f o r t h e i r t r e a t m e n t . ( 2 3 , 3 0 , 3 5 3 9 ) t h e r e a s o n s w h y d i d physicians over-prescribed antibiotics for r e s p i r a t o r y t r a c t i n f e c t i o n ( rt i ) , w a s o f u n c e r t a i n t y d i a g n o s i s , s o c i o c u l t u r e a n d economic pressures, prevention of secondary infection, shorten the duration of illness, lessen the illness severity, concerning over malpractice l i t i g a t i o n , a n d p a r e n t e x p e c t a t i o n s a b o u t antibiotics. ( 4 0 ) the quantitative systematic review of the treatment of uri concluded that the available evidence did not support antibiotic widgdo, mawardi, gandaputra, et al upper respiratory tract infection in children 175 universa medicina vol. 26 no.4 treatment of children with uri because of lack of efficacy and the risk of complications or illness progression.(32,34,41) the way to reduce bacterial resistance is through reducing the number of inappropriate antibiotic prescriptions given to children with uri.(30,42,43) thence the effort has been taken t h r o u g h t h e i n t e r v e n t i o n c o n s i s t i n g o f c o m m u n i t y d i s s e m i n a t i o n o f c o n s u m e r information on antibiotic use for uris and education of health professionals, resulted in statistically significant reductions in the range of 31-70% of the dispensing of 6 antibiotics.(44) in general, the recommended management for uri is symptomatic treatment that should be d i r e c t e d t o m a x i m i z e t h e r e l i e f o f m o s t prominent symptom(s), reduce the incidence of sequel such as deafness after otitis media, and minimize the inappropriate use of antibiotics in order to inhibit the development of antibiotic resistance and to converse the resources.(2) bed rest, and voice rest is necessary, and increased fluid intake are recommended for all uri. nasal decongestant, nsaid, anti-tussives a n d e x p e c t o r a n t s f r e e o f c o d e i n e , c a n b e administered to relieve the burden of uri.(2, 3) it is suggested that antibiotic treatment should be indicated for only a minority of patients with acute upper respiratory infections, included: (i) a c u t e o t i t i s m e d i a , ( i i ) m a s t o i d i t i s , ( i i i ) streptococcal pharyngitis, (iv) suppurative cervical lymphadenitis, (v) peritonsilar abscess, (vi) retropharyngeal abscess, and (vii) bacterial sinusitis.(2,45) particularly in the treatment of uri caused by group a beta hemolytic streptococci in approximately one third of cases,(34) mcisaac (1998) recommended the application of the sore throat score in rationalizing the use of antibiotics in treating the uris. to determine the score, the physician should assigns one score for each presence of the following 5 criteria: (i) history or measured temperature of > 380 c, (ii) absence o f c o u g h , ( i i i ) t e n d e r a n t e r i o r c e r v i c a l adenopathy, (iv) tonsillar swelling or exudates, and (v) age less than 15 years. the patients with a score of > 4 the have highest likelihood of getting infection, then initiation of antibiotics administration or throat swab should be taken. if the score is 2 or 3, throat swab is taken and antibiotic is given due to positive culture, and if the score is < 1, antibiotic and throat swab are not recommended. the sensitivity and specificity of the score in identifying group a streptococcus infection in children at academic center were 96.9% and 67.2%,(46) which significantly not different with the children of community based setting, i.e. 92.6% and 72.6%.(42) r e g a r d i n g t h e 5 s s c o r e o f m c i s a a c , navaz et al (2000) stated that the probability o f g r o u p a b e t a h e m o l y t i c s t r e p t o c o c c a l p h a r y n g i t i s w a s r e l a t e d t o n o d e s i z e a n d tenderness, tonsillar exudates and hypertrophy, and pharyngeal erythema and together these four predictors had a sensitivity of 71%, a specificity of 77%, and a positive predictive value of 46%, thence culture is still essential for the confirmation of the streptococci.(31) conclusion t h e m a n i f e s t a t i o n s o f u r i w e r e rhinopharyngitis (52%), pharyngitis (18%), rhinitis (12%), tonsilopharyngitis (10%), and t o n s i l l i t i s ( 8 % ) , e a c h o f w h i c h c o u l d b e a s s o c i a t e d w i t h t h e c o m p l i c a t i o n a n d a c c o m p a n y i n g d i s e a s e . t h e p a t h o g e n i c bacterial spectrum of the throat consisted of s. aureus, s. â hemolyticus, k. pneumonia, and c. diphtheriae. the bacteria were significantly different in distribution according to gender, age, and nutrition. these four pathogenic bacteria showed intermediate and resistant to 14 antibiotics tested, except c. diphtheriae w h i c h w a s s t i l l s e n s i t i v e t o a m o x y c i l i n c l a v u l a n a t e , c e f a d r o x i l , c e f i x i m e , a n d cotrimoxazol. 176 acknowledgments the authors would like to extend the appreciation and thank rector of trisakti university and dean of medical faculty for the support and providing the fund for this study. to the head of kalideres community health center we also addressed our gratitude for the permission to study and the use of facilities in health center. at last, we also highly appreciated the participation of the mothers and their children in the study. references 1. tim survei kesehatan nasional. survei kesehatan nasional 2001. laporan skrt 2001: studi morbiditas dan disabilitas. jakarta: 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widgdo, mawardi, gandaputra, et al upper respiratory tract infection in children c:\users\jurnal fkusakti\docume 181 *department of medical laboratory science school of basic medical sciences college of medical sciences university of benin correspondence: dr (mrs) e.o. osime department of medical laboratory science school of basic medical sciences college of medical sciences university of benin phone number: 08033607898 email: evarista.osime@uniben.edu date of first submission, may 7, 2018 date of final revised submission, august 23, 2018 date of acceptance, august 27, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina highly active antiretroviral therapy increases fibrinolytic and protein activity in pregnant women evarista odaburhine osime * and catherine obar * background various studies have examined optimal methods for prevention of mother to child transmission (pmtct) of human immunodeficiency virus (hiv) and subsequent outcome of response to highly active antiretroviral therapy (haart) as well as the impact of pregnancy on outcomes of hiv in the pre-haart era. little is known of the impact of pregnancy in response to haart in africa. this study is aimed to evaluate euglobulin lysis time (elt), protein c and protein s in hiv-positive pregnant women on haart. methods this was a cross-sectional study comprised of 150 participants attending ante-natal clinic (anc) in central hospital, benin city. pregnant women on haart (test subjects) (n=50, mean age 34 years), 50 pregnant newly diagnosed hiv-positive women that had not yet commenced haart (n=50, mean age 31 years) and 50 pregnant hiv-negative women (n=50, mean age 30 years) which served as controls. the elt was determined by methods described by bain, protein c and protein s were determined using enzyme linked immunosorbent assay (elisa). results there was a significant increase in elt in both pregnant women on haart and not on haart) when compared to hiv-negative pregnant women (p<0.05). there was a significant decrease in protein c in test subjects when compared with controls (p<0.05) and protein s increased significantly in hiv-positive pregnant women on haart when compared to those not on haart and hiv-negative pregnant women (p<0.05). conclusion there are changes in elt, protein c and protein s parameters with the introduction of haart in pregnancy. keywords: pregnancy, hiv, haart, pmtct, euglobulin lysis time, protein c, protein s. doi: http://dx.doi.org/10.18051/univmed.2018.v37.181-187 original article pissn: 1907-3062 / eissn: 2407-2230 september-december, 2018 vol.37 no.3 cite this article as: osime eo, obar c. highly active antiretroviral therapy increases fibrinolytic and protein activity in pregnant women. univ med 2018; 37:18 1 -7 . doi: 1 0.1 8051 /un ivmed. 2018.v37.181 -1 87 182 odaburhine, catherine highly active antiretroviral therapy introduction it has been ascertained that a significant proportion of human immunodeficiency virus (hiv)-positive adults who require antiretroviral therapy (art) are women of childbearing age.(1) more often, hiv infection in women is diagnosed for the first time during a pregnancy, and an estimated 1.38 million pregnant women in lowand middle-income countries of subsaharan africa are living with hiv.(2) human immunodeficiency virus can be transmitted from an hiv-positive mother to her child during pregnancy, childbirth and breast fee ding. mother-to-child transmission (mtct) accounts for over 90% of new hiv infections among children and an estimated 1.6 million new hiv infections among children have been prevented d ue t o t h e a va i la bi l i t y o f a nti r e tr ovi r a l medicines.(3) human immunodeficiency virus inf ection is a globa l burden and rapidly spreading. it is responsible for significant morbidity and mortality by various mechanisms and one among them is coagulation abnormalities. there are uncertainties in pathologies of coagulation abnormalities in hiv patients.(4) amongst these are hepatic damage which may be caused by the virus itself or by the administration of anti-retroviral (arv) drugs such as nevirapine which may also contribute to coagulation defects in these patients. platelets play an important role in the hemostatic process, however platelets have been shown to decrease in hiv infection due to autoimmune destruction, direct infection of megakaryocytes by the virus and consumption coagulopathy occurring in acquired immune deficiency syndrome (aids).(6) pregnancy is a state of hypercoagulation, which is an adaptive mechanism to reduce the risk of haemorrhage during and after the delivery. although a lot of studies have been carried out on the prevention of mt ct of hiv and subsequent response to haart, there is paucity of information on the impact of haart on pregnancy in africa. specifically no work has been done on elt, protein s and protein c in africa. the world health organization released new guidelines recommending lifelong art for all pregnant and breastfeeding women living with hiv.(7) the guidelines recommended option b, where lifelong art is provided to all pregnant and breastfeeding women living with hiv regardless of cd4 count or who clinical stage. the american national institute of health and o th e r o r gan iza t io n s e n c o ur a ge o f f e r i ng antiretroviral treatment to all patients with aids. un i t ed st a t e s o f a me r i ca a nd eu rop e a n guidelines strongly recommend antiretroviral drug initation if the cd4 count is less than 500 cells/mm3, with the urgency of art increasing as the cd4 count declines.(8,9) antiretroviral therapy has been found to improve haematocrit values (10-12) and presents a marked incre ase in tissue plasminogen activator antigen (t-pa) and plasminogen a c t i va t or i nh i b it o r t yp e i ( pai) l e ve l s, demonstrating that antiretroviral drugs lead to fibrinolytic changes contributing significantly to haemorrhage.(13) pregnancy is associated with widespread changes in haemostasis such that the procoagulant effect becomes dominant. these changes include increase in concentration o f mo st cl ot t i n g f a c t o r s , de c r e a s i ng c o nc e nt r ati o n s o f s o me of the n a t u r a l anticoagulants and diminishing fibrinolytic activity.(14) although a lot of studies have been carried out on the prevention of mtct of hiv and subsequent response to haart, there is paucity of information on the impact of haart on pregnancy in africa. specifically no work has been done on euglobulin lysis time (elt), protein s and protein c in africa. this study revealed the possible effect of the use of lifelong art by p r e gna n t w ome n o n so me h ae mos t a t i c parameters. methods research design this cross-sectional study was conducted in the central hospital in benin city, edo state and the central hospital in delta state which are 183 both situated in the south-south geopolitical zones. this study was carried out in central hospital pathology units and university of benin teaching hospital (ubth) pathology units between may and november 2016. research subjects this study comprised 150 participants consisting of 50 pregnant women on haart, 50 pregnant women newly diagnosed to be positive to hiv antibodies but have not commenced haart and 50 hiv-negative pregnant women who served as controls. the sample size required per group to compare means of elt at α = 0.05, power = 0.80 and effect size 0.50 was 50 per group.(15) the selection process using informed consent was initiated at the hiv prevention, treatment and care (pmtct) clinic where hiv seropositive pregnant women were enrolled after counselling for the test group and the anc for the control group over a period of seven months. the test and control participants were matched for age. lnclusion criteria were pregnant women confirmed to be hiv-positive and to have no previous history of coagulopathy, and have commenced haart. exclusion criteria were pregnant women with diabetes mellitus or hypertension. sample collection eight milliliters of venous blood was obtained from the cubital fossa of each participant using vacutainers. four milliliters each was dispensed into edta and sodium citrate vacutainers and well mixed. all samples were analysed within 4 hours post collection. laboratory analysis euglobulin lysis time (elt) was determined using the method described by bain et al. (14).to 9.5ml of 1% acetic acid in a test tube, 0.5ml of plasma was added and the tube kept at 4oc for 30 minutes to precipitate the euglobulin fraction. the tube was then centrifuged at 2000 rpm for 10 min. the supernatant was discarded and the tube was inverted to remove the acetic acid completely. the deposit was re-constituted with 0.5ml borate buffer (sodium borate 1g, sodium chloride 9g, and distilled water 100ml). the tube was pre-warmed at 37oc alongside calcium chloride (0.025m) for 2min, and 0.5ml of the calcium chloride was added to the tube containing the deposit and borate buffer. a stop watch was started after a clot was observed and the time for the euglobulin fraction to lyse completely was recorded in minutes. protein s and c were measured using elisa te chnique s, w her e anti-protein c fragments were adsorbed to polystyrene plates. the binding of serial dilutions of control and test plasma containing protein c was detected by incubation with peroxidase-labeled anti-protein cigg followed by the addition of hydrogen peroxide and o-phenylenediamine. a polyclonal antibody specific for human protein s was pre -coated onto a 96-well microplate with removable strips. protein s standard and samples were competed by a biotinylated protein s sandwiched by the immobilized antibody and streptavidin peroxidase conjugate. all unbound material was washed away and a peroxidase enzyme substrate was added. the colour development was stopped and the intensity of the colour was measured using an elisa reader. packed cell volume (pcv) was determined using the micro-haematocrit method. mixed blood sample from the edta container was introduced into a non-heparinized capillary tube filled to twothird capacity. it was flame-sealed at one end, placed in a microhaematocrit centrifuge, and spun at 10,000 rpm for 5 minutes. the volume of packed red blood cells was then read from a haematocrit reader. haemoglobin (hb) concentration was measured using the cyanmethaemoglobin method. five milliliters of drabkin’s solution (potassium cyanide and potassium ferricyanide) was mixed carefully with 0.5 ml of venous blood in a test tube, and the absorbance read at 540nm using a colourimeter. the hb concentration was then extrapolated from a standard calibration chart. univ med vol. 37 no.3 184 odaburhine, catherine highly active antiretroviral therapy statistical analysis data were analysed using the statistical package for social sciences (spss version 20) and expressed as mean ± standard deviation. student’s t test and analysis of variance (anova) were used in comparing mean values between and among groups, respectively. a post-hoc multiple comparison tukey test was used to determine which means differ. a p-value less than 0.05 at 95% confidence limit was considered as significant. ethical clearance verbal informed consent was obtained from all participants as well as ethical approval from the ethics and research committee of the edo state hospital management board, under reference number a732/t/33 and delta state hospital management board under reference number chw/ecc vol1/091. results demographic indices of hiv-positive pregnant women on haart, pregnant hivpositive women not on haart and pregnant hiv-negative women are indicated in table 1. all the participants were females within the age range of 27-36 years, of different ethnic groups and had no history of clotting disorders. the hiv-positive pregnant women on haart had a significantly higher age difference when compared to hiv-positive women not on haart and pregnant hiv-negative women. the mean systolic and diastolic blood pressure of hiv-positive pregnant women on haart and hiv-positive women not on haart was significantly higher than pregnant hiv-negative women (p=0.000; p=0.032). there was a significant reduction in pcv a nd h b i n hiv-p os it ive women o n haart a nd t hose not on haart when compared to hiv-negative pregnant women (p<0.05). the elt increased significantly (p<0.05), co agulatio n prote in c r educed significantly p<0.05 (table 2). in hiv-positive women on haart and hiv-p os i ti ve w ome n wh o h ad no t ye t commenced haart there were significant differences in pcv, haemoglobin concentration (hb) and protein c (p<0.05), while protein s was significantly reduced in pregnant women not on haart when compared to those on haart (p<0.05). there was no significant difference parameters hiv (+) women on haart (n=50) hiv (+) women not on haart (n=50) hiv (-) pregnant women (n=50) p value pcv (%) 26.6 ± 0.24 27.27 ± 0.07 34.34± 1.56 0.002 hb (g/dl) 8.87 ± 1.83 9.01 ± 0.62 11.48 ± 0.71 0.037 elt (mins) 354 ± 8.12 356 ± 11.6 296 ± 10.13 0.000 protein c(µg/ml) 1.34 ± 0.87 1.12 ± 0.75 2.02 ± 0.88 0.000 protein s (µg/ml) 13.82 ± 6.25 10.02 ±4.67 13.09 ± 5.36 0.010 table 2. distribution of studied parameters in test and control subjects variables hiv (+) pregnant women on haart (n=50) hiv (+) pregnant women not on haart (n=50) hiv-negative pregnant women (n=50) p value age (years) history of clotting disorder blood pressure (mmhg) systolic diastolic gestational age (weeks) 34.50 ± 0.70 0 134.70 ± 2.78 86.80 ± 1.46 22.12 ± 1.98 32.60 ± 0.41 0 128.68 ±1.71 81.24 ± 1.55 19.80 ± 1.46 31.40± 0.41 0 121.04 ± 2.14 79.12 ± 1.98 18.22 ± 1.04 0.000 0.000 0.032 0.672 table 1. distribution of feature variables by test and control group data presented as mean ± sd; pcv: packed cell volume, hb: hemoglobin; elt: euglobulin lysis time; hiv: human immunodeficiency virus; haart: highly active antiretroviral therapy 185 parameters mean differences p value pcv hiv(+) on haart hiv (+) not on haart hiv (-) hiv (+) not on haart hiv (-) hb hiv(+) on haart hiv (+) not on haart hiv (-) hiv (+) not on haart hiv (-) elt hiv(+) on haart hiv (+) not no haart hiv (-) hiv (+) not on haart hiv (-) protein c hiv(+) on haart hiv (+) not on haart hiv (-) hiv (+) not on haart hiv (-) protein s hiv(+) on haart hiv (+) not on haart hiv (-) hiv (+) not on haart hiv (-) -1.600 -7.800 -6.200 -1.600 -2.833 -2.233 -3.133 72.300 75.433 0.667 -0.600 -1.267 0.800 0.167 -0.633 0.004 0.000 0.000 0.003 0.000 0.000 0.952 0.000 0.000 0.017 0.035 0.000 0.000 0.597 0.001 table 3. tukey multiple comparison test note : pcv: packed cell volume, hb: hemoglobin; elt: euglobulin lysis time; hiv: human immunodeficiency virus; haart: highly active antiretroviral therapy in elt in hiv(+) on haart when compared to hiv (+) not on haart (p=0.952) (table 3). discussion human immunodeficiency virus infection is a global burden and rapidly spreading. it causes significant morbidity and mortality by various mechanisms and one among them is coagulation abnormalities. this is quite a serious complication especially in the late stage of hiv i nf e c t i on a n d t he r e a r e u n c e r ta i n t ie s i n pathogenesis of coagulation abnormalities in hiv subjects.(16) in this study, there was a significant increase in the ages of hiv pregnant women on haart and those newly diagnosed who had not yet commenced haart prior to sample collection when compared to the hivnegative women. this suggests that these groups of women are getting pregnant later than their seronegative counterpart. this may be attributed to the transient depression encountered by these wo me n w hen t he i r h iv s t a t u s i s f i r s t discovered. but as they receive counselling and monitoring, they get encouraged to want to have babies and live healthy normal lives. blood pressure was observed to increase significantly in hiv-positives on haart and those not on haart when compared to hiv seronegative wo me n. t hi s ma y be p h ys i ol ogi c a l a n d stigmatization may be a contributing factor especially in this part of the world. the transformation of the hiv epidemic over the last 20 years has been remarkable; with access to appropriate therapies, clinicians can now offer infected women a much improved prognosis as well as a very high likelihood of birthing children who will be hiv-uninfected. in this study, it was observed that hivinfected pregnant women on haart and those not on haart had significantly lower hb and pcv val ue s c ompa r e d wit h the c ontr ol s (p<0.05). although haart has been observed to increase pcv,(17) the reduced pcv observed in this study may be attributed to the hiv virus and non-compliance to the antiretroviral drugs regularly as observed from interaction with the participating women during sample collection. protein c levels were observed to reduce more in hiv-positive pregnant women not on haart ther a py tha n t hos e o n the r apy, but we re significantly reduced when both were compared univ med vol. 37 no.3 186 odaburhine, catherine highly active antiretroviral therapy to hiv seronegative pregnant women (p<0.05). this may be attributed to the large amounts of antiphospholipid antibodies produced during hiv infection which inactivates protein c.(18) protein c and i ts c ofa c t or p r ot e in s e x er t t he i r coagulation activity primarily through inactivation of coagulation factor va and viiia which are required for factor x activation and thrombin generation.(19) the catalytic activity of protein c is enhanced by the vitamin-k-dependent cofactor, deficiency of which distorts the delicate balance between procoagulant and anticoagulant proteins, thus introducing a prothrombotic state.(20) al s o in t hi s s tu d y, p r o te i n s wa s significantly increased in hiv-positive women on haart, when compared to those not on haart and hiv seronegative women. this may be attr ibuted to the direc t effect of antiretroviral therapy. there was a significant increase in elt in hiv-positive pregnant women on haart and those not on haart when compared with hiv seronegative women. this could be attributed t o t h e s id e e ff e c t s o f ha art on th o se individuals, some of which may be directly connected to hiv protease inhibitors. several studies have linked hiv protease inhibitors with the activation of endoplasmic reticulum stress and oxidative stress as well as increase in inflammatory cytokine production from several cell types including macrophages and intestinal epithelial cells. (21,22) a limitation of this study was the difficulty in having these women to participate in this study. their willingness to be enrolled for the study was very low. regular monitoring of elt, protein c and protein s levels is therefore recommended when hiv pregnant women are being treated with antiretroviral drugs. conclusion increased elt and decreased protein c levels were observed in hiv-positive pregnant women on haart and those not on haart, while protein s level is improved with the administration of haart. conflict of interest we declare that there is no conflict of interest. acknowledgement we acknowledge the management of central hospital, benin city and delta state hospital management board for granting us the ethical approval and allowing access to the patients. we are also grateful to the doctors and nurses who assisted in blood sample collection. financial disclosure this work was self-sponsored. contributors oe contributed to design supervision and write up of this manuscript. oc contributed to sample collection and analysis. all authors have read and approved the final manuscript. references 1. raman rt, manimaran p, rachakatla k, et al. study of basic coagulation parameters among hiv patients in correlation to cd4 and art status. j clin diagn res 2016;10:e-4-6. doi: 10.7860/jcdr/ 2016/17459.7718. 2. sturt as, dokubo ek, sint t. antiretroviral therapy (art) for treating hiv infection in arteligible pregnant women. cochrane database syst rev 2010;3:cd008440. doi: 10.1002/14651858. cd008440. 3. world health organization. guideline on when to start antiretroviral therapy and on pre-exposure prophylaxis for hiv. geneva: world health organization; 2015. 4. nasir ia, owolagba a, ahmad ae, et al. effects of first-line anti-retroviral therapy on blood coagulation parameters of hiv-infected patients attending a tertiary hospital at abuja, nigeria. malaysian j pathol 2016;38:103-9. 187 5. pau ak, george jm. antiretroviral therapy: current drugs. infect dis clin north am 2014;28: 371–402. doi: 10.1016/j.idc.2014.06.001. 6. simmonds mj, meiselman hj, baskurt ok. blood rheology and aging. j geriatr cardiol 2013;10: 291301. doi: 10.3969/j.issn.1671-5411.2013.03.010. 7. bositis cm, gashongore i, patel dm. updates to the world health organization’s recommendations for the use of antiretroviral drugs for treating pregnant women and preventing hiv infection in infants. med j zambia 2010;37:111–7. 8. bartlett ta, michael ft, demasi rh, et al. an updated systemic overview of triple combination therapy in antiretroviral-naive hiv-infected adults. aids;2010:20;2051-64. 9. thompson ma, aberg ja, hoy jf, et al. antiretroviral treatment of adult hiv infection: recommendations of the international antiviral society-usa panel. jama. 2012;308:387-402. doi: 10.1001/jama.2012.7961. 10. odunukwe n, idigbe o, kanki p, et al. haematological and biochemical response to treatment of hiv-1 infection with a combination of nevirapine +stavudne+lamivudine in lagos, nigeria. turkish j hematol 2009;22:125-31. 11. omoregie r, osakue si, ihemeje v, et al. correlation of cd4 count with platelet count, prothrombin time and activated partial thromboplastin time among hiv patients, benin city, nigeria. west indian med j 2009:58:437-40. 12. pintao m, garcia a, borgel d, et al. detection duplication in prost is relatively common in point mutation negative hereditary protein s deficiency. hum genet 2009:126:449-56. 13. marks km, clarke rma, bussel jb, et al. risk factors for thrombocytopenia in hiv-infected persons in the era of potent antiretroviral therapy. j acquir immune defic syndr 2009;52:595-9. doi: 10.1097/qai.0b013e3181b79aff. 14. bain bj, bates i, laffan am. dacie and lewis practical haematology. 12th ed. philadelphia: elsevier;2017. 15. browner ws, newman tb, hulley sb. estimating sample size and power: applications and examples. in: hulley sb, cummings sr, browner ws, grady dg, newman tb, editors. designing clinical research. 4th ed. philadelphia: lippincott williams & wilkins;2013. 16. bibas m, biava g, antinori a. hiv-associated venous thromboembolism. mediterr j hematol infect dis 2011;3:e2011030. doi: 10.4084/mjhid. 2011.030. 17. lbeh bo, omodamiro do, lbeh u, et al. biochemical and haematological changes in hiv subjects receiving winniecure antirethroviral drug in nigeria. j biomed sci 2013;20:73. https://doi.org/ 10.1186/1423-0127-20-73. 18. katz d, beilin y. disorders of coagulation in pregnancy .br j anaesth 2015;115 suppl 2:ii7588. doi: 10.1093/bja/aev374. 19. eyal a, veller m. hiv and venous thrombotic events. south african j surg 2009;47:93-5. 20. osime eo, onakewhor ju, kolade so. fibrinolytic changes in pregnant women on highly active antiretroviral therapy. saudi med j 2015;36:2003. 21. gahir s, anger gt, ibrahim m, et al. management of hiv positive pregnancies in ontorio: current status. canadian j clin pharmacol 2009;16:68-77. 22. danese s, vetrano s, zhang l, et al. the protein c pathway in tissue inflammation and injury: pathogenic role and therapeutic implications. blood 2010; 115:1121-30. doi: 10.1182/blood-200909-201616. univ med vol. 37 no.3 diana1 137 universa medicina juli-september 2007juli-september 2007juli-september 2007juli-september 2007juli-september 2007 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 vol.26 no.3 nyeri muskuloskeletal pada leher pekerja dengan posisi pekerjaan yang statis diana samara* *bagian anatomi fakultas kedokteran universitas trisakti korespondensi dr. diana samara, mkk bagian ilmu kesehatan kerja fakultas kedokteran universitas trisakti jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5662731 eks.2101 email: davidwp@cbn.net.id universa medicina 2007;26:137-42. abstrak nyeri leher pada pekerja pada umumnya lebih sering disebabkan oleh gangguan muskuloskletal di mana terjadi ketegangan dan peregangan otot dan ligamentum sekitar leher. sebuah studi menunjukkan prevalensi nyeri muskuloskeletal pada leher di masyarakat selama satu tahun besarnya 40% dan prevalensi ini lebih tinggi pada wanita. beberapa pekerjaan yang dapat memicu terjadinya nyeri leher antara lain bekerja dengan komputer dalam waktu yang lama atau bekerja di depan meja dengan posisi membungkuk dalam waktu lama. mengangkat, mendorong atau membawa barang, penari, dan pengemudi angkutan umum. gejala-gejala nyeri leher antara lain terasa sakit di daerah leher dan kaku, nyeri otot-otot leher, sakit kepala, dan migraine. nyeri bisa menjalar ke bahu, lengan, dan tangan disertai keluhan terasa baal atau seperti ditusuk jarum selain itu nyeri juga bisa menjalar ke kepala menyebabkan rasa sakit kepala. kebanyakan kasus nyeri leher dapat mengalami perbaikan dengan sendirinya. hal yang penting bagi pekerja yang mengalami nyeri leher adalah modifikasi pekerjaan termasuk manajemen administrasi dan pengaturan ergonomik. kata kunci: nyeri leher, posisi statik, faktor risiko, pekerja 138 samara nyeri muskuloskeletal pada pekerja neck musculoskletal among workers with static position diana samara* abstract *department of occupational health medical faculty trisakti university correspondence dr. diana samara, mkk department of occupational health medical faculty trisakti university jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5672731 ex.2101 email : davidwp@cbn.net.id universa medicina 2007; 26: 137-42. neck pain at worker is usually caused by musculoskletal disorders with strain and sprain of muscles and ligaments around the neck. a study shows that prevalence of musculoskletal neck pain in society for 1 year is 40% and prevalence of women is higher than men. some jobs which have risk to neck pain are working with computer for long time or working by sitting for long time. lifting, pushing or carrying things, dancer, truck drivers, also can caused neck pain. most of neck pain disappears without treatment. the most important for workers are job modification included administration management and ergonomic. keywords : neck pain, static position, risk factors, workers pendahuluan nyeri muskuloskeletal di leher merupakan masalah kesehatan pada masyarakat modern. sebuah studi menunjukkan prevalensi nyeri muskuloskeletal pada leher di masyarakat selama 1 tahun besarnya 40% dan prevalensi ini lebih tinggi pada wanita.(1) selama 1 tahun, prevalensi nyeri muskuloskelatal di daerah leher pada pekerja besarnya berkisar antara 6-76% d a n w a n i t a t e r n y a t a j u g a l e b i h t i n g g i dibandingkan pria.(1) di canada, sebanyak 54% dari total penduduk pernah mengalami nyeri di daerah leher dalam 6 bulan yang lalu.(2) pada perawat, prevalensi nyeri di daerah leher selama 1 tahun besarnya 45,8%. (3) yang dimaksud dengan nyeri muskuloskeletal di leher adalah rasa nyeri yang meliputi kelainan saraf, tendon, otot dan ligamen di sekitar leher. berbagai jenis pekerjaan dapat mengakibatkan nyeri leher terutama selama bekerja dengan posisi tubuh yang salah sehingga membuat leher berada dalam posisi tertentu dalam jangka waktu lama. misalkan pekerja yang sepanjang hari hanya duduk bekerja dengan komputer, dan pekerja yang sering menggunakan beban yang berat. penelitian yang dilakukan oleh palmer, et al(4) di inggris, skotlandia, dan wales pada 1 2 . 9 0 7 r e s p o n d e n b e r u m u r 1 6 6 4 t a h u n menunjukkan bahwa orang yang bekerja dengan lengan atas dan bahu lebih dari satu jam per hari m e m p u n y a i h u b u n g a n b e r m a k n a d e n g a n timbulnya nyeri leher {prevalens rasio (pr) = 1,3-1,7 pada wanita dan 1,2-1,4 pada pria}, tetapi tidak berhubungan dengan mereka yang b e k e r j a d e n g a n m e n g e t i k , m e n g a n g k a t , menggunakan alat-alat vibrasi, atau sebagai pengemudi profesional. nyeri leher yang kuat 139 berhubungan dengan responden yang sering mengeluh nyeri kepala (pr = 2,3 2,8) dan sering merasa letih atau mengalami stress (pr 2,2-2,5) daripada mereka dengan aktivitas bekerja. dari hasil penelitian ini disimpulkan bahwa nyeri leher terutama lebih disebabkan faktor psikososial daripada jenis pekerjaan yang dikerjakan. pekerja dengan gangguan nyeri pada leher memiliki jumlah waktu kerja yang hilang sama dengan pekerja yang nyeri pinggang, yang diukur dari tingkat absensi yang tinggi. (5 ) penelitian yang dilakukan alison, et al pada 1.162 perawat (dengan response rate = 74%) di baltimore, maryland menemukan bahwa 45,8% menderita nyeri leher (adjusted odds ratio = 4,33; 95% confidence interval = 2,85-6,56), 35,1% nyeri bahu (adjusted odds ratio = 4,83; 95% confidence interval = 3,00-7,77), dan 47,0% nyeri pinggang (adjusted odds ratio = 3,69; 95% confidence interval = 2,47-5,49).(6) pasien yang mengalami nyeri leher yang kronik menggunakan pelayanan kesehatan dua kali lebih banyak dibandingkan rata-rata penggunaan p e l a y a n a n k e s e h a t a n d i m a s y a r a k a t . ( 7 ) patofisiologi terjadinya nyeri muskuloskeletal di leher masih belum banyak diketahui, apakah disebabkan oleh trauma atau kondisi pekerjaan. jadi nyeri muskuloskeletal di leher bersifat multifaktorial. makalah ini bertujuan untuk membahas berbagai faktor penyebab nyeri leher, gejala-gejala, dan solusi pencegahannya. anatomi leher leher terdiri dari tujuh susunan vertebra servikal yang dimulai dari dasar kranium dan berakhir tepat di atas vertebra torakal atau setinggi batang tubuh bagian atas. vertebra servikal memiliki lengkung lordosis seperti yang t e r d a p a t p a d a v e r t e b r a l u m b a l i s . ve r t e b r a servikal lebih mudah bergerak dibandingkan vertebra lainnya.(8) tulang vertebra bersama d e n g a n l i g a m e n t u m m e n j a g a k e s t a b i l a n punggung. otot-otot yang ada di sekitar leher m e n u n j a n g d a n m e n g g e r a k k a n n y a . l e h e r memiliki gerakan yang khusus dan sangat fleksibel serta berperan menunjang berat kepala yang rata-rata 15 pounds.(9) selain itu leher melindungi saraf-saraf yang berasal dari otak dan berjalan ke seluruh tubuh. leher merupakan bagian tubuh yang perlindungannya lebih sedikit dibandingkan batang tubuh yang lain, sehingga leher lebih rentan terkena trauma atau kelainan yang menyebabkan nyeri dan gangguan gerakan terutama bila dilakukan gerakan yang mendadak dan kuat. bagi kebanyakan orang, nyeri leher merupakan kondisi yang sementara yang akan hilang dengan sendirinya. beberapa lainnya membutuhkan diagnosis dan penanganan yang tepat untuk membebaskannya dari nyeri leher tersebut. faktor risiko nyeri leher dalam pekerjaan beberapa jenis pekerjaan yang berpengaruh terhadap nyeri di leher adalah pergerakan lengan atas dan leher yang berulang-ulang, beban statis pada otot leher dan bahu, serta posisi leher yang ekstrem saat bekerja.(10) pada studi prosepektif ariens et al(11) mendapatkan bahwa pekerja yang bekerja dalam posisi duduk yang statis > 95% dari lamanya waktu bekerja per hari merupakan faktor risiko terjadinya nyeri leher. sebuah studi l o n g i t u d i n a l m e n u n j u k k a n l a m a k e r j a menggunakan tangan lebih tinggi dari bahu berhubungan dengan nyeri di leher.(12) pekerja yang sebagian besar waktunya selalu duduk menggunakan komputer mempunyai risiko lebih besar untuk mengalami nyeri leher.(13) selain dari karakteristik fisik pekerjaan, terdapat hubungan antara nyeri leher dan tuntutan pekerjaan yang tinggi, dukungan rekan-rekan kerja yang rendah, d u k u n g a n s u p e r v i s o r y a n g r e n d a h s e r t a kepuasan kerja yang rendah.(14) karakteristik individu yang merupakan faktor risiko terjadinya nyeri leher adalah usia (15) dan merokok. (16) rangkuman berbagai faktor risiko terjadinya nyeri leher disajikan pada tabel 1. universa medicina vol. 26 no.3 140 samara nyeri muskuloskeletal pada pekerja gejala-gejala nyeri leher gejala-gejala nyeri leher antara lain terasa sakit di daerah leher dan kaku, nyeri otot-otot leher yang terdapat di leher, sakit kepala dan migraine. nyeri leher akan cenderung merasa seperti terbakar. nyeri bisa menjalar ke bahu, lengan, dan tangan dengan keluhan terasa baal atau seperti ditusuk jarum. nyeri yang tiba-tiba dan terus menerus dapat menyebabkan bentuk leher yang abnormal, kepala menghadap ke sisi yang sebaliknya. yang dikenal dengan istilah torticolis.(19) n y e r i j u g a b i s a m e n j a l a r k e k e p a l a menyebabkan rasa sakit kepala pada satu sisi atau dua sisi. otot-otot leher menjadi tegang, bila disentuh terasa perih dan keras. nyeri pada dasar tengkorak bisa disertai dengan perasaan l e m a h p a d a b a h u d a n l e n g a n . b i s a j u g a dirasakan kesemutan pada lengan dan jarijari.(20) sebelum terjadi gejala-gejala tersebut di atas, pekerja perlu mengetahui adanya gejala alarm yang menyadarkan pekerja uintuk segera berobat ke dokter. gejala alarm meliputi nyeri leher yang disertai sakit kepala; nyeri dirasakan di salah satu belakang mata, terganggunya p e n g l i h a t a n , p e n d e n g a r a n , p e n g e c a p a t a u keseimbangan dan nyeri leher yang disertai dengan kekuatan otot lengan atau kaki yang melemah.(20) nyeri juga bisa menjalar ke kepala menyebabkan rasa sakit kepala pada satu sisi atau dua sisi. otot-otot leher menjadi tegang, bila disentuh terasa perih dan keras. nyeri pada dasar tengkorak bisa disertai dengan perasaan l e m a h p a d a b a h u d a n l e n g a n . b i s a j u g a dirasakan kesemutan pada lengan dan jarijari.(20) sebelum terjadi gejala-gejala tersebut di atas, pekerja perlu mengetahui adanya gejala alarm yang menyadarkan pekerja uintuk segera berobat ke dokter. gejala alarm meliputi nyeri leher yang disertai sakit kepala, nyeri dirasakan di salah satu belakang mata, terganggunya p e n g l i h a t a n , p e n d e n g a r a n , p e n g e c a p a t a u keseimbangan dan nyeri leher yang disertai dengan kekuatan otot lengan atau kaki yang melemah.(20) pemeriksaan fisik p e m e r i k s a a n d i l a k u k a n m u l a i d e n g a n palpasi pada bagian leher dan bahu pasien. karena tulang dan otot leher cukup dekat dengan permukaan, maka dokter yang berpengalaman akan dapat merasakan pembengkakan kelenjar, tumor, spasme otot, atau tonjolan yang abnormal dari vertebra. kemudian leher pasien ditolehkan tabel 1. faktor risiko terjadinya nyeri leher pada pekerja peneliti faktor risiko repetitive/recovery bernard(17) pergerakan ekstremitas atas ariens et al(10) pergerakan leher cassou et al(18) pekerjaan dengan waktu terbatas andersen et al(14) gerakan bahu > 15 kali per menit andersen et al(14) waktu istirahat terbatas akibat bekerja ≥ 80% jam kerjas posisi tubuh (posture) waktu bekerja ariens et al (11) duduk saat bekerja > 95% lama kerja ariens et al (11) fleksi leher > 20° dan > 70% lama kerja ariens et al (11) fleksi leher > 45° dan > 10% lama kerja andersen et al (1) fleksi leher > 20° dan > 66% lama kerja campeau b (13) kontraksi, beban dan posisi statik 141 ke kiri dan ke kanan untuk menentukan arah gerak dari leher tersebut. selain itu pemeriksaan mulut dan tenggorokan perlu dilakukan untuk mengetahui ada tidak gangguan pada glandula s a l i v a s e p e r t i d i t e m u k a n p a d a p e n d e r i t a parotitis.(4) pemeriksaan penunjang pemeriksaan x-ray roentgen anteroposterior /lateral pada vertebra servikalis dilakukan bila ditemukan suatu gejala serius. pemeriksaan radiologi seperti computed tomography (ct) scan atau magnetic resonance imagine (mri) bukan merupakan indikasi pada awal kasus kecuali dianggap ada masalah yang serius seperti a d a n y a g a n g g u a n p a t o l o g i s p a d a m e d u l a spinalis.(14) myelogram merupakan indikasi bila diperlukan untuk mengetahui adanya gangguan di medulla spinalis dan radix saraf. sedangkan e l e c t ro m y o g r a m ( e m g ) d i l a k u k a n b i l a merupakan indikasi untuk mengetahui gangguan pada saraf dan fungsi otot.(3) diagnosis banding diagnosis banding nyeri leher sangat rumit tidak hanya banyaknya kemungkinan tetapi juga k a r e n a k e n y a t a a n b a h w a b a n y a k p a s i e n menderita dua atau lebih gangguan pada saat yang sama.(4) pada pasien dengan nyeri yang t e r b a t a s p a d a d a e r a h i n t e r s c a p u l a r , p e r l u dipikirkan kemungkinan adanya tumor atau infeksi dari dorsal spinal. kekakuan tulang sepanjang prosesus vertebra perlu dicurigai d e n g a n p e m e r i k s a a n r o e n t g e n . ( 1 3 ) te s t elektrofisiologis dilakukan bila dicurigai adanya g a n g g u a n p a d a s a r a f s p i n a l a t a u u n t u k mengidentifikasikan kelainan otot. penatalaksanaan kebanyakan kasus nyeri leher menghilang dengan sendirinya tanpa penanganan yang berarti atau hilang dengan analgetik ringan. nyeri karena ketegangan atau keregangan dari otot maupun ligamentum tidak membutuhkan pemeriksaan dengan rontgen atau scanning. jika nyeri leher tidak berakhir setelah tiga bulan atau lebih, ini disebut nyeri leher kronik. nyeri leher k r o n i k m e m b u t u h k a n p e m e r i k s a a n d a n penanganan lebih lanjut yang lebih spesifik.(5) pengobatan secara konvensional untuk nyeri l e h e r m e l i p u t i o b a t o b a t a n , l a t i h a n f i s i k , massage, latihan otot-tot tubuh, heat packs, konsultasi ergonomi, traksi, transentameous e l e c t ro n e u ro s t i m u l a t o r ( t e n s ) , electromagneti treatment, magnetic therapy, pendidikan penderita, injeksi steroid, infrared light, ultrasound lasers, cooling spray dan strecthing.(21) untuk keluhan nyeri yang ringan dapat diberikan obat anti peradangan non steroid. namun untuk mereka yang bermasalah dengan lambungnya maupun asthma sebaiknya berhatihati dalam menggunakan obat ini.(12) pasien dianjurkan untuk menghindari duduk lama dengan leher dalam posisi menetap atau posisi ekstrem dari leher atau kepala, dan aktivitas yang menimbulkan gangguan leher, seperti menyetir yang kadang-kadang menimbulkan gerakan mendadak dan ekstrem.(13) terbukti bahwa akupuntur tidak efektif untuk mengobati nyeri leher kronik.(21) jika timbul nyeri leher, janganlah mengurangi a k t i v i t a s n o r m a l s e h a r i h a r i . j i k a p a s i e n d i b e r i k a n “ n e c k c o l l a r ” , u s a h a k a n j a n g a n memakainya lebih dari satu atau dua hari. menahan leher untuk imobilitas dalam periode yang cukup panjang, baik dengan kolar atau dengan kemampuan sendiri, akan menyebabkan nyeri yang lebih berat dan ketidakstabilan. nyeri leher akan hilang dengan sendirinya dalam s e m i n g g u . b i l a s e t e l a h s e m i n g g u m a s i h dirasakan nyeri leher, sebaiknya dikonsultasikan ke dokter.(19) universa medicina vol. 26 no.3 142 samara nyeri muskuloskeletal pada pekerja kesimpulan pekerja yang melakukan pekerjaan dengan posisi statik dalam waktu yang lama, atau posisi tubuh ektrim yang mengenai leher meningkatkan risiko terjadinya nyeri pada leher. berbagai teknik tentang cara menghilangkan stres pada leher perlu diinfomasikan kepada pekerja yang bekerja duduk secara statis pada periode yang lama. daftar pustaka 1. ariens gam, bongers pm, douwes m, miedema mc, hoogendoorn we, van der wal g, et al. are neck flexion, neck rotation, and sitting at work risk factors for neck pain? results of a prospective cohort study. occup environ med 2001; 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2000. increased matrix metalloproteinase-9 in male elderly 171 *department of clinical pathology, faculty of medicine, trisakti university, jakarta,indonesia **department of occupational medicine, faculty of medicine, trisakti university, jakarta, indonesia ***department of community medicine, faculty of medicine, trisakti university, jakarta, indonesia correspondence: dr. dr. pusparini, sppk department of clinical pathology, faculty of medicine, trisakti university jl. kyai tapa no 260, grogol, jakarta 11440, indonesia. phone: +62-21-5655786, fax: +62-21-5660706 email: pusparini_sppk@yahoo.com univ med 2016;35:171-80 doi: 10.18051/univmed.2016.v35.171-180 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina september-december, 2016september-december, 2016september-december, 2016september-december, 2016september-december, 2016 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 vol.35 no.3 increased matrix metalloproteinase-9 in male elderly with low 25-hydroxy-vitamin d pusparini*, lie tanu merijanti**, and novia i. sudharma*** background one of the extra-skeletal roles of vitamin d for health is associated with cardiovascular disease. poor vitamin d status has been associated with vascular endothelial dysfunction. there were controversial results about the association between vitamin d deficiency and matrix metalloproteinase 9 (mmp-9) concentration. the purpose of the present study was to determine the concentrations of 25(oh) vitamin d [25(oh)d] in an elderly population and to find any association between 25(oh)d and mmp-9 concentrations. methods this study was of cross-sectional design involving 160 male and female subjects aged 55–65 years, in south jakarta, indonesia. determination of mmp-9 and 25(oh)d concentrations was done concurrently on subjects who met the inclusion and exclusion criteria after all study subjects had been selected. 25(oh)d and mmp-9 concentrations were assessed by direct competitive chemiluminescence immunoassay (clia) and enzymelinked immunosorbent assay (elisa) respectively. statistical analysis used chi square and t tests. results mean 25(oh)d concentration in the study subjects was 14.4 ± 6.4 ng/ ml. a total of 68.8% of subjects had a 25(oh)d level of <20 ng/ml, and 31.2 % had a 25(oh)d level of >20 ng/ml.there was an increased mmp9 concentration in male subjects with a 25(oh)d level of <20 ng/ml compared with subjects with 25(oh)d level of >20 ng/ml (p=0.011), but not among female subjects (p=0.809). conclusion the mmp-9 concentration was increased among male subjects with low level of (oh)d. this study confirmed that 25(oh)d concentration may have a potential role in endothelial function. keywords: endothelial function, mmp-9, 25(oh)d, vitamin d deficient, elderly doi: http://dx.doi.org/10.18051/univmed.2016.v35.171-180 172 pusparini, merijanti, sudharma mmp-9 and 25(oh)d in elderly male introduction vitamin d deficiency is a health problem commonly found worldwide. vitamin d deficiency is found in around one-third to one-half of the population, from young adults to elderly. approximately 50% and 60% of elderly in north america and the rest of the world, respectively, do not have satisfactory vitamin d levels.(1) vitamin d concentrations are influenced by seasonal change, dietary intake, and clothing customs. lack of vitamin d is caused by inadequate exposure to sunlight and by dietary intakes low in vitamin d. vitamin d deficiency is most frequently associated with musculoskeletal disorders. in addition,vitamin d deficiency is also a risk factor for hypertension, various types of cancer, autoimmune diseases, type 2 diabetes mellitus, and cardiovascular diseases. (2-4) ecological studies have shown that coronary heart disease and hypertension are frequently found in regions far from the equator, with relatively low exposure to sunlight.(5,6) vitamin d receptors are widely distributed in the body, e.g. in vascular smooth muscle, vascular endothelium, and in cardiomyocytes. in vitro studies show that activation of 1,25 hydroxyvitamin d (1,25 oh d) directly inhibits renin gene expression and regulates the growth and proliferation of vascular smooth muscle cells and cardiomyocytes. studies in knockout rats prove that lack of activation of vitamin d receptors causes disorders of the renin-angiotensin system, leading to hypertension and left ventricular hypertrophy.(1,7) in the elderly, the prevalence of cardiovascular diseases increases exponentially with advancing age. the elderly have increased total cholesterol, low density lipoprotein cholesterol, and triglyceride concentrations, but slightly reduced high density lipoprotein cholesterol concentrations. in addition, there are changes in glucose metabolism, such that glucose concentrations tend to increase and insulin sensitivity to decrease. other changes are in the activation of coagulation with increasing fibrinogen and plasminogen activator inhibitor (pai-1) concentrations. in the elderly there occurs a process called endothelial dysfunction, with markers such as von willebrand factor (vwf), endothelial leukocyte adhesion molecule-1 (elam-1), intercellular cell adhesion molecule1 (icam-1), vascular cell adhesion molecule-1 (vcam-1) and matrix metalloproteinase-9 (mmp-9).(8,9) mmp-9 is one of the matrix metalloproteinases, a group of zinc-dependent extracellular enzymes, and plays a major role in the degradation of the extracellular matrix involving tissue remodelling. mmp-9 is also called gelatinase and has a molecular weight of 92kda. the mmp-9 levels are increased in several cardiovascular diseases, including hypertension, atherosclerosis and myocardial infarction.(10-13) several studies have reported on the association between vitamin d deficiency and mmp-9 concentration. the study by timm set al.(14) reported that vitamin d insufficiency is associated with increased circulating mmp-2, mmp-9 and c reactive protein (crp), correctable by supplementation of cholecalciferol. the study of wasse et al.(13) reported the finding of a significant inverse correlation between 25(oh)d concentration and mmp-9 concentration in patients with terminal renal failure. another study by shab-bidar et al.(15) reported that regular consumption of vitamin d-fortified yogurt drink (doogh) for 12 weeks resulted in a significant improvement in endothelin-1, endothelial selectin and mmp-9 in subjects with type 2 diabetes. the study of baker et al.(16) showed a significant inverse correlation between mmp-9 and vitamin d levels (r=-0.41; p=0.01). the study of lucey et al.(17) which was a randomized double blind placebo controlled trial, showed no effects of vitamin d supplementation on mmp-9 concentrations.the aim of the present study was to determine the 25(oh)d concentrations in the elderly and to find any association between 25(oh)d concentrations and mmp-9 concentrations in the elderly. 173 univ med vol. 35 no.3 methods study design this cross-sectional study was conducted from april 2013 until june 2013 at mampang prapatan district, south jakarta, indonesia. study subjects the study population used in this study comprised elderly residents of mampang prapatan district, south jakarta, aged 55-65 years and selected by simple random sampling. inclusion criteria in this study were: elderly persons aged 55-65 years, agreeing to participate in the study (by signed informed consent) after receiving explanations about the purpose of this study, ambulatory (capable of walking actively without walking aids), capable of verbal communication (capable of answering questions by themselves or aided by others). the exclusion criteria were: not being on corticosteroids, hormone replacement therapy, vitamin d supplements, not suffering from coronary heart disease, hypertension, diabetes mellitus, or stroke, ascertained from the history (anamnesis) of the subjects. to calculate the sample size, we used a previous study by wasse et al.(13) who reported the prevalence of low 25(oh)d was 0.43. the calculated sample size was 182, which was estimated using level of significance 0.05 and the precision 0.05. data collection the distributed questionnaires contained the following items: identity of respondent, health status, medical history, family history, history of habits, sports activities, sunlight exposure, clothing style, and use of sunblocking agents. study subjects who had been interviewed and met the inclusion and exclusion criteria by interview were asked to visit the health centre. the subjects were asked to fast overnight for 12 hours, previous to undergoing blood sample collection for routine laboratory tests. the following morning the subjects visited the health centre and underwent physical examinations, comprising blood sample collection and measurement of blood pressure using a mercury sphygmomanometer, pulse rate using a stopwatch, and body temperature. determination of blood pressure, temperature and pulse rate was done by 2 trained nurses. anthropometric measurements height was measured in cm using a portable microtoise with a precision of 0.1 cm, weight was measured in kg to the nearest 0.1 kg using sage portable scales. abdominal circumference was measured in cm to the nearest 0.1 cm, using a measuring tape. abdominal circumference was taken as the minimum circumference between the umbilicus and the xiphoid process and measured to the nearest 0.5 cm. body mass index (bmi) was calculated as the quotient of weight in kg and the square of height in m. determination of weight, height, and abdominal circumference was done by 2 trained nurses. laboratory analysis blood samples were collected after 10–12 hours of fasting and the laboratory tests were done on 5 ml venous blood samples. sera were collected and stored until all subjects had finished and had been screened on fasting blood glucose to rule out diabetes mellitus. determination of mmp-9 and 25(oh)d concentrations was done concurrently on subjects who met the inclusion and exclusion criteria after all study subjects had been selected. from the subjects who had fasted for 12 hours, 10 ml blood samples were collected for laboratory determination of 25(oh)d and mmp-9. the 25(oh)d concentrations were assessed by direct competitive chemiluminescence immunoassay (clia) using the diasorin liaison system and diasorin reagents (integral catalog number: en 310600liaison 36290 8/11). determination of mmp-9 concentrations was by enzyme-linked immunosorbent assay (elisa) using r&d system human mmp-9 reagents (minneapolis, mn 55413, usa, catalog number: dmp 900, lot number 307038). 25(oh)d and mmp-9 concentrations were expressed in ng/ml. 174 pusparini, merijanti, sudharma mmp-9 and 25(oh)d in elderly male ethical clearance the study was performed after approval of the procedure by the ethical committee, faculty of medicine, trisakti university, jakarta, under registration number 51/ker/fk/05/2013. at recruitment specially trained field workers explained the study protocol to the study subjects before requesting their written informed consent. statistical analysis tests on the normality of the data distribution using the kolmogorov-smirnov test showed that the data were normally distributed. subject characteristics were presented as mean and standard deviation. to evaluate factors affecting 25(oh)d concentrations in male and female subjects, the t and chi square tests were used. results a total of 225 respondents aged 55-65 years who were willing to participate in this study after receiving explanations about the purpose of this study were enrolled. among the 225 subjects who had filled in the study questionnaire, there were 52 who did not meet the criteria, among whom 17 subjects aged above 65 years, 7 subjects aged below 55 years, 2 subjects consuming antiplatelet agents such as aspirin, 2 subjects had suffered from stroke, 2 subjects were suffering from asthma, 6 subjects were suffering from heart disease, 2 subjects were suffering from goiter, 1 subject was suffering from liver disease and 13 subjects did not appear on the appointed days during the study, although they had expressed their willingness to participate in the study. there were 173 study respondents who came for physical and laboratory examinations and met the inclusion and exclusion criteria. among them were found 13 respondents with diabetes mellitus according to fasting glucose measurements, yielding 160 subjects meeting the inclusion and exclusion criteria. the flowchart of subject participation is shown in figure 1. figure 1. flow of subject participation during study 175 univ med vol. 35 no.3 characteristics of the study subjects are presented in table 1. the respondents participating in this study totalled 160 subjects, with a mean age of 59.4 ± 3.1 years. most study subjects were females (66.3%). with regard to education, the majority of the subjects, i.e. 53 respondents (33.8%), had finished primary school. most of the respondents, totalling 122 subjects (76.2%), were unemployed. the largest ethnic group in this study were the javanese with 82 subjects (51.3%). mean 25(oh)d concentration of the subjects was 14.4 ± 6.4 ng/ml, and 110 (68.8%) of subjects had a concentration of <20 ng/ml. mean mmp-9 concentration was 789.8 ± 414.7 ng/ml (table 1). in table 2 may be seen the subject activities associated with exposure to sunlight. the majority of subjects (141 subjects = 89.1%) stated that they were exposed to sunlight in the morning. the most common sunbathing period was >15 minutes in 72 subjects (51.1%). the most common sunbathing frequency per week was <3 times/ week in 72 subjects (51.1%). the most frequent sunbathing hour was before 9:00 am (90.7%). there were 93 subjects (58.1%) who used fullbody clothing and 94 subjects (58.7%) who did not use proctective measures against the sun. the mmp-9 level was significantly higher in male subjects with a 25(oh)d concentration of <20 ng/ml (p=0.011). in female subjects, bmi was significantly higher in study subjects with table 1. distribution of characteristics of study subjects (n=160) *mean ± s.d. table 2. distribution of characteristics of subject activities associated with sunlight exposure 176 pusparini, merijanti, sudharma mmp-9 and 25(oh)d in elderly male 25(oh)d <20 ng/ml (p=0.034). waist circumference was higher in females with 25(oh)d <20 ng/ml (p=0.045) (table 3). discussion the results of this study showed that the mean concentration of 25 (oh)d was 14.4± 6.4 ng/ml.this value represents the concentration of vitamin d below 20 ng/ml, indicating vitamin d deficiency. the majority of study subjects (110 or 68.8%) had 25(oh)d concentrations of ≤20 ng/ml or vitamin d deficiency, whereas 50 subjects (31.2%) had 25(oh)d concentrations of >20 ng/ml.to date there is no consensus about the optimal 25(oh)d concentration, but many experts define 25(oh)d deficiency as a 25(oh)d concentration of < 20 ng/ml. a 25(oh)d concentration in the range of 21-29 ng/ml is considered to indicate 25(oh)d insufficiency, while a 25(oh)d concentration of >30 ng/ml is termed a sufficient or normal concentration.(5,18,19) table 3. factor affecting vitamin d concentration in male and female *p value with significance at p< 0.05 (independent t test) **(chi square) mean ± s.d. the results of this study differ considerably from those of the study by setiati (20) conducted in jakarta and bekasi on 74 elderly females. the latter study showed a lower prevalence of vitamin d deficiency of only 35.1%, wereas in our study vitamin d deficiency was found in 82.8% subjects. the difference between these results may have been due to the different 25(oh)d assessment methods used. our study used direct competitive chemiluminescence immunoassay (clia) whereas setiati’s study used elisa. in addition, it has been only since the year 2009 that a standard calibrator for 25(oh)d measurement became available, such that the variability of measurements has decreased, from >30% in 1995 to 15% in 2011.(13,14) the 25 (oh)d is a difficult analyte to measure, since it is a hydrophobic molecule with several molecular forms and is strongly bound to vitamin d-binding protein (vdbp) and since there are no standardized reference materials and measurement procedures for 25(oh)d assessment.(18,21,22) 177 the study of green et al.(23) showed a prevalence of 63% for vitamin d deficiency in 18-40 year old females in jakarta. goswami et al.(24) reported a prevalence of up to 90% for hypovitaminosis d in india. these values are somewhat similar to those of our study. hypovitaminosis d in india is widely found in all age groups, including toddlers, children of schoolgoing age, pregnant women, and adult males and females in urban as well as in rural areas. this is caused by the skin type of asian indians, lack of exposure to sunlight, vegetarian habits, and absence of a vitamin d fortification program, although india belongs to countries with a hot climate and abundant sunshine.(24) in the present study, the high prevalence of 25(oh)d deficiency may have been due to the fact that on average the subjects took sunbaths for <15 minutes in the morning only, with 58.1% using full-body clothing and 41.3 % using protective measures against the sun (sun blocking agents and umbrellas). sunlight in the morning contains more ultraviolet a (uv-a) than ultraviolet b (uv-b) such that only small amounts of vitamin d are produced by the skin. the best time for uv-b irradiation is between 10.00 to 15.00. the use of full-body clothing and headscarfs also decreases the skin vitamin d producing capacity.(5,19) in addition, on average the subjects of this study, as do the majority of indonesians, seldom consume vitamin d containing foods, such as cow’s milk, butter, shrimps, salmon and sardines, all of which are not widely consumed by indonesian communities.(5) the price of vitamin d-containing foods tend also to be high, such that indonesians have difficulties in obtaining dietary sources of vitamin d. vitamin d in the circulation is found in two major forms, i.e. 25(oh)d, which is biologically inert, and 1,25(oh) 2 d, which is the biologically active form. in this study the concentrations of 25(oh)d were determined because 25(oh)d is the most abundant form of vitamin d in the circulation and 25(oh)d concentration is the best indicator of vitamin d status in the body.(18,25) the half life of 25(oh)d is 2-3 weeks, longer than that of 1,25(oh) 2 d, which has a half life of 4 hours only. the active form of vitamin d, i.e. 1,25(oh) 2 d, is not a good indicator for representing the vitamin d reserves in the body, because its concentrations are influenced by calcium concentrations and particularly decrease in cases of vitamin d deficiency, and more accurately reflect abnormalities of renal function.(25) in vitamin d deficiency the levels of parathyroid hormones are increased, subsequently inducing increased activity of 1á-hydroxylase, such that the concentration of 1,25(oh) 2 d becomes normal or is increased. in addition, its concentration is very low in the blood, being 1001000 fold lower than that of 25(oh)d, such that it is difficult to detect.(25,26) the factors influencing 25(oh)d concentrations were bmi and waist circumference in females, while mmp-9 concentration was a factor affecting 25(oh)d concentrations only in male subjects (table 3). with regard to bmi it may be seen that 83 subjects (51.9%) had bmi values of more than 25 kg/m2, which indicates obesity. in this study it was found that in female subjects the 25(oh)d concentration was significantly lower in subjects with higher bmi, but not in male subjects. vitamin d is a fat-soluble vitamin such that with increasing body fat more vitamin d is stored, thus reducing the circulating vitamin d levels. deposition of excess fat in the body depends on the location of the body fat, which may have an adverse influence on vitamin d status. obesity is more prevalent in women than in men. adipose tissue acts as a metabolic well for vitamin d, therefore obesity may decrease the bioavailability of vitamin d.(27-29) obesity is associated with increased risk of vitamin d deficiency. the inverse association between obesity and serum vitamin d concentration may have been caused by vitamin d deposition in the body fat compartment, by decreased release of vitamin d into the systemic circulation and by low sunlight exposure. increases in the number of subcutaneous and visceral adipocytes is associated with decreased vitamin d concentration.(27,29) the study conducted by univ med vol. 35 no.3 178 pusparini, merijanti, sudharma mmp-9 and 25(oh)d in elderly male salekzamani et al.(28) suggested an inverse association of vitamin d status with insulin resistance, the metabolic syndrome, and type 2 diabetes mellitus. serum levels of 25(oh)d are the best indicators to determine overall vitamin d status, since 25(oh)d is the most abundant form of vitamin d found in the human body and its concentrations are 1000-fold higher than those of 1,25 dihydroxy-vitamin d [1,25(oh) 2 d]. the circulation of 25(oh)d is inversely associated with abdominal obesity, hypertriglyceridemia and hyperglycemia.(28) mmp-9 belongs to a family of zincdependent proteases and has extracellular endopeptidase activity. the substrates for mmp9 are the extracellular matrix proteins and adhesion proteins. mmp-9 is important in the extracellular remodeling process and is the principal effector molecule of inflammatory cells. it also plays a key role in vascular diseases, including hypertension, and in the formation of aneurysms. in the present study the mean mmp9 concentration was 789.8 ± 414.7 ng/ml (table1). the study by welsh et al.(30) on patients with coronary heart disease showed a mean mmp9 concentration of 741.3 ± 274.7 ng/ml, whereas that of controls was 691.8 ± 245.0 ng/ml.the mean mmp-9 concentration in the controls of welsh’s study (30) was almost identical with the mean of 659.2 ± 262.9 ng/ml found in the present study among subjects with normal vitamin d concentrations (data not shown). on the other hand, in male subjects with 25(oh)d concentrations of <20 ng/ml, mean mmp-9 concentration was 909.0 + 455.6 ng/ml and in subjects with 25(oh)d concentration of >20 ng/ ml the mean mmp-9 concentration was 621.9 + 338.7 ng/ml (table 3). there was a significantly higher mean mmp-9 concentration in male subjects having deficient 25(oh)d concentrations in comparison with subjects having insufficient and normal vitamin d concentrations, although this difference was not found in female subjects. this statistically non-significant result in females may have been caused by the higher bmi in female subjects as compared with male subjests. mmp-9 is frequently associated with a susceptibility to vascular plaque rupture. the study of wasse et al.(13) reported the finding of a significant inverse correlation between 25(oh)d concentration and mmp-9 concentrations (r=0.29; p=0.004) in patients with terminal renal failure. in the study of wasse et al.(13) the mean mmp-9 concentration in all patients was 975.2 ± 662.9 ng/ml.the mean mmp-9 concentration in subjects with 25(oh)d concentrations of <15 ng/ ml was higher, namely 1155.7 ± 666.3 ng/ml, and in subjects with 25(oh)d concentrations of ≥15 ng/ml were lower, i.e. 839.8 ± 593.9 ng/ml with p=0.01. in wasse’s study (13) the subjects involved were subjects with terminal renal failure, whereas in our study the subjects were persons in normal health. although the results of wasse’s study showed a correlation, the correlation was low, being below 0.4. in contrast, the study of lucey et al.(17) which was a randomized double blind placebo controlled trial, showed no effects of vitamin d supplementation on mmp-9 concentrations. the factor with the strongest influence on 25(oh)d concentrations was time of contact with sunlight, but in our study this factor did not show any significant influence both in male and female subjects, which agrees with the pathophysiology of vitamin d synthesis. in this study most of the male and female subjects had a sunbathing frequency of <3 times/week and their sunbathing time was before 9:00 am. the main source of vitamin d is its synthesis in the skin, which is affected by exposure to uv-b radiation, initiating vitamin d synthesis with the formation of double bonds in the b ring, leading to opening of the b ring and the formation of previtamin d that is no longer rigid.(19,31) ultraviolet irradiation of the skin is measured as the minimal erythema dose (med) or the total amount of uv irradiation causing minimal erythema of the skin. exposure to 1 med is estimated to release 10.000 to 20.000 iu vitamin d into the circulation for 24 hours.(32) exposure of 40% of the body surface to ¼ med results in the formation of 100 iu vitamin d per day. these 179 values are minimum values of vitamin d synthesis required by the body. time of exposure to sunlight in the us to obtain 1 med at midday in the summer is 4-10 minutes for light-skinned individuals and 60-80 minutes for dark-skinned individuals.(32) vitamin d intake in this study was not associated with 25 (oh) concentration, both in male and female subjects. our study subjects were in the low vitamin d intake category, as compared with the recommended vitamin d intake. in indonesia vitamin d-containing foods are seldom consumed, because those foods are more expensive. one of the limitations of this study was the cross-sectional study design. in addition, it is necessary to conduct an experimental study on subjects receiving vitamin d supplementation. the clinical implication of this study is that the community and health personnel in areas of with high sunshine levels such as indonesia should be alert to the necessity of sunlight exposure as a source of vitamin d, since this study found 2 5 ( o h ) d d e f i c i e n c y i n t h e m a j o r i t y o f respondents. conclusion a deficiency of 25(oh)d was found in the majority (68.8%) of the subjects. the mmp-9 concentration was increased among male subjects with low 25(oh)d concentration. conflict of interest we hereby assure that the manuscript a) is an original work, b) has not been previously published in whole or in part, and c) is not under consideration for publication elsewhere. all authors have disclosed any actual or potential competing interests regarding the submitted article and the nature of those interests. all authors a) have read the manuscript, b) agree that the manuscript is ready for submission to the journal, and c) accept responsibility for the manuscript’s contents. acknowledgements we thank the participants in this study for their cooperation. this study was supported by the faculty of medicine, trisakti university, jakarta, indonesia. references 1. ku yc, liu me, ku cs, et al. relationship between vitamin d deficiency and cardiovascular disease. world j cardiol 2013;26:337-46. 2. alyami a, soares mj, sherriff jz, et al. vitamin d & endothelial function. indian j med res 2014;140:483-90. 3. norman pe, powell jt. vitamin d and cardiovascular disease. circ res 2014;114:37993. 4. mozos i, marginean o. links between vitamin d deficiency and cardiovascular diseases. biomed res int 2015;109275. doi:10.1155/ 2015/109275. 5. holick m. vitamin d deficiency. n eng j med 2007;357:266-81. 6. anderson jl, may ht, horne bd, et al. relation of vitamin d deficiency to cardiovasvular risk factors, disease status, and incident events in a general healthcare population. am j cardiol 2010;106:963-8. 7. wang tj, pencina mj, booth sl, et al. vitamin d deficiency and risk of cardiovascular disease. circulation 2008;117:503-11. 8. upadhyay rk. emerging risk biomarkers in cardiovascular diseases and disorders. j lipids 2015. article id 971453. doi: 10.1155/2015/ 971453. 9. teixeira bc, lopez al, macedo rco, et al. inflammatory markers, endothelial function and cardiovascular risk. vasc bras 2014;13:108-15. doi: 10.1590/vb.2014054. 10. yabluchanskiy a, ma y, iyer rp, et al. matrix metalloproteinase-9: many shades of function in cardiovascular disease. physiology 2013;28:391403. 11. garvin p, nilsson l, carstensen j, et al. circulating matrix metalloproteinase is associated with cardiovascular risk factors in a middle aged normal population. plos one 2008;3:e1774. doi: 10.137/journal.pone. 0001774. 12. jablonski kl, chonchol m, pierce gl, et al. 25-hydroxyvitamin d deficiency is associated with inflammation-linked vascular endothelial univ med vol. 35 no.3 180 pusparini, merijanti, sudharma mmp-9 and 25(oh)d in elderly male dysfunction in middle aged and older adults. hypertension 2011;57:63-9. 13. wasse h, cardarelli f, de staercke c, et al. 25hydroxyvitamin d concentration is inversely associated with serum mmp-9 in a cross sectional study of african american esrd patients. bmc nephrol 2011;12:24. doi: 14712369/12/24. 14. timms pm, mannan n, hitman ga, et al. circulating mmp-9, vitamin d and variation in the timp-1 response with vdr genotype: mechanisms for inflammatory damage in chronic disorders? q j med 2002;95:787-96. 15. shab-bidar s, neyestani tr, djazayery a, et al. regular consumption of vitamin d fortified yogurt drink (doogh) improved endothelial biomarkers in subjects with type 2 diabetes: a randomized double blind clinical trial. bmc medicine 2011;9:125. doi: 1741-7015/9/125. 16. baker a, wood cl, timms pm, et al. changes in vitamin d and matrix metalloproteinase-9 in submariners during a submerged patrols. occup environ med 2014;71:104-8. doi:10.1138/ oemed-2013-101793. 17. collins a. practice implications for preventing population vulnerability related to vitamin d status. j am ass nurse prac 2013;25:109-18. 18. wielders jpm, carter gf, ebert h, et al. automated competitive protein binding assay for total 25(oh) vitamin d, multicenter evaluation and practical performance. j clin lab anal 2014;00:1-14. doi 10.1002/jcla.21793. 19. mcgreevy c, williams d. new insights about vitamin d and cardiovascular disease: a narrative review. ann intern med 2011;155:820-6. doi: 10.7326/0003-4819-155-12-20112202-00004. 20. setiati s. vitamin d status among indonesian elderly women living in institutionalized care units. acta med indones 2008;40:78-83. 21. carter gd. 25-hydroxyvitamin d: a difficult analyte. clin chem 2012;58:486-8. 22. holmes ew, garbincius j, mckenna km. analytical variability among methods for the measurement of 25 hydroxyvitamin d. am j clin pathol 2013;40:550-60. 23. green tj, skeaff cm, rockell jep, et al. vitamin d status and its association with parathyroid hormone concentrations in women of child bearing age living in jakarta and kuala lumpur. eur j clin nutr 2008;62:373-8. 24. goswami r,ishra sk, kochupillai n. prevalence & potential significance of vitamin d deficiency in asian indians. indian j med res 2008;127: 229-38. 25. fraser wd, milan am. vitamin d assays: past and present debates, difficulties and developments. calcif tissue int 2013. doi: 10.1007/s00223-012-9693-3. 26. khan ka, akram pj, fazal m. hormonal actions of vitamin d and its role beyond just being a vitamin: a review article. int j med sci 2011;3: 65-72. 27. johnson lk, hofso d, aasheim et, et al. impact of gender on vitamin d deficiency in morbidity obese patients : a cross sectional study. eur j clin nutr 2012;66:83-90. 28. salekzamanis, neyestani tr, alavi-majd h, et al. is vitamin d status a determining factor for metabolic syndrome? a case control study. diabetes metab syndr obes 2011;4:205-12. 29. cheng s, massaro jm, fox cs, et al. adipocytes, cardiometabolic risk, and vitamin d status: the framingham heart study. diabetes 2010;59:2428. 30. welsh p, whincup ph, papacosta o, et al. serum matrix metalloproteinase-9 and coronary artery disease: a prospective study in middle aged men. qjm 2008;101:785-91. 31. lucey a, hill t, muldowney s, et al. no effect of vitamin d supplementation on circulating concentration of matrix metalloproteinase-9 (mmp-9) and tissue inhibitor of metalloproteinase-1 (timp-1) in adults aged 2040 and 64 years. proceed nutr soc 2009;68: e151. doi: 10.1017/s0029665109991078. 32. holick mf. sunlight and vitamin d for bone health and prevention autoimmune disease, cancers and cardiovascular disease. am j clin nutr 2004;80:1678s-88s. asiaticoside increases aquaporin-3 protein 25 *department of dermatovenereology, faculty of medicine, tarumanagara university correspondence: dr. dr. linda yulianti wijayadi, sp.kk., finsdv department of dermatovenereology faculty of medicine, tarumanagara university jl. letjen s. parman no. 1 jakarta barat 11440 email: lindajuliantiwijayadi@gmail.com univ med 2017;36:25-33 doi: 10.18051/univmed.2017.v36.25-33 pissn: 1907-3062 / eissn: 2407-2230 received october 17, 2016 accepted for publication april 7, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract asiaticoside increases aquaporin-3 protein expression in the cytoplasm of normal human epidermal keratinocytes linda yulianti wijayadi and hari darmawan introduction skin hydration decreases with aging. aquaporin-3 (aqp3) is a major protein that plays a role in skin hydration, therefore it is a novel target for skin moisturizing treatment. retinoic acid (ra) as a well-known active agent in antiaging treatment increases aqp3 expression, but frequently causes harmful side effects. asiaticoside, a saponin compound isolated from centella asiatica (ca) is also known as an antiaging cosmetic and plays a role in wound healing. the aim of this study was to evaluate and compare the effect of asiaticoside isolated from ca and the effect of ra on the aqp3 expression in normal human epidermal keratinocytes (nheks). methods an experimental laboratory study was performed using primary nheks that were derived from the foreskin of a boy. aqp3 expression in nheks was examined in vitro after the cells were incubated for 24 hours with asiaticoside or with ra at several concentrations. the aqp3 expression was evaluated by immunocytochemistry and quantitatively analyzed by image-j software. independent t-test and one-way anova were used to analyze the data, followed by post-hoc tukey test. results there was an increasing trend of aqp3 expression upon exposure to asiaticoside at all concentrations compared to the control group. however, ra exposure seemed to induce a higher level of aqp3 expression. asiaticoside effected a lower increase in aqp3 expression in nheks than did ra (p=0.042). optimal results were achieved at 1 mg/ml concentration of asiaticoside. conclusions asiaticoside isolated from ca can enhance the aqp3 expression in nheks. therefore it can be used as an active ingredient in cosmetic moisturizer formulation for dry skin treatment. keywords centella asiatica, asiaticoside, keratinocytes, aquaporin-3, retinoic acid doi: http://dx.doi.org/10.18051/univmed.2017.v36.25-33 universa medicinajanuary-april, 2017 vol.36 no.1 original article 26 wijayadi, darmawan asiaticoside increases aquaporin-3 protein introduction skin aging is a complex process controlled by genetic determinants (intrinsic aging) and under the influence of external factors (extrinsic aging).(1,2) the aging process reduces skin surface hydration, which then results in dry, dull, coarse and saggy skin. other visible signs of aging include pigmentation and the appearance of benign skin tumors. moreover, the epidermis becomes thinner with age and develops wrinkles as a result. the aquaporins (aqps) are a family of small transmembrane proteins that facilitate osmotically driven water transport. to date, 13 aqps (aqp 0-12) have been cloned from humans and are categorized into distinct subgroups in terms of their amino acid sequence and molecular functions.(3) aquaporin-3 (aqp3) as a water and glycerol channel is the key factor in the skin hydration mechanism as well as in keratinocyte proliferation and differentiation.(4,5) however, aqp3 expression in normal human epidermal keratinocytes (nheks) decreases along with the aging process. natural active compounds that can stimulate aqp3 expression would be effective as hydrating agents or moisturizers in anti-aging cosmetics.(6) therefore, aqp3 is a key protein target for future anti-aging treatment, in order to improve hydration, texture, and quality of the skin surface.(6) cosmeceutical anti-aging formulations contain active ingredients to achieve local therapeutical effects, which should not have harmful local and systemic side effects.(7) the well known anti-aging ingredients, retinoic acid (ra) and its derivatives, have harmful side effects by irritating the skin and causing dry, flaky skin, which is sensitive to light.(8) thus, it is necessary to develop innovative cosmeceutical anti-aging formulations with active ingredients which are effective, stable, safe and nontoxic. additionally, these ingredients should be compatible with other active ingredients and be delivered efficiently to the target cells.(9) a study found that all-trans retinoic acid attenuates ultraviolet radiation-induced down-regulation of aquaporin-3 and water permeability in human keratinocytes.(10) another study showed that trans-zeatin attenuates ultraviolet induced downregulation of aquaporin-3 in cultured human skin keratinocytes.(11) indonesian natural herbs promise a great potential for herbal cosmetics. centella asiatica (ca) (s y n o n y m : h y d ro c o t y l e a s i a t i c a), commonly known as gotu kola, is often found in indonesia and has been used for various medicinal purposes such as wound healing, treatment of asthma, ulcers, leprosy, lupus erythematosus, psoriasis, venous disorders, and c a n c e r s i n c e 1 0 0 0 y e a r s a g o . ( 1 2 1 4 ) t h e biologically active ingredients in ca are triterpenes, namely asiatic acid, madecassic acid, asiaticoside and madecassoside.(13,14) asiaticoside isolated from ca induces human dermal fibroblast (hdf) proliferation and type i and iii collagen synthesis in a timeand d o s e d e p e n d e n t p a t t e r n . ( 1 5 ) t h e r e f o r e , asiaticoside as an active component isolated from ca can improve wound healing.(16) asiaticoside displays the highest activity and has been widely used in wound healing treatment and anti-aging cosmetics, hence it also can be used as active mosturizing agent.(15) the study conducted by julianti et al.(17) showed that ca ethanolic extract encapsulated into chitosan nanoparticles can be used as moisturizer for antiaging activity by i n d u c i n g a q p 3 e x p r e s s i o n i n n h e k s . unfortunately there is a lack of research exploring the efficacy of asiaticoside as an active agent in ca that can increase aqp3 expression in nheks.(17) asiaticoside possesses interesting wound healing and angiogenic activities that are employed to stimulate tissue regeneration in wound healing applications. one study attempted to develop a chitosan-aluminum monostearate (alst) composite sponge containing asiaticoside for use as an absorbent medical dressing in c h r o n i c w o u n d s . t h e s t u d y f o u n d t h a t asiaticoside and asiaticoside-containing dressings 27 univ med vol. 36 no.1 exhibited dose-dependent angiogenic activity in the chick-chorioallantoic membrane model. therefore chitosan-aluminum monostearate c o m p o s i t e s p o n g e d r e s s i n g s c o n t a i n i n g asiaticoside may be of benefit for wound healing a n d a n g i o g e n e s i s p r o m o t i o n i n c h r o n i c wounds.(18) there has been no studies on the expression of aqp3 in nheks after treatment with asiaticoside as compared with that of ra. the aim of this study was to evaluate and compare the effect of asiaticoside isolated from ca and that of ra on the expression of aqp3 in nheks. methods research design an experimental laboratory study was conducted in the eijkman institute, jakarta, from january to april 2015. treatment ra was purchased from basf, germany, and chitosan with a deacetylation degree of >75% from sigma-aldrich, usa. asiaticoside was prepared in the center for pharmaceutical and medical technology, agency for the assessment and application of technology, center for research in science and technology (bppt puspiptek), serpong. ra and asiaticoside in step wise dilutions from 1 mg/ml, 0.5 mg/ml, to 0.25 mg/ml were used to determine aqp3 expression by immunocytochemistry. tissue samples and cell culture primary nheks were derived from the foreskin of a boy and cultured in roswell park m e m o r i a l i n s t i t u t e ( r p m i ) m e d i u m supplemented with 10% fetal bovine serum (fbs) and 1% penicillin-streptomycin. nhek cell cultures were prepared in the integrated laboratory of yarsi university, jakarta. immunocytochemistry i m m u n o c y t o c h e m i s t r y ( i c c ) w a s performed at eijkman institute, jakarta. icc is a specific cellular protein detection technique using a specific antibody (ab) to recognize cellular surface antigens. immunochemical staining was done using anti-aquaporin3 antibody ab125219. expression of aqp3 in nheks was then quantitatively analyzed by imagej software. statistical analysis data obtained in this study are presented as images, tables or graphs. to assess for significance across the different treatment groups, independent t-test and one-way anova were used, followed by post-hoc tukey test. the significance level was set at p<0.05 using spss software version 21. results aqp3 protein expression in keratinocytes following 24 hours exposure to asiaticocide in order to examine the expression of aqp3 protein, an immunocytochemical test with antiaquaporin3 antibody (ab125219) was done and visualized with the light microscope to obtain qualitative data. in this study we used three different concentrations of asiaticoside, i.e. 0.25, 0 . 5 , a n d 1 m g / m l . t h r e e d i ff e r e n t r a concentrations, namely 0.25, 0.5, and 1 mg/ml, w h i c h a r e c o m m o n l y u s e d i n t o p i c a l preparations, were applied as controls in this study. the study indicated an increased expression of aqp3 protein in keratinocytes upon exposure for 24 hours to asiaticoside at three different concentrations, namely 0.25 mg/ml, 0.5 mg/ml, and 1 mg/ml. the expression of aqp3 protein as indicated qualitatively increased linearly in relation with the concentrations of asiaticoside (figure 1). we found an increasing expression of aqp3 protein in nheks following 24 hours exposure to ra at three different concentrations (0.25 mg/ ml, 0.5 mg/ml, and 1 mg/ml). aqp3 protein expression increased proportionally with ra concentration (figure 2). 28 wijayadi, darmawan asiaticoside increases aquaporin-3 protein figure 1. aqp3 protein expression (brown colour, pointed by arrows) in nheks: (a) without treatment (control); and after 24 h exposure to asiaticoside at concentrations of: (b) 0.25 mg/ml; (c) 0.5 mg/ml; and (d) 1 mg/ml, at 400x magnification. immunocytochemical staining with anti-aquaporin3 antibody ab125219 figure 2. aqp3 protein expression (brown colour, pointed by arrows) in nheks: (a) without treatment (control); and after 24 h exposure to ra at concentrations of: (b) 0.25 mg/ml; (c) 0.5 mg/ml; and (d) 1 mg/ml, at 400x magnification. immunocytochemical staining with anti-aquaporin3 antibody ab125219 29 univ med vol. 36 no.1 comparison of aqp3 protein expression in nheks following 24 hours exposure to asiaticoside and ra using imagej software t h e q u a l i t a t i v e r e s u l t s f r o m t h e immunocytochemical analysis in the form of images were analyzed using the imagej program to obtain quantitative results for statistical analysis. following previous qualitative analysis, aqp3 expression was found to be increased with b o t h i n c r e a s i n g a s i a t i c o s i d e a n d r a concentration. qualitative analysis of different color densities using imagej program showed that expression of aqp3 was significantly highest on ra exposure as compared to asiaticoside (1 mg/ml) (table 1). there was an increasing trend of aqp3 expression upon exposure to asiaticoside at all concentrations compared to the control (no exposure) group. however, ra exposure seemed to induce a higher level of aqp3 expression (figure 3). figure 3. mean aqp3 expression in keratinocytes following 24 hours exposure to asiaticoside and ra using imagej program table 1. aqp3 expression after 24 hours exposure by treatment groups note: one way anova between control, asiaticoside and ra is statistically significant at all concentrations (p<0.05); post hoc tukey analysis between ra (1 mg/ml) and control is statistically significant (p=0.010); post hoc tukey analysis between ra (0.5 mg/ml) and asiaticoside (0.5 mg/ml) is statistically significant (p=0.024); post hoc tukey analysis between asiaticoside (1 mg/ml) and control is statistically significant (p=0.037) 30 wijayadi, darmawan asiaticoside increases aquaporin-3 protein discussion after 24 hours of incubation with three different asiaticoside concentrations (0.25 mg/ ml, 0.5 mg/ml, and 1 mg/ml), an increased expression of aqp3 protein in the cytoplasm of nheks was found. however, the expression of aqp3 protein was reduced upon exposure to the highest asiaticoside concentration (1 mg/ml). this result shows that the relation between e x p r e s s i o n o f a q p 3 a n d a s i a t i c o s i d e concentration is linear within a certain range. other studies showed that concentration and exposure time were important factors in determining the expression of aqp3 protein. pereda et al.(19) showed that green coffea arabica seed extract may increase aqp3 protein expression in normal human epidermis. there was a 6.5 fold increase in aqp3 mrna expression compared to negative controls following a 3–6 hours incubation with the optimal concentration of 25 mg/ml. another study done by pereda et al.(20) regarding the induction of aqp3 protein with piptadenia colubrina extract (10 and 20 mg/ml) in human skin cell cultures showed a statistically significant increase in aqp3 mrna expression, analyzed using realtime pcr. aqp3 started to increase after 2 hours and peaked at 6 hours, then further declined after 24 hours. in our study, the evaluation of increased a q p 3 p r o t e i n e x p r e s s i o n u s i n g immunocytochemistry was done after 24 hours of exposure to asiaticoside, as we hoped that the topical treatment can eventually be done once daily with its effect remaining up to 24 hours. this concept is similar to those of previous studies done by pereda et al.(20) and perez et al.(21) the latter evaluated the effect of 3% aquaxyltm solution on aqp3 expression in keratinocytes after 24 hours exposure, because a moisturizing cream should have a long lasting effect. aqp3 protein, as a water and glycerol channel, acts to transport water and glycerol to the skin, thus holding an important aspect in skin hydration.(4,22) a further study done by li et al.(23) regarding the reduced expression of aqp3 in keratinocytes and its relation to age indicates that aqp3 holds an important aspect in maintaining skin hydration and is reduced following an aging process.(24,25) aqp3 plays an important role in wound healing, since it acts as a water channel that helps the migration of nheks, while acting further to transport glycerol which may increase the p r o l i f e r a t i o n a n d d i ff e r e n t i a t i o n r a t e o f nheks.(26) a previous study done by ji et al.(11) showed that trans-zeatin increased aqp3 protein e x p r e s s i o n i n n h e k s , d e p e n d i n g o n i t s concentration and time of exposure, with the most optimal condition at 48 hours and 80 mol concentration. nowadays, there are many cosmetic moisturizers that are formulated with some active agent and claimed to increase the expression of aqp3 protein in nheks of the epidermis. the fact of increased expression of aqp3 is crucial in increasing resistance, skin barrier integrity, quality of the skin surface and in improving skin hydration. asiaticoside is one of the active components in ca. our in silico research has previously showed that asiaticoside has the highest bond energy in comparison to the other active ingredients in ca (asiatic acid, madecassic acid and madecassoside), at the same binding site at 234 alanine in aqp3.(27) the results of the present study indicated that aqp3 protein expression increased in n h e k s a f t e r 2 4 h o u r s t r e a t m e n t w i t h asiaticoside at concentrations of 0.25, 0.5 and 1 mg/ml. aquaporin-3 expression was 1.5 times greater as compared with negative controls, and the optimal concentration was found at 1 mg/ ml. these results indicate that asiaticoside can be used as a moisturizing cosmetic active ingredient to improve skin hydration. after nheks were incubated with all-trans retinoic acid (atra) for 24, 48, and 72 hours, the compound stimulated the entry of glycerol and increased expression of aqp3 followed by an increase in biological activity in human skin, such as the proliferation and differentiation of cells in the basal layer of the epidermis.(28) 31 retinol and ra are commonly used in antiaging cosmetics. retinol as one of the active ingredients can enhance in vivo repair of skin damage due to exposure to ultraviolet light (“photoaging”). topical application of retinol and ra achieves the treatment of “photoaging” by improving wrinkles, dry skin and delayed wound healing, but often causes side effects, such as skin irritation and dryness.(8,9) the effects of ultraviolet light causes a down regulation in aqp3 expression resulting in water permeability decrease and in reduced nhek migration and proliferation, which inhibits wound healing. this negative effect is reduced by atra therapy. these findings support the role of aqp3 in dehydrated skin due to sun exposure.(10) in our study, the expression of aqp3 in nheks increased after 24 hours treatment with ra at concentrations of 0.25, 0.5, and 1 mg/ ml. the intensity of aqp3 protein expression in nheks is directly proportional to the increase in concentration. atra increases aqp3 expression in nheks through retinoic acid receptor gamma (rar-γ). this is consistent with our finding that ra increased aqp3 exspression in nheks at concentrations of 0.25, 0.5 and 1 mg /ml. the quantitative analysis based on color density using imagej showed that exposure of nheks to asiaticoside concentrations of 0.25 and 0.5 mg/ml induces a slight increase in expression of aqp3 proteins. meanwhile, a significant increase in aqp3 protein expression was seen at 1 mg/ml after exposure to ra at all three test concentrations, the aqp3 expression increasing to a greater extent as shown in figure 3. in the present study one-way anova was used to determine significant differences in aqp3 expression in nheks after 24 hours exposure to asiaticoside, ra and in nheks without treatment at all test concentrations. the analysis was continued with the post hoc tukey test, demonstrating significant differences at a concentration of 0.25 mg/ml between the ra and control groups, at a concentration of 0.5 mg/ ml between ra and controls, also between asiaticoside and controls, and finally, at a concentration of 1 mg/ml between controls and ra. currently many research studies are being performed to investigate active compounds with the ability to increase aqp3 expression, thereby increasing the skin hydration endogenously. included in this list of active compounds are those from c o f f e a a r a b i c a , (17) p i p t a d e n i a colubrina,(18) and ajuga turkestanica.(19) from research on the induction of aqp3 b y p i p t a d e n i a c o l u b r i n a e x t r a c t a t concentrations of 10 and 20 mg/ml in cultured human skin cells it was found that the extract significantly increased the aqp3 mrna expression as analyzed by real timepolymerase chain reaction (rt-pcr).(18) aqp3 began to increase after 2 hours, reached its peak at 6 h o u r s , a n d d e c r e a s e d a f t e r 2 4 h o u r s . immunohistochemical examination showed increased expression of aqp3 in the membrane of nheks after exposure to the piptadenia colubrina extract at a concentration of 10 mg/ ml for 24 hours compared to the control group without treatment.(18) in another study by pereda et al.(20) an extract of green coffea arabica seed showed increased aqp3 expression in the normal epidermal layer of human skin. nheks were incubated for 3-6 hours with the optimal concentration of 25 mg/ml. aqp3 mrna achieved relative expression levels of up to 6.5 times than the negative controls. our study indicates that asiaticoside can i n c r e a s e t h e e x p r e s s i o n o f a q p 3 a t a l l concentrations tested, but at a lower level than ra. therefore, we may infer that asiaticoside at these concentrations does not cause dryness of the skin, which is a side effect of ra (due to over expression of aqp3). in our study, we only determined the aqp3 expression in nheks at a single point in time after 24 hours exposure to asiaticoside, so it was not exactly known when aqp3 expression began univ med vol. 36 no.1 32 wijayadi, darmawan asiaticoside increases aquaporin-3 protein to increase and decrease in nheks. asiaticoside can be used as an active ingredient in cosmetic moisturizers but still needs clinical trials to further determine its effectiveness and safety. conclusions asiaticoside increases aqp3 protein expression in nheks at concentrations 0.25, 0.5 and 1 mg/ml. the increase in aqp3 expression is directly proportional to the concentration. therefore asiaticoside can be used as an active ingredient in cosmetic moisturizers, to regulate skin hydration by increasing aqp3 expression. conflict of interest the authors have no conflict of interest. acknowledgement the authors wish thank intan razari and dani for laboratory assistance, dr. indra kusuma for nhek cell cultures and dr. prasetyawan yunianto for providing asiaticoside from the agency for the assessment and application of technology (badan pengkajian dan penerapan teknologi). contribution lyw contributed to drafting the manuscript and the design of the study. lyw and hd contributes to data collection, analysis and interpretation. lyw contributed revising manuscript for critically important content. all authors read and approved the final manuscript. references 1. rhein l. aging skin-general considerations. in: rhein l, fluhr jw, editors. aging skin: current and future theurapeutic strategies. usa: allured books; 2010.p.8-11. 2. rabe jh, mamelak aj, mcelgunn pj, et al. photoaging: mechanisms and repair. j am acad dermatol 2006;55:1-19. 3. sugiyama y, yamazaki k, kusaka-kikushima a, et al. analysis of aquaporin 9 expression in human epidermis and cultured keratinocytes. febs open bio 2014;4:611-6. 4. boury-jamot m, sougrat r, tailhardat m, et al. expression and function of aquaporins in human skin: is aquaporin-3 just a glycerol transporter? biochim biophys acta 2006;1758:1034-42. 5. draelos z. aquaporins: an introduction to a key factor in the mechanism of skin hydration. j clin aesthet dermatol 2012;5:53–6. 6. verkman as. a cautionary note on cosmetics containing ingredients that increase aquaporin 3 expression. exp dermatol 2008;17:871–2. 7. baumann l. skin ageing and its treatment. j pathol 2007;211:241–51. 8. rittie l, fisher gj, voorhess jj. retinoid therapy for photoaging. in: gilchrest b, krutmann j,editors. skin aging. berlin: springer;2006. p.143–56. 9. rawling av. retinoids and retinoic acid treatment of skin aging. in: rhein ld, fluhr jw,editors. aging skin: current and future therapeutic strategies. illinois: allured books; 2010.p.19–234. 10. cao c, wan s, jiang q, et al. all-trans acid attenuates ultraviolet radiation-induced downregulation of aquaporin-3 and water permeability in human keratinocytes.j cell physio 2008;215: 506-16. 11. ji c, yang y, yang b, et al. trans-zeatin attenuates ultraviolet induced down-regulation of aquaporin-3 in cultured human skin keratinocytes.int j mol med 2010;26:257-63. 12. bhavna d, jyoti k. centella asiatica: the elixir of life. int j res ayurveda pharm 2011;2:431–8. 13. gohil kj, patel ja, gajjar ak. pharmacological review on centella asiatica: a potential herbal cure-all. indian j pharm sci 2010;72:546–56. 14. zainol na, voo sc, sarmidi mr, et al. profiling of centella asiatica (l.) urban extract. malaysian j anal sci 2008;12:322–7. 15. lu l, ying k, wei s, et al. asiaticoside induction for cell-cycle progression, proliferation and collagen synthesis in human dermal fibroblasts. int j dermatol 2004;43:801–7. 16. yulianti l, mardliyati e, brahmono k, et al. asiaticoside induces cell proliferation and collagen synthesis in human dermal fibroblasts. univ med 2015;34.:96-103. 17. yulianti l, brahmono k, mardliyanti e, et al. effects of centella asiatica ethanolic extract encapsulated in chitosan nanoparticles on proliferation activity of skin fibroblasts and keratinocytes, type i and iii collagen synthesis 33 and aquaporin 3 expression in vitro. j pharm biomed sci 2016;6:315-27. 18. phaechamud t, yodkhum k, charoenteeraboon j, et al. chitosan-aluminum monostearate composite sponge dressing containing asiaticoside for wound healing and angiogenesis promotion in chronic wound. mat sci eng cmater 2015;50:210-25. 19. pereda mcv, dieamant gc, eberlin s, et al. effect of green coffea arabica l. seed oil on extracellular matrix components and waterchannel expression in in vitro and ex vivo human skin models. j cosmet dermatol 2009;8:56–62. 20. pereda mcv, dieamant gc, eberlin s, et al. expression of differential genes involved in the maintenance of water balance in human skin by piptadenia colubrina extract. j cosmet dermatol 2010;9:35–43. 21. perez c, dumont s, cattuzzo l. cosmetogenomics decodes hydrating action in cells. personal care 2013;6:79-82. 22. verdier-sevrain s, bonte f. skin hydration: a review on its molecular mechanisms. j cosmet dermatol 2007;6:75-82. 23. li j, tang h, hu x, chen m, et al. aquaporin 3 gene and protein expression in sun-protected human skin decreases with skin ageing. aus j dermatol 2010;51:106–12. 24. boury-jamot m, daraspe j, bonte f, et al. skin aquaporins: function in hydration, wound healing, and skin epidermis homeostasis. handb exp pharmacol 2009;190:205-17. 25. hara-chikuma m, verkman as. roles of aquaporin-3 in the epidermis. j invest dermatol 2008;128:2145–51. 26. hara-chikuma m, verkman as. aquaporin 3 facilitates epidermal cell migration and proliferation during wound healing. j mol med 2007;86:221–31. 27. yulianti l, brahmono k, mardliyanti e, et al. proceeding icmta. in silico molecular modeling and docking studies of aquaporin-3 with active compounds of centella asiatica. jakarta: icmta; 2013. 28. bellemere g, von stetten o, oddos t. retinoic acid increases aquaporin 3 expression in normal human skin. j invest dermatol 2008;128:5428. univ med vol. 36 no.1 c:\users\jurnal fkusakti\docume 19 *anatomy, physiology and cellular biology department, medical faculty, padjadjaran university **medical faculty, padjadjaran university ***department of internal medicine, medical faculty, padjadjaran university / dr. hasan sadikin general hospital, bandung correspondence: nur atik, dr., m.kes., ph.d anatomy, physiology and cellular biology department, medical faculty, padjadjaran university, jalan raya bandung-sumedang km.21, jatinangor, sumedang 45363 phone: +62812 8095 6825 email : n.atik@unpad.ac.id date of first submission, november 1, 2 01 7 date of final revised submission, february 15, 2018 date of acceptance, february 15, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinajanuary-april, 2018 vol.37no.1 guava leaf juice effect towards number of megakaryocytes in bone marrow of thrombocytopenic mice nur atik*, al hadi amrullah**, and andri reza rahmadi*** background dengue virus infection that most frequently occur in tropical and subtropical regions can cause many symptoms, one of which is a decrease in thrombocyte count. recent studies showed that guava leaf extract can increase the thrombocyte count in rats. the present study aimed to determine the effect of guava leaf juice in increasing the number of megakaryocytes in the bone marrow of thrombocytopenic mice. methods this study was of experimental design. the study subjects were 24 mice (mus musculus). the mice were randomly divided into four groups that were subjected to intervention for 14 days. group 1 was given guava leaf juice (56 mg/kg) and quinine (14 mg/kg), group 2 guava leaf juice (56 mg/ kg) only, group 3 was given quinine (14 mg/kg) and group 4 distilled water. after 14 days, from the bone marrow of the femoral bones of each of the mice, hematoxylin eosin stained histologic preparations were made. anova test was used to analyze the data. results the mean megakaryocyte count per field of view in group 1 (2.83), group 2 (3.30), group 3 (2.24) and group 4 (2.93). anova test results for all groups showed significant differences between groups (p<0.05). the mean megakaryocyte count was increased in group 1 compared to group 3, but the difference was statistically not significant (p=0.206). conclusion guava leaf juice can increase the megakaryocyte count in the bone marrow of thrombocytopenic mice. this suggests a potential role of guava leaf juice in improving the platelet count in thrombocytopenic disorders. keyword: guava leaf juice, megakaryocyte, mice, thrombocytopenia. doi: http://dx.doi.org/10.18051/univmed.2018.v37.19-24 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: atik n, amrullah al, rahmadi ar. guava leaf juice effect towards number of megakaryocytes in bone marrow of th romb ocyt open ic mice. univ med 2018;37:19-24. doi: 10.18051/univmed.2018.v37.19-24 20 atik, amrullah, rahmadi guava leaf juice effect towards number introduction dengue virus (denv) is transmitted by aedes aegypti mosquitoes, especially in tropical and subtropical regions. this virus can cause dengue fever, dengue hemorrhagic fever (dhf), and dengue shock syndrome. in 2010 there were 2.2 million case reports worldwide and in 2015 this number increased to 3.2 million cases. (1-3) there were approximately 50 to 100 million symptomatic cases every year in asia, followed by latin america and africa, and these countries became the regions with the greatest number of cases, which is 75% of total cases every year. (4,5) in indonesia there were 126,675 dhf cases in 2015, of which 1,229 died, whereas in 2014 there were 100,347 cases, of which 907 died.(6) thrombocytes come from megakaryocytes as precursors, which are giant discoid shaped cells with diameters of 1-3 micrometers and are usually found in bone marrow.(7,8) denv can cause thrombocytopenia by infecting endothelial cells in bone marrow. the infected cells become necrotic, release toxic cells and activate the coagulation and fibrinolytic systems that can reduce hemopoiesis a nd endothelial cell thrombogenicity.(3) in addition, denv infection induces platelet consumption due to disseminated intravascular coagulation, increases platelet destruction through apoptosis, lysis by the complement system and by the involvement of antiplatelet antibodies.(9-11) the early phase of dhf displays hypocellularity and attenuation of megakaryocyte maturation. this can be caused by direct lesions of progenitor cells by denv, infected stromal cells and changes in bone marrow regulation. in thrombocytopenia there are increased amounts of thrombopoietin (tpo), a cytokine that binds to the tpo receptor c-mpl (myeloproliferative leukemia virus onc ogene) to regulate megakaryopoiesis.(9) psidium guajava is a tropical plant of the myrtaceae family that can grow to 35 feet, and is recognizable by its brown and peeled off bark. guava leaves contain carotenoids, anthocyanins, essential fatty acids, lecithins, alkaloids, phenols, saponins, tannins, triterpenes, f lavonoids (particularly quercetin) and vitamin c.(12,13) quercetin acts as an antioxidant that affects cell differentiation. quercetin can also increase the cytokines granulocyte-macrophage-colonystimulating factor (gm-csf) and interleukin-3 (il-3) that stimulate megakaryopoiesis and can increase the number of megakaryocytes.(12,14,15) a previous study showed that guava leaf juice extract can increase the number of thrombocytes in rats.(14) another report showed that guava leaf extract increases the number of platelets in cyclophosphamide-induced thrombocytopenia in rats. however, the role of megakaryocytes in increasing the platelets is still poorly understood. (16,17) to evaluate the effect of guava leaf juice on the role of megakaryocytes in thrombocyte production, we performed this study to determine the megakaryocyte count in the bone marrow of the femoral bones of thrombocytopenic mice after guava leaf juice administration. methods study design the study was of experimental design and was carried out at the division of cell biology, department of anatomy, physiology and cell biology, faculty of medicine a nd at the biosystems laboratory, faculty of mathematics and science, padjadjaran university, from january-august 2017. study subjects the subjects of this study were 24 mice (mus musculus) divided into four groups. the number of subjects was obtained using federer’s formula (n-1)(t-1) 15 where n is the number of subjects and t is the number of intervention groups. the inclusion criteria of this study were eight to twelve-weeks old male mice weighing 20-30 grams. the exclusion criteria were mice that died or became ill during the adaptation period. the dropout criteria were mice that died or became ill during the intervention. 21 guava leaf juice administration six mice of each intervention group were placed in one cage in the biosystems laboratory, faculty of mathematics and science, padjadjaran university. the cages were equipped with chaff as bedding, ventilation, feed and water storage. the standard feed was given ad libitum. the mice were adapted for seven days, then they were randomly assigned to four groups. group 1 was given 56 mg/kg guava leaf juice and 14 mg/kg quinine, group 2 was given 56 mg/kg guava leaf juice, group 3 was given 14 mg/kg quinine, and group 4 was given 1 ml of distilled water.(14) the administration was done using orogastric tubes. quinine tablets were ground using pestle and mortar then dissolved in distilled water. the intervention was given for fourteen days, and then the mice were sacrificed by cervical dislocation preparation of guava leaf juice the guava leaf juice was made as previously described.(14) guava leaves were obtained from the arboretum, faculty of mathematics and sciences, padjadjaran university. the leaves were cleaned with water, rinsed in distilled water and blended in a juicer after addition of distilled water until smooth and the dregs were removed with a strainer. the ratio of distilled water and guava leaves was 1:1 (w/v), and the juice was kept at 4oc in the refrigerator. histologic analysis after the mice were terminated, their femor al bones wer e t aken for histologic preparations. the histologic preparations were made in the cellular biology preparation room, faculty of medicine, padjadjaran university. the histologic preparations were then stained with hematoxyli n-eosin. (1 2,18 ) the his tologic preparations were read under the microscope at 100 times magnification. every slide was read in three different random parts. from each slice, megakaryocytes were read in three different fields of views of the total visible area. statistical analysis data distributions were tested using kolmogorov-smirnov test and homogeneity of variance was tested using levene test. because the data were not normally distributed and had homogenous variance, we used the kruskalwallis test with the value of p<0.05 being considered statistically significant. since the kruskal-wallis test result was significant, we used the mann-whitney te st to determine the differences between groups. ethical clearance ethical clearance for this research study was given by the research ethics committee, faculty of medicine, padjadjaran university under number 972/un6/c1/3/2/kepk/pn/2016. results data obtained from histologic preparations of the femoral bones can be seen in table 1 and fi gu r e 1, s ho wi n g di f f e r e n ce s i n me a n megakaryocyte count. to determine whether the between-group differences were statistically significant or not, first we analyzed the data distribution and homogeneity of variance. the data distribution was analyzed using the kolmogorov smirnov test and the result was that the data was not normally distributed (p<0.05). the variance was analyzed using the levene test and the result was that data had homogenous variance (p>0.05). because the data were not normally distributed and had homogenous variance, we used a non-parametric treatment group p value g1 g2 g3 g4 megakaryocytes 2.83 ± 0.89 3.30 ± 0.29 2.00 ± 0.42 3.32 ± 0.98 0.206 table 1. megakaryocyte count by treatment groups g1=guava leaf juice and quinine; g2=guava leaf juice; g3=quinine; g4=distilled water univ med vol. 37 no.1 22 atik, amrullah, rahmadi guava leaf juice effect towards number a b c figure 1. histologic preparation of femoral bone marrow (a) group 1; (b) group 2; (c) group 3; (d) group 4 after guava leaf juice treatment, mice were sacrificed and the femoral bones were collected for he staining. quantitative observation of megakaryocytes in the bone marrow showed that the megakaryocyte count in the group receiving guava leaf juice and quinine (a) was increased compared to the quinine group (c). furthermore, there is no statistically difference between the guava leaf juice group (b) and the distilled water group (d). microscopic images at 10x magnification where red circles indicate megakaryocytes groups p value g1 g2 g3 g4 g2 g3 g4 g3 g4 0.671 0.206 0.644 0.022 1.00 0.020 table 2. mann whitney test g1=guava leaf juice and quinine; g2=guava leaf juice; g3=quinine; g4=distilled water test, i.e. the kruskal wallis test. the result of the kruskal wallis test was shown to be significant (p = 0.021). in table 1, the mean megakaryocyte count of group 1 (2.83) is greater than that of group 3 (2.24) and that the mean megakaryocyte count of group 2 (3.3) is greater than that of group 4 (2.93). to determine whether the differences between the means of group 1 and group 3 and between the means of group 2 and group 4 were significant or not we used the mann whitney test. the result of the mann whitney test shown in table 2 demonstrates that there was no significant difference between group 1 and group 3 (p>0.05) and also between group 2 and group 4 (p>0.05). discussion in this study quinine was used to induce t hr omb ocyt op e n i a a n d d e cr e a se t he megakaryocyte count in mice. quinine can cause thrombocytopenia by recognizing von willebrand 23 factor or the glycoprotein receptors ib/ix or iib/ iiia. these receptors are found on thrombocytes and megakaryocytes. quininedependent antibody can reduce megakaryocyte viability by apoptosis induction, and inhibit the expression o f ma r ke rs th a t d e c r e a se pro p la te l e t production.(19,20) reduction in megakaryocyte viability and apoptosis causes the decrease in megakaryocyte numbers in bone marrow. our present study showed that mean megakaryocyte count wreas decreased after quinine treatment (g1 and g3) (table 1). furthermore, there was an increase in me a n me ga ka r yo cyt e c o un t i n t he thrombocytopenia mice model after guava leaf administration (g2) (table. 1). this phenomenon could be explained by the fact that guava leaf contains the flavonoid quercetin, saponins and also vitamin-c.(17,21,22) quercetin could act as antioxidant affecting cell differentiation(14) and increasing granulocyte-macrophage colonystimulating factor (gm-csf) and interleukin-3 (il-3). finally, all these pathways stimulate megakaryopoiesis and are followed by increases in megakaryocyte count.(23,24) in addition, the mean megakaryocyte counts from the normal control group (g4) and the groups were given guava leaf juice (g1 and g2) did not show any significant differences. this indicates that guava leaf could not affect the number of megakaryocytes in normal mice. it is concluded that the components in guava l e a f do no t a f f e c t t h e ho meos t a s i s of megakaryocytes formation. on the other hand, megakaryocyte formation is influenced by systemic thrombopoietin, which then binds to cmpl on megakaryocytes to stimulate the f o r ma t i on of me gaka r yo c yte s. in t he thrombocytopenic condition, the levels of circulatory thrombopoietin are increased, so it binds to c-mpl to help improve megakaryocyte production. (9) on the contrary, in the normal condition, the platelet count is within normal limits a n d th e pl a t e l e t s bi n d to ci r c ul a t o r y thrombopoietin followed by no increment of megakaryocyte production.(22) the limitation of this study was that no previous study had shown the optimum dose, while our study only used one dose of guava leaf juice. another limitation was that the observations at the histological examination were restricted to counting the number of megakaryocytes in the femoral bone. although the results were not statistically significant, administration of guava leaf juice may still increase the number of megakaryocytes. this condition could inc rease the number of thrombocytes, and hopefully could reduce the risk of fatal cases in thrombocytopenia cases like dengue hemorrhagic fever. further studies are needed to find out the optimal dose and the toxicity of guava leaf. it is also necessary to conduct a new study to investigate the molecular mechanism of guava leaf in increasing the number of megakaryocytes. conclusion guava leaf j uic e can increase megakaryocyte number in bone marrow of thrombocytopenic mice model. this suggests a potential role of guava leaf juice in improving the platelet count in thrombocytopenic disorders. conflict of interest competing interest: no relevant disclosure. acknowledgement thanks are due to deden deni for assisting in mice maintenance and irfan anis ahmad for help during histologic slide preparation. contributors na contributed to design of the study, perfor med the experiment and wrote the manuscript. aha performed the experiment and wrote the manuscript. arr contributed to the design of the study. all authors read and approved the manuscript. univ med vol. 37 no.1 24 atik, amrullah, rahmadi guava leaf juice effect towards number references 1. guerdan br. dengue fever / dengue hemorrhagic fever. am j clin med 2010;7:51–3. 2. capeding mr, tran nh, hadinegoro sr, et al. clinical efficacy and safety of 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2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background diabetic nephropathy (dn) is the most serious complication of diabetes, causing end-stage renal disease throughout the world. recent studies have reported a direct role of vascular endothelial growth factor (vegf) and transforming growth factor-â (tgf-â) in dn pathogenesis. vegf and tgf-â are expressed early in glomeruli in response to hyperglycemia. active substances of phaleria macrocarpa (pm) pericarp are known to have nephroprotective effects. this study aimed to evaluate the effects of phaleria macrocarpa (scheff.) boerl pericarp extract on vegf and tgf-â expression in alloxan-induced diabetic rats. methods an experimental study was conducted on twenty five male albino (sprague dawley) rats divided into five groups (of five each): normal control; diabetic; diabetic + metformin 100 mg/kgbw; diabetic + methanolic pm extract 250 mg/ kgbw; and diabetic + aqueous pm extract 250 mg/kgbw. diabetes was induced by alloxan monohydrate 150 mg/bw intraperitoneally. treatment was given for 3 weeks. vegf and tgf-â expression analysis was performed by means of immunohistochemical technique. differences between groups were assessed by one-way anova. results vegf expression in the pm extract group was significantly lower than that in the diabetic group and even metformin group (p<0.01). tgf-â expression in methanolic pm extract group was significantly lower than in diabetic and metformin group (p<0.01), but aqueous pm extract group only showed significancy when compared with diabetic group (p< 0.01). conclusions phaleria macrocarpa pericarp extract reduces glomerular expression of tgf-â and vegf in alloxan-induced diabetic rats. key words: phaleria macrocarpa (scheff.) boerl., growth factor expression, alloxan, diabetic nephropathy, rats phaleria macrocarpa reduces glomerular growth factor expression in alloxan-induced diabetic rats evy sulistyoningrum* and setiawati** 72 sulistyoningrum, setiawati phaleria and diabetic nephropathy mahkota dewa menurunkan ekspresi glomerular pada tikus diabetik diinduksi aloksan latar belakang nefropati diabetik (nd) merupakan komplikasi diabetes paling serius yang menyebabkan penyakit ginjal tahap terminal di seluruh dunia. beberapa studi telah melaporkan peran vascular endothelial growth factor (vegf) dan transforming growth factor-â (tgf-â) dalam patogenesis nd. vegf dan tgf-â diekspresikan di glomerulus pada tahap awal nd sebagai respons terhadap kondisi hiperglikemia. susbtansi aktif pada daging buah mahkota dewa (phaleria macrocarpa (scheff.) boerl =pm) diketahui memiliki efek nefroprotektif. penelitian ini bertujuan untuk mengevaluasi efek ekstrak daging buah pm pada ekspresi vegf dan tgf-â glomerular tikus diabetik yang diinduksi aloksan. metode rancangan penelitian eksperimental dilakukan pada 25 tikus putih rattus norvegicus strain sprague dawley yang dibagi menjadi lima kelompok (masing-masing lima), yaitu: kontrol normal; diabetik; diabetik + metformin 100 mg/ kgbb; diabetik + ekstrak metanol pm 250 mg/kgbb dan diabetik + ekstrak air pm 250 mg/kgbb. kondisi diabetik diinduksi dengan injeksi alloksan monohidrat 150 mg/bb dosis tunggal secara intraperitoneal. perlakuan diberikan selama 3 minggu. ekspresi vegf dan tgf-â dianalisis secara semikuantitatif dengan teknik imunohistokimia. uji anova digunakan untuk membandingkan perbedaan antara kedua kelompok. hasil ekspresi vegf pada kelompok yang mendapatkan ekstrak pm lebih rendah dibandingkan kelompok diabetik dan bahkan kelompok diabetik yang mendapat metformin (p<0,01). ekspresi tgf-â pada kelompok yang mendapat ekstrak metanol pm lebih rendah dibandingkan kelompok diabetik dan metformin (p<0,01), sedangkan kelompok yang mendapatkan ekstrak air pm lebih rendah dibandingkan kelompok diabetik saja (p<0,01). kesimpulan ekstrak daging buah phaleria macrocarpa menurunkan ekspresi tgf-â dan vegf glomerular pada tikus diabetik yang diinduksi alloksan. kata kunci : phaleria macrocarpa (scheff.) boerl., ekspresi glomerular, aloksan, nefropati diabetik, tikus abstrak introduction a c c o r d i n g t o t h e wo r l d h e a l t h organization, the global prevalence of diabetes is estimated to increase from 2.8% in 2000 to 4.4% in 2030.(1) this number is predicted to rise due to longer life expectancy, urbanization, life style changes and higher prevalence of obesity. the increasing prevalence of diabetes is not only occurring in developed countries,but also in developing countries including asian countries.(2) diabetic nephropathy (dn) is one of the most serious complications affecting diabetes patients.(3) it is a major cause of morbidity and mortality in dm patients,(4,5) causing end-stage renal disease worldwide.(6) the diabetic milieu results in mesangial expansion and changes in the cellular and extracellular compartment of the glomerulus. diabetes induced by alloxan in rats 73 univ med vol. 32 no.2 results in development of severe hyperglycemia and reduced glomerular filtration rate similar to clinical dn.(7) the hyperglycemic milieu increases the expression of many growth factors in the kidney. recent evidence supports a direct role for vascular endothelial growth factor (vegf) and transforming growth factor-â (tgf-â) in the pathogenesis of dn. tgf-â action in glomerular mesangial cells leads to changes that appear to be central to the development of dn. e n h a n c e d r e n a l e x p r e s s i o n o f t g f â i s reported to be responsible for initiation and progression of dn. moreover, increased expression of tgfâ induces accumulation of extracellular matrix proteins and is related to renal hypertrophy in dn.(8) vegf expression is induced in glomerular cells by high glucose levels or advanced glycation end-products and reported to be increased in the early stages of dn. vegf may contribute to renal matrix accumulation, since treatment with anti-vegf antibodies attenuates glomerular basement membrane (gbm) thickening and mesangial expansion.(9) diabetes and its complications have been known to be amenable to control by natural p r o d u c t s . t h e r e f o r e , d i s c o v e r y a n d development of novel drugs for dm is very important. phaleria macrocarpa (scheff) boerl (thymelaeaceae) (pm), an endemic plant of indonesia, has been used in traditional medicine for treating several diseases. pm pericarp has been reported to contain phenolic and flavonoid compounds with antioxidant activity.(10) in addition, pm reportedly possesses hypoglycemic activity, through inhibition of á glucosidase.(11) in a previous study, pm was found to reduce renal hypertrophy and blood urea nitrogen level in diabetic rats.(12) the present study examines the effect of pm pericarp extracts on the glomerular expression of vegf and tgf-â in alloxan-induced dn in rats. this study is part of a larger study on the nephroprotective effect of pm pericarp extract for diabetic nephropathy. methods design of the study a post-test only laboratory experimental study with control, conducted from julydecember 2012 in the pharmacology laboratory, faculty of medicine and health sciences, jenderal soedirman university, purwokerto. plant materials r i p e f r u i t s o f p. m a c ro c a r p a w e r e p u r c h a s e d f r o m m e r a p i h e r b a l f a r m a , yogyakarta, indonesia. the plant species was identified by the plant taxonomy laboratory at the faculty of biology, jenderal soedirman university, purwokerto, indonesia. the pericarps were sliced, dried at 70°c and ground into a powder using a milling machine. to obtain the methanolic extract, about 900 g of the powdered material was added to 5.5 liters of methanol and left to stand for 24 h. the filtrate was collected using whatman no. 1 filter paper and the residue was re-extracted with 5.5 l methanol for 24 h and the re-extraction step was repeated for another 24 h. the filtrate from 3 days of m e t h a n o l e x t r a c t i o n w a s c o l l e c t e d a n d evaporated by rotary evaporation. an aqueous extract as a mimic of traditional use was obtained from 900 g of the ground material by boiling in water for 30 minutes, filtering through whatman no. 1 filter paper and evaporating the filtrate by rotary evaporation. the extracts were kept in the refigerator (at 4°c) from where a l i q u o t s w e r e w i t h d r a w n f o r t h e t e s t procedures.(12) experimental animals in accordance with the federer formula, this study used a total of 25 healthy male sprague dawley rats (rattus norvegicus) aged 6-8 weeks and weighing 160-200 grams. the rats were housed under standard laboratory conditions of 22-30°c temperature, 30% relative humidity, and 12 h light and dark cycle, with free access to standard pellet diet and water. rats were adapted for 10 days and then divided into 74 five intervention groups (of five each): normal control group receiving only distilled water; diabetic group; diabetic + metformin 100 mg/ kgbw, diabetic + pm pericarp methanolic extract (pmm) 250 mg/bw and diabetic + pm pericarp aqueous extract (pmw) 250 mg/kgbw. the treatments were conducted for 21 days, after which the animals were sacrificed.(12) the right kidney of each animal was taken for immunohistochemical staining. induction of diabetes after an overnight fast, rats were made diabetic with a single intraperitoneal injection of 150 mg/kgbw of alloxan monohydrate (sigma, germany) dissolved in distilled water.(12,13) after 72 h of alloxan administration, hyperglycemia was confirmed by measuring the fasting blood glucose level. rats with a fasting blood glucose level above 130 mg/dl were considered diabetic and employed for further studies.(12,14) immunohistochemical assay for immunohistochemical staining, a renal tissue block was deparaffinized and rehydrated in graded alcohols to tap water. kidney sections were incubated with h 2 o 2 3% for 15-20 minutes and washed and rinsed in distilled water. slides were transferred into solution for antigen retrieval at ph 6.0 and then microwaved for 10 min. after a water wash, slides were flooded with 1x pbs wash buffer and the protein blocker w a s a p p l i e d f o r 5 1 0 m i n u t e s a t r o o m temperature. after the protein blocker was drained, the primary antibody, polyclonal rabbit anti-rat vegf antibody (thermo scientific, usa) or tgf-â antibody (novocastra, uk) was added for 30-60 minutes. negative control sections were stained under identical conditions by omitting the antibody. slides were washed in 2 changes of 1x pbs wash buffer for 2 minutes e a c h . s l i d e s w e r e t h e n i n c u b a t e d w i t h biotinylated secondary antibody (biocare, uk) for 15 minutes. after washing in 2 changes of 1x pbs wash buffer for 2 minutes each, slides were incubated with streptavidin peroxidase (biocare, uk) for 10 minutes. after washing in 2 changes of 1x pbs wash buffer for 2 minutes each, slides were incubated with chromogen: dab for 3-5 minutes. after draining, the sections were counterstained with mayer ’s hematoxylin for 1 minute. to evaluate vegf and tgf-â staining, each glomerulus was graded semiquantitatively. each score reflects changes in the extent rather than the intensity of staining. five scores were awarded as follows; 0=very weak or absent staining and no localized increases in staining; 1=diffuse, weak staining with 1–25% of the glomerulus showing focally increased staining; 2=25–50% of the glomerulus demonstrating a focal, strong staining; 3=50– 75% of the glomerulus stained strongly in a focal manner; 4=more than 75% of the glomerulus stained strongly. for each sample, 50–60 glomeruli were evaluated, and the average score was calculated.(15) statistical analysis normally distributed data were expressed as mean ± sd and analyzed using one way anova whose results were further subjected to lsd post hoc test for multiple comparisons. all analyses were performed with spss 15.00 for windows version 15 and differences between means were accepted to be significant at p<0.05. ethical clearance ethical approval for this study was obtained from the research ethics committee, faculty of medicine and health sciences, jenderal soedirman university, purwokerto. results figure 1 shows significantly elevated glomerular expression of vegf in diabetic rats compared with normal rats (p<0.01). treatment with metformin and pm extracts (methanolic and aqeous) significantly reduced vegf expression (p<0.01), but treatment with pm extracts showed better reduction in vegf sulistyoningrum, setiawati phaleria and diabetic nephropathy 75 univ med vol. 32 no.2 expression as compared with metformin (p<0.01 for both extracts). no significant difference in vegf expression was found between diabetic rats treated with methanolic pm pericarp extract and those treated with aqueous pm extract. figure 2 shows a series glomeruli with various degrees of glomerular vegf expression. glomeruli of diabetic rats showed the highest score of vegf expression (figure 2b), while lower scores of vegf expression were shown by metformin and pm treated diabetic rats as well as normal rats (figure 2a, 2c, 2d). as shown in figure 3, glomerular tgf-â e x p r e s s i o n i n t h e d i a b e t i c r a t s s h o w e d significant elevation compared with normal rats (p<0.01). treatment with metformin and pm s i g n i f i c a n t l y r e d u c e d g l o m e r u l a r t g f â e x p r e s s i o n ( p< 0 . 0 1 ) . tr e a t m e n t w i t h methanolic pm extract significantly reduced glomerular tgf-â expression compared with metformin (p<0.01), whereas aqueous pm extract showed no significant difference with metformin in glomerular tgf-â expression. the various degrees of glomerular tgf-â expression are shown in figure 4. the highest glomerular tgfâ expression is shown in diabetic rats (figure 4b), while lower scores of tgfâ expression are shown by metformin and pm treated diabetic rats as well as normal rats (figure 4a, 4c, 4d). discussion diabetes produces several changes in the composition of the basement membrane. this a l t e r e d m a t e r i a l u n d e r g o e s a c c e l e r a t e d glycosylation and further rearrangement to form advanced glycation end-products (ages), which stimulate protein synthesis, further d e c r e a s e d e g r a d a b i l i t y o f t h e b a s e m e n t membrane, increase its permeability and cause endothelial dysfunction. consistent with previous studies,(9,16,17) this study showed that the untreated diabetic group showed significant elevation of glomerular vegf and tgf-â expression. both tgf-â and vegf may contribute to the cellular hypertrophy and enhanced collagen synthesis observed in diabetic nephropathy as glomerulosclerosis.(18) the hyperglycemic condition in diabetes enhances glucose uptake by mesangial cells and increases production of ages and reactive oxygen species (ros) that activate several responses, including inflammatory reactions through activation of protein kinase c (pkc) and erk pathways. these pathways will further stimulate synthesis of tgf-â and vegf.(19) in the present study, diabetic animals t r e a t e d w i t h p m s h o w e d r e d u c t i o n i n glomerular tgf-â and vegf expression. these results indicate that pm attenuates the progression of glomerular damage, i.e., pm has a nephroprotective effect in alloxan-induced diabetic rats. this study also reports that attenuation of renal damage by pm extract is better than that by metformin. the attenuation of renal damage by pm pericarp extract is presumably due to active substances contained in the pericarp, such as phenolic constituents, quercetin, kaempferol, and terpenoids.(10) the active substances in pm have biological actions mediated by the inhibition of oxidative stress a n d i n f l a m m a t i o n , ( 2 0 ) a n d a l s o h a v e antihypergycemic effects.(11) triastuti et al.(13) reported that pm extract restored decreased renal antioxidant enzymes such as superoxide d i s m u t a s e ( s o d ) , c a t a l a s e ( c at ) , a n d glutathione peroxidase (gpx) activities, and glutathione (gsh) level in alloxan-induced d i a b e t i c r a t s , a n d a l s o r e d u c e d r e n a l hypertrophy and blood urea nitrogen (bun) in diabetic rats. the role of oxidative stress in diabetes and diabetic tissue damage is reported e l s e w h e r e . ( 2 1 , 2 2 ) h y p e r g l y c e m i a n o t o n l y increases oxidative stress but also decreases antioxidant defenses in diabetic individuals.(22) administration of antioxidants in experimental diabetes reduced apoptotic cell loss, which could be a major cause of glomerular sclerosis in diabetic nephropathy.(23) our study also 76 sulistyoningrum, setiawati phaleria and diabetic nephropathy figure 1. semiquantitative analysis of glomerular expression of vegf, values are mean + standard deviation, **p<0.01 compared with diabetic group, ##p<0.01 compared with metformin group; anova followed by lsd test; vegf = vascular endothelial growth factor; pmm = methanolic extract of phaleria macrocarpa; pmw = water extract of phaleria macrocarpa figure 2. glomerular expression of vegf. a. normal glomerulus of control group showed no expression of vegf (score 0); b. glomerulus of diabetic group showed expression of vegf in 50-75% of the glomerulus (score 3); c. glomerulus of diabetic group treated with phaleria macrocarpa showed smaller area of expression of vegf of up to 25% of the glomerulus (score 1); d. glomerulus of diabetic group treated with metformin showed vegf expression in 25-50% of the glomerulus (score 2); (arrows indicate vegf) vegf = vascular endothelial growth factor a b c d 77 figure 3. semiquantitative analysis of glomerular expression of tgfâ; values are mean + standard deviation **p < 0.01 compared with diabetic group; ## p < 0.01 compared with metformin group; anova followed by lsd test; tgf-â = transforming growth factor-â; pmm = methanolic extract of phaleria macrocarpa; pmw = water extract of phaleria macrocarpa univ med vol. 32 no.2 figure 4. various degrees of tgf-â expression. a. normal glomerulus of control group showed no expression of tgf-â (score 0); b. glomerulus of diabetic group showed expression of tgf-â of more than 75% of the glomerulus (score 4); c. glomerulus of diabetic group treated with phaleria macrocarpa showed smaller area of expression of tgf-â up to 25% of the glomerulus (score 1); d. glomerulus of diabetic group treated with metformin showed tgf-â expression in 50–75% of the glomerulus (score 3); (arrows indicate tgf-â) tgf-â = transforming growth factor-â a b c d 78 s h o w e d t h a t p m e x t r a c t r e d u c e d r e n a l hypertrophy and glomerulosclerosis in diabetic rats.(12) in addition to the antioxidant properties of pm extract, recent studies also reported antihyperglicemic and anti-inflammatory p r o p e r t i e s o f p m . p m e x t r a c t h a s a n antihyperglycemic effect via intraand extrapancreatic mechanisms. alkaloid substances of pm regenerate pancreatic â cells and increase glucose utilitization and sensitivity of insulin receptors in peripheral tissues.(24) pm pericarp extract contains high amounts of flavonoids that inhibit nf-êb activation, which further inhibits proinflammatory cytokine synthesis.(25) there are differences in the nephroprotective effect of methanolic and aqueous pm extracts. the present study reports that methanolic pm extract reduces tgf-â expression better than the aqueous extract. an earlier anti-hyperglycemic study with serial crude extracts of phaleria macrocarpa (pm) fruits indicated the methanol extract as the most effective,(10) which might b e d u e t o t h e p r e s e n c e o f f l a v o n o i d compounds.(26) however, the aqueous extract as a mimic of its traditional use, which is more applicable to rural populations in developing countries, showed the same effect in reducing vegf expression. the present study shows that pm extract confers benefits in the early phases of dn development, and thus can be used as an alternative preventive agent for dn. however, our study was limited to two methods of extraction and two parameters of renal damage, therefore further investigations on pm pericarp extraction are still needed, as well as other parameters of nephroprotective effect to prevent diabetic complications. conclusions a l l o x a n i n d u c e d d i a b e t e s i n c r e a s e s glomerular expression of vegf and tgf-â, while treatment with phaleria macrocarpa (scheff.) boerl. is capable of decreasing glomerular vegf and tgf-â expression. sulistyoningrum, setiawati phaleria and diabetic nephropathy acknowledgements this research study was funded by a research grant from the health profession educational quality project. we thank mrs. eka prasasti nur rahmani for giving us support for this research. references 1. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes. estimates for the year 2000 and projections for 2030. diabetes care 2004;27: 1047-53. 2. ramachandran a, snehalatha c, samith sa, nanditha a. trends in prevalence of diabetes in asian countries. world j diabetes 2012;3:110-7. 3. kanwar ys, wada j, sun l, xie p, wallner ei, chen s, et al. diabetic nephropathy: mechanism of renal disease progession. exp biol med 2008; 233:4-11. 4. ohshiro y, lee y, king gl. mechanism of diabetic nephropathy: role of protein kinase c activation, adv stud med 2005;2:s10-9. 5. ghaisas mm, navghare vv, takawale ar, zope vs, phanse ma. antidiabetic and nephroprotective effect of tectona grandis linn. in alloxan induced diabetes. ars pharm 2010;51: 195-206. 6. tervaert twc, mooyart al, mann k, cohen ah, cook ht, crachenberg cb, et al. pathologic classification of diabetic nephropathy. j am soc nephrol 2010;1-8. doi: 10.1681/asn.2010010010. 7. di loreto, moreno hs, puche rc, locatto me. severe hyperglicemia: a determinant factor for hypofiltration in alloxan diabetic rats. acta diabetol 2004;41:56-63. 8. brosius fc. trophic factors and cytokines in early diabetic glomerulopathy. exp diab res 2003;4:22533. 9. flyvbjerg a, dagnaes-hansen f, de vriese as, schrijvers bf, tilton rg, rasch r. amelioration of long-term renal changes in obese type 2 diabetic mice by a neutralizing vascular endothelial growth factor antibody. diabetes 2002; 51:3090–4. 10. ali r, atangwho i, kaur n, abraika o, ahmad m, mahmud r, et al. bioassay-guided antidiabetic study of phaleria macrocarpa fruit extract. molecules 2012;17:4986-5002. 11. ali r, item ja, navneet k, elsnoussi ahm, ali jm, mohd. za, et al. hypoglycemic and antihyperglycemic study of phaleria macrocarpa fruit pericarp. j med plant res 2012;6:1982-90. 79 univ med vol. 32 no.2 12. sulistyoningrum e, setiawati, ismaulidiya fr. phaleria macrocarpa (scheff.) boerl improved renal histological changes in alloxan-induced diabetic rats. int j med plant altern med 2013;1:8792. 13. triastuti a, hee-jp, jong wc. phaleria macrocarpa suppress nephropathy by increasing renal antioxidant enzyme activity in alloxaninduced diabetic rats. nat prod sci 2009;15:16772. 14. sedigheh a, jamal ms, mahbubeh s, somayeh k, mahmoud rk, azadeh a, et al. hypoglycaemic and hypolipidemic effects of pumpkin (cucurbita pepo l.) on alloxan-induced diabetic rats. afr j pharm pharmacol 2011;5:2620-6. 15. cha dr, kang ys, han sy, jee yh, han kh, han jy, et al. vascular endothelial growth factor is increased during early stage of diabetic nephropathy in type ii diabetic rats. j endocrinol 2004;183:183–94. 16. gilbert re, cox a, wu ll, allen tj, hulthen ul, jerums g, et al. expression of transforming growth factor-â 1 and type iv collagen in the renal tubulointerstitium in experimental diabetes. diabetes j 1998;47:414–22. 17. langham rg, kelly dj, gow rm, zhang y, cordonnier dj, pinel n, et al. transforming growth factor â in human diabetic nephropathy. diabetes care 2006;29:2670–5. 18. teoh, sl, latiff aa, das s. histological changes in the kidney of experimental diabetic rats fed with momordica charantia (bitter gourd) extract. rom j morphol embryol 2010;51:91-5. 19. qian y, feldman e, pennathur s, kretzler m, brosius fc. from fibrosis to sclerosis: mechanism of glomerulosclerosis in diabetic nephropathy. diabetes j 2008;57:1439-45. 20. hendra r, ahmad s, oskoueian e, sukari a, shukor my. antioxidant, anti-inflammatory and cytotoxicity of phaleria macrocarpa (boerl.) scheff fruit. bmc complement altern med 2011; 17:4986-5002. 21. elmarakby aa, sullivan jc. relationship between oxidative stress and inflammatory cytokines in diabetic nephropathy.cardiovasc ther 2010;30: 1–11. 22. krishan p, chakkarwar va. diabetic nephropathy: aggressive involvement of oxidative stress. pharm educ res 2011;2:35-41 23. lee ey, lee my, hong sw, chung ch, hong sy. blockade of oxidative stress by vitamin c ameliorates albuminuria and renal sclerosis in experimental diabetic rats. yonsei med j 2007;48: 84755. 24. meiyanti hrd, fransiscus ds. efek hipoglikemik daging buah mahkota dewa (phaleria macrocarpa (scheff.) boerl.) terhadap kadar gula darah pada manusia sehat setelah pembebanan glukosa. univ med 2006;25:114-20. 25. gonzalez-gallego j, sanchez-campos s, tunon mj. anti-inflammatory properties of dietary flavonoid. nutr hosp 2007;22:287-93. 26. hendra r, ahmad s, sukari a, shukor my, oskoueian e. flavonoid analyses and antimicrobial activity of various parts of phaleria macrocarpa (scheff.) boerl fruit. int j mol sci 2011;12:3422-31. f:\36 no 2\haryono.pmd 123 *department of physiology, faculty of medicine, indonesia catholic university of atma jaya, jakarta, indonesia correspondence: nawanto agung prastowo department of physiology, faculty of medicine, catholic university of atma jaya, pluit raya street no 2, north jakarta, indonesia phone: +62216694366 mobile: +62813 1747 7849 fax: +6221 6606123 email: nawanto2005@yahoo.co.id date of first submission, january 15, 2017 date of final revised submission, august 7, 2017 date of acceptance, august 9, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 performance of osteoporosis self-assessment tool in detecting low bone mineral density in menopausal women ignatio rika haryono* and nawanto agung prastowo* background the osteoporosis self-assessment tool (ost) is a simple screening tool to assess risk of osteoporosis and to select high risk women for dual-energy x-ray absorptiometry (dxa) examination. this study aimed to evaluate ost performance in detecting low bone mineral density (bmd) in menopausal women. methods a cross-sectional study involving 60 menopausal women aged 50-65 years. the ost score was calculated from: [weight (kg) – age (yr)] x 0.2. subjects were classified by ost score into low risk (ost 2) and high risk (ost< 2) groups. bmd was determined by dxa at 3 bone locations (l1-l4, femoral neck, and total hip). dxa t-scores were categorized into: normal bmd (t-score >-1) and low bmd (t-score -1). independent t-test was used to compare subject characteristics between ost groups. diagnostic performance of ost was evaluated by measuring sensitivity, specificity, positive & negative predictive value (ppv, npv), positive & negative likelihood ratio (plr, nlr) and receiver-operating characteristic (roc). significance was set at p<0.05. results subject characteristics and bmd between groups were significantly different (p<0.05). most subjects (44/73.3%) had high risk of low bmd (ost < 2). low bmd (t score -1) was found in 43 subjects (71.7%) at l1-l4, 41 subjects (68.3%) at femoral neck, and 37 subjects (61.7%) at total hip. diagnostic performance of ost was significant at total hip bmd (sensitivity=0.946, auc=0.777). conclusion we conclude that use of the ost score in menopausal women is effective and has adequate sensitivity and specificity. the highest diagnostic performance of ost is on total hip bmd. keywords: bone mass, menopausal women, simple screening tools doi: http://dx.doi.org/10.18051/univmed.2017.v36.123-130 original article cit e th is a rtic le a s: haryono ir, pra stowo na. performance of osteoporosis self-assessment tool in detecting low bone mineral density in menopausal women. univ med 2017;36: 123-30. doi: 10.18051/univmed.2017. v36.123-130 pissn: 1907-3062 / eissn: 2407-2230 124 haryono, prastowo simple tool to assess risk of low bone mass introduction reduced bone mineral density, in the form of osteopenia or osteoporosis, frequently occurs in menopausal women. the prevalence of osteoporosis in menopausal women is up to 35.5%-44%,(1,2) and differs according to the location of the bones. in indonesia the prevalence of osteopenia is highest at the femoral neck (45.8%) and lowest at the distal radius (23.2%), whereas the prevalence of osteoporosis is highest at the distal radius (30%) and lowest at the femoral neck (4.9%).(3) bone mineral density is associated with several risk factors, i.e. age, menopause, body weight, and body mass index (bmi). the majority of studies show a correlation of body weight, bmi, and age with bone mineral density.(4,5) elderly females with low body weight and bmi have lower bone mass. another risk factor is socio-economic status. osteoporosis is more frequent in females of lower economic status.(6,7) to find patients with newly acquired osteoporosis in indonesia is not easy because of the still limited access to dual-energy x-ray absorptiometry (dxa). there are several factors limiting access dxa examination i.e. the r el at ively hi gh cost o f th e e xa mi na tion , particularly for community groups with low economic status, the limited number of hospitals possessing dxa equipment, their location in large cities only, and the relatively great distance to these hospitals. it has been estimated that there are only 65 hospitals in indonesia that possess dxa equipment. with the total size of the population of up to 250 million, the ratio of the number of the equipment to the population is less than 0.1 per million population. the limited access to dxa examination results in the actual number of patients with osteoporosis being much larger than reported by the studies. in addition, there should be a selection for females with low bone mineral density for dxa examination. several tools have been developed for evaluating the individual risk for osteoporosis that can be used for screening for dxa examination. the most frequent risk factors used in those tools are among others body weight, age, use of estrogens, and fractures.(8-10) on e o f t h e mos t e a s i l y us e d t oo l s t h a t nevertheless has relatively high sensitivity (up to 90%) is the osteoporosis self-assessment tool (ost).(11) the osteoporosis screening tools take only body weight and age into account.(8-10) there are several other tools that incorporate more risk factors, eg. the simple calculated osteoporosis risk estimation (score), osteoporosis risk assessment instrument (orai), osteoporosis index of risk (osiris), and who fracture risk assessment (frax). in addition to body weight and age, these tools also incorporate several other risk factors, e.g. height, gender, ethnicity, and use of hormones. frax is the tool comprising the highest number of risk factors of up to 11 items. in spite of this, both meta-analytic studies and systematic reviews have shown that the sensitivity of these tools is not higher than or equal to that of ost.(11,12) furthermore, the performance of ost does not show consistent results, its sensitivity being higher than its specificity or vice versa.(13) the present study was carried out to evaluate the performance of ost in a more specific manner i.e. by measuring the bmd of the bones according to location. referrals for dxa examination generally come from clinics or hospitals. in the present study referrals for dxa examination were obtained by directly visiting the community groups without access to dxa examination, so as to obtain data from a valid number of patients with low bone mass. methods design of the study this study of cross-sectional design was carried out at 3 posyandu (integrated health care posts) in tanah merah and luar batang villages, north jakarta, and in cengkareng, west jakarta from august 2014-january 2015. 125 subjects of the study the subjects were 60 menopausal women aged 50-65 years who were members of an elderly group under the guidance of the faculty of medicine, indonesia catholic university of atma jaya, jakarta. the sample size was determined using the formula for area-under-the curve (auc) diagnostics i.e.: where z=1.64, z=1.28, a uc1=0.7, auc2=0.8. with an estimated number of 10% drop-outs, the total sample size required was around 70. the sample selection flow may be seen in figure 1. from 112 subjects, 30 subjects did not meet the inclusion and exclusion criteria and 22 subjects did not agree to undergo bmd examination, for a variety of reasons, such as having no time and being afraid of the side effects of the examination. the inclusion criteria were menopausal women aged 50-65 years, having attended the posyandu for 3 months consecutively, and having experienced menopause for 12 months or more. the exclusion criteria were smoking, being under steroid and hormonal treatment, consuming vitamin d and/or calc ium supplements, having experienced fractures, and suffering from chronic disease. the inclusion and exclusion criteria, and demographic data were obtained through questionnaire-based interviews. measurements an t hr op omet r i c me a s ur eme nt s w e r e performed in the service ward of each posyandu by a physician. body weight was determined on the minimally clothed subjects, using seca digital portable scales (seca, germany) with an accuracy of 0.1 kg. height was measured in the frankfort position without footwear by means of a wall mounted stadiometer with an accuracy of 0.5 cm. body mass index (bmi) was determined by dividing the weight (in kilograms) by the square of the height (in meters) and expressed in kg/m2. 2 aucauc vvzβ2vzα n 21 211            the ost score was calculated with the formula: 0.2 x [body weight (in kilograms) age (in years)]. for example, a female person aged 50 years and weighing 65 kg, has an ost score of 0 .2 ( 65 -50) = 3. t h e ost s co r e s we r e categorized with the cut-off value of 2; the bmd was normal if the ost score was 2, and low if the ost score was <2.(8,11,12) bmd examination bmd examination was performed in siaga hospital, south jakarta, using standard dxa equipment (lunar prodigy df+301251, ge healthcare, usa). examination and reading of the results was performed by a radiologist. the locations of the bones to be examined were according to the recommendations of the international society for clinical densitometry, i.e. the vertebral column (l1-l4), left femoral bone (femoral neck), and total hip.(14) the bmd examination yielded the bmd (g/cm2) and t scores. the t-score was used as a criterion for determining the bmd classification according to who standards, i.e. osteopenia if t-score between between (-2.5) (-1.0) and osteoporosis if t-score <-2.5.(15) for statistical analysis the subjects were grouped according to the t-score, with a cut-off of -1; bmd was low if t-score 0.1, and bmd normal if t-score >-0.1. statistical analysis numerical data were expressed as mean (standard deviation) and categorical data were expressed as values and percentages. the unpaired-t test was used for comparison of the mean between the low-risk group (ost >-1) and the mod erate -high ris k gro ups ( ost< 2). evaluation of the ost performance was by calculating the sensitivity, specificity, positive likelihood ratio (plr), negative likelihood ratio (nlr) and area under the roc curve (area under the curve-auc). the positive likelihood ratio is the probability of a positive ost in subjects with low bmd in comparison with subjects with normal bmd, whereas nlr is the probability of a negative ost in subjects with normal bmd univ med vol. 36 no.2 126 haryono, prastowo simple tool to assess risk of low bone mass as compared to subjects with low bmd. the sensitivity, specificity, plr, and npr are considered to be good if the sensitivity and specificity is up to 90% or more, the plr more than 10 and the nlr less than 0.1. the auc value for diagnostic ability is considered to be sufficiently good if it is 0.700. the significance level was determined at p<0.05. statistical analysis was performed with spss version 17.0 for windows (spss inc., chicago, il.,usa). ethical clearance ethical clearance was granted by the ethics committee of the faculty of medicine, unversity of indonesia and cipto mangunkusumo hospital ( 6 7 9/ un 2.f1/ e t ik / 20 1 4) . t he su b j e c t s provided written and signed informed consent. the identity of the subjects was kept confidential and used only for the purpose of the study. results a total of 60 subjects met the inclusion and exclusion criteria and agreed to undergo dxa examination. the majority of the subjects (44 or 73.3%) were at high risk for low bone mass (ost score <2). subject characteristics and bmd between the two ost groups were significantly different (p< 0.05). in the low risk group, mean bmd at 4 bone locations was in the normal category, whereas in the high risk group, mean bmd was in the categories of osteopenia (femoral neck, trochanter, and total hip) and osteoporosis (lumbar spine). low bmd (t score -1) was most frequent in the lumbar spine (table 1). table 2 shows the ost performance in evaluating low bmd risk in post-menopausal women aged 50-65 years. ost sensitivity was table 1. comparison of subject characteristics and bmd between normal and at risk groups bmd= bone mineral density; ost= osteoporosis self-assessment tool table 2. validity of ost at each bone location bmd= bone mineral density; ci= confidence interval; nlr= negative likelihood ratio; plr= positive likelihood ratio sensitivity (95% ci) specifi city (95% ci) plr (95% ci) nlr (95% ci) bmd, lumbar spin e (l1-l4) 81.4 (66.6 -91.6) 47.1 (22.9-72.2) 1.54 (1.0-2.5) 0.39 (0.2-0.9) bmd, femoral neck 85.4 (70.8 -94.4) 52.6 (28.8-75.6) 1.81 (1.2-2.9) 0.28 (0.1-0.6) bmd, trochanter 92.9 (76.5 -99.1) 43.7 (26.4-62.3) 1.65 (1.2-2.3) 0.16 (0.0-0.7) bmd, total hip 94.6 (81.8 -99.3) 60.9 (38.5-80.3) 2.42 (1.4-4.1) 0.09 (0.0-0.4) normal ost (n=16) low ost (n=44) p value age (years) 53.87 ± 4.38 59.37 ± 4.87 0.000 age at menopause (years) 52.06 ± 3.60 54.39 ± 2.07 0.003 duration of menopause (years) 1.81 ± 1.76 4.99 ± 3.71 0.002 body weight (kg) 69.02 ± 5.46 53.18 ± 6.56 0.000 bmi (kg/m2) 29.74 ± 2.16 23.97 ± 3.03 0.000 bmd, lumbar spine (l1-l4) (g/cm2) 1.00 ± 0.13 0.88 ± 0.15 0.006 bmd, femoral neck (g/cm2) 0.86 ± 0.11 0.71 ± 0.14 0.000 bmd, trochanter (g/cm2) 0.73 ± 0.11 0.60 ± 0.11 0.000 bmd, total hip (g/cm2) 0.93 ± 0.11 0.77 ± 0.12 0.000 normal bmd low bmd bmd, lumbar spine (l1-l4) 17 (28.3%) 43 (71.7%) bmd, femoral neck 19 (31.7%) 41 (68.3%) bmd, trochanter 32 (53.3%) 28 (46.7%) bmd, total hip 23 (38.3%) 37 (61.7%) 127 sufficiently good for the bmd values of the trochanter and total hip. ost specificity was sufficiently good for total hip bmd. the plr and nlr values were low at all bmd locations. according to the area under the curve (auc), ost has lower diagnostic value in the bmd of the lumbar spine, trochanter and femoral neck. the diagnostic value of ost was significant only for total hip bmd (table 3). discussion the results of the present study show that ost functions as a fairly good tool for evaluating low bmd risk in menopausal women aged 50-65 years. ost performance showed a sensitivity in the range between 81-95%, specificity 47-61%, plr and nlr in the range between 1.54-2.42 and 0.09-0.39. however, according to the roc area, ost performance was only significantly different for total hip bmd (auc=77.7%). these study results differed from those of a study in t ha iland, which showed ost sensitivity and specificity of 36-48% and 7175%, respectively. the subjects of the thai study were menopausal women aged 45-87 years and the ost cut off value was -1.(13) similar results were found in argentinian studies, in which the subjects were menopausal women aged 50 years and older and the ost cut off score was 2. the results showed sensitivity and specificity values of 84% and 44%, respectively.(16) the sensitivity and specificity may have been influenced by the younger age of the subjects and the lower cut off value. in subjects less than 50 years old, the bone mass may not have decreased to a great extent, so that the majority of the subjects still had normal bmd values. negative ost scores may decrease the number of subjects who fall in the high risk category because the age of the subjects should be greater than their body weight or their body weight should be less than their age. the ost performance differed according to bone location. the results of the present study showed the highest sensitivity and specificity for total hip. several previous studies have found s i mi l a r r e su l t s. a s tu d y i n ma l a ys i a on me no p a us al su b je c t s me a s ur ed t he ost performance in the proximal femur and lumbar spine, and found that the sensitivity in the proximal femur was higher (87.5% vs 47.8%).(17) the aforementoned study in thailand also reported a higher sensitivity for bmd of the femur (40.6% vs 36.2%).(13) total hip in the present study comprised the femoral neck and trochanter, which are also parts of the proximal femur. the ost sensitivity was highest for bmd of the proximal femur or total hip because in these regions the cortical bone thinned out more rapidly as a result of aging and underwent fractures.(18) the number of subjects with low bmd in the present study was substantial. according to bone location, low bmd was most frequent in the lumbar spine. similar results were obtained in several previous studies.(13,17,18) however, the percentage of subjects with low bmd in the present study was relatively high as compared with the aforementioned studies, and even with a previous study in indonesia.(3) the difference may be the result of differences in t score cut off values, subject characteristics, type of dxa equipment and their operators. in addition to ost, there are several screening tools that are used for evaluating the risk of low bone mass and fractures. these screening tools have bee n developed by incorporating other risk factors besides age and body weight. the fracture risk assessment tool (frax) incorporates the greatest number of risk factors, among others gender, use of estrogens, table 3. area under the receiver operating characteristic curve auc= area under the receiver operating curve; bmd= bone mineral density; ci= confidence interval univ med vol. 36 no.2 auc 95% ci bmd, l umbar spine 64 .2% 0.479 0.806 bmd, femoral neck 69 .0% 0.536 0.844 bmd, trochanter 68 .3% 0.548 0.819 bmd, t otal hip 77 .7% 0.644 0.911 128 haryono, prastowo simple tool to assess risk of low bone mass glucocorticoids, smoking, alcohol, history of fractures, and rheumatoid arthritis. the simple calculated osteoporosis risk estimation (score) incorporates the factors of race, history of fractures, use of estrogens and rheumatoid arthritis. the osteoporosis risk assessment instrument (orai) incorporates only the use of estrogens.(8,10) several studies have been conducted to compare the efficacy of these tools. the study by pecina et al.(8) showed that the sensitivity and auc were 36% and 55% respectively for frax, 74% and 58% for score, 56% and 63% for ost, and 52% and 60% for orai.(8) the study by rubin et al, (10) comparing frax and other tools, found that the performance of frax and the other tools were not much different, with the auc ranging between 70.3%-72.2%.(10) these studies have demonstrated that the increase in added risk factors in these tools did not add to the performance in evaluating the risk of low bone mass. age and body weight are the most influential risk factors for osteoporosis (4,5,19) so that the other factors added to these screening tools could not much increase their performance. body weight is considered to have a protective effect on osteoporosis. the influence of body weight on bone mass is caused by physiological load, mechanical load, a nd endocrine effects.(20-22) however, the protective effect of body weight on reduced bone mass has to date been questioned. the study by greco et al (23) on overweight and obese subjects showed that 33% of female subjects and 45% of male subjects possess lower bmd.(23) it is thought that at a certain degree of obesity, because of the influence of interleukin and tumor necrosis factor ( t nf) , t he a di p oc yt e s ma y cha nge in t o osteoclasts that play a role in bone resorpstion and reduced bone mass.(20-22) the present study has several limitations. first, there are no data on several risk factors that affect bone formation and bone mass, such as the level of physical activity and exposure to sunlight. second, the subjects comprised a relatively homogenous group with regard to ethnicity and social status, which are risk factors for low bone mass. the screening tools for evaluating the risk for osteoporosis are important for the efficiency and efficacy of dxa examinations, which are still costly and unaffordable. there have been many studies on the performance of the tools in detecting the risk of low bone mass. the simple screening tools showed an identical or greater performance as compared with the more complex tools. the use of ost for screening of risk groups is very easy so that it can be carried out not only by medical personnel but also by trained health cadres. in addition, there are many different ethnic groups and socio-economic classes in indonesia. there is a need for studies on elderly groups in each province and of various socio-economic classes to find the prevalence of l ow bmd a nd si mu lt a n e o us l y t e s t t he performance of ost in subjects of different ethnic and socio-economic groups. conclusion this study demonstrated that use of the ost score in postmenopausal women was effective and had adequate sensitivity and specificity. conflict of interest the authors declare that there was no conflict of interests in any form and with any parties whatsoever. acknowledgment the authors express their gratitude to heydi md, nelson md, alfian prasetyo md, and alice angelina md for technical assistance. thanks are also due to ms. chamimah, ms. oon, and ms. pujawati for recruitment of the subjects. we also thank the faculty medicine, indonesia catholic university of atma jaya for the funding of this study. 129 contributors nap and rh contributed to drafting and revision of the manuscript. nap contributed to the study design and data collection. rh contributed to the statistical analysis and interpretation, all authors read and approved the manuscript. references 1. choi yj, oh hj, kim dj, et al. the prevalence of osteoporosis in korean adults aged 50 years or older and the higher diagnosis rates in women who were beneficiaries of a national screening program: the korea national health and nutrition examination survey 2008-2009. j bone miner res 2012;27:1879-86. 2. wright nc, looker ac, saag kg, et al. the recent prevalence of osteoporosis and low bone mass in the united states based on bone mineral density at the femoral neck or lumbar spine. j bone miner res 2014; 29:2520-6. 3. meiyanti. epidemiology of osteoporosis in postmenopausal women aged 47 to 60 years. univ med 2010;29:169-76. 4. montazerifar f, karajibani m, alamian s, et al. age, weight, and body mass index effect on bone mineral density in postmenopausal women. health scope 2014;3:e14075. doi: 10.17795/ jhealthscope-14075. 5. rexhepi s, bahtiri e, rexhepi m, et al. association of body weight and body mass index with bone mineral density in women and men from kosovo. mater sociomed 2015;27:259-62. 6. navarro mc, sosa m, saavedra p, et al. poverty is a risk factor for osteoporotic fractures. osteoporosis int 2009;20:393-8. 7. romero gt, rodriguez n, santana s, et al. prevalence of osteoporosis, vertebral fractures and hypovitaminosis d in postmenopausal women living in a rural environment. maturitas 2014;77:282-6. 8. pecina jl, romanovsky l, merry sp, et al. comparison of clinical risk tools for predicting osteoporosis in women ages 50-64. j am board fam med 2016;29:233-9. 9. ahmadzadeh a, emam m, rajaei a, et al. comparison of three different osteoporosis risk assessment tools: orai (osteoporosis risk assessment instrument), score (simple calculated osteoporosis risk estimation), and ost (osteoporosis self-assessment tool). med j islam repub iran 2014;28:94. 10. rubin kh, abrahamsen b, friis-holmberg t, et al. comparison of different screening tools (frax®, ost, orai, osiris, score and age alone) to identify women with increased risk of fracture: a population-based prospective study. bone 2013;56:16-22. 11. nayak s, edwards dl, saleh aa, et al. systematic review and meta-analysis of the performance of clinical risk assessment instruments for screening for osteoporosis or low bone density. osteoporosis int 2015;26:1543-54. 12. rubin kh, friis-holmberg t, hermann ap, et al. risk assessment tools to identify women with increased risk of osteoporotic fracture: complexity or simplicity? a systematic review. j bone miner res 2013;28:1701-17. 13. chaovisitsaree s, namwongprom sn, morakote n, et al. comparison of osteoporosis selfassessment tool for asian (osta) and standard assessment in menopause clinic, chiang mai. j med assoc thai 2007;90:420-5. 14. hans db, shepherd ja, schwartz en, et al. peripheral dual-energy x-ray absorptiometry in the management of osteoporosis: the 2007 iscd official positions. j clin densitometry 2008;11: 188-206. 15. siris es, adler r, bilezikian j, et al. the clinical diagnosis of osteoporosis: a position statement from the national bone health alliance working group. osteoporos int 2014;25:1439–43. 16. saraví fd. osteoporosis self-assessment tool performance in a large sample of postmenopausal women of mendoza, argentina. j osteoporos 2013; http://dx.doi.org/10.1155/2013/150154. 17. muslim daj, mohd ef, sallehudin ay, et al. performance of osteoporosis self-assessment tool for asian (osta) for primary osteoporosis in post-menopausal malay women. malay orthop j 2012;6:35-9. 18. chen h, zhou x, fujita h et al. age-related changes in trabecular and cortical bone microstructure. int j endocrinol 2013; http:// dx.doi.org/10.1155/2013/213234. 19. morin s, tsang jf, leslie wd. weight and body mass index predict bone mineral density and fracture in women aged 40 to 59 years. osteoporosis int 2009;20:363-70. 20. cao jj. effects of obesity on bone metabolism. j orthop surg res 2011;6:30. doi:10.1186/1749799x-6-30. 21. liu py, ilich jz, brummel-smith k, et al. new insight into fat, muscle, and bone relationship univ med vol. 36 no.2 130 haryono, prastowo simple tool to assess risk of low bone mass in women: determining the threshold at which body fat assumes negative relationship with bone mineral density. int j prev med 2014;5:152-63. 22. migliaccio s, greco ea, fornari r, et al. is obesity in women protective against osteoporosis? diabetes metab syndr obes 2011; 4:273-82. 23. greco ea, fornari r, rossi f, et al. is obesity protective for osteoporosis? evaluation of bone mineral density in individuals with high body mass index. int j clin prac 2010;64:817-20. yenny 46 resistensi antimikroba dari bakteri merupakan suatu masalah kesehatan masyarakat yang sifatnya global. masalah ini menjadi bertambah penting dalam hal pengobatan infeksi enterik. di negara-negara berkembang resistensi terhadap obat-obat lapis pertama (first-line drugs) telah dijumpai di antara luman-kuman patogen enterik, yang disebabkan oleh penggunaan antimikroba yang semenamena. pada beberapa kasus, derajat resistensi memaksa digunakannya obatobat lapis kedua dan ketiga, yang harganya lebih mahal. situasi ini juga mengancam timbulnya resistensi terhadap golongan obat-obat antimikroba golongan ini yang pada akhirnya menyebabkan beban ekonomi yang besar dan resistensi multipel. di banyak negara berkembang, data mengenai prevalensi resistensi antimikroba sedikit sekali ditemukan. kurangnya fasilitas laboratorium merupakan kendala besar dalam mengembangkan surveilans yang efektif. untuk mempertahankan jangka efektivitas suatu antimikroba, khususnya di negara berkembang, perlu dilakukan perbaikan sistem surveilans untuk memantau timbulnya resistensi antimikroba, perbaikan akses laboratorium, peraturan penggunaan antimikroba yang lebih baik, dan pendidikan masyarakat. kata kunci : resistensi, antimikroba, enteropatogen universa medicina januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007januari-maret 2007 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 vol.26 no.1 resistensi dari bakteri enterik : aspek global terhadap antimikroba yenny*a dan elly herwana* abstrak *bagian farmakologi fakultas kedokteran universitas trisakti korespondensi adr. yenny bagian farmakologi fakultas kedokteran universitas trisakti jl. kyai tapa no.260 grogol jakarta 11440 telp. 021-5672731 eks. 2801 email: stasia_mk@yahoo.com universa medicina 2007; 26: 46-56 47 universa medicina vol. 26 no.1 pendahuluan m e s k i p u n r e s i s t e n s i a n t i m i k r o b a d a r i bakteri telah dijumpai semenjak era ‘praantibiotika’ dan di lingkungan yang terpencil yang tidak mengalami paparan antimikroba, perkembangan resistensi antimikroba sejalan dengan perkembangan penggunaan obat-obat antimikroba yang semakin meningkat dan sejalan pula dengan ditemukannya obat-obat baru.(1) pemindahan resistensi (resistance transference) antara bakteria dilaporkan untuk pertama kali p a d a k u m a n e n t e r i k k e t i k a t e r j a d i w a b a h disenteri yang luas di jepang sekitar tahun 1950an. pemindahan resistensi ini tidak saja terjadi secara in vitro pada percobaan laboratorium antara kuman shigella dysenteriae dengan escherichia coli, namun juga dapat terjadi secara in vivo. penelitian-penelitian dengan berbagai kuman patogen lain juga menunjukkan bahwa pemindahan resistensi adalah suatu peristiwa yang umum di dunia mikroba dan dengan dicapainya perkembangan mutakhir di dalam biologi molekuler, pemindahan resistensi ini dapat dijelaskan secara rinci. kuman-kuman patogen telah mampu mengembangkan sejumlah besar mekanisme untuk menghindarkan diri dari efek antimikroba dengan cara membentuk enzim yang dapat merusak antimikroba sampai pada kemampuannya untuk melakukan modifikasi dari proses metaboliknya. situasi ini tidak statis, mekanisme yang baru yang lebih kompleks secara cepat dikembangkan mikroorgansime mengikuti diperkenalkannya dan digunakannya s u a t u a n t i m i k r o b a y a n g b a r u . p e r u b a h a n perubahan genetik yang menyebabkan terjadinya resistensi antimikroba mungkin terjadi melalui global aspects of antimicrobial – resistance of enteric bacterial yenny*a and elly herwana* abstract bacterial resistance to antimicrobial agents is a global public health problem and becoming important in the treatment of enteric infections. in developing countries, resistance to the first-line drugs has been observed among the enteric pathogens due to the uncontrolled usage of antimicrobial agents. in some cases, the level of resistance has forced a change to more expensive second or third-line drugs. resistance has emerged even to newer and more potent antimicrobial agents which caused economic burden and multiple resistance. in many developing countries, few data are available on the prevalence of antimicrobial resistance. the lack of laboratory facilities are major constrains to the development of effective surveillance. to maintain the useful life of antimicrobial drugs, especially in developing countries, there needs to be improved access to diagnostic laboratories, improved surveillance of the emergence of resistance, better regulation of the use of antimicrobial and better education of the public. keywords : resistence, antimicrobial, enteropathogen *department of farmacology medical faculty trisakti university correspondence adr. yenny department of farmacology medical faculty trisakti university jl. kyai tapa no.260 jakarta 11440 email: stasia_mk@yahoo.com universa medicina 2007; 26: 46-56. 48 mutasi, seperti misalnya multidrug resistance dari mycobacterium tuberculosis dan resistensi quinolon pada enterobacteriaceae. mekanisme genetik lain adalah transposon dan integron, yang memungkinkan gen pembawa plasmid menyatu ( integrated) ke dalam kromosom bakteri sehingga resistensi yang terjadi menetap d a n d i t u r u n k a n k e g e n e r a s i b a k t e r i berikutnya.(1) sejumlah penelitian telah menguraikan tentang meningkatnya prevalensi resistensi pada kuman-kuman patogen enterik seperti yang terjadi pada shigella, salah satu kuman enterik penyebab diare. hal ini penting karena diare akut memberikan kontribusi yang cukup besar sebagai penyebab kematian pada anakanak berusia ≤5 tahun di negara berkembang, termasuk indonesia. menurut survei kesehatan rumah tangga (skrt) tahun 2001(2) penyakit diare di indonesia menduduki urutan ke dua dari penyakit infeksi dengan angka morbiditas sebesar 4% dan mortalitas 3,8%. pada bayibayi, penyakit diare menempati urutan tertinggi sebagai penyebab kematian dengan angka sebesar 9,4% dari seluruh kematian bayi.(3) bakteri merupakan patogen yang paling penting sebagai penyebab penyakit diare. penggantian cairan dan elektrolit yang hilang dengan hidrasi oral atau terapi cairan intravena merupakan u p a y a p e n g o b a t a n u t a m a , n a m u n o b a t antimikroba yang diberikan bersamaan dengan rehidrasi memiliki peranan yang sangat penting dalam penanganan diare akut dan diindikasikan bagi diare infektif oleh beberapa bakteri enterik t e r t e n t u ( s h i g e l l a s p p , vi b r i o c h o l e r a e ) . penggunaan antimikroba pada diare tanpa indikasi yang jelas telah menyebabkan banyak j e n i s k u m a n e n t e r i k m e n j a d i r e s i s t e n . d i indonesia dilaporkan bahwa hampir semua spesies shigella, terlebih s. flexneri telah resisten terhadap antimikroba yang menjadi o b a t l a p i s p e r t a m a u n t u k d i a r e s e h i n g g a terpaksa digunakan obat-obat lapis kedua dan k e t i g a , y a n g h a r g a n y a l e b i h m a h a l , d a n menyebabkan beban ekonomi yang besar.(4) di banyak negara berkembang, juga di indonesia, data mengenai prevalensi resistensi a n t i m i k r o b a s e d i k i t s e k a l i d i t e m u k a n . kurangnya fasilitas laboratorium merupakan k e n d a l a b e s a r d i d a l a m p e n g e m b a n g a n surveilans yang efektif. untuk mempertahankan j a n g k a e f e k t i v i t a s s u a t u a n t i m i k r o b a , k h u s u s n y a d i n e g a r a b e r k e m b a n g , p e r l u dilakukan perbaikan sistem surveilans untuk memantau timbulnya resistensi antimikroba, p e r b a i k a n a k s e s l a b o r a t o r i u m , p e r a t u r a n penggunaan antimikroba yang lebih baik, dan pendidikan masyarakat. mekanisme resistensi antimikroba mekanisme utama dari populasi mikroba untuk bertahan hidup dalam situasi terancam adalah dengan cara mutasi genetik, ekspresi dari suatu gen resistensi yang laten, atau melalui gen yang memiliki determinan resistensi. ketiga mekanisme ini dapat berada bersama-sama dalam suatu bakterium. penggunaan antibiotika secara berlebihan dapat menimbulkan tekanan selektif yang mendorong perkembang-biakan mikroorganisme yang resisten (gambar 1).(5) te r d a p a t b u k t i k u a t b a h w a r e s i s t e n s i bakteri terhadap antibiotika sudah ada jauh s e b e l u m ’ e r a a n t i b i o t i k a ’ . ( 6 ) te l a h t e r j a d i ekspansi besar-besaran dari populasi bakteri yang resisten terhadap antibiotik selama ‘era antibiotika’. ekstraksi, purifikasi, sintesis dan pemberian antimikroba dalam jumlah besar yang dilakukan oleh manusia telah mempercepat evolusi bakteri dengan memberikan tekanan selektif terhadap bakteri yang harus memberikan respon untuk bertahan hidup dan menjadi resisten atau mati. mikroba yang semula peka terhadap suatu antimikroba, dapat berubah sifat genetiknya menjadi tidak peka (resisten) atau kurang peka. 49 universa medicina vol. 26 no.1 faktor yang menentukan sifat resistensi mikroba terhadap antimikroba terdapat pada elemen yang bersifat genetik. beberapa bakteri secara intrinsik resisten terhadap antimikroba t e r t e n t u . c o n t o h n y a b a k t e r i g r a m p o s i t i f , kuman ini tidak memiliki membran sel bagian l u a r ( o u t e r m e m b r a n e ) , s e h i n g g a s e c a r a intrinsik resisten terhadap polimiksin yang bekerja merusak membran sel setelah bereaksi dengan fosfat pada fosfolipid membran sel mikroba. kebanyakan resistensi antibiotika terjadi akibat mutasi atau transfer horizontal gen yang membawa sifat resisten. mutasi terjadi secara acak, spontan dan tidak tergantung dari adanya a n t i m i k r o b a . m u t a s i t e r j a d i b i l a t e r d a p a t kekeliruan dalam proses replikasi dna yang luput untuk diperbaiki oleh system dna repair. mutasi terjadi dalam kecepatan bervariasi (104-10-10 per pembelahan sel) dan meliputi proses delisi, substitusi atau adisi satu atau lebih p a s a n g a n b a s a n u k l e o t i d a , s e h i n g g a menghasilkan substitusi asam amino. proses m u t a s i y a n g d i k e n a l s e b a g a i s i n g l e s t e p mutations menyebabkan timbulnya resistensi tingkat tinggi dalam jangka waktu singkat dan cepat. contohnya, mutasi di dalam sistem yang mengatur kromosom yang mengkode (coded) produksi β-laktamase oleh enterobacter dan c i t ro b a c t e r s p p . h a l i n i m e n g a k i b a t k a n terjadinya produksi β-laktamase dalam jumlah yang sangat besar dalam waktu yang sangat s i n g k a t s e h i n g g a d a p a t m e n g h i d r o l i s i s antimikroba bahkan yang stabil terhadap βlaktamase seperti seftazidim dan sefotaksim (gambar 2).(6) gambar 1. resistensi antibiotika oleh mutasi genetik akibat perubahan pada: a. kode ikatan protein; b. ribosom; c. struktur membran; d. inaktivasi enzim(5) 50 sebaliknya, pada multistep mutation terjadi p e n i n g k a t a n r e s i s t e n s i s e c a r a b e r t a h a p . contohnya adalah mutasi dna gyrase (gyra dan p a r c ) p a d a k u m a n s a l m o n e l l a s p p a t a u staphylococcus aureus sehingga menimbulkan r e s i s t e n s i k l i n i s t e r h a d a p f l u o r o k u i n o l o n . kebanyakan informasi genetik bakteri dikode oleh kromosom, tapi tidak semuanya. beberapa kuman memiliki gen ekstrakromosom baik berupa plasmid (untaian dna ganda berpilin berukuran 2-200 kilobase tiap pasang) atau b a c t e r i o f a g ( v i r u s b a k t e r i y a n g d a p a t berintegrasi ke dalam kromosom bakteri). elemen genetik ekstrakromosom ini dapat ditransmisikan secara vertikal (dari bakteri ke keturunannya melalui pembelahan biner) dan y a n g l e b i h p e n t i n g l a g i t r a n s m i s i s e c a r a horizontal yang dapat melintasi spesies dan genus. faktor resistensi yang dipindahkan dapat berlangsung dari kromosom ke plasmid atau vice versa pada transposon dan integron. tidak setiap p l a s m i d d a p a t d i p i n d a h k a n . ya n g d a p a t dipindahkan adalah plasmid faktor r, disebut juga plasmid penular (infectious plasmids). faktor r sendiri terdiri atas dua unit: segmen re s i s t a n c e t r a n s f e r f a c t o r ( rt f ) d a n d e t e r m i n a n r ( u n i t r ) . s e g m e n rt f memungkinkan terjadinya perpindahan faktor r. masing-masing unit-r membawa sifat resistensi terhadap satu antimikroba. dengan demikian b e r b a g a i u n i t r p a d a 1 p l a s m i d f a k t o r r membawa sifat resistensi terhadap berbagai antimikroba sekaligus. faktor r ini ditularkan terutama di antara enterobakteria. transposon merupakan gen individual atau sekelompok kecil gen resisten yang terikat terbalik atau secara langsung mengulang rangkaian dna (like bookends). integron terdiri dari 2 segmen dna di mana pada salah satu sisi terdapat gen yang resisten terhadap antibiotika. bakteri yang resisten terhadap banyak antibiotika disebabkan oleh plasmid yang mengalami resistensi multipel atau terdapatnya gen dalam kromosom yang membawa sifat resistensi.(6) traktus gastrointestinal khususnya pada b a g i a n d i s t a l m e r u p a k a n t e m p a t u t a m a s e b a g i a n b e s a r d a r i f l o r a n o r m a l . a d a b e r m a c a m m a c a m p o p u l a s i b a k t e r i u s u s , bakteri gram negatif aerobik yang utama adalah escherichia coli (sekitar 107 colony-forming unit/g tinja).(6) ketika antibiotika diberikan, efek yang diharapkan adalah agar antibiotika itu mampu membunuh bakteri patogen yang merugikan dan pasien dapat disembuhkan. tapi yang jarang menjadi pertimbangan adalah bahwa banyak antibiotika diabsorbsi secara tidak sempurna atau akan di ekskresi kembali dalam bentuk utuh atau bentuk yang telah gambar 2. resistensi antibiotika β-lactam oleh β-lactamase 51 universa medicina vol. 26 no.1 mengalami modifikasi tapi masih mempunyai aktivitas antimikroba. dengan demikian setiap kali antibiotika digunakan, flora normal akan terpapar dalam konsentrasi dan lama pemberian obat yang bervariasi. pemakaian antibiotika, khususnya bila digunakan tanpa aturan yang jelas seperti yang banyak terjadi di negara berkembang akan menyebabkan penggunaan a n t i b i o t i k a s e c a r a t i d a k r a s i o n a l . a d a n y a p e n g g u n a a n a n t i b i o t i k a s e c a r a b e r l e b i h a n menyebabkan tingginya prevalensi resistensi p a d a f l o r a n o r m a l a e r o b i k . s t u d i y a n g dilakukan di inggris menunjukan pada 11% anak sehat bakteri koliform di dalam tubuhnya telah resisten terhadap kloramfenikol, dan sering kali juga berkaitan dengan resistensi t e r h a d a p a m p i s i l i n , s t r e p t o m i s i n d a n s p e k t i n o m i s i n . b a h k a n 3 , 2 % a n a k a n a k mengekskresi bakteri yang resisten terhadap seftazidim.(7) f l o r a k o m e n s a l u s u s d a p a t b e r p e r a n sebagai reservoir bagi gen yang resisten terhadap antimikroba. gen-gen ini seringnya berupa plasmid, transposon atau integron. keberadaan gen-gen ini merupakan faktor yang menentukan bagi timbulnya resistensi terhadap banyak obat. bila hal ini terjadi maka penggunaan satu antimikroba dapat menimbulkan resistensi pada beberapa jenis antimikroba lain yang tidak mempunyai hubungan sehingga terjadi resistensi s i l a n g ( c o s s re s i s t a n c e ) . f a k t o r y a n g menentukan resistensi pada banyak obat ini dapat dipindahkan pada bakteri patogen yang menginfeksi usus.(1) penyebaran resistensi antimikroba dari patogen enterik meningkatnya resistensi kuman enterik secara progresif di negara-negara berkembang menimbulkan keprihatinan yang besar pada b a n y a k p i h a k d a n m e n j a d i s u a t u m a s a l a h kesehatan masyarakat yang serius. meskipun data surveilans mengenai prevalensi resistensi terhadap berbagai obat antimikrobial di daerah t r o p i k d a n n e g a r a b e r k e m b a n g s e d i k i t jumlahnya, data yang dilaporkan menunjukkan adanya resistensi antimikrobial yang luas di d a e r a h i n i , b a h k a n p a d a b e b e r a p a k u m a n p a t o g e n e n t e r i k t e r t e n t u t e r c a t a t a d a n y a peningkatan prevalensi resistensinya.(1) penyakit diare akut di mana pemberian antibiotika merupakan upaya pengobatan yang efektif dan sangat dianjurkan adalah penyakitp e n y a k i t s e p e r t i s h i g e l l o s i s , d a n k o l e r a . sejumlah besar kuman-kuman enterik patogen y a n g d a p a t m e n y e b a b k a n i n f e k s i s a l u r a n gastrointestinal sudah mengalami resistensi. c a m p y l o b a c t e r j e j u n i d a n c . c o l i merupakan salah satu penyebab gastroenteritis pada manusia dan menyebar di seluruh dunia. mikroorganisme ini adalah flora normal dari usus unggas, sapi dan hewan ternak lainnya. penyebaran ke manusia biasanya terjadi karena konsumsi daging unggas mentah, kontaminasi silang dari daging unggas ke produk makanan l a i n , d a n m i n u m s u s u y a n g p r o s e s pasteurisasinya tidak sempurna atau minum air yang belum dimasak.(1) pada umumnya infeksi campylobacteriosis merupakan infeksi yang sifatnya self-limiting dan tidak memerlukan pengobatan antibiotika. dalam beberapa kasus infeksi dapat bersifat invasif dan sangat berat s e h i n g g a m e m e r l u k a n a n t i b i o t i k a y a i t u e r i t r o m i s i n a t a u f l u o r o q u i n o l o n . n a m u n , p r e v a l e n s i c . j e j u n i d a n c . c o l i y a n g mengalami resistensi terhadap antimikroba ditemukan meningkat di berbagai negara di dunia.(8-10) resistensi yang timbul ini ternyata berkaitan dengan penggunaan antimikroba di peternakan untuk memacu pertumbuhan ternak d a n m e n g o b a t i p e n y a k i t i n f e k s i p a d a ternak.(10,11) d i a m e r i k a s e r i k a t r e s i s t e n s i campylobacter terhadap quinolon meningkat secara tajam dalam waktu singkat. menurut food and drugs administration (fda) amerika 52 serikat, timbulnya resistensi campylobacter t e r h a d a p f l u o r o q u i n o l o n d i d a p a t k a n d a r i konsumsi ayam yang makanannya dicampur antibiotika agar ternak menjadi gemuk, atau antibiotika ini diberikan untuk mengobati penyakit infeksi unggas.(12) resistensi terhadap quinolon dilaporkan dari banyak negara di dunia, b a i k n e g a r a i n d u s t r i m a u p u n n e g a r a berkembang. paparan terhadap fluoroquinolon, baik pada manusia maupun pada hewan, dapat m e n g i n d u k s i t e r j a d i n y a r e s i s t e n s i p a d a campylobacter. resistensi campylobacter jejuni terhadap antimikroba bahkan ditemukan pada burung-burung liar di swedia.(13) s a l m o n e l l a e n t e r i c a yang terdiri atas serotipe atau serovar.typhi, dan ser. paratyphii a, b, c dan salmonella non-tifoid yang secara predominan menjadi penyebab penyakit diare, ada kalanya bersifat invasif dan menyebabkan infeksi ekstraintestinal terutama pada mereka y a n g d a y a t a h a n t u b u h n y a r e n d a h s e p e r t i penderita penyakit imun. serotipe salmonella non-tifoid yang paling sering dijumpai pada manusia adalah typhimurium, enteritidis, h a d a r, vi r c h o w, d a n c h o l e r a e s u i s . ( 1 4 ) pengobatan antibiotika tidak diperlukan untuk salmonella gastroenteritis, tetapi sangat esensial untuk demam tifoid, salmonellosis invasif, dan infeksi ekstraintestinal. diperkenalkannya kloramfenikol pada tahun 1948 sebagai standar pengobatan untuk tifoid telah membawa dampak yang luar biasa dalam pengobatan demam tifoid. kloramfenikol mampu menurunkan angka kematian yang disebabkan oleh penyakit tersebut secara sangat bermakna.(15) akan tetapi, dalam jangka waktu dua tahun setelah digunakan, telah terjadi resistensi terhadap antibiotika ini meskipun belum menjadi masalah besar. baru pada tahun 1972 resistensi tifoid terhadap kloramfenikol m e n j a d i s u a t u m a s a l a h y a n g b e s a r. ( 1 6 ) m u n c u l n y a g a l u r y a n g r e s i s t e n t e r h a d a p k l o r a m f e n i k o l d a n a n t i b i o t i k a l a i n n y a merupakan suatu kemunduran yang sangat besar.wabah tifoid yang resisten terhadap kloramfenikol dilaporkan terjadi di berbagai tempat seperti di meksiko, india, vietnam, korea, pakistan, bahkan beberapa kasus juga ditemukan di indonesia.(17) menjelang akhir tahun 1980-an, salmonella enterica ser. typhi menunjukkan resistensi secara simultan terhadap beberapa antibiotika yang digunakan sebagai f i r s t l i n e d r u g s s e p e r t i k l o r a m f e n i k o l , k o t r i m o k s a z o l , t e t r a s i k l i n , d a n a m p i s i l i n . masalah yang ditimbulkan oleh penyakit demam tifoid menjadi lebih rumit karena adanya galur k u m a n y a n g m u l t i r e s i s t e n ( m u l t i d r u g resistance). meskipun resistensi ini bervariasi dari satu daerah dan daerah lainnya serta berbeda dari tahun ke tahun, namun jelaslah bahwa kemungkinan akan bertambah luasnya galur multiresisten ini telah menimbulkan banyak kekuatiran. timbulnya multiple drug resistance (mdr) dari s. typhi dilaporkan dari d a e r a h d a e r a h d i a s i a ( i n d i a , p a k i s t a n , banglades, vietnam, korea),(18) dan negaranegara di timur tengah. adanya mdr juga dilaporkan berbagai negara di afrika seperti kenya(19) dan nigeria.(20) di indonesia, galur s. ty p h i y a n g r e s i s t e n k l o r a m f e n i k o l t i d a k persisten, setelah tahun 1978 hingga kini semua isolat s. typhi sensitif terhadap obat-obat antibiotika di atas.(4) te r j a d i n y a m d r t e r h a d a p o b a t o b a t antibiotika di atas menyebabkan digunakannya obat alternatif di dalam pengobatan demam tifoid, seperti misalnya golongan fluorokuinolon. p e n g g u n a a n f l u o r o k u i n o l o n ( m i s a l n y a siprofoksasin) pada mulanya menunjukkan hasil yang memuaskan namun penggunaan obat secara berlebihan akhirnya menyebabkan kepekaan s. ty p h i t e r h a d a p f l u o r o k u i n o l o n m u l a i menurun.(21) akhir-akhir ini, di asia ditemukan galur yang telah tidak peka lagi terhadap golongan fluorokuinolon sehingga menimbulkan masalah dalam pengobatan tifoid. suatu wabah 53 universa medicina vol. 26 no.1 yang disebabkan galur semacam terjadi di tajikistan, menyebabkan 8.000 orang sakit dengan 150 kematian.(22) mdr yang dialami oleh salmonella enterica ser typhi, typhimurium dan serovar lainnya dengan prevalensi yang rendah tampaknya mulai berkembang pesat dengan mendapatkan tambahan integron pada p l a s m i d a t a u k r o m o s o m d n a . ta m b a h a n plasmid yang mengkode β-laktamase mempunyai kemampuan untuk menghidrolisis antibiotik b e r s p e k t r u m l u a s s e p e r t i s e f o k s i t i n a t a u seftriakson. resistensi terhadap antibiotika yang t e r m a s u k d a l a m e x t e n d e d s p e c t r u m betalactamase (esbl) ini dilaporkan dari banyak tempat seperti afrika, asia, timur tengah, eropa dan amerika.(1,6) morbiditas dan mortalitas yang disebabkan o l e h s h i g e l o s i s c u k u p t i n g g i p a d a n e g a r a berkembang. dari 165 juta kasus diare yang disebabkan oleh shigella 99% terjadi di negara berkembang dan sebesar 69% episode diare dialami oleh anak balita. lebih dari 1,1 juta kematian disebabkan oleh infeksi shigella di negara berkembang, 60% kematian terjadi pada anak balita. terdapat empat spesies shigella yaitu s. dysentriae, s. flexneri, s. bodyii dan s. sonnei.(23) shigellosis merupakan salah satu penyebab diare akut di mana antimikroba di indikasikan. perkembangan resistensi kuman patogen e n t e r i k d i l a p o r k a n p e r t a m a t a m a p a d a s . dysenteriae. kuman ini menyebakan wabah yang luas di beberapa daerah di afrika seperti somalia, zaire, rwanda dan burundi. (1) di a s i a , w a b a h o l e h m u l t i p l e re s i s t a n t s . dysenteriae di india, bangladesh, myanmar dan thailand. selama beberapa dekade kumankuman ini secara progresif menjadi resisten terhadap first-line antimicrobial drugs, seperti ampisilin, kotrimoksazol, kloramfenikol dan tetrasiklin, akibat penggunaan obat antimikroba s e c a r a l u a s . ( 6 ) s h i g e l l a d y s e n t r i e t i p e i m e r u p a k a n s p e s i e s y a n g p a l i n g s e r i n g menunjukkan resistensi terhadap obat-obat yang disebutkan di atas. tahun 2003 laporan dari beberapa negara ditemukannya shigella d y s e n t r i e t i p e i y a n g r e s i s t e n t e r h a d a p siprofloksasin dan fluorokuinolon lain. pola resistensi yang berbeda ditunjukkan oleh s. flexneri dan s. sonnie. umumnya s. flexneri menunjukkan resistensi terhadap ampisilin, kloramfenikol dan tetrasiklin, sedangkan s. s o n n e i r e s i s t e n t e r h a d a p t r i m e t o p r i m s u l f a m e t o k s a z o l d a n t e t r a s i k l i n . o b a t antimikroba yang masih cukup efektif adalah mecillinam, fluoroquinolon, seftriakson dan azitromisin.(23) rehidrasi merupakan terapi utama yang paling penting dilakukan untuk penanganan penyakit kolera, namun penggunaan antimikroba yang tepat dapat mengurangi beratnya penyakit dan lamanya eksresi vibrio cholerae sehingga membatasi penyebaran penyakit. tetrasiklin, doksisiklin, furazolidin dan asam nalidiksat digunakan untuk mengatasi penyakit ini. sejak tahun 1994 ditemukan peningkatan v. cholerae o1 yang menunjukkan resistensi multipel t e r h a d a p a m p i s i l i n , k o t r i m o k s a z o l , a s a m nalidiksat.(24) di antara v. cholerae o1 dan o139 juga terdapat resistensi terhadap furazolidin, tapi secara umum prevalensi resistensi v. cholerae o139 lebih kecil dibandingkan o1. resistensi pada v. cholerae biasanya terjadi akibat penambahan plasmid atau integron pada flora normal. studi yang dilakukan taneja et al(25) di india menunjukkan v. cholerae masih peka terhadap terhadap tetrasiklin, kecuali asam nalidiksat (89,5% resisten) dan kotrimoksazol (77,8% resisten). escherichia coli dapat merupakan bakteri k o m e n s a l , p a t o g e n i n t e s t i n a l d a n p a t o g e n ekstraintestinal (infeksi traktus urinarius, meningitis, septicemia). traveler ’s diarrhea dapat disebabkan oleh berbagai etiologi, tapi y a n g p a l i n g s e r i n g d i s e b a b k a n o l e h enterotoxigenic escherichia coli (etec). di 54 negara berkembang etec juga merupakan penyebab diare akut pada anak-anak dan orang dewasa, umumnya oleh karena tidak cukupnya ketersediaan air bersih dan karena higiene yang buruk.(26) obat pilihan utama antimikroba untuk mengobati diare yang disebabkan etec adalah doksisiklin. resistensi yang meningkat dari etec terhadap antimikroba menyebabkan kotrimoksazol dan fluorokuinolon digunakan u n t u k m e n g a t a s i i n f e k s i i n i . b e l a k a n g a n diketahui e. coli dengan resistensi multipel (juga terhadap fluorokuinolon) pada beberapa k a s u s b e r k a i t a n d e n g a n p e n g g u n a a n antimikroba pada makanan ternak. k e p e k a a n k u m a n e n t e r i k d i indonesia st u d i p e n d a h u l u a n u n t u k m e n g e t a h u i organisme penyebab diare sudah dilakukan di indonesia. pada studi yang dilaksanakan dari tahun 1997-1999, s. flexneri ditemukan sebagai p e n y e b a b t e r s e r i n g p e n y a k i t d i a r e d i masyarakat yang berdomisili di jakarta. hasil tes kepekaan antimikroba menunjukkan bahwa kuman-kuman enterik patogen umumnya masih sensitif terhadap quinolon kecuali beberapa isolate c. jejuni yang menunjukkan resistensi terhadap siprofoksasin, asam nalidiksat, dan norfloksasin.(27) pada studi tahun 1995-2001, berhasil diisolasi 2.812 enterpatogen penyebab diare dari 8 rumah sakit di indonesia. (28) vibrio cholera o1 (37,1%) merupakan patogen yang paling tinggi frekuensinya diikuti oleh shigella spp (27,3%), sallmonella spp (17,7%), v. parahaemoliticu s (7,3%), s. typhi (3,9%), c. jejuni (3,6%), v. cholerae non-o1 (2,4%), dan s. paratyphi a (0,7%). dari 767 shigella spp y a n g b e r h a s i d i i s o l a s i , 8 2 , 2 % a d a l a h s . f l e x n e r i , 1 5 % s . s o n n e i , d a n 2 , 2 % s . dysentriae. tes kepekaan kuman enteropatogen t e r h a d a p a n t i m i k r o b a d i l a k u k a n d e n g a n m e n g g u n a k a n 8 a n t i m i k r o b a a n t a r a l a i n a m p i s i l i n , t r i m e t o p r i m s u l f a m e t o k s a z o l , k l o r a m f e n i k o l , d a n t e t r a s i k l i n , s e f a l o t i n , seftriakson, norfloksasin, dan siprofloksasin. s h i g e l l a s p p r e s i s t e n t e r h a d a p a m p i s i l i n , trimetoprim-sulfametoksazol, kloramfenikol, dan tetrasiklin. salmonella enterica ser. typhi d a n s . p a r a t y p h i a m a s i h p e k a t e r h a d a p antimikroba yang disebut di atas. sejumlah kecil v. cholera o1 resisten terhadap ampisilin, trimetoprim-sulfametoksazol, kloramfenikol, dan tetrasiklin; tapi masih sensitif terhadap seftriakson, norfloksasin, dan siprofloksasin. hal yang sama juga ditemukan pada v. cholera non-o1. campylobacter jejuni menunjukkan peningkatan resistensi terhadap seftriakson, norfloksasin, dan siprofloksasin, tapi masih s e n s i t i f t e r h a d a p e r i t r o m i s i n . s t u d i i n i m e n u n j u k k a n , s e l a i n c . j e j u n i d a n v. parahaemolyticus, tampaknya sebagian besar e n t e r i k p a t o g e n m a s i h s e n s i t i f t e r h a d a p fluorokuinolon.(29) meskipun di indonesia belum ditemukan galur s. typhi yang resisten terhadap asam nalidiksat, maupun terhadap siprofloksasin. k e n y a t a a n n y a s e m u a i s o l a t s . ty p h i d i indonesia masih sensitif terhadap semua jenis a n t i b i o t i k a , n a m u n d e n g a n a d a n y a g a l u r r e s i s t e n s i t e r s e b u t , t e t a p d i p e r l u k a n kewaspadaan. sebagai konsekuensinya, pada setiap penderita dengan dugaan demam tifoid harus dilakukan pemeriksaan mikrobiologis berupa pemeriksaan biakan kuman, sekaligus k e m u d i a n m e n e n t u k a n p o l a k e p e k a a n antibiotika dari isolat tersebut. pola kepekaan antibiotika pada etec sebagai penyebab diare akut sudah dilaksanakan di denpasar, bali dari tahun 2000-2001. ( 2 9 ) insiden etec paling tinggi ditemukan pada anak-anak usia 1-15 tahun. frekuensi resistensi etec terhadap antimikroba ditemukan paling banyak terhadap a m p i s i l i n , t r i m e t o p r i m s u l f a m e t o k s a z o l , kloramfenikol, tetrasiklin dan sefalotin. semua 55 universa medicina vol. 26 no.1 galur etec masih peka terhadap norfloksasin, siprofloksasin, asam nalidiksat.(29) studi yang dilakukan oleh agtini et al(30) di jakarta utara terhadap diare yang disebabkan shigellosis dan kolera menunjukkan anak berusia 1 2 t a h u n m e m p u n y a i i n s i d e n t e r t i n g g i shigellosis (32/1.000/tahun) dan s. flexneri m e r u p a k a n s p e s i e s y a n g p a l i n g s e r i n g ditemukan, yaitu dengan prevalensi sebesar 73% sedangkan 95% dari padanya menunjukkan resistensi terhadap ampisilin, trimetoprimsulfametoksazol, kloramfenikol dan tetrasiklin, tapi masih peka terhadap asam nalidiksat, siprofloksasin, seftriakson. resistensi v. cholera terhadap antimikroba belum menjadi masalah di indonesia. ti n g g i n y a r e s i s t e n s i e n t e r i k p a t o g e n terhadap obat pilihan pertama untuk pengobatan penyakit diare sudah terjadi juga di indonesia. besarnya persentase penderita diare yang mendapat antimikroba sangat mengkuatirkan k a r e n a a k a n m e n i m b u l k a n t e k a n a n u n t u k t e r j a d i n y a g a l u r b a k t e r i y a n g r e s i s t e n . p e n i n g k a t a n r e s i s t e n s i p r o g r e s i f t e r h a d a p s i p r o f o k s a s i n a k a n m e n i m b u l k a n m a s a l a h kesehatan besar di kemudian hari. kesimpulan r e s i s t e n s i a n t i m i k r o b a t e r h a d a p enteropatogen terus berlangsung di negara berkembang maupun negara maju. khususnya di negara berkembang keadaan ini didukung oleh banyak faktor. faktor-faktor tersebut antara lain: tersebar luasnya ketersediaan obat antimikroba yang dapat dibeli tanpa resep, regimen terapi yang sub-optimal, penggunaan p o l i a n t i m i k r o b a , k 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trial. antimicrob agents chemother 1993; 37: 1572-5. 16. mirza sh, beeching nj, hart ca. multi-drug resistant typhoid: a global problem. j med microbial 1996; 44: 317-19. 17. sanborn wr, lesmana m, dennis dt, trenggonowati r, kadirman, lita i, et al. antibiotic resistant typhoid in indonesia. lancet 1975; ii: 408-9. 18. lee k, yong d, yum jh. lim ys, kim hd, lee bk, chong y. emergence of multidrug-resistant sallmonella enterica serovar typhii in korea. antimicrob agents chemother 2004; 48: 41305. 19. kariuki s, revathi g, muyodi j, mwituri j, munyalo a, mirza s, et al. characterization of mulridrug-resistant thyphoid outbreaks in kenya. j clin microbiol 2004; 42: 1477-82. 20. akinyemi ko, smith si, oyefolu ao, coker ao. multidrug resistance in sallmonella enterica serovar typhi isolated from patients with typhoid fever complication in lagos, nigeria. public health 2005; 119: 321-7. 21. mohanty s, renuka k, sood s, das bk, kapil a. antibiogram pattern and seasonality of salmonella serotypes in a north indian tertiary care hospital. epidemiol infect 2006; 14:1-6. 22. mermin jh, villar r, carpenter j, robert l, samaridden a, gasanova l, et al. a massive epidemic of multi-drug resistant typhoid fever in tajikistan associated with consumption of municipal water. j infect dis 1999; 179: 1416-22. 23. niyogi sn. shigellosis. j microbiol 2005; 43: 13343. 24. garg p, cakraborty s, basu i. expanding multiple antibiotic resistance among clinical strains of vibrio cholerae isolated from 1992-7 in calcutta, india. epidemiol infect 2000; 94: 323-26. 25. taneja n, mohan b, khurana s, sharma m. antimicrobial resistance in selected bacterial enteropathogen in north india. indian j med res 2004; 120: 39-43. 26. qadri f, svennerholm am, faruque as, sack rb. enterotoxigenic escherichia coli in developing countries: epidemiology, microbiology, clinical features, treatment, and prevention. clin microbiol rev 2005; 18: 465-83. 27. oyono ba,subekti d, tjaniadi p, machpud n, komalarini s, setiawan b. enteropathogen associated with acute diarrhea in community and hospital patient in jakarta, indonesia. fems immunol med microbiol 2002; 34: 139-46. 28. tjaniadi p, lesmana m, subekti d, machpud n, komalarini s, santoso w, et al. antimicrobial resistance of bacterial pathogen associated with diarrhea patients in indonesia. am j trop hyg 2003; 68: 666-70. 29. subekti ds, lesmana m, tjaniadi p, machpud n, sriwati, sukarma, et al. prevalence of enterotoxigenic escherichia coli (etec) in hospitalized acute diarrhea patients in denpasar, bali, indonesia. diagn microbiol infect dis 2003; 47: 399-405. 30. agtini md, rooswanti s, lesmana m, punjabi nh, simanjuntak s, wangsaputra f, et al. the burden of diarrhea, shigellosis dan cholera in north jakarta, indonesia: findings from 24 month surbeillance. bmc infect dis 2005; 5: 89: 1-11. c:\users\jurnal fkusakti\docume 157 universa medicina september-december, 2017 vol.36 no.3 imminent threat of antibiotic resistance and the importance of diagnostic and antibiotic stewardship erlangga yusuf department of medical microbiology, antwerp university hospital (uza), university of antwerp, belgium antibiotics are one of the hallmarks of modern medicine. antibiotics have saved many lives that was unthinkable prior to their discovery. yet, antibiotic use is associated with the danger of bacterial resistance. bacterial resistance to antibiotics means simply that antibiotics cannot be used to treat infection, that a simple skin and soft tissue infection may lead to death. already in 1945, when alexander fleming received his nobel prize for his part in the discovery of penicillin, he delivered a speech where he warned about this danger.(1) in his speech, he mentioned the problem of underdosage of antibiotics. as the time passed by, we know that the problem is not only related to underdosage but also to other aspects of inappropriate use of antibiotics such as unnecessary use (for example when the infection is due to a virus), and extended duration of antibiotics.(2) the danger of resistance is imminent. in less than 20 years, extended spectrum beta-lactamase (esbl), i.e. resistance of gram-negative bacteria to third generation cephalosporines has become common in many medical institutions in the world.(3) extended spectrum beta-lactamase bacteria can still be treated with antibiotics from the carbapenem class, but resistance to this type of antibiotic has emerged too in the so-called carbapenemase-producing enterobacteriaceae (cpe). when cpe bacteria are found in a patient, not many other antibiotic options are left and resistance to last option antibiotics has been described. two years ago, the world was shocked when a group from china reported the finding of the genes that coded for the resistance to colistin.(4) while the speed of the emergence of bacterial resistance is high, the speed of the discoveries of new antibiotics is slow due to economical and scientific reasons.(5) unlike chemotherapeutic or anti-rheumatoid drugs for example, antibiotics are normally used for short term treatment and they are financially not promising for pharmaceutical industries. developing of new antibiotics is also hampered by the necessity to find targets in the bacteria that are not prone to rapid development of resistance and by problems with bacterial entry and efflux in gram-negative organisms. since the development of new antibiotics is so difficult, we have no choice at this moment except to conserve the antibiotics that we still have. to that end, diagnostic and antibiotic stewardship should be put in place. diagnostic stewardship is defined as coordinated guidance and interventions to improve appropriate use of microbiological diagnostics to guide therapeutic decisions. in other words, diagnostic testing is needed to establish the presence or absence of infection.(6) while the term diagnostic stewardship is relatively new, the term is antibiotic stewardship is already in place for a while. the idea of antibiotic stewardship is to have coherent actions wh i c h p r omo t e u s i ng a n t i mi c r ob i a l s responsibly.(7) the most important similarity of both terms is the ‘coordinated action’. to 158 universa medicina achieve the aim, all actors should have a role and work together in concerted action. the prescriber should make a diagnosis of infection based on clinical judgement. he should perform a diagnostic test, either culture or serology based. he can be aided by the clinical microbiologist who has the knowledge of the performance of a test. the clinical microbiologist has a very i mpo r t a n t r o le i n p r e ve n t in g mi s u se of microbiological tests. for example, taking a superficial swab for a skin and soft tissue infection should be discouraged since the finding of skin microorganisms will prompt unnecessary antibiotic therapy. when the clinician decides that an antibiotic is really needed, he should give the antibiotic in the correct dose, right duration and whenever possible by the oral mode of administration. the finding of the pathogen, and the antimicrobial susceptibility should be reported timely by the laboratory, within 48 to 72 hours. based on the laboratory results the empiric antibiotic that has been started should be de-escalated to a narrower spectrum. when multidrug resistant organisms (mdro) such as esbl and cpe are found in a patient, the patient should be isolated to prevent further spread of mdro. this action should be coordinated by infection prevention nurses and nurses of the ward. the pharmacologist should aid the clinician with the right dose of antibiotics. since all of this should be coordinated, the role of the hospital management is also important. they should ma i nt a i n the d i a gno s ti c a nd a n t i bi o ti c stewardship process. the process should be continuously present and not only as a short program. in some countries in europe the management is aided by an antimicrobial stewardship team as requested by law. clearly, this coordinated effort needs financial resources. in middle-income countries such as indonesia, the healthcare system with plenty of financial resources is the private one. however, in private healthcare, there is somehow the emphasis on pleasing the patient, so that antibiotics are perhaps prescribed more often than in public healthcare. the private healthcare system has also no financial benefit by containing mdro spread since the patients infected by mdro will stay longer in the hospital and ironically enough it will generate more money for the private hospital. in general, as a middle-income country, like many other countries in south east asia, indonesia’s healthcare system is also not bestowed with plenty of financial resources, and diagnostic and antimicrobial stewardship are also rare.(8) simply copying the implementation of diagnostic and antimicrobial stewardship from high income countries is surely not possible for a middle-income country. the diagnostic and antimicrobial stewardship in high-income countries are also merely oriented to second and third line healthcare, i.e. hospitals and not the first line care. first line care is perhaps the setting where the most antibiotics are prescribed in middle-income countries. some adaptation is clearly needed but the most important thing is political support. the ministry of health should put the fight against antimicrobial resistance as priority. awareness of the danger of antimicrobial resistance should be raised not only for the doctors but also for the pa tients. medic al faculties and scientif ic associations for the general practitioners and other medical specializations must be involved. social media should be used to promote the campaign to teach the patients that antibiotics are not always needed. regulation for sure is also needed on curbing and stopping over the counter and counterfeit antibiotics. it is also important to perform surveillance of antibiotics use and the prevalence of mdro. these data can perhaps be obtained by health authorities or statistics offices. these data are important in order to be able to react immediately when an increasing trend is found. antibiotic resistance is an imminent problem, the threat is real, and the solution to this problem is not easy. political will is needed and due to financial limitation and private oriented health care system, diagnostic and antimicrobial stewardship as performed in highincome countries should be adapted locally. 159 univ med 2017;36:157-9. doi: http://dx.doi.org/10.18051/univmed.2017.v36.157-159 september-december, 2017 vol.36 no.3 references 1. fleming a. nobel lecture: penicillin. nobel lect physiol or med 1945;83–93.available at: https:// www.nobelprize.org/nobel_prizes/medicine/ laureates/1945/fleming-lecture.pdf. accessed october 12, 2017. 2. morency-potvin p, schwartz dn, weinstein ra. antimicrobial stewardship: how the microbiology laboratory can right the ship. clin microbiol rev 2017;30:381–407. 3. gniadkowski m. evolution and epidemiology of extended-spectrum β-lactamases (esbls) and esbl-producing microorganisms. clin microbiol infect 2001;7:597–608. 4. liu yy, wang y, walsh tr, et al. emergence of plasmid-mediated colistin resistance mechanism mcr-1 in animals and human beings in china: a microbiological and molecular biological study. lancet infect dis 2016;16:161–8. 5. lewis k. challenges of antibiotic discovery. microbe 2015;10:363–9. 6. messacar k, parker sk, todd jk, e t al. implementation of rapid molecular infectious disease diagnostics: the role of diagnostic and antimicrobial stewardship. j clin microbiol 2017; 55:715–23. 7. dyar oj, huttner b, schouten j, et al. what is antimicrobial stewardship? clin microbiol infect 2017;11:793-8. 8. cox ja, vlieghe e, mendelson m, et al. antibiotic stewardship in low-and middle-income countries: ‘same, but different’? clin microbiol infect 2017; 11:812-8. c:\users\jurnal fkusakti\docume 216 *department of thoracic surgery, qom university of medical sciences, qom, iran correspondence : mohsen eshraghi qom university of medical sciences email: mohsen_eshraghi@yahoo.com orcid id: orcid.org/0000-00031231-1167 date of first submission, may 20, 2018 date of final revised submission, november 30, 2018 date of acceptance, december 3, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina hashimoto's thyroiditis is not a risk factor for thyroid cancer mohsen eshraghi* and seyed mojtaba mousavi* background thyroid carcinoma is the most common endocrine malignancy. chronic inflammation can be involved in tumorigenesis. it is estimated that more than 20% of all tumors are caused by persistent inflammatory conditions. the objective of the present study was to compare the inflammatory factor level of erythrocyte sedimentation rate (esr), c-reactive protein (crp), and thyroiditis between benign and malignant thyroid nodules. methods a cross-sectional study was conducted involving 94 patients (47 patients with benign tumors as controls and 47 patients with malignant tumors as cases). esr and crp were measured and analyzed. mean esr and crp in both groups was compared using independent t-test. the chi-square test was used to assess the risk of cancer in patients with hashimoto’s thyroidits and with significance level at p<0.05. results the mean age of the patients in the benign group was 42.28 ± 13.43 years and in the malignant group 42.20 ± 16.32 (20-85) years, which was not significantly different (p=0.350). independent t-test results were not significantly different between mean esr and crp in both groups (p=0.800 and p=0.993 respectively). hashimoto’s thyroiditis was not a risk factor for thyroid cancer (or=1.58; 95% ci:0.63-4.01). conclusion this study demonstrated that hashimoto’s thyroiditis was not a risk factor for thyroid cancer. factors such as esr and crp are acute phase reactants and their levels increase in cases of acute inflammation, but may not increase significantly in chronic inflammatory conditions and malignancies. keywords: thyroid nodules, crp, esr, thyroiditis subjects doi: http://dx.doi.org/10.18051/univmed.2018.v37.216-221 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: eshraghi m, mousavi sm. hashimoto's thyroiditis is not a risk factor for thyroid cancer. univ med 2018;37:2 1 6-2 1 . doi: 10.18051/ univmed.2018.v37.216-221 september-december, 2018 vol.37 no.3 217 univ med vol.37 no.3 introduction thyroid cancer (tc) is the most common endocrine malignancy, and the incidence has been increasing worldwide during the last decades.(1,2) thyroid cancer occurred in 8.7 per 100 000 individuals in 2002 according to the surveillance, epidemiology, and end results (seer) database.(3) papillary thyroid carcinoma (ptc) is the most frequent (64.21%) of all tcs, and follicular thyroid carcinoma (ftc) 6.37%) is the second thyroid malignancy according to frequency, followed by medullary thyroid carcinoma (mtc) (5.88%) and anaplastic thyroid carcinoma (atc) (5.39%).(4) most thyroid nodules are benign with ‘‘atypia of undetermined significance’’ (aus)/ ‘‘follicular lesion of unknown significance’’ (flus) cytology and benign follicular neoplasm.(5) thyroid gland lymphoma often occurs in the case of hashimoto’s thyroiditis. a rapidly growing thyroid mass should lead the physician to suspect this diagnosis.(6) the diffuse lymphoma with large cells is the most common type of thyroid lymphoma. on biopsy, there are laminae of lymphoid cells that are difficult to differentiate from small cell lung cancer or anatomical therapeutic chemical (atc) code system. these tumors often have a high sensitivity to external radiation.(7) surgical resection should be avoided as a primary treatment, as this may lead to the spread of the disease. otherwise, it remains limited to the thyroid gland. if staging indicates the presence of the disease outside the thyroid gland, treatment should be continued based on the treatment of other forms of lymphoma.(8) in a study by heikkila et al.(9) clinical and molecular aspe cts and the expre ssion of inflammatory genes were investigated in a large group of patients with papillary thyroid cancer (ptc) with and without related thyroiditis. in general, patients with cancer have been shown to have higher c-rea ctive protein (crp) concentra tions than healthy contr ols and participants with some benign diseases.(9) in conclusion, most of the studies attempting to evaluate the use of circulating crp in the diagnosis of various cancers did not present relevant statistical analyses and most of the vast literature published on the association of circulating crp with cancer has been based on studies of prevalent cancer cases, which cannot provide evidence for causality. the small number of prospective studies identified in this review did not provide strong evidence for a causal role of crp in malignancy, although there was some evidence that crp could be related to colorectal cancer in particular. further prevalent studies in this area will not add to what is already known; more large prospective studies and studies examining the association of crp functional genetic variants with cancer outcomes would be useful to determine the role of crp in the etiology of cancer. in a study by allin et al.,(10) the purpose of this study was to investigate the relationship between crp and risk of cancer, and whether increasing crp can be a cause of cancer. in addition, this study provides information on the response of the phase of chronic inflammation, molecular biology, performance and measurement of crp, cancer prognosis and cancer biomarkers. in a study in the general population of copenhagen (11) on around 63,500 people, about 97% of participants had a crp level below 10mg/ l. the mean plasma crp levels (iqr, 1.14-2.51) were 1.53 mg/l, and 34% had crp >2mg/l. epidemiological studies suggest that in patients with solid tumors, increased levels of crp are associated with poor prognosis, while in seemingly healthy people , elevated crp levels are associated with an increased risk of lung cancer and possibly colorectal cancer, but not prostate cancer. the lack of causality between increased levels of crp and increased risk of cancer does not invalidate the clinical application of a mild increase in crp levels to predict the risk of cancer types and treat cancer patients.(12) in a study by hou et al.,(13) 512 patients, consisting of 341 patients with ptc and 171 218 patients with nodular goiter, were included in the study. in patients with ptc, the mean esr level was11.35 ± 14.24 mm/h which was lower than in those with nodular goiter (16.90 ± 12.00 mm/h, p=0.006). the mean crp level was 1.81 ± 3.51 mg/l in the ptc group, which was lower than that in the nodular goiter group (2.09 ± 3.34 mg/ l, p=0.008). lee et al.(14) analyzed data from 38 published studies that assessed the association between thyroid cancer and hashimoto’s thyroiditis (ht). hashimoto thyroiditis is more common in papillary cancers (40%) than in benign thyroid nodules (21%), in female patients (23%) than in males (11%), and in papillary thyroid (17%) compared to other types of thyroid cancer (8%). this metaanalysis revealed that ptc who had ht had a lower chance of recurrence than patients with papillary congestion without ht. a study by repplinger et al.(15) investigating the association between hashimoto’s and ptc’s thyroiditis in women showed that ht was associated with an increased risk of ptc. a study by jankovic et al.(16) using data from population-based fine needle aspiration (fna) studies did not find a statistically significant correlation between ht and ptc. another study did not observe such an association between ht and thyroid cancer.(17) therefore, the recent notion that neoplastic thyroid transformation is capable of creating a small neurogenic proton inflammatory environment has tended to result in further studies on thyroid autoimmune ptc. therefore the goal of the present study was to compare the inflammatory factor level of esr, crp and thyroiditis between benign and malignant thyroid nodules. methods research design this observational study, which was a crosssectional analytical study of diagnostic type, involved patients with cold thyroid nodules referring to shahid beheshti hospital of qom in 2014. research subjects patients who were referred to the thyroid gland department were selected using a purposeb a s e d s a mpl i n g me t h od . e ryt h r o c yte sedimentation rate and crp was measured before surgery in patients who were candidates for thyroid surgery. thyroid nodules were divided into two groups: benign (control) and malignant nodules (the case group) according to the pathology report. the required number of samples in each group was 47, and the standard deviation in each group was considered to be 5.7 according to the same article.(18) the two groups were matched in terms of age and sex.the criteria for entry in this study were patients who had undergone surgery due to thyroid nodules, and whose crp and esr were measured before surgery. biochemical assays esr me asur eme nt was ac co rdin g to s t a nd a r d l a bo r a t o r y me t h od s o n t he sedimentation rate of red blood cells. a fixed amount of blood is drawn into a vertical tube with sodium citrate as anticoagulant. the wintrobe method is performed similarly to the westergren method except that the wintrobe tube is smaller in diameter than the westergren tube and only 100 mm long. the shorter wintrobe column makes this method less sensitive than the westergren method because the maximal possible abnormal value is lower. edta anticoagulated blood without extra diluent is drawn into the tube and the blood sample is thoroughly immediately prior to testing. the blood is then left to settle for 1 hour, after which the distance between the top of the blood column and the top layer of the red blood cells (rbcs) below is measured. the esr is thus expressed in millimeters/hour. crp was measured using the quantifies creactive protein (crp) by latex-enhanced nephelometry and reported in mg/l. the test requires no instrumentation and is available in two variants, one for serum, the other for ýblood. in each sample application position, the diluted eshraghi, mousavi hashimoto's thyroiditis and thyroid cancer 219 univ med vol.37 no.3 sample is applied on top of a membrane ýcoated with monoclonal antibodies directed against crp. the bound crp is then detected and ýmeasured using a gold-conjugated monoclonal crpantibody. the amount of this secondary ýantibody bound to the membrane is directly proportional to the crp concentration in the sample. crp was measured by immunoturbidimetry or nepholometry with a crp of 0.5 mg/dl being considered abnormal while esr was measured using the westergren method with >25 mm/h being considered as abnormal. more relevant clinical elevations were also examined, being defined as any measure which was twice the elevated cutpoint. each laboratory reading was compared on an individual basis as a mean for each patient and on a collective group basis. statistical analysis esr and crp were measured before surgery and were compared by independent ttest in two groups. factors such as age, sex, and history of thyroiditis were also compared in two groups. an independent-t test and chi-square test were used to analyze the data. a p value <0.05 was considered statistically significant. ethical clearance this investigation was approved by the research ethics committee shahid beheshti hospital of qom (ir.muq.rec.1395.121) and informed consent was obtained from the patients. the patients were assured that their information were to be kept confidential and would be used only for research purposes. results the number of patients in each group and the total by sex was 94 patients (78 women and 16 men) divided into two groups of 47 each, comprising 39 females and 8 males. the mean age of the patients in the benign group was 42.28 ± 13.43 (22-70) years and in the malignant group 42.20 ± 16.32 (20-85) years, which was not significantly different (p=0.350). mean esr in the benign and malignant groups was compared using the independent t-test. there was no significant difference in mean esr levels between the two groups (p=0.800). mean crp in the benign and malignant groups also showed no significant difference (p=0.993) (table 1). the rate of thyroiditis in both benign and malignant groups was evaluated and compared. in the benign group according to the pathological report, 15 patients (31.9%) had a history of hashimoto’s thyroiditis, while in the malignant group, a history of hashimoto’s thyroiditis was reported in 20 patients (42.6%). hashimoto’s thyroiditis (ht) increased the risk of cancer, but the increase was statistically not significant (o.r.=1.5;95% confidence interval 0.63-4.01). discussion the mean esr level was similar between the benign and malignant groups. contrary results were found in a study by hou et al.,(13) in that in patients with ptc the mean esr level was significantly lower than in those with nodular variable malignant group (n=47) benign group (n=47) p value age (years) sex, female (%) tumor size (cm) esr (mm/h) crp (mg/l) 45.20 ± 16.32 39 (83.0%) 17.64 ± 9.31 13.54 ± 8.09 5.88 ± 4.92 42.28 ± 13.43 39 (83.0%) 40.16± 28.81 13.03 ± 7.80 6.02 ± 4.91 0.350 0.560 0.009 0.800 0.993 table 1. demographic and laboratory characteristics according to pathological results of patients enrolled in the study esr : erythrocyte sedimentation rate; crp : c-reactive protein 220 nodular goiter. the mean crp level was also similar between the benign and malignant groups. in the study of hou et al.,(13) the mean crp level in the ptc group was significantly lower than that in patients with nodular goiter. the reason for the difference in the esr and crp results between the present study and that by hou et al.(13) may be due to differences in the samples and the genetic backgrounds of the two populations. in the hou study there was no homogeneity in the numbers of patients in the two groups, which can be one of the reasons for the difference between the results of the two studies. in the present study, homogeneity between the two groups was attained. hashimoto’s thyroiditis was not a risk factor for thyroid cancer in our study. this result was in line with the study of heikkila et al.(9) and anil et al.(17) however, the study of krátký al.(19) showed that ht was found to be higher in papillary cancer compared to benign thyroid nodule. however, a study conducted by j ankovic et a l. ( 16 ) in population-based fna studies did not find a statistically significant correlation between ht and ptc. at the present time, there is no valid established criterion to identify those patients with ht at a higher risk of developing ptc. careful observation and follow-up of ht patients is recommended, especially those with nodular variants.this study had a few limitations. first, the factors such as income level, alcohol consumption history, metastasis, diagnostic and treatment method are some factors affecting on the survival level, the effect of which may be a limitation in the present study. second, the tumor type in all patients was ptc in the malignant group, while the relation between esr/cpr level and other inflammatory factors such as il-1.6 and other thyroid cancers such as follicular, modular, anaplastic, and also thyroid lymphoma can be studied. it is also suggested that further studies are needed to investigate the relationship between the level of antithyroid antibodies such as thyroglobulin antibody (tgab) and thyroid peroxidase antibody (tpoab) with the type of nodule (benign with malignant) and the size of the nodule. and in the case of malignant nodules, the association of these antibodies with nodule invasion into thyroid capsules, blood vessels, lymph nodes, and prognosis and patient survival should be considered. conclusion according to this study, it can be concluded that factors such as esr and crp that are considered acute phase reactors and the levels of which incr ease in acute inflammatory conditions may not increase significantly in chronic inflammatory and malignant conditions. hashimoto’s thyroiditis was not a riskfactor of thyroid cancer. acknowledgment we thank all the staff at the hospital, including doctors, nurses and pathologists for helping us with this research. conflict of interest the authors declare no conflict of interest. contributors me wrote the paper and designed the study. smm contributed to data collection 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associated with increased risk of thyroid cancer in patients with thyroid nodules: a single-center prospective study. thyroid 2010;20:601-6. doi: 10.1089/thy.2009.0450.19. 18. baruah mp, bhattacharya b. significant role of serum crp in differentiating inflammatory from non-inflammatory causes of thyrotoxicosis. indian j endocrinol metab 2012;16:976-81. doi: 10.4103/2230-8210.103002. 19. kratky j, jezkova j, kosak m, et al. positive antithyroid antibodies and nonsuppressed tsh are associated with thyroid cancer: a retrospective cross-sectional study. int j endocrinol 2018, article id 9793850, 6 pages. doi: https://doi.org/10.1155/2018/9793850. c:\users\jurnal fkusakti\docume 143 *department of physiology, faculty of medicine, trisakti university **graduate student, biomedical science, faculty of medicine, university of indonesia ***department of physiology, faculty of medicine, university of indonesia ****department of p ath olo gical anatomy, faculty of medicine, university of indonesia correspondence: dr. sophie yolanda, m.biomed department of physiology, faculty of medicine, university of indonesia, jalan salemba raya no 6 jakarta phone: +62 816-1958-138 email: sophie.y@ui.ac.id date of first submission, june 21, 2018 date of final revised submission, august 7, 2018 date of acceptance, august 10, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina centella asiatica ethanol extract increases hippocampal brain derived neurotrophic factor in male wistar rats astri handayani*,**, sophie yolanda***, and ria kodariah**** background synaptic plasticity, which primarily takes place in the hippocampus, is the molecular basis of longterm memory formation. brain derived neurotrophic factor (bdnf), a member of the neurotrophin family, plays a significant role in synaptic plasticity and memory formation. when bdnf is released, it binds to its receptor and activates various intracellular signal transduction pathways leading to synaptic plasticity. several methods to improve memory function in humans have been studied, one of which is the use of herbal compounds, such as centella asiatica (cea), an herbaceous plant that has been used for improving memory. this study aims to examine the effects of cea ethanol extract on bdnf protein expression in the ca1 hippocampal region in adult male rats. methods a randomized experimental design was performed involving 18 adult male wistar rats. the rats were randomized into three groups: one control/ distilled water group and two groups treated with doses of cea ethanol extract of 300 mg/kgbw (cea300) and 600 mg/kgbw (cea600), respectively. cea ethanol extract was administered orally for 28 consecutive days with weekly weight-adjusted dose. after 28 days, the rats were decapitated, and the hippocampus was isolated from the brain. bdnf protein expression was assessed using immunohistochemistry. data was analyzed using kruskal-wallis test and continued with post-hoc analysis. results there was a significant increase in bdnf protein expression in the cea600 group compared to the control group (p<0.001). conclusion administration of cea ethanol extract increased bdnf protein expression in the ca1 hippocampal region of adult male rats. keywords: bdnf, centella asiatica, hippocampus, synaptic plasticity, adult male rats doi: http://dx.doi.org/10.18051/univmed.2018.v37.143-149 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cit e th is a rtic le a s: handa yani a, yolanda s, kodariah r. centella asiatica ethanol extract increases hippocampal brain derived neurotrophic factor in male wistar rats. univ med 2018;37: 143-9. doi: 10.18051/univmed.2018. v37.143-149 144 handayani, yolanda, kodariah centella asiatica increases hippocampal bdnf introduction as humans grow old, memory function declines. memory formation is a process based on activity-dependent modification of synapses, also known as synaptic plasticity. it is a condition by which the brain builds neuronal circuits during development and controls cognitive function and complex behaviors in the adult.(1) synaptic plasticity is input-specific or synapse-specific where structural and functional modification occurs only in synapses that experience changes in activity. activity-dependent changes in synaptic strength are considered to be the underlying mechanism of learning and memory. besides structural changes happening in hippocampal neuronal cells, molecular substances also affect activity-dependent synaptic strength and alter learning and memory.(2,3) one of these molecular substance s is brain derived neurotrophic factor (bdnf). this factor was first purified from the mammalian brain based on its survival-promoting action on dorsal root ganglion cells and was classified as the second member of the neurotrophin family of growth factors.(4) neurotrophins are a family of secreted proteins that have emerged as important regulators of synaptic plasticity. neurotrophins are also required for the development of the nervous system of vertebrates. the family also includes neurotrophin 3 (nt3), ne urotrophin-4/5, neurotrophin-6 and neurotrophin-7. brain derived neurotrophic factor is the member of the neurotrophin family that has been extensively studied and has been shown to modulate the development and function of synapses. some studies show that bdnf facilitates both earlyphase and late-phase long-term potentiation (ltp).(5) brain derived neurotrophic factor is a small dimeric protein, which will bind and activate, both preand post-synaptically, two differ ent transmembrane r ece ptor proteins, the tropomyosin related kinase-b (trkb) with high affinity and the pan neurotrophin receptor p75ntr with low affinity. brain derived neurotrophic factor and trkb are widely distributed across subregions of the hippocampus and the adult forebrain.(4,6) tropomyosin related kinase-b activation by bdnf follows the general pathway for tyrosine kinases receptors and initiates three maj or cascades of s ignaling pathways: phospholipase cγ (plcγ), phosphatidylinositol3-kinase (pi 3 k) and the cascade regulated by extracellular signal-regulated kinase (erk), a member of the mitogen-activated protein kinase (mapk) family.(7) several methods to increase bdnf expression have been studied, such as physical exercise and herbal supplementation, e.g. with centella asiatica (cea).(8,9) centella asiatica is a herbaceous plant, commonly found in south-east asian countries. the plant grows well in high-humidity areas and is dis tributed f rom the su btr opics to the tropics.(10) centella asiatica has been widely used for centuries as a medicinal herbal plant, for various purposes, such as wound healing, b r a i n -st i mul a t i n g e f f e c t s , t re a t me n t o f hypertension, microangiopathy, gastric ulcer, and as a potent antioxidant.(11,12) some important c h emic al c ons t it ue nt s fo u nd i n ce a a r e triterpenoids and flavonoids.(2,13) other studies also highlighted asiaticoside and asiatic acid which are part of the triterpenoids compounds in cea.(14,15) centella asiatica has been shown to be very useful in improving learning and memory; it is also used as a tonic to promote brain growth and improve memory. studies showed that administration of cea will enhance neuronal dendrites in rats undergoing growth spurts (16,17) and shows anti-inflammatory effects on hippocampal neuronal cells.(18) centella asiatica also functions as antioxidant, helps with the wound healing process, and is able to treat hypertensive microangiopathy.(19) centella asiatica also has neuroprotective effects by modulating antioxidant and mitochondrial pathways, and improving cognitive function in mice.(20,21) a previous study by sari et al.(2) showed that administration of cea ethanol extract at doses of 300 mg/kgbw and 600 mg/kgbw increases 145 bdnf level in serum. kamelia et al.(12) showed that administration of cea leaf extract increases neurogenesis and bdnf levels in hippocampal cell culture from 2-week-old mice. mirza et al.(22) also showed that administration of cea ethanol extract for 8 weeks will increase cognitive function in 2-month-old male wistar rats. these previous studies have shown that cea extract is capable of increasing bdnf levels in the blood and in hippocampal cell culture, and even improve cognitive and memory functions, but they have not showed the effect of cea ethanol extract on bdnf protein expression in the hippocampus of adult rats. our research used male wistar rats of 6 months of age, which is the equivalent of 18 years in humans.(23) the neurodegenerative process starts when humans reach the age of 18 years, thus it is necessary to take preventive action to reduce the decline of memory function. this study aimed to assess the effect of cea ethanol extract on the expression of hippocampal bdnf protein that plays a key role in synaptic plasticity and memory function. methods research design our research was an in-vivo experimental study, conducted at the laboratory of pathological anatomy, department of pathological anatomy, faculty of medicine, universitas indonesia from june until september 2017. this experiment was part of a larger research project that tried to explore the potential effect of cea ethanol extract on proteins that play a role in synaptic plasticity, one of them being bdnf. animals the experiments were conducted using eighteen 6-month-old adult male wistar rats weighing 300-400 grams, that were bred at biofarma laboratory, bandung. the subjects were divided randomly into three groups: one control/distilled water group and two groups treated with different doses (mg/kg) of cea ethanol extract: 300 mg/kgbw (cea300) and 600 mg/kgbw (cea600).(2) two animals were placed in the same cage with food and water available ad libitum and maintained on a 12-hour light and 12-hour dark cycle. the room temperature was maintained at 23 ± 1°c. animals were acclimatized for one week before treatment, followed by oral administration of cea ethanol extract and hippocampal isolation at the end of the treatment. sample size determination the sample size was calculated using the mead equation reference with the total number of animal subjects ranging from 13 to 23 subjects.(24) this experiment used 6 subjects in each group, with a total of 18 animals for 3 groups. intervention centella asiatica plants were obtained from cikabayan field, bogor agricultural institute. the extraction and formulation of cea was performed at the spices and medicinal plant research station (balai penelitian tanaman rempah dan obat, balittro), bogor. in order to prepare the doses of 300 mg/kgbw and 600 mg/ kgbw, the cea ethanol extract was freshly diluted with sterile distilled water. the cea ethanol extract was administered orally for 28 consecutive days with weekly weight-adjusted dose. measurement of bdnf protein expression after a 28-day-administration period of cea ethanol extract, the rats were decapitated, and the hippocampal tissue was isolated. bdnf protein expression was assessed with an immunohistochemical technique using anti-bdnf antibody (abcam, epr1292). bdnf protein expression was then calculated using the imagej program. statistical analysis between-group differences were analyzed by kruskal-wallis test and continued with post hoc analysis using the spss 22.0 software. the dif fer ences wer e consider e d statisti cally significant at a p value of <0.05. univ med vol. 37 no.2 146 handayani, yolanda, kodariah centella asiatica increases hippocampal bdnf a b c ethical clearance the experimental protocols were approved by the health research ethics committee, faculty of medicine, universitas indonesia, cipto mangunkusumo hospital under no. 824/ un.2f1/etik/2016. results the results of the immunohistochemical assay are presented in figure 1. cells expressing bdnf show a brownish color. it is apparent that bdnf protein expression level in the control group (figure 1.b) was the lowest of the three groups. the cea300 group showed a higher e x pr e s si on l e ve l o f bdn f ( fi gu r e 1 .c) compared to the control group (figure 1.d) the cea600 group showed the strongest expression level of bdnf in the ca1 region of the hippocampus. quantitatively, bdnf protein expression of each treatment group is presented in figure 2 as mean optical density score. statistical analysis was conducted to identify any significant difference in each group using kruskal-wallis test and continued with mannwhitney post hoc analysis. (figure 2) figure 2 shows that the control group has the lowest level of bdnf protein expression (1.94±041) compared to groups cea300 (2.22 ± 0.11) and cea600 (2.79 ± 0.08). post hoc analysis showed that there was a significant difference in bdnf protein expression level between the control group and the cea600 group (p=0.000) and also between the cea300 group and the cea600 group (p=0.000). figure 1. bdnf protein expression in hippocampal ca1 region using immunohistochemical staining: a. hippocampal ca1 region in negative control group; b. hippocampal ca1 region in control group, c. hippocampal ca1 region in cea300 group; d. hippocampal ca1 region in cea600 group 147 univ med vol. 37 no.2 discussion brain derived neurotrophic factor, a member of the neurotrophin family, is known as the main regulator in synaptic plasticity.(5,6,25) it is also known as a key molecule that has an important role in neuronal differentiation, neuronal survival, and formation of synapses.(26,27) centella asiatica extract has been shown to increase learning and memory.(28) memory is enhanced through facilitation of memory retention, the longer the retention of the memory the better it will be able to increase cognitive function.(29) the memory retention process involves growth and arborization of dendrites in hippocampal neurons.(16,17) learning and memory improvement might be connected with the antioxidant, antiinflammatory, and neuroprotective activities of cea.(22) centella asiatica has antioxidant potential, thus it can act as a neuroprotector and has been proven to be effective in protecting the rat brain against age-related oxidative damage.(30) our study showed that administration of cea ethanol extract increases bdnf protein expression in the hippocampus. centella asiatica significantly increased the bdnf protein expression in the cea600 group (compared to the control group, figure 2). this result is consistent with the results of previous studies conducted by sari et al.,(2) kamelia et al.,(12) and mirza et al.(22) some of the active ingredients contained in cea are the triterpenoids and flavonoids. asiatic acid and asiaticoside have also been highlighted and are parts of the triterpenoids of cea.(28,30,31) flavonoids in cea in previous studies have been shown to pass through the blood-brain barrier and enhance cortical bl ood f low. increased cerebrovascular function, especially in the hippocampus, a brain region important for memory, may facilitate adult neurogenesis. the enhancement of both short-term or long-term memory is controlled at the molecular level. four signaling pathways controlling this enhancement process are camp-dependent protein kinase (protein kinase a), calcium-calmodulin kinase, protein kinase c, and mitogen-activated protein kinase (mapk).(32,33) all four pathways converge to signal to the camp-response element-binding protein (creb), a transcription factor which binds to the promoter regions of many genes associated with memory and synaptic plasticity. with their ability to modulate creb, flavonoids may modulate the expression of neuronal genes which contain camp-response element (cre) sequences in their promoter region. one such a neuronal gene is the bdnf gene.(33) by increasing the activity of creb, bdnf protein expression figure 2. mean optical density score of bdnf protein immunohistochemistry. kruskal-wallis test p<0.001, #p<0.001 vs cea300 group, *p<0.001 vs control group 148 handayani, yolanda, kodariah centella asiatica increases hippocampal bdnf in the hippocampus will increase.(33,34) when bdnf is released from the presynaptic region, it will bind to its receptor tyrosine kinase b (trkb) and induce various intracellular signaling pathways. our study was able to demonstrate higher bdn f p r ote i n e x p r e s s io n in t he ca1 hippocampal region of the treated groups, but this higher bdnf protein expression does not equate to its activity of binding to its receptor trkb. thus, further exploration on bdnf activity, its mechanism of binding to its trkb receptor and its downstream signaling pathway is needed for further understanding about synaptic plasticity. next, a study on rats with induced neurodegeneration will also be useful t o o bs e r ve t he p ot e n t i a l r o l e of ce a i n neuroregenerative medicine. conclusions administration of cea ethanol extract increases bdnf protein expression in the ca1 hippocampal region. thus, the use of cea is promising for the treatment of neurodegenerative disorders. conflict of interest competing interests: no relevant disclosures. funding disclosure there is no financial conflict of interest to disclose. acknowledgment this research was partly funded by dana beasiswa tesis & disertasi lpdp kemenkeu 2017. contributors ah and sy contributed to the research concept and design of the study. rk contributed to drafting the manuscript. ah contributed to data acquisition and sy to data analysis and interpretation. all authors read and approved 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10.1016/j.exger.2005.06.001. 31. nurlaily a, noor baitee ar, musalmah m. comparative antioxidant and anti-inflammatory activity of different extracts of centella asiatica (l.) urban and its active compounds, asiaticoside and madecassoside. med heal 2012;7:62-72. 32. vauzour d, vafeiadou k, rodriguez-mateor a, et al. the neuroprotective potential of flavonoids: a multiplicity of effects. genes nutr 2008;3:11526. doi: 10.1007/s12263-008-0091-4. 33. spencer jpe. the interactions of flavonoids within neuronal signalling pathways. genes nutr 2007;2:257-73. doi: 10.1007/s12263-007-0056-z. 34. spencer jpe. flavonoids: modulators of brain function? br j nutr 2008;99:60-77. doi: 10.1017/ s0007114508965776. issn : 1907-3062 universa medicina table of contents volume 31 ─ september-december, 2012 ─ number 3 pages editorial the specter of resurging communicable disease epidemics………………………………… richard tjan research articles muc1 modulates gastric epithelial immune response to bacteria or inflammatory stimuli.......... shofyatul y. triyana, yong h. sheng and michael mcguckin increased bone calcium dissociation in lead-exposed rats..……………………………………. eko suhartono, yeni wahyu ulfarini, triawanti, warih anggoro mustaqim, rizky taufan firdaus and muhammad hafidz maulana setiawan low ki-67 gene expression in non-neoplastic proliferation of oral mucosal epithelium………. janti sudiono and irmawati hassan production of tumor necrosis factor- is increased in urinary tract infections…… …………… neni susilaningsih, bambang joni karjono and ratna damma purnamawati perception of non-communicable diseases predicts consumption of fruits and vegetables..…. charles surjadi, ismoyowati, dwidjo susilo and hernani djarir aminolevulinate dehydrogenase polymorphisms did not modified lead serum and memory relationship…………..……….……………….……………………………………………….. lantip rujito, arini dewi setyowati and saien gender impacts on motor skill proficiency-physical activity relationship in children……………… diana samara, nuryani sidarta, dian mediana and noviyanti low testosterone level increases fasting blood glucose level in adult males.…………………. suweino, ml edi parwanto and david tjahjadi author index…………………………………………………………………………………….. subject index…………………………………………………………………………………….. list of reviewers……………………………………………………………………………………. 139 141 151 159 167 175 184 192 200 208 209 210 oktavianus 11 *pharmacy department, faculty of medicine and health sciences jenderal soedirman university **medical department, faculty of medicine and health sciences jenderal soedirman university correspondence heny ekowati, m.sc., apt. pharmacy department medicine and health sciences faculty jenderal soedirman university jl. dr. soeparno karangwangkal purwokerto 53123 fax. +62281-642840 e-mail : heny240377@gmail.com univ med 2013;32:11-9 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 background doxorubicin as an anticancer drug has hepatotoxic side effects. curcuma xanthorrhiza, curcuma longa and zingiber officinale are commonly used as herbals in indonesia and around the world. several compounds in these plants have antioxidant activities and are known to exhibit protection against doxorubicin-induced toxicities. this study aimed to observe the hepatoprotective effect of a combination of c. xanthorriza, c. longa, and z. officinale extract on doxorubicin-induced hepatotoxicity in rats. methods a total of 28 wistar male rats were divided into four groups: 1) control group (0.9% nacl); 2) doxorubicin 5 mg/kg intraperitoneally (ip) four times in 14 days (days 1, 5, 9, 13); 3) doxorubicin + combination of c. xanthorriza, c. longa, and z. officinale (temulawak, kunyit, and jahe merah, designated as tekuja) 250 mg/kg/day orally for 14 days; and 4) doxorubicin + tekuja extract 500 mg/kg/day orally for 14 days. measurements of parameters based on liver histopathology and the parameters of serum alanine amino transferase (alt) and aspartate amino tranferase (ast). results doxorubicin caused significant elevation in serum alt and ast enzymes after 14 days of treatment. rats treated with doxorubicin + tekuja extract 250 mg/kg/ day showed no histological changes, but had decreased levels of alt and ast. conclusion this study indicates that the combination of c. xanthorriza, c. longa, and z. officinale has a protective effect in rats against liver damage induced by doxorubicin. key words : doxorubicin, curcuma xanthorriza, curcuma longa, zingiber officinale, hepatotoxicity, rats combination of three species of zingiberaceae prevents doxorubicin-induced hepatotoxicity heny ekowati*, sarmoko*, and retno widiastuti** 12 ekowati, sarmoko, widiastuti zingiberaceae prevent hepatotoxicity kombinasi tiga spesies zingiberacea mencegah hepatotoksitas yang diinduksi doksorubisin latar belakang doksorubisin adalah antikanker yang memiliki efek samping hepatotoksik. curcuma xanthorrhiza (temulawak), curcuma longa (kunyit), dan zingiber officinale (jahe), adalah herbal yang biasa digunakan di indonesia dan di dunia. senyawa-senyawa yang terkandung dalam ketiga herbal tersebut dilaporkan dapat menurunkan toksisitas yang disebabkan penggunaan doksorubisin. penelitian ini bertujuan untuk menilai efek hepatoprotektif dari kombinasi ekstrak c. xanthorriza, c. longa, and z. officinale (temulawak, kunyit dan jahe merah) pada tikus yang diinduksi doksorubisin. metode hewan tikus galur wistar jantan sejumlah 28 ekor dibagi dalam empat kelompok: 1) kelompok kontrol (0.9% nacl); 2) doksorubisin 5 mg/kg intraperitoneal, empat kali selama 14 hari (hari 1, 5, 9, 13); 3) doksorubisin + kombinasi ekstrak temulawak, kunyit dan jahe merah (tekuja) 250 mg/kg/hari per oral selama 14 hari; dan 4) doksorubisin + kombinasi ekstrak tekuja 500 mg/kg/hari per oral selama 14 hari. pengukuran parameter berdasarkan gambaran histopatologi organ hati dan parameter alanine amino transferase (alt) dan aspartate amino tranferase (ast). hasil doksorubisin menyebabkan peningkatan yang signifikan pada enzim alt dan ast setelah 14 hari perlakukan. hewan yang diberikan ekstrak tidak menunjukkan perbedaan pada gambaran histopatologi namun memberikan gambaran penurunan level alt dan ast. doksorubisin menyebabkan kenaikan jumlah yang signifikan enzim alt dan ast serum. kesimpulan studi ini menunjukkan bahwa kombinasi ekstrak tekuja memiliki efek protektif pada organ hati yang diinduksi doksorubisin. kata kunci : doksorubisin, curcuma xanthorriza, curcuma longa, zingiber officinale, hepatotoksisitas, tikus abstrak introduction n a t u r a l p r o d u c t s a n d t h e i r a c t i v e constituents as a source of new drugs for the treatment of disease have attracted attention in recent years. medicinal applications of spices or herbs have been gradually increasing in developed countries. curcuma xanthorriza, curcuma longa and zingiber officinale are commonly used as spices as well as herbals in indonesia and around the world. these spices are an indispensable component of curry, and belong to the zingiberaceae family.(1) several compounds in these plants have antioxidant activities and are known to exhibit protection against doxorubicin-induced toxicities.(1,2) c. xanthorrhiza is known as temulawak or javanese turmeric and its extract reportedly possesses anti-inflammatory and antitumor activities, particularly one of its active principles, xanthorrhizol,(3) z. officinale or ginger, is one of the most commonly used spices in indonesia and around the world. the active compounds of z. officinale are gingerol, paradol, shogaol, and 13 zingerone.(4) the major chemical constituent of c. longa rhizomes is a yellow pigment, 1,7-bis (4-hydroxy-3-methoxyphenyl) -1, 6-heptadiene3 , 5 d i o n e , k n o w n a s c u r c u m i n (diferulolylmethane). curcumin has shown a n t i o x i d a n t , a n t i n e o p l a s t i c a n d a n t i inflammatory properties.(5) doxorubicin (dox) is an anthracycline glycoside antibiotic that possesses a potent and broad spectrum antitumor activity against a variety of human solid tumors and hematological malignancies.(6) the dox antitumor effects include mechanisms related to alterations of dna and the production of free radicals.(7) however its use in chemotherapy has been limited, largely due to its diverse toxicities, including cardiac, hepatic, hematological and testicular toxicity.(6) several studies have shown that the combination of the inflammatory process, f r e e r a d i c a l o x i d a t i v e s t r e s s , a n d l i p i d peroxidation is frequently associated with liver damage, induced by toxic agents such as dox.(7) persistent and irreversible liver damage has been a well-known side effect of dox therapy. it has been observed that there is an increase in the apoptotic processes in liver tissues after a single dose of dox.(8,9) it was confirmed that the therapeutic dose of dox enhanced lipid peroxidation in microsomes and mitochondria in the liver, especially in the presence of fe3+ ions.(6) dox-mediated hepatotoxicity includes focal damage in hepatocytes, vascular damage and steatosis.(10) although the antioxidant activities of c. xanthorrhiza, z. officinale and c. longa were well-known, their protective effect against doxinduced hepatotoxicity has not yet been reported. several studies have shown that using a c o m b i n a t i o n o f p l a n t s i n c r e a s e s t h e i r effectiveness because of synergy with minimal s i d e e ff e c t s . ( 11 ) i n o u r s t u d y, w e u s e d a combination of three herbs, c. xanthorriza (temulawak), c . l o n g a (kunyit) a n d z . officinale ( j a h e m e r a h ) ( w h i c h w e w i l l designate as tekuja). a study on the combination of c. longa and c. xanthorrixa showed that it had a protective effect against doxorubicininduced liver damage in rats.(3,5) an earlier study, using z.officinale only, suggested that z. officinale protected against nephrotoxicity either by enhancing the renal antioxidant status that had been reduced by dox, or by exerting a direct antioxidant activity.(12) therefore the current study was aimed at evaluating the hepatoprotective activity of an aqueous ethanolic extract of a combination of c. xanthorrhiza, z. officinale and c. longa (tekuja) on doxorubicin-induced hepatotoxicity in rats. methods study design an experimental laboratory study was conducted from march to october 2012. a total of 28 male rats was randomized into 4 groups (control and experimental groups) of seven rats each. the minimum group size as derived from the formula (t-1) (r-1) >15, where t = number of treatments and r = number of rats, was 4 rats per group. animals from group i (control group) received nacl 0.9%; animals from group ii r e c e i v e d d o x o r u b i c i n ( d o x ) ( 5 m g / k g ) intraperitoneally (ip) 4 times in 14 days (on days 1, 5, 9,13); group iii was given tekuja extract 250 mg/kg/day orally for 14 days and dox (5 mg/kg) intraperitoneally (on days 1, 5, 9,13); animals from group iv were given tekuja extract 500 mg/kg/day orally for 14 days and dox (5 mg/kg) intraperitoneally (on days 1, 5, 9, 13). dox-treated groups received the drug every fourth day, while the other groups instead of dox received 0.9% sodium chloride (10 ml/ kg bw) intraperitoneally. the time intervals between two dox administrations were similar to the most frequently-used treatment schedules in humans. on day 15 (1 day after the last dose of dox) all animals were sacrificed.(12,13) experimental animals laboratory-bred wistar albino rats (8week-old and weighing 140 ± 30 g) were univ med vol. 32 no.1 14 ekowati, sarmoko, widiastuti zingiberaceae prevent hepatotoxicity obtained from medical faculty, gadjah mada university, yogyakarta, indonesia and approved by the animal care committee of gadjah mada university. the animals were maintained under standard laboratory conditions of 25o c and a photoperiod of 12 h dark and 12 h light. commercial pellet diet (clinical pharmacy laboratory rat and mouse feed, purwokerto, indonesia) and water were provided ad libitum. the animals were allowed to acclimatize for 10 days before beginning the experiments. extract preparation c. xanthorriza, c. longa and z. officinale rhizomes were purchased from and authenticated by the center for research and development of medicinal plants and traditional medicine/ b2p2to2t, tawangmangu, indonesia. the sundried rhizomes were ground separately to a powder, then 500 g each of the powders were combined and macerated in ethanol for 3 days at room temperature. the filtrate was air-dried and concentrated under reduced pressure. laboratory determination blood samples were withdrawn from the orbital vein using a capillary tube. the blood samples were left to clot at room temperature. the clotted blood was then centrifuged at 3,000 rpm for 15 min to obtain blood serum, and analyzed for the biochemical parameters serum alanine amino transferase (alt) and aspartate amino tranferase (ast). all serum biochemical testing was conducted at biofit laboratory, purwokerto, indonesia using a sysmex kx 21 serum analyzer. h i s t o p a t h o l o g i c e x a m i n a t i o n o f l i v e r tissues at autopsy, the liver of each rat was removed and fixed in 10% buffered formalin. after 12–24 h of fixation, 3–5 mm tissue slices were embedded in paraffin, and stained with h e m a t o x y l i n ( h ) a n d e o s i n ( e ) f o r histopathologic examination. ethical clearance animals were handled according to the rules and regulations of the animal care committee of the faculty of medicine, gadjah mada university. data analysis all results were expressed as mean and standard deviation. differences between groups were assessed by one-way analysis of variance (anova), followed by tukey hsd. statistical significance was defined as p<0.05. results observation was terminated at 14 days a f t e r t h e l a s t d o x t r e a t m e n t . macroscopic observation of the livers showed no perceptible differences between groups (figure 1). the histopathological appearance of the livers in rats treated with dox and dox + tekuja extract also showed no differences between groups (figure 2). however, we found a significant increase in alt and ast levels (p<0.05) in the group treated with dox only, compared with the control group (table 1). administration of tekuja (250 and 500 mg/kg bw) plus dox significantly (p < 0.05) attenuated the decrease in alt and ast levels observed after administration of dox alone. the alt levels were restored to normal in the tekuja plus dox groups (tukey hsd test, p<0.05) (table 2). discussion the results of the present study indicate that the aqueous ethanolic extract of the combination of c. xanthorrhiza, c. longa and z. officinale (tekuja) significantly protected rats against dox–induced hepatotoxicity. a study on the combination of c. longa and c.xanthorrixa showed that it had a protective effect against dox-induced liver damage in rats.(3,5) another study using c. longa alone 15 figure 1. absence of morphological differences in livers of rats treated with c. xanthorrhiza, z. officinale and c. longa combination and doxorubicin (a) nacl control group; (b) doxorubicin group; (c) doxorubicin+250 mg/kgbw c. xanthorrhiza, z. officinale, and c. longa combination; (d) doxorubicin+500 mg/kgbw c. xanthorrhiza, z. officinale, and c. longa combination figure 2. histological evaluation in liver tissues of control and experimental group. (a) nacl control group; (b) doxorubicin group; (c) doxorubicin + 250 mg/kgbw c. xanthorrhiza, z. officinale, and c. longa extract combination; (d) doxorubicin + 500 mg/kgbw c. xanthorrhiza, z. officinale, and c. longa extract combination univ med vol. 32 no.1 16 ekowati, sarmoko, widiastuti zingiberaceae prevent hepatotoxicity table 1 mean of on alt and ast bases on treatment groups in rats each value represents mean ± s.d. of seven animals table 2. multiple comparison test (tuckey hsd) of alt (u/l) and ast (u/l) levels between treatment groups showed that c. longa has multiple therapeutic activities by blocking cardiac, hepatic,and renal toxicities induced by doxorubisin.(14) an earlier study using z.officinale only, suggested that z. officinale protected against nephrotoxicity either by enhancing the renal antioxidant status that had been reduced by dox, or by exerting a direct antioxidant activity.(12) the present study explored the effect of a combination of c. xanthorrhiza, c. longa and z. officinale extract (tekuja) on doxinduced hepatotoxicity in rats. the tekuja extract was given during the dox induction period. treatment with the combination of c. xanthorrhiza , z. officinale and c. longa extract was designed to prevent metabolic activation of dox and suppress liver damage. the liver is the central organ of metabolism and acts as a storage organ. liver cells metabolize toxic agents, including dox. the liver is extremely vulnerable to damage by chemical agents, presumably as a result of its central role in the metabolism of foreign substances.(15) in our study, histopathological e x a m i n a t i o n o f t h e l i v e r s s h o w e d n o appreciable differences between groups. these results may be interpreted as indicating that administration of dox does not result in liver damage, although the serum transaminase was elevated. dox in the form of dox semiquinone has been suggested to play a major role in its hepatotoxic action. semiquinones are unstable under aerobic conditions, thereby generating superoxide anion radicals by reacting with molecular oxygen.(16) hepatocytes are the likely targets of attack by reactive oxygen species (ros) in the failing liver. it is conceivable that free radicals cause damage at their formation. consequently the mitochondria as the major source of ros production, could also be the major target susceptible to attack by ros.(16) 17 serum transaminases have long been considered a sensitive indicator of hepatic injury. injury to the hepatocytes alters their transport function and membrane permeability, leading to leakage of enzymes from the cells, causing a decrease in the levels of alt and ast in hepatic cells but an increase of these enzymes in serum.(17) in this study, administration of dox to rats significantly increased serum alt and ast levels. ast is a more liverspecific enzyme. on the other hand, an increase in alt activity is usually proportional to the extent of cardiac damage. treatment with the combination of c. xanthorrhiza, z. officinale and c. longa (250 and 500 mg/kg) resulted in a significant decrease in enzyme activities in dox-treated animals, thus offering considerable protection against hepatotoxicity. the data presented in this study demonstrate that dox increased serum indices of liver function including alt and ast. these elevations of alt and ast are attributable to hepatocellular damage and decreased liver functions.(15,18) these elevated levels of serum indices for hepatocellular damage have been p r e v i o u s l y r e p o r t e d i n a d o x i n d u c e d hepatoxicity model.(18,19) the effectiveness of c. xanthorrhiza in lowering the serum enzyme levels of ast, alt, and glutamate transferases demonstrates the hepatoprotective effect of this plant against cisplastin induced hepatotoxicity.(20,21) the plant also acts to prevent fatty degeneration of the liver, which can cause irreversible functional breakdown, inevitably leading to death.(22) yemitan et al.(23) tested the effect of an ethanol extract z. officinale rhizomes against carbon tetrachloride (ccl 4 ) and acetaminopheni n d u c e d l i v e r t o x i c i t i e s i n r a t s . c a r b o n tetrachloride and acetaminophen induced many histopathological changes and increased the activities of alt, ast, alp, ldh and sdh in serum. the protective effect of z. officinale extract against carbon tetrachlorideand acetaminophen-induced damage was confirmed by histopathological examination of the liver. c . x a n t h o r r i z a , c . l o n g a a n d z . officinale all contain the active compounds curcumin, xanthorrizhol and oleoresin.(3-5) curcumin has shown antioxidant, anti-neoplastic and anti-inflammatory activity.(5,24,25) many of the activities associated with curcumin relate to its ability to suppress acute and chronic inflammation.(26) an in vivo study showed that rats fed curcumin for 7 days prior to being treated with cyclophosphamide to induce lung injury, exhibited an increase in antioxidant defense mechanisms. thus, curcumin exhibits substantial antioxidant properties in a wide variety of experimental settings.(25) the antioxidant properties of [6]-gingerol, which is a very effective agent for anticipation of ultraviolet b (uvb)-induced reactive oxygen species production and cox-2, and a promising therapeutic agent against uvb-induced skin disorders, have been studied both in vitro and in vivo. the compound also has a protective role against the toxicity and lethality induced by a g e n t s s u c h a s c a r b o n t e t r a c h l o r i d e a n d cisplatin.(27,28) the present study provides the information that tekuja extract protected against the hepatotoxicity of dox. tekuja was shown to p r o t e c t l i v e r t i s s u e , a s i n d i c a t e d b y t h e histopatological profile, and alt and ast levels. these results suggest the possible use of tekuja as a novel agent against dox-induced organ toxicities and a potential candidate to be further evaluated. however, the parameters in our present study could not explore the activity of dox in dox-induced decline in hepatic antioxidant status or its direct antioxidant activity. therefore further studies should be conducted to explore hepatic antioxidant status and direct antioxidant activity. conclusion the results of the present study indicate that the combination of c. xanthorriza, c. longa, and z. officinale has a protective effect against liver damage induced by dox. univ med vol. 32 no.1 18 ekowati, sarmoko, widiastuti zingiberaceae prevent hepatotoxicity acknowledgements the authors wish to express their sincere gratitude to the directorate of higher education, ministry of education and culture, and to the jenderal soedirman university for financial support by institutional research grant 20112012. references 1. ali bh, blunden g, tanira mo, nemmar a. some phytochemical, pharmacological and toxicological properties of ginger (zingiber officinale roscoe): a review of recent research. food chem toxicol 2008;46:409-20. 2. ghosh ak, banerjee s, mullick hi, banerjee j. zingiber officinale: a natural gold. int j pharma bio sci 2011;2:283-94 3. deasywaty, mangunwardoyo w, usia t. antimicrobial and identification of active compound in curcuma xanthorrhiza roxb. ijbas-ijens 2012;12:69-78 4. ravindran pn, babu kn. ginger the genus zingiber. new york: crc press; 2005. 5. mohanty ir, arya ds, gupta sk. dietary curcuma longa protects myocardium against isoproterenol-induced hemodynamic, biochemical and histopathological alternations in rats. int j appl res nat prod 2008;1:19-28. 6. balmer cm, valley am, iannucci a. cancer treatment and chemotherapy in pharmacotherapy: a pathophysiologic approach. new york: the mcgraw-hill companies; 2005. 7. yagmurca m, bas o, mollaoglu h, sahin o, nacar a, karaman o. protective effects of erdosteine on doxorubicin-induced hepatotoxicity in rats. arch med res 2007;38:380-5. 8. pedrycz a, wieczorski m, czerny k. increased apoptosis in the rat liver after a single dose of adriamycin administration. annales umcs sect d 2004;59:313-8. 9. pedrycz a, boratynski z, wieczorski m, visconti j. ultrastuctural and immunohistochemical evaluation of apoptosis in fetal rat liver after adriamycin administration. bull vet inst pulawy 2005;49:475-9 10. pedrycz a, wieczorski m, czerny k. the influence of a single dose of adriamycin on the pregnant rat female liver histological and histochemical evaluation. ann univ mariae curie sklodowska 2004;59:319-23. 11. beinfield h, kornglod e. chinese medicine and cancer care. altern ther 2005;9:38-52. 12. ajith ta, aswathy ms, hema u. protective effect of zingiber officinale roscoe against anticancer drug doxorubicin-induced acute nephrotoxicity. food chem toxicol 2008;46:3178–81. 13. kolarovic j, popovic m, mikov m, mitic rgl. protective effects of celery juice in treatments with doxorubicin. molecules 2009;14:1627-38. 14. mohamad rh, el-bastawesy am, zekry zk, almehdar ha, al-said mg, aly ss, et al. the role of curcuma longa against doxorubicin (adriamycin)-induced toxicity in rats. j med food 2009;12:394-402. 15. ibrahim ss, barakat ma. and helmy hts. modulating effect of carvedilol on doxorubicininduced cardiomyopathy and hepatic damage. j am sci 2010;6:20-32. 16. kalender y, yel m, kalender s. doxorubicin hepatotoxicity and hepatic free radical metabolism in rats: the effects of vitamin e and catechin. toxicology 2005;209:39-45. 17. yadav np, dixit vk. hepatoprotective activity of leaves of kalanchoe pinnata pers. j ethnopharmacol 2003;86:197-202. 18. injac r, perse m, boskovic m, djordjevic-milic v, djordjevic a, hvala a. cardioprotective effects of fullerenol c60(oh)24 on a single dose doxorubicin induced cardiotoxicity in rats with malignant neoplasm. technol cancer res treat 2008;7:15–26. 19. alshabanah oa, hafez mm, al-harbi mm, hassan zk, al rejaie ss, asiri ya, et al. doxorubicin toxicity can be ameliorated during antioxidant l-carnitine supplementation. oxid med cell longev 2010;3:428-33. 20. sakr sa, mahran ha, lamfon ha. protective effect of ginger (zingiber officinale) on adriamycin-induced hepatotoxicity in albino rats. j med plants res 2010;5:133-40. 21. seong hk, kyoung oh, won yc, jae kh, kwang kp. abrogation of cisplatin-induced hepatotoxicity in mice by xanthorrhizol is related to its effect on the regulation of gene transcription. toxicol appl pharmacol 2004:196: 346–55. 22. devaraj s, ismail s, ramanathan s, marimuthu s, fei ym. evaluation of the hepatoprotective activity of standardized ethanolic extract of curcuma xanthorrhiza roxb. j med plants res 2010;4:2512-17. 23. yemitan ok, izegbu mc. protective effects of zingiber offcinale (zingiberaceae) against carbon tetrachloride and acetaminopheninduced 19 hepatotoxucity in rats. phytother res 2006;20:9971002. 24. kelkel m, jacob c, dicato m, diederich m. potential of the dietary antioxidants resveratrol and curcumin in prevention and treatment of hematologic malignancies. molecules 2010;15: 7035-44. 25. hatcher h, planalp r, cho j, torti mf, torti sv. curcumin: from ancient medicine to current clinical trials. cell mol life sci 2008;65:1631-52. 26. shishodia s, sethig, aggarwal bb. curcumin: getting back to the roots. ann n y acad sci 2005; 1056 :206-17. 27. jagetia gc, baliga ms, venkatesh p, ulloor jn. influence of ginger rhizome (zingiber officinale rosc.) on survival, glutathione and lipid peroxidation in mice after whole-body exposure to gamma radiation. radiat res 2003;160:584-92. 28. kim hw, murakami a, abe m, ozawa y, morimitsu y, williams mv, et al. suppressive effects of mioga ginger and ginger constituents on reactive oxygen and nitrogen species generation, and the expression of inducible proinflammatory genes in macrophages. antioxid redox signal 2005;7:1621-29. univ med vol. 32 no.1 c:\users\jurnal fkusakti\docume 65 *department of medical laboratory sciences, school of basic medical sciences, college of medical sciences, university of benin, benin city, nigeria **department of chemical pathology, toxicology and micronutrient metabolism unit, college of medicine, university of ibadan, ibadan, nigeria ***department of chemistry and basel convention coordinating centre for training and technology transfer for the africa region, university of ibadan, oyo state, nigeria correspondence: osaretin godwin igharo department of medical laboratory sciences, school of basic medical sciences, college of medical sciences, university of benin, benin city, nigeria email: osaretin.igharo@uniben.edu phone: +23480 3866 4896 date of first submission, january 15, 2018 date of final revised submission, april 18, 2018 date of acceptance, april 19, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina endocrine disrupting metals lead to alteration in the gonadal hormone levels in nigerian e-waste workers osaretin godwin igharo*,**, john i. anetor**, oladele osibanjo***, humphrey benedo osadolor*, emmanuel c. odazie*, and zedech chukwuemelie uche** background electronic waste (e-waste) is known to contain thousands of toxic chemicals and metals, many of which have identified endocrine disruption potentials even at low blood concentrations resulting from occupational and environmental exposures. e-waste crude reprocessing in nigeria is massive and a growing number of nigerians are occupationally exposed. the present study aimed to determine changes in gonadal hormone levels associated with occupational crude e-waste reprocessing in nigerian male e-waste workers. methods this cross-sectional study which was carried out in benin city, southsouth nigeria recruited male e-waste workers (n=63); and apparently healthy non e-waste workers (n=41), as exposed and unexposed participants respectively. male fertility hormones which includes luteinizing hormone (lh), follicle stimulating hormone (fsh), testosterone (testo) and prolactin (prol); as well as progesterone (prog), estrogen (est) and inhibin (inh) were determined using enzyme linked immunosorbent assay (elisa) methods; whole blood levels of chromium, cadmium, arsenic and mercury were determined using inductively coupled plasma mass spectrometry. results levels of testo, prog, lh and fsh; as well as prol and est were significantly lower in e-waste workers compared with unexposed participants (p<0.05). in contrast, serum inh was significantly higher in ewaste exposed population compared with the unexposed. in addition, chromium correlated significantly and negatively with testosterone, progesterone and estrogen, while inhibin correlated positively with chromium. similar correlation patterns were observed for cadmium, arsenic and mercury. conclusion nigerian e-waste workers experienced reduced levels of key gonadal hormones and this may be associated with occupational exposure to endocrine disrupting metals in e-waste. keywords: endocrine disruptors, e-wastes, toxic metals, male reproductive hormones doi: http://dx.doi.org/10.18051/univmed.2018.v37.65-74 original article pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 cite this article as: igharo og, anetor ji, osibanjo o, et al. endocrine disrupting metals lead to alteration in the gonadal hormone levels in nigerian ewaste workers. univ med 2018;37:6574. doi: 10.18051/univmed.2018.v37. 6574 66 igharo, anetor, osibanjo, et al endocrine disrupting metals lead and gonadal introduction electrical and electronic waste (e-waste) which is also known as waste electrical and electronic equipment (weee) can be referred to as any end-of-life equipment that is dependent on electrical currents or electromagnetic fields in order to function properly. this end-of-life equipment include small and large household a p pl i a n c e s; i n f o r ma t i on te c hn o lo gy a nd t el ec o mmuni ca ti ons e qu ipment ; li ght in g equipment; electrical and electronic tools, toys, and leisure and sports equipment; medical devices; monitoring and control instruments; and automatic dispensers. components of electrical and electronic equipment such as batteries, circuit boards, plastic casings, cathode-ray tubes, activated glass, and lead capacitors are al so gro uped as e -was te . ( 1) e-wa ste a lso includes used electronics which are destined for reuse, resale, salvage, recycling, or disposal.(2) it is estimated that 20–50 million tons of e-waste are produced annually worldwide in which the united states, western europe, china, japan, and australia are the major producers.(3,4) ni ger i a i s cu r r e n t ly un d e r goi n g a ra p id advancement in information and communication technology (ict). a very significant proportion of ict users in nigeria rely on second-hand equipment from developed countries, primarily f r om eu r ope a n d n or t h amer i c a . t h e management of e-waste in nigeria is a serious environmental and health issue that requires serious consideration. it was reported that nigeria is the major e-waste dumping ground in africa.(5) e-waste products contain intricate blends of plastics and chemicals, which when not properly handled can be harmful to humans and pose environmental hazard.(6) e-waste contains more than 1000 different chemical substances which fall under ‘hazardous’ and ‘non-hazardous’ categories. the presence of toxic elements like lead, mercury, arsenic, cadmium, selenium and hexavalent chromium and flame retardants in ewaste classifies them as hazardous waste. many environmental chemicals of concern both globally and across africa are categorized as endocrinedisrupting chemicals (edcs).(7) occupational exposure to heavy metals predisposes workers to metal toxicity.(7) the mechanism of toxicity of some heavy metals still remains unknown, although e nzymatic i nhibition, impaired antioxidants metabolism, and oxidative stress may play a role. heavy metals generate many of their adverse health effects through the formation of free radicals, resulting in deoxyribonucleic acid (dna) damage, lipid peroxidation, and depletion of protein sulfhydryl (for example, glutathione).(8) a substantial number of environmental pollutants such as heavy metals (arsenic, lead, cadmium, mercury), polychlorinated biphenyls, dioxin, polycyclic aromatic hydrocarbons, phthalates, bisphenol a, pesticides and alkylphenols have been shown to cause endocrine disruption and are thus referred to as endocrine disruptors.(9) these endocrine disruptors can cause adverse effects in organisms or their progeny following the disruption of endocrine systems.(10) the endocrine system is involved in normal growth and sexual development. the endocrine system is comprised of key organs such as the hypothalamus, the pituitary gland, the pineal gland, the thyroid gland, the parathyroids, the thymus gland, the pancreas, the adrenal glands, the ovaries, and the testes. these glands secrete substances known as hormones, which are chemical messengers re leased into the bloodstream, where they travel to other organs and stimulate these or their receptors.(11) there are three general classes of hormones which are proteins and polypeptides, steroids and amino acidderived hormones.(12) proteins and polypeptides include hormones secreted by the anterior and posterior pituitary glands, the pancreas (insulin and glucagon), the parathyroid gland (parathyroid hormone), and many others. steroids are secreted by the adrenal cortex (cortisol and aldosterone), the ovaries (estrogen and progesterone), the testes (testosterone), and the placenta (estrogen and progesterone). derivatives of the amino acid tyrosine, are secreted by the thyroid (thyroxine 67 and triiodothyronine) and the adrenal medullae (epinephrine and norepinephrine.(12,13) previous studies have shown changes in the concentration of hormones in workers occupationally exposed to heavy metals such as lead (pb). these changes were proposed to be mediated at the hypothalamic-pituitary level. the reproductive toxicity of lead was documented by the greeks and romans centuries ago and is known to cause reproductive and developmental toxicity at different levels in an organism.(14) lead toxicity in the male reproductive system is long known despite the controversies on the effect of low to moderate doses or route and duration of exposure involved.(15) human evidence suggests that lead may cause reduced semen quality without any effect on endocrine function as determined by studies of men occupationally exposed to lead (16) cadmium (cd), an extremely toxic element has also been reported to have deleterious effects on the reproductive process, causing retardation of growth, sterility and embryotoxic effects. it was demonstrated that the harmful effects of cadmium are associated with interaction with other toxic metals such as lead and environmental pollutants such as fossils and petroleum by-products.(17-19) testicular changes due to cadmium toxicity have been seen in a variety of animal models at different stages of growth and maturity. cadmium-induced testicula r pathogeni city includes severe haemorrhage, oedema, necrosis and atrophy, as well as reduction in counts and motility of sperm and decreases in the testosterone concentrations in plasma and testes.(15) the daily pattern release of plasma testosterone and luteinizing hormone (lh) levels was also found to be affected by cadmium. in addition, plasma levels of pituitary hormones, for example, lh, follicle stimulating hormone (fsh), pro lactin (prol) and adrenocorticotropic hormone (acth), were also modified after cadmium exposure.(15) arsenic-induced male reproductive toxicity has also been documented. other studies have reported that arsenic caused decrease in plasma levels of lh and fsh.(20) it was also reported that chromium (vi) is present in e-waste and that chromium (vi) compounds are more toxic than chromium (iii).(21) it was repor te d that wor kers exposed to chromium (vi) had significantly higher serum fsh concentration and lower sperm count and motility, lower seminal plasma zinc levels, lactate dehydrogenase (ldh), and ldh-c4 (22), and significantly higher percentage of abnormal sperm than control workers, but other semen quality parameters did not differ between them.(32) a further report showed that blood chromium positively correlated with the percentage of abnormal sperm in the exposed workers.(22) in 2014, nigeria generated about 219 kilotons of e-waste, which is largely recycled by the informal sector.(24) most studies on e-waste in nigeria were mainly on environmental and animal studies. there has been paucity of human population based studies. the present study aimed to determine changes in gonadal hormone levels associated with occupational crude e-waste reprocessing in nigerian male e-waste workers. methods research design the study was designed as a cross-sectional study with purposive approach in the recruitment of participants into the study groups. the groups comprised of e-waste occupationally exposed participants (e-wast e workers) and nonoccupationally and minimally environmentally exposed; appar ently he althy par ticipa nts (unexposed or control participants). the study was carried out from june 2014 to june 2016. other aspects of this extensive study were concluded in 2017. study area t hi s s t udy w a s c a r r i e d ou t in t he metropolitan city of benin which is the capital of edo state located in south-south nigeria. it is a city approximately 25 miles north of the benin river and situated 200 miles by road east univ med vol. 37 no.1 68 igharo, anetor, osibanjo, et al endocrine disrupting metals lead and gonadal a b c of lagos. it has an estimated population of 1,147,188 according to the 2006 census. study sample the study sample consisted of male workers (n=63) occupationally exposed to e-waste in benin city with duration of 5 years and above. the control subjects (n=41) were recruited from apparently healthy non-exposed male subjects in ugbowo campus community, benin city. the subjects in the exposed group were adult male workers who were above 18 years old with no serious health challenge in recent years and had been engaged in the work for at least 5 years and are currently doing the work. the five years duration of exposure is based on the e-waste risk assessment report of adaramodu and colleagues.(6) the control subjects were recruited from the ugbowo community, had not been exposed to e-waste and were apparently healthy without medical complication in the past 5 years. the control subjects were males aged 18 years and above. individuals who were below 18 years of age, engaged in the e-waste work for less than 5 years, apparently unhealthy or with serious health challenge in recent years and have worked for 5 years but currently not engaged in the work, wer e excluded from the exposed group. individuals who were below 18 years of age, exposed to e-waste, unhealthy or on therapy were excluded from the control group. e-waste workers m al e wa st e ele c t r ic an d el e ctr oni c equipment (weee) workers who were daily involved with four main work tasks, namely, purchasing/reception (i.e., buying and/or receiving second-hand electrical and electronic equipment (eee)); dismantling (i.e., all work tasks involving manual dismantling of the goods); repair (i.e., all work tasks involving repair, like circuit board cleaning, soldering, rewiring, etc.) and resale (reselling repaired or inadvertently functional weee/eee, or components thereof), formed the group of e-waste workers enrolled into the study. unexposed participants (controls) non-occupationally and minimally environmentally exposed, and apparently healthy age-and sex-matched participants formed the unexposed or control group. these included fortyone (41) participants recruited from the ugbowo campus community of the university of benin, benin city, edo state, nigeria. ethical approval the protocol for this pilot study was approved by the hea lth research ethics committee of the university of ibadan/university college hospital, ibadan, nigeria (ui/uch ec registration number: nhrec/05/01/2008a). subjects for this study were adults who were adequately briefed on the research protocol and informed consent was obtained prior to sample collection. sample collection approximately 5 milliliters of venous blood was collected from test subjects (e-waste workers) and control subjects using standard phlebotomy techniques. blood samples obtained were dispensed into plain (anticoagulant-free) specimen bottles to obtain serum after clotting and centrifugation at 3000 revolutions per minute for 3 minutes. analysis of samples for the generation of research data was carried out using the well-preserved and labeled samples. laboratory analysis hormonal assays were done with elisa and the results were read with the awareness technology product stat fax® model 4700 microstrip reader, serial number: 4700-1333. (awareness technology inc., palm city, fl 34990, usa.). the inh assay was done by immunoassay method using beckman coulter elisa kit. immunoassays of lh, testo, prol, prog, and est were carried using accubind elisa microwells kits produced by monobind inc., lake forest, ca 92630, usa. whole blood levels of chromium, cadmium, arsenic and mercury were determined using 69 inductively coupled plasma mass spectrometry based on the method of fong et al. (25) statistical analysis statistical analyses including descriptive statistics were carried out using the statistical package for social scientists (spss) version 16.0. all values were to be expressed as mean ± standard deviation. the independent t-test was used to determine significant differences in test parameters in the test and controls groups. differences were considered significant when p<0.05. results the mean age of the e-waste workers is 31.0±2.0 years while that of the non-exposed respondents is 29.0±2.0 years. two-thirds of the e-waste workers are within the age range of 20-29 years, while most controls (non-e-waste workers) are within the age range of 30-39 years. for smoking status of the respondents, ‘never smoked’ was high for both the exposed and non-exposed study groups. for alcohol use, ‘never used’ was high for both the exposed and non-exposed respondents. levels of male fer tility hormones in exposed and unexposed participants are shown in table 2. correlation between heavy blood metals and male fertility hormones are shown in table 3. levels of testo, prog, lh and fsh; a s w e l l a s pro l a nd e st we r e significantly lower in e-waste workers compared with unexposed participants (p<0.05). in contrast, serum inh was significantly higher in e-waste exposed population compared with the unexposed. in addition, a significant negative correlation of chromium with testo, prog and est was observed (p=0.006, 0.042 and 0.000 respectively). inhibin positively correlated with chromium (p=0.001). similar correlation univ med vol. 37 no.1 variables study participants p-value e-waste workers n=63(%) controls n=41(%) age group (years) 20-29 42 (66.7) 4 (9.8) <0.001* 30-39 19 (30.2) 34 (82.9) 40-49 2 (3.2) 3 (7.3) mean age 31 ± 2.0 29 ± 1.1 smoking status ever smoked 22 (34.9) 12 (29.3) 0.548 never smoked 41 (65.1) 29 (70.7) alcohol use ever used 21 (33.3) 11 (26.8) 0.482 never used 42 (66.7) 30 (73.2) table 1. sociodemographic characteristics of the respondents by study groups * significant at p<0.05 male fertility hormone exposed (n=63) unexposed (n=41) p value lh (iu/l) 2.62 ± 0.25 3.66 ± 0.29 0.009 fsh (iu/l) 3.61 ± 0.28 4.73 ± 0.31 0.011 testo (ng/ml) 1.56 ± 0.14 3.09 ± 0.18 0.000 prol (ng/ml) 3.14 ± 0.48 4.87 ± 0.66 0.035 prog (ng/ml) 0.09 ± 0.02 0.18 ± 0.03 0.008 est (pmol/l) 47.25 ± 4.63 133.84 ± 9.35 0.000 inh (ng/l) 18.89 ± 1.85 6.94 ± 1.39 0.008 lh (iu/l) 2.62 ± 0.25 3.66 ± 0.29 0.009 table 2. levels of male fertility hormones in exposed and unexposed participants lh luteinizing hormone; fsh: follicle stimulating hormone; testo: testosterone : prol: prolactin; prog: progesterone; est: estrogen; inh: inhibin 70 igharo, anetor, osibanjo, et al endocrine disrupting metals lead and gonadal patterns were observed for cadmium, arsenic and mercury. discussion the present study demonstrates that demographic and lifestyle indicators in the form of age, alcohol and tobacco use were comparable in the exposed and control groups, ruling out their confounding influences on the data obtained. additionally, the observed significantly lower levels of testo, prog, lh and fsh; as well as prol and est in e-waste workers compared with unexposed participants may be associated with endocrine disrupting effects of known ewaste-borne chemicals / metals. this observation may further be fortified by the observed significant negative correlation of chromium with testo, prog and est in the e-waste exposed population. electronic waste (e-waste) is known to contain thousands of toxic chemicals and metals, many of which have identified endocrine disruption potentials even at low blood concentrations resulting from occupational and environmental exposures. (1-3). it is well known that exposure to toxic levels of metals causes pathological conditions to living organisms.(5) in human, metals may cause damage to different body organs and systems. (9) according to pizent et al.,(21) metals may affect the male reproductive system directly, when they target specific reproductive organs, or indirectly, when they act on the neuroendocrine system. these effects can be long lasting and irreversible if sertoli cells are disrupted during foetal development. the number of sertoli cells determines the number of sperm produced in adulthood, because each sertoli cell can support only a finite number of germ cells that develop into sperm. sertoli cells proliferate during the foetal, neonatal and pre-pubertal period, and each of these periods is particularly sensitive to the adverse effects of metals.(6) whole blood heavy metals concentration of the subjects studied in this work has previously been reported (25) in the study, it was observed that heavy metals in the exposed participants were significantly elevated. accumulation of metals in the epididymis, prostate, vesicular seminalis or seminal fluid may impair progressive sperm motility especially by causing the formation of varicocele.(21) in addition, metals can cause hormonal imbalance by aff ecting the neuroendocrine system, disrupting the secretion of androgens from leydig cells or inhibin b from sertoli cells.(26) the disruption of spermatogenesis in men at any stage of cell differentiation can decrease the total sperm count, increase the abnormal sperm count, impair the stability of sperm chromatin or damage sperm dna. there is growing evidence that oxidative stress is implicated in the pathogenesis of male infertility.(27) as observed in this study, the differences in the male fertility hormones analyzed between the exposed and unexposed participants may indicate the influence of toxic metals on the reproductive system of the exposed participants. for example, lh and fsh are anterior pituitary hormones, in males; lh stimulates the interstitial (leydig) cells in the testes to produce testosterone while fsh stimulates the sertoli cells of the testes to produce heavy metals lh fsh testo prol prog est inh pb cr cd as hg -0.018® -0.053 0.367 -0.155 0.189 -0.018 0.005 -0.040 -0.145 0.038 0.049 -0.304** -0.439** -0.299** -0.470** -0.017 0.161 0.144 -0.028 -0.093 -0.090 -0.226* -0.305** -0.145 -0.201 -0.163 -0.456** -0.523** -0.255* -0.459* 0.063 0.367** 0.432** 0.189 0.189 table 3. correlation between heavy metals and male fertility hormones among participants *significant (p<0.05); **highly significant (p<0.001). ®: coefficient of correlation (r); note: pb: plumbum; cr: chromium; cd: cadmium; as: arsenic; hg: mercury; lh: luteinizing hormone; fsh: follivcle stimulating hormone; testo: testosterone; prol: prolactin; prog: progesterone; est: estrogen; inh: inhibin 71 inhibin which provides the main direct negative feedback control of fsh biosynthesis and release from the anterior pituitary, it also has little effect on lh release. a moderate exposure to lead has been reported to be associated with changes in the male endocrine functions, particularly affecting the hypothalamic-pituitary axis. since the release of these hormones is contr olled by the hypothalamic-pituitary axis, the decrease observed in the levels of lh, fsh, testo, prol, prog and est may therefore be attributed to the effect of lead and other metals with similar chemica l reactivity on the hypothalamic-pituitary axis of the occupationally exposed subjects. inhibin which is produced by the sertoli cells of the testes in response to fsh stimulation also provides the main direct negative feedback control of fsh biosynthesis and release from the anterior pituitary.(12) this may further reduce the level of fsh in the exposed subjects. the repression of synthesis and release of lh and fsh have the secondary effect of repressing the release of remaining hormones down-stream the hormonal regulatory pathway. further, lh and fsh also show negative correlations with lead, however, these correlations were statistically insignificant even at confidence limit of 95%. testosterone, secreted by the leydig cells located in the interstitium of the testis, is essential for growth and division of the testicular germinal cells, which is the first stage in the formation of sperm cells. fsh stimulates the sertoli cells; without this stimulation, the conversions of the spermatids to sperm cells (the process of spermiogenesis) will not occur.(12) similarly, oestr ogens, f ormed from testosterone by the sertoli cells when they are stimulated by fsh, are probably also essential for spermiogenesis.(12) oestrogen was drastically lowered in the exposed participants as compared to the mean concentration in the unexposed participants. these three key hormones controlling spermatogenesis were significantly lowered in the exposed studied group, the decrease in the hormones controlling spermatogenesis in this group may result in low sperm count and infertility. endocrine disruptors are natural products or synthetic chemicals that interfere with the synthesis, secretion, transport, binding, action or elimination of natura l hormones that are responsible for the maintenance of homeostasis, development, reproduction and behaviour. dose, duration and timing of exposure at critical periods of life are important considerations for assessing the adverse effects of endocrine disruptors. several reports suggest that many chemicals released into the environment can affect normal endocrine function.(28) heavy metals are known endocrine disruptors, for example, arsenic, lead, boron, mercury, cadmium, antimony, aluminum, cobalt, chromium, lithium) have been found to exert adverse effects on the reproductive axis of human and experimental animals. men working in battery plants and exposed to toxic levels of lead demonstrated adverse effects on their reproductive ca pacity.(6) plasma levels of pituitary hormones, for example, lh, fsh, prol and adrenocorticotropic hormone (acth), were also modified after cadmium exposure. (15) the endocrine disrupting properties of metals may be the sole cause of the differences in the hormonal concentrations betw een the exposed and unexposed participants, this is also supported by the correlation of the male fertility hormones and toxic metals (table 3). as observed in these tables, most of the male fertility hormones correlated negatively with toxic metals, only inhibin correlated positively with all metals analysed. correlations of cr, cd and hg with inhibin were highly statistically significant while others were not. this increase in inhibin concentration caused by toxic metals may impair hormonal function as inhibin has negative feedback effect on fsh and lh as earlier stated. strong negative correlations were observed between testo and cr, cd, as and hg, these correlations were statistically significant (table 3). it was reported that workers exposed to chromium (vi) had significantly higher serum fsh concentration and lower sperm count and motility, lower seminal plasma zinc levels, lactate dehydrogenase (ldh), and ldh-c4. cadmiumuniv med vol. 37 no.1 72 igharo, anetor, osibanjo, et al endocrine disrupting metals lead and gonadal induced testicular pathogenicity includes severe haemorrhage, oedema, necrosis and atrophy, as well as reduction in counts and motility of sperm and decreases in the testosterone concentrations in plasma and testes. the daily pattern of release of plasma testo and lh levels were also found to be affected by cadmium.(15) arsenic caused decrease in plasma levels of lh and fsh. paternal exposure to mercury in humans d oe s n o t ap pe a r t o c a u s e i n f er t i l i t y or malformations, but a study of pr egna ncy outcome among the wives of men occupationally exposed to hg showed an increased incidence of spontaneous abortion.(29) additionally, est showed strong negative correlations with cr, cd, as and hg (p = 0.000, 0.000, 0.021 and 0.000 respectively). est is also essential for spermiogenesis.(13) the strong negativity observed in the correlation of testo and est with toxic metals shows the effect of the toxic metals on the endocrine functions of the e-waste workers. the workers are therefore at risk of developing pathological conditions that are associated with reproductive endocrine d ys f u nc tio n, t h e e f f e c t o f w hi c h ma y cumulatively lead to sterility and cancer. metal toxicity exacerbates oxidative stress which causes different pathological processes including lipid peroxidation, dna oxidation and enzyme inactivation. peroxidation of lipids leads to the formation of reactive products including acrolein, malondialdehyde (mda) and 4hydroxyl-2-nonenal as major products.(30) mda concentration was also found to increase in liver and kidney after cadmium exposure.(30) these lipid peroxidation products have the ability to bind to virtually all kinds of biomolecules. when they bind to dna, the binding may result in obstructed and abnormal replication during cell division, especially in actively dividing cells during spermatogenesis. dna damage in testis ma y ac cel erat e t he pr oc ess of ger m ce ll apoptosis, also known as programmed cell death.(50) this can lead to a decline in sperm counts resulting in infertility. several studies have indicated a significant increase in the levels of apoptotic spermatozoa in the semen of infertile men.(51,52) the oxidative damage to mitochondrial dna (mtdna) is also known to occur in all aerobic cells that are rich in mitochondria, including sper matozoa. multiple mtdna deletions in spermatozoa could arise through a free radical-driven event occurring at the spermatogonial cell stage and can account for reproductive failure in some men.(31) also, the binding of lipid peroxidation p r o du c t s to t i s s ue p r o te in s ma y le a d t o autoimmune disease. modification of self antigens in the course of oxidative stress, by adduct formation with reactive products of lipid peroxidation, 4-hydroxy-2-nonenal (hne) being one of the most commonly involved, is generally r e ga r d e d t o a s a me c ha ni s m b y wh i c h concomitant modification of self and neoantigen formation may lead to the breaking of tolerance to self antigens and, thus, to the pathogenesis of autoimmune disease.(54) abnormally high levels of hne-protein adducts have been detected in the sera of children affected by autoimmune diseases.(32) according to this view, cross-linking of hne with self antigens would be instrumental in creating neoantigens from formerly tolerated autoantigens and, thus, initiating autoimmunity.(32) fo r ma t i on o f a d du c t s w i th sp e r ma t o zoa structural protein may be the implicating pathogenic mechanism in the production of antisperm antibodies in women; however this has not been demonstrated.(32) the e-waste workers studied in this work are exposed to toxicity from many metals, it can therefore be said that they are experiencing synergistic toxicity as a result of the exposure. the limitations of this study include the fact that most e-waste workers in the study area did not meet the inclusion criteria for the study, a factor that limited us to only carry out a pilot study with a subset of the exposed population. from the outcome of this pilot study, the future directions and implications are that extensive studies on the toxicology of e-waste in the growing exposed human population in nigeria should be undertaken. 73 conclusion t hi s s tud y s e e ms t o d e mon s t r a t e a r emar kab l e as s oc iat io n be tw ee n e -wa ste occupational exposure and disruption of male fertility hormone status in the studied population. acknowledgement the study was financed by the ministry of education for support of research in nigeriat (tertiary education trust fund) conflict of interest none declared. contributors this work was carried out in collaboration between all authors. iog, aji. and oo designed the study, wrote the protocol, and wrote the first draft of the manuscript. iog, ohb, oec and uzc managed the literature searches, the experimental process and data analysis. all authors read and approved the final manuscript. references 1. united nations environment programme (unep). basel convention on the control of transboundary movements of hazardous waste and their disposal. châtelaine, switzerland: united nations environment programme; 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63: 35-46. doi: 10.2478/10004-1254-63-20122151. 22. marouani n, tebourbi o, mahjoub s, et al. effects of hexavalent chromium on reproductive functions of male adult rats. reprod biol 2012;12: 119–33. 23. kumar s, sathwara ng, gautam ak, et al. semen quality of industrial workers occupationally exposed to chromium. j occup health 2005;47:424430. 24. ogungbuyi o, nnorom ic, osibanjo o, et al. ewaste country assessment nigeria. secretariate of basel convention; geneva, switzerland: 2012. 25. fong bm, lee ts, tam s. determination of mercury in whole blood and urine by inductively coupled plasma mass spectrometry. j anal toxicol 2007;31:281-7. 26. igharo go, anetor ji, osibanjo oo, et al. toxic metals in nigerian electronic waste workers indicates occupational metal toxicity associated with crude electronic waste management practices. biokemistri 2014;26:107-133. 27. jensen tk, bonde jp, joffe m. the influence of occupational exposure on male reproductive function. occup med 2006;56:544-53. 28. li x, gao y, wang j, et al. exposure to environmental endocrine disruptors and human health. j public health emerg 2017;1:8. doi: 10.21037/jphe.2016.12.09. 29. bashir mr, suresh s. developmental and reproductive disorders: role of endocrine disruptors in testicular toxicity. reproductive and developmental toxicology. elsevier. london. 2011: pp.904. 30. ayala a, munoz mf, arguelles s. lipid peroxidation: production, metabolism, and signaling mechanisms of malondialdehyde and 4-hydroxy-2-nonenal. oxidative med cell longevity 2014; article id 360438, 31 pages. doi: http://dx.doi.org/10.1155/2014/360438. 31. aprioku js. pharmacology of free radicals and the impact of reactive oxygen species on the testis. j reprod infertil 2013;14:158-72. 32. pizzimenti s, ciamporcero, daga m, et al. interaction of aldehydes derived from lipid peroxidation and membrane proteins. front physiol 2013;4:242. doi: 10.3389/fphys.2013.00242 c:\users\jurnal fkusakti\docume 165 ageing, longevity and fitness: towards new paradigms in healthy ageing nugroho abikusno department of nutrition, faculty of medicine, trisakti university universa medicina september-december 2018 vol.37 no.3 we have nearly reached the end of the second decade of the millennium. within 5 years we hope the third ageing summit will be held to discuss lessons learned and future directions in the field of ageing and longevity. i mention the word longevity since many countries especially in the asia pacific region, the second hub of ageing after the western world countries, have experienced this phenomenon since the early years of this millennium.(1) looking back at the previous two ageing summits, the first was held in the early eighties in vienna and had nursing homes as the main paradigm in solving the issues related to an ageing population mostly in the post-industrial western world. two decades later the second ageing summit was held in madrid well known for the declaration of madrid international plan of action on ageing (mipaa) endorsed by 57 countries in the world.(2) this declaration in a nutshell consisted of 3 pillars to solving the issues of ageing namely 1) participation of older persons in development, 2) provision of quality social and health services for the elderly, and 3) creating an age-friendly environment that would support a society for all ages and in the case of the elderly the right to age in a place which would provide a supporting environment for them. meanwhile at the global level most of the countries in the world were battling poverty in all forms and hence the millennium development goals (mdgs) was the overall direction taken by all countries in the world. this was followed by the present sustainable development goals (sdgs) where issues of poverty eradication remained the ultimate goal especially focused on women, children and elderly. besides the issue of poverty in the elderly, this vulnerable group is mostly affected not only by famine but also natural disasters as frequently experienced by countries due to earthquakes and most recently tsunamis such as the great asian tsunami in 2004. indonesia, as an example of an ageing society in the asia pacific region, has entered its demographic bonus phase 1 period. this is an ideal period for it to develop infrastructure and age-friendly human resources at all tiers that provide services for the older population as well as those disabled so that they become not a burden but an asset in development as espoused by the 3 pillars of mipaa. the second phase of the demographic bonus which follows will be around the 100th anniversary of indonesia where the development process will reach the first cycle of its national development as the largest island archipelago nation-state in the world. at that moment the older population will be around 25 percent of its total population. the total number of older people will be around 50 million and the age dependency ratio will be around 25 where 1 older person is supported by only 4 young people. the age dependency ratio is around 12 presently so 1 older person is still supported by 10 young people who are mainly family members. in the future the burden of young people to support their elders, specifically caregiving services will increase because 1) there are not many young people available, 2) the present baby boomers are 166 univ med 2018;37:165-6. doi: http://dx.doi.org/10.18051/univmed.2018.v37.165-166 entering the last phase of their lives where life expectancy at 60 years is around 20 years but the healthy life expectancy is only around 15 years; meaning the last years of their lives will be mostly at home and bedridden, and 3) the next batch of baby boomers have entered their own pension age (the 3-generations in one roof concept). the main issue in this situation is how do we attempt to extend their healthy life expectancy by shortening the period where they are living at home and bedridden. presently, population-wise as related to healthy ageing concept starts from the very beginning of one’s life known as the life course approach. every stage of vulnerability prevention such as detection of anemia in female adolescents is followed by other specific program interventions in the stages of pregnancy, infancy, childhood, adulthood and lastly old age. in 2012, this concept has be en espoused at the international conference on healthy ageing held in kuala lumpur and at the world health organization/south-east asia regional office region the regional strategy for healthy ageing 2013-2018 became a major guidance for all thirteen of its member nations.(3) in ageing we are aware of our chronological age as measured by our birth certificate and the date of birth in our identity card or passport. the biological age is observed by signs of ageing such as white or gray hair, coarse skin, being over or undernourished or suffering from various noncommunicable diseases such as hypertension, diabetes, hypercholesterolemia, to name a few. all these signs and many more will affect our physical appearance as an image of being older than one’s actual age. what is more important and often overlooked is one’s fitness age. assessment of fitness status can apply the following measures such as maximal oxygen volume, waist circumference, duration of exercise or even grip strength.(4) these measurements are important when we deal with fitness in the elderly together with nutrition.(5) since most of the elderly will follow 2 distinct prototypes, namely obesity mostly in women or frailty mostly in men above 70 years. what is more distressing is the underlying disorder suffered by the elderly in this condition namely sarcopenia of their muscle cells which are less in number as well as size eventually leading to the general condition of frailty and signs of old age. geriatric syndromes can be in the form of incontinence, insomnia, delirium (or confusion) eventually followed (if undetected) by dementia, falls, osteoporosis and weight loss. thus, in this situation the life course approach will be the paradigm of ageing in the future.(6) healthy life style behavior should begin at the earliest life stage as possible and it is never too late to start this type of lifestyle. it consists of eating a healthy balanced diet, routine simple exercise such as walking, running, bicycling or swimming, meditation or daily deep breathing exercise, sufficient sleep especially between 10 pm to 3 am, brain exercise such as reading, comprehension, writing and discussion or learning new languages, and last but not least social interaction among family, friends, neighbors and/ or colleagues. references 1. united nations. world population report 2015. new york. 2. united nations. the madrid international plan of action on ageing and the political declaration. new york: united nations;2002. 3. world health organization/south-east asia regional office. regional strategy for healthy ageing. delhi: world health organization, regional office for south-east asia;2014. 4. roberts hc, denison hi, martin hi, et al. a review of measurement of grip strength in clinical a nd ep ide mio lo gica l stud ie s: to war d s a standardized approach. age ageing 2011; 40: 423-9. doi: 10.1093/ageing/afr051. 5. trouwborst i, verreijen a, memelink r. exercise and nutrition strategies to counteract sarcopenic obesity. nutrients 2018;10:605. doi: 10.3396/nu/ 10050605. 6. kuruvilla s, sadana r, montesinos ev, et al. a life-course approach to health: synergy with sustainable development goals. bull who 2018;96:42-50. doi: http://dx.doi.org/10.2471/ blt.17.198358. universa medicina september-december 2018 vol.37 no.3 oktavianus 61 *medical profession program, faculty of medicine, atma jaya catholic university of indonesia, jakarta **department of psychiatry, faculty of medicine, atma jaya catholic university of indonesia, jakarta ***department of radiology, faculty of medicine, atma jaya catholic university of indonesia, jakarta ****dharmais cancer hospital, jakarta correspondence jean valeria medical profession program, faculty of medicine, atma jaya catholic university of indonesia jl. pluit raya no.2 jakarta utara 14440 mobile: +6285641332222 email: jean.valeria93@hotmail.com univ med 2015;34:61-7 doi: 10.18051/univmed.2016.v35.61-67 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background women with hiv/aids (wlwha) have a complex psychosocial burden and a tendency to negative self-esteem, possibly resulting in mental and emotional problems. they need family support to deal with the hiv/aids infection and its psychosocial burden. the purpose of this study was to determine chacteristics of family support, self-esteem, and depression of wlwha and the relationship between family support and self-esteem and depression. method this was a cross-sectional study of 99 wlwha infected through their husbands/partners, with no history of drug abuse. the data was taken by a consecutive sampling of two proportions test at dharmais cancer hospital from november 2013 – january 2014. the instruments comprised a demographic questionnaire, the rosenberg self-esteem questionnaire, the hamilton depression rating scale (hdrs), and a family support questionnaire. the data was analyzed by binary logistic regression. results there were 99 respondents with mean age of 36 years, of whom 44.4% were high school graduates, 54.5% unemployed, and 91.9% had hiv/ aids for more than a year. binary logistic regression analysis showed no significant relationship between family support and self-esteem (p=0.700) and depression (p=0.396). good family support has a protective effect of 1.3 times (or=0.772; 95%ci: 0.138-3.770) towards increasing self-esteem, whereas poor family support increases the risk of depression 1.5 times (or=1.477; 95%ci: 0.598-3.645) in wlwha infected with hiv/aids from their husband/partner. conclusions good family support tend to have a protective effect towards increasing self-esteem, whereas poor family support increases the risk of depression in wlwha infected with hiv/aids from their husband/partner. keywords: hiv/aids, self-esteem, depression, family support, women family support is not a risk factor of negative self-esteem in hiv/aids women jean valeria*, surilena**, yanto budiman***, samsuridjal djauzi****, and haridana indah**** doi: http://dx.doi.org/10.18051/univmed.2015.v34.61-67 62 valeria, surilena, budiman, et al negative self-esteem in hiv/aids women dukungan keluarga bukan merupakan faktor resiko terhadap self-esteem pada perempuan dengan hiv/aids pendahuluan perempuan yang terinfeksi hiv/aids (odha perempuan) memiliki beban psiko-sosial kompleks dan cenderung dengan self-esteem negatif yang dapat berlanjut dengan gangguan mental emosional. dalam menghadapi infeksi dan beban psikososial hiv/aids, odha perempuan membutuhkan dukungan keluarganya. tujuan dari penelitian ini adalah untuk mengetahui gambaran dukungan keluarga, self-esteem, dan depresi pada perempuan dengan hiv/aids, serta hubungan antara dukungan keluarga dengan self-esteem dan depresi. metode desain penelitian ini adalah cross-sectional pada 99 perempuan yang terinfeksi hiv dari suami/pasangannya, tidak ada riwayat penyalahgunaan napza, secara consecutive sampling dari uji dua proporsi di rs kanker dharmais, november 2013 – januari 2014. instrumen penelitian adalah kuesioner demografi, rosenberg self-esteem, hamilton depression rating scale (hdrs), dan dukungan keluarga (keluarga suami/pasangannya). data dianalisis dengan binary logistic regression hasil terdapat 99 responden dengan rerata usia 36 tahun, 44,4% pendidikan sma, 54.5% tidak bekerja (ibu rumah tangga), dan 91,9% menderita infeksi hiv/aids lebih dari satu tahun. analisis binary logistic regression menunjukkan tidak ada hubungan signifikan antara dukungan keluarga dengan self-esteem (p=0,700) dan depresi (p=0,396). dukungan keluarga yang baik memiliki efek protektif sebesar 1,3 kali (or=0,772; 95%ci: 0,1383,770) terhadap peningkatan self esteem dan dukungan keluarga yang buruk meningkatkan resiko untuk terjadinya depresi sebesar 1,5 kali (or=1,477; 95%ci: 0,598-3,645) pada odha perempuan yang terinfeksi dari pasangan /suaminya. kesimpulan dukungan keluarga yang baik cenderung memiliki efek protektif terhadap peningkatan self esteem dan dukungan keluarga yang buruk meningkatkan resiko akan terjadinya depresi pada odha perempuan yang terinfeksi dari pasangan /suaminya. kata kunci: hiv/aids, self-esteem, depresi, dukungan keluarga, perempuan abstrak introduction acquired immunodeficiency syndrome (aids) is an end stage manifestation of human immunodeficiency virus (hiv) infection which causes opportunistic infection due to immune deficiency.(1) world health organization data for 2011 show that 49% of people living with hiv/ aids (plwha) worldwide are women, with the highest mode of transmission being sexual intercourse.(2) in indonesia, the highest mode of transmission in 2012 was 48.7% through sexual intercouse and 42.4% through injection. in 2012, the indonesian health minister stated that housewifes are threatened with hiv/aids infection because the majority of men using the services of sexual workers do not use condoms. (3) the united nations programme on hiv/aids (unaids) report that 34 million people worldwide are infected with hiv/aids, among whom 16.7 million women, while 22% of new cases are women.(4) 63 according to who criteria, hiv/aids stages are determined from the severity of the disease. the first stage is asymptomatic, the second shows a decrease in immune system functioning, and clinical manifestations begin to start, such as weight loss (<10%), recurrent pulmonary infection, herpes zoster, oral ulcer, etc. in the third stage, the symptoms grow worse, with weight loss of >10%, persistent diarrhea, persistent fever, persistent oral candidiasis, hairy leukoplakia, tuberculosis, anemia (<8 g/dl), etc. the fourth stage is the final stage, with wasting s y n d r o m e , p n e u m o c y s t i s p n e u m o n i a , extrapulmonary tuberculosis, chronic herpes simplex, kaposi’s sarcoma, toxoplasmosis, encephalopathy, meningitis, etc.(4) h i v / a i d s c a u s e s p h y s i c a l a n d psychosocial impacts which vary between individuals. women living with hiv/aids (wlwha) have a heavier psychosocial burden compared to men. the psychosocial burden faced by wlwha is complex, comprising changes in self-esteem, care of hiv/aids-infected family members, death of family members, and working to financially support the family if their husband dies, losing their custody, infecting their children and guilt. women living with hiv/aids also face the stigma and discrimination from the neighbourhood and family, and other issues regarding social aspects such as social relations with their friends and family.(5,6) the psychosocial burden of wlwha may cause a negative selfesteem, mental and emotional health problems which could lead to a mental emotional disorder.(7-9) depression is one of the mental disorders encountered in wlwha, with an incidence of around 5% 48%.(10) family support has a role in self-esteem development in wlwha. the family plays a role in giving emotional support in the form of empat hy, aff ection, aff or ding tr eatment, motivating a healthy life style, and encouraging them to take their medicine regularly.(11) research shows that family support in wlwha is needed in several conditions, such as decision making, going through treatment and rehabilitation, facing infection and the psychosocial impact of hiv/aids, and facing their future.(12) li et al.(11) stated that family support gives a positive impact t o w lw h a i n v a r i o u s f o r m s , t h e m o s t significant coming from their partner and siblings. the aim of the present research was to determine the relationship between family support and self-esteem and between family support and depression, in women infected with hiv/aids from their partner/husband. methods research design the design used in this research was a cross sectional and the research was conducted between november 2013 – january 2014 at dharmais cancer hospital, jakarta. research subjects the subjects were 99 women infected with hiv/aids from their partner/husband, aged 1660 years, with no history of drug abuse, with family, able to read and write, never having engaged in sexual intercourse with others than their husband/partner. they were excluded if they had a record of serious mental health issues, such as psychosis and mental retardation. the diagnoses had been established previously by a specialist in internal medicine and stated in the medical records. the sample were obtained through consecutive sampling by two proportions test. measurements the instruments used in this research were a demographic questionnaire on age, educational, marital, and occupational status, number of children, and number of children with hiv/ aids, economic status, duration of illness, hiv/ aids stage, and cd4+ titer. self-esteem was measured by the rosenberg self-esteem questionnaire of ten questions with four response options, with a different score on each option, giving one total score; self-esteem was positive if the total score was >15 and negative if the univ med vol. 34 no.1 64 valeria, surilena, budiman, et al negative self-esteem in hiv/aids women score was <15.(13) the hamilton depression rating scale (hdrs) of 17 questions was used to measure depression, with each question having a different score. total scores are interpreted as follows: 0-7 = no depression (normal mental state); 8-13 = mild depression; 14-18 = moderate depression; 19-22 = severe depression; and >23 = very severe depressions. a family support questionnaire of 20 questions with five different response options was used to measure family support, stated as supportive if the total score is >60, and not supportive if the total score is <60. statistical analysis the data were analyzed using binary logistic regression analysis for the relationship between family support and self-esteem, and between family support and depression. multiple logistic regression analysis was used to examine the relationship between determinant factors and family support. ethical clearance this research had been approved by ethical clearance committee faculty of medicine of atma jaya catholic university of indonesia at april 29th, 2014. results the results showed that the 99 women infected with hiv/aids from their partner/ husband had a mean age of 36 years, 44.4% were graduated from high school, 54.5% were unemployed (housewife), 98% were married, 82.8% had children, and 57.6% had a satifactory economical status with an income of more than rp 2,200,000 per month. the research also showed that 91.9% respondents had been infected with hiv/aids for more than a year, 39.4% were in the third stage, and 92.9% had a cd4+ count of <200 cell/mm3 (table 1). the results of this research indicate that as many as 93.9% of respondents have a positive table 1. demographic characteristics, selfesteem, family support, and depression of hiv/aids women 65 self-esteem. the depression status showed that 72.7% had depression, with 32.3% having mild depression, 17.2% moderate depression, 13.1% severe depression, and 10.1% very severe depression the family support data showed that 57.6% of respondents had a supportive family. the results of the binary logistic test showed no significant relationship (p=0.700) between family support and self-esteem and between family support and depression (p=0.396) in wlwha. good family support has a protective effect of 1.3 times (or=0.772; 95%ci, 0.1383.770) towards an increase in self-esteem and poor family support increases the risk of depression 1.5 times (or=1.477; 95%ci, 0.5983.645) in wlwha infected with hiv/aids from their husband/partner (table 2). discussion the results of this study indicate that as many as 93.9% of respondents have a positive self-esteem. these results differ from other studies, which showed that 84.62% injecting drug users (idu) with hiv/aids had a negative self-esteem.(14,15) a study on 48 hiv/aids inpatients (30 males and 18 females) in brazil showed that both genders had depression and anxiety.(16) another study was conducted in thailand on 409 people living with hiv, consisting of 112 (27.4%) men and 297 (72.6%) women, among whom there were 207 couples (married or living together) with on average 1.4 children aged 6 to 17 years. the study confirmed the importance of family support for people living with hiv in thailand.(17) the present study showed that 72.7% of the respondents had depression, with 32.3% table 2. relationship between family support and self-esteem and between family support and depression in wlwha having mild depression, 17.2% having moderate depression, 13.1% having severe depression, and 10.1% very severe depression. another study stated that hiv/aids poses a complex problem, comprising physical, social, and emotional problems. a weak physical condition, threats of death, and the psychosocial burden of people living with hiv are likely to cause mental emotional or psychosocial problems.(18) the psychosocial burden of wlwha is greater than that of men. douaihy stated that one of the greatest emotional issues experienced by wlwha was depression. their number was higher than the general population prevalence of d e p r e s s i o n , w h i c h w a s a b o u t 5 1 0 % . ( 1 9 ) mascolini (20) stated that 17.9% of women and 14.3% of men living with hiv/aids suffered from depression. wlwha must be a care giver for their husband or children infected with hiv/ aids, become the “head of the family”, and face social stigma and discrimination. another research showed that 54% of wlwha had a psychopathological disorder, 20% had major depression, 18% had a maladaptive behavior, and 74% had drug abuse.(21) holmes et al.(22) showed that depression affects patient self care. depression can contribute to a decrease in physical and mental health, causes a reluctance to perform daily self-care routines and to undergo treatment, lack of appetite, lack of excercise, and i n s o m n i a t h a t m a y c a u s e c o m p l i c a t i o n s aggravating the disorders. li et al.(11) stated that depression can lead to reluctance to seek help in the form of treatment, care, and to look for information about the disease, which in turn could exacerbate the degree of health. the results of our research showed that 57.6% of respondents had good family support. univ med vol. 34 no.1 66 valeria, surilena, budiman, et al negative self-esteem in hiv/aids women these results are consistent with those of a previous research in 2012 which showed that plwha had good family support.(16,19) jones and weissmam (21) stated that family support is necessary for plwha as a major support system so that they may develop adaptive responses in dealing with stressors and/or infection-related impacts in facing hiv/aids. li et al,(11) reported that family support can have a positive impact on the quality of life of plwha. our results are consistent with other research showing that there was no significant relationship between family support and selfesteem in wlwha.(13) this might be because 93.9% of the respondents of the present research had a positive self-esteem and 57.6% had good family support. these results are consistent with unnikrishnan et al. (23) study showing that there was no significant relationship between family support and depression. li et al.(11) suggested that plwha experiencing life situations where they often face their own condition without the support of friends or family, could suffer from anxiety, depression, guilt, and suicidal behavior or thoughts. li et al.(11) also stated that significant hiv/aids -related stigma and discrimination had a negative impact on psychological wellbeing, and that social support was shown to lower the level of depression in plwha after controlling for gender, age, economic status, and level of education. family support had a positive influence toward the psychosocial aspect as a whole.(24) a limitation of this study was the short time for interview with each respondent, since most of them could give only a little of their time for the interview, so in-depth interviews were not possible. in addition, there was no private room for the interviews, so that some of them had to be done in open space where people passed by. t h i s u n c o m f o r t a b l e s i t u a t i o n m a d e t h e respondents feel insecure to give information about their health status and related problems. this study hopes for the comprehensive treatment of wlwha, on either physical or emotional aspects. wlwha need support from their family, partner, friends, and social relations to deal with hiv/aids infection, medication, and psychosocial impact. family support of wlwha today is still low. this condition can lead to mental emotional problems among wlwha, such as depression and anxiety, and can reduce their treatment adherence and quality of life. (25,26) further studies should be done on wlwha infected for less than a year and on the assessment of support from the women’s family, so increasing the probability of determining the true relationship between family support, self-esteem, and depression. conclusion family support is an important factor needed by wlwha in their recovery process. good family support has a protective effect towards increasing self-esteem, whereas poor family support increases the risk of depression in wlwha infected with hiv/aids from their husband/partner. acknowledgment the authors would like to thank the dharmais cancer hospital for the opportunity to carry out this research. references 1. komisi penanggulangan aids. strategi dan rencana aksi nasional penanggulangan hiv dan aids tahun 2010-2014. jakarta: komisi penanggulangan aids;2010. 2. united nations programme on hiv/aids (unaids). global aids response: country progress report. reporting period january 2010 to december 2011. geneva: united nations programme on hiv/aids; 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11:298–307. 18. palmer ak, duncan kc, ayalew b, et al. “the way i see it”: the effect of stigma and depression on self-perceived body image among hivpositive individuals on treatment in british columbia, canada. aids care 2011;23:145666. doi: 10.1080/09540121.2011.565021. 19. douaihy r. the association between self-esteem and anxiety. am j psychiatry 2005;1:135-9. 20. mascolini m. more depression in hiv+ women than men, regardless of art, in 15-country study. 2nd international workshop on hiv & women. maryland; 2012. 21. jones mj, weissmam m. anxiety greatly impairs treatment adherence. am j psychiatry 2008;127: 118-22. 22. holmes bd, marzillier ebc, ulman g,et al. self care, psychological distress and hiv diseases. j assoc nurse aids care 2007;4:1514-20. 23. unnikrishnan b, jagannath v, ramapuram jt, et al. study of depression and it’s associated factors among women living with hiv/aids in coastal south india. retrovirol 2012;9 suppl 1:s137. doi:10.1186/1742-4690-9-s1-p137. 24. hartzell jd, janke ie, weintrob ac. impact of depression on hiv outcomes in the haart era. j antimicrob chemother 2008;62:246-55. 25. kusuma h. hubungan antara depresi dan dukungan keluarga dengan kualitas hidup pasien hiv/aids yang menjalani perawatan di rsupn cipto mangunkusumo [t esis]. jakarta: universitas indonesia; 2010. 26. ria ht, wirawan he. gambaran stres perempuan yang terinfeksi hiv dalam menjalani perannya berumah tangga. arkhe 2007;12:12632. univ med vol. 34 no.1 c:\users\jurnal fkusakti\docume 197 *faculty of medicine duta wacana christian university, yogyakarta **internship doctor dr. efram harsana air force hospital, east java correspondence : rizaldy pinzon, md, phd faculty of medicine duta wacana christian university kaliurang street km.7, yogyakarta, indonesia phone : +62 274 888682, +62 81294638229 email : drpinzon17@gmail.com date of first submission, july 26, 2017 date of final revised submission, november 13, 2017 date of acceptance, november 13, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 complications as important predictors of disability in ischemic stroke rizaldy taslim pinzon* and rosa de lima renita sanyasi** background stroke is the main cause of disability and death in many countries. the high incidence of disability in stroke survivors requires special attention to determine various predictive factors of disability. this study aimed to identify the various predictive factors of disability in ischemic stroke. methods this study was a cross sectional study on 4510 ischemic stroke patients. each patient’s data had been recorded in the electronic stroke registry of bethesda hospital. ischemic stroke diagnosis was confirmed by brain ct scan, which was interpreted by a neurologist and a radiologist. disability was assessed using the modified rankin scale. predictors of disability were assessed. multiple logistic regression analysis was used to analyse the data. results the subjects were predominantly males, >60 years of age, and suffered stroke for the first time. the incidence of disability was 31.5% (1420/4510). multiple logistic regression analysis showed that the presence of complications (or: 6.43; 95% ci: 4.74-8.73; p<0.001), decreased level of consciousness (or: 4.82; 95% ci: 3.95-5.90; p <0.001), onset 3 hours (or: 1.93; 95% ci: 1.52-2.45; p<0.001), recurrent stroke (or: 1.63; 95% ci: 1.391.90; p<0.001), and age >60 years (or: 1.55; 95% ci: 1.35-1.79; p<0.001) were independent predictive factors of disability. conclusion we demonstrated that a substantial proportion of patients with ischemic stroke become disabled. and the presence of complications was the most predictive factor of disability in ischemic stroke. keywords: stroke, ischemic, cohort study, predictive factors, disability doi: http://dx.doi.org/10.18051/univmed.2017.v36.197-204 original article cite this article as: pinzon rt, sanyasi rdlr. complications as important predictors of disability in ischemic stroke. univ med 2017;36:197-204. d oi: 10.18051 /univmed.2 017 .v36 . 1 9 7 -2 0 4 pissn: 1907-3062 / eissn: 2407-2230 198 pinzon, sanyasi complications predict disability in ischemic stroke introduction the four main non-communicable diseases according to the world health organization (who) are cardiovascular disease (coronary heart disease and stroke), cancer, chronic respiratory disease, and diabetes.(1) in many countries, stroke is the main cause of disability and death.(2-4) disability is defined as a lack of ability to perform activities or tasks in the range considered normal for a human being.(5) stroke causes negative impacts for most of the stroke survivors.(6) about 25% to 74% of 50 million stroke survivors in the world experience physical, cognitive, and emotional decline. they also require partial or full assistance to perform activities of daily living (adl).(7) ninety percent of stroke patients showed residual symptoms. one-third of them were not able to continue their adl as usual.(8) cross-sectional research by hong et al.(9) showed that 35% of stroke survivors are dependent on caregivers to perform adl. post-stroke disability exerts a negative impact on the socioeconomic status,(10) because stroke survivors with disability become a burden for their family.(11) the high incidence of disability in stroke survivors requires special attention to find out various predictive factors of disability. there have been only a few studies on stroke disability in indonesia and the research on predictive factors of disability in indonesia is not yet satisfactory. previous studies in indonesia were concerned with the degree of disability among patients with degenerative disease (including stroke),(12) the psychological burden caused by post-stroke disability,(13) and citicoline injections to improve disability in stroke patients,(14) but not on the factors contributing to post-stroke disability. the present study is a new study on stroke disability in indonesia, involving a large number of subjects. by knowing the predictive factors, we may make a program to reduce the incidence of disability among stroke survivors. this study aimed to identify the various predictive factors of disability in ischemic stroke. methods research design in this cross-sectional design we analyzed the data from 4510 patients who were ischemic stroke during an approximately 7.5-year period (from june 2009 to december 2016) at bethesda hospital, yogyakarta, indonesia. each subject had been followed up from the first day until they were discharged from the hospital. their data were recorded in the electronic stroke registry of bethesda hospital. study subjects the subjects were ischemic stroke patients who met the inclusion criteria i.e: (i) ischemic stroke, (ii) hospitalized at bethesda hospital, yogyakarta, indonesia, (iii) hospitalized between june 2009 to december 2016, and (iv) their data had been recorded in the electronic stroke registry of bethesda hospital. the exclusion criteria were: (i) incomplete data and (ii) death during hospitalization. measurements ischemic stroke diagnosis was confirmed by brain ct scan, interpreted by a neurologist and a radiologist. ischemic stroke diagnosis was established using the trial of org 10172 in acute stroke treatment (toast) classification as a reference, i.e: (i) large-artery atherosclerosis, (ii) cardioembolism, (iii) small-vessel occlusion, (iv) stroke of other determined etiology, and (v) stroke of undetermined etiology. (15) ischemic stroke subjects were assigned to two groups: subjects with disability and subjects without disability (as controls). assessed variables comprised gender, age, stroke history, disability s t a t us , st r oke s ymp to ms , c omor bi d i ty, complications during hospitalization, and onset. based on age, the subjects were grouped into: 60 years old and >60 years old. based on stroke history, the subjects were divided into those with first-time stroke and those with recurrent stroke. 199 disability status was defined as limitations in pe r for ming a ctivities a nd tasks on an acceptable level of social norm (5) and on this basis the subjects were differentiated into those without disability and those with disability. disability status was measured on a modified rankin score (mrs) scale.(16) subjects without d is a b i l i ty i n c l ud e d s ub j e c ts wh o we r e independent without any sequelae and no significant disability (mrs score 0 and 1). subjects with disability included subjects with moderate disability, moderately severe disability, and severe disability with a home care program (mrs score 2 to 5). the subjects who had died (mrs score 6) were excluded from this study. decreased level of consciousness was the only stroke symptom observed, and the subjects were differentiated into subjects with decreased level of consciousness and without decreased level o f c o ns c io us n e s s. de c r e a s e d l e ve l of consciousness was defined as any state of a r o u sa l oth e r th a n c o mpo s ment i s ( i . e . somnolence, stupor, coma, and delirium), a p pe a r i n g s ud d e nl y, a n d c o nf i r me d b y neurological and physical examination. based on comorbidity the subjects were differentiated into subjects with comorbidity and without comorbidity. comorbidities assessed in this study included type 2 diabetes mellitus (dm2), hypertension, dyslipidemia, ischemic heart disease (ihd), and atrial fibrillation. hypertension was diagnosed based on the joint national committee on prevention, detection, evaluation, and treatment of high blood pressure8 (jnc 8) criteria. hypertension was defined as systolic blood pressure 140 mmhg or diastolic blood pressure 90 mmhg or consumi ng an tihyperte nsive dru gs. dm2 d ia gn os i s w a s ma de b as e d o n a me r ic a n diabetes association criteria. dm2 was defined as fasting plasma glucose 126 mg/dl, or 2-hour plasma glucose 200 mg/dl during oral glucose tolerance test, or random plasma glucose 200 mg/dl with classic symptoms of hyperglycemia, or a1c 6.5%, or consuming antidiabetic drugs. the diagnosis of dyslipidemia was made based o n a me r i c a n as s oci a t i o n of cl i ni c a l endocrinologists criteria. dyslipidemia was defined as an any increase in level of blood lipids: low density lipoprotein cholesterol level 140 mg/ dl, high density lipoprotein cholesterol <40 mg/ dl, triglyceride level >200 mg/dl, and/or total cholesterol level >200 mg/dl. ihd diagnosis was made based on american heart association criteria. ihd included stable angina pectoris; which was confirmed by cardiac stress test; unstable angina pectoris, st-elevation myocardial infarction (stemi), and non stemi, confirmed by electrocardiography (ecg). atrial fibrillation (af) was defined as an irregularly irregular heart beat confirmed by ecg test. ba s e d on c omp l ic a t io ns du r i n g h os p it a l i za t i o n t he su b j e c t s we r e a l s o differentiated into subjects with complications and without complications. complications assessed in this study included urinary tract infection (uti), pneumonia, gastrointestinal bleeding, and decubitus ulcer. uti was defined as an infection in any part of the urinary tract during hospitalization, confirmed by physical examination and urinalysis. pneumonia was defined as pulmonary infection confirmed by physical examination, blood test, and chest radiograph. gastrointestinal bleeding was d e f i n e d a s bl e e d i n g i n a n y pa r t of t he gastrointestinal tract with hematemesis and/or melena as the symptom(s). decubitus ulcer was defined as any change in the color of the skin and the presence of a wound in the skin and underlying soft tissues covering bony areas compromized by pressure. on the basis of stroke onset the subjects were grouped into those with an onset of <3 hours and those with an onset of 3 hours. stroke onset was defined as the beginning of stroke symptom(s). statistical analysis univariate analysis was performed to identify subject characteristics. bivariate analysis was conducted by analyzing disability status with univ med vol. 36 no.3 200 pinzon, sanyasi complications predict disability in ischemic stroke a b c age, gender, stroke history, decreased level of co ns ci ous ne ss, pr e se nce o f c omorb idit y, presence of complications, and stroke onset. multiple logistic regression analysis was pe rfo rme d to det er mine th e i ndepe nde nt predictive factor(s) of disability. statistical significance was set at p<0.05. ethical clearance this study was verified by duta wacana ch r i st i a n un i ve r si t y e t hi c a l re s e a r c h committee. the ethical clearance number was 418/c.16/fk/2017. results from june 2009 to december 2016, there were 6526 subjects who had suffered from ischemic stroke, among whom 331 had died and 1685 had incomplete data and were excluded from this study. table 1 shows the basic characteristics of the remaining 4510 subjects. the subjects were predominantly males, >60 years old, and suffered stroke for the first time. the incidence of disability was 31.5% (1420/ 4510). most of the subjects had no decreased level of consciousness, had a comorbidity, and did not experience any complication during hospitalization. a total of 68.5% subjects were independent without any sequelae after discharge from the hospital. most of the subjects came to the hospital 3 hours after stroke onset. bivariate analysis indicated that the presence of complications was the strongest predictive factor of disability (or: 2.83; 95%ci: 2.63-3.05; p<0.001) (table 2), followed by decreased level of consciousness and recurrent stroke. male gender (or: 0.88; 95%ci: 0.81-0.96; p=0.008) and the presence of comorbidity (or: 0.80, 95%ci: 0.73-0.88, p<0.001) were predictive factors of disability. bivariate analysis was followed by multiple logistic regression analysis (table 3), which showed that the presence of complications (or: 6.43; 95%ci: 4.74-8.73; p<0.001), decreased level of consciousness (or: 4.82, 95%ci: 3.95-5.90; p<0.001), onset 3 hours (or: 1.93, 95%ci: 1.522.45; p<0.001), recurrent stroke (or: 1.63; 95%ci: 1.39-1.90; p<0.001), and age >60 years (or: 1.55; 95% ci: 1.35-1.79; p<0.001) were characteristics n (%) gender male female age (years) 60 >60 stoke history first stroke recurrent stroke decreased level of consciousness yes no comorbidity yes no complication yes no onset (hours) <3 3 disabilty yes no 2680 (59.4) 1830 (40.6) 2015 (44.7) 2495 (55.3) 3379 (74.9) 1131(25.1) 604 (13.4) 3906 (86.6) 3430 (76.1) 1080 (23,9) 306 (6.8) 4204 (93.2) 538 (11.9) 3972 (88.1) 1420 (31.5) 3090 (68.5) table 1. distribution of characteristics and clinical features of the subjects (n=4510) characteristics with disabilty without disability or 95% ci p value gender: male age: 60 years stroke history: recurrent stroke decreased level of consciousness: yes comorbidity: yes complication: yes onset 3 hours 803 935 486 420 1021 243 1302 1877 1560 645 184 2409 63 2670 0.88 1.55 1.55 2.71 0.80 2.83 1.49 0.81-0.96 1.41-1.70 1.42-1.69 2.52-2.92 0.73-0.88 2.63-3.05 1.26-1.76 0.008 <0.001 <0,001 <0.001 <0.001 <0.001 <0.001 table 2. risk factors of various clinical parameters of disability 201 independent predictive factors of disability. in this study, the presence of comorbidity was not significant for increased disability risk. gender was not a predictive factor of disability. discussion stroke is one of the common causes of morbidity worldwide, as has been proved by many previous studies. the present study showed that the incidence of disability was 31.5%, for which several studies had also indicated similar results. research by sridharan et al. (17) showed that 42.4% of stroke subjects suffered from mild disability, 43% were moderately disabled, and 14.6% were bedridden. in malaysia, about 87% of post-stroke patients needed assistance for ambulation of varying degree and 40% still required assistance after 3 months.(18) in the present study, the reduced incidence of disability among stroke survivors was the result of stroke management in the multidiciplinary unit of the hospital where this study was conducted. recent studies have shown that stroke-related disability may be substantially reduced by emergency treatment of acute stroke in dedicated stroke units.(19) a guideline for systematic stroke management is highly recommended to prevent complications, thus reducing the incidence of poststroke disability.(9) good stroke prevention and treatment may reduce the mortality and morbidity rates due to stroke.(20) our study showed that the subjects were predominantly males, >60 years old, and suffered stroke for the first time, which was similar to previous studies. the incidence of stroke increases sharply with age.(21) a study by ghani et al.(22) showed that the prevalence of stroke increases sharply at the age >45 years. the vascular health and risk management study (23) showed that the incidence of stroke is 1.25 times higher in males. most of the subjects in the present study experienced stroke for the first time (74.9%). in 2015, there were about 795,000 people in the united states who continued to experience a new or recurrent stroke and approximately 610,000 of these were first events.(24) in our study, most of the subjects did not experience a decreased level of consciousness, contrary to the study by lisabeth et al.,(25) who stated that decreased level of consciousness was the most frequent non-traditional stroke symptom (p<0.03). however, the result of our study corresponds to some earlier studies, suggesting that decreased level of consciousness was the rarest, whereas the most frequent symptom was limb weakness.(20,26) most of the subjects in our study had comorbidity (76.1%), which is similar to many previous studies. since stroke is a multifactorial disease, stroke patients frequently have various comorbidities such as hypertension, diabetes mellitus, heart disease, and others.(27-29) about 31.5% of our subjects experienced disability after stroke. of the various factors that trigger disability analyzed in this study, the presence of complications was the strongest predictive factor of disability, followed by decreased level of consciousness and recurrent str oke . male gender a nd the pre sence of comorbidity did not increase the disability risk. our study showe d th at the presence of complications increased the risk of disability 6.43 times, while decreased level of consciousness increased the risk of disability 4.828 times. these univ med vol. 36 no.3 characteristics aor 95% ci p value age: 60 years stroke history: recurrent stroke decreased level of consciousness: yes comorbidity: yes complication: yes onset: ≥3 hours 1.55 1.63 4.82 0.74 6.43 1.93 1.35-1.79 1.39-1.90 3.95-5.90 0.63-0.86 4.74-8.73 1.52-2.45 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 table 3. factors associated with risk of disability by multiple logistic regression analysis aor : adjusted odds ratio 202 pinzon, sanyasi complications predict disability in ischemic stroke results are similar to those of earlier studies. tong et al.(30) state that post-stroke complications worsen outcome. complications aggravate the degree of disability and increase the risk of recurrent stroke. complications are related to poor outcomes in the 3 months post-stroke.(28) disability-adjusted life year (daly) is a parameter to measure the morbidity and mortality rate due to a disease. the more complications experienced by stroke patients, the more they are likely to become disabled.(9) a study by nadeau et al.(31) conducted in cana da showe d that decreased leve l of consciousness was an independent predictive factor of poor outcome. early disturbance of consciousness in stroke increases the risk of poststroke disability 3.372 times in 3 months.(32) decreased level of consciousness and low glasgow coma scale (gcs) score are associated with a poor outcome in ischemic stroke patients.(33) other independent predictive factors of our study were onset 3 hours, recurrent stroke, and age >60 years. this result was also similar to previous studies. ischemic stroke sufferers who receive treatment within 3 hours of onset will gain 4.4 disability-free years of life.(34) thrombolytic therapy given within 3 hours after the onset increases good outcome 1.75 times.(35) stroke history is the main predictive factor of disability.(6) a total of 50% of patients with recurrent stroke suffer permanent disability. (3 6) t he perth community stroke study of 1989-1990 also identified stroke recurrence as a prognostic factor of disability, using logistic regression analysis after adjusting for sex.(37) good outcome of stroke sufferers is highest in the age range of 18 to 35 years and declines by 3.1 to 4.2% per decade until the age of 75 years.(35) stroke sufferers aged 65 to 84 years are at risk of disability in the next 5 years. stroke sufferers aged 75 to 84 years have a 5.7 times higher risk of disability.(37) the present study indicates that male gender is not a predictive factor of disability. the prevalence of stroke is higher in males, but the incidences of disability and dependence in performing adl are higher in females,(39,40) who are 4.3 times more dependent than males after a stroke attack.(41) the finding that the presence of comorbidity is a predictive factor of disability, is consistent with the results of previous research. the degree of disability will be severe if accompanied by various comorbidities such as diabetes mellitus,(42) heart disease (atrial fibrillation and ischemic heart disease), and hypertension.(43) a limitation of our study is that mrs is likely not the best measure of minor disability and future studies should consider other measures of disability. we also did not find out what made these patients disabled and this would be of interest in designing future studies. another limitation is that the electronic stroke registry in bethesda hospital is a secondary data source, so there is the possibility of errors in the data input process by the operator. the strength of this research is the large sample size and the fact that the types of variable listed in the electronic stroke registr y were relatively complete. conclusion this study demonstrated that the presence of complications was the most important predictive factor of disability in ischemic stroke. knowledge of the predictive factors of disability increases the alertness and awareness of medical personnel to conduct real efforts to reduce the number of post-stroke disabilities. conflict of interest competing interests: no relevant disclosure. acknowledgement the authors are grateful to all patients who took part in this study, to the faculty of medicine duta wacana christian university ethical committee who gave ethical clearance, and to 203 univ med vol. 36 no.3 the bethesda hospital as the center where this study was conducted. contributors rtp contributed to collect the subjects’ data, managed ethical clearance, and completed the manuscript. rdlrs contributed to data analysis, literature search, and writing the draft. all authors have read and approved the final manuscript. references 1. badan penelitian dan pengembangan kesehatan. riset kesehatan dasar. jakarta: kementerian kesehatan republik indonesia; 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thereby constituting major health problems in resident commercial sex workers (csws) who temporarily reside in brothels for sex business. hence, we investigated the associations between behavioural risk factors and cervical lesions in resident commercial sex workers. methods we conducted a cross-sectional study among 119 resident commercial sex workers using questionnaires as the data gathering tool, liquid-based cytology for cervical lesions screening, and microscopy for examination of slides. statistical analyses were conducted using epiinfo™ version 6.4 and statistica (statsoft, version 5, 2009 edition, atlanta, ga 303294027 usa). data were analysed using multivariate and simple logistic regression analyses, while the level of significance was set at p 0.05. results the behavioural risk factors, like smoking and hard drugs, alcohol intake, commercial sex duration, client’s daily patronage and sex frequency were not significant apart, but regular condom use tends to decrease the cervical lesions in csws (or=0.91;95% ci 0.92-1.06), including anal and oral sex (or=0.99 (95% ci 0.29-3.60). the patterns of cervical lesions further showed acute and chronic cervicitis with a prevalence of 10.1%, including lowgrade squamous intraepithelial lesion (lsil) and atypical squamous cells of undetermined significance (ascus). we recorded 8/12 (66.7%) for acute cervicitis, 4/12 (33.3%) chronic, and 82.4% negative. conclusion we suggest that behavioural risk factors are associated with cervical lesions and that regular condom use tends to decrease cervical lesions among resident csws. keywords: cervical lesions, behavioural risk factors, resident commercial sex workers, sub-saharan africa doi: http://dx.doi.org/10.18051/univmed.2018.v37.39-49 original article pissn: 1907-3062 / eissn: 2407-2230 january-april, 2018 vol.37no.1 cite this article as: odigie eb, achukwu pu. regular condom use tends to decrease the cervical lesions in residentcommercial sex workers in sub-saharan africa. univ med 2018;37:39-49. doi: 10.18051/univmed.2018.v37.39-49 40 odigie, achukwu cervical lesions in sex workers introduction resident commercial sex workers are females living temporarily in brothels to exchange sex for money from male clients. their activities may lead to exposure to transmitted sexual infections, which could have uneven impacts on both sexual and reproductive health.(1) this group of women is occupationally exposed to diverse forms of infection making them at increased risk of cervical lesions. in many societies today, due to the variety of social and legal discriminatory issues, sex workers are usually poorly identified and do not have regular pap smears or have their health status checked in the hospital.(2) in a country like iran, voluntary counseling and testing (vct) centers offer services to women at high risk due to their offensive sexual behaviors; yet, a large number of them are unidentified (3) and unscreened.(2) sex business is rampant in edo state, nigeria without adequate government interventions to cater or combat the spread of unforeseen diseases,(1) which may be recycled between the female sex workers and their male clients. there exist different methods for cervical lesions screening and the most successful in the reduction of the incidence and mortality in commercial sex workers is the screening by papanicolaou smear.(4,5) early detection of cervical lesions has excellent prognosis (4) and is one of the most efficient ways of diagnosing the disease at an early pre-cancerous stage.(5) cervical lesions in some region revealed a prevalence of 16.5% in resident commercial sex workers in iran,(2) 12.46% in hong kong(3) and 21% in another survey reported in shenyang, china.(4) various studies reported the behavioral risk factors as age at having first sexual intercourse, oral sex, anal intercourse, smoking, hard drugs and substance abuse (1), alcoholism,(2) multiple sex partners, and present and previous history of sexually transmitted diseases or infections.(3,4) however, they were not associated with cervical lesions in the general sex worker’s population as reported in another literature.(6) although a dearth of studies examining behavioral risk factors for cervical lesions among sex workers remains, data are available on patterns of cervical lesions in the women’s population (2,4) but not in resident sex workers. among sex workers in hong kong;(3) difficulty in accessing resident sex workers, failure to keep up with appointments, withdrawal from screening, and the fear of the screening procedure has been reported. an observational study in shenyang, china (4) reported on a range of behavioral factors to cervical lesions among female sex workers. it includes contraception method, failure to attend regular pap tests screening, bleeding after coitus, a lack of education surrounding cervical lesions, educational status of sex workers and numerous partners. other factors like the avoidance of health care due to fear of stigmatisation (1) and policing due to the fear of arrest,(6) have also been identified as barriers to screening exercise by resident csws; in addition to the fact that they deprive themselves of the routine health care access for fear of identity exposures. these events necessitated the need to explore further the association between behavioral risk factors and cervical lesions in sex workers. there is, therefore, a need for full-scale behavioral and disease pattern studies on these occupationally exposed women, to u nderstand the best preventive and or treatment plans available to the clinicians. against this backdrop, we investigated the behavioral risk factors associated with cervical lesions in resident csw methods research design we conducted a cross-sectional study using questionnaires as the data gathering tool, liquidbased cytology for cervical lesion screening, and microscopy (7) for examination of slides. the study lasted from january 2016 to september 2017. study area t hi s st u dy wa s c o nd uc te d a mo ng commercial sex workers plying their trade within 41 the four local government areas (lgas) that make up benin city in edo state, nigeria [figure 1]. benin city is the most extensive urban center and state capital, while nigeria is the most extensive country in sub-saharan africa.(8) research subjects the subjects were females (aged 13-42), who took commercial sex as occupation and were resident in brothels strictly for sex business. the resident csws were adequately briefed on the conduct and protocol of the research. informed consent before sample collection was obtained after the research had been explicitly explained to the participants in english, vernacular (pidgin english), and local dialects (where possible). participation was voluntary without any form of inducement or coercion. female csws with healthy or unhealthy cervix and those with a suspected malignancy, which may be a visible growth with an irregular surface, were included in this study. meanwhile, pregnant csws and those who had undergone hysterectomy, including those who received any prior treatment relating to cervical neoplasia, were excluded. sample size determination the sample size was determined using the formula where n = sample size, p= prevalence of cervical lesions in resident csws from a previous study and q=1–p, while l is 5% level of precision adopted from odigie et al. (8) initially, 178 subjects were recruited for the study. however, 59 subjects were unable to continue due to a clash of interest (clashes between sampling time and day, and sex business, which subjects were unable to forego). some could not abide by rules governing cervical screening, i.e. avoidance of vaginal intercourse, all forms of douching (use of tampons, medicinal vaginal cream or contraceptive cream) for the duration of 24-48hrs before pap smear uptake. january-april, 2018 vol.37no.1 figure 1. showing geographical location of the four lgas in benin city, nigeria 2l q p 4 n  42 odigie, achukwu cervical lesions in sex workers a b c data collection a probabilistic simple random sampling method was undertaken in benin metropolis, nigeria, in which a cross-sectional approach was used. a structured paper questionnaire was privately administered for data gathering by t r a i n e d int e r vi e we r s , c ove ri n g s o c i odemographic characteristics and sexual behavior of the subjects. the research questions were predominantly in a multiple-choice format and o pe n -e n d ed i n s o me a s p e c t s . t he s t ud y questions also include the health-care-seeking activities and sexual performances, together with the number of sexual contacts and history of current and former symptoms. questionnaires were completed voluntarily and conducted in the vernacular (pidgin english) where necessary. sample collection we collected samples using the liquidbased cytology (lbc) method (surepath™; bd tripath, burlington, nc, usa) involving the speculum for inspection of the cervix for lesions or inflammation in the sampled areas.(5,8) a consultant gynecologist examined the cervix for lesions or inflammation and was assisted by a trained gynecological nurse for the collection of the lbc smears. clinical signs potentially indic ating cer vical lesions, e.g., cervical discharge, pus on the cervical swab, bleeding after sampling, and inflammatory cervix were noted. details of the data and sample collection, smear preparation [using the cytospin (iec 61010 thermo scientific cytospin 4, gmi, inc. usa)], sample processing and staining methods in the present study have been documented.(5,9) examination of slides were conducted blindly according to nayar and wilbur,(7) by more than two consultant pathologists during routine slides reading. data analysis a descriptive statistical method was used in analysing the results, which were represented as whole numbers and percentages in tabular form. all statistical analyses were conducted using epi-infotm version 6.4 and statistica (statsoft, version 5, 2009 edition, atlanta, ga 30329-4027 usa). data were analysed using multivariate and simple logistic regression analyses, and the differences were considered significant at p<0.05. ethical clearance we obtained ethics permit from the edo state ministry of health (protocol number: ha. 577/vol.11.165). we also obtained written informed consent from the subjects before data and sample collections. the brothel managers gave the consent on behalf of teenage sex workers as they are below the age of consent. teenagers in this study fell within the age range of 13-19 years, which was adopted from odigie et al.(8) our study was conducted in compliance with the ethical principles for medical research involving human subjects stated in the helsinki’s declaration of world medical association. results one hundred and nineteen (119) resident commercial sex workers (csws) were sampled and screened in the present study. sixteen sex workers from one hundred and nineteen subjects ( 1 6 /119 ) we r e c e r vi c a l l e s io n-po s i ti ve , representing 13.4% of the study population suffering from cervical lesions in benin city alone. the patterns of cervical lesions showed that cervicitis (12/119 or 10.1%) is common among resident csws in this part of the subsaharan africa. our findings further revealed that acute cervicitis (8/119 or 6.7%) is the leading type of cervical lesion affecting sex workers and is closely followed by chronic cervicitis (4/12 or 33.3%). cytology profiles of sex workers revealed that 98/119 (82.4%) of the resident csws are negative for cervical lesion; 04/119 (3.4%) are abnormal having 2 cases of a low-grade squamous intraepithelial lesion (lsil) and atypical squamous cells of undetermined significance (ascus) each. inflammatory smears (12/119 or 10.1%), and 43 january-april, 2018 vol.37no.1 unsatisfactory smears (5/119 or 4.1%) are observed within the confines of 37 brothels across the 4 lgas in benin city (table 1, figure 2). multivariate and simple logistic regression was conducted to understand the effect of age, customer daily patronage, sex duration, condom usage, consumption of alcohol, hard drugs, anal intercourse, oral sex practices and age at first exposure as the likelihood that resident csws in benin city may be exposed to cervical lesions. age range 13-19 years was selected as baseline comparison as a dummy variable. commercials sex workers within the age category of 20-39 years have odds of developing cervical lesions that was 1.254 (95% ci 0.452–2.684) of the odds of someone who is within the age range of 13-19 table 1. distribution of participants’ characteristics (n=119) note: resident csws: commercial sex workers who are mainly resident in brothels. teenage csws: commercial sex workers who are teenagers and as such, they are below the age of consent and are resident in brothels for sex business. migrant csws: commercial sex workers who are from other countries but are resident in brothels as sex workers. ascus: atypical squamous cells of undetermined significance. lsil: low-grade squamous intraepithelial lesion variable number (%) age (years) 13-19 20-39 40-59 60 and above levels of education primary school drop out primary education secondary school drop out secondary education and more brothels visited per lga (n=37) egor ikpoba-okha oredo ovia north-east type of sex workers resident teenage migrant cervical lesions cervicitis acute cervicitis chronic cervicitis ascus lsil classification of smears normal abnormal inflammatory unsatisfactory behavioural factors condom usage smoking cigarette & hard drugs usage cigarette alone hard drugs alone drinking habit (alcohol intake) anal and oral sex practices anal intercourse only oral sex only mean commercial sex duration mean client’s daily patronage mean sex frequency 34 (28.57) 63 (52.94) 17 (14.29) 5 (4.2) 27 (22.69) 56 (47.06) 30 (25.21) 6 (5.04) 11 (29.73) 7 (18.92) 14 (37.84) 5 (13.51) 119 (100) 34 (28.57) 89 (74.79) 12 (10.1) 8 (6.7) 4 (3.4) 2 (1.7) 2 (1.7) 98 (82.4) 4 (3.4) 12(10.1) 5 (4.1) 93 (78.2) 79 (66.4) 37 (31.1 ) 3 (2.5) 107 (89.9) 87 (73.11) 19 (15.97) 13 (10.92) 52.64 months 11.26 clients per day 17.12 rounds of sex per day 44 odigie, achukwu cervical lesions in sex workers years, but statistically not significant. consequently, the respondents within the age range of 40-59 years (or=1.254; 95% ci 0.452-2.689)] were more variables cervical lesions or (95% ci) yes (n=16) no (n=103) age (years) 13-19* 05 27 1.000 20-39 08 52 0.208 (0.242–1.467) 40-59 02 18 1.254 (0.452–2.684) 60 and above 01 06 0.104 (0.646–2.339) levels of education primary school dropout* 04 16 1.000 primary education 07 39 0.330 (0.567–2.527) secondary school dropout 03 31 0.871 (0.231-2.668) secondary education and more 02 17 1.140 (0.428–2.251) commercial sex duration 0-24 months* 06 46 1.000 25-84 months 07 33 0.961 (0.411–1.764) 85 months and above 03 24 0.932 (0.232-1.624) client’s daily patronage 0-10* 07 68 1.000 11-20 06 19 0.238 (0.232–1.212) 21 and above 03 16 1.200 (0.425–1.463) sex frequency 0-12* 08 57 1.000 13-25 05 31 0.283 (0.561–2.687) 26 and above 03 15 2.024 (0.463–2.682) age at first exposure 0-10 05 06 1.000 11-15 09 93 0.063 (0.291–2.164) 16 and above 02 04 1.379 (0.532–1.763) table 2. simple logistic regression analysis of risk factors for cervical lesions in csws (n=119) figure 2. patterns of cervical lesion in resident csws in 4 lgas in benin city, edo state, nigeria (a= negative for cervical lesion, stained with the modified pap staining method at x40 magnification, b = negative lbc slide at x10 magnification, c = chronic cervicitis, d = low grade squamous intraepithelial lesions, e = atypical squamous cells of undetermined significance and f = acute cervicitis likely to develop cervical lesion than the ones within the reference age, but statistically not significant. csws with a primary school degree have odds of 45 january-april, 2018 vol.37no.1 behavioral risk factors cervical lesions cor (95% c.i) yes (n=16) no (n=103) regular condom use yes no 9 7 83 20 0.91(0.09-1.06) smoking and use of hard drugs yes no 11 5 71 32 0.99 (0.29-.4.60) drinking habit (alcohol intake) yes no 16 0 101 2 1.02 (0.99-1.05) anal and oral sex practices yes no 13 3 79 24 1.32 (0.31-6.32) note : all the behavioral risks factors are dichotomous variables coded as yes and no. condom usage assumes two levels, i.e. “regular condom usage” [yes or 1] and “occasional condom usage” [no or 2]. the default value, (i.e. yes) was used as the reference category. since the p-value for regular condom usage was not significant, it could be suggested that the sex workers who use condoms occasionally are more likely to develop cervical lesions compared with those that do not. it further implies that regular condom users are 0.91 less likely to develop cervical lesions compared to those using condoms occasionally table 3. multivariate logistic regression analysis of behavioral risk factors for cervical lesions in resident csws (n=119) decreased cervical lesions that was (or=0.30 95% ci 0.563-2.527) of the odds of a primary school dropout sex worker. for example, sex workers with secondary school dropouts tend to decrease cervical lesion compared to primary school drop out.but statistically not significant (or=0.871; 95% ci 0.231-2.682) (table 2). the demographics (age and education) and some risk factors (customer daily patronage, sex duration, consumption of alcohol, hard drugs, and age at first exposure) were not significant (p>0.005) (table 2), except that regular condom use tended to decrease the risk of cervical lesions ( o r= 0. 9 1; 95 % ci 0. 09 – 1. 06 ), bu t w a s statistically not significant in the exposure of resident csws to cervical lesions (table 3). discussion the prevalence of cervical lesion has been repor ted to be incre asing in females or transactional sex workers,(10) which indeed calls for concerns. our study showed that cervicitis (12/119 or 10.1%) is on the rise amongst the resident csws. it, however, supersedes the lowgrade squamous intraepithelial lesions and the atypica l squamous cells of undetermined significance both having 4/119 (3.4%); which aligns with the report by vorsters et al.(9) that cervical cancer is commoner among women who have been in transactional sex work for a long time and women who have multiple sex partners.(11) in buttress, we found that the majority of the affected women have been engaged in sex work for a longer duration than their counterparts who are not affected (table 1). however, our finding is not consistent with the report in enugu state, nigeria, (1 ) with cervi citis a mong commercial sex workers amounting to 7.6%. the disparity may be due to an uncommon screening of cervical lesions in resident commercial sex workers in edo state, compared to enugu,(1) nigeria and other areas in sub-saharan africa(9,12) that already have cervical lesion or pap smear screening programmes for sex workers.(13) bhar et al.(13) reported that pap screening with hpv testing is one of the sure ways of detecting cervical cancer. blood or smear hpv testing (serology) and molecular methods are commonly used methods in this era, which complements the result from pap smears. according to singh et al.,(14) examination by lbc technique remains the standard gold method for cancer screening worldwide. we also observed a case of a low-grade squamous intraepithelial lesion (lsil) and atypical 46 odigie, achukwu cervical lesions in sex workers squamous cells of undetermined significance (ascus) representing 04/119 (3.4%), which did not corroborate with the prevalence that was reported in india.(14) singh et al.(14) found abnormalities in the form of hsil and ascus relating to 2 (1.6%) in female sex workers, as subsequently confirmed by biopsy. one each was reported in two females, and both of them were positive for hpv 16. this procedure may explain the slight variation in our study compared to singh et al.(14) in that we did not examine by biopsy and hpv testing, thus confirming the need for periodic screening among sex workers to identify early cervical abnormalities. conversely, the abnormalities observed in the present study have been reported by vafaei et al.(2) where they detected the emergence of lsil, hsil and ascus amongst female sex workers in iran, which is comparable with our study in benin city, nigeria (table 1, figure 2). other results showed that 84.2% of our study population was negative for cervical lesion. this information partially agrees with the rate observed in another study among hungarian female sex workers,(15) where 82.4% are negative for cervical lesion but dissimilar to the 88% reported in obstetrics and gynaecology clinics in nnewi, nigeria.(16) we also disagree with the study conducted among women with unhealthy cervix in a rural population of a developing country (17) in which normal smear showed (58%) from the entire participants screened. the disparity may be due in part to sample size and study population, and have been reported.(8) odigie et al.(8) argued on sample and population sizes that these factors play a vital role in research involving sex workers in that the subjects believe their identity may not be adequately protected and as such indirectly affects the study output. also, pathologists’ bias as to what quantity/extent of the presence of inflammatory cells in a cervical smear constitutes an infection seems not to be in favour of this study. susceptibilities to bias from misclassification of the results by the pathologists in massive screening exercises have been reported.(17) according to moriarty et al.(18) about the tahoe study, when cytologists review a pap test result with the knowledge of positive hpv status, epithelial cell abnormality is more likely to be identified than without knowledge of the hpv status; this is another type of reportage bias. to ensure accuracy and conformity to standard research roles and procedures, we repeated the examination of subjects with unsatisfactory smears after 3 months, and we found all to be negative for cervical lesion. although, some subjects were sited at entirely different locations other than the scene at first contact /screening; in addition to an observation that the younger a commercial sex worker is, the more patronage in the sales of sex that she gets, which translates to increase chances of acquiring sexually transmitted diseases.(19) again, the larger the family size of residentcsws, the more tendency to having different social characters in the family. our study, however, is in agreement with gaya et al.(20) where poverty as a result of large family sizes has been linked to prostitution and could serve as reservoirs for diseases that may lead to cervical lesion. we also observed that the oredo lga in benin city, nigeria had the highest influx of both migrant 27/89 (30.3%) and teenage 13/ 34 (38.2%) resident csws (table 1). this information slightly disagrees with odigie and achukwu,(1) who screened csws for cervicitis and came up with the report that 47.4% were tee nagers and were r esiden t in br othe ls. however, this is near consonance with the 40.8% of the teenage resident csws being migrants from other states of the country. sample size and floating of the resident csws have been reported to be responsible for the variations in the reports of different kinds of literature.(21) in the present analysis sample size may not play a role in determining the accuracy of our report, but this could be attributed to the meandering way in which sex workers operate in order to protect their identity. recall that the former (1) screened 118 sex workers, while the present study investigated 119 subjects both from the r e s i d e nt co mme r c i a l s e x w or ke r s’ r i ng. 47 additionally, more of the subjects are barely educated with basic education, and from an impoverished background (table 1). our study, thus, substantiates the finding in kano,(19) nigeria where a significant part of the people screened for cervical cancer have low literacy level, and some have an existing socio-cultural practice of polygamy, which predisposes to multiple sexual partners, with the women tending to have a large number of children. in support of this, hazra et al.(15) reported that carcinomas of the cervix have not only a biomedical spectrum but also have an e x t e ns ive c u l t ur a l a nd s o c i o e c o n omi c background associated with poverty. the main focus of this study is dissimilar to the report by vafaei et al.(2) whose finding showed a non-significant association between cervical inf ection and age at f ir st intercourse, contraception method, bleeding after coitus, number of partners, educational status, or drug abuse, and are found to have no correlation with cervical abnormalities.(2) our result demonstrated a different trend in the demographics and behavioural risk factors where age, education, daily customer patr onage, sex period, consumption of alcohol, hard drugs, and age at first exposure have an insignificant association, except that regular condom use tends to decrease cervical lesions and thus conforms with the findings by vafaei et al.(2) to corroborate our result, jia et al.(4) revealed in their study a significant association for age of first sexual intercourse and a host of other variables that are not similar to the present result. several kinds of literature have reported on the key behavioural risk variables, which are in association with cervical lesion.(10,14,17) for clarity, we refer to age at first exposure to sexual intercourse as the age at which the subjects started to have sex either willfully, forcefully or otherwise. commercial sex duration relates to the entire months or years in which subjects have spent in the commercial sex business.(19) client’s daily patronage is the average number of customers patronising the subjects in a single day.(21) sex frequency is the average number of performances by male clients that sex workers can accommodate in one day.(22) condom usage is the total number and percentages of safe sex by sex workers involving commercial sex and regular sex with partners.(19) the use of hard drugs amounts to the act of taking banned substances like marijuana(23, 24) and heroin(24, 25). drinking and smoking in this study were also defined as oral consumption of alcohol either socially, indiscriminately or regularly (26) and the habit of smoking cigarettes in like manner (23) to either ease tension or surpass depression.(27) anal sex practices in our context are the use of the anus for sexual activities(25) and are used on demand by clients or as an alternative to vaginal sex whenever the subject is in her menstrual flow.(21, 25) laslett et al. (22) suggest that drug users may engage in sex work as a way to either purchase drugs or exchange services for drugs, while some sex workers may come to use hard drugs as means to cope with social problems. the study results of coleman-cowger et al. (23) conform to our study (table 2), where resident csws are seen buying and selling hard drugs and making indiscriminate use of it. the floating and relocation pattern exhibited by resident sex workers is thought to be restricted to the street sex workers (25) alone due to the nature of their business (moving from one part of the city to another searching for male clients). however, floating and relocation have continually been the tactics that are commoner among sex workers. it is, therefore, necessary to note this attribute a s s oc i a t ed wi t h f e ma l e se x wor ke r s, ( 1 7 ) transactional,(43) commercial,(19) street (21, 25) and resident commercial sex workers (8) as it poses t he mo st si gni fi c a nt c h al le nge f a ce d b y researchers. financial constraints limited our study to cytopathology assessment of cervicitis and cervical cytology using the lbc method only. however, a more comprehensive range of tests would have to be done, including hiv, syphilis, herpes, chlamydia, gonorrhoea and trichomonas testing to complement the present findings. january-april, 2018 vol.37no.1 48 odigie, achukwu cervical lesions in sex workers secondly, this study dealt solely with resident commercial sex workers; we had difficulties in assessing the street sex workers, which is also due to the roaming nature of this particular group of sex workers. our study was approached in the context o f a r i ght s -b a s e d r e p r od u c ti ve h e a l t h intervention for the resident commercial sex wor ke rs , whi ch a lso c over s a sp ec ts l ike contraception (condom usages). in the future, researchers are encouraged to include a wide range of tests with the use of molecular pathology and immunohistochemistry for the assessment of cervical lesion in the general population of sex workers. conclusions we conclude that the behavioral risk factors like regular condom users tending to decrease cervical lesion among resident csws, especially cervicitis. however, early detection of lsil and ascus in our study may help reduce mortality among sex workers. financial support and sponsorship this survey is self-sponsored. conflicts of interest no conflicts declared. acknowledgements the authors appreciate the members of sexual health concerns for commercial sex wor ke r s ( shccsw ) re s e a r ch gr o up , university of benin for providing a workforce for the administration of questionnaires. contributors all authors contributed to the conception and design of the study, field exercise, acquisition of samples and data, laboratory and data analysis, and interpretation. all authors critically revised the manuscript for valuable intellectual content. beo contributed to the drafting of the manuscript. pua read and approved the final draft. all authors made significant contributions that warrant authorship. beo acted as the corresponding author on behalf of others. all authors have read and approved the final manuscript funding te r ti a r y e du c a ti on tr us t fun d s (tetfund), university of benin research and publication committee (urpc), sexual health co nc e r ns f or comme r c i a l s e x wo r ke r s (shccsw) research group, uniben, edo state ministry of health, dr p.u. achukwu and mr b.e. odigie respectively. references 1. odigie be, achukwu pu. cytopathological examination and epidemiological study of cervicitis in commercial sex workers (csws) in coal city (enugu), nigeria. ethiopian j health sci 2015;25:227-32. doi: http://dx.doi.org/10.4314/ ejhs.v25i3.5. 2. vafaei h, asadi n, foroughinia l, et al. comparison of abnormal cervical cytology from hiv positive women, female sex workers and general population. intl j community based nurs midwifery 2015;3:76-83. 3. leung km, yeoh gp, cheung hn, et al. prevalence of abnormal papanicolaou smears in female sex workers in hong kong. hong kong med j 2013;19:203-6. doi: 10.12809/hkmj133917. 4. jia h, wang x, long z et al. human papillomavirus infection and cervical dysplasia in female sex 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high-risk human papillomavirus and abnormal pap smears in female sex workers compared to the general population in antwerp, belgium. bmc public health 2016;16:477. doi: 10.1186/s12889016-3099-5. 11. soohoo m, blas m, byraiah g, et al. cervical hpv infection in female sex workers: a global perspective. open aids j 2013;7:58–66. 12. ting j, mugo n, kwatampora j, et al. high-risk human papillomavirus messenger rna testing in physicianand self-collected specimens for cervical lesion detection in high-risk women, kenya. sex transm dis 2013;40:584-9. doi: 10.1097/ olq.0b013e31828e5a91. 13. bhar vs, gupta n, singh mp, et al. human papillomavirus (hpv) types 16 and 18 in liquidbased cervical cytology samples. virchows arch 2015;466:711-5. doi: 10.1007/s00428-015-1750-7. 14. singh mp, kaur m, gupta n, et al. prevalence of high-risk human papillomavirus types and cervical smear abnormalities in female sex workers in chandigarh, india. ind j med microbiol 2016;34: 328-34. doi: 10.4103/0255-0857.188325. 15. hazra sk, maiti s, chaudhuri a, et al. cervical cancer in women with unhealthy cervix in a rural population of a developing country. j basic clin reprod sci 2013;2:97-100. doi: 10.4103/2278960x.118648. 16. nwosu ob, okafor ci, obiechine nj, et al. the pattern of cervical cytology in obstetrics and gynaecology clinics in nnewi, south-eastern nigeria. orient j med 2010;22:25-8. doi: http:// dx.doi.org/10.4314/ojm.v22i1-4.63578. 17. marek e, dergez t, d2 cruz g, et al. human papillomavirus infections among hungarian female sex workers. eur j cancer care 2014;23:6575. doi: 10.1111/ecc.12110. 18. moriarty at, nayar r, arnold t, et al. the tahoe study: bias in the interpretation of papanicolaou test results when human papillomavirus status is known. arch path lab med 2014;138:1182-85. doi: 10.5858/arpa.2012-0115-cp. 19. moeen ud, irum s, umaira z. knowledge, attitude and practices of female sex workers about hiv/ aids prevention in lahore, pakistan. intl j perceptions public health 2017;1:148-156. 20. gaya sa, yakasai ia, muhammad az, et al. cancer of the cervix in unscreened west african women. j basic clin reprod sci 2012;1:44 8. doi: 10.4103/2278-960x.104296. 21. odigie be, achukwu pu. street sex occupational exposure to viral infections in benin city, nigeria: a call for global awareness. j appl sci res 2017;13: 1-10. 22. laslett am, jiang h, room r. alcohol’s involvement in an array of harms to intimate partners. drug alcohol rev 2017;36:72-79. doi: 10.1111/dar.12435. 23. coleman-cowger vh, schauer gl peters en. marijuana and tobacco co-use among a nationally representative sample of us pregnant and nonpregnant women: 2005–2014 national survey on drug use and health findings. drug alcohol dependence 2017;177:130-135. doi: 10.1016/j.drugalcdep.2017.03.025. 24. oguntayo ao, zayyan m, kolawole ao, et al. cancer of the cervix in zaria, northern nigeria. ecancer med scie 2011;5:219-25. doi: 10.3332/ ecancer.2011.219. 25. neal n, schrader t, hyndman j, et al. street-based sex workers need assessment for toronto, barrie and oshawa. street health 2014;1-24. 26. macintyre akj, montero var, sagbakken m. from disease to desire, pleasure to the pill: a qualitative study of adolescent learning about sexual health and sexuality in chile. bmc public health 2015;15:945. doi: 10.1186/s12889-015-22539. 27. do lat, boonmongkon p, paek sc. hu hong’ (bad thing): parental perceptions of teenagers’ sexuality in urban vietnam. bmc public health 2017;17:226. doi: 10.1186/s12889-017-4133-y. f:\36 no 2\mae.pmd 102 *faculty of medicine universitas gadjah mada, yogyakarta, indonesia, ** faculty of pharmacy, universitas gadjah mada, yogyakarta, indonesia, ***graduate school of biological sciences, nara institute of science and technology, ikoma, japan correspondence: mae sri hartati wahyuningsih department of pharmacology and therapy, faculty of medicine, universitas gadjah mada, yogyakarta, indonesia. phone and fax: +62274 511103 email: maeshw@ugm.ac.id/ maeshw98@gmail.com date of first submission, may 29, 2017 date of final revised submission, august 4, 2017 date of acceptance, august 7, 2017 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicinamay-august, 2017 vol.36 no.2 5-oleandrin reduce bcl-2 protein and increase bax protein expression on hela cervical cancer cell mae sri hartati wahyuningsih*, sofia mubarika*, ibnu g. ganjar**, subagus wahyuono**, and tatsuo takeya*** background the leaves of nerium indicum mill. have been utilized traditionally to cure cancer. by using bioassay guided extraction and isolation method, three compounds (nio-1, nio-2, nio-3) were isolated from an active fraction of the chcl 3 extract of n. indicum leaves. nio-2 identified as 5-oleandrin is the best cytotoxic compound on hela cervical cancer cell in vitro among the other two. however mechanism of action of the compound hasn’t been evaluated yet. the aims of this study were to determine the mechanism of action at molecular level. methods the action mechanism of 5-oleandrin on hela cervical cancer cells was analyzed by staining the cells with hoechst 33342; the agarose gel electrophoresis was aimed to determine the dna fragmentation and the western blotting was aimed to determine the bcl-2 and bax protein expression. results incubation of hela cervical cancer cell with 5-oleandrin at the concentration 3,47x10-4 mm (24 hours) followed by staining with hoechst 33342, a broken up light blue color of nucleus was observed (compared with intensive color of untreated control). by gel electrophoresis (at the same concentration of the tested compound), a smear band at about 200 bp was observed. in addition, cells treated with 5-oleandrin displayed a decreasing of the bcl-2 protein expression and increasing of the bax protein expression conclusion 5-oleandrin induced hela cervical cancer cells dna fragmentation observed by the presence of a smear band at about 200 bp indicative the apoptotic occurrence. 5-oleandrin induces apoptosis by reducing the bcl-2 protein expression but the bax protein expression increases. keywords: nerium indicum, 5-oleandrin, cytotoxicity, dna, anticancer doi: http://dx.doi.org/10.18051/univmed.2017.v36.102-109 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: wahyuningsih ms, mubarika s, ganjar ig, wahyuono s, takeya t. 5a-oleandrin reduce bcl-2 protei n an d in crea se bax p rotein expression on hela cervical cancer cell. uni v med 20 17;3 6:10 2-9. doi: 1 0 . 1 8 0 5 1 / un i vm ed . 2 0 1 7 . v 3 6 . 1 0 2 1 0 9 103 univ med vol. 36 no.2 introduction cervical cancer is the fourth most common cancer worldwide, among females in 2012. there were estimated about 528 000 new cases of cervical cancer, of which around 85% occurred in less developed regions. around 266.000 females died of cervical cancer, accounting for 7.5% of all female cancer deaths. about 87% of cervical cancer deaths occurred in the less developed regions.(1) cervical cancer is still the second most frequent cancer among indonesian women, thus screening program is still critically important to prevent it. prevalence of visual inspection with acetic acid (via) test-positive is 4.7% in jakarta population. the findings of precancerous lesions and cervical cancers are not only between thirty and fifty years old, but also below the thirty years old and after fifty years old.(2) cervical cancer appears as the most frequent cancer among women, and its position was the first rank, followed by breast, ovary, skin, thyroid, rectum, lymph nodes, uterus, colon, nasopharynx.(3) targeted cancer therapies is substance that block the growth and spread of cancer by interfering with molecular targets that are involved in the growth, progression, and spread of cancer. during the past few decades, targeted therapy has emerged as a promising approach for the development of selective anticancer agents.(4) several molecular targets for anticancer drug discovery and development have been identified including genes associated with cell cycle control and apoptosis. it is well known that two main apoptosis pathways, the intrinsic and extrinsic pathways, are involved in the regulation of tumorigenesis. apoptosis is one method the body uses to get rid of unneeded or abnormal cells, but cancer cells have strategies to avoid apoptosis. apoptosis inducers can get around these strategies to cause the death of cancer cells.(5) hela cervical cancer cell is a cell culture model of cervical cancer, which is generally used for the study because it is quite safe, grows faster and easy to be handled.(6,7) the le aves of nerium indicum mill. containing cardiac glycosides possibly having a role in cancer management, cardiac arrhythmias, and anti-viral.(8-10) current evidence showed that oleandrin from n. indicum inhibited proliferation and suppress the export of fibroblast growth factor-2 in prostate cancer cells, also induce apoptosis in human colorectal cancer cells via the mitochondrial pathway.(11) our previous study showed that the major compound isolated from the leaves of nerium indicum mill. was identified as 5-oleandrin was cytotoxic on several human cancer cells, but this compound was also cytotoxic to normal cells in vitro.(12) sonawane et al.(13) stated that safe delivery of effective antitumor agents is the main goal in cancer therapy. this is understandable that only a limited amount of the functional drug reaches tumor cells whereas it also acts on the normal healthy tissues resulting in serious adverse effects. further isolated compound identified as 5-oleandrin (16-acetyl-3-oleandrosa-14hydroxy-5-card-20(22)-enolide), possessed the remarkable cytotoxic effect on hela cervical c a n c e r ce ll s ( ic 5 0 , 8 . 38 x1 0 -6 mm) , a n d surprisingly was not cytotoxic to normal cell.(12) till today the mechanism of action of 5oleandrin compound hasn’t been evaluated yet. therefore, this study was aimed to determine the mechanism of action at molecular level especially the bcl-2 and bax protein expression using western blotting method. methods research design this study was an experimental laboratory study conducted at the integrated research and testing laboratory (irtl), universitas gadjah mada, yogyakarta and also at the division of molecular oncology, gr aduate school of biological sciences, nara institute of science and technology, japan. hela cervical cancer cell line, treated with 200 ng/ml of 5-oleandrin compound was used in this study. 104 study subjects culture of hela cervical cancer cell line, obtained from prof. tatsuo takeya (naist, japan), which were recultured by the integrated research and testing laboratory (irtl), universitas gadjah mada yogyakarta. research material 5-oleandrin was isolated by prof. dr. mae sri hartati w., m.si.,apt. (department of pharmacology and therapy, faculty of medicine, un i ve r s ita s g a dj a h m a d a , yogya ka r t a , indonesia). the compound is presented in figure 1. preparation of stock solution 5-oleandrin (1.0 mg) was dissolved in 100,0 l of dimethyl sulfoxide (dmso) (merck, germany) and then rpmi-1640 (medium) was added to obtain 10 mg/ml of stock solution. preparation of cells culture he la c e r vi c a l c a n c e r c e l l l in e wa s maintained in vitro in rpmi-1640 medium ( si gma che mic a l co ., usa) wh i c h w a s supplemented with 10% fetal bovine serum (g ibco invitr ogen , usa), 100 g/ ml of streptomycine (gibco invitrogen,usa), 100 unit/ ml of penicillin (gibco invitrogen, usa), and 2 mm of glutamin in tissue culture flask. the cells were incubated in 5% of co 2 incubator set at 37oc. p re para t io n of f l uore sc e nt s ta i ning of apoptotic cells using hoechst 33342 hela cervical cancer cells 1x105 cell/well in rpmi 1640 media were placed on 6 well plate, put on cover slips and incubated overnight at 37°c, 5% co2. the medium was removed and added 5-oleandrin and 5-oleandrin (200 ng/ ml) of each well in rpmi 1640 medium 10% fbs plate was incubated for 24 hours at 370c, 5 % co 2 . t h e y w e r e f i xe d i n 4% paraformaldehyde, washed with pbs and stained with 10 mm hoechst 33342 (molecular probes, eugene, or, usa) for 10 min. the stain was then removed and the cells were covered with mou n ti n g me d i a ( immu no t e c h ) p r i o r t o observation using fluorescence microscopy. preparation of gel electrophoresis analysis dna fragmentation was assessed by gel electrophoresis of extracted genomic dna from hela cervical cancer cells as described by mishra et al.(14) with slight modifications. experimental treatment using 5b-oleandrin (no. 3), 5-oleandrin (no.4), and nio3 (no.5) 200 ng/ml of each in rpmi 1640 medium 10% fbs, was incubated for 24 hours at 370c, 5% co 2 ; dna as a marker (no.1) and negative control (no.2). after treatment using those samples, cells were lysed in a buffer, then isopropanolol (0,5 ml) was added. the precipitate (dna) was transferred to a new eppendorf containing 500 ml 10 mm tris hcl, 0.1 mm edta, ph 7.5, and then incubated overnight at 37 °c stirred slowly. a total of 13 ml dna was added into 2 l of loading buffer mixed until homogeneous mixture was obtained. the mixture was inserted into the wells and run for 75 minutes at 50 volts. after electrophoresis was completed, the gel was transferred into a clean plastic, ethidium bromide was added and observed under uv light. preparation of western blot analysis the bcl-2 and bax protein expression was assessed by western blot analysis as described by hidayat et al.(15) and flanagan et al.(16) with slight modifications. after treatment with 5αoleandrin (3,47x10-4 mm) and followed by incubation for period of time, cells were lysed in a buffer containing 50 mm tris–hcl (ph 8), 450 mm nacl, 1% triton x-100, 5 mm edta, 1% (v/v) of protease inhibitor cocktail, and 1% (v/v) phosphatase inhibitor cocktails i and ii (sigma, st. louis, mo). protein (30–40 mg per lane) was separated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (sds-page) and transferred to a polyvinylidene difluoride (pvdf) membrane (bio-rad laboratories, hercules, ca). the membrane was blocked by 5% non-fat dry milk in tris-buffered saline and wahyuningsih, mubarika, ganjar, et al 5oleandrin on hela cervical cancer cell 105 univ med vol. 36 no.2 0.1% tween-20 (tbs-t) before incubation with a designated primary antibody (pharmingen, san diego, ca). after washing the membrane with tbs-t, the residue was incubated in secondary antibody and the immunoreaction was visualized using an ecl western blotting kit (amersham corp., arlington heights, il). results 5-oleandrin induces chromatin condensation in hela cervical cancer cells to observe whether 5-oleandrin could induce the chromatin condensation in hela cervical cancer cells, we assessed by hoechst 33342 staining. the result indicated that the nuclei of hela cervical cancer cells presented the changing morphology of the c ells like chromatin condensation in response to an treatment with 5boleandrin and 5-oleandrin (200 ng/ml) for 24 hours incubation. these results is typical of cell apoptosis (figure 2) 5-oleandrin induces dna fragmentation response in hela cervical cancer cells to investigate whether 5-oleandrin could induce dna fragmentation in hela cervical cancer cells, we assessed by gel electrophoresis of extracted genomic dna from hela cervical cancer cells. experimental treatment using 200 ng/ml of 5boleandrin (no.3); 5α-oleandrin (no.4); and nio3 (no.5) of each was incubated for 24 hours is presented in figure 3. the 5-oleandrin showed a clear observed by the presence of a smear band at about 200 bp on agarose gel, however this band was not observe d when 5-olea ndrin at equal concentration was applied. nio3 compound is one of other two compound isolated from the leaves of n. indicum mill at the equal concentration with 5-oleandrin (200 ng/ml) showed a smear band similar to 5-oleandrin, but this band was not as clear as showed by 5-oleandrin. 5-oleandrin decreased bcl-2 protein and increased bax protein activities in hela cervical cancer cells. to examine the bcl-2 and bax protein expression in 5-oleandrin-induced apoptosis, we used by western blot analysis. the data presented in figure. 4 demonstrate that treatment of hela cervical cancer cell with 5-oleandrin ( 3 , 47 x1 0 -4 mm) c omp ar e d wi t h β -a c t in , incubation for period of time, it can be seen that bc l-2 e xpr e ssion d ec re a ses c omp are d to untreated control. protein band of treated cells was similar to that of bcl-2 protein at 26 kda. figure 1. 5-oleandrin 106 figure 2. hela cervical cancer cell treated with 200 ng/ml, control (a), 5b-oleandrin (b), 5a-oleandrin (c) incubation for 24 hours, staining with hoechst 33342, fluorescens microscop (100x). note: (i) chromatin condensation; (ii) normal cells figure 3. gel electrophoresis of hela cervical cancer cells treated with 200 ng/ml incubation for 24 hours (1) dna marker 180 bp; (2) control; (3) 5b-oleandrin; (4) 5a-oleandrin; (5) nio3; note: (i) dna fragmentation (ii). dna leader control the 5a-oleandrin at the same concentration (3,47x10-4 mm) treated hela cervical cancer cells increases bax protein expression presented in figure 4. protein band of treated cells was similar to that of bax protein at 23 kda. as expected, the results of the western blot assays demonstrated that 5a-oleandrin-induced apoptosis was associated with the upregulation of cyt c and bax, the down regulation of bcl-2 proteins in hela cervical cancer cells. overall, these results indicate that 5±-oleandrin-induced apoptosis is associated with the mitochondrial apoptosis-related signaling pathway. discussion incubation of hela cervical cancer cells with 5-oleandrin (200 ng/ml) for 24 hours wahyuningsih, mubarika, ganjar, et al 5oleandrin on hela cervical cancer cell 107 univ med vol. 36 no.2 followed by staining with hoechst 33342 (figure 2), showed a clear broken up light blue color of nucleus occurred. the color produced was different to the color intensity of the untreated control. the staining hoechst 33342 is able to enter the living cells without causing toxicity to the cells, and that fluorescence occurs due to complex formation with native dna. the fluorescence occurs due to interaction between dna chromatin of the cells and hoechst 33342. this method is characterized as the non-toxic specif ic vital sta in for dna. chr omat in condensation can therefore be used to distinguish apoptotic cells from healthy cells or necrotic cells. ( 17 ) apo ptosis ha s a key r ole in the mechanism by which chemotherapy drugs cause cancer cell death. apoptotic can be recognized from the changing morphology of the cells, plasma membrane destruction, and chromatin condensation.(11) similar result was also obtained from the dna fragmentation, treated hela cervical cancer cells with 5-oleandrin (200 ng/ml) for 24 hours (figure 3), showed a clear observed by the presence of a smear band at about 200 bp on agarose gel. this band is as a signal or indicative of the apoptotic occurrence; however, this band was not observed when 5-oleandrin at equal concentration was applied. this indicated that 5b-oleandrin did not induce hela cervical cancer cells dna fragmentation, it was suggested that the cells was lysis due to the cytotoxicity of 5oleandrin. nio3 is one of other two compound isolated from the leaves of n. indicum mill at the equal concentration with 5a-oleandrin (200 ng/ml) showed a smear band similar to 5oleandrin, but this band was not as clear as s howe d by 5-o le a nd r in . bi oc h e mi ca l l y a p op t ot i c c a n b e r e c o gn i ze d b y dna fragmentation, internucleosomal of the dna fragments which are variable in between 180200 base pair (bp) to form dna ladder on the electrophoresis agarose gel. dna fragmentation is one reason of tumorigenesis, and most of tumors have genetic dna defects, while normal cells usually does not have these problems.(18) the dna ladder formation on the electrophoresis agarose gel indicates that apoptotic occurs in vitro. the mecha nism of a ction study was conducted at molecular level by determining the bcl-2 and bax protein expression. bcl-2 protein is a trans membrane protein having molecular weight around 26 kda produced by gen bcl-2 (6kb). bcl-2 antagonists are novel compounds that sensitise cancer cells to apoptosis by mimicking bh3-only proteins. in the intrinsic apoptotic pathway, bcl-2 family proteins, which include the anti-apoptotic proteins bcl-2 and bclxl, as well as the pro-apoptotic proteins bax, bak, bad and bim, are primary up-stream molecules that respond to apoptosis signals.(16,19) when cells figure 4. bcl-2 protein expression (a); bax (b); and β-actin (c) hela cervical cancer cells treated with 5-oleandrin (3,47x10-4 mm), incubation for period of time 108 a re s t imu la te d by a po pto si s signal s , the aforementioned apoptosis-associated proteins rapidly react to the stress and dramatically induce ros generation meanwhile, a high concentration of ros in mitochondria may reduce the mmp that enhances the mitochondrial permeability, and induce cytochrome c released from the mitochondria to the cytoplasm. in the cytoplasm, cytochrome c is involved in the formation of cytochrome c/apaf-1/caspase-9-containing apoptosome, which subsequently results in the a c t i va t i on o f c a sp a s e -9 a nd ca s pa se s -3 . activation of caspase-3, the last step of the cascade, leads to the degradation of specific cellular substrates and induces cell apoptosis.(19) the protein bands yield was visualized by bc l -2 a nti bo d y f o l lo wi n g t ra n s f e r t o polyvinylidene difluoride (pvdf) membrane (figure 4), it can be seen that bcl-2 expression decreases due to addition of 5-oleandrin (3,47x10-4 mm) for 2 24 hours compared to untreated control. protein band of treated cells was similar to that of bcl-2 protein at 26 kda. the decreasing of the bcl-2 expression level was suggested to be indicative to the death of cancer cells. similar procedure for bcl-2 protein was applied in the bax protein expression of the cells. the 5-oleandrin (3,47x10-4 mm) treated hela cervical cancer cells increases bax protein expression (figure 4). protein band of treated cells was similar to that of bax protein at 23 kda. it was reported that the presence of an moleculer antiapoptotic (bcl-2) can inhibit the activation of bax following a death signal. our r e s u l t s s t u dy sh o we d t ha t 5-o l e a n dr in significantly up-regulated the expression of bax but down-regulated the level of bcl-2 with the increase of treating time, which implied an increase of bax/bcl-2 ratio in 5-oleandrintreated hela cervical cancer cell line. other study on cardiac glycosides inhibited cell proliferation renal cell carcinoma by increasing the number of cells in the g2/m cell cycle phase. this results may provide a basis to study combined treatment consisting of cardiac glycosides and radio-/chemotherapy for rcc in the future.(20) 5-oleandrin is a polyphenolic component of cardiac glycosides. conclusion the 5α-oleandrin induced hela cervical cancer cells dna fragmentation observed by the presence of a smear band at about 200 bp indicative the apoptotic occurrence. 5-oleandrin induced apoptosis by reducing bcl-2 protein e x pr e s si on a n d i n c r e a si n g ba x pr o t e i n expression. conflicts of interests authors declare that there are no conflicts of interest. acknowledgements this study is financed by the overseas education cooperative foundation, those are appreciated. we are grateful to the takuya ogawa, division of molecular oncology, graduate school of biological sciences, nara institute of science and technology, japan for his technical assistance and especially to dr. dwi aris an., m.sc., ph.d, dept. of pharmacology and therapy, faculty of medicine ugm for insighful comments on the molecular level test. contributors msh contributed to the preparation of manuscript and research design, data collection, a n a l ys i s an d i n t e r p r e t a t i on . sm a nd t t contributed to revision of the manuscript especially on the mechanism of molecular action. igg and sw contributed to revision of the manuscript especially on the isolated compounds from n. indicum. msh wrote the manuscript and all the authors read and approved the final manuscript. wahyuningsih, mubarika, ganjar, et al 5oleandrin on hela cervical cancer cell 109 univ med vol. 36 no.2 references 1. ferlay 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ijms17111950. 20. nolte e, wach s, silva it, et al. a new semisynthetic cardenolide analog 3b-[2-(1amantadine)-1-on-ethylamine]-digitoxigenin (amantadig) affects g2/m cell cycle arrest and mirna expression profiles and enhances proapoptotic survivin-2b expression in renal cell carcinoma cell lines. oncotarget 2017;8:1167691. oktavianus 153 *department of biochemistry, faculty of medicine, diponegoro university, semarang, indonesia **department of biochemistry and molecular carcinogenesis, chiba cancer center research institute, japan correspondence dr. amallia n. setyawati, msi.med. department of biochemistry, faculty of medicine, diponegoro university jl. prof. soedharto, sh, tembalang, semarang p.o.box.: 1269 email: amallia.setyawati@fk.undip.ac.id univ med 2014;33:153-62 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background neuroblastoma (nb) is one of the most common extracranial solid tumors occurring in infancy and childhood with highly variable outcomes. polycomb group (pcg) proteins are epigenetic gene silencers. enhancer of zeste homolog 2 (ezh2) is a member of the polycomb repressor complex 2 (prc2) group, with the main function to catalyze the polycomb repressor complex by methylating lysine 9 and 27 of histone h3. this study aimed to investigate the biological functionality of ezh2 in nb. methods this was an experimental study with an analysis of correlation initially of the known prognostic factors of nb patients’ outcomes, by comparing the expression of v-myc avian myelocytomatosis viral oncogene neuroblastoma (mycn) with that of ezh2, on the basis of the patients’ overall and relapse free survival rates. this was followed with a biological functional study to assess the role of ezh2 expression in nb. results ezh2 knockdown induces neurite extension and differentiation marker growth associated protein 43 (gap43) in nb cells, although it does not affect cell cycle. by ectopic expression of ezh2, all-trans retinoic acid (atra) induced neurite extension was suppressed and gap43 was decreased. overall, ezh2 seems to have an important role in nb cell differentiation. although ezh2 did not alter cell proliferation, in the soft agar colony formation assay there was a significant increase in total colony number and number of large colonies. conclusion our result clarified the potential role of ezh2 in the regulation of cell differentiation and proliferation, which subsequently may play an important role in the poor prognosis of nb patients. keywords: neuroblastoma, ezh2, mycn enhancer of zeste homolog 2 regulates cell differentiation and proliferation in neuroblastoma amallia n. setyawati*,** and takehiko kamijo** 154 setyawati, kamijo enhancer of zeste homolog 2 regulates enhancer of zeste homolog 2 berperan pada diferensiasi dan proliferasi sel neuroblastoma pendahuluan neuroblastoma (nb) merupakan salah satu penyakit keganasan tumor solid ekstrakranial yang terjadi pada bayi dan masa kanak-kanak dengan luaran yang beragam. protein kelompok gen polycomb (pcg) merupakan silencer gen epigenetic. enhancer of zeste homolog 2 (ezh2) diketahui merupakan anggota kelompok polycomb repressor complex 2 (prc2) dengan fungsi utamanya yaitu katalisasi subunit kompleks repressor polycomb dengan cara metilasi lisin 9 dan 27 dari histon h3. beragam jenis kanker menunjukkan ekspresi tinggi ezh2. penelitian ini bertujuan untuk menyelidiki fungsi biologis ezh2 pada nb. metode penelitian ini merupakan penelitian experimental dengan melakukan analisis korelasi menghubungkan faktor prognostik luaran pasien nb, dengan membandingkan ekspresi v-myc avian myelocytomatosis viral oncogene neuroblastoma (mycn) dibandingkan dengan ezh2 berdasarkan tingkat kesintasan dan tingkat bebas penyakit pasien nb. selain itu dilakukan pemeriksaan regulasi diferensiasi nb cell oleh ezh2 dan studi fungsional biologis untuk mengetahui peranan ekspresi ezh2 pada neuroblastoma. hasil knockdown ezh2 menginduksi ekstensi neurit dan marker diferensiasi growth associated protein 43 (gap43) pada sel nb, walaupun tidak berpengaruh terhadap siklus sel. pada ekspresi ektopik ezh2, terjadi penekanan ekstensi neurit yang diinduksi all-trans retinoic acid (atra) dan gap43 mengalami penurunan. secara keseluruhan, ezh2 tampak berperan terhadap diferensiasi sel nb. walaupun ezh2 tidak memiliki efek pada proliferasi sel, namun demikian pada study soft agar colony formation assay didapatkan peningkatan jumlah koloni dan sejumlah koloni berukuran besar. hal ini menunjukkan bahwa ezh2 berperanan di dalam tumorigenesis nb. kesimpulan ezh2 memiliki peranan di dalam pengaturan diferensiasi dan proliferasi sel neuroblastoma, sehingga dapat berpengaruh terhadap luaran buruk pasien nb. kata kunci: neuroblastoma, ezh2, mycn abstrak introduction neuroblastoma is one of the most common extracranial solid tumors occurring in infancy and childhood, derived from sympathetic neuroblasts. clinical outcomes are highly variable, from spontaneous regression, caused by neuronal differentiation and/or apoptotic cell death, to malignant progression.(1) cytogenetic and molecular genetic studies identified that genetic abnormalities such as loss of the short arm of chromosome 1 (1p), amplification of vmyc avian myelocytomatosis viral oncogene neuroblastoma (mycn) and 17q gain are frequently observed and often associated with poor clinical outcome.(2) epigenetic alterations such as altered dna methylation, misregulation o f c h r o m a t i n r e m o d e l i n g b y h i s t o n e modifications, and aberrant expression of polycomb group (pcg) genes proteins, have emerged as common hallmarks of many nbs,(3,4) other than genetic changes that occur in cancer 155 such as the deletion of tumor suppressor genes (tsgs), amplification/activation of oncogenes, and loss of heterozygosity or gene mutations in tumor associated genes.(5) p o l y c o m b g r o u p g e n e p r o t e i n s a r e epigenetic gene silencers and are involved in the maintenance of embryonic and adult stem cells, w h i c h a r e i m p l i c a t e d i n n e o p l a s t i c development.(6) polycomb group gene proteins construct multiprotein complexes known as polycomb repressive complexes (prcs). polycomb group gene proteins are classified into two groups. one group, prc2, contains enhancer of zeste homolog 2 (ezh2), embryonic ectoderm development (eed), suz12, and rbap48. while the other, prc1, consists of >10 subunits including the oncoprotein b lymphoma mo-mlv insertion region 1 homolog (bmi1), and the hpc proteins, namely hph1-3, ring1-2 and scml.(3) previously, there were reports that prc1 bmi1 has an important role in nb tumorigenesis and that bmi1 transcription was induced by mycn.(7,8) enhancer of zeste homolog 2, also called histone lysine methyltransferase (hkmt), was cloned as one of the polycomb group genes. the function of ezh2 is to catalyze the polycomb repressor complex by methylating lysine 9 and 27 of histone h3. enhancer of zeste homolog 2 has recently been identified to be involved in essential celullar processes, namely cell fate decision, cell cycle regulation, senescence, cell differentiation and cancer. various cancer types also highly express ezh2.(9) cancer initiation and progression has also been associated with overexpression of ezh2. overexpression of ezh2 contributes not only to cancer stem cell formation but also to expansion of an aggressive cancer stem cell population that promotes cancer progression.(10) ezh2 plays a crucial role in stem cell maintenance and tumor development. it was identified that reducing ezh2 expression using sirna or treatment with a small molecule, s-adenosylhomocysteine hydrolase inhibitor 3-deazaneplanocin (dznep), i n h i b i t s m e t h y l t r a n s f e r a s e s a n d i n d u c e s degradation of ezh2, which subsequently result in cell growth inhibition and reduced tumor formation in various cancers.(11) a l t h o u g h p r e v i o u s s t u d i e s i n d i c a t e involvement of ezh2 in various cancers, however, specifically in nb, the biological molecular mechanisms underlying this remain poorly understood. in view of the above mentioned background, this study aimed to identify the association of ezh2 expression with the classical poor prognosis outcome of nb and its biological translation. methods research design the present study using an experimental design was conducted from january to december 2012 at the laboratory of biochemistry and molecular carcinogenesis, chiba cancer center research institute. cell cultures we selected thirteen primary neuroblastoma cell lines (nb-69, sk-n-sh, sh-sy5y, sk-nas, sk-n-be, sk-n-dz, nb39-nu, ngp, nb19, nb9, imr32, tgw, sms-san) which represent mycn single-copy and mycnamplified cells. the cells were maintained in rpmi 1640 or dulbecco’s modified eagle’s medium (wako, osaka, japan) supplemented with 10% heat-inactivated fetal bovine serum (invitrogen, carlsbad, ca, usa), ~50 µg/ml penicillin/streptomycin (sigma-aldrich, st louis, mo, usa), l-glutamine, phenol red supplemented with 10% fetal bovine serum (fbs), and 1% antibiotics under humidified 5% co 2 in air at 37oc. rna extraction and reverse transcription polymerase chain reaction (rt-pcr) analyses the quantitative rt-pcr (qrt-pcr) was performed with a sybr green real time quantitative rt-pcr assay kit (qiagen) on rna extracts obtained from neuroblastoma cancer stem cell lines using real time pcr applied univ med vol. 33 no.3 156 setyawati, kamijo enhancer of zeste homolog 2 regulates biosystem 7500. the primer set used was 1096f 5’-tataagcggaagaacacagaaaca-3’ and 1171r 5’-tccctccaaatgctggtaac ac-3’. a s a n i n t e r n a l c o n t r o l , t h e l e v e l o f glyceraldehyde-3-phosphate dehydrogenase (gapdh) was analyzed. a master mix (50 µl) of the following reaction components was prepared to the indicated end reaction: 2xsybr green master mix (sybr de), 10µl of dyell (reff dye ii), 0.4µl of 200 nm reverse primer (10µm), 0.4µl of 200 nm forward primer (10µm), and h 2 o 6.8µl. western blot analyses cells were lyzed in a buffer containing 120 mm nacl, 50 mm tris–hcl (ph 8.0), 50 mm m g c l 2 , 4 0 µ g / m l a p r o t i n i n , 1 0 m m βglycerophosphate, 1mm phenylmethylsulfonyl fluoride, 1 mm naf, 250 u/ml benzonase® (novagen, san diego, ca, usa), 0.1mm na 3 vo 4 , and 0.5% nonidet p-40. western blot analysis was performed as previously reported.(12) after transferring to an immobilon-p membrane (millipore, bedford, ma, usa), proteins were reacted with either anti-bmi1 mouse monoclonal (229f6; upstate, charlottesville, va, usa), anti-ezh2 (rabbit polyclonal, millipore 07-681), or anti-ring1b mouse monoclonal antibody as described in a previous report.(13) semi quantitative rt-pcr semi-quantitative rt-pcr was conducted as previously described.(12) total cellular rna to prepare rt-pcr templates was extracted from nb cell lines using isogen (nippon gene k, tokyo, japan) and subsequently cdna was synthesized from 1 µg total rna template according to the manufacturer’s protocol (river tra ace-α, rt pcr kit, toyobo, osaka, japan). lentiviral infection the packaging cell line hek 293t (4x106) was plated and transfected the next day, when 1.5 µg of the transducing vectors containing the gene or shrna and 2.0 µg of the packaging vectors (sigma-aldrich) were cotransfected by the fugene6 transfection reagent (roche applied science, indianapolis, in, usa), according to the manufacturer’s protocol. the medium was changed the next day and cells were cultured for another 24 hours. conditioned medium was collected and cleared from the debris by filtering through a 0.45µm filter (milipore). thereafter, 1x105 nb cells were seeded in each well of a 6 w e l l p l a t e a n d t r a n s d u c e d b y l e n t i v i r a l conditioned media. transduced cells were analyzed by western blot and rt pcr. overexpression and knockdown of ezh2 for the overexpression of ezh2, flagtagged hezh2 was subcloned into lentivirus phr-sin-csgw. for the knockdown of ezh2, the five plko.1-puromycin based lentiviral vectors containing shrna sequences targeting ezh2 (refseq nm_004456) were obtained from the mission trc-hs 1.0 (human) shrna library (sigma aldrich). virus production, infection and selection were performed according to the manufacturer’s protocol. one week post infection, cells were collected and knockdown efficiency was examined by western blot. we checked ezh2 knockdown by the five lentiviral shrnas and used two for experiments. cell proliferation assay nb cells were seeded in 96-well plates at a density of 750 cells per well in a final volume of 100 µl. the culture was maintained under 5% co 2 and 10 µl wst-8 labeling solution (cell counting kit-8; dojindo, kumamoto, japan) was added, then the cells were returned to the incubator for 2 h. the absorbance of the formazan products formed was detected at 450nm in a 96-well spectrophotometric plate reader, according to the manufacturer’s protocol. soft agar colony formation assay imr32 cells were seeded at 2,000 cells in semi-solid noble agar medium in a 2-cm dish and evaluated after 2 weeks for colony numbers. 157 colony number was automatically counted using winroof software. statistical analysis all data were tested statistically using the 2-tailed t test. p <0.05 was considered to indicate statistical significance. ethical clearance ethical clearance was granted by the institutional review board at chiba cancer center research institute after the study protocol had been carefully reviewed. results to identify the association of ezh2 expression and patient’s outcome, we analyzed the correlation of the known prognostic factor of nb patients’ outcome, mycn, with ezh2 expression (figure 1a). subsequently kaplanmeier analysis was used to determine overall and relapse free (event free) survival rate of nb patients, who were divided into high and low ezh2 expression groups. (figure 1b and 1c). r2: microarray analysis and visualization platform openware database was used for these analyses. figure 1. clinical relevance of ezh2 expression in nb patients. ezh2 is associated with a poor prognosis in neuroblastoma. (a) average ezh2 expression in 88 neuroblastoma tumors according to their inss stage and correlation with mycn expression (p=0.01). (b,c) kaplan-meier analysis for event free and overall survival of neuroblastoma patients, divided into high and low ezh2 expression groups for all neuroblastoma patients (n=88). univ med vol. 33 no.3 158 setyawati, kamijo enhancer of zeste homolog 2 regulates figure 2. endogenous ezh2 expression in nb cell lines (a) western blot analysis and (b) qrt-pcr of ezh2 in neuroblastoma cell lines the study was initiated by comparing ezh2 expression among nb cell lines both at protein and transcriptional level (figure 2). the result suggested that there is an enhancement of ezh2 expression in mycn-amplified nb cell lines at protein level. to identify the association of prc1 group member with ezh2, we overexpressed ring1b in sk-n-be and nb69 cells and examined the molecules as indicated (figure 3). the results suggested that prc1 group ring1b expression does not induce ezh2 expression, but induces bmi1, another prc1 molecule. next, we overexpressed ezh2 in these nb cell lines. ezh2 expression did not induce either bmi1 or ring1b proteins. a significant increase in total colony number and number of large colonies, was also observed in soft agar colony formation assay of ectopically expressed ezh2 (figure 4). discussion in the knockdown study of ezh2, ezh2 knockdown induces neurite extension and differentiation marker gap43 in nb cells. however, it does not affect cell cycle and this is in contrast to previous results, in which silencing of ezh2 decreases nb cell proliferation and induces neurite extension.(14) by the ectopic expression of ezh2, atra-induced neurite extension was suppressed and gap43 was decreased. together, ezh2 seems to have an important role in nb cell differentiation. it is known that methylation of h3k27 by ezh2 in neural stem cells is required to maintain a proper 159 figure 3. effects of ezh2 on prc1 polycomb proteins flag-ezh2 and myc-ring1b were expressed by lentivirus as described in methods (a) western blot analysis (b) semi-quantitative rt-pcr results analyzed molecules are shown in the left margin of the panels stem cells into oligodendrocytes. while ezh2 is highly expressed in proliferating neural stem c e l l s , i t s e x p r e s s i o n i s d e c r e a s e d u p o n differentiation into neurons and abolished during differentiation into astrocytes. however, ezh2 balance between self-renewal of neural stem cells and differentiation into neurons and to prevent massive neurogenesis and depletion of the neural stem cell pool that would eventually result in a decreased number of neurons.(15) further, we studied the effect of ezh2 on cell proliferation. although ezh2 did not alter cell proliferation, in the soft agar colony formation assay, which is known to be the h a l l m a r k o f c e l l u l a r t r a n s f o r m a t i o n a n d uncontrolled cell growth, we identified a significant increase both in total and large colony numbers. these results suggest that ezh2 may have role in tumorigenesis of nb. however we cannot elucidate the exact mechanism on how ezh2 regulates prc target versus nontarget methylation in neuroblastoma. our finding reveals that ezh2 has a strong association with tumor progression and cancer survival. the histon methyl transferase (hmt) ezh2 is needed for proper in vitro specification of mouse neural figure 4. soft agar colony assay, day 15 univ med vol. 33 no.3 160 setyawati, kamijo enhancer of zeste homolog 2 regulates expression remains high in oligodendrocytes.(16) a comparative chip-seq analysis of ezh2 target genes in neural stem cells and in premyelinating oligodendrocytes (pols) reports the presence of ezh2 on oligodendrocyte, neuronal and astrocytic lineage specific genes in neural stem cells, whereas in pols, ezh2 is not anymore present on the oligodendrocyte lineage specific genes but is still found on astrocytic and neuronal lineage specific genes.(17) individual nb tumors can be classified histologically into nb (undifferentiated, schwannian stroma poor, and high risk), ganglioneuroblastoma, and ganglioneuroma (differentiated ganglion cells). the latter two are schwannian stroma rich and low risk. (18) overexpression of ezh2 is found in a number of cancers, including prostate(19) and breast c a n c e r s ( 2 0 ) a s w e l l a s e w i n g s a r c o m a , ( 2 1 ) g l i o b l a s t o m a , ( 2 2 ) a n d m e l a n o m a . ( 2 3 ) t h e mechanisms responsible for hypermethylation of groups of genes in specific types of human cancers have not yet been identified. there have been researches conducted on the involvement of polycomb repressive complexes (prcs) in dna methylation, and particularly on the ezh2 gene, which is the catalytic component of the prc2 and prc3 complexes.(24) previous studies indicate that ezh2 is a prognostic biomarker in breast and prostate cancer. it is known that gene silencing is associated with a well-known histone mark, the methyltransferase ezh2 of which are histone acetylation for gene activation and the trimethylation of histone-3 on lysine-27 (h3k27me3). ezh2 has been shown to control dna methylation through its ability to produce t h e n u c l e o s o m a l h i s t o n e h 3 l y s i n e 2 7 trimethylation mark (h3k27me3) that provides a p l a t f o r m f o r r e c r u i t i n g d n a methyltransferases.(25) polycomb repressive complex (prc) activation is linked to cell typespecific patterns of gene repression and dna methylation through the h3k27me3 markings that are produced in stem cells. thus, prc gene targets in cancer progenitor cells may be preprogrammed for dna hypermethylation that is triggered later during cell transformation.(26,27) ezh2 overexpression is associated with aggressive clinical behavior in prostate, breast, and bladder cancers. in this study we found a relatively high expression of ezh2 at protein level of mycnamplified neuroblastoma cells compared to mycn-single copy cells. however, the mycn copy status does not show a strong effect on the expression level of ezh2 at mrna level. previously, there was a report that mycn induces bmi1 transcription in nb cells.(7) mycn-related prc1 increase may also have some effects on ezh2 protein stabilization. we could conclude that ezh2 knockdown or ectopically expressed ezh2 caused not only d i ff e r e n t i a t i o n i n n e u r i t e e x t e n s i o n a n d significant changes in differentiation markers of gap43, but also a prominent increment in total colony numbers and the presence of large colonies in our soft agar colony formation assay. however, our study is at an early stage, therefore we still could not identify the mechanism of ezh2 downregulation and the potential molecular mechanism of nb tumorigenesis. conclusion enhancer of zeste homolog 2 contributes to the maintenance of the differentiation stage of the nb phenotype and may be important in n b t u m o r i g e n e s i s . h o w e v e r, t h e e x a c t mechanism and targeted pathway remain to be elucidated. acknowledgement we thank dr. kamijo for the opportunity to conduct this study, dr. takenobu for the supervision, mentoring and helpful discussion, and k sakurai for technical assistance. thanks to a takeda science foundation grant for fiscal year 2012, the authors were able to complete this study course. 161 references 1. paffhausen t, schwab m, westermann f. targeted mycn expression affects 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ezh2 in nevi and melanoma. j cutan pathol 2007;34: 597-600. 24. rajasekhar vk, dalerba p, passegue e, lagasse e, najbauer j. stem cells, cancer, and context dependence. stem cells 2007;26:292-8. 25. vire e, brenner c, deplus r, blanchon l, frage m, didetot c, et al. the polycomb group protein ezh2 directly controls dna methylation. nature 2006;439:871–4. univ med vol. 33 no.3 162 setyawati, kamijo enhancer of zeste homolog 2 regulates 26. keshet i, schlesinger y, farkash s, rand e, hecht m, segal e, pikarski e, et al. evidence for an instructive mechanism of de novo methylation in cancer cells. nat genet 2006;38: 149–53. 27. schlesinger y, straussman r, keshet i, farkash s, hecht m, zimmerman j, et al. polycombmediated methylation on lys27 of histone h3 pre-marks genes for de novo methylation in cancer. nat genet 2007;39:232–6. c:\users\jurnal fkusakti\docume 221 *institute of research and development for biomedicine papua **mimika aids commision correspondence : mirna widiyanti institute of research and development for biomedicine papua ministry of health, republic of indonesia jl. kesehatan no.10 dok ii jayapura phone: (+62967) 534389 fax (+62967) 534697 mobile: +6281344388000 email: ninawidhy@gmail.com date of first submission, april 3, 2017 date of final revised submission, november 27, 2017 date of acceptance, november 27, 2 01 7 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 low body mass index increases risk of anemia in patients with hiv-aids receiving antiretroviral therapy mirna widiyanti*, reynold ubra**, and eva fitriana* background in acquired immunodeficiency syndrome (aids) cases, cytopenias of all major blood cell lines were increasingly recognized in patients with hiv infection. anemia commonly occurs during hiv infection and has been associated with increased progression to aids and decreased survival. the aim of this study was to determine the prevalence of anemia and associated risk factors in adults with hiv-aids receiving antiretroviral therapy (art). methods a cross sectional study was conducted involving 90 adults with hivaids. socio demographic data were collected using a questionnaire. in all patients, cd4+ lymphocyte counts were performed by means of a pima analyzer, while hemoglobin was determined using a sysmex hematology analyzer. the who cut-off value of hemoglobin was adjusted to altitude to define anemia. mild to moderate anemia was defined as hemoglobin 8-13 g/dl for men and 8-12 g/dl for women. severe anemia was defined as hemoglobin 8 g/dl. data was analyzed by using logistic regression test. results there were 90 subjects with hiv-aids in this study. anemia was found in 50 subjects (55.6%), consisting of 12.2% with mild anemia and 43.4% with moderate to severe anemia. multiple logistic regression showed that an important risk factor for anemia was low body mass index (bmi) between <17-18.5 (or=4.20; 95% ci=1.23-14.36). however, the cd4 <350 cells/mm3 was not a significant risk factor for anemia (or=1.03; 95% ci=0.21-5.04). conclusions low bmi increases the risk of anemia in subjects with hiv receiving art. better screening for anemia and infectious diseases and modified harm reduction strategy are primary needs in hiv patients. keywords: anemia, body mass index, cd4+, hiv-aids doi: http://dx.doi.org/10.18051/univmed.2017.v36.221-227 original article cite this article as: widiyanti m, ubra r, fitriana e. low body mass index increases risk of anemia in adults with hiv-aids receiving antiretroviral therapy. univ med 2017;36:221-7. doi: 1 0 . 1 8 0 5 1 / un i vm e d . 2 0 1 7 .v 3 6 . 2 2 1 2 2 7 pissn: 1907-3062 / eissn: 2407-2230 222 widiyanti, ubra, fitriana anemia in hiv-aids introduction acquired immune deficiency syndrome (aids) is an infectious disease caused by the human immunodeficiency virus (hiv ), a retrovirus that attacks and destroys t-helper lymphocytes (cd4+), resulting in an immunocompromised condition of the patient. hiv causes a decrease in the body’s immunity and increases the incidence of various cognitive impairment and aids dementia.(1) hiv-aids cases in indonesia continue to increase and until 2014 the number reached 150 285 cases of hiv, while aids cases reached 55 799. the number of hiv cases in papua reached 16 051 and in timika regency reached 3 900.(2) anemia is one of the most common blood abnormalities in persons with hiv disease. up to 90% of adults can develop anemia during an hiv infection, especially individuals with advanced disease and low cd4+ t-cell count. persons with anemia have a decreased quality of life that potentially increases the chance of mortality. the incidence of anemia has been found to be strongly associated with the progression of hiv to aids.(3,4) even among persons initiating antiretroviral therapy, anemia has been demonstrated to be a strong risk factor for disease progression and subsequent death independent of cd4 count and viral load.(5) several causes of anemia have been described in hiv-positive patients, such as changes in cytokine production with subsequent e f f e c t on h e ma t op oi e si s , d e c r e a se d erythropoietin concentration, opportunistic infections agents such as mycobac terium a v ium c omp l e x a n d a d min i s t r a t i on of chemotherapeutic agents such as zidovudine and ganciclovir.(6) the more advanced the disease the higher the incidence of anemia and also the morbidity and mortality. anemia has been proved to be an independent risk factor for death. according to the world health organization (who) mild to moderate anemia is defined as hemoglobin 813 g/dl for men and 8-12 g/dl for women. severe anemia is defined as hemoglobin 8 g/ dl.(7) according to a study in sub-saharan africa, which has the largest burden of hiv in the world, anemia is common as patients are more likely to be malnourished, have severe i mmuno suppr essi o n, a nd high e r r a t es o f comorbidities (especially tuberculosis and malaria) than those in high-income countries.(8) a study in iran on the prevalence of anemia and related factors in hiv positive patients showed that among 212 hiv positive patients the proportion of anemia was 71%, with the majority of patients having mild to moderate anemia, while 17 patients (8%) had a positive history of tuberculosis. the investigators found a strong association between anemia and death.(9) a cross sectional study in india showed that proper etiological diagnosis could be reached in 46 patients. among them the correlation between hb% and cd4 count was statistically nonsignificant (p=0.074, r=0.47) whereas it was significant between absolute lymphocyte and cd4 counts (p=0.006, r=0.41).(10) the major cause of anemia is impaired erythropoiesis resulting from the release of i nf l a mma to r y c yt oki ne s a n d d e c r e a s e d production of hematopoietic growth factors, coupled with malabsorption and impaired recycling of iron. t hi s s t ud y a i me d t o d e t er mi n e t h e prevalence of anemia and the risk factors associated with the incidence of anemia in hiv patients receiving antiretroviral (arv) therapy. methods research design an analytic cross sectional study was conducted on hiv/aids patients undergoing routine care at mitra masyarakat hospital, mimika, papua, from march-may 2015. research subjects the study was conducted by reviewing hospital data of patients who were receiving 223 univ med vol. 36 no.3 highly active antiretroviral therapy (haart) at the hiv clinic of mimika. the sample size was estimated using a single population formula, 3% level precision (d) with 95% confidence interval. patients who were pregnant at baseline or during the follow up visits were excluded from the study. measurements a d e t a i l e d h i s to ry i nc l u di n g s o c i od e mo gr a p hic c h a r a c t e r i s t i c s , p r e s e n t in g symptoms, presence of symptoms related to anemia, nutritional history, past medical history, previous and current drug history, caretaker characteristics (including education level and occupation) was assessed with a pretested and standardized questionnaire. after the interview, a detailed review of t he me d i c al r e c o r ds of a ll s ub j e c t s w a s performed, noting such details as concurrent opportunistic infections and who clinical staging of hiv disease. blood samples for determination of hemoglobin level and cd4+ count were collected and processed. laboratory analysis three ml of venous blood was collected from the median cubital vein of each subject by an experienced laboratory technologist for cd4+ count dete rmin ation using a pima analyzer (alere, germany) and for hemoglobin determination using a hematology analyzer (sysmex, japan). an e mi a wa s de f i ne d b a se d o n hematological reference values for anemia for the adult indonesian population. accordingly, mild to moderate anemia was defined as a hemoglobin concentration of 8-13 g/dl for men and 8-12 g/dl for women. severe anemia was defined as hemoglobin 8 g/dl. the cd4 count was reported as the number of cd4 cells present in 1 mm3 of blood (cells/ mm3). the classification of cd4 cell counts of <350 cells / mm3 and 350 cells / mm3 was based on the who recommendation that hiv patients starting arv therapy should be patients with cd4 <350 cells / mm3.(11) statistical analysis logistic regression analysis was used to analyze the data, and odds ratio with 95% confidence interval was used to quantify the strength of association between anemia and its potential predictors. a p-value of less than 0.05 was considered significant. ethical clearence this study was conducted after obtaining approval from the ethical commission of the national institute of health research and development, ministry of health, under number: lb.02.01/5.2/ke/156/2015 results table 1 shows that among hiv-positive respondents in mimika, 25 (27.8%) were male while 65 (72.2%) were female. the largest age group of <35 years consisted of 55 subjects (61.1%) and the group of >35 years comprised 35 (38.9%). hiv patients who were ethnic papua consisted of 65 (72.2%) individuals, while ethnic non-papuans consisted of 25 (27.8%) individuals. with respect to educational level, there were more individuals who had been enrolled in school i.e. 81.1%, compared to 18.9% who had not attended school. most hiv-positive individuals (75 individuals or 83.3%) were employed, compared with 16.7% who were nonemployed. hiv-positive respondents who were married comprised 80 individuals (88.9%), whereas unmarried respondents comprised 10 individuals (11.1%). nearly all respondents (83 individuals or 92.2%) had heterosexual risk factors and only 7 (7.8%) had other risk factors. according to clinical characteristics, the number of subjects at clinical stage iii/iv was 74 individuals (82.2%) and at clinical stage i and ii 16 individuals (17.8%). the cd4 count of >350 cells/mm3 included a total of 71 individuals (78.9%), body mass index (bmi) of 18.5-25 c o mp r i s e d 70 ( 7 7. 8 % ) i n di vidu a l s . t he prevalence of anemia was 50 (55.6%). a total of 74 patients (82.2%) had tuberculosis as 224 widiyanti, ubra, fitriana anemia in hiv-aids a b c opport unis ti c in fe ct ion. t he dur at io n of azidothymidine treatment of >24 months was found in 59 (65.6%) subjects. the relationship between gender and the incidence of anemia showed that there was no significant difference between the percentages of anemia in males and females (p=0.237). similarly, for the variables age, marital status, risk factors, opportunistic variable n % gender male 25 27.8 female 65 72.2 ethnicity papua 65 72.2 non-papua 25 27.8 age (years) <35 55 61.1 35 35 38.9 educational status no education 17 18.9 education 73 81.1 employment status yes 75 83.3 no 15 16.7 marital status never married 10 11.1 married 80 88.9 risk factor heterosexual 83 92.2 other 7 who clinical stage stage i/ ii 74 82.2 stage iii/iv 16 17.8 cd4 cell count (cells/mm3) < 350 19 21.1 350 71 78.9 body mass index (kg/m2) <17-18.5 20 22.2 18.5-25 70 77.8 anemia yes no opportunistic infection 50 40 56.7 43.3 tuberculosis 74 82.2 other 16 17.8 therapy azt-based 58 64.4 other 32 35.6 duration of therapy (months) <24 31 34.4 24 59 65.6 table 1. socio-demographic and clinical characteristics and art duration in hiv subjects (n=90) azt = azidothymidine (zidovudine) infections, clinical staging and therapy, there was no significant relationship with the occurrence of anemia (p>0.05). the variables that showed a significant association with anemia were bmi (or: 4.23; 95% ci: 1.28-13.94) and cd4 count (or: 3.52; 95% ci: 1.31-9.69) (table 2). table 3 shows the logistic regression model with anemia as the dependent variable and body mass index and numbers of cd4+ cells as independent variable. the results of the analysis showed that the risk of anemia in hiv subjects with low bmi was 4.20 times higher compared to those with normal bmi (or: 4.10; 95% ci: 1.23-14.36). subjects with low cd4+ count (<350 cells/mm3) had a 1.03 times higher risk for anemia compared to subjects with cd4+ counts of 350 cells/mm3, but the relationship was statistically not significant (or:1.03; 95% ci: 0.21-5.04). discussion our study showed that the prevalence of anemia among hiv-infected patients in timika was 55.6%. this result is in line with another study at hasan sadikin hospital as the referral hospital for hiv in west java, showing that the prevalence of anemia among hiv-infected p a t i e nt s wa s 4 9. 6 % .( 1 2 ) ho we ve r, t hi s prevalence is higher than the prevalence of anemia among hiv-infected patients in south africa of 18.0%.(5) since hemoglobin measurements are simple and inexpensive, monitoring hemoglobin levels over the first 6 months of art among patients at high risk of anemia may allow for improved detection of hiv-related anemia. a cross-sectional study showed that the prevalence of anemia in hiv-aids patients is prominent and that the important risk factors for anemia are oral candidiasis and cd4, while bmi and administration of arv drugs are protective factors.(13) research in south india reported an anemia prevalence of 22% in patients with cd4 >500 cells / mm3, while the prevalence increased to 225 univ med vol. 36 no.3 variables anemic non-anemic p value or (95% ci) gender male female 11 39 14 26 0.237 0.52 (0.21-1.33) age (years) <35 32 23 0.664 1.31 (0.56-3.08) 35 18 17 body mass index (kg/m2) <17-18,5 18.5-25 16 34 4 36 0.020* 4.23 (1.28-13.94) marital status married 45 35 0.746 1.28 (0.34-4.79) not married 5 5 risk factor heterosexual 46 37 1.000 0.93 (0.19-4.43) other 4 3 opportunistic infection tuberculosis 42 32 0.782 1.31 (0.49-3.87) other 8 8 who clinical stage stage i/ii 40 34 0.590 0.70 (0.23-2.14) stage iii/iv cd4 cell count 10 6 <350 27 10 0.005* 3.52 (1.31-9.69) 350 23 30 therapy azt-based 33 25 0.826 1.16 (0.48-2.77) other 17 15 duration of therapy (months) <24 18 13 0.825 1.16 (0.48-2.81) 24 32 27 table 2. association of socio-demographic and other clinical variables with anemia among hiv subjects (n=90) azt : azidothymidine (zidovudine) b p value adjusted 95% ci or or lower upper cd4 0.037 0.963 1.03 0.21 5.04 bmi 1.436 0.022 4.20 1.23 14.36 table 3. factors associated with anemia resulting from multiple logistic regression analysis 60% in patients with cd4 <100 cells/mm3. this study concluded that anemia is associated with severe immune suppression in subjects with hiv infection.(14) in addition, studies in 11 sub-saharan african countries showed that in hiv-infected women the mean ane mia prevalence was 56.7%, with a range between 33 and 2%.(15) ramezani et al.(16) in iran found that mild-tomoderate hemoglobin concentrations between 8-14 g/dl was present in 46% of hiv-infected patients, but there was no difference between anemic and non-anemic patients in terms of age, sex, stage of disease, cd4 and antiretroviral treatment. we also found that low bmi increased the risk of anemia. this result was in line with the results of the study in south africa showing that low bmi was associated with anemia in hiv subjects.(5) the association between anemia and low bmi is likely to reflect malnutrition, as a number of nutritional deficiencies contribute to anemia. the association of anemia with low bmi is of specific relevance to india, where the rate of chronic malnutrition is among the highest in the world.(17) interestingly, even 226 widiyanti, ubra, fitriana anemia in hiv-aids individuals with a normal bmi had a two and half times increased odds of anemia as compared to those who were overweight. low bmi is associated with many nutrient deficiencies including iron, folate and b12 that contribute directly to anemia. the increased risk in anemia among patients with immunosuppresion is in agreement with previous published reports.(18) our study found that low cd4+ cell count was not a risk factor for anemia in hiv subjects. another study in mexico found different results, i.e. that low cd4+ cell count was positively correlated with hemoglobin level in hiv subjects.(19) it wa s pos t ul a t e d t ha t , apa r t f r o m immunological status and viral load, other factors might play major roles in the pathogenesis of anemia. detecting normocytic normochromic morphology as the major type of anemia was in tune with the existing data. despite conflicting reports worldwide, in a few studies strong correlation has been shown between absolute lymphocyte and cd4 counts.(20) therefore the absolute lymphocyte count can sometimes be used as a surrogate marker of immunological status in art naive hiv patients, particularly in resource poor areas. being on an azt containing regimen was also a risk factor for developing anemia. studies have shown the short-term tolerability related to the use of azt, in particular the development or worsening of anaemia.(21) finally, the high prevalence of anemia in patients with cd4 cell counts of <200 cells/μl has implications for the choice of initial haart regimen in developing countries. since anemia is an adverse effect of zidovudine (azt), use of this drug is contraindicated in patients with preexisting anemia.(22) the main limitation of this analysis is its cross sectional design, which precludes definite determination of the temporal relationship between anemia and its associated factors. also, our database does not c apture laboratory findings that could clarify the frequency of anemia due to particular nutrient deficiencies. other inflammatory parameters such as creactive protein (crp), interleukin-6 (il-6), and tumor necrosis factor alpha (tnf-alpha) should be studied as well in the future. conclusion low bmi increases the risk of anemia in subjects with hiv. better screening for anemia is the primary need in hiv patients. conflict of interest there was no conflict of interest between the authors and the study subjects acknowledgements this study was supported financially by the institute of health research and development for biomedicine papua, ministry of health, republic of indonesia. we also acknowledge all the staff who assisted with blood and data collection at mitra masyarakat hospital, mimika, papua. contributors mw designed the study and was involved in sampling and performing the experiment. mw, ef interpreted the data. mw, ef, ru+ contributed to writing and finalizing the manuscript. all authors read and approved the manuscript. references 1. sharp pm, hahn bh. origins of hiv and the aids pandemic. cold spring harb perspect med 2011;1: 1-22. 2. direktorat jenderal pemberantasan penyakit menular dan penyehatan lingkungan. 2014 statistik kasus hiv/aids di indonesia. jakarta: kementerian kesehatan ri;2014. 3. kulkarni mb, bhalerao mm, mungal su, et al. anemia in subjects living with hiv/aids: a cross sectional study from india. iosr j dent med sci 2015;14:4-8. 227 univ med vol. 36 no.3 4. ndlovu z, chirwa t, takuva s. incidence and predictors of recovery from anemia within an hivinfected south african cohort 2004-2010. pan african med j 2014;19:114-20. 5. takuva s, maskew m, brennan at, et al. anemia among hiv-infected patients initiating antiretroviral therapy in south africa: improvement in hemoglobin regardless of degree of immunosuppresion and the initiating art regimen. j trop med 2013. article id 162950. 6. jam s, ramezani a, sabzvari d, et al. a crosssectionel study of anemia in human immunodeficiency virus-infected patients in iran. arch iranian med 2009;12:145-50. 7. world health organization. hemoglobin concentrarion for the diagnosis of anemia and assesment of severity. geneva: world health organization;2011. 8. world health organization. worldwide prevalence of anemia 1993-2005: who global database on anemia. geneva: world health organization;2008. 9. meidani m, rezaei f, maracy mr, et al. prevalence, severity, and related factors of anemia in hiv/aids patients. j res med sci 2012;17:13842. 10. pande a, bhattacharyya m, pain s, et al. anemia in antiretroviral naive hiv/aids patients: a study from eastern india. j health allied sci 2011;10:14. 11. world health organization. who issues new hiv recommendations calling for earlier treatment. geneva : world health organization;2013. 12. wisaksana r, sumantri r , indrati ar, et al. anemia and iron homeostasis in a cohort of hiv-infected patients in indonesia. bmc infect dis 2011;11:213. 13. sumantri r, wicaksana r, ariantana ar. prevalensi dan faktor risiko anemia pada hivaids. mkb 2009;41:187-92. 14. subbaraman r, anemia among hiv-infected in south india [dissertation]. new haven: the yale university of medicine;2007. 15. bradley sek, mishra v. hiv and nutrition among women in sub-saharan africa. dhs analytical studies no. 16. calverton, maryland, usa: macro international inc.;2008. 16. ramezani a, aghakhan a, sharif mz, et al. anemia prevalence and related factors in hiv-infected patients; a cohort study. iranian j phathol 2008;3: 128-8. 17. gragnolati m, shekar m, gupta m. india’s undernourished children: a call for reform and action. washington, d.c : the world bank ; 2005. 18. takuva s, louwagie g, zuma k, et al. durability of first line antiretroviral therapy: reasons and predictive factors for modification in a swaziland cohort. j antivir antiretrovir 2012;4:14-20. 19. mata-marin ja, gaytan-martinez je, martinezmartinez re, et al. risk factors and correlates for anemia in hiv treatment-naïve infected patients: a cross-sectional analytical study. bmc res notes 2010;3:230. 20. alavi sm, ahmadi f, farhadi m. correlation between total lymphocyte count, hemoglobin, hematocrit and cd4 count in hiv/aids patients. acta medica iranica 2009;47:1-4. 21. zhou j, jaquet a, bissagenene e. short-term risk of anaemia following initiation of combination antiretroviral treatment in hiv-infected patients in countries in sub-saharan africa, asia-pacific, and central and south america, j int aids society 2012;15:5. 22. subbaraman r, devaleena b, selvamuthu p, et al. factors associated with anemia in hiv infected individuals in southern india. int j std aids 2009;20:489-92. oktavianus 68 *department of anatomy and occupational health, faculty of medicine, trisakti university **department of community medicine, faculty of medicine, university of indonesia ***department of allergy and immunology, department of otorhinolaryngology, national general hospital cipto mangunkusumo correspondence dr. magdalena wartono, mkk department of anatomy and occupational health, faculty of medicine, trisakti university jl. kyai tapa no 260 grogol jakarta 11440 phone :+6221-5672731 ext.2106 email: univ med 2015;34:68-76 doi: 10.18051/univmed.2016.v35.68-76 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2015january-april, 2015january-april, 2015january-april, 2015january-april, 2015 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 vol.34 no.1 background few studies have investigated the impact on olfactory functioning of occupational exposure to toluene, an industrial solvent used in paints and cleaning fluids. the estimated olfactory dysfunction prevalence is 0.5– 5%. patients frequently do not complain about olfactory dysfunction. however, occupational exposure to chemicals may affect workers’ health and safety, because of their continuous inhalation. this study aimed to examine the relationship between toluene exposure and olfactory dysfunction in furniture workers. methods this was a cross-sectional study involving 65 workers. data collection was by observation and interview on demographic characteristics, history of habits, and symptoms of chronic rhinitis. risk of exposure scores were evaluated from potential hazard, exposure level, duration of employment, type of work, use of masks, ventilation of work space, and education and training. olfactory function was tested using sniffin’ sticks, and determination of environmental toluene level was by personal sampling. the odds ratio was used to test correlations between variables. results only 44 subjects could be analyzed, 37 (84.1%) of whom had olfactory dysfunction. workers with high toluene exposure had a significantly 12.5fold risk of olfactory dysfunction in comparison with those with low exposure (or=12.5; ci 95% 1.35 – 115.79). conclusions toluene exposure increases risk of olfactory dysfunction in furniture workers. olfactory function testing should be considered for initial screening or periodic testing of furniture workers. low toluene levels with a high proportion of olfactory dysfunction indicate that olfactory dysfunction is an early negative impact of chemical inhalation. keywords: olfactory dysfunction, toluene, sniffin sticks, furniture workers high toluene exposure risk increases risk of olfactory dysfunction in furniture workers magdalena wartono*, herqutanto**, and niken lestari*** doi: http://dx.doi.org/10.18051/univmed.2015.v34.68-72 69 univ med vol. 34 no.1 risiko pajanan toluen meningkatkan gangguan penghidu pada pekerja mebel latar belakang terdapat sedikit studi yang meneliti dampak pajanan toluen di tempat kerja terhadap fungsi penghidu. toluen merupakan bahan pelarut cat dan bahan pembersih. prevalensi gangguan penghidu akibat pajanan bahan kimia pada pekerjaan diperkirakan antara 0.5 – 5%. gangguan penghidu sering tidak dikeluhkan oleh penderitanya, tetapi dapat mempengaruhi kesehatan dan keselamatan. penelitian ini bertujuan untuk menentukan adanya hubungan antara besarnya risiko pajanan toluen dengan kejadian gangguan penghidu pada pekerja mebel. metode penelitian ini menggunakan desain potong lintang mengikut sertakan 65 orang. pengumpulan data menggunakan observasi dan wawancara yang meliputi karakteristik demografi, riwayat kebiasaan dan gejala rinitis kronik. skor besar risiko pajanan dinilai berdasarkan tingkat bahaya potensial, tingkat pajanan, masa kerja, jenis pekerjaan, penggunaan masker, ventilasi ruang kerja, edukasi dan pelatihan. pemeriksaan fungsi penghidu dengan menggunakan sniffin’ sticks dan kadar toluen lingkungan diukur dengan menggunakan sampel perorangan. uji rasio odds digunakan untuk menguji hubungan antara berbagai variabel. hasil hanya 44 subjek yang dapat dianalisis, dengan 37 (84,1%) yang mengalami gangguan penghidu. kelompok pereja yang mengalami risiko pajanan toluene yang tinggi meningkatkan risiko terjadinya gangguan penghidu sebesar 12,5 kali secara bermakna dibandingkan kelompok dengan risiko pajanan yang rendah (ro=12,5; 95% interval kepercayaan 1,35 – 115,79). kesimpulan pajanan toluen meningkatkan risiko gangguan penghidu pada pekerja mebel. pemeriksaan fungsi penghidu perlu dipertimbangkan sebagai salah satu skrining awal atau pemeriksaan berkala bagi pekerja di industri mebel. kadar toluen yang rendah dengan proporsi gangguan penghidu yang besar, menandakan bahwa gangguan fungsi penghidu merupakan dampak negatif dini dari pajanan inhalasi bahan kimia. kata kunci: gangguan penghidu, toluen, sniffin sticks, pekerja mebel abstrak introduction airborne chemicals and volatile molecules e n t e r t h e n o s e a n d c a n i n t e r a c t w i t h chemoreceptors in the nasal cavity, especially trigeminal and olfactory receptors. exposure to pollutant or toxic substances is known to induce adverse health effects but few studies have been devoted to the impact on olfactory functioning. actually, effects of toluene inhalation on human health have been extensively described.(1,2) toluene is used as an additive in gasoline mixtures to increase octane ratings, in benzene production, and as a solvent in paints, coatings, inks, adhesives, and cleaners. additionally, toluene is used in the production of nylon, plastics, and polyurethanes. workers in many occupations, including painting and paint manufacturing, lumber and furniture fabrication, machining operations, equipment cleaning and degreasing, and floor and carpet laying, are exposed over time to significant levels of 70 solvents, such as acetone, methyl ethyl ketone, and toluene.(3) the olfaction in catalonia (olfacat) survey was carried out in the general population of catalonia in spain and showed that the prevalence of smell dysfunction was 19.4% for detection (0.3% anosmia, 19.1% hyposmia), 43.5% for recognition (0.2% anosmia, 43.3% hyposmia) and 48.8% for identification (0.8% anosmia, 48% hyposmia).(4) the exact prevalence of chemically-induced occupational olfactory dysfunction is unknown, but one proposed estimate is 0.5–5% of all olfactory dysfunctions, including use of pharmaceutical drugs. the real relevance of this p r o b l e m i s p o s s i b l y o v e r l o o k e d , s i n c e occupational exposure may account for a significant part of so-called idiopathic olfactory dysfunction, i.e. 10–25% of all olfactory problems within the general population.(5) olfactory dysfunction may be caused by various factors, such as mechanical, infectious, traumatic, iatrogenic, metabolic, neurologic, exposure to toxic substances such as organic solvents, metals, and alcohol.(6) in contrast to other sensory dysfunctions, olfactory dysfunction is frequently undetected and not complained of by the patient. fom the standpoint of safety, olfactory dysfunction may reduce a person’s alertness to the hazards of fire, chemical vapors, and spoiled food.(7,8) in a study conducted to detect olfactory dysfunction, where the subjects were exposed to toluene inhalation, there was an increase in olfactory sensitivity after toluene inhalation.(9) in contrast, a study involving workers in a plastics factory who were exposed to organic solvents, particularly toluene, found a significant reduction in olfactory threshold scores before and after working hours, with a hyposmia prevalence of 4%.(7) an animal study involving rats exposed to 1000 ppm toluene for 5 hours per day, 5 days per week, for 4 weeks, found in the fourth week a reduction in olfactory epithelial thickness, that returned to normal 2 weeks after cessation of the exposure.(10) to date, several olfactory function testing methods of higher sensitivity have been developed and subjected to cultural validation, such as the university of pennsylvania smell identification test (upsit), brief smell identification test, japanese odor stick identification test (osit), scandinavian odor identification test (soit), and sniffin sticks.(11) the present study aimed to determine any association between risk of toluene exposure in furniture workers and olfactory dysfunction (including olfactory threshold, discrimination, and individuality). the risk of toluene exposure in this study was determined semiquantitatively, to obtain estimates of toluene uptake by the workers.(12) methods design of the study this study was of cross-sectional design and was conducted in a number of furniture workshops in east jakarta from april to may 2013. subjects of the study the subjects who participitated in the study were furniture workers who had been employed for more than 3 months and were less than 55 years old. the exclusion criteria based on history and anterior rhinoscopy were as follows: workers who had acute or chronic rhinosinusitis, deviation of the anterior superior septum, or a tumor mass or polyps in the nasal cavity, history of functional endoscopic sinus surgery (fess), currently using steroid nasal sprays, history of head injury, and history of diabetes mellitus. the sample size was calculated from the cross-sectional study formula with a chemically induced hyposmia prevalence of 5% (5) and α=0.05, giving a minimal sample size of 62 subjects. it was decided to recruit the 65 subjects by total sampling of funiture workers in a factory complex.(13) wartono, herqutanto, lestari toluene exposure and olfactory dysfunction 71 univ med vol. 34 no.1 data collection each study subject was interviewed using a questionnaire comprising items on age, education, marital status, smoking habit, alcohol consumption, symptoms of chronic rhinitis, and history of atopy. determination of risk of exposure to determine risk of exposure scores, seven components were evaluated: i) potential hazard level, ii) exposure level, iii) duration of employment, iv) type of work, v) use of masks, vi) ventilation of work space, and vii) education and training. potential hazard level: the score for toluene exposure had been predetermined from hazard rating tables, giving a score of 3.(14) exposure level: the score was calculated by comparing the mean weekly exposure level (e) with the permissible exposure limit (pel) for toluene of 50 ppm or 18 mg/m3.(18) the value for e was obtained by multiplying duration of exposure (d) with the exposure frequency (f), and the measured toluene level (m) and dividing the product by total working hours per week (w). the exposure level scores ranged from 1-5.(12) w mxdxf e = duration of employment: the score was determined from the duration of employment in the furniture production sector, with the following scores: score = 1 if employed for less than or equal to 1 year; score = 2 if employed for 1-5 years, score = 3 if employed from 5 to 10 years, and score = 4 if employed for more than 10 years. type of work: the score was determined from the task or department in the furniture workshop, as follows: working in the cutting department, score = 1, working in the assembly department, score = 2, and working in the finishing department, score = 3. use of masks: the scores were given according to continuity of using masks per week of work. if always using masks when working, the score was 1, if occasionally using masks, the score was 2 and if never used masks the score was 3. ventilation of work space: the scores were given b a s e d o n t h e e v a l u a t i o n o f a d e q u a c y o r inadequacy of work space ventilation, by comparing ventilated areas with floor areas. if ventilated area >15%, the score was 1, if <15% the score was 2, and if completely without ventilation, the score was 3. education and training: the scores were given on the basis of having ever or never received official education and training on management of chemicals and use of personal protective devices. if ever received, the score given was 1, and if never received, the score given was 2. scores for the seven components were totalled to give an exposure risk score. the range of scores was 9-23. the workers were considered to be at high risk if the score was 15 -23 and at low risk if the score was less than or equal to 14. olfactory function testing the selected subjects underwent olfactory function testing by the investigators using sniffin’ sticks under the supervision of an ent specialist. the sniffin’ sticks test kit is a validated and commonly used tool for assessment of olfactory function in subjects with normal sense of smell and in individuals with loss of smell. the kit consists of 32 or 16 pens, 14 cm in length and 1.3 cm in diameter, that can be filled with an odorant to a total volume of 4 ml and then capped, the odorant being perceptible at the tip of the pen.(15) determination of odor threshold (t), was performed by preparing a 4% solutions of pure butanol or phenyl ethyl alcohol as odorant, diluted in deionized water in a ratio of 1:2. from this odorant solution, 16 dilutions were prepared in a geometric series. three pens were placed in random order, with 2 pens containing the solvent only and 1 pen containing the odorant at a given dilution. when testing, the cap was taken off the p e n , a n d t h e t i p o f t h e p e n w a s p l a c e d approximately 2 cm from the nostrils of the subject for around 3 seconds. the task of the 72 subject was to identify the pen containing the odorant. the test was done using a singlestaircase, 3-alternative forced-choice procedure. if the odorant was identified on two consecutive tests, this triggered a reversal of the staircase. testing was then performed to a total of 7 reversals. the olfactory threshold value was estimated as the mean of the last 4 staircase reversal points, with scores of 1 to 16.(14,16) determination of odor discrimination (d) was performed using 3 pens, with 2 pens containing the same odorant and 1 pen containing a different odorant. the testing used the technique of 3-alternative forced-choice, in which the subject had to identify the one pen that had a different odor. a true identification was given 1 point. the total score ranged from 0 to 16 for a set of 16 pens containing odorants.(14,16) in the t e s t i n g f o r o d o r t h r e s h o l d a n d o d o r discrimination, the subject was blindfolded to prevent visual identification of the pens.(15) the odor identification (i) test used 16 odorants with the multiple forced-choice design method. the subject was asked to identify odors by chosing from a list of 4 descriptors. the score for this test also ranged from 0-16. the odors used were of citrus fruit, peppermint, turpentine, cloves, leather, banana, garlic, rose, fish, lemon, coffee, anise, cinnamon, licorice, apple, and pineapple.(14,16) the values of the three scores were summed and categorized as positive (+) for olfactory dysfunction, if the total of threshold (t), discrimination (d) and identification (t) (tdi) < 29.53 and negative (-) if tdi > 29.53.(15) measurement of toluene level in the work environment this was carried out by personal diffusive sampling of toluene vapour in breathing zone air. in this method the monitor device is placed in the area of respiration of an individual, to measure the total exposure of the worker from inhalation.(17) the measurement was carried out according to the procedures of the industrial hygiene and occupational health agency, jakarta special metropolitan area (balai hiperkes dki jakarta), with toluene detection limits of 0.001 – 0.01 mg per sample, allowing the results to be expressed as parts per million (ppm). the measurement was done by an expert from the industrial hygiene and occupational health agency, jakarta special metropolitan area (balai hiperkes dki jakarta). measurement of air temperature and relative humidity of the work environment this was carried out by means of a wetbulb globe thermometer at points in the workplace considered to be representative of the temperature and relative humidity of the work environment. the measurement was done by an e x p e r t f r o m t h e i n d u s t r i a l h y g i e n e a n d occupational health agency, jakarta special metropolitan area (balai hiperkes dki jakarta) data analysis the odds ratio test was used to test for correlations between independent variables and olfactory dysfunction. ethical clearance this study obtained ethical clearance in february 2013 from the health research ethics committee, faculty of medicine, university of indonesia. results of the 65 workers, for only 44 were the data complete and analyzed, because for 21 w o r k e r s n o p e r s o n a l t o l u e n e e x p o s u r e measurements could be done. all subjects were male, the majority totaling 21 (47.7%) were 1830 years old and 19 (432%) subjects had finished primary school. a total of 40 workers (90.9%) were smokers, 23 (53.5%) had symptoms of chronic rhinitis and 22 (50.0%) admitted to a history of atopy (table 1). measurement of toluene levels by personal sampling yielded a mean toluene level of 0.94 ± 1.47 ppm and a total of 31 (70.5%) had toluene wartono, herqutanto, lestari toluene exposure and olfactory dysfunction 73 univ med vol. 34 no.1 table 1. distribution of demographic characteristics and offactory function of the study subjects (n=44) levels below the mean. based on the risk of exposure scores that were obtained, the subjects were subsequently assigned to 2 categories, i.e. those with a high risk of exposure and those with a low risk of exposure. high values for risk of toluene exposure were obtained in 26 (59.1%) subjects, whereas 18 (40.9%) subjects belonged to the group of low risk of toluene exposure. the results of olfactory function testing by means of sniffin’ sticks showed that 37 (84.1%) subjects had olfactory dysfunction (tdi score < 29.53). the descriptors of the olfactory dysfunction comprised an odor threshold of 75.0%, odor discrimination of 25.0% and odor identification of 63.6% (table 1). results of bivariate analysis showed that the group at high risk of toluene exposure had a 12.5-fold increased risk of olfactory dysfunction (or=12.5; 95% confidence interval 1.3511 5 . 7 9 ) . t h e t o l u e n e l e v e l i n t h e w o r k environment was not significantly correlated with o l f a c t o r y d y s f u n c t i o n ( o r = 0 . 4 9 4 ; 9 5 % confidence interval 0.094-2.608) (table 2). discussion very few studies have tried to determine the exposure of furniture workers to toluene, probably because of the relatively small population. our study results showed that the proportion of olfactory dysfunction was 84.1%, which was far higher than the prevalences of 0.5– 5% for olfactory dysfunction from chemical exposure in the workplace according to gobba.(5) however, our results were similar to those of table 2. correlation of risk of toluene exposure and toluene level with olfactory dysfucntion 74 t h e s t u d y b y c h e n g e t a l . ( 7 ) w h o f o u n d prevalences for mild and moderate hyposmia in plastic workers of 60.0% and 4.0%, respectively. with regard to descriptors of these olfactory dysfunctions, our study showed a threshold of 7 5 . 0 % , d i s c r i m i n a t i o n o f 2 5 . 0 % a n d identification of 63.6%. these results differed from the olfacat study results, with values of 19.4% for detection (0.3% anosmia, 19.1% hyposmia), 43.5% for recognition (0.2% anosmia, 43.3% hyposmia) and 48.8% for identification (0.8% anosmia, 48% hyposmia).(4) however, the instrument used in the olfacat study also differed from that used in our study. the olfacat study used scratch papers as odor instruments and each study subject was asked to scratch and sniff each odor and then answer three questions, i.e. on odor detection (did you smell any scent? yes, no); odor recognition/ memory (have you ever smelt this scent? yes, no); forced-choice odor identification (which name defines the scent you have smelt?). the percentages of subjective complaints of olfactory dysfunction in the present study were not representative of the proportion of olfactory dysfunction. in the present study, the percentage of subjects complaining of olfactory dysfunction was only 10.8%. these results underline the s t a t e m e n t t h a t o c c u p a t i o n a l o l f a c t o r y dysfunction is subclinical and can be determined only by objective testing using devices for evaluation of olfactory function.(5) risk of exposure scores in subjects with olfactory dysfunction were higher than those in subjects without olfactory dysfunction, with near-significant differences. the risk scores were obtained from assessment of exposure level (total working hours per week), duration of each exposure, frequency of exposure per week, m e a s u r e d t o l u e n e l e v e l s , d u r a t i o n o f employment, type of work, control measures such as use of masks, ventilation of the work space, and education and training. among the components of exposure level, although no significant differences were found, exposure frequency exceeded those in subjects without olfactory dysfunction, whereas duration of employment was shorter. in the present study involving informal workers, it was rather difficult to determine duration of employment objectively, since the workers were employed on a demand basis. in times of great demand, they worked continuously for several months without taking free days off. on the other hand, in times of no demand, they were unable to work for several weeks or months. in addition, the workers frequently moved to other workshops needing their services, possibly with less conducive work environments. therefore it was difficult to determine duration of employment, since equal durations of employment may not necessarily represent identical total working hours and working days. therefore to determine the cumulative dose of exposure in such workers, a more accurate way is to determine total working hours, frequency and duration of exposure per week.(18) our study results showed that subjects belonging to the group at high risk of toluene exposure had a higher risk for olfactory dysfunction, in comparison with the group at low risk of toluene exposure. improvements in paint materials have caused a substantial reduction of emissions,(19) but these are still potentially harmful. particulate matter and gas emitted from toluene is small enough to reach the lower respiratory system (20) and the brain, via the olfactory nerves.(21) especially in the case of benzene and toluene, long-term exposure has negative effects on both the cardiovascular and the respiratory systems. our study results showed that among those at high risk of toluene exposure 59.09% subjects were in the category of high risk for olfactory dysfunction, since the bivariate test results showed a 12.51-fold higher risk of olfactory dysfunction in workers at high risk of toluene exposure. the toluene levels measured in the present study were relatively low and far below the toluene pel value of 50 ppm.(22) although no significant differences were found, the impact of toluene on olfactory dysfunction at high levels of exposure cannot be ruled out. toluene is also wartono, herqutanto, lestari toluene exposure and olfactory dysfunction 75 univ med vol. 34 no.1 known as toluol, phenylmethane, methylbenzol, methylbenzene, monomethyl benzene, and methacide. it is commonly used as a solvent in paints, coatings, inks, ahesives, and cleaners. o u r s t u d y p o i n t s o u t t h a t o l f a c t o r y dysfunction is an early negative impact of exposure by inhalation of chemicals and other irritants, which has also been demonstrated by magagnotti et al.(24) therefore, olfactory function testing should be considered for initial screening or periodic testing of workers in the furniture or other industries that use irritant substances. in addition, a revision of the safe levels of toluene in chronic exposure should be considered, particularly with regard to olfactory functions. although olfactory dysfunction is not lifethreatening by itself, it may have serious consequences, since inability to detect some olfactory warning signals (such as spoiled food, gas leaks, etc.) can have a significant impact on nutritional status and other aspects related to the quality of life. among the limitations of the study we would like to point out that we were not able to evaluate occupational risks related to a particular type of activity, primarily due to the rather small number of subjects in each restoration department. another limitation of this study was the inability to control for other risk factors, such as smoking habit, since more than 90% of subjects were smokers. the study by katotomichelakis et al.(25) found decreased olfactory function, marked by lower scores for threshold, discrimination, and identification, than in the control group and proportional to the number of cigarettes smoked and duration of smoking. another risk factor that could not be controlled for was the presence of wood dust as another irritant in the work place. in the present study no measurement of wood dust was performed, but wood dust also cannot be placed in the exclusion criteria, since all workers in the furniture department are certain to be exposed to wood dust. with the unaided eye it could be seen that the floors in all the workshops that were studied always were covered with moderate to high amounts of wood dust. similarly, the air in the work environment was mildly to moderately dusty. wood dust by itself can cause rhinitis, so as to induce mucosal edema that can decrease olfactory function.(26) however, the subjects in this study were those who on anterior rhinoscopy had no visible mucosal pathologies such that rhinitis from irritation by wood dust can be eliminated. the minimal complaints accompanying s e v e r e o l f a c t o r y d y s f u n c t i o n d e s e r v e consideration, because in persons with olfactory dysfunction, the nose has reduced protective functions against toxic substances, which eventually impact on their health. subsequent studies investigating the effects of toluene exposure on olfactory dysfunction should include determination of the toluene levels in the body in a larger sample, nasal examination by endoscopy, and control for other risk factors such as smoking. the relevance of these findings for the future clinical course of occupational or work-exacerbated rhinitis, as well as the underlying mechanisms, should be elucidated through further studies. conclusion a high risk of toluene exposure increases the risk of olfactory dysfunction in furniture workers. olfactory impairment seems to be highly dependent on chemicals, therefore olfactory function testing should be considered for initial screening or periodic testing for workers in the furniture industry. acknowledgements we are indebted to dr.dr.herqutanto, mph,mars and dr. niken lestari, spthtkl(k), as tutors during this study who provided time, suggestions and energy for this study, and to dr. dr. dewi s soemarko, ms, spok, as head of the occupational medicine post graduate program. we also thank the owners and workers of furniture workshops in rw 01, k e l u r a h a n p o n d o k b a m b u , d u r e n s a w i t subdistrict, east jakarta. 76 references 1. kang sk, rohlman ds, lee my, et al. neurobehavioral performance in workers exposed to toluene. environ toxicol pharmacol 2005;19:645–50. 2. muttray a, spelmeyer u, hommel g, et al. acute exposure to 50 ppm toluene does not increase sleepiness. environ toxicol pharmacol 2005;19: 665–9. 3. u.s. environmental protection agency. toxicological review of toluene. washington d.c.: u.s. environmental protection agency; 2005. 4. mullol j, alobid i, marino-sanchez f, et al. furthering the understanding of olfaction, prevalence of loss of smell and risk factors: a population-based survey (olfacat study). bmj open 2012;2:e001256. doi:10.1136/ bmjopen-2012-001256. 5. gobba f. olfactory toxicity: long-term effects of occupational exposures. int arch occup environ health 2006.doi 10.1007/s00420-005-0043-x. 6. zoni s, giulia b, lucchini r. olfactory functions at the intersection between enviromental exposure to manganese and parkinsonism. j trace elem med biol 2012;26:179-82. 7. cheng sf, chen ml, hung pc, et al. olfactory loss in poly (acrylonitrile-butadiene-styrene) plastic injection-moulding workers. occup med 2004;54:469-74. 8. shelton kl, hernandez gl. characterization of an inhaled toluene drug discrimination in mice: effect of exposure conditions and route of administration. pharmacol biochem behav 2009;92:614-20. 9. cometto-muniz je, cain ws, abraham mh, et al. psychometric functions for the olfactory and trigeminal detectability of butyl acetate and toluene. j appl toxicol 2002;22:25–30. 10. jacquot l, pourie g, buron g, et al. effects of toluene inhalation exposure on olfactory functioning: behavioral and histological assessment. toxicol letters 2006;165:57-65. 11. simmen d, briner hr. olfaction in rhinologymethods of assessing the sense of smell. rhinology 2006;44:98-101. 12. tan kt, na g, shaik s, et al. a semi-quantitative method to assess occupational exposure to harmful chemicals (manual). occupational safety and health division, ministry of manpower, singapore;2012. 13. charan j, biswas t. how to calculate sample size for different study designs in medical research?. indian j psychol med 2013;35:1216. doi: 10.4103/0253-7176.116232. 14. haehner a, mayer am, landis bn, et al. high test-retest reliability of the extended version of the “sniffin’ sticks” test. chem senses 2009;34: 705-11. 15. zibrowski em, robertson jmd. olfactory sensitivity in medical laboratory workers occupationally exposed to organic solvent mixtures. j occ med 2006;56:51-4. 16. becker s, pflugbeil c, groger m, et al. olfactory dysfunction in seasonal and perennial allergic rhinitis. acta otolaryngol 2012;132:763-8. 17. soeripto m. higiene industri. jakarta: balai penerbit fkui;2008. 18. seidler a, mohner m, berger j, et al. solvent exposure and malignant lymphoma: a population -based case-control study in germany. j occupational med toxicol 2007;2. doi: 10.1186 /1745-6673-2-2. 19. olsson ac, gustavsson p, kromhout h, et al. exposure to diesel motor exhaust and lung cancer risk in a pooled analysis from case-control studies in europe and canada. am j respir crit care med 2011;183:941-8. 20. moskal a, makowski l, sosnowski tr, et al. deposition of fractal-like aerosol aggregates in a model of human nasal cavity. inhal toxicol 2006;18:725-31. 21. oberdorster g, sharp z, aturei v, et al. translocation of inhaled ultrafine particles to the brain. inhal toxicol 2004;16:389-99. 22. american conference of governmental industrial hygienists, inc. toluene: documentation of the threshold limit values (tlvs) and biological exposure indices (beis). 6th ed. (manual); 2008. 23. park ej, kim h, kim y, et al. carbon fullerenes (c60s) can induce inflammatory responses in the lung of mice. toxicol appl pharmacol 2010;244: 226-33. 24. magagnotti n, picchi g, sciarra g, et al. exposure of mobile chipper operators to diesel exhaust. ann occup hyg 2014;58:217-26. doi: 10.1093/annhyg/met059. 25. eire ma, pineda f, losada sv, et al. occupational rhinitis and asthma due to cedroarana (cedrelinga catenaeformis ducke) wood dust allergy. case report. j investig allergol clin immunol 2006;16:385-7. wartono, herqutanto, lestari toluene exposure and olfactory dysfunction c:\users\jurnal fkusakti\docume 195 *department of pharmacology and medical pharmacy, faculty of medicine, trisakti university, jakarta **department of physiology, faculty of medicine, trisakti university, jakarta ***department of clinical pathology, faculty of medicine, trisakti university, jakarta †department of occupational health, faculty of medicine, trisakti university, jakarta correspondence: dr. meiyanti, spfk department of pharmacology and medical pharmacy, faculty of medicine, trisakti university jalan kyai tapa no 260 – grogol, jakarta indonesia phone: +6221-5672731 ext 2805 email: meiyanti_suhardi@yahoo.com orcid id: orcid.org/0000-00021770-5504 date of first submission, august 14, 2018 date of final revised submission, november 14, 2018 date of acceptance, november 14, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina hypoglycemic effect of phaleria macrocarpa (scheff.) boerl dry extract in healthy adults meiyanti*, eveline margo**, pusparini***, lie tanu merijanti†, and alvina lesmana*** background the prevalence of diabetes in indonesia continues to increase and causes many complications. fruits of phaleria macrocarpa (scheff.) boerl. (pm) are used in the traditional health system of the indonesians, as effective remedy in the management of diabetes mellitus. the aim of this study was to evaluate the effect and magnitude of doses of pm dry fruit extract (pme) on blood glucose levels in healthy adults. methods this research was of pre-posttest design involving 30 healthy volunteer subjects aged 20-55 years receiving glucose loads. at pre-test the subjects were given orally 75 g glucose, and at post-test they were given pme at doses of 125 mg and 250 mg. data collection included anthropometric examination and blood glucose level. the area under the curve (auc) of blood glucose levels was measured at 0, 15, 30, 60, 120, and 180 minutes. data analysis was performed using paired t-test with p<0.05. results the percentage of reduction in auc between preand post-test after administration of pme 125 mg was 12.1% and significant (p=0.000). but the percentage of reduction in auc between preand post-test after administration of pme 250 mg was 4.07% and not significant (p=0.06). the reduction in auc between post-test pme 125 mg vs pme 250 mg showed significant results (p=0.011). conclusion this study demonstrated that a dose of 125 mg phaleria macrocarpa (scheff.) boerl. dry extract has a hypoglycemic effect in healthy adults, and may therefore be suitable for use as traditional anti-diabetic drug. keywords: phaleria macrocarpa, extract, blood glucose, healthy adult doi: http://dx.doi.org/10.18051/univmed.2018.v37.195-202 original article pissn: 1907-3062 / eissn: 2407-2230 september-december, 2018 vol.37 no.3 cite this article as: meiyanti, margo e, pusparini, et al. hypoglycemic effect of phaleria macrocarpa (scheff.) boerl dry extract in healthy adults. univ med 201 8;37 :1 9 5 -2 0 2 . doi: 10.1 805 1/ univmed.2018.v37.195-202 196 meiyanti, margo, pusparini, et al hypoglycemic effect of phaleria macrocarpa introduction the use of traditional medications of plant origin (herbals or jamu) is steadily increasing in indonesia, as indicated by the increasing number of herbal-producing industries. some of the reasons for the increasing use of herbals are their ready availability, lower prices and traditional medicinal effects. on the other hand, the use of traditional herbals in the community is as yet empirical, so that their quality, medicinal effects, and safety are in doubt. to raise the status of herbals to that of phytopharmaceutical or phytotherapeutic medications, it is imperative to test these herbals on humans through clinical trials. one of the medicinal plants that is widely used in the community is phaleria macrocarpa (scheff.) boerl. (pm), locally known as mahkota dewa. phaleria macrocarpa belongs to the division of spermatophyta, subdi vision angiospermae, class dicotyledonae, order celastrales, family thymelaceae and genus phaleria. this plant originates from papua and is also known as phaleria papuana warb. var. wichannii (val.) back.(1) four parts of the plant are used in herbal medicine, namely the stem, leaf, seed coat and fruit. the stem is used for bone cancer, while the leaf is for impotence, blood disorders, allergy and diabetes mellitus. the fruit of pm contains alkaloids, saponins, flavonoids, and polyphenols that are frequently used as antioxidants.(2,3) phaleria macrocarpa extracts prepared from different parts of the plant have a number of medicinal pr ope rties, such as anticancer, antidiabetic, antihyperlipidemic, antiinflammatory, anti-bacterial, anti-fungal, and antioxidant.(4) in the community pm is frequently used as alternative medication for the treatment of diabetes mellitus, in addition to oral antidiabetics.(5) diabetes mellitus (dm) is at present a global health problem. data from several global studies state that dm is a serious health problem, which is caused by the increasing numbers of patients with dm from year to year. in 2011 around 366 million adults suffered from dm, a fourfold increase from the 108 million in the 1980s, and it was estimated that in the year 2030 there would be a very large increase of up to 551 million in the number of patients with dm.(6) several in vitro studies and in vivo studies on animals indicate pm’s effect as an antidiabetic. in the study by ali et al.(3) on rats it was concluded that a methanol extract of pm fruit pericarp has hypoglycemic effects. the study of triastuti et al.(7) concluded that pm has nephroprotective effects through pm’s anti-hyperglycemic and antioxidant effects. the study by ali et al. (8) concluded that pm fruit pericarp methanol extract is able to decrease hyperglycemia in vitro as well as in vivo through inhibition of carbohydrate hydrolyzing enzymes. from several studies it is apparent that by using methanol as solvent, a higher content of active compounds may be obtained from pm, but since methanol is known to be toxic, it cannot be used as a commonly recognized safe solvent.(9) in contrast to the foregoing studies, the study conducted by hending (10) states that the leaves of pm have antidiabetic effects. the study of meiyanti(11) concluded that pm pericarp powder at a dose of 500 mg can decrease the blood glucose level of healthy persons after a glucose load. although there are many studies on evaluation of the hypoglycemic effect of pm fruit pericarp extract (pme) in animals, there is scanty information on the hypoglycemic effect of pme in healthy adult humans. in addition, to date the extracts of pm have not yet been sufficiently standardized to yield de tailed quantitative information of their phytoconstituents.(12) the preparation of pm from the grinding and drying, up to the selection of extraction method and solvent to be used, will affect the efficacy and phytochemical contents of pm.(13,14) accordingly there is a need to conduct a study for evaluation of the hypoglycemic effect of pme in healthy human adults using a different solvent, with administration of the extract at several doses. 197 methods research design this study was of experimental pre-post test design and was conducted in the kelurahan (village) of angke, kecamatan (district) of tambora, jembatan dua, west jakarta from february until april 2018. study subjects this study used a sample size of 30 subjects, which was calculated using α=5%, zα=1.96, β=20%, and zβ=0.842, with sd=17.86 and mean difference (“x)=9.84 mg/dl.(11) the minimum number of subjects required was 26, but in anticipation of a drop-out level of 15%, the total number of subjects was increased to 30. the subjects were selected by consecutive non-random sampling. the inclusion criteria were as follows: i) males and females aged 18-55 years, ii) with body mass index of 18.5 25 kg/m2, iii) not suffering from chronic diseases such as hypertension and diabetes mellitus, iv) not consuming certain herbal preparations (jamu) or vitamin supplements, from 1 week prior to the start of the study up to the completion of the study, v) not consuming caffein-containing beverages and fruit juice and not smoking within the last 24 hours, vi) agreeing to participate in the study and to sign a declaration of informed consent. the exclusion criteria were: i) being currently pregnant or breast-feeding, ii) consuming other medications, such as corticosteroids, that may affect the blood glucose level, from 1 week prior to the start of the study up to the completion of the study, iii) having participated in other studies in the 3 months prior to the present one. plant material collection and preparation of extracts the preparation used in this study consisted of capsules containing a dry extract of pm pericarp (pme). the pm pericarp (without seed coat and seed) was obtained from plant nurseries in the region of semarang, central java. the harvested pericarp was cut up and oven-dried at 40 p c for 1 day. the dried pericarp was then ground into a powder, from which an extract was prepared with water and ethanol as solvents.(15) the prepared extract was placed in gelatin capsules, with half their number containing 125 mg pme and the remaining capsules containing 250 mg pme, but of identical appearance. intervention the intervention given consisted of the administration of one of the capsules containing pm dry pericarpextract (pme) from semarang, central java. at each visit the subjects received 400 calor ies (or 75 gram of glucose), in accordance with the glucose oral tolerance test procedure.(16) at the first visit the subjects received a glucose load only, whereas at the following visit on day 4, each subject received 125 mg pme. after a wash-out period of 1 week, at the next visit on day 11 the subjects received 250 mg pme. the doses used in this study were based on a previous study, which found that 500 mg of pm fruit powder has hypoglycemic effects and that one gram of pm fruit powder yields 250 mg of extract, so that the present trial was conducted with the doses of 125 mg and 250 mg pme.(11) parameter for evaluation of hypoglycemic effect the parameter used to determine the subjects’ responses to glucose loading was the a r e a u nd e r t h e c u r ve ( au c) . ( 1 7 ) t h e h yp ogl yc e mi c e f f e c t o f the pme w a s determined by using the auc of the serum glucose level of healthy volunteers who did not receive the pme and acted as controls, to be compared to the auc of the serum glucose level of subjects receiving the pme at doses of 125 mg and 250 mg. the aucs were calculated with the trapezium formula (18) for each respective intervention: auc 0-n = t 1 -t 0 /2 .(c 0 +c 1 ) + t 2 -t 1 /2. (c 2 +c 1 ) +……+t n -t n-1 /2 (c n +c n-1 ) univ med vol. 37 no.3 198 meiyanti, margo, pusparini, et al hypoglycemic effect of phaleria macrocarpa measurement of blood glucose level the blood glucose level was determined in accordance with the oral glucose tolerance test standard procedure. each of the blood samples was drawn from the cubital vein and then tested prior to the oral glucose load and respectively at 15, 30, 60, 120 and 180 minutes (16) after the oral glucose load and administration of the pmecontaining capsules. the serum glucose level was measured in duplicate using the glucose oxidase-phenol and 4-aminophenazone (godpap) enzymatic colorimetric assay that had been validated with a perkin-elmer lambda 3b spectrophotometer. t he reagent used was glucose god fs from diasys, germany. prior to the study, calibration curves were constructed and validation tests were performed on the measurement method, consisting of tests of accuracy, precision, and stability. statistical analysis the auc of the blood glucose level was calculated using the trapezoidal rule for a period of 3 hours (auc 0-3 hours). the kolmogorov test of normality of the data distribution showed that the data were normal ly distribute d. t he characteristics of the subjects were presented as mean ± sd. the absorbed amount of the ingested glucose with or without pme was statistically compared with the paired ttest. differences at p<0.05 were considered significant. ethical clearance this study obtained ethical clearance from the faculty of medicine, trisakti university, under no. 123/ker/fk /xii/2017. results the subjects participating in this study totaled 30 males and females with mean age of 34.2 ± 10.5 years, with 19 (63.33%) female subjects. the subject characteristics may be seen in table 1. tests of accuracy and precision of the daily (within run) glucose level showed that the coefficient of variation (cv) for the normal controls (merck qualitrol® hsn) was 0.95% with accuracy (recovery) of 98.21%, whereas for the high controls (merck qualitrol® hsp) the cv was 1.5 % and accuracy 97.12%. the tests of accuracy and precision of the day-to-day glucose level showed that for hsn the cv was 2.16% with accuracy of 98.35% whereas for hsp the cv was 3.05% with accuracy of 95.23%. the mean blood glucose levels of the study subjects at 0, 15, 30, 60, 120 and 180 minutes after glucose loading, without administration of pme (pre-test), as compared with post-test administration of pme at doses of 125 and 250 mg, may be seen in figure 1. peak blood glucose level with or without administration of pme was reached within 30 minutes after glucose loading, whereas the lowest level was reached within 180 minutes (3 hours) after glucose loading. pre-test glucose level within the first 30 minutes (glucose peak) was 122.45 ± 20.98 mg/ dl, while for post-test pme doses of 125 mg and 250 mg the results were 107.09 ± 20/98 mg/ dl and 117.6 ± 21.80 mg/dl, respectively. the decrease in glucose level at the glucose peak was 12.5% and 3.96% for post-test pme doses of 125 mg and 250 mg, respectively, as compared with pre-test levels. the area under the curve for blood glucose level up to 3 hours (auc 0-3 hours) was calculated using the trapezoidal rule. for the post-test pme dose of 125 mg there was a decrease in blood glucose level, with mean decrease in auc 0-3 hours of 12.1%, which was characteristic n age (years) 34.17± 10.5 sex female 19 (63.3) male 11 (36.7) weight (kg) 54.62 ± 8.17 height (m) 1.57 ± 0.09 body mass index (bmi) kg/m2 22.06 ± 2.12 table 1. distribution of features characteristics of the subject (n=30) data were presented as mean ± sd, except for sex n (%) 199 higher than the decrease for the post-test pme dose of 250 mg. the post-test pme dose of 125 mg resulted in a significant decrease in auc (p=0.000), whereas the pre-test auc and the post-test pme dose of 250 mg did not differ significantly (p=0.06) (table 2). a significant difference in auc was also obtained for the post-test pme dose of 125 mg as compared with the dose of 250 mg (p= 0.011). prior to ingesting the pme, each of the subjects underwent physical examinations with respect to blood pressure, pulse rate, and clinical condition of the heart, lungs and abdomen. these examinations were also performed at each subsequent visit to obtain a continuous picture of the effect of pm on the vital functions and on complaints emerging during the administration of pme. there were 2 subjects who complained of a bloated stomach after receiving pme at 250 mg, whereas after administration of 125 mg of pme there were no subjects who had complaints during the course of the study. a number of subjects who received pme showed a reduction in blood pressure. after administration of 125 mg pme there was a reduction in blood pressure of 5-7% in 4 out of the 30 subjects, whereas after administration of 250 mg pme there were 10 out of the 30 subjects who had a blood pressure reduction of 10-12%. univ med vol. 37 no.3 figure 1. the effect of phaleria macrocarpa extract on blood glucose level (mg/dl) of subjects data were expressed as mean ± sd 0.00 20.00 40.00 60.00 80.00 100.00 120.00 140.00 0 15 30 60 120 180 b lo o d g lu c o se l e v e l (m g /d l ) time (min) pre-test post-test 125 post-test 250 variable pre-test post-test pme 125 mg % diff p post-test pme 250 mg % diff p blood glucose* (mg/dl) 122.45 ± 20.98 107.09 ± 20.98 12.5 0.04** 117.6 ± 21.80 3.96 0.384 auc (mg.min/dl) 18034.83 ± 2688.97 15660.18 ± 2707.28 12.1 0.000** 17092.9 ± 2444.57 4.07 0.06 table 2. effect of phaleria macrocarpa extract on blood glucose level and area under the curve *blood glucose at 30 minutes; * % difference between pre-test and post-test; ** p <0.05 significant difference (paired t-test) 200 meiyanti, margo, pusparini, et al hypoglycemic effect of phaleria macrocarpa discussion our study results show that within 30 minutes after administration of the pme dose of 125 mg there was a higher mean reduction in blood glucose leve l as compared with administration of 250 mg pme. differing results were obtained in the study of ali et al.(3) on rats, showing that administration of pmes in petroleum ether, methanol, or water did not have an effect in the acute dose hypoglycemic test, but that the methanol and aqueous extracts showed a nonsignificant decrease in blood glucose between 30 and 120 minutes in the intra-peritoneal glucose test. the evaluated parameter was the area auc because the effect to be evaluated was the response of the blood glucose level on administration of pme. in our study there was a better hypoglycemic effect at a pm dose of 125 mg as compared with a dose of 250 mg. this shows that the methods of pre-extraction and extraction, as well as the solvent used in the extr action process, will inf luence the concentration and phytochemical constituents of pm. in addition, the point to be considered is the region from which the pm fruits come. the active constituents contained depend very much on the plant parts used (leaf, stem, or fruit) and on the type of solvent used in the preparation of the extract.(12,19) region of origin, type of soil, growing medium, and fertilizers used will affect the mineral content of the fruit.(20) the four parts of the pm plant that are frequently used in the community are the stem, leaf, pericarp, and fruit. previous studies on the chemical composition of the pericarp and pericarpof pm found alkaloids, saponins, flavonoids, and polyphenols.(21-23) the decrease in blood glucose is effected by the action of saponins and flavonoids. the effect of saponin on the cell membrane inhibits the absorption of the smaller nutrient molecules that should be absorbed, such as glucose, abnormalities in cell structure are thought to cause disturbances in the glucose transporter system, leading to inhibition of glucose absorption. (15) the study of sabina et al.(24) concluded that the decrease in blood glucose level after administration of pme is presumably due to inhibition of αglucosidase, which is an intestinal enzyme that converts disaccharides into glucose. this enzyme acts to inhibit glucose absorption in the small intestine. the antidiabetic activity of pm through inhibition of α-glucosidase was also concluded by elya et al.(5) the fruit of pm is thought to contain flavonoids that can increase insulin secretion, alter the metabolism of ca2+ (25) and regenerate the pancreatic islets of langerhans, particularly the β-cells. (26) similar findings were obtained in the study that was conducted by arjadi and mustafa (27) who concluded that pme can decrease the blood glucose level in diabetic rats, and regenerate the cells of the pancreatic islets of langerhans of diabetic rats. the decrease in blood glucose level as a result of pme administration is presumably effected through 2 main mechanisms, i.e. intrapancreatic by repairing the β-cells of the islets of langerhans and by stimulating insulin release. the increased insulin secretion is due to sympathomimetic stimulation by the alkaloid, causing increases in insulin secretion. the flavonoids contained in pme have antioxidant effects that protect against damage to the cells of the pancreas from free radicals. the second mechanism is extra-pancreatic, through inhibition of intestinal glucose absorption by the alkaloids.(28) the complaint found among the subjects was a sense of bloating in the stomach after administration of the pme dose of 250 mg, which may be caused by the rather high saponin content (20.4%) (11) in pm fruits that causes gastric irritation. in addition, there was also a decrease in blood pressure of the subj ects after administration of both the pme doses of 125 mg and 250 mg. this decrease in blood pressure is the result of the action of pm fruit as ace inhibitor.(29) the main limitation of this study is its design as a pre-post test experimental study without controls, so that further studies should be 201 conducted in accordance with the standards of a good clinical trial (randomized, using controls, and conducted on patients with diabetes). the clinical implication of this study is that dry pm fruit extract has antidiabetic effects, so that it can be used both as medication for the prevention of diabetes and as additional medication in patients with diabetes. the recommendation for further studies is to conduct a clinical trial in patients with diabetes comparing the extract to standard antidiabetics, so as to find more on the efficacy of pm as an antidiabetic. biologically active plant extracts may be further exploited in the future by pharmaceutical and nutraceutical industries. conclusions phaleria macrocarpa extra ct has hypoglycemic effects on blood glucose level in healthy adult humans. the pme dose of 125 mg has better hypoglycemic efficacy than the dose of 250 mg. conflict of interest the investigators state that there is no conflict of interest in this study. acknowledgments the investigators thank the faculty of medicine trisakti university and trisakti university resea rch institute (lemb aga penelitian universitas trisakti) for the funding of the study. t hanks ar e also due to the dhammasavana foundation and all healthy volunteers who agreed to participate in this study. contributors m contributed to study concept, design and drafting manuscript. em and p contributed to acquisition of data and critical revision of manuscript for intellectual content. al and ltm contributed to data analysis and critical revision of manuscript for intellectual content. all authors have read and approved the final manuscript. references 1. govindappa m. a review on role of plant(s) extracts and its phytochemicals for the management of diabetes. j diabetes metab 2015; 6:565. doi: 10.4172/2155-6156.10000565. 2. lee sy, mediani a, nur ashikin ah, et al. antioxidant and α-glucosidase inhibitory activities of the leaf and stem of selected traditional medicinal plants. ifrj 2014;21:165-72. 3. ali rb, atangwho ij, kuar n, et al. hypoglycemic and antihyperglycemic study of phaleria macrocarpa fruits pericarp. j med plant res 2012; 6:1982-90. doi: 10.5897/jmpr11.1683. 4. othman snam, sarker sd, nahar l, et al. the ethnomedicinal, phytochemical and pharmacological properties of phaleria macrocarpa (scheff). boerl. tang humanitas med 2014;4:1-11. doi: http://dx.doi.org/10.5667/ tang2014.0018. 5. elya b, handayani r, sauriasari r, et al. antidiabetic activity and phytochemical screening of 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nn. a review on the extraction methods use in medicinal plants: principle, strength, and limitation. med aromat plants 2015;4:3. doi : http://dx.doi.org/10.4172/2167-0412.1000196. 15. rahmi e. wahyuni wt, darusman lk, et al. combination of ethanolic extract of α-glucosidase inhibitory activity of (phaleria macrocarpa (scheff.) boerl.) fruits and annona muricata linn. leaves. trad med j 2016;21:63-8. doi: https:// doi.org/10/22146/tradmedj.12819. 16. purnamasari d. diagnosis dan klasifikasi diabetes mellitus. in: setiati s, alwi i, sudoyo aw, editors. buku ajar ilmu penyakit dalam jilid ii edisi vi. jakarta: interna publishing; 2014.p.2325-9. 17. scheff jd, almon rr, dubois dc, et al. assessment of pharmacologic area under the curve when baselines are variable. pharm res 2011;28: 1081-9. doi: 10.1007/s11095-010-0363-8. 18. chotimah c, sutrisna em, wahyuni as. water extract of jaka tuwa (scoparia dulcis l) herb activity on lowering blood glucose on male rabbit that loaded with glucose. pharmacon 2008;9:4651. 19. muhtadi a, hendriani r, mustarichie r. pharmacological screening of various indonesian herbal potentially used as antidiabetic. irjpas 2013;3:90-5. doi: 10.1038/ncomms9482. 20. anggraeni dn. pengaruh pemberian variasi dosis pupuk kandang terhadap pertumbuhan tanaman mahkota dewa (phaleria macrocarpa (scheff.) boerl.). biogenesis 2014;2:16-20.doi:https:// doi.org/10.24525/bio.v.2i1.463. 21. ali rb, atangwho ij, kuar n, et al. bioassayguided antidiabetic study of phaleria macrocarpa fruits extract. molecules 2012;17: 4986-5002. doi: 10.3390/molecules17054986. 22. azmir j, zaidul ism, sharif km, et al. supercritical carbon dioxide extraction of highly unsaturated oil from phaleria macrocarpa seed. frin 2014; 65:394-400. doi: 10.1016/j.foodres2014.06.049. 23. andrean d, prasetyo s, kristijarti ap, et al. the extraction and activity test of bioactive compounds in phaleria macrocarpa as antioxidants. procedia chem 2014;9:94-101. doi: 10.1016/j.proche.2014.05.012. 24. sabina e, zaidul ism, ghafoor k, et al. screening of various part of macrocarpa plant for αglucosidase inhibitory activity. j food biochem 2016; 40:201-10. doi: https://doi.org/10.1111/ jfbc.12212. 25. ramachandran b, rajasekaran s. blood glucose lowering effect of tectona grandis flowers in type 2 diabetic rats: a study on identification of active constituents and mechanisms for antidiabetic action. j diab 2014;6:427-37. doi: 10.1111/17530407.1212. 26. cicero l, yenshou l, arlene pb, et al. herbal therapies for type 2 diabetes mellitus: chemistry, biology, and potential application of selected plants and compounds. evidence-based comp alt med 2013;33-6. doi: http://dx.doi.org/ 10.1155/2013/378657. 27. arjadi f, mustofa. ekstrak daging buah mahkota dewa meregenerasi sel pulau langerhans pada tikus putih diabetes. biogenesis 2017;5:27-33. doi: http://dx.doi.org/10.24252/bio.v4i2.3430. 28. aziz mta, el-asmar mf, rezq am. the effect on a novel curcumin derivative on pancreatic islet regeneration in experimental type 1 diabetes in rats (long term study). diabet metab synd 2013;5: 75. doi: 10.1186/1758-5996-5-75. 29. hendra r, achmad s, sukari s, shukor ym. flavonoid analyses and antimicrobial activity in various parts of phaleria macrocarpa (scheff.) boerl fruit. int j mol sci 2011:3422-3.doi:10.3390/ ijms12063422. c:\users\jurnal fkusakti\docume 10 *department of emergency medicine, faculty of medicine, mashhad university of medical sciences, mashhad, iran **department of community medicine, faculty of medicine, mashhad university of medical sciences, mashhad, iran ***school of persian and complementary medicine, faculty of medicine, mashhad university of medical sciences, mashhad, iran †resident of emergency medicine, emergency medicine department, faculty of medicine, mashhad university of medical sciences, mashhad, iran ‡university of arizona, college of medicine, tucson, az, usa correspondence: mahdi foroughian department of emergency medicine, faculty of medicin e, mash had university of medical sciences, imam reza hospital, imam reza square, mashhad, iran postal code: 9137913316 phone: 05138525312; 09151240686 email: foroughianmh@mums.ac.ir orcid id : 0000-0002-3944-9361 date of first submission, september 27, 2 01 8 date of final revised submission, february 9, 2019 date of acceptance, february 11, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina acupressure has not an analgesic effect in patients with renal colic: a randomized controlled trial ehsan bolvardi*, bahram zarmehri*, seyed mojtaba mousavi bazzaz**, ali khorsand vakilzadeh***, mahdi foroughian*, hasan hoseini faegh†, and neema john mehramiz‡ background easing renal colic (rc) pain is of the most important issues in the emergency departments. this study was conducted to investigate the analgesic effects of intravenous paracetamol in comparison to acupressure and intramuscular diclofenac in patients with rc. methods this randomized clinical trial was conducted on 96 eligible patients with rc, visiting the emergency department of imam reza hospital of mashhad, iran. the participants were selected by consecutive non-random sampling method, and were randomized in three groups, each comprising of 32 participants. the first and second groups received 75 mg intramuscular diclofenac and 1g intravenous paracetamol, respectively. in the third group, three acupressure points were manually stimulated for six minutes. the pain score was measured by visual analog scale for pain at baseline and after 10, 30, and 60 minutes. collected information analyses by spss version 20 and interpreted using descriptive statistics (frequency, percentage) and inferential statistics (chi-square test). results the mean age was 31.60 ± 7.42 years (range:18-50). the two methods (diclofenac and paracetamol groups) were significantly better in pain reduction compared to acupressure after 10 minutes (p<0.001). ninety four percent of participants in diclofenac group, 87% of paracetamol group and none of acupressure group were completely satisfied with their treatment method (p<0.001). conclusion this study demonstrated that acupressure did not effectively reduces renal colic pain. it seems that contrary to acupressure, intravenous paracetamol is a safe and effective analgesic medicine for patients with rc. keywords: renal colic, satisfaction, paracetamol, acupressure, diclofenac original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.10-16 january-april, 2019 vol.38no.1 cite this article as: bolvardi e, zarmehri b, bazzaz mm, et al. acupressure has not an analgesic effect in patients with renal colic: a randomized controlled trial. univ med 2019;38:10-6. doi: 10.18051/univmed.2019.v38.10-16 11 univ med vol. 38 no.1 introduction pain is a physiological response to a harmful stimulation. the expression of pain depends on many factors including emotional, physical, and cultural states of an individual.(1) as pain presents 75-80% of emergency primary compliant, proper pain management is an important task of physicians in the emergency department (ed.(1) patients with renal colic (rc) comes to ed with severe pain in flank or abdomen which needs immediate treatment.(2) the urinary system consists of the kidneys, ureters, bladder, and the urethra (urinary meatus). renal colic pa in is the most common urinary system disorder.(3) renal colic is associated with severe pain, which begins from flanks and spreads to the groin. it is a recurring pain with the chance of exacerbation. the standard rc treatment includes relieving pain and correcting water and electrolyte imbalanc e. the only definite treatment is the elimination of the pain source, i.e., the cause of obstruction. however, the majority of initial treatments at admission are bound to temporary pain relief.(1,4) morphine and some other opiate compounds are the most effective analgesics; however, some concerns regarding their complications, such as physical dependence, substance abuse, inhibition of respiratory center, and sensitivity reactions, have signified the need for alternative methods. as a result, there is currently a greater tendency t ow a r d s the u se of n on s t e r oid a l a nt i inflammatory drugs (nsaids).(5-8) on the other h a nd , n said s h a ve c omp li ca ti o ns a s gastrointestinal bleeding and renal failure.(9) according to aforementioned complications, it is necessary to use less complicated and more effective medications for rc. the efficacy of intravenous paracetamol for pain control in therapeutic dose (1 gr four times a day in adult patients) can be compared with morphine or ketorolac, and because of fewer side effects it can be a treatment option in renal colic for patients presenting to ed.(10) re ga r d i ng t h e a b un d a nt pr ob l e ms a nd complications of pharmacotherapy, the use of non-medical methods that results in pain reduction in rc patients seems logical. in recent years, the application of non-medical methods, known as alternative medicine, has been given special attention. acupressure is a non-medicinal, non-aggressive, and relatively inexpensive method, which has almost no side effect. it is an effective technique for pain reduction without any complication. the acupressure helps to rejuvenate the body merely through applying a pressure on a certain body points by hands. it is a derivative of the chinese medicine.(11-13) in other words, acupressure is a type of massage that stimulates certain body points to increase c i r c ul a t i on a nd r e d uc e mus c le t e n si o n. acupressure is a means of stimulation of the same acupoints as used in acupuncture but without the use of needles or other medical instruments.(14) some studies have reported positive effects of pain reduction for some medical conditions like dysmenorrhea, neck pain, and fractures.(15-17) however, to the best of our kno wl e d ge, n o s t u dy h a s foc us e d on effectiveness of acupressure on pain reduction in rc. in addition to the financial burden, the use of medicines, such as opiates and nsaids, is associated with many side effects including h yp ove n t il a t i o n, n a u se a , pur p ur a , a nd bleeding.(18) according to many complications from nonsteroidal and morphine medications, it is necessary to use less complicated and more effective rc medications. so, we intended to compare the analgesic effects of intravenous paracetamol, acupressure, and intramuscular diclofenac in patients with rc. methods research design t hi s r a ndo mi ze d c l i ni c a l t r i a l w a s performed at the emergency departments of imam reza hospital in mashhad, iran. this study performed from january to june 2017. 12 research subjects the participants were selected from 1850 years old patients visiting the emergency department (ed) of imam reza hospital of mashhad, iran. inclusion criteria were patient coming to ed with rc, accepting the study and completing the informed consent form. exclusion criteria were the history of drug dependence, patients with allergy to medications of the present study, pregnant women, and patients who had used other analgesics before admission. also, patients who developed any unwanted medical complication during the study were excluded. the sample size was calculated using the formula f o r c omp ar i s on o f me a n bet we e n t wo i nd e p e n den t sa mp l e s c o n si d er i n g t he se parameters: alpha error=0.05, beta error=0.2, x 1 =9.1, sd 1 =2.1, x 2 =8.5, sd 2 =2.1, x 3 =5.1, sd 3 =1.2. the optimal sample was 32 for each group.(19) intervention the patients were randomized into three equal-sized groups using a random number sequence and were matched regarding gender. the randomization scheme was generated by using the web site randomization.com (http:// www.randomization.com). the first and second group received 75 mg intramuscular diclofenac and 1g intravenous paracetamol, respectively. in the third group, three acupoints (first web of fingers, the second web of toes, and a point on the medial side of distal tibia, above the medial malleolus) were selected according to the chinese medicine. these points were stimulated under a certain amount of pressure (4-5kg) every 2-3 seconds for 5 seconds. this process was performed for an overall time of six minutes. the researcher who performed the acupressure (akv) had a certified training in clinical acupressure by china beijing international acupuncture training center) cbiatc (, and have experiences in acupressure for more than 10 years. it is worth noting that an alternative method (morphine therapy) was used if the patients did not respond d e s ir a bl y t o p a i n 1 5 mi n ut e s a f t e r t he intervention. the pain level of all groups was measured after 10, 30, and 60 minutes between the three groups. pain score was categorized into three levels: severe pain (score: 7-10), moderate pain (score: 4-6) and mild pain (score: 0-3). besides, the level of patient satisfaction (not satisfied, moderately satisfied, and completely satisfied) and possible side effects in each method were also recorded. pain measurements the demographic information including gender, age, and weight were obtained. the research colleague, who was blinded to the patients’ groups, measured the pain level of all patients, using visual analog scale at baseline and after the intervention. statistical analysis then, the collected information was analyses by spss version 20 and interpreted using descriptive statistics (frequency, percentage) and inferential statistics (chi-square test). the significance level was considered below 0.05. ethical clearance first, the advantages and disadvantages of all three study methods were explained to the participants, and their informed written consent was collected. this study was approved by mashhad university of medical sciences (940520) and ethical committee of mashhad university of m edic al sciences (ir.mums.fm.rec.1395.79). this study was also registered at iranian registry for clinical trials (irct2016072129023n1). results in this study 134 patients assessed for eligibility. thirty-eight patients excluded from the study (not meeting inclusion criteria =26 patients, declined to participate =12 patients). finally, we randomized 96 patients with rc bolvardi, zarmehri, bazzaz, et al analgesic effect and renal colic 13 univ med vol. 38 no.1 visiting the ed to three groups of intervention (figure 1). the mean age was 31.60 ± 7.42 years (range: 18-50). half of admitted patients were between 27-34 years old (50%). most patients were male (84.4%, 81 individuals). concerning bmi, 55.2% of patients had normal weight. table 1 shows that there was no statistical difference between groups regarding basic characteristics and baseline pain. t his means that the randomization was successfully distributed the other variable between the three groups. as the acupressure group have severe pain after 10 min morphine therapy substituted and for this reason, we did not compare it to other groups that have pain relief in 30 and 60 minutes. as table 2 shows, 96.9% of participants in acupressure group still had severe pain after 10 minutes, this was 6.2% for diclofenac group and 9.4% for paracetamol group (p<0.001). it is should be reminded that due to the lack of pain reduction in acupressure group after 15 minutes, the morphine therapy was substituted for the acupressure. no side effects were observed for any participants. finally, while 93% (30) of participants in diclofenac group and 87.5% (28) of participants in paracetamol group were completely satisfied, 96.9% (31) of participants in acupressure group were not satisfied with their treatment method for pain reduction (p<0.001) (table 2). discussion this study showed no significant difference in pain reduction between the paracetamol and diclofenac groups after 10, 30, and 60 minutes, and the pain was relieved with passing time in both groups. in recent years, many studies have been conducted on the application of intravenous paracetamol. for example, mofidi et al.(20) a b c figure 1. flow diagram of the participants 14 compared the effectiveness of intravenous paracetamol and tramadol for rc patients. their findings suggested the greater effectiveness of the former medication in relieving pain in renal patients. serinken et al.(6) and bektas et al.(7) compared the effects of paracetamol a nd morphine in rc patients and did not find any significant difference. in iran, baghi et al.(21) compared the effectiveness of low-dose and highdose intra venous pa r acetamol-morphine combinations in treating rc patients. results did not show any significant difference in pain reduction and side effects. van aken et al.(22) in a study compared the effectiveness of a singledose intravenous paracetamol-intramuscular morphine and its repetition in improving analgesia after dental surgery. they did not find any specific statistical and clinical difference between the paracetamol and morphine groups regarding pain reduction; however, there were fewer side effects in the paracetamol group. comparison of our findings with results from other studies showed that paracetamol is a safer and more effective medication in pain reduction for patients with renal pain. this medication can produce desirable analgesic effects without significant complications. regarding the lack of access to intravenous nsaids in some medical centers and many complications associated with opiates (nausea, vomiting, seizure, etc.), the intravenous paracetamol can used as a suitable substitute for pain reduction in rc patients. characteristics intervention p value diclofenac (n=32) paracetamol (n=32) acupressure (n=32) gender male female age (years) 18-26 27-34 35-42 43-50 bmi (kg/m2) underweight normal overweight obese pain severe moderate mild 27 (84.4) 5 (15.6) 4 (12.5) 18 (56.2) 8 (25.0) 2 (6.3) 4 (12.5) 17 (53.1) 11 (34.4) 0 (0.0) 28 (87.5) 3 (9.4) 1 (3.1) 28 (87.5) 4 (12.5) 6 (18.8) 14 (43.8) 11 (34.3) 1 (3.1) 5 (15.6) 14 (43.8) 11 (34.4) 2 (6.2) 27 (84.4) 4 (12.5) 1 (3.1) 26 (81.2) 6 (18.8) 3 (9.4) 16 (50.0) 11 (34.3) 2 (6.3) 3 (9.4) 22 (68.8) 5 (15.6) 2 (6.2) 26 (81.2) 6 (18.8) 0 (0.0) 0.789 0.860 0.217 0.708 table 1. base-line characteristics of the three intervention groups data presented as n (%); bmi : body mass index outcome criteria intervention p value diclofenac (n=32) paracetamol (n=32) acupressure (n=32) pain after 10 minutes severe moderate mild satisfaction completely moderately not satisfied 2 (6.2) 16 (50.0) 14 (43.8) 30 (93.8) 2 (6.2) 0 (0.0) 3 (9.4) 22 (68.8) 7 (21.9) 28 (87.5) 4 (12.5) 0 (0.0) 31 (96.9) 1 (3.1) 0 (0.0) 0 (0.0) 1 (3.1) 31 (96.9) < 0.001 < 0.001 table 2. comparison of analgesic effects and satisfaction score between the intervention groups after 10 minutes data presented as n (%) bolvardi, zarmehri, bazzaz, et al analgesic effect and renal colic 15 univ med vol. 38 no.1 results from the current study showed a significant difference in the pain score between the diclofenac and acupressure groups, and also between the paracetamol and acupressure group 10-minute after the interventions. previous s t ud i e s wh i c h we re c on du c t e d o n t he effectiveness of acupressure have reported controversial findings. for example, there are studies about the effectiveness of acupressure on dysmenorrhea,(15) neck pain,(16) pain after distal radial fractures,(17) and pain caused by bone marrow aspiration in cancer patients.(23) results of these studies do not comply with our findings. this contradiction can be attributed to the different effect of acupressure on different types and levels of pain: regarding the high-intensity level of rc pain, acupressure may not have a considerable effect on rc pain. this study was not without limitations. first, we used a subjective tool for measuring pain. second, personal characteristics and even belief to modern or alternative medicine can influence the perception of pain. third, the natural difference between the acupressure therapy and the two pharmacological therapies did not allow us to perform a participant blinding. however, d esi gni n g a n rct f o r c o mp a ri s on th re e treatment methods in one of the most painful conditions in medicine is one of the strengths of this study. future studies with patients in other painful conditions can improve the results. conclusion acupressure was not found to be an effective analgesic in rc. regarding the side effects of opioid analgesics and nsaids, it seems that intravenous paracetamol can be considered as a n e ffective and safe pain medication in emergencies. acknowledgements this study was financially supported by mashhad university of medical sciences and we thank our patients for their cooperation. conflict of interest none to be declared. contributors study design, data gathering and analysis performed by eb, bz, sm-mb, akv, mf, hh f, nj m . ac u pr e s su r e d o ne b y ak v. manuscript drafting done by all authors, and all authors accepted the final version of manuscript. references 1. tintinalli je, stapczynski js, ma oj, et al. tintinalli’s emergency medicine : a comprehensive guide. 8th ed. new york: mc graw hill; 2016. 2. soylu a, sarier m, altunoluk b, et al. comparison of the efficacy of intravenous and intramuscular lornoxicam for the initial treatment of acute renal colic: a randomized clinical trial .urol j 2018;1:1-6. doi: 10.22037/uj.v0i0.4496. 3. bultitude m, rees j. management of renal colic. bmj 2012;345:e5499. doi: https://doi.org/10.1136/ bmj.e5499. 4. frassetto l, kohlstadt i. treatment and prevention of kidney stones: an update. am fam physician 2011;84:1234-42. 5. talebi doluee m, reihani h, zarmehri b, et al. pain management in emergency department: intravenous 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of analgesia effect between intravenous propacetamol and tramadol. j mazandaran univ med sci 2007;17:81-7. 21. morteza-bagi hr, amjadi m, mirzaii-sousefidi r. the comparison of apotel plus low dose of morphine and full dose of morphine in pain relief in patients with acute renal colic. addict health 2015;7:66-73. 22. van aken h, thys l, veekman l, buerkle h. assessing analgesia in single and repeated administrations of propacetamol for postoperative pain: comparison with morphine after dental surgery. anesth analg 2004;98:15965. 23. bao t, ye x, skinner, et al. the analgesic effect of magnetic acupressure in cancer patients undergoing bone marrow aspiration and biopsy: a randomized, blinded, controlled trial. j pain symptom manage 2011;41:995-1002. doi: 10.1016/ j.jpainsymman.2010.08.012. bolvardi, zarmehri, bazzaz, et al analgesic effect and renal colic c:\users\jurnal fkusakti\docume 164 *postgraduate biomedical science program, medical faculty, sultan agung islamic university, semarang, indonesia **department of postgraduate biomedical science, medical faculty, sultan agung islamic university, semarang, indonesia ***department of pathological anatomy, medical faculty, sultan agung islamic university, semarang indonesia †stem cell and cancer research laboratory, medical faculty, sultan agung islamic university correspondence dr. dr. h. agung putra, m. si. med. chairman of stem cell and cancer research (sccr) laboratory, medical faculty, universitas islam sultan agung semarang, jl. raya kaligawe km. 4 semarang, jawa tengah 50112 phone.+628164251646 email: dr.agungptr@gmail.com orcid id : 0000-0003-4261-9437 date of first submission, october 10, 2 01 8 date of final revised submission, october 11, 2019 date of acceptance, october 16, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina hypoxia enhances self-renewal properties and markers of mesenchymal stem cells vivi yustianingsih*, titiek sumarawati**, and agung putra***,† background mesenchymal stem cells (mscs) are multipotent stromal cells that express cd73, cd90, and cd105 surface markers, but not cd14, cd45, cd34, cd11b, and hla-dr. mscs under hypoxic conditions have the essential role of maintaining the stemness capacity by releasing several growth factors into their medium, known as hypoxia conditioned medium (hcm). this study was performed to compare the effect of percentage of hcm to normoxic medium (nm) in increasing msc proliferation marked by proliferation rate and surface marker expression. methods this study was of post-test only control group design using human umbilical cord-mscs (huc-mscs) as subjects. the hcm treatment group was obtained by culturing mscs under 5% o 2 , whereas the nm control group was grown under 20% o 2 . the huc-mscs were divided into 4 groups with different dose ratios of hcm to nm (25%:75%; 50%:50%; 75%:25% for p1, p2 and p3, respectively and 100% of nm for the controls). all of these groups were maintained at 37oc and the data was collected after 72 hours incubation. msc marker expression of cd73, cd90 and cd105 was analyzed using flow cytometry and msc proliferation by trypan blue assay. result there were significant differences in msc marker expression of cd73, cd90 and cd105 and proliferation at all dose ratios of hcm to nm (p<0.05). conclusion low oxygen concentration promotes msc proliferation and stemness thus it might be beneficial for maintaining the msc physiologic niche in-vitro. keywords: msc, msc-hcm, proliferation, stemness original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.164-171 september-december, 2019 vol.38no.3 cite this article as: yustianingsih v, sumara wati t, putra a. hypoxia enhances self-renewal properties and markers of mesenchymal stem cells. univmed 201 9;38 :1 6 4-7 1 . doi: 10 .18 05 1/ univmed.2019.v38.164-171 165 univ med vol. 38 no.3 introduction mesenchymal stem ce lls (mscs) are multipotent stromal cells with the capacity for selfrenewal and differentiation into mesodermal lineages such as chondrocytes, osteoblasts, adipocytes, and neurons.(1) according to the international society for cellular therapy (isct) mscs’ main characteristics are adherence to plastic, expression of cd73, cd90, and cd105 and lack of expression of cd14, cd45, cd34, cd11b, and cd31 and hla-dr. (2 ) mesenchymal stem cells have been widely used for tissue engineer i ng and treatment of degenerative diseases and immune disorders, due to their trophic activity, immunomodulatory and angiogenic propertie s and multipotent differentiation capacity.(3) mesenchymal stem cells can be isolated from a wide range of source tissues such as the umbilical cord, bone marrow, adipose tissue and dental pulp.(4-6) moreover, an essential goal for expansion of mscs in vitro is to obtain sufficient cell numbers for research and clinical application. this depends mainly on tissue source of mscs and processing method. in umbilical cord-derived mscs (uc-mscs) the stemness is highest in comparison with bone marrow and adiposederived mscs, due in part to their higher telomerase activity, and pluripotent properties under certain conditions.(7–9) a previous study has shown that the major concerns of in vitro msc propagation are poor growth kinetics, early senescence and dna damage during expansion.(6) therefore, isolation techniques, culture medium, oxygen tension, supplements, cell seeding density and their modification such as under hypoxia and tnf-α are essential for msc propagation.(10,11) one previous study reported that oxygen concentration is a critical environmental factor for maintaining in vitro stem cell plasticity and proliferation.(12) other studies also demonstrated that hypoxic conditions (o 2 pressure under 5% have positive effects on mscs in terms of in vitro survival and self-renewal, particularly in prese rving the stemness and enhancing proliferation.(13–15) there was an enhancement of msc proliferation in hypoxic conditions compared to normoxia even following 2 weeks of incubation of the cultures.(16) however, this study did not report the use of the hypoxia chamber as a culture device. in contrast, another study reported that continued hypoxia reduced the clonogenic ability and differentiation potential of mscs.(7) another study described that 24-h hypoxia is beneficial for the functional properties of mscs.(17) since hypoxia is the most similar to the physiological conditions occurring in living organisms, we decided to examine msc culture both in normoxic and hypoxic conditions. in this study, we analyzed the comparative effect of hypoxia-conditioned medium (hcm) obtained after 24-h hypoxia incubation to nm (with hcm:nm ratios of about 25%:75%; 50%:50% and 75%:25%, respectively) in enhancing msc stemness through analyzing msc proliferation and cd73, cd90 and cd105 expression for 72 hours incubation. methods research design this post-test only control group design was conducted in the stem cell and cancer research laboratory, faculty of medicine, universitas islam sultan agung (unissula), semarang from july-september 2018. research subjects human umbilical cord-mscs (huc-msc) were used as the subjects and divided into 4 groups with different doses of msc-hcm. the total number of huc-mscs was 1.2 x 106 cells and was equally divided into 4 groups. the control group was treated with normal medium, whereas the treatment groups were supplemented with 25% hcm (p1), 50% hcm (p2) and 75% hcm (p3), respectively. mesenchymal stem cell isolation mononuclear ce ll s (mncs) from an umbilical cord were collected and separated by 166 ficoll-paque solution (density 1.077 g/ml; ge) in centrifuge tubes (15 ml). the mononuclear cells were transferred to a new tube and washed twice through centrifugation at 2000 rpm for 10 minutes. they were then seeded into 25-cm2 flasks at a density of 1 × 107–108 cells/cm2. the flasks contained dulbecco’s modified eagle’s medium– low glucose (dmem-lg, gibco invitrogen) supplemented with 1% l-glutamine 200 mm, 1% antibiotic—antimycotic comprising 10.000 u/ml sodium penicillin, 1% 10.000 μg/ml streptomycin sulfate, 25 μg/ml amphotericin b (gibco/ invitrogen corporation), and 10% fetal bovine serum ( gibco/invitr oge n corporation). umbilical cord blood (ucb)-msc cultures were maintained at 37 °c in 5% co 2 and non-adherent cells were removed after 48 h. the medium was changed every other day. the experiments were performed at the fourth passage of the cultures with approximately 80–90% confluence. ucbmsc lineages were established in culture up to the fourth passage. characterization of mscs t he c h a ra c t e r i s t ic s o f mscs w e r e analyzed by flow cytometric analysis at the fourth passage. the cells were subsequently incubated at room temperature and in the dark wi th f lu or e s c ei n i sot h i ocya n a te ( fitc) conjugated, allophycocyanin (apc)-conjugated or phycoerythrin (pe)-conjugated monoclonal antibodies, including cd73, cd90 and cd105. apc-, pe-, and fitc-conjugated isotypes were used as negative controls. the analysis used bd pharmingentm (bd biosciences, franklin lakes, nj, usa) at 4°c for 30 min. the cells were washed twice with 1% bsa/pbs, resuspended in 200 µl 1% bsa/pbs and analyzed using a flow cytometer (bd biosciences, san jose, ca, usa). in-vitro differentiation for the differentiation test, mscs were grown on 24-well plates at densities of 1x104 cells/ well, with dmem supplemented with osteogenicinduction medium containing 10-7 mol/l/0.1 μm dexamethasone, 10 mmol/l β-glycerophosphate, 50μmol/l ascorbate-2-phosphate (sigma-aldrich, louis st, mo) and 10% fetal bovine serum (fbs). the cells were rinsed in pbs and fixed with cold 70% ethanol (v/v) for 1 hour at room temperature, then rinsed three times with twicedistilled water after 21 days of induction. a volume of 1 ml 2% alizarin red solution (w/v) (ph 4.14.3) was added and the cells incubated for 30 minutes at room temperature, then rinsed four times in twice-distilled water. hypoxia conditioned medium (hcm) a previous protocol (18) was adapted for use in this study. briefly, hmscs were incubated until confluent, after which they were seeded in a t25 flask (2 × 106 cells), washed twice with endothelial basal medium (ebm-2; lonza), and then placed in a hypoxic chamber (anaerobic environment; thermoforma, waltham, ma, usa) containing 5 ml ebm-2 for 12 hours. the airtight humidified hypoxic chamber was maintained at 37°c and continuously supplied with 5% co 2 , 10% h 2 , and 85% n 2 . the oxygen level in the chamber was ~0.5%. after incubation, the medium was collected and centrifuged at 1000 rpm for 10 minutes at 4°c. then the hcm was added to the treatment groups at concentrations of 25, 50% and 75%, respectively. mesenchymal stem cell proliferation essay and stemness the cells were counted in a hemocytometer for analyzing cell proliferation. in brief, following treatment, the cells are harvested and an aliquot of cells is combined with trypan blue dye solution and loaded into the hemocytometer chamber. the cells are then viewed under the microscope and the number of live cells present within a specific area are counted. the number of cells counted per area multiplied by the dilution factor will determine the number of live cells per milliliter. the stemness of mscs was analyzed after 72-h induction using a bd pharmingentm flow cytometer (bd biosciences, franklin lakes, nj, usa). yustianingsih, sumarawati, putra effect of msc-hcm on msc self-renewal 167 univ med vol. 38 no.3 statistical analysis all values are expressed as the mean ± sd. comparisons between the four groups were analyzed using anova and then followed by posthoc fisher’s lsd. a p value of <0.05 was considered significant. all analyses were performed with spss 16.0. ethical clearance all research activities were approved by the commission on test animal ethics (komisi etik hewan uji), faculty of medicine, universitas islam sultan agung, semarang, under no. 233/ vii/2018/komisi bioetik. a b c results mesenchymal stem cell characterization the mscs were successfully isolated from the umbilical cord, based on their adherence to plastic. after reaching 80% confluence, the cells were harvested for 4 passages. the mscs showed a fusiform appearance, became confluent after 5-7 days in culture and were regularly passaged. flow cytometry analysis showed that the mscs were positively expressing 75.8% of cd73, 84.1% of cd90 and 55.2% of cd105 (figure 1). figure 1. characteristics of uc-mscs expressing cd73, cd90 and cd105 by flow cytometry 168 in vitro differentiation the msc differentiation assay was done by means of osteogenic medium. the results s ho we d tha t m scs w e r e c ap a b l e of differentiating into osteogenic cells as indicated by their red color from alizarin red staining (figure 2b). mscs self-renewal and stemness the msc appearance was analyzed using the light microscope at 24, 48, and 72 hours after msc-hcm induction (figure 3). the results showed significant between-group differences in msc self-renewal appearance (p=0.000). the highest result was shown by p3 with a high concenteration of msc-hcm (75%) (table 1). this is in line with the expression of msc positive markers (cd 73, cd90, cd105). discussion several studies have reported that hypoxic conditions may modulate the paracrine activity of mscs by up-regulating the various secretable factors into a medium such as hcm. these soluble molecules are associated with the figure 2. (a) uc-msc-like from in vitro culture showing fibroblast-like and polygonal cells, at 40x magnification, (b) osteogenic differentiation test with alizarin red staining appearing in msc population 0 hours 24 hours 48 hours 72 hours control p1 p2 p3 figure 3. appearance under light microscope of mscs after hcm induction yustianingsih, sumarawati, putra effect of msc-hcm on msc self-renewal 169 univ med vol. 38 no.3 increased proliferation rate and stemness properties of mscs. whereas the effects of hypoxia have been investigated previously, the comparison between hcm and nm in enhancing msc prolif eration in vitro by constantly maintaining stemness pr ope rties rema ins unknown. in this study we introduced various hcm to nm dose ratios in mscs using the hypoxic condition chamber as in a previous study,(17) then analyzed the increase in msc proliferation rate and their expression of cd73, cd90, and cd105. our results showe d that there were significant differences between all hcm to nm dose ratios in cell proliferation rate after 72 hours. this is in accordance with a previous study that found that hypoxic conditions may increase the proliferation and stemness in mscs.(18) we suggest that the hypoxic state induces hypoxiainducible factor-1a (hif-1a) as transcription factor for inducing hif-1a survival target genes including self-renewal and proliferation.(19) this is in line with one other study reporting that mscs under hypoxic conditions also have the ability to express the transcription embryonic factors such as sox2.(18) in undifferentiated mouse and human embryonic stem cells (es), oct3/4 and sox2 also interact with the nanog promoter to strengthen the stemness of the cells.(18,20) these factors serve as candidate molecules for mastering the regulation of initiation, maintenance, and differentiation of pluripotent cells. this is also in accordance with our finding that there are significant differences between all hcm to nm dose ratios in msc positive markers (cd73, cd90, cd105) with the highest result in group p3 (75%:25%). we suggest that the secretome released by mscs under hypoxic conditions may induce the in vitro msc progeny cells to grow over with maintaining their stemness. variable treatment groups p value control (n=3) p1 (n=3) p2 (n=3) p3 (n=3) proliferation (cell) 411666.67 ± 12583.05 461666.67 ± 10408.33 535000.00 ± 13228.75 571666.67 ± 10606.60 0.000* cd73 (%) 76.41 ± 0.70 86.5 ± 1.20 90.86 ± 0.40 99.26 ± 0.50 0.000* cd90 (%) 84.16 ± 0.30 90.83 ± 0.45 93.73 ± 0.45 96.43 ± 0.55 0.000* cd105 (%) 55.41 ± 0.26 60.81 ± 0.45 63.86 ± 0.40 66.80 ± 0.30 0.000* table 1. distribution of msc proliferation and stemness after 72 hour incubation by treatment groups note: p1 medium with 25% hcm, p2 medium with 50% hcm, p3 medium with 75% hcm. *significant at p<0.05 groups mean differences p value proliferation control p1 p2 p3 p1 p2 p3 p2 p3 cd73 control p1 p2 p3 p1 p2 p3 p2 p3 cd90 control p1 p2 p3 p1 p2 p3 p2 p3 cd105 control p1 p2 p3 p1 p2 p3 p2 p3 -50000.00 -123333.33 -155833.33 -73333.33 -105833.33 -32500.00 -10.033 -14.43 -22.83 -4.40 -12.80 -8.40 -6.66 -9.56 -12.26 -2.90 -5.60 -2.70 -5.40 -8.46 -11.40 -3.06 -6.00 -2.93 0.001 0.000 0.000 0.000 0.000 0.020 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 0.000 table 2. multiple comparisons of msc proliferation and stemness note: p1 medium with 25% hcm, p2 medium with 50% hcm, p3 medium with 75% hcm 170 our findings indicate that hcm plays an important role in stemness potentials of the adult stem cell (asc) including uc-msc under hypoxic conditions. the limitation of this study is that we did not analyze the transcription marker molecules such as sox2, oct3/4 and nanog. conclusions low oxygen c once ntra tion in m scs promotes cell proliferation and stemness, thus it might be beneficial for maintaining the msc physiologic niche in-vitro. conflict of interest co mp e t i ng i n t e r e s t s : n o r e l e va nt disclosures. acknowledgement we would like to thank the stem cell and cancer research laboratory, medical faculty, universitas islam sultan agung, semarang and the department of postgraduate biomedical science, medical faculty, universitas islam sultan agung, semarang for all the facilities needed to finish this research. contributors vy, ap and ts contributed to the basic concept and design of the study. vy and ap contributed to writing the manuscript and p e r f o r mi ng th e e x pe r i me nt . v y a nd a p contributed to sample preparation and data collection. vy contributed to the statistical analysis. all authors have read and approved the final manuscript. references 1. kagami h, agata h, tojo a. bone marrow stromal cells (bone marrow-derived multipotent mesenchymal stromal cells) for bone tissue engineering: basic science to clinical translation. int j biochem cell biol 2011;43:286-9. doi: https:/ /doi.org/10.1016/j.biocel.2010.12.006. 2. sharma a, rani r. do we really need to 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cell pluripotency. stem cell res ther 2010;1:39. doi: 10.1186/scrt39. oktavianus 165 *division of gastroenterology and hepatology, department of internal medicine, medical faculty, north sumatera university, medan correspondence dr. ester morina silalahi division of gastroenterology and hepatology, department of internal medicine, medical faculty, north sumatera university, jl. dr. mansur no. 5 medan sumatera utara 20155 email: silalahiester70@yahoo.com univ med 2013;32:165-71 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background carcinoembryonic antigen (cea) and carbohydrate antigen (ca) 19-9 are tumor markers for colorectal cancer (crc), both having prognostic value. according to the colorectal working group of the american joint committee on cancer, increases in cea and ca 19-9 levels of >5 µg/l and > 37 u/ml, respectively, are considered abnormal. increased serum cea may be encountered in postoperative crc patients from recurrences or metastases. there are no research data in indonesia on the characteristics of cea and ca 19-9 levels according to preoperative crc cellular differentiation. the objective of this study was to determine differences in serum cea and ca 19-9 levels according to crc cellular differentiation. methods this was a cross-sectional study conducted on 40 crc subjects from july 2012 until may 2013. determination of serum cea and ca 19-9 levels and histopathological (cellular) differentiation grades in crc biopsies was done in all subjects. results the study involved forty crc patients, consisting of 22 males and 18 females, with mean age of 51.93 ± 11.63 years, cea levels of 51.93 ± 84.07 ng/ml and ca 19-9 levels of 33.81 ± 62.39 u/ml. carcino-embryonic antigen levels tended to decrease with decreasing crc histopathological grade, while ca 19-9 levels increased in well-differentiated crc. however, both relationships were statistically not significant (with p=0.314 and p=0.787, respectively). conclusions carcinoembryonic antigen (cea) levels tend to decrease with decreasing histopathological grade of crc, and ca 19-9 levels tend to increase in welldifferentiated crc. key words: carbohydrate antigen 19-9, carcinoembryonic antigen, cellular differentiation, colorectal cancer serum carcinoembryonic antigen tends to decrease in poorly-differentiated colorectal cancer ester morina silalahi*, lukman hakim zain*, and rustam effendi* 166 silalahi, zain, effendi serum carcinoembryonic antigen karsinoembrionik antigen serum cenderung menurun pada sel kanker kolorektal diferensiasi buruk latar belakang carcinoembryonic antigen (cea) dan carbohydrate antigen 19-9 (ca 19-9) adalah penanda tumor untuk kanker kolorektal (kkr), keduanya memiliki nilai prognostik. berdasarkan colorectal working group of the american joint committee on cancer dikatakan nilai abnormal bila peningkatan nilai cea di atas normal yaitu >5 µg/l dan peningkatan nilai ca 19-9 >37 u/ml. peningkatan cea dalam serum bisa dijumpai pada penderita kkr pasca operasi bila terdapat rekurensi atau metastasis. belum ada data penelitian di indonesia mengenai karakteristik kadar cea dan ca 19-9 berdasarkan diferensiasi sel penderita kkr preoperatif. tujuan penelitian ini untuk membedakan kadar cea dan ca 19-9 dalam serum berdasarkan diferensiasi sel yang berbeda pada kkr. metode penelitian ini merupakan desain potong-silang mengikutsertakan 40 penderita kkr yang dilakukan antara bulan juli 2012 hingga mei 2013. pemeriksaan cea dan ca 19-9 dalam serum serta diferensiasi sel (histopatologi) di jaringan biopsi tumor dilakukan pada penderita kkr. hasil hasil penelitian didapatkan 40 penderita kkr 22 pria dan 18 wanita dengan umur rata-rata 51,93 ± 11,63 tahun, kadar cea 51,93 ± 84,07 ng/ml, dan kadar ca 19-9: 33,81 ± 62,39 u/ml. kadar cea cenderung semakin menuurn dengan semakin rendahnya grade histopatologi dari sel kkr, tetapi secara statistik tidak bermakna (p=0,314). kadar ca 19-9 semakin meningkat pada sel kkr berdiferensiasi baik, tetapi tidak bermakna secara statistik (p=0,787) kesimpulan kadar cea cenderung semakin menurun dengan semakin rendahnya grade histopatologi sel kkr, sedangkan kadar ca 19-9 cenderung semakin meningkat pada sel kkr berdiferensiasi baik. kata kunci: carbohydrate antigen 19-9, carcinoembryonic antigen, diferensiasi sel, kanker kolorektal abstrak introduction world-wide, colorectal cancer (crc) ranks third among cancers in males and second among cancers in females, with more than 1.2 million new cases annually and 608.700 deaths in 2008.(1) in the us a total of 146.970 new crc cases are found annualy, and an estimated total of 49.920 deaths from crc.(1) in general the prevalence of crc shows a sharp increase after the age of 50 years, which is associated with exposure to various carcinogens and lifestyle.(2) in indonesia, various reports indicate an increase in the number of crc cases. to date crc is a public health problem that ranks third among the most frequently occurring cancers in indonesia. the age-standardized incidence of crc in indonesia per 100.000 persons was 19.1 in males and 15.6 in females.(3) a study on crc profile at pirngadi hospital in medan found 197 crc patients among a total of 760 patients examined by colonoscopy (25.9%), while 101 (51.3%) of the crc patients were female. around one-third (28.9%) of the crc patients were in the age range of 51-60 years, 46.1% were ethnic batak, 167 univ med vol. 32 no.3 and 74.6% of the crc were located in the rectum.(4) carcinoembryonic antigen (cea) is the most common tumor marker for crc, and its levels are of value in continuous monitoring and as prognostic markers.(5,6) the american society of clinical oncology (asco) recommend the preoperative assessment of cea levels to assist in crc staging, operative planning, or in m o n i t o r i n g t h e r a p e u t i c r e s p o n s e d u r i n g treatment.(6,7) s e r i a l c e a d e t e r m i n a t i o n t o d e t e c t recurring crc has a sensitivity of 80%, a specificity of 70% and may persist for 5 months. one study concluded that assessment of preoperative serum cea and ca 19-9 levels may be used prognostically for 5-year recurrence free survival (rfs) in crc. (8) elevated cea concentration is associated with grade 1 and 2 tumors, late stages of crc and metastases to i n t e r n a l o rg a n s . a l t h o u g h s e r u m c e a concentration is an independent prognostic factor, serum cea values may be called significant only upon continuous postoperative monitoring.(9) in spite of the limited specificity and sensitivity of the cea test, it is frequently suggested to find early recurrences. preoperative cea testing is very useful as a prognostic factor and to determine any connection between elevated c e a a n d t h e p r i m a r y t u m o r. e l e v a t e d preoperative cea is of benefit for early identification of metastases, since metastatic tumor cells frequently result in elevated cea.(9) there are many factors influencing the prognosis of crc, one of them being histopathological grade determined from biopsies. apart from b e i n g a d e t e r m i n a n t o f p r o g n o s i s , histopathological grade is also an important determinant of crc etiology and management. p a r t i c u l a r l y f o r c r c m a n a g e m e n t , histopathological examination of crc is mandatory, because histopathological grade is important for determining further management o f c r c . a m o n g t h e h i s t o p a t h o l o g i c a l characteristics evaluated are tumor species and degree of differentiation. the histopathological picture may determine the degree of malignancy of a neoplasm. in addition to being a diagnostic determinant, the histopathological picture is also of great influence in determining tumor prognosis and recurrence.(10) the study carried out by sudoyo et al. (11) concludes that there are significant differences in the histopathological picture comprising crc grade and stage. poorly differentiated crc is associated with genetic mutations, but this has yet to be proven. around 20% of crcs are poorly differentiated, and have a poor prognosis.(12) histopathological grade significantly affects patient survival in addition to tumor stage. patients with well-differentiated carcinomas (grades 1 and 2) have better 5-year survival rates than those with poorly differentiated carcinomas (grades 3 and 4).(9) one study showed that welldifferentiated crc have higher cea levels in comparison with poorly differentiated crc.(13) on the basis of the abovementioned information, the purpose of the present study was to find d i ff e r e n c e s i n s e r u m c e a a n d c a 1 9 9 c o n c e n t r a t i o n s a s s o c i a t e d w i t h c e l l u l a r differentiation (tumor histopathological grade) in subjects with crc. methods design of the study this study was a comparative analytical study of cross-sectional design. the study was carried out at h. adam malik and pirngadi medan hospitals between july 2012 and may 2013. study subjects the target population comprised patients w i t h s u s p e c t e d c r c a n d d i a g n o s e d b y colonoscopy and biopsy. the study sample consisted of patients with crc meeting the i n c l u s i o n c r i t e r i a , ( p a t i e n t s w i t h histopathologically established diagnosis of crc, both females and males >18 years of age) and the exclusion criteria (crc patients with 168 silalahi, zain, effendi serum carcinoembryonic antigen cancer of the head of the pancreas, pancreatitis, or pulmonary, ovarian, or hepatic tumors). the study subjects were selected by non-random consecutive sampling. data collection all study subjects were interviewed by means of a questionnaire whose items included the subject’s name, age and gender. the subject’s weight was measured using omron scales and his/her height was determined by a kenko height measuring instrument. laboratory analysis the laboratory investigations comprised determination of cea and ca 19-9 levels. a 5 ml venous blood sample was divided equally into 2 tubes of 2.5 ml each. the samples were left to clot, centrifuged at 1500 rpm for 5 minutes, then put into a cobas 6000 analyzer. the cea levels were expressed in ng/ml, while the ca 19-9 levels were expressed in u/ml. the cutoff values for cea and ca 19-9 were 5 ng/ ml and 37 u/ml, respectively.(14) histopathologic examination after colonoscopy, biopsy specimens were o b t a i n e d f r o m t h e c o l o n o r r e c t u m f o r histopathologic determination of cellular differentiation. the cellular differentiation of the h i s t o p a t h o l o g i c s p e c i m e n s w a s g r a d e d microscopically by a pathologist. grade 1 (welldifferentiated): atypical glands, disorganization with disorganized epithelium, pleiomorphic hyperchromatic nuclei, coarse chromatin, eosinophilic cytoplasm. grade 2 (moderately d i ff e r e n t i a t e d ) : p r o l i f e r a t i v e g l a n d s , disorganization with disorganized epithelium, pleiomorphic hyperchromatic nuclei, irregular nuclear membrane, eosinophilic cytoplasm. grade 3 (poorly differentiated): proliferative glands, disorganization with disorganized epithelium, pleiomorphic hyperchromatic nuclei, irregular nuclear membrane, eosinophilic cytoplasm. data analysis to compare cea and ca 19-9 levels for each crc cellular differentiation grade, one-way anova was used, followed by the chi-square test to find a relationship between cea and ca19-9 levels on the one hand and crc cellular differentiation on the other. statistical analysis was performed using spss version 15.0, with the level of significance set at p<0.05. ethical clearance the study protocol was approved by the health research ethical committee of north sumatra university, faculty of medicine. all study subjects signed written informed consent after having been informed about the aims and benefits of the study. results there were in total 40 crc patients who p a r t i c i p a t e d i n t h i s s t u d y. t h e c l i n i c a l characteristics of the crc patients, crc location, crc cellular differentition, and cea and ca 19-9 levels, are presented in table 1. table 1. distribution of clinical characteristics of the subjects (n=40) cea= carcinoembryonic antigen; ca= carbohydrate antigen * values are mean ± sd 169 univ med vol. 32 no.3 table 3. comparison of cea and ca 19-9 groups by crc histopathological grade crc=colorectal cancer; * normal cea (carcinoembryonic antigen) ≤ 5 ng/ml, high > 5 ng/ml; ** normal ca (carbohydrate antigen) 19-9 ≤ 37 u/ml, high ca 19-9 > 37 u/ml; # chi-square test: non significant table 2. comparison of cea and ca 19-9 levels by crc histopathological grade crc=colorectal cancer; cea=carcinoembryonic antigen; ca=carbohydrate antigen * values are mean ± sd; ** one way anova test: non significant among the 40 subjects, 22 (55.0%) were males and 18 (45%) were females. mean age of the subjects was 51.931 ± 11.63 years. the colonoscopically diagnosed crcs according to location consisted of 30 cases (75%) of rectal carcinoma, 4 cases (10%) of sigmoid carcinoma, 3 cases (7.5%) of carcinoma of the descending colon, 2 cases (5%) of carcinoma of the ascending colon, and 1 case (2.5%) of cecal carcinoma. the results of cellular differentiation grading was dominated by 28 samples (70%) of well-differentiated crc, while moderately and poorly differentiated crc comprised 6 samples (15%) each. one-way anova was used to compare cea and ca 19-9 levels according to cellular differentiation. the anova results showed that c e a l e v e l s d e c r e a s e d w i t h d e c r e a s i n g histopathological grade of the crc cells, but the differences was statistically not significant (p=0.314). the results for ca 19-9 levels did not show a decreasing tendency with decreasing histopathological grade of the crc cells. there was no significant difference in ca 19-9 levels between the three histopathological grade categories (p=0.787) (table 2). the chi-square test results showed that elevated cea levels were more frequently found in well-differentiated crcs, in comparison with those in poorly differentiated crcs, but the relationship was statistically not significant (p=0.475). increased ca 19-9 levels were found in all well-differentiated crcs, but this relationship was also statistically not significant (p=0.247) (table 3). discussion cea and ca 19-9 are the most frequently used tumor markers for crc, both of which have prognostic value. the use of cea and ca 19-9 as tumor markers and for prognostic evaluation in various tumor stages is according to the colorectal working group of the american joint committee on cancer (ajcc). serial cea determination to detect recurring crc has a sensitivity of 80%, a specificity of 70% and may p e r s i s t f o r 5 m o n t h s . s e r i a l c a 1 9 9 170 silalahi, zain, effendi serum carcinoembryonic antigen determination to detect recurring crc has a sensitivity of 70-80% and a specificity of 8090%. according to ajcc, a cea level is considered to be elevated if it is >5 µg/l, while a ca 19-9 level is elevated if it is >37 u/ml.(15) our study showed decreasing cea levels with decreasing histopathological differentiation of the crc cells, but the relationship was s t a t i s t i c a l l y n o t s i g n i f i c a n t . s i m i l a r b u t statistically significant results were found by duffy, showing that well-differentiated crc produced significantly higher cea levels than poorly differentiated crc. this may be due to the much larger sample size used in duffy’s study.(13) in addition, in our study there were s e v e r a l s a m p l e s o f w e l l d i ff e r e n t i a t e d histopathology with normal cea levels (<5 ng/ ml). in a study by cerna et al.(16) on cea levels in tumor tissues, the most significant finding was that cea was present in practically all tissue samples. this is in contrast with the current belief that serum cea is not elevated in all primary crcs or even in the metastases. therefore, to obtain significant results in studies such as ours, in addition to determination of serum cea levels, t h e c e a l e v e l s i n t u m o r t i s s u e s s h o u l d presumably be assessed. in our study, the tendency for well-differentiated crcs to result in higher ca 19-9 levels in comparison with poorly differentiated crcs, is to be considered as not proven, because we found that the above relationship was statistically not significant. similarly, the study by chen et al.(5) on the relationship between cea and ca 19-9 levels on the one hand and histopathologically determined cellular differentiation on the other, also did not find a significant relationship. with regard to crc location, our study found that the most frequent location was the rectum (75%) as compared with other sites, and that the least frequent location was the cecum (2.5%). this agrees with the results of a previous study by effendi et al.(4) who stated that the most frequent location of crc was the rectum (74.6%). the study conducted by sudoyo et al.(11) also found the most frequent crc location to be the rectum (72.7%). in connection with crc histopathological differentiation, the most frequently found was well-differentiated crc (70%), with moderately and poorly differentiated crcs each accounting for 15%. however, these results differ from those found in the study by stewart et al.(10) conducted i n t h e u s f r o m 1 9 9 8 t o 2 0 0 1 , w h e r e a d e n o c a r c i n o m a s w e r e m o r e f r e q u e n t l y moderately differentiated. therefore, our findings cannot as yet be used as a prognostic measure for comparing preoperative and postoperative status. the present study still has a number of limitations. firstly, the small size of the study population, being restricted to 2 central hospitals, resulting in the lack of significant differences in c e a a n d c a 1 9 9 l e v e l s i n r e l a t i o n t o histopathological cellular differentiation. secondly, in this study no tumor staging was done, so that cea and ca 19-9 cannot be used as prognostic indicators of crc. therefore multicenter studies are needed with larger samples and tnm staging based on duke’s classification.(17) conclusions cea levels tend to decrease with decreasing histopathological grade of crc, and ca 19-9 levels tend to increase in histopathologically welldifferentiated crc. acknowledgments we thank all study subjects who agreed to participate in the present study and all colleagues in our laboratory who assisted in the analysis of biopsy specimens. references 1. jemal a, bray f, center m. global cancer statistic. ca cancer j clin 2011;61:69-90. 2. abdullah m. tumor kolorektal: distribusi kanker kolorektal menurut lokasi di kolon. in: sudoyo 171 univ med vol. 32 no.3 aw, setiyohadi b, alwi i, marcellus sk, setiati s, editors. buku ajar ilmu penyakit dalam. 5th ed. jakarta: pusat penerbitan ilmu penyakit dalam fakultas kedokteran indonesia; 2009. p. 567-75. 3. abdullah m, sudoyo aw, utomo ar, fauzy a, rani aa. molecular profile of colorectal cancer in indonesia: is there another pathway gastroenterol hepatol bed bench 2012;5:71-8. 4. effendi r, efendi d, dairy lb, sembiring j, sihombing m, marpaung b, et al. profile of colorectal cancer patients in endoscopic unit at pirngadi hospital medan. indonesian j gastroenterol hepatol dig endosc 2008;9:758. 5. chen cc, yang sh, lin jk, lin tc, chen ws, jiang jk, et al. is it reasonable to add preoperative serum level of cea and ca 19-9 to staging for colorectal cancer? j surg res 2005; 124:169-74. 6. locker gy, hamilton s, harris j, jessup jm, kemeny n, macdonald js, et al. asco 2006 update of recommendation for use of tumor markers in gastrointestinal cancer. j clin oncol 2006;24:5313-27. 7. weissenberger c, von plehn g, otto f, barke a, momm f, geissler m. adjuvant radio chemotherapy of stage ii and iii rectal adenocarcinoma: role of cea and ca 19-9. anticancer res 2005;25:1787-94. 8. yang xq, chen c, wang fb, peng cw, li y. preoperative serum carcinoembryonic antigen, carbohydrate antigen 19-9 and carbohydrate antigen 125 as prognostic factors for recurrencefree survival in colorectal cancer. asian pacific j cancer prev 2011;12:1251-6. 9. casciato da. manual of clinical oncology. 7th ed. philadelphia: lippincott williams & wilkins; 2012. 10. stewart sl, wike jm, kato i, lewis dr, michaud f. a population based study of colorectal cancer histology in the united stated 1998-2001. cancer 2006;107:1128-41. 11. sudoyo aw, hernowo b, krisnuhoni e, reksodiputro ah, hardjodisastro d, sinuraya es. colorectal cancer among young native indonesians: a clinicopathological and molecular assessment on microsatellite instability. med j indonesia 2010;19:245-51. 12. capple ms. the pathophysiology, clinical presentation, and diagnosis of colon cancer and adenomatous polyps. med clin north am 2005; 89:1-42. 13. duffy mj. carcinoembryonic antigen as a marker for colorectal cancer: is it clinically useful? clin chem 2001;47:624-30. 14. morita s, nomura t, fukushima y, morimoto t. hiraoka n, shibata n. does serum ca19-9 play a practical role in the management of patients with colorectal cancer. dis colon rectum 2004;47:227-32. doi: 10.1007/s1035000 3-0041-6. 15. compton c, fenoglio-preiser cm, petigrew n, fielding cp. american joint committee on cancer prognostic factors consensus conference: colorectal working group. cancer 2000;88:173957. 16. cerna m, holubec l, pesta m, kormunda s, topolcan o, cerny r. quantitative estimation of cea and ck20 expression in tumour tissue of colorectal cancer and its liver metastases with reverse transcription and real-time pcr. anticancer res 2006;26:803-8. 17. duffy mj, van dalen a, haglund c, hansson l, klapdor r, lamerz r, et al. clinical utility ofbiochemical markers in colorectal cancer: european group on tumour markers (egtm) guidelines. eur j cancer 2003;39:718-27. c:\users\jurnal fkusakti\docume 172 *department of chemical pathology, edo university, iyamho, edo state, nigeria **department of haematology and blood transfusion and acting dean, faculty of clinical sciences, edo university, iyamho, edo state, nigeria ***department of chemical pathology, university of benin teaching hospital, benin city, edo state, nigeria †department of chemical pathology and immunology, faculty of basic medical sciences, university of ilorin, ilorin, nigeria correspondence augustine onovuakpo eguvbe department of chemical pathology, edo university, iyamho, edo state, nigeria. e-mail: austinoforlife@yahoo.com orcid id: https://orcid.org/ 00000002-2722-2186 date of first submission, june 22, 2019 date of final revised submission, november 16, 2019 date of acceptance, november 19, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina the role of urine albumin creatinine ratio and serum β2 microglobulin as biomarkers of chronic kidney disease augustine onovuakpo eguvbe*@, marcellinus uchechukwu nwagu**, eshiotseme sylvester idogun***, and adeyinka abdulrasaq akande† background chronic kidney disease (ckd) is an increasing burden on individuals and on the healthcare system. the need to identify more sensitive and specific markers of ckd cannot be overemphasized to facilitate detection and appropriate intervention. β2 microglobulin is one of such markers of ckd. the aim of this study was to investigate the sensitivities and specificities of serum β2 microglobulin and major biochemical markers of ckd, namely creatinine and urine albumin. methods this was a hospital-based cross-sectional study involving 124 subjects with ckd and 124 healthy controls. participants were categorized in two groups : group 1 the ckd based on persistent reduction in gfr <60 ml/ min/1.73 m2 and group 2 healthy subjects as controls. blood (serum) samples of participants were analyzed for serum creatinine and serum β2 microglobulin while their urine samples were analyzed for creatinine and albumin. urine albumin creatinine ratio (uacr) was calculated from the results of the analyses. results there was a very strong positive correlation of serum β2 microglobulin with serum creatinine (r=0.750; p=0.000) and uacr (r=0.775; p=0.000), respectively. also, there was a very strong negative correlation between serum β2 microglobulin and egfr (r=-0.866; p=0.000). uacr had the highest sensitivity and specificity as shown by receiver operating curve characteristics (roc) analysis. conclusion in ckd, uacr and serum β2 microglobulin had the best diagnostic value. periodic renal assessment of renal patients is mandatory as they may be affected by hidden renal dysfunction. keywords: β2 microglobulin, serum creatinine, urine albumin creatinine ratio, chronic kidney disease original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.172-178 september-december, 2019 vol.38no.3 cite this article as: eguvbe ao, nwagu mu, idogun es, akande aa, et al. the role of urine albumin creatinine ratio and serum â2 microglobulin as biomarkers of chronic kidney disease. univmed 201 9;38 :1 7 2-8 . doi: 10. 1805 1/ univmed.2019.v38. 172-178 173 univ med vol. 38 no.3 introduction chronic kidney disease (ck d) is an increasing burden on individuals and on the healthcare system, with an increasing prevalence and incidence. it is linked with a poor outcome and high cost of treatment.(1) chronic kidney disease is defined as “an abnormality of kidney structure or function that has been present for three months or more and that has implications for health”. the diagnostic criteria for ckd include a gfr of <60 ml/min/1.73 m2, the presence of kidney damage (proteinur ia, albuminuria, urinary sediment abnormalities, pathological imaging abnormalities, genetic disorders, or a history of renal transplantation).(2) the prevalence of ckd in nigeria varies between 7.8% and 18.8%. hospital based prevalence is 8–10%. ckd stages 1, 2, 3, 4 and 5 accounted for 2.4%, 0.6%, 3.0%, 1.2% and 0.3% of participants respectively.(3-6) mortality r ate of ckd in nigeria varies between 40–50%.(7) ckd was documented to be the 18th highest cause of death worldwide in 2010.(8) β2 microglobulin , an 11.8-kda protein, is the light chain of the major histocompatibility complex class i (mhc i) molecule that is present on the cell surface of all nucleated cells. it is freely filtered by the glomeruli and reabsorbed and broken down by proximal tubular cells.(9) it is not affected by age, gender, or muscle mass. however, it is influenced by infectious or inflammatory process, proliferative syndromes, and hepa tic and autoimmune illnesses.(10,11) in a study assessing the biomarker value of serum β2 microglobulin, cystatin c, and lipocalin-2 in detecting renal impairment in fabry disease, serum β2 microglobulin was found to be the best biomarker.(12) lack of further studies in the last decade however has limited the utility of b2m in clinical practice. cr e a ti n ine i s a 11 3 da ami no a c i d derivative, is easily filtered by the glomerulus, and is the most commonly used for assessing gfr. also, it is produced at an approximately constant rate from muscle creatine. however, it is secreted at variable rates by the proximal tubule. consequently, creatinine clearance exceeds the gfr.(13,9) microalbuminuria is the main laboratory indicator for kidney structure damage.(14,15) it was initially defined as the presence of small quantities of albumin in urine, too little to be detected by standard “dip stick” methods.(16) it is now defined as “urinary albumin excretion between 30 and 300 mg/day, if measured in a 24 hour urine collection, 20–200 ug/min, if measured in a timed urine collection or 30–300 mg/g, if measured with the use of urinary albumin to creatinine ratio in a spot urine collection”.(17) furthermore, early diagnosis of ckd can provide an opportunity to modify some of the risk factors and reduce the rate of progression to end stage renal disease. this study was to investigate the diagnostic value of serum b2m and major biochemical markers of ckd such as serum creatinine and urine albumin. methods research design this was a hospital-based cross-sectional study carried out between april and june 2016 at the university of benin teaching hospital. research subjects participants diagnosed with ckd based on persistent reduction in gfr <60 ml/min/1.73 m2, albuminuria and abnormalities based on histology or imaging studies, at the renal unit of the university of benin teaching hospital, were included in the study. consecutive sampling was used to recruit participants. the number of subjects in each group required to complete the study with α= 0.05 and β=0.20, and standardized effect size of 0.40 and to account for dropouts was 124 subjects per group.(18) therefore, the study comprised 124 participants with ckd and 124 healthy controls. the exclusion criteria for t hi s s t u dy we r e c h r o n ic h e mo d i a l ys i s, lymphoproliferative and myeloproliferative 174 disorders, glucocorticoid therapy, inflammatory conditions and patients who have had kidney transplant. written informed consent was obtained from each study participant. the study used a structured interviewer-administered questionnaire, which included identification number, age, gender, weight, height, medical history and laboratory results sampling blood sample about five to ten milliliters (5–10 ml) of blood was c ollected from each pa rticipant by venipuncture into a plain bottle and allowed to clot. the specimen was centrifuged at 3000 rpm. the serum was separated from the cells using a pasteur pipette and stored at -200c until analysis. am and pm temperature monitoring was used to monitor the temperature of the freezer. serum level of â2 microglobulin was analyzed by enzyme linked immunosorbe nt assay (elisa), according to the manufacturer’s protocol, while serum creatinine and urine creatinine were analyzed on a spectrophotometer using the kinetic modification of the jaffe procedure.(19) urine sample about ten milliliters of random urine was collected into plain bottles for one week for batching at 2 to 8 until analysis for urine albumin and creatinine. urine albumin was analyzed on a spectrophotometer using the immunoturbidimetric assay.(20) urine albumin creatinine ratio (uacr) was calculated by dividing urine albumin concentration in milligrams by urine creatinine concentration in grams. statistical analysis data was analyzed with spss version 16. results were expressed as proportions, mean and standard deviation. student’s independent t-test was used to compare numerical variables while chi-square and fisher’s tests were used to compare qualitative variables. pearson correlation coefficient was used to correlate serum β2 microglobulin with other markers of r e n a l d i se a se . a r e c e i ve r op e r a t in g characteristic (roc) curve with a 95% ci was used to determine the sensitivity and specificity of the renal markers. the p value was set at 0.05. ethical clearance this study was approved by the ethics and research committee (protocol number: adm/e 22/a/vol. vii/11200) of the university of benin teaching hospital, benin city. results a total of 248 participants enlisted for this study consisting of 124 participants with ckd and 124 healthy controls. the mean and standard deviation of serum β2 microglobulin, serum creatinine and uacr are contained in table i. mean serum creatinine was significantly higher in ckd participants than in controls (147.67 ± 81.38 µmol/l versus 96.45 ± 12.78 µmol/l; p<0.001). similarly, β2 microglobulin was higher in ckd participants than in controls (4.51 ± 2.74 mg/l versus 1.77 ± 0.86 mg/l; p<0.001) (table 1) . ur in e al bu mi n an d ua cr wer e al so significantly higher among participants with ckd than in controls. serum β2 microglobulin had a very strong ne gati ve c orr elat ion wit h eg fr in ckd participants (r=-0.866; p<0.001). conversely, serum creatinine had a very strong positive correlation with β2 microglobulin in participants with ckd (r=0.758; p<0.001). uacr had a ve r y s tr on g posit ive cor rel ation w ith β2 microglobulin in ckd participants (r=0.775; p<0.001) (table 2). figure 1 and table 3 show the performance of various markers. the sensitivities of uacr, β2 microglobulin and serum creatinine in detecting ckd were 98.6%, 95.7% and 87.8% respectively while their specificities were 94.4%, 79.0% and 13.7% respectively. this is based on the following cut-offs: an egfr of 0.87 ml/s/ m2; uacr of 3.4 mg/mmol; β2 microglobulin of 2 mg/l; serum creatinine of 115 µmol/l. eguvbe, nwagu, idogun, et al biomarkers of chronic kidney disease 175 univ med vol. 38 no.3 discussion in t h e pr e s e nt s t ud y, t h e s e r u m concentrations of β2 microglobulin, creatinine and uacr wer e higher in ck d patients compared to controls, implying that they can all s er ve a s ma r ke r s o f r e n al in su ff i ci e n cy. however, their sensitivities differ, which is the fo cu s of th is study. t he re as ons f or t he differences in their sensitivities may be attributed t o th e f ac t t ha t i n c r e a si n g l e ve l s of microalbuminuria has been associated with an increased cardiovascular mortality in subjects with normal renal function.(16) β2 microglobulin maybe a better marker of ckd than serum creatinine because of its low molecular weight, constant production by all nucleated cells and a b c its independence of age, gender or muscle mass.(21) β2 microglobulin, a low molecular weight protein, has been assessed as a potential marker of ckd. (9) in the present study, mean serum levels of β2 microglobulin were significantly higher in patients with ckd than in the control group and its level progressively increased with decreasing gfr. many studies have confirmed the relationship between β2 microglobulin and egfr. in a study involving forty biomarkers for the prediction of rapid decline in renal function in type 2 diabetes, β2 microglobulin and kidney injury molecule 1 showed the most consistent effects.(22) t he p r e s e n t st u dy sh o we d a st r on g association between serum β2 microglobulin and serum creatinine in both ckd participants and healthy controls. β2 microglobulin also had a strong positive association with uacr and a strong negative association with egfr. from these results, it is obvious that serum creatinine is a less sensitive marker of renal injury. the results also show that β2 microglobulin is a more sensitive marker of ckd. these findings are supported by a cross-sectional study on the correlation of β2 microglobulin with serum variable ckd (n=124) controls (n=124) p value age (years) <30 30 – 39 40 – 49 50 – 59 ≥60 gender male female serum cr* (µmol/l) 3 (2.7) 34 (27.4) 34 (27.4) 23 (18.9) 30 (24.3) 54 (43.2) 70 (56.8) 147.67 ± 81.38 17 (13.7) 36 (29.0) 33 (26.6) 19 (15.3) 19 (15.3) 63 (50.8) 61 (49.2) 96.45 ±12.78 0.762 0.303 0.000 β2 microglobulin (mg/l) 4.51 ± 2.74 1.77 ± 0.86 0.000 urinary cr (µmol/l) 5244.00±1126.56 9768.70±1286.56 0.018 urine albumin(mg/l) 67.00 ± 40.01 0.75 ± 0.20 0.000 uacr* (mg/mmol) 13.31 ± 8.04 0.08 ± 0.03 0.000 egfr (ml/s/m2) 0.53 ± 0.19 0.77 ± 0.02 0.000 *data presented as mean ±sd, except for age and gender (n,%); abbreviations: sd: standard deviation, cr: creatinine; uacr: urinary albumin creatinine ratio; egfr: estimated glomerular filtration rate table 1. comparison of demographic and biochemical parameters between the ckd and control groups variable β 2 microglobulin r p value serum cr (µmol/l) 0.758* 0.000 uacr (mg/mmol 0.775* 0.000 egfr (ml/s/m2) -0.866† 0.000 table 2. correlation of serum β2 microglobulin with other biochemical markers *strong positive correlation, †strong negative correlation; abbreviations: cr: creatinine; uacr: urinary albumin creatinine ratio; egfr: estimated glomerular filtration rate 176 creatinine and creatinine clearance in patients with different levels of renal function, which reported that β2 microglobulin was a more sensitive and accura te biomarker for the assessment of renal function as compared to creatinine.(23) furthermore, a study conducted among sickle cell disease patients reported that β2 microglobulin had a higher sensitivity and specificity than serum creatinine.(24) similarly, in the present study, β2 microglobulin had a higher sensitivity and specificity than serum creatinine. the results of the roc analysis revealed that serum β2 microglobulin had a better diagnostic accuracy than serum creatinine. the auc for serum β2 microglobulin was also higher than that of serum creatinine. it h a s be e n we ll d oc ume nte d t h a t albuminuria is a sensitive marker of ckd caused by diabetes mellitus, hypertension and glomerular diseases.(25) in such cases, the first sign of glomerular disease is increased urinary excretion of albumin.(26) the results of the present study confirm the reports that urinary albumin is a sensitive marker of ckd. the results show that diagonal segments indicate ties figure 1. receiver operator curve comparing uacr, β2 microglobulin and serum creatinine, based on egfr as markers of ckd auc* se† p value 95% ci sensitivity specificity uacr 0.996 0.003 0.000 0.991 – 1.001 98.6% 94.4% β2m 0.884 0.027 0.000 0.830 – 0.937 95.9% 79.0% cr 0.809 0.036 0.000 0.739 – 0.879 87.8% 13.7% egfr 0.171 0.031 0.000 0.109 – 0.232 1.4% 83.1% table 3. diagnostic accuracy of creatinine, β2 microglobulin, uacr, and egfr as diagnostic tools for chronic kidney disease *area under the curve, †standard error of the mean; abbreviations: cr: creatinine; uacr: urinary albumin creatinine ratio; β2m: β2-microglobulin; egfr: estimated glomerular filtration rate eguvbe, nwagu, idogun, et al biomarkers of chronic kidney disease 177 univ med vol. 38 no.3 mean uacr was much higher in ckd than in controls. also, the uacr was elevated in all the stages of ckd. in addition, the roc plots confirmed that the uacr was the most sensitive marker of ckd. the present study also reported that the uacr was signif icantly higher in patients with advanced ckd compared to those with early disease. in a cross-sectional study of north american children with ckd, it was shown that the level of proteinuria tended to be higher as the level of iohexol gfr decreased irrespective o f t he c a u se of ck d. ( 2 7 ) a si gni f i c a nt relationship between uacr and end stage renal disease was reported in a cohort study. in the latter, higher uacr and lower egfr were associated with increased risk of end stage renal disease.(28) furthermore, in a collaborative meta-analysis of egfr and albuminuria on outcome of ckd, it was reported that higher albuminuria was associated with higher risk of end stage renal disease, independent of lower egfr and of traditional cardiovascular disease risk factors.(29) although serum β2 microglobulin is not as good as uacr as a biomarker of ckd, its clinical value is justified because of its analytical r e l i a b i l ity. m e a s ur e me nt o f s e r u m β 2 microglobulin along with serum creatinine for renal evaluation should be considered. one limitation of this study was that elevation of serum β2 microglobulin can occur in certain conditions, which may limit its use as a biomarker of ckd. conclusion in ckd, uacr had the best diagnostic value, followed by serum β2 microglobulin and serum creatinine. periodic renal assessment of renal patients are mandatory as they may be affected by hidden renal dysfunction. conflict of interest the authors declare that they have no conflict of interest concerning this article. acknowledgement the authors thank the staff of the renal unit of university of benin teaching hospital. contributors ao e a nd m un ma de s igni f i c a nt c o nt r ib u ti o ns to c o n c e p t io n, d e s i gn , experimentation, acquisition and interpretation of data and writing of manuscript. esi and aaa made substantial contribution in interpretation of data and revising the manuscript for intellectual content. all authors read and approved the final manuscript. references 1. national kidney foundation. guidelines and commentaries. new york: national kidney foundation; 2019. 2. lamb ej, levey as, stevens pe. the kidney disease improving global outcome (kdigo) guideline update for ckd: evolution not revolution. clin chem 2013;59:462-5. doi: 10.1373/clinchem.2012.184259. 3. ulasi ii, ijoma ck. the enormity of ckd in nigeria: the situation in a teaching hospital in south-east nigeria. j trop med 2010; article id 501957, 6 pages. doi:10.1155/2010/501957 501957. 4. egbi og, okafor uh, miebodei ke, et al. prevalence and correlates of ckd among civil servants in bayelsa state, nigeria. niger j clin pract 2014;17:602-7. doi: 10.4103/11193077.141426. 5. oluyombo r, ayodele oe, akinwusi po, et al. a community study of the prevalence, risk factors and pattern of ckd in osun state, south-west nigeria. west afr j med 2013;32:8592. doi: 10.4103/1119-3077.175960. 6. okwuonu cg, chukwuonye ii, adejumo oa, et al. prevalence of ckd and its risk factors among adults in a semi-urban community of south-east nigeria. niger postgrad med j 2017;24:81-7. doi: 10.4103/npmj.npmj_34_17. 7. odubanjo mo, oluwasola ao, kadiri s. the epidemiology of end stage renal disease in nigeria: the way forward. int urol nephrol 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of renal function. gomal j med sci 2011;9:178-82. 24. el-gamasy ma, el-naghy ws. early predictors of renal dysfunction in paediatric patients with sickle cell disease. indian j nephrol 2019;29:2833. doi: 10.4103/ijn.ijn_40_18. 25. baumgarten m, gehr t. ckd: detection and evaluation. am fam physician 2011;84:1138-48. 26. narva as, bilous rw. laboratory assessment of diabetic kidney disease. diabetes spectr 2015;28:162–6. doi: 10.2337/diaspect.28.3.162. 27. wong cs, pierce cb, cole sr, et al. association of proteinuria with race, cause of chronic kidney disease and glomerular filtration rate in the chronic disease in children study. clin j am soc nephrol. 2009;4:812-9. doi: 10.2215/cjn.01780408. 28. nitsch 1, grams m, sang y, et al. for chronic kidney disease prognosis consortium. association of egfr and albuminuria with mortality and renal failure by sex: a meta-analysis. bmj 2013;346:f324. doi: 10.1136/bmj.f324. 29. gansevoort rt, matsushita k, van der velde m, et al. for chronic kidney disease prognosis consortium. lower egfr and higher albuminuria are associated with adverse kidney outcome. kidney int 2011;80:93-104. doi: 10.1038/ ki.2010.531. eguvbe, nwagu, idogun, et al biomarkers of chronic kidney disease c:\users\jurnal fkusakti\docume 202 *department of occupational medicine, medical faculty, trisakti university **department of clinical pathology, trisakti university ***department of pharmacy, trisakti university †department of public health, medical faculty, trisakti university ‡center of education and training, ministry of industry indonesia republic of indonesia correspondence: lie t merijanti s, dr. mkk department of occupational medicine, faculty of medicine, trisakti university jl. kyai tapa no 260 grogol, jakarta 11440 phone: 021-5672731 ext 2802 email: liemerijanti@trisakti.ac.id orcid id : 0000-0001-8203-0753 date of first submission, september 18, 2 01 9 date of final revised submission, december 18, 2019 date of acceptance, december 19, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina heavy mental workload increase poor sleep quality in informal garment workers lie t. merijanti s*, pusparini**, meiyanti***, alvina**, novia i sudharma†, and muljadi tjahjadi‡ background sleep is needed by the human body so that the performance of body remains optimal when the body is awake, especially during work. informal workers often work without clear regulations, with inadequate equipment and poor work environment, and pay little attention to occupational health and safety factors. the existence of large work demands with unclear work system arrangements will certainly result in a mental workload in these workers. the objective of this study was to determine the relationship between mental workload and sleep patterns of workers in the informal garment sector. methods a cross-sectional study involving 225 informal garment workers was conducted between december 2018 and may 2019. data collection included respondents’ demographics, job characteristics, measurement of mental burden and disturbances in sleep patterns. the sleep pattern used the psqi (pittsburgh sleep quality index) instrument and the mental burden used the rating scale mental effort (rsme). a multiple linear regression analysis was used to analyze the data. results the mean sleep quality was 5.40 ± 2.54. multiple linear regression test found that mental workload correlated significantly with sleep quality of workers (=0.016, p=0.012). conclusion this study demonstrated that heavy mental workload decreases the quality of sleep in informal garment workers. a good work system management is required so that workers are assigned that portion of the task that is commensurate with their capacity. keywords: mental workload, informal garment workers, sleep quality original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.202-208 september-december, 2019 vol.38no.3 ci te t his arti cle as: mer ijan ti lt, pusparini, meiyanti, alvina, sudharma ni, tjahjadi m. heavy mental workload increase poor sleep in informal garment workers. univmed 2019;38:202-8. doi: 10.18051/univmed.2019.v38.202-208 203 univ med vol. 38 no.3 introduction sleep is needed by the human body so that the performance of the body remains optimal when awake, especially during work. during sleep, the body will form and regenerate its cells, support brain function, and replenish bodily energy. sleep quality is an important clinical construct for being healthy. poor sleep quality can be an important symptom of many sleep disorders and other medical diseases and may even directly lead to increased mortality.(1) workers with a sleep duration of less than five hours will experience more stress than do those with an adequate sleep duration. there is a greater awareness of high stress in younger workers, specialized workers, office workers and those with mid-high household income.(2) the centers for disease control and prevention, after an analysis of the data from the behavioral risk factor surveillance system (brfss) for the years 2013 and 2014 in 29 states of the usa, found that the highest prevalence of inadequate sleep duration was in production workers and health personnel.(3) sleep disorders cause the loss of 1.2 million workdays, so that it is estimated that for each worker who has less than six hours of sleep, a total of 226 us dollars must be added to the us economic budget.(4) sleep deprivation and drowsiness are the workers’ main health problems that may result in increased morbidity, work-related errors, oc cupational accidents, absenteeism, and decreased work productivity.(1,2) the precipitating factors of sleep disorders may arise from the work environment, such as the shift work system, work requirements or workloads, and also from individual factors, such as age, gender, life style, current illness, inadequate resting time on workdays, and family and social environment, such as marital status and activities of daily living.(5,6) in the modern worker ’s life, there is increased work insecurity and mental workload. mental workload is a set of factors that affect the mental processing of information, providing decision making and individual reactions in the workplace.(7) a study involving 656 us students showed that a higher mental workload is associated with lower overall sleep quality.(8) a study on 243 healthcare office workers indicated that mental workload is significantly associated with sleep quality.(9) a japanese study on 5951 male and 1500 f emale government office workers found that the gender difference in insomnia depended on the type of work and the f a mi l y c ha r a c t e r is t i c s o f th e wo r ke r s themselves, where significant gender differences in insomnia were not found among shift workers, subjects engaged in 6 h per day or more of vdt work, those with high job strain, those who did household tasks for 1 h per day or more, and those living with persons who needed care and support.(10) in indonesia, a study on 259 firemen found 111 persons (42.9%) with insomnia associated with excessive qualitative and quantitative workloads and residential areas near sources of noise.(11) heavy workloads, in addition to a delay of sleep onset, also cause a homeostatic sleep response by increasing subjective fatigue and drowsiness, and reducing the time of awakening after sleep.(12) individuals with a constant heavy workload will have a poorer sleep quality than worke rs with a low/ moderate workload. however, the sleep quality does not decrease and work-related perseverative cognition does not increase over time for individuals experiencing stable high job demands.(13) according to the indonesian central bureau of statistics, there are in total 73.98 million (58.22%) informal workers and 41.78 million formal workers. of 127.07 million working persons, 7.64% are c ategorized a s semiunemployed and 23. 83% are part-time worke rs. (1 4) previous studies have more frequently focused on formal sector workers, and there is a lack of information on studies involving informal workers. the present study aimed to determine the relationship between mental workload on sleep quality in informal garment workers. 204 methods design of the study this observational cross-sectional study was conducted in one village (kelurahan) of west jakarta, from december 2018 to may 2019. study subjects the study subjects were informal garment workers from one kelurahan of west jakarta who met the inclusion criteria and agreed to participate in the study. the exclusion criteria wer e: per sons with aff ective disorders (depression), anxiety, or chronic disease and those consuming antidepressants, narcotics, diuretics, beta blockers, or caffeine. the required sample size was calculated using the formula for the sample size of a cross-sectional study, with α of 0.05, prevalence of poor sleep quality of 42.9% (15) and anticipated drop-out of 10%, yielding a total of 225 workers. the subjects were selected by consecutive sampling and all subjects signed an informed consent form. data collection da ta we r e c ol l ec t e d u si n g t h r e e questionnaires as assessment instruments, n a me l y a de mo gr a ph i c c ha r a ct e r i s t i c s questionnaire, the rating scale mental effort (rsme) questionnaire for mental workload, and the pittsburgh sleep quality index (psqi) questionnaire for sleep quality. the demographic questionnaire comprised questions such as gender, age, education, type of employment, work duration (work period), length of employment, resting time and work wages. the pittsburgh sleep quality index (psqi), developed in 1989 by buysse and coworkers,(16) has seven domains for evaluating sleep quality or sleep degradation during one month. the domains are subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleep medications and daytime dysfunction. the total psqi score has a value of 0 to 21, being the sum of the point values for the seven domains, each question yielding point values of 0 to 3. high scores indicate bad sleep quality and low scores indicate adequate sleep quality. a person with a total score of more than 5 has a clinically poor sleep quality. the psqi has been used successfully in a recent study.(17) the mental wo r kl oa d i s e va l ua t e d s u bj ec t i ve l y b y constructing a psychometric scale to measure the mental workload based on the worker’s or respondent’s subjective perception, using the rating scale mental effort (rsme) instrument, in which the respondent is asked to evaluate his or her effort to complete the assigned work on a scale of 0 -150, the score of 0 indicating no effort whatsoever and the score of 150 indicating extreme effort.(18) statistical analysis the data were entered, coded, processed and analyzed using spss version 21. the results of the test on normality of the data showed that the collected data were normally distributed. for the categorical variables, the frequency and percentage was calculated, whereas continuous variables were summarized using the mean and standard deviation and analyzed by multiple linear regression. a p value of <0.05 was considered statistically significant. ethical clearance this study received ethical clearance from the commission on research ethics, faculty of medicine, trisakti university, under no. 139/ ker/fk. results the respondents were informal garment workers, comprising 136 males (60.4%) with mean age of 33.31 ± 11.14 years. the education of the workers was predominantly elementary school (48.4%) and junior high school (40%), with mean rsme of 79.64 ± 27.55, and mean psqi of 79.64 ± 27.55 (table 1). table 2 above of the results of the multiple linear regression analysis shows that mental merijanti lt, pusparini, meiyanti, et al mental workload and sleep quality 205 univ med vol. 38 no.3 workload is significantly associated with sleep quality, but gender and age are not significantly associated (β=0.016; p=0.012). discussion our study results showed that heavy mental workload reduced sleep quality in informal garment workers. psychological occupational hazards such as long work duration, heavy mental workload, inadequate work control, inadequate wages, discomfort during work and problems in regulation of work organization, are associated with increased sleep disorders in workers throughout the world, potentially causing various cardiovascular, cerebrovascular and mental health disorders among the workers.(19) heavy mental workloads and long work duration interferes with health and causes the workers to leave the workplace.(20) a b c according to the present study there was a positive correlation between performance/work results and drowsiness, so that this should become a matter for consideration of the work assignment to be given, and the specific capacity of workers in completing the work, because of its individual nature. if the workers are incapable of completing the assignment then there will be a cumulative physical work fatigue in the form of reduced capacity of the muscular system and mental fatigue in the form of reduced alertness.(21) in variable n (%) gender male 136 (60.4%) female 89 (39.6%) age (years) 33.31 ± 11.14 education elementary school 109 (48.4%) junior high school senior high school 90 (40%) 26 (11.6%) work unit manual sewing 137(60.9%) cutting 6 (2.7%) piping overlock machine sewing 22 (9.8%) 3 (1.3%) thread trimming 32 (14.2%) ironing 7 (3.1%) finishing 18 ( 8%) length of employment (years) 8.047 ± 7.74 work duration per working day (hours) 11.413 ± 1.99 resting time during work (hours) 1.18 ± 0.44 wages rp 200.000 – 500.000/week 79 (35.1%) rp 600.000 – 1.000.000/week 146 (64.9%) occupational accident ever never 143 (63.6%) 82 (36.4%) rsme 79.64 ± 27.55 psqi 5.40 ± 2.54 table 1. distribution of demographics, work characteristics, rsme, and psqi of the respondents (n=225) data presented as n(%) except for age, length of employment, work duration per day, rsme and psqi (as mean ±sd) respondent’s characteristic β p gender -0.268 0.477 age -0.004 0.839 work duration 0.085 0.350 length of employment -0.006 0.829 resting time -0.094 0.809 rsme 0.016 0.012* occupational accident 0.169 0.644 table 2. multiple linear regression between occupational variables and sleep quality 206 the present study it can be seen that with the large reported number of cases of occupational accident, where 143 workers (63.6%) ever had an occupational accident, but the results of the analysis did not show a significant association with the sleep quality of the workers. the majority of the workers had extremely long working hours of more than 8 hours per day, so that they worked until late in the night, which if occurring continually, will interfere with the body’s circadian rhythm, such as disturbing the sleep pattern. continual physical activity during work causes musculoskeletal pain and interferes with sleep.(22) a different study showed that older workers do not have sleep problems with the passage of time, in contrast with younger workers. aging has a positive effect on the occurrence of fatigue and sleep, both of which are related, where the need for sleep decreases with age.(23,24) fr o m th e r e s u l t s o f mul t i pl e l i n e a r regression analysis, the characteristic of the respondents, in this case gender in relation to sleep quality, turned out to be not significantly associated. similar results were found in a japanese study, where the effect of gender on t he o cc ur re nc e of s le ep dis or de r is a ls o influenced by the shift work system, computerrelated work, household work, and care for the family, that must be performed more by women, in comparison with the biological differences between males and females.(10) another study obtained different results, where women showed decreased sleep quality, possibly caused by high sensitivity to psychosocial stress and changes in the body’s circadian rhythm.(24) a study on hospital nurses in china found that sleep disorder is statistically associated with gender, age, work duration, and night shift.(9) the high degree of stress will decrease sleep quality due to increased sleep disturbance and interference with activities of daily living.(25) the role of polymorphism of the receptor gene 5-htr2a is also associated an individual’s sleep quality, and will add to the cumulative effect on sleep quality. (26) work load increases the risk of poor sleep quality,(9) and its effect will be wo r s e i n wo r ke r s w it h a sp ec i f i c ge n e polymor phism. (23 ) low sleep quality was significantly associated with lower morning cortisol secretion. while job strain had no main effects on the cortisol reactivity there was a significant interaction effect between the input variables on morning cortisol secretion. these findings tentatively support the hypothesis that lack of sleep for workers with high job strain may result in a flattened diurnal cortisol reactivity.(27) however, other studies state that sleep quality does not decrease and work-related perseverative cognition did not increase over time for individuals experiencing stable high job d e ma nd s . ( 1 3 ) t he swe di s h lo n gi t ud i n a l occupational survey of health states that the findings of this cross-lagged analysis indicate a we a k e f f ec t of de ma n d s on la t e r sl e e p disturbances, an effect of sleep disturbances on later ratings on social support, and reciprocal relationship between awakening problems and work characteristics, they suggests that there are reverse and reciprocal causal relationships, in addition to associations in the usually h yp ot h e s ize d d i r e c t i o n, be tw e e n w or k characteristics and sleep problems based on a 2-year time lag.(27) a decrease of 25% of weekly work duration without reducing work pay, had positive benefits on increase in sleep duration, reduced drowsiness and incidence of stress during work days and holidays.(28) the work condition should be improved and mental workloads should be adjusted so as to not disrupt their sleep status.(6) this study has several limitations, firstly, the cross-sectional design of the study did little to describe the cause-and-effect relationship between mental workload and sleep disorder. secondly, the measurement of mental workload and sleep quality was subjective, although rsme is a good index to evaluate the mental workload because of its acceptable correlation with another such as nasa tlx (task load index).(29) psqi reflects mainly sleep quality on workdays.(30) further studies using objective merijanti lt, pusparini, meiyanti, et al mental workload and sleep quality 207 univ med vol. 38 no.3 measurements of sleep quality could be a next step to understand how these relationships are intertwined. additionally, studies on sleep disorders of workers that investigate how sleep quality differs on workdays when compared with work-free days might help us to understand how these patterns might be clinically relevant. finally, this understanding might foster purposeful solutions to improve sleep quality and health. conclusions th is s tudy f ound t ha t he avy me ntal workload increases poor sleep quality in informal garment workers. to improve the health and pr oductivity of the workers, requires the education and the realization of both the workers and the proprietors in the informal sector about adequate sleep for the workers. conflict of interest there was no conflict of interest between both the investigators and the respondents in the conduct of this study. acknowledgments the investigators express their utmost gratitude to all parties who provided aid and assistance in the course of this study, to the trisakti university as funding provider, to the p r o pr i e t o rs o f h ome i nd us t r ie s a nd a l l r e s p on d e nts wh o p a r t i c i p a t e d u p to t he completion of this study. contributors ltm contributed to the study design. ltm and p contributed to the construction of the questionnaire. ltm, p, m, a,n, mt contributed to recruitment of the subject sample and data collection. ltm and n performed analysis and data analysis. ltm and mt contributed to writing of the manuscript of this study. all authors read and approved the final manuscript. references 1. yazdi z, sadeghniiat-haghighi k, loukzadeh z, elmizadeh k, abbasi m. prevalence of sleep disorders and their impacts on occupational performance: a comparison between shift workers and non-shift workers. sleep disorders 2014:1-5. http://dx.doi.org/10.1155/2014/870320. 2. choi dw, chun sy, lee sa, han kt, park ec. association between sleep duration and perceived stress: salaried worker in circumstances of high workload. int j environ res public health 2018;15,796:1-11 doi:10.3390/ijerph15040796. 3. shockey tm, wheaton ag. short sleep duration by occupation group 29 states, 2013-2014. us department of health and human service/centers for disease control and prevention. mmwr 2017;66:207-13. 4. hafner m, stepanek m, taylor j, troxel wm, van stolket c. why sleep matters the economic costs of insufficient sleep: a cross-country comparative analysis. santa monica, ca: rand corporation; 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doi:10.3390/ijerph 10115863. 27. hanson llm, leineweber c, persson v, hyde m, heorell t, westerlund h. cohort profile: the swedish longitudinal occupational survey of health (slosh). int j epidemiol 2018;691–2i.doi: 10.1093/ije/dyx260. 28. schiller h, lekander m, rajaleid k, et al. the impact of reduced worktime on sleep and perceived stress-a group randomized intervention study using diary data. scand j work environ health 2017;43:109-16. doi: 10.5271/sjweh.3610. 29. ghanbary sa, ashnagar m, habibi e, sadeghi s. evaluation of rating scale mental effort (rsme) effectiveness for mental workload assessment in nurses. johe 2016;5:211-7. doi:10.18869/ acadpub.johe.5.4.211. 30. pilz lk, keller lk, lenssen d, roenneberg t. time to rethink sleep quality: psqi scores reflect sleep quality on workdays. sleep j 2018;41:1–8. doi: 10.1093/sleep/zsy029. merijanti lt, pusparini, meiyanti, et al mental workload and sleep quality renal cortex echogenicity increases degree of 19 *division of nephrology and hypertension, department of internal medicine, faculty of medicine, sumatera utara university, medan **division of endocrinology and metabolic diseases, department of internal medicine, faculty of medicine, sumatera utara university, medan correspondence dr. indah maulidawati division of nephrology and hypertension department of internal medicine faculty of medicine sumatera utara university jl. bunga lau no.17 medan sumatera utara 20136 fax: +6261 8363009 mobile: +62813 7073 3033 email: imw093@gmail.com univ med 2015;35:19-25 doi: 10.18051/univmed.2016.v35.19-25 pissn: 1907-3062 / eissn: 2407-2230 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license abstract universa medicina january-april, 2016january-april, 2016january-april, 2016january-april, 2016january-april, 2016 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 vol.35 no.1 renal cortex echogenicity increases degree of retinopathy in diabetes mellitus indah maulidawati*, abdurrahim rasyid lubis*, and dharma lindarto** background the number of people with diabetes mellitus (dm) is increasing due to population growth, aging, and increasing prevalence of obesity. diabetic retinopathy and diabetic nephropathy are two main complications of dm. some studies suggest a correlation between diabetic nephropathy and diabetic retinopathy. however, other studies found that renal cortex echogenicity is associated with chronicity of kidney disease and renal histopathology. the aim of this study was to determine whether there is a correlation between renal cortex echogenicity as determined by renal ultrasonography and degree of retinopathy as determined by funduscopy in subjects with dm. methods a cross sectional study was conducted on 41 dm subjects from september to november 2014. data obtained by anamnesis, physical examination, and examination of ureum, creatinine, urinalysis, glycated hemoglobin (hba1c), renal and urinary tract ultrasonography and funduscopy, were collected from all subjects. blood samples were taken from the median cubital vein for biochemical measurements using cobas automated analyzers. normality of data distribution was tested using the shapirowilk test. to determine the relationship between variables the spearman correlation test was used. results using the spearman correlation test, a strongly significant correlation was found between degree of renal cortex echogenicity and degree of retinopathy (r=0.773; p=0.0001). a significant relationship was also found for the degree of retinopathy with age (r=0.317; p=0.044), duration of diabetes mellitus (r=0.639; p=0.0001) and hba1c (r=0.681; p=0.001). conclusion this study found that renal cortex echogenicity increased the degree of diabetic retinopathy in diabetic subjects. renal ultrasonography for patients with type 2 dm has a great role in diagnosing and grading diabetic retinopathy. keywords: renal cortex echogenicity, retinopathy, subjects with diabetes mellitus doi: http://dx.doi.org/10.18051/univmed.2016.v35.19-25 20 maulidawati, lubis, lindarto renal cortex echogenicity and retinopathy introduction various epidemiological studies have shown an increasing trend in the incidence and prevalence of diabetes mellitus type 2 (dm) in various parts of the world. the world health organization (who) predicts a substantial increase in the number of people with diabetes in the coming years. for indonesia, the predicted increase will be from 8.4 million in 2000 to approximately 21.3 million in 2030.(1) diabetic retinopathy (dr) and diabetic nephropathy (dn) a r e t w o m a i n c o m p l i c a t i o n s o f d m . approximately 40% of diabetic patients have renal involvement.(2) diabetic nephropathy is the most common renoparenchymatous disease, accounting for 40% of cases of renal disease. there are studies showing that ultrasonographically determined kidney size and cortex echogenicity are associated with chronicity and renal histopathology. (3,4) increase in echogenicity is proportional to the increase in renal failure. after 5 years, approximately 25% of type 1 patients will have retinopathy. after 10 years, almost 60% have retinopathy, and after 15 years, 80% have retinopathy. of type 2 patients over the age of 30 years who have a known duration of diabetes of less than 5 years, 40% of patients taking insulin and 24% of those not taking insulin have retinopathy.(5) proliferative dr and proteinuria secondary to dn are late complications of overt dn, usually occur 10 to 15 years after the establishment of the dm, and are closely related to each other. the relationship between dn and dr has been described, with the finding of an association between the presence of gross proteinuria in the basic examination and 96% increased risk of proliferative retinopathy.(6) there are some similarities in the appearance of dr and dn, both being microvascular disorders with microscopic evidence of thickening of the capillary basement membrane. (7) the pathophysiology of dn approximates that of dr, beginning with an increase in vascular permeability. increased selective permeability for albumin in early dn is caused by the loss of polarity along the glomerular basement membrane and mechanisms of disorders of the eye because of the possibility of damage to the connections between the cells. the occurrence of proteinuria and proliferative retinopathy are both associated with poor glycemic control, duration of diabetes, and hypertension.(6,8) most of previous studies found a relationship between renal microvascular complications of diabetes characterized by microalbuminuria and the degree of retinopathy. however, to date no studies have been conducted linking renal cortex echogenicity with retinopathy. some recent studies have proved that the presence of retinopathy may indicate risk of nephropathy.(6) based on the above, the objective of this study was to determine the correlation between renal cortex echogenicity and the degree of retinopathy in diabetic patients. methods research design a cross-sectional study was conducted at h adam malik hospital, medan, between september and november 2014. research subjects subjects were recruited from the outpatient internal clinics, the inclusion criteria were: male or female subjects with dm, aged >18 years, being in stable condition and willing to participate in this research. patients who were not willing to undergo the screening examinations, pregnant women, patients with anemia, hypertensive retinopathy, or other eye diseases (such as cloudiness of the eye) on funduscopy, were excluded from the study. based on a significance level of 0.05 and power of 85%, the size of the sample was found to be 40. interviews all subjects were interviewed using a questionnaire consisting of the subject’s name, age, gender, duration of diabetes, smoker or non smoker status, a history of either disease in the patients and their families. 21 measurements the subject’s weight was measured using omron scales and height was determined by a kenko height measuring instrument. a kidney and urinary tract ultrasound was carried out and classified as follows: grade 0: normal (renal cortex echogenicity less than that of liver); grade i: renal cortex echogenicity equal to that of liver; grade ii: renal cortex echogenicity more than that of liver, but less than that of the renal sinuses; grade iii: renal cortex echogenicity is equal to that of the renal sinuses. direct fundoscopy was carried out and the results classified as normofunduscopy, non-proliferative diabetic retinopathy (npdr) and proliferative diabetic retinopathy (pdr). laboratory analysis laboratory studies included ureum (mg/dl), creatinine (mg/dl), urinalysis, and glycated haemoglobin (hba1c,%). blood samples were taken from the median cubital vein for biochemical measurements using cobas automated analyzers in the clinical pathology laboratory of h. adam malik hospital, medan, indonesia. statistical analysis normality of the data distribution was tested using the shapiro-wilk test. to determine the relationship between variables, the spearman correlation test and fischer’s exact test were used. statistical analysis was performed using spss, where p<0.05 was declared to be significant. ethical clearance the study protocol was approved by the health research ethical committee of the faculty of medicine, sumatera utara university. all study subjects signed written informed consent after having been informed about the aims and benefits of the study. results this study involved 41 patients with diabetes mellitus (dm) who had met the inclusion and exclusion criteria. most patients were female, comprising 22 people (53.7%). the mean age of the patients involved in this study was 62.49 ± 9.73 years. respondents who were smokers was 12 people (29.3%) and 24 dm patients involved in this study had a family history of diabetes. mean duration of diabetes for all patients was 10.71 ± 7.92 years (table 1). mean ureum and creatinine concentrations were 30.81 ± 12.4 mg/dl and 1.02 ± 12.4 mg/ dl, respectively. mean gfr value was 56.96 ± 21.65 ml/min. mean body mass index (bmi) of the respondents was 24.82 ± 4.10 kg/m2. the mean hba1c concentration was 9.08 ± 1.97%. a total of 34 patients (82.9%) had +1 urinary protein (table 1). univ med vol. 35 no.1 table 1. distributions of demographic characteristics and laboratory data (n=41) *mean ± sd 22 maulidawati, lubis, lindarto renal cortex echogenicity and retinopathy using the spearman correlation test, a strong and significant correlation was found between the degree of renal cortex echogenicity and degree of retinopathy (r= 0.773; p=0.0001) (table 2). a weakly positive correlation was obtained between age and degree of retinopathy (r=0.317; p=0.044). furthermore, the variables duration of diabetes and hba1c had a moderate positive correlations of 0.639 and 0.681, respectively, with the degree of retinopathy (table 2). thus a longer suffering from diabetes results in a more severe degree of retinopathy. similarly, a higher hba1c concentration results in a more severe degree of retinopathy in patients with dm. discussion d i a b e t i c r e t i n o p a t h y a n d d i a b e t i c nephropathy are two major microvascular complications of diabetes. prevention of the occurrence of this complication is very necessary t o r e d u c e t h e m o r b i d i t y a n d mortality of the number of people with diabetes that are quite large from year to year. this study aimed to determine the correlation between the degree of renal cortex echogenicity and degree of retinopathy in diabetic patients. this study found a significant and strongly positive correlation between the degree of renal cortex echogenicity and degree of retinopathy. the positive value of the correlation shows that the higher the degree of renal cortex echogenicity, the more severe the degree of retinopathy. results of previous studies showed an increase in renal cortex echogenicity in line with the increase in renal failure.(3,9) in addition, these studies also found that cortex echogenicity correlated positively with the parameters of renal histopathology, the significant one being glomerulosclerosis which is a glomerular lesion observed in diabetic nephropathy grades iii and iv. thus, apart from albuminuria, we can also predict the occurrence of renal microvascular complications in diabetic patients by assessing cortex echogenicity. in our study, the mean duration of diabetes of the study subjects was 10.7 years, while according to the literature, patients suffering from diabetes for >10 years are likely to have stage iii diabetic nephropathy (diabetic incipient nephropathy), and are already showing early signs of nephropathy such as microalbuminuria and thickening of the basement membrane.(2,10,11) most of previous studies searched for a relationship between diabetic kidney disease characterized by microalbuminuria and the degree of retinopathy, with a positive correlation being found in two studies. this correlation may be explained by assuming that the appearance of microalbuminuria is a sign of a general vascular dysfunction, including retinal vascular dysfunction.(12,13) in our study, 34 (82.9%) of the patients entering the study had proteinuria of +1, which could explain the positive correlation between the degree of cortex echogenicity and the degree of retinopathy. albuminuria and associated complications appear to be primarly caused by enzymes with a susceptibility to hyperglycemia, that are involved in the metabolism of anionic components of the extracellular matrix (eg heparan sulfate proteoglycan).(12) thickening of the basement membrane, both in the retinal and glomerular capillaries, are seen in advanced stages of diabetic retinopathy and nephropathy. in addition to the above mechanisms, kidney damage may also accelerate retinopathy table 2. correlation of age, laboratory data, and renal cortex echogenicity with the degree of retinopathy gfr= glomerular filtration rate; hba1c= glycated hemoglobin 23 where it is associated with higher levels of fibrinogen and serum lipoproteins.(14) in the statistical analysis of the correlations of all basic characteristics and the degree of renal cortex echogenicity, two variables were found that had a significant correlation with the degree of renal cortex echogenicity, namely duration of diabetes and hba1c. the positive correlation obtained between duration of diabetes and degree of kidney cortex echogenicity was of moderate, meaning that a longer duration of diabetes also increases the degree of cortex echogenicity. this is similar to the results of a study by inassi et al.(15) which confirmed that the increasingly frequent occurrence of microproteinuria as a marker of diabetic nephropathy was associated with increased duration of diabetes. similar results were observed in the studies of chowta et al.(16) and klein et al.(7) furthermore, the variable hba1c correlated m o d e r a t e l y w i t h t h e d e g r e e o f c o r t e x echogenicity. thus we concluded that the higher the concentration of hba1c, the more severe the degree of cortex echogenicity in the diabetic p a t i e n t s i n t h i s s t u d y. t h e m e a n h b a 1 c concentration was 9.08%, indicating that the study subjects were in a state of poor glycemic control. this positive correlation was also found in a study by java et al.(17) and can be explained by the mesangial extensions/expansions that are directly induced by hyperglycemia resulting in increased matrix production or matrix protein glycosylation. in vitro studies show that hyperglycemia stimulates mesangial cell matrix formation. based on the data analysis looking for a correlation of all basic characteristics with the degree of retinopathy, there were three variables having a significant correlation with the degree of retinopathy, namely: age, duration of diabetes, and hba1c. meanwhile other variables, namely ureum, creatinine, gfr, bmi and proteinuria did not correlate significantly with the degree of retinopathy. the correlation obtained between age and degree of retinopathy was a weak correlation, meaning that with age the degree of retinopathy will also increase. furthermore, duration of diabetes and hba1c had a moderate correlation with the degree of retinopathy. thus we concluded that the longer the patient suffers from diabetes, the higher the degree of retinopathy. s i m i l a r f i n d i n g s w e r e f o u n d i n a s t u d y by thomas et al.(18) who found that the incidence of referable retinopathy was positively and independently associated with the known duration of type 2 diabetes. for participants on dietary treatment with a duration of diabetes of less than five years, the cumulative incidence of referable diabetic retinopathy at one, two, and three years was 1.83, 3.66, and 5.45 per 1000 people, respectively. similarly, the higher the concentration of hba1c, the more severe the degree of retinopathy in the diabetic patients in this study. identical findings were found in a study by ng et al.(19) where the investigators found a positive correlation between hba1c levels and the degree of retinopathy. this can be explained by the mechanism of the increasing transduction activation of the diacylglycerol (dag) protein kinase c (pkc) pathway identified in the vascular tissue of diabetic animals, and in vascular cells exposed to a high glucose level. this would trigger abnormalities of the blood vessels, including changes in blood flow, extracellular matrix deposition, thickening of the basement membranes, increased permeability, and neovascularization. in addition it was that found the relation between hyperglycemia and apoptosis of capillary pericytes of the retina, where the pericytes containing glutathione, w h i c h a c t s a s a b a s i c d e f e n s e a g a i n s t peroxidation, are reduced in conditions of high glucose. high glucose levels followed by fluctuations in glucose levels will trigger the death of retinal capillary pericytes.(17) one limitation of this study is the relatively small sample size, involving only one health care c e n t e r. a n o t h e r l i m i t a t i o n i s t h a t n o h i s t o p a t h o l o g i c a l e x a m i n a t i o n a n d n o quantitative assessment of albuminuria were carried out, since previous studies found that the univ med vol. 35 no.1 24 maulidawati, lubis, lindarto renal cortex echogenicity and retinopathy state of microalbuminuria (30-300mg/24 hours) c o u l d h a v e p r e d i c t e d t h e o c c u r r e n c e o f microvascular complications.(20) the clinical implication of this study is that the retinopathy parameter can be used as a predictor of the start of nephropathy in diabetic patients, and vice versa. and it can improve the prevention and treatment as early as possible to reduce the worsening of the disease. further studies are needed, using larger samples in m u l t i c e n t e r s e t t i n g s , t h a t i n c l u d e r e n a l histopathology and quantitative albuminuria examination of the research subjects. conclusion this study found that a higher degree of renal cortex echogenicity increased the degree of retinopathy in subjects with diabetes mellitus. conflict of interest the authors who have taken part in this study declare that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript. acknowledgement we thank all study subjects who agreed to participate in the present study and all colleagues who gave their advice to improve the manuscript. references 1. perkumpulan endokrinologi indonesia. konsensus pengelolaan dan pencegahan diabetes melitus tipe 2 di indonesia. jakarta: pb perkeni; 2011. 2. lubis hr. penyakit ginjal diabetik. dalam: setiati s, editor. buku ajar ilmu penyakit dalam. edisi vi, jilid ii. jakarta: interna publishing; 2014. hal. 2102-5. 3. moghazi s, jones e, schroepple j, et al. correlation of renal histopathology with sonographic findings. kidney int 2005;67:151520. 4. araújo n, rioja l, rebelo m. a clinical predictor index for renal survival. j bras nefrol 2010;32: 27-32. 5. american academy of ophthalmology retina/ vitreous panel. preferred practice pattern guidelines : diabetic retinopathy. san francisco (ca): american academy of ophthalmology; 2014. 6. aroca pr , ballart jf, soler n, et al. review of the relationship between renal and retinal microangiopathy in type 1 diabetes mellitus patients. curr diabetes rev 2010;6:88-101. 7. klein r, zinman b, gardiner r, et al. the relationship of diabetic retinopathy to preclinical diabetic glomerulopathy lesions in type 1 diabetic patients: the renin-angiotensin system study. diabetes 2005;54:527–33. 8. al wakee js, hammad d, al suwaida a, et al. microvascular and macrovascular complications in diabetic nephropathy patients referred to nephrology clinic. saudi j kidney dis transplant 2009;20:77-85. 9. tervaert t, mooyaart a, amann k, et al. pathologic classification of diabetic nephropathy. j am soc nephrol 2010;21:556–63. 10. roshan b, stanton rc. a story of microalbuminuria and diabetic nephropathy. j nephropathol 2013;2:234–40. 11. quaia e, editor. radiological imaging of the kidney. berlin: springer verlag;2011. 12. rani pk, rama r, gupt a, et al. albuminuria and diabetic retinopathy in type 2 diabetes mellitus sankara nethralaya diabetic retinopathy epidemiology and molecular genetic study (sn-dreams, report 12). diabetol metab syndr 2011;3:1-8. 13. chen yh, chen hs, tang dc. more impact of microalbuminuria on retinopathy than moderately reduced gfr among type 2 diabetic patients. diabetes care 2012;35:803–8. 14. kundu d, osta m, mandal t, et al. serum magnesium levels in patients with diabetic retinopathy. j nat sci biol med 2013;4:113–6. 15. inassi j, vijayalakshmy r. role of duration of diabetes in the development of nephropathy in type 2 diabetic patients. nat j med res 2013;3:58. 16. chowta nk, pant p, chowta mn. microalbuminuria in diabetes mellitus: association with age, sex, weight, and creatinine clearance. indian j nephrol 2009;19:53–6. 17. java a, kcomt j, fonseca v. diabetic nephropathy and retinopathy. med clin n am 2004;88:1001–36. 25 18. thomas rl, dunstan f, luzio sd, et al. incidence of diabetic retinopathy in people with type 2 diabetes mellitus attending the diabetic retinopathy screening service for wales: retrospective analysis. bmj 2012;344:e874. 19. ng zx, chua kh, tajunisah i, et al. attenuated levels of pro-inflammatory markers in diabetic retinopathy patients undergoing treatment with antihyperglycemic and antihypertensive drugs. clinics 2013;68:185-91. 20. samatha p, venkateswarlu m, prabodh s. role of biochemical markers in the prediction of microvascular complications in type-2 diabetes mellitus. j clin diagn res 2011;5:1154-7. univ med vol. 35 no.1 c:\users\jurnal fkusakti\docume 1 the role of occupational health services in the universal halth coverage era in indonesia muchtaruddin mansyur*,** *occupational medicine division, faculty of medicine universitas indonesia **southeast asian ministers of education regional center for food and nutrition (seameo-recfon) in accordance to act no 24 of the year 2011 on the social security administrating body, the indonesian national social security program is managed by two national organizations, namely: soc ial sec urity health age ncy (badan penyelenggara jaminan sosial kesehatan = bpjs ke sehatan) a nd soc ial sec urity administering body on employment (badan penyelenggara jaminan sosial ketenaga kerjaan = bpjs ketenagakerjaan). the bpjs kesehatan is responsible for providing health coverage for all indonesians through the national social health insurance scheme known as the jaminan kesehatan nasional/jkn. the bpjs ketenagakerjaan is responsible for providing the worker’s social security consisting of provident fund benefit, accident benefit, pension benefit, and death benefit.(1) the indonesian government has been continuously improving the health service program towards better national universal health coverage and has set the 2019 functional achievement target of 95% of the population enrolled in the program. to ensure that this target of the program will be achieved, the government pays the premium of bpjs kesehatan of the poor and near poor.(2) however, bpj s kes ehat an has be en facing a financial problem since the first year of operation due mainly to the discrepancy between the expenditure and the collected membership premium. the expenditure of healthcare cost they paid to the healthcare facilities was much higher than the funding they collected from the membership premium, and the compliance rate of the members in regular premium paying was not good enough. in addition, around ten to twelve million members did not pay their premium regularly. the bpjs claimed that their potential financial deficit for the fiscal year 2018 projected up to idr 16.5 billion including the 2017 carryover of the fina ncial deficit. (3 ) in contrast, the bpjs ketenagakerjaan has shown a more stable financial standing with a surplus of idr 1,299,524 million in 2017.(4) this does not mean, however, that the bpjs ketenagakerjaan has been successful in providing the national workers social security as their responsibility to assure the eligible workers to get the benefit of occupational diseases compensation is still less. t he c o mp en sa t e d c a s e s of o cc u pa ti o n a l diseases were far below of the expected total number of occupationa l disease cases in indonesia. this means that bpjs kesehatan has been paying much unnecessary health care service cost due to underdiagnosed occupational diseases. the majority of indonesian physicians do not pay any attention to the diagnosis of occupational diseases, and there is also no r e gu la t io n t o a l l ow f o r t r a n sf e r of t he occupational diseases care funding responsibility universa medicina january-april 2019 vol.37 no.1 2 universa medicina f r om t h e bp j s ke se h at a n t o the bp j s ke t e n ag ak e r ja a n no r i s t he r e a c l e a r responsibility transfer procedure of handing over the occupational diseases management costs from the former to the latter organization. the continuous deficit of the bp js kesehatan is not surprising for many health insurance experts as there are some principal policies that ha ve not been implemented consistently. moreover, the medical care cost went to the curing and treatment for some cases of chronic disease, malignancy, and terminal disease. the indonesian global burden disease study 2016 found that the leading causes of disability-adjusted life-years (dalys) in 2016 are the six non-communicable diseases (ncds) comprising ischemic heart attack, cerebrovascular diseases, diabetes, low back and neck pain, sense organ diseases, and road injuries. these are the common prevalent cases among the working age group. furthermore, occupational risk is one of the ten leading risk factors for premature mortality.(5) the data of the high rank of the occupational risk for premature mortality supports the assumption of potential unnecessary financial load of bpjs kesehatan due to under-diagnosed occupational disease. in addition the data of ncds burden also supports the opinion that implementing the comprehensive health care approach is an urgent matter in the era of universal health coverage and national social security. the target of making the comprehensive health care approach into a universal coverage program is not only to improve health care services but also to solve public health problem issues, including the equity and efficiency of the health services, which are strongly correlated with health promotion and disease prevention.(6) occupational disease management and workplace-based health promotion are the ways to build a bridge between the bpjs kesehatan and the bpjs ketenagakerjaan to transfer the health care cost proportionally. the occupational health program through workplace health p romo t io n a nd di s ea se pr e ve nt i on ha s a potentially high leverage to reduce the financial burden of noncommunicable diseases and malignancy. the other strategic program is transferring the bpjs kesehatan’s financial hardship due to under-diagnosed occupational diseases to the bpjs ketenagakerjaan. in this case, some approaches to be undertaken include the improvement of occupational diseases management at the levels of health service facilities, policy and regulation, and public education. the primary care physicians and other health professionals should have training on how t o id en ti f y, dia gnose an d re por t occupational diseases along with improvement of equipment and health facilities to support determination of occupational disease causality. it is good to know that there is a new regulation in presidential decree no 7 of the year 2019 concerning occupational diseases which defines the compensable occupational diseases categories. in addition, the indonesian medical as s oc ia t io n ( im a) w it h t he ind on e s i a n occupational medicine association and other indonesian medical specialist organizations just launched the guidelines on occupational diseases management. however, without the policy and regulation as the basis for a clear point of departure of transferring the management of o c c u pat i o na l d i se a s e s f r o m t he bp j s kesehatan to the bpjs ketenagakerjaan, the presidential decree and the ima guidelines might be useless. it is equally important to give proper attention to public education about occupational disease identification, diagnosis, and compensation to increase the worker ’s awareness and participation towards better occupational disease management. universal health coverage as part of the national social security in indonesia has been challenging for years in relation to the continuous deficit bpjs kesehatan has been experiencing. one option to minimize the deficit is to implement comprehe nsive health care including the occupational medicine and health approach to improve occupational disease management, and n on c o mmu ni c a b l e di s e a s e c o n t r ol a n d prevention. 3 january-april 2019vol.38 no.1 references 1. ministry of law and human rights, republic of indonesia. act no 24 of the year 2011 concerning the social security administrating body. supplement of the state gazette of the republic of indonesia number 5256. 2. ministry of national development planning. indonesian health sector review. jakarta: kementerian ppn/bappenas; 2014. 3. social security health agency. peserta program jkn per 30 september 2018. social security health agency/bpjs kesehatan; 2018. univ med 2019;38:1-3. doi: http://dx.doi.org/10.18051/univmed.2019.v38.1-3 4. badan penyelenggara jaminan sosial ketenegakerjaan. laporan keuangan konsolidasian tahun 2017. jakarta: bpjs ketenegakerjaan;2018. 5. mboi n, surbakti im, trihandini i, et al. on the road to universal health care in indonesia, 1990 – 2016: lancet 2018;392:581–91. doi: 10.1016/s01406736(18)30595-6. 6. mansyur m. health finance policy and its impact to the occupational health and safety in indonesia: asian-pacific newsletter occup health safety 2012;9:48-51. oktavianus 80 *department of biochemistry, faculty of medicine, atma jaya catholic university **department of parasitology, faculty of medicine, atma jaya catholic university correspondence dr. ayly margaret, m.biomed department of biochemistry faculty of medicine atma jaya catholic university jl. pluit raya no.2 jakarta, 14440 phone : +6221 6693168 ext 203-204 email : margaret_ayly@yahoo.com univ med 2013;32:80-5 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background aspergillosis is an infectious disease due to inhalation of aspergillus spores. drug resistance to aspergillosis has been reported since 14 years ago. therefore, alternative treatment using herbals began to attract the attention of the medical profession and the community, because herbals are relatively safe and have minimal side effects. neem (azadirachta indica) is an herb with broad spectrum biological activity, but studies on the antifungal activity of neem leaves are lacking. the objective of this study was to evaluate the activity of neem leaves on aspergillus flavus (a. flavus). methods this was a laboratory experimental study using a. flavus samples obtained from bronchial washing specimens of inpatients. neem leaf extract in ethanol (nle) was prepared in various concentrations. aspergillus. flavus antifungal activity of nle were assessed by disc diffusion (using nle at 1.0; 0.75; 0.50; 0.25 g/dl) and macrodilution (using nle at 1.0; 0.75; 0.5; 0.25; 0.125 g/dl). ketoconazole 0.1 g/dl was used as antifungal positive control. disc diffusion and macrodilution were conducted using sabouraud dextrose agar and broth, to measure inhibition zone diameter and minimal inhibitory concentration (mic), respectively. statistical analysis was performed using mann-whitney test from spss 15. results there were significant differences in inhibition zone diameter (p = 0.034) between a. flavus samples exposed to nle (1 g/dl) and the control group exposed to ketoconazole (0.1 g/dl). the mic of nle against a. flavus was 0.5 g/dl. conclusions neem leaf ethanol extract has antifungal activity against a.flavus in vitro. key words : a. flavus, neem, azadirachta indica, antifungal antifungal activity of neem leaf ethanol extract on aspergillus flavus ayly margaret*, hanna yolanda**, and lies k wibisono* 81 aktivitas antijamur ekstrak daun mimba dalam etanol terhadap aspergillus flavus latar belakang aspergilosis adalah penyakit infeksius yang disebabkan oleh inhalasi spora aspergilus. resistensi obat terhadap aspergilosis telah dilaporkan sejak 14 tahun yang lalu. oleh sebab itu, alternative pengobatan menggunakan tanaman obat mulai menjadi perhatian di kalangan medis dan masyarakat awam karena sifatnya relatif aman dan efek samping yang minimal. mimba (azadirachta indica) merupakan tanaman obat yang memiliki aktivitas biologis yang sangat banyak, namun demikian informasi mengenai aktivitas antijamur dari daun mimba masih jarang. tujuan penelitian ini adalah untuk mengevaluasi aktivitas daun mimba terhadap pertumbuhan a.flavus. metode desain penelitian adalah eksperimental in vitro. a. flavus berasal dari bilasan bronkus pasien. ekstrak etanol daun mimba (eedm) dibuat dalam berbagai konsentrasi. metode yang digunakan adalah tes disc diffusion eedm (1,0; 0,75; 0,5; 0,25 g/dl) dan makrodilusi eedm (1,0; 0,75; 0,5; 0,25; 0,125 g/dl). antifungi yang digunakan sebagai kontrol positif adalah ketoconazole 0,1 g/dl. medium yang digunakan untuk tes disc diffusion dan makrodilusi adalah sabouroud dextrose agar (sda) dan sabouraud dextrose broth (sdb). tes tersebut untuk mendapatkan diameter zona inhibisi dan konsentrasi inhibisi minimal. analisa statistik menggunakan mann-whitney dari program spss 15. hasil terdapat perbedaan zona inhibisi yang bermakna (p=0,034) antara a. flavus yang terpajan kelompok eedm (1 g/ dl) dan kelompok ketoconazole (0,1 g/dl). konsentrasi inhibisi minimal eedm terhadap pertumbuhan a. flavus adalah 0,5 g/dl. kesimpulan ekstrak etanol daun mimba memiliki aktivitas antijamur terhadap pertumbuhan a. flavus pada percobaan in vitro. kata kunci : a. flavus, mimba, azadirachta indica, antijamur abstrak univ med vol. 32 no.2 introduction aspergillosis is a fungal disease caused by inhalation of aspergillus spores.(1) aspergillosis morbidity and mortality have been increasing in recent years. aspergillosis varies in clinical manifestations according to its non-invasive or i n v a s i v e n a t u r e . ( 1 4 ) a m p h o t e r i c i n b a n d itraconazole are the drugs of choice that are currently widely available for treatment of aspergillosis.(2-4) however, these drugs can cause side effects, such as dry mouth, nausea, vomiting, extreme drowsiness, muscle pain or weakness, more or less than normal urinary output, fever, and bleeding.(2-4) the mortality rate of invasive aspergillosis is 50% in patients who have received antifungal therapy. cases of drug resistance have been reported since 14 years ago.(3-4) aspergillus flavus is the second leading cause of invasive aspergillosis after aspergillus fumigatus. experiments in mice showed that invasive infection caused by a. flavus was 100 times more virulent than infection by a. 82 figure 1. neem leaf ethanol extract (nle) margaret, yolanda, wibisono neem leaves on aspergillus flavus fumigatus.(5) recently, drug resistance has attracted the attention of the medical profession and the public. these have turned to the use of medicinal plants as alternative therapy for treating infectious diseases,(1) and the use of medicinal plants has increased due to their minimal side effects.(6) for more than 2000 years, neem (azadirachta indica a. juss., meliaceae) has been known as an herb with a broad spectrum of biological activity.(6) neem leaves have been used for their antimicrobial, antifungal, antiviral, or anthelminthic activities, and also to lower blood glucose levels, for healing of skin diseases, for their gastroprotective effect, and to lower total serum cholesterol.(7-8) in this study, we examined the antifungal activity of an ethanolic extract of neem leaves (which were obtained from karyasari botanical garden, indonesia) on a. flavus samples from humans. this is in contrast to most of the other studies that used a. flavus from plants.(9-11) methods research design this was a laboratory experimental study carried out at the laboratory of chemistry, faculty of medicine, university of indonesia, and the laboratories of biochemistry and parasitology, school of medicine, atma jaya catholic university of indonesia. the study began in september and was completed in december 2012. preparation of neem extract fresh neem leaves were cut into small pieces and then allowed to dry for at least 2-3 weeks (no direct sunlight), then blenderized into powder. one hundred grams of dried neem leaf powder was extracted with 500 ml of 90-95% alcohol for 8 hours using a soxhlet extractor. the extracted solution was evaporated by distillation (by heating in a water bath at a temperature of 70-750c). the extract, still containing traces of alcohol, was dried in a dessicator with silica gel. one hundred grams of dried neem leaf powder produced about 10 grams of neem leaf extract (figure 1). the pure neem leaf extract was diluted with sabouraud dextrose broth (sdb) for assessment of the minimal inhibitory concentration (mic) and with twice-distilled water for disc diffusion, in both cases to concentrations of 1.0 g/dl, 0.75 g/dl, 0.5 g/dl, 0.25 g/dl, and 0.125 g/dl. in vitro tests a.flavus samples were collected at the parasitology department, faculty of medicine, university of indonesia, from bronchial washing specimens and then cultured. a.flavus samples were incubated at 370c for 48 hours before the tests. the 5-mm diameter discs were made from whatman paper no 42 and impregnated with nle. a fungal suspension equivalent to a mcfarland no. 0.5 standard was prepared in 2 ml nacl 0.9%.(12) the diameters of the inhibition zones were measured to the nearest millimeter. mics were determined visually. all test groups were incubated for 24 hours. all experiments were performed in triplicate. in the disc diffusion method to detect antifungal activity, 6 groups were used: a negative control group consisting of a. flavus culture (afc) in sabouraud dextrose agar (sda) only, group a (afc and neem leaf ethanol extract (nle) discs at 1.0 g/ dl), group b (afc and nle 0.75 g/dl), group c (afc and nle 0.50 g/dl), group d (afc and nle 0.25 g/dl), and a positive control group (afc and ketoconazole 0.1 g/dl discs). 83 univ med vol. 32 no.2 ba figure 2. disc diffusion test results of nle (a) at concentrations of 1.0 g/dl; 0.75 g/dl; 0.50 /dl; 0.25 g/dl and ketoconazole (b) at concentrations of 0.1 g/dl (1); 0.05 g/dl (2); 0.025 g/dl (3); 0.0125 g/dl (4) there were 7 groups in the macrodilution method: a negative control group (afc in sdb), group a (afc and nle 1.0 g/dl), group b (afc and nle 0.75 g/dl), group c (afc and nle 0.50 g/dl), group d (afc and nle 0.25 g/dl), group e (afc and nle 0.125 g/dl), and a positive control group (afc and ketoconazole 0.1 g/dl). data analysis statistical analysis was performed using the mann-whitney test in spss 15, for analyzing disc diffusion results between the groups exposed to nle 1.0 g/dl and ketoconazole 0.1 g/dl, respectively. results disc diffusion test disc diffusion test results of nle against a. flavus showed no inhibition zone (figure 2a), compared to the control group which showed inhibition zones 35-40 mm in diameter (figure 2b). statistical analysis by mann-whitney test showed significant differences in inhibition zone diameters (p=0.034) between groups of a. flavus exposed to 1.0 g/dl nle and those exposed to 0.1 g/dl ketoconazole (table 1). macrodilution test at the concentrations of 1.0 g/dl, 0.75 g/ dl, and 0.50 g/dl nle (tubes number 1-3 in figure 3), the test showed no turbidity visually. at the concentrations of 0.25 and 0.125 g/dl nle, the test revealed turbidity at the surface o f t h e m e d i u m . a l l o f t h e k e t o c o n a z o l e concentrations revealed no turbidity. therefore the minimum concentration of nle needed to inhibit a. flavus growth was 0.5 g/dl. discussion in this study, the final neem leaf ethanol e x t r a c t ( n l e ) w a s a d a r k g r e e n s t i c k y solution. the extraction process used ethanol as a polar solvent, which has the ability to extract most of the compounds contained in neem leaves, such as triterpenoids (nimbin, nimbidin, azadirachtin).(9,13) mondali et al.(9) reported that neem extracted with ethanol had a greater the inhibitory effect on a. flavus than neem extracted with water. in the disc diffusion test for nle against a. flavus, no inhibition zones were found, which were however present in the test for ketoconazole (figure 2b). an inhibition zone of more than 10 mm in diameter indicates that the test substance effectively 84 figure 3. macrodilution test results (from left to right) of nle at 1.0 g/dl; 0.75 g/dl; 0.50 g/dl; 0.25 g/dl; 0.125 g/dl; negative control margaret, yolanda, wibisono neem leaves on aspergillus flavus table 1. median a. flavus inhibition zone (mm) difference between 1.0 g/dl nle and 0.1 g/dl ketoconazole *mann-whitney test inhibits fungal growth.(14) the present study differs from a previous study conducted by rai e t a l . w h o c o n c l u d e d t h a t n e e m e x t r a c t expressed an inhibition zone of 0.4 cm in diameter.(15) however, these authors did not s p e c i f y w h i c h s o l v e n t t h e y u s e d i n t h e preparation of the neem extract. among the many factors that could influence the results of the disc diffusion test are the ability of the test substance to diffuse into the medium, interaction between test substance and test microbes, the amount of inoculated microbes, the growth rate of the microbes, and the sensitivity of the microbes to the test substance.(12,16-19) in our experimental study, the reason why no inhibition zones were produced by nle in disc diffusion method was probably the low ability of nle to diffuse. the differences in results between our study and that of rai et al. were presumably caused by differences in the human specimens used and the origin of the neem plants. rai et al. used a.flavus obtained from otomycotic patients.(15) observations in the macrodilution test were made after incubating the tubes for 48 hours. no turbidity was seen in the 1.0 g/dl, 0.75 g/ dl, and 0.50 g/dl tubes (tubes 1-3 in figure 3), signifying absence of visible fungal growth at t h e s e c o n c e n t r a t i o n s . t h e s e r e s u l t s a r e consistent with the findings of mahmoud et al., where 5% neem extract in ethanol yielded i n h i b i t i o n o f a b o u t 4 4 % . ( 2 0 ) t h e h i g h e r concentrations of neem extract in ethanol inhibited a.flavus growth to a larger extent.(20) the limitations of this study were the difficulties in the preparation of the various concentrations of neem extract, because of the extremely sticky nature of nle, thus hindering the achievement of consistency of fungal suspension turbidity for each triplicate. the clinical implication of this study is to increase the use of natural resources for healing diseases. we hope that neem leaf ethanol extract can be used as an alternative treatment for aspergillosis. further studies are needed to determine the antifungal activity of n e e m e x t r a c t i n o t h e r s o l v e n t s , a c h i e v e 85 univ med vol. 32 no.2 consistency of fungal suspension turbidity by spectrophotometry, and use more diverse and more numerous fungal samples. conclusion neem leaf ethanol extract has antifungal activity against a. flavus. the minimum concentration of nle to inhibit a. flavus growth was 0.50 g/dl. acknowledgement this work was supported by the health professional education quality project. references 1. bansod s, rai m. antifungal activity of essential oils from indian medicinal plants against human pathogenic aspergillus fumigatus and a. niger. world j med sci 2008;3:81-8. 2. subapriya r, nagini s. medicinal properties of neem leaves: a review. curr med chem anticancer agents 2005:5:149-56. 3. thompson gr, patterson tf. pulmonary aspergillosis. semin respir crit care med 2008;29: 103-10. 4. hedayati mt, pasqualotto ac, warn pa, bowyer p, denning dw. aspergillus flavus: human pathogen, allergen and mycotoxin producer. microbiol 2007;153:1677-92. 5. rasmin m, syarifuddin pk, setiawati a, soemitro d, hamid a. sistem pernapasan. dalam: budimulyati u, bramono k, menaldi sl,editor. konsensus fkui dan pmki tentang tatalaksana mikosis sistemik. jakarta: balai penerbit fkui; 2001.p.5-16. 6. paul d, kevin k. pulmonary aspergillosis: clinical presentation, diagnosis, and therapy. brit j biomedical sci 2001;58:197-205. 7. ofusori da, falana ba, ofusori ae, abayomi ta, ajayi sa, ojo gb. gastroprotective effect of aqueous extract of neem on induced gastric lesion in rats. int j biol med res 2010;1:219-22. 8. chattopadhyay rr, bandyopadhyay m. effect of azadirachta leaf extract on serum lipid profile changes in normal and streptozocin induced diabetic rats. afr j biomed res 2005;8:101-4. 9. mondali nk, mojumdar a, chatterje sk, banerjee a, datta jk, gupta s. antifungal activities and chemical characterization of neem leaf extracts on the growth of some selected fungal species in vitro culture medium. j appl sci environ manage 2009;13:49-53. 10. niharika a, aquicio jm, anand a. antifungal properties of neem (azadirachta indica) leaves extract to treat hair dandruff. e-isrj 2010;2:24452. 11. cappuccino jg, sherman n. microbiology a laboratory manual. new york: benjamin cummings;2002. 12. klancnik a, piskernik s, jersek b, mozina ss. evaluation of diffusion and dilution methods to determine the antibacterial activity of plant extracts. j microbiol methods 2010;81:121-6. 13. mahmoud da, hassanein nm, youssef ka, abou zeid ma. antifungal activity of different neem leaf extracts and the nimonol against some important human pathogens. braz j microbiol 2011;42:1007-16. 14. nwachukwu eo, umechuruba c.i. antifungal activities of some leaf extracts on seed-borne fungi of african yam bean seeds, seed germination and seedling emergence. j appl sci environ mgt 2001; 5:29-31. 15. da costa cl, geraldo mrf, arrotéia cc, kemmelmeier c. in vitro activity of neem oil [azadirachta indica a. juss (meliaceae)] on aspergillus flavus growth, sporulation,viability of spores, morphology and aflatoxins b1 and b2 production. adv biosci biotechnol 2010;1:292-99. 16. gujar j, talwankar d. antifungal potential of crude plant extract on some pathogenic fungi. wjst 2012;2:58-62. 17. rai s, padhye s, narula n. in vitro sensitivity of otomycotic agents against synthetic antifungals and natural herbs. rrjmb 2013;2:1-3. 18. abyaneh mr, allameh a, tiraihi t, shamsghahfarokhi m, ghorbanian m. morphological alterations in toxigenic aspergillus parasiticus exposed to neem (azadirachta indica) leaf and seed aqueos extracts. mycopathologia 2005;159: 565-70. 19. kustimur s, kalkanci a, mansuroglu h, senel k. evaluation of the disc diffusion method with a comparison study for fluconazole susceptibility of candida strains. chinese med j 2003;116:63336. 20. kumar r, shrivastava sk, chakraborti a. comparison of broth dilution and disc diffusion method for the antifungal susceptibility testing of aspergillus flavus. am j biomed sci 2010;2:2028. c:\users\jurnal fkusakti\docume 228 *biomedical magister program, faculty of medicine, university of sumatera utara **faculty of nursing and midwifery, university of prima indonesia ***laboratory of molecular biology, faculty of medicine, university of prima indonesia †department of pharmacology, faculty of pharmacy, university of sumatera utara †† department of pathology anatomy, faculty of medicine, university of sumatera utara †††department of pharmacology and therapeutics, faculty of medicine, university of sumatera utara ‡department of histology, faculty of medicine, university of sumatera utara cor re spo ndence : sunarti faculty of nursing and midwifery, university of prima indonesia jln. manggis kel. limau sundai binjai mobile: +62812 6408 0414 email: sunartibiomed@gmail.com date of first submission, june 8, 2017 date of final revised submission, november 6, 2017 date of acceptance, november 8, 2017 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicinaseptember-december, 2017 vol.36 no.3 hepatoprotective effect of red ginger rhizome extract in deep frying oil-fed male wistar rats sunarti*,**, edy fachrial***, urip harahap†, delyuzar††, tri widyawati†††, and lokot donna lubis‡ background non-alcoholic steatohepatitis (nash) is a chronic liver disease associated with increased free fatty acids, trans fatty acids and free radical compounds due to the consumption of deep frying oil (dfo). ginger has hypolipidemic and antioxidant properties, and acts as an insulin sensitizer. the present study was conducted to evaluate the hepatoprotective effect of an ethanol extract of zingiber officinale roxb var rubrum (eezo) in dfo-fed male wistar rats. methods a study of experimental design was conducted involving 24 non-alcoholic steatohepatitis (nash) male wistar rats that were randomly divided into 4 groups. group k1 (control) was given dfo-3 (3 times heated dfo) at a dose of 10 μl/g bw/day. the treatment groups (p1, p2, p3) were given dfo-3 and red ginger rhizome extract at doses of 100, 200, and 400 mg/kg bw orally for 30 days. the histopathology examination assessed fatty degeneration, and degree of fibrosis. the data were analyzed with the kruskal-wallis and mann-whitney test. results the kruskal-wallis test showed that fatty degeneration (steatosis, lobular inflammation, hepatocyte ballooning and nash) were significantly different among the four groups (p<0.05). mann-whitney test proved that fatty degeneration was not significantly different between control and treatment groups (p>0.05). conclusion deep frying oil increased fatty degeneration and nash, and none of red ginger rhizome extract doses was able to inhibit fatty degeneration significantly. for the future research it is intended to prolong the induction time and the administration of red ginger rhizome extract. keywords: nash, red ginger rhizome extract, fatty degeneration, fibrosis, rats doi: http://dx.doi.org/10.18051/univmed.2017.v36.228-235 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: sunarti, fachrial e, harahap u, et al. hepatoprotective effect of red ginger rhizome extract in deep frying oil-fed male wistar rats. univ med 2017;36: 228-35. doi: 10.18051/ univmed.2017.v36.228-235 229 univ med vol. 36 no.3 introduction non-alcoholic fatty liver disease (nafld) is a chronic condition that is histopathologically characterized by mor e than 5 to 10% of macrovesicular liver steatosis and is not due to alcohol consumption. non-alcoholic fatty liver disease is categorized into non-alchoholic fatty liver (nafl) and non-alchoholic steatohepatitis (nash). the nash is a chronic condition that is histopathologically characterized by hepatic steatosis and inflammation with hepatocyte injury (ballooning) with or without fibrosis.(1,2) the prevalence of nafld worldwide is estimated to be between 6.3% and 33%.(2) in the united states the incidence of nafld in 2012 in the united states was up to 27-34%, in europe it reached 20-30% and in asian countries such as sri lanka, malaysia, and indonesia it reached 15-20%, while 2-3% of the general population is estimated to have nash.(3) non-alcoholic steatohepatitis is considered to be one of the causes of chronic liver disease. the hazards of fatty liver are hepatic cirrhosis and liver cancer, due to liver inflammation from steatosis.(4) one of the causes are free fatty acids, free radical compounds and trans fatty acids, which can be produced by deep frying oil (dfo).(5–7) trans fatty acids are able to increase lipid peroxidation, and the lipid peroxides formed will damage the rough endoplasmic reticulum so that the synthesis of lipoprotein-forming proteins is impaired. low lipoprotein levels cause some of the fat in the liver to not bind to lipoproteins to be transported to other parts of need. the accumulation of fat in the liver causes fatty degeneration of the liver cells. degeneration is a change in cell morphology due to injury. cell injury causes changes in intracellular metabolism that subsequently alter the cell structure.(8) lipid peroxidation is characterized by incr ease d malondia lde hyde (mda) and decreased antioxidant enzymes such as catalase (cat ), superoxide dismutase (sod) and glutathione peroxidase (gpx), tha t cause oxidative stress.(9) the resulting oxidative stress or reactive oxygen species (ros) destroys mitochondrial deoxyribonucleic acid (dna) and increases the expression of proinflammatory cytokines [tumor necrosis factor alpha (tnf-α), transforming growth factor beta (tgf-β), interleukin-8 (il-8)], so increasing the formation of mallory bodies and collagen synthesis in the stellate cells. the ros will also cause apoptosis directly through the nuclear factor-κb (nf-κb) activation, thereby causing injury to the liver, inflammation and development of fibrosis.(10,11) zingiber officinale roxb var rubrum (zo) reportedly contains flavonoids, polyphenols and tannins, that are known to have antioxidant activity.(12) and is able to inhibit lipid peroxidation, to increase antioxidant enzymes, to regulate the low density lipoprotein (ldl) receptors and 3hydroxy-3-methylglutaryl coenzyme-a (hmgcoa) that affect the absorption of cholesterol in the liver.(13) the zo component zingerone is particularly effective as a free radical scavenger of superoxide, hydroxyl, nitric oxide (no), peroxyl and peroxynitrite radicals in vitro. gingerol is also an effective peroxyl radical scavenger and inhibits no formation. therefore, 6-gingerol (the most abundant gingerol) and zingerone effectively inhibit lipid peroxidation due to the generation of ros.(14) phytochemical studies showed that zo is rich in a large number of substances, including gingerols and shogaols, the dehydrated form of gingerols. these compounds display diverse biological activities such as antioxidant, anti inflammatory, and anticarcinogenic properties.(15) a study on 30 male wistar rats given dfo at a dose of 0.42 ml per day for 14 days found increased steatosis, leading to the development of fatty liver.(16) another study, in which 48 rats were fed a high cholesterol diet and received an aqueous infusion of ginger orally at doses of 100, 200 and 400 mg/kg bw in comparison with atorvastatin, found that the doses of 200 and 400 mg/kg bw lowered cholesterol more effectively than did atorvastatin.(17) yet another study showed that oils from z. officinale (200 mg/kg) exhibited 230 hepatoprotective activity in acute ethanol-induced fatty liver in male wistar rats.(18) a study on male wistar rats that were fed used frying oil showed that there were a large number of dark-red patches, necrosis, and bleeding in the liver, while the renal cells were also obviously dama ged. (1 9) however, no information could be obtained from previous studies on the effects of an ethanol extract of red ginger rhizome on nash. the objective of the pre sent study was to eva luate the hepatoprotective effect of an ethanol extract of red ginger rhizome on the histopathology of nash in dfo-fed male wistar rats. methods research design this study was of a randomized post-test only control group design, in which the control and treatment group s were each treated differently. the study was conducted in the animal breeding house in the pharmacy laboratory, while the histopathology examination was conducted in the laboratory of histology, medical faculty, universitas sumatera utara from september 2016 to january 2017. plant material red ginger rhizomes were collected from raya simalungun and have been identified in the herbarium medanese laboratory of universitas sumatera utara under number 871/meda/2016. preparation of red ginger rhizome extract red ginger rhizomes were washed and then dried in a drying oven at a temperature of 600c. after drying, the rhizomes were then crushed in an electric blender. the simplicia homogenate powder was macerated in 96% ethanol. after filtration, the extract was evaporated using a rotary evaporator at a temperature range of 40600c until it had thickened. the solutions made from the extract were prepared freshly each time and were administered orally once daily for 30 days to the treatment groups. standardization of simplicia and extract was based on the indonesian pharmacopeia 2014,(20) to calculate the water content, water soluble extract, ethanol soluble extract, total ash content and acid soluble ash content.(20) identification of the phytochemicals in red ginger rhizome ethanol extract was based on yang et al.(21) to identify flavonoids, alkaloids, tannins, saponins, steroids and terpenoids as well as glycosides. preparation of deep frying oil deep frying oil was made by frying 5 kg catfish 3 times in 2.5 liter cooking palm oil at 200ºc (measured with a cooking thermometer) for 15 minutes. after each frying the oil was left to cool for ± 5 hours at room temperature. the 3-times heated dfo preparation will subsequently be referred to as dfo-3. experimental animals the experimental animals were 24 healthy male wistar rats aged 2-3 months and weighing between 200 and 250 g. the rats were divided by simple randomization into 4 groups of 6 animals per group, by using the formula of federer: (t-1) (n-1) 15.(22) the animals were acclimatized at room temperature (25-300c) for one week under a 12 hour light-and-dark cycle. food was given orally ad libitum in the form of the cp 551 standard animal feed concentrate from pt. charoen pokphand, tanjung morawa, medan. experimental procedure the inter ventions were based on the treatment group. group k1 (control) was given standard diet and dfo-3 + distilled water + 1 ml of 1% sodium carboxymethyl cellulose (nacmc), and treatment groups p1, p2, p3 were given orally standard diet and dfo-3 + distilled water + 1 ml of 1% na-cmc and red ginger rhizome extract at stratified dosages of 100, 200 and 400 mg/kg bw, administered daily for 30 days. at the end of the treatment on the 37th day (of acclimatization and treatment), the rats were anesthetized by using ketamine xylazine at doses of 75-100 mg/kg + 5-10 mg/kg intraperitoneally sunarti, fachrial, harahap, et al hepatoprotective effect of red ginger rhizome 231 univ med vol. 36 no.3 for 10-30 minutes. then surgical laparatomy was performed to remove the liver, which was prepared for histological analysis. histological analysis the slide preparations for histological analysis were stained with hematoxylin and eosin (h&e) for the assessment of lipid degeneration and with masson’s trichrome stain for the assessment of fibr osis, then examined microscopically. the preparations successively underwent fixation, washing, dehydration, clearing, embedding, sectioning, affixing, staining, mounting and labeling.(23) the stained slides were analyzed under the light microscope at 400x magnification. blind labeling was performed randomly on 24 slides. fat droplet-containing liver cells were counted in four areas around the central veins of randomly selected liver lobules and then the mean number of fat droplets was calculated from the total number of fat droplets in all areas. the degree of fibrosis was determined according to brunt.(24) statistical analysis the data were not normally distributed. the kruskal-wallis test was followed by the mannwhitney test to analyze the data. significance was set at p<0.05. ethical clearance the study was conducted according to the ethical clearance from the animal research ethics committee of the faculty of mathematics and natural sciences, universitas sumatera utara (fmipa usu), medan, under number 589/ keph-fmipa/2016. results the standardization of the zo ethanol extract (eezo) yielded the following results: water content of simplicia (7.50%), extract (4.90%), water soluble content (21.70%), ethanol soluble concentration (10.43%), total ash content (3.42%) and acid soluble ash (1.32%). the phytochemical screening of simplicia and eezo traced the presence of alkaloids, saponins, tannins, flavonoids, steroids, triterpenes and glycosides. fatty degeneration was found in 24 male wistar rat liver tissue samples with h & e staining, as shown in figures 1a and 1b. the histologic sections in some groups showed steatosis, lobular inflammation and hepatocyte ballooning after 30 days of dfo administration. figure 1c shows decreased lobular inflammation with neutrophils in normal sinusoids after administration of red ginger extract at the dose of 200 mg/kg bw. a b c figure 1. histopathology of lipid degeneration; si=sinusoid, he=hepatocyte, vs=central vein, a. steatosis b. ballooning of hepatocyte c. lobular inflammation (h&e stain), d. arrows show collagen fibers clearly visible surrounding the central vein (f0) (masson’s trichrome stain) a c d b 232 determination of the degree of fibrosis was performed on 24 male wistar rat liver tissue samples with masson’s trichrome staining, as shown in figure 1d. the image shows that the masson’s trichrome staining was successful, with a perfect color of collagen fibers clearly visible between the central veins. therefore it can be concluded that in this study the collagen fibers were normal (f0=no fibrosis). the result of kruskal-wallis test showed that the number of hepatocytes with steatosis (p=0.001), lobular inflammation (p=0.001), hepatocyte ballooning (p=0.000) and nash (p=0.001) were significantly different between the four groups (table 1). the result of mann-whitney test showed the comparison between ki (control) and treatment groups (p1, p2, p3) with regard to t he e f f e ct o f d fo-3 o n th e n u mb e r of hepatocytes with steatosis, lobular inflammation, hepatocyte ballooning and nash grade, there was no significant difference between the four groups (p>0.05). there was only a significant difference in the amount of steatosis between the control group and the treatment group p1 at the dose of 100 mg/kgbw. it can be seen from the result that the amount of steatosis was greater in the treatment group than in the control group (data not shown). discussion this study showed that the administration of deep frying oil can increase the amount of steatosis, lobular inflammation, hepatocyte ballooning and nash grade. although the administration of red ginger rhizome ethanol extract did not significantly decrease the histopathologic abnormalities, at a dose of 200 mg/kg bw the result was better than that in the o th e r gr oup s . in a no t he r s tu dy o n t h e administration of aqueous extract of ginger to sprague dawley rats at a dose of 400 mg/kg bw, it was found that eezo can be used as an a n t i -i nf l a mma t or y a ge nt . ( 2 5) in a n o t he r histopathological study on acute and chronic models of inflammation in wistar rats, it was found that administration of an aqueous ginger e x t r a c t de c r e a s e d gr a n ul a t ion t i s s ue , inflammatory cells and fibroblasts as compared to controls.(26) this is supported by another study, which found that red ginger rhizome can decrease fatty degeneration which is influenced by anti oxidant compounds such as flavonoids and tannins as free radical scavengers that can decrease the formation of proinflammatory cytokines and increase the formation of lipoprotein as a fat carrier in the liver.(27) in previous studies the doses of 100, 200, and 400 mg/kg bw could decrea se fatty degeneration of the liver using an assessment of the enzymatic system, but in the present study only the dose of 200 mg/kg bw was able to reduce fatty degeneration of the liver. the present study was directly assessed on the gold standard of liver histology, especially hepatocyte degeneration (amount of steatosis, lobular inflammation, hepatocyte ballooning), nash and fibrosis. decreased fatty degeneration is due to the fact that the red ginger rhizome has an antioxidant content of flavonoids. flavonoids are compounds data presented as median; *significant at p< 0.05 by mann-whitney test; nash : non-alcoholic steatohepatitis; k1: normal diet +dfo-3 (10 μl/g/day) as control; p1: normal diet + dfo-3 + eezo (100 mg/kg once daily); p2: normal diet + dfo-3 + eezo (200 mg/kg once daily); p3: normal diet + dfo-3 + eezo (400 mg/kg once daily) table 1. effect of ethanol extract of red ginger rhizome on liver histopathology in dfo-fed male wistar rats sunarti, fachrial, harahap, et al hepatoprotective effect of red ginger rhizome histopathology k1 (n=6) p1 (n=6) p2 (n=6) p3 (n=6) p value steatosis 0.00 1.00* 0.00 1.00 0.001 lobular inflammation 1.00 1.00 0.00 1.00 0.001 hepatocyte ballooning 0.00 0.00 0.00 0.00 0.000 nash 0.50 1.00 0.00 1.00 0.001 233 univ med vol. 36 no.3 belonging to the phenolic group. the main phenolics in red ginger are gingerol and shogaol, that are more effective than quercetin.(28) a phenolic has one aromatic ring a, one aromatic ring b, and an oxygen-containing middle ring and contains one or more hydroxyl groups.(29) the hydroxyl group in phenolics is a group that has antioxidant activity and plays an important role as free radical scavenger, due to its ability to donate hydrogen atoms, thereby stabilizing free radical compounds.(30,31) high temperature frying at 2000c converts trans fatty acids through thermal polymerization and oxidation reactions after a second repeat frying for 30 minutes.(32) trans fatty acids (tfa) inh ibit the a c tivity of e nzymes f or li pid metabolism, such as fatty acid desaturase elongase and lecithin cholesterol acyl transferase (lcat); the latter plays a role in the metabolism of high-density lipoprotein cholesterol (hdl), i.e. the transport of cholesterol from the tissues to the liver, to be reconverted into very low density lipoprotein.(33) in our study the formation of fatty acids in the liver was due to excessive accumulation of fat after administration of dfo3 in volumes of 10 μl/g bw for 30 days. heating oil at the high temperatures for frying will decrease the quality of the oil and can induce nafld.(34) deep frying oil has a high trans fatty acid ( t fa ) a n d mo no -u ns a t u r a t e d f a t t y a c i d (mufa) content.(35) it decreases the efficiency of antioxidant enzyme systems by increasing pro-inflammatory signals, thus increasing oxidative stress. it can cause failure of the liver in synthesizing apolipoprotein b-100 (apo b-100) to transport fat from the liver and prevent triglyceride and ldl accumulation.(36) when viewed from the effect of the eezo dose, it was found that a dose of 200 mg/kg bw/ day was effective in reducing steatosis, lobular i nf l a mma ti o n a nd de gr e e of na sh i n comparison with the control group (k1). the effectiveness of eezo at the dose of 200 mg/ kg bw/day, proves the concept of drug or vitamin consumption, because each drug or vitamin has different dosage rules so as not to harm or cause new diseases. previous results also stated that eezo at the dose of 200 mg/kg bw significantly decreased pro-cytokines such as (il-1β, il-2, il-6 and tnf-α).(37) the results of our study in using multilevel dosages in the administration of eezo with dfo-3 showed no significant difference in the decrease in steatosis, lobular inflammation, hepatocyte ballooning, nash and fibrosis (f0). this may be induced and it requires a longer duration of therapy to affect a more optimal improvement of the liver tissue. therefore, high doses of eezo increase the intake of gingerol in the body. the gingerol compound is spicy, so high doses cause irritation which has the potential to increase liver injury, thus exacerbating hepatocyte steatosis. this study did not measure the duration of eezo administration, so further studies need to consider the duration of treatment to evaluate the hepatoprotective effect of eezo. conclusions ethanol extract of z. officinale effectively red uc es t he amount o f stea tosis , lobular inflammation and nash. however, there was no significant difference in the treatment at the doses of 100, 200 and 400 mg/kg bw. this study concludes to favor ginger but further research is required before its efficacy can be claimed. conflict of interest the authors declare that there was no conflict of interest between the authors and the study subjects. acknowledgments the authors are especially grateful to the directorate general of higher education (ditjen dikti) who has provided fund support in this study. thanks are also due to the biomedical master program, medical faculty, sumatera utara university. 234 contributors sn contributed to the design of the study, drafting of the manuscript and was responsible for the final content. sn, ef, uh, and dy contributed to data collection, analysis and 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sartika rad. effects of saturated, unsaturated and trans fatty acids on health. kes mas 2008;2: 154-60. 34. susianti. the effect of cooking oil refined with noni fruit (morinda citrifolia) on the histopathology of rat liver and heart. mka 2014; 37:54-60. 35. bruhl l. fatty acid alterations in oils and fats during heating and frying. eur j lipid sci technol 2014;116:707–15. 36. labonte me, jenkins dja, lewis gf, et al. adding mufa to a dietary portfolio of cholesterollowering foods reduces apoai fractional catabolic rate in subjects with dyslipidaemia. br j nutr 2013; 110:426–36. 37. fouda a, berika m. evaluation of the effect of hydroalcoholic extract of zingiber officinale rhizomes in rat collagen-induced arthritis. basic clin pharmacol toxicol 2009;104:262-71 . c:\users\jurnal fkusakti\docume 222 *department of haematology and blood transfusion, faculty of clinical medicine, delta state university, nigeria correspondence: marcellinus uchechukwu nwagu department of haematology and blood transfusion, faculty of clinical medicine, delta state university, abraka, delta state, nigeria email:unwagu@yahoo.com phone:+2348033851263 date of first submission, may 29, 2018 date of final revised submission, august 14, 2018 date of acceptance, august 23, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina successful reatment of a patient with herbal drug-induced immune thrombocytopenic purpura using freshly donated blood marcellinus uchechukwu nwagu* background drug-induced thrombocytopenia (dit p), which also includes thrombocytopenia induced by beverages, foods, and herbal remedies, is an important clinical problem for haematologists. drug-induced thrombocytopenia is often misdiagnosed as immune thrombocytopenic purpura with resulting inappropriate treatment. immune thrombocytopenic purpura (itp) is a clinical disorder that leads to easy bruising (purpura), excessive bleeding or extravasation of blood from capillaries into skin and mucous membranes (petechiae). the bleeding tendency is due to decreased number of circulating platelets (thrombocytopenia). there is production of antibodies against the platelets by the patient’s immune system. this case report was conducted to introduce the effectiveness of freshly donated blood and steroids on patients with immune thrombocytopenic purpura (itp) after ingestion of herbal drugs. case description dt was a 30-year old female who presented with bleeding per vagina, gum bleeding and weakness. the patient did not have any systemic disease that would cause any spontaneous hemorrhage. the patient was referred to a hematologist urgently and her thrombocyte count was found to be 2000/μl. other test results were in normal range. full blood count revealed severe thrombocytopenia. freshly donated whole blood was given to the patient and then the changes in her general condition were analyzed, as well as the blood test results. conclusion in the absence of platelet concentrate especially in rural settings and resource-poor countries, freshly donated whole blood can be used in the management of a case of severe thrombocytopenia from itp. keywords: freshly donated whole blood, immune thrombocytopenic purpura, herbal drugs doi: http://dx.doi.org/10.18051/univmed.2018.v37.222-226 case report pissn: 1907-3062 / eissn: 2407-2230 september-december 2018 vol.37no.3 cite this article as: nwagu mu. successful reatment of a patient with herbal dru g-in duced immune thrombocytopenic purpura using freshly donated blood. univ med 2018;37:222-6. doi: 1 0 . 1 8 0 5 1 / un i vm e d . 2 0 1 8 . v3 7 . 2 2 2 2 2 6 223 univ med vol. 37 no.3 introduction immune thrombocytopenic purpura (itp) is an acquir ed haemat ological disorder characterized by platelet destruction mediated by auto-antibody. the re sultant clinical manifestations include purpuric lesions of the skin, mucosa and other sites.(1) there are usually two distinct clinical syndromes namely, an acute condition in children and a chronic condition in adults. the acute type follows an infection and has a spontaneous resolution within few months while chronic immune thrombocytopenia usually persists more than six months with an unknown specific cause.(2) the antibodies implicated are immunoglobulin g (igg) directed against platelet membrane antigens, specifically glycoproteins ibix or iib-iiia. these immunoglobulins can be detected in about 60% of cases.(3) the coating of the platelets by these auto-antibodies makes them to be opsonised and removed both by the splenic macrophages and hepatic kuppfer cells. the destruction of mega kar yocytes by these antibodies is also thought as one of the mechanisms of platelet destruction. impaired production of thrombopoietin has also been demonstrated as a contributor to thr ombocytopenia. t his has led to the development of thr ombopoietin receptor agonists.(4) the activity of the abnormal tlymphocytes leads to stimulation of the autoantibody production.(5) these t-lymphocytes are also affected by drugs which are targeted against b-lymphocytes such as rituximab.(6) if the platelet count falls below 20,000 per μl, any of these signs and symptoms may occur, namely, formation of spontaneous petechiae (tiny bruises) on the limbs, bleeding from the gums/ nostrils and menorrhagia. a platelet count of below 10,000 per μl may result in oral and mucous membrane haematomas. there is prolonged bleeding time from minor cuts or lacerations.(7) at extremely low platelet counts (<5,000 per μl) there may be serious complications such as subarachnoid and intracerebral blee ding, gastrointestinal haemorrhage and various types of internal bleeding following blunt abdominal injury, eg following a car crash. normally, there is no enlargement of the spleen despite destruction of platelets by splenic macrophages. an enlarged spleen should lead to finding other causes of the thrombocytopenia. diagnosis of itp is made by exclusion. it must be established that there are no other blood disorders apart from thrombocytopenia and no other physical findings except bleeding. about 510% of cases are caused by secondary factors which must be excluded. such secondary causes include leukemia, medications (heparin, quinine), hepatitis c, hiv, lupus erythematosus and antiphospholipid syndrome. other secondary causes include cirrhosis, antiphospholipid syndrome and von willebrand factor deficiency.(7,8) usually the bleeding time is prolonged. however, the use of bleeding time in making a diagnosis of itp is discouraged by american society of hematology guidelines,(9) a normal bleeding time does not exclude platelet disorder. bone marrow examination is performed when the diagnosis is in doubt or for patients who are not responding to treatment.(8) examination of the bone mar row shows incr eased megakar yocytes. analysis of anti-platelet antibodies has about 80% specificity and hence its clinical usefulness not universally accepted. its use is a matter of choice by the physician.(8) indication for treatment is not based on just platelet counts except in rare situations. currently, the guidelines on treatment are based on severity of bleeding. however, there are diffe rent indications of treatment in both adults and children. (10) steroids are usually the first line of treatment. these steroids are medications which suppress the immune system. the determinants of mode of administration and dose include the platelet count and severity of bleeding. in urgent situations and in cases of severe bleeding, infusions of methylprednisolone or dexamethasone may be used. in less severe cases, oral prednisone may suffice. once there is improvement in platelet counts, the dosage of steroids is reduced while looking out for the possibility of relapse. about 224 60-90% of patients will experience relapse following dosage reduction or cessation.(8,9) steroids are avoided being used for a prolonged period due to its potential side effects and complications which include osteoporosis and cataract. (1 0)apart f rom steroids, there is increasing use of steroid-sparing agents such as azathioprine. in chronic refractory cases vinca alkaloids may be used, (1 1-13 ) a nd chemotherapeutic agents such as vincristine may be tried.(14,15) other treatment options include use of intravenous immunoglobulins, thrombopoietin receptor agonists, anti-d immunoglobulin, surgery (splenectomy) and the use of rituximab, which is however very expensive.(16) treatment with platelet transfusion alone is not usually successful in producing a long-term increase in platelet count, hence it is not usually recommended except in an emerge ncy due to se vere bleeding. t he explanation is that the underlying autoimmune mechanism that is destroying the patient’s platelets will also destroy donor platelets.(17) case report miss dt is a 30-year old single lady who presented to the accident and emergency (a & e) section of delta state university teaching hospital oghara, south-south nigeria. her presenting complaints were seven months history of irregular prolonged vaginal bleeding, two months duration of gum bleeding and one week of general body weakness. the prolonged vaginal bleeding was said to have been provoked by the ingestion of herbal drugs, chukatrin herbal powder, which she mixed with lime for treatment of severe dysmenorrhea. on ingesting the medication, she developed the bleeding which progressively worsened with passage of clots. physical examination revealed a young fair lady not in any obvious distress, she was pale, anicteric and not febrile to touch. there were petechial haemorrhages on the abdomen, chest, and the anterior parts of both upper and lower limbs. there were also blood clots in the buccal cavity. her pulse was 96 beats per minute, blood pressure was 100/80 mmhg and there was neither splenomegaly nor any enlarged organ in the abdomen. a preliminary diagnosis of bleeding disorder was made. series of laboratory investigations were requested and the results were as follows: 1) full blood count showed haemoglobin 8.72g/dl; white blood cell count 5,740/µl; and platelet count 1,650/ µl. urine analysis showed presence of red blood cells (haematuria). clotting profiles were normal as well as the electrolytes, urea and creatinine. the erythrocyte sedimentation rate (esr) was also within normal range. the peripheral blood film was remarkably normal except for the presence of very few platelets. our centre does not have the facility to detect platelet autoantibodies. the final diagnosis was adult (chronic) thr ombocytopenic pu rpura. the line of management was as follows: the patient was admitted and the offending herbal preparation was stopped forthwith. the hospital does not have an aphaer esis machine to prepare platelet concentrates. alternatively, she was commenced on fresh whole blood (transfused within six hours of donation). her relations and well-meaning individuals donated blood for her. she received a total of eight units of freshly donated whole blood while on admission, which raised the platelet count to 27,000/µl during the four weeks of admission. this stopped all forms of bleeding and she became better clinically. in addition to blood transfusion products, she was placed on injections of methylprednisolone 500mg daily. the platelet count has been above 25,000 per µl for a period of two weeks after the last blood transfusion and the bleeding per vagina, petechiae and gum bleedings have stopped. she was discharged home on oral prednisolone 60mg daily and was given a one-week appointment for a follow up visit at the haematology clinic. two months later, her platelet count was normal (375,000 per µl) and all the presenting c ompl ai nts w er e no lo nge r pr es e nt . t he prednisolone was reduced to 30mg daily and was given a four-week follow up. she was seen hejazi, tekantapeh, maddahi immune thrombocytopenic purpura 225 univ med vol. 37 no.3 a b c again one month later and her platelet count was 392,000 per µl. this case report was reported per the tenets of the declaration of helsinki and was approved by the institutional review board and ethical committee of my university. i obtained written informed consent from the patient. discussion immune thrombocytopaenic purpura (itp) is rare among africans and people of african descent living in other countries compared to caucasians. the overall prevalence rate in nigeria is 0.005% of hospital cases.(18) no case has ever been reported in the niger-delta region of nigeria. there is no clear cause of itp in most cases of the disease. however, only 5-10% of cases are linked to secondary factors or causes,(7,8) none of these secondary causes was associated with native preparations as in this case. this patient ingested an aloe vera product named chukatrin which elicited her immune reaction and itp. nigeria is one of the countries wh e r e t he us e of va r i ou s na ti ve / he r b a l preparations is rampant and has been on the increase.(19-21)these herbal preparations are not without harmful effects. some of these harmful effects are not even reported because of the high cost of western healthcare. many cases do not present to the hospital. worse still, marketers of these herbal drugs make lots of claims in the media and public places without being checked by constituted authorities and thereby endangering lots of our populace. the r ole of freshly-donated blood is e mph a s i ze d i n th i s c a se . se ve r e thrombocytopenia is preferably treated using platelet concentrate or platelet-rich plasma. however, the development of component therapy is still rudimentary in our hospital setting and we resorted to using freshly-donated blood to salvage the thrombocytopenia in this patient. a freshly-donated blood one unit of platelet c o nc e nt r ate f r o m a p ha e r e s i s o r p oo l e d leucocyte depleted would be e xpe cted to increase the platelet count of a 70kg adult by 20,000-40,000 per μl. in this case 8 units of fresh blood raised the platelet count to about 25,350 per μl, giving an average of 3,000 per unit of fresh whole blood. the exact mechanism by which fresh blood assists in itp is unclear. however, the dilutional effect of the freshly donated blood on the circulating anti-platelet antibodies may play a significant role. conclusion this is the first reported case of successful management of severe thrombocytopenia using freshly donated whole blood transfusion rather t ha n p l a t el e t c on c e n t r a t e . it i s be in g recommended that in the absence of platelet concentrate, fresh whole blood can be of benefit. government and constituted authorities should d is c o u r a ge p u bl i c pr omo ti o n o f he r b a l preparations especially when the efficacy and safety of the use of such products have not been verified. acknowledgement my acknowledgement goes to goldpin deborah akpotie (mrs), a senior administrative officer in the department of haematology and blood transfusion, delta state university, abraka, for her role in typing and setting of this manuscript. conflict of interest the author confirms that this article content has no conflict of interest. references 1. stasi r. pathophysiology and therapeutic options in primary immune thrombocytopenia. blood transfus 2011;9:262–73. doi: 10.2450/2010.008010. 2. rodeghiero f, stasi r, gernsheimer t, et al. standardization of terminology, definitions and outcome criteria in immune thrombocytopenic 226 purpura of adults and children: report from an international working group. blood 2009;11:2386– 93. doi: 10.1182/blood-2008-07-162503. 3. mcmillan r. antiplatelet antibodies in chronic immune thrombocytopenia and their role in platelet destruction and defective platelet production. hematol oncol clin north am 2009;23:1163–75. doi: 10.1016/j.hoc.2009.08.008. 4. schwartz rs. immune thrombocytopenic purpura—from agony to agonist. n. engl j med 2007;357:2299–301. doi: 10.1056/nejme0707126. 5. siegal d, crowther m, cuker a. 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maddahi immune thrombocytopenic purpura c:\users\universa medicina\docu 55 *department of public health & community medicine, faculty of medicine, jenderal soedirman university purwokerto **magetan district health office, east java correspondence: dr. yudhi wibowo, m.ph department of public health & community medicine, faculty of medicine, jenderal soedirman university purwokerto jl. dr. gumbreg no. 1, mersi, east purwokerto 53112 phone: +62281-622022 email: dryudhiwibowo2005@gmail.com orcid: 0000-0001-7523-2170 date of first submission, december 12, 2 01 8 date of final revised submission, april 10, 2020 date of acceptance, april 10, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina spatial modeling of the spread of scabies and environmental sanitation as a risk factor among schoolchildren: a case-control study yudhi wibowo*, nendyah roestijawati*, diah krisnansari*, and agoes y purnomo** background scabies is a world health organization neglected tropical disease common in children in low-and middle-income countries. the prevalence of scabies, especially in tropical countries, is still quite high at more than 200 million cases per year. transmission of scabies is predominantly via skin-to-skin contact. the purpose of the study was to determine the epidemiology of clinical scabies among schoolchildren and spatial modeling using geographic information systems (gis). methods a study of unmatched case-control design with a ratio of 1: 1 was performed to identify specific risk factors of scabies. cases were patients with clinical scabies and controls were healthy people in the area. a total of 100 students were involved in the study. simple and multiple logistic regression was used to test any association between the variables. gis modeling was used to determine the spatial distribution of clinical scabies based on 69 cases. result there was a significant association between the level of environmental sanitation and the prevalence of clinical scabies (or = 2.53;95% c.i. 1.115.74). multivariate analysis showed that the level of environmental sanitation was significant (aor = 3.05;95% c.i. 1.19-7.81). risk factors for developing clinical scabies increased to 3 times after considering other risk factors, namely gender, age group and respondent education level. analysis of average nearest neighbor distance showed that the spatial distribution of clinical scabies was clustered. conclusions the distribution of clinical scabies was spatial clustering, and prediction of transmission in a clockwise direction with southeast and northwest ellipses. keywords: risk factors, spatial analysis, scabies, schoolchildren original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.55-62 january-april, 2020 vol.39no.1 cit e th is article as: wib owo y, r oestij a wa t i n, kri sn a n sari d, purnomo ay. spatial modeling of the spread of scabies and environmental san itat ion as a risk fa ctor among schoolchildren: a case-control study. uni v med 2020;3 9:5 2 -6 2 . doi: 10.18051/univmed.2020.v39.55-62 56 wibowo, roestijawati, krisnansari, et al spatial modeling and environmental sanitation introduction the prevalence of scabies, especially in tropical countries, is still quite high at more than 200 million cases per year. the five countries with the largest scabies burden were indonesia (agestandardized dalys 153.86), china (138.25), timor-leste (136.67), vanuatu (131.59), and fiji (130.91). (1 ) the pr evalence of scabies in indonesia, according to the indonesian ministry of health, has declined from year to year, as seen from the prevalence data for 2008, 2009 and 2013 of 5.60% 12.96%, 4.9-12,95%, and 3.9 6%, respectively. although there has been a decline in scabies prevalence, indonesia is still not free from this disease, which still is one of the infectious diseases in indonesia that constitute a public health problem.(2) scabies transmission is apparently easy due to environmental factors and unhealthy behavior. the incidence of scabies has risen to a higher level than 20 years ago and the disease is mostly found in orphanages, dormitories of islamic boarding schools, prisons, hospitals, and places with poor sanitation.(3) in classical population-based and ecological studies, the description of disease-navigating webs is based on the analysis of differences in the temporal and spatial distribution of disease prevalence between different host species, sexes, ages, and geographical locations.(4,5) there was a study on the spatial distribution of scabies, but this was done during a nosocomial outbreak in an inpatient ward for cases of senile dementia.(6) spatial analysis of the distribution and spread of clinical scabies in human hosts in the broader area has not been performed until recently. therefore, the present study aimed to identify the distribution and spread of clinical scabies by means of spatial analysis and to examine whether there is a relationship between demographic characteristics, level of knowledge about scabies, and environmental sanitation. methods research design this was an unmatched case-control design with a ratio of 1:1. the study was conducted in 8 months from 1 march to 28 november 2018 in banyumas district, in the southern region of central java, indonesia. research subjects the subjects in this case study comprised clinical scabies patients who had been diagnosed by doctors in the last three months based on the cardinal signs of scabies among students obtained from secondary data at the jatilawang sub-district health center, banyumas district, and the results of medical examinations in boarding schools (elementary, junior high and senior high) of tinggarjaya village, jatilawang sub-district. the control group consisted of young people without clinical scabies from one sub-district. all schoolchildren from a given boarding school were the target population of this study. the sample size of 50 subjects per group was determined based on or = 2.56,(7) significance level α=0.05, and power of the test =80%. the subjects were randomly selected using cluster and simple random sampling. spatial analysis was based on 69 clinical scabies cases. data collection data were collected by interview, using a va lidate d semi-struc tured questionnaire comprising items on socio-demographic characteristics (sex, age, level of education), level of knowledge, and environmental sanitation associated with scabies. the age of the subjects was divided into two categories, namely <12 years and 12 year s. the educ ation level was categorized into elementary school, junior high school, and senior high school. the level of knowledge and environmental hygiene was categorized as deficient (score <20) and good (score >20). 57 univ med vol. 38 no.1 spatial analysis this study used the geographic information system (gis) to determine the distribution and spatial modeling of clinical scabies cases in jatilawang sub-district, banyumas district. the spatial analysis included average nearest neighbor distance, kernel density, and analysis of the standard deviational ellipse (sde).(8) the nearest neighbor analysis was an approach to look at the pattern of the spread of clinical scabies cases. the calculated average nearest neighbor (ann) score was used to determine the distribution pattern of clinical scabies cases. an average nearest neighbor may be calculated using the formula: ann= ïo / ïe, where ïo is the observed mean distance between each feature and their nearest neighbor, and ïe is the expected mean distance for the features given a random pattern. however, in practice, ann values are calculated using geographical information systems (gis) software. ann values are inferred based on the value of the expected mean distance. they are represented by (i) ann = 1 indicating the occurrence of a random pattern, (ii) ann <1 indicating the incidence of crowding (clustered), and (iii). ann >1 indicating the incident spread (dispersed).(9) estimated kernel density is a method for estimating the density of a point in an area. therefore, estimation of kernel density can be used to map the risk of the area affected by the d is e a se by kno wi n g t he de nsi t y of t he patients.(10) the standard deviational ellipse (sde) was used to analyze trends and patterns of dispersion of clinical scabies. the standard deviational ellipse is a summary of central tendency and dispersion in two dimensions, as well as a directional trend. two points were used as the basis for the distribution of point locations on sde, namely central tendency and dispersion. central tendency uses the center of the mean and dispersion refers to the spread of the mean ce nt er bounde d by th e el li ps e. s ta nd ar d deviational ellipse models can be used to gain a better understanding of the geographical aspects of the phenomenon and identify the cause of an event, based on specific geographic patterns.(11) data analysis an odds ratio was used to examine the association between variables, with significant variables being included in a model for multiple logistic regression. ethical clearance the study was approved by the commission on health research ethics, faculty of medicine, jenderal soedirman university with ref. no: 1412/ un23.07.5.1/pn.1/2018. results the results of the analysis of demographic relations, level of knowledge and environmental sanitation with the incidence of scabies are presented in table 1. table 1 shows that the results of the bivariate analysis were statistically significant on the level of environmental sanitation (p=0.025). a multivariate analysis was conducted to find out the most influential risk factors for the occurrence of scabies. risk factors that had a p-value of <0.25 were included in the multivariate analysis, consisting of gender, age, education level, and level of environmental sanitation. the results of the multivariate analysis are presented in table 2. table 2 shows that the risk factors for environmental sanitation are statistically significant for the pr evalence of clinical scabies (aor=3.05;95% ci 1.19-7.81). the center of cases, with a buffer radius of 1 km, was presented in figure 1. based on the average nearest neighbor distance analysis, the distribution of the prevalence of clinical scabies was clustered as a z score of -15.888 (p<0.05). the study also reveals that kernel density analysis shows the low-density category (0 165.796.010,7 square map units), as presented in figure 2. the results of the standard deviational ellipse (sde) analysis to determine the trend of 58 wibowo, roestijawati, krisnansari, et al spatial modeling and environmental sanitation distribution of clinical scabies in the jatilawang sub-district in 2018 were presented in figure 3. the direction of the spread of clinical scabies in jatilawang sub-district was clockwise with angle of y-axis rotation 83.25°, 1st sde: y-axis length: 391 m, x-axis length: 1.683 m and ellipse area: 517 km²; 2nd sde: y-axis length: 783 m, xaxis length: 3.366 m and ellipse area: 2.071 km². the tendency of the spread of clinical scabies will follow the pattern of the sde results if the risk factors exist in the area and are uncontrolled. the results of the spatial analysis show that the highest distribution of cases is in tinggarjaya village, jatilawang sub-district. as many as 50 subjects with scabies were in the boarding schools in the village. the clinical distribution of scabies was clustered, with the central cluster in the boarding schools, and had a low density within a radius of 1 km. however, not all cases were identified, so that they did not provide a near-real picture in the field. the sixty-nine cases of scabies consisted of 50 subjects who lived in the boarding school, and 19 young people residing in the surrounding village areas. discussion with regard to the characteristics of the respondents in the case group, most were of female gender (60%). this finding was different from the other studies which reported that 41.2% to 61.8% of cases were male.(12-16) this was understandable since the percentage of students in these studies was 58.1% females, identical with other previous reports.(17–20) the male-to-female ratio on scabies prevalence index, mostly may be a c variable case control or* 95% ci p value n (%) n (%) sex female male 33 17 60 40 26 24 52 48 1.79 0.80-4.01 0.156 age group (years) 12 >12 17 33 34 34 24 26 48 52 0.55 0.24-1.25 0.155 level of education elementary school junior high school senior high school 11 16 23 22 32 46 12 24 14 24 48 28 0.55 0.40 ref 0.19-1.60 0.16-1.01 0.276 0.052 level of knowledge low 22 44 24 48 0.85 0.38-1.87 0.688 good 28 56 26 52 environmental sanitation low 26 52 15 30 2.52 1.11-5.74 0.025* good 24 48 35 70 table 1. demographic analysis, level of knowledge and environmental sanitation of the subjects * or : odds ratio variable aor 95% ci p value sex 0.62 0.21-1.86 0.402 age group 0.95 0.26-3.38 0.938 level of education elementary school 1.02 0.17-6.55 0.948 junior high school 2.11 0.46-5.26 0.821 senior high school environmental sanitation ref 3.05 1.19-7.80 0.020* table 2. multiple logistic regression analysis of sex, age, level education and sanitation with clinical scabies 59 univ med vol. 38 no.1 figure 1. the map of clinical scabies analyzed using average nearest neighbor figure 2. the map of clinical scabies on kernel density 60 wibowo, roestijawati, krisnansari, et al spatial modeling and environmental sanitation influenced by the number of students at the school. it is not really related to gender, but to health sanitation and daily behavior. poor sanitation facilitates the development of scabies larvae, whereas poor health behavior worsens the situation and facilitates the transmission of worms.(21) the results showed that as much as 66% of scabies cases was >12 years old. this result is in accordance with the research result of ugbomoiko et al. (19) which amounted to 46.4% of cases of scabies aged 11-15 years. other studies showed 44.2% of cases of scabies aged 13-18 years.(15) the high prevalence of scabies at age >12 years can be associated with length of stay of the students in boarding schools so that they have a higher chance of being exposed to or being infected with scabies in the boarding school. however, the present study did not examine the relationship between length of stay in islamic boarding schools and incidence of scabies. respondents in the scabies group had the highest level of education, namely senior high school or equivalent, or were > 15 years of age (46%), while according to previous data by liu et al.,(15) the majority of people with scabies were between 13-18 years of age, this being the junior and senior high school age. we also found that other studies have encountered the highest prevalence in pre-schools, elementary schools and junior high schools.(16,17,19) the school as a place for social community is a suitable place for transmission and movement of scabies larvae. unlimited relationships and access to games with poor sanitation can make things worse. schools with a boarding system without proper health management lead to high risks of development of diseases. the high number of students or insufficient space for the school population increases the risk of developing scabies. fifty-two percent of subjects in the scabies gr ou p h a ve p o or b e h a vi o r i n t e r ms of environmental hygiene. improper daily habit a c t i vit i e s h a ve a si gni f i c a nt a s p e c t of transmitting scabies to other people. this includes the use of towels alternately, using the figure 3. pattern tendency of clinical scabies 61 same prayer tools or accessories, and sleeping together in the same bed or using the same pillows. these factors can increase the risk for suffering from scabies to 2.5 times, and may increase to 3 times after considering other risk factors, including gender, age group, and education level of respondents following multivariate analysis. the risks would increase in overcrowded residential conditions.(20–23) t he d is t ri b ut i o n o f c l i ni c al s c a b ie s prevalence appears to be clustered. the cluster center is in the boarding school (figures 2 and 3) and is related to the risk factors for scabies transmission, namely the density of occupancy and improper behavior of sharing clothes, beds, and others. the pattern of clockwise direction with southeast and northwest ellipse appears as the prediction of transmission. if the risk factors remain high in the area and there is no attempt to cope with scabies, there is a tendency for clockwise transmission of scabies with a slope between the directions southeast and northwest. to prevent transmission, many efforts are urgently needed in the form of counseling about clean and healthy living behaviors and also treating the patients to cut off the sources of infection. the possibility of monitoring parameters such as temperature and air humidity should be considered, particularly if there are scabies outbreaks. conclusions environmental sanitation was a risk factor of clinical scabies among schoolchildren. the distribution of clinical scabies in jatilawang was clustered, and the spatial distribution of cases was in a clockwise direction with southeast and northwest ellipses. conflict of interest the authors declare there is no conflict of interest in developing and submitting this manusccript. acknowledgment the authors thank the jenderal sudirman university, the institute for research and community service, faculty of medicine, banyumas district health office and community health care for their cooperation and assistance. contributors yw and ayp contributed to the conception and study design; ns and pk contributed to statistical analysis and data interpretation; yw contributed to writing the draft preparation; ayp contributed to editing. all authors have read and approved the ûnal manuscript. references 1. karimkhani c, colombara d v, drucker am, et al. the global burden of scabies: a cross sectional analysis from the global burden of disease study 2015. lancet infect dis 2017;17:1247–54. doi: 10.1016/s1473-3099(17)30483-8. 2. kementerian kesehatan. profil kesehatan indonesia tahun 2013. jakarta: kementerian kesehatan ri; 2014. 3. engelman d, steer ac. control strategies for scabies. trop med infect dis 2018;3:98. doi: 10.3390/tropicalmed3030098. 4. alasaad s, permunian r, gakuya f, mutinda m, soriguer rc, rossi l. sarcoptic-mange detector dogs used to identify infected animals during outbreaks in wildlife. bmc vet res 2012;8:110. https://doi.org/10.1186/1746-6148-8-110. 5. alasaad s, granados je, fandos p, cano-manuel f-j, soriguer rc, pérez jm. the use of radiocollars for monitoring wildlife diseases: a case study from iberian ibex affected by sarcoptes scabiei in sierra nevada, spain. parasit vectors 2013;6:242. doi: 10.1186/1756-3305-6-242. 6. tsutsumi m, nishiura h, kobayashi t. dementiaspecific risks of scabies: retrospective epidemiologic analysis of an unveiled nosocomial outbreak in japan from 1989–90. bmc infectious diseases 2005;5:85. 7. ejigu k, haji y, toma a, tadesse bt. factors associated with scabies outbreaks in primary schools in ethiopia: a case–control study. res rep trop med 2019;10:119-27. doi: 10.2147/ rrtm.s214724. univ med vol. 38 no.1 62 wibowo, roestijawati, krisnansari, et al spatial modeling and environmental sanitation 8. elliott p, wakefield j, best n, briggs d. spatial epidemiology: methods and applications. oxford: oxford university press;2009. doi: 10.1093/ acprof:oso/9780198515326.001.0001. 9. diggle p. applied spatial statistics for public health data. j am stat ass 2012;100:702-3. https:/ /doi.org/10.1198/jasa.2005.s15. 10. sabel ce, lötöne m. cluster of disease. in: gis in public health practice. maheswaran r, craglia. eds. boca raton: crc press;2014.p:51-68. 11. lai pc, so fm, chan kw. spatial epidemiological approaches in disease mapping and analysis. boca raton: crc press;2009. p.58–65. 12. korte lm, bowen ac, draper adk, et al. scabies and impetigo in timor-leste: a school screening study in two districts. plos negl trop dis 2018; 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p=0.025), while younger age (aor 0.39; p=0.006) and aneurysm treatment (aor 0.34; p=0.006) were independent protective factors against in-hospital death. conclusion patients with higher mean arterial pressure on initial measurement had higher risks of mortality. more endovascular neurointervention facilities are needed to decrease the mortality rate of aneurysmal sah. keywords : cerebral aneurysm, mortality, subarachnoid hemorrhage, stroke original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.153-161 september-december 2020 vol.39no.3 cite this article as: ramadhania nn, darmawan af, sani af. in-hospital mortality in aneurysmal subarachnoid hemorrhage. univ med 2020;39:15361. doi: 10.18051/univmed.2020.v39. 1 5 3 -1 6 1 *neurology department, faculty of medicine, airlangga university – dr soetomo general hospital, surabaya correspondence: achmad firdaus sani, dr., sp.s(k)., fins margorejo tangsi v/5a, surabaya phone and fax: +62 878 5231 3200 email: achmad_sani@yahoo.co.id orcid id: 0000-0001-8623-5975 date of first submission, december 16, 2019 date of final revised submission, september 20, 2020 date of acceptance, september 23, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 154 ramadhania, darmawan, sani predictors of mortality in aneurysmal sah introduction subarachnoid hemorrhage (sah) is a form of bleeding that occurs in the subarachnoid space between the pia mater and arachnoid mater, whereas its most fr equent cause is f rom traumatic injury. even so, spontaneous sah is also commonly found as one of the forms of stroke. (1) the reported incidence rate of sah varies from 6.2 in finland,(2) nigeria 6.51,(3) us 7.7 (4) per 100,000 population each year and japan (5) 27.63 per 100,000 person-years, with women still predominating, namely 71.9% in kenya (6) and 61.6% in china. (7) the majority of spontaneous sah is caused by ruptured cerebral aneurysm, which accounts for 70 to 80% of cases.(8) cerebral aneurysm occurs in 1 to 3% of the population.(9) the detection rate of unruptured cerebral aneurysm worldwide has been growing due to advancement of ct and mr angiographies and its treatment has been widely discussed to prevent future aneurysmal sah (asah).(8) some countries have even started screening of first-degree relatives of asah patients.(10,11) this has not yet been carried out in indonesia, because the use of ct scan and mri in nonsymptomatic patients is still not routinely done. prior studies regarding predictors of mortality and poor outcome in asah have been done. aneurysm re-bleeding and delayed cerebral ischemia in the course of this disease have been t he s t r o n ge s t in d e pe nd e n t p r ed i c t or s o f unfavorable outcome.(12,13) higher age was found to be associated with death and worse outcomes in various studies.(14-16) patients having in-hospital complications such as nosocomial i nf e c t i on a nd p n eumo ni a e xp e r i e n c e d significantly worse outcomes.(17-19) radiological findings includin g the c har acte ristic s of aneurysms (14,19) and other space-occupying lesions on ct-scan (14,20) and laboratory findings such as red cell distribution width (rdw) (21) and leukocyte counts (22) were also found to be associated with patients’ outcome. witsch et al.(16) and ahn et al.(23) have even developed s c o r e s t h at a r e u s e d t o pr e d ic t pa ti e nt outcome.(16, 23) an understanding of the pathophysiology of asah and evidence-based recommendations concerning its acute management might have important roles in decreasing asah mortality rates;(24,25) however, it remains unclear whether this decrease in mortality can be attributed to a specific cause. the key to further improving survival after asah is to identify and eliminate fact ors or events that p redict a nega tive outcome. this study aimed to identify the factors associated with in-hospital mortality in asah patients. methods research design this was an observational analytic crosssectional study conducted in dr. soetomo general hospital, surabaya, from march 2013 to july 2018. research subjects the population in this study consisted of patients with sah hemorrhage who had been admitted to dr. soetomo general hospital, surabaya. the inclusion criterion was patients with icd-10 code for non-traumatic sah (i60.9). exclusion criteria were patients with known arteriovenous malformation, those with incomplete medical records and those with mis s ing da t a i n r e co r de d i ni ti a l c lin i ca l assessments and history-taking. a total sample of 264 subjects was used in this study. subjects were then divided into two groups, survivors and non-survivors. measurements data were all obtained from subjects’ medical records. we defined sah as patients with symptoms of acute onset of thunderclap headache, seizures, focal neurologic signs, or d e c r e a se of c o n sc i o u sn e s s tha t s ho we d subarachnoid high attenuation on ct-scan. patients’ ages were taken from their national 155 id card. degree of consciousness in glasgow coma scale (gcs) and blood pressure were measured in the emergency department during a d mi s s i on t o o ur ho s pi t a l . hi s t or y of hypertension, diabetes mellitus, cerebrovascular diseases and smoking were taken by asking the patients and their families. aneurysm treatment data was obtained from the procedure report in the medical record. t he i n de pe n de n t va r ia bl e s we r e a ll dichotomous, patients’ age was categorized into 45 years and < 45 years. we used 135mmhg as the cutoff point for the mean arterial pressure (map). patients’ degree of consciousness that was scored in the glasgow coma scale (gcs) was divided into 13 and <13. the other variables were categorized into present (yes) and absent (no) according to history-taking and medical records. statistical analysis simple logistic regression analysis was used to analyze the relationship between dependent and independent variables. the results of the analysis were expressed as odds ratio (or), 95% confidence interval (ci) and p-value. significant results are the ones with a p-value of less than 0.05. the independent variables that had p<0.25 in the bivariate analysis were included in the multivariate logistic regression analysis. the results were expressed as adjusted odds ratio (aor), 95% confidence interval (ci) and pvalue, which was considered significant if pvalue<0.05. ethical clearance the ethical committee of dr. soetomo general hospital issued ethical clearance no. 1573/kepk/x/2019 on october 9th, 2019. results subjects in this study were divided into two groups, those who survived and those who died during hospitalization. hundred and forty patients (53.1%) did not survive hospital treatment. the median age of the subj ects was 55 years (interquartile range [iqr] 47–63), with initial mean arterial pressure of 116.45±22.69 mmhg. degree of consciousness was measured with gcs score, with median of 11 (iqr 9 -15). more than half (57.2%) of the subjects had a history of hypertension, and most of the subjects did not have a history of diabetes mellitus (88.3%), cerebrovascular diseases (89.4%), and smoking (90.9%). during hospitalization, aneurysm treatment was only administered to 39 patients (14.8%), in which 36 of them underwent endovascular coiling, two patients underwent surgical clipping and one patient underwent both procedures (data not shown). simple logistic regression analysis showed that the risk of mortality was lower in younger patients than in the older ones (or= 0.36; 95% ci= 0.19-0.69; p=0.002). similar finding was found in patients with higher gcs score during admission (or= 0.42; 95% ci = 0.25-0.71; p=0.001). patients with higher initial map had 2.82 times higher risk of death (or= 2.82; ci 9 5% 1. 44 -5 .5 2; p = 0 .0 02 ) . as e xp e c t e d , aneurysm treatment was found to be a protective factor against mortality (or= 0.27; 95% ci = 0.13-0.58; p<0.001). history of hypertension, diabetes mellitus, smoking and cerebrovascular disease were not found to be associated with mortality in asah (table 1). multivariate analysis found that age, map, and aneur ysm tre atment were the factors associated with mortality in asah. younger subjects had a smaller risk of death (aor 0.39; ci95% 0.20-0.76; p=0.006), those with higher mean arterial pressure had a higher risk of death (aor 2.29; ci 95% 1.10-4.39; p=0.025) and the ones who underwent aneurysm treatment were more likely to survive (aor 0.34; ci 955 0.160 .7 4; p= 0. 00 6) ( ta bl e 2 ) . de s p i te i t s significance, this logistic regression model had area under roc of 0.63 (ci 95% 0.57-0.68) (figure 1). univ med vol. 39 no.3 156 ramadhania, darmawan, sani predictors of mortality in aneurysmal sah a b c discussion in this study, we found that the case fatality r at e of a sa h wa s 53.1 %. t hi s re su lt i s considered very high compared to developed countries such as australia and new zealand (27.2%),(26) the netherlands (30.0%),(25) england (24.3%),(24) and canada (21.5%) (27) and still higher compared to other studies in developing countries such as india (38.5%) (28) and iran (44.2%).(29) our study shows that subjects with older age were less likely to survive. similar results have been found in other studies, in which higher age also correlates with higher risk of death.(30,31) a recently conducted study by kanamaru et al.(32) showed that older patients had more comorbidities, presented with poor world non-survivors (n=140) survivors (n=124) or 95% ci p-value n % n % age 0.36 0.19-0.69 0.002 <45 years 17 12.2 34 117.5 ≥45 years 123 87.8 90 72.5 gcs 0.42 0.25-0.71 0.001 13-15 45 35.1 64 54.7 < 12 87 64.9 53 45.3 map 2.82 1.44-5.52 0.002 ≥135mmhg 37 26.4 14 11.3 < 135mmhg 103 73.6 110 88.7 hypertension 1.12 0.69-1.83 0.631 yes 82 58.6 69 55.7 no 58 41.4 55 44.3 diabetes mellitus 1.08 0.51-2.31 0.830 yes 17 12.1 14 11.3 no 123 87.9 110 88.7 cerebrovascular diseases 1.42 0.63-3.21 0.388 yes 17 12.1 11 8.9 no 123 87.9 113 91.1 smoking 0.73 0.31-1.71 0.459 yes 11 7.8 13 10.5 no 129 92.2 111 89.5 aneurysm treatment 0.27 0.13-0.58 <0.001 yes 11 17.9 28 12.8 no 129 92.1 90 77.1 table 1. relationship between clinical predictors and in-hospital mortality, estimated using simple logistic regression analysis gcs: glasgow coma scale; map: mean arterial pressure aor 95% ci p-value age <45 years 0.39 0.20-0.76 0.006 map > 135mmhg 2.29 1.10-4.39 0.025 aneurysm treatment 0.34 0.16-0.74 0.006 table 2. multivariate logistic regression model aor: adjusted odds ratio 157 univ med vol. 39 no.3 federation of neurological societies (wfns) grades, that resulted in poor outcomes. an interesting study in the netherlands showed that from 1997 to 2006 there was a decline of 1.6% in the yearly mortality rate of aneurysmal sah due to improvements in health facilities, but not in the older age group, in which the mortality rate remained constant.(33) even in a group of patients that underwent aneurysm clipping, older patients still had worse outcomes compared to younger ones.(34) however, not all studies had similar results. a study by yue et al.(35) showed that age was not associated with prognosis in asah patients who underwent endovascular coiling. a low initial degree of consciousness measured using the gcs score on admission was found to be associated with a higher risk of mortality. this result is in line with a study in uruguay where patients with the lowest gcs score of 3 had the highest mortality rate (82%) that was significantly different with the higher gcs scores.(36) a study in the usa also showed that 77.5% of deaths occurred in the group of patients with a gcs of 3 to 8.(37) loss of consciousness at onset was found to be one of the risk factors of poor outcome in asah.(38) the degree of consciousness that is measured with the gcs score is commonly used as a predictor of outcomes in patients with both traumatic and non-traumatic brain injuries.(39) in the present study, the higher mean arterial pressure (map) that was measured was found to be associated with higher risk of death. there were not many studies that we could find which used map as a prognostic factor. zafar et al.(40) stated that maximum map measured within the first three admission days predicted outcome. other studies used the systolic blood pressure (sbp) as a prognostic factor. a study in finland showed that an increase of 21.4 mmh g i n sys t ol i c b l oo d p r e ssu r e c o u ld contribute to a higher risk of sudden death caused by sah ( rh 1.34; ci 95% 1.091.65).(41) rodriguez et al.(42) also showed that patients who had a maximum systolic blood pressure of more than 160mmhg within the first four hours of admission had worse outcomes. figure 1. receiver operating characteristics graph of the final prediction mode 158 ramadhania, darmawan, sani predictors of mortality in aneurysmal sah a survey of members of the neurocritical ca r e so c i e t y ( ncs) s h owe d t ha t mo r e neurointensivists (neurology and non-neurology) preferred to use sbp as a target of aneurysm treatme nt in asah compared to map. (4 3) however, the ability of the human auto-regulation s ys t e m t o in c r e a s e m a p i n re s po ns e t o increased intracranial pressure (icp) to maintain cerebral perfusion should be taken into account, therefore map can also be used to monitor the patient’s condition. patients with effective autor e gula ti o n ha d a highe r c ha n c e of goo d outcome.(44) the pressure reactivity index (prx) is sometimes used to monitor patients’ autoregulation. this measurement calculates the correlation of map fluctuation with changes in icp, which might be more important in the initial phase of asah. a study in a neurological intensive care unit (nicu) in austria showed that a higher prx value was associated with the occurrence of delayed cerebral ischemia.(45) treatment of high blood pressure in patients with sah has to be done carefully to prevent secondary ischemia.(46,47) aneurysm treatment was found to be a protective factor against mortality in aneurysmal sah. the number of aneurysm treatment procedures done in our hospital was as low as 14.8% of all aneurysmal sah patients, with mos t of th e pr oc e du r e s ( 9 2.3% ) be in g endovascular coiling. re-bleeding is one of the contributors of death in aneurysmal sah, which can be prevented by aneurysm treatment. a study in the netherlands showed that the decrease in deaths caused by re-bleeding (arr 0.68; ci 95% 0.52-0.90) paralleled the shift of earlier aneurysm treatment from day-4 after sah t o d a y-1 a f t e r s ah. ( 2 5 ) t h e 1-ye a r mortality rate of untreated ruptured aneurysms in helsinki (finland) was as high as 65%, varying with admission delay and clinical status.(48) aneurysm treatment does not only decrease patients’ mortality, since a us study showed that e a r l y a neu r ys m t r e a t me n t a ls o r e d u c e d hospitalization cost.(49) these studies showed that endovascular neurointervention facilities are e s s e nt i a l t o d e c r e a se mo r t a l it y r a t e i n aneurysmal sah. treatment in high-volume centers have been proved to increase functional outcome and decrease mortality, whereas delayed transport to neurosurgical facilities was also associated with increased mortality.(50,51) this data can be used as reference to encourage the families of patients that are to be referred to endovascular neurointervention facilities. we did multivariate analysis on 3 variables, n a me l y a ge , me a n a r t e r i a l p re s su r e a n d aneurysm treatment, and this model proved to be statistically significant. however, its area under roc was 0.63, signifying that the combination of these three variables was incapable of predicting mortality in aneurysmal sah patients. one way to make a better model for predicting mortality in aneurysmal sah patients would be the addition of the other variables used in previous studies, such as aneurysm characteristics including aneurysm location, size and type; patients’ clinical scores such as the wfns score or the hunt-hess score on admission; and occurrence of rebleeding, delayed cerebral ischemia and cerebral vasospasm. there are a number of limitations of our study. this study was a retrospective analysis of archived data. during data collection, we could not find all medical records that were listed as subarachnoid hemorrhage in the register. moreover, the severity of aneurysmal sah in individual cases was not recorded. better medical record management is suggested for the medical records center in dr. soetomo general hospital. the findings in this study have clinical implications in terms of predicting patients’ outcome at the time of a dmission and of supporting clinical decisions of physicians wh e t h e r or no t t o a dmi ni s t er ma x i mu m treatment. this information is also needed by the families to know the possible outcomes of patients’ current conditions. the results showing aneurysm treatment to be a strong independent protective factor could be used to encourage 159 univ med vol. 39 no.3 stable patients with a good prognosis to be referred to a higher neurosurgical unit for a n e u r ys m tr e a t me n t de s p i te ind on e s i a ’s geographical challenges. this study raised the question on how map as a blood pressure parameter could affect patients’ outcome. further studies on this matter should be done to evaluate the prognostic values of various blood pressure parameters. however, as stated before, the final logistic regression model in this study was not as accurate as expected in predicting outcome. therefore, further research on other possible predictors should be done to develop a more accurate predictive model for asah mortality. conclusions patients with higher mean arterial pressure on initial measurement had higher risks of mortality. but younger age and any form of aneurysm treatment had lower risks of mortality. more endovascular neurointervention facilities are needed to decrease the mortality rate of aneurysmal sah. conflict of interest the authors have no conflicts of interest to declare. acknowledgement we thank dr. budi utomo, dr., m.kes. and j. eko wahono, dr., sp.s, m.kes. for suggestions of the method and statistical analysis used in this study. contributors nnr and afd built the database and did the data collection. nnr did all the analysis. afs supervised the study and revised the article. all authors have read and approved the final manuscript. references 1. catapano j, lawton mt. subarachnoid hemorrhage. in: joaquim af, ghizoni e, 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update on treatment. eur cardiol rev 2019;14:111-5. doi: 10.15420/ecr.2019.11.1. 47. connolly es jr, rabinstein aa, carhuapoma jr, et al. guidelines for the management of aneurysmal subarachnoid hemorrhage: a guideline for healthcare professionals from the american heart association/american stroke association. stroke 2012;43:1711–37. doi: 10.1161/str.0b013e3182587839. 48. korja m, kivisaari r, jahromi br, lehto h. natural history of ruptured but untreated intracranial aneurysms. stroke 2017;48:1081–4. doi: 10.1161/ strokeaha.116.015933. 49. sonig a, shallwani h, natarajan s, et al. better outcomes and reduced hospitalization cost are associated with ultra-early treatment of ruptured intracranial aneurysms: a us nationwide data sample study. neurosurgery 2018;82:497-505. doi: 10.1093/neuros/nyx241. 50. rush b, romano k, ashkanani m, mcdermid r, celi l. impact of hospital case-volume on subarachnoid hemorrhage outcomes: a nationwide analysis adjusting for hemorrhage severity. j crit care 2017;37:240-3. doi: 10.1016/ j.jcrc.2016.09.009. 51. van lieshout jh, bruland i, fischer i, et al. increased mortality of patients with aneurysmatic subarachnoid hemorrhage caused by prolonged transport time to a high-volume neurosurgical unit. am j emerg med 2017;35:45–50. doi: 10.1016/ j.ajem.2016.09.067. univ med vol. 39 no.3 oktavianus 108 *department of biochemistry, faculty of medicine diponegoro university correspondence dr. amallia nuggetsiana setyawati, m.si.med. department of biochemistry, faculty of medicine diponegoro university jl. prof. soedharto, sh, tembalang, semarang p.o.box.: 1269 email: dramallia@undip.ac.id univ med 2013;32:108-17 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 introduction regular physical exercise and healthy life style have been demonstrated to improve health status of a number of populations. in adherence, counselling on life style and dietary nutrition have been one of the investigated methods to determine its effectiveness toward metabolic syndrome predictors. the aim of this study was to evaluate the effect of lifestyle change and dietary counselling on glucose level, body mass index (bmi), waist circumference, blood pressure, total cholesterol, triglycerides, low density lipoprotein (ldl), and high density lipoprotein (hdl) in type 2 diabetes mellitus (t2dm) patients. methods a randomized controlled trial was conducted and 26 diagnosed subjects with t2dm were enrolled in the study. after initial screening, they were randomized into the control group and the intervention group, following stratification by gender and age. the intervention group received weekly counselling and education, while the control group received counselling and education only once at the initial meeting. independent t-test was used to compare the glucose level between the two-groups. results lifestyle counselling and dietary modification effected a significant improvement in blood glucose (p=0.002), but not in bmi, waist circumference, total cholesterol, triglycerides, ldl and hdl. in addition, a positive time effect was found in the intervention group before and after treatment on bmi (p=0.009), waist circumference (p=0.0014), and glucose level (p=0.001). the intervention group also showed a significant positive attitude; almost all of the respondents tried to control their food intake and perform exercise regulary. conclusion dietary nutrition and lifestyle counselling improve blood glucose control in the short term. key words : nutrition counselling, life style modification, blood glucose, t2dm weekly lifestyle counselling improves glucose level in type 2 diabetes mellitus patients amallia nuggetsiana setyawati*, inggar octa pusthika*, and kusmiyati tjahjono dk* 109 univ med vol. 32 no.2 konseling gaya hidup menurunkan kadar gula darah pada pasien diabetes melitus tipe 2 pendahuluan latihan fisik secara reguler dan gaya hidup sehat diketahui berpengaruh di dalam perbaikan status kesehatan masyarakat. konseling gaya hidup dan diet gizi merupakan satu dari sekian metode yang diteliti untuk mengetahui pengaruhnya terhadap prediktor sindroma metabolik. penelitian ini bertujuan untuk menilai pengaruh perubahan gaya hidup dan konseling gizi terhadap kadar gula, indeks massa tubuh (imt), lingkar pinggang (lp), tekanan darah (td), kolesterol total, low density lipoprotein (ldl), dan high density lipoprotein (hdl) pada penderita diabetes melitus tipe 2 (dmt2). metode sebuah penelitian eksperimental secara acak menggunakan kontrol mengikutsertakan 26 subjek dengan dmt2. setelah skrining awal, subjek secara acak dibagi dalam kelompok intervensi dan kontrol. kelompok intervensi mendapatkan konseling dan edukasi intensif mingguan dan kelompok kontrol hanya mendapatkan sekali pada pertemuan awal. uji t-independent digunakan untuk membandingkan kadar gula darah antara kedua kelompok. hasil konseling gaya hidup dan modifikasi diet memperbaiki kontrol kadar gula darah pada kelompok intervensi bila dibandingkan dengan kelompok kontrol (p=0,002), akan tetapi tidak demikian dengan imt, lp, sistolik, diastolik, kolesterol total, trigliserida, ldl, dan hdl. efek positif seiring waktu ditemukan pada kelompok intevensi sebelum dan sesudah perlakuan pada imt (p=0,009), lp (p=0,014), tds (p=0,031), tdp (p=0,014), kadar gula (p=0.001). pada kelompok intervensi ditemukan adanya hubungan positif; di mana seluruh responden mengatur intake makananannya dan melakukan olahraga fisik dengan teratur. kesimpulan diet asupan nutrisi dan konseling gaya hidup memperbaiki kontrol kadar gula dalam jangka waktu pendek. kata kunci : konseling nutrisi, modifikasi gaya hidup, kadar gula darah, dmt2 abstrak introduction type 2 diabetes mellitus (t2dm) is a metabolic disorder that is characterized by high blood glucose due to insulin resistance and relative insulin deficiency. ( 1 ) improperly m a n a g e d t 2 d m m a y c a u s e s e v e r e complications, including renal failure, blindness, s l o w h e a l i n g o f w o u n d s , c a r d i o v a s c u l a r diseases, and erectile dysfunction.(2) changes in lifestyle, such as reduced physical activity, an unhealthy diet, and subsequent obesity, are the main causes of t2dm.(3) moreover, the global burden of diabetes is rising dramatically worldwide and is having as impact a double burden of poor health, i.e. continued high rates of infectious diseases and rapidly growing noncommunicable disease prevalence, in lowand middle income countries, as well as in indonesia. the indonesian basic health survey 2007 reported that the prevalence of t2dm patients in urban areas was 5.7% nationally, and that estimated by population size in 2030 there will be 14.7% (12 million) of the population with t2dm in urban areas and 7.2% (8.2 million) in rural areas.(4,5) 110 diet, exercise and weight counselling with follow-up for patiens with diabetes have been reccomended in many dm guidelines including the consensus on management and prevention of diabetes mellitus type ii published by p e r k e n i ( i n d o n e s i a n a s s o c i a t i o n o f e n d o c r i n o l o g i s t s ) i n 2 0 11 . ( 6 ) p r e v i o u s researches showed that an energy-wise diet, coupled with moderate levels of physical activity, favorably prevents t2dm and delays the onset of diabetic complications.(7,8,9) although the application of lifestyle counselling is promising, there remains the question of its efficacy in everyday clinical practice, considering the involvement of resource-intensive interventions and the duration of the intervention itself. consequently, further evidence is required to establish that application of lifestyle counselling and dietary nutrition modification in routine care will improve the outcomes of patients with t2dm. we therefore conducted a short term study (8 weeks) to examine the effect of routine lifestyle counselling and dietary nutrition modification on t2dm patients. methods study design this study was an 8-week randomized controlled trial conducted on east semarang residents and was performed between march and july 2011. research subjects diabetes mellitus type 2 was defined as high fasting (>200 mg/dl) or high random plasma glucose (>126 mg/dl).(6) patients with t2dm were recruited after being diagnosed previously by primary care physicians (pcps) affiliated with primary health care (phc) in east semarang. to select the participants, the researchers performed a preliminary screening of 65 medical records of patients who had been diagnosed with t2dm. sample size was determined for two independent samples with effect size of 0.3, an α of 0.05 and a â of 0.2, which resulted in 26 subjects to be enrolled in the study. the study subjects were selected using inclusion criteria as follows: i) previously diagnosed with t2dm; ii) body mass index (bmi) >25 kg/m2; iii) having abdominal obesity with a waist circumference of >90 cm for males and >80 cm for females (10); iv) having resided in east semarang for at least 5 years; and v) being 50-60 years old. patients were excluded from the study if they had i) alcohol consumption; and ii) t2dm complications (stroke, diabetic gangrene, etc). subsequently the subjects were randomly assigned to the intervention and control group for an 8-week study period. to our knowledge, this 8-week period is considerably longer than the 4 weeks used in previous published studies on therapeutic life style modification.(11) d i e t a r y i n t e r v e n t i o n a n d l i f e s t y l e modification the 8-week intervention was given during weekly home visit sessions (one hour per session for counselling and one hour for exercise). the intervention consisted of 5 components: (1) health screening (blood p r e s s u r e a n d b o d y w e i g h t c h e c k ) , ( 2 ) counseling, (3) exercise, (4) counseling, and (5) diet. all of the data sampling was undertaken according to standards as described elsewhere. the intervention for the intervention group is described in table 1, while the control group received only an educational booklet and counselling at the first meeting and no further education or intervention for the subsequent 8 weeks. education participants in the intervention group r e c e i v e d h e a l t h i n f o r m a t i o n f r o m t h e researchers, including a definition of t2dm, and information on exercise, diet, risk factors, related disease (e.g., hypertension, hyperlipidemia, obesity) and self-care. details of this intervention is presented in table 1. an educational booklet which contained this information was also given to the participants. setyawati, pusthika, tjahjono lifestyle improves glucose level 111 univ med vol. 32 no.2 exercise participants attended one supervised session per week and were encouraged to do home-based walking exercises for 45 minutes. s u p e r v i s e d t r a i n i n g i n c l u d e d w a r m u p , stretching, strength training, rhythmic dancing, and cool dancing. the training was modeled after the consensus statement of the american diabetes association.(12) diet subjects were suggested to have more fruit, vegetables, reduce intakes of carbohydrate, saturated fat, total fat and cholesterol. we provided a daily food diary for each subject. during the home visits, we calculated total calories based on the participants’ food diary. to assess the intakes of carbohydrate, fat, protein and minerals, we used the food frequency questionnaire (ffq) and calculated these using nutrisurvey software.(11) health counselling o n e a c h h o m e v i s i t , 2 0 m i n u t e s o f counselling time was allocated for the diet-based food diary, exercise adherence, and health status (blood pressure, body weight). problems in applying the intervention were also discussed. we obtained primary data for sample identity including name, date of birth, and occupation, which were collected by interviewing every subject using a standardized questionnaire. anthropometric indicators body weight was measured with a highprecision scale of 0.001 kg. weights were taken at the same time at each session; prior to weight measurement, the participants were asked to remove their shoes. waist circumference was measured midway between the lowest rib and the iliac crest. the sphygmomanometer was applied at the standard location for blood pressure measurement, which is the upper arm, with the stethoscope at the elbow crease over t h e b r a c h i a l a r t e r y. ( 1 3 ) t h e a v e r a g e o f 2 measurements was used, taken at a 2or 3minute interval with the subject in a sitting position after resting for at least 15 minutes. biochemistry panels blood samples were obtained from the antecubital vein with the subject in a seated position. samples were collected and analyzed at the prodia laboratory with an autoanalyzer. to minimize the influence of any incidental acute phase reaction, participants were requested not table 1. eight-week dietary nutrition and life style modification intervention 112 table 2. baseline descriptive statistics of the subjects by treatment groups expressed in mean + sd; idr=rupiah; bmi=body mass index; sbp=systolic blood pressure; dbp=diastolic blood pressure to perform any major physical activity before collection of blood samples. statistical analysis statistical analysis was performed using spss 17.0 for windows. descriptive analysis was used to evaluate the demographic and clinical characteristics of the subject. we used the shapiro-wilks test to evaluate the normality of data with p>0.05. the t-test was used to compare all variables between the two groups. all p values are 2-sided, and a p value of less than 0.05 was considered statistically significant. ethical clearance this study was approved by the ethics c o m m i t t e e o f t h e f a c u l t y o f m e d i c i n e , diponegoro university/dr kariadi hospital (no:072/ec/fk/rsdk/2011). proper informed consent was obtained from each participant. results characteristics of the participants the characteristics of the participants in the intervention and control groups are presented in table 2. their mean age was 51.8 years setyawati, pusthika, tjahjono lifestyle improves glucose level 113 univ med vol. 32 no.2 (sd=3.8), and most (26.9%) of the respondents worked in the private sector, with monthly incomes of less than 1,000,000 idr. their educational status was relatively moderate, with 9 participants being senior high school graduates (34.62%). during the study course, there were no drop-outs or cases lost to follow up, because the researchers were engaged in the study by home visiting. effects of the dietary counselling and lifestyle modification intervention the means of outcome measures for the treatment groups at 8 weeks post baseline are presented in table 3. there was a significant reduction in the intervention group for blood glucose compared to the control group. at week 8 we could not identify any improvement for total cholesterol (p=0.486), triglycerides (p=0.20), ldl (p=0.951), and hdl (p=0.87). results of an analysis of changes in health behavior (food control, weight control, exercise >3 times per week) are shown in table 4. it was shown that the subjects in the control group did not practice food control, weight control, or adherence to exercise with a frequency of at least 3 times per week. this is different from the situation in the intervention group which showed improvement in exercise habit and a more positive attitude on food intake control. discussion o u r r e s u l t s i n d i c a t e t h a t i n t h e implementation of short-term (8 weeks) dietary nutrition and lifestyle counselling program, which consisted of screening, education, exercise, diet and counseling, a positive effect in glucose control and improved health behavior was obtained. this suggests that despite the short duration of the intervention (8 weeks, with 8 counselling sessions and 8 times structured exercises), the program can improve glycemic control over a relatively short period. this study confirmed the previous study conducted by oh et al., which table 4. changes in health behavior after dietary nutrition and lifestyle modification table 3. mean of bmi, waist circumference, blood pressure, and glucose level, by treatment group after 8 weeks intervention sbp=systolic blood pressure; dbp=diastolic blood pressure; ldl=low density lipoprotein; hdl=high density lipoprotein 114 showed that comprehensive therapeutic lifestyle modification can improve metabolic syndrome within 4 weeks.(11) other published literature also suggested the beneficial influence of lifestyle counselling on glucose control.(8,12) our result was supported b y t h e p o s i t i v e b e h a v i o r s h o w n i n t h e intervention group, which contributed to the glycemic control performance. it is known that glycemic control education for t2dm patients c o n s i s t s o f m e a l p l a n n i n g a n d d i e t a r y modification,(14) which was also provided in each home counselling session for the intervention group. daily incorporation of low glycemic index (gi) carbohydrates in meal planning can be an effective diabetes self-management strategy not only for glycemic control but also for weight management..(15) however, in our study there was no significant effect post-intervention in the intervention group vs the control group with r e s p e c t t o b m i r e d u c t i o n . m o s t o f t h e respondents in the intervention group made effort to exercise regularly, which showed a time positive effect and positive health behavior from the baseline data in the intervention group. it has been found that regular exercise without weight loss results in a reduction in skeletal muscle lipid content coincident with an increase in skeletal muscle mass in t2dm populations.(16) these results might explain the insignificant differences in body weight in our study. moreover, a study conducted by kim et al. suggested that regular exercise (3 times per week) was associated with a significant reduction in intramuscular triglycerides and an increase in glucose transporter 4 protein (glut 4 ) protein expression and fatty acid o x i d a t i o n c a p a c i t y ( â h y d r o x y a c y l c o a dehydrogenase) as measured by percutaneous biopsy.(17) taken together, our study show that although there was no significant body weight reduction, which ultimately affect bmi, it had demonstrated the utility of exercise without weight loss as a strategy to improve skeletal muscle morphology, independent of age, race, and obesity. waist circumference is a well-known predictor for the risk of t2dm.(18) abdominal obesity is a stronger risk factor than overall obesity often expressed as body mass index (bmi) for the future development of t2dm.(19) although we could not find a significant effect on waist circumference in our intervention group, there was a decremental tendency (reduction of 0.7 ± 0.75 cm). previously, a study published by morrison et al.,(8) concluded that lifestyle counselling in a primary setting is associated with a faster i m p r o v e m e n t i n b l o o d p r e s s u r e . i t i s recommended that controlling blood pressure must be a priority in the management of persons with t2dm, due to the fact that up to 80% of patients will develop microvascular disease or die from it. the development of nephropathy and retinopathy was contributed significantly to by the blood pressure increment, therefore blood pressure control is a must in t2dm patients.(20) in our intervention group, we had a significant reduction of 7.67 ± 2.52 mmhg (p=0.031) from the baseline data. although we could not find a significant difference between intervention and control group, there was a positive effect from engagement of participants in exercise, which could explain the diminishment of exercise which reduces sympathoadrenergic activity and enhance prostaglandin mechanism.(21) moreover increased production of nitric oxide results in endothelium dependent-vasodilatation which lowers bp.(22) endothelial dysfunction also characterizes individuals with impaired glucose tolerance, insulin resistance and t2dm, suggesting that insulin resistance and endothelial (vasodilatory) dysfunction are intimately linked.(23) i n t e n s i v e c o u n s e l l i n g r e s u l t e d i n a significant change in glucose level in the intervention group (p=0.002), in contrast to the control group. several trials in clinical setting have previously documented the benefit of setyawati, pusthika, tjahjono lifestyle improves glucose level 115 univ med vol. 32 no.2 lifestyle counselling on control of glucose.(8,11) application of carbohydrate-restricted diets (crd) and diets comprised of foods with a low gi is considered to improve insulin resistance and metabolic syndrome, potentially preventing the development of t2dm.(24) integration of lifestyle modification using low carbohydrate interventions is effective for improving and reversing t2dm.(25) regarding the effectiveness of the intervention in our study, introduction of low glucose intake was successful in lowering the blood glucose level in the time frame of 8 weeks. recent studies have shown that under conditions of carbohydrate restriction, fuel sources are shifted from glucose and fatty acids t o f a t t y a c i d s a n d k e t o n e s , l e a d i n g t o i m p r o v e m e n t i n s u r r o g a t e m a r k e r s o f cardiovascular disease.(26) the 8-week dietary counselling and lifestyle modification program could not identify group and time interactions in total cholesterol, ldl and hdl. however, there was a positive time effect, as we found a decrement tendency (preand postcounselling cholesterol: 220 ± 42 mg/dl and 209.08 ± 37.03 mg/dl, respectively; preand postcounselling ldl: 132.5 ± 38.6 mg/dl and 113.46 ± 38.6 mg/dl, respectively; preand post counselling triglycerides: 263.15 ± 49.12 mg/dl and 221.8 ± 22.2 mg/dl) respectively. a longer study time would be beneficial to address this question. thus our data show that intensive lifestyle counseling, which involved glycemic control of diabetes mellitus, is effective in reducing the glucose level of study subjects, which is in support of the current treatment guidelines for patients with diabetes. moreover, a study conducted by egede, who examined lifestyle modification in t2dm, revealed that patients with diabetes appeared to modify behavior in response to the advice of physicians.(27) the duration of intervention period remains questionable. a few clinical trials of lifestyle counselling with a follow-up of longer than 12 months provided evidence for lasting effects of lifestyle counseling.(28) however, there are some studies which suggest that the effect of intensive diet and exercise may not be durable.(29,30) taken these studies into consideration, we should also assess the importance of life style counselling and its positive impact on patient behavior and health outcomes. lifestyle counselling is time consuming, therefore time limitations of practice sessions from the health practitioner remains cumbersome. in that context, implementation of current guidelines may require modification of the prevalent physician-patient treatment care, with more involvement of mid-level providers, such as nurse practitioners, physician assistants, nutritionists, or exercise physiologists. another alternative is to implement group counselling sessions. this experimental study involved 26 subjects with intensive (weekly) lifestyle modification. it was a population-based study, where the researchers visited each house and performing dietary counselling and lifestyle modification intervention. therefore, we were able to develop a more interpersonal relationship to the patients and share the decision making in participatory research approaches. home visits to diabetic people by a trained and educated health care provider will improve adherence to dietary advice, lifestyle intervention, and physical activity promotion. among the limitations of this study that may first be mentioned is the small sample size and the limited geographic region in which this study was conducted. thus, the interpretation of this study should be done with caution, and a larger study is needed in the future. second, the intervention period in our study was short. there is a need to conduct a study of longer duration to examine whether the effects are sustainable over time. conclusions this finding suggests that a systematic weekly therapeutic life style modification program can improve blood glucose level even though it is only provided for a short-term period (8 weeks). other studies are needed to identify whether this positiveness of patients’ attitude in following dietary counselling and lifestyle 116 modification is durable and eventually will lead to long-term outcomes, such as mortality, morbidity and improvement in quality of life (qol) in t2dm patients. acknowledgement this research was funded by the health research unit (up3), faculty of medicine, diponegoro university, for the year 2010. references 1. suyono s, purnamasari d, yunir em, soebaardi s, shahab a. dalam: sudoyo a w, setiyohadi b, alwi i, simadibrata m, setiati s, editors. buku ajar ilmu penyakit dalam. edisi v. jilid iii. jakarta: pusat penerbitan departemen ilmu penyakit dalam fkui;2009:hal.1852-6. 2. fowler mj. microvascular and macrovascular complications of diabetes. clin diab 2008;26:7782. 3. steyn np, mann j, bennet ph, temple n, zimmet p, tuomilehto j, et al. diet, nutrition and the prevention of type 2 diabetes. public health nutr 2004;7:147-65. 4. wild s, roglic g, green a, sicree r, king h. global prevalence of diabetes: estimates for the year 2000 and projections for 2030. diabetes care 2004;27:1047-53. 5. badan penelitian dan pengembangan kesehatan departemen kesehatan, republik indonesia. laporan nasional riset kesehatan dasar (riskesdas). jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan;2008. 6. perkumpulan endokrinologi indonesia. konsensus dan penatalaksanaan diabetes mellitus di indonesia. jakarta: pb perkeni;2011. 7. boden g, sargrad k, homko c, mozzoli, m, stein tp. effect of a low-carbohydrate diet on appetite, blood glucose levels, and insulin resistance in obese patients with type 2 diabetes. ann intern med 2005;142:403–11. 8. morrison f, shubina m, turchin a. lifestyle counseling in routine care and long-term glucose, blood pressure, and cholesterol control in patients with diabetes. diabetes care 2012;35:334–41. 9. colberg sr, sigal rj, fernhall b, regensteiner jg, blissmer bj, rubin rr, et al. exercise and type 2 diabetes: the american college of sports medicine and the american diabetes association: joint position statement executive summary. diabetes care 2010;33:2692-6. 10. sang yl, hye sp, dae jk, jee hh, seon mk ,guem jc, et al. appropriate waist circumference cutoff points for central obesity in korean adults. diabetes res clin pract 2007;75:72-80. 11. oh eg, hyun ss, kim sh, bang sy, chu sh, jeon jy, et al. a randomized controlled trial of therapeutic lifestyle modification in rural women with metabolic syndrome: a pilot study. metabolism 2008;57:255-61. 12. sigal rj, kenny gp, wasserman dh, seppa cc. white rd. physical activity/exercise and type 2 diabetes: a consensus statement from the american diabetes association. diabetes care 2008;31:s61-s78. 13. pickering tg, hall je, appel lj, falkner be, graves s, hil mb, et al. reccomendations for blood pressure in humans & experimental animals. circulation 2005;111:697-716. 14. bantle jp, wylie-rosett j, albright al, apovian cm, clark ng, franz mj, et al. nutrition recommendations and interventions for diabetes. a position statement of the american diabetes association. diab care 2008;31 suppl 1:s61-78. 15. burani j, longo pj. low-glycemic index carbohydrates: an effective behavioral change for glycemic control and weight management in patients with type 1 and 2 diabetes. diabetes educ 2006;32:78-88. 16. lee sj, kuk jl, davidson le, hudson r, kilpatrick k, graham te, et al. exercise without weight loss is an effective strategy for obesity reduction in obese individuals with and without type 2 diabetes. j appl physiol 2005;99:1220-5. 17. kim hj, lee js, kim ck. effect of exercise training on muscle glucose transporter 4 protein and intramuscular lipid content in elderly men with impaired glucose tolerance. eur j appl physiol 2004;93:353-8. 18. siren r, eriksson jg, vanhanen h. waist circumference a good indicator of future risk for type 2 diabetes and cardiovascular disease. bmc public health 2012;12:631. 19. lindström j, tuomilehto j. the diabetes risk score: a practical tool to predict type 2 diabetes risk. diabetes care 2003;26:725-31. 20. vijan s, hayward ra. treatment of hypertension in type 2 diabetes mellitus: blood pressure goals, choice of agents, and setting priorities in diabetes care. ann intern med 2003;138:593-602. 21. frisoli tm, schmieder re, grodzicki t, messerli fh. beyond salt: lifestyle modifications and blood pressure. eur heart j 2011;32:3081-7. setyawati, pusthika, tjahjono lifestyle improves glucose level 117 univ med vol. 32 no.2 22. goto c, higashi y, kimura m, noma k, hara k, nakagawa k, et al. effect of difference intensities of exercises on endothelium-dependent vasodilation in humans: role of endotheliumdependent nitric oxide and oxidative stress. circulation 2003;108:530-5. 23. caballero ae. endothelial dysfunction in obesity and insulin resistance: a road to diabetes and heart disease. obes res 2003;11:1278–89. 24. wood rj, fernandez ml. carbohydrate-restricted versus low-glycemic-index diets for the treatment of insulin resistance and metabolic syndrome. nutr rev 2009;67:179-83. 25. westman ec, yancy ws jr, mavropoulos jc, marquart m, mcduffie jr. the effect of a lowcarbohydrate, ketogenic diet versus a lowlycemic index diet on glycemic control in type 2 diabettes mellitus. nutr metab 2008;5:36. doi:10.1186/1743-7075-5-36. 26. al-sarraj t, saadi h, calle mc, volek js, fernandez ml. carbohydrate restriction, as a first-line dietary intervention, effectively reduces biomarkers of metabolic syndrome in emirati adult. j nutr 2009;139:1667-76. 27. egede le. lifestyle modification to improve blood pressure control in individuals with diabetes. diabetes care 2003;26:602-7. 28. he j, whelton pk, appel lj, charleston j, klag mj. long-term effects of weight loss and dietary sodium reduction on incidence of hypertension. hypertension 2000;35:544-9. 29. turner rc, cull ca, frighi v, holman rr. uk prospective diabetes study (ukpds). group. glycemic control with diet, sulfonylurea, metformin, or insulin in patients with type 2 diabetes mellitus: progressive requirement for multiple therapies (ukpds 49). jama 1999;281: 2005–12. 30. kirk a, mutrie n, macintyre p, fisher m. effects of a 12-month physical activity counselling intervention on glycaemic control and on the status of cardiovascular risk factors in people with type 2 diabetes. diabetologia 2004;47:821–32. c:\users\jurnal fkusakti\docume 145 aspirin for primary prevention of cardiovascular events: what are the evidences? yenny department of pharmacology, faculty of medicine, trisakti university cardiovascular disease (cvd) is the leading cause of mortality in the united states, accounting for about 1 in 3 deaths.(1) in view of the healthcare and economic burdens of cardiovascular disease, the public health aim should be the prevention of cardiovascular e vents thr ough risk factor optimization. aspirin is essentia l for antiplatelet pharmacotherapy in patients with cardiovascular diseases because it plays a well-established role in secondary prevention and is widely available and affordable. it has been suggested that aspirin doses <75 mg daily may be more effective than higher doses because they spare prostacyclin (an antiplatelet and vasodilator) and cause less gastrointestinal toxicity.(2) however, the use of aspirin in primary prevention of cardiovascular events is controversial. several large scale placebo-controlled randomized studies have been conducted to determine the effect of low-dose aspirin (100 mg/day) in the prevention of primary cardiovascular events, among others the aspirin to reduce risk of initial vascular event (arrive) trial,(3) a study of cardiovascular events in diabetes (ascend),(4) and the aspirin in reducing events in the elderly (aspree) trial,(5) with durations of 2.5, 7.4, and 4.7 years, respectively. of the three above-mentioned studies, it was only ascend (4) that showed the efficacy of aspirin in the prevention of primary cardiovascular events, i.e. in subjects with moderate cardiovascular risks. the increase in risk of bleeding in the group of subjects using aspirin was reported in all three studies. to date there is no strong evidence with regard to the specific dose of aspirin to be used in primary prevention of cardiovascular events, but the low dose aspirin used in in clinical practice is 75 to 100 mg/day. the use of higher doses does not increase the efficacy, but increases the risk of gastrointestinal hemorrhage. a meta-analysis carried out by lin et al.(6) showed that the dose of aspirin used in studies on the effect of aspirin in primary cardiovascular prevention ranges from 100 mg every other day to 650 mg per day. this is due to the fact that even though the anti-thrombotic effect of aspirin is apparently not doserelated, since inhibition of platelet cox-1 by aspirin is saturated at low doses, the gastrointestinal toxicity is doserelated.(7) the controversy on the results of the largescale studies is also reflected in the various guidelines used in clinical practice. the european society of cardiology (esc) guideline (8) does not recommend the use of aspirin for primary prevention of cardiovascular events. the us preventive service task force guideline (9) recommends the use of aspirin in individuals 50– 59 years of age who have a 10-year risk of cv disease of 10%. the american college of cardiology/american heart association (acc/ aha) task force on clinical practice guideline (10) recommends aspirin only in individuals with atherosclerotic cardiovascular disease (ascvd) ages 40-70 years who are at high risk for ascvd, but are not at high risk for gastrointestinal hemorrhage. t he american college of universa medicina september-december 2019 vol.38no.3 146 cardiology/american heart association do not recommend aspirin in subjects >70 years of age, or at any age who are at high risk for hemorrhage. based on existing evidence the benefit of low-dose aspirin in the prevention of primary cardiovascular events is still a controversial subject that impacts on the variety of existing clinical guidelines. in a meta-analysis of 13 trials with 164,225 participants without cardiovascular disease, aspirin use was associated with a lower risk of cardiovascular events, defined as cardiovascular death, nonfatal myocardial infarction, and nonfatal stroke (hazard ratio [hr], 0.89; absolute risk reduction, 0.41%) and an increased risk of major bleeding (hr, 1.43; absolute risk increase, 0.47%).(11) from a practical point of view, should healthy people take a daily aspirin? the logical answer should be no, unless recommended by your physician. although it is an inexpensive over-thecounter drug, aspirin may cause bleeding, the risk increasing with age. therefore, healthy individuals with no history of heart disease should not just start taking aspirin without medical advice. however, for healthy individuals at very high risk of heart attack, based on current smoking, family history of premature heart attacks, or very high cholesterol level with intolerance to statins, the preventive use of low-dose aspirin might be justified, after consultation with the cardiologist. references 1. mozaffarian d, benjamin ej, go as, et al. american heart association statistics committee and stroke statistics subcommittee heart disease and stroke statistics—2015 update: a report from the american heart association. circulation 2015; 131:e29-322. doi: 10.1161/cir.0000000000000 152. 2. gargiulo g, windecker s, vranckx p, gibson cm, mehran r, valgimigli m. a critical appraisal of aspirin in secondary prevention: is less more? circulation 2016;134:1881–1906. doi: 10.1161/ circulationaha.116.023952. 3. gaziano jm, brotons c, coppolecchia r, et al. use of aspirin to reduce risk of initial vascular events in patients at moderate risk of cardiovascular disease (arrive): a randomised, double-blind, placebo-controlled trial. lancet 2018;392:1036-46. doi: 10.1016/s0140-6736(18) 31924-x. 4. bowman l, mafham m, wallendszus k, et al. effects of aspirin for primary prevention in persons with diabetes mellitus. new engl j med 2018;379:1529-39. doi: 10.1016/j.jvs.2018.10.072. 5. mcneil jj, wolfe r, woods rl, et al. effect of aspirin on cardiovascular events and bleeding in the healthy elderly. new engl j med 2018;379:1509-18. doi: 10.1056/nejmoa1805819. 6. lin mh, lee ch, lin c, et al. low-dose aspirin for the primary prevention of cardiovascular disease in diabetic individuals: a meta-analysis of randomized control trials and trial sequential analysis. j clin med 2019;8:pii.e609. doi: 10.3390/ jcm8050609. 7. rothwell pm, cook nr, gaziano jm, et al. effects of aspirin on risks of vascular events and cancer according to bodyweight and dose: analysis of individual patient data from randomised trials. lancet 2018;392:387-99. doi: https://doi.org/ 10.1016/s0140-6736(18)31133-4. 8. piepoli mf, hoes aw, agewall s, et al. european guidelines on cardiovascular disease prevention in clinical practice: the sixth joint task force of the european society of cardiology and other societies on cardiovascular disease prevention in clinical practice (constituted by representatives of 10 societies and by invited experts) developed with the special contribution of the european association for cardiovascular prevention & rehabilitation (eacpr). eur heart j 2016;37:2315-81. doi: 10.1093/eurheartj/ehw106. 9. bibbins-domingo k. us preventive services task force. aspirin use for the primary prevention of cardiovascular disease and colorectal cancer: us preventive services task force recommendation statement. ann inter med 2016;164:836-45. doi: 10.7326/m16-0577. 10. arnett dk, blumenthal rs, albert ma, et al. 2019 acc/aha guideline on the primary prevention of cardiovascular disease. circulation 2019: cir0000000000000677. doi: 10.1161/cir. 0000000000000677. 11. zheng sl, alistair j. roddick aj. association of aspirin use for primary prevention with cardiovascular events and bleeding events: a systematic review and meta-analysis. jama 2019; 321:277-87. doi: 10.1001/jama.2018.20578. universa medicina september-december 2019 vol.38no.3 univ med 2019;38:145-7. doi: http://dx.doi.org/10.18051/univmed.2019.v38.145-146 c:\users\jurnal fkusakti\docume 156 *trauma research center, baqiyatallah university of medical sciences, tehran, iran correspondence: marzieh lak anesthesiologist, trauma research center, baqiyatallah university of medical sciences, tehran, iran tel: +989125262585 e-mail: marziehlak@yahoo.com orcid id: 0000-0003-3498-4653 date of first submission, october 24, 2 01 8 date of final revised submission, october 9, 2019 date of acceptance, october 14, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina preemptive analgesic effect of magnesium sulfate on postoperative pain in patients undergoing lumbar fusion surgery abasali delavari*, marzieh lak*, hasan araghizadeh*, and babak salatini* background reducing postoperative pain can improve patient satisfaction and hospital cost. intravenous magnesium sulfate is one of the proposed drugs for preemptive analgesia. the study aimed to evaluate the effect of magnesium sulfate on postoperative pain in patients undergoing lumbar fusion surgery. methods a double-blind randomized clinical trial was conducted on candidates for vertebral fusion surgery with american society of anesthesiologists (asa) class i-ii. one hundred and two patients were randomized into the magnesium sulfate group and control group. the magnesium sulfate group received magnesium sulfate at a dose of 50mg/kg in 20 ml volume and infused during 15-30 minutes pre-operation, while the control group received 20 ml normal saline. the severity of the pain was assessed by the visual analogue scale (vas) at 2, 4, 6, 12 and 24 hours after patients entering the post-anesthetic care unit. after the operation, the patients’ relaxation rate was assessed based on ramsay sedation score (rss). results there was no significant difference at 2, 4, 6, 12 and 24 hours post-operation between the two groups in the mean severity of pain (p>0.05). there was no significant difference at 2, 4, 6, 12 and 24 hours post-operation between the two groups in the mean of pethidine consumption. there was no significant difference in the relaxation of the patients according to the rss criteria in the magnesium sulfate and control groups (p=0.162). conclusion pre-operative administration of magnesium sulfate does not affect reduction in postoperative pain and opioid consumption of patients undergoing lumbar fusion surgery. keywords: pain, magnesium sulfate, vertebral fusion surgery original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.156-163 september-december, 2019 vol.38no.1 ci te t his arti cle as: dela vari , la k, araghizadeh, et al. preemptive analgesic effect of magnesium sulfate on postoperative pain in patients undergoing lu mbar fusion surgery. un iv m ed 201 9;38 :1 5 6-6 3 . doi: 10 .18 05 1/ univmed.2019.v38.156-163 157 univ med vol. 38 no.3 introduction postoperative pain is one of the most c o mmo n c omp li c a t io n s of a l l s ur gi c a l p roc e du r es . t he r e a r e cur re n tly va r i ous methods to control postoperative pain,(1) one of which is preemptive analgesia. in this method prior to surgery and tissue damage, the medicine i s a d min i s ter e d t o t he p a ti e n ts . ( 2) t h e s e medications through controlling the pain pathways and preventing the release of pain mediators can better control postoperative pain.(3) preemptive analgesia is the strategy of preventing the establishment of pain signals and is designed to prevent central sensitization. to reduce acute pain after tissue damage and preventing postoperative pain and its spread is the main purpose of preemptive analgesia.(4) intravenous magnesium sulfate administration is one of the most ef fect ive me th ods of preemptive analgesia.(5) the second most important intracellular ion after potassium is magnesium that is effective in several physiological functions and enzymatic activities.(6,7) other magnesium tasks are protein synthesis, vasomotor system adjustment, and neurotransmitter.(6,8) magnesium also plays a role as a non-competitive n-methyl d-aspartate (ndma) glutamate receptor antagonist that is involved in the acute pain process as well as postoperative pain. the mechanism of the analgesic effect of magnesium sulfate is by interfering with calcium channels and nmda receptors, as well as reducing the release of c a t e c ho l a min e . m agn e s i um s ul f a t e b y increasing the pain threshold also helps prevent pain.(9-13) since 1990, the effect of magnesium sulfate has been studied on postoperative analgesia and decreased postoperative opioid consumption.(6,14-16) several studies have reported contradictory results of the effect of magnesium sulfate on postoperative pain relief. some of these studies suggest that pre-operative magnesium sulfate significantly reduced postoperative pain.(17,18) however, some other studies do not support that.(16,19) the aim of the present study was to evaluate the effect of magnesium sulfate on postoperative pain in patients undergoing lumbar fusion surgery. methods research design a double-blind randomized clinical trial was conducted in baqiyatallah hospital between february 2018 and january 2019. research subjects all patients who were candidates for vertebral fusion surgery with american society of anesthesiologists (asa) class i-ii and aged between 20 to 70 years were included in the study. by a computer-generated randomization table, patients were divided into two groups: magnesium sulfate (ms) group (n=51) and control group (normal saline group) (n=51). the exclusion criteria included a history of previous spinal surgery, major systemic disorders in vital organs such as liver, kidney and heart, magnesium sulfate susceptibility, use of calcium b loc ke rs , p re gna nt w ome n , ne ur ol ogic a l disorders, opiate addiction patients, a history of psychological illness or history of taking psychiatric drugs, bmi less than 30. patients who consumed a non-protocol drug during the study were also excluded. sample size determination t he s a mpl e s i ze d e t e r mi na ti o n w a s according to a moderate effect size of 0.5, a statistical power of 80%, and a confidence coefficient of 95%, and yielded 51 cases per group. for this sample size determination, gpower-3.1.7 software and its sample size formula for comparing two means between two groups were used. interventions a special code was assigned to each syringe using a random number table. the syringes containing magnesium sulfate and 158 control were completely uniform in terms of color and specification, and the patients and anesthesiologist who injected anesthetic drugs were not aware of the type of syringe content. one of the authors was aware of the number of cases and grouping, and also the syringe content and this author delive red syringes to the anesthesiologist. the magnesium sulfate group received magnesium sulfate at a dose of 50mg/kg in 20ml volume and infused during 15-30 minutes preoperation, while the control group received 20 ml normal saline. the main outcomes were dose of propofol, remifentanil, and pethidine, vas score, and ramsay sedation score. regular monitoring was performed every five minutes on blood pressure, pulse oximetry and electrocardiogram (ecg) during the injection of magnesium sulfate, perioperatively and during recovery. during syringe injection, patients were monitored for adverse drug reactions every five minutes. in the presence of complications of magnesium sulfate, such as dizziness, drowsiness, hypotension or flushing, the rate of drug injection was reduced. in case of hypotension when mean arterial pressure (map) <65 mm hg or there was a reduction in systolic blood pressure of 30% relative to the initial blood pressure, ephedrine at a dose of 5 to 10 mg was used. after completion of magnesium sulfate administration, the patients were pre-oxygenated and pre-medicated with 2 mg midazolam and 100 mcg fentanyl. then anesthesia was induced with nesdonal at a dose sufficient to eliminate the eyelid reflex, and atracurium at a dose of 0.5 mg/ kg was used to facilitate intubation and muscle relaxation. we recorded the nesdonal dose in the checklist. to maintain anesthesia, o 2 and n 2 o ratios of 50% and propofol with an appropriate dosage were used. if no anesthetic depth had been achieved despite the injection of propofol at a dose of 100 mcg /kg/min, then remifentanil was also used. the dose of propofol and remifentanil was also recorded in the checklist. 30 minutes before the end of surgery, methadone at a dose of 5 mg was injected intramuscularly in all patients. at the end of the opera tion after complete awakening, the patient was given 40 mcg/kg neostigmine and 20 mcg/kg atropine for reversal of neuromuscular blockade and the tracheal tube was removed. durations of all surgeries in the two groups were between 2.5 to 4 hours. assessment of pain the severity of the pain was assessed by the visual analogue scale (vas) at 1, 2, 4, 6, 12 and 24 hours after patients entering the postanesthetic care unit (when the patient can express the intensity of pain according to the criteria). during the first postoperative 24 hours, vital signs were assessed at 1, 2, 4, 12 and 24 hours after the operation. in the case of vas>4, pethidine was administered intravenously at a dose of 0.5 mg/kg. assessment of the relaxation rate after the finishing surgery, the patients’ relaxation rate was assessed based on the ramsay sedation score. the method of rating patients’ relaxation was as follows: 1patient awake, anxious, agitated or restless, 2patient awake, cooperative, orientated and tranquil, 3patient drowsy with response to commands only, 4patient asleep, brisk response to stimulus, 5patient asleep, sluggish response to stimulus, for example patient reacts with difficulty to a tap on the forehead or loud stimulus, 6no response to firm nail-bed pressure or other noxious stimuli such as a tap on the forehead or loud stimulus.(20) statistical analysis data were analyzed by spss-23. normality of data distribution was investigated using kolmogorov smirnov test. comparison of qualitative variables or variables with non-normal distribution was performed using nonparametric statistical tests such as chi-square, fisher exact test, and mann-whitney u test. independent samples t-test was used in the case of quantitative data with normal distribution. a p-value less than 0.05 was considered statistically significant. delavari, lak, araghizadeh, et al magnesium sulfate in lumbar fusion surgery 159 univ med vol. 38 no.3 ethical clearance the protocol of the study was approved by ethical committee of baqiyatallah university of medical sciences under no. ir.bmsu.rec.1396. 604 cod. the informed consent form was received from each patient. results all 102 patients were divided into two groups, i.e. magnesium sulfate group (n=51) and normal saline group (n=51). all patients completed the trial (figure 1). at base-line there were no significant differences between the two groups regarding the age, sex and weight (p=0.546, p=0.529 and p=0.086 respectively). also, there were no significant differences between the two groups regard ing the hea rt rate, vas, pethi dine consumption, duration of surgery (p=0.546, p=0.186, p=0.132, and p=0.932 respectively) (table 1). there was no significant difference at 2, 4, 6, 12 and 24 hours post operation between two groups in the mean of vas (pain). in the magnesium sulfate group 40 (78.4%) and in the a b c figure 1. flow diagram of the patients characteristics treatment groups p value magnesium (n=51) saline (n=51) age (years) sex (male/female) weight (kg) heart rate vas† pethidine consumption (mg) duration of surgery (minutes) 53.25 ± 13.87 15/36 78.50 ± 7.78 80.98 ± 10.20 4.96 ± 1.35 23.72 ± 9.78 194.54 ± 120.21 51.72 ± 13.81 19/32 75.29 ± 7.64 79.79 ± 9.51 5.25 ± 1.21 22.58 ± 8.10 198.46 ± 111.26 0.546* 0.529** 0.086* 0.546*** 0.186*** 0.132* 0.932* data presented as mean ± sd, except for gender; †vas: visual analog scale; * t-test, **chi-square test, and ***mannwhitney test table 1. comparison of demographic variables (age and sex), heart rate, blood pressure, vas, pethedin consumption and duration of surgery between the two groups at base-line 160 control group 37 (72.5%) patients were awake, cooperative, orientated and tranquil. there was no statistically significant difference between the two groups (p=0.393) (table 2). there was no significant difference at 2, 4, and 6 hours post operation between the two groups in the mean of pethidine consumption (table 3) discussion th er e wa s no si gni fi ca nt dif f er en ce between the two groups in pain and injection rate of pethidine. finally, based on the rss criteria, there was no significant difference in the level of relaxation between the two groups. oguzhan et al.(17 ) and srivastava et al.(1 8) demonstrated that magnesium sulfate was effective for postoperative opioid use and pain in patients following lumbar disc surgery and gynecological operation. but, in their study, they applied a continuous or repeat bolus in addition to the primary bolus of magnesium sulfate in opposition to single bolus preoperatively in the current research. it is essential to consider that there are some significant subjects associated with the studies performed on magnesium sulfate that could be the source of differences between their outcomes: various methods of anesthesia, techniques of “single dosage” versus “bolus plus infusion” dosage, and a complete dosage of intravenous magnesium sulfate.(9) our research has been carried out to assess the effectiveness and safety of preventive single-dose intravenous magnesium sulfate for postoperative pain reduction and analgesia. furthermore, pain relief is multi-fa ceted and de pends on culture, table 2. postoperative vas scores at 2, 4, 6, 12 and 24 hours and the relaxation rate between the two groups data presented as mean ± sd, except for relaxation rate. * t-test, and ** fisher exact test.; †vas: visual analog scale; §1patient awake, anxious, agitated or restless, 2patient awake, cooperative, orientated and tranquil, 3patient drowsy with response to commands, 4patient asleep, sluggish response to stimulus table 3. comparison of pethidine consumption 2, 4, 6, 12 and 24 hours post operation between the two groups data presented as mean ± sd. *t-test and **mann-whitney u test delavari, lak, araghizadeh, et al magnesium sulfate in lumbar fusion surgery pethidine consumption (mg) magnesium sulfate (n = 51) control (n = 51) p value 2 h 11.76 ± 12.60 13.92 ± 11.32 0.441* 4 h 2.15 ± 6.72 1.47 ± 5.12 0.688* 6h 0.21 ±1.90 0.39 ± 2.80 0.317** magnesium sulfate (n=51) control (n=51) p value vas† 2 h 4 h 6 h 12 h 24 h 3.56 ± 1.34 2.52 ± 1.12 1.92 ± 0.68 1.50 ± 0.67 1.56 ± 0.67 3.82 ± 1.12 2.41 ± 0.96 1.90 ± 0.75 1.68 ± 0.70 1.35 ± 0.56 0.175* 0.175* 0.175* 0.165* 0.087* relaxation rate§ 1 2 3 4 6 40 3 2 8 37 6 0 0.393** 161 univ med vol. 38 no.3 genetics, sex, r ace, socioeconomic state, cognition, and previous pain concept. taheri et al.(10) and rezae et al.(4) assessed the effect of single dose magnesium sulfate in patients with hysterectomy and cesarean section. they s ho we d t ha t pe t h i di n e c on s ump ti o n a n d postoperative pain were lower in the magnesium sulfate group. pains following hysterectomy are multivariable and different from other pains e.g. surgery pain, visceral constructions pain, and dynamic pain (while straining, coughing and mobilizing). so, differences in the type of pains may be the sources of conflict between these studies. a meta-analysis showed that magnesium sulfate is capable of decreasing pain severity in early-stage and anesthetic consumption following laparoscopic cholecystectomy.(15) a systematic review in patients undergoing orthopedic operation demonstrated that the studies did not give persuasive evidence of useful impacts on postoperative pain severity and the time to first analgesic demand.(16) due to the heterogeneity of orthopedic surgeries that were reported in their studies comparison of the duration of surgeries between the two studies (15,16) is impossible. in another study manaa et al.(9) examined the effect of magnesium sulfate on postoperative anesthesia and analgesia in patients undergoing neurosurgical procedures. in this study, patients received magnesium sulfate (20 mg/kg) pre-operation then magnesium sulfate infusion (10 mg/kg/h) continued until the end of surgery. results showed that postoperative pain score, as well as a total analgesic requirement of morphine, was significantly lower in the magnesium group compared to the control group. their results were in disagreement with the current study. patients in magnesium group underwent bolus dose 20 mg/kg over five mi nu t e s b ef o r e i nd u c t i on o f an e s th e s i a , continued through 10 mg/kg/h as an infusion, in contrast to our study where the magnesium sulfate group received magnesium sulfate at a dose of 50mg/kg in 20ml volume and infused during 15-30 minutes pre-operation. a study by el r a hma n e t al . ( 2 1 ) e val u a t e d t h e r ole o f p r e o pe r a ti ve ma gn e s iu m s ul f a t e gui d e d transversus abdominis plane block in augmenting postcesarean analgesic effects. it started in the r e c ove r y u ni t w he r e p a t i e n ts r e c e i ve d lornoxicam, which is a non-steroidal substance. the mean pain at 6 and 12 hours postoperatively was significantly lower in the magnesium sulfate group than in the control group. the current study confirmed their results regarding the nonsignificant between-group differences in mean pain at other times. in the studies of taheri et al.(10) and elrahman et al.(21) the fact that analgesics were not used in both groups and/or non-steroidal analgesics were used to control postoperative pain was probably due to the potent effect of the opioid used. in the present study and studies consistent with the current study, the effectiveness of magnesium sulfate has been overshadowed by an opiate. so, differences in opioid consumption and pain in the two groups were not significant. however, in studies that did not use analgesics for postoperative pain control or where non-steroidal analgesics were u se d f o r ana l ge s i a , t he e f f e c ti ve ne ss of magnesium sulfate was clearly seen in reducing pain and opioid consumption. however, in the studies of rezae et al.(4) and manaa et al.(9) who used opioids for postoperative analgesia, the d if f e r e nce b e t we e n t h e tw o gr o up s w a s significant. kiran et al.(2) reported that a single dose of intravenous magnesium sulfate could decrease post-operative pain in patients after inguinal operation. also, the degree of relaxation was lower in patients in the group receiving magnesium sulfate than the control group. they also showed that patients receiving magnesium s ul f a t e woul d b e l e s s l ike l y to r e c e i ve postoperative analgesics. their results were in disagreement with the current study. it may be because of differences between type of patients and surgical conditions in the two groups. ghaffaripour et al.(22) investigated the effects of intravenous magnesium sulfate on postoperative analgesia after laminectomy. magnesium sulfate was administered as a bolus followed by a maintenance dose up to the end 162 of the surgery. they showed that the infusion of magnesium sulfate during laminectomy had n o e f f e c t o n p a t i e n t s’ p a i n a nd o p io i d requirement postoperatively. kahraman et al.(23) showed that an infusion of magnesium sulfate has no effect on morphine consumption and postoperative pain in patients undergoing abdominal hysterectomy under spinal anesthesia. in the studies of frassanito et al.,(13) kahraman et al.(23) and ghaffaripour et al.(22) whose results were consistent with the present study, morphine was used for postoperative analgesia, as we also used methadone for postoperative analgesia. also, in the study of frassanito et al.(13) and kahraman et al.,(23) patients were spinally anesthetized. there were some limitations in our study. fi r s t , we a p p li e d on l y o ne do s e of magnesium sulfate, while different regimens may have yielded different outcomes. second, the infusion was restricted to the intraoperative interval only. third, we did not evaluate the relationships between the magnesium level and the total anesthetic and analgesic demands. finally, we did not assess the economical aspects of magnesium sulfate administration compared t o o t he r an e s t he t i c me di c a t io n s. it i s recommended to conduct more comprehensive s t ud i e s t o e va l u a t e t he e f f e c ti ve ne s s of magnesium sulfate in patients undergoing lumbar fusion surgery and in patients for whom the use of opiates is harmful (such as obese patients, etc.). conclusion the results showed that pre-operative administration of magnesium sulfate has no effect on reduction of postoperative pain and opioid consumption in patients undergoing lumbar fusion surgery. conflict of interest we declare that there is no conflict of interest in this study. acknowledgement we would like to thank the staff of the trauma research center, baqiyatallah university of medical sciences for their kind cooperation in this research. contributors all authors have contributed to the concept, design of methods, data gathering, data analysis and preparation of the final manuscript. all authors have read and approved the final manuscript. references 1. dabbagh a, elyasi h, razavi s, et al. intravenous magnesium sulfate for post-operative pain in patients undergoing lower limb orthopedic surgery. acta anaesthesiol scand 2009;53:108891. doi: 10.1111/j.1399-6576.2009.02025.x. 2. kiran s, gupta r, verma d. evaluation of a singledose of intravenous magnesium sulphate for prevention of postoperative pain after inguinal surgery. indian j anaesth 2011;55:31-5. doi: 10.4103/0019-5049.76605. 3. vadivelu n, mitra s, schermer e, et al. preventive analgesia for postoperative pain control: a broader concept. local reg anesth 2014;7:17-22. doi: 10.2147/lra.s62160. 4. rezae m, naghibi k, taefnia am. effect of preemptive magnesium sulfate infusion on the postoperative pain relief after elective cesarean section. adv biomed res 2014;3:164. doi: 10.4103/ 2277-9175.139127. 5. do sh. magnesium: a versatile drug for anesthesiologists. korean j anesthesiol 2013;65:4. doi: 10.4097/kjae.2013.65.1.4. 6. al alawi am, majoni sw, falhammar h. magnesium and human health: perspectives and research directions. int j endocrinol 2018; 9041694. doi: 10.1155/2018/9041694 7. mishra ak, afzal m, mookerjee ss, et al. preemptive analgesia: recent trends and evidences. indian j pain 2013;27:114-20. doi: 10.4103/09705333.124582. 8. yamanaka r, shindo y, oka k. magnesium is a key player in neuronal maturation and neuropathology. int j mol sci 2019;20:3439. doi:10.3390/ijms20143439. delavari, lak, araghizadeh, et al magnesium sulfate in lumbar fusion surgery 163 univ med vol. 38 no.3 9. manaa e, alhabib a. effect of magnesium sulfate on the total anesthetic and analgesic requirements in neurosurgery. j neurol neurophysiol 2012; s11001. doi: 10:2155-9562. 10. taheri a, haryalchi k, ghanaie m, et al. effect of low-dose (single-dose) magnesium sulfate on postoperative analgesia in hysterectomy patients receiving balanced general anesthesia. anesthesiol res pract 2015;2015:306145. doi: 10.1155/2015/306145. 11. ryu jh, kang mh, park ks, et al. effects of magnesium sulphate on intraoperative anaesthetic requirements and postoperative analgesia in gynaecology patients receiving total intravenous anaesthesia. br j anaesth 2008;100:397-403. doi: 10.1093/bja/aem407. 12. lysakowski c, dumont l, czarnetzki c, et al. magnesium as an adjuvant to postoperative analgesia: a systematic review of randomized trials. anesth analg 2007;104:1532-9. doi: 10.1213/ 01.ane.0000261250.59984.cd. 13. frassanito l, messina a, vergari a, et al. intravenous infusion of magnesium sulfate and postoperative analgesia in total knee arthroplasty. minerva anestesiol 2015;81:1184-91. 14. cavalcanti il, de lima fl, da silva mj, et al. use profile of magnesium sulfate in anesthesia in brazil. front pharmacol 2019;10:429. doi: 10.3389/ fphar.2019.00429. 15. chen c, tao r. the impact of magnesium sulfate on pain control after laparoscopic cholecystectomy: a meta-analysis of randomized controlled studies. surg laparosc endosc percutan tech 2018;28:349-53. doi: 10.1097/ sle.0000000000000571. 16. peng yn, sung fc, huang ml, et al. the use of intravenous magnesium sulfate on postoperative analgesia in orthopedic surgery: a systematic review of randomized controlled trials. medicine 2018;97:e13583. doi: 10.1097/md.00000000000 13583. 17. oguzhan n, gunday i, turan a. effect of magnesium sulfate infusion on sevoflurane consumption, hemodynamics, and perioperative opioid consumption in lumbar disc surgery. j opioid manag 2008;4:105-10. doi: 10.5055/ jom.2008.0015. 18. srivastava vk, mishra a, agrawal s, e al. comparative evaluation of dexmedetomidine and magnesium sulphate on propofol consumption, haemodynamics and postoperative recovery in spine surgery: a prospective, randomized, placebo controlled, double-blind study. adv pharm bull 2016;6:75-81. doi: 10.15171/ apb.2016.012. 19. demiroglu m, un c, ornek dh, et al. the effect of systemic and regional use of magnesium sulfate on postoperative tramadol consumption in lumbar disc surgery. biomed res int 2016, article id 3216246, 5 pages. http://dx.doi.org/10.1155/2016/ 3216246. 20. mottahediantabrizi e, tadrissi sd, mohammad yari a, et al. validity and reliability of ramsay sedation scale in intensive care unit adult patients. iranian j critical care nursing spring 2010;3;39-44. 21. elrahman tna, youssry ma. the impact of single low dose iv magnesium sulphate adjuvant to ultrasound guided transverses abdominis plain block for control of postcesarean pain. open j obstet gynecol 2017;7:269-80. doi: 10.4236/ ojog.2017.73029. 22. ghaffaripour s, mahmoudi h, eghbal h, et al. the effect of intravenous magnesium sulfate on post operative analgesia during laminectomy. cureus 2016;8:e626 . doi: 10.7759/cureus.626. 23. kahraman f, eroglu a. the effect of intravenous magnesium sulfate infusion on sensory spinal block and post operative pain score in abdominal hysterectomy. biomed res int 2014, article id 236024, 5 pages. http://dx.doi.org/10.1155/2014/ 236024. c:\users\jurnal fkusakti\docume 167 *medical biotechnology research center, ashkezar branch, islamic azad university, ashkezar, yazd, iran correspondence: dr.mahmood dehghani ashkezari medical biotechnology research center, ashkezar branch, islamic azad university, ashkezar, yazd, iran email: drmdehghani31@gmail.com orcid id : orcid.org/0000-00025349-2083 date of first submission, june 28, 2018 date of final revised submission, september 12, 2018 date of acceptance, september 13, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina wnt7a (rs104893832) polymorphism increases the risk of recurrent spontaneous abortion in iranian women manouchehr mazdapour*, mahmood dehghani ashkezari*, and seyed morteza seifati* background recurrent spontaneous abortion is defined as the occurrence of three or more clinical miscarriages in one woman. several factors, including genetics and environmental factors, are involved in this kind of infertility, in which wnt7a (rs104893832) polymorphism plays a major role. the aim of the present study was to determine the association between a common polymorphism of wnt7a (rs104893832) with recurrent spontaneous abortion in females. methods in the present case-control study, the wnt7a (rs104893832) polymorphism was investigated in 70 women with recurrent spontaneous abortion as cases and 100 women with at least one child and no history of infertility or abortion as controls. polymerase chain reactionrestriction fragment length polymorphism (pcr-rflp) was used to investigate the wnt7a (rs104893832) polymorphism in both case and control groups. the data were subsequently analyzed using the chi-square and logistic regression tests by spss software (version 18.0). results a significant association was found between the wnt7a (rs104893832) polymorphism and recurrent spontaneous abortion (or=25.00, 95% ci=5.52-157.09; p<0.0001). our finding showed that g allele frequency in women with recurrent spontaneous abortion was significantly different compared to the control group. (or=6.42, 95% ci=2.82-15.16; p<0.0001).therefore, genetic variation in wnt7a (rs104893832) polymorphism may play a role in recurrent spontaneous abortion. conclusion this study revealed that wnt7a (rs104893832) polymorphism increased the risk of recurrent spontaneous abortion. knowledge of these mutations and polymorphisms can provide an insight into the prognosis for individual patients. therefore, further studies are necessary to establish the association of wnt7a (rs104893832) polymorphism with recurrent spontaneous abortion in a larger population. keywords: recurrent spontaneous abortion, wnt7a, polymorphism, infertility female doi: http://dx.doi.org/10.18051/univmed.2018.v37.167-172 original article pissn: 1907-3062 / eissn: 2407-2230 september-december, 2018 vol.37 no.3 cit e this arti cle as : mazdapour m, ashkezari md, seifati sm. wnt7a (rs1048 9383 2) p olymorph ism increases the risk of recurrent spontaneous abortion in iranian women. univ med 2018;37:167-72. doi: 10.18051/ univmed.2018.v37.167-172 168 mazdapour, ashkezari, seifati wnt7a polymorphism and abortion introduction recurrent spontaneous abortion (rsa) is associated with the recurring loss of pregnancy (three times or mor e) and cause s many difficulties for couples.(1) there are many factors involved in rsa, including anatomical factors (approximately 10–15%),(2) endocrine-related factors (approximately 17-20%),(3) infections (approximately 0.5%),(4) immunological factors (approximately 3-5%),(5) thrombotic factors (approximately 15%),(6) environmental factors (approximately 5-10%), and unexplained etiologies (approximately 20-30%).(7) genetic factors are one of the most important factors involved in rsa (approximately 2-4%) and many different kinds of genes are involved in this process. gene polymorphism is associated with infertility and plays a central role in rsa.(8) in addition, parental chromosome rearrangement e.g. robertsonian translocations, chromosomal inversions, and insertions is one of the main causes of rsa. for human fertility several genes are required which are so conserved. several infertility disorders are associated with these genes. each gene has a different role in the process of fertility and any change in the sequence of these genes can lead to infertility. the most important genes in this regard are hoxa10 (fr equent embryo loss pr ior to implantation),(9) hoxa11 (uterine defects),(10) ptx3 (ovulation defects),(11) nos3 (compromised ovulation, delayed meiotic progression from meta phase i), (1 2) and wnt 7a ( abnormal development of oviducts and uterus).(13) the wnt7a gene is located on chromosome 3 (p25.1 region). wnt7a is a member of the wnt gene family and plays a role in ensuring successful pregnancy by controlling fertilization, implantation and progression. the gene is related to many other developmental processes such as cell fate specification, cell proliferation, cell migration and pregnancy. this gene encodes secreted signaling proteins, which in turn are associated with oncogenesis and developmental processes such as pregnancy.(14) therefore, the investigation of the wnt7a gene can throw new light on rsa. notably, the available evidence indicates that the wnt7a gene has a critical role in the survival and maintenance of oocytes; thus with regard to its roles, these likely manifest in fertility. this research is the first to report that wnt7a (rs104893832) polymorphism has an intense effect on rsa; however, further studies are necessary to confirm the association in other populations. the aim of the present case-control study was to estimate allele and genotype frequencies of wnt7a (rs104893832) polymorphism, in order to investigate the potential association between this polymorphism and spontaneous abortion in our population. therefore, the current study was planned to investigate the role of wnt7a (rs104893832) polymorphism in rsa in iranian females. methods research design a case-control study was performed at tehran women’s general hospital (mirza kochak khan), tehran, iran, between january 2015 and october 2016. research subjects seve nty women with more than 2 miscarriages (18–35 years old) and 100 healthy women with a history of at least one live birth and no history of infertility or abortion as controls. in this study, for the sample size determination, we used an online sample size program, https:// www.stat.ubc.ca/~rollin/stats/ssize/by clicking of “comparing proportions for two independent samples”, proportion in the population to be sampled (both control and case). based on related previous studies, p1=0.7, p2=0.9, α=0.05, and power=0.8. the required sample size (for each sample separately) was 62. the patients were also examined for known factors affecting rsa, including for anatomical abnorma lities, immunological factors and endocrine factors and testosterone, factor v leiden and prothrombin mutations, and were indicated to be normal with 169 regard to such factors. all patients and their partners had a normal karyotype. the control group was healthy women with no history of abortion and at least one live birth. genotyping of wnta genomic dna from venous blood samples (5ml) was isolated using a dna extraction kit (genid gmbh, germany) according to the manufacturer’s instructions. the purity and concentration of genomic dna were evaluated using a nanodrop spectrophotometer and a concentration of 50 ng/μl was prepared for the working tubes. amplification was done using specific primers, 5´ccgacgccatcatcgt cata -3´ as f o r w a r d pr i me r, a nd 5´gtagcggatgtcggcagag -3´ as reverse primer. the reactions were prepared in a tube containing 2 l of forward primer, 2 l of reverse primer, 5.5 l of sterilized distilled water, 3 µl of dna (50 ng/ml), and 12.5 µl of master mix. the pcr cycling conditions were maintained with an initial denaturation step for 5 min at 94 ºc, followed by 35 cycles of 45 sec at 94 oc, 40 sec at 59oc and 45 sec at 72oc and the final extension step at 72oc for 5 min. the wnt7a (rs104893832) polymorphism was determined on the basis of product size-band using bsrdi restriction enzyme digestion. the amplified fragment after digestion with bsrdi restriction enzyme, created two fragments (145 bp and 163 bp) that indicated the presence of g/g genotype, and the appearance of one undigested 308 bp fragment indicated the presence of a/a genotype, while the presence of three fragments (308bp, 145bp and 163bp) indicated a/g genotype (figure1). the pcr products were separated by electrophoresis on 3% agarose gel. in addition, 50bp dna marker was used in this experiment and the gel was visualized on a gel documentation system using gelred dye. statistical analysis statistical analysis was performed using spss s oft war e (vers ion 18. 0; spss in c, chicago, il, usa). the odds ratios (ors) and 9 5% c o nf i de n c e i nt e r va l s ( cis ) we r e determined using binary logistic regression, and the genotype and allele fr equencies were compared using the χ2 test. a p<0.05 was considered to indicate statistical significance. ethical clearance the study has been approved by the resea rch committee at the islamic azad university, yazd (ir.iau.yazd.rec.1396, 15). all subjects were informed of the study protocol in written and verbal form and provided written informed consent. results the genotype and allele frequencies of wnt7a (rs104893832) polymorphism in women with rsa and normal controls are shown in table i. the results demonstrated that the frequencies of aa, ag, ag genotypes were 92.8%, 2.9% and 4.3% respectively in cases and 52%, 40% and 8% in controls. the frequency of ag genotype was significantly different in cases and controls (or=25.00, 95% ci=5.52-157.09; p<0.0001). in addition, the frequencies of a and g alleles were 94. 2% and 5.8% in cases, and 72% and 28% in controls and g allele was significantly different between the case and control groups (or=6.42, 95% ci=2.82-15.16; p<0.0001). wnt 7a (rs104893832) polymorphism significantly increased the risk of rsa in our research subjects. therefore, wnt7a (rs104893832) polymorphism might be related to rsa in our research subjects. discussion this study was designed to determine the association between wnt7a (rs104893832) polymorphism and rsa. our results indicate that there are significant relations between this polymorphism and rsa. this research is the first to report that the wnt7a (rs104893832) polymorphism has an intense effect on rsa; however, further studies are necessary to confirm univ med vol. 37 no.3 170 mazdapour, ashkezari, seifati wnt7a polymorphism and abortion the association in other populations. the literature review demonstrated that the majority of the studies assessed the role wnt7a in the pathogenesis of rsa. a study by vainio et al.(15) suggested that the wnt family including wnt4 and specially wnt7a gene is certainly involved i n s e xu a l d e ve l op me nt a l pr oc e s s e s a n d maintenance of pregnancy. a study by fan et al.(16) showed that wnt7a expression plays a very important role in the human endometrium and in infertility. in another study, tepekoy et al.(17) reported that wnt7a plays a key role in pre-implantation and implantation and guides uterine development. van amerongen et al.(18) demonstrated that wnt signaling plays a part in fertility. however there is a vast list of genes involved in infertility. among them, wnt7a is one of the most essential genes that affect fertility. mutations and polymorphisms in wnt7a gene can cause different kinds of infertility and biological functions of wnt7a are vital in the regulation of pregnancy. our results demonstrated that there was an association between this polymorphism of wnt7a gene and rsa. our finding in this research can be a sign of this fact that the polymorphism plays an important role in rsa. in addition, results obtained by dunlap et al.(19) revealed that the deletion of the wnt7a gene inhibits uterine gland morphogenesis and causes infertility in genotype wnta7a case (n,%) control (n,%) or (95% c.i.) p value aa ag gg 65 (92.8) 2 (2.9) 3 (4.3) 52 (52.0) 40 (40.0) 8 (8.0) 1 (reference) 25.00 (5.52-157.09) 3.33 (0.75-16.00) <0.0001 0.086 allele a g 132 (94.2) 8 (5.8) 144 (72.0) 56 (28.0) 6.42 (2.82-15.16) < 0.0001 table 1. genotype and allele frequencies of wnt7a (rs104893832) polymorphism in women with rsa and in controls the data was analyzed with binary logistic regression and chi-square test. (p<0.0001) figure 1. pcr-rflp for wnt7a (rs104893832) polymorphism using bsrdi restriction enzyme digestion. lane 1 shows 50 bp dna markers on 3% agarose gel electrophoresis. lane 2 represents a/a genotype, lane 3 represents heterozygous (a/g) and lane 4 is g/g genotype 171 adult mice. gilbert (20) also indicated that the wnt7a gene is one of the most important genes that control fertilization, implantation and pregnancy. based on the previously reported functions of the wnt7a gene, our results in this study show that there is a relation between the polymorphism of wnt7a gene and rsa. the findings in this research can be an indication that the wnt7 gene plays a key role in rsa. therefore, the wnt7a (rs104893832) polymorphism may be a genetic risk factor for rsa in our population. with the increasing recognition of genetic-environmental interactions in the etiology of rsa, it is necessary to include genetic information into epidemiological studies to identify how some polymorphic traits are associated with several phenotypic traits. therefore, the indicated association of the wnt7a (rs104893832) polymorphism may be p op ul a t i on-s p e c i f i c a n d i nfl u e n c e d b y environmental factors. however, our knowledge about the genetic factors determining rsa in general population is still limited. and this association might be population specific and highly influenced by environmental factors. however further studies are necessary to confirm the association in other populations. conclusion the results conclusively indicated that w n t 7 a ( r s1 04 8 93 83 2 ) p ol ymo r p hi s m increased the risk of rsa in our research subjects. conflict of interest there is no conflict of interest in this report. acknowledgment special thanks are due to tehran women’s general hospital (mirza kochak khan), and medical biotechnology research center of ashkezar branch for financial support. authors’ contributions mm was responsible for writing of the article. mda was responsible for submission of the article. sms was the advisor. all authors have read and approved the final manuscript. references 1. doubilet pm, benson cb, bourne t, et al. diagnostic criteria for nonviable pregnancy early in the first trimester. n engl j med 2013;369:144351. doi: 10.1056/nejmra1302417. 2. larsen ec, christiansen ob, kolte am, et al. new insights into mechanisms behind miscarriage. bmc med 2013;11:154-9. doi: 10.1186/17417015. 11.154. 3. kaur r, gupta k. endocrine dysfunction and recurrent spontaneous abortion: an overview. int j appl basic med res 2016;6:79-83. doi: 10.4103/ 2229-516x.179024. 4. nigro g, mazzocco m, mattia e, et al. role of infections in recurrent spontaneous abortion. j matern fetal neonatal med 2011;24:983-89. doi: 10.3109/14767058.2010.547963. 5. wong lf, porter t, scott jr. immunotherapy for recurrent miscarriage. cochrane database of systematic reviews 2014;10:112-8. doi: 10.1002/ 14651858.cd000112.pub3. 6. battinelli em, marshall a, jean m. the role of 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abortion 12. tiwari m, prasad s, pandey na, et al. nitric oxide signaling during meiotic cell cycle regulation in mammalian oocytes. front biosci 2017;9:307-318. doi: 10.2741/s489. 13. garavelli l, wischmeijer a, rosato s, et al. alawadi-raas-rothschild (limb/pelvis/uterus— hypoplasia/aplasia) syndrome and wnt7a mutations: genetic homogeneity and nosological delineation. am j med genet 2011;155:332-6. doi: 10.1002/ajmg.a.33793. 14. sondereggera s, pollheimerb j, knoflerb m. wnt signalling in implantation, decidualisation and placental differentiation – review. placenta 2010;31:839–47. doi: 10.1016/j.placenta.2010. 07.011. 15. vainio s, heikkila m, kispert a, et al. female development in mammals is regulated by wnt-4 signalling. nature 1999;397:405-409. 16. fan x, krieg s, hwang j, et al. dynamic regulation of wnt7a expression in the primate endometrium: implications for postmenstrual regeneration and secretory transformation. endocrinol 2012:153: 1063-1069. doi: 10.1210/en.2011-1826. 17. tepekoy f, akkoyunlu g, demir r. the role of wnt signaling members in the uterus and embryo during pre-implantation and implantation. j assist reprod genet 2015;32:337-348 doi: 10.1007/s10815-014-0409-7. 18. dunlap ka, filant j, hayashi k, et al. postnatal deletion of wnt7a inhibits uterine gland morphogenesis and compromises adult fertility in mice. biol reprod 2011;85:386–96. doi: 10.1095/ biolreprod.111.091769. 19. van amerongen r, nusse r. towards an integrated view of wnt signaling in development. development 2009;136:3205-14. doi: 10.1242/ dev.033910. 20. gilbert sf. developmental biology. 9th ed. sunderland: mass sinauer associates;2010. oktavianus 128 *department of parasitology faculty of medicine trisakti university **department of parasitology faculty of medicine university of indonesia correspondence dr. suriyani tan, m.biomed department of parasitology, faculty of medicine, trisakti university jl kyai tapa no.260, jakarta-11440 phone: 6221-5672731 ext.2211 email : suriyanitan@gmail.com univ med 2013;32:128-34 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background according to the world health organization, malaria is the major cause of death from tropical infections in children and pregnant women. severe anemia is a common complication of malaria, particularly malaria caused by plasmodium falciparum. the purpose of the present study was to determine any association between plasmodium falciparum infection and anemia in school children. methods this was an observational study with cross-sectional approach. a total of 540 whole blood samples of school children after administration of anthelminthic treatment for 30 days, were collected for measurement of hemoglobin concentration and preparation of giemsa-stained thin and thick blood smears. the species of plasmodium was confirmed by real-time polymerase chain reaction (pcr). chi-square and prevalence ratio (pr) tests were used to determine the relationship between plasmodium falciparum infection and anemia. results the prevalence of plasmodium infection in school children of malarial endemic areas was 3.51%. there were 43 cases of anemia with 41 cases of mild anemia and 2 cases of moderate anemia. among the 41 mild cases of anemia, plasmodium falciparum infection was found in only 3 cases. the 2 cases of moderate anemia and the remaining 38 cases of mild anemia were not caused by plasmodium falciparum infection. subjects with plasmodium falciparum infection had a 1.91-fold higher risk of suffering from anemia than subjects without plasmodium falciparum infection, but not significant (pr=1.91; 95% c.i.=0.65-5.62). conclusions plasmodium falciparum infection did not significantly increase the risk of anemia in school children. efforts to improve strategies for controlling anemia and malaria are needed. key words: plasmodium falciparum, anemia, school children plasmodium falciparum infection and the risk of anemia in school children suriyani tan*, taniawati supali**, and heri wibowo** 129 univ med vol. 32 no.2 infeksi plasmodium falciparum dan risiko terjadinya anemia pada anak sekolah latar belakang world health organization menyatakan bahwa malaria merupakan penyebab kematian utama penyakit infeksi tropis pada anak-anak dan wanita hamil. anemia berat merupakan komplikasi yang umum dari malaria, terutama malaria yang disebabkan oleh plasmodium falciparum. penelitian ini bertjuan untuk menentukan adanya hubungan antara infeksi plasmodium falciparum dan anemia pada anak sekolah. metode penelitian ini merupakan penelitian observasional dengan pendekatan cross-sectional. sejumlah 540 sampel darah anak sekolah yang telah mendapat terapi kecacingan selama 30 hari, diambil untuk pengukuran kadar hemoglobin dan pembuatan preparat malaria darah tebal serta darah tipis dengan pewarnaan giemsa. spesies plasmodium dipastikan dengan menggunakan real time polymerase chain reaction (pcr). digunakan uji chi-square dan rasio prevalensi (rp) untuk menentukan hubungan antara infeksi plasmodium falciparum dengan anemia. hasil tingkat infeksi plasmodium pada anak sekolah di daerah endemis malaria sebesar 3,51%. terdapat 43 kasus anemia dengan kasus 41 anemia ringan dan 2 kasus anemia sedang. dari total 41 kasus anemia ringan, infeksi plasmodium falciparum hanya ditemukan pada 3 kasus. dua kasus anemia sedang dan 38 kasus anemia ringan yang dialami oleh subyek ternyata bukan disebabkan oleh infeksi plasmodium falciparum. subyek yang terkena infeksi plasmodium falciparum mempunyai resiko 4,22 kali untuk mengalami anemia dibandingkan dengan subyek yang tidak terinfeksi, tetapi tidak bermakna (rp=1,91;95% c.i.=0,65-5,62). kesimpulan infeksi plasmodium falciparum tidak mampu secara bermakna meningkatkan risiko terjadinya anemia pada anak sekolah. diperlukan upaya peningkatan strategi pengendalian anemia dan malaria. kata kunci : plasmodium falciparum, anemia, anak sekolah abstrak introduction to date malaria is still the major infectious disease leading to high rates of disability and mortality in tropical and subtropical countries. t h e wo r l d h e a l t h o rg a n i z a t i o n ( w h o ) recorded in 2008 an annual total of 247 million malaria cases worldwide, with one million deaths and 85% of these deaths occurring in the under-fives. this makes malaria a health problem in endemic countries that urgently needs attention and integrated management.(1) a total of 3.3 milliard people throughout the world live in 450 malarial endemic countries.(1) the highest mortality rate, especially in the under-fives, occurs in the region of sub-saharan africa, while the second highest mortality rate is in southeast asia.(2) the manifestations of clinical malaria is variable, from asymptomatic parasitemia in semi-immune populations to severe malaria in susceptible populations, such as the under-fives, pregnant women, and n o n i m m u n e i n d i v i d u a l s . in susceptible populations, clinical malaria manifests itself in 130 tan, supali, wibowo plasmodium falciparum infection and a nemia various forms, such as cerebral malaria, severe anemia, metabolic acidosis, hypoglycemia, and other symptoms. (3,4) cerebral malaria and severe anemia are the major causes of death in children. cerebral malaria occurs more frequently in children older than 2 years in areas of low transmission. on the other hand, severe anemia commonly occurs in children under 2 years old in areas of high transmission.(5,6) severe anemia is a frequent complication o f m a l a r i a , p a r t i c u l a r l y m a l a r i a d u e t o plasmodium falciparum. the pathogenesis of anemia in malaria is at present unclear, since it is complex and multifactorial. anemia results in a mortality rate of 15-20% among patients with malaria, in spite of their having received anti-malarial drugs. damage to erythrocytes infected with plasmodium is a minor factor contributing to the pathogenesis of malaria. it has been demonstrated that parasite density is not directly associated with the degree of anemia.(7,8) presumably other factors contribute to the pathogenesis of malarial anemia, such as abnormal erythropoiesis in the bone marrow as a result of proinflammatory cytokines, excessive destruction of plasmodium-infected o r n o n i n f e c t e d e r y t h r o c y t e s b y s p l e n i c macrophages, and intravascular hemolysis of erythrocytes infected with the parasite.(9) in sub-saharan africa more than 3.8 million children below the age of 10 years s u f f e r f r o m f a l c i p a r u m m a l a r i a a n d a n estimated 2.6 million among these become anemic. anemia in children with malaria is still a public health problem, since it affects their s u r v i v a l a n d d e v e l o p m e n t o f c o g n i t i v e functions. low hemoglobin levels lead to impaired intellectual and motor functions of the children. younger children will have a more severe anemia.(10) t h e s u b d i s t r i c t ( k e c a m a t a n ) o f nangapanda, nusa tenggara timur, is an area e n d e m i c f o r m a l a r i a . t h e p r e v a l e n c e o f plasmodium falciparum and plasmodium v i v a x i s 1 2 % . t h e t o t a l p o p u l a t i o n o f kecamatan nangapanda is 22,000 and 27% of them are children of school-going age who are a t r i s k f o r m a l a r i a l i n f e c t i o n . ( 11 ) wi t h a population of school children constituting onefourth of the total population of kecamatan nangapanda, it was considered necessary to conduct a study to determine any relationship between plasmodium falciparum infection and anemia in school children. methods design of the study a study of observational cross-sectional design was conducted from june 2011 until january 2013. study subjects the subjects of this study were all of the 540 school children aged 5 to 18 years attending primary schools (sd), junior high schools (smp) and senior high schools (smu) in k e c a m a t a n n a n g a p a n d a , n u s a te n g g a r a timur. measurement of hemoglobin concentration hemoglobin (hb) concentration was measured with a sysmex kx-21 hematology analyzer, using tubes containing 3 ml of blood with added edta as anticoagulant. subjects were categorized as having anemia if the hb concentration was <11g/dl.(12) microscopic examination of blood detection of plasmodium spp. in thin and thick blood smears was done conventionally by giemsa staining. real-time pcr dna was isolated from 200 µl whole blood using the qiaamp® dna mini kit (qiagen, jerman), according to the qiagen manual. realtime pcr was performed with the hotstartag mastermix kit (qiagen, jerman). specific primers for each gene were obtained from genbank accession. p. falciparum xs-probe was labelled with yakima yellow, and p.vivax 131 univ med vol. 32 no.2 table 1. association between plasmodium falciparum infection and anemia category *chi-square test : non significant non-anemia; hb ≥11.0 g/dl; moderate anemia: hb : 9.5-10.9 g/dl; severe anemia: hb :8.0-9.4 g/dl xs-probe was labelled with fam. amplification of the respective dna samples was done by real-time pcr. ethical clearance this study obtained ethical clearance from the faculty of medicine, university of indonesia. the ende district health service was informed of this study and approved this study to be c o n d u c t e d a t 2 1 s c h o o l s i n k e c a m a t a n nangapanda. all school children participating in this study returned the informed consent form signed by their parents or wards and received treatment against soil transmitted helminths. statistical analysis chi-square and prevalence ratio (pr) tests were used to determine the relationship between plasmodium falciparum infections and anemia, at a significance level of 0.05. results there were in total 540 subjects, consisting of 417 male children and 123 female children. the age range of the subjects was from 5 to 18 years, with mean age of 10 ± 2.80 years. the mean weight of the subjects was 25.08 ± 8.47 kg, the lowest weight being 12 kg and the highest 62 kg. mean hb concentration of the 540 study subjects was 11.93 ± 0.75 g/dl. the lowest hb concentration of all study subjects was 8.0 g/dl and the highest hb concentration was 12.9 g/ dl. mean hb concentration of the male subjects was 12.09 ± 0.70 g/dl, with their lowest hb concentration being 8.8 g/dl and their highest 12.9 g/dl. mean hb concentration of the female subjects was 11.37 ± 0.64 g/dl, while the lowest hb concentration was 8.0 g/dl and the highest hb concentration was 11.9 g/dl. among the 540 study subjects from whom b l o o d s a m p l e s h a d b e e n c o l l e c t e d f o r identification of plasmodium falciparum by real-time pcr, 19 subjects (3.3%) were infected. there were 16 subjects (3.2%) with plasmodium falciparum infection but without anemia, whereas 3 subjects (6.8%) had both plasmodium falciparum infection and mild anemia. there was no significant association between plasmodium falciparum infection and anemia category (p=0.4497) (table 1). the risk o f a n e m i a i n s u b j e c t s w i t h p l a s m o d i u m falciparum infection was found to be 1.91-fold higher than the risk in subjects without anemia, b u t t h e i n c r e a s e w a s n o t s i g n i f i c a n t (pr=1.91;95% confidence interval = 0.65-5.62) (table 2). discussion our study results indicated that the risk of anemia in subjects with plasmodium falciparum infection was 1.9.1 fold higher than the risk in subjects without anemia. essentially similar results were obtained in a kenyan study showing that infection with plasmodium was the major cause of childhood anemia in the study area.(13) 132 tan, supali, wibowo plasmodium falciparum infection and a nemia table 2. association between plasmodium falciparum infection and severe anemia *pf = plasmodium falciparum these mild plasmodium falciparum infections were associated with anemia in school children. halliday et al.(14) also found similar results indicating a strong association between p. falciparum and anemia in areas with moderate malarial transmission. malaria is a public health problem that may result in deaths, particularly in high risk groups, such as infants, the under-fives, and pregnant women, in addition to being a direct cause of a n e m i a a n d c a p a b l e o f d e c r e a s i n g w o r k productivity. the disease is still endemic in most areas of the indonesian archipelago.(15,16) the focus of the present study was to determine the c o n t r i b u t i o n o f p l a s m o d i u m f a l c i p a r u m infection to anemia in school children in malarial endemic areas. to eliminate possible bias from soil-transmitted helminths, especially mine worms, as a cause of iron-deficiency anemia, with clinical manifestations similar to anemia from malaria, all school children received anthelminthic treatment before inclusion in this study. more than half of the deaths from malaria, particularly in children and pregnant women, are t h e r e s u l t o f s e v e r e a n e m i a . o f a l l pathophysiological processes underlying severe malaria, the mechanism of anemia as one of the clinical signs of malaria is still not clearly understood. there have been many studies and hypotheses to explain anemia in malaria, but none of the suggested pathophysiological mechanisms of severe anemia are satisfactory.(8) the who defines severe anemia in m a l a r i a a s a n e m i a w i t h a h e m o g l o b i n concentration of <5 g/dl or a hematocrit of <15%.(12) anemia in malaria leads to poor oxygenation at tissue or cellular levels, due to poor oxygen uptake by the mitochondria and the resulting dysfunction of the whole cell. in our study, mild and moderate anemia were almost uniformly distributed over the whole age range of the school children. this is in contrast with the results of a study conducted by ngui et al. in west malaysia barat, where anemia was more common in preschool children. this was due to a low prevalence of anemia among school children in the study area, resulting f r o m s t a t e s u p p o r t e d s c h o o l n u t r i t i o n programs.(17) t h e p r o g r a m f o r e n h a n c e m e n t o f community nutrional status and the nutrional supplementation program carried out by the ende district health service may presumably not yet h a v e t h e s c h o o l c h i l d r e n i n k e c a m a t a n nangapanda under coverage. there are to our k n o w l e d g e n o p u b l i s h e d s t u d i e s o n t h e occurrence of anemia among preschool children in kecamatan nangapanda. the majority of the subjects with positive results for plasmodium falciparum had no anemia. this may be firstly due to mild infections which do not result in anemia, and secondly, there is the possibility that the majority of subjects are adapted to malaria in the study area, which is endemic for malaria. assuming that the possibilty of the anemia being caused by mine worm infection has been eliminated, the factors presumably playing a role in cases of moderate anemia are nutrition and undiagnosed infections. the low socioeconomic status of kecamatan nangapanda may predispose to poor nutritional intake.(11) the weights of all school 133 univ med vol. 32 no.2 children involved as study subjects are normal according to the z-score (data not shown), therefore nutritional factors presumably play a minor role in anemia incidence among these children. however, there are no reports of studies on the relationship of nutrition to anemia in school children in kecamatan nangapanda. other possible causes of anemia in general are other infections, such as pyogenic infections by gram-positive or gram-negative bacteria, m y c o b a c t e r i a l i n f e c t i o n s , e s p e c i a l l y b y mybacterium tuberculosis, brucellosis or even non-infectious factors such as arthritis.(18) although indonesia is the third country in t h e w o r l d w i t h a h i g h p r e v a l e n c e o f tuberculosis,(19) anemia in the school children of kecamatan nangapanda cannot with certainty be assumed to originate from infection with mycobacterium tuberculosis, since all study subjects have a good nutritional status according to their z-score. among the school children of kecamatan nangapanda, the causes of mild and moderate anemia cases that are not the result of plasmodium infection, are as yet unknown and need to be further investigated. all study subjects are original inhabitants of the district, with a high probability of harboring malarial infection. however, few subjects were found who were positive for plasmodium faciparum, either by microscopy or real-time pcr. using microscopy or real-time pcr on the 540 school children as study subjects, the infection rate of plasmodium falciparum was found to be very low, even in areas endemic for malaria. the present study did not collect data on nutrition of the study subjects and data on other infections besides those by plasmodium, thus the other possible causative factors of anemia could not be evaluated. conclusions plasmodium falciparum infection did not increase the risk of anemia in school children. efforts to improve strategies for controlling anemia and malaria are needed. acknowledgements the authors wish to thank dr. dra taniawati supali as project leader, for providing financial support for this study and the study data. our thanks are also due to the nangapanda district health service (dinas kesehatan kecamatan nangapanda), nusa tenggara timur. references 1. world health organization. regional guidelines for the management of severe falciparum malaria in large hospitals. india: world health organization;2006. 2. hotez pj, molyneux dh. tropical anemia: one of africa’s great killers and a rationale for linking malaria and neglected tropical disease control to achieve a common goal. plos negl trop dis 2008; 2:1-4. 3. gimez nd, safekui i, addelani aa, tewari r, reddy jk, rao s, et al. deletion of a malaria invasion gene reduces death and anemia in model hosts. plos one 2011;6:1-8. 4. rantala am, taylor sm, trottman pa, luntamo m, mbewe b, maleta k, et al. comparison of realtime pcr and microscopy for malaria parasite detection in malawian pregnant women. malaria j 2010;9:1-9. 5. melo gc, lecca rcr, silva sv, monteiro wm, martins m, benzecry sg, et al. concurrent helminthic infection protects school-children with 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health 2012;17:532-49. doi:10.1111/j.1365-3156.2012.02971. 15. cox feg. history of the discovery of the malaria parasites and their vectors. parasite vectors 2010; 3:5. doi:10.1186/1756-3305-3-5. 16. kementerian kesehatan republik indonesia. epidemiologi malaria di indonesia. jakarta: kementerian kesehatan republik indonesia;2011. 17. totino prr, magalhaes ad, silva la, banic dm, daniel-ribeiro ct, ferreira-da-cruz mf. apoptosis of non-parasitized red blood cells in malaria: a putative mechanism involved in the pathogenesis of anaemia. malaria j 2010;9:1-5. 18. rivera s, ganz t. animal models of anemia of inflammation. semin hematol 2009;46:351–7. doi:10.1053/j.seminhematol.2009.06.003. 19. world health organization. global tuberculosis report 2012. geneva: world health organization; 2012. c:\users\jurnal fkusakti\docume 173 *department of medical laboratory science, faculty of health sciences and technology nnamdi azikiwe university, nigeria correspondence: okeke okechukwu chizoba department of medical laboratory science, faculty of health sciences and technology, college of health sciences, nnamdi azikiwe university, nnewi campus, p.m.b. 5001, anambra state, nigeria. e-mail: ochizoba93@yahoo.com phone: +2347033484245 date of first submission, april 3, 2018 date of final revised submission, october 7, 2018 date of acceptance, october 9, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina duration of dialysis increases risk of hepatitis c virus infections among hemodialysis patients in anambra state, nigeria okeke okechukwu chizoba* and ajulu a chibuogwu* background sexually transmitted infections and syphilis are a major public health concern. hemodialysis patients are at an increased risk of acquiring hepatitis b virus (hbv) and hepatitis c virus (hcv) infection. the aim of this study was to determine the seroprevalence of hbv, hcv, syphilis, and the association between these infections and hemodialysis among hemodialysis patients. methods a cross-sectional study was conducted involving 90 hemodialysis patients. blood samples were collected and analysed for hbv, hcv and syphilis using immunochromatographic test kits. all subjects completed a questionnaire on demographic characteristics and other risk factors. a chisquare test was used to analyse the data. results the prevalence of hbv, hcv and syphilis infections was 4.4%, 6.7% and 2.2% respectively. highest prevalence of hbv, hcv and syphilis were found in patients whose duration of dialysis were >1 year, >1 year, and 4 months to 1 year, respectively. similarly, those who had undergone dialysis for > 10 times had the highest prevalence of these infections. the major risk factor the patients was exposed to was blood transfusion (100%), with those who had been transfused for 5 times having the highest prevalence of hbv and hcv and those transfused twice for syphilis. a significant association was seen between duration of dialysis and hcv infection (p<0.05). conclusion this study has demonstrated that duration of dialysis increased hcv infection in hemodialysis patients. the prevalence of hbv, hcv and syphilis in the hemodialysis unit is a warning that universal precautions will be the next challenge for decentralised hemodialysis services. keywords: hepatitis b virus, hepatitis c virus, syphilis, hemodialysis patients doi: http://dx.doi.org/10.18051/univmed.2018.v37.173-180 original article pissn: 1907-3062 / eissn: 2407-2230 september-december, 2018 vol.37 no.3 cit e th is a rtic le a s: c hizoba oo, chibuogwu aa. duration of dialysis increases risk of hepatitis c virus infections among hemodialysis patients in an ambr a st ate, nigeria . un iv m ed 201 8;37 :17 3 -8 0 . doi: 10.1 805 1/ univmed.2018.v37.173-180 174 chizoba, chibuogwu dialysis and risk of hepatitis c virus infections introduction hemodialysis, commonly called kidney dialysis or simply dialysis, is a process of purifying the blood of a patient with diseased kidneys. nigeria has the third highest number (6.3 per million population) of patients on hemodialysis after south africa and kenya.(1) it is estimated that one third of the world’s population are infected with hepatitis b virus (hbv) and more than 350 million persons have chronic hepatitis c virus (hcv) infection with about three to four million new infections occurring each year, with more than 2 million and 350 thousand people dying yearly from hepatitis b and hepatitis c-r e lated diseases and complications. the highest frequencies however have been reported in the developing countries of sub saharan africa such as nigeria where it is hyperendemic and pandemic.(2) both infections share common means of transmission through contact with infected blood or body fluids. risk factors for both infections include working in a health care setting, transfusions, dialysis, tattooing, sharing razors or tooth brushes with an infected person, acupuncture, intravenous drug use, and unsafe sex.(3,4) both viruses can produce acute (rapid onset) and chronic liver injury, although hcv has greater chances of chronicity and a longer incubation period.(5) syphilis is a sexually transmitted infection caused by the bacterium treponema pallidum subspecies pallidum. syphilis has been known as “the great imitator” as it may cause symptoms similar to many other diseases.(6) approximately 90% of all syphilis is sexually transmitted and can also be transmitted through infected blood and blood products (blood transfusion). in 2013, syphilis infected about 315,000 people.(7) hepatitis b and hepatitis c virus and syphilis infections may occur in patients with end-stage renal disease on renal replacement therapy (dialysis and/or transplantation). the prevalence of hbv and hcv infection among hemodialysis cases is high and varies between countries. a major risk factor for hbv and hcv is nosocomial routes of transmission which includes the use of contaminated equipment and patient to patient exposure. a study among hemodialysis patients showed that the risk factors for hcv included blood transfusion, multiple visits to different h e mo d i a l ys i s u ni ts a nd f r e qu e n c y o f hemodialysis.(8) another study showed that risk factors for hcv infection were dialysis duration, blood transfusion, and attending more than one dialysis unit. hbv infection was independently associated with age, family member with hepatitis infection, gender, and surgery.(9) a systematic review and meta-analysis showed that duration and frequency of dialysis and exposure to blood transfusions were the most commonly reported risk factors for hcv infection among hemodialysis patients in the middle east and north africa. (10) however, a study among 104 hemodialysis patients showed no association between blood transfusion and duration of hemodialysis with hbv and hcv infections.(11) a negative impact of hbv and hcv infection on survival after renal transplantation has been linked to extrahepatic complications, including chronic glomerulonephritis, sepsis, chronic allograft nephropathy, post-transplantation diabetes mellitus, and abnormal metabolism of calcineurin-inhibitors.(12) this study was not just aimed at determining the seroprevalence of syphilis, hepatitis b and c but also determine the risk factors that predisposed these subjects undergoing dialysis to these infections. methods study sites the study was carried out at nnamdi azi ki we un i ve r si t y te a c hi n g ho s pi t a l (nauth), st. charles borromeo specialist hospital, onitsha and iyienu mission hospital, ogidi, all being located in anambra state, south eastern nigeria. these are hospitals with wellequipped hemodialysis units. 175 research design a cross-sectional study was conducted among hemodialysis patients in some hospitals in anambra state. the study was conducted between february and july 2016. study subjects a total of 90 subjects (both male and females) undergoing hemodialysis in the three selected hospitals within the stipulated time were consecutively recruited for the study based on voluntarism of the patients. both male and female adult individuals undergoing hemodialysis were included in the study. however, subjects with renal disease but not undergoing hemodialysis were excluded. data collection a close-ended questionnaire was used to collect relevant socio-demographic data and other information such as duration and number of dialysis, history of blood transfusion and other possible risk factors. specimen collection three millilitres of whole blood sample was collected from each of the subjects with a sterile syringe through vene-puncture. it was dispensed into a plain container and was allowed to clot and centrifuged to separate the serum. this sample was screened for hbv, hcv and syphilis infections. laboratory analysis hepatitis b assay was performed using hbv one-step lateral r apid immunochromatographic strip test. the pouch was brought to room temperature and opened to remove the test strip. the test strip was then immersed vertically in the serum with arrows pointing downwards for 10-15 seconds, making sure that the maximum (max) line on the test strip was not exceeded. the strip was placed on a non-absorbent flat surface and the result read at 15 minutes. the test was read positive when two distinct red lines appear, one on the control region and the other on the test region. the test was read negative when only one red line appears on the control region. the test was recorded as invalid when the control line failed to appear. a procedural control is included in the test. a coloured line appearing in the control region (c) is the internal procedural control. it confirms suff icient specimen volume and corr ect procedural technique. hepatitis c assay was performed using hcv test strip. also, positive and negative hepatitis c virus sera were used to pre-control batches of the test strips so as to ascertain the workability of the strips. syphilis assay was performed using syphilis ultra-rapid test strip. the test was read positive when two distinct red lines appear, one on the control region and the other on the test region. the test was read negative when only one red line appears on the control region. data analysis the results obtained from the study were presented in tables and subjected to statistical analysis using statistical package for social science [spss] version 20. chi square was used to analyse associations between variables. a p<0.05 was considered statistically significant. ethical clearance and informed consent ethical approval was obtained from the nnamdi azikiwe university teaching hospital ethics committee (nauth/cs/66/vol9/65) and written permissions were obtained from the medical directors of the other hospitals. also, informed consent of the individuals was obtained. results of the 90 subjects recruited, 52 (57.8%) were males, and 26 (28.9%) were single. also, 26 (28.9%) were aged between 20-30 years, and 49 (54.4%) obtained secondary education. and out of the 90 subjects, 90 (100%) were transfused with blood, 8 (8.9%) had previous infection, 13 (14.4%) had history of hbv vaccine, 5 (5.6%) had multiple sex partners in the past while 6 univ med vol. 37 no.3 176 chizoba, chibuogwu dialysis and risk of hepatitis c virus infections (6.7%) and 4 (4.4%) were seen to be having unprotected sex and sharing of personal items respectively (table 1). four (4.4%) subjects were positive for hbv, 6 (6.7%) were positive for hcv while 2 (2.2%) were positive for syphilis infection. however, only 1(1.1%) subject tested positive for hbv and c co-infection but there was no case of co-infection between the two viruses and syphilis infection (table 2). table 3 shows that 7.7% (2) of the subjects from the age range of 20-30 years was positive for hbv. similarly, 15.4% (2) of the subjects from the age ranges of 51-60 years and greater than 60 years was positive for hcv. however, 12.5% (2) of the subjects from the age range of 31-40 years were positive for syphilis. chi-square analysis showed that there was no statistically significant association between hbv, hcv, syphilis and age (p>0.05). table 4 shows that 18.2% (2) of the subjects with duration of dialysis greater than 1 year was positive for hbv. similarly, 36.4% (4) of the subjects with duration of dialysis greater than 1 year was positive for hcv. interestingly, chisquare analysis showed a significant statistical association between hcv and duration of dialysis (p<0.05). however, there was no statistically significant association between hbv, syphilis and duration of dialysis (p>0.05). there was no statistically significant association between hbv, hcv, syphilis and number of blood transfusions. and there was no statistically significant association between hbv, hcv, syphilis and number of dialyses. discussion hepatitis b virus, hcv and syphilis infections are important causes of morbidity and mortality in hemodialysis patients and pose problems in the management of the patients in renal dialysis units. hemodialysis patients may likely be at high risk for hepatitis viral infections due to the high number of blood transfusions and the potential for exposure to infected patients and contaminated equipment. socio-demographic parameters n % marital status married 64 71.1 single 26 28.9 educational status primary 13 14.4 secondary 49 54.4 tertiary 28 31.1 age groups (years) 20 – 30 26 28.9 31 – 40 16 17.8 41 – 50 20 22.2 51 – 60 15 16.7 >60 13 14.4 gender male 52 57.8 female 38 42.2 risk factors blood transfusion yes no previous infection (hbv, hcv or syphilis) yes no family history of infection (hbv, hcv or syphilis) yes no hbv vaccine history yes no multiple sex partner yes no unprotected sex yes no sharing personal items yes no 90 0 8 82 5 85 13 77 5 85 6 84 4 86 10.0 0.0 8.9 91.1 5.6 94.4 14.4 85.6 5.6 94.4 6.7 93.3 4.4 95.6 table 1. socio-demographic parameters and risk factors of the hemodialysis subjects (n=90) infection/co-infection positive (n, %) negative (n, %) hepatitis b virus 4 (4.4) 86 (95.6) hepatitis c virus 6 (6.7) 84 (93.3) syphilis 2 (2.2) 88 (97.8) hbv / hcv 1 (1.1) 89 (98.9) hbv/hcv/syphilis 0 (0.0) 90 (100.0) table 2. sero-prevalence of hepatitis b and c virus and syphilis infections among hemodialysis patients 177 in our present study, a prevalence of hbv, hcv and syphilis infe ctions among the hemodialysis patients was observed with values of 4.4%, 6.7% and 2.2% respectively. the prevalence of hbv, hcv and syphilis infections among hemodialysis patients is high and varies between countries (2% to 60%) and between dialysis units within a single country.(13) the prevalence of hbv (4.4%) among hemodialysis patients obtained in our study is lower than the prevalence of 34.9% in libya(14) and 7.0% in china,(15) but higher than that reported in india which is 1.4%. (1 6) moreover the prevalence of hcv (6.7%) among hemodialysis patients obtained is lower than the prevalence of 31.1% in libya(14) and 15% in north-eastern nigeria,(17) but higher than the 6.1% reported in china, (15) while the prevalence of syphilis (2.2%) among hemodialysis patients is lower than the prevalence of 4.5% in france.(18) hepatitis b virus and hcv share a common route of transmission and can coexist with each other. hepatitis b virus and hcv co-infection prevalence of 1.1% among hemodialysis patients in our study is lower than that reported in india of 3.7%.(16) there was no case of co-infection between the two viruses and syphilis in this study. and another study found that out of the total 262 patients, 88 (33.5%) were found to be having hcv infection, 4 (1.5%) were found to be positive for hbv infrction and dual infection was observed in 2 (0.8%) patients. (17) the result obtained showed that infection can occur at any age. however, the age of 20-30 years recorded the highest percentage frequency of occurrence at 7.7% for hbv, while for hcv the age of >60 years recorded the highest percentage (15.4%) and for syphilis the age group of 31-40 years had the highest percentage (12.5%). in contrast to our own finding, ummate et al.(18) reported that individuals aged 40-49 years had the highest prevalence of hcv with an infection rate of 6% in their own study, however this could be due to geographical differences as theirs was carried out in the north-eastern part of nigeria. there was a significant association between duration of dialysis and hcv infection. a systematic review and meta-analysis showed similar results that among patients who were under hemodialysis for more than 5 years the prevalence of hepatitis c was higher than those who were treated for a shorter time period. (18) it was observed that individuals with longer 9duration of dialysis of >1 year had the highest prevalence of hepatitis b and c virus at a rate of 18.2% and 36.4% respe ctively while the prevalence of syphilis was seen highest at 5% with patients that have undergone dialysis for 4 months to 1 year. similarly, this study noted that individuals with the highest number of blood transfusions (5 times) had the highest prevalence of hbv and hcv infection. also, the result showed that patients who were dialysed for more than 10 times had the highest prevalence of hbv, hcv and syphilis infections. in other words, this research noted blood transfusion, duration of dialysis, and number of times dialysis was done as common possible risk factors of transmission of hbv, hcv and univ med vol. 37 no.3 hbv hcv syphilis age group (years) +ve -ve p value +ve -ve p value +ve -ve p value 20-30 (n=26) 2 (7.7) 24 (92.3) 0.476 0 (0.0) 26 (100) 0.690 0 (0.0) 26 (100) 0.822 31-40 (n=16) 0 (0.0) 16 (100) 2 (12.5) 14 (87.5) 2 (12.5) 14 (97.4) 41-50 (n=20) 1 (5.0) 19 (95.0) 1 (5.0) 19 (95) 0 (0.0) 20 (100) 51-60 (n=15) 1 (6.7) 14 (93.3) 1 (6.7) 14 (93.3) 0 (0.0) 15 (100) >60 (n=13) 0 (0.0) 13 (100) 2 (15.4) 11 (84.6) 0 (0.0) 13 (100) table 3. seroprevalence of hepatitis b and c virus and syphilis infections based on age groups of hemodialysis subjects p-value is significant if (p<0.05) ; +ve = positive; -ve= negative 178 chizoba, chibuogwu dialysis and risk of hepatitis c virus infections t ab le 4 : s er o p re v al en ce o f h ep at it is b a n d c v ir u s an d s y p h il is i nf ec ti o ns b a s ed o n d u ra ti o n o f d ia ly si s, n um b er o f b lo o d tr an sf u s i o n s an d n u m b er o f d ia ly se s p -v al u e is s ig n if ic an t if ( p < 0 .0 5 ) an d i s n o t si g n if ic an t if ( p > 0 .0 5 ) + v e = p o si ti v e -v e. = n eg at iv e 179 syphilis among hemodialysis patients. this is in agreement with the findings by alashek et al.(14) and dahmani et al.(20) our finding of a nonstatistically significant difference between the risk factors and infection with hbv, hcv and syphilis could be attributed to the limited number of hemodialysis subjects used in this study. there was a limited number of patients undergoing hemodialysis in the study sites. the need for adequate precautions in handling hemodialysis subjects is evident from this study and mandatory pre-screening for syphilis, he pa ti ti s b an d c ma y be nec essa ry f or hemodialysis patients to ensure a holistic management of infected patients. it is therefore recommended that appropriate screening of blood before transfusion to hemodialysis patients should be done. also, hemodialysis patients should undergo appropriate screening before dialysis and infected patients should be handled with care to avoid cross-infections among patients and staff. further studies will focus on how these infections influence the clinical outcome of renal disease patients. conclusion among dialysis patients, longer duration of dialysis, higher number of blood transfusions, and increased number of dialysis times constituted the major risk factors for hbv, hcv and syphilis infection. acknowledgement the authors acknowledge the management and staff of the hemodialysis section of nnamdi azikiwe university teaching hospital nnewi, st. charles borromeo specialist hospital, onitsha and iyienu mission hospital, ogidi, all in anambra state. conflict of interest the authors declare that no conflict of interest exists in this study. contributors oco contributed to the research design, statistical and laboratory analysis as well as manuscript preparation, aca contributed to the design, did the literature search, wrote proposal and carried out laboratory analysis while oco and aca played a role in interpr etation, manuscript editing and review. all authors have read and approved the final manuscript. references 1. pozo me, leow jj, groen rs, et al. an overview of renal replacement therapy and 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10.3350/cmh.2012.18.3.249. 7. kent me, romanelli f. reexamining syphilis: an update on epidemiology, clinical manifestations, and management. ann pharmacother 2008;42: 226–36. doi: 10.1345/aph.1k086. 8. global burden of disease study 2013 collaborators. global, regional, and national incidence, prevalence, and years lived with disability for 301 acute and chronic diseases and injuries in 188 countries. lancet 2015;386:743– 800. doi: https://doi.org/10.1016/s0140-6736(15) 60692-4. 9. duong cm, olszyna dp, mclaws ml. hepatitis b and c virus infections among patients with end stage renal disease in a low-resourced hemodialysis center in vietnam: a cross-sectional 180 chizoba, chibuogwu dialysis and risk of hepatitis c virus infections study. bmc public health 2015;15:192. doi: 10.1186/s12889-015-1532-9. 10. su y, yan r, duan z, et al. prevalence and risk factors of hepatitis c and b virus infections in hemodialysis patients and their spouses: a multicenter study in beijing, china. j med virol 2013;85:425–32. doi: https://doi.org/10.1002/ jmv.23486. 11. harfouche m, chemaitelly h, mahmud s, et al. epidemiology of hepatitis c virus among hemodialysis patients in the middle east and north africa: systematic syntheses, metaanalyses, and meta-regressions. epidemiol infect 2017;145:3243–63. doi: 10.1017/s095026881700 2242. 12. luma hn, halle mp, eloumou safb, et al. seroprevalence of human immunodeficiency virus, hepatitis b and c viruses among hemodialysis patients in two newly opened centres in cameroon. pan african medical j 2017;27:235. doi: 10.11604/pamj.2017.27.235.13121. 13. fabrizi f, martin p, messa p. hepatitis b and hepatitis c virus in chronic kidney disease. acta gastroenterol belg 2010;73:465-71. 14. delarocque-astagneau e, baffoy n, thiers v, et al. outbreak of hepatitis c virus infection in a hemodialysis unit: potential transmission by hemodialysis machine. infect control hosp epidemiol 2002; 23:328-34. 15. alashek wa, mcintyre cw, taal mw. hepatitis b and c infection in hemodialysis patients in libya: prevalence, incidence and risk factors. bmc infect dis 2012;12:265. doi: 10.1186/14712334-12-265. 16. reddy ga, dakshinamurthy kv, neelaprasad p, et al. prevalence of hbv and hcv dual infection in patients on hemodialysis. indian j med microbiol 2005;23:41-43. 17. malhotra r, soin d, grover p, galhotra s, khutan h, kau n. hepatitis b virus and hepatitis c virus co-infection in hemodialysis patients: a retrospective study from a tertiary care hospital of north india. j nat sci biol med 2016;7:72–7. doi: 10.4103/0976-9668.175076. 18. ummate i, kida im, baki b, et al. prevalence of hepatitis c virus infection among hemodialysis patients in north-eastern nigeria. tropical j nephrol 2013;8:7-11. 19. ashkani-esfahan s, alavian sm, salehimarzijarani m. prevalence of hepatitis c virus infection among hemodialysis patients in the middle-east: a systematic review and metaanalysis. world j gastroenterol 2017;23:151-66. doi: 10.3748/wjg.v23.i1.151. 20. dahmani o, belkhalfa s, ayad ka, et al. late latent syphilis in two hemodialysis units. saudi j kidney dis transpl 2013;24:124-127. oktavianus 29 *department of biochemistry, faculty of medicine, universitas padjadjaran, bandung **health research unit, faculty of medicine, universitas padjadjaran, bandung ***department of obstetrics and gynaecology, hasan sadikin hospital/ faculty of medicine, universitas padjadjaran, bandung correspondence dr. ani melani maskoen, drg, mkes. department of biochemistry / health research unit faculty of medicine, universitas padjadjaran, bandung, indonesia jl. eijkman no. 38 bandung phone: + 62 22 2038218 email : amelani@yahoo.com univ med 2013;32:29-36 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 introduction cervical cancer cases are rising and many women are infected with human papillomavirus (hpv). interferon gamma (ifn-ã) is one of the key regulatory cytokines that influence the hpv clearance. the production and the function of ifn-ã may impaired by the defect of the ifng gene leading to the cervical malignant progression. this study aimed to examine the association between ifng+874 t>a polymorphism and cervical cancer in women methods in a case-control study design, consecutive untreated women with cervical cancer who showed for the first time in hasan sadikin hospital bandung were enrolled (n=98) and for controls women who came for pap smear (n = 81). controls were not matched in ages and ethnicities. dna extracted from blood was amplified by amplification refractory mutation system polymerase chain reaction method (arms – pcr) to detect ifng+874 t>a polymorphism. results the distribution of ifng genotypes tt, ta and aa for women with cervical cancer who met the inclusion criteria (n= 64) and with negative intraepithelial lesion or malignancy (n=42) were 14.1%, 50.0%, 35.9% and 7.1%, 52.4%, 40.5%, respectively. no significant differences could be observed between both groups (p=0.64). stratifying the cervical cancer women into a group of squamous cell carcinoma (n = 54) revealed no statistical different. conclusion ifng +874 t>a polymorphismseems not to contribute in susceptibility to cervical cancer. identification of other variants in ifng gene signaling and its role in the development of cervical cancer diseases need to be further examined. key words: ifng gene, single nucleotide polymorphism, cervical cancer ifng polymorphism (+874 t>a) is not a risk factor for cervical cancer ani melani maskoen*,**, herman susanto***, samsudin surialaga*, and edhyana sahiratmadja*,** 30 maskoen, susanto, surialaga, et al polymorphism risk of cervical cancer polimorfisme ifng+874 t>a bukan merupakan faktor risiko untuk kanker serviks pendahuluan walaupun banyak wanita yang terinfeksi virus hunan papilloma (hpv) tetapi hanya sebagian saja dari wanita yang secara kronik terinfeksi oleh hpv tipe risiko tinggi akan berkembang menjadi kanker serviks. interferon gamma (ifn-ã) merupakan salah satu sitokin yang berperan dalam mengeliminasi hpv.produksi dan fungsi ifn-ã dapat mengalami penurunan bila terjadi perubahan pada gen ifng yang menjadi sitokin ifn-ã sehingga proses eliminasi hpv terganggu yang dapat menyebabkan kanker serviks dikemudian hari. peneitian ini bertujuan untuk menentukan adanya asosiasi antara polimorfisme ifng+874 t>a dan kanker serviks pada wanita. metode penelitian ini merupakan penelitian kasus-kontrol yang mana kasus adalah wanita yang dating ke poli kebidanan rumah sakit hasan sadikin untuk pertama kali dengan kasus kanker serviks baru. kontrol diambil dari wanita yang dating untuk pemeriksaan pap smear. kontrol tidak dipasangkan dengan usia dan etnisnya. darah edta diambil untuk diektraksi dna nya, kemudian diamplifikasi dengan menggunakan metoda amplification refractory mutation system polymerase chain reaction (arms – pcr) untuk mendeteksi polimorfisme pada ifng+874 t>a. hasil distribusi genotype ifng tt, ta dan aa pada wanita dengan kanker serviks adalah 14,1%, 50,0%, 35,9%, dibandingkan dengan kontrol dengan pap smear negatif sebesar 7,1%, 52,4%, 40,5%. secara statistik tidak didapatkan perbedaan yang bermakna (p=0,64). dengan melakukan stratifikasi untuk kanker serviks hanya kelompok squamous cell carcinoma juga tidak didapatkan perbedaan yang bermakna. kesimpulan pada penelitian ini, polimorfisme ifng +874 t>a tampaknya bukan merupakan faktor risiko yang berkontribusi pada kerentanan terhadap kanker serviks. identifikasi variasi-variasi yang lain pada gen ifng perlu di pelajari dalam hubungannya dengan perkembangan kanker serviks. kata kunci: gen ifng, polimorfisme, kanker serviks abstrak introduction c e r v i c a l c a n c e r i s t h e s e c o n d m o s t prevalent female cancer after breast cancer, affecting annually more than 150.000 women w o r l d w i d e . (1) t h i s c a n c e r h a s b e c o m e a significant health burden in lowand mediumresourced countries in sub-saharan africa, latin america, south and south east asia, as 80% of cervical cancers occur in developing countries.(1) in indonesia, cervical cancer ranks first among gynecological cancers.(2) persistent infection with high-risk types of human papillomavirus (hpv) is strongly associated with development of cervical cancer.(3) in immunocompetent individuals hpv infection is a self-limited disease that can be cleared by the immune system, as hpvs have a unique mechanism in limiting the infection to the basal cells of stratified epithelium, the only place where they replicate.(4) metaanalyses on hpv genotypes distribution have shown that many women with normal cytology 31 can also be infected with high-risk types of hpv in single or multiple infections. (5) interestingly o n l y f e w w o m e n w i l l e v e r d e v e l o p cervical cancer. a recent study using a mouse cervicovaginal challenge model has proven that the virus cannot initially bind to keratinocytes in vivo.(6) immunological components in cellular mediated immunity, in particular interferon g a m m a ( i f n ã ) , p l a y a k e y r o l e i n t h e development of cancers(4) and intracellular infections e.g. m. tuberculosis, m. leprae, and salmonella.(7) ifn-ã is one of the key regulatory cytokines, produced by activated t cells and natural killer (nk) cells, that enhances cellular i m m u n e r e s p o n s e s b y i n c r e a s i n g tc e l l cytotoxicity and nk-cell activity. ifn-ã plays a role in antiproliferative, antitumor and antiviral a c t i v i t i e s . f u r t h e r m o r e , i f n ã i s a p r o inflammatory cytokine playing a role in both innate and adaptive immune responses that may influence hpv clearance.(4) the susceptibility to hpv infection that leads to cervical cancer may be influenced by the gene that encodes ifn-ã. studies on hpv-infected patients point out the role of ifn-ã in the control of the infection, for example low production or impaired function of ifn-ã may lead to malignant progression of cervical hpv lesions.(8) moreover, there is a direct relationship between ifn-ã and the severity of cervical intraepithelial neoplasia.(8) clearly, ifn-ã plays a pivotal role in defense against viruses and intracellular pathogens and in the induction of immunemediated inflammatory responses. furthermore, intralesional ifn-ã concentrations are detectable during hpv infections and may therefore be used as a valuable prognostic marker for c l e a r a n c e o f h i g h r i s k h p v. (9) i n t h e abovementioned study, after a 12-month followup period women who tested positive for the presence of ifn-ã were significantly more likely to be free of hpv infection compared with women who were negative for ifn-ã.(9) the ifn-ã gene (ifng) encodes the cytokine ifn-ã, thus host genetic differences in the immune response to hpv infection may play a role in the susceptibility to disease.(10) a single nucleotide polymorphism located in the first intron of the ifng gene can influence the s e c r e t i o n o f t h e c y t o k i n e . ( 1 1 ) the i f n g genotype tt determines high ifng production, and the ifng genotypes ta and aa are for intermediate and low production of ifn-ã, respectively. (11) interestingly, ethnic background may influence the distribution of cytokine gene polymorphisms.(12) for example, studies in v a r i o u s p o p u l a t i o n s h a v e s h o w n a c l e a r correlation between ethnicity and distribution of ifng polymorphisms across different population groups.(13) ifng+874t>a polymorphisms are frequently studied in intracellular infectious disease such as tuberculosis (14) and also in cancers.(15) a l t h o u g h m e t a a n a l y s e s o f t h e ifng+874t>a polymorphism have found it not to be associated with cancer in general, it still was associated with cervical cancer. ( 8 , 1 5 ) h o w e v e r, t h e v a r i o u s s t u d i e s o n i f n g polymorphisms and cervical cancers have shown conflicting results. for example, a study from south africa revealed no significant differences b e t w e e n c e r v i c a l c a n c e r p a t i e n t s a n d controls. (13) the aim of our study was to i n v e s t i g a t e t h e a s s o c i a t i o n b e t w e e n t h e fng+874t>a polymorphism and cervical cancer. methods research design a case-control study was carried out from june 2010 to december 2010 in the gynecology outpatient clinic of hasan sadikin hospital, bandung, indonesia. research subjects this study enrolled consecutive new female patients with cervical cancer (n=98), diagnosed by the gynecologists on duty as cervical carcinoma stage iia/iib, according to the fédération internationale de gynécologie et univ med vol. 32 no.1 32 maskoen, susanto, surialaga, et al polymorphism risk of cervical cancermaskoen, susanto, surialaga, et al polymorphism risk of cervical cancer d’obstétrique (figo) staging system. we limited enrollment to cervical cancer patients who presented to the clinic from june 2010 to december 2010. excluded were women who were less than 18 years old, pregnant, previously treated with chemotherapy or radiation for any cancer, and also those lacking histological data. in the same period, consecutive females who came for pap smears (n=81) were included as controls. the control group was not matched to the cervical cancer group for age and ethnicity. both cervical cancer patients and controls were interviewed for recording purposes, using the standardized medical questionnaire in the clinic. dna extraction and genotyping dna was isolated from venous blood collected in ethylenediaminetetraacetic acid (edta) tubes according to the manufacturer’s protocol (qiagen blood mini kit, germantown, md). bi-allelic ifng+874 t>a polymorphism w a s a n a l y z e d b y a m o d i f i c a t i o n o f t h e amplification refractory mutation systempolymerase chain reaction techtique (arms – pcr). in brief, pcr was performed using combinations of primers consisting of a sense primer for allele a or t and a common antisense p r i m e r. to a s s e s s t h e s u c c e s s o f p c r amplification in both reactions, an internal control was amplified using a pair of primers designed from the nucleotide sequence of the human growth hormone (hgh). the pcr conditions were as follows: hold at 95ºc for 1 min, 10 cycles of denaturation at 95ºc for 15 s, annealing at 62ºc for 50 s, extension at 72ºc for 40 s, followed by 20 cycles of denaturation at 95ºc for 20 s, annealing at 56ºc for 50 s, and extension at 72ºc for 50 s, and final extension at 72ºc for 10 min then at 4ºc for a indefinite time for short-term storage. the pcr products were analyzed by electrophoresis on 1.5% agarose gel, showing pcr products of human growth hormone (426 bp) as internal controls. each individual showed the t and a alleles separately, identified by the 261 bp band. ethical clearance written consent was obtained from all subjects, and the study protocol was approved by the institutional review board of padjadjaran university, bandung. statistical analyses the allelic and genotypic frequencies were obtained by direct counting. the hardywe i n b e rg e q u i l i b r i u m ( h w e ) o f e a c h polymorphism was checked using the program hwe. the program conting was used to calculate ÷ 2 a n d a s s o c i a t e d v a l u e s f o r contingency tables. data from the questionnaire and genotyping were analyzed using spss version 13.0 (spss inc., chicago, il, usa). all statistical analyses were two-sided and p values <0.05 were considered as statistically significant. the odds ratio (or) was determined to calculate possible significant differences in genotypes. results of the 98 subjects with cervical cancer, 29 had missing histological data, 5 had no malignant cells found in the samples (designated as “others” in table 1), thus, only 64 participants were further enrolled (table 1). squamous cell carcinomas (scc) were more prevalent in the study population (n = 53 of 64; 82.8%), of which most were non-keratinizing (n=43 of 53; 81.1%). the patient characteristics could not be described in this study, as some of the medical records were lacking in data on age, marital status, number of children, smoking status, and high risk sexual behavior. this caused difficulties in analyzing further data, therefore, the analysis was only based on the histopathological data. in the control group the excluded subjects were as follows: 18 had missing histological data, 21 showed abnormal findings on cytological examination such as low-grade (n=16) and highgrade squamous intraepithelial lesions (n=5) (table 1). 33 tabel1. histopatological distribution of the subjects note: *were not included in the study, this includes:others and missing data for cervical cancer patients and hsil, lsil and missing data for control group. others designated for biopsies contained no abnormalities; hsil= high-grade squamous intraepithelial lesion; pap= papanicolau smear; lsil=low-grade squamous intraepithelial lesion; nilm=negative intraepithelial lesions or malignancy ifng +874 t>a polymorphism in the arms-pcr method using primer combinations, dna samples were of good quality and positive controls using primers for human growth hormone (hgh) were shown in the gel electrophoresis (figure 1). the genotypes of the ifng +874 t>a polymorphism were in hardy – weinberg equilibrium in the total group of individuals as well as in the patient and control groups. the distribution of ifng genotypes tt, ta and aa for women with cervical cancer who met the inclusion criteria (n=64) and had negative intraepithelial lesions or malignancy (nilm) (n = 42) were 14.1%, 50.0%, 35.9% and 7.1%, 52.4%, 40.5%, respectively, and no significant differences could be observed between both groups (p=0.64) (table 2). as the scc group was the most prevalent in our study population, these cases were stratified further, to make the groups more homogenous. the distribution of ifng genotypes for patients with scc (n~53) was as follows: tt (15.1%), ta (54.7%), aa (30.2%), and no significant differences could be observed between the scc group and nilm group (p=0.29) (data now shown). discussion the susceptibility to hpv infection that leads to cervical cancer may be influenced by variations in the ifng gene that codes for the table 2. distribution of alelle and genotype ifng t874a in cervical cancer and control note: nilm=negative intraepithelial lesión or malignancy univ med vol. 32 no.1 34 maskoen, susanto, surialaga, et al polymorphism risk of cervical cancer figure 1. electrophoresis of ifng t874a polymorphisms upper band is an internal control using primer hgh (500bp). every individual has 2 lanes, i.e. lane t and lane a. person no. 1 has genotype ta, person no. 2 has genotype aa etc. 500 bp 300 bp cytokine ifn-ã. our study showed no significant difference between cervical cancer patients and their controls with negative intraepithelial lesions (nilm). the distribution of the t and a allelic and genotypic frequencies in our study results is in concordance with the study in brazil.(16) however, in contrast to the latter, we found no significant differences even after stratifiying the patients into a more homogenous group e.g. only scc and nilm. interestingly, the frequency of the tt genotype, denoting high-level ifn-ã producers, was lower than that of the aa genotype, i.e. the prevalence of low-level ifnã producers in our population was similar to those in the indian and brazilian studies.(8,16) ethnic background differences may play a role in the distribution of ifng +874 genotypes. c e r v i c a l c a n c e r i s t h e s e c o n d m o s t common cancer in women worldwide and persistent infection with high risk types of hpv, e . g . h p v 1 6 a n d h p v 1 8 , a r e s t r o n g l y associated with development of cervical cancer.(17) although many women with normal cytology are also infected with high-risk types of hpv,(5) only few women will ever develop cervical cancer, as ifn-ã production may influence hpv clearance and prevent malignant progression.(1) the majority of hpv infections are cleared without further consequences for the host and only a small percentage of women, who are persistently infected with oncogenic genotypes, will develop cancer. it is suggested that ifn-ã is crucial to control of hpv. ifn-ã production and function are encoded by the ifng gene, and the +874 a allele of ifng may influence the secretion of the cytokine.(18) a m e t a a n a l y s i s t o d e t e r m i n e t h e association of ifng gene polymorphisms and various types of cancer showed that although in general the +874 t allele of ifng showed no protective significant association, yet a strong association was shown with cervical cancer.15 for example, in two studies at different sites in india, variation in the ifng +874 aa genotype as a low ifn-ã producer was associated with a s i g n i f i c a n t l y i n c r e a s e d r i s k o f cervical cancer.(8,19) clearly, studies in human viral diseases point out a complex immune system response in controlling the infection. ifn-ã may not be the only cytokine influencing hpv clearance or cervical cancer development. other th2 cytokines such as il-4(18) and il-10 (21,22) may also play a role in hpv clearance, e.g. the il10 -1082g polymorphism has been shown to be associated with clearance of hpv infection (21) and increased il-10 production.(22) other studies showed that interferon alpha-17 (ifn17) ile184arg polymorphism may also play a s i g n i f i c a n t r o l e . ( 2 3 ) m o r e o v e r, t h e t u m o r suppressor gene tp53 is associated with cervical cancer as a genetic co-factor.(24) however, a pooled analysis of studies on polymorphism in codon 72 of tp53 has indicated that that excessively high risks were most likely not due to clinical or biological factors, but to errors in study methods.(25) better study methods and larger numbers of participants need to be underlined. we acknowledge that our study has several limitations, among others missing data in medical records, such that age and ethnicity were not matched between cancer patients and their control group. furthermore, the small number of cases and controls is another limitation of this study that has prevented us to come to any major 35 conclusions. the distribution of ifng +874 t/ a polymorphisms in our study population may not reflect that of cervical cancer in indonesia per se; however, this study may provide direction for studies involving much larger groups of patients. ifng genotyping in larger numbers of participants from the indonesian archipelago with different ethnic backgrounds may also reveal more valuable information. further understanding of the role of this cytokine may contribute to the development of a biomarker of hpv infection and result in the improvement of the treatment of squamous intraepithelial lesions. conclusions ifng polymorphism at +874 t>a seems not to contribute to susceptibility to cervical cancer. identification of other variants in ifng gene signaling and its role in the development of cervical cancer need to be further examined to provide information on biological markers that would be useful for the development of diagnostic and therapeutic strategies. acknowledgements we are grateful to dr. maringan tobing sp og (k), dr. bethy hernowo, sp pa (k) and dr. birgitta, sp pa for their kind help. we thank nurul setia rahayu, yeni rendieni and tri yuli siswanti from the health research unit laboratory fk unpad for their technical support. this work has been financially supported by hibah andalan grant from universitas padjadjaran 2010 (project no.672/h6.26/lppm/ pl/2010). references 1. castellsagué x. natural history and epidemiology of hpv infection and cervical cancer. gynecol oncol 2008;110 suppl 2:s4-7. 2. aziz mf. gynecological cancer in indonesia. j gynecol oncol 2009;20:8-10. 3. coutlée f, ratnam s, ramanakumar av, insinga rr, bentley j, escott n, et al. distribution of human papillomavirus genotypes in cervical intraepithelial neoplasia and invasive cervical cancer in canada. j med virol 2011; 83:1034-41. 4. stanley m. hpv-immune response to infection and vaccination. infect agent cancer 2010;20:19. 5. bruni l, diaz m, castellsagué x, ferrer e, bosch fx, de sanjosé s. cervical human papillomavirus prevalence in 5 continents: meta-analysis of 1 million women with normal cytological findings. j infect dis 2010;202:1789-99. 6. schiller jt, day pm, kines rc. current understanding of the mechanism of hpv infection. gynecol oncol 2010;118 suppl 1:s12-7. 7. van de vosse e, ottenhoff th. human host genetic factors in mycobacterial and salmonella infection: lessons from single gene disorders in il-12/il-23-dependent signaling that affect innate and adaptive immunity. microbes infect 2006;8: 1167-73. 8. gangwar r, pandey s, mittal rd. association of interferon-gamma +874a polymorphism with the risk of developing cervical cancer in a north indian population. bjog 2009;116:1671-7. 9. song sh, lee jk, lee nw, saw hs, kang js, lee kw. interferon-gamma (ifn-gamma): a possible prognostic marker for clearance of high-risk human papillomavirus (hpv). gynecol oncol 2008;108: 543–8. 10. calhoun es, mcgovern rm, janney ca, cerhan jr, iturria sj, smith di, et al.host genetic polymorphism analysis in cervical cancer. clin chem 2002;48:1218-24. 11. pravica v, perrey c, stevens a, lee jh, hutchinson iv. a single nucleotide polymorphism in the first intron of the human ifn-gamma gene: absolute correlation with a polymorphic ca microsatellite marker of high ifn-gamma production. hum immunol 2000;61:863–6. 12. hoffmann sc, stanely em, cox ed, di mercurio bs, koziol de, harlan dm. ethnicity greatly influences cytokine gene polymorphism distribution. am j trans 2002;2:560-7. 13. govan va, carrara hr, sachs ja, hoffman m, stanczuk ga, williamson al. ethnic differences in allelic distribution of ifn-ã in south african women but no link with cervical cancer. j carcinog 2003;2:3-11. 14. amim lh, pacheco ag, fonseca-costa j, loredo cs, rabahi mf, melo mh, et al. role of ifngamma +874 t/a single nucleotide polymorphism in the tuberculosis outcome among brazilians subjects. mol biol rep 2008;35:563-6. 15. mi yy, yu qq, xu b, zhang lf, min zc, hua lx, et al. interferon gamma +874 t/a polymorphism contributes to cancer susceptibility: a metauniv med vol. 32 no.1 36 maskoen, susanto, surialaga, et al polymorphism risk of cervical cancer analysis based on 17 case-control studies. mol biol rep 2011;38:4461-7. 16. von linsingen r, bompeixe ep, maestri ca, carvalho ns, da graça bm. ifng (+874 t/ a) polymorphism and cervical intraepithelial neoplasia in brazilian women. j interferon cytokine res 2009;29:285-8. 17. sjoeborg kd, tropé a, lie ak, jonassen cm, steinbakk m, hansen m, et al. hpv genotype distribution according to severity of cervical neoplasia. gynecol oncol 2010;118:29-34. 18. gey a, kumari p, sambandam a, lecuru f, cassard l, badoual c, et al. identification and characterization of a group of cervical carcinoma patients with profound downregulation of intratumoral type 1 (ifn gamma) and type 2 (il-4) cytokine mrna expression. eur j cancer 2003;39: 595–603. 19. kordi tmk, sobti rc, shekari m, mukesh m, suri v. expression and polymorphism of ifngamma gene in patients with cervical cancer. exp oncol 2008;30:224–9. 20. el-sherif am, seth r, tighe pj, jenkins d. quantitative analysis of il-10 and ifn-gamma mrna levels in normal cervix and human papillomavirus type 16 associated cervical precancer. j pathol 2001;195:179-85. 21. farzaneh f, roberts s, mandal d, ollier b, winters u, kitchener hc, et al. the il-10 -1082g polymorphism is associated with clearance of hpv infection. bjog 2006;113:961–4. 22. stanczuk ga, sibanda en, perrey c, chirara m, pravica v, hutchinson iv, et al.cancer of the uterine cervix may be significantly associated with a gene polymorphism coding for increased il-10 production. int j cancer 2001;94:792-4. 23. kim jw, roh jw, park nh, song ys, kang sb, lee hp. interferon, alpha (ifn17) ile184arg polymorphism and cervical cancer risk. cancer lett 2003;189:183-8. 24. agorastos t, lambropoulos af, constantinidis tc, kotsis a, bontis jn. p53 codon 72 polymorphism and risk of intra-epithelial and invasive cervical neoplasia in greek women. eur j cancer prev 2000;9:113-8. 25. klug sj, ressing m, koenig j, abba mc, agorastos t, brenna sm, et al. tp53 codon 72 polymorphism and cervical cancer: a pooled analysis of individual data from 49 studies. lancet oncol 2009;10:772-84. editorial 71 antagonism of vitamin c and vitamin e on action of quinolones julius e. surjawidjaja department of microbiology, faculty of medicine, trisakti university univ med vol. 31 no.2 the quinolone antibiotics are potent drugs for combating infections caused by various bacterial species with satisfactory results and relatively minimal adverse effects. antioxidant dietary supplements, such as vitamin c (ascorbic acid) and vitamin e (α -tocopherol), are occasionally prescribed along with quinolone antibiotics during the course of treatment of an infection. therefore it is important to understand the effects of these antioxidants on the antibacterial action of quinolone antibiotics. pfister et al.(1) conducted a study using 4 groups of 12 young wistar rats. group i was given normal feed, group ii was given vitamin e-enriched feed and group iii magnesium-enriched feed, while group iv was given feed enriched with magnesium and vitamin e, all for 10 days. all groups received ciprofloxacin (cpfx) at a dosage of 600 mg. vitamin e was shown to reduce the number of abnormalities in the pelvic and femoral bones. gurbay et al.(2) studied the cytotoxic effects of cpfx in fibroblasts given pretreatment with vitamin e, in comparison with those given no vitamin e pretreatment. vitamin e pretreatment of the cells provided significant protection against cpfx-induced cytotoxicity; lowered the level of lipid peroxidation significantly. it was concluded that cpfx cytotoxicity was due to oxidative stress in the fibroblasts and that pretreatment with vitamin e was capable of protecting the cells. a subsequent study conducted by gurbay et al.(3) on cpfx cytotoxicity in rat astrocytes also concluded that cpfx-induced cytotoxicity is related to oxidative stress. masadeh et al.(4) conducted a study on the effects of cpfx-induced oxidative stress on a number of reference bacterial strains and the protective effects of vitamin c and vitamin e on these bacteria. the bacteria used were p. aeruginosa atcc 27853, e. coli atcc 25922, s. aureus atcc 29213, and clinical isolates of mrsa (methicillin resistant s. aureus). the inhibitory activity of cpfx on the test bacteria was determined by calculating the minimal inhibitory concentration (mic), while oxidative stress was assessed through measurement of h 2 o 2 production by means of facscan flow cytometry. the bacteria pretreated with vitamin c and vitamin e were found to be protected from cpfx-induced cytotoxicity. this study demonstrates the antagonistic effect of vitamin e and vitamin c administration on the bactericidal effects of cpfx, when given concurrently during treatment of infections. more recently, the antioxidant ubiquinone (coenzyme q10) demonstrated protective effects on human achilles tendon cells exposed to ciprofloxacin and moxifloxacin.(5) oxidative stress frequently occurs in the mitochondria, and fluroquinolone-induced oxidative damage to mitochondria in tenocytes and chondrocytes has been reported. fluoroquinolones are important antibiotics with immense therapeutic value, and 72 univ med vol. 31 no.2 the effectiveness of treatment by these drugs may be affected by dietary intake and cellular levels of these antioxidants. overall, these antibacterial agents can be considered safe and well tolerated drugs. patients should always be counseled regarding the risk associated with fluoroquinolones, even when they are prescribed according to standard recommendations. references 1. pfister k, mazur d, vormann j, stahlmann r. diminished ciprofloxacin-induced chondrocytotoxicity by supplementation with magnesium and vitamin e in immature rats. antimicrob agents chemother 2007;51: 1022-7. 2. gurbay a, garrell c, osman m, richard mj, favier a, hincal f. cytotoxicity in ciprofloxacin-treated human fibroblast cells and protection by vitamin e. hum exper toxicol 2002;12:635-41. 3. gurbay a, gonthier b, barret l, favier a, hincal f. cytotoxic effect of ciprofloxacin in primary culture of rat astrocytes and protection by vitamin e. toxicol 2007;229:54-61. 4. masadeh mm, mhaidat nm, alzoubi kh, al-azzam si, shaweesh ai. ciprofloxacin-induced antibacterial activity is reversed by vitamin e and vitamin c. curr microbiol 2012;64:457-62. 5. hall mm, finnoff jt, smith j. musculoskeletal complications of fluroquinolones: guidelines and precautions for usage in the athletic population. pmr 2011;3:132-42. oktavianus 175 *faculty of medicine atmajaya catholic university **indonesian society for health promotion education ***world health organization indonesia correspondence prof. charles surjadi, md, mph, ph.d. faculty of medicine atmajaya catholic university jl. pluit raya no 2 jakarta utara email: kotasehat@hotmail.com univ med 2012;31:175-83 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background nutrition has come to the fore as one of the major modifiable determinants of chronic disease. establishing healthy eating habits during adolescence is important given that fruit and vegetable consumption has long-term healthprotective benefits. the objective of this study was to investigate the determinant factors of fruit and vegetable consumption habits among padang inhabitants methods we conducted a questionnaire-based rapid assessment of 150 respondents who came from different settings: the questionnaire consisted of items on personal characteristics such as age, working status, gender, and personal knowledge of the subjects about the cause of non-communicable diseases (ncds) and their activities to prevent ncds. bivariate analysis was applied to look for variables significantly related to healthy eating (vegetable and fruit consumption). we applied multiple logistic regression to look for the best model to explain factors related to regular fruit and vegetable consumption. results the age range of the subjects was 14 to 76 years, 60% of subjects were women, and 40% were men. the study indicated that 64.7% of the respondents perceived that eating habits relate to ncd, while 67.3% consumed fruits and vegetables regularly. multivariate logistic regression analysis indicated that gender (o.r.=2.74; 95% c.i. 1.54-5.27) and perception of ncd as being related to healthy eating (o.r.=5.62;95% c.i. 2.93-10.76) were significantly related to regular fruit and vegetable consumption. conclusion this study demonstrated that perception of ncd was the most determinant factor of regular fruit and vegetable consumption. activities to improve practice of regular fruit and vegetable consumption are part of control of ncd risk factors. keywords: non-communicable diseases, perception, fruit and vegetable consumption perception of non-communicable diseases predicts consumption of fruits and vegetables charles surjadi*, ismoyowati**, dwidjo susilo**, and hernani djarir*** 176 surjadi, ismoyowati, susilo, et al fruit and vegetable consumption persepsi tentang penyakit tidak menular memprediksi konsumsi buah dan sayur latar belakang gizi merupakan salah satu faktor determinan penyakit tidak menular yang dapat dimodifikasi. perilaku makan yang sehat semasa remaja dan dewasa merupakan hal yang penting karena konsumsi buah dan sayur merupakan faktor kesehatan protektip yang berjangka panjang. tujuan dari studi ini adalah menentukan faktor determinan dari kebiasaan konsumsi buah dan sayur pada penduduk kota padang. metode dilakukan penilitian secara cepat dengan menggunakan kuesioner pada 150 responden dari berbagai tatanan kehidupan. kuesioner terdiri dari beberapa karakteristik penting seperti umur, status kerja, jender, pengetahuan yang berkaitan dengan sebab penyakit tidak menular serta kegiatan mencegah penyakit tidak menular. analisis bivariat dilakukan untuk menilai variabel yang berkaitan secara signifikan dengan perilaku konsumsi buah dan sayur secara teratur. lebih lanjut dilakukan analisis regresi logistik ganda untuk menentukan model terbaik yang menjelaskan faktor yang berkaitan dengan konsumsi buah dan sayur secara teratur. hasil umur dari subjek berkisar antara 14 sampai 76 tahun, 60% responden adalah wanita, dan 40% pria. sebanyak 64.7% subjek mempunyai persepsi bahwa makanan sehat berkaitan dengan kejadian penyakit tidak menular, 67.3% responden mengkonsumsi buah dan sayur secara teratur. analisis regresi logistik ganda menunjukkan bahwa jender (o.r.=2.74;95% c.i. 1.54-5.27) dan persepsi tentang penyakit tidak menular (o.r.=5.62;95% c.i. 2.9310.76) secara signifikan berkaitan dengan konsumsi buah dan sayur secara teratur. kesimpulan persepsi tentang penyakit tidak menular merupakan faktor utama yang menentukan perilaku makan buah dan sayur secara teratur. kegiatan untuk meningkatkan persepsi tersebut perlu dilakukan sebagai bagian dari program penanggulangan faktor risiko penyakit tidak menular. kata kunci: penyakit tidak menular, persepsi, konsumsi buah dan sayur abstrak introduction non-communicable diseases (ncds), primarily cancers, cardiovascular diseases, chronic respiratory diseases, and diabetes are a l e a d i n g t h r e a t t o h u m a n h e a l t h a n d development in indonesia. the number of deaths caused by ncds increased significantly from 41% in 1995 to 59.5% in 2007. the basic health research in 2007 revealed the following prevalences of ncds as follows: hypertension (31.7%), arthritis (30.3%), heart diseases (7.2%), tumor/cancer (4.3%), asthma (3.5%), diabetes mellitus (1.1%), stroke (0.83%), and injuries from traffic accidents were 25.9%.(1) ncds are mostly related to four behavioral risk factors that are linked to globalization, rapid urbanization, economic transition, and poor lifestyles: a) tobacco use, b) poor diet, c) insufficient physical activities, and d) the use 177 o f a l c o h o l . ( 2 ) i n i n d o n e s i a , t o b a c c o u s e , unhealthy diet (including low vegetable and fruit c o n s u m p t i o n ) , a n d i n s u ff i c i e n t p h y s i c a l activities are common among the population and have a high prevalence nationwide. on the other hand, alcohol consumption is unusual among indonesians. the national prevalence of alcohol consumption was only 4.6%(1) and thus will not be discussed in this study. nutrition has come to the fore as one of the major modifiable determinants of chronic disease.(3) to prevent the development of chronic disease, decreasing the consumption of energydense foods and increasing the consumption of fruits and vegetables during adolescence are important targets for nutrition interventions. t h e d e v e l o p m e n t o f e ff e c t i v e n u t r i t i o n interventions requires a detailed understanding of the determinants of target eating behaviors.(4) the world health organization (who) has indicated that a low intake of fruits and vegetables is one of the top ten risk factors for global mortality.(5) the basic health research indicated that 93.6% of indonesians have low fruit and vegetable consumption. among 33 provinces in indonesia, riau had the lowest fruit and vegetable consumption with a prevalence of 97.9%, followed by west sumatra with a prevalence of 97.8%.(1) a low degree of physical activity is also an issue in indonesia. almost half of indonesians (48.2%)(1) are lacking in physical activities. people are categorized as having sufficient physical activities, if they regularly perform physical activities for 30 minutes per day and for at least a cumulative total time of 150 minutes five days per week. they are categorized as having insufficient physical activities (being less physically active) if the cumulative total time for activities is less than 150 minutes in a week. the highest prevalence of insufficient physical activities was in the provinces of east kalimantan (61.7%), riau (60.2%), banten (55.0%), jambi (57.8%) and west sumatera (54.8%).(1) at padang panjang, people have a habit to eat fatty food, and perform few physical activities. these unhealthy lifestyle habits are a major cause of ncds in the community of padang panjang. therefore, 11.62% of the community has obesity, 10.19% heart disease, 8.14% hypertension, 1.42% stroke, and 2.11% diabetes mellitus.(1) padang panjang is a small municipality in west sumatra with a population of 51,233 inhabitants. the non-communicable disease prevention programs have been implemented since 2007 and were included in the 2008-2013 mid-term plan of the city as well as the 20082 0 1 3 c i t y h e a l t h o ff i c e s t r a t e g i c p l a n . sustainability prevention and control of ncds is reinforced by the social health insurance act (local act 7/2009),(3) smoke-free zone (local act 8/2009) and mayor regulation 10/2009).(6) there is also a total ban on cigarette sponsorship and outdoor advertising, and integrated community coaching posts for ncds (pos pembinaan terpadu penyakit tidak menular), have been established in every village since 2009 to function as a place for socializing ncds prevention efforts (communication, information and education), and conducting screening tests for e a r l y d e t e c t i o n o f n c d s , n u t r i t i o n a l consultation, and simple gym exercises. however, the determinant factors of community knowledge and practices related to control ncds risk factors are still undefined. these data are important for pinpointing the activities to be done for changing community perception and behavior to prevent ncds, and can be valuable for ncds prevention and control at padang panjang. therefore, the objective of this study was to investigate the d e t e r m i n a n t f a c t o r s r e l a t e d t o f r u i t a n d vegetable consumption among the people of west sumatra padang city. methods research design this study was done from february 2011 to september 2011 at padang panjang, west sumatra. as part of the case study to look for univ med vol. 31 no.3 178 surjadi, ismoyowati, susilo, et al fruit and vegetable consumption socio ecological determinants of health related to ncds funded by who,(7) we conducted a rapid assessment of respondents from five different community settings, namely from the religious community (mosques), student community (high schools), restaurants, public servant (government offices) and integrated community coaching posts for ncds. research subjects t h e r e s p o n d e n t s w e r e p e r s o n s w h o understand the indonesian language and were older 18 years (except for students, where those aged 15 years were included), while persons having non communicable disease (cancer, diabetes mellitus, hypertension and stroke, cardiac disease), vegetarian or frequently practiced fasting during the last year before the interview were excluded from this study. data collection the purpose of this data collection was to determine the knowledge and practices of the subjects in relation to ncd risk factors and to analyzes the determinant factors of activities for prevention of ncds. at each of the above community settings two trained interviewers asked a given set of questions to 30 people attending these places, who were selected randomly on the day of interview, to give a total 150 respondents. we did not calculate the minimal sample size for this study, since it was not our aim to measure the prevalence of healthy life practice. this study aimed specifically at comparing the knowledge and practice of above five community groups to pinpoint what had b e e n n e g l e c t e d i n t h e n c d a w a r e n e s s campaign. the questionnaire contained items on personal characteristics, such as age, working status, and gender. in addition to demographical q u e s t i o n s , t h e s t u d y r a i s e d q u e s t i o n s t o respondents as follows: a. on ncd knowledge: what causes a person to be exposed to ncds (hypertension, stroke, cardiovascular disease, diabetes)? possible answers for ncds knowledge: i) insufficient exercise; ii) smoking habits; iii) eating habits (“eat vegetables and fruits regularly”) b. on ncds prevention: what have you done to prevent ncd? possible answers for ncds prevention: i) exercise regularly yes or no; if yes specify every day, every two days, every three day, every four days, every five days, every six days; ii) reduce/ q u i t s m o k i n g ; i i i ) h e a l t h y e a t i n g specifically eat fruits and vegetables regularly yes or no; if yes specify every day, every two days, every three days, every four days, every five days, every six days. data analysis the filled-in questionnaires were checked for errors and consistency, and input into the computer for univariate and bivariate analysis. in the bivariate analysis we applied the chisquare test to look for variables significantly related to eating habits (eat vegetables and fruits) and for the variables which were significantly related to eating habits (p<0.10). we applied multiple logistic regression to look for the best model to explain factors related to regular fruits and vegetables consumption using spss software version 17.0. results table 1 showed that 60% of respondents were women, 40% were men, within the age range of 14 to 76 years. a total of 27.3% of respondents were older than 30 years, while the groups above 40 years of age amounted to 28.7%. regarding their occupational status, 20.8% of respondents were housewifes, 34.2% government workers, 24.8% students, and 20.1% self-employed (farmers, laborers and others which we grouped as non-government workers). only less than 5% persons were not doing any of the three prevention practices, and about one third (35.5%) did these three prevention practices to prevent ncds. 179 women were significantly more likely to eat fruits and vegetables than men (p=0.002), while housewifes were more likely have healthy food eating habits than government workers, although the difference was statistically not significant (p=0.054) subjects with perception of ncd significantly eat fruits and vegetables more regularly compared to persons who did not have that perception (p=0.000) (table 2). table 1. characteristics and practice of subjects to prevent ncds (n=150) § ncds=non-communicable diseases table 2. factors related to regular fruits and vegetables consumption (n=150) § ncds=non-communicable diseases (ncds) univ med vol. 31 no.3 180 surjadi, ismoyowati, susilo, et al fruit and vegetable consumption further analysis using multiple logistic regression (table 3) indicated that the practice of regular consumption of vegetables and fruits w a s s i g n i f i c a n t l y r e l a t e d t o g e n d e r a n d perception of ncds. women were 2.74 times (95% confidence interval 1.43 5,27) more likely than men to practice regular consumption of fruits and vegetables to prevent ncds. in addition, persons who perceived that ncds are related to healthy eating were 5.62 times (95% confidence interval 2.93 to 10.76) more likely to practice regular consumption of fruits and vegetables than persons who did not perceive that ncds is related to healthy food. discussion this main purpose of the present study was to investigate determinant factors related to the habit of eating vegetables and fruits regularly to control risk factors of ncd. the results of this study indicated that the habit of regular fruit and vegetable consumption was affected by gender. women were significantly more likely than men to have regular fruit and vegetable consumption. similar results were obtained in an australian study, where the habit of fruit and vegetable consumption was significantly found among a larger number of women as compared to men.(4) in contrast, in studies conducted in bangladesh and india, women were more likely than men to have inadequate fruits and vegetables consumption.(8) this suggests that gender also plays a role in food patterns, possibly related to broader cultural differences within the study site. as pointed out by narain,(9) ncds are a global health and developmental emergency, as they cause premature deaths, exacerbate poverty and threaten national economies. activities to control ncds will require a broad r a n g e o f i n t e g r a t e d , p o p u l a t i o n b a s e d interventions as well as measures focused on the individuals at high risk.(10) our study results demonstrated that subject perception of ncds play a major role in determining regular fruits and vegetables consumption. the latter is encompassed in an individual’s perception on healthy foods, by w h i c h i s m e a n t c o n s u m p t i o n o f f r u i t s , vegetables and meat, and limitation of sugar, f a t s a n d s a l t s , a n d a l s o v a r i a t i o n a n d moderation.(11) data on several important ncds risk factors pointed out by several authors,(12,13) such as blood pressure, fasting blood sugar, and blood cholesterol, were not collected in our study, because of our limited resources. on the other hand, data on healthy eating and physical activities, reported by many researchers in developing countries as important factors of n c d s , a r e e a s i l y c o l l e c t e d b y o r d i n a r y persons.(14,15) this study would like to look for what strategies had been used in empowering communities for prevention and control of n c d s , s p e c i f i c a l l y f o c u s e d o n d i e t a n d individual attitude toward ncds. in urban areas diet receives much attention, since the majority of people in cities have to buy vegetables at markets, while vegetables are more expensive than snacks. in addition to the work done in other studies, we would like to analyze why some table 3. multiple logistic regression of genders and perception of ncds on regular fruits and vegetables consumption § ncds=non-communicable diseases; *b= regression coefficient 181 p e r s o n s h a v e h e a l t h y p r a c t i c e s , s u c h a s consuming a healthy diet and performing physical activity, while others do not.(16) we realize that there are others factors that contribute to physical activity in urban areas. these factors are grouped into the micro and macro factors, such policy and ecological condition, .and should also be analyzed(17) people can be encouraged to increase their physical activity, especially by walking and bicycling, and by using public transport and not their own motorized transport,(18,19) as well as related to diet such as agricultural policy.(20,21) the important role of gender is also pinpointed by the world health organization (who) commission of social determinants, which set up gender for analysis of health inequity.(22) gender has an important effect on the determinants and consequences of noncommunicable diseases and conditions in both developing and industrialized countries.(23) this indicates that a different approach is needed as discussed by beaglehole et al.(24) we need a comprehensive health system that integrates all common disorders, irrespective of cause and course over time, including ncds. in the case of padang, the health agency should facilitate healthy eating in the community to convince people that healthy eating (eating vegetables and fruit regularly) may prevent ncds in the community through primary health care activities in the community and at public health centers as advised by some researchers.(25 ) at city level this may be done under the leadership of the mayor in collaboration with associations of restaurants and culinary societies and may be integrated into the agricultural agency as part of improving the consumption of agricultural products, which in turn increase income of farmers. the latter activities will be more likely to be successful if there is strong national policy, such as strong support for the presence of traditional markets. these usually sell fresh vegetables and fruits and can give better access to vegetables and fruits, and thus may be considered an alternative to supermarkets. among ministry of health policy makers, padang panjang is well known for its health promotion activities at community level and the role of healthy city forum. however, the results of this study indicates the importance to explore action on social determinants of health through intersectoral health actions and action at city, district and community level through distal factors such as availability of fresh vegetables and food, social support at community level, leadership at city and district as well as community level for healthy eating with support from agriculture, traditional markets etc. this kind of action has already been discussed e l s e w h e r e a s p a r t o f a c t i o n s o n s o c i a l determinants of health.(26-28) lifestyles and life changes are complex, and depend on the interaction of many different factors over time. once health problems occur, they last throughout life. ncd prevention requires strategies that are directed at different target age groups. apart from all of the above factors, community perception and attitude are also important factors for controlling some disease problems, such as communicable diseases. it is pointed out how irrational knowledge and p e r c e p t i o n b e h a v i o r t h a t t h r e a t e n s t h e effectiveness of public health programs can be tied to public perceptions created by media portrayals of health risk, which is applied to communicable diseases and possibly also to non communicable disease, however this study indicates that the perception of ncds as being related to healthy eating is an important factor for the practice of healthy eating. this idea to collect data on people’s perception to determine rational thinking is in line with the study on communicable diseases which is pointed out the i m p o r t a n c e o f t h i s i s s u e t o w a r d p u b l i c practices.(29) the present study has some limitations. first, the data are not representative of the population, because we did not calculate the minimum sample size of the respondents. second, we did not apply random sampling to univ med vol. 31 no.3 182 surjadi, ismoyowati, susilo, et al fruit and vegetable consumption the population groups, because our aim was to make comparisons among the five urban groups about their health knowledge and practice. other limititations of this study was in the collection of data on the practice of fruits and vegetables consumption, which was only qualitative, by asking whether the respondents consumed fruits and vegetables regularly, and how frequent, using the questionnaire used by the basic health research survey. however, the majority of the respondents did not specify the frequency of fruit and vegetable consumption, since the method of data collection was a rapid interview in the field. therefore we only analyzed if the respondents “eat fruit and vegetables regularly”. this limitation is acceptable due to the nature of data collection as a participatory rapid assessment by the community, with the aim to discuss the results with the community for further action, to validate the results and plan for improvement. as a matter of fact one month after the rapid survey, the community groups from each setting discussed the results and made plans for improvement under supervision of the healthy city forum. conclusion women and perception of ncds were important factors of healthy eating practice (“eat fruits and vegetables regularly”) for the prevention of ncds. based on this result it is important to implement activities to improve people’s perception on the importance of regular fruit and vegetable consumption to control risk factors of ncds. acknowledgements t h a n k s a r e d u e t o w h o s e a r o , specifically dr suvajee good, for her support and advice to conduct this study.we are also grateful for the cooperation of the center for health promotion of ministry of health and the city of padang. finally, we thank all anonymous reviewers for their advice to improve the manuscript. references 1. national institute of health research and development. report of basic health research 2007. jakarta: ministry of health republic of indonesia;2008. 2. world health organization. global status report on non communicable diseases 2010. geneva: world health organization;2011. 3. world health organization. diet, nutrition and the prevention of chronic diseases: report of a joint who/fao expert consultation. who technical report series no. 916. geneva: who;2003. 4. pearson n, ball k, crawford d. predictors of changes in adolescents’ consumption of fruits, vegetables and energy-dense snacks. br j nutr 2011;105:795–803. 5. world health organization. global strategy on diet. physical examination and health. geneva: world health organization;2006. 6. padang panjang municipality. regulation of mayor on local health insurance 10/2009; padang panjang;2009. 7. surjadi c, ismoyowati, susilo d. changing lifestyle movement and non communicable disease: a case study in padang panjang municipality: final report to who searo;2011. 8. kanungsukkasem u, ng n, van minh h, razzaque a, ashraf a, juvekar s, et al. fruit and vegetable consumption in rural adults population in indepth hdss sites in asia. global health action suppl 2009;1:35-43. doi: 10.3402/gha.v2i0.1988 9. narain jp, garg r, fric a. non-communicable diseases in the south-east asia region: burden, strategies and opportunities. nat med j india 2011;24:280-7. 10. unwin n, alberti kg. chronic noncommunicable diseases. ann trop med parasitol 2006;5:455-64. 11. paquette mc. perception of healthy eating: state of knowledge and research gaps. can j public health 2005;96 suppl 3:s15-9. 12. ikeda n, inoue m, iso h, ikeda s, satoh t, node m, et al. adult mortality attributable to preventable risk factors for non-communicable diseases and injuries in japan: a comparative risk assessment. plos med 2012;9:e1001160. doi:10.1371/journal.pmed.1001160. 13. msyamboza kp, ngwira b, dzowela t, mvula c, kathyola d, harries ad, et al. the burden of selected chronic non-communicable diseases and their risk factors in malawi: nationwide steps 183 survey. plos one 2011;6:e20316. doi:10.1371/ journal.pone.0020316. 14. singh k, reddy k, prabhakaran d. what are the evidence based public health interventions for prevention and control of ncds in relation to india. indian j comm med 2011;36 suppl 1:s23–31. 15. mathur p, b shah b. research priorities for prevention and control of non communicable diseases in india. indian j com med 2011;36 suppl 1: s72–7. 16. bauman ae, reis rs, sallis jf, wells jc, loos rjf, martin bw for the lancet physical activity series working group. correlates of physical activity: why are some people physically active and others not? lancet 2012;380:258-71. 17. popkin bm, duffey k, gordon-larsen p. environmental influences on food choice, physical activity and energy balance. physiol behav 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2005;19:407–21. univ med vol. 31 no.3 c:\users\jurnal fkusakti\docume 108 *department of surgery, atma jaya school of medicine and health science, jakarta **department of surgery, atma jaya hospital, jakarta correspondence: daniel ardian soeselo department of surgery, atma jaya school of medicine and health science, jl. pluit raya no.2, north jakarta 14440 phone: +62813 8193 7250 email: ardsoeselo@yahoo.com orcid id: orcid.org/0000-00033051-665x date of first submission, july 31, 2018 date of final revised submission, june 17, 2019 date of acceptance, june 18, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina adequate fluid resuscitation decreases incidence of acute renal failure in burn patients daniel ardian soeselo*,**and etheldreda alexandria stephanie suparman* background burns constitute a severe health problem in many countries. in indonesia burns rank 4th of all trauma-related diseases and are a burden on the country’s health system. adequate fluid resuscitation is the initial management of burns that determines the success of treatment. this study aimed to determine the relationship between adequate fluid resuscitation and incidence of acute renal failure in burn patients. methods a retrospective study of cross-sectional design was conducted on 30 burn patients who came to the emergency unit (er) from january 2015-december 2017. medical records were reviewed to examine the data on fluid resuscitation according to the parkland formula and the laboratory data. acute renal failure was defined as a creatinine level of more than 2.1 mg/dl after 7 days. hypoalbuminemia was defined as an albumin level of less than 3.4 g/dl. fisher’s exact test was used to analyze the data. results twenty-two subjects received fluid resuscitation according to the parkland formula and 8 did not. twenty-five experienced complications such as acute renal failure (arf) (13.3%), hypoalbuminemia (46.7%) and a combination of arf and hypoalbuminemia (23.3%). one person died. adequate fluid resuscitation was significantly associated with decrease incidence of arf (p=0.015), but not significantly with hypoalbuminemia (p=0.214) and with mortality (p=0.267). conclusion adequate fluid resuscitation decreased the incidence of arf in burn patients. consensus protocols for initial burn resuscitation and treatment are crucial to avoid the consequences of arf after burn injury. keywords: burns, fluid resuscitation, acute renal failure, hypoalbuminemia, parkland formula original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.108-113 may-august, 2019 vol.38no.2 ci te t his art icle as: soes elo da, suparman eas. adequate fluid resuscitation decreases incidence of acute renal failure in burn patients. univ med 201 9;38 :1 0 8-1 3 . doi: 10 .18 05 1/ univmed.2019.v38.108-113 109 univ med vol. 38 no.2 introduction burns are one of the health problems in ma ny c o unt r i e s , ( 1 ) a n d c o ntr i b ut e t o approximately 1% of the disease burden in the world. they also cause disability in nearly 18 million persons and more than 265,000 deaths globally each year..(2,3) more than 95% of burns occur in low-income developing countries.(4,5) in addition, burns rank fourth of all trauma-related disorders and are also a burden for a country’s health system because of the high healthcare costs as a result of prolonged hospital stays.(2) data from the who show that burns occupy ninth rank of all-cause mortality in the age group of 5-14 years, with an estimated total number of deaths of 41,575; and fifteenth rank in the age groups of 0-4 years and 15-29 years, with an estimated total number of deaths of 62,655 and 49,067, respectively.(2,6) in indonesia, burns cause approximately 195,000 deaths each year. cipto mangunkusumo hospital, as the national burn referral center in indonesia, receives more than 130 patients each year from all over the country. according to the indonesian department of health, burns occupy sixth rank as a c ause of inj ury.(2) recent research on the mortality of burn patients in cipto mangunkusu mo hospital, j akar ta and in soetomo hospital, surabaya, found that the mortality rate from burns is still high.(4,7,8) a study on burns at soetomo hospital, surabaya, in 2007-2011 showed that the maleto-female ratio of burns was 2:1. the most frequent cause of burns is thermal injury to a total of 48.3%.(8) one study at the burns unit of cipto mangunkusumo hospital in 2011-2012 showed that the most frequent cause was thermal injury (heat), to a total of 78%, while the majority of burns are second-degree burns, with the most frequent cause of mortality being sepsis.(4) another study from the burns unit of ciptomangunkusumo hospital in 2013-2015 showed that most burn patients were >18 years old and that the most frequent cause was thermal injury.(2) t he stud y of san c he z e t a l. ( 9) usi ng hydroxyethyl starch found a high incidence of acute renal failure (arf) in burn patients, as evaluated using the criteria of the risk/injury/ f a i l u r e / lo ss / e n d-s t a ge ki d ne y ( rifle ) classification and the acute kidney injury network (akin). the study by michael et al. (10) using crystalloids showed a high incidence of arf from inadequate resuscitation as evaluated with the rifle criteria, leading to a high mortality risk in burn patients. other studies also state that one of the risk factors of arf in burn patients is inadequate administration of rescuscitation fluid.(11-13) the study of kumar et al.(14) on urea creatinine after hospitalization for 5 days states that the total amounts of crystalloid resuscitation fluid administered determine the arf incidence in burn patients. the study by thalji (15) states that arf is associated with the morbidity and mor ta l it y r a t e s of bu r n p a t i en ts du r i ng hospitalization and at one year post-trauma. the study of schneider et al.(16) using a classification and regression tree (cart) analysis and rifle criteria showed a high incidence of arf in burn patients. the difference between previous studies and the present study is that the latter describes the characteristics of burn patients who attended the atma jaya emergency unit (er) hospital and evaluated the mortality and morbidity rate by determining the presence or absence of acute kidney failure, hypoalbuminemia and death as a result of burns. methods design of the study this was an analytical observational crosssectional study carried out at atma jaya hospital (ajh), jakarta, by reviewing the medical records of patients hospitalized from january 2015 to december 2017. study sample the study sample was obtained from the medical record data, using purposive sampling. 110 the subjects in this study were all patients who came to the ajh er within the first 24 hours after injury and who were hospitalized between january 2015 and december 2017. the exclusion criteria in this study were patients who were discharged against medical advice, patients with incomplete medical records, and patients with a history of renal failure. the formula used in calculating the sample size was n = (zα)2 pq / d2 , where zα = conversion area under the standard normal curve at the 96% confidence level, p = categorical proportion, q = 1 p, and d = 10% deviation (confidence interval). all patients with the diagnosis of burns were entered into this study. according to the formula the minimal sample size was 28. a total of 40 subjects was found, but 10 subjects were excluded because of incomplete medical record data, so that in total only 30 subjects were analyzed. definitions the parameter of acute kidney failure used was one of the criteria of the kidney disease: global improvement outcome (17) i.e. an increase in blood creatinine concentrations of 1.5 times the normal value after the 7th day, this being >2.1 mg/dl. the parameter of hypoalbuminemia used was a blood albumin concentration of <3.4 g/dl on day 7 of hospitalization. the parameter of fluid therapy used was the agreement of the administered rescuscitation fluid with the parkland formula, i.e. 4 ml x bw x % burn area, using crystalloids. (18) data collection in this study, the data obtained from the medical records were among others age, gender, and area, degree, cause, and total number of b ur ns a nd typ e o f r e su s c i t at io n f lu i d administered, blood creatinine and albumin concentrations in the er and at day 7 of hospitalization, and mortality data. in addition, this study evaluated the agreement with the parkla nd formula of the total amount of resuscitation fluid administered (4 ml x bw x % burn area) and its type (crystalloids).(18) statistical analysis t he s t a ti st i c a l t e s t us ed wa s t he kolmogorov-smirnov test of normality to evaluate the distribution of the study data. the relationship of administered resuscitation fluid with morbidity and mortality of the burn patients was evaluated with the fisher test. ethical clearance this study obtained ethical clearance from the ethics commission, faculty of medicine, atma jaya catholic university of indonesia, jakarta, under no. 02/06/kepfkuaj/2018. results the characteristics of burn patients at ajh (table 1) showed that the majority were males, to a total of 18 persons (60%). the most frequent age group, according to the world health organization (who) classification, was the age group of 18 65 years, totalling 27 persons (90%). the largest burn area (as percentage of total body surface) was 21-49 %, totalling 19 persons (63.4%). the most frequent degree of burn was iia with a total of 18 persons (60%). the most frequent cause of burns was heat, with a total of 28 persons (93.3%). the percentage of burn patients who received resuscitation according to reference was 73.3%. the number of morbidity events in the form of arf and hypoalbuminemia was found to be 25. four persons (13.3%) had arf only, 14 per sons (46.7% ) had hypoalbuminemia only, and 7 persons (23.3%) had arf and hypoalbuminemia. one person died after administration of re suscitation fluid and hospitalization for 22 days as a result of hospitalacquired pneumonia (table 1). analysis of the as sociation be tween resuscitation fluid and morbidity of burn patients in ajh was performed with fisher’s test. the morbidity of burn patients in this study was divided into two, i.e. due to arf and hypoalbuminemia. analysis of the association of arf with resuscitation fluid in burn patients found a p value of 0.015 (p<0.05), so that there was a significant soeselo, suparman fluid resuscitation in burn trauma 111 univ med vol. 38 no.2 association between adequate administration of r es usci ta ti on f luid an d de cr ea se i n arf incidence. in the analysis of the association of hypoalbuminemia with resuscitation fluid in burn patients, a non-significant association was found (p=0.214) (table 2). analysis of the association between resuscitation and mortality of burn patients found a non-significant association between administration of resuscitation fluid and incidence of mortality of burn patients (p=0.267) (table 2). discussion this study showed that the most frequent gender was male, in a male-to-female ratio of 1.5:1. a study in the last 5 years stated that the most frequent cases of burns were in males as compared with females. this is due to the gre ater mobility and activity of males as compared to females.(8) a study carried out at rscm also found a predominance of males as compared to females in burn cases.(2)a b c total n % gender male 18 60 female 12 40 age  17 years 2 6.7 18 65 years 27 90 66 79 years 1 3.3  80 years 0 0 burn area  20% 5 16.7 21 49 % 19 63.3 ≥ 50 % 6 20 degree of burn grade i 0 0 grade ii a 18 60 grade ii b 11 36.7 grade iii 1 3.3 cause of burn thermal 28 93.3 electrical 1 3.3 chemical 1 3.3 agreement of fluid therapy yes 22 73.3 no 8 26.7 morbidity none 5 16.7 acute renal failure 4 13.3 hypoalbuminemia 14 46.7 acute renal failure + hypoalbuminemia 7 23.3 mortality live 29 96.7 dead 1 3.3 table 1. distribution of characteristics of burn patients (n=30) resuscitation p value in agreement (n) not in agreement (n) arf yes 5 6 0.028 no 17 2 hypoalbuminemia yes 14 7 0.374 no 8 1 mortality live 22 7 0.267 dead 0 1 table 2. analysis of the association of resuscitation fluid with arf incidence, hypoalbuminemia and mortality of burn patients (n=30) arf: acute renal failure 112 in the present study, the highest number of burn cases was in the age group of 18 65 years (90% of 27 cases). this is in agreement with existing research data on the age of burn patients, showing that these mostly occur in the first to the fifth decade of life.(2,8) these age groups are the productive age groups, whose members are working a nd more frequently exposed to hazardous situations, so that the incidence of burns is highest in the productive age (68%).(2) the study results show that the number of burn cases occurring in the age interval of 18-65 years was in total 27 (90%). the highest age of the burn cases in this study was 65 years (one case) and the youngest age was 6 years (one case). the study results show that the most frequent burn area was 20%, with 5 cases (16.7%). this differs from the study carried out at rscm where the burn area was 20-50%.(2,8) in our opinion, this is due to a difference in hospital type. rscm is the reference burns centre, so that the burn cases managed over there are certainly more complicated and severe. the study results show that the most frequent cause of burns was heat, with a total of 28 cases (93%). this is in line with the study carried out at rscm where the most frequent cause of burns was heat (due to explosion of lpg bottles).(2,7) fluid resuscitation therapy in burn patients is varied. in this study we used the parkland formula as reference and found that 22 patients (73.3%) received therapy according to the parkla nd formula. t his for mula , even if appropriately applied, still has several limitations, such as errors in determining body weight, burn degree, and burn area. these errors may result in the overor underestimation of the total amount of fluid actually required by the patients. this study found a significant association between adequate administration of resuscitation fluid and decreased incidence of arf. this is in line with the results of other studies, stating that adequate administration of resuscitation fluid may prevent arf, by improving tissue perfusion, particularly in the kidneys.(16,19,20) adequate administration of resuscitation fluid to burn patients may maintain the homeostatic balance in the blood vessels and tissue perfusion. this study found no significant association between administration of resuscitation fluid according to the parkland formula and incidence of hypoalbuminemia. this is in line with other studies stating that even adequate administration of resuscitation fl uid ma y still cause hypoalbuminemia, as seen from the serum albumin concentration.(20) in addition, this is also one of the pathophysiological mechanisms of burns, where vasodilation of the blood vessels is still present, even after adequate administration of resuscitation fluid. this causes extravasation of protein and albumin as a result of capillary permeability.(21) a limitation of this study was that it was not designed to demonstrate the effectiveness and safety of fluid administration according to the parkland formula and therefore had no control group. in addition, this study comprised many confounding factors, so that a larger number of patients would be needed to draw appropriate conclusions. the clinical impact of the findings of this study is that administration of fluid according to the parkland formula may avoid arf in burn patients. further research is expected to involve prospective studies with a larger sample size. conclusions adequate administration of resuscitation fluid may reduce arf incidence in burn patients. although the parkland formula may prevent arf, it has several limitations in its application. there is a need for strict supervision during fluid resuscitation, even when the parkland formula is used. conflict of interest there is no conflict of interest in this study. soeselo, suparman fluid resuscitation in burn trauma 113 univ med vol. 38 no.2 acknowledgement the authors wish to thank all staff members in the medical record section who provided the medical records of burn patients in atma jaya hospital, jakarta. contributors das dan eass contributed to composing and writing all manuscript drafts. all authors contributed information for the manuscript, participated in revising the manuscript, and approved the final version for publication. references 1. yasti ac, senel e, saydam m, et al. guideline and treatment algorithm for burn injuries. turk j trauma emerg surg 2015;21:79-89. doi: 10.5505/ tjtes.2015.88261. 2. wardhana a, basuki a, prameswara adh, et al. the epidemiology of burns in indonesia’s national referral burn center from 2013 to 2015. burns open 2017;1:67–73. doi: https://doi.org/ 10.1016/j.burnso.2017.08.002. 3. world health organization. global health estimates 2016: disease burden by cause, age, sex, by country and by region, 2000-2016. geneva: world health organization; 2018. 4. martina nr, wardhana a. mortality analysis of adult burn patients. j plast rekonstr 2013;2:497– 524. doi: https://doi.org/10.14228/155. 5. van der merwe ae, steenkamp wc. prevention of burns in developing countries. ann burns fire disasters 2012;25:188-91. 6. world health organzation. who health estimates 2014 summary tables: deaths and global burden of disease. geneva: world health organzation; 2014. 7. pujisriyani, wardana a. epidemiology of burn injuries in cipto mangunkusumo hospital from 2009 to 2010. j plast rekonstruksi 2012;1:528-31. doi: https://doi.org/10.14228/110. 8. hidayat tsn, noer ms. five years retrospective study of burns in dr. soetomo general hospital surabaya. j rekonstr estet 2013;1:123-30. 9. sánchez-sánchez m, garcia-de-lorenzo a, cachafeiro l, et al. acute kidney injury in critically burned patients resuscitated with a protocol that includes low doses of hydroxyethyl starch. ann burns fire disasters 2016; 29:183–8. 10. michael j, tam n, matthew b, et al. early acute kidney injury predicts progressive renal dysfunction and higher mortality in severely burned adults. j burn care res 2010;31:83–92. doi: 10.1097/bcr.0b013e3181cb8c87. 11. wu g, xiao y, wang c, et al. risk factors for acute kidney injury in patients with burn injury: a metaanalysis and systematic review. j burn care res 2017;38:271-82. doi: 10.1097/bcr.0000000000 000438. 12. clark a, neyra j, madni t, et al. acute kidney injury after burn. burns 2017;43:898-908. doi: https://doi.org/10.1016/j.burns.2017.01.023. 13. coca sg, bauling p, schifftner t, et al. contribution of acute kidney injury toward morbidity and mortality in burns: a contemporary analysis. am j kidney dis 2008;49:517-23. 14. kumar ab, andrews w, shi y, et al. fluid resuscitation mediates the association between inhalational burn injury and acute kidney injury in the major burn population. j crit care 2017;38:62-67. doi: 10.1016/j.jcrc.2016.10.008. 15. thalji sz, kothari an, kuo pc, et al. acute kidney injury in burn patients: clinically significant over the initial hospitalization and 1 year after injury: an original retrospective cohort study. ann surg 2017;266:376–82. doi: 10.1097/sla.000000000000 1979. 16. schneider df, dobrowolsky a, shakir ia, et al. predicting acute kidney injury among burn patients in the 21st century: a classification and regression tree analysis. j burn care res 2012; 33:242-51. doi: 10.1097/bcr.0b013e318239cc24. 17. international society of nephrology. guidelines – kdigo. kidney disease improving global outcomes (kdigo). kidney international supplements 2017;7:1–59. 18. wardhana a, hardya h, rezza m. parkland formula vs restricted fluid in burn resucitation a systematic review of cohort studies. critical care med 2018;46:34. doi: 10.1097/01.ccm.0000528323. 46970.09. 19. watkins sc, shaw ad. fluid resuscitation for acute kidney injury: an empty promise. curr opin crit care 2016;22:527–32. doi: 10.1097/mcc. 0000000000000363. 20. pérez-guisado j, de haro-padilla jm, rioja lf, et al. serum albumin levels in burn people are associated to the total body surface burned and the length of hospital stay but not to the initiation of the oral/enteral nutrition. int j burns trauma 2013;3:159–63. 21. becerra oaa, garibay ct, amezcua mdm, et al. serum albumin level as a risk factor for mortality in burn patients. clinics (sao paulo) 2013;68:9405. doi: 10.6061/clinics/2013(07)09. c:\users\jurnal fkusakti\docume 159 *tuberculosis and lung disease research center, imam reza hospital, division of respiratory and critical care medicine, department of internal medicine, medical faculty, tabriz university of medical sciences, tabriz, iran **medical student, imam reza hospital, division of respiratory and critical care medicine, department of internal medicine, medical faculty, tabriz university of medical sciences, tabriz, iran correspondence: sepideh tahsini tekantapeh tuberculosis and lung disease research center (lung.center@yahoo.com), imam reza hospital, division of respiratory and critical care medicine, department of internal medicine, tabriz university of medical sciences, tabriz, iran email: tahsinis@tbzmed.ac.ir date of first submission, december 20, 2017 date of final revised submission, may 16, 2018 date of acceptance, june 4, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina bronchoscope saline injection method (me hejazi method) in ruptured peripheral and central pulmonary hydatid cysts: a case series mohammad esmaeil hejazi*, sepideh tahsini tekantapeh*, and saeede maddahi** background this paper mainly focuses on patients with ruptured pulmonary echinococcus granulosus infections (alveolar hydatid disease), who suffered from ruptured alveolar hydatid cyst. in this study we aimed to remove these ruptured central and peripheral pulmonary hydatid cysts by the bronchoscopic saline injection method (me hejazi method). case description in this retrospective study, we evaluated eight patients from an endemic area who were non-surgically treated for ruptured pulmonary hydatid cysts at imam reza hospital between 2016-2017. by the bronchoscopic saline injection method (me hejazi method), we extracted the entire hydatid cysts of all patients by fiber optic bronchoscopy for the detachment of the underlying membrane from the cavity wall. there were three female and five male patients, with a mean age of 40 ± 23 years (range 17–63 years). ruptured cysts were located in the peripheral (2) and central (6) parts of the lungs. all of our experiences have been successful without any complications and residual cyst membrane. during the follow-ups, clinical and radiological recovery were seen in these patients. conclusions in the peripheral ruptured hydatid cysts, accurate recognition and location of the cyst is essential and the blind approach is not recommended because it needs several bronchoscopic interventions. therefore the hejazi method will be a beneficial and suitable alternative method for surgery in the treatment of patients with ruptured pulmonary hydatid cyst with cyst membrane adhesions. keywords: bronchoscope saline injection method, me hejazi method, pulmonary hydatid disease, ruptured hydatid cysts doi: http://dx.doi.org/10.18051/univmed.2018.v37.159-164 case report pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 ci te this arti cle as: heja zi m e, tekantapeh st, maddahi s. bronchoscope saline injection method (me hejazi method) in ruptured peripheral and central pulmonary hydatid cysts: a case series. univ med 2018;37:159-64. d oi : 10.1 805 1/univm ed. 201 8.v3 7. 1 5 9 -1 6 4 160 hejazi, tekantapeh, maddahi me hejazi method results in bronchoscopy introduction human cystic and alveolar echinococcosis (hydatid disease) is still the cause of morbidity and mortality in some parts of the world including eastern asia, middle east, north europe, and south america. somehow it is a public health problem in the mediterranean and middle east area.(1,2) hydatid disease is a chronic parasitic disease with an animal-human cycle. it is caused by the larval (metacestode) stages of the dog tapeworm belonging to the genus echinococcus (taeniidae). there are three recognized forms o f ec hi n o coc c os is: c yst i c , ca us e d b y e. g ra n ul o su s , a l ve ol a r, c a u s e d by e. multilocularis, and polycystic, caused by e. vogeli.(2,3) among the three forms of hydatid disease, alveolar echinococcosis is associated with poor response and cyst rupture is an expected probability which complicates patient management and treatment.(4,5) de sp i t e c ur r e n t c o n tr ol me t ho ds , elimination of this pathogen is difficult. hydatid control campaigns have made impressive technological improvements in the prevention, diagnosis and treatment of human and animal cystic echinococcosis. although using these new methods increased the efficiency of hydatid control programs, hydatid disease is still common, the treatment is difficult enough and it will be more complex when we face a ruptured hydatid cyst. (2) human cystic echinococcosis is the most co mmon p res enta ti on whic h is estimated to be more than 95% of whole global cases (2-3 million) compared to alveolar echinococcosis which is less than 0.5 million cases (all reported in the northern hemisphere). the lungs are involved in about 30% of cystic echinococcosis.(3) t he ma i n wa y t o d i a gn os e h uma n echinococcosis is by imaging techniques (e.g. computed tomography, magnetic resonance imaging, ultrasound and radiography) which can accurately detect space occupying lesions. laboratory-based techniques is the other useful way for confirmation of clinical infection by the aid of specific serum antibodies (igg antibodies and recombinant antigen b subunits).(1,2) in cystic echinococcosis, fluid-filled cysts develop and grow in potential body cavities, mainly in the liver and lungs or other locations. cystic larvae grow slowly, thus they are well-tolerated by the host. in contrast, alveolar echinococcosis invades the surrounding parenchyma and does not have well-defined external margins.(4,5) the treatment of cystic lesions is difficult because most develop rapidly in the lungs. therefore surgical removal is the principal therapeutic approach to treat ruptured and chronic pulmonary hydatid cysts, but on the other side medicosurgical approaches are expanding, along with percutaneous drainage for hepatic cystic echinococcosis.(3-5) generally the outlook for these patients is unclear and rarely predictable, so long-term imaging follow-ups seems to be essential to manage their condition and achieve favorable treatment outcomes.(6) pulmonary hydatid cysts are often asymptomatic and can be diagnosed incidentally on plain chest radiographs, but computed tomography (ct) is the preferred i ma gi n g mo da l i t y. ( 3 ,7 ,8 ) t he a i m o f th i s retrospective study was to share our experiences o f r e mo vi ng t he se r up t u r e d ce nt r a l a nd peripheral pulmonary hydatid cysts by the bronchoscopic saline injection method (me hejazi method). case series the eight patients described in this report were seen and evaluated in the respiratory and critical care medicine ward of imam reza hospital between 2016 to 2017 for complaints of exacerbation of respiratory manifestations (table 1). al l ei ght pati ents wer e succes sfully managed nonsurgically by the me hejazi method (figure 1) and all were asymptomatic at oneyear follow-up (figures 2-5). this research was approved by the tuberculosis and lung disease research center ethics board. 161 univ med vol. 37 no.2 cases 1 2 3 4 5 6 7 8 gender age (years) cyst type history m 51 peripheral chronic cough with acute exacerbation m 17 peripheral recurrent purulent cough f 63 central chest wall pain and productive cough f 34 central fever, chills and pneumonia m 23 central abrupt purulent cough and dyspnea m 29 central chronic cough with recent exacerbation m 31 central increased sputum and dyspnea f 38 central pneumonia and purulent cough table 1. pertinent details of ruptured pulmonary hydatid cyst subjects note: m: male; f: female figure 1*. stepwise bronchoscopic saline injection method to extract the ruptured hydatid cyst. (a) ruptured hydatid cyst was seen with adhesion and inflammation in cyst base that could not be removed only with bronchoscopic suction. (b) a thin catheter was entered to the space between cyst and lung tissue. (c) fugarti balloon was inflated to release the adhesion of hydatid cyst. (d) a thin bronchoscope was entered to release the space between cyst and lung tissue. (e) the whole cyst contents was extracted by bronchoscope and the empty cyst was seen. (f) the procedure was performed with two bronchoscopes simultaneously * figure 1; reproduced from the clinical respiratory journal 2016; 1752-6981, with permission of the clinical respiratory journal ltd and courtesy of me hejazi et al. a novel bronchoscope method (saline injection method) for complete extraction of ruptured pulmonary hydatid cyst (case report article) figure 2. hydatid cyst white membrane as seen in orifice and in formalin 162 hejazi, tekantapeh, maddahi me hejazi method results in bronchoscopy a b c figure 3. peripheral hydatid cyst in a 51-year old man before (a) and after (b) me hejazi method figure 4. (a) peripheral hydatid cyst in a 17-year old young man who was referred with recurrent purulent cough. (b) complete removal of ruptured peripheral hydatid cyst 1 month after me hejazi method figure 5. ruptured central hydatid cysts of 6 patients which were resolved by interventional bronchoscopy method (a-f) 163 univ med vol. 37 no.2 discussion hydatid cyst infestation commonly involves the liver (55% to 70%), the lungs (18% to 35%), or both the liver and the lungs (5% to 13%).(9) a pulmonary hydatid cyst can spontaneously rupture into the pleura or bronchus due to superinfection, chest trauma, or erosion of cyst wall into a bronchus. (10) the diagnosis of complicated pulmonary hydatid cysts may not be easy because hydatid cyst disease mimics t ub e r c ul o si s , l un g c a nc e r, emp ye ma , or abscess.(11) pulmonary hydatid cysts may rupture into the bronchial tree or peritoneal cavity and produce cough, chest pain or hemoptysis.(12) surgery has traditionally been the principal definitive method of treatment. as mentioned above there are some choices for treatment of hydatid cyst but the main treatment of ruptured chronic lung hydatid cyst is surgery, which based on previous review studies, is associated with some complications such as pneumonia, lung atelectasis, deep vein thrombosis, hospitalization, and morbidity. in case that the patient was not a proper candidate for surgical operation or did not agree with surgery because of complications, the routine treatment is a combination of medications which has been proved not so effective.(3) in recent years several case reports were presented of incidental hydatid cyst removal by bronchoscope, but there was not any systematic and specific plan for proper clinical action.(5,13) since it seemed an efficient way to use, we started to think about making a welldefined method with a classified plan (as explained above) to remove ruptured hydatid cyst by bronchoscope. based on mahomed and jayaguru’s (14) method, we designed our saline injection method by bronchoscopy (me hejazi method) for removing the ruptured pulmonary hydatid cysts with adhesions which cannot be removed only by bronchoscopic suction. finally, for the first time, we tried our method on two patients and the results were satisfying. there was not any sign of complications, recurrence and remaining cyst membrane on the bronchial tree wall. so, we decided to introduce and describe this novel treatment method in a case report published in 2016 which had not been reported until then.(1) due to the satisfying results, we expanded our study and tried it on more patients with ruptured hydatid cyst (table 1). during the last three years (2015-2017), we did this method on 8 other patients with the same condition (figures 3-5). fortunately it was effective in all cases without any complications and residual cyst membrane. during the followups, clinical and radiological recovery were seen in these patients. based on cyst membrane position against the main bronchus, hydatid cysts are divided into the two groups of central and pe ripheral cysts. obser vation of the cyst me mbr a ne is a n imp or t a nt pr in c i p l e f or intervention, especially in the peripheral lesions. in these patients use of the fogarty balloon is not possible. with the aid of a catheter we are able to reach the margins of the cyst and inject the saline, which is more difficult and needs several bronchoscopic interventions. in two of the patients this method was used without fail. the important point about peripheral lesions which should be considered is that if we were not able to see the hydatid cyst membrane, normal saline injection will not be useful. therefore we recommend that: i) this method should be used before surgical operation in central ruptured hydatid cysts;ii) in the peripheral ruptured hydatid cysts, accurate recognition and location of the cyst is essential and the blind approach is not recommended because it needs several bronchoscopic interventions;and iii) in all of the cases medical treatment is necessary. conclusions this novel bronchoscopic saline injection method is curative, cost-effective, minimally invasive and needs no general anesthesia and hospitalization, with less complications in comparison with surgery. therefore it will be a beneficial and suitable alternative method for 164 hejazi, tekantapeh, maddahi me hejazi method results in bronchoscopy surgery in treatment of patients with ruptured pulmonary hydatid cysts. however, to extract f u r t h e r inf or ma t i o n, mor e st u di e s a r e recommended to be done on this method. conflicts of interest the authors declared no competing and conflict of interests. acknowledgments we a c kno wl e d ge w i th t h a nks t he contribution of all staff of the respiratory and critical care medicine ward of imam reza hospital. funding/support the authors declare that they have no competing financial interests in relation to the work described and although this research was done in respiratory and critical care medicine ward of internal medicine department of imam reza hospital, tabriz university of medical sciences, iran, it was not financially supported by any specific center. contributors all authors contibuted equally to this work. al l a uth or s re ad a nd ap pr oved t he f in al manuscript. references 1. hejazi me, tekantapeh st, hasani s. a novel bronchoscope method (saline injection method) for complete extraction of ruptured pulmonary hydatid cyst. clin respir j 2018;12:312-6. doi: 10.1111/crj.12484. 2. craig ps, mcmanus dp, lightowlers mw, et al. prevention and control of cystic echinococcosis. lancet infect dis 2007;7:385-94. 3. morar r, feldman c. pulmonary echinococcosis. eur respir j 2003;21:1069-77. doi: 10.1183/ 09031936.03.00108403. 4. santivanez s, garcia hh. pulmonary cystic echinococcosis. curr opin pulm med 2010;16: 257–61. doi: 10.1097/mcp.0b013e3283386282. 5. dziri c, haouet k, fingerhut a, et al. management of cystic echinococcosis complications and dissemination: where is the evidence? world j surg 2009;33:1266-73. doi: 10.1007/s00268-0099982-9. 6. turgut at, altinok t, topcu s, et al. local complications of hydatid disease involving thoracic cavity: imaging findings. eur j radiol 2009;70:49–56. doi: 10.1016/j.ejrad.2008.01.002. 7. madan k, singh n. bronchoscopic diagnosis of pulmonary hydatid cyst. cmaj 2012;184: e158. doi: 10.1503./cmaj.111185. 8. yilmaz a, tuncer ly, damadoglu e, et al. pulmonary hydatid disease diagnosed by bronchoscopy: a report of three cases. respirology 2009;14:141-3. doi: 10.1111/j.14401843.2008.01390.x. 9. basavana gh, siddesh g, jayaraj bs, et al. ruptured hydatid cyst of lung. j assoc physicians india 2007;55:141–5. 10. puri d, mandal ak, kaur hp, et al. ruptured hydatid cyst with an unusual presentation. case rep surg 2011; article id 730604, 4 pages. doi: 10.1155/2011/7306042011:730604. 11. yasar z, acat m, turgut e, et al. diagnosis of pulmonary hydatid cyst by bronchoscopy. j bronchology interv pulmonol 2015;22:343–6. doi: 10.1097/lbr.0000000000000165. 12. somasundaran s, kane d, rao s, et al. anaesthetic management of a case of bilateral hydatid cysts of the lungs with hepatic hydatid cysts posted for cyst excision. internet j anesthesiol 2006;13:14. 13. sharif a, ansarin k, rashidi f, et al. bronchoscopic diagnosis and removal of a ruptured hydatid cyst. j bronchology interv pulmonol 2011;18:362–4. doi: 10.1097/lbr. 0b013e31823504ac. 14. mahomed k, jayaguru as. extra-amniotic saline infusion for induction of labour in antepartum fetal death: a cost effective method worthy of wider use. br j obstet gynaecol 1997;104:1058– 61. c:\users\jurnal fkusakti\docume 33 department of clinical psychology, school of medicine, shahid beheshti university of medical sciences, tehran, iran correspondence: abbas masjedi arani *department of clinical psychology, school of medicine, shahid beheshti university of medical sciences, tehran, iran email: doctormasjedi@yahoo.com, tel: +09125752870 date of first submission, january 13, 2 01 9 date of final revised submission, march 20, 2019 date of acceptance, march 22, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina psychometric properties of persian version of beck depression inventory in coronary patients seyed mojtaba ahmadi, abbas masjedi arani*, maryam bakhtiari, mohamad hasan davazdah emamy, and rasul mohamadian background obtaining psychometric properties regarding specific populations increases diagnostic accuracy and reduces economic health burdens. beck depression inventory-second version (bdi-ii) is useful for the screening and assessment of depression in clinical and research settings. the aim of the present study was to investigate the psychometric properties of the persian (farsi) version of bdi-ii in patients with coronary heart disease (chd). methods a cross-sectional study was conducted on 284 patients with chd admitted to imam ali hospital in kermanshah. they were first given a structured clinical interview (scid-i) and then were asked to complete the beck depression inventory-ii (bdi-ii), patient health questionnaire 9 (phq-9) and generalized anxiety disorder 7 (gad-7). data were analyzed by descriptive statistics, cronbach’s α, spearman’s correlation coefficient, exploratory factor analysis and receiver operating characteristic curve (roc). results the internal consistency measured using cronbach’s alpha was 0.90. the obtained correlation of bdi-ii with phq-9 and gad-7 was 0.74 and 0.65, respectively (p< 0.001). factor analysis of the inventory revealed 5 factors, namely cognitive, somatic , impaired performance , negative attitude to self and ,and self punishment respectively. the cut-off point for chd patients was 11 with sensitivity of 0.78 and specificity of 0.81 according to the youden index and 10 with sensitivity of 0.80 and specificity of 0.77 according to the two-stage approach. the area under the curve was 0.86 (95% confidence interval 0.82-0.90). conclusion the persian version of the bdi-ii possesses the acceptable psychometric properties that can be used to screen depression in chd patients. keywords: beck depression inventory-ii, depression, coronary disease, psychometrics, sensitivity and specificity original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.33-40 january-april, 2019 vol.38no.1 cit e th is article as: ah ma d i sm , masjedi arani a, bakhtiari m, et al. psychometric properties of persian version of beck depression inventory in coronary patients. univ med 2019; 38:33-40. doi: 10.18051/univmed. 2019.v38.33-40 34 ahmadi, masjedi arani, bakhtiari, et al psychometric properties bdi-ii in chd introduction depression is a comorbid disorder to severe physical problems,(1) such as cancer (2) and cardiovascular problems.(3,4) depression is highly witnessed among coronary patients. a study in china estimated the prevalence to be between 34.6% to 45.8%.(5) research carried out in iran reported the prevalence of depression among coronary patients to be 66%.(6) depression is one of the cardiovascular risk factors which has been reported to be linked to negative treatment results and even to increased death rates among such patients.(7,8) in case patients with cardiovascular problems are diagnosed with depression, there is an urgent need for psychotherapy.(9) rapid and accurate diagnosis of depression in coronary patients is one of the pressing priorities. interviews which lead to diagnosis impose time and cost restrictions on the healthcare system so the need for an accurate and specialized inventory to help with diagnosis is strongly felt. various depression inventories are available, such as beck depression inventory (bdi), properties patient health questionnaire 9 (phq-9), cardiac depression scale (cds), and each displa ys diffe rent psychometric specifications.(9,10) regarding the major importance attached to the case of coronary patients, the need is felt to consider different depression inventories so that the best inventory with the most accurate sensitivity can be singled out and introduced as the appropriate choice for such patients. beck depression inventory is an acceptable one, approved and suggested by the national heart foundation of australia. (9) although this questionnaire has shown acceptable reliability and validity in other countries f or measuring depression in cardiac patients such as indonesia (11) and canada,(12) the need to investigate the reliability, validity, cut-off point, sensitivity, and specificity of bdi-ii in iranian cardiac patients is strongly felt. as it is well proved that depr ession symptoms can surface differently in differing cultures,(13) it is necessary to investigate the reliability and validity of the bdi in different countries and with different populations. beck depression inventory -second edition (bdi-ii) has been frequently used in various groups, including heart patients in iran.(14-16) however, in all of the studies, the psychometric properties obtained in the general population or the general properties of the questionnaire have been used. that is the rea son why the psychometric properties of various groups may be different and one of the limitations we encounter is the restricted ability to generalize data for example. wang et al.(17) showed that the cutoff points for screening for depression in various populations are different, and the cut-off point for the bdi in the general population is in the lower range (from 10-16) compared to the clinical population (from 7-20) and the psychiatric population (from 19-31). in iran, there is no study that has addressed the psychometric properties of this questionnaire in patients with coronary artery disease. it is thus mandatory to investigate the psychometric properties and the diagnostic cutoff points of the inventory among coronary patients in iran. accordingly, the present research aims at investigating the reliability, validity, and cut-off point of the bdi-ii in iranian coronary patients. methods participants and procedures among patients referred to imam ali hospital in kermanshah from june 22, 2018 to november 22, 2018, 289 patients with coronary heart disease were selected as samples. excluded were patients with thyroid dysfunction, cognitive disorders such as dementia or alzheimer ’s disease, records of b rain str okes, severe psychological problems like psychotic symptoms and dissociative identity disorder as well as those who, due to severe heart problems, could not fill out questionnaires. following briefing of the participants and acquiring their consent, clinical structured interviews were conducted by two 35 psychologists. the participants then completed the phq-9, gad-7, and bdi-ii questionnaires. measurements structured clinical interview for dsm-5 (scid): the structured clinical interview for dsm-v is the semi-structured interview for main diagnosis of dsm-5 (previously placed on the first axis). this instrument needs to be implemented by a clinical psychologist or a trained psychological health specialist familiar with diagnostic scales and classification of disorders in dsm-5.(18) depression beck depression inventory (bdi-ii) is a 21question inventory that measures the severity of depression. the inventory uses a 4-point likert scale that assigns a value of 0 to 3 to every answer. the higher the score, the more severe the depression would be. the total score ranges from 0 to 63. scores 0 to 13 indicate minimal depression, scores 14 to 19 indicate mild depression, scores 20 to 28 indicate moderate depression and scores 29 to 63 indicate severe depression.(19) patient health questionnaire-9 (phq-9) is a self-report questionnaire which is designed to diagnose based on the diagnostic and statistical manual for mental disorders-iv (dsm-iv). it has 9 questions and is derived from the complete version of phq. participants need to report how they have felt over the past two weeks on a 4point likert scale ranging from not at all (0), several days (1), more than half the days (2), almost every day (3) and the total score ranges from 0 to 6.(20) anxiety generalized anxiety disorder 7 (gad-7) is a self-report seven-item questionnaire that was devised by spitzer et al.(21) it basically includes 9 items and is based on dsm. it is scored according to a 4-point likert scale from 0 to 3 (not at all = 0, several days = 1, more than half the days=2, and nearly every day=3). the obtained total score is between 0 to 21 and the questionnaire is of a sensitivity of 0.89 and specificity of 0.82 at the cut-off point of 10. data analysis to analyze the data spss software version 21 was implemented. cronbach’s alpha was used to monitor the reliability. to investigate the validity, criterion validity based on the correlation between phq-9 and gad-7 was used and to do so, pearson correlation coefficient was calculated. to investigate the construct validity, exploratory factor analysis was performed. to interpret the roc curve and obtain the best cut-off point, two methods suggested by lowe et al.(22) were used. in the first method, maximal youden index (sensitivity + specificity-1) was implemented. the second me thod implemented a two-stage approach for screening depression disorder. in this method, the cut-off point is obtained with maximum sensitivity and specificity, higher than 0.75. the two-stage approach sounds more congenial to the clinical situation where the positive results of screening are usually followed by long diagnostic interviews and treatment in case they are rendered necessary. the area under the curve (auc) was considered as the accuracy of scale. ethical considerations this research project was approved by the ethics committee of the shahid beheshti university of medical silences under the code of ethics ir.sbmu.msprec.1396.286. as for those patients showing severe depression in clinical interviews, psychiatric counseling was requested after informing the cardiologic section of the hospital and the related specialist. results characteristics of the subjects two hundred eighty-four patients admitted to kermanshah’s imam ali hospital who were diagnosed with chd filled out the questionnaires. the age range of the participants was 59.78 ± 10.19 years and there was an almost equal gender distribution, with 155 male participants making up univ med vol. 38 no.1 36 ahmadi, masjedi arani, bakhtiari, et al psychometric properties bdi-ii in chd a b c table 1. item analysis of bdi-ii number item description scale mean if item deleted scale variance if item deleted corrected item-total correlation squared multiple correlation cronbach's alpha if item deleted bdi 1 sadness 9.2784 66.745 0.739 0.601 0.885 bdi 2 pessimism 9.5804 69.016 0.577 0.408 0.890 bdi 3 past failure 9.4510 68.359 0.612 0.474 0.889 bdi 4 loss of pleasure 9.3451 65.605 0.540 0.415 0.891 bdi 5 guilty feelings 9.5412 70.438 0.421 0.389 0.894 bdi 6 punishment feelings 9.6627 70.516 0.442 0.390 0.893 bdi 7 self-dislike 9.2588 65.232 0.495 0.426 0.894 bdi 8 self-critical 9.5490 69.123 0.560 0.442 0.890 bdi 9 suicidal thoughts 9.8392 72.411 0.399 0.310 0.894 bdi 10 crying 9.3922 69.200 0.432 0.268 0.894 bdi 11 agitation 9.3333 66.900 0.651 0.534 0.887 bdi 12 loss of interest 9.5176 67.896 0.727 0.603 0.886 bdi 13 indecisiveness 9.6314 70.541 0.555 0.417 0.891 bdi 14 worthlessness 9.6431 69.876 0.554 0.429 0.891 bdi 15 loss of energy 9.2902 69.057 0.550 0.474 0.890 bdi 16 changes in sleep pattern 9.2667 67.740 0.484 0.360 0.893 bdi 17 irritability 9.5882 70.613 0.486 0.345 0.892 bdi 18 changes in appetite 9.5647 70.711 0.394 0.372 0.894 bdi 19 concentration difficulties 9.6706 71.206 0.486 0.368 0.892 bdi 20 tiredness and fatigue 9.3294 69.175 0.555 0.534 0.890 bdi 21 loss of interest in sex 9.0118 67.933 0.418 0.280 0.895 54.6% of the sample. forty six participants were clerks (16.2%), 108 were business people (38%) and 130 were unemployed (45.8%). ten percent of the population had bachelor or higher university degrees, 42% had diploma and associate degrees, 48% had some school education. reliability the reported cronbach’s alpha on the internal consistency of the bdi-ii was 0.90 and the correlation of each item with the total score ranged from 0.34 to 0.79 (p<0.001). the results of item analysis also indicated that alpha was reduced after illuminating items and it was consistent over all items so it was concluded that every item was suitable. (table 1) validity the correlation of bdi-ii with phq-9 and gad-7 was reported to be 0.74 and 0.65 respectively. factor analysis was implemented to investigate the construct validity and the result of kaplan-meier-olkin test was 0.90. the bartlett test proved significant which is an indication of the data being appropriate for factor analysis. factor analysis results indicated that the questionnaire is of 5 factor loadings where the first factor explains 37.63% of the variation, the second factor explains 6.10%, the third factor 5.83% and the fourth and fifth factors explain 5.38% and 4.97%, respectively, of the variation. the factors totally explain 59.93% of the variation. (table 2) specification of the test for screening to obtain accuracy and the optimal cut-off point, the roc test was implemented. the results indicated that beck depression inventory covers 0.86 (95% ci=0.82 -0.90) of the area under the curve (figure 2). this data indicates that the second version of beck depression inventory can recognize depressed coronary patients from the non-depressed. according to the youden index, the optimal cut-off point to diagnose depression in this study, and for research purposes, is 11 with sensitivity 37 univ med vol. 38 no.1 table 2. factor analysis of bdi-ii present study confirmed previous research carried out on coronary patients in indonesia that came up with an alpha coefficient similar to ours (α=0.87).(11) in a study in the general and hospital population in the dominican republic, cronbach’s alpha coefficients were reported between 0.70 to 0.89.(24) the validity of t he inventory was investigated through pearson correlation of bdi with phq-9 and gad-7, which turned out to be 0.74 and 0.65 respectively, indicating the inventory as valid. the above-mentioned study in indonesia figure 1. screen plot for beck depression inventory of 0.78 and specificity of 0.81. the cut-off point of 10 with sensitivity of 0.80 and specificity of 0.77 is recommended for clinical purposes (table 3). discussion the internal consistency of bdi-ii based on cronbach’s alpha was 0.90. generally, cronbach’s alpha coefficients higher than 0.70 are considered as significant and coefficients equal or higher than 0.70 are trusted.(23) the total % of variance cumulative % questions 7.90 37.63 37.63 13 0.61 12 0.44 7 0.77 3 0.56 2 0.52 1 0.52 cognitive factor 1 1.28 6.01 43.71 21 19 18 17 16 11 somatic factor 2 0.49 0.63 0.50 0.67 0.49 0.61 1.22 5.83 49.57 20 15 4 impaired performance factor 3 0.79 0.65 0.47 1.13 5.38 54.95 14 9 8 negative attitude to self and worthlessness factor 4 0.69 0.72 0.50 1.04 4.97 59.57 10 6 5 self punishment factor 5 0.52 0.80 0.74 38 ahmadi, masjedi arani, bakhtiari, et al psychometric properties bdi-ii in chd (11) on coronary patients, and depressed and healthy people investigated the correlation of bdi with questionnaires of type d personality scale (ds14) , beck anxiet y inventory ( bai), multidimensional scale of perceived social support (mspss), and life orientation test-revised version (lot-r), yielding correlation coefficients of 0.52, 0.52, -0.039 and -0.46, respectively, which are lower than the values of the present study. factorial analysis of bdi implemented for coronary patients led to 5 major factor loadings listed here under the headings ccognitive, somatic, impaired performance, negative attitude to self and, and self punishment. previous studies, carried out on differing populations, have reported diff erent factor loadings, while pr evious researches on coronary patients share cognitive, emotional and physical factor loadings with ours.(11) with distinct samples of hiv patients factorial structures of cognitive/emotional and physical as well as factorial structures of negative thoughts, dysfunction and physical signs were table 3. sensitivity, specificity, predictive values, and likelihood ratios at various cut-off scores of the bdi-ii *recommended cut-off points for a two-stage screening (maximal sensitivity and 75% specificity) **optimal cut-off points according to maximal youden index (sensitivity+specificity-1). figure 2. receiver operating characteristic curve for bdi-ii cut-off point sensitivity specificity ppv npv lr+ lr bdi-ii ≥7 0.78 0.66 0.63 0.87 2.52 0.19 ≥8 0.78 0.69 0.66 0.88 2.81 0.18 ≥9 0.84 0.74 0.69 0.86 3.20 0.22 ≥10* 0.80 0.77 0.71 0.84 3.48 0.25 ≥11** 0.78 0.81 0.74 0.83 4.12 0.27 ≥12 0.73 0.84 0.75 0.81 4.47 0.31 ≥13 0.70 0.88 0.80 0.80 5.73 0.34 ≥14 0.66 0.89 0.8 0.85 5.69 0.39 39 reported.(13) considering the fact that depression surfaces differently in differing communities, the observed differences can be due to distinct communities. the results of the roc curve demonstrated that the auc of the bdi-ii was 0.86, which is considered good according to the conventional classification system.(25) in the indonesian study on heart disease mentioned earlier, the auc was 0.81, which is similar to that of the present study.(11) the obtained cut-off point in this study was 11 with sensitivity of 0.78 and specificity of 0.81 which indicates the instrument to be suitable for diagnosis and screening purposes. confirming the present study results, a study on heart patients in canada reported the optimal cut-off point, independent of demographic variables, as 10 with sensitivity of 83% and specificity of 73%.(12) in yet another research in lithuania a cut-off point of 14 with sensitivity of 0.89 and specificity of 0.74 was reported.(26) as well, in a study in indones ia on d epr e ssed , no ndepressed and coronary patients, the cut-off point of 17 was obtained with sensitivity and specificity of 0.73 which is a bit different from the results of the present study as well as the two aforementioned studies.(11) it is worth mentioning that the population in indonesian study was a combination of healthy, depressed and coronary patients and it aimed at diagnosing minimal depression which could explain the different reported cut-off point. the present study has some limitations that should be taken into consideration.the present sudy investigated the bdi-ii in a sample of the patients with chd; therefore, the findings should be generalized to other populations with caution. due to the limitations of this study, it is recommended that these questionnaires be studied in a different group of patients. conclusion this study indicated that bdi was of a suitable reliability and validity in patients with univ med vol. 38 no.1 coronary heart disease in iran. in iran, the cutoff point of 10 could be implemented for clinical purposes and the cut-off point of 11 suits research purposes better. acknowledgments this article has been extracted from the thesis written by seyed mojtaba ahmadi in school of medicine of shahid beheshti university of medical sciences (registration no: 377m). the authors of this article would also like to thank all patients who participated in this study. conflict of interest there is no conflict of interest between the authors. contributors sma and ama contributed to the study concept and de sign. sma, ama, mb, mhde,and rm contributed to the analysis and interpretation of data. ama contributed to the drafting of the manuscript and critical revision of the manuscript for important intellectual content. mb and mhde contributed to the study supervision. all authors have read and approved the final manuscript. references 1. kang hj, kim sy, bae ky, et al. comorbidity of depression with physical disorders: research and clinical implications. chonnam med j 2015;51:818. doi: 10.4068/cmj.2015.51.1.8. 2. smith hr. depression in cancer patients: pathogenesis, implications and treatment. oncol lett 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patients. j psychosom res 2012;72:22-5. doi: 10.1016/j.jpsychores.2011.10.006. c:\users\jurnal fkusakti\docume 97 #joint first authors *department of oral biology, faculty of dentistry, universitas padjadjaran, bandung, indonesia **department of pediatrics, dr. hasan sadikin general hospital/ faculty of medicine, universitas padjadjaran, bandung, indonesia ***biotechnology master program, post graduate school, universitas padjadjaran, bandung, indonesia †department of biochemistry and molecular biology, faculty of medicine, universitas padjadjaran, bandung, indonesia correspondence: edhyana sahiratmadja, md, phd department of biochemistry and molecular biology, faculty of medicine, universitas padjadjaran jl. eijkman no. 38 bandung phone: +62816 99 6309 email: e.sahiratmadja@unpad.ac.id date of first submission, january 29, 2018 date of final revised submission, may 19, 2018 date of acceptance, may 24, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina high ferritin in homozygous and heterozygous -thalassemia tends to decrease oxidative stress levels ani melani maskoen*#, lelani reniarti**#, nur imaniati sumantri***, and edhyana sahiratmadja† background ineffective erythropoiesis and multiple blood transfusions may cause iron overload, leading to high level of ferritin in β-thalassemia patients. iron has the ability to catalyze the production of reactive oxygen species (ros), which can be prevented by an adequate activity of superoxide dismutase (sod) and glutathione peroxidase (gpx). the aim of this study was to explore the association between high ferritin levels and oxidative stress among β-thalassemia patients by measuring sod and gpx levels. methods this was a cross sectional study among β-thalassemia major patients. ferritin, sod, and gpx were measured and compared between β-thalassemia patients with homozygous ivs1nt5 and heterozygous ivs1nt5/hbe mutations, which were the most prevalent mutations found in our previous study. the spearman correlation test was used to analyze the data. results the ferritin levels in all β-thalassemia major patients were very high (range: 791-12,340 μg/l). however, no significant differences were observed (p=0.318) between homozygous ivs1nt5 (n=45) compared to heterozygous ivs1nt5/hbe (n=13). the oxidative stress markers sod and gpx were not significantly different between homozygous ivs1nt5 and heterozygous ivs1nt5/hbe (p=0.450 and p=0.323, respectively). the correlations between ferritin and sod and gpx oxidative stress levels were not significant in both homozygous ivs1nt5 and heterozygous ivs1nt5/hbe mutations. conclusion high ferritin levels in β-thalassemia patients tend to decrease the gpx level in all thalassemia patients and sod level in half of the patients, indicating that gpx and sod may play a role in the occurrence of oxidative stress among thalassemia patients. the mechanism of oxidative stress in thalassemia needs to be further explored. keywords: ivs1nt5, hbe, superoxide dismutase, glutathione peroxidase, thalassemia patients doi: http://dx.doi.org/10.18051/univmed.2018.v37.97-104 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 ci te t his art icle as: m a s k oen am , reniarti l, sumantri ni, sahiratmadja e. high ferritin in homozygous and heterozygous -thalassemia tends to decrease oxidative stress levels. univ med 2018;37:97-104. doi: 10.18051/ univmed.2018.v37.97-104 98 maskoen, reniarti, sumantri, et al ferritin and oxidative stress in -thalassemia introduction thalassemia is an inherited blood disorder due to a mutation in the αor β-globin gene, leading to unbalanced globin chain synthesis. the αand β-globin proteins are encoded by α and β genes that lie on chromosomes 16 and 11, respectively.(1) thalassemia is the most frequent monogenic disease in the world, and about 1.5% of the global population are heterozygotes or carriers of β-thalassemia.(2) half of these reside in southeast asian countries, where indonesia harbors 0.1%, with ivs1nt5 is being the most common β-thalassemia mutation.(3) beta-thalassemia major patients have mutations in both chains of the beta globin gene (homozygous), leadi ng to a decr ease in erythrocyte production, therefore requiring regular blood transfusion.(4) iron is absorbed in the intestinal tract and circulated in the blood. about half of the body iron makes up part of the red blood cell (rbc) composition and the other half is stored in ferritin or hemosiderin that is recycled during rbc destruction.(5) multiple transfusions increase iron absorption, leading to iron overload and its complications. body iron and proteinbound iron such as serum iron, ferritin, and non– transferrin-bound iron (ntbi) may reflect the iron status of thalassemia patients. for example, ferritin is a strong indicator of iron deposit level. high levels of ferritin indicate iron overload, inflammation, malignancy, collagen disease, or liver disease.(6) therefore, thalassemia patients receiving multiple transfusions will have increased ferritin levels. in β-thalassemia, ineffective erythropoiesis occurs as a result of unstable globin chains. unpaired β-globin chains will form heinz bodies. hemoglobin subunit oxidation will cause h e mi c hr o me f or ma t i o n t ha t inf l ue n c e s hemolysis. hemichrome then binds to band 3 proteins, ankyrins, spectrins, or other erythrocyte c o mp o ne n ts . af te r t h a t , h e mi c h r ome i s precipitated, heme is disintegrated, and ntbi is released, leading to the formation of reactive oxygen species (ros).(7) thus, iron plays a role as a catalyst in redox reactions, including the formation of ros, such as superoxide (o 2 •-), hydrogen peroxide (h 2 o 2 ), and hydroxyl radical (ho•), mainly through the fenton reaction.(8) at low levels, ros play a role as a defense system against pathogens. meanwhile, high levels of ros exceed the antioxidant capacity, which is known as oxidative stress. this causes oxidative damage in lipids, proteins, dna, or other cellular compartment damage. antioxidant complexes will prevent oxidative damage by the cryopreservative cell system.(8) intracellular antioxidants prevent cell damage by oxygen and iron combination. antioxidants are classified into enzymatic and non-enzymatic antioxidants. superoxide dismutase (sod), glutathione peroxidase (gpx), catalase (cat), and glutathione reductase (gr) are the major enzymatic antioxidants involved in scavenging ros.(8) previous studies have measured sod, gpx, cat, malonyldialdehyde, ferritin, transferin, hb, vitamin a, and vitamin e to determine the relationship between oxidative stress and β-thalassemia major, however, results were conflicting, ranging from lower to higher compared with normal controls.(9-11) the combination of sod and gpx is the main mechanism for superoxide detoxification in the cell, where sod is a first-line defense endogenous enzyme that converts ros, from superoxide (o 2 -) to h 2 o and h 2 o 2 .(12) glutathione plays an important role in erythrocyte defense, which catalyzes h 2 o 2 to h 2 o or o rganic hydroperoxide to alcohol. furthermore, gpx also has wide affinity range, making it an important system against ros.(12) the activity of sod and gpx in βthalassemia patients may be lower than that in the control group.(11) this is caused by high production of ros that depletes those enzymes. moreover, ros also has the capability to damage dna, proteins, and lipids in the cell, which includes antioxidant molecules.(12) the activity of sod may decrease 1.5 times in homozygous βthalassemic children compared to non-hemolytic anemia controls.(10) superoxide radicals can trigger autooxidation of hemoglobin chains, which 99 plays a role in hemolysis. ther efore, the measurement of sod and gpx levels in βthalassemia patients is important to assess the presence of oxidative stress.(11) the aim of this study was to explore the association in β-thalassemia patients between high ferritin levels on the one hand and oxidative stress level sod and gpx on the other. in this study, we have compared homozygous ivs1nt5 and heterozygous ivs1nt5/hbe mutations of the β-globin gene, as these were prevalent in our thalassemia patients from bandung, indonesia.(13) methods research design this study was a cross sectional analysis among β-thalassemia patients recruited in the thalassemia clinic, dr. hasan sadikin general hospital in bandung, indonesia, during the year 2017. research subjects the β-thalassemia patients were clinically diagnosed from the clinical history and the regular blood transfusions, and from laboratory tests, as well as deoxyribonucleic acid (dna) screening, as described in a previous study.(13) total sampling was performed, thus all β-thalassemia patients with dna mutation data in the clinic were asked to participate in this study upon written consent of their parents, since these patients were considered as a vulnerable group. patients with heart failure, hepatitis, renal failure, nephrotic syndrome, and diabetes mellitus were excluded. β-thalassemia patients were then grouped into homozygous ivs1nt5 and heterozygous ivs1nt5/ hbe, and ferritin level and antioxidant activity were measured and compared between the two groups. hematological parameters a volume of 3 ml of blood was withdrawn aseptically from each of the subjects before they received blood transfusion. blood was collected in a tube containing ethylenediaminetetraacetic acid (edta) for various regular standardized tests in the dept. of clinical pathology of dr. hasan sadikin general hospital, including complete blood count (using a sysmex xp-100 automated hematology analyzer, japan) and determination of ferritin concentrations (sigmaaldrich, usa). measurement of gpx and sod antioxidant activity enzymatic tests were performed at the molecular genetic laboratory, faculty of medicine, universitas padjadjaran. about 0.5 ml of whole blood was centrifuged at 3000 rpm for 10 minutes to separate the serum and the red blood cells (rbcs). the rbcs were washed using 3 ml of 0.9% nacl and centrifuged 3 times at 3000 rpm for 10 minutes. the pellet was diluted with 2 ml of disti lled wa ter f or sod measurement (ransod, randox, antrim-uk). the diluted sample for gpx measurement used 0.02 ml of whole blood mixed directly with the reagent from the kit (ransel, randox, antrim-uk). the samples were then run on a uv-visible spectrophotometer and the antioxidant activity was calculated. statistical analysis the kruskal-wallis test was used to compare the level of oxidative stress (sod and gpx) between the two β-thalassemia mutations i.e. homozygous ivs1nt5 and heterozygous ivs1nt5/hbe. a p-value of less than 0.05 was considered as the limit for the level of significance. the spearman correlation test was used to analyze the relationship between the activity of both antioxidants. ethical clearance the protocol of this study was approved and ethical clearance was granted by the health research ethics committee, medical faculty, university of padjadjaran no.172/un6.c10/pn/ 2017. univ med vol. 37 no.1 100 maskoen, reniarti, sumantri, et al ferritin and oxidative stress in -thalassemia a b c results in total, we have included 58 patients βthalassemia in our study, consisting of patients with homozygous iv s1nt5 (n=45) and heterozygous ivs1nt5/hbe (n=13) mutations, with age ranges between 1 to 18 years and 2 to 26 years, respectively. the hemoglobin (hb) levels in the β-thalassemia subjects were generally found to be low, with a mean hb level of 6.8 g/ dl, ranging from 4.53 to 8.28 g/dl. however, there was no significant difference in the severity of anemia between homozygous ivs1nt5 and heterozygous ivs1nt5/hbe, since the hb level and age in both groups were not significantly different (table 1). furthermore, the frequency of transfusion in homozygous ivs1nt5 was higher compared to heter ozygous ivs1nt5/hbe (p=0.019). deferiprone (dfp) was the most used iron chelator in both groups. the ferritin level in both homozygous and heterozygous groups was high (table 2). all patients had low gpx levels, with a median value of 264.98 u/ml (range: 8.41-1,753 u/ml) which was under the normal range of 4,171-10,881 u/ ml. the median sod level among the subjects was 170.65 u/ml (range: 54.51-276.26 u/ml), which was within the normal range of 164-240 u/ml. however, when the individual values were dichotomized into low or normal value, it was homozygote ivs1nt5 (n=45) heterozygote ivs1nt5/hbe (n=13) p-value age (year); mean(sd) 11.0 (4.2) 13.4 (5.8) 0.459a hb level (g/dl); mean (sd) 6.8 (0.8) 6.9 (0.7) 0.647a frequency of transfusion; n (%) every 2 weeks 8 (17.8%) every 3 weeks 19 (42.2%) 3 (23.1%) 0.019b 4 times weekly 18 (40.0%) 10 (76.9%) iron chelator; n (%) deferoxamine (dfo) 1 (2.2%) deferasirox (dfx) 15 (33.3%) 4 (30.8%) deferiprone (dfp) 29 (64.4%) 9 (69.2%) table 1. characteristics of β-thalassemia subjects with homozygote ivs1nt5 mutation and heterozygote ivs1nt5/hbe mutation note: a t-test analysis, b chi-square comparison <4 vs. >4 times weekly; p value < 0.05 is significantly different ivs1nt5 homozygote ivs1nt5/hbe heterozygote p-value ferritin (μg/l) median# 3,784 3,555 0.318 range (791-12,340) (1,785-8,135) sod (u/ml) median## 172.12 167.55 0.450 range 54.51-276.26 94.31-228.94 low; n (%) 17 (37.8) 6 (46.2) 0.749 normal; n (%) 28 (62.2) 7 (53.8) gpx (u/ml) median### 227.1 319.6 0.323 range (8.4-1,329) (16.8-1,753) table 2. ferritin concentration and oxidative stress levels among β-thalassemia patients with homozygote ivs1nt5 and heterozygote ivs1nt5/hbe note: #normal range for ferritin (12-300 μg/l); ##normal range for sod (164-240u/ml) and for gpx (4,171-10,881 u/ml); sod: superoxide dismutase; gpx: glutathione peroxidase 101 univ med vol. 37 no.1 figure 1. correlation between superoxide dismutase (sod) and glutathione peroxidase (gpx) activity in β-thalassemia patients with: (a) homozygous ivs1nt5 mutation and (b) heterozygous ivs1nt5/hbe mutation figure 2. correlation between ferritin levels and sod activity in β-thalassemia patients with: (a) homozygous ivs1nt5 mutation or (c) heterozygous ivs1nt5/hbe mutation; and correlation between ferritin levels and gpx activity in βthalassemia patients with: (b) homozygous ivs1nt5 mutation or (d) heterozygous ivs1nt5/hbe mutation (spearman correlation analyses). note. sod: superoxide dismutase; gpx : glutathione peroxidase 102 maskoen, reniarti, sumantri, et al ferritin and oxidative stress in -thalassemia observed that half of the patients had low sod levels. the levels of oxidative stress (gpx and sod) did not differ significantly in homozygous ivs1nt5 compared to heterozygous ivs1nt5/ hbe β-thalassemia patients (table 2). there was no association between sod and gpx activities, both in the homozygous ivs1nt5 and heterozygous ivs1nt5/hbe groups (figure 1). furthermore, there was also no association between the ferritin level and sod or gpx antioxidant level in both groups (figure 2). discussion in t h e pr ovi nc e o f we s t j a va , t h e homozygous and heterozygous forms of ivs1nt5 a r e th e mos t f r e qu e n t mu t a tio n s, wi t h h omo zygou s iv s1nt 5 ( 4 9 .2 5% ) a n d heterozygous ivs1nt5/hbe (8.89%) being the two most prevalent, as described in a previous study.(13) patients with thalassemia have chronic a n e mi a , a nd t h e r e f or e r e qu i re r e p e a t e d transfusions. the frequency of transfusion in homozygous ivs1nt5 is higher compared to that in heterozygous ivs1nt5/hbe (p=0.019). this is the opposite of the results from another study showing that the co-inheritance of ivs1nt5 in hbe patients may lead to more severe symptoms compared to homozygous ivs1nt5.(14) furthermore, repeated transfusions can result in iron overload. the ferritin levels in patients receiving repeated transfusions in this study is comparable in both groups, i.e. in the homozygous ivs1nt5 and the more severe heterozygous ivs1nt5/hbe groups. since thalassemia patients have undergone repeated blood transfusions, the ferritin levels are higher when the rbcs are depleted, which does not necessarily depend on the type of mutation, resulting in a similar range of blood parameters such as ferritin between the two groups.(15) one study in indonesia showed that ferritin levels in heterozygous ivs1nt5/hbe patients were higher than in homozygous ivs1nt5 or without hbe, confirming that the heterozygous type is the more severe.(16) the more severe type of ivs1nt5 in heterozygous form was also discussed elsewhere.(17) interestingly, ferritin levels in heterozygous hbe/β-thalassemia can be 40 times higher compared to that in the control group.(18) these conflicting results between the various studies require further explorative and confirmative studies. as in β-t ha l a sse mi a ma jo r p at ie nt s , ineffective erythropoiesis, apoptosis, and o xi d a t i ve d a ma ge c a u se s a s ho r t e n e d erythrocyte lifespan among hbe/β-thalassemia patients.(18) the levels of sod and gpx activity in heterozygous hbe/β-thalassemia are higher than in homozygous β-thalassemia and controls, and the increased sod activity in β-thalassemia and hbe/β-thalassemia occurs as a response against superoxide radicals, forming more hydrogen peroxide.(19) another study showed that sod activity in β-thalassemia was higher than in controls, while that of gpx was lower than in controls.(20) in contrast, all patients in our study, including those with homozygous ivs1nt5 β-t ha l a s s e mi a a nd he te r o zygou s h be / βthalassemia had normal gpx levels. on the other hand, although sod levels in β-thalassemia are generally normal, approximately half of the subjects had low sod levels (table 2). the lack of comparison with healthy control subjects was a limitation in our study, as the levels of sod and gpx were not evaluated in healthy controls, which is a notion for future studies. this study showed that there was no association between sod and gpx activity, both in the homozygous ivs1nt5 and the heterozygous ivs1nt5/hbe groups (figure 1). furthermore, there was also no correlation between the ferritin levels and sod or gpx activity in both groups. interestingly, other studies had shown a negative correlation between ferritin levels and sod as well as gpx activity in anemic patients receiving repeated transfusions.(21,22) the limitations of this study are related to the fact that the activities of sod and gpx in t he c on t ro l gr o u p w e r e n o t me a su r e d . furthermore, we were hampered by several 103 univ med vol. 37 no.1 other limitations, such as the lack of data on iron chelator adherence, duration of treatment with chelating agents, and the degree of disease severity. moreover, as this was a cross sectional study, age was not further explored in relation to the oxidative stress level. the low sod level in thalassemia patients suggests its role in thalassemia, therefore, studies should be directed towards determining the role of sod as a potential therapeutic agent in thalassemia. conclusion high ferritin levels in β-thalassemia patients tend to decrease the gpx level in all thalassemia patients and the sod level in half of the patients, indicating that gpx and sod may play a role in the occurrence of oxidative stress among t ha la ss e mi a pa t ie nt s. t h e me cha ni sm of oxidative stress in thalassemia needs to be further explored. acknowledgements we would like to thank all participants, nurses, and the organization of thalassemia pa t i e n t s and pa r e nt s ( popt i) f or t he i r participation in this study. competing interests the authors declare that they have no competing interests. funding this study was financially supported by the fundamental research grant university of padjadjaran for the year 2017. authors’ contributions amm and lr contributed equally to this paper. amm and lr conceived and designed the experiments. amm, lr, nis, and es participated in the design of the study and the interpretation of the data. nis performed the experiments, nis and es conducted data analysis. amm and nis wrote the first draft, and reviewed by es. all authors read and approved the final manuscript. references 1. kumar r, sagar c, sharma d, et al. β-globin genes: mutation hot-spots in the global thalassemia belt. hemoglobin 2015;39:1-8. doi: 10.3109/03630269. 2014.985831. 2. colah r, gorakshakar a, nadkarni a. global burden, distribution and prevention of betathalassemias and hemoglobin e disorders. expert rev hematol 2010;3:103-17. doi: 10.1586/ehm. 09.74. 3. departemen penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia. riset kesehatan dasar (riskesdas) 2007, laporan nasional 2007. jakarta: departemen penelitian dan pengembangan kesehatan departemen kesehatan republik indonesia; 2008. 4. galanello r, origa r. beta-thalassemia. orphanet j rare dis 2010;5. doi: 10.1186/1750-1172-5-11. 5. kohgo y, ikuta k, ohtake t, et al. body iron metabolism and pathophysiology of iron overload. int j hematol 2008;88:7-15. doi: 10.1007/ s12185-008-0120-5. 6. gozzelino r, arosio p. iron homeostasis in health and disease. int j mol sci 2016;17:e130. doi: 10.3390/ijms17010130. 7. patel m, ramavataram dv. non transferrin bound iron: nature, manifestations and analytical approaches for estimation. indian j clin biochem 2012;27:322-32. doi: 10.1007/s12291-012-0250-7. 8. pham-huy la, he h, pham-huy c. free radicals, antioxidants in disease and health. int j biomed sci 2008;4:89-96. 9. mahdi ea. relationship between oxidative stress and antioxidant status in beta thalassemia major patients. acta chim pharm indica 2014;4:137-45. 10. rujito l, mulatsih s, sofro asm. status of superoxide dismutase in transfusion dependent thalassaemia. n am j med sci 2015;7:194-8. doi: 10.4103/1947-2714.157480. 11. asif m, manzoor z, farooq ms, et al. status of oxidant, antioxidant and serum enzymes in thalassaemic children receiving multiple blood transfusions. j pak med assoc 2015;65:838-43. 12. lu jm, lin ph, yao q, et al. chemical and molecular mechanisms of antioxidants: experimental 104 maskoen, reniarti, sumantri, et al ferritin and oxidative stress in -thalassemia approaches and model systems. j cell mol med 2010;14:840-60. doi: 10.1111/j.1582-4934.2009. 00897.x 13. maskoen am, rahayu ns, reniarti l, et al. mutation spectrum of β-globin gene in thalassemia patients at hasan sadikin hospital west java indonesia. cell mol biol (noisy-legrand) 2017;63:22-4. doi: 10.14715/cmb/2017.63. 12.6. 14. fucharoen s, weatherall dj. the hemoglobin e thalassemias. cold spring harb perspect med 2012;2:a011734. 15. jones e, pasricha sr, allen a, et al. hepcidin is suppressed by erythropoiesis in hemoglobin e β-thalassemia and β-thalassemia trait. blood 2015; 125:873-80. doi: 10.1182/blood-2014-10-606491. 16. wahidiyat pa, gatot d, tjitrasari t, et al. phenotypic diversity in beta-hbe thalassemia patients. pediatr indones 2006;46:82-6. doi: 10.14238/pi46.2.2006.82-6. 17. sripichai o, makarasara w, munkongdee t, et al. a scoring system for the classification of βthalassemia/hb e disease severity. am j hematol 2008;83:482-4. doi: 10.1002/ajh.21130. 18. aphinives c, kukongviriyapan u, jetsrisuparb a, et al. impaired endothelial function in pediatric hemoglobin e/β-thalassemia patients with iron overload. southeast asian j trop med public health 2014;45:1454-63. 19. yanpanitch ou, hatairaktham s, charoensakdi r, et al. treatment of β-thalassemia/hemoglobin e with antioxidant cocktails results in decreased oxidative stress, increased hemoglobin concentration, and improvement of the hypercoagulable state. oxid med cell longev 2015;2015. article id 537954, 8 pages. doi: 10.1155/2015/537954. 20. fernandes ms, rissi tt, zuravski l, et al. oxidative stress and labile plasmatic iron in anemic patients following blood therapy. world j exp med 2014;4:38-45. doi: 10.5493/wjem.v4.i3.38. 21. abdalla my, fawzi m, al-maloul sr, et al. increased oxidative stress and iron overload in jordanian β-thalassemic children. hemoglobin 2011;35:67-79. doi: 10.3109/03630269.2010. 544624. 22. dhawan v, kumar kr, marwaha rk, et al. antioxidant status in children with homozygous thalassemia. indian pediatr 2005;42:1141-5. oktavianus 172 *department of clinical pathology, faculty of medicine, hasanuddin university correspondence dr. liong boy kurniawan, m.kes, sp.pk department of clinical pathology, faculty of medicine, hasanuddin university jl. perintis kemerdekaan, unhas hospital, building a, 4th floor makassar, 90245 sulawesi selatan email: liongboykurniawan@yahoo.com univ med 2013;32:172-8 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 introduction blood urea nitrogen and creatinine levels are routine laboratory tests for evaluating renal function. renal dysfunction has been related to worse prognosis of cardiovascular diseases. the purpose of this study was to determine the relationship between admission blood urea nitrogen and creatinine levels with in-hospital mortality in acute myocardial infarction patients. methods a cross-sectional study was performed using secondary data of 80 acute myocardial infarction patients hospitalized in intensive cardiac care unit of dr. wahidin sudirohusodo hospital, makassar, from june 2010 to july 2011. admission blood urea nitrogen and creatinine levels were analyzed with mann whitney and chi-square tests. results mortality risk in the patients with blood urea nitrogen levels >50 mg/dl was 3.58 higher compared with those with blood urea nitrogen levels <50 mg/dl [or=3.58; 95% confidence interval (ci) =1.27-10.11, p=0.013]. mean admission creatinine levels in surviving and nonsurviving patients were 0.99 ± 0.30 mg/dl and 1.70 ± 1.99 mg/dl, respectively (p=0.043). mortality risk in patients with creatinine levels >1.1 mg/dl was 3.0-fold higher compared to patients with creatinine levels <1.1 mg/dl [or=3.00; 95% confidence interval (ci) =1.13-7.92, p=0.024]. multiple logistic regression showed blood urea nitrogen to be a better predictor of mortality than creatinine (or= 3.583, p=0.016 vs or 1.844, p=0.317). conclusions patients with high levels of blood urea nitrogen and creatinine had higher mortality risks than patients with normal levels. blood urea nitrogen was a better predictor of mortality than creatinine. key words: blood urea nitrogen, creatinine, myocardial infarction, mortality blood urea nitrogen as a predictor of mortality in myocardial infarction liong boy kurniawan*, uleng bahrun*, fitriani mangarengi*, darmawaty er*, and mansyur arif* 173 univ med vol. 32 no.3 kadar ureum darah sebagai prediktor mortalitas infark miokard pendahuluan ureum dan kreatinin merupakan tes laboratorium rutin yang sering digunakan untuk menilai fungsi ginjal. disfungsi ginjal telah lama dihubungkan dengan prognosis buruk penyakit kardiovaskuler. tujuan penelitian ini adalah untuk menentukan adanya hubungan antara kadar ureum dan kreatinin saat masuk rumah sakit dengan mortalitas selama perawatan di rumah sakit.pada pasien infark miokard akut. metode penelitian ini merupakan studi potong silang dengan menggunakan data sekunder dari rekam medik sebanyak 80 pasien infark miokard akut yang dirawat di unit perawatan jantung intensif rumah sakit dr. wahidin sudirohusodo makassar selama bulan juli 2010 hingga juni 2011. kadar ureum dan kreatinin yang diteliti adalah kadar saat pasien masuk rumah sakit. uji statistik dilakukan dengan uji mann whitney dan chi-square. hasil risiko terjadinya mortalitas pasien infark miokard dengan kadar ureum >50 mg/dl adalah 3,58 kali lebih besar dibandingkan kadar ureum <50 mg/dl (or=3,58; interval kepercayaan = 1,27 10,11, p=0,013). rerata kadar kreatinin pada penderita infark miokard akut yang survive dan yang meninggal masing-masing adalah 0,99 ± 0,30 mg/dl dan 1,70 ± 1,99 mg/dl (p=0,043). risiko mortalitas pasien infark miokard dengan kreatinin >1,1 mg/dl 3,0 kali lebih besar dibandingkan <1,1 mg/dl (or=3,00; 95% interval kepercayaan = 1,13 7,92, p=0,024). analisis regresi logistik ganda kadar ureum merupakan prediktor mortalitas yang lebih baik dibandingkan kreatinin (or= 3,583, p=0,016 vs or 1,844, p=0,317). kesimpulan pasien infark miokard akut dengan kadar ureum dan kreatinin dalam darah yang tinggi memiliki risiko mortalitas lebih besar dibandingkan pasien dengan kadar normal. kadar ureum dalam darah merupakan prediktor mortalitas yang lebih baik dibandingkan kreatinin. kata kunci: ureum, kreatinin, infark miokard, mortalitas abstrak introduction acute myocardial infarction is caused by the interruption of blood supply to cardiac muscle, resulting in gross necrosis of the myocardium. the diagnosis of acute myocardial i n f a r c t i o n a c c o r d i n g t o wo r l d h e a l t h organization criteria should be established on the basis of at least two of these criteria, including a history of chest pain, evolving changes on the electrocardiogram and/or elevations of serial cardiac markers.(1) urea is the major nitrogen-containing metabolic product of protein catabolism in humans, and more than 90% of this compound is excreted through the kidney. creatinine is an endogenous substance produced by muscle from creatine and creatine p h o s p h a t e , w i t h a r a t e o f p r o d u c t i o n proportionate to the muscle mass. creatinine is filtered freely by the glomerulus, is not 174 reabsorbed by the renal tubulus, and is secreted by the renal tubules only in small amounts. urea and creatinine are both used for evaluation of kidney function.(2) the levels of blood urea and creatinine may increase in some conditions, including decreased cardiac output from cardiac failure.(3) carter et al.(4) found that low spot urine creatinine is associated with cardiovascular disease risk. some studies have suggested that patients with renal failure have poorer outcomes in the setting of unstable angina and myocardial infarction.(5) cakar et al.(6) observed the effect of admission creatinine levels on one-year mortality of myocardial infarction patients and found that the mortality rate in the group with elevated creatinine levels was higher than that in the group with normal levels. kirtane et al.(7) also found that blood urea nitrogen (bun) could be used as an independent marker of mortality among patients with acute coronary syndromes in six months of follow up. sorensen et al.(8) observed that decreased creatinine clearance was related to an increased mortality rate among myocardial infarction patients in six years of follow up. worsening of renal function (defined as an increase in creatinine level of > 0.3 mg/dl) during the first 2 weeks after myocardial infarction also showed an increased risk of cardiovascular death.(9) smith et al.(10) found that in older myocardial infarction patients, adjusted mortality increased by 3% per 5u increase in blood urea nitrogen. limited information is available about the effect of acute kidney injury during hospitalization on clinical outcomes after myocardial infarction (mi).(11,12) the objective of the present study was to evaluate the effect of admission bun and creatinine levels on in-hospital mortality among myocardial infarction patients. in this study, we also analyzed whether bun or creatinine levels could be used as better markers of mortality risk in acute myocardial infarction patients during hospitalization, which had not been mentioned in previous studies. methods research design a cross-sectional study was conducted using secondary data of 80 acute myocardial infarction patients hospitalized in the intensive cardiac care unit of dr. wahidin sudirohusodo hospital, makassar, from june 2010 to july 2011. research subjects during the abovementioned period, 146 myocardial infarction patients were hospitalized, but only 80 patients had their admission bun and creatinine levels recorded. admission bun and creatinine levels in surviving and nonsurviving patients were then analyzed. t h e d i a g n o s i s o f a c u t e m y o c a r d i a l infarction was established by a cardiologist using world health organization criteria. acute myocardial infarction patients who had no admission bun and creatinine levels in their medical records were excluded from analysis. patients who survived hospitalization and were allowed by the clinician to have outpatient care were defined as surviving and those who died during hospitalization were defined as nonsurviving. laboratory analysis blood urea nitrogen, creatinine, total cholesterol, low density lipoprotein, high density lipoprotein and triglycerides were determined with an abx pentra 400. statistical analysis kolmogorov-smirnov normality tests showed that bun and creatinine levels were not normally distributed, so that the data were analyzed with the mann whitney test. we also used a bun level of >50 mg/dl as the cut-off for high bun levels and >1.1 mg/dl for high creatinine levels; values below the cut-off were defined as normal. mortality risks between high kurniawan, bahrun, mangarengi, et al blood urea nitrogen in myocardial infarction 175 univ med vol. 32 no.3 table 2. differences in blood urea nitrogen and creatinine levels between surviving and non-surviving groups * student t test; **mann whitney test table 1. characteristics distribution of the subjects (n=80) hdl = high density lipoprotein; ldl = low density lipoprotein and normal bun and creatinine levels were then analyzed using the respective cut-offs. all p values were two-sided, and a p value of < 0.05 w a s c o n s i d e r e d t o i n d i c a t e s t a t i s t i c a l significance. multiple logistic regression analysis of bun and creatinine levels was then performed to determine which of the two was the better predictor of mortality. all data were analyzed using spss version 21. ethical clearance this study was performed after obtaining permission from the educational and training unit (diklat) of wahidin sudirohusodo hospital to access the medical records. results during the study, we found 80 subjects with admission bun and creatinine levels recorded in the medical records. there were 57 male patients (71.3%) and 23 female patients (28.7%). fifty two patients (65.0%) survived and 28 (35.0%) patients did not survive hospitalization. the characteristics of the subjects are shown in table 1. there were significant differences in bun and creatinine levels between surviving and non-surviving groups (table 2). using 50 mg/dl as the cut-off value for bun, we found significant mortality risks among patients with high as compared with normal 176 table 3. mortality risks of patients using blood urea nitrogen and creatinine group table 4. multiple logistic regression analysis of blood urea nitrogen and creatinine on mortality of acute myocardial infarction patients levels of bun. logistic regression tests showed that patients with bun levels of >50 mg/dl had mortality risks 3.58 times higher than those with bun levels of <50 mg/dl. similarly, using 1.1 mg/dl as the cut-off value for creatinine, we found significant mortality risks among patients with high as compared with those with normal creatinine levels. patients with creatinine levels of >1.1 mg/dl had mortality risks 3.00 times than those with levels of <1.1 mg/dl (table 3). multiple logistic regression analysis showed that bun had more influence on mortality than creatinine (or= 3.583, p=0.016 vs or 1.844, p=0.317) (table 4). discussion in this study we found significant difference of bun levels between survive and non survive groups. patients with bun levels >50 mg/dl had mortality risks 3.58 times compared with bun levels <50 mg/dl. kirtane et al.(7) found that myocardial infarction patients with bun levels >25 mg/dl had mortality risks 3.2 times and patients with bun 20-25 mg/dl had 1.9 times of mortality risks compared with patiens with bun <20 mg/dl in six months. aronson et al.(13) found that elevated admission bun (>25 mg/ dl) was an independent predictor of mortality in median follow up of 27 months in myocardial infarction patients and an increase in bun 50% above admission value was associated with increased risk of mortality (hazard ratio 1.7; 95% ci 1.3 to 2.2). another study found that serum blood urea was significantly higher in mi patients.(14) furthermore, ostfield et al.(15) found that admission serum bun was associated with an increased burden of coronary artery disease on cardiac catheterization. we also found that creatinine levels of nonsurviving patients were higher than in surviving patients. patients with high creatinine levels (>1.1 mg/dl) had a threefold higher mortality risk than patients with normal levels (<1.1 mg/ dl). this was concordant with the study of gibson et al.,(16) who found that 30 days mortality increased stepwise among patients with normal (creatinine <1.2 mg/dl), mildly impaired (creatinine >1.2 to 2 mg/dl) and severely impaired (creatinine >2mg/dl) renal function. patients with mild, moderate, and severe impairment had respectively 1.38, 2.06 and 3.81 times higher mortality risks compared with normal ones. jose et al.(9) found that in patients with systolic dysfunction after myocardial infarction, worsening renal function (an increase of creatinine >0.3 mg/dl measured from baseline after 2 weeks) showed an increased risk for kurniawan, bahrun, mangarengi, et al blood urea nitrogen in myocardial infarction 177 univ med vol. 32 no.3 cardiovascular death (hazard ratio 1.62; 95% ci 1.14 to 2.30) during 42 months follow up. cakar et al.(6) observed that the mortality rate in acute myocardial infarction patients with elevated creatinine (defined as creatinine levels >1.3 mg/ dl) was higher than in the normal creatinine group (25.9% vs 6.8%) after 1 year follow up. in our study, multiple logistic regression analysis also showed that blood urea nitrogen had more influence on mortality than creatinine. renal insufficiency has been related to adverse cardiovascular outcomes in patients with cardiovascular disease. sachdeva et al.(17) found that patients with renal dysfunction had a significantly increased risk (almost 4 times) of developing adverse outcomes after acute myocardial infarction. a l i m i t a t i o n o f t h i s s t u d y w a s t h e unavailability of blood urea nitrogen and creatinine levels before the onset of myocardial infarction, preventing the exclusion of patients with nonsymptomatic renal dysfunction, which may have introduced bias into the statistical analysis. as a follow-up to this study, a cohort study should be performed about the effect of blood urea nitrogen and creatinine levels on short and long term mortality risks among patients with acute myocardial infarction. further studies about evaluation of blood urea nitrogen creatinine changes during hospitalization and their effect on mortality are also necessary. admission blood urea nitrogen and creatinine levels could be used as predictors of mortality in acute myocardial infarction patients during hospitalization. conclusions blood urea nitrogen and creatinine levels in non-surviving patients with acute myocardial infarction were higher than in surviving patients. patients with high levels of blood urea nitrogen and creatinine had higher mortality risks than patients with normal levels. blood urea nitrogen was a better predictor of mortality than creatinine. acknowledgement we thank the medical record officers who helped us find the data of patients. we also would like to thank all reviewers for their advice to improve the manuscript. references 1. apple fs, jaffe as. cardiovascular disease. in: burtis ca, ashwood er, bruns de, editors. tietz fundamentals of clinical chemistry. 6th ed. philadelphia: saunders elsevier;2008.p.61430. 2. lamb ej, price cp. creatinine, urea and uric acid. in: burtis ca, ashwood er, bruns de, editors. tietz fundamentals of clinical chemistry. 6th ed. philadelphia: saunders elsevier;2008.p.363-72. 3. delaney m, price cp, lamb ej. kidney function and disease. in: burtis ca, ashwood er, bruns de, editors. tietz fundamentals of clinical chemistry. 6th ed. philadelphia: saunders elsevier;2008.p.631-54. 4. carter, ce, gansevoort rt, scheven l, heerspink hjl, shlipak mg, de jong p, et al. influence of urine creatinine on relationship between the albumin-to-creatinine ratio and cardiovascular events. clin j am soc nephrol 2012;7:595-603. doi:10.2215/cjn.09300911. 5. french wj, wright rs. renal insufficiency and worsened prognosis with stemi: a call for action. j am coll cardiol 2003;42:1544-6. 6. cakar ma, gunduz h, vatan mb, kocayigit i, akdemir r. the effect of admission creatinine levels on one-year mortality in acute myocardial infarction. scientific world journal 2012. doi:10.1100/2012/186495. 7. kirtane aj, leder dm, waikar ss, chertow gm, ray kk, pinto ds, et al. serum blood urea nitrogen as an independent marker of subsequent mortality among patients with acute coronary syndromes and normal to mildly reduced glomerular filtration rates. j am coll cardiol 2005;45:1781-6.doi:10.1016/j.jacc.2005.02.068. 8. sorensen cr, brendorp b, rask-madsen c, kober l, kjiller e, torp-pedersen c. the prognostic importance of creatinine clearance after acute myocardial infarction. eur heart j 2002;23:948-2. doi:10.1053/euhj.2001.2989. 9. jose p, skali h, anavekar n, tomson c, krumholz hm, rouleau jl, et al. increase in creatinine and cardiovascular risk in patients 178 with systolic dysfunction after myocardial infarction. j am soc nephrol 2006;17:2886-91. 10. smith gl, shlipak mg, havranek ep, foofy jm, masoudi fa, rathore ss, et al. serum urea nitrogen, creatinine, and estimators of renal function: mortality in older patients with cardiovascular disease. arch intern med 2006; 166:1134-42. 11. lazaros g, tsiachris d, tousoulis d, patialiakas a, dimitriadis k, roussos d, et al. in-hospital worsening renal function is an independent predictor of one-year mortality in patients with acute myocardial infarction. int j cardiol 2012; 155:97-101. 12. amin ap, spertus ja, reid kj, lan x, buchanan dm, decker c, et al. the prognostic importance of worsening renal function during an acute myocardial infarction on long-term mortality. am heart j 2010;160:1065-71. 13. aronson d, hammerman h, beyar r, yalonetsky s, kapeliovich m markiewich w, et al. serum blood urea nitrogen and long term mortality in acute st-elevation myocardial infarction. int j cardiol 2008;127:380-5. 14. zhang b, gao c, hou q, yin j, xie l, pu s, et al. different independent susceptibility markers for first-ever cerebral infarction and myocardial infarction in young patients. j neurol 2012; 259:1420–5.doi 10.1007/s00415-011-6368-3. 15. ostfeld r, spinelli m, mookherjee d, holtzman d, shoyeb a, schaefer m, et al. the association of blood urea nitrogen levels and coronary artery disease. ejbm 2010;25:3-7. 16. gibson cm, pinto ds, murphy sa, morrow da, hobbach hp, wiviott sd, et al. association of creatinine and creatinine clearance on presentation in acute myocardial infarction with subsequent mortality. j am coll cardiol 2003; 42:1535-43. doi:10.1016/s0735-1097(03)01067 -2. 17. sachdeva s, singh np, saha r. impact of renal dysfunction on the outcome of acute myocardial infarction. jiacm 2010;11:277-81. kurniawan, bahrun, mangarengi, et al blood urea nitrogen in myocardial infarction c:\users\universa medicina\docu 105 abstract universa medicina propranolol decreases drd3 and slc1a2 gene expression in patients with essential tremor nefise kandemir*@, murat gultekin**@, mehmet kara***, arslan bayram†‡ nazife tascioglu*, meral mirza**, and munis dündar* background essential tremor (et) is the most common disease among movement disorders. genes such as essential tremor 1-4 (etm 1-4), hs1-binding protein-3 (hs1bp3), dopamine receptor d3 (drd3), leucine-rich repeat and ig domain containing 1 (lingo1), glial high affinity glutamate transporter member 2 (slc1a2), fus, high temperature requirement a2 (htra2) and tenm4 had been shown to be responsible for the genetic inheritance of the disease. the aim of the present study was to investigate the effect of propranolol on the expression of drd3, slc1a2, and htra2 genes in patients with et. methods a study of non-randomized experimental design was conducted involving 76 subjects. they were divided into two groups: 38 patients with et in the patient group (group 1) and 38 healthy people in the control group (group 2). drd3, slc1a2 and htra2 gene expressions were assessed before and after 8 weeks of propranolol treatment. fahn-tolosa-marin tremor scale results were compared before and after propranolol administration. kruskal wallis test was used to determine differences in gene expressions between the groups. results d3 dopamine receptor and slc1a2 gene expression in the patient group appeared to be lower than in the control group (p<0.001). however, the htra2 gene expression level was significantly higher in the patient group (p<0.001). conclusion d3 dopamine receptor and slc1a2 gene expressions were decreased in et patients which at first glance can be explained in relation to etiology, but after the treatment it was not increased as expected but decreased even more. key words: essential tremor, drd3, gene expression, htra2, propranolol, slc1a2 original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.105-112 may-august 2020 vol.39no.2 @these authors have made an equal contribution to this study and should be considered as co-first authors. *department of medical genetics, erciyes university faculty of medicine, kayseri, turkey **department of neurology, erciyes university faculty of medicine, kayseri, turkey ***ministry of health, republic of turkey †department of medical genetics, etlik zubeyde hanim women's diseases education and research hospital, ankara, turkey ‡department of medical genetics, deta-gen genetic diagnosis centre, kayseri, turkey *this study was funded by erciyes university, project no ttu-20166761. correspondence: dr. nefise kandemir, md erciyes university medical school, department of medical genetics talas kayseri / turkey telephone: +90-(352) 2076666 mobile: +90-(533) 3013827 e-mail: dr.nefise86@gmail.com orcid id: 0000-0003-2151-7300 date of first submission, march 16, 2020 date of final revised submission, july 21, 2020 date of acceptance, july 23, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license cite this article as: kandemir n, gultekin m, kara m, et al. propranolol decreases drd3 and slc1a2 gene expression in patients with essential tremor. univ med 2020;39:105-12. doi: 10.18051/univmed.2020.v39.105-112 106 kandemir, gultekin, kara, drd3, slc1a2 and essential tremor introduction essential tremor (et) is the most common movement disorder with motor and non-motor symptoms. although the disorder can occur at any age, the incidence increases with age.(1, 2) positive family history in individuals with et varied between 17.4% and 100%.(2) most of the patients with et were not diagnosed correctly and had missed the appropriate treatment options. the pathophysiology of the disease is still unknown. the clinical presentation of et was the 6-12 hz kinetic and postural tremor, which occurred in the hands and arms by the voluntary movements. other anatomical parts affected by tremors were head, vocal cords, trunk, legs and face regions. essential tremor is currently revealed as a heterogeneous disease.(3, 4) the etiology of et is mostly unexplained. about half of the cases of et appear to result from a genetic mutation, although a specific gene has not been identified. this form is referred to as familial tremor and is an autosomal dominant disorder. the variability in age of onset, the presence of sporadic cases, and incomplete concordance of et among monozygotic twins suggest that environmental factors play a role.(58) the relationship of the disease to non-motor symptoms of such entities as parkinsonism, dystonia, c erebe llar dysfunction, sleep disturbances, mild cognitive impairment, and cognitive disorder, personality and temperament disorder, showed that the disease included not only the motor but also non-motor dysfunction.(911) essential tremor is autosomal dominantly inherited with an incomplete penetration. however, some families may also have polygenic inheritance.(12) a study at yale university found that et patients and their families knew the most basic, observable features of et (e.g., the presence of hand tremors). however, 16.7% of the affected did not endorse genes as a cause for et.(13) treatment of patients with et can be divided into two parts, i.e. medical and surgical parts. pharmacological agents such as propranolol, pirimidone, topiramate, and gabapentin are preferred as a medical treatment, whereas surgical treatment may include thalamotomy, lesion surgery, or deep brain stimulation.(14, 15) a randomized controlle d tr ia l showed that propranolol reduced tremor amplitudes as measured by accelerometry by a mean of 55%.(14) the dopamine receptor d3 (drd3) gene is localized on chromosome 3q13.31 and has been shown to be associated with et together with the etm1 locus. the d3 receptors are primarily expressed in mesolimbic areas associated with cognitive functions and motivated behaviour and have been implicated in the antipsychotic effects of neuroleptics as well as in the conditioning to psychostimulants.(16) the glial high affinity glutamate transporter member 2 (slc1a2) gene as a member of the glutamate transport system, also known as glutamate transporter 1, contains five different members with variable expression patterns in the central nervous system. due to the physiological and pathological effects of possible mutations in the solute carrier family 1 member 2 (slc1a2) gene, these mutations were assumed to cause glutamatergic defects in the central nervous system of et patients. although the slc1a2 expression was shown in peripheral organs, the symatlas results showed that the slc1a2 gene is mostly expressed in the brain. this astroglial specific high-affinity glutamate transporter has been reported for expression alterations in psychiatric disorders.(17,18) the high-tempe rat ure -regulate d a2 (htra2) gene is a serine protease that is released during apoptosis from the mitochondria to the cytosol and plays role in many neurodegenerative diseases. (19-21) high-temperature-regulated a2 has already been designated as park13 which may cause parkinson’s disease, although there are still discrepancies among the study results.(22) a case control study involving 45 subjects with et and 13 subjects without tremor showed that the genotype frequencies did not differ significantly between subjects with et and healthy controls. (23) 107 to the best of our knowledge this is the first study aiming to present the expression change in drd3, slc1a2, and htra2 genes before and after the treatment of et patients with propranolol. methods research design this non-randomized experimental study was carried out in the departments of medical genetics and neurology, faculty of medicine, erciyes university between july 2016 and january 2019. research subjects a total of 76 subjects with the diagnosis of et and healthy subjects were recruited. they were stratified into the et group and the control group consisting of the healthy subjects. the sample sizes were determined using effect size =0.6; α=0.05 and β=0.2, resulting in 38 subjects for each group. the inclusion criteria of the et patients presenting with an involuntary rhythmic sinusoidal oscillation of a body part without any known causes, were age between 18 and 60 years, having no additional disease and being only on propranolol treatment at 40 mg twice per day for 8 weeks. exclusion criteria were hyperthyroidism, psychiatric disorders such as depression, anxiety, psychosis , etc. , cere brova scular disea se, diabetes mellitus, other forms of tremor in the patient including neuropathic tremor, dystonic tremor, orthostatic tremor, etc. and being on t re a tme nt tha t i nc r ea s es tr e mo r se ve r it y (valproic acid, etc.). the study included 38 patients with et and 38 healthy individuals as a control group. the control group included healthy people with the age of 18-60, chosen from the epidemiological area of the eumf hospital, who had no other neurological disorder or chronic medical disease as a result of the evaluation done by an experienced neurologist. patients admitted to the neurological movement disorders outpatient clinic of erciyes university, faculty of medicine and diagnosed with et by an experienced neurologist were included into the study using the fahn-tolosa-marin clinical rating scale for tremor scale (ftm). (24) tremor severity was given a score between 0 and 4 in ftm. in order to determine the tremor severityfrequency of the patients with the ftm, the device was given to patients to measure the tremor frequency with both hands and they were asked to hold for 10 sec. p r i m e r se que nc es us e d f o r ge ne expression the rna was isolated by leukocyte isolation using red cell lysis solution (sigma-aldrichý, st. louis, missouri, united states) and trizol, a chemical solution used in the extraction of dna, rna, and proteins from cells (invitrogen, thermo fisher scientific, california, united states). quantitative real-time pcr which is a pcrbased technique that compares amplification of a target dna sequence with quantification of the concentration of the selected refe rence (housekeeping) dna were performed on light cycler 480 ii (roche diagnostics ltd., rotkreuz, switzerland). the expression levels of drd3, slc1a2 and htra2 genes were measured via specific real time ready catalogue primary-probe kits (roche diagnostic gmbh, mannheim, germany) (table 1). the β-actin gene was used as the reference housekeeping gene. data were analyzed via the light cycler 480 software (version 1.5.0 sp4) (roche diagnostics ltd., rotkreuz, switzerland) using the ‘delta-delta ct’ formula to calculate the relative fold gene expression. gene expression results were compared between control, preand posttreatment groups. statistical analysis statistical analysis was performed using graphpad prism 6.0 (graphpad software, ca, usa). the histogram, quantile-quantile plot and shapiro-wilks tests were used to evaluate the normal distribution of the data and an independenttest was used. in the case of an abnormal data univ med vol. 39 no.2 108 kandemir, gultekin, kara, drd3, slc1a2 and essential tremor distribution being found, the nonparametric mannwhitney test was us ed to determine the difference between the groups. a chi square test was used for comparison of distribution regarding gender of participants. p<0.05 was considered as the significance level. ethical clearance ethical approval (no: 2016/297) was obtained from the ethical committee of erciyes university medical faculty. results a total of 76 subjects participated in the study, and were stratified into the et group and the healthy control group. the et subjects were given propranolol for 8 weeks (figure 1). at base line there was no significant difference in age and gender between the et and control groups. but the gene expression was significantly different between the et and control groups. the drd3 and slc1a2 expressions a b c gene assay id primer sequence (5’-3’) slc1a2 111792 f: cctgccaacagaggacatc r: gactgaagttctcatcctgtcca drd3 113554 f: ttcgggagaagaaggcaac r: gcagccagcagacaatga htra2 145466 f: tcagcatggtgtactcatcca r: agcttcataa table 1. primer sequences used in expression figure 1. flow diagram of the participants 109 were significantly lower in the et group in comparison to the healthy group (p<0.001), but the htra2 gene was significantly higher in the et group (p<0.001) (table 2). preand post-treatment expression levels of the drd3, slc1a2 and htra2 genes were compared in the et gr oup. propranolol significantly decreased slc1a2 expression, but the decrease in htra2 and drd3 ge ne expressions were not significant. we did not compare gene expression after 8 weeks between the et group and the control group, as the latter did not receive any treatment. discussion our results showed reduced drd3 and slc1a2 gene expression and increased htra2 gene expression levels in the patient group. the drd3 receptor is a protein consisting of 446 amino acids. (25) the drd3 receptor is found widely in the brain in the control centers for sensing, affect and motor functions, nucleus accumbens, ventral tegmental area, substantia nigra, limbic region, and basal ganglia.(26, 27) in the present study, the expression level of the drd3 gene was lower in the patients with et than in the control group. expression of the drd3 gene was lower after treatment with propranolol. since the dopamine level was low in the patients with et, the low mrna level might be due to polymorphisms in this gene.(28) a further decrease in drd3 e xpression unde r the propranolol treatment observed in our study possibly indicates that propranolol does not affect the expression of this gene and that decreased level of mrna is a feature of et. jiménez-jiménez et al.(23) in a study with drd3 rs6280, slc1a2 rs3794087, and mapt rs1052553 gene variants, which was carried out on 11 spanish families with et clinical signs and the risk of these genes for et inheritance, concluded that no relationship exists between drd3 and slc1a2 gene variant and et. although an increased risk for the mapt variant was detected, this result was not confirmed by a family-based study. slc1a2, which encodes the glutamate transporter and exhibits high affinity especially in astroglial cells, is a brain-specific gene. (23) the present study found that the expression level of the slc1a2 gene was lower in the patient group. the expression level of the slc1a2 gene was found to be even lower in the post-propranolol univ med vol. 39 no.2 table 2. distribution of demographic characteristics and ftm-trs grade within the et and control groups table 3. gene expression comparison between the treatment (before and after 8 weeks propanorol treatment) and control groups characteristics treatment group (n=38) control group (n=20) p value age (years) 36.7 ± 12.2 37.35 ± 1.1 >0.05 gender male female 25 13 12 8 >0.05 ftm-trs grade before treatment after treatment <0.0001 2.7 ± 1 2.1 ± 0.8 et diagnosed patient group control group p value before treatment after treatment drd3 1.2 ± 1.6 0.7 ± 0.6 5.3 ± 4,7 <0.0001 slc1a2 2.3 ± 3 0.4 ± 4 10.6 ± 8.2 <0.0001 htra2 6.2 ± 7.5 4.3 ± 6 1.8 ± 0.98 >0.05 110 kandemir, gultekin, kara, drd3, slc1a2 and essential tremor treatment period. as the glutamate level is high in et patients, the low level of slc1a2 mrna, which is known as one of the glutamate carriers, may be due to polymorphisms in this gene. in another study on exome sequencing done by gulsuner et al. (29) a rare variant of the etassociated htra se ri ne peptidase 2 gene (htra2) was described in a family of 6 generations. the htra2 gene encodes a 458 amino acid serine pr ote ase found in the intermembrane region of mitochondria. the hightemperature-regulated a2 is known to be eff ective in the d evelopment of ma ny neurodegenerative diseases as well as in apoptosis. (30, 31) in the present study, the expression level of the htra2 gene was higher in the patient group. despite a slight decrease in the post-propranolol treatment period, there was a higher expression in the patient group than in the control group. however, the statistically insignificant difference in htra2 gene expression seen in the present study could be in parallel with the fact that the effect of propranolol on apoptosis has not yet been fully elucidated. propra nolol is a nonselective betaadrenergic receptor antagonist. the mechanism of action suggests that it blocks a protein target in the form of peripheral beta-2 receptors. there are different studies in the literature about the effect of propranolol on genes.(32) the mechanisms underlying the properties of propranolol in gene expression are unclear at present. to explain these mechanisms more comprehensive investigations (in vivo and in vitro) should be carried out with different doses of propranolol and the protein levels in 3d cultures should be determined in future. although htra2 expression level was found to be increased in the et group, the increase was not statistically significant. therefore further investigations are needed with larger samples to find out whether our results occurred by chance or there actually was a statistically significant increase in gene expression level. as a limitation of this study, only the sample size can be pointed out. for validation of obtained results other similar projects with larger samples must be carried out. also, the effects of different dosages of propranolol could not be compared because of budget limitations. conclusion the gene expression study previously performed with propra nolol showed that propranolol generally suppresses protein synthesis and gene expression. according to our findings, drd3 and slc1a2 gene expressions were deceased in et patients which at first glance can be explained in relation to etiology, but after treatment it was not increased as expected but decreased even more. acknowledgements this study wa s f unded by erciyes university, project no ttu-2016-6761. conflict of interest there is no conflict of interest between authors. contributors nk conceived the concept and wrote the first draft of the manuscript. mg analyzed the data. mk contributed to the writing of the manuscript. ab, nt, mm jointly developed the structure and arguments for the paper. md made critical revisions. all authors reviewed and approved the final manuscript. references 1. kuhlenbäumer g, hopfner f, deuschl g. genetics of essential tremor: meta-analysis and review. neurology 2014;82:1000-7. doi: 10.1212/ wnl.0000000000000211. 2. louis ed, ferreira jj. how common is the most common adult movement disorder? update on the worldwide prevalence of essential tremor. mov disord 2010;25:534-41. doi:10.1002/mds.22838. 111 univ med vol. 39 no.2 3. pahwa r, dhall r, ostrem j, et al. an acute randomized controlled trial of noninvasive peripheral nerve stimulation in essential tremor. neuromodulation 2019;22:537–45. doi: 10.1111/ ner.12930. 4. prasad s, bhalsing ks, jhunjhunwala k, lenka a, binu v, pal pk. phenotypic variability of essential tremor based on the age at onset. can j neurol sci 2019;46:192-8. doi: 10.1017/ cjn.2018.384. 5. agarwal s, biagioni mc. essential tremor. treasure island (fl): statpearls publishing; 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parkinson’s disease; evidence-based; case report case report pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.63-70 january-april, 2020 vol.39no.1 cite this article as: mochtar i, mansyur m. parkinson's disease and the welding job: are both related?. univ med 2020; 39:6 3 -7 0 . doi: 10 .18 051/ uni vmed. 2020.v39.63-70 *division of occupational medicine, department of community medicine, universitas indonesia, indonesia **department of community medicine, universitas indonesia, indonesia correspondence: iqbal mochtar division of occupational medicine, department of community medicine, universitas indonesia, indonesia phone: 0974-66554913 email: muhammad.mochtar03@alumni. imperial.ac.uk orcid id: 0000-0002-1057-3916 date of first submission, june 7, 2019 date of final revised submission, march 8, 2020 date of acceptance, march 16, 2020 this open access article is distributed under a creative commons attribution-non commercial-share alike 4.0 international license 64 mochtar, mansyur parkinson's disease and the welding job introduction parkinson’s disease (pd) is the second most common neurodegene rative disease affecting 6.1 million people worldwide in 2016.(1) the pathological basis of the disease is related to the aggregation of the alpha-synuclein protein of lewy bodies and the loss of pigmented mel a nin in t he mi d br a in, le a d ing t o th e degeneration of dopaminergic neurons in the substantia nigra and dopamine insufficiency in the striatum.(2) it is the most common cause of parkinsonism, a syndrome characterized by postural instability, tremor, bradykinesia and rigidity.(3) pe s t i c i de e xp os u r e , da ir y p r o du c t consumption, and brain trauma have been associated with an increased risk of pd, while smoking, caffeine intake and exercise have been linked with reduced risk of the disease. (4,5) some studies linked pd with the welding job, due to manganese exposure in the welding works. those studies identified neuropsychomotor symptoms and manganese deposition on mri in manganese-exposed workers. other studies refuted the finding and argued that pd was not related to welding works. it still remains c o nt r ove rs i a l wh e t h e r we l d er s h a ve a n i nc r e a s e d r i sk of pd a nd whe t he r t h e neuropsychomotor symptoms observed in manganese-exposed workers are related to pd.(6) the aim of this evidence-based case report was to obtain evidence-based information regarding the relationship between parkinson’s disease and the welding job through an evidencebased case report derived from a literature review. case illustration a 55-year-old male qatari patient came with resting tremor in both hands, r igid movement, slow and unstable gait since two yea rs ago. due to the symptoms, he had difficulty in doing work and daily activities. the initial symptom was only tremor in the left hand, which was mild in quality and on-off. later, the tremor occurred in both hands and was more severe and frequent. his general movement was bradykinesia without slurred speech. he was a non-smoker and had controlled diabetes and hypertension. he had been working as a welding inspector in an oil and gas company for the last 12 years. previously, he worked as a welding technician for 8 years. his latest work was to supervise welding activities, to test welding materials, to inspect and control welding equipment and consumables such as filler wire, fluxes and electrodes. he had under gone treatment in some hospitals, including one in germany. the final diagnosis was idiopathic pd and he had been given some medications, including co-beneldopa and co-careldopa. following se ver al mont hs of tre atment , the worker observed some improvement in his symptoms. the worker believed that his condition was due to his job and therefore he planned to claim financial compensation. the clinical question for this case was: in the workers, is the welding job, compared to the non-welding jobs, associated with pd? evidence following the search of articles using the keywords (table 1), the selection was performed using the inclusion and exclusion criteria (figure 1). the inclusion criteria included human studies, publications within the last 10 years, in the english language, using adult subjects, and relevant to the clinical question. exclusion criteria included pathophysiological, phar macological and radiological studies as well as opinion papers. applying the inclusion and exclusion criteria, the article list was shortened, and eventually only three articles remained for appraisal, consisting of one systematic review (mortimer et al.(9)) and two observational studies (racette et al.(10) and racette et al.(11) the selecte d ar ticl es were critic ally appraised using the appraisal tools f or a systematic review (7) and for cross-sectional and 65 prospective cohort studies.(8) the results of the appraisal are presented in tables 2, 3 and 4. mortimer et al.(9) performed a meta-analysis a n d s c r u t ini ze d 10 5 s t ud i e s to se e k t he association of the welding job and manganese exposure with pd.(9) the authors analyzed two main questions, namely the association between the welding job and pd and the association between manganese exposure and pd. the authors demonstrated that the pooled relative risks of the welding job to pd disease was 0.86 (95% ci: 0.80-0.92) and that of manganese exposure to pd was 0.76 (95% ci: 0.41-1.42). the authors concluded that both the welding job and manganese exposure were not related to an increased risk of pd. racette et al.(10) compared the prevalence of parkinsonism in welding-exposed workers to the reference group. the study utilized clinical assessment including the use of the unified parkinson’s disease rating motor scale 3 (updrs3) to categorize the subjects into parkinsonism (score >15), intermediate (score 614) and normal (score <6). the exposure assessment was performed using a questionnaire. the prevalence of parkinsonism in the welding figure 1. the process of article selection univ med vol. 38 no.1 66 mochtar, mansyur parkinson's disease and the welding job job was 15.6% and in the non-welding jobs 0%. the study concluded that welding-exposed workers had a high prevalence of parkinsonism compared to the non-exposed workers. in anothe r ar ticle , racette et al. (1 1) assessed the progression of parkinsonism in the manganese-exposed welders in three worksites. they measured the clinical assessment of 886 workers using updrs3 questionnaire and clinical evaluation while the exposure assessment was measured by questionnaire. the authors followedup the patients, with a mean follow up of 4.2 years and revealed that in all examined workers, there was a change of 0.24 (0.10-0.38) in updrs3 score. the study concluded that the progression of parkinsonism increased with increased cumulative manganese exposure. up to now, there has been a controversy on whether welding-exposed workers are associated with pd. some studies supported and other studies refuted the association. park pointed out that one of the factors that induced these conflicting findings were the pitfalls in various methodological issues in the studies, including a healthy worker effect bias, statistical modeling and the use of litigation studies. in addition, a number of studies also did not carefully consider confounder factors in manganese work such as the exposure to carbon-monoxide, aluminum and heat stress.(6) couper fir st reported the association b e t we e n ma n ga nes e e x po s ur e a n d neurobehavioral symptoms in 1873.(12) the report was the first to mention the toxicity of manganese in humans, which is now known as manganism. manganism is a neurobehavioral syndrome due to an excessive accumulation of manga nese in t he brain fr om manganese exposure. manganism has clinical features resembling pd, and therefore many physicians mi sd i a gn ose ma n ga n i s m a s pd. in f a c t , manganism and pd are two different entities wi t h d i f f er e n t c l i n i c a l a nd pat h o lo gi c a l ma ni f e s ta ti o ns . ( 1 3 ) t he di s t i ngui s h in g characteristics of manganism are the absence of resting tremor, the appearance of gait disorder as an initial symptom and the lack of response to l-dopa. also, in contrast to pd, neuroimaging in patients with manganism does not reveal any degeneration of dopaminergic neurons in the substantia nigra and dopamine insufficiency in the striatum.(14) comparing the three selected articles, the study of mortimer et al.(9) is more relevant and appropriate for answering the clinical question. first, mortimer used a meta-analytical method in scrutinizing relevant studies. the authors applied strict inclusion and exclusion criteria and excluded studies that potentially led to bias effects, lacked validity, and inadequate statistical method. the authors also calculated the heterogeneity of study effects to argue that the existing variation in the studies was due to chance. unlike mortimer, racette et al.(10) supported the relationship between welding or manganese exposure and parkinsonism. in their article, the authors showed an increased prevalence of parkinsonism in welding-exposed workers compared to the reference population (15% vs. 0%). however, racette’s study has some potential bias, mainly due to the use of the questionnairebased metric for exposure calculation. recall bias might occur because the subjects might provide inadequate information when estimating the type, quantity and the duration of exposure. a selection bias might also occur because the authors utilized the reference group sample provided by the trade union without clear subject criteria. in another article racette et al.(11) followed up the manganese-exposed workers to observe the progression of parkinsonism. during a mean follow-up period of 4.2 years, the authors found that in the manganese-exposed workers there a b c database keyword hits filter pubmed “welding” or “welder” and “parkinson” 26 title/abstract cochrane library “welding” or “welder” and “parkinson” 88 title/abstract jstor “welding” or “welder” and “parkinson” 3 title/abstract table 1. search strategy using keywords 67 article mortimer ja, et al. association of welding and manganese exposure with parkinson’s disease – review and meta-analysis. (9) level 1 (systematic review of clinical trials, cohort, case-control studies) (7) questions does the systematic review address a focused question (pico) and use it to direct the search and select articles for inclusion? yes the authors analyzed the association between welding and pd and the association between manganese exposure and pd. the authors applied the focused questions to select the most relevant articles. find did the research find all the relevant evidence? yes the authors searched pubmed and cochrane databases, published reviews and abstracts of scientific meetings. appraise have the studies been critically appraised and was the overall quality adequate? yes the authors indicated some appraisal criteria for the focused questions, such as types of exposure, outcomes, and number of person-years followed up. synthesize have the results been synthesized with appropriate summary tables and plot? were the results similar between studies? yes yes the authors listed the details of each study in the appropriate tables and plot/pooled relative risk. the results of the studies were similar, in that the relative risk (rr) for both welding and manganese exposure to pd were less than 1. result what measure was used, how large was the effect (could it have been due to chance?) uncertain the rr between welding and pd was 0.86 (0.80-0.92) and between manganese exposure and pd 0.76 (0.41-1.42). the association between welding and pd lacked heterogeneity, indicating that the different results of the reviewed studies were due to chance. table 2. critical appraisal checklist for systematic review was a progression of parkinsonism evidenced by a 0.32 (sd 2.1) change in updrs3 score. however, this article also has potential bias, such as information, recall and selection bias, mainly due to the use of questionnaire findings that were converted into weighted exposure years. in relation to the patient concerned in this case illustration, there are some reasons to argue that his pd is not related to his job. first, aging – not manganese exposure is the largest contributing factor for pd; pd affects 1% of the population above 60 years and 5% of the population over 85 years. around 5% of pd, however, occurs at the age below 60 years and this is associated with mutations in a number of genes such as pink-1, parkin, dj-1 and alphanuclein.(15) second, pd is a multifactorial disease, where various risk factors may play a role in the occurrence of the disease. the patient has a number of risk factors, such as diabetes and hypertension, which may play a role in the onset of his pd. third, based on current and relevant evidence-based medicine publications available, including meta-analytical studies, no increased pd risk is observed in welders or welding-exposed workers.(9) univ med vol. 38 no.1 68 mochtar, mansyur parkinson's disease and the welding job article racette ba, et all. increased risk of parkinsonism with welding exposure. (10) level level 2 c (case-control study) (8) did the study address a clearly focused question? yes to determine the prevalence and phenotype of parkinsonism in shipyard and fabrication welders and compare these with non-exposure subjects comprising pd patients from movement disorders center, washington. is the research method (study design) appropriate for answering clinical questions? yes the study type (cross-sectional and nested casecontrol) is appropriate for the prevalence estimation. is the method of selection of the subjects (employees, team, division, organization) clearly described? uncertain the authors did not mention the reason for the recruitment from the places. could the way the sample was obtained introduce (selection) bias? yes in the study subjects, the authors took the list of welders and contacted them for participation. in the reference group 1, the subjects were recruited by the local trade union and the authors were not given the list of the workers. in the reference group 2, the subjects were taken from the clinical database of newly diagnosed untreated pd at movement disorders center, without any subject selection criteria. was the sample of subjects representative with regard to the population to which the findings will be referred to? yes for the study group but not for the reference groups in the study group, the subjects represented the population to which the finding referred. in the reference groups 1 and 2, the recruited samples did not represent the reference population. was the sample size based on prestudy considerations of statistical power? no no predetermined sample size. was a satisfactory response rate achieved? yes 811 workers out of 1,612 welding-exposure workers (50.3%) agreed to participate. were the measurements (questionnaires) likely to be valid and reliable? for the clinical diagnosis they were valid and reliable but for the exposure assessment, they lacked validity and reliability. clinical diagnosis of parkinsonism was based on a neurological examination that included updrs3 questionnairre. the clinical assessment is considered valid and reliable. exposure assessment utilized welding exposure questionnaire. based on the questionnaire, weighted exposure-years was calculated for each participant. table 3. critical appraisal checklist for a cross-sectional study 69 table 4. critical appraisal checklist for a prospective cohort study article racette ba, et all. dose-dependent progression of parkinsonism in manganese-exposed welders. (11) level prospective cohort (2b) (8) are the results of the study valid? was the defined representative samples of patients assembled at a common (usually early) point in the course of their diseases? yes baseline examination on the study subjects was conducted from january 2006 to september 2013. was patient follow up sufficiently long and complete? yes each subject had a different period or number of follow up. 61% had 1 follow-up, 26% had 2 follow-ups, 12% had 3 follow-ups and 1% had 4-5 follow-ups. mean time period between baseline and any follow-up was 3.9 (1.8) years. were outcomes criteria either objective or applied blindly? yes the primary outcomes were the rates of changes of updrs3 score. the secondary outcomes were the annual rates of changes of updrs3 sub-score. the exposure assessment was blinded to the neurologists who examined the subjects. if subgroups with different prognosis are identified, did an adjustment for important prognostic factors take place? yes the authors adjusted the prognostic factors; e.g. duration of exposure to baseline examination was divided into <5 years and ± 5 years. what are the results? how likely are the outcomes over time? yes the authors provided the estimation of expected additional increase of updrs3 score over a 10-year time period how precise are the prognostic estimates? uncertain the precision of prognostic value can be based on the confidence interval. the confidence intervals of findings were mixed, some of them were narrow and the rest were wide. can i apply this valid, important evidence about prognosis to my patient? no the study subjects were from the shipyard and fabrication unit while our patient was from a gas and petroleum company. the race also matters, since the study subjects were non-hispanic caucasian and our patient was arabic. our patient had consumed pd medication and therefore the estimation of pd deterioration using a time-based estimation cannot be applied. learning points based on the selected evidence-based resources, it is concluded that: (i) parkinson’ disease is not related to the welding job; (ii) the welding job, however, may produce clinical symptoms and signs resembling pd, known as manganism. univ med vol. 38 no.1 70 mochtar, mansyur parkinson's disease and the welding job conflicts of interest none. acknowledgements we would like to thank the staff of the department of community medicine, universitas indonesia for the support for this publication. contributors im contributed to conceptualization and methodology. im and mm contributed to preparing and writing the original draft. mm contributed to supervision. im contributed to review and editing. all authors have read and approved the final manuscript. references 1. rocca wa. the burden of parkinson’s disease: a worldwide perspective. lancet neurol 2018;17: 928-9. doi: 10.1016/s1474-4422(18)30355-7. 2. oertel wh. recent advances in treating parkinson’s disease. f1000res 2017;6:260. doi: 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2012;79:1174-80. 10. racette ba, criswell sr, lundin ji, et al. increased risk of parkinsonism with welding exposure. neurotoxicology 2012;33:1356-61. 11. racette ba, nielsen ss, criswell sr, et al. dosedependent progression of parkinsonism in manganese-exposed welders. neurology 2017;88: 344-51. 12. couper j. on the effects of black oxide of manganese when inhaled in the lungs. br ann med pharmacol 1873;1:41–2. cited by: blanc pd. t he early history of manganese and the recognition of its neurotoxicity, 1837-1936. neurotoxicology 2018;64:5-11. doi: 10.1016/ j.neuro.2017.04.006. 13. kwakye gf, paoliello mmb, mukhopadhyay s, et al. manganese-induced parkinsonism and parkinson’s disease: shared and distinguishable features. int j environ res public health 2015;12: 7519-40. 14. guilarte tr. manganese and parkinson’s disease: a critical review and new findings. environ health perspect 2010;118:1071-81. 15. reeve a, simcox e, turnbull d. ageing and parkinson’s disease: why is advancing age the biggest risk factor? ageing res rev 2014;14:1930. c:\users\jurnal fkusakti\docume 25 *department of medical laboratory science, school of basic medical sciences, college of medical sciences, university of benin, benin city, nigeria **medical laboratory unit, cottage hospital, auchi polytechnic, auchi, edo state, nigeria. correspondence: helen oroboghae ogefere department of medical laboratory science, school of basic medical sciences, college of medical sciences, university of benin, benin city, nigeria e-mail: helenogefere@yahoo.com phone number: +2348023509447 date of first submission, september 17, 2 01 8 date of final revised submission, march 19, 2019 date of acceptance, march 20, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina molecular characterization of methicillin-resistant staphylococci among apparently healthy students helen oroboghae ogefere* and lawrence ayodele ogunleye*,** background staphylococcus aureus are widely considered a major factor of nosocomial and community-acquired infections. this work was aimed at determining the prevalence of methicillin-resistant s. aureus (mrsa) among apparently healthy students. methods a cross-sectional study was conducted involving 400 nasal swab samples randomly collected from students using sterile swab sticks and processed to recover s. aureus using standard microbiological techniques. conventional methods were used to identify the isolates and antibiotic susceptibility tests were performed using kirby-bauer disc diffusion method according to performance standards of clinical and laboratory standard institute guidelines. methicillin-resistance was detected phenotypically using cefoxitin 30µg discs. bacterial deoxyribonucleic acid (dna) extraction was done on cefoxitin-resistant staphylococci isolates only using zymoresearch (zr) fungal/bacterial dna minipreptm kit. a polymerase chain reaction assay targeting the 16s rrna, nuc, and meca genes on 1.0% agarose gel electrophoresis stained with ethidium bromide was used to identify s.aureus and detect methicillin resistance. results the overall prevalence of mrsa was 5.8% using phenotypic methods. pcr amplification of the 23 phenotypically confirmed mrsa using 16s rrna and nuc genes identified staphylococci 23/23(100%) and s. aureus 23/23(100%) at band size 886bp and 225bp respectively. however, 16(69.6%) were positive for meca gene at band size 532bp by pcr method. poor level of susceptibility was recorded among the mrsa namely to erythromycin (26.6%), cloxacillin (0%), augmentin (0%), cefuroxime (0%), ceftriaxone (0%) and ceftazidime (0%). ofloxacin was the most effective antibiotic (60.9%). conclusion active antimicrobial surveillance of pathogenic staphylococci is important to analyze the infections and transmission rate for possible control measures. keywords: methicillin-resistance, staphylococci, students, antibiotics, meca, nuc. original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.25-32 january-april, 2019 vol.38no.1 cit e th is a rtic le a s: ogefere ho, ogunleye la. lmolecular characterization of methicillin-resistant staphylococci among apparently healthy students. univ med 2019;38:25-32. doi: 10.18051/univmed.2019.v38.25-32 26 ogefere, ogunleye methicillin-resistant staphylococci introduction the widespread resistance across various species of staphylococci has been reported in recent years.(1) the most notable example is the e me r ge n c e o f me t h ic il l i n -r e s i s ta nt staphylococcus aureus (mrsa), which was reported just one year after the launch of methicillin in the early 1960s and has now become a major public health concern with its prevalence increasing globally.(2) mrsa can cause the same types of infections as other s. aureus isolates such as: skin and soft tissue infections, including impetigo, folliculitis, furunculosis, abscesses, cellulitis, and wound infections.(2) the mrsa has also been reported to cause invasive infections such as: pneumonia, endocarditis, osteomyelitis, septic arthritis, meni n gi t i s, se pt i ce mia , tox i c sh oc k and staphylococcal scalded skin syndromes in both infants and adults.(2) the mrsa strains are resistant to nearly all beta-lactam antibiotics by producing an alternative penicillin-binding protein (pbp) known as pbp-2a.(3) this protein is encoded by the meca gene and has a low affinity to many beta-lactam antibiotics. mrsa strains are not o nl y r e s ist a n t t o b e t a -l ac t a ms a nd cephalosporins, but also often show resistance to a wide range of antibiotics.(3) the meca is p ar t o f a mo bi le ge n et i c e le me n t ca lle d s t a ph ylo co c c a l c a s s e t t e ch r o mo s ome (sccmec). the sccmec is flanked by cassette chromosome recombinase genes (ccra or ccrb or ccrc) that permit intraand interspecies horizontal transmission of sccmec.(3, 4) s. aureus has four pbps, (pbp1, pbp2, pbp3, and pbp4), with pbp2a/2' and pbp2 being responsible for peptidoglycan synthesis with transpeptidase and transglycosylase activities.(3) in nigeria, the prevalence of hospital associatedmrsa in clinical samples also varies from one region to another; 19.2% in ekiti, 28.6% in kano, 12.5% in maiduguri and 38.0% in benin.(5-9) several reports of studies on prevalence of community-associated mrsa (ca-mrsa) are also emerging in nigeria; 10.8% in apparently healthy school children in okada, edo state, 41% and 56.7% in apparently healthy university students in edo and ogun states respectively, and 60.7% in otherwise healthy inhabitants of uturu communities in abia state.(10-12) characterization of the 16s rrna gene is now well-established as a standard method for identifying and classifying species, genera and families of bacteria including staphylococci.(13) the nuc gene, which encodes thermonuclease, is widely used as a specific target for the identification of s. aureus by polymerase chain reaction (pcr).(14) numerous in-house pcr based assays targeting the nuc gene alone or in combination with the meca gene have been designed for fast screening or identification of methicillin-resistant s. aureus (mrsa) and methicillin-susceptible s. aureus (mssa).(14) in recent years, detection of meca by pcr is also considered as the gold standard for identification of mrsa.(9, 14) cefoxitin, being a cephamycin, is a stronger inducer of the meca regulatory system than oxacillin. the cefoxitin disc test is easier to read than the oxacillin disc diffusion test and more accurate for detection of meca mediated resistance.(15) many pcr based molecular methods were developed as alternative ways for accurate identification of mrsa.(16) most reports in our locality concentrate on phenotypic methods of identifying methicillin-resistant staphylococci especially among apparently healthy subjects and neglecting molecular characterization to identify the specific bacteria a n d ge ne s r e s p on si b l e f or a nti mic r ob i a l resistance. this study therefore aimed to use the pcr assay to target three genes: a genusspecific 16s rrna to detect staphylococcal dna; nuc, which encodes the s. aureus— specific region of the thermonuclease gene and meca, a determinant of methicillin resistance among apparently healthy students of a tertiary institution in auchi, edo state, nigeria. 27 methods research design this study was of cross-sectional design and was conducted using 400 apparently healthy students (consisting of 167 males and 233 females) of auchi polytechnic, auchi, edo state, nigeria. informed consent was obtained from all participants prior to specimen collection. institutional ethical approval was obtained before study commencement. the duration of the study was from july 2016 to november 2017. specimen collection and processing a t o t a l of 4 0 0 a n t e r i o r na sa l s w a b specimens were collected from the participants. all samples were collected from the left anterior nares using sterile swab stick soaked in sterile saline, labelled, packaged and transported immediately to the microbiology laboratory for analysis. staphylococcus aureus isolates were identified using api-staph system (api system; biomerieux, paris, france). antimicrobial susceptibility testing antimicrobial susceptibility testing was carried out following the recommendation of the british society for antimicrobial chemotherapy (bsac) method.(17) the test colonies were emulsified in sterile distilled water and the turbidity matched with 0.5 mcfarland. once matched, a sterile cotton wool swab was dipped in the organism suspension and excess liquid was removed by turning the swab on side of the test tube. the entire surface of mueller–hinton agar plate was seeded by swabbing in three directions with the swab. the antibiotic discs were placed on the plate with the use of a sterile forceps. the antimicrobial agents tested were ofloxacin ( 5 µg) , a mox i c i l li n -c l a vul a n at e ( 30 µg) , ceftazidime (30g), erythromycin (5µg), cloxacillin (5µg), ceftriaxone (30µg), cefuroxime (30µg) and gentamicin (10µg). the plates were incubated at 370c for 18 – 24 hours. detection of cefoxitin resistance all staphylococcus aureus isolated were screened for methicillin-resistance by following clsi guidelines using 30 μg cefoxitin discs ( a bt e k u .k ) . ( 1 5 ) pla te s we r e r e a d a f te r incubation at 35°c for 18 h. zone diameter d” 21mm was dee me d to in di ca te c ef ox it in resistance. bacterial dna extraction bacterial dna extraction was done on the 23 cefoxitin-resistant staphylococci isolates only. the dna of the representative bacterial isolates was extracted using zr fungal/bacterial dna mini prep tm kit (zymo research corporation, usa) following the manufacturer’s instruction. pcr amplification of 16s rrna, nuc and meca genes the 23 cefoxitin-resistant staphylococci eluted dna extracts were amplified for 16s rrna, nuc and meca genes by pcr using their respective primers as shown in table 1. briefly, a vol ume of 25μl pcr r ea c ti on mi xt ur e consisting of 12.5μl of pcr master mix, primers (1.25μl each), double distilled water (5μl), and dn a te mp la t e ( 5μl ) was us e d f or pcr. univ med vol. 38 no.1 primer code oligonucleotide sequence (5‘3‘) annealing temp (oc) expected amplicon size (bp) 16s rrna 16s-1 (f) 5’gtgccagcagccgcggtaa 3’ 16s-2 (r) 5’agacccgggaacgtattcac 3’ 57 886 nuc (f) 5’tcagcaaatgcatcacaaacag3’ (r) 5’cgtaaatgcacttgcttcagg 3’ 57 225 meca meca f 5’aaaatcgatggtaaaggttggc 3’ meca r 5’agttctgcagtaccggatttgc 3’ 53 532 table 1: list of primers amplified by pcr 28 ogefere, ogunleye methicillin-resistant staphylococci amp l if ic a t i on s w e r e pe r f o r me d u s in g geneamp* pcr system 9700 thermocycler (applied biosystems, usa) beginning with an initial denaturation at 94º c for 3 min followed by 30 cycles of 30 s denaturation step at 94ºc, annealing at 57º c for 30 s, extension at 72º c for 30 s, and a final 7 min extension at 72ºc followed by a hold at 4oc. the 10µl of pcr products was electrophoresed on 1% trisa c e ta te -edta a ga r os e ge l sta i n e d wi t h ethidium bromide for 60 min with 90v current and viewed under uv trans illuminator (dolphindoc plus, wealtec corporation).(18) statistical analysis statistical analysis was by the chi (x2) square test and fischer ’s exact test where appropriate using instat® software. a p value of <0.05 was deemed statistically significant. ethical approval ethical approval for this study was sought and obtained from auchi polytechnic health ethical committee, auchi with registration number: ap/hc.14/vol.1/69. results out of 400 apparently healthy students screened, 229 (57.3%) were culture positive for s. aureus. twenty-three (7.7%) of these isolates were methicillin resistant using cefoxitin disc. this comprised 8 (8.1%) males and 15 (11.5%) females. gender did not however significantly affect the distribution of mrsa (p=0.5219) (table 2). the prevalence of specific genes 16s rrna, nuc and meca among cefoxitin-resistant s . a ure us wa s 10 0 % , 1 00 % , a nd 69 . 6% a b c gender s. aureus (n,%) cefoxitin resistance (n,%) positive negative p value positive negative p value gender male female 99 (59.3) 130 (55.8) 87 (40.7) 103 (44.2) 0.553 8 (8.1) 15 (11.5) 159 (91.9) 218 (87.5) 0.5219 table 2. prevalence rate of mrsa isolated from the nares of apparently healthy students using cefoxitin disc figure 1. polymerase chain reaction of 16s rrna gene in cefoxitin-resistant staphylococci isolates using 1.0% agarose gel electrophoresis stained with ethidium bromide to identify staphylococci. left-hand lane is 100bp1517bp dna ladder (molecular marker) while nc is a no dna template control 29 univ med vol. 38 no.1 figure 3.polymerase chain reaction results for detection of meca gene from confirmed cefoxitin-resistant staphylococcus aureus isolates on 1.0% agarose gel electrophoresis stained with ethidium bromide. l is 100bp-1517bp dna ladder (molecular marker) while nc is a no dna template control figure 2. polymerase chain reaction of nuc gene in cefoxitin-resistant staphylococci isolates using 1.0% agarose gel electrophoresis stained with ethidium bromide to identify staphylococcus aureus. l is 100bp1517bp dna ladder (molecular marker) while nc is a no dna template control respectively. polymerase chain reaction results for detection of these genes can be seen in figure 1, 2, and 3 respectively. gender also did not significantly affect the distribution of meca gene among cefoxitin-resistant s. aureus as 16 (69.6%) isolates harbored the gene, in a maleto-female ratio of 6:10 (p=0.975). methicillin resistant s. aureus showed poor susceptibility to commonly available antibiotics a s 0% s u sc e pt i bi l i t y wa s o bs e r ve d f or ceftazidime, cefuroxime, ceftriaxone, cloxacillin and amoxicillin-clavulanate (augmentin). a poor level of susceptibility was recorded among the mrsa namely to erythromycin (26.6%), the most active antibiotic was ofloxacin (60.9%). discussion the prevalence rate of mrsa in this study was 5.8% (using cefoxitin disc). this finding is 30 ogefere, ogunleye methicillin-resistant staphylococci in agreement with the work of mahde et al.(19) which reported community-associated mrsa among healthy university students (4.2%). also, okwu et al.(10) reported a prevalence of 10.8% of community-associated mrsa in apparently healthy school children in okada, edo state, nigeria. a study carried out in colombia by bettin et al. showed that the mrsa carriage status by medical students in their clinical rotations was 1.6%.(20) a study in thailand reported 1% prevalence among university students while shadi recorded 6.7% mrsa colonization of nares among medical students in jeddah, saudi arabia.(21, 22) on the contrary, eke et al.(11) reported 41% mrsa in apparently healthy university students in ekpoma, edo state, nigeria. the difference in prevalence rates recorded could be attributed to method of identification of s. aureus isolates used to detect mrsa. most other studies in nigeria reported above used phenotypic identification as against the genotypic method used in this study. ayeni and odumosu had reported 85% misidentification rate of other microorganisms as staphylococus aureus in southern nigeria when comparing phenotypic methods with genotypic (16s rrna).(13) po l yme r a s e c h a in r e a c t i o n ( pcr) amplification of 16s rrna genes to identify staphylococci among the 23 mrsa of apparently healthy subjects was positive (100%). this is in line with work of degaim et al.(23) where all isolates were positive for both 16s rrna genes and meca genes (100%). ayeni and odumosu similarly showed the reliability of this genotypic test when compared with phenotypic tests in identification of s. aureus.(13) this current study results agreed with makgotlho,(24) who showed that all isolates 97/97 (100%) have 16s rrna gene while meca gene was detected in 96/97 (99%) of the mrsa isolates, the 1/97(1%) which did not show the presence of meca gene was, however, phenotypically identified as mrsa. in this study, the pcr amplification of nuc gene to detect staphylococcus aureus among mrsa showed 100% positive results. according to vremera et al.(25) the nuc gene in s. aureus encodes the ther monuclease e nzyme and amplification of nuc gene is a potential method for rapid diagnosis of s. aureus infection. theref ore, the primer used in this study confirmed the ability of pcr as a fast and reliable method for detection of the nuc gene to identify s. aureus strains. also, of the 23 mrsa (using phenotypic method), 16 (69.6%) were confirmed to be meca positive by the pcr method. this is also similar to the work of ibadin et al. (9) where sensitivity and specificity of meca gene in detection of mrsa was 89.5% and 90.3% respectively. phenotypically methicillin-resistant strains without meca gene and methicillin sensitive strains harboring meca gene have been shown in previous studies.(13) in this study, phenotypic and genotypic methods for detection of mrsa were used. the results of this study showed that 5.8% and 4.0% of s. aureus isolates were recognized as mrsa by cefoxitin disc diffusion test and pcr method respectively. considering that detection of the meca gene by pcr method is a gold standard method for identifying methicillin-resistance in s. aureus isolates, therefore the prevalence of mrsa in this study was 4.0%. in this study, a poor level of susceptibility was recorded among the mrsa namely to er ythromycin (2 6.6 %), cl oxa cill in (0% ), augmentin (0%), cefuroxime (0%), ceftriaxone (0%) and ceftazidime (0%). this implies that mrsa isolates are generally resistant to betalactam antibiotics as previously reported.(9,10) however, some mrsa isolates were susceptible to gentamicin (47%) and ofloxacin (60.9%). the sensitivity to the non-beta-lactam antibiotics was also similar to previous findings and it is recommended that non-beta-lactam antibiotics should be the preferred drugs for the treatment o f c o mmu ni t y-a cq ui r e d ( ca) mrsa infections.(8, 9) the moderately high resistance (52.2%) recorded for gentamicin was similar to the report on s. aureus from clinical samples (44.4%).(9) ofloxacin (with sensitivity of 60.9%) 31 appeared to be the best antibiotic of choice for the treatment of mrsa infections in auchi, edo state and its surrounding communities. a limitation of this study is hinged on the fact that only phenotypically identified mrsa strains were subjected to molecular analysis. more far reaching recommendations for routine microbiological practice may have been reached if all phenotypically identified s. aureus were screened for the nuc gene. the study however holds important clinical implications as the role of carriers of mrsa is continually being explored in op portun istic infec tions and nosocomial outbreaks. further studies that explore nasal carriage of hospital-acquired and community-acquired mrsa strains among a ppa r e ntl y h ea l thy s u bj e ct s may de e pe n understanding of the superbug-mrsa in our region. conclusions t hi s p r e se n t st u dy e s ta bli s he d t he phenotypic and genotypic prevalence rates of mrsa among apparently healthy subjects in the auchi polytechnic to be 5.8% and 4.0%, respectively. it showed that the pcr technique is a very useful tool in detecting and identifying mrsa using 16s rrna, nuc and meca genes. in comparison with conventional methods, genotypic analysis of these three genes is still t he go l d s t a nd a r d f o r d e t e ct i n g m rsa. ofloxacin was found to be most effective antibiotic against mrsa. conflict of interest the authors declare no conflicts of interest acknowledgement the author s are gratef ul to all study participants for their cooperation and to the management of auchi polytechnic, auchi, for the approval to carry out this research. univ med vol. 38 no.1 contributors hoo took part in the conception and design of the study. lao took part in data generation. hoo and lao contributed to data analysis and interpretation. both authors participated in writing and approving the final draft of the manuscript. references 1. peng q, hou b, zhou s, et al. staphylococcal cassette chromosome mec (sccmec) analysis and antimicrobial susceptibility profiles of methicillinresistant staphylococcus aureus (mrsa) isolates in a teaching hospital, shontou, china. afr j microbiol 2010;4:844-8. 2. rongpharpi sr, hazarika nk, kalita h. the prevalence of nasal carriage of staphylococcus aureus among healthcare workers at a tertiary care hospital in assam with special reference to mrsa. j clin diagn res 2013;7:257–60. doi: 10.7860/jcdr/2013/4320.2741. 3. garza-gonzález e, morfin-otero r, llaca-diaz jm, et al. staphylococcal cassette chromosome (sccmec) in methicillin-resistant coagulase negative staphylococci. a review and the experience in a tertiary-care setting. epidemiol infect 2010;138:645–54. doi: https://doi.org/ 10.1017/s0950268809991361 4. treesirichod a, hantagoo s, prommalikit o. nasal carriage and antimicrobial susceptibility of staphylococcus aureus among medical students at the hrh princess maha chakri sirindhorn medical center, thailand: a follow-up study. j infect public health 2014;7:205-9. doi: 10.1016/ j.jiph.2012.12.004. 5. olowe oa, kukoyi oo, taiwo ss, et al. phenotypic and molecular characteristics of methicillin-resistant staphylococcus aureus isolates from ekiti state, nigeria. infect dr resist 2013;6:87–92. doi: http://dx.doi.org/10.2147/ idr.s48809 6. nwankwo bok, sale a, magagi a, et al. methicillin-resistant staphylococcus aureus and their antibiotic susceptibility pattern in kano, nigeria. afr j clini 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in cartagena, colombia. brazil j infect dis 2012;16:329–34. doi: 10.1016/j.bjid.2012. 06.017. 21. kitti t, boonyonying k, sitthisak s. prevalence of methicillin-resistance staphylococcus aureus among university students in t hailand. southeast asian j trop med pub hea 2011;42: 1498-504. 22. zakai sa. prevalence of methicillin-resistant staphylococcus aureus nasal colonization among medical students in jeddah, saudi arabia. saud med j 2015;36:807-12. https://doi.org/10.15537/ smj.2015.7.11609. 23. degaim zd, shani ws, hamim ss. in virulence factors of methicillin resistant staphylococcus aureus (mrsa) isolated from burn patients. inter j curr microbiol appl sci 2015;4:898-906. 24. makgotlho pe, kock mm, hoosen a, et al. molecular identification and genotyping of mrsa isolates. fems immun med microbiol 2009;57:104–15. 25. vremera t, iancu ls, logigan c, et al. optimization of triplex real time pcr for detecting staphylococcus aureus meca, pvl and nuc genes. roman arch microbiol immun 2011;70:69– 73. c:\users\jurnal fkusakti\docume 150 *infertility & reproductive health research center, shahid beheshti university of medical sciences, tehran, iran **shohadaye tajrish hospital, shahid beheshti university of medical sciences, tehran, iran ***besat hospital, aja university, tehran, iran correspondence: fatemeh salehi infertility & reproductive health research center, shahid beheshti university of medical sciences, tehran, iran email: dr.salehi1389@gmail.com date of first submission, may 20, 2018 date of final revised submission, august 9, 2018 date of acceptance, august 10, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina cervical consistency index is a valid predictor of preterm birth in low-risk pregnant women parichehr pooransari*, fatemeh salehi*, maryam afrakhteh**, masoumeh mirzamoradi**, soraya saleh gargari**, and omid ghatreh samani*** background preterm birth (ptb) remains a global problem associated with perinatal morbidity, including low birth weight, growth retardation and irreversible damage to the nervous system. the objective of this study was to determine the predictive value of cervical consistency index (cci) to indicate the occurrence of ptb in mid trimester screening of low risk pregnant women. methods this was a prospective study conducted on low-risk pregnant women at 14-28 weeks of pregnancy. the cervical length (cl) and ap cervical diameter were measured and the cci determined according to the formula ap2/ ap1*100. the roc curves were drawn according to gestational age and the sensitivity and the specificity were calculated for optimal cut-off for 1st, 5th, 10th, 21th, and 24th centiles of cci. the inter-observer agreement was validated by interclass correlation coefficients (icc). results a total of 149 participants were enrolled in this study. among them, 12.08% had ptb (<37weeks) and 87.92% had normal birth. mean cci for all subjects was 68.60%, but was lower in ptb (48.9%). the best cutoff for predicting ptb based on cci was 59.46% with 99.18% sensitivity and 85% specificity. the auc for cci for prediction of ptb <37weeks was 0.936. there was no inter-observer difference in measurement of cci and cl (icc values for cci and cl were 0.997 and 0.990 respectively). conclusion in women with normal cl the cci could predict ptb in the second trimester in low risk women. so, cci is a valid predictor of ptb in low risk women. keywords: pre term birth, cervical consistency index, low risk pregnancy doi: http://dx.doi.org/10.18051/univmed.2018.v37.150-158 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cite this article as: pooransari p, salehi f, afrakhteh m, et al. cervical consistency index is a vali d pr edic tor of preterm birth in low-risk p regn ant women. univ med 2018;37:150-8. doi: 1 0 . 1 8 0 5 1 / un i vm ed . 2 0 1 8 . v 3 7 . 1 5 0 1 5 8 151 univ med vol. 37 no.2 introduction preterm birth (ptb) is defined as a delivery of an infant before 37 weeks of pregnancy.(1) according to the who, each year about 15 million infants are born preterm and this number is rising every year, despite some progression in the medical field.(2) the overall incidence of ptb ranges from 5 to18% of infants born annually.(2) so this complication becomes a major concern in gynecology and obstetrics. preterm birth has been divided into three categories: extremely preterm (less than 28 weeks), very preterm (28-32 weeks) and moderate preterm (32-37 weeks).(2,3) preterm birth is responsible for neonatal morbidity and complications. these c o mp l i c a ti on s a r e du e t o t he i mma t u r e development of organs like the lungs, which is associated with respiratory distress syndrome (rds), hence, the neonates may have cerebral p a l sy, visi o n p r o bl e ms , ga s t r oi n t e s t in a l complications, etc.(4,5) the neonatal mortality rate has been decreasing in recent years, but ptb contributes to the death of 1.5 million infants annually.(6) the mortality rate increases with decreasing gestational age.(7) more than 75% of preterm births occurs during the 32th to 36th weeks of pregnancy. (8 ) some risk factors including; the maternal age at delivery, weight, the introduction of assisted reproductive technology (art), have increased the incidence of ptb recently.(9) risk assessment like; history o f pr e vi ous a b or t io n o r ptb, m ül l er ia n malformation, and cervical conization, multiple gestation, bleeding in the second trimester, and l ow pr e gna nc y we i ght , a r e some of t h e predispositional factors for the occurrence of ptb in the current pregnancy, although their sensitivity is not reliable.(10) in normal term, the cervix becomes soft but the uterus is closed until term, but in preterm birth the cervix shortens.(11) cervical length (cl), which is measured via transvaginal ultrasound (tvu), is a good predictor for ptb, especially in symptomatic women,(12) although the accuracy of this technique is limited in asymptomatic women.(13) it should be noted that about 50% of ptb occurs in asymptomatic women.(14) hence, despite determination of the cl index for the screening of ptb the rate has not decreased yet, especially in low-risk women.(15) in the study of parra-saavedra et al.(16) an ultrasonographic marker was investigated to me a s ur e ce r vi x c o ns i s t e nc y t hr ou gh o ut pregnancy quantitatively by tr ansvaginal ultrasonography. recently, a new technique based on measurement of the anteroposterior diameter of the cervix before and after cervical compression and calculating the cervical consistency index (cci), has been administered for prediction of ptb.(16) it is shown that cci is lower in women with preterm delivery and is a l s o d e c r ea se d i n t h e t h i r d t r ime s t e r o f pregnancy. so, it could be a good indicator for ptb in low-risk women.(16) previous studies conducted on low risk women demonstrated that cci was the better indicator of ptb in the second trimester, although women with cl under 25 mm were enrolled in previous studies where cl<25mm is the predictor of ptb. in the current research, women with cl<25 mm were excluded, because medical intervention is needed in order to prevent complications such as ptb. so for the first time we evaluated normal pregnant women who were referred for routine screening and the association of cci with the occurrence of ptb in this group was evaluated, and compared with the cervical length.(17) although cci perf ormed better than sonographic cl in predicting ptb, due to the still limited predictive capacity of these two measurements, other tools are needed to better identify women at increased risk of preterm delivery.(17) both cci and cl measurements were significantly reduced in the high-risk compared with the low risk pregnancies. however, the diagnostic accuracy of both measurements was better in the low-risk population than in the highrisk population.(18) 152 the novelty of the study is that in the previous studies, in addition to healthy pregnant women with normal cervical length, pregnant women with reduced cervical length were studied. the present study did not include women with cervical length lower than 25, who are at risk for early delivery. in addition, the cervical congestion index in healthy pregnant women with normal cervical length was studied. because of the inclusion of normal cervical length and early delivery in the healthy pregnant women group, the present study is better for confirming the greater efficiency of cci as compared to cervical length in predicting early delivery. the objective of this study was to determine the predictive value of cervical c o nsi s t e nc y in d e x (cci) t o i n di c a t e t he occurrence of ptb in mid trimester screening of low risk pregnant women. methods research design this was a prospective study conducted in shohadye tajrish hospital, tehran, iran from april 2017 to march 2018. research subjects the sample size was determined according to cazanas et al.(19) (effect size2=0.082) and the gestational age and cci in linear multiple regression model for prediction of ptb were considered. the optimum sample size was 140. singleton pregnant women with low risk of preterm labor in the 14-28 weeks of pregnancy, including women who do not have the risk factors for preterm labor. the exclusion criteria were multiple pregnancies and use of drugs for the prevention of preterm labor (such as non-steroidal anti-inflammatory dr ugs, progesterones, magnesium sulfate, and c alc ium channel blockers), use of cerclage pessary, cervical length lower than 20mm that requires the intervention to prevent preterm labor. women with one of the preterm labor risk factors (previous preterm labor, loss of pregnancy due to cervical incompetency), cervical cone biopsy, and presence of molar anomalies in the reproductive system, were excluded from the study. measurements women with gestational ages in the range o f 14 -2 8 w e e ks u nd e r w e nt t ra ns va gi n a l sonography examination. the cervical length and anterioposterior diameter were measured twice during pregnancy and the cci index was calculated. gestational age was calculated according to reliable last menstrual period recall data or first trimester sonographic examination. ultrasound examination the sonographic examination was carried out t wic e f or eac h p ar tic ipa nt usi ng ge voluson e8 (ge healthcare ultrasound, milwaukee, wi, usa) ultrasound machine which is equipped with a transvaginal probe. cervical length, anterioposte rior cervical diameter , gestational age, maternal weight, body ma s s i nd ex ( bmi) , a ge a nd pa r i ty we r e determined during the examination. determination of cervical consistency index (cci) in order to determine the cci, the following steps were performed. cervical length was measured according to the standard technique described in a previous study.(17) the measurement of anterioposterior cervical diameter was carried out with an empty bladder and the cervical length was measured without any pressure. then the midpoint of the line running along the longitudinal cervical axis was marked as (c/2), then a line perpendicular to the longitudinal cervical axis through the point c/2 was drawn from the anterior lip to the posterior lip of the cervix. this length was designated the ap1 diameter. then pressure was applied with the vaginal probe progressively until it reached the highest pressure at which there was no further shortening of the anterioposterior cervical diameter. then the ap2 diameter was measured in a same way as described for ap1 pooransari, salehi, afrakhteh, et al cervical consistency index and preterm birth 153 univ med vol. 37 no.2 a b c (figure 1). to calculate the cci the ap2 was divided by ap1 then multiplied by 100: cci= (ap2 d ap1) ×100 the measurement were performed twice and the lowest number was used for statistical analysis and inter-observer agreement.(16) the patients were followed up to end of pregnancy and the outcome and date of delivery were recorded. statistical analysis all data were analyzed with spss v.22 (ibm copr, armonk, ny). the gestational age in the second trimester (14-28 weeks) was evaluated to determine the ability of cci to predict the ptb (<37weeks). a multivariate logistic regression model for cci and cl was constructed to assess the independent relation of each variable with ptb. the relationship between quantitative va r i a b l e s w a s a ss e s s e d wi t h pe a r s on ’s correlation coefficient test. distribution of cci and cl were analyzed in different percentiles according to gestational age at scan time. receiver operating characteristic (roc) curves were drawn for cci as predicting variable. sensitivity, specificity, positive predictive value (ppv), negative predictive value (np v), as well as positive a nd negat ive likelihood ratios (lr+ and lr-) were analyzed at 95% confidence interval to predict the ptb <37 weeks and the optimal cut-off point based on the roc curves was calculated. all data obtained from the roc curves demonstrate the diagnostic ability of cci and cl to predict preterm birth. the area under the roc curves (aus) also was calculated.(18) reliability of measurement the agreement between two measurements determined by different observers is called interobserver agreement. so, each participant was examined twice at one-hour interval by two observers. the differences were calculated using a bland and altman plot and intraclass correlation coefficient (icc) (20) limits of agreement (mean differences ± 1.96) (standard deviation*1.96) ± mean) were calculated with the one-sample t-test and the systematic bias between two measurements was calculated at 95% ci for mean difference. ethical clearance each participant signed an informed consent letter and the study was approved by the ethics committee of shohadye tajrish in f e r ti l i ty re s e a r c h ce n te r ( u nd e r no . ir.sbmu.retech.rec.1397.306). figure 1. ultrasound image of cervix before (a) and after maximal compression with probe (b). the anterioposterior diameters (ap1, ap2) and cervical length (cl1, cl2) were measured in each position. the cci index was determined from ap2/ap1*100 for each participant 154 results about 200 pregnant women, who were referred to shohadye tajrish hospital, tehran, iran for routine examination, were enrolled in this study. a total of 51 women were excluded from the study due to several factors as follows. about 12 women were excluded due a maternal indication or a iatrogenic disorder before the 37th week of pregnanc y, such as fetal growth retardation, preeclampsia, non reassuring fetal assessment, 3 women with a history of multiple pregnancy, 8 women with a history of abortion (20 wee ks), and 28 women receiving progesterone for prevention of ptb. participants with cervical length under 25mm and having a non-horizontal cervix were also excluded from the study. all participants were followed up to delivery. about 149 pregnant women who were referred for a routine check-up in the second trimester were enrolled in this study. among 149 pregnancies, 18 cases had ptb before 37 weeks (12.08%) and 131 cases had a normal birth (87.92%). the prevalence of normal delivery was relatively higher than that of cesarean section (53.7% vs. 46.3%).the characteristics of participants in the normal and preterm birth groups are summarized in table 1. there was no statistical difference in maternal age among women who had a normal birth and those with ptb(p>0.05). sonography assessment results demonstrated that the mean cervical consistency index was 68.60% (71.32 to 48.9%) in which the percentage of cci was lower in ptb (48.9% vs. 71.32%) (p=0.00) (table 1). in order to determine the best cut-off value of cci, the roc curves were used and the specificity, sensitivity, positive and negative predictive values and positive and negative likelihood ratio for best cut-off values to predict the ptb before 37 weeks were calculated. the best cut-off for predicting the ptb based on cci was 59.46% (sensitivity 99.18%, specificity characteristic n = 149 normal birth (n=131) preterm birth (n=18) p maternal age (years) 28.93 (16-44) 29.4(17-44) 25.2 (16-36) >0.05 bmi before pregnancy 25.06 (20.2-35.56) 25.1 (20.2-35.56) 24.17 (20.3-29.62) >0.05 weight gain (kg) 14.49 ( 4-30) 14.70 (4-30) 12.94 (6-22) >0.05 gestational age at scan (weeks) 19.42 (14-27) 19.29 (14-27) 20.3 (17-28) >0.05 gestational age at delivery (weeks) 38 (31-41) 38.47 (36-41) 34.9 (31-36) 0.000 infant weight (g) 3139 (1870-4100) 3218 (2500-4100) 2563 (1870-3640) 0.031 mode of delivery noninvasive delivery 79 (53.7%) 67 (51.1%) 12 (66%) ns cs 69 (46.3%) 63 (48.1%) 6 (33%) ns cci (%) 68.60 (34.86-93.02) 71.32 (50-93.02) 48.9 (34.8-64.61) 0.000 cl (mm) 32.89 (20.05-44.8) 33.28 (24.1-44.8) 30.63 (20.05-36.50) >0.05 table 1. distribution of characteristics of patients enrolled in the study (n=149) bmi: body mass index; cs: cesarean; cci; cervical consistency index; cl: cervical length; *one case was excluded due to abortion in 17th week of gestation cut-off* sensitivity specificity lr+ lrppv npv 47.75 p(1) 1 (100%) 50% 2 0.5 21.56 100 54.6 p(5) 0.969 ( 99.04%) 65% 2.768571 0.361197 27.99 99.79 57.32 p(10) 0.868 (99.131%) 70% 5.526667 0.180941 31.22 99.82 59.46 p(21)** 0.829 (99.18%) 85% 5.526667 0.180941 47.61 99.87 62.675 p(24) 0.775 (99.23%) 95% 15.5 0.064516 73.6 99.88 table 2. cervical consistency index cutoff based on roc curve to predict ptb before 37 weeks value lower than cutoff indicates high risk;**optimal cutoff based on roc curve corresponds to 21th centile lr+, positive likelihood ratio, lr-, negative likelihood ration, ppv, positive predictive value, npv, negative predictive value, p1, 1st centile; p5, 5th centile; p10, 10th centile; p21, 21th centile; p24, 24th centile pooransari, salehi, afrakhteh, et al cervical onsistency index and preterm birth 155 univ med vol. 37 no.2 85%), which according to the distribution of cci in gestational ages at scan was located in percentile 21 (table 2). the roc curves for cci are shown in figure 2. auc for cci in relation to predicting the ptb before 37 weeks was 0.936 (95%ci 0.891-0.982;p=0.000). the specificity, sensitivity, positive and negative predictive values and positive and negative likelihood ratio for cci and cut-offs of cci in regard to predicting the. to ensure the reliability of the cci and cl measurements by one specialist, the icc test was performed. the bland-altman plots (figure 2) illustrate that there are no inter-observer differences in the measurement of cci and cl. the inter-observer icc values for cci and cl were 0.997 (95%ci, 0.996-0.998) and 0.990 (95%ci, 0.993-0.997) respectively. the interobserver agreement reliability of the cci and cl (data was not shown). discussion the purpose of this study was to determine the diagnostic value of the cervical consistency index (cci) in the prediction of preterm birth (ptb) (under 37 weeks) in low-risk pregnant women. our results indicated that the cci could be a reliable value for predicting ptb in a lowrisk population in the second trimester. figure 2. receiver operating characteristics (roc) curve for cervical consistency index(cci) for prediction of ptb before 37 weeks auc: area under roc curve. optimal cut-off based on roc curve 156 this was a first study in iran to assess the cci in low-risk pr egnant wome n using transvaginal sonogra phy. evaluating the percentage of cci in the second trimester (1428 weeks), demonstrated that low cci in this period of pregnancy may lead to preterm labor (delivery before 37 weeks).(16) in the current study, the ptb rate was 12.8% which was higher than that of a previous report from iran,i.e. 5.6 %.(21) the ptb rate in the united states is about 12%, which is similar to the current study.(22) preterm birth (ptb) is one of the major causes of neonatal mortality in the population.(23) according to the who the ptb rate ranges from 5 to 18% and about 5-7% of infants die due to ptb in developed countries.(2) this indicates that the ptb rate is higher in developing countries and because the current study was just about one hospital in tehran, some socio-economic differences may also have an effect on the prevalence of ptb.(24) in our study, in order to predict the ptb risk, the cervical length was determined.(18) however, a cl of less than 25mm indicates the need for medical intervention in order to prevent the complications of ptb.(10) in low-risk women with a cl higher than 25mm no medical intervention is necessary, but as demonstrated in this study the rate of ptb in pregnant women with cl above 25mm was noticeably high (12%). so it is clear that cl could not be a reliable factor for predicting ptb. according to a previous study, changes in cervical length may be a late outcome and softness of the cervix occurs be fore shortening, and water concentration during the ripening process may not cause any clear symptoms and could not be detected in the first trimester.(25) the assessment of cervical consistency by pelvic examination did not have any clinical value for predicting ptb. for the first time, cervical consistency was measured by transvaginal sonography. the cci index in our study was lower in women with preterm delivery than in those with ter m del ivery, as wa s a lso demonstrated in other studies. the low cervical consistency index indicates weakness of the cervix and is directly related to preterm birth.(26) in order to determine the predictive value of cci for detection of ptb, the cutoff value is needed. according to our study, the best cutoff for predicting ptb before 37 weeks was 59.46% with 85% specificity, 99.18% sensitivity, lr+ 5.52, and lr0.18, which is connected with percentile 21 of the gestational week. the study by baños et al.(18) found the best cutoff to predict the ptb before 37 weeks in percentile 20 (corresponding to cci 64.6 %, sensitivity 72%, specificity 61.2% lr+1.9 and lr0.4). in order to confirm the inter-observer differences in the measurement of cci and cl, the icc value was calculated and was 0.99 in both measurements. so the measures obtained were reliable and sufficient for clinical use in both cci and cl. the main limitation of our study is the limited number of ptb (n=18) cases, due to the low risk population selection of the study. however, in the current study, in which only low risk people were selected and women with cl under 25mm were excluded from the study, a good relation between cci and ptb in the low risk population has been obtained, so indicating the validity of the current study. this study was conducted in one hospital, but in order to confirm the reliability of our results, it is better to repeat it in other centers with large populations. finally, due to lack of persons with expertise for measuring the anterioposterior diameter of cervix and determining the cci, we did not have inter-observer agreement. with regard to the increase of the expert people in the measurement of the cervical compression index, it is better that the present study be performed in a larger and multi-centre study by a number of people, allowing conclusions to be made on interobserver reliability. conclusion determination of cci in asymptomatic pregnant women could prevent occurrence of pooransari, salehi, afrakhteh, et al cervical consistency index and preterm birth 157 univ med vol. 37 no.2 pt b a n d me di c a l i mpl i c a t i o n f o l l ow in g complications in the newborn, hence cci should be calculated in routine sonography examination. acknowledgement we thank our colleagues from shohadaye tajrish hospital for their assistance with sonograpghic examination and we are also immensely grateful to dr hanieh rezaee for her support on manuscript preparation. conflict of interest all authors declare that they do not have any conflict of interest. contributors pp contributed to the basic concept and design of the study. fs and ma contributed to writing the manuscript and performing the experiment. mm and ssg contributed to sample preparation and data collection. ogs contributed to the statistical analysis. all authors read and approved the final manuscript. references 1. kemp mw. preterm birth, intrauterine infection, and fetal inflammation. front immunol 2014;5:574. doi: 10.3389/fimmu.2014.00574. 2. world health organization. preterm birth. geneva: world health organization;2017. 3. blencowe h, cousens s, oestergaard mz, et al. national, regional, and worldwide estimates of preterm birth rates in the year 2010 with time trends since 1990 for selected countries: a systematic analysis and implications. lancet 2012; 379:2162-72. doi: 10.1016/s0140-6736(12)60820-4. 4. saigal s, doyle lw. an overview of mortality and sequelae of preterm birth from infancy to adulthood. lancet 2008;371:261-9. doi: 10.1016/ s0140-6736(08)60136-1. 5. hafstrom m, kallen k, serenius f, et al. cerebral palsy in extremely preterm infants. pediatrics. 2018;141:e20171433. doi: 10.1542/peds.2017-1433. 6. lawson mj. the role of inflammation in the pathogenesis of preterm birth [thesis].cincinnati, ohio: university of cincinnati; 2017. 7. gargari ss, kashanian m, zendedel h, et al. survival and risk factors of extremely preterm infants (<28 weeks) in the three iranian hospitals. acta medica iranica 2018;56:181-8. 8. simmons le, rubens ce, darmstadt gl, et al. preventing preterm birth and neonatal mortality: exploring the epidemiology, causes, and interventions. semin perinatol 2010;34:408-15. 9. tamura n, hanaoka t, ito k, et al. different risk factors for very low birth weight, term-small-forgestational-age, or preterm birth in japan. int j environ res public health 2018;15:369. doi: 10.3390/ijerph15020369. 10. koullali b, oudijk ma, nijman ta, et al. risk assessment and management to prevent preterm birth. semin fetal neonatal med 2016;21:80-8. doi: 10.1016/j.siny.2016.01.005. 11. myers km, feltovich h, mazza e, et al. the mechanical role of the cervix in pregnancy. j biomech 2015;48:1511-23. doi: 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samani ro, et al. prevalence and determinants of preterm birth in tehran, iran: a comparison between logistic regression and decision tree methods. osong public health res perspect 2017;8:195-200. doi: 10.24171/j.phrp.2017.8.3.06. 22. martin j, hamilton b, ventura s, et al. births: final data for 2010. natl vital stat rep 2012;61:1–72. 23. liu l, oza s, hogan d, et al. global, regional, and national causes of child mortality in 2000–13, with projections to inform post-2015 priorities: an updated systematic analysis. lancet 2015;385: 430-40. doi: 10.1016/s0140-6736(14) 61698-6. 24. goldenberg rl, culhane jf, iams jd, et al. epidemiology and causes of preterm birth. lancet 2008;371:75-84. doi: 10.1016/s0140-6736(08)600744. 25. donoghue d, lincoln d, morgan g, et al. influences on the degree of preterm birth in new south wales. aust n z j public health 2013;37: 562–7. 26. myers km, feltovich h, mazzad e, et al. the mechanical role of the cervix in pregnancy. j biomech 2015;48:1511–23. doi: 10.1016/j.jbiomech. 2015.02.065. pooransari, salehi, afrakhteh, et al cervical consistency index and preterm birth oktavianus 99 *department of biology, faculty of mathemathics and science, semarang state university **department of pathological anatomy, medical faculty, diponegoro university ***department of parasitology, medical faculty, diponegoro university ****department of clinical nutrition, medical faculty, diponegoro university correspondence dr. dr. nugrahaningsih wh, m.kes department of biology, mathemathics and science faculty, semarang state university email: nugrahaningsihwh@yahoo.com univ med 2013;32:99-107 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background apoptosis plays an important role in tumorigenesis. induction of apoptosis is a strategy for developing cancer therapy. in vitro study found that andrographolide isolated from andrographis paniculata has anticancer activity by an apoptotic mechanism in cancer cell lines. the aim of the present study was to prove the effect of andrographis paniculata extract administered orally on apoptosis of mammary adenocarcinoma in c3h mice. methods this study was of post test randomized control group design. twenty four c3h mice with transplanted mammary adenocarcinomas were divided into four groups. to three groups andrographis paniculata extract was administered orally for 14 days, at doses of 5, 10 and 15 mg/day, respectively, whereas to the control group no andrographis paniculata extract was administered. on day 15 the mice were terminated. the mammary adenocarcinomas were examined by the terminal deoxynucleotide transferase dutp nick end labeling (tunel) method. the values of the apoptotic index were expressed as mean±sd and analyzed using anova and pearson’s correlation test. results the mean apoptotic index values differed significantly among the experimental groups (p=0.001). the highest value was found in the group receiving andrographis paniculata extract 15 mg/day, while the lowest was in the control group, the values being significantly correlated (r=0.974). conclusions oral administration of andrographis paniculata extract induced apoptosis in c3h mice with mammary adenocarcinoma key words: andrographis paniculata, apoptosis, mammary adenocarcinoma, mice andrographis paniculata extract induced apoptosis of adenocarcinoma mammae in c3h mice nugrahaningsih*, sarjadi**, edi dharmana***, and hertanto wahyu subagio**** 100 nugrahaningsih, sarjadi, dharmana, et al andrographis paniculata induced apoptosis ekstrak andrographis paniculata menginduksi apoptosis adenokarsinoma mamma pada mencit c3h latar belakang apoptosis mempunyai peran penting pada tumorigenesis. induksi apoptosis merupakan strategi yang dikembangkan untuk terapi kanker. andrografolid yang diisolasi dari tanaman andrographis paniculata memiliki aktivitas antikanker melalui mekanisme apoptosis sel line kanker pada penelitian in vitro. penelitian ini bertujuan untuk menilai pengaruh ekstrak andrographis paniculata yang diberikan secara oral terhadap apoptosis sel adenokarsinoma mamma mencit c3h. metode penelitian dilakukan dengan desain post test randomized control group design. sebanyak 24 mencit yang telah tumbuh adenokarsinoma mamma dibagi menjadi 4 kelompok. diberikan ekstrak andrographis paniculata secara oral dengan variasi dosis 5, 10 dan 15 mg/hari selama 14 hari. satu kelompok sebagai kontrol tidak mendapat ekstrak. hari ke 15 mencit diterminasi, jaringan kanker diambil untuk duperiksa apoptosisnya dengan metode terminal deoxynucleotide transferase dutp nick end labeling (tunel). data dihitung rerata dan penyimpangannya dan dianalisis dengan uji beda anova serta uji korelasi person. hasil terdapat perbedaan rerata indeks apoptosis antara keempat kelompok perlakuan (p=0,001). apoptosis terbanyak didapatkan pada mencit yang mendapat ekstrak 15 mg/hari dan paling sedikit pada mencit kontrol dan berhubungan secara bermakna (r=0,974). kesimpulan pemberian ekstrak andrographis paniculata secara oral dapat menginduksi apoptosis sel adenokarsinoma mamma pada mencit c3h. kata kunci: andrographis paniculata, apoptosis, adenocarcinoma mammae, mencit abstrak introduction apoptosis is a mechanism of cell death for the purpose of maintaining a stable cell population and plays an important role in tumorigenesis. inhibition of apoptosis results in uncontrolled growth, such as occurs in the development of malignancy. apart from influencing growth, apoptosis also facilitates cancer metastasis. this is through the inhibition of a type of apoptosis called anoikis [celld e t a c h m e n t i n d u c e d a p o p t o s i s ] , w h i c h normally eliminates cells that are not in their appropriate environment/extracellular matrix. inhibition of the apoptotic process is also c o n n e c t e d w i t h r e s i s t a n c e t o b o t h chemotherapy and radiotherapy.(1) the occurrence of apoptosis, which is not accompanied by an inflammatory reaction, is a n i m p o r t a n t p a r t o f c a n c e r t h e r a p y. apoptosis is responsible for a reduction in the mass of solid tumors. induction of apoptosis is one of the strategies has been developed for cancer therapy through manipulation of the 101 genes and proteins responsible for apoptosis, as well as through manipulation of inhibitory apoptosis protein (iap).(2) breast cancer is a type of malignancy frequently found in women throughout the world, and ranks second as a cause of death from cancer. worldwide, more than 1.2 million women are diagnosed with breast cancer annually.(3) each year 250,000 new cancer cases are found in europe and around 175,000 in america. in 2005 there were 40,870 deaths from breast cancer in america, comprising 40,410 women and 460 men. according to data from the who in 2008, 460,000 of deaths are caused by breast cancer.(4) the department of health of the republic of indonesia stated that the number of breast cancer patients in indonesia ranks first among malignancies. in the year 2004, a total of 29,740 b r e a s t c a n c e r c a s e s w e r e r e p o r t e d f r o m hospitals, while in 2005 16,671 cases were r e p o r t e d , c o n s i s t i n g o f 8 , 8 2 9 c a s e s o n ambulatory care and 7,844 hospitalized ones. in 2006 the number of hospitalized cases increased to 41,736.(5) standard treatments of breast cancer according to who guidelines(3) are surgery, radiotherapy, cytostatic drugs and hormonal therapy. these treatments do not yet yield satisfactory results, as indicated by the high mortality rate among breast cancer patients. in order to obtain better therapeutic results, herbal preparations have been used. sambiloto (andrographis paniculata) is one of the traditional medications widely used i n a s i a n c o u n t r i e s . a q u e o u s e x t r a c t s o f andrographis paniculata contain alkaloids, amino acids, flavonoids, glycosides, saponins and tannins.(6) andrographis paniculata has various activities, viz. gastroprotective,(7) hepatoprotective,(8,9) antiviral, antipyretic, i m m u n o s t i m u l a t o r y a n d a n t i c a n c e r. ( 1 0) isoandrographolide, 3,19-isopropylideneandrographolide and 14-acetylandrographolide isolated from andrographis paniculata have tumor suppressant effects.(11) andrographolide possesses anticancer activity through an apoptotic mechanism on hela cancer cells with an ic 50 of 109.90 µg/ml,(12) and can also induce apoptosis of the td-47 human breast cancer cell line. (13) an aqueous extract of andrographis paniculata applied to cell cultures of mammary adenocarcinoma from c3h mice showed increased apoptosis starting from a concentration of 1 mg/l.(14) all three of the aforementioned studies used acridine orange staining to identify apoptotic cells, which is however less sensitive than the terminal deoxynucleotide transferase dutp nick end labeling (tunel) method.(15) the previous studies were in vitro studies, therefore oral administration of andrographis paniculata extract may yield differing results, because of the roles of the microenvironment a n d t h e p r o c e s s e s o f d r u g a b s o r p t i o n , d i s t r i b u t i o n , a n d m e t a b o l i s m . t h e microenvironment influences the growth of cancer tissue and is associated with therapeutic success.(16) fibronectins in the extracellular matrix can induce apoptosis, thus the different result may found between in vitro and in vivo study.(17) the in vitro studies demonstrating the potential of andrographis paniculata extract as an anticancer drug need to be proven by in vivo studies. the objective of the present study was to evaluate the effect of orally administered andrographis paniculata extract on apoptosis in c3h mouse mammary adenocarcinoma cells. methods design of study this study was of posttest randomized control group design. the experimental animals were kept at the experimental laboratory, department of pathologic anatomy, faculty of medicine, university of indonesia. the assays for apoptosis were performed at the pathologic anatomy laboratory, dr.sardjito hospital/ faculty of medicine, gadjah mada university. t h e s t u d y w a s c o n d u c t e d f r o m a p r i l t o december 2012. univ med vol. 32 no.2 102 nugrahaningsih, sarjadi, dharmana, et al andrographis paniculata induced apoptosis calculation of sample size the sample size was calculated according to federer’s formula, resulting in 6 animals per group or 24 animals for 4 groups. the federer’s formula: (k-1) (n-1) >15 k = number of experimental group (4) n = number of sample induction of mammary adenocarcinomas in c3h mice the c3h mice were obtained from the department of pathologic anatomy, faculty of medicine, university of indonesia. a total of 24 tumor-free c3h mice aged 2-3 months and weighing 17-25 grams were prepared for tranplantation of mammary adenocarcinoma. three c3h mice with established mammary adenocarcinoma tumors around 1.5 cm in diameter were prepared as donors. the donor mice were terminated with ether, then dissected for collection of cancer tissue. the cancer tissue was minced to pulp in a petri dish, 5 ml of normal saline was added, and 0.2 ml of the cancer suspension was injected subcutaneously into each of the experimental animals. the site of injection was palpated daily to determine tumor growth. on the seventh day the tumors were about the size of a pea, when the experimental animals were ready to receive interventions. preparation of andrographis paniculata extract t h i s e x t r a c t m a d e f r o m l e a v e s o f andrographis paniculata plant proccessing in lansida laboratory yogyakarta. the fresh leaves was dried in the fresh dryer at 300 c. the dried leaves was blended in hammer mills, and then extracted by distilled water for about 24 hours. the filtrat of extract was evaporated in rotavapor at 400c to get powder form. the aqueous andrographis paniculata leaf extract (aaple) powder was prepared in solution by added aquades as solvent. intervention the 24 c3h mice with transplanted tumors were assigned by simple random sampling into 4 groups of 6 animals. one group as the control group did not receive andrographis paniculata e x t r a c t . t h e o t h e r t h r e e g r o u p s a s t h e intervention groups were given andrographis paniculata extract at doses of 5, 10 and 15 mg per animal per day. the oral dose was based on t h e s t u d y b y s h e e j a e t a l . , w h o u s e d andrographis paniculata extract at 10 mg/ animal/day to determine its antiangiogenic effects.(18) administration of the andrographis paniculata extract was performed by means o f a g a v a g e t u b e . t h e a n d ro g r a p h i s paniculata extract was given in the form of a solution in distilled water, once daily for 14 days. on the fifteenth day the mice were terminated with ether. the cancer tissue specimens were formed into paraffin blocks for apoptosis testing by means of the terminal deoxynucleotide transferase dutp nick end labeling (tunel) method. during experimental periodes the mice were given animal feed pellets and drinking water ad libitum. apoptosis assay by the tunel method tunel is one of the methods for detecting apoptosis by examining for dna fragmentation. it is based on the presence of nicks in dna, w h i c h c a n b e i d e n t i f i e d b y t e r m i n a l deoxyribonucleotidyl transferase. this is an enzyme catalyzing the addition of labelled dutp, which will label cells with damaged dna. in this study, apoptosis in cancer tissue was detected by means of the apo-brdu-ihc in situ dna fragmentation assay kit (biovision k403). the paraffin sections were deparaffinized and rehydrated. proteins were digested with 100 ìl proteinase k solution for 20 minutes at room temperature. proteinase activity is blocked with 100 ì l of 3% h 2 o 2 , then incubated at room temperature for 5 minutes. after a rinse in phosphate buffered saline (pbs), the specimens were flooded with 1 0 0 ì l of dh 2 o reagent buffer, then incubated for 10-30 minutes at room 103 temperature. the reagent buffer is carefully blotted once, then 50 ìl of labelling reagent mixture is added. the specimens were covered with thin paraffin sheets of wider dimension than the specimens and incubated at 370c for 1 to 1.5 hour. the paraffin cover was removed and the slides were rinsed in pbs. all specimens were covered with 100 ìl blocking buffer then incubated at room temperature for 10 minutes. after blotting the blocking buffer, the specimens were covered with 100 ìl antibody solution. the antibody solution was prepared for 25 slides by dissolving 125 ìl anti-brdu-biotin in 2,375 ì l of blocking buffer. the slides were covered with aluminum foil then incubated in a dark room for 1-5 hours at room temperature, after which the slides were rinsed in pbs. all specimens were then covered with 100 ìl blocking buffer, counterstained with 100 ìl methyl green, and incubated at room temperature for 3 minutes. the slides were twice immersed in 100% ethanol, blotted with absorbent tissue and immersed in xylene. the excess xylene under the slides and around the specimens was removed, then the slides were mounted and covered with a cover glass. the apoptotic index was counted under a b i n o c u l a r m i c r o s c o p e . t h e s l i d e s w e r e examined at 40 x magnification for selection of 5 areas for reading of the results. each area was examined up to a total of 200 cancer cells at 400 x magnification. examination of the apoptotic index was done by counting positive cells among 1000 cancer cells in the 5 reading areas.(19) ethical clearance ethical clearance was obtained from the commission on medical and health research ethics, medical faculty, diponegoro university/ dr.kariadi general hospital, semarang. data analysis for each group, the mean and standard deviation of apoptotic indices was calculated. test of normality of the data was performed with the shapiro-wilk test and homogeneity of data was by means of the lavene test. differences in apoptotic index in each study group was evaluated by anova, followed by an lsd post hoc to test for differences in between-group means. the level of significance was set at 0.05. results the apoptotic cells were identified by their brown-colored nuclear region (figure 1). the lowest number of apoptotic cells was in the control group (a) and the highest in the group r e c e i v i n g 1 5 m g / d a y o f a n d ro g r a p h i s paniculata extract (d). the test of normality and homogeneity showed that the data were normally distributed and homogenous. the collected apoptotic indices are presented in table 1. the highest apoptotic index was obtained for the group receiving andrographis paniculata extract at 15 mg/day, and the lowest for the control group. the analysis was then followed by anova to test for differences between groups. the results of anova showed significant differences in the means of the apoptotic indices between the four groups (p=0.001). the results of lsd post hoc shows significant betweengroup differences in apoptotic indices for all study groups. the pearson’s correlation test showed a very strong correlation between dose o f a n d ro g r a p h i s p a n i c u l a t a e x t r a c t a n d apoptotic index (r=0.974) (data not shown). tabel 1. mean apoptotic indices and results of anova *significance level 0.05 ap = andrographis paniculata univ med vol. 32 no.2 104 nugrahaningsih, sarjadi, dharmana, et al andrographis paniculata induced apoptosis discussion the results of this study indicated that administration of andrographis paniculata extract to c3h mice was able to induce apoptosis in cancer cells. the effect of apoptosis was already apparent in the group on andrographis paniculata extract of 5 mg/day, showing a significant difference with the control group not receiving the extract. there was more apoptosis in the group receiving the extract at 10 mg/day in comparison with the group receiving the extract at 5 mg/day. similarly there was more apoptosis in the group receiving the extract at 15 mg/day in comparison with the groups receiving 5 and 10 mg/day. this indicates that a p o p t o s i s i n c r e a s e d p r o p o r t i o n a l l y w i t h increasing dose of andrographis paniculata extract. apoptosis can be detected by various methods, such as by identification of proteins involved in apoptosis, identification of apoptosisregulating genes, or identification of apoptotic cells.(20) these results are consistent with those of previous in vitro studies, which demonstrated that andrographis paniculata extract increased a p o p t o s i s o f c 3 h m o u s e m a m m a r y adenocarcinoma cells cultured in rpmi-1640 medium(14) and that of hela cells. (12) the results of the present study show that oral administration of andrographis paniculata extract is capable of increasing apoptosis of m a m m a r y a d e n o c a r c i n o m a c e l l s a f t e r u n d e r g o i n g t h e p r o c e s s e s o f a b s o r p t i o n , distribution, metabolism, and exctretion (adme). systemic absorption of a preparation or a drug figure 1. apoptosis in mammary adenocarcinoma cells without andrographis paniculata extract (a) and after administration of andrographis paniculata extract at doses of 5 mg/day (b), 10 mg/day (c), dan 15 mg/day (d) in the tunel assay a b c d 105 from the gastrointestinal tract or other absorption sites is dependent on the physicochemical characteristics and form of the preparation, and on the anatomical and physiological features of the absorption site. factors such as surface area of the gastrointestinal tract, gastric emptying time, gastrointestinal motility, drug metabolism by the intestinal microflora, and blood supply of the absorption site, may all influence the absorption rate and the amounts absorbed.(21,22) absorption and distribution of a preparation or a drug throughout the body takes place via several mechanisms, via passive diffusion, transport by convection, active transport, facilitated transport, ion pair transport, and pinocytosis. on arriving in the blood stream, a large fraction of the preparation is bound to plasma proteins and only a small fraction remains unbound and is called the free drug fraction, the form in which the drug is distributed to all tissues. drug distribution occurs by diffusion or transport from the blood stream into the interstitial spaces and the cells in tissues and organs. drug distribution is not always uniform throughout the tissues, because it depends on several factors, such as the physicochemical characteristics of the drug, the ratio of the proteinor biomaterial-bound fraction of the drug i n t h e b l o o d a n d t h a t i n t h e t i s s u e s , vascularization and blood velocity in the tissues, the chemical characteristics of the tissues, and the presence of carrier proteins (transporter) in the tissues. another factor affecting dose variations in the blood and tissues is the quality of the subject, which in turn is affected by intrinsic and extrinsic factors. therefore, administration of the same preparation or substance to an individual, using an identical dose and identical route of administration, may at different time points result in different drug concentration profiles in the blood.(21) on oral administration of andrographis paniculata extract to rats, the preparation reaches its peak plasma concentration in the relatively short time of around 2 hours and starts t o d i m i n i s h a f t e r t h r e e h o u r s . (23) o r a l l y consumed andrographolide accumulates in visceral organs, including the brain, kidneys, h e a r t a n d l u n g s . a m a j o r f r a c t i o n o f andrographolide is bound to plasma proteins and only a small fraction enters the cells. around 80% of the absorbed andrographolide is rapidly excreted from the body in 8 hours, and 90% in 48 hours.(24) the diterpenoids from andrographis p a n i c u l a t a i n c r e a s e a p o p t o s i s t h r o u g h induction of tumor necrosis factor-related apoptosis-inducing ligand (trail), which is one of the important types of tumor necrosis factor (tnf) having a high potential in cancer therapy. andrographis paniculata extract sensitizes cancer cells to trail-induced a p o p t o s i s a n d i n c r e a s e s t r a i l i n d u c e d apoptosis mainly by promoting caspase 8 activation. the activated caspase 8 promotes flice-like inhibitory protein (flip) cleavage and downregulates inhibitor of apoptosis p r o t e i n s ( i a p ) . ( 2 5 ) t h e a b o v e m e n t i o n e d sensitization of cancer cells to trail-induced apoptosis occurs through p53-dependent t r a n s c r i p t i o n a l r e g u l a t i o n r e s u l t i n g i n upregulation of death receptors 4 (dr4). andrographis paniculata extract stabilizes and activates p53 functions through activation of reactive oxygen species (ros)-dependent cjun n-terminal kinase (jnk). apoptosis occurs because of the presence of extrinsic and intrinsic signals. apoptosis in cancer cells does not proceed as in normal cells, because of systemic abnormalities resulting in inhibition of proapoptotic gene expression. the a p o p t o t i c p r o c e s s r e q u i r e s a d e n o s i n e triphosphate (atp) as cofactor. atp breakdown produces energy that is used for releasing cytochrome-c from the mitochondria. atp is obtained from nutrients that are transported through blood vessels in cancer tissue. if no atp is available, the cell may proceed to necrosis, inducing an inflammatory reaction. t h e a n d r o g r a p h o l i d e c o n t a i n e d i n andrographis paniculata extract may induce apoptosis through intrinsic as well as extrinsic univ med vol. 32 no.2 106 nugrahaningsih, sarjadi, dharmana, et al andrographis paniculata induced apoptosis pathways. it can initiate apoptosis through p53, which functions primarily in the intrinsic pathway.(13,25) the presence of an intrinsic signal releases bound apoptosis activating factor 1 (apaf-1) from bcl-2. apaf-1 then complexes with cytochrome-c and forms an apoptosome with caspase 9 and atp. this apoptosome complex activates pro-caspase 3 to become caspase 3, which is the effector of the caspase cascade. andrographolide may also act on the extrinsic apoptotic pathway, thereby activating caspase 8, which in turn activates caspase 3 as the effector caspase. the extrinsic signal is formed by complementary binding of apoptosis stimulating fragment (fas) and tnf which form the death activator.(26) the signal from the death activator is transmitted to the cytoplasm, where it activates caspase 8. the latter initiates the caspase pathway, resulting in the caspase cascade and apoptosis. activation of caspase 8 also causes bcl-2-interacting domain (bid) to become active and initiate the apoptotic program through increased activity of the pro-apoptotic proteins bcl-2-associated x-protein (bax)and bak. one limitation of this study was the absence of a positive control group receiving a cytostatic drug. conclusions oral administration of andrographis pani cul at a e x t r a c t i n d u c e s a p o p t o s i s o f mammary adenocarcinoma cells in c3h mice. a dose of 15 mg/day yielded the best results in this study, but it cannot as yet be determined if this is the optimal dose. acknowledgements this study received support from may sides. i am grateful to drs. kusmardi,ms and d r a . p u s p i t a e k a wu y u n g , m s o f t h e department of pathologic anatomy, faculty of medicine, university of indonesia, who assisted in the care of the experimental animals. i also thank dr.ahmad gozali,sp.pa, dr.harijadi sp.pa and ms. agustin of the department of pathologic anatomy, dr. sardjito hospital/faculty of m e d i c i n e , g a d j a h m a d a u n i v e r s i t y, f o r assistance in tissue processing and reading of the results. references 1. williamson ke, el din os, o’kane hf. apoptosis. in: the cancer handbook. 2nd ed. alison mr, editor. new york: john wiley & sons ltd; 2007.p.51-63. 2. ferreira cg, epping m, kruyt fae, giaccone g. apoptosis: target of cancer therapy. clin cancer res 2002;8:2024-34. 3. world health organization. guidelines for management of breast cancer. emro technical publication series; 2006. 4. world health organization. estimated. agestandardised incidence and mortality rate. available at: http://www.globocan.iarc.fr/. accessed june 25, 2013. 5. departemen kesehatan ri.. profil kesehatan indonesia tahun 2006. jakarta: departemen kesehatan r.i.;2007. 6. pandey mk, singh gn, sharma rk, lata s. physicochemical standardization of andrographis paniculata (nees): an ayurvedic drug. ijprd 2011;3:81-9. 7. saranya p, geetha a, selvamathy n. a biochemical study on the gastroprotective effect of andrographolide in rats induced with gastric ulcer. res paper 2011;73:550-7. 8. sivaraj a, vinothkumar p, sathinyraj k, sundarsen s, devi k, senthilkumar b. hepatoprotective potential of andrographis paniculata aqueous leaf extract on ethanol induced liver toxicity in albino rats. japs 2011;1:204-8. 9. ventriselvan s, subasini u, rajamanikam vc, thirumuruja s. hepatoprotective activity andrographis paniculata in ethanol induced hepatotoxicity in albino wistar rats. ijcp 2011; 1:1-4. 10. suebsana s, pongnaratorn p, sattayasai j, arkaravichien t, tiamkao s, aromdee c. analgesic, antipyretic, anti-inflammatory, and toxic effect of andrographolide derivates in experimental animals. arch pharmacol res 2009; 32:1191-200. 11. chao ww, lin bf. isolation and identification of bioactive compounds in andrographis paniculata (chuanxinlian). cmj 2010;5:17. doi: 10.1186/1749-8546-5-17. 107 12. sukardiman, rahman a, ekasari w, sismindari. induksi apoptosis senyawa andrographolida dari sambiloto (andrographis paniculata nees) terhadap kultur sel kanker. media kedokteran hewan 2005;21:105-10. 13. sukardiman, harjotaruno, widyawariyanti a, sismindari, zaini nc. apoptosis inducing effect of andrographolide on td-47 human breast cancer cell line. afr j tradit cam 2007;4:345-51. 14. nugrahaningsih, tjahjono, dharmana e. apoptosis sel adenokarsinoma mamma mencit c3h setelah pemberian ekstrak sambiloto (andrographis paniculata) (penelitian in vitro). media medika indonesiana 2003;38:121-4. 15. martins cf, dode mn, báo sn, rumpf r. the use of the acridine orange test and the tunel assay to assess the integrity of freeze-dried bovine spermatozoa dna. genet mol res 2007;6: 94-104. 16. shojaei f, ferrara n. role of the microenvironment in tumor growth and in rerfactoriness/resistance to anti-angiogenic therapies. drug resist updat 2008;11:219-30. 17. natal c, osés-prieto ja, pelacko b, iraburu mj, lopez-zabaiza mj. regulation of apoptosis by peptides of fibronectin in human monocytes. apoptosis 2006;11:209-19. 18. sheeja k, guruvayoorappan c, kuttan g.. antiangiogenic activity of andrographis paniculata extract and andrographolide. int immunopharmacol 2007;7:211-21. 19. al-dissi an, haines dm, singh b, kidney ba. immunohistochemical expression of vascular endothelial growth factor and vascular endothelial growth factor receptor-2 in canine simple mammay gland adenocarcinoma. can vet j 2010;51:1109-14. 20. vaculova a, zhivotovsky b. caspases: determination of their activities in apopotic cells. methods enzymol 2008;442:157-81. 21. hakim l. farmakokinetik klinik.yogyakarta: bursa ilmu kedokteran;2012. 22. shargel l, wu-pong s, yu abc. applied biopharmaceutics and pharmacokinetics. 5th ed. boston:mcgraw-hill medical publishing division;2005. 23. naidu sr, ameer oz, salman iw, venkatesh g, sadikun a, asmawi mz. pharmacokinetic study of androhraphis paniculata chloroform extract in rats. pharmacol 2009;1:309-19. 24. jarukamjorn k, nemoto n. pharmacological aspect of andrographis paniculata on health and its major diterpenoid constituent andrographolide. health sci 2008;54: 370-81. 25. zhou j, lu gd, ong cs, ong cn, shen hm. andrographolide sensitizes cancer cells to trail–induced apoptosis via p53-mediated death receptor 4 up-regulation. mol cancer ther 2008;7:2170-80. 26. wong hc, sagineedu sr, lajis nh, loke sc, stanslas j. andrographolide induces cell cycle arrest and apoptosis in pc-3 prostate cancer cell. afr j pharm pharmacol 2011;5:225-33. univ med vol. 32 no.2 c:\users\jurnal fkusakti\docume 17 *department of medical laboratory science, school of basic medical sciences, college of medical sciences, university of benin, nigeria correspondence: odigie bolaji efosa (phd) department of medical laboratory science (histopathology unit), school of basic medical sciences, college of medical sciences, university of benin, benin city, edo state, nigeria. email: bolaji.odigie@uniben.edu gsm: +2348023345132 orcid id: http://orcid.org/00000002-1233-0491 date of first submission, august 14, 2 01 8 date of final revised submission, march 19, 2019 date of acceptance, march 20, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina life style as a risk factor of human papillomavirus co-infections with trichomonas vaginalis in female sex workers efosa bolaji odigie* and micheal awol okungbowa* background asymptomatic trichomonas vaginalis (tv) and human papillomavirus (hpv) co-infection is perceived to have a devastating combined effect on victims. hence, we investigated tv/ hpv co-infection and determined the risk factors associated with it in female sex workers (fsw). methods a cross-sectional study was used to recruit 338 subjects from 6 local government areas (lgas) in nigeria for 18 months. agglutination tests for blood samples, and direct and wet examination of urine and vaginal swabs were conducted in the laboratory. life style of fsws was surveyed; data were analyzed with chi-square test (x2), paired and unpaired t-test and logistic regression analysis with statistical significance set at p< 0.05. results life style characteristics were significantly associated with hpv and tv co-infection (p < 0.001). the model explained 49.9% (nagelkerke r2) of the variance in infections among fsws and effectively classified 93.2% of cases. those involved in oral sex were 1.795 times more likely to be infected compared to those not involved (or: 1.795; 95% c.i.: 1.005 – 33.525; p=0.001). street and residential fsws all have increased likelihood of being infected. hard drug usage was also associated with an increased likelihood of being infected with the disease (or: 3.582; 95% c.i.: 1.573 11.756; p=0.001). conclusions life style (hard drug usage and oral sex practices) was observed in association with hpv/ tv co-infection in fsws with a preponderance of 49.9%. this study demonstrated the value of screening for genital infections in hpv positive subjects in order to reduce the probable synergistic effects of co-infections. keywords: co-infection, female sex workers, human papillomavirus, trichomonas vaginalis original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.17-24 january-april, 2019 vol.38no.1 cite this article as: odgie, okungbowa. life style as a risk factor of human pap illomavi rus co-i nfec tion s wi th trichomonas vaginalis in female sex workers. univ med 2019;38:17-24. doi: 10.18051/univmed.2019.v38. 1724 18 odigie, okungbowa hpv/tv in female sex workers introduction se x ua l c ont a c t i s t h e ma in f or m o f transmission of human papillomavirus (hpv), which causes one of the most common cervical infections in the world. as such, female sex workers (fsw) who engage in unwholesome activities are cut up with different kinds of infection.(1) female sex workers constitute a vulnerable group for the development of cervical ca nc er pr ec ur sor le si ons , since t hey a re constantly exposed to risk factors that facilitate the spread. it has been revealed that sex work is a predisposing factor for acquiring sexually transmitted diseases (stds).(2) however, stds are commonly available in the general population irrespective of age, sex, tribe, socioeconomic class, and types of exposure.(1, 3) epidemiology of viral co-infection with other forms of infective organisms in nigeria has been studied among sex workers.(4) it is likely that the prevalence of viral co-infection in nigeria may be the same irrespective of the occupation in different sectors of the nigerian population.(2) a report has it that the rate of co-infection amongst the different infective organisms in nigeria may be higher compared to other countries in the west-african sub-region due to her population.(4) co-infection may differ according to populations at risk (e.g. fsw); geographical locations, and individual lifestyle, which are important factors for consideration.(5) transactional sex is a critical factor for acquiring trichomonas vaginalis (tv) and human papillomavir us (hpv) infections. combined health effects from tv and hpv coinfection in a host could be devastating and may result in a more harmful health implication. trichomonas vaginalis, a common motile protozoan parasite, is the most prevalent non-viral sexually transmitted infection (sti) in the world, having a complex social and public health implications, in addition to their medical significance.(6) in combination with an hpv infection, the combined effects may become more harmful and devastating on the sufferers. human papillomavirus in turn is a type of viral infection that has been associated with cervical cancer and with substantial evidence for cancers in other anogenital regions, like the anus, vulva, penis and vagina in recent times.(7) very similar to the disposition of tv in women, hpv is also the most common sexually transmitted infection of viral origin and with over 100 hpv types, which have been reportedly identified and of which over 40 of the species can infect the genital areas.(7, 8) investigations revealed that females with tv infection are more predisposed to hpv coinfection. combined hpv and tv infection has been reported in tanzania (9), which resulted in tv having a strong association with hpv [or=4.2;95% ci. 1.7-10.3)]. subjects infested with tv were reported to be 6.5 times potentially exposed to hpv type 16 than those negative for tv (50% vs. 13.3%) [or= 6.5;95% c.i. 1.13.7)]. a study conducted in belgium involving 62,944 liquid-based papanicolaou smears with polymerase chain reaction for detecting both infections, observed that about 15.1% of women had an hpv infection and 0.37% had co-infection with tv.(10) also, working in the sex industry had an increased risk of hpv co-infection with tv compared to other women in the general population (or= 8.6; 95% c.i. 4.4-16.9).(10) life styles, which has been termed behaviorally risk factors in this study, include characteristics of a woman’s sexual history (number of partners, characteristics of the partners, contraceptive use) and substance use history (alcohol, cigarettes, illicit drugs), including oral and anal sex practices, have been well discussed in some studies.(9,10) of the sexual history characteristics, increased number of lifetime sexual partners and having had a recent, new sexual partner are two factors that are unswervingly associated with an increased risk of hpv infection.(10) sex workers are most disadvantaged by their occupation in a tv/hpv asymptomatic coinfection due to poor access to proper healthcare. (1 ) as socia tion b etwee n differe nt st is is frequent, but there are few studies on the prevalence of hpv co-infection with tv in the 19 high risk group of fsws. we assume that these women have a higher number of risk factors for acquiring the infection, which ranges from hard drugs, irregular condom usage and oral sex practices of the affected women. this study aimed at investigating the risk factors associated with hpv and tv co-infections in fsws. methods study area the study focuse d on the af emai communities in etsako local government areas (lgas) within the northern part of edo state, which is located in the south-southern region of nigeria. it is located about 103 miles north-east of benin city and geographically located on latitudes 6o 101n and 6o 201n and longitudes 6o 101e and 6o 151e (figure 1). cloth weaving, farming, and fishing are essential occupations of the afe mai clan who are known as the ivbiosakon. research design we used a cross-sectional study design to explor e tv /hpv co-e xistence including relationship between tv and hpv in fsws through neighbourhood outreach. the study was conducted between june 2016 and december 2017. study subjects and sample size determination participants were a combination of street and resident female sex workers who are engaged in the commercial sex business for a minimum of 1 year. pregnant women and women with bloody discharge were excluded. we investigated apartments, red light junctions, and guest houses where the e xchange of sex for money is transacted. we adopted the sample size determination according to the study by odigie and achukwu (13) in which the authors used the fisher’s formula (n = z α 2.p.q/d2) to determine their sample size. the desired sample size is n, the standard deviation (1.96) is z α , and p is the marked proportion of the population, which was estimated to have a particular characteristic (i.e., coinfection of tv/hpv). however, since no reasonable estimate exists, we assume 50% (i.e., 0.5) for p with regards to another study in a different location.(14) a total of 352 subjects who understood the research aims were included into the study. data collection a reliable and validated questionnaire was distributed amongst the subjects prior to sample collection. it was completed voluntarily and conducted in the vernacular in some instances.(13) one on one consulta tion was also used sometimes, while the question types were more of multiple-choice and open-ended options. data gathering information focused on the sociobehavioral background, which includes working duration per day, commercial sex frequency, and irregular use of condoms. health-care related issues, sexual exploitation, and history of previous or present infections (tv and/or hpv), including current and former symptoms (if known) were also part of the questionnaire. (1) laboratory analysis about 3-4 ml of venous blood was aseptically collected using a 5ml syringe and emptied into a steri le container without anticoagulant with tab gel and centrifuged (broadbent, uk) at 3000 rpm for 5 min to obtain the serum content. thereafter, serum separated from whole blood was used for serology/agglutination tests using hpv (12) and tv(15) kits in line with the manufacturer ’s instructions. high vaginal swabs (hvs) and midstream urine (msu) specimens were collected for microscopy (direct and wet preparation). other screening methods were used, which include: point-of-care trichomonas assay,(6) rapid antigen testing for trichomoniasis,(6, 11) rapid diagnostic method,(15) and osom trichomonas rapid test.(16) in addition, uncertain hpv results wer e confirmed wi th enzyme-linked immunosorbe nt assa y ( high-risk human papillomavirus testing kits).(17) univ med vol. 38 no.1 20 odigie, okungbowa hpv/tv in female sex workers data analysis we analyzed the empirical data with chisquare test (x2), paired and unpaired t-test and logistic regression analysis using minitab version 17.0. x2 tests. statistical tests were considered significant if p<0.05. ethical clearance in f o r ma t io n o n t h e a pp r o ve d e t h ic s clearance from the edo state ministry of health is available in protocol numbers (ha. 577/vol. 11. 165). the subjects were adequately briefed on the research protocols, while the informed c o ns e n t s w e r e ob t a i ne d b e f or e s a mp le collections with adequate explanations of the risk involved in the procedures. results out of 352 initial subjects, data from only 338 subjects were accepted for analyses due to inadequacy or quality of the specimen in addition to some missing or incomplete data observed for tv (08) and hpv (06). missing values were randomly distribut ed among the fsws (asymptomatic, residential, street etc.), which are unlikely to have introduced bias. therefore, there exists a preponderance of 1:10.2 representing 33/ 338 of fsws who play host to hpv co-infections with tv in the study areas (table 2). life style of fsws (hard drug usea and oralsex practices) was highly implicated and significantly expressed in hpv/tv co-infections (table 2). the result of the multiple logistic regression model, which was conducted to as certain lif e styles characteristics associated with hpv and tv coinfection among female sex workers (fsws) in the affected lgas of edo state, nigeria was statistically significant, χ2(7) = 90.97, p<0.001. the model explained 49.9% (nagelkerke r2) of the variance in infections among sex workers and correctly classified 93.2% of cases. those involved in oral sex were 1.795 times more likely to be infected compared to those not involved (or:1.795; ci: 1.005 – 33.525; p=0.001). street fsw and residential fsws all have increased likelihood of being infected. and hard drug use was associated with an increased likelihood of being infected with the disease (or: 3.582; ci: 1.573 11.756; p=0.001). a b c parameters tv infection hpv infection tv (-) (n=262) tv (+) (n=76) pvalue hpv (-) (n=279) hpv (+) (n=59) pvalue mean age (yrs) 26.8 ± 3.05 28.4 ± 3.11 28.6 ± 2.18 28.2 ± 3.09 mean work duration 2.84 ± 0.42 2.88 ± 0.16 2.84 ± 0.12 3.16 ± 0.25 sex work (12hrs) per day 14.75 ± 1.12 16.22 ± 1.28 14.16 ± 1.63 15.22 ± 1.82 irregular condom use 253/262 (96.6%) 21/76 (27.6%) 0.021 * 253/279 (90.7%) 18/59 (30.5%) 0.011 * hard drug use 81/262 (30.9%) 53/76 (69.7%) 0.644 88/279 (31.5%) 45/599 (76.3%) 0.644 anal sex practice 91/262 (34.7%) 13/76 (17.1%) 0.043 * 101/279 (36.2%) 11/59 (18.6%) 0.043 * alcohol intake 128/262 (48.9%) 43/76 (56.6%) 0.004 * 136/279 (48.7%) 21/59 (35.6%) 0.002 * oral sex practice 71/262 (27.1%) 29/76 (38.2%) 0.112 93/279 (33.3%) 28/59 (47.5%) 0.112 street fsw 172/262 (65.6%) 18/76 (23.7%) 0.204 107/279 (38.4%) 31/59 (52.5%) 0.204 residential fsw 90/262 (34.4%) 58/76 (76.3%) 0.001 * 172/279 (61.6%) 28/59 (47.5%) 0.001 * table 1. distribution of socio-behavioral characteristics of fsw in tv infection and hpv infection (n=338) note: data presented as mean ± sd; n (%); x2 test with yate’s correction was used in obtaining the p-values 21 parameters infection 95% c.i of odds ratio hpv (n= 33) tv (n=305) p-value aor lower upper irregular condom use yes 22 204 0.990 1.639 0.014 12.683 no 11 101 1.000 hard drug use yes 29 96 0.001* 3.582 1.573 11.756 no 4 209 1.000 anal sex practice yes 20 214 0.254 1.795 0.002 61.442 no 13 91 1.000 alcoholism yes 19 233 0.018* 0.734 1.0226 33.682 no 14 72 1.000 oral sex practice yes 22 77 0.001* 2.227 1.0050 33.525 no 11 228 1.000 street fsw yes 14 59 0.002* 1.377 1.0311 42.449 no 19 246 1.000 residential fsw yes 19 246 0.003* 2.849 1.0001 17.566 no 14 59 1.000 table 2: multiple logistic regression analysis of life styles characteristics associated with hpv and tv co-infection in fsws note: ci = confidence interval; fsw = female sex worker; hpv = human papillomavirus; aor = adjusted odds ratio; tv = trichomonas vaginalis figure 1: map is showing focal areas within afamai clans in etsakko lgas of edo state, nigeria univ med vol. 38 no.1 22 odigie, okungbowa hpv/tv in female sex workers discussion many factors have been linked to sales of sex including poverty,(13) which is a characteristic of a depressed economy.(18,19) we observed that oral sex practices have a significant bias in acquiring hpv/tv co-infections by fsws. oral sex particularly has been reported as a typical root of spreading genital hpv through sustained direct skin-to-skin contact with anal sex being the most common mode of transmission.(13) also observed are the degree of tv infection and the number of hpv/tv co-infections amongst residential fsws, while hpv infection is commoner in street sex workers, with the majority being asymptomatic carriers of the infection. in our study, residential fsws are resident in a particular brothel used for commercial sex activities waiting for male client’s patronage.(13) street fsws in turn, are seen as female commercial sex workers who move from one location to another searching for clients.(1) the difference here is the male “clients”; in the case of residential fsws, male clients are in search of fsws, but in the case of street sex workers, fsws scout for male clients.(1) street sex workers (13) and resident commercial sex workers (5) have been reported to be active carriers of co-infections in women compared to their counterparts in the general population. this statement expresses the that all fsws whether residential or street-based are considered as high-risk individuals.(2, 5) our result agrees with samarawickrema et al.(14) in which co-infection resulting from tv, hpv, chlamydia trachomatis, and neisseria gonorrhoeae were significantly observed in a sexual health clinic setting in urban sri lanka. co-infection with viruses,(13) bacteria,(20, 21) helminths,(22) yeast/fungi,(23) and other forms of infections has been documented.(7,23) to further corroborate our finding, ogunkan et al.(19) reported that hpv infection in fsws co-exists with other infections apart from viruses, with the prevalence varying from one region to another. this is not unanticipated, as it has been argued that sex works play a crucial role in the realization of hiv/hpv co-existence with some infective organisms thereby l eading to a massive immunological breakdown in host systems.(24) the mechanism by which tv and hpv shares a common ground in a host has been explained.(23) cell-mediated immunity generated against an invading organism involves recruitment of leucocytes in large numbers.(24) trichomonas often imbibes nutritious elements like fatty acids and iron by the destruction of host red blood cells.(23) this is caused by cytotoxic trypsin-like substances called cell detaching factor (cdf) and n-nitrosamines liberated during infection, which subsequently promote the process of epithelial atypia and dysplasia that is observable in cervical cytology.(24, 25) our study is comparable with the reports by taj and jamil (20) and gomih-alakija et al. (25) in which co-infec tions of mycoplasma genitalium and hpv and of hiv and tv were observed in fsws. however, it was reported (24) that a prolonged hpv/tv co-infection may enhance the risk of cervical intraepithelial neoplasia grade 3 (cin 3). human papillomavirus positive women co-infected with tv had a higher risk of cervical cancer in a study on the association between tv and high-risk hpv in rural tanzanian women undergoing cervical cancer screening.(9) irregular condom usage has been revealed in notable studies,(26, 27) which is dissimilar to the finding in this study. we suggest that there is a likelihood of not using condoms regularly in certain situations like the non-availability of condoms at the time of sexual encounter, (26) or in a situation where the last condom gets burst at the point of affixing it. the present report differed from some studies probably due to bias responses from the subjects, which may have introduced the differences observed as against o th e r r e s e a rc h o ut c o me s. ( 1 ) t h e ma j o r drawbacks in the present study centres on population size (number of participants), the limited time frame to consult with fsws and meandering pattern of sex workers (residential fsws), as well as changes in location, which is 23 commoner with street fsws in nigeria.(1) the implication of this study is that co-infection could be detrimental to human health if proper attention is not promptly apportioned. an understanding of the nature and consequences of an hpv/tv co-infection is vital for accurate estimates of infectious disease burden especially in fsws living in a depressed economic society. in particular, more holistic data on infectious diseases would help to quantify the size of the effects of hpv/tv co-infection on human health. from the foregoing, it therefore appears that a long-term assessment of cohort studies may be required to adequately evaluate the i nf l ue n c es of hpv / t v c o -inf e c t i on , pathogenesis, combined role and interactions of a dual infection (hpv-tv) on their host. in the future, f urthe r studies should investigate the carcinogenic effects of coinfection on the uterine cervix in fsws. it should endeavour to look critically into the association/ co-existence of stds in a large-scale research usin g the po lyme ra se cha in r e ac ti on f or assessment. conclusions we therefore conclude that hpv co-exists wi t h t v in f e ma l e s e x wo r ker s wi t h a prepondera nce of 1:10.2 and was highly implicated by life-style (hard drug use and oral sex practices) of the subjects. this study demonstrated the value of screening for genital infections in hpv positive patients in order to reduce the probable synergistic effects of coinfections. financial support and sponsorship: selfsponsored. conflicts of interest: none. acknowledgements all members of sexual health concerns for commercial sex workers (shccsw) research group, university of benin are well appreciated for the providence of adequate workforce in field exercises. contributors all authors immensely contributed to the drafting of the manuscript. oma and oeb indorsed the conception and design of the study. all authors participated in acquisition of data and field exercises. oeb and oma funded the data analysis and interpretation. all authors critically revised the manuscript for valuable intellectual content. oeb acted as the corresponding author on behalf of the first author. all authors have read and approved the final draft. references 1. odigie eb, achukwu pu. regular condom use tends to decrease the cervical lesions in residentcommercial sex workers in sub-saharan africa. univ med 2018;37:39-49. doi: http://dx.doi.org/ 10.18051/univmed.2018.v37.39-49. 2. duron s, panjo h, bohet a, et al. prevalence and risk factors of sexually transmitted infections among french service members. plos one 2018;13:e0195158. doi: https://doi.org/10.1371/ journal.pone.0195158. 3. szwarcwald cl, damacena gn, borges de souzajunior pr et al. factors associated with hiv infection among female sex workers in brazil. medicine 2018;97 suppl:s54-61. doi: 10.1097/ md.0000000000009013. 4. umego cf, mboto ci, mbim en, et al. epidemiology of hepatitis b virus infection in south-south nigeria: a review. int std res rev 2018;7:1-17. doi: 10.9734/isrr/2018/36989. 5. nasirian m, kianersi s, hosseini sg, et al. prevalence of sexually transmitted infections and their risk factors among female sex workers in isfahan, iran: a cross-sectional study. j int assoc provid aids care 2017;16:608–14. doi: https:// doi.org/10.1177/2325957417732836 6. gaydos ca, klausner jd, pai np, et al. rapid and point-of-care tests for the diagnosis of trichomonas vaginalis in women and men. sex transm infect 2017;93:s31-5. doi: 10.1136/ sextrans-2016-053063. 7. bruni l, barrionuevo-rosas l, albero g, et al. for ico information centre on hpv and cancer (hpv univ med vol. 38 no.1 24 odigie, okungbowa hpv/tv in female sex workers information centre). human papillomavirus and related diseases in the world: summary report 27 july 2017. barcelona, spain: ico/iarc hpv information centre;2017. 8. noel jc, fayt i, romero-munoz mr, et al. high prevalence of high-risk human papillomavirus infection among women with trichomonas vaginalis infection on monolayer cytology. arch gynecol obstet 2010;282:503-5. doi: 10.1007/ s00404-009-1291-x. 9. lazenby gb, taylor pt, badman bs, et al. an association between trichomonas vaginalis and high-risk human papillomavirus in rural tanzanian women undergoing cervical cancer screening. clin ther 2014;36:38-45. d oi:10.1016/ j.clinthera.2013.11.009. 10. depuydt ce, leuridan e, van damme p, et al. epidemiology of trichomonas vaginalis and human papillomavirus infection detected by realtime pcr in flanders. gynecol obstet invest 2010;70:273-80. doi: 10.1159/000314017. 11. postenrieder nr, reed jl, hesse e, et al. rapid antigen testing for trichomoniasis in an emergency department. pediatrics 2016;137:e20152072. doi 10.1542/peds.2015-2072. 12. saslow d, andrews ks, manassaram-baptiste d, et al. human papillomavirus vaccination guideline update: american cancer society guideline endorsement. ca cancer j clin 2016; 66:375-85. doi: 10.3322/caac.21355. 13. menon s, davy vb, rodolfo r, et al. multiple hpv infections in female sex workers in western kenya: implications for prophylactic vaccines within this subpopulation. infect agents can 2017;12:2. doi: 10.1186/s13027-016-0114-5. 14. samarawickrema na, tabrizi sn, young e, et al. prevalence of trichomonas vaginalis, chlamydia trachomatis, neisseria gonorrhoeae and human papillomavirus in a sexual health clinic setting in urban sri lanka. int j std aids 2015; 26: 733-9. doi: 10.1177/0956462414552813. 15. gaydos ca, hobbs m, marrazzo j, et al. rapid diagnosis of trichomonas vaginalis by testing vaginal swabs in an isothermal helicasedependent amplivue assay. sex transm dis 2016; 43:369-73. 16. madhivanan p, li t, trammell s, et al. performance of the osom trichomonas rapid test for diagnosis of trichomonas vaginalis infection among women in mysore, india. sex health 2013;10:320-4. doi: 10.1071/sh13015. 17. huh wk, ault ka, chelmow d, et al. use of primary high-risk human papillomavirus testing for cervical cancer screening: interim clinical guidance. obstet gynecol 2015; 125:330-7. doi: 10.1097/aog.0000000000000669. 18. ojo od, ugochukwu nda. impacts of societal prejudice on attainment of life/personal goals of physically challenged persons in nigeria. int res j human 2012;4:12-35. 19. ogunkan dv, omoruan a, fawole o. environmental and socio-economic dimensions of prostitution in ogbomoso, oyo state. ife res publ geogr 2010;9:282-300. 20. taj a, jamil n. viral and bacterial coinfection in patients with neurological disorders: an analytical cross-sectional study from karachi. clin infect dis 2018;2:108-13. 21. el baroudy nr, el refay as, abdel hamid ta. respiratory viruses and atypical bacteria coinfection in children with acute respiratory infection. open access maced j med sci 2018;6:1588-93. doi: https://doi.org/10.3889/ oamjms.2018.332. 22. okungbowa ma, omorodion tn. environmental and clinical implications of helminthic infection: a brief review. am j biomed sci 2018;10:45-8. doi:10.5099/aj180100045. 23. mcardle aj, turkovab a, cunningtonc aj. when do co-infections matter? curr opin infect dis 2018;31:209–15. do i:10.1097/ qco.0000000000000447. 24. ghosh s, mandal r, kundu p, et al. association of genital infections other than human papillomavirus with pre-invasive and invasive cervical neoplasia. j clin diagn res 2016;10:xe01 6. doi: 10.7860/jcdr/2016/15305.7173. 25. gomih-alakija a, ting j, mugo n, et al. clinical characteristics associated with mycoplasma genitalium among female sex workers in nairobi, kenya. j clin microbiol 2014;52:3660-6. doi: 10.1128/jcm.00850-14. 26. johnston lg, bonilla l, caballero t, et al. associations of hiv testing, sexual risk and access to prevention among female sex workers in the dominican republic. aids behav 2017;21:2362–71. doi: 10.1007/s10461-017-1751-4. 27. nadol p, hoang tv, le lv, et al. high hiv prevalence and risk among male clients of female sex workers in hanoi and ho chi minh city, vietnam. aids behav 2017;21:2381-393. doi: 10.1007/s10461-017-1751-4. editorial 135 occupational noise exposure and cardiovascular disease in male workers lie t. merijanti s department of occupational medicine, trisakti university, jakarta univ med vol. 32 no.3 noise is a common occupational hazard worldwide. the impact of noise on human health is emerging as an important public health issue. the most well-known effect of high noise levels at work (>85 dba) is hearing loss that is significantly associated with the period of exposure.(1) in addition, noise exposure can cause non-auditory effects, such as sleep disturbance, annoyance, psychological stress, and cardiovascular disease (cvd). in a study among korean blue collar workers, won et al. found that the prevalence of cvd risk was 32 cases per 100 workers.(2) however, the relationship between occupational noise and cardiovascular disease remains unsettled.(3) people who work in loud environments for at least one year and a half have a twoto threefold increase in prevalence of angina pectoris, myocardial infarction, coronary heart disease and isolated diastolic hypertension, especially men aged less than 50 years and current smokers.(2) one study found that workers in noisy workplaces without hearing protectors were 53% and 75% more likely to have prevalent cvd and angina, respectively.(4) however, another study found that cumulative occupational noise of more than five years did not increase the mortality risk from ischemic heart disease.(5) cvd risk is closely linked to individual and work-related factors, including noise, job stress and chemical hazards. these occupational factors particularly affect blue collar workers, who are more likely to be exposed to hazardous work environments, occupational stressors and cigarette smoking. this may explain why male workers are at greater actual cvd risk than female workers, because most of blue collar workers are men.(3) according to the hypothesis of noise stress (as a physical stressor in the workplace) and stress physiology responses in the defense reaction, the responses of the sympathetic adrenal neuroendocrine system are normally transient. repeated or chronic activation of the system, however, might increase blood pressure and change the structure of the heart and vessels, which are regarded as important pathophysiological changes in the development of cvd.(6) integrating worksite intervention, including promoting individual behavior change (such as smoking, dietary pattern, physical activity, and weight control), changing and monitoring the work environment (noise exposure, chemical hazards, psychosocial and organizational) and the family-community interface, may be synergistic to reduce cvd risk factors in the workplace. these prevention efforts require a partnership among public policymakers, employers, workers, health professionals, and the local community.(7) 136 univ med vol. 32 no.3 references 1. dube kj, ingale lt, ingle st. hearing impairment among workers exposed to excessive levels of noise in ginning industries. noise health 2011,13:348-55. 2. won ju, hong os, hwang wj. actual cardiovascular disease risk and related factors. a cross-sectional study of korean blue collar workers employed by small businesses. workplace health safety 2013;61:16371. doi:10.3928/21650794-20130327-17. 3. gan wq, davies hw, demers pa. exposure to occupational noise and cardiovascular disease in the united states: the national health and nutrition examination survey 1999–2004. occup environ med 2011;68:183-90. doi: 10.1136/oem.2010.055269. 4. gopinath b, thiagalingam a, teber e, mitchell p. exposure to workplace noise and the risk of cardiovascular disease events and mortality among older adults. prev med 2011;53:390-4. doi:10.1016/ j.ypmed.2011.10.001. 5. suadicani p, hein ho, gyntelberg f. occupational noise exposure, social class, and risk of ischemic heart disease and all-cause mortality: a 16-year follow-up in the copenhagen male study. scand j work environ health 2012;38:19-26. doi:10.5271/sjweh.3200. 6. al absi m, arnett dk. adrenocortical responses to psychological stress and risk for hypertension. biomed pharmacoth 2000;54:234-44. 7. sorensen g, landsbergis p, hammer l, amick bc, linnan l, yancey a, et al. preventing chronic disease in the workplace: a workshop report and recommendations. am j public health 2011;101suppl 1:s196207. c:\users\universa medicina\docu 27 *department of clinical pathology faculty of medicine trisakti university **department of pharmacology and pharmacy, faculty of medicine trisakti university ***department of occupational medicine faculty of medicine trisakti university corespodence: dr. alvina, sppk department of clinical pathology of medicine trisakti university phone: 6221-5672731 ext 2604 email: vina_march_dr@yahoo.com date of first submission, june 24, 2019 date of final revised submission, march 23, 2020 date of acceptance, march 23, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina glycated albumin is a better indicator for glucose levels than glicated hemoglobin in patients with diabetes mellitus on insulin therapy alvina*, pusparini*, meiyanti**, and lie t.merijanti*** background diabetes mellitus (dm) is a metabolic disease with a large incidence in the world and constitutes a global health problem. by 2030 it is estimated that there will be around 439 million people suffering from dm. diabetes mellitus is a metabolic disease caused by a lack or absence of the hormone insulin. in type 2 dm pharmacotherapy can be given one of which is insulin. to monitor therapy, random blood glucose, glycated hemoglobin (hba1c) and glycated albumin (ga) levels can be examined. the objective of this study was to determine the relationship of glycated albumin and glycated hemoglobin (hba1c) with random blood glucose in insulin-treated diabetics. methods a cross-sectional study was conducted involving 92 type 2 diabetic patients treated with insulin. the study used a questionnaire and blood samples. we measured the ga , hba1c and random blood glucose levels. a multiple linear regression was used to analyze the data. results mean hba1c was 9.21 ± 2.15%, mean glycated albumin was 24.4 ± 8.65%, and mean blood glucose was 229.47 ± 98.7 mg/dl. multiple linear regression tests showed that hba1c (b= 5,544; β=0.121; p=0.420) and ga (b=5.899; β=0.517; p=0.001) was signigicantly corelated with random blood gucose, respectively, indicating that glycated albumin is significantly related to and has the greatest influence on glucose level. conclusion glycated albumin is correlated with and has greater influence on glucose level than does hba1c. glycated albumin could be a better marker for glycemic control than glycated hemoglobin in diabetic patients treated with insulin. keywords: hba1c, glycated albumin, blood glucose, diabetic type 2 patients original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.27-33 january-april, 2020 vol.39no.1 cite this article as: alvina, pusparini, mei yant i, merijan ti lt. glycat ed albumin is a better indicator for glucose levels than glycated hemoglobin in pat ients wi th d iabetes mellitus on insulin therapy. univ med 2020;39:2733. doi: 10.18051/univmed.2020.v39. 2733 28 alvina, pusparini, meiyanti, et al. glycated albumin and diabetes mellitus introduction diabetes mellitus (dm) is a metabolic disorder with a high worldwide incidence that constitutes a global health problem. by 2030 it is estimated that there will be around 439 million people suffering from dm.(1) diabetes mellitus is a metabolic disease caused by a lack or absence of the hormone insulin.(2) the general picture of diabetes is chronic hyperglycemia that is associated with organ damage, such as renal failure, blindness, or amputation, which may increase its morbidity and mortality rates.(2,3) diabetes mellitus may be classified as type 1 dm, type 2 dm, gestational dm, and other specific types of diabetes (e.g. monogenic diabetes). type 1 dm is caused by autoimmune destruction of the pancreatic beta cells, type 2 dm is caused by a defective insulin secretion, gestational dm is diabetes occurring in pregnancy, while the specific types of diabetes have varied etiologies.(4) type 2 diabe tes mellitus has a high prevalence. this is associated with urbanization, in which the type 2 dm population increases five to ten-fold because of changes in behavior, from traditional rural to urban. the risk factors that are epidemiologically altered are also increased, such as more and longer lasting obesity, lack of physical activity, a nd presence of hyperinsulinemia.(5) to establish the diagnosis of diabetes, several examinations are required, among others random blood glucose level, fasting blood glucose level, the oral glucose tolerance test, and glycated hemoglobin (hba1c) level.(6) in type 2 dm pharmacotherapy may be administered to control the blood glucose level. the oral antihyperglycemic drugs that may be administered are the biguanids, glitazone, sulphonylureas, glinides, alpha glucosidase inhibitors, and combination therapy. in the case that oral antihyperglycemic therapy cannot control the blood glucose level, insulin may be administered. for therapeutic monitoring purposes, blood glucose, hba1c, and glycated albumin may be measured, glycated albumin being the most recent addition.(6,7) glyc ate d hemoglob in ( hba1c) is an important marker for evaluating the fasting or postprandial plasma glucose over the preceding 120 days.(8) glycated hemoglobin is formed from the reaction between hemoglobin and glucose through a non-enzymatic reaction step between the n-terminal valine on the hemoglobin a beta chain and glucose.(9,10) glycated hemoglobin was discovered in the late 1960’s as a glycemic control marker. glycated hemoglobin is used for monitoring glycemic control in patients with diabetes and has been routinely used in the clinical laboratory since around 1977.(8) since 1988, glycated hemoglobin has been recommended by the american diabetes association for routine monitoring of patients with diabetes.(11) there are a number of factors that may influence the hba1c level, such as anemia, chronic renal failure, and variant hemoglobin.(11) it is because of these factors possibly influencing its level that hba1c does not reflect short-term glycemic changes and that its accuracy is reduced in diabetic patients with anemia or end-stage renal disease.(9) another method for evaluating glycemic control is the determination of glycated albumin (ga), which is not influenced by disturbances in hemoglobin metabolism. glycated albumin reflects the blood glucose status of the preceding 2-4 weeks, whereas hba1c illustrates the blood glucose status of the preceding 3 months. therefore ga reflects the glycemic status over a relatively shorter period of time than does hba1c, and does it more rapidly. the ga level is not affected by the serum protein level, since it is expressed as the glycated albumin to total serum albumin ratio.(9,12) the ga level for therapeutic monitoring is beginning to be widely used. in the study carried out by hamaguchi et al.(13) that aimed to evaluate the glycated albumin level following sitagliptin therapy, the ga level was actually reduced within one month of treatment. intensive insulin therapy performed as initial treatment of 8 patients with type 2 diabetes mellitus 29 performance liquid chromatography (hplc),(17) which is based on the ion exchange principle. the sample is automatically mixed and diluted in the variant ii sampling station and injected into the analytical cartridge. the chromatographic station dual pumps deliver a programmed buffer gradient of increasing ionic strength to the cartridge, where the hemoglobins are separated based on their ionic interaction with the cartridge material. the separated hemoglobins pass through the flow cell of the filter photometer that measures changes in the absorbance at 415 nm. an additional filter at 690 nm corrects the background absorbance. the variant ii clinical data management program processes the data resulting from the respective analyses. two-level calibration is used for adjusting the calculated hba1c level. the sample results include the retention time of the detected peak and the chromatogram produced by the clinical data management program for the respective samples. the a1c peaks are shaded (rendered in a darker color). the a1c peak area is calculated by means of an exponentially modified gaussian algorithm that excludes the labile a1c and carbamylated a1c peak areas from the a1c peak area.(17) the glycated albumin assessment method used was enzymatic colorimetry (lucica ga-l; asahi kasei pharma co., tokyo, japan). the principle of the glycated albumin assay is the measurement of glycoalbumin, involving the conversion of glycated albumin (glycoalbumin) into glycated amino acid, then glycated amino acid + o 2 + h 2 o becomes glucosone + amino acid + h 2 o 2 , and finally h 2 o 2 + 4-aa + todb (n,n’bis(4-sulfobutyl)-3-methylanilin, disodium salt) yields a bluish-purple pigment + h 2 o. the glycated amino acid resulting from glycoalbumin is determined by photometric measurement of the absorbance of the bluish-purple dye.(14) the measured variables hba1c and ga are expressed in %. the measurement principle of blood glucose is automatic enzymatic photometry using the god-pap method. the variable measured is the random blood glucose level. and poor glycemic control, merely led to a small reduction in mean hba1c from 10.9% to 10.0%, whereas mean glycated albumin was actually reduced from 35.6% to 25.0%.(14) yoon et al.(15) reported that the deterioration in pancreatic beta cell function was correlated with the duration of dm and also with increased ga and the ga to hba1c ratio, but not with hba1c alone. based on the inconsistent results of previous studies, the aim of the present study was to determine the relationship of blood glucose and hba1c with glycated albumin in patients with diabetes mellitus receiving insulin therapy. methods research design the design of this study was cross-sectional. the study was carried out at the diabetes polyclinic of sumber waras hospital and the prodia clinical laboratories from january until march 2019. research subjects the inclusion criteria were male and female patients with dm, aged 30-80 years, duration of dm >1 year, receiving insulin for >1 year. the exclusion criteria were patients with renal failure/ undergoing hemodialysis, liver cirrhosis, and hypothyroidism/hyperthyroidism. the sample size was determined based on the correlation coefficient of 0.75 between glycated albumin and fasting blood glucose level as found in a previous study.(16) from zα=1.96, zβ= 0.84 and drop-out = 20%, the number of subjects required was 92. laboratory analysis the blood sample s were drawn by venipuncture and the blood was collected in vacutainers with and without edta anticoagulant. the blood with edta was directly examined for hba1c, whereas the blood without edta was centrifuged for 10 minutes at 2000 rpm to obtain the serum, which was examined for glycated albumin. the hba1c level was assessed by high univ med vol. 39 no.1 30 alvina, pusparini, meiyanti, et al. glycated albumin and diabetes mellitus data analysis the test of normality was done with the kolmogorov-smirnov test, which showed that the data were normally distributed. multiple linear regression analysis was used to determine the variable with the largest effect on glycated albumin. the level of significance used was 0.05. ethical clearance this study was approved by the research ethics commission, faculty of medicine, trisakti university, under no. 134/ker/fk/xii/2018. results in this study it was found that the majority of the respondents (59.8%) were female, and had a mean age of 55.88 ±11.83 years. mean duration of dm was 11.04 ± 7.62 years and mean duration of insulin use was 3.88 ± 4.00 years. it was also found that mean hba1c level was 9.21 ± 2.15 %, mean ga level 24.4 ± 8.65 % and mean random blood glucose 229.47 ± 98.70 mg/dl. in table 2 for the simple linear regression analysis it was found that hba1c, gender and gawere correlated with random blood glucose (p<0.05). the variables that were significantly correlated with random blood glucose were subsequently entered into the multiple linear regression analysis model. in table 3 for the results of the multiple linear regression analysis, it was found that ga had a larger effect on random blood glucose (beta = 0.517; p=0.000) than had hba1c (beta = 0.121; p=0.420). discussion in this study it was found that the majority of respondents (59.8%) were female, with mean age of 55.88 ±11.83 years. there were thus more patients with diabetes mellitus among the female respondents. this is in line with the study by miharja et al. (18) in that the prevalence of diabetes mellitus and abnormal glucose tolerance in indonesia was found to be more prevalent in females as compared to males. these results differ from those of chinese and japanese studies where the prevalence of dm was higher in males than in females. this may be due to differences in dietary intake, physical activity, and other habitual activities. the basic health research (riset kesehatan dasar, riskesdas) 2013 of the ministry of health (kementrian kesehatan) also found that according to gender, most of the patients with dm were women.(19) the prevalence of diabetes mellitus and abnormal glucose tolerance in indonesia increases with age. elderly subjects are at higher risk of hyperglycemia, which is associated with a decrease in pancreatic functions, because the pancreas becomes less effective in producing insulin.(18) the riskesdas 2013 also found that the proportion of patients with dm increases with advancing age and was highest in the age group of 65-74 years. the proportion of the group with an abnormal glucose tolerance test was found to be increased with age, so that the highest proportion was in the age group of 65-74 years, then decrea sed somew hat. similarly, the a b c table 1. distribution of subject characteristics (n=112) data presented as mean ± sd, except for gender in n (%) table 2. simple linear regression between respondent characteristic and blood glucose characteristic n age (years) 55.88 ± 11.83 gender male 37 (40.2) female 55 (59.8) duration of dm (years) 11.04 ± 7.62 duration of insulin use (years) 3.88 ± 4.00 random blood glucose level (mg/dl) 229.47 ± 98.7 hba1c (%) 9.21 ± 2.15 glycated albumin (%) 24.4 ± 8.65 variable b p value age -1.411 0.107 duration of dm -0.385 0.283 duration of insulin therapy -2.938 0.258 hba1c 25.727 0.000 glycated albumin 7.174 0.000 gender 51.976 0.012 31 proportion of abnormal fasting blood glucose also increased with age, with the highest proportion in the age group of 55-64 years, then decreasing somewhat in the next age groups.(19) in the present study it was found that mean duration of dm was 11.04 ± 7.62 years, since dm is a chronic metabolic disease, due to the fact that the pancreas is incapable of producing insulin in adequate amounts or the body cannot effectively utilize the produced insulin. diabetes is also one of the diseases that are known as the silent killers, because the patient with diabetes does not know or realize that he or she is suffering from diabetes and only knows when he or she suffers from complications.(19) in simple regression analysis it was found that hba1c, gender, and glycated albumin were correlated with random blood glucose (p<0.05), but the results of multiple regression analysis showed that ga had the largest effect on random blood glucose, which may be due to the fact that serum albumin is strongly bound to glucose and the glycation reaction proceeds ten times as rapid as glycation of hemoglobin, so that ga is capable of more rapidly capturing fluctuations and changes in glycemic status than does hba1c.(20) glycation depends on the degree and duration of hyperglycemia. the glucose level and the duration of exposure between protein and sugar is a factor in glycation for the lifespan of the protein.(2) mean albumin glycation rate is 9-10 times that of hemoglobin.(2,21) the serum protein level does not influence the ga, because the ga is calculated from the ratio of glycated albumin level to total serum albumin.(20) in the present study there was also found a significant correlation between ga and blood glucose. this may be have been caused by the shortened erythrocyte lifespan in diabetic patients with poor glycemic control, by delayed gl ut 1-med i a t e d gl u c o se upt a ke b y erythrocytes, resulting in lower degree of increase in a1c, by differences in glycation rates between hemoglobin and albumin in the blood, and possibly the involvement of a direct insulin effect on serum albumin metabolism. intensive insulin therapy reduces glycemic excursions, stressing the need for minimizing fluctuations in glucose level.(22) human serum albumin is of benefit to patients with various disorders, where albumin limits oxidative damage. the albumin antioxidant properties are the result of limitation of reactive oxygen species (ros) production by ligandbinding capacities as well as ros scavenging through free radical trapping. in connection with ligand binding capacity, albumin binds readily to molecules such as metal ions, fatty acids, etc. free cu (ii) and fe (ii) are very strong ros generators when reacting with oxygen.(23) glycation and oxidation are the most common principal non-enzymatic mechanisms. glycation, sometimes termed non-enzymatic glycosylation, is a simple process in which excess sugar molecules such as fructose or glucose are attached to normal protein or lipid molecules in the blood without enzymatic intervention.(23) t he s tu d y c on d uc t e d b y ue da a n d matsumoto on in-vitro glycation rates of human albumin and hemoglobin from three volunteers found that the production of ga was around 4.5 times higher than that of hba1c, after the administration of the same amount of glucose to the samples. this shows that under the same in-vitro glycation conditions, glycated albumin is produced more rapidly than hba1c.(24) univ med vol. 39 no.1 table 3. multiple linear regression between several respondent parameters and random blood glucose note : b: regression coefficient; beta: standardized regression coefficient variable b beta p value hba1c glycated albumin gender 5.544 5.899 5.844 0.121 0.517 0.029 0.420 0.001 0.743 32 alvina, pusparini, meiyanti, et al. glycated albumin and diabetes mellitus according to whaley-connell and sowers, within the individual glycemic measures, ga and hba1c are strongly associated, but ga tends to trend with individual glucose profiles.(25) won et al.(26) demonstrated a significant correlation between changes in ga over a 3-week period and in hba1c over a 3-month period following intensive oral antidiabetic or insulin therapy. the study of ake et al.(9) also reports a strong positive correlation between ga and hba1c.(9) in the study conducted by lee et al.(27) it was found that the mean gan/hba1c ratio tends to increase with increasing hba1c level. the ga serum level increases 3-4% for each 1% increase in hba1c level. therefore, the increased ga/ hba1c ratio with high hba1c may have been caused by an increase in ga level rather than hba1c level, especially in patients with poor glycemic control. the study of koga et al.(22) showed tha t the ga/hba1c ratio was significantly higher in patients on insulin therapy than in patients on dietary treatment or oral hypoglycemic drugs. according to the study of koga et al.(12) ga decreases more rapidly than hba1c in cases of improved glycemic control and increases more rapidly in cases of poor short-term glycemic control. t here are several situations and conditions that require ga as determinant of glycemic control status, such as fulminant type 1 dm onset, type 1 dm, type 2 dm under insulin therapy, patients with postprandial hyperglycemia, hemolytic anemia, variant hemoglobin, chronic renal failure under hemodialysis, liver cirrhosis, iron deficiency anemia and patients treated with iron preparations, pr egnant women and premenopausal women.(12) the glycemic index may also be classified as long-term, intermediateterm and short-term glycemic indicator. the use of hba1c as long-term glycemic indicator is due to the long erythrocyte lifespan of 120 days. for intermediate-term glycemic indices, glycated serum protein and albumin may be used to determine glycemic status over intermediate periods (2-4 weeks) that reflect the half-life of the respective molecules in serum. glycated albumin with a half-life of 12-19 days may be a good glycemic index of recent ambient glycemia.(22) glycated albumin is at present a popular glycemic control index during intensive treatment.(27) one limitation of the present study is that it did not investigate the factors that may affect hba1c and ga, such as hb level, red cell count, body mass index, blood pressure, lipid profile, protein intake, and albumin level. it is suggested that future studies should evaluate abovementioned factors in diabetic patients on oral antihyperglycemic therapy as well as in diabetic patients on insulin therapy. conclusion there is a significant role and association between ga and random blood glucose among diabetic patients treated with insulin.. conflict of interest the authors declare no conflict of interest. acknowledgments our heartfelt tha nks to the diabetes polyclinic team of sumber waras hospital and the personnel of the sumber waras hospital laboratory. contributors a wrote the proposal and coordinated the study locations. a, p, m, and lmt performed the data collection. a and p performed the statistical analysis, drafted the manuscript, and were responsible for data collection and implementation. all authors have read and approved the final manuscript. references 1. chamnan p, simmons rk, forouhi ng, et al. incidence of type 2 diabetes mellitus using 33 proposed hba1c diagnostic criteria in the european prospective investigation of cancernorfolk cohort. diabetes care 2011;34:950-6. doi: 10.2337/dc09-2326. 2. freitas pac, ehlert lr, camargo jl. glycated albumin: a potential biomarker in diabetes. arch endocrinol metab 2017;61:296-304. doi: 10.1590/ 2359-3997000000272. 3. american diabetes association: standard of medical care in diabetes-2016. diabetes care 2016; 39 suppl 1:s1-s112. doi: 10.2337/dc14-s081. 4. baynest hw. classification, pathophysiology, diagnosis and management of diabetes mellitus. j diabetes metab 2015;6:1-9. doi: 10.4172/21556156.1000541. 5. international diabetes federation. diabetes risk factors. brussels: international diabetes federation; 2019. 6. american diabetes association. classification and 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2016;39:1299-306. doi: 10.2337/dc15-2727. 12. koga m, kasayama m. clinical impact of glycated albumin as another glycemic control marker. endocrine j 2010;57:751-62. 13. hamaguchi t, koga m, murai j, et al. estimation of hba1c response to sitagliptin by change in glycated albumin level for 2 weeks. j diabetes investig 2012;3:175-8. doi: 10.1111/j.204011242011.00167x.2012. 14. kohzuma t, koga m. lucica ga-l glycated albumin assay kit: a new diagnostic test for diabetes mellitus. mol diagn ther 2010;14:49-51. doi: 10.2165/11317390-000000000-00000. univ med vol. 39 no.1 15. yoon hj, lee yh, kim kj, et al. glycated albumin levels in patients with type 2 diabetes increase relative to hba1c with time. biomed res int 2015; 2015:576306. doi: 10.1155/2015/576306. 16. paroni r, ceriotti f, galanello r, et al. performance characteristics and clinical utility of an enzymatic method for the measurement of glycated albumin in plasma. clin biochem 2007;40:1398-405. doi: 10.1016/j.clinbiochem.2007.08.001. 17. bio rad. d-10 hemoglobin testing system for hemoglobin a1c. bio-rad laboratories, inc.;2019. 18. mihardja l, soetrisno u, soegondo s. prevalence and clinical profile of diabetes mellitus in productive aged urban indonesians. j diabetes invest 2014;5:507-12. doi: 10.1111/jdi.12177. 19. kementerian kesehatan ri infodatin pusat data and informasi. situasi and analisis diabetes. jakarta : kementerian kesehatan ri infodatin pusat data and informasi;2014. 20. simanjuntak s, darmanta b, kartikasari ey, et al. hubungan glycated albumin dengan hba1c pada pasien diabetes mellitus tipe ii. jurnal kedokteran methodist 2017;9:631-8. 21. rondeau p, bourdon e. the glycation of albumin: structural and functional impacts. biochimie 2011; 93:645-58. doi: 10.1016/j.biochi.2010.12.003. 22. koga m, murai j, saito h, et al. glycated albumin and glycated hemoglobin are influenced differently by endogenous insulin secretion in patients with type 2 diabetes. diabetes care 2010;33:270-2. doi: 10.2337/dc09-1002. 23. kim kj, lee bw. the roles of glycated albumin as intermediate glycation index and pathogenic protein. diabetes metab j 2012;36:98-107. doi: 10.4093/dmj.2012.36.2.98. 24. ueda y, matsumoto h. recent topics in chemical and clinical research on glycated albumin. j diabetes sci technol 2015;9:177-82. doi: 10.1177/ 1932296814567225. 25. whaley-connell a, sowers jr. implications for glucose measures in the diabetes control and complications trial/epidemiology of diabetes interventions and complication study. diabetes 2014;63:45-7. doi: 10.2337/db13-1497. 26. won hk, kim kj, lee bw, et al. reduction in glycated albumin can predict change in hba1c: comparison of oral hypoglycaemic agent and insulin treatments. diabet med 2012;29:74-9. doi: 10.1111/j.1464-5491.2011.03386.x. 27. lee ey, lee bw, kim d, et al. glycated albumin is a useful glycation index for monitoring fluctuating and poorly controlled type 2 diabetic patients. acta diabetol 2011;48:167-72. doi: 10.1007/ s00592-010-0242-0. c:\users\jurnal fkusakti\docume 147 *department of emergency medicine, faculty of medicine, mashhad university of medical sciences, mashhad, iran **department of neurology, faculty of medicine, mashhad university of medical sciences, mashhad, iran ***department of community medicine, mashhad branch, islamic azad university, mashhad, iran correspondence: dr. mahdi foroughian department of emergency medicine, faculty of medicine, mashhad university of medical sciences, mashhad, iran tel: 05138525312; email: foroughianmh@mums.ac.ir orcid id: 0000-0002-3944-9361 date of first submission, october 1, 2 01 8 date of final revised submission, september 9, 2019 date of acceptance, september 11, 2 01 9 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina predicting outcomes 3 months after traumatic brain injury in patients admitted to emergency department ehsan bolvardi*, maryam ahmadzadeh*, hamideh feiz disfani*, fariborz rezaeitalab**, yalda ravanshad***, and mahdi foroughian* background traumatic brain injury (tbi) is among the leading causes of mortality and long-term disability. prognosis assessment is a primary factor of clinical decision-making by emergency physicians. this study aimed to investigate the prognostic factors of tbi in the patients admitted to a typical emergency department. methods this prospective cohort study was conducted involving 100 tbi patients. baseline characteristics, including age, gender, glasgow coma scale (gcs), and vital signs were recorded. blood tests and brain ct scans were collected. the patients were followed-up three months after the date of admission. the observed outcomes were categorized as recovery without complication, recovery with complication, or death. statistical analysis was performed using the simple and multivariate binary logistic regression in the software ibm spss version 19. results the most common brain ct scan findings were subarachnoid hemorrhage (21.0%) and epidural hemorrhage (20.0%). in the follow-up performed three months after the admission, 47 patients (47.0%) had died, 39 (39.0%) were suffering from complications, and 14 patients (14.0%) were recovering without complications. simple binary logistic regression showed that older age (or=3.28, 95% c.i.=1.27-8.41), minor/moderate head trauma (or=13.93, 95% c.i.=1.73-112.11), severe head trauma (or=54.40, 95% c.i.=5.71-517.56) and presence of deep skull fracture (or=8.92, 95% c.i.=1.04-75.53) were statistically significant predictors of mortality. multivariate logistic regression showed that mortality chance was higher in elderly (or=7.45, 95% c.i.=2.02-27.36), minor/moderate head trauma (or=26.87, 95% c.i.=2.42-298.25) and severe head trauma (or=127.97, 95% c.i.=9.111796.28). conclusion this study demonstrated that severe head trauma was the most predicted risk factor of poorer clinical outcomes after tbi. keywords: traumatic brain injury, prognosis, emergency, mortality, trauma original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.147-155 may-august, 2019 vol.38no.3 cit e th is article as: bolvard i e , ahmadzadeh m, disfani hf, et al. predicting outcomes 3 months after traumatic brain injury in patients admitted to emergency department. univ med 201 9;38 :147 -55. doi : 10. 1805 1/ univmed.2019.v38.147-155 148 bolvardi, ahmadzadeh, disfani, et al. prognostic factors of traumatic brain injury introduction traumatic brain injury (tbi) is one of the leading causes of mortality and long-term disability, especially among young people, and accounts for about 30% of accident-related deaths.(1,2) every year, about 2.5 million people sustain tbi, of which about 50,000 die and more than 80,000 become permanently disabled.(2) in iran, trauma, and especially tbi, is one of the leading causes of death following a hospital a d mi ss i on . ( 3 ,4 ) be c a us e of i t s s e ve r e p hysi o l ogic a l , e mo t i on a l , a nd ph ys i c a l complications, this injury imposes significant direct and indirect financial burdens that are hard to bear, especially in lowand middleincome countries, where more than 90% of tbi c a s e s oc c ur. ( 1 ,5 ) in t h e l a s t d e c a d e , t h e hospitalization rate has shown no notable change and the mortality rate has declined, but there has been an increase in the number of tbirelated admissions to emergency departments (ed).(6) this trend calls for better management strategies for predicting, preventing, and a d dr e s si n g th e s hor t a nd l o ng-t e r m complications of tbi. prognosis assessment is one of the primary factors of clinical decision-making and selection of therape utic str ategy for t bi patients, especially in emergency departments.(7,8) studies have shown that more than two-thirds of physicians consider prognosis as the most important step in making a clinical decision particularly about ventilation, use of analgesics, or serum therapy.(4) in addition to helping physicians decide on the type and strength of treatment, having a confident prognosis is very important for answering the questions of patient’s family members and companions about his or her condition. in re c ent de c ade s, r ese ar ch er s h ave proposed several models, including the glasgow outcome scale (gos) and its extended version glasgow outcome scale extended (gose), rancho los amigos scale revised (rlas-r), and clinical randomization of an antiûbrinolytic in signiûcant hemorrhage (complete crash2 ) , f or pr e di c t i n g t h e o ut c ome s o f t bi patients.(2,5) in these models, factors such as age, glasgow coma score (gcs), hypoxia and hypotension upon arrival to the ed, cause of the accident, biochemical parameters, and ct-scan findings have been used as the determinants of p rogno si s. ( 9 -11 ) ho we ve r, c on s i de r in g th e d if f e r e nc e s in t he s e r vi c e s of ed s, epidemiological patterns of tbis, driving safety levels, and the demographics of tbi patients (e.g. age and gender) in different parts of the world, these models may not be uniformly applicable to all countries.(11,12) in iran, which has a particularly high rate of traffic and occupational accidents, only one retrospective study has investigated the prognostic factors of tbi;(3) therefore, more prospective studies are needed to develop a localized model for predicting the outcomes of tbi for iranian patients. although gcs is a useful tool for tbi evaluation, it has some limitations and gcs documentation have some differences and pitfalls in patients.(13,14) generally, in gcs level documentation, gcs in admission to ed and highest or lowest gcs in 24 hours of admission can be used. moreover, some interventions that can change the gcs level are medical intervention, administration of drugs such as sedatives or paralytic drugs, drug toxicity, and facial trauma.(15,16) the objective of this study was to investigate the prognostic factors of tbi in the patients admitted to a typical emergency department. methods research design t hi s p r os pe c ti ve c oh o r t s t u dy w a s conducted on tbi patients admitted to the emergency department of hashe minej ad hospital, mashhad, iran from january 2015 to december 2016. 149 research subjects patients with moderate or severe tbi and age of more than 15 years were included in the study. exclusion criteria were (i) simultaneous trauma in other organs, (ii) discharge from the emerge ncy departmen t be fore the final evaluation, and (iii) lack of cooperation in subsequent follow-ups. the sample size was calculated based on the results of yang et al.(17) using the formula for comparison of a quantitative variable in two groups, with α and β set to 0.05 and 0.2 respectively. accordingly, the minimum sample size was calculated as n=138. patients were entered into the study consecutively until the target sample size was reached. data collection an e me r ge n c y me di c i n e s pe c i a l i st recorded the basic information of patients including age, gender, gcs, and vital signs, upon their admission to the ed. blood pressure and oxygen saturation were measured by a digital pressure meter and a pulse oximeter respectively. mean arterial pressure (map) was calculated using the formula [(2 × diastolic blood pressure) + systolic blood pressure]/3. hypotension was defined as a systolic pressure of less than 70mm hg, and hypoxia was defined as an oxygen saturation of less than 90 percent. for all patients, arterial blood samples were taken to measure arterial blood gas and venous blood samples were taken to measure hematocrit (hct). the normal range of ph was 7.35-7.45 acidemia was defined as a serum ph of less than 7.35, and alkalemia as ph>7.45. the normal range of hct was 38-42% and anemia was defined as hematocrit of less than 42% for men and less than 38% for women. the normal range of hco3 was defined as 22 to 28 milliequivalents per liter; metabolic acidosis as hco3<22 milliequivalents per liter; metabolic alkalosis as hco3>28 milliequivalents per liter. the normal range of pco2 was defined as 35–45 mmhg; respiratory acidosis as pco2>45 mmhg; and respiratory alkalosis as pco2<35 mmhg and ph 7.35 7.45. vital signs were categorized as follows: bradycardia: hr<60; normal hr: 0hr100; tachycardia: hr>100; bradypnea: rr<12; normal: 12rr20; tachypnea: rr>20; normal gcs=15; minor or moderate head trauma 8gcs<15; seve re he a d tr auma gcs<8; children: age <16 years; adults: 1 6  a g e < 6 5 years; elderly: age 65 years. a brain ct scan with an axial cross sec tion wa s taken on admission to the ed (less than 6 hours after trauma) and its result was interpreted by a radiologist. clinical outcomes follow-up was performed three months after admission to the ed and the outcome at that date was categorized as (i) recovery without complications, (ii) recovery with complications (emergence of new neurological symptoms after tbi), or (iii) death. ethical considerations the research plan was fully explained to the patient’s legal guardian and written informed consent was acquired before collecting any information. personal information of all patients remained confidential and was handled and processed in a codified form. the research proposal was authorized by the research deputy department of the mashhad university of medical sciences and received approval from t he e t hi c s co mmi t t ee of t h is un ive r si t y (registration code: 920748). data analysis descriptive statistical analyse s were performed using spss forwindows, version 19 (armonk, ny: ibm corp.). simple binary logistic regression was used to find predictor variables for mortality. then all predictors that were significant were entered into a multivariate logistic regression. findings were represented a s od ds ra t i o ( or) a l o ngsi de wi t h 9 5% confidence interval (ci). in all tests, p-values of less than 5% were considered statistically significant. using the per-protocol analytical approach, the analyses were restricted to those univ med vol. 38 no.3 150 bolvardi, ahmadzadeh, disfani, et al. prognostic factors of traumatic brain injury patients whose information up to the final stage of follow-up was available. results baseline characteristics a total of 138 patients entered the study, of which 38 patients were excluded because of simultaneous trauma in other organs (31 patients), discharge from the emergency department before the final evaluation (4 patients), and lack of cooperation during the follow-up (3 patients). ultimately, the study was performed on 100 patients (figure 1). participants (n = 100) were predominately male (85%) with a mean age of 43.7± 23.0 years. in 76.0% of patients, gcs on admission was more than 8. the most common disorders of vital signs were tachycardia (67.0%) and hypotension (42.0%). the most common brain ct scan findings were subarachnoid hemorrhage (21.0%) and epidural hemorrhage (20.0%). in the followup performed three months after admission, 47 patients (47.0%) had died, 39 (39.0%) were suffering from complications, and 14 patients (14.0%) were recovering without complications. other results regarding the background variables, physical examination findings, vital signs, and paraclinical findings are presented in table 1. variables associate d w it h the out come (death or survival) simple binary logistic regression showed that older age group (or=3.28, 95% c.i.= 1.27-8.41), minor/moderate head trauma (or=13.93, 95% c.i.=1.73-112.11), se vere head trauma (or=54.40, 95% c.i.=5.71-517.66) and presence of deep skull fracture (or=8.92, 95% c.i.=1.0475.53) were statistically significant predictors of mortality (table 2). cox and snell r square for multivariate binary logistic regression was 37% showing an acceptable overall prediction. in this model, only severe head trauma, minor/moderate head trauma and elderly age group remained statistically significant predictors (table 3). discussion the findings of this study showed that older age, minor/moderate head trauma, and severe head trauma on admission were the factors associated with the death of these patients. the first factor that this study found to be related to the outcome of death following a tbi was age. the mean age of the subjects of our study was about 44 years. on average, patient who died during the follow-up period were about 10 years older than those who survived this period with or without complications. the presence of a b c figure 1. flow diagram of traumatic brain injury 151 similar reports in other studies suggests that age is likely to be a key factor of the clinical outcome of tbi, and younger people have a lower chance of mortality following a tbi. (3,10) it has also been reported that each year of increase in age is associated with 5% higher chance of postconcussion syndrome and 2% higher chance of a gcs of less than 7.(10,18,19) both quantitative and qualitative analyses of our study showed that reduced gcs is another important factor in the adverse clinical outcomes of tbi. the frequency of gcs of less than 8 among the patients who did not survive the followup period was significantly higher than among other patients. other studies have also reported an association between gcs and the prognosis of tbi patients.(20,21) although our finding in regard to the association of gcs with the clinical outcome of tbi is not new and has been previously mentioned in other studies,(9-11) this is the first prospective study that reports such an association in iran, although a cross-sectional study in iran found age, gender, gcs, pupillary reflex, hypernatremia, and increased intracranial pressure (iicp) as the predictors of death in severe tbi.(3) among the vital signs, only the heart rate exhibited an association with undesirable clinical outcomes. however, this association was only observed in quantitative analyses (there was no relationship between tachycardia and outcome in the qualitative analysis). since tachycardia, as the prominent sign of hemodynamic instability, can be caused by other underlying disorders such as cerebral hemorrhage, its association with the adverse clinical outcomes in tbi patients can be explained from this perspective. while primary vital signs are rare among the main factors of prognostic models, our findings suggest that vital signs can serve as a prognostic factor for the assessment of the clinical outcome of tbi. however, it should be noted that the number of studies in this area is still very low to draw a definite conclusion. the last factor that our study found to be associated with the final condition of tbi univ med vol. 38 no.3 table 1. distribution of demographic, clinical findings and outcomes of tbi subjects (n=100) variable n (%) age children adults elderly gender male female o2 saturation normal hypoxia rr normal bradypnea tachypnea hr normal bradycardia tachycardia map normal hypotension head trauma normal minor/ moderate severe hco3 normal metabolic acidosis metabolic alkalosis pco2 normal respiratory acidosis respiratory alkalosis ph normal acidemia alkalemia hct normal anemia subdural hemorrhage yes no subarachnoid hemorrhage yes no epidural hemorrhage yes no depressed skull fracture yes no intracranial hemorrhage yes no pneumocephalus yes no contusion 67 (67.0) 6 (6.0) 27 (27.0) 85 (85.0) 15 (15.0) 90 (93.8) 6 (6.3) 88 (92.6) 3 (3.2) 4 (4.2) 83 (85.6) 2 (2.1) 12 (12.4) 87 (90.6) 9 (9.4) 17 (17.3) 59 (60.2) 22 (22.4) 41 (42.3) 48 (49.4) 8 (8.2) 48 (49.5) 18 (18.5) 31 (31.9) 46 (47.4) 38 (39.1) 13 (13.4) 14 (19.4) 58 (80.6) 18 (18.0) 82 (82.0) 24 (24.0) 76 (76.0) 21 (21.0) 79 (79.0) 8 (8.0) 92 (92.0) 10 (10.0) 90 (90.0) 4 (4.0) 96 (96.0) # normal gcs=15; minor or moderate head trauma 8gcs<15; severe head trauma gcs<8 152 bolvardi, ahmadzadeh, disfani, et al. prognostic factors of traumatic brain injury or (95%ci) p value age children 3.28(0.55-19.23) 0.188 adults elderly 1 3.28(1.27-8.41) 0.013 gender male female 1 0.69(0.22-2.13) 0.530 o2 saturation normal hypoxia 1 2.45(0.42-14.07) 0.315 rr normal 1 bradypnea 2.46(0.21-28.16) 0.469 tachypnea 1.23(0.16-8.14) 0.839 hr normal bradycardia 1 0.999 tachycardia 2.43(0.67-8.71) 0.172 map normal hypotension 1 1.95(0.44-8.70) 0.377 head trauma normal minor/moderate head trauma 1 13.93(1.73-112.11) 0.013 severe head trauma 54.40(5.71-517.65) 0.001 hco3 normal acidemia alkalemia 1 1.61(0.70-3.66) 0.255 pco2 normal acidosis 1 1.28(0.57-2.87) 0.540 ph normal acidemia 1 0.92(0.41-2.07) 0.858 hct normal anemia 1 1.23(0.38-3.96) 0.728 sdh no yes 1 1.96(0.69-5.58) 0.205 sah no yes 1 1.60(0.62-4.11) 0.323 edh no yes 1 0.52(0.18-1.45) 0.215 dsf no yes 1 8.92(1.04-75.53) 0.045 pneumocephalus no yes 1 0.35(0.0-3.53) 0.377 contusion no yes 1 1.70(0.27-10.67) 0.569 table 2. simple binary logistic regression to find predicting factors of mortality or: odds ratio; aor: adjusted odds ratio; rr: respiratory rate; hr: heart rate; sbp: systolic blood pressure; dbp: diastolic blood pressure; gcs: glasgow coma scale; hct: hematocrit; sdh: subdural hemorrhage; sah: subarachnoid hemorrhage; edh: epidural hemorrhage; dsf: deep skull fracture 153 patients was the findings of cerebral ct scan taken on admission. analysis of the ct scan findings suggests that non-surviving patients had a higher frequency of subdural hematoma ( sd h) , i nt r a c r a n ia l h e mor r ha ge ( ich ) , depressed skull fracture (dsf), and cerebral contusion, while the patients who recovered with complications had a higher frequency of epidural hematoma (edh). one of the most common consequences of brain trauma is extra-axial hemorrhages, which are categorized as epidural and subdural hemorrhages and subarachnoid hemorrhage (sah). it has been reported that 58% of all patients undergoing hematoma removal and 21% of all patients with severe head trauma have subd ura l he matomas. (2 2) the prevalence of subdural hematoma among our subjects was close to the figures reported in other studies, which suggests that the pattern of brain pathologies following tbi is likely to be independent of the country.(9,11) the association of the discovery of a lesion in ct scan with the outcome of treatment has been already reported in other studies and our results further support these reports.(10,22,23) in the present study, which was conducted on a reasonably large-sized sample, there were significantly more (almost five times more) male tbi patients than female tbi patients. this difference in the gender distribution can be attributed to the men’s riskier social activities and occupations.(24) this finding is consistent with the majority of other studies, which report that men are more likely to sustain tbi than women, although the reported male to female ratio varies between 1.5 and 4.(8,12,25) interestingly, the other study conducted on tbi in iranians reported that its subjects were 85% men and 15% women, figures that are identical to the gender ratio of our patients.(3) however, our comparison of clinical outcomes of tbi shows that gender has no effect on the incidence of death following a tbi. considering that several other studies have reported a similar conclusion,(3,7,26) it can be claimed with sufficient confidence that gender has no impact on the outcome of tbi. our study showed that hypoxemia was not associated with the clinical outcome of tbi. however, we believe that this lack of significant association is purely statistical because while 34% of the patients in the non-surviving group had hypoxia, 30% of the patients who recovered with complications and only 7% of those who recovered without complications had hypoxia, univ med vol. 38 no.3 aor (95%ci) p value age children 0.48(0.007-32.29) 0.735 adults elderly 7.45(2.02-27.36) 0.002 hr normal bradycardia 1 tachycardia 2.51(0.42-15.01) 0.311 head trauma normal minor/moderate head trauma 1 26.87(2.42-298.25) 0.007 severe head trauma 127.97(9.11-1796.28) 0.001 sdh no yes 1 1.86(0.49-6.98) 0.353 dsf no yes 1 28.71(0.99-835.41) 0.051 table 3. multivariate binary logistic regression to find predicting factors of mortality aor: adjusted odds ratio; hr: heart rate; sdh: subdural hemorrhage; dsf: deep skull fracture 154 bolvardi, ahmadzadeh, disfani, et al. prognostic factors of traumatic brain injury which seems clinically significant. in this regard, a study in the united states has shown that brain hypoxia exacerbates the short-term outcome of tbi independent from the raise in intracranial pressure. in contrast, manley et al.,(21) like us, found that hypoxia is not associated with increased mortality in the early stages of recovery. however, we found that factors other than hypoxia, namely acidosis and anemia also lack any effect on the outcome of tbi, a claim that have many opponents and proponents in the literature. for example, while one study has reported that anemia with cerebral hypoxia is a prognostic factor for negative outcome,(19) another study has found that elevation of hematocrit above 28% is not associated with reduce d mortality fr om head trauma; (2 6) moreover, another study in taiwan (20) has shown that initial anemia does not increase the risk of mortality in tbi patients. the present study suffers from several limitations. first, this study did not examine the parameters such as the type of accident leading to tbi, the time between accident and admission, pupil reflex condition, extracranial damage, and patient’s economic status, whereas many studies have claimed that these parameters play a significant role in predicting the clinical outcomes of tbi patients. in addition, the follow-up of this study was performed via phone calls, and this may have led to a higher number of patients being categorized as recovering with complications, because t bi-r ela ted complic ations were recorded as stated by the patients or their family members. we recommend in-depth management of patients with a gcs of less than 8 presenting to the emergency department. conclusion our study demonstrated that severe head trauma in the initial examinations are associated with poorer clinical outcomes after tbi. considering the factors identified to be associated with the outcomes of tbi among iranians, emergency departments are recommended to give a higher priority to the older tbi patients with lower gcs and unstable hemodynamics. acknowledgments this article is derived from the dissertation prepared by dr. maryam ahmadzadeh as a requirement for the completion of the emergency medicine specialty program at the mashhad university of medical sciences (code: 920748). this study was performed with the financial and moral support of t he research deputy department of the mashhad university of medical sciences under a proposal authorized with the permission code 920748. conflict of interest the authors declare that there are no conflicts of interest regarding the publication of this manuscript. author contributions all authors contributed to compilation of the subject matter, writing of the manuscript drafts, data collection, and data analysis. hfd and ma contributed to manuscript finalization. yr and mf contributed to the study concept and design. eb, mf, and fr contributed to manuscript revision. all authors have read and approved the final manuscript. references 1. abou el fadl mh, o’phelan kh. management of traumatic brain injury: an update. neurosurgery clinics north america 2018;29:213-21. doi: 10.1016/ j.nec.2017.11.002. 2. joseph b, khan m, rhee p. non-invasive diagnosis and treatment strategies for traumatic brain injury: an update. j neurosci res 2018;96:589-600. doi: 10.1002/jnr.24132. 3. ziaeirad m, alimohammadi n, irajpour a, et al. association between outcome of severe traumatic brain injury and demographic, clinical, injuryrelated variables of patients. j nursing midwifery res 2018;23:211-6. doi: 10.4103/ijnmr.ijnmr_65 _17. 155 4. zand s, rafi’ei m. causes and duration of hospitalization and mortality rate in intensive care units in markazi province. iranian j crit care nurs 2010;3; 63-7. 5. mrc crash trial collaborators, perel p, arango m, clayton t, et al. predicting outcome after traumatic brain injury: practical prognostic models based on large cohort of international patients. bmj 2008;336:425-9. doi: 10.1136/bmj.39461. 643438.25. 6. centers for disease control and prevention. tbis and injuries : prevent tbis and future unintentional injuries by understanding the risks. atlanta, usa: centers for disease control and prevention;2017. 7. brennan pm, murray gd, teasdale gm. simplifying the use of prognostic information in traumatic brain injury. part 1: the gcs-pupils score: an extended index of clinical severity. j neurosurg 2018;128:1612-20. doi: 10.3171/ 2017.12.jns172780. 8. murray gd, brennan pm, teasdale gm. simplifying the use of prognostic information in traumatic brain injury. part 2: graphical presentation of probabilities. j neurosurg 2018;128:1621-34. doi: 10.3171/2017.12.jns172782. 9. einarsen ce, van der naalt j, jacobs b, et al. moderate traumatic brain injury: clinical characteristics and a prognostic model of 12month outcome. world neurosurg 2018;114: e1199-e1210. doi: 10.1016/j.wneu.2018.03.176. 10. silverberg nd, gardner aj, brubacher jr, et al. systematic review of multivariable prognostic models for mild traumatic brain injury. j neurotrauma 2015;32:517-26. doi: 10.1089/neu. 2014.3600. 11. baum j, entezami p, shah k, et al. predictors of outcomes in traumatic brain injury. world neurosurg 2016;90:525-9. doi: 10.1016/j.wneu. 2015.12.012. 12. lee sh, lim d, kim dh, et al. predictor of isolated trauma in head: a new simple predictor for survival of isolated traumatic brain injury. j emerg med 2018;54:427-34. doi: 10.1016/j.jemermed.2018. 01.001. 13. amyot f, arciniegas db, brazaitis mp, et al. a review of the effectiveness of neuroimaging modalities for the detection of traumatic brain injury. j neurotrauma 2015;32:1693-721. doi: 10.1089/neu.2013.3306. 14. the brain trauma foundation. the american association of neurological surgeons. the joint section on neurotrauma and critical care. guidelines for the management of severe traumatic brain injury 4th ed. the american univ med vol. 38 no.3 association of neurological surgeons and the congress of neurological surgeons;2017. 15. durant e, sporer ka. characteristics of patients with an abnormal glasgow coma scale score in the prehospital setting. western j emerg med 2011;xii: 30-6. 16. kodliwadmath h, koppad sn, desai m, et al. correlation of glasgow outcome score to glasgow coma score assessed at admission. int surg j 2016;3:1959-63. doi: http://dx.doi.org/10.18203/ 2349-2902.isj20163172. 17. yang cj, hsiao ky, su ic, et al. the association between anemia and the mortality of severe traumatic brain injury in emergency department. j trauma 2011;71:e132-5. 18. jacobs b, beems t, stulemeijer m, et al. outcome prediction in mild traumatic brain injury: age and clinical variables are stronger predictors than ct abnormalities. j neurotrauma 2010;27:655-68. doi: 10.1089/neu.2009.1059. 19. savola o, hillbom m. early predictors of postconcussion symptoms in patients with mild head injury. eur j neurol 2003;10:175-81. 20. maloney-wilensky e, gracias v, itkin a, et al. brain tissue oxygen and outcome after severe traumatic brain injury: a systematic review. crit care med 2009;37:2057-63. doi: 10.1097/ccm. 0b013e3181a009f8. 21. bao l, chen d, ding, et al. fever burden is an independent predictor for prognosis of traumatic brain injury. plos one 2014;9:e90956. doi: 10.1371/journal.pone.0090956. 22. githinji kj, kithikii pk, ontita ap, et al. outcome after acute traumatic subdural haemoatoma in kenya: a single-centre experience. african j neurol sci 2010;9:1-3. 23. subodh r, gupta dk, mehta vs. predictors of outcome in acute subdural hematoma with severe head injurya prospective study. indian j neurotrauma 2004;1:37-44. doi: 10.1016/s09730508(04)80008. 24. mousavi bazzaz m, zarifian a, emadzadeh m, et al. driving behaviors in iran: a descriptive study among drivers of mashhad city in 2014. glob j health sci 2015;26;7:39-45. doi: 10.5539/gjhs. v7n7p39. 25. stenberg m, koskinen l-od, jonasson p, et al. computed tomography and clinical outcome in patients with severe traumatic brain injury. brain inj 2017;31:351-8. doi: 10.1080/02699052.2016. 1261303. 26. yuan q, yu j, wu x, et al. prognostic value of coagulation tests for in-hospital mortality in patients with traumatic brain injury. scand j trauma resusc emerg med 2018;26:3. doi: 10.1186/s13049-017-0471-0. oktavianus 60 *department of public health and community medicine, medical faculty, jenderal soedirman university **department of obstetrics and gynecology, faculty of medicine, gadjah mada university ***department of psychiatry, faculty of medicine, gadjah mada university correspondence dr. yudhi wibowo *department of public health and community medicine, medical faculty, jenderal soedirman university phone: email : dr_yudhi_wibowo@yahoo.com univ med 2013;32:60-68 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 introduction pre-eclampsia is a multisystem complication that occurs after 20 weeks of pregnancy and can cause considerable maternal and fetal morbidity and mortality. mental health is influenced by social support, and emotional distress during pregnancy may cause pre-ecclampsia/ecclampsia (pe/e). the objective of this study was to determine the association of social support and common mental disorders (cmd) with pe/e in pregnant women. methods this study was a matched case-control study. cases were pregnant women who had been diagnosed with pe/e. controls were those with normal pregnancies or not diagnosed as pe/e. instruments social support questionnaire-6 (ssq-6) was used to measure social support and self-reporting questionnaire 20 (srq-20) items for measuring cmd. conditional logistic regression was used to estimate matched odds ratios (ors) and 95% confidence intervals (95% ci). results low social support was not directly associated with pe/e. compared with women with high social support, those with low social support had a 26-fold increased risk of cmd (+) (or=26.4, 95% ci: 10.67 to 77.20, p=0.001). several variables significantly associated with pe/e were cmd (+) (or=6.11, 95% ci: 2.99 to 14.07, p=0.001), low family income (or=2.93, 95% ci: 1.56 to 5.82, p=0.001), history of chronic hypertension (+) (or=7.67, 95% ci: 2.32 to 39.89, p=0.001), history of pe/e (+) (p=0.001), and history of hereditary pe/e (+) (or=6, 95% ci: 1.34 to 55.20, p=0.013). conclusion low social support was not directly associated with pe/e but was associated with cmd. to prevent cmd in pregnant women, there is a need for social support from the family. key words: social support, common mental disorders, preeclampsia, eclampsia, pregnancy common mental disorders increase pre-eclampsia/ eclampsia risks in pregnancy y. wibowo*, m hakimi**, and c.r marchira*** 61 gangguan jiwa ringan meningkatkan risiko dengan pre-eklampsia/ eklampsia pada ibu hamil pendahuluan pre-eklamsia merupakan komplikasi multisistem yang terjadi setelah kehamilan 20 minggu dan dapat menimbulkan tingkat morbiditas dan mortalitas maternal dan fetal. dukungan sosial mempengaruhi kesehatan mental maka distres selama kehamilan kemungkinan mengarah kepada pre-eklamsia/eklamsia (pe/e). tujuan penelitian ini adalah untuk menganalisis hubungan dukungan sosial dan gangguan jiwa ringan (cmd) pada ibu hamil dengan pe/e. metode penelitian ini merupakan matched case-conrol. kasus adalah ibu hamil yang telah didiagnosis pe/e. kontrol adalah ibu dengan kehamilan normal atau tidak didiagnosis sebagai pe/e. instrumen penelitian adalah social support questionnaire-6 (ssq-6) yang digunakan untuk mengukur dukungan sosial and self-reporting questionnaire20 (srq-20) untuk mengukur cmd. kondisional regresi logistik digunakan untuk mengestimasi matched odds ratio (or) dan confidence interval 95% (ci 95%). hasil dukungan sosial yang rendah tidak berhubungan langsung dengan pe/e. dibandingkan dengan kelompok dukungan sosial tinggi, kelompok dukungan sosial rendah memiliki 26 kali peningkatan risiko gangguan jiwa ringan (cmd +) (or=26,4, 95% ci: 10,67 to 77,20, p=0,001). variabel yang berhubungan signifikan dengan kejadian pe/e adalah cmd (+) (or=6,11, 95% ci: 2,99 to 14,07, p=0,001), pendapatan keluarga rendah (or=2,93, 95% ci: 1,56 to 5,82, p=0,001), riwayat hipertensi kronik (+) (or=7,67, 95% ci: 2,32 to 39,89, p=0,001), riwayat pe/e (+) (p=0,001), dan riwayat keturunan pe/e (+) (or=6, 95% ci: 1,34 to 55,20, p=0,013). kesimpulan dukungan sosial rendah tidak berhungan langsung dengan pe/e akan tetapi berhubungan dengan cmd. untuk pencegahan cmd pada kehamilan diperlukan dukungan sosial dari keluarga. kata kunci: dukungan sosial, gangguan jiwa ringan, preeklamsia, eklamsia, kehamilan abstrak introduction the maternal mortality rate (mmr) in indonesia of 228/100000 live births (lb) is higher than in other asean countries.(1) in sukoharjo district in 2010, mmr of 152.28/100000 lb was over the national target of 117.1/100000 lb. the direct cause of maternal death is the high preeclampsia/eclampsia (pe/e) (47.62%) and since the years 2007-2011, pe/e has been the direct cause of the highest maternal mortality rate. pe is a multi-systemic syndrome usually recognized by new-onset hypertension and proteinuria in the second half of pregnancy.(2,3) common mental disorders (cmd) in pregnancy are depressive and anxiety disorders that are classified in icd10 as “neurotic, stress-related and somatoform disorders” and “mood disorders”.(4,5) distress conditions (depression, anxiety and stress) during pregnancy can induce pe.(6) family structure modifies the association but, contrary to expectations, spousal emotional support is a stronger correlate of antenatal depression in traditional rather than nuclear family settings.(7) univ med vol. 32 no.1 62 depression and anxiety in early pregnancy are a s s o c i a t e d w i t h a 2 . 5 a n d 3 . 2 f o l d r i s k , respectively, for subsequent pe. mental stress is frequently associated with pe.(8) the world health organization has estimated that the prevalence of mental disorders in pregnant women in low-to-middle income countries is 1041.2%.(9) in brazil, prevalence of cmd in pregnant women in low social economic is 20.2%.(10) the prevalence of mental emotional disorders in indonesia’s population aged >15 years is 11.6%.(11) chronic hypertension is an i n d e p e n d e n t r i s k f a c t o r f o r s e v e r e preeclampsia.(12) subjects living alone or as couples are significantly more likely to suffer from severe hypertension in comparison to those living in extended families.(13) sukoharjo district was the 4th district with a high prevalence of p r i m a r y h y p e r t e n s i o n o f 1 0 . 8 9 % . ( 1 4 ) a particularly important potential consequence of global population expansion and trans-national and rural-urban migration has been the disruption of traditional family-based support structures. the association between social support, psychological stress and pregnancy outcome is undoubtedly complex. the importance and implications of social support during pregnancy remain incompletely understood.(15) therefore this study was conducted to analyze the association of social support and cmd with pe/ e in pregnancy. methods research design this study was a facility-based matched case-control study in sukoharjo district, conducted from january 2010-december 2011. study subjects cases were pregnant women who had been diagnosed as having pe/e by hospital doctors (blood pressure >140/90 mmhg, with proteinuria and or seizures). controls were pregnant women with normal pregnancies or not diagnosed as pe/ e by health personnel (doctors, midwives or nurses) in primary health care, based on medical records (mr), register cohort of mothers and maternal & child health (mch) book. cases and controls were matched on maternal age and parity. calculation of the sample size was based on the results of previous studies and odds ratio (or) discordant proportion (ð) =0.4, using 95% confidence level with á =5%, power (1-â) =80% and or gained 2.0 to 3.2; at least 82 respondents for both case and control groups. (16) measurements for interview of participants a structured questionnaire was used, comprising items on age, family structure, parity, inter-pregnancy interval, history of antenatal care (anc), history of contraception used, history of abortion, history of pe/e, history of hereditary pe/e, and history of chronic hypertension. total family income is the income of all family members per month, and high if higher than or equal to the minimum regional salary. the social support questionnaire-6 (ssq6) is one’s perception that he endeared, appreciated and be a part community, and a score is considered high if it is higher than or equal to the median value. the validity of the ssq-6 in indonesia for measuring social support has been tested, as has been the validity of selfreporting questionnaire 20 items (srq-20) to measure common mental disorders (cmd).(17,18) the srq-20 consists of twenty questions, which have to be answered by ‘yes’ (1) or ‘no’ (0). item scores are summarized to obtain a total score. a score higher than or equal to the cutoff point of 6 indicates the existence of a mild mental disorder. the sensitivity and specificity o f s r q 2 0 a r e 8 4 . 8 % a n d 8 3 . 7 % , respectively.(19) data analysis all data analyses were performed using stata version 11. to analyze the association of social support and cmd, bivariate analysis was done by the chi-square test. to identify the factors associated with pe/e, bivariate matched wibowo, hakimi, marchira common mental disorders pregnancy 63 analysis was done by comparing the cases and controls (1 case: 1 control) for each variable of interest and crude matched odds ratio and their 95% confidence intervals along with p values were calculated using mcnemar ’s test. in multivariate analysis, matched analysis in logistic regression was performed to identify associated factors of pe/e while adjusting for other variables. finally any variable with p-value > 0.05, and not a confounder or interacting with other variables, were removed from the model to obtain a parsimonious and biologically meaningful model that best explains the phenomena of pe/e. ethical clearance written informed consent was obtained from all women, and the study was approved by the ethical review board of gadjah mada university. results the socio-demographic, obstetric and antenatal characteristics of all participants are presented in table 1. the actual sample size achieved was a total of 143 cases matched on maternal age and parity with 143 controls participating in the study of preeclampsia risk factors. there were 103 nulliparous/primiparous women and 40 multiparous women with single pregnancy. no significant differences were observed between cases and controls with regard to family structure, social support, maternal age, inter-pregnancy interval, parity, history of abortion, pre-pregnany bmi, history of anc, and history of contraception used. a total of 45.10% of respondents had low social support, and 27.27% of respondents were positive for cmd (table 1). table 2 shows that some of the variables significantly associated with the incidence of pe/ e are: cmd (+) (p<0.01, or =6.11, 95% ci: 2.99 to 14.07), low family income (p<0.01; or =2.93, 95% ci: 1.56 to 5.82), history of chronic hypertension (+) (p<0.01, or =7.67, 95% ci : 2.32 to 39.89), and history of hereditary of pe/ e (+) (p=0.01, or=6. 95% ci: 10.34 to 55.20). multivariate analysis was performed to obtain any factors associated with pe/e (table 3). the second model is better than the first model, as can be seen from the difference bic1bic2 =-4,625, which means a positive support t h e s e c o n d m o d e l . h o w e v e r, p s e u d o r 2 =0,3254, which means that the independent variable in the equation of the second model is able to explain the relationship between the pe/ e incidence of 32,54% and while the remaining 67.46% is explained by the other variables discussion low family income per month is significantly associated with incidence of pe/e. low socioeconomic status is a strong risk factor for incident pe. (20) another study found that socioeconomic status is not associated with the o c c u r r e n c e p e . ( 5 , 2 1 ) s o m e r e s e a r c h e r s concluded that women with more advanced socioeconomic rarely infected with pe, whereas in epidemiological studies well controlled, found that the incidence of pe did not differ between s o c i o e c o n o m i c c l a s s e s . (3) i n l o w e r socioeconomic groups is likely to have lifestyle/ behavior of a lack of exercise and poor nutritional intake. lack of exercise has been associated with cardiovascular health, diabetes and others leading to poor of pregnancy outcomes.(22) r e g u l a r e x e r c i s e c a n r e d u c e t h e r i s k o f hypertensive disorders in pregnancy.(23) lower socioeconomic groups are also related to the daily nutrient intake, weight gain patterns during pregnancy and what to eat contribute to a poorer pregnancy outcomes.(24) in countries with lowand middle-income, pregnant women are at higher risk for experiencing cmd because of the increased burden of risk factors such as lack of economic, domestic violence, drug abuse and lack of support sosial.(10) the results of this study showed no direct relationship with the incidence of social support pe/e. psychosocial assets have a buffering univ med vol. 32 no.1 64 table 1. comparability between cases and controls by characteristics of respondents *cmd=common mental disorders; @ pe/e= pre-eclampsai/eclampsia; $ bmi= body mass index; # anc = ante natal care effect on the events of life, pregnant women who report many change of life events during pregnancy, low psychosocial assets had a higher complication rate compared to women with high psychosocial assets. psychosocial assets are measured in five dimensions: ego strength, quality of marriage, relationships with family, social resources, including patterns of friendship and feelings about the pregnancy. however, mothers with many changes of life and low support had the highest complication rates. the assumption that the tangible support alone is not wibowo, hakimi, marchira common mental disorders pregnancy 65 table 2. relation between maternal socio-demographic characteristics and obstetrical history with pe/e * mc nemar test; ** cannot be calculated because all cells have zero values ; @ bmi =body mass index; # anc : ante natal care; $ pe/e =pre-eclampsia-eclapmsia; ¶cmd= common mental disorders table 3. modeling of conditional logistic multiple regression between social support plus external variables and pe/e *aor=adjusted odds ratio by family income, history of hypertension, hereditary pe/e, and inter-pregnancy interval; @ cmd=common mental disorders; # pe/e =pre-eclampsia/eclampsia; $ lr= likelihood ratio; ¶ bic=bayesian information criterion a significant predictor because it is always combined with the emotional support for the prediction of pregnancy outcome, this is consistent with the buffering effect stres. another study found that systolic and diastolic blood pressure higher score was positively related to higher depressive symptoms, but did not correlate with the score of the support sosial.(25) while other studies have found that lack of social support lead to greater depressive symptoms, has also been associated with high blood pressure among american afrika.(26) chronic hypertension is an independent risk factor for the incidence of pe/e, especially superimposed preeclampsia.(27-29) hypertension can be caused by vasospasm or vasoconstriction. vasospasm itself may cause damage to blood vessels. this change will cause endothelial damage and leakage in sub-endothelial cells that cause blood constituents, including platelets and univ med vol. 32 no.1 66 fibrinogen precipitate in subendotel.(3) results of multivariable analysis showed that low social support increased the risk 1.38 times the incidence of pe/e after controlling for a history of chronic hypertension variable, although still not statistically significant. in this study it was found that the cases reported to have high social support. the case of pregnant women with pe, including high risk and will be monitored closely by a midwife during anc services including m o r e f r e q u e n t a n c v i s i t s , h o m e v i s i t s , monitoring by phone, measurement of blood pressure and asks for the development through changes in symptoms and examination of urine protein. in addition, there are habits of pregnant women, especially for a first pregnancy at 8 months of gestation aged, pregnant women will be reunited with their parents or their families for childbirth preparation. this is likely to contribute to the perception of high social support. although there is no direct relationship between social support with the incidence of pe/ e, but there are differences in low social support in pregnant women with and without cmd ( 9 2 . 3 % v s . 3 1 . 3 % ) . l o w s o c i a l s u p p o r t increased the risk 26.4 times for the event cmd.the results of a prevalence study of the prevalence of other cmd increased in pregnant w o m e n w h o d o n o t h a v e r e l a t e s i n t h e community and lived in crowded household.(10) crowded household as a chronic stressor adverse psychological well-being, contribute to psychological distress, unhappiness, irritability can even cause a person to think of suicide. lack of privacy and the perceived demands in the crowded household, is also strongly and consistently associated with welfare. families with psychosomatic complaints have redundant protection characteristics (overprotection), excessive involvement (over involvement) family members with one another and excessive pressure (overemphasis) in the role of social nurture support.(30) social support have an influence on depressive symptoms in mothers pregnant.(31) to clarify why social support affects physical and mental health conditions, more study is needed to determine how life events experienced by respondents and specify social support in the form of emotional support, instrumental support, the support judgment or support information as well as how the quality of relationships within the family. it is estimated that social support can affect physical and mental health through its influence on emotions, cognition and behavior. good social support can act as a buffer against stress, thus preventing of diseases.(32) combination of oxytocin and social support showed the lowest cortisol concentration and calmness and decreased anxiety during stress.(33) the results obtained that cmd increases the risk six times more to the incidence of pe/ e. the results of other studies found depression and anxiety in early pregnancy is associated with risk pe. similarly, a history of anxiety and mood disorders in pregnant women increases the risk 2.13 times the incidence of pe after controlling for the variables age, race, and bmi before pregnant.(34) the relationship between cmd with the incidence of pe/e can be proved biologically plausible. conditions of distress (depression, anxiety and stress) during pregnancy leads to the incidence of pe with elevated levels of cortisol and changes in the sensitivity of lymphocytes to glukokortikoid. high levels of cortisol during stress conditions may also induce hypertension and dysfunction endotel..(6) several limitations must be considered when interpreting the results from our study. first of all recall bias and inability to establish temporality between preeclampsia and certain variables are inherent due to the matched case control study design. we cannot exclude the possibility that our results could be partially confounded by unidentified risk factors. prepregnancy weights and body mass index (bmi) were assessed by subtracting the average weight gain from the full term maternal weight which might not be a good proxy measure as well as social support. wibowo, hakimi, marchira common mental disorders pregnancy 67 conclusions low social support is not directly associated to the incidence of pe/e. cmd is more common in pregnant women with pe/e. future study is needed to clarify the influence of low social support on the incidence of pe/e by way of improved methodology, specify social support into the emotional support and instrumental support and the quality of relationships within the family. acknowledgements i am thanksful to post-graduate of public health, gadjah mada university, sukoharjo health district, community health centers, n i r m a l a s u r i h o s p i t a l , ya r s i s h o s p i t a l , sukoharjo hospital and moewardi hospital. references 1. badan perencanaan dan pembangunan nasional (bappenas). laporan pencapaian tujuan pembangunan milenium di indonesia 2010. jakarta: kppn/bappenas;2010. 2. trogstad l, magnus p, stoltenberg c. preeclampsia: risk factors and causal models. best pract res clin obstet gynaecol 2011;25:329–42. 3. cunningham fg, kenneth jl, bloom sl, hauth jc, rouse dj, et al. williams obstetrics. 23rd ed. new york: mcgraw-hill companies inc;2010. 4. patel v, kleinman a. poverty and common mental disorders in developing countries. bull world health organ 2003;81:609–15. 5. santos is, matijasevich a, tavares bf, da cruz lima ac, riegel re, lopes bc. comparing validity of edinburgh scale and srq20 in screening for post-partum depression. clin pract epidemiol ment health 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calderon-margalit r, cripe sm, sorensen tk. preeclampsia risk in relation to maternal mood and anxiety disorders diagnosed before or during early pregnancy. am j hypertens 2009;22:397–402. wibowo, hakimi, marchira common mental disorders pregnancy oktavianus 155 *department of physiology, faculty of medicine, trisakti university **department of physiology, faculty of medicine, university of indonesia correspondence dr. fransisca chondro department of physiology, faculty of medicine, trisakti university, jl. kyai tapa no.260 grogol jakarta phone : +6221-5672731 ext. 2804 email: fransisca.chondro@gmail.com univ med 2013;32:155-64 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background intercellular communication in cardiac muscle is effected by connexin, particularly connexin43 (cx43), forming gap junctions in cardiomyocytes. aerobic physical exercise may result in increased left ventricular size and function. the purpose of the present study was to assess the effect of aerobics and detraining on c43 expression and distribution in rats. methods this was an in vivo experimental study on 32 young male wistar rats. the animals were divided into the following 8 groups and their interventions: i : 4 weeks of aerobics (a4); ii: exercise control for 4 weeks (c4); iii: 4 weeks of aerobics plus 4 weeks of detraining (a4d4); iv: 8-week control (c8); v: 12 weeks of aerobics (a12); vi: 12-week control (c12); vii: 12 weeks of aerobics plus 4 weeks of detraining (a12d4); viii: 16-week control (c16). rat cardiac tissue was subjected to immunohistochemical assay to determine total cx43, intercalated disc cx43, and lateral cx43. independent t-test was used to compare all cx43 parameters between control and treatment groups. results after aerobics, there were significant differences in total cx43, intercalated disc cx43, lateral cx43, and intercalated disc cx43 and lateral cx43 percentages between control and treatment groups (p<0.05). between-group comparison in treatment groups did not find significant differences between exercise-only groups and groups with exercise plus detraining. cx43 surface area tended to decrease after detraining. conclusions aerobics significantly increases cx43 levels in rat hearts. detraining decreases intercalated disc cx43 and lateral cx43. key words: connexin43, gap junction, lateralization, aerobic physical exercise, rat. aerobic exercise increases connexin43 expression in rat cardiac muscle fransisca chondro*, minarma siagian**, and dewi is santoso** 156 chondro, siagian, santoso exercise increases connexin43 in rat latihan fisik erobik meningkatkan ekspresi protein connexin43 pada sel otot jantung tikus latar belakang komunikasi antar sel otot jantung terjadi dengan bantuan protein connexin, terutama connexin43 (cx43), yang merupakan protein utama penyusun gap junction pada sel otot jantung. latihan fisik erobik dapat mengakibatkan peningkatan ukuran dan kerja ventrikel kiri. penelitian ini bertujuan untuk menilai pengaruh latihan fisik erobik dan detraining terhadap ekspresi dan distribusi protein c43 pada tikus. metode studi eksperimental secara in vivo pada 32 ekor tikus jantan dewasa muda strain wistar. subjek penelitian dibagi menjadi 8 kelompok, yakni : kelompok i : tikus diberi perlakuan erobik selama 4 minggu; ii kontrol 4 minggu; iii erobik 4 minggu + detraining 4 minggu; iv kontrol 8 minggu; v erobik selama 12 minggu; vi kontrol 12 minggu; vii erobik 12 minggu + detraining 4 minggu; dan viii kontrol 16 minggu. pada organ jantung tikus dilakukan pemeriksaan imunohistokimia untuk mengukur kadar total cx43, cx43 diskus interkalaris, dan cx43 lateral. uji t-independen digunakan untuk membandingkan semua parameter cx43 antara kedua kelompok perlakuan. hasil setelah diberikan latihan erobik, terdapat perbedaan bermakna pada parameter total cx43, cx43 diskus interkalaris, cx43 lateral, dan presentase cx43 diskus interkalaris dan lateral antara kedua kelompok perlakuan (p<0,05). pada perbandingan antara kelompok perlakuan, tidak terdapat perbedaan bermakna antara kelompok yang diberi latihan fisik saja dengan kelompok yang diberi latihan fisik dan detraining. namun terjadi kecenderungan terjadi penurunan luas area cx43 pada pemberian detraining. kesimpulan latihan fisik erobik meningkatkan kadar cx43 secara bermakna pada organ jantung tikus. proses detrain mengakibatkan penurunan cx43 diskus interkalaris dan lateral.. kata kunci : connexin43, gap junction, lateralisasi, latihan fisik erobik, tikus abstrak introduction in the human heart there are around 2 to 3 milliard cardiac muscle cells. the functioning of the heart is extremely complex. one of the factors enabling the functioning of the heart is intercellular communication, e.g. via gap junctions. communication between cardiac muscle cells occurs through gap junction channels, allowing the rapid transmission of impulses and the synchronous contraction of the heart as a whole. therefore the maintenance of gap junction functions is essential to guarantee the normal functioning of the heart.(1,2) the gap junction channels of the ventricular myocardium (especially in the intercalated discs) are formed m a i n l y b y c o n n e x i n 4 3 ( c x 4 3 ) , a l l o w i n g electrical coupling and communication between a d j a c e n t c a r d i o m y o c y t e s . ( 3 ) i n p o s t n a t a l development of the ventricle, the connexin proteins of the gap junctions, which in the prenatal period are laterally located, accumulate a t t h e i n t e r c a l a t e d d i s c s t o g e t h e r w i t h desmosomes and adherens junctions. the mean size of the gap junctions also increases with the postnatal ventricular development.(4,5) 157 univ med vol. 32 no.3 one of the characteristic cardiac structural abnormalities is the lateral redistribution of cx43 proteins and decreased cx43 protein expression. decreased colocalization of cx43 with zonula occludens-1 (zo-1) proteins and cadherins, and morphologic heterogeneity of the l a t e r a l i z e d g a p j u n c t i o n s i n d i c a t e s t h e occurrence of changes in their structure and function.(6) decreased cx43 expression results in increased resistance at the gap junction, decreased transmission velocity of electrical impulses, and increased duration of action potential formation, all three of which lead to arrhythmia.(7) the study by molmen-hansen et al. showed that regular high-intensity physical exercise is necessary to induce changes in substrate and energy source utilization by the heart.(8) high-intensity physical exercise also increases mitochondrial respiration in the cardiomyocytes. high-intensity physical exercise is expected to induce cardiac adaptations in the form of increases in contractility, glucose oxidation, and mitochondrial functions.(9) at the onset of hypertrophy, the amount of regulated connexin is increased. however, in longstanding hypertrophy with impending heart failure, the amount of connexin is decreased. to date the precise mechanism and the signalling pathway regulating connexin expression is still not known with certainty. kostin et al. found that cx43 expression is increased in the compensated hypertrophic stage, but decreases and becomes heterogeneous in decompensation. (10) the d e c r e a s e d c x 4 3 p r o t e i n e x p r e s s i o n i s accompanied by decreased cx43 mrna, so that the decreased cx43 expression presumably involves transcription factors.(11) aerobic physical exercise results in two types of stress, i.e. oxidative stress and vascular strain. to date there have been a few studies to investigate the effect of aerobic physical exercise on the expression of connexin43. in the study conducted by bearden et al., the investigators came to the conclusion that chronic physical exercise does cause changes in connexin43 mrna expression. however, it could not be concluded yet that chronic physical exercise causes changes in connexin protein expression.(12) in view of the inconsistent study results, the present study was carried out to evaluate the effect of aerobic physical exercise and detraining on cx43 protein expression in rat myocardiocytes. methods design of the study an experimental study conducted from november 2012 until march 2013 at the pathological anatomy laboratory, university of indonesia. subjects of the study the subjects in this study consisted of young adult male wistar rats weighing 150– 250 grams, 8 weeks old at the start of the study, healthy, and fit to participate in the aerobic physical exercise program. the sample size was determined with the federer formula: (t-1)(n1) >15, where t = number of experimental groups (8); n = number of animals per group. the calculated sample size was 4 animals per group, giving a total of 32 animals. intervention the study subjects were divided into 8 groups, consisting of 4 control groups (coded c4, c8, c12, c16) and 4 treatment groups (a4, a12; a4d4, a12d4). the treatment groups were subjected to a single daily aerobics exercise of 20 minutes, at a treadmill velocity of 20 m/minute, with a rest period of 90 seconds every 5 minutes. as to the control exercise program, the animals were also put into the treadmill for 20 minutes, but with the treadmill inactive. in the detraining phase, the aerobics exercise program was stopped for a given period, during which the animals were left to themselves in their cages, without being placed in the treadmill. the treatment schema was as 158 chondro, siagian, santoso exercise increases connexin43 in rat follows: group i: 4 weeks of aerobics only (a4); group ii: 4 weeks of control (c4); group iii: 4 weeks of aerobics plus 4 weeks of detraining (a4d4); group iv: 8 weeks of control (c8); group v: 12 weeks of aerobics (a12); group vi: 12 weeks of control (c12); group vii: 12 weeks of aerobics plus 4 weeks of detraining (a12d4); group viii: 16 weeks of control (c16). cx43 immunohistochemical assay detection of cx43 protein in the intercalated discs and the lateral domains of cardiomyocytes was done by immunohistochemical assay. paraffin-embedded rat cardiac tissue blocks were sectioned with a microtome and mounted on glass slides. the sections were then deparaffinized by immersing in xylol three times, each time for 5 minutes. rehydration was done by immersion in graded alcohols (absolute, 96%, and 80%), for 4 minutes each. the rehydrated tissue sections were washed under running water for 5 minutes. subsequently, endogenous peroxides were blocked with 0.5% h 2 o 2 in methanol for 30 minutes. after again washing the slides under running water for 5 minutes, they were subjected to pretreatment with citrate buffer in a microwave oven and left to cool. the cooled slides were washed in phosphate buffered saline (pbs) at ph 7.4 for 5 minutes, followed by background sniper protein blocker (biocare medical) using donor horse serum 3% solution for 30 minutes at room temperature. after 30 minutes, the donor horse serum 3% solution was drained, and to the s l i d e s w a s a d d e d 2 0 0 ì l p r i m a r y a n t i connexin43/gja1 antibody (abcam ab11370) at a dilution of 1/5000, and the slides incubated at room temperature for 1 hour. then the slides were washed in pbs at ph 7.4 for 5 minutes, a n d 4 d r o p s o f tr e k k i e u n i v e r s a l l i n k biotinylated secondary antibody (part of starr trek detection system from biocare medical) was added to the slides, which were then incubated for 15 minutes at room temperature. thereafter the slides were was washed in pbs at ph 7.4 for 5 minutes, dried, and 5 drops of h o r s e r a d i s h p e r o x i d a s e ( h r p ) l a b e l e d streptavidin (trekavidin-hrp label, biocare medical) were added and the slides again incubated at room temperature for 15 minutes. after washing in pbs at ph 7.4 for 5 minutes, the slides were dried and 200 ì l of chromogen (diaminobenzidine solution) was added to each slide and left to react for 2 minutes. after 2 minutes, the slides were washed under running water for 10 minutes, then counterstained with hematoxylin for 1 minute, followed by washing under running water for 5 minutes, to be reacted with a saturated solution of lithium carbonate (5% in distilled water) for 1 minute, and again washed under running water for 3 minutes. this was followed by dehydration of the slides in graded alcohols (80%, 96%, absolute) for 5 minutes each. the slides were cleared three times with xylol, for 5 minutes each. entellan mounting fluid was added and the slides provided with a coverslip. image processing using imagej program the immunohistochemical slides were s u b s e q u e n t l y e x a m i n e d u n d e r t h e l i g h t microscope, photographed, and the photographic images processed using the imagej program. for cx43, all images were recorded using the same settings and the pixel intensities were quantified using imagej software. the whole surface area o c c u p i e d b y t h e c x 4 3 p r o t e i n i n t h e cardiomyocytes was calculated to determine the total cx43 area and the intercalated disc cx43 and lateral domain cx43 (expressed in pixels). statistical analysis for subgroup comparison of control versus treatment groups, data analysis was performed using the independent t-test, while for overall comparison of all control and treatment groups (c4, c12, c16, a4, a12, a4d4, a12d4), the data were analyzed by means of one-way anova. the analysis was done at a significance level of 95% (á =0.05), and differences were considered significant at p<0.05. 159 univ med vol. 32 no.3 ethical clearance the protocol for the present study was approved by the ethical committee, faculty of medicine, university of indonesia. results initially the cx43 levels of the 4-week aerobic exercise control group (c4) versus the treatment group (a4) were compared, as was also done with cx43 levels of c8 and a4d4 (treatment group with 4 weeks of aerobics followed by 4 weeks of detraining) (table 1). from the results it was concluded that there were significant differences between control and treatment groups with regard to levels of total cx43, intercalated disc cx43, lateral cx43, and lateral cx43 percentage. there was a 16.1% reduction in total cx43 levels in group a4d4, in comparison with group a4 (4 weeks of aerobics without detraining) (table 1). after 12 weeks of aerobics and 12 weeks of aerobics followed by 4 weeks of detraining, there were significant differences in cx43, intercalated disc cx43, lateral cx43, and intercalated disc cx43 and lateral cx43 percentages (table 2). table 1. comparison of cx43 levels between control and treatment groups after 4 weeks of aerobic physical exercise and 4 weeks of detraining cx = connexin; c4 = 4 weeks of control; a4 = 4 weeks of aerobics only ; a4d4 = 4 weeks of aerobics plus 4 weeks of detraining; c8 = 8 weeks of control after 12 weeks of aerobics followed by 4 weeks of detraining, there was a 7.1% decrease in total cx43 levels, in comparison with cx43 levels in the group performing only aerobics for 12 weeks. in the comparison of the 4 control groups for total cx4r3, there was only a significant difference between 8 weeks and 12 weeks of control exercise, at p=0.002 (p<0.05). similarly for intercalated disc cx43, there was only a significant difference between 8 weeks and 12 weeks of control exercise, at p=0.001 (p<0.05). for the parameters of lateral cx43, intercalated disc cx43 and lateral cx43 percentages, there were no significant differences between the 4 control groups. regarding the between-group comparison of treatment groups, a comparison was carried out between group a4 and group a4d4. also compared were group a12 and a12d4, as well a s g r o u p a 4 a n d g r o u p a 1 2 d 4 . f o r t h e parameter of total cx43, a significant difference was found between group a4 and group a4d4. total cx43 between groups with a4 and a12 did not show significant differences (table 3). 160 chondro, siagian, santoso exercise increases connexin43 in rat table 2. comparison of cx43 levels between control and treatment groups after 12 weeks of aerobic physical exercise and 4 weeks of detraining cx : connexin c12 : 12 weeks of control; a12 : 12 weeks of aerobics only; c16 :16 weeks of control; a12d4: 12 weeks of aerobics plus 4 weeks of detraining discussion from the results of an overall comparison between treatment groups and control groups, i t m a y b e c o n c l u d e d t h a t a l t h o u g h t h e experimental animals were of the same age, physical exercise had a substantial effect on the amount of cx43 protein present in their cardiac tissues. in all aerobic physical exercise groups, there was an increase in the amounts of total cx43, intercalated disc cx43, and lateral cx43, a s w e l l a s a n i n c r e a s e i n l a t e r a l c x 4 3 p e r c e n t a g e . f o r i n t e r c a l a t e d d i s c c x 4 3 percentage, lower values were found in the treatment groups than in the control groups. this reduction in intercalated disc cx43 percentage may be due to the fact that although t h e r e w a s a n i n c r e a s e i n t h e a m o u n t o f intercalated disc cx43, there was also a significant increase in the amount of lateral cx43, leading to a far greater increase in total cx43. the intercalated disc cx43 percentage was obtained by comparing intercalated disc cx43 and total cx43, and because the increase in total cx43 was far higher than the increase in disc cx43, the calculated cx43 percentage was lower. therefore, in spite of decreased intercalated disc cx43 percentage, there was an increase in the amounts of intercalated disc cx43. this finding is in agreement with the results of the study by bao et al.(12) in vascular smooth muscle and endothelial cell cultures, stating that stress from aerobic physical exercise causes increases in cx43 mrna and protein. however, this is in contrast with the view of bearden et al.(13) who state that chronic physical exercise does not cause changes in cx43 mrna expression and that it cannot be concluded that chronic physical exercise causes changes in connexin protein expression. from the subgroup comparison of all control groups by one-way anova, it may be concluded that age differences between the control groups did not lead to significant differences in the amounts of lateral cx43, intercalated disc cx43 percentages, and lateral cx43 percentages. although significant age differences were found between total cx43 and intercalated disc cx43, these were present only in some groups, namely in the comparison between the groups with 8 weeks and 12 weeks of aerobics. therefore it cannot be concluded that increasing age leads to increased cx43 protein. this does not agree with the findings 161 univ med vol. 32 no.3 t ab le 3 . a n al y si s o f v ar ia n ce a n d m u lt ip le c o m p ar is o n s o f c x 4 3 le v el s b et w ee n a ll c o n tr o l a n d tr ea tm en t g ro u p s c x : co nn ex in a 4 : 4 w ee ks o f ae ro bi cs o nl y; c 4 : 4 w ee ks o f co nt ro l; a 4d 4 : 4 w ee ks o f ae ro bi cs p lu s 4 w ee ks o f de tr ai ni ng ;: c 8 : 8 w ee ks o f co nt ro l; a 12 : 1 2 w ee ks o f ae ro bi cs o nl y; c 12 : 1 2 w ee ks of c on tr ol ; a 12 d 4 : 12 w ee ks o f ae ro bi cs p lu s 4 w ee ks o f de tr ai ni ng ; c 16 : 1 6 w ee ks o f co nt ro l. t he d if fe re nc e in s up er sc ri pt s in di ca te s a si gn if ic an t di ff er en ce f ro m t he c on tr ol ( p< 0. 05 ) 162 chondro, siagian, santoso exercise increases connexin43 in rat of barker et al., who stated that in post-natal development of the cardiac ventricles there is an increase in intercalated disc cx43.(14) on further consideration of the data on intercalated disc cx43 and lateral cx43 percentages, although no statistically significant differences were found between control groups, intercalated disc and lateral cx43 values in the control tended to be stable. therefore we may conclude that increasing age of the experimental animals in the control groups did not result in significant cx43 differences between the control groups. comparison of the treatment groups was limited to find any effects of duration of physical exercise and detraining, therefore only the following pairs of groups were compared: groups a4 and a12; groups a4 and a4d4; and groups a12 and a12d4. in the comparison of groups a4 and a12 to find any effect of exercise dose on amount of cx43, no significant differences were found between all parameters. after 12 weeks of aerobics plus 4 weeks of detraining, the reduction in total cx43 (7.1%) was lower than after 4 weeks of aerobics plus 4 weeks of detraining (16.1%). with increasing duration of exercise, there was a proportionally diminishing effect of detraining on total cx43 levels. the data also show that 12 weeks of aerobic physical exercise results in increased intercalated disc cx43 in comparison with 4 weeks of exercise, so that we may conclude that long-term aerobics is very beneficial for health. in the comparisons to evaluate the effect of detraining on aerobic physical exercise, significant differences were found only in the comparison of total cx43 between group a4 and a4d4. for the parameters intercalated disc cx43 protein, lateral cx43, intercalated disc cx43 percentage, and lateral cx43 percentage, no significant between-group differences were found. on the basis of these data it may be concluded that the 4-week detraining process after 4 weeks of aerobic physical exercise yielded significant differences in the amounts of total cx43. this is consistent with the studies by benito et al.(15) and pelliccia et al.(16) who stated that the cardiac structural and functional changes occurring during the adaptation process caused by physical exercise disappears within 8–13 weeks. although for the other parameters no significant differences were found, from a consideration of the results on surface area and percentages of intercalated disc and lateral cx43, we can see that the 4-week detraining process causes a reduction in the amounts of intercalated disc cx43, lateral cx43, and lateral cx43 percentage. for the intercalated disc cx43 there was a rebound, which is similar to the findings of gamelin et al.(17) and sloan et al.,(18) who revealed that chronic adaptation due to physical exercise disappears upon detraining. our study results showed that after 12 weeks of aerobic physical exercise followed by 4 weeks of detraining, there were no significant differences in cx43 levels as compared to 4 weeks of aerobic physical exercise followed by 4 weeks of detraining. our study results are essentially in agreement with those of previous studies showing that the changes after detraining may be seen after 28 days.(19) however, upon a closer look at the obtained data, it may be c o n c l u d e d t h a t , a l t h o u g h t h e r e w e r e n o significant differences among the parameters, 4 weeks of detraining still leads to a reduction in the areas of total cx43, intercalated disc cx43, lateral cx43 lateral, and in lateral cx43 percentage. as for intercalated disc cx43 percentage, there is an increase after the detraining process. this is because the reduction in the amount of lateral cx43 is proportionally greater that that in the amount of intercalated disc cx43, so that in spite of a reduced amount of intercalated disc cx43, its percentage is increased after the detraining process. this is in agreement with the conclusions of previous studies, revealing similar effects, in that chronic adaptation due to physical exercise disappears if the individual is subjected to the detraining process.(15,20) in this connection it is of interest to note that in postinfarction ischemic cardiomyopathy 163 and other types of heart failure, the expression of cx43 is downregulated.(21) one of the limitations of our study is the use of an ihc assay using anti-connexin43 only, so that all of the cx43 proteins are detected, namely those active in gap junction formation as well as those that are inactive. since active cx43 proteins are those phosphorylated at their carboxyl terminal end, the accuracy of the cx43 protein measurements may be increased by the addition of another antibody, that is capable of detecting phosphorylated cx43. the use of two types of antibody allows us to find the amount of actively gap junction forming cx43 protein, both in the intercalated discs and in the lateral domains of the cardiomyocytes. in this way we m a y a s c e r t a i n w h e t h e r p h y s i o l o g i c a l hypertrophy due to aerobic physical exercise is able to increase the amount of cx43 protein in the lateral domains of the cardiomyocytes. in other words, whether it results in increased numbers of gap junctions in the lateral regions o f t h e c a r d i o m y o c y t e s , t h u s c a u s i n g a transversal propagation of electrical impulses that is one of the causes of arrhythmia. conclusions this study demonstrates that cx43 has an important and previously unknown modulatory effect in myocardial energy metabolism, as evidenced by the increased levels of protein expression after 4 and 12 weeks of aerobic physical exercise. these changes in cx43 expression and distribution as a result of physical activity will diminish in the detraining process. the occurrence of these significant differences indicate the need for further in-depth investigation of the cardioprotective role of cx43 proteins and its adaptation to ischemic conditions. references 1. tirziu d, giordano f, simons m. cell communications in the heart. circulation 2010; 122:928-37. 2. danik sb, yu lf, jie z, suk hj, morley ge, fishman gi, et al. modulation of cardiac gap junction expression and arrhythmic susceptibility. circ res 2004;95:1035-41. 3. desplantez t, dupont e, severs nj, weingart r. gap junction channels and cardiac impulse propagation. j membr biol 2007;218:13–28. 4. hesketh gg, shah mh, halperin vl, cooke ca, akar fg, yen te, et al. ultrastructure and regulation of lateralized connexin43 in the failing heart. circ res 2010;106:1153-63. 5. sato t, ohkusa t, honjo h, suzuki s, yoshida m, ishiguro ys, et al. altered expression of connexin43 contributes to the arrhythmogenic substrate during the development of heart failure in cardiomyopathic hamster. am j physiol heart circ physiol 2007;294:1164-73. 6. palatinus ja, o’quinn mp, barker rj, harris bs, jourdan j, gourdie rg. zo-1 determines adherens and gap junction localization at intercalated disks. am j physiol heart circ physiol 2011;300:583-94. 7. polontchouck l, haefliger ja, ebelt b, schaefer t, stuhlmann d, mehlhorn u, et al. effects of chronic atrial fibrillation on gap junction distribution in human and rat atria. j am coll cardiol 2001;38:883-91. 8. molmen-hansen he, stolen t, tjonna ae, aamot il, ekeberg is, tyldum ga, et al. aerobic interval training reduces blood pressure and improves myocardial function in hypertensive patients. eur j prev cardiol 2012;19:151-60. 9. hafstad d, boardman nt, lund j, hagve m, khalid am, wislof u, et al. high intensity interval training alters substrate utilization and reduces oxygen consumption in the heart. j appl physiol 2011;111:1235-4 10. kostin s, dammer s, hein s, klovecorn wp, bauer ep, schaper j. connexin 43 expression and distribution in compensated and decompensated cardiac hypertrophy in patients with aortic stenosis. cardiovasc res 2004;62: 426–36. 11. teunissen bej, jongsma hj, bierhuizen mfa. review: regulation of myocardial connexins during hypertrophic remodeling. eur heart j 2004;25:1979-8. 12. bao x, clark cb, frangos ja. temporal gradient in shear-induced signaling pathway: involvement of map kinase, c-fos, and connexin43. am j physiol heart circ physiol 2000;278:1598–605. 13. bearden se, linn e, ashley bs, looft-wilson rc. age-related changes in conducted vasodilation: effects of exercise training and role 164 chondro, siagian, santoso exercise increases connexin43 in rat in functional hyperemia. am j physiol regul integr comp physiol 2007;293:1717-21. 14. barker rj, price rl, gourdie rg. increased association of zo-1 with connexin43 during remodeling of cardiac gap junctions. circ res 2002;90:317-24. 15. benito b, gay-jordi g, serrano-mollar a, guasch e, shi y, tardif jc, et al. cardiac arrhythmogenic remodeling in a rat model of a long-term intensive exercise training. circulation 2011;123:13-22. 16. pelliccia a, maron bj, deluca r, dipaolo fm, spataro a, culasso f. remodeling of left ventricular hypertrophy in elite athletes after long-term deconditioning. circulation 2002;105: 944-9. 17. gamelin fx, berthoin s, sayah h, libersa c, bosquet l. effect of training and detraining on heart rate variability in healthy young man. int j sports med 2007;28:1-7. 18. sloan rp, shapiro pa, demeersman re, bagiella e, brondolo en, mckinley ps, et al. impact of aerobic training on cardiovascular reactivity to and recovery from challenge. psychosom med 2011;73:134-41. 19. maass k, shibayama j, chase se, willecke k, delmar m. c-terminal truncation of connexin43 changes number, size, and localization of cardiac gap junction plaques. circ res 2007;101:128391. 20. bosquet l, mujika i. detraining. in: endurance training – science and practice. vitoria-gasteiz: inigo mujika s.l.u.; 2012. p.99–106. 22. severs nj, coppen sr, dupont e, yeh hi, ko ys, matsushita t. gap junction alterations in human cardiac disease. cardiovasc res 2004;62: 368–77. c:\users\jurnal fkusakti\docume 85 new tuberculosis vaccine to support tuberculosis elimination fransisca chondro department of physiology faculty of medicine, trisakti university the sustainable development goals have prioritized ending the epidemic of tuberculosis by 2030.(1) around one-third of the world population is infected with mycobacterium tuberculosis (mtbc), but is asymptomatic, a conditon known as late nt t b. (2 ) according to the global tuberculosis report, in 2017 there were 5-15% of 1.7 billion persons infected with mtbc, who will develop tb in their lives.(1) around 10% of latent tb will develop into active tb disease, with a higher risk in patients with immunodeficiencies such as hiv, undernutrition, diabetes mellitus, smoking, and habitual alcohol consumption. as a result there are annually an estimated 8.8 million new tb cases, with a tb mortality of 1.1 million and deaths from tb and hiv coinfection of 350 thousand cases.(1,3) the world health organization has planned a number of strategies for tb elimination in the year 2030, such as developing rapid and accurate diagnostic tests, new regimens for the treatment of drug-susceptible or drugresistant tb, prevention of transmission of m. tuberculosis through infection control, new vaccine candidates to prevent the development of tb, and to help improve the outcomes of treatment for tb disease.(1) based on the estimated incidence of tb, the who published a list of countries with the highest numbers of cases of tb, tb-hiv, and multi-drug resistant tb (mdr-tb). indonesia is included in all categories; in other words, indonesia belongs to the 20 countries with the highest incidence of tb, tb-hiv, and mdrtb.(1) natural infection induces the production of antibodies against mycobacterium tuberculosis (mtbc). studies show that 90% of tb patients have a higher serum immunoglobulin titer against mtbc at the onset of the clinical symptoms of the disease. however, there are a wide variety of antigens that produce the target antibody. one of the studies show that among 10 culture results of mtbc protein filtrates, there was not one antigen that is found in the serum of patients with active mtbc.(1) in the last few decades, several dna fingerprinting techniques have been used to differentiate between a number of mtbc s t r a i n s. re st r i c t io n f r a gment l e n gt h polymorphism (rflp) is based on the number of dna copies and the genomic location of the is6110 insertion sequence. this technique is the gol d st a n da r d f o r e x a min i n g t he a c t u a l transmission stage of mtbc bacteria in the patient’s body, for differentiating between relapse and reinfection in the patient, and for detecting cross-contamination in the laboratory. is6110 is an insertion sequence of 1361 base pairs, with inverted repeats of 28 base pairs at e a c h e nd . re s tr i c t io n f r a gme n t l e n gt h polymorphism analysis of is6110 depends on the d ige s t i on of d na ge n e t i c mat e r i a l b y endonucleases followed by electrophoretic separation of the dna fragments, which are then transferred to a nylon or nitrocellulose membrane. the fragments on the membrane are then subjected to southern blot hybridization with a labeled dna probe, which will adhere to the universa medicina may-august 2018 vol.37 no.2 86 universa medicina is6110-specific dna fragment. two limitations of this technique are the difficulty of obtaining comparable results in different laboratories and the need for dna of good quality in large a mou n t s. t h e r e f or e , s e ve r a l pcr-b a s e d methods have been developed to assist in the identification of mtbc strains using relatively small amounts of dna. in this category belong spoligotyping and mycobacterial interspersed repeat units (mirus) typing, both of which are considered the new gold standards in molecular epidemiological studies of mtbc.(3,4) to date many studies seek to evaluate the effect of mtbc strain variations on virulence and immunogenicity. virulence of tb may be influenced by the ability of the bacteria to survive after facing the host immune response, to damage the lungs, to survive aerosolization outside the host, and to be transmitted to healthy persons. studies in humans and experimental animals have shown that tnf-alpha, gamma interferon, il-12 and il17 are important mediators for the formation of the immune response of the patients against mtbc.(4) other mediators such as il-10 are also thought to play a role in limiting the development of mtbc at the initial stages of the immune response. previously it was concluded that the more severe the inflammatory response, the more severe the damage to the lungs, increasing the efficiency of mtbc transmission. however, several studies found that the increase in mtbc virulence was associated with a decreased and/ or delayed inflammmatory response, possibly as a result of considerable bacterial proliferation in the early stages of infection and leading to increased mtbc virulence in the final stages.(5,6) the ba cille ca lme tte-guérin (bcg) vaccine, which was developed almost 100 years ago and has been shown to prevent severe forms of tb in children, is still widely used. however, there is currently no vaccine that is effective in preventing tb disease in adults, either before or after exposure to tb infection. the bcg vaccine is considered to be less effective, since it protects only against tb infection in childhood, but does not influence the development of tb infection or reinfection in adults. in addition, the vaccine shows variable efficacy due to differences in the bcg strain used. therefore, there are currently several initiatives, such as the development of a better vaccine than the present bcg vaccine, the administration of a booster with a second dose of bcg or the administration of a bcg vaccine containing a different strain. to date there are 12 types of vaccine under study. for phase i studies, there are 3 vaccines, i.e. i). ad5 ag85a – adenovirus serotype 5 expre ss ing ag85 a. t his vac c ine will be administered by the intramuscular route after it has been demonstrated to be safe, is tolerated, and increases the patient’s immune level with a higher immunogenicity if administered to patients wh o h a ve pr e vi o us l y r e c e i ve d t he bcg vaccine;ii). chadox185a – mva85a. this vaccine is a combination of a simian adenovirus and a recombinant pox virus, both of which express the 85a antigen. the chadox185a vaccine is for intramuscular administration and has been tested in a phase i trial. at present the phase i trial for its administration in aerosol form is ongoing;iii) mtbvac. this is a live mtbc strain attenuated through deletions in the genes phop and fadd26. the main target population of this vaccine are the neonates (for replacing the current bcg vaccine). in addition, this vaccine may also be administered to adolescents and adults as repeat vaccine (booster). for phase ii and iii studies, there are 9 vaccines under development: i). dar-901 b oo s te r is a va c c i n e t ha t con s i st s of nontuberculous mycobacteria, in whole-cell and heat-inactivated form;ii). h4:ic31 is a booster vaccine containing a fusion protein of ag85b and tb10.4 with ic31 adjuvant. this vaccine is under trial to evaluate its efficacy in preventing infection in adolescents at high risk for mtbc infection, and has been tested for administration to infants;iii). h56:ic31 is a vaccine comprising a combination of three mtbc antigens (ag85b, esat-6, and rv2660c) with ic31 adjuvant. this vaccine has been tested, proven to be safe and 87 may-august 2018 vol.37 no.2 possesses substantial immunogenicity;iv). id93 + gla-se is a vaccine consisting of 4 mtbc antigens associated with virulence (rv2608, rv3619 and rv3620) or latency (rv1813), and the adjuvant gla-se;v). m72/as01e is a subunit vaccine containing 2 mtbc antigens (32a and 39a) with an adjuvant (as01e);vii). ruti® is a therapeutic vaccine containing mtbc cell-wall fragments for co-administration with short-term antibiotics. the main targets of this vaccine are patients with mdr-tb;vii). tb/ flu -04l i s a muc osa l-ve ct or e d va cc ine containing influenza virus with a replication d ef i ci e nc y tha t has be e n a tt e nua t ed an d expresses the ag85a and esat-6 antigens. this vaccine is intended as prophylactic boost vaccine for infants, adolescents, and adults; viii). vac cae™ is an immunothera peutic agent intended to shorten the therapeutic period in patients with mtbc infections that are still susceptible to the basic mtbc therapy; and ix) . v pm 10 02 is a r ec ombi na nt vac ci ne containing live mtbc bacteria. this vaccine is u nd e r tr i al t o e va l u a t e i t s s a f e ty a n d immunogenicity in neonates exposed to the hiv virus and those unexposed to hiv.(1) it is hope d that with the increased understanding and knowledge about the host and the mtbc bacteria, the goal of developing a suitable vaccine may be achieved, so that tb may be expected to be eliminated in the year 2030. references 1. world health organiztaion. global tuberculosis report 2017. geneva: world health organization; 2017. 2. achkar jm, casadevall a. antibody-mediated immunity against tuberculosis: implications for vaccine development. cell host microbe 2013; 13:250-62. doi: http://dx.doi.org/10.1016/j.chom. 2013.02.009. 3. fogel n. tuberculosis: a disease without boundaries. tuberculosis 2015;95:527-31. doi: http://dx.doi.org/10.1016/j.tube.2015.05.017. 4. coscolla m, gagneux s. consequences of genomic diversity in mycobacterium tuberculosis. seminars immunology 2014;26:431– 44. doi: http://dx.doi.org/10.1016/j.smim.2014.09. 0121044-5323. 5. mbugi ev, katale bz, siame kk, et al. genetic diversity of mycobacterium tuberculosis isolated from tuberculosis patients in the serengeti ecosystem in tanzania. tuberculosis. 2015;95: 170-8. doi: http://dx.doi.org/10.1016/j.tube.2014. 11.006. 6. cadmus s, hill v, van soolingen d, et al. spoligotype profile of mycobacterium tuberculosis complex strains from hiv-positive and -negative patients in nigeria: a comparative analysis. j clin microbiol 2011;49:220-6. doi: 10.1128/jcm.01241-10. univ med 2018;37:85-7. doi: http://dx.doi.org/10.18051/univmed.2018.v37.85-87 c:\users\jurnal fkusakti\docume 188 *department of parasitology, school of medicine and health sciences, atma jaya catholic university of indonesia **department of dermatology, school of medicine and health sciences, atma jaya catholic university of indonesia ***school of medicine and health sciences, atma jaya catholic university of indonesia correspondence : sem samuel surja department of parasitology, school of medicine and health sciences, atma jaya catholic university of indonesia jalan pluit raya 2 jakarta utara 14440 phone : +6221-6694366, 6693168 fax : +62216606123 email : semsurja@gmail.com orcid id: orcid.org/0000-00015981-0014 date of first submission, april 25, 2018 date of final revised submission, october 26, 2018 date of acceptance, october 26, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina uncontrolled blood sugar tends to increase prevalence of dermatomycosis in diabetic type 2 patients sem samuel surja*, melyawati hermawan**, meiliyana wijaya*, pramanta***, and hanna yolanda* background the prevalence of type 2 diabetes mellitus (dm) is increasing. diabetic patients have a higher risk of getting dermatomycosis. dermatomycoses, although a common health problem amongst dm, is often misdiagnosed and consequently undertreated. studies on the association between dermatomycosis and type 2 diabetes are lacking, especially in indonesia. therefore, the aim of this study was to determine the prevalence, etiology, and association of dermatomycosis with diabetic control of type 2 dm. methods a cross-sectional study was performed involving 87 subjects with type 2 dm. demographic and clinical data, including age, sex, and blood glucose level, were collected. if a dermatomycosis lesion was found, a specimen would be taken for identification. determination of serum glucose level was conducted using roche c111 analyzer®. statistical analysis was performed with the chi-square test and kolmogorov-smirnov twoindependent sample test. results seventeen (19.55%) subjects had dermatomycosis. the predominant age group affected was 51 60 years (42.4%). the number of clinically apparent dermatomycosis was greater in the uncontrolled than in the controlled blood sugar group, but the difference was statistically not significant (p > 0.05). the lesions were mostly found on the nails (74%) and the most common etiology was candida (50%) followed by dermatophyte (25%) and non-dermatophyte molds (25%). conclusion uncontrolled blood sugar tends to increase the risk of dermatomycosis in type 2 dm patients. fungal skin infections are common in type-2 dm patients, especially in those with poor glycemic control. keywords: candida, dermatomycosis, dermatophytes, diabetes mellitus type 2 doi: http://dx.doi.org/10.18051/univmed.2018.v37.188-194 original article pissn: 1907-3062 / eissn: 2407-2230 cite this article as: surja ss, hermawan m, wijaya m, et al. uncontrolled blood sugar tends to increase prevalence of dermatomycosis in diabetic type 2 patients. univ med 2018;37:184-94. doi: 10.18051/univmed.2018.v37.188-194 september-december, 2018 vol.37 no.3 189 univ med vol.37 no.3 introduction diabetes mellitus (dm) describes a group of metabolic disorders characterized by high blood glucose level.(1) in type 2 dm, this condition is caused by both insulin resistance and insulin s e c r e ti o n d e f e c t t ha t a f f e ct gl uc os e metabolism.(2) the international diabetes federation (idf) estimated that there were 451 million people (age 18-99 years) with diabetes in the world in 2017. this number is predicted to increase to 693 million by 2045. nearly 9.9% of the population aged 20-79 years is estimated to have dm in 2045. (1) the world health organization (who) predicted that there will be an increase from 8.43 million in 2000 to 21.3 million patients in 2030 in indonesia.(2) dermatomycosis is a skin disease caused by fungi. it might appear under various clinical pictures, such as dermatophytosis, candidiasis, piedra, pytir ia sis versicolor, tinea nigra, onyc homycosis, a nd paronychia. (3 ) dermatomycosis affects more than 20-25% of the world population.(3,4) these numbers continue to increase along with demographic and social economic changes, and the emerging of comorbid diseases and treatment options. the causative agents of dermatomycosis are dermatophytes, yeasts, and non-dermatophyte molds (ndm).(5,6) people with type 2 dm might be more susceptible to dermatomycosis because of the state of their immune system.(5,7,8) one study showed that patients with type 2 dm tended to have a higher prevalence of dermatomycosis than patients with type 1 dm, which could be possibly explained by the delay in the disease detection and following glycemic control.(9) a chronic hyperglycemic condition affects cellular immunity and disrupts the function of phagocytes throughout the body, including the skin. this is one of the reasons why the skin becomes more easily colonized by the fungi.(10) most of the dermatomycosis features found in people with type 2 dm are dermatophytosis (66%) and candidiasis (24.1%).(11) the agents causing the clinical manifestations are changing according to time and place.(3) and the fact that these skin infections are more prevalent in people with uncontrolled type 2 dm raises the need of attention to their presence.(12) specifically, onychomycosis is one of the most frequent form of dermatomycosis found in asia.(13) this condition is more prevalent in people with dm, about 1.9 – 2.8 times higher than in normal people.(14) the most common agents causing onychomycosis are the dermatophytes, chiefly trichophyton rubrum. (5) ninety percent of onychomycosis in toenails is caused by d e r ma t op hyt e s , wh i l e on yc h omyc o si s in fingernails is caused by yeasts, especially candida albicans.(15,16) in the last two decades, t he r e h a s b e e n a n i nc r e a s i ng tr e n d o f nondermatophyte molds (ndm). some species s uc h a s s c op u la r i op s is bre v i c a ul i s , aspergillus spp., fusarium spp., scytalidium spp, alternaria alternata, and many more were found to be the cause of onychomycosis. (17-19) this may be the result of an increasing population with an immunocompromized state, peripheral vascular disorders, and other conditions.(20) dat a re late d to the epid emio logy of dermatomycosis in diabetic patients in our country are scarce. this study was developed in order to find the prevalence and the most frequent causative agents of dermatomycosis in people with type 2 dm. therefore, the aim of this study was to determine the prevalence, etiology, and association of dermatomycosis with the diabetic control of type 2 dm patients. methods research design the design of this study was cross-sectional. this study was conducted in the inte rnal medicine wards and the parasitology laboratory, school of medicine and health sciences, atma j a ya ca t h ol i c un i ve r s it y o f in do ne si a , from october 2016 march 2017. 190 study subjects patients with type 2 dm who underwent hospitalization were included in the study. the inclusion criterion was type 2 dm patients who were hospitalized in the internal medicine wards of atma jaya hospital from october 2016 until january 2017. a total of 91 patients were hospitalized during that period. of these, 4 patients were excluded due to insufficient data or because they had been admitted twice to the hospital, leaving 87 patients included in this study. data collecting demographic and clinical data such as age, sex, patient’s diagnosis, blood glucose level and microbiological test results were collected. age and sex were collecte d f rom the medical records. the diagnosis of type 2 dm was made b y a n i n t er n i st , wh i l e t he dia gn os i s of dermatomycosis was made by a dermatologist. the blood glucose level was collected from the medical records (collection of blood glucose samples was described below). mycological tests were conducted in all patients suffering from dermatomycosis. laboratory analysis a venous blood sample was collected from each patient. serum was then separeated from other blood components. determination of blood glucose level was conducted on serum using a roche c111 analyzer®. the patient’s blood glucose level was grouped into uncontrolled and uncontrollable blood glucose. controlled blood glucose was defined by fasting blood glucose (fbg) <130 mg/dl and postprandial blood glucose (pbg) <180 mg/dl.(2) isolation and identification of isolates each patient underwent a thorough skin examination by a dermatologist to determine the presence of dermatomycosis clinical lesions. dermatomycosis lesions were wiped with alcohol swabs then scraped with sterile glass slides. the samples from skin and nail scrapings were inoculated onto three types of media, i.e. sabouraud dextrose agar ( sda) (oxoid, ha mp s hi r e , e n gl a nd) , sda c on t a i n in g c h l or a mp he ni c o l ( ind of a r ma , j a ka r t a , indonesia), and sda containing 0.4 mg/ml chloramphenicol and 0.5 mg/ml cycloheximide (amresco, ohio, usa).(21,22) if pytiriasis was suspected, the sample would be inoculated onto sda containing chloramphenicol and overlaid with 2% olive oil. (23) inoculated samples were incubated at 25°c 30°c. the onychomycosis samples were inoculated at 16 points in those media. if no dermatophyte or candida was detected, the cause of dermatomycosis was determined from the most-growing colonies.(24) the cultures were examined daily until the growth of the fungus was observed and was declared negative if there was no growth within 4 weeks. if the isolates formed mold colonies, the samples were continued in slide culture with sda. but, if the isolates formed yeast colonies, the samples were continued in slide culture with rice cream and culture on hi-chrom agar (himedia laboratories, mumbai, india) for identification.(22) statistical analysis the statistical tests used were chi-square and kolmogorov-smirnov 2-independent sample test, to analyze the relationship between several variables, such as gender, age, blood glucose level, and dermatomycosis case. the software used was spss for windows version 17. ethical clearance this study has obtained ethical approval from the institute of ethics studies of atma jaya and has received permission from atma jaya hospital. the number of ethical clearance was 01/02/kep-fkuaj-2016. results of the 87 diabetic patients, 50 (57.5%) were female and the predominant age group affected was 51 60 years (42.4%) (table 1). from the clinical examination, 17 (19.55%) surja, herwaman, wijaya, et al dermatomycoses in type-2 dm patients 191 univ med vol.37 no.3 patients had dermatomycosis, and no significant difference between dermatomycosis and sex or a ge ( ta b le 2 ) . t he n umbe r of c l i ni c a l dermatomycosis was greater in uncontrolled fbg or pbg than in controlled fbg or pbg, but the difference was statistically not significant (p=0.394 and p=0.658 respectively) (table 2). of t h e bo dy s i te s a f f e c t e d wi t h dermatomycosis 74% were nails and out of all d e rma t omyc os is c a se s 50% we re d ue t o candida. candida glabrata was the most commonly found candida species (40%) and the most common dermatophyte was trichophyton rubrum (75%), followed by trichophyton mentagrophytes (25%). other fungi that have been isolated were ndm and rhodotorulla glutinis. the ndms found were aspergillus, ony ch oc o la c ana de ns is, neo scy ta li diu m dimidiatum, and geotrichum. discussion this study showed that the prevalence of dermatomycosis in females was higher than in males, but the difference was statistically not significant. other studies showed that men are more likely have dermatomycosis than women, b ut t h e d if f e r e nc e wa s n ot s t at i st i c a l l y confirmed.(8,9,25) dermatomycosis is associated with the physical activities and habits of the patient, such as water-related work, and physical activities that cause injuries.(12,25,26) the hyperglycemic state disrupts skin h ome o st a s is by i nh i bi t i ng ker a t i no c yte proliferation or migration, protein biosynthesis, inducing apoptosis, reducing nitric oxide synthesis, disturbing phagocytosis and cell characteristic n (%) sex male 37 (42.5) female 50 (57.5) age (years) 57.93 ± 9.14 40 4 (5) 41 50 15 (17.1) 51 60 37 (42.4) >60 31 (35.5) dermatomycosis yes 17 (19.55) no 70 (80.05) type of dermatomycosis* onychomycosis 13 (72.22) tinea 2 (11.11) candidiasis 3 (16.67) blood glucose (mg/dl) fasting 169.59 ± 69.66 controlled 103.63 ± 11.5 uncontrolled 184.45 ± 68.66 2 h-postprandial 217.84 ± 104.37 controlled 127.94 ± 24.99 uncontrolled 238.09 ± 104.81 table 1. distribution of features characteristic of research subjects (n=87) *based on clinical diagnosis by dermatologist (1 patient could have more than 1 diagnosis) variables dermatomycoses p value yes no sex male female age group (years) 40 41–50 51–60 >60 fbg uncontrolled controlled pbg uncontrolled controlled 6 (16.2%) 11 (22%) 0 (0%) 3 (20%) 9 (24.3%) 5 (16.1%) 13 (22%) 4 (14.3%) 10 (21.3%) 7 (17.5%) 31 (83.8%) 39 (78%) 4 (100%) 12 (80%) 28 (75.7%) 26 (83.9%) 46 (78%) 24 (85.7%) 37 (78.7%) 33 (82.5%) 0.472* 1.000# 0.394* 0.658* table 2. the relationship between dermatomycoses and sex, age, and blood glucose control *: chi-square test; #: kolmogorov-smirnov 2-independent sample test; fbg: fasting blood glucose; pbg: 2-h postprandial blood glucose 192 chemotaxis. these conditions promote skin disorders in diabetes, including skin fungal infection.(27) the results of our study showed that the prevalence of dermatomycosis tends to be higher in uncontrolled diabetic patients than in controlled ones. this result is in concordance with sugandhi et al.(28) in that poor glycemic control was significantly associated with fungal infection. type 2 diabetes was previously known as p re d isp os ing fa c t or f or f un ga l i nfe c t io n especi al l y ca ndi da i n f e c t i o n a n d dermatophytosis of the foot and nail.(7,9,29) akkus et al.(12) stated that tinea pedis and o nyc h omyc os i s w e re mo r e f r e q ue nt i n uncontrolled diabetic patients and patients with vascular disturbance. tzar et al.(30) also found that the most frequent fungal infection is onychomycosis. uncontrolled blood glucose level is also associated with onychomycosis.(7) the result of the present study is in concordance with other previous studies, in that the most frequent dermatomycosis was onychomycosis. this study found that candida was the most common cause of dermatomycosis, followed by dermatophytes, ndm, and r. glutinis. the high prevalence of candida infection was supported by many studies stating that the most frequent etiology of dermatomycosis in dm was candida infection, followed by dermatophytes, especially in nails and their surroundings.(31,32) candida is part of the normal flora in humans that is a potential opportunistic pathogen. in the diabetic condition, there are an increased blood glucose level and an impaired immune system. the high blood glucose level provides the optimal condition for fungal growth. the impaired immune system results in the incapability of the host immune s ys t e m t o c ou nt e r ca nd i da i n c ut a ne ou s invasion.(32) different results were obtained by nenoff et al.(7) who found that the most common etiology of dermatomycosis was dermatophytes, rather than candida. the most common dermatophyte found in this study was t. rubrum, followed by t. mentagrophytes. this result is in concordance with previous knowledge that dermatophytosis is commonly caused by t. rubrum and t. mentagrophytes.(7) studies in asian countries such as india and japan, as reviewed by hayette et al.,(5) also confirmed that these species are the main agents causing dermatophytosis. parada et al.(9) a lso found that t. rubrum and t. me nt a grop hy te s we r e t he c a us e o f dermatomycosis in the lower limb of patients with type 2 diabetes. reddy et al.(25) stated that the high incidence of t. rubrum is related to its a b i li t y t o f or m col o n ies i n h ar d ke ra t i n structures. it is also known that t. rubrum more commonly affects people with predisposing factors such as dm.(7) of the 30% sample in this study it was found that the etiological agents were nondermatophyte and non-candida fungi, such as aspergillus, r. glutinis, o. canadensis, n. dimidiatum, and geotrichum, the most common b e ing as p e rgi l l us . be si d e s c a n di d a a n d dermatophytes, the other fungi have also been kno wn a s t h e c a us a t i ve age n t s of o nyc h omyc os is, s uc h a s s c op u la r i op s is brevicaulis, aspergillus spp., fusarium spp., scytalidium spp, alternaria alternata, and many more.(17-19) nondermatophyte molds may invade nails damaged by occupational trauma, such as in young persons, immunocompromized individuals, those with impaired peripheral c i r c ul a t i on , a n d th o se wi t h p e r i ph e r a l neuropathy.(20) however, ndm may also present as contaminants that grow saprophytically in nails. to diagnose ndm as the etiology of onychomycosis, is challenging. there are several methods that may be u se d t o de c i de nd m a s t he et i o lo gy of onychomycosis cases. in this study, samples have been cultured on 16 spots in media. if candida or dermatophytes appeared, they would be decided as the etiology of onychomycosis. but, if they did not, the etiology would have been decided from the most populous colony of fungi that grew in the media.(23 ) gupta et al.(2 4) suggested 6 criteria for diagnosing ndm as the etiology of onychomycosis cases accurately: surja, herwaman, wijaya, et al dermatomycoses in type-2 dm patients 193 univ med vol.37 no.3 d e t e c t i ng n dm b y d i r e c t mi c r os c o p ic examination from clinical samples with potassium hydroxide (koh), positive culture of ndm, repositive culture of ndm (at different points in ti me) , the den sity of c olony, abse nce of dermatophytes, and histologic examination. minimally 3 of those criteria have to be obtained. it is important to bear in mind some limitations of this study. first, the true frequency of onychomycosis in the group of patients might have been underestimated due to negative cultures for fungi. second, the cross-sectional design of this study prevents the finding of the t r u e c a us a l r e l a t i on s hi p be t w e e n dermatomycosis and poor blood glucose control. since dermatomycosis is common in dm patients, the management of type 2 dm should also address this problem to improve the patient’s quality of life. future research studies with similar objectives should be designed as cohort studies with larger sample sizes to find a more precise causal relationship between dermatomycosis and diabetic control. conclusions uncontrolled blood sugar tends to increase dermatomycosis in patients with type 2 diabetes me l l it u s . t he mos t c o mmo n c a s e s of dermatomycosis are the onychomycoses and the most common etiology is candida. conflict of interest the authors declare they have no conflict of interest. funding disclosure this study was funded by atma jaya catholic university of indonesia. acknowledgement the authors thank vilia budi prasetio, k e yn e chr is t a m on i n tj a , an ge l a m e i ke indrayani, josephine, richard firmansyah, ivon setiawan, and linda widyawati for the ir contribution during this study. contributors sss contributed to conceiving and presenting the research idea, conducting laboratory work, conducting statistical analysis, constructing article. mh contributed to collecting research data, constructing article. mw contributed to collecting research data, conducting laboratory work, conducting statistical analysis, constructing article. p contributed to collecting research data. hy contributed to finding research grant, conducting statistical analysis, constructing article. all authors have read and approved the final manuscript. references 1. cho nh, shaw je, karuranga s, et al. idf diabetes atlas: global estimates of diabetes prevalence for 2017 and projections for 2045. diabetes res clin pract 2018;138:271–81. doi: 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infections in subjects with diabetes mellitus. int j res dermatol 2017;3:55-8. doi: http://dx.doi.org/10.18203/issn.2455-4529. int j res dermatol 2016;44:12. 32. santhosh y, ramanath k, naveen m. fungal infections in diabetes mellitus: an overview. int j pharm sci rev res 2011;7:221-5. surja, herwaman, wijaya, et al dermatomycoses in type-2 dm patients c:\users\jurnal fkusakti\docume 209 *molecular biology research center, systems biology and poisonings institute, baqiyatallah university of medical sciences, tehran, iran **department of microbial biotechnology, payame noor university, tehran, iran ***department of biology, payame noor university, tehran, iran †department of veterinary microbiology, faculty of animal husbandry and veterinary sciences, sindh agriculture university, tandojam 70060, pakistan ††dvm, faculty of veterinary medicine, karaj branch, islamic azad university, karaj, iran correspondence reza ranjbar molecular biology research center, systems biology and poisonings institute, baqiyatallah university of medical sciences, tehran, iran tel/fax: 00982188039883 email: ranjbarre@gmail.com orcid id: orcid.org/0000-00018593-1514 date of first submission, may 30, 2018 date of final revised submission, november 26, 2018 date of acceptance, november 30, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina antibiotic resistance and prevalence of class 1 and 2 integrons in escherichia coli isolated from hospital wastewater reza ranjbar*, maryam zeynali**, nooshin sohrabi***, asghar ali kamboh†, and arman moshaveri†† background hospital wastewaters may contain antibiotic resistant bacteria such as escherichia coli. these e. coli harbor integron genes that are responsible for antibiotic resistance. the purpose of the current study was to evaluate the frequency of class 1 and 2 integrons in environmental antibiotic resistant e. coli strains isolated from the hospital wastewaters in tehran, iran. methods as a descriptive cross-sectional study, this research was performed from april to september 2015 on hospital wastewaters in tehran. bacterial isolation and identification was performed by standard biochemical and bacteriological procedures. susceptibility testing was done by employing the disk diffusion method using different antibiotics. total dnas were extracted to evaluate the presence of class 1 and 2 integrons by using the polymerase chain reaction (pcr) method with specific primers. results fifty e. coli strains were isolated and identified from the wastewaters of 25 hospitals in tehran. the phenotype results showed that 46 isolates (92%) were resistant to at least one antibiotic and 27 isolates (54%) were multidrug resistant. pcr showed that 35 (70%) and 20 (40%) of the isolates had class 1 and 2 integrons respectively and 14 isolates (28%) had both class 1 and class 2 integrons. conclusion this study has shown a considerable presence of class 1 and class 2 integrons in e. coli strains isolated from hospital wastewaters in tehran. proper antibiotics prescription and appropriate hospital wastewater treatment can prevent resistance genes in e. coli from circulating in the environment. keywords: escherichia coli, integron, antibiotic-resistant, hospital wastewaters doi: http://dx.doi.org/10.18051/univmed.2018.v37.209-215 original article pissn: 1907-3062 / eissn: 2407-2230 september-december, 2018 vol.37 no.3 cite this article as: ranjbar r, zeynali m, sohrabi n, et al. antibiotic resistance and prevalence of class 1 and 2 integrons in escherichia coli isolated from hospital wastewater. univ med 2018;37:209-15. doi: 10.18051/univmed.2018.v37.209-215. 210 ranjbar, zeynali, sohrabi, et al class 1 and 2 integrons in escherichia coli introduction water that has been produced for specific consumptions and the quality of which has deteriorated after use, is called wastewater. in hospitals, clean water is used in various sections to safeguard patients’ health. as a result, water quality diminishes and is therefore converted to wa st e wa t e r s . ( 1) t he wi d e sp r e a d us e o f antibiotics has led to an increase in the global dispersion of bacteria that are resistant to antibiotics. furthermore, due to an increase in the use of antibiotics in hospitals, hospital wastewaters now contain large numbers of bacteria that are resistant to antibiotics, including e.coli.(2) drug-resistant strains of e.coli can lead to problems in the management and control of intraand extra-intestinal infections caused by these bacteria. e.coli is a commensal bacterium and is part of the normal flora of the human gut. the gram-negati ve bacterium e. coli i s nonpathogenic but can cause diseases and diarrhea when virulence factors like adhesins and endotoxin are available.(3-5) intestinal infection pathotypes of e.coli comprise enteroaggregative e.coli (eaec), enterotoxigenic e.coli (etec), enteropathogenic e.coli (epec), enteroinvasive e.coli (eiec), enter ohemorrhagic e.coli (ehec), cell-detaching e.coli (cdec), and diffusely adherent e.coli (daec). extraintestinal pathogenic e.coli (expec) can cause serious and dangerous diseases such as urinary tract infection, meningitis and septicemia.(6-8) the development of drug resistance can occur by the horizontal transfer of antimicrobial res istanc e gen es thr ough mobile gene tic elements.(9) integrons are one of the mobile genetic elements that play a significant role in the acquisition and dissemination of antimicrobial resistance genes.(10 ) t hey have been also associated with other mobile genetic elements such as transposons and plasmids. the role of integrons and gene cassettes in the development of drug resistance in gram-negative bacteria has been proven.(11) all integrons consist of three sections, including the gene for the integron i nt e gra s e ( i nt i ) , i n t e gr on-a s s oc i a te d recombination site (atti) and integron-carried promoter (pc). integrons have been associated wi t h o t he r mo bi l e ge ne t i c e le me nt s a nd resistance genes which cause their spread among bacteria in the environment that are exposed to antibiotics. five classes of integrons have been established that are known based on integrase enzyme coding sequence. class 1, 2 and 3 integrons are more frequent in multiple drug resistance.(12) the class 1 integron has two conserved segments (52 -cs and 32 -cs). the 32 -cs region has the qace”1 gene (which causes resistance to quaternary ammonium compounds) and also has the sul1 gene which causes resistance to sulfonamides. class 2 integrons are related to transposon tn7 and carry six resistance gene cassettes.(13) gene cassettes that encode antibiotic resistance genes have 59b a s e e l e me n ts , t h e s pe c i f i c s i t e s f o r recombination. gene cassettes related to class 1, 2 and 3 integrons are associated with resistance to aminoglycosides, chloramphenicol, β -l a c t ama s e , t r ime t h op r i m a n d o t h e r antibiotics.(14,15) some limited previous studies have reported antibiotic resistance of e. coli strains isolated from sewage or wastewaters. reinthaler et al.(16) reported that 12% of e. coli strains isolated from sewage and sludge wer e r esistant to trimethoprim/sulfamethoxazole and 57% to tetracycline. elmanama et al.(17) reported that 31.9% of e.coli strains isolated from hospital wastewater were resistant to tetracycline, 10.6% to amikacin, 25.8% to co-trimoxazole and 12.8% to chloramphenicol. low resistance rates to trimethoprim/sulfamethoxazole (12.24%) and chloramphenicol (32.65%) are reported in e. coli strains isolated from hospital wastewater in hamadan, iran by hadi et al.,(18) while gündogdu et al.(19) reported high rates of resistant isolates against imipenem (84%) and tetracycline (94%). to the best of our knowledge, little is known about the integrons and their associated gene cassettes in e.coli strains isolated from hospital wastewaters specifically and in tehran city 211 generally. the aim of this study was to appraise antibiotic resistance and the presence of class 1 and 2 integrons in e.coli strains isolated from hospital wastewaters. methods study design a descriptive cross-sectional study was performed from april to september 2015 on the hospital wastewaters in various parts of tehran city, iran. bacterial isolates the hospital wastewater samples were collected from 25 hospitals located in various parts of tehran city, iran. e.coli isolates were identified and isolated by standard microbiological methods.(20) wastewater samples were cultured on chrom agar (merck, germany) and plates were incubated at 370c for 18 hours, then suspected purple colonies were subcultured to emb agar (merck, germany) and incubated at 370c for 18 hours to confirm the presence of e. coli isolates, and finally the isolates were kept as e. coli at -200c for more investigations. antimicrobial susceptibility testing antimicrobial susceptibility testing of total e. coli isolates was done by the disc diffusion method according to the clinical laboratory standard institute (clsi) instructions.(21) antibiotic discs that were used in this study included tetracycline (te, 30μg), co-trimoxazole (sxt, 1.25/ 23.75 μg), chloramphenicol (c, 30 μg), amikacin (an, 30 μg), nitrofurantoin (fm, 300 μg), imipenem (ipm, 10 μg) and streptomycin (s, 10 μg). bacterial suspensions of isolates were prepared based on mcfarland standard no. 0.5 and were cultured on mueller-hinton agar (merk, germany). afterwards the plates were kept at 37oc overnight, the inhibition zone diameter was measured and the results were reported based on the clsi table as sensitive, intermediate and resistant. polymerase chain reaction (pcr) total dna samples were extracted by the boiling method. class 1 and class 2 integrons were detected by using the pcr method with specific primers. primer sequence, sizes of pcr products and pcr programs are shown in table 1. the pcr was performed in a total volume of 20 μl, comprising 10 μl master mix ( taq dna polymerase, dntp and mgcl 2 ; ampliqon, finland), 1 μl template dna, 1 μl of each primer and 7 μl sterile deionized water. the amplified products were then examined by electrophoresis on 1.5% agarose gel (merck, germany) in tbe (tris base-boric acid-edta) 1x buffer and gels were stained with simplysafe stain (eurx, finland) then visualized under ultraviolet light. univ med vol. 37 no.3 gene class primer sequence size (bp) pcr programs reference class 1 integron (int1) f:5׳-gggtcaaggatctggatttcg ׳3 r:5׳-acatgggtgtaaatcatcgtc ׳3 484 initial denaturation at 95° c for 5 min., 30 cycles of denaturation at 94° c for 50 s, primer annealing at 52° c for 50 s, and primer extension at 72° c for 1 min. final extension at 72° c for 5 min. roe et al.(22) class 2 integron (int2) f:5׳-gcaaatgaagtgcaacgc-3׳ r:5׳-acacgcttgctaacgatg-3׳ 466 initial denaturation at 95° c for 4 min., 30 cycles of denaturation at 94° c for 50 s, primer annealing at 52° c for 50 s, and primer extension at 72° c for 1 min. final extension at 72° c for 5 min. sunde (23) table 1. primer sequence and size, and pcr programs 212 ranjbar, zeynali, sohrabi, et al class 1 and 2 integrons in escherichia coli results fifty strains of e.coli were isolated from the different 25 hospital wastewater samples collected from tehran city. the results obtained from antibiotic susceptibility of isolates showed that 46 isolates (92%) were resistant to at least one antibiotic and 27 isolates (54%) of the 46 isolates were multidrug resistant. also the highest percentages of antibiotic resistance in isolates were related to streptomycin (58%), imipenem (48%) and co-trimoxazole (48%) while the lowest percentages were associated with nitrofurantoin (14%) and amikacin (2%) (table 2). the results obtained from pcr showed that 35 (70%) isolates had class 1 integrons and 20 isolates (40%) had class 2 integrons. in addition, 65.7%, 57.1% and 17.1% of isolates harboring class 1 integrons were resistant to streptomycin, imipenem and nitrofurantoin, respectively. also 70%, 55% and 10% of isolates harboring class 2 integrons were resistant to streptomycin, cotrimoxazole and amikacin. antibiotic agent total susceptibility of isolates (n=50) r integron negative (n=9) n (%) r integron positive (n=41) n (%) r integron class 1 (n=35) n (%) r integron class 2 (n=20) n (%) r n (%) i n (%) s n (%) chloramphenicol 13 (26) 3 (6) 34 (68) 2 (22.2) 11 (26.8) 8 (22.9) 7 (35) tetracycline 22 (44) 5 (10) 23 (46) 1 (11.1) 21 (51.2) 18 (51.4) 9 (45) streptomycin 29 (58) 8 (16) 13 (26) 3 (33.3) 26 (63.4) 23 (65.7) 14 (70) amikacin 1 (2) 1 (2) 48 (96) 1 (11.1) co-trimoxazole 24 (48) 4 (8) 22 (44) 5 (55.5) 19 (46.3) 17 (48.6) 11 (55) nitrofurantoin 7 (14) 5 (10) 38 (76) 1 (11.1) 6 (14.6) 6 (17.1) 2 (10) imipenem 24 (48) 14 (28) 12 (24) 4 (44.4) 20 (47.8) 20 (57.1) 8 (40) table 2. distribution of antibiotic susceptibility and resistance r: resistant, i: intermediate, s: sensitive figure 1. pcr detection of class 1 and 2 integrons. pcr products of class 1 integron (lane 2 and 3, integron positive, and lane 1, integron negative); lane 4: molecular marker (100 bp); pcr products of class 2 integron (lane 6 and 7, integron positive, and lane 5, integron negative) 213 discussion among gram-negative bacteria, e. coli is known to be a leading cause of nosocomial infections globally. control of e.coli illnesses has become especially challenging due to emergence of multidrug resistant strains.(24) previous research has suggested that antibiotic resistant bacteria enter the water environment from the various ecosystems like humans or animals  soil  water (25) and that unsafe water has contributed to 3.2% deaths worldwide.(26) the fecal bacteria in the water environment might transfer antibiotic resistance to autochthonous bacteria through lateral transfer of movable genetic elements.(27) furthermore, indirect evidences of the transmission of antibiotic resistance genes in aquatic habitats are also extant.(28) in the current study we have explored the antimicrobial profile of e.coli isolates of hospital wastewaters. the results of the antibiotic susceptibility testing showed that 26% of the isolates were resistant to chloramphenicol, 44% to tetracycline, 58% to streptomycin, 2% to amikacin, 48% to co-trimoxazole, 14% to nitrofurantoin and 48% to imipenem. reinthaler et al.(16) noted that 12% of their isolates were resistant to trimethoprim/ sulfamethoxazole and 57% to tetracycline, therefore in comparison with our study, the resistance to tetracycline in their study was the highest. another research study was done by elmanama et al.(17) who reported that 31.9% of e.coli isolates were resistant to tetracycline, 10.6% to amikacin, 25.8% to cotrimoxazole and 12.8% to chloramphenicol. they also noted that 0% of isolates were resistant to imipenem, whereas in our study 48% of isolates were resistant to imipenem. hadi et al.(18) observed that the resistance rates to trimethoprim/ sulfamethoxazole and chloramphenicol were 12.24% and 32.65%, respectively, which was considerably low in comparison with our study. however, gündogdu et al.(19) observed that 84% and 94% of isolates were resistant to imipenem and tetracycline, which was markedly high in comparison with our current study. in the present study it was observed that 70% of isolates had class 1 integrons and 40% of isolates had class 2 integrons. fourteen isolates (28%) jointly had class 1 and class 2 integrons, and 41 (84%) isolates were integronpositive. the prevalences of isolates with class 1 i nt e gr ons t h a t w e r e r e si st a n t t o chloramphenicol, tetracycline, co-trimoxazole, imipenem, streptomycin and nitrofurantoin among 35 e. coli isolates were 22.9%, 51.4%, 48.6%, 57.1%, 65.7%, and 17.1%, respectively. also the prevalences of antibiotic-resistant i s ol a t e s w i th c la ss 2 i n te gr on s t o chloramphenicol, tetracycline, co-trimoxazole, imipenem, streptomycin and nitrofurantoin among 20 e. coli isolates were 35%, 45%, 55%, 40%, 70%, and 10%, respectively. mokracka et al.(29) reported that e. coli isolated from one wastewater treatment plant in poland were resistant to trimethoprim/sulfamethoxazole, amikacin, imipenem, chloramphenicol and tetracycline with percentages of 70.6%, 41.3%, 3.7%, 52.3%, and 73.4%, respectively. as compared to our results, the authors reported a relatively high occurrence of antimicrobial resistance to all antibiotics except imipenem that exhibited the lowest resistance percentage. the authors also noted that 10.8% of isolates had class 1 integrons and only 2% of isolates had class 2 integrons, whereas 2% of isolates harbored both class 1 and 2 integrons. their integron results were also very low as compared with our current results. likewise, kotlarska et al.(30) reported that 20% of e. coli strains isolated from wastewater were resistant to trimethoprim/sulfamethoxazole. among those, 32.6% of isolates had class 1 integrons and just 3.5% of isolates had class 2 integrons, whereas 0.38% of isolates had jointly class 1 and 2 integrons. diwan et al.(31) reported the antibiotic resistance in e. coli isolates from hospital waste water in india, showing the highest resistance to amikacin. it was also observed that antimicrobial resistance rates were significantly higher in hospital sewage isolates than in hospital clinical isolates in taiwan.(32) univ med vol. 37 no.3 214 ranjbar, zeynali, sohrabi, et al class 1 and 2 integrons in escherichia coli integrons contain mobile gene cassettes that encode resistance to antibiotics. we did not investigate the gene cassettes within the integrons studied, which is a limitation of the current study. the use of molecular typing of the isolates may be able to elucidate genetic relationships between the strains under study. conclusion this study demonstrated a high number of class 1 and class 2 integrons among e.coli strains isolated from hospital wastewaters in tehran city. therefore, the use of appropriate strategies for the elimination and decrease of antibiotic resistance is necessary. acknowledgments we would like to thank the clinical research development center of baqiyatallah hospital for their kind cooperation. this study was supported in part by a grant from the clinical research development center of baqiyatallah hospital. conflict of interest the authors declare that they have no conflicts of interests contributors rr designed the study and analyzed the data. mz performed laboratory work. ns contributed to data analysis and aak revised the manuscript. all authors have read and approved the final manuscript. references 1. li l, dechesne a, he z, et al. estimating the transfer range of plasmids encoding antimicrobial resistance in a wastewater treatment plant microbial community. environ sci tech let 2018;5:260-5. doi: 10.1021/acs.estlett.8b00105. 2. farshad s, anvarinejad m, tavana am, et al. molecular epidemiology of escherichia coli strains isolated from children with community acquired urinary 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28. tajbakhsh e, khamesipour f, ranjbar r, et al. prevalence of class 1 and 2 integrons in multidrug resistant escherichia coli isolated from aquaculture water in chaharmahal va bakhtiari province, iran. ann clin microbiol antimicrob 2015;14:37. doi: 10.1186/s12941-015-0096-y. 29. mokracka j, koczura r, jablonska l, et al. phylogenetic groups, virulence genes and quinolone resistance of integron-bearing escherichia coli strains isolated from a wastewater treatment plant. antonie van leeuwenhoek 2011;99:81724. doi: 10.1007/ s10482-011-9555-4. 30. kotlarska e, luczkiewicz a, pisowacka m, et al. antibiotic resistance and prevalence of class 1 and 2 integrons in escherichia coli isolated from two wastewater treatment plants, and their receiving water (gulf of gdansk, baltic sea, poland). environ sci pollut res 2015;22:2018-30. doi 10.1007/s11356-014-3474-7. 31. diwan v, tamhankar aj, khandal rk, et al. antibiotic and antibiotic resistant bacteria in water associated with a hospital in ujjain, india. bmc public health 2010;10:414. doi: 10.1186/1471-245810-414. 32. yang cm, lin mf, liao pc. comparison of antibiotic resistance patterns between clinical and sewage isolates in a regional hospital in taiwan. lett appl microb 2009;48:560-5. https://doi.org/ 10.1111/j.1472-765x.2009.02572.x. univ med vol. 37 no.3 oktavianus 75 *department of anatomy, faculty of medicine, yarsi university **department of biochemistry, faculty of medicine, yarsi university correspondence prof. dr. endang purwaningsih, ms, pa department of anatomy, faculty of medicine, yarsi university jln. let.jend. suprapto, cempaka putih, jakarta 10510 phone +6221 4206674-76 email: endpurwaning@gmail.com / endang.purwaningsih@.yarsi.ac.id univ med 2014;33:75-82 abstract universa medicina may-august, 2014may-august, 2014may-august, 2014may-august, 2014may-august, 2014 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 vol.33no.2 background cancer is one of the causes of high mortality. breast and cervical cancers are two of the most frequent cancers affecting women around the world, including indonesia. natural materials such as rodent tuber (typhonium flagelliforme) have anticancer potentials. the rodent tuber extract contains ribosome inactivating proteins (rips) capable of cutting the dna or rna of cancer cells and blocking the growth of cancer cells. the purpose of this study was to evaluate the cytotoxic effects of typhonium flagelliforme lodd extract on hela cervical cancer and michigan cancer foundation-7 (mcf-7) breast cancer cells. methods subjects were cultured cell lines of hela cells in rosswell park memorial institute (rpmi) and of mcf-7 cells in dulbecco’s minimum essential medium (dmem). rodent tuber ethanolic extract was diluted in dimethyl sulfoxide (dmso). the cytotoxicity assay used the 3-(4,5-dimethyl thiazol-2-yl,5-diphenyl) tetrazolium bromide (mtt) method. absorbance was read in an elisa reader at a wavelength of 595 nm. results rat tuber extract at all dilutions (500; 250; 125; 62.5; 31.25; 15.625;7.81; 3.9 ì g/ ml) showed cytotoxic effects against hela and mcf-7 cells. higher concentrations of the extract gave a higher proliferation inhibition effect. calculated ic50 values of the extract by probit analysis were 30.19 ìg/ml against hela cells and 5.586 ì g/ml against mcf-7 cells. conclusions ethanolic extract of typhonium flagelliforme lodd has cytotoxic effects against hela cells and mcf-7 cells. the cytotoxic effects against mcf-7 cells are greater than the cytotoxic effects against hela cells. key words : typhonium flagelliforme, tetrazolium bromide, cytotoxic, hela cells, mcf-7 cells cytotoxicity assay of typhonium flagelliforme lodd against breast and cervical cancer cells endang purwaningsih*, etty widayanti*, and yulia suciati** 76 purwaningsih, widayanti, suciati cytotoxicity of typhonium flagelliforme uji sitotoksisitas ekstrak typhonium flagelliforme lodd terhadap sel kanker payudara dan rahim latar belakang kanker menjadi salah satu penyebab kematian yang cukup tinggi. kanker payudara dan kanker leher rahim menempati dua urutan terbanyak diderita wanita di seluruh dunia termasuk indonesia. bahan alam seperti keladi tikus (typhonium flagelliforme) memiliki potensi sebagai anti kanker. di dalam ekstrak keladi tikus mengandung ribosom inactivating protein (rips) yang mampu memotong dna atau rna sel kanker dan dapat memblokir pertumbuhan sel kanker. tujuan penelitian adalah untuk menilai efek sitotoksik ekstrak tanaman keladi tikus (typhonium flagelliforme l) terhadap sel kanker rahim hela dan sel kanker payudara michigan cancer foundation-7 (mcf-7). metode subyek penelitian adalah kultur sel (cell line) hela dalam medium rosewell park memorial institute (rpmi) dan sel mcf-7 dalam dulbecco’s minimum essential medium (dmem). ekstrak etanolik tanaman keladi tikus diencerkan dengan dimethyl sulfoxide (dmso). uji sitotoksisitas menggunakan metode 3-(4,5-dimetil tiazol-2-il,5-difenil tetrazolium bromide (mtt). serapan warna dibaca dengan elisa reader pada panjang gelombang 595 nm. hasil dari beberapa pengenceran ekstrak yang digunakan yaitu 500; 250; 125; 62,5; 31,25; 15,625; 7,81; dan 3,9 µg/ml masing-masing menunjukkan adanya efek sitotoksik ekstrak keladi tikus terhadap sel hela dan sel mcf-7. konsentrasi ekstrak yang lebih tinggi memberikan efek hambatan proliferasi yang lebih tinggi. perhitungan nilai ic50 ekstrak keladi tikus dengan analisis probit diperoleh nilai ic50 30,19 µg/ml terhadap sel hela dan 5,59 µg/ml terhadap sel mcf-7. kesimpulan ekstrak etanol keladi tikus (typhonium flagelliforme lodd) memiliki efek sitotoksik terhadap sel hela dan sel mcf7. efek sitotoksik terhadap sel mcf-7 lebih besar dibandingkan efek sitotoksik terhadap sel hela. kata kunci: typhonium flagelliforme, tetrazolium bromide, sitotoksik, sel hela, sel mcf-7 abstrak introduction cancer is a multistep process occurring in stages, consisting of initiation (acquisition of irreversible genetic changes), promotion (increase in the population of initiated cells), and finally development of malignancy. cancer causes considerable mortality. it has been estimated that each year there are 190.000 new cancer patients, one-fifth of whom will die.(1) among the various types of cancer, two with the highest ranking are cervical cancer and breast cancer. modern or conventional (medical) treatments are considered to be extremely expensive, having significant side effects, while the results may not always be satisfactory. to date, tumor or cancer management still has many constraints, such as insensitiveness of the cancer towards antiproliferation signals and their capability of evading the apoptotic program.(2) in indonesia various herbals have been applied as alternative drug therapy to aid in the treatment of cancer. many herbals are known to possess anticancer properties due to their antiproliferative effects against cancer cells, thus inhibiting cancer cell growth. one of the plants 77 with a potential for development into an anticancer drug is rodent tuber [typhonium flagelliforme (lodd) blume]. the extracts of this plant contain antineoplastic or anticancer compounds.(3) rodent tuber of the species typhonium flagelliforme has long been known to be a potent traditional medication against cancer cells. according to a number of studies conducted in malaysia, rodent tuber is capable of reducing pain and decreasing the occurrence of metastases or dissemination of cancer cells, including breast, nasopharyngeal, and cervical cancers, and cancers of the prostatic gland, pancreas, and lungs.(4-5) in addition, this plant is found growing in large numbers in indonesia and malaysia, and is used to treat hemorrhoids, skin disorders, and to neutralize narcotic drugs. r o d e n t t u b e r a l s o h a s a n t i b a c t e r i a l a n d a n t i o x i d a n t a c t i v i t i e s . ( 4 6 ) s e v e r a l h e r b a l preparations use typhonium flagelliforme for cancer treatment.(7) there are few data on the chemical composition of rodent tuber. phytochemical analyses at the research station for medicinal a n d a r o m a t i c p l a n t s ( b alai p enelitian tanaman obat and aromatik) demonstrated the presence of alkaloids, saponins, steroids and glycosides.(8) however, the active substances in rodent tuber that specifically play a role in the healing of cancers are still unknown. rodent t u b e r s a l i n e e x t r a c t s c o n t a i n r i b o s o m e inactivating proteins (rips). these are proteins that are capable of cutting cancer cell dna or rna, so as to inactivate cancer cells without damaging the surrounding tissues. in addition, rips can also block cancer cell growth,(9) and play a role in triggering apoptosis through cutting dna and rna, and through n-glycosidase activity.(10-11) in a previous study, the dichloromethane fractions of typhonium flagelliforme tuber extract were capable of increasing caspase-3 and p21 expression in the michigan cancer foundation-7 (mcf-7) breast cancer cell line that has a deletion of the caspase 8 gene apoptosis-related cysteine peptidase (casp8).(2) however, the abovementioned study did not involve cytotoxicity tests of the rodent tuber extract against the mcf-7 cell line. another study using chloroform and ethyl acetate extracts of typhonium divaricatum (l) decne, demonstrated that the extracts had cytotoxic effects on hela cells, but with differing ic50 values.(12) therefore the purpose of the present study was to test the cytotoxicity of rodent tuber (ty p h o n i u m f l a g e l l i f o r m e l o d d ) a g a i n s t cultures of mcf-7 cells as an in vitro model for breast cancer, and of hela cells as an in vitro model for cervical cancer. methods study design this was a laboratory experimental study conducted from april 2013 until november 2013. plants materials rodent tuber plants were obtained from medicinal herb gardens in the jagakarsa area, south jakarta. tumor cell lines the hela and mcf-7 cell lines were supplied by the parasitology laboratory, faculty of medicine, gadjah mada university. the reagents used for culture media and cell cultures were 0.5% fetal bovine serum (fbs), rosswell park memorial institute culture medium [(rpmi) (gibco)], dulbecco’s minimum essential medium (dmem), 70% ethanol, sodium bicarbonate, hydroxyethyl piperazineethanesulfonic acid (hepes), sodium dodecyl sulphate (sds), and dimethyl sulfoxide [(dmso) (sigma)]. preparation of culture media and cell growth media culture media for hela and mcf-7 cells were prepared by dissolving rpmi 1640 and dmem powder, respectively, in 800 ml twicedistilled water, with the addition of 2.0 grams of sodium bicarbonate and 2.0 grams of hepes, univ med vol. 33 no.2 78 purwaningsih, widayanti, suciati cytotoxicity of typhonium flagelliforme made up to a volume of one liter with twicedistilled water. the solution was mixed with a magnetic stirrer for about 10 minutes until homogenous, than neutralized with 1 n hcl to a ph of 7.2 – 7.4. cell growth media were prepared by mixing 19 ml fbs, 2 ml penicillin streptomycin, and 0.5 ml fungizone, and diluting with rpmi 1640 and dmem culture medium, respectively, to a volume of 100 ml. the solution was then aseptically filtered through a sterile 0.2 µm polyethylene sulfone filter and stored in the refrigerator in stoppered bottles. the cell c u l t u r e s w e r e o b s e r v e d u n d e r t h e l i g h t microscope (olympus). preparation of rodent tuber extract rodent tuber whole plant extract was prepared by washing fresh whole plants, cutting them into strips, and air drying. the dried strips were then pulverized in a blender, then the powder was macerated with 80% ethanol in an erlenmeyer flask, under continuous shaking. the ethanolic extract was decanted into a rotary flask and concentrated in a rotary evaporator. the concentrated rodent tuber extract was serially diluted with dmso and filtered through an 0.2 µm filter. the prepared serial dilutions were: 500 µg/ml, 250 µg/ml, 125 µg/ml, 62.5 µg/ml, 31.25 µg/ml, 15.625 µg/ml, 7.81 µg/ml, and 3.9 µg/ml. 3-(4,5-dimethylthiazole-2-il,5-diphenyl tetrazolium bromide) (mtt) assay hela and mcf-7 cells, grown on a 96well microplate to a density of 1 x 104 cells per well, were incubated in rpmi medium (hela cells) or dmem medium (mcf-7 cells) for 48 hours to obtain optimal growth. after a change of medium, rodent tuber extract serial dilutions in dmso (sigma) were added to the respective wells, then the microplate was incubated in a 5% co2 incubator at 37 oc for 48 hours. at the end of the incubation period, the media and extracts were discarded and the cells washed in phosphate buffered saline (pbs, sigma). to each microplate well, 100 µl of a 5 mg/ml mtt (sigma) solution was added. the cells were then again incubated for 4-6 hours in a 5% co2 incubator at 37oc. the mtt reaction was stopped with 4 n hcl-isopropanol (stopper reagent, 1:100), and gently agitated on a shaker for 10 minutes or incubated overnight at room temperature. absorbance was read in an elisa reader (biorad) at 595 nm wavelength. data analysis calculation of ic50 values was performed by probit analysis using spss version 16.0. results the calculated percentage inhibition values of rodent ulcer extract against growth of hela and mcf 7 cancer cells are presented in tables 1 and 2. dilution of rodent ulcer extract (µg/ml) inhibition (%) 500 250 125 62.5 31.25 15.625 7.8125 3.90 1.78 ± 0.25 0.96 ± 0.39 0.58 ± 0.17 1.98 ± 1.03 88 .08 ± 6.87 90 .32 ± 3.99 91 .80 ± 4.58 79 .86 ± 3.29 table 1. percentage inhibition of rodent ulcer extract against hela cells *values are mean ± sd dilution of rodent ulcer extract (µg/ml) inhibition (%) 500 250 125 62.5 31.25 15.625 7.8125 3.90 67.85 ± 4.66 84.69 ± 10.58 86 .87 ± 0.17 89 .97 ± 1.11 91 .39 ± 4.83 90 .52 ± 4.44 94.61 ± 10.21 82 .18 ± 3.37 table 2. percentage inhibition of rodent ulcer extract against mcf-7 cells *values are mean ± sd 79 figure 1. probit curves for rodent tuber extract against hela cells note: higher doses tend to result in greater inhibitory effects figure 2. probit curves for rodent tuber extract against mcf-7 cells note: the highest dose (500 µg/ml) showed lower inhibition than the dose of 250 µg/ml. for the other doses, lower inhibition was associated with lower doses univ med vol. 33 no.2 80 purwaningsih, widayanti, suciati cytotoxicity of typhonium flagelliforme inhibition of hela and mcf-7 cells tended to increase with higher dilutions of rodent tuber extract. maximum inhibition of hela cells was 91.8 ± 4.58% at a dilution of 7.1825 µg/ml (table 1), while for mcf-7 cells, maximum inhibition was also at a dilution of 7.1825 µg/ ml, the percent inhibition being 94.61 ± 10.21% (table 2). calculation of ic50 values by probit analysis, from a probability of 0.01 up to 0.99, gave ic50 values of 30.19 µg/ml for hela cells and 5.59 µg/ml for mcf-7 cells. the probit curves for rodent tuber extract are presented in figures 1 and 2. discussion r o d e n t t u b e r e t h a n o l i c e x t r a c t demonstrated inhibitory effects on the growth of the test cancer cells. the cytotoxicity assay of rodent tuber extract against hela and mcf7 cells used dmso at a concentration of 0.1% a s t h e s o l v e n t . a p r e v i o u s s t u d y o n t h e cytotoxicity of another rodent tuber species, typhonium divaricatum l, against hela cells reported that the use of dmso as a solvent at concentrations of up to 0.1% or below 3%, had no effects on cancer cells. (12) the results obtained from our cytotoxicity assays showed a tendency of the rodent tuber ethanolic extract to have a cytotoxic potential against both hela and mcf-7 cancer cells. essentially similar results were obtained in an in vivo study, s h o w i n g t h a t ty p h o n i u m f l a g e l l i f o r m e ethanolic extract was capable of decreasing tumor cell proliferation in mice, as shown by the expression levels of ki67 as a proliferation marker.(13) the cytotoxic effect of rodent tuber extract in the present study, i.e. inhibition of hela and mcf-7 cell proliferation, tended to increase with increasing rodent tuber concentration. this is in agreement with the abovementioned study on typhonium divaricatum l cytotoxicity against hela cells, using chloroform and ethyl acetate extracts of the plant, where the highest dose used was 250 µg/ml and the lowest dose 5 µg/ ml.(12) from phytochemical analyses by previous investigators, it is known that rodent tuber contains alkaloids, saponins, steroids, phenols, and glycosides, but the specific active principle of rodent tuber that plays a role in the healing of cancer is still unknown.(8) these studies showed that phenolic compounds in rodent tuber are responsible for its anticancer potential. in addition, it has been reported that phenolic compounds in herbal extracts can play a role as chemopreventive agents or may promote inhibition of the cell cycle and trigger apoptosis of the cells.(4,14) other studies demonstrated that phenolic compounds of the flavonoid group are able to inhibit cancer cell growth. flavonoids containing phenolic groups are capable of inhibiting the growth of rat h4iie cells at low doses and also promote apoptosis at these low doses.(12) furthermore, it is known that rodent tuber saline extract contains ribosome inactivating proteins (rips), which are capable of cutting cancer cell dna or rna, thus inhibiting cancer cell growth without damage to the surrounding tissues; in addition, rips are also able to block cancer cell growth. ribosome inactivating proteins are in general of plant origin and are cytotoxic against mammalian cells.(9) rodent tuber extract ic50 against hela cells and that against mcf-7 cells are different in value, the ic50 against mcf-7 cells being lower than that against hela cells. this shows that rodent tuber extract is more toxic against mcf-7 cells than against hela cells. as is well known, hela cells are commonly used for in vitro assays of cervical cancers, whereas mcf7 cells are commonly used for assays of breast cancer cells, in addition to t47d cells. thus rodent tuber extract has a greater anticancer potency against breast cancer cells than against cervical cancer cells. rodent tuber is reportedly capable of inhibiting the growth of breast cancer cells and enhance apoptosis.(15,16) previous investigators have reported that chloroform and 81 hexane extracts of rodent tuber roots and tubers had ic50 values of 15.0 µg/ml and 6.0 µg/ml, respectivley, against p288 leukemia cells, while chloroform and hexane extracts of rodent tuber stalks and leaves had ic50 values of 8.0 µg/ml and 65.0 µg/ml, respectively.(17) another study has demonstrated that the dichloromethane extract fraction number 7 (dcm/f7) of typhonium flagelliforme inhibited leukemia cancer-cell proliferation selectively, via the induction of apoptosis.(18) dichloromethane and ethyl acetate extracts of typhonium flagelliforme inhibit the in vitro proliferation of the cem-ss human t4-lymphoblastoid cell line.(19) these study results indicate that typhonium flagelliforme possesses anticancer potential, for which further studies are necessary. the present study is still in its early stages, therefore more advanced studies are required in connection with the potential of rodent tuber plants as anticancer agents, e.g. through an antitelomerase mechanism, in view of the fact that cancer cells possess high telomerase activity. conclusions the study results indicate that the dmso extract of rodent tuber plants (typhonium flagelliforme lodd) have cytotoxic potential against hela and mcf-7 cells. the cytotoxic effect against mcf-7 cells is greater than the cytotoxic effect against hela cells. further investigations are necessary on the effects of rodent tuber extract against telomerase activity or expression in mcf-7 and hela cancer cells. acknowledgements the authors express their gratitude to the directorate general of the ministry of higher education (direktorat jendral dikti) for granting the necessary funds for the conduct of this study, through the research funds of desentralisasi riset unggulan perguruan tinggi. references 1. greenlee rt, hill-harmon mb, murray t, thun m. cancer statistics. cancer j clin 2001;51:15-36 2. putra a, tjahjono, winarto. ekstrak keladi tikus (typhonium flagelliforme) fraksi diklorometanolik dan ekspresi caspase-3 dan p21 cell line kanker payudara mcf-7. m med indones 2011;45:95104. 3. kainsa s, kumar p, rani p. medicinal plants of asian origin having anticancer potential: short review. asian j biomed pharm sci 2012;2:1-7. 4. mankaran s, dinesh k, deepak s, gurmeet s. typhonium falgelliforme: a multipurpose plant. int res j pharm 2013;4:45-8. 5. mohan s, abdul ab, wahab sia, al-zubairi as. antibacterial and antioxidant activities of typhonium flagelliforme (lodd) blume tuber. am j biochem biotech 2008;4:402-7. doi: 10.3844/ ajbbsp.2008.402.407. 6. mohan s, abdul ab, abdelwahab si, al-zubairi as, aspollah sukari m, abdullah r, et al. typhonium flagelliforme inhibits the proliferation of murine leukemia wehi-3 cells in vitro and induced apoptosis in vivo. leuk res 2010;34:148392. 7. yasmin a, mudahar h. penapisan fitokimia, klt, serta uji toksisitas ekstrak etanol 70% umbi keladi tikus dengan metode bslt. laporan penelitian, badan libangkes; 2005. 8. syahid, kristina nn. induksi dan regenerasi kalus keladi tikus (typhonium flagelliforme lodd) secara in vitro. j littri 2007;13:142-6. 9. de virgilio m, lombardi a, caliandro r, fabbrini s. ribosome inactivating protein: from plant defense to tumor attack. toxin 2010;2:2699-737. doi: 10.3390/toxin2112699. 10. narayanan s, surendranath k, bora n, surolia a, karande a. ribosome inactivating protein and apoptosis: minireview. febs letters 2005;579: 1324-31. 11. das mk, sharma rs, mistra v. induction of apoptosis by ribosome inactivating proteins: importance of n-glycosidase activity. appl biochem 2012;166:1552-61. 12. da’i m, fiveri a, meiyanto e. efek sitotoksik ekstrak tanaman keladi tikus (typhonium divaricatum, l) terhadap sel hela. j farm ind 2007; 3:163-7. 13. chodidjah, dharmana e, susanto h, sarjadi. typhonium flagelliforme decreases tyrosine kinase and ki67 expression in mice. univ med 2013;32:146-54. 14. sujatha r, norhanom aw, nurhayati za, suguraman m. effect of extract from phyllanthus univ med vol. 33 no.2 82 purwaningsih, widayanti, suciati cytotoxicity of typhonium flagelliforme watsonii airy shaw on cell apoptosis in cultured human breast cancer mcf-7 cells. j exp toxicol pathol 2013;65:341-9. 15. lai cs, mas rh, nair nk, majid mi, mansor sm, navaratnam v. typhonium flagelliforme inhibits cancer cell growth in vitro and induces apoptosis: an evaluation by the bioactivity guided approach. j ethnopharmacol 2008;118:14-20. 16. widowati l, mudahar h. uji aktivitas ekstrak etanol 50% umbi keladi tikus (typhonium flagelliforme lood) terhadap sel kanker payudara mcf-7 in vitro. media litbang kes 2009;19:9-14. 17. choo cy, chan kl, takeya k, itokawa h. cytotoxic activity of typhonium flagelliforme (araceae). phytotherapy res 2001;15:260-2. 18. mohan s, bustamam a, ibrahim s, al-zubairi as, aspollah m, abdullah r, et al. in vitro ultramorphological assessment of apoptosis on cemss induced by linoleic acid-rich fraction from typhonium flagelliforme tuber. evid based complement altern med 2011. article id 421894, 12 pages. doi:10.1093/ecam/neq010. 19. mohan s, bustaman a, ibrahim s, al-zubairi as, aspollah m. anticancerous effect of typhonium flagelliforme on human t-4 lymphoblastoid cell line crm-ss. j pharmacol toxicol 2008;3:449-56. oktavianus 187 *department of anatomy, faculty of medicine, trisakti university **department of physical medicine and rehabilitation, faculty of medicine, university of indonesia ***department of community medicine, faculty of medicine, university of indonesia ****department of orthopedics and traumatic surgery, faculty of medicine, university of indonesia ***** department of physical medicine and rehabilitation, faculty of medicine, university of airlangga correspondence dr. dr. maria regina rachmawati, pa(k), spkfr department of anatomy, faculty of medicine, trisakti university jl. kyai tapa no.260 grogol jakarta phone : +6221-5672731 ext. 2101 email: rachmawati@trisakti.ac.id univ med 2013;32:187-96 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background foot pronation causes biomechanical abnormalities in the form of functional leg-length disparity. foot orthoses are often used in the treatment of abnormal pronation. the aim of the study was to evaluate the effect of foot orthoses on abnormal kinematic chain the differences of pelvic height, step length, and walking distance on walking test in young women with biomechanical abnormality. methods a randomized double blind controlled clinical trial was conducted on 27 young adult women having abnormal biomechanical abnormalities. by random allocation the subjects were divided into the intervention group (14 subjects) receiving correction of foot pronation using foot orthoses, and the control group (13 subjects) receiving no orthoses. before and during use of foot orthoses, we determined pelvic height difference (mm), step length difference (cm), and walking distance at maximal walking speed for 15 minutes. results correction of foot pronation resulted in decreased pelvic height difference from 4.7 ± 2.1 mm to 1.7 ± 1.3 mm (p<0.001) and in a reduction in step length difference, from 4.9 ± 2.9 cm to 2.1 ± 1.5 cm (p=0.002). walking test distance of the intervention group was 1318.5 ± 46.3 m, as compared with that of the control group of 1233 ± 114.7 m (p = 0.05). walking distance of the intervention group rose steadily in the second test to 1369.3 ± 27 m, and in the third test to 1382.14 ± 10.5 m (p<0.001). conclusions foot orthoses improved the kinematic chain, resulting in a more symmetrical pelvic height, reduced step length difference, and increased functional walking ability. key words: foot orthoses, biomechanical disorder, kinematic chain, young women foot orthoses improve kinematic measurement in young women with biomechanical abnormality maria regina rachmawati*, angela bm tulaar**, muchtarudin mansyur***, ferial hadipoetro idris**, ismail****, and ratna darjanti haryadi***** 188 rachmawati, tulaar, mansyur, et al foot orthoses and kinematic measurement ortosis kaki memperbaiki pengukuran kinematik pada wanita muda dengan kelainan biomekanikal latar belakang adanya kelainan biomekanik berupa kaki pronasi dapat berhubungan dengan terjadinya disparitas panjang tungkai fungsional.. ortosis kaki seringkali digunakan untuk mengatasi kaki pronasi abnormal. penelitian ini bertujuan untuk menilai pengaruh penggunaan ortosis kaki terhadap kelainan rantai kinematik selisih tinggi pelvis, panjang langkah, serta jarak tempuh saat uji jalan pada perempuan muda dengan kelainan biomekanik. metode rancangan eksperimental tersamar ganda ini mengikutsertakan 27 perempuan muda dengan kelainan pronasi. randomisasi menempatkan 14 subjek pada kelompok intervensi yang menggunakan ortosis kaki, dan 13 subjek kelompok kontrol yang tidak menggunakan ortosis kaki. ukuran kinematik berupa selisih tinggi pelvis (mm), selisih panjang langkah (cm) dan jarak tempuh saat berjalan pada kecepatan maksimal selama 15 menit diukur sebelum dan selama penggunaan ortosis kaki. hasil penggunaan ortosis kaki menurunkan selisih tinggi pelvis secara bermakna dari 4,7 ± 2,1 mm menjadi 1,7 ± 1,3 mm (p<0,001). penurunan secara bermakna juga terjadi pada selisih panjang langkah dari 4,9 ± 2,9 cm menjadi 2,1 ± 1,5 cm (p=0,002). uji jalan pada kelompok perlakuan jarak tempuhnya sebesar 1318,5 ± 46,3 m, yang lebih jauh dibandingkan kelompok kontrol sebesar 1233,0 ± 114,7 m (p=0,05). jarak tempuh uji jalan pada kelompok intervensi meningkat secara bermakna menjadi 1369,3 ± 27,0 m pada uji jalan kedua dan 1382.14 ± 10.5 m pada uji jalan ketiga (p<0,001). kesimpulan penggunaan ortosis kaki mampu memperbaiki kelainan biomekanik yang menghasilkan tinggi pelvis yang simetris, penurunan selisih panjang langkah dan meningkatkan kemampuan berjalan. kata kunci : ortosis kaki, kelainan biomekanik, rantai kinematik, perempuan muda abstrak introduction according to previous studies, the most prevalent biomechanical abnormality in the population is functional leg-length disparity (60% to 90%). previous studies have also found an association between functional leg-length disparity and foot pronation. the prevalence of flexible foot pronation in the population is 2030%.(1) foot pronation is a combination of subtalar eversion, anterior abduction and dorsiflexion of the foot, and flat foot.(2) foot pronation may result in other biomechanical abnormalities, i.e. increased knee q angle, pelvic tilt, and lumbar postural scoliosis. pelvic tilt causes functional leg-length disparity, which may lead to abnormal walking. the latter may be due to walking kinematics in the sagittal plane, in the form of asymmetry in right and left step lengths.(3) foot pronation accompanied by f u n c t i o n a l l e g l e n g t h d i s p a r i t y w i t h o u t subsequent intervention may lead to structural joint damage, such as damage to cartilage of the subtalar, knee, and facet joints, along with 189 univ med vol. 32 no.3 damage to the intervertebral discs.(4,5) these findings support other studies demonstrating that s u b j e c t s w i t h l o w b a c k p a i n h a d h i g h e r prevalence of functional leg-length disparity than controls. a disparity of more than 5 mm was found in 75% of patients with low back pain, and in 44% of the control group.(6) previous studies also found that cases of back pain and l e g p a i n w e r e c l o s e l y a s s o c i a t e d w i t h asymmetrical leg-length.(7,8) individuals with foot pronation and functional leg-length disparity may experience v e r t e b r a l p e l v i c h i p k i n e m a t i c c h a i n abnormalities. the kinematic chain abnormalities may increase the activity of skeletal muscles around the joints during activity, increasing the mechanical tension of the muscular tissue of the lower limbs, and possibly resulting in skeletal muscle injury.(7,8) correction of foot pronation is indicated to p r e v e n t m u s c l e i n j u r y i n b i o m e c h a n i c a l disorders. conservative management of flexible foot pronation consists in advising the patient to frequently walk without footwear on a soft surface, such as sand, turf, or a thick carpet, to perform active and passive contraction exercises on the achilles tendon, and to use foot orthoses. the latter is the most effective conservative management of foot pronation, as it ensures the stability of correction of foot pronation posture in the performance of daily activities. correction of foot pronation may result in pelvic equality, which will improve functional leg-length disparity, thus improving the kinematic measures of walking, i.e more symmetrical right and left step lengths.(9,10) the assessment of foot pronation is performed by means of foot prints, pelvic height difference by means of bodyworks body scan, and kinematic evaluation of walking activity in the sagittal plane by means of the biodex gait trainer 2 (230 vac).(11-12) based on the above description, there is a need for a study to evaluate the effect of foot orthoses on kinematic m e a s u r e m e n t s i n y o u n g w o m e n w i t h biomechanical abnormalities. gender and age affect the occurrence of muscle injury and degenerative processes, thus to obtain a homogenous sample of subjects without degenerative problems, this study was conducted on young adult women aged 20-29 years.(13-15) methods research design this study was a randomized double blind controlled trial and was conducted from october 2010 to february 2011 in three locations. history taking (anamnesis), physical examination, and fitting of foot orthoses were performed at trisakti medical center (pusat medis trisakti). walking tests were conducted by means of the biodex gait trainer 2 at a private hospital in east jakarta. three-dimensional body scans were performed at the ergonomics laboratory, faculty of industrial technology, university of indonesia, depok. study subjects the study subjects were female students of universities in west jakarta with biomechanical abnormalities in the form of functional leg-length disparity accompanied by foot pronation. the sample size calculation was according to the formula for the difference between two means, to detect a 25% difference of mean change in kinematic measures, and the obtained estimation of sample size was 14 subjects for each group. intervention research subjects were randomly divided into two groups, i.e. the control group and the intervention group. the intervention group was given foot orthoses for correction of foot pronation whereas the control group was not. we evaluated the differences in pelvic alignment with and without foot orthoses. the assessment was conducted using three-dimensional body scans to meassure pelvic heights. subjects in both groups performed the walking test to assess step length differences and walking distance on the biodex gait trainer 2. the intervention group 190 rachmawati, tulaar, mansyur, et al foot orthoses and kinematic measurement figure 1. (a) foot orthoses; (b) height of medial arch measured by digital caliper; (c) type of shoes used; (d) inserting orthosis into shoe a b c d performed the walking test twice, both with and without foot orthoses. to meassure step length differences, the walking test was performed at normal speed for 2 minutes, whereas to meassure walking speed, the test was performed by both groups at the maximum speed that could be tolerated by the subjects for 15 minutes. the walking test to measure walking distance in the intervention group was performed three times, i.e at the start of the study, at day seven, and at day fourteen, whereas walking distance in the control group was assessed only once. the type of ready-made polyethylene orthoses used in this study as shown in figure 1 are relatively expensive and not freely sold on the indonesian market. mean height of the medial arch supports in these foot orthoses is 1.30 ±0.15 cm. prescribing foot orthoses with identical heights of medial arch supports resulted in elevation of both subtalar joints to a relatively similar level in all subjects of the intervention group.(9,10) protocol of assessment a s s e s s m e n t o f f o o t p r o n a t i o n w a s performed by means of foot prints to find the medial contour of the foot prints, from which the grade of foot pronation was determined (grades 1-3).(4) assessment of pelvic alignment was by means of body scans obtained by laser beam technology projected on the human body. the light sensors act concurrently on the simple geometric principle of triangulation, and will measure the surface of the human body. a special optical system and glass are used to obtain a linear reflection of the single laser beam. the laser scanning unit, consisting of the laser, optical system, and light sensor, moves across the human body to obtain digital measures of the body surface. three scanning units move vertically and synchronously along three posts and are aimed at the center where the subject is standing. the light sensor, optical system and electric motor can be detached and reattached. the scanning unit has to be calibrated such that the positions of the geometric elements may be accurately determined. a disadvantage of this instrument is that it is expensive and rare. when scanning is performed, the body has to be immobilized for a few seconds. the values o b t a i n e d b y d i g i t a l i z a t i o n c o n s i s t o f anthropometric measurements expressed in centimeters and millimeters. the use of the scanner for anthropometric measurements of the human body has been demonstrated to be valid and reliable.(11) 191 univ med vol. 32 no.3 a b c figure 2. (a) right subtalar position without orthoses; (b) right subtalar position with orthoses; (c) difference in right subtalar height with and without orthoses a b c the anthropometric measurement used for assessment of pelvic height is the distance from the hip (iliac crest) to the ball of the foot. the pelvis is considered to be straight when the difference between the right and left pelvic heights is small. the assessment of walking step length was performed in subjects with foot pronation and functional leg-length disparity, both without and during use of foot orthoses. the computer of the biodex gait trainer 2 automatically measures the distance between two contralateral heel strikes and calculates the mean right and left step lengths.(12) assessment of step lenght by means of the biodex gait trainer 2 was performed in our previous study investigating the effect of step figure 3. (a) left subtalar position without orthoses; (b) left subtalar position with orthoses; (c) difference in left subtalar height with and without orthoses 192 rachmawati, tulaar, mansyur, et al foot orthoses and kinematic measurement figure 4. flowchart of the study subjects length on body posture and low back pain in 77 young adults.(8) the walking test was evaluated by a research assistant was blinded to the use of foot orthoses by the subjects. in the present study, improvement of subtalar joint height when using foot orthoses was determined from x-ray radiographs. the radiographs were taken from the medial sides of both feet, with the foot supporting the weight of the body on the stance phase in the walking cycle. the radiographs were taken both with and without foot orthoses. both radiographs were then compared to find the difference in the heights of the subtalar joints with and without using foot orthoses, as shown in figures 2 and 3. data analysis tests of normality for all variables were performed according to the saphiro-wilk method. intervention groups were compared with respect to baseline demographic and clinical characteristics. independent t test was performed to compare all variables at baseline the kinetic measurements after foot correction. repeated anova was performed to evaluate the effect of the intervention on pulse rate and walking distance. analyses were conducted at the 0.05 á level. all analyses were performed with spss software (version 17: spss inc, chicago, il). ethical clearance in this study all components of ethical validity have passed the test of ethics by the research ethics committee of the faculty of medicine, university of indonesia. results screening of the 177 subjects obtained 27 subjects meeting the inclusion criteria figure 2. 193 univ med vol. 32 no.3 table 1. baseline characteristics of the subjects table 2. effect of foot orthoses on kinematic variables in the two experimental groups the baseline characteristics of the study subjects are shown in table 1. the mean age of the intervention group was 20.5 ± 0.5 years, and the mean age of the control group was 20.3 ± 0.5 years. the mean height was 1.5 ± 0.4 m in the intervention group and was 1.5 ± 0.5 m in the control group. at the beginning of the study, there were no significant differences of the various physical and kinematic variables between the intervention and control groups. this suggested that randomization had been performed successfully to spread evenly all variables between the two groups, except the intervention variable. the fitting of foot orthoses decreased significantly the differences in pelvic height (1.7 ± 1.3 cm) in the intervention group as compared with the control group (4.7 ± 2.1 cm) (p <0.001). the same effect is shown in the significantly smaller differences between right and left step lengths in the intervention group (2.1 ± 1.5 cm) compared with the control group (4.9 ± 2.9 cm) (p = 0,002) (table 2). to determine walking intensities performed by the subjects on walking tests, the achieved of walking distance was measured. the measurement results of walking distance achieved in walking tests 1, 2, and 3 are presented in table 3. to determine walking distance difference in walking test 1 between both groups, the mannwhitney test was performed. the results showed that in walking test 1 the walking distance of 1318.5 ± 46.3 m in the intervention group was longer than the walking distance of 1233.1 ± 114.7 m in the control group (p=0.05). analysis of walking distance in walking tests 1, 2, and 3 in the group with foot orthoses, was conducted by means of the friedman test and found a significant difference in walking distance (p<0.001). subsequently the wilcoxon test was performed, with the results indicating that walking distance in walking test 3 was longer than that in walking test 1 (p=0.001). similarly, walking distance in walking test 2 was longer than that in walking test 1 (p=0.002), while walking distance in walking test 3 was longer than that in walking test 2 (p=0.048). 194 rachmawati, tulaar, mansyur, et al foot orthoses and kinematic measurement table 3. distance of the walking test between the intervention and control groups discussion the fitting of foot orthoses in young adult women with foot pronation and functional leglength disparity was demonstrated to improve pelvic alignment (pelvic alignment in the horizontal plane), as the pelvic height difference became smaller, from 4.7 ± 2.1 mm to 1.7 ±1.3 mm. the results of measurement of left and right pelvic height, with and without foot orthoses showed that the left pelvic height increased from 98.3 ± 3.6 cm to 101.0 ± 3.4 cm, and right pelvic height increased from 98.6 ± 3.6 cm to 101.0 ± 3.5 cm. the presence of functional leg-length disparity due to foot pronation, which results in pelvic height difference, has been demonstrated in previous studies conducted by knutson, lenvinger et al., and rothbart.(1,4,5) knutson states that the mean functional leg-length disparity was 5.2 ± 4.1 mm.(1) our study results showing that the pelvic becomes more aligned when using foot orthoses, demonstrates the occurrence of a biomechanical improvement of the feet, knees, and of the sacroiliac joints. the results of this study support those of a study conducted by shorten and bishop et al., in that the use of foot orthoses in foot pronation is capable of enhancing foot posture, which may improve rotation in the knee and sacroiliac joints.(9,11) t h e o c c u r r e n c e o f i m p r o v e d p e l v i c alignment from use of foot orthoses is due to the fact that foot orthoses support the low medial arches of the feet in foot pronation. the use foot orthoses elevates the medial arch of the foot to the level of the orthotic arch.(4,5) the use of foot orthoses with identical support heights of the medial arches on both sides causes the subtalar heights to be more symmetrical. the improved subtalar position leads to improvement of tibial and sacroiliac rotation. the ensuing correction of joint position results in better alignment of the subtalar, knee, and sacroiliac joints and promotes more symmetrical pelvic heights. in foot pronation, the subtalar joint is in a valgus position, causing rotation of the knee and sacroiliac joints. the resulting series of biomechanical abnormalities causes tilting of the pelvis and functional shortening of one of the lower extremities. the pelvic tilt promotes the occurrence of functional lumbar scoliosis, with its convexity in the direction of the shorter one of the lower extremities. the occurrence of functional lumbar scoliosis leads to structural imbalance of the vertebrae and promotes the formation of a compensatory curve towards the longer one of the lower extremities. the resulting r o t a t i o n o f t h e j o i n t s i n t h e s e r i e s o f biomechanical abnormalities in uncorrected foot pronation, may cause damage to the cartilage of the knee and sacroiliac joints, and of the intervertebral disks.(4,5) the results of this study support the study conducted by weiner et al.(7) and nourbakhsh et al.,(8) who state that the presence of biomechanical abnormalities in the lower extremities and the vertebral column increases the risk of back pain. the occurrence of back pain in biomechanical abnormalities is due to tension and strain in the ligaments and m u s c l e s , d u r i n g a c t i v i t i e s o f t h e l o w e r extremities, such as standing, walking, and running, thus stimulating nociceptors for the afferent pain-conducting nerve fibers to the brain.(1,13) the study results demonstrated that the use of foot orthoses improves pelvic alignment (alignment of the pelvis in the horizontal plane) 195 univ med vol. 32 no.3 in young adult women with foot pronation and functional leg-length disparity. the use of foot orthoses in young adult women with foot pronation and functional leglength disparity resulted in more symmetrical right and left step lengths, due to decreased step length difference, from 4.9 ± 2.9 cm without foot orthoses to 2.1 ± 1.5 cm with foot orthoses. in addition, with foot orthosis use there was an increase in mean left and right step lengths, where mean left step length increased from 41.6 4 ± 5.1 cm to 49.2 ± 5.7 cm, while mean right step length increased from 45.6 ± 3.6 cm to 50.9 ± 6.5 cm. assessment of the difference in left and right step lenght, with and without foot orthoses, was performed using the biodex gait trainer 2.(12) the computer system of the biodex gait trainer 2 automatically calculates mean step length, thus allowing the evaluation of the mean differences in left and right step lengths, with and without foot orthoses. functional leg-length disparity, manifesting as a difference in left and right step lengths on walking, will increase the risk of back pain. improved step length symmetrical will improve biomechanical vertebral-pelvis-hip abnormalities, and may decrease the incidence of back pain. this conclusion supports a previous study on 77 young adults, that there is a relationship between step length differences, i n c i d e n c e o f b a c k p a i n a n d p o s t u r a l abnormalities.(6) according to the previous study, a more symmetrical step length decreases the risk of back pain.(6-8) murley et al.(15) demonstrated decreased activity of the tibialis anterior, tibialis posterior, and peroneal muscles on heel strike while using orthoses. shorten (12) and bishop et al.(13) obtained an increase in dorsiflexion and a decrease in shifts between the bones of the foot at heel strike, when using special shoes with bulit-in foot orthoses. the decreased work intensity of the muscles, increased kinematics symmetrical, and decreased shifts between the soles of the foot on walking with foot orthoses, may decrease the risks of muscle injury.(14,15) the symmetrical step lenghts that occur during using foot orthoses, is due to the fact that the foot orthoses support the medial arches. this support improves the kinematic chain at the knees and hips, to yield a more aligned pelvic posture, and also improves foot posture, thus improving the gravitational axis. a better aligned pelvic posture and normal foot posture improves functional leg-length disparity, and improves musculoskeletal system balance on walking. improvement of musculoskeletal system balance in the foot-hip-pelvis-vertebral chain causes a more symmetrical and longer step length. the use of foot orthoses also results in elevation of right and left pelvic heights, thus increasing walking distance.(5) an increase in walking distance of 1319.3 ± 44.6 m occurred in the group of foot orthosis users, as compared with a walking distance of 1233.0 ± 114.7 m in controls. these study results indicate that the use of foot orthoses improved the effectiveness of intrinsic and extrinsic muscular activity of the lower extremities on walking activity. these results support the results of previous studies indicating that using foot orthoses in foot pronation decreases muscular activity and improves kinematic measures.(9,10) the clinical implication of the results of this study is that young adult women with the biomechanical abnormality of foot pronation, accompanied by functional leg-length disparity, are recommended to use foot orthoses to improve their walking ability. a limitation of the study was the assessment of kinematic measurements in one plane only, because of limitations of the equipment. conclusions correction of foot pronation accompanied by functional leg-length disparity, through the use of ready-made polyethylene foot orthoses, results in more symmetrical pelvic height and step length. the use of foot orthoses in foot pronation enhances the functional capacity of the lower extremities, as evidenced from the longer 196 rachmawati, tulaar, mansyur, et al foot orthoses and kinematic measurement step lenghts and walking distance. further research needs to be done to assess kinematic m e a s u r e m e n t s i n o t h e r p l a n e s a n d t h e effectiveness of foot orthoses in young adult men. acknowledgements we thank the ergonomics laboratory, faculty of industrial technology, university of indonesia for the use of their three-dimensional body scan facilities, private hospital at east jakarta for the use of their biodex gait trainer 2 and the trisakti medical center (pusat medis trisakti) for enabling us to conduct the clinical examinations and orthoses fitting. thanks are also due to the faculty of medicine, trisakti university for funding this study. last but not least, we thank the all participants of this study for their cooperation in completing the studies. references 1. knutson ga. anatomic and functional leglength inequality: a review and recommendation for clinical decision-making. part i, anatomic leg-length inequality: prevalence, magnitude, effects and clinical significance. chiropr osteopathy 2005;13:12. doi:10.1186/1746134013-12. 2. keenan am, redmond ac, horton m, conaghan pg, tennant a. the foot posture index: rasch analysis of a novel, foot-specific outcome measure. arch phys med rehabil 2007; 88:88-93. 3. fattah am, hassanin mm, felembane fa, nassaane mt. flat foot among saudi arabian army recruits: prevalence and risk factors. east mediter health j 2006;12:211-7. 4. levinger p, murley gs, barton cj, cotchett mp, mcsweeney sr, menz hb. a comparison of foot kinematics in people with normal and flat-arched feet using the oxford foot model. gait posture 2010;32:519-23.doi: 10.1016/j.gaitpost.2010. 07.013. 5. rothbart ba. relationship of functional leglength discrepancy to abnormal pronation. j am pod med ass 2006;96:499-504. 6. rachmawati mr, samara d, tjhin p, wartono m, bastian y. musculoskeletal pain and posture decrease step length in young adults. univ med 2009;28:92-9. 7. weiner dk, sakamoto s, perera s, breuer p. chronic low back pain in older adults: prevalence, reliability, and validity of physical examination findings. j am ger soc 2006;54:1120. 8. nourbakhsh mr, arab am. relationship between mechanical factors and incidence of low back pain. j orthop spor phys ther 2002;32: 447-60. 9. shorten mr. running shoe design: protection and performance. in: pedoe dt,editor. marathon medicine. london; royal society of medicine; 2000.p.159-69. 10. bishop m, fiolkowski p, conrad b, brunt d, horodyski m. athletic footwear, leg stiffness, and running kinematics. j athlet train 2006;41:38792. 11. pepper mr, graves jhf, yu w, stanforth pr, cahill jm, mahometa m, et al. validation of a 3-dimensional laser body scanner for assessment of waist and hip circumference. j am coll nutr 2010;29:179-88. 12. biodex. gait trainer 2 application/operation manual. available at: http://www.biodex.com/ rehab/manuals/950385man.pdf. accessed december 29, 2010. 13. caselli ma. evaluation and management of leglength discrepancy. podiatr manage 2006: 14756. 14. murley gs, menz hb, landorf kb. methodology article: a protocol for classifying normaland flat-arched foot posture for research studies using clinical and radiographic measurements. j foot ankle res 2009;2:1-13. 15. murley gs. do foot orthoses change lower limb muscle activity in flat-arched feet towards a pattern observed in normal-arched feet? clin biomech 2010;24:89-98. 16. kirby ka. subtalar joint axis location and rotational equilibrium theory of foot function. j am pod med assc 2001;91:465-87. 17. khamis s, yizhar z. effect of feet hyperpronation on pelvic alignment in a standing position. gait posture 2007;25:127-34. oktavianus 141 *griffith university, brisbane, australia **department of microbiology, faculty of medicine, islamic university of indonesia, yogyakarta ***immunity, infection and inflammation program, mater medical research institute and university of queensland, brisbane, australia correspondence dr. shofyatul y. triyana, msc department of microbiology faculty of medicine, islamic university of indonesia jl. kaliurang km 14.5 yogyakarta email: shofyatul@uii.ac.id univ med 2012;31:141-50 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background cell surface mucin glycoproteins are expressed on the mucosal surface. one of these cell surface mucins is mucin-1 (muc1), which plays a role as a physical barrier and limits inflammation. however, its functional role in modulating responses to pathogens, particularly with respect to intracellular signaling, needs to be investigated. therefore, the aim of this study was to characterize the modulation of responses of human gastric epithelial cells by muc1 to common mucosal pathogens and inflammatory stimuli. methods human gastric epithelial cell lines (mkn7) were co-cultured with campylobacter jejuni (c. jejuni). in order to investigate the effect of muc1 expression on c. jejuni-induced cytokine production, mkn7 cells were transfected with 1:1 small interfering rna (sirna) to knock down the muc1 gene using muc1 targeting sirna or non targeting sirna as a control. the read out of the experiment was interleukin (il)-8 concentration as a result of the inflammation process. this cytokine concentration was measured using elisa and compared between the two groups. results this study demonstrated that by using sirna transfection, knockdown of muc1 expression in mkn7 human gastric epithelial cells suppressed il-8 production at the early phase of incubation, but promoted an increase in il-8 production at the late phase, in response to c. jejuni. conclusion knockdown of the muc1 gene in mkn7 cells reduced il-8 levels both in the cells with and without exposure to c. jejuni. this study provides direction for exploration of the intracelular mechanisms by which cell surface mucins modulates inflammation in the response of gastrointestinal epithelial cells to pathogens or other inflammatory stimuli. keywords: muc1, epithelial cells, pathogen, inflammation muc1 modulates gastric epithelial immune response to bacteria or inflammatory stimuli shofyatul y. triyana*,**, yong h. sheng***, and michael mcguckin*** 142 triyana, sheng, mcguckin, et al muc1 modulates gastric epithelial immune muc1 memodulasi respon imun sel epitel lambung akibat stimulus bakteri dan peradangan abstrak latar belakang glikoprotein musin seluler adalah musin yang diekspresikan di permukaan mukosa jaringan epitel. musin-1 (muc1) adalah musin seluler yang banyak diekspresikan di permukaan mukosa lambung dan berperan penting sebagai sawar fisik serta mencegah inflamasi. meskipun demikian, peran fungsional dalam memodulasi respon terhadap patogen, perlu diteliti secara mendalam. hal tersebut penting untuk memperoleh pemahaman yang baik tentang terjadinya respon alamiah terhadap infeksi. tujuan penelitian ini adalah untuk menentukan modulasi respon imun sel epitel lambung dari muc1 terhadap bakteri patogen dan stimulus inflamasi lainnya. metode penelitian in vitro menggunakan sel epitel lambung manusia berupa mkn7 cell line dilakukan ko-kultur bersama dengan campylobacter jejuni (c. jejuni). untuk menilai pengaruh ekspresi gena muc1 pada produksi sitokin yang diinduksi oleh c. jejuni, pada sel mkn7 dilakukan transfeksi small interfering rna (sirna) menggunakan muc1 targeting sirna untuk membuat gena muc1 non aktif dan transfeksi dengan non targeting sirna digunakan sebagai kontrol. elisa digunakan untuk mengukur konsentrasi interleukin (il)-8 dan kemudian dibandingkan antara kedua kelompok yakni kelompok dengan gena muc1 non aktif dan kelompok kontrol. hasil dengan transfeksi sirna, penonaktifan ekspresi gena muc1 pada sel epitel lambung mengakibatkan produksi il8 dihambat pada awal fase inkubasi namun produksi il-8 tersebut menjadi meningkat di akhir fase inkubasi ketika sel dipaparkan patogen c. jejuni. kesimpulan gena muc1 berpengaruh pada sekresi il-8. konsentrasi il-8 menurun ketika gena tersebut di-non-aktifkan, baik pada kelompok tanpa perlakuan maupun kelompok yang dipaparkan patogen c. jejuni. penelitian ini dapat memberikan gambaran awal untuk eksplorasi lebih lanjut tentang mekanisme intraseluler musin dalam mengubah respon inflamasi sel epitel gastrointestinal terhadap paparan patogen atau paparan inflamasi lainnya. kata kunci : muc1, sel epitel, pathogen, inflamasi introduction being a portal of entry to large numbers of bacteria, mucosal epithelial cells lining the gastrointestinal tract have mucins which can act a s a p h y s i c a l b a r r i e r a n d p l a y a r o l e i n modulating the immune response.(1) mucin 1 (muc1) is expressed by most mucosal tissues including stomach and intestinal tissue, and expressed by hematopoietic cells.(2) the muc1 molecule has been estimated to be 200-500 nm in length, so that it will exceed other molecules attached to the plasma membrane. the molecule are surface contains a complex array of o-linked oligosaccharides that have been shown to bind to microbial molecules. for example, distinct sets of carbohydrates play an important role in adhesion of helicobacter pylori (h. pylori) to epithelial cells, attenuation of h. pylori colonisation and facilitation of inflammatory 143 response following h. pylori infection.(1,3) there is evidence that muc1 can limit infection and inflammation. the extracellular domain of muc1 protects the epithelial cells by inhibiting bacterial adhesion to the mucosal surface by steric hindrance or by acting as a releasable decoy so that the severity of pathology caused by pathogen invasion such as h. pylori and campylobacter jejuni ( c. jejuni ) can be reduced.(4-6) in addition to limiting adhesion of bacteria to the cell surface, muc1 can modulate immune responses to bacterial components and toll-like receptor (tlr) ligands.(7-9) such immune modulation has been demonstrated in relation to the involvement of phosphorylated cytoplasmic domains in nf-κb activation and attenuation of tlr-signalling.(8,10-12) the severity of pathology following pathogen infection is known due to the increased bacterial binding to the cells in the absence of muc1. in a mouse model study, it has been demonstrated that muc1 limits h. pylori colonisation.(5,9) mice lacking muc1 had more bacterial colonization compared to wild type mice. additionally, following h. pylori infection the degree of gastric inflammation was compared between mice lacking muc1 and wild type and the results were consistent showing that mice lacking muc1 developed more severe gastric inflammation. co-culture of primary gastric epithelial cells with h. pylori showed that there was an increased adherence of h. pylori to murine primary gastric epithelial cells which lack muc1. muc1 appears to have a key role in limiting h. pylori colonisation and gastritisassociated pathologies.(5) however, it is possible that there is an alteration of immune response resulting from altered cell signalling as a result of interaction between pathogen and host epithelial cells. therefore, it is important to explore the immune response modulation by muc1 in vitro. the o b j e c t i v e o f t h e p r e s e n t s t u d y w a s t o characterize the response of gastrointestinal epithelial cells to live pathogenic bacteria in the presence and absence of muc1. methods research design this was an experimental in vitro study using co-culture technique between human gastric epithelial cell lines and c. jejuni and was conducted from february to october 2010. moreover, tnf á was also co-cultured with the cell lines as a trigger for inflammatory stimuli o t h e r t h a n p a t h o g e n i c b a c t e r i a . s m a l l interference rna transfection technique was used to knockdown the gene of interest (muc1). cell culture the human gastric epithelial cell line mkn7 (riken cell repository, japan) was stored in liquid nitrogen. the cells were then propagated in a complete culture media with added antibiotics, containing rpmi-1640 media, 10% heat-inactivated fetal bovine serum (fbs), 2 mm l-glutamine (invitrogen), 100 units/ml penicillin g sodium and 100µg/ml streptomycin (invitrogen). muc1 knockdown muc1 was knocked down using small interfering rna transfection technique. small interfering rna (sirna) specific for muc1 as well as non-targeted control sirna were chemically synthesized. mkn7 cells were transfected with either 100nm of muc1 sirna or control sirna using lipofectamine 2000 (life technologies, carlsbad, ca) according to the manufacturer ’s instructions. after 48 h of transfection, the cells were treated with c. jejuni and tnf-á respectively for the indicated time. the level of knockdown of muc1 protein was measured using flow cytometry. c. jejuni culture the pure glycerol stocks of bacteria (c. jejuni strains 81-176 and ∆cdt81-176) stored at -75°c were thawed and then mixed with the appropriate volume of brucella broth. twenty µl of the bacterial suspension was aseptically spread using a bacterial spreader onto selective univ med vol. 31 no.3 144 triyana, sheng, mcguckin, et al muc1 modulates gastric epithelial immune agar plates (c. jejuni plates: 2% columbia agar, 1% bacteriological agar, 5% defibrinated horse blood, skirrow selective supplement (oxoid), incubated under microaerophilic conditions (5% o2, 15% co2, 80% n2) in an anaerobic jar using campygen, a microaerophilic gasgenerating kit (oxoid) for 2 days at 42°c. the mutant strain 81-176 ∆cdt was also grown under the same conditions as the parental strain. co-culture the co-culture experiments were performed on confluent mkn7 cells with c. jejuni.the cells were transferred to microaerobic conditions (5% o2, 15% co2, and 80% n2) at the start of the co-culture. before introducing bacteria, the media was changed into antibiotic-free media containing rpmi-1640, 10% fbs and 2 mm lglutamine. analysis of il-8 by elisa the concentrations of il-8 secreted by mkn7 in various treatments were determined by human il-8 elisa (bd biosciences, san jose, ca, usa) following the manufacturer’s instructions. flow cytometry mkn7 cells were harvested using trypsine and incubated with the anti-muc1 antibody bc2 (ct2) or isotype control 401.21 at 3 µg/ml in 5% fcs for 60 min at 4°c and then with antimouse antibody conjugated to alexa fluor 488 (life technologies, carlsbad, ca). after s t a i n i n g , a l l c e l l s w e r e f i x e d w i t h 1 % paraformaldehyde. assessment of staining was performed on fcs 500 flow cytometer (bd biosciences, san jose, usa). data analysis the mann-whitney u-test was used to compare means of il-8 concentrations in the presence and absence of muc1. all analyses were conducted using graphpad prism version 5. p values less than 0.05 were considered significant. results muc1 gene was first knocked down to investigate the effect of muc1 expression on c. jejuni-induced cytokine production. figure 1 shows the expression of cell surface muc1 after knockdown using non-target sirna and muc1 sirna. in this experiment, transfection of muc1 sirna into mkn7 cells reduced cell surface muc1 expression by 91%. then, il-8 levels in culture media was measured after co-culture with c. jejuni. initially, the il-8 concentration was measured after 4 h and 24 h-bacterial exposure. it seems that at the short term co-culture, a higher bacterial density is needed to elicit significant il8 production. it was also demonstrated that at the same lower bacterial density but with a longer incubation time, the il-8 production was much higher (figure 2). the knockdown of muc1 gene in mkn7 cells had an effect on il-8 secretion. the il-8 levels decreased in the absence of muc1 both in cells with and without exposure to c. jejuni. the production of il-8 after c. jejuni treatment could be partially due to the presence o f m i c r o b i a l t o x i n . t h e r e f o r e , t h e n e x t experiment was done using parent c. jejuni 81176 and ∆cdt-c. jejuni 81-176. in this experiment, mkn7 cells transfected with a muc1-targeting sirna exhibited 92% reduction in muc1 protein levels as depicted in figure 3. comparison between parent and ∆cdt c. jejuni 81-176 demonstrated that there is no significant difference in il-8 level between these two treatment groups either in muc1-expressing cells or in muc1-knocked down-cells. the timing of incubation also does not influence the difference in il-8 concentration between treatment with parent and treatment with ∆ cytolethal distending toxins (cdt) c. jejuni 81176 (figure 4). therefore, probably cdt is not involved in inducing il-8 production in response to this particular bacterial strain. focusing on il-8 production, it is likely that at short term incubation, the muc1 145 knockdown causes decrease in il-8 secretion but with the longer incubation time, the concentration of il-8 increases although in s o m e c o n d i t i o n s t h e s e c h a n g e s a r e n o t statistically significant (figure 4). tnf-α was used as a positive control and it was shown that the concentration of il-8 induced by this pro-inflammatory cytokine varies with length of incubation time in the absence of muc1. tnf-α induces a higher il8 concentration at 4 h but a lower il-8 concentration at 24 h, although in some conditions such changes were not significant. discussion although there are some variations in the results obtained from this study, it has been demonstrated that muc1 knock down in mkn7 cells tends to cause a decrease in il-8 production figure 1. cell surface muc1 expression after knockdown using 1:1 sirna transfection. representative histograms from the flow cytometry analysis showing median fluorescence intensitiy (mfi). mfi was determined after subtraction of the mfi using a negative control antibody 401.21. results from 3 replicates univ med vol. 31 no.3 146 triyana, sheng, mcguckin, et al muc1 modulates gastric epithelial immune in the short term but that a higher concentration is achieved in the long term. because in this study the il-8 production was only measured at endpoints (4 h and 24 h), the probable fluctuation of il-8 secretion over the incubation time cannot be determined accurately. therefore the factual timing of il-8 production by epithelial cells in a certain period needs to be elucidated further by observing il-8 secretion over a full time course. the variations in il-8 production may also be influenced by actual bacterial growth. this hypothesis is based on a certain set of data of this study. in the same experiment with the same time course (24 h) and same multiplicity of figure 2. the effect of muc1 knock down on il-8 production in response to pathogenic bacteria. mkn7 cells were transfected with a muc1 targeting sirna or non-targeting control sirna for 48 hours and then treated with campylobacter jejuni (moi 10 and 100) or tnf-α (100ng/ml) for a further 4 h (a) or 24 h (b) and il-8 levels in culture media were measured by elisa. statistics: box plots show mean, quartiles and range. mann-whitney u-test, p values shown, n=4 147 infection (moi 100), the results are opposite. knockdown of muc1 expression causes an increase in il-8 production in one experiment and a decrease in il-8 production in the other (figures 2b and 4b). this difference may be due to the variation of actual bacterial growth. although the moi provides the average number of bacteria per cell, the actual number of bacteria that infect any given epithelial cells can vary in different experiments, for example, if the moi is 1, some cells will get infected with one bacteria but some cells may be infected with 0 bacteria and other cells infected with 2 b a c t e r i a . h e n c e , t h e p r o p o r t i o n o f c e l l population infected by a specific number of bacteria should have been controlled to get the number of bacteria evenly in each experiment. however, in dealing with the dynamic nature of bacterial culture, it is a challenge to control the same actual bacterial growth with the number of moi. a variation in the number of actual bacterial growth compared to the moi may be one of the limitations of this study that might influence the variation in il-8 production. the increase in il-8 production in the absence of muc1 in the late phase of incubation s u g g e s t s t h a t m u c 1 h a s a r o l e i n t h i s p r o i n f l a m m a t o r y c y t o k i n e e x p r e s s i o n i n response to c. jejuni. with regard to cytolethal distending toxins (cdts) and its role in inducing an inflammatory response, it seems that there is no strong evidence that this toxin plays a role in inducing il-8 production, given that there was no significant difference in il-8 levels between parent c. jejuni 81-176 and ∆ cdts c. jejuni 81-176-treated figure 3. cell surface muc1 expression after knockdown using 1:1 sirna transfection. results from 4 replicates univ med vol. 31 no.3 148 triyana, sheng, mcguckin, et al muc1 modulates gastric epithelial immune figure 4. knockdown of muc1 expression tends to decrease il-8 production in short term incubation but increases il-8 secretion in long term incubation. deletion of cdt does not change the il-8 levels significantly (see blue line). mkn7 cells were transfected with a muc1 targeting sirna or non-targeting control sirna for 48 hours and then treated with campylobacter jejuni (moi 100) or tnf-α (5ng/ml) for a further 4 h (a) or 24 h (b) and il-8 levels in culture media were measured by elisa. statistics: box plots show mean, quartiles and range. mann-whitney u-test, p values shown, n=4 cells. other previous studies have established that the ability of c. jejuni to induce il-8 production is determined by many factors such as a process that is related to bacterial adhesion or invasion and a process that depends on cdts.(13) it has been demonstrated by zheng et al. that c. jejuni can induce il-8 secretion depending on cdt and nf-κ b activation mediated by tlr.(14) however, the role of cdts in inducing il-8 production and the modulation of cellular response to cdts by muc1 needs further investigation. 149 with end-point analysis it is difficult to explain what is really happening in this proinflammatory secretion regulation and what action is performed by the mucins. several possibilities may help us to understand such variation in il-8 production with regard to incubation timing first, there is potential modulation by a steric hindrance mechanism or releasable decoy to inhibit bacterial attachment to the cell surface by the mucins(6) and second, there is potential intracellular signal transduction by the mucins.(8-10) regarding the timing of these potential modulations, it is not known which one will happen at the early phase of incubation and which one will happen at later on. the timing of certain mechanisms of such modulation probably influences the variation of il-production in response to pathogenic bacteria. in untreated cells, the muc1 knockdown causes variations in il-8 production. there is a c o m p l e x u n d e r l y i n g m e c h a n i s m o f i l 8 production in epithelial cells. epithelial cells are able to secrete il-8 constitutively but such production can be enhanced once bacteria enter the cell.(15) however, in the absence of bacteria, the sirna transfection itself causes not only a decrease in muc1 expression but also influences bacterial adherence to the epithelial cells. this may explain why there is a variation in il-8 secretion. this study has demonstrated that muc1 knockdown mostly causes an increase in il-8 production in response to tnf-α, although one of the results was just the opposite. tnf-α is an important pro-inflammatory cytokine in both acute and chronic inflammation states and an activator of the transcription factors nf-κb and activator protein-1 (ap-1), two key modulators of the inflammatory response. the increase in il-8 in the absence of muc1, suggests that muc1 has an anti-inflammatory effect on tnfα. in a previous in vivo study, it has been demonstrated that muc1 knock out mice exhibit increased levels of tnf-α following h. pylori infection.(9) this anti-inflammatory effect is not only exhibited against bacterial but also viral infections. a previous in vitro study has revealed that muc1 knockdown resulted in greater tnfα production following respiratory syncytial virus (rsv) infection. it also showed that the increase in tnf-α upregulates muc1 which in turns suppresses further increase in tnf-α induced by rsv. hence, muc1 controls the level of tnf-α during rsv infection, thus preventing the potential harmful effects of excessive tnfα.(17) regarding the variations in il-8 results in this study, it has been demonstrated that possibly the sirna transfection used can exert an effect on the cells that might influence the adherence of bacteria to the cell surface which eventually will have an impact on il-8 production. conclusion this study has shown that knockdown of muc1 expression in mkn7 human gastric epithelial cells suppressed il-8 production at the early phase of incubation, but promoted an increase in il-8 production at the late phase, in response to c. jejuni.furthermore, with respect to the dynamic nature of bacterial/cell culture, the actual growth of bacteria in relation with moi number needs to be worked out more accurately. acknowledgement we t h a n k p r o f . j a m e s g. f o x (massaschusetts institute of technology, cambridge, massachusetts, usa), for the c. jejuni strains 81-176 and ∆cdt81-. and to prof. s. gendler, scottsdale, usa) for the gift of ct2. references 1. linden sk, sutton p, karlsson ng, korolik v, mcguckin ma. mucins in the mucosal barrier to infection. mucosal immunol 2008;1:183-97. 2. hattrup cl, gendler sj. structure and function of the cell surface (tethered) mucins. annu rev physiol 2008;70:431-57. univ med vol. 31 no.3 150 triyana, sheng, mcguckin, et al muc1 modulates gastric epithelial immune 3. kobayashi m, lee h, nakayama j, fukuda, m. roles of gastric mucin-type o-glycans in the pathogenesis of helicobacter pylori infection. glycobiology 2009;19:453-61. 4. mcauley jl, linden sk, ping cw, king rm, pennington hl, gendler sj, et al. muc1 cell surface mucin is a critical element of the mucosal barrier to infection. j clin invest 2007;117:231324. 5. mcguckin ma, every al, skene cd, linden sk, chionh yt, swierczak a, et al. muc1 mucin limits both helicobacter pylori colonization of the murine gastric mucosa and associated gastritis. gastroenterology 2007;133: 1210-8. 6. linden sk, sheng yh, every al, miles km, skoog ec, florin th, et al. muc1 limits helicobacter pylori infection both by steric hindrance and by acting as a releasable decoy. plos pathog 2009;5:e1000617. 7. lu w, hisatsune a, koga t, kato k, kuwahara i, lillehoj ep, et al. cutting edge: enhanced pulmonary clearance of pseudomonas aeruginosa by muc1 knockout mice. j immunol 2006;176: 3890-4. 8. ueno k, koga t, kato k, golenbock dt, gendler sj, kai h, et al. muc1 mucin is a negative regulator of toll-like receptor signaling. am j respir cell mol biol 2008;38:263-8. 9. guang w, ding h, czinn sj, kim kc, blanchard tg, lillehoj ep. muc1 cell surface mucin attenuates epithelial inflammation in response to a common mucosal pathogen. j biol chem 2010;285:20547-57. 10. lillehoj ep, kim h, chun ey, kim kc. pseudomonas aeruginosa stimulates phosphorylation of the airway epithelial membrane glycoprotein muc1 and activates map kinase. am j physiol lung cell mol physiol 2004;287:l809-15. 11. thompson ej, shanmugam k, hattrup cl, kotlarczyk kl, gutierrez a, bradley jm, et al. tyrosines in the muc1 cytoplasmic tail modulate transcription via the extracellular signal-regulated kinase 1/2 and nuclear factorkappa b pathways. mol cancer res 2006;4:48997. 12. ahmad r, raina d, trivedi v, ren j, rajabi h, kharbanda s, et al. muc1 oncoprotein activates the i kappa b kinase beta complex and constitutive nf-kappa b signalling. nat cell biol 2007;9:1419-27. 13. hickey te, mcveigh al, scott da, michielutti re, bixby a, carroll sa, et al. campylobacter jejuni cytolethal distending toxin mediates release of interleukin-8 from intestinal epithelial cells. infect immun 2000;68:6535-41. 14. zheng j, meng j, zhao s, singh r, song w. campylobacter-induced interleukin-8 secretion in polarized human intestinal epithelial cells requires campylobacter-secreted cytolethal distending toxinand toll-like receptor-mediated activation of nf-kappa b. infect immun 2008; 76:4498-508. 15. wajant h, pfizenmaier k, scheurich p. tumor necrosis factor signaling. cell death differ 2003; 10:45-65. 16. bradley jr. tnf-mediated inflammatory disease. j pathol 2008;214:149-60. 17. li y, dinwiddie dl, harrod ks, jiang y, kim kc. anti-inflammatory effect of muc1 during respiratory syncytial virus infection of lung epithelial cells in vitro. am j physiol lung cell mol physiol 2010;298:l558-63. c:\users\jurnal fkusakti\docume 131 *edo state school of health technology, benin city, nigeria **department of medical laboratory science, school of basic medical science, college of medical sciences, university of benin, nigeria correspondence: dr. (mrs.) e. o. osime department of medical laboratory science school of basic medical science college of medical sciences university of benin, nigeria email: evarista.osime@uniben.edu orcid id: https://orcid.org/00000003-4972-9735 date of first submission, november 21, 2 01 8 date of final revised submission, july 2, 2 01 9 date of acceptance, july 10, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina haemostatic properties of vernonia amygdalina and chromolaena odorata leaf extracts using wistar rat model lucy omokhegbe matthew* and evarista odaburhine osime** background the leaves of african plants are widely employed in nigeria to control bleeding from wounds. this work is aimed at evaluating the haemostatic effects of vernonia amygdalina (va/bitter leaf) and chromolaena odorata (co/sunflower) leaves on some haemostatic parameters. methods a laboratory study of experimental design was conducted involving 35 wistar rats that were randomized into seven groups. groups 1 and 2 received leaf extracts of va at concentrations of 150 and 250mg/kg bw, respectively; groups 3 and 4 received leaf extracts of co at 150 and 250mg/kg bw; groups 5 and 6 received combined leaf extracts of va and co at 150 and 250mg/kg bw, respectively, for 30 days. the last group 7 as control group received only water. parameters investigated were clotting time (ct), fibrinogen concentration, prothrombin time (pt), activated partial thromboplastin time test (aptt), factor vii, protein c and d – dimer. results there was a significant reduction in ct, fibrinogen concentration and pt in the intervention groups compared to controls (p<0.01). there was no significant difference in aptt, factor vii and protein c in the intervention groups compared to the controls (p>0.05). d-dimer levels were observed to increase significantly in rats treated with 150mg/kg bw of va and 150mg/ kgbw of the combined leaf extracts (va/co) compared to the controls (p<0.05). conclusion the va and co extracts reduced ct, pt, fibrinogen concentrations and increased d-dimer levels in rats. this study suggests the possible incorporation of the leave extracts of va and co in bleeding diathesis as well as in coagulation studies. keywords: haemostatic parameter, vernonia amygdalina, chromolaena odorata, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.131-138 may-august, 2019 vol.38no.2 cite this article as: matthew lo, osime eo. haemostatic properties of vernonia amygdalina and chromolaena odorata leaf extracts using wistar rat model. univ med 2019;38:131-8. doi: 1 0 . 1 8 0 5 1 / un i vm ed . 2 0 1 9 . v 3 8 . 1 3 1 1 3 8 132 matthew, osime vernonia amygdalina and chromolaena odorata on haemostasis introduction whenever blood vessels are damaged, there is an immediate physiological response designed to minimize and arrest blood loss.(1) the sum total of this response is encompassed by the term haemostasis and involves the interaction of the vascular system, platelets, plasma proteins, inhibitors and the fibrinolytic system.(2) the functions of these components are divided into primary and secondary haemostatic mechanisms. the primary haemostatic mechanism involves the vascular system and platelets, while the secondary mechanism involves the plasma protein responses of the coagulation process to injury. the combined eff ects of these mec hanisms lead to the formation of a stable fibrin-platelet plug, vessel healing and dissolution of the plug.(3) vernonia amygdalina (va) commonly called bitter leaf is a perennial shrub, 2.5m in height, that grows throughout tropical africa.(4) it belongs to the family asteraceae and has elliptic leaves that are about 6mm in length. the leaves are green and have a characteristics odor and bitter taste.(5) the macerated leaves of the plant are consumed as vegetables and condiments and a source of green leafy vegetable for culinary application as well as for topical treatment of wounds.(6) in many parts of nigeria the plant has been domesticated.(7) different ethnic groups in nigeria ascribe various names to this plant such as ewuro, etidot, onugbu, chusa-dike, “ebeoriwo” and others.(8) chromolaena odorata (co) is a tropical species of flowering shrub in the sunflower family asteraceae. in western africa, it prevents regeneration of tree species in areas of shifting cultivation. the extract has been used to stop bleeding and in wound healing in many tropical countries. it also contains a master regulator of genes with defensive anti-inflammatory and detoxifying functions.(9) in many communities, individuals squeeze and paste these leaves regularly on the affected portion of the injured vessels which stops the bleeding in a short time. the nutritional and medicinal benefits of these vegetables assist in combating malnutrition, prevention of many diseases as well as contributing to the food security system in rural areas. (4) medicinal plants have formed the basis of health care throughout the world since the earliest days of humanity and have remained relevant in both the developing and developed nations for various chemotherapeutic purposes.(10) the use of plantderived natural compounds as part of herbal preparations form an alternate source of medication. the leaf extr act of vernonia amygdalina ha s be en found to possess ethnomedical and pharmacological properties, such as antidiabeti c, antimalarial and antihelminthic effects.(11,12) previous studies done on this leaf extract investigated the first-line routine investigation in bleeding episodes and the healing process.(12) our study went further to investigate specific clotting factors and added that increase in d-dimer levels was responsible for the reduction in prothrombin time (pt), stoppage of bleeding which will invariably enhance wound healing. the combination of these two extracts in bringing this about has not been previously investigated. against this backdrop, this work was carried out to evaluate the effect of vernonia amygdalina and chromolaena odorata leave extracts on some haemostatic parameters using wistar rats. methods research design the extracts of both leaves were analyzed to identify the active ingredients using methods described by sofowora.(13) acute toxicity studies using ld 50 was carried out before the leaf extracts were administered to the rats. this work was carried out between january and august, 2017 in benin city, edo state, nigeria. collection and preparation of plant extract fresh specimens of vernonia amygdalina and chromolaena odorata leave s wer e 133 harvested in a farm land in benin city and identif ie d by a qua lif ie d pha rmacist/ pharmacologist. the extract of the leaves was prepared at the pharmacognosy laboratory, faculty of pharmacy, university of benin. the registration numbers of the leaves were ubw/ pcg/1024 and ubw/pcg/1025 for vernonia amygdalina and chrom olae na od orata respectively. acute toxicity testing the median lethal dose (ld 50 ) is a common test used to evaluate acute toxicity. this was done on twenty (20) rats, using two (2) rats for each concentration by the standard method. phytochemical screening on both leaves was d on e b y me t ho ds de s c r ib e d by va r i ou s researchers.(13,14) animal treatment a total of 35 apparently healthy wistar rats of both sexes weighing between 140-180 g, o bt a i n e d f r o m t he a ni ma l h ou s e of t he department of anatomy, university of benin, were sorted for the haemostatic evaluation. all experimental animals were fed with standard animal feed and water ad libitum. sample size determination this was calculated using the resource equation e = total number of animals “ total number of groups where e is the degree of freedom of analysis of variance (anova). the value of e should lie between 10 and 20. if e is less than 10 then adding more animals will increase the chance of getting more significant results.(15) experimental animals group 1 received 150mg/kg body weight extract of vernonia amygdalina (va). group 2 received 250mg/kg body weight va. group 3 r e c e i ve d 15 0mg/ kg b o dy w e i gh t e xt r a c t chromolaena odorata (co). group 4 received 250mg/kg body weight extract chromolaena odorata (co). group 5 received 150mg/kg body weight extract combined extract va/co. group 6 received 250mg/kg body weight extract combined extract va/co leaves. group 7 served as control group and received water. leaf extracts were administered once daily via orogastric intubation for 30 days. collection of blood samples the rats were euthanized under mild c o nc e nt r ati o n of c h l or of or m va p or a n d sacrificed. whole blood was collected via cardiac puncture using sterile syringes into edta bottles for full blood count and into trisodium citrate bottles for haemostatic factor assay. determination of haemostatic parameters quantitative estimation of protein c the enzyme linked immunosorbent assay (elisa) method was used. the micro elisa plate was pre-coated with an antibody specific to protein c. standards and samples were added to the appropriate micro-elisa plate wells and combined with the specific antibody. a biotinylated detection antibody specific for protein c and avidin-horseradish peroxidase (hrp) conjugate was added to each micro plate successively and the plate incubated. free components were washed away. the substrate solution was added to each well. the enzyme-substrate reaction was terminated by the addition of a stop solution (sulphuric acid solution) to form a yellow colour.(16) estimation of factor vii one hundred microlitres of standard and sample were added to separate tubes, 100μl of prepared detection reagent a was added and incubated for 1 hr at 37o c. they were washed three times using normal saline, 90μl of substrate solution was added and incubated for 30 minutes at 37oc. finally 50μl stop solution was added a n d t h e op t i c a l d e ns i t y ( o d) r e a d spectrophotometrically at 540nm.(16) univ med vol. 38 no.2 134 matthew, osime vernonia amygdalina and chromolaena odorata on haemostasis estimation of d-dimers fifty microlitres of standard and sample was measured in separate test tubes. forty microlitres of biotinylated detection antibody was added immediately and incubated for 45 minutes at 37o c, then washed three times using normal saline. ninety microlitres of substrate reagent was added and incubated at 370c for 15minutes. fifty microlitres of stop solution was added and the optical density read at 450nm immediately.(16) estimation of plasma fibrinogen concentrate (fc) this was done using ingram’s clot weight methods. when calcium chloride is added to citrated plasma, the intrinsic pathway is triggered, resulting in‘ the formation of a fibrin clot, which can be collected and dried; the weight of fibrinongen obtained is directly proportional to the amount of fibrinogen present.(16) determination of clotting time (ct) the principle of clotting time is based on the time required for a sample of blood to coagulate in vitro under standard conditions. blood samples collected were immediately placed in test tubes which were already pre-warmed to a temperature of 37°c in a water bath, and timing was done immediately with a stop watch. the tubes were swirled gently at 30 second intervals to observe for clot formation. as soon as a clot was observed, the timer was stopped and the time recorded in minutes. determination of prothrombin time (pt) the diagen diagnostic reagent was used. the pt test measures the clotting time of plasma in the presence of optimal concentrations of thr omboplastin and indic ate s the overall efficiency of the extrinsic clotting system. in the presence of calcium ions, tissue thromboplastin initiates the extrinsic coagulation pathway by the direct activation of factor vii to viia. this culminates in the conversion of soluble fibrinogen to insoluble fibrin by the direct a c t i o n of th r o mbi n . a r e du c ti on i n t he concentration of clotting factors of the extrinsic and common pa thways will result in the prolongation of the pt, the degree of which is proportional to the level of concentration reduction. act ivat ed parti al t hrom bopl asti n ti me (aptt) this was also done using diagen diagnostic reagent. it is a measure of the combined effect of the clotting factors of the intrinsic and common coagulation pathways. it represents the ultimate refinememt in which platelet activity is standardized by the use of platelet substitutes and contact activation is standardized by preincubation of the plasma with the kaolin platelet substitute mixture for a standard time before recalcification.(16) statistical analysis result obtained were expressed as mean ± standard error of mean (sem). differences were dete rmined by one-way analysis of variance (anova). computer software used was graph pas instat version 2.05 software/uk. level of significance was at p<0.05. ethical clearance all the experiments were performed in accordance with the guidelines for care and use of laboratory animals of the faculty of pharamacy, university of benin. the registration numbers of the leaves were ubw/pcg/1024 and ubw/ pcg/1025 for va and co respectively. results there was a significant decrease in pt (p<0.05), ct and fc (p<0.01) in rats treated with 150mg/kgbw of the combined extracts when compared to controls (table 1). d-dimer levels were observed to increase significantly (p<0.05) in rats treated with 150mg/kgbw of va and va/co extracts when compared with the controls (p<0.05) (table 2). h b c 135 t ab le 1 . c o m p ar is o n o f so m e h ae m o st at ic p ar am et er s tr ea te d w it h d if fe re n t co n ce n tr at io n s o f v a a n d c o e x tr ac t w it h c o n tr o ls a= p > 0 .0 5 ; b = p < 0 .0 5 ; c= p < 0 .0 1 v a : v er n o n ia a m yg d a li n a ; c o : c h ro m o la en a o d o ra ta ; v a /c o : v er n o n ia a m yg d a li n a / c h ro m o la en a o d o ra ta t ab le 2 . c o m p ar is o n o f so m e co ag u la ti o n f ac to rs a n d i n h ib it o rs t re at ed w it h d if fe re n t co n ce n tr at io n s o f v a a n d c o w it h c o n tr o ls a= p > 0 .0 5 ; b = p < 0 .0 5 ; c= p < 0 .0 1 v a : v er n o n ia a m yg d a li n a ; c o : c h ro m o la en a o d o ra ta ; v a /c o : v er n o n ia a m yg d a li n a / c h ro m o la en a o d o ra ta univ med vol. 38 no.2 136 matthew, osime vernonia amygdalina and chromolaena odorata on haemostasis discussion in this study, the haemostatic activities of va and co leaf extract was evaluated. there was a significant reduction in the pt and ct at 1 50 mg/ kgbw an d 2 40mg/kgbw of b ot h extracts. this may be attributed to the high levels of calcium, vitamin c, proteins and saponins present in the leaves. although calcium is the principal active ingredient present in plasma and bones, it is the free calcium ions that are physiologically active in the coagulation mechanism. this agrees with another research finding,(9) which demonstrated that the leaves of va would stop bleeding if squeezed and placed on cuts and wounds of injured vessels. calcium is essential in the conversion of prothrombin to thrombin. vernonia amygdalina and co leave extracts have been reported to be rich in antioxidant and flavonoids.(17,18) the high antioxidant levels present in the extracts may act in synergy with other phytochemicals which activate endothelial cells to synthesize tissue factors thus promoting haemostasis. plant e x t r a c t s ar e a dmi ni s t e r e d t o hu ma ns i n u nco n tr ol l ed d os e s by th e e th n ome d ic a l practitioners. these activities of leaf extracts on blood parameters provides some physiological information on the blood assessment in the body.(18) there was significant reduction in fibrinogen levels with the extract concentrations at 150mg/ kgbw of va and co and at 250mg/kgbw of combined extracts when compared with the controls. fibrinogen is an acute phase protein and its concentration rises in a variety of non-specific conditions such as inflammation, trauma and myocardial infarction. the reduction of fibrinogen in this study indic ates that va and co administration reduces and prevents inflammation which may be largely attributed to the presence of flavonoids as contained in the phytochemical constituents of the leaf extracts. high levels of fibrinogen are associated with thrombotic complications. it has been observed that individuals with a fibrinogen level greater than 4g/l have a two-fold increased risk of venous thrombosis.(24) methanolic extracts of va of 100mg/kgbw and 200mg/kgbw could reduce 40% and 50% inhibition against thrombosis in mice.(20,21) administration of the extracts did not significantly alter the concentrations of factor vii and protein c when compared with the controls. this may indicate that the leaf extract in enhancing blood clotting does not directly lead to increased production of these clotting factors. ddimers are present in the blood of most healthy individuals in only negligible amounts. elevated blood levels of d-dimers is evidenced by increase fibrinolysis and thrombotic episodes.(22) in this study, administration of 250mg/kg bw of va and 150mg/kgbw of combined va/ co showed a significant increase in d-dimer levels when compared with the controls. this suggests that the extract enhances fibrinolysis thereby facilitating the healing of an injured vessel. extracts of va have been shown to have antimicrobial and antiseptic properties.(23) this may also act as a protective mechanism enhancing the complete healing of injured vessels with the administration of these extracts. the combined effect of va and co to injuries especially superficial wounds would reduce hemorrhage. this also has revealed a channel to phar mace utical companies to incorporate these extracts in drugs used in preventing haemorrhage. although the present findings suggest the presence of hemostatic compounds in leaf extract of va and co, the precise mechanism of its hemostatic action is still speculative and requires further studies for appropriation elucidation. conclusion the leave extracts of va and co shorten the protrombin time, clotting time and decrease fibrinogen levels while d-dimer levels are increased. this implies that the administration of these leave extracts goes a long way in the stoppage of bleeding and aids fibrinolysis. 137 conflict of interests we declare that there is no conflict of interest acknowledgement we acknowledge the non-accademic staff of the department of anatomy for helping to take care of the animals during the research duration. authors contribution mol conceived th e study and was responsible for the experimental design and practical implementation. oeo contributed to the experimental design and did the manuscript writeup. all authors have read and approved the final manuscript. references 1. schiller am, howard jt, convertion va. the physiology of blood loss and shock: new insights from a human laboratory model of hemorrhage. exp biol med 2017;242:874-83. doi: 10.1177/ 1535370217694099. 2. aleman mm, walton bc, byrnes jr, et al. fibrinogen and red blood cells in venous thrombosis. thromb res 2014;133:538-40. doi: 10.1016/j.thromres.2014.03.017. 3. yun s, sim e, goh r, et al. platelet activation: the mechanisms and potential biomarkers. biomed res int 2016;2016:9060143. doi: 10.1155/2016/ 9060143. 4. kadiri o, olawoye b. vernonia amygdalina: an underutilized vegetable with nutraceutical potentials – a review. turk j agric food sci technol 2016;4:763-8. 5. audu sa, taiwo ae, ojuolape ar, et al. a study review of documented phytochemistry of vernonia amygdalina (family asteraceae) as the basis for pharmacologic activity of plant extract. j natural sci res 2012;2:1-8. 6. shokunbi os, anionwu oa, sonuga os, et al. effect of post-harvest processing on the nutrient and anti-nutrient compositions of vernonia amygdalina leaf. afr j biotechnol 2011;10: 1098085. doi: 10.5897/ajb11.1532. 7. krausi i, tobin g. discovery, development of natural products in using old solutions to new problems. in: kulka m,editor.natural drug discovery in the 21 century. 2nd ed. london, uk: intech open; 2013.p.3-36. 8. lobo vc, phatak n, chandra n. acute toxicity studies of some indian medicinal plants. phcog j 2010;2:207-10. 9. pandith h, zhang x, liggett j, et al. hemostatic and wound healing properties of chromolaena odorata leaf extract. isrn dermatology 2013; article id 168269, 8 pages. doi: http://dx.doi.org/ 10.1155/2013/168269. 10. abay m, lucantoni l, dahiya n, et al. plasmodium transmission blocking activities of vernonia amygdalina extracts and isolated compounds. malar j 2015;14:288. doi: 10.1186/s12936-015-08122. 11. chinedu e, arome d, ameh fs. a new method for determining acute toxicity in animal models. toxicol int 2013;20: 224–6. doi: 10.4103/09716580.121674. 12. oguwike fn, offor cc, onubeze dpm, et al. evaluation of activities of bitterleaf (vernonia amygdalina) extract on haemostatic and biochemical profile of induced male diabetic albino rats. iosr j dent med sci 2013;11:60-4. 13. sofowora a, ogunbodede e, onayade a. the role and place of medicinal plants in the strategies for disease prevention. afr j tradit complement altern med 2013;10:210-29. doi: http://dx.doi.org/ 10.4314/ajtcam.v10i5.2. 14. olorunfemi ea, arnold ic, igbo c, et al. effects of the leaf extract of vernonia amygdalina on the pharmacokinetics of dihydroartemisinin in rat. pharmacologia 2012;3:713-8. doi: 10.5567/ pharmacologia.2012.713.718 15. charan j, kantharia nd. how to calculate sample size in animal studies? j pharmacol pharmacother 2013;4:303–6. doi: 10.4103/0976-500x.119726. 16. bain bj, bates i, laffan m. dacie and lewis practical haematology. 12th ed. elsevier ltd.;2016. 17. alaraa or, abdurahman nh, mudalip ska, et al. phytochemical and pharmacological properties of vernonia amygdalina: a review. j chem eng ind biotechnol 2017;2:80-96. doi: https://doi.org/ 10.15282/jceib-v2-07.29/9/2017/2.2. 18. sirinthipaporn a, jiraungkoorskul w. wound healing property: review of siam weed, chromolaena odorata. pharmacogn rev 2017;11:35–8. doi: 10.4103/phrev.phrev_53_16. 19. yeap s, ho w, beh bk, et al. vernonia amygdalina, an enthnoveterinary and ethnomedical used green vegetable with multiple bioactivites. j med plants res 2010;4:2787-812. 20. asomugha rn, okafor pn, ijeh ii, et al. hepatic effects of aqueous extract of chromolaena univ med vol. 38 no.2 138 matthew, osime vernonia amygdalina and chromolaena odorata on haemostasis odorata in male wistar albino rats. pharmacol ol 2014;1:127-36. 21. owolabi ms, ogundajo a, yusuf ko, et al. chemical composition and bioactivity of the essential oil of chromolaena odorata from nigeria. rec nat prod 2010;4:72-8. 22. lippi g, bonfanti l, saccenti c, et al. causes of elevated d-dimer in patients admitted to a large urban emergency department. eur j intern med 2014;25:45-8. doi: 10.1016/j.ejim.2013.07.012. 23. lyumugabe f, uyisenga jp, bayingana c, et al. antimicrobial activity and phytochemicals analysis of vernonia aemulans, vernonia amygdalina, lantana camara and markhamia lutea leaves as natural beer preservatives. am j food technol 2017;12:35-42. doi: 10.3923/ajft.2017. 35.42. c:\users\jurnal fkusakti\docume 48 *department of histology, faculty of medicine, universitas islam indonesia, yogyakarta **department of physiology, faculty of medicine, universitas islam indonesia, yogyakarta ***faculty of medicine, universitas islam indonesia, yogyakarta †department of medical education, faculty of medicine, universitas islam indonesia, yogyakarta correspondence: @umatul khoiriyah,md,m.med.ed,phd faculty of medicine, universitas islam indonesia jl. kaliurang km14,5 sleman yogyakarta, indonesia phone: (62)81328801247 fax: (62)274898444 ext.2007. email: umakhoiriyah@uii.ac.id orcid id: https://orcid.org/0000-0001-8645-8482 date of first submission, november 17, 2 01 8 date of final revised submission, march 29, 2019 date of acceptance, march 29, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina mild anxiety and depression related to elevated dopamine level in young adults ika fidianingsih*, titis nurmasitoh**, adika zhulhi arjana***, ninda devita***, and umatul khoiriyah†@ background dopamine is an important neurotransmitter that plays a role in the pathogenesis of anxiety and depression. dopamine secretion occurs when there is a pleasurable stimulus. blood dopamine levels have the potential to be developed as biomarkers of depression or anxiety, but previous studies related to the relationship between dopamine levels and levels of anxiety and depression are still controversial. the purpose of this study was to determine the correlation between anxiety and depression score with dopamine level in young adults. methods this was an observational cross sectional study. a total of 43 subjects aged 18 to 40 years were recruited by consecutive non-random sampling according to inclusion and exclusion criteria. anxiety and depression were assessed using the beck anxiety inventory and beck depression inventoryii questionnaire. dopamine plasma level was measured using the enzymelinked immunosorbent assay (elisa). the spearman rho correlation test was used to analyze the data and p<0.05 was considered significant. results the median dopamine levels in respondents with mild, moderate and severe anxiety were respectively: 77.79; 63.43; 62.51 ng/ml (p=0.043). the median dopamine levels in respondents with reasonable depression, mood disorder, moderate and severe depression were respectively: 79.2; 61,32; 62,51; 60.24 (p=0.001). correlation test results showed a weak correlation between dopamine and anxiety (r=0.310) and a moderate correlation for depression (r=-0.505). conclusion mild anxiety and depression were associated with elevated dopamine level in young adults. further studies are required to confirm the role of dopamine metabolism in anxiety and depression in young adults. keywords : anxiety, depression, dopamine, elisa, young adults original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.48-55 january-april, 2019 vol.38no.1 cit e th is a rtic le a s: fidia ningsih i, nurmasitoh t, arjana az, et al. mild anxiety and depression related to elevated dopamine level in young adults. un iv m ed 2 019; 38:4 8-55 . doi: 10.18051/univmed.2019.v38.48-55 49 univ med vol. 38 no.1 introduction in line with technological advances, the public becomes more individualistic, and the level of depression and anxiety increases. worldwide, the number of persons experiencing anxiety and depression from 2005 to 2015 increased by 15% and 18%, respectively.(1) in indonesia, the number of mental cases is also on the increase, impacting on an increased national burden and decreased long-term human productivity. data from riskesdas 2013 showed that the prevalence of mental-emotional disorders as shown by the symptoms of depression and anxiety for the age group of 15 years and above attained around 14 million persons or 6% of the total indonesian population,(2) whereas globally there was in 2015 an estimated 264 million inhabitants (3.6% of the population) experiencing anxiety and 322 million (4.4%) experiencing depression. depression results in a 40 % higher early mortality than in the general population. worldwide, a total of 788,000 persons in 2015 committed suicide due to depression.(1) there are various manifestations of anxiety disorder, depending on the type of anxiety. according to the diagnostic and statistical manual of mental disorders (dsm-5), anxiety disorder may be in the form of mutism, phobia, social anxiety disorder, panic disorder, generalized anxiety disorder, and anxiety from certain illnesses and medications. the complaint of anxiety may be accompanied by somatic symptoms such as palpitations, shortness of breath, pain in the thorax, nausea, perspiration, tremors, choking, and anesthesia.(3) the symptom of anxiety frequently also occurs in patients with depression, although not invariably so. depression is a mood disorder that causes sadness and affects feeling, thinking, and activities of daily living such as sleeping, eating, or working.(4) up to the present, the diagnosis of anxiety and depression is based on subjective symptoms only and there are yet no specific biomarkers to be found, so that there are still patients with undiagnosed depression.(5) any uncertainty of diagnosis results in the patients not receiving good management and treatment, so that their condition becomes chronic and they are at risk of more severe mental disorders. the biomarkers that are suitable for neuropsychiatric disorders may frequently be obtained from brain tissue or cerebrospinal fluid, but the procedure is relatively more invasive than drawing a blood sample. therefore, there is a need for the development of blood biomarkers of depression or anxiety.(6) biomarkers in the form of micrornas (mirnas) from blood samples of patients with schizophrenia have been identified to be significantly associated with the diagnosis and clinical manifestations of this disorder.(7) administration of antidepressants also showed a significant increase in the expression of 28 types of mirnas and a significant decrease in 2 types of mirna.(8) the causative factors of anxiety and depression are nearly identical, comprising biologic (e.g. genetics, age, gender), psychological (e.g. cognitive factors, problem solving ability, and convictions), and social factors such as trauma, social support, and social skills. depression may occur concomitantly with other serious disorders, such as cancer, heart disease, and parkinson’s disease.(4) the precipitating factor of anxiety may be other diseases such as diabetes mellitus. the pathophysiology of anxiety and depression is also much associated with neurotransmitter imbalance in the brain.(3) one of the essential neurotransmitters in the behavioral response to environmental stimulants that plays an important role in the genesis of anxiety and depression is dopamine. dopamine is a precursor of norepinephrine (noradrenaline), and is the neurotransmitter involved in the regulation of movement, feeling, and motivation, so that an abnormality of the neurotransmitter will be associated with anxiety and depression. dopamine is produced upon stimulation of pleasure from ltyrosine, that is c onver ted to l-3,4dihydroxyphenylalanine (l-dopa) to form dopamine by means of the enzyme dopa decarboxylase, also known as aromatic l-amino acid decarboxylase, which is secreted and binds 50 to the dopamine receptors dr1, dr2, dr3, dr4, and dr5. dopamine can be degraded by means of monoamine oxidase (mao) and catechol-omethyltransf erase (comt) into 3,4dihydroxyphenylacetic acid (dopac) and 3methoxytyramine (3mt).(9) deficient production of dopamine results in decreased responsiveness to pleasure stimulation. the neurotransmitter known up to now, i.e. serotonin, has been recommended as the target for treatment of depression by selective serotonin reuptake inhibitors (ssri), but large-scale clinical trials failed to show improvement.(10) therefore, the involvement of dopamine in anxiety and depression constitutes an important and novel perspective that should be studied further. the results of existing studies on dopamine and anxiety are still variable, such as that lack of dopamine may induce anxiety, (11) whereas administration of l-dopa, which is the precursor of dopamine, may improve anxiety.(4) medications that may lower dopamine function, such as dopamine antagonists, have been reported in connection with the occurrence of symptoms of social anxiety disorder.(12) there are many studies on the involvement of dopamine in depression and anxiety that are still conducted in experimental animals, since they involve the dopaminergic system in the brain. depression is frequently associated with decreased dopamine secretion in the synaptic cleft of the accumbent nucleus or the ventral tegmental area, leading to decreased response to pleasure stimuli.(13) although other studies showed the opposite, i.e. rats with depression and psychiatric disorders, experience an increase in dopamine level in certain regions of the brain, in comparison with normal rats.(14) the relationship between blood dopamine level and depression in humans is still subject to controversy. studies on patients with depression showed that blood dopamine levels are lower than in persons without depression.(15) however, other studies showed that on the contrary dopamine levels are higher in persons experiencing depression.(16) still other studies showed that dopamine levels are similar in patients with and without depression. (1 7) administ ra tion of methyldopa, which is the enzyme for synthesizing dopamine, may induce depression.(3) on the other hand there is evidence that administration of dopamine agonists may improve depression.(10) therefore, there is still a need for further studies in connection with the possibility of dopamine from blood samples being used as a biomarker of the level of depression or anxiety. the present study aimed to determine any correlation of blood dopamine level with the level of anxiety and depression in young adults. methods study design this study was of observational crosssectional design, using young adult subjects. the study was conducted from may to september 2018 in the yogyakarta special region (daerah istimewa yogyakarta). study subjects the study subjects were adult males and females who agreed to participate in this study. the inclusion criteria were males or females, 18 up to 40 years of age, healthy, without severe illness. the exclusion criteria were lactating and pregnant females, smokers, consumption of ldopa or methyldopa. the condition of being healthy was determined from anamnesis that the respondent was in a condition of not having any symptom complaints or illness, and not having chronic disease. determination of sample size the sample size for this study was calculated from the formula for sample size for correlation tests, which is {(zα + zβ) / 0.5 ln [(1+r)/(1-r)]}2 +3, with an estimated moderate degree of correlation (r =0.6) and confidence level of 95%, power of 80%, with the addition of an anticipated number of drop-outs of 10%. the result of the calculation showed that the minimal sample size was 32. recruitment of study subjects was by fidianingsih, nurmasitoh, arjana, et al anxiety depression and elevated dopamine 51 univ med vol. 38 no.1 non-random consecutive sampling until the required minimal sample size was obtained. the investigators published the call for voluntary participation in the study through the whatss app social media up to a given time limit and obtained 48 subjects. however, 5 subjects were prevented at the time of blood sample collection and elisa assay, so that only data on 43 subjects were available for analysis. determination of anxiety and depression scores the anxiety score is determined by means of the beck anxiet y inventor y (bai) questionnaire. the bai score has been validated and its reliability tested, and may be used to determine the anxiety level in the clinic as well as in studies.(18) the bai questionnaire consists of 21 items, where each item uses a 4-point likert scale (0-1-2-3). the scores range from 0 to 63. scores between 0-21 indicate mild anxiety, scores 22-35 indicate moderate anxiety, while scores 36 and over indicate severe anxiety. the depression scores are determined with the beck depression inventory-ii. this instrument has been proven to be of good validity and reliability and may be used to assess the depression level in populations of healthy individuals as well as in populations of individuals with disorders.(15,16) the questionnaire consists of 21 items on a 4-point likert scale of 0, 1, 2, 3 and total scores between 0 and 63. interpretation of the scores is as follows: scores 1-10 are normal scores, scores 11-16 indicate mild mood disorder, scores 17-20 mild depression, scores 21-30 moderate depression, scores 31-40 severe depression and scores higher than 40 extreme depression. assessment of dopamine to each of the patients’ venous blood samples edta was added, then the samples were centrifuged at 1000g for 20 minutes, and stored at -800c prior to the elisa assay. the microplate wells were filled with an equal volume of serum sample and assayed according to the standard protocol of da (dopamine) elisa kit catalog no.u0392d028 (finetest, wuhan, hupei, china). the results of the elisa assay were read on an elisa reader at a wavelength of 450 angstrom. eight different concentrations of purified da (0, 1.562; 3.125; 6.25; 12.5; 25, 50, and 100 ng/ml were used to construct the standard curve. the dopamine concentration was calculated with the formula (y-0.893)/ 0.009. statistical analysis data on subject characteristics such as gender, education, and occupation were presented as total and frequency. numerical data such as age, anxiety score, depression score, and dopamine score were presented as mean and standard deviation. the kolmogorovsmirnov test showed that the dopamine scores were normally distributed, but that the depression scores and anxiety scores were not. therefore, the anxiety and depression scores were converted to an ordinal scale in accordance with the bai guidelines, i.e. mild, moderate, and severe, and with the bdi guidelines, i.e. normal mood, mood disorder, and mild, moderate, severe, and extreme depression. correlation was tested with the spearman rho test. the level of significance used was 0.05. ethical clearance the present study obtained ethical clearance from the ethics committee, faculty of medicine, universitas islam indonesia, under number 48/ ka.kom.et/70/ke/v/2018. prior to the start of the study, the study subjects signed written informed consent. results among the subjects participating in this study there were more females (62.8%) with mean age of 27 years. the most frequent educational level was s1 (48.8%), and the most frequent occupation was employee (27.9%) (table 1). although the dopamine concentrat ions were normally distributed, the results of the kolmogorova b c 52 smirnov test showed that the anxiety and depression scores were not normally distributed (p>0.05). there was a tendency showing that the higher the anxiety and depression, the lower the dopamine concentrations. this was proven from the spearman rho, which showed a negative correlation between blood dopamine level and degree of depression as well as between blood dopamine level and degree of anxiety, with a low coefficient of correlation for anxiety and a moderate correlation for depression. (table 2). discussion the results of this study showed that there was a correlation between blood dopamine concentrations and severe degree of anxiety, although the correlation was weak. dopamine is released when a person performs a pleasurable activity. on the other hand, a person with anxiety cannot feel pleasure.(20) the results of the present study were in agreement with previous studies in experimental animals. rat models of anxiety have lower dopamine levels than do normal rats.(21) however, other studies obtained different results, since in patients with internet addiction disorder and concomitant anxiety, the blood dopamine levels were not significantly different from those of healthy controls.(17) in contrast, studies in dogs found that plasma dopamine levels were higher in dogs with anxiety than in controls.(22) dopamine is involved in the regulation of pleasure, but it is also involved in the regulation of other factors such as addiction, motivation, and motor movement. there are four dopaminergic pathways in the brain, i.e. the nigro-striatal, mesolimbic, mesocortical, and tuberoinfundibular pathways. the mesolimbic pathway is involved characteristic n (%) mean (±sd) gender female male 27 (62.8) 16 (37.2) education senior high school d3 s1 15 (34.9) 4 (9.3) 21 (48.8) s2 3 (7) occupation employee student artist unemployed entrepreneur 12 (27.9) 10 (23.2) 5 (11.6) 8 (18.6) 8 (18.6) age (years ) 27.6 ± 6.58 anxiety score mild moderate severe 34 (79.0) 6 (14.0) 3 (7.0) 15.44 ± 12.04 depression scores 12.56 ± 33.32 reasonable mood disorder mild depression moderate depression severe depression extreme depression 32 (74.4) 7 (16.3) 0 (0.0) 3 (7.0) 1 (2.3) 0 (0) dopamine concentration (ng/ml) 73.19 ± 12.07 table 1. characteristics of study subjects (n=43) r* p value degree of anxiety -0.310 0.043 degree of depression -0.505 0.001 table 2. correlation of dopamine level with degree of anxiety and depression *spearman correlation : r fidianingsih, nurmasitoh, arjana, et al anxiety depression and elevated dopamine 53 univ med vol. 38 no.1 in the regulation of pleasure.(23) the blood dopamine level varies in the above-mentioned studies, because plasma dopamine level is also influenced by the dopamine secretion of sympathetic neurons, adrenal medulla, and neuroendocrine cells (amine precursor uptake and decarboxylation cells or apud cells). these cells are found in the kidneys, leukocytes, and in both endocrine and exocrine glands of the pancreas. the blood dopamine level may increase due to stress, physical exercise, standing posture, and hypovolemia. this means that there are many factors that influence the blood dopamine level. indeed, dopamine also plays an important role in glucose metabolism, weight regulation, and is said to play a role in protection against the proliferation of cancer.(24) although there is evidence that anxiety may increase in patients with parkinson’s disease after the cessation of dopaminergic medications, not all patients with parkinson’s disease experience improvement of anxiety after consumption of ldopa (the precursor of dopamine). (25) increased dopamine in the synaptic cleft as a result of administration of dopamine reuptake inhibitors causes behavioral effects such as anxiety.(26) in addition, other studies show that blood dopamine level cannot be equated with dopamine level in the brain. this is because dopamines in the blood cannot pass the blood brain barrier,(27) except in the form of dopamine sulfate. the existing studies are more about central dopamines. further studies ne ed to be conducted in conne ction with peripheral dopamines. the results of our study showed a moderate correlation between low dopamine level and severe depression. prior studies on patients with post-stroke depression also showed lower dopamine levels as compared with those without depression ( or:0.64 , 95%ci: 0.45-0.91, p=0.014).(15) however, other studies showed no correlation between bdi scores in 50 subjects with depression and blood dopamine level(28) and no correlation of blood dopamine level with depression in patients with internet addiction disorder and concomitant depression in a study involving 20 patients and 15 controls.(17) in contrast, in a study on 342 women postpartum, blood dopamine level in those with depression was higher than in those without depression.(16) similarly, studies on patients with major depressive disorder showed that the dopamine concentration as determined by gas chromatography-mass spectrome tr y (gc-ms) and liquid chromatography tandem mass spectrometry (lcms/ms) was higher than in healthy controls.(6) the existing studies on blood dopamine level and depression have still yielded variable results. in spite of this, there is evidence that administration of medications capable of increasing blood dopamine levels, such as tyrosine, amphetamine, and bupropion, may reduce the symptoms of depression.(23) the regulation of dopamine secretion in the brain is rather complex. dopamine is secreted by the pedunculopontine tegmental nucleus and the laterodorsal tegmentum by reward or appreciation stimuli. activation of the ventral subiculum (vsub) of the hippocampus also induces increased dopamine secretion through the nucleus accumbens that inhibits the action of the ventral pallidum. however, if there is hyperactivity of the infralimbic subregion (ilpfc), this will activate the ventral pallidum via the basolateral amygdala, leading to a reduction in dopamine secretion, such as occurs in experimental animal models of depression.(13) dopamine neurotransmitters in the blood as biomarkers of depression are important for diagnosis and relatively simple to determine, but further studies are still necessary, because previous studies also showed that patients with medication-resistant depression who are treated with transcranial magnetic stimulation, experience increases in the level of dopamine metabolites in the brain, but there is no correlation between clinical improvement of depression and blood dopamine level. t he mea surement of neuroendocrine biomarkers such as dopamine is rather complex, in view of the rather widespread role of dopamine in the physiology of the body.(29) the limitation of the present study is that it did not determine brain dopamine levels, that so 54 far has been demonstrated to be more associated with the pathophysiology of anxiety and depression. further studies need to be conducted in connection with peripheral dopamines and their connection with anxiety. conclusion low blood dopamine level in young adults is correlated with a higher degree of anxiety and depression, but the correlation is weak for anxiety and moderate for depression. acknowledgments the investigators thank the ministry of research and technology directorate of higher education (kemenristek dikti) for the funding of this study in the framework of the basic research grant scheme for higher education (skema hibah penelitian dasar perguruan tinggi). the investigators also thank ms ernadita budiatuti, technician in the physiology laboratory, faculty of 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tp, kostiainen r. permeation of dopamine sulfate through the blood-brain barrier. plos one 2015;10:e0133904. 28. ambade v, arora mm, singh p, et al. adrenaline, noradrenaline and dopamine level estimation in depression: does it help? med j armed forces india 2009;65:216–20. doi: 10.1016/s03771237(09)80006-3. 29. fidalgo t, morales-quezada l, muzy g, et al. biological markers in non-invasive brain stimulation trials in major depressive disorder: a systematic review. j ect 2015;30:47–61. doi: 10.1097/yct.0b013e31828b34d8. c:\users\universa medicina\docu 113 abstract universa medicina assessment of needlestick injuries and hepatitis b and c infection among surgeons shaban mehrvarz*, seyed masoud khatami, shahram manoochehry*@, hossien khedmat**, mahdi arefinia***, and hamid reza rasouli* background specialist surgeons are at high risk of exposure to hepatitis viruses through occupational exposure to blood or body fluids. protective measures against occupational exposure to the hepatitis b virus (hbv) and hepatitis c virus (hcv) must be taken in order to prevent infection in surgeons. we aimed to determine the needlestick injuries, and markers hbv and hcv in iranian surgeons. methods this study was cross-sectional research, performed in baqyatallah, shohada, rasoul akram, sina, taleghani, and emam hossein hospitals (all university hospitals) of tehran, iran. overall 318 eligible surgeons were included. anonymous questionnaires were used containing data about demographic characteristics, self-reported blood and needlestick contacts, occasional exposures, risk behaviors and vaccination. also, the blood samples were taken and tested for hepatitis b surface antigen (hbsag), antibody against hepatitis b surface antigen (antihbs) and hcv antibody (hcvab). fisher exact test and kruskal wallis test were used to analyze the data. results the mean age of the surgeons was 47.76  8.95 years and 177 of them (55.66%) were male. the average number of needle–sticks was 28.28 ± 16.58 during the surgeons’ working life. among them, 5 cases (1.59%) were positive for hbsag and 2 cases (0.66%) were positive for hcvab. conclusion in spite of the high needlestick rate in iranian surgeons, prevalence of hepatitis b and c is not very high among them. a high degree of vigilance and a careful surgical technique is the only means available to prevent the transmission of the viruses. keywords: hepatitis b virus, hepatitis c virus, surgeon original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.113-120 may-august 2020 vol.39no.2 cite this article as: mehrvarz s, khatami sm , manoochehry s, khedmat h, arefinia m, rasouli hr. assessment of needlestick injuries and hepatitis b and c infection among surgeons. univ med 202 0;39 :1 1 3 -2 0 . doi : 10 .18 05 1/ univmed.2020.v39.113-120 *trauma research center, baqiyatallah university of medical sciences, tehran, ir iran. **baqiyatallah research center for gastroenterology and liver diseases, baqiyatallah university of medical sciences, tehran, ir iran. ***department of general surgery, faculty of medicine, baqiyatallah university of medical sciences, tehran, ir iran. correspondence: @shahram manoochehry, trauma research center, baqiyatallah university of medical sciences, tehran, ir iran. email: shahram.manoochehry@yahoo.com tel/fax: +982188053766 orcid id: 0000-0002-0424-1763 date of first submission, january 19, 2020 date of final revised submission, august 9, 2020 date of acceptance, august 11, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 114 mehrvarz, khatami, manoochehry, et al hepatitis b and c in iranian surgeons introduction viral hepatitis is a systemic infection that involves the liver. severe viral hepatitis cases occur as types a, b, c, d, and e.(1) all the viral hepatitis agents are rna viruses, except for hepatitis b which is a dna virus.(1) these agents are characterized by different molecular and antigenic characteristics, but all create similar clinical signs in humans. (1,2) the hepatitis b prevalence in various areas in iran is estimated to be about 1.3%-2.4%.(3) hepatitis c prevalence is estimated to be around 0.13%-0.89% in iran. (4-6) compared to similar studies in other countries, the prevalence of hepatitis b virus (hbv) and hepatitis c virus (hcv) infection in iran is low.(79) hepatitis b is the 10th cause of death in the world.(4) annually, 0.5-1.2 million people die because of hepatitis b infection in the world, of whom 75% are in asia.(4, 5) cirrhosis and hepatocellular carcinoma are the leading causes of death in hbv infected patients. annually more than half a million people are infected with hbv, and if they receive no treatment about 15%-40% of them ar e at high risk of cirrhosis or hepatocellular carcinoma.(5) this imposes high expenses to the patients and the health systems of countries. in the united states, admission fee of these patients is about one billion dollars.(6) injection, exposure to blood products and transfusion, organ transplantation, hemodialysis and unsafe sex are major transmission pathways of hcv.(7, 8) sharp injuries and needlestick injuries are the most important occupational routes of hbv, hcv and human immunodeficiency virus (hiv) inf ections in health-car e wor kers (hcws).(9-11) the risk of transmission via percutaneous injury with hcv infected blood is estimated to be 3% to 10% and 7% to 30% for hbv.(12,13) hepatitis b infection has a higher prevalence in physicians, dentists, dialysis unit staff and laboratory workers compared with the general population.(14,15) among hcws, surgeons have the highest risk of hbv infection from their patients.(16) surgeons are at risk of infection with blood-borne pathogens, including hbv, hcv and hiv.(17, 18) specialist surgeons are at high risk of occupational exposure to these viruses through blood and body fluids. a systematic review showed that the highest incidence of needlestick injuries was seen in nurses and contraction of hepatitis b or hepatitis c from work-related needlestick injuries is one of the most common occupational hazards among health care workers.(19) a comprehensive national survey of surgical residents showed that needlesticks occurred frequently.(20) there is no previous study regarding evaluation of markers of hbv in surgeons with different specialties. we therefore aimed to determine the needlestick injuries, and markers of hbv and hcv in iranian surgeons. methods research design this cross-sectional study was conducted at six hospitals of tehran from january to may 2013. research subjects surgeons of baqiyatallah, shohada, rasoul akram, sina, taleghani, and emam hossein hospitals of tehran, iran, who had a history of needlestick injury, were included in this study. the sample size was calculated based on an odds ratio of 0.7 and using the formula for determination of prevalence in one group, with á and â set to 0.05 and 0.2 respectively. accordingly, the minimum sample size was calculated as n=318. among 318 eligib le sur geons who participated in the study, surgeons of the all fields (general, orthopedic, urology, neurosurgery, gynecology, ent and etc.) were included in this study. surgeons with a previous history of exposure to blood or blood products, those who had undergone major surgeries or had a history of traumatic injuries, and those who had a history of contact with b or c hepatitis patients, were excluded from the study. 115 data collection we made use of anonymous questionnaires. information regarding subjects’ demographics, number of their surgeries per month, medical history, occupationa l history, number of needlestick contacts, occasional exposures, risk behaviors and vaccination history were recorded after detailed interviews with the subjects. we also investigated the surgeons’ reaction to needle– stick injuries or to positive viral markers of themselves or their patients. we assessed the average number of needle stick in the whole experience work of iranian surgeons. a high risk exposure was defined if the number of needle stick > average number of needle stick and low exposure was defined if the number of needle stick d” average number of needle stick. laboratory tests under aseptic conditions, about 5 ml of venous blood was obtained from the subjects using standard procedures and the serum was stored frozen at -20°c before tests. blood samples were coded according to coding of the questionnaires. all samples were first screened for hbv and hcv through immune-chroma togr aphic technique. we us ed enzyme-linked immunosorbent assay (elisa) to carry out hbs antibody (hbsab), hcv antibody (hcvab), hbsag and anti-hbs tests. testing for hcv antibodies was performed using a third generation enzyme immunoassay. data analysis descriptive statistics was used to summarize the variables and distribution of variables in the samples. the relationship between these variables was assessed using fisher’s exact test and the kruskal-wallis test was used to analyze the data. data were analyzed using spss 20 software (ibm corp, armonk, ny, usa). a p-values less than 0.05 were considered significant. ethical clearance this study was approved by the medical research ethics committee of baqyatallah university under no. ir.bmsu.rec.1395.2. the selected hospitals had been notified and visited to offer administrative assistance before the questionnaires were given to the participants. participation in the study was strictly voluntary. informed consent sheets were obtained from all participants, each assured of maximum confidentiality. results among the 318 eligible surgeons who participated in the study, 177 surgeons (55.66%) were male. 93 general surgeons, 93 gynecologists, 37 orthopedists, 40 urologists, 34 ent surgeons and 21 were neurosurgeons. table 1 depicts demographics and distribution of selected variables in the surgeons of different specialties participating in the study. there was a significant lower number of monthly operations in orthopedists compared with other groups of surgeons (p<0.01). among the 318 surgeons, 291 surgeons (91.51%) had the complete vaccination for hb. and the highest duration of hb vaccination was among the urology ( p<0.001) the mean numbe r of needlesticks was 28.28 ± 16.58 during the surgeons’ working life. although orthopedists had the lowest number of surgeries per month, they had the highest number of needlesticks compared with ge neral surge ons, ent surgeons, gynecologists and neurosurgeons. then the highest rate of needlesticks also occurs in the orthopedist group (p<0.001). (table 1). mean hbsab concentration was 149.05 ± 288.03 iu/ml in surgeons and 15.7% of surgeons had antibody concentrations of less than 10 iu/ ml such that they were not immune against hepatitis b virus. the hbsab level in ent surgeons was lowest. (p<0.001) (table 2). among the 318 surgeons, 5 cases (1.59%) were positive for hbsag and 2 cases (0.66%) had a positive hcvab. the positive hbsag cases consisted of two orthopedists, two gynecologists and one general surgeon, while the hcvab positive case s consisted of one univ med vol. 39 no.2 116 mehrvarz, khatami, manoochehry, et al hepatitis b and c in iranian surgeons a b c t ab le 1 . d is tr ib u ti o n o f d em o gr ap h ic c h ar ac te ri st ic s an d s el ec te d v ar ia b le s in p ar ti ci p an ts o f th e st u d y d at a p re se n te d a s n ( % ) an d m ea n ± s d 117 univ med vol. 39 no.2 t ab le 2 : d is tr ib u ti o n o f se le ct ed v ar ia b le s o f d if fe re n t s p ec ia lt y gr o u p s in th e st u d y p o p u la ti o n *d at a p re se n te d a s n ( % ) an d m ea n ± s d 118 mehrvarz, khatami, manoochehry, et al hepatitis b and c in iranian surgeons orthopedist and one gynecologist. none of these surgeons had positive hbsag and hcvab results simultaneously. generally, high-risk exposure was 18.0% in the orthopedic group, 40.0% in gynecologists, 26.0% in urologists, 18.0% in general surgeons, 4.0% in neurosurgeons and 4.0% in ent surgeons (p=0.055) (table 2). discussion in the current study, hbsag positive prevalence was 1.59% and hcvab positive prevalence was 0.60%. this study shows that surgeons in iran do not have a higher hbv and hcv infection prevalence than the general population. in a study in lagos, nigeria, there was a higher prevalence of hbv infection in surgeons than in the general population. the study reported that the prevalence of surface antigen in the surgeons was found to be 25.7%.(21) another study in poland on medical students reported that anti-hepatitis core antigen (hbc) was positive in 5.3%, and hbsag was positive in 0.7 %.(22) the prevalence of hepatitis b in various areas of iran is a bout 1.3-2.4% which is considered a low prevalence in comparison to other countries.(3) anti-hcv positive prevalence is 0.20-0.89% in iran.(23-25) according to a nother study on 3411 orthopedists, the percentages of hepatitis b infection and positive hcvab were 13% and less than 1%, respectively. (24) in the orthopedic group of our study, the percentages of positive hbsag and positive hcvab were 5.55% and 2.7%, respectively. in the gynecology group, the percentages of positive hbsag and positive hcvab were 2.171 and 1.14%, respectively. needle–stick injury is one of most prevalent modes of transmission for hepatitis b and c.(12) in our study, numbers of needle-stick in orthopedists group was more than other groups; it’s because of type of instruments and tissues which orthopedists work with. in our study, all surgeons had needle-stick history and high risk exposures were seen in 15.72%. one of the groups at high risk is orthopedic surgeons. apart from needles, they frequently use other sharp materials, such as wires, drills and saws.(26) in our study, in spite of the lower number of surgeries in the orthopedic and gynecology groups, these groups were at higher risk of needlesticks and had more positive hbsag and hcvab results in comparison to the other surgeons. in this study, the most complete vaccination coverage was seen in the neurosurgery and orthopedic groups, followed by the gynecology group. the least complete vaccination coverage was seen in the ent group. the neurosurgeons, orthopedists and gynecologists also had highest level of serum hbsab. nevertheless, in this study there was no significant relationship of positive hbsab, needlesticks, high-risk exposure and the number of surgeries with any of the surgeons’ groups. despite the availability of hepatitis b vaccine, orthopedic surgeons remain at risk for acquiring blood-borne viral infection. also, there is a relationship between positive hcvab results and high-risk exposure. one limitation of this study was the difficulty in convincing the faculty surgeons to undergo laboratory tests needed for the research. more studies are recommended to be done for better understanding about prevention and treatments in those high risks groups. conclusion the results showed that in spite of the high needlestick ra te in irania n surgeons, the prevalence of hepatitis b and c is not very high among them. most needlestick and positive cases were in the groups of gynecologists. acknowledgement we would like to thank the deputy research director of baqiyatallah hospital for their kind cooperation in this research. 119 univ med vol. 39 no.2 conflict of interest all authors declare that there is no conflict of interest in this research. contributors sm and sm contributed to conceptualization, data handling, and experimental design. smk, hk, ma contributed to data analysis, provision of study materials and equipment. sm, ma and hrr contributed to study validation, supervision, data presentation, draft preparation, study consultation, writing and reviewing, and project administration. all authors have read and approved the final manuscript. references 1. center for disease control and prevention. what is viral hepatitis? atlanta : center for disease control and prevention;2020. 2. 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24. merat s, rezvan h, nouraie m, jafari e, abolghasemi h, radmard ar, et al. seroprevalence of hepatitis c virus: the first population-based study from iran. int j infect dis. 2010;14 suppl 3:e113–6. doi: 10.1016/j.ijid.2009. 11.032. 25. zamani f, sohrabi m, poustchi h, et al. prevalence and risk factors of hepatitis c virus infection in amol city, north of iran: a population-based study 2008-2011. hepat mon 2013; 13: e13313. doi: 10.5812/hepatmon.13313. 26. fisher wd. hepatitis c and the surgeon. j can chir 2013;56:80-1. doi: 10.1503/cjs.000912. c:\users\user\documents\editori 69 universa medicina may-august 2019 vol.38 no.2 stunting: a multisectoral challenge and unfinished nutrition agenda elvina karyadi senior health specialist, world bank indonesia it was great news that during the presidential e lectio n campaign, both the presidential and vice-presidential candidates addressed the stunting problem during the debate. it shows that stunting is regarded as a national priority nutrition problem. stunting is part of a wider nutrition crisis in indonesia: wasting, anemia and the double burden of malnutrition. according to the 2018 natio nal health sur vey (riskesdas), 30.8 percent of indonesian children under 5 years of age were stunted (almost 8 million children), 17.7 percent were underweight, 10.2 percent were wasted (low weight-for-height).(1) although the prevalence of stunting declined significantly since 2013 (37.2%), the stunting rates remain unacceptably high. a child that is wasted faces double the risk of mortality associated with stunting. a child that is both stunted and wasted faces an even higher risk. frequent episodes of wasting also increase the risk of stunting. stunted growth refers to low height for age, indicating that a child is short for his/her age. this reflects chronic malnutrition and carries long-term development risks. stunting, with its associated impacts on child cognitive and physical development, occurs in the first 1,000 days of life and is largely irreversible. the first 1,000 days represent a life window when growth rates and neuroplasticity of the brain are at their peak and where nutritional deficiencies can exert their most devastating impacts. poor maternal nutrition, indicated by low birth weight, is a strong predictor of stunting, and analysis of growth faltering in indonesia shows that about 20 percent of children are considered stunted at birth. there is relatively little variation in growth faltering across regions, gender, and birth order. however, it is worse in rural areas, among the bottom 20 percent (income), and among less educated women.(1) stunting has intergenerational effects. a child's risk of developing chronic malnutrition starts in the womb. maternal short stature (less than 145 cm) is associated with an increased risk of underweight and stunted offspring. poor maternal diet not only can lead to inadequate weight gain for mothers during pregnancy but it can impact the growth of the fetus. nutrition needs are increased during pregnancy and dietary requirements are higher; it is estimated that a pregnant woman requires about 75,000 calories extra during pregnancy and an increased intake of micronutrients such as iron, zinc and calcium.(2) it is estimated that today about eight million indonesian children under the age of five are stunted. they are not growing well and are at high risk of becoming ill. they are less likely to benefit from education because their brain development has been compromised. and they are less likely to earn a good income and contribute to the overall economy. lastly, they also are more likely to develop chronic disease such as diabetes and obesity in adulthood. the unicef framework to tackle chronic malnutrition was started by unicef in 1990 and is used worldwide. the model has been 70 universa medicina adapted (3) and it operates by dealing with the four main drivers of malnutrition through the following steps: (1) adequate access to care and age-appropriate feeding practices (c), (2) access to adequate health car e services (h), (3) environmental health, hygiene, sanitation and access to safe water (e), and (4) access to a diversity of safe food (f) chef. in indonesia, the absence or poor quality of all four chef drivers have contributed directly to the country's high rates of stunting. what is the context situation with indonesia? firstly, food security can be patchy across the indonesian archipelago. adequate amounts of quality food are not always available, particularly to the poor in rural areas. moreover, high prices of nutritious food such as fruits, vegetables and animal protein, make it difficult for poor families to purchase a diverse diet. secondly, access to adequate health services tends to be lower among poor and rural women, including pregnant women and mothers. although women attend their antenatal visits regularly, the quality of the health services is not always optimal, resulting in low impact of such visits. for example, the availability of iron folic acid (ifa) supplements is very good (a round 94% availability at puskesmas level), but counselling on how to take ifa supplements and face the side-effects is often poor, resulting in low compliance. of the 73.2% of pregnant women who received ifa supplements only 24% received ?90 ifa tablets, whereas the remaining 76% received ?90 tablets. of the women receiving 90 ifa tablets, only 38.1% consumed all ifa tablets (90) and 48.9% suffered from anemia in pregnancy.(4) access to health services is also measured by immunization rates. complete immunization among children 1223 months decreased from 59.2% in 2013 to 57.9% in 2018.(4) it remains below the rencana pembangunan jangka menengah nasional (rpjmn) target of 93% in 2019. thirdly, feeding practices are also not always what they should be to ensure the healthy growth and development of children. the current rate of exclusive breastfeeding in infants was 37.3% in 2018.(4) there remain many obstacles and constraints to exclusive breastfeeding practices, ranging from lack of enforcement of laws and regulations regarding marketing of breastmilk substitutes, insufficient attention to cultural barriers and more women entering the workforce without adequate baby-friendly policies in place. complementary feeding practices are also important to prevent stunting. many children are vulnerable to stunting after the age of 6 months. children are not fed a diverse age-appropriate diet, nutritious food is not given and meals are given too infrequently. fourthly, poor sanitation, lack of access to safe water and unhygienic feeding practices also contribute to stunting. children are very susceptible to infection such as diarrhea, resulting from unclean water and unhygienic behavior. especially children at very young age, who start exploring their environments, still come too easily into contact with an unsafe environment and are at risk for becoming ill. research shows the importance of having adequate access to as many of the four nutrition drivers as possible. the probability that children between zero and three years of age will be stunted is lower when they have access to adequate levels of two of the chef drivers. in 2013, some 23% of children between zero and five years of age did not have access to adequate levels in any of the four nutrition drivers in indonesia.(5) indonesia has been internationally recognized for its successful steps forward in public health nutrition. it was an early adopter of an universal salt iodization (usi) law and received the helen keller award for significantly decreasing the prevalence of xeropthalmia in the past decades. in 1986, the posyandu (health post) were officially established, as integrated community health posts in villages to improve maternal and child health with a focus on growth monitoring and promotion, nutrition, immunization and micronutrient distribution. also, in the 1980s, the sistim kewaspadaan pangan gizi (skpg) or food and nutrition surveillance program was introduced. 71 may-august 2019 vol.38 no.2 the skpg monitored not only nutrition status in posyandu, but also looked at climate, economic changes at all levels (national, provincial, district and sub-district). it raised alerts in multiple sectors to prevent severe malnutrition. however, past success has not yet translated into sustained success. much more needed to be done until recently. there is a growing body of experiences in other developing countries that a "convergence approach" to tackle stunting can be effective, in which multisectoral interventions are coordinated to jointly target priority geographic areas and beneficiaries.(6) stunting reduction in indonesia involves a multisectoral approach, including nutrition-specific interventions and nutritionsensitive interventions. nutrition-specific interventions have been implemented mostly in the primary health care services for pregnant mothers, lactating mothers and children aged 023 months, adolesc ents and women of reproductive age, and children aged 24-59 months. indonesia has a multitude of nutritionsensitive programs that have been proven to improve early childhood outcomes, including stunting. the ongoing nutrition-sensitive programs support stunting reduction, such as conditional cash transfer (program keluarga harapan/pkh, family hope program), national health insurance (jkn: jaminan kesehatan nasional), non-cash food assistance (bpnt: bantuan pangan non tunai), water, sanitation and hygiene (wash), early child educat ion ( eced) paud: pendidikan anak usia dini. there are still challenges for the quality of these nutritionsensitive intervention programs, which need to be improved and monitored intensively. inspired by the peru's success to reduce stunting, indonesian high-level officials from the ministry of health and the ministry of finance visited peru and learned from the peruvian example about how to accelerate stunting reduction. in august 2017, vice president jusuf kalla launched the implementation of the national strategy on stunting (stranas stunting). this new national strategy to accelerate stunting prevention will ensure that all "1,000-day households"-those with pregnant women or children under two years of age can access the complete package of services essential to prevent stunting.(1) the stranas stunting involves 22 ministers and consists of five pillars that aim to raise public awareness of stunting, secure nationwide commitments to stunting reduction, and manage, implement, and converge the delivery of the priority nutrition interventions across three levels of government, which notably are (1) pillar 1: national leadership and commitment; (2) pillar 2: national public awareness campaign, (3) pillar 3: national, regional, and community program convergence, coordination, and consolidation, (4) pillar 4: nutritional food security and (5) pillar 5: monitoring and evaluation. the stranas stunting includes a very strong monitoring system with annual surveys measuring progress and reporting at annual summits. the projection of estimated outcomes will also allow better planning and budgeting, as well as underpinning national commitments to drive down rates of stunting. indonesia is optimistic to be able to reduce stunting by more than 1% per year (as business as usual), because many factors support the stranas stunting such as high political commitment from our president and vice president, formal regulation on the acceleration of stunting reduction (uu no 42/2014) and regulation of food (uu pangan no 18/2012). indonesia has joined the global scaling up nutrition (sun) movement, and current supporting programs. under bappenas, the sun movement has involved all stakeholders including business groups, civil society organizations (csos), un/ donor and academia to move and actively support the stunting prevention programs. as was hoped for, indonesia has launched an ambitious stunting reduction strategy which aims not only to achieve national coverage by 2021 but also commits the country to implement all the key elements (nutrition-specific and nutrition-sensitive interventions) required for success. if peru can do it, so can indonesia! 72 references 1. rokx c, subandoro a, gallagher p. aiming high: indonesia's ambition to reduce stunting. washington dc: world bank group; 2018. 2. karakochuk cd, whitfield kc, green tj, kraemer k, editors. the biology of the first 1,000 days. boca raton: crc press;2017. 3. the lancet. maternal and child nutrition: executive summary of the lancet maternal and child nutrition series; 2013. 4. kementerian kesehatan badan penelitian dan pengembangan kesehatan. hasil utama riskesdas 2018. jakarta: kementerian kesehatan badan penelitian dan pengembangan kesehatan;2018. 5. world bank group. operationalizing a multisectoral approach for the reduction of stunting in indonesia: an application using the 2007 and 2013 riskesdas. washington dc: world bank group;2017. 6. levinson fj, balarajan y, marini a on the peru case study. addressing malnutrition multisectorally: what have we learned from recent international experience? unicef nutrition working paper. new york: unicef and mdg achievement fund; 2013. univ med 2019;38:69-72. doi: http://dx.doi.org/10.18051/univmed.2019.v38.69-72 univ med vol. 38 no.2 c:\users\jurnal fkusakti\docume 194 *department of medical physiology, faculty of medicine, universitas indonesia **master program in biomedical science, faculty of medicine, universitas indonesia ***department of histology, faculty of medicine, universitas indonesia †department of physiology, faculty of medicine universitastrisakti *department of medical physiology, faculty of medicine university of indonesia correspondence: dewi irawati soeria santoso department of medical physiology, faculty of medicine university of indonesia email: dewi.irawati.1307@gmail.com orcid id: https://orcid.org/00000001-7390-5298 date of first submission, december 26, 2 01 8 date of final revised submission, december 3, 2019 date of acceptance, december 11, 2 01 9 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina low vitamin b12 diet increases liver homocysteine levels and leads to liver steatosis in rats imelda rosalyn sianipar*, irena ujianti**, sophie yolanda*, ahmad aulia yusuf***, neng tine kartinah*, patwa amani†, krishna aditya murti**,† and dewi irawati soeria santoso*a background nonalcoholic fatty liver disease (nafld) is one of the most widespread chronic liver diseases, caused by the development of insulin resistance. one of the mechanisms involved is a disturbance in insulin signaling by certain toxic substances that interact with one of the proteins responsible for the insulin signaling pathway. increased homocysteine level, upon disruption of the methionine pathway, is associated with insulin resistance. the aim of this study was to evaluate the effect of hyperhomocysteinemia and insulin resistance (homa-ir level) induced by dietary vitamin b12 restriction on liver steatosis. methods a study of laboratory experimental design was conducted involving 18 male sprague dawley rats (age 36-40 weeks, bw 300-350 g), that were randomly divided into 3 groups: control, 8-week treatment, and 16-week treatment. standard ain-93 diet was administered to the control group, whereas rats in the treatment groups were fed vitamin b12 deficiency-ain93m. at the end of treatment, liver homocysteine levels were determined by elisa, homa-ir values were calculated, and steatosis degree of the liver was determined histologically. statistical analysis was performed using independent t-test. results a significant increase in liver homocysteine levels was found between the control and both the 8and 16-week treatment groups (p<0.001). homair levels were significantly higher in both treatment groups compared to controls (p<0.001). the area of liver steatosis in both treatment groups was significantly larger than that of the control group (p<0.001). conclusion increased homocysteine levels due to dietary vitamin b12 deficiency induces liver steatosis due to insulin resistance in rats. keywords: homocysteine, vitamin b12 deficiency, nafld, insulin resistance, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.194-201 september-december, 2019 vol.38no.3 cite this article as: sianipar ir, ujianti i, yolanda s, et al. low vitamin b12 diet increases liver homocysteine levels and leads to liver steatosis in rats. univmed 201 9;38 :19 4 -2 0 1 . doi: 10 .18 05 1/ univmed.2019.v38.194-201 195 univ med vol. 38 no.3 introduction nonalcoholic fatty liver disease (nafld) is defined as the presence of liver steatosis, as demonstrated using imaging or histology, in patients who ingest less than 20 mg of alcohol per day. it is the most prevalent liver disease worldwide, with an estimated global incidence of 25.24%.(1,2) different metabolic co-morbidities are often associated with nafld, including central obesity, insulin resistance, type 2 diabetes mellitus, hyperlipidemia, hypertension, and metabolic syndrome.(3) nonalcoholic fatty liver disease is an umbrella term which includes a wide spectrum of disorders, ranging from a simple form of steatosis to a progressive form known as nonalcoholic steatohepatitis (nash), which is characterized by hepatocellular injury and inf lammation. furt hermore, nafld is considered to be an important cause of cryptogenic cirrhosis.(4) vitamin deficiency/hypovitaminosis is a general term for a widespread condition which has reached epidemic proportions in developing and developed countries alike, mainly due to lifestyle and dietary habits. although subclinical hypovitaminosis can occur in well-nourished individuals, it is most often the result of limited dietary intake or vitamin malabsorption. several studies have shown that chronic liver diseases are associated with lowered levels of vitamins such as vitamin d, e, b6, and a.(5,6) despite the indications in the literature regarding the association between chronic liver diseases and hypovitaminosis, little is known about any specific correlation between hypovitaminosis and nafld. vitamin b12 (cobalamin) is a water-soluble cobalt-containing vitamin with an important role in biochemical processes referred to as single carbon transfers.(7) vitamin b12 is a cofactor for at least 3 enzymes that catalyze these reactions, and acts as a transitional carrier of a single carbon group. it has been reported that several liver diseases such as acute hepatitis, cirrhosis, hepatocellular carcinoma, and metastatic liver disease are associated with major changes in plasma vitamin b12 concentrations.(8) although these pathologies can lead to alterations in plasma levels of vitamin b12 via different pathways, no clear effect of nafld has been determined. several studies have shown the correlation between vitamin b12 deficiency and the incidence of steatosis.(1,7,8) a study conducted by mahamid et al.(9) demonstrated a correlation between low levels of folate and vitamin b12 with the condition of nash seen histologically. li et al.(10) reported elevated serum homocysteine in cases of nafld in animals induced by high-fat diet, due to decreased transsulfuration pathway regulation. several studies have reported that there might be an as sociat ion b etwee n insu lin resistance and levels of vitamin b12 and nafld.(9-11) this might be explained by the multiple hit theory which states that insulin resistance is the primary basic pathology in liver steatosis.(3) however, the mechanism of vitamin b12 deficiency resulting in liver steatosis via the insulin resistance pathway correlated with increased plasma and liver homocysteine levels is still not widely reported, thus the objective of this study was to investigate the potential correlation between vitamin b12 serum levels and nafld, with focus on the insulin resistance pathway as one of the underlying mechanisms. methods study design this study was an in vivo experimental study and part of a larger research project at the department of medical physiology faculty of medicine universitas indonesia. this study was conducted at the health rese arch and development center (pusat penelitian dan pengembangan kesehatan, puslitbangkes) faculty of medicine universitas indonesia (fmui) from october 2017 to may 2018. animals this study used male sprague dawley rats aged 36-40 weeks with body weight ranging from 196 300-350 grams, which were obtained from badan penelitian dan kesehatan ri. sample size was calculated using federer’s formula, yielding a total of 18 rats.(12) rats were placed in individual cages in a r o o m wi t h a d e q ua te ve n t il a ti o n. roo m temperature was maintained between 18-26°c with humidity 30-70% and a 12-hour light and dark cycle. rat cages were cleaned every day to ensure maintenance of healthy rats. after one week of acclimatization, they were randomly divided into three groups. the first group was given an ain-93m diet (research diets inc., usa) ad libitum and was considered as the control group (group 1, n=6, designated as c). the second group was given a modified ain93m diet ad libitum (ain-93m with restricted vitamin b12) with additional pectin/cellulose as a source of fiber for eight weeks (group 2, n=6, designated as t8). the third group was given the same modified ain-93m diet ad libitum for 16 weeks (group 3, n=6, designated as t16). the vitamin b12 content in the modified diet was much lower than that in the control diet (0.016 vs. 28 mcg/kg diet). the modified diet also contained 50 g pectin/kg to bind to the intrinsic factor in the intestine and to reduce the bioavailability of vitamin b12. food intake was recorded daily, and body weight was measured once weekly. after eight and 16 weeks of f e e di n g, le ve l s o f p l a sma vit a mi n b12 , homocysteine and glucose were measured. biochemical parameters at the end of eight and 16 weeks of diet administration, blood was collected from the supraorbital sinus, after overnight fasting. liver homocysteine levels were determined using a standard elisa kit (rat homocysteine elisa, mybiosource, usa). liver homocysteine assays were performed at the immuno-endocrinology laboratory, faculty of medicine, universitas indonesia. insulin resistance indicator values, namely homa-ir (h omeostasis model assessment values for insulin resistance) were calculated using the formula: homa-ir = fasting glucose (mmol/l) x fasting insulin (μiu/l)/22.5.(13,14) histological analysis of the liver af t e r e igh t a n d si x t e e n we e ks o f intervention respectively, rats were sacrificed and their livers were fixed in 10% buffered formalin and embedded in paraffin. liver sections (5 μm) were stained with hematoxylin and eosin (h&e), and the steatotic degrees of individual liver samples were determined by subjecting sections to blind examination by a pathologist; sections were scored depending on the percentage of hepatocytes containing lipid droplets.(15,16) liver histology preparations were carried out at the histology laboratory fmui. ethical clearance rats were treated in accordance with the helsinki convention. ethical approval was obtained from the health research ethics committee faculty of medicine universitas indonesia/cipto mangunkusumo hospital (fkui/ rscm) under number: 184/un2.f1/etik/2017. statistical analysis statistical analysis was performed using oneway anova followed by post-hoc analysis for homocysteine levels and liver steatosis, while independent t-test and paired t-test were used for homa-ir, in which the treatment group was compared to the control group in their respective treatment week, and the homa-ir value was also assessed between treatment groups. data were previously analyzed for normality using the shapiro-wilk test. data processing was performed using the spss 12 software (statistical social sciences program version 12). results liver homocysteine levels based on the one-way anova test, group t16 showed significantly higher levels of liver homocysteine (p<0.001) compared to both control and t8 groups (table 1). sianipar, ujianti, yolanda, et al low vitamin b12 and liver homocysteine level 197 univ med vol. 38 no.3 homa-ir values based on the independent t-test, there was a significant difference in homa-ir values between treatment groups t8 and t16 (p<0.001), with group t16 showing higher homa-ir values. based on the paired t-test, there were significant differences in homa-ir values between control and treatment groups in both week 8 and week 16 (table 1). liver histopathology there was no obvious steatosis in the livers of the control group. the area of hepatic steatosis in both treatment groups were significantly larger compared to controls (p<0.001), with the largest area of hepatic steatosis found in group t16 (p<0.001 vs t8) (table 1). discussion nafld is a widespread chronic liver disease and its incidence is increasing in developed countries. our previous study showed that vitamin b12 restricted diet caused lower vitamin b12 plasma levels.(17) the study also showed that the decrease in plasma vitamin b12 levels caused increased plasma homocysteine levels.(17) in this study, there was a significant increase in liver homocysteine levels in the treatment groups, namely approximately twice as high as in the control group. the study supports the use of vitamin b12 deficient rats as an animal model for hyperhomocysteinemia. in vitamin b12 deficient rats, methionine metabolism is impaired. the methyl group of b12 a b c functions as a coenzyme of methionine synthase which catalyzes the methyl transfer from methyltetrahydrofolate to homocysteine, resulting in the donation of a methyl group to homocysteine, forming methionine.(18) methionine synthase is important to re-synthesize methionine and to metabolize methyltetrahydrofolate. in patients with vitamin b12 deficiency leading to decreased liver transsulfuration pathways in homocysteine metabolism, an increase in plasma homocysteine level was observed. (1 9) inc reased pla sma homocysteine causes oxidative stress, resulting in insulin resistance through the ros pathway.(20,21) according to the literature, oxidative stress produced by the conversion of homocysteine (hcy) to hcy thiolactone leads to the inhibitory activity of the tyrosine kinase insulin receptor. a study by najib and sanchez-margalet (22) showed that homocysteine thiolactone, the active form of homocysteine, inhibited the insulinstimulated tyrosine phosphorylation of insulin receptor β-subunit and its substrates (insulin receptor substrate-1 and p60-70) in rat hepatoma cells. in addition, they showed that homocysteine thiolactone decreased the p85 regulatory subunit of phosphatidylinositol 3-kinase activity, inducing a reduction in insulin-stimulated glycogen synthesis. insulin signaling disorder results in an increase in plasma blood glucose. this is in accordance with our previous study, in that the significantly higher fasting blood glucose levels in rats receiving fed restricted b12 diet indicated that the restriction of vitamin b12 could lead to the development of insulin resistance. this is table 1. homocysteine levels and percent of steatosis from liver tissue and plasma hom-ir by treatment groups values are expressed as mean ± sd; dietary groups: c: control; c8: control at week 8;t8: ain-93m with restricted vitamin b12 for 8 weeks; c16: control at week 16;t16: ain-93m with restricted vitamin b12 for 16 weeks; ***p<0.001 compared to control; ###p<0.01 compared to t8; @one-way anova; ^independent and paired t-tests c (n=6) t8 (n=6) t16 (n=6) liver homocysteine @ 1.52 ± 0.05 1.6 ± 0.05 2.48 ± 0.20***### % of steatosi @ 3.33 ± 0.25 53.83 ± 4.56*** 69.64 ± 2.43***### c8 t8 c16 t16 homa-ir^ (μiu/l) / 22.5 2.27 ± 0.07 3.91 ± 0.13*** 3.03 ± 0.05 5.87 ± 0.09***### 198 consistent with several studies that suggest a link between increased homocysteine due to vit a mi n b1 2 d e f i c i e n c y a nd i n s ul i n resistance.(11,23) our study also showed similar results to these studies, which was shown by the homa-ir values. another study suggested that hepatic insulin resistance can perturb the regulation of lipid metabolism and it is a common feature observed in human and rodent models of nafld.(24) the regulation of glucose and lipid production is disrupted when insulin action is compromised in the liver. a study by abdul-ghani and de fronzo (25) have documented 35–50% reductions in glucose disposal, a measure of whole-body insulin sensitivity, and an impaired ability of i ns u l i n t o s up pr e s s e n do ge nou s gl u c o se production, indicative of hepatic insulin resistance additionally, subjects with nafld exhibit a defect in insulin suppression of free fatty acids (ffa), in keeping with insulin resistance at the level of the adipocyte.(26) compared with controls, subjects with nafld also demonstrate a blunted inhibition of fatty acid oxidation, reflecting the decreased uptake and use of glucose as a source of fuel. these findings suggest the possibility that insulin resistance may be an intrinsic defect in nafld, in which diminished insulin responsiveness at the level of the adipocyte may contribute to hepatic steatosis by excess ffa flux to the liver.(27) expression of genes involved in the de novo l i po ge ne s is pr o c e s s i n t h e l ive r i s a l so significantly increased in nafld patients. in healthy conditions, lipogenesis contributed 5% of the total amount of liver triglycerides. but this number is increased by 26% in patients with nafld. this biosynthetic increase contributed greatly to nafld patients.(28) although there was an increase in regulation of fatty acid oxidation and export of triglycerides by vldl in nafld patients, this process cannot restore homeostasis in the liver.(29) besides via insulin resistance, vitamin b12 deficiency contributes to the development of steatosis through hyperhomocysteinemia. vitamin b12 is a very important molecule for the process of synthesizing s-adenosylmethionine (sam) a b c figure 1. liver histology of a normal rat (c16) (a) and nafld rat at week 8 (t8); (b) and week 16 (t16); (c) (magnification 40x) sianipar, ujianti, yolanda, et al low vitamin b12 and liver homocysteine level 199 univ med vol. 38 no.3 from methionine. phosphatidylethanolamine nmethyltransferase (pemt) uses sam as a methyl donor to ca ta lyze methylation of phosphatidyletha nolamine (pe) to phosphatidylcholine (pc), which is needed for vldl formation in hepatocytes. phosphatidylcholine synthesis decreases in vitamin b12 deficiency. phosphatidylcholine affects lipid export by vldl, and causes accumulation of liver lipids.(8,30) in addition, disorders of homocysteine metabolism due to vitamin b12 deficiency cause a decrease in the transsulfuration pathway in the liver, increase the sam/sah comparison index causing liver steatosis.(31) due to an increase in the prevalence of risk factors for vitamin b12 deficiency, such as advanced age, vegetarian lifestyle, or patients with gastrointestinal problems which result in impaired absorption of vita min b12, one of the consequences of these conditions is liver steatosis.(32) this study explores one of the pathways of liver steatosis due to vitamin b12 deficiency via the insulin resistance pathway. it is hoped that the results of this study may help in preventing the adverse effects of vitamin b12 deficiency in high-risk populations. one of the limitations of this study is that although adverse eff ects of vitamin b12 deficiency have been observed, the rats were clinically not yet categorized as having vitamin b12 deficiency. it is hoped that future studies will address this issue through modification of the length of the feeding or an increase in the percentage of pectin which can lead to vitamin b12 deficiency. conclusion this study demonstrated that vitamin b12 restricted diet results in the occurrence of liver s t e a t os i s vi a i n su l i n r e s i st a n c e a n d hyperhomocysteinemia in rats. and elevated homocysteine was a critical factor in the pathogenesis of nafld. acknowledgement this study was funded by a grant provided by the ministry of research, technology and higher education, republic of indonesia, hibah pasca sarjana 2018 with contract number 551/un2.r3.1/hkp05.00/2018. conflict of interest the authors declare no conflict of interest. contributors diss and irs contributed to the concept and design. iu and irs contributed to writing the original draft. iu and sy contributed to literature search. diss and irs contributed to the investigation. aay, pa and kam contributed to data collection and analysis. irs, diss, and contributed to writing the revision and editing. all authors have read and approved the final manuscript. references 1. lindenmeyer cc, mccullough aj. the natural history of nonalcoholic fatty liver disease: an evolving view. clin liver dis 2018;22:11–21. doi: 10.1016/j.cld.2017.08.003 2. abd el-kader sm, el-den ashmawy ems. nonalcoholic fatty 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10.3390/ijms15058591. c:\users\universa medicina\docu 47 *department of pharmacology and pharmacy, faculty of medicine, trisakti university **department of clinical pathology, faculty of medicine, trisakti university *** department of medical checkup, mintoharjo hospital correspondence: dr. dr. elly herwana, m.biomed department of pharmacology and pharmacy, faculty of medicine, trisakti university jl. kiyai tapa, grogol, (kampus b), jakarta 11440 phone : +62-5672731 ext.2801 email: elly.herwana@trisakti.ac.id orcid: 0000-0003-1814-1189 date of first submission, march 3, 2 01 9 date of final revised submission, april 8, 2020 date of acceptance, april 9, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina high dietary daidzein intake lowers cholesterol levels among post-menopausal women elly herwana*, pusparini**, and audria graciela*** background menopause is associated with changes in metabolic profile. although hormone replacement therapy (hrt) has been shown to have beneficial effects on lipid metabolism, its adverse effects have indicated a need for alternative estrogen-based treatments. several investigations have evaluated the effects of isoflavones on serum lipid levels in postmenopausal women, but the results were ambiguous. the objective of this study was to determine the relationship of dietary daidzein, genistein, and glycitein levels with lipid profile in postmenopausal women. methods a cross-sectional study was conducted involving 186 post-menopausal women. a food recall questionnaire was used to measure dietary genistein, daidzein, and glycitein levels. serum total cholesterol (tc), low density lipoprotein (ldl) cholesterol, high density lipoprotein (hdl) cholesterol, and triacylglycerol (tag) were measured using the enzymatic colorimetric method. simple and multivariate linear regression were used to analyze the data. results genistein intake was significantly associated with tc (=145.48, p=0.023) and hdl cholesterol levels (=48.80, p=0.032). daidzein intake was significantly associated with tc (=-204.60, p=0.003), ldl cholesterol (=160.81, p=0.014) and hdl cholesterol levels (=-67.118, p=0.032). glycitein was not significantly associated with tc (=232.78;p=0.133), hdl (=43.59;p=0.428), and ldl (=235.84;p=0.116). dietary daidzein had a more lowering effect on tc (beta=-2.80) and hdl cholesterol (beta=-2.67) than had genistein on tc (beta=2.66) and hdl cholesterol (beta=2.03). conclusions high dietary daidzein level has a significant lowering effect on tc, ldl cholesterol and hdl cholesterol in post-menopausal women. our study supports the advice given to the public to increase soy isoflavone intake in post-menopausal women. keywords : daidzein, genistein, glycitein, serum lipid, postmenopausal women original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.47-54 january-april, 2020 vol.39no.1 cit e th is article as : her wa n a e, pu sp arin i, gra ci ela i. high dietary daidzein intake lowers cholesterol levels among post-menopausal women. uni v med 2020;3 9:4 7 -5 4 . doi: 10.18051/univmed.2020.v39.47-54 48 herwana, pusparini, graciela daidzein intake and post-menopausal women introduction menopause is a physiological condition in the female life cycle that is marked by the cessation of menstruation and the end of r e p r o d uc t ive c a pa c it y. t h e c on di t i on of menopause is associated with decreased ovarian function, thereby causing a reduction in the circulatory estrogen level. the lower mortality f r om c a r di o va s c u l a r d i se a s e ( cv d ) i n premenopausal women as compared to males is associated with the protective effects of e s t r o gen a n d pr oge s te r o ne . ( 1 -3 ) es t r o ge n demonstrates cardioprotective effects through maintaining high high-density lipoprotein (hdl), high low-density lipoprotein (ldl) and low triacylglycerol (tag) levels. the decreased estrogen level increases dyslipidemia and thus the risk of cvd.(3-5) increasing age is associated with increased cvd risk. the differences between the premenopausal, perimenopausal, and postmenopausal phases lie in the increases in body mass index (bmi), systolic blood pressure, diastolic blood pressure, and ldl and tag levels. the increased ldl and tag levels are demonstrable after the age of 45 years and increase sharply in the perimenopausal and postmenopausal phases.(6) hormone replacement therapy for estrogen, which had been expected to be able to return the cardioprotective effects of estrogen, turned out not to show the eff ects in question. the administration of ex ogenous estr ogen is associated with increased r isk of venous thrombosis, resulting in myocardial infarction, stroke, and peripheral arterial disease. the possibility of estrogen being associated with prothrombotic effects is still a subject of controversy and is as yet incompletely understood.(7) a study conducted by the women’s health initiative (whi) that evaluated and compared the effects of conjugated equine estrogens (cee), singly or concurrently with medroxyprogesterone acetate (mde), reported that there was an increase in the risk of cvd, stroke, and breast malignancies in both groups.(8,9) the differ ence in cardiovascular disease prevalence between asian and caucasian women had led to opinions on the difference in dietary patterns. isoflavones that are present in high levels in soybean-derived foods, are consumed in higher amounts by asian than by caucasian women.(10) soybe ans contain ina ctive glycosides of isoflavones. the isoflavones in the gut are hydrolyzed by intestinal bacterial β-glucosidases to yield bioactive aglycones that can be detected in the blood. the isoflavone derivatives with a molecular structure similar to that of estrogen, are believed to act as phytoestrogens. genistein, daidzein, and glycitein that are aglycones of soy isoflavone, have a molecular structure similar to that of 17β-estradiol and show estrogenic effects. the phenolic ring group in soy phytoestrogenic isoflavones is the binding site for estrogen receptors, which comprise two types, namely estrogen-α (erα) and estrogen-β (erβ) receptors. isoflavones as partial agonists show an affinity to the estrogen receptors, particularly to erβ.(11,12) another mechanism of the biological effects of isoflavones is through activation of peroxisome proliferator activated receptors (pparα and pparγ), resulting in decreased hepatic lipid synthesis, bile acid synthesis, and cholesterol absorption.(13,14) many studies have been conducted on the effects of isoflavones on lipid profile, but the results are still inconsistent. one study found that phytoestrogens derived from both soy and red clover have a positive metabolic effect on serum lipids of postmenopausal women. both soy and red clover supplements were found to have an almost equal impact on serum lipid levels.(15) a meta-analysis performed by tokede et al.(16) found that soy protein is beneficial for dyslipidemia that is marked by decreased total cholesterol, ldl cholesterol, and tag, and increased hdl concentrations. the effects are stronger on subjects with hypercholesterolemia. whole soy foods are more beneficial than soy supplements, and isoflavone supplements are not beneficial for the lipid profile. differing results were shown in a study on isoflavone supplementation at 60 mg/ 49 univ med vol. 38 no.1 day for three months, in which the supplement did not cause changes in ldl cholesterol levels of postmenopausal women.(17) the still inconclusive results of previous studies need to be investigated further. therefore the present study aimed to determine the relationship between dietary intakes of isoflavone derivatives and lipid profile in postmenopausal women. methods design of the study this was a cross-sectional study conducted in the district health center, mampang prapatan, south jakarta, from january 2010 until february 2011. study subjects females aged 47-60 years meeting the criterion of natural menopause (cessation of menstruation for 1-10 years and not resulting from hysterectomy or medications) were recruited as study subjects. females with a history of malignancies of the breast or reproductive organs, renal failure, diabetes mellitus, heart disease, hyperthyroidism, post stroke, currently on estrogen replacement therapy, or consuming isoflavone-containing supplements, were excluded from the study. a pre liminary study by one of the investigators (eh) showed a correlation between daidzein intake and tc level of -0.25 (data not shown). on the basis of the preliminary study results, with α=0.05 and β=0.1 and a drop-out of 15%, the required sample size was 186. determination of isoflavone intake isoflavone intake was calculated from food recall, especially for isoflavone-containing foods. the subjects were asked to record the foods they consumed during one whole day. food recall was performed twice, with an interval of one week. food recall was done using interviews to confirm the food record and using food models as aids in recall. the evaluation of food recall was aimed at determining soy isoflavone intake in the foods consumed by the subjects. anthropometric measurements weight was measured in kg using portable scales with an accuracy of 0.1 kg. height was measured in cm using a portable microtoise with accuracy of 0.1 cm. body mass index (bmi) was determined from calculation of weight in kg divided by height in m2. the bmi values were categorized using the criteria determined by the department of health of the republic of indonesia, namely: underweight (<18 kg/m2), normal weight (18-25 kg/m2), overweight (2527 kg/m2), and obese (>27 kg/m2).(18) laboratory analysis laboratory examinations were initiated with the collection of 10 ml venous blood samples from the subjects after a fast of 10-12 hours before the collection. for the determination of the blood lipid profile the total cholesterol, ldl cholesterol, hdl cholesterol, and tag levels were measured by means of an enzymatic colorimetric method, using the roche cobas c111 analyzer. statistical analysis simple linear regression and multivariate regression was used to evaluate the relationship between variables. the level of significance used was 0.05. ethical clearance all subjects agreed to sign informed consent and the proposal for this study obtained approval from the ethics commission, faculty of medicine, trisakti university, under no. 140/ ker/fk/ix/2009. results a total of 186 postmenopausal women meeting the inclusion and exclusion criteria participated in this study. mean age of the subjects was 53.59 ± 3.54 years. for the 50 herwana, pusparini, graciela daidzein intake and post-menopausal women isoflavone intakes determined from total intake of isoflavone derivatives per day, it was found that the highest intake was that of daidzein. the lipid profile showed that total cholesterol level was slightly above normal, while ldl, hdl and tag levels were within normal limits (table 1). the results of simple linear regression analysis showed that total cholesterol level was significantly associated with genistein level and bmi (β=145.48, p= 0.023; β = 2.03, p=0.000, respectively) and inversely and significantly associated with daidzein level (β=-204.06, p=0.003). ldl cholesterol was significantly associated with bmi and inversely associated with daidzein level (β=1.90, p=0.000; β=-160.81, p=0.014). hdl cholesterol was significantly associated with genistein level and inversely associated with daidzein level and bmi (β=48.80, p=0.03; β=-67.11; p=0.006; β=-0.43, p=0.021, respectively). tag level was significantly associated with bmi and inversely associated with employment (β=4.011, p=0.000; β=21.98, p=0.033) (table 2). the risk factors that were significantly associated with cholesterol level were entered into the multiple linear regression model. the results of the multiple regression analysis of the relationship between lipid profile and several risk factor variables showed that total cholesterol was significantly associated with genistein level and bmi (β=167.83, p=0.008; β= 2.06, p=0.000, respectively) and inversely and significantly associated with employment and daidzein level (β=-11.18, p=0.036; β=-148.45, p=0.006, respectively). ldl cholesterol was significantly associated with bmi (β=1,83, p=0,000). hdl cholesterol hdl was significantly associated with genistein level (β=44.99, p=0,044) and inversely associated with daidzein and bmi (β=-49.741, p=0.008; β=-0.43, p=0,02, respectively). tag level was significantly associated with bmi (β=4.28, p=0.000) and inversely associated with employment (β=-24.41, p=0.018) (table 3). genistein intake was significantly associated with increases in cholesterol and hdl cholesterol levels. daidzein intake was significantly associated with decreased total cholesterol, ldl cholesterol, and hdl cholesterol level. discussion the results of this study showed that higher daidzein intakes were associated with total lower c h ol e s t e ro l , l dl c ho l e s t e r ol , a n d h dl c hole st e ro l le ve l s. g eni st ei n int ake wa s associated with higher total cholesterol and hdl cholesterol levels. the results of this study differ from those of a meta-analytic study by tokede et al.(16) who report that the effects of soy products are of benefit in significantly lowering the total cholesterol, ldl cholesterol, and tag level by 2%, 3%, and 4%, respectively, and increasing the hdl level by 3%, the effects being more pr onounc ed in subj ec ts with hypercholesterolemia. whole soy food is more a b c characteristic age (years)a) 53.59 ± 3.54 educational level b) low 166 (89.20%) middle 20 (10.80%) employmentb) employed 59 (31.70%) unemployed 127 (68.30%) duration of menopause (years)a) 4.40 ± 2.23 bmi (kg/m2)a) 26.82 ± 4.67 blood pressurea) systolic 124.87 ± 20.44 diastolic 78.63 ± 11.93 isoflavone intake (g/day)a) genistein 0.11 ± 0.12 daidzein 0.19 ± 0.14 glycitein 0.04 ± 0.04 lipid profile (mg/dl)a) total cholesterol 207.18 ± 35.62 ldl cholesterol 129.87 ± 33.51 hdl cholesterol 56.91 ± 12.16 triglycerides 117.42 ± 68.49 table 1. distribution of socio-demographic characteristics and other important variables in postmenopausal women (n=186) a)values are mean ± standard deviation; b) number of subjects (%) 51 univ med vol. 38 no.1 characteristic (n=186) β p value total cholesterol (mg/dl) age (years) 1.50 0.057 employment -11.22 0.035* education 11.08 0.171 genistein (g/day) 145.48 0.023* daidzein (g/day) -204.06 0.003* glycitein (g/day) 232.78 0.133 duration of menopause (years) -0.52 0.673 bmi (kg/m2) 2.03 0.000* systolic bp (mmhg) 0.06 0.760 diastolic bp (mmhg) 0.07 0.823 hdl cholesterol (mg/dl) age (years) 0.15 0.580 employment 0.59 0.751 education 3.32 0.249 genistein (g/day) 48.80 0.032* daidzein (g/day) -67.11 0.006* glycitein (g/day) 43.59 0.428 duration of menopause (years) -0.55 0.211 bmi (kg/m2) -0.43 0.021* systolic bp (mmhg) 0.09 0.177 diastolic bp (mmhg) -0.20 0.098 ldl cholesterol age (years) 0.70 0.354 employment -7.17 0.162 education 10.70 0.172 genistein (g/day) 104.02 0.091 daidzein (g/day) -160.81 0.014* glycitein (g/day) 235.84 0.116 duration of menopause (years) 0.05 0.966 bmi (kg/m2) 1.90 0.000* systolic bp (mmhg) -0.07 0.696 diastolic bp (mmhg) 0.17 0.603 triacylglycerol age (years) 1.99 0.193 employment -21.98 0.033* education -10.41 0.507 genistein (g/day) 2.14 0.986 daidzein (g/day) 91.91 0.482 glycitein (g/day) -227.85 0.448 duration of menopause (years) 0.53 0.825 bmi (kg/m2) 4.01 0.000* systolic bp (mmhg) 0.24 0.534 diastolic bp (mmhg) 0.49 0.470 table 2. relationship of various important variables with cholesterol in postmenopausal women p values calculated by simple linear regression; *significant; bp = blood pressure; hdl = high density lipoprotein; ldl = low density lipoprotein; bmi = body mass index; educational level: low = no schooling, elementary school, and junior high school; middle = senior high school beneficial than isoflavone supplementation and the effects of soy protein are stronger in the postprandial than the fasting state. (19) the study of j assi et al.(17) c ompared the effects of supplementation of 30 g/day soy protein powder (containing 60 mg isoflavone) and 60 mg/day soy isoflavone tablets on the lipid profile, with 3 0g/ da y of c a s e i n a s c o nt r ol . t h e supplementation was administered for 12 weeks and was evaluated at 4 and 12 weeks. the results 52 herwana, pusparini, graciela daidzein intake and post-menopausal women of this study showed that in the group on soy protein there was a significant reduction in total cholesterol, tag, and ldl cholesterol levels, accompanied by increased hdl cholesterol, whereas soy isoflavone supplementation was only significant in reducing tag. the results of another study found that supplementation of 100 mg isoflavone for 6 months significantly de creased ldl cholesterol level, but the de crease became non-signif icant after 12 months.(20) the isoflavones contained in soy-derived foods compr ise inact ive glucoside s that subsequently undergo hydrolysis by intestinal bacterial β-glucosidases into the bioactive aglycones genistein, daidzein, and glycitein. the isoflavone intake in the present study, as determined by food recall, showed mean ± sd values for genistein, daidzein, and glycitein of 0.11 ± 0.12 g, 0.19 ± 0.14g, and 0.04 ± 0.05g, respectively. mean isoflavone intake in the present study was found to be higher than mean isoflavone intake in asian populations (japan, china, korea) that vary from 17.7 to 47.2 mg/day, whereas the isoflavone intake in caucasian populations are extr emely low, namely <2mg/day. (1 0) th e indonesian communities consume great amounts of soy foods, particularly those that are processed into tempeh, tofu, soy milk, and kecap (ketchup). ethnic differences may affect the bioavailability and pharmacokinetics of isoflavone. individual capacities in metabolizing the inactive glucosides into the bioactive aglycones genistein, daidzein, and glycitein depend on the hydrolyzing capacities of intestinal bacterial β-glucosidases. daidzein may be metabolized into dihydrodaidzein, and subseque ntly into equol and odesmethyla ngolens in ( o-dma). equol demonstrates a potentially higher effect than daidzein. only the aglycones genistein, daidzein, glycitein, equol, and o-dma can enter the circulation and reach the cells. there are interindividual differences in the capacity to metabolize daidzein into equol and this plays a role in the variability of the inter-individual effects of isoflavone, so that the individuals in question may be distinguished into equol producers and nonequol producers. (11) there are differences in the proportions of equol producers between the asian and caucasian populations. one study also reported that isoflavone intake may increase the individual capacity to become equol producers. (21) there is the possibility that the inconsistent results of the studies on the effects of isoflavones on lipid profile were associated with interindividual pharmacokinetic variability. risk factor b beta p value total cholesterol (mg/dl) employment -11.18 -2.11 0.036* genistein (g/day) 167.83 2.66 0.008* daidzein (g/day) -148.45 -2.80 0.006* bmi (kg/m2) 2.00 3.79 0.000* hdl cholesterol (mg/dl) genistein (g/day) 44.99 2.03 0.044* daidzein (g/day) -49.74 -2.67 0.008* bmi (kg/m2) -0.43 -2.33 0.020* ldl cholesterol (mg/dl) daidzein (g/day) -6.98 -0.03 0.672 bmi (kg/m2) 1.83 0.25 0.000* triacylglycerol (mg/dl) employment -24.41 -0.16 0.018* bmi (kg/m2) 4.28 0.29 0.000* table 3. multiple linear regression analysis of the relationship of various risk factor variables with cholesterol level in postmenopausal women p values calculated by multiple regression; *significant; hdl = high density lipoprotein; ldl = low density lipoprotein; bmi = body mass index 53 meanwhile chilibeck et al. (22) reported that isoflavone supplementation reduced ldl cholesterol level, but showed no effects on the levels of the other lipids. the isoflavone effects on the risk of cardiovascular disease are still controversial. the european food safety au t ho r i t y h a s e va l u a t e d t h e e f f e c t s of isoflavones on the risk of cardiovascular disease, with a focus on the effects on lipid profile.(23) several previous studies have investigated the important clinical effects of isoflavones on the decreases in total cholesterol, ldl cholesterol, and tag levels and on the increase in hdl cholesterol levels. these studies that were r e p o r t e d in t h e l a s t t e n ye a r s f a il e d t o demonstrate that isoflavones exert significant clinical effects, and failed to find the active isoflavone compounds that play a role in lipid metabolism. intake of soy protein as a nutrient ma y i n c r e as e pr ot e i n i nt a ke a nd r e d uc e carbohydrate and fat intakes, so showing a better effect on lipid profile. the direct benefit of soy protein or isoflavones in cardiovascular health is minimally the best.(24) in spite of this, there are still many studies and meta-analyses that have found significant effects of soy protein in reducing ldl cholesterol and tag levels by 4-6% and 5%, respectively, and in increasing hd l c h ol es t e r o l b y 1 % -3 % . al t ho ugh apparently the effects are not large, they are still relevant for the clinical effects in the population, because epidemiological and interventional data suggest that each 1% reduction in ldl cholesterol is associated with a 1-2% reduction in cardiovascular events and that each 2%-3% increase in hdl cholesterol is associated with a 2%-4% reduction in cardiovascular events.(24) the present study has several limitations. firstly, the cross-sectional study design cannot show any causal relationship. secondly, the measurement of isoflavone intake using food recall depends on the subjects’ memory of the types and amounts of soy-containing foods consumed, and in addition the food processing method was not analyzed. further studies of prospective design are necessary to confirm our study results. conclusions high daidzein intake is associated with low total cholesterol, ldl cholesterol, and hdl cholesterol levels. genistein intake is associated with high total cholesterol and hdl cholesterol levels. phytoestrogen intake has no side-effects, so the dietary approach could be considered safe. conflict of interest the investigators did not have conflicts of interest for the results of this study. acknowledgement we are grateful to all postmenopausal women who participated in this study, to the enumerators who assisted in this study and to the faculty of medicine, trisakti university, for the funding of this study. contributors eh contributed to writing the original draft. eh and p contributed to review and editing. ag contributed to data collection and analysis. all authors have read and approved the 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the effect of soy protein on cardiovascular disease risk compared with the fasting lipid profile. lipids 2010;45:112738. doi: 10.1007/s11745-010-3487-z. 20. yenny, pusparini. isoflavone supplementation reduces low-density lipoprotein cholesterol levels in postmenopausal women. univ med 2013;32:197-207. 21. herwana e, setiabudy r, soegondo s, et al. soy isoflavone supplementation increases equolproducing capability in postmenopausal women with osteopenia. univ med 2012;31:120-30. 22. chilibeck pd, vatanparast h, pierson r, et al. effect of training combined with isoflavone supplementation on bone and lipids in postmenopausal women: a randomized clinical trial. j bone min res 2013;28:780-93. doi: 10.1002/ jbmr.1815. 23. efsa panel on dietetic products, nutrition and allergies (nda); scientific opinion on the substantiation of health claims related to soy isoflavones and maintenance of bone mineral density (id 1655) and reduction of vasomotor symptoms associated with menopause. efsa j 2012;10:2847. doi:10.2903/j.efsa.2012.2847. 24. messina m. soy and health update: evaluation on the clinical and epidemiologic literature. nutrients 2016;8:754. 42 pages. doi: 10.3390/ nu8120754. oktavianus 118 *department of obstetrics and gynecology faculty of medicine trisakti university correspondence dr. dr. raditya wratsangka, spog(k) department of obstetrics and gynecology faculty of medicine trisakti university jl. kyai tapa no.260 grogol jakarta 11440 phone: +6221-5672731 ext.2706 email : rwratsangka@ymail.com univ med 2013;32:118-27 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background menopausal symptoms in postmenopausal women tend to decrease healthrelated quality of life (hrqol). the present study’s objective was to determine the effect of daily supplementation of 100 mg soy isoflavones on menopausal symptoms of healthy postmenopausal women. isoflavones are phytoestrogens abundantly found in soy beans, and several studies have demonstrated that isoflavones are the best among the phytoestrogens. methods the study was a community-based double blind randomized controlled trial involving 60 healthy postmenopausal women, aged between 48–60 years, in the mampang prapatan district, south jakarta. participants were block-randomized to receive either 100 mg soy-isoflavone + 500 mg calcium carbonate (intervention group) or 500 mg calcium carbonate only (control group). both supplements were taken daily for 12 weeks, from january to april 2010. menopausal symptoms (measured by menopause rating scale questionnaire) were assessed at baseline and after supplementation. chi-square test was used to examine the effect of soy isoflavone supplementation on menopausal symptoms. results fifty-six (93.3%) of participants completed the study. there were no statistically significant differences (p>0.05) in the prevalence of menopausal symptoms between the isoflavone group and the control group. however, supplementation of soy isoflavones for 12 weeks tended to decrease the prevalence of menopausal symptoms in women with normal body mass index and adequate daily dietary isoflavone intake. conclusion daily supplementation of 100 mg soy isoflavones for 12 weeks tend to decrease the incidence of menopausal symptoms in postmenopausal women. key words : soy isoflavones, menopausal symptoms, postmenopausal soy-isoflavone supplementation tends to reduce menopausal symptoms in postmenopausal women raditya wratsangka* and atut cicih mayasari* 119 suplementasi soy isoflavon cenderung meringankan simtom menopause pada perempuan pascamenopause latar belakang simtom menopause yang berkaitan dengan penurunan kadar estrogen dapat menurunkan kualitas hidup yang berkaitan dengan kesehatan (health-related quality of life) perempuan pascamenopause. penelitian ini bertujuan untuk menilai pengaruh suplementasi harian soy-isoflavon 100 mg terhadap simtom menopause pada perempuan pascamenopause yang sehat. isoflavon merupakan salah satu jenis fitoestrogen, dan beberapa penelitian menunjukkan bahwa isoflavon merupakan jenis fitoestrogen yang terbaik, banyak ditemukan dalam kedelai (soy) yang banyak dikonsumsi oleh masyarakat indonesia dan penduduk asia lainnya. metode rancangan community-based double blind randomized controlled trial yang digunakan melibatkan 60 perempuan pascamenopause berusia antara 48–60 tahun di kecamatan mampang prapatan – jakarta selatan. subyek penelitian secara randomisasi blok dibagi menjadi kelompok yang mendapat suplemen soy-isoflavon 100 mg + kalsium karbonat 500 mg dan yang mendapat kalsium 500 mg karbonat saja selama 12 minggu, yaitu antara januari dan april 2010. penilaian simtom menopause dilakukan dengan menggunakan kuesioner menopause rating scale (mrs) terhadap subyek penelitian sebelum dan sesudah suplementasi. analisis statistik yang digunakan adalah uji chisquare. hasil sebanyak 56 subyek (93,3%) menyelesaikan penelitian ini. tidak ada perbedaan yang bermakna secara statistik (p>0,05) dalam hal kejadian simtom menopause (gangguan psikologik, vasomotor dan urogenital) antara kelompok subyek yang mendapatkan isoflavon dan kelompok kontrol, tapi pemberian suplementasi isoflavon cenderung menurunkan angka kejadian simtom menopause pada perempuan pascamenopause dengan indeks massa tubuh yang normal dan asupan isoflavon dalam makanan harian yang cukup. kesimpulan suplementasi 100 mg soy-isoflavon selama 12 minggu cenderung menurunkan angka kejadian simtom menopause pada perempuan pascamenopause. kata kunci : soy-isoflavon, simtom menopause, pascamenopause abstrak univ med vol. 32 no.2 introduction the decrease in sex hormone levels in the body, in particular estrogens, as occurs in menopausal women, not only changes the menstrual pattern but also impacts on general health. these health changes comprise mainly somatic, urogenital and vasomotor symptoms, such as loss of skin elasticity leading to a wrinkled appearance of the skin, dryness of the v a g i n a ( c a u s i n g d y s p a r e u n i a ) , d y s u r i a , palpitations, migraine, hot flushes [or hot flashes], n o c t u r n a l s w e a t s , i n s o m n i a , a n d s e x u a l symptoms, the whole being known as the menopausal symptoms.(1) the prevalence of menopausal symptoms in european and american women is up to 5075%, whereas in asian women it is only around 120 wratsangka, mayasari isoflavone and menopausal sy mptoms 20-25%. (2) the prevalences of vasomotor symptoms in indonesia among premenopausal, perimenopausal and postmenopausal women are 6%, 30%, and 8%, respectively.(3) hormone therapy (ht), in the form of e s t r o g e n s o n l y o r i n c o m b i n a t i o n w i t h progesterones, has in the last few years still been t h e m o s t p r e s c r i b e d t r e a t m e n t f o r postmenopausal women in industrialized countries.(4) administration of ht forms part of several measures to attain and maintain quality of life in menopausal women. there is a considerable body of evidence regarding the benefi ci al ef f ects of ht on menopausal symptoms, lipid profile, osteoporosis, dementia and colorectal cancer. most women use ht to relieve menopausal symptoms and to prevent chronic disease, such as osteoporosis, coronary heart disease (chd) and alzheimer’s dementia. on the basis of recommendations issued by the north american menopause society (nams), the indications for ht administration are the v a r i o u s m e n o p a u s a l s y m p t o m s , s u c h a s vasomotor and urogenital symptoms.(4) as with other medications, in addition to its many beneficial effects, ht may cause various adverse reactions, from mild (nausea, mastalgia, headaches) to severe (uterine bleeding). studies have associated the use of ht with increased risk of several diseases, such a s c o r o n a r y h e a r t d i s e a s e , v e n o u s t h r o m b o e m b o l i s m ( v t e ) a n d o v a r i a n malignancies.(5) the use of ht in asia is still very limited and ht is given only to menopausal patients with complaints related to troublesome estrogen deficiency, or to the hazard of osteoporosis and fractures.(6) the women’s health initiative randomized trial reported on the increased risk of breast cancer, coronary heart disease, stroke and venous thromboembolism (vte) in users of ht. this has caused some women to refuse using ht and to look for alternative treatments capable of fulfilling their need for alleviation of menopausal symptoms.(7) phytoestrogens are natural substances from plants of differing chemical structure than estrogens, but having very similar actions, so that they are called estrogen-like substances and are also known as herbal estrogens. these compounds can bind to estrogen receptors (er-á and er-â) and possess estrogenic activity, but are also weakly antie s t r o g e n i c . (8) t h e v a r i o u s t y p e s o f phytoestrogens occurring in plants have two important effects on humans, i.e. estrogenic and anti-estrogenic, depending on endogenous estrogen and estrogen receptor concentrations, so that these phytoestrogens are also known as selective estrogen receptor modulators (serm), having both agonistic and antagonistic effects.(9) isoflavones are phytoestrogens abundantly found in soy beans, and several studies have demonstrated that isoflavones are the best among the phytoestrogens. soy beans are c o n s u m e d i n c o n s i d e r a b l e q u a n t i t i e s i n i n d o n e s i a n c o m m u n i t i e s a n d o t h e r a s i a populations. the prevalence of chronic and degenerative diseases were significantly lower in asian populations with a high consumption of foods rich in soy beans, in comparison with american or european populations.(10-12) the results of a systematic review in the cochrane collaboration did not yield conclusive f a c t s o r e v i d e n c e o n t h e b e n e f i t s o f phytoestrogens on vasomotor symptoms. in spite of this, several of the most recent studies reported that phytoestrogens (particularly soy isoflavone extract) can significantly relieve hot flushes, improve plasma lipid profile, and inhibit the development of atherosclerosis, thus d e c r e a s i n g t h e r i s k o f c a r d i o v a s u l a r disease.(13,14) the aim of the present study was t o e v a l u a t e t h e e f f e c t o f s o y i s o f l a v o n e supplementation on menopausal symptoms in healthy postmenopausal women in indonesia. methods design of the study this study was a community-based double blind randomized controlled trial conducted in south jakarta from january to april 2010. 121 univ med vol. 32 no.2 study subjects the inclusion and exclusion criteria for recruitment of the postmenopausal women aged 48-60 years for this study, have been reported previously.(15) sample size this study was part of a larger investigation examining the effects of soy isoflavone supplementation on specific immune responses i n p o s t m e n o p a u s a l w o m e n . s a m p l e s i z e determination and sampling techniques have been reported previously.(15) intervention subjects were randomly assigned to two groups, the isoflavone group receiving 250 mg of 40% soybean isoflavones (equivalent to 100 mg soy isoflavone) + 500 mg calcium carbonate, and the control group receiving 500 mg calcium c a r b o n a t e o n l y. t h e i n t e r v e n t i o n w a s administered continuously for 12 weeks at a dosage of 1 x 1 tablet daily. the supplement tablets were prepared by pt ikapharmindo putramas pharmaceutical company on special license of the board for supervision of drugs and foods of the republic of indonesia (badan pengawasan obat dan makanan republik indonesia, bpom-ri). assessment of menopausal symptoms the subjects were asked to fill in the menopause rating scale (mrs) questionnaire to assess their menopausal symptoms. the mrs questionnaire used in this study is an official indonesian translation following the international methodological recommendations for the linguistic & cultural adaptation of hrqol measures, and has been validated in indonesia in a trial involving 1000 respondents. the menopausal symptoms evaluated by the mrs i n s t r u m e n t c o m p r i s e 3 d i m e n s i o n s , v i z . psychological (score > 2), somato-vegetative/ vasomotor (score > 3) and sexual/urogenital (score > 1). furthermore, using the established criteria, the subjects are categorized into a group without or a few symptoms (score 0–4) and one with symptoms (score > 5). the results of a reliability test on mrs yielded a total score á=0.84 (internal consistency coefficient) and a test-retest correlation coefficient of 0.8.(16,17) assessment of dietary intake recording and calculation of dietary intake (carbohydrate, fat, protein and isoflavones) was done by a trained nutritional enumerator using the 24-hour food recall method. the dietary intake was also assessed using the food record method on 3 days of the week, namely 2 work days and 1 holiday (saturday or sunday). assessment of the quality and quantity of dietary i s o f l a v o n e i n t a k e w a s b a s e d o n t h e completeness of soy isoflavone-containing foods and the weekly frequency of consumption using t h e s e m i q u a n t i t a t i v e f o o d f r e q u e n c y questionnaire (sq-ffq), which comprises a list of soy isoflavone-containing foods. the subjects were divided into quintiles, viz. i) q 1 low isoflavone intake in foods, and ii) q 2 – q 5 adequate isoflavone intake in foods. anthropometric measurements to assess nutritional status, the body mass index (bmi) was calculated using the formula bmi = weight in kilograms divided by height in square meters. height was measured to the nearest 0.1 cm using a portable microtoise, while weight was measured to the nearest 0.1 kg using portable sage scales. the criteria for bmi were according to the guidelines of the world health organization (who), namely normal (18.5-25.0 kg/m2), mildly overweight (25.1-27.0 kg/m2) and severely overweight (>27 kg/m2).(18) ethical clearance this study was approved by the research ethics committee, faculty of public health, university of indonesia. data analysis data processing and analysis for this study was performed using the software statistical 122 package for social sciences (spss) version 17. data were tested for normality of distribution using the kolmogorov-smirnov test of normality. a comparability test was performed between subject characteristics in the isoflavone and control group. the chi-square test or fisher exact test was performed to determine the presence or absence of statistically significant differences (significance level p<0.05) in the number of subjects with menopausal symptoms b e f o r e a n d a f t e r s u p p l e m e n t a t i o n i n t h e isoflavone group and the control group. results mrs scores at baseline table 1 below lists the total mrs score, number of subjects with menopausal symptoms a n d t h e t h r e e s y m p t o m d i m e n s i o n s (psychological, somatic/vasomotor and sexual/ urogenital). at baseline mean total mrs score in the isoflavone group was 7.4 ± 6.6 and in the c o n t r o l g r o u p 7 . 4 ± 6 . 3 , t h u s n o t b e i n g significantly different (p=0.996). there were also no statistically significant differences in the three symptom dimensions between both treatment groups. e f f e c t o f i s o f l a v o n e s o n m e n o p a u s a l symptoms in table 2 below, it is apparent that after daily supplementation of 100 mg soy isoflavones for 12 weeks, in spite of the greater number of subjects with menopausal symptoms in the isoflavone group, there were no statistically significant differences between the isoflavone group and the control group. there were also no statistically significant differences in the n u m b e r o f s u b j e c t s w i t h p s y c h o l o g i c a l , vasomotor, and urogenital symptoms between the isoflavone group and the control group, although there were more subjects in the isoflavone group with symptoms in the three dimensions. this indicates that supplementation of 100 mg soy isoflavones for 12 weeks was unable to relieve the menopausal symptoms suffered by the postmenopausal women. table 3 below shows that in the overall analysis of subjects, the number of subjects with menopausal symptoms after supplementation decreased both in the isoflavone group and control group, but the decrease was lower in the isoflavone group in comparison with the control group (p=0.103). in subgroup analysis, the overall number of subjects with menopausal table 1. distribution of menopausal symptoms at baseline by treatment group mean (standard deviation), *independent t-test, values are expressed as percentage of subjects, **chi-square test mrs = menopause rating scale wratsangka, mayasari isoflavone and menopausal sy mptoms 123 univ med vol. 32 no.2 symptoms also decreased after supplementation, both in the isoflavone group and the control group. regarding the subjects with menopausal symptoms who also had adequate dietary isoflavone intake, the number decreased slightly more in the isoflavone group (31.8%) than in the control group (27.3%) (p=0.322). i n t h e s u b g r o u p w i t h p s y c h o l o g i c a l symptoms, for all subjects after supplementation there was a decrease in their numbers in the isoflavone group as well as the control group, without a statistically significant difference between the two groups (p=0.440). there was in general also a decrease in psychological symptoms after supplementation in the isoflavone and control groups, except in subjects with inadequate dietary isoflavone intake (40%) (p=0.222). similarly, in all subjects with somatic or v a s o m o t o r s y m p t o m s , t h e r e w a s a f t e r supplementation no statistically significant decrease in their numbers, both in the isoflavone and the control group (p=0.411). in the subgroup analysis, the isoflavone group had no significantly larger decreases in the number of subjects with vasomotor symptoms and above normal baseline bmi or inadequate dietary isoflavone intake in comparison with the control group. however, subjects with normal baseline bmi and those with adequate dietary isoflavone intake, had a larger decrease of respectively 45.4% and 45.5% in number of subjects with vasomotor symptoms in the isoflavone group than in the control group, although there was no statistically significant difference between the two groups (p=1.000). this demonstrates a tendency for isoflavone supplementation at a dosage of 100 mg for 12 weeks to decrease vasomotor symptoms in postmenopausal women of both of above subgroups. it cannot be ascertained why precisely in subjects with inadequate isoflavone intake, the decrease in the number of subjects with menopausal symptoms was greater in the control group (57.1%) than in the isoflavone group (p>0.05). in table 3 below it may also be seen that in the analysis of all subjects with urogenital symptoms, the differences in decreased number of subjects in the isoflavone and control groups were statistically not significant (p=0.629). in the subgroup analysis, there was an 18.2% decrease in subjects with urogenital symptoms after receiving isoflavone supplements, this being t w i c e t h e d e c r e a s e i n t h e c o n t r o l g r o u p (p=0.531). discussion this study shows that administration of soy isoflavone supplementation at a dosage of 100 mg per day for 12 weeks to postmenopausal women tended to decrease the incidence of menopausal symptoms, in comparison with the control group, but the results were statistically not significant. in the subgroup analysis there was a tendency for the administration of supplementation to decrease the incidence of menopausal symptoms in several subgroups. table 2. menopausal symptoms and the three symptom dimensions in postmenopausal women after supplementation, by treatment group values are expressed as percentage of subjects *chi-square test 124 wratsangka, mayasari isoflavone and menopausal sy mptoms t ab le 3 . p er ce n ta g es o f re la ti v e d if fe re n ce s (ä ) in m en o p au sa l sy m p to m s, a n d p sy ch o lo g ic al , v as o m o to r, a n d u ro g en it al s y m p to m d im en si o n s, b ef o re a n d a ft er s u p p le m en ta ti o n , b y t re at m en t g ro u p i n a ll s u b je ct s an d s u b g ro u p s o f p o st m en o p au sa l w o m en * r es ul ts o f si gn if ic an ce t es ts o n th e nu m be r of s ub je ct s w it h m en op au sa l sy m pt om s an d w it h ps yc ho lo gi ca l, v as om ot or , an d ur og en it al s ym pt om s, a ft er s up pl em en ta ti on b et w ee n th e is of la vo ne g ro up a nd t he c on tr ol g ro up , p er fo rm ed w it h ch i sq ua re t es t or f is he r’ s ex ac t te st . v al ue s ar e ex pr es se d as p er ce nt ag es o f re la ti ve d if fe re nc es i n th e nu m be r of s ub je ct s w it h m en op au sa l sy m pt om s an d w it h ps yc ho lo gi ca l, v as om ot or , a nd u ro ge ni ta l sy m pt om s (+ ) b ef or e an d af te r s up pl em en ta ti on , co m pu te d ac co rd in g to t he f or m ul a: p er ce nt ag e of s ub je ct s w it h sy m pt om s (+ ) af te r su pp le m en ta ti on m in us [ pe rc en ta ge o f su bj ec ts w it h sy m pt om s (+ ) be fo re su p p le m en ta ti o n ] b m i : bo dy m as s in de x 125 univ med vol. 32 no.2 these subgroups comprised the subjects in the e a r l y p o s t m e n o p a u s a l g r o u p ( u r o g e n i t a l symptoms), in those subjects receiving adequate daily dietary isoflavone intakes (psychological, urogenital, and vasomotor symptoms) and in those having a normal body mass index (bmi) (vasomotor symptoms), although there were no statistically significant differences between the isoflavone group and the control group. essentially similar results were obtained in the soy estrogen alternative study, which involved women aged 45–55 years experiencing one vasomotor symptom in 24 hours. this study found that the decrease in vasomotor symptoms was not due to isoflavones.(19) in contrast, differing results were obtained in a placebo-controlled trial involving 80 postmenopausal women, where the menopausal symptoms were assessed by the kupperman index. significant improvement in menopausal s y m p t o m s w a s f o u n d w i t h a d a i l y supplementation of 100 mg isoflavones (70% genistein, 19% daidzein, and 11% glycitein) for 4 months, in comparison with a placebo. however, the daily frequency of hot flashes decreased only from 11.3 ± 0.3 to 8.2 ± 0.5 per day in the isoflavone group, while the reduction in hot flash frequency in the control group was from 10.4 ± 0.4 to 9.9 ± 0.4 per day.(20) in another study it was found that soy isoflavones w e r e a b l e t o a f f e c t h o t f l a s h e s o n l y i n combination with polyunsaturated fatty acids.(21) statistically significant results were found in a meta-analysis of 17 randomized clinical trials on isoflavone supplementation and hot flash frequency.(22) this was in contrast with an earlier meta-analysis of 17 randomized isoflavone trials w h i c h s h o w e d v a r i a b l e r e s u l t s f o r s o y isoflavones in their effects on hot flashes.(23) a third meta-analysis of 11 studies found that b e n e f i c i a l e f f e c t s w e r e o n l y f o u n d w i t h isoflavone supplements having a markedly higher genistein content, and not with those rich in daidzein.(24) in view of the currently inconclusive evidence regarding the effect of soy isoflavone supplementation on menopausal symptoms, it is worth while to speculate on the thus far unexplained mechanism of action of isoflavones in the reduction of vasomotor symptoms. one of the explanations may be that isoflavones act on estrogen receptors, which are able to bind compounds such as natural estrogens and other chemically similar isoflavones.(9,14) a cochrane systematic review and metaanalysis performed by jacobs et al.(25) and bolanos et al.(26) on a number of studies aiming to evaluate the effectiveness of isoflavones on v a s o m o t o r s y m p t o m s u s i n g v a r i o u s m e t h o d o l o g i e s , d i d n o t c o m e t o a c l e a r conclusion, although there were indications of beneficial effects of soy isoflavones on the frequency and severity of hot flashes. the many soy isoflavone studies are heterogenous with regard to intervention and outcome assessment. many questions arise as to the optimal dosage and duration of supplementation required to achieve the intended clinical response. some experts call into question the duration of soy isoflavone administration used in a number of studies, which is considered to be too short for yielding a clinical response. similarly to estrogens, isoflavones also undergo several biochemical processes in the body. the activity of isoflavones at the cellular level depends on target tissue, tissue receptor status, and endogenous estrogen level. in premenopausal women with high circulatory endogenous estrogen levels, isoflavones may act as estrogen antagonists. on the other hand, in postmenopausal women with lower endogenous estrogen levels, isoflavones may exert the opposite effect, acting as estrogen agonists. in the present study, no assessment was done on endogenous estrogen and isoflavone levels after supplementation, thus it was not possible to determine their relationship with menopausal symptoms. the high genistein, daidzein and 126 g l y c i t e i n c o n t e n t i n t h e s o y i s o f l a v o n e supplementation may presumably modify the effects. genistein is considered to be the most p o t e n t a m o n g t h e t h r e e s o y i s o f l a v o n e s (genistein, daidzein, and equol). the study conducted by ferrari concludes that in ordinary living conditions, a high dosage of isoflavones (60 mg genistein in 80 mg isoflavones) given for 12 weeks, can be used to treat hot flashes in postmenopausal women, because it gives better effects in comparison with placebo at a high level of safety.(27) one limitation of the present study was that the data collected using the mrs questionnaire as a diagnostic instrument for the presence of menopausal symptoms, were self-reported on the basis of presence or absence and severity of the subjects’ menopausal symptoms, thus tending to induce bias. other studies used differing scoring systems, i.e. the kupperman vasomotor scale and the simple severity scale. these instruments are more detailed in assessing f r e q u e n c y a n d s e v e r i t y o f m e n o p a u s a l symptoms. conclusion supplementation of 100 mg soy isoflavones for 12 weeks in postmenopausal women tends to reduce menopausal symptoms. further studies are indicated to examine any corelations of amount (dosage) and route of administration (consumption) of soy isoflavones with their effects on menopausal symptoms. acknowledgements we thank all study subjects, the staff at the mampang prapatan community district health center and all village health centers (puskesmas kelurahan), and all parties who assisted in the conduct and completion of this study, especially the faculty of medicine of trisakti university, for providing funds and research facilities. references 1. armitage gd, suter e, verhoef mj, bockmuechi c, bobey m. women’s needs for cam information to manage menopausal symptoms. climacteric 2007;10:215-24. 2. chen y, lin sq, wei y, gao hl, wu zl. menopause-specific quality of life satisfaction in comunity-dwelling menopausal women in china. gynecol endocrinol 2007;23:166-72. 3. freeman ew, sherif k. prevalence of hot flushes and night sweats around the world: a systematic review. climacteric 2007;10:197-214. 4. north american menopause society (nams). estrogen and progestogen use in postmenopausal women: 2010 position statement of the north american society. menopause 2010; 17:242-55. 5. shapiro s. recent epidemiological evidence relevant to the clinical management of the menopause. climacteric 2007;10:2-15. 6. gold eb, block g, crawford s, lachance l, fitzgerald g, miracle h, et al. lifestyle and demographic factors in relation to vasomotor symptoms: baseline results form the study of women’s health across the nation. am j epidemiol 2004;159:1189-99. 7. anderson gl, judd hw, kaunitz am, barad dh, beresford saa, mary pettinger m, et al. effects of estrogen plus progestin on gynecologic cancers and associated diagnostic procedures. the women’s health initiative randomized trial. jama 2003;290:1739-48 8. ryan-borchers ta, park js, chew bp, mcguire mk, fourmier lr, beerman ka. soy isoflavones modulate immune function in healthy postmenopausal women. am j clin nutr 2006;83: 1118-25. 9. rosett jw. menopause, micronutrients, and hormone therapy. am j clin nutr 2005;8:1223-31. 10. nagata c, takatsuka n, shimizu h. soy and fish oil intake and mortality n a japanese community. am j epidemiol 2002;156:824–31. 11. van der schouw yt, kreijkamp-kaspers s, peeters ph, keinan-boker l, rimm eb, grobbee de. prospective study on usual dietary phytoestrogen intake and cardiovascular disease risk in western women. circulation 2005;111:465– 71. 12. hogervorst e, sadjimim t, yesufu a, kreager p, rahardjo tb. high tofu intake is associated with worse memory in elderly indonesian men and women. dement geriatr cogn disord 2008;26:50– 7. doi: 10.1159/000141484. wratsangka, mayasari isoflavone and menopausal sy mptoms 127 13. lethaby a, marjribanks j, kronenberg f. phytoestrogens for vasomotor menopausal symptoms (cochrane review). chichester, united kingdom: john willey & sons; 2007. 14. nedrow a, miller j, walker m, nygren p, huffman lh, nelson hd. complementary and alternative therapies for the management of menopauserelated symptoms. arch intern med 2006;166: 1453-65. 15. wratsangka, r. soy-isoflavone supplementation tends to improve specific immune response in postmenopausal women. univ med 2011;30:16272. 16. heinemann laj, potthoff p, schneider hpg. international version of the menopause rating scale (mrs). health qual life outcomes 2003;1:28 17. heinemann laj, dominh t, strelow f. the menopause rating scale (mrs) as outcome measure for hormone treatment? a validation study. health qual life outcomes 2004;2:67. 18. world health organization. bmi classification; 2006. available at: http://apps.who.int./bmi/ index.jsp?intro page=intro_3.html. accessed november 14, 2011. 19. vitolins mz, case ld, morgan tm, miller ma, burke gl. soy use and vasomotor symptoms: soy estrogen alternative follow-up study. intern j womens health 2010:2 381–6. 20. han kk, soares jm jr, haidar ma, de lima gr, baracat ec. benefits of soy isoflavone therapeutic regimen on menopausal symptoms. obstet gynecol 2002; 99:389–94. 21. campagnoli c, abba c, ambroggio s, peris c, perona m, sanseverino p. polyunsaturated fatty acids (pufas) might reduce hot flushes: an indication from two controlled trials on soy isoflavones alone and with a pufa supplement. maturitas 2005;51:127–34. 22. howes lg, howes jb, knight dc. isoflavone therapy for menopausal flushes: a systematic review and meta-analysis. maturitas 2006;55:203– 11. 23. nelson hd, vesco kk, haney e, fu r, nedrow a, miller j, et al. nonhormonal therapies for menopausal hot flashes: systematic review and meta-analysis. jama 2006;295:2057–71. 24. williamson-hughes ps, flickinger bd, messina mj, empie mw. isoflavone supplements containing predominantly genistein reduce hot flash symptoms: a critical review of published studies. menopause 2006;13:831–9. 25. jacobs a, wegewitz u, sommerfeld c, grossklaus r, lampen a. efficacy of isoflavones in relieving vasomotor menopausal symptoms – a systematic review. mol nutr food res 2009:53:1084-97. 26. bolanos r, des castillo a, francia j. soyisoflavones versus placebo in the treatment of climacteric vasomotor symptoms: systematic review and meta-analysis. menopause 2010;17: 660-6. 27. ferrari a. soy extract phytoestrogens with high dose of isoflavones for menopausal symptoms. j obstet gynaecol 2009;35:1083-90. univ med vol. 32 no.2 c:\users\jurnal fkusakti\docume 139 *department of haematology and blood transfusion, faculty of clinical sciences, edo university, iyamho, edo state, nigeria **department of haematology and blood transfusion, faculty of clinical sciences, edo university, iyamho, edo state, nigeria correspondence: marcellinus uchechukwu nwagu faculty of clinical sciences, edo university, iyamho, edo state, nigeria email: unwagu@yahoo.com phone: +2348033851263 orcid id: https://orcid.org/00000002-2942-4849 date of first submission, october 22, 2 01 8 date of final revised submission, apil 24, 2019 date of acceptance, april 29, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina advanced metastatic breast carcinoma in sickle cell disease marcellinus uchechukwu nwagu*, ologo thompson**, and akinola oyekemi** background breast cancer is the leading cancer in women leading to over 400,000 deaths per year worldwide. it begins in the breast tissue and can metastasize to other organs if early diagnosis and treatment is not instituted. women with sickle cell disease are usually spared from breast cancer and other solid tumours due to the tumoricidal effect of sickled erythrocytes. breast cancers are rare among these group of patients. despite its rare occurrence, this paper was to emphasize the need for breast cancer screening among female sickle cell disease patients who have positive family history of breast cancer. case description oo was a 30-year old woman with sickle cell disease who presented to the hospital one and half years ago with a seven months history of right breast swelling and pains. she had lost her mother to breast cancer about 15 years ago. mammography and histology of breast biopsy confirmed diagnosis of invasive ductal carcinoma of the right breast. financial constraint was a major challenge in managing this patient as she was unable to buy her chemotherapy. she developed features suggestive of metastasis such as seizures and hepatomegaly. she was stabilized and discharged home but we lost her to follow up. she died at home. conclusion breast cancer is rare among females with sickle cell disease; any of them with a family history should be routinely screened for early diagnosis and treatment. keywords: breast carcinoma, sickle cell disease, woman case report pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.139-143 may-august, 2019 vol.38no.2 cite this article as: nwagu mu, thompson o, oyekemi a. advanced metastatic breast carcinoma in sickle cell disease. univ med 2019;38:139-43. doi: 10.18051/univmed.2019.v38.139-143 140 nwagu, thompson, oyekemi breast carcinoma in sickle cell disease introduction sickle cell disease (scd) refers to a spectrum of inherited haemoglobin disorders a s s oc i a t e d wi t h r e d c e l l si c kli ng i n t h e deoxygenated state or in low oxygen tension. the presence of sickle haemoglobin (hbs) leads to the formation of sickle-shaped erythrocytes. th e pre valen ce is highes t i n af ric a a nd especially nigeria. other affected regions include the middle east, central india and countries bordering the mediterranean sea, especially italy and greece. the complications of scd are broad and chronic in nature with affectation of virtually every body organ/system. widely documented complications include nephropathy, retinopathy, chronic leg ulcer, heart failure, priapism, stroke,(1) to mention but a few. a recent study in california showed an increased risk of leukemia among scd patients but reduced risk of solid tumours.(2) breast carcinoma is a cancerous growth that begins in the breast tissues. it is the most common cancer in women with an annual incidence of about 1 million (3) and more than 500,000 women died in 2011 due to breast cancer.(4) as of january 2019, there are more than 3.1 million women with a history of breast cancer in the united states.(5) in nigeria, about 15% of cancer-related deaths is due to breast cancer, making it the leading cause of cancer-related deaths among nigerian women.(6,7) risk factors associated with breast cancer include female gender, increasing age, family history (especially mothers and siblings), early menarche or late menopause, overweight, prolonged oestrogen exposure and use of oral contraceptive pills, not having children or having children after 35years.(6) breast cancer, even though very common in women, is not usually seen in women with sickle cell disease because of the reason earlier mentioned. this case is reported to draw attention of medical practitioners and healthcare providers to the need for vigilance and caution in respect of breast cancer among sickle cell disease patients. for every female scd patient, attention should be paid towards detailed family history, as a positive family history is a strong associated factor for breast cancer. this would create room for routine screening, early diagnosis and timely treatment. case report ms oo was a 30-year old woman with scd patient who presented to the surgical outpatient clinic of delta state university teaching hospital (delsuth) on 15th of may 2017 with complaints of right breast swelling, right breast pain, and right axillary swelling of 7 months duration. her menarche was at the age o f 1 3 ye a r s a n d sh e w a s no t on o r a l contraceptive pills. her mother died 15 years ago to breast cancer. physical examination revealed a young woman who was pale and jaundiced with right axillary lymphadenopathy. there was a nontender mass on the right breast measuring 3cm x 5cm that was associated with skin dimpling and nipple retraction but showed no nipple discharge. mammography showed features of right breast malignancy (figure 1). histology (730 x 550 magnification) showed a stained haematoxylin and eosin section of breast tissue (figure 2). it consists of invading sheets of tumor cells having moderate nuclear pleomorphism in a desmoplastic stroma. in a focus are malignant neoplastic cells penetrating through the duct wall. within its supporting stroma are few dysplastic cells. the patient was counselled on various treatment modalities. due t o l a c k o f f u n ds s he c ou l d not p r o c u r e chemotherapeutic agents and was therefore discharged against medical advice. she was lost to follow up until two months later when she was rushed back to the hospital with severe bone pains /vaso-occlusive crisis. she was markedly pale, jaundiced with a hepatomegaly of 9 cm below the right costal margin. she had one episode of seizures. abdominal ultrasound revealed hepatomegaly with hepatic metastasis 141 from the breast primary. electrolytes showed h yp on a t r e mi a ( so di u m 12 5 mmo l/ l ) a n d hypokalemia (potassium 2.8mmol/l). she was stabilized and discharged from hospital. patient was lost again to follow up. she died at home! discussion sickle cell disease is associated with many systemic complications which ultimately lead to increased morbidity and mortality with reduced life expectancy. no work has been done to determine the incidence of cancer in scd patients compared to the general nigerian population. however, a study in california, usa, showed that scd patients had a 72% increased risk of haematologic malignancies and a 38% reduced risk of solid tumours, with lower risk of breast cancer and male genital cancers (prostate, testis, penis, and other male genital organs).(2) the characteristic pathologic features of scd inclu de chro nic in fla mma tio n, i ncr eas ed univ med vol. 38 no.2 figure 1. mammography of right breast. showing abnormal breast tissue. note the (arrowed) area of white and high-density tissue figure 2. histology: invasive ductal carcinoma showing breast tissue with abnormal malignant neoplastic cells penetrating through the duct wall into the stroma 142 nwagu, thompson, oyekemi breast carcinoma in sickle cell disease susceptibility to infections, iron overload from multiple transfusions, erythroid hyperplasia and bone marrow cellular turnover. studies have shown that these identified factors play a multidimensional role in cancer development.(8-12) these common complications may explain the increased risk of leukemia. however, the 38% reduced risk of solid tumours can be explained from the sickling of the red blood cells.(13) te r ma n e t a l . ( 1 3 ) a p p li e d s i c kl e -sh a p e d erythrocytes to target oxygen-deprived breast cancer cells in infected mice. they found that sickled erythrocytes occluded the blood vessels surrounding the tumour. sickled erythrocytes may be acting as anti-angiogenic agents and thus inhibiting solid tumour formation. this patient had seizures which is not uncommon in scd patients. the seizure in our patient was aborted using diazepam injections. the cause of seizure in this patient could be due to electrolyte changes especially hyponatremia which was seen in this patient. it could also be due to metastasis of cancer cells to the brain from the breast. about 15-30% of patients with metastatic breast cancer will develop brain metastases.(14) electrolytes showed hyponatremia and hypokalemia which were corrected with full-strength darrow’s solution and normal saline. the patient was also transfused with 2 units of packed red blood cells. limitations in managing this patient revolved around financial constraints. scd, on its own, has been established to cause an enormous financial burden on parents and households.(15) t he pat i e n t w a s un a b l e t o pr oc ur e chemotherapeutic agents leading to worsening of the disease and eventual metastasis. with appropriate chemotherapy, breast cancer in sickle cell disease could be well managed with minimal side effects to the patient.(16) again, lack of funds was a hindrance to carrying out a brain computed tomography (ct) or magnetic resonance imaging (mri) to confirm brain metastasis. other investigations we would have loved to do such as immunohistochemistry was also thwarted by financial constraints. lack of funds is a humanitarian issue of public health significance that needs to be addressed by government and relevant authorities. the national health insurance scheme (nhis) would be of tremendous assistance in this regard; unfortunately more than 70% of nigerians are yet to register with the scheme.(17) t he na ti o na l he a l t h in su r a n c e scheme (nhis) was established under act 35 of 1999 by the federal government of nigeria to improve the health of all nigerians a t an affordable cost. all efforts should be made by necessary government agencies to increase the awareness of the populace and encourage them to enrol into the health insurance scheme. agencies such as national orientation agency, established in 2005, can do a lot in this regard. there was a family history of breast cancer in the index case making the patient to be at high risk of breast cancer.(18) routine screening methods such as self breast examination, mammography and breast sonography could have assisted in early diagnosis and prevention of late disease. conclusion solid tumours such as breast cancer is not common in female patients with scd when compared with the general population. routine breast cancer screening methods are necessary and should be recommended to those patients with high risk of breast cancer such as positive family history. conflict of interest there is no conflict of interest. acknowledgement we t h a n k t he t e c hni c a l s t a ff o f t he departme nts of anatomic pathology and radiology for their assistance in providing the histology slides and mammographs, respectively. a b c 143 contributors mun contributed to writing the manuscript a n d r e t r i evi ng t he hi s t ol o gy s l i de s a n d mammographs from the concerned departments. ot retrieved the case file from the records department and wrote the summary of the case. ao made a substantial modification to the manuscript and added some references. all authors have read and approved the final manuscript. references 1. nwagu mu. sickle cell disease: review of complications, current and emerging trends in management. ebonyi med j 2011;10:84-92. 2. brunson a, keegan thm, bang h, et al. increased risk of leukemia among sickle cell disease patients in california. blood 2017;130:1597-9 . doi: 10.1182/ blood-2017-05-783233. 3. ghoncheh m, pournamdar z, salehiniya h. incidence and mortality and epidemiology of breast cancer in the world. asian pac j cancer prev 2016; 17:43-6. 4. world health organization. global health estimates. geneva: world health organization; 2013. 5. breastcancer.org. u.s breast cancer statistics. ardmore, pa. us; breastcancer.org;2019. 6. osaro e. breast cancer in nigeria: diagnosis, management and challenges. bloomington, in, usa: author house;2016. 7. okobia mn, bunker ch, okonofua fe, et al. knowledge, attitude and practice of nigerian women towards breast cancer: a cross-sectional study. world j surg oncol 2006;4:11–5. 8. kundu jk, surh yj. emerging avenues linking inflammation and cancer. free radic biol med 2012;52:2013-37. doi: 10.1016/j.freeradbiomed. 2012.02.035 9. crusz sm, balkwill fr. inflammation and cancer: advances and new agents. nat rev clin oncol 2015;12: 584-96. doi: 10.1038/nrclinonc.2015.105. 10. aggarwal bb, shishodia s, sandur sk, et al. inflammation and cancer: how hot is the link? biochem pharmacol 2006;72:1605-21. 11. chung ws, lin cl, lin cl, et al. thalassaemia and risk of cancer: a population-based cohort study. j epidemiol community health 2015;69:1066-70. doi : 10.1136/jech-2014-205075. 12. zacharski lr, chow bk, howes ps, et al. decreased cancer risk after iron reduction in patients with peripheral arterial disease: results from a randomized trial. j natl cancer inst 2008;100:996-1002. 13. terman ds, viglianti bl, zennadi r, et al. sickle erythrocytes target cytotoxics to hypoxic tumor microvessels and potentiate a tumoricidal response. plos one 2013;8:e52543. doi: 10.1371/ journal.pone.0052543. 14. tabouret e, chinot o, metalus p, et al. recent trends in the epidemiology of brain metastasis: an overview. anticancer res 2012;32:4655-62. 15. olatunwa o, ogundare eo, fadare j. the financial burden of sickle cell disease on households in ekiti, southwest nigeria. clinicoecon outcomes res 2015;7:545-53. doi: 10.2147/ceor.s86599. 16. al zaman as. breast cancer in patients with sickle cell disease can be treated safely with weekly paclitaxel. saudi med j 2013;34:199-201. 17. brown bj, okereke jo, lagunju ia, et al. burden of health-care on carers of children with sickle cell disease in nigeria. health soc care community 2010;18:289-95. doi: 10.1111/j.1365-2524.2009. 00903.x. 18. boyd nf, martin lj, yaffe mj, et al. mammographic density and breast cancer risk: current understanding and future prospects. breast cancer res 2011;13:223. doi: 10.1186/ bcr2942. univ med vol. 38 no.2 c:\users\jurnal fkusakti\docume 114 *department of neurology, faculty of medicine, trisakti university, jakarta **department of gynecology and obstetrics, faculty of medicine, trisakti university, jakarta ***department of physiology, faculty of medicine, trisakti university, jakarta correspondence: dr yudhisman imran, sp.s. department of neurology, faculty of medicine, trisakti university jln kyai tapa no 260, grogol –jakarta barat 11440 email: yudhisman.imran@trisakti.ac.id date of first submission, january 21, 2019 date of final revised submission, july 17, 2019 date of acceptance, july 19, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina low 25-hydroxyvitamin d level increases carotid intima-media thickness in elderly women yudhisman imran*, andini aswar*, irmiya rachmiyani**, and donna adriani*** background cerebrovascular disease occurs due to atherosclerosis of the carotid artery triggered by endothelial dysfunction, which can be assessed by measuring the carotid intima-media thickness (cimt). there are several risk factors contributing to endothelial dysfunction, such as 25-hydroxyvitamin d [25(oh)d] deficiency. a number of research studies have shown variable results on the relationship between 25(oh)d deficiency and endothelial dysfunction. the objective of the present study was to determine the relationship between 25(oh)d and cimt in elderly women. methods a cross-sectional study was conducted involving 45 elderly women aged 60 years. the inclusion criteria were: able to communicate well and able to walk actively without any help. the exclusion criteria were: not having diabetes mellitus, stroke, heart disease, kidney disease, not consuming vitamin d or medicines containing corticosteroids, and not using hormone replacement therapy. laboratory examination was conducted for fasting blood sugar, lipid profile, and 25(oh)d. cimt was measured using carotid doppler ultrasonography at the left carotid artery. simple linear regression analysis was used to analyze the data. results mean 25(oh)d level was 11.753 ± 4.027 ng/ml, and mean cimt 0.61 ± 0.10 mm. simple regression analysis showed a significant relationship between 25(oh)d level and cimt (=-0.001; 95% c.i.=-0.020-0.003; p=0.009). conclusion our data suggest an inverse association between 25(oh)d level and cimt in elderly women. this study supports the protective role of vitamin d against subclinical atherosclerosis in elderly women. keywords: endothelial dysfunction, 25(oh) vitamin d, carotid intima-media thickness, elderly women original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.114-120 may-august, 2019 vol.38no.2 cite this article as: imran y, aswar a, rac hmiyani i, et al. low 2 5hydroxyvitamin d level increases carotid intima-media thickness in elderly women. univ med 2019;38:114-20. doi: 10.18051/univmed.2019.v38.1141 2 0 115 univ med vol. 38 no.2 introduction atherosclerosis is a process that progresses slowly in stages, starting in childhood and advancing up to adult age at varying rates and is prone to cause complications. the initial structural change that can be detected in atherosclerosis is an increase in carotid intimamedia thickness (cimt),(1) which leads to the presence of atherosclerosis of the carotid arteries, an important factor in patients with ischemic stroke,(2) and is a strong predictor for future vascular conditions of the patients.(3) at h e r o s c le r o si s i s a s s oc ia t e d wi t h inflammation resulting from several factors, such as vitamin d deficiency.(4) vitamin d receptors are present in all cells of the body and influence the atherosclerotic process, i.e. maintaining endothelial function of the blood vessels, inhibiting the proliferation of smooth muscle cells and playing a role as natural immunomodulators.(5) vitamin d has a protective effect against endothelial dysfunction and the i nf l a mma to r y pr o c e s s t ha t p r e c e de s atherosclerosis. vitamin d stimulates nitric oxide (no) synthesis (6) and decreases oxidative stress in the endothelium.(7) the anti-atherogenic effect of 25-hydroxyvitamin d [25(oh)d] constitutes a specific influence on the immune system, by exerting a direct effect on cd4 cells and stimulating an increase in type 2 t-helper (th2) lymphocytes, and is responsible for interleukin 10 (il-10) synthesis by inhibiting the macrophage activation that is the key to the atherogenic process. in addition, it also inhibits the transcription of interferon-γ (ifn-γ).(8) in its protection against atherosclerosis, 25(oh)d also represses the expression of matrix metalloproteinases (mmps), which are tissue enzymes that are secreted by activated macrophages in the inflammatory process and play a role in remodeling of the blood vessel wall and the myocardium. in addition, mmps also damage the collagenous tissues in the atherosclerotic lesion and cause atherosclerotic rupture that subsequently results in thrombosis.(9) lesions in the carotid arteries may cause embolism in the brain. half of all strokes is caused by atherosclerosis. atherosclerosis in the carotid arteries can be seen with carotid duplex ultrasonography and the flow rate of the circulation can be seen with transcranial doppler ultrasonography.(10) vitamin d deficiency influences the activity o f ma c r op ha ge s a n d l ymp ho c yt e s in atherosclerotic plaques and causes chronic inflammation of the arterial wall.(3) in addition, there occurs endothelial dysfunction in the form of a decrease in total nitric oxide (no) and an increase in reactive oxygen production.(11) vi t a min d d e f i c i e n c y a l s o i nh i bi t s t h e proliferation and differentiation of cd4 cells into th1, th17, and th2.(5) de f i c i e nc y of 2 5( oh) d i s f o un d i n approximately 50% of the world population and in 25% to 57% of that of the united states.(5) in indonesia according to the study results of oemar et al.(12) the prevalence of 25(oh)d deficiency is 50% in women aged 45-55 years. the study of setiati et al.(13) in jakarta and bekasi encountered 25(oh)d deficiency in 35.1% of women aged 60-75 years. other investigators, namely arifin et al.(14) showed that 25(oh)d deficiency in postmenopausal women attending the obstetrics polyclinic of rsupn cipto mangunkusumo jakarta attained 81%. research conducted by carrelli et al.(3) using a cross-sectional study involving 203 subjects aged 50 93 years indicates that low 25(oh)d concentrations are associated with increased cimt. essentially identical results were found in a cross-sectional study carried out by reis et al., involving 654 community-dwelling older adults aged 55-96 years,(15) where in subgroup analyses, low levels of 25(oh)d were inversely associated with cimt among those with hypertension. differing from the study results of carelli et al.(3) and reis et al.,(16) a cohort study by deleskog et al.(16) on 3430 adult and elderly subjects found that low vitamin d was not independently associated with increased cimt. therefore the results of the various 116 studies on the relationship between 25(oh)d a n d cim t a s ma r ke r o f s ub c l i ni c a l atherosclerosis and predictor of cardiovascular a n d c e r e br ova s c ul a r d i se a s e a r e s t i l l contradictory. 25-hydroxyvitamin d deficiency is an emerging mayor public health problem and has important implications toward cardiovascular and cerebrovascular morbidity. based on the still inconsistent study results, further studies are needed with the aim to determine the relationship between 25(oh)d concentration and cimt in elderly women. methods study design this was an analytical cross-sectional study to determine the relationship between 25(oh)d and cimt in elderly women in 5 villages (kelurahan) in mampang prapatan district, south jakarta, consisting of the villages of kuningan barat, mampang prapatan, tegal parang, pela 1 and pela 2. the study was carried out between november 2018 and january 2019. study subjects the study subjects were females aged 60 years and over with the inclusion criteria of being capable of good communication and capable of active locomotion without walking aids. the exclusion criteria used were: suffering from heart disease, stroke, kidney disease, consumption of 25(oh)d supplements, or corticosteroids and hormone replacement therapy. the subjects were interviewed by 10 health center cadres using a questionnaire consisting of a number of items, including age. all participants signed informed consent. the calculated optimal sample size was 27 subjects, but in this study 45 subjects were used. the sample size was determined from a type 1 error (alpha) of 5%, for a one sided hypothesis so that zα =1.64. the type 2 error was set at 10%, so that zβ=1.28 and the correlation r=0.6.(17) the study subjects were selected by cluster and simple random sampling. m ea sur em ent o f car ot id inti mame di a thickness ca r o t i d-i n t i ma me d i a t h ic kn e s s measurement was by means of carotid duplex usg, with the subject in the supine position and the neck slightly extended and rotated away from the side to be examined. the 10 mhz linear transducer was placed on the neck and moved upwards from the supraclavicular region to the r i ght a n d l e f t ma nd i b ul a r angl e s . t he measurement was performed on the distal part of the common carotid artery, i.e. on a 1-cm long segment of the carotid bulb. on the screen a double-line would be visible, indicating the simultaneous capture of the near-wall and the far-wall. the cimt measurements were done in duplicate at each visit, and the readings were reported to the nearest 0.1 mm. the mean of the two measurements was used in the analysis. the instrument used was the mindray z6 digital ultrasonic imaging system. the cimt value used was that for the left carotid artery, since t hi s i s mo r e c los el y co r r e l at e d wi th the biochemical parameters, whereas the right carotid artery is more closely correlated with hemodynamic parameters.(18) measurement of body mass index after ascertaining that the barefooted subject was standing upright and not slouching, height was measured by means of a portable microtoise with a precision of 0.1 cm. weight was measured using sage portable scales and expressed in kg with a precision of 0.1 kg. body mass index (bmi) was calculated from the weight in kg divided by height in meters squared and categorized as follows: underweight (<18.5 kg/m2), normal (18.5-22.9 kg/m2), overweight (23.0-27.5 kg/m2) and obesity (>27 kg/m2). blood pressure was measured with a riester mercury sphygmomanometer and the results expressed in mmhg. imran, aswar, rachmiyani 25-hydroxyvitamin d level and carotid intima-media 117 univ med vol. 38 no.2 measurement of lipid profile blood samples were collected from each subject to a volume of 10 ml by means of venipuncture from the cubital vein, after a 12 14 hour overnight fast, then the blood samples were centrifuged at 3000 rpm for 15 minutes. the serum was separated and placed in an automatic analyzer. the normal total cholesterol concentration was defined at <200mg/dl, low density lipoprotein (ldl) cholesterol at <100mg/ dl, high density lipoprotein (hdl) cholesterol >40mg/dl, and tr iglycerides <150mg/dl. determination of total cholesterol concentration was performed by the cholesterol oxidase/ peroxidase aminophenazone method (chodpap reagent), with a coefficient of variation of 1.1 9. fo r hdl and ldl ch olest erol the coefficients of correlation were 1.43 and 2, respectively. triglycerides were measured with the glucose dehydrogenase (gdh)-nicotinamide adenine dinucleotide (nad) method, with a coefficient of correlation of 1.27.(19) measurement of blood glucose this was done with the godpap method, with coefficient of variation of 1.05. the normal value of fasting blood sugar was <100 mg/dl.(20) measurement of 25(oh)d the examination used 200 µl of the serum sample with the addition of 500 µl acetonitrile containing vitamin as internal standard for the precipitation of the protein, homogenized by vortexing for 2 minutes, then centrifuged at 14.000 rpm for 10 minutes. a volume of 100 µl of the supernatant was transferred into an amber-colored hplc vial and tightly capped. the standard was prepared as follows: standard stock solution of vitamin d in methanol was serially diluted vitamin d3 and d2: 10-50 ng/ml. the standa rd was diluted using the same procedure as for the serum sample by adding acetonitrile, then homogenized by vortexing for 2 minutes. the prepared sample was put into an hplc vial and a volume of 2 µl was injected into the lc-ms/ms system. the result of the analysis was read based on the molecular retention time from hplc separation and the peak area as read from ms/ms. a value of 25(oh)d <10ng/ml was designated deficiency, 10-<30 ng/ml insufficiency, 30-100 ng/ml sufficiency, >100 ng/ml toxicity.(21) data analysis analysis of background characteristics and normality of data distribution was performed using the kolmogorov-smirnov test. normal data distributions were presented as mean, standard d evi at i on and p e rc ent age . simple lin ea r regression analysis was done to determine the correlation between cimt and 25( oh)d concentration. initially simple linear regression analysis was performed, then multiple linear r e gr e s s i on a na l ys is of t h e a ss o c i a t i on s presenting p values of <0.20 in the simple regression analysis was to be carried out. the statistical analysis was by means of the spss software for windows, version 17 (spss, chicago), and a p value of <0.05 was declared statistically significant. ethical clearance ethical clearance in this study was issued by the ethics commission, faculty of medicine, trisakti university under no. 140/ker/fk/1/ 2019. results mean age of the subjects was 65.64 ± 4.25 years and mean bmi 24.98 ± 3.93 kg/m2. mean total cholesterol was 203.22 ± 30.77 mg/dl, mean ldl cholesterol 144.20 ± 34.12 mg/dl, mean hdl cholesterol 53.44 ± 12.49 mg/dl and mean triglycerides 123.28 ± 48.07 mg/dl. mean 25 (oh)d concentration was 11.71 ± 3.93 ng/ ml, categorized as vitamin d insufficiency, mean left cimt was 0.61 ± 0.10 mm. there were 15 ( 3 3 .3 3% ) e ld e r ly w ome n w i t h 25 ( o h) d deficiency (table 1). h b c 118 initially simple linear regression analysis was performed, in which the results showed that only the 25(oh)d concentration as the independent variable was significantly associated with cimt (β=-0.011 95% c.i. = -0.020 to -0.003). it turned out that there was no other independent variable with p <0.20, so that the multiple linear regression could not be performed (table 2). discussion in this study an inverse relationship was found between 25(oh)d concentration and cimt. similar results were obtained in the study of hao et al.(22) who found a negative relationship between 25(oh)d concentration and cimt in chinese post-menopausal women. a different team of investigators, carreli et al.,(3) also found a negative relationship between 25(oh)d concentration and cimt. differing results were obtained in the study conducted by monteiro junior et al.(23) who showed a significant inverse characteristic mean ± sd (n=45) age (years) 65.64 ± 4.25 body mass index (kg/m2) 24.98 ± 3.93 total cholesterol (mg/dl) 203.22 ± 30.77 ldl cholesterol (mg/dl) 144.20 ± 34.12 hdl cholesterol (mg/d l) 53.44 ± 12.49 triglycerides (mg/dl) 123.28 ± 48.07 fasting blood glucose (mg/dl) 97.07 ± 11.09 systolic blood pressure (mmhg) 125.95 ± 24.52 diastolic blood pressure (mmhg) 77.60 ±12.56 25(oh)d concentration (ng/ml) 11.71 ± 3.93 25(oh)d deficiency (n,%) 15 (33.33) cimt (mm) 0.61 ± 0.10 table 1. characteristics present in all elderly women (n=45) 25(oh)d = plasma concentration of 25-oh-vitamin d; cimt: carotid intima-media thickness linear relationship between serum 25(oh)d c o nc e nt r a t io n a n d cim t. t h e i nve r s e association between serum levels of 25(oh)d and cimt, initially observed in monteiro’s study, became non-significant after the inclusion of the variable age in the multivariate analysis model. on the other hand, the study supported the role of traditional risk factors for atherosclerosis, with independent associations of the variables age, male gender, smoking, systolic blood pressure, fasting glucose and ldl cholesterol with cimt. the role of vitamin d in normal physiological conditions has cardioprotective effects by inhibiting cholesterol uptake and preventing the development of atherogenesis. our study results showed that there were 15 (33.33%) elderly women with vitamin d deficiency. similar results were obtained in the study of setiati et al.(13) in jakarta and bekasi, who found 25(oh)d deficiency in 35.1% of women aged 60-75 years. consistent results were obtained in a french study involving 125 patients aged 75 years and older, where 43% had vitamin d deficiency.(24) other study results in both tropical and temperate zone countries showed a sufficiently high proportion of 25(oh)d, ranging from 33.3 to 43%. lack of physical activity and sun exposure are most likely the key reasons behind this relevant vitamin d deficiency in elderly people.(25) vitamin d deficiency plays an important role in the development and progress of a th e r osc l er os is . lo w 25 ( o h) vit a min d concentrations influence the activities of lymphocytes and macrophages in causing chronic inflammation in the arterial wall.(26) al mheid et al.(27) in their study on 554 healthy subjects found that low vitamin d concentrations independent variable β 95.0% confidence interval for β p value age (years) total cholesterol ldl cholesterol hdl cholesterol triglycerides bmi 25(oh)d 0.001 -0.002 0.002 0.002 0.001 0.001 -0.011 -0.077 – 1.468 -0.008 – 0.008 -0.005 – 0.010 -0.007 – 0.010 -0.001 – 0.002 -0.008 – 0.009 -0.020 – -0.003 0.076 0.586 0.503 0.719 0.564 0.901 0.009 table 2. independent factors of cimt identified by simple linear regression analysis (n=45) imran, aswar, rachmiyani 25-hydroxyvitamin d level and carotid intima-media 119 univ med vol. 38 no.2 are associated with rigidity of blood vessels and endothelial function. the study of pilz et al.(28) found that low serum 25(oh) vitamin d was an independent predictor of fatal stroke in >3000 subjects who were routinely examined by coronary angiography. hypovitaminosis d is independently associated with myocardial infarction, stroke, peripheral vascular disease, and cardiovascular mortality.(27) other influences of vitamin d on lipids are decreases in ldl cholesterol and triglyceride levels.(29) deleskog et al.(12) found that vitamin d does not play a role in combating subclinical atherosclerosis. this may be due to dietary differences, vitamin d supplementation, genetic factors, and habitual exposure to sunlight.(3) the limitation of this study is that the crosssectional design of the study does not allow for the determination of an increasing or decreasing effect of vitamin d on cimt. moreover, the reproducibility of the cimt measurements, performed by a single experienced examiner, was not tested. the results of this study show that elderly women have low vitamin d concentrations, so that the patients can be informed and pay attention to their vitamin d level, especially the elderly, among others by increasing the intake of vitamin d-rich foods and by doing regular physical exercise, particularly for exposure to ultraviolet rays of the morning sun, in order to delay thickening of the carotid artery tunica intima and media. it is hoped that experimental studies may be conducted to determine the effect of vitamin d administration to elderly women. in addition, into this future study should be entered a larger number of risk factors that are associated with vitamin d, such as obesity and diabetes mellitus, so as to obtain more complete results. conclusions there is a significant inverse relationship between 25(oh) vitamin d and cimt in elderly women. these findings in older women suggest a potential role for vitamin d in the development of subclinical atherosclerosis. conflict of interest none declared. acknowledgments the investigators express their utmost gratitude to the dean and vice-deans of the faculty of medicine, trisakti university, for their moral and material support of this study. we also thank the head of mampang health center and staff and all participants of this study. references 1. zhong w, cruickshanks kj, schuberg cr, et al. carotid atherosclerosis and 10-year changes in cognitive function. atherosclerosis 2012;224:50610. doi: 10.1016/j.atherosclerosis.2012.07.024. 2. singh as, atam v, patel ml, et al. carotid intima media thickness as areflextion of generalized atherosclerosis is related to body mass index in ischemic stroke patients. am j med sci 2013;5:22834. doi: 10.4103/1947-2714.109200. 3. carrelli al, walker md, lowe h, et al. vitamin d deficiency is associated with subclinical carotid atherosclerosis. stroke 2011;42:2240-45. doi: 10 1161/strokeaha 110 608 539. 4. park kh, park wj. endothelial dysfunction: clinical implications in cardiovascular disease and therapeutic approaches. j korean med sci 2015; 30:1213-25. 5. pusparini. defisiensi vitamin d terhadap penyakit. indonesian j clin pathol med lab 2014;21:90-5. 6. molinary c, uberti f, grossini f, et al. 25dihydroxy cholecalciferol induces nitric oxide production in cultured endothelial cells. cell physiol biochem 2011;27:661-8. 7. polidoro i, properzi g, marampon f, et al. vitamin d protecs human endothelial cells from h2o2 oxidant injuri through the mek/erk sirtl axis activations. j cardiovasc transl res 2013;6:22131. 8. mandarino nr, monteiro junior fc, salgado jvl, et al. is vitamin d deficiency a new risk for cardiovascular disease? open cardiovasc med j 2015;9:40-9. doi: 10.2174/187419201509010040. 9. kaufman jm. benefits of vitamin d supplementation. j am phy surg 2009;14:38-45. 120 10. byrnes kr, ross cb. the current role of carotid duplex ultrasonography in the management of carotid atherosclerosis: foundations and advances. int j vasc med 2012;2012. doi: http:/ /dx.doi.org/10.1155/2012/187872. 11. kassi e, adamopoulus c, basdra ek. role of vitamin d in atherosclerosis. circulation 2013;128: 2617-31. doi: 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relation between vitamin-d status and subclinical atherosclerosis. adv res biol sci 2015;2:73-81. 18. luo x, yang y, cao t, et al. differences in left and right carotid intima-media thickness and the associated risk factors. clin radiol 2011;66:393-8. doi: 10.1016/j.crad.201012.002. 19. arsana pm, rosanda r, manaf a, et al. panduan pengelolaan dislipidemia di indonesia. jakarta: pb perkeni 2015. 20. soelidtijo sa, novido h, rudijanto a, et al. konsensus pengelolaan dan pencegahan diabetes melitus tipe 2 di indonesia. jakarta: pb.perkeni; 2015. 21. bianchi s, maffei s, prontera c, et al. preanalytical, analytical (diasorin liaison) and clinical variables potentially affecting the 25 oh vitamin d estimation. clin biochem 2012;45:16527. 22. hao y, ma x, luo y, et al. inverse association of serum vitamin d in relation to intima-media thickness in chinese postmenopausal women. plos one 2015;10:1-10. doi: 10.1371/journal. pone.0122803. 23. monteiro junior fc, mandarino nr, santos em, et al. correlation between serum 25hydroxyvitamin d levels and carotid intima media thickness in a brazilian population descended from african slaves. braz j med biol res 2018;5:2-8. doi: 10 1590/1414-431x20177185. 24. kweder h, eidi h. vitamin d deficiency in elderly: risk factors and drugs impact on vitamin d status. avicenna j med 2018;8:139-46. doi: 10.4103/ajm.ajm_20_18. 25. wyskida m, wieczorowska-tobis k, chudek j. prevalence and factors promoting the occurrence of vitamin d deficiency in the elderly. postepy hig med dosw 2017;71:198-204. 26. wang l, song y, manson je, et al. circulating 25-hydroxy-vitamin d and risk of cardiovascular disease: a meta-analysis of prospective studies. circ cardiovasc qual outcomes 2012;5:819-29. doi: 10.1161/circoutcomes112967604. 27. al mheid f, patel r, murrow j. vitamin d status is associated with arterial stiffness and vascular dysfunction in healthy humans. j am coll cardiol 2011;58:186-92. doi: 10.1016/j.jacc.2011.02.051. 28. pilz s, dobing h, fischer je. low vitamin d levels predict stroke in patient referred to coronary angiography. stroke 2008;39:2611-13. doi: 10. 1161/strokeaha 107 513655. 29. lupoli r, vaccaro a, ambrosino p, et al. impact of vitamin d deficiency on subclinical carotid atherosclerosis: a pooled analysis of cohort study. j clin endocrinol metab 2017;102:214653. imran, aswar, rachmiyani 25-hydroxyvitamin d level and carotid intima-media erna12 81 *biomedical study program, faculty of medicine, brawijaya university, malang **academy of pharmaceutics and food analysts “putra indonesia malang”, malang ***endocrine and metabolic division, faculty of medicine, brawijaya university §physiology laboratory, molecular physiology division, faculty of medicine, brawijaya university, malang correspondence erna susanti academy of pharmaceutics and food analysts “putra indonesia malang” jl. barito no. 5 malang phone: +62341–491132 email: rna_far@yahoo.co.id univ med 2012;31:81-7 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background a catechin isolate from the green tea clone gmb 4, which shows antioxidant activity, may be a candidate drug for prevention of atherosclerosis. the aim of this study was to analyze the effect of catechin on endothelial nitric oxide synthase (enos) and p110 phosphoinositide 3-kinase inhibitor (pi3k) expression and on p38 mitogen activated protein kinase (mapk) activity in male rats fed a high fat diet. methods twenty five male wistar rats were divided into the following five groups: rats on standard diet; rats on high fat diet; rats on high fat diet + catechin 3 mg/ day; rats on high fat diet + catechin 6 mg/day; and rats on high fat diet + catechin 24 mg/day. enos and p110 pi3k expression and p38 mapk activity were measured by means of elisa. results high fat diet significantly increased enos expression, decreased p110 pi3k expression, and increased p38 mapk activity in male rats, in comparison with standard diet (p<0.05). administration of 3 mg/day catechin decreased enos expression compared to that in the high fat diet group without catechin (p<0.05). the administration of catechin increased p100 pi3k expression to a similar extent as that in the high fat diet groups with catechin 6 mg/day and 24 mg/day. administration of catechin at all doses decreased p38 mapk activity to the level of the standard diet group. conclusions high fat diet increases enos expression, decreases pi3k expression, and increases p38 mapk activity. administration of catechin decreases enos expression, increases pi3k expression, and decreases p38 mapk activity. keywords: catechin, enos, p110 pi3k, p38 mapk, high fat diet, male rats catechins inhibit atherosclerosis in male rats on a high fat diet erna susanti*,**, achmad rudijanto***, and retty ratnawati§ abstract 82 susanti, rudijanto, ratnawati catechins inhibit atherosclerosis katekin menghambat proses aterosklerosis pada tikus jantan dengan diet lemak tinggi latar belakang isolat golongan senyawa katekin dari teh hijau klon gmb 4, yang mempunyai aktivitas antioksidan, dapat dikembangkan sebagai kandidat preventif aterosklerosis. penelitian ini bertujuan menilai efek katekin terhadap penghambatan penurunan ekspresi p110 phosphoinositide 3-kinase inhibitor (pi3k) dan endothelial nitric oxide synthase (enos) serta penghambatan peningkatan aktivitas p38 mitogen activated protein kinase (mapk) pada tikus jantan dengan diet tinggi lemak. metode dua puluh lima ekor tikus wistar jantan yang terbagi dalam lima kelompok perlakuan, tikus dengan diet pakan standar; tikus dengan diet tinggi lemak; tikus dengan diet tinggi lemak + katekin 3 mg/hari; tikus dengan diet tinggi lemak + katekin 6 mg/hari; dan tikus dengan diet tinggi lemak + katekin 24 mg/hari. ekspresi enos, p110 pi3k, dan aktivitas p38 mapk dianalisis dengan teknik elisa. hasil diet tinggi lemak meningkatkan ekspresi enos, menurunkan ekspresi p110 pi3k, meningkatkan aktivitas p38 mapk secara bermakna dibandingkan kelompok diet standar (p<0,05). pemberian katekin dosis 3 mg/hari meningkatkan ekspresi enos secara bermakna dibandingkan diet standar dan diet tinggi lemak (p<0,005). pemberian katekin meningkatkan ekspresi p110 pi3k secara bermakna dibandingkan diet tinggi lemak (p<0,05), akan tetapi baru dapat mencapai ekspresi pada diet standar di dosis 6 mg/hari dan 24 mg/hari. pemberian katekin berbagai dosis terbukti menurunkan aktivitas p38 mapk secara bermakna dibandingkan diet tinggi lemak (p<0,01), bahkan mencapai aktivitas p38 mapk diet standar. kesimpulan diet tinggi lemak meningkatkan ekspresi enos, menurunkan ekspresi pi3k, dan meningkatan ekspresi p38 mapk. pemberian katekin dapat menurunkan ekspresi enos, meningkatkan ekspresi pi3k, dan menurunkan aktivitas p38 mapk. kata kunci: isolat katekin, enos, p110 pi3k, p38 mapk, diet tinggi lemak, tikus jantan abstrak introduction according to who estimates, annually 3.8 million males and 3.4 million females worldwide die from coronary heart disease. atherosclerosis is the principal contributor to the pathomechanism of coronary heart disease.(1) one of the risk factors causing the progressivity of atherosclerosis is dyslipidemia resulting from a high fat diet and obesity. a high fat diet may t r i g g e r t h e d e v e l o p m e n t o f c h r o n i c i n f l a m m a t i o n l e a d i n g t o e n d o t h e l i a l dysfunction. the contribution of endothelial d y s f u n c t i o n t o t h e p a t h o g e n e s i s o f atherosclerosis is via uncoupling (deregulation) of endothelial nitric oxide synthase (enos). uncoupling of enos triggers the formation of superoxide anions (o 2 -) and decreases no release. superoxide anions may react with no to form peroxynitrites for inactivation of tetrahydro biopterin (bh 4 ), which in turn i n c r e a s e s a c c u m u l a t i o n o f a s y m m e t r i c dimethylarginine (adma), the endogenous inhibitor of enos.(2) in addtion to causing 83 univ med vol. 31 no.2 endothelial dysfunction, accumulation of toxic lipid metabolites, i.e. fatty acyl coenzyme a (coa), diacylglycerol, and ceramides in tissues and arteries causes the development of insulin resistance.(3) the latter condition triggers a b n o r m a l i n s u l i n s i g n a l t r a n s d u c t i o n i n endothelial cells, involving two main pathways, i.e. the phosphoinositide 3-kinase inhibitor (pi3k) and mitogen activated protein kinase (mapk) pathways.(4) insulin resistance decreases pi3k signaling and increases mapk signaling. continuous stimulation of mapk activity causes vascular smooth muscle cell (vsmc) proliferation, increased collagen synthesis, overproduction of growth factors and pro-inflammatory cytokines, thereby accelerating the development of atherosclerosis.(3) among the naturally occurring substances that have antioxidant potential are the catechins isolated from the tea plant (camellia sinensis). the gambung research center for tea and quinine have developed green tea gmb4 clones with higher levels of catechins (14% 16%) extracted from the third level leaf buds.(5) the number of hydroxyphenolic and galloyl groups and the epimerization structure of the isolates affect their activity despite similar levels of total catechins. there are several studies in support of the activity of tea catechins on vascular function, such as the study conducted by anter et al. which shows that the polyphenol fraction of black tea stimulates enos catalytic activity. the mechanism involves stimulation-mediated a c t i v a t i o n o f p 3 8 m a p k a n d p i 3 k / a k t pathways leading to enos phosphorylation at serine 177 and dephosphorylation at thr-495. this activity results in the activation of calmodulin-dependent enos and increased no bioactivity.(6) other studies have found strong evidence of no involvement in the induction of vasorelaxation by tea polyphenols.(7) catechins with their galloyl groups are natural inhibitors of tyrosine kinases that can modify the activity of various signaling kinases such as extra cellular signal-regulated kinase 1 and 2 (erk1/2), protein kinase b (akt), pi3k and p38 mapk.(8) the hydroxyphenolic groups of catechins contribute free radical scavengers, inhibition of lipid peroxidation and hydrolysis of fat, while their galloyl groups contribute to the production of prostacyclins, reduction in vascular cell adhesion molecule-1 (vcam-1) expression and inhibition of vsmc proliferation.(9) o n t h e b a s i s o f t h e r e s u l t s o f abovementiomed studies, the aim of the present study was to analyze the effect of administration of a catechin isolate from the green tea clone gmb4 on the expression of enos and p110 pi3k, and on p38 mapk activity in male wistar rats on a high fat diet. methods design of the study a laboratory experimental study with controlled post test design, conducted from june 2 0 1 0 t o m a r c h 2 0 11 i n t h e p h y s i o l o g y l a b o r a t o r y o f t h e f a c u l t y o f m e d i c i n e , brawijaya university, malang. animals in acccordance with the federer formula, a total of 25 male wistar rats (rattus norvegicus) aged 7-8 weeks and weighing 130 155 grams, were divided into five intervention groups, which were fed the following diets: standard diet, high fat diet, high fat diet + catechin 3 mg/day, high fat diet + catechin 6 mg/day, and high fat diet + catechin 24 mg/day.(10) before undergoing the interventions, the rats were adapted to laboratory conditions for 7 days. the interventions were conducted for 60 days, after which the rats were sacrificed to obtain their aortas. preparation of animal feed the standard feed consisted of pars chicken feed (containing water, protein, lipid, fiber, ash, calcium, phosphorus, antibiotics, and coccidiostatics, to a total content of 66.6%, and 33.4% wheat flour). the high fat diet was a combination of 57.3% standard feed (pars) and 84 susanti, rudijanto, ratnawati catechins inhibit atherosclerosis table 1. mean enos and p110 pi3k expression in the intervention groups 31.8% wheat flour), with the addition of cholesterol 1.9%, cholic acid 0.1% and pork fat 8.9%.(11) measurement of p110 pi3k and enos expression and p38 mapk activity the elisa assay was performed as follows: after determination of the required number of microtiter wells, a standard curve was prepared, for which 100 ì l of assay buffer was pipetted into the blank well, and 100 ì l phosphorylated p38 mapk or p110 pi3k standards 1-7 were pipetted into the designated treatment wells. protein from aortic tissue samples isolated by means of radio immuno precipitation assay (ripa) buffer was used for measurement of p38 mapk and p110 pi3k concentrations. for the treatment samples, 100 ì l of treatment sample was pipetted into each well. the microtiter plates were incubated at 370c for 2 hours. then the wells were washed with wash buffer 3 x 400 ì l. next, each of the wells, with the exception of the blank, was filled with 100 ì l conjugate. after further incubation of the microtiter plates at 370c for 30 minutes, the wells were each washed again with wash buffer 3 x 400 ì l. subsequently the substrate, i.e. 100 ìl 3,3’,5,5’ -tetramethylbenzidine (tmb), was pipetted into each well, and the microtiter plates were again incubated for 30 minutes at room temperature. to stop the conjugation reaction, 100 µl hcl was pipetted into each well and left to react for 5 minutes. absorbance readings were performed at an optical density (od) of 492 nm.(12) for nos and p110 pi3k expression, the measurements were in ìg/ml and pg/ml, respectively, while p38 mapk activity was expressed in pg/ml. statistical analysis normally distributed data on p38 mapk activity and p110 pi3k expression from each intervention are presented as mean and standard deviation. non-normally distributed data are presented as the median, or as the mean and s t a n d a r d d e v i a t i o n a f t e r a p p r o p r i a t e transformation. data analysis was by means of one-way anova followed by the tukey test, using spss for windows version 17. ethical clearance this study was accorded ethical clearance by the commission of medical research ethics, faculty of medicine, brawijaya university, malang. results based on the anova results, there was a significant difference in aortic enos expression between intervention groups (p<0.05). from the results of the tukey test it was concluded that high fat diet significantly increased enos expression in comparison with the standard diet group (p<0.05). catechin administration at a dose of 3 mg/day significantly increased enos expression in comparison with the standard and high fat diets (p<0.05). administration of catechin at a dose of 6 mg/day significantly r e d u c e d e n o s e x p r e s s i o n ( p < 0 . 0 5 ) i n comparison with the high fat diet, although the reduction did not reach the level of the standard diet. administration of catechin at a dose of 24 mg/day did not significantly reduce enos levels as compared with the high fat diet (p>0.05) (table 1). 85 univ med vol. 31 no.2 table 2. mean p38 mapk activity in the intervention groups according to anova, mean aortic p110 pi3k expression showed significant differences between intervention groups (p<0.05). from tukey’s test it was concluded that the high fat d i e t s i g n i f i c a n t l y d e c r e a s e d p 11 0 p i 3 k expression, in comparison with the standard diet ( p < 0 . 0 5 ) . a d m i n i s t r a t i o n o f c a t e c h i n s significantly increased p110 pi3k expression in comparison with the high fat diet (p<0.05), but reached the expression level of the standard diet only at the doses of 6 mg/day and 24 mg/ day (table 1). as shown in table 2, aortic p38 mapk activities differed significantly between intervention groups (p<0.05). tukey’s test indicated that the high fat diet significantly increased p38 mapk activity in comparison with the high fat diet (p<0.05). administration of catechins at various doses was shown to effect an exceedingly significant reduction in p38 mapk activity in comparison with the high fat diet (p<0.01), and even reached the p38 mapk activity level of the standard diet. discussion nitric oxide (no) has vasodilator effects, prevents platelet adhesion, aggregation, and recruitment, inhibits migration and growth of smooth muscle, regulates interaction of platelets and blood vessels, and inhibits oxidation of low density lipoprotein.(13) in the present study, a high fat diet significantly triggered increased expression of enos, as compared with a standard diet. increased enos expression is a compensatory mechanism to raised blood cholesterol concentrations,(13,14) and may be triggered by akt-dependent calcium mobilizing or phosphorylating agents. in this study, a high fat diet was the stimulus for no production via phosphorylation of enos on serine 177 through the pi3-kinase/akt pathway.(15,16) administration of catechins at a dosage of 3 m g / d a y s i g n i f i c a n t l y i n c r e a s e d e n o s expression as compared with that caused by the high fat diet. this indicates that this dose increases the stimulus for no production via phosphorylation of enos on serine 177 through the pi3 kinase/akt pathway, apart from the stimulus of the high fat diet. by contrast, in a previous study administration of feed containing catechins 0.3 % (w/w) for 10 days did not increase enos expression in aorta homogenates in comparison with controls.(17) these differing results were presumably due to differences in duration of catechin administration. administration of catechins at a dosage of 6 mg/day decreased enos expression as a result of the high fat diet, although not to the same extent as with a normal diet. catechins may inhibit increased enos expression, because of their ability to inhibit the decrease in stimulus for increased no requirement, by means of their antioxidant mechanism to prevent low density lipoprotein (ldl) oxidation, also known as their hypocholesterolemic effect. in addition, catechins also increase phosphorylation of enos and akt.(18) catechins even increase the bioavailability of no through inhibition of nadph oxidase.(19) the results of this study differ from those of ou et al.,(20) where catechins did not trigger changes in enos expression. at a catechin dose above 6 mg/day enos was not reduced in comparison to that due to the high fat diet. this indicates that 6 mg/day was the effective dose of catechins for counteracting the 86 susanti, rudijanto, ratnawati catechins inhibit atherosclerosis decreased aortic enos expression due to the high fat diet. in the study of ou et al.(20) on human umbilical vein endothelial cells (huvec) it was found that oxidized ldl activates oxidized low density lipoprotein (lox-1) to trigger the formation of reactive oxygen compounds. furthermore, reactive oxygen compounds block pi3k, leading to subsequent increase in enos expression. high fat diet significantly decreased p110 pi3k expression, in comparison with the group on standard diet. the decreased expression of pi3k in this study was caused by increased oxidized ldl that activated lox-1 to trigger the formation of reactive oxygen compounds to block pi3k. administration of catechins significantly increased the expression of p110 pi3k, as compared with the high fat diet (p<0.05), but could only equal pi3k expression on the standard diet at a dose of 6 mg/day and 24 mg/day. the increased pi3k expression was due to the antioxidant catechins inhibiting both oxidized ldl and scavengers of reactive oxygen compounds. m a p k h a s i m p l i c a t i o n s o n v a r i o u s physiological processes, among others cell proliferation, differentiation, and apoptosis. there are three main types of mapks in mammalian cells, viz. erk1/2, jnk, and p38 mapks. activation of mapk modulates several steps in the inflammatory cascade.(21) the high fat diet significantly increased p38 mapk activity in comparison with the standard diet. increased p38 mapk activity is due to increased oxidized ldl in endothelial cells or hepatocytes, as a result of the high fat diet.(20,22) administration of catechins at various doses was shown to most significantly decrease aortic p38 mapk activity in comparison with the high fat diet, and even equalled the activity level of p38 mapk on standard diet. these findings are consistent with those of the study of choi et al.,(23) who found that (-) epigallocatechin gallate inhibited phosphorylation of p38 mapk in endothelial cells. the mechanism of catechininduced inhibition of mapk activity is based on the metal-chelating activity of catechins.(24) the activity of kinase receptors are dependent on divalent cations, which are removed through chelation by catechins, thus inactivating the receptors. in addition, the antioxidant activity of catechins is also capable of decreasing p38 mapk activity.(20) the administration of catechins to obese patients reduces insulin resistance, thus preventing progessivity of atherosclerosis. this may be caused by decreased expression of enos, which acts on the vasodilator no, and by decreased p38 mapk activity, which inhibits vsmc proliferation. one limitation of this study was that it did not investigate other signaling pathways apart from pi3k and mapk, which play a role in the development of insulin resistance and atherosclerosis, respectively. conclusions a high fat diet incrases enos expression, decreases pi3k expression, and increases p38 mapk expression. administration of catechins is capable of decreasing enos expression, increasing pi3k expression, and decreasing p38 mapk activity. acknowledgements t h i s r e s e a r c h s t u d y w a s f u n d e d b y incentive program for basic research, ministry of research and technology. we thank ms. ciptati, ms. rohayati suprihatini and mr. satuman who have given us support for this research. references 1. world health organization. deaths from coronary heart disease. geneva: world health organization; 2003. 2. yang z, ming xf. recent advances in understanding endothelial dysfunction in atherosclerosis. clin med res rev 2006;4:5365. 87 univ med vol. 31 no.2 3. defronzo ra. insulin resistance, lipotoxicity, type 2 diabetes and atherosclerosis: the missing link. diabetol rev 2010;53:1270–87. 4. muniyappa r, montagnani m, kon kk, guon mj. cardiovascular actions of insulin. endocrine rev 2007;28:463-91. 5. ratnawati r, ciptati, satuman. isolasi egcg dari teh hijau klon gmb4 jawa barat. laporan penelitian program insentif riset dasar, ristek kementerian negara riset dan teknologi. 2009. 6. anter e, thomas sr, schulz e, shapira om, vita ja, keaney jf. activation of endothelial nitric oxide synthase by the p38 mapk in response to black tea polyphenols. am soc biochem mol biol 2004;279:4637-43. 7. lorens m, wessler s, follmann e, michaelis w, dusterhoft t, baumann g, et al. a constituent of green tea, egcg, activates enos by a phosphatidylinositol-3ohkinase, campdependent protein kinase, and akt-dependent pathway and leads to endothelial dependent vasorelaxation. j biol chem 2003;279:6190-5. 8. stangl v, dreger h, stangl k, lorenz m. moleculer targets of tea polyphenols in the cardiovascular system. cardiovasc res 2007;73: 348-58. 9. velayutam p, babu a, liu d. green tea catechins and cardiovascular health: an update. curr med chem 2008;15:1840-50. 10. nagao t, komine y, soga s, meguro s, hase t, tanaka y, tokimitsu i. ingestion of a tea rich in catechins leads to a reduction in body fat and malondialdehyde modified ldl in men. am j clin nutr 2005;81:122-9. 11. ali m, muliartha ik. optimization of high-fat diet in rats models on atherogenik diet. ub medical journal 2004;3:15-21. 12. aulanni’am. principles and techniques of biomolecular analysis 2003.p.78-81. 13. salimi s, firoozrai m, mourmohammadi i, shabani m, mohebbi a. endothelial nitric oxide synthase gene intron4 vntr polymorphisms in patients with coronary artery disease in iran. indian j med res 2006;124:683-8. 14. li p, kim sw, li x, datta s, pond wg, wu g. dietary supplementation with cholesterol and docosahexanoic acid increases the activity of the arginine-nitric oxide pathway in tissues of young pigs. nitric oxide 2008;19:259-65. 15. jagnandan d, sessa wc, fulton d. intracellular location regulate calcium-calmodulin-dependent activation of organelle-restricted enos. am j physiol cell physiol 2005;289:c1024-c33. 16. ohkita m, sugii m, kitamura a, mori t, hayashi t, takaoka m, et al. differential effects of different status on endothelin-1 gene expression and endothelial nos phosphorylation in porcine aortic endothelial cells. exp biol med 2006;231: 772-6. 17. benito s, lopez d, saiz mp, buxaderas s, sanchez j, puig-parellada p, et al. a flavonoidrich diet increases nitric oxide production in rat aorta. br j pharmacol 2002;135:910-6. 18. yamakuchi m, bao c, ferlito m, lowenstein cj. epigallocathechin gallate inhibits endothelial exocytosis. biol chem 2008;389: 935-41. 19. steffen ym, schewe t, sies h. (-) epicathecin elevates nitric oxide in endothelial cells via inhibition of nadph oxidase. bioch biophys res comm 2007;359:828-33. 20. ou h, song t, yeh y, huang c, yang s, chiu t, et al. egcg protects against oxidized ldlinduced endothelial dysfunction by inhibiting lox-1-mediated signaling. j applied physiol 2010;108:1745-56. 21. joo s, song y, park y, myung e, chung c, park k, et al. epigallocathecin-3-gallate inhibits lpsinduced nf-κb and mapk signalling pathways in bone marrow-derived macrophages. gut liver 2012;6:188-96. 22. hemi r, yochananov y, barhod e, kashermeron m, karasik a, tirosh a, et al. p38 mitogen-activated protein kinase-dependent transactivation of erbb receptor family. a novel common mechanism for stress-induced irs-1 serine phophorylation and insulin resistance. diabetes 2011;60:1134-45. 23. choi y, jeong y, lee y, kwon h, kang y. (-) epigallocathecin gallate and quercetin enhance survival signalling in response to oxidantinduced human endothelial apoptosis. j nutr 2005;135:707-13. 24. kim hs, kim mh, jeong m, hwang ys, lim sh, shin ba, et al. ecgc blocks tumor promoter-induced mmp-9 expression via suppression of mapk and ap-1 activation in human gastric ags cells. anticancer res 2004; 24:747-54. oktavianus 179 *department of anatomical pathology, faculty of medicine, tarumanagara university **department of histology, faculty of medicine, tarumanagara university ***department of public health, faculty of medicine, tarumanagara university **** department of anatomical pathology, faculty of medicine, university of indonesia. **** stem cell and cancer institute correspondence dr. irmawati hassan, sp.pa department of anatomical pathology, faculty of medicine, tarumanagara university jl. let. jend. s. parman no. 1 jakarta 11440 phone: 6221-5636943 email: dr_irma0706@yahoo.com univ med 2013;32:179-86 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background breast cancer is an important health problem in the world. uncontrolled cell proliferation represents a malignant characteristic of neoplasia such as breast cancer, and can be examined immunohistochemically by measuring the ki-67 proliferative marker. the objective of this study was to determine the role of ki-67 for classification of the degree of malignancy in women with invasive ductal breast cancer. methods a cross-sectional study was conducted on 20 women with invasive ductal breast cancer. the samples were immuno-histochemically tested for ki-67 using antiki-67 primary antibody. the ki-67 proliferative index was determined by enumerating the proportion of ki-67 positive nuclei among the total number of cells in ten areas observed at 400x magnification, using a 20% cut-off value to distinguish between low and high proliferative indices. statistical analysis was by means of the chi-square test. results seventy five persent of the high grade malignancies had a high ki-67 proliferative index (>20%), while only 12.5% of the low grade malignancies had a high ki-67 index (>20%). the difference in grade malignancy was statistically significant (p=0.022), whereas tumor size was not associated with a statistically significant difference in ki-67 index (p=0.648). conclusion the study showed that invasive ductal breast cancer with high ki-67 index was significantly associated with high grade of malignacy. the high ki-67 marker index can be used for classification of the grade of malignancy of invasive ductal breast cancer. keywords: ki-67 proliferative index, grading, ductal invasive breast cancer ki-67 marker useful for classification of malignant invasive ductal breast cancer irmawati hassan*, twidy tarcisia**, agnestina ***, santoso cornain****, and i made nasar**** 180 hassan, tarcisia, agnestina, et al ki-67 marker usefull for breast cancer petanda ki-67 mampu mengklasifikasi derajat keganasan kanker payudara duktal invasif abstrak kanker payudara merupakan masalah kesehatan penting dunia. tidak terkendalinya proliferasi sel merupakan ciri ganas suatu neoplasia termasuk kanker payudara. proliferasi sel dapat diperiksa secara imunohistokimia dengan petanda ki-67. penelitian ini bertujuan untuk menentukan peran petanda proliferasi ki-67indeks untuk klasifikasi derajat keganasan pada perempuan dengan kanker payudara duktal invasif. metode rancangan penelitian potong silang digunakan pada 20 perempuan dengan kanker payudara duktal invasif. sampel diuji secara imunohistokimiawi terhadap keberadaan ki-67, dengan antibodi primer anti-ki-67. indeks ki-67 dihitung berdasarkan proporsi jumlah sel ki-67 positif dengan semua jumlah sel pada area yang diobservasi dengan pembesaran 400 x. digunakan nilai cut-off 20% untuk membedakan indeks proliferasi rendah dan tinggi, dan analisis data dilakukan menggunakan uji chi-square. hasil tujuhpuluh lima persen dari karsinoma payudara duktal invasif dengan derajat keganasan tinggi mempunyai indeks ki-67 yang tinggi (>20%), dan hanya 12,5% dari karsinoma payudara dengan derajat keganasan rendah mempunyai indeks ki-67 tinggi (>20%). perbedaan derajat keganasan tersebut bermakna secara statistik (p=0,022). namun besar tumor tidak terkait dengan perbedaan indeks ki-67 yang bermakna (p=0,648). kesimpulan hasil penelitian menunjukkan bahwa kanker payudara duktal invasif dengan indeks ki-67 yang tinggi secara bermakna terkait dengan derajat keganasan yang tinggi. indeks petanda ki-67 yang tinggi dapat digunakan untuk klasifikasi derajat keganasan kanker payudara duktal invasif. kata kunci: indeks proliferasi ki-67, derajat keganasan, kanker payudara duktal invasif abstrak introduction breast cancer is still an important health problem both in developed and developing countries. according to global cancer statistics, breast cancer is the most frequently diagnosed cancer and the major cause of death in females, accounting for 23% of all cancer cases and causing 14% of deaths from cancer.(1) in indonesia, breast cancer has risen from second to first rank among the 10 most frequent cancers in females, replacing cervical cancer.(2) the most frequent type of breast cancer is ductal invasive breast cancer.(3) the diagnosis of a tumor is based on its morphological features on histopathological examination. histopathologically, the degree of differentiation of breast cancer is determined by 3 components, i.e. nuclear morphology (nuclear pleomorphism), differentiation of tubules, and frequency of mitoses.(3) cell proliferation is a concrete parameter that can be determined by examination of ki-67, which indicates agressiveness of tumor growth.(4) for many years cell proliferation was determined by enumeration of the number of mitoses on routine histomorphological preparations. however, with this type of examination it is 181 difficult to distinguish between mitotic and pycnotic nuclei. c u r r e n t l y m a n y e x p e r t s u s e immunohistochemistry as an early indicator of malignant tumor growth. ki-67 is a nuclear protein antigen that stimulates the production of an antibody that is a marker for a tumor a n t i g e n f o u n d o n b r e a s t c a n c e r c e l l s . 5 identification of a tumor biomarker can be used for early detection of cancers, for monitoring the progress of the disease, and as prognostic and predictive markers for individualized patient management.(6) trihia et al. state that the ki-67 assay is easy to perform immunohistochemically on tumor samples obtained by multiple core biopsies and on cytological specimens.(4) according to urruticoechea et al., immunohistochemical assessment of ki-67 as a cell proliferation marker is easy and economical to perform, highly reproducible, and able to detect ki-67 in biopsied tissues.(7) the specific tumor cell proliferation marker ki-67 is an antigen expressed in all active phases of the cell cycle, from phases g1-s-g2 to m (mitosis), except in the resting phase (g0). in addition, ki-67 can be used to enumerate the proportion of actively dividing tumor cells, to assess tumor growth, and to determine the grade of malignancy. from the results of the study by trihia et al., it was concluded that detection of ki-67 is a reliable method for objective assessment of mitosis to be used in the grading system, and for predicting the degree of histological differentiation. ki-67 is also a prognostic parameter in addition to vascular i n v a s i o n , t u m o r s i z e , a n d l y m p h n o d e involvement, and is a predictor of survival of breast cancer patients. similarly, barnard et al. found a positive correlation of ki-67 score with mitotic index, and a weakly positive correlation with histologic tumor type.(8) t h e u s e o f t h e k i 6 7 p r o l i f e r a t i o n biomarker as predictive and prognostic markers for breast cancer has been widely studied.(9-11) therefore the expert panel at the st. gallen consensus conference considered that in addition to the estrogen receptor (er), progesterone receptor (pr) status, and the human epidermal growth factor receptor 2 (her-2) status, the ki67 label index is an important prognostic parameter for breast cancer.(9) using the ki-67 assay, a tumor may be classified as a low-grade, intermediate, and high-grade malignancy, on the basis of a ki-67 label index of <15%, 16%-30%, and >30%, respectively.(9) in the study by nishimura et al., it was shown that a higher ki67 index (>20%) was significantly correlated with a higher grade of malignancy.(12) several studies have argued that, apart from the presence o r a b s e n c e o f s t a n d a r d s o f p a t h o l o g i c a l evaluation of ki-67, the acceptance of ki-67 as a s t a n d a r d m a r k e r s h o u l d b e f u r t h e r investigated.(6,13) the objective of this study was to determine the role of ki-67 for classification of the degree of malignancy in women with invasive ductal breast cancer. the objective of this study was to determine a relationship between ki-67 proliferation marker index scores and degree of malignancy of invasive ductal breast cancer. methods study design this study used a cross-sectional design and w a s c o n d u c t e d f r o m j a n u a r y 2 0 0 8 u n t i l december 2011. study subjects the study subjects comprised 20 women hospitalized at the tomang clinic and royal taruma hospital with the diagnosis of invasive d u c t a l b r e a s t c a n c e r, o n w h o m b i o p s i e s extirpation had been performed. immunohistochemistry the hematoxylin-eosin (he) stained s e c t i o n s p r e p a r e d a t t h e d e p a r t m e n t o f pathological anatomy, faculty of medicine, tarumanagara university, and diagnosed histopathologically as invasive ductal breast univ med vol. 32 no.3 182 hassan, tarcisia, agnestina, et al ki-67 marker usefull for breast cancer cancer, were sent to the stem cell and cancer institute for immunohistochemical analysis of the ki-67 proliferative marker, using anti-ki-67 m o n o c l o n a l a n t i b o d y. ( 11 ) i n t h e immunohistochemical assay, ki-67 primary antibody was added to the sections, followed by chromogen to visualize the antigen-antibody reaction. in the negative control group the sections were counterstained with he without addition of ki-67 antibody. the assay was considered to be positive for ki-67 if the cell nucleus had a brown color. ki-67 negative nuclei did not express the brown color. the ki-67 proliferation marker index is expressed as percentages, determined from the number of brown-colored ki-67 positive cell nuclei divided by the total cell population in the observation fields. for reading of the results, 10 high power fields were selected containing many ki-67 positive cells.(12,14) the immunohistochemical preparations of ki-67 markers were evaluated according to abovementioned criteria and confirmed by 3 independent evaluators. in cases of differing r e s u l t s , t h e e v a l u a t o r s r e e x a m i n e d t h e preparations together for coming to a consensus. histopathological examination the histopathological sections, stained r o u t i n e l y w i t h h e , w e r e e v a l u a t e d morphologically according to who histologic typing. (3) tu m o r a g g r e s s i v e n e s s w a s histopathologically categorized as welldifferentiated, moderately differentiated, and poorly differentiated, using a modification of the nottingham system of bloom-richardson. this categorization depended on a total score of 3 components, viz. number of tubules (glands), nuclear pleomorphism, and number of mitoses in 10 high power fields (400x magnification). each component was evaluated and accorded a score from 1-3. the total score of the three components was 3-9. a score of 3-5 was categorized as grade 1 (well-differentiated), a score of 6-7 indicated grade 2 (moderately d i ff e r e n t i a t e d ) , a n d a s c o r e o f 8 9 w a s categorized as grade 3 (poorly differentiated).(3) tumor diameter was divided into 3 groups, n a m e l y <2 c m , > 2 5 c m , a n d > 5 c m , respectively.(3) ethical clearance this study was approved by the committee for research ethics of the gading pluit hospital, jakarta. statistical analysis statistical analysis was performed using spss for windows version 17.0 and medcalc. the analysis was based on differences in ki-67 scores at a cut-off point of 20%, for invasive ductal breast cancers with low and high grades of malignancy and a tumor diameter of 5 cm. the data were analyzed by means of the chisquare test. results the 20 samples were grouped according to ki-67 index, grade of malignancy, tumor size, and age of the patient. table i shows 10 samples (50%) with a ki-67 index of <20% and 10 samples (50%) with a ki-57 index of >20%. tumors with a low degree of malignancy comprised 8 samples (40%), and those with high degree of malignancy comprised 12 samples (60%). based on tumor size, tumor diameters of <5cm were found in 8 samples (40%), and tumor diameters of >5 cm were found in 12 samples (60%). the age of the patients was in the range of 31 up to 78 years, with mean age of 45.35 ± 12.19 years. i n t h e g r o u p w i t h a h i g h d e g r e e o f malignancy, 75% showed a high (>20%) ki-67 index compared to 12.5% in the group with a low degree of malignancy. the difference was statistically significant (p=0.022). (see also figures 1 and 2). but the difference in ki-67 index was not statistically significant between tumor sizes (p=0.648) (table 1). 183 univ med vol. 32 no.3 b figure 1. (a) invasive ductal breast cancer, low-grade/well-differentiated (he) (at x 100 magnification); (b) ki-67 = 4% <20% (brown-colored nuclei) (at x 400 magnification) a a b figure 2. (a) invasive ductal breast cancer, high-grade/poorly differentiated (he). solid, markedly pleomorphic nuclei; mitoses ++; (b) grade 3: ki-67 = 60% >20%. (both at x 400 magnification) discussion according to trihia, for an objective determination of the number of mitoses, the ki67 proliferation marker assay may be used,(4) since it is correlated with the clinical picture. this statement is supported by the results of studies by trihia et al., urruticoechea et al., and nishimura, showing that a high ki-67 score indicates a poor prognosis, but a good response to chemotherapy. (4,7,17) the lack of standards for the ki-67 cut-off value, as a result of the use of different antibodies and the absence of standards for the minimum number of cells to be counted, explains the findings of different ki-67 scores among the various studies. nishimura et al. and wiesner et al. use the ki-67 cut-off value of 20%.(14,17) a ki-67 index of 20% indicates a high level of cell growth and division.(12) because a high grade of malignancy also shows a high ki-67 score,(13,14) the ki-67 index is considered to be of significant positive value for differentiating among tumors with low and high malignancy grades, by using the categories of low, medium, and high proliferation indices of respectively <20%, 20%<50%, and 50%.(14) the assessment of the ki67 index according to the st. gallen consensus of 2009 is important to select prospective patients for chemotherapy as an adjunct to endocrine 184 hassan, tarcisia, agnestina, et al ki-67 marker usefull for breast cancer therapy. tumors are classified as having low, intermediate, and high proliferation rates, on the basis of ki-67 indices of <15%, 16%-30%, and >30%, respectively.(9) de azambuja et al. use a cut-off value of 10%, whereas other investigators use the mean, median, and optimum cut-off value, or varying values, and it is these differing values that cause difficulties in determining a t h r e s h o l d v a l u e a s a s t a n d a r d f o r d a i l y practice,(11) but some authors use cut-off points that are dependent on objective clinical data. the use of the ki-67 cut-off point of 10% is to exclude patients with slow proliferation rates from the chemotherapy protocol, so as to prevent excessive treatment for these patients. on the other hand, nishimura et al. are of the opinion that it is better to administer adjunctive chemotherapy to patients with high cell proliferation rates and ki-67 values of >25%.(18) in this study, the authors used the cut-off value of 20%, considering the small number of tumor samples and the fact that the cases were at an advanced stage. on the basis of the ki-67 score, the samples were divided into two groups, i.e. the group with a low ki-67 score of <20%, and the group with a high ki-67 score of >20%. the samples with a ki-67 index of <20% accounted for 50%, which was almost equal to the value of 46.5% obtained by nishimura et al.(14) this was also the case with the samples with a ki-67 index of 20%/>, with 10 cases (50%) in our study and 53.5% in the study by nishimura et al.(17) our s t u d y f o u n d 8 c a s e s ( 4 0 % ) w i t h a histopathological picture of well differentiated or low grade of malignancy, and 12 cases (60%) of moderately to poorly differentiated or high grade malignancies. a total of 90% of invasive ductal breast cancers with a higher ki-67 index (>20%) also had a high degree of malignancy, while 75% of tumors with a high degree of malignancy also had a high ki-67 index (>20%). similarly, 87.5% of tumors with a low grade of malignancy, had a low ki-67 index (<20%). statistically, there was a significant association between ki-67 proliferation index and grade of malignancy of the tumors (p<0.05). the majority of cases (7 or 70%) with a ki-67 index of <20% showed a histopathologic picture of well-differentiated tumors of low degree of malignancy, whereas the remaining 3 cases (30%) were tumors with an intermediate or low level of differentiation. samples with a ki-67 proliferation index of 20% > showed 9 cases (90%) with a higher proliferation rate and morphologically these showed a moderate to poor level of differentiation or high grade of malignancy. only one case (10%) showed a histopathologic picture of well-differentiated tumors of low degree of malignancy. loly found that invasive breast cancers with a poor degree of differentiation tended to have a ki-67 expression of >10%.(20) the results of the present study also showed that the ki-67 proliferation index was correlated with the degree of malignancy. high ki-67 indices showed tumor aggressiveness, in agreement with the results obtained by nishimura et al. and trihia et al.(14) our study also supports the necessity of assessing tumor proliferation markers as indicators of tumor growth, based on the cell divisions table 1. distribution of ki-67 index by grade of malignancy and tumor size 185 occurring in all phases of the cell cycle, except in the g0 phase. the ki-67 indices can be used as indicators of prognosis and as predictors of therapeutic reponse.(4,11, 14,18) recently, zabaglo published the results of a study comparing ki-67 to the sp6 antibody, which is more suitable for analytic imaging, while both antibodies are also markers for monitoring tumor proliferation.(21) the age of the patients in this study was in the range of 31 up to 78 years, with mean age of 45.35 ± 12.9 years, and the most frequent age was between 41 and 50 years, comprising 9 cases. the study results of nishimura showed that the youngest age was <31 years and the oldest 95 years, with mean age of 56.2 years, while the most frequent age was >50 to <65 years. the differences in youngest and oldest ages between our results and those of nishimura et al. were probably the result of differences in life span, and in genetic and hormonal factors. it is also possible that the patients in the study by nishimura et al. were more conscious of any abnormalities of their body and immediately sought treatment.(14) the mean age of the patients in our study was lower, and the factors affecting development of disease are unclear. possibly these comprise genetic and economic factors, because loly et al. mentioned in their study that in west sumatra the majority of patients come from the economically disadvantaged, with a peak at ages 40-50 years,(20) similar to our data. in this regard, a larger population is needed, to find factors that play a role in the development of breast malignancies at younger ages. tumors with a diameter between 3 and 12 cm (mean 5.925 cm) comprised 50% of samples, while in the study of nishimura et al. the mean tumor diameter was 2.2 cm.(14) the majority of tumors were >5 cm in diameter, comprising 12 samples (60%). the results of our statistical analysis showed that tumor size did not correlate with ki-67 score and degree of differentiation. this is in contrast with the findings obtained by nishmura and trihia, showing a significant correlation, with a higher ki-67 index indicating a higher degree of malignancy of the tumors.(4,17) the different results may be due to the low number of samples; our data showed that ki-67 index was more correlated with degree of tumor differentiation, depending on morphology and biological behavior of the tumors, as evaluated by number of mitoses. the implication of this study is that immunohistochemical determination of the ki67 proliferation index should be performed in cases of breast cancer, to obtain clinically useful information on tumor aggressiveness as reflected in their proliferative rate, which is essential to the management of the patients on an individual basis. there are other assays of markers for monitoring tumor proliferation, namely the kis2 and sp6 antibody assays, but these were not performed in our study due to limited funds. conclusions based on the results of this study, invasive ductal breast cancer with high ki-67 index was significantly associated with high grade of malignancy. the high ki-67 marker index can be used for classification of the grade of malignancy of invasive ductal breast cancer. further studies are required on a larger number of samples, using a ki-67 cut-off of say 10%, to be compared with the existing study results, in t h e e x p e c t a n c y o f b e i n g u s e f u l t o o t h e r investigators. acknowledgments the authors are deeply indebted to dr. tom surjadi, mph as dean of the faculty of medicine, tarumanagara university, and to ir. jap tji beng, mmis, phd, as chairman of the i n s t i t u t e o f r e s e a r c h a n d d e v e l o p m e n t (lemlitbang) of tarumanagara university, who provided us with the opportunity and the funds to conduct this study. thanks are also due to dr.dr. meily kurniawidjaja, mph, for her directions, and to the stem cell and cancer institute, in particular prof. dr. dr santoso univ med vol. 32 no.3 186 hassan, tarcisia, agnestina, et al ki-67 marker usefull for breast cancer cornain and prof. dr. i made nasar sp.pa(k), for their guidance until the completion of this study. we would also like to thank the committee on research ethics of gading pluit hospital, and the tomang clinic and royal taruma hospital who provided the study samples, and all of those who were involved in this study. references 1. jemal a, bray f, center mm, ferlay j, ward e, forman d. cancer global statistic. ca cancer j clin 2011;61:69-90. 2. direktorat jendral pelayanan medik departemen kesehatan r.i. badan registrasi kanker perhimpunan dokter spesialis patologi indonesia, yayasan kanker indonesia. kanker di indonesia tahun 1988, 1998, 2008: data histopatologik. jakarta: direktorat jendral pelayanan medik departemen kesehatan r.i.;2009. 3. allen dc. histopathology reporting: guidelines for surgical cancer. 3rd ed. london: springer; 2013. 4. trihia h, murray s, price k, gelbert rd, golouh r, goldhirsch a, et al. ki-67 expression in breast carcinoma: its associations with grading systems, clinical parameters, and other prognostic factors – a surrogate marker? cancer 2003;97:1321-31. 5. keam b, im sa, lee kh, han sw, oh dy, kim jh, et al. ki-67 can be used for further classification of triple negative breast cancer into two subtypes with different response and prognosis. breast cancer res 2011;13:r22. 6. jonat w, arnold n. is the ki-67 labelling index ready for clinical use? ann oncol 2011;22:5002. doi:10.1093/annonc/mdq732. 7. urruticoechea a, smith ie, dowsett m. proliferation marker ki-67 in early breast cancer. j clin oncol 2005;23:7212-20. 8. barnard nj, hall pa, lemoine nr, kadar n. proliferative index in breast carcinoma determined in situ by ki-67 immunostaining and its relationship to clinical and pathological variables. j pathol 2005. doi: 10.1002/path. 1711520407. 9. goldhirsch a, ingle jn, gelber rd, coates as, thürlimann b, senn hj. thresholds for therapies: highlights of the st gallen international expert consensus on the primary therapy of early breast cancer 2009. annal oncol 2009;20:1319-29. 10. de azambuja e, cardoso f, de castro jr g, colozza m, mano ms, durberq v, et al. ki-67 as prognostic marker in early breast cancer: a meta-analysis of published studies involving 12,155 patients. br j cancer 2007;96:1504-13. 11. yerushalmi r, woods r, ravdin pm, hayes mm, gelmon ka. ki-67 in breast cancer: prognostic and predictive potential. lancet oncol 2010;11: 174-83. 12. bonnefoi h, underhill c, inggo r, cameron d. predictive signatures for chemotherapy sensitivity in breast cancer: are they ready for use in clinic? eur j cancer 2009;45:1733-43. 13. colloza m, sidoni a, piccart-gebhart m. value of ki-67 in breast cancer: the debate is still open. lancet oncol 2010;11:414-5. 14. nishimura r, osaka t, okumura y, hayashi m, toyozumi y, arima n. ki-67 as a prognostic marker according to breast cancer subtype and a predictor of recurrence time in primary breast cancer. exp ther med 2010;1:747-54. 15. tavassoli fa, devilee p, editors. world health organization classification of tumours, pathology and genetics of tumours of the breast and female genital organs. lyon: iarc;2003. 16. wiesner fc, magener a, fasching pa, wesse j, bani mr, rauh c, et al. ki-67 as a prognostic molecular marker in routine clinical use in breast cancer patients. breast 2009;18:135-41. 17. nishimura r, osaka t, okumura y, hayashi m, arima n. clinical significance of ki-67 in neoadjuvant chemotherapy for primary breast cancer as a predictor for chemosensitivity and for prognosis. breast cancer 2010;17:269-75. 18. patil av, singhai r, bhamre rs, patil vw. ki67 biomarker in breast cancer of indian woman. n am j med sci 2011;3:119-28. 19. loly d. hubungan imunoekspresi ki-67 dengan beberapa parameter prognostic histopatologik karsinoma payudara invasif di tiga laboratorium patologi anatomi sumatera barat (thesis). padang: f.k. unandalas;2011. 20. zabaglo l, salter j, anderson h, quinn e, hills m, detre s, et al. comparative validation of the sp6 antibody to ki-67 in breast cancer. j clin pathol 2010;63:800-4. oktavianus 137 *biomedical department and medical genetics laboratory, biomolecular research center, faculty of medicine, wijaya kusuma university, surabaya **department of public health, faculty of medicine, diponogoro university correspondence putu oky ari tania, s.si biomedical department and medical genetics laboratory, biomolecular research center, faculty of medicine, wijaya kusuma university, surabaya email: putu.oky@gmail.com univ med 2013;32:137-45 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background increasing industrial activity is causing many problems in reproductive health, such as infertility. the factors causing infertility are hormones, infection, radiation, drugs and chemicals. one of these is 2-methoxyethanol (2-me). the objective of this study was to evaluate the effect of mucuna pruriens (mp) seed fraction on spermatogenesis, including number of spermatogenic cells and spermatozoal membrane protein profiles after exposure to 2-me in mice. methods this study was of experimental design. thirty mice were randomized into 5 groups, i.e. 2 control groups and 3 treatment groups. all mice were injected subcutaneously with 2-me doses of 100mg/kg body weight (bw)/day, for a period of 12 days. positive (pc) and negative controls (nc) were treated with carboxy methyl cellulose (cmc). the three treatment groups were given mp seed fraction in doses of 14 (t1), 28 (t2) and 56 mg/kg bw (t3), respectively, from day 13 until day 64. data on spermatogenic cells were collected from histological cross-sections and analyzed with one-way anova. the spermatozoal membrane protein profile was identified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) with silver staining and were analyzed descriptively. results administration of mp seed fractions resulted in significantly different numbers of spermatocytes i and round spermatids between pc and t1, t2, and t3. a protein with the molecular weight of 41.6 kda was expressed in all groups except the positive controls, while a 24.5 kda protein was expressed in the positive controls only. conclusion mp seed fraction effectively recovers spermatogenesis loss in mice due to 2me exposure. key words : mucuna pruriens, 2-methoxyethanol, spermatogenic cells, mice mucuna pruriens restores spermatogenesis in mice after exposure to 2-methoxyethanol putu oky ari tania* and sri winarni** 138 tania, winarni mucuna pruriens restores spermatogenesis mucuna pruriens memulihkan spermatogenesis pada mencit setelah paparan 2-methoxyethanol latar belakang peningkatan aktivitas industri mengakibatkan masalah kesehatan, khususnya mengenai kesehatan reproduksi, yaitu infertilitas (kemandulan). faktor yang menyebabkan infertilitas antara lain hormon, infeksi, radiasi, obat dan bahan kimia baik alami maupun sintetik, yang dapat berinteraksi dengan sistem endokrin. salah satu bahan kimia tersebut adalah 2-methoxyethanol (2-me). penelitian ini bertujuan untuk menilai pengaruh fraksi biji mucuna pruriens (mp) terhadap spermatogenesis meliputi jumlah sel spermatogenik dan profil protein membran spermatozoa setelah pemaparan 2-me pada mencit. metode sebuah rancangan eksperimental digunakan pada studi ini. tiga puluh ekor mencit jantan dibagi dalam 5 kelompok, 2 kelompok kontrol, yaitu kontrol positif (pc) diberikan 2-me dan kontrol negatif (nc) diberikan carboxy methyl cellulose (cmc), serta 3 kelompok perlakuan diberikan fraksi biji mp dosis 14 (t1), 28 (t2) dan 56 mg/kg berat badan (t3). semua mencit diinjeksi secara subkutan dengan 2 mp sebanyak 100 mg/kg berat badan 12 hari sebelum perlakuan. pengambilan data sel spermatogenik dilakukan dengan pembuatan sediaan histologi, dan dianalisis dengan oneway anova. profil protein membran spermatozoa diidentifikasi dengan sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page), silver staining dan dianalisis jumlah pita secara deskriptif. hasil pemberian fraksi biji mp meningkatkan jumlah sel spermatosit i dan spermatid bundar secara bermakna pada kelompok t1, t2, dan t3 dibandingkan kelompok pc. terdapat 13 pita yang muncul pada gel elektroforesis untuk semua kelompok, pita dengan bm 41,6 kda muncul di semua kelompok, kecuali kontrol positif sedangkan pita dengan bm 24,5 kda hanya muncul pada kontrol positif. kesimpulan fraksi biji mp mampu memulihkan spermatogenesis mencit akibat pemaparan 2-m. kata kunci : mucuna pruriens, 2-methoxyethanol, sel spermatogenik, mencit abstrak introduction in recent years, there are serious problems in reproductive health, including infertility and sterility. the factors that cause infertility are hormones, infection, radiation, and natural and synthetic chemicals, which can interact with the endocrine system.(1) several studies are currently investigating the causes for infertility. one of these are chemical agents that are toxic to the human body and impair fertility. 2-methoxyethanol (2-me) is a chemical agent that is widely distributed in the environment. it is a glycol ether derived from phthalate esters. the latter compounds are used globally as water-based organic solvents in industry and household appliances.(2) i n t h e m a m m a l i a n b o d y, 2 m e i s metabolized into methoxyacetic acid (maa), which has more toxic effects than others.(3) s e v e r a l s t u d i e s s u g g e s t e d t h a t t h e r e a r e abnormalities in the reproductive system caused by 2-me. administration of 2-me at a dose of 100 mg / kg body weight (bw) can decrease 139 univ med vol. 32 no.3 testosterone levels in male mice,(4) and also inhibit the secretion of androgen-binding protein (abp).(5) normally, the process of spermatogenesis is regulated by endocrine and paracrine signals. gonadotropine releasing hormone (gnrh) plays an important role. pulsatile secretion of gnrh produces follicle-stimulating hormone (fsh) and luteinizing hormone (lh). production of testosterone is stimulated by lh, and fsh stimulates sertoli cells to secrete abp.(6) the decrease in testosterone and abp levels due to 2-me is associated with decreased functioning of the hypothalamus and pituitary that regulates the secretion of lh and fsh. testosterone and abp levels decreased by 2 m e w o u l d c a u s e a n i n t e r r u p t i o n o f s p e r m a t o g e n e s i s . r e d u c e d t e s t o s t e r o n e autoregulatively inhibits expression of some testicular proteins, including those that play a role in spermatogenesis.(7) spermatogenesis disruption caused by 2m e c a n a ff e c t p r o t e i n s y n t h e s i s i n t h e spermatozoal membrane. exposure to 2-me at a dose of 200 mg / kg bw 3 times weekly significantly reduces the width of the 19 kda and 24 kda protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (sdspage).(8) in order to promote a life style of using natural products and to manage the negative impact of 2-me, we need natural or herbal substances, such as mucuna pruriens (mp). seeds of mp contain highly beneficial natural substances, and mp seeds have been reported to possess anti-diabetic, anti-neoplastic, anti-microbial, aphrodisiac, and learning and memory enhancing properties. (9) mucuna pruriens seed extract acts as a herbal medicine that reduces stress and improves sperm quality.(10) according to singh et al., mp efficiently repairs spermatogenic cell damage caused by ethinyl estradiol (ee).(11) there have been many previous studies on the effect of mp seed fraction on spermatogenic cells, but not on its effect on exposure of spermatogenic cells to 2-me in mice. the most important component of mp seed fraction that improves spermatogenesis is l-3,4 dihydroxyphenyl alanine (l-dopa).(12) l-dopa undergoes biotransformation into dopamine, which is mostly converted to norepinephrine (ne) by dopamine-β-hydroxylase and undergoes methylation to epinephrine (e) in b r a i n v e n t r i c l e s . n o r e p i n e p h r i n e a ff e c t s hypothalamic pulsatile release of gnrh, and frequency and amplitude of gnrh stimulation. as a result, there is increased gnrh stimulation of fsh and lh, ( 1 3 ) r e s u l t i n g i n n o r m a l spermatogenesis. in this study, an mp seed extract containing l-dopa was expected to induce normal spermatogenesis after exposure to 2-me. the purpose of this study was to evaluate the effect of mp seed fraction on spermatogenesis in mice after exposure to 2-me, particularly the number of spermatogenic cells and the spermatozoal membrane protein profile. methods research design t h i s s t u d y w a s o f a n e x p e r i m e n t a l completely randomized design. this study was conducted at airlangga university (surabaya), brawijaya university (malang), and gadjah mada university (yogyakarta), from january to july 2011. experimental animals thirty 6-8 week old adult male balb/c mice (mus musculus) weighing 25-35 g and purchased from gadjah mada university, yogyakarta, were used in this study. the animals were divided into 5 groups, comprising positive controls (pc) and negative controls (nc), and 3 treatment groups (t1, t2, t3). plant material the plant material used in this study was mp seeds from banyakprada village, central java, obtained from and verified by the institute for research on pulses and root crops (balai penelitian tanaman kacang-kacangan/umbi140 tania, winarni mucuna pruriens restores spermatogenesis figure 1. experimental procedures for treatment (t1-3) and control groups (nc, pc) for 63 days umbian), malang. acetone, ascorbic acid, ethanol, 2-methoxyethanol, and distilled water were used for mp seed fractionation in the phytopharmaca-pharmacognosy laboratory, airlangga university. histologic examination histological sections of testicular tissue were prepared by the paraffin method with harris’s hematoxylin-eosin staining at the airlangga university. preparation of histological sections of testicular tissues by the paraffin method comprises the following steps: fixation, washing, dehydration, clearing, infiltration, embedding, sectioning, staining and mounting.(14) intervention during the first twelve days all groups except the negative control group were injected subcutaneously with 2-me. from day 13 until day 64, all mice in the treatment groups were treated with mp seed fraction at oral doses of 14, 28 and 56 mg /kg bw/day, respectively, whereas the control groups were given 0.5% carboxy-methyl cellulose (cmc) orally (figure 1). after completion of the treatment, one testicle of each animal was used for preparation of hematoxylin-eosin paraffin sections and the other for sperm membrane protein isolation.(14) spermatogenic cell count determination of spermatogenic cell numbers in the histological preparations was done by hand counter under a light microscope at 400 x magnification, at the faculty of science and technology, airlangga university. the n u m b e r o f s p e r m a t o g e n i c c e l l s i n c l u d e s spermatogonia (cell number per seminiferous tubule), spermatocytes i (cell number per seminiferous tubule) and round spermatids (cell n u m b e r p e r s e m i n i f e r o u s t u b u l e ) . t h e s e m i n i f e r o u s t u b u l e s w e r e o b s e r v e d i n accordance with the criteria of spermatogenic cell stage 7 of epithelium.(15) electrophoresis i s o l a t i o n a n d p u r i f i c a t i o n o f s p e r m membrane protein using nacl, potassium phosphate buffer (ppb), mgso 4 , octyl-beta-dthioglucopyranoside (osgp) (1%), glycerol (10%), tris, and complete mini protease i n h i b i t o r, w a s p e r f o r m e d a t b r a w i j a y a university, malang, according to a centrifugal method. isolation of sperm membrane proteins was performed in several stages. first the spermatozoa were collected from the testicles by enumeration techniques, followed by sperm membrane protein isolation. finally, the membrane protein profile was determined using 141 univ med vol. 32 no.3 electrophoresis sds-page, silver staining, and v i s u a l i z a t i o n o f t h e p r o t e i n b a n d s . electrophoresis of sperm membrane protein bands by sds-page with silver staining was completed at airlangga university. histological preparations preparation of histological sections of testicular tissues by the paraffin method comprises the following steps: fixation, washing, dehydration, clearing, infiltration, embedding, sectioning, staining and mounting.(14) data analysis data were analyzed by anova and least significant differences (lsd), with p< 0.05. proteins from the spermatozoal membrane profile were analyzed descriptively. ethical clearance ethical clearance was issued by the faculty of medicine, airlangga university. results the mean number of spermatogenic cells and lsd test results can be seen in table 1. the differing effects of 2-me on the number of spermatogenic cells can be seen in the pc and nc. the mean of numbers of spermatogonia, spermatocytes i and round spermatids were lower in pc as compared with nc, but there was no difference between pc and nc in the number of spermatogonia. the effect of mp seed fraction on the recovery of spermatogenesis after administration of 2-me was seen in pc and t1, t2 and t3. the number of spermatocytes i and round spermatids differed significantly between pc and t1, t2, t3, but the number of spermatogonia showed a gradual but non-significant increase in numbers in t1, t2, and t3. the number of round spermatids also did not differ significantly between t1, t2 and t3. the effect of administration of mp seed fraction on the seminiferous tubules of mice exposed to 2-me can be seen in the histological cross sections in figure 2. the effect of mp toward spermatozoal membrane protein profile can be seen in table 2. the protein profile was described by the molecular weight (kda) of the proteins in testicular tissues, and was visualized by bands in gel electrophoresis. the sperm membrane protein profile in the control groups and the treatment groups showed 13 protein bands on gel electrophoresis and was analyzed descriptively. in pc, the proteins were visualized by 13 bands of molecular weight 120; 114; 70.6; 66.2; 56.4; 53.4; 49.5; 38.5; 32.2; 28.4; 24.5; 22.7 and 19.4 kda. while in nc and t1, t2, t3, there were 13 protein bands with molecular weight 120; 114; 70.6; 66.2; 56.4; 53.4; 49.5; 41.6; 38.5; 32.2; 28.4; 22.7, and 19.4 kda. the protein with molecular weight of 24.5 kda was expressed in pc, but not in the other groups. the 41.6 kda protein was present in all groups except pc. table 1. mean numbers of spermatogenic cells in the control and treatment groups values represent mean ± sd pc = positive controls receiving 2-me; nc = negative controls; t1 = treatment group receiving mp seed fraction at a dose of 14 mg/kg bw/day; t2 = treatment group receiving mp seed fraction at a dose of 28 mg/kg bw/day; t3 = treatment group receiving mp seed fraction at a dose of 56 mg/kg bw/day. superscripts (a,b,c,d,e) indicate a significant difference by lsd test 142 tania, winarni mucuna pruriens restores spermatogenesis figure 2. cross sections of seminiferous tubules of mus musculus. a = nc; b = pc ; c, d, and e = treatment groups t1, t2, and t3, receiving mp seed fraction at doses of 14, 28 and 56 mg/kg, respectively discussion in this study mp seed fraction was shown to induce the recovery of spermatogenic cells after exposure to 2-me. this finding has previously not been made in other studies. in the treatment groups (t1, t2, t3) the number of spermatogenic cells were shown to be greater than in the negative controls (nc). 2-me decreases the number of spermatogenic cells, especially spermatocytes i, which are the main targets of 2-me.(5) methoxyacetic acid (maa), a metabolite of 2-me, can damage spermatogenic cells, particularly pachytene spermatocytes. ( 1 6 ) spermatocytes are also the main target of apoptosis induced by maa.(17) the effect of 2-me on spermatogenesis can be seen in the nc and pc. the number of spermatogenic cells in pc was always lower than in nc, and this decrease in spermatogenic c e l l c o u n t i n d i c a t e s d i s r u p t i o n o f spermatogenesis by 2-me. t h e r e s u l t s o f o n e w a y a n o va o n number of spermatogonia in t1, t2 and t3 showed significance differences, but no effect of mp treatment at different doses on the number of spermatogonia. spermatogonia are resistant to 2-me and its metabolites, (16) implying that administration of 2-me does not e ff e c t a d e c r e a s e i n t h e n u m b e r o f spermatogonia. spermatocytes are the first cells to be affected by exposure to 2-me. oxidative stress in spermatocytes due to maa triggers release of cytochrome c, activation of caspase and apoptotic cell death.(18) spermatocytes i are the cells that are the most sensitive to 2-me.(16) twelve hours after exposure to 2-me, almost 143 univ med vol. 32 no.3 50% of spermatocytes i undergo apoptosis, indicating that spermatocytes are the main targets of 2-me. the effect of 2-me on the histology of the seminiferous tubules can be seen in figure 1b, where the density of spermatocytes i is greatly diminished. exposure to 2-me in laboratory animals causes decrease in testicular weight, histopatological changes in the testes, decreased sperm count and motility, and increased proportion of epididymal sperms with abnormal morphology.(18,19) the effect of mp seed fraction at different doses on the number of spermatocytes i are shown by the results of the lsd test. in t1, t2 and t3, the number of the cells increased significantly as compared with pc. thus the decrease in the number of spermatocytes i caused by 2-me can be dramatically restored by giving mp seed fraction. the effect of mp seed fraction on recovery of the number of round spermatids was shown on lsd testing. the treatment groups given mp seed fraction at various doses showed a significant increase in number of spermatogenic cells compared with pc. this indicates that mp seed fraction has a significant enhancing effect on the number of round spermatids that have been exposed to 2-me. mp seed fraction contains 14.7% l-3,4 dihydroxyphenyl alanine (l-dopa) and it is estimated that l-dopa can recover the number of spermatogenic cells that had previously been e x p o s e d t o 2 m e . l d o pa u n d e rg o e s b i o t r a n s f o r m a t i o n i n t o d o p a m i n e a n d i s converted into norepinephrine (ne). ne affects the increase in hypothalamic pulsatile gnrh, and the frequency and amplitude of gnrh stimulation, which ultimately increases fsh and lh.(13) protein bands appearing in pc showed an effect of 2-me on the spermatozoal membrane protein profile. in pc a 24.5 kda protein band was visualized that did not appear in the other groups. table 2. molecular weight of spermatozoal membrane proteins in pc, nc, and treatment groups t1, t2 and t3 pc = positive controls receiving 2-me; nc = negative controls; t1 = treatment group receiving mucuna pruriens (mp) seed fraction at a dose of 14 mg/kg bw/day; t2 = treatment group receiving mp seed fraction at a dose of 28 mg/kg bw/ day; t3 = treatment group receiving mp seed fraction at a dose of 56 mg/kg bw/day. 144 tania, winarni mucuna pruriens restores spermatogenesis in a study by honda et al.(20) testicular proteins with molecular weights of 14-18 kda and 41-42 kda were found on the surface of spermatozoal membranes. the proteins act as serine proteases in the penetration of the zona pellucida (zp). non-expression of serine proteases in pc can lead to infertility via blocking of acrosine, an enzyme in the acrosome of the spermatozoal head, activated at the time of zp penetration in this study, the 41.6 kda protein band was not expressed in the pc and was also c o r r e l a t e d w i t h d e c r e a s e d n u m b e r s o f spermatocytes i and round spermatids after administration of 2-me. because this protein is supposedly a serine protease and estimated to play an important role in fertilization, this suggests that exposure to 2-me could lead to infertility due to non-expression of protein serine proteases. administration of mp seed fraction at different doses was capable of inducing recovery of spermatogenesis, as indicated by the increase in number of spermatocytes i and round spermatids. however, mp seed fraction has no effect on spermatogonia, as indicated by the non-significant increase in their number. mucuna pruriens seed fraction is also capable of inducing the expression of the 41.6 kda protein. one limitation of this study was its sole focus on the effect of mp seed fraction on spermatogenesis after exposure to 2-me, especially on spermatogonia, spermatocytes i and round spermatids. another limitation was that the molecular weights of the sperm membrane proteins were not exact, but only rough estimates. in the near future we expect that the mp seed fraction will become an alternative medicine for infertile males, especially those at risk of exposure to 2-me. conclusions this study showed that administration of mp seed fraction caused increases in the number of spermatocytes i and round spermatids, but not that of spermatogonia. there were 13 protein bands which appeared in all groups. a 41.6 kda protein was expressed in all groups except pc, while a 24.5 kda protein was expressed in pc only. acknowledgements we thank allah swt for the blessings given so that this research could be done and be beneficial to us and others. upon completion of this study we would like to thank: the b i o l o g y te a m , f a c u l t y o f s c i e n c e a n d te c h n o l o g y, a i r l a n g g a u n i v e r s i t y ; t h e phytopharmaca-pharmacognosy laboratory, airlangga university; integrated research and testing laboratory (laboratorium penelitian dan pengujian terpadu, lppt) unit i, gadjah mada university; agency for nuclear energy ( b ata n ) l a b o r a t o r y, yo g y a k a r t a ; f o o d chemistry laboratory of research station for pulses and root crops (balai penelitian tanaman kacang-kacangan/umbi-umbian), malang, and experimental animal maintenance section of the faculty of biotechnology, gadjah mada university who have helped in the implementation of this research, and all those who have helped but cannot be mentioned one by one. references 1. pasqualotto ff, lucon am, sobreiro bp, pasqualotto eb, arap s. effects of medical therapy, alcohol, smoking, and endocrine disruptors on male infertility. rev hosp clin 2004;59:375-82. 2. starek a, miranowicz-dzier¿awska k, starekœwiechowicz1 b. interactive effect of combined exposure to ethylene glycol ethers and ethanol on hematological parameters in rats. health 2010;2:1054-64. 3. irnidayanti y. expression of vimentin protein and neurofilaments on forelimb buds of black-6 mice on gestation day 12 induced by 2methoxyethanol by rt-pcr. biosci 2010;2:11620. 145 4. supadmi. efek 2-methoxyethanol terhadap kadar testosteron dan histologis testis mencit (mus musculus l) [tesis]. surabaya: universitas airlangga; 2009. 5. tirado om, martinez ed, rodriguez oc, danielsen m, selva dm, reventos j, et al. methoxyacetic acid disregulation of androgen receptor and androgen-binding protein expression in adult rat testis. biol reprod 2003; 68:1437-46. 6. william hw, cheng j. fsh and testosterone signaling in sertoli cells. reprod 2005;130:15– 28. 7. mikkila tfm, toppari j, paranko j. effects of neonatal exposure to 4-tert-octylphenol diethylstilbestrol, and flutamide on steroidogenesis in infantile rat testis. toxicol sci 2006;91:456–66. 8. hayati a, mahmuda a, ray pidada ib. 2methoxyethanol exposure affects protein profile of testicular sperm membrane in mice (mus musculus). folia medica indonesiana 2010;46: 253-26. 9. sharma bk, ahmad s, singh r, verma rk, kumar n. a review on mucuna pruriens: its phyto-constituents and therapeutic uses. int j pharm sci 2012;1:308–12. 10. shukla kk, mahdi aa, ahmad mk, jaiswar sp, shankwar sn, tiwari sc. mucuna pruriens reduces stress and improves the quality of semen in infertile men. evid based complement alternat med 2010;7:137–44. 11. singh ap, sarkar s, tripathi m, rajender s. mucuna pruriens and its major constituent ldopa recover spermatogenic loss by combating ros, loss of mitochondrial membrane potential and apoptosis. plos one 2013;8:e54655. doi: 10. 1371/journal.pone.0054655. 12. pugalenthi m, vadivel v, siddhuraju p. alternative food/feed perspectives of an underutilized legume mucuna pruriens var. utilis: a review. plant foods hum nutr 2005;60: 201-18. 13. speroff l, fritz ma. clinical gynecology endocrinology and infertility. 7th ed. philadelphia: lippincot williams & wilkins; 2005. 14. sudiana ik. teknologi ilmu jaringan dan imunohistokimia. jakarta: penerbit sagung seto; 2005. 15. hess ar, renato de franca l. spermatogenesis and cycle of the seminiferous epithelium. adv exp med biol 2008;636:1-15. doi: 10.1007/9780-387-09597-4_1. 16. hayati a, any d, rai pidada ib. spermatozoa motility and morphological recovery process in mice after the induction of 2-methoxyethanol. folia medica indonesia 2005;41:90-5. 17. barone f, aguanno s, agostino d. modulation of maa-induced apoptosis in male germ cells: role of sertoli cell p/q-type calcium channels. reprod biol endocrinol 2005;3:13-23. 18. bagchi g, waxman dj. toxicity of ethylene glycol monomethyl ether: impact on testicular gene expression. int j androl 2008;31:269–74. 19. office of environmental health hazard assessment (oehha). proposition 65 maximum allowable dose level (madl) for reproductive toxicity for ethylene glycol monomethyl ether; 2004. 20. honda a, yamagata k, sugiura s, watanabe k, baba t. a mouse serine protease tesp5 is selectively included into lipid rafts of sperm membrane presumably as a glycosylphosphatidy linositol-anchored protein. biol chem 2002;227: 16976-84. univ med vol. 32 no.3 oktavianus 37 *department of cardiology and vascular medicine faculty of medicine lambung mangkurat university/ulin general hospital, banjarmasin ** department of pharmacology faculty of medicine brawijaya university, malang *** department of cardiology and vascular medicine faculty of medicine airlangga university/ dr. soetomo district general hospital, surabaya **** department of cardiology and vascular medicine faculty of medicine brawijaya university/ saiful anwar general hospital, malang correspondence : dr. dwi laksono adiputro department of cardiology and vascular medicine faculty of medicine lambung mangkurat university/ ulin general hospital jl. a. yani km 2 no. 43 banjarmasin, south kalimantan e-mail: d_adiputro@yahoo.com univ med 2013;32:37-43 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 background in cardiovascular medicine, garcinia mangostana has been used as an antioxidant to inhibit oxidation of low density lipoproteins and as an antiobesity agent. the effect of garcinia mangostana on hyperlipidemia is unknown. the aim of this study was to evaluate the effect of an ethanolic extract of garcinia mangostana pericarp on lipid profile in rats fed a high lipid diet. methods a total of 40 rats were divided into five groups control, high lipid diet, and high lipid diet + ethanolic extract of garcinia mangostana pericarp at dosages of 200, 400, and 800 mg/kg body weight. the control group received a standard diet for 60 days. the high lipid diet group received standard diet plus egg yolk, goat fat, cholic acid, and pig fat for 60 days with or without ethanolic extract of garcinia mangostana pericarp by the oral route. after 60 days, rats were anesthesized with ether for collection of blood by cardiac puncture. analysis of blood lipid profile comprised colorimetric determination of cholesterol, triglyceride, low density lipoprotein (ldl), and high density lipoprotein (hdl). results from the results of one-way anova it was concluded that there were significant between-group differences in cholesterol, trygliceride, ldl, and hdl levels (p=0.000). ethanolic extract of garcinia mangostana pericarp significantly decreased cholesterol, trygliceride, and ldl levels, starting at 400 mg/kg body weight (p=0.000). ethanolic extract of garcinia mangostana pericarp significantly increased hdl level starting at 200 mg/kg body weight (p=0.000). conclusion ethanolic extract of garcinia mangostana pericarp has a beneficial effect on lipid profile in rats on a high lipid diet. key words: mangosteen, lipid profile, high lipid diet, rats extract of mangosteen increases high density lipoprotein levels in rats fed high lipid dwi laksono adiputro*, m. aris widodo**, rochmad romdoni***, and djanggan sargowo**** 38 adiputro, widodo, romdoni, et al mangosteen and high density lipoprotein ekstrak kulit buah manggis meningkatkan kadar high density lipoprotein pada tikus diet tinggi lemak latar belakang di bidang kardiovaskuler, buah manggis bersifat antioksidan untuk menghambat oksidasi low density lipoprotein (ldl) dan antiobesitas. efek buah manggis terhadap hiperlipidemia belum diketahui. tujuan penelitian ini adalah untuk mengevaluasi efek ekstrak etanol dari kulit buah manggis (garcinia mangostana) terhadap profil lipid pada tikus yang diberikan diet tinggi lemak. metode empat puluh ekor tikus terbagi menjadi lima kelompok meliputi kontrol, kelompok diet tinggi lemak, dan kelompok diet tinggi lemak + ekstrak etanol kulit buah manggis dosis 200; 400; dan 800 mg/kgbb. kelompok kontrol mendapat diet standar selama 60 hari. kelompok diet tinggi lemak mendapat diet standar ditambah kuning telur, minyak kambing, asam kolat, dan minyak babi selama 60 hari dengan atau tanpa ekstrak etanol kulit buah manggis per oral. setelah 60 hari, tikus dianestesi dengan eter dan dilakukan pungsi darah dari jantung. analisis profil lipid yang meliputi kadar kolesterol, trigliserida, ldl, dan high density lipoprotein (hdl) dilakukan dengan metode kolorimetrik. hasil uji anova menyimpulkan bahwa terdapat perbedaan bermakna kadar kolesterol, trigliserida, ldl, dan hdl pada berbagai kelompok perlakuan (p=0,000). ekstrak etanol kulit buah manggis menurunkan kadar kolesterol, trigliserida, ldl secara bermakna dimulai pada dosis 400 mg/kgbb (p=0,000). ekstrak etanol kulit buah manggis meningkatkan kadar hdl secara bermakna dimulai pada dosis 200 mg/kgbb (p=0,000). kesimpulan disimpulkan bahwa ekstrak etanol kulit buah manggis mempunyai efek menguntungkan terhadap profil lipid pada tikus yang mendapat diet tinggi lemak. kata kunci: manggis, kulit buah, profil lipid, diet tinggi lemak, tikus abstrak introduction t h e m a n g o s t e e n t r e e h a s l o n g b e e n cultivated in tropical countries. the tree presumably originated from southeast asia or indonesia, and may also be found in the malay peninsula, myanmar, thailand, kambodia, and vietnam. the mangosteen pericarp is 0.6-1 cm t h i c k a n d c o n t a i n s a v i o l e t p i g m e n t . (1) communities of the abovementioned countries have utilized the pericarp or the fruit flesh in traditional medicine against abdominal pain, d i a r r h e a , d y s e n t e r y, w o u n d i n f e c t i o n s , suppuration, and chronic ulcers.(2) a variety of studies have revealed the potential of the active principles of mangosteen, among others as antioxidant, antibacterial, antifungal, antimalarial, anti-inflammatory, c y t o t o x i c , a n d i n h i b i t o r o f t h e h u m a n immunodeficiency virus (hiv), aromatase inhibitor, and inducer of quinone reductase.(3) in cardiovascular medicine, the mangosteen can potentially inhibit the oxidation of low density lipoprotein (ldl). in obese individuals, the mangosteen is capable of reducing c-reactive protein level.(4) in addition, the active substances in mangosteens can also decrease oxidative stress due to cardiac reperfusion.(5) 39 on the basis of the concept of natural intelligence, the potential of active substances of plant origin becomes more limited if the pharmacological actions are only displayed by one active substance. the substances isolated from mangosteen pericarps contains many xanthones, among others á -mangostin, â m a n g o s t i n , ãm a n g o s t i n , g a r t a n i n , 8 d e o x y g a r t a n i n , a n d m a n g o s t a n o l . (6,7) αmangostin is the principal active substance and has been frequently used in traditinal medicine throughout the world as an anti-inflamatory,(8) antibacterial,(9) and anticancer agent.(10) to date it has been demonstrated that mangosteen pericarps contain more than 85 active substances. a study by nakatani et al.(12) stated that ethanolic extracts of mangosteen pericarp contains 7.7% á-mangostin and 39% ã-mangostin. a study by tewtrakul et al.(13) found higher ic 50 values for nitric oxide (no) inhibition and prostaglandin-e 2 (pge 2 ) release in isolates of á -mangostin and ã-mangostin i n c o m p a r i s o n w i t h e t h a n o l i c e x t r a c t o f mangosteen pericarp. however, none of the abovementioned s t u d i e s h a v e i n v e s t i g a t e d t h e e f f e c t o f manosteen pericarp on the lipid profile of rats on a high lipid diet. therefore the purpose of the present study was to analyze the role of an ethanolic extract of mangosteen pericarp on on the lipid profile of rats on a high lipid diet. the hypothesis of this study was that ethanolic extracts of mangosteen pericarp is capable of reducing total cholesterol, triglyceride, and ld levels in rats receiving a high lipid diet. in addition, it was expected that ethanolic extracts of mangosteen pericarp may increase high density lipoprotein (hdl) levels in rats on a high lipid diet. methods study design t h e p r e s e n t s t u d y w a s a n i n v i v o experimental study using animal models, conducted at the pharmacological and the biomedical laboratories of the faculty of medicine, brawijaya university from march 2010 to june 2010. experimental animals the experimental animals were albino rats (rattus norvegicus) o b t a i n e d f r o m t h e pharmacological laboratory of the faculty of medicine, brawijaya university. the rats used were male rats aged 6-8 weeks and weighing 100-150 grams. the rats were kept in open ventilated cages and were acclimatized for 2 weeks before being subjected to the intervention. the rats were kept in a single caging system, where each cage contained one rat. the cages were plastic boxes measuring 45 cm x 35.5 cm x 14.5 cm, with wire covers. the cages had beddings of rice hulls that were changed daily. the animal feed given daily was feed for adult rats composed of confeed par-s 66%, wheat flour 33%, and water. this was the composition of the feed for the standard diet. the feed given to each rat was ± 40 gram per day and renewed daily. for the high lipid diet, the composition of the feed was comfeed par-s 53%, wheat flour 26.5%, goat fat 0.1%, cholic acid 0.0013% and pig fat 3.22%. the experimental animals were divided into one group of rats receiving the standard diet, one group receiving the high lipid diet, and three groups receiving high lipid diet + ethanolic extract of mangosteen pericarp at dosages of 200, 400, and 800 mg/kgbw, respectively. the intervention was conducted for 60 days. each intervention group consisted of 8 rats, making up a total of 40 rats. p r e p a r a t i o n o f e t h a n o l i c e x t r a c t o f mangosteen pericarp the preparation of the ethanolic extracts of mangosteen pericarp was performed in three stages, comprising the processes of drying, extraction, and evaporation.. drying was done by washing the mangosteen pericarps obtained from forests at marabahan, south kalimantan, and subsequently comminuting the cleansed univ med vol. 32 no.1 40 adiputro, widodo, romdoni, et al mangosteen and high density lipoprotein table 1. increase in body weight and feed consumption of the various groups values are mean material. the pieces of pericarp were then either heated in an oven at 80oc or placed to dry in the sun. extraction was performed by blenderizing the material and weighing off a portion of 100 grams. the 100 gram portion of the blenderized pericarp material was put into an erlenmeyer flask of 1 liter capacity, then 900 ml of 70% ethanol was added. the soaked mixture was shaken well, then left to settle overnight. evaporation was done by decanting the upper layers of the ethanol-pericarp mixture into an evaporating flask. the latter was placed in an evaporator, the water bath was filled completely, the device was installed and connected to an electricity source (220 v). the ethanolic pericarp extract dripping into the receiver flask was collected and transferred to a plastic bottle for storage in a freezer. collection of blood samples blood samples were obtained from the heart of each rat after the intervention period of 60 days. the blood collecting procedure was started by putting the rat into a glass container into which ether-saturated cotton wads had been p l a c e d f o r i n h a l a t i o n a n e s t h e s i a . t h e anesthesized rat was sectioned by opening the abdomen up to the chest. after exposing the heart, the cardiac apex was punctured and the blood slowly withdrawn using a 5 ml syringe until the maximum amount of blood was obtained. the blood sample was subsequently transferred itno an eppendorf tube without added edta. the tube was then code-labelled and transported to the clinical pathology l a b o r a t o r y o f t h e f a c u l t y o f m e d i c i n e , brawijaya univeristy, for analysis of serum lipid profile, comprising total cholesterol, triglyceride, ldl, and hdl levels. the analysis was performed according to the procedural steps outlined in the kit. data analysis to determine differences in cholesterol, triglyceride, ldl, and hdl levels, anova and p o s t h o c t e s t s w e r e d o n e . t h e l e v e l o f significance was set at p<0.05. the statistical analysis was performed with spss version 13. ethical clearance this study was approved for ethical clearance by the committee on research ethics, faculty of medicine, brawijaya university, malang. results after administration of the intervention for 60 days, the highest increase in body weight was found in the group receiving a high lipid diet and ethanolic extract of mangosteen pericarp at a dosage of 800 mg/kg bw, the increase being 1.84 gram/day. mean feed consumption did not differ in the five groups (p>0.05), making them comparable (table 1). rats receiving the high lipid diet for 60 days had significantly increased total cholesterol, triglyceride, and ldl levels, as compared with rats receiving the standard diet (p=0.000). in addition, rats on the high lipid diet also had a significant reduction in hdl levels, as compared to rats on the standard diet (p=0.000). administration of ethanolic extract of mangosteen pericarp at dosages of 400 mg/ 41 table 2. total cholesterol, triglyceride, ldl, and hdl levels in various group values represent mean ± sd; ldl= low density lipoprotein; hdl= high density lipoprotein; mg/kgbw= milligram/kilogram body weight; mg/dl: milligram/deciliter; athere was a significant difference with the standard diet (p<0.05); bthere was a significant difference with high lipid diet without ethanolic extract of mangosteen pericarp (p<0,05); cthere was a significant difference with high lipid diet + extract at 200 mg/kgbw (p<0.05); dthere was a significant difference with high lipid diet + extract at 400 mg/kgbw (p<0.05) kgbw and 800 mg/kgbw, respectively, to the high lipid diet group, resulted in decreased cholesterol levels. administration of ethanolic extract of mangosteen pericarp at the dosage of 800 mg/kgbw to the high lipid diet group was capable of reducing cholesterol levels down to the levels in the standard diet group. similar results were obtained for triglyceride levels. for ldl levels, administration of ethanolic extract of mangosteen pericarp was capable of reducing ldl levels, starting with the dosage of 200 mg/ kgbw and restoring the ldl levels to standard diet levels at a dosage of 800 mg/kgbw. similarly, administration of ethanolic extract of mangosteen pericarp significantly increased hdl levels, starting at a dosage of 200 mg/ kgbw and approaching standard diet hdl levels at a dosage of 800 mg/kgbw (table 2). discussion the present study found increases in cholesterol, triglyceride, and ldl levels, with a reduction in hdl level in the rats receiving the high lipid diet. this was caused by the intake of saturated fatty acids contained in goat fat, pork fat, egg yolk, and cholic acid. these results were consistent with those of previous studies on high lipid diets in rats.(14,15) consumption of long-chain saturated fatty acids (c>10) led to increases in cholesterol and triglyceride levels. these increases caused increases in ldl levels, necessary for transporting the cholesterol to peripheral tissues for oxidation or to adipose tissues for storage.(16) a higher proportion of lipids to proteins in the lipoproteins is associated with a lower density of the lipoproteins. thus the higher the ldl levels, the lower the hdl levels. furthermore, increases in triglyceride levels lead to increases in chylomicron and very low density lipoprotein (vldl) levels, as transporters of triglycerides. ldl is the last stage of vldl catabolism, therefore raised v l d l l e v e l s a l s o i n c r e a s e l d l l e v e l s . i n c r e a s e d c h o l e s t e r o l l e v e l s r e s u l t i n downregulation of native ldl receptors, thus inducing ldl uptake by receptor scavengers, ultimately leading to foam cell formation.(17) administration of ethanolic extract of mangosteen pericarp reduced blood cholesterol and triglyceride levels at dosages of 400 mg/ kgbw and 800 mg/kgbw. the mechanism of the reduction in blood cholesterol levels is due to inhibition of cholesterol formation. in cholesterol formation there is a stage of squalene synthesis before becoming cholesterol. a this stage there is the combination of two molecules of farnesyl pyrophosphate and elimination of pyrophosphate radicals. the combination of two molecules of farnesyl pyrophosphate is marked b y t h e c o m b i n a t i o n o f t w o f a r n e s y l pyrophosphate radicals. this combination is univ med vol. 32 no.1 42 adiputro, widodo, romdoni, et al mangosteen and high density lipoprotein inhibited by the antioxidant properties of the ethanolic extract of mangosteen pericarp, thus inhibiting squalene formation. the antioxidant properties of ethanolic extract of mangosteen pericarp has been proven by several studies. the study of chonmawanang et al.(18) demonstrated the potential of mangosteen pericarp as inhibitor of superoxide radicals through the mechanism of transfer of electrons or hydrogen atoms. a s t o t h e m e c h a n i s m o f i n c r e a s e d cholesterol katabolism, cholesterol is converted into bile salts. the process of conversion of cholesterol into bile salts requires the availability of oxygen, nadph, and cytochrome p450. the study of foti et al.(19) demonstrated that the active substances in mangosteens are capable of inhibiting cytochrome p450. therefore, the m e c h a n i s m o f c h o l e s t e r o l r e d u c t i o n b y mangosteen pericarp in the present study was acused by inhibition of cholesterol synthesis. the reduction in triglyceride lebels in the present study was caused by increased vldl and chylomicron katabolism. vldl is a triglyceride transporter originating in the liver, while chylomicrons are triglyceride transporters from the intestines. vldl and chylomicron katabolism is effected by lipoprotein lipases that hydrolyze triglycerides into free fatty acids and glycerol. administration of ethanolic extract of mangosteen pericarp increased hdl levels, starting at a dosage of 200 mg/kgbw. the increase in hdl in the present study is due to increased synthesis and secretion by the liver and intestines. the main function of hdl is as a storage site of apolipoproteins c and e, which are needed in the katabolism of chylomicrons and vldl, where apolipoprotein c is a cofactor of lipoprotein lipase and apoliprotein e is a ligand for ldl receptors.(20) the clinical implication of these study r e s u l t s a r e t h a t t h e p o t e n t i a l i n r a t s o f mangosteen pericarp extract may be applied to human subjects. a limitation of this study was that the supporting parameters associated with lipid metabolism were not studied in detail. conclusion ethanolic extract of mangosteen pericarp has beneficial effects on the lipid profile of rats receiving a high lipid diet, in the form of reduced total cholesterol,, triglyceride, and ldl levels and increased hdl levels. the benefits of mangosteen supplementation in patients with hypercholesterolemia need to be further investigated. references 1. akao y, nakagawa y, linuma m, nozawa y. anticancer effect of xanthones from pericarps of mangosteen. int j mol sci 2008;9:355-70. 2. cui j, hu w, cai z, liu y, li s, tao w, et al. new medicinal properties of mangostins: analgesic activity and pharmacological characterization of active ingredients from the fruit hull of garcinia mangostana l. pharmacol biochem behav 2010; 95:166-72. 3. han ar, kim ja, lantvit dd, kardono lbs, riswan s, chai h, et al. cytotoxic xanthone constituents of the stem bark of garcinia mangostana (mangosteen). j nat prod 2009;72: 2028-31. 4. udani jk, singh bb, barret ml, singh vj. evaluation of mangosteen juice blend on biomarkers of inflammation in obese subsjects: a pilot, dose finding study. nutr j 2009;8:48. 5. buelna-chontal m, correa f, hernandez-resendiz s, zazueta c, pedraza-chaverri j. protective effect of α-mangostin on cardiac reperfusion damage by attenuation on oxidative stress. j med food 2011;14:1370-74. 6. suksamrarn s, suwannapoch n, ratananukul p, aroonlerk n, suksamrarn a. xanthones from the green fruit hulls of garcinia mangostana. j nat prod 2002;6:761-3. 7. nilar lj, harrison lj. xanthones from the heartwood of garcinia mangostana. phytochemistry 2002;60:541–8. 8. moongkarndi p, kosem n, kaslungka s, luanratana o, pongpan n, neungton n. antiproliferation, antioxidation and induction of apoptosis by garcinia mangostana (mangosteen) on skbr3 human breast cancer cell line. 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inflammatory mediators in raw264.7 macrophage cells. j ethnopharmacol 2009;121:379-82. 14. lee yj, choi dh, cho gh, kim js, kang dg, lee hs. arctium lappa ameliorates endothelial dysfunction in rats fed with fat/cholesterol diets. bmc compl alternative med 2012;12:116 doi: 10.1186/1472-6882-12-116. 15. silva maa, vechetti-junior ij, do nascimento af, furtado ks, azevedo l, ribeiro da, et al. effects of swim training on liver carcinogenesis in male wistar rats fed a low-fat or high-fat diet. appl physiol nutr metab 2012;37: 1101-9. 16. edwar z, suyuthie h, yerizel e, sulastri d. pengaruh pemanasan terhadap kejenuhan asam lemak minyak goreng sawit dan minyak goreng jagung. j indonesian med assoc 2011;61:248-52. 17. mclaren je, michael dr, ashlin tg, ramji dp. cytokines, macrophage lipid metabolism and foam cells: implications for cardiovascular disease therapy. prog lipid res 2011;doi:10.1016/j.plipres. 2011.04.002. 18. chomnawang mt, surassmo s, nukoolkarn vs, gritsanapan w. effect of garcinia mangostana on inflammation caused by propionibacterium acnes. fitoterapia 2007;78:401–8. 19. foti rs, pearson jt, rock da, wahlstrom jl, wienkers lc. in vitro inhibition of multiple cytochrome p450 isoforms by xanthone derivates from mangosteen extract. drug metab disp 2009; 37:1848-55. 20. ansell bj, watson ke, fogelman am, navab m, fonarow gc. high-density lipoprotein function: recent advances. j am coll cardiol 2005; 46:1792– 8. univ med vol. 32 no.1 c:\users\jurnal fkusakti\docume 186 *department of medical laboratory science, school of basic medical sciences, university of benin, benin city, nigeria **pathology department, medical services and hospital, 81 division, dodan barracks, ikoyi, lagos state, nigeria ***medical microbiology division, medical laboratory services, university of benin teaching hospital, benin city, nigeria correspondence: @helen oroboghae ogefere department of medical laboratory science, school of basic medical sciences, university of benin, benin city, nigeria email: helen.ogefere@uniben.edu date of first submission, july 24, 2019 date of final revised submission, november 28, 2019 date of acceptance, december 4, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina detection of shv and tem-type extended spectrum β-lactamase in bacterial isolates in military hospitals helen oroboghae ogefere*@, samuel e. iriah*,**, and ephraim ehidiamen ibadin*** background multi-drug resistant bacterial strains have been increasingly implicated in clinical infections worldwide and beta-lactamase production is one of the commonest mechanisms of resistance in these strains. this study investigated the prevalence of extended spectrum β-lactamase (esbl)producing isolates and determined the temoneira (tem) and sulfhydryl variable (shv) types implicated in two military hospitals in south-south nigeria. methods three-hundred and eighty (380) consecutive non-duplicate bacterial isolates (gram negative bacilli) recovered from clinical samples were identified following standard techniques. antimicrobial susceptibility tests were performed for each isolate following the clinical laboratory standards institute guidelines. bacterial isolates recovered which comprised escherichia coli, klebsiella spp, proteus spp and pseudomonas aeruginosa were screened for esbl using a phenotypic method (double disc synergy test). all positive isolates were screened for tem and shv genes by pcr method. results sixty-five isolates (17.1%) were esbl producing using phenotypic method, e. coli showed the highest esbl prevalence (24.3%). one isolate was shv positive (1.5%), 8 (12.3%) were tem positive while 3 (4.6%) isolates harbored both shv and tem genes. fluoroquinolone ofloxacin showed marked activity against esbl-producing isolates (90.8%) while the least active were ceftriaxone (9.2%), ceftazidime (3.1%) and ampicillin (1.5%). conclusion this study demonstrated that 17.1% of gram-negative bacilli were esbl producers. screening of clinical isolates for esbl should be implemented. the findings of this study suggest the need for caution in the use of antimicrobial agents in order to curb the incidence of antimicrobial resistance. keywords: extended spectrum β-lactamase, resistance, gram negative bacilli, antimicrobial original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.186-193 september-december, 2019 vol.38no.3 cite this article as: ogefere ho, iriah se, ibadin ee. detection of shv and tem -typ e ext ended spectru m lactamase in bacterial isolates in military hospitals. univmed 2019;38:1863. doi: 10.18051/univmed.2019.v38. 186-193 187 univ med vol. 38 no.3 introduction the rising prevalence of drug-resistant bacteria causing clinical infections is an alarm suggesting that therapeutic options for patients a r e co n st a n t l y be i n g r e duc e d . ex t e n de d spectrum beta-lactamase (esbl), a β-lactamase enzyme type produced by some bacteria, is capable of hydrolyzing the amide bond of the four-membered β-lactam ring of the penicillins, t he f i r s t , s e c o n d a nd t hi r d ge ne r a t i on c e p h a l os p or i n s , a n d a zt r e on am, t he r e b y inactivating them.(1) its genetic determinants are largely plasmid-borne and highly transmissible between bacterial strains, being of the sulfhydryl va r i a b l e ( shv ) , te mo ne i r a ( t e m ) a nd cefotaxime-munich (ctx -m) types, with oxacillin-hydrolyzing (oxa), vietnamese esbl (veb) and pseudomonas extended resistance (per) types being increasingly implicated worldwide.(1-3) although shv variants have not been as rapidly disseminated worldwide as the ctx-m variants, they have been increasingly detected in enterobacteriaceae strains besides escherichia coli and klebsiella pneumoniae in which they were first detected in the 1970s and 1980s.(1,3) indeed, outbreaks of shv-pr oducing acinetobacter spp and pseudo monas aeruginosa have also been reported worldwide from clinical spec imens and hospital environments.(3) similarly, over 100 variants of tem have been det ected in several enterobacteriaceae since their first detection in k. pneumoniae and k. oxytoca in the 1970s.(4) in nigeria, literature is growing on the increased association between multidrug resistance and esbl production among enterobacteriaceae causing clinical infections.(5-7) previous studies in south-western nigeria reported a high prevalence of shv, tem and ctx-m genes among gram negative bacterial isolates causing clinical infections from tertiary hospitals.(6) another study in benin city, south-south nigeria reported an esbl prevalence of 41.7% among clinical isolates.(8) military hospitals are secondary hospitals tending to the health needs of military personnel and community dwellers where they are located. to the best of our knowledge, no previous nigerian study has explored esbl prevalence among clinical isolates from military hospitals. this study ther efor e sought to determine the prevalence of esbl-producing isolates among clinical isolates from two military hospitals in south-south nigeria as well as detect the tem and shv types implicated by using polymerase chain reaction method (pcr). methods study area this study was ca rried out in the metropolitan cities of benin city, edo state and port-harcourt in rivers state south-south nigeria. research design a hospital based cross-sectional study was conducted in the microbiology section of military hospital, benin city and military hospital, portharcourt in edo and rivers states, nigeria, respectively from 5th june 2019 to 4th september, 2019. both hospitals are a 150 bed secondary care hospital and serve as referral center for primary health care with outpatient clinics, medical, surgical, gynecological, pediatric and geriatric wards. bacterial isolates consecutive non-duplicate bacterial isolates recovered from clinical samples were gram stained. gram negative bacilli were identified following standard techniques.(9) these were thereafter preserved on nutrient agar slants at 4ºc for further tests. antimicrobial susceptibility tests antimicrobial susceptibility tests were performed for each isolate following the clinical la bo r a t o ry st a n d a r d s ins t i tu t e ( cl si) guidelines.(10) briefly, test organisms were 188 emulsified in sterile water and the turbidity matched with 0.5 mcfarland standard. once matched, a sterile cotton-wool swab was dipped in the organism suspension and excess liquid was removed by turning the swab on the side of the test tube. the entire surface of mueller-hinton agar plate was seeded by swabbing in three directions with the swab. the antibiotics included; ofloxacin (5 µg), ceftriaxone (30 µg), gentamicin (10 µg), amoxicillin-clavulanate (30 µg), ampicillin (30 µg), cefproxil (30 µg), imipenem (10 µg), nitrofurantoin (100 µg) and ceftazidime (30 µg) (all from abtek biologicals limited, u.k). the control strain escherichia coli atcc 25922 was also included. phenotypic detection of esbl all gram negative bacilli isolates were screened for esbl. the double disc synergy test was used in detecting the presence of esbl.(11) briefly, the test organisms were emulsified in sterile distilled water and the turbidity matched with 0.5 mcfarland standard. a sterile cotton wool swab was then dipped in the organism suspension and excess liquid was drained off by turning the swab on the side of the test tube. the entire surface of mueller– hinton agar (mha) plate was then seeded by swabbing in three directions and allowed to dry for 3-10 mins. a 30 µg amoxicillin–clavulanate (oxoid, u.k) disc was placed at the center of the agar plate. this was flanked at opposite sides by 30 µg ceftazidime and cefotaxime discs (abtek biologicals ltd, liverpool, u.k) at a dista nce o f 25mm fr om the amox icilli nclavulanate disc. the plates were thereafter i nc ub a t e d a t 3 7°c f o r 18 -2 4 hr s. esbl production was inferred as positive if there was an expansion of the zone of inhibition between amoxicillin–clavulanate disc and any of the indicator cephalosporins (ceftazidime and cefotaxime) or both. dna extraction and amplification the dna used for pcr was extracted from pure isolates grown overnight on nutrient agar using the quick-dna™ fungal/bacterial miniprep kit according to the manufacturer’s protocol. the concentration and purity of the extracted dna was estimated using a nanodrop spectrophotometer. the gene primers which included tem (for ward primer) 5´atgagtattcaacatttccgc-3´; reverse primer 5´-tgacagttaccaatgct-3´ and shv (forward primer) 5´-ccgcagccgcttg agcaaa-3´; reverse primer 5´gctggccgg ggtagtggtgtc-3´ were used for pcr amplifications as previously described.(12) a molecular size marker (thermo scientific™ sm0241 effective size range: 100 to 1000 kb) was used to assess pcr product size. then, pcr products were thereafter analyzed by agarose gel electrophoresis and photographed using an ultraviolet illuminator. the pcr products for tem and shv genes were detected at 867bp and 477bp respectively. esbl-positive klebsiella pneumoniae atcc 700603 and esbl-negative e. coli atcc 25922 were included as control strains. statistical analysis the data obtained were analyzed with chi (x2) square test or fischer’s exact test where appropriate using instat® software. a p value of < 0.05 was deemed statistically significant. ethical clearance ethical approval was sought from the ministry of health, edo state. this was approved with protocol number ha.577/vol.11/18/201. strict patient confidentiality was observed throughout the study and patients that declined were not included. results a total of 380 bacterial isolates (gram negative bacilli) were recovered from various clinical specimens during the study period. sixtyfive isolates (17.1%) were esbl producing using phenotypic method, while e. coli showed the highest esbl prevalence (24.3%) and ogefere, iriah, ibadin shv and tem-type extended spectrum lactamase 189 univ med vol. 38 no.3 showed statistical significance (p=0.0016). similarly, among 65 esbl producing isolates which were screened using pcr technique to detect shv and tem genes, one was shv positive (1.5%), a total of 8 (12.3%) were tem positive while 3 (4.6%) isolates harbored both shv and tem genes (figure 1 and 2) (table 1). in relation to clinical specimens, isolates recovered from urine specimens showed the highest esbl prevalence with 29 (23.5%), 4 of which harbored tem gene and one isolate being positive for shv + tem. the findings were however not statistically significant (p=0.2335). all gram negative bacteria recovered from tongue swabs showed absence of esbl activity (table 2). a b c the fluoroquinolone ofloxacin showed marked activity against esbl-producing isolates of e. coli (86.5%), klebsiella spp (100%), proteus spp (100%) and p. aeruginosa (92.3) in comparison with other antibiotics while the l e a s t a c ti ve w e r e c e f t r i a x on e ( 9. 2% ) , ceftazidime (3.1%) and ampicillin (1.5%). moderate activity was observed for imipenem (58.5%) and ciprofloxacin (58.5%) (table 3). discussion for this study, the prevalence of esbl producing bacteria causing infection in military hospitals in south-southern nigeria was 17.1%. this value is lower than that of previous studies in a tertiary hospital in benin city, nigeria figure 1. polymerase chain reaction results for bacterial isolates analyzed with 1.0% agarose gel electrophoresis stained with ethidium bromide. l is 100bp-1517bp dna ladder (molecular marker). samples number 3, 34 and 54 are positive for shv resistance gene with bands at 477bp while samples 17, 100, 70, 85, 112, 29 and 89 are negative for shv resistance gene. nc is a no dna template control figure 2. polymerase chain reaction results for bacterial isolates analyzed with 1.0% agarose gel electrophoresis stained with ethidium bromide. l is 100bp-1517bp dna ladder (molecular marker). samples number 35, 31, 133 and 161 are positive for tem resistance gene with bands at 867bp. nc is a no dna template control 190 (41.7%) and a chadian hospital, where 47.42% prevalence was observed.(7,13) it is however similar to that of another study in a tertiary hospital in kano, nigeria (15.0%).(14) the finding is also strikingly lower when compared with a ugandan study which used the same phenotypic method where a prevalence of 89.0% was observed.(15) the prevalence of esbl has been shown to vary according to geographical location and over time.(1,3) our study thus shows the existence of esbl phenotypes among bacterial isolates causing clinical infections at the secondary healthcare level. it is noteworthy however that isolates recovered are not routinely screened for this enzyme despite the simplicity of the double disk diffusion technique and the implication of such results for patient management. escherichia coli showed the highest prevalence (24.3%) in this study. this finding differs with earlier mentioned studies in nigeria where providencia spp and klebsiella spp r a n ke d hi ghe st i n be ni n a n d k a no respectively.(7,14) escherichia coli however is of great interest to researchers because of its ability to cause opportunistic infections, nosocomial infections as well as acquire plasmids which confer antimicrobial resistance or may harbor virulence determinants to the detriment of their host. moreover, esblproducing e. coli has been implicated in clinical infections in several studies.(5-7) the prevalence of shv and tem genes among esbl producing isolates was 1.5% and 12.3% respectively, while 4.6% of isolates showed positivity for both genes. this finding suggests that some other genetic determinants for esbl are at play besides the aforementioned two. recent studies have documented the role of ctx-m esbl variants in clinical infections in nigeria.(5,16) previous studies in southorganisms no. tested esbl positive (phenotypic detection) pcr detection shv tem tem + shv e. coli 152 (40) 37(24.3) 1 (2.7) 6 (16.2) 2 (5.4) klebsiella spp 95 (25) 8 (8.4) 1 (12.5) proteus spp 76 (20) 7 (9.2) p. aeruginosa 57 (15) 13 (22.8) 1 (7.7) 1 (7.7) total 380 65 (17.1) 1 (1.5) 8 (12.3) 3 (4.6) table 1. prevalence of esbl producers among the gram negative bacteria isolates isolates vs phenotypic esbl; p=0.0016; abbreviations: pcr-polymerase chain reaction, esbl-extended spectrum betalactamase, number in brackets = value in percentage specimen no. of isolates esbl positive (phenotypic method) pcr detection tem shv tem+shv urine 123 29 (23.5) 4 (13.8) 1 (3.4) wound 99 15 (15.2) 1 (6.7) sputum 68 11(16.2) 2 (18.2) stool 42 6 (14.3) 1 (16.7) 1 (16.7) hvs 22 2 (9.1) 1 (4.5) tongue swab 14 semen 12 2 (16.7) 1 (8.3) specimen vs phenotypic esbl; p = 0.2335; hvs-high vaginal swab, ddstdouble disk synergy test, pcr-polymerase chain reaction, number in brackets = value in percentage table 2. prevalence of esbl among gram negative bacteria isolated from different clinical specimens ogefere, iriah, ibadin shv and tem-type extended spectrum lactamase 191 univ med vol. 38 no.3 western nigeria showed that a high percentage of isolates causing clinical infections were esbl-producing and of the ctx-m type.(5,16) the researchers equally showed high fecal carriage of ctx-m type esbl-producing bacteria among out-patients in south-western nigeria, though shv, tem and oxa types were equally implicated in both studies.(5,17) also, novel genetic determinants of esbl namely veb and ox a typ es h a d be e n d e t e ct e d a mon g providencia spp in a tertiary hospital in lagos; the veb and ctx-m-15 variants have also been markedly detected among isolates causing urinary tract infections in south-western nigeria.(2,17) we therefore surmise that the low detection of shv and tem genes in known esbl strains points to the fact that other esbl genetic determinants are at play. further studies exploring the role of the ctx-m, veb, oxa, per and other novel genes among esblproducing isolates in our region would give clarity to this assertion as well as aid epidemiological surveys. interestingly, isolates recovered from urine of patients with significant bacteriuria had a higher esbl prevalence when compared with any other sample. this finding is similar to two previous nigerian studies and one ugandan study.(7,12,15) it however differs from another study in rajasthan, india in which respiratory specimens had the highest prevalence.(18) urine specimens are one of the most frequently received specimens in diagnostic microbiology laboratories.(7) their connection with high prevalence of esbl-producing bacterial strains may be due to the widespread availability of antibacterial agents notably the third generation cephalosporins in hospital and community settings where blind prescription and abuse is r i fe . ( 6 ,7 ,1 6 ) suc h pr a c t i c e s e xe r t s e l e c t i ve pressure, induce resistance mechanisms notably esbl production and ensure survival and subsequent spread of these resistant bacterial strains.(5-7) the fluoroquinolone-ofloxacin showed high activity against esbl-producing strains in comparison with any other antibacterial agent. the fluoroquinolones are however contraindicated in children and pregnant women and their use in various formulations has been restricted in european countries owing their serious, disabling and potentially permanent side effects.(19) bacterial resistance to fluoroquinolones increased drastically not long after the introduction of ofloxacin and ciprofloxacin.(20) other antibacterial agents used in this study were also poor against esbl-producing bacterial strains. this therefore highlights the limited therapeutic options faced by clinicians in patient management as it is an established fact that esbl enzymes inactivate the penicillins and the second and third generation cephalosporins, some of which were used in this study.(1) similarly, plasmids harboring esbl determinants have been known to harbor genes organism antibacterial discs (µg/disc) cpr cro ofl aug nit caz gen amp imp e. coli n=37 22 (59) 5 (13.5) 32 (86.5) 15 (41) 26 (70) 2 (5.4) 11 (30) 1 (2.7) 24 (65) klebsiella spp n=8 4 (50) 0 8 (100) 4 (50) 6 (75) 0 (0) 4 (50) 0(0) 6 (75) proteus spp. n=7 4 (57) 2 (28.6) 7 (100) 4 (57) 4 (57) 0 3 (43) 0 7 (100) p.aeruginosa n=13 8 (62) 0 12 (92.3) 0 (0) 13 (100) 0 3 (23) 0 1 (8) total (n=65) 38 (58.5) 6 (9.2) 59 (90.8) 23 (35.4) 49 (75.4) 2 (3.1) 21 (32.3) 1 (1.5) 38 (58.5) table 3: susceptibility profiles of esbl producing isolates abbreviations: n=number of isolates, number in brackets=value in percentages, cpr-ciprofloxacin, cro-ceftriaxone, ofl-ofloxacin, aug-amoxicillin-clavulanate, nit-nitrofurantoin, caz-ceftazidime, gen-gentamicin, amp-ampicillin, imp-imipenem 192 conferring resistance to other antibacterial classes.(1,5) these reasons may explain our finding. although ofloxacin was the most active antibacterial agent against esbl-producing bacteria, considering the brewing resistance to the carbapenems, our study harps on prudence in the use of antimicrobial agents. there is therefore need for institutional, local and national surveillance in order to monitor and curb the menace of antimicrobial resistance. conclusion our study documented a comparatively high prevalence of esbl genes (shv and tem) among e. coli isolates in comparison to other gram-negative bacterial isolates in military hospitals in south-south nigeria. the relatively low prevalence of the aforementioned genes among esbl-producing isolates suggests the need to determine the prevalence of a wider array of genes implicated in esbl production. acknowledgement the authors are grateful to the laboratory staff of the benin and port-harcourt military hospitals, for their cooperation during the course of this research. conflict of interest the authors declare no conflicts of interest contributors hoo, ise and eei conceived the study. ise and eei contributed to data collection and data analysis. hoo assisted in data interpretation. all authors participated in writing and approved the final draft of the manuscript. references 1. malloy amw, campos jm. extended-spectrum beta-lactamases: a brief clinical update. pediatr infect dis j 2011;30:1092–3. doi: 10.1097/ inf.0b013e31823c0e9d. 2. aibinu ie, pfeifer y, ogunsola f, et al. emergence of β-lactamases oxa-10, veb-1 and cmy in providencia species from nigeria. j antimicrob chem 2011;66:1931–2. https://doi.org/10.1093/jac/ dkr197. 3. shaikh s, fatima j, shakil s, rizvi smd, kamal ma. antibiotic resistance and extended spectrum beta-lactamases: types, epidemiology and treatment. saud j biol sci 2015;22:90–101. 4. jeannot k, fournier d, müller e, et al. clonal dissemination of pseudomonas aeruginosa isolates producing extended-spectrum βlactamase shv 2a. j clin microb 2013;51:673-5. doi: 10.1128/jcm.02313-12. 5. abreu ag, marques sg, monteiro-neto v, et al. nosocomial enterobacteriaceae infection and characterization of extended-spectrum βlactamases-producing in northeast brazil. rev soc bras med trop 2011;44:441-6. http:// dx.doi.org/10.1590/s0037-86822011000400008. 6. ogbolu do, daini oa, ogunledun a, et al. high levels of multidrug resistance in clinical isolates of gram-negative pathogens from nigeria. int j antimicrob agents 2011;37:62-6. doi: 10.1016/ j.ijantimicag.2010.08.019. 7. ibadin ee, omoregie, r, enabulele oi. prevalence of extended spectrum β-lactamase, ampc βlactamase and metallo-β-lactamase among clinical isolates recovered from patients with urinary tract infections in benin city, nigeria. n z j med lab sci 2018;72:11-6. 8. ibadin ee, omoregie r, igbarumah oi, et al. prevalence of extended spectrum β-lactamase, ampc β-lactamase and metallo-β-lactamase among gram negative bacilli from clinical specimens in a tertiary hospital in benin city, nigeria. int j ent path 2017;5:85-91. doi: 10.15171/ ijep.2017.20. 9. barrow gi, feltham rka. cowan and steel’s manual for the identification of medical bacteria. 3rd ed. cambridge: cambridge university press; 2003. 10. clinical and laboratory standards institute. performance standards for antimicrobial susceptibility testing: twenty-fourth informational supplement m100-s24. wayne, p.a., usa:clsi;2014 11. kaur j, chopra s, sheevani, mahajan g. modified double disc synergy test to detect esbl production in urinary isolates of escherichia coli and klebsiella pneumonia. j clin diagn res 2013;7:229–33. doi: 10.7860/jcdr/2013/4619.2734. 12. monsi tp, abbey sd, wachukwu ck, et al. plasmid-mediated resistance in extended spectrum beta–lactamase–producing klebsiella pneumoniae isolates at rivers state university ogefere, iriah, ibadin shv and tem-type extended spectrum lactamase 193 univ med vol. 38 no.3 teaching hospital. eur j pharm med res 2019;6: 75-82. 13. mahamat oo, lounnas m, hide m, et al. high prevalence and characterization of extendedspectrum ß-lactamase producing enterobacteriaceae in chadian hospitals. bmc infect dis 2019;19:205. doi: 10.1186/s12879-0193838-1. 14. yusuf i, haruna m, yahaya h. prevalence and antibiotic susceptibility of ampc and esbl producing clinical isolates at a tertiary health care center in kano, north-west nigeria. afr j clin exper microb 2013;14:109-19. http://dx.doi.org/ 10.4314/ajcem.v14i2.12. 15. andrew b, kaginta a, bazira j. prevalence of extended-spectrum beta-lactamases-producing microorganisms in patients admitted at krrh, southwestern uganda. int j microb 2017; 3183076. doi.org/10.1155/2017/3183076. 16. ogbolu, do, alli o, webber ma, et al. ctx-m15 is established in most multidrug-resistant uropathogenic enterobacteriaceae and pseudomonaceae from hospitals in nigeria. eur j microb immun 2018;8:20-4. doi: 10.1556/ 1886.2017.00012. 17. ogbolu do, alli oa, olanipekun lb, ojo oi, makinde oo. faecal carriage of extendedspectrum beta-lactamase (esbl)-producing commensal klebsiella pneumoniae and escherichia coli from hospital out-patients in southern nigeria. int j med medic sci 2013;5:97105. doi: 10.5897/ijmms12.0005. 18. sharma m, pathak s, srivastava p. prevalence and antibiogram of extended spectrum β-lactamase (esbl) producing gram negative bacilli and further molecular characterization of esbl producing escherichia coli and klebsiella spp. j clin diagn res 2013;7:2173-7. 19. european medicines agency. disabling and potentially permanent side effects lead to suspension or restrictions of quinolone and fluoroquinolone antibiotics. amsterdam: european medicines agency;2019. 20. redgrave ls, sutton sb, webber ma, piddock ljv. fluoroquinolone resistance: mechanisms, impact on bacteria, and role in evolutionary success. trends microbiol 2014; 22: 438–45. doi: 10.1016/j.tim.2014.04.007. c:\users\jurnal fkusakti\docume 56 *department of medical biology, faculty of medicine, universitas trisakti, jakarta **graduate student for biomedical science, faculty of medicine, universitas indonesia, jakarta ***department of medical biology, faculty of medicine, universitas indonesia, jakarta correspondence: dwi ari pujianto, m.biomed, phd department of medical biology, faculty of medicine, universitas indonesia, jalan salemba raya no.6 jakarta phone : +62 811 1100 944 email : dwi.ari@ui.ac.id orcid id :0000-0001-7546-3892 date of first submission, september 4, 2 01 8 date of final revised submission, march 29, 2019 date of acceptance, april 1, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina progesterone decrease plasma membrane in human sperm with subnormal hypoosmotic swelling test scores sisca*,**, luluk yunaini***, and dwi ari pujianto*** background progesterone (p4) is known as a female hormone affecting oocyte maturation and developing uterine wall. a proteomic study identified several receptors including p4 receptors on human sperm. the role of p4 in human sperm cells remains unknown as to whether p4 has non-genomic effects on human sperm. the present study aims to determine the effect of progesterone (p4) on the hyperactivated motility and membrane integrity of human sperm cells. methods semen from normal individuals was obtained from donors. the semen was washed by gradient density centrifugation. p4 was added to each semen sample to final concentrations of 0 (control), 250, 500, 750 and 1000 ng/ml. after the sample treatment was completed, the sperm membrane integrity was assessed with the hypoosmotic swelling test (sodium citrate dihydrate and d-fructose) and the hyperactivated sperm motility parameter was determined with the computer assisted sperm analyzer [casa] (hamilton thorne, ivos ii, usa). the percentage was then compared between the treatment groups and the control group. the percentage differences were analyzed with the sigmastat version 2.0 statistical program. results administration of p4 increased sperm hyperactivated motility when compared with the control group at a concentration of 500 ng/ml, but the increase was statistically not signicant (p>0.05). in contrast, p4 decreased sperm membrane integrity significantly (p=0.042). and the mean of plasma membrane integrity in all groups was subnormal hypoosmotic swelling test score. conclusion progesterone administration tends to increase sperm hyperactivated motility. the integrity of plasma sperm membrane was affected by progesterone. keywords: progesterone, fresh human sperm,membrane integrity, motility original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.56-62 january-april, 2019 vol.38no.1 cite this article as: sisca, yunaini l, pujianto da. progesterone decrease plasma membrane in human sperm with subnormal hypoosmotic swelling test scores. univ med 2019;38:56-62. doi: 10.18051/univmed.2019.v38.56-62 57 univ med vol. 38 no.1 introduction progesterone (p4) is a steroid hormone which is released by the cumulus cells surrounding the ovum.(1) ovulation, implantation, and pregnancy are processes that involve p4.(2)this steroid hormone causes the sperm cells to move towards the ovum then helps them to penetrate into the egg.(1) calcium influx and chloride efflux are induced by p4 as an essential way for successful fertilization, with effects that include sperm hyperactivation, acrosome reaction, and chemotaxis towards the ovum.(1,3) the latter is described as irregular movement of the sperm head and flagellum to penetrate the ovum. one study reports that an insufficient amount of p4 is related to infertility,(3) which may be due to the fact that p4 is a calcium mobilizing agent for sperm by generating ca2+ release, causing a biphasic increase in ca2+, and mediating p4induced ca2+ influx in human sperm. lack of p4 in sperm causes asthenozoospermia and is highly related to infertility problems.(3,4) the human sperm proteome study by baker et al. (5) proved that sperm have progesterone receptors on their plasma membrane,(5) and that there are several complex arrays of receptors including extragenomic p4 receptors. the study by sayme et al.(6) showed that p4 increased hyperactivated motility in human sperm cryopreserved by slow freezing and vitrification, at p4 concentrations of 0, 25, 50 nm. the present study was different from that of sayme et al.(6) in that our study used fresh human sperm. the aim of the present study was to evaluate the effect of various p4 concentrations on hyperactivated motility and membrane integrity of fresh human sperm. methods research design this was an experimental in vitro study using normozoospermia donor semen at the molecular biology laboratory, universitas indonesia, over a period of 1 year from march 2017 to march 2018. semen samples this study used semen samples obtained from donors by masturbation after abstinence for 3-5 days. the samples were divided into five groups, comprising one group without p4 as control and four groups with different doses of p4 (250, 500, 750, 1000 ng/ml). each group contained a total of 10 million sperm. the experiment was repeated nine times using different semen donors. sperm preparation and intervention sperm was obtained from healthy normal donors. the sperm was washed out from the seminal plasma by putting 3 ml of the sperm sample on top of a percoll gradient and centrifuging it at 1900 rpm for 30 minutes. after centrifugation, the sperm was collected and separated from the supernatant. then the sperm pellet was put into 1 ml of warm biggerswhitten-whittingham (bww) medium. this medium consists of 0.21 g sodium hydrogen carbonate, 0.1 g d-glucose, 0.1 g polyvinyl alcohol (pva), 0.05 ml sodium pyruvate stock, 2 ml 4(2-hydroxyethyl)-1-piperazineethanesulfonic acid (hepes), and 0.37 mlsodium lactate. after that, the sample was checked to determine its normality. a volume of 95µl sperm was added to 5 µl sperm diluting fluid, which is made from 2.5 g sodium hydrogen carbonate, 0.5 ml formaldehyde, and 50 ml h 2 o. a volume of 10 µl diluted sample was added to a hemocytometer, left to stand for 2-3 minutes, then the number of sperm cells was counted in the five squares of the hemocytometer at 40x magnification. if the sample was normal, the sperm was incubated with progesterone (p4). five hundred µl aliquots of the sperm sample, containing 10x106sperm cells, were put into five eppendorf tubes. then p4 was added into those five tubes, at concentrations of 0, 250, 500, 750, and 1000 ng/ml, respectively. after that, the samples were incubated at 37ºc 58 for 2 h. before being subjected to membrane integrity and motility testing. measurem ent of sperm hyperact ivate d motility the evaluation of sperm concentration and motility was performed using the computer assisted sperm analyzer [casa] (hamilton thorne, ivos ii, usa). sperm hyperactivated motility measures include total motility (tm) (%), forward progressive motility (fpm) (%), and velocity parameters such as curvilinear velocity (vcl) (m/sec), straight-line velocity (vsl, µm/ s), linearity of forward progression, which is the vsl to vcl ratio (linearity coefficient, lin, %), amplitude of sperm cell lateral deviation about its axis of progression or average path (amplitude of lateral head displacement, alh, µm).(7) measurement of sperm membrane integrity sperm membrane integrity was assessed using the hypo-osmotic swelling (hos) test.(8) ten microliters of sperm were mixed with 100 µl of hos solution. tail morphology was evaluated under a light microscope at 400x magnification. sperm cells with bended/curly tails were considered as sperm with an intact plasma membrane, whereas those with straight tails were regarded as sperm with a disrupted plasma membrane. the percentages of intact and disrupted sperm plasma membranes were obtained by counting a total of 100 curly and straight-tailed sperm cells. according to who, the membrane integrity of normal sperm is more than or equal to 58% (5th centile, 95% ci 55– 63). (9) statistical analysis mean values of movement parameters and labeled sperm were compared between control and test samples by one way anova. the post hoc differences between groups were analyzed by the tukey hsd test. a value of p< 0.05 was considered statistically significant. ethical clearance this research has been approved by the health research ethics committee, faculty of medicine, universitas indonesia, under no. 303/ un2.f1/etik/iii/2018. results the p4 dose-dependent effects on sperm motility and progressivity are illustrated in table 1. in group i (control group), the percentage of motile sperm during the observation period was 30.14 ± 16.25%. the control group had a lower percentage of motile sperm compared to the p4 treatment groups. the highest percentage of motile sperm was in the p4 250 ng/ml group, amounting to 36.03 ± 17.69%. there were no significant differences between the control group and the p4 treatment groups (p>0.05). in the control group, the percentage of progressive sperm during the observation period was 10.02 ± 9.01%. the control group showed a lower perce ntage of pr ogr essive sperm compared to that observed in the p4 treatment groups, but the result was not statistically s i gn i f i c a nt . t he hi ghe s t p e rc e n t a ge of progressive sperm was in the p4 500 ng/ml group, namely 15.46 ± 15.27%. sisca, yunaini, pujianto p4 increases hyperactivated motility parameter progesterone concentration (ng/ml) p value i ii iii iv v motile (%) progressive (%) 30.14 ± 16.25 10.02 ± 9.01 36.03 ± 17.69 12.20 ± 10.98 35.32 ± 23.95 15.46 ± 15.27 32.02 ± 21.27 14.11 ±13.64 33.57 ± 6.88 13.20 ± 9.79 0.173 0.216 table 1. the percentages of motile sperm and progressive movement parameter in the control group and after incubation with progesterone i: control; ii: incubation with progesterone 250 ng/ml; iii: incubation with progesterone 500ng/ml; iv: incubation with progesterone 750ng/ml; v: incubation with progesterone 1000 ng/ml. values are presented as mean ± sd 59 univ med vol. 38 no.1 after 2 h of p4 incubation, vcl in the control group (52.13 ± 12.12 μm/sec) was observed to be lower compared with the p4 treatment groups (59.18 ± 8.73 μm/sec 62.26 ± 9,59 μm/sec) (p=0.074), but this result was not statistically significant (p>0.05). the lin in the control group (40.10 ± 8.06%) was lower than that in the p4 treatment groups (>42%) (p=0.113). the amplitude of the head lateral displacement in the control group (3.00 ± 0.82 μm) was lower than in the p4 treatment groups (>3 μm). furthermore, no statistical differences were found for alh at any p4 dose (table 1). in all groups, the mean of the plasma membrane integrity had a subnormal hypoosmotic swelling test (host) scores (<50%). p4 addition to sperm slightly decreased membrane integrity of the sperm cells in the p4 250 ng/ml and 500 ng/ml groups but increased sperm membrane integrity in the p4 750 ng/ml and 1000 ng/ml groups compared to untreated sperm (p=0.042; table 2), the increase being statistically significant. a posthoc tukey test showed that the control group and p4 treatment groups at doses of 250, 500, and 750 ng/ml differed significantly at p<0.05, with overall mean differences of respectively -3.03, -4.18 and 2.71; the control group was not significantly different from the p4 1000 ng/ml group (table 3). discussion the p4 treatment samples had different motile sperm percentages even though all fulfilled normozoospermia c ri ter ia (motile sperm percentage > 32%). (1 0,11 ) in cont rast to progressive sperm percentages, the minimum semen criteria were achieved after p4 500 ng/ ml treatment. this data indicates that p4 increases the percentage of progressive and motile sperm. progesterone is a steroid hormone with a non-genomic action that causes increased intracellular ca2+ to trigger sperm motility and hyperactivation, and acrosome reaction.(12) therefore it is expected that the addition of an optimum dose of p4 can exert potential effects on sperm motility. the control group of this study showed the lowest hyperactivity of sperm compared with after p4 treatment. this is consistent with a previous study by sumigama et al.(13) who reported that fertilization increased when the sperm were exposed to a medium containing p4 compared to the control (bww medium). this was because the administration of p4 stimulated an increased ca2+ concentration in the sperm. a b c parameter progesterone concentration (ng/ml) p value i ii iii iv v vcl (µm/sec) lin (%) alh (µm) membrane integrity (µm/sec) 52.13 ± 12.12 40.10 ± 8.06 3.00 ± 0.82 43.00 ± 1.15 59.18 ± 8.73 43.6 ± 6.82 3.62 ± 0.70 34.00 ± 1.00 61.03 ± 15.81 43.49 ± 6.41 3.49 ± 0.85 37.00 ± 3.00 60.75 ± 23.17 43.25 ± 6.30 3.54 ± 1.09 44.00 ± 15.00 62.26 ± 9.59 42.22 ± 5.12 3.63 ± 0.84 49.00 ± 16.00 0.074 0.113 0.126 0.042 table 2. sperm motion parameter and membrane integrity in the control group and after incubation with progesterone i: control; ii: incubation with progesterone 250 ng/ml;iii: incubation with progesterone 500ng/ml; iv: incubation with progesterone 750ng/ml; v: incubation with progesterone 1000 ng/ml values are presented as mean ± sd; p= significance level, p<0.05. vcl: curvilinear velocity; lin: linearity coefficient; alh: amplitude of head lateral displacement treatment groups mean difference p value i ii iii iv v ii iii iv v iii iv v iv v 3.0333 4.186 2.710 0.930 -1.153 5.743 3.963 6.896 5.116 -1.780 0.015 0.002 0.026 0.392 0.293 0.000 0.003 0.000 0.001 0.017 table 3. plasma sperm membrane integrity with tukey hsd post-hoc comparison note : mean difference is significant at the 0.05 level 60 the elevation of intracellular ca2+ from ionic influx and detachment from the intracellular reserve is very important in sperm function, resulting in changes in sperm motility and lipid membrane remodeling which causes the sperm to form bonds with the pellucid zone and induce acrosomal reactions.(4,14) curvilinear velocity, lin and alh are important indicators of hyperactivity for successful fertilization, so it can be concluded that p4 can be a mediator for in vitro fertilization.(15,16) based on table 2, it can be seen that sperm membrane integrity has slightly decreased for 2 h incubation, based on who criteria (normal 58%). to determine whether there was a significant difference in sperm membrane integrity in each p4 treatment group, statistical analysis was performed. samples from two donors had different percentages between the control and experimental groups. lodi et al.(17) demonstrated that there is a relationship between motility, viability and membrane integrity. however, this is not in accordance with our results. the study of duru et al.(18) revealed that p4 has no effect on the hos test to improve sperm membrane integrity after postfreezing. this is also supported by research by smith et al.(19) showing that p4 can disrupt the integrity of the sperm membrane. postthaw sper m cells are alive but have low membrane integrity. the integrity of the sperm membrane is determined by several factors. henning et al.(20) reported that washing by density gradient centrifugation can cause sperm membrane integrity disruption due to the several stages of the washing process that can cause sperm cell membrane damage. mohammadi and mahdion (21) stated that the sperm membrane is biochemically disrupted during the process of freezing and thawing. in the present study, it appears that the p4 treatment has an important effect, but perhaps the study has an inadequate number of samples for this difference to be statistically significant. another limitation is that semen donor current disease was underreported, which may have c a u s e d a n un de r e s t i ma t i o n of t he t r u e association. our study was able to demonstrate that p4 in vitro increased the hyperactivated motility in sperm cells, and this can be used to improve the success rate of intra-uterine insemination.(22) next, a study on time-dependent p4 effects should be carried out to determine the time required for sperm incubation with p4. conclusions the addition of progesterone in vitro tends to increase sperm motility, but the increase is statistically not significant. plasma sperm me mbr a ne i nt e gr i t y d e c r e a se sd so th a t p r o ge s t e ro ne ha ve a n e f f e c t o n sp e r m membrane integrity. conflict of interest co mp e t i ng i nt e r e s ts : n o r e le va n t disclosures. funding disclosure there is no financial conflict of interest to disclose. acknowledgment this study was funded by competencebased cluster r ese a rch gra nt 2010 f rom universitas indonesia. this study was funded by competence-based cluster research grant 2010 from universitas indonesia. this study was funded by rese arch grant for indexed international publication for student final assignment 2017 from the research council and community services of universitas indonesia. contributors ss a nd dap we r e r e s p on si b l e f o r ma na gi n g r e s e a r c h d e s ign s, c ol l e c t e d sisca, yunaini, pujianto p4 increases hyperactivated motility 61 univ med vol. 38 no.1 manuscripts and funding. ss contributed to data acquisition and ly to data analysis. all authors have read and approved the final manuscript. references 1. lishko pv, botchkina il, kirichok y. progesterone activates the principal ca2+ channel of human sperm. nature 2011;471:387-91. doi: 10.1038/ nature09767. 2. meharvan s, chang s, selena n. non-genomic mechanisms of progesterone action in the brain. front neurosci 2013;7:159. doi: 10.3389/ fnins.2013.00159. 3. alasmari w, costello s, correia j, et al. ca2+ signals generated by catsper and ca2+ stores regulate different behaviour in human sperm. j biol chem 2013;288:6248-58. doi: 10.1074/jbc. m112.439356. 4. strünker t, goodwin n, brenker c, et al. the catsper channel mediates progesterone induced ca2+ influx in human sperm. nature 2011;471:3826. doi: 10.1038/nature09769.11. 5. baker ma, hetherington l, reeves gm, et al. the mouse sperm proteome characterized via ipg strip prefractionation and lc-ms/ms identification. proteomic 2008;8:1720-30. doi: 10.1002/pmic.200701020. 6. sayme n, dite l, krebs t, et al. positive effect of progesterone on motility and velocity of fresh, vitrified without permeable cryoprotectants and frozen with permeable cryoprotectans human spermatozoa. andrologia 2018;e13133:1-7. doi: 10.1111/and.13133 7. wiser a, sachar s, ghetler y, et al. assessment of sperm hyperactivated motility and acrosome reaction can discriminate the use of spermatozoa for conventional in vitro fertilisation or intracytoplasmic sperm injection: preliminary results. andrologia 2013;46:313-5. doi: 10.1111/ and.12068. 8. ramu s, jeyendran rs. the hypo-osmotic swelling test for evaluation of sperm membrane integrity. in: carrell d, aston k, editors. spermatogenesis. methods in molecular biology (methods and protocols). totowa nj: humana press;2013.p.21-5. 9. world health organization, department of reproductive health and research. who laboratory manual for the examination and processing of human semen. 5th ed. geneva: world health organization, department of reproductive health and research;2010. 10. perdrix a, saidi r, menard jf, et al. relationship between conventional sperm parameters and motile sperm organelle morphology examination (msome). int j androl 2012;35:491-8. doi: 10.1111/j.1365-2605.2012.01249.x. 11. cooper tg, noonan e, von eckardstein s, et al. world health organization reference values for human semen characteristics. hum reprod update 2010;16:231-45. doi: 10.1093/humupd/ dmp048. 12. lishko pv, botchkina il, kirichok y. progesterone activates the principal ca2+ channel of human sperm. nature 2011;471:387–91. doi: 10.1038/ nature0976710. 13. sumigama s, mansell s, miller m, et al. progesterone accelerates the completion of sperm capacitation and activates catsper channel in spermatozoa from the rhesus macaque. biol reprod 2015;93:1-11. doi: 10.1095/ biolreprod.115.129783. 14. rahman ms, kwon ws, pang mg. calcium influx and male fertility in the context of the sperm proteome: an update. biomed res int 2014;1-13. doi: 10.1155/2014/841615. 15. agarwal a, virk g, ong c, et al. effect of oxidative stress on male reproduction. world j mens health 2014;32:1-17. doi: 10.5534/wjmh.2014. 32.1.1. 16. breznik bp, kovacic b, vlaisavljevic v. are sperm dna fragmentation, hyperactivation and hyaluronan-binding ability predictive for fertilization and embryo development in in vitro fertilization and intracytoplasmic sperm injection? fertil steril 2013;99:1233-41. doi: 10.10.16/j. fertnstert.2013.11.048. 17. lodhi la, zubair m, qureshi zi, et al. correlation between hypo-osmotic swelling test and various conventional semen evaluation parameters in fresh nili ravi buffalo and sahiwal cow bull semen. pak vet j 2008;28:186–8. 18. duru nk, morshedi m, schuffner a, et al. semen treatment with progesterone and/or acetyl-lcarnitine does not improve sperm motility or membrane damage after cryopreservationthawing. fertil steril 2000;74:715-20. 19. smith jf, syritsyna o, fellous m, et al. disruption of the pricipal, progesterone-activated sperm ca2+ channel in a catsper2-deficient infertile patient. proc natl acad sci usa 2013;110:6823-8. 20. henning h, ngo tt, waberski d. centrifugation stress reduces the responsiveness of spermatozoa to a capacitation stimulus in in vitroaged semen. andrology 2015;3:834-42. doi: 10.1111/andr.12064. 62 21. mohammadi g, mahdion h. evaluation of membrane integrity of bull frozen-thawed sperm using water and hypo osmotic swelling test. bas j vet res 2017;16:131-43. 22. youn js, cha sh, park cw, et al. predictive value of sperm motility characteristics assessed by computer assisted sperm analysis in intrauterine insemination with superovulation in couples with unexplained infertility. clin exp reprod med 2011. 38:47-52. doi: 10.5653/cerm.2011.38.1.47. sisca, yunaini, pujianto p4 increases hyperactivated motility 1 prevention of self-harm and suicide in adolescents erita istriana department of psychiatry faculty of medicine trisakti university adolescence is a transitional stage in physical and psychological development, and in this stage multiple physical, emotional and social changes, can make adolescents vulnerable to mental health problems like self-harm and suicide. self-harm and suicide are serious problems in children, adolescents and young people with the highest rates seen in those aged 16-24 years,(1) and self-harm is highly correlated with the presence of anxiety disorders and depression.(2) the term self-harm can be used to refer to acts of self-poisoning or self-injury (eg, cutting, scratching, breaking bones and burning) carried out intentionally regardless of motive or suicidal intent.(3) there is a strong association between self-harm and risk of future suicide, with approximately 50% of adolescents who die by suicide having previously self-harmed, and selfharm increasing the risk of death by suicide approximately tenfold.(4) predicting suicide and repetition of selfharm in adolescents is a challenge as it is usually a secretive and hidden behaviour the most common reasons why adolescents participate in self-injury are as a coping mechanism, a means of relief, for the regulation of feelings, self-punishment, attention seeking and sensation seeking.(5) there is growing evidence that perfectionism and selfcriticism, low self-esteem, social isolation, impulsivity, hopelessness, poor parent-child attachment, and exposure to negative life events are associated with self-harm in clinical and community populations.(5,6) but only a small proportion of individuals who self-harm present to hospitals, meaning that this behaviour is largely hidden (at least from clinical services) at the community level. given the heterogeneity and complexity of pathways deaths to suicide, an approach that includes effective universal, secondary, and tertiary prevention strategies will likely yield the greatest benefits at a population level. for tertiary prevention among adolescents who have already shown self-harm behavior or tendencies, intensive community treatment strategies that reduce unnecessary exposure to hospitalization and crisis services may be an important component.(6) as a place where many young people spend a considerable proportion of their time, schools are one of the most important institutions that are in a position to help-self injurers, and are logical and natural settings for youth or adolescents suicide prevention efforts.(7) suicide prevention has not typically been a priority for schools unless there has been a suicide death. the programmes which have aim of enhancing emotional wellbeing among a broader population of students by using a variety of mental health promotion and mental illness prevention activities are important to cultivate the development of a set of protective factors. school-based programme can be considered to have a role to play in youth or adolescents suicide prevention strategies by using surveillance and instructional methods to enhance the identification and referral, through many activities that should take account of the particular characteristics of adolescents suicide and self-harm (for example, target issues around the social transmission of suicide and self-harm, sexual orientation and bullying in young people, promote help-seeking behaviour, and foster selfesteem and resilience), and also improve the distress-coping skills, of students who may be at risk for suicide.(7,8) study by wyman et al. (9) also showed that strengthening social integration could prevent suicidal behavior and have implications for developing network-informed suicide prevention (nisp) approaches in schools and other education settings. network-informed suicide prevention interventions will involve youth and adults and is likely to most be effective and safe by incorporating multiple focuses: maximizing 2 protective bonds across school populations, increasing opportunities for group cohesion including integrated youth-adult networks, and promoting influence of youth with healthy coping.(9) the identification of successful prevention initiatives aimed at adolescents and those at specially high risk, and the establishment of effective treatments for those who self-harm, are also paramount needs. in addition the presence of social support such support includes good peer relations, support from teachers, and a warm and stable relationship with their parents, also plays an especially important protective role to prevent self-harm and suicidal behavior among adolescents. references 1. chan mky, bhatti h, meader n, et al. predicting suicide following self-harm: systematic review of risk factors and risk scales. br j psychiatry 2016;209:277–83. 2. mars b, heron j, crane c, et al. differences in risk factors for self-harm with and without suicidal intent: findings from the alspac cohort. j affect disord 2014;168:407-14. doi: 10.1016/j.jad.2014.07.009. hawton k, bergen h, kapur n, et al. repetition of self-harm and suicide following self-harm in children and adolescents: findings from the multicentre study of self-harm in england. j child psychol psychiatry 2012;53:1212–9. 3. harris im, beese s, moore d. predicting future self-harm or suicide in adolescents: a systematic review of risk assessment scales/tools. bmj open 2019;9:e029311. doi:10.1136/bmjopen2019-029311. 4. yakeley j, james wb. psychodynamic approaches to suicide and self-harm. b j psych advances 2018, vol. 24, 37–45 doi: 10.1192/bja. 2017.6. 5. asarnow jr, mehlum l. practitioner review: treatment for suicidal and selfharming adolescents-advances in suicide prevention. j child psychol psychiatry 2019, 60:10, pp 1046– 1054 doi:10.1111/jcpp.13130. 6. singer jb, erbacher ta, rosen p. school‑based suicide prevention: a framework for evidence‑based practice. school mental health 2019;11;54-71. https://doi.org/10.1007/s12310018-9245-8. 7. hawton k, saunders kea, o’connor rc. selfharm and suicide in adolescents. lancet 2012;379:2373-82. doi: 10.1016/s0140-6736(12) 60322-5. 8. wyman pa, pickering ta, pisani ar, et al. peer-adult network structure and suicide attempts in 38 high schools: implications for network-informed suicide prevention. journal of child psychology and psychiatry 2019,60:10, pp 1065–1075 doi:10.1111/jcpp.13102. c:\users\universa medicina\docu 34 *department of physiology, faculty of medicine, trisakti university, jakarta **department of neurology, faculty of medicine, trisakti university, jakarta ***magister program in biomedical sciences, faculty of medicine, universitas indonesia, jakarta correspondence: dr. donna adriani, m.biomed departement of physiology faculty of medicine, trisakti university, jakarta jl. kyai tapa no.260 grogol jakarta, 11440 phone: +628991777777 email: donna.adriani@trisakti.ac.id orcid id: 0000-0003-4595-4097 date of first submission, april 16, 2019 date of final revised submission, march 27, 2020 date of acceptance, march 28, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina effect of brain gym ® exercises on cognitive function and brain-derived neurotrophic factor plasma level in elderly: a randomized controlled trial donna adriani*, yudhisman imran**, martiem mawi*, patwa amani*, and ermita i.i.ilyas*** background cognitive impairment and dementia are some of the major health concerns in the aging population. many studies showed positive effects of physical exercise in delaying or preventing these conditions. brain gym ® exercises is a structured aerobic exercise involving head, eyes and crossing movements of the extremities in order to stimulate both brain hemispheres. this study was conducted to evaluate the effect of brain gym ® exercises sessions on cognitive function and plasma brain-derived neurotrophic factor (bdnf) in the elderly. methods a non-blinded randomized controlled study involving 64 healthy women aged >60 years who were randomized into brain gym ® exercises treatment group (n=32) and control group (n=32). treatment group joined 60 minutes of brain gym exercises sessions twice a week for 12 weeks. the measured outcomes were cognitive function (assessed by mini-mental state examination [mmse] questionnaire) and plasma bdnf levels. the outcomes were measured at base-line and after the intervention. an independent ttest was used to analyze the data. results the bdnf levels were increased in both groups after 12 weeks, and there was a significant difference between treatment group (41.26 ± 6,82 ng/ml) and control group (37.10 ± 8.11 ng/ml)(p=0.040). however, the mmse score was not significantly different between the two groups (p=0.200). conclusion brain gym ® exercises sessions significantly increase plasma bdnf level in the elderly population. in practical terms, we may suggest evaluation of the effects of brain gym ® exercises as a strategy in the treatment of disorders associated with central degenerative changes. keywords: bdnf, brain gym exercises, elderly population original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.34-41 cite this article as: muhammad dak, imran y, mawi m, amani p, ilyas eii. effect of brain gym ® exercises on cognitive function and brain-derived neurotrophic factor plasma level in elderly: a randomized controlled trial. un iv m ed 2 020; 39:3 4-41 . doi: 10.18051/univmed.2020.v38.34-41. january-april, 2020 vol.39no.1 35 january-april, 2020 vol.39no.1 introduction the increase in longevity impacts on an increase in the elderly population (60 years and above).(1) in 2010 there were 18.1 million elderly or around 7.6 % of the total population of indonesia.(2) by the year 2025, it is estimated that the number of elderly will increase to 33.7 million, or 11.8 percent of the total population. in the year 2035, the number of elderly will increase to 48.2 million, or 15.8 percent of the total population.(2) the increase in the number of elderly requires measures for improving the quality of life of the elderly.(2) the prevalence of age-related health problems has attracted attention as a result of the increased growth of the elderly population worldwide.(3) cognitive disturbances such as mild cognitive disorder and dementia are some of the health problems encountered in the elderly.(3) the number of patients with dementia is estimated to increase two-fold every 20 years.(3) in 2050 the number of patients with dementia is projected to increase to 70.5%.(3) the more advanced a person’s age, the higher the decrease in cognitive function.(3) cognitive disturbances may cause a reduction in the quality of life of the elderly and become an economic burden for the family.(4) one of the strategies for preventing cognitive disturbances is the prevention of the modifiable risk factors, such as poor physical activity.(3,4) physical activity is associated with increased cognitive function and decreased risk of dementia in several meta-analytical studies.(5) regular and measurable physical exer cise has been demonstrated to increase the expression of brainderived neurotrophic factor (bdnf) in the hippocampus that is directly associated with improved plasticity of the brain synapses and cognitive function.(6) the improvement in synaptic plasticity has an important role in the learning process and memory functions. .physical exercise triggers increases in cellular and molecular processes, such as angiogenesis, neurogenesis, and brain synaptogenesis. in addition, there is a biological mechanism, namely increased blood flow, increased synthesis and utilization of neurotransmitters, and increased synthesis and release of bdnf. brain-derived neurotrophic factor is a protein that increases the survival of neurons and synapses that play a role in the learning process and memory.(6) although physical exercise is beneficial in delaying dementia, frequently aerobic sports or resistance training are difficult to perform by the elderly population who have a limited range of motion (rom) and have comorbidities (hypertension, coronary heart disease (chd), diabetes mellitus (dm)). brain gym ® (bg) exercises is one of the alternative forms of physical exercise that may be performed by the elderly. studies that compared three types of exercise; 1) physical exercise, 2) virtual realitybased exercise, and 3) brain exercise, concluded that brain exercise is better in increasing cognitive ability in the elderly.(7) computer-based brain exercise and games are also known to significantly increase cognitive function.(8,9) fur thermor e, th e lowered bdnf concentrations, especially in the elderly, are associated with atrophy in the hippocampus and may contribute to memory impairment, which is possibly associated with cognitive challenges in alzheimer’s disease.(10) the results of a korean study on the impact of a 12-week senior brain health exercise (sbhe) program in elderly women as compared to basic active physical fitness, on cognitive function and bdnf, showed that mini-mental state examination korean version (mmse-k) scores and bdnf concentrations, were significantly increased in the exercise group as compared with the control group.(11) the results of a study carried out by huang et al. (1 2) showed that physical exercise significantly increased bdnf concentrations. however, different results were obtained in the study conducted by schega et al.(13) on 38 elderly, in that 30 minutes of aerobic training at the rate of three times per week for four consecutive weeks did not increase the vo2max and serum bdnf level. 36 these inconsistent study results need to be further investigated. therefore the aim of the present study was to evaluate the effect of bg exercises on cognitive function and plasma bdnf concentration in the elderly. methods research design this non-blinded randomized controlled study was conducted at the mampang public health center, south jakarta. the laboratory examinations were performed in the prodia laboratories. the study was carried out from january to may 2018. research subjects elderly males and females who met the inclusion and exclusion criteria were selected for this study. participants were recruited from the mampang area, south jakarta, among those meeting the inclusion criteria, namely age 60 years and older, capable of walking unaided, and agreeing to participate in the study (by signing the informed consent form) after receiving particulars on this study. the exclusion criteria were: suffering from psychotic and/or neurologic disturbances, consuming antidepressants or antipsychotics, having malignancies, or not participating until completion of the study. the sample size was determined from an effect size of 0.7, =0.05 and =0.2, so that the optimal sample size was 26 per group, which with the addition of an anticipated drop-out rate of 20%, yielded the required total sample size of 32 per group. therefore the 64 participants who were qualified for the study were blockrandomized with a block size of four into the control group (n=32) and the brain gym group (n=32). data collection the demographic data (age, sex, job, and educational level) of the participants were c o l l e c t e d du r i n g a n i n t e r vie w u si n g a questionnaire. the interview also obtained data on regular physical exercise, history of past illness, current illness and smoking. measurements hight wa s measur ed with a portable microtoise with accuracy of 0.1 cm and weight was measured with portable sage scales with accuracy of 0.1 kg. the body mass index was determined by dividing the weight in kilograms by the square of the height in meters. the blood pressure was measured using a pre-calibrated mercury sphygmomanometer. the blood pressure was measured as follows: the respondents were seated or at rest for 10 minutes, the right arm was positioned on the table at heart level, the cuff was wrapped around the upper right arm at a distance of 3 cm above the cubital fossa, the “on” button was pressed so that the instrument automatically inflated the cuff, and the resulting systolic and diastolic blood pressure became visible on the screen. the results of the first blood pressure measurement was recorded, followed by a waiting period of 1-2 minutes, then a second blood pressure measurement was performed, followed by another waiting period of 1-2 mi nu t e s , t h e n a t h i r d bl o od p r e s su r e measurement was done. the results of the three blood pressure measurements were added and divided by three to obtain the mean blood pressure. in case there were differences of >10 mmhg between the three systolic or diastolic blood pressures, the blood pressure examination had to be repeated. the participant performed the six-minute walk test, during which he or she evaluated his or her own fitness using the borg scale, which measures dyspnea and fatigue scores from 6 (signifying no exertion at all or zero exertion) to 20 (maximal exertion). the procedure of the sixminute walk test is as follows: before starting the test, the participant’s weight, height, blood pressure, pulse rate per minute, oxygen saturation, and borg dyspnea and fatigue score are measured. then the participant is tested for static and muhammad dak, imran y, mawi m, et al. brain gym ® exercises and elderly population 37 january-april, 2020 vol.39no.1 dynamic balance. the subjects are instructed to walk along a path at the highest speed possible for six minutes, after which they are to come to a stop. the total distance walked from the start to the stop sign is measured in meters. the subjects are then asked to sit down and immediately afterward their blood pressure, pulse rate per minute, oxygen saturation and borg dyspnea and fatigue score are again measured. subsequently the predicted vo2 max is calculated using a standard formula. measurement of cognitive function the mini-mental state examination (mmse) is a widely used cognitive screening test, where scores from 24 to 30 are considered to be within the normal r ange. items address orientation, memory, recall, attention, naming objects, following verbal and written commands, writing a sentence, and copying a figure.(14) intervention the treatment group joined bg sessions twice a week for 12 weeks. each session lasted 60 minutes with 10 minutes warming up, 40 minutes bg, and 10 minutes cooling down. the exercise was led by a professional who made sure every partic ipa nt made the corr ect movements. the control group did not participate in any course. the bg protocol used in the present study followed the super body, super brain procedure of michael gonzales-wallace.(15) the exercise was preceded by a warming-up period of 10 minutes , consisted of core movements for 40 minutes, and was followed by a cooling-down period of 10 minutes. a b c figure 1. flow diagram of the subjects included in this study 38 blood sample collection after overnight fasting, venous blood samples were collected between 8:00 and 10:00 a.m. blood samples were taken at the very beginning and at the end of the bg program. blood samples were drawn from a vein in the cubital fossa region to a volume of 10 ml and immediately put in the edta tubes. the blood samples were then centrifuged at 3000 rpm and the plasma was obtained. plasma aliquots were made in 1 ml vacutainer tubes and were kept at -800c until required for examination. serum bdnf concentration was measured using an elisa kit provided by mybiosource, usa. the principle of the procedure was indirect competitive enzyme immunoassay, where the bdnf c oncentratio n was determined spectrophotometrically at 450 nm in a microplate elisa reader and compared with the standard curve. t he color inte nsity was inversely proportional to the bdnf concentration. blood glucose was measured spectrophotometrically using the advia 1800 instrument and bayer advia 1800 74024 glucose reagent. statistical analysis normally distributed data were presented as mean ± sd. the kolmogorov-smirnov test (p>0.05) was used to check sample normality an independent t-test analysis was used to verify difference in bdnf and cognitive function (mmse-k) between groups (exercise group and control group). statistical significance for all tests was accepted at the level below 0.05.table 1. distribution of the general characteristics of the participants at base-line ethical clearance ethical clearance for this study was issued by the ethics commission, faculty of medicine, trisakti university, under no. 119/ker/fk/xii/ 2017. results at the beginning of the program, 64 participants met the criteria and volunteered to join the program (control group n=32 and treatment group n=32). however, 6 participants from the control group could not finish the program. a total of 58 subjects finished the program and the data were analyzed. there was no report of injury or medical concern during the bg sessions. at base-line there were no significant differences between the treatment and control groups. this showed that the block randomization that had been performed was successful in equalizing all other variables in both groups, apart from the treatment variable (table 1). after 12 weeks of training there was a significant difference in plasma bdnf levels between the treatment and control groups (p=0.040). however, there was no significant difference in mmse scores between treatment and control groups (p=0.200) (table 2). muhammad dak, imran y, mawi m, et al. brain gym ® exercises and elderly population characteristics brain gym treatment (n=32) control (n=32) p value age (years) height (cm) body weight (kg) bmi (kg/m2) sbp (mmhg) dbp (mmhg) pulse rate bdnf (ng/ml) mmse 65.48 ± 4.51 151.59 ± 5.75 56.99 ± 6.67 25.75 ± 2.13 124.65 ± 18.31 79.84 ± 9.87 80.32 ± 5.19 39.43 ± 10.98 25.032 ± 2.99 64.77 ± 4.31 150,80 ± 7.12 60.150 ± 8.16 26.73 ± 2.78 130.87 ± 25.50 82.00 ± 13.26 79.55 ± 4.99 33.61±14.52 23.42 ± 3.43 0.530 0.633 0.100 0.091 0.145 0.102 0.824 0.091 0.324 table 1. distribution of the general characteristics of the participants at base-line *bmi: body mass index; sbp: systolic blood pressure; dbp: diastolic blood pressure; bdnf: brain-derived neurotrophic factor; mmse: mini mental state examination 39 january-april, 2020 vol.39no.1 discussion our study showed that bg exercise for 12 weeks was capable of increasing serum bdnf concentrations in the elderly. these study results were in agreement with the results of the study of szuhany et al.(5) on 1111 subjects, in which the investigators stated that bdnf concentrations had a statistically significant increase in the physical exercise group. the increase in plasma bdnf concentration in the group with regular physical exercise was higher than that in the group with a single exercise. the increase in plasma bdnf concentration was relatively associated with exercise dose.(5) in the study conducted by zoladz et al.(16) it was also found that aerobic physical exercise may increase bdnf concentration. brain-derived neurotrophic factor is capable of regulating longterm potentiation (ltp) in the hippocampal area by strengthening the synaptic responses toward tetanic stimulation. long-term potentiation as a form of synaptic plasticity that plays a role in longterm memory formation.(16) the human brain is a structure that easily undergoes functional adaptation, so as to affect cognitive function. brain plasticity is affected by experience, which can be stimulated by physical activity. physical activity may make various changes in the brain. the physical exercise that is capable of affecting cognitive function is aerobic physical exercise, since it can provide the best environment for cortical activity, hemodynamics, and metabolism.(17) aerobic physical exercise is an activity that is planned, structured, and performed repeatedly, to improve physical fitness.(18) physical exercise can increase neuroplasticity. in humans, the indicator that plays a role in changing to brain structure is the brain-derived neurotrophic factor that is correlated with increased cognitive function. the biological mechanism that causes this is aerobic physical exercise that increase blood flow to the brain, increases oxygen in the cerebral tissues, and increases neuroplasticity.(19) this increase in cognitive function is caused by bdnf activity. brain-derived neurotrophic factor is a protein found at high concentrations in the central nervous system, particularly in the hippocampus, cerebral cortex, hypothalamus, and cerebellum. bdnf is capable of penetrating the blood-brain barrier so that its concentrations can be detected in the blood.(5) in the study of rasmussen et al.(20) it was found increased plasma bdnf concentrations during aerobic physical exercise. bdnf plays an important role in neuronal growth and endurance. brain-derived neurotrophic factor also affects learning and memory. synthesis of bdnf is mediated and influenced by physical activity. physical exercise increases the transcription of bdnf in the brain. physical exercise increases circulatory bdnf concentrations. physical exercise increases the concentrations of bdnf, which is a trophic factor associated with increases in cognitive function. in a study on the effect of exercise on bdnf expression in the laboratory mouse (mus musculus), it was found that exercise resulted in the release of an endogenous molecule, namely â-hydroxybutyrate, which increases the activity of bdnf promoters and leads to an increase in neurotransmitter release that is dependent on the trkb receptor. this explains the endogenous mechanism of increased bdnf by physical exercise.(21) the results of our study showed that after performing bg exercise for 12 weeks, there were no significant differences in cognitive function between the treatment and control groups. differing results were obtained in a study brain gym (n=32) control (n=26) p value bdnf (ng/ml) mmse 41.26 ± 6.82 25.93 ± 2.78 37.10 ± 8.11 24.50 ± 3.36 0.040 0.200 table 2. bdnf and mmse levels after 12 weeks of bg exercises bdnf: brain-derived neurotrophic factor; mmse: mini mental state examination 40 involving 24 women the age range of 65-79 years, who underwent a sbhe program for 12 weeks. it turned out that cognitive function was underwent a statistically significant increase in the experimental group when compared with the control group.(11) a different study found that a physical exercise and cognitive program for 12 weeks could increase the efficiency of brain activation during cognitive tasks in elderly aged more than 60 years. the group performing aerobic physical exercise had significantly increased memory and executive functions as compared with the control group.(22) according to a number of systematic reviews, aerobic-type training in the elderly has mild but heterogeneous effects on cognition and memory capacities.(23,24) this may have been the result of the necessity for minimal duration, frequency and intensity in a program of short duration, to induce changes in global cognition. brain-derived neurotrophic factor is the main regulator of neurogenesis that is associated with synaptic transmission and long-term potentiation. aerobic physical exercise performed for 4 weeks may increase memory function in individual who previously were in the habit of performing aerobic physical exercise.(25) the aging process is associated with a gradual decrease in cognitive function and increas ed risk of cognitive disturbances.(26) the aerobic physical exercise and cognitive program (brain gym) for 12 weeks is expected to be capable of preventing decreased cognitive function in the elderly.(20) our study has several limitations. we cannot exclude that participants performed additional unrecorded physical exercise outside the program, constituting an additional confounding factor affecting the results. other limitations include unknown group differences in the presence of ge netics f actors, such as bdnf gene polymorphism, which could influence bdnf synthesis. in addition, further research is also needed to determine whether peripheral plasma bdnf reflects central bdnf in the elderly. conclusions based on the results of this study it can be concluded that bg exercise performed regularly twice weekly for 12 weeks by healthy elderly women can significantly increase plasma bdnf concentrations. conflict of interest compe ting inte re sts: no re levant disclosures. acknowledgement the authors wish to express their gratitude to the faculty of medicine, trisakti university, for the funding of this study. contributors dakm, yi, mm, pa, eiii contributed to the concept and design of the study, data acquisition, data analysis, and/or interpretation. dakm and yi drafted and revised the manuscript critically for important intellectual content. all author s had read and approved the final manuscript. references 1. kementerian kesehatan ri. analisis lansia di indonesia. jakarta: pusat data dan informasi kementerian kesehatan ri;2017. 2. united nations population fund (unfpa) indonesia. indonesia on the threshold of population ageing. jakarta : united nations population fund (unfpa) indonesian;2014. 3. mavrodaris a, powell j, t horogood m. prevalences 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ludyga s, gerber m, brand s, holsboer-trachsler e, pühse u. acute effects of moderate aerobic exercise on specific aspects of executive function in different age and fitness groups: a metaanalysis. psychophysiology 2016;53:1611–26. doi: 10.1111/psyp.12736. 18. world health organization. global recommendations on physical activity for health. geneva; world health organization; 2010. 19. mandolesi l, polverino a, montuori s, et al. effects of physical exercise on cognitive functioning and wellbeing: biological and psychological benefits. front psychol 2018;9: 1– 11. doi: 10.3389/fpsyg.2018.00509. 20. rasmussen p, brassard p, adser h, evidence for a release of brain-derived neurotrophic factor from the brain during exercise. exp physiol 2009;94:1062-9. doi: 10.1113/ expphysiol.2009. 048512. 21. sleiman sf, henry j, al-haddad r, et al. exercise promotes the expression of brain derived neurotrophic factor (bdnf) through the action of the ketone body ?hydroxybutyrate. elife 2016;5: pii: e15092. doi: 10.7554/elife.15092. 22. nishiguchi s, yamada m, tanigawa t, et al. a 12week physical and cognitive exercise program can improve cognitive function and neural efficiency in community-dwelling older adults: a randomized controlled trial. j am geriatr soc 2015;63:1355-63. doi: 10.1111/ jgs.13481. 23. sanders lmj, hortobágyi t, la bastide-van gemert s, van der zee ea, van heuvelen mjg. dose-response relationship between exercise and cognitive function in older adults with and without cognitive impairment: a systematic review and meta-analysis. plos one 2019;14:e0210036. doi: 10.1371/journal.pone. 0210036. 24. öhman h, savikko n, strandberg te, pitkälä kh. effect of physical exercise on cognitive performance in older adults with mild cognitive impairment or dementia: a systematic review. dement geriatr cogn disord 2014;38: 347-65. doi: 10.1159/000365388. 25. basso jc, suzuki wa. the effects of acute exercise on mood, cognition, neurophysiology, and neurochemical pathways: a review. brain plast 2017;2:127-52. doi: 10.3233/bpl-160040. 26. brown bm, peiffer jj, sohrabi hr, et al. intense physical activity is associated with cognitive performance in the elderly. transl psychiatry 2012;2:e191. doi: 10.1038/tp.2012. 118.10.1038/ tp.2012.118. oktavianus 163 *department of anatomy, faculty of medicine, sultan agung islamic university **department of biochemistry, faculty of medicine, sultan agung islamic university ***department of chemistry, faculty of medicine, sultan agung islamic university **** department of pharmacology, faculty of medicine, sultan agung islamic university correspondence dr. hj. chodidjah, m.kes, pa department of anatomy, faculty of medicine, sultan agung islamic university jl. raya kaligawe km.4, semarang 50112 email: chodiab@yahoo.com univ med 2014;33:163-70 abstract universa medicina september-december, 2014september-december, 2014september-december, 2014september-december, 2014september-december, 2014 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 vol.33 no.3 background breast cancer treatment is still ineffective, having also various side effects. breast cancer growth is affected by human epidermal growth factor receptor 2 (her2/neu) and b cell lymphoma 2 (bcl2) expression. in vitro studies on continuous culture of continuous culture of human lymphoblasts (cems) showed that typhonium flagelliforme (tf) increases apoptosis. the objective of this study was to evaluate whether tf syrup (tfs) could decrease her2/ neu and bcl2 expression as well as breast cancer volume (bcv) in mice. methods an experimental post-test only control group design was conducted on 18 c3h mice with breast cancers, randomly allocated to 3 groups of 6. group 1 received 0.2 ml of distilled water. group 2 and 3 animals were each given 0.2 ml of 40 mg/ml and 80 mg/ml tfs, respectively. the treatment was given orally once daily for 25 days. assessment of her2/neu and bcl2 expression was by immunohistochemistry, whereas bcv was measured by caliper. anova and lsd were used for data analysis. results there was a significant difference in her2/neu and bcl2 expression as well as in bcv among the treatment groups. lsd analysis showed that her2/neu and bcl2 expression in group 3 (51.60%; 24.60%) was significantly lower than in group 1 (245.40%; 114.40%) as well as group 2 (235.50%; 54.20%) (p=0.000). bcv in group 3 (4392.33 mm3) was significantly greater than bcv in group 1 (253.87 mm3) (p=0.002), but was not significantly different from bcv in group 2 (3667.16 mm3) (p=0.306). conclusion suplementation with tfs decreases her2/neu and bcl2 expression. tf appears to be a promising plant demonstarting anti cancer activity. keywords: breast cancer, bcl2, her2/neu, apoptosis, mice typhonium flagelliforme decreases protein expression in murine breast cancer chodidjah*, taufiqurrahman nasihun**, eni widayati***, and edijanti goenarwo**** 164 chodidjah, nasihun, widayati, et al typhonium flagelliforme in murine breast cancer typhonium flagelliforme menurunkan ekspresi protein pada kanker payudara mencit pendahuluan pengobatan kanker payudara (kp) belum efektif, masih menyisakan efek samping. perkembangan kp dipengaruhi oleh ekspresi human epidermal growth factor receptor 2 (her2)/neu dan b cell lymphoma 2 (bcl2). penelitian pada sel continuous culture of human lymphoblasts (cems) menunjukkan, umbi keladi tikus (ukt), meningkatkan sekresi cytochrome c, ekspresi caspase3, fragmentasi dna, dan apoptosis. tujuan penelitian ini untuk membuktikan, sirup ukt mampu menurunkan ekspresi her2/neu, bcl2, dan volume kanker payudara. metode rancangan eksperinmental post test only control group design mengikut sertakan 18 mencit c3h dengan kp’ mereka recara random alokasi sederhana dibagi dalam 3 kelompok (6 mencit setiap kelompok). kelompok 1, mendapat aqua 0.2 ml. kelompok 2 dan kelompok 3, diberi 0.2 ml (40 mg/ml) dan 0,2 ml (80 mg/ml) sirup ukt. perlakuan dilakukan sekali sehari secara oral selama 25 hari. pemeriksaan ekspresi her2/neu dan bcl2 dilakukan dengan immunohistokimia, sedangkan volume kanker diukur dengan kaliper. analisis statistik menggunakan oneway anova dan lsd, dengan tingkat kemaknaan <0,05. hasil terdapat perbedaan ekspresi her2neu, bcl2, dan volume kp yang bermakna antara ketiga kelompok perlakuan (p=0,000). analisis lsd menunjukkan ekspresi her2/neu dan bcl2 (51,60%; 24,60%) pada kelompok 3 lebih rendah bermakna dibanding kelompok 1 (245,40%; 114,40%) (p=0,000). demikian pula dibanding dengan kelompok 2 (235,50%; 54,20%) (p=0,000). volume kp pada kelompok 3 (4392,33 mm3) lebih tinggi bermakna dibanding kelompok 1 (2563,87 mm3) (p=0,002), dibanding dengan kelompok 2 (366,16 mm3) tidak menunjukkan perbedaan bermakna (p=0,306). kesimpulan pemberian sirup ukt mampu menurunkan ekspresi her2/neu dan bcl2. umbi keladi tikus nampaknya mempunyai aktivitas anti kanker. kata kunci: kanker payudara, bcl2, her2/neu, apoptosis, mencit abstrak introduction breast cancer (bc) is a complex cancer that is most frequent among women after cancer of the cervix, in indonesia and asia, as well as globally.(1,2) several factors, genetic as well as environmental, play a role in bc pathogenesis. among the various types of bc, invasive ductal carcinoma is the most frequent.(3) the modern modes of treatment of bc still rely on surgery, irradiation, and cytostatics. these treatment m o d e s , b e s i d e s r e q u i r i n g s u b s t a n t i a l expenditures, also do not yet give satisfactory results, and even leave various harmful side e ff e c t s , s u c h a s h a i r l o s s a n d s k i n hyperpigmentation. in view of this situation, it is essential to search for alternative treatments of bc that are more effective, more affordable, and safer, particularly treatments using natural or herbal substances. traditionally, communities have used typhonium flagelliforme [tf] (rodent tuber, umbi keladi tikus) for treatment of bc. on the other hand, there has been no definitive scientific evidence for the curative action of tf on bc. the curative effect of tf on bc needs to be proven, in view of the increasing numbers 165 of bc patients. it is estimated on the basis of global data that there are more than 1 million women with the diagnosis of bc, with the number of deaths amounting to more than 410.000.(4,5) so far, there have been only a few studies on tf. one of these is the in vitro study on continuous culture of human lymphoblasts (cems) treated with 10 and 20 µg/ml of tf. the results of this study showed that tf could increase cytochrome c secretion, caspase 9 and caspase 3 expression, dna fragmentation, and a p o p t o s i s . ( 6 ) a n i n v i v o s t u d y o n o r a l administration of tf to balb/c mice with induced leukemia found that tf was able to reduce the numbers of immature granulocytes. the treatment with tf was carried out for 28 days, using doses of 200, 400, and 800 mg/kg bw/day.(7) the abovementioned results of increased cytochrome c secretion, caspase 9 and caspase 3 expression, dna fragmentation, and apoptosis, may provide clues to the curative action of tf on bc. tr a d i t i o n a l l y, t f h a s b e e n u s e d b y communities for the treatment of cancers. the compounds present in tf that are considered to have curative effects on cancer are the flavonoids. epidemiological studies of lung cancer have shown that flavonoids are beneficial for its treatment.(8) the study conducted by sghaire et al.(9) also showed that flavonoid-containing plants are capable of inducing apoptosis and inhibiting the proliferation of human leukemia cells. furthermore, a study using the flavonoids genkwanin and chrysin, in combination with the enzyme cyp1 (polypeptide 1, subfamily a, family 1, of the cytochrome p450 group), that plays a role in the metabolism of xenobiotics, showed an increased antiproliferative effect on bc cells.(10) on the other hand, trastuzumab, an anti-bc agent that has been widely and effectively used as chemotherapeutic adjuvant, being a monoclonal antibody derived from humanized recombinant dna, is able to bind to the extracellular domain of the human epidermal growth factor receptor-2 (her2)/neu. this binding prevents the binding of the natural ligand to the receptor and reduces the number of receptors by a process of endocytosis.(11) in addition, trastuzumab is also able to directly inhibit the activity of tyrosine kinase as messenger protein. in consequence, these two processes lead to decreased proliferation of bc cells.(12) with reference to this mechanism, trastuzumab is also effectively used in the treatment of bc metastases and expresses her2/ neu.(13-15) the flavonoid-containing tf, which has been shown to induce apoptosis and to be antiproliferative, is expected to be able to reduce bc volume, by a mechanism similar to that of trastuzumab, using decreased her2/neu and bcl2 expression as indicators. therefore the present study was conducted with the objective to evaluate the effect of administration of tf syrup against bc, with as indicators the reduction in expression of her2/neu and bcl2, and bc volume in female mice. methods study design the study design used was the experimental post test only control group design. the study was conducted from march to april 2013 in the experimental laboratory, faculty of medicine, university of indonesia, jakarta, for the intervention and preparation of paraffin blocks. immunohistochemical assays for her2/neu and bcl2 expression were done in the laboratory of pathologic anatomy, prof dr sardjito hospital, jogyakarta. experimental animals the sample used in this study comprised 3 month-old female ch3 mice, weighing around 20-25 grams, obtained from the experimental laboratory, faculty of medicine, university of indonesia, jakarta. the sample size was determined according to who guidelines to be 6 mice per group. the mice were adapted for one week, after which the tumor pulp was transplanted by subcutaneous injection of 0.2 ml tumor pulp into each mouse (to a total of 18 univ med vol. 33 no.3 166 chodidjah, nasihun, widayati, et al typhonium flagelliforme in murine breast cancer mice). the tumor pulp was prepared from a tumor taken from a donor mouse that had been killed by ether anesthesia. the tumor was cleaned from blood vessels and necrotic tissue, minced, then physiologic saline was added in a 1 : 1 ratio. one week after transplantation, ± 2 mm x 2 mm x 1mm palpable tumors had grown in all mice. intervention after the growth of the tumors, the mice were randomly allocated to three groups of 6 mice each. group 1 was given 0.2 ml distilled water, group 2 was given 0.2 ml of 40 mg/ml tf syrup, and group 3 was given 0.2 ml of 80 mg/ ml tf syrup. all interventions were given orally, once daily, for 25 days. preparation of typhonium flagelliforme syrup the fresh tf rhizomes were washed in running water, then sliced. six kilograms of tf rhizome was then extracted by maceration using 60 l of 90% ethanol as solvent (1:10), the resulting extract weighing 50 g. simple syrup was prepared from saccharose 130 g and nipagin 0.25% b/v. the mixture was stirred on a low flame until homogenous, then hot distilled water was added to a volume of 200 ml. the 40 mg/ml tf syrup (tfs) was made by adding 100 ml of simple syrup to 4 g tf extract, while the 80 mg/ ml tfs was prepared from 8 g tf extract plus 100 ml simple syrup. t h e t f s y r u p w a s p r e p a r e d i n t h e laboratory of chemistry, faculty of medicine, sultan agung islamic university, semarang, from tf rhizomes obtained from west semarang, central java. measurement of breast cancer volume at the end of the study (day 26), the size of the tumors was measured (length, width, height in mm) using calipers. determination of bcl-2 and her2/neu expression after the tumor had been measured, a tissue s a m p l e w a s t a k e n f o r p r e p a r a t i o n o f histopathology slides, which were stained immunohistochemically to determine her2/neu and bcl2 expression. the reagents used for preparation of the histopathology slides comprised: h 2 o 2 3%; trypsin 0.25% in phospate buffered saline (pbs); 3,3’-diaminobenzidine (dab) solution (as color indicator) prepared from 1 ml distilled water, 50 drops of h 2 o 2 buffered substrate, 1 drop of dab stock solution; x y l o l , a b s o l u t e e t h a n o l , a n d e t h a n o l a t concentrations of 70%, 80%, and 95%; antiher2/neu/ erbb2 (us biological); and anti-bcl2. the histopathology slides were prepared by deparaffining, i.e. immersion of the tissue sections consecutively for 2 minutes in xylol, then in absolute ethanol, 95% ethanol, 80% ethanol, and 70% ethanol, respectively, for 1 minute, and running water for 10-15 minutes. the slides were then immersed in 3% h 2 o 2 for 30 minutes, washed three times in pbs for 2 minutes each, placed in 0.25% trypsin for 6 minutes at 370c, then again washed three times in pbs for 2 minutes each. after this the slides were placed in the monoclonal antibody solutions of anti bcl2/anti her2/neu for 30 minutes, washed three times in pbs for 2 minutes each, placed in peroxidase-labeled secondary antibody solution, washed three times in pbs for 2 minutes each, placed in chromogen substrate solution for 5 minutes, washed three times in pbs for 2 minutes each, then rinsed in distilled water. the slides were then immersed in mayer hematoxylin stain for 6 minutes, washed in running water, and dehydrated, cleared, and mounted. bcl-2 and her2/neu expression was determined by light microscopy at 400x magnification, using the hot spot area method in 10 fields of view. in each field 100 cells were examined, and the numbers counted of the cells expressing her2/neu (brown colored cell membrane), and bcl2 (brown colored cytoplasm).(23) statistical analysis statistical analysis was by one-way anova followed by lsd, using a significance level of 0.05. 167 ethical clearance the study was conducted after ethical clearance from medical/health bioethics commission, faculty of medicine, sultan agung islamic university, semarang. results during the intervention, one mouse from group 2 (the 40 mg/ml group) died on day 15 and one mouse from the 80 mg/ml group died on day 21. up to the end of the study, the number of mice in the control group was still complete (6 animals), whereas in groups 2 and 3 there only remained 5 animals in each. the death of the animals is believed to be caused by lowered immune defenses as a result of tumor growth. after administration of the intervention for 25 days, the c3h mice with bc showed differences in her2/neu expression between the three groups (figure 1). the lowest her2/neu expression was in group 3 (40 mg/ml), followed by group 2 (20 mg/ml), and the highest expression was in group 1 (controls). results of anova indicated that the differences were significant (p=0.000). furthermore, lsd analysis showed that her2/neu expression in group 3 was significantly lower than that in group 1 (p=0.000). in comparison with group 2, her2/ neu expression in group 3 was also significantly lower (p=0.000). this was also the case with her2/neu expression in group 2, which was significantly lower than that in group 1 (p=0.011) (table 1). the bcl2 protein assay results also found differences in expression between the 3 groups (figure 2). bcl2 expression was lowest in group 3 (40 mg/ml), followed by group 2 (20 mg/ml), while the highest expression was in group 1 (controls). results of anova indicated that the differences were significant (p=0.000). lsd statistical analysis yielded results indicating that bcl2 expression in group 3 was significantly far lower than that in group 1 (p=0.000). n comparison with group 2, bcl2 expression in group 3 was also significantly lower (p=0.000). this was also the case with bcl2 expression in group 2 which was significantly lower than that in group 1 (p=0.000) (table 1). figure 2. bcl2 protein expression (brown colored cytoplasm) in the treatment groups group 1 group 2 group 3 univ med vol. 33 no.3 figure 1. her2/neu expression (brown colored cell membrane) in the treatment groups group 1 group 2 group 3 168 chodidjah, nasihun, widayati, et al typhonium flagelliforme in murine breast cancer table 1. mean her2/neu and bcl2 expression and mean breast cancer volume discussion the results of this study showed that administration of tfs at doses of 20 and 40 mg/ ml for 25 days was able to significantly reduce her2/neu expression in breast cancer cells in comparison with the control group. these results are in accord with the results of chen’s study, proving that flavonoids are able to effect degradation of her2neu receptors, in spite of t h e u n e x p l a i n e d m e c h a n i s m o f s i g n a l transduction.(17) the results are also in agreement with other studies, providing evidence that flavonoids, compounds frequently found in plants, are able to inhibit the in vivo proliferation of both hematopoietic cancer cells and bc cells.(9,10) this proliferation-inhibiting effect of flavonoids on cancer cells is strongly believed to be similar to that of trastuzumab or tamoxifen. the results of a study involving 3 randomized controlled trials (rcts) showed that the addition of trastuzumab, a monoclonal antibody against the her2/neu receptor, to anthracyclin and taxan used as chemotherapeutic adjuvants, was found to be of benefit in improving the survival rate of women with her2/neu-expressing bcs.(13) in addition, administration of the anti-estrogen tamoxifen as chemotherapeutic adjuvant in postmenopausal patients with phase 1 bc expressing estrogen receptors, was also of substantial benefit, and could even reduce the risk of recurrences by 35–50% 15 years after surgery.(13) both these facts illustrate how her2/ neu and estrogen receptors in bc cells may be manipulated with monoclonal antibodies as well as receptor agonists to cure cancer. in the reduction of her2/neu expression by flavonoids, these compounds do not involve monoclonal antibodies, but function as agonists of the her/ neu receptor. the activity of flavonoids as her2/ neu agonists causes a decrease in the expression o f h e r 2 / n e u r e c e p t o r s , s o t h a t n o c e l l p r o l i f e r a t i o n t a k e s p l a c e . f u r t h e r m o r e , flavonoids are strongly believed to be able to play the role of estrogen receptor agonists in cancer cells. they are thought to cause a decrease in the numbers of estrogen receptors, so leading to cessation of cell proliferation. there is evidence that the flavonoid genistein is a strong estrogen agonist and acts to inhibit tumor cell proliferation in vitro.(18,19) referring to these various facts, it may be deduced that flavonoids contained in tf can act as agonists of her2/ neu as well as estrogen receptors, although this requires further studies. the results of the present study also prove that administration of tf at a dose of 0.2 ml (40 mg and 80 mg/ml) was capable of reducing bcl2 expression in bc cells. these study results are consistent with those of a study by mohan,(7) showing that administration of tf was able to decrease bcl2 expression, dna fragmentation, and apoptosis. bcl2 is an anti-apoptotic protein, involved in the survival and proliferation of cancer cells. bcl2 acts as anti-apoptotic protein by preventing cytochrome c and diablo/smac her=human epidermal growth factor; bcl=b cell lymphoma; bcv= breast cancer volume 169 release from the mitochondria. diablo/smac is a protein that plays an important role in binding to the inhibitor of apoptosis protein (iap), so that once released by the mitochondria, it will bind to iap, inducing the cell to undergo apoptosis, resulting in tumor regression.(20) with reference to the roles of bcl2 and the her2/neu receptor as mentioned above, the reduction in cancer volume is a result of decreased expression of the bcl2 protein and the her2/neu receptor in breast cancer cells, through the mechanisms of apoptosis and antiproliferation. however, the present study was shown to yield the opposite result, because an i n c r e a s e i n b c v o l u m e w a s f o u n d a f t e r administration of tf. this contradictory result is strongly believed to be caused by the type of flavonoids contained in tf. flavonoids have in general positive roles in human health. various studies show that flavonoids have anti-ischemic, anti-platelet, anti-inflammatory, and antilipoperoxidant effects.(9) in addition, flavonoids have also been shown to inhibit the activities of various enzymes in the oxidation system, such a s l i p o x yg e n a s e s, c y c l o o x yg e n a s es, monoxygenases, and xanthine oxidases. the activities of these enzymes are closely related to the antioxidant properties of flavonoids. the antioxidant effect of flavonoids is due to neutralization of ros, which initiates lipid peroxidation and lipid peroxides by binding metal ions and the system of enzymes that is responsible for the formation of free radicals. on the other hand, flavonoids, particularly quercetins, in addition to possessing antioxidant functions, also have mutagenic and carcinogenic effects.(21,22) with reference to this fact, the increase in bcv after receiving tf is strongly thought to be related to the mutagenic and carcinogenic properties of flavonoids. these properties are substantially the result of the pro-oxidant activity of flavonoids (quercetin) that is related to that of cathecol or pyrogallol, which may produce free radicals in the presence of metal ions, such as fe and zn.(9) in consequence, although there is a reduction in her2/neu receptor and bcl2 protein expression, the cells do not undergo apoptosis, and there is even an increase in proliferation, so that the volume of the cancer is increased. the mutagenic and carcinogenic properties are not found in all flavonoids. an analysis of tf content by a study team before starting their study, showed that the majority of flavonoids present in tf is of the quercetin type. this agrees with the evidence reported by sghaire et al.,(9) who showed that flavonoids with mutagenic and carcinogenic properties are found in the quercetin group. in connection with this fact, one limitation of the present study is the omission to measure the fe and zn concentrations, that may promote the mutagenic effect of the quercetin flavonoids on breast cancer growth. therefore further studies are required, where in addition to measurement of fe and zn concentrations during the administration of flavonoids, it should be determined which flavonoids have mutagenic and carcinogenic properties, so that only safe flavonoids will be consumed by the community. another limitation of this study was that it did not measure other variables that play a role in proliferation or apoptosis. conclusion administration of typhonium flagelliforme syrup was able to decrease expression of the her2neu receptor and the bcl2 protein in mice, but did not decrease breast cancer volume. acknowledgments the investigators wish to extend their gratitude to the ministry of higher education (dikti) for the funding of this study, to dra puspita ekawuyung, msi and mr. slamet of the laboratory of pathologic anatomy, faculty of medicine, university of indonesia, and ms. agustin, amd of the laboratory of pathologic anatomy, prof dr sardjito hospital, yogyakarta, for their assistance in the successful completion of this study. univ med vol. 33 no.3 170 chodidjah, nasihun, widayati, et al typhonium flagelliforme in murine breast cancer references 1. kimman m, norman r, jan s, kingston d, woodward m. the burden of cancer in 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2008;55:297-302. 17. azimi i, robert-thomson sj, montith gr. calcium influx pathway in breast cancer: opportunities for pharmacological intervention. br j pharmacol 2014;171:945-60. 18. galluzzo p, marino m. nutritional flavonoids impact on nuclear and extranuclear estrogen receptor activities. genes nutr 2006;1:161-76. 19. kang mh, reynolds p. bcl2 inhibitors: targeting mitochondrial apoptotic pathway in cancer therapy. clin cancer res 2009;15:112632. 20. caretta a, mucignat-caretta c. protein kinase a in cancer. cancers 2011;3:913-26. doi:10. 3390/cancers3010913. 21. resende fa, vilegas w, santos lc, varanda ea. mutagenicity of flavonoids assayed by bacterial reverse (ames) test. molecules 2012;17:525568. 22. blessing eo, francis m. dose–related effect of quercetin in the human breast carcinoma mcf7 cell line. am j sci ind res 2010;1:242-61. 23. sudiana ik. teknologi ilmu jaringan dan imunohistokimia. jakarta: sagung seto;2005. oktavianus 92 *department of public health, faculty of medicine, university of lambung mangkurat, banjarmasin **department of pharmacology, faculty of medicine, university of lambung mangkurat, banjarmasin ***department of embryology, faculty of veterinary medicine, university of airlangga, surabaya ****department of obstetrics and gynecology, faculty of medicine, dr. soetomo general hospital, university of airlangga, surabaya correspondence dr. meitria syahadatina noor department of public health, faculty of medicine, university of lambung mangkurat jl. veteran, banjarmasin, kalimantan selatan phone: +6281391739795 email: drmeitria79@gmail.com univ med 2013;32:92-8 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background indonesia has the third largest tobacco consumption in the world after china and india. nicotine as the main component of cigarette smoke has negative effects on the reproductive system, such as oocyte maturation, ovulation, and fertilization, and increasing the diploidy of oocytes. the goal of this research was to evaluate the effect of nicotine on oocyte maturation in rattus norvegicus. methods this was an experimental study with post test only control group design. the subjects were 40 rats selected homogenously and randomly. they were divided into a control group (receiving carboxy-methyl-cellulose sodium and 3 treatment groups (i-iii) receiving nicotine subcutaneously for 7 days at dosages of 21 mg/ kgbw, 41 kg/kgbw and 84/kgbw, respectively. the observations comprised oocyte maturation stage, viz. germinal vesicle (gv), germinal vesicle breakdown (gvbd), metaphase i and metaphase ii. data were analyzed by one-way anova with á=0.05, followed by tukey’s hsd test. results one-way anova showed significant differences in oocyte maturation in all groups. tukey’s hsd test showed that for gv, the differing groups were control and i, control and ii, i and iii. for gvbd, the differing groups were control and i, i and ii, i and iii. for metaphase i, the differing groups were control with i, ii, and iii, i and ii, i and iii. for metaphase ii, the differing groups were control versus i, ii, and iii, i and ii, i and iii. conclusion low dose of nicotine is capable of affecting oocyte maturation in rattus norvegicus. key words: nicotine, maturation, oocyte, rattus norvegicus nicotine supplementation blocks oocyte maturation in rattus norvegicus meitria syahadatina noor*, h.m. bakhriansyah**, widjiati***, and budi santoso**** 93 pemberian nikotin menghambat maturasi oosit pada rattus norvegicus latar belakang indonesia adalah negara konsumen tembakau terbesar di dunia dan menempati urutan ketiga setelah cina dan india. nikotin sebagai komponen terbesar dari rokok memiliki efek terhadap sistem reproduksi, antara lain dapat menghambat maturasi oosit, menurunkan ovulasi dan fertilisasi, serta meningkatkan jumlah oosit yang diploidi. tujuan penelitian ini adalah untuk menilai efek nikotin terhadap maturasi oosit pada rattus norvegicus. metode penelitian ini adalah penelitian eksperimental dengan post test only control group design dengan menggunakan 40 ekor tikus putih. kelompok hewan coba secara acak dibagi menjadi kontrol diberikan carboxy-methyl-cellulose sodium, kelompok i-iii yang masing-masing diberikan injeksi nikotin subkutan selama 7 hari dengan dosis 21 mg/ kgbb, 42 mg/kgbb, dan 84 mg/kgbb. hasil yang diamati adalah germinal vesicle (gv), germinal vesicle breakdown (gvbd), metafase i dan ii. anova satu arah dengan taraf kepercayaan 95% digunakan untuk membedakan anatar keempat kelompok, bila berbeda bermakna maka dilanjutkan dengan uji tukey hsd. hasil ada perbedaan semua fase maturasi oosit antara keempat kelompok (p<0,05). hasil uji tuckey hsd untuk fase gv menunjukkan terdapat perbedaan antara kontrol dan i, kontrol dan ii, i dan ii, i dan iii, serta ii dan iii. pada fase gvbd, terdapat perbedaan antara kontrol dan i, i dan ii, serta i dan iii. pada fase metaphase i, terdapat perbedaan antara kontrol dengan i, ii, dan iii, i dan ii, serta i dan iii. sedangkan pada fase metaphase ii terdapat perbedaan antara kontrol dengan i, ii, dan iii, i dan ii, serta i dan iii. kesimpulan pemberian nikotin dalam dosis rendah mampu menghambat maturasi oosit pada rattus novergicus. kata kunci: nikotin, maturasi, oosit, tikus putih abstrak introduction according to a statement by the who there were 1.3 billion smokers in the world in 2003 and their numbers will increase to 1.7 billion in 2010. it has been estimated that around one billion people died from smoking in the 21st century.(1) nicotine is the main component of cigarette smoke and the cause of the smoking habit or addiction.(2) exposure to cigarette smoke affects both active and passive smokers.(3) the effects of cigarette smoke on the reproductive system are to influence the production and function of gametes, ovulation, the reproductive cycle, f e r t i l i z a t i o n , a n d e m b r y o t r a n s p o r t a n d i m p l a n t a t i o n . (4) t h e m a i n c o m p o n e n t o f c i g a r e t t e s i s n i c o t i n e ( c 10 h 14 n 2 ) , w h i c h constitutes 50% of all components.(5) nicotine is capable of forming free radicals, thus being a pro-oxidant. nicotine exerts adverse effects on follicle growth, number of follicles, thickness of the endometrium and uterine glands.(6) it also a d v e r s e l y a f f e c t s c u m u l u s c e l l s a n d t h e noor, bakhriansyah, widjiati, et al nicotine blocks oocyte maturation 94 organization of microtubules and microfilaments in the oocyte during meiosis. in vitro experiments showed that nicotine induced meiotic blockage in metaphase-i of mouse oocytes, while administration of nicotine to mice in vivo resulted in reduced numbers of ovulated oocytes.(7) the study by dwirahayu found that nicotine blocks oocyte maturation in rattus norvegicus at dosages of 35, 52.5, and 70 mg/kgbw. this is because nicotine reduces the size of ovarian follicles, thus affecting the oocyte maturation process.(8) in view of the above, it was thought necessary to conduct further studies on the effects of nicotine on oocyte maturation, using lower doses of nicotine, in order to determine the minimal dose capable of affecting fertility in females. the doses used were based on those of kakisina who used doses of 3, 6, and 12 mg/ k g b w f o r d e t e r m i n i n g d e v e l o p m e n t a l abnormalities in mouse embryos. the doses had been validated in preliminary studies and were far below the lethal dose.(9) these doses were then converted into doses of 21, 42, and 84 mg/ kgbw for use in the norway rat (rattus norvegicus). methods design of the study this study used an experimental laboratory method with post test only control group design. it was conducted at the embryology laboratory, faculty of veterinary medicine, university of airlangga, surabaya, from june to july 2011. experimental animals the study subjects were adult female norway rats (rattus norvegicus). the rats were randomly assigned to one control group and three treatment groups (i-iii). the sample size was 9 rats per group, based on á =0.05, â =0.2 and effect size = 0.3.(11) to anticipate a reduction in numbers through death, the size of the groups was increased by 20%, so that each group contained 11 rats. preparation of nicotine the nicotine doses of 21 mg/kgbw, 42 mg/ kgbw and 84 mg/kgbw, were adjusted to the weight of individual rats. liquid nicotine of 70% purity was diluted with twice-distilled water (aqua bidestillata) and the calculated dose for each rat was administered. treatment the rats were given a subcutanous injection of nicotine at 21 mg/kgbw (i), 42 mg/kgbw (ii) and 84 mg/kgbw (iii) for 7 days. these d o s e s w e r e s e v e n t i m e s l a r g e r t h a n t h e corresponding dose for mice (7 x mouse dose).(10) the controls were given an injection of carboxy-methyl-cellulose sodium (cmc-na) using a comparable technique and duration of treatment as used for the treatment groups. the injections were performed by experienced personnel using disposable syringes and needles for each injection. harvesting of oocytes the rats were given an injection of 10 iu of pregnant mare serum gonadotropin (pmsg), and 48 hours later an injection of 10 iu human chorionic gonadotropin (hcg). subsequently the rats were mated with vasectomized male rats to induce ovulation. after 17 hours each female was examined for the presence of a vaginal plug. rats with a vaginal plug were sacrified by euthanasia for harvesting of oocytes. harvesting of oocytes was done by lifting the uterus under a dissecting microscope and looking for the fertilization pouch. the oocytes were released by rupturing the fertilization p o u c h . t h e r e l e a s e d o o c y t e s w e r e t h e n transferred by means of a modified pipette into a petri dish containing phosphate buffered saline (pbs) as washing medium. staining of oocytes the oocytes were placed on a glass slide that was ringed with vaseline and covered with a cover slip. subsequently the oocytes were fixed noor, bakhriansyah, widjiati, et al nicotine blocks oocyte maturation 95 in fixing solution (acid alcohol : absolute methanol = 1:3) for at least 24 hours. the slide was then removed from the fixing solution and air-dried. the oocytes were stained in 1% aceto-orcein for 2-3 seconds and washed in decolorizing solution. they were examined under the microscope for germinal vesicles (gv), germinal vesicle breakdown (gvbd), metaphase i and metaphase ii. examination of the oocytes was done at the embryology laboratory, faculty of veterinary medicine, university of airlangga, surabaya. data analysis the data on oocyte maturation were obtained from the stages of gv, gvbd, metaphase i and metaphase ii. statistical analysis was performed by means of one-way anova, followed by tukey’s hsd test. the level of significance wasnet at 0.05. ethical clearance this study was given ethical clearance by the research ethics committee, faculty of medicine, university of lambung mangkurat, banjarmasin. results the total number of oocytes collected in each treatment group are presented in figure 1. t h e n u m b e r o f o o c y t e s w a s i n v e r s e l y proportional to the nicotine dose, i.e. larger doses of nicotine resulted in reduced number of oocytes. the results of one-way anova showed significant differences in the numbers of oocytes between all four groups (p=0.000). the control group had a significantly larger number of oocytes in comparison with the three treatment groups (p=0.00). the reduction in the number of oocytes in the group receiving nicotine at a dose of 21 mg/bw (i) was significantly smaller than that in the groups receiving nicotine at doses of 42 mg/kg bw(ii) and 84 mg/kg bw (iii) (p=0.00). the nicotine dose of 42 mg resulted in a smaller reduction in the number of oocytes than the dose of 84 mg, but the difference was statistically not significant (p=0.74). t h e m a t u r a t i o n o f t h e o o c y t e s w a s subsequently observed, with the results shown in figure 2, depicting oocyte maturation stages in the control group and the groups exposed to nicotine at various doses. exposure to nicotine at a dose of 21 mg/kgbw significantly reduced the numbers of oocytes in the gv stage as compared with controls (p<0.05). exposure to doses of 42 and 84 mg/kgbw did not significantly alter the numbers of gv compared with controls (p>0.05). in the gvbd stage, exposure to nicotine at 21 mg/kgbw significantly reduced the number of oocytes in that stage as compared figure 1. effect of nicotine exposure at various doses on number of mature oocytes in rattus norvegicus univ med vol. 32 no.2 96 figure 2. effect of exposure to nicotine at various doses on oocyte maturation in rattus norvegicus. gv=germinal vesicle; gvbd=germinal vesicle breakdown with controls (p<0.05). exposure to doses of 42 and 84 mg/kgbw did not significantly alter the numbers of oocytes in the gvbd stage as compared with controls (p>0.05). in metaphase i, a significant reduction in the number of oocytes was found on exposure to nicotine doses of 42 and 84 mg/kgbw in comparison with controls (p<0.05), which was not found with the dose of 21 mg/kgbw (p>0.05). in metaphase ii, a significant reduction in the number of oocytes was found on exposure to nicotine at all doses in comparison with controls (p<0.05). tukey’s hsd found significant differences between controls and the three nicotine dosage groups with respect to all observed maturation stages, with p=0.000 for all comparisons. for the gv stage, there were significant differences between controls and i, controls and ii, and between i and ii, i and iii, ii and iii. for gvbd, the differing pairs of groups were controls and i, i and ii, i and iii. for metaphase i, the differences were between controls on the one hand and i, ii, and iii on the other, and also between i and ii, and between i and iii. in metaphase ii stage, the differing groups were controls versus i, ii, and iii, i and ii, i and iii. discussion in this study there were differences in the number of oocytes released from the ovaries between controls, treatment groups i (21 mg/ kg bw), ii (42 mg/kg bw) and iii (84 mg/kg bw). our study results are consistent with the study conducted by mokhtar et al., where it was shown that nicotine adversely affects the n u m b e r a n d q u a l i t y o f o o c y t e s a n d t h e fertilization rate in animal models.(12) a recent study using confocal microscopy on oocytes from mice exposed to cigarette smoke revealed that these oocytes had significantly thicker zona pellucida and shorter and wider meiotic spindles. approximately 25% of these oocytes had errors in chromosomal congression or abnormally shaped spindles.(13) in the present study, observations on the m a t u r a t i o n s t a g e s o f r e t r i e v e d o o c y t e s , comprising gv, gvbd, metaphase i and metaphase ii, showed significant differences at all maturation stages between the control group and the three treatment groups. this study was an improvement over the study of dwirahayu, who made similar observations with different noor, bakhriansyah, widjiati, et al nicotine blocks oocyte maturation 97 d o s e s . t h e s t u d y o f d w i r a h a y u s h o w e d significant differences for metaphase ii only.(8) in contrast, for gv and gvbd there were no significant differences, and for metaphase i no statistical tests could be performed because all results were zero. (6) another study using nontoxic doses of nicotine of 1.0, 2.5, 5.0 and 10.0 mmol/l, respectively, showed nicotine to have no adverse effects on gv breakdown.(14) free radical or reactive oxygen species (ros) production by nicotine is the result of inhibition of anti-oxidant enzyme and subsequent lipid peroxidation. oxidative stress from free radicals or ros may damage the cell membrane a n d a l s o i n d u c e d n a f r a g m e n t a t i o n . ( 1 5 ) oxidative stress also leads to chromosomal instability and programmed cell death, the latter being the main mechanism of oocyte death. when fully developed or mature oocytes are released from the follicle for ovulation, the meiotic process is completed as shown by their being in metaphase ii. if the meiotic process is not completed, it will stop at any given stage, e i t h e r g v, g v b d o r m e t a p h a s e . i f t h e developing oocytes have not reached their full size when released from the follicle, they cannot become mature, being in the gv and gvbd stages. medium-sized oocytes may reach maturity although they have not yet completed the meiotic process and have stopped at metaphase i.(10) the mechanism of oocyte maturation blockage as a result of exposure to nicotine is by systemic oxidative stress and oxidative stress in the follicular fluid. intrafollicular oxidative stress may cause apoptosis of the follicular granulosa cells, thus impeding the process of f o l l i c u l o g e n e s i s a n d r e d u c i n g f o l l i c u l a r diameter.(4) the size of the follicle affects oocyte development because the capacity of oocytes to complete meiosis for full maturation depends on follicular size. if the follicle becomes smaller in size, the maturation process of the oocytes within the follicle will be blocked. the number of oocytes from small follicles are lower than that of mature oocytes from large follicles.(16,17) immature oocytes are characterized by the gv stage, gvbd and metaphase i, whereas mature oocytes are characterized by metaphase ii.(16,17) the results of the present study showed that each group differs significantly from the others, signifying that low doses of nicotine is capable of affecting oocyte maturational development. increasing the dose severely affects the development resulting in fewer mature oocytes. exposure to nicotine from cigarette smoke at any dose significantly affects oocyte development in metaphase i and ii. if there are no oocytes in metaphase ii, there are no mature oocytes. ultimately, this results in female infertility because only mature oocytes can be fertilized by sperm. these findings demonstrate the extreme sensitivity of human oocytes to cigarette smoke, and underline the need for experimental animal data to clarify the causes of meiotic blockage. one limitation of this study was the inability to perform nicotine exposure by inhalation in order to mimic smoking. the implication of the study is that the dose of 21 mg/kgbw, which was capable of reducing the number of oocytes in rattus norvegicus, can be used to calculate the minimal dose in humans, i.e. 1176 mg. one cigarette contains 0.3-2 mg of nicotine, therefore 588-3920 cigarettes smoked actively or passivley, are sufficient to impair fertility in women. conclusion nicotine, the major alkaloid in tobacco, is capable of blocking oocyte maturation in the norway rat (rattus norvegicus). immature oocytes cannot be fertilized by sperm, invariably leading to infertility. acknowledgement we thank the staff at the embryology laboratory, faculty of veterinary medicine, university of airlangga, surabaya and bambang setiawan, s. ked., m. biomed for enabling us to complete and publish this study. univ med vol. 32 no.2 98 references 1. rabinoff m, caskey n, rissling a, park c. pharmacological and chemical effects of cigarette additives. am j public health 2007;97:1981-8. 2. hukkanen j, jacob p iii, benowitz nl. metabolism and disposition kinetics of nicotine. pharmacol rev 2005;57:79-115. 3. vasquez j. human rights and health: persons exposed to second-hand tobacco smoke. washington dc: pan american health organization; 2008. 4. bordel r, laschke mw, menger md, vollmar b. nicotine does not affect vascularization but inhibits growth of freely transplanted ovarian follicles by inducing granulosa cell apoptosis. hum reprod 2006;21:610-6. 5. talbot p, riveles k. smoking and reproduction: the oviduct as a target of cigarette smoke. reprod biol endocrinol 2005;3:52. doi:10.1186/1477-78273-52. 6. bordel r, laschke mw, menger md, vollmar b. nicotine does not affect vascularization but inhibits growth of freely transplanted ovarian follicles by inducing granulosa cell apoptosis. hum reprod 2006;21:610-7. 7. mailhes jb, young d, caldito g, london sn. sensitivity of mouse oocytes to nicotine induced perturbations during oocyte meiotic maturation and aneuploidy in vivo and in vitro. mol hum reprod 2000;6:232-7. 8. dwirahayu y. pengaruh nikotin terhadap tingkat maturasi oosit pada tikus (rattus novergicus) [thesis]. surabaya: universitas airlangga; 2010. 9. kakisina p. pengaruh nikotin terhadap perkembangan embrio mencit (mus musculus) [tesis]. surabaya: universitas airlangga;2003. 10. kusumawati d. bersahabat dengan hewan coba. yogyakarta: gadjah mada university press; 2004. 11. shah h. how to calculate the sample size for animal studies.? nat j physiol pharm pharmacol 2011;1: 35-9. 12. mokhtar n, rajikin m, zakaria z. role of tocotrienol-rich palm vitamin e on pregnancy and preimplantation embryos in nicotine-treated rats. biomed res 2009;19:181-4. 13. jennings pc, merriman ja, beckett el, hansbro pm, jones kt. increased zona pellucida thickness and meiotic spindle disruption in oocytes from cigarette smoking mice. hum reprod 2011;26:878-84. 14. zenzes mt, bielecki r. nicotine-induced disturbances of meiotic maturation in cultured mouse oocytes: alterations of spindle integrity and chromosome alignment. tob induced dis 2004;2:151-61. 15. asadi e, jahanshahi m, golalipour mj. effect of vitamin e on oocytes apoptosis in nicotine-treated mice. iran j basic med sci 2012;15:880-7. 16. son wy, lim jh. fertilization, cleavage and blastocyst development according to the maturation timing of oocytes in invitro maturation cycles. hum reprod 2005;20:3204-7. 17. mrazek m, fulka jr j. failure of oocyte maturation: possible mechanisms for oocyte maturation arrest. hum reprod 2003;18:2249-52. noor, bakhriansyah, widjiati, et al nicotine blocks oocyte maturation oktavianus 20 *department of internal medicine, faculty of medicine, trisakti university **division of infectious and tropical diseases, department of internal medicine, faculty of medicine, university of indonesia ***division of gastro-enterohepatology, department of internal medicine, faculty of medicine, university of indonesia correspondence dr. ronald irwanto, sppd, kpti department of internal medicine, faculty of medicine, trisakti university jl. kyai tapa grogol, jakarta 11440 phone: +6221-5672731 email: ronald.irwanto@yahoo.com univ med 2013;32:20-8 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 background data collected in 2010 from cipto mangunkusumo hospital indicate that complicated skin and soft tissue infections accounted for more than 10% of cases. etiological diagnoses are based on the findings on bacterial culture and thus evaluation of the effectiveness of bacterial culture becomes a necessity. the purpose of this study was to evaluate the operational effectiveness of bacterial culture for etiological diagnosis of complicated skin and soft tissue infections. methods this was a historical cohort study using secondary data of patients with complicated skin and soft tissue infections admitted for hospitalization to cipto mangunkusumo hospital, jakarta from july 2011 to july 2012. the 90 subjects meeting the inclusion and exclusion criteria were divided into 2 groups of 45 patients each. group 1 comprised patients who received initial antibiotic therapy according to cultural results, while the patients in group 2 received initial antibiotic therapy without reference to cultural results. successful diagnostic culture was assessed by the absence of therapeutic failure. therapeutic failure was determined using 3 parameters that had to be fulfilled, viz. absence of antibiotic escalation, repeat operations, and clinical deterioration. the latter parameter was assessed by clinical judgement of the attending physician. results after controlling for confounding variables (age, severity of infection, comorbidity), there was no statistical difference in therapeutic success between culture-based and non-culture based initial antibiotic therapies (or=0.45, p=0.085). conclusion this study demonstrates the ineffectiveness of bacterial culture as a diagnostic criterion for appropriate antibiotic therapy of complicated skin and soft tissue infections. key words : culture, antibiotic, diagnostic, skin and soft tissue infection, complicated culture-and nonculture-based antibiotics for complicated soft tissue infections are comparable ronald irwanto*,**, suhendro**, khie chen**, and murdani abdullah*** 21 pengobatan antibiotika sesuai dan tidak sesuai kultur pada infeksi jaringan lunak komplikata adalah sebanding latar belakang data yang diperoleh di ruang rawat inap penyakit dalam rumah sakit cipto mangunkusumo menunjukkan lebih dari 10% kasus infeksi kulit dan jaringan lunak komplikata sepanjang tahun 2010. diagnostik kausatif penyebab infeksi ditegakkan melalui temuan kultur, oleh karenanya sangatlah penting untuk menilai efektifitas kultur. tujuan penelitian ini adalah untuk menilai efektifitas operasional kultur sebagai media diagnostik kausatif pada infeksi kulit dan jaringan lunak komplikata. metode penelitian merupakan studi historical cohort menggunakan data sekunder pada pasien-pasien dengan infeksi kulit dan jaringan lunak komplikata yang masuk ke rawat inap penyakit dalam di rumah sakit cipto mangunkusumo jakarta antara bulan juli 2011 juli 2012 . sebanyak 90 subjek yang memenuhi kriteria inklusi dan eksklusi, dibagi dalam 2 kelompok, yaitu kelompok yang diberikan antibiotik awal sesuai kultur tidak sesuai kultur, dengan jumlah 45 subjek untuk masing-masing kelompok. keberhasilan diagnostik kultur dilihat dari ada tidaknya kegagalan antibiotik yang penilaiannya dilakukan menggunakan 3 parameter yang semuanya harus dipenuhi, yaitu tidak adanya eskalasi antibiotik, tidak adanya operasi ulang, dan tidak adanya perburukan klinis dinilai melalui clinical judgement oleh dokter yang merawatnya. hasil setelah mengontrol variabel perancu usia, beratnya infeksi dan komorbid, secara statistik tidak ada perbedaan keberhasilan antara antibiotik awal yang diberikan sesuai kultur dengan antibiotik awal yang diberikan tidak sesuai kultur (or=0,45, p=0,085). kesimpulan studi ini menunjukkan pemeriksaan kultur untuk diagnostik penggunaan antibiotik yang sesuai untuk pengobatan infeksi kulit dan jaringan lunak komplikata tidak efektif. kata kunci : kultur, antibiotik, diagnostik, infeksi kulit dan jaringan lunak, komplikata abstrak introduction data collected in 2010 from the internal diseases ward of cipto mangunkusumo hospital indicate that complicated skin and soft tissue infections accounted for more than 10% of cases. similarly, cases of sepsis also amounted to around 10%. several studies showed that 4.3%-10.5% of cases hospitalized for sepsis were due to skin and soft tissue infections.(1,2) the high prevalence of infections results in correspondingly high antibiotic usage.(3) this impacts on increased prevalence of bacterial antibiotic resistance, ultimately leading to significant increases in morbidity and mortality. marwick et al.(4) in a study on patients at ninewells hospital in scotland found that empirical administration of antibiotics according to uk guidelines failed to significantly reduce patient morbidity. this was presumably caused b y i n a p p r o p r i a t e e m p i r i c a l a n t i b i o t i c administration with regard to the bacteria causing the infections. the uk guidelines have recorded that inappropriate antibiotic usage in univ med vol. 32 no.1 22 patients with complicated skin and soft tissue infections resulted in a 20% higher morbidity rate in comparison with patients receiving appropriate antibiotic therapy.(5,6) there is a need for the evaluation of the effectiveness of bacterialculture-based etiological diagnosis, since in fact antibiotic administration based on bacterial cultural results does not completely guarantee a reduction in patient morbidity and mortality, made possible by the multiplicity of errors in the collection of specimens and reporting of the results. in contrast, administration of antibiotics that is at variance with cultural findings may frequently yield substantial clinical improvement. this is connected with a variety of factors, such as development of bacterial colonization or inappropriate specimen collection methods. the purpose of the present study was to evaluate the effect of cultureand non-culture-based antibiotic administration on the success rate of treatment of complicated skin and soft tissue infections. methods research design this was a historical cohort study using secondary data from the medical records of the internal disease ward of cipto mangunkusumo hospital for the period of july 2011 july 2012. study subjects patients admitted to the internal disease ward with complicated skin and soft tissue infection and a variety of clinical manifestations were recruited into this study, with as inclusion criterion completeness of data, including data on bacterial culture. exclusion criteria were: patients with complicated skin and soft tissue infection referred from other hospitals, who had received antibiotics prior to transfer to cipto m a n g u n k u s u m o h o s p i t a l ; p a t i e n t s w i t h complicated skin and soft tissue infection showing severe focal infection at other sites; patients from whom pus had been collected after 7 days of hospitalization; and patients with polymicrobial cultures and variable resistance to the administered antibiotic. the sample size was calculated by means of the formula for a difference between 2 proportions, yielding 90 patients meeting the inclusion and exlusion criteria, who were divided into 2 groups of 45 patients each. group 1 comprised patients who received initial antibiotic therapy according to cultural results, while the patients in group 2 received initial antibiotic therapy without reference to cultural results. data collection c o n f o u n d i n g c o m p o n e n t s t h a t w e r e considered to affect host responses to infection were age, disease severity (based on the presence or absence of systemic involvement or sepsis), and comorbidity, such as diabetes m e l l i t u s , m a l i g n a n c y, h i v / a i d s , a n d autoimmune disease, according to the clinical diagnosis of the attending physician. assessment of cultural effectiveness therapeutic success was determined from the absence of antibiotic failure, using three parameters that had to be fulfilled, viz. absence of antibiotic escalation to indicate antibiotic failure and defined as the administration of antibiotics with increasingly broader spectra, repeat operations, and clinical deterioration. the latter parameter was assessed by clinical judgement of the attending physician. statistical analysis a simple logistic regression was used to determine success of cultureand noncultureb a s e d t r e a t m e n t s . m u l t i v a r i a t e l o g i s t i c regression was used to control for confounding variables. all analyses were performed using spss for windows version 15.0. research ethics this study was approved by the medical ethics commission, faculty of medicine, university of indonesia. irwanto, suhendro, chen, et al culture and nonculture based antibiotics 23 table 1. clinical characteristics of study subjects based on therapeutic success results in this study data from 90 patient records were collected. with regard to gender. there were 51 male subjects (56.7%) and 39 female subjects (43.3%). according to age, the younger person group comprised 27 subjects (30.0%). the number of subjects categorized as older person comprised 26 subjects (28.9%). subjects with diabetic comorbidity were 63 in number. while those with nondiabetic comorbidity comprised 27 subjects. the most frequent cause of nondiabetic comorbidity among the study subjects was malignancy, found in 15 out of 27 subjects (55.6%). there were no differences of clinical characteristic between the two groups expect for sepsis (table 1). confounding factors were considered significant at p<0.25. a variety of microorganisms were found upon culture of swabs taken from lesions. among the microorganisms found on culture, gram-negative bacteria were the dominant group. overall, pseudomonas sp were the most numerous, being found in 22 cultures (19.5%), followed by escherichia coli in 20 cultures (17.7%) and klebsiella pneumoniae in 17 cultures (15.0%). gram-positive bacteria c o m p r i s e d st a p h y l o c o c c u s a u re u s a n d staphylococcus epidermidis, found in equal numbers, namely in 13 cultures (11.5%) each. g r a m n e g a t i v e b a c t e r i a f o u n d i n appreciable numbers were acinetobacter sp., present in 10 cultures (8.8%), whereas the microorganism found in lowest numbers in cultures was stenothrophomonas maltophilia, present in 1 culture (0.9%). univ med vol. 32 no.1 24 table 2. patterns of antibiotic sensitivity and resistance of gram-positive bacteria *r=resistant; @s=sensitive; #i=intermediate the possibility of a high resistance rate of m e t h y c i l l i n r e s i s t a n t st a p h y l o c o c c u s epidermidis (mrse) was supported by the finding of a relatively high resistance rate of s. epidermidis against beta-lactam antibiotics, amounting to an overall rate of more than 50%. in contrast to s.epidermidis, in the case of s. aureus the proportion of representative isolates of methycillin resistant staphylococcus aureus (mrsa) against beta-lactam antibiotics was not sufficiently high. however, the r e s i s t a n c e r a t e w a s q u i t e h i g h a g a i n s t levofloxacin, attaining 61.5% (table 2). the possibility of a high prevalence of extended spectrum beta-lactamase (esbl) producers is indicated by the high resistance rate of e. coli and k. pneumoniae against beta lactam antibiotics. the resistance rate of e.coli against cephalotin was up to 70%, while the resistance rate against cefotaxime, ceftriaxone and cefoperazone were 55%, 60% and 60%, respectively. e. coli showed a high sensitivity toward aminoglycoside antibiotics, with the sensitivity of e. coli for gentamycin and amikacin attaining 60% and 70%, respectively. the highest level of sensitivity of e. coli was for the carbapenem antibiotics, the sensitivity for meropenem and imipenem attaining 100%. the resistance rate of k. pneumonia against beta-lactam antibiotics was also substantially high. in contrast, this species was highly sensitive for the cephalosporins, with the sensitivity for ceftazidime attaining 64.7%. the sensitivity for aminoglycoside antibiotics was also high, that for amikacin attaining 76.5%. however, the sensitivity for gentamycin was only 47.1%. the sensitivity for meropenem and imipenem was also high, being 94.1% for each of them (table 3). the resistance rate of pseudomonas sp against cephalosporins was found to be high, being on average above 50%. the lowest rate of resistance against cephalosporins was shown by cefepime, with a resistance rate of only 27.3% and a sensitivity of 54.5%. a relatively high sensitivity was shown for carbapenems, with a sensitivity for meropenem and imipenem of 68.2% and 72.7%, respectively. cultural findings for acinetobacter sp showed an extremely high antibiotic resistance both against beta-lactam antibiotics, antib e t a l a c t a m a s e s , a m i n o g l y c o s i d e s a n d quinolones, attaining rates of 80%-100%. irwanto, suhendro, chen, et al culture and nonculture based antibiotics 25 table 3. antibiotic sensitivity and resistance patterns of gram-negative bacteria resistance against meropenem and imipenem was 40% for each, with a sensitivity of up to 60%. after controlling for confounding factors, the success and failure rates of culture-based i n i t i a l a n t i b i o t i c t r e a t m e n t a n d t h o s e o f nonculture-based initial antibiotic treatment in complicated skin and soft tissue infections are as shown in table 4. in this study, significant confounding factors (at p<0.25) were diabetes mellitus and sepsis, with a valid model having the smallest or (0.01) and the narrowest precision of 0.94 with p=0.085 and or=0.45 (table 4). therefore, on the basis of the above calculations, there was no statistically significant difference in success *r=resistant; @s=sensitive; #i= intermediate; values represent percentages table 4. comparison of success and failure rates between cultureand nonculture-based initial antibiotic treatment in complicated skin and soft tissue infections rate between culture-based and nonculturebased initial antibiotic administration. discussion in this study, from the viewpoint of demographic and clinical characteristics, subjects in the age group of 46-55 years were the most numerous in comparison with other age groups. with regard to gender, there were more males (56.7%) than females in this study. the demographic characteristics in our study were similar to those obtained by moran et al.,(7) where 62% of subjects were male. this was in contrast with a previous study by irwanto et al. on patients with complicated soft tissue infections in the emergency wards of three univ med vol. 32 no.1 26 hospitals in jakarta, in which there were more female than male subjects. (8) this gender difference is presumably due to differing time and location of the studies. regarding the results of bacterial culture, t h e r e w e r e 7 4 . 3 % g r a m n e g a t i v e microorganisms among the cultural findings, indicating a predominance of gram-negative microorganisms among the cultural findings. on this point our study differs from several previous studies, where it was demonstrated that in complicated skin and soft tissue infections the cultural results should be dominated by grampositive microorganisms.(9-12) the resistance rate of s. epidermidis against oxacillin was relatively high in our study. as oxacillin is representative of methicillin, this high resistance rate of s. epidermidis may be indicative of a high probability of mrse. this resistance rate is quite significant when c o m p a r e d w i t h t h e r e s i s t a n c e r a t e o f s . epidermidis against the penicillins (ampicillin) and cephalosporins, which are on average greater than 50%. previous studies have shown similar results. in the selection of subjects in the s e t t i n g o f e m e r g e n c y d e p a r t m e n t s , t h e resistance rate of s. epidermidis against both oxacillin and beta-lactams (represented by cefoxitin) was identical (62.5%).(8) the resistance rate of s. aureus against oxacillin was categorized as low, being only 15.4%, or found in only 2 of 13 cultures of s. aureus. however, this resistance rate was not in accord with the resistance rate against ampicillin of up to 84.6%. the reason for this discrepancy is unclear. the findings of miller et al.(13) in los angeles showed a considerably high rate of mrsa isolates. however, these isolates came from subjects with necrotizing fasciitis. therefore, it may be concluded that the subjects in the los angeles study had different clinical characteristics when compared with our subjects. an indication of esbl producers is found in the resistance of e.coli and k. pneumoniae against cephalosporins. the study of rodriguezbano et al. ( 1 4 ) s h o w e d a r e l a t i v e l y h i g h proportion (65%) of esbl-producing microorganisms in isolates from urine and blood samples. petkovsek et al.(15) also state that relatively virulent isolates of e. coli are frequently found in skin and soft tissue infections. the high resistance rate of pseudomonas sp. against beta-lactam antibiotics in this study may presumably be used as an indicator for the presence of a high number of multi drug resistant (mdr) pseudomonas. gillespie (16) states that the carbapenem group of antibiotics may be used as treatment option for administration to cases of infection by mdr pseudomonas or other gram-negative bacteria. the present study i n d e e d f o u n d t h a t t h e s e n s i t i v i t y o f pseudomonas sp. for carbapenems is still high, with the sensitivity for meropenem and imipenem attaining 68.2% and 72.7%, respectively. fernandez et al.(17) in an in vivo experiment explains that that ceftobiprole, a fifth-generation cephalosporin that acts by binding to the penicillin b i n d i n g p r o t e i n 3 ( p b p 3 ) , w a s a b l e t o significantly inhibit the growth of p. aeruginosa. this opens new perspectives for eradication of infections caused by p. aeruginosa.(17,18) the number of isolates of acinetobacter sp in this study was up to 8.8%, but based on their resistance patterns, the proportion of mdr-panresistant acinetobacter sp. was v e r y h i g h . a s a r u l e m d r p a n r e s i s t a n t acinetobacter sp are found among cases of extended care with prolonged application of medical devices. however, in the present study, the high proportion of mdr-panresistant acinetobacter sp was in spite of the fact that one of the exclusion criteria was duration of hospitalization of more than seven days. the possiblity of contamination or colonization acquired in hospital cannot be ruled out. sebeny et al.(19) found in their study that skin and soft tissue infections from trauma with application of medical devices, were a significant risk factor associated with mdr acinetobacter. a history of trauma accompanied by cellulitis with irwanto, suhendro, chen, et al culture and nonculture based antibiotics 27 edema or vesicles, also indicates a significant probability of acinetobacter as the cause of the infection. however, several studies found that skin and soft tissue infections had a r e l a t i v e l y h i g h i n c i d e n c e o f m r s a , i n comparison with other infections. therefore, there should be a high degree of suspicion of the presence in the community of a high proportion of mrsa that are resistant to penicillins and cephalosporins.(20-22) in our study there was no statistical difference in therapeutic success rates between culture-based and nonculture-based initial antibiotic treatments for complicated skin and soft tissue infections. this may have been due to presence of sepsis as a confounding variable affecting the relationship between both types of treatment. the proportion of cases without sepsis was as high as 72.5% in the group of patients with successful therapeutic outcomes, as compared with 27.5% in the groups with therapeutic failures. one of the limitations of this study was its historical cohort design, making the study a retrospective one. therefore the data collected cannot provide detailed information on patient presentations, approaches to treatment or clinical outcomes. conclusions in this study, a relatively high antibiotic resistance rate was found among gram-positive as well as gram-negative microorganisms. after controlling for confounding factors, culturebased initial antibiotic administration did not yield significant success rates in comparison with nonculture-based initial antibiotic administration. acknowledgments we wish express our gratitude to the faculty of medicine, university of indonesia and cipto mangunkusumo hospital, for providing the facilities for our study. thanks are also due to the experts for their guidance in the conduct of this study and to the faculty of medicine, trisakti university for funding part of this study. references 1. shen hn, lu cl. skin and soft tissue infections in hospitalized and critically ill patients: a nationwide population-based study. bmc infect dis 2010;10:151. 2. vincent jl, rello j, marshall j, silva e, anzueto a, martin cd, et al. international study of the prevalence and outcomes of infection in intensive care units. jama 2009 302:2323-9. 3. setiawan bnl. prinsip penggunaan antibiotika. bunga rampai penyakit infeksi. 1st ed. jakarta: divisi penyakit tropik dan infeksi, departemen ilmu penyakit dalam, fkui; 2004.p.104-15. 4. marwick c, broomhall j, mccowan c, phillips g, gonzalez-mcquire s, akhras k, et al. severity assessment of skin and soft tissue infections: cohort study of management and outcomes for hospitalized patients. j antimicrob chemother 2011;66:387-97. 5. eron lj, lipsky ba, low de, nathwani d, tice ad, volturo ga. managing skin and soft tissue infections: expert panel recommendations on key decision points. j antimicrob chemother 2003; suppl 1:13-17. 6. crest. guidelines on the management of cellulitis in adults. northern ireland: fulton r; 2005. 7. moran gj, krishnadasan a, gorwitz rj, fosheim ge, mcdougal lk, carey rb, et al. methicillin-resistant s. aureus infections among patients in the emergency department. n engl j med 2006;355:666-74. 8. irwanto r, suhendro, khie chen, yeva r. pattern of sensitivity and resistance of aerobic microorganism of complicated soft tissue infection at three hospital’s emergency room in jakarta. in: sungkar s, basuki b, kurniawan a, editors. 5th malaysia indonesia brunei medical sciences conference; july 23-25, 2009; jakarta, indonesia. jakarta, indonesia: faculty of medicine university of indonesia;2009. p.103. 9. dryden ms. complicated skin and soft tissue infection. j antimicrob chemother 2010;65 suppl 3:iii35-44. 10 gordon rj, lowy fd. bacterial infections in drug users. n engl j med 2005;353:1945-54. 11. sumpio be. foot ulcer. n eng j med. 2005;343: 787-93. univ med vol. 32 no.1 28 12. grayson ml. the treatment triangle for staphylococcal infections. n eng j med;2006: 724-7. 13. miller lg, perdreau-remington f, rieg g, mehdi s, perlroth j, bayer as, et al. necrotizing fasciitis caused by community-associated methicillin-resistant staphylococcus aureus in los angeles. n engl j med 2005;352:1445-53. 14. rodriguez-bano j, navarro md, romero l, martinez-martinez l, muniain ma, perea ej, et al. epidemiology and clinical features of infections caused by extended-spectrum betalactamase-producing escherichia coli in nonhospitalized patients. j clin microbiol 2004; 42:1089-94. 15. petkovsek z, elersic k, gubina m, zgur-bertok d, starcic erjavec m. virulence potential of escherichia coli isolates from skin and soft tissue infections. j clin microbiol 2009;47:18117. 16. gillespie sh. management of multiple drugresistant infections. humana press; 2004 17. fernandez j, hilliard jj, abbanat d, zhang w, melton jl, santoro cm, et al. in vivo activity of ceftobiprole in murine skin infections due to staphylococcus aureus and pseudomonas aeruginosa. antimicrob agents chemother 2010;54:116-25. 18. habif tp. pseudomonas celulitis. n eng j med 2005;353:380-91. 19. s e b e n y p j , r i d d l e m s , p e t e r s e n k . acinetobacter baumannii skin and soft-tissue infection associated with war trauma. clin infect dis 2008;47:444-9. 20. amir nh, rossney as, veale j, o’connor m, fitzpatrick f, humphreys h. spread of community-acquired meticillin-resistant staphylococcus aureus skin and soft-tissue infection within a family: implications for antibiotic therapy and prevention. j med microbiol 2010;59:489-92. 21. muttaiyah s, coombs g, pandey s, reed p, ritchie s, lennon d, et al. incidence, risk factors, and outcomes of panton-valentine leukocidin-positive methicillin-susceptible staphylococcus aureus infections in auckland, new zealand. j clin microbiol 2010;48:3470-4. 22. donnio py, preney l, gautier-lerestif al, avril jl, lafforgue n. changes in staphylococcal cassette chromosome type and antibiotic resistance profile in methicillin-resistant staphylococcus aureus isolates from a french hospital over an 11 year period. j antimicrob chemother 2004;53:808-13. 23. kumar a, roberts d, wood ke, parrillo je, sharma s, suppes r, et al. duration of hypotension before initiation of effective antimicrobial therapy is the critical determinant of survival in human septic shock. crit care med 2006; 34:1589–96. irwanto, suhendro, chen, et al culture and nonculture based antibiotics oktavianus 86 *department of biochemistry, faculty of medicine, atma jaya catholic university **department of molecular biology and biochemistry, faculty of medicine, university of indonesia correspondence dr. maria dara novi handayani, m.biomed department of biochemistry, faculty of medicine atma jaya catholic university pluit raya no.2 jakarta utara, 14440 jakarta indonesia phone: +6221-6694366 ext. 203 email: dara.sudarya@gmail.com univ med 2013;32:86-91 abstract universa medicina may-august, 2013may-august, 2013may-august, 2013may-august, 2013may-august, 2013 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 vol.32 no.2 background red fruit oil (rfo) is popular in indonesia especially in papua. it is consumed by people for maintaining health and to treat diseases. red fruit (pandanus conoideus lam) oil is reported to contain the antioxidants á-tocoferol and âcarotene. the aim of this study was to confirm the protective effect of rfo against decreased glutathione levels in the plasma and liver of rats induced by 2n-(2-fluorenyl) acetamide (2-faa). methods wistar male rats weighing 200 ± 20 g were randomly assigned into a control group (receiving distilled water only) and three intervention groups, designated rfo, faa, and rfo-faa (n=6 rats per group). rfo was given rfo 10 ì l/g/ bw/day, faa received fluoroenylacetamide (faa) at 40ìg/day, while rfofaa received both rfo and faa. at 4 weeks blood samples were taken from the tail. at 8 weeks the rats were sacrified for collection of blood and liver tissues. ellman’s method was employed to determine the parameters of antioxidant glutathione (glutahione sulfhydryl/gsh). one-way anova and tukey post hoc test were used to compare glutathione levels between groups. results this study showed that liver and plasma glutathione levels were not significantly lower in the rfo-faa group than in the faa group. glutathione levels were significantly lower in plasma and liver homogenates of the rfo group compared with the control group and were not significantly different from those in the faa group. conclusion administration of rfo in rats does not protect against decreased glutathione but is a potential source of oxidative stress. key words : red fruit oil, oxidative stress, n-(2-fluoroenyl) acetamide, glutathione, rats red fruit oil supplementation fails to prevent oxidative stress in rats maria dara novi handayani*, parwati abadi soekarno**, and septelia inawati wanandi** 87 minyak buah merah tidak mampu mencegah terjadinya stres oksidatif pada tikus latar belakang minyak buah merah (rfo) banyak dikonsumsi masyarakat indonesia khususnya papua untuk kesehatan dan pengobatan beberapa penyakit. rfo dilaporkan mengandung antioksidan á-tokoferol dan â-karoten. penelitian ini bertujuan menilai efek perlindungan rfo untuk mencegah penurunan kadar antioksidan glutation dalam plasma dan jaringan hati tikus yang diinduksi 2n-(2-fluorenyl) acetamide (2-faa). metode tikus jantan wistar dengan berat 200 ± 20 g dibagi secara acak dalam 4 kelompok perlakuan eksperimental (n=6 tikus/kelompok) yaitu kelompok yang hanya diberi aquades (kontrol), 10ì l/g/bb/hari rfo, diinduksi 40ì g/hari fluorenylacetamide (faa) dan kelompok yang diberi 10ì l/g/bb/hari rfo dilanjutkan dengan induksi 40ì g/ fluorenylacetamide (rfo+faa). setelah perlakuan 4 minggu, darah diambil dari ekor tikus dan pada minggu ke-8 semua tikus dimatikan. pemeriksaan kadar glutation (glutathione sulfhydryl/gsh) menggunakan metode ellman. one-way anova dilanjutkan dengan uji post hoc tukey digunakan untuk membandingkan kadar glutation antara keempat kelompok perlakuan. hasil hasil pengukuran dalam plasma dan jaringan hati menunjukkan bahwa kadar gsh plasma dan jaringan hati tikus kelompok rfo+faa menurun tidak berbeda bermakna dibandingkan faa. dari penelitian ini juga terungkap bahwa kadar glutation dalam plasma dan jaringan hati kelompok rfo lebih rendah bermakna dibandingkan kontrol dan tidak berbeda bermakna dibandingkan kelompok faa. kesimpulan pemberian rfo pada tikus ternyata tidak memiliki kemampuan mencegah turunnya kadar glutation dan justru berpotensi menyebabkan stres oksidatif. kata kunci : minyak buah merah, stres oksidatif, n-(2-fluoroenyl) acetamide, glutation (gsh), tikus abstrak univ med vol. 32 no.2 introduction an imbalance in the production of free radicals and antioxidant defenses in the body is termed oxidative stress, causing oxidative damage in cellular macromolecules.(1,2) however, dietary manipulation with antioxidant-containing foods may prevent oxidative stress.(2-4) red fruit oil (rfo) and other traditional medications as antioxidant supplements for the prevention of various degenerative diseases are n o w b e g i n n i n g t o b e c o m e p o p u l a r i n indonesia.(4-6) rfo supplements are already w i d e l y c i r c u l a t e d a n d c o n s u m e d b y t h e community. rfo is reported to contain a number of antioxidants, such as á -tocopherol, â -carotene, and other phenolic and flavonoid compounds.(6,7) in vitro and in vivo studies have demonstrated the antioxidant activity of rfo.(8,9) glutathione (glutathione sulfhydryl/gsh) is one of the intracellular antioxidants produced by the body and acts to prevent oxidative damage due to free radicals.(11,12) a previous study induced oxidative stress in rats by means of n-2-fluorenylacetamide (2-faa) 40 ì g/day 88 for 8 weeks, resulting in a significant decrease in glutathione concentrations as compared to controls.(10) the objective of the present in vivo experimental study was to evaluate the effect of rfo supplements in preventing decreases in plasma and hepatic tissue gsh levels in rats w i t h o x i d a t i v e s t r e s s i n d u c e d b y n 2 fluorenylacetamide (2-faa). study design this experimental study was conducted in the biochemistry laboratory of the department of biochemistry and molecular biology, faculty of medicine, university of indonesia, from september until november 2006. experimental animals the number of animals required (24) was calculated according to frederer’ formula, viz. (n-1)(k-1) >15. the animals were two-month old male wistar albino rats (rattus sp) weighing 200 ± 20 g, obtained from the veterinary research institute (balai penelitian veteriner), bogor, west java. the animals were kept in the animal house of the department of biochemistry and molecular biology, faculty of medicine, university of indonesia. interventions the wistar rats were randomized into a control group (receiving distilled water only) and three intervention groups, designated rfo, faa, and rfo+faa. group rfo received rfo at 1 0 ìl/gbw/day and group faa received fluoroenylacetamide (faa) at 40ìg/day, while group rfo+faa received both rfo (10 ìl/g bw/day) and faa (40ìg/day). in the last group, faa was given 2 hours after administration of rfo. the interventions were administered daily for 8 weeks, except for rfo administration in the rfo+faa group. in this group rfo was given one week before induction with faa, in order to ensure the protective effect of rfo. all interventions were given by gavage. the r f o d o s a g e o f 1 0 ì l / g b w / d a y w a s a conversion of the usual dose for human consumption of 3 spoonfuls per day for administration to the rats. biochemical measurements a t t h e e n d o f t h e f o u r t h w e e k o f intervention, blood samples were collected from the rats by snipping off the tips of their tails, after an antiseptic swab to prevent infection, and collecting the blood into tubes containing ethylene diamine tetraacetic acid (edta). in the eighth week of intervention, all rats were sacrified using ether. the anesthesized rats had their legs fully extended and were fixated by pinning the legs to a dissecting board. after antisepsis of the thorax and abdomen, the thoracic cavity was opened and the heart wetted with 3 drops of heparin solution, followed by cutting the cardiac apex with scissors and collecting the blood by means of a pasteur pippette. subsequently the liver was removed from the surrounding tissues by blunt dissection. the blood in the edta tubes was centrifuged at 3000 rpm for 10 minutes to separate the plasma, which was stored in a freezer at -20°c until required. for measurement of gsh concentrations, one gram of rat liver was ground in nacl 0.9% into a homogenous mass. gsh concentrations in plasma and the homogenized liver tissue were then determined by a modification of ellman’s method in the biochemistry laboratory of the department of biochemistry and molecular biology, faculty of medicine, university of indonesia. the principle of gsh measurement is to react the test material with 5,5’-dithiobis(2-nitrobenzoic acid) (dtnb). if the test solution contains a compound with sulfhydryl (sh) groups, the compound will reduce the dtnb, causing the latter to be converted into 2-nitro-5-thionitrobenzoate, which in an alkaline environment absorbs light maximally at a wavelength of 412 nm, thus giving a yellow color.(10) handayani, soekarno, wanandi red fruit oil fails prevent oxidative stress 89 univ med vol. 32 no.2 figure 1. plasma gsh levels at 4 and 8 weeks *p<0.05 compared to control group; rfo = red fruit oil; faa = n-2-fluorenylacetamide figure 2. hepatic gsh levels at 8 weeks * p<0.05 compared to control group; rfo : red fruit oil; faa : n-2-fluorenylacetamide data analysis the data on glutathione concentrations were tested for normality of distribution using a standard test of normality. the normally distributed data were then tested using oneway anova at a significance level of p=0.05. in the case of significant between-group differences in plasma and hepatic glutathione levels being found at 4 and 8 weeks, the analysis was to be followed by a post hoc tukey test. ethical clearance the study protocol was approved by the research ethics commission, faculty of medicine, atma jaya university. results induction with n-2-fluorenylacetamide in g r o u p s faa a n d r f o + fa a r e s u l t e d i n significantly lower concentrations of hepatic glutathione as compared with controls (p=0.004). similar results were obtained for plasma glutathione at 4 and 8 weeks (p=0.001, p=0.027) (figure 1). the glutathione concentrations in the liver and plasma of group rfo+faa were not significantly different from those of group faa at each time of observation. the results even showed that the liver glutathione concentrations of group rfo at 8 weeks were significantly lower than those of and were not significantly different from those of group faa (figure 2). 90 the decreases in glutathione levels in the liver and plasma were similar, indicating that damage of liver tissues was reflected systemically in the plasma. discussion the decrease in glutathione concentrations in group faa was caused by the glutathione neutralizing the free radical arylnitrenium, which was formed in the bioactivation process of n-2fluorenylacetamide. glutathione conjugates with arylnitrenium either spontaneously or by means of a reaction catalyzed by gsh-s transferase. the lower levels of glutathione in group faa provides evidence that induction with n-2fluorenylacetamide at 40 µg/animal resulted in oxidative stress.(10-12) t h e r e s u l t s o f t h i s s t u d y s h o w t h a t administration of rfo in the group of rats r e c e i v i n g n 2 f l u o r e n y l a c e t a m i d e w a s evidently not capable of preventing a decrease in glutathione concentrations, either at 4 weeks or at 8 weeks, in both plasma and liver. moreover, this study also reveals that rfo supplementation was associated with lower glutathione levels as compared with controls and that these levels were not significantly different from those of the group that had been given faa. thus the present study yielded results that are at variance with the reported antioxidant effects of rfo in previous in vitro (8) and in vivo studies.(9,13) on the other hand, the results of the present study are similar to those of an in vitro study comparing the antioxidant potency of several kinds of rfo in sheep erythrocytes that had been subjected to oxidative stress by i n d u c t i o n w i t h t h e o x i d a t o r t e r b u t y l hydroperoxide (tbhp).(14) the decrease in the glutathione antioxidant levels on administration of rfo may possibly have been caused by the oxidants originally present in rfo or oxidants formed by the prolonged high temperatures employed in the extraction of the oil.(15) the glutathione antioxidants may have been used to neutralize these oxidants, leading inevitably to decreased glutathione levels. the decreased glutathione in group rfo may also have been caused by impaired gsh synthesis as a result of liver damage.(16,17) other studies have shown that rfo supplementation increases plasma sgpt concentrations.(18,19) to account for the oxidative stress due to rfo it may be assumed that the dosage of the rfo administered in the present study was relatively high and that the high levels of vitamins, minerals and antioxidants present in rfo may together act as prooxidants by inducing fenton and haber-weiss reactions, thus increasing the production of hydroxyl radicals and decreasing the antioxidant levels in the blood.(20-22) another explanation may be that the high amounts of fatty acids and carotenoids in rfo can activate m i c r o s o m a l o x i d a s e s t h u s r e s u l t i n g i n bioactivation of toxic substances or fatty acid degradation products caused by the prolonged heating of the red fruits for oil extraction.(23-25) the present study demonstrates the potential for oxidative stress of rfo and therefore further studies on rfo are highly desirable. conclusion administration of commercially available red fruit oil at 10ìl/gbw/day to rats induced with n-2-fluorenylacetamide at 40mg/animal/ day for 8 weeks was unable to prevent oxidative stress, but was in contrast potentially capable of causing oxidative stress through decreased plasma glutathione levels. acknowledgements the authors thank atma jaya university (unika atma jaya) for the funding of this study. 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characterization of red fruit (pandanus conoideus lam) oil. int food res j 2012;19:563-7. 8. rohman a, riyanto s, yuniarti n, saputra wr, utami r, mularsih w. antioxidant activity, total phenolic and total flavonoid of extracts and fractions of red fruit (pandanus conoideus lam). int food res j 2010;17:97-106. 9. syarkiah, fitri le, pudjirahayu a. the effect of red fruit (pandanus conoideus lam) oil toward the formation of foam cells in aorta of wistar strain rat (rattus norvegicus) with atherogenic diet. j kedokter brawijaya 2008;23:6. 10. sukmarini l. konsentrasi glutation tereduksi (gsh) plasma tikus putih (rattus norvegicus l.) jantan galur wistar yang mengalami karsinogenesis sebagai akibat induksi n-2fluorenilasetamida. [skripsi]. jakarta: universitas indonesia;2004. 11. mei zh, hua rh, tao lh. progress in the research of gsh in cells. chi sci bulletin 2011;56:3057-63. 12. townsend dm, tew kd, tapiero h. the importance of glutathione in human disease. biomed pharmacother 2003;57:145-55. 13. mun’im a, andrajati r, susilowati h. tumorigenesis inhibition of water extract of red fruit (pandanus conoideus lam) on spraguedawley rat female induced by 7,12 dimetilbenz(a) antrasen (dmba). j pharm sci 2006;3:153-61. 14. yulhaeka s. perbandingan daya antioksidan beberapa minyak buah merah (pandanus conoideus lam.) pada eritrosit domba yang mengalami stres oksidasi secara in vitro. [skripsi]. jakarta: universitas indonesia;2006. 15. liu p, guo ws, pu hc, feng cn zhu xk, peng yx. effects of high temperature on antioxidant enzymes and lipid peroxidation in flag leaves of wheat during grain filling period. agric sci china 2006;5:425-30. 16. james lp, mayeux pr, hinson ja. acetaminophen-induced hepatotoxicity. drug metabol dispos 2003;31:1499-506. 17. thomson rw, valentine hl, valentine wm. in vivo and in vitro hepatotoxicity and glutathione interactions of n-methyldithiocarmbate and n, n-dimethyldithio-carbamate in the rat. toxicol sci 2003;70:269-80. 18. mukhyarjon, septelia, parwati, nurjati. antikarsinogenesitas vs hepatotoksisitas minyak buah merah (mbm) pada tikus yang diinduksi fluoroenilasetamida (faa). j ilmiah kedokter 2007; 1:1-8. 19. djuartina t. pengaruh pemberian minyak buah merah (pandanus conoideus lam.) pada hati tikus yang cedera akibat d-galaktosamin. [thesis]. jakarta: universitas indonesia;2006. 20. thomas c, mackeymm, diaz aa, cox dp. hydroxyl radical is produced via the fenton reaction in submitochondrial particles under oxidative stress: implications for diseases associated with iron accumulation. redox rep 2009;14:102-8. 21. oliveira bf, veloso ca, machado ja, chaves m. high doses of in vitro beta-carotene, alphatocopherol and ascorbic acid induce oxidative stress and secretion of il-6 in peripheral blood mononuclear cells from healthy donors. curr aging sci 2012;5:148-56. 22. bjelakovic g, nikolova d, gluud ll, simonetti rg, gluud c. mortality in randomized trials of antioxidant supplements for primary and secondary prevention. jama 2007;297:842-57. 23. he y, wang k, wang l. effect of tocopherol and carotene supplementation on meat quality and antioxidant capacity of pigs fed high-linseed oil diet. j anim plant sci 2010;20:180-8. 24. mathew m, tay e, cusi k. elevated plasma free fatty acids increase cardiovascular risk by inducing plasma biomarkers of endothelial activation, myeloperoxidase and pai-1 in healthy subjects. cardiovasc diabetol 2010;9:1-9. 25. tripathy d, mohanty p, dhindsa s, syed t, ghanim h, alda a. elevation of free fatty acids induces inflammation and impairs vascular reactivity in healthy subjects. diabetes 2003;52: 2882-7. c:\users\jurnal fkusakti\docume 90 *national institute of health research and development, ministry of health, republic of indonesia correspondence sulistyowati tuminah national institute of health research and development, ministry of health, republic of indonesia email: watidarjoko@gmail.com date of first submission, september 21, 2 01 8 date of final revised submission, may 10, 2019 date of acceptance, may 14, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina high carbohydrate intake increases risk of coronary heart disease in adults: a prospective cohort study sulistyowati tuminah darjoko*, tri wahyuningsih*, and sudikno* background coronary heart disease (chd) ranks second after diabetes mellitus (dm) based on hazard rate, and after stroke (based on number of deaths caused). our aim was to determine the risk factor and magnitude of chd among adults. methods a cohort study on risk factors of non-communicable diseases (cs-rfncd) was conducted on subjects aged 25years. initiated by screening, followup (fu) was done 3 times yearly and complete health examination every 2 years. chd cases screened by electrocardiographic examination on subjects aged 40 years and <40 years with history of hypertension and/ or heart disease. screening results found 840 of 5690 subjects with chd diagnosis who were excluded from cohort study sample. non-chd subjects and those aged <40 years without a history of hypertension and/or heart disease, totalling 4840 people, were included in study sample and followed up for 6 years. data were analyzed using cox regression. results carbohydrate intake of 60% of total energy had a 2.8-fold higher chd risk [hr=2.790; 95% ci: 1.962 3.967; p=0.000] than that of an intake of <60% of total energy. age of 55 years had 2.6-fold higher chd risk [hr=2.573; 95% ci: 1.803 3.671; p=0.000] than age of <55 years. blood total cholesterol of 200 mg/dl had 1.9-fold higher chd risk [hr=1.893; 95% ci: 1.319 2.715; p=0.001] than that of <200 mg/dl. conclusion higher intake of carbohydrate increases chd incidence among adults. efforts in controlling chd risk factors are still needed especially in consumption behavior through a family approach. keywords: carbohydrate intake, predictor, coronary heart disease, aged 25 years original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.90-99 may-august, 2019 vol.38no.2 cit e th is a rtic le a s: darjoko st, wahyuningsih t, sudikno. high carbohydrate intake increases risk of coronary heart disease in adults: a prospective cohort study. univ med 2019;38: 90-9. doi: 10.18051/univmed.2019. v38.90-99 91 univ med vol. 38 no.2 introduction coronary heart disease (chd) is a major cause of death throughout the world and is responsible for more than 7 million deaths each year. the latest estimate from the global burden of disease (gbd) 2010 shows that death from chd between the years 1990 and 2010 in southeast asia increased by 77.7%, ranking third after east and south asia.(1) deaths from chd in china, india, sub-saharan africa, latin america and the middle east are estimated to increase from 9 million in 1990 to 19 million in 2020.(2) chd is caused by an accumulation of plaques in the coronary arteries that supply oxygen to the cardiac musculature. this causes the blood flow to become blocked and the cells to become deficient in oxygen, resulting in death of the cardiac musculature, that is called myocardial infarction.(3) in indonesia, chd is still an important health problem with a socio-economic impact, b e c a u se o f t he r e l a ti ve l y h igh c o s t o f medications, the length of care and treatment and the additional examinations needed for treatment.(4) the results of the indonesian basic health research (riset kesehatan dasar, riskesdas) of 2013 for bogor city, based on diagnosis/ symptoms, show that prevalences of chd a nd st roke we re 2. 2% an d 14.8 ‰, r e s p e c ti ve ly, whe r ea s th e pr e va le nc e o f hyper tension based on measur ement was 28.6%. these prevalences are higher than the national prevalences of chd, stroke, and hypertension of 1.5%, 12.1‰ and 25.8%, respectively.(5,6) according to the sample registration system (srs) survey, the mortality rate of chd is 12.9% of total deaths.(4) the results of a cross-sectional analysis performed by pradono and werdhasari (7) using a subset of the cs-rfncd baseline data (from 2011-2012) showed that the determinant factors of chd were stroke, hypertension, and hyperglycemia. to prevent an increase in chd morbidity and mortality, there is a need for knowledge of the risk factors playing a role in chd incidence. the novelty of the present article lies in the inclusion of carbohydrate intake as independent variable in the analysis to determine its influence on chd incidence. the aim of the present study was to determine the most dominant risk factor on the chd incidence in adults. methods study design the present study was a prospective cohort study of individuals who did not have the major ncd (in this case chd) and was performed between january 2011 and december 2017 in five villages in central bogor district, bogor city. subjects in the cs-rfncd, monitoring was carried out on three major non-communicable diseases (ncds) as outcomes, i.e. chd, stroke and dm. during baseline data collection, screening was done to select respondents in accordance with the respective outcomes. subjects who were diagnosed with chd at screening, were excluded from the cohort study sample. subjects who did not have chd at screening (aged 40 years with “normal” ecgs or with ecgs that were “not normal but not chd”), and respondents aged <40 ye ars (25-39 ye ars) without histor y of hypertension and/or heart disease, were included in the cohort sample and their health condition monitor ed f or 6 years (2011-2017). chd incidence, in this case new chd cases, was determined from the results of examination of csrfncd electrocardiograms (ecgs) that was performed once every 2 years. respondents who in the cs-rfncd were diagnosed with chd for the first time, subsequently were still considered to be patients with chd. the sample collection was performed c o ns e c u t i ve l y, i .e . s ub j e c t s me e ti n g t h e requirements were selected as respondents until the required number of subjects was reached. the sample consisted of males and females aged 25 years and above, who did not have difficulties in verbal communication and did not have severe 92 illness. the inclusion criterion was agreeing to follow all of the cs-rfncd activities and the exclusion criterion was respondents who were diagnosed with chd from the results of the examination of ecgs at cs-rfncd screening. a total of 4840 individuals became respondents of the cohort study of risk factors for coronary heart disease (cs-rfchd). collection of follow-up (fu) data the major exposures of the present study were: i) the physical condition (obesity and central obesity; ii) hypertension ; iii) blood glucose concentration (hyperglycemia) and blood lipid concentration (hypercholesterolemia); and iv) behavior (intake of sodium/fat/carbohydrate, smoking habit, alcohol consumption, insufficient physical activity). data collection for the cs-rfncd was p e r f o r me d a t i nt e gr a t e d c o a ch i ng p os t s (posbindu) and the agenc y for nutrition research and development of bogor city [litbang gizi – kota bogor]. the study was initiated by screening (baseline) and continued by monitoring (follow-up/fu) three times yearly. for ease of reference, each of the fus was given a sequence number. in fus 1-5, 7-11 and 13-17 blood pressure and anthropometric measurements were performed at the posbindu. particularly in fu 3, 9, and 15 (1-year fus) blood glucose and blood cholesterol determinations on capillary blood was added to the examinations, and also interviews on nutrition and risk factors (smoking habit, alcohol consumption and physical activity). final monitoring for the 2-year fus i.e. fu 6 (final monitoring for the first 2 years), fu 12 (final monitoring for the second 2 years / in the 4th year) and fu 18 (final monitoring for the third 2 years /the complete 6-year study period) was performed at litbang gizi. the 2year fu activities comprised: i) interviews using questionnaires; ii) anthropometric and blood p r e s s ur e me a su r e me n t s ; ii i ) la bo r a t o r y examinations (blood glucose and blood lipid concentrations); and iv) examination of ecgs. interviews interviews were performed for collecting information on the following: i) health, comprising characteristics of the respondents (age, gender, education, occupation, and marital status), and behavioral risk factors (smoking habit, physical activity, and mental/emotional disorder); ii) nutrition, i.e. dietary pattern of the respondents, comprising eating habits and types of foods consumed by the respondents. health interviews were performed by enumerators who were nursing/obstetrics gr aduates. nutritional interviews were performed by enumerators who were nutrition science graduates. prior to performing their activities, the enumerators were given training. age was the age of the respondents in the 6th year of monitoring, being the difference in years between the last birthday and the date of birth. to determine the age characteristics of the respondents, age was divided into 4 groups, i.e. i.e. 34 years, 35 44 years, 45 54 years and 55 years. in the analysis, however, age was divided into 2 groups only, i.e. <55 years and 55 years, since according to the study results, the percentage of chd increases twofold at age 55 years.(8) gender was grouped into 2, i.e. male and female. education was categorized into 3 groups, i.e. low, middle, and high. occupation was divided into 5 categories, i.e. house wife, entrepreneur/service provider/trader, employee, laborer, and other. marital status was grouped into 4 categories, i.e. single, married, divorced, and widowed. degree of smoking was calculated from the number of cigarettes smoked per day multiplied by the duration of smoking in years (brinkman index), and was categorized into 3 groups, i.e. non-smokers, moderate smokers (<200), and heavy smokers (200).(9) the level of physical activity was based on composite calculation of the type and duration of the activity (days per week and minutes per day), including sports. both vigorous physical activity and vigorous sports had a weight of 8, moderate activity or moderate darjoko, wahyuningsih, sudikno coronary heart disease in adults 93 univ med vol. 38 no.2 sports had a weight of 4, while light activity had a weight of 2. subjects were categorized as having poor physical activity if they had a total activity of <600 metabolic equivalents (met) per week.(8) detection of mental/emotional disorder was per for med using the self-reporting questionnaire (srq) consisting of 20 items. the respondents were declared to have mental/ emotional disorder if they answered “yes” to at least 6 of 20 questions.(10) dietary intakes of the respondents, in this case sodium, fat, and carbohydrate, were based on general guideline for balanced nutrition (ggbn) (pedoman umum gizi seimbang, pugs). sodium intake was divided into 2 categories, i.e. <2000 mg and 2000 mg. fat intake was categorized into <25% of total energy and 25% of total energy. carbohydrate intake was categorized into <60% of total energy and 60% of total energy.(11) nutritional interviews were performed by means of the food frequency questionnaire (ffq) and food recall questionnaire. measurements anthropometric measurements comprised weight, height, and waist circumference (wc). weight (kg) was determined using and type uc-322 digital scales of 150 kg capacity and 50 g accuracy, with the respondent standing upright without footwear and being lightly clothed. height (cm) was determined with a multifuctional fiberglass stature meter, with the respondent in the upright position. body mass index (bmi) was calculated from the ratio of weight (kg) and height (m2). bmi status was categorized into non-obese (bmi <25.0) and obese (bmi 25.0). (1 2) measurement of wc (cm) was performed with “hrp282” measuring tape with centimeter graduations and the respondent in the standing position. the clothing covering the area around the abdomen was rolled up so that the measuring tape was in direct contact with the abdominal skin. subsequently the midpoint between the lowest rib and the pelvic bone was determined and the measuring tape was placed horizontally round the abdomen through this point. if the abdomen was distended, the measuring tape was placed around the greatest diameter of the abdomen. the wc was grouped by gender, i.e. abdominal obesity 90 cm (males) and 80 cm (females).(13,14) blood pressure (bp) measurement (mm/hg) was done with an and type ua-852 digital blood pressure monitor. t he measur ement was performed on the right arm 2 times in succession with an interval of 3 minutes. if there was a difference in blood pressure of 10 mmhg between the first and second measurements, for systolic as well as diastolic pressures, a third measurement was performed after a resting period of 10 minutes, then the mean of the three measurements was taken, both for systolic and diastolic blood pressures. according to the joint national committee (jnc vii), the criteria of hypertension are systolic bp 140 mmhg and diastolic bp 90 mmhg.(15) determination of anthropometric measurements and blood pressure in the 2-year monitoring was performed by health personnel from the primary health center (pusat kesehatan masyarakat, pkm) of central bogor district, while measurements at the posbindu were performed by health personnel from the respective public health centers and by their cadres. the health personnel from the public health centers as well as the cadres were given training prior to the fu. laboratory investigations blood examination comprised blood fasting blood glucose (fbg) and 2-hr postprandial blood glucose (2hppbg) concentrations and blood lipid concentrations (total cholesterol [tc], high density lipoprotein [hdl], low density lipoprotein [ldl], and triglycerides [tg]). prior to blood sample collection, the subjects were asked to fast for around 12-14 hours and not to perform vigorous activity. the subjects received 75 grams of glucose dissolved in 250 ml of plain water, whereas respondents with dm did not receive glucose but instead received 300 calories of food. for determination of fbg, tc, hdl, ldl and tg, a volume of 8 ml of venous blood was drawn a b c 94 for each subject, whereas for determination of 2hppbg, the venous blood volume was 1 ml. the blood samples were examined with an automatic analyzer. blood collection and examination was performed by a team from a clinical laboratory in bogor city. tc and tg were each divided into 2 categories, namely <200 mg/ dl and 200 mg/dl for tc and <150 mg/dl and 150 mg/dl for tg. ldl was categorized into <100 mg/dl and 100 mg/dl, whereas hdl was categorized on the basis of gender into <40 mg/ dl and 40 mg/dl for males and <50 mg/dl and 50 mg/dl for females.(11,16) examination of electrocardiograms examination of electrocardiograms was performed on the subjects at rest, using an electrocardiograph and the minnesota code. the diagnosis of chd was established if there was a picture of myocardial ischemia and infarction, such as the presence of abnormal q waves, t wave inversion, and st depression.(17) for chd, the minnesota code (mc) in appendix c on p. 325 states that mc 1-1 indicates a major q wave abnormality, whereas mc 1-2 indicates an old myocardial infarction. with regard to st-t abnormalities, the st segment is involved if the ecg meets mc criteria 4-1 or 4-2 and the t wave if the ecg meets mc criteria 5-1 or 5-2. these st-t abnormalities are categorized as ischemic heart disease. examination and interpretation of ecgs was performed by a clinical laboratory team from bogor city, to be confirmed subsequently by a specialist from the department of cardiology and vascular medicine, faculty of medicine, ui, at the harapan kita cardiology and vascular medicine hospital/ national cardiac center. all abnormal ecgs were read and coded by a senior cardiologist and verified by two others. if the verificators had a difference of opinion, the diagnosis was concluded after intensive discussion. the focus of research of the three senior cardiological investigators is on the investigation of ecgs in coronary heart disease [mc 1-1, 1-2; 4-1, 4-2; 5-1, 5-2] and the rhythm of atrial fibrillation [mc 8-3-1] that are considered to be of great impact on community health. examination of ecgs was performed on subjects who were 40 years of age and over, and on subjects who were under 40 years of age (2539 years) but having “a history of hypertension and/or heart disease”. data analysis to determine the characteristics of the respondents, cross-tabulation (crosstabs) was performed. to determine the risk factors affecting chd incidence during the 6 years of observation, bivariate and multivariate analysis was performed using cox regression. the dependent variable (chd) and the independent variables (age and gender) were taken from the fu-18 data (final monitoring in the 6th year). the other independent variables were taken from the previous fu data that had been filled in (not missing). ethical clearance before starting its yearly activities, the csrfncd always tendered a request for ethical clearance. the ethical clearances that have been obtained from the national health ethics commission national institute of health research and development, ministry of health [komisi nasional etik penelitian kesehatan, knepk badan pene litian dan pengembangan keseh atan, kementerian kesehatan], are the following: no.ke.01.08/ec/ 485/2011 dated 10 august 2011; no.ke.01.05/ ec/394/2012 dated 11 may 2012; no.lb.02.01/ 5.2/ke.215/2013 dated 30 may 2013; no.lb02.01/5.2/ke.143/2014 dated 17 april 2014; no.lb.02.01/5.2/ke.135/2015 dated 9 march 2015; no.lb.02.01/5.2/ke.042/2016 dated 3 february 2016; no.lb.02.01/2/ke.108/2017 dated 27 march 2017. results among the total number of 5690 candidate subjects, 840 were diagnosed with chd (at screening) so that they were excluded from this cohort sample. subjects who had no chd at darjoko, wahyuningsih, sudikno coronary heart disease in adults 95 univ med vol. 38 no.2 cohort study activities because of their occupation (having no time for the study due to having to trade, or having some difficulty in obtaining permission from their superiors). more than half of the respondents had middle education, i.e. junior and senior high school graduates, and only few respondents had higher education (8.4%). the majority of the respondents were married (84.8%) and their occupation was house wife (47.7%), followed by entrepreneur/service provider/trader (30.5%). prior to multivariate analysis, bivariate analysis was performed to determine the relationship of each independent variable with the dependent variable (chd). the results of the bivariate analysis was that age, gender, education, central obesity, hypertension, total cholesterol, ldl, hdl, mental/emotional disorder, brinkman index, and intakes of sodium, fat, and carbohydrate, met the requirements (p0.25) for multivariate analysis (table 2). from the final results of the multivariate analysis, it was found that age of 55 years (hr=2.57; 95% ci: 1.803 3.671; p=0.000); carbohydrate intake of 60% of total energy (hr=2.79; 95% ci: 1.962 3.967; p=0.000); and total cholesterol concentration of 200 mg/dl (hr=1.893; 95% ci: 1.319 2.715; p=0.001) played a significant role in chd (table 3). carbohydrate intake of 60% of total energy had the greatest role in increasing the risk for chd. discussion from the results of the analysis it was found that the respondents aged 55 years and over had a 2.6-fold risk for having chd than had the respondents aged under 55 years. this agrees with the study results of delima et al.(8) who found that the risk of heart disease tended to increase 2.2-fold in the age group of 55 years and over. similarly the results of an analysis by pradono(7) showed an inc reas ed chd prevalence particularly in the age group of 55-64 years. chd is highly correlated with age, and is the major cause of death in europe and the united states, characteristic n % age group 34 years and below 1283 26.5 35 44 years 1546 31.9 45 54 years 1283 26.5 55 years and over 728 15.0 gender male 1807 37.3 female 3,033 62.7 education high 406 8.4 middle 2892 59.8 low 1537 31.8 marital status single 378 7.8 married 4102 84.8 divorced 99 2.0 widowed 254 5.2 occupation house wife 2308 47.7 entrepreneur/service provider/trader 1474 30.5 employee 526 10.9 laborer 340 7.0 other 187 3.9 table 1. distribution of sociodemographic characteristics of subjects at baseline screening time (“normal” or “not normal but not chd”), were <40 years of age (25-39 years) with no history of hypertension and/or heart disease, and totalling 4840 individuals, were included in the sample and their health condition was monitored for 6 years. in the initial 2 years, 103 new chd cases were found, in the second 2-year period 50 new chd cases, and in the third 2-year period 9 new chd cases, resulting in a cumulative total of 162 new chd cases in 6 years. in table 1 are presented the baseline characteristics of the respondents who composed the cohort study sample with the outcome of chd. the percentage of chd in the age group of 35— 44 years was twice the percentage in the age group of 55 years and over, whereas the age groups of 34 years and below and 45-54 years had identical percentages. the percentage of females was almost twice that of males, which was due to the fact that there were more male respondents who could not participate in the 96 table 2. bivariate cox regression model showing the relationship between risk factors and coronary heart disease hr: hazard ratio; c.i.: confidence interval risk faktor unadjusted hr 95% ci p value age (years) <55 1.000 ≥55 3.503 2.566 4.783 0.000 gender male 1.000 female 3.245 1.651 6.379 0.001 education high 1.000 low 1.949 1.348 2.819 0.000 central obesity no 1.000 yes 1.354 0.978 1.875 0.068 hypertension no 1.000 yes 2.384 1.738 3.270 0.000 total cholesterol (mg/dl) <200 1.000 ≥200 2.148 1.556 2.964 0.000 triglycerides (mg/dl) <150 1.000 ≥150 1.113 0.754 1.643 0.588 ldl (mg/dl) <100 1.000 ≥100 1.824 1.128 2.948 0.014 hdl (mg/dl) ≥40 (males), ≥50 (females) 1.000 <40 (males), <50 (females) 0.787 0.555 1.116 0.179 2-hr postprandial blood glucose (mg/dl) <200 1.000 ≥200 1.317 0.773 2.244 0.311 physical activity adequate 1.000 inadequate 0.840 0.607 1.162 0.293 mental/emotional disorder no 1.000 yes 2.691 1.887 3.837 0.000 brinkman index non-smoker 1.000 mild smoker (<200) 1.740 1.146 2.643 0.009 heavy smoker (≥200) 1.197 0.725 1.974 0.482 sodium (mg) <2000 1.000 ≥2000 0.650 0.451 0.936 0.021 fat (% of total energy ) <25 1.000 ≥25 2.348 1.562 3.529 0.000 carbohydrate (% of total energy ) <60 1.000 ≥60 3.008 2.133 4.242 0.000 since with increasing age there is an increase in arterial wall thickening and stiff ness (atherosclerosis) that may cause hypertension, stroke and arterial fibrillation.(18,19) from the results of the analysis in the present study found that respondents with a carbohydrate intake of 60% of total energy had a 2.8-fold risk for having chd than had respondents with darjoko, wahyuningsih, sudikno coronary heart disease in adults 97 univ med vol. 38 no.2 carbohydrate intake of <60% of total energy. essentially similar results were found in the study of minoura et al.,(20) who stated that subjects with carbohydrate intake in the highest tertile (>58.1% total energy) had a higher 10-year odds ratio for chd after controlling for alcohol consumption, physical activity, and daily intake of protein and fat. the glycemic index (gi) is an indicator of how rapid carbohydrates can be absorbed in the form of glucose. since the amount of carbohydrate in foods is variable and has diverse effects on gi response, the glycemic load (gl) is used.(21) replacement of intake of saturated fats with carbohydrate of high gi increases the risk of cvd, but replacement with carbohydrate of low gi (fruits, vegetables, pulses and nuts) decreases the risk.(22,23) according to yu et al.,(24) a positive relationship between glycemic consumption load and chd incidence is found in males and females. when carbohydrate intake (particularly processed carbohydrate such as sugar) replaces intake of saturated fat, this has unfavorable effects on lipid profile i.e. total cholesterol tends to increase, hdl to decrease, and triglycerides (that are also associated with chd) tend to increase. human as well as animal studies show a variety of metabolic risk factors for chd with foods high in sugar, such as abnormal glucose tolerance, insulin resistance, increased uric acid concentration, and altered platelet functions.(21,25,26) siri-tarino et al.(27) state that replacement of intake of saturated fatty acids with carbohydrate actually increases the risk of cvd. the results of the analysis in the present study show that respondents with blood tc concentration of 200 mg/dl had a 1.9-fold risk for having chd than did respondents with blood tc concentration of <200 mg/dl. this agrees with the results of the korean national health syste m pros pec tive coho rt stu dy whi ch reported that serum cholesterol is positively associated with myocardial infarction (mi). an increase of 1 mmol/l cholesterol increases the risk of mi 1.48-fold.(28) however, this differed from the study results of ramsden et al.(29) that showed a 22% higher risk of death for each 30 mg/dl reduction in serum cholesterol. similarly, the results of the framingham heart study showed that chd is associated with total cholesterol in subjects aged <50 years, but not in subjects aged 50 years.(30) according to utami et al., (11) theoretically ther e should be a c orrelation between carbohydrate intake and lipid pr ofile. carbohydrate intake and tc concentration are influenced by the confounding factor of physical activity. this is because active or aerobic physical activity that raises the heart rate and needs rhythmic body movements and deep breathing, tends to increase hdl cholesterol. high blood hdl concentrations may increase clearance of free cholesterol in the blood, so reducing the risk of atherosclerosis. based on the results of the present study, there is still a need for controlling the risk factors of chd, particularly with regard to eating behavior, through family approaches. in addition, table 3. multivariate cox regression model showing the relationship between risk factors and coronary heart disease hr: hazard ratio; c.i.: confidence interval; unadj: unadjusted; adj: adjusted risk factor unadj hr 95%ci p value adj hr 95% ci p value age group <55 years 1.000 1.000 reference ≥55 years 3.503 2.566 4.783 0.000 2.573 1.803 3.671 0.000 carbohydrate intake <60% dari total energy 1.000 1.000 reference ≥60% dari total energy 3.008 2.133 4.242 0.000 2.790 1.962 3.967 0.000 total cholesterol <200 mg/dl 1.000 1.000 reference ≥200 mg/dl 2.148 1.556 2.964 0.000 1.893 1.319 2.715 0.001 98 there should be routine monitoring of the r e sp on d ent s ’ he a lt h c on di t io n by h e al t h personnel outside of the rfncd cohort study. the strenght of this article lies in the indepth analysis on subjects with “undiagnosed chd” at the time of screening (baseline), by taking the contribution of time into consideration. the limitation of the present study is that several of the subject characteristics (such as education, occupation, and marital status) in the baseline data were incomplete, so that the number of subjects was less than 4840. family history of chd was not analyzed, because many subjects did not know for certain at what age their family members were diagnosed with or died from chd. there were also subjects who did not know for certain the ncds of their family members. conclusion the predictor for chd incidence is an intake of carbohydrate of 60% of total energy after controlled by age and tc. conflict of interest there was no conflict of interest in this study. acknowlegdments we express our utmost gratitude: to all parties (the rfncd cohort study [studi kohor frptm] team), the heads of the primary health centers (puskesmas), the cadres and respondents, the prodia team, and the team from harapan kita national cardiac center hospital [rs pusat jantung nasional harapan kita]) for their cooperation in the conduct of the rfncd cohort study. to prof. dr. dr. budhi setianto, sp.jp(k), prof. dr. dr. bambang budi siswanto, sp.jp(k), and dr. ade meidian sp.jp (k), who provided help as consultant in the fields of cardiology and vascular medicine. to dr. dra. woro riyadina, m.kes who provided guidance and directions for writing this article. contributors std contributed to literature search, data analysis and writing of the draft. tw contributed to 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2015;12: 1-15. doi: 10.1186/s12986-015-0001-x. 27. siri-tarino pw, chiu s, bergeron n, krauss rm. saturated fats versus polyunsaturated fats versus carbohydrate for cardiovascular disease prevention and treatment. annu rev nutr 2015;35:517-43. doi:10.1146/annurev-nutr-071714034449. 28. hata j, kiyohara y. epidemiology of stroke and coronary artery disease in asia. circ j 2013;77: 1923-32. doi: 10.1253/circj.cj-13-0786. 29. ramsden ce, zamora d, majchrzak-hong s, et al. re-evaluation of the traditional diet-heart hypothesis: analysis of recovered data from minnesota coronary experiment (1968-1973). bmj 2016;353:i1246. http://dx.doi.org/10.1136/ bmj.i1246. 30. ashmaig me, ashmeik k, ahmed a, et al. association of lipids with coronary heart disease in a saudi population. j vasc bras 2011;10:131-6. doi: 10.1590/s1677-54492011000200007. c:\users\universa medicina\docu 12 *department of pharmacology, faculty of medicine, universitas jenderal soedirman **department of physiology, faculty of medicine, universitas jenderal soedirman ***department of clinical pathology, faculty of medicine, universitas jenderal soedirman correspondence: afifah department of pharmacology, faculty of medicine, universitas jenderal soedirman jl. dr. gumbreg, mersi, purwokerto jawa tengah, indonesia 53112 mobile: +6282242637171 email: afifah2403@gmail.com orchid id: 0000-0002-5703-7061 date of first submission, december 17, 2 01 8 date of final revised submission, january 22, 2020 date of acceptance, january 31, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina the protective effect of celery (apium graveolens l.) ethanol extract on anemia in 5/6 subtotal nephrectomy rat model afifah*, khusnul muflikhah**, tri lestari***, eman sutrisna*, ajeng sekar kirana*, and sindy devi prastiwi* background anemia is a frequent complication of chronic kidney disease (ckd). anemia in ckd is associated with reduced quality of life, increased cardiovascular disease, cognitive impairment, and mortality. therefore it is necessary to find an alternative agent for preventing anemia in ckd. celery is one of the natural substances that have anti-inflammatory, antioxidant, and antihypertensive pharmacological effects. based on the mechanism of ckd and its progression, celery is thought to prevent anemia in ckd. this research was aimed at evaluating the protective effect of celery extract against anemia in a ckd rat model. methods this was an experimental laboratory study using 25 male sprague dawley rats, aged 2-3 months, they were randomized into 5 groups, namely group a, sham operation; group b, subtotal nephrectomy; group c, d, e, subtotal nephrectomy + 250, 500, 1000 mg/kg bw ethanol extract of celery, respectively. the administration of celery extract was performed 14 days before and 14 days after induction of 5/6 subtotal nephrectomy. the hematological parameters (hb, rbc, ht, mcv, mch, mchc) and serum creatinine level were measured at the end of the study. data were analyzed with one way anova and kruskal-wallis test followed by mann-whitney test at p<0.05. results there were no significant differences between groups in hb, rbc, ht, mcv (p>0.05) and significant differences between groups in mch and mchc (p<0.05). the highest levels of hb, rbc, and ht were found in group c. conclusion celery ethanol extract at a dose of 250 mg/kg bw/day may prevent anemia in the ckd rat model. keywords: 5/6 subtotal nephrectomy, celery, chronic kidney disease, hematological parameter, rats original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.12-18 cite this article as: afifah, muflikhah k, lestari t, sutrisna e, kirana as, prastiwi sd. the protective effect of celery (apium graveolens l) ethanol extract on anemia in 5/6 subtotal nephr ectomy rat model. un iv m ed 2020;39:12-8. doi: 10.18051/univmed. 2020.v39.12-18 13 january-april, 2020 vol.39no.1 introduction anemia is a frequent complication of chronic kidney disease (ckd).(1) anemia is defined as the condition in which the concentration of hemoglobin (hb) in the blood is below normal. anemia is also a decrease in red blood cells (rbc) or lowered ability of rbc to carry oxygen. the prevalence of anemia is twice as high in people with ckd (15.4%) as in the general population (7.6%). the prevalence of anemia increases with stage of ckd from 8.4% at stage 1 to 53% at stage 5.(2) anemia in ckd is associated with reduced quality of life and increased cardiovascular disease, hospitalizations, cognitive impairment, and mortality.(3) the main cause of anemia in ckd is the inadequate production of endogenous erythropoietin. other contributory factors include deficiency of iron, folate, and vitamin b12, blood loss, and infection or inflammation. erythropoietin is a hormone that acts on the differentiation and maturation of the red blood cell precursors.(4) chronic kidney disease is a major public health problem affecting 13.5% of adult australians and 11% of the population in the us.(5) the prevalence of ckd in indonesia was 89% of those who underwent hemodialysis in 2015.(6) treatment of anemia in ckd consist of various therapeutic agents, such as iron, erythropoiesis-stimulating agent, and red cell transfusion. the cost incurred for treatment in ckd patients with anemia is higher than in ckd patients without anemia.(7) therefore it is necessary to develop an alternative agent for preventing anemia in ckd patients. one strategy that can be done to prevent anemia in ckd is using natural substances. celery is a natural substance that we can find around us and many people have used it as food. celery is recognized to have anti-inflammatory,(8) antioxidant,(9,10) and antihypertensive pharmacological effects.(11) the anti-inflammatory effects of celery are related to the presence of flavonoids and saponins in celery. (8 ) other ingr edien ts that provide antioxidant effects from celery are iridoids, coumarins, and phenolic acids.(9) based on the mechanism of ckd and its progression, celery is thought to prevent anemia which is a ckd complication. there is no information about the protective effect of celery ethanol extract to prevent anemia in a ckd rat model. the 5/6 subtotal nephrectomy is a disease model for ckd and has been a mainstay of studies of progressive renal disease. induction of 5/6 subtotal nephrectomy in mice for two weeks leads to systemic arterial hypertension and to functional and morphological damage of the remnant kidney, which is considered the main characteristics of chronic kidney disease.(12) based on the background above, the objective of this study was to evaluate the effect of celery in preventing anemia in 5/6 subtotal nephrectomy. methods research design this study was an experimental laboratory study with a post-test only control group design. the study was conducted in the pharmacology laboratory, while laboratory measurements of the variables were performed in the biofit laboratory, purwokerto from may until july 2018. plant material fresh celery was collected from pratin, purbalingga, central java and was identified in the environmental laboratory, faculty of biology, universitas jenderal soedirman. preparation of celery extract celery was washed and then dried in a drying oven at a temperature of 45oc. after drying, the celery was then crushed in a disk mill, with a sieve of 60 mesh. it was then put into the extractor container, with the addition of 70% ethanol. the mixture was extracted with the ultraturrax disperser (ika werke gmbh, staufen, germany) at a speed of 1000 rpm, and macerated for 24 hours. filtration of the mixture was performed with a büchner funnel attached to a vacuum pump. for evaporation of the filtrate a 14 va cuum rotar y evapor ator was used at a temperature of 45oc and rotary speed of 90 rpm. the extract was transferred into a porcelain dish and placed in a digital oven at 45oc. the extract was dried 3 times to a constant weight. the solutions made from the extract were prepared freshly each time and were administered orally once daily for 14 days before and 14 days after 5/6 subtotal nephrectomy to the treatment groups. experimental animals the sample size was determined based on the federer formula. a total of 25 healthy male sprague dawley rats aged 2-3 months and weighing between 150 and 250 grams were included into the study. the rats were divided by simple randomization into 5 groups of 5 animals each. the animals were acclimatized at room temperature (25-30oc) for 7 days under a 12hour light and dark cycle with standard rat feed and water ad libitum. induction of experimental chronic kidney disease chronic kidney disease was induced by the 5/6 subtotal nephrectomy technique. the animals underwent either 5/6 subtotal nephrectomy or sham surgery under ketamine anesthesia. briefly, the left kidney was exposed, and the upper and lower poles were tied with silk, followed by right nephrectomy. the peritoneum and skin were then sutured, and the animals were returned to their cages. (12) experimental procedure the rats were randomized into five groups: group a, sham operation; group b, subtotal nephrectomy; group c, subtotal nephrectomy and 250 mg/kg bw ethanol extract of celery orally; group d, subtotal nephrectomy and 500 mg/kg bw ethanol extract of celery orally; group e, subtotal nephrectomy and 1000 mg/kg bw ethanol extract of celery orally. the administration of celery extract was performed 14 days before and 14 days after 5/6 subtotal nephrectomy. the blood samples were collected by retro-orbital puncture to measure the hematological parameters (hb, rbc, ht, mcv. mch. mchc) and serum creatinine. statistical analysis statistical analysis was performed using oneway anova and kruskal-wallis test followed by mann-whitne y test. differences were considered to be significant at p<0.05. ethical clearance the study was conducted after obtaining ethical clearance from the ethics committee of the medical faculty, universitas jenderal soedirman, purwokerto, indonesia, under no. 1664/kepk/iv/2018. results this study used 5/6 subtotal nephrectomy as a ckd model. the impaired kidney function due to ckd was assessed based on serum creatinine levels. serum creatinine levels after nephrectomy and administration of celery extract are presented in table 1. we used a normality test with the shapiro-wilk test, and levene test to know the homogeneity of variances. the serum creatinine levels were normal (p=0.121) a n d ho mo ge no u s ( p= 0 . 48 2) . t h e s e r u m creatinine levels showed significant differences between all groups (p=0.007). the serum creatinine level in group b (1.46 ± 0.09 mg/dl) was significantly higher than in group a (1.32±0.06 mg/dl), indicating that 5/6 subtotal nephrectomy increased serum creatinine level. the serum creatinine levels in group c (1.31 ± 0.06 mg/dl), d (1.38 ± 0.04 mg/dl), and e (1.38 ± 0.05 mg/dl) were lower than in group b and there were significant differences in serum creatinine level between group b and groups c and e, respectively. t he h e ma to l ogi c a l pa r a me te r s a f t e r administration of celery extract 14 days before and 14 days a fter induction of ckd are presented in table 1. the data was subjected to afifah, muflikhah, lestari, et al celery ethanol extract on anemia 15 january-april, 2020 vol.39no.1 normality test with the shapiro-wilk test and the levene test to know the homogeneity of variances. the results of the normality test were normal for hb, rbc, and ht, but not for mcv, mch and mchc. furthermore, a one-way anova statistical test was performed on hb, rbc, and ht, and kruskal-wallis test on mcv, mch, and mchc. the results of the one-way anova test showed that there were no significant differences in mean hb, rbc, and ht levels (p>0.05). the kruskalwallis test showed that mch and mch were significantly different between all groups (p=0.024 and p=0.025, respectively). the mann-whitney test on mch and mchc showed significant differences between group c and groups b, d, and e, respectively. discussion serum creatinine level is one parameter that has been used to represent kidney function. this study showed that serum creatinine level in the 5/6 subtotal nephrectomy group was significantly higher than that in the sham operation group. this result showed that the induction of 5/6 subtotal nephrectomy increased the serum creatinine level, indicating a decline in renal function. according to gava et al.,(12) and layal et al.,(13) 5/6 subtotal nephrectomy increases the level of serum creatinine, showing the presence of renal disturbances. the serum creatinine levels in groups c, d, and e were lower than in group b, and group c had the lowest serum creatinine level, signifying that the administration of ethanol extract of celery decreased the serum creatinine level. an o th e r fun c t i on o f t h e ki d ne ys i s producing erythropoietin (epo), a molecule that stimulates the formation of rbc. disorders in the kidneys disrupt the formation of rbc, causing a n e mi a , w hi c h i s a n a l mo st ir r e du c i b le complication of ckd. anemia occurs more frequently in patients with advanced kidney dysfunction and relates to the quality of life and mortality in ckd patients.(14) anemia in ckd is mainly due to the inadequate amount of epo production of injured kidneys, and the epo deficiency is proposed to be the central feature of ckd associated anemia.(3) the means of hb, rbc, and ht in this study showed no significant differences between all groups, but it can be seen that the means of hb, rbc, and ht were lower in group b compared to group a. this shows that ckd induction using 5/6 subtotal nephrectomy decreased the level of hb, rbc, and ht. corresponding to the research of cahyawati et al.,(15) 5/6 subtotal nephrectomy decreases the hematological parameters at 2 weeks after induction. according to sun et al.,(16) in a ckd mouse model with adenine induction, erythropoietin levels began to decline in the first week of ckd induction, but the hemoglobin level decreased significantly in the fourth week, even though kidney damage already looked significant in the second week. a b c group a (n=5) group b (n=5) group c (n=5) group d (n=5) group e (n=5) p-value creatininea 1.32 ± 0.06* 1.46 ± 0.09 1.31 ± 0.06* 1.38 ± 0.04 1.38 ± 0.05* 0.007 hb (g/dl)a 10.82 ± 1.9 10.04 ± 0.6 10.20 ± 0.8 10.12 ± 1.6 9.94 ± 0.9 0.823 rbc (x106/μl)a 5.75 ± 1.9 4.18 ± 1.0 6.40 ± 1.0 4.49 ± 1.4 4.48 ± 0.4 0.067 ht (%)a 33.1 ± 11.1 25.72 ± 5.8 36.62 ± 5.5 25.90 ± 8.0 25.42 ± 2.1 0.079 mcv(fl)b 60.20 60.80 58.30 57.60 58.10 0.386 mch (pg)b 17.20 20.10# 15.20 20.40# 22.80# 0.024 mchc (g/dl)b 28.50 33.10# 26.00 36.30# 40.30# 0.025 table 1. distribution of creatinine and hematological parameters by treatment groups data presented as mean±sd, except for mcv, mch and mchc as median aone-way anova and post-hoc independent -t test. bkruskal-wallis and post hoc mann-whitney test. *=p<0.05 vs group b; #=p<.0.05 vs group c. group a/sham operation: group with sham operation + distilled water; group b/nephrectomy: ckd model + distilled water; group c: ckd model+ethanol extract of celery 250 mg/kg bw; group d: ckd model+ethanol extract of celery 500 mg/kg bw; group e: ckd model+ethanol extract of celery 1000 mg/kg bw. 16 the mean hb and rbc levels of group c, d, and e (ckd rat model given ethanol extract of celery) tended to be higher than in group b (ckd rat model without ethanol extract of celery), showing the function of active substances in celery in preventing a decrease in hb and rbc levels. celery contains active ingredients that have antioxidant and anti-inflammatory properties such as saponins, flavonoids (quercetin, tannins), polyphenols, iridoids, phenolic acids, and coumarins which can prevent the progression of ckd by inhibiting the occurrence of inflammation and oxidative stress.(17) as an antioxidant, flavonoids prevent cellular component damage due to chemical reactions involving free radicals. some mechanisms are to deactivate ros and stimulate the production of antioxidants.(18) antioxidant properties are also shown by phenolic acids which have the effect of preventing the formation of ros.(19) anemia of ckd has also been reported to be associated with the suppressive effects of pro-inflammatory cytokines on erythropoiesis, which cause the anemia of inflammation.(20) anti-inflammatory eff ects of celery ar ise through sever al mechanisms. flavonoids and saponins have antiinflammatory effects by inhibiting the formation of cyclooxygenase 2 (cox-2), while iridoids act by inhibiting pro-inflammatory nf-κb p65 gene expression.(21,22) coumarin inhibits the formation of superoxide anions and can inhibit the production of cox. by inhibiting the progression of ckd by celery active substances, the decline in kidney function that causes ckd complications such as anemia can be prevented.(23-26) besides flavonoids, especially quercetin types can inhibit 2,2-azo-bis (2-amidinopropane) dihydrochloride (aaph) (free radicals that cause oxidative damage to erythrocyte membranes causing erythrocyte hemolysis). pre vention of hemolysis of erythrocytes can prevent a decrease in the number of erythrocytes and hemoglobin levels in the blood.(19) although the difference in mean hemoglobin levels between groups c, d, and e was not significant, among the 3 groups it was found that group c, receiving celery ethanol extract at a dose of 250 mg/kg bw/day orally, had the highest mean hb, rbc and ht levels. the results in the d and e groups are influenced by the dose factor. the serum creatinine level at the dose of 250 mg/kg bw/day of celery extract, was the lowest compared to the doses of 500 mg/kg bw/day and 1000 mg/kg bw/day. in patients with kidney disease such as ckd, celery should not be given at too high doses because it can have adverse effects.(27) at low doses, flavonoids have antioxidant effects, but at high doses, the flavonoids in celery can produce prooxidative effects by stimulating the formation of reactive oxygen species (ros).(19) the mean mch and mchc in this study were significantly different between groups c and b. the mcv, mch, and mchc values were characteristic for the type of anemia, which in ckd is typically normocytic, normochromic and hypoproliferative. it is most commonly caused by decreased erythropoietin production and/or iron deficiency.(3) a similar study needs to be carried out to assess the erythropoietin and iron levels with a duration of at least 4 weeks after ckd induction.(15) one limitation of this study was the relatively short time from operation until termination in which to obtain samples. therefore, anemia, the complication of ckd, was not yet apparent. the implication of this study is that it can be applied to ckd high risk populations, such as patients using nephrotoxic drugs, patients with diabetes mellitus, and patients with hypertension, to prevent ckd and its complication. conclusions celery ethanol extract at a dose of 250 mg/ kg bw may prevent anemia in the 5/6 subtotal nephrectomy rat model. conflict of interest the authors declared no conflict of interests. afifah, muflikhah, lestari, et al celery ethanol extract on anemia 17 january-april, 2020 vol.39no.1 acknowledgement this study was supported by research grants (blu) from unsoed. contributors aa, km, tl, and es contributed to the basic concept and design of the study. aa, km contributed to writing the manuscript and performing the experiment. aa, km, ask, sdp contributed to sample preparation and data collection. aa contributed to the statistical analysis. all authors have read and approved the final manuscript. references 1. cases a, egocheaga m, salvador t, et al. anemia of chronic kidney disease: protocol of study, management and referral to nephrology. nefrologia 2018;38:8–12. doi: 10.1016/j.nefroe. 2018.01.007. 2. staufer m, fan t. prevalence of chronic kidney disease in the united states. plos one 2014;9:e84943. doi:10.1371/journal.pone.0084943. 3. 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**department of postgraduate biomedical science, faculty of medicine, universitas islam sultan agung, semarang ***stem cell and cancer research laboratory (sccr), faculty of medicine, univeritas islam sultan agung, semarang correspondence : dr. dr. agung putra, m. si. med. candi prambanan utara raya kav. 1135-1136 kelurahan kalipancur, manyaran, kecamatan ngaliyan, semarang phone: +62816 4251 646 email: dr.agungptr@gmail.com orcid id: orcid.org/0000-00029822-3119 date of first submission, april 27, 2018 date of final revised submission, july 23, 2018 date of acceptance, july 24, 2018 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina tumor necrosis factor-α-activated mesenchymal stem cells accelerate wound healing through vascular endothelial growth factor regulation in rats aditya nugraha* and agung putra**,*** background wounds are areas of physical or thermal damage of the epithelial layer of skin or mucosa. the wound healing process consists of hemostasis, inflammation, proliferation, and remodeling. mesenchymal stem cells (mscs) play a role in wound healing by suppressing potent proinflammatory molecules, such as tumor necrosis factor-α (tnf-α), leading to macrophage polarization from the pro-inflammatory type to the proregeneration type characterized by increasing vascular endothelial growth factor (vegf) production. mscs are able to increase vegf level in-vivo correlated with collagen synthesis. the objective of this study was to assess the role of tnf-α-activated mscs on vegf in rat wounds. methods an experimental animal study with post-test only control group design was performed involving 24 wistar rats. the rats were randomized into four groups consisting of one control (k) and three treatment groups (p) (activated mscs at doses of 3x105, 6x105, and 12x105 cells, respectively). the measurement of vegf levels was done using elisa assay while the collagen analysis was performed by light microscopy. one way anova and post hoc lsd were used to analyze the data. results the results showed a significant increase in vegf levels (p <0.05) on day 3 and then a significant decrease on day 5 along with a significant increase in the amount of collagen on day 7 (p<0.05). conclusion this study demonstrated that tnf-α-activated mscs were able to regulate vegf levels and collagen synthesis in wound healing in rats. the molecular mechanism by which tnf-α-activated mscs stimulate cutaneous wound healing should be clarified further. keywords: mesenchymal stem cells, tumor necrosis factor-α, vascular endothelial growth factor, collagen, rats doi: http://dx.doi.org/10.18051/univmed.2018.v37.135-142 original article pissn: 1907-3062 / eissn: 2407-2230 may-august, 2018 vol.37no.2 cite this article as: nugraha a, putra a. tumor necrosis factor--activated mesenchymal stem cells accelerate wound healing through vascular endothelial growth factor regulation in rats. univ med 2018;37:135-42. doi: 10.18051/ univmed.2018.v37.135-142 136 nugraha, putra activated mscs regulate vegf & collagen introduction wounds are defined as areas of physical or thermal damage in the cutaneous or mucosal epithelium that can be categorized as acute and chronic. (1) the wound healing process consists of the phases of hemostasis, inflammation, proliferation, and remodeling.(2) the inflammatory phase is initiated by neutrophil migration to the wound site as a result of stimulation by various chemoattractant molecules, such as tumor necrosis factor-α (tnf-α) or interleukin-1 (il1). the proliferative phase is marked by an increase in a number of growth factors, such as vascular endothelial growth factor (vegf).(3,4) the increase of these molecules may stimulate fibroblast proliferation that results in increased collagen synthesis, in addition to angiogenesis.(1– 3) several studies report that mesenchymal stem cells (mscs) play an important role in wound healing, particularly in the inflammatory phase, because of their immunoregulator y characteristics, so that they are able to control the inflammatory process.(5) mesenchymal stem cells are adult stem cells with the characteristics of adhering to plastic culture dishes, having various surface markers, such as cd73(+), cd90(+), cd105(+), cd45(), cd34(-), cd14(-) or cd11 (-), cd79(-) or cd19 (-) and hla-dr(-),(6) and being able to differentiate into various cells, such as neurons and adipocytes.(6,7) mesenchymal stem cells also play a role in immunoregulation of inflammation, by suppressing the release of pro-inflammatory molecules from various inflammatory cells, so accelerating the transition from the inflammatory phase into the regenerative phase.(1,8) several potent pro-inflammatory cytokines, particularly tnf-α, play a n important role in mscs activation.(9,10) activated mscs release various molecules, such as prostaglandin e 2 (pge 2 ) that will bind to their ep4 and ep2 receptors on macrophages, so effecting the polarization of macrophages from the inflammatory into the regenerative type.(11) this is marked by the release of the anti-inflammatory interleukin-10 (il-10).(12) specifically, mscs activation occurs through binding of tnf-α molecules to msc receptors that impact on the activation of the nuclear factor kappa-b (nf-κb) pathway, so that several anti-inflammatory and proliferative molecules are expressed.(11) theoretically, the tnf-α-activated mscs are able to express va rious pro-r egenera tive growth factors, particularly vegf, platelet-derived growth factor (pdgf), and fibroblast growth factor (fgf), that can activate fibroblasts and impact on collagen synthesis.(9) previous studies have reported that mscs activated by the pro-inflammatory cytokine interferon gamma (ifn-γ) are able to increase wound regeneration and tensile strength that is correlated with the amount of collagen.(13) on the other hand, mscs that are activated in vitro by tnf-α, lipopolysaccharide (lps) and hypoxia, are able to increase the production of growth factors, particularly vegf,(14) but the role of tnf-αactivated mscs on vegf concentrations and their relationship with collagen are as yet unknown. the aim of the present study was to evaluate the effect of mscs that are activated by tnf-α at a dose of 10 ng/ml on vegf and collagen concentrations in rats with incised wounds. methods research design this study used a post-test only control group design and was conducted in the stem cell and cancer research laboratory, faculty of medicine, universitas islam sultan agung (unissula), semarang, from 2 april to 25 august 2017. animals the subjects of this study were 4 to 6-month old wistar rats weighing 250-300 grams, that were certified healthy by the agricultural and fishery service (dinas pertanian dan perikanan) of salatiga city under no. 524.3/0211/421. the sample size calculation was according to the 137 federer formula for determining the required sample size: (n-1) x (t-1) 15. the sample used in this study comprised 6 animals per group with an estimated drop-out rate of 10%, so that the total sample size was 24 rats (n=6 rats/group). the rats were randomly divided into 4 groups (3 intervention groups p1-p3 and 1 control group k). the inclusion criteria were: purebred rats aged 4 to 6 months, weighing 250-300 grams, and without apparent anatomical defects. the exclusion criteria were: not active during the 7day adaptation period and/or having apparent anatomical defects. the drop-out criterion was d yi ng i n th e c our se of t he e xper imen ta l interventions. the incised rats were injected with different doses of mscs. the control group was injected with phosphate buffered saline (pbs). groups p1, p2, and p3 received subcutaneous injections of activated mscs at doses of 3x 105, 6 x 105 and 12 x 105 cells, respectively. the interventions were applied once daily in the morning. surgical procedure paravertebral full-thickness dorsal skin incisions were made aseptically with a no. 15 scalpel (b. braun aesculap ag, tuttlingen, germany) under general anesthesia in the longitudinal axis at least 1 cm apart a nd perpendicular to the skin cleavage lines. as a result, minimal strain was applied to the skin, allowing for direct visual control of the depth of the incision. for each incision, the dorsal muscles were the limits at which to stop.(15) isolation of mscs the umbilical cord of a female rat at 19 days of pregnancy was washed in pbs. the umbilical blood vessels were removed, then the umbilicus was cut into lengths of 2-5 mm using a sterile knife and the sections distributed evenly on a 66-mm culture dish. the medium used was dulbecco’s modified eagle’s medium (dmem) (sigma-aldrich, st. louis, mo) mixed with 10% pbs, 100 iu/ml penicillin/streptomycin (gibco, invitrogen), then incubated at 37oc with 5% co 2 . the medium was refreshed once in 3 days.(16) characteristics of the msc phenotype the expression of the mscs isolated from the umbilical cord of the female rat was evaluated by immunocytochemistry using msc-positive markers. after the mscs were 60-80% confluent, they were harvested and grown on coverslips, then fixed with 4% paraformaldehyde in 90% ethanol for 15 minutes at 4oc. the cells were incubated with cd73 and cd105 primary antibody as msc-positive markers (1:100; bd pharmingen, san diego, ca, usa) for 60 minutes at room temperature, washed in pbs for 10 minutes then secondary antibody (1:2500) was added, the cells left to stand for 15 minutes at room temperature, then counterstained with diaminobenzene (dab) (santa cruz biotech) and observed under the microscope.(16) in-vitro differentiation the mesenchymal stem cells were grown in culture dishes at densities of 5x103 and 1x104 cells/well, to which was added osteogenic induction medium containing 10 mmol/l βglycerophos phate, 10 -7 mol/l/ 0.1 µm dexamethasone, 50µmol/l ascorbate-2-phosphate (sigma-aldrich, louis st, mo) and 10 % fetal bovine serum (fbs) in dmem. osteogenic differentiation was observed by alizarin red staining after 21 days of induction to find calcium deposits. the cells were rinsed in pbs and fixed with cold 70% ethanol (v/v) for 1 hour at room temperature, then rinsed three times with twicedistilled water. a volume of 1 ml 2% alizarin red solution (w/v) (ph 4.1-4.3) was added and the cells incubated for 30 minutes at room temperature, then rinsed four times in twicedistilled water.(17) tnf-α induction of mscs mscs (1x104 cells/well) were supplemented with 10 ng/ml recombinant tnf-α (biolegend, san diego, ca) on a 24-well plate using alphamem medium (sigma-aldrich, louis st, univ med vol. 37 no.2 138 nugraha, putra activated mscs regulate vegf & collagen a b c mo), then incubated for 24 hours at 37o with 5% co 2 . rat vegf elisa assay the serum vegf assay by means of an elisa kit (fine test, china) was performed at room temperatur e according to the manufacturer’s instructions. the elisa plate was coated with capture antibody and incubated overnight at 4oc. the wells were washed, then blocked for 1 hour, after which they were incubated with rat vegf standard solution and the intervention and control rat sera for 2 hours, then diluted to 1:1 00 so that the vegf concentrations could be determined on the standard curves. after washing, the wells were incubated with detection antibody for one hour, then washed again several times. the wells were incubated with avidin-hrp for 30 minutes, washed thoroughly, and incubated with substrate solution for 15 minutes, which was followed by the addition of the stop solution. the results were analyzed at a wavelength of 450 å using a microplate reader. the vegf concentrations (pg/ ml) were measured on days 3 and 5 of the experiment. preparation of collagen slides skin tissue was collected and placed in 10% neutral-buffered formalin then embedded in paraffin or histogel (thermo scientific, watham, ma, usa). subsequently the tissue paraffin block was cut on a microtome to a thickness of 5-10 µm then stained with masson trichrome and observed under the microscope. the collagen density was calculated from the area of the collagenous tissue formed on each slide (%). statistical analysis the amount of collagen and the vegf concentration were presented descriptively. data processing was performed using spss 23.0 for windows. the test of normality used the shapirowilk test and the test of homogeneity used levene’s statistical test. subsequently a parametric difference test was performed followed by anova and lsd post-hoc test. ethical clearance all research activities were in accordance to and approved by the commission on test animal ethics (komisi etik hewan uji), faculty of medicine, universitas islam sultan agung, semarang, under no. 204/vi/2017/komisi bioetik. results characteristics of mscs the characteristics of the mscs isolated by in-vitro dish culture until the cell population was at 80% confluence were as follows. the cells were spindle-shaped, resembling fibroblasts (figure 1a). the confluent mscs that were tested using an immunocytochemical technique showed positive expression of various protein surface markers, particularly cd105(+) (figure 1b) and cd73(+) (figure 1c), as indicated by the development of a brown color in the population of mscs. tests for differentiating the mscs were figure 1. characteristics and differentiation of cultured mesenchymal stem cells legend: (a) umbilical cord candidate mscs from in-vitro culture (b) cd105 expression, (c) cd73 expression. mscs are brownish in color (d) osteogenic differentiation with alizarin red staining 139 univ med vol. 37 no.2 performed by administration of osteogenic medium and showed that mscs were capable of differentiating into osteogenic cells, as indicated in the cell population by a red color from alizarin red staining (figure 1d). vegf concentration and collagen density the mean vegf concentration increased on day 3, then decreased on day 5. the vegf concentration on day 3 showed a significant increase (p<0.05) (table 1) with the following mean values: k = 29.17 ±11.67 pg/ml; p1 = 87.38 ±17.38 pg/ml; p2 = 112.99 ±19.91 pg/ml; p 3= 98.52 ± 43.88 pg/ml. however, on day 5 a non significant decreased was found (p>0.05) (table 1). the mean collagen density on day 7 differed significantly between the control group and the intervention groups (p<0.05). mean collagen density was highest in group p3 (2.83 ± 0.80) and lowest in the control group, group p1, and group p2 (1.33 ± 0.51, 1.50 ± 0.54, and 2.50 ± 0.54, respectively) (table 1). these results were also supported by the microscopic picture of collagen preparations with masson trichrome staining. the collagen was colored blue and filled the field of view (figure 2). the results of the post-hoc analysis showed that mean vegf concentration on days 3 and 5 differed significantly between the control group and groups p1, p2 and p3 (p<0.05). however, mean vegf concentration on days 3 and 5 showed no significant difference between groups p1, p2 and p3 (table 2). mean collagen density on day 7 differed significantly between the control group and groups p1 and p2, between groups p1 and p3, and between groups p2 and p3. discussion our study results showed that serum vegf concentration increased significantly on day 3 in rat wounds treated with tnf-α activated mscs, giving the impression that mscs are able to accelerate the transition from the inflammatory phase into the proliferative phase through the polarization of type m1 proinflammatory day intervention group (mean±sd) p value control (n=6) p1 (n=6) p2 (n=6) p3 (n=6) vegf on day 3 (pg/ml) 29.17 ± 11.67 87.38 ± 17.38 112.99 ± 19.91 98.52 ± 43.88 0.001 vegf on day 5 (pg/ml) 67.45 ± 53.65 5.09 ± 3.42 22.05 ± 8.23 15.93 ± 34.49 0.012 collagen on day 7 (%) 1.33 ± 0.51 1.50 ± 0.54 2.50 ± 0.54 2.83 ± 0.80 0.000 table 1. mean vegf concentration on days 3 and 5 and collagen density on day 7 *significant at p<0.05; note: p1: activated mscs at dose of 3x 105 cells; p2: activated mscs at dose of 6 x 105 cells; p3: activated mscs at dose of 12 x 105 cells figure 2. microscopic preparations showing collagen density, with masson trichrome staining. collagen is bluish in color. magnification 100x legend : panels a,b, c and d represent controls, p1, p2 and p3, respectively 140 nugraha, putra activated mscs regulate vegf & collagen ma cr o pha ge s i n to typ e m 2 pr o l if e r a ti ve macrophages.(18) this is in line with previous studies stating that mscs activated by 50 ng/ml of tnf-α are able to increase vegf expression significantly in 24 hours post-induction,(14) indicating that the mscs are induced by the t nf-α i n f l a mma to r y s ign a l s t o s e c r e te proliferative molecules, such as vegf, in addition to anti-inflammatory molecules.(19) other studies have reported that in normal wound healing the serum vegf concentration starts to increase on the fourth day and continues until the seventh day of the prolifer ative phase.(1,20) t his gives the impression that activated mscs are more potent in suppressing inflammation in wounds and promote the activation of various pro-proliferative cells, so accelerating the wound healing process. on the other hand, our study also found that the vegf concentration decreased significantly on day 5 (p<0.05). we surmise that this decrease in vegf concentration on day 5 was due to the more rapid transition from the proliferative to the remodeling phase, as a result of the administration of tnf-α-activated mscs. this agrees with previous studies stating that in the remodeling phase the vegf concentration decreases significantly, marking the end of the proliferative phase.(21) a high vegf concentration in the remodeling phase may cause an interference with wound healing that is marked by tissue fibrosis.(22) our study also found a significantly increased collagen density in wounds receiving tnf-α-activated mscs. the collagen density is shown by the microscopic picture of a bluish color in preparations with masson’s trichrome staining. this collagen density correlates with the number of tnf-α-activated mscs administered at each of the interventions. these results are supported by previous studies, in that msc administration is able to accelerate wound regeneration by modulating collagen synthesis, in addition to inhibiting apoptosis.(23) we think that the increase in collagen density on day 7 is related to the ability of tnf-α-ac tivated mscs for pa racrine expression of various growth factors, such as vegf, so accelerating the transition from the inflammatory to the proliferative phase, in addition to differentiating into fibroblasts and activating endogenous stem cells to promote fibroblast formation and collagen synthesis. this agrees with previous studies stating that fibroblasts that express large amounts of vegf may trigger collagen formation.(24) one limitation of the present study is that we did not determine the concentrations of pdgf and transforming growth factor (tgf) that are potent mediators of fibroblast activation in collagen synthesis. we also did not measure vegf concentrations at baseline and at the end o f t h e i n te r ve nt i o ns t o d e t e rmi ne t he i r r e l a ti o ns hi p w it h t he in f l a mma t or y a n d remodeling phases. this study has the potential to become in the future one of the solutions to the problem of accelerating wound healing, particularly chronic wounds. mean difference p value vegf on day 3 controls p1 p2 p3 p1 p2 p3 p2 p3 62.36 45.40 51.51 16.96 10.848 6.114 0.002* 0.018* 0.009* 0.339 0.587 0.727 vegf on day 5 controls p1 p2 p3 p1 p2 p3 p2 p3 57.21 82.82 68.35 25.61 11.13 14.47 0.003* 0.000* 0.001* 0.144 0.513 0.398 collagen on day 7 controls p1 p2 p3 p1 p2 p3 p2 p3 1.00 1.33 0.16 0.33 0.16 1.500 0.003* 0.000* 0.576 0.269 0.001* 0.000* table 2. post-hoc test for vegf on days 3 and 5 and collagen on day 7 *significant at p<0.05; note : p1 : activated mscs at dose of 3x 105 cells; p2 : activated mscs at dose of 6 x 105 cells; p3 : activated mscs at dose of 12 x 105 cells 141 univ med vol. 37 no.2 conclusion this study demonstrated that tnf-αactivated mscs are able to regulate vegf concentrations and collagen density in wound healing. the vegf concentrations expressed by activated mscs accelerate the transition from the inflammatory to the regenerative phase and trigger collagen synthesis in wound healing. conflict of interest competing interests: no relevant disclosures financial disclosure the authors did not receive any specific grant from any public funding agency. acknowledgement we would like to express our heartfelt gratitude to the staff members of the stem cell and cancer research laboratory (sccr), faculty of medicine, universitas islam sultan agung, semarang an d the postgra duate biomedical program, faculty of medicine, universitas islam sultan agung, for their assistance in this study. references 1. landen nx, li d, stahle m. transition from inflammation to proliferation: a critical step during wound healing. cell mol life sci 2016;73:3861– 85. doi: 10.1007/s00018-016-2268-0. 2. nuschke a. activity of mesenchymal stem cells in therapies for chronic skin wound healing. 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http://dx.doi.org/ 10.1155/2016/7357096. 24. bauer sm, bauer rj, liu zj, et al. vascular endothelial growth factor-c promotes vasculogenesis, angiogenesis, and collagen constriction in three-dimensional collagen gels. j vasc surg 2005;41:699–707. oktavianus 159 *department of pathological anatomy, faculty of dentistry, trisakti university **department of pathological anatomy faculty of medicine tarumanagara university correspondence prof. dr. janti sudiono, mdsc. department of pathological anatomy faculty of dentistry universitas trisakti jakarta 11440 email: jantish@hotmail.com univ med 2012;31:159-66 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background neoplastic and non-neoplastic oral mucosal growths often have a variety of clinical manifestations according to their biological nature. immunohistochemical diagnostic markers, such as ki-67, are used to detect their proliferation and differentiation. ki-67 is expressed in all phases of the cell cycle, except g 0 . the objective of this study was to determine ki-67 expression in benign, malignant and non-neoplastic proliferation of oral mucosal epithelium. methods a laboratory study of cross sectional design was conducted using samples from excised oral mucosa diagnosed as inflammatory gingival hypertrophy (n=5); epulis (n=6); gingival polyps (n=5); pulpal polyps (n=5); papilloma (n=3) and squamous cell carcinoma (n=2). the antigen retrieval endogenous peroxidase block method was used in the application of ki-67 primary antibody and chromogen to display the antigen antibody reaction, with positive cells showing brown nucleoplasm staining. the ki-67 positive index was calculated by dividing the number of positive epithelial cells with the total number of epithelial cells in the areas observed at 400x magnification. one-way anova was used to compare the ki-67 indexes of neoplastic and non-neoplastic lesions. results the highest ki-67 positive index was for squamous cell carcinoma (64.55 ± 23.55%) followed by papilloma (23.33 ± 6.94%), gingival polyps (7.06 ± 7.43%) and gingival hypertrophy (1.40 ± 2.80%). one-way anova showed significant differences in ki-67 expression between neoplastic and non-neoplastic samples (p<0.05). conclusions the high ki-67 expression in neoplasms is proportional to the grade of malignancy. in non-neoplastic processes ki-67 expression is merely an adaptive response and does not indicate increased ki-67 proliferative gene expression. keywords: ki-67, proliferative, non-neoplastic, benign, malignant low ki-67 gene expression in non-neoplastic proliferation of oral mucosal epithelium janti sudiono* and irmawati hassan** 160 sudiono, hassan ki-67 gene expression in non-neoplastic ekpresi gen ki-67 rendah pada proliferasi non-neoplastik epitel mukosa oral latar belakang pertumbuhan proliferatif mukosa mulut non neoplastik dan neoplastik banyak dijumpai dengan manifestasi klinis bervariasi sesuai sifat biologisnya. petanda diagnostik imunohistokimiawi banyak digunakan untuk mengetahui proliferasi dan diferensiasi sel serta membantu memahami sifat biologis lesi. ki-67 merupakan petanda proliferasi terekspresi dalam semua fase siklus sel kecuali istirahat, memberikan pewarnaan kuat pada sediaan parafin. penelitian in bertujuan untuk menentukan ekspresi gen ki-67 pada pertumbuhan proliferatif non-neoplastik, neoplastik jinak dan ganas pada epitel mukosa mulut. metode penelitian laboratoris rancangan potong silang dilakukan pada sampel hasil terapi eksisi mukosa mulut dengan diagnosis hipertrofi gingiva (n=5); polip gingiva (n=5); epulis (n=6); polip pulpa (n=5); papiloma (n=3); dan karsinoma sel skuamosa (n=2). pemberian antibodi primer ki-67 dan kromogen untuk ekspresi warna reaksi antigen antibodi. sel positif bila nukleoplasma berwarna coklat. indeks ki-67 diperoleh dengan membagi jumlah sel epitel positif dengan jumlah sel epitel pengamatan pembesaran 400x. uji anova one-way digunakan untuk membandingkan indeks ki-67 antara lesi non-neoplastik dan neoplastik. hasil hasil penelitian menunjukkan indeks ki-67 tertinggi didapatkan pada karsinoma sel skwamosa (64,55 ± 23,55%) disusul papiloma skwamosa (23,33 ± 6,94%), polip gingiva (7,06 ± 7,43%) dan hipertrofi gingiva (1,40 ± 2,80%). analisis one-way anova menunjukkan adanya perbedaan indeks ki-67 yang bermakna antara lesi neoplastik dan non neoplastik (p<0.05). kesimpulan ekspresi ki-67 tinggi didapatkan pada proses neoplastik sesuai tingkat keganasannya, dan rendah pada proses proliferatif non neoplastik. adanya ekspresi ki-67 pada proses proliferatif non neoplastik hanya merupakan respon adaptif, tak cukup untuk mengindikasikan peningkatan gen proliferatif ki-67. kata kunci: ki-67, proliferatif, non-neoplastik, jinak, ganas abstrak introduction studies of cell cycle regulation have revealed that malignant growth is caused by cumulative damage to specific cell growthregulating genes. histopathologic diagnosis of premalignant lesions in the oral cavity increases in quality if accompanied by quantitative measurement of specific diagnostic biomarkers, a s t h i s w i l l i n c r e a s e t h e a c c u r a c y o f identification.(1) increased cell proliferation as a result of modifications in the genes regulating cell growth, namely the proto-oncogenes and the tumor suppressor genes, is believed to play a role in the induction of human cancers. these genetic changes are closely associated with the development of neoplasms, and can be used as a marker for spesific changes that play a role in tumor formation.(2-4) excessive growth is a cellular response to signals or to higher functional requirements caused by increased expression of genes of differentiation.(5) a frequently encountered example of excessive growth with manifestations 161 of gingival inflammation is inflammatory e n l a rg e m e n t o f t h e g i n g i v a o r g i n g i v a l hypertrophy due to inflammation.(6) in addition to inflammation, other examples of abnormal growth frequently found in the gingiva are gingival polyps and epulis. one type of epulis that constitutes a real neoplasm is congenital epulis, which is a granular cell myoblastoma.(7) to date many experts have used methods for detecting diagnostic markers that can function as early indicators of abnormal growth, including m a l i g n a n c y. t h e s e m e t h o d s i n c l u d e immunohistochemical detection of genetic markers of cell proliferation, cell differentiation, gene mutation, and tissue vascularization, which are expected to help in further understanding the disease process. immunohistochemical methods can localize specific intracellular proteins in fixed tissue samples, based on the principle of the binding of specific antibodies to antigens present in these tissues. immunohistochemical markers, such as p53, proliferating cell nuclear antigen (pcna), and ki-67, can be used in paraffin sections to show the presence of proliferative genes or cellular proliferation capacity and possible early malignant changes.(8) the ki-67 antibody is of great diagnostic benefit only for mammalian species, in contrast to the pcna antibody, which can be used for all species of animals. the ki-67 protein is expressed in all phases of the cell cycle, except in the resting (g 0 ) phase, and is a good proliferation marker, as it stains strongly in paraffin sections. therefore, the use of ki-67 as a proliferation marker for diagnostic as well as research is developing rapidly. ki-67 is a nuclear protein expressed in the g 2 and m phases of the actively dividing cell.(3) this antigen is a proliferation marker that is correlated with epithelial dysplasia and the grade of the dysplasia.(3) excessive expression of gene protein products that are associated with the cell cycle, such as the p53, p16 and ki-67 (identifiable with the monoclonal ki-67 a n t i b o d y ) i s h i g h l y s i g n i f i c a n t . (9) ki-67 expression can significantly differentiate between normal mucosa, benign neoplastic disease, and mild dysplasia.(9) ki-67 antigen expression also appears when dna synthesis is stopped or when the cell undergoes apoptosis. however, according to other investigators, the proliferation markers that are in use recently, such as ki-67, can only yield a limited picture of its involvement in the cell cycle.(10) in addition, the value of ki-67 as a p r o g n o s t i c f a c t o r i s s t i l l i n c o m p l e t e l y documented and the evidence on the relationship between cell proliferation and the clinical course of disease is still contradictive.(11,12) ki-67 expression does not clearly reflect the precise biological nature of the lesion. one study showed a slightly lower ki-67 expression in oral cancers (80%) in comparison with precancerous lesions (85.1%).(8) this indicates that the functions of the ki-67 protein are still not known with certainty, and this protein is considered not to be a key element in cell proliferation. (13) nevertheless, ki-67 can be used to measure the growth fraction, both in normal, premalignant, and malignant tissues.(14) some are also of the opinion that ki-67 as marker for tumor growth stimulating genes and p53 as tumor suppressor gene marker are closely associated with their prognostic value.(15) although there are many studies on ki-67 gene expression in benign and malignant neoplastic oral lesions in relation to the grade of malignancy or dysplasia, few studies have been conducted on ki-67 proliferative gene expression in non-neoplastic oral lesions. these considerations led us to conduct the present study on immunohistochemical determination of ki-67 protein expression in relation to the b i o l o g i c a l n a t u r e o f t h e s a m p l e s , w h i c h consisted of benign, malignant, and nonneoplastic proliferative oral lesions. methods study design a cross-sectional laboratory study was conducted from january to december 2007. univ med vol. 31 no.3 162 sudiono, hassan ki-67 gene expression in non-neoplastic tissue samples a total of 26 specimens were obtained as follows. sixteen tissue samples resulting from surgery at the department of periodontology (5 s a m p l e s o f g i n g i v a l h y p e r t r o p h y d u e t o inflammation, 5 of gingival polyps, and 6 of epulis); 5 samples (pulpal polyps) from the department of pedodontics of the educational dental clinic, faculty of dentistry, trisakti university, and 5 paraffin sections (3 squamous papillomas and 2 squamous cell carcinomas of the tongue) from the department of pathological anatomy, faculty of medicine, tarumanagara university. on the basis of their biological nature, the samples were categorized as nonneoplastic, benign neoplastic, and malignant neoplastic. all non-neoplastic samples had been diagnosis on the basis of their clinical features only, whereas the benign and malignant n e o p l a s t i c p a r a ff i n s e c t i o n s h a d b e e n histopathologically diagnosed. immunohistochemistry analysis the specimens were sent to the pathological anatomy laboratory of dharmais cancer hospital for examination by hematoxylin-eosin (he) staining and immunohistochemical staining using ki-67 monoclonal antibody. ki-67 monoclonal antibody staining was also used for the positive control preparations using special glass slides. in the negative control group counterstaining was performed by means of he staining, without ki-67 antibody staining. interpretation of specimens the histopathological preparations stained with he were evaluated according to the following characteristics: thickness of the e p i t h e l i a l l a y e r, c a t e g o r i z e d a s n o r m a l , hyperplastic, hyperplastic and ulcerative; presence of signs of epithelial dysplasia or epithelial malignancy, i.e. hyperchromatic and pleiomorphic nuclei, increased nuclear to cytoplasmic ratio, prominent nucleoli, bizzare nuclei. the presence of inflammation was also figure 1. gingival hypertrophy due to inflammation. shown are positive ki-67 cells (arrow) with brown nucleoplasm (magnification 400x) figure 2. squamous cell papilloma of the tongue. shown are positive ki-67 cells (arrow) with brown nucleoplasm (magnification 400x) figure 3. squamous cell carcinoma of the tongue. shown are positive ki-67 cells (arrow) with brown nucleoplasm (magnification 400x) 163 evaluated, based on the intensity of inflammatory cell infiltrates in subepithelial connective tissue, categorized as negative, mild, moderate, and severe. this was done because the presence of inflammatory cells reflects the responses of the b o d y t o p a t h o l o g i c a l c o n d i t i o n s , e i t h e r inflammation or malignant conditions, which also affect the overlying epithelium. epithelial cells were considered to be ki67 positive if there was brownish staining of the nucleoplasm (figures 1, 2, 3). determination of the ki-67 positive score was done by selecting 3 areas containing the greatest number of p o s i t i v e c e l l s f r o m o b s e r v a t i o n a t 4 0 0 x magnification. the percentage of ki-67 positive scores (labelling index) was calculated by dividing the number of positive epithelial cells by the total number of epithelial cells in the respective areas counted at 400x magnification. there is no standard value for the ki-67 index that is considered to positively indicate the presence of proliferative gene markers with a potential or at high risk of undergoing gene mutation or neoplastic transformation. one study showed that ki-67 expression in oral squamous cell carcinomas is in the range of 560%.(16) in the present study the ki-67 positive index was divided into 3 categories: scores of <10%, scores of >10 20%, and scores of >20%. data analysis statistical analyses were performed using spss version 17.0 software. on the basis of the mean difference in ki-67 expression between non-neoplastic proliferative, benign neoplastic, and malignant neoplastic samples, the data were analyzed by one-way anova. table 1. mean ki-67 expression in all samples *values are mean ± standard deviation results a total of 15 (57.7%) specimens showed the presence of ki-67 expression. mean ki-67 expression in gingival hypertrophy due to inflammation, gingival polyps, squamous papillomas, and squamous cell carcinomas is shown in table 1. the highest ki-67 expression was found in squamous cell carcinomas (64.55 ± 23.55), followed by squamous papillomas (23.33 ± 6.94), gingival polyps (7.06 ± 7.43), and gingival hypertrophy due to inflammation (1.40 ± 2.80). the results of one-way anova showed a significant difference between all four groups of specimens (p=0.000) (table 1). bonferroni’s multiple comparison test (post hoc test) revealed highly significant differences between neoplastic samples (squamous papilloma and squamous cell carcinomas) and non-neoplastic samples (gingival polyps and gingival hypertrophy due to inflammation), with the respective p values of 0.001, 0.001, 0.001, and 0.007. the proliferative state of the oral mucosal epithelial cells of the samples was categorized as normal, hyperplastic, and hyperplasticulcerative (table 2). there was no statistically significant difference (p=0.104) between ki-67 score and proliferative state of oral mucosal epithelial cells. the intensity of inflammatory cells of the lesions was categorized as negative, mild, moderate, and severe. statistically, there was a significant difference (p=0.00) in mean ki-67 expression between neoplastic lesions with mildto-severe inflammatory cell intensities and nonn e o p l a s t i c l e s i o n s w i t h m i l d t o s e v e r e inflammatory cell intensities (table 3). univ med vol. 31 no.3 164 sudiono, hassan ki-67 gene expression in non-neoplastic table 3. distribution of ki-67 expression in biological nature of lesions and inflammatory cell intensity according to ki-67 expression category *values are mean ± standard deviation discussion the results of this study showed that the neoplastic samples also had moderate to severe inflammatory cell intensities on histological examination (table 3). this is supported by the results of statistical tests indicating that inflammation affects cell proliferation and ki67 proliferative gene expression, both in nonneoplastic and neoplastic samples. gingival mucosal epithelial proliferation in epulis and pulpal polyps yielded negative ki-67 scores. however, in gingival polyps and gingival hypertrophy due to inflammation, which histologically also showed epithelial proliferation categorized as epithelial hyperplasia, the mean ki-67 score was found to be 7.06 ± 7.43%. this may be due to the fact that epithelial hyperplasia is an adaptive response to non-neoplastic stimuli and mainly caused by chronic irritation or inflammation, such that ki-67 proliferative gene expression is incompletely detected. this was demonstrated by the statistical analysis which revealed no significant differences between mean ki-67 score and oral mucosal epithelial hyperplasia, but instead showed a significant difference between mean ki-67 score and inflammatory cell intensity. however, the f r e q u e n t l y e n c o u n t e r e d n o n n e o p l a s t i c proliferative lesions of the oral mucosa have the potential to undergo neoplastic transformation, because these lesions are associated with stimuli capable of inducing proliferative growth (epithelial hyperplasia) as a cellular adaptive response.(5) in the neoplasms of this study, particularly malignant neoplasms (squamous cell carcinoma), the ki-67 score was found to be high, because table 2. mean ki-67 expression by epithelial cell proliferation (hyperplasia) and ki-67 expression category *values are mean ± standard deviation 165 cell proliferation in neoplasms is caused by mutations in the genes regulating cellular growth patterns. in this study, the ki-67 scores of the two squamous cell carcinomas were found to be 41% and 88.1%, respectively. these scores are slightly lower than the mean score of 80% obtained by raju et al.(8) from 11 oral cancer samples, but higher than those in the study of koelbl et al.,(16) who reported ki-67 scores between 5% and 60% for squamous cell carcinomas. there are no standards for ki-67 scores, but some have suggested a value of 20% as positively indicating the presence of proliferative gene markers with a potential or at high risk of u n d e rg o i n g g e n e m u t a t i o n o r n e o p l a s t i c transformation. this is demonstrated in the present study, where beign and malignant neoplastic samples showed ki-67 indexes in the range of 23.33 ± 6.94 to 64.55 ± 23.55%, with a mean index of >20%. the number of ki-67 positive cells (ki67 labeling index) in the neoplastic samples of this study was correlated with the clinical course of disease. the results of this study showed that high ki-67 scores are proportional to grade of malignancy, as indicated by the severe dysplasia found in the squamous cell carcinomas. these results are also consistent with those of angiero et al.(9) and soares et al.,(1) who found that the ki-67 score increased with increasing dysplasia. our study also supports the statement that there are strong indications that ki-67 protein expression is an absolute requirement for progressive growth by means of cell division. our study is also consistent with the statement on the high significance of excessive expression of proteins associated with the cell cycle, such as the ki-67 protein (identified by monoclonal ki-67 antibody).(3) the present study has as limitation the unequal numbers of neoplastic and nonneoplastic samples. there is also the possibility of variable epithelial thickness in the nonneoplastic samples, which may possibly influence ki-67 proliferative gene expression. conclusions ki-67 proliferative gene expression in nonneoplastic conditions, was low, varying nonsignificantly with cellular hyperplasia, since it is merely an adaptive process and does not satisfactorily indicate increased ki-67 gene e x p r e s s i o n . to o b t a i n m o r e a p p r o p r i a t e prognostic ki-67 scores in non-neoplastic lesions, it is recommended that further studies should be conducted using a larger number of samples of relatively equal epithelial thickness. acknowledgement we wish to express our gratitude to dr.evlina suzanna sp.pa (k) of the department of pathological anatomy, dharmais cancer hospital, for her valuable contribution to this study, particularly in the preparation of the immunohistochemical preparations and their evaluation. references 1. soares cp, zuanon jas, teresa db, fregonezi pa, neto1 cb, oliveira mrb, et al. quantitative cell-cycle protein expression in oral cancer assessed by computer-assisted system. histol histopathol 2006;21:721-8. 2. sudiono j, editor. pemeriksaan patologi untuk diagnosis neoplasma. in: pemeriksaan patologi untuk diagnosis neoplasma mulut. jakarta: egc;2008.p.68-71. 3. kovesi g, szende b. changes in apoptosis and mitotic index, p53 and ki-67 expression in various types of oral leukoplakia. oncology 2003;65:331-6. 4. whyte da, broton ce. the unexplained survival of cells in oral cancer: what is the role of p53? j oral pathol med 2002;31:25-33. 5. rubin e, strayer d. cell injury. in: rubin e, gorstein f, rubin r, scwarting r, strayer d, editors. rubin‘s pathology clinicopathologic foundations of medicine. 4th ed. baltimore: lippincott;2005.p.6. 6. klokkevold pr, mealey bl. influence of systemic disorders and stress on the periodontium. in: newman mg, takei hh, klokkevold pr, carranza fa jr, editors. univ med vol. 31 no.3 166 sudiono, hassan ki-67 gene expression in non-neoplastic carranza’s clinical periodontology. 10th ed. st.louis: saunders;2006.p.284-311. 7. eghbalian f, monsef a. congenital epulis in the newborn, review of the literature and a case report. j pediatr hematol oncol 2009;31:198-9. 8. raju b, mehrotra r, jordsbakken g, al-sharab ai, vasstrand en, ibrahim so. expression of p53, cyclin d1 and ki-67 in pre-malignant and malignant oral lesions: association with clinicopathological parameters. anticancer res 2005;25:4699-706. 9. angieroi a, berenzi a, benetti a, rossi e, del sordo r, sidoni a, et al. expression of p16, p53 and ki-67 proteins in the progression of epithelial dysplasia of the oral cavity. anticancer res 2008;28:2535-40. 10. szelachowska j, dziegiel p, krzeszewska jj, jelen m, matkowski r, pomiecko a. mcm-2 protein expression predicts prognosis better than ki-67 antigen in oral cavity squamocellular carcinoma. anticancer res 2006;26:2473-8. 11. dziegiel p, forgacz j, suder e, surowiek p, kornafel j, zabel m. prognostic significance of metallothionein expression in correlation with ki-67 expression in adenocarcinomas oflarge intestine. histol histopathol 2003;18:401-7. 12. dziegiel p, salwa-zurawska w, zurawski j, wojnar a, zabel, m. prognostic significance of augmented metallothionein expression correlated with ki-67 antigen expression in selected soft tissue sarcomas. histol histopathol 2005;20:83-9. 13. souza pea, mesquita ra, gomez rs. evaluation of p53, pcna, ki-67, mdm2 and agnor in oral peripheral and central giant cell lesions. oral dis 2008;6:35-9. 14. iamaroon a, khemaleelakul u, pongsiriwe s, pintong j. coexpression of p53 and ki 67 and lack of ebv expression in oral squamous cell carcinoma. j oral pathol med 2004;33:30-6. 15. schliephake h. prognostic relevance of molecular markers of oral cancer a review. int j oral maxillofac surg 2003;32:233-45. 16. koelbl o, rosenwald a, haberl m, muller j, reuther j, flentje m. p53 and ki-67 as predictive markers for radiosensitivity in squamous cell carcinoma of the oral cavity? an immunohistochemical and clinicopathologic study. int j radiat oncol biol phys 2001;49:14754. c:\users\universa medicina\docu 128 abstract universa medicina bipolar disorder increases the risk of poor sleep quality among senior high school students surilena hasan* and christopher yovan junardi** background patients with bipolar disorder commonly experience sleeping problems. on the other hand, sleep disturbances have been suggested as one of the symptoms that indicate the development of bipolar disorder. considering the detrimental effect of bipolar disorder, especially in young people, this study aimed to determine the relationship between sleep quality and risk of bipolar disorder in senior high school students. methods a cross-sectional study was conducted involving 725 senior high school students. the instruments used were a demographic questionnaire, young mania rating scale (ymrs), mini international neuropsychiatric interview (mini icd-10), hypomania/mania symptom checklist (hcl-32), and pittsburgh sleep quality index (psqi). univariate and bivariate (simple logistic regression) analyses were performed to evaluate the relation between sleep quality and the risk of bipolar disorder. results around 28% of subjects were at risk of bipolar disorder, of which 96.6% were prone to type i, and the rest were type ii. most of the participants with bipolar disorder (64%) had poor sleep quality. the percentage of participants with type i bipolar disorder that had poor sleep quality was higher than that of type ii (65.3% and 28.6%, respectively). there was a significant relationship between bipolar disorder and sleep quality (or = 2.2; 95% ci=1.581-3.087; p=0.000). conclusions our study demonstrated that in senior high school students, bipolar disorder increased the risk of poor sleep quality. assessment of sleep disturbances routinely in psychiatric interviews may improve sleep quality. keywords: bipolar disorder, sleep quality, senior high school student original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.128-134 may-august 2020 vol.39no.2 cite this article as: hasan s, junardi cv. bipolar disorder increases the risk of poor sleep quality among senior high school students univ med 2020;39:1283 4. d oi: 10 .180 51/univmed .202 0. v39.1 28 -13 4 *department of psychiatry and behavioural sciences, school of medicine and health sciences, atma jaya catholic university of indonesia, jakarta **study program of medicine school of medicine and health sciences, atma jaya catholic university of indonesia, jakarta correspondence: surilena school of medicine and health sciences, atma jaya catholic university of indonesia jl. pluit raya no. 2, jakarta utara 14440 mobile: +6281289769996 email: surilena@atmajaya.ac.id orcid id : 0000-0001-6164-1117 date of first submission, december 30, 2019 date of final revised submission, august 17, 2020 date of acceptance, august 19, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license 129 univ med vol. 39 no.2 introduction bipolar disorder is considered a chronic and detrimental condition. bipolar disorder is common in clinical psychiatric practice, and several studies have estimated its prevalence to range from 0.5 to 5% in community-based samples.(1) in south east asia, the proportion of type i bipolar disorder is 0.6% and that of type ii is 0.4%.(2) however, establishing the diagnosis in both children and adolescents is still challenging. in fact, the prevalence of paediatric bipolar has been a subject of debate in published literature. a meta-analytical study stated that the prevalence rate of bipolar disorder in children and adolescents across 12 studies was 1.8%.(3) the international classification of diseases (icd 10) defines bipolar disorder as a medical condition characterised by two or more episodes of significant disturbance in mood and activity level, which consists of some occasions of an elevated mood, increased energy and activity (hypomania or mania), and others of a lowered mood and decreased ener gy and activity (depression).(4) there are two types of bipolar disorder, namely type i bipolar disorder with manic and type ii bipolar disorder with hypomanic episodes as the essential features. while a major depressive episode is essential for the diagnosis of type ii bipolar disorder, it is not required for type i bipolar disorder.(5) clinical studies on patients with bipolar disorder have suggested that sleeping problems and sleep disturbances are symptoms indicating the development of bipolar disorder.(6,7) a recent study by ritter et al.(8) indicated that poor sleep quality significantly increases the risk of bipolar disorder among adolescents and young adults. the study of levenson et al.(9) suggested that those who are at risk, such as offspring of bipolar disorder patients, are more likely to develop the same diagnosis when they are poor sleepers. while a wide range of longitudinal studies have established insomnia to be a risk factor for the subsequent onset of major depression no such evidence is available with regard to bd so far.(10) the need to gain a deeper understanding on bipolar disorder, particularly in children and adolescents, is commonly acknowledged.(11) it is considered important to add studies from nonwestern countries since most published literature come from the northern hemisphere.(2) since mental health among children and adolescents will impose a serious health burden on developing countries like indonesia, research from our country to gain more comprehensive insight concerning paediatric bipolar disorder would be very strategic and valuable. as a developing nation indonesia needs quality human resources. research that contributes to improving the continuum of care in mental health disorders for the young generation should have urgency and priority.(12) therefore, the present study aimed at providing a new insight on bipolar disorder and its relationship with sleep quality among senior high school students. methods research design this was a cross-sectional study involving children and adolescents in the age range of 14 to 20 years, aimed at evaluating sleep quality among adolescents at risk of bipolar disorder. this study was performed from november to december 2017 at four high schools in north penjaringan district, north jakarta. research subjects the participants of this study were students from the selected senior high schools. a total of 725 participants responded to the questionnaire and were included into the study. the inclusion criterion was all year 10, 11, and 12 students. the exclusion criteria were students who were absent, unwilling to be a participant, and refused to sign informed consent. measurements several instruments were used to assess the risk of bipolar disorder, namely young mania rating scale (ymrs), mini international 130 neuropsychiatric interview (mini icd-10), and hypomania symptom checklist (hcl-32). the ymrs is an 11-item questionnaire to assess symptoms of mania. it has proven to be reliable {interrater reliability (irr) = 0.93, p<0.001) and valid (concurrent validity with global rating, r=0.86, p<0.001) to measure the severity of mania symptoms. each item of the ymrs assesses a different component of mania, namely elevated mood, increased motor activity or energy, sexual interest, sleep, irritability, speech (rate and amount), language (thought disorder), content, disruptive or aggressive behaviour, appearance, and insight. the responses to these items are on a five-point likert scale. the total score from all 11 items is used to determine the condition of mania. score 12 or above means that the person has mania.(13) the mini is a short-structured diagnostic interview for mental disorders listed in icd-10, and has a good reliability for the assessment of major depressive disorder (test/retest kappa = 0.76). the questions in the mini for depression are grouped into two parts. the first part consists of questions asking about symptoms of major depressive disorder, namely depressed mood, lack or loss of interest, and lack of energy, experienced by a person in the last two weeks. if a respondent answers ‘yes’ to two or more items, than they should continue to the second part, which is a continuation of the first part. questions in the second part ask about other symptoms, such as loss of weight, sleeping pattern, slow speech and movement, self-confidence, guilt feelings, lack of concentration, and self-harm or suicide. each ‘yes’ answer receives one point, such that a total score of four or above means depression. in other words, a respondent who answers ‘yes’ to four or more questions is considered as having depression.(14) the hcl-32 was d eveloped to help clinicians in assessing the symptoms of hypomania and establish the diagnosis of type ii bipolar disorder. the sensitivity for type ii bipolar disorder of the hcl-32 was 80% in comparison to unipolar depression or other non-bipolar disorders, while it was 65.3% and 57.3% compared to the unipolar depression and other diagnoses, respectively. it consists of 32 items, which should be responded to with ‘yes’ (score=1) or ‘no’ (score=2). the total score of 16 or above reflects hypomania.(15) the measurements were performed in sequences. first, the symptoms of mania were determined using the ymrs. the participants who did not have mania (ymrs total score <12) were further evaluated using the mini icd-10 to determine the depression symptoms. finally, those who had depression symptoms (mini icd10 total score 4) were evaluated using the hcl2. the risk of type i bipolar disorder was concluded for the participants who had a ymrs score of 12 (mania) and a mini icd-10 score of 4 (depression). the risk of type ii bipolar disorder was concluded for those who had a mini icd-10 total score of 4 (depression) and a hcl-32 score of e”16 (hypomania).(13-15) the pittsburgh sleep quality index (psqi) is used to measure sleep quality and the presence of sleep disturbances at 30-day intervals. the psychometric properties of the psqi are good in comparing good and poor sleepers, with sensitivity of 89.6% and specificity of 86.5% (kappa=0.75, p001). it can be used to assess sleeping problems among clinical and non-clinical populations. this questionnaire consists of seven components, namely the quality of sleep, the length of time required to fall asleep, the duration of sleep, the efficiency of sleep, the presence or absence of sleep disturbances, the use of sleeping pills, and work functions at productive hours. the total score of all seven components determines the sleep quality. a total score of five or above means poor sleep quality, while below five means good sleep quality.(16) statistical analysis the sample group was divided into two groups according to psqi score. in order to find the predictors of subjects with clinically significant sleep disturbance, simple logistic regression analysis was used. all p values were two-tailed, and statistical significance was set as p<0.05. hasan, junardi bipolar disorder and risk of poor sleep quality 131 univ med vol. 39 no.2 a b c ethical clearance the ethical research committee of the school of medicine and health sciences, atma jaya catholic university of indonesia, had approved this study on november 20th, 2017 under no. 10/11/kep-fkuaj/2017. results most participants were female (55.7%,), and in year 12 (37.1%). the mean age of the participants was 16.5 years. slightly more than half of them (50.1%) had poor sleep quality. almost a third (28%) of the participants were at risk of bipolar disorder, of which 96.6% were prone to type i bipolar disorder, and the rest were type ii bipolar disorder (table 1). more than half of those who were at risk of type i bipolar disorder characteristic n (%) mean ± sd age (years) range 14 15 16 17 18 19 20 14-20 2 (0.3) 128 (17.7) 218 (30.1) 281 (38.8) 84 (11.6) 9 (1.2) 3 (0.4) 16.5 ± 0.988 gender male female 321 (44.3) 404 (55.7) high school grade 10 th 11 th 13 th 217 (29.9) 239 (33,0) 269 (37.1) sleep quality poor good bipolar disorder no risk risk: type i type ii 363 (50.1) 362 (49.9) 522 (72) 203 (28) 196 (96.6) 7 (3.4) table 1. characteristics, bipolar disorder and type of bipolar disorder or 95% c.i. for or p value low high bipolar disorder bipolar disorder type 2.209 4.706 1.581 0.889 3.087 24.898 0.000 0.111 table 2. logistic regression analysis to predict bipolar disorder and type for poor sleep quality or : odds ratio were female (55.1%), while 57.1% of those at risk of type ii bipolar disorder were male (data not presented). most of the participants with bipolar disorder (64%) had poor sleep quality. there was a significant relationship between bipolar disorder and sleep quality (or=2.2; 95% ci=1.581-3.087; p=0.000). those who were at risk of bipolar disorder had 2.2 times poorer sleep quality compared to students without bipolar disorder. this study showed that 65.3% participants with type i bipolar disorder had poor sleep quality, while the percentage of type ii bipolar disorder with poor sleep quality was only 28.6%. there was no significant correlation between the type of bipolar disorder and sleep quality. (or=4.7; 95% ci=0.889-24.898; p=0.111) (table 2). discussion this study found that the majority of students who were at risk of bipolar disorder were at risk of type i bipolar disorder, and were more likely to have sleeping problems compared to those who were not at risk. second, those who were at risk of type i bipolar disorder were more likely to have poor sleep quality compared to those who were at risk of type ii bipolar disorder. the prevalence of students who were at risk of bipolar disorder (28%) was muc h higher than the global prevalence reported in previous studies (0% 7%).(2,4) the present study used ymrs, mini icd-10 and hcl-32 i nstruments without structured psychiatric interviews, in contrast to the pre vious studies that used struc tured psychiatric interviews. the results of the present study indicate that adolescents had a high risk of bipolar disorder (28%). this shows that early onset of bipolar disorder is more common in 132 adolescents and therefore it is necessary to do early detection of bipolar disorder in adolescents, especially in educational institutions so that interventions can be carried out as early as possible to prevent the effects that may arise. the findings of the present study were similar to those of a previous study, which showed that sleeping problems commonly occur in adolescents at risk of bipolar disorder.(17) one study suggested that sleep onset latency occurs more often in the offspring of patients with bipolar disorder compared to the control group.(18) similar findings were found in another study that showed poorer sleep quality in adolescents who had one parent with bipolar disorder than in the control group.(19) importantly, they also found that adolescents at risk of bipolar disorder who had sleeping problems were more likely to develop bipolar disorder than their counterparts,(18) and sleep disturbances were recognised as one of prodromal symptoms of paediatric bipolar disorder.(17) considering that most of our participants who were at risk also experienced sleeping problems, it is essential to regularly evaluate their condition and provide relevant intervention to prevent the development of bipolar disorder. there are no published studies reporting the difference in sleep quality between type i and type ii bipolar disorder among adolescents. indeed, steinan et al.(20) reported that hypersomnia was related to type i bipolar disorder, and this relation was more likely to occur in the younger age group. however, they performed their study among adult participants (mean age=38). one previous study evaluated sleeping patterns between adolescents with type i bipolar disorder and bipolar disorder not otherwise specified (bipolar nos), and found no significant difference, although the percentages of insomnia and any sleep disorder were higher in the latter.(21) in type i bipolar disorder problems in sleeping are associated with more severe conditions. for example, longer latency period and more frequent awakening during the night were associated with depressed mood, while the variability in sleeping duration was related to impulsive behaviour.(22) hence, further studies should evaluate whether sleep quality in type ii bipolar disorder can influence the progression of the disorder. in our research, the risk in adolescents for type i bipolar disorder and type ii was not significantly associated with poor sleep quality. this may have been due to the f act that the number of adolescents who were at risk for type i bipolar disorder was much greater than those at risk for type ii bipolar disorder. there are several limitations of this study that need to be pointed out. first, we performed the study in only four high schools of one district in jakarta. therefore, the generalizability of the findings could be questionable. however, considering that this is the first study that explored the association between sleep quality and risk of bipolar disorder among high school students, the findings provide recommendation for future studies to evaluate other aspects of bipolar disorder, particularly in adolescents. second, we did not assess other factors that might contribute to the risk of bipolar disorder and sleep quality, such as history of mental disorder in the family, parenting functions, or individual coping skills. third, our study measured sleep quality using a self-report instrument, which may produce a measurement bias. m or e research using objective measurement of sleep should be pursued to support these data and to further investigate the causal relationship between sleep quality and bipolar disorders. finally, in this study ymrs, mini icd-10 and hcl-32 instruments were used without str uctur ed psychiatric interviews, whereas the other studies used structured psychiatric interviews. future studies should evaluate the aforementioned factors to provide more understanding about bipolar disorder in adolescents. conclusion our study confirmed that bd increased the risk of poor sleep quality in high school students. importantly, a prevention program is highly hasan, junardi bipolar disorder and risk of poor sleep quality 133 univ med vol. 39 no.2 recommended at school to improve students’ mental health. finally, detection and intervention programs for students at risk are required for early diagnosis and treatment. conflict of interest competing interests: no relevant disclosures acknowledgement the author thanks the headmasters and teachers of the four high schools in penjaringan district, north jakarta who had permitted this study and all high school students that were willing to participate and signed informed consent. author’s contributions s contributed to write the draft of the and design the study. cyj supervised and/or performed the data management and data entry. s a nd scyj cont ribu ted to a nalysis and interpretation of the data.. all authors have read and approved the final manuscript. references 1. carlson ga, dubicka b. very early onset bipolar disorder international differences in prevalence, practice or language? child adolesc ment health 2019;24:86-7. https://doi.org/10.1111/camh.12315. 2. melo mca, garcia rf, neto vbl, et al. sleep and circadian alterations in people at risk for bipolar disorder: a systematic review. j psychiatr res 2016;83:211–9. https://doi.org/10.1016/ j.jpsychires.2016.09.005. 3. van meter a, moreira alr, youngstrom e. updated meta-analysis of epidemiologic studies of pediatric bipolar disorder. j clin psychiatry 2019;80:18r12180. doi: 10.4088/jcp.18r12180. 4. world health organization. international statistical classification of diseases and related health problems 10th revision. geneva :world health organization; 2016. 5. bobo wv. the diagnosis and management of bipolar i and ii disorders: clinical practice update. mayo clinic proceedings 2017;92:1532-51. https:/ /doi.org/10.1016/j.mayocp.2017.06.022. 6. melo mca, abreu rlc, neto vbl, de bruin pfc, de bruin vms. chronotype and circadian rhythm in bipolar disorder: a systematic review. sleep medicine rev 2017;34:46-58. doi: 10.1016/ j.smrv.2016.06.007. 7. skjelstad dv, malt uf, holte a. symptoms and signs of the initial prodrome of bipolar disorder: a systematic review. j affect disord 2010;126:1. doi: 10.1016/j.jad.2009.10.003 8. ritter ps, höfler m, wittchen hu, et al. disturbed sleep as risk factor for the subsequent onset of bipolar disorder – data from a 10-year prospectivelongitudinal study among adolescents and young adults. j psychiatr res 2015;68:76-82. doi: 10.1016/j.jpsychires.2015.06.005. 9. levenson jc, soehner a, rooks b, et al. longitudinal sleep phenotypes among offspring of bipolar parents and community controls. j affect disord 2017;215:30-6. https://doi.org/ 10.1016/j.jad.2017.03.011. 10. baglionia c, battaglieseb g, feigea b, et al. insomnia as a predictor of depression: a metaanalytic evaluation of longitudinal epidemiological studies. j affect disord 2011;135:10-9. https://doi.org/10.1016/ j.jad.2011.01.011. 11. findling rl, stepanova e, youngstrom ea, young as. progress in diagnosis and treatment of bipolar disorder among children and adolescents: an international perspective. evidence based mental health 2018;21:177. http:/ /dx.doi.org/10.1136/eb-2018-102912. 12. maramis mm, karimah a, yulianti e, bessing yf. screening of bipolar disorders and characteristics of symptoms in various populations in surabaya, indonesia. anima indonesian psychol j 2017;32:90-8. https://doi.org/10.24123/ aipj.v32i2.587. 13. cazorla p, zhao j, mackle m, szegedi a. asenapine effects on individual young mania rating scale items in bipolar disorder patients with acute manic or mixed episodes: a pooled analysis. neuropsychiatr dis treat 2013;9:409– 13. doi: 10.2147/ndt.s38390. 14. øhre b, saltnes h,von tetzchner s, falkum e. psychometric properties of a sign language version of the mini international neuropsychiatric interview (mini). bmc psychiatry 2014; 14: 148. doi: 10.1186/1471-244x-14-148. 15. soares ot, moreno dh, de moura ec, angst j, moreno ra. reliability and validity of a brazilian version of the hypomania checklist (hcl-32) compared to the mood disorder questionnaire (mdq). rev bras psiquiatr 2010;32:416–23. doi: 10.1590/s1516-44462010000400015. 134 16. buysse dj, reynolds iii cf, monk th, berman sr, kupfer dj. the pittsburgh sleep quality index: a new instrument for psychiatric practice and research. psychiatry res 1989;28:193-213. doi: 10.1016/0165-1781(89)90047-4. 17. hernandez m, marangoni c, grant mc, estrada j, faedda gl. parental reports of prodromal psychopathology in pediatric bipolar disorder. curr neuropharmacol 2017;15:380-5. doi: 10.2174/ 1570159x14666160801162046. 18. sebela a, novak t, kemlink d, goetz m. sleep characteristics in child and adolescent offspring of parents with bipolar disorder: a case control study. bmc psychiatr 2017;17:199. https:// doi.org/10.1186/s12888-017-1361-8. 19. baroni a, hernandez m, grant m, faedda g. sleep disturbances in pediatric bipolar disorder: a comparison between bipolar i and bipolar nos. front psychiatry 2012;3. doi: 10.3389/fpsyt.2012. 00022. 20. steinan mk, scott j, lagerberg tv, et al. sleep problems in bipolar disorders: more than just insomnia. acta psychiatr scand 2016:133:368–77. doi: 10.1111/acps.12523. 21. roybal dj, chang kd, chen mc, howe me, gotlib ih, singh mk. characterization and factors associated with sleep quality in adolescents with bipolar i disorder. child psychiatry hum dev 2011;42:724-40. doi: 10.1007/s10578-011-0239-0. 22. gershon a, johnson sl, thomas l, singh mk. double trouble: weekend sleep changes are associated with increased impulsivity among adolescents with bipolar i disorder. bipolar disord 2019;21:132-41. doi: 10.1111/bdi.12658. hasan, junardi bipolar disorder and risk of poor sleep quality oktavianus 200 *department of biochemistry, faculty of medicine, trisakti university **department of biology, faculty of medicine, trisakti university ***department of histology, faculty of medicine, trisakti university correspondence dr. suweino, sp.bkm department of biochemistry, faculty of medicine, trisakti university jl. kyai tapa no. 260 grogol jakarta 11440 phone: +6221-5672731 ext. 2204 email: suweino@trisakti.ac.id univ med 2012;31:200-207 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background total testosterone (tt) levels in males decrease with age. there has been a vigorous debate on the extent to which low testosterone causally contributes to diabetes and its complications. the aim of the present study was to determine the relationship between sex hormones and blood glucose levels in adult males. methods a cross-sectional study involving 259 males aged 41 70 years was conducted at cilandak subdistrict, south jakarta. sex hormone binding globulin (shbg) and testosterone levels were measured by means of electro–chemiluminescent immunoassay (eclia), while blood glucose levels were measured enzymatically using a spectrophotometer. free testosterone index (fti) and body massa index (bmi) were calculated. inter-variable relationships were tested by pearson correlation analysis, followed by multiple linear regression analysis to determine the most influential factor on fasting blood glucose levels. results bmi was positively correlated with fasting blood glucose, but the correlation was statistically not significant (r=0.105; p=0.106). in contrast, total testosterone (tt) (r=-0.258; p=0.000) and shbg (r=-0.193; p=0.02) had a significant negative correlation with fasting blood glucose level. multiple linear regression showed that tt was the most influential factor on fasting blood glucose level (â=-0.044; p=0.008). conclusions low total testosterone level may increase fasting blood glucose level in adult males. shbg levels did not predict fasting blood glucose levels.assessment of testosterone in middle-aged men may allow early intervention for diabetes mellitus. keywords: total testosterone, sex hormone binding globulin, blood glucose, male low testosterone level increases fasting blood glucose level in adult males suweino*, ml edy parwanto**, and david tjahjadi*** 201 univ med vol. 31 no.3 kadar testosteron yang rendah meningkatkan kadar gula darah puasa pada laki-laki dewasa latar belakang pada laki-laki, seiring dengan meningkatnya usia terjadi penurunan kadar kadar testosteron total (tt). masih banyak perbedaan pendapat mengenai kontribusi kausal dari kadar testosteron yang rendah terhadap diabetes dan komplikasinya. penelitian ini bertujuan untuk menentukan adanya hubungan antara hormon seks dan kadar glukosa darah puasa pada pria dewasa. metode studi potong lintang dengan mengikut sertakan 259 pria yang berumur 41 – 70 tahun dilakukan di kecamatan cilandak, jakarta selatan. pengukuran kadar sex hormone binding globulin (shbg) dan testosteron dilakukan dengan teknik electro–chemiluminescent immunoassay (eclia), dan glukosa diukur secara enzimatis menggunakan spektrofotometer. dilakukan perhitungan indeks testosteron bebas (itb) dan indeks massa tubuh (imt). hubungan antar variabel diuji menggunakan analisis korelasi pearson, yang dilanjutkan dengan analisis regresi ganda linear untuk menetukan faktor yang paling berperan terhadap kadar glukosa darah puasa. hasil imt berkorelasi positif dengan kadar gula darah puasa, tetapi secara statistik tidak bermakna (r=0.105; p=0.106). sebaliknya tt (r=-0.258; p=0.000) dan shbg (r=-0.193; p=0.02) berkorelasi negatif secara bermakna dengan kadar gula darah puasa. analisis regresi ganda linear menunjukkan kadar tt yang paling berperan secara bermakna terhadap kadar gula darah puasa (â=-0.044; p=0.008). kesimpulan kadar testosteron total yang rendah dapat meningkatkan kadar gula darah puasa pada pria dewasa. shbg tidak dapat memprediksi kadar gula darah puasa. pengukuran testosteron pada pria setengah baya memungkinkan intervensi dini terhadap diabetes melitus. kata kunci: testosteron total, sex hormone binding globulin, glukosa darah, pria abstrak introduction in males, the age-related reduction in total testosterone and adrenal androgens such as dehydroepiandrosterone sulfate (dheas) has been documented and linked to changes in physiological functions, including abdominal obesity, insulin resistence, lung function, and cardiovascular disease.(1-3) testosterone levels in males are low before puberty and increase with puberty, reaching a peak in the age group of 40 – 49 years and decreasing gradually with advancing age.(4) testosterone is synthesized by leydig cells in the testis and is the main sex hormone in males. part of the testosterone in the testes is used for spermatogenesis and the rest is secreted into the circulation. the total amount of testosterone in the circulation is called total testosterone. in general, total testosterone (tt) in the circulation is divided into 4 main parts, viz. testosterons bound to sex hormone binding globulin (shbg) (44%), t e s t o s t e r o n e b o u n d t o a l b u m i n ( 5 0 % ) , testosterone bound to cortisol binding globulin (4%), and free or unbound testosterone (2%). one third of us men older than 65 years have type 2 diabetes, and a similar percentage have low or subnormal testosterone levels, compared 202 with reference ranges based on healthy young men.(5) dhindsa et al.(6) reported that 43% of males with type 2 diabetes had low total testosterone levels, with a decrease in 57% of the free testosterone. on the other hand, among patients with type 1 diabetes, 1.7% had decreased tt and 20% had low free testosterone levels. a high prevalence of hypogonadism was reportedly found in patients with type 2 diabetes. the variable prevalences of hypogonadism found in several studies was presumably caused by differences in diagnostic criteria and the instruments used to determine testosterone levels.(7) one study reported a high prevalence of hypogonadism in type 2 diabetes, on the basis of free testosterone, and showed that one-third of males with type 2 diabetes (aged between 31 – 75 years) had a low free testosteron level.(7) supplementation of testosterone have been found to improve body composition and insulin sensitivity.(8) in hypogonadal male diabetics older than 30 years, testosterone improved insulin resistance, glycemic control, and visceral obesity. o n t h e o t h e r h a n d , a b r u p t c e s s a t i o n o f testosterone supplementation in men with idiopathic hypogonadotropic hypogonadism caused worsening of insulin sensitivity, without detectable changes in body fat content (9) another study reported that lower testosterone levels may promote insulin resistance through impairment of mitochondrial function.(10) because is is still uncertain whether insulin resistance and diabetes may be causing male hypogonadism or vice versa, the mechanisms regulating any preventive effect of conserved testosterone levels need to be investigated further. it has been observed that low testosterone is inversely associated to both diabetes and the metabolic syndrome, and may be a predictor of these disorders. these associations are of moderate strength, corresponding to a decrease of 2–3 nmol/l in total testosterone.(11) total testosterone has a stronger association with diabetes than does free testosterone. since changes in total testosterone (but not free testosterone) are proportional to those of shbg, it may be suggested that shbg also plays a causal role in the above associations. however, the possibility of a reverse causation or a common third factor cannot be excluded, because of the strong inverse association of shbg with insulin resistance and visceral adiposity. further study is required to shbg levels add additional information to conventional risk factors for diabetes and related complications and whether s h b g m e d i a t e s r i s k v i a a n d r o g e n i c o r testosterone-independent mechanisms. in the meta-analytical study conducted by ding et al.(12) on 20 cross sectional studies, involving 850 diabetic men and 2000 nondiabetic controls, it was found that in each study, total testosterone levels were lower in diabetic men compared with nondiabetic controls. however, the meta-analysis did not address the association of free testosterone levels with future diabetes, this has been reported by six prospective studies.(13) based on the variable results of the abovementioned studies, it was felt necessary to conduct a study aiming to determine any relationships between sex hormone levels and other determinant factors, such as bmi and shbg, and blood glucose levels in adult males. methods study design this study used a cross-sectional design and was conducted between february 2011 and october 2011 at cilandak subdistrict, south jakarta. study subjects the study subjects were healthy males who were at least 40 years old and were residents of cilandak subdistrict, south jakarta. inclusion criteria were a good health status as evidenced by physical examination by the investigators, and normal serum albumin level (3.5 5.0 g/dl). exclusion criteria were: i) liver cirrhosis, hypogonadal or hypergonadal status; ii) consumption of drugs related to liver functions, suweino, parwanto, tjahjadi testosteron increases blood glucose 203 univ med vol. 31 no.3 e.g. phenytoin; iii) consumption of drugs affecting reproductive organs, e.g. androgens, growth hormones, high-dose glucocorticoids; iv) c o n s u m p t i o n o f d r u g s a ff e c t i n g s t e r o i d production and metabolism, e.g. diazoxide; and v) consumption of drugs affecting insulin production, e.g. epinephrine, cortisol, progestins, sulfonylureas. the subjects gave written informed consent before participating in the study. subjects were selected by cluster random s a m p l i n g o f t h e k e l u r a h a n ( v i l l a g e s ) i n cilandak subdistrict, south jakarta. in each selected rw (hamlet), subject selection was by simple random sampling. measurement of body mass index (bmi) height and weight were measured by means of weighing scales with a built-in device for measuring height. bmi was computed as the ratio of weight to the square of height (kg/ m2). the anthropometry was performed in standing subjects wearing light clothes and no shoes. laboratory measurements venous blood samples were collected in the morning from the subjects after a 10-hour fast. fasting blood samples were drawn from the cubital vein in the supine position. serum aliquots were prepared for immediate analysis and for storage at 80°c for further analysis. b l o o d g l u c o s e l e v e l s w e r e m e a s u r e d enzymatically using a spectrophotometer and were expressed in mg/dl. total testosterone levels were determined by electrochemiluminescence (eclia) immunoassay (elecsys 2010; roche diagnostics, indianapolis, in, usa), and levels of shbg were measured by chemiluminescent immunometric assay (immulite, dpc, los angeles, ca, usa). free testosterone index (fti) values were calculated using the formula tt/shbg x 100 and were expressed in percentages, in accordance with vermeulen’s method. (14) fti was used to determine the testosterone status, to indicate binding abnormalities between shbg and testosterone. determination of shbg and total testosterone levels was performed at the prodia laboratories, jakarta. testosterone levels were expressed in nmol/l. ethical clearance the study protocol was approved by the research ethical committee, faculty of medicine, trisakti university, jakarta. data analysis descriptive statistics, means and sd for continuous variables were used to describe the study population. the correlations between age, bmi, serum hormone levels and blood glucose levels were tested by pearson’s test. multivariate regression linear analysis was conducted in order to identify the most influential variable on fasting blood glucose levels. all p-values were twotailed, and p< 0.05 was considered statistically significant. all analyses were performed with spss 17.0. results a total of 237 males participated in this study, with mean age of 53.19 ± 8.39 years and mean bmi of 24.35 ± 3.74 kg/m2. mean fasting blood glucose level was 106.59 ± 33.78 mg/dl, tt 529.76 ± 190.75 nmol/dl, and shbg 40.59 ± 17.58 nmol/l (table 1). characteristics all subjects (n = 428) age (years) 53.19 ± 8.39 bmi (kg/m2) 24.35 ± 3.74 glucose (mg/dl) 106.59 ± 33.78 tt (nmol/dl) 529.76 ± 190.75 shbg (nmol/l) 40.59 ± 17.58 fti (%) 48.17 ± 14.15 table 1. demographic and clinical characteristics of the study subjects (n=237) data are expressed as mean ± sd; bmi : body mass index; tt: total testosterone; shbg: sex hormone binding globulin; fti: free testosterone index 204 ta b l e 2 a b o v e s h o w s t h a t b m i w a s positively correlated with fasting blood glucose, b u t s t a t i s t i c a l l y t h e c o r r e l a t i o n w a s n o t significant (r=0.105; p=0.106). in contrast, tt (r=-0.258) and shbg (r=-0.193; p=0.002) had a significant negative correlation with fasting blood glucose. it may be concluded that high tt and shbg levels will result in decreased fasting blood glucose levels, while low tt and shbg levels will result in increased fasting blood glucose levels. although bmi did not show a significant relationship with fasting blood glucose level, theoretically bmi affects fasting blood glucose level, which was the reason for its inclusion in the multiple linear regreassion model. multiple linear regression analysis showed that tt was the most infuential factor on fasting blood glucose, in comparison with bmi and shbg levels (â=-0.044; p=0.008). this means that low tt levels result in high fasting blood glucose levels (table 3). table 2 . correlation between several covariates and fasting blood glucose level covariate fasting blood glucose p age bmi tt shbg fti 0.077 0.105 -0.258 -0.193 -0.068 0.224 0.106 0.000* 0.002* 0.283 bmi: body mass index; tt: total testosterone; shbg: sex hormone binding globulin; fti: free testosterone index table 3. multiple linear regression analysis of several covariates and fasting blood sugar level bmi: body mass index; tt: total testosterone; shbg: sex hormone binding globulin discussion our study results showed reduced tt levels in the study subjects, accompanied by increased fasting blood glucose levels. this is consistent with the results of other studies reporting that decreases in the levels of androgenic hormones, i.c. testosterone, is one of the causal factors for increased blood glucose levels in the subjects,(15) with zinc deficiency and adipokines (such as leptin and tumor necrosis f a c t o r a l p h a ) b e i n g t w o o f t h e p o s s i b l e causes.(16) increased insulin levels decrease shbg levels leading to a decrease in tt. the correlation between low testosterone levels and diabetes may also be the result of decreased functioning of leydig cells. reductions in tt and shbg levels are closely associated with male abdominal obesity.(17,18) although its mechanism is uncertain, abdominal osetity in males may be caused by increased lipid uptake and increased accumulation of visceral fat. o t h e r s t u d i e s f o u n d t h a t t e s t o s t e r o n e replacement therapy significantly decreases insulin resistence and improves blood glucose levels. in addition, it has been documented that men with impaired glucose tolerance (igt) have low tt levels and that tt levels are inversely associated to fasting blood glucose.(19) tt levels in male diabetics were lower than those in normal males, possibly because free testosterone (ft) levels in the male subjects with diabetes were also lower than in normal males. furthermore, ft has autoregulatory functions for a feedback mechanism to both the hypothalamus and the pituitary gland. suweino, parwanto, tjahjadi testosteron increases blood glucose 205 univ med vol. 31 no.3 our findings that shbg levels were inversely related to fasting blood glucose are consistent with the results of the studies of ding et al.(20) as well as lakshman et al.(21) similar results were obtained in male diabetics, showing a negative correlation of shbg with tt.(22,23) the current evidence about the impact of shbg on the prediction of later diabetes suggests that previous data on the impact of tt on later diabetes might have been confounded by shbg. therefore, we repeated the analyses intending to explore the independent contribution from total testosterone and shbg when included in the same multivariate models. tt but not shbg was still independently predictive of subsequent diabetes. our study showed that bmi was not correlated with blood glucose level. kaplan et al.(23) clearly demonstrated that obesity and fasting hyperglycemia are the key components of the metabolic syndrome that exacerbates the age-related decrease in tt levels. it is well recognized that obesity and type 2 dm are both conditions associated with low tt levels. (5,25) decreased testosterone levels are known to be associated with increased glucose concentration, and our study confirmed this association. the clinical implication of our findings is that administration of testosterone may be beneficial to males. testosterone replacement therapy may have beneficial effects on insulin sensitivity in older diabetic men with late hypogonadism. since testosterone replacement therapy results in reduced fat mass and consequently decreased circulating free faty acids, the end result may be improved insulin sensitivity.(16) however, routine administrtaion of testosterone therapy to diabetic men with low-normal testosterone levels should await the outcomes of well-conducted clinical trials. males with subnormal testosterone levels should implement lifestyle measures such as weight reduction and exercise, which may raise testosterone levels and provide multiple health benefits. some limitations should be recognized in the current study. first, ft were calculated from tt and shbg, using the vermeulen’s equation. second, we used a single blood sample to determine tt and shbg levels. increasing the number of samples would have yielded greater precision in measurement. daily testosterone levels may fluctuate greatly, and therefore repeated tests are indicated for a diagnosis of hypogonadism. our use of a single blood sample did not allow us to account for the intrai n d i v i d u a l d a i l y v a r i a t i o n i n o u r s t u d y. observational studies of cross-sectional design as used in our study cannot explain the temporal nature of the association between testosterone and blood glucose levels. conclusions to t a l t e s t o s t e r o n e l e v e l i s t h e m o s t influential factor on fasting blood glucose level. a low total testosterone level may increase fasting blood glucose level in adult males. shbg levels did not predict fasting blood glucose levels. however, a follow-up study is needed to confirm these associations in men. assessment of testosterone in middle-aged men may allow early intervention for diabetes mellitus. acknowledgements we would like to express our gratutude to the dean and vice-deans of the faculty of medicine, trisakti university for providing the funding of this study. we also thank the male subjects who participated in this study for their willingness and cooperation. references 1. kaufman jm, vermeulen a. the decline of androgen levels in elderly men and its clinical and therapeutic implications. endocr rev 2005; 26:833–76. 2. laaksonen de, niskanen l, punnonen k, nyyssonen k, tuomainen tp, salonen r, et al. sex hormones, inflammation and the metabolic syndrome: a population-based study. eur j endocrinol 2003;149:601–8. 206 3. mawi m, nirmalasari. high free testosterone index increases lung function in adult males. univ med 2012;31:113-9. 4. agledahl i, skjærpe pa, hansen jb, svartberg j. low serum testosterone in men is inversely associated with non-fasting serum triglycerides: the 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van der schouw yt. endogenous sex hormones in men aged 40–80 years. eur j endocrinol 2003;149: 583–9. 20. halmenschlager g, rhoden el, riedner ce. the influence of age on bioavailable and free testosterone is independent of body mass index and glucose levels. world j urol 201;29:541–6. doi 10.1007/s00345-011-0724-x. 21. ding el, song y, manson je, hunter dj, lee cc, rifai n, et al. sex hormone-binding globulin and risk of type 2 diabetes in women and men. n engl j med 2009;361:1152–63. 22. lakshman km, bhasin s, araujo ab. sex hormone-binding globulin as an independent predictor of incident type 2 diabetes mellitus in men. j gerontol a biol sci med sci 2010;65: 503–9. 23. bremner wj, vitiello mv, prinz pn. loss of circadian rhythmicity in blood testosterone levels with aging in normal men. j clin endocrinol metabol 2006;56:1278–81. 24. bolelli g, muti p, micheli a, sciajno r, franceschetti f, krogh v. correlation studies of testosterone hormones level in serum with the development of diabetes. epidemiol biomark prev 2005;4:509–13. 25. kaplan sa, meehan ag, shah a. the age related decrease in testosterone is significantly suweino, parwanto, tjahjadi testosteron increases blood glucose 207 univ med vol. 31 no.3 exacerbated in obese men with the metabolic syndrome. what are the implications for the relatively high incidence of erectile dysfunction observed in these men? j urol 2006;176:1524– 8. 26. kapoor d, aldred h, clark s. clinical and biochemical assessment of hypogonadism in men with type 2 diabetes. diabetes care 2007;30:911– 7. oktavianus 167 *department of histology, faculty of medicine, diponegoro university **department of internal medicine, dr. kariadi central hospital/ faculty of medicine, diponegoro university correspondence dr. neni susilaningsih, m.si department of histology, faculty of medicine, diponegoro university jl. dr. sutomo 18 semarang email: nsusilaningsih@gmail.com univ med 2012;31:167-74 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background urinary tract infection (uti) is a common source of bacteriemia. the most common cause of uti is escherichia coli (e. coli). tumor necrosis factor (tnf)-á gene polymorphism has been reported to be responsible for an excessive production of tnf-á and eventual disruption of pro-inflammatory cytokine regulation. the aim of this study was to compare tnf-á serum levels and tnf-á allele polymorphisms in patients with uti due to e.coli and in nonuti controls. methods a cross-sectional study was conducted at dr. kariadi central hospital and the center for biomedical research, faculty of medicine, diponegoro university, semarang. in 68 patients with uti the tnf-á serum levels were determined by means of elisa and compared to those of non-uti controls (n=55). tnfá-308g>a gene polymorphism was analyzed by polymerase chain reaction restriction fragment length using the ncoi enzyme. fragments were visualized on polyacrylamide gel with silver staining. results tnf-á serum level in patients with uti had a median of 8.9 pg/ml, which was significantly higher than the median of 3.7 pg/ml in the control group (p<0.001). tnf-á-308g>a gene polymorphisms found in the patient group were g/g=61 (90%), g/a=7(10%) and a/a=0, while in the control group were g/g=48 (87%), g/a=7 (13%) and a/a =0. there was no significant differences (p=0.578) in gene polymorphisms between the two groups. conclusions tnf-á serum levels in patients with uti due to e. coli were significantly higher than in non-uti controls, but for the tnf-á-380 gene polymorphisms no significant difference was found between the two groups. there are presumably more important factors than host genotype that influence uti pathogenesis. keywords: urinary tract infection, e. coli, serum tnf-á, tnf-á gene polymorphism production of tumor necrosis factor-á is increased in urinary tract infections neni susilaningsih*, bambang joni karjono**, and ratna damma purnawati* 168 susilaningsih, karjono, purnamawati tumor necrosis factor-α is increased produksi tumor nekrosis faktor-á meningkat pada penderita infeksi saluran kemih latar belakang infeksi saluran kemih (isk) merupakan sumber tersering bakteremia. penyebab isk terbanyak adalah escherichia coli (e. coli). tumor necrosis factor (tnf)-á merupakan sitokin penting pada inflamasi akut yang paling sering dihubungkan dengan sepsis. polimorfisme gen tnf-á bertanggung jawab terhadap produksi tnf-á yang berlebihan dan akhirnya mengacaukan pengaturan kerja sitokin-sitokin serta menyebabkan terjadinya penyakit. tujuan penelitian ini adalah untuk membedakan kadar tnf-á serum dan polimorfisme gen tnf-á g308a antara penderita isk karena e. coli dan kontrol non isk. metode penelitian ini adalah cross sectional dilakukan di rsup dr. kariadi dan center for biomedical research fk undip semarang. enam puluh delapan penderita isk karena e. coli dan 55 kontrol non isk dukur kadar tnf-á serum dengan metode elisa. polimorfisme tnf-á-308 g>a ditentukan dengan metode polymerase chain reaction restriction fragment length menggunakan enzim ncol. fragmen divisualisasikan pada gel poliakrilamid dan diwarnai dengan silver. hasil median kadar tnf-á serum pada penderita isk karena e.coli didapatkan sebesar 8,9 pg/ml lebih tinggi secara bermakna dibanding kontrol dengan median 3,7pg/ml (p<0,001). polimorfisme gen tnf-á-308g>a pada kelompok penderita isk karena e.coli didapatkan g/g=61(90%), g/a=7(10%) dan a/a=0. kelompok kontrol didapatkan g/g =48 (87%), g/a=7(13%) dan a/a=0. analisis dengan fisher’s exact test tidak didapatkan perbedaan bermakna (p=0,578) antara penderita isk karena e.coli dan kontrol. kesimpulan kadar tnf-á serum penderita isk karena e. coli lebih tinggi secara bermakna dibanding kontrol non isk, tetapi tidak didapatkan perbedaan bermakna polimorfisme gen tnf-á antara kedua kelompok. kata kunci: infeksi saluran kemih e.coli, tnf-á serum, polimorfisme gen tnf-á abstrak introduction urinary tract infection (uti) is one of the most frequently encountered bacterial infections, particularly in women.(1) the majority of uti cases occur in the age range of 16-35 years.(1,2) the female-to-male ratio is 4:1, with 10% of females having uti annually and 60% having several infections during their lifetime.(1,2) uti is the most frequent source of bacteriemia.(3) the most frequent cause of uti is escherichia coli (e. coli), found in 80% to 90% of uti cases.(4-7) the body response in uti is initiated by an immune response at the time of bacterial contact with urothelial cells. subsequently various b a c t e r i a l f a c t o r s ( f i m b r i a e , p i l i a n d lipopolysaccharide/lps) interact with epithelial receptors, leading to activation of several pathways. lps stimulates the phagocytic cells of the innate immune system through interaction with toll-like receptor 4 (tlr-4), resulting in release of cytokines, viz. tumor necrosis factor (tnf)-á, interferron (ifn)-γ, interleukin (il)8, il-1, and il-6.(7-9) 169 tnf-á is an important mediator in the acute inflammatory response against gram-negative bacteria and other infectious microbes, and is responsible for the systemic complications of severe infections. production of tnfá is induced by lps from the cell walls of gramnegative bacteria. tnf is produced in large amounts in severe infections and causes systemic abnormalities.(10) tnfá is a cytokine that is most frequently associated with sepsis or septic shock.(11) several of the underlying reasons are: 1) tnfá plasma concentrations increase at the time of sepsis, 2) the increase in tnfá is associated with increased mortality, 3) administration of exogenous tnfá in humans and experimental animals induces sepsis and organ failure, and 4) neutralization of tnf-á in experimental animals results in improvement from sepsis and increases survival.(9,12) genetic and environmental factors play a role in determining illness in an individual. a person may have been programmed to respond to infection in various ways. some individuals have a strong immune system and are able to overcome the infection before the appearance of physical signs, whereas others more frequently become ill.(13) the influence of genetic factors on this response to disease has been demonstrated by studies with adopted children and twins. genetic factors are the major determinants of the tendency towards death from infection. the mortality risk in patients with sepsis is related to gene polymorphisms of tnf-α and tnfβ.(14,15) tnf-á gene polymorphism causes variation in tnf-á production. tnf-á gene polymorphism is responsible for overproduction of tnf-á, such as occurs in sepsis and septic shock.(11) one of the identified tnf-á gene polymorphisms is localized in the promoter region at position -308, where the common allele g (guanine) is replaced by the a (adenine) allele, known as tnf-á-308 g>a.(16) tnf-á gene polymorphism in the promoter region at position -308 is reportedly strongly associated with induction of disease, including uti.(17,18) however, investigations on the tnf-á gene in the promoter region -308g>a in cases of uti due to e. coli have never been reported in indonesia as well as internationally. the objective of this study was to compare serum tnf-á levels and tnf-á-308g>a gene polymorphisms in patients with uti due to e. coli and in non-uti controls. methods study design this was a cross-sectional observational study conducted at dr. kariadi central hospital and the cebior laboratory, faculty of medicine, diponegoro university, from march 2009 until november 2010. study subjects patients with uti were identified on the basis of leukocyturia on urine examination and an e. coli count of ≥100.000 colony forming unit (cfu)/ml on bacterial culture of urine. inclusion criteria were age 14 years and older, presence of e. coli infection as evidenced by bacterial culture of urine, and agreeing to become a study subject. subjects with a past history of urinary tract obstruction (prostate h y p e r t r o p h y, s e n i l e v a g i n i t i s ) , u s e o f instruments in the urinary tract lasting ≥3 days, disturbances of miction (chronic urinary incontinence, etc.), and immunocompromized patients (organ transplantation, renal failure, diabetes mellitus), were excluded from the study. controls were selected from healthy volunteers not suffering from uti, based on the absence of leukocyturia and sterile bacterial culture of urine. a total of 68 patients with uti due to e. coli and 55 persons without uti as controls comprised the subjects of this study. measurement of tnf-á serum levels serum was separated from venous blood samples and stored pending measurement of tnf-á concentration by means of elisa, using the quantikine® human tnf-á elisa kit univ med vol. 31 no.3 170 susilaningsih, karjono, purnamawati tumor necrosis factor-α is increased (r&d systems). measurements were carried out according to the manufacturer’s instructions. dna extraction dna extraction was done by the salt saturation method as described by previous investigators.(19) blood samples were placed in tubes containing ethylenediamine tetraacetate (edta), then 5-10 ml nh 4 cl lysis buffer was added to the tubes, which were left to stand for 10-30 minutes at room temperature. the tubes were centrifuged for 5 minutes at 3000-3500 rpm. the supernatant was discarded, nh 4 cl lysis buffer was added, and the tubes were centrifuged once more. to the white pellets was added 2 ml telesys, 30-50 µl proteinase k and 100 µl 10% sodium dodecyl sulfate, then the samples were kept at 500c overnight. subsequently 6 m nacl was added at 1/3 of the sample volume, and then the sample was shaken and centrifuged at 4000 rpm for 10 minutes. the supernatant was transferred to a new tube, and 100% ethanol was added at twice the sample volume. the dna in the form of a white substance was taken out and washed in 70% ethanol, left to dry, then dissolved in tris-edta (te) buffer. analysis of gene polymorphism analysis of tnf-α gene polymorphism was performed by amplification of the tnf-α gene in the promotor region at position -308, using a pcr cycler (applied biosystems). the total volume of each sample mixture (20 µl) contained 100 ng dna, 4 pmol/µl of forward primer and reverse primer, respectively, 0.2 mm dntp, 1 mm mgcl 2 and 0.5 units of the taq enzyme. the polymerase chain reaction (pcr) cycle consisted of 5 minutes at 950c for initial denaturation, followed by final extension for 7 minutes at 720c. the pcr product of 107 bp was then cut by restriction fragment length polymorphism (rflp), by incubation at 550c for 18 hours (overnight) with the ncoi restriction enzyme. the cut pcr product was visualized by silver staining on 10% polyacrylamide gel. in individuals homozygous for the -308 (a/a) allele, the pcr product was not cut, resulting in a band on the gel of 107 bp. in contrast, homozygous g/g yielded fragments of 87bp and 20 bp, while heterozygous g/a yielded fragments of 107 bp, 87 bp and 20 bp.(20) data analysis data were analyzed by descriptive statistics to show the frequency distribution of the data. tests of normality of tnf-α concentrations indicated that the data were non-normally distributed. the mann whitney was used for comparing serum tnf-α levels in patients with uti and in controls. fisher’s exact test was used to compare tnf-α gene polymorphism events in patients with uti and in controls. all analyses were performed by means of the spss program version 11 for windows, at a significance level of 5%. ethical clearance the present study was approved by the commission on research ethics of the faculty of medicine, diponegoro university/dr kariadi central hospital, semarang, with ethical clearance no 16/ec/fk/rsdk/2009. results this study found that the 68 respondents with uti due to e. coli were in the age range of 16-86 years, while the 55 controls were in the age range of 18-84 years, and a median in the age range of 65-69 years. the group with uti due to e. coli consisted of 46 females (67.6%) and 22 males (32.4%), while the control group had 29 females (52.7%) and 26 males (47.3%). the results of the analysis on age and gender showed no significant difference between the uti group and the control group (p=0.093). the subjects with uti due to e. coli were taken from respondents suffering from uti with leucocyturia and positive bacterial culture of urine (>100.000 cfu/ml). the subjects with uti were taken from subjects whose urinary bacterial cultures were positive for e. coli only 171 and for e. coli in combination with other bacteria, as seen in table 1. in 51 (75.0%) subjects the cause of uti was e.coli. serum tnf-á measurements yielded a median of 8.9 pg/ml in the group with uti due to e. coli and a median of 3.36 pg/ml in the control group. the mann whitney test found significantly higher tnf-á levels in patients with uti due to e coli than in controls (p<0.001). the results of the analysis are shown as box plots in figure 1. the tnf-α-308g>a gene polymorphism indicated that 7 (10%) of the 68 respondents with uti and 7 (13%) of the 55 controls had the heterozygous genotype g/a, while the majority of both patients with uti and controls had the homozygous genotype g/g. no homozygous a/ a genotype was found in patients with uti or in controls (figure 2). fisher’s exact test showed no significant difference in tnf-α gene polymorphism events in patients with uti due to e. coli and in controls (p=0.578) (table 2). discussion tnf-á is an important mediator of the acute inflammatory response against gram-negative bacteria and other infectious microbes, and is responsible for various systemic complications of severe infection. tnfá production by macrophages is induced by lps from the cell walls of gram-negative bacteria. tnfá is a cytokine most frequently associated with sepsis or septic shock.(10,21) the results of the present study showed that the tnf-á serum level was higher in the group of patients with uti due to e.coli. this indicates that the inflammation occurring in uti due to e. coli activates tlr4 macrophages and induces tnf-á production.(22) the tnf-á produced plays a role in inducing the activation of integrins in leukocytes and integrin ligands in endothelial cells, so that the leukocytes adhere to the endothelium and subsequently migrate into the extra vascular tissues, where they engage in fighting the microbes. t n f á as a potent pro-inflammatory cytokine has been reported to be increased in sepsis. this cytokine is reportedly increased in the urine in uti (pyelonephritis), and also in the urinary bladder during uti, particularly at the onset of the disease.(10,23) sadeghi(24) reported that during bacteriuria in renal transplant patients there was an increase in urinary cytokine content, including tnf-á. the production of tnf-occurs after activation of toll-like receptors (tlr) by bacterial products, including endotoxins.(9) genetic and environmental factors play a role in determining illness in an individual. a person may have been programmed to respond to infection in various ways. some individuals have a strong immune system and are able to overcome the infection before the appearance of physical signs, whereas others more frequently become ill.(13) genetic factors are the major univ med vol. 31 no.3 table 2. distribution of tnf-α 308g>a gene polymorphisms in uti* and control groups *uti= urinary tract infection *uti= urinary tract infection bacterial species n % e. coli 51 75.0 e. coli and pseudomonas spp 2 2.9 e. coli and enterococcus spp 1 1.5 e. coli and staphylococcus a ureus 8 11.8 e. coli and enterobacter spp 6 8.8 tabel 1. distribution of bacterial causes of uti* (n=68) 172 susilaningsih, karjono, purnamawati tumor necrosis factor-α is increased determinants of the tendency towards death from infection. the mortality risk in patients with sepsis is related to gene polymorphism of tnfα and tnf-β.(14,15) the present study found the occurrence of tnf-á 308g>a gene polymorphisms, with the g/a genotype present in 7 (10%) of 68 patients with uti due to e. coli, and in 7(13%) of 55 controls, but did not find the homozygous p o l y m o r p h i s m a l l e l e a / a . t n f á g e n e polymorphism causes variation in the production of tnf-á in the defense mechanism of the body figure 2. pcr results for tnf-á 308g>a lanes a1 and b1= markers, lanes a2 and b2 = blanks. lanes a4,5,6 and b9 = heterozygous g/a (sample nos 49, 51, 56 and 147) all other lanes = homozygous g/g a 1 2 3 4 5 6 7 8 b 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 figure 1. box plots of tnf-á serum levels. uti due to e. coli (n=68); non-uti controls (n=55) 173 against infectious diseases. t n f á g e n e p o l y m o r p h i s m i s r e s p o n s i b l e f o r t h e overproduction of tnf-á and ultimately disrupts the regulation of cytokine activity, thus resulting in disease. the a allele has been reported to increase tnf-á production to a higher degree t h a n t h e g a l l e l e , a n d t h e h o m o z y g o u s polymorphism a/a is called a high producer of tnf-á.(11,25) the homozygous polymorphism g/g was found in dominant numbers, both in patients with uti due to e. coli (90%) and in non-uti controls (87%), while the homozygous polymorphism a/ a was not found in this study. the results of this study showed that there was no significant difference between the group of patients with uti due to e. coli and the non-uti control group. the genotypic distribution in the tnf-á gene polymorphism was dominated by the g/g polymorphic allele, which is consistent with the various studies conducted by sallakci,(25) krasowska(26) in 2005, moffet et al.(27) in 2005, and the study by cabantau et al.(28) in mali in 2006. a limitation of this study was the small size of the control group in comparison with t h e u t i g r o u p , a l t h o u g h n o s i g n i f i c a n t difference was found in age range and gender between the two groups. furthermore, this study was conducted on hospitalized patients, who are thus not representative of non-hospitalized patients. conclusions serum tnf-á level was higher in patients with uti due to e. coli than in non-uti controls. no significant difference was found for the tnfá -380 gene polymorphism between patients with uti due to e. coli and controls. further studies should be done with a better matching between uti cases and controls, and the inclusion of nonhospitalized patients. there are presumably more important factors than host genotype that influence uti pathogenesis. acknowledgements we wish to express our gratitude to the indonesian ministry of research and technology for the funding of this study through the basic research incentives program (program insentif riset dasar) for 2009-2010. we also thank prof.dr. sultana mh faradz, phd and staff at the center for biomedical research (cebior), faculty of medicine, diponegoro university, for their inputs and facilitation of this study. references 1. salvatore s, salvatore s, cattoni e, siesto g, serati m, sorice p, et al. urinary tract infections in women. eur j obstet gynecol reprod biol 2011;156:131-6. 2. nicolle le. uncomplicated urinary tract infection in adults including uncomplicated pyelonephritis urol clin north am 2008;35:1– 12. 3. bingen-bidois m, clermont o, bonacorsi s, terki m, brahimi n, loukil c, et al. phylogenetics analysis and prevalence of urosepsis strain of escherechia coli bearing phatogenecity island like-domains. infect immun 2002;70:3216-26. 4. ronald a. the etiology of urinary tract infection: traditional and emerging pathogens. am j med 2002;113 suppl 1a:s14–9. 5. nicolle le. complicated urinary tract infection in adults. can j infect dis med microbiol 2005; 16:349-60. 6. ronald a. the etiology of urinary tract infection: traditional and emerging pathogens dis mon 2003;49:71–82. 7. lee s-j, kim sw, cho y-h, yoon ms. antiinflammatory effect of an escherichia coli extract in a mouse model of lipopolysaccharide-induced cystitis. world j urol 2006;24:33-8. 8. kucheria r, dasgupta p, sacks sh, khan ms, sheerin ns. urinary tract infections: new insights into a common problem. postgrad med j 2005;81:83-6. 9. ramesh g, kimball sr, jefferson ls, reeves wb. endotoxin and cisplatin synergistically stimulate tnfproduction by renal epithelial cells. am j physiol renal physiol 2007;292: f812–9. 10. mohkam m, asgarian f, fahimzad a, sharifian m, dalirani r, gorgi fa. diagnostic potential univ med vol. 31 no.3 174 susilaningsih, karjono, purnamawati tumor necrosis factor-α is increased of urinary tumor necrosis factor-alpha in children with acute pyelonephritis. iranian j kidney dis 2009;3:89-92. 11. wong hr. genetics and genomics in pediatric septic shock. crit care med 2012;40:1618-26. 12. knuefermann p, remoto s, baumgarten g, misra a, sivasubramanian n, carabello b, et al. cardiac inflammation and innate immunity in septic shock. is there a role for toll like receptors? chest 2002;121:1329-36. 13. holmes cl, russell ja, walley kr. genetic polymorphisms in sepsis and septic shock: role in prognosis and potential for therapy. chest 2003;124:1103-15. 14. hotchkiss r, karl i. the pathophysiology and treatment of sepsis. n engl j med 2003;348:13850. 15. sipahi t, pocan h, akar n. efect of various genetic polymorphisms on the incidence and outcome of sever sepsis. clin appl thromb hemost 2006;12:47-54. 16. mossböck g, renner w, el-shabrawi y, faschinger c, schmut o, wedrich a, et al. tnfá –308 g>a and –238 g>a polymorphisms are not major risk factors in caucasian patients with exfoliation glaucoma. mol vis 2009;15:518-22. 17. haukim n, bidwell j, ajp ajps, keen lj, gallagher g, kimberly r, et al. cytokine gene polymorphism in human disease: on-line databases, supplement 2. genes immun 2002;3: 313–30. 18. hollegaard m, bidwell j. cytokine gene polymorphism in human disease: on-line databases. genes immun 2006;7 suppl 3:s269– 76. 19. ngestiningsih d, faradz smh. polimorfisme alel tnf-alfa pada penderita dhf di semarang. m med indones 2006;41:46-51. 20. moukoko ce, el-wali n, saeed ok, mohamedali q, gaudart j, dessein aj, et al. no evidence for a major effect of tumor necrosis factor alpha gene polymorphisms in periportal fibrosis caused by schistosomamansoni infection. infect immun 2003;71:5456-60. 21. knuefermann p, remoto s, baumgarten g, misra a, sivasubramanian n, aabello ba, et al. cardiac inflammation and innate immunity in septic shock. is there a role for toll like receptors? chest 2002;121:1329-36. 22. svanborg c, bergsten g, fischer h, godaly g, gustafsson m, karpman d, et al. uropathogenic escherichia coli as a model of host-parasite interaction. curr opin microbiol 2006;9:33-9. 23. engel d, dobrindt u, tittel a, peters p, maurer j, gutgemann i, et al. tumor necrosis factor alphaand inducible nitric oxide synthaseproducing dendritic cells are rapidly recruited to the bladder in urinary tract infection but are dispensable for bacterial clearance. infect immun 2006;74:6100-7. 24. sadeghi m, daniel v, naujokat c, wiesel m, hergesell o, opelz g. strong inflammatory cytokine response in male and strong antiinflammatory response in female kidney transplant recipients with urinary tract infection. transpl int 2005;18:177-85. 25. sallakci n, akcurin g, koksoy s, kardelen f, uguz a, coskun m, et al. tnf-alpha g-308a polymorphism is associated with rheumatic fever and correlates with increased tnf-alpha production. j autoimmun 2005;25:150-4. 26. krasowska d, chodorowska g, koziolmontewka m, ksiazek a, buraczynska m. the -308 promoter polymorphism in the tumour necrosis factor gene in patients with lichen planus. acta derm venereol 2005;85:400-3. 27. moffett s, zmuda j, oakley j, beck t, cauley j, stone k, et al. tumor necrosis factorá polymorphism, bone strength phenotypes, and the risk of fracture in older women. j clin endocrinol metab 2005;90:3491–7. 28. cabantou s, doumbo o, ranque s, poudiougou b, traore a, hou x, et al. alleles 308a and 238a in the tumor necrosis factor alpha gene promoter do not increase the risk of severe malaria in children with plasmodium falciparum infection in mali. infect immun 2006;74:7040–2. c:\users\universa medicina\docu 19 *department of molecular biology, faculty of medicine universitas jenderal soedirman, purwokerto **department of paediatrics, faculty of medicine universitas jenderal soedirman, purwokerto correspondence: lantip rujito department of molecular biology, faculty of medicine universitas jenderal soedirman jl gumbreg no 1 purwokerto 53112, jawa tengah, indonesia email : l.rujito@unsoed.ac.id orcid id : 0000-0001-6595-3265 date of first submission, july19, 2018 date of final revised submission, february 1, 2020 date of acceptance, february 4, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina glutathione s transferase and catalase gene polymorphisms did not tend to influence the severity of hemoglobin e/-thalassemia lantip rujito*, yundandhika rizki widodo*, ghaida sakina*, qodri santosa**, and ariadne tiara hapsari** background thalassemia, a monogenic genetic disease of red blood cells, is spread widely throughout the world. glutathione s transferase (gst) enzymes have an antioxidant role in detoxification processes of toxic substances this study aimed to determine the role of the genetic modifier genes gstt1 and gstm1, and the catalase (cat) gene in clinical degrees of hemoglobin (hb)e/β thalassemia. methods sixty hbe/β thalassemia patients were examined to determine their clinical pictures. clinical score was based on age when thalassemia symptoms appeared, time of diagnosis, time of first blood transfusion, pre-transfusion hemoglobin concentration, frequency of transfusions, and enlargement of spleen. ferritin concentration was also obtained from medical records. gene polymorphisms of gstt1, gstm1, and cat were measured using pcr and pcr-rflp methods. clinical scores were categorized into mild (03.5), moderate (4-7), and severe (7.5-10) degrees, while ferritin level was expressed in mg/dl. one way anova was used to analyze the data. results the clinical appearance showed that severe, moderate, and mild degrees accounted for 42%, 45%, and 13%, respectively. the majority had a high ferritin level of more than 5000 mg/dl (67%). gstt1 null, gstm1 null, and cat minor allele genotypes were 21.7%, 33.3%, and 12.1%, respectively. gstt1, gstm1, and cat genotypes had no impact on the severity of thalassemia patients (p=0.091, p=0.082, and p=0.141, respectively). conclusion gstt1, gstm1, cat gene polymorphisms tend to be a minor aspect of severity of clinical outcome for hbe/β thalassemia patients and should be not considered a routine laboratory check. keywords: gstt1, gstm1, cat, hbe/β thalassemia, clinical score original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.19-26 january-april, 2020 vol.39no.1 cite this article as: rujito l, widodo yr, sakina g, santosa q, hapsari at. glutathione s transferase and catalase gene polymorphisms did not tend to influence the severity of hemoglobin e/b-thalassemia. univ med 2020;39: 19-26. doi: 10.18051/univmed.2020. v39.19-26 20 rujito, widodo, sakina gstt1, gstm1, and cat in hbe/ β-thalassemia introduction clinical pictures of hemoglobin e (hbe)/β thalassemia have been reported widely, ranging from the type with a healthy appearance needing no transfusion to the regular transfusiondependent type. the phenotype modification of hbe/ β th a l a s s e mia h as be e n de sc r i be d extensively concerning the types of beta gene mutations, and the presence of the secondary modifiers xmn1, bcl11a, and hbs1l-myb.(1) the other findings reported that genes in the redox system are thought to play a role in the clinical conditions of thalassemia patients. it relates to the system that regulates the oxidantantioxidant reaction especially against free radicals from routine transfusions and lack of iron chelation. glutathione-s-transferase mu (gstm1) and glutathione-s-transferase theta ( g st t 1) , t he ge ne s r e s p on s ib l e f or t h e production of glutathione s-transferase, affect the degree of cardiac siderosis as one of the factors related to the death of thalassemia patients.(2) on the other hand, gstt1 and gstm1 a l so h ave an e f f e c t on ir on o ve rl oa d in thalassemia patients.(3) the genotypes of gstm1 and gstt 1 are polymorphic. the “null” genotype of the gst genes results in increased susceptibility to oxidative reactions, with the increased risk of tumors of the prostate and cardiac disorders.(4) likewise, with catalase (cat) genes, the polymorphism of exon nine at the rs769217 locus was allegedly related to low catalase activity resulting in reducing the effect of the catalase enzyme.(5) research on gstm and gstt1 against many antioxidant factors has been widely reviewed in various articles. in general, the data shows that gstm and gstt1 affect the molecular level. however, there is less knowledge in the literature on the internet especially on the clinical aspects of thalassemia. the present study was intended to add data about the effect of gstm, gstt1 and cat gene polymorphisms in clinical issues, especially hbe/thalassemia. methods research design the study was of cross-sectional design and used secondary clinical data from a previous study that were retrieved through the medical records of banyumas hospital.(1) genotyping was carried out in the research laboratory of the faculty of medicine, universitas jenderal soedirman, in the 2017-2018 period. research subjects using the formula of slovin for a total population of 350, 60 patients aged 10-12 years were recruited to participate in this study. the subjects who came from the three categories of mild, moderate, and severe types, underwent genotyping for the genes of interest. hbe/β thalassemia status was acquired from the previous genotyping study.(1) genotyping genotyping for each gene was carried out according to the following description: gstt1 and gstm1 were obtained by the polymerase chain reaction (pcr) technique, whereas cat was determined using the polymerase chain reactionrestriction fragment length polymorphism (pcrrflp) method. table 1 contains the details of primers and enzymes used in this study. the pcr cocktails containing 50 ng template dna, pcr mix kit (invitrogen corp., carlsbad, ca, usa), h 2 o, and primers were subjected to a cycle at 94°c for 10 minutes, then to 30 cycles each at 9°c for 55 seconds, 69.5°c for 55 seconds, and 72°c for 90 seconds. ten iu of restriction enzyme in rflp application was incubated overnight at 37°c with 15 μl of pcr product. we then used gel documentation procedure for band visualization. clinical score the mahidol score was used to determine the clinical appearances of the hbe/β thalassemia patients as described elsewhere.(6) this score included the age at which thalassemia symptoms 21 the gel documentation system showed pcr and rflp visualization. bands were presenting the genotyping of gstt, gstm1, and cat genes on the agarose gel labeled with ethidium bromide (figure 1). the genotype distribution of the gstt, gstm1, and cat gene polymorphisms and the correlation picture for clinical scores are depicted in tables 3 and 4. the normal gstm1 and the gstm1 null alleles both had a frequency of 33.3% (20/60), which was higher than that of gstt1 null( (21.7%)(13/60)), while in the cat gene, the t/t minor genotype accounted for 12.1%. discussion hbe/β-thala ssemia is a variant of thalassemia which has a phenotype spread from mild to severe. mild conditions, in general, are manifestations of the mild combination of hbe and beta thalassemia genotypes.(7) hemoglobin e itself in the heterozygous and homozygous states gives rise to mild clinical conditions, and generally without the need for transfusion. however, some patients may require transfusion since hbe mutations are a kind of cryptic mutations.(8) clinical symptoms in hbe/β-thalassemia patients appear in extensive variations, stretching in the age range 1-15 years.(9) similar to the data in the present study, generally, transfusion in hbe/ β-thalassemia patients follows the type of mutant in β-thalassemia. the more severe the mutant type, the more frequent transfusions must be carried out, varying from 2 weeks to sporadic in months. the present study obtained the same data table 1. primers and enzymes used for genotyping the genes *cyp gene was used as a control of multiplex pcr appeared, time of diagnosis, the time of first transfusion, pre-transfusion hemoglobin, the frequency of transfusion, and enlargement of the spleen. iron level represented by ferritin was also obtained from the medical records. in brief, the score represents mild (0-3.5), moderate (4-7), and severe (7.5-10) types. the present study included the three types of patients (mild, moderate, and severe) in equal proportions. statistical analysis one way anova was used to determine the differences in mean clinical score for each genotype. a p<0.05 was considered to indicate statistical significance. ethical clearance the medical ethi cs committee of universitas jenderal soedirman approved the ethics of the study protocol for the research, under registry number 1328/kepk/iii/2018. results the majority of the severe patients were diagnosed with anemia at the age of 1-5 years. in the less severe types, usually the symptoms appeared at an older age. because of the limitations of the previously documented data, we obtained data for pre-transfusion hemoglobin for three months, where the concentration was between 7 to 9 g/dl. some patients had been splenectomized, but most of the spleens were only mildly enlarged. ferritin level was also higher in the severe type of patients (table 2). rujito, widodo, sakina gstt1, gstm1, and cat in hbe/ β-thalassemia gene primers enzymes (rflp) pcr product interpretation gstm1 5’ aggaactccctgaaaagctaaa 3’ 215 bp + =presence =null 5’ tgggctcaaatatacggtggag 3’ gstt1 5’ cttactggtcctcacatctcctt 3’ 480 bp 5’agc tca ccggat catggc 3’ cat 5'-gccgcctttttgcctatcct-3 ' bstxi 202 bp c=remaining 202 bp t=digested 108, 94 bp 5'-tcccgcccatctgctccac-3' cyp* 5’-gaactgccacttcagctgtct-3’ 312 bp 5‘-tcaccggatcatggccagca-3’ 22 rujito, widodo, sakina gstt1, gstm1, and cat in hbe/ β-thalassemia as the previous study. there are patients with moderate and severe symptoms with initial transfusions occurring at the age of 10 years, which may possibly be due to delayed diagnosis or inc rease d blood needs such as during menstruation in female patients.(10) the iron levels represented by ferritin also showed significant differences between groups of patients. the number of transfusions in each group correlated with iron levels stored in the body. in general, there were clear differences in the time of clinical appearance between groups (table 2). genetic modification is one of the essential topics to be studied considering the diversity of clinical symptoms that appear in patients. the primary modifier is a mutation in the beta genes, whereas the subsequent modification is the involvement of hemoglobin f coding genes and co-inheritance with alpha genes mutations.(11) repeated transfusion has been known as the cause of redox reaction disturbance due to iron overload. gstm1 and gstt1 genes are among the possible factors that play a role in the clinical modification process of thalassemia patients. this study revealed that the genotype of gstm1 null had the highest percentage of patients with thalassemia major, as high as 33.3%, while the frequency of gstt1 null was 21.7% (table 4). it was in an equilibrium state according to previous data on the snp database. according to the liter ature review, the gstm1 null frequency reaches a range of 23-62% in different a b c figure 1. genotyping of gstm1, gstt1, and cat genes lane 2 is gstt1 and gstm1 positive (480 bp and 215 bp). lane 3 and lane 4 are gstt1+/gstmand gstt-/ gstm1+ respectively. lanes 5 and 7 are the cc genotype in the cat gene (uncut 202 bp), whereas lane 6 depicts the ct heterozygote (202, 108, 94 bp). the tt alleles of the cat gene are depicted in lane 8 (108, 94 bp), while the 312 bp lane is a control for the pcr process table 2. clinical appearance of hbe/β thalassemia *cyp gene was used as a control of multiplex pcr clinical parameters mild (n=20) moderate (n=20) severe (n=20) p value first diagnosis (age in years) 15.50±3.25 5.25±4.75 1.50±1.50 0.032 first transfusion (age in years) 17.25±2.05 6.50±2.05 1.25±1.50 0.026 pretransfusion hb (g/dl) 9.15±0.25 8±1.45 7±1.75 0.052 splenomegaly (cm) no 4.68±3.25 10.75±4.25 splenectomized : 2 0.001 ferritin (mg/dl) 358.75±153.50 5752.00±134.75 9370.75±1250.50 0.024 clinical score 3.05±0.25 7.25±1.65 8.75±1.00 0.042 23 populations worldwide. meanwhile, gstt1 null has been found to be 15-30% in caucasians and more than 50% in mongoloid races.(12) the present study also showed that gstt1 and gstm1 alleles did not determine the clinical severity of the groups. each group has this type of allele gene polymorphism. this demographic distribution, combined with the previous spreading data illustrate that gstm1 and gstt1 alleles are types of genes that have uniform distribution among world popula tions so that the polymorphisms of these genes become random and cannot be a reference for the determination of certain ethnic groups. our study indicated that there was no significant relationship between gstt1 and gstm1 gene polymorphisms against the clinical de gree of hbe/thalassemia β patients in indonesia (table 3). the data has used both comprehensive outcome data, namely mahidol score and using ferritin levels only. ferritin is used as a protein marker of iron in thalassemia patients who undergo regular transfusions. the ferritin level increases with the number of transfusions and lack of compliance with iron chelation medications.(13) ferritin in the gstt1 and gstm1 null groups showed higher mean levels than the normal group, but this difference was not significant. likewise in the cat gene, groups containing allele mutants also showed higher values than those in the wild-type group, but did not show data significance (table 4). differing genotype disease severity p value mild (n=20) moderate (n=20) severe (n=20) gstt1 null non-null gstm1 null non-null gstt1+gstm1 null non-null cat genes c/c c/t t/t 4 16 7 13 2 18 13 4 3 4 16 8 12 2 18 15 3 2 5 15 5 15 3 17 13 5 2 0.906 0.591 0.850 0.912 table 3. frequency distribution of gst gene polymorphisms among hbe/β thalassemia patients table 4. genotype frequencies of gstt, gstm1, and cat genes and the genotypephenotype relationship with severity of disease and the ferritin level genotype frequency clinical score ferritin level (mg/dl) mild moderate severe total (%) (x±sd) p value (x±sd) p value gstt and gstm genes normal 7 6 7 20 (33.3) 6.27±1.19 0.09 4726.42±71.67 0.08 gstt1 null and gstm1 null 2 2 3 7 (11.7) 5.14±1.31 5954.57±54.46 gstm1 null 7 8 5 20 (33.3) 6.15±0.74 5723.50±140.72 gstt1 null 4 4 5 13 (21.7) 5.8±1.52 5224.85±98.24 cat gene c/c 13 15 13 41 (70.7) 5.4±1.40 0.08 5473±102.92 0.10 c/t 4 3 5 12 (17.21) 5.9±0.6 5002±48.38 t/t 3 2 2 7 (12.1) 8.5±1.15 5974±218.46 univ med vol. 39 no.1 24 rujito, widodo, sakina gstt1, gstm1, and cat in hbe/ β-thalassemia data were reported previously by sharma and colleagues who concluded that gstt1 and gstm1 genotype deletions worsened the clinical outcome in maj or th alassemia patie nts characterized by elevated serum iron and serum ferritin levels compared with major thalassemia patients with normal gst genotypes.(14) for major thalassemia, these data did not explain in detail the type of mutation involved. differences in primary mutants may cause different results considering that there are mor e than 300 variations recorded in the human beta gene variation database.(15) this fact may explain why there are differences between studies. observation of ferritin levels in these patients showed no different levels in various types of genotypes. this indicates that ferritin iron levels are not affected by gstt1 and gstm1 alleles, although on average the minor allele group shows an increase over other groups. the sharma (14) study was based on iron parameters only whereas the present study used the general clinical outcome and also the serum ferritin level. in some hematological diseases, these genes may constitute independent risk factors, such as in acute myeloid leukemia (aml), with no impact on prognosis.(16) the gstt and gstm1 null also have an impact on the pro-hepcidin level on iron overload and organ dysfunction. however, these mutants were not as good a biomarker as ferritin for the iron stores. another report in sickle cell patients was also following this issue, especially in iron overload status.(17,18) glutathione s transferase expression levels are an essential factor in determining the sensitivity of cells to a broad spectrum of toxic chemicals, but it does not appear to be a vital element in influencing the clinical state of thalassemia patients.(19) however, comparison of gstm1 and gstt1 in healthy and affected individuals shows that deletion in both gst genes influences gst enzyme activity which was demonstrated by low gst levels in the group of individuals with double deletions in both genes. the results is a deficiency of the enzyme in favor of free radical reduction processes due to excessive iron deposits.(3,20) studies of polymorphisms in exon nine at the locus rs769217 cat genes in thalassemia are limited. however, in some other chronic diseases such as diabetes mellitus, it is reported that the minor alleles cc and c have decreased activity of catalase and hemoglobin a1c and allow the risk of complications to increase.(21) in other chronic diseases such as chronic hepatitis and hepatocarcinoma, the polymorphism of the cat gene was also reported to influence disease severity.(22) we found a report in the pubmed database that was allegedly associated with low catalase activity and reduced catalase protection against free radicals in the body.(5,23) however, the data of the present study did not show a significant association between the cat gene and the clinical condition of the hbe/thalassemia β patients. in the case of minor thalassemia, there is also a change in oxidative reactions, which are thought to be associated with hypochromic microcytic anemia, similar to iron deficiency anemia. however, other previous findings of catalase activity in hypochromic microcytic anemia reported conflicting results.(24,25) many factors influence the resulting nonsignificant relationship in the present study. the type of mutation in the globin gene may play a role in the clinical state of major thalassemia patients. additionally, the presence of xmni+ site polymorphisms in the β globin gene cluster, and the secondary modifier bcl11a and hbs1lmyb in patients with thalassemia major has been reported to be advantageous since it may reduce clinical manifestations although belonging to thalassemia major.(1,26) this study used hbe/-t hala ssemia patients with a long transfusion history. routine transfusion bias can obscure the patient’s clinical history. it is necessary to use patients who are in an early stage of diagnosis and follow the next clinical developments to ensure a causative relationship. the study showed that the gstt1, gstm1, and cat genes were not important modifiers of hbe/thalassemia, so that they cannot be a routine checking procedure. this study needs further exploration to find modifiers 25 related to clinical variation, other than primary mutations of beta genes. conclusion polymorphisms of gstt 1, gst m1, colia1, and cat genes did not modify outcome pictures of hbe/thalassemia β. acknowledgments the authors thank shinta prima and eli yuliya for technical help of laboratory and data management. this research was supported by the ministry of research, technology, and higher education of indonesia through the hibah unggulan universitas jenderal soedirman. conflict of interest the authors declare no conûict of interest. contributors lr contributed to the research design, conceptual framework, laboratory experiment, data analysis, and manuscript development. yrw and gs contributed to laboratory experiment and data analysis. qs and ath contributed to the research design, data analysis, and manuscript development. all authors have read and approved the final manuscript. references 1. rujito l, basalamah m, siswandari w, et al. modifying effect of xmni, bcl11a, and hbs1lmyb on clinical appearances: a study on βthalassemia and 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erythrocytes of iron deficiency anemic patients supplemented with vitamins. iran biomed j iran biomed j 2014;18:82-7. doi: 10.6091/ibj.12282.2013 26. yamsri s, singha k, prajantasen t, et al. a large cohort of beta(+)-thalassemia in thailand: molecular, hematological and diagnostic considerations. blood cells mol dis 2015;54:1649. doi: 10.1016/j.bcmd.2014.11.008. c:\users\jurnal fkusakti\docume 41 *cancer molecular pathology research center, department of pathology, faculty of medicine, mashhad university of medical sciences, mashhad, iran **department of pathology, faculty of medicine, mashhad university of medical sciences, mashhad, iran correspondence nema mohamadian roshan, md pathology department, school of medicine, mashhad university of medical sciences, mashhad, iran email: roshann@mums.ac.ir orcid id : 0000-0002-7862-7140 date of first submission, december 14, 2 01 8 date of final revised submission, march 27, 2019 date of acceptance, march 28, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina cd10 expression in gastric carcinoma is correlated with tumor grade and survival amir hosein jafarian*, melika kooshki forooshani**, leila takallou**, and nema mohamadian roshan** background gastric carcinoma (gc) is the most common non-skin malignancy in iranian men and the second leading cause of cancer-related mortality. invasion and metastasis are considered as the major causes of cancer-related morbidity and mortality. proteinases such as matrix metalloproteinases play an important role in tumor progression and mediating extracellular matrix remodeling. cd10 is a 90-110kd cell surface zinc-dependent metalloproteinase and there is evidence that this membrane protein may facilitate invasion and/or metastasis of tumoral cells. the objective of this study was to determine the frequency of cd10 expression in the stromal cells of gc and determine its relationship with survival and clinicopathological factors. methods a cross-sectional study was performed involving 50 patients with histopathologic diagnosis of gc. cd10 expression was determined by immunohistochemistry (ihc). survival of the patients as well as the grade and stage of the tumors and demographic variables were documented. the kaplan-meier test was used for data analysis. results stromal cd10 was detected in 46% of the gc stromal cells. no immunoreactivity was identified in the stromal cells of normal adjacent tissue. stromal cd10 expression in gastric carcinoma did not correlate with the age and gender of the cases as well as the size and location of the tumor, and lymph node involvement but correlated with tumor stage (p=0.01), tumor grade(p=0.01) and patients’ survival (p=0.02). conclusion stromal cd10 expression is correlated with tumor differentiation, clinical stage and survival in gc. cd10 expression could be considered as a negative prognostic factor for gastric carcinoma. keywords: cd10, immunohistochemistry staining (ihc), gastric carcinoma, stromal cell original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.41-47 january-april, 2019 vol.38no.1 cit e th is a rtic le a s: jafarian ah, forooshani mk ta kallou l, et al. cd10 expression in gastric carcinoma is correlated with tumor grade and survival. univ med 2019;38:41-7. doi: 10.18051/univmed. 2019.v38.41-47 42 jafarian, forooshani, takallou, et al cd10 expression in gastric carcinoma introduction gastric carcinoma (gc) is considered a deadly tumor and is the second most common c a u s e of ca n c e r-a s so c i a t e d mo r t a l i t y. ( 1 ) approximately 990,000 patients are diagnosed with gc worldwide annually, of whom about 738,000 die from this malignancy.(2) gastric carcinoma also causes one of the highest cancer burdens, as measured by disability-adjusted life years lost.(3,4) cd10, a common zinc-dependent metalloendoprotease that cleaves and inactivates a variety of signaling peptides, is now widely known for its involvement in various cancers. its role in tumor progression, however, seems to differ in a tissueor cancer-specific manner.(5) in addition, previous studies on lymphoma, lung adenocarcinoma and invasive ductal carcinoma of the breast have shown that the expression of cd10 in primary tumors can serve as an independent predictor of poor prognosis. fo r e x a mp l e i n o n e st u dy, i t wa s d e mo n st r at e d t ha t cd 10 e xp re s s i on in melanoma increased with tumor progression and predicted poor patient survival.(6) moreover, cd10 could have been widely used in cancer diagnosis while it recently has been proposed that cd10 is associated with thera peutic resistance.(7) re c e n t re s e a r c h, f o c us i ng o n t h e importance of stromal cells in pancreatic carcinomas, showed that pancreatic stellate cells which expressed cd10 were related to more aggressive nature. it has been suggested that cd10 could be a target for the treatment of pancreatic carcinomas using agents that inhibit metalloproteinase. for instance, fish oil which has some inhibitory effects on metalloproteinase, has been found useful in this regard.(8) in a recent study, murakami et al.(9) tried t o e l uc i d at e t he c l i ni c o pa tho lo gic a nd immunohistochemical featur es of ga stric a d e n oc a rc i no ma wi t h e n t er o bl a s t i c differentiation (gaed), by analyzing 29 cases. immunohistochemistry for alpha-feto protein (afp) , glypican 3, sall4, an d p53 and i mmu no s t a ini n g wi t h a n t i bo d ie s a ga i ns t muc5ac, muc6, muc2, cd10, and caudaltype homeobox 2 (cdx2) were performed. cd10 was diffusely or focally expressed in all gaed cases while invasive areas with hepatoid or enteroblastic differentiation were negative for cd10, but they did not study the correlation between the mentioned items and the prognostic clinical factors of patients namely age, stage and gra de o f dis ea se . th i s is w ha t has bee n approached by our study. another study on cd10 revealed that cd10 down expression was frequently observed along with an additiona l distinct fe ature of gastrointestinal follicular lymphoma (gi-fl). cd10-downexpressed gastrointestinal follicular lymphoma (cd10 down gi fl) frequently involved the stomach or large intestine. however, there was no data on gastric carcinoma.(10) in our study, not only the pathological features of the tumor (stage, grade, lymph node involve ment) but al so the surviva l was investigated. moreover, size and location of the tumor were also considered in comparison with other similar studies.(11) in this regard, we investigated whether cd10 expression predicted gc prognosis and whether it corre lated with other clinicopathological factors of this disease namely age, gender, tumor size, tumor grade, tumor stage, lymph node metastasis and survival of the patients. methods research design this cross-sectional study was performed at the departments of pathology and oncology, ghaem hospital, mashhad, iran. the diagnosis of gc was made between april 2004 and april 2010. research subjects the study population consisted of patients with the diagnosis of intestinal type invasive gc 43 (in which the tumor cells pass the stromal layer) who underwent gastrectomy at our medical center from 2004 to 2010. inclusion criteria were patients having complete information of the required clinicopathological factors including age, gender, and immunohistochemistry (ihc) for cd10. those with insufficient sample for h&e and ihc staining or doubtful diagnosis or incomplete clinicopathological factors were excluded from study. to estimate the sample size, based on the study of huang et al.,(11) we considered a confidence interval of 19 and confidence level of 99%, the calculated sample size was 46 and for more confidence, 50 cases were entered into the study. data collection a checklist of personal and paraclinical information, past medical history and prognosis was provided. the gathered variables include age, gender, tumor size, tumor grade, tumor stage, tumor location, lymphadenopathy, prognosis and cd10. immunohistochemistry subsequently, h&e staining of the slides of all cases were controlled, paraffin blocks with sufficient tumoral and stromal tissue were taken and two pathologists checked and confirmed the ihc results of the samples to identify the expression of cd10. for the ihc study, 3×4 micron slides were provided and stained by rat monoclonal antibody [anti cd10, igg1, 56c6 clone, novocastra, uk]. we used renal cell carcinoma tissue as positive control and primary antibody as negative control. in terms of the percentage of staining, the cells were divided into 2 subsets: under 10% as negative and over 10% as positive. statistical analysis the chi-square test was used to compare the studied variables between the two groups of patients with positive and negative cd10 expression. the student t-test was applied to compare between these two groups. kaplanmeier analysis was used to compare overall survival time between negative cd10 and positive cd10 expression groups. significance level was set a t 0.05. al l analyse s were performed using spss software (ver. 16.0, ibm). ethical clearance the study protocol was fully supported by the research council ethics committee of our medical university (t-2852). gastrectomy was indicated for the patients. the study was in conformity with the declaration of helsinki. results of 50 included patients, 43 (86%) were male and seven (14%) were female. mean (±sd) age of the patient was 65.2 ± 9.4 years (range, 43 to 80 years). nine tumors were located in the cardia (18%), 27 in the body (54%), two in the fundus (4%), and 12 in the pyloric antrum (24%). the maximum tumor diameter was between two and ten cm with an average of 5 ±2 cm. three patients (6%) had tumor stage i, eight cases (16%) had stage ii, 16 had stage iiia, 12 cases had stage iiib, and 11 patients had stage iiic. in 20 samples (40%), the tumor was well differentiated. poorly differentiated tumors were observed in 12 samples (24%) and in 18 cases (36%), the tumor was moderately differentiated. mean (±sd) overall survival in the studied patients was 26.1± 9.3 months. there was no s i gn i f i c a nc e d i ff e r e n c e of a ge , ge nd e r, lymphadenopathy and tumor location by cd10 (table 1). ihc staining twenty-three samples (46%) were positive for cd10 expression. surrounded dysplastic mucus was positive in 5 (10%) cases and stroma of normal tissue was negative. at the last followup, 41 patients (82%) had died. univ med vol. 38 no.1 44 jafarian, forooshani, takallou, et al cd10 expression in gastric carcinoma a b c the mean age of patients with cd10expressing tumors was 64 years which was not different from the mean age of 27 patients with negative cd10 expression which was 66 years (p=0.435). of 23 patients with positive cd10 expression, 19 were male and four were female (p=0.681). no significant relationship was found between cd10 expression and tumor location (p=0.413). likewise, no relationship was found between cd10 expression and lymph node metastasis (p=0.508) (table 2). however, a significant relationship was seen between cd10 expression and tumor grade (p=0.016), tumor stage (p=0.011), and survival (p=0.020) (table 2). average overall survival in the cd10 positive group was 16 months and in the cd10 negative group group 34 months (log rank mantel-cox, p=0.020) (figure 1). discussion in our study, there was a significant correlation between cd10 expression and tumor differentiation as well as overall survival. however, age, gender, tumor location and lymph node metastasis seem not to be statically related to cd10 expression. moreover, in our study in contrast to the results of a similar study,(11) a statistically significant correlation was detected between tumor stage and cd10 expression. despite the reduction in the incidence of gc worldwide as seen in the recent years, this malignancy is still the fifth most common malignant tumor with high mortality rate. generally, the prognosis of gc is poor despite observed improvements in surgical as well as adjuvant therapy of gc. these facts render it necessary to determine the prognosis of gc and to identify cd10 positive cd10 negative p value age (years) 66.4 ± 9.1 64.2 ± 8.9 0.435 gender male 19 (44,2%) 24 (55,8%) 0.681 female 4 (57.1%) 3 (42,9%) lymph node involvement + 16 (32%) 22 (44%) 0.508 7 (14%) 5 (10%) tumor location cardia 5 (10%) 4 (8%) 0.413 body 13 (26%) 14 (28%) fundus 0 2 (4%) pyloric antrum 5 (10%) 7 (14%) table 1. distribution of age, gender, lymphadenopathy and tumor location by cd10 cd 10 expression p value positive (n=23) negative (n=27) tumor stage i ii iiia iiib iiic tumor grade poorly differentiated moderately differentiated well differentiated survival alive dead 2 2 6 9 4 5 3 15 1 22 1 6 10 3 7 7 15 5 8 19 0.011 0.016 0.020 table 2. relationship between tumor stage, tumor grade and survival with cd10 expression in patients with gastric adenocarcinoma (n=50) 45 univ med vol. 38 no.1 in the normal colorectal mucosa. significant correlation was found between caf and high cd10 expression.(17) it has been shown that extracellular proteinase may contribute to promotion of regulated growth factors and cytokines. these alterations can result in tumor progression. since cd10 is structurally very s i mi l a r to ma tr i x me t a l l op r o te in a s e a nd stromelysin, it might facilitate cancer cell invasion and/or metastasis. the aim of one study was to determine the rate of cd10 expression in the stromal cells of invasive ductal breast carcinomas and the immunohistochemical aspects. stromal cd10 expression in intraductal carcinoma (idc) is closely correlated with invasion and metastasis and might play an important role in the pathogenesis of idc.(18,19) finally another research pointed out that the cd10 antigen has been shown to be lost as an early event in prostate cancer. loss of cd10 expression appears to be assoc iated with increasing tumor grade in carcinoma of the prostate and this can potentially be useful in the stratification of such patients.(20) its prognostic factors, including histopathologic markers such as cd10, a nd any possible correlation between cd10 and other present prognostic factors namely, age, tumor location, tumor stage , and gr ade and lymph node metastasis. (1 2-14 ) recent studie s of cancer research have been focusing on new targets being able to predict prognosis the prognosis or explain the invasive as well as the metastatic mechanism or highlight new strategies for treatment. the cd10 antige n is a membr ane glycoprotein that is normally expressed in early lymphoid progenitors and normal germinal center b cells. therefore, cd10 expression has gained attention especially in b cell lymphoma.(15) furthermore, a study claimed that in stage i lung adenocarcinoma, tumoral cd10 correlates with high-grade histology and is an independent predictor of recurrence in intermediate-grade tumors.(16) another study on cd10 expression was on the cancer-associated fibroblasts (caf) and their effects on colorectal cancer initiation and progression. cd10 expression was not detected figure 1. overall survival curves for negative cd10 and positive cd10 expression groups among patients with gastric cancer 46 jafarian, forooshani, takallou, et al cd10 expression in gastric carcinoma a study by kim et al. (21) showed that there was no significant association between survival rate and the expression of other mucins (i.e. muc2, muc6, cd10) or mucin phenotypes. obviously, dozens of cd10 expression effects may be seen in different types of cancers, but gc is not one of them. therefore, we intended to examine the correlation of cd10 expression with other clinicopathological and prognostic factors in gc. the main limitation we had was the lack of enough cancerous tissue for staining which made us exclude some samples from our study. in this study, we showed that cd10 expression is associated with the aggressive biological behavior of the tumor, so it seems that using medications to reduce its expression would help overall survival to a large degree. in the future, some cohort studies in this regard will definitely help to reach a more effective conclusion. conclusion t his s tud y de monst r a te d t ha t cd1 0 expression was correlated with tumor grade and survival rate. cd 10 expression can be used to predict outcomes in gc. acknowledgments the study was supported financially by the vice chancellor for research of mashhad university of medical sciences, mashhad, iran. the biospecimens for this study were provided by the surgeons of ghaem hospital. this study was the result of the thesis to obtain specialty degree in pathology by dr. leila takallou. conflict of interest none. contributors ahj contributed to the conception of the work, conducting the study, revising the draft, approval of the final version of the manuscript, and of all aspects of the work. mkf contributed to drafting and revising the draft. lt contributed to conducting the study. nmr contributed in conception development and statistical analysis of the study. all authors read and approved the final manuscript. references 1. karimi p, islami f, anandasabapathy s, et al. gastric cancer: descriptive epidemiology, risk factors, screening, and prevention. cancer epidemiol biomarkers prev 2014;23:700-13. doi: 10.1158/1055-9965.epi-13-1057. 2. ferlay j, shin hr, bray f, et al. estimates of worldwide burden of cancer in 2008: globocan 2008. int j cancer 2010;127:2893–917. doi: 10.1002/ ijc.25516. 3. van cutsem e, sagaert x, topal b, et al. gastric cancer. lancet 2016; 388: 2654-64. doi: 10.1016/ s0140-6736(16)30354-3. 4. soerjomataram i, lortet-tieulent j, parkin dm, et al. global burden of cancer in 2008: a systematic analysis of disability-adjusted life-years in 12 world regions. lancet 2012; 380: 1840–50. doi: 10.1016/s0140-6736(12)60919-2. 5. maguer-satta v, besancon r, bachelard-cascales e. concise review: neutral endopeptidase (cd10): a multifaceted environment actor in stem cells, physiological mechanisms, and cancer. stem cells 2011;29:389–96. doi: 10.1002/stem.592. 6. oba j, nakahara t, hashimoto-hachiya a, et al. cd10-equipped melanoma cells acquire highly potent tumorigenic activity: a plausible explanation of their significance for a poor prognosis. plos one 2016;11:e0149285. doi: 10.1371/journal.pone.0149285. 7. fukusumi t, ishii h, konno m, et al. cd10 as a novel marker of therapeutic resistance and cancer stem cells in head and neck squamous cell carcinoma. br j cancer 2014;111:506-14. doi: 10.1038/bjc.2014.289. 8. salibay cj, rewerska j, gupta s, et al primary carcinosarcoma of the pancreas with cd10positive sarcoma component. j investig med high impact case rep 2017;5:2324709617740906. doi: 10.1177/2324709617740906. 9. murakami t, yao t, mitomi h, et al. clinicopathologic and immunohistochemical characteristics of gastric adenocarcinoma with enteroblastic differentiation. gastric cancer 2016;19:498-507. doi: 10.1007/s10120-015-0497-9. 47 10. ohnishi n, takata k, miyata-takata t, et al. cd10 down expression in follicular lymphoma correlates with gastrointestinal lesion involving the stomach and large intestine. cancer sci. 2016;107:1687-95. doi: 10.1111/cas.13031. 11. huang wb, zhou xj, chen jy, et al. cd10positive stromal cells in gastric carcinoma: 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2015;89:329-36. doi: 10.1016/j.lungcan. 2015.06.003. univ med vol. 38 no.1 17. zhu y, zheng jj, yang f, et al. expression of cd10 in cancer-associated fibroblasts and its effect on initiation and progression of colorectal carcinoma. chin j pathol 2016;45:859-65. doi: 10.3760/cma.j. issn.0529-5807.2016.12.009. 18. taghizadeh-kermani a, jafarian ah, ashabyamin r, et al. the stromal overexpression of cd10 in invasive breast cancer and its association with clincophathologic factors. iran j cancer prev 2014;7:17-21. 19. puri v, jain m, thomas s. stromal expression of cd10 in invasive breast carcinoma and its correlation with er, pr, her2-neu, and ki67. int j breast cancer 2011, article id 437957, 4 pages. doi: http://dx.doi.org/10.4061/2011/437957. 20. kaur m, verma s, gupta r, et al. cd10 expression pattern in prostatic adenocarcinoma: elucidation of differences between gleason’s grades. malays j pathol 2018 40:57-60. 21. kim dh, shin n, kim gh, et al. mucin expression in gastric cancer: reappraisal of its clinicopathologic and prognostic significance. arch pathol lab med 2013;137:1047–53. doi: 10.5858/arpa.2012-0193-oa. c:\users\user\documents\39 no 2 81 *department of emergency medicine, health science university, antalya training and research hospital, antalya, turkey **department of radiology, burdur state hospital, antalya, turkey ***department of radiology, health science university, antalya training and research hospital, antalya, turkey †department of emergency medicine, karaman state hospital, karaman, turkey correspondence: cihan bedel health science university antalya training and research hospital, kazim karabekir street postal zip code: 07100, muratpasa, antalya, turkey phone: +905075641254 orcid id: 0000-0002-3823-2929 date of first submission, march 9, 2020 date of final revised submission, june 24, 2020 date of acceptance, june 27, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina computed tomography and magnetic resonance cholangiopancreatography in the assessment of acute pancreatitis mustafa korkut*, cihan bedel1*, kürsat erman**, alpaslan yavuz***, sinan ulgen***, and ali avci† background acute pancreatitis (ap) is a disease associated with sudden onset of abdominal and back pain in a band-like pattern, nausea, and vomiting. in patients with ap, the gold standard, i.e., the initial imaging modality to be used is ultrasonography (us). however, in cases where evaluation is insufficient, computed tomography (ct) and magnetic resonance cholangiopancreatography (mrcp) are other modalities to be applied. in this study, we aimed to demonstrate the diagnostic accuracy of ct and mrcp in patients with acute pancreatitis. methods seventy-five patients diagnosed with acute pancreatitis were evaluated using ct and, as well as patients who underwent contrast-enhanced mrcp within 48 hours. ct and mrcp were examined in terms of the biliary tract and pancreatic duct structure, gallbladder, common bile duct stones and complications. chi-square test and the mann-whitney u test were used to analyze the data at significance level of p<0.05. results of all the included patients, 44 were male (58.7%), and the mean age was 59.27 ± 17.37 years. in ct findings, the percentage of ap complication findings such as pancreatic necrosis, extrapancreatic complications, the pancreatic or peripancreatic fluid collection was significantly higher than the mrcp group (p<0.05). mrcp showed biliary tract findings such as cholelithiasis, gallstone, common bile duct dilatation, intrahepatic bile duct dilatation, choledochal stone and pancreatic duct dilatation at a higher percentage than ct. conclusion mrcp was superior to ct in gallbladder and choledochal stone and biliary tract dilatation but not in ap complications. therefore, non-invasive and radiation-free mrcp can be used more widely in ap patients, especially in emergency departments. keywords: acute pancreatitis, computed tomography, magnetic resonance cholangiopancreatography original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.81-87 may-august, 2020 vol.39no.1 cite this article as: korkut m, bedel1 c, erman k, yavuz a, ulgen s, avci a. computed tomography and magnetic resonance cholangiopancreatography in the assessment of acute pancreatitis. uni v med 202 0 ;3 9 :8 1 -7 . doi: 10.18051/univmed.2020.v39.81-87 82 korkut, bedel, erman, et al ct and mrcp in patients with acute pancreatitis introduction acute pancreatitis (ap) is a reversible acute inflammatory condition, accompanied by suddenonset of abdominal and back pain in a band-like pattern, nausea, and vomiting, where local tissue or organ systems may also be affected.(1) the annual incidence of ap is about 5–80/100,000 people, and the most common etiological causes are alcohol and gallstones.(2) amylase and lipase are the most commonly used biochemical parameter s. trypsin, phospholipase-a, carboxypeptidase-a, serum elastase-1 and lipase isoforms are also used for diagnosis.(3) radiological examinations are used in ap to determine both the etiology of the disease and local complications occurring in the treatment and follow-up processes of the disease.(4) the gold standard in patients with ap, i.e., the initial imaging modality to be used, is ultrasonography (us).(5) computed tomography (ct) examination with intravenous contrast agent is the initial imaging modality applied in emergency departments, especially in cases where us is not sufficient for evaluation. computed tomography is a more useful method than us both for diagnosis and demonstration of ap complications, but ct often fails to evaluate the biliary tract and gallbladder and is currently replaced by other imaging modalities due to exposure to high radiation and contrast agent.(6) on the other hand, with the advances in technology, the biliary tract can be easily monitored by rapid magnetic resonance imaging and thus, can serve as a guide for diagnosis and treatment. therefore, non-invasive and r adiation-free magnetic re sonance cholangiopancreatography (mrcp) has been increasingly used in examinations of the biliary tract and pancreatic duct.(7) magnetic resonance cholangiopancreatography has the unique capability of providing noninvasive images of the pancreatic ducts and can demonstrate possible communication of a pancreatic pseudocyst with pancreatic ducts.(8) in order to recognize the cause and complications of ap, especially in emergency departments, it is important to exclude alternative causes of abdominal pain and to evaluate the degree of acute pancre atitis as wel l as ct imaging for preoperative planning.(9,10) although both imaging methods have advantages and disadvantages to each other, the use of mrcp in patients with ap is highly limited in emergency departments. thus, in this study, we aimed to evaluate the diagnostic performance and superiority of mrcp and ct, compared to each other, in the ap evaluation. methods research design a retrospective study was conducted in health science university, antalya training and research hospital, antalya, turkey between january 2016 and december 2018. research subjects during the study period a total of 274 patients suffering from ap, who applied to our emergency department were assessed for eligible into the study. patients who are younger than 18 years, pregnant, have acute, chronic pancreatitis exacerbation, have ap-related malignancy or are transferred to another hospital, those who have not performed cts in the emergency room or mrcp in 48 hours in the hospital or those with incomplete data were excluded from the study. a total of 199 patients were excluded from the study after exclusion criteria. consequently seventyfive patients complying with these criteria were selected for this study. all subjects underwent contrast-enhanced abdominal tomography (ct), as well as patients who underwent contrast enhanced mrcp within 48 hours of hospitalization for the diagnosis of ap. all images were evaluated separately by the radiologist. abdominal tomography and mrcp were performed in all patients included in the study. clinical variables demographic characteristics, prognosis, length of hospital stay, ranson scores at admission, laboratory parameters {white blood 83 cell, amylase, lipase, alanine transaminase (alt) and aspartate transaminase (ast)} of the patients were recorded. balthazar scores were calculated from a to e according to the necrosis and fluid collection in the pancreas. mild pancreatitis grouped as balthazar score (a, b, c); severe pancreatitis grouped as balthazar score (d, e). contrast-enhanced abdominal computed tomography abdominal tomography of the cases included in the study was performed with eclos 16 slice ct scanner (hitachi medical systems, tokyo, japan). the scanning area was the area located between the diaphragm and the iliac crest and the images were taken with a collimation section thickness of 0.5 mm at kvp 120, 150-200 mas, reconstruction interval of 0.3 mm, fov width (30 cm), pitch value 1-1.5, window width 350 (200600) and window level: 50 (30-60). all patients received the contrast agent at a rate of 3ml/sec from the antecubital vein, and images were taken within a single breath-hold period of 65 seconds. none of the patients developed allergic reactions during the administration of the contrast agent. intra-extrahepatic biliary tract, pancreatic parenchyma, pancreatic duct, surrounding fat planes and other possible pathological findings of the patients were examined (figure 1-2). in the pancreas, parenchymal lesion, pancreatic duct enlargement, peripancreatic adipose tissue contamination, and fluid presence were examined and the diameter (width) of pancreatic duct was measured at its widest point. abdominal tomography and mrcp imaging findings of the patients were compared. magnetic resona nce cholangiopancreatography mrcp was performed on all patients on days 0-2 following hospitalization with 1.5 tesla philips achieva mr device (best, t he netherlands). hd 8 channel body array coil was used. images were first obtained with a breath-hold axial 2d fiesta sequence, and then, data were obtained from breath-triggered, coronal oblique 3d t2-weighted fast spin-echo (frfse – xl) sequence, followed by thick-slab, breathhold t2 ssfse (haste) sequence. besides, breath-hold ssfse (haste) thin-section coronal oblique images were obtained. univ med vol. 39 no.2 figure 1: axial enhanced ct shows that multipl acute necrotic collection in pancreatic bed (asterisks) figure 2: axial enhanced ct shows that edematous pancreatitis with bowel involvement (arrow) figure 3: coronal t2w mri shows that common bile duct is dilated with mild intrahepatic duct dilatation (asterisk). filling defect within the distal common bile duct keeping with choledocholithiasis (arrow) 84 korkut, bedel, erman, et al ct and mrcp in patients with acute pancreatitis a b c the parameters of the mrcp sequences used are given in table 1. only intravenous contrast agent was used during the examination. the resulting sections were examined in terms of the biliary tract and pancreatic duct structure, gallbladder, common bile duct stones and complications (figure 3). statistical analysis the statistical analysis of all variables was made using spss version 23.0. continuous variables were expressed as mean ± standard deviation in the analysis of data. frequency and percentage (%) were used for categorical data. pearson’s chi-squared test was used to evaluate the variables. in the comparison of the positive findings in ct and mrcp groups, student’s ttest was used f or va riables with normal distribution, and the mann-whitney u test was used for variables without normal distribution. the value of p<0.05 was considered statistically significant. ethical clearance this study was approved by the ethics committee antalya training and research hospital under no.2020/135 and patients signed a consent form before inclusion to the study results seventy-five patients who met the inclusion criteria were included in the study. of all the included patients, 44 were male (58.7%), and the mean age was 59.27 ± 17.37. patients were divided into two groups according to their imaging findings: positive on ct and positive on mrcp. although the mean age was higher in the ct group compared to mrcp, it was not statistically significant (p=0.792). there was no significant difference between the groups in terms of gender (p=0.621). in ct findings, the percentage of ap complication findings such as pancreatic necrosis, extrapancreatic complications, the pancreatic or peripancreatic fluid collection was significantly higher than the mrcp group (p<0.05). biliary results such as cholelithiasis, gallstone, common duct dilatation, intra-hepatic biliary duct (ihbd) dilatation, choledochal stone and pancreatic duct dilatation had a higher percentage in mrcp compared to ct (p <0.05 for all parameters). no significant differences were found in serum wbc, amylase, lipase, ast, alt and calcium levels between the groups. demographic data, imaging findings and laboratory values of the groups are compared in table 2. discussion the present results show that mrcp is a reliable alternative to ct for assessing ap. while mrcp is superior to ct in cases such as cholelithiasis, gallstone, common duct dilatation, ihbd dilation, choledochal stone and pancreatic dilatation; ct was superior in complications such table 1: magnetic resonance sequence parameters using in mrcp 2d fiesta: two-dimensional fast imaging employing steady state acquisition, frfse-xl: fast recovery fast spin echoaccelerated, ssfse: single-shot fast spin-echo, haste: half-fourier single-shot turbo spin-echo, tr: repetition time, te: echo time, fov: field of view axial 2d fiesta coronal oblique t2-weighted 3d frfse-xl thick-slab t2-weighted ssfse (haste) thin slice, coronaloblique t2-weighted ssfse (haste) tr (msec) 428 1324 466 1324 te (msec) 80 700 80 700 matrix 276x242 256x256 268x232 256x256 slice thickness (mm) 5 37 5 2,5 gap (mm) 0 0,3 0,3 0 fov(mm) 385 252 375 260 number of signals acquired 1 1 2 1 flip angle 90 90 90 85 as pancreatic necrosis, extr apancreatic complications, pancreatic or peripancreatic fluid collection. unlike our study, it was found in a study that ct showed bile and choledochal stone as effectively as mrcp, it was reported that it was not so effective in showing the peripancreatic fluid collection.(11). in another study, similar to our findings, it was emphasized that ct is a superior imaging method for pancreatic necrosis and extrapancreatic complications than mrcp.(9) radiological imaging methods are of great importance in the evaluation of patients with ap or biliary problems admitted to the emergency department. therefore, although us is the first choice as it is both easily accessible in almost all emergency departments and fast, there are some limitations, the first one of which is its administrator -dependent natur e and low sensitivity. besides, although it can easily detect dilatation in the biliary tract, it may be insufficient for determining the cause.(12) even though ercp is the gold standard for biliary imaging in these examinations, ct and mrcp stand out as an alternative to us due to the need for experienced personnel, being invasive and the low possibility of emergency application.(13) univ med vol. 39 no.2 different reasons play a role in the etiology of ap and gallstone and alcohol accounts for approximately 90% of these cases. other most f r e qu e n t e ti o l ogi c c a u se s ar e t r a u ma , hyperlipidemia, drugs, endoscopic retrograde chol angi ography or surgi cal procedures.(14) another study reports that ap is most frequently diagnosed in the sixth decade and alcohol, gallbladder stones and idiopathic causes account for the etiology. this study also indicates that alcohol is the primary cause in european countries.(15) a study conducted in turkey reports that gallbladder stones are diagnosed in 72% of patients etiologically with mild ap, while the figure increases to 58% in patients with severe ap.(16) in our study, the most common causes were found to be gallbladder stones and idiopathic causes. magnetic resonance imaging is sensitive to detect subtle changes of ap; especially minor peripancreatic inflammatory changes; even in a morphologically normal pancreas state in ct imaging; it may appear normal in 15% to 30% of patients with clinical features of ap. the sensitivity of magnetic resonance imaging exceeds the sensitivity of ct imaging and table 2. comparison of demographic characteristics and imaging findings between ct and mrcp data presented as mean ± sd, percentage and range, a:mann-whitney u test b: chi-square test; ast : aspartate transaminase; alt : alanine transaminase ct findings positive mrcp findings positive p value age (years)a 61.39 ± 18.05 60.4 2± 17.78 0.792 gender; male (%)b 29 (38.7) 33 (44.6) 0.621 presence of cholelithiasis n(%) b 9 (12) 11 (14.7) 0.328 gallstoneb 18 (24) 27 (36) <0.001 common bile duct dilatationb 20 (26.7) 23 (30.7) 0.003 ihbd dilatationb 19 (25.3) 21 (28) 0.043 choledochal stone a 8 (10.7) 22 (29.3) 0.022 pancreatic duct dilatationb 11 (14.7) 14 (18.7) 0.033 ranson scorea balthazar scoreb 1.59 ± 1.07 1.49 ±1.08 0.368 0.014 mild pancreatitis (a, b, c) 61 (81.3) 57 (76) severe pancreatitis (d, e) 14 (18.7) 18 (24) pancreatic necrosisb 17 (22.7) 5 (6.7) <0.001 extrapancreatic complicationsb 14 (18.7) 8 (10.7) 0.005 pancreatic or peripancreatic fluid collectionb 28 (37.3) 17 (22.7) <0.001 white blood cell a 12.54 ± 5.07 12.13 ± 4.81 0.74 alt a 75.5 (8-791) 80 (8-791) 0.449 ast a 63.5 (18-578) 69 (18-683) 0.682 calcium a 8.75 ± 0.94 8.77 ± 0.78 0.977 amylase a 444 (153-4673) 438 (53-4622) 0.635 lipase a 598 (59-8157) 585 (66-9626) 0.528 86 korkut, bedel, erman, et al ct and mrcp in patients with acute pancreatitis emphasizes its role in the evaluation of patients with clinically suspected ap. (17) as ct can conveniently demonst rate bile ducts and surrounding tissue, it can quickly determine metastasis or vascular invasion in malignant cases.(18) ct images of gallstones, especially cholesterol stones, may sometimes be impossible to detect depending on their chemical contents. in a recent study, chemical analyzes of stones were compared and cholesterol-containing stones were reported to be isodense with bile, while those with high calcium content were reported to be dense.(19) another recent study indicates that ct examination of ap patients within the first 72 hours is useful in the diagnosis of pancreatic necrosis, but the examinations within the first 24 hours may be false negative.(20) similarly, our study revealed that ct was superior to mrcp in detecting complications, but we think that ct, together with mrcp, may be able to determine the baltazar score at a higher rate in patients with severe ap. magnetic resonance cholangiopancreatography will soon change the approach to patients presenting with biliary obstruction that is because it can totally replace the diagnostic role of ct. noninvasive and radiation-free mrcp imaging method should be used more and more in emergency departments in the examination of the biliary tract. ou r s t ud y i s a si n gl e -c e n te r e d , retrospective study, which constitutes the most important limitations of our study. failure to ascertain the minute of applying ct and mrcp imaging methods and interpretation of images by a single radiologist are other limitations. besides, the effects of clinic, symptoms and additional diseases of patients on mortality are not included in the study, which is another limitation. it is suggested that future studies should evaluate mrcp is a superior radiological imaging method in ap patients, especially in emergency departments. further well-designed prospective studies enrolling larger populations are needed. conclusion magnetic resona nce cholangiopancre atogr aphy was superior to ct in gallbladder and choledochal stone and biliary tract dilatation but not in ap complications. therefore, non-invasive and radiation-free mrcp can be used more widely in ap patients, especially in emergency departments. conflict of interest the authors declare that there are no conflicts of interest regarding the publication of this manuscript. acknowledgement special thanks to the antalya training and research hospital radiology department for their support. contributors all authors contributed to compilation of the subject matter, writing of the manuscript drafts, data collection, and data analysis. cb and mk contributed to manuscript finalization. ke and aa contributed to the study concept and design. cb, mk and ke contributed to manuscript revision. all authors have read and approved the final manuscript. references 1. crockett sd, wani s, gardner tb, falck-ytter y, barkun an, american gastroenterological association institute clinical guidelines committee. american gastroenterological association institute guideline on initial management of acute pancreatitis. gastroenterology 2018;154:1096-101. doi: 10.1053/j.gastro.2018.01.032. 2. tsuji y, takahashi n, isoda h, et al. early diagnosis of pancreatic necrosis based on perfusion ct to predict the severity of acute pancreatitis. j gastroenterol 2017;52:1130-9. doi: 10.1007/s00535017-1330-5. 87 resonance cholangiopancreaticography (magnetic resonance imaging). int j sci stud 2015; 3:149-53. doi: 10.17354/ijss/2015/325. 13. chen w, mo jj, lin l, li cq, zhang jf. diagnostic value of magnetic resonance cholangiopancreatography in choledocholithiasis. world j gastroenterol 2015;21:3351-60. doi: 10.3748/ wjg.v21.i11.3351. 14. leppäniemi a, tolonen m, tarasconi a, et al. wses guidelines for the management of severe acute pancreatitis. world j emerg surg 2019;13:1427. doi: 10.1186/s13017-019-0247-0. 15. roberts se, morrison-rees s, john a, williams jg, brown th, samuel dg. the incidence and aetiology of acute pancreatitis across europe. pancreatology 2017;17:155-65. doi: 10.1016/j.pan.2017.01.005. 16. önmez a, bilir e, torun s. the relationship between acute pancreatitis severity of platelet lymphocyte ratio, neutrophil lymphocyte ratio, erythrocyte distribution width and mean platelet volume. konuralp med j 2019;11:24-9. doi: 10.18521/ktd.492274. 17. busireddy kk, alobaidy m, ramalho m, et al. pancreatitis-imaging approach. world j gastrointest pathophysiol 2014;5:252-70. doi:10.4291/wjgp.v5.i3.252. 18. khanna ak, meher s, prakash s, et al. comparison of ranson, glasgow, moss, sirs, bisap, apacheii, ctsi scores, il-6, crp, and procalcitonin in predicting severity, organ failure, pancreatic necrosis, and mortality in acute pancreatitis. hpb surg 2013; article id 367581, 10 pages. http://dx.doi.org/10.1155/2013/36758. 19. vege ss, dimagno mj, forsmark ce, martel m, barkun an. initial medical treatment of acute pancreatitis: american gastroenterological association institute technical review. gastroenterology 2018;154:1103-39. doi: 10.1053/ j.gastro.2018.01.031. 20. yadav ak, sharma r, kandasamy d, et al. perfusion ct: can it predict the development of pancreatic necrosis in the early stage of severe acute pancreatitis? abdom imaging 2015;40: 48899. doi: 10.1007/s00261-014-0226-6. univ med vol. 39 no.2 3. staubli sm., oertli d, nebiker ca. laboratory markers predicting severity of acute pancreatitis. crit rev clin lab sci 2015;52:27383. doi: 10.3109/10408363.2015.1051659. 4. ural koç, ýsa çam. revised atlanta classification and roadmap of radiologist türk radyoloji derg 2017;36:46-51. doi: 10.5152/turkjradiol.2017.532. 5. williams e, beckingham i, el sayed g, et al. updated guideline on the management of common bile duct stones (cbds). gut 2017;66:765-82. doi: 10.1136/gutjnl-2016-312317 6. mcgillicuddy ea, schuster km, brown e maxfield mw, davis ka, longo we. acute cholecystitis in the elderly: use of computed tomography and correlation with ultrasonography. am j surg 2011;202:524-7. doi: 10.1016/j.amjsurg.2011.06.012. 7. mandelia a, gupta ak, verma dk, sharma s. the value of magnetic resonance cholangiopancreatography (mrcp) in the detection of choledocholithiasis. j clin diagn res 2013;7:1941– 45. doi: 10.7860/jcdr/2013/6158.3365. 8. grassedonio e, toia p, grutta ll, et al. role of computed tomography and magnetic resonance imaging in local complications of acute pancreatitis. gland surg 2019;8:123-32. doi: 10.21037/gs.2018.12.07. 9. sawarkar k, shaha p, narayan r, et al. role of computed tomography in predicting severity of acute pancreatitis and its correlation with clinical outcome. int j contemp med res 2019;6:10-7. doi:10.21276/ijcmr.2019.6.2.45. 10. mir ma, bali bs, mir ra, et al. assessment of the severity of acute pancreatitis by contrastenhanced computerized tomography in 350 patients. ulus travma acil cerrahi derg 2013;19: 103–8. doi: 10.5505/tjtes.2013.12080. 11. zhang j, li n, huang b, et al. the value of performing early non-enhanced ct in developing strategies for treating acute gallstone pancreatitis. j gastrointest surg 2016;20: 604–610. doi: 10.1007/s11605-0153066-y. 12. kushwah a, jain s, agarwal r, tomar s. biliary tract obstructive diseases: a comparative evaluation by ultrasonography and magnetic oktavianus 184 *department of biotechnology medical faculty and health sciences, jenderal soedirman university, purwokerto **department of anatomy , medical faculty and health sciences, jenderal soedirman university, purwokerto ***department of soil science, agriculture faculty of jenderal soedirman university, purwokerto correspondence dr. lantip rujito, m.si.med. department of biotechnology medical faculty and health sciences jenderal soedirman university jl. gumbreg no 1 purwokerto 53146 email: l.rujito@unsoed.ac.id univ med 2012;31:184-91 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background lead accumulation in the blood widely known affecting the formation of heme and oxygen transport processes in vital organs, leading to organ failure including the brain synapses. lead affinity has been recognized influenced by constitutional genotype of aminolevulinate dehydrogenase (alad), which encodes for heme synthesis. this research aimed to determine the relationship between plumbum (pb) and short term memory on each alad gene genotyping (alad 1-1, alad 1-2 or alad 2-2) in gas station workers. methods seventy six probands from gas station workers were recruited to participate in this research. each probands was carried out alad genotyping using polymerase chain reaction-restriction fragment length polymorphism (pcrrflp) method, lead serum level using atomic absorbent spectrophotometer (aas), and short term memory was measurement by intelligence structure test (ist). results proportion of δ alad 1-1, 1-2, and 2-2 were 91.8%, 8.2% and 0% respectively. lead serum showed 15.84 ppb in homozygous 1-1, and 20.79 ppb in heterozygous. short term memory in the probands varied from 85 until 117, with average in 99.71. there was significant negative relationship between lead serum and short term memory (r=-0.24; p=0.038). however, we could not find any significant correlation in each δ alad genotypes. conclusion the δ alad genotypes did not modified the relationship between serum lead level and short term memory in gas station workers. keywords: lead, short term memory, ä alad, gas station workers aminolevulinate dehydrogenase polymorphisms did not modified lead serum and memory relationship lantip rujito*, arini dewi setyowati**, and saien*** 185 univ med vol. 31 no.3 polimorfisme aminolevulinate dehidrogenase tidak memodifikasi hubungan antara kadar timbal dan memori abstrak pendahuluan akumulasi timbal (pb) dalam darah mempengaruhi proses transportasi heme dan oksigen pada organ-organ vital, termasuk aktivitas sinaps di otak. afinitas pb dalam darah dipengaruhi oleh konstitusi genotip gen ä aminolevulinate dehydrogenase (alad), yang mengkode protein untuk sintesis heme. penelitian ini bertujuan untuk mencari hubungan antara menentukan adanya hubungan antara kadar pb dan memori jangka pendek disetiap genotipe gen ä alad (alad 1-1, 1-2 alad atau alad 2-2 pada pekerja pompa bensin. metode tujuh puluh enam responden dari pekerja pompa bensin direkrut untuk berpartisipasi dalam penelitian ini. setiap subyek dilakukan genotyping δ alad menggunakan metode polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp). kadar serum timbal diukur menggunakan atomic absorbent spectrophotometer (aas), sedangkan pengukuran memori jangka pendek menggunakan intelegence scoring test (ist). hasil proporsi genotip δ alad 1-1, 1-2 dan 2-2 masing-masing adalah 91,8%, 8,2% dan 0%. rerata pb serum menunjukkan angka 15,84 ppb pada subyek homozigot δ alad 1-1, dan 20,79 ppb pada subyek heterozigot. memori jangka pendek responden bervariasi mulai dari 85 sampai 117, dengan rerata 99,71. terdapat hubungan negatif yang bermakna antara kadar serum pb dengan memori jangka pendek (r=-0,24; p=0,038), tetapi tidak ada korelasi yang signifikan untuk setiap genotip ä alad. kesimpulan genotip ä alad tidak dapat memodifikasi hubungan antara kadar pb dalam serum dan memori jangka pendek pada pekerja pompa bensin. kata kunci: timbal, memori jangka pendek, ä alad, pekerja pompa bensin h e a v y m e t a l p o i s o n i n g b e c o m e s government worry especially in environmental issues. latent effect and its potential to decrease human resources quality have attracted much concern. nowadays, heavy metal contamination is increasing because of the development of industry, the increase of fuel use, the household modernization, and the change of environment support ability.(1) data showed that heavy metal poisoning symptoms like lead or plumbum (pb) were growing in some countries. usa government showed the fact that 17% household equipments content hazardous pb. (2) in china, it was reported that about 53.7% citizens who were screened had blood lead level above the threshold,(3) and mostly in children. (4) indonesia is also a risky country, moreover in the big cities with its pollution cases. world bank record showed the impact of lead air contamination in indonesia caused 350 cases of heart disease, 62.000 cases of hypertension, and 340 deaths per year.(5) augmentation of pollution in big city l i k e j a k a r t a r e a c h e d 3 6 . 6 % a b o v e t h e 186 threshold.(6) lead contamination in indonesia is caused by high consumption of fuel. from 1996, there are more than 9 million kiloliters fuels used with 7% annual growth. it’s about 36 million kiloliters of fuels in 2010 burns into gas in indonesia.(7) the escalating of blood lead level can c a u s e m u l t i e ff e c t t o m a n y o rg a n s l i k e cardiovascular system, hemolymphatic, urinary tract, immune system, reproductive system, and neural system.(8) neural system is the most sensitive system for lead intoxication.(9) lead over accumulation cause neuron and brain damage,(10,11) and also autism.(12) other research showed that lead poisoning more than 4 years could cause cancer and disturb cognitive functioning.(13) short term memory is one of the f a c t o r s t h a t a f f e c t c o g n i t i v e f u n c t i o n . mechanism of short term memory involves transient modification in the function of preexisting synapses. it is saved in the modification of specific ca2+ channels.(14) lead has ability to substitute ca2+ in general neurotoxic mechanism that can disturb short term memory aptitude.(15) in addition, lead can give rise to interact with aminolevulinate dehydrogenase (alad) enzyme which disturbs heme synthesis, lacking in brain function if it is occurred in long phase.(8) alad enzyme itself has three genotyping: homozygous alad 1-1, homozygous alad 1-2, and heterozygous alad 1-2 causing by substitution of nitrogen base g into c in position 177 (g177c).(16) lead is a potent inhibitor of alad in the biosynthesis of heme.(17) this research aimed was to determine the relationship between pb and short term memory on each alad gene genotyping (alad 1-1, alad 1-2 or alad 2-2). methods research design the observational study using cross sectional design was used in this research and conducted from october to november 2011. research subjects seventy six gas station workers from seven gas stations in banyumas were recruited to participate in this research. inclusion criterias for the subjects were javanese etnicity, age range from 20-60 years old, and have been a worker in gas station for minimum 1 year. as an exclution parameters, subject must be free from kidney disease history, and having no medication treatment for at least 1 month. informed consent and interview were taken individually to collect the basic information about identity and medical history. blood lead measurements blood lead measurements were performed b y a q u a n t i f i e d u s i n g a t o m i c a b s o r b e n t s p e c t r o p h o t o m e t e r ( a a s ) o n 1 7 , 0 n m wavelength. alad genotyping about 10 cc venous blood was drawn from each probands. δ alad genotyping was performed using polymerase chain reactionrestriction fragment length polymorphism (pcr-rflp) method with the following primer f o r w a r d a n d p r i m e r r e v e r s e u s e d : 5 ’ agacagacattagctcagta-3’ and 5’ggcaaagaacaggtccattc-3’. pcr profile was as follow: initial denaturation 95oc for 30’’, amplicon multiplication with 30 cycles (denaturation 95oc for 30’’, annealing 57oc for 30’’, and synthesis 72oc for 30’’), and polishing step in temperature 72oc for 5’ and 26oc for 10’’. primer product then was cut with rflp technique using msp1 enzyme.. assessment of memory function short term memory measurement was using intelligence structure test (ist) subtest t h a t d o n e b y a p s y c h o l o g i s t . i s t i s a n intelligence test constructed for subjects aged 14-60 years. the ist is an intelligence test battery that measures verbal, numerical and figural intelligence. rujito, setyowati, saien aminolevulinate dehydrogenase polymorphisms 187 univ med vol. 31 no.3 figure 1. genotyping of ä alad polymorphism pattern in gel electrophoresis. digestion of pcr product using msp1 result two band available, 582 and 511 bp reflecting alad 1 and alad 2 alleles statistics analysis descriptive statistics was used to calculate the frequencies and means of variables, including lead serum levels, short term memory score, ä alad gene polymorphism, sex, age, smoking habit, and alcohol consumption data. the spearman correlation was used to test the relationship between lead serum levels and short t e r m m e m o r y s c o r e s w i t h p < 0 . 0 5 w a s considered as statistically significant. computer program stata was used for the data analysis. ethical clearance this study was performed after getting ethical clearance from ethic committee and conducted inline with helsinski declaration. results a total of 76 subjects were recruited into this study, with a mean age was 33.22 ± 10.25 years and most of gas station workers (81.6%) are male. there was also a tendency that most of gas station workers were having smoking habit (60%) but not consuming alcohol (93.4%). mean blood lead level of all subjects was 14.16 ± 17.51 ppb and the mean score of short term memory was 99.97 ± 9.77. the results showed 45 probands (91.8%) had homozygous alad 1-1 and only 4 probands (8.2%) had heterozygous alad 1-2. there was no proband that recorded with homozygous alad 2-2 (table1). however, 27 subjects could not be determined due to technical issues, then we excluded these subjects for further analysis the pattern of alad gene polymorphism was presented in figure 1. the serum lead levels in workers with the alad 1-1 genotype were lower than those with the alad 1-2 genotype, and the difference was not statistical significance (p=0.595). table 1. characteristics of study participants (n=76) ¶ mean ± sd 188 table 2 represents the relationship between lead serum level and short term memory score in all subjects and subgroups. the results indicated that there was a significant negative correlation between the serum lead level with s h o r t t e r m m e m o r y ( r = 0 . 2 4 ; p = 0 . 0 3 8 ) . however, there was a different result when we tested the relationship between lead serum level and short term memory in each genotype of ä alad. the study also reported that there were no significant relationship on different sex, age group, smoking habit, and alcohol consumption (table 2). there were no significant relationship between serum lead level and short term memory in subjects <40 years old and in >40 years old worker group, the coefficient correlation were r=-0.16 (p=0.271) and r=-0.12 (p=0.581) respectively. the relationship between serum lead level and short term memory in smoking and in non smoking worker were not statistically significant. discussion lead is well known material that affects many organs, including the nervous system. in this research, the effect of lead towards short term memory was investigated in gas station workers. the ä alad gene polymorphism was conducted as a modifier effect. the results showed that most of gas station workers (81.6%) are male. there was also a tendency that most of gas station workers were having smoking habit (60%) but not consuming alcohol (93.4%). all female workers were not smoking and not consuming alcohol. the previous research on gas station worker in purwokerto didn’t give the detail about the demographic characteristics such as sex, smoking habit, or alcohol consumption.(18) the result showed that 45 probands (91.8%) had homozygous alad 1-1 and only 4 probands (8.2%) had heterozygous alad 1-2. there was no probands that recorded with homozygous alad 2-2. this result was consistent with previous researches. yang et al.[16] found that in caucasian, allele alad-1 (88%) was greater than allele alad-2 (12%). kamel et al.(11) also showed 78% of the respondent had alad 1-1, 20% of alad 1-2, and only 2% had alad 2-2. this result showed that serum lead level serum was lower in worker with alad 1-2 than alad 1-1. it was also consistent with previous research that the person with alad 1-2 and alad 2-2 had higher susceptibility to have higher degree of serum lead level than alad 11.(19-22) *significant p<0.05 table 2. correlations between serum lead level and short term memory in all subjects and subgroup rujito, setyowati, saien aminolevulinate dehydrogenase polymorphisms 189 univ med vol. 31 no.3 it is increasing the fact that alad plays an important role in the bioaccumulation of lead; however it is still an open question for research. there are two established scenarios to explain these evidences. first, alad (particularly alad -2) act as the distributor of lead in target organs such as kidneys and brain structures. second, it is possible that alad can serve as a sink, separate from the interaction of lead in blood. alad-2 could serve as a substrate with high affinity, lead levels are maintained in blood and protecting other organs. alad-2 could serve as a highaffinity substrate, retaining lead in the blood and therefore protecting other organs. in this case, people who have the alad-2 allele could experience less severe effects of lead on kidney and brain and lesser accumulation of lead in bone while at the same time having higher blood lead levels than alad-1 homozygote exposed to the same doses of lead .(23) it may be also possible that alad-2 allele boost the retention of lead in blood and decreases the amount of chelatable lead. treatment with dmsa (chelator agent of lead), therefore, would not be as effective in alad-2 carriers. other investigators found that alad genotype may influence kidney function, which may also affect excretion of lead. (24) the short term memory score mean was 99.97. based on ist score categorization, the short term memory score mean for gas station worker was low. this low level of short term memory score could be affected by a lot of factor, such as genetic, intellectual stimulation, physical activities, nutrition, and also toxic exposure. this study showed a significant negative relationship between serum lead level and short term memory in all subjects. it was a weak relationship. but this relationship could give a reason why even low level of lead in the blood could affect cognitive function. kosnett(25) already showed that accumulation dose of lead could be a predictor in poor performance of cognitive test in adult. it could be explained from atkinsonshiffrin model about short term memory. short term memory is important in learning process because information should be stored in short term memory first before it could be stored in long term memory which bigger in capacity.(26) other significant relationship showed between serum lead level serum and short term memory in non alcohol consumer. it was assumed that the significant relationship could happen because almost all subjects are non alcohol consumer. this study showed that there was no significant relationship found in the analysis between lead level serum and short term memory in alad 1-1 and alad 1-2. the results might happened because the small number of sample that analyzed in the group. it was statistically insignificant since proband with alad 1-1 only 45 and even in alad 1-2 only 4. it was c o n s i s t e n t w i t h l i d s k y a n d s c h n e i d e r statement.(15) the insignificant relationship between lead level serum and short term memory in male in our study was inconsistent with weisskopf(27) and payton et al (28) studies. weisskopf showed that low blood lead level could worse cognitive test result in elderly men. payton et al also found that men with higher levels of blood and bone lead copied spatial figures less accurately. the relationship in female worker also showed insignificant relationship. it was consistent with weuve study that relation only significant between bone lead and cognitive performance, not with blood lead level.(29) smoking and age were two factors that could affect short term memory. it was said that smoking could affect cardiovascular and respiratory systems so it could hamper the oxygen and nutrition to the brain. the age itself affected the brain function since neural cell b e c a m e l e s s o p t i m a l i n o l d e r p e o p l e . ( 3 0 ) however, there were no significant result when lead level serum and short term memory relationship in different group age and different smoking habit. inabilities to record and control genetic factor that affect brain function, especially in short term memory may be important factor that could affect this study. 190 one limitation of this study was the lack of statistical power and the consequent inability to reproduce associations in the present study to a larger population. another limitation was the observational nature of the study, making confounding a concern. conclusions there was significant relationship between lead serum level and short term memory. this study also showed that lead serum level in gas station worker with alad 1-2 was higher than alad 1-1. however there was no modification effect of ä alad genotypes on the relationship between serum lead level and short term memory among gas workers. the present findings as well as lead toxicity complications will be 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lead poisoning – a case report. indian j clin biochem 2007;22:162-3. 24. chia se, zhou h, tham mt, yap e, viet dong n, hong t, et al. possible influence of äaminolevulinic acid dehydratase polymorphism and susceptibility to renal toxicity of lead: a study of a vietnamese population. environ health perspect 2005;113:1313–7. 25. kosnett mj. health effects of low dose lead exposure in adults and children, and preventable risk posed by the consumption of game meat harvested with lead ammunition. in: watson rt, fuller m, pokras m, hunt wg, editors. ingestion of lead from spent ammunition: implications for wildlife and humans. boise, idaho: the peregrine fund;2009.p.24-33. 26. atkinson rl, atkinson rc, hilgrad er. pengantar psikologi. edisi kedelapan jilid 1 jakarta: penerbit erlangga;2005. 27. weisskopf mg, proctor sp, wright ro, schwartz j, spiro iii a, sparrow d, et al. cumulative lead exposure and cognitive performance among elderly men. epidemiol 2007;18:59-66. 28. shih ra, hu h, weisskopf mg, schwartz bs. cumulative lead dose and cognitive function in adults: a review of studies that measured both blood lead and bone lead. environ health perspect 2007;115:483–92. 29. weuve j, korrick sa, weisskopf ma, ryan lm, schwartz k, et al. cumulative exposure to lead in relation to cognitive function in older women. environ health perspect 2009;117:574-80. 30. suprenant am, neath i, brown gda. modeling age-related differences in immediate memory using simple. j mem lang 2006;55:572-86. c:\users\universa medicina\docu 42 *clinical pathology department, medical faculty, jenderal soedirman university **bioethics and humaniora department, medical faculty, jenderal soedirman university correspondence: vitasari indriani clinical pathology department, medical faculty, jenderal soedirman university gumbreg street no 1, purwokerto, central java indonesia email: vita.indriani@gmail.com orcid id: 00000001-8563-1781 date of first submission, december 11, 2 01 8 date of final revised submission, march 31, 2020 date of acceptance, april 1, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina duration of diabetes as an important risk factor of microalbuminuria in type 2 diabetes vitasari indriani*, tri lestari*, and vidya dewantari** background microalbuminuria is the earliest evidence of diabetic nephropathy and a major predictor of end stage renal disease (esrd). the objective of the study was to determine the influence of several risk factors on the presence of microalbuminuria in type 2 diabetics. methods this observational cross-sectional study was done on 73 patients with type 2 diabetes, who attended the prolanis program in primary health care from may to november 2018. detailed medical histories including duration of diabetes and relevant clinical examinations including fasting blood sugar (fbs), post-prandial blood sugar (ppbs), hba1c, serum creatinine, blood urea and urinary microalbumin were recorded for each patient. a multiple regression analysis was used to analyze the data. the analysis was assessed at 5% level of significance. results mean age of study population was 51.89 ± 6.78 years with female preponderance (51.1%). mean fbs, ppbs, hba1c, duration of diabetes, systolic blood pressure, microalbuminuria and serum creatinine was 182.51 ± 74.63 mg/dl, 186.25 ± 26.72 mg/dl, 8.8 ± 1.83%, 9.37 ± 5.96 years, 118.44 ± 4.13 mmhg, 30.32 ± 3.2 mg/day and 1.33 ± 0.64 mg/dl respectively. duration of diabetes and hba1c were positively correlated with microalbuminuria (β=0.052; beta =0.367; p<0.001 and β=0.058; beta=0.363; p<0.001) respectively. conclusions duration of diabetes was the most important risk factor of microalbuminuria in type 2 diabetes patients. therefore microalbuminuria can predict diabetic nephropathy earlier, as a warning to prevent further worsening of diabetic complications. keywords: diabetic nephropathy, hba1c, microalbuminuria, type 2 diabetes original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.42-46 c it e t h is ar t ic le a s: in d ri a ni v, lestari t, dewantari v. duration of d ia betes as a n import an t ri sk factor of microalbuminuria in type 2 diabetes. univ med 2020;39:426. d oi : 10.18051/univmed.2020. v38 .42-46. january-april, 2020 vol.39no.1 43 introduction diabetes mellitus (dm) is a metabolic disorder characterized by persistent hyperglycemia resulting from defects in insulin secretion, action or both. chronic hyperglycemia initiates microvascular complications including nephropathy, retinopathy and neuropathy. poor glycemic control plays an important role in the development and progression of nephropathy with associated increase in morbidity and mortality. (1) the presence of albuminuria is an indicator of renal impairment. microalbuminuria, defined as an elevation of urinary albumin excretion in the range of 30-300 mg/24 hr or 20-200 µg/min, is associated with adverse health outcomes in adults. (2) microalbuminuria is strongly associated with endothelial dysfunction, which increases the risk of nephropathy and other complications in diabetes. (3) glycosylated hemoglobin (hba1c) has been used as a measure of long-term glycemic control and has proven to be a more accurate and stable measure than fasting blood sugar level (fbs) and post-prandial blood sugar (ppbs).(4)an annual assessment of kidney function by the determination of urinary albumin excretion and of hba1c for glycemic control is recommended for patients with diabetes.(5) chen et al. (6) identified the relationships of high normal albuminuria and glycemic control on microalbuminuria development among type 2 diabetes patients. previous studies had revealed that various factors such as glucose level(7), glycemic variability(8) and insulin resistance impact development of microalbuminuria in type 2 diabetes. (9) in the present study the population comprised patients with type 2 diabetes mellitus attending the primary health care system under the chronic disease management program (prolanis). only fbs and ppbs were checked every month. since increased risk of nephropathy must be detected earlier, further correlations between the various factors were investigated in our study. the present study was performed to determine the relationship of hba1c with microalbuminuria by considering the following factors: age, gender, fbs, ppbs, blood pressure, blood urea level, serum creatinine level and duration of diabetes. methods research design an observational study of cross-sectional design was done in the prolanis program in primary health care (fktp klinik tanjung purwokerto) from may 2018 to november 2018. research subjects an observational study and a randomized sample of 73 clinically diagnosed cases of type 2 diabetes aged >18 years. based on a previous finding that serum creatinine was positively correlated with microalbuminuria (r=0.33) (10) and using =0.05, =0.20, and drop-out rate of 20%, the total calculated sample was 73 subjects thirty seven female and thirty six male clinically diagnosed cases of type 2 diabetes who attended the prolanis program were included. patients suffering from acute and chronic inflammatory conditions, pre-existing chronic kidney disease, glomerulonephritis, nephrotic syndrome, smokers, alcoholics, patients on nephrotoxic drugs, and primary hypertensives were excluded from the study. medical records of the patients comprising age, gender, duration of diabetes, glycated hemoglobin, blood urea, serum creatinine, and urinary microalbumin levels were analyzed for each patient. laboratory analysis blood samples were collected after 10-12h of fasting. taking all aseptic and antiseptic precautions, 5 ml of blood was drawn from the antecubital vein and a 24-hour urine sample was collected in a sterilized container. the following measurements were done: fbs, ppbs, blood urea, serum creatinine by enzymatic method (dimension exl 200), hba1c measured by clover a1c self with boronate affinity method, and microalbuminuria by fluorescent immunoassay (fia) method (ichroma microalbumin). statistical analysis all analyses were performed using ibm spss version 20. results on continuous measurements were presented as mean ± standard deviation (sd) and results on categorical measurements as numbers (%). a determine the relationship between several risk factors and microalbuminuria. the analysis was assessed at 5% level of significance. 44 table 1. distribution of characteristics and laboratory parameters of patients with type 2 diabetes *data presented as mean ± sd, except age in % ethical clearance the study has been approved by the health research ethics commission (komisi etik penelitian kesehatan, kepk), medical faculty, jenderal soedirman university, under no. 1792/kepk/iv/2018. all subjects were informed of the study protocol in written and verbalform and provided informed consent. results the mean age of the subjects was 51.89 ± 6.78 years, and most subjects were female (51.1%). the mean serum creatinine was 1.33 ± 0.64 mg/dl, mean duration of diabetes was 9.37 ± 5.96 years, mean hba1c was 8.8 ± 1.83 (table 1). discussion our study showed that duration of diabetes was the most potential risk factor of microalbuminuria. a similar result was shown by a study involving 227 subjects with type 2 dm in kathmandu, in that diabetes duration was the most important risk factor for the development of microalbuminuria in diabetes. (11) another study showed consistent results in that duration of diabetes was an independent risk factor for microalbuminuria.(12) microalbuminuria is the most sensitive marker of early recognition of diabetic nephropathy. monitoring microalbuminuria and other risk factors associated with this condition is important to take measures to prevent or postpone overt nephropathy.(13) more importantly, tight glycemic control with insulin during the microalbuminuria stage has been shown to prevent the development of overt diabetic nephropathy.(14) the level of hba1c has been widely accepted as an indicator of mean daily blood glucose concentration over the preceding 8-12 weeks. our study also found a significant relationship between hba1c and microalbuminuria. bhoomika et al. (15) studied risk factors for microalbuminuria in type 2 diabetes mellitus patients and reported significantly high hba1c levels in patients with microalbuminuria. another study also highlights that there was a significant correlation between microalbumin levels and hba1c in diabetic type 2 subjects. (16) however, another study in iran did not find a significant association between hba1c and microalbuminuria.(12) table 2. multiple linear regression analysis of several risk factors and microalbuminuria beta p value age (years) 0.946 0.086 0.033 gender 0.876 0.112 0.064 systolic blood pressure (mmhg) 0.270 0.081 0.034 diastolic blood pressure (mmhg) 0.278 0.085 0.043 diabetes duration (years) 0.052 0.367 <0.001* fbs (mg/dl) 0.045 0.091 0.059 ppbs (mg/dl) 0.48 0.086 0.033 hba1c (%) 0.058 0.363 <0.001* blood urea (mg/dl) 0.054 0.087 0.059 serum creatinine (mg/dl) 0.048 0.076 0.023 multiple linear regression showed that diabetes duration was the most potential risk factor of microalbuminuria ( beta=367; p<0.001) followed beta=0.363; p<0.001) parameter diabetic type 2 patients (n=73) age (yr) gender (%) male female fasting blood sugar (mg/dl) post prandial blood sugar (mg/dl) systolic blood pressure (mmhg) diastolic blood pressure (mmhg) hba1c (%) microalbuminuria (mg/day) serum creatinine (mg/dl) blood urea (mg/dl) duration of diabetes (yr) 51.89 ± 6.78 36 (48.9%) 37(51.1%) 182.51 ± 74.63 186.25 ± 26.72 118.44 ± 4.13 84.44 ± 19.25 8.8 ± 1.83 30.32 ± 3.2 1.33 ± 0.64 43.2 ±15.0 9.37 ± 5.96 45 relationship of duration of type 2 diabetes mellitus with microalbuminuria and high hba1c level has been reported. chronic hyperglycemia is responsible for the pathogenesis of diabetic nephropathy. the possible mechanism is that hyperglycemia leads to glycation of virtually all proteins, resulting in the formation of advanced glycation end products. the interaction of advanced glycation end products and their receptors and increased activity of the polyol pathway have been implicated as mediators of increased microvascular permeability, ischemia and angiogenesis.(16) baig et al.(17) studied 60 patients and concluded that in patients with type 2 diabetes mellitus, long duration of diabetes and poor glycemic control significantly correlated with high level of microalbumin. another study reported that higher grades of hba1c are associated with higher severity of microalbuminuria and so can be used as a marker of good glycemic control.(18) microalbuminuria usually comes out 5-15 years after the diagnosis of type 2 diabetes mellitus. combination of glomerular hypertension and glomerular hyperperfusion the remaining nephrons. these changes occur in the absence of uremia. in later stages the glomerular filtration rate (gfr) decreases with the appearance of macroproteinuria along with clinical signs of nephropathy and finally leads to end stage renal disease (esrd).(19) liu et al. (3) and kondaveeti et al.(20) also reported that good glycemic control was the strong influencing factor which played a key role in the transition of non-microalbuminuric subjects to microalbuminuric. several limitations were encountered in this study. first, the study was not populationbased and only patients who presented at diabetes centers were included. this may have introduced referral bias and it would therefore be difficult to extend our findings to the general population of diabetes patients. second, we have also not examined the role of hba1c variability on microvascular complication development, and we had difficulties in determining the duration of diabetes. third, the cross-sectional nature of the study design limits the reliability of the observed associations between risk factors and diabetic nephropathy. because microalbuminuria is widely used as a sensitive risk marker to identify those at risk for renal dysfunction, screening programs should be implemented at an early stage to prevent or postpone end-stage renal disease (esrd). so, it may be suggested that determination of microalbumin levels in urine is an easy method of screening diabetic patients, especially diabetic patients with long-term diabetes. further well-designed prospective studies enrolling larger populations are warranted. conclusions duration of diabetes and hba1 level were important risk factors of microalbuminuria in patients with type 2 diabetes, with duration of diabetes being the most important. . conflict of interest there is no conflict of interest in their report. acknowledgement special thanks are due to prolanis program in primary health care and medical faculty of jenderal soedirman university for financial support. contributors vi was responsible for writing of the article. tl was responsible for submission of the article. vd was the advisor. all authors have read and approved the final manuscript. references 1. kumar r, abbas a, fausto n, aster jc, eds. robbins and cotron pathologic basis of disease. 8th ed. philadelphia: saunders elsevier publishing division; 2010. 2. lee jm, eason a, nelson c, et al. screening practices for identifying type 2 diabetes in adolescents. j adolesc health 2014;54:139 43. 3. liu z, fu c, wang w, xu b. prevalence of chronic complications of type 2 diabetes mellitus in outpatients a cross-sectional hospital based survey in urban china. health qual life outcomes 2010;8:62. doi: 10.1186/ 1477-7525-8-62. 4. jun m, perkovic v, cass a. intensive glycemic control and renal outcome. contrib nephrol 2011;170:196 208. doi: 10.1159/000325664. 46 5. haller h, ito s, izzo jr jl, et al. olmesartan for the delay or prevention of microalbuminuria in type 2 diabetes. n engl j med 2011;364:907 17. doi: 10.1056/nejmoa1007994. 6. chen wz, hung cc, wen yw, ning hc, gau br, huang yy. effect of glycemic control on microalbuminuria development among type 2 diabetes with high-normal albuminuria. ren fail 2014; 36: 171 5. doi: 10.3109/0886022x. 2013.832312. 7. murussi m, campagnolo n, beck mo, gross jl, silveiro sp. high-normal levels of albuminuria predict the development of micro and macroalbuminuria and increased mortality in brazilian type 2 diabetic patients: an 8-year follow-up study. diabet med 2007;24:1136 42 8. hsu cc, chang hy, huang mc, et al. hba1c variability is associated with microalbuminuria development in type 2 diabetes: a 7-year prospective cohort study. diabetologia 2012; 55:3163 72. doi 10.1007/s00125-012-2700-4. 9. hsu cc, chang hy, huang mc, et al. association between insulin resistance and development of microalbuminuria in type 2 diabetes: a prospective cohort study. diabetes care. 2011;34:982 7. doi: 10.2337/dc10-1718. 10. gupta m, singh jp. correlation of microalbuminuria with glycosylated haemoglobin in patients of diabetes having nephropathy. int j adv med 2017;4:805-8. doi: http://dx.doi.org/10.18203/2349-3933. ijam20172276. 11. thakur sk, dhakal sp, parajuli s, sah ak, nepal sp, paudel bd. microalbuminuria and its risk factors in type 2 diabetic patients. j nepal health res counc 2019;17:61-5. doi: 10.33314/ jnhrc.1620. 12. ansar mm, shahrokhirad r, lebady mk. risk factors of microalbuminuria and macroalbuminuria in type 2 diabetic patients in north of iran rasht. nephrourol mon 2017; 9:e40031.doi: 10.5812/numonthly.40031. 13. pasko n, toti f, strakosha a, et al. prevalence of microalbuminuria and risk factor analysis in type 2 diabetes patients in albania: the need for accurate and early diagnosis of diabetic nephropathy. hippokratia 2013;17: 337-41. 14. parchwani dn, upadhyah aa. diabetic nephropathy: progression and pathophysiology. int j med sci public health 2012; 4: 7-11. doi: 10.5455/ijmsph.2012.1.59-70. 15. dadhania bp, aravat ah, dhruva ga. study of microalbuminuria in type 2 diabetes patients as a marker of morbidity (a study of 100 cases in rajkot city). jbar 2012;3:763-6. 16. kundu d, roy a, mandal t, bandyopadhyay u, ghosh e, ray d. relation of microalbuminuria to glycosylated hemoglobin and duration of type 2 diabetes. niger j clin pract 2013;16:216-20. 17. baig ja, asif n, sarfaraz a, alam jm. correlation of microalbuminuria with glycosylated hemoglobin (hba1c) and duration of type 2 diabetes mellitus (t2dm) in male and female patients. middle-east j sci res. 2016; 24:2900-3. 18. bagzai a, bagzai ds. correlation between severity of microalbuminuria with hba1c in type ii diabetic patients. jmscr2019;7:310-3 19. sabatini s, kurtzman na. role of hyperfiltration in the pathogenesis of diabetic nephropathy. in: advances in pathogenesis of diabetic nephropathy. new york: nova science publishers, inc; 2012.p.:1-27. 20. kondaveeti sb, kumaraswamy d, mishra s, kumar ar, shaker ia, evaluation of glycated albumin and microalbuminuria as early risk markers of nephropathy in type 2 diabetes mellitus. j clin diagn res 2013;7: 1280 3. doi: 10.7860/jcdr/2013/5145.3117. oktavianus 51 department of biochemistry, faculty of medicine, diponegoro university, semarang correspondence dr. kusmiyati tjahjono dk department of biochemistry, faculty of medicine, diponegoro university, semarang e-mail : kusmiceria@gmail.com univ med 2013;32:50-8 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 background the prevalence of diabetes in indonesia is increasing due to various factors, including life style changes such as trans fatty acid (tfa) intake. high tfa intake is known to be related to blood lipid profile changes resulting in cardiovascular disorders. this study was to identify the effect of tfa on nitric oxide (no) production and on necrosis of pancreatic beta cells. methods a study of randomized pre-test post–test design with control group. thirty sprague dawley rats were divided into 3 groups, i.e. group k (control), group p1 receiving a diet with 5% tfa, and p2 receiving 10% tfa. the intervention was performed for 8 weeks. no level and pancreatic beta-cell necrosis were analyzed using pearson’s chi square test. results after 4 weeks of treatment there was no change in no levels in group k, but increased no in p2 (2.6-3.8 ì m). at 8 weeks after treatment, no levels in groups p1 and p2 increased to 2.6-3.4 ìm and 4.2-14.3 ìm, respectively, while in group k only 2 rats had increased no levels of 2.8-2.9 ìm. with pearson’s chi-square test, there was a signifant difference in the proportions of necrotic pancreatic beta cells after 4 weeks and 8 weeks (p= 0.000). no necrosis of beta cells was found in group k, mild necrosis in group p1 (1-25%) and moderate necrosis in group p2 (26-50%). conclusion tfa consumption significantly increases no levels in sprague dawley rats and also results in moderate grades of necrosis of pancreatic beta cells. keywords : trans fatty acids, nitric oxide levels, necrosis of pancreatic beta cells trans fatty acids increase nitric oxide levels and pancreatic beta-cell necrosis in rats kusmiyati tjahjono dk*, santoso*, and dwi ngestiningsih* 52 tjahjono, santoso, ngestiningsih trans fatty acids and nitric oxide asam lemak trans meningkatkan kadar oksida nitrit dan neksrosis sel beta pancreas pada tikus latar belakang prevalensi diabetes melitus di indonesia semakin tahun semakin meningkat. asupan tinggi asam lemak trans selama ini dihubungkan dengan memburuknya profil lipid, yang berakibat terjadinya penyakit kardiovaskuler. tujuan penelitian ini untuk membuktikan asupan tinggi asam lemak trans terhadap stres oksidatif, produksi oksida nitrit (no), dan nekrosis sel beta pankreas. metode penelitian ini merupakan penelitian eksperimental laboratorik dengan randomized pre-test post –test control group design dengan rancangan acak lengkap. tiga puluh tikus spargue dawley dibagi dalam 3 kelompok kontrol (k), kelompok perlakuan (p1) 5% asam lemak trans, kelompok perlakuan (p2) dengan pakan 10%, perlakuan diberikan selama 8 minggu. kadar no dianalisis dengan uji pearson-chi square, nekrosis sel beta pankreas dengan analisis deskriptif hasil pengukuran kadar no diklasifikasikan menjadi kelompok dengan kadar no < 2,5 ìm, dan meningkat bila > 2,5 ìm. kelompok kontrol tidak terdapat perubahan, p1 terdapat peningkatan pada 1 tikus (2,6) ìm, p2 terjadi peningkatan pada semua tikus (2,6-3,8 ) ìm. setelah 8 minggu, kelompok k, 2 tikus mengalami peningkatan (2,8-2,9) ìm, p1 terdapat peningkatan pada semua tikus (2,6-3,4) ìm, p2 terjadi peningkatan pada semua tikus (4,2-14,3). pada p1 dan p2 dengan uji beda pearson chisquare, setelah 4 minggu dan 8 minggu terdapat perbedaan proporsi yang bermakna (p=0,000). kelompok k tidak dijumpai nekrosis sel beta pankreas, p1 semua tikus mengalami nekrosis sel beta pankreas dengan derajat ringan (1-25%) dan p2 dengan derajat sedang (26-50%). kesimpulan terjadi peningkatan kadar no secara bermakna pada p1 dan p2 dan terjadi nekrosis derajat ringan pada p1 dan derajat sedang pada p2. kata kunci: asam lemak trans, nitric oxide, nekrosis sel beta pankreas abstrak introduction the prevalence of diabetes mellitus (dm) is steadily increasing worldwide. in 2009 there were 285 million patients with dm and for the year 2030 the prevalence is projected to increase to 438 juta patients, 60% of whom will be asians. epidemiologically, type 2 dm may be found in developed as well as developing c o u n t r i e s . (1 , 2) t h e p r e v a l e n c e o f d m i n indonesia is estimated to be up to 5.7% of the total population.(3) one of the risk factors for dm is the changing dietary pattern, from the traditional diet to a western diet, including increased consumption of fast foods.(3,4) the results of various studies have revealed that currently a large amount of fast foods is consumed by indonesian communities. fast foods are not only high in calories and fats but also high in trans fatty acids (tfa).(5) 53 trans fatty acids are naturally present in small amounts in products from ruminants, such as beef, milk, cheese and butter.(6) most of the t fa c o m e s f r o m t h e p r o c e s s i n g o f c i s unsaturated fatty acids which are abundantly found in nature. in indonesian communties the m a j o r s o u r c e s o f t fa a r e a v a r i e t y o f hydrogenated plant oil products, such as margarine, shortening, hydrogenated vegetable oils (hvo) and other products processed with industrially hydrogenated fats, such as breads, packaged snacks (chips, cereals and biscuits) and deep fried foods. these ready-made products are in high demand in the communities, because they are tasty, crispy, practical, not easily becoming rancid, and easily transported, since they are in semi-solid form.(5,7) currently tfa are attracting attention because they have more negative effects on health in comparison with saturated fatty acids, formerly considered to be bad for health.(8) thus far, studies have shown that high intakes of tfa have a negative effect on the lipid profile, which is a predictor of the development of atherosclerosis (9) and a major cause underlying cardiovascular disease (cvd).(10,11) theoretically a high tfa intake may cause oxidative stress, resulting in the formation of intracellular reactive oxygen species (ros), (12,13) thus causing cellular damage and dysfunction.(12-15) one of the ros compounds formed by oxidative stress is nitric oxide or nitrogen monoxide (no).(12,16) no is an important intracellular signaling and messenger molecule in humans. no in normal concentrations is important for protecting organs against ischemia. on the other hand chronic expression of no may kill healthy host cells, particularly in chronic inflammation, and may be associated with the development of degenerative disease. mitochondria play a role in no-induced cell death. this occurs through inhibition of respiration and reactive nitrogen s p e c i e s ( r n s ) i n d u c e d m i t o c h o n d r i a l permeability transition (mpt), thus resulting in depletion of adenosine triphosphate (atp), ultimately leading to necrosis. production of no in large amounts may also cause dna damage, have toxic effects, and cause necrosis.(17) similar processes presumably take place in pancreatic beta cells as a result of high tfa intake. the present study used high dosages of tfa of 5% and 10% of total calories, with reference ot previous studies, where at these dosages tfa may result in nutritional metabolic disturbances.(18) the limit for tfa intake based on studies up to date is 1% of total calories. the pathophysiology of high tfa intake is still controversial and unclear. the objective of the present study was to evaluate the effect of administration of high dosages of tfa on no levels, as a result of oxidative stress, and in connection with the hypothesis of an in vitro study that increased no levels causes necrosis of pancreatic beta cells (17,19) in male sprague dawley rats as experimental animals. methods research design this a true experimental laboratory study, with completely randomized pre-test post-test design with control group. simple randomization was done using a computer. the study was conducted in 2012 at the integrated research and development laboratory of the preclinical r e s e a r c h s e r v i c e f o r d e v e l o p m e n t o f experimental animals (lpptlp3hp), gajah mada university, yogyakarta. animals and experimental protocol t h i s s t u d y w a s c o n d u c t e d o n t h e e x p e r i m e n t a l a n i m a l s f o r 8 w e e k s , w i t h reference to the study conducted by dorfman18 on the effect of tfa on nutrient metabolism in liver and adipose tissues using male sprague dawley rats with the following inclusion criteria: i) aged 7 weeks, ii) weighing 200-295 grams, iii) being in healthy condition, actively moving, and without anatomical abnormalities, iv) not suffering from weight loss in the adaptation period, and v) having an initial fasting blood glucose of <110 mg/dl. the exclusion criteria univ med vol. 32 no.1 54 tjahjono, santoso, ngestiningsih trans fatty acids and nitric oxide were: i) animals suffering from diarrhea during the intervention period, marked by unformed stools, ii) weight changes of >10% in the adaptation period, and iii) rats dying during the study period. all rats were obtained from the i n t e g r a t e d r e s e a r c h a n d d e v e l o p m e n t laboratory of the preclinical research service for development of experimental animals ( l p p t l p 3 h p ) , g a j a h m a d a u n i v e r s i t y, yogyakarta. this study was conducted on sprague dawley rats, comprising 3 groups. group k was the control group receiving standard feed, group p 1 was the first intervention group receiving rat feed pellets containing tfa corresponding to 5% of the total daily energy requirement, and group p 2 was the second intervention group receiving rat feed pellets tfa corresponding to 10% of the total daily energy requirement. calculation of the sample size was based o n t h e w h o g u i d e l i n e s o n t h e u s e o f experimental animals for evaluation of the safety and effectiveness of herbal medications,(20) with a mininum of 5 animals per group and including a control group. the calculation was also based on a previous study conducted by dorfman,(18) using 8 sprague dawley rats per group. the present study used 10 rats per group. care of experimental animals the rats were placed in individual cages in a well-ventilated at a temperature between 28 32oc, with cycles of 12 hours light and 12 hours darkness, and were acclimatized to adapt them to the weather and environment. the cages were cleaned daily. the rats were daily given pelleted feed and water ad libitum. the pelleted feed was produced by research diet, nj (usa).(18) assessment of no levels no levels were assessed indirectly by s p e c t r o p h o t o m e t r y, f o l l o w e d b y n i t r i t e quantitation using griess’ reagent at an absorbance of 450 nm. no measurements were classified into a group without increased no levels (<2.5 ì m ), and a group with increased no levels (>2.5 ì m ). histopathological examination of pancreatic â-cells after completion of the interventions, the sprague dawley rats were terminated in accordance with the principles set forth in the medical ethics manual 2005.(21) the rats were anesthesized with ether by group and terminated by cervical dislocation. the pancreas was extracted from the abdomen, weighedpan, and processed into paraffin blocks. the blocks were m a d e i n t o h e m a t o x y l i n e e o s i n ( h e ) histopathologic preparations. from each slide five fields were examined at 400x magnification for necrotic pancreatic â-cells. the criteria used for determining the grade of necrosis were according to the study by matveyenk et al.(22) the necrosis was considered to be severe when more than 50% of the â-cells were necrotic. in diabetes a more severe grade of necrosis (>65%) was expected. the criteria to be used in this case was according to a study conducted at the bogor agricultural institute (institut pertanian bogor, ipb).(23) the necrosis was considered moderate when there was necrosis in 26-50% of â-cells and mild when there was necrosis in 1-25%, of â-cells, while in normal rats there are no necrotic â-cells (0%). data analysis the collected primary data were tabulated for further analysis. the p1 and p2 data were analyzed using statistical difference tests (pearson’s chi square). data on necrosis in cells are on the categorical (ordinal) scale and therefore the pearson chi-square test was used to find differences in proportions between groups. ethical clearance ethical clearance for this study was issued under no. 173/ec/fk/rsdk/2011 by the commission on medical research ethics, 55 faculty of medicine, diponegoro university, semarang. results initially all rats had no levels of 2.5 ì m, and after a four-week intervention no changes in no levels were found in the control group. the pearson chi-square test for differences b e t w e e n p r o p o r t i o n s f o u n d s i g n i f i c a n t differences in no levels in the intervention groups at the end of the fourth and eighth weeks, with p=0.000. n e c r o s i s o f p a n c r e a t i c â c e l l s o n histopathologic examination on histopathologic examination of the pancreas at the end of the intervention period no necrosis was found in the control cells (group k). in group p1 all rats had mild necrosis of the pancreatic â-cells (1-25%), while in group p2 all rats had moderate necrosis of the pancreatic â-cells (26-50%). the results of pearson’s chi-square test showed a significant difference between p1 table 1. between-group differences in proportions of no levels at weeks 0, 4, and 8 *pearson chi-square test table 2. between-group difference in proportions of necrosis in pancreatic â =cells *pearson’s chi square test and p2 with a p value of 0.000. the proportions of necrotic events by group is presented in table 2 and the difference in necrosis is shown graphically in figure 1. discussion tfa and no levels nitric oxide (no) or nitrogen monoxide is an important signaling molecule in humans and an important messenger molecule in the cell. no in normal concentrations is important for protecting the organs against ischemia, on the other hand chronic and excessive no expression may result in the death of healthy cells,(24) particularly in chronic inflammation and i n c o n n e c t i o n w i t h t h e d e v e l o p m e n t o f degenerative diseases such as cancer and diabetes. chronically raised no levels induce inhibition of mitochondrial respiration, through rns-induced mitochondrial permeability transition (mpt), thus causing depletion of atp and ultimately cell necrosis. production of large amounts of no may also lead to dna damage, toxic effects, and necrosis.(17) univ med vol. 32 no.1 56 tjahjono, santoso, ngestiningsih trans fatty acids and nitric oxide a (a) no necrosis in islets of langerhans (x400) b (b) mild (10%) necrosis in islet of langerhans as indicated by cells without nuclei (x 400) (c) moderate (20%) necrosis in islet of langerhans as indicated by cells without nuclei (x 400) c d (d) moderate (30%) necrosis in islet of langerhans as indicated by many cells without nuclei (x 400) (e) moderate (40%) necrosis in islet of langerhans as indicated by scarcity of cells (x 400) e figure 1. necrosis of pancreatic â-cells (arrows indicate cell nuclei) 57 a t p h y s i o l o g i c a l c o n c e n t r a t i o n s (approximately 1-100 nm), no tends to be nonreactive and most of its physiological functions is mediated by no bound to fe2+ by guanylate cyclase causing no activation and cgmp production. no may be converted into many derivatives, designated reactive nitrogen species (rns). at high concentrations in the cell or the cell, no reacts directly with oxygen producing no 2 , which subsequently reacts with no form n 2 o 3 . the results of this study show that no levels after 4 weeks was <2.5 µm in all control rats, identical with baseline values (signifying no change in no levels). in group p1 only one rat (10%) showed no levels of >2.5 ì m, while in group p2 all rats (100%) had no levels of >2.5 ì m. after 8 weeks 2 rats (20%) in the control group had no levels of >2.5 ì m (these being 2.8 and 2.9 ì m, respectively), in group p1 all rats (100%) had no levels of >2.5 ìm, as did the rats in group p2. these findings are in agreement with an in vitro study by downar et al. stating that high tfa intakes may cause oxidative stress, leading to formation of intracellular ros and may thus cause cellular damage and ultimately cellular dysfunction.(14,15) one of the ros formed by oxidative stress is no.(12,25) the study conducted by mukhopadhyay (26) in animal experiments showed that the increase in no due to oxidative stress may cause myocardial cell death. this may presumably also occur in pancreatic â-cells as a result of high tfa intakes. the study of rao et al.(27) on the nervous system has reported that free radicals produce lipid peroxides and more prominent is the formation of ros and no free radicals resulting in ischemic attacks. high tfa intakes result in the formation of ros/rns causing oxidative stress, such that it causes lipid peroxidation which triggers no formation. continuous peroxidation of lipids will cause higher increases in no levels. cells exposed to no or cells producing no show inhibition of respiration in cytochrome oxidase that occurs rapidly but is reversible. after exposure to no, an irreversible i n h i b i t i o n o c c u r s , m a d e p o s s i b l e b y t h e c o n v e r s i o n o f n o t o r n s , w h i c h c a u s e irreversible inhibition of respiration in other enzymes. the resulting peroxynitrites are strong antioxidants and can cause dna damage, induce lipid peroxidation, produce oxidants and cause death of cells, including pancreatic â-cells. for the abovementioned reasons, high tfa intakes may cause pancreatic â-cell necrosis through no formation. trans fatty acids and pancreatic â-cell necrosis necrosis is cell death, with karyolysis and severe damage to the cell membrane (resulting in loss of membrane integrity). in addition, the lysosomes release enzymes that enter the cytoplasm and cause damage the cell’s own organelles, resulting in irreversible necrosis. cell necrosis cause an efflux of cellular contents through the damaged plasma membrane and thus result in an inflammatory reaction. necrosis of pancreatic â-cells may be caused by a variety of factors, such as genetics, infection, toxic diabetogenic substances, oxidative stress and nutrition. tfa are among the factors capable of causing pancreatic â-cell necrosis. on histopathological examination of pancreatic tissues in the present study, no pancreatic â-cell necrosis was found at the end of the eighth week in the control group k (0.0% necrosis). however, in group p1 all rats had pancreatic â-cell necrosis in varying degrees, viz. 6 rats had 5% necrosis and 4 had 10% necrosis, both of which are considered to represent mild necrosis. in group p2, 3 rats had mild necrosis (20% necrosis), while 1 rat had 30% necrosis and 6 rats had 40% necrosis, both being categorized as moderate necrosis. the cause of pancreatic â-cell necrosis in this study as a result of the high tfa intakes presumably occurs through inflammation (leading to crp production) and oxidative s t r e s s . b o t h i n d u c e n o s y n t h e s i s , t h u s univ med vol. 32 no.1 58 tjahjono, santoso, ngestiningsih trans fatty acids and nitric oxide increasing no levels. oxidative stress in group p1 with administration of 10% tfa causes more severe cell necrosis in comparison with group p2 with administration of 5% tfa. previous studies by szmitko et al.(15) and malhi (28) have advanced the lipotoxicity theory, stating that cell death may be due to abnormal l i p i d a c c u m u l a t i o n . f r e e f a t t y a c i d s a r e hydrophobic and therefore able to pass the cell membrane directly or through fatty acid transport protein (fatp) or fatty acid transporter cd36. saturated fatty acids may increase apoptosis (programmed cell death) and thus may cause cell damage and ultimately cellular dysfunction. however, in this study no apoptosis was found, possibly because tfa are more toxic than free fatty acids and thus did not cause programmed cell death, which is a physiological process to kill unwanted cells. the study of stumfold et al.(29) focussed o n a n e x p l a n a t i o n o f â c e l l n e c r o s i s b y gl ucot oxi city, as glucose metabolism in pancreatic â-cells is oxidative and thus causes ros formation which in turn damages the âcells. pancreatic â-cells have limited amounts of catalase and superoxide dismutase for neutralizing ros. ros induces the production of nf-êb which causes the proapoptotic effect of hyperglycemia in the pancreas and decreases the expression of the duodenal homeobox-1 gene. this is a regulator of insulin transcription and causes abnormalities in the final stages of insulin exostosis. the pancreatic â-cell necrosis occurring in the present study may presumably be produced by an inflammatory process through the activation of cytokines such as il-6 and tnf-á or through induction of crp production. these processes occur continuously because the administration of high tfa feed was conducted daily, and thus continuously inducing no production. another mechanism of â-cell necrosis in this study is through increased ros/ rns free radical production, resulting in oxidative stress which in turn triggers lipid peroxidation a n d t h u s i n c r e a s i n g n o p r o d u c t i o n a n d pancreatic â-cell necrosis. although this study did not perform an indepth analysis of the relationship between severity of tfa intake by the experimental animals on the one hand, and no production and pancreatic cell necrosis on the other hand, it may still be concluded that tfa cause increased no production and necrosis of pancreatic â-cells. conclusions administration of tfa at the higher dosage of 10% within the longer intervention period of 8 weeks reults in increased no levels and more severe necrosis of pancreatic â-cells. acknowledgements we wish to express our sincere gratitude to prof. siti fatimah muis,dr, msc, sp.gk, prof. dr. hertanto ws, dr, ms, sp.gk, and dr. ratu ayu dewi sartika apt, for their valuable guidance and advice and to all staff of the department of biochemistry, faculty of m e d i c i n e , d i p o n e g o r o u n i v e r s i t y, f o r facilitating the conduct of this study. references 1. international diabetes federation western pasific regional. plan of action (2006-2010) for the western pacific declaration on diabetes: from evidence to action. singapore: international diabetes federation western pasific regional;2008. 2. hu fb. globalization of diabetes. the role of diet, lifestyle, and genes. diabetes care 2011; 34:124957. 3. badan penelitian dan pengembangan kesehatan departemen kesehatan, republik indonesia. laporan nasional riset kesehatan dasar (riskesdas). jakarta: badan penelitian dan pengembangan kesehatan departemen kesehatan;2008. 4. krishnan s, coogan pf, boggs da, rosenberg l, palmer jr. consumption of restaurant foods 59 and incidence of type 2 diabetes in african american women. .am j clin nutr 2010;91:46571. 5. sartika rad. pengaruh asupan asam lemak trans terhadap profil lipid darah (disertasi). jakarta: fakultas kesehatan masyarakat universitas indonesia; 2007. 6. mauger jf, lichtenstein ah, ausman lm, jalbert sm, jauhiainen m, ehnholm c, et al. effect of different forms of dietary hydrogenated fats on ldl particle size. am j clin nutr 2003;78:370-5. 7. baer dj. what do we really know about the health effects of natural sources of trans fatty acids? am j clin nutr 2012;95:267-8. 8. us departement of health and human services, food and drug administration. guidance for industry: trans fatty acids in nutrition labeling, nutrient content claims, health claims; small entity compliance guide. college park: center for food safety and applied nutrition;2003. 9. lemieux i, lamarche b, couillard c, pascot a, cantin b, dagenais gr, et al. total cholesterol/ hdl cholesterol ratio vs ldl cholesterol/ hdl cholesterol ratio as indices of ischemic heart disease risk in men. arch intern med 2001;161: 2685-92. 10. roos r. atherosclerosis – an inflamatory disease. nejm 2005;340:115-26. 11. motard-bélanger a, charest a, grenier g, paquin p, chouinard y, lemieux s, et al. study of the effect of trans fatty acids from ruminants on blood lipids and other risk factors for cardiovascular disease1,2,3. am j clin nutr 2008;87:593-99. 12. downar dz, kosmider a, naruszewicz m. trans fatty acids induce apoptosis in human endothelial cells. j physiol pharmacol 2005;56:611-25. 13. chatgilialoglu c, ferreri c, lykakis in, wardman p. tran-fatty acids dan radical stress: what are the real culprits? j bmc 2006;5:52. 14. mccord jm. oxidative stress and aging: advances in basic science, diagnostics and intervention. cutler rg, rodriguez h. singapore: world scientific 2003: ii: p.883-5. 15. szmitko pe, wang ch, weisel rd, de almeida jr, anderson tj, verma s. new markers of inflamation and endothelial cell activation: part i. circulation 2003;108:1917-23. 16. hole ps, darley rl, alex tonks a. do reactive oxygen species play a role in myeloid leukemias? blood 2011;117:5816-26. 17. brown gc, borutaite v. nitric oxide, mitochondria, and cell death. iubmb life 2001; 52:189-95. 18. dorfman se, laurent d, gounarides js, li xue, mullarkey tl, rocheford ec. metabolic implication of dietary transfatty acids. obesity j org 2009;17:1200-7. 19. borutaite v, brown g. what else has to happen for nitric oxide to induce cell death. biochem soc trans 2005;33:1394-5. 20. who world health organization regional office for the western pasific. research guidelines for evaluating the safety, and efficacy of herbal medicines. 1993; available at: http://apps.who. int/medicinedocs/en/d/jh2946e/. accessed at: june 12, 2012. 21. william jr. medical ethics manual. ethics unit of the world medical association, 2005. 22. matveyenk av, velduis jd, buttler pc. mechanisms of igt and ifg induce by 50% pancreatectomy diabetes 2006;5:234-56. 23. suarsana, i nyoman. perubahan histopatologi jaringan pankreas tikus diabetes dengan pemberian ekstrak metanol tempe. repository ipb pada aktivitas hipoglikemik dan antioksidatif ekstrak metanol tempe pada tikus diabetes. 2009; available at: http://repository.ipb. ac.id/ handle/123456789/54971. accessed at: august 11, 2012. 24. bal-price a, brown gc. nitric-oxide-induced necrosis and apoptosis in pc12 cells mediated by mitochondria. neurochem. 2000;75:1455-64. 25. kuhnt k, wagner a, kraft j, basu s, jahreis g. dietary supplementation with 11transand 12trans-18:1 and oxidative stress in humans1,2,3. am j clin nutr 2006;84:981-8. 26. mukhopadhyay p, rajes m, batkai s, kashiwaya y, haskó g, liaudet l, et al. role of superoxide, nitric oxide, and peroxynitrite in doxorubicin-induced cell death in vivo and in vitro. am j physiol heart circ physiol 2009;296:h14668. 27. rao am. lipid alterations in transient forebrain ischemia: possible new mechanisms of cdp choline neuroprotection. j neurochem 2000;75: 2528-35. 28. malhi h, gores gj. molecular mechanism of lipotoxicity in fatty liver disease. semin liver dis 2008;28:360-9. 29. stumfold m, goldstein bj, van haeten tw. pathogenesis of type 2 dm. in: goldstein bj, weiland. dm type 2 diabetes principles and practice. human press 2008;13-27. univ med vol. 32 no.1 oktavianus 3 *department of pharmacy faculty of medicine and health sciences jenderal soedirman university purwokerto **faculty of pharmacy gadjah mada university yogyakarta correspondence hanif nasiatul baroroh, s.farm, apt., m.sc department of pharmacy faculty of medicine and health sciences universitas jenderal soedirman jl. dr. soeparno karangwangkal, purwokerto jawa tengah 53146 email: h_baroroh@yahoo.co.id univ med 2014;33:3-10 abstract universa medicina january-april ,2014january-april ,2014january-april ,2014january-april ,2014january-april ,2014 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 vol.33 no.1 background jatropha curcas leaves have been proven to be anti-inflammatory and antioxidant. in this study we examined the antiarthritic effects of ethanolic extract of j. curcas leaves using adjuvant induced arthritis (aia) in rats. methods male wistar rats were divided into 6 groups (n=8), consisting of normal group (0.9% nacl), control group (complete freund’s adjuvant/cfa 1 mg/ml), sodium diclofenac group at a dose of 6.75 mg/kg (p.o), ethanolic extract of j.curcas groups at doses of 150 mg/kg (p.o), 300 mg/kg (p.o) and 600 mg/kg (p.o). each group was induced by 0.2 ml cfa on day 1 and a booster injection on day 5. extracts of j. curcas were administered on days 14-28. arthritic scores were determined, then analyzed using kruskal wallis followed by mann whitney tests. mobility scores were analyzed using one way analysis of variance, followed by least significant difference multiple comparison test. arthritic joint histopathology was observed on day 29. results the results showed that the ethanolic extract of j. curcas leaves at doses of 150 mg/kg, 300 mg/kg and 600 mg/kg significantly reduced arthritis scores (p<0.05) compared to control group (cfa). the j. curcas leaf extract at doses of 150 and 300 mg/kg bw decreased mobility scores. histopathology studies showed that the j. curcas extract reduced edema and cartilage destruction in arthritic joints. conclusions the j. curcas leaf extract had anti-arthritic effects by reducing arthritis scores and mobility scores. the extract should be further examined as a potential candidate for anti-arthritic therapies. keywords : antiarthritic, jatropha curcas, adjuvant induced arthritis, rats jatropha curcas leaves exert anti-arthritic activity on adjuvant-induced arthritis in rats hanif nasiatul baroroh*, iskandar sobri*, eka prasasti nur rachmani*, triana hertiani**, and zullies ikawati** 4 baroroh, sobri, rachmani, et al jatropha curcas for arthritis daun jatropha curcas memiliki aktivitas antiartitis terhadap adjuvant-induced arthritis pada tikus latar belakang daun jarak pagar (jatropha curcas) merupakan salah satu tanaman herbal indonesia yang telah terbukti sebagai antioksidan dan antiinflamasi. penelitian ini bertujuan untuk menilai aktivitas antiartritis ekstrak etanol daun jarak pagar pada tikus dengan model adjuvant induced arthritis (aia). metode tikus wistar jantan dibagi menjadi 6 kelompok (n=8), terdiri dari kelompok normal (nacl 0,9%), kelompok kontrol (complete freund’s adjuvant/cfa), natrium diklofenak dengan dosis 6,75 mg/kg bb (p.o), dan kelompok ekstrak etanol daun j. curcas dengan dosis 150 mg/kg bb (p.o), 300 mg/kg bb (p.o) dan 600 mg/kg bb (p.o). setiap kelompok diinduksi dengan 0,2 ml cfa (1 mg/ml) pada hari ke-1 dan diberikan injeksi booster dengan 0,1 ml cfa (1 mg/ml) pada hari ke-5. ekstrak j. curcas diberikan pada hari ke 14-28. skor artritis diukur, kemudian dianalisis dengan uji kruskal wallis dilanjutkan uji mann whitney. skor mobilitas diukur dan dianalisis dengan one way analysis of variance dilanjutkan dengan least significant difference multiple comparison test. histopatologi sendi tibioarsal diamati pada hari ke-29. hasil hasil penelitian menunjukkan bahwa ekstrak etanol daun j. curcas pada dosis 150 mg/kg, 300 mg/kg dan 600 mg/ kg mampu menurunkan skor artritis secara signifikan (p<0,05) dibandingkan dengan kelompok kontrol. ekstrak j.curcas dosis 150 mg/kg dan 300 mg/kg mampu menurunkan skor mobilitas. hasil histopatologi sendi menunjukkan bahwa j.curcas mampu menghambat edema dan destruksi kartilago pada jaringan sendi. kesimpulan ekstrak j. curcas memiliki aktivitas menurunkan artritis dan mobilitas sendi sehingga perlu diteliti lebih lanjut potensinya sebagai antiartritis. kata kunci : antiartritis, jatropha curcas, adjuvant induced arthritis, tikus abstrak introduction many plants have long been recognized as important sources of therapeutically effective medicines. traditional medicine using plant extracts continues to provide health coverage for over 80% of the world’s population, especially in the developing world. recently, the use of extracts from plants for arthritis treatment has been promoted, especially after the withdrawal of food and drug administration (fda) approved anti-inflammatory drugs. one such plant that is widely used in asia but that has not been studied in a well-controlled experimental trial to date, is the species jatropha curcas.(1) j.curcas belongs to the family euphorbiaceae and is used in asian traditional medicine to cure various ailments. compounds that have been isolated from j.curcas leaves include the flavonoids apigenin and its glycosides vitexin and isovitexin, the sterols stigmasterol, 3-d-sitosterol and its 3-d-glucoside.(2) this plant is traditionally 5 univ med vol. 33 no.1 used in various disorders, such as ulcers, edema, rheumatism, pain, and as a mouthwash.(3) previous studies have reported that the plant has been shown to possess anti-inflammatory and analgesic effects that can be used as therapy in managing inflammatory conditions or as complementary therapy allowing patients to take smaller doses of conventional anti-inflammatory drugs.(4,5) previous investigations reported that ethanolic extracts of j.curcas leaves at a dose of 500 mg/kg exert their anti-inflammatory activity i n wi s t a r r a t s b y d e c r e a s i n g n e u t r o p h i l recruitment.(6) the leaves of j.curcas had antioxidant activity by inhibition of inducible nitric oxide synthase (inos).(7) arthritis is an inflammatory disorder which affects multiple joints and causes cartilage erosion. it is a lifelong progressive disease which produces significant morbidity and premature mortality. various inflammatory mediators produce joint inflammation resulting in pain, loss of function, joint destruction and permanent deformity after a certain time if the condition is left untreated.(8,9) this disease has a world wide distribution but its pathogenesis is not clearly understood. although there are a few antirheumatic drugs showing effectiveness on the treatment of rheumatoid arthritis, the side effects and toxicity call for new and more effective natural drugs.(10) adjuvant-induced arthritis is a chronic crippling, musculoskeletal disorder that is the nearest approximation to human rheumatoid arthritis for which there is at present no medicine available effecting a permanent cure.(11) the modern steroidal and nonsteroidal anti-inflammatory drugs are used for the amelioration of the symptoms of the disease, but also produce severe side effects. over the years, an increasing proportion of p a t i e n t s w i t h a r t h r i t i s a r e r e s o r t i n g t o complementary and alternative medicine for their health needs. the aim of this study was to evaluate the effect of an ethanolic extract of j. curcas leaves on adjuvant-induced arthritis in rats. methods research design this study used a completely randomized design (crd) of unidirectional pattern. the research was carried out in the laboratory of p h a r m a c e u t i c a l b i o l o g y, d e p a r t m e n t o f pharmacy, faculty of medicine and health sciences, jenderal sudirman university, purwokerto for preparation of the extract; the laboratory of pharmacology and toxicology, pharmaceutical faculty of gadjah mada university for the animal experimental study; and the laboratory of pathology and anatomy, medical faculty of gadjah mada university, yogyakarta for the histopathology study. the study was conducted from juni to october 2013. animals studies were conducted on 48 male wistar rats weighing 110-170 g obtained from the animal house in the faculty of pharmacy, gadjah mada university. the number of rats was determined by the formula of federer; with the anticipation of deaths (drop outs) of 30%, thus the resulting total number of rats was 48. animals were acclimatized to experimental conditions in cages and kept under standard environmental conditions (22 ± 3°c; 12/12 h light/dark cycle). rats were allowed to feed and water ad libitum. plant material l e a v e s w e r e c o l l e c t e d f r o m karangwangkal, purwokerto. taxonomic identification of the plant was made by the laboratory of taxonomy, faculty of biology, jenderal soedirman university. preparation of extract the simplicia were cleaned and oven-dried at the controlled temperature of 70°c, then powdered by homogenizer. the crude drug powder was macerated in 96% ethanol for 3x24 hours. the macerate was evaporated in a rotary 6 baroroh, sobri, rachmani, et al jatropha curcas for arthritis evaporator for ± 90 minutes in the temperature range of 70-80°c, then evaporated over a water bath to produce a thick extract, which was stored in a refrigerator until used in the study. induction of arthritis freund’s adjuvant induced arthritis model was used to assess the anti-arthritic activity of the ethanolic extract of j.curcas in wistar rats. each group was induced by 0.2 ml complete freund’s adjuvant (cfa 1 mg/ml) on day 1 and a booster injection of 0.1 ml cfa (1 mg/ml) on day 5 by intradermal injection.(12) intervention on adjuvant-induced arhritis forty-eight wistar rats were divided into six groups (n=8). they were fed a standard diet and water was given ad libitum. the animal subjects were assigned randomly into groups, consisting of: (i) normal group, injected with 0.9% nacl, (ii) negative control group, induced by cfa, (iii) positive control group, given sodium diclofenac at a dose of 6.75 mg/kg, and (iv-vi) treatment groups, given ethanol extracts j. curcas leaves at doses of 150 mg/kg, 300 mg/ kg and 600 mg/kg. the treatment extracts and sodium diclofenac were administered orally on days 14-28. on day 29, the animals were killed by cervical dislocation. arthritis and mobility scores the arthritis and mobility scores are indicators of the severity of arthritis. the arthritis and mobility scores were measured on days 0, 1, 4, 8, 12, 16, 20, 24, and 28.(13) each paw was scored on a scale of 0-4 for the degree of swelling, erythema and deformity (maximum score 16 per animal) as follows: 0 = normal, 1 = slight erythema and/or swelling of the ankle or wrist, 2 = moderate erythema and/or swelling of ankle or wrist, 3 = severe erythema and/or swelling of ankle or wrist and 4 = complete erythema and swelling of toes or fingers and ankle or wrist and inability to bend the ankle or wrist. whole animal mobility was scored between 0 and 4 according to the following definitions: 0 = normal, 1 = slightly impaired, 2 = major impairment, 3 = does not step on paw and 4 = no movement.(13) histological processing and assessment of arthritis damage the rats were killed by cervical dislocation. knee joints were removed and fixed for 4 days in 10% formaldehyde. after that the specimens were processed for paraffin embedding and preparation of tissue sections ( 7 ìm thick) and were stained with hematoxylin and eosin. statistical analysis the data were analyzed using spss software version 17.0. all variables were checked for normal distribution using the kolmogorovsmirnov one sample test. the arthritis scores were analyzed using kruskal wallis followed by mann whitney test (abnormal distribution). mobility scores were analyzed using one way analysis of variance, followed by least significant difference multiple comparison test. values of p<0.05 were regarded as statistically significant. ethical clearance this study was accorded ethical clearance by the commision for research ethics for the medical and sciences, faculty of medicine and health sciences, jenderal soedirman university. results throughout the 28-day study, all arthritic rats showed persistent increases in both arthritis and mobility scores. following the injections of cfa, the rats developed arthritis beginning from the 8th day. in arthritic controls (group ii), there was an increase in arthritis scores in the rats until the 28th day (table 1). in the 28-day study it was found that sodium diclofenac significantly decreased the arthritic condition from the 20th day after induction by freund’s adjuvant (p<0.05) (table 1). the extract at a dose of 150 mg/kg significantly decreased the arthritis scores after the 20th day (p<0.05) and there were 7 univ med vol. 33 no.1 table 1. effect of j.curcas extracts on mobility scores in freund’s complete adjuvant induced arthritis in rats values expressed as mean ± s.e.m of 8 rats; a = p<0.05 compared with arthritic controls (cfa-induced); b=p<0.05 compared with standard drug (sodium diclofenac) table 2. effect of j.curcas extracts on mobility scores in freund’s complete adjuvant induced arthritis in rats values expressed as mean ± s.e.m of 8 rats; a = p<0.05 compared with normal controls; b = p<0.05 compared with arthritic controls (cfa-induced); c = p<0.05 compared with standard drug (sodium diclofenac) significant differences in arthritis scores compared with sodium diclofenac on the 20th day. rats treated with j.curcas extract at doses of 300 and 600 mg/ kg showed significant decreases in arthritis scores (p<0.05) from the 20th to the 28th day compared to arthritic control rats (table 1). i n a r t h r i t i c r a t s t h e m o b i l i t y s c o r e s increased (60%) on the 28th day compared with the mobility scores on the 12th day (table 2). the mobility scores on the 12th day in arthritic rats (control and treatment groups) were increased compared with normal controls (p<0.05). the j. curcas leaf extract at doses of 150, 300 and 600 mg/kg decreased mobility scores on the 28th day and there were significant differences with the arthritic control group (p<0.05). sodium diclofenac significantly decreased mobility scores (p<0.05). there were no significant differences in the activity of j. curcas leaf extracts on mobility score at doses of 150 and 300 mg/kg compared with sodium diclofenac (table 2). t h e j . c u rc a s e x t r a c t d e c r e a s e d inflammation, edema, and cartilage destruction in arthritic joints. figure 1 shows the histological changes in joints of control and experimental animals, as follows: standard drug section showing joint cavity with synovial membrane lining and normal joint space in between two articular cartilages. section of joint cavity of arthritic rats showing proliferation with granulation tissue adjacent to the damaged articular cartilage. section of joint cavity of 8 baroroh, sobri, rachmani, et al jatropha curcas for arthritis figure 1. histopathological representation of joints stained with h&e (a) normal rat (b) control rat (c) diclofenac (6.75 mg/kg) treated rat (d) j. curcas 150 mg/kg treated rat, (e) j. curcas 300 mg/kg treated rat, (f) j. curcas 600 mg/kg treated rat. images (x 40 magnification) are typical and representative of each study group, (i) section of joint cavity of arthritic rats showing inflammation and (p) proliferation with granulation tissue adjacent to the damaged articular cartilage. section of joint cavity of rats on standard drug, j. curcas 150 mg/kg, 300 mg/kg and 600 mg/kg showing decreasing inflammation, edema in arthritic joints and cartilage destruction arthritic rats showing pannus. section of joint cavity of j. curcas treated rats showing normal architecture of both cartilages. discussion this investigation on arthritic rats showed joint swellings that were noticeable around the ankle joints during the acute phase of arthritis and were due to edema of periarticular tissues such as ligaments and joint capsules. the progress of the arthritic condition was evident around day 12 which indicated systemic inflammation.(14) the joint swellings were found t o b e i n c r e a s i n g i n t h e i n i t i a l p h a s e o f inflammation and becoming constant in 2 weeks (beginning on day 8). these increases in arthritis and mobility scores have been found to be associated with chronic inflamation. j. curcas extract at doses of 150 mg/kg, 300 mg/kg and 600 mg/kg significantly suppressed the arthritis of the paws in the chronic phase which may be due to the suppression of inflammatory mediators released as a result of induction by freund’s adjuvant. release of various inflammatory mediators including cytokines (il-1b and tnf-alpha), interferons and platelet derived growth factor (pdgf) are responsible for the initiation of pain along with swelling of the limbs and joints, bone deformations and disability of joint function.(10) this is because the activation of macrophages results in the production of several cytokines including il 1, il 6, interferon ã (ifn ã) and tnf á which have been implicated in immune arthritis. these mediators are responsible for the pain, destruction of bone and cartilage that can lead to severe disability.(10) pro-inflammatory cytokines such as il-1â are potent inducers of reactive oxygen species, including nitric oxide a n d i n f l a m m a t o r y m e d i a t o r s s u c h a s prostaglandin e2, via enhanced expression of inducible nitric oxide synthase.(15) nitric oxide can modulate adhesion of leukocytes to the 9 univ med vol. 33 no.1 vascular endothelium and the activity of numerous enzymes, all of which can have an impact on inflammatory process.(16) several studies have shown the antioxidant and anti-inflammatory properties of flavonoids. recent studies have also shown that certain flavonoids, especially flavone derivatives, express their anti-inflammatory activity at least in part by modulation of proinflammatory gene expression such as cyclooxygenase-2, inducible nitric oxide synthase, and several pivotal cytokines.(17) oskoueian et al.(7) found that the methanol extract of j. curcas at a concentration of 3.1 up to 200 ug/ml had inhibitory activity on inos. the leaves of j. curcas were strong inos inhibitor contributing to an anti-inflammatory effect with an ic 50 value of 93.5 ìg/ml and no scavenging activity of samples correlated well with the levels of phenolics, flavonoids and saponins present in the leaves.(7) similar findings on the antiinflammatory effect of extracts from different parts of j. curcas plant have been reported. mujumdar and misar (18) observed the anti-inflammatory activity of topical application of j. curcas root powder paste, on 12-otetradecanoylphorbol-13-acetate (tpa) induced ear inflammation in albino mice. similarly, uche and aprioku (19) reported the inhibitory activity of j. curcas leaf extract (10-80 mg/kg bb), on egg-albumin induced inflammation in wistar albino rats. j. curcas extract (150 mg/kg) had anti-inflammatory effects on carrageenaninduced arthritis in rats. saxena et al.(2) reported that the compounds which were found to be antiarthritic are the flavonoids apigenin and its glycosides vitexin and isovitexin, the sterols stigmasterol, 3-d-sitosterol and its 3-dglucoside. t h o u g h t h e a c t u a l m e c h a n i s m o f suppressing the arthritic condition is not known, it can be correlated with the presence of flavonoids in suppressing the inflammation and exerting antioxidant activity.(4) synergistic activity between polyphenolic compounds in the extract may possibly contribute to the antioxidant activity.(20) the mechanism of action of these compounds is not known. jatropha curcas leaves also are antioxidants and free-radical scavengers, which may aid in suppressing reactive oxygen species (ros) that stimulate inflammatory r e s p o n s e s . t h e l e a v e s o f j . c u rc a s a l s o demonstrated potent nitric oxide and superoxide radicals scavenging (antioxidant) activity.(21) i g b i n o s a e t a l . ( 2 2 ) s h o w e d t h a t p h e n o l i c compounds have the ability to absorb and neutralize free radicals, and disable oxygen species and hydroxyl radicals. from the results observed in the current investigation, it may be concluded that the j. curcas extract (at doses of 150 mg/kg, 300 mg/kg and 600 mg/kg) can be employed in potential anti-arthritic formulations since it was active in adjuvant-induced arthritis. a potential limitation of this study that must be considered is that only a limited number of rats were used for this study as indicated by the animal ethical clearance. it would provide better conclusions if validated with a larger sample size. conclusion the results showed that j. curcas leaf had anti-arthritic activities that should be further examined as a potential candidate to be exploited for anti-arthritic therapies. acknowledgement the authors thank to directorate general of higher education for financial support, gadjah mada university for providing necessary facilities to carry out the research work, primawati kusumaningrum and nadhifa j. ali for helping in animal experimental. references 1. funk jl, frye jb, oyarzo jn, kuscuoglu n, wilson j, mc caffrey g, et al. efficacy and mechanism of action of turmeric supplements in the treatment of experimental arthritis. arthritis rheum 2006;54:3452-64. 2. saxena s, sharma r, batra a, rajore s. isolation and identification of flavonoid “vitexin” from 10 baroroh, sobri, rachmani, et al jatropha curcas for arthritis jatropha curcas l. j plant sci res 2005;21:1167. 3. hutapea jr. inventaris tanaman obat indonesia. edisi 3. jakarta: departemen kesehatan ri, badan penelitian dan pengembangan kesehatan;2004.p.29-30. 4. olukunle jo, adenubi ot, oladele gm, sogebi ea, oguntoke pc. studies on the antiinflammatory and analgesic properties of jatropha curcas leaf extract. acta vet brno 2011;80:259-62. 5. nayak bs, patel kn. anti-inflammatory screening of jatropha curcas root, stem and leaf in albino rats. rom j biol plant biol 2010;55:913. 6. baroroh hn, warsinah. antiinflammatory activity of ethanolic extract of leaves of jarak pagar (jatropha curcas) and neutrophils profile in rats foot induced carrageenan. proceedings of the international conference on medicinal plants, the future of medicinal plants: from plant to medicine; july 21-22; surabaya, indonesia;2010.p.490-4. 7. oskoueian e, abdullah n, saad wh, omar ar, ahmad s, kuan wb, et al. antioxidant, antiinflammatory, and anticancer activities of methanolic extracts from jatropha curcas linn. j med plant res 2011;5:49-57. 8. schuna aa. rheumatoid arthritis. in : dipiro jt, talbert rl, yee gc, matzke gr, wells bg, posey lm, editors. pharmacotheraphy: a phatophysiologic approach. 7th ed. new york: the mcgraw-hill companies;2008.p.1505-16. 9. national collaborating centre for chronic conditions. rheumatoid arthritis: national clinical guideline for management and treatment in adults. london: national institute for health and clinical excellence; 2009. 10. patil mvk, kandhare ad, bhise sd. antiarthritic and anti-inflammtory activity of xanthium srtumarium l. ethanolic extract in freund’s complete adjuvant induced arthritis. biomed aging pathol 2012;2:6-15. 11. bevaart l, vervoordeldonk mj, tak pp. evaluation of therapeutic in animal models of arthritis, how does it relate to rheumatoid arthritis? review, arthritis and rheumatism. am coll rheumatol 2010;62:2192-205. 12. snekhalatha u, anburajan m, venkatraman b, menaka m. evaluation of complete freund’s adjuvant-induced arthritis in a wistar rat model, comparison of thermography and histopathology. z rheumatol 2012;72:375-82. 13. ablin jn, entin-meer m, aloush v, oren s, elkayam o, george j, et al. protective effect of eotaxin-2 inhibition in adjuvant-induced arthritis. clin exp immun 2010;161:276-83. 14. venkatesha sh, rajaiah r, berman bm, moudgil kd. immunomodulation of autoimmune arthrits by herbal cam. evid based complementary altern med 2010;11:1-13. 15. ramprasth vr, shanthi p, sachdanandam. immunomodulatory and antiinflammatory effects of semecarpus anacardium linn. nut milk extract in experimental inflammatory conditions. biol pharm bull 2006;29:693-700. 16. wallace jl. nitric oxide as a regulator of inflammatory process. mem inst oswaldo cruz 2005; 100 suppl 1: s5-9. 17. kim hp, son kh, chang hw, kang ss. antiinflammatory plant flavonoids action mechanism. pharmacol sci 2004;96:229-45. 18. mujumdar am, misar av. anti-inflammatory activity of jatropha curcas roots in mice and rats. j ethnopharmacol 2004;90:11-5. 19. uche fi, aprioku js. the phytochemical constituents, analgesic and anti-inflammatory effects of methanol extract of jatropha curcas leaves in mice and wister albino rats. j appl sci environ manage 2008;12:99-102. 20. panchal ah, patel rk, bhandari a. antiarthritic and synergetic activity of wedelia calendulacea l. with methotrexate in adjuvant induced arthtitis with cardioprotective activity in at. pharmacologyonline 2011;3:175-87. 21. okoli co, akah pa, ezike ac, udegbunam s, nworu sc, okoye tc. ethnobiology and pharmacology of jatropha curcas l. ethnopharmacol 2008;101-25. 22. igbinosa oo, igbinosa ih, chigor vn, uzunuigbe oe, oyedemi so, odjadjare ee, et al. polyphenolic contents and antioxidant potential of stem bark extracts from jatropha curcas (linn). int j mol sci 2011;12:2958-71. c:\users\universa medicina\docu 74 *department of obstetrics and gynecology, faculty of medicine, universitas sebelas maret / dr. moewardi general hospital, surakarta correspondence: uki retno budihastuti jl. penggung jatinom km 1 jatiwiro, blanceran, karang anom, klaten mobile: +62812 2656 140 email: ukiretno@staff.uns.ac.id orcid id: 0000-0003-0611-7464 date of first submission, december 16, 2 01 9 date of final revised submission, april 23, 2020 date of acceptance, may 20, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina mucin-1 expression in endometrium is higher in polycystic ovary syndrome than in normal women uki retno budihastuti*, sri sulistyowati*, eriana melinawati*, yohan pamuji marbun* background the polycystic ovary syndrome (pcos) is caused by endocrine system dysfunction in women. mucin-1 (muc-1) expression is found in endometrial tissues, which leads to implantation process dysfunction because of imbalance of trophoblast adhesion process. this study was conducted to compare endometrial muc-1 expression between pcos and normal women considering all existing external variables. methods this cross-sectional study was conducted in general hospital dr. moewardi surakarta. endometrial samples were obtained from 30 infertile pcos women based on rotterdam criteria, and 30 normal women. life style and reproductive data such as age, menstrual problems, menstrual cycle, age at menarche, and bmi were collected. subjects underwent endometrial biopsy in luteinizing hormone (lh) secretion phase lh + 5 days to lh + 10 days for immunohistochemistry (ihc) of muc-1 expression. an independent-t and multiple linear regression test were used to analyze the data at significance level of p<0.05. results mean muc-1 expression in the pcos endometrium (49.66 ± 47.79) was significantly higher than in normal women (7.66 ± 14.55) (p=0.03). multivariate linear regression model of life style and reproductive variables with muc-1 showed that pcos (=29.54; 95% ci 9.57-49.49; p=0.004) and bmi (= 29.99; 95% ci 5.91-54.07; p=0.001) significantly increase muc-1 expression. pcos (beta=0.37) was more important than bmi (beta=0.30) in increasing the muc-1 expression. conclusion expression of muc-1 levels in the pcos endometrium was higher than in normal women. this suggests that muc-1 contributes to the unexplained reproductive failure in pcos. keywords : endometrium, muc-1 expression, pcos, normal women original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.74-80 cite this article as: budihastuti ur, sulistyowati s, melinawati e, marbun yp. mu cin1 expressi on in endometrium is higher in polycystic ovary syndrome than in normal women. univ med 2020;39:74-80. doi: 10.18051/ univmed.2020.v39.74-80 may-august 2020 vol.39no.2 75 may-august, 2020 vol.39no.2 introduction infertility in patients with polycystic ovary syndrome (pcos) is caused by the dysfunction in endometrial receptivity contributing to failure of embryo implantation, since it is estimated that almost 75% of miscarriages are caused by implantation failure. (1–3) for a succ essful pregnancy, competent blastocyst implantation into a receptive endometrium is needed.(4,5) abnormal endometrial receptivity will cause failure of embryo implantation and lowers the mean number of pregnancies in women with pcos. in pcos, f a c t o r s t ha t a f f e c t implantation undergo some changes causing the requirements for implantation to be incompletely fulfilled.(6) mucin 1 (muc-1) is a protein that is highly expressed in endometrial luminal epithelium and glands, and is the first molecule on the surface of the uterus where the embryo is being implanted. typically, muc-1 is expressed in various luminal epithelial and glandular cells, provides protection to the underlying epithelium, and plays a role in cell signaling.(5,7,8) there is a need for research to determine whether the expression of muc-1 changes during implantation. in addition, are there external variables that contribute to changes in m uc-1 e x pr e s si o n t ha t i nt e r f e r e wi t h endometrial receptivity. in normal women, muc-1 is produced in functional endometrium, e s p e c i a l l y t o i nc r e a s e t he a p po s i ti o n t o endometrial stroma.(7,9) at present, endometrial receptivity studies in pcos are still needed to find out the causes o r ma r ke r s t ha t c a n be u se d t o de t e c t implantation failures in those who are infertile, especially in pcos. since implantation failure is almost 75%,(2) it is important to determine whether these markers affect the implantation failure rate in pcos. the novelty of this study lies in the effect of external factors on changes in muc-1 expression. the endometrium in pcos patients has a higher expression of muc-1 than in normal wo me n. ( 1 0 ) a s ys t e ma t i c r e vi e w s ho we d controversy with respect to whether mic-i is significantly higher or lower in women with pcos compared to healthy women.(11) a study on 78 women with recurrent implantation failure found that age, body mass index (bmi), and menstrual cycle length were not significantly associated with endometrial muc-1. (7) the presence of muc-1 can cause failure of embryo i mpl a n t a ti on d ur i n g t he app os i t i on phase.therefore it is necessary to discover how exactly the expression of muc-1, a glycoprotein on epithelial cells, and other modulators are expected to play a maj or role in embryo implantation in women with pcos resulting in implantation dysfunction because of imbalance in the trophoblast apposition process.(7) the objective of this study was to investigate differences in endometrial muc-1 expression between women with pcos and normal women, and to determine the effect of pcos on muc1 expression by considering all existing external variables. methods research design a cross-sectional study was conducted in the outpatient clinic of dr. moewardi general hospital, surakarta, from june until november 2019. research subjects the study sample was taken by a nonrandom consecutive sampling, with the subjects signing the participation agreement form with regard to the research activities (informed consent). the sample size was determined with effect size = 0.7, α=0.05 and β=0.2, and was 30 for each group. the subjects were divided into two groups of 30 pcos women and 30 normal women, respectively, who were between 25 and 40 years old. the inclusion criteria were infertile pcos women who met 2 of the 3 rotterdam criteria:(12-14) 1) oligo-ovulation or anovulation defined as <8 menstrual cycles in the past year, 76 2) hyperandrogenism (hirsutism or acne), with hirsutism defined by a ferriman-gallwey (fg) self -reported score of >5 (for asian) , 3) polycystic ovaries (pco) as dete cted by transvaginal ultrasound (tvus) with 12 follicles of 2–9 mm diameter, and age 23-40 years. the controls were 30 normal women who were undergoing sterilization and/or routine via (visual inspection with acetic acid) screening, history taking and gynecological examinations, and not suspected of suffering from pcos (no pelvic pain, no history of dysmenorrhea or dyspareunia, normal clinical gynecological examination). exclusion criteria were normal women with malignancies, not agreeing to become research subjects, using hormonal contraceptives, having androgen excess disorder, being at risk of hypertension, endometriosis and diabetes mellitus. exposure data da ta o n lif e s t yl e a nd r e pr o du c t i ve exposures, as well as anthropometric measures, were collected, including age, age at menarche, menstrual problems, menstrual cycles. and bmi. immunohistochemistry the expression of muc-1 in pcos was deter mined by endometria l biopsy in the luteinizing hormone (lh) secretory phase, at lh + 5 days until lh + 10 days by endometrial sampling with the pipelle curette, 2-3 cm below the uterine fundus. the endometrial biopsy was then put into a bottle with buffered formalin and sent to the pathological anatomy department of dr. sa r d j it o ho sp i t a l i n yogya ka r ta f or immunohistochemical examination (ihc) of m uc-1 e x pr e ssi on u si n g mous e m uc-1 monoclonal antibody (novocastra ncl-muc1 from vision biosystems). in the present study, the points in time of lh + 5 days to lh + 10 days were used, because at these time points there are surges in endometrial receptivity after ovulation, at which the endometrial secretory phase and receptivity for optimal implantation can occur. the expression of muc-1 was calculated based on observations of 200 luminal epithelial and glandular cells, using a microscope at 40x10 magnification. the number of positive (=brown-colored) cells was counted, then the results were divided by 200 and expressed as a percentage. statistical analysis an independent t-test and multivariate linear regression was used to analyze the data. for statistical analysis the ibm-spss statistics program version 21 was used, with significance set to a p value of <0.05. ethical clearance ethical clearance was obtained from the health research ethics commission of dr. moewa rdi hospital/fac ulty of medicine, universitas sebelas maret, surakarta, central java, indonesia under no.744/x/hrec/2018 dated 29 october 2018. results table 1 shows the mean values of the variables of age, menstrual problems, menstrual cycle, age at menarche, bmi, and muc-1 for the groups of pcos and normal women. there were significant differences between the two groups with regard to menstrual cycle (27.88 ± 2.39 vs 28.06 ± 2.40 days; p=0.040); age at menarche (14.31 ± 2.19 vs 13.36 ± 1.21 years; p=0.028), and muc-1 (49.66 ± 47.79% vs 7.66 ± 14.55%; p=0.03). the differences in age, me ns t r u a l pr ob l e ms, a n d bm i we r e n o t significant (table 1). all exte r na l var iab l es , s uc h a s a ge , menstrual problems, menstrual cycle, age at menarche, bmi and pcos were entered into the multivariate linear regression model. there was a statistically significant association of pcos (β=29.54; 95% ci 9.57-49.49; p=0.004), a n d bm i ( β= 2 9. 9 9; 95 % ci 5 . 91 -54 .0 7 ; p=0.016) with muc-1. however, there were no significant differences in age, menstrual problems, menstrual cycle, and age at menarche. budihastuti, sulistyowati, melinawati, et al mucin-1 expression in endometrium 77 may-august, 2020 vol.39no.2 pcos (beta=0.37) was more important than bmi (beta=0.30) in increasing the muc-1 (table 2). furthermore, endometrial tissue samples wer e e xamined by i mmunohistoch emical methods in pcos and normal women, then observations were made using a microscope at 40 x 10 magnification to obtain a visual representation of muc-1 levels as shown in figure 1 (a) and (b). in figure 1 (a) the cytoplasm and glandular stroma are clear (show no color), while in figure1 (b) they are brown in color. thus, the results of immunohistochemical expression of muc-1 in luminal endometrial epithelium and glandular stroma in the secretory phase showed t ha t pcos wo me n ha d a hi gh er m uc-1 e x pr e s si on t ha n no r ma l w ome n . m uc-1 expression appears brown in the cytoplasm and glandular stroma. discussion the results showed that there was an increase in endometrial muc-1 expression in pcos compared to normal women, resulting in a decrease in endometrial receptivity. the high l e ve l s of m uc-1 a l s o c a u se d i mpa ir e d endometrial reception which will affect the embryo implantation adhesion process in the endometrial wall. thus, it could be concluded that muc-1 had an effect on endometrial receptivity. therefore, it is necessary to conduct early detection of muc-1 as biomarker of endometrial receptivity to increase the numbers of successful implantations. in addition, estrogen in pcos also causes endometrial dysfunction associated with increased muc-1 levels.(11,12) a previous study showed that failure of embryo i mpl a n t a ti on w a s r e l a t e d t o a bn o r ma l endometrial expression of muc-1.(13) a higher than normal muc-1 level causes a decrease in endometrial receptivity. excessive muc-1 expression in the endometrium in pcos will influence the changes in embryo adhesion to the endometrial wall during implantation. muc-1 is one of the many biomarkers needed during blastocyst adhesion to the endometrial wall and muc-1 also plays an important role in the embryo during the blastocyst stage.(11) other studies have also shown in their research results that muc-1 is only one of several biomarkers that are deregulated in the endometrium of patients with pcos.(9) a b c variables means of several p value pcos (n=30) normal (n=30) age (years) menstrual problem menstrual cycle (days) age at menarche (years) bmi (kg/m2) muc-1 (%) 33.75 ± 5.38 34.00 ± 11.33 27.88 ± 2.39 14.31 ± 2.19 23.18 ± 3.02 49.66 ± 47.79 31.70 ± 4.71 28.77 ± 5.25 28.06 ± 2.40 13.36 ± 1.21 22.24± 2.19 7.66 ± 14.55 0.058 0.718 0.040* 0.028* 0.052 0.03* table 1. means of several variables in pcos and normal women table 2. multivariate linear regression model of demographic and clinical characteristics with muc-1 independent variables β beta 95% c.i. p value age menstrual problems menstrual cycle age at menarche bmi pcos 1.35 -0.19 7.81 2.72 29.99 29.54 0.01 -0.01 0.07 0.03 0.30 0.37 -18.78-21.49 -25.49-25.10 -17.90-33.53 -17.92-23.38 5.91-54.07 9.57-49.49 0.893 0.988 0.545 0.792 0.016* 0.004* 78 infertile patients may suffer recurrent implantation failure, since embryo implantation needs a series of molecular interactions between the embryo and the maternal uterus.(15) the event needed during the implantation process is endometrial reception, so that the embryo can function normally at the blastocyst stage resulting in the emergence of the complex interaction dialog between the embryo and the endometrium. this dialog can occur if there is a synchronization between oocyte and endometrial maturation, followed by blastocyst orie ntation in the endometrial wall. in the early stages of implantation involving bi-directional molecules between the blastocyst and endometrium about 6-12 days after ovulation, this dialog is very important for success.(16) the implantation includes apposition, adhesion, and invasion through the endometrial wall. this means that embryo implantation is the result of many well-functioning processes, including cellular adhesion, invasion, and decidualization of the endometrium.(15,17) part of the immune system mechanism is regulated by genetic expression of ovarian hormones. during most of the menstr ual cycle, the endometrium basically rejects the embryo. in order to reverse this endometrial condition and make an implantation process possible, a physiological effort is needed. during the beginning of the menstrual cycle, the estrogen level increases to enhance endometrial cell proliferation. when ovulation occurs, the progesterone secreted by the luteinizing follicle leads to cell differentiation. therefore, the endometrium is in a mature and optimal condition for embryo implantation.(7,18) endometrial receptivity is physiologically mar ked by pinopodes, specific projections on the surface of the endometrial epithelial membrane, as a morphologic sign of endometrium receptivity. expression of pinopodes is limited to a brief period, with a maximum of two days in the menstrual cycle during the implantation window. blastocyst attachment is seen to be located on the top of endometrial pinopodes.(19) the results of the present study showed that there was an increase in endometrial muc-1 expression in pcos compared to normal women, causing a decrease in endometrial receptivity. this increased muc-1 will also cause impaired endometrial reception, which will affect the embryo implantation adhesion process in the endometrial wall. thus, it could be concluded that muc-1 has an effect on endometrial receptivity. therefore, it is necessary to conduct early detection of muc-1 as biomarker of endometrial receptivity to increase implantation success rate. in addition, estrogen in pcos also causes endometrial dysfunction associated with increased muc-1 levels.(11,12) figure1. immunohistochemistry of muc-1 expression in endometrium. (a) normal group, (b) pcos group. black bar: 10 µm.; : muc-1 expression budihastuti, sulistyowati, melinawati, et al mucin-1 expression in endometrium 79 may-august, 2020 vol.39no.2 during implantation, muc-1 reduction is localized on specific receptor area. mucin-1 expression rate on the uterine surface greatly decreases during the conception attachment period and the endometrium is exposed to stimulation by progesterone.(20) there is a correlation between muc-1 expression and progesterone receptors due to the presence of the embryo going to be implanted. (21) then, muc-1 will disappear followed by the loss of progesterone receptors. muc-1 epithelial synthesis is stimulated by progesterone, it will cause the loss of epithelial progesterone from t he ut e r u s . as a c o ns e q ue n c e , m uc-1 production decreases and enables a receptive condition for conception attachment.(9) increased m uc-1 e xp r e s s i on i n pcos c a us e s t he endometrial sur face to be non-receptive, because of the anti-adhesive characteristic of m uc-1 . hen c e , a n i n c r e a se i n m uc-1 e x pr e s si o n d e c r e a s e s t he o c cu r r e nc e of implantation or is anti-adhesive. a higher than normal increase in muc-1 expression will interrupt embryo attachment to the endometrial wall, and this will cause higher progesterone receptor needs and impair embryo adhesion.(17) the limitation of this study were lutheal phase length, prolaktin levels, ovulation confirmation, uterine cavity shape or level progesterone were not measured. the clinical implication of this study is that in the application of assisted reproductive technology (art) attention should be paid to the muc-1 marker, to increase the implantation success rate. directions in further studies are the provision of adequate anti-muc-1 and progesterone to improve endometrial receptivity so that implantation can occur. conclusions t he l e ve l of e nd o me tr i a l mu c-1 expression in pcos was higher than in normal women. the higher expression of muc-1 in pcos causes an endometrial receptivity defect and decreases embryo implantation. conflict of interest competing interests: no relevant disclosures. acknowledgement we would like to express our heartfelt thanks to the director of dr. moewardi hospital, dr. suharto wij anarko, dr., sp.u for his permission and cooperation for the conduct of this research in above-mentioned hospital. contributors all authors contributed to conception and design of the study; ypm contributed to data acquisition; all authors contributed to analysis and/or interpretation of data. urb drafted the manuscript; urb, ss and em contributed to revising the manuscript critically for important intellectual content. all authors have read and approved the version of the manuscript to be published. references 1. savaris rf, groll jm, young sl, et al. progesterone resistance in pcos endometrium: a microarray analysis in clomiphene citrate-treated and artificial menstrual cycles. j clin endocrinol metab 2011;96:1737-46. doi: 10.1210/jc.2010-2600. 2. li sy, song z, song mj, qin jw, zhao ml, yang zm. impaired receptivity and decidualization in dhea-induced pcos mice. sci rep 2016;6:1–10. http://dx.doi.org/10.1038/srep38134. 3. bashiri a, halper ki, orvieto r. recurrent implantation failure update overview on etiology, diagnosis, treatment and future directions. reprod biol endocrinol 2018;16:1–18. doi: 10.1186/s12958-018-0414-2. 4. cha j, sun x, dey sk. mechanisms of implantation: strategies for successful pregnancy. nat med 2012;18:1754–67. http://dx.doi.org/ 10.1038/nm.3012. 5. davidson lm, coward k. molecular mechanisms of membrane interaction at implantation. birth defects res part c embryo today rev 2016;108: 19–32. 6. piltonen tt. polycystic ovary syndrome: endometrial markers. best pract res clin obstet 80 gynaecol 2016 ;37:66-79. doi: 10.1016/j.bpobgyn. 2016.03.008. 7. wu f, chen x, liu y, et al. decreased muc-1 in endometrium is an independent receptivity marker in recurrent implantation failure during implantation window. reprod biol endocrinol 2018;16:1–7. doi: 10.1186/s12958-018-0379-1. 8. zeng y, zhang q, zhang y, et al. muc-1 predicts colorectal cancer metastasis: a systematic review and meta-analysis of case controlled studies. plos one 2015;10:e0138049. doi:10.1371/ journal.pone.0138049. 9. margarit l, taylor a, roberts mh, et al. muc-1 as a discriminator between endometrium from fertile and infertile patients with pcos and endometriosis. j clin endocrinol metab 2010;95:5320–9. doi: 10.1210/jc.2010-0603. 10. lopes im, maganhin cc, oliveira-filho rm, et al. histomorphometric analysis and markers of endometrial receptivity embryonic implantation in women with polycystic ovary syndrome during the treatment with progesterone. reprod sci 2014;21:930-8. 11. baracat mcp, serafini pc, simões rds, maciel gar, soares jm, baracat ec. systematic review of cell adhesion molecules and estrogen receptor expression in the endometrium of patients with polycystic ovary syndrome. int j gynecol obstet 2015;129:1–4. doi: 10.1016/j.ijgo.2014.10.022. 12. hu m, li j, zhang y, et al. endometrial progesterone receptor isoforms in women with polycystic ovary syndrome. am j transl res 2018;10:2696-705. 13. bastu e, mutlu mf, yasa c, et al. role of mucin 1 and glycodelin a in recurrent implantation failure. fertil steril 2015;103 : 1059-64. http://dx.doi.org/ 10.1016/j.fertnstert.2015.01.025 21. 14. yang y, han y, wang w, et al. assessing new terminal body and facial hair growth during pregnancy: toward developing a simplified visual scoring system for hirsutism. fertil steril 2016;105: 494–500. doi: https://doi.org/10.1016/ j.fertnstert.2015.10.036. 15. fukui y, hirota y, matsuo m, et al. uterine receptivity, embryo attachment, and embryo invasion: multistep processes in embryo implantation. reprod med biol 2019;18:234–40. doi: 10.1002/rmb2.12280. 16. parks jc, mccallie br, patton al, et al. the impact of infertility diagnosis on embryoendometrial dialogue. reproduction 2018;543–52. doi: 10.1530/rep-17-0566. 17. raheem ka. cytokines, growth factors and macromolecules as mediators of implantation in mammalian species. int j vet sci med 2018;6 sup1:s6–14. https://doi.org/10.1016/j.ijvsm.2017. 12.001. 18. craciunas l, gallos i, chu j, et al. conventional and modern markers of endometrial receptivity: a systematic review and meta-analysis. hum reprod update 2019;25:202–23. doi: 10.1093/humupd/ dmy044. 19. altmäe s, koel m, võsa u, et al. meta-signature of human endometrial receptivity: a meta-analysis and validation study of transcriptomic biomarkers. sci rep 2017;7: 10077. doi: 10.1038/ s41598-017-10098-3. 20. redzovic a, laskarin g, dominovic m, haller m, rukavina d. mucins help to avoid alloreactivity at the maternal fetal interface. clinl develop immunol 2013, article id 542152, 9 pages. http:// dx.doi.org/10.1155/2013/54215. 21. shen f, yan c, liu m, feng y, chen y. decreased expression of mucin-1 in endometriosis endometrium correlated with progesterone receptor b involved in infertility. arch gynecol obstet 2015;291:439–45. doi: 10.1007/s00404014-3419-x. budihastuti, sulistyowati, melinawati, et al mucin-1 expression in endometrium c:\users\jurnal fkusakti\docume 81 *atrosclrose research center, baqiyatallah university of medical sciences, tehran, iran **truma research center, baqiyatallah university of medical sciences, tehran, iran ***department of immunology, faculty of medicine, isfahan university of medical sciences, isfahan, iran correspondence: marzieh lak truma research center, baqiyatallah university of medical sciences tehran, iran email: marziehlak@yahoo.com orcid id: orcid.org/0000-00033498-4653 date of first submission, october 24, 2 01 8 date of final revised submission, may 24, 2019 date of acceptance, june 17, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina brain oximetry is not a good monitor on reducing neurological complications after cardiac surgery seyed mohammadreza amouzegar zavareh*, hasan araghizade**, nahid eskandari*** and marzieh lak** background cerebral deoxygenation is related to several adverse systemic consequences. we aimed to assess the effect of noninvasive monitoring of cerebral oxygenation on reducing neurological complications after cardiac surgery. methods in this randomized clinical trial, subjects were randomized into two groups: intervention group (111 subjects with monitoring of cerebral oximetry) and control group (112 subjects without any monitoring of cerebral oximetry). monitoring of regional cerebral oxygen saturation (rsco2) was performed in the intervention group without any monitoring of cerebral oxygenation. the rsco2 was not recorded in the control group and no specific treatments were employed. any neurological complications such as hallucinations, delirium, stroke, and length of stay in icu after surgery were recorded. a pvalue less than 0.05 was used as a cut-off for statistical significance. results after surgery, in the intervention group one (1/111=0.09%) patient suffered from stroke and one patient had delirium, while in the control group three patients had stroke and three (3/112=2.6%) had delirium. there was no significant difference between the two groups regarding complications (p=0.527). the length of stay in icu was 3.49 ± 0.96 days in the case group and 3.40 ± 0.80 days in the control group and there was no significant difference in the two groups (p=0.477). conclusion monitoring of rsco2 does not seem to be a good monitor for brain oxygenation. further studies are needed to judge the usefulness of rsco2 for monitoring brain oxygenation. keywords: regional cerebral oxygen saturation, cardiac surgery, neurological complications original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.81-89 may-august, 2019 vol.38no.2 cite this article as: zavareh sma, hasan araghizade h, eskandari n, et al. brain oxi metr y is not a good mon itor on reducing neurological complications after cardiac surgery. univ med 2019; 38:8 1 -9 . doi: 10 .18 051/ uni vmed. 2018.v38.81-89 82 zavareh, araghizade, eskandari, et al brain oximetry and neurological complications introduction advancing age and associated diseases in patients undergoing cardiac surgery raise postoperative neurological complications such as str oke and other a dverse per ioperative outcomes.(1-3) the prevalence of cognitive impairment after coronary artery bypass graft (cabg) surgery of more than 50%, as well as the relationship of hypoxia of the brain with nervous disorders, increased length of stay in icu and hospital stay are described in several studies.(4,5) during cardiopulmonary bypass the blood supply to the brain is impaired, due to cerebral auto-regulatory dysfunction, arterial embolism, cerebral venous obstruction, technical problems, as well as the patient’s underlying disease (carotid stenosis).(6,7) cardiac surgery under cardiopulmonary bypass leads to an imbalance in brain oxygenation and oxygen consumption. (8 -10 ) reducing oxygenation of the brain is associated with neuropsychiatric disorders after surgery (11-13) although the technology of transcranial nearinfrared spectroscopy (nirs) was introduced 25 years ago for the measurement of cerebral oxygen saturation, until recently there was little interest in using it during surgery.(14) brain oximetry is a non-invasive method for monitoring brain tissue oxygenation, which is probably the benchmark for total brain oxygenation.(15) in a review article, zheng et al.(16) concluded that at present, studies have not been able to prove that intervention to modify the regional cerebral o 2 saturation leads to a reduction in post-operative neurological complications. there is no universal definition of brain oxygen desaturation as abnormal findings. in various reviewed studies, both the relative reduction of brain oxygen saturation from baseline, and its absolute reduction (e.g., less than 50%) were used.(17-19) oximetry of the brain may be valuable in heart surgery in detecting abnormality in cerebral blood flow and its correction. in this study, we aimed to assess the effect of noninvasive monitoring of cerebral oxygenation on reducing neurological complications after cardiac surgery. methods research design a randomized clinical trial was carried out on 223 patients who underwent cardiac surgery with cardiopulmonary bypass (cpb) pump in tehran, iran, between march 2017 and october 2018. research subjects using a computer-generated randomization list, patients were divided into two groups: intervention group (111 patients with monitoring of cerebral oximetry), and control group (112 without any monitoring of cerebral oximetry). sample size determination the calculation of sample size was based on a presumed effect size of 0.4, a statistical power of 80%, and a type i error of 5%, which yielded 100 patients per group. however, for more confidence at least 112 subjects per group was considered in this study. intervention in the intervention group, before anesthesia patients had bilateral oximetry probe, and regional brain oxygenation were recorded; during the operation, rsco 2 was continuously monitored and recorded. if the absolute threshold of rsco 2 was less than 50%, interventions were performed to maintain rsco 2 over 50%. the rsco 2 was not recorded in the control group and no specific treatments were employed. before induction of anesthesia and endotracheal intubation, all patients in the case group had invos cerebral oximeter sensors (invos 5100c; somanetics corp, troy, mi) placed bilaterally on the forehead and baseline regional cerebral oxygen saturation values were measured. performing of cerebral oximetry is not compulsory and using it is not routine in our 83 hospital due to its cost. this is the reason why cerebral oximetry was not performed in the control group. induction of anesthesia was performed using fentanyl (7 µg/kg), diazepam (0.15 mg/kg), and atracurium (0.5 mg/kg). after intubation for maintenance, anesthesia comprised of fentanyl (0.07 µg/kg/min), midazolam (0.5 µg/kg/min), atracurium (0.8 µg/kg/min) and, if necessary, propofol infusion (20–200 mg/h) was used. central venous pressure (cvp) was recorded in the right internal jugular vein. patient’s vital signs we re r ecord ed eve ry f ive minut es, including blood pressure (bp) by the invasive method, pulse rate (pr), core temperature by the nasal method, oxygen saturation with pulse oximetry (spo 2 ) and end-tidal co 2 (et co 2 ) and cvp. also, we maintained systolic blood pressure at 100 to 110 mm hg by nitroglycerin (tng) and inotrope. heparin was inj ected after sternotomy and discharging of the internal thoracic artery and saphenous vein. activated clotting time (act) was checked three to five minutes after injection of heparin. if the act was equal to or more than 480 seconds, we connected the patient to cpb pumps (pump type was stockert siii with head type roller pump, and prime fluid included: mannitol, ringer, heparin, bicarbonate, voluven and transamine. the temperature of the patients was kept at around 32-34° c during the cpb pump. blood flow rate of cpb pump was calculated based on the body surface area (bsa) (2.4 l/min/ bsa). by changing blood flow rate of cpb pump and using noradrenaline or tng, mean arterial pressure during cardiopulmonary bypass was maintained between 50 to 80 mmhg. paco2 was maintained between 35-45 mmhg with changing of oxygen flow of cpb pump and monitored with measurement of arterial blood ga se s. art er ial blo od gase s (a bg) we re determined after installing the arterial line, after induction of anesthesia, after starting of cpb pump, half an hour after the cpb pump, at end of cpb and each time that there was a decrease in the oxygen saturation of the boundary. an absolute threshold of brain saturation (e.g., less than 50%) was used in the various studies.(17) interventions that occurred during the loss of rsco 2 below 50%, included increasing partial pressure of carbon dioxide (paco 2 ) to 45mmhg, increasing blood pressure by increasing blood flow pump, use of inotropes, and lowering o xyge n c ons ump t i on of t h e br a i n u s in g barbiturates and repositioning the patient’s head. percentile of desaturation below 50% and minutes of desaturation were recorded. patients were separated from the cpb pump after establishing satisfactory conditions such as hematocrit (hct) equal to or more than 22%, normal electrolytes and acid-base status. we started protamine sulfate for the reversal of heparin. after the operation, patients were transferred to the open heart intensive care unit. weaning and extubation were administered according to the standards of the open heart i nt e ns i ve c a r e un i t , s uc h as c ompl e te c o ns c i o usn e s s, h e mod yn a mi c s t a bi l i t y, appropriate drainage volume and normal chest x-ray. all patients had elective surgery and emergency patients have not been investigated. intramuscular morphine (five mg) and one mg oral lorazepam one hour preoperative were administered to each patient. we conducted the patients to the operating room with standard monitoring. vital signs and invasive blood pressure monitoring by the left radial artery were recorded. measurements the serum fasting blood sugar (fbs), blood urea nitrogen (bun), creatinine, sodium, potassium, hb, and hct of each patient were measured preoperatively. patients with a history of psychological disorder as well as patients who were taking psychiatric drugs were excluded. an y n e u r ol ogic a l si d e e f f e ct s su c h a s hallucinations, delirium, and stroke and so on up to length of stay in icu after surgery were recorded. univ med vol. 38 no.2 84 zavareh, araghizade, eskandari, et al brain oximetry and neurological complications statistical analysis da ta wa s pr e s e nt e d a s me a n , sd , frequency and percentage. the normality of the data was assessed using the kolmogorov– smirnov test. independent t-test was used for comparison of variables with normal distribution between the two groups. for the quantity variables with non-normal distribution, the mann whitney test was used. also, qualitative data were analyzed by chi-square and fisher exact test depending on circumstance. spss version 17 software was used to analyze findings. the p -va l u e le ss t ha n 0. 0 5 wa s c o ns i de r e d statistically significant. ethical clearance this study was registered in the iranian re gis t r y o f cl i ni c a l tr ia ls un d e r no . irct20161022030421n2 cod. the protocol of the study was confirmed by the ethics committee of baqiyatallah university of medical sciences and informed consent was obtained from each patient. results two hundred thirty three patients were screened for inclusion in the study. no one was excluded, and a total of 233 patients was randomized into the intervention group (111 patients) and 112 patients into the control group. the primary analysis included all randomized patients (figure 1). they were followed up for 3-4 days post-surgery. at base line age, gender and type of surgery were similar between the two groups (table 1). also, there was no statistically significant dif ference between the groups based on preoperative clinical factors and carotid artery stenosis (table 1 and table 2). comparison of the two groups in terms of intraoperative clinical factors indicated that there was no significant difference between the two groups in terms of cpb time and cross-clamp time (table 2). in the intervention group, one patient suffered from a stroke (a man 61 years old with a history of diabetes, hypertension, smoker and opium a b c figure 1. the flow diagram of the subjects 85 addiction. in the control group there were three patients with stroke and three patients with delirium. there was no significant difference between the two groups. the length of stay in icu was 3.49 ± 0.96 days in the intervention group and 3.40 ± 0.80 days in the control group and there was no between difference between the two groups (p=0.447) (table 2). intervention (n=111) control (n=112) p value mean (sd) mean(sd) age (years) 62.02 (10.34) 59.98 (11.19) 0.171 ejection fraction ( %) 45.95 (9.24) 46.71 (8.15) 0.109 n (%) n (%) sex female 32 (28.8) 28 (25.2) 0.546 2 male 79 (71.2) 83 (74.8) type of surgery cabg 92 (86.0) 92 (83.6) 0.631 2 mvr 19 (17.8) 15 (13.5) 0.388 2 avr 14 (13.1) 19 (17.1) 0.406 2 risk factors hypertension 73 (65.8) 61 (54.5) 0.101 2 hyperlipidemia 58 (52.7) 46 (41.1) 0.106 2 diabetes mellitus 48 (43.2) 40 (35.7) 0.250 2 thyroid dysfunction 1 (1) 2 (1.8) 0.503 3 kidney dysfunction 8 (7.2) 8 (7.1) 0.985 2 transient ischemic attack 3 (2.7) 2 (1.8) 0.683 3 cerebrovascular accident 11 (9.9) 10 (8.9) 0.802 3 cigarette smoking 11 (9.9) 5 (4.5) 0.115 2 pulmonary dysfunction 11 (9.9) 10 (8.9) 0.802 3 opioid addiction 21 (18.9) 15 (13.4) 0.262 2 myocardial infarction 12 (10.8) 8 (7.1) 0.338 3 chemical lung injury 1 (0.9) 2 (1.8) 0.503 3 table 1. demographics, ejection fraction, type of surgery and pre-operative clinical factors by treatment groups at base line 1: independent sample t-test 2: pearson chi-square test 3: fisher exact test intervention (n=111) n (%) control (n=112) n (%) p value carotid 43 (38.7) 40 (35.7) 0.640 1 cva 1 (0.9) 3 (2.7) 0.622 2 delirium 1 (0.9) 3 (2.7) 0.622 2 neurological 2 (3.6) 6 (5.4) 0.527 2 mean (sd) mean (sd) length of stay in icu (days) 3.49 (0.96) 3.40 (0.80) 0.477 3 cpb 56.95 (17.92) 59.85 (24.89) 0.326 4 cct 45.90 (16.23) 40.88 (20.36) 0.058 4 1: pearson chi-square test, 2: fisher exact test, 3: mann whitney test, 4: independent sample t-test icu : intensive care unit; cva :cerebro vascular accident; cpb : cardiopulmonary bypass ; cct: cross clamp time table 2. comparison of the two groups in terms of the number of patients with carotid artery stenosis, neurological complications after surgery, length of stay in icu univ med vol. 38 no.2 86 zavareh, araghizade, eskandari, et al brain oximetry and neurological complications side and 40 minutes in left side, in pump: 15 minutes in right side and 20 minutes in left side, post pump: 20 minutes in right side and 10 minutes in left side (table 4). discussion neurological problems of cardiac surgery have a huge effect on patient outcomes. in adding to a decrease in the prevalence of in the intervention group, 73 patients had at least one stage rsco2 less than 50% in one side, 56 patients had at least five minutes of rsco2 less than 50% in the left side and 57 patients in the right side. a total of 38 patients had never rsco2 of less than 50%. table 3 shows the average time of rsco2 less than 50% in d if f e r e nt s ta ge s . t he c e r e br a l ox yge n desaturation at different stages follows the following steps; pre-pump: 45 minutes in right number of patients mean (sd) (min) pre pump right 30 26.67 (16.05) left 23 26.30 (17.14) on pump right 52 26.35 (20.32) left 49 27.04 (21.29) post pump right 23 23.48 (22.38) left 22 20.23 (22.55) table 3. the average time(minutes) of rsco 2 less than 50 in different stages in intervention group table 4. the number of patients and duration of cerebral oxygen desaturation at different stages duration of rsco2<50 pre pump n (%) on pump n (%) post pump n (%) right left right left right left 0 min 81 (73) 88 (79.3) 59 (53.2) 62 (55.9) 87 (78.4) 88 (79.3) 5 min 3 (2.7) 1 (0.9) 10 (9) 9 (8.1) 5 (4.5) 6 (5.4) 10 min 4 (3.6) 7 (6.3) 6 (5.4) 6 (5.4) 3 (2.7) 3 (2.7) 15 min 2 (1.8) 3 (2.7) 7 (6.3) 6 (5.4) 2 (1.8) 3 (2.7) 20 min 4 (3.6) 1 (0.9) 4 (3.6) 7 (6.3) 4 (3.6) 5 (4.5) 25 min 5 (4.5) 0 5 (4.5) 2 (1.8) 5 (4.5) 1 (0.9) 30 min 3 (2.7) 3 (2.7) 3 (2.7) 0 2 (1.8) 35 min 2 (1.8) 1 (0.9) 6 (5.4) 1 (0.9) 0 0 40 min 1 (0.9) 1 (0.9) 5 (4.5) 2 (1.8) 0 1 (0.9) 45 min 3 (2.7) 1 (0.9) 2 (1.8) 4 (3.6) 2 (1.8) 0 50 min 1 (0.9) 1 (0.9) 2 (1.8) 3 (2.7) 0 0 55 min 0 1 (0.9) 2 (1.8) 3 (2.7) 1 (0.9) 0 60 min 1 (0.9) 0 0 0 0 0 65 min 1 (0.9) 0 0 0 0 0 70 min 0 0 1 (0.9) 1 (0.9) 0 0 75 min 0 0 0 0 0 0 80 min 0 0 0 0 0 0 85 min 0 0 0 0 0 0 90 min 0 0 0 1 (0.9) 0 0 95 min 0 0 1 (0.9) 1 (0.9) 0 0 100 min 0 0 0 0 0 0 105 min 0 0 0 0 1(0.9) 0 110 min 0 0 0 0 0 1(0.9) 87 neurological damage and length of hospital stay, intr a -ope ra ti ve use of ce re br al oximet ry monitoring has been accompanied by early detection of potentially severe brain damage demanding fast mediation.(18,19) th er e wa s no si gni fi ca nt dif f er en ce between the two groups regarding age, sex, type of surgery, preoperative clinical factors, carotid artery stenosis, cpb time and crossc l a mp t i me . in t e r ms of n e ur o lo gi c a l complications after surgery, in the intervention group, one patient suffered from a stroke and one patient had delirium, while in the control group there were three patients with stroke and three patients with delirium, but there was no significant difference between the two groups. although in the case group 73 patients had brain oxygen desaturation, neurological complications were rare. there was no significant difference between the two groups in terms of the length of stay in icu. in contrast to this study, parnia et al.(20) indicated that in patients who had an in-hospital cardiac arrest, it was proved that monitoring of brain oxygen saturation could predict the return of spontaneous circulation (rosc). they also demonstrated that patients with rosc had oxygen saturation above 30% for more than 50% of the duration of cardiac arrest, while those who died had oxygen saturation below 30% for more than 50% of their cardiac arrest duration. in addition, other research performed brain oximetry in patients who went to the intensive care unit after resuscitation from cardiac arrest. they did not find significant differences in the oximetry of the brain in surviving patients compared to those who died during 24-48 hours post resuscitation.(21) in a study on patients undergoing cardiac surgery with cpb pump, it was concluded that patients who had rsco 2 drops had post-operative neuropsychological disorders. but whether intervention to maintain rsco 2 can prevent neuropsychological disorders, has not been investigated.(22) moreover, the effect of brain oximetry on neurocognitive function of patients after coronary surgery is that the cognitive outcome in the oximetry group is significantly better than that in the control group.(23) in post hoc analysis in diabetic patients, murkin et al.(1) concluded that monitoring and maintaining oxygen saturation in diabetic patients reduce postoperative complications and the length of stay in the icu. slater et al.(4) divided 265 patients who underwent coronary artery bypass grafting to control and interven tion groups. in the intervention group, if rsco 2 dropped 20% from the base, intervention was performed to maintain it. in the control group, rsco 2 was recorded, but there was no intervention to maintain it. the investigators observed that patients with an rsco 2 desaturation score above 3000%-second had a higher risk of neurological complications after surgery, and longer hospitalization periods. subramanian et al. (3) in a multicenter research showed that 50% to 75% of cardiac surgery patients have one or more rsco 2 desaturations whil e undergoing cpb. approximately 10% of desaturation events were not recognized by clinicians, recommending that suitable alert systems should be implemented to warn clinicians of such happenings. we tried to maintain pre-pump systolic blood pressure between 100 to 110 mm hg by using tng and inotrope and during cardiopulmonary bypass by changing rate of cpb pump blood flow, and using noradrenaline or tng to maintain mean arterial pressure at 50 to 80 mm hg. during the operation, patients in the case group had continuous monitoring of the rsco 2 during the entire period of the operation. if the absolute threshold of rsco 2 was less than 50%, intervention was performed to maintain regional rsco 2 at, or above, the baseline value. as shown in tables 2 and 3, the intervention failed to quickly reach the brain’s rsco 2 of 50%. the monitor rsco 2 does not seem to be a good mon i t or f or b r a i n o xyge na t i o n; i ns t e a d , maintaining hemodynamics in the various stages of the operation is more important in reducing neurological complications. univ med vol. 38 no.2 88 zavareh, araghizade, eskandari, et al brain oximetry and neurological complications the major limitation of this study was that we did not restore hemodynamic stability with the use of vasoactive drugs, when we could have prevented neurological complications in both groups. the use of rsco 2 has a high cost for medical service. although our findings did not show any differences between the two groups, more studies are required to reach conclusions on the application of rsco 2 for monitoring brain oxygenation. conclusion the monitor rsco 2 does not seem to be a good monitor for brain oxygenation. further studies are needed to judge the usefulness of rsco 2 for monitoring brain oxygenation. conflict of interest the authors declare there is not conflict of interest. acknowledgement we thank the staff of trauma research center of baqiyatallah university of medical sciences for their kind cooperation in this research. contributors smaz and ml contributed to the study concept, design, analysis and interpretation of data. ha and ml contributed to drafting of the manuscript. all authors have read and approved the final manuscript. references 1. murkin jm, adams sj, pardy e, et al. monitoring brain oxygen saturation during coronary bypass surgery improves outcomes in diabetic patients: a post hoc analysis. heart surg forum 2011;14:e16. doi: 10.1532/hsf98.20101065. 2. deschamps a, hall r, grocott h, et al. cerebral oximetry monitoring to maintain normal cerebral oxygen saturation during high-risk cardiac surgery: a randomized controlled feasibility trial. anesthesiology 2016;124:826-36. doi: 10.1097/ aln.0000000000001029. 3. subramanian b, nyman c, fritock m, et al. a multicenter pilot study assessing regional cerebral oxygen desaturation frequency during cardiopulmonary bypass and responsiveness to an intervention algorithm. anesth analg 2016;122: 1786-93. doi: 10.1213/ane.0000000000001275. 4. slater jp, guarino t, stack j, et al. cerebral oxygen desaturation predicts cognitive decline and longer hospital stay after cardiac surgery. ann thor surg 2009;87:36-44. doi: 10.1016/j.athoracsur. 2008.08.070. 5. green dw, kunst g. cerebral oximetry and its role in adult cardiac, non cardiac surgery and resuscitation from cardiac arrest. anaesthesia 2017;72:48-57. doi: 10.1111/anae.13740. 6. tovedal t, thelin s, lennmyr f. cerebral oxygen saturation during pulsatile and non-pulsatile cardiopulmonary bypass in patients with carotid stenosis. perfusion 2016;31:72-7. doi: 10.1177/ 0267659115586280. 7. aktuerk d, mishra pk, luckraz h, et al. cerebral oxygenation monitoring in patients with bilateral carotid stenosis undergoing urgent cardiac surgery: observational case series. ann card anaesth 2016;19:59-62. 8. scheeren tw, kuizenga mh, maurer h, et al. electroencephalography and brain oxygenation monitoring in the perioperative period. anesth analg 2019;128:265-77. doi: 10.1213/ane. 0000000000002812. 9. kirkman ma, smith m. brain oxygenation monitoring. anesthesiol clin 2016;34:537-56. doi: 10.1016/j.anclin.2016.04.007. 10. okonkwo do, shutter la, moore c, et al. brain oxygen optimization in severe traumatic brain injury phase-ii: a phase ii randomized trial. crit care med 2017;45:1907-14. doi: 10.1097/ccm. 0000000000002619. 11. langer t, santini a, zadek f, et al. intraoperative hypotension is not associated with postoperative cognitive dysfunction in elderly patients undergoing general anesthesia for surgery: results of a randomized controlled pilot trial. j clin anesth 2019;52:111-8. doi: 10.1016/j.jclinane. 2018.09.021. 12. goettel n, burkhart cs, rossi a, et al. associations between impaired cerebral blood flow autoregulation, cerebral oxygenation, and biomarkers of brain injury and postoperative cognitive dysfunction in elderly patients after major noncardiac surgery. anesth analg 2017; 124:934-42. doi: 10.1213/ane.0000000000001803. 13. erdem af, kayabasoglu g, tuna at, et al. effect of controlled hypotension on regional cerebral 89 oxygen saturation during rhinoplasty: a prospective study. j clin monit comput 2016; 30:655-60. doi: 10.1007/s10877-015-9768-6. 14. kobayashi k, kitamura t, kohira s, et al. cerebral oximetry for cardiac surgery: a preoperative comparison of device characteristics and pitfalls in interpretation. j artif organs 2018;21:412-8. doi: 10.1007/s10047-018-1052-3. 15. yoshitani k, kawaguchi m, ishida k, et al. guidelines for the use of cerebral oximetry by nearinfrared spectroscopy in cardiovascular anesthesia: a report by the cerebrospinal division of the academic committee of the japanese society of cardiovascular anesthesiologists (jscva). j anesth 2019;33:167-96. doi: 10.1007/ s00540-019-02610-y. 16. zheng f, sheinberg r, yee ms, et al. cerebral near-infrared spectroscopy (nirs) monitoring and neurologic outcomes in adult cardiac surgery patients and neurologic outcomes: a systematic review. anesth analg 2013;116. doi: 10.1213/ ane.0b013e318277a255. 17. lewis c, parulkar sd, bebawy j, et al. cerebral neuromonitoring during cardiac surgery: critical appraisal with an emphasis on near-infrared spectroscopy. j cardiothorac vasc anesth 2018;32:2313-22. doi: 10.1053/j.jvca.2018.03.032. 18. wang y, li l, wang t, et al. the efficacy of nearinfrared spectroscopy monitoring in carotid endarterectomy: a prospective, single-center, observational study. cell transplant 2018;28:1705. doi: 10.1177/0963689718817760. 19. jonsson m, lindstrom d, wanhainen a, et al. near infrared spectroscopy as a predictor for shunt requirement during carotid endarterectomy. eur j vasc endovasc surg 2017;53:783-91. doi: 10.1016/j.ejvs.2017.02.033. 20. parnia s, nasir a, shah c, patel r, mani a, richman p. a feasibility study evaluating the role of cerebral oximetry in predicting return of spontaneous circulation in cardiac arrest. resuscitation 2012;83:982-5. doi: 10.1016/ j.resuscitation.2013.12.007 21. ahn a, yang j, inigo-santiago l, et al. a feasibility study of cerebral oximetry monitoring during the post-resuscitation period in comatose patients following cardiac arrest. resuscitation 2014; 85:522-6. doi: 10.1016/j.resuscitation.2012.01.039 22. yao fs, tseng cc, ho cy, et al. cerebral oxygen desaturation is associated with early postoperative neuropsychological dysfunction in patients undergoing cardiac surgery. j cardiothorac vasc anesth 2004;18:552-8. doi: 10.1053/j.jvca.2004.07.007 23. colak z, borojevic m, bogovic a, et al. influence of intraoperative cerebral oximetry monitoring on neurocognitive function after coronary artery bypass surgery: a randomized, prospective study. eur j cardiothorac surg 2014;47:447-54. doi: 10.1093/ejcts/ezu193. univ med vol. 38 no.2 oktavianus 151 * department of chemistry/ biochemistry, faculty of medicine, lambung mangkurat university **study group for free radicals and utilization of natural substances ***graduate of public health study program, faculty of medicine, lambung mangkurat university ****smf orthopedic surgery, ratu zalecha general hospital, martapura ***** mutiara bunda maternal and child health hospital, martapura correspondence drs. eko suhartono, m.si department of chemistry/ biochemistry, faculty of medicine, lambung mangkurat university jl. a.yani km 36 banjarbaru hp: +6281251126368 email: ekoantioksidan@yahoo.com univ med 2012;31:151-8 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background lead is still a major environmental and occupational health hazard, since it is extensively used in the production of paints, gasoline and cosmetics. this causes the metal to be ubiquitous in the environment, being found in the air, soil, and water, from which it can enter the human body by inhalation or ingestion. absorbed lead is capable of altering the calcium levels in bone. the aim of this study was to demonstrate the effect of lead on bone calcium levels by measuring the reaction constant, gibbs free energy, and enthalpy. methods this study was of pure experimental design using 100 male albino rats (rattus norvegicus). the experimental animals were assigned by simple randomization to two groups, one group receiving lead acetate orally at a dosage of 100 mg/ kgbw, while the other group did not receive lead acetate. the intervention was given for 4 weeks and the rats were observed weekly for measurement of bone calcium levels by the permanganometric method. results this study found that k1 (hydroxyapatite dissociation rate constant) was 0.90 x 10-3 dt-1, and that k2 (hydroxyapatite association rate constant) was 6.16 x 10-3 dt-1 for the control group, whereas for the intervention group k1 = 26.20 x 10-3 dt-1 and k2 = 16.75 x 10-3 dt-1. thermodynamically, the overall reaction was endergonic and endothermic (∆g > 0 and ∆h > 0). conclusions lead exposure results in increased dissociation rate of bone in comparison with its association rate. overall, the reaction was endergonic and endothermic (∆g > 0 and ∆h > 0). key words: rate constant, hydroxyapatite, gibbs free energy, enthalpy, rats increased bone calcium dissociation in lead-exposed rats eko suhartono*,**, yeni wahyu ulfarini***, triawanti*, warih anggoro mustaqim****, rizky taufan firdaus*****, and muhammad hafidz maulana setiawan***** 152 suhartono, ulfarini, triawanti, et al bone calcium dissociation in lead penguraian kalsium dalam tulang lebih cepat pada tikus terpajan timbal abstrak latar belakang timbal masih tetap merupakan risiko kesehatan asal lingkungan atau akibat kerja, karena digunakan secara luas dalam pembuatan cat, keramik, bensin, dan kosmetik. dengan demikian logam ini terdapat di mana-mana dalam lingkungan, karena ditemukan di dalam udara, tanah, air, dan tumbuhan. kemudian logam tersebut dapat memasuki tubuh manusia melalui inhalasi atau melalui makanan dan minuman. timbal yang diabsorpsi dapat mempengaruhi kadar kalsium pada tulang. penelitian ini bertujuan untuk menilai pengaruh timbal terhadap kadar kalsium tulang dengan mengukur konstanta laju reaksi, energi gibbs, dan entalpi. metode sebuah rancangan eksperimental murni dengan prospektif desain mengikutsertakan 100 ekor tikus putih jantan (rattus norvegicus). hewan coba secara acak sederhana dibagi dalam 2 kelompok, yaitu kelompok tikus dengan pemberian larutan timbal asetat per oral 100 mg/kg bb dan kelompok tikus tanpa pemberian larutan timbal asetat. pemberian perlakuan dilakukan selama 4 minggu dan tikus diamati setiap minggu untuk diukur kadar kalsium. kadar kalsium tulang diukur dengan metode permanganometri. hasil hasil penelitian diperoleh k1 (tetapan laju penguraian hidroksiapatit) 0,90 x 10-3 dt-1dan k2 (tetapan laju pembentukan hidroksiapatit) 6,16 x 10-3 dt-1 untuk kontrol sedangkan untuk perlakuan k1 = 26,20 x 10-3 dt-1 dan k2 = 16,75 x 10-3 dt-1. secara termodinamika, keseluruhan reaksi tergolong reaksi endergonik dan endotermis (∆g > 0 dan ∆h > 0). kesimpulan pajanan timbal menyebabkan penguraian kalsium pada tulang lebih cepat dibandingkan dengan pembentukannya. secara keseluruhan reaksi tergolong reaksi endergonik dan endotermis (∆g > 0 dan ∆h > 0). kata kunci: konstanta laju, hidroksiapatit, energi gibbs, entalpi, tikus introduction lead (pb) is a heavy metal belonging to period iv of the periodic table. this element is grey in color, malleable, soft, and can be extracted from the mineral galena (pbs). lead and its compounds is widely utilized by man, e.g. in lead-acid batteries and lead solder, for waterproofing (as roofing material and in lead sheath cables), for shielding against radioactive material and x rays. lead is also used as an additive in gasoline, although the increased production of lead-free gasoline is steadily reducing the use of lead-based additives.(1,2) the increasing use of lead makes that this element may become an environmental pollutant and pose a health hazard to man. lead may enter the body through the digestive and respiratory tracts and the skin. ingested lead is distributed to bones (60%), liver (25%), kidneys (4%), reticuloendothelial system (3%), intestinal wall (3%), and other tissues.(3,4) accumulation of lead in abovementioned organs may cause organ disorders that are mediated by several mechanisms, such as i n a c t i v a t i o n o f e n z y m e s a n d o t h e r macromolecules through binding of sulfhydryl, phosphate, and carboxyl groups.(5) in addition, 153 l e a d m a y i n t e r a c t w i t h v a r i o u s c a t i o n s , particularly calcium, zinc, and iron. the pathological process may be located within the c e l l m e m b r a n e a n d m i t o c h o n d r i a , i n neurotransmitter synthesis and function, heme synthesis, cellular redox status, and nucleotide metabolism. lead toxicity causes physiological, biochemical and structural changes in the skeletal, nervous, urinary, hemopoietic and cardiovascular systems. it may also exert carcinogenic and mutagenic effects on the gastrointestinal and reproductive systems.(6-11) the adverse effects of lead have been amply demonstrated in previous studies. the study by vahedian et al.(12) demonstrated the occurrence of lead-induced inflammatory changes in the rat proventriculus. a study in iran found an increased risk of aplastic anemia associated with lead exposure.in children.(13) furthermore, the study conducted by todorovic et al.(14) revealed that rats exposed to 100 mg pb2+/kg body weight for 30 days may have a 16% decrease in bone calcium. this is due to the greater affinity of pb2+ for bone as compared with that of ca2+, causing pb2+ to substitute for ca2+ in the bone mineral matrix, thus lowering the ca2+ content of the bone. however, the above study did not cover the kinetic aspects a n d e n e rg y c h a n g e s a c c o m p a n y i n g t h e biochemical or bioenergetic reactions. there have been numerous previous studies on bioenergetics. the study by martin et al.(15) has demonstrated the kinetic and t h e r m o d y n a m i c a s p e c t s o f i n h i b i t i o n o f serotonin transporter binding. studies on the effects of lead on bone are also numerous, but no studies have focused on the kinetics and thermodynamics of decreased calcium in bone caused by lead exposure. there is therefore a need to conduct a study on the pathomechanism of the lead-induced decrease in calcium content of bone, by measurement of the reaction rate constant, gibbs free energy (äg) and enthalpy (äh). methods study design this study was of pure experimental prospective design, conducted for 4 weeks and observed at weekly intervals from october to december 2011. study subjects the subjects were male albino rats (rattus norvegicus) assigned to an intervention group receiving lead acetate orally at a dosage of 100 mg/kgbw, and a control group receiving no lead acetate. selection of the animals was by simple random sampling, while the sample size was calculated by means of federer ’s formula, yielding a sample size of 50 rats per group. experimental animals this study used male albino rats (rattus norvegicus) aged 2-3 months, weighing 80-200 grams, obtained from airlangga university, surabaya. before intervention, the rats were acclimatized for 7 days, and kept in separate cages according to their assigned group. during the acclimatization period the animals were given exactly the same animal feed pellets and tap water as were to be used during the i n t e r v e n t i o n . i m m e d i a t e l y b e f o r e t h e intervention, the rats were fasted for 1-2 hours to ensure that their proventriculus was empty. during the 4 weeks of intervention, the rats were given oral lead acetate daily for one week at a dosage of 100 mg/kg, after which 5 of the rats were killed with ether and their bones taken for determination of calcium content by the permanganometric method, as performed in a previous study.(9) this procedure was repeated in the following 3 weeks. the collected bones were cleaned and washed, then left to dry in an oven. the dried bones were then pulverized and passed through a 4 0 m e s h s i e v e . t h e b o n e p o w d e r w a s dissolved in hcl (1:4), the resulting solution univ med vol. 31 no.3 154 suhartono, ulfarini, triawanti, et al bone calcium dissociation in lead was concentrated by evaporation and then placed in a water bath for 1 hour. the dry residue was redissolved in 5 ml concentrated hcl and 50 ml distilled water, then again placed in the water bath for several minutes, and filtered through #52 whatman filter paper. the filtrate was collected in a 200 ml volumetric flask. the precipitate still remaining on the filter paper was washed in distilled water and the wash water was added to the collected filtrate. distilled water was added to the mixture of filtrate and wash water to yield a volume of 200 ml. this solution was labeled aliquot a. a volume of aliquot a equivalent to 2 g bone ash was pipetted into a 300 ml beaker and diluted to 200 ml with distilled water. the solution was made slightly alkaline by the addition of nh 4 oh (1:4) using methyl orange as indicator. hcl (1:4) was then added to slightly acidify the solution, after which 10 ml 0.5 n hcl and 10 ml 2.5 % oxalic acid solutions were added, and the mixture heated to boil. under continuous stirring, 15 ml saturated ammonium-oxalate solution was added. the mixture was further heated until a granular precipitate was formed, then the mixture was left to cool. eight ml of 20% sodium acetate was added and the mixture left for 12 hours. next the mixture was filtered and washed with hot water until free of chloride [washed with hot water and then with a small amount of hcl (1:4) and finally with hot water until free of chloride]. the residue was then put into a beaker by perforating the tip of the filter paper cone with a glass stirrer, then sprinkled with just sufficient hot water until all precipitate had fallen into the beaker. next, 10 ml h 2 so 4 (1:4) was added and the dissolved precipitate heated to near-boiling, left to cool, then titrated with 0.1 n kmno 4 . the calcium content was calculated using the formula: determination of equilibrium constant and reaction rate constants bone contains calcium in the form of h y d r o x y a p a t i t e , w h i c h h a s t h e f o r m u l a ca 10 (po 4 ) 6 (oh) 2 . therefore, in the presence of metal ions of equal charge (valence), the reaction may be simplified as follows (equation 1): where m is a bivalent metal ion (m+2), while k1 is the hydroxyapatite dissociation constant and and k2 the hydroxyapatite association constant. the equilibrium constant is calculated using equation 2: the total reaction rate constant is determined with a first-order reversible kinetics model as in equation 3: where [ca]t is the calcium concentration at time t and [ca]e is the the calcium concentration at equilibrium. determination of thermodynamic parameters ∆∆∆∆∆g and ∆∆∆∆∆h energy changes as well as heat of reaction are expressed as the thermodynamic parameters gibbs free energy (endergonic energy) (∆g) and enthalpy (endothermic internal energy) (∆h). these parameters can be determined using eyring’s equation as follows (equation 4):(16) x1n t 1 [ca]e[ca]t [ca]t 1n t 1 k2k1k = − =+= (3) k2 k1 (oh)])(po[ca (oh)])(po[m k 345 345 == (2) ++ ++ 2345 2 344 ca(oh))(pom k2 k1 m(oh))(poca (1) rt *∆g b e h tk k − = (4) 155 the amount of enthalpy (∆h) is expressed using the van’t hoff equation (equation 5): ∆h = -rt ln k where k=reaction rate constant, k b =boltzman constant (1.381x10 -23 j k -1) , t = a b s o l u t e temperature (310 k), r=ideal gas constant (8.314 j·k-1·mol-1) and h=planck’s constant (6.626 x 10 -34 j s). ethical clearance the present study was approved by the research ethics commission of the faculty of medicine, lambung mangkurat university. ao a tk h b ln data analysis the obtained data were converted into a linear graph of as a function of time, using microsoft exel 2007. the slope of the curve indicates the ∆g value. results the results of bone calcium concentration as a function of time may be seen in figure 1. figure 1 shows that at equilibrium the bone calcium content of the albino rats in the control g r o u p w a s 8 5 . 2 5 % , w h e r e a s t h a t i n t h e intervention group was 39%. the reaction rate constant may be determined by means of a graph as illustrated in figure 2. figure 2. bone calcium dissociation rate as a function of time (� = bone calcium in intervention group; * = bone calcium in control group) (5) univ med vol. 31 no.3 figure 1. bone calcium concentration as a function of time (� = calcium concentrations of intervention group; � = calcium concentrations of control group) 156 suhartono, ulfarini, triawanti, et al bone calcium dissociation in lead figure 2 shows that the bone calcium dissociation rate in the intervention group had a steeper slope than that in the control group. using the expressions in equations (2) and (3), the values of k, k1, and k2 can be calculated, as presented in table 1. table 1 shows that in the control group k2 > k1, signifying that the rate of association of hydroxyapatite in the control group was greater than its dissociation rate. this is in contrast with the intervention group, where k1>k2, signifying that the presence of lead may increase the dissociation rate of hydroxyapatite in comparison with its rate of association. using the data in ta b l e 1 a n d e q u a t i o n s ( 4 ) a n d ( 5 ) , t h e thermodynamic parameters shown in table 2 may be calculated. thermodynamically, the values of ∆g1 and ∆g2 in the control group were larger than those in the intervention group (table 2), indicating that the presence of lead may increase the frequency of spontaneous association and dissociation reactions in hydroxyapatite. discussion the present study shows that the reaction rate constant is a kinetic parameter that can be used to evaluate the reaction rate. the rate of dissociation of calcium in the intervention group was greater than that in the control group. moreover, the association rate of hydroxyapatite in the control group was greater than its table 1. kinetic parameters and equilibrium constants table 2. thermodynamic parameters of bone calcium dissociation in albino rats dissociation rate, while the reverse was true for the intervention group. kinetically, these findings may be explained by the fact that lead and calcium have similar properties. however, lead has a larger electron density than calcium, such that it can be precipitated in the metabolically active parts of bone.(17,18) at present, two compartments may be identified in bone, namely labile trabecular bone and stable cortical bone. lead accumulation in bone continues throughout life, as shown by the occurrence of intrauterine lead deposition in bone. this lead accumulation increases with each exposure, indicating that there is no threshold for lead absorption in bone. approximately 95% of the lead content is stored in bone. a high lead content of bone is associated with bone turnover rate or its demineralization rate, such as occurs i n h y p e r t h y r o i d i s m a n d i m m o b i l i z a t i o n osteoporosis.(17,18) the ability to deposit lead in bone is thought to be a defense mechanism of the body, since the deposited lead cannot react with sulfhydryl groups, thus preventing it from inactivating enzymes and other proteins. nevertheless, the minerals present in the skeletal system are in dynamic equilibrium with minerals from other parts of the body. with each occurrence of bone calcium mobilization, lead is also mobilized. the present study also shows that the free energy changes were lower in the intervention group than in the control group, whereas the enthalpy changes were larger in the intervention group in 157 comparison with the control group. however, in general the reactions could be categorized as being endergonic and endothermic (∆g > 0 and ∆h > 0), signifying that the calcium dissociation reaction did not proceed spontaneously or at most very slowly. t h e i n s p o n t a n e i t y o f t h e c a l c i u m dissociation reaction is presumably due to various factors, one of them being diet, especially dietary intake of calcium. calcium contained in foods is thought to interact with lead in several ways, such as (a) binding of lead in the intestines, causing the lead to become unabsorbable by the small intestine (b) modulating the avidity of intestinal cells for lead, and (c) modulating the affinity of target tissues for lead. these changes in avidity and affinity are regulated by the cholecalciferol endocrine system via 1,25h y d r o x y c a l c i f e r o l a n d c a l c i u m b i n d i n g proteins.(19,20) the results of this study support the report of todorovic et al.(14) that the decrease in bone ca2+ may be caused by the presence of lead. this i s i n d i c a t e d b y t h e g r e a t e r v a l u e o f t h e hydroxyapatite dissociation rate constant (k2) in comparison with its association constant (k1), which had not been revealed by previous studies. therefore, the results of the present study may b e u s e d i n f o r m u l a t i n g a m o d e l f o r t h e development of new drugs that act by decreasing the hydroxyapatite dissociation rate. conclusions lead exposure results in a greater rate of dissocation of calcium as compared with its association rate. overall the reaction can be categorized as being endergonic and endothermic (∆g > 0 and ∆h > 0). in view of this, further studies are indicated to evaluate the effect of lead on bone calcium in men. acknowledgements we thank the dean of the faculty of medicine, lambung mangkurat university for the funding of this study through the dipa pnbp for the year 2012 of the faculty of medicine, lambung mangkurat university. references 1. soemirat j, editor. toksikologi lingkungan. yogyakarta: gadjah mada university press; 2004. 2. drum da. are toxic biometals destroying your children’s future? bio metals 2009;22:679-700. 3. patrick l. lead toxicity, a review of the literature. part i: exposure, evaluation, and treatment. altern med rev 2006;11:2-22. 4. tan m, meiri i. hubungan kadar timbal dalam darah dengan kadar hemoglobin dan hematokrit pada petugas pintu tol jagorawi. j kesehatan masyarakat nasional 2011;6:35-41. 5. devi os, suhartono e, akbar iz. korelasi antara kadar glukosa darah dengan kadar kalsium tulang pada model tikus (rattus novergicus) hiperglikemia. j kedokter yarsi 2010;18:11420. 6. patrick l. lead toxicity part ii: the role of free radical damage and the use of antioxidants in the pathology and treatment of lead toxicity. altern med rev 2006;1:114-27. 7. prozialeck wc, edwards 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piehl ll, et al. lead bone toxicity in growing rats exposed to chronic intermittent hypoxia. bull environ contam toxic 2012;89:693-8. 19. fan g, feng c, li y, wang c, yan j, li w, et al. selection of nutrients for prevention or amelioration of lead-induced learning and memory impairment in rats. ann occup hyg 2009;53:341–51. 20. mohamed he, alhaidary a, beynen ac. calcium metabolism in rats fed diets containing various concentrations of magnesium. res j biol sci 2010;5:215-8. c:\users\jurnal fkusakti\docume 63 *dr. soetomo general hospital, department of orthopedic and traumatology, medical faculty, airlangga university, surabaya, indonesia **resident, dr. soetomo general hospital, department of orthopedic and traumatology, medical faculty, airlangga university, surabaya, indonesia ***jombang general hospital, internship doctor correspondence: komang agung irianto dr. soetomo general hospital, department of orthopaedic and traumatology, medical faculty, airlangga university jalan mayjend prof. dr. moestopo 68 surabaya, indonesia phone: +6231 5501481 email: komang168@yahoo.com orcid id: 0000-0001-6625970x date of first submission, october 5, 2 01 8 date of final revised submission, january 29, 2019 date of acceptance, january 30, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina open reduction in neglected elbow dislocation in children: a case series komang agung irianto*, raymond parung**, and william putera sukmajaya*** background elbow deformity in children due to neglected proper fracture management is a devastating condition. the stiffness and pain complicated the function in daily activity. successful management of neglected elbow dislocation is a challenging problem for orthopedic surgeons. in this study, we aimed to evaluate results of open reduction for neglected elbow dislocation in children. case description this is a case series of 13-14 years old neglected elbow dislocations, for up to 15 months. open reduction after external distractor and followed by intensive rehabilitation was implemented. clinical and functional outcome were evaluated within 4-7 years. initial average elbow flexion was 53,3°, extension was 0°, arc of flexion was 53,3°, arc of pronation-supination was 150° and mayo elbow performance index (mepi) was 80. clinical and functional outcome were evaluated within 4-7 years. at follow-up after open reduction, the improvement in whole range of movement was significant. average elbow flexion was 118,3°, extension was 36,67°, arc of flexion was 81,67°, arc of pronation-supination was 133°. the average improvement of flexion was 65°, arc of flexion was 31,67°, and arc of pronation-supination was 8,3°. the average loss of flexion was 15,5%, arc of flexion was 44,2%, and arc of pronation-supination was 10,7% compared with uninjured side. the average mayo elbow performance index (mepi) was 96,67; all with excellent results. conclusion planned and well execution open reduction in pediatric neglected elbow dislocation may bring back the painless movement within normal daily function. keywords: neglected, elbow dislocation, children, open reduction case report pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.63-68 january-april, 2019 vol.38no.1 cite this article as: irianto ka, parung r, sukmajaya wp. open reduction in neglected elbow dislocation in children: a case series. univ med 2019;38:63-8. doi: 10.18051 /univmed.2018.v38.636 8 64 irianto, parung, sukmajaya treatment of neglected elbow dislocation introduction pediatric elbow deformity mostly caused by unreduced dislocation following neglected proper f racture ma nagement. (1-3) fracture around elbow, especially in children, often caused by fall (domestic, school, playground), not by traffic accident;(4-6) this is the main reason why usually they were taken to bonesetter and not to hospital.(7,8) unfortunately, this type of fracture often associated with elbow dislocation wh i c h pr a ct ic a l ly f a i l t o b e r e du c e d in incompetent hand.(9,10) neglected elbow dislocation limit the range of motion (rom) especially the flexion to nearly 0 -50; the patients could not function their hand for simple daily function (comb, button, feed) or fine motoric (writing).(10-12) open reduction is a good choice of management even when the unreduced joint was untreated for more than three weeks.(7,10,11) several reported cases of several months up to two years elbow neglected dislocation were successfully treated.(7,8,12) however, in pediatric where the open physes is the issue, the precondition and intense post rehabilitation should be considered. we report thre e cases of neglected pediatr ic elbow dislocation treated by external distractor to precondition the soft tissue, open reduction with or without v-y plasty triceps lengthening, kwire stabilization, cast, and intense 6 weeks post-surgery rehabilitation. the mayo elbow performance index (mepi) (13) was used to evaluate the subjective, objective, and functional characteristics prior to the surgery and at follow-up. this scoring system has four parameters: pain, motion, stability, and performance of five activities of daily living (comb, feed, personal hygiene, put on shirt, and shoe (table 1). stability of the elbow is rated as stable (no apparent varus/valgus instability), mod e r a te in s ta bi l i t y ( < 1 0 º var u s / va l gus instability), or gross instability (10º varus/valgus instability). depending on the score, the mepi was classified as excellent (90–100), good (75– 89), fair (60–74), or poor (<60). case presentation the research protocol of this case series was a pproved by institutional ethics and r e vi e w b o a r d i n d r. s o e t o mo g e n e r a l hospital. we report three cases of pediatric neglected elbow dislocation whom underwent o pe n r e d u c t io n i n d r. so e t omo g e ne r a l hospital during january 2012may 2014. the age of patients was 11, 14, and 14 years old. in all cases, the mode of injury was fall with outstretched hand. all patients were presented with the history of bone-setter treatment in the f o r m o f ma s sa ge or ma n i pu l a t i on a n d immobilization for up to fifteen months. elbow stiffness in extension and pain were the principal indication for surgical treatment. the range of motion of flexion, extension, pronation, and supination were examined with a handheld goniometer. patient 2 (figure 1) and patient 3 has associated lateral condyle fracture. the initial average elbow flexion was 53,3° (900, 200, and 500). the average of arm rotation (arc of pronation-supination) was 150° (1300, 1600, and 1600); pronation being more limited than supination; and average of mepi was 80 (85, 65, and 90). the deformity itself did not trouble all male patients but the pain in trying to table 1. mayo elbow performance index (mepi) (2) function points definition (points) pain 45 none (45) mild (30) moderate (15) severe (0) motion 20 arc > 1000 (20) arc 500-1000 (15) arc< 500 (5) stability 10 stable (10) moderate instability (5) gross instability (0) function 25 comb hair (5) feed (5) perform hygiene (5) don shirt (5) don shoe (5) total 100 excellent >90; good: 75-89; fair: 60-74; poor <60 65 do their daily activity is the main reason to seek for surgical cor rection. patient 2 got the complication of ulnar numbness and the mepi was worst (65) (table 2). prior to surgery all patients were put on external distractor for at least 2 weeks, to stretch the soft tissues slowly to achieve easier reduction during surgery. elbow distraction was performed until ulno-humeral joint were in the same level, where the triceps contracture were gradually loosen and no longer resist and pin down the joint. open reduction was performed with or without vy-plasty of triceps lengthening depend on the success perseverance of the distraction. the intraoperative findings of the fibrosis and calcification of the soft tissue determine the further surgical expansion. patient 1 did not need triceps lengthening. but they all need k-wire stabilization and cast afterward. k-wire was extracted after 2 weeks. patient 2 need ulnar nerve transposition to release the trapped nerve. the nerve fibrosis was not severe. rehabilitation was carried soon after kwire removal and wound healed. intensive rehabilitation physiotherapy was maintained for 6 weeks. none of patients got post-surgery skin infection. clinical and functional outcome of all three patients (mepi) were evaluated after 4 years to 6 years. all patients were satisfied and could perform functional range of motion for daily activity (mepi average 96.67; excellent) although deformity is not fully corrected. all three patients showed none of any sign of instability (score:10), pain free (score: 45), and able in most of five activities of daily living. the average elbow flexion was 118,3° (range 110°– 125°), arc of flexion was 81,67° (range 70°– 105°), arc of pronation-supination was 156.67° (range 145°–170°). the improvement in the whole range of move me nt wa s ve r y we l l . n one t he l e s s , compared to the uninjured side, the average loss of flexion was still 15,5% (range 10.7%–18.5%), arc of flexion was 44,2% (range, 27.58%– 53.3%). patient 1 regained functional range of movements and two other patients regained optimum range of moveme nts (tab le 2). hypoesthesia of the hand over the ulnar nerve distribution without appreciable motor weakness was still present in patient 2. all patients in this study had no trouble in performing functional task. discussion for the patients, cosmetic is not the major concern, but the limitation of function is. not only that it is predictable and could be avoided, it also decreases the child’s self-esteem and quality of life when they could not function normal in daily activity especially for personal hygiene. patient 1 came with flexion 00 – 900 after treatment gain almost full flexion into 50 – 1100 (1050 arc of flexion). he gains a perfect function and good cosmetic as well and he was very satisfied with mepi score 100. but patient 2 and 3 still deform (flexion 500 and 550), not a cosmetic but functionally they gain a lot (1200 and 1250). the biomechanical study of normal functional elbow motion from mayo clinic notion 1300 flexion as the normal functioning range of motion where a person could reach the occiput, to comb, to feed, doing personal hygiene, and a patient 14-year-old boy with 4-month-old unreduced complex dislocation of the left elbow: (a) preoperative clinical presentation, (b) lateral view preoperative showing complex dislocation of the left elbow. (c) lateral view postoperative at 6-years follow-up (d), (e), and (f) at 6 years follow-up, the patient has optimum range of movement of the left elbow and has no difficulty in performing functional task for activities of daily living univ med vol. 38 no.1 66 irianto, parung, sukmajaya treatment of neglected elbow dislocation tie his shoe. (10) some of the activities need less flexion arc since there are compensatory motion of adjacent joints. for example, while tying the shoe, they flex the hip. re ga r d l ess d ur a t i on of ne gl e c t e d , regardless the age of the patient, open reduction offers better motion, better stabilisation for fu nct io n. cas e rep ort s a nd ne w sur gic al approach showed good to excellent result.(14) but if there are associated fracture around the elbow, the outcome of the open reduction is not satisfying. (9,15,16) in our cases, patient 2 and 3 with a ssociated late ral co ndyle frac ture, complicating the soft tissue fibrosis, not the alignment of fracture healing. that is the reason for vy plasty triceps muscle lengthening. the contracture of the triceps muscle locks the joint and limit the motion, conversely triceps muscle need to be loosen. more than three decades ago when surgeon reluctant to operate neglected elbow dislocation especially in children, conservative external fixator (hinged elbow distraction device) were applied for 6-8 weeks to gradually reconstruct. this technique was not advocate anymore owe to high failure outcome.(8) in our protocol of treatment, we use external distractor for 2 weeks to gradually distract the soft tissue responsible for the contracture. we used the device as adjunct to open reduction. other reports the similar way prior to surgical reduction either with or without triceps muscle lengthening.(9,12-14) anderson et a l . ( 9 ) d e ve l o p n e w t e c hn i qu e o f t r i c e p s lengthening without vy plasty, by repeated piercing the tendon percutaneous with 18 g nee dle, only for selected case for simple dislocation and without associated fracture. kirschner wire was placed to stabilized and the cast was applied for 2 weeks to allow early rehabilitation which is very important to maintain the reduced joint and avoid further contraction. (12,15,16) the two weeks period was also the reason to prevent skin infection. in our report, none of the patients have skin infection. the a b c table 2. data before and after open reduction for neglected pediatric elbow dislocation patient 1 patient 2 patient 3 gender male male male age (years) 14 14 11 mode of injury fall fall fall injured side left left right first treatment at bonesetter bonesetter bonesetter time of neglected 5 months 4 months 15 months associated fracture none lateral condyle lateral condyle associated lesion none ulnar palsy none treatment: elbow distraction yes yes yes open reduction yes yes yes k-wire stabilization yes yes yes ulnar transposition no yes no triceps lengthening no no yes cast yes yes yes rehabilitation yes yes yes surgery complication none none none follow-up time 4 years 6 years 5 years uninjured elbow: flexion 1450 (-100 1350) 1450 (00-1450) 1500 (-1001400) pronation/supination 1700 (850850) 1800 (900-900) 1650 (750900) injured elbow; before: flexion 900 (00-900) 200 (00-200) 500 (00-500) pronation/supination 1300 (500-800) 1600 (800-800) 1600 (750-850) injured elbow; on follow-up flexion 1050 (50-1100) 700 (500-1200) 700 (550-1250) pronation/supination 1450 (600-850) 1700 (850-850) 1550 (700-850) mepi before / after 85/100 65/95 90/95 67 other technique to stabilize the reduced joint was reported in india by garg et al.(17) by adding cruciate ligament like from semitendinous autologous graft. excellent mepi result was achieved. almost all authors in the last decades advice an earlier rehabilitation for excellent result d is r e ga r di n g t h e pe r i od o f un r e d u c e d e l bow. ( 4 ,1 8,1 9 ) al t h ou gh a l l o ur p a t ie nt s accomplished similar intense rehabilitation, only patient 1 without vy plasty triceps muscle avoid futher contracture (excellent result). patient 2 and 3 managed to benefit enough rom for functional goal and that goal is enough for the patient render the poor cosmetic. conclusions open reduction is a necessity for neglected elbow dislocation in children regardless of the time of the injury. the outcome of the treatment is to gain enough rom for useful hand to achieve painless daily activity. conflict of interest the authors declared no competing and conflict of interests. acknowledgments w e t ha nk a n d a c kn ow l e dge t he contribution of all patients involved in this study. contributors kap contributed to study concept, design and drafting manuscript. rp contributed to acquisition of data. wps contributed to critical revision of the manuscript. all authors have read and approved the final manuscript. references 1. herring ja, ho c. upper extremity injury. in: herring ja, editor. tachdjian’s pediatric orthopaedics: from the texas scottish rite hospital for children.5th ed. philadelphia: elsevier saunders; 2014.p.1264-319. 2. mahaisavariya b. neglected dislocation of the elbow. in: jain ak, kumar s, editors. neglected musculoskeletal injuries. 1st ed. new delhi: jaypee brothers medical publishers (p) ltd;2010.p.31825. 3. mehta s, sud a, tiwari a, et al. open reduction for late-presenting posterior dislocation of the elbow. j orthop surg (hong kong) 2007;15:15– 21. 4. ivo r, mader k, dargel j, et al. treatment of chronically unreduced complex dislocations of the elbow. strat traum limb recon 2009;4:49–55. doi: 10.1007/s11751-009-0064-1. 5. elzohairy mm. neglected posterior dislocation of the elbow. injury 2009;40:197–200. doi: 10.1016/ j.injury.2008.05.034. 6. o’driscoll sw. elbow dislocations. in: morrey bf, sanchez-sotelo j, editors. the elbow and its disorders. 4th ed. philadelphia: w.b. saunders 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doi: 10.5435/ jaaos-d-14-00460. 13. morrey bf. chronic unreduced elbow dislocation. in: morrey bf, sanchez-sotelo j, editors. the elbow and its disorders. 4th ed. philadelphia: w.b. saunders co.; 2008.p.463-71 14. coulibaly nf, tiemdjoa h, sanea ad, et al. posterior approach for surgical treatment of neglected elbow dislocation. orthop traumatol surg res 2012; 98:552-8. doi: 10.1016/j.otsr.2012. 03.006. univ med vol. 38 no.1 68 irianto, parung, sukmajaya treatment of neglected elbow dislocation 15. kapukaya a, ucar by, gem m. open reduction and kirschner wire fixation with triceps lengthening for neglected elbow dislocations. j orthop surg 2013;21:178-81. 16. kachnerkar ni, lakde n, salokhe s. neglected old posterior dislocation of elbow: treatment and results of open reduction. int j orthop sci 2017; 3:1062-6. doi: https://doi.org/10.22271/ortho. 2017.v3.i3o.154. 17. garg p, paik s, sahoo s, et al. a new technique for surgical management of old unreduced elbow dislocations: results and analysis. j orthop allied sci 2014; 2:45-51. doi: 10.4103/2319-2585.145599. 18. kembhavi rs, james b, sugirtharaj j, et al. old unreduced posteromedial elbow dislocation: a rare case report. webmed central orthopaedics 2015;6:wmc004823. 19. maru nd, sayani k, ramesh. a rare case of pediatric neglected elbow dislocation treated with speed procedure. gujarat med j 2015;70:103-4. c:\users\jurnal fkusakti\docume 4 *biomedical sciences doctoral program, faculty of medicine, universitas indonesia **department of anatomy, faculty of medicine, trisakti university ***department of anatomy, faculty of medicine, universitas indonesia †department of biochemistry and molecular biology, faculty of medicine, universitas indonesia ‡departmen t o f obstetrics and gynecology, faculty of medicine, universitas indonesia correspondence tjam diana samara department of anatomy, faculty of medicine, trisakti university jl. kyai tapa no.260-grogol jakarta phone: +6221 5672731 ext.2106 e-mail: dianasamara2@gmail.com date of first submission, september 26, 2 01 8 date of final revised submission, january 29, 2019 date of acceptance, january 30, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina cullin 1 is not associated with late-onset preeclampsia tjam diana samara*,**, isabella kurnia liem***, ani retno prijanti †, and andrijono‡ background late-onset preeclampsia (pe) is preeclampsia occurring after 34 weeks of gestational age or later. cullin 1 (cul1), a proangiogenic protein, is expressed in the placenta, where an imbalance between proangiogenic and antiangiogenic proteins during gestation can cause disturbance of trophoblast invasion. this defect results in vascular ischemia that may produce preeclampsia. the objective of this study was to determine the correlation between cul1 as proangiogenic factor and late-onset preeclampsia. methods this study was of analytical observational cross-sectional design and involved 44 preeclampsia patients with 34 weeks of gestational age (lateonset pe). the cul1 level in the subjects’ sera, taken before they gave birth, and in homogenates of their placenta, obtained per vaginam or by cesarean section, were examined by the elisa technique. statistical analysis was performed with the spearman correlation test with significant p value of <0.05. results median maternal age was 31 years and median gestational age was 37 weeks. median serum cul1 was 41.78 pg/ml and median placental homogenate cul1 was 32.24 pg per milligram of total placental tissue protein. there was no significant correlation between serum cul1 level and late-onset preeclampsia (r=-0.281; p=0.065). there was also no significant correlation between placental cul1 level and late-onset preeclampsia (r=-0.166; p=0.281). conclusion serum cul1 and placental cul1 were not correlated with late-onset preeclampsia. however, this study indicated that low serum cul1 tends to prolong gestational age in preeclampsia. keywords: cul1, proangiogenic, serum, placenta, late-onset, preeclampsia original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.4-9 january-april, 2019 vol.38no.1 cite this article as: samara td, liem ik, prijanti ar, et al. cullin 1 is not associated with late-onset preeclampsia uni v med 20 19;3 8:4 -9 . doi: 10.18051/univmed.2018.v38.4-9 5 univ med vol. 38 no.1 introduction preeclampsia (pe) is the occurrence of hypertension with proteinuria at a gestational age of more than 20 weeks or immediately after delivery.(1) preeclampsia is the main cause of morbidity and mortality, both in mother and fetus. the global incidence of pe is around 28% of all pregnancies, with a maternal mortality rate of around 10%-15%.(2) the most recent pe classification is based on the categories early-onset and late-onset. ear ly-onset pe (< 34 weeks) is generally associated with abnormal uterine arterial doppler findings, fetal growth restriction (fgr), and adverse outcomes to both mother and newborn.(3,4) on the other hand, late-onset pe (34 weeks) is mostly associated with a normal or slightly increased uterine resistance index, a low rate of fetal involvement, and has a more favorable perinatal outcome.(3,5) there are several hypotheses on pe etiology, such as genetic factors, immunological d is o r d e r s , p a t e r n a l f a c t or s , end o t he l i a l dysfunction, and placental ischemia due to trophoblast invasion of the spiral arteries. placental ischemia causes various problems in pregnant women, such as hypertension and proteinuria and abnormal fetal growth. the ischemia is due to functional imbalance between proangiogenic and antiangiogenic factors, resulting in trophoblast invasion, and ultimately causing reduced perfusion of the fetoplacental unit.(6) the distribution of angiogenic factors depends on gestational age.(7) at the onset of a normal pregnancy, the proangiogenic factors are predominant, whereas at the end of pregnancy, the antiangiogenic factors take over, being r eq uir e d f or de li ve r y. t he imba l ance o f angiogenic factors in pe results in a shallow cytotrophoblast endovascular invasion and inadequate vascular formation of the uterine vessels as compared with a normal pregnancy.(6) as a result of the disturbed trophoblast invasion, there is no dilation of the spiral arteries, so causing increased vascular resistance, leading to the signs of hypertension and proteinuria in pe.(8) examples of proangiogenic factors that play a role in trophoblast invasion are vascular endothelial growth factor (vegf),(8) placental growth factor (plgf),(8) transforming growth factor-beta1 (tgf-β1),(9) insulin-like growth factor 2 mrna binding protein 3 (igf2bp3),(10) matrix metalloproteinase (mmp),(11) cullin 1 (cul1),(12) cullin 3 (cul3),(13) cullin 4a (cul4a ),(14,15) and cullin 7 (cul7).(16) with regard to the association between angiogenic factors and late-onset pe, soto et al.(17) found an imbalance between proangiogenic and antiangiogenic factors in patients with lateonset pe, with or without placental lesions and consistent with maternal under perfusion. in contrast, schaarschmidt et al.(18) found increased antiangiogenic factors in early-onset pe, with sflt-1 being higher than in late-onset pe, and a sharp decline in proangiogenic plgf levels as compared with late-onset pe, and yielding a higher soluble fms-like tyrosine kinase-1 (sflt-1)/plgf ratio as compared with late-onset pe. meanwhile, escudero et al.(19) found increases in placental angiogenic factors in both early and late-onset pe. cul1 is a proangiogenic protein in humans from the cullin gene family and is an essential component of the skp1-cul1-f-box protein (scf) e3 ubiquitin ligase complex. cul1 consists of 776 amino acids and has a molecular weight of 89679 daltons. the protein is found in the cytosol and nucleus, and in small amounts in the cytoskeleton, and is localized in chromosome 7q36.1.(20,21) cul1 belongs to the group of cullinbased ubiquitin ligases, that are a subtype of the ring-type ubiquitin ligases and form multisubunit complexes.(22) cul1 plays an important role in protein degradation and ubiquitination, and mediates protein ubiquitination for involvement in cell cycle progression, signal transduction, and transcription. in the scf complex, cul1 serves as a rigid scaffold for assembling of the skp1 and rbx1 subunits.(23) knockout of cul1 in mice produces 6 early embryonic lethality. therefore cul1 plays an essential role in embryonic development and contributes significantly to normal and malignant cellular processes.(24) there is only a single study on cul1 in connection with pe, namely the study of zhang et al.(12) showing that cul1 stimulates human trophoblast invasion and dysregulation of cul1 expression in patients with pe. to date there is no information about studies on the association between cul1 concentration and pregnancies with late-onset pe. the aim of this study was to determine the association between cul1 concentration and gestational age in late-onset pe. methods design of the study this study was of analytical observational cross-sectional design and was conducted in cipto mangunkusumo hospital (rscm) and budi kemuliaan hospital, jakarta, indonesia from april 2017 – january 2018. subjects of the study the study sample comprised women with late-onset pe and gestational age of 34 weeks, who were more than 15 years old, and agreed to participate in this study by signing a declaration of informed consent. the exclusion criteria of the study subjects were diabetes mellitus and chronic renal failure. venous blood samples and placentas were collected to a total of 44 samples from women with pe at gestational ages of 34 weeks who were delivered per vaginam or by cesarean section. pe is defined and classified based on the criteria recommended by the american college of obstetrics and gynecologists (acog) in 2013, namely hypertension appearing and developimg after 20 weeks of pregnancy (systolic pressure 140 mmhg and/or diastolic pressure 90 mmhg), and proteinuria (300 mg in 24-hr urine) or with protein-positive 1 (+) reading from midstream urine. preeclampsia may be without proteinuria but must have one of the following symptoms: thrombocytopenia (<100.000/ microliter), renal insufficiency (serum creatinine >1.1 mg/dl), abnormal liver function (two-fold increases in transaminases as compared to normal), pulmonary edema, and cerebral or visual disturbances.(1) serum cul1 measurement a volume of 5 ml of maternal blood was collected less than 24 hours before delivery and subseque ntly pr oce ssed into ser um by centrifugation at 3500 rpm for 10 minutes. the serum was collected and stored at a temperature of -800c. after the required number of blood samples was reached, the cul1 level was determined (mybiosource mbs921353) by means of the elisa technique according to the protocol from mybiosource. placental cul1 measurement the placental cotyledons (maternal side) were cut up into cubes of 0.5x0.5x0.5 cm immediately after vaginal delivery or delivery by cesarean section. a total of 100 mg of placental tissue was washed in pbs and then processed to obtain a homogenate in 1 ml of pbs and stored at -800c. after the required number of placental samples was reached, the cul1 level was determined (mybiosource mbs921353) by means of the elisa technique according to the protocol from mybiosource. ethical clearance this study obtained ethical clearance from the health research ethics committee, faculty of medicine, universitas indonesia, under no. 34/ un2.f1/etik/2017. statistical analysis the normality of the data was determined using the kolmogorov-smirnov test. since the data turned out to be non-normally distributed, the non-parametric spearman correlation test was used to determine the association of serum cul1 and placental cul1 levels with late-onset pe. a p-value of <0.05 was considered significant. samara, liem, prijanti, et al cullin 1 and late-onset preeclampsia 7 univ med vol. 38 no.1 results table 1 shows that the median age was 31 years and the median gestational age 37 weeks. median systolic blood pressure was 160 mmhg and median diastolic blood pressure 100 mmhg. median qualitative urinary protein reading was 2. median serum cul1 was 41.78 pg/ml and median placental homogenate cul1 was 32.24 pg/mg of placental tissue protein. median total placental tissue protein was 9.82 mg/ml. placental cul1 level was calculated from homogenate cul1 divided by total placental tissue protein, to obtain pure cul1 protein per total placental tissue protein. in table 2 it is apparent that there was no significant association between pe maternal serum cul1 concentration and gestational age of 34 weeks (r=-0.281; p=0.065). similarly, there was no significant association between placental cul1 concentration and gestational age of 34 weeks (r=-0.166; p=0.281). discussion late-onset pe is pe occurring at 34 weeks of pregnancy or later. there are few studies on late-onset pe in connection with angiogenic f a c t or s , i n c l ud i ng st u d ie s on cul1 a s proangiogenic factor in pregnancy. there are indeed several studies on cul1, but not in connection with pe in pregnancy. the study conducted by choudhury et al.(25) showed that cul1 is part of the scf complex that mediates ubiquitin-dependent cyclin g1 degradation and cyclin-dependent kinase inhibitors. the scf complex consists of skp, cullin, and the f-box containing complex. staropoli et al.(26) proved that the f-box and wd40 domain protein 7 (fbxw7) and cul1 are located inside a distinct ubiquitin ligase complex that targets its ligase activity on cyclin e in the regulation of neuronal apoptosis. deficiency of the scf-skp2 e3 ligase complex results in breast cancer metastases, impaired rhoa expression, cell migration, and cell invasion.(27) our study comprises 44 samples from pe study subjects with gestational age of 34 weeks. according to the categories of earlyand late-onset pe, a gestational age of 34 weeks b e l on gs to t h e l a te -on s e t c a t e go r y. ( 4 ) theoretically, in a normal pregnancy, the later the gestational age, the lower the concentrations of proangiogenic proteins, followed by increased concentrations of antiangiogenic proteins. in pe t he c o nd i ti on s a r e r e ve r s e d , wh e r e t he antiangiogenic concentrations are increased at the onset of pregnancy.(6) however, there are as yet no further insights in connection with the changes in proangiogenic protein concentrations in patients with pe. fu et al. found another member of the cullin family, i.e. cul7, that in the form of the cul7 e3 ligase increases trophoblast migration and induces trophoblast epithe lial-mesenchymal transition (emt) through upregulated expression of zeb1 and sl ug, t wo r e pr e s so r s of e-c a d h e r i n transcription.(16) zhang et al.(13) in their . study a b c variable n median maternal age (years) gestational age (weeks) systolic pressure (mmhg) diastolic pressure (mmhg) urinary protein (qualitative) serum cul1 (pg/ml) placental cul 1 (pg/mg) placental protein (mg/ml) 44 44 43 43 32 44 44 44 31 37 160 100 2 41.78 32.24 9.83 table 1. distribution of features characteristics of the subjects variables r p-value serum cul1 – gestational age of 34 weeks homogenate cul1 – gestational age of 34 weeks -0.281 -0.166 0.065 0.281 r = spearman correlation coefficient table 2. association between cul1 concentration and gestational age of 34 weeks 8 on cul3 found that cul3 plays a role in increased trophoblast invasion and migration and the occurrence of cul3 dysregulation may cause pe. however, there is just a single study on cul1 in connection with trophoblast invasion in pe. zhang et al.(12) found by means of realtime q pcr and western blot assays, that cul1 expression significantly decreases in primary h uma n p l a ce n t a l c yt o t r op h ob la s t d ur in g s yn c yti a li za t i o n. cu l1 e xp re s s i on w a s s i gn i f i c a nt ly l ow e r i n p l a c e n ta l vi l li i n preeclampsia, as compared with equivalent placental controls. these investigators also found that cul1 plays a role in enabling trophoblast invasion. the study subjects used by zhang et al. were pe patients aged 23-35 years with mature pregnancies (36-40 weeks). in contrast, our study used study subjects of age 20-43 years and did not find any association between pe gestational age of 34 weeks and cul1 concentration, both in serum and placental homogenate. since zhang et al.(12) did not determine the serum cul1 concentration in patients with pe, there can be no comparison of serum cul1 concentration in patients with pe with those of controls. the theory expressed by tomimatsu et al.(6) does not mention decreased proangiogenic protein concentration as one of the causes of pe, but that imbalances in angiogenic factors are one of the causes of pe, either through ear ly or excessive antiangiogenic protein production. further studies are needed to deter mine the extent of proangiogenic factor involvement in pe angiogenic factor imbalance. our study did not include the gestational age of <34 weeks, so that it is not possible to compare the changes in cul1 concentration between late-onset (34 weeks) and early-onset pregnancy. in our study no normal pregnancies were used, which is one of the limitations of this study. the knowledge about the association of cul1 concentration with gestational age is required to allow the use of the cul1 concentration as a marker for monitoring pe development in connection with gestational age. conclusions the serum cul1 concentration as well as the placental cul1 concentration are not associated with pe gestational age of 34 weeks. further studies are needed using pe gestational age of 34 weeks to determine the presence or absence of differences in cul1 concentrations between these differing pregnancy groups. these studies are needed to determine the extent of the role of angiogenic factors, in this case cul1, in connection with pe development by gestational age. conflict of interest competing interest: no relevant disclosures. acknowledgment this study was funded by the directorate of research and community service, directorate general of researc h and development reinforcement, indonesian ministry of research, technology, and higher education (no: 011/km/ /bap/i/pnt/2018). contributors tds contributed to compilation of the subject matter, writing of the manuscript drafts, data collection, and data analysis. ikl contributed to manuscript finalization. arp contributed to the study concept and design. a contributed to manuscript revision. all authors have read and approved the final manuscript. references 1. woelkers d, barton j, von dadelszen p, et al. 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*department of public health, faculty of medicine, hitit university, çorum, turkey **department of nursing faculty of medicine, hitit university, corum, turkey correspondence @gulay yilmazel, assoc.prof.dr. department of public health, hitit university faculty of medicine, samsun cd. 19000 corum, turkey email: dryilmazelgul@gmail.com orcid id: http://orcid.org/00000002-2487-5464 date of first submission, april 24, 2019 date of final revised submission, november 26, 2019 date of acceptance, november 27, 2 01 9 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina limited health literacy is associated with poorer clinical outcomes in elderly with type 2 diabetes mellitus gulay yilmazel*@ and remziye cici** background diabetes mellitus is a significant global public health concern. poor knowledge of disease and healthcare utilization is associated with poor health outcomes, leading to increasing burden of diabetes in many developing countries. the present study aimed to assess diabetes health literacy and clinical outcomes in elderly patients registered to the home health agency. methods a cross-sectional study was conducted in the city of çorum, turkey, with 160 type 2 diabetic patients of both sexes and aged between 50-91 years. to identify health literacy, the rapid estimate of adult literacy in medicine test was administered to the patients. clinical parameters were based on routine medical examinations by measuring blood pressure levels, glycosylated hemoglobin, and lipoprotein levels. in order to identify the risk of depression, the beck depression scale was used. results of the patients, 85.0% had limited health literacy. the majority of patients (95.0%) had poor glycemic control and limited health literacy was associated with having high level of hba1c (p<0.05). adequate health literacy was associated with regular foot care (p<0.05). also patients with limited health literacy were more likely to have depression (p<0.001). limited health literacy increased the risk of poor glycemic control (or=6.82;95% ci=1.34-9.78) and retinopathy (or=6.91;95% ci=1.23-9.44). conclusion limited health literacy is consistently associated with poorer diabetes clinical outcomes in elderly type 2 diabetes melltius contents of diabetes education should be arranged according to patients’ health literacy level which requires visual and auditory teaching materials for patients with limited health literacy. keywords: health literacy; diabetes mellitus; outcomes; home health agency original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.179-185 may-august, 2019 vol.38no.3 cite this article as: gilmazel g, cici r. limited health literacy is associated with poorer clinical outcomes in elderly with type 2 diabetes mellitus. univ med 2019;38:179-85. doi: 10.18051/ univmed.2019.v38.179-185 180 yilmazrl, cici limited health literacy and type 2 diabetes introduction health literacy is a broad concept that includes abilities for reading, understanding and navigating health information in the health system to make relevant appropriate decisions.(1) health literacy plays a vital role in self-management of chronic disease which accounts for 44% of the global burden of disease.(2) evidence suggests that limited health literacy is a common condition which has considerable impact on chronic conditions such as type 2 diabetes mellitus (dm), asthma, acquired immune deficiency syndrome (aids) and hypertension.(3-6) according to the global status report on non-communicable disease, 89 million disability-adjusted life years (dalys) was attributed to dm with estimated prevalence of 9% in 2014.(7) patients with diabetes are at increased risk of poor health outcomes (cardiovascular disease, strokes, amputations, blindness, and end-stage renal disease) and poor control of clinical outcomes (e.g., blood pressure, lipoprotein levels, glycemic control).(8-10) type 2 diabetes is a classic public health problem affecting 6,503,027 turkish people with 13.7% prevalence.(11) in turkey, the home health agency is a part of public health services for patients who are in a disadvantageous position due to severe illness. the population of home health care are mostly elderly. patients with asthma, paralysis, ser ious r espirator y insufficiency, those completely bed-ridden or physically disabled, and those with terminal cancer and severe muscle disease utilize the services in their homes. diabetic patients with any complications can also utilize these services. elderly individuals are one of the groups at risk of limited health literacy to implement tasks for disease control individually.(12,13) aging results in declined cognitive functions and therefore elderly patients feel stressful about managing diabetes tasks. studies give an impression that elderly patients with diabetes do not appear to be receiving diabetes care at optimum level.(14) several studies have reported that low health literacy is related to negative health outcomes such as poor health status (15) and inadequate disease management.(16) evidence examining the link between low health literacy and glycemic outcomes are mixed. some studies have demonstrated links between higher levels of health literacy and better glycemic control, while others have failed to demonstrate an association.(17,18) the distinguishing aspect of the present study is that participants were taking regular home care services. the two hypotheses of this study are that patients with diabetes have limited health literacy and that these patients would be less likely to control clinical outcomes such as glycosylated hemoglobin (a1c), blood pressure and low density lipoprotein (ldl) level. the present study aimed to assess health literacy and clinical outcomes in elderly type 2 diabetic patients registered to the home health agency. methods research design this cross-sectional study was conducted between february-march 2019 in the city of çorum in turkey. study subjects patients were recruited from the home health agency. in çorum city, 1670 patients utilize this service and 187 diabetic patients were included in the study. the calculated minimum sample size was 156 patients, based on the prevalence of dm in turkey of 13.7%.(11) among those diabetic patients, 27 were excluded because of illiteracy, hearing impairment, psychotic disorder, dementia, blindness, aphasia, end-stage cancer and renal disease (these conditions may be a barrier to accurately measure health literacy). a total of 160 type 2 diabetic patients of both sexes and aged between 50-91 were enrolled in the study. pa t i e nt s we r e o r al l y a d mi ni s te r e d a questionnaire form regarding self-reported sociodemographic information (age, educational level, monthly income, marital status, self-reported 181 health, smoking status, history of hypertension) and self-management behaviors (measuring blood glucose, adherence to diet and treatment, foot care). information on diabetes condition (current treatment, complications) was collected from the patients’ files. measurements health literacy to identify health literacy, the patients were then administered the rapid estimate of adult literacy in medicine (realm) test, which is a widely acknowledged test and is a practical way of measuring reading, pr onunciation and comprehension of terms. it was developed by davis et al.(19) and converted into and validated as the turkish version by özdemir et al.(20) the test scoring is based on individual responses and correct responses take “1” point. health literacy level is structured according to total score (066) as follows: inadequate (0-44), marginal (4560), adequate (61-66). in this study, health literacy was grouped in two levels as adequate and limited (marginal and inadequate) health literacy. depression in order to identify the risk of depression and level of depressive symptoms, the beck depression scale, including 21 self-assessment items, was used. (21) the scale was developed by beck (21) and validated as the turkish version. (22) each item takes a score between 0-3 and the total score range is 0-63. the depression score was categorized into d”16 and e”17. clinical measurements following the questionnaire, systolic and diastolic blood pressure levels were measured. t he n , th e p r o c e s s o f c ol l e c ti n g cl i ni c a l parameters was carried out for routine medical examinations by home health nurses. blood samples were obtained to measure hba1c and low density lipoprotein (ldl) levels. after testing, measurement values were obtained from online-laboratory systems. poor glycemic control was defined as hba1c >7%. statistical analysis data management and analysis were performed using spss 17.0. for categorical va r i a b l e s, fi sh e r ’s e x a c t t e st wa s us e d . multivariate logistic regression was used to predict the clinical outcomes of limited health literacy. a p<0.05 value was accepted as significant. ethical approval t he pr oce d u r e s o f t h i s st u dy w e r e performed in accordance with the helsinki principles and approved by the hitit university non-interventional research ethics committee (under no. 2019-198). informed consent was obtained from all individual participants included in the study. results a total of 160 elderly diabetic patients participated in this study with a mean age of 65.6 (sd:12.6) years. of those, 47.5% were women and 52.5% were men. in the study group, 88.1% had primary education or lower. table 1 shows patient characteristics according to health literacy level. of the patients, 85.0% had limited (20.6% marginal, 64.4% inadequate), and 15.0% had adequate health literacy. there were no differences in patients’ gender, marital status, perceived health, smoking status, duration of diabetes, treatments, history of hypertension ( h tn) , l dl, b l oo d p r es s ur e l e ve l s a n d macrovascular complications according to health literacy level (p>0.05). limited health literacy was significantly associated with being older, ha ving less education, and lower income (p<0.05). the majority of patients (95.0%) had poor glycemic control (hba1c>7). limited health literacy was associated with a high level of hba1c (p<0.05). conversely, adequate health literacy was associated with regular foot care (p<0.05). approximately three in four patients univ med vol. 38 no.3 182 yilmazrl, cici limited health literacy and type 2 diabetes a b c all n=160 (100.0%) limited n=136 (85.0%) adequate n=24 (15.0%) p* age, mean (sd), years 65.6 (12.6) 66.7 (12.7) 59.5 (10.9) 0.014 sex female 76 (47.5) 64 (47.1) 12 (50.0) 0.790 male 84 (52.5) 72 (52.9) 12 (50.0) education 0.000 primary and below 141(88.1) 128 (94.1) 13 (54.2) secondary and over 19 (11.9) 8 (5.9) 11 (45.8) marital status 0.156 married 114 (71.3) 94 (69.1) 20 (83.3) not married 46 (28.8) 42 (30.9) 4 (16.7) income 0.025 ≤$500 32 (20.0) 23 (16.9) 9 (37.5) >500 128 (80.0) 113 (83.1) 15 (62.5) perceived health 0.576 good 28 (17.5) 22 (16.2) 6 (25.0) moderate 45 (28.1) 39 (28.7) 6 (25.0) poor 87 (54.4) 75 (55.1) 12 (50.0) smoking status 0.287 current 33 (20.6) 27 (19.9) 6 (25.0) former 36 (22.5) 28 (20.6) 8 (33.3) none 91 (56.9) 81 (59.6) 10 (41.7) duration of diabetes (mean years) 12.4 (8.8) 12.8 (8.8) 9.7 (8.9) 0.052 treatments for diabetes 0.483 only oral hypoglycemic 77 (48.1) 68 (50.0) 9 (37.5) only insulin regimen 37 (23.1) 31 (22.8) 6 (25.0) insulin and oral hypoglycemic 46 (28.8) 37 (27.2) 9 (37.5) self-management behaviors adherence to diet 58 (36.3) 51 (37.5) 7 (29.2) 0.167 adherence to treatment 104 (65.0) 92 (67.6) 12 (50.0) 0.059 regular foot care 94 (58.8) 73 (53.7) 21 (87.5) 0.008 reported history of htn 43 (26.9) 38(27.9) 5 (20.8) 0.469 clinical outcomes hba1c, mean (sd) 8.7 (3.1) 8.9 (3.2) 7.3 (1.8) 0.016 ldl cholesterol, mean (sd) 113.3 (43.4) 114.1 (39.8) 109 (61.0) 0.334 systolic blood pressure, mean (sd) 126.5 (19.3) 126.3 (20.0) 127.9 (15.0) 0.645 diastolic blood pressure, mean (sd) 75.1 (11.8) 74.5 (12.2) 74.8 (8.5) 0.320 microvascular complication retinopathy 52 (32.5) 50 (36.8) 2 (8.3) 0.006 nephropathy 20 (12.5) 18(13.2) 2 (8.3) 0.503 neuropathy 65 (40.6) 57 (41.9) 8 (33.3) 0.430 macrovascular complication cerebrovascular disease 47 (29.4) 40 (29.4) 7 (29.2) 0.981 coronary artery disease 11 (6.9) 10 (7.4) 1 (4.2) 0.486 bds depression score 17 119 (74.4) 119 (87.5) 0 (0.0) 0.000 depression score 16 41 (25.6) 17 (12.5) 24 (100.0 table 1. general features of the subjects by health literacy *fisher’s exact test was used for categorical variables; the kruskal-wallis test was used for continuous variables. abbreviations: hypertension (htn), glycated hemoglobin (a1c); low density lipoprotein (ldl); beck depression scale (bds) h a d d e p r es s io n a nd t h e me a n s c o r e f or depression was 30.7 (sd:16.8). patients with limited health literacy were more likely to have depression (p<0.001). table 2 shows associations between clinical outcomes and limited health literacy. limited health literacy was 6.82 times significantly higher in patients with poor glycemic control 183 (hba1c>7%) and 6.91 times significantly higher in patients with retinopathy (or=6.82;95% ci=1.34-9.78 and or=6.91;95% ci=1.23-9.44, respectively) (table 2). there was no signiûcant association between limited health literacy and other diabetes complications. discussion as mentioned in the reviewed literature, a chain of evidence links health literacy and diabetes care.(23-25) this is the first study reporting the effect of limited health literacy on poor clinical outcomes in turkish diabetic home health care patients. in the current study, the majority of diabetic patients (85%) had limited health literacy. the results of this study are consistent with international studies. (26,27) socioeconomic factors (such as aging, education, income, etc.) have a dominant role in the maintenance of adequate health literacy. prior studies have noted the problem of limited health with advanced age, less education and low income.(28,29) the current study supports the previous studies, in that 94.1% of patients with less education and 83.1% of patients with low income had limited health literacy. these factors can contribute to high rates of poor health literacy, severe complications and poor clinical outcomes in diabetic patients. diabetes complications remain a major challenge despite current clinical success. patients have to suffer from multiple complications especially foot ulcers, vision and renal problems. maintaining relevant food hygiene and adhering to medical treatment and diet are at the heart of univ med vol. 38 no.3 limited health literacy aor cl p poor glycemic control (hba1c >7%) 6.82 1.34-9.78 0.017 retinopathy 6.91 1.23-9.44 0.019 nephropathy 1.63 0.17-3.31 0.576 neuropathy 1.12 0.38-3.56 0.820 table 2. analysis of multiple binary logistic regression between several risk factors and limited health literacy aor : adjusted or for age and sex quality self-management of diabetes care. however, self-management behaviors in chronic disease vary depending on patients’ health literacy skills.(30-32) this study demonstrated that regular foot care was more common in patients with adequate health literacy. glycosylated hemoglobin is an leading clinical indicator of diabetic complications.(33) many guidelines for diabetes recommend a target hba1c level of less than 7%.(34-36) however, studies reported that diabetic patients with limited health literacy have poorer glycemic control and higher levels of retinopathy than have those with adequate health literacy level.(37) one important and similar finding is that poor glycemic control and retinopathy was more frequent in patients with limited health literacy. among clinical outcomes hba1c and retinopathy had a significant correlation with limited health literacy. depression is an obvious accompanying psychiatric problem in patients with diabetes.(38,39) in the present study depression was more common in diabetic patients with limited health literacy. depression can be an aggravating circumstance for adverse outcomes which also may dash patients’ hopes and restrict diabetes care. one of the limitations of this study was that it included only home care patients. health literacy should be seen as an important component of health education in preventing complications and sequelae in diabetic patients and low health literacy skills should not be ignored in patients receiving home care. the first step of home care services for diabetes should be to increase patients’ health literacy skills. 184 yilmazrl, cici limited health literacy and type 2 diabetes conclusions patients with limited health literacy had poorer glycemic control and foot care, also had more retinopathy and higher 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literacy and the use of an internetbased patient portal in an integrated health system—results from the diabetes study of northern california (distance). j health commun 2010;15 suppl 2:183-96. doi: 10.1080/ 10810730.2010.499988. 27. anbarasan s, gurtoo a, srinivasaan m , ap mk. level of health literacy among type 2 diabetic persons and its relation to glycemic control. j assoc physicians india 2019;67:59-62. oktavianus 146 *department of anatomy, faculty of medicine, sultan agung islamic university **department of parasitology, faculty of medicine, diponegoro university ***department of anatomy, faculty of medicine, diponegoro university ****department of pathologic anatomy, diponegoro university correspondence dr. chodidjah, m.kes. department of anatomy, faculty of medicine, sultan agung islamic university jl. raya kaligawe km.4, semarang 50112 email: chodiab@yahoo.com univ med 2013;32:146-54 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background worldwide, breast cancer is the most frequent cancer in women after lung cancer. treatments include surgery, radiation, immunotherapy and chemotherapy, but are not effective. tyrosine kinase and ki67 protein are markers of proliferation. typhonium flagelliforme ethanol extract (tfee) has been shown to inhibit proliferation of michigan cancer foundation-7 (mcf7) cells in culture. the aim of the present study was to examine the effect of administration of tfee on tyrosine kinase and ki67 expression in mice. methods this experimental study using post test randomized design with control group was conducted in 24 tumor-bearing ch3 mice. they were randomly divided into 4 groups, consisting of one control and 3 treatment groups (ti, t2, t3) treated daily for 30 days with 0.2 ml tfee at dosages of 200, 400, and 800 mg/kgbw, respectively. on day 31 the tumor tissues were collected and their tyrosine kinase and ki67 expression were levels assessed using elisa and immunohistochemical staining, respectively. tyrosine kinase and ki67 expression levels were analyzed, respectively using kruskal wallis test and one-way anova followed by bonferroni post hoc test. results mean tyrosine kinase level was highest in the control group, followed by t3, t2 and t1 (p=0.019). mean level of ki 67 expression was highest in the control group, followed by t2, t3 and t1 (p=0.000). conclussions oral administration of tfee at a dose of 200 mg/kgbw decreases tyrosine kinase levels and ki 67 expression. key words: typhonium flagelliforme, tyrosine kinase, ki67 expression, mammary adenocarcinoma, mice typhonium flagelliforme decreases tyrosine kinase and ki67 expression in mice chodidjah*, edi dharmana**, hardhono susanto***, and sarjadi**** 147 umbi keladi tikus menurunkan kadar enzim tirosin kinase dan protein ki67 pada mencit latar belakang kanker payudara merupakan jenis kanker terbanyak pada wanita di seluruh dunia setelah kanker paru. pengobatan kanker payudara dengan obat, radioterapi, immunoterapi dan kemoterapi masih belum memuaskan. enzim tirosin kinase dan protein ki67 merupakan parameter proliferasi sel kanker. umbi keladi tikus (typhonium flagelliforme) diketahui dapat menghambat proliferasi kultur sel michigan cancer foundation-7 (mcf7/adenokarsinoma mamma). tujuan penelitian ini adalah untuk menilai efek ekstrak etanol umbi keladi tikus terhadap kadar enzim tirosin kinase dan ekspresi protein ki67 pada mencit. metode studi eksperimen dengan post test randomized design with control group digunakan pada 24 ekor mencit c3h bertumor adenokarsinoma mamma. secara random mencit dikelompokkan menjadi kelompok kontrol (k), kelompok perlakuan 1 (p1), kelompok perlakuan 2 (p2), dan kelompok perlakuan 3 (p3) masing masing diberi 0,2 cc ekstrak umbi keladi tikus dosis 200, 400 dan 800 mg/kgbb peroral perhari selama 30 hari. hari ke 31, jaringan tumor mencit diambil, sebagian diperiksa kadar enzim tirosin kinase dengan metode elisa, sebagian dibuat preparat blok parafin serta dilakukan pengecatan imunohistokimia. data kadar enzim tirosin kinase di uji dengan uji kruskal wallis. data ekspresi protein ki67 diuji menggunakan one way anova dilanjutkan dengan uji benferoni. hasil terdapat perbedaan penurunan kadar enzim tirosin kinase yang bermakna antara kelompok kontrol dengan t1, t2 dan t3 (p=0,019). rerata ekspresi ki67 tertinggi pada kelompok kontrol, diikuti t2, t3 dan yang paling rendah kelompok t1 (p=0,000). kesimpulan pemberian ekstrak umbi keladi tikus dosis 200 mg/kgbb dapat menurunkan kadar enzim tirosin kinase dan ekspresi protein ki67. kata kunci: typhonium flagelliforme, tirosin kinase, ki67, adenokarsinoma mamma, mencit abstrak introduction breast cancer is the most frequently found cancer in women worldwide after lung cancer. the number of new cancer cases in the us was 1,638,910 in 2012, with a mortality of 577,190 persons.(1) in indonesia, breast cancer is the most commonly found cancer after cervical malignancies.(2) the mortality rate of patients with breast cancer worldwide is from 6 to 9 per 100,000 women. in europe, in the period of 1998 to 2002 there were 420,800 cases in the age range of 50 to 60 years, with a mortality rate of 129,300 persons. in europe, in the year 2008, among 238.8 million inhabitants 7.5% had breast cancer.(3) radiotherapy following mastectomy for treatment of breast cancer carries the risk of e s o p h a g e a l c a n c e r a f t e r 5 1 0 y e a r s . ( 4 ) chemotherapy administered after mastectomy serves mainly as adjuvant therapy to reduce r e c u r r e n c e . t h e m o d e o f a c t i o n o f chemotherapeutic drugs is to decrease the enzyme tyrosine kinase in cellular proliferation univ med vol. 32 no.3 148 pathways, e.g. irresa/gefitinib (tyrosine kinase receptor inhibitor). (5) the side effects of chemotherapeutic drugs include depression of the immune system, nausea, vomiting, loss of hair, changes in the menstrual cycle, infections of the oral cavity, and fatigue.(6) immune and cytokine therapy are aimed at strengthening the immune system against cancer, but the results are still unsatisfactory. tumor cell proliferation is marked by increases in tyrosine kinase and vascular endothelial growth factor (vegf) levels, which are produced by the tumor cells and may be m e a s u r e d b y m e a n s o f e l i s a . histopathologically, the levels of tumor cell proliferation markers may be seen from the expression of the ki67 protein found in the nucleus. the ki67 protein in normal tissues is expressed at very low levels, but is increased in breast cancer and can be measured by immunohistochemical methods.(7,8) bioflavonoids, alkaloids, and other natural plant substances are known to be able to inhibit cell proliferation, thus constituting potential anticancer agents. for example, flavonoids are reportedly capable of inhibiting tyrosine kinase activity and tumor growth in vivo.(9) an extract of the tubers of typhonium flagelliforme (rodent tuber, umbi keladi tikus) is known to inhibit cancer cell proliferation.(10,11) an ethanol extract of typhonium flagelliforme added to m i c h i g a n c a n c e r f o u n d a t i o n 7 ( m c f 7 ) mammary cell cultures at a concentration of 89.15 µg/nl can inhibit cell proliferation by 50% within 24 hours. (9) the hexane and dichloromethane fractions of typhonium flagelliforme tubers are known to inhibit the growth of ncl-h23 cells.(11,12) an extract of t h e d i c h l o r o m e t h a n e ( d c m ) f r a c t i o n o f typhonium flagelliforme administered orally at dosages of 200, 400 and 800 mg/kgbw to leukemic balb/c rats for 28 days resulted in d e c r e a s e d n u m b e r s o f p e r i p h e r a l b l o o d immature granulocytes.(12) to date, there have been few invitro and invivo studies on ethanol extracts of typhonium flagelliforme. therefore, there is a need to study the invivo effect of typhonium flagelliforme ethanol extracts on cancer cell proliferation. the aim of the present study was to evaluate the effect of a typhonium flagelliforme ethanol extract (tfee) on t y r o s i n e k i n a s e l e v e l s a n d k i 6 7 p r o t e i n e x p r e s s i o n l e v e l s i n c 3 h m i c e b e a r i n g mammary adenocarcinoma tumors. methods study design an experimental study using post test randomized design with control was conducted from october 2012 up to april 2013 at the l a b o r a t o r y f o r e x p e r i m e n t a l p a t h o l o g i c a n a t o m y a n d t h e b i o m o l e c u l a r a n d b i o c h e m i s t r y l a b o r a t o r i e s , f a c u l t y o f medicine, university of indonesia, jakarta, and at the prof dr sardjito hospital, yogyakarta. experimental animals the subjects of this study were 25 tenweek old c3h mice weighing 19-20 grams kept at the laboratory for experimental pathologic anatomy, faculty of medicine, university of indonesia. the animals were adapted for one w e e k , t h e n i n o c u l a t e d w i t h m a m m a r y adenocarcinoma cells. tumors were already p a l p a b l e a t d a y 1 0 , a n d o n e m o u s e w a s sacrificed for preparation of tumor tissue paraffin blocks with hematoxyline-eosin staining. preparation of typhonium flagelliforme ethanol extract one-year old typhonium flagelliforme plants were obtained from the area around jalan sri rejeki, west semarang, for utilization of their tubers. tfee was prepared at the center f o r d r u g d e v e l o p m e n t f r o m n a t u r a l substances, diponegoro university research institute, semarang. fresh tubers weighing 1 kg were washed in running water, cut into small pieces and left in a drying chamber at 400c for 48 hours. the chodidjah, dharmana, susanto, et al typhonium flagelliforme and marker proliferation 149 univ med vol. 32 no.3 dried material was ground to powder in a mill grinder. the powder was extracted in a soxhlet apparatus with ethanol as the solvent, then evaporated to a concentrate in a rotary vacuum evaporator at 700c. the concentrate was diluted with water to yield 20 mg/ml, 40 mg/ml and 80 mg/ml, respectively. intervention the prepared dosages were administered at the laboratory for experimental pathologic anatomy, university of indonesia. a total of 24 tumor-bearing mice were assigned randomly into 4 groups, each consisting of 6 animals. the control group was left untreated. experimental group t1 was given orally 0.2 ml of tfee at a daily dosage of 200 mg/kgbw for 30 days. experimental groups t2 and t3 also received oral 0.2 ml tfee for 30 days, at daily dosages o f 4 0 0 m g / k g b w a n d 8 0 0 m g / k g b w, respectively. laboratory analysis at day 31, tumor tissue was taken for determination of tyrosine kinase levels by the elisa method and ki67 protein expression using immunohistochemically stained paraffin sections. ki67 protein expression was assessed under a nikon eclipse e400 light microscope by the hot spot method, which uses 5 high power fields containing 1000 adenocarcinoma cells, so that each high power field contains 200 cells. assessment of tyrosine kinase levels was carried o u t a t t h e d e p a r t m e n t s o f m o l e c u l a r biochemistry and biology, faculty of medicine, university of indonesia. immunohistochemical assessment of ki57 protein expression was done at sardjito hospital, yogyakarta. preparation of tumor tissue samples for homogenization from each tissue tumor sample, 50 mg was w e i g h e d , l y s e d a n d d i s s o l v e d i n 2 5 0 µ l extraction buffer in a test tube. each sample w a s m i x e d a t 4 0c f o r 3 m i n u t e s i n a homogenizer at 1000 rpm to yield soluble material (the test tubes with the tissue samples were put in a jar containing ice cubes). the insoluble tissue remnants at the bottom of the tube were transferred to a different test tube. all samples were centrifuged at 1000 g for 10 minutes, then stored at – 800 c until required for determination of tyrosine kinase levels. elisa assay for tyrosine kinase the prepared tissue sample solution was diluted more than five-fold with kinase reacting solution (genway biotech, san diego, ca). a total volume of 7200 ml kinase reacting solution was prepared, with the addition of 10 mm mercapto-ethanol just before use (5 µl mercapto-ethanol + 7195 µl kinase reacting buffer). one vial of lyophilized protein tyrosine kinase (ptk) control was dissolved in 100 µl twice-distilled water, then 400 µl kinase reacting buffer solution (at a concentration of 108 x 10 units per microliter) was added, resulting in a five-fold dilution of ptk control, and subjected to serial dilution. phosphorylation of tyrosine was carried out in duplicate by micropipetting 40 µl of ptk control or tissue sample into each microplate well (containing immobilized ptk substrate). then 10 µl of 40 nm atp-2na solution was added to each well and well-mixed, after which the microplate was i n c u b a t e d f o r 3 0 m i n u t e s a t 3 7 0 c . t h e remaining sample solution was removed and the plate washed 4 times in washing buffer (pbs 0.1 m + 0.05 % tween). to each well of the 7 pairs of duplicate wells (in total 14 wells) 100 µl blocking solution was added, then the wells were again incubated for 30 minutes at 370 c. the blocking solution was discarded, 50 µl antiphosphotyrosine-horse radish peroxidase (genway biotech) was added, then incubated for 30 minutes at 370 c. the remaining antibody solution was discarded and washed four times, then hrp substrate was added, consisting of 100 µl tetramethyl-benzidine dihydrochloride (tmbz [2hcl]). after the plate was covered with aluminum foil, it was incubated for 15 minutes at 370 c. after the addition of 100 µl 150 table1. comparison of tyrosine kinase dan ki67 expression by experimental group t1 = tfee at a daily dosage of 200 mg/kgbw; t2 = tfee at a daily dosage of 400 mg/kgbw; t3 = tfee at a daily dosage of 800 mg/kgbw; values are mean ± sd; *kruskal wallis; **one-way anova stop solution, the assay result was read at 400 nm wavelength using a plate reader. immunohistochemical ki67 protein assay (direct method) in this method, the monoclonal antibody used to detect ki67 protein is directly labelled with an enzyme. the following chemicals were used: h 2 o 2 3% for inactivation of endogenous peroxidase; trypsin 0.025% in pbs for clearing protein debris presumably covering epitopes of the substance to be detected); dab working solution, consisting of 50 drops of h 2 o 2 substrate buffer and 1ml of distilled water, as color indicator of the enzymatic reaction. briefly, the assay procedure was as follows: deparaffinizing of tissue sections in xylol (2 minutes) and graded alcohols (absolute, 95%, 8 0 % , 7 0 % ) f o r 1 m i n u t e e a c h . t h e deparaffinzed sections were then immersed in h 2 o 2 3% (30 minutes at room temperature), trypsin 0.025% (6 minutes at 37 0c), enzymelabelled anti-ki67 monoclonal antibody (60 minutes), with a rinse in pbs (3 x 2 minutes) after each step. after the last pbs rinse and addition of chromogen substrate (5 minutes), the sections were rinsed in pbs (3x 2 minutes), then in distilled water, immersed in mayer’s haematoxylin (6 minutes), rinsed in running water until free of excess stain, dehydrated, cleared, and mounted. data analysis tyrosine kinase concentrations and ki67 protein expression levels were tested for normality using the kolmogorov smirnov test and for homogeneity using the levene test. these tests indicated that the tyrosine kinase levels were non-normally distributed and nonhomogenous, therefore the data were subjected to the non-parametric kruskal wallis test. since the ki67 protein expression levels were normally distributed and homogenous, they were analyzed by one-way anova, followed by the bonferroni post hoc test. ethical clearance the study protocol was approved by the commission on health research ethics, faculty of medicine, diponegoro university and dr kariadi central general hospital, semarang. results during the whole intervention period until the completion of the study, there were no deaths among the test animals. mean tyrosine kinase levels by experimental group are presented in table 1. the results showed that the highest mean tyrosine kinase level was found in the control group, followed by treatment group t3 (receiving tfee 800 mg/kgbb), t2 (400mg/ kgbb), while the lowest mean level was in t1 (200 mg/kg bw). the kruskal wallis test results on tyrosine kinase levels showed that tyrosine kinase levels were significantly different between the four groups (p=0.019). on the basis of these results, it can be stated that the greatest reduction in tyrosine kinase levels in tumor-bearing c3h mice was found at a dose of 200 mg/kgbb tfee. one-way a n o v a r e s u l t s o n k i 6 7 e x p r e s s i o n w e r e chodidjah, dharmana, susanto, et al typhonium flagelliforme and marker proliferation 151 univ med vol. 32 no.3 table 2. post hoc benferroni test results on mean between-group ki67 expression t1 = tfee at a daily dosage of 200 mg/kgbw; t2 = tfee at a daily dosage of 400 mg/kgbw; t3 = tfee at a daily dosage of 800 mg/kgbw figure 1. ki67 expression in the control group (brown-colored nuclei [arrow]) magnification 1000x s i g n i f i c a n t l y d i ff e r e n t b e t w e e n t h e f o u r experimental groups (p=0.000). the greatest reduction in ki67 expression was found in group t1 (receiving 200 mg/kgbb tfee) (table 1). the results of the bonferroni post hoc test showed that administration of tfee at dosages of 200 mg/kgbb, 400mg/kg bw and 800 mg/ kg bw, respectively, to tumor-bearing c3h mice for 30 days, was able to decrease the expression of ki67, in comparison with the control group (p<0.05) (table 2). discussion the highest tyrosine kinase level was found in the control group, followed by t3 (receiving tfee at a dose of 800mg/kgbb) and t2 (400 mg/kg bw), while the lowest level was found in t1 (200 mg/kgbb). tfee at dosages of 200, 400 and 800 mg/kg bw, respectively, was able to decrease tyrosine kinase levels, indicating that abovementioned dosages still contain the flavonoids, alkaloids and other substances capable of decreasing tyrosine kinase levels invivo. typhonium flagelliforme was reported to be capable of inhibiting tyrosine kinase activity invitro.(13) a study on the flavonoidand alkaloidcontaining tubers of curcuma zedori (temu putih) also showed that these substances were capable of inhibiting tyrosine kinase activity invitro.(14) tyrosine kinase belongs to the protein kinases that function in signal transduction pathways of cell proliferation. inhibition of tyrosine kinase prevents transduced signals from entering the nucleus, leading to inhibition of tumor cell proliferation, which presumably also occurred in the present study. there are many pathways for signal transduction in cell proliferation, such as the pathway in which vegf binds to its receptor (vegfr). this will activate receptor-associated tyrosine kinases, resulting in autophosphorylation and activation of the tyrosine domain, leading to activation of the ras transduction factor. then the growth factor receptor bound protein-2 (grb-2) and son of sevenless (sos) proteins effect the conversion of guanosine diphosphate (gdp) in the ras protein into guanosine triphosphate (gtp). binding of gtp to the ras protein results in ras activation, leading to phosphorylation of the mapkkk-mapkk-mapk cascade, after which these activated kinases in turn activate transcription factors.(16,17) at the dosages of 400 mg/kgbw and 800 mg/kg bw, the decrease in tyrosine kinase levels was minimal and almost equal to that in the control group. this may be due to saturation of the tumor cell receptors at 152 the dosages of 400 and 800 mg/kgbw, preventing maximal inhibition of proliferation. this was presumably also caused by the anti-apoptotic protein hsp 70, a chaperone molecule capable of inhibiting apoptosis. the role of hsp 70 in malignancies is to effect tumor cell survival and is involved in stabilization of the akt kinase, a cytoplasmic protein regulating various control pathways in cell metabolism, growth, death and mobility. akt kinase dysfunction leads to increased cell growth, metabolism and mobility, while inhibiting apoptosis.(22) the fact that the inhibition of cell proliferation was not maximal in the present study, is presumably due to the effect of a number of proteins. the cooperation between stem cells and cancer cells in the regulation of cell proliferation may become the target of cancer treatment. the ephb receptor contributes to epithelial cell proliferation, but also functions as a tumor suppressor in the development of colonic carcinoma. how one protein can play the different roles of cell proliferation and tumor suppression of the same tissue is unclear.(15) the results of the ki67 assay showed that ki67 had the highest level of expression in the control group (indicating increased tumor cell proliferation), followed by t2 (400 mg/kgbb), t3 (800 mg/kgbb) and t1 (200 mg/kg bw). statistically there were significant differences in ki67 expression between groups t1, t2, and t3 on the one hand and the control group on the other. this indicates that tfee at dosages of 200 mg/kgbb, 400 mg/kgbb and 800 mg/kg bw w a s c a p a b l e o f d e c r e a s i n g t u m o r c e l l proliferation, as shown by the expression levels of ki67 as a proliferation marker. the greatest decrease in cell proliferation (shown by increased ki67) was in t1 at a dosage of 200 mg/kgbw. quercetin, a ubiquitous flavonoid, is also known to decrease ki67 protein expression in cancer, e.g. nasopharyngeal carcinoma invitro.(21) chodidjah, dharmana, susanto, et al typhonium flagelliforme and marker proliferation figure 2. ki67 expression (brown-colored nuclei [arrow]). magnification 400x. a, control group. b, group t1. c, group t2. d, group t3 153 thanks are also due to dr harjadi, sp pa, and ms. agustina supriyanti, amd of the laboratory for patologic anatomy, faculty of medicine, gadjah mada university/dr sardjito hospital, yogyakarta, for permitting the conduct of this study and for their assistance in the p r e p a r a t i o n o f t u m o r t i s s u e s e c t i o n s , immunohistochemical staining, and reading of the results of this study. references 1. siegel r, naishadam d, jemal. cancer statistic ca cancer j clin 2012;62:10-29. 2. tjindarbumi d, mangunkusumo r. cancer in indonesia: present and future. jpn j clin oncol 2002;32 suppl 1:s17-21. 3. curado mp. breast cancer in the world: incidence and mortality. salud publica mex 2011;53:372– 84. 4. zablotska lb, chak a, das a, neugut ai. inceased risk of squamous cell esophageal cancer after adjuvant radiation. am j epidemiol 2005; 161:330-7. 5. azab s, al hendy a. signal transduction pathways in breast cancer: drug target and challenges. in: gunduz m, gunduz e, editors. breast cancer: carcinogenesis, cell growth and signalling pathways. intech;2011.p.109-38. isbn. 970-953-307-714-7. available at: http:// www.intechopen.com/books/breast-cancer carcinogenesis-cell-growth-and-signallingpathways/signal-transduction-pathways-inbreast-cancer-drugtargets. accessed november 10, 2013. 6. punglia rs, kuntz k, lee jh, recht a. radiation therapy plus tamoxifen versus tamoxifen alone after breast conserving surgery in postmenopausal therapy for primary breast cancer. am j epidemiol 2005;161:330-37. 7. urruticoechea a, smith ie, dowsett m. proliferation marker ki-67 in early breast cancer. j clin oncol 2005:23:7212-20. 8. weisner fg, magener a, fasching pa, wesse f, bani mr, rauh c, et al. ki-67, a prognostic nucleolar marker in routine clinical use in breast cancer patients. breast 2009;18:135-41. 9. widowati l, mudahar h. uji aktivitas ekstrak etanol 50% umbi keladi tikus (typhonium flagelliforme) terhadap sel tumor payudara (mcf7 cell line) secara in vitro. media litbang kesehatan 2009;xix:9-14. univ med vol. 32 no.3 the ki67 protein is a proliferation marker in malignancies, where inhibition of proliferation leads to decreased ki67 expression. the gene for the ki 67 proliferation marker protein is located at the long arm of chromosome 10 (10q25). ki67 expression varies in intensity during the cell cycle and is very low (<3%) in normal mammary or epithelial tissues.(18) ki67 is used for the evaluation of mammary cancer proliferation, although its prognostic role in mammary cancer is unclear. according to a metaanalysis, ki67 is positively associated with early recurrence of mammary cancer.(20) bcl2 is an anti-apoptotic protein that is correlated to ki67. ki67 expression is associated with invasive in situ ductal mammary carcinoma.(20) a limitation of this study was that it did not assess other proliferation parameters, such as cyclin-dependent kinase (cdk), mitogenactivated protein kinases (mapks) and others. hopefully this study may be extended to a clinical trial, so that it may be of benefit for the prevention of malignancies in the community. conclusions o r a l a d m i n i s t r a t i o n o f ty p h o n i u m flagelliforme ethanol extract is capable of decreasing tyrosine kinase levels and ki57 e x p r e s s i o n i n c 3 h m i c e w i t h m a m m a r y adenocarcinoma. it is recommended that further studies be conducted on the effect of typhonium flagelliforme ethanol extract on cdk and mapk levels. acknowlegdments t h a n k s a r e d u e t o d r a p u s p i t a e k a wuyung, msi, head of the laboratory for experimental pathologic anatomy, department of pathologic anatomy, faculty of medicine, university of indonesia, jakarta, and to dr febriana catur iswanti, m. biomed and hanifah rahmi, ssi, m. biomed, staff of the departments of biochemistry and molecular biology, faculty of medicine, university of indonesia. 154 10. nobakt gm, kadir ma, stansias j. analysis of preliminary phytochemical screening of typhonium flagelliforme. afr j biotechnol 2010; 9:1655-7. 11. lai cs, mas rh, nair nk, majid mi, mansor sm, navarasmanv. typhonium flagelliforme inhibits cancer cell growth in vitro and induces apoptosis: an evaluation by the bioactivity guide approach. j. ethnopathomol 2008;118:14-20. 12. mohan s, abdul ab, abdalwahab si, al-zubairi as, sukari ma, abdullah r, et al. typhonium flagelliforme inhibits the proliferation of murine leukemia wehi-3 cells invitro and induces apoptosis invivo. leuk res 2010;34:1483-92. 13. dyah i, syahbirin g, affandi y. keladi tikus (typhonium flagelliforme) sebagai anti kanker: mekanisme inhibisi ekstrak etanol dan ekstrak air terhadap aktivitas enzim tirosinkinase. prosiding seminar hki, departemen kimia fmipa ipb: bogor; 2006. 14. iswantini d, syahbirin g, pratiwi w. daya inhibisi ekstrak air dan etanol temu putih (curcuma zedori) terhadap aktivitas enzim tirosin kinase secara in vitro. prosiding seminar nasional sains. penemuan sains dalam kebangkitan pertanian. fmipa ipb: bogor; 2008. 15. genander m, halford m, xu n, eriksson m, ye z. dissociation of ephb signaling pathways mediating progenitor cell proliferation and tumor suppressor. cell 2009;139:679-92. 16. osaki l, gama p. mapks and signal transduction in the control of gastrointestinal epithelial cell proliferation and differentiation. int j mol sci 2013;14:10101-43. 17. burcombe r, wilson g, dowsett m, khan i, richman pi, dalley f, et al. evaluation of ki67 proliferation and apoptotic index before, during and after neoadjuvant chemotherapy for primary breast cancer. breast cancer res 2006;8: r31. doi:10.1186/bcr1508. 18. urruticoechea a, smith ie, dowsett m. proliferation marker ki-67 in early breast cancer. j clin oncol 2005;23:7212-20. 19. kanadaswami c, lee l, lee p, hwang j, ke f, huang y. the antitumor activities of flavonoids. invivo 2005;19:895-919. 20. nishimura r, osako t, hayashi m, toyosumi y, arima n. ki67 as a prognostic marker according to breast cancer subtype an a predictor of recurrence time in primary breast cancer. exp ther med 2010;1:747-54. 21. daker m. ahmad m, khoo as. quercetininduced inhibition and synergistic activity with cisplatin a chemotherapeutic strategy for nasopharyngeal carcinoma cells. cancer cell int malaysia 2012;12:34. 22. yoeli-lerner m, toker a. akt/pkb signaling in cancer: a function in cell motility. cell cycle 2006;5:603-5. chodidjah, dharmana, susanto, et al typhonium flagelliforme and marker proliferation f:\39 no 2\setyo trisnadi.pmd 97 *department of postgraduate biomedical science, medical faculty, sultan agung islamic university (unissula), semarang, central java, indonesia **department of surgery, medical faculty, universitas sumatera utara (usu), medan, indonesia ***department of pathological anatomy, medical faculty, sultan agung islamic university (unissula), semarang, central java, indonesia †stem cell and cancer research (sccr), medical faculty, universitas islam sultan agung (unissula), semarang, central java, indonesia ‡department of pediatrics, medical faculty, universitas islam sultan agung (unissula), semarang, central java, indonesia correspondence : agung putra, md chairman of stem cell and cancer research (sccr) laboratory, faculty of medicine, sultan agung islamic university, semarang jl. raya kaligawe km. 4 semarang, central java 50112 phone +628164251646, fax. 0246594366 email: dr.agungptr@gmail.com orcid id: 0000-0003-4261-9437 date of first submission, october 10, 2 01 9 date of final revised submission, july 8, 2 02 0 date of acceptance, july 9, 2020 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina hypoxia-preconditioned mesenchymal stem cells attenuate peritoneal adhesion through tgf- inhibition setyo trisnadi*, adi muradi muhar**, agung putra*,***,†, and azizah retno kustiyah †,‡ background peritoneal adhesions (pas) are generally described as fibrous bands between intra-abdominal organs following an abdominal surgical operation. the definitive treatments of pas are currently ineffective yet. hypoxiamesenchymal stem cells (h-mscs) have a higher capability to survive at the site of injury than normoxia-mscs (n-mscs) to repair injured tissue without fibrosis. this study aimed to analyze the effect of h-mscs in controlling formation of pas by reducing tgf-β level in a rat model. methods a study of post-test only control group design was conducted, involving eighteen pa rat models weighing 250 ± 25 g that were randomly assigned into 3 groups, comprising control group (c), and groups t1 and t2 receiving h-msc treatment at doses of 3 x 106 and 1.5 x 106, respectively. to induce h-mscs, mscs were incubated in hypoxic conditions at 5% o 2 and 37oc for 24 hours. expression level of tgf-β was analyzed by enzyme-linked immunosorbent assay (elisa) at 450 nm and adhesion formation was described macroscopically. the kruskal-wallis variance analysis was used to analyze significant differences among the groups. results the results of this study showed that h-mscs in group t1 inhibited tgfβ expression significantly on day 8 (p<0.001) and day 14 (p<0.05). moreover, there was almost no adhesion apparent following h-msc administration in group t1. conclusions based on this study, we conclude that h-mscs may attenuate pa formation following inhibition of tgf-β expression in the pa rat model. keywords: peritoneal adhesions, hypoxia, h-mscs, tgf-β, rat original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2020.v39.97-104 may-august, 2020 vol.39no.1 cite this article as: trisnadi s, muhar am, putra p, kustiyah ar. hypoxiapreconditioned mesenchymal stem cells attenuate peritoneal adhesion through tgf- inhibition. univ med 2020;39:97-104. doi: 10.18051/univmed.2020.v39. 9 7 -1 0 4 98 trisnadi, muhar, putra, et al hypoxia-preconditioned mesenchymal stem cells introduction peritoneal adhesions (pas) are fibrous bands between two or more intra-abdominal organs or between these organs and the inner abdominal wall, due to secondarily prolonged inf lammation of an abdominal surgical operation.(1) the occurrence of pas approaches 63-97% and approximately one-third of such cases are readmitted due to complications. pas may cause complications such as abdominal or pelvic pain, small bowel obstructions, and infertility.(2) although the definitive treatment of pas such as laparoscopic adhesiolysis and/or laparotomy adhesiolysis has the benefit of reducing the pas, those treatments have the potential to induce several complications such as small bowel obstructions and the risk of pa recurrence.(3-5) therefore, more effective and safer treatment of pas is needed. mesenchymal stem cells (mscs) are a population of pleiotropic cells with self-renewing capacity that have the ability to differentiate into various canonical mesenchymal cells, namely adipocytes, chondrocytes, and osteocytes.(6) mscs express a number of markers, such as cd29, cd44, cd73, cd90, cd105, cd166, but are lacking in cd14, cd34, cd45, or cd11b, cd79a or cd19, and hla class ii expression.(7) a recent study reported that hypoxia offers benefits to mscs for increasing their capability of self-renewal, proliferation, and survival gene modulation.(8) hypoxia-mesenchymal stem cells (h-mscs) can mimic a physiological niche in the bone marrow that potentially offers the benefits of a wound healing process. this is in line with a previous study reporting that one of the therapeutic failures of msc administration was poor engraftment ca pacity and low cell survivability during the transplantation process at the hypoxia injury site.(9) this fact showed that the competence of mscs to survive under hypoxic conditions in preventing pa formation was crucial to be explored.(10) furthermore, hmscs are more robust cells in controlling inflammation than n-mscs through increasing il-10 production to tightly compete with tgf-β for fibroblast activation.(11) the pathogenesis of pas involves several inflammatory factors to stimulate the prolonged release of tgf-β leading to the activation of fibroblasts and adhesion formation.(12) a previous study revealed that mscs cultured in hypoxia exhibit higher rates of proliferation and better retain their stem cell properties, including pas.(13) our previous study showed that h-mscs can survive in injured tissues and have the capacity to r educe pa f orma tion and suppr ess inflammatory conditions by releasing il-10 as a pleiotropic anti-inflammatory cytokine.(14) however, the role of h-mscs in decreasing tgf-β as the main cytokine of the main fibrosis mechanism in pa formation remains unclear. therefore, to decrease tgf-β through h-msc administration for inhibiting pa formation was crucial to be investigated. in this study, we aimed to analyze the effect of h-mscs in controlling pa formation by reducing tgf-β levels. methods research design this study of post-test only control group design was carried out in the stem cell and canc er rese arch lab oratory, fa culty of medicine, universitas islam sultan agung (unissula), semarang from august-october 2018. pa animal model the sample size was calculated according to the federer formula. (15) this study used eighteen wistar albino rats which were randomly divided into 3 groups, consisting of the control group, the high dose group (t1) and the low dose group (t2) (n=6 rats per group) with 10% estimated drop-out rate. the wistar albino rats weighing between 250 and 300 g were purchased from the animal holding unit, faculty of veterinary medicine, gadjah mada university. rats were acclimatized and housed in 12 h light-dark cycle standard cages at 24oc with food and water ad libitum. after fasting for 12 h, rats were 99 anesthetized by intraperitoneal administration of ketamine and xylazine (90 and 10 mg per kg body weight; r espectively). t he surgical p r o c e du r e w a s p e r f or me d a s d e s c r i b e d previously.(14) the operations were performed under aseptic conditions and took less than 20 minutes for each rat to minimize room air tissue dying effect. the musculoperitoneal layer was incised and opened over a length of 3 cm at the linea alba after cutaneous ventral midline incision. in order to induce pas, the standardized surgical injuries were applied to one side of the terminal ileum (3 cm from the cecum) at a size of 0.5 × 0.5 cm on the right sidewall of the ileum by brushing with a cytobrush (gynobrush, langenbrink, emmendingen, germany) until punctuate red spots were observable as visual indicator of ileum trauma. following the intervention, the abdominal incision was closed with 3–0 polyglactin suture and the rats were reared in battery cages for 14 days. msc isolation culture and h-msc induction mesenchymal stem cells were isolated from the umbilical cord (uc) obtained from pregnant single wistar albino rats and expanded as described previously.(8) briefly, ucs were chopped into smaller pieces and transferred into a t25 culture flask (corning, tewksbury, ma, usa) containing dmem (gibco™ invitrogen, ny, usa) which was supplemented with 10% fbs (gibco™ invitrogen, ny, usa), 1% penicillin (100 u/ml) and 0.25% streptomycin (100 µg/ml) (gibco™ invitrogen, ny, usa) and incubated at 37°c in 5% co 2 and e”95% humidity. the medium was replaced every 3 days and the cells harvested after reaching 80% confluence (14 days). the 4-6th passage msclike cells were employed for the experiments. to induce h-mscs, mscs derived from the 4th passage were incubated under 5% o2 condition in a hypoxia incubation chamber (stemcell technologies, biopolis, singapore) for 24 h at 37°c and 5% co2, then collected for the following experiment. h-msc characterization hypoxic-mesenchymal stem cell surface markers at the 4-6th passage were analyzed by flow cytometry analysis according to company protocols. briefly, the cells were subsequently incubated in the darkroom with allophycocyanin (apc) mouse anti-human cd73, fluorescein isothiocyanate (fitc) mouse anti-human cd90, percp-cy5.5.1 mouse anti-human cd105, and phycoerythrin (pe) mouse anti-human lin negative (cd45/cd34/cd11b/cd19/hla-dr) antibodies. h-msc cells were stained with msc specific antibody for 30 minutes at 4°c, then examined and analyzed with a bd accuri c6 plus flow cytometer (bd biosciences, san jose, ca, usa). h-msc osteogenic differentiation assay the h-mscs were cultured on 24-well plates (1.5 x 104 cells/well density) with standard medium containing dmem (sigma-aldrich, louis st, mo), supplemented with 10% fbs (gibco™ invitrogen, ny, usa), 1% penicillin (100 u/ml) and 0.25% streptomycin (100 µg/ml) (gibco™ invitrogen, ny, usa) at 37°c, 5% co2, and e”95% humidity. after reaching 95% confluence, the standard medium was aspirated and replaced with osteogenic differentiation medium containing human mesencult™ osteogenic differentiation basal medium (stem cell technologies, singapor e), augmented with 20% human mesencult™ osteogenic differentiation 5x supplement (stem cell technologies, singapore) and 1% l-glutamine (gibco™ invitrogen, ny, usa). the differentiation medium was replaced every 3 days. the bone matrix was formed after 15 days and was visualized by 2% alizarin red staining. h-msc administration after the surgical procedure of pa induction, the rats were treated once with 3x106 cells for the t1 group and 1.5 x106 cells for the t2 group via submucosal injection, while the control group received the omental patch treatment. univ med vol. 39 no.2 100 trisnadi, muhar, putra, et al hypoxia-preconditioned mesenchymal stem cells a b c elisa assay at days 8 and 14 post-pa surgical procedure, elisa was used to measure the level of tgf-β released in rat peripheral blood in the treatment and control groups. the analysis was done according to the manufacturer’s instructions (fine test, wuhan, china) using a standard curve and was performed in five replications. the colorimetric absorbance wavelength used was 450 nm. macroscopic analysis the induced pa rat animal models were euthanized using co2 inhalation on the 14th day after treatment. the adhesion formation was observed, and categorized using nair’s adhesion grade (table 1). (16) statistical analysis the statistical analysis was performed using the spss ver. 23 (spss inc., chicago, il, usa). descriptive data were expressed as mean ± standard error. for intergroup analysis, the kruskal-wallis variance analysis was used to analyze significant differences among the groups. and mann whitney test to compare between gr oups. a p-value of less than 0.05 was considered statistically significant. ethical clearance the study was approved by the health research ethical committee, medical faculty, universitas sumatera utara, medan, indonesia, under no. 541/tgl/kepk fk usu-rsup ham/2019. grade 0 no adhesion grade 1 only one adhesion band between abrasion site and abdominal wall grade 2 two adhesion bands between abrasion site and abdominal wall grade 3 more than two adhesion bands between abrasion site and abdominal wall grade 4 adhesion of all viscera to the abdominal wall table 1. nair’s macroscopic adhesion grade (16) figure 1. (a) h-mscs from the in-vitro culture showing spindle shape and fibroblast-like characteristics (10x magnification) ; (b) osteogenic differentiation analysis using alizarin red staining showing calcium deposition marked by red color ; (c) flow cytometry analysis of h-mscs confirmed that h-mscs expressed high levels of cd90, cd105, cd73 and lacked the expression of lin 101 results h-msc characterization and differentiation the h-mscs that were incubated from the 4th passage at 80% confluence under hypoxic conditions (5% o 2 ) showed denser fibroblast and spindle-like shape characteristics (figure 1a). after undergoing the osteogenic differentiation assay the h-mscs showed calcium deposition under 2% alizarin red staining (figure 1b). the flow cytometry analysis confirmed that h-mscs expressed high levels of cd90 (99.9%), cd105 (95.9% ), cd73 (99. 2%) and lacked the expression of lin(2.0%) thereby demonstrating a characteristic immunophenotype of mscs (figure 1c). macroscopic assessment the nair macroscopic adhesion grade was used to describe adhesion occurrence in this study. there was grade 4 adhesion in the control group, no adhesion in group t1 (grade 0), and less adhesion in group t2 (grade 1) (figure 2). tgf-β expression analysis we analyzed tgf-β levels as an inflammatory marker of the ileum tissue. tgf-β univ med vol. 39 no.2 expression was detected using elisa assay. the results showed that the tgf-β expression level in groups t1 and t2 were significantly lower than in the control group on both the 8th and 14th days (p<0.05) (table 2). discussion pas are commonly fibrous bands occurring after abdominal surgery that have severe complications of life quality.(17) the pathology of peritoneal fibrinolysis reduction is thought to play a key role in pa pathogenesis.(18) inflammatory cytokines such as il-17 may also promote pas by increasing the activation of fibroblasts along with tgf-β1 stimulation.(19) a previous study reported the potential of mscs to treat pa formation.(20) however, although mscs were able to reduce peritoneal inflammation, they were ineffective in controlling pas.(21) hypoxia-mscs have positive effects on the in vitro stemness, survival capacity, and angiogenic potential that is mediated by hypoxia-inducible factors (hifs), such as hif-1α and hif-2α.(22) to analyze the effect of h-mscs in preventing pa formation, we induced abrasion in rats as the established animal model of pas according to a previous figure 2. macroscopic appearance showing severe pa in control group at grade 4 (red arrow) (a). treatment groups showing fewer adhesions in the t2 group (b); and almost no adhesions in the t2 group (black arrow) (c) treatment groups p value* t1 t2 c tgf-β level (pg/ml) 8th day 14th day 492.04±53.50 450.88±20.86 645.05±59.89 539.50±43.79 699.68±18.75 563.55±26.24 t1 = 0.008; t2 = 0.008 t1 = 0.008; t2 = 0.032 table 2. tgf-β levels according to treatment groups at 8th and 14th days t1: h-msc 3x106 cells; t2: h-msc 1.5x106 cells; c: nacl; *kruskal-wallis and mann whitney test 102 trisnadi, muhar, putra, et al hypoxia-preconditioned mesenchymal stem cells study.(23) in this study, we transplanted the hmscs into pa rat models directly to the ileum by submucosal injection, then analyzed the tgf-β on day 8 and 14. our study found that there was a significant decrease in tgf-β levels in the t1 and t2 groups on days 8 and 14. our results indicate that there was complete wound healing in the h-msc groups as compared with the control group. this finding was in line with our nair macroscopic analysis in which there were pas in the control group but no adhesion formation in the h-msc treatment groups. a previous study reported that mscs could attenuate peritoneal adhesion through increasing the level of tnf-α stimulating gene6 (tsg-6) to reduce the inflammatory process.(24) this is in line with our previous study revealing that h-mscs could increase the level of il-10 as a major anti-inflammatory cytokine to control adhesion formation.(14) this indicated that hmscs could accelerate wound healing and attenuate pa formation through the decrease in tgf-β levels. a recent study found that proinflammatory cytokines such as tnf-α and il1β may stimulate tgf-β production to induce the activation of myofibroblasts (mfs) for pa formation.(25) a previous study also reported that h-mscs could decrease the expression levels of various pro-inflammatory cytokines, such as tnf-α, il-1β, and mip-2, and promote the secretion of anti-inflammatory molecules, including il-10 and prostaglandin e 2 (pge 2 ). (26) the il-10 as anti-inflammatory molecules could suppress the tgf-β level which might be attributed to the inactivation of mf that have crutial roles in adhesion formation.(27) this study also demonstrated the dosedependent effect of h-mscs. this was in accordance with a previous study that found a progressive increase in the number of altered cytokines at higher doses of mscs.(28,29) the interac tion betwe en these proand antiinflammatory molecules at the injury site following h-msc administration may explain the tgf-β lowering effect of h-mscs. this was supported by our morphological findings, in which there were fibrous bands of pas in the control group. these findings indicated that inflammation could be optimally controlled by high doses of h-mscs. the fibrin matrix contained in the initial adhesion was replaced by extracellular matrix (ecm) under prolonged inflammation due to the ecm synthesis being more active than the degradation process, leading to the formation of new endothelial layers connecting to other organs.(30,31) the activated mfs that were characterized by α-smooth muscle actin (α-sma) were the main actors of these processes to produce ecm in excessive amounts due to the incompetence of il-10 of the control group to suppress the inflammation.(14,21) this study has a limitation, as we did not observe the expression of α-sma i n myofibroblasts, thus we could not confirm the role of h-mscs in reducing pa formation. in addition, the number of lymphocytes as a hallmark of prolonged inflammation in the wound area was not investigated, which is the other limitation of this study. this study provides new insights on the benefits of h-msc administration for inhibiting pa formation post abdominal surgical operation. further studies are warranted to confirm the effect of h-mscs in inhibiting mf activity that might provide important evidence for their potential clinical applications. conclusions in conclusion, our data demonstrate that hmscs attenua te 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mesenchymal stem cell treatment on systemic cytokine levels in a phase 1 dose escalation safety trial of septic shock patients. crit care med 2019;47:918–25. doi:10.1097/ccm.000000000000 3657. 31. filová e, brynda e, riedel t, et al. improved adhesion and differentiation of endothelial cells on surface-attached fibrin structures containing extracellular matrix proteins. j biomed mater res a 2014;102:698-712. doi:10.1002/jbm.a.34733. oktavianus 44 *department of biology, faculty of mathematics and natural sciences, islamic university of malang, malang, east java **department of pathology, faculty of medicine, islamic university of malang, malang, east java correspondence nour athiroh as, s.si., m.kes. department of biology, faculty of mathematics and natural sciences, islamic university of malang, jl. mayjen haryono 193 malang, east java phone: +62341-575461 email: nur_athiroh_mlg@yahoo.co.id univ med 2013;32:44-50 abstract universa medicina january-april, 2013january-april, 2013january-april, 2013january-april, 2013january-april, 2013 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 vol.32 no.1 background hypertension is the most prevalent chronic disease and has an impact on one billion people. production of superoxide radicals and endothelial dysfunction are involved in hypertension. scurrula atropurpurea (bl.) dans. is a tea plant parasite. this study aimed to evaluate the role of scurrula atropurpurea (bl.) dans. on nitric oxide (no) as a marker of endothelial dysfunction and malondialdehyde (mda) as a marker of oxidative stress in hypertensive rats. methods this study subjected rats to deoxycorticosterone acetate (doca)-induced hypertension. the experimental groups consisting of the control group and 3 hypertension groups receiving scurulla atropurpurea extract at a dosage of 50; 100; and 200 mg/kgbw. scavenging activity of scurrula atropurpurea (bl.) extract was analyzed by the 1,1-diphenyl-2-picrylhydrazyl (dpph) method. the levels of arterial nitric oxide (no) and pulmonary malondialdehyde (mda) were analyzed by spectrophotometry. anova and a post hoc test were applied to find the difference of arterial no and pulmonary mda levels between groups. results the level of arterial no was significantly decreased in the hypertension groups as compared with the control group, while the level of pulmonary mda was significantly increased (p<0.05). scurulla atropurpurea significantly increases the no level at a dosage of 200 mg/kgbw, compared with the hypertension groups (p<0.001). scurulla atropurpurea significantly decreases pulmonary mda level at a dosage of 100 and 200 mg/kgbw compared with the hypertension groups (p<0.05). conclusion scurulla atropurpurea extract increases arterial no and decreases pulmonary mda in hypertensive rats, thus playing an important role in endothelial dysfunction and oxidative stress. key words: mistletoe, arterial no, pulmonary mda, hypertension, rats scurrula atropurpurea increases nitric oxide and decreases malondialdehyde in hypertensive rats nour athiroh as* and erna sulistyowati** 45 univ med vol. 32 no.1 benalu teh meningkatkan kadar nitrit oksida dan menurunkan kadar malondialdehid pada tikus hipertensi latar belakang hipertensi merupakan penyakit kronik yang paling kerap ditemukan dan memberikan pengaruh kepada sekitar satu milyar individu. hipertensi berhubungan dengan peningkatan produksi radikal superoksida dan disfungsi endotel. scurrula atropurpurea (bl.) dans. merupakan tanaman parasitik yang menyerang teh. penelitian ini bertujuan untuk mengevaluasi peranan benalu teh terhadap disfungi endotel yang duikur menggunakan kadar nitrit oksida (no) dan stres oksidatif diuukr menggunakan kadar mondialdehid (mda) pada tikus model hipertensi. metode subyek penelitian adalah tikus model hipertensi yang diinduksi oleh deoxycorticosterone acetate (doca). kelompok perlakuan terdiri atas kelompok kontrol, kelompok tikus hipertensi, kelompok ekstrak benalu teh dosis 50; 100; dan 200 mg/kgbb. analisis aktivitas scavenging dari benalu teh dilakukan dengen metode dpph. analisis kadar no arteri dan kadar mda paru dilakukan dengan metode kolorimetrik dengan spektrofotometer. uji anova dan post hoc dilakukan untuk melihat perbedaan kadar no arteri dan mda paru pada berbagai kelompok perlakuan. hasil terdapat penurunan kadar no arteri dan peningkatan kadar mda paru secara bermakna pada tikus hipertensi dibandingkan kontrol (p<0,05). ekstrak benalu teh dosis 200 mg/kgbb dapat meningkatkan kadar no arteri secara bermakna dibandingkan tikus hipertensi (p<0,001). ekstrak benalu teh dosis 100 dan 200 mg/kgbb dapat menurunkan kadar mda paru secara bermakna dibandingkan tikus hipertensi (p<0,05). kesimpulan ekstrak benalu teh meningkatkan kadar no arteri dan menurunkan kadar mda paru pada tikus hipertensi. jadi berperan penting pada disfungsi endotel dan stress oksidatif. kata kunci: benalu teh, no arteri, mda paru, tekanan darah tinggi, tikus abstrak introduction hypertension is the end result of a complex interaction between genetic and environmental factors affecting the physiological systems regulating blood pressure. throughout the world hypertension is the most frequently encountered chronic disease and affects around one billion individuals.(1) death resulting from hypertension m a y b e c a u s e d b y c e r e b r o v a s c u l a r a n d cardiovascular complications, such as stroke, end-stage renal disease, congestive heart failure, myocardial infarction, and cardiac standstill.(2) various lines of evidence reveal the involvement of reactive oxygen species and oxidative stress in hypertension and the development of its complications. hypertension is associated with increased production of s u p e r o x i d e r a d i c a l s a n d e n d o t h e l i a l dysfunction. (3) superoxide radicals have a negative effect on endothelial function by reacting directly with nitric oxide (no), such that they decrease no bioavailabily. in addition, peroxynitrites as the products of the reaction of superoxide radicals with no, also have negative effects on endothelial cells.(4,5) hydroxyl radicals 46 p r o d u c e d b y t h e d e c o m p o s i t i o n o f h y d r o p e r o x y n i t r i t e s m a y t r i g g e r l i p i d p e r o x i d a t i o n , m a r k e d b y i n c r e a s e d malondialdehyde (mda) levels. for the treatment of hypertension and its complications, many drugs of plant origin have been developed, comprising digitoxin from digitalis purpurea, reserpine from rauwolfia s e r p e n t i n a , a s p i r i n f r o m s a l i x a l b a , t e t r a m e t h y l p y r a z i n e f r o m j a t h ro p h a podagrica, and tetrandrine from stephenia tetradra.(2) scurrula atropurpurea (bl.) dans. is a parasitic plant attacking tea plants and t h e r e f o r e k n o w n a s t h e t e a p a r a s i t e . i n indonesia, especially on the island of java, the stems and leaves of this plant have been traditionally used, among others for the treatment of cancers.(6) no studies have been found for evaluating the potential of the tea parasite for the treatment of hypertension. therefore the objective of the present study was to evaluate the effect of tea parasite leaf extract (tple) on the levels of no as marker of endothelial dysfunction and on the levels of mda as a marker of oxidative stress in a rat model of hypertension. methods study design this was an laboratory experimental study conducted from february to july 2012. preparation of rat hypertension models the study subjects were wistar rats aged 3-5 months and weighing 250-300 grams. the r a t s w e r e i n j e c t e d s u b c u t a n e o u s l y w i t h deoxycorticosterone acetate (doca) at a dosage of 10 mg/kgbw, 2 times weekly for 6 weeks. the rats were given 2% nacl instead of drinking water. the blood pressure and the weights of the rats were then determined.(7) the treatment groups consisted of the control group, the group of non-tple hypertensive rats, three groups of hypertensive rats receiving tple at dosages of 50, 100, and 200 mg/kgbw. the rats were assigned randomly into the groups, with each group containing five rats. preparation tea parasite crude extract botanical determination of the leaves of the tea parasite was done at the indonesian scientific institute (lipi) at purwodadi, pasuruan, east java. the leaves were washed, left to dry in an oven at 40-600c, then ground into a powder. one hundred milligrams of tea parasite leaf powder was steeped in methanol in an erlenmeyer flask of 1 l capacity. the mixture was shaken for 30 mnutes to distribute t h e p o w d e r u n i f o r m l y i n t h e m e t h a n o l . subsequently the mixture was left to stand overnight until a precipitate was formed. the supernatant, being a mixture of methanol and the active constituents, was subjected to evaporation. the extract was labelled and stored in a freezer.(8) the tple was administered daily by the oral route using a catheter, this being continued for 6 weeks. determination of antioxidant activity the antioxidant activity of the extract was analyzed by a modification of the dpph free scavenging activity method.(9,10) the sample was dissolved in methanol (at concentrations of 10100 ppm), reacted with 0.2 mm dpph, and incubated for 30 minutes at room temperature. a b s o r b a n c e w a s m e a s u r e d a t 5 1 5 n m . antioxidant activity was calculated as a percentage of inhibition of dpph (percentage of scavenging effect), i.e. % inhibition = [1(absorbance of sample /absorbance of blank)] x 100%. the ic 50 is the concentration of the sample required for yielding 50% inhibition. determination of arterial no and pulmonary mda the specimens used for the analysis of no levels were arterial tissues from the tails of the rats. the method used was the griess reaction. the analysis was performed according to the procedural instructions included in the kit. no concentrations were expressed in µmol.(2) athiroh, sulistyowati scurrula atropurpurea and hypertension 47 univ med vol. 32 no.1 figure 1. no concentration (µmol) in the treatment groups. data are presented as mean ± standard deviation. dosage unit of tple is mg/kgbw; *significantly different in comparison with control group (p<0.05); # significantly different in comparison with non-tple hypertension group (p<0.05) t h e m e t h o d u s e d f o r d e t e r m i n i n g pulmonary mda levels was developed at the pharmacology laboratory, faculty of medicine, brawijaya university, malang, east java. one hundred milligrams of tissue was homogenized with 2 ml of phosphate buffer at ph 7.4. then 50 ml of 0.1% triton-x was added and the mixture was vortexed. thereafter, 250 ml of 1 n hcl was added to the mixture, which was again vortexed. the mixture was heated to 1000 c for 30 minutes, then centrifuged at 3000 rpm for 10 minutes until a colloid precipitate formed. t h e a b s o r b a n c e w a s r e a d w i t h a spectrophotometer at a wavelength (ë) of 532 nm. the mda concentration was expressed in ng/200 mg. data analysis to examine the differenceas in arterial no and pulmonary mda, the data were tested with anova and a post hoc test. a value p<0.05 was considered to indicate a significant difference. the statistical analysis was done using spss version 13. ethical clearance this study was approved by the committee on research ethics, faculty of medicine, brawijaya university, malang. results on the basis of dpph radical scavenging activity, tple was a strong antioxidant with an ic 50 of 11.64%. figure 1 presents arterial no levels in the treatment groups. post hoc testing found a significant reduction in arterial no in the nontple hypertension group, as compared with the control group (p<0.001). to raise arterial no, tple was administered daily for 6 weeks. administration of tple at a dosage of 200 mg/ kgbw was able to significantly increase arterial no, as compared with non-tple hypertensive rats (p<0.001), although the increase did not attain the levels in control rats. in figure 2 are presented the pulmonary mda concentrations in the treatment groups. post hoc testing found a significant increase in pulmonary mda concentration in rats with nontple hypertension as compared with control rats (p<0.05). administration of tple at the dosages of 100 and 200 mg/kgbw was able to significantly reduce mda levels in comparison with non-tple hypertensive rats, although not attaining the levels in control rats. 48 figure 2. mda concentration (ng/200 mg) in the treatment groups. data are presented as mean ± standard deviation. dosage unit of tple is mg/kgbw; *significantly different in comparison with control group (p<0.05); # significantly different in comparison with the non-tple hypertension group (p<0.05) discussion the present study demonstrates that in the rat model of hypertension there is a significantly reduced arterial no concentration and a significantly increased pulmonary mda concentration, in comparison with the control group. the reduction in arterial no level i n d i c a t e s t h e o c c u r r e n c e o f e n d o t h e l i a l dysfunction in hypertension. the increase in pulmonary mda indicates an increase in oxidative stress in hypertension. endothelial dysfunction and pulmonary oxidative stress in h y p e r t e n s i o n m a y b e m o d u l a t e d b y t h e administration of tple. the reduced arterial no in hypertension m a y b e d u e t o d e c r e a s e d e x p r e s s i o n o f endothelial nitric oxide synthase (enos) or by decreased no bioavailability.(11) the latter may be the result of a reaction between no and superoxide radicals to form peroxynitrites.(12) subsequently peroxynitrites may trigger the formation of hydroxyl radicals leading to lipid peroxidation, which is marked by an increase in mda levels. administration of crude tple at a dosage of 200 mg/kgbw is able to significantly i n c r e a s e a r t e r i a l n o c o n c e n t r a t i o n , a s c o m p a r e d w i t h t h e g r o u p o f n o n t p l e hypertensive rats, but is not able to attain the no levels in the control rats. the ability of tple to modulate no levels is due to its active constituents, among others flavonol glycosides (quercetin and rutin), monoterpene glucosides (icariside b), lignan glycosides (aviculin), flavans (catechin, epicatechin, epicatechin-30 g a l l a t e , e p i g a l l o c a t e c h i n 3 0 g a l l a t e , g a l l o c a t e c h i n , a n d e p i g a l l o c a t e c h i n ) . ( 6 ) quercetin diffuses directly into endothelial cells and increases no production.(13) catechin increases enos phosphorylation and no bioavailability by inhibition of nadph oxidase.(14) in the present study, pulmonary oxidative stress increased significantly in comparison with t h e c o n t r o l s . t h i s i n d i c a t e s p u l m o n a r y involvement in the rat model of hypertension induced by doca. in the vascular system, one of the mechanisms for increases in reactive oxygen compounds is the high activity of nadph oxidase.(15) we suppose that this mechanism is also at work in the lungs. administration of tple at dosages of 100 and 200 mg/kgbw is capable of significantly d e c r e a s i n g p u l m o n a r y m d a l e v e l s i n comparison with the group of non-tple athiroh, sulistyowati scurrula atropurpurea and hypertension 49 univ med vol. 32 no.1 hypertensive rats, but cannot attain the mda levels in the control rats. this indicates that tple has antioxidant properties and inhibits pulmonary oxidative stress. based on its dpph radical scavenging activity, tple is a strong antioxidant with an ic 50 value of 11.64%. quercetin has a high scavenging ability for dpph radicals (16) and may also modulate endogenous antioxidants.(17) the ability to inhibit oxidative stress is also found in rutin and catechin.(18,19) in the pathomechanism of hypertension, the antioxidant properties of tple may inhibit oxidative stress at several points. first, tple scavenges superoxide radicals such that the l a t t e r d o n o t r e a c t w i t h n o t o f o r m peroxynitrites. second, tple scavenges h y d r o x y l r a d i c a l s f o r m e d a s a r e s u l t o f decomposition of hydroperoxynitrites. one l i m i t a t i o n o f t h i s s t u d y i s t h a t n o h i g h performance liquid chromatography (hplc) analysis was performed to determine the active constituents present in scurrula atropurpurea such that its antihypertensive action may be revealed in detail. conclusion tple increases arterial no and decreases pulmonary mda in hypertensive rats, thus playing an important role in endothelial dysfunction and oxidative stress. acknowledgements we wish to express our gratitude to: 1). the national ministry of education for the provision of research grant in the framework of competitive research grants (penelitian hibah bersaing, phb) for the year 2011/2012 (year i), no. 0032/sp2h/pp/k7/kl/ii/2012; 2). prof. dr. m. aris widodo, ms., spfk., phd, professor at the faculty of medicine, islamic university of malang (unisma) and the faculty of medicine, brawijaya university (fkub), for his valuable scientific information and suggestions; 3). the umbrella research team at the faculty of mathematics and natural sciences (fmipa) and the faculty of medicine, islamic university of malang (unisma) for their cooperation. references 1. zhou w, longhurst jc. neuroendocrine mechanisms of acupuncture in the treatment of hypertension. evid based complement altern med 2012;9 pages. doi:10.1155/2012/878673 2. iwalokum ba, hodonu sa, nwoke s, ojo o, agomo pu. evaluation of the possible mechanisms of antihypertensive activity of loranthus micranthus: an african mistletoe. biochem res int 2011;9 pages. doi:10.1155/2011/ 159439. 3. szasz t, lindr ae, davis rp, burnett r, fink gd, watts sw. allopurinol does not decrease blood pressure or prevent the development of hypertension in the development of hypertension in the doca-salt rat model. j cardiovasc pharmacol 2010;56:627-34. 4. schulz e, jansen t, wenzel p, daiber a, münzel t. nitric oxide, tetrahydrobiopterin, oxidative stress, and endothelial dysfunction in hypertension. antioxid redox signal 2008;10:1115–26. 5. szasz t, watts sw. uric acid does not affect the acetylccholine-induced relaxation of aorta from mormotensive and deoxycorticosterone acetatesalt hypertensive rats. j pharmacol exp 2010;333:758-63. 6. ohashi k, winarno h, mukai m. inoue m, prana ms, simanjuntak p, shibuya h. cancer cell invasion inhibitory effects of chemical constituetnts in the parasitic plant scurrula atropurpurea (loranthaceae). chem pharm bull 2003;51:343-5. 7. badyal h, lata h, dadhich ap. animal models of hypertension and effect of drugs. indian j pharmacol 2003;35:349-62. 8. fard sg, shamsabadi ft, emadi m, meng gy, muhammad k, mohamed s. ethanolic extract of eucheuma cottonii promotes in vivo hair growth and wound healing. j anim vet sci 2011;10:601-5. 9. duan xj, zhang ww, li xm, wang bg. evaluation of antioxidant property of extract and fractions obtained from a red alga, polysiphonia urceolata. food chem 2006;95:37–43. 10. rosenblat m, volkova n, aviram m. pomegranate phytosterol (β-sitosterol) and polyphenolic antioxidant (punicalagin) addition to statin, 50 significantly protected against macrophage foam cell formation. atherosclerosis 2013; 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(-)-epicatechin elevates nitric oxide in endothelial cells via inhibitins of nadph oxidase. biochem biophys res comm 2007;359:828-33. 15. kiel ec, benkirane k, javeshghani d, touyz rm, schiffrin el. xanthine oxidase and mitochondria contribute to vascular superoxide anion generation in doca-salt hypertensive rats. am j physiol heart circ physiol 2008;295:281-8. 16. hong co, lee ha, rhee ch, choung sy, lee kw. separation of the antioxidant compound quercitrin from lindera obtusiloba blume and its antimelanogenic effect on b16f10 melanoma cells. biosci biotechnol biochem 2013;1:58-64. 17. piao mj, yoo es, koh ys, kang hk, kim j, kim yj, et al. antioxidant effects of the ethanol extract from flower of camellia japonica via scavenging of reactive oxygen species and induction of antioxidant enzymes. int j mol sci 2011;12:261830. 18. nakayama m, aihara m, chen yn, araie m, tomita-yokotani k, iwashina t. neuroprotective effects of flavonoid on hypoxia-,glutamate-, and oxidative stress-induced retinal ganglion cell death. mol vis 2011;17:1784-93. 19. stangl v, dreger h, stangl k. moleculer target of tea polyphenols in the cardiovascular system. cardiovasc res 2007;739:348-58. athiroh, sulistyowati scurrula atropurpurea and hypertension c:\users\jurnal fkusakti\docume 100 *master program of biology, department of biology, faculty of mathematics and natural sciences, universitas negeri malang **department of biology, faculty of mathematics and natural sciences, universitas negeri malang correspondence: sri rahayu lestari department of biology, faculty of mathematics and natural sciences, universitas negeri malang jl semarang no. 5, malang 65145, jawa timur, indonesia phone : +62341 588077 email: srirahayulestari@um.ac.id orcid id: orcid.org/0000-00032208-4156 date of first submission, december 2, 2 01 8 date of final revised submission, may 14, 2019 date of acceptance, may 15, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina single bulb garlic oil improves interleukin-6 via decreased reactive oxygen species (ros) in high-fat diet male mice riza rahayu ilmawati*, abdul gofur**, and sri rahayu lestari** background high-fat diet (hfd) is highly responsible for the development of excessive reactive oxygen species (ros). ros and low-density lipoprotein (ldl) then trigger macrophage activation to secrete interleukin-6 (il-6). single bulb garlic or in indonesian called bawang lanang is traditional medicine that possesses strong antioxidant properties. this study aimed to evaluate the effect of single bulb garlic oil (sbgo) on ros, il-6, and lymphocyte density in hfd-fed male mice. methods this was an experimental study on 24 male mice randomly subdivided into 6 groups: one group was fed a normal diet, whereas the remaining five groups were fed hfd for 45 days and were then treated with single bulb garlic oil 0 mg/kg, simvastatin 26 mg/kg bw, single bulb garlic oil 12.5 mg/ kg bw, 25 mg/kg bw, and 50 mg/kg bw respectively for the next 35 days. at the end of treatment, the mice were dissected for collection of (i) serum in order to measure ros and il-6 levels using elisa, (ii) aortas to measure il-6 expression using immunohistochemistry-fluorescence (ihc-f) and to obtain lymphocytes from bone marrow and spleen which were then counted using a light microscope. results our results indicated that sbgo decreased the ros level, il-6 level, il-6 expression, and lymphocyte density in hfd mice. sbgo 12.5 mg/kg bw is the optimum dose in our study to reduce inflammation in hfd male mice. conclusion sbgo is useful to reduce inflammation and improve antioxidant imbalance, thus might be a potential antiatherogenic agent. keywords: single bulb garlic, ros, il-6, lymphocyte, male mice original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.100-107 may-august, 2019 vol.38no.2 cite this article as: ilmawati rr, gofur a, lestari sr. single bulb garlic oil improves interleukin-6 via decreased reactive oxygen species (ros) in highfat diet male mice. univ med 2019; 38:1 0 0 -7 . doi: 10 .18 051/ uni vmed. 2018.v38.10 0-1 07 101 univ med vol. 38 no.2 introduction atherosclerosis is a chronic inflammatory disease of the arterial wall due to oxidation of lipoprotein that stimulates the adaptive immune response,(1) the disease ultimately causing coronary heart disease.(2) the prevalence of coronary heart disease in 2013 in indonesia was about 883,4 47 people. (3 ) death from cardiovascular disease, especially coronary heart disease, is estimated to increase by 23.3 million in 2030. the primary cause of atherosclerosis is an unhealthy lifestyle.(4) the high-fat diet causes fat deposits to increase leading to excessive production of lowdensity lipoprotein (ldl) and reactive oxygen species (ros), including superoxide, peroxyl, and hydroxyl radicals.(5) reactive oxygen species causes ldl to be easily oxidized into oxidized low-density lipoprotein (ox-ldl) thus causing vascular injury and organ dysfunction.(6) oxidized low-density lipoprotein will activate endothelial cells in blood vessel walls. thus monocytes are activated into macrophages and cause scavenger receptor (sr) expression. macrophage activation triggers the a ctivation of cd4 + t-ce lls. macrophages and th17 lymphocytes secrete the interleukin-6 (il-6) pro-inflammatory cytokine(7) which induces intercellular adhesion molecule (icam) in endothelial cells. further, il-6 triggers inflammation by increasing the production of neutrophils in infected sites.(8) simvastatin is the general treatment for atherosclerosis. the use of simvastatin has side eff ects called simvastatin-associa ted symptoms.(9) nowadays, herbal medicines are preferable to synthetic drugs. herbal therapy is expected to be safe with fewer side effects on people with atherosclerosis.(10) herbal medicine offers great opportunities for exploration since indonesia has many plant natural resources. one of the plants which are traditionally used as medicine is single bulb garlic. single bulb garlic, or bawang lanang in indonesian, is a traditional medicine for which the plant is grown in extreme conditions. single bulb garlic has a characteristically stronger odor than common garlic. (11) another advantageous effect is that single bulb garlic has a 3.5-fold greater total phenolic content than common garlic.(12) two of the most critical organosulfur compounds in garlic are the non-volatile amino acid γ-glutamyl-s-alk(en)yl-l-cysteine and essential oil of s-alk(en)yl-cysteine sulphoxides or alliin.(13) the enzymatic reaction of γ-glutamyls-alk(en)yl-l-cysteine produces allicin.(14) allicin is the primary active compound in garlic which has been reported to inhibit ros and nadph oxidase in huv ecs. (15 ) a previous study reported that aqueous garlic extract exerts antioxidant activity by scavenging ros.(16) in addition, garlic oil extract was reported to improve regulatory t cells in hfd mice.(17) however, there are as yet few reports about the beneficial effect of using oil extract of single bulb garlic to directly scavenge ros and ameliorate interleukin-6. single bulb garlic is expected to inhibit the formation of ros as the free radicals, thus inhibiting the secretion of il-6 and decreasing the number of lymphocytes in highfat diet (hfd)-fed mice. methods research design this was experimental research using a completely randomized design. the research was carried out at the human and animal physiology laboratory, universitas negeri malang; the physiology laboratory, faculty of medicine, central life science laboratory, and the bioscience institute, brawijaya university, malang, from april to november 2017. single bulb garlic oil preparation single bulb garlic oil was prepared by the soxhlet method base d on the proc edure conducted at the phytochemical laboratory of the batu materia medica technical implementing unit (upt materia medica batu) as follows: 1 kg of air-dried single bulb garlic was extracted with 4.5 l of n-hexane (emsure®, cat no: 102 104367). the resulting extract was 3 ml then evaporated using a rotary evaporator (ika® rv 10 basic v). the single bulb garlic oil stock (3 ml) was th en p r ep ar ed f or tr ea tme nt t o constitute doses of 12.5 mg/kg bw, 25 mg/kg bw, and 50 mg/kg bw, respectively, diluted in corn oil (tropicana slim®, nutrifood, indonesia). study subject and sample size determination mice were obtained from cv. galaxy science, jember. the mice were balb/c strain males aged 8 weeks and weighing 28 g ± 2 g. the number of required mice was determined based on federer’s formula. twenty-eight male balb/c mice housed in individual plastic cages were acclimatized for 1 week. the mice were randomly divided into two major groups, i.e. one group on normal diet (nd) (n=4) and five groups on hfd (n=20). the normal diet concentrate consisted of 63% carbohydrate, 16% protein, 3% fat, 21% vitamins and minerals (pt. comfeed phokpand, indonesia). the hfd consisted of hi-gro medicated 551 chicken feed (300 g), corn flour (200 g), duck egg yolk (100 g), wheat flour (50 g), cholic acid (0.1 g), and waste cooking oil (0.3 l). the mice were fed the hfd at a dose of 7 g/day/mouse and ad libitum drinking water for 45 days. the hfd groups were then divided randomly and equally (n = 4) into hfd, hfd + simv (hfd + simvastatin 26 mg/kg), and hfd + p1-p3 (hfd + sbgo 12.5, 25, and 50 mg/kg bw r e sp ect ive ly). aft er 45 da ys of di et manipulation, the three concentrations of sbgo and simvastatin were administered orally for 35 days consecutively. measurement of ros and il-6 at the end of the experiment, the mice were sacrificed through euthanasia (isoflurane 4%). blood was collected (± 0.5 ml) from the heart and incubated for 30 minutes at 37oc, then centrifuged at 10000 rpm for 10 minutes, and the serum collected. ros and the il-6 level we r e me a s ur e d us in g el isa a t 45 0 n m according to the protocol. briefly, 20 μl serum was mixed with coating buffer (1:20) then incubated at 4°c overnight. the well plate was then washed with pbs-tween 0.2%, coated with anti-il-6 primary antibody (cat. no. sc73319, santa cruz biotechnology, usa) or ros (e1654mo: bt-lab) (1:1000) in assay buffer, and incubated for 2 hours at room temperature. after that, the well plate was washed with pbstween-0.2%, then coated with secondary antibody, i.e. alkaline phosphatase (ap)conjugated goat anti-rabbit igg in assay buffer ( 1 : 20 0 0) , i nc u b a t e d f or 1 hour a t r oo m temperature, and washed with pbs-t 0.2%. to the well plate was added streptavidin horseradish peroxidase (sa-hrp) in assay buffer (1:2000) and incubated for 1 hour at room temperature. the well plate was washed with pbs-tween0.2%, then 3,3’,5,5’-tetramethylbenzidine (tmb) chromogenic reagent for peroxidase was added, and incubated for 20 minutes at room temperature. finally, 1n hcl as the stop solution was added and the results read immediately with an elisa reader (bio-rad benchmark, japan) at 450 nm. immunohistochemistry from each mouse, the aorta was separated from the heart and fixed in buffered formalin 10%. each aorta was then prepared into slides for immunohistochemistry-fluorescence (ihcf) with il-6 as the primary antibody. firstly, the aorta slides were incubated with primary antibody and 2% bovine serum albumin (bsa) in the ratio of 1:1000 for one hour. then the slide was treated with the secondary antibody, na me ly goat anti-mous e igg fl uo res ce in isothiocyanate (fitc) in 2% complete bsa (1:1500), and incubated for one hour. the slides were then washed using complete pbs ph 7.4 for three times, dried and covered with cover glasses. the slides were observed using a fluorescence microscope (fsx-100), and il-6 expression was measured (intensity/mm2).(18) measurement of lymphocyte density lymphocytes were taken from the spleen and bone marrow. briefly, spleen lymphocytes imawati, gofur, lestari reactive oxygen species in high-fat 103 univ med vol. 38 no.2 were minced using a syringe holder. the homogenate was put into a 15 ml polypropylene tub e. ( 19 ) bone ma rr ow lymphocyt es we re isolated by cutting both ends of the femur then flushing out using 1 ml of phosphate buffered saline (pbs). the suspension was then put into a 15 ml polypropylene centrifuge tube.(20) the lymphocyte density was calculated under the light microscope using a hemocytometer and trypan blue staining.(21) the number of lymphocytes wa s c ou n te d in 5 la r ge s qu ar e s of t he hemocytometer. the lymphocyte density was calculated according to the formula in dwijayanti et al.(21): σ cells = σ cells counted x 5 to correct for the 1:5 dilution from the trypan blue addition x 104 cells/ml. statistical analysis data were expressed as mean ± standard d e vi a t i on ( sd ) . o ne -wa y an ova wa s performed to assess significant differences (p<0.05) between groups after sgbo treatment followed by duncan multiple range test (dmrt) as post hoc test. one-way anova was performed using spss 16.0 for windows (spss inc., chicago, il, usa). the number of lymphocytes was analyzed using the kruskalwallis h test at the level of confidence of 95% (=0.05) and continued with the mann-whitney u test. ethical clearance the research had obtained approval from the ethics committee of brawijaya university under no. 880-kep-ub. results single bulb garlic oil effects on ros, il-6 level, expression of il-6 in the aorta, and lymphocyte density in the spleen and bone marrow in male mice fed on the high-fat diet. our results showed that ros level was significantly increased in hfd mice compared to the normal ones (p<0.05) (table 1). sbgo treatment at a dose of 12.5 mg/kg bw significantly reduced the ros le vel compar ed to hfd (p<0.05). interestingly, sbgo 25 and 50 mg/kg bw decreased ros level but did not significantly differ from hfd. our findings showed that sbgo at 12.5 mg/kg bw reduced ros towards near normal levels. the il-6 level increased significantly in hfd mice compared to the normal group (p<0.05). sbgo treatment at a dose of 12.5 mg/kg bw significantly reduced the il-6 level compared to hfd (p<0.05). interestingly, sbgo 25 and 50 mg/kg bw failed to decrease il-6 level and were not significantly different compared to hfd. our findings showed that sbgo at 12.5 mg/kg bw reduced il-6 towards near normal levels (table 1). a b c table 1. ros and il-6 level, il-6 expression in aorta, and lymphocyte density of mice fed a normal or high fat diet after treatment with/without sbgo ros : reactive oxygen species; il : interleukin; n = normal diet, k+ = hfd + simvastatin 26 mg/kg bw, k= hfd only, p1=p3 = hfd + sbgo 12.5, 25, and 50 mg/kg bw respectively. the same notation indicates no differences between groups based on dmrt test (p<0.05). * = data analysis based on kruskal-wallist test followed by mann-whitney test (p0.05) treatment group p value n (n=4) k+ (n=4) k(n=4) p1 (n=4) p2 (n=4) p3 (n=4) ros level (pg/ml) 286.59a ± 6.71 292.43ab ± 9.24 302.47b ± 4.02 286.12a ± 4.99 298.88b ± 4.54 300b ± 6.85 0.05 il-6 level (pg/ml) 225.91a ± 56.98 275.92a ± 87.72 404.04b ± 23.51 279.53a ± 86.77 445.61b ± 115.81 396.12b ± 52.46 0.05 il-6 expression in the aorta (intensity/mm2) 58.57a ± 2.99 59.61a ± 4.77 70.6b ± 3.24 59.8a ± 2.6 66.13b ± 4.77 66.97b ± 2.1 0.05 lymphocyte density (1x104)* 906a ± 28.12 933.25a ± 72.75 3788a ± 241.27 962a ±105.48 968a ± 47.07 984.75a ± 73.84 0.05 104 t he e x pr e s si o n o f il-6 in c r e a s e d significantly (p<0.05) in hfd mice compared to the normal group (p<0.05) (table 1, figure 1). sbgo treatment reduced the il-6 expression compared to hfd alone. interestingly, sbgo 25 and 50 mg/kg bw reduced il-6 expression but were not significantly different compared to hfd. our findings showed that sbgo at 12.5 mg/kg bw reduced il-6 expression towards near normal levels. the lymphocyte density both in bone marrow and spleen decreased significantly in hfd mice compared to the normal group (p<0.05). sbgo treatment decreased lymphocyte density compared to hfd. interestingly, sbgo 12.5, 25 and 50 mg/kg bw decreased lymphocytes but not significantly different compared with the normal group (table 1). discussion our recent findings suggest that sbgo suppresses the production of ros in mice fed a high-fat diet. these findings are in line with a previous study conducted by basu and sur (22) which reported that s-allyl cysteine protected cells from h 2 o 2 -induced oxidative damage. moreover, organosulfur compounds in garlic oil may act as radical scavengers of ros (16,23) and possess antioxidant activity to inhibit lipid peroxidation.(24) garlic oil increases superoxide dismutase (sod), catalase, and glutathione peroxidase (gpx) in the endothelial cells of blood vessels.(23) single bulb garlic acts as a chainbreaking antioxidant for scavenging f ree radicals.(12) our recent finding suggests that exoge nous antioxida nt is needed to help neutralize ros during hfd administration. the decrease of ros will inhibit oxidation thus reducing the amounts of ox-ldl followed by inhibition of macrophages in the immune system. allicin decreases the level of cholesterol in the blood by reducing cholesterol synthesis, through inhibition of hydroxymethyl-glutaryl-coenzyme a (hmg-coa) reductase.(25) allicin and hmgcoa have similar structures, thus allicin is suspected to be a competitive inhibitor of hmgcoa reductase enzyme. sulfur-containing groups in allicin play an antioxidant role that could decrease ox-ldl.(15) figure 1. il-6 expression in the aorta of mice at the end of study. blue arrow indicates the expression of il-6 in the aorta. n = normal diet, k+ = hfd + simvastatin 26 mg/kg bw, k= hfd only, p1 = hfd + sbgo 12.5 mg/kg bw, p2 = hfd + sbgo 25 mg/kg bw, p3 = hfd + sbgo 50 mg/kg bw imawati, gofur, lestari reactive oxygen species in high-fat 105 univ med vol. 38 no.2 relationship with the development of oxidative stress. also, we did not measure erythrocyte progenitors, such as ter-119+cd34+, ter119+, and ter+vla-4+. our study is expected to be one of the solutions for the treatment of metabolic disease, particularly that caused by hfd, since garlic is easy to find, is commonly used daily, and is an effective functional food. however, further research still needs to be conducted before single bulb garlic oil can be applied to h uma n s by givi ng r e c omme n da t i o ns o n appropriate doses. conclusions single bulb garlic oil decreased ros and il-6 levels, and lymphocyte density in mice fed a high-fat diet. based on our research, sbgo 12.5 mg/kg bw is the effective dose to reduce ros and il-6 levels, and lymphocyte density in mice fed a high-fat diet. conflict of interests co mp e t i ng i n t e r e s t s : n o r e l e va nt disclosures. acknowledgement the authors thank all staff of animal physiology laboratory, central life science laboratory, and bioscience institute, brawijaya university, for the use of facilities. the research was part of research by dr. sri rahayu lestari, m.si with contract number of drpm 3.4.8/ un32.14/lt/2017. contributors rri contributed to writing the manuscript, data analysis and interpretation of the data. ag contributed to data collection and processing. srl contributed to the concept, supervision and critical review. all authors have read and approved the final manuscript. the inhibition of hmg-coa reductase causes inhibition of cholesterol synthesis in the liver thus the liver secretes lower amounts of cholesterol into the blood. the liver will activate ldl receptors to bind ldl in the blood, thus decreasing cholesterol and ldl in the blood.(25) the decreased ldl level could suppress the secretion of il-6 by macrophages and th17. our findings suggest that sbgo treatment r e d u c e s bot h il-6 s e c r e t i on a n d il -6 expression. this is in accordance with the research that garlic oil could suppress the production of the proinflammatory cytokine il6 through the il-6/mek5/erk5 signaling pathway.(26) furthermore, garlic components restore the lymphocyte balance during hfd. our result is consistent with that of a previous study which reported that hfd leads to an imbalanced blood production due to an increase in gr-1 expression and a decrease in erythrocyte progenitor markers, such as ter-119+cd34+, ter119+, and ter+vla-4+ during a high-fat f r uc t o se d ie t. ( 1 9 ) we a s s ume th a t ga r l i c components maintain the balance of blood production and restore the lymphocyte density to close to normal. our research indicates that the sbgo dose of 12.5 mg/kg bw is the effective dose to inhibit the production of the il-6 inflammatory cytokine in mice fed a high-fat diet. sbgo may be used as an alternative agent to reduce free radicals and inflammation caused by hfd. further research is still needed to explain the detailed me c h a ni s m o f sbgo i n ove r c o min g inflammation caused by hfd. our findings suggest that sbgo at higher doses cannot suppress the ros and il-6 levels. hamlaouigasmi et al.(27) reported that garlic at high doses triggers a prooxidative state characterized by the fenton reaction between h 2 o 2 and free iron inducing tissue calcium repletion. one limitation of the present study is that we did not measure ros end-products such as malondialdehyde (mda). we also did not mea s ur e a n t iox ida nt s t o d e te r mine t 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of biology), faculty of medicine, yarsi university jln. letjend suprapto, cempaka putih, jakarta 10510 phone: 4206674-76 email: endpurwaning@gmail.com univ med 2012;31:73-80 universa medicina may-august, 2012may-august, 2012may-august, 2012may-august, 2012may-august, 2012 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 vol.31 no.2 background curcumin inhibits steroidogenesis in luteal cell cultures by inhibiting progesterone secretion. the site of action of curcumin on steroidogenesis in luteal cell cultures is as yet unkown. the purpose of this study was to determine the influence of curcumin on phosphorylation of extracellular signal regulated kinase (erk) in steroidogenesis of luteal cell cultures. methods an experimental study with control was conducted to investigate the site of action of curcumin by measurement of erk phosphorylation in luteal cell cultures (lcc) after administration of luteinizing hormone (lh) and/or prostaglandin f2 alpha (pgf2á). the subjects of this study were luteal cell cultures derived from the corpus luteum of sprague dawley rat with superovulation induced by pregnant mare serum gonadotropin. the luteal cell cultures were devided into 16 groups, both with and without the addition of forskolin and each group contained 4 replicates samples. curcumin was administered immediately following lh and/or pgf2á stimulation with or without addition of forskolin. the cell culture was then incubated for 24 h. erk phosphorylation was measured by immunohistochemistry. data on erk phosphorylation was analyzed using one-way anova, followed by multiple comparison tests. results lh significantly increased erk phosphorylation, whereas pgf2á significantly reduced erk phosphorylation. forskolin significantly increased erk phosphorylation to a similar degree as lh. curcumin inhibited erk phosphorylation in both lh and forskolin-stimulated luteal cell cultures. conclusion curcumin inhibits erk phosphorylation in luteal cell cultures by suppressing signal transduction upstream of erk. key words: curcumin, signal transduction, forskolin, luteal cells culture curcumin inhibits luteal cell steroidogenesis by suppression of extracellular signal regulated kinase endang purwaningsih*, sri kadarsih soejono**, djaswadi dasuki§, and edy meiyanto§§ abstract 74 purwaningsih, soejono, dasuki, et al curcumin inhibits luteal cell kurkumin menghambat fosforilasi extracellular regulated kinase pada steroidogenesis kultur sel luteal latar belakang senyawa kurkumin dapat menghambat steroidogenesis kultur sel luteal dengan cara menghambat sekresi progesteron. letak kerja kurkumin pada steroidogenesis kultur sel luteal belum diketahui. tujuan penelitian ini adalah untuk menentukan pengaruh pemberian kurkumin terhadap sinyal fosforilasi extracellular regulated kinase (erk) pada steroidogenesis kultur sel luteal metode sebuah penelitian eksperimental dengan kontrol digunakan untuk menilai efek kurkumin terhadap fosforilasi erk setelah diberikan luteinizing hormone (lh) dan/atau prostaglandin f2 alpha (pgf2á) pada kultur sel luteal tikus subjek penelitian adalah kultur sel luteal yang diperoleh dari tikus sprague dawley yang diinduksi dengan pregnant mare serum gonadotropin (pmsg). kultur sel luteal dibagi menjadi 16 kelompok dengan atau tanpa diberi forskolin, dan setiap kelompok terdiri atas 4 replikasi. kurkumin diberikan segera setelah distimulasi lh dan atau pgf2á dengan atau tanpa penambahan forskolin. kemudian kultur sel luteal diinkubasi selama 24 jam. fosforilasi erk diukur dengan metode immunohistochemistry. data fosforilasi erk dianalisis menggunakan anova satu arah dilanjutkan dengan uji perbandingan berganda. hasil lh meningkatkan fosforilasi erk secara bermakna, sedangkan pgf2á mengurangi fosforilasi erk secara bermakna. forskolin meningkatkan fosforilasi erk secara bermakna dan hampir sama dengan yang terstimulasi lh. kurkumin menghambat fosforilasi erk pada kultur sel luteal yang terstimulasi lh maupun forskolin. kesimpulan kurkumin menghambat fosforilasi erk pada kultur sel luteal dengan cara menekan sinyal transduksi di up stream erk. kata kunci: kurkumin, sinyal transduksi, forskolin, kultur sel luteal abstrak introduction regulation of steroidogenesis in luteal cells has an exceedingly complex mechanism, and produces progesterone (p4) as a major component. the regulation of steroidogenesis in luteal cells involves several hormones, such as luteinizing hormone (lh) and prostaglandin f2 alpha (pgf2á ). lh secretion continuously s t i m u l a t e s t h e c o r p u s l u t e u m t o s e c r e t e progesterone,(1) by involving signal transduction through the activity of adenylate cyclase within the luteal cell membrane, the formation of cyclic adenosine 3’,5’ monophosphate (camp) and the activation of protein kinase a (pka) and steroidogenic enzymes, such as cytochrome p450 side chain cleavage (p450scc), and 3âhydroxysteroid dehydrogenase (hsd).(2) the role of pgf2á as an antigonadotropic is among other things to interfere with the binding of lh to its receptor and decreasing the number of l h r e c e p t o r s , i n h i b i t i n g t h e a c t i v i t y o f adenylate cyclase and the accumulation of camp, reducing the transcription of cytochrome p450scc and 3â-hsd, reducing the secretion of progesterone via the protein kinase c (pkc) 75 univ med vol. 31 no.2 pathway,(3) and inhibiting the phosphorylation of extracellular signal regulated kinase mitogen activated protein kinase (erk map-kinase). p k a i s i n v o l v e d i n t h e t r a n s c r i p t i o n a l regulation of the steroidogenic acute regulatory (star) gene and the expression of cytochrome p-450 side chain cleavage (p-450 scc) by increasing the phosphorylation of camp response element binding protein (creb) and extracellular signal regulated kinase (erk).(4) star plays a role in cholesterol transport from the cytosol into the mitochondrial outer membrane, while cytochrome p-450sec within the mitochondria converts the cholesterol into pregnenolone. pregnenolone is then converted into progesterone by 3â-hsd in the smooth endoplasmic reticulum.(1) the compound curcumin (1,7 bis (4hydroxy-3-methoxyphenyl)-1-6-heptadine -3-5dion) has been isolated from the extract of the curcuma rhizome and synthesized in the l a b o r a t o r y, a n d i t s a n a l o g s h a v e b e e n constructed by modification of its terminal aromatic methylenated residues.(5) one of the curcumin analogs that has been patented as an anti-inflammatory drug and introduced as n a t i o n a l m o l e c u l e ( m o l e k u l n a s i o n a l , molnas) is pentagamavunon-0 (pgv-0). curcuma extract can cause anovulation and reduce testicular weight, testosterone concentration, and spermatogenesis. in vitro studies on the reproductive system have reported an effect of curcumin on luteal cell cultures following stimulation by human chorionic gonadotropin (hcg).(6) the role of curcumin and pgv-0 in the production of progesterone with or without the administration of theophylline, a phosphodiesterase inhibitor, has also been reported. curcumin has also been shown to be capable of inhibiting progesterone production in cultured granulosa cells.(7) on the other hand, curcumin is known to inhibit cox-2, an enzyme that plays a role in the synthesis of pgf2á from arachidonic acid. the results of abovementioned studies indicate that even without pgf2á , curcumin inhibits the production of progesterone. thus curcumin inhibits signal transduction in luteal cell steroidogenesis, but its site of action is still not known. the objective of this study was to e v a l u a t e t h e e ff e c t o f c u r c u m i n o n steroidogenesis in luteal cells by measurement of erk phosphorylation. methods design of the study an experimental study was conducted f r o m s e p t e m b e r 2 0 0 7 t o a p r i l 2 0 0 8 , t o investigate the effect of curcumin on erk phosphorylation in luteal cell cultures (lcc) after administration of lh and/or pgf2á. the study involved the following 16 groups, both with and without the addition of forskolin: 1. lcc + methanol 1 % (as control), 2. lcc + curcumin ; 3. lcc + lh ; 4. lcc + pgf2á ; 5. lcc + lh + pgf2 á; 6. lcc + lh + curcumin; 7. lcc + pgf2 á + curcumin; 8. lcc + lh + pgf2á + curcumin. groups 9-16 are identical to groups 1-8 except for the addition of forskolin. each group contained 4 replicates samples. materials the subjects of this study were luteal cells derived from the 4-day old corpus luteum of a premature female sprague dawley rat (rattus norvegicus l) with superovulation induced by pregnant mare serum gonadotropin/pmsg (gestyl, organon). the luteal cells were mechanically and enzymatically separated from the corpus luteum. reagents used for the luteal cell cultures were minimum essential medium (mem), fetal bovine serum (fbs), penicillins t r e p t o m y c i n ( p e n s t r e p ) , g e n t a m y c i n , fungizone, phosphate buffered saline (pbs), collagenase, trypan blue (gibco, brl, life technologies, rockville, md). reagents for assessment of erk phosphorylation were rabbit phosphorylated erk1/erk2 pab (promega, uk), streptavidine-biotin, decaminobenzidine tetrahydrochloride (dab), theophylline and 76 purwaningsih, soejono, dasuki, et al curcumin inhibits luteal cell forskolin (both from sigma aldrich inc, st. louis, mo). chemicals required for treatment and tests were synthesized curcumin and pentagamavunon-0 (lab. molnas, fakultas farmasi ugm), lh, pgf2á , and forskolin (sigma aldrich inc, st. louis, mo). preparation of mem culture medium m e m p o w d e r f r o m t h e s a c h e t s w a s dissolved in an erlenmeyer flask containing one liter of sterile twice-distilled water using a magnetic stirrer. to the mem solution was added 2.2 gram sodium bicarbonate, imparting a red color to the solution. the ph of the medium was then measured by means of a electric ph meter. the ph was adjusted to 7.4 by the addition of 0.1 m sodium hydroxide or 0.1 m hydrochloric acid as required. the mem solution was filtered using a peristaltic pump in a sterile room. from the erlenmeyer flask the medium was passed into the pump through a plastic pipe, at the end of which a sterile 0.22 ì m millipore filter was attached. the filtered solution was collected in a sterile medium flask and stored in the refrigerator at 2-8o c. to prepare a dispersion medium, to the mem solution was added pen-strep 5%, fungizone 1% and collagenase 1mg/ml. for a washing medium, to the mem solution was added serum 10 %, while for a growth medium to the mem solution was added penstrep 5%, fungizone 0.7% and fbs 10%. preparation of lcc the corpus luteum was surgically removed from the ovary in a cool steril laminar-airflow room. after dressing, the corpus luteum was enzymatically dispersed in a medium containing collagenase/dispase 1 mg/ml and 5% penstrep. the preparation was subsequently incubated in co 2 5% at 370c for 30 minutes, then dispersed mechanically by vortexing and shaking for 3 minutes. this procedure of incubation and subsequent dispersion was repeated three times. the cell supension was then filtered through sterile gauze, and cleansed in washing mem containing fetal bovine serum [fbs] 10% (gibco), then it was centrifuged for 10 minutes 4oc in a sorvall super t21 centrifuge at a speed of 720 g (2.000 rpm). the washing was repeated t h r e e t i m e s . a t t h e t h i r d w a s h i n g , t h e supernatant was discarded and growth mem was added, containing fbs 10%, penstrep 5%, gentamycin 5% and fungizone 0.7%. before seeding, viable cells were counted in a hemocytometer after the addition of trypan blue. the suspension was diluted with growth medium to obtain a luteal cell concentration of 105-106 cells/ml. luteal cells were then seeded in 24-well culture plates at a volume of 0.5 ml per well or on 96-well culture plates at a volume of 0.1 ml per well, and incubated in co 2 5% at 37oc for 72 hours. the culture medium was renewed once in 24 hours and checkcd by observation of the cell cultures under an inverted microscope. viable cells adhered to the bottom of the wells, while dead cells floated in the medium and were removed on renewal of medium. the cells were then treated with a single dose of curcumin at a dosage of 100 ìm/ l in accordance with the study design. before treatment the cells in one of the sample wells were also counted. determination of erk phosphorylation after treatment, coverslips were placed in the wells and the luteal cell cultures incubated for 24 hours. the luteal cells adhering to the coverslips were immunohistochemically stained in the pathology laboratory of dr. sardjito central hospital, yogyakarta. the procedure used to measure erk phosphorylation by immunohistochemistry (ihc) in this study was a modification of the method of cicero & herrup,(8) as follows: the treated cell culture on the coverslip was incubated in 5% co 2 at 37oc for 24 hours, after which the coverslip was placed on a poly-l-lysine slide, and fixed in acetone or methanol for 10 minutes at -200c. the slide was then washed three times in pbs, 77 univ med vol. 31 no.2 each time for 5 minutes, and h 2 o 2 0.3 % was added dropwise for 20 minutes. normal mouse serum (1:50) was also added dropwise for 15 minutes. the liquid covering the slide was discarded but the slide was not rinsed. this was followed by the addition of the primary rabbit anti-phosphorylated erk antibody erk1/ erk2 pab, at a dilution of 1:200 for 60 minutes. the slide was washed three times in pbs, each washing lasting 5 minutes, then it was incubated with biotinylated secondary antibody for 5-10 minutes, and again washed three times 5 minutes in pbs. this was followed by incubation with streptavidin-peroxidase for 5-10 minutes, washing in pbs 3 x 5 minutes, i n c u b a t i n g w i t h t h e c h r o m o g e n deaminobenzidine tetrahydrochloride for 5-10 minutes, at a chromogen-substrate ratio of 1: 20, and finally washing in distilled water. the preparation was then immersed in hematoxylin for 3-5 minutes as counterstain, rinsed in distilled water, dehydrated with ethanol 95%, and immersed in xylene for 10 minutes. to the preparation was added mounting medium (canada balsam), then it was covered with a coverslip. erk phosphorylation was observed by light microscopy. phosphorylation-positive c e l l s h a d a d a r k b r o w n c o l o r, w h e r e a s phosphorylation-negative cells were violet. a total of 200 luteal cells were counted and the results were expressed in percentages. data analysis the percentages of erk phosphorylation were analyzed by one-way anova, followed by l e a s t s i g n i f i c a n t d i ff e r e n c e m u l t i p l e comparison tests. ethical clearance this study on rat luteal cell cultures obtained ethical clearance from the commission for research ethics for the medical and health sciences, faculty of medicine, gadjah mada university, yogyakarta. results erk1/erk2 phosphorylation in luteal cell cultures was detected by immunohistochemstry (ihc). the calculation was based on the percentage of phosphorylation-positive cells obtained by ihc. the phosphorylation value considered positive was the erk1/erk2 detected by ihc analysis, erk1/erk2 cells having a brown-colored nucleus and cytoplasm after staining with dab chromogen (figure 1). the results of calculation of erk1/erk2 phosphorylation (percentage of phosphorylationpositive cells) in all groups after administration of curcumin are presented in table 1. there was a significant differences between treatment groups. control (solvent) solvent + curcumin figure 1. ihc erk phosphorylation results in control and treatment groups. magnification 400x brown color (arrows) in nucleus and cytoplasm indicates positive erk phosphorylation 78 purwaningsih, soejono, dasuki, et al curcumin inhibits luteal cell erk phosphorylation after curcumin administration in the solvent lcc group with or without forskolin and in the lcc groups stimulated by lh, pgf2á and lh + pgf2á showed significantly lower values (p<0.05) compared with the groups without curcumin. administration of forskolin to the solvent lcc group significantly increased erk1/erk2 phosphorylation (p<0.05) compared with the solvent-only group. discussion l h s t i m u l a t i o n i n c r e a s e d e r k phosphorylation, similar to previous studies.(9) in previous studies forskolin was used for detetcting erk phosphorylation, because f o r s k o l i n i s c a p a b l e o f i n c r e a s i n g e r k phosphorylation by increasing adenyate cyclase activity.(10,11) erk1/erk2 phosphorylation in the groups receiving forskolin was almost equal that in the groups with lh stimulation only. luteinizing hormone can activate erk directly from g. protein-coupled receptor (gpcr).(9) the increase in erk1/erk2 phosphorylation by lh may also be the result of increased camp concentration through pka activation. both lh and camp are capable of activating erk phosphorylation. forskolin increases erk phosphorylation in the same manner as lh, because both substances increase adenylate cyclase activity. (12) the administration of pgf2á is antigonadotropic, as it inhibits erk phosphorylation, which is consistent with the opinion of previous investigators. pgf2á in luteal cells can influence erk phosphorylation through the ca 2+/calmodulin pathway by inducing the expression of the nur 77 gene.(13) administration of curcumin causes a significantly lower erk phosphorylation in comparison with the solvent group. curcumin shows antigonadotropic activity by inhibiting the increase in erk phosphorylation by lh alone or by lh+pgf2á . inhibition of erk phosphorylation by pgf2á was stronger after addition of curcumin. curcumin also causes a significant inhibition of erk phosphorylation after the addition of forskolin. this indicates that curcumin has antigonadotropic activity against the increase in erk phosphorylation b y f o r s k o l i n . t h e i n c r e a s e i n e r k phosphorylation by forskolin is almost equal t o t h a t b y l h . ( 1 2 ) i n h i b i t i o n o f e r k phosphorylation by curcumin is the result of table 1. erk phosphorylation in each group after curcumin administration with and without forskolin values are mean ± standard deviation 79 univ med vol. 31 no.2 inhibition of camp accumulation, because erk is situated downstream to camp/pka. pka phosphorylation can activate erk and the transcription factor creb. (14) there is a possibility that curcumin also inhibits erk phosphorylation via signal transduction of the gpcr complex. previous studies reported that p4 concentration in lh-stimulated lcc was slightly higher than p4 concentration due to forskolin.(15) this indicates that apart from signal transduction through camp/pka, c u r c u m i n a l s o u s e s t h e d i r e c t s i g n a l transduction pathway from gpcr. another possibility is that curcumin interferes with gá i , one of the gá members from the gpcr complex that activates signal transduction directly to erk. there are four gá members, namely gá q/11 which activates signal transduction from gpcr to plc/pkc; gás, which activates signal transduction via c a m p / p k a ; g á i w h i c h a c t i v a t e s s i g n a l transduction directly from gpcr to erk, and gá 12/13 . (16,17) erk phosphorylation can be a c t i v a t e d f r o m t h e g p c r c o m p l e x v i a cooperation between gá q and gá i . whether curcumin can inhibit the cooperation of both gá members is unclear. the obtained results i n d i c a t e t h a t c u r c u m i n i n h i b i t s e r k phosphorylation both in the control group and the groups stimulated by lh with or without pgf2á . curcumin exhibits an antigonadotropic effect on steroidogenesis in lcc, especially on erk phosphorylation. based on its activity as revealed in this study, curcumin may be developed into an antifertility agent with antiimplantation action by inhibiting p4 production through inhibition of signal transduction in steroidogenesis of luteal cells. apart from its anti-inflammatory activity, curcumin may be developed as an antiovulatory agent. previous studies have demonstrated that curcumin inhibits p4 and e2 production in granulosa cell cultures by stimulating fsh.(7) it is hoped that this investigation may b e c o m e a v a l u a b l e c o n t r i b u t i o n t o t h e community and the pharmaceutical industry as a reference on the use of curcumin as an a l t e r n a t i v e c o m p o n e n t f o r d e v e l o p i n g medications for fertility control, such as contraceptives. one limitation of the present study is that it did not study the factors playing a role on signal transduction upstream to erk, such as adenylate cyclase activity, expression of pka and expression of transcription factors. further studies are necessary to study the effect of curcumin on these parameters. conclusion curcumin inhibits signal transduction for steroidogenesis in luteal cells by acting on a site upstream to erk. acknowledgment the authors are grateful to the chairman of the governing board (bph) of yarsi foundation, prof. dr. h. jurnalis uddin, pak and the chairman of bpps dirjen dikti for financial support of this study. references 1. stauffer ri. progesterone as mediator of gonadotropin action in the corpus luteum: beyond steroidogenesis, hum reprod update 2003;9:94–117. 2. chin ec, harris te, abayasekara dre. changes in camp dependent protein kinase (pka) and progesteron secretion in luteinizing human granulose cells. j endocrinol 2004;183: 39-50. 3 wiltbank mc, diskin mg, flores ja, niswender gd. regulation of the corpus luteum by protein kinase c. ii. inhibition of lipoprotein stimulated steroidogenegenesis by prostaglandin f2α. biol reprod 1990;42:239-45. 4. wettschureek n, offermanns s. mammalian g protein and their cell type specific functions. physiol rev 2005;85:1159-204. 5. niswender gd. molecular control of luteal secretion of progesterone. reproduction 2002; 123:333-9. 6. zulkhah n, soejono sk, suwono. pengaruh kurkumin sintetik terhadap produksi progesteron 80 purwaningsih, soejono, dasuki, et al curcumin inhibits luteal cell oleh kultur sel luteal tikus dengan perangsangan hcg dan pgf2α. sains kedokteran 2000;13:5362. 7. nurcahyo h, soejono sk. effect ct of curcumin and pentagamavunon-0 on the steroidogenesis, proliferation activity and apoptosis in cultured porcine granulosa cells at varying stages of follicular growth. in : pudjono gig, sismindari, riyanto s, meiyanto e, susidarti ra, ikawati, nugroho ak, ritmaleni, editors. recent development in curcumin pharmaco-chemistry. proceedings of the international symposium on recent progress in curcumin research; 2007, 11-12 september 2006, yogyakarta. faculty of pharmacy gadjah mada university;2007.p.22741. 8. cicero s, herrup k. cyclin dependent kinase-5 is essential for neuronal cell cycle arrest and differentiation. j neurosci 2005;25:9658-68. 9. shiraishi k, and ascoli m. activation of the lutropin /choriogonadotropin receptor in ma10 cells stimulates tyrosine kinase cascades thatactivate ras and the extracellular signal regulated kinase (erk1/2). endocrinol 2006; 147:3419-27. 10. dewi da, abayasekara dre, wheeler-jones rd. requirement for erk1/2 activation in the regulation of progesteron production in human granulosa-lutein cells is stimulus specific. endocrinol 2002;143:877-88. 11. tajima k, yoshii k, fukuda s, orisaka m, miyamoto k, amsterdam a, et al. lh-induced erk activation differently modulates progesterone and androstenedione production in bovine theca cells. endocrinol 2005;146:290310. 12. salvador lm, maizels e, hales db, miyamoto e,yamamoto h, hunzicker-dunn m. acute signaling by the lh receptor is independent of protein kinase c activation. endocrinol 2002; 143:2986-94. 13. stocco co, lau lf, gibori g. a calcium / calmodulin dependent activation of erk 1/2 mediates jun d phosphorylation and induction of nur 77 and 20 á-hsd genes by prostaglandin f2á in ovarian cells. j biol chem 2005;27:3293302. 14. needle e, piparo k, cole d, worral c, whitehead i, mahon g, et al. protein kinase aindependent camp stimulation of progesterone in a luteal cell model is tyrosine kinase dependent but phosphatidylinositol-3 kinase and mitogen activated protein kinase independent. biol reprod 2007;77:147-55. 15. purwaningsih e, meiyanto e, dasuki d, soejono sk. efek kurkumin sintesis dan pentagamavunon-0 terhadap produksi progesteron kultur sel luteal dengan pemberian forskolin. j kedok yarsi 2007;15:171-7. 16. new dc, wong yh. molecular mechanisms mediating the g-protein coupled receptor regulation of cell cycle progression. j mol signal 2007;2:2. doi:10.11871760-2128-2-2. 17. belcheva mm, coscia cj. diversity of g-protein coupled receptor signaling pathways to erk/ map kinase. neurosignals 2002;11:34-44. oktavianus 192 *department of anatomy, facultas of medicine, trisakti university correspondence dr. diana samara, mkk department of anatomy, facultas of medicine, trisakti university jl. kyai tapa grogol, jakarta barat phone: +6221-5672731 ext: 2101 email: dianasamara2@gmail.com univ med 2012;31:192-9 abstract universa medicina september-december, 2012september-december, 2012september-december, 2012september-december, 2012september-december, 2012 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 vol.31 no.3 background physical activity is the greatest contributor to achievement of adequate physical activity. children performing adequate daily physical activity will get positive benefits from their activity. several studies indicate a difference in motor skills between boys and girls. to understand the development of motor skill proficiency and physical activity in boys and girls, a study was conducted to determine the role of gender on motor skill proficiency and physical activity in children aged 6-12 years. methods a cross-sectional observational study was conducted and a total of 162 children were included at a primary school in the grogol area, west jakarta. data collection was by questionnaire-based interviews, covering age, gender, and physical activity (watching tv, playing games, and outdoor play). assessment of motor skills was performed using the bruininks-oseretsky test–second edition (bot-2). data analysis was performed using spss for windows release 17.0 and level of significance was set at 0.05. results multiple linear regression results showed that in boys the strength subset was the most influential factor on tv watching activity, with the higher scores for strength indicating a lower tv watching activity (â=-0.125;p=0.021). age was the most influential factor on outdoor playing activity in girls, with older girls having lower outdoor playing activity (â=-0.375;p=0.016). conclusions this study revealed that gender difference impacts on motor skills and physical activity in children. higher motor proficiency increases outdoor playing activity only in boys. primary school pupils should be given opportunities for performing outdoor playing activities to improve their motor proficiency. keywords: motor proficiency, strength subset, outdoor play. boys, girls gender impacts on motor skill proficiency-physical activity relationship in children diana samara*, nuryani sidarta*, dian mediana*, and noviyanti* 193 univ med vol. 31 no.3 gender berdampak terhadap hubungan antara kemampuan motorik dan aktivitas fisik pada anak-anak latar belakang aktivitas fisik merupakan penyumbang terbesar untuk mencapai kesehatan yang baik. anak-anak yang melakukan aktivitas fisik setiap hari dengan baik akan mendapat manfaat yang positif. beberapa penelitian menunjukkan adanya perbedaan ketrampilan motorik antara anak laki-laki dan perempuan. untuk dapat memahami mengenai perkembangan kemampuan motorik dan aktivitas fisik pada anak laki-laki dan perempuan, maka perlu dilakukan penelitian yang bertujuan untuk menentukan peran gender terhadap hubungan antara kemampuan motorik dan aktivitas fisik pada anak-anak berusia 6-12 tahun. metode penelitian menggunakan studi observasional dengan desain potong lintang yang mengikutsertakan 162 murid sekolah dasar di daerah grogol jakarta barat. data dikumpulkan dengan cara wawancara menggunakan kuesioner yang meliputi usia, jenis kelamin, dan aktivitas fisik (berupa menonton tv, bermain game, dan bermain di luar rumah). pengukuran kekuatan motorik dilakukan dengan menggunakan metode bruininks-oseretsky test–second edition (bot-2). analisis data dengan menggunakan spss for windows versi 17.0 dan tingkat kemaknaan yang digunakan bersarnya 0,05. hasil analisis regresi linear ganda menunjukkan pada anak laki-laki strength subset merupakan faktor yang paling berperan terhadap kegiatan menonton televisi, semakin tinggi skor strength subset, semakin berkurang kegiatan menonton televisi (â=-0,125; p=0,021). usia merupakan faktor yang paling berperan terhadap kegiatan main di luar pada anak perempuan, semakin bertambah usianya maka kegiatan main di luar akan semakin berkurang (â=0,375; p=0,016). kesimpulan penelitian ini menunjukkan perbedaan jender berdampak terhadap hubungan antara kemampuan motorik dan aktivitas fisik pada anak-anak. pada anak laki-laki kemampuan motorik yang tinggi meningkatkan aktifitas bermain di luar. murid-murid sekolah dasar harus diberikan kesempatan bermain di luar untuk memperbaiki ketrampilan motoriknya. kata kunci: kemampuan motorik, strength subset, bermain di luar, anak laki, anak perempuan abstrak introduction physical activity is the greatest contributor to achievement of good health. according to the u.s.surgeon general, children with moderate to vigorous daily physical activity will get positive benefits from their activity.(1) there are many children who perform less than the recommended amount of physical activity and their physical activity will worsen as they grow older.(2) there are many factors that play a role in physical activity of children, such as perception about physical activity, enjoyment of physical activity, p a r e n t s , s i b l i n g s , a n d f r i e n d s . a n o t h e r determinant of physical activity in children is their motor competence or mastery of basic movements used in physical activity.(3) children with high motor competence perform physical activity with ease and prefer to do it in groups. in contrast, children with low motor competence prefer sedentary play, because they cannot perform the difficult movements. 194 motor competence is the ability to execute d i ff e r e n t m o t o r m o v e m e n t s , i n c l u d i n g coordination of fine and gross movements (static and dynamic balance). some children experience considerable difficulties coordinating and controlling their body movements and are often described as having developmental coordination disorder.(4,5) motor proficiency in childhood appears to be an important factor in developing a positive self-perception of competence in the physical domain and seems to be connected to increased physical activity and fitness in adolescence.(6) recent findings suggest that children with low motor competence demonstrate significantly poorer performance on important components of physical fitness, such as aerobic and anaerobic endurance and muscular strength, than their peers who are developing typically.(7,8) a s s e s s m e n t o f m o t o r s k i l l s m a y b e performed by one of the several standardized, comprehensive tests in existence. most of these tests include multiple balance tasks and some even offer a specific balance subscale score, e.g. the bruininks-oseretsky test second edition (bot-2) for 4 to 21years old,(9) the peabody developmental motor scales second edition (pdms-2) for 0 to 5 years old,(10) and the movement assessment battery for children second edition (m-abc-2) for 3 to 16 years old.(11) in the present study we used the bot-2 instrument for assessment the motor skills. several previous studies have shown a significant positive relationship between motor s k i l l s , v i s u a l c o o r d i n a t i o n , g r o s s m o t o r development, and physical activity in youth.(12,13) children who perform physical activities also tend to perform these as adults. thus physical activity in children is indirectly associated with their future health status.(14) several studies showed a difference in motor skills between boys and girls.(15,16) to understand the development of motor ability and physical activity in both boys and girls, it is necessary to conduct a study with the objective of determining the role of gender in motor skills and physical activity among children aged 6-12 years. methods study design this was a cross-sectional observational study conducted from january to march 2012 at a primary school in the grogol area, west jakarta. study subjects all first to sixth grade form primary school pupils aged between 6 and 12 years who agreed to be tested with permission of their parents, and who gave informed consent were included in this study. assessments data were collected by questionnaire-based interviews, covering age and gender. the bruininks-oseretsky test second edition (bot2)(9) was used to assess motor skills of the children. the bot-2 is a standardized, normreferenced measure of fine and gross motor skills for individuals from 4 through 21 years of age. the test consists of 8 subset to assess gross motor development, including running speed and agility, balance, bilateral coordination, and strength; gross and fine motor development, including upper limb coordination, fine motor precicion, fine motor integration, and manual dexterity. a total standard score, adjusted for child age, was used to interpret test performance. in the present study. physical activity was measured by a questionnaire covering the following items: watching tv, playing games, and playing outdoors. if the answer was yes, the frequency of physical activity was asked (every day, every 2 days, every 3 days, every 4 days, every 5 days) expressed as the frequency of physical activity in one week. data analysis the independent t-test was used to compare age, motor proficiency subsets and bot scores, physical activity between boys and girls. pearson product-moment correlations were computed to determine relationships between age, physical samara, sidarta, mediana, et al gender and motor skill proficiency 195 univ med vol. 31 no.3 activity, motor proficiency subsets, and bot score in all subjects, boys and girls. multiple linear regression analysis was then used to whether motor proficiency subsets and bot scores accounted for a significant amount of child activity after controlling for age and gender. data analysis was performed using spss for windows release 17.0 and level of significance was set at 0.05. ethical clearance this study received ethical approval from the research ethical committee, faculty of medicine, trisakti university. parents of all participants gave written informed consent. results t h e s t u d y i n v o l v e d 1 6 2 s u b j e c t s , comprising 69 (42.6%) boys and 93 (57.4%) girls. there was no significant difference in mean age of boys (8.4 ± 1.6 years) and girls (8.4 ± 1.7 years). mean scores for fine motor precision and motor strength were significantly different between boys and girls. mean fine motor precision score in girls of 18.6 ± 4.2 was significantly higher than the mean in boys of 16.7 ± 4.3. on the other hand, mean strength score in girls (11.5 ± 3.8) was significantly lower in comparison with that in boys (12.7 ± 3.6). the means of upper limb coordination scores, bilateral coordination scores and total bot scores did not differ between boys and girls (table 1). in all subjects there was a negative correlation between gender and frequency of playing games (r=-0.19; p=0.016), with girls performing games less frequently than boys. age and fine motor precision had a significant negative correlation with frequency of outdoor play, with r=-.017; p=0.034, and r=-0.16; p=0.036, respectively, as seen in table 2. there was no significant relationship between age and motor proficiency subsets (fine motor precision, upper limb coordination, bilateral coordination and strength) on the one hand and frequency of game playing. gender, upper limb coordination, bilateral coordination and strength had no significant relationship with frequency of outdoor play. in boys the bot score had a linear correlation with outdoor play, with higher bot score indicating higher frequency of outdoor play (r=0.28; p=0.020). strength subsets had a significant negative correlation with watching television (r=-0.37; p=0.002). in girls, age had a significant negative c o r r e l a t i o n w i t h o u t d o o r p l a y ( r = 0 . 2 6 ; p=0.012), while the upper limb coordination subset had a linear correlation with outdoor play, table 1. distribution of children’s characteristics by gender § bot: bruininks-oseretsky test 196 (r=0.22; p=0.035). the strength subset had a linear correlation with watching television (r=0.26; p=0.011). there was a very clear difference in the correlation of the strength subset with television watching activity. boys with high strength score showed a lower frequency of watching tv, whereas girls showed a higher frequency of watching tv (table 2). multiple linear regression showed that in boys the strength subset was the most influential factor on tv watching activity, with higher strength score indicating lower frequency of watching television (â=-0.125; p=0.021). age was the most influential factor on outdoor play in girls, with higher age indicating lower frequency of outdoor play (â=-0.375; p=0.016) (table 3). discussion the results of this study indicate that there was no difference in bot scores between boys and girls. our study results were similar to those of previous studies indicating no difference in table 2. correlation between age, gender, and motor proficiency subsets with playing games, outdoor play and watching tv § bot : bruininks-oseretsky test samara, sidarta, mediana, et al gender and motor skill proficiency 197 univ med vol. 31 no.3 table 3. regression model predicting outdoor play and tv watching § bot: bruininks-oseretsky test of motor proficiency; β = regression coefficient; beta = standardized coefficient bot scores between boys and girls.(3) taken individually, fine motor precision scores and strength scores were significantly different between boys and girls. boys had significantly higher strength scores in comparison with girls. in contrast, girls had higher scores on fine motor precision skills in comparison with boys. previous studies have demonstrated gender differences in motor skills, with boys performing better than girls in motor tasks of strength.(17) these results are in agreement with the study by krombholz, who showed significant differences in motor proficiency between boys and girls, with boys scoring better on strength and girls on fine motor precision.(18) gender differences in motor proficiency can be explained by environmental influences, biological factors, or their interaction. before puberty, the physical characteristics of boys and girls are similar, and environmental influences are more likely to explain gender differences in motor proficiency. the type of sports and games that boys and girls are drawn to participate in give them more opportunity to practice and refine their motor skills and may contribute to gender differences. despite the important role of the environment in motor skill development in childhood, some biological factors may also be present for certain skills, such as throwing. for example, compared with girls, boys have more mid arm muscle tissue and a greater shoulder/ hip ratio.(19) in our study we found that girls scored higher than boys on fine motor precision (filling in shapes-circle). the study conducted by wrotniak yielded similar results, viz. that girls sorted a greater number of shape cards.(3) in boys there was a significant positive correlation of bot scores with outdoor play. children who play out of doors have better gross motor development, in comparison with children with a higher weekly television viewing frequency.(20) outdoor play also offers children opportunities to explore their community; enjoy sensory experiences with dirt, water, and sand; find or create their own places for play; collect objects and develop hobbies; and increase their liking for physical activity. such vigorous movements and play activities can not only enhance muscle growth, but also support the growth of the child’s heart and lungs as well as all other vital organs essential for normal physical development.(21) activities such as playing outside in rain, or muddy areas give children a sense of adventure. self-esteem is also increased when parents and teachers act as a positive audience for the child’s imaginative outdoor play activities. however, our study revealed that boys who watched television more frequently had lower strength subset scores. today children spend more time in watching television than in outdoor play. a study in the usa involving 492 fifthand sixthgraders at six elementary schools found that most of them performed indoor activities the results showed that 96% of these children watched television regularly; 81% played computer games; 74% participated in reading; 61% played 198 video games; and 60% played board games indoors.(21) many mothers are concerned with the personal safety of their children and give them opportunities for indoor play, such as watching television and playing games, thus constituting factors that prevent their children from playing outdoors. some children may actually prefer to sit and watch events on television, instead of playing outdoors and creatively thinking of ways to entertain themselves. outdoor play positively impacts on development of children’s physical and motor skills, and are a positive stimulus for cognitive skills. the main limitation of our study is that physical activity was assessed on the basis of self-report and did not use more objective means of assessment, such as accelerometers. we were unable to assess the bot-2 manual dexterity subset because of techical problems in the field. another limitation of this study was that we used a cross-sectional design, and thus were unable to determine possible causal relationships between motor proficiency and physical activity. conclusions this study revealed that gender difference impacts on motor skills and physical activity in children. higher motor proficiency increases outdoor playing activity only in boys. girls have higher fine precision scores than boys. primary school pupils should be given opportunities for performing outdoor playing activities to improve their motor proficiency. therefore further studies of prospective design are necessary to determine causality between motor proficiency and physical activity in children. acknowledgements we thank the primary school pupils and their parents who agreed to participate in this study. thanks are also due to the dean and vicedeans of the faculty of medicine, trisakti university for providing the funding of this study. references 1. u.s. department of health and human services. physical activity guidelines for americans. washington dc;2008. available at: http:// www.health.gov/paguidelines/guidelines/ summary.aspx. accessed april 12, 2012. 2. pate r, freedson p, sallis j, taylor w, sirad j, trost sg, et al. compliance with physical activity guidelines: prevalence in a population of children and youth. ann epidemiol 2002;12:303–8. 3. wrotniak bh, epstein lh, dorn jm, jones ke, kondilis va the relationship between motor proficiency and physical activity in children. pediatrics 2006;118:e1758-65. doi: 10.1542/ peds.2006-0742. 4. polatajko h, cantin n. developmental coordination disorder (dyspraxia): an overview of the state of the art. semin pediatr neurol 2006; 12:250–8. 5. summers j, larkin d, dewey d. activities of daily living in children with developmental coordination disorder: dressing, personal hygiene, and eating skill. hum mov sci 2008; 27:215–29. 6. barnett lm, morgan pj, van beurden e, beard jr. perceived sports competence mediates the relationship between childhood motor skill proficiency and adolescent physical activity and fitness: a longitudinal assessment. int j behav nutr phys act 2008;5:40. doi: 10.1186/14795868-5-40. 7. scott n, alof v, hultsch d, meemann d. physical fitness in children with developmental coordination disorder. res q exerc sport 2007; 78:438–50. 8. haga m. physical fitness in children with movement difficulties. physiotherapy 2008;94: 253–9. 9. bruininks rh, bruininks bd. bruininksoseretsky test of motor proficiency manual.2nd ed. minneapolis, mn: ncs pearson inc; 2005. 10. folio mr, fewell rr. peabody developmental motor scales-2. austin, tx:pro-ed; 2000. 11. henderson se, sugden da, barnett al. movement assessment battery for children: examiner’s manual. 2nd ed. london, united kingdom: pearson assessment inc; 2007. 12. graf c, koch b, kretschmann-kandel e, falkowski g, christ h, coburger s, et al. correlation between bmi, leisure habits and motor abilities in childhood (chilt-project). int j obes relat metab disord 2004;28:22–6. 13. taylor w, sallis j, dowda m, freedson p, eason k, pate r. activity patterns and correlates among samara, sidarta, mediana, et al gender and motor skill proficiency 199 univ med vol. 31 no.3 youth: differences by weight status. pediatr exerc sci 2002;14:418–31. 14. kristensen pl, moller nc, korsholm l, wedderkopp n, andersen lb, froberg k. tracking of objectively measured physical activity from childhood to adolescence: the european youth heart study. scand j med sci sports 2008;18:171–8. 15. barnett lm, van beurden e, morgan pj, brooks lo, beard jr. gender differences in motor skill proficiency from childhood to adolescence: a longitudinal study. res q exerc sport 2010;81: 162-70. 16. roberton et, langendorfer s. does the throwing “gender gap”occur in germany? res q exerc sport 2005;76:488-93. 17. okely a, booth m. mastery of fundamental movement skills among children in new south wales: prevalence and sociodemographic distribution. j sci med sport 2004;7:358–72. 18. krombholz h. physical performance in relation to age, sex, birth order, social class, and sports activities of preschool children. percept mot skills 2006;102:477-84. 19. harrell j, pearce p, markland e, wilson k, bradley c, mcmurray r. assessing physical activity in adolescents: common activities of children in 6th-8th grades. j am acad nurse pract 2003;15:170–8. 20. graft c, koch b, kretschmann-kandel e, falkowski g, christ h, coburger s, et al. correlation between bmi, leisure habits and motor abilities in childhood. int j obes 2004; 28:22-6. 21. clements r. an investigation of the status of outdoor play. contemp issues early child 2004; 5:68-80. c:\users\jurnal fkusakti\docume 73 *department of anatomy, faculty of medicine, universitas islam indonesia, yogyakarta **faculty of medicine, universitas islam indonesia, yogyakarta correspondence: dr. kuswati, msc department of anatomy, faculty of medicine, universitas islam indonesia jl kaliurang km 14,5 yogyakarta phone (0274) 898444 email: kuswati@uii.ac.id date of first submission, december 17, 2 01 8 date of final revised submission, june 17, 2019 date of acceptance, june 18, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina propolis inhibited bax expression and increased neuronal count of hippocampal area ca1 in rats receiving sodium nitrite kuswati*, ety sari handayani*, zainuri sabta nugraha*, fishella aprista rahmanti**, zulfikar loka wicaksana**, and muhammad zhafirrahman** background sodium nitrite induces hypoxia and oxidative stress in the hippocampus, decreasing the number of neurons in the hippocampus and cognitive function. propolis contains chrysin that has antioxidant effects that are expected to inhibit neuronal damage in the hippocampus. this study aims to determine the effects of propolis on the expression bcl-2-associated x protein (bax) and the number of neurons in the rat hippocampus receiving sodium nitrite. methods this study of laboratory experimental design was conducted on 18 male wistar strain rats (rattus norvegicus), they were randomized into 3 groups: one control group (k) received sodium nitrite and two intervention groups (p1 and p2) received sodium nitrite and propolis at doses of 100 and 200 mg/kgbw. treatment with sodium nitrite and propolis were given for 60 days, followed by termination. the number of neurons and bax expression in the hippocampal ca1 area were measured. one-way anova was used to analyze the data. results there were significant differences in bax expression between group k and groups p1 and p2 (p<0.001). the lowest number of neurons in the hippocampal ca1 area was in the k group. there were significant differences in the number of neurons between control (k) group and groups p1 and p2 (p<0.001). conclusion propolis inhibited the expression of bax and decreased the number of neurons in the hippocampal ca1 area of rats receiving sodium nitrite. this study provides information about the benefits of propolis as an antioxidant in the brain. keywords: propolis, bax, hippocampus, sodium nitrite original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.73-80 may-august, 2019 vol.38no.2 cite this article as: kuswati, handayani es, nugraha zs, et al. propolis inhibited bax expression and increased neuronal count of hippocampal area ca1 in rats receiving sodium nitrite. univ med 2019;38:73-80. doi: 10.18051/ univmed.2019.v38.73-80 74 kuswati, handayani, nugraha, et al propolis inhibited bax and sodium nitrite introduction exposure to sodium nitrite results in structural and functional changes in various organs of the body. in the brain, the exposure results in congestion of the blood vessels and bleeding in the meninges, ventricles, brainstem and cerebellum. exposure to sodium nitrite also results in perineuronal edema, vacuolation, neuronal degeneration, neurophagy and necrosis of the purkinje cells in the cerebellum.(1) the study conducted by jebasingh et al.(2) showed that intraperitoneal administration of sodium nitrite at a dose of 60 mg/kgbw in male wistar rats for 30 days decreased the capacity for learning and memory and increased anxiety. in addition, administration of sodium nitrite resulted in changes in the morphology of the cortex, hippocampus, thalamus, and cerebellum. these changes are among others pyknotic nuclei and fragmented and vacuolated neurons resulting from apoptosis and neuronal degeneration. sodium nitrite is a compound that converts hemoglobin into methemoglobin, which causes h yp ox i a , t h us l e a di n g t o i sc he mi a a nd inflammation of the brain. (3) according to halim and ibrahim(4) the hypoxia increases reactive o xyge n s pec ie s ( ro s) , r e s ul ti n g i n t h e degradation of lipid membranes and enzymes, and in dna damage. oxidative stress disturbs metabolic activity, inhibits expression of the f a c t or s of ne u r o n a l r e ge n e ra t i o n a nd proliferation in the hippocampus and decreases the number of receptors in the hippocampus.(5) the hippocampus is one of the brain areas that are susceptible to oxidative stress. exposure to stress in the hippocampus results in a decreased number of neurons in the hippocampus and amygdala and decreased memory and cognitive function.(6) to protect the neurons in the hippocampus from hypoxia and oxidative stress as a result of exposure to sodium nitrite, a compound is needed, such as propolis, that possesses neuroprotective effects and prevents damage and death to the neurons. propolis contains chrysin and has a nt io xid a nt a nd n eur opr o t ec t i ve e ff e c ts. administration of propolis at a dose of 200 mg/ kgbw in rats exposed to stress and showing increases in h 2 o 2 has the effect of decreasing the h 2 o 2 concentration.(7) administration of propolis to rats with cerebral ischemia reduces apoptosis with increased expression of bcl-2 and decreased expression of bax and caspase-3.(8) propolis-containing polyphenols may prevent the death of dopaminergic neurons in the substantia nigra as a result of exposure to 6-ohda (6hydroxydopamine), may increase the expression of brain-derived neurotrophic factor (bdnf), increase the antioxidant activity of enzymes such as superoxide dismutase (sod), glutathione peroxidase (gpx), and glutathione reductase (gsr), and decrease the concentration of malondialdehyde that may cause oxidative stress in the neurons.(9) in neuronal cell culture, administration of propolis at a dose of 25 μg/ml may prevent oxidative stress, inhibit release of ldh and increase the viability of the neurons as a result of exposure to h 2 o 2 . (10) in rats with neurotoxicity induced by lead (pb) for four weeks, administration of propolis may inhibit the neurotoxic effect of pb. this is shown in the n or ma l a c ti vi ty of a c e t ylc ho l in e s t e r a s e , decreased mda concentrations in the brain, and i nc r e a s e d a c ti vi t y of na dh , su c c i n a t e dehydrogenase (sdh) and cytochrome c oxidase in mitochondria.(11) a previous study had evaluated the effect of propolis on the neuronal count in the hippocampus.(12) caffeic acid phenethyl ester (cape) is a phenolic chemical compound present in numerous plants and is extracted from honeybee hive propolis.(13) the study of wang et al.(14) states that cape may inhibit the apoptosis of neurons as a result of sevoflurane exposure. this is shown by the decreased expression of the pro-apoptotic proteins bax and bcl 2 associated agonist of cell death (bad) and the increased expression of the antiapoptotic proteins b cell ccl/lymphoma 2 (bcl 2) and bcl 2 like 1 (bcl xl). cape inhibits neuronal apoptosis in the hippocampus that is marked by decreased numbers of terminal 75 deoxynucleotidyl transferase dutp nick end labeling (tunel) positive cells and decreased expression of activated caspase-3.(14) in contrast with all of the above-mentioned studies, the present study aimed to evaluate the neuroprotective effect of propolis in inhibiting bax expression and the decrease in hippocampal neuronal count of rats exposed to sodium nitrite. methods research design this was an experimental laboratory study of posttest only control group design, conducted a t t h e re se a r c h l a b or a t or y, fa c ul t y of medicine, universitas islam indonesia (fk uii) from july to december 2017. research subjects the subjects of this study were four-monthold male wistar strain rats (rattus norvegicus) weighing 200-300 grams. the required number of subjects were calculated from the formula of charan and biswas (15) as follows: e = total number of animals total number of groups e= 10 to 20 total number of animals = 20 total number of groups total number of animals = 20 3 from above calculation the required number of subjects was 17 rats. to obtain 6 subjects per group, the total number of subjects u sed was 1 8 r a ts . al l e xp e ri me nt s we r e pe rfo rmed acc ording t o t he ins tit ut ion al guidelines of the experimental animal center and in accordance with the principles outlined in the guide for the care and use of laboratory animals. preparation of propolis extract the propolis used in this study was a product available in the open market, in which 1 ml of the material contains 600 mg propolis. from this propolis preparation 0.1 ml was taken and 0.9 ml distilled water was added, so as to obtain a concentration of 60 mg/ml. the diluted propolis solution was then administered to the rats in the appropriate doses, i.e. 100 mg/kgbw and 200 mg/kgbw, respectively, in accordance with the study by lee et al.(16) expo s ur e t o s odi um ni t r i t e an d administration of propolis in the sodium nitrite only group (group k) sodium nitrite at a dose of 50 mg/kgbw/day was administered orally for 60 days by means of a gastric tube. in the group receiving sodium nitrite and propolis (groups p1 and p2), sodium nitrite at a dose of 50 mg/kgbw/day and propolis at d os e s of 1 00 a nd 20 0 mg/kgbw / d a y, respectively, were administered by the oral route for 60 days by means of a gastric tube. sample preparation on day 61, when the rats were terminated and underwent transcardial perfusion, the rats were anesthesized by intramuscular injection of ketamine (100 mg/kg). the transcardial perfusion was done with a solution of nacl at a volume of 100-200 ml until the emerging perfusion fluid was clear. after perfusion, the brain was carefully excised, the part containing the hippocampus was taken and fixed in phosphateb uf f e r e d fo r ma l i n f o r 2 4 ho ur s. it w a s subsequently made into paraffin blocks and sectioned at 3 μm thickness. from each rat brain one slice was taken for immunohistochemical staining and one slice for hematoxylin-eosin staining. immunohistochemical staining was initiated by deparaffinizing with xylol and alcohol in decreasing concentrations. the tissue was then incubated with 3% h 2 o 2 in 10% methanol for 20 minutes and washed three times with distilled water followed with three times phosphatebuffered solution (pbs). subsequently antigen retrieval was carried out in citrate buffer at ph 6 in a microwave oven. for about 10 minutes, the slices were heated to a high temperature (1000c), followed by med-low temperature for 20 minutes. after cooling, the slices were again univ med vol. 38 no.2 76 kuswati, handayani, nugraha, et al propolis inhibited bax and sodium nitrite washed three times with pbs. subsequently the slices were blocked with background sniper for 10 minutes. without washing again, the tissue was dripped with bax primary antibody (ab) and incubated overnight at 40c, then washed three times in pbs and incubated with trekkie universal link for 10 minutes. after washing three times with pbs, the tissue was incubated wi t h ho r s er a d i s h p e r o x i da s e -c o n j uga t e d streptavidin (sa-hrp complex) for 10 minutes, then washed again three times with pbs. baxlabeled pyramidal cells were visualized with 3,3’diaminobenzidine (dab) (1 : 100) for 5 minutes. the tissue was then washed five times with distilled water, followed by counterstaining with mayer’s hematoxylin for one minute. after washing in running water for 2 minutes, the slices were dehydrated in ethanol 70%, 80%, 90%, 95% and 100%, for one minute, respectively, cleared in xylol and covered with canada balsam and coverslip. hematoxylin-eosin staining was performed according to the protocol of the research laboratory of the faculty of medicine, uii. briefly, the protocol was as follows: the slides with the tissue sections were placed in the staining jar, deparaffinized, then washed twice with distilled water. then the slides were placed in hematoxylin solution for 10 minutes, washed three times with distilled water, and checked under the microscope. if the hematoxylin staining was adequate, the slides we re washed in running water. subsequently the slides were placed in eosin and washed twice with distilled water. the slides were then placed in 70%, 80%, 90%, 95% and 100% alcohol, and dried with tissue paper. the dried slides were placed in xylol i, ii and iii for 5 minutes, respectively, followed by mounting and were then observed under the microscope. observation of bax-expressing cells observation of bax-expressing cells was performed in the hippocampal area ca1. the slide preparations were observed under the light microscope at 400 x magnification. the whole hippocampal area ca1 was observed and the bax expression interpreted using the allred score, which evaluates the proportions and staining intensities of cells with positive bax expression. the allred score is semi-quantitative, using the proportion of positive cells (scores 0-5) and staining intensity (scores 0-3), which are summed to yield the total score (0-8).(17) as per the allred score for bax-expression, the proportion score (ps) and the % positive tumor cells are respectively, (-) (0%), (+) (<1%), (++) (1–10%), (+++) (11–33%), (++++) (34– 66%), and +++++ (67–100%). the intensity of staining (is) for the nuclear positivity of the cells graded as 0, 1, 2, and 3 was as negative, weak, mild, intermediate, and strong, respectively. allred score is obtained by adding up the proportion score and intensity score. hippocampal neuronal count the neuronal count was performed in area ca1 of the hippocampus. the slides were observed under the light microscope at 400 x magnification. the cells were counted in the whole hippocampal area ca1. then the mean number of neurons per field of view for each slide was calculated with the formula: mean number of cells = number of cells in area under observation/number of fields of view. data analysis to compare the mean allred score for bax expression and neuronal count between groups in the hippocampus, one-way anova was used, followed by tukey’s multiple comparison post hoc test to determine the differing groups. a p value of less than 0.5 shows a significant difference. ethical clearance this study obtained ethical clearance from the committee on research ethics, faculty of medicine, universitas islam indonesia, under no. 03/ka.kom.et/70/ke/vii/2017. a b c 77 results the results of the evaluation of bax expression in area ca1 of the hippocampus using the allred score are presented in table 1. group k receiving treatment with sodium nitrite had the highest mean allred score. analysis using one-way anova showed significant r e s u l t s (p= 0. 00 0 ) . po st -h oc a n a l ys i s demonstrated significant differences between group k and groups p1 and p2. there was no significant difference between groups p1 and p2 (table 2). the results of the pyramidal neuronal count in hippocampal area ca1 of the control group (k), treatment group 1 (p1), and treatment group 2 (p2) are presented in table 1. analysis with one-way anova showed significant results (p= 0.000). the post-hoc analysis also showed significant differences between group k and groups p1 and p2. however, there were no significant differences between group p1 and group p2 (table 2). discussion in this study, exposure to sodium nitrite increased bax expression in area ca1 of the hippocampus, which agrees with the study of zaidi (18) stating that exposure to sodium nitrite causes degeneration in the hippocampus in areas ca1ca4 and the gyrus dentatus, showing the presence of pyknotic nuclei, darkly stained cells, cells with condensed nuclei, and vacuolation. the neuronal count in the hippocampus also underwent a decrease as a result of the exposure to sodium nitrite. in another study zaidi (19) found that sodium nitrite causes damage to the purkinje cells in the cerebellum. morphological changes in the purkinje cells comprised swollen, autolyzed dark and shrunken nuclei. this shows that purkinje cells of the cerebellum are susceptible to the hypoxia that results from the exposure to sodium nitrite so that the cells degenerate. amin et al. (1) report that exposure to sodium nitrite decreases the number of red blood cells and the hemoglobin concentration. hypoxia may cause leakage of the mitochondrial membrane so that cytochrome-c will enter the cytosol and activate pro-apoptotic proteins followed by apoptosis of the cell. (20) in the present study the pyramidal neuronal count in area ca1 of the hippocampus underwent a greater reduction in group k as compared with groups p1 and p2. this result is similar to that of the study by amin et al.(1) who found congestion of blood vessels and bleeding in meninges, cerebrum and cerebellum. in addition, apparently the purkinje cells of the cerebellum undergo degeneration and necrosis. exposure to sodium nitrite in the brain results in neurotoxicity that is marked by an increase in serum biochemical parameters, such as nitrates, thiobarbituric acidreactive substances (tbars) and a decrease in glutathione and dopamine concentrations.(21) the study conducted by jebasingh et al.(2) shows that intraperitoneal administration of sodium nitrite at 60 mg/kgbw in male wistar rats for 30 days table 1. mean bax expression and pyramidal neuronal count in the hippocampus k: sodium nitrite; p1: sodium nitrite + propolis at 100 mg/kgbw; p2: sodium nitrite + propolis at 200 mg/kgbw mean difference p value bax expression k p1 p2 p1 p2 pyramidal neuronal count k p1 p2 p1 p2 3.20 3.21 0.01 20 23 3 0.001 0.000 0.989 0.000 0.000 0.309 table 2. results of tukey’s multiple comparison test k: sodium nitrite; p1: sodium nitrite + propolis at 100 mg/ kgbw; p2: sodium nitrite + propolis at 200 mg/kgbw univ med vol. 38 no.2 intervention p value k p1 p2 bax expression pyramidal neuronal count 7.27 ± 0.43 26.0 ± 2.53 4.07 ± 0.65 46.0 ± 4.27 4.06 ± 0.72 49.0 ± 3.05 0.000 0.000 78 kuswati, handayani, nugraha, et al propolis inhibited bax and sodium nitrite of neurons in the cerebrum and cerebellum. in rats receiving ethanol and cape, expression of bax and caspase-3 was lower than in rats exposed to ethanol only. expression of bcl-2 was higher in rats exposed to ethanol and then given cape. propolis contains flavonoids with strong antioxidant and anti-inflammatory effects. administration of propolis at doses of 100 and 200 mg/kgbw decreases apoptosis in rat brain tissue examined by tunel immunohistochemistry, decreases expression of tnf-α in brain tissues of a rat model of traumatic brain injury (tbi) and decreases the area of necrosis.(25) annis et al.(26) report that administration of propolis at doses of 50, 100 and 200 mg/kgbw for 7 days may decrease apoptosis and increase expression of bdnf in the brain of a rattus norvegicus traumatic model. decrease in apoptosis and increase in bdnf expression is proportional to dose increases. the most effective dose of propolis is 200 mg/kgbw/day. the dose of 50 mg/kgbw is less effective in increasing bdnf expression. the study of khalil et al. (27) shows that propolis may reduce the toxic effect of methotrexate in the liver and brain. propolis increases the glutathione (gsh) concentration, increases the antioxidant activity of superoxide dismutase (sod), glutathione peroxidase (gpx), and glutathione reductase (gsr) in the liver and brain tissues and decreases the malondialdehyde (mda) concentration. propolis is capable of modulating enzyme antioxidant activity and reduce the oxidative dama ge ca used by methotrexate. administration of propolis in a rat model of spinal cord injury was able to decrease the concentrations of caspase 3 and cathepsin b.(28) the results of the present study differ from those of the study by hemieda et al. (29) who tested the effect of the administration of propolis in a rat model of diabetes. diabetic rats were given propolis orally in two separate doses, 50 or 100 mg/kg bw/day for 6 weeks. the obtained results show that administration of propolis induced apoptosis and cell cycle arrest in the pancreas of diabetic rats, that are marked by decreases learning ability and memory and increases the anxiety level in tests with the elevated plus maze. in addition, induction of hypoxia with subcutaneous administration of sodium nitrite at a dose of 50 mg/kgbw d e c r e a se s l oc omot o r a c t i vi t y a nd mo t or coordination in male wistar rats.(22) according to jeb asingh et al., (2 ) morphological examination of the cortex, hippocampus, thalamus, and cerebellum shows changes resulting from exposure to sodium nitrite. the changes comprise pyknotic and rounded nuclei, with fragmented dead neurons and vacuolation. the afore-mentioned brain areas play an important role in the regulation and coordination of movement and behavior. the morphological changes are caused by apoptosis and neuronal degeneration. mohammed (23) reports that administration of sodium nitrite at a dose of 30 mg/kgbw causes histopathological damage in various brain areas, including area ca1 of the hippocampus that is marked by loss of pyramidal cells and incr ease in vascular endothelial growth factor (vegf). the results of the present study show that administration of propolis at doses of 100 and 200 mg/kgbw may inhibit the decrease in pyramidal neuronal count in area ca1 of the hippocampus. this condition is consistent with previous studies showing that propolis inhibits the decrease in neuronal count in area ca1 of the hippocampus and the cortex prefrontalis in rats with induced stress. (12) propolis may inhibit oxidative stress with increased activity of antioxidant enzymes in rats that were induced with 6-hydroxydopamin to trigger parkinson’s disease. (9) propolis contains caffeic acid phenyl ester (cape) as antioxidant, that may inhibit neurotoxicity and neuronal degeneration in the cerebrum and cerebellum as a result of exposure to ethanol.(24) in this study, administration of propolis in groups p1 and p2 decreased bax expression in area ca1 of the hippocampus. the results of this study are in agreement with the study by basarslan et a l. (2 4) who repo rte d that administration of cape may inhibit the apoptosis 79 increased p53 and caspase-3 in the g0/g1 phases; and decreased bcl-2, as compared with the untreated diabetic rats. in spite of this, the administration of propolis markedly reduced oxidative stress, as seen in the decreased concentrations of malondialdehyde (mda) and carbonyl proteins in the liver and pancreas. administration of propolis increased the capacity of the antioxidant defense system by increasing the content of glutathione (gsh), superoxide dismutase (sod), catalase (cat) and total antioxidants (tac) in the liver and pancreas. administration of propolis significantly improved inflammation as shown in the decrease in serum markers of inflammation such as c-reactive protein (crp), interleukin 10 (il-10) and transforming growth factor β (tgf-β), in comparison with untreated diabetic rats. one of the clinical implications of the results of this study is the use of propolis to inhibit the neurodegeneration resulting from exposure to sodium nitrite. one of the limitations of this study is that only one piece of the brain was taken as the sample. further studies should take a larger number of samples using stereology. this study only determined the expression of bax as one of proapoptotic proteins. further studies may be pe rformed by deter mining a ll markers of apoptosis, such as caspases (caspase 3, 8 and 9), bad, bcl 2 and bcl-xl and count the number of cells undergoing apoptosis with the tunel assay. conclusion administration of propolis is capable of inhibiting bax expression and the decrease in neuronal count in area ca1 of the hippocampus in rats receiving sodium nitrite. this study provides information on the benefits of propolis as a brain antioxidant. conflict of interest there was no conflict of interest in this study. acknowledgment the authors thank the research and community service unit, faculty of medicine, universitas islam indonesia, for the funding of the study through the lecturer grant schema (skema hibah dosen).. contributors k designed the study, wrote the protocol and the first draft of the manuscript. zsn revised the manuscript. esh revised the protocol and manuscript. far, zlw, mz carried out the study. all authors have read and approved the final manuscript. references 1. amin ra, elsabagh ra, amin a. protective effects of ascorbic acid and garlic oil against toxic effects induced by sodium nitrite as meat additive in male rats. global veterinaria 2016;16:508-24. doi: 10.5829/idosi.gv.2016.16.06.10393. 2. jebasingh d, jackson dd, venkataraman s, et al. the protective effects of cyperus rotundus on behavior and cognitive function in a rat model of hypoxia injury. pharm biol 2014;52:1558–69. doi: 10.3109/13880209.2014.908395. 3. mcginnis wr, audhya t, edelson sm. proposed toxic and hypoxic impairment of a brainstem locus in autism. int j environ res public health 2013;10:6955-7000. doi: 10.3390/ijerph10126955. 4. halim hdp, ibrahim n. efek neuroprotektif ekstrak akar acalypha indica 500 mg/kgbb terhadap perubahan inti sel saraf hipokampus pascahipoksia serebri. ejki 2013;1:113-7. https:// doi/org/10.23886/ejki.1.2057.113-117. 5. sunarno, manalu w, nastiti, et al. pengoptimalan kinerja motorik pada penuaan fisiologis dan penuaan akibat stres oksidatif dengan alaninglutamin dipeptida dan hubungannya dengan perbaikan fungsi hippocampus. j kedokteran hewan 2012;6:56-9. doi: https://doi.org/10.21157/ j.ked.hewan.v6i1.354. 6. fitranto a, soejono sk, maurits ls, et al. jumlah sel piramidal ca3 hippocampus tikus putih jantan pada berbagai model stres kerja kronik. mkb 2014;46:197-202. doi: 10.15395/mkb.v46n4.337. 7. seven pt., yilmaz s, seven i, et al. the effect of propolis in animals exposed oxidative stress. in: oxydative stress environmental induction and univ med vol. 38 no.2 80 kuswati, handayani, nugraha, et al propolis inhibited bax and sodium nitrite dietary antioxidants. lushchak v, editor. rijeka, croatia: intech europe;2012.pp.267-88. doi: 10.5772/34850. 8. fei yq, dong bd. effect of propolis flavonoids on the apoptosis of neuron and expression of caspase-3, bcl-2 and bax following focal cerebral ischemia reperfusion in rats. chinese j clin 2010;4:1023-27. 9. safari m, sameni hr, badban l, et al. protective effect of water extracy of propolis on dopaminergic neurons, brain derived neurotrophic factor and stress oxidative factors in the rat model of parkinson’s disease. int j pharmacol 2015;11:300-08. doi: 10.3923/ijp.2015. 300.308. 10. jeong ch, jeong hr, kim do, et al. phenolics of propolis and in vitro protective effects against oxidative stress induced cytotoxicity. j agric life sci 2012; 46:87-95. 11. el-masry ta, emara am, shitany na. possible protective effect of propolis against lead induced neurotoxicity in animal model. j evol biol res 2011;3:4-11. 12. kuswati, handayani es, nugraha zs. propolis increases neuronal count in hippocampal area ca1 and prefrontal cortex in stressed rats. univ med 2017;36:214-20. doi: 10.18051/univmed.2017. v36.214-220. 13. demestre m, messerli sm, celli n, et al. cape (caffeic acid phenethyl ester)-based propolis extract (bio 30) suppresses the growth of human neurofibromatosis (nf) tumor xenografts in mice. phytother res 2009;23:226–30. doi: 10.1002/ ptr.2594. 14. wang ly, tang zj, han yz. neuroprotective effects of caffeic acid phenethyl ester against sevoflurane induced neuronal degeneration in the hippocampus of neonatal rats involve mapk and pi3k/akt signaling pathways. mol med rep 2016;14:3403-12. doi: 10.3892/mmr.2016.5586. 15. charan j, biswas t. how to calculate sample size for different study designs in medical research? indian j psychol med 2013;35:121–6. doi: 10.4103/ 0253-7176.116232. 16. lee ms, kim yh, park ws. novel antidepressantlike activity of propolis extract mediated by enhanced glucocorticoid receptor function in the hippocampus. evidence-based complementary alternative medicine 2013, article id 646039, 10 pages. doi: http://dx.doi.org/10.1155/2013/217853. 17. allred dc, harvey jm, berardo m, et al. prognostic and predictive factors in breast cancer by immunohistochemical analysis. mod pathol 1998;11:155–68. 18. zaidi zf. sodium nitrite-induced hypoxic injury in rat hippocampus. pak j med sci 2010;26:532-7. 19. zaidi zf. effects of sodium nitrite-induced hypoxia on cerebellar purkinje cells in adult rats: pak j med sci 2010;26:261-6. 20. hidayat a, wiradisastra k, hernowo bs, et al. ekspresi bcl-2 dan caspase-3 pascapaparan hipoksia hipobarik intermiten. mkb 2011;43:16670. doi: http://dx.doi.org/10.15395/mkb.v43n4.64 21. rao vp, kumar ack, kumar jv, et al. evaluation of neuroprotective activity of aegle marmelos (fruit) against sodium nitrite induced neurotoxicity in albino wistar rats. int j innovative pharmac res 2014;5:374-7. 22. dimitrova d, getova d. effect of cholinesterase inhibitors tacrine and metrifonate on exploratory activity in rats with induced hypoxia. sci technol 2015;5:400-4. 23. mohammed ff. comparative neuropathological study of sodium nitrite and nisin exposed rats with special reference to expression of glial fibrillar acidic protein and vascular endothelial growth factor. egypt j comp path clinic path 2013;26:6171. 24. basarslan sk, osun a, senol s, et al. protective effects of intralipid and caffeic acid phenyl esther (cape) on neurotoxicity induced by ethanol in rats. turk neurosurg 2017;27:66-73. doi: 10.5137/ 1019-5149.jtn.14463-15.2. 25. chandra a, ali m, purnomo h. efek ekstrak propolis terhadap ekspresi tnf-α, apoptosis dan nekrosis jaringan otak tikus model traumatic brain injury (tbi). j kedokter brawijaya 2016;29:11724. 26. annis f, purnomo h, balafif f. pengaruh ekstrak propolis terhadap apoptosis melalui ekspresi brain-derived neurotrophic factor (bdnf) pada sel otak tikus model cedera otak traumatic. j kedokter brawijaya 2017;29:209-15.doi: http:// dx.doi.org/10.21776/ub.jkb.2017.029.03.5. 27. khalil fa, el-kirsh aaa, kamel ea, et al. beneficial effect of propolis extract (bee glue) against methotrexate induced stress in liver and brain of albino rats. indian j med res pharmac sci 2016;3:24-34. doi: 10.5281/zenodo.54549. 28. ozkara e, durmaz r, kanbak g, et al. the effect of propolis following experimental spinal cord injury. wscj 2014;5:6-11. 29. hemieda fae, el-kholy wm, al-habibi esm, et al. influence of propolis on oxidative stress, inflammation and apoptosis in streptozotocininduced diabetic rats. int j adv res 2015;3:83145. oktavianus 197 *department of pharmacology, faculty of medicine, trisakti university **department of clinical pathology, faculty of medicine, trisakti university correspondence dr.yenny,spfk department of pharmacology, faculty of medicine, trisakti university jl. kyai tapa, grogol, (kampus b) jakarta 11440 phone: 6221-5672731 ext. 2801 email: yenfarmako@gmail.com univ med 2013;32:197-207 abstract universa medicina september-december, 2013september-december, 2013september-december, 2013september-december, 2013september-december, 2013 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 vol.32 no.3 background cardiovascular disease is the main cause of death in postmenopausal women. this study aimed at evaluating the effect of soy isoflavone supplementation on plasma lipid profile in postmenopausal women, since this effect is still unclear. methods a double-blind randomized placebo-controlled trial was conducted from january 2010 until february 2011. in total 180 postmenopausal women were randomized into an isoflavone group and a control group of 90 subjects each. the isoflavone group received tablets containing 100 mg soy isoflavones and 500 mg calcium carbonate, while the control group received 500 mg calcium carbonate only. supplementation was given once daily for 1 year. plasma lipid levels [triacylglycerol, total, low-density lipoprotein (ldl), and high-density lipoprotein (hdl) cholesterols] were assessed at baseline, and after 6 and 12 months of supplementation using an enzymatic colorimetric method (cobas c 111, roche). independent t-test was used for data analysis. results baseline subject characteristics and lipid profile in the two groups were comparable. in the isoflavone and control groups after 6 months of supplementation ldl cholesterol levels were 124.9 ± 35.2 mg/dl vs 112 .7 ± 29.7 mg/dl (p=0.013*), respectively, and after 12 months 116.9 ± 31.7 mg/dl vs 109.1 ± 29.8 mg/dl (p=0.086). there were no significant differences in the other lipid levels at 6 and 12 months. conclusions soy isoflavone supplementation for 6 months was capable of significantly reducing ldl cholesterol levels in postmenopausal women. no significant changes in total cholesterol, triacylgycerol, and hdl cholesterol were found after isoflavone supplementation. key words: soy isoflavone, serum lipids, postmenopausal isoflavone supplementation reduces low-density lipoprotein cholesterol levels in postmenopausal women yenny* and pusparini** 198 yenny, pusparini isoflavone and cholesterol levels suplementasi isoflavon menurunkan kadar kolesterol low-density lipoprotein pada perempuan pascamenopause latar belakang penyakit kardiovaskular merupakan penyebab utama mortalitas pada perempuan pascamenopause. efek suplementasi isoflavon kedelai terhadap profil lipid plasma masih bersifat kontradiktif. studi ini bertujuan untuk menilai pengaruh suplementasi isoflavone kedelai terhadap profil lipid dalam plasma pada perempuan pascamenopause. metode sebuah uji klinik acak tersemar ganda dengan menggunakan kontrol dilakukan antara bulan januari 2010 – februari 2011. sebanyak 180 perempuan pascamenopause dirandomisasi menjadi grup isofalvone (n = 90 subjek) memperoleh tablet berisi 100 mg isoflavone and 500 mg kalsium karbonat, and grup kontrol memperoleh 500 mg kalsium karbonat. suplementasi diberikan 1x/hari selama 1 tahun. kadar lipid plasma {kolesterol total, triacylglycerol, kolesterol low-density lipoprotein (ldl), and kolesterol high-density lipoprotein (hdl) diukur pada awal studi, bulan ke-6 and 12 pascasuplementasi dengan menggunakan enzymatic colorimetric method (cobas c 111, roche). uji t independen digunakan untuk analisis data. hasil tidak terlihat adanya perbedaan karakteristik subjek and profil lipd pada awal studi antara kedua grup. setelah suplementasi isoflavon selama 6 and 12 bulan menunjukkan kadar kolesterol total, triacylcholesterol and kolesterol hdl lipid serum tidak ada perbedaan yang bermakna antara kedua kelompok perlakuann. kadar kolesterol ldl pada kelompok isoflavon and kontrol setelah suplementasi 6 bulan besarnya 124.9 ± 35.2 mg/dl vs 112 .7 ± 29.7 mg/dl (p= 0,013*), and 116,9 ± 31,7 mg/dl vs 109,1 ± 29,8 mg/dl (p= 0,086). kesimpulan suplementasi isoflavone kedelai selama 6 bulan mampu menurunkan kadar kolesterol ldl secara bermakna pada perempuan pascamenopause. tidak terlihat adanya penurunan yang bermakna dari suplementasi isoflavone terhadap kadar kolesterol total, triacylgycerol, and kolesterol hdl. kata kunci: isoflavon kedelai, lipid serum, pascamenopause abstrak introduction cardiovascular disease is the main cause of death in women throughout the world. the us national vital statistics reports indicate that 24% of women aged 45–74 years die from cardiovascular disease.(1) menopause is one of the risk factors for cardiovascular disease. estrogen deficiency in menopause is associated with significant c h a n g e s i n l i p o p r o t e i n m e t a b o l i s m , characterized by increased plasma lipid levels (dyslipidemia) during the postmenopausal y e a r s . ( 2 ) t h e d y s l i p i d e m i a i s m a r k e d b y increased total cholesterol (tc), low density lipoprotein (ldl) cholesterol and/or decreased high density lipoprotein (hdl) cholesterol.(3) t h e p r e v a l e n c e o f d y s l i p i d e m i a i n postmenopausal women is 39%.(4) preventive measures to reduce the increase in plasma lipids is frequently associated with a decreased risk of cardiovascular disease.(2) 199 univ med vol. 32 no.3 deaths from cardiovascular disease are lower in asian countries, as compared with those in western countries, with their very different dietary patterns; one outstanding difference is t h a t a s i a n p o p u l a t i o n s c o n s u m e m o r e soybeans.(5) soybean products are beginning to attract interest because of their pleiotropic effects and their utilization for prevention of many disease conditions, such as metabolic diseases, chronic inflammatory diseases, and cancers, due to their isoflavone content.(6) estimated soybean isoflavone intake in asian countries is 25-50 mg per day (expressed as aglycone equivalent),(7) whereas isoflavone intake of american women is less than 1 mg per day.(8) this difference in dietary pattern leads to the suggestion of a role of isoflavones on cardiovascular events. i s o f l a v o n e s a r e p l a n t e s t r o g e n s (phytoestrogens), most abundantly found in soybeans, and composed mainly of genistein, daidzein, and glycitein. they are structurally similar to 17-â -estradiol, causing them to bind to estrogen receptors (er), thus mimicking the effects of estrogens on target organs. there are 2 types of estrogen receptor expressed in different tissues, with erα receptors being expressed in the uterus, hypotalamus/pituitary, and skeleton, whereas erâ receptors are expressed in the ovaries, cardiovascular system, and the brain. the affinity of genistein for erâ is from 20 to 30 times higher than for erα, and is comparable to the affinity of 17-â -estradiol for these receptors.(9) the abovementioned properties of soybean isoflavones enable these micronutrients t o r e p l a c e t h e f u n c t i o n s o f e s t r o g e n s i n postmenopausal women. t h e r e a r e v a r i o u s t h e o r i e s o n t h e mechanism of action of soy isoflavones in lowering the plasma lipid levels. according to one theory, soy isoflavones activate erβ and decrease lipoprotein lipase activity, thus d e c r e a s i n g l i p o g e n e s i s a n d a d i p o c y t e differentiation. another mechanism may be through peroxisome proliferator activated receptors (ppar α and pparγ) that control the transcription of genes involved in the regulation of fatty acid metabolism, and as tyrosine kinase inhibitors that inhibit phosphorylation of several e l e m e n t s r e q u i r e d f o r a d i p o c y t e differentiation.(10,11) the soy isoflavones vary in their effect on prevention of cardiovascular disease, particularly reduction of plasma lipid levels. a meta-analysis conducted by zhan et al. showed that an intake of soy proteins with an isoflavone content of > 80 mg/day may reduce total cholesterol, ldl cholesterol, and triacylglycerol levels by 3.8%, 5.3%, and 7.3%, respectively, while increasing hdl cholesterol by 3%.(12) a meta-analysis by prediger et al.(13) showed that isoflavonecontaining soy proteins result in a significant decrease in total cholesterol levels (by 5.34 mg/ dl, or 2.4%). however, no significant effect was shown with regard to ldl cholesterol, hdl cholesterol, or triglycerides. a systematic review s h o w e d t h a t i s o f l a v o n e s h a v e a s l i g h t l y significant effect on triglycerides in comparison with placebo (mean difference – 0.46 mmol/l), but have no statistically significant effect on total c h o l e s t e r o l , l d l c h o l e s t e r o l , a n d h d l cholesterol. (14) the abovementioned metaanalyses included all subjects and were not exclusively for postmenopausal women. the objective of the present study was to evaluate the effect of soy isoflavone supplementation for 12 months o n p l a s m a l i p i d l e v e l s ( t o t a l cholesterol, triacylglycerol, ldl cholesterol, and hdl cholesterol) in postmenopausal women. methods design of the study this experimental study was designed as a double-blind randomized placebo-controlled clinical trial. the study was conducted in the catchment area of the district health center, mampang prapatan, south jakarta, from january 2010 until february 2011. study subjects the inclusion criteria used in this study were: women 47 – 60 years of age, being at post200 yenny, pusparini isoflavone and cholesterol levels menopause (having had no menstrual periods for minimally 1 year and maximally 10 years), the menopause being a natural menopause (not induced by total hysterectomy, bilateral oophorectomy, radiation, or chemotherapy), not taking drugs and supplements during the last 6 months (hormonal drugs such as glucocorticoids, anticoagulants, antihyperlipidemic drugs, antihypertensive drugs, isoflavone-containing supplements, and oral antidiabetics), agreeing to participate in the study by signing informed consent, capable of walking unassisted, and able to communicate. the subjects were excluded from the study if they had malignant disease (such as mammary, cervical, and endometrial cancers) or severe psychotic disorders, such as schizophrenia. the calculated sample size per group was 90, which was estimated to be adequate to detect a 30% difference in the mean lipid profile values between the treatment groups using a two-tailed test, an alpha of 0.05, and a power of 80%. the mampang prapatan district in south jakarta consists of five kelurahan (villages), from which four were selected by multistage cluster random sampling. from the four selected kelurahan, two rukun warga (rw) were chosen, and from each rw five rukun tetangga (rt, a kind of neighborhood association) were taken. in total 40 rts were selected. from each rt a list of postmenopausal women was made. by simple random sampling the study subjects were selected from among the postmenopausal women in each of the 40 rts. the sample sizes in each rt was determined proportionally according to the available numbers of postmenopausal women. intervention the soybean isoflavone extract was imported from hui song pharmaceuticals, china. the supplement tablets used in this study were prepared, packed, and labelled by pt. ikapharmindo putramas, indonesia. each supplement tablet contained 250 mg soybean extract, equivalent to 100 mg isoflavone aglycones (comprising genistein 56%, daidzein 41%, and glycitein 3%) and 500 mg calcium carbonate. the placebo (control) tablets contained 500 mg calcium carbonate. the form, color, and flavor of the supplement and control tablets were identical. the supplements were assigned to the two groups in a double-blind manner. the tablets in each group were given once daily by the oral route after breakfast, between 07.00 – 10.00 we s t e r n i n d o n e s i a n ti m e ( w i b ) . t h e supplementation was given by cadres at home visits, when the supplements were taken directly in front of the cadres. a checklist was used to note daily tablet consumption and any complaints arising during supplementation. measurement of physical characteristics height in kg was measured by means of a portable microtoise at an accuracy of 0.1 cm. weight in kg was determined using sage portable scales at an accuracy of 0.1 kg. body mass index (bmi) was calculated by dividing the weight in kg by the square of the height in m. threshold values for bmi were according to the criteria issued in 2013 by the department of health of the republic of indonesia (depkes ri). the depkes ri bmi threshold criteria for women are as follows: underweight (<18 kg/m2), normal weight (18-25 kg/m2), overweight (25–27 kg/m2), obese (>27 kg/m2). in the present study, bmi values were categorized into normal/ underweight <24.9 kg/m2 and overweight ≥ 25 kg/m2.(15) laboratory analysis a 10 ml venous blood sample was collected from each subject after a 12-hour fast for determination of lipid levels (total cholesterol, triacylglycerol, hdl cholesterol, and ldl cholesterol). normal lipid levels according to the adult treatment panel iii (atp iii) guidelines are: total cholesterol <200 mg/dl, triacylglycerol <150 mg/dl, ldl cholesterol <130 mg/dl, hdl cholesterol > 40 mg/dl.(16) blood samples were collected three times, i.e. before soy isoflavone supplementation (baseline), after 6 months of 201 univ med vol. 32 no.3 supplementation, and after 12 months of supplementation. the venous blood samples were centrifuged at 2000 rpm for 10 minutes. the obtained serum was frozen at -70 oc pending laboratory investigations, which were performed simultaneously for samples of all subjects before supplementation and after 6 and 1 2 m o n t h s o f s u p p l e m e n t a t i o n . to t a l cholesterol, triacylglycerol, ldl cholesterol, and hdl cholesterol were assessed by means of an enzymatic colorimetric method (cobas c 111, roche). assessment of compliance subject compliance in this study was determined by observing the subjects taking the supplementation tablets in front of the cadres, by counting the tablets remaining in the bottle at the end of each month, and by measuring blood soy isoflavone levels at baseline and at the completion of the study. the subjects were categorized as drop-outs if they failed to take supplements for 7 consecutive days or if the total tablets consumed was < 90% (151 tablets) for the 6-month trial. for the 12-month trial, the criteria were 14 consecutive days or < 90% (328 tablets), respectively. statistical analysis n o r m a l i t y o f d a t a d i s t r i b u t i o n w a s determined by means of the kolmogorovsmirnov test. the independent t-test was used to find differences between the isoflavone and control groups in subject characteristics, total cholesterol, triacylglycerol, ldl cholesterol and hdl cholesterol at baseline, after 6 months, a n d a f t e r 1 2 m o n t h s . a p o f < 0 . 0 5 w a s c o n s i d e r e d s t a t i s t i c a l l y s i g n i f i c a n t . t h e software used for statistical analysis was the statistical program for social sciences (spss) version 17. ethical clearance the study protocol was approved by the committee on research ethics of the faculty of medicine, trisakti university. figure 1. flow of subject participation during study 202 yenny, pusparini isoflavone and cholesterol levels table 1. distribution of demographic and physical characteristic of the subjects at baseline by treatment groups a)values are mean ± standard deviation; b) number of subjects (%); c) p values calculated by independent t-test results at the start of the study, 180 women postmenopausal women meeting the inclusion and exclusion criteria were randomized into two groups, i.e. the isoflavone group and the control group, each comprising 90 women. after 6 months of supplementation, one subject in the isoflavone group dropped out, as she moved out of the study area, whereas no drop-outs occurred in the control group. at the completion of the study, after 12 months of supplementation, there were no dropouts in the soy isoflavone group, whereas in the control group one subject dropped out, because she returned to her native village to get married. the data collected for statistical analysis were from 178 subjects (89 in each group). the flow of subject participation may be seen in figure 1. the distribution of subject characteristics at baseline between the isoflavone and control groups is presented in table 1. mean age was 53.4 ± 3.5 years in the isoflavone group vs 53.5 ± 3.5 years in the control group, duration of menopause was 4.5 ± 2.1 years vs 4.4 ± 2.4 years, most subjects were married (65% vs 71%), and of low educational level (66.7% vs 68.9%). in the isoflavone group 54.5% were employed, while in the control group 61.1% were unemployed. the independent t-test did not find any significant differences between the isoflavone group and the control group in the baseline distribution of the various variables, either demographic or physical, including plasma lipids, with all p values being above significance level. this indicates that the randomization performed in this study had successfully distributed all variables uniformly between the two groups, except for the treatment variable. table 2 presents the total cholesterol, triacylglycerol, ldl cholesterol, and hdl cholesterol levels after supplementation for 6 203 univ med vol. 32 no.3 and 12 months. the total cholesterol level in the i s o f l a v o n e g r o u p v s c o n t r o l g r o u p a f t e r supplementation for 6 months was 208.2 ± 37.4 mg/dl vs 198.2 ± 35.6 mg/dl (p=0.070), while after 12 months it was 197.0 ± 37.3 mg/dl vs 188.5 ± 34.8 mg/dl (p=0.114). there was therefore a reduction in total cholesterol levels at 6 and 12 months, both in the isoflavone group and control group, but the reduction was statistically not significant. as for triacylglycerol and hdl cholesterol levels in the isoflavone and control groups after 6 months and 12 months of supplementation, they increased at 6 months, but decreased again at 12 months approaching baseline levels, both in the isoflavone and control groups, but the changes were statistically not significant. only for ldl cholesterol levels in the isoflavone vs control groups was there a s i g n i f i c a n t d e c r e a s e a f t e r 6 m o n t h s o f supplementation (124.9 ± 35.2 mg/dl vs 112.7 ± 29.7 mg/dl (p= 0.013*). however, a further reduction after 12 months of supplementation was statistically not significant (116.9 ± 31.7 mg/dl vs 109.1 ± 29.8 mg/dl (p= 0.086) (figure 2). table 2. mean lipid profiles after 6 months and 12 months of supplementation by treatment groups values are mean ± s.d.; p values calculated by independent t-test; *significance hdl = high density lipoprotein; ldl= low density lipoprotein figure 2. levels of ldl cholesterol at baseline, at 6 months and 12 months in the isoflavone group and control group. a significant reduction is seen at 6 months 204 yenny, pusparini isoflavone and cholesterol levels adverse events there were no life-threatening adverse events during the conduct of this study. there were no subjects who dropped out for clinical reasons. the complaints reported in both groups w e r e k n e e p a i n , l e g p a i n a n d l e g a c h e , paresthesia, back pain, headache, and increased appetite. discussion the results of this study showed that administration of soy isoflavones at 100 mg/day to postmenopausal women significantly reduced their ldl cholesterol levels after 6 months of supplementation (9.2 mg/dl or 6.86%). after 12 months of supplementation ldl cholesterol decreased steadily but nonsignificantly. there were no reductions in other serum lipid levels at 6 and 12 months. these results did not markedly differ from those of the studies by yang et al.(17) and taku et al.(18) the study conducted by yang et al.(17) on 130 healthy postmenopausal taiwanese women showed that soy isoflavone supplementation for 6 months succeeded in significantly reducing the levels of total cholesterol in the isoflavone and control groups by 4.5 and 3.06%, respectively, and the ldl levels by 4.67 and 5.09%, respectively, in patients with total cholesterol levels of >200 mg/dl. in our study the reduction in ldl levels was higher than that in the study by yang et al.(17) (6.86% vs 5.09%), which presumably was the result of the larger dose of soy isoflavones which we used (100 mg vs 70 mg). apart from ldl, our study did not find any effect of isoflavones on other serum lipid levels. the marked differences of yang’s study with ours were in the open labeled study design, which was randomized but used no controls, and involved women of normal weight. the metaanalytic study performed by taku et al.(18) evaluated the effects of soy protein intake on lipid profiles in men and in premenopausal as well as postmenopausal women. the meta-analysis indicated that soy isoflavones significantly reduced serum total cholesterol and ldl cholesterol levels, but not hdl cholesterol and triacylglycerol levels. the study by mangano et al. (19) was conducted on 131 healthy women over 60 years of age, who were overweight, and had moderate hypercholesterolemia. the aim of the study was to evaluate the long term effects on serum lipids and inflammatory markers of soy protein s u p p l e m e n t a t i o n ( 1 8 g / d a y ) a n d / o r s o y isoflavones (105 mg/day aglycone equivalent), either separately or in combination. after a oneyear intervention no significant effect was seen of the soy protein and isoflavone interventions on serum lipids, lipids ratio, or inflammatory markers. in subjects who were equol producers, significant effects of respectively -5.9% and 7.2% were found on the total cholesterol/hdl and ldl/hdl ratios. t h e o p t i m a l e ff e c t i v e d o s e o f s o y isoflavones capable of exerting a therapeutic effect is to date still not known with certainty. various studies on the effect of isoflavones in reducing serum lipids have used varying values for the dose and duration of therapy, e.g. 35 mg (6 months),(17) 60 mg (12 weeks),(20) and 164 mg (10 weeks).(21) the dose of soy isoflavone extract used in our study was 100 mg/day (aglycone equivalent). the studies investigating the effect of isoflavones on reduction of serum lipids in postmenopausal women generally use soy proteins with a certain isoflavone content, thus leading to uncertainty whether the reduction in serum lipids are due to the soy protein itself or to its isoflavone content. one of such studies was performed by jassi et al.,(20) where in spite of a reduction in serum lipids of the controls after soy protein supplementation, no significant changes were seen in the isoflavone group, indicating that apart from isoflavones, soy protein has other components that are responsible for the hypocholesterolemic effect after soy protein administration. our study found no reduction in serum lipid 205 univ med vol. 32 no.3 levels other than ldl cholesterol, either after 6 months or 12 months of supplementation, similar to the findings of yang et al.(17) and taku et al.(18) the nonsignificant reduction in ldl cholesterol after 6 months of supplementation in our study may be the result of the unsupervised dietary intake of the subjects, or of the longer duration of supplementation, which was continued after plateau levels had been attained. this view is supported by a review performed by manach et al.(22) on studies evaluating the bioavailability of soy isoflavones for 1-3 months, indicating that continuous supplementation does not increase blood isoflavone levels. the absence of isoflavone effects on serum lipids is also seen with several other studies. the study by ho et al.(23) was a one-year study conducted on 203 postmenopausal chinese women. these investigators are of the opinion that the weakly estrogenic effect obtained from an isoflavone dose of 40-80 mg/day or from the usual intakes in asian populations is inadequate to counteract the effects of postmenopause in increasing cholesterol levels. the study was conducted on postmenopausal women with mean age of 54 years and mean duration of menopause of 4 years, thus resembling our study subjects. the study of ho et al.(23) differed from ours in the source of isoflavones (the netherlands) and the percentages of the isoflavone components (genistein 14.7%, daidzein 46.4%, and glycetein 38.8%). furthermore, although their study used the same duration of supplementation as in our study (1 year), the study by ho et al.(23) did not determine plasma lipids at 6 months. therefore, any significant changes in ldl cholesterol levels at 6 months would escape observation. the study conducted by rios et al.(24) on 47 postmenopausal women aged 47–66 years, receiving 40 mg isoflavones vs casein for 6 months, aimed at evaluating the effect of soy isoflavone supplementation on lipid profile, also did not show any significant effects. the small sample size of the study and the occurring changes in dietary pattern and in physical exercise may presumably account for its nonsignificant results. no life-threatening adverse events were found in our study, in agreement with the metaanalytic results of qin et al.(14) the reported complaints in our study subjects were knee pain, leg pain and leg ache, paresthesia, back pain, headache, and increased appetite, whereas in the meta-analysis by qin et al.(14) the complaints were gastrointestinal (bloating and constipation). in the study conducted by steinberg et al.(25) using soy hypocotyl isoflavones at 80–120 mg, a significant increase in blood urea nitrogen was found after a 2-year supplementation. the strengths of our study are in the doubleblind randomized controlled design, sufficiently large sample size, low drop-out rate (1%), and high compliance of participants up to the completion of the supplementation (99.9%). a limitation of this study is the lack of control on daily food consumption by the subjects, so that the study results may have been affected by the consumption of foods high in isoflavones or fatty acids. it should also be borne in mind that the isoflavone extract used in our study was from china, with a presumably different composition than isoflavone extracts from other countries, while the processing method may also affect the isoflavone content.(26) f u r t h e r p h a r m a c o k i n e t i c s t u d i e s o n i s o f l a v o n e s a r e r e q u i r e d i n i n d o n e s i a n postmenopausal women, since the bioavailability o f d r u g s i s a ff e c t e d b y a b s o r p t i o n a n d metabolism, leading to variable effects due to polymorphisms in the genes encoding metabolic enzymes.(27) in addition, studies are needed to detect genetic polymorphisms of the erβ gene in indonesian postmenopausal women. according to one study, (28) the genotype deriving benefits from isoflavones are the aa alui genotype of erβ (associated with increased risk for cardiovascular disease) and the tsp509i snp in the erβ ex splice variant of the aa genotype, with increased plasma hdl cholesterol levels after soy isoflavone supplementation.(28) 206 yenny, pusparini isoflavone and cholesterol levels the reduction in ldl cholesterol in our study is greater than that in the statement of the american heart association (aha) advisory paper (6.86% vs 3%), but the effect lasted only up to 6 months postsupplementation. our study supports the aha advisory statement that the effect of isoflavones in reducing cardiovascular risk factors (serum lipids) in postmenopausal women is “minimal at best”.(29) conclusions soy isoflavone supplementation reduced ldl cholesterol levels in postmenopausal women s i g n i f i c a n t l y a f t e r s i x m o n t h s , b u t n o n significantly after twelve months. no significant difference was found on the effect of isoflavones in decreasing total cholesterol, triacylgycerol, and hdl cholesterol levels, both at 6 months and at 12 months. acknowledgements t h e i n v e s t i g a t o r s t h a n k a l l s t u d y participants for their cooperation, and the dean of the faculty of medicine, trisakti university, for the funding of this study. we also thank ikapharmindo pharmaceuticals for preparing the supplements for this trial. references 1. heron m. deaths: leading causes for 2009. national vital statistics reports 2012;61:1-96. 2. cignarella a, kratz m, bolego c. emerging role of estrogen in the control of cardiometabolic disease. trends pharmacol sci 2010;31:183-9. 3 the task force for the management of dyslipidaemias of the european society of cardiology (esc) and the european atherosclerosis society (eas). esc/eas guidelines for the management of dyslipidemias. eur heart j 2011:32:1769-818. 4. tandon vr, mahajan a, sharma s, sharma a. prevalence of cardiovascular risk factors in postmenopausal women: a rural study. j med life health 2010;1:26-9. 5. gil-izquierdo a, penalvo jl, gil ji, medina s, horcajana mn, lafay s, et al. soy isoflavones and cardiovascular disease epidemiological, clinical and omics perspectives. curr pharm biotechnol 2012;13:624-31. 6. nagaraju gp, zafar sf, el-rayes bf. pleiotropic effects of genistein in metabolic, inflammatory, and malignant diseases. nutr rev 2013;71:56272. 7. messina m, nagata c, wu ah. estimated asian adult soy protein and isoflavone intakes. nutr cancer 2006;55:1-12. 8. de kleijn mjj, van der schouw yt, wilson pwf, adlercreutz h, mazur w, grobbee de, et al. intake of dietary phytoestrogens is low in postmenopausal women in the united states : the framingham study. j nutr 2001;154:434-41. 9. pilsakova l, riecansky i, jagla f. the physiological action of isoflavone phytoestrogen. physiol res 2010;59:651-64. 10. orgaard a, jensen l. the effects of soy isoflavones on obesity. exp biol med (maywood) 2008;233:1066-80. 11. patel rp, barnes s. isoflavones and ppar signaling: a critical target in cardiovascular, metastatic, and metabolic disease. ppar res 2010. article id 153252, 10 pages. doi:10.1155/ 2010/153252. 12. zhan s, ho sc. meta-analysis of the effects of soy protein containing isoflavones on the lipid profile. am j clin nutr 2005;81:397-408. 13. prediger ccc, olinto mta, nacul lc, ziegler dr, pattussi mp. effect of soy protein containing isoflavones on women’s lipid profile: a metaanalysis. rev nutr campinas 2011;24:161-72. 14. qin y, niu k, zeng y, liu p, yi l, zhang t, et al. isoflavones for hypercholesterolaemia in adults. cochrane database syst rev. 2013 jun 6;6:cd009518. doi: 10.1002/14651858. cd009 518.pub2. review. 15. kementerian kesehatan. status gizi dewasa menurut indeks massa tubuh: riset kesehatan dasar (riskedas) 2010. jakarta: badan penelitian dan pengembangan kesehatan kementerian kesehatan ri tahun 2010. available at: http://www.riskesdas.litbang. depkes.go.id/download/tabelriskesdas2010.pdf. accessed august 6, 2013. 16. national choletsrol education program national institute of health. detection, evaluation, and treatment of high blood cholesterol in adults. (adult treatment panel iii). national institute of health;2002. 17. yang ts, wang sy, yang yc, su ch, lee fk, chen sc, et al. effects of standardized phytoestrogen on taiwanese menopausal women. taiwan j obstet gynecol 2012;51:22935. 207 18. taku k, umegaki k, sato y, taki y, endoh k, watanabe s. soy isoflavones lower serum total and ldl cholesterol in humans: a metaanalysis of 11 randomized controlled trials. am j clin nutr 2007;85:1148-56. 19. mangano km, hutchins-wiese hl, kenny am, walsh sj, abourizk rh, bruno rs, et al. soy proteins and isoflavones reduce interleukin6 but not serum lipids in older women: a randomized controlled trial. nutr res 2013;33: 1026-33. 20. jassi hk, jain a, arora s, chitra r. effect of soy proteins vs soy isoflavones on lipid profile in postmenopausal women. indian j clin biochem 2010;25:201-7. 21. shidfar f, ehramphosh e, heydari i, haghighi l, hosseini s, shidfar s. effects of soy bean on serum paraoxonase 1 activity and lipoproteins in hyperlipidemic postmenopausal women. int j food sci nutr 2009;60:195-205. 22. manach c, williamson g, morand c, scalbert a, rémésy c. bioavailability and bioefficacy of polyphenols in humans: review of 97 bioavailability studies. am j clin nutr 2005;81: 230s-42s. 23. ho sc, chen ym, ho ss, woo jl. soy isoflavone supplementation and fasting serum glucose and lipid profile among postmenopausal chinese women: a double-blind, randomized, placebo-controlled trial. menopause 2007;14: 905-12. 24. rios dr, rodrigues et, cardoso ap, montes mb, franceschini sa, toloi mr. lack of effects of isoflavones on the lipid profile of brazilian postmenopausal women. nutrition 2008;24:1153-8. 25. steinberg fm, murray mj, lewis rd, cramer ma, amato p, young rl, et al. clinical outcomes of a 2-y soy isoflavone supplementation in menopausal women. am j clin nutr 2011;93:356-67. 26. north american menopause society. the role of soy isoflavones in menopausal health: report of the north american menopause society/wulf h. utian translational science symposium in chicago, il (october 2010). menopause 2011;18:732-53. doi: 10.1097gme.0b013e31821 fc8e0. 27. lampe jw, chang jl. interindividual differences in phytochemical metabolism and disposition. semin cancer biol 2007;17:347-53. 28. hall wl, vafeiadou k, hallund j, bugel s, reimann m, koebnick c, et al. soy isoflavone enriched foods and markers of lipid and glucose metabolism in postmenopausal women: interactions with genotype and equol production. am j clin nutr 2006;83:592-600. 29. sack fm, lichtenstein a, van horn l, harris w, harris w, kris-etherton p, et al. soy protein, isoflavones and cardiovascular health: an american heart association science advisory for professionals from the nutrition committee. circulation 2006;113:1034–44. c:\users\jurnal fkusakti\docume 121 *monitoring and evaluation coordinator at plan international ethiopia, bahir dar, ethiopia **lecturer, research and publication officer, graduate program coordinator, rift valley university, lancha campus, addis ababa, ethiopia ***monitoring, evaluation and research, amref health africa in ethiopia, addis ababa, ethiopia correspondence: mulualem endeshaw lecturer, research and publication officer, graduate program coordinator of rift valley university, lancha campus, addis ababa, ethiopia email: emulualem@gmail.com orcid id: orcid.org/0000-00017773-9469 date of first submission, november 8, 2 01 8 date of final revised submission, june 21, 2019 date of acceptance, june 24, 2019 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina severe acute malnutrition as a major predictor for poor nutritional outcome among adult hiv patients treated with fpb program: a case-control study nigest abebaw*, mulualem endeshaw**, and mamaru ayenew*** introduction different studies had explained the treatment outcomes of the food by prescription (fbp) program among people living with hiv (plhiv). this study aimed to assess factors affecting nutritional treatment outcome among plhiv using fbp. methods an institution-based unmatched case control study was conducted from august 1 to september 30, 2017 in public health facilities. a total of 566 samples (283 cases and 283 controls) was drawn using random sampling technique. data were collected from fbp, art/pre-art registers and client’s follow up charts. bivariate and multivariate logistic regression analyses were conducted to estimate the relationship of the independent variables with the outcome variable, and a p-value <0.05 was considered as statistically significant at 95% confidence level. all statistical analysis was performed using spss version 23. results in this study severe acute malnutrition at admission (aor=4.45. 95% ci: 2.63–7.55), moderate acute malnutrition at admission (aor=3.8, 95% ci: 2.18–6.67), lack of regular follow up (aor=3.81, 95% ci: 2.18–6.67), low cd4 count below 100 (aor= 2.93, 95% ci: 1.19–7.20), hemoglobin value below 10mg/dl (aor=2.72, 95% ci: 1.17, 6.35), and male sex (aor=1.77, 95% ci: 1.19, 2.63) were found to be significant predictors for poor nutritional treatment outcome. conclusions severity of malnutrition was the major predictor for poor nutritional treatment outcome of patients who were on fbp program. the policy makers can use the information to improve hiv and nutrition interventions based on the result. keywords: food by prescription, plwha, treatment outcome, bahir dar original article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.121-130 may-august, 2019 vol.38no.2 cite this article as: abebaw n, endeshaw m, ayenew m. severe acute malnutrition as a major predictor for poor nutritional outcome among adult hiv patients treated with fpb program: a casecontrol study. univ med 2019;38:12130. doi: 10.18051/univmed.2019.v38. 12 1 1 3 0 122 abebaw, endeshaw, ayenew severe acute malnutrition and nutritional outcome introduction the twin global epidemics of hiv infection and food scarcity disproportionately affect subsaharan africa, and a significant proportion of patients who require antiretroviral therapy are malnourished because of a combination of hivassociated wasting and inadequate nutrient intake.(1) in resource-limited settings where poverty and food insecurity are common, provision of therapeutic and supplementary food products is a critical component of comprehensive care and support interventions.(2) nutritional interventions have been successful in the management of hiv and aids, and many patients enrolled into such programs have markedly improved both their body weight and general health status.(3) globally, an estimated 35.3 (32.2–38.8) million people were living with hiv at the end of 2015 and 70% of people living with hiv (plhiv) are located in sub-saharan africa.(4,5) according to the national aids resource center 2014, adult hiv prevalence in ethiopia is estimated at 1.2 percent.(6) currently in ethiopia, there are about 789,960 adults living with hiv/aids. of these, 204,000 (38.7%) are found in amhara national regional state.(7) malnutrition among people living with hiv remains a major challenge in achieving the full impact of interventions aimed at improving the quality of life, productivity and survival.(2) over 850 million people worldwide and 200 million adults in sub-saharan africa suffer from malnutrition.(8) malnutrition is also associated with faster disease progression, higher mortality rates, and suboptimal response to antiretroviral therapy (art).(9) individuals at all stages of hiv disease are at risk of nutritional deficiency and nutritional status is a strong predictor of disease progression, survival and functional status during the course of the disease.(10) counseling and supporting people living with hiv is indicated as a solution to improve their nutritional status and their health. attainment of good nutrition will contribute to the adoption of a positive attitude, which normally improves the quality of life for adults living with hiv.(11) diffe rent studies have reported that nutritional supplementation among plhiv taken concurrently with art have improved body mass index (bmi),(12-14) survival,(15) cd4 count,(13) hiv viral load,(16) immune response,(13,14) quality of life,(14) and adherence to antiretroviral therapy(17) one way of addressing malnutrition among hiv/aids patients is through the food by pre scription ( fbp) progr a m. (1 8) t he fbp program is one of the strategies that addresses u nd e r -n u t r i t io n amon g pl hiv t hr ou gh nutritional assessment, counseling, and support (nacs).(19) the fbp program targets beneficiaries who are diagnosed to have either moderate acute malnutrition (mam) or severe acute malnutrition (sam) with two different products for a maximum of three or six months. ready to use supplementary food (rusf) is given for three months for mam clients and ready to use therapeutic food (rutf) is given for sam cases.(3) in amhara region, the fbp program has been implemented in 125 public health facilities since 2011 including four health facilities in bahir dar town. even though the fbp nutrition treatment program is designed with the objective to improve the nutritional and functional outcomes of plhiv the intended outcome (recovery from malnutrition) is low.(3,20) studies conducted in amhara region,(7) addis ababa (18) and kenya (21) have reported low fbp recovery rates of 41%, 36.3% and 40.6% respectively. all these studies revealed that the recovery rate from malnutrition was below 50% and still remained to be a challenge. in relation to this there are previous studies c onducte d to assess the effectiveness of prescribed food on recovery f r om ma l nut r i t i o n, ( 3) f a c t o r s i n f l u e nc i ng a d he r e n c e t o t h e f o o d b y p re s c r ip t i on program,(18) nutrition outcomes of hiv-infected malnourished adults treated with ready-to-use therapeutic food (20) and food insecurity and 123 factors associated with malnutrition.(22) the above studies focused on the outcome of patients treated with fbp and factors associated with malnutrition but not specifically on factors associated with treatment failure. besides, studies that explore factors associated with nutritional treatment failure after fbp among adult plhiv are limited in ethiopia. therefore this study tried to find out the factors contributing to treatment failure among adult plhiv in bahir dar town health facilities with a different study design. methods study design and period this institution-based unmatched case control study was conducted from august 1, 2017 – september 30, 2017. study area the study was conducted in four public health facilities of bahir dar town, that have been implementing fbp nutritional treatment program to plhiv from march 2017 to december 2017. bahir dar is the capital of the amhara region, located 560 km from addis ababa. the fbp nutritional treatment program was started since 2011 in the felege hiwot referral hospital (fhrh) and bahir dar health center. the other two han and abay health centers have started fbp since 2012. the felege hiwot referral hospital serves about 15,961 plhiv, of whom 3200 were enrolled into the fbp nutrition treatment program. there were 4, 524 plhiv in bahir dar health center among whom 746 were enrolled into fbp. the numbers of plhiv and fbp beneficiaries in han hc and abay hc were 3301,1514 and 254,321 respectively. study population the study population were all adult nonpregnant, non-lactating plhiv above the age of 18 years whose bmi was <18.5 kg/m2 and who were enrolled in to the fbp nutritional treatment program and had completed the treatment regime for three months (for mam ) or six months (for sam). cases were plhiv who did not recover from malnutrition (bmi <18.5kg/m2) after treatment with rutf/rusf for a maximum of three months (mam) or six months (sam). controls were plhiv who recovered from malnutrition (bmi 18.5) after treatment with rutf/rusf for a maximum of three (mam) or six months (sam). sample size determination the sample size was determined using epi info stat-calc calculator for two populations by selecting power of 80%, a significance level of 5%, control to case ratio of 1: 1 and taking poor art adherence as a risk factor of recovery in the study conducted in amhara region with aor 2.14 for the proportion of exposure among recovered subjects of 9.7%.(7) based on the above assumption, the total sample size required for cases and controls including 10% anticipated dropouts was 566 or 283 pairs. sampling technique after all cases and controls were sorted out from each health facility fbp register, simple random sampling technique was applied to select the required number of samples from each facility using a random number generator. the total sample size was proportionally allocated to the four health facilities based on their total number of recovered and non-recovered plhiv. study variables the outcome of the study was nutritional treatment outcome (recovered =0, unrecovered =1). variables of interest collected were socioeconomic status, age, sex, marital status, religion, household size/number of people in the house, employment, educational status, residence, disclosure status about adherence to the fbp. the health status included cd4 count at entry, type of malnutrition, presence of opportunistic infections (oi’s/tb, chronic diarrhea, candidiasis of the esophagus or lower respiratory tract, recurrent pneumonia), and other conditions such univ med vol. 38 no.2 124 abebaw, endeshaw, ayenew severe acute malnutrition and nutritional outcome a s k a po s i’s s a r c o ma , l ymph o ma , a n d unexplained severe weight loss, who clinical stages i-iv, antiretroviral therapy (art) adherence, art status, hemoglobin at admission, other fbp related factors, regular follow-ups, and type of prescribed commodity. in who clinical stage i the patient is asymptomatic and has persistent generalized lymphadenopathy, while in clinical stage ii there is moderate unexplained weight loss of less than 10% of presumed or measured body weight. clinical stage iii is characterized by unexplained severe weight loss greater than 10% of presumed or measured body weight, unexplained chronic diarrhea for longer than one month, unexplained persistent fever above 37.6°c intermittent or constant, for longer than one month, while stage iv is characterized by hiv wasting syndrome or unexplained severe wasting, recurrent severe pneumonia, and extra pulmonary tuberculosis. data collection method a s ta nd ar d c he c kl i st wa s u s e d f o r extracting information from fbp registration book, preart, art, and patient follow up c a r ds . t he c h e c k l i st wa s che c ke d f or components that could capture the important information from the register and follow up cards. this form was developed using the standardized fbp follow up, pre art, art, and patient follow up cards employed by the art clinic. the laboratory results of cd4 count recorded before enrolled in to fbp was used as a base line values. for those with no pretreatment laboratory test, however, results obtained within one month of enrolment into fbp were considered as baseline values. data analysis de sc r i pt i ve a n alys i s ( f r e qu e n c y distribution, mean (±sd) and percentages) were calculated to describe each socio-demographic va r i a b l e . ba s e d o n t h i s, u ni va r i a t e a n d multivariate logistic regressions were done to investigate associations of outcome of treatment (i.e. recovered and unrecovered) and different independent variables. p-value less than 0.05 was considered as statistically significant throughout the study. ethical considerations ethical clearance was gained from the ethical review committee of the institute of public health, college of medicine and health science, university of gondar. permission was also received from the regional health bureau under no. arhb-307/17 and a copy of the letter given to the four health facilities, namely felege hiwot referral hospital (fhrh), bahir dar health center, han health center, and abay health center. confidentiality was ensured from the investigator by using code numbers instead of names and the information collected was kept in a secured place. results socio-demographic characteristics of the study participants the study included a total of 566 (283 cases and 283 controls) eligible patients living with hiv/ aids recorded on food by prescription nutrition treatment in bahir dar town health facilities. among those 566 patients, three hundred twelve (55.1%) were female. of these, 49.5% were cases and 60.8% were controls. the majority of the study participants (83.4%) were christians and the rest 16.6% were muslims. regarding the marital status, 232 (41.0%), of patients were married. the residence of patients in the group of cases were similar to that of patients in the group of controls, 89.8% of the cases and 90.8% of the controls were urban dwellers. as to educational status the majority of patients in both case (72.8%) and controls (73.8%) had received primar y education. concerning employment, 72.8% of cases and 73.9% of controls were formally employed in private and governmental institutions. finally the majority of participants in both the cases and controls group were living with a family size of 1–4 people (table 1). a b c 125 factors associated with treatment outcome the bivariate analysis in this study revealed that there were several factors as predictors of food by prescription treatment outcome, i.e. sex, malnutrition status, failure of regular follow up, who clinical stage, cd4 count, and hemoglobin level, which were found to be predictors of food by prescription treatment success. when we look at the crude odds ratio (cor) of the covariates, males were 1.58 times more likely not to recover than females (cor=1.58, 95% ci: 1.13–2.21). malnutrition status is the other independent factor identified in this study, since patients who were diagnosed to have sam at base line were 8.1 times more likely not to respond to therapeutic food than patients who had a diagnosis of mild malnutrition (cor=8.1, 95% ci: 3.84–17.01). similarly, patients who were diagnosed to have moderate malnutrition were at higher risk of not recovering than did those with mild malnutrition (cor=6.25, 95% ci: 4.25–9.18). participants who had a cd4 count below 100 were 2.30 times more likely n ot to re s pond to th er a pe u ti c f oo d t ha n participants who had a cd4 count above 350. furthermore, patients who failed to have regular follow up, who clinical stage three, hemoglobin level below 10mg/dl (cor=3.63, 95% ci: 2.24– 5 .8 7) , ( cor= 1.6 8, 95 % ci: 1.0 9– 2. 6 0) , (cor=2.77, 95% ci: 1.35–5.67) respectively, were also associated with increased risk of no recovery from malnutrition (table 2). variable cases n=283 (%) controls n=283 (%) p value sex male 143 (50.5) 111 (39.2) 0.007 female 140 (49.5) 172 (60.8) age (years) 34 170 (60.1) 152 (53.7) 0.111 35-44 71 (25.1) 91 (32.2) 45-54 31 (11.0) 35 (12.4) 55+ 11 (3.9) 5 (1.8) residence urban 254 (89.8) 257 (90.8) 0.672 rural 29 (10.2) 26 (9.2) marital status not married 68 (24.0) 66 (23.3) 0.982 married 114 (40.3) 118 (41.7) separated 18 (6.4) 16 (5.7) divorced 59 (20.8) 61 (21.6) widowed 24 (8.5) 22 (7.8) education primary 206 (72.8) 209 (73.8) 0.863 secondary 60 (21.2) 60 (21.2) higher 17 (6.0) 14 (5.0) employment status employed 206 (72.8) 209 (73.9) 0.394 un employed 77 (27.3) 74 (26.1) religion christian 261 (84.1) 264 (81.5) 0.822 muslim 22 (15.9) 19 (18.5) household size 1 – 4 236 (83.4) 241 (85.2) 0.641 4 8 45 (15.9) 40 (14.1) 8 2 (0.7) 2 (0.7) table 1. socio-demographic characteristics of cases versus controls * christian (orthodox, protestant, catholic) univ med vol. 38 no.2 126 abebaw, endeshaw, ayenew severe acute malnutrition and nutritional outcome table 2. bivariate and multivariate logistic regression analysis of covariates with treatment outcome of fbp program participants variable cases (%) controls (%) cor aor pvalue age (years) <34 170 (60.1) 152 (53.7) 1 35–44 71 (25.1) 91 (32.2) 0.7 (0.48, 1.02) 45–54 31 (11.0) 35 (12.4) 0.79 (0.47, 1.35) >55 11 (3.9) 5 (1.8) 1.97 (0.67, 5.79) sex male 143 (50.5) 111 (39.2) 1.58 (1.13,2.21) 1.77 (1.19, 2.63) 0.005 female 140 (49.5) 172 (60.8) 1 residence urban 254 (89.8) 257 (90.8) 1.13 (0.65,1.98) rural 29 (10.2) 26 (9.2) 1 education primary 206 (72.8) 209 (73.9) 1 secondary 60 (21.2) 60 (21.2) 1.02 (0.68, 1.52) higher 17 (6.0) 14 (5.0) 1.23 (0.59, 2.56) disclosure yes 224 (79.2) 225 (79.5) 1 no 59 (20.8) 58 (20.5) 1.0 2(0.68,1.54) type of malnutrition severe acute malnutrition 34 (12.0) 10 (3.5) 8.10 (3.84,17.01) 4.45 (2.63, 7.55) 0.0001 moderate acute malnutrition 160 (56.5) 61 (21.6) 6.25 (4.25,9.18) 3.81 (2.18, 6.67) 0.0001 mild malnutrition 89 (31.4) 212 (74.9) 1 type of commodity rutf 159 (56.2) 141 (49.8) 1.29 (0.93,1.79) rusf 124 (43.8) 142 (50.2) 1 presence of oi yes 22 (7.8) 22 (7.8) 1 (0.54, 1.85) no 261 (92.2) 26 (92.2) 1 regular follow up yes 207 (73.1) 257 (90.8) 1 no 76 (26.9) 26 (9.2) 3.63 (2.24,5.87) 3.81 (2.18, 6.67) 0.0001 employment yes 206 (72.8) 209 (73.9) 1 no 77 (27.3) 74 (26.1) 1.06 (0.73,1.53) who stage who stage 1 55 (19.4) 75 (26.5) 1 who stage 2 80 (28.3) 85 (30.0) 1.28 (0.81,2.04) who stage 3 127 (44.9) 103 (36.4) 1.68 (1.09,2.60) † who stage 4 21 (7.4) 20 (7.0) 1.43 (0.71, 2.89) art status on art 221 (78.1) 219 (77.4) 1.04 (0.70, 1.55) pre art 62 (21.9) 64 (22.6) 1 art adherence (n=440) yes 202 204 1 no 18 16 1.13 (0.61, 5.41) † cd4 (cells/mm3 ) < 100 12 (4.24) 26 (9.20) 2.30 (1.05,5.07) 2.93 (1.19, 7.20) 0.019 100 200 51 (18.02) 48 (16.96) 2.35 (1.11, 4.96) 3.79 (1.61, 8.92) 0.002 200 350 89 (31.45) 82 (28.98) 2.24 (1.08, 4.62) 4.20 (1.82, 9.71) 0.001  350 131 (45.33) 127 (44.88) 1 1 hb level < 10g/dl 29 (10.25) 11 (3.88) 2.77 (1.35, 5.67) 2.72 (1.17, 6.35) 0.021 10-11.99g/dl 30 (10.60) 37 (13.07) 0.85 (0.51, 1.42) 0.88 (0.46, 1.61) 0.65 > 12g/dl 224 (79.15) 235 (83.01) 1 1 † dropped in the final model, rusf: ready to use supplementary food; rutf: ready to use therapeutic food. cor=crude odds ratio, aor= adjusted odds ratio, oi = opportunistic infection, art = antiretroviral therapy 127 results of multivariate analysis variables that were significant at p-value 0.2 in the simple binary logistic regression analysis were further examined in the multiple binary logistic regressions to determine the relative effects of predictors of treatment outcome among attendants of fbp. factors such as male sex, malnutrition status, failure of regular follow up, lower cd4 count, and hemoglobin value below 10mg/dl remained significant in the multivariate analysis in this study. males were 1.77 times more at risk of non-recovery compared to females (aor 1.77, 95% ci: 1.18–2.6). participants who were diagnosed as sam during admission had an almost 4.5 times increased risk of not responding to therapeutic food compared to participants with mild malnutrition (aor 4.45, 95% ci: 2.63–7.55). in addition, lower cd4 count had a statistically significant association with non-recovery after treatment with fbp, cd4 c o unt < 1 00 , 10 0 –2 00 , 20 0 –3 50 , (aor=2.93, 95% ci: 1.19, 7.20), (aor=3.79, 95% ci: 1.61–8.92), (aor=4.20–95% ci: 1.82– 9.71) respectively. on top of this, patients with hemoglobin value below 10mg/dl were at almost three times higher risk of no recovery after treatment with fbp than their counterparts with a normal hemoglobin value. unlike other studies, the present study found that factors such as age, residence, education level, disclosure status, type of commodity, presence of oi’s, employment status, functional status and art status did not show any significant association with treatment outcome (table 2). discussion the main finding of this study was that severity of malnutrition and low cd4 count were the two major predictors for poor nutrition treatment response. the results of our study are in line with those of other similar studies conducted in mekele,(23) amhara region (7) and sub-saharan countries.(20) in our study, patients who were diagnosed as sam during admission were at almost 4.5 times increased risk of not responding to therapeutic food compared to participants with mild malnutrition. this highlights the need to reach malnourished hiv patients early with nutritional support in order to maximize the recovery rate from malnutrition.(3) in addition, lower cd4 counts of <200 cells/mm3 and 200– 350 cells/mm3 were 3.79 and 4.2 times more at risk of non-response, respectively, compared to the higher cd4 count of >350 cells/mm3. the result of this study was scientifically plausible, since our immunity is nutrition-dependent, plhiv have a compromised immunity due to the effect of hiv, so that they are at risk for opportunistic inf ection which f urt her e xacer bates the malnutrition status and end up with poor response to nutritional supplement. in addition, the low cd4 count results in recurrence of opportunistic inf ection, with imba lance of nutritional requirement and uptake and thus ineffective nutritional supplementation response.(18) in our study, males were 1.77 times more at risk of non-recovery from malnutrition compared to females. the result of this study is in agreement with that of previous studies done in other parts of ethiopia, such as in mekele.(23) subsaharan african counties also showed that males were at increased risk of non-recovery from malnutrition.(20) this may be due to the fact that males tend to access hiv care at a more advanced stage of the disease than females. in addition, females may have good compliance to nutrition and hiv care that predisposes females to improve the uptake.(3) in contrast to this, other studies done in kenya (20) and amhara region (7) reported that females are at increased risk of nonresponse to nutrition treatment. this difference may be attributed to the difference in study design, educational status, or temporal variation in patient characteristics. patients who failed to have regular followup during the fbp treatment were 3.8 times more at risk of nonrecovery from malnutrition than those having regular follow-up. this finding is consistent with that of other studies conducted in univ med vol. 38 no.2 128 abebaw, endeshaw, ayenew severe acute malnutrition and nutritional outcome amhara region, showing that patients with poor rutf adherence were at 11 times lower risk of bmi improvement.(7) other studies also reported that missing follow-up appointments increased the risk of poor nutrition treatment outcome,(18,20,24) since patients missing appointments also miss their monthly doses and the counseling that is provided by the health workers, leading to poor treatment adherence. patients with a hemoglobin value below 10mg/dl were at almost 2.7 times higher risk of non-recovery after treatment with fbp than their counterparts with a normal hemoglobin value. the result of this study is in line with the established facts in many ways: anemia is a condition characterized by inadequate red blood cells (erythrocytes) or hemoglobin. (26) anemia occurs when the body lacks sufficient amounts of iron, resulting in reduced production of the protein hemoglobin. plhiv with poor diet are most susceptible to the disease since malnutrition is known to be a leading factor for the occurrence of anemia. an inadequate intake of macroand micronutrients can play a well-documented role in chronic anemia pathophysiology due to the aforementioned facts together with the result of this study, in that there is a strong association between anemia and malnutrition.(25) in this study, age was not found to be a predictor of fbp treatment outcome. however, other studies undertaken in different settings have documented that old age is associated with increased risk of non-response.(23,24) art status and number of months on art also did not show any significant association with fbp treatment outcome in this study. but patients on pre-art were at risk of non-recovery compared to those on art, and those patients who stayed on art for more than 6 months were at increased risk of non-recovery compared to patients on preart.(21,25) education level of patients and presence of opportunistic infections (oi’s) did not show an association with treatment of fbp outcome in this study, however patients with low level of education were 1.82 times more at increased risk of non-recovery compared to educated o ne s , b e s ide s p a t i e nt s wi t h pr e s e n c e of opportunistic infections were at higher risk of non-recovery from malnutrition compared to those without opportunistic infections.(25) this may be due to the fact that the higher the level of education, the higher the awareness about management of hiv, opportunistic infections and malnutrition. who clinical stage of disease did not show a significant association with fbp treatment outcome in this study, however a study done in mekele northern ethiopia reported that who clinical stages i and ii were four times more likely to respond to ready-to-use therapeutic food than patients with who stages iii and iv.(25) t he r e st of t he f a c t or s s uc h a s t yp e of commodity, employment status, disclosure status, a n d r e si d en c e we r e n o t a s s oci a te d wi t h nutritional treatment outcome in this study and other studies conducted so far. the main limitation of this study was its retrospective nature that limits its ability to gather data about factors that may influence failure to respond to ready-to-use food therapy. it is recommended that clinicians ought to encourage regular screening of nutritional status among plhiv in order to identify malnourished individuals early to improve the outcome of fbp. further longitudinal research is needed to understand the effect of dosage and compliance of individuals on fbp. conclusion severity of malnutrition and lower cd4 count were the most influencing factors of the outcome of ready-to-use therapeutic food by prescription. hence encouraging regular and comprehensive nutritional screening on plhiv and maximizing efforts to reach malnourished hiv patients early with nutritional support for recovery, besides strengthening the nutritional adherence counseling for patients on ready-touse therapeutic food, are crucial elements to improve the outcome of fbp. 129 acknowledgment the authors would like to thank the rift valley university for sponsoring this study. the authors are also grateful to the amhara regional health bureau, felege hiwot referral hospital, bahir dar health center, han health center and abay health center for facilitating the data c o l l e c t i on pr oc e ss . we a l s o e xt e n d ou r appreciation to our data collectors for their contribution in the data collection process. authors’ contributions na has contributed to the design, data collection and write up of the study protocol, me has contributed to the design, data analysis, and manuscript development and revisions, and ma has contributed to the revision of the manuscript. all authors have read and approved the final draft. competing interests the authors declare that they have no competing interests. references 1. koethe jr, heimburger dc. nutritional aspects of hiv-associated wasting in sub-saharan africa. am j clin nutr 2010;91:1138s-42s. doi: 10.3945/ajcn.2010.28608d. 2. food and nutrition technical assistance ii project (fanta-2). review of kenya’s food by prescription program. connecticut ave., nw washington, dc: food and nutrition technical assistance ii project (fanta-2);2009. 3. sadler k, bontrager e, rogers b, et al. food by prescription: measuring the impact and costeffectiveness of prescribed food on recovery from malnutrition and hiv disease progression among hiv+ adult clients in ethiopia. boston: feinstein international center;2012. 4. unaids. global report: unaids report on the global aids epidemic. geneva: unaids; 2013. 5. unaids. people living with hiv: the gap report. geneva: unaids; 2014. 6. ethiopian health and nutrition research institute federal ministry of health. hiv related estimates and projections for ethiopia. addis ababa, ethiopia: ethiopian health and nutrition research institute;2012. 7. gedefaw m, tariku m. determinates of regain in body mass index among malnourished aids patients on therapeutic food in amhara national regional state, northwest ethiopia: a retrospective cohort study. open j epidemiol 2015;5:122-8. doi: 10.4236/ojepi.2015.52016. 8. sicotte m, langlois év, aho j, et al. association between nutritional status and the immune response in hiv + patients under haart: protocol for a systematic review. syst rev 2014;3: 9. doi: 10.1186/2046-4053-3-9. 9. swaminathan s, padmapriyadarsini c, yoojin l, et al. nutritional supplementation in hiv-infected individuals in south india: a prospective interventional study. clin infect dis 2010;51:51– 7. doi: 10.1086/653111. 10. ivers lc, cullen ka, freedberg ka, et al. hiv/ aids, undernutrition, and food insecurity. clin infect dis 2009;49:1096-102. doi: 10.1086/605573. 11. bontrager e, sadler k. early stages of a ‘food by prescription’ programme for hiv infected adults. enn strategy; 2011. 12. ndekha mj1, van oosterhout jj, zijlstra ee, et al. supplementary feeding with either ready-to-use fortified spread or corn-soy blend in wasted adults starting antiretroviral therapy in malawi: randomized, investigator blinded, controlled trial. bmj 2009; 338:b1867. doi: 10.1136/bmj.b1867. 13. olsen mf, abdissa a, kæstel p, et al. effects of nutritional supplementation for hiv patients starting antiretroviral treatment: randomised controlled trial in ethiopia. bmj 2014;348:g3187. doi: 10.1136/bmj.g3187. 14. evans d, mcnamara l, maskew m, et al. impact of nutritional supplementation on immune response, body mass index and bioelectrical impedance in hiv-positive patients starting antiretroviral therapy. nutr j 2013;12:111. doi: 10.1186/1475-2891-12-111. 15. lategan r, steenkamp l, joubert g, et al. nutritional status of hiv-infected adults on antiretroviral therapy and the impact of nutritional supplementation in the northern cape province, south africa. s afr j clin nutr 2010;23:197-201. 16. rawat r, kadiyala s, mcnamara pe. the impact of food assistance on weight gain and disease progression among hiv-infected individuals accessing aids care and treatment services in uganda. bmc public health 2010;10:316. doi: https://doi.org/10.1186/1471-2458-10-316. 17. tafese z, birhan y, abebe h. nutritional care and support among adults living with hiv at hawassa referral hospital, southern ethiopia: a qualitative univ med vol. 38 no.2 130 abebaw, endeshaw, ayenew severe acute malnutrition and nutritional outcome study. afr j aids res 2013;12:105-11. doi: 10.2989/ 16085906.2013.825301. 18. kebede ma, haidar j. factors influencing adherence to the food by prescription program among adult hiv positive patients in addis ababa, ethiopia: a facility-based, cross-sectional study. infect dis poverty 2014;3:20. doi: 10.1186/ 2049-9957-3-20. 19. moench-pfanner r, van ameringen m. the global alliance for improved nutrition (gain): a decade of partnerships to increase access to and affordability of nutritious foods for the poor. food nutr bull 2012;33(4suppl):s373-80. doi: 10.1177/ 15648265120334s313. 20. ahoua l, umutoni c, huerga h, et al. nutrition outcomes of hiv-infected malnourished adults treated with ready-to-use therapeutic food in subsaharan africa: a longitudinal study. j int aids soc 2011;14:2. doi: 10.1186/1758-2652-14-2. 21. nagata jm, cohen cr, young sl, et al. descriptive characteristics and health outcomes of the food by prescription nutrition supplementation program for adults living with hiv in nyanza province, kenya. plos one 2014;9:e91403. doi: 10.1371/journal.pone.0091403. 22. gebremichael d, hadush k, kebede e, et al. food insecurity, nutritional status, and factors associated with malnutrition among people living with hiv/aids attending antiretroviral therapy at public health facilities in west shewa zone, central ethiopia. bio med res intl 2018, article id 1913534. doi: 10.1155/2018/1913534. 23. maldey b, haile f, shumye a. outcome of ready to use food therapy among patients on hiv/ aids care in mekelle hospital, northern ethiopia: retrospective cohort study. j aids clin res 2014;5:1-6. doi: 10.4172/2155-6113.1000268. 24. bahwere p, deconinck h, banda t, et al. effective therapeutic feeding with chickpea sesame based ready-to-use therapeutic food (cs-rutf) in wasted adults with confirmed or suspected aids. world j aids 2011;1:169-81. doi: 10.4236/wja.2011. 14025. 25. janz tg, johnson rl, rubenstein sd. anemia in the emergency department: evaluation and treatment. emerg med pract 2013;15:1-15. c:\users\jurnal fkusakti\docume 209 *department of medical physiology, faculty of medicine, universitas indonesia correspondence: dewi irawati soeria santoso department of medical physiology, faculty of medicine universitas indonesia jl. salemba raya no. 6, jakarta pusat, indonesia email: dewi.irawati.1307@gmail.com phone: +6281296440098 orcid id: https://orcid.org/00000001-7390-5298 date of first submission, january 11, 2 01 9 date of final revised submission, september 19, 2019 date of acceptance, september 20, 2 01 9 this open access article is distributed under a creative commons attributionnon commercial-share alike 4.0 international license abstract universa medicina the benefits and physiological changes of high intensity interval training dewi irawati soeria santoso* and hafizh ahmad boenjamin* physical inactivity have been linked with many major non-communicable diseases and as many as 27.5% of adults globally are considered inactive. physical activity has been proven to be beneficial in the prevention of many chronic diseases and may reduce the risk of premature death. high intensity interval training (hiit) has been gaining popularity as a timeefficient alternative for regular exercise training. current studies show that hiit is more efficient in improving cardiorespiratory fitness, increasing insulin sensitivity and reducing blood pressure than moderate intensity continuous training (mict). the advantage of hiit in fat loss compared to mict is still unclear, but hiit might be more efficient in the obese population. the effect of hiit on increasing aerobic fitness could be caused by increase in stroke volume due to the increase in cardiac contractility, capillary density and mitochondrial adaptation. fat loss during hiit could be caused by increased fat oxidation and elevated hormones that drive lipolysis and reduce appetite. while vigorous physical activity may transiently increase the risk of cardiac events. the effect of hiit on increasing aerobic fitness could be caused by increase in stroke volume due to the increase in cardiac contractility, increased of capillary density and mitochondrial adaptation. while fat loss during hiit could be caused by an increased fat oxidation, elevated hormones that drives lipolysis and reduces appetite. while vigorous physical activity may transiently increase the risk of cardiac event. high intensity interval training is generally safe even in the elderly population and in people with coronary heart disease. keywords: high intensity interval training, physiological changes, fat loss, cardiorespiratory fitness review article pissn: 1907-3062 / eissn: 2407-2230 doi: http://dx.doi.org/10.18051/univmed.2019.v38.209-216 may-august, 2019 vol.38no.3 ci te t his article as: sa n t oso dis, b oen j ami n ha. th e b enefit s a nd physiological changes of high intensity interval training. univ med 2019;38: 209-16. doi: 10.18051/univmed.2019. v38.2 09 -21 6 210 santoso, boenjamin benefits & physiological changes of hiit introduction physical inactivity is now the fourth highest risk factor in global mortality.(1) it is associated with many non-communicable diseases such as obesity, hypertension, cardiovascular diseases, type 2 diabetes and cancer.(2) inactivity is thought to be responsible for 9% of premature mortality worldwide in 2008.(3) physical activity has been proven to be beneficial in the prevention of many chronic diseases and may reduce the risk of premature death.(4) it is recommended by the world health organization and u.s. department of health and human services that adults should perform 150-300 minutes of moderate intensity or 75-150 minutes of vigorous intensity aerobic physical activity per week.(1,5) however, a recent study has found that globally as many as 27.5% of adults are considered inactive,(6) and one of the main perceived barriers to exercise is the lack of time.(7) to solve this problem, one of the exercise options that has been gaining popularity is the practice of high intensity interval training (hiit).(8) high intensity interval training is considered to be more time-efficient and effective than moderate intensity continuous training (mict). here we will examine the benefits of hiit and the current supporting evidence. high intensity interval training high intensity interval training is a form of exercise in which there is a short burst of vigorous activity followed by a period of recovery by rest or low-intensity exercise before starting another bout.(9) depending on the presence of a lactate steady state, hiit can be categorized as aerobic or anaerobic.(10) the high intensity during hiit is completed above the anaerobic threshold. however recently researchers have also begun differentiating the varying level and duration of the high intensity in hiit with sprint or supramaximal-intensity interval training (sit). the intervals in hiit last 1-4 minutes above the anaerobic threshold and up to vo 2 max, while sit intervals last 20-30 seconds above 150% vo 2 max power.(11) the target intensity in hiit is usually measured by the heart rate; often the target is based on the heart rate reserve (hrr) or the maximum heart rate (mrr).(12) the interval nature of hiit allows a period of recovery so that multiple rounds of high intensity activity can be performed. there is no main or official protocol for hiit, but some of the mor e popula r pr otocols of hiit /sit summarized by cress et al.(12) are listed in table 1. high intensity interval training can be done with various modalities and equipment, and often cycle ergometers are used in this activity. the benefit of using a cycle ergometer is that the total amount of work and power can be measured. running tracks, swimming pools, arm ergometers, body weight and resistance training are also commonly used for this activity. (12 ) it is recommended to measure the heart rate to make sure that the targeted intensity is achieved. one of the benefits of hiit compared to other activities is its time efficiency, the whole activity of hiit can be done in a relatively short time compared to traditional forms of exercise. so hiit is considered an attractive exercise modality for people with a busy lifestyle. benefits of hiit there are a lot of studies on the beneficial effects of hiit. one of the most common targeted outcomes of hiit is fat loss. while there are many protocols for hiit, a study has shown that both short and long periods of high intensity exercise during hiit have resulted in a reduction in fat mass, fat percentage as well as an increase in physical endurance (vo2max) with no significant differences between the two groups.(13) studies have also compared the amount of fat loss between hiit and continuous exercise. while it is suggested that hiit is more efficient in causing fat loss, a meta-analysis by keating et al.,(14) who analyzed 31 studies that compared the effect of fat loss by hiit / sit and mict, showed no significant difference in the amount of reduced total body fat and fat mass when similar time 211 commitment and energy is used. however, a different meta-analysis that examined 13 studies about the effect of hiit on an obese population showed that hiit produces similar effects of fat loss compared to mict, even when the time committed for hiit in this study was 40% less (shorter) than that in the mict group.(15) one study on obese young women found that there was a comparable decrease of abdominal visceral fat in the hiit group and the mict group after 12 weeks of exercise even when the duration of the mict sessions was almost double that of the hiit sessions.(16) this result may show that when the targeted outcome is the amount of fat loss, hiit is more efficient in the obese population, but in the general population it has the same effectiveness as mict. a targeted outcome for which hiit might be more beneficial than mict is cardiorespiratory fitness. a meta-analysis that compared the increase in vo 2 max, as an indicator for increased aerobic fitness, between endurance training and hiit showed a greater vo 2 max gain in the hiit group.(17) another meta-analysis in a special population with coronary artery disease also showed that hiit is more beneficial in increasing cardiorespiratory fitness compared to mict.(18) even a study on the elderly population, with a mean age of over 60, showed a positive effect of hiit on their overall cardiorespiratory fitness.(19) with different protocols, a meta-analysis showed that long interval, high volume and moderate to long-term hiit gave the greatest benefit in improving vo 2 max.(20) another documented effect of hiit is the reduction in blood pressure. one meta-analysis that compared the effect of hiit and mict on resting blood pressure in individuals with prehypertension and hypertension, showed that both groups had a positive and similar effect on the reduction of both resting systolic and diastolic blood pressure.(21) however, in some of those studies in the meta-analysis, the exercise time was less in the hiit than in the mict groups. another meta-analysis of 16 studies on the 24hour blood pressure reduction effect between hiit and mict showed that hiit had a greater decrease of night-time diastolic blood pressure compared to mict. (22) individuals with type 2 diabetes also seem to benefit more from hiit. a meta -analysis showe d that compared to continuous training, hiit was more effective in reducing insulin resistance and showed significant reductions in hba1c.(23) one of the advantages of hiit is that it needs less time than conventional continuous training. therefore it is hoped that more people will adhere to the programs, but the results of the studies on adherence have been mixed. one meta-analysis examined the acute affective and univ med vol. 38 no.3 name work interval and intensity rest interval and intensity series modality total time (including warm-up and cooldown) tabata 20 seconds 170% vo2 max 10 seconds rest or very low intensity 8 times cycle ergometer, track, treadmill, body weight or resistance exercises 4 minutes wingate 30 seconds all out against constant force 4 minutes active recovery low intensity 4-6 times mechanically braked cycle ergometer 18-27 minutes conventional 60 seconds >90% hrr 60 seconds rest or active recovery 10 times cycle ergometer, track, treadmill, body weight or resistance exercises 20 minutes clinical 4 minutes 85%-95% mhr 3 minutes 60%-70% mhr 4 times treadmill with handrails, cycle ergometer, arm ergometer 25 minutes table 1. popular hiit / sit protocols (12) 212 santoso, boenjamin benefits & physiological changes of hiit enjoyment response of hiit and mict on different scales of interest. high intensity interval training had beneficial effects in most of the studies but showed similar or identical results on some scales of interest, and small and trivial increases on other scales compared to mict.(24) several short-term studies (4 to 8 weeks) have shown that compared to mict, participants in hiit expr essed gr e ater e nj oyment and adherence for the program.(25,26) one study that combined both supervised and independent rounds of hiit compared to mict for 8 weeks in obese adults, showed no differences between the level of enjoyment and adherence of hiit compared to mict. (2 7) one long-ter m study on the adherence of unsupervised hiit in an obese population for 12 months showed that there was a significant decrease in adherence from 60.8% at baseline to 19.6% at the end of the study.(28) in different age groups, studies on young adults in one university have also shown low adherence to the programs.(29) meanwhile a study on the aging population have shown good adherence to the program.(19) one of the possible biases in these studies might come from the nature of the supervision during the research. the level of intensity may also affect the level of enjoyment; hiit performed at higher intensity with low recovery time ratio may bring a negative effect on the overall enj oyment of hiit. (3 0) to effectively know the real adherence rate of hiit, more long-term unsupervised studies on different populations have to be conducted. physiological changes in hiit several acute physiological responses that were observed after a session of hiit include increase in heart rate, cortisol, catecholamines, growth hormone, glucose and plasma lactate levels, glycer ol, de pletion of adenosine triphosphate (atp), phosphocreatine (pcr) and glycogen stores as well as a decrease in parasympathetic reaction.(31) the increase in heart rate may differ depending on the hiit protocol used, however higher peak heart rate is observed in long exercises compared to the short ones. (3 2) hormone s such as epin ephrine, norepinephrine, growth hormone, interleukin 6, free cortisol and adrenocorticotropic hormone (act h) a re all incr ea sed after hiit. (3 3) compared to moderate intensity exercise, the increases in adrenocorticotropic hormone (acth), free cortisol and growth hormone were higher after hiit.(33) this may be significant, as catecholamine, cortisol and growth hormone play a role in lipolysis.(34) the lactate level also increases during hiit and the increase seems to be higher in trained athletes. (35) another study that compared hiit to high volume training (hvt), also found that 5 weeks of hiit intervention significantly increase maximal lactate concentration while hvt decreases it. a higher rate of lactate accumulation correlates with higher anaerobic energy yield, which could lead to better performance.(36) hiit also seems to increase the release of fatty acids, as indicated by the increased levels of glycerol.(35) blood glucose is increased after hiit and is higher than after moderate intensity exercise.(33) parasympathetic activation also seems to decrease after repeated sprint exercise; this might be caused by the increase in sympathetic activity as well as the continued elevation of adrenergic factors and other local metabolites (epinephrine, norepinephr ine, h +, lactate, e tc.) during recovery.(37) in the metabolic response, first atp and pcr were decreased in hiit followed with a decrease in glycogen stores by anaerobic glycolysis.(31,38,39) the eff ect of hiit in incr easing cardiorespiratory fitness may be caused by multiple factors. the beneficial effect for aerobic fitness by hiit is likely to be caused by central and peripheral adaptation.(40) central adaptation includes increase in stroke volume caused by the increase in cardiac contractility.(31,41) peripheral adaptations include increased capillary density and mitochondrial adaptation.(40) a study in skeletal musc le a daptation showed an improvement in mitochondrial content and activity.(40) another contribution to aerobic fitness might be the result of the degradation of a b c 213 recruitment and use of glycogen.(48) the increase in insulin-independent glucose transporter type 4 (glut4) translocation or muscle glut4 content may also play a part.(48) the blood pressure reduction in hiit may come from the improvement in endothelial function. one study has shown that hiit in older hypertensive patients increased apelin and nitric oxide, a factor in endothelial vasodilatation, and decreased endothelin-1.(49) better vascular function outcome in hiit may also be caused by increased shear stress in the arterial wall.(22) adverse effects the high intensity in hiit ha s been considered for adverse effects, since vigorous physical activity may transiently increase the risk of cardiovascular events.(50) however, current evidence reveals that hiit is relatively safe, even in individuals with a cardiac condition. a metaanalysis that compared the effect of hiit and mict in cardiac rehabilitation showed that there were no recorded events of death or cardiac events that required hospitalization in 953 participants (465 hiit and 488 mict).(18) another study that examined the use of mict and hiit in 4846 coronary heart disease patients, showed that the risk of any cardiovascular event was low in both groups, with only a single fatal incident reported in the mict group after over 129,456 exercise hours and two non-fatal incidents in the hiit group after over 46,364 exercise hours.(51) conclusion when compared to mict, hiit is superior in improving cardiorespiratory fitness, reducing blood pressure and increasing insulin sensitivity. while some have been cautious about the nature of the high intensity, hiit is generally safe even in special populations, such as elderly people or individuals with coronary heart disease. the advantage of adherence in hiit compared to mict is not clear, since some studies have shown greater enjoyment and adherence levels but univ med vol. 38 no.3 phosphocreatine during repeated hiit.(31) in anaerobic fitness, a study showed an increase in anaerobic capacity after two weeks of sit.(42) the theorized mechanism for fat loss during hiit includes an increased in high intensity exercise, fat oxidation and the loss of appetite.(31) repeated high intensity exercise may cause an inhibition of anaerobic glycogenolysis, so that atp is der ived mainly from intramuscular triacylglycerol storage and pcr degradation.(43) increased venous glycerol after hiit could be caused by the release of fatty acids from adipose and intramuscular triacylglycerol stores.(35) other studies have also shown that after a number of sessions in hiit there was an increased capacity for the oxidation of fatty acids in skeletal muscle and the whole body.(44) elevated hormones such as growth hormone, catecholamine and cortisol may also play a role in driving lipolysis.(33,34) in rats, hard exercise has been repeatedly reported to reduce food intake and reduce food intake by facilitating the release of corticotropin releasing factor (crf) a potent anorectic peptid.(45) animal studies in rats have also shown that high intensity activities such as running facilitate the release of corticotrophin releasing factor, a potent anorectic peptide.(45) a recent study on individuals with type 2 diabetes has also found that hiit influences appetite after 12 weeks by stimulating appetite hormones, reducing tnf-α, peptide yy (pyy) and ghrelin and increasing glucagon-like peptide1 (glp-1). however, the changes in appetite hormones may differ between lean and obese participants, with obese patients with type 2 diabetes being more likely to elicit appetite hormones in hiit.(46) the mechanism of insulin sensitivity in hiit is still unclear. increased insulin sensitivity might be caused by succ essful we ight loss. 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